key: cord- -msz au b authors: gershan, william m. title: cough date: - - journal: practical strategies in pediatric diagnosis and therapy doi: . /b - - - - . - sha: doc_id: cord_uid: msz au b nan the previous response or lack of response to some therapies for recurrent and chronic cough can provide important information (see table - ). furthermore, some coughs may be caused or worsened by medications (table - ) . a history of accompanying signs or symptoms, whether localized to the respiratory tract (wheeze, stridor) or elsewhere (failure to thrive, frequent malodorous stools) can give important clues (table - ; see tables - and - ) . it is essential to remember that the daily language of the physician is full of jargon that may be adopted by parents but with a different meaning from that understood by physicians. if a parent says that a child "wheezes" or "croups" or is "short of breath," it is important to find out what the parent means by that term. because many disorders of childhood have genetic or nongenetic familial components, the family history can provide helpful information: are there older siblings with cystic fibrosis (cf) or asthma? is there a coughing sibling whose kindergarten class has been closed because of pertussis? similarly, the key to today's problems may be found in the past: was the child premature and, if so, did he or she spend a month on the ventilator, and does he or she now have chronic lung disease (bronchopulmonary dysplasia)? did the toddler choke on a carrot or other food months ago? did the child receive a bone marrow transplant a year ago? is the child immunized? did the infant have a tracheoesophageal fistula repaired in the neonatal period? *infections include upper (pharyngitis, sinusitis, tracheitis, rhinitis, otitis) and lower (pneumonia, abscess, empyema) respiratory tract disease. † anatomic abnormality includes tracheobronchomalacia, tracheoesophageal fistula, vascular ring, abnormal position, or take-off of large bronchi. common; less common; much less common. cf, cystic fibrosis; ger, gastroesophageal reflux. initial inspection often reveals the seriousness of an illness: is the child struggling to breathe (dyspnea)? does the child have an anxious look? can the child be calmed or engaged in play? is the child's skin blue (representing cyanosis) or ashen? does the child appear wasted, with poor growth that may indicate a chronic illness? the respiratory rate is often elevated with parenchymal lung disease or extrathoracic obstruction. respiratory rates vary with the age of the child (fig. - ) and with pulmonary infection, airway obstruction, activity, wakefulness and sleep, fever, metabolic acidosis, and anxiety. odors may also give helpful clues. does the examining room or the clothing smell of stale cigarette smoke? is there a foul odor from a diaper with a fatty stool, which may suggest pancreatic insufficiency and cf? is the child's breath malodorous, as can be noticed in sinusitis, nasal foreign body, lung abscess, or bronchiectasis? it has been said that the examination of the lungs begins at the fingertips. cyanotic nail beds suggest hypoxemia, poor peripheral circulation, or both. the examiner looks for the presence of digital clubbing ( fig. - ) , which makes asthma or acute pneumonia extremely unlikely. the absence of digital clubbing but a history of severe chronic cough in an older child makes cf unlikely. the shape of the chest gives information. is the anteroposterior (ap) diameter increased, which indicates hyperinflation of the lungs from obstruction of small airways (asthma, bronchiolitis, cf)? is this diameter small, as can be seen with some restrictive lung diseases with small lung volumes (muscular dystrophy, spinal muscular atrophy)? the normal infant has a "round" chest configuration, with the ap diameter of the chest about % of the transverse (lateral) diameter. with growth, the chest becomes more flattened in the ap dimension, and the ap-to-transverse ratio is closer to % to %. although obstetric calipers can be used to give an objective assessment of the ap diameter of the chest, most clinicians rely on their subjective assessment of whether the diameter is increased: does the patient look "barrel-chested"? intercostal, subcostal, suprasternal, and supraclavicular retractions (inspiratory sinking in of the soft tissues) indicate increased effort of breathing and reflect both the contraction of the accessory muscles of respiration and the resulting difference between intrapleural and extrathoracic pressure. retractions occur most commonly with obstructed airways (upper or lower), but they may occur with any condition leading to the use of the accessory muscles. any retractions other than the mild normal depressions seen between an infant's lower ribs indicate a greater-than-normal work of breathing. less easy to notice than intercostal retractions is their bulging out with expiration in a child with expiratory obstruction (asthma). contraction of the abdominal muscles with expiration is easier to notice and is another indication that a child is working harder than normal to push air out through obstructed airways. inspection of the spine may reveal kyphosis or scoliosis. there is a risk of restrictive lung disease or static pneumonia if the curvature is severe. palpating the trachea, particularly in infants, may reveal a shift to one side, which suggests loss of volume of the lung on that side or extrapulmonary gas (pneumothorax) on the other side. placing one hand on each side of the chest while the patient breathes may enable the examiner to detect asymmetry of chest wall movement, either in timing or in degree of expansion. the former indicates a partial bronchial obstruction, and the latter suggests a smaller lung volume, voluntary guarding, or diminished muscle function on one side. palpating the abdomen gently during expiration may allow the examiner to feel the contraction of the abdominal muscles in cases of expiratory obstruction. palpation for tactile fremitus, the transmitted vibrations of the spoken word ("ninety-nine" is the word often used to accentuate these vibrations), helps determine areas of increased parenchymal density and hence increased fremitus (as in pneumonic consolidation) or decreased fremitus (as in pneumothorax or pleural effusion). the percussion note determined by the examiner's tapping of one middle finger on the middle finger of the other hand, which is firmly placed over the patient's thorax, may be dull over an area of consolidation or effusion and hyperresonant with air trapping. percussion can also be used to determine diaphragmatic excursion. the lowest level of resonance at inspiration and expiration determines diaphragmatic motion. because lung sounds tend to be higher-pitched than heart sounds, the diaphragm of the stethoscope is better suited to pulmonary auscultation than is the bell, whose target is primarily the lower-pitched heart sounds (table - ). the adult-sized stethoscope generally is far superior to the smaller pediatric or neonatal diaphragms, even for listening to small chests, because its acoustics are better. the two-headed stethoscope enables the user to hear homologous segments of both lungs simultaneously in order to identify instances in which there is a delay in air entry or exit. the traditional single-headed stethoscope is adequate in most children with cough. the ability to recognize normal breath sounds comes with practice ( fig. - ). adventitious sounds come in a few varieties, namely, stridor, crackles, rhonchi, and wheezes. other sounds should be described in clear, everyday language. stridor is a continuous musical sound usually heard on inspiration and is caused by narrowing in the extrathoracic airway, as with croup or laryngomalacia. crackles are discontinuous, representing the popping open of air-fluid menisci as the airways dilate with inspiration. fluid in very common and suggests the diagnosis; ++, common; +, uncommon; -, almost never and makes examiner question the diagnosis. respiratory disorders larger airways causes crackles early in inspiration (congestive heart failure); crackles that tend to be a bit lower in pitch ("coarse" crackles) than the early, higher-pitched ("fine") crackles are associated with fluid in small airways (pneumonia). although crackles usually signal the presence of excess airway fluid (pneumonia, pulmonary edema), they may also be produced by the popping open of noninfected fibrotic or atelectatic airways. fine crackles are not audible at the mouth, whereas coarse crackles may be. crackles is the preferred term, rather than the previously popular "rales." rhonchi, or "large airway sounds," are continuous gurgling or bubbling sounds typically heard during both inhalation and exhalation. these sounds are caused by movement of fluid and secretions in larger airways (asthma, viral uri). rhonchi, unlike other sounds, may clear with coughing. wheezes are continuous musical sounds (lasting longer than milliseconds), caused by vibration of narrowed airway walls, as with asthma, and perhaps vibration of material within airway lumens. these sounds are much more commonly heard during expiration than inspiration. the chest radiograph is often the most useful diagnostic test in the evaluation of the child with cough. table - highlights some of the radiographic features of the most common causes of cough in pediatric patients. radiographic findings are often similar for a number of disorders, and thus these studies may not indicate a definitive diagnosis. chest films are normal in children with psychogenic (habit) cough and in children with sinusitis or gastroesophageal reflux (ger) as the primary cause of cough. a normal chest radiograph indicates the unlikelihood of pneumonia caused by respiratory syncytial virus (rsv), influenza, parainfluenza, adenovirus, chlamydia species, or bacteria. although children with cough resulting from cf, mycoplasma species, tuberculosis, aspiration, a bronchial foreign body, or an anatomic abnormality usually have abnormal chest radiographs, a normal radiograph does not exclude these diagnoses. hyperinflation of the lungs is commonly seen on chest films of infants with rsv bronchiolitis or chlamydia pneumonia, and a lobar or round (coin lesion) infiltrate is the radiographic hallmark of bacterial pneumonia. the diagnosis of sinusitis cannot be sustained with normal sinuses on radiograph or computed tomography (ct) scan. the white blood cell (wbc) count may help exclude or include certain entities for a differential diagnosis, but, with the possible exception of pertussis, can seldom establish a diagnosis with certainty. a wbc count of , with % lymphocytes strongly suggests pertussis, but not every child with pertussis presents such a clear hematologic picture. the presence of a high number or large proportions of immature forms of wbcs suggests an acute process, such as a bacterial infection. immunoglobulins provide supportive evidence for a few diagnoses, such as chlamydial infection, which rarely occurs without elevated serum concentrations of immunoglobulins g and m. specific bacteriologic or virologic diagnoses can be made in a number of disorders causing cough, including rsv, influenza, parainfluenza, adenovirus, and chlamydia pneumonia. in most cases, these diagnoses are based on culturing the organism from nasopharyngeal washings. in some cases, the viruses can be rapidly identified with immunofluorescence or amplification of viral genome through polymerase chain reaction (pcr). in bacterial pneumonia, the offending organism can be cultured from the blood in a small proportion ( %) of patients. a positive culture provides definitive diagnosis, but a negative culture specimen is not helpful. throat cultures are seldom helpful (except in cf) in identifying lower respiratory tract organisms. sputum cultures and gram stains may help guide initial empirical therapy in older patients with pneumonia or purulent bronchitis, but their ability to identify specific causative organisms with certainty (again with the exception of cf) has not been shown clearly. infants and young children usually do not expectorate but, rather, swallow their sputum. specimens obtained via bronchoscopy may be contaminated by mouth flora, but heavy growth of a single organism in the presence of polymorphonuclear neutrophils certainly supports the organism's role in disease. if pleural fluid or fluid obtained directly from the lung via needle aspiration is cultured, the same rules apply: positive cultures are definitive, but negative cultures are not. bacterial antigen detection in serum or urine by various techniques (latex agglutination) can help identify pneumococcus and haemophilus influenzae type b. a number of specific tests can help to establish diagnoses in a child with cough (see table - ). these include a positive response to bronchodilators in a child with asthma; visualizing the red, swollen epiglottis in epiglottitis (to be done only under very controlled conditions, as described later); the bronchoscopic visualization of the peanut, plastic toy, or other offender in foreign body aspiration; a positive purified protein derivative in tuberculosis; and several studies of the esophagus in ger. several imaging techniques, such as ct or magnetic resonance imaging (mri), can help to delineate various intrathoracic anatomic abnormalities. finally, multiple tests can be employed to confirm the diagnosis of cf (table - ). infections are the most common cause of acute cough in all age groups and are responsible for some chronic coughs. the age of the patient has a large impact on the type of infection. viral upper respiratory infections (common cold); croup (laryngotracheobronchitis); viral bronchiolitis, particularly with rsv; and viral pneumonia are the most frequently encountered respiratory tract infections and hence the most common causes of cough in infancy. viral illness may predispose to bacterial superinfection (croup and staphylococcus aureus tracheitis or influenza and h. influenzae pneumonia). cold symptoms and signs usually include stuffy nose, with nasal discharge (rhinorrhea); sore throat and sneezing frequently occur. there may be fever, constitutional signs (irritability, myalgias, headache), or both. cough is common and may persist for to days. the mechanism by which upper respiratory infections cause cough in children is undetermined. in adults, it is generally thought that "postnasal drip"-that is, nasal or sinus secretions draining into the posterior nasopharynx-causes cough and, in fact, may be one of the most frequent causes of cough. indeed, sinus ct in older patients , or the phalangeal depth ratio, is normally less than but increases to more than with finger clubbing. the dpd/ipd ratio can be measured with calipers or, more accurately, with finger casts. the hyponychial angle is measured from lateral projections of the finger contour on a magnifying screen and is normally less than degrees but greater than degrees with finger clubbing. schamroth's sign is useful for bedside assessment. the dorsal surfaces of the terminal phalanges of similar fingers are placed together. with clubbing, the normal diamond-shaped aperture or "window" at the bases of the nail beds disappears, and a prominent distal angle forms between the end of the nails. in normal subjects, this angle is minimal or nonexistent. ( whispering pectoriloquy produces clearer sounding whispered words (e.g., "ninety-nine"). pmi, point of maximal impulse. with colds often reveals involvement of the sinus mucosa. whether this is true in children remains undetermined. other authorities believe that cough in a child with a cold indicates involvement (inflammation or bronchospasm) of the lower respiratory tract. the physician's bias on this matter will probably influence how to treat the child with cough accompanying a cold. in adults, the cough of the common cold may respond to a combination antihistaminedecongestant preparation, presumably from the decreased postnasal drip. it is uncertain whether such treatment is effective or indicated in children, particularly young infants, in whom toxicity of the drugs may be a greater concern than in adults. common viral pathogens include rhinovirus, rsv, and parainfluenza virus. the differential diagnosis includes allergic rhinitis, which often demonstrates clear nasal secretions with eosinophils and pale nasal mucosa, in contrast to mucopurulent nasal secretions with neutrophils and erythematous mucosa. infectious croup (see chapter ) is most common in the first years of life. its most dramatic components are the barking ("croupy") cough and inspiratory stridor, which appear a few days after the onset of a cold. in most cases, the patient has a low-grade fever, and the disease resolves within a day or two. in severe cases, the child can be extremely ill and is at risk for complete laryngeal obstruction. there may be marked intercostal and suprasternal retractions and cyanosis. stridor at rest signifies significant obstruction. diminishing stridor in a child who is becoming more comfortable is a good sign, but diminishing stridor in and of itself is not necessarily good: if the child becomes fatigued because of the tremendous work of breathing through an obstructed airway and can no longer breathe effectively, smaller-than-needed tidal volumes make less noise. low yield on culture in paroxysmal stage. pleural effusion relatively common. blood culture positive in %; needle aspiration of pleural fluid or lung fluid may yield organism; bacterial antigen in urine. in older infants and children, common pathogens include pneumococci and group a streptococci; staphylococcus aureus is rare and may be associated with pneumatoceles or empyema. localized hyperinflation is common; localized atelectasis in common; inspiratory-expiratory films may show ball-valve obstruction. esophageal biopsy specimen shows esophagitis. multilobular or multisegmental, dependent lobes. (?) lipid-laden macrophages from bronchoscopy or gastric washings; barium swallow or radionuclide study showing aspiration. right-sided arch, mass effect on airways, mass identified; magnetic resonance imaging (mri). bronchoscopy; computed tomography; mri. +++, almost always-if not present, must question diagnosis; ++, common; +, less common; -, seldom-if present, must question diagnosis. +bld cult, blood culture may be positive; bronch, bronchoscopy can reveal the foreign body; diff, diffuse or scattered; ↑ eos, increased eosinophil count; esoph ph, prolonged esophageal ph probe monitoring; ge, gastroesophageal; hyper, hyperinflated; ig, immunoglobulin; infil, infiltrates; ↑ ly, increased lymphocyte count; +np cult, nasopharyngeal culture positive for specific organism; paraflu, parainfluenza virus; pcr, polymerase chain reaction; ↑ pmn, increased polymorphonuclear neutrophil count; ppd, purified protein derivative (tb); rad, reactive airways disease; rsv, respiratory syncytial virus; tb, tuberculosis; ↑ wbc, increased white blood cell count. it is important to distinguish croup from epiglottitis in the child with harsh, barking cough and inspiratory stridor because the natural histories of the two diseases are quite different (see table - ). epiglottitis occurs more commonly in toddlers than in infants (see chapter ). treatment of mild croup is usually not needed. for decades, pediatricians have recommended putting a child with croup in a steamy bathroom or driving to the office or emergency room with the car windows rolled down. (it is likely that these remedies are effective because of the heat exchange properties of the upper airway; air that is cooler or more humid than the airway mucosa will serve to cool the mucosa, thus causing local vasoconstriction and probably decreasing local edema.) in a child who has stridor at rest, hospitalization is indicated. symptomatic, often dramatic relief through decreased laryngeal edema can usually be achieved with aerosolized racemic epinephrine ( . % solution, . to . ml/dose). it is essential to remember that the effects of the epinephrine are transient, lasting only a few hours, although the course of the illness is often longer. the result is that when the racemic epinephrine's effect has worn off, the child's cough and stridor will probably be as bad or even worse than before the aerosol was administered. this is not a "rebound" effect: the symptoms are not worse because of the treatment but, rather, because of the natural progression of the viral illness. repeating the aerosol will probably again have a beneficial effect and reduce the likelihood of requiring a tracheotomy or endotracheal intubation. a child who responds favorably to such an aerosol needs to be observed for several hours because further treatment may be needed. a single dose of dexamethasone ( . mg/kg orally, intramuscularly, or intravenously) reduces the severity and hastens recovery. aerosolized steroids (budesonide) may also be effective in patients with mild to moderately severe croup. bronchiolitis is a common and potentially serious lower respiratory tract disorder in infants (see chapters and ). it is caused usually by rsv but on occasion by parainfluenza, influenza, or adenovirus. it occurs in the winter months, often in epidemics. rsv bronchiolitis is seen uncommonly in children older than years. typically, "coldlike" symptoms of rhinorrhea precede the harsh cough, increased respiratory rate, and retractions. respiratory distress and cyanosis can be severe. the child's temperature is seldom elevated above °c. the chest is hyperinflated, widespread crackles are audible on inspiration, and wheezing marks expiration. the most striking laboratory abnormalities are in the chest radiograph, which invariably reveals hyperinflation, as depicted by a depressed diaphragm, with an enlarged retrosternal air space in as many as % of patients, peribronchial thickening in approximately %, and consolidation and/or atelectasis in % to %. the diagnosis is confirmed with demonstration of rsv by immunofluorescent stain or pcr of nasopharyngeal washings. in most cases, no treatment is needed because the disease does not interfere with the infant's eating or breathing. apnea is a common complication of rsv bronchiolitis in young infants and may necessitate close monitoring. in severe cases, often those in which there is underlying chronic heart, lung, or immunodeficiency disease, rsv can be life-threatening. in these cases, hospital care with supplemental oxygen and intravenous fluids is indicated. the effect of aerosolized bronchodilators is not clear but is probably beneficial in some infants. the aerosolized antiviral agent ribavirin may be beneficial for the sickest infants. it is expensive and difficult to administer; it needs to be given to hours per day (some studies advocate hours three times a day) and may block ventilator tubing and valves. ribavirin may improve oxygenation but should not be used in lieu of mechanical ventilation in patients with hypoxia and hypercarbia (respiratory failure). viral pneumonia can be similar to rsv bronchiolitis in its manifestation, with cough and tachypnea, after a few days of apparent upper respiratory infection. there can be variable degrees of fever and of overall illness. infants and children with viral pneumonia may appear relatively well or, particularly with adenovirus, may have a rapidly progressive course, which ends in death within a few days after the onset of illness. frequent symptoms include poor feeding, cough, cyanosis, fever (some patients may be afebrile), apnea, and irt, immunoreactive trypsinogen. respiratory disorders rhinorrhea. frequent signs include tachypnea, retractions, crackles, and cough. cyanosis is less common. the most common agents causing viral pneumonia in infancy and childhood are rsv, influenza, and parainfluenza. adenovirus is less common, but it is important because it can be severe and leave residua, including bronchiectasis and bronchiolitis obliterans. adenovirus pneumonia is often accompanied by conjunctivitis and pharyngitis, in addition to leukocytosis and an elevated erythrocyte sedimentation rate (esr); the esr and leukocyte count are usually not elevated in other types of viral pneumonia. additional viral agents include enteroviruses and rhinovirus. radiographs most often reveal diffuse, bilateral peribronchial infiltrates, with a predilection for the perihilar regions, but occasionally lobar infiltrates are present. pleural effusions are not common. on occasion, if an infant is extremely ill, bronchoscopy with bronchoalveolar lavage may be indicated to isolate the virus responsible for the pneumonia. treatment is largely supportive, with oxygen and intravenous fluids. mechanical ventilation may be necessary in a small minority of infants. in young infants, the afebrile pneumonia syndrome may be caused by chlamydia, ureaplasma, or mycoplasma species; cytomegalovirus; or pneumocystis carinii. in this syndrome, cough and tachypnea are common. severe pneumonia may develop in neonates as a result of herpes simplex. pertussis is an extremely important cause of lower respiratory tract infection in infants and children. the causative organism, bordetella pertussis, has a tropism for tracheal and bronchial ciliated epithelial cells; thus, the disease is primarily bronchitis, but spread of the organism to alveoli, or secondary invasion by other bacteria, can cause pneumonia. the disease can occur at any age, from early infancy onward, although its manifestations in young infants and in those who have been partially immunized may be atypical. most commonly, pertussis has three stages: • catarrhal, in which symptoms are indistinguishable from the common cold • paroxysmal, dominated by repeated forceful, paroxysmal coughing spells; many spells may be punctuated by an inspiratory "whoop," post-tussive emesis, or both • convalescent, in which the intensity and frequency of coughing spells gradually diminish each stage typically lasts to weeks, except the paroxysmal stage, which lasts many weeks. the chinese term for pertussis translates to " days of cough." most children are entirely well between coughing spells, when physical findings are remarkably benign. infants younger than months of age may have the most severe illness, and in this age group, the rate of mortality from pertussis is as high as %. diagnosis can be difficult because the definitive result-namely, culturing the organism from nasopharyngeal secretions-requires special culture medium (bordet-gengou, which must be prepared fresh for each collection). culture specimens are much less likely to be positive during the paroxysmal stage than during the catarrhal stage, when the diagnosis is not being considered. fluorescent antibody stains (for the antigen) of secretions are also helpful if they are positive, but, similarly, they are more likely to be positive before the paroxysmal stage. serum antibodies against b. pertussis may occasionally be helpful, although they are often difficult to interpret in immunized individuals. perhaps the laboratory test that is most helpful is the wbc count, which is typically elevated; values are as high as , to , , with lymphocytes predominating. pcr is also useful. chest radiographic findings are nonspecific. infants with severe disease may require hospitalization. treatment is largely supportive, with oxygen, fluids, and small frequent feedings for patients who do not tolerate their normal feedings. treatment with erythromycin estolate ( mg/kg/day for days, every hours, orally) decreases infectivity and may ameliorate the course of the disease if given during the catarrhal stage. studies suggest that azithromycin or clarithromycin may also be effective. in some patients, aerosolized bronchodilators (albuterol) or systemic steroids may help, although such treatment is controversial. cough suppressants are not helpful, but good hydration, oxygenation, and nutrition, in addition to not disturbing the infant, are important. complications include those related to severe coughing (table - ) and those specific to pertussis, such as seizures and encephalopathy. pertussis is prevented by three primary immunizations (at , , and months of age) and regular booster immunizations at to months and to years of age. pertussis infection produces lifelong immunity. chlamydia trachomatis can cause pneumonia in young infants, particularly those aged to weeks. cough, nasal congestion, lowgrade or no fever, and tachypnea are common. conjunctivitis is an important clue to chlamydial disease but is present in only % of infants with chlamydial pneumonia at the time of presentation. affected infants may have a paroxysmal cough similar to that of pertussis, but post-tussive emesis is less common. crackles are commonly heard on auscultation, but wheezing is much less common than the overinflated appearance of the lungs on radiographs would suggest. the organism may be recovered from the nasopharynx by culture or antigen testing. the complete blood cell count may reveal eosinophilia. chlamydial infection responds to oral erythromycin therapy ( to mg/kg/day, every hours for to days). ureaplasma urealyticum pneumonia is difficult to diagnose but causes cough in some infants. there are no particularly outstanding features to distinguish this relatively uncommon infection from viral pneumonias. bacterial pneumonia is relatively less common in infants than is viral pneumonia but can cause severe illness, with cough, respiratory distress, and fever. chest films are strikingly abnormal; the wbc count is elevated. treatment is with broad-spectrum intravenous antibiotics effective against pneumococci, group a (possibly b) streptococci, and, if illness is severe, s. aureus. cefotaxime with or without nafcillin may be effective. in early childhood, as children attend day care and nursery schools, they are constantly exposed to respiratory viruses to which they have little or no immunity (e.g., rhinoviruses, adenoviruses, parainfluenza, and coxsackievirus). such children may have as many as to or even more colds in a year. the remarks concerning colds and cough in infants (see previous discussion) apply to this older age group. the differential diagnosis of rhinorrhea is noted in table - . the sinuses may become the site for viral and subsequent secondary bacterial infection spreading from the nasopharynx (fig. - ) . the signs and symptoms are usually localized, including nasal congestion, a feeling of "fullness" or pain in the face ( fig. - ) , headache, sinus tenderness, day or night cough, and fever. maxillary toothache, purulent nasal discharge for more than days, a positive transillumination (opacification), and a poor response to oral antihistamines or nasal decongestants are important clues. sinus radiographs or (more accurate) ct scan may facilitate the diagnosis of sinusitis by demonstrating opacification of the sinus with mucosal thickening. sinusitis is thought to be a cause of cough in adults and can probably be listed, with lower certainty, as a cause of cough in children. sinusitis is frequently seen in other conditions known to cause cough, especially cf, asthma, and ciliary dyskinesia. it may be difficult to ascertain whether the cough is a direct result of the sinus infection or the underlying problem (purulent bronchitis in the child with cf or ciliary dyskinesia, exacerbation of asthma). in the first two situations, it may not matter because treatment is the same. in the case of the child with asthma, it is important to treat the asthma with bronchodilating and antiinflammatory agents, as well as to treat the infected sinuses with antibiotics. the treatment of sinusitis involves the use of oral antibiotics active against the common pathogens (streptococcus pneumoniae, nontypable h. influenzae, moraxella catarrhalis, and, in rare cases, anaerobic bacteria or streptococcus pyogenes). treatment regimens include the use of amoxicillin, amoxicillin-clavulanate, cefuroxime, cefpodoxime, or cefdinir. amoxicillin is considered the initial agent of choice. oral (pseudoephedrine, phenylephrine) or topical (phenylephrine, oxymetazoline) decongestants may be of benefit by increasing the patency of the sinus ostia, which permits drainage of the infected and obstructed sinuses. oral antihistamines may benefit patients with an allergic history. treatment with antimicrobial agents should continue for at least days after the patient has responded. this may require to days of therapy. many patients with presumed sinusitis recover without antibiotic therapy. complications of acute sinusitis include orbital cellulitis, abscesses (orbital, cerebral), cranial (frontal) osteomyelitis (pott puffy tumor), empyema (subdural, epidural), and thrombosis (sagittal or cavernous sinus). see previous text and chapter . viral pneumonia. the features discussed for viral pneumonia in infants are relevant for viral pneumonia in older children. the differentiation of viral or atypical pneumonia from classical bacterial pneumonia is noted in table - . bacterial pneumonia. bacterial pneumonia is more common in toddlers and older children than in infants. the most common pathogen is s. pneumoniae; other bacterial causes vary with age ( table - ) . cough may not be as prominent a presenting symptom or sign as tachypnea and grunting, sometimes (especially in infants) with vomiting (see table - ). raised respiratory rates (≥ in infants to months old, ≥ in children to years old) plus retractions and grunting with or without hypoxia (oxygen saturation < %) have a high specificity and sensitivity for pneumonia. chest pain, abdominal pain, headache, or any combination of these symptoms may occur. upper lobe pneumonia may produce meningeal signs, and lower lobe involvement may cause abdominal pain and an ileus. examination of the chest shows tachypnea but may be otherwise surprisingly normal. in older children, there may be localized dullness to percussion, with crackles or amphoric (bronchial) breath sounds over a consolidated lobe. the chest film may be normal in the first hours of the illness, inasmuch as the radiographic findings often lag behind the clinical manifestations. nonetheless, both posteroanterior and lateral views are the main diagnostic tools; lobar consolidation is usual, with or without pleural effusion. in infants, the pattern may be more diffuse and extensive. some clinical and radiographic features may be suggestive of the bacterial cause of pneumonia. children (especially infants) with staphylococcal pneumonia are more likely to have a rapid overwhelming course. staphylococcal pneumonia may be accompanied by more extensive radiographic abnormalities, including multilobar consolidation, pneumatocele formation, and extensive pleural (empyema) fluid. the presence of a pleural effusion is not helpful in indicating the specific bacterial diagnosis because other bacterial pneumonias may be accompanied by pleural effusion. pleural effusions may represent a reactive parapneumonic effusion or an empyema. pleural fluid may be characterized as transudate, exudate, or complicated empyema, the latter necessitating closed chest drainage with a chest tube (table - ). if the effusion is of sufficient size, as demonstrated by a lateral decubitus film or ultrasonography, a thoracentesis is indicated to differentiate the nature of the effusion and to identify possible pathogens. differentiating among the causes of bacterial pneumonia can be done with certainty only with positive cultures from blood, pleural fluid, fluid obtained by direct lung tap, or, in rare cases, sputum. current or previous antibiotic treatment diminishes the yield of such cultures. the presence of bacterial antigens in the urine for s. pneumoniae or h. influenzae provides strong evidence of the causative agent. bronchoscopy with or without lavage may yield helpful specimens from the progressively ill child or the child who has not responded promptly to empirical antibiotics. treatment is with antibiotics. cefotaxime or ceftriaxone is the drug of choice for the previously healthy child who requires hospitalization with lobar pneumonia. for the critically ill child, vancomycin may be considered for possible resistant s. pneumoniae. many children with pneumonia do well with oral antibiotics (amoxicillin, amoxicillin-clavulanate, oral cephalosporins) and respond within hours to the first dose. a smaller number may require hospitalization and intravenous antibiotics along with supportive measures (e.g., oxygen and intravenous fluids). repeated or follow-up chest films may remain abnormal for to weeks after pneumonia and are not indicated for a single episode of uncomplicated pneumonia (i.e., no effusion, no abscess, and good response to treatment). children with suspected pneumococcal pneumonia must be monitored carefully because of the possibility of resistance to penicillin and cephalosporin. mycoplasma pneumoniae is a common cause of pneumonia among school-aged children. the disease often occurs in community outbreaks in the fall months. the illness typically begins with table - ); atypical in terms of extrapulmonary manifestations, low-grade fever, patchy diffuse infiltrates, poor response to penicillin-type antibiotics, and negative sputum gram stain. sars, severe acute respiratory syndrome. extrapulmonary symptoms (i.e., sore throat, myalgias, headache, fever), which then progress to include worsening cough, paroxysmal at times. patients do not often appear acutely ill, but cough may persist for weeks. there may be no specific abnormalities on the chest examination, although a few crackles may be heard, and about one third of younger patients wheeze. the radiographic findings in mycoplasmal pneumonia can mimic almost any intrathoracic disease; scattered infiltrates with nonspecific "dirty" lung fields, predominantly perihilar or lower lobes, are common, and lobar infiltrates and pleural effusion are occasionally seen. laboratory data (complete blood cell count, esr, sputum culture) may not be helpful. a rise in antimycoplasmal immunoglobulin g over to weeks may be demonstrated but is seldom helpful in guiding therapy. a positive immunoglobulin m response may be useful, although it can persist in serum for several months and, consequently, may not indicate current infection. pcr may be helpful. the cold agglutinin test yields positive results in about % of patients with mycoplasmal pneumonia, but they are also positive in other conditions, including adenovirus infection. the more severe the illness is, the greater is the frequency of positive cold agglutinins. the diagnosis is often made from the history of an older child who has a lingering coughing illness in the setting of a community outbreak, unresponsive to most (non-erythromycin) antibiotic regimens. treatment is with erythromycin (azithromycin or clarithromycin are alternatives in children younger than years, whereas tetracycline or doxycycline can be administered to older children), which usually shortens the course of the illness. extrapulmonary complications of mycoplasmal infection include aseptic meningitis, transverse myelitis, peripheral neuropathy, erythema multiforme, myocarditis, pericarditis, hemolytic anemia, and bullous otitis media (myringitis). in patients with sickle cell anemia, severe respiratory failure and acute chest syndrome may develop. infection with chlamydia pneumoniae mimics respiratory disease resulting from m. pneumoniae, inasmuch as it occurs in epidemics, is seen in older children, and produces an atypical pneumonia syndrome and pharyngitis. the incidence of tuberculosis is increasing as a result of acquired immunodeficiency syndrome, homelessness, urban poverty, and immigration from endemic countries. tuberculosis must be considered in the child with chest disease that is not easily explained by other diagnoses, especially if the child has been exposed to an adult with active tuberculosis. nonetheless, tuberculosis is an infrequent cause of cough in children, even in those with active disease. the diagnosis is made primarily by skin testing (purified protein derivative [ppd]); a history of contact with a person who has tuberculosis; and recovery of the organism from sputum, bronchoalveolar lavage, pleural fluid or biopsy, or morning gastric aspirates (table - ). the yield from these procedures is relatively low, even from children with active pulmonary tuberculosis. the patterns of disease in normal hosts include primary pulmonary tuberculosis, with subsequent inactivation usually noted in young children and reactivation pulmonary disease among adolescents. primary pulmonary disease is often noted as a lower or middle lobe infiltrate during the period of t lymphocyte reaction to the initial infection. before resolution, the mycobacterium tuberculosis infection may disseminate to the better oxygenated upper lobes and extrathoracic sites, such as bone, or the central nervous system. if the immune response contains the initial infection, the radiographic findings may be indistinguishable from those of any other pneumonic process. with altered immune function, however, there may be progressive local disease, dissemination to miliary pulmonary disease, or early reactivation (months to years) at distal sites, which produces tuberculous meningitis or osteomyelitis. reactivation of upper lobe pulmonary disease may produce cavities that are similar to the disease among adults. cavitary and endobronchial lymph node involvement are highly infectious, in contrast to the much less contagious nature of the hypersensitivity reaction noted in primary pulmonary disease. treatment of active disease, especially in regions of multidrugresistant tuberculosis, consists of three or four drug regimens, including isoniazid, rifampin, pyrazinamide with or without streptomycin, and ethambutol. risk of infection based on history and ppd induration size are presented in table - . inhaling food, mouth or gastric secretions, or foreign bodies into the tracheobronchial tree causes acute, recurrent, or chronic cough. interference with normal swallowing disrupts the coordination of swallowing and breathing that prevents aspiration. structural causes of disordered swallowing include esophageal atresia (in neonates), strictures, webs, or congenital stenoses. mediastinal lesions (tumors, lymph nodes), including vascular rings, may compromise the esophageal lumen and esophageal peristalsis, increasing the likelihood of aspiration. functional disorders include central nervous system dysfunction (e.g., coma, myopathy, neuropathy) or immaturity, dysautonomia, achalasia, and diffuse esophageal spasm. prior neck surgery, including tracheostomy, may alter normal swallowing. tracheoesophageal fistula and laryngeal clefts are congenital malformations with direct physical connections between the tracheobronchial tree and the upper gastrointestinal tract; oral contents enter the lungs directly. making the diagnosis of aspiration as the cause of cough may be difficult. barium contrast studies during swallowing may help characterize these disorders if barium enters the trachea. because most patients aspirate sporadically, a normal barium swallow does not rule out aspiration. radionuclide studies can be helpful if ingested radiolabeled milk or formula is demonstrated over the lung fields at several-hour intervals after the meal. bronchoscopy and bronchoalveolar lavage that recover large numbers of lipid-laden macrophages suggest that milk aspiration has taken place; however, the finding is neither sensitive nor specific for aspiration. treatment depends largely on the cause of aspiration. because many patients who aspirate do so because of lack of neurologic control of swallowing and breathing, it is often difficult to prevent. even gastrostomy feedings cannot prevent aspiration of oral secretions. in extreme cases, tracheostomy with ligation of the proximal trachea has been employed. this prevents aspiration but also prevents phonation, and it must be considered only in unusual situations. aspiration pneumonia is often treated with intravenous penicillin or, preferably, clindamycin to cover mouth flora of predominant anaerobes. additional coverage against gram-negative organisms (an aminoglycoside or cefepime) may be indicated if the aspiration is nosocomial. any child with cough of abrupt onset should be suspected of having inhaled a foreign body into the airway. toddlers, who by nature put all types of things into their mouths and who have incompletely matured swallowing and airway protective mechanisms, are at high risk. infants with toddlers or young children in the household who may "feed" the baby are also at risk. in older children, it is usually possible to obtain an accurate history of the aspiration event. these events are described as choking, gagging, and coughing while something (e.g., peanuts, popcorn, small toys, sunflower seeds) is in the mouth. the child may come to the physician with cough and wheeze immediately after the event, with a clear history and a straightforward diagnosis. in many children with a tracheobronchial foreign body, however, the initial episode is not recognized; these children may not come to medical attention for days, weeks, or even months (fig. - ). the initial episode may be followed by a relatively symptomless period lasting days or even weeks, until infection develops behind an obstructed segmental or lobar bronchus. at this point, cough, perhaps with hemoptysis, with or without wheeze, recurs. on physical examination early after an aspiration episode, there is cough, wheeze, or both, often with asymmetry of auscultatory findings. there may be locally diminished breath sounds. later, localized wheeze or crackles may be detected. in some cases, the two-headed stethoscope may enable the examiner to recognize that a lobe or lung has delayed air entry or exit in comparison with the other side. the triad of wheezing, coughing, and decreased breath sounds is present in fewer than % of patients. the presence of laryngotracheal foreign bodies often manifests with stridor, retractions, aphonia, cough, and normal radiographs. chest radiographs may be normal in % of patients with intrathoracic foreign bodies but should be obtained in both inspiration and expiration, because in some cases the only abnormality is unilateral or unilobar air trapping, which is occasionally more clearly identified with a view in expiration. in this view, an overdistended lung that had appeared normal on the inspiratory view does not empty, but the normal, unobstructed lung empties normally. this phenomenon causes a shift of the mediastinum toward the emptying lung (away from the side with the obstructing foreign body). in other patients, localized infiltrate or atelectasis may be present behind the obstructing object. in a few patients, it may be possible to identify the foreign body itself; nonetheless, most inhaled food particles are not radiopaque and cannot be seen on radiographs. aspiration is usually unilateral ( %); % to % of the objects are in the right lung (the lobe depends on body position-supine versus standing-but is often the right middle lobe). the definitive diagnostic and therapeutic maneuver is bronchoscopy; either the flexible or rigid open-tube bronchoscope enables direct visualization of the object; the rigid instrument also enables its removal. ger is a common cause of cough in all age groups (see chapter ). the typical patient is an infant in the first months of life who spits up small amounts of milk frequently after feedings. this "regurgitant reflux" most commonly resolves by year of age. however, many toddlers and children continue to have reflux, although it may be "silent" or nonregurgitant (without spitting up). in most people with ger, it is merely a nuisance or not noticed. in some there are sequelae, and this condition is designated gastroesophageal reflux disease (gerd). one manifestation is cough; the mechanisms for the cough are not fully understood. aspiration of refluxed material is one mechanism for cough but is probably not very common in neurologically intact children. a major mechanism for gerd with cough is mediated by vagal esophagobronchial reflexes (bronchoconstriction), stimulated by acid in the esophagus. whether acid in the esophagus is sufficient stimulus to cause bronchoconstriction by itself or whether it merely heightens bronchial reactivity to other stimuli is not yet clear (see chapter ). many children with reactive airways disease have cough or wheeze that is difficult to control until their concurrent ger is also treated. many episodes of cough caused by gerd occur in children with asthma that is difficult to control. the diagnosis of gerd must also be considered in the child with chronic or recurrent cough with no other obvious explanation. the child who coughs after meals or at night, when the supine position may provoke ger, should be evaluated for ger. if ger is confirmed, the next step is a therapeutic trial of antireflux therapy (see chapter ). if the results of the therapeutic trial are negative or equivocal, it may make sense to establish a causal relationship between the ger and the cough, by using the modified bernstein test. during this test, hydrochloric acid and saline are alternately infused into the esophagus through a nasoesophageal tube while the child is observed for cough or wheeze or while the older child undergoes serial pulmonary function tests. if the symptoms occur or if pulmonary function deteriorates during acid but not saline infusion, it is likely that esophageal acidification through ger is the cause of the child's cough or wheeze. treatment in a child whose cough is related to ger may be accomplished by treating the reflux (see chapter ) or by a combination of antireflux and antiasthma treatment (see chapter ). theophylline may worsen ger by lowering the tone of the lower esophageal sphincter, and some drugs that increase lower esophageal sphincter tone may cause bronchoconstriction. on occasion, the cough may be abolished by stopping all antiasthma medications. in such cases, the cough was a manifestation of reactive airways with esophageal acidification as the trigger for bronchospasm; the esophageal acidification was caused by the bronchodilator effects on the lower esophageal sphincter. inhaled bronchodilators are less likely than oral or intravenous drugs to cause ger. cough is frequently the sole or most prominent manifestation of asthma; wheezing may be entirely absent. in fact, reactive airway disease or asthma (see chapter ) is almost certainly the most common cause of recurrent and chronic cough in childhood. some of the features that characterize the cough of a child with asthma are listed in table - . treatment for asthma manifesting as cough is the same as the treatment for asthma (see chapter ). cf is a common cause of recurrent or chronic cough in infancy and childhood. cf occurs in in live births among white persons, is far less common among african americans ( in , ), and is rare among native americans and asians. early diagnosis improves the poor prognosis for untreated cf; if untreated, many patients die in infancy or early childhood. with current state-of-the-art care, median length of survival is to age . cf is a genetic disorder, inherited as an autosomal recessive trait. the cf gene is on the long arm of chromosome ; more than mutations have been identified at the cf locus. of these mutations, one (Δf , indicating a deletion, Δ, of a single phenylalanine, f, at position of the protein product) is the most common, responsible for % to % of all cf chromosomes. the currently recognized mutations account for approximately % of cases. the mutation affects the gene's protein product, termed cystic fibrosis transmembrane regulator, which acts as a chloride channel and affects other aspects of membrane transport of ions and water. not all the consequences of the defective gene and protein have been determined. most explain the long-observed clinical manifestations of the disease, including thick, viscid mucus in the tracheobronchial tree, leading to purulent bronchiolitis and bronchitis with subsequent bronchiectasis, pulmonary fibrosis, and respiratory failure; pancreatic duct obstruction, leading to pancreatic insufficiency with steatorrhea and failure to thrive; and abnormally high sweat chloride and sodium concentrations. the airway disease in cf is characterized by infection, inflammation, and endobronchial obstruction. the infection begins with s. aureus, h. influenzae, escherichia coli, klebsiella species, or combinations of these organisms but eventually is dominated by nonmucoid or mucoid pseudomonas aeruginosa. other organisms, such as burkholderia cepacia, xanthomonas maltophilia, alcaligenes xylosoxidans, aspergillus fumigatus, or nontuberculous mycobacteria may also appear; their significance remains undetermined. in some patients, b. cepacia has been associated with rapid deterioration and death, and in others, aspergillus species has caused allergic bronchopulmonary aspergillosis. the airway inflammation in all patients with cf appears to be the result of toxic substances, including elastase, released by neutrophils as they respond to the endobronchial infection and by similar enzymes released by the invading organisms. cf may manifest at birth with meconium ileus ( % to % of patients) or later, with steatorrhea and failure to thrive despite a voracious appetite, in an apparent effort to make up for the calories that are lost in the stool (see chapter ). the most common presenting symptom is cough, which may appear within the first weeks of life or may be delayed for decades. the cough can be dry, productive, or paroxysmal. cough may respond to antibiotics or perhaps steroids, but it is less likely to improve with bronchodilators (see tables - and - ). although cf is a genetic disease, there is often no family history. furthermore, in atypical cases, patients may not have pancreatic insufficiency (usually present in % to %) and thus may not demonstrate steatorrhea and failure to thrive. in addition, malabsorption may not be evident in the neonatal period. there is no such thing as a child who looks "too good" to have cf; common abnormalities found on physical examination are noted in table - . one of the most important physical findings is digital clubbing. in most patients with cf, clubbing develops within the first few years of life. although the list of conditions associated with digital clubbing (table - ) is long, they are less common than cf, or the incidence of digital clubbing with these conditions is low. there is some relationship between the degree of pulmonary disease severity and the degree of digital clubbing. a child who has had years of severe respiratory symptoms without digital clubbing is not likely to have cf. the diagnosis is confirmed by a positive sweat test or confirming the presence of two cf mutations on chromosome . the sweat test, if not performed correctly in a laboratory with extensive experience with the technique (as, for example, in an accredited cf center), yields many false-positive and false-negative results. the proper technique is to use the gibson-cooke method, with quantitative analysis of the concentration of sodium, chloride, or both, in the sweat produced after pilocarpine iontophoretic stimulation. chloride (and sodium) concentrations higher than meq/l are considered positive indications, and those lower than meq/l are negative (normal). healthy adults have slightly higher sweat chloride concentrations than do children, but the same guidelines hold for positive tests in adults. the non-cf conditions yielding elevated sweat chloride concentrations are listed in table - . false-negative results of sweat tests can be seen in cf children presenting with edema or hypoproteinemia and in samples from children with an inadequate sweat rate. sweat testing can be performed at any age; newborns within the first few weeks of life may not produce a large enough volume of sweat to analyze ( mg minimum), but in those who do (the majority), the results are accurate. indications for sweat testing are noted in table - . in patients for whom sweat testing is difficult (e.g., because of distance from an experienced laboratory, small infants who have not produced enough sweat, patients with extreme dermatitis, or patients with intermediate-range sweat chloride concentrations), dna testing can be useful. demonstration of two known cf mutations confirms the diagnosis. finding one or no known mutation makes the diagnosis less likely but is not exclusive, inasmuch as there are patients with not-yet-characterized mutations. furthermore, commercial laboratories do not identify all of the -plus mutations. recovery of mucoid p. aeruginosa from respiratory tract secretions is strongly suggestive of cf. similarly, pansinusitis is nearly universal among cf patients but is quite uncommon in other children. some states are using a neonatal screen for cf. the cf screen is for immunoreactive trypsinogen (irt) levels, which are elevated in most infants with cf for the first several weeks of life. (some states do genetic testing on dna.) because of the very high sensitivity of this test (almost no one with cf has normal irt levels) and because early institution of treatment is beneficial, this test may come into wider use. its main drawback is that it has relatively poor specificity; as many as % of the positive results on the initial screen are false-positive results. if an infant's irt screen is positive, the test should be repeated; at to weeks of age, which is when the test is repeated, the false-positive rate has fallen dramatically but is still quite high ( %). definitive testing needs to be carried out on infants with two elevated irt levels. in the unusual older child whose appendix is removed and examined carefully by a knowledgeable pathologist, the diagnosis may be suggested by the typical histologic appearance of the appendix (the mucus-secreting glands are overdistended with eosinophilic material). laboratory data that may support the diagnosis of cf include absence of stool trypsin or chymotrypsin. this suggests pancreatic insufficiency, which occurs most commonly in cf but can be seen in other diseases. the test is not perfect even for confirming pancreatic insufficiency, because intestinal flora may produce or destroy trypsin. pulmonary function test findings with an obstructive pattern, incompletely responsive to bronchodilators, are consistent with cf but, of course, can be seen in other conditions. conversely, some patients with cf also have asthma and may show a marked response to a bronchodilator. complications of cf that should suggest the diagnosis are noted in table - . the treatment of patients with cf requires a comprehensive approach, best performed in, or in conjunction with, an approved cf center. several studies have shown survival to be significantly better in center-based care than in non-center-based care. the treatment of the pulmonary aspects of cf involves approaching the obstruction, infection, and inflammation that cause the cough. cough should be monitored closely, and any increase in frequency or in the intensity should be taken as an indication that there is worsening endobronchial infection, inflammation, or both. because active infection and inflammation lead to irreversible lung damage, such changes need to be taken seriously and treated aggressively. this is just as true for the appearance of a mild morning cough in the child who was previously cough free as it is for severe coughing spells that keep a child awake through the night. obstruction is treated with physical means (chest physical therapy, with percussion, vibration, and postural drainage) to dislodge the mucus into the large central airways, where cough can then effectively clear it. studies have shown this rather crude and timeconsuming procedure to be effective in helping to maintain lung function acutely and over a period of years. variations on the physical maneuvers to help with mucus clearance include forced expiratory technique, positive airway pressure face masks, and masks with expiratory flutter valves. the frequency with which any of these physical means of expelling mucus should be used varies but should be increased with signs of active infection and obstruction. other approaches to relieving obstruction in the bronchial tree include the use of inhaled bronchodilators (despite a paucity of studies showing their long-term efficacy) and mucolytic agents. n-acetylcysteine (mucomyst) has been available for years, but it may cause tracheobronchial irritation, with bronchorrhea, bronchospasm, or both, in an unacceptably high proportion of patients. an inhaled drug, dornase alfa, or recombinant human dnase (pulmozyme), is clearly effective in the test tube for liquefying the thick mucus associated with cf. this occurs because % of the mucus viscosity in cf is attributed to dna released from dying polymorphonuclear cells. another drug that appears promising is amiloride. long available as a diuretic, amiloride can bring about a partial correction of the membrane transport defects in cf. amiloride aerosols seem to decrease sputum viscosity and increase cough clearance. in a small -month study, aerosolized amiloride appeared to slow the decline in lung function. the approach to endobronchial infection in children with cf includes prevention and treatment. prevention involves immunizing patients with cf against preventable respiratory pathogens, particularly influenza, measles, and pertussis. prevention also means avoiding unnecessary exposure to respiratory viruses (e.g., at day care centers). it should not mean avoiding school or other social functions and settings, because this approach is invariably futile and can cause severe emotional damage. however, spread of bacteria, especially resistant organisms such as b. cepacia, between patients is a concern. treatment of infection usually proceeds in a stepwise manner. if colonies of h. influenzae or s. aureus are present, appropriate antibiotics should be initiated. if no recent throat or sputum specimens for culture are available, and if the patient is young with very mild lung disease, empirical therapy can also be directed at those organisms. in the older or sicker patient who has any sign of chronic pulmonary involvement, such as pulmonary overinflation, infiltrates on a chest film, digital clubbing, or severe coughing spells, it makes sense to include antibiotics effective against p. aeruginosa (fig. - ; table - ) . treatment of the inflammation associated with cf is evolving. some patients benefit from short-term oral prednisone. a -year study of alternate-day prednisone showed improved pulmonary function but unacceptable side effects (e.g., glucose intolerance, growth failure) in those taking mg/kg/day and similar side effects (although less severe) in those taking mg/kg/day. the beneficial role of oral nonsteroidal antiinflammatory agents has been demonstrated; the role of inhaled topical steroids and α -antitrypsin is being investigated. table - lists the main anatomic abnormalities (most of them congenital) that cause cough. vascular rings and slings are often associated with inspiratory stridor because the abnormal vessels compress central airways, most commonly the trachea (see chapter ). the patient may also have difficulty swallowing if the esophagus is compressed. the diagnosis may be suspected from plain films of the chest, especially those showing tracheal deviation and a right-sided aortic arch. further support for the diagnosis can be found at bronchoscopy (which shows extrinsic compression of the trachea or a mainstem bronchus), barium swallow study (which shows esophageal compression), or both. the definitive diagnosis is made with magnetic resonance imaging, angiography, or magnetic resonance angiography. treatment is surgical. pulmonary sequestration is relatively unusual, occurring in in , children. it occurs most commonly in the left lower lobe and can manifest in several ways (fig. - ; see table - ) . the chest radiograph usually shows a density in the left lower lobe; this density often appears to contain cysts. the feature distinguishing a sequestered lobe from a complicated pneumonia is that the blood supply arises from the aorta and not the pulmonary circulation. doppler ultrasonography and angiography provide the definitive diagnosis. the treatment is surgical removal. cystic adenomatoid malformation is a rare condition. it manifests in infancy with respiratory distress in nearly % of cases; the other half may manifest as cough with recurrent infection later in childhood or even adulthood. the chest film reveals multiple cysts, separated by dense areas. chest ct scans can help make the diagnosis with near certainty. surgical removal is the treatment. congenital lobar emphysema occurs in one of , live births. it can manifest dramatically with respiratory distress in the neonatal period or later (fig. - ) , with cough or wheeze, or as an incidental finding on a chest radiograph. radiography shows localized overinflation, often dramatic, with compression of adjacent lung tissue and occasionally atelectasis of the contralateral lung because of mediastinal shift away from the involved side. the appearance on chest ct scan is typical, with widely spaced blood vessels (as opposed to congenital cysts, for example, which have no blood vessels within the overinflated area). bronchoscopy can document patent bronchi and should probably be performed in older children, in whom congenital lobar emphysema can be confused with acquired overinflation of a lobe as the result of bronchial obstruction, as with a foreign body. if the disease is symptomatic, treatment is surgical. tracheoesophageal fistula is common, with an incidence of about one in live births. of these fistulas, the large majority ( %) are associated with esophageal atresia; only % are the isolated, h-type fistula (a patent esophagus with fistulous tract connecting the esophagus and trachea). a neonate with esophageal atresia experiences respiratory distress, excessive drooling, and choking and gagging with feeding. the h-type fistula causes more subtle signs and may be undiagnosed for months or even years. the child may have only the diagnosis is not challenging in the infant with esophageal atresia; a nasogastric tube cannot be passed, and swallowed barium outlines the trachea. in the older child with h-type fistula, a barium esophagogram may or may not reveal the fistula. bronchoscopy and esophagoscopy should permit direct visualization of the fistula; however, the opening may be hidden in mucosal folds. treatment is surgical. many children born with tracheoesophageal fistula have recurrent cough and lower respiratory tract infection for many years, even after successful surgical correction. the cough is characteristically the harsh cough of tracheomalacia, which is present at the site of the fistula. the infections result from several causes, including ger, with or without aspiration, and altered mucociliary transport. treatment involves regular chest physical therapy and early and aggressive use of antibiotics whenever there is evidence of increased pulmonary symptoms. hemangiomas may be present within the airway and can cause cough, rarely with hemoptysis; stridor (if the hemangioma is high in the airway) and respiratory distress (if the hemangioma is large) may also occur. in rare cases, with very large airway hemangiomas, there may even be dysphagia from extrinsic compression. about % of children with airway hemangiomas have cutaneous hemangiomas as well. the diagnosis is made with bronchoscopy. as with cutaneous hemangiomas, these lesions may resolve spontaneously over the first year or so. however, if they cause symptoms, it may not be advisable or possible to wait for them to resolve. many airway hemangiomas regress with steroid treatment, although others have been shown to respond to interferon-α. laser ablation may be indicated in some refractory cases. in the case of a large subglottic hemangioma, a tracheostomy is frequently performed and maintained until the mass regresses. enlarged mediastinal lymph nodes, such as those resulting from tuberculosis, leukemia, other hematologic malignancies, or other infections, are occasionally a cause of cough in children (tables - and - ). these nodes are usually seen on plain films of the chest. the x-ray study or bronchoscopy may show extrinsic compression of the trachea. treatment is directed at the underlying cause. occasionally bronchial stenosis, either congenital or acquired, may cause cough. the diagnosis is made with bronchoscopy, after suspicion has been raised by the child's having recurrent infiltrates in the same lobe, especially with localized wheeze. treatment may be difficult. in some cases, endoscopic balloon dilatation or airway stent placement is successful; in others, surgical resection of stenotic areas may be necessary. bronchogenic cysts are uncommon, but they can cause cough, wheeze, stridor, or any combination of these. they may also cause recurrent or persistent pneumonia if they block a bronchus sufficiently to interfere with normal drainage of the segment or lobe. radiography may show localized overinflation if the cyst causes a ball-valve-type obstruction. the cyst itself may or may not be seen on plain films. bronchoscopy reveals extrinsic compression of the airway. ct studies often definitively show the lesion. surgical removal is indicated. on occasion, a school-aged child may develop a cough that lasts for weeks, often after a fairly typical cold. this cough occurs only during wakefulness, never during sleep. in many cases, the cough is harsh and foghorn-like. it often disrupts the classroom, and the child is asked to leave. the child is otherwise well and may seem rather unbothered by the spectacle created. there has been no response to medications. it seems that this type of cough, often termed "psychogenic," or "psychogenic cough tic," but perhaps more accurately and humanely thought of as habit cough, has given the child valuable attention. this attention then serves as the sustaining force, and the cough persists beyond the original airway inflammation. in the small minority of cases, there may be deep-seated emotional problems, of which the cough is the physical expression. during the history or physical examination, the child appears completely well and may cough when attention is drawn to the child or when the word "cough" is uttered. the physical examination findings are otherwise completely normal, as are laboratory values. because this may occur in any child, evidence of mild reactive airways disease (history or pulmonary function testing) does not rule out the diagnosis. once a physician has seen a child with this problem, it is usually possible to make the diagnosis with certainty on entering the examining room or, indeed, from the hallway outside the room. treatment can prove more difficult. there are several treatment schools, summarized in tables - and - . one approach is "the bed sheet," in which the child is told that he or she coughs because of weak chest muscles. a bed sheet is wrapped tightly and uncomfortably around the chest "to serve as added support for the muscles … [and] with this support, the muscles would then be able to suppress [the] cough." the child is to go to school wearing the bulky bed sheet under his or her clothes and may not remove the sheet until he or she is certain that the cough will not return. the authors who describe this method call it a "reinforcement suggestion technique." some view this approach as demeaning to the child. whatever its mechanism of action, this method was reported to have been successful in of patients. postnasal drip is thought to be a major cause of cough in adults. the mechanism by which this occurs is unclear, and most pulmonologists believe that this must remain a diagnosis of exclusion for explaining cough in children. bronchiectasis is defined as an abnormal dilation of the subsegmental bronchi and is usually associated with chronic cough and purulent sputum production. it occasionally occurs after severe pneumonias (bacterial or viral); it eventually develops in nearly all patients with cf. diagnosis may, on occasion, be made with plain chest radiographs, but high-resolution ct scanning is the diagnostic procedure of choice, replacing bronchography. treatment of bronchiectasis consists of chest physiotherapy and postural drainage, occasionally bronchodilators and mucolytic agents, and antibiotic therapy during exacerbations. surgical resection may be indicated in cases that are progressive and localized when medical therapy has failed. the prognosis of bronchiectasis depends on the underlying cause. bronchiectasis associated with cf is fatal, although many cases of bronchiectasis remain stable or may even regress with therapy. conditions in which the cilia do not function properly (dysmotile cilia or ciliary dyskinesia) lead to cough, usually because infection (and bronchiectasis) occurs in the absence of normal mucociliary transport. treatment is similar to that for cf, with regular chest physical therapy and frequent and aggressive use of antibiotics at the first sign of airways infection, most commonly increased cough. there are several varieties of interstitial lung disease: desquamative, lymphoid, and "usual" (see chapter ). all are very uncommon in childhood, and little is known about their causes, courses, or treatment. one type of pediatric interstitial lung disease, the lymphoid type, is becoming much more common, inasmuch as it is seen in human immunodeficiency virus (hiv) infection. interstitial lung disease manifests with cough, dyspnea, and crackles on examination. because the diagnosis is based on histologic findings, lung biopsy is required. the only exception to this may be in the child with documented acquired immunodeficiency syndrome who has new pulmonary infiltrates and symptoms, in whom bronchoscopy and bronchoalveolar lavage are initially used to diagnose infection (p. carinii, cytomegalovirus). interstitial lung disease in a patient with a chronic seborrhea-like dermatitis should suggest the diagnosis of histiocytosis x. heart failure can cause cough but seldom as its sole clinical manifestation. pulmonary hemosiderosis is a rare, and often fatal, condition of bleeding into the lung that can manifest with cough. if sputum is produced, it is often frothy and blood-tinged. there may be frank hemoptysis. however, the cough may be nonproductive, or the sputum may be swallowed. some cases are associated with milk perform all possible tests can tell patient and family: "we've reinforces the idea of a physical cause ruled out all physical problems." in one study, resolution followed bronchoscopy ("aversive stimulus"?) apply an aversive stimulus has worked in some patients by definition, this treatment is unpleasant (e.g., an electric shock to forearm) try the "bed sheet" (see text) seems to work in most patients demeans the patient try placebo drugs probably works in some patients is a dishonest technique provide psychotherapy may work in some patients is unnecessary in most patients; labels the patient as having a psychological problem gently explain that there is no physical works in some patients is resented by some families focused on cause and this is a habit that the body an organic cause or treatment has sustained prevent mouth breathing by holding a has worked in some patients not known button between the patient's lips apply speech therapy techniques (see works in many patients: can be presented is resented by some families focused on table - ) as specific therapy; nonthreatening organic cause or treatment treatment is often unsatisfactory; the mortality rate is as high as %. milk products should be eliminated from the patient's diet, and underlying collagen vascular disease should be treated. some affected children seem to respond to corticosteroids or cytotoxic drugs (e.g., azathioprine, cyclophosphamide, and chlorambucil), but the episodic nature of the disease, with some clear cases of spontaneous resolution, makes it difficult to evaluate therapies. see chapter . tumors, which fortunately are rare in childhood, can cause cough, usually because of bronchial blockage, either extrinsic or endobronchial (see table - ). the diagnosis is usually made from bronchoscopy, chest ct, or both. treatment depends on the cell type, but it usually involves at least some surgical removal. chemotherapy or radiation may be used in some cases. isolated tracheomalacia or bronchomalacia is uncommon but can cause cough in some children. the cough of tracheomalacia is typically harsh and brassy. treatment is difficult but, fortunately, is seldom needed. some children, usually preschoolers, may episodically awaken at night with stridor and a harsh, barking cough indistinguishable from that of viral croup. this entity is termed spasmodic croup and is of unclear origin. viral and allergic causes have been postulated. ger may be the cause in some patients. treatment with cool mist or racemic epinephrine is effective in most patients. if ger is the underlying cause, antireflux treatment is beneficial. obliterative bronchiolitis is very rare except in lung transplant recipients. in other instances, it may arise after adenovirus, measles, or influenza pneumonia; after exposure to certain toxins; or in other rare circumstances. children may exhibit cough, respiratory distress, and exercise intolerance. the diagnosis is suggested by the pulmonary function or radiographic evidence of small airways obstruction; however, these findings are not always present. not all chest films show overinflated lungs, and not all pulmonary function tests show decreased small airways function. the definitive diagnosis is histologic via open or transbronchial biopsy. no specific treatment is available. most children with obliterative bronchiolitis recover, but many progress to chronic disability or death. the child who coughs out blood or bloody mucus presents special diagnostic and therapeutic challenges. although hemoptysis is relatively uncommon in children, particularly among those without cf, many conditions can cause it (table - ) . it is important (and not always easy) to distinguish cases in which blood has originated in the tracheobronchial tree (true hemoptysis), the nose (epistaxis), and the gastrointestinal tract (hematemesis). table - gives some guidelines to help localize sites of origin of blood that has been reported or suspected as hemoptysis. none of these is foolproof, partly because blood that has originated in one of these sites might well end in another before being expelled from the body; for instance, blood from the nose can be swallowed and vomited or aspirated and coughed out. infection is among the most common causes of hemoptysis. lung abscess and tuberculosis need to be considered. bronchiectasis can readily cause erosion into bronchial vessels, often made tortuous by years of local inflammation, and produce hemoptysis. other infectious settings are less common and include necrotizing pneumonias and fungal and parasitic lung invasion. foreign bodies in the airway can cause hemoptysis by direct irritation, by erosion of airway mucosa, or by secondary infection. pulmonary embolus is uncommon in children and adolescents, but it needs to be considered in the differential diagnosis of an adolescent with hemoptysis of unclear origin. clues to the diagnosis of pulmonary embolus include a positive family history, severe dyspnea, chest pain, hypoxia, a normal chest film, an accentuated second heart sound, an abnormal compression ultrasonographic study of the leg veins, a positive homans sign, a positive helical ct scan, and a high-probability lung ventilation-perfusion scan. the diagnosis of several causes of hemoptysis is straightforward. for example, hemoptysis that occurs immediately after a surgical or invasive diagnostic procedure in the chest should suggest an iatrogenic problem. the chest film can help suggest lung abscess, pulmonary bronchoscopy can sometimes localize a bleeding site, identify a cause (e.g., a foreign body or endobronchial tumor), or recover an offending bacterial, fungal, or parasitic pathogen. in many instances, bronchoscopy does not help except by excluding some possibilities, because either no blood or blood throughout the tracheobronchial tree is found. bronchial artery angiography may help to identify the involved vessel or vessels. treatment of hemoptysis depends on the underlying cause. it can be a terrifying symptom to children and their parents, and a calm, reassuring approach is essential. because hemoptysis is seldom fatal in children, reassurance is usually warranted. furthermore, hemoptysis most often resolves, and treatment of the bleeding itself is not often needed. what is required is treatment of the underlying cause of the hemoptysis, such as therapy for infection, removal of a foreign body, or control of collagen-vascular disease. when death occurs from hemoptysis, it is more likely to be from suffocation than from exsanguination. in cases of massive bleeding, the rigid open-tube bronchoscope may help suction large amounts of blood while ventilating and keeping unaffected portions of lung clear of blood. interventional radiologists treat as well as localize a bleeding site by injecting the offending vessel with occlusive substances, such as gelfoam or silicone coils. in extremely rare instances, emergency lobectomy may be indicated. cough itself seldom necessitates specific treatment. nonetheless, cough is not always completely benign (see table - ). most complications are uncommon, and most accompany only very severe cough, but some are serious enough to justify treatment of the cough itself. cough suppressants include codeine and hydrocodone (two narcotics) and dextromethorphan (a nonnarcotic d-isomer of the codeine analogue of levorphanol). such agents should be used only for severe cough that may produce significant complications (see table - ). for most diseases, suppressing the cough offers no advantage. disadvantages include narcotic addiction and loss of the protective cough reflex with subsequent mucus retention and possible superinfection. demulcent preparations (sugar-containing, bland soothing agents) temporarily suppress the cough response from pharyngeal sources, and decongestant-antihistamine combinations may reduce postnasal drip and thus cough in adults. evaluation of chronic or recurrent cough the cough and the bedsheet managing cough as a defense mechanism and as a symptom. a consensus panel report of the american college of chest physicians bacterial tracheitis: report of eight new cases and review computed tomographic study of the common cold epidemiology, pathogenesis, and treatment of the common cold does this patient have sinusitis? diagnosing acute sinusitis by history and physical examination report of the committee on infectious diseases chlamydial respiratory infections infant pneumonitis associated with cytomegalovirus, chlamydia, pneumocystis, and ureaplasma: follow-up value of radiological follow-up of childhood pneumonia etiology and treatment of pneumonia report of a workshop on respiratory viral infections: epidemiology, diagnosis, treatment, and prevention pulmonary manifestations of acquired immunodeficiency syndrome standardized diagnosis of pneumonia in developing countries the causes of hospital-treated acute lower respiratory tract infection in children hypoxaemia in young kenyan children with acute lower respiratory infection the evaluation of the child with recurrent chest infections bronchoscopic removal of aspirated foreign bodies in children tracheobronchial foreign bodies subglottic foreign bodies in pediatric patients tracheobronchial foreign bodies cystic fibrosis the cystic fibrosis genotype-phenotype consortium. correlation between genotype and phenotype in patients with cystic fibrosis prospects for gene therapy in cystic fibrosis prediction of mortality in patients with cystic fibrosis wheezing in infants with cystic fibrosis: clinical course, pulmonary function, and survival analysis physiotherapy in cystic fibrosis efficacy and safety of short-term administration of aerosolised recombinant human dnase i in adults with stable stage cystic fibrosis the diagnosis of cystic fibrosis: a consensus statement emerging therapies for cystic fibrosis pseudomonas cepacia: pulmonary infection in patients with cystic fibrosis effect of long-term treatment with inhaled budesonide on adult height in children with asthma a clinical index to define risk of asthma in young children with recurrent wheezing airway responsiveness in early infancy predicts asthma, lung function, and respiratory symptoms by school age low-dose inhaled corticosteroids and the prevention of death from asthma third international pediatric consensus statement on the management of childhood asthma. international pediatric asthma consensus group venous thromboembolic complications in children clinical assessment and management of massive hemoptysis massive haemoptysis: medical management will usually arrest the bleeding pathogenesis and management of hemoptysis in children cough is important because it is a symptom and sign of underlying disease that frequently merits treatment. in the acute setting, severe disease, including massive hemoptysis or profound dyspnea or hypoxemia, warrants immediate attention, rapid diagnosis, and rapid management. certain chronic conditions, including those that suggest cf and those in which symptoms have persisted and interfere with a child's daily activities and quality of life, warrant further evaluation and treatment. finally, a child whose cough fails to respond to what should have been reasonable treatment should be referred to a pulmonary specialist (table - ) . key: cord- -jy j sh authors: huang, ting; wang, wei; bessaud, mael; ren, peijun; sheng, jun; yan, huajie; zhang, jing; lin, xin; wang, yongjin; delpeyroux, francis; deubel, vincent title: evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: jy j sh background: human rhinoviruses (hrvs) are a highly prevalent cause of acute respiratory infection in children. they are classified into at least three species, hrv-a, hrv-b and hrv-c, which are characterized by sequencing the ′ untranslated region (utr) or the vp /vp region of the genome. given the increased interest for novel hrv strain identification and their worldwide distribution, we have carried out clinical and molecular diagnosis of hrv strains in a -year study of children with acute respiratory infection visiting one district hospital in shanghai. methodology/findings: we cloned and sequenced a -nt fragment that covered part of the ′utr and the vp /vp capsid genes. sixty-four hrv-infected outpatients were diagnosed amongst children with acute low respiratory tract infection. two samples were co-infected with hrv-a and hrv-b or hrv-c. by comparative analysis of the vp /vp sequences of the hrvs, we showed a high diversity of strains in hrv-a and hrv-b species, and a prevalence of . % of strains that belonged to the recently identified hrv-c species. when analyzing a fragment of the ′ utr, we characterized at least two subspecies of hrv-c: hrv-cc, which clustered differently from hrv-a and hrv-b, and hrv-ca, which resulted from previous recombination in this region with sequences related to hrv-a. the full-length sequence of one strain of each hrv-ca and hrv-cc subspecies was obtained for comparative analysis. we confirmed the close relationship of their structural proteins but showed apparent additional recombination events in the a gene and ′utr of the hrv-ca strain. double or triple infections with hrv-c and respiratory syncytial virus and/or bocavirus were diagnosed in . % of the hrv-infected patients, but no correlation with severity of clinical outcome was observed. conclusion: our study showed a high diversity of hrv strains that cause bronchitis and pneumonia in children. a predominance of hrv-c over hrv-a and hrv-b was observed, and two subspecies of hrv-c were identified, the diversity of which seemed to be related to recombination with former hrv-a strains. none of the hrv-c strains appeared to have a higher clinical impact than hrv-a or hrv-b on respiratory compromise. human rhinoviruses (hrvs) are a highly p revalent cause of the acute respiratory infection (ari) defined as the common cold [ , , ] , which is frequently associated in children with bronchitis, bronchiolitis, wheezing, pneumonia, asthma and otitis [ , , , , , ] . hrvs are classified in genus enterovirus (hevs) in family picornaviridae [ ] . hrvs are non-enveloped, single-stranded, positive-sense rna viruses of approximately nt, composed of a untranslated region (utr), followed by a long open reading frame coding for capsid proteins vp , vp , vp and vp , and seven non-structural proteins a, b, c, a, b, c and d, and terminated by a short utr and poly a tract. more than serotypes of hrv are known, which have been classified into two species, hrv-a and hrv-b, according to comparative alignment of nucleotide fragments of vp [ , ] , vp /vp [ ] and utr [ , ] , and more recently, on complete genome nucleotide sequences [ ] . moreover, some genomic sequences have been found not to cluster with hrv-a and hrv-b species, which suggests the existence of other species (hrv-c and hrv-d) [ ] . a new species of hrv-c was recently identified worldwide by comparative analysis of vp or vp /vp genes [ , , , , , ] and utr [ , ] . however, discrepancies have appeared in the classification of some of the new hrv-a or hrv-c strains, depending on the size and location of the nucleotide sequence in the viral genome and on the phylogenetic methods used for direct analysis of hrv sequences [ , , , , , , , , , ] . these recent data underline the lack of knowledge about the biodiversity of hrv strains and their worldwide distribution [ , ] . moreover, little is known about the characteristics and diversity of hrvs circulating in a given area in a short period of time. in the present study, we looked for hrvs in a -year collection of nasopharyngeal swabs (npss) of children with ari visiting a district hospital in shanghai, and compared sequences in two regions previously defined for genetic classification of hrv serotypes [ , ] . our study showed a high diversity of hrv species and genotypes, and a prevalence of the novel hrv-c species in npss of children with bronchitis and pneumonia. this biodiversity appeared to result partly from recombination events in the utr, between hrv-c strains and those close or similar to hrv-a species, which led to the suggested classification of hrv-c into at least two subspecies. eight hundred and twenty-seven samples were collected from a group of children consulting the shanghai nanxiang hospital during a -year period, and tested for respiratory viruses using a multiplex rt-pcr (mrt-pcr). sixty-four samples ( . %) were positive for hrv, according to the length of the amplified fragment in the vp /vp region visualized on agarose gel (data not shown). a larger fragment of nt, including part of the utr (starting at nt ) and the vp /vp genes (ending at nt ), was amplified and cloned into plasmid vectors for genetic analysis (table ) . only one sample, n , could not be amplified and was amplified in two steps in the utr (nt - ) and in the vp /vp region (nt - ), respectively. analyses of different clones for each sample allowed the identification of multiple infections: sample n was shown to contain one hrv-a and one hrv-b (n a and n b, respectively), while n contained one hrv-a and one hrv-c (n a and n c, respectively) (see below). in order to further characterize the hrvs identified in the samples, the nucleotide sequences located in the utr ( nt) and the vp /vp genes ( nt) were chosen to allow comparative alignment with sequences of reference serotypes and field strains available in genbank. we first compared and classified shanghai strains according to their vp /vp sequences. the pairwise nucleotide divergence in the vp /vp region ranged from to . %. twenty-seven hrvs ( . %) showed . % nucleotide identity with the closest hrv-a clusters, and five hrvs ( . %) showed . . % nucleotide identity with hrv-b clusters ( table ). the remaining strains ( . %) diverged from hrv-a and hrv-b species by . . % in their vp /vp nucleotide sequence ( table ) . these strains showed from . to % nucleotide identity with each other and were related to the recently described hrv-c strains, nat and nat , isolated in california, usa [ ] , c , c and c in hong kong [ ] , and qpm in australia [ , ] ( fig. ). strains n , n and n were closely related to the recently identified strains c and nta with . . % nucleotide identity, whereas the remaining hrv-c strains showed only . - . % nucleotide identity with six other recent strains (qpm, nat , nat , c , c and c ; table ), which were classified tentatively as hrv-c species [ , , , ] . classification of the strains into three different species was also demonstrated by construction of a phylogenetic tree using aligned vp /vp sequences (fig. ) . to characterize and classify further the chinese hrv strains, utr sequences were considered (table , fig. ). they were compared to the utr of all reference hrvs, to those of new strains identified in children with respiratory illness in wisconsin (indicated as w) [ ] , and to those of other recently identified hrv-c strains [ ] . pairwise nucleotide divergence between the three hrv species was . - . %, and a limit of , % divergence between genotype pairs was chosen for similar genotype assignment in one species [ ] . new genotypes were identified when they had - % pairwise nucleotide divergence from the nearest serotype in the same species (table ) . fifty-five hrvs shared . . % nucleotide identity with strains already identified, and hrvs showed . - . % nucleotide divergence with the nearest known hrvs. they may represent newly discovered genotypes. these strains clustered with hrv-a (n ) or hrv-c species (n , n , n , n , n , n , n , n , n and n ) ( table ) . most surprisingly, of the strains classified as hrv-c by comparative analysis of vp /p sequences ( table ) were related more closely to hrv-a strains when their utrs were analyzed, and showed incongruent clustering in phylogenetic trees (figs. and ). the nucleotide sequences in the utr of these strains were related closely to those of formerly identified qpm, nat , nat , c , c and c hrv-c strains, and to some of the w strains recently identified as hrv-a [ ] (fig. ) . however, our strains clustered together with these six strains in two major branches in the phylogenetic tree constituted a subspecies of hrv-c called hrv-ca (table , figs. and ) . the fourteen other hrv-c strains formed another unique branch of hrv-c subspecies, called hrv-cc, which clustered differently from other species of hrv-a and hrv-b, and from subspecies hrv-ca in the phylogenetic tree based on utr sequences (table ; figs. and ). these strains were related closely to some w strains of hrv that were classified as hrv-c [ ] (fig. , table ) in order to characterize more precisely the differences observed in the utr between hrv-ca and hrv-cc subspecies, and to localize possible recombination sites in the utr of the genome of hrv-ca subspecies, bootscanning and similarity plot analyses were conducted in the gene fragment of nt that included the utr and adjacent capsid genes. hrv-ca nucleotide sequences were scanned against sequences of n , r and r , which are considered as representative strains of hrv cc subspecies and hrv-a and hrv-b species, respectively. stretches of nucleotide sequences that were closer to hrv-a (r ) than to hrv-cc (n ), flanked by sequences related to hrv-c could be detected in the utr of hrv-ca strains, as exemplified with hrv-ca n strain ( fig. a and b) . these hrv-a-related nucleotide stretches were thus flanked by putative recombination sites. these sites were located differently among the hrv-ca strains, delimiting hrv-a-related stretches that ranged from to nt in length (fig. c ). while variable among the strains, the identified recombination sites were all located inside the utr and none of them was identified in the downstream vp /vp coding sequence. hrv-a-related nucleotide sequences and putative recombination sites were also found in the utr of the previously described hrv-c strains c , c , c , nat , nat and qpm (fig. ) . the results corroborated the clustering observed in the phylogenetic tree based on utr sequences (fig. ) , since strains gathered in the same hrv-ca subcluster (for example n , n , n and n , or n , n , n , n , n and n ) displayed the same recombination pattern. these subclusters revealed different recombinant lineages, each of which originated from independent recombination events. in order to further characterize the genome of the hrv-cc subspecies, for which no full-length sequence was yet available, we sequenced the remaining genes that covered the whole coding sequence and utr of n strain, which was chosen as the representative of this subspecies (tables and ). the full-length n genome sequence was compared to those of the hrv-ca subspecies strains c , c and c , and to that of n strain, which was sequenced as the representative of the hrv-ca subspecies. the genome sequences were also compared to those of the hrv-a strains n and r , and to the hrv-b strains r and r (table ). the full-length nucleotide sequence of n strain contained nt, excluding the poly(a) tract, which was shorter than sequences from hrv-a and hrv-b strains, but similar to those of hrv-ca strain n and other related strains (c , c and c ). the aa lengths of the polyprotein and of each of the individual proteins of n were slightly different from those of hrv-a and hrv-b species, but similar to those of other hrv-c strains. the most divergent amino acid length between hrv was observed for the vp protein that was shorter in hrv-c species ( table ). the unique putative cleavage (m/s) site between vp and vp protein identified previously for qpm, c , c and c strains [ ] was also observed for n and n strains. it was different from those of the hrv-a strains n and r (q/s), and from those of the hrv-b strains r and r (n/s) (data not shown). alignment of the vp amino acid sequence of hrv-cc strain n with those of other hrv-a and hrv-b species and hrv-ca subspecies, designated in table , showed structural features typical of hrv-c species [ , , ] (data not shown). in particular, footprints including deletions in the bc, de and hi loops and conserved amino acids potentially involved in inter-cellular adhesion molecule (icam- ) receptor binding [ , , , ] were conserved within the hrv-c species (data not shown). bootscanning and similarity plot analysis conducted on the genomic sequences of n (hrv-ca), n (hrv-cc), r (hrv-a) and r (hrv-b) confirmed that n featured a utr sequence that was related to the r sequence (stretch i), followed by a capsidic sequence related to the n sequence. n nonstructural sequence ( a to utr) was related more closely to n than to r and r sequences. however, in stretch ii (nt - according to n numbering), n strain (hrv-ca) was closer to r (hrv-a) than to r (hrv-b) or n (hrv-cc), which resulted in high bootstrap values between n and r a sequences (fig. ) . this may have been the result of a recombination event that occurred in the part of the a-encoding sequence of the parental strain n , and which involved an hrv-a strain. nevertheless, the hrv-a parental strain or ancestral strain could not be identified since the closest hrv-a a nucleotide sequence available was , % identical to that of n in this region. in contrast from nt , to the end (stretch iii in fig. ) , the n strain genome was found to be closely related to that of n , with nucleotide identity . %. this result is corroborated in figure , which shows a phylogenetic analysis of the utr sequences of n and n compared to those of hrv-ca subspecies and hrv-a and hrv-b species. this suggests that n and n strains share a common recent ancestor through recombination. and ) and on local recombination in utr (see fig. ). b strains closely related with more than % identity. c these strains clustered differently when based on vp /vp sequences (see table and figs. and ) . doi: . /journal.pone. .t clinical outcome from hrv strains isolated from pediatric outpatients among the pediatric patients, were males and females, and their age ranged from months to years. the majority of hrv infections were diagnosed between and years of age ( . %). bronchitis ( . %) and pneumonia ( . %) were highly prevalent in children with comparable incidence in hrv-a and hrv-c infections (table ) . moreover, the ratio of pneumonia over bronchitis ( . %) was comparable to that in the whole cohort of children ( . %). only one child among the hrv-positive patients had asthma and was co-infected with hrv-c, influenza a virus (iav) and respiratory syncytial virus (rsv) ( table ) , whereas of the patient were diagnosed with asthma. hrvs were isolated throughout the years, with a predominance of hrv-c viruses in the cold season (table ) . interestingly, different hrv genotypes were detected within the same period (for example, n and n , n and n /n , n /n and n , and n /n and n /n ), with a larger diversity and distribution of individual or paired hrv-a genotypes compared to hrv-c strains, which clustered in closely-related genotypes (fig. , tables and ) . conversely, n and n strains of samples collected at months interval showed . % identity (fig. ) . single hrv infection was diagnosed in children and coinfections were identified in patients (table ) , with double and five triple infections. the viruses most often identified in hrv co-infection were rsv (six cases) and human bocavirus (hbov; four cases), and two patients were co-infected with hrv, hbov and rsv (table ). there was no difference between hrv-ca or hrv-cc subspecies and any of the clinical or epidemiological data (data not shown). in this report, we looked for hrvs in a -year collection of npss from children with ari visiting a district hospital in shanghai, and found a high diversity of hrv strains that belonged to different species and genotypes. we characterized by rt-pcr and sequenced hrvs, among them hrv-a, five hrv-b, and hrv-c strains. when sequencing the vp /vp region of the hrv genome, several recent studies have identified new strains of viruses from children and adults with ari, asthma, or otitis, which are clustered differently from hrv-a and hrv-b, and have been classified into a novel hrv-c species [ , , , , , , , , , , ] . other groups have also identified novel hrv-c strains by sequencing the vp gene [ ] or the utr [ , ] . the different sizes and locations of the regions amplified in the hrv genomes renders difficult comparative genetic analysis. recently, palmenberg et al. ( ) have finalized the full-length genome sequences of all hrv-a and hrv-b reference strains, and identified structural features of these two species and the novel hrv-c species [ ] . in our study, we identified hrvs ( . %) that clustered differently from hrv-a and hrv-b in a phylogenetic tree that was established on the basis of vp /vp sequences, which were related to recent strains classified in the novel hrv-c species (fig. , table ). fourteen hrv-c strains ( . %) segregated from the other strains ( . %) that were closely related to hrv-a in their utr sequence (fig. ) . this led us to propose a classification of two hrv-c subspecies, hrv-cc and hrv-ca. in previous studies [ ] . these strains clustered with our field strains within the hrv-cc subspecies. moreover, strains that clustered with hrv-a, and had - % pairwise nucleotide divergence from the nearest reference serotype [ ] , clustered within the two major branches of hrv-a and hrv-ca strains (fig. ) . therefore, we cannot ensure that some of the strains were hrv-a or hrv-ca strains. kiang et al. ( ) have identified five novel hrvs out of clinical samples ( . %), which segregated from hrv-a and hrv-b, and were classified as hrv-c, and three additional strains ( . %) that also clustered with qpm, c , c , c , nat and nat [ ] (fig. ) . however, the field hrv strains of these previous studies were sequenced using a utr that did not match fully our sequence and that of lee et al. ( ) [ ] , and could not be included in the present study for comparative analysis. interestingly, the five strains identified in california in [ ] and n and n from our study were closely related to strain w isolated in wisconsin in the late s [ ] , and to nat isolated in the winter of in california [ ] , which confirms that similar genotypes of hrv-ca are widespread [ ] . the strains of hrv-c species identified in the present study were characterized by analyzing the utr, vp , and part of vp (fig. ) . this approach showed the advantages of covering only ncr, vp /vp , vp or d genome fragments. analyzing sequences that covered the utr and the downstream vp / vp capsid region allowed identification of co-infections when several clones were sequenced, and helped to locate the recombination sites in strains of the hrv-ca subspecies. thus, this region of the genome may be useful for building a database of the novel strains that are circulating worldwide. the genome of hevs is subject to frequent recombination [ , , , , , , ] , with interspecies exchanges observed in the utr [ ] . palmenberg et al. ( ) have observed intraspecies recombination in three hrv-a, with structural characteristics and phylogenetic evidence that suggests a novel clade d classification [ ] . tapparel et al. ( ) observed phylogenetic incongruities in utr, vp and cd sequences of two clinical isolates of hrv-a related to recombination [ ] . we observed incongruent clustering of n , n and n strains of hrv-a species in the phylogenetic trees based on the utr or the vp /vp regions of their genomes (figs. and , table ), which suggests intraspecies recombination in the utr. we observed one co-infection with hrv-a and hrv-b (n a and n b), one with hrv-a and hrv-c (n a and n c), and three co-infections of hrv-a and hrv-b with hevs that may favor recombination events. previous comparison of genome sequences between hrvs showed only limited recombination events and a pattern of genetic diversity lower than that observed with other picornaviruses [ ] . the presence in hrv-c subspecies of sequences that share . - . % identity with hrv-a strains (table ) suggests that recombination events occurred between hrv-c and hrv-a. bootscanning of the utr of hrv-c strains also showed different sites and lengths of recombination (fig. c) , which suggested that there were several independent events that led to several groups of hrv-ca genotypes, which formed clusters in the phylogenetic tree (fig. ) . comparative analysis of the full-length nucleotide sequences of two field strains of different hrv-c subspecies (n and n ) with those of other hrv species suggested that multiple interspecies recombination events occurred in the utr and in the ns a protein gene, and that recombination also occurred in the utr between n and a strain close to n . these findings are in agreement with those observed for other hevs, for which recombination events in the capsid-encoding sequence are very rare, probably because of structural constraints that restrict the functioning of chimeric capsids [ ] . this result appeals for the full-length genome sequencing of the major representatives of the hrv-c species, in order to establish a clear understanding of the evolution and classification of the novel virus into subspecies. comparison of the coding sequences of n hrv-cc with other strains of hrv-ca subspecies [ ] , including our field strain n , showed high similarities in the lengths of the proteins, their cleavage sites, and the structural features of vp . these characteristics and the absence of growth in cell culture, noted in our laboratory and by others (data not shown), support the classification of the novel strains into a unique hrv-c subspecies. our clinical specimens all originated from npss from pediatric outpatients. the remarkable outcome of the study is the large diversity of genotypes that has circulated in a relatively small group of people in a district of shanghai during a -year observation. although some clusters of similar genotypes in a limited period of time were observed, co-circulation of different genotypes and hrv species and subspecies, and co-infections with two hrv species were observed. the prevalence of the novel hrv-c in our specimens ( . %) differed from previous studies that associated the prevalence of the novel variant with severe disease outcomes, which ranged from influenza-like illness or infection of the low respiratory tract [ , ] to asthma exacerbation, bronchiolitis, and febrile wheeze [ , , , , , , , ] . all our patients showed bronchitis or pneumonia, with no etiological correlation with any of the species or subspecies of hrv. only one patient co-infected with hrv-c, iav and rsv was diagnosed with asthma among the hrv-positive patients ( . %), whereas of the children had asthma ( %). the difference observed with previous studies, . % [ ] and % [ ] asthma in hrv-positive patients, may be related to the criteria for enrolment. moreover, none of the patients in our study were hospitalized, which makes comparison with hospitalized children difficult [ , ] . another criterion to consider in the trend to correlate clinical symptoms with hrv infection is the presence of co-infecting pathogens. in our study, four strains of hbov and six strains of rsv ( . %) were identified in association with hrv-c ( . %). hbov and rsv are common viruses diagnosed in ari, which are often associated with hrv [ , ] , and hbov was identified in . % of children co-infected with hrv [ ] . nevertheless, the incidence of hbov in ari and in severe outcomes remains elusive [ ] . more studies need to be carried out on large numbers of samples from severe and mild diseases, to identify any obvious role of hrv sequence diversity and association with other pathogens in disease severity. since a large diversity of recombination in hrvs has become obvious, we must be aware of the occurrence of novel hrvs that may become highly virulent. this study was approved by the ethical committee of shanghai nanxiang hospital and written informed consent was obtained from the parents of the children. clinical specimens (n = ) from npss were collected from children under years old, who experienced a lower respiratory tract infection, and who were consulting the pediatric department of shanghai nanxiang hospital during the period october to october . total rna was extracted from nps specimens using qiaamp viral rna mini kit (qiagen, hilden, germany), and stored at uc. rna was amplified using the qiagen one step rt-pcr kit. a five-tube mrt-pcr was used for virus identification as previously described [ , ] . tube targeted iav, influenza b virus, rsv, and human metapneumovirus; tube , parainfluenza viruses to ; tube , hrv and influenza c virus; tube , human coronaviruses (hcovs) e-hcov, oc -hcov, nl -hcov and hku -hcov; and tube , adenovirus and hbov. amplified products were analyzed in . mg/ml ethidium bromide/ % agarose gel. samples that showed positive results for hrv were amplified again using specific primers p - f and vp / r, located in the utr and vp gene, respectively (table ) . one strain of hrv-c (n ) could not be amplified using the p and vp extreme primers and was amplified using primers in utr and vp / vp , respectively (table ). in brief, . ml of extracted rna was mixed with buffer and . mm dntps, . mm of each of the primers, and ml of enzyme mix, and diethylpyrocarbonatetreated ultrapure water was added to a final volume of ml. amplification programs included reverse transcription at uc for min, inactivation at uc for min, followed by cycles at uc for s, uc for s, uc for s, and final extension at uc for min. the amplified dna products were detected by ethidium bromide-agarose gel electrophoresis. the lengths of p -vp , vp -vp and p -p amplicons were , and nt, respectively. dna products were extracted from agarose gels by using qiaquick gel extraction kit (qiagen), and were ligated into pmd -t vector (takara biotechnology, dalian, china), and at least two recombinant plasmids were sequenced in biosune sequence company and life biotechnology in shanghai, china. sequences of different clones of n and n showed identities for either hrv-a or hrv-b strains. more plasmids were sequenced for these strains to confirm that the two patients were originally coinfected with two different hrv species. sequences of three complete genomes of hrv were obtained for strains n (reference r collected on december , ) , n (r collected on june , ) and n (r collected on july ). primers used for the amplification of viral genomes were designed after multiple alignments of sequences from the genomes of different hrvs available in genbank (table ) . overlapping amplified dna products were obtained after pcr of cdna that was obtained using oligodt and a transcriptor high fidelity cdna synthesis kit (roche, mannheim, germany), following the manufacturer's protocols. briefly, . ml viral rna was mixed with ml oligodt and heated at uc for min, and then kept on ice for min. after addition of ml buffer, . ml protector rnase inhibitor, ml dntps, ml dtt, and ml rt enzyme, the reaction was incubated at uc for h, inactivated at uc for min, and stored at uc. amplification of a d region of n , n and n hrv strains was carried out by nested-pcr using takara extaq (takara biotechnology) and specific primers (table ) , for cycles of s at uc, s at uc, and s at uc. to amplify vp (upstream of a) sequences of n and n strains, nested pcr was carried out using takara lataq with gc buffer i(takara biotechnology), specific primers (table ) , and incubation for cycles of s at uc, s at uc, and min at uc. the fragment vp - d of n hrv strain was obtained by seminested pcr and specific primers vp f, and d inner and outer reverse primers (table ) , using takara lataq with gc buffer ii, for cycles of s at uc, s at uc, and min at uc. the terminal part of the whole genome was obtained by rapid amplification of cdna ends using / rapid amplification of the cdna kit, following the manufacturer's protocol (roche). to perform terminal race, ml of cdna buffer, ml dntps, . ml specific primer ( mm), . ml rna, ml control primer neo /rev ( . mm), ml control rna, ml rt enzyme, and . ml rnase inhibitor (roche) were mixed and incubated for uc for min, followed by inactivation at uc for min, and stored on ice. the product was purified using the qiagen pcr purification kit and eluted with ml deionized distilled water. a polya tail was added to the cdna, by mixing . ml dna with . ml reaction buffer, . ml ( mm) datp, and after incubation at uc for min, the reaction was chilled on ice for min. after addition of . ml terminal transferase, the reaction was incubated at uc for min, inactivated at uc for min, and kept on ice. nested pcr was performed by using the expend high fidelity pcr kit (roche). a mixture of . ml poly-da-tailed cdna, . ml oligodt-anchor primer . mm, . ml sp primers ( mm) (table ) , . ml control neo /rev primer ( . mm), . ml dntp, . ml enzyme, . ml buffer, and ml ddh o was incubated for cycles of s at uc, s at uc, and s at uc. to perform terminal race, the method was similar to normal two-step rt-pcr using d inner and outer f primers (table ) . sequence alignment, phylogenetic analyses and recombination analysis dna sequences used for p -p gene analysis were based on hrv- nt - and those used for vp /vp gene analysis were based on hrv- nt - . multiple sequences were aligned using clustal x [ ] . the multiple-sequence alignment was subjected to phylogenetic analyses using programs in the phylip package (v . ). bootstrap analysis was performed using seqboot, with a replicate number of . then, dnadist and neighbor were used to obtain distance matrices with the f parameter, and a transition/transversion ratio of . consensus trees were computed by consense, and then re-rooted with retree. the final tree was visualized and edited with mega version [ ] . recombination analysis was carried out by using recombination detection program v. . . manual bootscanning was performed by using the juke-cantor algorithm and the neighbor-joining method [ ] , with a window size of nt, a step size of nt and replicates. pairwise identities between sequences were determined with simplot software method [ ] ,with a window size of nt and a step size of nt. pairwise homology matrices were obtained by using clc combined workbench . software (clc bio, aarhus, denmark). the original p -vp sequences described in this study were deposited in genbank under accession nos. gq to gq . the vp -vp sequences were deposited under the nos gq to gq , and the p -p sequences under the nos gq to gq . the full length genomes sequences of n , n and n strains were deposited under the nos. gq , gq , gq , respectively. rhinovirus and the lower respiratory tract the common cold human rhinoviruses: the cold wars resume new developments in the epidemiology and clinical spectrum of rhinovirus infections association of respiratory picornaviruses with acute bronchiolitis in french infants respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with 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type genomes from subtype cinfected seroconverters in india, with evidence of intersubtype recombination key: cord- - yvyiiuy authors: nikas, jason b. title: inflammation and immune system activation in aging: a mathematical approach date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: yvyiiuy memory and learning declines are consequences of normal aging. since those functions are associated with the hippocampus, i analyzed the global gene expression data from post-mortem hippocampal tissue of old (age ≥ yrs) and young (age ≤ yrs) cognitively intact human subjects. by employing a rigorous, multi-method bioinformatic approach, i identified genes that were the most significant in terms of differential expression; and by employing mathematical modeling, i demonstrated that of the genes were able to discriminate between the old and young subjects with high accuracy. remarkably, % of the known genes from those most significant genes are associated with either inflammation or immune system activation. this suggests that chronic inflammation and immune system over-activity may underlie the aging process of the human brain, and that potential anti-inflammatory treatments targeting those genes may slow down this process and alleviate its symptoms. i n the absence of any neurodegenerative disease, the aging process of the human brain is inevitably and quintessentially characterized by memory and learning impairments. unlike in the case of a neurodegenerative disease, normal aging has not been associated with neuronal loss [ ] [ ] [ ] . rather, it has been observed that the impairments induced by normal aging are associated with synaptic remodeling, and that they are more likely to affect functions that are associated with the hippocampus, i.e. several areas of memory and learning , , . in order to study the process of human normal aging, this study focuses on the most vulnerable target of that process, namely, the hippocampus. given the long, gradual course of the normal aging process, i arbitrarily defined the boundaries of the two groups as follows: ) old subjects (o) with age $ years and ) young subjects (y) with age # years. this -yr age gap, i theorized, would accentuate the contrast between the two groups in connection with this otherwise continuous and overlapping process. i analyzed the global gene expression data from post-mortem hippocampal tissue (harvested from the body of the hippocampus at the level of the lateral geniculate nucleus) of old and young cognitively intact human subjects, posted at the gene expression omnibus (gse ) . demographical information pertaining to all subjects is shown in supplementary table . having employed three different and independent methods of statistical significance, namely, roc curve analysis, fold change, and p-value, i was able to identify genes that were the most significant in terms of differential expression. fig. b depicts the results of k-means clustering analysis based on the expression of the top most significant genes. all k-means clustering analysis results (with respect to both the housekeeping genes and the most significant genes) are shown in supplementary table . as can be seen in both fig. b and supplementary table , there is a clear separation of the two groups. fig. depicts the heat map that resulted by plotting the expression of those genes for all subjects ( young and old). as can be seen by the relative intensities, all of the most significant genes are over-expressed (red color) in the case of the old subjects as compared with the case of the young subjects (blue color). the direction of the differential expression of those genes also appears in table . moreover, fig. provides a d representation of the differential expression of those genes between the two groups in a surface-contour plot. classify the subjects with a high accuracy. such a model would be valuable in future studies of global gene expression analysis of post-mortem hippocampal tissue investigating biological and chronological aging. to that end, i randomly selected approximately % of the subjects [ / young subjects and / old subjects] for the development of the function (henceforward referred to as super variable), and i used the remaining subjects ( young and old) solely for the purpose of validating the super variable. employing a general methodology that i have previously introduced , , i was able to generate a super variable (function) that, based on the input of genes from the most significant genes, was able to identify/classify accurately all but one of the old subjects {subject # [ yrs (f)]} [sensitivity ( / ) . ] and all of the young subjects [specificity ( / ) . ]. those overall results of the performance of the f super variable were obtained by combining the results from the development and the validation phases. according to the rank that appears in table , the seven genes that provide the input to the f super variable are: c a (c b), adora , ms a , bcl , cd , c ar , and hla-drb . all of those seven genes are, in terms of biological function, either genes of inflammation or genes of immune system activation. supplementary fig. shows the f super variable function in relation to its input gene variables. fig. table show the overall results of the f super variable, i.e. the f scores of all subjects used in this study, as well as their respective classification. fig. and supplementary table were created by combining the results from the development phase (the f scores of all subjects that were randomly selected and used exclusively for the development of the model) with the results from the validation phase (the f scores of all subjects that were randomly selected and used exclusively for testing purposes). the results of the f super variable in the development phase are shown in supplementary fig. and supplementary table , whereas the results in the validation phase are shown in supplementary fig. and supplementary table . it is interesting to note here that, assessing and comparing the performance of the f super variable (supplementary table ) with that of the supervised k-means clustering (supplementary table ), one can see that the former yielded one misclassification as opposed to four yielded by the latter. finally, it should also be noted here that, owing to the constraints of this study, namely, the paucity of healthy, normal human brain tissue samples and respective available data, the f super variable needs to be further validated with a larger, independent cohort. biovariability of aging. it has long been observed empirically that aging is not a steady-state, uniformly continuous process; that it is characterized by a relatively wide biovariability; and that biological age may not necessarily coincide with chronological age. the results of my study corroborate those observations. looking at the expression of the most significant hippocampal genes of all subjects table ). moreover, the aforementioned observations about the biovariability of the aging process were also supported by the results of hierarchical clustering analysis performed on the f scores of all subjects (supplementary fig. ) . young subjects (y) with respect to the house-keeping genes (a), and with respect to the most significant genes (b). in (a), in connection with the house-keeping genes, the two groups are inseparable and indistinguishable; whereas in (b), in connection with the most significant genes, the two groups are separated and are clearly distinguishable. d is subject distance from the centroid of cluster , and d is subject distance from the centroid of cluster . inflammation and immune system activation in aging. remarkably, of the known genes out of the most significant genes, were -in terms of function -either genes of inflammation or genes of immune system activation (table ). this suggests that -to a large extent, and insofar as it pertains to the hippocampal area of the brain -the dual process of a chronic inflammation and the elicited chronic immune-system response and activity can differentiate between old and young brains with a high accuracy. this is further supported by the fact that the aforementioned seven genes employed by the f super variable, all of which are genes of inflammation or genes of immune system activation, can discriminate between old and young brains with almost a perfect accuracy [sensitivity ( / ) . and specificity ( / ) . ]. the seven genes [c a (c b), adora , ms a , bcl , cd , c ar , and hla-drb ], which are the constituent input variables of the model (f super variable), and all of which are -in terms of function -inflammation or immune system activation genes (table ) , were all found to be over-expressed in the old subjects compared with the young subjects (table ) . c a (c b) has been observed to be over-expressed in patients with huntington disease and alzheimer disease , in mice with rheumatoid arthritis , etc. adora has been found to be over-expressed in the hippocampus of patients with parkinson disease , in patients with astrocytomas , etc. ms a has been observed to be over-expressed in mice with rheumatoid arthritis . bcl has been observed to be over-expressed in patients with huntington disease , with ischemic stroke , with rheumatoid arthritis , with b-cell lymphoma , etc. cd has been found to be over-expressed in patients with systemic lupus erythematosus , with immune thrombocytopenia , with schwannomas , with huntington disease , and numerous other diseases and conditions. over-expression of c ar has been observed in patients with severe acute respiratory syndrome , with asthma , etc., while overexpression of hla-drb has been observed in patients with multiple sclerosis , with rheumatoid arthritis , with duchenne muscular dystrophy , etc. previous studies using animal models have observed associations between aging and inflammation in connection with the hippocampus, the neocortex, and the cerebellum , . using animal models or human subjects with early-stage neurodegenerative diseases, such as alzheimer, other studies have observed a link between neuroinflammation and deficits in synaptic plasticity, especially long-term potentiation (ltp) in the hippocampus, which is associated with longterm memory consolidation [ ] [ ] [ ] [ ] [ ] [ ] . the fact that definitive causality cannot be established here notwithstanding -in other words, whether it is the normal aging process that induces inflammation/ immune-system-overactivity, or whether the vice versa occurs, or whether another, hitherto unspecified, process engenders the normal aging process, which in turn induces inflammation/immune-systemoveractivity, or whether that unspecified process engenders inflammation/immune-system-overactivity, which in turn induces the normal aging process -the results of my study support a direct causal link between the normal aging process and the process of inflammation/immune-system-overactivity. when considered collectively, therefore, the results of my study and all of the above observations from the other aforementioned studies point to a plausible theory on the normal aging process. at some point in time, chronic, low-level inflammation establishes itself and elicits a corresponding chronic immune response and activity. these two conjugate processes ultimately are responsible for a gradual loss of synaptic plasticity, particularly ltp in the hippocampus, accompanied with a minimal neuronal loss , , [ ] [ ] [ ] . it is this loss of synaptic plasticity -at least in the hippocampus part of the brain -that is associated with the phenotypical changes of normal aging. the results of my study, in addition to providing evidence for this dual process of chronic, low-level neuroinflammation/immune-system-activation in connection with normal aging, suggest a means of a potential treatment. regardless of the exact causal sequence of the events, administration of anti-inflammatory drugs/chemicals that can normalize the expression of the aforementioned genes of inflammation/immune-system-activation may decelerate the onset of the aging process, as well as the aging process itself, and mitigate its symptoms by restoring synaptic plasticity throughout the hippocampus and possibly throughout the rest of the brain. supplementary table lists all those most significant genes as possible targets for the development of such an anti-inflammatory treatment, along with potential candidate drugs/chemicals that are known (via ingenuity pathway analysis) to interact with those genes. it is worth noting here that various anti-inflammatory drugs have been used in an effort to slow down the progression of neurodegenerative diseases, such as alzheimer, with various degrees of success , . the magnitude of the neuroinflammatory processes in the case of alzheimer disease or other neurodegenerative diseases, however, cannot be compared to that of the neuroinflammation in the normal aging process; and by virtue of the same argument, the task of . the f uses of the most significant genes as its input variables. using the expression value of those genes for a particular subject, the f yields the f score of that subject; and, based on the determined cut-off score of . , the f classifies that subject as young if the f score is , . or as old if the f score is $ . . as can be seen by the overall performance, the f classified correctly all subjects except one old one [sensitivity ( / ) . and specificity ( / ) . ]. the mean f score of the y subjects was . (top of the blue bar) and their standard deviation (whiskers above or below the top of the blue bar) was . . the mean f score of the o subjects was . (top of the red bar) and their standard deviation (whiskers above or below the top of the red bar) was . . the significance level was set at a . (two-tailed), and the probability of significance for the f was p . (independent t-test with t-value . ). the f is parametrically distributed with respect to both groups. the f scores of all subjects are shown in supplementary table . www.nature.com/scientificreports scientific reports | : | doi: . /srep halting neuronal cell loss during the course of a neurodegenerative disease cannot be compared to that of restoring synaptic plasticity during the course of the normal aging process. it would stand to reason, therefore, that anti-inflammatory treatment strategies may be more successful and efficacious than those employed against the progression of neurodegenerative diseases. finally, i should point out that, based on increased evidence over the last twenty years or so, neuroinflammation seems to be the common denominator of normal aging and neurodegenerative diseases, such as alzheimer. understanding the causal circumstances under which a chronic, low-level neuroinflammatory process can transition to a major neuroinflammation conducive to neuronal degeneration and death would be of paramount importance. according to the latest evidence , even a single substitution mutation on a single inflammation gene might suffice to trigger that transition in a small percentage of the population. data acquisition. i downloaded the raw intensity microarray data (cel files) of old subjects (age $ years) and young subjects (age # years) posted at the gene expression omnibus (accession number: gse ) . data processing. i processed the original raw intensity data (cel files) using the expression console software by affymetrix with the library for the hg-u plus . microarray chip, and choosing the rma algorithm ( (k) fda approved) with the standard settings. statistical methods. i first assessed the quality of the data by examining the expression of all housekeeping genes by all subjects ( old and young). with regard to the housekeeping genes, there was no statistically significant differential expression between the two groups (supplementary table ). as can be seen by the results of k-means clustering analysis in fig. a , the two groups cannot be discriminated based on the expression of the housekeeping genes. having, thus, established the quality of the data, i investigated for any differential expression among all gene variables using three different and independent methods. ) using a methodology that i have developed and introduced previously - , i performed roc curve analysis on all gene variables in order to assess their discriminating power with respect to the two groups (old vs. young), and with respect to this method, i set statistical significance at roc auc $ . . table . in order to minimize the number of false negatives in the case of the third method , , for the final selection of significant variables, i imposed the condition that if a given gene variable met the significance criteria of all three methods, or those of the first method and those of only one of the other two methods, it would be deemed significant. excluding multiplicities (different transcripts that corresponded to the same genes), thirty six genes made up the final list of the most significantly differentially expressed genes between the two groups, as assessed by the aforementioned three different and independent methods of statistical significance (table ) . in greater detail, to assess statistical significance, i used to assess statistical significance, i used the following three different and independent methods. ) roc curve analysis. i performed roc curve analysis on all gene variables in order to assess their discriminating power with respect to the two groups (old vs. young), and with respect to this method, i set statistical significance at roc auc $ . . ) fold change. for all gene variables, i defined fold change (fc) as the mean expression value of the old subjects over the mean expression value of the young subjects, and i set statistical y) . ) p-value. i used the independent t-test for parametric gene variables (both normality and homogeneity of variance conditions were met); the aspin-welch unequal-variance test (aw) for gene variables that met the normality condition but not the homogeneity of variance condition; and the mann-whitney u test (mw) for the non-parametric gene variables, i.e., for those variables that i) the normality condition was not met or ii) the normality and the homogeneity of variance conditions were not met. taking into account that there are , probe sets (including those of the housekeeping genes) in the affymetrix hg-u plus . chip, and using the bonferroni correction, i set the significance level for the entire study at a . . therefore, in order for any variable to be deemed significant according to the p-value method, the following condition must be met: p , a. regarding the mann-whitney u test (mw), since none of the non-parametric variables had any sets of ties (a subject from one group having the same expression value as a subject from the other group), i used the exact probability for all mw tests. incorporating the three aforementioned independent methods of statistical significance assessment, and in order to minimize the number of false negatives in the case of the third method , , i set the overall significance criterion as follows: in order for any variable to be included in the final list of the most significant variables, it would have to meet the significance criteria of the first method (roc auc $ . ) and those of at least one of the other two methods [fc $ . (or fc # . ) and/or p , . ]. mathematical modeling. utilizing the final most significant genes, i wanted to explore the possibility of developing -via mathematical modeling -a function that could identify as correctly as possible the age status (o or y) of an unknown subject based on the expression of any combination of those most significant genes. to that end, i randomly selected approximately % of the subjects [ / young subjects and / old subjects] that could be used only for the development phase of such function. in other words, a function could be developed only by the exclusive use of those subjects. the remaining subjects ( young and old ones) were designated unknown (test) subjects and were used solely for the purpose of validating any promising function generated in the development phase. this split into two fixed sets, whereby one is used only for training and the other only for validation, represents the simplest implementation of k-fold cross validation , . a function was deemed promising in the development phase only if it exhibited a sensitivity $ . and a specificity $ . in connection with the subjects of the development phase. pertaining to the validation phase, and in connection with the unknown subjects, a promising function would have to exhibit the same minimum classification accuracy (a sensitivity $ . and a specificity $ . ) in order to be accepted. i was able to generate one such function (f -henceforward also referred to as super variable) that fulfilled all of the aforementioned criteria. supplementary fig. shows the equation of f as a function of genes. the cut-off score of the f was determined by taking into account the results of the following two analyses: ) calculation of the optimal point on the roc curve based on the f scores of the subjects used in the development phase [optimal point is defined as the point with the highest sensitivity and the lowest false positive rate ( specificity)] and ) calculation of the . % confidence intervals for the mean f scores of the two groups (o and y) of those subjects and their respective standard deviations. the . % confidence intervals were calculated based on a bootstrap sample size of , . taking into account the aforementioned roc optimal point, as well as the relative overlap of mo and my [mo llo -sdo and my uly sdy] (llo: the . % confidence lower limit for the mean of the o group; sdo: standard deviation of the o group; uly: the . % confidence upper limit for the mean of the y group; sdy: standard deviation of the y group), the cut-off score of the f super variable was determined to be . . if a subject's f score is , . , then that subject is classified as y (young); otherwise, if the f score is $ . , then that subject is classified as o (old). it should be pointed out here that, based on the equation of the f ( supplementary fig. ), a given f score is just a numerical value and does not signify age or number of years. in addition to the main validation method explained above, and in order to further assess the performance of the f super variable, i employed two other and different cross validation methods: ) a -fold cross validation and ) a leave-one-out cross validation . both of those methods yielded a misclassification rate of . and a mean-squared error of . in connection with the f super variable. the results of those methods, along with the confusion matrices generated by them, are shown in supplementary table . as can be seen in supplementary table , each one of those two and different validation methods resulted in a correct classification of all of the young subjects and in a correct classification of all but two of the old subjects. computer software. all analyses in this study were carried out with custom software written in matlab r b. all computer programs in connection with the model were also created using matlab r b. the ageing cortical synapse: hallmarks and implications for cognitive decline neural mechanisms of ageing and cognitive decline aging and the human neocortex genomic and proteomic strategies to identify novel targets potentially involved in learning and memory gene expression changes in the course of normal brain aging are sexually dimorphic mathematical prognostic biomarker models for treatment response and survival in epithelial ovarian cancer prognosis of treatment response (pathological complete response) in breast cancer regional and cellular gene expression changes in human huntington's disease brain complement activation by beta-amyloid in alzheimer disease identification of arthritis-related gene clusters by microarray analysis of two independent mouse models for rheumatoid arthritis therapeutic potential of adenosine a( a) receptor antagonists in parkinson's disease a adenosine receptors in human astrocytoma cells: agonist-mediated desensitization, internalization, and down-regulation gene expression in huntington's disease skeletal muscle: a potential biomarker the future of genomic profiling of neurological diseases using blood inducible bronchus-associated lymphoid tissue (ibalt) in patients with pulmonary complications of rheumatoid arthritis primary mediastinal b-cell lymphoma: high frequency of bcl- mutations and consistent expression of the transcription factors oct- , bob. , and pu. in the absence of immunoglobulins phosphorylated erm is responsible for increased t cell polarization, adhesion, and migration in patients with systemic lupus erythematosus microrna regulate immunological pathways in t-cells in immune thrombocytopenia (itp) cd expression is aberrant in benign schwann cell tumors possessing mutations in the neurofibromatosis type , but not type , gene expression profile of immune response genes in patients with severe acute respiratory syndrome complement regulates inhalation tolerance at the dendritic cell/t cell interface genome, epigenome and rna sequences of monozygotic twins discordant for multiple sclerosis autoimmune diseases cdna microarray analysis of individual duchenne muscular dystrophy patients immune response gene expression increases in the aging murine hippocampus gene expression profiling of aging using dna microarrays synaptic correlates of increased cognitive vulnerability with aging: peripheral immune challenge and aging interact to disrupt theta-burst late-phase long-term potentiation in hippocampal area ca age-related neuroinflammatory changes negatively impact on neuronal function neuroinflammation and synaptic loss circuitspecific alterations in hippocampal synaptophysin immunoreactivity predict spatial learning impairment in aged rats gene regulation and dna damage in the ageing human brain gene-expression profile of the ageing brain in mice preserved neuron number in the hippocampus of aged rats with spatial learning deficits aging, spatial learning, and total synapse number in the rat ca stratum radiatum protective effects of nsaids on the development of alzheimer disease relation of nsaids to incident ad, change in cognitive function, and ad pathology variant of trem associated with the risk of alzheimer's disease comparison of analytical mathematical approaches for identifying key nuclear magnetic resonance spectroscopy biomarkers in the diagnosis and assessment of clinical change of diseases roc-supervised principal component analysis in connection with the diagnosis of diseases application of clustering analyses to the diagnosis of huntington disease in mice and other diseases with well-defined group boundaries linear discriminant functions in connection with the micro-rna diagnosis of colon cancer apobec b is an enzymatic source of mutation in breast cancer what's wrong with bonferroni adjustments do multiple outcome measures require p-value adjustment? an introduction to the bootstrap asymptotic statistical theory of overtraining and cross-validation the elements of statistical learning this study was supported by the dept. of neurosurgery and the masonic cancer center at the university of minnesota. the author would like to thank douglas yee and walter low for their support. j.b.n. conceived, designed, and carried out all aspects of this study and wrote and edited the manuscript. key: cord- - bw jbzq authors: borlak, jürgen; singh, prashant; gazzana, giuseppe title: proteome mapping of epidermal growth factor induced hepatocellular carcinomas identifies novel cell metabolism targets and mitogen activated protein kinase signalling events date: - - journal: bmc genomics doi: . /s - - -z sha: doc_id: cord_uid: bw jbzq background: hepatocellular carcinoma (hcc) is on the rise and the sixth most common cancer worldwide. to combat hcc effectively research is directed towards its early detection and the development of targeted therapies. given the fact that epidermal growth factor (egf) is an important mitogen for hepatocytes we searched for disease regulated proteins to improve an understanding of the molecular pathogenesis of egf induced hcc. disease regulated proteins were studied by de maldi-tof/tof and a transcriptomic approach, by immunohistochemistry and advanced bioinformatics. results: mapping of egf induced liver cancer in a transgenic mouse model identified n = (p < . ) significantly regulated proteins of which n = were tumour-specific. to unravel molecular circuits linked to aberrant egfr signalling diverse computational approaches were employed and this defined n = key nodes using n = disease regulated proteins for network construction. string analysis revealed protein-protein interactions of > % disease regulated proteins with individual proteins being validated by immunohistochemistry. the disease regulated network proteins were mapped to distinct pathways and bioinformatics provided novel insight into molecular circuits associated with significant changes in either glycolysis and gluconeogenesis, argine and proline metabolism, protein processing in endoplasmic reticulum, hif- and mapk signalling, lipoprotein metabolism, platelet activation and hemostatic control as a result of aberrant egf signalling. the biological significance of the findings was corroborated with gene expression data derived from tumour tissues to evntually define a rationale by which tumours embark on intriguing changes in metabolism that is of utility for an understanding of tumour growth. moreover, among the egf tumour specific proteins n = were likewise uniquely expressed in human hcc and for n = proteins regulation in human hcc was confirmed using the publically available human protein atlas depository, therefore demonstrating clinical significance. conclusion: novel insight into the molecular pathogenesis of egf induced liver cancer was obtained and among the newly identified proteins several are likely candidates for the development of molecularly targeted therapies and include the nucleoside diphosphate kinase a, bifunctional atp-dependent dihydroyacetone kinase and phosphatidylethanolamine-binding protein , the latter being an inhibitor of the raf- kinase. electronic supplementary material: the online version of this article (doi: . /s - - -z) contains supplementary material, which is available to authorized users. liver malignancies are common cancers worldwide and are responsible for approximately one million deaths each year with most hcc patients having poor prognosis as a result of rapid disease progression. the relative -year survival rate is about % and can be attributed to an advanced stage of disease at the time of diagnosis, the occurrence of cirrhosis and of other co-morbidites [ ] . detection of early stages of disease is essential for an improved prognosis and overall survival. however, apart from alpha-fetoprotein (afp) only a few serological markers are available in clinical practice (such as glypican- , mir- , fucosylated gp , α-fucosidase) with afp diagnostics remaining unsatisfactory because of its low sensitivity and the non-specific correlation between the clinical behavior of hcc and afp blood levels. for this reason, new biomarkers are in strong demand [ , ] and more selective markers, such as soluble interleukin- receptor levels, are evaluated [ ] . importantly, research into the molecular pathogenesis of hcc identified several signalling pathways as deregulated. this inspired the development of molecularly targeted therapies such as the multikinase inhibitor sorafenib that inhibits signalling of c-raf- , mek, erk, vegfr, pdgfr and other kinases, effectively [ ] . given that epidermal growth factor is an important mitogen for hepatocytes we were particularly interested in an understanding of the consequences of its targeted overexpression in liver. in our initial study we reported the oncogenomics and pathology of egf induced hepatocarcinogenesis and an identification of molecular circuitries linked to exaggerated egfr signalling [ ] . furthermore, we investigated the serum proteome of egf-tumourbearing mice to obtain information on disease-regulated proteins and to search for novel biomarkers at different stages of disease [ ] . note, a regulatory loop was proposed whereby egf induces transcriptional activation of hdac by ck Α/akt activation in liver cancer cells [ ] . additionally, inhibition of egfr in different animal models by erlotinib was shown to attenuate liver fibrosis and the development of hepatocellular carcinoma [ ] thus suggesting new therapeutic intervention strategies in the prevention of hcc. indeed, the egf receptor tyrosin kinase plays a much wider role in the immortalization of different cell types as originally anticipated [ ] , and is highly expressed in a number of solid tumours and egfr over-expression correlates well with tumour progression, resistance to chemotherapy and poor prognosis. the present study aimed at an identification of disease regulated proteins to facilitate an improved understanding of its complex signalling networks and to search for cross-talk amongst other pathways while an identification of disease regulated proteins would aid the development of molecularly targeted therapies. for this purpose, a two-dimensional electrophoresis and maldi-tof/tof ms strategy was employed to identify disease regulated proteins in an egf transgenic mouse model of hcc. this resulted in an identification of statistically significant regulated proteins of which are uniquely expressed in liver cancer. importantly, out of mouse tumour specific proteins were likewise uniquely expressed in human hcc and disease regulated proteins identified in egf induced liver cancer were similarly regulated in human hcc, as determined by immunhistochemistry using different antibodies and the information given in the publically available human protein atlas depository. clinical significance of the identified proteins could be demonstrated and a total of so far unkown proteins could now be related to egf induced liver cancer, several of which are likely candidates for the development of molecularly targeted therapies. this includes the nucleoside diphosphate kinase a, bifunctional atp-dependent dihydroxyacetone kinase, phosphatidylethanolamine binding protein , i.e. an inhibitor of the raf- kinase as well as aldo-keto reductase family proteins, members c and c , interleukin and the v-crk sarcoma virus ct oncogene homolog. finally, to gain insight into the molecular circuitries of egfr induced hepatocarcinogenesis diverse computational approaches were employed. this revealed master regulatory proteins and permitted network constructions of disease regulated proteins with protein-protein interactions being confirmed for > % of regulated proteins in string analysis. their regulations were also studied by immunohistochemistry in egf transgenic hcc. we also compared the serum and liver proteomes of hcc bearing mice and found proteins to be similarly regulated, thus evidencing leakage of tumour proteins that can be detected in serum. obviously, these are highly interesting biomarker candidates, of which were also regulated in human hcc as determined by immunohistochemistry. all animal work followed strictly the public health service (phs) policy on humane care and use of laboratory animals of the national institutes of health, usa. formal approval to carry out animal studies was granted by the animal welfare ethics committee of the state of lower saxony, germany ('lower saxony state office for consumer protection and food safety' (laves)). the approval id is az: . - - - / . a total of n = c /bl non-transgenic and n = egf transgenic mice (aged - months), weighing - g, were housed in makrolon® type iii cages. water and food (v - , ssniff, the netherlands) was given ad libitum. the temperature and relative humidity was set to ± °c and - %, respectively and a -h day and night cycle was used. tris, urea, thiourea, chaps, dithiothreitol, bromophenol blue, glycerin, sodium dodecyl sulfate, glycine, temed, ammoniumperoxodisulfate, ammonium sulfate, ammonium bicarbonate, colloidal coomassie blue, and acrylamide were purchased from roth (karlsruhe, germany). iodacetamide was obtained from serva (heidelberg, germany) and benzonase was purchased from novagen (darmstadt, germany). ampholytes (biolyte - ) were purchased from bio-rad laboratories (münchen, germany) and destreak was obtained from amersham bioscience (freiburg, germany). mice were anesthetized with ketamine % μl/ g and xylazine % μl/ g, and after surgical removal the liver was perfused and rinsed with ice cold ringer solution until free of blood. approximately . g of the liver sample was ground in a mortar under liquid nitrogen flow. then, the samples were processed with . ml of a buffer containing mm tris base, m urea, % chaps, mm dtt, and . % (v/v) biolyte - first (lb ). the suspensions were homogenized by sonication ( × s) and after addition of μl of benzonase (endonuclease that degrades dna and rna) were left at room temperature for min. the samples were then centrifuged at , g for min. the pellets were washed and sonicated for min with a further . ml of lb and centrifuged at , g for another min, and the resulting two fractions of supernatant were collected (extract a). finally, the pellets were redissolved with . ml of buffer containing mm tris base, m urea, m thiourea, % chaps, mm dtt, and . % (v/v) biolyte - (lb ), sonicated, and centrifuged at , g for min. the pellet was collected, and the supernatant was marked as extract b. from the same animals, a further . -g portion was ground in a mortar, but was now treated with . ml of lb . the suspensions were sonicated, incubated with benzonase, and centrifuged. the pellets were then washed with another . ml of lb , sonicated and centrifuged, and the supernatants were collected (extract c). proteome mapping was done under a variety of conditions, e.g. extraction with lysis buffers and . in addition, proteins were separated at two different ph ranges [ ] [ ] [ ] [ ] [ ] [ ] . a total of independent experiments were carried out, and duplicate measurements were run for each experiment. the protein concentration of all extracts was determined using the bradford assay. liquid-phase ief pre-fractionation was performed in the rotofor cell system (bio-rad) following the supplier's instructions. ion exchange membranes were equilibrated overnight in the appropriate electrolyte (anion exchange membranes in naoh . m and cation exchange membranes in h po . m). after four runs ion exchange membranes were always discarded and new membranes were replaced for the other samples. for each run, the electrode chambers were filled with appropriate fresh electrolytes ( ml) . initially, the cell was filled with pure water and run for min at watts constant power to remove residual ionic contaminants from the membrane core and ion exchange membranes. approximately ml of lb were used to fill the cell. a total of mg of total proteins in approximately ml of lb were added to the cell to reach the maximum loadable volume ( ml) . focusing started at watts constant power. after approximately hours the voltage increased to v and the wattage decreased to w. the focused proteins were harvested in ~ . ml fractions, and ph values were checked. fractions having ph values between and . were collected and denoted "a-a" (acid). fractions having ph values > . were collected and denoted "a-b" (basic). again, the protein concentration was determined for both fractions (a-a and a-b) by the bradford method. approximately mg of protein were recovered at the end of the liquid-phase ief pre-fractionation from an initial -mg load. the losses are accounted for by the multi-step pre-fractionation procedure, but are not the result of a precipitate that could not be dissolved in our lysis buffer. after each run the membrane core was cleaned with naoh . m overnight and sonicated for min in water before the new focusing. isoelectric focusing (ief) -first dimension ief was performed using precast linear ipg strips. the -cm ipg strips - and - were loaded with . mg of proteins by active rehydration ( h, v) . samples destined to be separated by ipg strips - received an excess of hydroxyethyldisulphide (hed) (destreak™) prior to the focusing run. focusing began at v for min in rapid mode, , v for h in linear mode and , v for , vh in rapid mode (for the ipg strips - ). ief for the strips - was carried out at v for min in rapid mode, , v for h in linear mode and , v for , vh in rapid mode. each sample was analysed in duplicate. control and hcc samples were run always at the same time ( control and hcc samples). after ief, the ipg strips were either stored at − °c or transferred to ml of equilibration buffer ( m urea, % w/v glycerin, % w/v sds, mm tris-hcl ph . ) with % w/v dtt and . % v/v bromophenol blue solution ( . % w/v bromophenol blue, . m tris-hcl ph . , . % w/v sds) and incubated for min at room temperature. strips were removed and incubated in equilibration buffer with % w/v iodoacetamide and . % v/v bromophenol blue solution for further min at room temperature. finally, the strips and μl sds-page molecular weight standard on filter paper were placed on top of the cm x . cm % second-dimension gel ( % v/ v acrylamide/bis solution, mm tris, ph . , . % v/v sds, / temed, . % v/v aps). both were fixed in place with a . % w/v agarose overlay. gels were run in protean plus dodeca cell (bio-rad) at v for approximately h, followed by v until the bromophenol blue dye reached the bottom of the gel. the running buffer ( mm tris, . m glycin, . % sds) was cooled externally to °c. gels/proteins were fixed overnight in % ethanol, % phosphoric acid, and washed x min with % phosphoric acid. the gels were equilibrated with % ammoniumsulfate, % ethanol, % phosphoric acid for min and finally stained with colloidal coomassie blue for h. after staining, gels were washed min with pure water and scanned on a molecular fx scanner (bio-rad) at μm resolution. protein spots were imaged first automatically and then manually and analysed using the pdquest™ software (bio-rad). the normalization was carried out in total density in gel mode according to the manufacturer's recommendation. gels were excised using the spot cutter of bio-rad and placed into -well microtiter plates. excised gel spots were washed manually with μl of water for min and destained twice, first with μl ammonium bicarbonate mm for min and then with μl % ammonium bicarbonate mm - % acetonitrile for min. finally, the gel particles were covered by acetonitrile until gel pieces shrunk and left to dry for min. all gels/proteins were digested manually in situ with μl of ammonium bicarbonate mm containing ng trypsin (sequencing grade modified trypsin, promega, germany). after min each gel piece was re-swelled with μl of ammonium bicarbonate mm and incubated for h at °c. after h the reaction was stopped by adding μl of trifluoroacetic acid % containing . % (w/v) n-octyl-beta-d-glucopyranoside (ogp) (applichem). for the application of the samples, μl of peptide solution were loaded onto an mtp anchor chip target / (bruker daltonics) previously prepared with a saturated solution of matrix, alpha-cyano- -hydroxy-cinnamic acid (alpha-hcca) (bruker daltonics, bremen, germany). maldi-ms was performed on an ultraflex ii maldi-tof/tof (bruker daltonics) mass spectrometer equipped with a smartbeam™ laser and a lift-ms/ms facility. the instrument was operated in positive ion reflectron mode and an acceleration voltage of kev for the peptide mass fingerprint (pmf) mode. typically, spectra, acquired at hz, were summed and externally calibrated. in the case of ms/ms-cid the lift device was used for selection and fragmentation of the ions; the acceleration voltage in the ion source kv, the timed ion selector was set to . % (relative to parent mass), and argon was used as collision gas (~ - × - mbar). resulting fragments were further accelerated in a second source by kv and analysed by a two-stage gridless reflectron. typically, shots were accumulated for the parent ion signal and shots for the fragments. flexcontrol™ . , and flexanalysis™ . were used as instrument control and processing software (bruker daltonics, bremen, germany). a calibration standard was used for the external calibration of spectra (peptide calibration standard for mass spectrometry, which covered the mass range~ - da internal calibration was achieved using trypsin autolysis products (m/z's . , . and . ) resulting in a mass accuracy of ≤ ppm. spectra were collected by the flexcontrol software without smoothing or baseline subtraction and a peak resolution higher than or a.u. in case of dhb and chca matrixsample preparation, respectively. the spectra were sent to the flexanalysis software which labeled the peaks for protein identification by proteinscape . or biotools . (bruker daltonics). trypsin autolysis products, tryptic peptides of human keratin and matrix ions were automatically discarded by proteinscape (mass control list). proteinscape score booster feature was used to improve database search results by automatic iterative recalibrations and background eliminations. protein scores greater than were considered significant (p < . , mascot) and an annotation as mouse protein as the top candidates was requested in the search when no restriction was applied to the species of origin. identified proteins were checked individually for further considerations. for pmf peak picking the snap peak detection algorithm, a signal to noise threshold of , maximal number of peaks , a quality factor threshold and baseline subtraction tophat was applied. peptide masses were searched against the swiss-prot database (download - sequences, residues) employing the mascot server (in-house mascot-server, matrix sciences ltd., http://www.matrixscience.com/, revision . . ), taking into account carbamidomethyl of cysteines -carbamidomethyl (c)-as fixed modification and possible oxidation of methionine -oxidation (m)-as a variable modification but allowing one missed cleavage. based on initial data, ion precursors were selected by proteinscape for tandem ms data acquisition (by lift-tof/tof, bruker daltonics, bremen, germany). in the mascot ms/ms ions search, the restriction mammalia was applied with peptide tolerance of ( ppm and ms/ms tolerance of ( . da (fixed and variable modifications as pmf). the acceptance criteria for pmf-based identification were an individual ions score > , at least five matching peptides and % peptide coverage of the theoretical sequences. livers, dissected from egf-overexpressing mice aged between - months, were fixed in % buffered paraformaldehyd and embedded in paraffin. μm thick sections were deparaffinized and rehydrated through a descending alcohol series followed by a min washing step in destilled h o. subsequently, antigen retrieval was performed in citrate buffer (ph ) by autoclaving the sections min at °c. the envision kit (dako, hamburg, germany) was used for immunohistochemistry. the slides were rinsed with destilled h o and after a min incubation step in tris-buffered saline (washing buffer), endogenous peroxidase activity was blocked with dako peroxidase blocking reagent for min followed by a second washing step. thereafter, the sections were blocked for min with protein-block serum free (dako) and incubated with primary antibodies for min. details of antibody dilutions with washing buffer are given in additional file : table s . in the case of goat primary antibodies a rabbit-anti-goat bridging antibody (dako) was employed. specifically, the bound primary antibodies or bridging antibodies were detected by use of labelled polymer hrp anti-rabbit secondary antibody (envision kit; dako) and the immunoreactivity was visualized by dako liquid dab substrate chromogen system in a min incubation. finally, the sections were counterstained with harris haematoxylin for min, dehydrated in an ascending alcohol series, coverslipped and examined under a light microscope (leica, jülich, germany). a total of n = disease regulated proteins were filtered for statistical significance at p < . (table ). this yielded n = statistically significantly regulated proteins two of which had identical accession number, i.e. aah = atp synthase h+ transporting mitochondrial f complex, subunit d and bac = apoa but differed in their spot ids as a result of posttranslational modifications. the statistically significantly regulated proteins were grouped into four different categories to yield tumour specific (to), up-regulated (ur), down-regulated (dr) and proteins only expressed in healthy non-transgenic control livers (co). categorization of tumour regulated proteins based on ontology terms non-redundant tumour proteins covering to, ur and dr categories were considered and analysed for ontologies using the genexplain software (v. . . ), the biological pathways tools reactome (http://www.reactome.org) and kegg (http://www.genome.jp/kegg) and wikipathways (http://wikipathways.org). the tumour regulated proteins (to + ur + dr) were subjected to functional classification based on ontology terms and a p-value of < . was considered to be significant. moreover, disease regulated proteins were analysed with the cytoscape software version . . using the function go-tree levels and number or % of proteins for a given term (see additional file : table s ). master regulatory proteins were searched based on the designated workflow of the genexplain software. it is designed to find master regulatory molecules upstream of an input list of regulated tumour proteins. after annotation of the input datasets the tool for master regulator finding over geneways network (http://www.genexplain.com) was applied. specifically, the geneways software is used to automatically extract, analyse, visualise and integrate molecular pathway data from the published peer reviewed literature. it is based on document sorting, term identification, term meaning disambiguation, information extraction, ontology, visualization and system integration [ ] . the following filtering threshold was used, i.e. score cutoff ( . ), search collection (geneways hub), maximum radius [ , ] , fdr cutoff ( . ), z-score cutoff ( . ), penalty ( . ) and decay factor ( . ) (additional file : table s ). protein network for disease regulated proteins were also constructed using the string software (http:// string-db.org/). the underlying database informs on known and predicted protein-protein interaction and the constructed networks are based on active prediction methods of neighborhood, gene fusion, co-occurrence, co-expression, databases and textmining. eventually, confidence scores were calculated for each interaction pair and only those above default cutoff scores ( . ) were selected. finally, mapping of pathways information from reac-tome, kegg and wikipathways have been implemented the proteins are sorted in alphabetical order, and the ncbi annotation is given in the accession number column. molecular weight, pi, and mascot scores are also given. the column "gels", "c" (c = control) and "t" (t = tumour) indicate the frequency of positive identification of proteins in a total of independent gels, whereas "lb " and "lb " (lb = lysis buffer) refers to the different lysis buffers employed. furthermore, references are given for those proteins which have already been described as hcc-associated whereas those marked with a star (*) are so far unknown as egfr disease regulated in hepatocellular carcinoma. over protein networks using information of known pathways and sustained proteins connecting these pathways in a given network. the histopathology and oncogenomics of egf induced liver cancers was previously reported [ ] and an important finding of the study was the % incidence of malignant tumour formation in less than one year after birth. notably, a sequence of events was observed that initially consisted of diffuse large cell dysplasia followed by multiple dysplastic foci and nodules and growth of hcc. figure a and b depict the histopathology of healthy non-transgenic control liver and egf induced tumours, respectively. after protein extraction de was performed. subsequently, the gels were scanned on a bio-rad molecular fx scanner at a μm resolution. image analysis was done with the pdquesttm software and spots were detected automatically. a total of proteins differed in expression or were de novo expressed when de gels of non-transgenic controls and hcc mice were compared (see table for detailed information on the proteins identified and figure e -g depicting examples of zoomin-gels of some regulated proteins). among them are statistically significantly regulated proteins (p ≤ . ) of which were significantly up-regulated (ratio hcc/control ≥ ) and included fibrinogen and subunits of it, vimentin, cu/zn superoxide dismutase, and apolipoprotein e ( figure f (i-iv), while proteins were repressed in expression (ratio hcc/control ≤ . ) and included arginase , dhdh protein, glutathione peroxidase and agmatine ureohydrolase ( figure g (i-iv) and table ). a reference -de map of mouse liver and serum proteins was constructed that consists of more than n = proteins [ , ] . note, in our previous efforts we identified n = serum proteins as regulated in the egf transgenic disease model. among them were alpha-fetoprotein, clusterin, fibrinogen-α and -γ, serum amyloid component p and several apolipoproteins all of which were significantly up-regulated. based on the combined use of de and maldi-ms a total of n = differentially expressed proteins were identified (table ) and included isoforms as well as post translational modifications of albumin ( up-regulated spots), alpha enolase ( down-regulated spots), apoliproptein a-i ( up-regulated spots), atp synthase h+ transporting mitochondrial ( down-regulated spots), fibrinogen beta ( up-regulated spots), glycine n-methyltransferase ( spots, in controls only), hsp ( down-regulated spots), nit protein ( down-regulated spots), peroxiredoxin ( up-regulated spot and down-regulated spot), and c rik ( up-regulated spots) (see table ). importantly, a total of n = so far unknown disease regulated proteins were identified that can now be related to egf induced liver cancer. these are marked with an asterisk in table . furthermore, a comparison of serum and liver proteoms revealed n = proteins to be regulated in common, thus evidencing leakage of tumour proteins into systemic circulation (table ) . among them was serum afp; it's up-regulation and that of others was confirmed by western blot analysis (figure a-e) . likewise, apolipoprotein e was up-regulated both in serum and tumour samples, the ratio hcc/control being . and . , respectively. in a previous study on human hcc increased expression of apoe was observed in % of study cases; however, gene apoe expression and serum levels were unchanged to suggest its accumulation and impaired secretion [ ] . two isoforms of alpha- -macroglobulin were up-regulated in serum of hcc-bearing mice (spot : ratio hcc/control = . ; spot : ratio hcc/control = . ). its expression was exclusively associated with tumours. finally, serum amyloid component p was up to -fold up-regulated in serum and its tissue expression was tumour specific ( table ) . to further evidence disease regulated proteins and to provide information on their subcellular localization a total of n = proteins were studied by immunohistochemistry. five of them were selected for their novelty (see table ) while amphiregulin and epiregulin were chosen for their importance in the egf-signalling pathway. furthermore, hnf α was studied for its pivotal role in liver cancer [ ] . depicted in figure are immunohistochemistry stainings performed with egf transgenic livers to confirm regulation and predominant cytoplasmic expression of arginase ii. note, arg is only expressed in hcc and recent evidence suggest modulation of arginine levels in the extracellular milieu to be part of an immune escape mechanism whereby lack of local arginine weakens tumour-infiltrating lymphocytes as t cells require adequate argine levels [ ] . likewise, the tumour specific and cytoplasmic expression of the f-actin capping protein α subunit (capza ) and the predominant nuclear expression of tubulin β that was particularly visible beneath the liver capsule may possible promote microtubule stability and interactions of microtubules with endogenous proteins. furthermore, the induced and predominat cytoplasmic expression of the gdp dissociation inhibitor (gdi ) protein is part of the control of vesicular trafficking. this protein is known to regulate gdp-gtp exchange amongst members of the rab family of proteins. the tumour specific and cytoplasmic expression of amphiregulin supports the notion of a switch in autocrine signalling and it has been reported that amphiregulin is a prognostic marker for poor outcome of a variety of malignancies including colorectal liver metastasis [ ] . finally, the repressed nuclear expression of hnf a was not unexpected and confirms earlier findings [ ] . based on the information given in table the human protein atlas depository (www.proteinatlas.org, version ) was interrogated. as shown in additional file : table s out of mouse liver cancer regulated proteins were likewise regulated in human hcc. it should be noted that for some proteins several antibodies were used to study their expression; only representative data were considered. importantly, out of the proteins uniquely expressed in mouse liver tumours n = were likewise uniquely expressed in human hcc thus evidencing clinical significance of our findings. we compared our previously published transcriptomic data of egf induced liver cancers with the proteomic data obtained in the present study. such comparisons revealed n = genes to be significantly regulated of which n = are in common regulated whereas for n = genes transcript expression was opposite to that of the coded proteins (see additional file : table s ). of the significantly regulated proteins were mapped to different biological processes (see figure a ) of which prominent examples are regulation of arginine metabolism and amino acid import, regulation of cdc protein signal transduction', cellular response to oxidative stress, hydrogen peroxide and superoxide, glycolysis and gluconeogenesis, regulation of cholesterol transport, protein-lipid complex and plasma lipoprotein particle remodeling, positive regulation of steroid metabolic process, negative regulation of calcium ion transmembrane transporter activity and release of sequestered calcium ion into cytosol by sarcoplasmic reticulum, (see additional file : table s ). in figure a -c the go biological process, cellular components and molecular functions are depicted. note, some of the ontology terms could be grouped, i.e. chaperone-mediated protein complex assembly and folding, endoplasmic reticulum unfolded protein response, er-nucleus signalling pathway and response to er oxidative stress as well as hypoxia, blood coagulation, developmental growth and regulation of programmed cell death. as depicted in figure b significantly regulated proteins were mapped to cellular components (see additional file : table s ), i.e. mitochondrial crista, matrix and inner membrane, endoplasmic reticulum lumen, early endosome and cytoplasmic membrane-bounded vesicle, chylomicron, very-low and high density lipoprotein particle, proteasome accessory complex, peroxisome, extracellular vesicular exosome and extracellular membrane-bounded organelle. furthermore, significantly regulated proteins were mapped to molecular functions (see figure c ) and included arginase activity, fructose-bisphosphate aldolase activity, hydrolase and oxidoreductase activity, acting on carbon-nitrogen (but not peptide) bonds, acting on aldehyde, ch-oh group or oxo group of donors, nad or nadp as acceptor as well as steroid dehydrogenase activity. in addition, phosphatidylcholine-sterol o-acyltransferase activator activity, cholesterol transporter activity, sterol transporter, antioxidant and lipid transporter activity as well as electron carrier and serine-type endopeptidase inhibitor activity were prominent functions. finally, proteins functioning in metal ion and purine ribonuleoside triphosphate binding, lipoprotein particle receptor binding, chaperone and oxygen binding, binding of magnesium ion and nad, protease and single-stranded dna binding were observed as disease regulated (additional file : table s ). in all, significantly regulated proteins were classified by the reactome, kegg and wikipathway databases, respectively. the different databases provided similar information with the majority of tumour proteins acting in major metabolic pathways (see figure and information derived from cluego and cluepedia). for example, the proteins aldoa, aldoc, fbp and pkm function in glycolysis and gluconeogenesis whereas akr c , aldoa, aldoc and fbp are part of the fructose and mannose metabolic pathway. likewise, atp h and ndufv are part of the oxidative phosphorylation pathway and mdh and pkm contribute to pyruvate metabolism. similarly, the proteins akr c , akr c , akr c , alb, apoa , apoa , apoe, fdps, gpx , hacl and plg take part in the metabolism of lipids, arachidonic acid and lipoproteins whereas the proteins agmat, arg , arg , bckdha, ckb, cps , haao and phgdh are specified for arginine and proline metabolism. in the same manner the proteins gpx , itpa and nme contribute to the metabolism of nucleotides and related to this are the proteins itpa, pkm and psmc which are part of the purine metabolic pathway. apart from these pathways a highly significant regulation of the blood coagulation cascade, figure western blotting of serum proteins in control and egf transgenic mice. for the commonly regulated proteins in serum and tumours their regulation in liver tissue was confirmed by de and maldi-tof/ms (see table ). depicted are western blots for serum proteins. note, with the exception of egf the regulated serum proteins were already reported in our earlier publication [ ] . platelet activation and fibrinolysis was observed as defined by the proteins crk, fga, fgb, fgg, plg and sod all of which were highly significantly regulated. furthermore, trna aminoacylation (aars, gars and sars), advanced glycosylation endproduct receptor signalling (alb, capza and lgals ), peroxisome (ech , hacl and sod ), protein processing in endoplasmic reticulum (ganab, hyou and pdia ), proteasome (psmc and psmd ) and activation of chaperone genes by xbp (s) and 'unfolded protein response' (hyou and lmna) are pathways significantly perturbed in liver cancer induced by egf (see additional file : table s and additional file : table s ). using the designated workflow of the genexplain platform (see methods section) we searched for master regulatory proteins. the software is designed to identify molecules upstream of regulated tumour proteins to assist in the construction of molecular circuitries. after annotation of the input datasets the tool "find master regulators in networks (geneways)" was used to identify key nodes amongst proteins exclusively expressed in tumours (to). this revealed upstream regulatory molecules. among them five were selected for their link to the egfr signalling pathway, i.e. plaur, fgfr , ptbp and agtrap while the protein s a was chosen for its importance in the plaur/egfr network, (see additional file : figure s a -e). in additional file : table s and additional file : table s , the tumour regulated proteins distributed amongst the selected master regulatory molecules are summarized. in support of its biological significance the constructed networks were enriched with gene expression data from transgenic non-tumour and tumour tissues. thus, the gene and protein data were merged and hybrid networks for each master regulatory protein were constructed. subsequently, these were merged into one (see figure ) and the integrated hybrid network consisted of n = network proteins of which n = were tumour specific. in support, the genes coding for lmna, i.e. a component of the nuclear lamina that is frequently up-regulated in cancers and mvp that codes for multidrug resistance were up-regulated (ur-t) whereas nme, a suppressor of metastasis was repressed in expression (dr-t). likewise, the genes coding for igals , i.e. a beta-galactoside-binding protein frequently overexpressed in cancers and pcbp that is involved in transcription and functions as an inhibitor of invasion [ ] were up-regulated in transgenic nontumour livers (ur-tr-nt) whereas transcript expression of aars, a member of trna synthases and anaxa , a calcium-dependent, phospholipid-binding protein with important roles in the tumour microenvironment and metastasis were repressed (dr-tr-nt). finally, the entire network was enriched with expression data of and genes, respectively that were significantly regulated in tumour and non-tumour transgenic livers. next, we searched for master regulatory molecules by considering regulated tumour proteins obtained from the comparison tumour specific or up-and down regulated as compared to healthy non-transgenic controls (to + ur + dr). this revealed filtered (threshold radius of ) upstream regulators. among these were selected as candidates because of their regulation in liver tumours and their link to egfr signalling. notably, in the constructed network all master regulators were significantly up-regulated and included pdia , apeh, pebp and apoe while the protein expression of arg , fbp and haao was repressed (see additional file : figure s a-g) . note, in the case of arg transcript expression was equally repressed. in additional file : table s and additional file : table s the tumour regulated proteins distributed amongst the selected master regulatory molecules are summarized. in support of its biological significance the fused hybrid network was enriched for gene expression data derived from transgenic non-tumour and tumour tissues. thus, the integrated hybrid network consisted of out of regulated proteins and gene expression calls evidenced of the up-regulated tumour (to + ur) proteins to be regulated at the transcript level as well whereas among the down-regulated tumour proteins (dr) the gene arg was repressed in expression. likewise, gene expression data from non-tumour transgenic livers evidenced genes out of networks partners to be increased in expression (ur-tr-nt) and among the down-regulated networks proteins the gene phb was repressed (dr-tr-nt). thus, when the tumour gene expression data of the entire network was considered a total of genes were regulated, of which were up-regulated and were repressed in expression, (see figure ) . based on the information of the hybrid master regulatory network and in addition to other disease regulated proteins summarized in table (note, some of the proteins were not part of the networks) a total of n = disease regulated proteins were considered for network construction. after filtering for non-connected proteins the string database informed on n = protein-interactions of which n = were disease regulated as identified in the present study. among these , and were either up-, down-or not statistically significantly regulated. furthermore, gene expression calls for up-regulated proteins were supported by up-and down-regulated genes identified in tumours and up-and down-regulated genes in transgenic non-tumour livers. likewise, gene expression calls for down-regulated proteins were supported by and downregulated genes in tumours and transgenic non-tumour livers, respectively. therefore, the entire network was supported by induced and repressed tumour specific gene expression changes and up-regulated and downregulated genes observed in transgenic non-tumour livers. as depicted in figure the proteins of the fusion network displayed functional associations via the egf/egfr network and included out of ( %) significantly regulated tumour proteins with out of master regulators being connected to egfr through the network's proteins (see additional file : table s for possible protein-protein interactions and related scores). of the network proteins could be mapped to distinct pathways. after removal of non-relevant terms such as alzheimer disease a total of proteins were mapped to pathways with meaningful associations (see figure ) and consisted of 'platelet activation, signalling and aggregation (platelet degranulation)' , 'lipoprotein metabolism' , 'mapk signalling pathway' , 'glycolysis and gluconeogenesis', 'metabolism of amino acids and derivatives (arginine and proline metabolism)', 'apoptosis' and 'egfr signalling pathway'. additionally, a total of and tumour regulated proteins were mapped to the hif- signalling and protein processing in endoplasmic reticulum pathways, respectively. the pathway mapping was also supported by gene expression data with up-and down-regulated genes in tumours and up-and downregulated genes in transgenic non-tumour livers. note, two of the significantly regulated tumour proteins, i.e. crk and pebp are members of the egfr signalling pathway with pebp also functioning as a master regulator while the other regulated proteins are connected to egfr signalling through cross-talk among the pathways (see additional file : table s ). figure integrated master regulatory network for proteins uniquely expressed in tumours. based on network information obtained for the different master regulators an integrated hybrid network was constructed. the network contained proteins including with connectivity to egfr signalling (yellow coloured inner node). the master regulator, the connecting proteins (network elements) and regulated proteins are given as red, green and blue coloured inner node, respectively. furthermore, each node is partitioned into four segments whereas the first segment seen from left refers to tumour specific proteins and is red-coloured. the second, third and fourth segments refer to either up-and down-regulated proteins, tumour specific gene expression changes and gene regulations in transgenic non-tumour liver tissue, respectively. increased expression of either proteins or genes is given in red, whereas the blue colour denotes repressed expression. recent research into the molecular pathogenesis of hcc evidenced significant alterations in signalling pathways. given the fact that the epidermal growth factor is an important mitogen for hepatocytes we were particularly interested in investigating the consequences of its targeted overexpression in the liver. in our previous study we employed chromatin immunoprecipitation followed by cloning and sequencing of dna to search for tumour associated gene regulations targeted by novel hnf alpha p and p promoter-driven isoforms. this identified egf-receptor substrate (eps r) and eps as regulated by the p promoter-driven hnf alpha splice variant in mouse and human hcc. a molecular circuitry was proposed whereby eps and eps r mediate internalization of activated egfr to stimulate receptor recycling, therefore responding to mitogenic signalling of egf [ ] . in the present study disease proteomics was performed to further investigate the role of egf in liver cancer. this identified regulated proteins of which are novel and have not been reported so far. a total of proteins were significantly up-regulated (table ) . among these were extra-cellular or secreted proteins and included albumin and isoforms of it, apolipoproteins (apoe, apoa and apoai), α-, β-, γfibrinogen, plasminogen as well as interleukin receptor antagonist (il- ra). note, an isoform of apoa was already proposed as serum marker of hcc [ ] and based on ihc staining il- ra expression was confirmed in about % of mouse liver adenoma and carcinoma cases; however preneoplastic foci as well as normal hepatocytes surrounding the lesions were negative. furthermore, rt-pcr analysis confirmed mouse hepatic tumours to contain both secreted and intracellular forms of il- ra [ ] and figure integrated master regulatory network for hcc regulated proteins. based on network information obtained for different master regulators an integrated hybrid network was constructed. the network contained proteins including with connectivity to egf/egfr signalling (yellow coloured inner node). the master regulator, the connecting proteins (network elements) and regulated proteins are given as red, green and blue coloured inner node, respectively. furthermore, each node is partitioned into four segments whereas the first segment seen from left refers to tumour specific proteins and is red-coloured. the second, third and fourth segments refer to either up-and down-regulated proteins, tumour specific gene expression changes and gene regulations in transgenic non-tumour liver tissue, respectively. increased expression of either proteins or genes is given in red, whereas the blue colour denotes repressed expression. serum levels of il- ra were monitored to assess therapeutic efficacy of radiofrequency ablation in hcc patients [ ] . an important finding of the present study is the statistically significant regulation of mitochondria associated proteins of which were repressed while were upregulated. similar results were reported by chignard and wei sun with mitochondrial proteins being the second largest proportion of regulated proteins in human viral hcc [ , ] . among the repressed proteins were nadh dehydrogenase (ubiquinone) alpha subcomplex and prohibitin, a mitochondrial chaperone. this protein, when deleted (prohibitin ko mice) induced fibrosis, bile duct metaplasia, liver dysplasia and eventually multifocal hcc. however, its overexpression in tumour cell lines inhibited cell proliferation to demonstrate tumour suppressor function [ ] . likewise, glutathione peroxidase (response to oxidative stress) and argininosuccinate synthetase (ass, urea cycle) were repressed. note, ass is the first of two enzymes to convert citrulline to arginine and this pathway allows cells to synthesize arginine from citrulline to function in no production, ammonia detoxification and synthesis of polyamines. several reports suggest ass deficiency to be common in tumour cell lines [ ] [ ] [ ] [ ] [ ] [ ] , and the present study confirms ass expression to be confined to healthy non-transgenic control liver, but ass was absent in tumour tissue extracts (see table ). ablation of ass in diverse tumours suggests a tumour suppressor function and the fact that forced expression of ass in osteosarcoma cell lines suppresses growth adds weight to this notion [ ] . another example of tumour specific ablation of proteins refers to glycine n-methyltransferase (gnmt). the enzyme catalyzes the transfer of a methyl group from s-adenosylmethionine (sam) to glycine thereby generating s-adenosylhomocysteine and n-methylglycine. this protein was completely downregulated in liver tumours. gnmt is known to play a role in the maintenance of genetic stability [ , ] , and a novel tumour suppressor function was recently reported that is independent of its catalytic activity but does require its nuclear localization [ ] . several of the proteins listed in table were already reported for their tumour specific regulation while proteins so far unknown for their regulations in hcc, are marked with an asterisk (table ) . these function in diverse biological processes including metabolism, translation and signalling. specifically, changes in carbohydrate metabolism are commonly observed in tumours where energy production relies on glycolysis rather than mitochondrial oxidative phosphorylation. in the present study induced expression of several glycolytic enzymes was observed, most notable [ ] pyruvate kinase that catalyzes the transfer of a phosphate group from phosphoenolpyruvate to adp and was shown to be a target of mi-rna in hcc [ , ] aldolase, an enzyme that converts fructose , -bisphosphate into dihydroxyacetone phosphate (dhap) and glyceraldehyde -phosphate and was reported to be a sensitive marker for benign and malignant liver disease [ ] and [ ] alpha glucosidase , a hydrolase that cleaves glycosidic bonds with the release of alpha glucose from carbohydrates. further important findings include the tumour specific expression of alanyl-, glycyl-and seryl-trna synthetases which catalyze the transfer of specific amino acids to trna, as well as regulation of eukaryotic translation elongation factor and poly(rc) protein that binds to oligo dc. note, knowledge on the role of aminoacyl-trna synthetases in cancer is just emerging [ ] and through the use of a lentiviral mediated shrna vector, a link between aminoacyl-trna synthetases [aars]-interacting multifunctional protein (aimp ) and repressed egfr signalling was established that resulted in repressed glucose uptake [ ] . we also observed induced expression of heterogeneous ribonucleoprotein (hnrnp) that takes on diverse functions in the processing of mrna. its expression was reported to be increased in serum of hcc patients . in contrast, proteins involved in the synthesis and degradation of cholesterol, lipids, steroids and fatty acid were in part oppositely regulated and included induced expression of the aldo-keto reductase family . regulation of this protein has been reported for lung and pancreatic cancers [ ] , and gene silencing of aldo-keto reductase family b resulted in growth inhibition of colorectal cancer cells that might be of therapeutic utility [ ] . the repressed expression of figure pathways mapping of fussed network proteins. cytoscape . . with plugins (see methods section) are used to generate functionally grouped network of pathways. grouping of significant pathway terms (p ≤ . ) were based on kappa score threshold of . , initial group size of and sharing group percentage of . the pathway network consisted of significantly and non-significantly regulated proteins involved in distinct pathways which are colour-coded. note, the two individual terms are grey-coloured. up and down-regulated proteins are coded as orange and green small discs, respectively. up-and down-regulated as well as non-significantly regulated proteins and connecting proteins of the network are given as orange, green, yellow and blue coloured discs, respectively. the network depicts protein-protein interactions in liver tumours of egfr transgenic mice and their relation to various pathways under the influence of egfr signalling. egfr is highlighted as blue triangle in this network. certain proteins may also be considered as an adaptive response and includes the enzyme enoyl coenzyme a hydratase . its activity was shown to contribute to lymphatic spread of liver tumours as was evidenced in gene silencing studies [ ] . likewise, we observed repressed expression of dihydrodiol dehydrogenase in tumours. this enzyme plays an important role in the metabolism of steroids that leads to inactivation of circulating androgens, progestins and glucocorticoids and was repeatedly reported to be overexpressed in non-small cell lung cancer. amongst patients with high dhd expression the incidence of early tumour recurrence and distant metastasis is significantly higher and patients are highly resistant to chemo and radiotherapy [ ] . intriguingly, complete ablation of mitochondrial butyryl coenzyme a synthetase , a gtp-dependent lipoateactivating enzyme was observed in tumours of egf transgenic mice. little is known about the possible link between butyrate metabolism and liver cancer. however, butyrate is well known to inhibit proliferation of human colon carcinoma cells in an epigenetic manner that involves histone acetylation [ ] . note, it was recently reported that due to the warburg effect butyrate-mediated histone acetylation and cell proliferation is dictated [ ] . several lines of evidence therefore suggest butyrate to act as a cytosolic sensor for histone acteylation and when transformed to intermediates by butyryl coenzyme a synthetase is unable to escape the mitochondria. moreover, we observed a highly significant repression of -hydroxyphytanoyl-coa-lyase. this peroxisomal thiamine pyrophosphate-dependent enzyme is rate limiting in the breakdown of -hydroxy fatty acids. the biological role of -hydroxy fatty acids has only recently become apparent [ ] and cumulative evidence suggests intermediates of energy metabolism to specifically activate g-protein coupled receptors which are now classified as hydroxy carboxylic acid receptors (hca - ). the hca receptor is involved in a complex negative feed-back loop whereby ketone bodies derived from fatty acid oxidation are sensed by hca via the activity of -hydroxybutyrate that leads to inhibition of lipolysis and to restriction of further fatty acid supply. in this way triglyceride use is diverted and energy demands for tumour growth are met more efficiently. specifically, during rapid tumour growth and the herewith associated ischemia the yield of high energy bonds (atp) from glucose oxidation is about twice that of fatty acid oxidation. our observation that proteins involved in the ß-oxidation of fatty acids were either repressed or unchanged agrees well with this principle (see also discussion below). the reduced expression of lysophosphopholipase signifies an adaptive response; it catalyses the production of lysophosphatidic acid, i.e. a second messenger known to contribute to tumour cell motility, survival and proliferation [ ] . additionally, the repressed expression of mitochondrial acyl-coa thioesterase in liver tumours which hydrolyzes acyl-coas to free fatty acids and coenzyme a, will influence the supply of ligands for nuclear receptors and the regulation of fatty acid oxidation in mitochondria and peroxisomes. equally, the regulation of farnesyl diphosphate synthetase, i.e. a key enzyme in the isoprenoid biosynthetic pathway is highly interesting and this enzyme is explored as a drug target of bisphosphonates to treat tumour growth [ ] . it's up-regulation in colon cancers was reported [ ] . in the present study repressed expression of the ribosom-compononent rps and enzymes of amino acid metabolism like branched chain ketoacid dehydrogenase e as well as dimethyl glycine dehydrogease was observed. conversely, expression of the proteasome s atpase subunit (p /sug) and its non-atpase regulatory subunit (psmd ) was confined to tumour tissues (see table ); the latter subunit is known to display high activity in embryonic stem cells. this multicomplex molecular machinery degrades intracellular proteins marked up by ubiquitin chains. psmd was reported to be up-regulated in breast cancer cells [ ] . enhanced expression of cytoskeletal proteins such as tubulin β and capza was also confirmed by ihc staining (see figure ). differences in the localization of these proteins were obvious with tubulin ß expression being primarily associated with cells proximal to the liver capsule, whereas expression of capping protein zline α (capza ) was strongly associated with tumour foci and this protein is known to play a pivotal role in cytoskeletal networks to support cell mobility, invasion and metastasis. additionally, gdi , a protein functioning in the cycling of rab gtpases and arginase ii, i.e. a non-liver isoform of the urea cycle were up-regulated in tumours of egf transgenic mice (see figure ). regulation of arginase ii was observed in various malignancies including lung cancer [ ] . besides, the actin-binding protein lasp was uniquely expressed in tumours and is also up-regulated in breast cancer [ ] to possibly support migration of cancer cells [ ] . furthermore, pdia , a disulfide bond isomerase and master regulator of the constructed networks (see below) was up-regulated as was kininogen that is part of the blood coagulation system and functions as a precursor of kinin. conversely, the serinproteinase inhibitor serpinb a was repressed in expression to possible limited immunological responses in tumour growth and to influence inflammatory cytokine production by infiltrating monocytes [ ] . the significant regulation of the calcium binding protein sorcin and nucleobindin are further highly interesting results. sorcin is associated with multidrug-resistance in human leukemia cells [ ] and nucleobindin is evaluated as a biomarker of colon cancer [ ] . in egf induced liver tumours transthyretin was also up-regulated. this protein is involved in the transport of thyroid hormones and was reported to be aberrantly regulated in thyroid cancer [ ] . among the newly identified proteins is v-crk sarcoma virus ct . this oncoprotein interacts with several tyrosinephosphorylated proteins and is part of the intracellular signalling cascades notably the phosphoinositide -kinase (pi k)/akt pathway [ ] . likewise, regulation of the kda glucose-regulated protein grp is of great importance. this lumenal endoplasmic reticulum plays a role in immunoglobulin folding as was confirmed by coimmunoprecipitation in four different b cell hybridoma cell lines [ ] . in our previous study several immunoglobulins were found to be either repressed or absent in serum of egf tumour bearing mice and this was particularly obvious for the ig k and l classes [ ] . it remains to be determined whether repression of immunoglobulins can be attributed to aberrant grp activity. a summary of the biological functions in addition to their previous reported tumour association is given in additional file : table s while the regulation of genes coding for newly identified proteins and of genes coding for commonly regulated proteins in liver tumours and serum of egf b-transgenic mice is given in additional file : table s and additional file : table s . initially the network construction was based on proteins exclusively expressed in tumours and by selecting master regulatory proteins linked to egfr signalling. thereafter, a fused hybrid network was developed in which tumour specific proteins were part of it. subsequently, the search was extended to all significantly regulated proteins (table ) . this revealed master regulatory proteins and its associated networks and encompassed proteins of which were disease regulated. eventually a fused network was developed; however not all disease regulated proteins are part of it. the performed pathway mapping over fused networks (see string analysis) defined protein interactions and grouped disease regulated proteins into distinct pathways of which platelet activation, signalling and aggregation is a major one (see figure ) . specifically, the glycoprotein fibrinogen is a multimeric protein and consists of α, ß and y subunits. it is synthesized by hepatocytes and an essential blood coagulation factor with all polypeptide chains being highly regulated in tumours of egf transgenic mice. note, an association between coagulation factors and malignancies was established whereby fibrinogen functions as an extracellular matrix protein to interact with integrin receptors in the control of cell proliferation and cell migration [ ] . accordingly, induced gene expression of the integrin receptors itgb , itga and itgav was observed in egf induced liver tumours. in cancer progression a regulatory loop between fibrinogen, platelets and tumour cells has been determined that is activated by platelet cytosolic ca +. this second messenger induces integrin receptor complex formation through an association of platelet glycoprotein chains iib and iiia (cd /cd ) thereby creating an active binding site for fibrinogen. an association of tumour regulated proteins with the regulatory loop was confirmed in string analysis ( figure ) and fibrinogen was reported to be an important determinant for metastasis of circulating tumour cells [ ] . it is therefore of no surprise that elevated blood fibrinogen is a poor prognostic factor. haemostatic complications are commonly observed in cancer patients and future therapeutic strategies may focus on the hemostatic system by targeting tumour stroma. in this regard the tumour specific induction of plasminogen is of great importance. this zymogen [ ] is converted to plasmin by urokinase (upa), a serine protease which itself was unchanged; however, gene expression of its receptor was significantly up-regulated in transgenic non-tumour livers. one report suggests the urokinase receptor to prime cells for proliferation in response to egf by promoting tyr phosphorylation and stat b activation; nonetheless, this depended on intracellular c-src levels [ ] . further studies established a link between induced expression of plasminogen activator, upa receptor and plasminogen activator inhibitor type- (pai- ) and invasiveness and metastasis of hcc [ , ] . indeed, a fine balance exists between the plasminogen activating system and its inhibition by pai- and pai- . based on transcriptomic data a highly significant induction of pai-i (up to -fold) in large tumours of egf transgenic mice was observed [ ] ; consequently, the regulation of components of the plasminogen activating system may be considered as part of a strategy to degrade extracellular matrix thereby facilitating invasion and metastasis [ , ] . to meet energy demands efficiently different sources are utilized and the induction of the proteins aldoa, aldoc, eno , pkm and fbp is testimony to an altered glycolytic and pentose phosphate pathway. however, with the exception of acyl-coa thioesterase that was below the limit of detection and functions in the hydrolysis of myristoyl-palmitoyl-, stearoyl-and arachidoyl-coa esters the regulation of enzymes linked to fatty acid metabolism in mitochondria and peroxisomes was hardly observed. in pursue of tumour growth and to sustain organelle and membrane biogenesis lipids are de novo synthesized and mobilized from stores and while the complex interaction of hepatic lipid and glucose metabolism in liver disease is the subject of intense research [ ] the present study evidences significant regulation of several apolipoproteins, i.e. apoe, apoa , apoa and isoforms of albumin. apart from lipid transport apolipoproteins play a wider role in cancers and are known to interact with diverse receptors to elicit cellular events as demonstrated for apoe to cause sustained proliferation and survival of cancer cells [ ] . a further group of highly regulated proteins are aldoketo reductases. their quantitative evaluation in different hepatocellular carcinoma (hcc) cell lines was recently reported [ ] . this superfamily of proteins comprises nad (p)(h)-dependent enzymes which catalyze oxidoreduction of a variety of prostaglandins, steroids and toxic aldehydes. their involvement in tumorigenesis is supported by several studies and they are explored as drug targets to overcome chemoresistance. in the present study the aldo-keto reductases akr c , akr c and akr c were uniquely expressed in tumours, however glutathione peroxidase was repressed to % of healthy control livers to possibly support hif- signalling. indeed, the redox state and therefore glutathione participates in the hypoxic induction of hif- [ ] , and two proteins of the glycolytic pathway, i.e. aldoa and eno , which respond to hif- signalling, were regulated. moreover, glutathione peroxidase was shifted in the gel as shown in figure panel g iii as a result of post translational modifications that most likely involved c-abl and arg kinase activity at tyr of gpx [ ] . likewise, the genes coding for aldo and eno were significantly up-regulated in egf induced liver tumours. a complex interaction exists between egfr and rage signalling. this receptor for advanced glycation end-products is a member of the immunoglobulin family of cell surface molecules and was reported to significantly influence hepatic tumour growth in murine models of colorectal carcinoma [ ] . there is strong evidence for rage to promote cancer growth upon ligand dependent activation and several proteins of the s family bind to the extracellular domain of rage [ , ] . it is of considerable importance that gene expression of s a and s a was up to -fold induced in tumours of egf transgenic mice, however expression of s a was repressed. likewise the tumour specific expression of the rage binding proteins lectin, galactoside-binding, soluble, and capza in tumours of egf transgenic mice is highly suggestive for a sustained crosstalk between rage and egfr [ ] . although the precise mechanism by which s proteins stimulate egfr signalling remains to be elucidated binding of s a to egf and to other egfr ligands was reported to possibly facilitate interaction with the receptor [ ] . similarly, the binding of s a /a to rage was shown to promote migration and invasion of human breast cancer cells through actin polymerization and epithelial-mesenchymal transition [ ] . conversely, advanced glycation endproduct (age) receptor suppressed oxidant stress-dependent signalling via the egfr and shc/grb /ras pathway [ ] . as depicted in figure the amino acid metabolism was another distinct pathway to which several of the regulated proteins could be mapped to. note, the tumour specific regulations of arginine and as well as the regulation of subunits of the proteasome s atpase (psmc and psmd ) were already discussed (see above). in the following additional proteins regulated in this pathway are briefly summarized. specifically, -hydroxyanthranilate- , -dioxygenase (haao) catalyzes oxidation of -hydroxyanthranilate to quinolinate and this intermediate functions as a precursor in nad and pyridine biosynthetic pathways. expression of haao was significantly repressed in tumours of egf transgenic mice and hypermethylation of the coding gene was observed in ovarian cancer [ ] . due to the fact that haao is significantly repressed at the gene and protein level in at least two different tumour entities (ovarian and liver cancer) the protein may function as a tumour suppressor that appears to be repressed by an epigenetic mechanism. a significant finding is the tumour specific expression of -phosphoglycerate-dehydrogenase which catalyses the production of -phosphoglycerate. this intermediate of glycolysis is an essential precursor of the serine biosynthetic pathway. importantly, a recent metabolomic study evidenced -phosphoglycerate to be diverted into serine and glycine metabolism and repressed expression of -phosphoglyceratedehydrogenase resulted in impaired tumour cell proliferation [ ] . in support of tumour growth the diversion of intermediate of glycolysis affects protein, membrane lipid and nucleotide synthesis. moreover, the observed induction of creatine kinase in tumours of egf transgenic mice creates a circuitry for cellular energy homeostasis in conditions of high metabolic demands [ ] . the enzyme catalyses the reversible transfer of phosphate from phosphocreatine to adp to yield atp and creatine. its induction has been observed in many cancers including liver cancer cell lines [ , ] and a further study suggested a possible interplay between p mutations, hcc, ck expression with growth-inhibitory effects of cyclocreatine in hcc [ ] . while the rationale of tumour cells in embarking on abnormal metabolism had already been discussed (see above) the finding that agmatine ureohydrolase was strongly repressed in egf induced liver tumours to about % of non-transgenic healthy livers is of great importance. this enzyme hydrolyzes agmatine (= decarboxylated arginine) to form putrescine and urea and repression of the enzyme will significantly increase agmatine tissue concentration to influence diverse cellular control mechanisms. importantly, in the study of battaglia and coworkers [ ] mm agmatine induced large amounts of superoxide production in rat liver mitochondria; however, it did not affect mitochondrial respiration or redox levels of thiols and glutathione. furthermore, atp synthesis remained normal and prevented ca( +)-induced mitochondrial permeability transition in the presence of phosphate to suggest an intriguing regulatory loop whereby h o induces hypoxia signalling that is linked to abberant metabolism, nonetheless by selecting interconnected physiological pathways tumour cells are equipped to avoid programmed cell death [ , ] . thus, arginine deprivation is evaluated for its utility in cancer therapy [ ] . a further enzyme repressed to % of healthy nontransgenic liver is carbamoyl phosphate synthetase (cps ), i.e. a liver specific ligase to function in ammonia detoxification. it is perplexing that tumour cells disable such an important pathway of the urea cycle. however, a recent study demonstrated dna hypermethylation as a key mechanism of silencing cps gene expression in human hcc. note, forced expression of cps induced cell proliferation and the observed repression in human hcc may simply be the result of genomic instability as was observed in tumour cells [ ] . the present study identified novel disease regulated proteins induced by overexpression of egf to provide new insight into the complex signalling events in hcc. six major pathways perturbed by egfr hyperactivity were identified and several of the regulated proteins are interesting drug target candidates and this includes tumour specific expression of kinases as well as proteins involved in aberrant metabolism. an identification of commonly regulated proteins in tumour and sera will be of great utility in the development of biomarkers to monitor disease progression and responses to therapy. the following additional data are available with the online version of this paper. aminoacyl-trna synthetases interacting multifunctional protein ; alpha-hcca: alpha-cyano- -hydroxycinnamic acid; aps: ammonium persulfate; ass: argininosuccinate synthetase ; atp: adenosine triphosphate; ck: creatine kinase; ck Α: casein kinase ii subunit alpha; co: control specific protein dr: down-regulated protein; dr-t: down-regulated tumour; dr-tr-nt: down-regulated transgenic non-tumour; egf: epidermal growth factor egfr: epidermal growth factor receptor; eps : epidermal growth factor receptor substrate ; eps r: epidermal growth factor receptor substrate r; er: endoplasmic reticulum; erk: extracellular signalling regulated kinase; fdr: false discovery rate; fgfr : fibroblast growth factor receptor go: gene ontology; grp : kda glucose-regulated protein; gtp: guanosine triphosphate h po : orthophosphoric acid; hca: hydroxy carboxylic acid; hcc: hepatocellular carcinoma; hdac : histone deacetylase ; hed: bis( -hydroxyethyl) disulfide; hrp: horseradish peroxidase ihc: immunohistochemistry; il- ra: interleukin- receptor antagonist ko: knockout mouse mek: mitogen-activated protein kinase kinase; nad: nicotinamide adenine dinucleotide; nad(p)(h): nicotinamide adenine dinucleotide phosphate nadp: nicotinamide adenine dinucleotide phosphate; no: nitric oxide; ogp: n-octyl β-d-glucopyranoside pai- : plasminogen activator inhibitor type- pdgfr: platelet-derived growth factor receptors; plaur: plasminogen activator, urokinase receptor; pmf: peptide mass fingerprinting; ptbp : polypyrimidine tract binding protein ; rage: receptor for advanced glycation endproduct s a : s calcium binding protein a ; sam: s-adenosylmethionine short hairpin rna; stat b: signal transducer and activator of transcription b; temed: tetramethylethylenediamine; to: tumour specific protein; trna: transfer rna; tyr : tyrosine residue ; upa: urokinase-type plasminogen activator; ur: up-regulated protein up-regulated transgenic non-tumour; vegfr: vascular endothelial growth factor receptor; xbp (s): x-box binding protein isoform surgery and ablative therapy for hepatocellular carcinoma improved method for proteome mapping of the liver by -de maldi-tof ms identification of specific protein markers in microdissected hepatocellular carcinoma soluble interleukin- receptor levels in hepatocellular cancer: a more sensitive marker than alfa fetoprotein discovery and development of 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• thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we gratefully acknowledge support from the virtual liver network (grant ) of the german federal ministry of education and research (bmbf) to jb. additional file : table s . antibodies and dilutions used to study disease regulation by immunohistochemistry.additional file : table s . statistics and selection criteria for functional grouping of pathways terms of disease regulated proteins. a hypergeometric test followed by bonferroni correction was used with p-value ≤ . . for grouping of pathway terms, the kappa score threshold was set to . . additional file : table s . different master regulators and associated network for proteins expressed in tumours only (to) or significantly regulated when compared to healthy liver of non-transgenic control animals (t + ur + dr).additional file : table s . comparison of disease regulated proteins in mouse and human hcc. data were taken from 'the human protein atlas' database.additional file : table s . comparison of transcriptomic and proteomic data. this comparison revealed significantly regulated genes of which are regulated in common whereas for genes transcript expression was opposite to that of the coded proteins.additional file : table s . biological processes ontology for significantly regulated proteins. additional file : table s . cellular component ontology for significantly regulated proteins.additional file : table s . molecular function ontology for significantly regulated proteins.additional file : table s . biological pathways and their cluster. cytoscape . . with plugins (cluego and cluepedia) were used to generate functionally grouped network of pathways based on reactome, kegg and wikipathways databases. the grouping of significant pathway terms (p ≤ . ) is based on a kappa score of . , initial group size of and sharing group percentage of .additional file : table s . biological pathways information based each significantly regulated protein (to + ur + dr). this table depicts pathway terms of nearly % of regulated proteins (to + ur + dr) and are taken from reactome and kegg databases.additional file : figure s . master regulatory networks for proteins uniquely expressed in tumours with link to egfr signalling: (a) the plaur network consists of proteins of which are tumour-specifically regulated, (b) the fgfr consists of proteins of which are tumour-specifically regulated, (c) the ptbp network consists of proteins of which are tumour-specifically regulated, (d) the agtrap network consists of proteins of which are tumour-specifically regulated and (e) the s a network consists of protein of which are tumour-specifically regulated. note all networks display connectivity to egfr signalling (yellow coloured inner node) and in the case of the s a master regulatory protein egfr s ignalling is via the plaur/egfr network.the master regulator, the connecting proteins (network elements) and regulated proteins are given as red, green and blue coloured inner node, respectively. furthermore, each node is partioned into four segments whereas the first segment seen from left refers to tumour specific proteins and is red-coloured. the second, third and fourth segments refer to either up-and down-regulated proteins, tumour specific gene expression changes and gene regulations in transgenic non-tumour liver tissue, respectively. increased expression of either proteins or genes is given in red, whereas the blue colour denotes repressed expression.additional file : table s . integrated hybrid network with master regulator information for proteins expressed in tumours only (to) and significantly regulated proteins when compared to heathy liver of non-transgenic control animals (t + ur + dr).additional file : figure s . master regulatory networks for regulated proteins with link to egfr signalling: (a) the pdia network consists of proteins including significantly regulated proteins, (b) the apeh network consists of proteins including significantly regulated proteins, (c) the pebp network consists of proteins including significantly regulated proteins, (d) the apoe network consists of proteins including significantly regulated proteins, (e) the arg network consists of proteins including significantly regulated proteins, (f) the fbp network consists of proteins including significantly regulated proteins, (g) the haao network consists of proteins including significantly regulated proteins. note, all networks display connectivity to egf protein (yellow coloured inner node). the master regulator, the connecting proteins (network elements) and regulated proteins are given as red, green and blue coloured inner node, respectively. furthermore, each node is partioned into four segments whereas the first segment seen from left refers to tumour specific proteins and is red-coloured. the second, third and fourth segments refer to either up-and down-regulated proteins, tumour specific gene expression changes and gene regulations in transgenic non-tumour liver tissue, respectively. increased expression of either proteins or genes is given in red, whereas the blue colour denotes repressed expression.additional file : table s . protein interaction information of the fused network. given are interacting proteins with association score from different prediction methods, i.e. neighborhood, gene fusion, co-occurrence, co-expression, databases and textmining.additional file : table s . biological pathways and their cluster for fused network proteins. reactome, kegg and wikipathways database information was used as input data file for cytoscape . . . the table shows grouping of significant pathway terms (p ≤ . ) and is based on a kappa score of . , initial group size of and sharing group percentage of .additional file : table s . biological function of newly identified proteins and their previously reported tumour association.additional file : table s . regulation of genes coding for newly identified proteins in egf b-transgenic liver tumours.additional file : table s . regulation of genes coding for common proteins in tumour tissue and serum of egf transgenic mice. the authors declare that they have no competing interests.authors' contributions jb conceived the study and contributed the reagents, gg performed the experiments, ps performed the bioinformatics analysis. jb, gg and ps analysed the data, jb wrote the manuscript and all authors read and approved the final manuscript. key: cord- -zlqh d authors: chasman, deborah; walters, kevin b.; lopes, tiago j. s.; eisfeld, amie j.; kawaoka, yoshihiro; roy, sushmita title: integrating transcriptomic and proteomic data using predictive regulatory network models of host response to pathogens date: - - journal: plos comput biol doi: . /journal.pcbi. sha: doc_id: cord_uid: zlqh d mammalian host response to pathogenic infections is controlled by a complex regulatory network connecting regulatory proteins such as transcription factors and signaling proteins to target genes. an important challenge in infectious disease research is to understand molecular similarities and differences in mammalian host response to diverse sets of pathogens. recently, systems biology studies have produced rich collections of omic profiles measuring host response to infectious agents such as influenza viruses at multiple levels. to gain a comprehensive understanding of the regulatory network driving host response to multiple infectious agents, we integrated host transcriptomes and proteomes using a network-based approach. our approach combines expression-based regulatory network inference, structured-sparsity based regression, and network information flow to infer putative physical regulatory programs for expression modules. we applied our approach to identify regulatory networks, modules and subnetworks that drive host response to multiple influenza infections. the inferred regulatory network and modules are significantly enriched for known pathways of immune response and implicate apoptosis, splicing, and interferon signaling processes in the differential response of viral infections of different pathogenicities. we used the learned network to prioritize regulators and study virus and time-point specific networks. rnai-based knockdown of predicted regulators had significant impact on viral replication and include several previously unknown regulators. taken together, our integrated analysis identified novel module level patterns that capture strain and pathogenicity-specific patterns of expression and helped identify important regulators of host response to influenza infection. to combat infections from diverse pathogens, mammalian immune systems must be able to mount appropriate and specific responses to pathogenic infections. a key challenge in current infectious disease research is to understand the molecular mechanisms that make the host immune system more or less susceptible to a particular strain of a pathogen, for example, different influenza a virus strains, than another. transcriptional regulatory networks that connect regulatory proteins to target genes are central players in how mammalian cells mount appropriate responses to different pathogenic infections. because the components and connectivity of these networks are largely not known, a significant amount of effort has been invested to collect high-throughput datasets that provide a comprehensive molecular characterization of host response to multiple viruses at multiple levels, including the transcriptome and proteome [ ] [ ] [ ] [ ] . these genomic datasets provide unique opportunities to identify molecular network components of host response that are conserved across multiple viruses or specific to a virus of a particular pathogenicity. such networks can be used to prioritize regulators for follow up validation studies that provide greater insight into the mechanisms by which the host cell perceives and responds to different pathogenic infections. network-and module-based approaches for analyzing omic datasets have been powerful for dissecting mammalian cellular response to different environmental perturbations, including response to various pathogenic infections [ , ] . the majority of these approaches have used genome-wide transcriptomic data and can be grouped into those that infer correlational networks [ , [ ] [ ] [ ] [ ] [ ] [ ] , or module networks, in which regulators are inferred for groups of coexpressed genes called modules [ , , ] . a few approaches have integrated additional data types, e.g. physical protein-protein or signaling networks with transcriptional data [ ] [ ] [ ] [ ] [ ] [ ] , that vary in the number of samples needed to infer networks. while these approaches have provided important insights into the host response, they have not integrated multiple types of omic measurements (e.g. mrna and protein levels), which can differ in quality and sample size. a second challenge is that experimental validation of large-scale predictions is expensive and most of the generated predictions have not been validated experimentally. although several network-based prioritization methods have been proposed, the results of a prioritization scheme have been followed up with experimental validation in a handful of studies [ ] . to gain a more complete understanding of the molecular networks driving host response, we integrated transcriptomic and proteomic measurements of mammalian host response with existing protein-protein interactions. these omic measurements capture human and mouse cellular response to multiple influenza a viruses exhibiting different levels of pathogenicities. our starting point is an expression-based regulatory network connecting transcription factors and signaling proteins to target genes and gene modules. we then use proteomic measurements to predict additional regulators of gene modules by applying a structured sparsity-inducing regression approach, multi-task group lasso, to find proteins whose levels are predictive of mrna levels of entire modules. by decoupling the mrna and protein-based regression into two steps, our approach is less sensitive to varying sample size for each type of omic data. finally, we predict physical regulatory programs connecting mrna and proteinbased regulators through a small number intermediate nodes using integer linear programming (ilp)-based network information flow. we used our integrated regulatory networks to study the host response across the different viruses. we tested prioritized regulators using small interfering rnas and found several regulators that significantly impact viral replication, five of which have not been previously associated with influenza related response. we examined host response dynamics at the network level, identifying regulatory network components that are active or missing under different viral treatments. our inferred gene modules capture strain-and pathogenicity-specific patterns of mammalian immune response to influenza infections that recapitulate and expand upon known immune response pathways. in particular, we identified one module that was enriched for interferon signaling and exhibited repressed expression in the high-pathogenicity wild-type h n , but was induced in low pathogenicity h n strains. another module suggests that apoptosis-related pathways might be down-regulated in low-pathogenicity viruses. our findings of host regulatory modules together with their upstream regulatory programs suggest that our network-based approach is a powerful way to systematically characterize immune response to diverse pathogenic infections. to perform a systematic, integrative analysis of transcriptome and proteome measurements of host response to influenza virus infections, we began by inferring a regulatory module network in two stages, followed by three major downstream analyses (fig ) . we ( ) infer a regulatory module network based on changes in mrna abundance under viral infection and ( ) predict protein regulators whose abundances are predictive of gene expression in the modules. this integrated regulatory module network enabled ( ) prioritization of regulators for validation of their ability to modulate viral replication, ( ) an examination of network dynamics across virus treatments, and ( ) a further integration with external protein-protein interactions to predict directed physical connections between the mrna, protein-based regulators and known influenza host response genes. the first component of our approach, a regulatory network inference algorithm, was needed because mammalian regulatory networks are incomplete for most biological processes. we used a recently developed network inference algorithm, 'modular regulatory network learning with per gene information' (merlin [ ] ) that uses genome-wide mrna levels from multiple biological samples (time points or treatments) to predict regulatory relationships between regulators (e.g. transcription factors or signaling proteins) and target genes. our rationale for selecting merlin was to enable the study of host response regulatory networks both at the individual gene level and at the module level. alternative methods either infer the regulators for individual genes [ ] or for gene modules [ , ] , but not both. we applied merlin within a stability selection framework to the host transcriptional measurements of mouse lungs and human bronchial epithelial cells (calu- cell line) infected with one of six influenza virus strains exhibiting a range of pathogenicity levels (materials and methods). on the human calu- cell line (fig ) , we identified consensus modules of at least genes comprising a total of , genes (~ % of the input, table ). on the mouse data, we identified modules, encompassing , genes ( % of the input, table ). the average expression patterns in each inferred module revealed commonalities and differences between strains and pathogenicity levels (fig , calu- ; s fig, mouse) . based on a hypergeometric test with fdr correction (fdr< . ), out of the human calu- modules ( of mouse modules) exhibited enrichment in one or more of the annotation categories representing gene ontology processes, kegg pathways, and influenza related gene sets identified from high-throughput rnai studies and viral-host protein-protein interaction screens (fig , s fig, s table, s table) . moreover, of the human modules were enriched specifically for immune response related overview of human influenza response module expression patterns. shown are human calu- modules with at least genes. the red-blue heat map shows mean expression of all genes in each module. the more red an entry the higher the expression in infected versus mock, while the more blue the entry, the higher the expression in mock compared to the infected sample. under "viruses and time points (h)", each virus treatment's time series is shown separately, and viruses are ordered from low to high pathogenicity. low-pathogenicity samples labeled h n used a/ca/ / h n unless labeled '(nl)' for a/netherlands/ / . samples labeled h n used a/vn/ / h n and laboratory mutants thereof. the "genes" column shows the size of each module; larger values are shown in darker blue. under "enrichment", a red box indicates module enrichment with any msigdb motif, any msigdb gene set, any gene ontology process, any influenza screen set, or any immune response gene set (methods). doi: . /journal.pcbi. .g processes ( fig a) suggesting that the modules were biologically coherent and relevant to immune response to influenza infections. importantly, compared to ordinary expression-based gene clusters (identified by gaussian mixture modeling, clusters), merlin modules exhibited greater fold enrichment in innate immune system categories and motif-based targets of transcription factors (s table) . in particular, merlin modules had enrichment for targets of immune response-relevant regulatory elements irf , , and isre and targets of inflammatory response regulator nf-kb, while expression-based clusters were not enriched or enriched at a lower level. in addition to the modules, we defined consensus regulatory networks for the calu- and mouse transcriptome data by selecting regulatory edges with a confidence at least . (materials and methods). the consensus networks predicted regulatory connections between , regulators (signaling proteins and tfs) and , target genes in human, and , regulators and , target genes in mouse (s table, s table) . both calu- and mouse regulatory networks were significantly enriched in transcription factor target interactions cataloged in msigdb ( fig b, conserved motifs (msigdb)) suggesting that the predicted regulatory-target connections are supported by sequence specific motifs. we also compared the inferred merlin mouse network to two additional networks: a pathogen-responsive regulatory network inferred from gene expression profiles after rnai-based transcription factor knockdowns ( fig b, mouse pathogen, sirna [ ] ), and a computationally constructed regulatory network for th cellular response to lps stimulation ( fig b, mouse th , yosef, [ ] ). the mer-lin mouse network significantly overlaps with both of these networks (fold enrichment > . , fig b) , indicating that merlin's predicted regulatory network interactions are recapitulated in other immune response regulatory networks. to more directly test the predicted edges of merlin, we compared the predicted targets of four (irf , nmi, stat , tceb ) of our top ranked regulators using two published experimentally generated networks obtained from genome-wide expression profiles in single-gene knockdown sirna screens in other cell lines [ , ] . we found significant overlap (fdr< . ) between the merlin and independently identified targets of three regulators (irf , nmi, stat ), suggesting that the edges predicted in the merlin network are associated with functional changes in expression. as a final type of evaluation we compared the extent of conservation of immune response of our two host systems to influenza infection (materials and methods). at the module level, we estimated the significance of overlap of genes for all module pairs between the two species according to a hypergeometric test. we identified pairs of modules that significantly overlapped in the gene content (p-value< . ) between the two species ( fig a) . the number of genes involved in this overlap was fairly modest, illustrating the challenges of integrating data fig b) , including a core set of interactions, of which form subnetworks with at least genes ( fig b) . this conserved regulatory network contained many key players from the interferon production and jak-stat pathways (stat , nmi, irf ; [ , ] ) as well as regulators about which little is known, perhaps representing new relevant host processes. one conserved regulator with many conserved targets was znhit (also known as trip ), a zinc finger hit domain-containing protein that binds to thyroid hormone receptor [ ] , but which is otherwise poorly characterized in the pathway databases. in summary, the enrichment of immune related functions, motif instances of known immune response tfs, agreement with existing computational and experimentally generated immune response related networks, and the conservation of the key immune related modules and networks between two distinct host systems is indicative of valid regulatory programs that can be explored with further experimental analysis. we used the merlin inferred networks to develop a regulator prioritization strategy wherein regulators were ranked according to the loss in the merlin model's predictive accuracy when a regulator was omitted from its targets' regulatory programs (materials and methods; s table) . in comparison to three other ranking schemes (outgoing regression weight, out-degree, and right eigenvector centrality; s text), this strategy identified the most known influenza host genes among high-ranking regulators (s fig) . we next used these rankings to guide our experimental validation (materials and methods). we selected regulators based primarily on the human rankings, the known annotations of the regulators, expression of the regulator's module and to exclude well-studied immune response regulators (irf , nmi, stat ). to experimentally validate our networkbased prioritization scheme, we measured h n virus replication in human lung epithelial cells (a ) following knockdown of predicted human regulators of host response by sirna (materials and methods, s table) . for each gene, four sirnas were used, in order to mitigate off-target or cytotoxic effects of single sirnas. we called a gene a high confidence hit using a stringent criteria requiring at least two sirnas (out of four used for each gene) to yield a statistically significant (t-test p-value < . ) and high-magnitude ( fold) change in virus titer compared to negative controls, and if none of the sirnas yielded a significant change in the opposite direction. using these strict criteria, three out of the twenty tested regulators were called hits: bola , hcls , and hoxa ( table ) . three additional regulators were mediumconfidence hits with > fold change but significant and consistent effects in multiple sirnas: conservation between human and mouse subnetworks. a. modules conserved between human and mouse. there were fifteen (red shading) significantly overlapping module pairs (hypergeometric p-value < . ), including human modules and mouse modules. b. a core set of interactions conserved between the human and mouse consensus regulatory networks. shown are the of shared interactions that belong to subnetworks with at least three nodes. shading indicates calu- isgs (violet) and nodes identified both as known host genes and isgs (magenta). star indicates a gene that significantly impacted viral replication on knockdown as identified in this study. fgfr , irak , and ythdc . knockdown of all six of the above consistently resulted in reduced virus titer, suggesting that the genes are important for virus production. other tested regulators had multiple significant sirnas, but conferred lower fold changes or showed divergent changes in viral titer between different sirnas for the same gene. we note that some of the genes in our study for which multiple sirnas had statistically significant but inconclusive direction or magnitude of effects were identified as hits by genome-wide screens (s table) . while understanding the detailed role of these predicted regulators in viral replication will require further experiments, these results suggest that our network inference and prioritization method can successfully identify important regulators of host response. to integrate proteomic measurements with the host transcriptional response, we used a predictive modeling approach to identify proteins whose levels are predictive of the mrna levels of gene modules. in theory, the merlin network inference algorithm could be used to integrate these proteins as additional regulators of a target gene's expression levels. however, there were three reasons that prevented us from doing this. first, entire time courses of protein measurements were missing, and integration into the initial network inference step would require either extensive interpolation of entire time courses or excluding many mrna measurements. second, only~ % of our candidate regulators (signaling proteins and tfs) with available mrna levels were measured at the protein levels (s table, s table) . third, compared to mrna levels of regulators, protein levels were not good as predictors of target gene mrna level, likely because of the smaller dynamic range of proteomic measurements (s fig) . our predictive modeling approach used a structured-sparsity based regression framework, called multi-task group lasso (mtg-lasso, fig a) [ , ] . our approach is based on using group lasso for multi-task feature selection [ , ] . unlike lasso [ ] , which solves one regression problem at a time, mtg-lasso aims to solve multiple regression problems simultaneously, one for each gene in a module. our regression formulation has two properties: (a) multi-task regression (where each task is the regression problem of each gene in a module) and (b) group lasso, to enable selection of the same regulators (with possibly different regression weights) for all genes in a module. lasso-based approaches have been applied extensively for mrna-based regulatory network inference [ , ] ; however, to our knowledge, our mtg-lasso approach is the first to employ grouping structure in order to integrate sparse protein-level data with comparatively higher-coverage mrna-level data. a module-based regression enables us to pool information from all genes in the module to select regulators that are informative for all genes in the module. the mtg-lasso regression problem is illustrated in fig a. the full protein data matrix, x consists of m samples for p proteins. all p proteins are used as covariates. the target gene expression matrix for a module, y is an m x n matrix, each column representing the expression profile of a gene in the module. the regression weight matrix, w is a p x n matrix, each row representing the regression weight of a protein for all n genes. in the group lasso framework, a pre-defined grouping structure of covariates is used to select or de-select a group of coefficients together. we define a group as the set of regression weights for a single protein's association to all module genes, resulting in p groups. the framework uses a mixed l /l -norm penalty to impose smoothness and sparsity: the number of proteins (groups) with any nonzero regression weights should be small, and the weights within a group should be similar. we compared the performance of the mtg-lasso models to models learned from randomized protein data (using one-sample z-tests; materials and methods). about half of the human modules, and all but two mouse modules, were predicted better than chance for multiple λ values. this result was consistent for both rmse and pearson correlation measures of predictive quality. from this analysis, we concluded that the protein data does indeed contain predictive signal for many modules; however, it should be used conservatively as predictive quality is not equally good across modules. the alternative to our mtg-lasso approach is to perform traditional lasso for each gene independently, ignoring the module structure. to assess the advantage of mtg-lasso over regular lasso, we applied both methods to each module separately. we compared the methods on the basis of (i) sparsity of the model measured by the average number of regulators chosen for a module across the folds, and (ii) prediction quality measured by pearson's correlation and the root mean square error (rmse) between the measured and predicted set using -fold cross-validation. both mtg-lasso and lasso have a regularization term, λ, which controls the tradeoff between the model complexity penalty and a model's predictive power. we examined five settings of λ, between . (highest penalty imposed, requiring few regulators) and . (least penalty imposed, allowing many regulators). the regularization term λ is a number between and . it denotes a fraction of λ max (maximum possible regularization before reaching a solution), and is comparable between mtg-lasso and lasso (see materials and methods). first, we observe that lasso identified more regulators per module at every value of λ compared to mtg-lasso ( fig b, s a fig) . the proteins selected by mtg-lasso were contained within and important to the lasso models. in particular, when the lasso proteins were ranked by their average absolute outgoing weight for the same λ, the mtg-lasso regulators appeared at the top of the list. we quantified the ranking by the area under roc curve (auroc), treating mtg-lasso as the positive class and the additional lasso regulators as the negative class. the auroc gives the probability that a randomly chosen mtg-lasso regulator ranks above a randomly chosen lasso regulator. for human, auroc ranged between . - . , and for mouse . - . when considering all genes together. the high ranking of mtg-lasso regulators in the lasso selected regulators is observed on a per-module level as well (s d and s e fig) . because mtg-lasso learned a subset of the lasso regulators, we asked if the regulators that were identified by lasso but not mtg-lasso were known to be important based on existing sirna screening studies. focusing on modules that were predicted better than random ( in human and in mouse), we defined consensus regulators per-module as regulators that were selected in at least of folds of cross-validation after determining a module-specific value of λ (materials and methods, fig d and e ). our analysis identified a total of human consensus protein regulators for modules in human ( - protein regulators per module, table ; selected regulators shown in fig a) and a total of consensus protein regulators predicted for modules in mouse ( - regulators per module, table , s table) . a similar analysis for lasso identified different regulators for human and for mouse, which contained the mtg-lasso regulators entirely (s table) . comparable proportions of the consensus regulators identified by each method have been found as hits in published influenza screening studies (for human, mtg-lasso . % and % lasso; for mouse, mtg-lasso % and lasso %). because the mtg-lasso regulators were included in the lasso rankings we also computed the precision after excluding the mtg-lasso regulators. in human the precision was markedly lower ( . %), and slightly lower in mouse ( . %). thus for the human cell line data, there is a distinct advantage of using mtg-lasso to quickly identify the important regulators. the mouse data is more challenging likely due to the heterogeneous nature of the cell populations. together, these results suggests that mtg-lasso is able to learn as good a predictive model as regular lasso, and is particularly advantageous for identifying regulators at the module level. below we further experimentally validate the mtg-lasso regulators. we first qualitatively examined our regulators based on known literature and annotation of these regulators. several of our mtg-lasso protein regulators are known to be associated with immune response pathways, cellular membranes and intracellular transport, rna splicing machinery, mitochondrial inflammation, and may be involved in viral replication, entry or transport within the cell (tables and ) . we experimentally validated seven of the human mtg-lasso regulators for which we already had sirna libraries (table , s table) , transfecting cells with sirnas per regulator. for all seven regulators at least two of the four sir-nas resulted in a significant change in virus titer, and had at least one sirna resulted in a fold-change far beyond -fold. unlike in the mrna case, where most of the significant and high magnitude changes were in one direction, for several of the protein regulators, different sirnas targeting the same gene resulted in both significant and high magnitude increase and decrease in viral replication. we therefore used a majority rule on the significant changes to phenotypically characterize the effect of knockdown of a particular protein regulation. specifically, a gene was called "pro" viral replication if the number of significant decreases in viral replication was greater than the number of significant increases. similarly, a gene was called "anti" viral replication if knocking it down resulted in more significant increases than decreases in viral replication. among the seven proteins that were tested, four were classified as "pro" viral replication with more significant decreases in viral replication (hist hb, isg , prpf , thbs ), while three were called as "anti" viral replication with significant increase in viral replication (app, itgb , serpina ). prpf , which had the strongest, consistent effect across all significant sirnas, is a pre-mrna splicing factor that was previously implicated in viral replication by a genome-wide screen. it is known that the host rna splicing machinery is hijacked by influenza virus [ ] . isg is a ubiquitin-like protein that is stimulated by interferon alpha and beta and is associated with diverse cellular functions including cell-to-cell signaling and anti-viral activity. hence, isg 's pro-viral phenotype was surprising. however, this protein has been observed to be attached to both host and viral proteins [ ] and has been previously shown to reduce viral replication on knockdown in a previous study [ ] . thbs is a ligand of cd and an inhibitor of t and dendritic cells and may play a role in inhibiting inflammation [ ] . serpina and app in particular are interesting candidates as potential inhibitors of viral replication. the serine protease inhibitor serpina (module ) and its mouse homologs serpina k (two modules) and serpina m (eight modules) were identified independently for both species. serpina belongs to the family of serine protease inhibitors that have diverse roles in innate immunity [ ] [ ] [ ] . while serpina has not been shown to affect viral replication, another member of this family, serpine , was shown to reduce the infectivity of the virus particle [ ] . app has been mostly studied for its role in neurodegenerative diseases: beta amyloid plaques are associated with neuronal cell death in alzheimer's disease [ ] but a recent study also suggests it may have an antiviral role, based on observations of decreased influenza a replication (h n and h n ) in cells treated with beta amyloid [ ] , consistent with the increase in viral titer observed in this study. taken together, our results indicate that mtg-lasso can identify regulator candidates that are relevant to immune responses to viral infections. overall mtg-lasso's ability to highly rank the most relevant regulators is an important factor for tractable downstream interpretation and validation of selected regulators. we next used the integrated mrna and protein-based networks to examine the temporal dynamics of host response. this type of analysis can be used to predict which network components may be repressed or differentially wired in response to different viruses. we built time point-specific active networks by overlaying time point-specific expression on the integrated regulatory network connecting both mrna and protein regulators to their target genes. for each time point and virus, we obtained a time point-specific regulatory network by including inferred edges between regulator nodes (mrna or proteins) and target genes (mrna) that were significantly up-regulated (z-score ! , compared to all expression values at that time point). for this analysis we focused on the calu- data set. examination of the active network size (number of edges, regulators and targets) over time showed that the size of networks for each viral treatment tended to change by adding more edges over time (s fig), with the h n mutants and wild-type achieving the largest networks. interestingly, h n nl's network grew more slowly than that of h n ca (s fig), although the networks for the two viruses were highly similar in terms of which nodes and edges they contained by the last time point (s fig) . to identify network components that were common to a subset of viruses or time-points, we clustered the edges from the active networks according to their presence-absence pattern across all samples (materials and methods, fig ) . we obtained five clusters of edges ( fig a) four of which included edges active at multiple early time points (fig a; clusters a-d) and a fifth containing edges that were only present in the -hour (very late) time points for medium or high-pathogenicity viruses (fig a; cluster e) . further, clusters a-c exhibited a sustained pattern of edge presence with edges present through the entire time course, while cluster d was associated with edges present during the later time points. clustering was strongly driven by presence-absence pattern of edges in viruses rather than individual time points. edges from the two low-pathogenicity h n strains (ca and nl) tended to be placed in the same cluster (fig , clusters a, b) , and edges from the mediumpathogenicity (pb - e) and two high-pathogenicity strains (pb -f del, h n wildtype) did as well (cluster b, c, d). the networks for the medium and high pathogenicity mutants additionally distinguished activation responses that occurred at later time points (cluster a, d) from those that were present at all time points (clusters b, c). to identify specific processes that were associated with these edge clusters, we tested them for enrichment of pathways ( table , materials and methods). cluster a was significantly enriched with immune response processes, namely anti-viral and interferon response. this cluster was active for h n infections as well as h n ns trunc. predicted regulators in this cluster included key immune response regulators at the mrna (e.g. irf , stat , trim , nmi) and the protein level (isg p). in terms of pathogenicity, the ns trunc virus is more lethal than the h n strains; however, truncated ns protein disrupts the virus' ability to effectively suppress host immune response. the h n ns trunc mutant in particular displayed an interesting clustering pattern across the other clusters. it was represented in two clusters that represent late response to other medium-and high-pathogenicity viruses (clusters d, e), but absent from a cluster that represents early-onset sustained edges among h n strains (cluster c) as well as from cluster b, which included edges from all other viral strains. cluster b and cluster c, which are associated with the two highly pathogenic viruses are associated with gpcr signaling and muscle contraction and include some of our sirna based regulators (ythdc , cluster b) and (hcls , hoxa , cluster c). both clusters d and e, which include active edges primarily from the high and medium pathogenicity viruses, are associated with signaling pathways including wnt signaling and the circadian clock. wnt signaling has been implicated in influenza infections [ , ] , while disruptions in circadian clocks have been shown to increase susceptibility of the immune system to infections [ ] . in summary, our active network analysis identifies different subnetwork components that are specific to different viruses, and implicates additional processes that might be relevant in a strain-specific manner. our analyses thus far inferred two sets of regulators for each module: ( ) mrna-based regulators, signaling proteins and tfs, predicted by merlin based on transcriptome changes, and ( ) protein-based regulators predicted by mtg-lasso based on proteome and transcriptome changes. both these types of regulators were predicted based on patterns of co-variation of regulators and targets (at the individual gene or module levels). what is missing is information about the underlying physical mechanisms that connect the signaling proteins to transcription factors, and the protein-level regulators to the mrna-level regulators. to gain insight into the underlying physical mechanisms that connect the regulators, and to link them to host genes identified from functional screening studies, we integrated the predicted regulators with physical protein-protein interactions, transcription factor-target interactions, and metabolic reactions from multiple public databases (materials and methods). typically, the interaction data do not provide evidence for direct interactions between regulators, requiring the identification of additional intermediate nodes that connect these regulators. however, even allowing only one intermediate node can result in large subnetworks that are unwieldy for interpretation (see for example fig a) . furthermore, because available interactions are not condition-specific, they may contain false positive interactions and may be missing relevant connections. to identify high-confidence physical subnetworks that are easily interpretable, we formulated a constrained optimization problem to find directed paths that connect merlin-identified signaling proteins to transcription factors and mtg-lasso-identified proteins to merlin regulators using a minimal set of intermediate nodes (materials and methods), prioritizing the inclusion of host genes that were recently identified by watanabe et al. [ ] using both . nodes without borders are candidate intermediates between two regulators and were not given special treatment by the method. node color indicates host genes involved in influenza obtained from sirna or protein interaction studies and calu- isgs (as in fig ) ; this information is for visualization purposes and is not used in the subnetwork inference method. rnai and co-immunoprecipitation with viral proteins. such nodes are important in the virusspecific protein-protein interaction network and provide additional context for interpreting our predicted transcriptome and proteome-based regulators. we applied an integer linear programming (ilp) approach to solve this optimization problem, which has been shown to find more precise solutions compared to heuristic algorithms [ ] [ ] [ ] . using our ilp approach we identified high-confidence physical subnetworks for human modules, which included between - intermediate nodes, including a subset of the watanabe hits and protein interaction partners (s text, s table, supporting website). nine of the module subnetworks included predicted protein regulators. the high-confidence subnetworks were able to connect more true regulators with fewer intermediate nodes than subnetworks inferred from random regulators with the same degree distribution, suggesting that the sparsity of the high-confidence physical subnetworks was not merely due to the degree of the input regulators in the physical interaction network (s text). we also used the random input subnetworks to compute an empirical fdr for each protein by measuring the frequency at which the protein appears randomly, and found that it ranged from - . , demonstrating that many of the proteins used in the subnetworks are unlikely to be identified by chance (materials and methods). a low fdr is a conservative measure of a protein's importance, as many relevant proteins are network hubs and are likely to be identified by chance. these modules, together with their subnetworks, provide an integrated view of different types of regulators that are interacting to drive the downstream expression pattern of the host response. we discuss several below and provide all others on a supporting website. our integrated network analysis identified several modules that captured different components of the host immune response machinery. one of these modules was module , which was associated with immune response-related processes by many of our computational analyses (figs and ) . module exhibited a particularly interesting strain-specific pattern of induced expression under infection with low-pathogenicity viruses and the medium pathogenicity virus, h n -ns trunc, and repressed expression under infection with high pathogenicity viruses (fig ) . module is also enriched for genes associated with interferon signaling, which are critical for mounting the innate immune response. the influenza ns ('non-structural') protein is already known to inhibit the host's antiviral type i interferon response [ ] , suggesting that this module would be a good candidate for further investigation into the mechanism of action of ns . furthermore, the genes in module overlapped significantly with two mouse modules (fig a) , and its regulators featured prominently in the conserved regulatory network identified by the intersection of the human and mouse consensus networks ( fig b) . this module is associated with nfs , app, serpina , and itgbp protein regulators, and irf , nmi, and stat mrna regulators, which are well-known members of the interferon response and jak-stat antiviral response pathway [ , ] . the subnetwork analysis applied to the regulators of this module highlighted hspa , also known as hsp , as a hub that connects gene members from multiple immune response pathways (fig b) . hspa has been proposed as both an antiviral factor [ ] as well as a chaperone required for viral replication [ ] . hspa 's direct subnetwork connections include members and modulators of the antiviral jak-stat pathway (isg [ ] , fgfr , stat ) and also inflammation (app). fgfr was a hit in our merlin-network based prioritization sirna study and acts as a modulator of the jak-stat pathway in growth disorders such as achondroplasia (omim). app was identified as an inhibitor of viral replication in our sirna validation. other important genes in this subnetwork are the protein regulator thbs and an mrna-based regulator, mlkl. thbs is involved in apoptosis [ ] and potentially inhibiting inflammation [ ] , and our sirna validation results classify it as a pro-viral replication gene, while mlkl induces necroptosis (inflammatory cell death; [ ] ). as a whole, the subnetwork for module ties together multiple immune response pathways, namely, antiviral interferon signaling, inflammation and apoptosis. in contrast to module , module ( fig a) shows a pattern of repressed expression in response to low-pathogenicity viruses, and increased expression over time in response to high-pathogenicity viruses. the subnetwork analysis revealed connections between the mrna (ankdrd , set) and protein-based regulators (hist hb , thbs ) of this module via intermediate nodes, sirt and elavl and the watanabe host gene, tp (fig b) . the sirt family of proteins have been identified as antiviral factors for multiple viruses [ ] . the subnetwork suggests that part of its antiviral activity is mediated through interactions with anti-apoptotic signaling protein set [ ] as well as through direct interactions with histones. the other proteins in the subnetwork have roles in apoptosis and the p pathway: tp (p ) itself, elavl (which stabilizes p mrna; [ ] ), ankrd [ ] , and thbs . in summary, module identified interactions between histone proteins and various pro-and anti-apoptotic factors, some of which may explain the independently observed antiviral activity of sirt . a third characteristic pattern was exhibited by module (s fig). genes in this module were associated with a general pattern of induced expression but differed in the intensity of induction (stronger induction in the high pathogenicity strains compared to the low pathogenicity strains). this module was predicted to be regulated by several transcription factors (ankrd , emx , en , foxc , sox , rlx , znf ) and two signaling proteins, grin and sik . sik is a protein kinase which is involved in phosphorylation of hdacs (histone deacetylases) which can in turn modulate innate anti-viral responses [ ] and interferon signaling genes [ ] . moreover, the subnetwork analysis linked sik to the predicted protein regulator thbs (an inflammation inhibitor) through the intermediate node fyn, a tyrosine kinase with potential role in nfkb-mediated adaptive immunity. finally, a fourth module of interest was module (s fig), which exhibited a pattern of high expression in the low pathogenicity viruses compared to both high and medium pathogenicity viruses. both the candidate and minimal subnetworks for this module provided useful information for generating hypotheses about mechanistic interactions between the regulators. the module was associated with both mrna and protein regulators as well as hits from the watanabe study: snw , which interacts with viral ns protein, and psmc , which interacts with viral ha, m , na, pa, pb , and pb proteins. the subnetwork analysis connects predicted mrna regulators (src, btrc, ppm f) and protein-based regulators (thbs , ehd ) through snw and psmc (s c fig). ehd is a regulator of endocytosis [ ] , suggesting a role in virus entry, and src is a tyrosine kinase that has been identified to be involved in antiviral signaling [ ] . taken together, our integrated analysis identified the major regulatory modules of host transcriptional response and predicted mechanistic regulatory programs associated with these in the "regs" columns, consensus regulators for each gene are marked with purple boxes. in "motifs", genes containing msigdb regulatory motifs, including mirna motifs, are marked in green. motifs shown are only those that are enriched in the module. next, colored boxes indicate host genes identified from screening studies (salmon) and immune response gene sets (violet). below the rows for module genes are rows for merlin-predicted mrna regulators and mtglasso-predicted protein regulators. gene expression values are scaled (- , ); protein values are scaled (- , ) to improve visibility. time courses entirely missing at the protein level are indicated as "no protein data". doi: . /journal.pcbi. .g modules. the modules exhibited pathogenicity or strain-specific patterns and were enriched in immune related processes and predicted to be regulated by genes from diverse pathways including innate immune response and apoptosis. these module case studies support our regulators as important players of host response and provide an integrated view of how host response may be regulated at multiple levels, from mrna, to protein, to interaction networks. identification of the molecular networks that underlie host response to different pathogenic infections is important to understand both the mechanisms of immune response as well as to design better therapeutics. towards this end, we performed an integrative regulatory networkbased analysis that combines transcriptomic, proteomic and existing molecular interaction datasets to identify important genes, modules and subnetworks. we found that the key components of innate immune response processes namely, interferon production and signaling, and important regulators of immune response (stat , nmi, irf ), were conserved between a human cell-line (in vitro) and mouse lung (in vivo) model system. our approach was able to predict novel regulators at the mrna and protein level and implicate molecular pathways that may drive virus-specific host responses. prioritization of predictions, including important genes, interactions and networks, is important in systems biology studies, which can easily generate a large number of hypotheses. while a large number of prioritization methods, including network-based strategies, have been proposed [ ] and computationally validated, relatively few have been used to inform experimental validation in a medium to high-throughput manner [ , ] . towards this end we used our inferred merlin networks to prioritize important regulators of host response and test regulators using sirna. six of the regulators exhibited highly significant and consistent effects on viral replication and included novel mrna regulators (bola , hoxa , hcls , fgfr ) in addition to irak and ythdc , which were identified by genome-wide studies using a different influenza virus [ , ] . mice lacking irak have an increased mortality rate compared to wild type in influenza-induced pneumonia [ ] . bola is a mitochondrial protein, which helps maintain mitochondrial morphology and oxidative stress [ ] . mitochondria provide important innate immune functions, including cellular response to double-stranded viral dna by induction of cytokines through the mavs (mitochondrion antiviral signaling) protein [ ] . influenza a virus proteins have also been shown to translocate to mitochondrial membranes and promote mitochondrial fragmentation [ ] . hcls , which is a hematopoetic lineage specific protein [ ] , is also interesting due to its role in signal transduction pathways in b and t cells [ , ] . hoxa is a member of the homeobox family of transcription factors, known to have critical roles in differentiation and embryonic development. the hoxa gene has to our knowledge not been associated with specific immune related functions, however, a closely related gene, hoxa was shown to be involved in lymphoid and b cell development, which are important cell types of the immune system [ ] . the hoxa gene was also shown to code an antigen in specific tumor types [ ] and could be involved in differentiation programs of immune cell types in response to influenza infections. finally, fgfr , a fibroblast growth factor receptor gene is known to have diverse roles in multiple cellular functions [ ] [ ] [ ] . the fgfr - genes were investigated for their role in influenza a viruses [ ] . fgfr was shown to significantly impact cellular internalization of two influenza a viruses but fgfr was not expressed in the cell line tested, leaving open the possibility of its potential role in the influenza life cycle. to our knowledge, these regulators have no previously known role in influenza response, but serve as promising leads for further in-depth validation studies using in vivo models. while approaches to infer and examine networks from mrna are routinely used in systems biology studies of complex responses [ , , ] , examining proteomic datasets and especially integrating them with transcriptomic data to gain insight into regulatory mechanisms is an open challenge that has been addressed by relatively few approaches [ , , ] . this is because, unlike mrna levels, proteomic technologies are still maturing and datasets have lower genome coverage and higher frequency of missing values [ ] . to tackle this challenge we introduced a novel structured sparsity inducing approach, multi-task group lasso (mtg-lasso), which enabled us to leverage the overall signature of expression at the level of modules. our experiments confirmed that while the mtg-lasso and lasso achieved comparable prediction performance on held-aside data, the mtg-lasso approach identified a sparser set of regulators per module. proteins with the strongest contributions to module expression prediction were involved in innate immune response pathways, rna splicing, membrane organization and transport, that are relevant to different parts of virus life cycle. experimental validation of these regulators further associated pro or anti-viral replication functions with them. both directions are interesting from the point of view of understanding the mechanisms of immune response as well as for designing vaccines that could disrupt viral replication and growth. prpf , which is involved in rna splicing, was particularly notable as an example of a pro-viral replication regulator, given the emerging role of post-transcriptional process in diverse pathogenic infections including bacterial pathogens [ ] . serpina (anti-viral, serine protease inhibitor) is also interesting as a candidate anti-viral drug target due to the known roles of serpins in innate immunity [ ] [ ] [ ] . we also predict a pro-viral role of isg , an interferon stimulated protein and that is involved in diverse processes including anti-viral activity. importantly, several predictive protein regulators were not identified as differentially expressed at the mrna level ( / for human; / for mouse; s table, s table) , emphasizing the importance of measuring multiple types of cellular components. these results were further bolstered with our subnetwork analysis that combined the mrna and protein regulators through physical interactions (identified as interaction partners in the physical subnetwork). even though no information about known relevant pathways was provided as input, our approach was able to give interaction-driven predictions for how these different regulators are coordinated. a notable example was a newly identified gene, hspa , in module 's regulatory program, that connected fgfr (discussed above), stat (a member of the jak-stat signaling pathway), and inflammation and cell death pathways (represented by thbs and app). without the subnetwork analysis, it would not be possible to identify hspa , as it was not part of the input set of mrna and protein regulators. our approach identified several important modules that exhibit strain and pathogenic specific patterns of expression. in particular, module , which was associated with interferon signaling and interferon-stimulated genes, exhibited a striking pattern of differential expression of repression in the wild-type h n virus. our results are consistent with those of [ ] , who observed a differential pattern of expression of the isg genes and showed that the differential expression of these genes were inherently tied to host response. another module, module exhibited an opposite pattern of expression and is associated with cell-cell signaling. one caveat to the validation was that we tested the sirnas only in pandemic h n and therefore we do not know how impact of these regulators in different virulent strains. the physical regulatory programs together with the expression phenotype associated with these modules enables us to make intriguing hypothesis of potential mechanisms by which upstream regulatory networks drive context-specific expression, which could be followed by further validation. some of the same mrna time courses have been previously studied with computational network approaches [ , ] , offering additional points of reference against which to compare our inferred multi-virus regulatory network. mitchell et al. [ ] prioritized influenza host genes from wild-type h n and h n samples. they learned a correlational network from which they identified central nodes as well as modules. they also used a regularized regression approach (inferelator, [ ] ) to identify sparse regulator sets that predict the average module expression. our top predicted regulators (both protein and mrna) intersected with theirs on only one gene (nmi). the limited overlap may not be surprising as the bulk of our prioritized regulators were restricted to transcription factors and signaling proteins; however, similar gene families were present in our list and theirs (including ddx, hox, and isg genes). additionally, mcdermott et al. [ ] previously analyzed the h n mrna time courses using a similar approach: first identifying modules through hierarchical clustering, followed by identification of regulators for the modules' average expression. that study identified a set of conserved clusters across human, mouse, and macaque, and a list of prioritized regulators. we found significant overlap of several of those clusters with several our modules (s fig) , but no overlap in the presented prioritized regulators. however, both our study and theirs identified shared functional processes (cytokine signaling and production, inflammation, apoptosis, and cell cycle regulation) and gene families (irf genes). our work can be extended in several ways. one limitation of the current subnetwork approach is that the protein-protein interactions employed are not necessarily functionally relevant to the tissues or conditions under study; a future direction of work is to integrate tissuespecific interactions at this step, such as from large-scale computationally-inferred compendia [ , ] . another direction of future work is to jointly learn modules and their physical regulatory programs using an iterative framework while integrating proteomic measurements. we anticipate that as systems biology studies expand to more viruses, host systems and diseases, approaches such as ours are going to be increasingly useful to characterize host responses at multiple omic levels, prioritize genes and subnetworks for validation. the outcomes from such studies will be important to assemble a comprehensive picture of the mechanisms responsible for healthy and disease states, and ultimately guide the design of effective therapeutics. we obtained background corrected and between-arrays quantile normalized host mrna response data from multiple strains and dosages of influenza virus in calu- human cells (geo accessions gse , gse , gse , gse , gse , gse ) and -week old c bl/ mice (gse , gse , gse , gse , gse , gse , gse ) (full details, [ ] ). all experiments were performed using agilent microarrays. in the original work, each array was subject to quality control, background correction, and quantile normalization. we directly used the processed data available. the viruses include three wild-type and four mutant strains. wild-type strains include a/ california/ / (h n ), a/netherlands/ / (h n ), and a/vietnam/ / (h n ). the mutant strains of h n each affect different aspects of the virus life cycle [ ] . haavir, used in the mouse experiments only, has restricted tissue tropism due to a mutated cleavage site in the hemagglutinin glycoprotein. the wild-type h n virus has a lysine at position in the pb polymerase protein that is associated with the adaptation of h n viruses to mammals. mutation of this amino acid to glutamic acid (pb - e) reduces polymerase activity in mammalian cells and pathogenicity in mice. ns trunc has a shortened version of the ns protein, thereby interfering with the virus' ability to suppress host antiviral responses through the rig- pathway. pb -f del is missing viral protein pb -f , which is involved in many aspects of virus pathogenicity, including polymerase activity and host immune regulation. for each virus infection in human cell line, six or nine time points were collected, spanning hours for low-pathogenicity viruses (at , , , , , , , , hours) and for medium and high (at , , , , , hours). each time point had at least three biological replicates. an additional four-time-point replicate series was collected for h n (hours , , , ) and two additional two-point series were collected for h n (hours , ). in the mouse system, multiple dosages were available for some viral treatments. all time points were taken at days , , , and after infection, with the exception of the highest dosage of h n , which omitted day due to complete lethality. we collapsed replicates of each time point using the median value of a gene's expression level. in total, after collapsing replicates, there were samples per human gene and per mouse gene. using mld values [ ] , we classified the viruses into high, medium and low pathogenicity groups: high pathogenicity included wt h n and h n -pb -f del; medium pathogenicity included h n -ns -trunc and h n -pb - e, and low pathogenicity included h n -haavir and h n . instead of haavir, the calu- data included a different strain of wild-type h n (a/netherlands/ / , or nl); both viruses have the same low level of pathogenicity. having viruses exhibiting similar extents of pathogenicity enabled us to perform a systematic comparison of host response divergence under the same type of perturbation. because our focus was to compare findings between in vivo (mouse) and in vitro (human cell lines) we started with genes that were conserved (had orthologs) between human and mouse, and exhibited differential patterns of expression between high, medium and low pathogenicities. we first obtained the relative expression value of a gene to the same gene's expression in an untreated mock sample from the same time point. we included a gene if its relative expression profiles compared to mock in either species were significantly different between any two pathogenicity groups (assessed by t-test, p-value< . for human and p-value< . for mouse). the resulting gene set comprised , genes in the human cell line and , genes in mouse lung. as regulators, we selected transcription factor and signaling proteins [ , , ] that were present in the differentially expressed gene set. this included a total of , encoded candidate regulators in human and , candidate regulators in mouse. we used protein level data that was available for a subset of the same samples as the mrna data from [ , , , , ]. briefly, peptide-level abundances were obtained by liquid chromatography-mass spectrometry (lc-ms) and matched to protein levels following normalization and quality control. for human, the h n nl time course and a short replicate time course of h n ca were missing (see gray boxes in protein regulator heatmap at the bottom of figs , , s a and s a); for mouse, the missing samples spanned h n haavir and two of four dosages of h n ca . these data provided unique samples for human and for mouse. we selected proteins with fewer than % missing values (resulting in , for human and , for mice), and imputed remaining missing values using the mean value for existing samples (within the same time course). to prepare data for input into the mtg-lasso method, we normalized both protein and mrna data by their row means. learning consensus merlin regulatory module networks for human and mouse using stability selection we used merlin [ ] , a network inference algorithm, to learn regulatory module networks for the human and mouse datasets separately. the input of merlin is a matrix of gene expression data and a list of candidate regulators (e.g., transcription factors and signaling proteins); the output is a regulatory network and a set of regulatory modules. this dual output is a unique feature of merlin compared to other network inference methods. it uses an iterative procedure that alternates between learning the network structure using a greedy search for regulators of individual targets (giving a regulatory program per gene) and performing hierarchical clustering on the target genes based on both co-expression and the similarity in their current assigned regulator sets. the module assignments are also used in the network learning step to provide a prior preference for adding regulators to a gene's regulatory program if the regulator is already assigned to another gene in the module. we embedded merlin in a stability selection framework [ ] , in which merlin networks are learned independently for random sub-samples of the expression data. each subsample consisted of about % of the total samples ( / for human, / for mouse). the resulting ensemble of networks provides a confidence value for each edge in the regulatory network, thereby enabling the identification of a robust consensus regulatory module network. for each individual network, we set merlin's three parameters according to recommendations from the original publication based on simulated data [ ] , setting p = - , r = , h = . . the parameter p controls regulatory network sparsity (more negative values, fewer edges), r controls network modularity (higher values, stronger preference for sharing regulators in a module), and h specifies a threshold on the distance used to cut a hierarchical clustering into gene modules (lower values, more modules). we derived a consensus regulatory module network in several steps from the ensemble of networks that were produced under stability selection. first, to derive a consensus network of regulator-target edges, we applied a threshold of . confidence to the confidence-weighted regulator-target edges produced by stability selection. this threshold was picked based on its fdr, assessed by comparing to a random consensus regulatory network generated by running the approach on randomizations of the expression data. we calculated fdr as the ratio of the fraction of edges from the random network that would be accepted at the threshold, over the fraction of edges from the true network that were accepted by the threshold. the fdr of human and mouse networks were . and . , respectively. next, to independently derive co-expressed, co-regulatory modules, we began by hierarchically clustering the genes, defining the similarity between any pair of genes as the frequency at which the two genes were clustered together across the separate module assignments. we then applied a distance threshold of . on this new clustering to define consensus modules. finally, we identified consensus regulators for each module (at the module level) by assessing the significance of overlap of each regulator's consensus targets within each consensus module, as measured by the hypergeometric test (fdr < . ). we evaluated the modules based on their enrichment with various sources of gene sets and pathways. we call each gene set or pathway an annotation category, and performed enrichment testing independently on groups of annotation categories coming from the same source. to assess significance of the enrichment of the modules with an annotation category, we computed a hypergeometric p-value specifying the probability of observing k or more genes from a module with n genes to have an annotation a, given that there are a total of m genes with annotation a among a total of n genes. considering together all of the annotation categories for a module, we applied the benjamini-hochberg procedure to control fdr at . and accepted corrected p-values < . . the groups of annotation categories that we tested include gene ontology biological process [ ] , targets of transcription factors from msigdb [ ] [ ] [ ] as well as those determined by scanning the promoters of genes using known motifs in the jaspar database [ ] with fimo [ ] . we additionally included gene sets available from msigdb, reactome [ ] , biocarta (http://www.biocarta.com) and kegg [ ] . in addition to the above general curated pathways, we also used experimental, literature-based, and manually curated gene sets that were specifically associated with influenza and with innate immune response. we assembled hit sets from a group of rnai and protein-protein interaction screens [ , , , , , [ ] [ ] [ ] [ ] [ ] . we also created an immune response group of annotation categories consisting of calu- interferon stimulated genes [ ] , curated targets of the nf-kb transcription factor (http://www.bu.edu/nf-kb/ gene-resources/target-genes), genes differentially expressed in response to inflammatory interleukins il- or il- [ ] and members of curated immune response pathways from innatedb [ ] , downloaded november ). for the influenza and immune response gene sets, we obtained human-mouse gene orthologs from the mouse genome database [ ] . we used a hypergeometric test and a fold enrichment to assess the significance of the overlap in edges between the merlin consensus regulatory network and other immune response and transcriptional regulatory networks described in fig b ( msigdb motifs, [ ] mouse pathogen (amit) [ ] , mouse th (yosef) [ ] ). we refer to the merlin network as the "query" network, and the other network as the "test" network. because the networks were directed, we first defined the shared universe of regulators and the universe of targets as the intersections of the respective node sets from the two networks. then, we defined the shared universe of edges as all possible edges between regulators and targets. the size of the universe, u, is calculated as the product of the numbers of regulators and targets in the universe. we measure overlap, o, as the number of edges in the universe that are common to both query and test. we measure the size of the query and test networks, q and t respectively, as the number of edges in each network restricted to the shared universe. to test significance of the size of the overlap, we use the hypergeometric distribution to assess the probability of identifying o or more overlapping edges in a random draw of q edges from a universe of size u that contains t test edges. fold enrichment is defined as the ratio of observed to expected fraction of edge overlap between the two networks, or (o/q)/(t/u). a module was considered to exhibit an interesting strain or pathogenicity-specific pattern of expression (module catalogs, s table, s table) if the mean expression of genes in that module was significantly higher or lower for any two pairs of conditions. conditions were defined based on high vs low, high vs medium, and low vs high pathogenicities. in addition, we considered those modules that exhibited different patterns of expression between the two wild-type viruses h n (ca ) and h n (vn ). for significant expression we used a t-test p-value < . for human and . for mouse. we relaxed the threshold for mouse lung because the data represents transcriptional response from a more heterogeneous collection of cells as compared to the human cell line; we also omitted day from all mouse data due to lack of data (due to lethality) for the high pathogenicity viruses. to classify the modules based on expression differences between different pathogenicities, we used an additional criteria of selecting modules whose mean expression in one pathogenicity type was different in sign compared to mean expression in the second pathogenicity type. we implemented mtg-lasso using the mtleastr function available as part of the sparse learning with efficient projections package for matlab (slep . ; [ ] ). the objective function for mtg-lasso is defined as the first term in the objective function is the least squares loss obtained by the difference between the observed gene expression data matrix, y, and the predicted values from the product of the protein data x and learned regression weight matrix w (fig a) . the second term is the group lasso norm penalty on the complexity of the weight matrix. this norm penalizes the number of groups (according to the one-norm) and encourages smoothness among the weights within each group (according to the euclidean two-norm). the parameter λ controls the trade-off between loss and the regularization term. we evaluated the mtg-lasso and lasso methods over a range of λ, expressed as a fraction of its maximum possible value λ max , above which the coefficient vector will be forced to zero. the slep package calculates λ max for each model as follows. for mtg-lasso, λ max is the largest two-norm of rows in x'y, where x' is the matrix of protein data (transposed from fig a, now with proteins/groups on rows, samples on columns) and y is the matrix of mrna data for one module (samples on rows, genes/tasks on columns). for lasso, λ max is the largest absolute element in the vector x'y, where x' is the protein data and y is the expression vector for one gene. we varied λ between several values from its minimum ( . , almost no sparsity imposed) to its maximum ( . , significant sparsity imposed). the complete set of tested values were { . , . , . , . , . , . }. because λ is normalized by the λ max it represents a comparable regularization strength between both regression techniques. we obtained predicted mrna values for all protein-matched, mrna samples of all module genes using a -fold cross-validation approach, where a consecutive set of about % of samples were held aside from each fold (comprising most of a time course). for the very largest modules (modules , in human), mtg-lasso was computationally intractable, and therefore we could not identify any regulators. to select regulators for each module, we first chose a setting of λ for each module based on λcorrelation curves that plotted correlation of the predicted values and the true data against λ for each module (fig d and e, s dataset) . surprisingly, we observed that the curve did not have the same shape for each module. among human modules, we found three categories of modules based on these curves. the first consisted of modules for which correlation is roughly constant across all values of λ; that is, all predictive performance on the held-aside data was entirely due to a very small set of regulators. the second consisted of another modules for which performance improved as mtg-lasso was allowed to use more regulators (as sparsity decreased). a final third category contained those modules that could not be predicted more accurately than random for more than one setting of λ, usually the highest or lowest tested value (all remaining modules). in contrast to the curves for the human modules, many mouse modules yielded λ-correlation curves that showed a visible inflection point, with high correlation before a particular λ and low correlation after. we grouped the modules based on a visual determination of the inflection point. only two mouse modules were not predicted more accurately than random for multiple values of λ (based on either rmse or pearson). we only considered λ values for which accuracy was significantly greater than random based on z-tests described in the next section. plots for pearson correlation are shown for example human and mouse modules in fig d and e , with stars indicating the chosen values. all curves are available in s dataset. for human modules, we chose λ = . for modules with constant correlation (such as module , fig d) , and λ = . for modules with correlation that decreased as λ increased (such as module , fig d) . for the mouse modules, the curves were not so obviously matched into 'constant' and 'decreasing' categories. we chose based on the visible inflection point in the curve, preferring the next higher (sparser) λ if the drop in correlation was not dramatic. after choosing λ, we defined consensus regulators using both frequency in cross-validation for the specific λ value and the magnitude of regression weights. first, we considered regulators that received nonzero regression weight in at least of the folds. next, we applied a threshold on average absolute regression weights (across all genes, across folds with nonzero weight), followed by a bonferroni-corrected significance z-test (p-value< . ) to assess whether the same protein would be given a weight above that threshold by chance. we used a x = . for human regulators and x = . for mouse regulators, choices that resulted in approximately - regulators per module. mean and standard deviation for the z-test were estimated from the random regression weights. see s table, s z-test to assess significance of mrna prediction quality we evaluated the significance of mtg-lasso and lasso predictive quality using one-sample z-tests [ ] . to assess module-level predictive quality, we obtained one statistic, x (pearson's correlation, rmse), for a module from all predictions from folds of cross validation. we then assembled a null distribution of statistics by running the method on n = random permutations of protein data and real module gene expression data, obtaining the null mean μ and variance σ . we calculated the z-score for the statistic as x Àm s = ffiffi ffi n p and obtained a one-sided pvalue from the normal distribution. we developed a regulator prioritization score that is based on the loss in predictive power of the consensus regulatory network under in silico perturbations. for each regulator r, we held aside the regulator from the consensus regulatory network n, creating a "lesioned" network, n-{r}. we then re-learned regulator-target regression weights for the lesioned network using fivefold cross-validation. we use this network to score each regulator according to the average increase in prediction error when that regulator is removed from each of its targets' regulatory programs: where targets(r,n) is the set of r's target genes as predicted by the consensus regulatory network n, e n t is the mean squared prediction error for the expression profile of gene t using network n (obtained by cross-validation), and e nÀfrg t is the mean squared prediction error for the same gene t given by the lesioned regulatory network n-{r}. we tested regulators from merlin prioritization using sirna. these regulators were selected based on their rankings in human and were additionally informed by their rankings in mouse. nineteen of these regulators were in the top for human. an additional candidate, fig (rank ) was added because it was ranked in the top of the mouse regulator list ( ) . other regulators that were in the top of mouse included well-studied regulators (irf , nmi, stat ) and were already in the top of human rankings. for sirna transfections, human lung epithelial cells (a ) were seeded into -well plates ( x cells/well) and allowed to settle for hours before transfection with nm sirna (final concentration) and μl of lipofectamine rnaimax reagent (invitrogen). for each candidate regulator a gene-specific package of four preselected sirnas were used (flexitube genesolution sirna, qiagen) (s table) . the following sirnas were used as controls: a cell death inducing blend of sirnas (allstars hs cell death, catalog number , qiagen) for visual confirmation of efficient sirna delivery, a validated nontargeting sirna (allstars negative control, cataglog number , qiagen) as a negative control and a previously described sirna targeting influenza virus np mrna (np- ; synthesized by qiagen) as a positive control. each sirna was evaluated in triplicate. cells were incubated for h before infection with plaque forming units of a/oklahoma/vir - / (pandemic h n ) per well. supernatants were collected from each well h post infection and viral titers were determined by plaque assay in madin-darby canine kidney epithelial (mdck) cells. results for each sirna were statistically assessed separately. first, we log-transformed the virus titers obtained by suppressing the expression of the candidate genes using sirnas. next, we compared the replicates of each candidate sirna to the replicates of the negative control (all-star sirna), using one sided, unpaired t-tests. the p-values were not adjusted because the number of candidates was small and by adjusting the p-values we would likely lose true positives [ ] . finally, we calculated the fold-change and the log-fold change for each sirna candidate compared to the negative control, and used these two measures (significance, foldchange) to identify hits that significantly changed the virus titers in the cells. to identify coarser temporal and virus-specific patterns among the active regulatory subnetworks derived from each sample, we clustered the edges according to the samples in which they were active. in order to focus on early stage immune response rather than late-stage cell death responses, we held aside the -hour time point from the medium and high-pathogenicity virus treatments (h n and all mutants), placing them in their own cluster (cluster e, fig ) . we performed average-linking hierarchical clustering using manhattan distance, specifying the number of clusters k. we performed clustering for k = , , , , , and inspected the resulting clustering by eye and by silhouette index. while k = gave the highest silhouette index (which decreased with k), we chose k = because it made a distinction between a sustained, nearly pan-virus cluster (cluster b, fig ) and a sustained medium/high pathogenicity cluster (cluster c, fig ) . we used hypergeometric test-based enrichment ( . fdr corrected p-value < . ) to interpret the clusters using annotated pathways from msigdb [ ] [ ] [ ] , reactome [ ] , bio-carta (http://www.biocarta.com) and kegg [ ] ; results are summarized in table , "enriched pathways". we also used a hypergeometric test to identify whether any regulators were cluster-specific, rather than common to all clusters ( table , "enriched regulators"). first, we took the union of the sample-specific subnetworks and identified the union target set of every regulator. for each of those regulators, we tested whether any cluster subnetwork was enriched for the targets of the regulator relative to the union (fdr corrected p-value < . ). to integrate the signaling proteins, transcription factors and protein regulators predicted for each merlin module, we used an integer linear programming-based (ilp) method for extracting subnetworks from a background network, similar to previous work [ ] [ ] [ ] . the ilps were modeled using gams modeling system v. . . and the ilog cplex solver v. . . . we applied this method separately to each human module. the subnetworks that are extracted by this approach are composed of paths through a background physical interaction network (provenance described below). we searched for two kinds of paths: (i) paths that begin with merlin signaling proteins and terminate in merlin tfs (sinks) that share at least three targets in common, and (ii) paths that begin with mtg-lasso-based protein regulators (sources) and terminate in any merlin regulator (signaling protein or tf, sinks). for all paths, we allowed only one intermediate node between the source and sink. for several modules, the sources and sinks were not sufficiently close (or represented) in the background network to allow for the generation of any paths. we also excluded the largest two modules ( and ), which had a large number of regulators, and only a few regulators could be included in short paths. for some modules, the union of candidate paths resulted in small and visually interpretable subnetworks, as in fig . however, for most modules, the resulting subnetwork was not visually interpretable (as in fig ) . our ilp-based method can identify high-confidence interpretable subnetworks for all modules that had paths, regardless of size. the ilp-based approach (described in detail in s text) finds an ensemble of connecting subnetworks and assigns confidence values to paths according to how important they are for connecting the regulators using a small number of additional nodes. we extract a subnetwork that connects all regulators by solving an integer linear program in which instructions for how paths may be chosen are expressed as linear constraints, and the objective function optimizes a global property of the subnetwork. our constraints require the inclusion of reachable predicted regulators, and specify that each protein-protein interaction may only be used in one direction within the subnetwork. in the objective function, we encoded a preference that the subnetwork should use the influenza host genes from watanabe et al. [ ] as intermediates whenever possible, and to otherwise minimize the use of intermediate nodes. because many subnetworks may satisfy these constraints, we combine multiple solutions to the ilp into an ensemble. we score each path by the fraction of solutions that contain that path. we defined a high confidence subnetwork as the paths that received at least . confidence over the ensemble. to estimate the false discovery rate of nodes and edges by this approach, we generated a null distribution of subnetworks by running the method on randomized input data. randomization was performed as follows. we first replaced the consensus merlin regulators with randomly drawn tfs and signaling proteins from the set that was provided as input to merlin, maintaining the degree distribution (in the background network) of the entire group. we also drew random replacements for the protein regulators from the input set of proteins. then, for each module, we mapped the original pairs of merlin sources and sinks, and protein sources and merlin sinks, to their randomized counterparts, searched for paths to connect them, and assigned confidence values using the ilp approach as we did for the true predicted regulators. we performed randomizations, and calculated the fdr of a protein or an edge at a particular confidence level as the fraction of the random subnetworks that included it in a path of that confidence level. we assembled a human background network composed of protein-protein, protein-dna, and metabolic interactions from the string database v . ([ ] ; excluding interactions labeled as 'expression'), high-confidence interactions from hippie ( [ ] ; downloaded september , using high-confidence threshold of . as recommended on the website), low-throughput physical interactions from biogrid ( [ ] ; downloaded september ), and a human kinase-substrate network [ ] . each resulting background network consists of both directed (e.g., post-translational modifications such as phosphorylation and ubiquitination) and undirected (e.g., binding) interactions. we then removed all interactions involving ubiquitin (ubb, ubc, ubd), sumo (sumo - ), and additional ubiquitin fusion proteins. these proteins are used as post-translational modifiers and are recorded as binding partners for large proportions of proteins in the background network. the ubiquitin and sumo systems are still represented in the background network in the form of directed ubiquitination and sumoylation events between ligases and substrates. networks in figures were developed using cytoscape [ ] and supporting website visualizations were developed with cytoscape.js (http://cytoscape.github.io/cytoscape.js) we provide the inferred modules with their mrna-based regulators, protein-based regulators, and physical subnetworks (for human only) as a navigable resource at http://pages.discovery. wisc.edu/~sroy/integrative_influenza/. code for mtg-lasso and physical subnetwork identification is available from our repository at https://bitbucket.org/roygroup/integrative_networks supporting information s table. frequencies of human protein regulators across -fold cross validation. (pdf) s table. frequencies of mouse protein regulators across -fold cross validation. (pdf) s fig. overview of mouse influenza response modules identified by merlin. expression patterns of mouse modules with at least genes. the red-blue heat map shows mean expression of all genes in each module for each sample, compared to mock treatment value (similar to fig ) . blocks of columns are time series (in days) from different viruses and dosages (pfu: particle forming units). viruses are ordered from low to high pathogenicity; different dosages of the same virus are placed next to each other. the "genes" column shows the size of each module; larger values are shown in darker blue. under "enrichment", a red box indicates module enrichment with any msigdb motif, any curated gene set from gene ontology, kegg, reactome or biocarta (msigdb curated gene sets), any influenza screen set, or any immune response gene set (described in materials and methods). each column is a sample from one of the virus treatment time courses. rows in each heatmap are genes in the intersection of the 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comparison of several mammals jaspar : an extensively expanded and updated open-access database of transcription factor binding profiles fimo: scanning for occurrences of a given motif reactome: a database of reactions, pathways and biological processes data, information, knowledge and principle: back to metabolism in kegg systems-based candidate genes for human response to influenza infection drosophila rnai screen identifies host genes important for influenza virus replication the use of random homozygous gene perturbation to identify novel host-oriented targets for influenza the interactomes of influenza virus ns and ns proteins identify new host factors and provide insights for adar playing a supportive role in virus replication. chanda sk, editor. plos pathogens generation and comprehensive analysis of an influenza virus polymerase cellular interaction network identification of interleukin- and interleukin- -responsive genes in human monocyte-derived macrophages using microarrays innatedb: systems biology of innate immunity and beyond-recent updates and continuing curation mouse genome database group. the mouse genome database (mgd): facilitating mouse as a model for human biology and disease handbook of parametric and nonparametric statistical procedures. chapman and hall/ crc string v . : proteinprotein interaction networks, with increased coverage and integration hippie: integrating protein interaction networks with experiment based quality scores the biogrid interaction database: update construction of human activitybased phosphorylation networks cytoscape: a software environment for integrated models of biomolecular interaction networks we thank dylan pozorski for constructing the supporting website, michael ferris for sharing computational resources for ilp modeling and solving, and center for high-throughput computing for computational resources for bootstrap and stability selection analysis. key: cord- -qzkcwrit authors: cochran, christina l.; soni, parul p. title: neonatal emergencies date: - - journal: clinical pathways in emergency medicine doi: . / - - - - _ sha: doc_id: cord_uid: qzkcwrit newborns may present with vague signs and symptoms that result from a large variety of aetiologies. • respiratory distress syndrome (rds) is caused by insuffi cient surfactant production, most commonly in preterm infants. -insuffi cient surfactant causes inappropriate alveolar expansion. -decreased alveolar expansion leads to respiratory distress. • bronchiolitis is the result of a viral infection affecting the lower respiratory tree. -bronchioles become infl amed and increase secretion production. -common viral aetiologies include respiratory syncytial virus, human metapneumovirus, rhinovirus, enterovirus, and coronavirus [ ] . • meconium aspiration syndrome (mas) results from aspiration of meconium at the time of delivery. • initial signs of respiratory distress include tachypnoea and increased work of breathing (table . ) • as distress progresses, newborns are at risk of developing respiratory failure and apnoea. • rds presents in the fi rst days of life (table . ) • bronchiolitis is a clinical diagnosis based on physical exam and history [ ] . -newborns will have increased work of breathing with crepitations and rhonchi on exam. • consider rds, bronchiolitis, sepsis, bronchopulmonary dysplasia (bpd), pulmonary hypertension, pneumonia, mas, and pertussis. • history of meconium presence in amniotic fl uids should raise concern for mas. • when considering pulmonary hypertension, conduct thorough cardiac evaluation. • chest radiograph (cxr) should be obtained in patients suspected to have rds, pneumonia, and foreign body aspiration (table . ). • cxr is not indicated in patients with bronchiolitis [ ] . • obtain nasal secretions to confi rm pertussis infection. • if suspected, infectious workup should be completed including blood, urine, and cerebrospinal fl uid studies (csf). • intervention can be tailored to a degree of respiratory distress (table . ). • surfactant should be provided to patients with rds [ ] . • supportive care, including suctioning and supplemental oxygen, is the treatment of choice for bronchiolitis. -salbutamol, normal saline, and hypertonic saline nebulisers have not been shown to improve outcome in bronchiolitis [ ] . • for patients without clear viral aetiology, consider antibiotic coverage. • if suspicious for pertussis, start prophylactic antibiotics. • congenital heart defects are a result of abnormal embryogenesis • defects can be classifi ed as cyanotic (table . ) and non-cyanotic (table . ). • cyanosis is a result of right to left shunting of non-oxygenated blood into systemic circulation. • newborns may present with a variety of fi ndings (table . ). • assess colour of the newborn and central/peripheral pulses ( fig. . ). • measure four extremity blood pressures and pulse oximetry. -in the setting of coarctation, lower extremity blood pressures and pulse oximetry will be decreased compared to right upper extremity. • cxr should be obtained to assess the cardiac silhouette. • obtain electrocardiogram to assess for rhythm disturbances. • echocardiogram is used to assess cardiac anatomy and function. • consider blood gas to assess ph and arterial oxygenation. • detailed maternal and prenatal history, including ultrasound results, should be gathered. -many maternal conditions and medications can be associated with congenital heart disease [ ] . • supplemental oxygen should be provided. • haemodynamic support should be provided with fl uids, inotropes, and chronotropes as clinically indicated. -the degree of fl uid resuscitation is dependent on the underlying cardiac defect. • prostaglandins should be initiated to delay closure of the ductus arteriosus in patients suspected of having a shunt-dependent defect. -initial dose -prostaglandin e : . mcg/kg/min • initiate inhaled nitric oxide if pulmonary hypertension is suspected. • cardiology consult should be obtained as soon as cardiac defect is suspected. • improved management of maternal diabetes and hypertension may decrease congenital heart defects. any newborn with concern for infectious process should be evaluated and treated immediately. sbis in neonates include, but not limited to, urinary tract infections (utis), bacteraemia, sepsis, meningitis, pneumonia, pyelonephritis, and cellulitis. neonates with bacterial infection are at high risk for long-term morbidity and mortality [ ] . low threshold for workup of an sbi should be maintained when evaluating children less than days old. • newborns have reduced defence against bacterial infections secondary to an immature immune system. • the two most common bacteria responsible for infection in newborns are group b streptococcus (gbs) and escherichia coli ( e. coli ) [ ] . • utis are the most common bacterial infection in this patient group. • necrotising enterocolitis (nec) is a result of intestinal ischaemia, leading to bacterial overgrowth and gut necrosis [ ] . • rectal temperature greater than ° c is suggestive of infection. • infants may present with hypothermia, lethargy, increased sleeping, poor feeding, jaundice, vomiting, changes in their respiratory pattern, or abnormal vital signs. • detailed prenatal and antenatal history should be obtained. -risk factors for infection include prolonged rupture of membranes, maternal fever, maternal infection, and lack of appropriate treatment for maternal gbs. • abdominal tenderness, distension, vomiting, bloody stool, and lethargy may indicate nec. • redness, tenderness, or drainage from the umbilicus is concerning for omphalitis. • consider hypoglycaemia, metabolic dysfunction, hyperbilirubinemia, congenital heart conditions, and neurologic dysfunction when assessing a patient with the above features. • workup should include cbc, blood culture, urinalysis, urine culture, and csf studies including cell count, differential, glucose, protein, and culture. • consider obtaining herpes simplex virus (hsv) tests. • in the setting of omphalitis, send culture of umbilical discharge. • obtain cultures prior to antibiotic initiation unless patient is clinically decompensating. • for patients with dehydration or concern for metabolic abnormalities, obtain electrolytes and liver function panel. • cxr should be considered in newborns with respiratory distress, and hypoxia. • nec diagnosis and management is reviewed in table . [ ] . • prompt antibiotic therapy is essential. • initiate broad-spectrum antibiotic coverage with ampicillin and gentamicin and/ or cefotaxime. • continue antibiotics until csf, blood, and urine cultures have resulted. • nec management is reviewed in table . . • appropriate treatment of maternal gbs infections reduces the likelihood of newborn infection [ ] . there are a variety of gastrointestinal (gi) emergencies that may present in the newborn period. the majority of diagnoses can be distinguished based on physical exam and radiologic evaluation. omphalocele and gastroschisis are not discussed in depth, but ed physicians should be aware of these complications. • most newborns develop clinical hyperbilirubinemia [ ] . • most cases of hyperbilirubinemia are physiologic, or secondary to normal delayed conjugation and excretion of bilirubin in the newborn, though pathologic aetiologies must be considered. • factors contributing to higher bilirubin levels are listed in table . . • patients can be classifi ed into low risk, medium risk, or high risk for complications of bilirubin (table . ) [ ] . • newborns with hyperbilirubinemia will appear jaundiced and may have decreased activity and desire to feed. • jaundice occurs in a cranial to caudal progression and presents within the fi rst week of life, with peak from day to . • jaundice presenting within the fi rst h of life is likely pathologic. • consider underlying genetic abnormalities, including gilbert, crigler-najjar, and dubin-johnson, when diagnosing unconjugated hyperbilirubinemia. • also consider causes of elevated direct/conjugated bilirubin, including biliary atresia. • infection and sepsis can lead to increased bilirubin levels. • laboratory workup will demonstrate elevated unconjugated bilirubin levels. • consider blood type testing, direct bilirubin level, complete blood count, blood smear, liver function panel, and infectious workup. • treatment for hyperbilirubinemia is based on age, risk factors, and level of bilirubin (table . ). • the goal of treatment is to prevent encephalopathy secondary to bilirubin deposition in the brain or kernicterus [ ] . • initial intervention includes oral hydration, intravenous (iv) hydration, and phototherapy. • for high-risk cases or concern for kernicterus, exchange transfusion is employed [ ] . • overall prognosis of hyperbilirubinemia is very good [ ] . • the incidence of kernicterus is declining with the advent of phototherapy and exchange transfusion, though patients with kernicterus continue to have longterm neurologic morbidities [ , ] . • prevention of hyperbilirubinemia focuses on appropriate hydration of the newborn and appropriate treatment of maternal infections at the time of delivery. • awareness of risk factors will assist in early recognition and intervention. • kernicterus is an entirely preventable disease though not reversible [ ] . • duodenal atresia occurs when there is an occlusion of the duodenum, restricting passage of materials through the small bowel. • overall incidence is reported at in , births [ ] • this diagnosis is highly associated with trisomy . • patients will present in the fi rst days of life with bilious vomiting. • physical exam may reveal a scaphoid abdomen. • newborn may pass meconium but is unlikely to have any bowel movements. • prenatal history is often positive for polyhydramnios. • infection, oesophageal atresia, tracheaoesophageal fi stula, pyloric stenosis, and refl ux should be considered. • vomiting in the setting of duodenal atresia is bilious in nature, distinguishing this diagnosis from many others. • abdominal x-ray will reveal a double bubble sign indicating trapped air in the stomach and proximal duodenum. • electrolytes, including glucose, should be obtained to assess hydration and nutritional status. • management of duodenal atresia is reviewed in table . . • complications may include dependence of parenteral nutrition, poor growth, perforation of the duodenum, and stricture at the site of anastomosis [ ] . • hirschsprung's disease is the result of failed migration of neural crest cells leading to a lack of innervation in a section of the colon. • the aganglionic colon is unable to relax, leading to constriction of that segment. • hirschsprung's disease is more common in males with a : male to female predominance [ ] . • most newborns present with delayed passage of meconium and stool. -ninety-eight percent of normal newborns pass meconium within h. • newborns may present with abdominal distension, vomiting, or stringy stools • physical exam reveals a tight anal sphincter and explosive stooling with rectal exam. • patients may present with toxic megacolon -extreme dilation of the colon leading to distension, pain, perforation, and shock. • consider constipation, meconium ileus, duodenal atresia, or other intestinal obstruction. • rectal suction biopsy of the narrowed section of the colon is gold standard for diagnosis [ ] . • anal manometry and barium enema can assist with diagnosis. • if toxic megacolon is suspected, obtain abdominal x-ray, electrolytes, complete blood count, and blood culture. • management of hirschsprung's disease is reviewed in table . • in the case of toxic megacolon, provide resuscitation as clinically indicated and iv antibiotics • long-term complications of hirschsprung's disease include constipation, bowel dysfunction, and toxic megacolon [ ] . metabolic disorders can masquerade as many different diagnoses on initial presentation. in newborns, always consider underlying metabolic disorders such as • metabolic emergencies in a newborn result from a range of disorders and defi ciencies. • pathophysiology is entirely dependent on the underlying disorder. • newborns may present with poor weight gain, feeding diffi culties, lethargy, emesis, diarrhoea, and decreased movement. • female and male infants with cah present with slight variation [ ] . -females will likely have ambiguous genitalia with enlarged clitoris. -males tend to present with salt wasting and electrolyte abnormalities including hyponatremia and hypokalaemia. • consider sepsis, genetic disorders, and cardiac abnormalities in these patients. • newborn screening results should be reviewed. • glucose levels should be obtained immediately. • blood gas should be obtained to assess for signs of metabolic acidosis or alkalosis. • multiple metabolic disorders can present with electrolyte derangement. • obtain acth, cortisol, and -hydroxyprogesterone levels if concerned for cah [ ] . • consider urine organic acids, serum amino acids, acylcarnitine profi le, lactate, and pyruvate in non-emergent phase. • consider thyroid studies in patients with clinical signs or maternal history of antithyroid antibodies. • provide dextrose for patients with hypoglycaemia defi ned as less than . mmol/l or symptomatic. -oral feeds with dextrose (milk, formula) if patient tolerates. -iv dextrose % bolus of ml/kg. -if blood sugar remains low, consider an iv infusion of % dextrose. • consider stress-dose steroids if cah and adrenal crisis are suspected [ ]. -hydrocortisone iv: - mg/m • correct electrolyte abnormalities as indicated. -cah patients may require sodium chloride supplementation [ ] . -consider sodium benzoate for sodium replacement after discussion with an endocrinologist. • many metabolic disorders require long-term dietary supplementation or restriction. • newborn screening should be administered on all newborns in the fi rst days of life. bruising, bleeding, and petechiae are not common presenting issues in newborns, though when present, should raise concern. broad differentials should be maintained for these clinical features. • in neonatal alloimmune thrombocytopenia, maternal antibodies cross the placenta and target paternally derived antigens, causing destruction of the platelet. • autoimmune thrombocytopenia occurs when maternal antibodies target maternal and neonatal platelets. • patients may present with easy bleeding, petechiae, and purpura. -assess if males experienced prolonged bleeding with circumcision. • mental status changes could be indicative of intracranial haemorrhage. • consider vitamin k defi ciency, neonatal alloimmune thrombocytopenia, and autoimmune thrombocytopenia. • also consider nonaccidental trauma, infection, underlying coagulopathy, necrotizing enterocolitis, haemophagocytic lymphohistiocytosis, and leukaemia. • gather thorough history including maternal medications, illnesses, birth history, and vitamin k administration. • obtain complete blood count, coagulation panel. • immediately administer vitamin k if not previously given or coagulopathy is suspected. -vitamin k: . - mg intramuscularly, subcutaneously, or intravenously [ ] • provide platelet transfusion if patient is thrombocytopenic and actively bleeding (table . ). • consider fresh frozen plasma in setting of moderate to severe bleeding. • if concerned for an antibody-mediated process, consider intravenous immunoglobulin (ivig) and/or platelet transfusion [ , ] . -ivig: . - g/kg/day [ , ] . -steroid infusion can be considered as adjunct therapy [ , ] . seizures may present in the neonatal period as a secondary process or as a primary seizure disorder. the primary goal after stabilisation of the patient should be to uncover and manage the underlying aetiology of the seizure. • seizures are more frequent in preterm infants and infants with hypoxic ischemic encephalopathy (hie) [ ] . • abnormal movements occur secondary to withdrawal from maternal drug exposure, commonly opioids. • seizures may manifest with abnormal extremity or eye movements. • withdrawal symptoms are listed in table . [ ] . -neonatal abstinence scoring may be utilised to assess for risk of neonatal withdrawal. • aetiologies of seizure range from encephalopathy, stroke, hie, metabolic disorders, infection, to genetic disorders [ ] . • electrolyte abnormality can present abnormal movements or behaviour. • nonaccidental trauma should always be considered. • obtain glucose and electrolytes in infants with abnormal movements. • gather thorough history, including maternal history, maternal medications, maternal infections, and birth history [ ] . • obtain head imaging • complete infectious workup, including csf studies, should be performed. • send urine and meconium toxicology screens to assess for withdrawal syndrome. • if hypoglycaemia is present, correct with iv dextrose. • consider treating a seizing neonate with phenobarbital or phenytoin • if seizures are nonresponsive to the above medications, please consider lorazepam or levetiracetam • in cases of neonatal withdrawal, provide supportive care and tailor medical management based on maternally abused drug (table prognosis • prognosis depends on the underlying aetiology and duration of symptoms american heart association guidelines for cardiopulmonary resuscitation and emergency cardiovascular care prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis surfactant replacement therapy at birth: fi nal analysis of a clinical trial and comparisons with similar trials association between maternal chronic conditions and congenital heart defects: a population-based cohort study. canadian perinatal surveillance system (public health agency of canada) neonatal meningitis in england and wales: sequelae at years of age neonatal meningitis in england and wales: years on pathology of neonatal necrotizing enterocolitis: a ten-year experience microbiology and management of neonatal necrotizing enterocolitis division of bacterial diseases, national center for immunization and respiratory diseases. prevention of perinatal group b streptococcal disease -revised guidelines from cdc american academy of pediatrics subcommittee on hyperbilirubinemia. management of hyperbilirubinemia in the newborn infant or more weeks of gestation outcome of neonatal hyperbilirubinemia in a tertiary care hospital in bangladesh duodenal atresia. fundamentals of pediatric surgery the role of parenteral nutrition following surgery for duodenal atresia or stenosis hirschsprung's disease in japan: analysis of patients based on a nationwide survey in years diagnostic tests in hirschsprung disease: a systematic review congenital adrenal hyperplasia due to steroid -hydroxylase defi ciency: an endocrine society clinical practice guideline management of severe alloimmune thrombocytopenia in the newborn alternative therapies for neonatal autoimmune thrombocytopenia platelets and neonatal transfusion study group. prospective, observational study of outcomes in neonates with severe thrombocytopenia the epidemiology of clinical neonatal seizures in newfoundland: a population-based study committee on drugs, committee on fetus and newborn proposal of an algorithm for diagnosis and treatment of neonatal seizures in developing countries long-term prognosis in children with neonatal seizures: a population-based study • intracranial haemorrhage and lower platelet levels are associated with increased risk of morbidity and mortality [ ] . • majority of newborns with thrombocytopenia have good outcomes [ ] . • administration of vitamin k immediately following birth • close monitoring of infants born to moms with idiopathic thrombocytopenia • maternal education on the harms of drug use key: cord- -os faovj authors: peghin, maddalena; danziger-isakov, lara title: prevention and treatment of respiratory virus infection date: - - journal: infectious diseases in solid-organ transplant recipients doi: . / - - - - _ sha: doc_id: cord_uid: os faovj there is increasing recognition of infections caused by respiratory viruses (rvs) as a major cause of morbidity and mortality in solid organ transplant (sot) recipients, especially within the thoracic and pediatric population. in addition to their direct, cytopathic, and tissue-invasive effects, rvs can create an inflammatory environment, autoimmune responses, resulting in acute and chronic rejection, although this relationship remains controversial. a laboratory diagnosis in sot with respiratory syndrome should be performed with nucleic acid amplification tests on respiratory specimens, mainly nasopharyngeal swabs (nps) and bronchoalveolar lavage (bal). treatment options remain limited and consist of supportive care, reduction of immunosuppression, and, if available, antiviral therapy. the use of immunomodulatory agents remains a clinical dilemma. since treatment options for rvs are limited, maximizing prevention measures against viral infections in sot is mandatory. the main preventive strategy against influenza remains the administration of yearly inactivated influenza vaccine in all sot. the aim of this review is to summarize the evidence-based recommendations on the diagnostic, preventive, and therapeutic strategies to decrease the burden of rv infections in sot recipients. respiratory viruses (rvs) are increasingly recognized as a major cause of morbidity and mortality in solid organ transplant recipients, especially within the lung transplant population. respiratory viral infections are typically caused by rhinovirus (rhvs), coronavirus (cov), respiratory syncytial virus (rsv), influenza (flu), parainfluenza (piv), human metapneumovirus (hmpv), and adenovirus (adv) ( table . ). respiratory infections can also be caused by viruses less commonly associated with the respiratory tract such as cytomegalovirus (cmv), human herpesviruses (hsv , hsv ), and varicella zoster virus (vzv) that will be discussed in another chapter (chap. ) . a detailed discussion of other newer respiratory viruses (table . ) is beyond the scope of this chapter, since they have not been widely studied in immunocompromised patients and their clinical impact is not fully understood. however, these viruses should be considered in the differential diagnosis of patients presenting with severe lower tract disease, especially if clinical history indicates potential exposure. the newer rvs are more challenging to diagnose since they are not included in the routinely available diagnostic tests and optimal management has not been defined. the definition of rv disease includes ( ) a new onset of symptoms and ( ) at least one respiratory symptom and ( ) the clinician's judgment that the illness is due to an infection [ ] . an upper respiratory tract infection (urti) is defined with the onset of sore throat, rhinorrhea, or hoarseness. a lower respiratory tract infection (lrti) is defined as new onset of shortness of breath, cough, sputum, rales, hypoxemia, and/or wheezing. when symptoms of lrti are associated with a new pulmonary infiltrate (on chest radiograph or chest computed tomography), pneumonia is distinguished from tracheobronchitis. many common respiratory viral infections in sot patients are mild, self-limiting upper respiratory tract infection (urti) and do not require hospitalization. however, compared to immunocompetent hosts and due to alterations in cellular and humoral immunity, infections can cause protracted symptoms with greater risk of progression to lrti, prolonged periods of viral shedding, and increased mortality. in sot, lrtis have been associated with increased risk of adverse complications and subsequent development of fungal, viral, and bacterial superinfections [ ] . although these complications may appear in the context of any type of transplantation, pediatric, lung, and heart-lung transplantation recipients appear to have the greatest risk of respiratory viral infections with more severe courses and complications [ ] [ ] [ ] . in addition to their direct, cytopathic, and tissue-invasive effects, rvs can create an inflammatory environment that leads to local and systemic microbially determined immune modulation (mdim) [ ] . mdim may increase the alloimmune and autoimmune responses that increase susceptibility to other opportunistic infections and are associated with the development of acute and chronic rejection. the greatest risk appears from data in lung transplant recipients, although data on this topic in the literature are conflicting [ , , ] . in transplantation overall, rhv and cov are the most common etiological agents, causing mostly mild urti, with lrti less frequently described. in contrast, flu and other paramyxovirus (rsv, piv, and hmpv) have a greater association with lrti and particularly acute and chronic rejection in adult lung transplant recipients [ , ] (tables . and . ). outcomes of infection are associated strongly with site of involvement, net state of immune suppression, and availability and use of antiviral agents. the clinical diagnosis of rvs can be difficult, since sot recipients often present with mild or atypical symptoms and signs, which are often overlapping and not always specific for any one viral agent. fever can be absent in sot with pneumonia or can be the sole presenting sign. in addition bacterial and fungal coinfections may occur. the distribution of rv infections throughout the year suggests that seasonal patterns of rv circulation in sot are similar to those circulating in the general [ , ] . consequently, vigilance regarding circulating community rv infections is required while caring for sot recipients. rapid and reliable laboratory diagnosis is required in sot with respiratory syndrome to significantly impact on patient care and management. the ideal method of sampling has also come into question, as the yield of viral specimen may differ depending on the specimen source. all sots with suspected rv infection should have a nasopharyngeal sample tested by pcr, including nasopharyngeal swab (nps), wash, or aspirate. between common respiratory specimens collected from the upper respiratory tract, nps are preferred, since they are practical for widespread use and comparable in sensitivity to nasopharyngeal aspirates or bronchoalveolar lavage (bal) for the detection of all major rvs [ , , ] . nps should be collected diligently by trained staff, using the standardized procedures of the centers for disease control and prevention (cdc) (https://www.cdc.gov/urdo/downloads/ speccollectionguidelines.pdf; https://www.youtube.com/watch?v=dvjnwefmhje) [ ] . if upper tract samples fail to document the rv cause of the respiratory illness and clinical or radiologic evidence of lower tract involvement exists, bal should be performed for rv testing [ ] . the array of diagnostic tools for rvs in immunocompromised patients has greatly increased over the last few years, and diagnosis can be performed using realtime pcr (rt-pcr) techniques, antigen detection, and serology (table . ) [ ] . the sensitivities of contemporary molecular diagnostic techniques have been substantially improved, allowing for the rapid simultaneous detection of a wide variety of conventional and emerging rvs in respiratory samples. at present, realtime multiplex nucleic acid amplification testing (multiplex nat) based on the rt-pcr technology is the preferred diagnostic tool for studying rvs in immunocompromised patients and is incorporated into many of the current guidelines [ , ] . both laboratory-developed and commercial rt-pcr assays are currently available, differing in specificity and sensitivity (ranges from % to %, with best sensitivity seen for flu and lower sensitivities for adv and piv). with the aim of overcoming technical complexity of pcr-based testing, fully automated rt-pcr instrument for rapid detection of rv has been tested in immunocompromised patients with promising results with a turnaround time of approximately - h [ ] . therefore, clinicians should be aware of the performance characteristics of the assay performed (https://www.cdc.gov/urdo/downloads/speccollectionguidelines. pdf). of note that regarding adv, negative testing from the upper or lower airway may not exclude infections particularly for sot with disseminated disease if there is limited to no involvement of the respiratory tract. rt-pcr should be applied on respiratory specimen, blood (quantitative viral load testing), and other compartments depending on clinical presentation (urine, cerebrospinal fluid). it is important to remember, however, that despite the excellent sensitivity, poorly collected samples may yield false-negative results, and results may greatly vary depending on the quality of the swab. the high sensitivity of these methods also has drawbacks, such as frequent detection of viruses in asymptomatic individuals and prolonged detection of viruses in patients who have already clinically recovered [ , ] . the major challenge is to determine association between the presence of microbial nucleic acids and a clinical syndrome in individual patients. quantification of the virus may be a helpful result interpretation, since high viral loads are associated with the presence of symptoms and may be related to the severity of the clinical symptoms [ ] . antigen detection techniques, which include immunofluorescence (if) and immunoassay (ia), are fast and have high specificity but are only available for specific viruses and their sensitivity less than molecular methods. this technique is not available for viral respiratory infections caused by rhv or cov and is moderately complex, and interpretation of results is subjective [ ] . a number of commercial ia are available for rsv and flu (a and b) and require little technical expertise. however, false-negative and false-positive results can be generated. a low prevalence of circulating virus within the community decreases the positive predictive value of the test. for flu, rapid ia has shown high specificity but low sensitivity ( - %) as compared to other assays, making them suboptimal for sot recipient, particularly in clinical decision-making for antiviral therapy [ ] . antiviral susceptibility testing for rvs is primarily focused on influenza, and both phenotypic and genotypic assays can be tested, although such testing is not widely available in local or commercial labs. antiviral resistance is of considerable concern among immunocompromised patients infected with influenza virus, and testing should be strongly considered in sot undergoing treatment who fails to have an appropriate clinical response within - days of initiating antiviral therapy or who has a relapsing course despite ongoing therapy. in the absence of available treatment options and of strong evidence of effectiveness for any particular therapy, treatment strategies differ widely among centers [ ] . limited understanding of ( ) risk factors for progression to severe lrti and poor outcomes and ( ) indirect inflammatory effects of viral infection impact opinions on appropriate interventions for respiratory viral infections. rv infections, particularly cause by influenza virus, are a risk factor for subsequent bacterial and fungal superinfections. in cases of lrti, secondary infections must be ruled out and appropriately treated, and initiation of oral or nebulized antifungal prophylaxis to prevent invasive fungal infections should be evaluated in high-risk patients [ , , ] . management in transplant patients is generally focused on reduction of immunosuppression feasible to speed resolution of viral infection. treatment options for rvs are limited (tables . and . ). resistance patterns may change and affect recommended antiviral strategies. consequently, clinicians should consult national health authority regularly for updated recommendations, especially for influenza. in our opinion treatment efforts should be always performed in any sot with lrti or in lung transplant and heart-lung transplantation recipients both with urti and with lrti, due to increased morbidity and mortality [ ] . reconstitution of the immune system appears to be important in overcoming rv infections. clearly, the currently available treatment option is a clinical dilemma [ , , ] . there are numerous reports in the literature citing the use of intravenous immunoglobulin (ivig) as part of therapy for viral infections in immunocompromised patients. hypogammaglobulinemia has been associated with an increased risk of opportunistic infections in sot, but not to community-acquired rvi. however, some experts recommend considering the addition of ivig for severe rv infection in sot [ ] . the use of monoclonal antibodies is limited to the treatment of rsv. immunotherapy including transfer of rv-specific t lymphocytes from healthy donors is under investigation and has been reported to be safe and effective when performed early in the course of the infection for hmpv, adenovirus, rsv, and piv. at the same time, virus-associated immune modulation may sometimes be deleterious in rvs due to local inflammatory responses. adjunctive therapy with corticosteroids has been purposed for sot with influenza and rsv and for lung transplant recipients with any rvs with lrti because of the risk of both acute and chronic rejection [ ] . treatment options for rvs are limited, and maximizing prevention measures against viral infections in sot is mandatory. rvs are potential community and nosocomial pathogens that can be spread by staff or visitors with mild upper respiratory illness. overall awareness among sot, healthcare personnel, family members, and caregivers about the potential deleterious outcomes of rv infections in sot and the importance of early detection of infection may have a significant impact on the incidence of rv infections and risk of transmission [ ] . strict adherence to hand hygiene, contact precautions, and respiratory droplet isolation are required to reduce rv nosocomial spread and outbreaks during hospitalization (table . ) (https://www.cdc.gov/infectioncontrol/guidelines/isolation/). the appropriate length of isolation for patients with laboratory proven rvs is debated, as prolonged shedding is a common finding in sot patients, but viral load thresholds for infectivity are unknown. infection control measures should be maintained until the patient is discharged home or until pcr is negative. stringent hygiene precautions should be also applied in community settings, where sot recipients should avoid close contact with individuals with respiratory tract infections [ ] . the influenza virus is currently the only carv that can be prevented with vaccination [ ] . prevention and treatment of specific rvs three main viral strains have been recently associated with human infection, namely, influenza a/h n , influenza a/h n , and influenza b. influenza infection in sot causes significant morbidity and mortality compared to general population [ ] . in studies performed in h n pandemic, the proportion of patients who required hospitalization varied between % and %, and one of every five patients suffered severe complications with - % mortality [ ] . the mainstay of treatment for influenza a and b are the neuraminidase inhibitors (nai), mainly oseltamivir (tables . and . ) [ ] . doubling the treatment dose of oseltamivir in hospitalized patients with influenza does not seem to increase virologic efficacy, except perhaps for influenza b infections or in case of oral absorption concerns, with no evidence of emergence of oseltamivir resistance [ , ] . zanamivir is used less frequently than oral oseltamivir, likely due to the inhaled delivery route, although it has shown better activity against influenza b and few cross-resistance with oseltamivir. regarding intravenous formulations, if available, intravenous zanamivir or peramivir can be considered in sot recipients who are severely ill despite oral oseltamivir, in case of concerns with oral absorption, although experience with these drugs in sot recipients is lacking [ ] . parenteral zanamivir is currently available in europe, and a single dose intravenous peramivir has been approved in the united states for treatment of uncomplicated influenza infections. however, peramivir use in sot likely would require repeated dosing or switching to oral oseltamivir to complete therapy. nai resistance is currently uncommon ( . - . % of isolates), especially for influenza a/h n and influenza b viruses, but remains an area of growing concern. in case of high-level oseltamivir resistance (such as h n viruses strains with h y substitution), peramivir usually preserves reduced susceptibility, but zanamivir is usually active. another common resistance mutation (h y in h n ) confers resistance to both oseltamivir and peramivir, but not zanamivir. therefore, peramivir should not be used in patients with oseltamivir resistance unless the isolate is proven to be susceptible [ ] (tables . and . ). das , an inhaled sialidase potentially inhibiting influenza and parainfluenza infection, has shown promising in vitro results of activity against oseltamivir-resistant influenza strains but failed to show superiority compared to placebo in previous studies in healthy subjects with influenza infection [ ] . treatment should be initiated as soon as possible since antiviral therapy is most likely to provide benefit when initiated within the first h of illness in sot, with a reduced rate of influenza-associated complications (admission to icu, use of invasive ventilation, and death) [ ] . however, benefit has been demonstrated even with delayed treatment, and most experts endorse influenza-specific antiviral treatment at any point in the illness. further, treatment should not be delayed while awaiting diagnostic testing results or if a rapid antigen ia test is negative when clinical symptoms are suggestive of infection due to the poor sensitivity of rapid antigen tests (table . ) [ ] . in general, duration of antiviral therapy should be at least days for sot patients although some data suggest that longer duration (≥ days) may be required, particularly in critically ill patients, those with pneumonia and persistent viral shedding. aside from advances in supportive care, no specific adjunctive therapies are routinely recommended. corticosteroids have been shown to decrease the need for mechanical ventilation and progression to lrti but at the cost of prolonged viral shedding and risk for invasive fungal coinfection. corticosteroids are not routinely recommended but should be used if indicated for another reason such as concurrent acute rejection [ ] . the main preventive strategy against influenza in sot recipients remains the administration of yearly inactivated influenza vaccine. all transplant recipients and candidates, as well as family members, close contacts, and healthcare workers, should receive the influenza vaccine to provide herd immunity [ , ] (table . ). influenza vaccines are available in inactivated (intramuscular or intradermal administration) and live-attenuated (intranasal) formulations. the liveattenuated vaccine is not recommended for immunocompromised recipients and close contacts, due to a potential risk of dissemination of the vaccine [ , ] . current guidelines recommend the standard injected inactivated influenza for sot starting - month posttransplantation with option for administration as early as month posttransplantation in an outbreak setting. if influenza vaccine was administered earlier than months posttransplantation, when it is likely to be less effective, consideration may be given to administering a second dose of vaccine later in the influenza season [ , ] . an association between vaccination and the development of the de novo antibodies and graft rejection is unproven. a higher-dose vaccine in pediatric sot and a booster strategy weeks after standard influenza vaccination in adult sot have shown to induce an increased antibody response compared with standard single dose. whether or not protection is increased by use of higher-dose vaccine, adjuvants, booster doses, or quadrivalent versus trivalent vaccines constitutes an area of active research [ ] . clinical failure of influenza vaccination in sot recipients has not been extensively studied, but most of the studies clearly suggest a reduced immune response in sot, with a seroconversion rate that varies between % and %, although this is also dependent on the match between the vaccine and the circulating strains [ ] . vaccination has shown to attenuate adverse outcomes among sot recipients with a lower incidence of pneumonia and shorter length of hospital stay [ , ] . beyond influenza vaccination, pre-exposure or postexposure chemoprophylaxis with either oseltamivir or zanamivir is approved (tables . and . ) and may be considered [ ] . caution should be used with prescribing oseltamivir for prophylaxis in patients exposed to an index case because prophylaxis has been associated with emergence of resistant mutants; therefore, monitoring and empiric therapy are generally recommended in these cases [ ] . respiratory syncytial virus has long been recognized as a concerning pathogen in immunocompromised hosts. in sot, rsv infection typically manifests as an urti with progression to lrti in - %. risk factors for more severe disease after organ transplantation include infection in children under a year of age or lung transplantation [ , ] . the use of ribavirin (rbv) for the treatment of rsv infection is controversial. in immunocompromised patients (mainly hematopoietic stem cell transplant recipients), rbv has been shown to decrease progression to lrti when given to patients with urti. among sot, the greatest experience with rbv is with lung transplant recipients. based on published reports as well as self-reported treatment strategies in surveys from sot centers, lung and heart-lung recipients often receive rbv for both rsv-related urti and lrti [ ] . due to lack of clear evidence of efficacy, wide variation in the management of rsv exists including variability often dependent on availability of the inhaled, intravenous, and oral rbv formulations [ ] . intravenous and inhaled rbv are not available in most european countries. oral ribavirin appears to be an effective, well-tolerated alternative to intravenous or inhaled ribavirin, providing potential cost savings and reducing length of hospital stay [ ] (tables . and . ). aln-rsv , a small interfering rna that targets the rsv nucleocapsid messenger rna, has shown some early promise in potentially preventing chronic rejection in lung transplant recipients with rsv; this agent is no longer being developed clinically. in addition, there are a number of other small molecule therapies in various stages of development including early clinical trials [ ] . immunomodulators have also been investigated. experts recommend considering the addition of an antibody preparation (palivizumab) and ivig with or without corticosteroids for severe rsv infection in sot, although data are limited to support this recommendation [ , ] . a systematic review reported that any form of rbv, alone or in combination with an immunomodulatory agent, was effective in preventing progression from urti to lrti, with a trend toward better outcomes with inhaled rbv plus an immunomodulatory with monoclonal (palivizumab) or polyclonal antibody preparations (ivig) ( table . ). prevention in addition to the general preventive measures, the only fda-approved agent for the prevention of severe rsv infection in high-risk patients under the age of years is palivizumab [ , ] . survey data suggest that antibody-based prophy-laxis is used among pediatric transplant centers in young candidates and recipients. however, guidelines regarding the use of this agent in older children and adults do not exist, and the high combined with a lack of clear evidence of efficacy in sot recipients precludes its wide-scale use (table . ). in sot patients piv, most commonly piv , is able to cause more serious and even fatal infections, which mostly occur in patients after lung transplantation [ ] . an outbreak of piv infections in a kidney transplant unit demonstrated that all infections were mild and symptoms resolved spontaneously without associated mortality [ ] . treatment there are no currently approved antiviral treatments for parainfluenza disease. treatment is supportive and includes reduction in immunosuppression. oral, aerosolized, and intravenous rbv and/or ivig and corticosteroids have been used off-label in piv with variable results and no impact on mortality [ ] . das has been used to treat piv infections in immunocompromised patients and has shown encouraging results including reduction in piv quantitative viral load and overall outcomes [ ] . clinical trial results are pending. prevention outbreaks caused by piv have been reported previously [ ] , and patients with known or suspected piv should be isolated with standard contact precautions. there are no approved vaccines or prophylactic antiviral agents. human metapneumovirus has a clinical pattern similar to rsv and is a significant cause of disease in transplant recipients [ ] . hmpv has been associated with lrti (pneumonia) and high hospitalization rates [ ] . treatment there is no approved drug for the treatment for hmpv respiratory infection. supportive therapy is the main treatment although rbv alone or with ivig could be considered for the management of lrti and severe cases of hmpv in sot [ ] . prevention there are no approved vaccines or prophylactic antiviral agents. rhinovirus has more than serotypes in different species: a, b, and the more recently characterized c. rhinoviruses are the leading cause of community-acquired rv infections, and that finding is in agreement with the knowledge that this rv is the primary cause of acute viral respiratory illnesses [ , ] . infections with rhinovirus are usually mild and self-limiting urti, although significant lrti has been described in lung transplant recipients [ , ] . prolonged shedding for over months with minimal symptoms has been reported in lung transplant recipients. treatment no specific treatment is approved for rhinovirus infection. prevention there are no approved vaccines or prophylactic antiviral agents. coronavirus generally results in self-limited disease but may progress to lrti. the most common types of hcov are oc , e, hku , and nl . severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) are novel coronavirus that have been responsible for recent acute respiratory syndrome epidemics. treatment there are no antivirals licensed for the treatment of hcov infections, and therapy consists of supportive care. rbv has been used for the treatment of lrti caused by coronavirus during the outbreak of sars, and the use of rbv in combination with interferon-α- a on mers-cov has been reported. however, this combination has not been reported in sot, and there are no specific data to recommend rbv for the treatment of cov infection in sot recipients [ ] . prevention there are no approved vaccines or prophylactic antiviral agents. adenovirus is a double-stranded dna virus of the family adenoviridae, with subgroups (a-g) and serotypes. in contrast to many of the other community-acquired rvs, adenoviral infection can occur from primary acquisition or through reactivation. the transplanted organ is typically the site of infection, and pneumonia is most frequent in lung transplant recipients [ ] . of note, commercial rt-pcr assays differ in sensitivity and specificity for adenovirus (adv), and quantitative adv pcr from blood may also be obtained to aid in diagnosis (tables . and . ). treatment treatment is supportive and includes reduction in immunosuppression. the optimal timing for therapeutic intervention during the course of illness is unclear. existing data suggests that cidofovir and brincidofovir, an orally bioavailable lipid conjugate of cidofovir, may provide the highest likelihood of antiviral efficacy. brincidofovir appears to have increased in vitro and in vivo efficacy against adv for treatment of serious infections with less renal and bone marrow toxicity than cidofovir (table . ). rbv does not appear to have significant anti-adv activity in humans and is generally not recommended to treat serious adv infections. the use of ivig remains controversial because it does not appear to have a clear benefit at this time. adoptive t-cell transfer has generally been limited to a few centers (predominantly in hematopoietic stem cell transplantation) and has been reported to be safe and effective when performed early in the course of the infection [ ] . prevention there are no approved vaccines or prophylactic antiviral agents. longitudinal prospective surveillance using molecular diagnostics is needed to understand the true epidemiology and clinical spectrum of respiratory viral diseases in sot, particularly in non-lung population. optimal timing, duration, and treatment indication for rvs are a dilemma that needs to be clarified in clinical practice. the efficacy of adjuvant immunogenic therapies remains controversial. maximizing prevention and infection control measures against rvs in sot is essential (table . ). table . key points for rv infections in sot epidemiology and clinical presentation • there is increasing recognition of infections caused by rvs as a major cause of morbidity and mortality in sot • in addition to their direct, cytopathic, and tissue-invasive effects, rvs can create a microbially determined immune modulation the impact of rvs in acute and chronic rejection remains controversial, with the greatest risk in lung transplant recipients • pediatric solid organ, lung transplant, and heart-lung transplantation recipients appear to have the greatest risk of both rvs infections and more severe complications • rhinovirus and coronaviruses are the most common etiological agents • influenza and other paramyxovirus (rsv, piv, and hmpv) have a greater propensity to produce lrtid diagnosis • diligent collection of respiratory specimens and knowledge of the limitations of the assay used by your laboratory are essential for interpreting the results • nasopharyngeal swabs (nps) are preferred for the detection of all major rvs • bronchoalveolar lavage is the preferred specimen for diagnostic testing in lrtid with negative nps • laboratory diagnostic methods include virus culture, rapid antigen detection tests, the reverse-transcriptase polymerase chain reaction (rt-pcr) and other nucleic acid amplification assays, and serology • nucleic acid amplification tests, mainly rt-pcr, are the best diagnostic tools for studying rvs in sot treatment • treatment options remain limited and consist of supportive care, reduction of immunosuppression and, if available, antiviral therapy • the use of immunomodulatory agents (intravenous immunoglobulin, monoclonal antibodies, rv-specific t lymphocytes or augmented corticosteroid therapy) is a clinical dilemma • sot should receive antiviral therapy with a neuraminidase inhibitor (nai, either oseltamivir or zanamivir) when influenza is suspected, before laboratory confirmation • treatment of influenza with m inhibitors (amantadine and rimantadine) is not recommended for high resistance rates • treatment for influenza with nai should be initiated as soon as possible (< h) for optimal benefit, however and all symptomatic patients should receive antiviral therapy, irrespective of symptom onset • duration of therapy should be minimum of days, but longer duration of therapy (≥ days) may be required in critically ill patients • double dosing of oseltamivir is not recommended but may be considered in severe cases or in case of insufficient response to therapy • the use of intravenous zanamivir or peramivir can be considered in patients not responding to oseltamivir therapy or for whom oral absorption is a concern • in case of high-level oseltamivir resistance, zanamivir is usually active • the efficacy of ribavirin (aerosolized, intravenous, or oral) for the treatment of rsv infection in sot recipients has not been determined • in severe cases of lrtis with rsv, piv, and hmpv infections in sot recipients, therapy with ribavirin (aerosolized, intravenous, or oral) may be used, alone or in combination with an immunomodulatory agents prevention • maximizing prevention infection control measures against rvs in sot is mandatory • sot recipients should avoid close contact with individuals with respiratory tract infections • the main preventive strategy against influenza remains the administration of yearly trivalent inactivated influenza vaccine in all sot recipients and their relatives • influenza vaccination is safe in sot recipients, even in the early posttransplant period • oseltamivir may be used as pre-exposure and postexposure prophylaxis in selected patients • the use of palivizumab for prevention of rsv infection is not recommended in adult sot recipients flu influenza, hmpv human metapneumovirus, lrtid lower respiratory tract infectious disease, nps nasopharyngeal swab, piv parainfluenza, rbv ribavirin, rsv respiratory syncytial virus, rvs respiratory viruses, sot solid organ transplant table . (continued) influenza and other respiratory virus infections in solid organ transplant recipients epidemiology and immediate indirect effects of respiratory viruses in lung transplant recipients: a -year prospective study incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study respiratory viral infections in pediatric solid organ and hematopoietic stem cell transplantation respiratory viruses in lung transplant recipients: a critical review and pooled analysis of clinical studies from the classic concepts to modern practice rna respiratory viruses in solid organ transplantation nasopharyngeal viral pcr in immunosuppressed patients and its association with virus detection in bronchoalveolar lavage by pcr respiratory virus detection in immunocompromised patients with filmarray respiratory panel compared to conventional methods antiviral agents for the treatment and chemoprophylaxis of influenza -recommendations of the advisory committee on immunization practices (acip) current practices for treatment of respiratory syncytial virus and other non-influenza respiratory viruses in high-risk patient populations: a survey of institutions in the midwestern respiratory virus collaborative update in the treatment of non-influenza respiratory virus infection in solid organ transplant recipients vaccination in solid organ transplantation outcomes from pandemic influenza a h n infection in recipients of solid-organ transplants: a multicentre cohort study global update on the susceptibility of human influenza viruses to neuraminidase inhibitors antiviral treatments a prospective intervention study on higher-dose oseltamivir treatment in adults hospitalized with influenza a and b infections a five-year prospective multi-center evaluation of influenza infection in transplant recipients influenza vaccination in solid-organ transplant recipients a comprehensive review of immunization practices in solid organ transplant and hematopoietic stem cell transplant recipients reduced incidence of pneumonia in influenza-vaccinated solid organ transplant recipients with influenza disease rsv prevention and treatment in pediatric lung transplant patients: a survey of current practices among the international pediatric lung transplant collaborative oral ribavirin for respiratory syncytial virus infection after lung transplantation: efficacy and cost-efficiency updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection parainfluenza virus infection in adult lung transplant recipients: an emergent clinical syndrome with implications on allograft function parainfluenza infections early after kidney or simultaneous pancreas-kidney transplantation parainfluenza virus in the hospitalized adult successful outcome of human metapneumovirus (hmpv) pneumonia in a lung transplant recipient treated with intravenous ribavirin key: cord- -qwh ei authors: knopf, harry l.s.; hierholzer, john c. title: clinical and immunologic responses in patients with viral keratoconjunctivitis date: - - journal: am j ophthalmol doi: . / - ( ) - sha: doc_id: cord_uid: qwh ei nan ocular viral infections manifest them selves in a variety of clinical pictures. most commonly, the clinician finds follicular con junctivitis, with or without accompanying keratitis. epithelial and subepithelial corneal infiltrates are frequently observed with adenovrrus mwnons. hut -also occur with hprppg simpw and nfhpr sriraijnduced kerajilis-ia&ctions. ' in addition to keratoconjunctivitis, viruses can (directly or in directly) cause deep ocular involvement, in cluding uveitis and optic nerve inflamma tion. in an effort to learn more about the etiolo gies of ocular viral infections and the pathogenesis of viral keratitis, we studied two groups of patients with epidemic and nonepidemic viral infections. all of them had follicular conjunctivitis, and many suffered from keratitis during at least one stage of their illness. we had three major goals: to determine the rate of viral recovery from cases of nonepidemic acute or subacute follicular conjunc tivitis; to compare the accuracy of clinical diagnoses with that achieved by culture or serologic studies, or both; and to explore the possible role of serum antibody in the forma tion and maintenance of corneal subepithelial infiltrates. we examined one group of patients who were involved in an epidemic caused by a single viral agent, and a second group of patients who were infected with many differ ent viruses. our results indicate that a variety of agents can cause similar illness patterns, and that clinical observation cannot always distinguish one from another. furthermore, serologic tests are a more accurate diagnostic method than clinical course in ocular viral illness, as is the case in systemic viral illnesses. finally, viral anti body titers may correlate directly with spe cific clinical changes in adenovirus-induced keratoconjunctivitis. patients-we studied two groups of pa tients by the following methods. the first was a population of industrial employees who had epidemic keratoconjunctivitis. these patients were described in detail elsewhere, but the data concerning their ocular findings were not presented. the second group of patients comprised a heterogeneous population seen during an -month period. the only criterion for se lection was follicular conjunctivitis. all but nine patients were seen in the emergency room, clinic, or staff offices at barnes hos pital from november to december . the other nine were followed at the national eye institute from july to june . one of us (h.l.s.k.) saw every patient during the acute or subacute phase of the illness and followed them until signs and symptoms abated. there were males and females, ranging in age from to years old (mean, . years). twenty-nine patients were black, were white, and two were orientals. all were ambulatory and in good health. collection of virus culture specimens-fifty-eight of the nonepidemic patients had viral cultures. a cotton swab was rolled in the inferior cul-de-sac and transferred to a carrier medium (sodium chloride, potas sium chloride, dipotassium phosphate, char coal, and agar, ph . ). the contents of this tube were either stored at - °c for a short period or immediately transferred to tissue culture tubes of human diploid fibroblast, primary human embryo kidney cell, or human epidermoid carcinoma. stan dard culture methods have been described previously.* all viruses isolated in this initial attempt were passed and compared to ref erence prototype virus strains for indication of atypicality. the methods for viral culture in the epidemic keratoconjunctivitis patients have been described elsewhere. a single patient was cultured for trachoma inclusion con junctivitis (tric) agent by other meth ods. serologic studies-of the original study patients, did not return for a convalescent serum. therefore these serologic results are based on patients. we collected sera dur ing the acute phase and once or twice during the convalescent phase. there were two family groups in which a specimen from one uninf ected member was considered acute and another infected member's specimen was considered convalescent. we took a con valescent specimen from of the pa tients within days of the initial visit; within days; and three patients had speci mens that were separated by more than days. the sera were tested for antibodies to the prototype strains of many respiratory and other viruses in order to survey all of the likely causes of follicular conjunctivitis (table ) . complement-fixation, hemagglutination-inhibition, indirect hemagglutination, or serum neutralization tests were employed as appropriate for each particular virus. the sera were heat-inactivated ( °c, minutes) for all serologic tests. antibodies to influenza a, b, and c, parainfluenza , , and , mumps virus "viral antigen," rubeola, respiratory syncytial virus (rs v), coronavirus oc- and e, herpes simplex , varicella, mycoplasma pneumoniae, bedsonia (tric agent), cytomegalovirus, and adeno virus group-specific hexon-antigen were as sayed by the standardized complement-fixa tion test with overnight fixation of units of complement. ' antibodies to influenza a and b, parainfluenza , , , a, and b, newcastle disease virus, mumps virus, rubeola, coronavirus oc- , vaccinia, and adenovirus types to were assayed by the standardized hemagglutination-inhibition test with . m phosphate-buffered saline diluent and spectrophotometrically standard ized . % mammalian or . % avian red blood cells. antibodies to coronavirus e were additionally measured by an indirect hemagglutination test. neutralizing anti bodies to rubeola, rs v, selected adenoviruses, the six coxsackie b viruses, echovirus types and , and coxsackievirus a- were assayed by serum neutralization tests in human epidermoid carcinoma or monkey kidney tissue culture.' neutralizing vol. , no. viral keratoconjunctivitis antibody titers to vaccinia, herpes, coxsackievirus a- , a- , and a- , and four strains of acute hemorrhagic conjunctivitis virus were similarly assayed by serum neutraliza tion tests in human diploid fibroblast tissue culture. serum neutralization antibodies in coxsackievirus a- and a- were assayed in suckling mouse brain. for diagnostic purposes a fourfold titer change (either rise or fall) was consid ered significant for any of the serologic tests. in addition a stable titer of > : in any serologic test (hemagglutination-inhibition, serum neutralization, or complement-fixa tion) suggested infection with that agent if the clinical course and other serologic tests were confirmatory. when serologic comparisons were carried out, as with the adenovirus groups described below, all of the testing was done in a stan dardized manner. we used only the hemag glutination-inhibition test, with . m phos phate-buffered saline and standardized mam malian or avian red blood cells, to compare adenovirus types. antigens for types to were maintained at the center for disease control and were used for similar serologic studies in the past. other serologic test re sults did not enter into the calculations for significance of titer levels. we tested all sera simultaneously and in duplicate with the same cell lots whenever possible so that test ing conditions and results would be equiv alent. clinical examinations-those patients who had epidemic keratoconjunctivitis were ex amined during the initial phase of their ill ness and approximately one month after res olution of all symptoms. visual acuity was re corded and, by slit-lamp microscopy, we made a determination of presence or absence of subepithelial infiltrates. there was no at tempt to quantitate these or to judge sever ity. the patients in the nonepidemic group were treated in a different manner. all pa tients were seen at least twice for evaluation of subjective and objective evidence of ocu lar illness. during each examination, we at tempted to document severity of symptoms, visual acuity, the presence of pre-auricular nodes (or other adenopathy), and the appear ance of the conjunctiva, cornea, and anterior chamber by slit-lamp microscopy. detailed drawings of the cornea were made in keratitis cases, but the exact number of lesions was not recorded. instead, we attempted to depict accurately the areas of cornea in volved for future evaluation. each patient was followed until keratitis showed signs of stability or actual regression. when possible, those patients with persistent lesions were followed for a longer time period. at the end of the study period, all of the drawings were re-examined. for each pa tient we estimated the percentage of cornea involved with infiltrates during the peak phase of the illness. we assigned values be fore the serologic data were available. when all of the serologies were complete, the adeno virus groups were investigated separately. we calculated the severity of keratitis vs. sero logic titer by hemagglutination-inhibition test values and the percentage of corneal involve ment arrived at previously. therapeutic regimen-almost all of the patients were treated with topical antibiotics, compresses, or astringents. two of the pa tients in the adenovirus group received « corticosteroids after cessation of the acute phase and when visual acuity decreased. pa tients in the herpes group were given various antiviral preparations. although these were generally effective in controlling the acute disease, we did not attempt statistical corre lations to determine efficacy. ancillary studies-we took swabs for rou tine bacterial cultures in nonepidemic pa tients simultaneously with the viral cultures. although most of these were taken at ran dom, occasionally patients presented with signs and symptoms suggestive of bacterial conjunctivitis complicating the viral illness; these patients were cultured specifically to identify possible infectious agents. in addi tion, conjunctival scrapings were examined american journal of ophthalmology october, s in patients. these were processed with giemsa or gram stains, or both. tear studies-tears were collected from ten patients with acute and chronic, nonepidemic viral infections or chronic noninfectious conjunctivitis. the details for collect ing, processing, and quantitating immunoglobulins in tears have been described pre viously. ' the viral serologic studies on the tear specimens were similar to those done on the sera from these individuals, but the tear specimens were not heat-inactivated and were diluted starting at : instead of : . (other studies involving the epidemic keratoconjunctivitis group have been de scribed previously. ) although more than % of the infected eyes were cultured, the yield of positive viral cul tures was low. table * shows the difference between the number of positive isolates ob tained by culturing before or after the eighth * all of the data for the epidemic keratoconjunc tivitis group were published elsewhere, except for the corneal quantitative material in clinical studies.' day of illness. culturable virus tended to dis appear after the first week of illness in most of the virus groups examined. adenovirus isolates (types , a, , and ) were typical prototype-like strains but three of the adenovirus type a viruses were not; these were neutralized or inhibited less well with prototype reference antisera than with the patient's own convalescent serum. this was not clinically significant. serologic studies (nonepidemic patients)-since positive viral cultures were sparse, the restrospective study of acute and convales cent sera constituted the major means of diag nosis. if cultures were positive or if clinical evidence pointed to a specific diagnosis-the presence of herpetic dendrites-the total bat tery of serologic tests (table ) was not al ways carried out. we compared the clinical and serologic diagnoses in the patients whose sera we examined (table ). all patients with posi tive cultures showed the expected serologic changes. two patients appeared to have ex perienced infections with two agents, either simultaneously or in close temporal proxim ity. in one patient, the hemagglutination-inhibition titer to adenovirus type was stable at : , indicating a recent infection; at the same time, the culture for tric agent was positive. the second patient had an ill ness characterized by initial upper respiratory infection and secondary chronic uniocular keratitis, with comeal ulceration and recur rences. over the three months of observation during the chronic phase, he developed a gradual fourfold rise in adenovirus type hemagglutination-inhibition antibody ( : to : ). almost eight months after his initial illness he demonstrated signs of an terior uveitis after an upper respiratory ill ness (nonspecific) and increased his herpes simplex antibody titer fourfold ( : to : ) in both complement-fixation and serum neutralization tests. these data sup port a premise of double infections in both patients. five additional patients demonstrated sero-vol. , no. viral keratoconjunctivitis logic changes suggestive of either sequential or simultaneous infection with two or three agents. however, in these patients we could not differentiate clinically among the various agents. therefore, the serology that best fitted the clinical picture and time sequence of infection was chosen as the final diagnosis. most of the clinical misdiagnoses were made in the cases of presumed epidemic keratoconjunctivitis. adenovirus type was the most prominent masquerader, and herpes simplex passed for epidemic keratoconjunctivitis in one case. we listed the geometric mean titers for the major adenovirus groups encountered in this study in table . although the average fold-rise in geometric mean titers was simi lar, the final absolute titers were different by more than twofold. this difference was sta tistically significant. in an attempt to explain the higher final titer of the group adenovirus-infected pa tients, we looked at two different factors. the first was a consideration of a possible differ ence in the acute and convalescent phase se rum samples ( table ). the two groups were similar in makeup in that nine of group patients and ten of group were examined during the acute phase of their illness: they showed a fourfold rise in their titer to the infecting adenovirus. there was a moderate disparity between patient history and the serologic data. three group patients (cases , , and ) claimed they were ill for a few days, but their titer reflected a long-term illness (> initial or no change with time, or both). we ob served one patient (case ) with an un changing : titer throughout his illness, and we could not explain the lack of change in titer. this disparity was less evident in group (case ). the difference in acute phase geometric titer for each group was nearly significant . < p < . ). however, when we con sidered only patients proven to be in the acute phase, the difference was not signifi cant ( . vs. . ; p > . ). there was a difference in the number of days between acute and convalescent samples for the two groups. overall, the mean num ber of days was for group and for group (not significant). however, if only the patients in the acute phase were again considered, the averages were days and days, respectively (p <. ; t = . ; df, ). (from these data, a higher titer should have been expected for group be cause of the longer interval between acute phase and serum peak.) thus, neither the titer of acute phase sera nor the sampling time of convalescent phase sera seemed to ac count for the difference in final antibody levels. we also considered the degree of upper respiratory or generalized illness among pa tients in both groups. of the patients with serologic evidence of group infection, only gave accurate histories; nine of these reported an associated upper respiratory in fection with conjunctivitis. in contrast, one (with adenovirus type ) of the patients in group exhibited signs or symptoms of upper respiratory infection. this differ ence was again significant (p < . , \ test). thus, those with group nonepidemic adenovirus infection were afflicted with more upper respiratory infections, but these in- fections were not necessarily responsible for the significantly higher serologic titers. in fact, there was no significant relationship be tween the presence of an upper respiratory infection and a serologic response that equaled or exceeded : (table ). this negative correlation held true for changes in both homologous and heterologous virus titers. therefore, the explanation for the higher serum titers in patients with group infections may not be related directly to the concomitant upper respiratory infections. we compared the sensitivity of the two most commonly employed serologic tests (table ) . hemagglutination-inhibition tests demonstrated fourfold rises in many cases where complement-fixation tests failed to show significant changes. hence, primary de pendence on the usual complement-fixation hexon-antigen test would have resulted in fewer positive diagnoses, even in acute infec tions. ancillary laboratory studies (nonepidemic patients)-we microscopically examined conjunctival smears from patients, and routine bacterial cultures were taken from of these patients plus ten other individuals. cultures from five patients showed coagulasepositive staphylococci; these patients had various diagnoses: adenovirus type epi demic keratoconjunctivitis, adenovirus type pharyngoconjunctival fever, herpes sim plex keratitis, and a case of chronic "fac titious" conjunctivitis. the smears were helpful only in cases of herpetic infections. in three patients with herpes simplex and one with herpes zoster, twenty-eight patients showed signs of keratitis during their illness and had chronic subepithelial infiltrates when examined one month after resolution of their illness ( of these experienced some degree of decreased visual acuity as well). the remaining patients, who were also infected during the first wave of the epidemic were seen at the same time and had no subepithelial infiltrates. the peak geometric mean hemagglutination-inhibition titer of the for mer group with residual infiltrates was : . (range : to : ). the data for the un affected group were : . geometric mean titer (range : to : ). this difference was significant (p < . ). (the collection of sera and intervals between examinations were similar for patients because they were part of an epidemic group. the serologic tests were exactly the same as those carried out in the nonepidemic group, and often were done simultaneously t estimated maximum percent of cornea with epithelial or subepithelial infiltrates. * titers did not change significantly (> fourfold) during the observation period (compare with table ) analysis of corneal disease. group was composed of five patients with adenovirus type and patients with adenovirus type infections. group contained patients with adenovirus type and one each with adenovirus type and adenovirus . the data concerning the corneal involve ment and antibody levels of both groups were compiled (table , compare with table ). analysis by x testing showed several sig nificant correlations. first, the number of eyes involved with corneal changes differed significantly in the two groups. in group , there were five patients with no involve ment, seven with one eye, and four with both eyes affected. group had only one patient without corneal changes, three pa tients with a single eye involved, and nine patients with both eyes involved (x = . ; df, ; ps . ). when this same factor vol. , no. (number of eyes) was used, and the antibody levels were compared for all adenovirus infections (groups and ) , a more signifi cant difference was noted. at a level of : , there were doubly involved patients, four with one eye, and three with no corneal in volvement. at a level of : , only one pa tient showed infiltrates in both eyes, six had single eye involvement, and three had no ocu lar involvement. the difference between the number of patients in these three categories proved to be statistically significant (x = . ;df, ;p<. ). correlations of the degree of corneal in volvement with antibody level were also car ried out. there was no difference between the two groups in the number of patients with central lesions vs. peripheral lesions of the cornea. this was reflected in the patients with decreased visual acuity (table ) . we devised a comparison of the two groups in terms of fold-rise in antibody titer (fig ure) . (the fold-rise was a reflection of the rate of antibody production as well as the amount of antibody.) as evidenced by the graph, the responses of the two groups were almost diametrically opposed. for each foldrise in titer, the group patients had pro gressively less corneal involvement. for the same change in titer, the group patients showed progressively more corneal infiltra tion. however, the final level of antibody achieved was lower in group (compare with table ). a similar difference in the two groups was noted at the % level of corneal infiltration. only two patients (of ) in group had infiltrates that involved > % of the corneal surface area. in group , six of patients were affected in the same way ( x = . ; df, ; p<. ). finally, age was not a factor differentiat ing the two groups. the average age of group patients was . years; group patients averaged . years. the difference was not statistically significant (t = . ; df, ). a comparison of the percent corneal involvement with fold-rise in antibody titer among patients infected with group (av ) and (av ) adenoviruses. r represents the correlation coefficient of the least-squares line calculated to fit the data. only patients with at least a fourfold rise are included. group)-rises in titers to heterologous vi ruses were observed in groups and infec tions. thus, in patients infected with adeno virus type (group ), a significant change in titer to this agent occurred, as well as smaller changes to such related types as , , and (table ). however, rises in heterologous titers were more consistently seen in patients with group infections. for example, adenovirus type infections produced rises to related viral types in the group series. this was reflected in the higher heterologous average titers (table ). in particular, we noted a tendency for heterologous responders within family groups. in one of these families, all of the blood relatives showed changes in titer to several other group subtypes after adeno virus type infection. however, the spouse of one member, exposed to the same virus, had a serologic change to adenovirus type alone (homologous responder). american journal of ophthalmology october, we observed patients with keratoconjunctivitis from other causes during this study (table ) , but only adenovirus patients formed homogenous groups that lent them selves to statistical analysis. the herpes group was large (eight herpes simplex, two herpes zoster, two possible mixed herpes simplex-zoster, and three presumed cytomegalovirus), but heterogenous in terms of acute and chronic illness, treatment with var ious antiviral drugs or corticosteroids, and poor serologic responses. variability in his tory, follow-up, and other factors occurred among the patients with adenovirus so the number of patients who met the criteria for various analyses often differed from one test to another. in terms of treatment, however, almost all of the patients with adenovirus required only therapy with topical vasocon strictors or antibiotics, or both. only two patients (one each from groups and ) required corticosteroids for symptomatic re lief, and this was instituted after achieving maximum corneal scores. analysis of tears-ocular external secre tions (tears) were analyzed in ten patients. in general, there were low antibody titers. four patients with serologic diagnoses of herpetic infection represented chronic, re current keratoconjunctivitis or kerato-uveitis. only one patient with disci form keratitis and uveitis had detectable tear-borne neutralizing antibody to herpes simplex. his serum titer, however, was not greater than : in a simul taneous neutralization test. one other patient showed tear antibody to herpes zoster and se rologic changes consistent with simplex. whether this represented true simultaneous infection or the presence of cross reaction was not determined. two patients had high titers ( : , : ) to coronavirus oc- in their external secretions. upper respiratory infection has produced antibody in tears and this may represent that kind of relationship. the predominant immunoglobulin species in the tears was iga, in agreement with pre vious work. ' however, we made no at tempt to prove that the measurable hemagglutination-inhibition or serum neutralization antibodies were unique to iga. discussion because all patients in the second part of this study were seen during a nonepidemic time period, the cases of keratitis were varied in their etiologies (table ). this observa tion agrees with previous studies that de scribed a similar variety of viral agents re sponsible for epidemic keratoconjunctivitis keratitis. " the behavior of the individual viral infec tions also conformed to expected patterns. thus, group adenoviruses caused more upper respiratory and generalized illness, but less keratitis, than group infections. ' - [ ] [ ] [ ] herpes viruses caused more severe keratitis and kerato-uveitis, but clinical illnesses cor related poorly with serologic changes in anti body titer. ' we also encountered difficulty in culturing any virus after seven to ten days of symptoms. this has been reported before for epidemic keratoconjunctivitis, but it is probably true for many other viral syndromes. , - we, like others, also found the hemagglutination-inhibition test to be supe rior to complement-fixation as a serum diag nostic procedure in adenovirus infection. no procedure gave consistent results with tears. there were differences in the extent of corneal involvement observed in group and infections. the correlations indicate that group viruses produced a lower level of specific antibody titer (despite a longer time interval between the collection of acute and convalescent sera), and induced more corneal disease in the affected indi viduals. these data suggest that the higher anti body titer in group protected the cornea, supported by examination of the keratocon junctivitis epidemic of adenovirus type , a group virus. in the latter patients, a range of antibody was observed that was somewhat higher than that of the nonepidemic group . (the reason for this difference is not readily apparent, but does not lie in testing differences, since both sets of sera were ex amined under identical conditions and often simultaneously.) what is more important, however, is the range of corneal involve ment with this single viral agent. once again, the patients with higher antibody titer ex perienced less corneal involvement with this usually virulent agent. ' the corneal infiltrates may only represent a pattern of infection and the antibody re sponse to it. certainly more than one viral type can cause this pattern, as shown in the nonepidemic group. also, the same virus can cause different degrees of involvement (epi demic study), perhaps depending on the level of antibody illicited by the infection. since delayed hypersensitivity was not spe cifically assayed, the elevated humoral anti body in epidemic group or nonepidemic group patients may reflect a higher degree oi overall immunity, and cellular immunity may be the chief deterrent of corneal dis ease. however, systemic antibody alone may protect the cornea (and even the fellow eye) from severe involvement. whether it was humoral or cellular, or both, the nonepidemic and epidemic groups of patients displayed a reasonably similar pattern of protection that correlated with high antibody titer. we only speculate about the pathogenesis of the subepithelial infiltrates. in studies of immune-complex disease, for instance, rheu matoid arthritis and aleutian mink disease, antigen-antibody complexes in blood vessel walls can attract complement and inflamma tory cells that cause the damage. ' these complexes are made to persist by maintain ing a low titer antibody, and the disease may be completely eliminated by raising the titer. the cornea is similar to the collagenous tis sue in the walls of blood vessels, and these tissues can absorb certain antigens. ' therefore, it seems feasible that viral par ticles might be bound to the stromal collagen, form complexes with low levels of antibody diffusing within the cornea, and thereby act as a nidus for inflammation. the subsequent fixation of complement and attraction of in flammatory cells completes the subepithelial infiltrate seen by slit-lamp microscopy. corticosteroids would disrupt the inflammatory response, but it would not remove the com plexes. hence, discontinuation of the drug would allow inflammatory cells to reappear. a similar set of circumstances was proposed in the pathogenesis of stromal disease in herpes simplex keratitis. based on these suppositions, one may con clude that the sometimes permanent visual damage observed in epidemic keratoconjunctivitis might be decreased or eliminated by enhancing the immune response of the in fected patient, perhaps during the acute in fection. of individuals suffering from follicular conjunctivitis, with or without keratitis, had epidemic keratoconiunctivitis caused by adenovirus type . the remaining pa tients had keratoconjunctivitis produced by several different viruses, including herpes simplex, adenoviruses types , , , , , and , and others. positive viral cultures were difficult to obtain after the first week of illness in most viral infections. serologic tests were far more successful in identifying causative agents. hemagglutination-inhibition tests in adenovirus infections proved to be fast, accurate, and more sensitive than other serologic tests. among the epidemic and nonepidemic adenovirus groups, the de gree of corneal involvement in the infections appeared to be correlated with the level anti body against the infectious agent. patients with low antibody titers had more severe keratitis than those with high titers. this correlation was true for both the epidemic and nonepidemic patients. microbiology of the eye adenovirus type keratoconjunctivitis in the united states. . epidemiologic, clini cal and microbiological features epidemic keratoconjunctivitis. a severe industrial outbreak of adenovirus type adenoviruses simplified irradiated mccoy cell culture for iso lation of chlamydia standardized diagnostic com plement fixation method and adaptation to micro test, no. standardized viral hemagglutination and hemagglutination-inhibition tests. . description and statistical evaluation detection of coronavirus e antibody by indirect hemagglutination antibody in tears following intranasal vaccination with in activated virus. . enhancement of tear antibody production by the use of polyinosinic: polycytidilic acid (poly i:c) epidemic keratoconjunctivitis on taiwan. etiological and clinical studies adenovirus type infections in the united states adenovirus infection of the conjunctiva and cornea association of adenovirus type with epidemic keratoconjunctivitis diagnosis of adenovirus infections by use of fluorescent antibody technique corneal im mune response to herpes simplex virus antigens the pathogenesis of chronic inflam mation in experimental antigen induced arthritis. . preferential binding of antigen antibody complexes to collagenous tissues the patho genesis of corneal inflammation due to herpes sim plex virus key: cord- -n sih authors: villard, viviane; agak, george w.; frank, géraldine; jafarshad, ali; servis, catherine; nébié, issa; sirima, sodiomon b.; felger, ingrid; arevalo-herrera, myriam; herrera, socrates; heitz, frederic; bäcker, volker; druilhe, pierre; kajava, andrey v.; corradin, giampietro title: rapid identification of malaria vaccine candidates based on α-helical coiled coil protein motif date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: n sih to identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. the corresponding synthetic peptides are expected to mimic structurally “native” epitopes. indeed the chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. these antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. this strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens. human plasmodium falciparum (pf) infection is a dramatic public health problem. today approximately forty percent of the world's population is at risk of malaria. malaria causes more than million acute clinical cases, and at least one million deaths annually. ninety percent of malaria deaths occur in sub-saharan african countries mostly among young children and pregnant women (http://rbm.who.int). thus, there is an urgent need of a malaria vaccine. however, vaccine discovery, in general and particularly in malaria, is still a very empirical process. in fact, protective antigens do not bear any structural, physico-chemical or sequence-related characteristics that would allow their identification. for protective humoral responses, the only recognized characteristics of antigens are their antigenicity/immunogenicity and accessibility. in addition, the lack of surrogate markers of protection renders the vaccine discovery process difficult and time consuming. hence, in spite of a constant and impressive progress in molecular biology techniques and antigen identification and expression [ , ] , vaccine discovery is still labor-intensive, making the approach fastidious, costly and poorly adapted to highthroughput screening. proper protein folding and solubility remain a limitation in numerous cases. thus, overcoming the bottlenecks of manufacturing and identification of fragments/proteins, as possible targets of a protective immune response still constitute a scientific and technical challenge. we addressed this challenge by combining bioinformatics, chemical peptide synthesis and functional protection assays. we focused on the search for a-helical coiled coil motifs that, in general, do not exhibit a folding problem, and are a target of effective antibodies for several current malaria vaccine candidates (eg. lsa- [ ] , lsa- [ ] , msp- [ ] , and msp- [ ] and other pathogens [ ] . msp- , another leading malaria vaccine [ ] , also contains predicted a-helical coiled coil regions (unpublished results). our choice was based on the following considerations. first, the a-helical coiled coil motif bears a characteristic seven amino acid residue repeat (abcdefg) n with hydrophobic residues located in a and d positions and hydrophilic residues generally elsewhere. this motif can be easily identified by bioinformatic analysis. secondly, an important known characteristic of the ahelical coiled coil domains is that, taken separately from the whole protein, they frequently and readily fold into the same stable oligomeric structure [ ] . thirdly, for this reason, the a-helical coiled coil fragments are frequently recognized by conformational dependent antibodies, and can similarly elicit antibodies reactive with structurally ''native'' epitopes. in addition, these domains are short (about residues) and can be rapidly produced by chemical synthesis. furthermore, when the antibodies have an anti-parasite biological activity, this designates the corresponding proteins/ fragments as potential, novel vaccine candidates to be further developed and assessed. here, we focus on the pf parasite erythrocytic stage, a target of protective antibodies and describe a straightforward, rapid procedure based on bioinformatic analysis of a-helical coiled-coil motifs and peptide synthesis. the screening of the pf genome [ ] using generalized sequence profiles [ ] identified several hundred proteins containing putative a-helical coiled coil motifs. through proteome and transcriptome data [ ] [ ] [ ] we assessed which of these molecules are expressed in the pf parasite erythrocytic stage. the combined analysis/assessment identified over segments associated with this stage and displaying the putative a-helical coiled coil motifs with high probability score (table s ) . out of these a-helical coiled coil fragments, in general - amino acids long, present either in the same protein or in different ones, were chemically synthesized and hplc purified. among them, longer peptides (up to amino acids), which contained one or more a-helical coiled coil domains, were also synthesized (antigens , and ; table s ). the selected antigens were then tested in elisa assays for reactivity with three panels of sera obtained from adult donors from burkina faso, tanzania and colombia, respectively. to our surprise, all of the a-helical coiled coil fragments were antigenic, though the prevalence of responders varied greatly (tables and s ). in this manner, proteins were identified whose lengths varied from to , amino acids. twenty-one peptides with the highest prevalence of responders and elisa mean od value were selected for further studies. variation in recognition among the three panels of sera may be due to differences in the genetic background of the hosts, of the parasites and, most likely, to distinct malaria transmission conditions in the three regions. the high level of recognition of the a-helical coiled coil motifs may be explained by the fact that taken separately from the whole protein these fragments readily fold into the same stable structure in aqueous solution. indeed, circular dichroism (cd) studies of selected peptides associated with biological activities (tables and ) indicate that they predominantly assume an a-helical conformation in water. peptides , and ( figure s a ) exhibit a cd pattern characteristic of a high a-helical content, whereas the remaining peptides show cd profiles similar to that shown for peptide ( figure s b ) or intermediate between those shown in figures s a and s b characteristic of a partial a-helical organization. when analyzed by size exclusion chromatography on fplc columns, peptides presented elution profiles between those exhibited by chymotrypsin and ribonuclease (mw and kda, respectively). the cd and size exclusion chromatography results suggest that peptides adopt an a-helical coiled-coil structure, which need to be unambiguously ascertained by nmr and ultra-centrifugation studies. to test the biological activity of peptide-specific antibodies, the latter were purified by affinity chromatography using three serum pools obtained from papua new guinean adults. the serum pools were first tested in elisa assays against peptides that were the most antigenic (table ) ; from these, peptide-specific antibodies were purified from the most positive serum pool and tested again in elisa. these antibodies all reacted with parasite native proteins in infected red blood cells as shown by ifat ( figure a ; table ). reactivity was restricted to blood stages, since the antibodies did not react with sporozoites stages (data not shown), and this reactivity was also peptide-specific as shown by ifat competition assays with the corresponding peptide ( figure a ). the specificity of the antibodies obtained was investigated in detail, particularly since several peptides contain glutamic acid (glu)-rich sequences which are known to generate cross reactivity among several malarial glu-rich proteins [ ] . cross-reactions were systematically investigated using each of the affinitypurified antibodies on each of the peptides. results show thatwith few exceptions-each antibody preferentially recognizes the peptide against which antibodies were affinity-purified, i.e. they are specific for the corresponding peptide (table s ) . to determine if non-specific antibody binding to solid phase-adsorbed antigens could be responsible for the rare cross-reactivities detected, elisa competition assays were performed. to this end, binding of antibodies to the solid phase-adsorbed antigen was competed against increasing concentrations of the homologous or cross-reacting peptides. only homologous peptides competed best whereas peptides having sequence similarity did not (figures a, b , and s b), or at a much higher concentration ( figure s a ) including the shorter glu-rich peptides derived from the cterminus of peptide and (figures a and b) . finally, the pattern of recognition of peptides by the various sera tested, which differ markedly from one to the other (data not shown), confirms the above results i.e., specificity of antibodies to the corresponding peptide. antibodies corresponding to the selected peptides were tested for direct and cell-mediated anti-parasite activity. clinical experiments have shown that the antibody-dependent cellmediated inhibition (adci) of p. falciparum malaria represents one of the mechanisms controlling parasitemia and thereby clinical manifestations in humans [ ] . twelve peptide-specific antibodies proved able to induce a strong (more than %) and intermediate (lower than %) monocyte-dependent parasite killing (table ) , whereas, in the absence of monocytes, no direct effect of antibodies on parasite growth was observed. the effects were in the range observed with antibodies from african adults who have the highest natural protection known against malaria. therefore peptidespecific, human affinity-purified antibodies were functionally effective as shown by their ability to react with parasite proteins and to inhibit parasite growth. thus, in vitro functional assays show that peptide-specific antibodies elicited by natural exposure to the parasite can induce protective mechanisms effective against malaria. sixteen peptides -twelve targeted by adci positive antibodies and four controls-were used to immunize cb f mice ( table ) . eleven of them elicited an intermediate or high antibody response, four of which also recognized the parasite protein in infected erythrocytes as determined by ifat ( figure b ; table ). as seen before for human antibodies, recognition was restricted to blood stages since sporozoites were negative in ifat assays (data not shown) and by ifat competition assays with the corresponding peptide ( figure b ). anti-peptide mouse antibodies, which are positive in ifat, are also specific for the homologous peptide but not for the sequence related peptides , and (table s ) . thus, peptides, which were chosen for their propensity to form ahelical coiled coil, can induce the production of antibodies that recognize epitopes present in the native protein. improvement of the immunogenicity and structural specificity of the remaining peptides might be achieved in the future by a) a short elongation at the n-and c-terminal ends, b) stabilizing the a-helix as suggested by cooper et al. and lu and hodges [ , ] and/or c) use of other adjuvants. genetic polymorphism in current vaccine candidates is a major limitation to vaccine development. however available genotyping studies of our peptide sequences in parasite isolates of worldwide origin indicate very limited polymorphism (plasmodb . , and unpublished sequencing data). a few peptide dna sequences show deletion of one entire heptad repeat so that the shorter region still preserves its potential for the a-helical coiled coil formation. with regard to the structural features and cellular location prediction of the proteins corresponding to the peptides selected for adci assays ( table ) , of the proteins contain a pentapeptide conforming to the pexel consensus [ , ; , ] , but that none of these have a position within the amino acid sequence that conforms to the location of known active pexel motifs (see materials and methods and membrane segments, and none of them has a gpi anchor. only one protein contains a signal sequence. fourteen proteins are predicted to be in the cytoplasm, one in the nucleus, one in the mitochondria, and one in the peroxysomes (table s ). the prediction of the sub-cellular localization of these proteins should be taken with caution because gene annotation is being constantly updated and/or protein trafficking of the parasite is complex and not fully elucidated [ ] . further investigations will be required to determine the actual localization of the corresponding antigens. the predicted localization is a priori surprising for molecules able to trigger an adci activity. however, recent studies have shown that in addition to merozoite surface proteins, soluble proteins released at the time of schizont rupture were equally effective at triggering adci provided they defined at least two epitopes [ ] , which is the case for a-helical coiled coil heptad repeats. therefore, molecules expressing a trans-membrane domain that can be exported to the parasite or host cell membrane, as well as molecules present in the cytoplasm of maturing schizonts and released by bursting schizonts can trigger antibodies to cross-link fc-c receptors on monocytes to achieve pf parasite killing. in conclusion, an approach combining a genome-wide search by bioinformatics of a-helical coiled coil protein motifs and chemical synthesis can lead to the rapid identification and development of new malaria vaccine candidates. in fact, this approach is straightforward and easy to scale up; vaccine formulations may comprise mixtures of peptides or single constructs made up of several epitopes. in principle, this strategy can be extended to the discovery of proteins and vaccine candidates in other complex pathogens. the pf d genome [ ] was used for the bioinformatics analysis. the generalized sequence profile method and the pftools package [ ] were used to search for the short a-helical coiled coil domains. the coiled coil profiles were constructed using an alignment of several amino acid sequences corresponding to the known coiled coil domain. two profiles containing four and five heptad repeats were used for the analysis. the cut-off levels of the profiles were chosen by tests performed against sequence database of proteins with the known d structures. subsequently, the coiled coil figure . immunofluorescence microscopy analysis of pf d parasites with peptide specific antibodies. acetone/methanol-fixed schizonts and merozoites were reacted with a: human peptide specific, affinity purified antibodies obtained with peptides and (table ) and b: sera from mice immunized with peptide (table ) . grey: bright field images; blue staining: indicates dapi nuclear staining of schizont stage parasites; red staining shows labeling of peptide specific antibodies by cy -conjugated anti-human or anti-mouse igg specific antibody. merge picture is an overlay of the blue and red fluorescence channel. doi: . /journal.pone. .g regions selected by this approach were tested manually for the presence of the characteristic heptad repeats. these proteins were also analyzed by the coils program [ ] . the selected a-helical coiled coil containing proteins were further tested on their possible surface location and gpi anchoring by using the following programs: identification of potential signal peptides, secretomep and signalp (http://www. cbs.dtu.dk/services/) [ ] ; transmembrane spanning regions (tmpred http://www.ch.embnet.org/software/tmpred_ form.html and tmhmm http://www.cbs.dtu.dk/services/ tmhmm; [ , ] ), and gpi-anchored proteins (http://mendel. imp.univie.ac.at/sat/gpi/gpi_server.html; [ ] ) and prediction of sub-cellular localization (ptarget http://bioinformatics. albany.edu/,ptarget; [ ] ). to identify the pexel-like motifs in sequences of the selected proteins we used the following pattern [ [deq] that represents a combination of the pexel patterns indicated in recent papers [ , ] . the presence of the identified proteins in the asexual erythrocytic stages was also checked using the published data on the transcriptome and proteome of this stage of development of p. falciparum (www.plasmodb.org; [ ] ). peptides were synthesized on the advanced chemtech (hatley st george, uk) ac t omega multi channel synthesizer and the applied biosystem synthesizer a and a (foster city, ca) using solid-phase fmoc chemistry. crude peptides were purified by rp-hplc (c preparative column) and analyzed by mass spectrometry (maldi-tof; applied biosystem, foster city, ca). chemicals and solvents used for peptide synthesis were purchased from fluka (buchs, switzerland) and novabiochem (laufelfinger, switzerland). circular dichroism (cd) spectra of peptides were recorded on a jasco j- spectrometer (jasco corporation, tokyo, japan) equipped with a temperature controller and a . cm path length cuvette. the measurements were made in water at ph . and uc and at a peptide concentration of . mg/ml. the sera from burkina faso were collected in the village of goundry located in the central mossi plateau, between and km north of the capital ouagadougou, in the province of oubritenga. the climate is characteristic of areas of sudanese savannah, with a dry season from november to may and a rainy season from june to october. malaria transmission is very high during the rainy season and markedly seasonal. ethical clearance was obtained from the ministry of health, burkina faso. after obtaining informed consent from parents and caretakers, heparinized venous blood samples were collected during a crosssectional survey during the malaria low transmission season . the tanzanian sera came from a large-scale community based study undertaken in kikwalila village, kilombero district, morogoro region from to . blood samples from adults (. years) were taken by finger prick and the serum was kept at - uc until use. research and ethical clearance for the study was obtained by the tanzanian commission for science & technology. the colombian sera were collected in buenaventura the main port on the colombian pacific coast after human informed consent, during a cross sectional survey carried out from february to may within the framework of a project supported by the colombian research council, colciencias. the area has unstable transmission of both p. falciparum and p. vivax malaria. ethical clearance to draw blood from human volunteers was human purified antibodies were used at mg/ml; ifat was not performed on ring stages. valle. blood was taken by venipuncture into tubes containing edta and sera fractionated and stored frozen until use. the sera from adults from papua new guinea (png) pooled for affinity purification were collected in the maprik district of the east sepik province, during a cross sectional survey in july within the framework of the malaria vaccine epidemiology and evaluation project (mveep) supported by the united states agency for international development [ ] . the area is highly endemic for malaria. ethical clearance for mveep was obtained from the png medical research advisory committee. blood was taken by venipuncture into tubes containing edta. the pool of immune african globulins (piag) used for adci was prepared from immune individuals living in endemic areas and negative control igg (n-igg) was obtained from a pool of more than french adult donors with no history of malaria. briefly, the igg fractions from both positive and negative controls were purified using a size exclusion trisacrylh gf m (pall bioseprah; pall life sciences, ny) column followed by an ionic exchange deae ceramic hyperdh f column (pall bioseprah). purified igg were then extensively dialyzed against rpmi and kept at uc until use. cb f mice were injected times with mg of the indicated peptide in montanide isa at the base of the tail on day , and . bleeding was performed days after the second and third immunization. elisa was performed according to lopez et al. [ ] and anti human igg-or anti mouse igg conjugated to alkaline phosphatase was used (sigma, st louis, mo) as second antibody. individual human sera from , and adults donors from burkina faso, tanzania and colombia respectively were used at : dilution. serum was considered positive if the optical density (od) reading was higher than the mean od value+ standard deviation (sd) of the negative controls (individual serum samples from to naïve swiss donors) or if the od ratio of the mean of duplicate experimental values to the mean od of the negative control was higher than . for mouse sera, the end point value was determined as the last dilution of the mean od value+ standard deviation (sd) of the negative control (non immune sera). elisa competition assays were performed by incubating either each of the selected human affinity-purified antibodies, or antibodies elicited in mice, together with each of the antigens over the indicated range of concentrations for minutes at room temperature prior to addition to the elisa peptide-coated plate wells. antigen-sepharose conjugate preparation: mg of antigen was dissolved in ml of coupling buffer ( . m nahco containing . m nacl, ph . ). the cnbr-sepharose b (amersham bioscience ab, uppsala, sweden) was activated by swelling in mm hcl and then washed with coupling buffer. the antigen solution was added to the gel and the mixture was stirred for h at rt. after the coupling reaction, excess antigen was washed away with coupling buffer. the unreacted activated groups were blocked by treatment with ethanolamine ( . m; ph . ) for min at rt. the gel was then washed with sodium acetate buffer ( . m; ph . ), followed by coupling buffer. the antigensepharose beads were either used or stored at uc in pbs ( x) containing mm azide. isolation of specific antibody: pooled human serum was diluted five times with pbs ( x) containing . m sodium chloride and mixed with antigen-sepharose conjugate. this mixture was then stirred gently on a wheel o/n at uc. after centrifugation the supernatant was collected and stored at uc for further use. the antigen-sepharose beads were then washed with ml of trizma base tris ( mm containing . m nacl, ph . ) then with ml of tris ( mm, ph . ). the elution of bound antibody was achieved with glycine ( . m, ph . ). the fractions obtained were instantly neutralized with tris ( m, ph . ), dialyzed against phosphate buffer ( . m, ph . ) and the antibody concentration was determined by the absorbance of the solution at nm. slides coated with pf sporozoites were dried at rt for minutes, fixed with % acetone at uc for minutes, washed times in pbs- . % tween , dried carefully and blocked with ml/ well of pbs- % bovine serum albumin (bsa) for minutes at rt. slides coated with pf merozoites were fixed with % acetone at uc for minutes and dried o/n at rt. the appropriate antibody or serum dilutions prepared in pbs- % bsa were distributed ( ml/well) and incubated for h at rt in a humid chamber. after washing with pbs- . % tween- , goat anti-human or goat anti-mouse polyvalent immunoglobulins conjugated to cy (molecular probes) diluted / in pbs- % bsa or anti-human igg (fc specific) fitc conjugate (sigma) diluted / in evans blue solution ( / ) was added ( ml/slide) and incubated for h at rt in a humid chamber in the dark. slides were washed as above, covered with % glycerol, sealed and read using a fluorescence microscope (leica dmirb dc ). the uganda palo alto strain (fup/c) was cultured in rpmi- supplemented with . % albumax i (gibcobrl-invitrogen, san diego, ca). for adci assays, blood stage parasite cultures were synchronized by at least two successive sorbitol treatments followed, after maturation over h, by floatation on % porcine skin gelatin type a (sigma). blood monocytes (mn) were prepared from cytapheresis samples obtained from healthy blood donors with no previous history of malaria (lecourbe blood bank, paris, france). peripheral blood mononuclear cells (pbmc) were separated on ficoll density gradients j prep (techgen, les ulis, france) and washed in ca + and mg + free hbss buffered with mm hepes (both from gibcobrl-invitrogen). cells were then distributed on polystyrene -well flat-bottomed culture plates (tpp, trasadingen, switzerland) and adherent mn were selected by incubation for h at uc, in a humidified % co atmosphere. more than % of the adherent cells obtained in this manner were mn as estimated by the non-specific esterase test (a-naphtyl acetate esterase; sigma). mn from each donor were tested prior to adci assays and only those without direct inhibitory effect were used in assays. to wells containing mn purified as described above, ml of an asynchronous parasite culture at . % parasitemia and % hematocrit were added. wells were then supplemented with test or control antibodies (ab) and the total volume adjusted to ml with culture medium. after h and h, ml of culture medium were added to each well and after h the adci assay was stopped and the final parasitemia was determined by light microscopy on giemsa-stained smears by counting $ , red blood cells. for each ab tested, duplicate wells included the following controls ) non-specific monocytic inhibition, both mn+parasite, and mn+n-igg+parasites and ) direct inhibition by control or test igg, both n-igg+parasites, and test abs+parasites. piag and n-igg were used at a final concentration of mg/ml as positive and negative controls respectively. immunopurified tests abs were used at mg/ml. the specific growth inhibitory index (sgi) which considers the parasite growth inhibition due to the effect of test abs cooperating with mn was calculated as follows: sgi = [ (% parasitemia with mn and test abs/% parasitemia test abs)/(% parasitemia with mn and n-igg/% parasitemia n-igg)]. figure s cd spectra of the peptides (s a) and (s b) found at: doi: . /journal.pone. .s ( . mb tif) figure s elisa inhibition assay using anti-human peptide specific antibodies. binding of peptide specific antibodies to peptides (s a) and (s b) absorbed on elisa plates was inhibited by incubating specific antibodies ( - mg/ml) with peptides , and (s a) and peptides and (s b), respectively (see material and methods). peptides , and share nnm or mnn as sequence similarity while peptides and do not exhibit any apparent sequence similarity. table s structural feature and cellular location prediction of the proteins containing the peptides whose specific antibodies were tested in adci (table ) . found at: doi: . /journal.pone. .s ( . mb doc) identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing reverse vaccinology and genomics plasmodium falciparum liver stage antigen- is well conserved and contains potent b and t cell determinants protection against plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen phase i malaria vaccine trial with a long synthetic peptide derived from the merozoite surface protein antigen plasmodium falciparum merozoite surface protein displays multiple targets for naturally occurring antibodies that mediate monocyte-dependent parasite killing template-based coiled-coil antigens elicit neutralizing antibodies to the sars-coronavirus phase randomized double-blind safety and immunogenicity trial of plasmodium falciparum malaria merozoite surface protein fmp vaccine de novo design of alpha-helical proteins: basic research to medical applications genome sequence of the human malaria parasite plasmodium falciparum a flexible motif search technique based on generalized profiles transcriptomics and proteomics: tools for the identification of novel drug targets and vaccine candidates for tuberculosis a proteomic view of the plasmodium falciparum life cycle the transcriptome of the intraerythrocytic developmental cycle of plasmodium falciparum crossreactive antigens between life cycle stages of plasmodium falciparum antibodies that protect humans against plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes mapping of conformational b cell epitopes within alpha-helical coiled coil proteins a de novo designed template for generating conformation-specific antibodies that recognize alpha-helices in proteins targeting malaria virulence and remodeling proteins to the host erythrocyte a host-targeting signal in virulence proteins reveals a secretome in malarial infection proteomic analysis identifies novel proteins of the maurer's clefts, a secretory compartment delivering plasmodium falciparum proteins to the surface of its host cell multi-character population study of the vir subtelomeric multigene superfamily of plasmodium vivax, a major human malaria parasite a maurer's cleft-associated protein is essential for expression of the major malaria virulence antigen on the surface of infected red blood cells a novel antibody-dependent cellular cytotoxicity mechanism involved in defense against malaria requires costimulation of monocytes fcgammarii and fcgammariii predicting coiled coils from protein sequences improved prediction of signal peptides: signalp . tmbase-a database of membrane spanning proteins segments predicting transmembrane protein topology with a hidden markov model: application to complete genomes prediction of potential gpimodification sites in proprotein sequences ptarget [corrected] a new method for predicting protein subcellular localization in eukaryotes plasmodb: the plasmodium genome resource. a database integrating experimental and computational data the malaria vaccine epidemiology and evaluation project of papua new guinea: rationale and baseline studies a synthetic malaria vaccine elicits a potent cd (+) and cd (+) t lymphocyte immune response in humans. implications for vaccination strategies the authors wish to thank luis rodrigues and florela penea for the synthesis and purification of peptides and thomas smith for discussion and all of the blood donors. key: cord- -iaktm a authors: soto-quintero, albanelly; guarrotxena, nekane; garcía, olga; quijada-garrido, isabel title: curcumin to promote the synthesis of silver nps and their self-assembly with a thermoresponsive polymer in core-shell nanohybrids date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: iaktm a this work presents a simple one-pot protocol to achieve core-doped shell nanohybrids comprising silver nanoparticles, curcumin and thermoresponsive polymeric shell taking advantage of the reducing properties of phenolic curcumin substance and its ability to decorate metallic surfaces. silver nanoparticles were synthesized, via sodium citrate and silver nitrate addition into a boiling aqueous solution of curcumin, monomers and surfactant. curcumin and sodium citrate promoted silver nucleation, acting as reducing and stabilizing agents. these curcumin-capped agnps enabled, after adding the radical polymerization initiator, the assembling of the growing polymer chains around the hydrophobic agnp surface. the resultant core-doped shell nanohybrids exhibit plasmonic, luminescent and volume thermoresponsive properties, with improved possibilities to be used as successful therapeutic platforms. in fact, the possibility to nanoconfine the synergistic antioxidant, antiviral, antibacterial features of silver and curcumin in one bioavailable hybrid paves the way to promising applications in the biomedical field. synthesis of ag@cur-p(meo ma) core-doped shell hybrid nps. ag@cur-p(meo ma) hybrid nanoparticles, from now on also called as (ag@cur-g), were synthesized through one-pot two-steps method (fig. ) . letter g denotes to thermoresponsive p(meo ma) polymer participation. a typical procedure for the synthesis is described below for sample ag@cur-g b (table ). in the first step (fig. b) , ag@cur nps (silver nps decorated with curcumin (table ) (fig. b) . the mixture was vigorously stirred for an additional min at reflux and then allowed to cool down slowly to room temperature. in the second step (fig. c) , p(meo ma) shell formation around ag@cur core was endowed by free radical precipitation polymerization (frpp) of meo ma and tegdma crosslinker. the solution was degassed by n gas for min at room temperature and polymerization was initiated by adding μl of aps solution ( . m) after temperature was raised to °c. the reaction was allowed to proceed for h at °c under stirring and n gas inlet (fig. c ). after that, the mixture was cooled down in an ice-cold water bath while it was exposed to the air. the resultant core-doped shell nps (ag@cur-g b) were purified by three centrifugation cycles by using successive decreasing centrifugal rates ( , and rpm) at °c for min. subsequent centrifugation rendered three supernatant fractions (f , f and f ) and a fourth fraction (f ) corresponding to the final precipitate from the third cycle. this procedure ensured the complete removal of empty polymer particles and excess of reactants. finally, nps pellet was redispersed in milli-q water and kept at °c in a refrigerator immediately prior to use. during the synthesis, different reaction parameters such as reaction temperature (first step, fig. b ) and curcumin and sodium citrate concentration were considered in order to study their influence on size, shape and concentration of agnps. ag@citrate nps without curcumin, as control samples, were also synthetized. synthesis of cur-p(meo ma) nps or (cur-g). the p(meo ma) crosslinked nanogels in the presence (cur-g) and absence (g , control sample) of curcumin were synthesized through precipitation polymerization strategy in water. a typical procedure for the synthesis is described below for sample cur-g (table ). in a pyrex tube equipped with a magnetic stirrer, μl of a solution of curcumin in ethanol ( . mmol), meo ma monomer ( . mmol), μl of tegdma crosslinker solution in ethanol ( . mmol), μl of sds aqueous solution ( . - mmol) and ml of milli-q water were added. after min n gas purge, polymerization was initiated by heated up to °c, followed by addition of μl of aps solution ( . m). after around min, the solution turned cloudy, indicating that polymerization started, and the solution was left to react for h. to stop the reaction, the solution was cooling down in an ice-cold water bath while the tube was opened to air. afterward, the sample was purified by centrifugation ( rpm, min, °c) and then the pellet redispersed in water. the final product was stored at °c until further use. methods for characterization. ftir spectra were recorded by perkin elmer spectrum two spectrometer using attenuated total reflectance (atr) accessory. uv-vis absorption spectra, at controlled temperature, table . summary of curcumin concentration, ζ-potential, z average diameter and polydispersity (pdi) by dls, volume temperature induced phase transition (vptt), swelling ratio (q), and maximum of curcumin absorbance (λ abs ) of cur-p(meo ma) nanogels. (a) curcumin wt% is the feed curcumin/polymer ratio, (b) ζ values were determined at °c were carried out in a cary bio-varian uv-vis spectrophotometer equipped with a peltier temperature control device. the particle size and zeta potential (ζ) of core-shell nps and agnps were determined by a zetasizer nano zs instrument (malvern instruments ltd, uk) equipped with a mw he-ne laser operating at a light source wavelength of nm and a fixed scattering angle of ° for detection. malvern dispersion software was used for data acquisition and analysis, applying the general purpose algorithm for calculating the size distribution. transmission electron microscopy (tem) images were recorded with a field emission scanning electron microscope (fesem) hitachi su- operated at kv in transmitted electron imaging mode (s-tem). energy-dispersive x-ray spectroscopy (edx) analysis to determine the elemental composition of the hybrid samples was performed using a bruker nano with x-flash detector coupled to the sem. data were recorded at an accelerating voltage of kv. the progress of sample purification by centrifugation was verified by uv-vis spectroscopy (nanodrop one thermo-scientific spectrometer). the centrifugation was performed by refrigerating micro centrifuge r (eppendorf ™ ) to obtain ag@cur-g and cur-g nanoparticle pellets. to determine the luminescence features, uv-vis absorption and fluorescence spectra were recorded on a perkin elmer lambda- and perkin elmer ls ob spectrophotometer, respectively. emission spectra of dilute solutions of ag@cur-g core-doped shell nanohybrids and cur-g nanogels in water and dilute solutions of fluorescein in naoh . m were recorded keeping absorbance at the excitation wavelength (λ exc = nm) less than . . quantum yields were obtained by comparing the studied samples with fluorescein standard by the following equation applicable for dilute solutions. where Φ f and Φ s are the photoluminescence qy of the sample and that of the standard fluorescein (Φ s = . ) , respectively; i e and i e(s) are the integrated intensity of the emission curves corresponding to the sample and standard; η and η (s) are the refractive indices of the sample and reference solution; and i a and i a(s) are the fraction of light absorbed by the sample and standard respectively estimated as: where a is the absorbance at λ exc . synthesis and characterization of ag@cur-p(meo ma) core-doped shell nanohybrids. the ag@cur-p(meo ma) core-doped shell hybrid nps were prepared by free radical precipitation polymerization (frpp) of the stimuli-responsive meo ma monomer in the presence of tegdma (crosslinking agent), using curcumin-decorated ag@cur nps as seeds (fig. ) . the presence of sds guaranteed the control of np size. among all the reagents involved in the process, silver nitrate and sodium citrate followed standard concentration procedures , . however, the dual key-role of curcumin, as reducing agent and growth-polymerization promoter in this specific synthesis, required additional investigation to understand and optimize the chemical variables (solubility, concentration and reaction temperature) in order to achieve homogeneous, monodisperse and mononuclear ag@cur-p(meo ma) core-shell nanohybrids. indeed, frpp is a useful and common strategy to synthesize particles of thermoresponsive polymers, as long as monomers are soluble at the reaction temperature and polymer precipitates when their chains reach a critical size. then, since water solubility of curcumin is rather poor; pure curcumin compounds needed to be first dissolved into an organic phase of ethanol, meo ma monomer and crosslinker (fig. a) . a subsequent injection of aqueous solution of sds surfactant followed by dropwise addition of water resulted in a good dispersion of monomers and curcumin. a color change from cloudy (at room temperature) to transparent yellowish dispersion (at the reaction temperature), indicated that both curcumin and monomers had been dissolved. note that curcumin solubility increases with temperature . when the desired temperature was reached, sodium citrate solution was added to the reaction mixture already containing curcumin; and finally, in the presence of the two reducing agents, silver nitrate was added to the reaction (fig. b) . agnps synthesis (outlined in fig. b ) was completed at two different temperatures, °c and °c (series -a and -b in table , respectively). after min of reaction, the flask was removed from the silicon bath and allowed to cool to room temperature. the as-synthesized silver nanocores had an average size around nm ( °c, series -a) and nm ( °c, series b) in diameter, respectively. to elucidate the role of curcumin as stabilizing agent, the sample obtained in this first step was purified by centrifugation to eliminate the excess of reagents that are not protecting the silver core. the presence of curcumin close to the agnp surface, attained in this first step, was confirmed by atr-ftir spectroscopy (fig. s ). as it can be observed in this figure, the spectrum corresponding to ag@cur (green line) is very similar to that of curcumin (blue line). the most prominent vibration band at ∼ cm − assigned to highly mixed vibration including c=o stretching appears in both spectra as well as other characteristic vibration bands of curcumin at cm − (mixed c=c and c=o stretching), cm − (deformation of ch ), cm − (deformation of phenyl rings), cm − (c-h out-of-plane aromatic motions). in this fig. s , spectra corresponding to sodium citrate and ag@citrate nps are also displayed. as it can be seen, two strong bands corresponding to the asymmetric and symmetric stretching of co − groups at cm − and at cm − , respectively appear with nearly similar intensity for the free sodium citrate spectrum (violet line); whereas for ag@citrate spectrum (orange line), the most prominent band corresponds to the symmetric stretch at cm − . in the spectrum corresponding to ag@cur nps (green line), this vibration band at cm − is www.nature.com/scientificreports www.nature.com/scientificreports/ absent and only the asymmetric stretching (∼ cm − ) can be hardly intuited overlapped with vibration bands from curcumin (blue line); these facts indicate that the affinity of sodium citrate for the silver surface is lower than that of curcumin. in addition, the bands attributed to c-h ( - cm − ) and c=o stretching vibrations of meo ma appear very weak, also suggesting a low interaction of the monomer with the silver surface. figure c outlines the polymer shell preparation. this second step was done without previous ag@cur sample purification. for the purpose of thermoresponsive-shell polymerization, ag@cur was first flushed with n for min at the lowest possible temperature (room temperature), with the aim of reducing water evaporation during nitrogen flow. subsequently, aps initiator was added and incubated for h in a closed flask under stirring at °c. the presence of hydrophobic curcumin nearby the metallic surface led to precipitation-polymerization of p(meo ma) around the agnps previously formed (fig. b,c) ; and the resulting ag@cur-p(meo ma) nanoparticles were born negatively charged due to the persulfate groups from the aps initiator, which promotes their colloidal stability. the successful encapsulation of ag@cur nps within p(meo ma) nanogels can be anticipated by the atr-ftir spectrum shown in fig. s corresponding to f of ag@cur-g b sample (table ) , which displays typical vibration bands attributed to both curcumin and p(meo ma). it should be noted that an attempt to encapsulate agnps synthesized solely using sodium citrate caused irreversible aggregation in the second polymerization step (fig. s ). this fact evidences the role of hydrophobic curcumin to provide both a protection to the agnps and a good interface between the polymer and the metal surface. well-defined ag@cur-p(meo ma) nanohybrids (table ) were obtained by selective removal of the silver-free nanogels by centrifugation (see experimental section). it should be taken into account that the number of empty polymer particles created during the polymerization process exceeded the number of agnps in the solution by about two orders of magnitude; and they should be removed from. as illustration, uv-vis spectra at different purification steps by centrifugation of the ag@cur-g b crude sample are displayed in fig. . initially, the spectrum of the crude shows an intense band at nm resulting from a combined contribution of curcumin absorbance and surface plasmon resonance (spr) due to the collective oscillations of conductions electrons of silver nps. note that spr maximum for spherical agnps can be typically tuned from to nm depending on their np size and shape as well as their surrounding dielectric medium. then, after the first and second centrifugation cycles, the supernatant fractions (f and f respectively) revealed absorption maxima around nm, indicating that the most predominant absorbance is due to curcumin embedded in p(meo ma) nps. the third centrifugation cycle, however, on the basis of the supernatant fraction (f ) and its corresponding precipitate (f ) evidenced only core-doped shell ag@cur-p(meo ma) nanoparticles content (absorption www.nature.com/scientificreports www.nature.com/scientificreports/ maxima at nm). therefore, the core-doped shell nanohybrids used for further characterization and analysis correspond to f fraction from each crude sample (table ) . moreover, curcumin trapped-polymeric nanogels (cur-p(meo ma)) were synthesized through a similar precipitation-polymerization strategy reported for polymer encapsulating of ag@cur (fig. c , ag@ cur-p(meo ma)) and using similar curcumin/polymer ratios (see experimental section and table ). the chemical structure of both, polymeric nps (cur-g) and hybrid nps (ag@cur-g) can be elucidated from the atr-ftir spectra shown in fig. . typical vibration bands (c=o stretching at cm − and c-o stretching at cm − ) corresponding to p(meo ma) are present in both types of nps, in addition to vibration bands at , , and cm − attributed to curcumin molecule. surprisingly, when comparing both cur-g and ag@cur-g nps, for similar curcumin/polymer ratios (tables and ), qualitative differences were observed in their respective spectra evolution (fig. ) . consistently with curcumin data (tables and ) , curcumin related vibration bands seemed to be differently affected by its content. cur-g series exhibited an increase tendency of intensity (fig. a,b) ; whereas ag@cur-g series (fig. c,d) barely varied. this observation could be explained through the key-implication of curcumin in our nuclei-growth approach (fig. ) which determines, at the end, the morphology of ag@cur-g nanohybrids. in fact, the specific silver-nucleation strategy (fig. b) makes feasible that certain amount of curcumin be preferably confined at the metal surface, being subsequently enclosed at higher concentration during p(meo ma) polymer growing shell onto silver core nps (ag@cur-g) than during polymer assembling along silver-free p(meo ma) nanogels formation (cur-g). s-tem micrographs in fig. evidence a core-shell morphology of the ag@cur-g hybrid nps with silver core formed at °c (series ag@cur-g#a, fig. a ) and at °c (series ag@cur-g#b, fig. b) . a mere glance to s-tem pictures revealed almost concentric nanostructuration of polymer shell coverage around metal core for both series of nanohybrids. both series were synthesized with similar curcumin/polymer ratios ( . , . , . and . wt%, table ). the resultant mean values of ag-core diameter ( nm at °c and nm at °c) for both series are collected in table . these differences can be attributed to the faster nucleation rate with increasing temperature that leads to smaller nps. edx spectra in fig. s confirm the presence of ag in two representative hybrid samples with nm (ag@cur-g a) and nm (ag@cur-g b) core diameter, the determined elemental composition is in agreement with ther core size of both nanohybrids. tables and . moreover, it seems that samples synthesized with the two highest curcumin ratios ( . wt%, fig. aiv -biv and . wt%, fig. av -bv) exhibit a more homogeneous spherical shape and a quasi monodistribution, especially, when synthesized at °c (fig. biv-v) . this is in contrast to what happens with the lowest curcumin concentration ( fig. aii and bii) where two silver np populations appeared during the synthesis. these morphological changes were found to affect the spr of the resulting core-doped shell au@cur-g hybrid nps. spr bands of ag@cur-g#a samples synthesized at °c (uv-vis, fig. ai ) are broader and red-shifted compared to those of ag@cur-g#b hybrid nps obtained at °c (uv-vis, fig. bi ). in fact, these wavelengths variations resulted from changes of refractive index of the medium surrounding the metal surface, due to polymer/curcumin reorganization around the two different ag-core sizes. correlative changes in color were also observed when different sizes of metal core were used by keeping all the other reaction parameters constant (table and uv-vis insets of fig. ). larger silver nps exhibited a visual reddish color and a wavelength red-shift. in fact, the λ max for ag@citrate of and nm are and nm respectively , so that red shifts of about - nm collected in table are in accordance to the polymer coating with shell thickness values shown in table . aware of the importance of chemical variables, as curcumin and citrate reducing agents, on the silver-nuclei and their impact on the polymer-growth, an additional experiment with a higher amount of sodium citrate was programmed. at this time, μl of a citrate solution . m was added to the reaction mixture instead of . m, in the presence of . wt% of curcumin, at °c. as expected, it resulted in two highly differentiated ag-core populations ( nm and nm) shown in fig. s . interestingly, agnps (∼ nm) were mostly formed at the limit around the polymeric vesicle boundary but also closely packed inside polymer networking-shell of nm of size (fig. s ). this behavior is attributed to the high nucleation of the nanoparticles due to the high concentration of citrate that causes a greater ion exchange and increases the total ionic strength in the solution, promoting the formation of particle agglomerates in some cases. then, in the regime of low curcumin concentration, phase separation can take place, being the smallest spherical cores produced by sodium citrate and the almost rod-like agnps promoted by curcumin. so, we hypothesize that an appropriate balance among both reducing agents (sodium citrate and curcumin) is needed to obtain more uniform agnp population. thermoresponsive properties. micro/nanogels of crosslinked p(meo ma) in aqueous solution exhibit a reversible volume decrease when raising temperature above the lower critical solution temperature (lcst) of linear p(meo ma). this temperature is known as temperature-induced volume phase transition (vptt) which can be followed by measuring the particle hydrodynamic size by dls. in fig. representative z-average vs temperature curves are shown for the series of ag@cur-g#b nps synthesized at °c ( fig. ai and table ) and of the cur-g# nanogels (fig. aii and table ). as can be observed the vptt values, calculated from the inflection www.nature.com/scientificreports www.nature.com/scientificreports/ point of the curve (fig. ai) , are around °c for the two hybrid nps series (ag@cur-g, table ), showing no dependence on the curcumin content. these results are in good agreement with the atr-ftir spectra (fig. ) which indicated rather similar curcumin content for ag@cur-g hybrids nps. on the contrary, cur-g nanogels (fig. aii ) exhibit slightly decreasing vptt values with increasing the curcumin content (table ). this implication is clearly evidenced at low temperature, where the decrease of the nanogel size is affected by the hydrophobicity feature of curcumin (fig. aii) . interestingly, hybrid nanoparticles exhibit a higher nps size than polymeric nanogels. a plausible explanation of this behavior can lie on the impact of metal core which acts as seed during the precipitation-polymerization; since hybrid nps may need to grow more in order to decrease the polymer-metal interfacial energy and to reach then the stabilization. swelling ratio (q) is an important parameter affecting nanogel properties that can be determined by dls. it can be directly calculated from the ratio between the nanogel volume (v) at swollen state and at the collapsed state : while for polymeric nanogels the volume is directly calculated from the particle radius (r) as v = / π r , for spherical hybrid nps, shell volumes at both temperatures are calculated as v(shell) = / π [r p -r c ]. r p is the hydrodynamic radius of the hybrid particle and r c the hydrodynamic radius of the ag core. table shows the swelling ratios for the silver-polymer (ag@cur-g) hybrid nps and table for the polymeric (cur-g) nanogels investigated. the observed temperature-responsive swelling ratios (q) were close to for the most of the hybrid nps and slightly higher for polymeric nanogels. indeed, for polymeric nanogels ( table ) a decrease of q results from the increase of curcumin in the polymeric nanogels, which could be attributed to a lower swelling ability since the hydrophobic balance increases. on the other hand, the fact that for ag@cur-g hybrid nps, q values show no dependence on the feed composition, could indicate that the final composition of the polymer p(meo ma) shell is rather similar, corroborating again the atr-ftir results (fig. d) . some cyclic heating-cooling experiments were done (data not shown), after observing the reversible collapse-swelling behavior of the samples (fig. ) . at low temperature, the steric protection by the hydrophilic (table ) and (aii) cur-p(meo ma) nanogels (table ) with different curcumin content. (b) ζ-potential determined by dls at °c and °c for colloidal samples of (bi) p(meo ma) nanogels (g ), (bii) agnps functionalized with . wt% curcumin content (ag@cur) before and (biii) after (meo ma) polymerization, ag@cur-p(meo ma) hybrid nps (ag@cur-g b). (c) uv-vis spectra (below and above vptt) of ag@cur-p(meo ma) hybrid nps with ag core synthesized at °c (ci) and °c (cii), and cur-p(meo ma) nanogel (ciii). all samples shown (c) were synthesized with . wt% curcumin/polymer ratio (table and table www.nature.com/scientificreports www.nature.com/scientificreports/ p(meo ma) shell provides good stability in the aqueous medium; whereas at higher temperature, the driving force of colloidal stability is the electrostatic repulsion among the particles revealed by ζ-potential measurements. tables and show ζ-potential values corresponding to ag@cur, ag@cur-g hybrid nps and cur-g nanogels at °c. all types of nps synthesized with curcumin exhibit a fairly high negative ζ-potential that would confer stability against aggregation, especially at temperature above the vptt, where the increase of hydrophobic balance in the polymer shell suddenly produces water expelling from the nanogel. the negative ζ-potential of the synthesized nps can be attributed to several origins. it is well well-known that during polymerization process, incorporation of initiator groups (anionic persulfate) into polymer chains introduces negative charges into micro/nanogel particles synthesized by frpp. additionally, in the specific synthesis of the nanohybrids with silver core explored in this work, the contribution of the remanent sodium citrate cannot be neglected. even more, the negative ζ-potential may arise from curcumin, as it has been reported by some authors , . certainly, in previous works, we found an increase of the ζ-potential at temperature above polymer collapse for both, thermoresponsive polymeric nanogels and hybrid nps with metallic au-core . this effect was attributed to the decrease of the particle size, which increases the charge density at the np surface. nevertheless, in the present investigation, we only observed this temperature-dependent effect on nanoparticles without curcumin (g in fig. bi) . indeed, hybrid nps exhibited a tunable ζ-potential temperature-dependent behavior before (ag@ cur) and after polymerization (ag@cur-g). so, initial ζ-potential diminution values with increasing temperature (fig. bii ) rapidly transited to high and almost invariant ζ-potential values with temperature (fig. biii) as polymerization progressed. these observations (table and fig. b ) so far can be explained through the antagonistic tendencies of the polymer shell and curcumin. the ability of the polymer shell to swell or collapse as function of temperature can tune the optical and luminescent properties of the synthesized nanoparticles. figure c displays representative uv-vis spectra (below and above vptt) for two nanohybrid samples with different core size (ag nm@cur-g a, fig. ci and ag nm@ cur-g b, fig. cii ) and nanogels (cur-g , fig. ciii ) synthesized with . wt% of curcumin content (table ). in uv-vis absorbance spectra, spr band of the nanohybrid with a higher silver diameter (fig. ci) red-shifted while broadening considerably with respect to spr band of the nanohybrid with a smaller silver diameter (fig. cii) . obviously, the spr band position and width was affected by the core-size and the temperature-induced variation of the electromagnetic field in its neighboring environment. the cur-g sample containing silver-free nanogel (fig. ciii) only showed the curcumin effect on nanogel swelling behavior which determines the refractive index of the polymer-gel with the surrounding aqueous medium. in addition, a reversible red-blue shift was observed, when the temperature increase-decrease the vptt of the polymer shell (fig. ) . when the external temperature rises above the vptt, the nanogel shell expels water dictated by the strengthening of hydrophobic interactions giving rise to (i) a decrease of the polymer shell thickness and (ii) an increase of the refractive index. at that point, the effect of refractive index augmentation exceeds to that of the shell thickness diminution; therefore a red-shift should be observed above the vptt for plasmonic-core@thermoresponsive-shell nanohybrids. fig. ci and cii illustrate this behavior for our ag-based thermoresponsive-polymer (core@shell) hybrid nps which correlates well with similar results reported for au-core based thermoresponsive core@shell nanogels . furthermore, the change of the spr maximum with temperature is more pronounced for larger silver core (fig. ci) as previously observed for core-shell nanohybrids with gold core . indeed for the nanohybrids with silver core of nm (series ag@cur-g#a), the Δλlspr max is about - nm whereas for the nm silver core ones (series ag@cur-g#b) the Δλlspr max is about - nm ( table ). the cur-g nanogel sample, without metallic core intervention (fig. ciii) , exhibits no so remarkable λ max absorption change with temperature, at similar curcumin/polymer ratio. www.nature.com/scientificreports www.nature.com/scientificreports/ luminescent properties. curcumin is a tautomeric compound that can occur in diketo and keto-enol forms which ratio strongly varies with the solvent. according to manolova et al. , in solvents as ethanol, only the enol-keto tautomer is present; whereas the addition of water leads to appearance of a new spectral band (about nm), which was attributed to the diketo tautomeric form. the keto-enol form, due to a strong intramolecular hydrogen bond, undergoes transformation into a totally delocalized π-system. due to the hydrophobic character of curcumin, it presents luminescence properties in organic solvents but it is almost non-fluorescent in aqueous solution. however, by complexation through hydrophobic interactions with proteins or with amphiphilic polymers as pluronic , or encapsulating curcumin in hydrophobic polymer nanoparticles , fluorescence enhancement in aqueous medium has been reported. previous to the luminescence investigation of the ag@cur-g hybrid nps, we investigated the absorption and emission of curcumin encapsulated in the thermoresponsive nanogels, cur-g (table and fig. s ). hydrophobic curcumin was engulfed by thermoresponsive nps during the polymerization process, based on the precipitation of the growing polymer at °c due to its unfavorable interaction with the water solvent above lcst (fig. ) . absorbance spectra for curcumin embedded in p(meo ma) nanogels (cur-g) at the polymer concentration of mg ml − show an increase of intensity proportional to the curcumin concentration (fig. s ). in agreement with previous reports of curcumin entrapped in hydrophobic or amphiphilic block copolymers , a main absorption band at - nm in addition to a shoulder about nm appear in fig. s . some authors attributed the signal at nm to the keto-enol tautomer , , whereas the band at nm, associated to the keto form and that typically appears in water, is absent in the present case. fig. s inset shows the linear dependence of the curcumin absorption maxima at nm vs molar concentration of curcumin according to the beer-lambert law for cur-g nanogels. table ). samples were excited at nm. ( fig. a emission spectra in water for the synthesized cur-g nanogels excited at nm evidence remarkable luminescence due to curcumin. in table , the emission maxima (λ em ) and qy, calculated with fluorescein standard, are collected. the emission maxima are about - nm in good agreement with the values reported by banerjee et al. for curcumin encapsulated in polymeric nanoparticles. the position of the emission maxima of curcumin is strongly dependent on the solvent; so by increasing the polarity from chloroform to water, the emission maximum is red-shifted and broadened from nm to nm . both the qy values and the emission maxima position for the present cur-g nanogels seem to indicate that curcumin is well protected by the hydrophobic environment provided by the polymer. with respect to the qy values, two facts have been observed: first, the qy value increases with the decrease of curcumin concentration in the nanogels (fig. a and table ) ; and, second, an amazing fluorescence enhancement is noticed by the temperature increase (fig. b) . the effect of curcumin concentration could be related to the protection that hydrophobic domains of the polymer offer to curcumin, which is enhanced at low curcumin/polymer ratio. the increase of luminescence with increasing temperature should be associated to the water expelled out of the nanogels when temperature rises above vptt. indeed, at lower curcumin ratio (fig. b) , the remarkable increase in absorbance at °c can be attributed to the higher polymer contribution. this reversible change in luminescence with temperature has table ). samples were excited at nm. ( www.nature.com/scientificreports www.nature.com/scientificreports/ been previously reported for fluorescent dyes as bodipy in thermoresponsive p(meo ma) linear polymers and hydrogels . in fig. and fig. s a different behavior can be found for hybrid nps. in first place, analyzing the qy values collected in table , it can be seen that nanohybrids´ fluorescence values are lower than those measured for cur-g nanogels. on the other hand, for both series of ag@cur-g hybrid nps, an increase of luminescence with increasing of curcumin concentration is detected, in opposite to what happened in cur-g nanogels (table and fig. a ). however we know that it can be an ¨apparent increase¨ since the absorption band consideration for estimating the qy values was not properly drawn up. note that this absorption band reflects the additive contribution from the overlapped silver and curcumin spr bands. then, on the basis of these considerations, at lower curcumin concentration, the real curcumin contribution to the final absorbance will be lower. the same miscalculation could arise when comparing qys of hybrid nps with those of polymeric nanogels, because for hybrids an important part of the absorbance arises from the spr of agnps. in last place, the temperature effect also departs from that effect observed for cur-g nanogels, where an increase of fluorescence occurred after polymer collapse on account of hydrophobicity augmentation. in the case of hybrid nps, a slight decrease of fluorescence is detected by moving away from room temperature. this experimental evidence suggests a change on the curcumin-polymer interaction due to the silver presence, or in this specific case, that the interaction of curcumin with silver played a major role, since the specific silver-nucleation strategy causes that certain amount of curcumin be confined at the metal surface. the fluorescence diminution that accompanies the temperature decrease may find its origin in the hydrophilicity-gain experienced by the thermoresponsive polymer shell and the subsequent quenching of curcumin emission induced by water. whereas, when increasing temperature, the polymer increases its hydrophobic balance, which should contribute to protect curcumin against water; nevertheless this positive effect could be overcome by the fact that after polymer collapse, curcumin can get too close to the metal surface and this effect should lead to a further emission quenching. similar observation was reported on the strong curcumin emission-quenching for other silver np-based hybrid systems by other authors , . conclusion in this work, we focused on the knowledge and application of one-pot protocol to obtain core-shell hybrid nanoparticles with the ability to encapsulate hydrophobic, bioactive, antioxidant, and low bioavailability molecules. the aim is to draw on the reductive capacity of some bioactive molecules as curcumin for the in situ formation of metallic nps, meanwhile their hydrophobic character leads the polymer nanostructuration in aqueous medium in the presence of the inorganic metal-core. therefore, new core-doped shell nanohybrids based on silver plasmonic core, thermoresponsive polymer shell and embedded luminescence and bioactive curcumin are easily obtained with potential bioapplications such as antimicrobial systems. furthermore, polymeric nanogels encapsulating curcumin were also achieved, which luminescent properties are strongly enhanced by increasing temperature. this last property could be tuned even with the minimum ratio of bioactive compound. functional microgels and microgel systems temperature-and ph-tunable plasmonic properties and sers efficiency of the silver nanoparticles within the dual stimuli-responsive microgels stimuli-responsive nanogel composites and their application in nanomedicine facile synthesis of gold/polymer nanocomposite particles using polymeric amine-based particles as dual reductants and templates a simple approach to obtain hybrid au-loaded polymeric nanoparticles with a tunable metal load versatile thiolated thermosensitive polymers synthesized by atrp of meo ma and acsema, a new methacrylic monomer with a protected thiol group plasmonic gold-poly(n-isopropylacrylamide) core-shell colloids with homogeneous density profiles: a small angle scattering study encapsulation and growth of gold nanoparticles in thermoresponsive microgels controlled nucleation for regulation of particle-size in monodisperse gold suspensions a study of the nucleation and growth processes in the synthesis of colloidal gold au@pnipam colloids as molecular traps for surface-enhanced, spectroscopic, ultra-sensitive analysis au@pnipam thermosensitive nanostructures: control over shell cross-linking, overall dimensions, and core growth microgels loaded with gold nanorods: photothermally triggered volume transitions under physiological conditions synthesis of au@polymer nanohybrids with transited core-shell morphology from concentric to eccentric emoji-n or optical and swelling stimuli-response of functional hybrid nanogels: feasible route to achieve tunable smart core@shell plasmonic@polymer nanomaterials smart core-shell hybrid nanogels with ag nanoparticle core for cancer cell imaging and gel shell for ph-regulated drug delivery catalytic reduction of -nitrophenol using silver nanoparticles with adjustable activity core-shell hybrid nanogels for integration of optical temperature-sensing, targeted tumor cell imaging, and combined chemo-photothermal treatment silver-decorated polymeric micelles combined with curcumin for enhanced antibacterial activity in situ formation of curcumin stabilized shape-selective ag nanostructures in aqueous solution and their pronounced sers activity green synthesis and catalytic application of curcumin stabilized silver nanoparticles rapid green synthesis and characterization of silver nanoparticles arbitrated by curcumin in an alkaline medium in situ synthesis and surface functionalization of gold nanoparticles with curcumin and their antioxidant properties: an experimental and density functional theory investigation synthesis of au nanorods through prereduction with curcumin: preferential enhancement of au nanorod formation prepared from ctab-capped over citrate-capped au seeds encapsulation of curcumin nanoparticles with mmp -responsive and thermos-sensitive hydrogel improves diabetic wound healing antibacterial adhesive injectable hydrogels with rapid self-healing, extensibility and compressibility as wound dressing for joints skin wound healing multisite inhibitors for enteric coronavirus: antiviral cationic carbon dots based on curcumin nanocurcumin is superior to native curcumin in preventing degenerative changes in experimental cerebral malaria curcumin modified silver nanoparticles for highly efficient inhibition of respiratory syncytial virus infection effect of encapsulation of curcumin in polymeric nanoparticles: how efficient to control esipt process? fluorescence quantum yields and their relation to lifetimes of rhodamine g and fluorescein in nine solvents: improved absolute standards for quantum yields ¶ adsorption and surface-enhanced raman of dyes on silver and gold sols raman response of dithiolated nanoparticle linkers temperature-dependent spectroscopic evidences of curcumin in aqueous medium: a mechanistic study of its solubility and stability dft and experimental studies of the structure and vibrational spectra of curcumin a rapid method to estimate the concentration of citrate capped silver nanoparticles from uv-visible light spectra interfacial tension of turmeric nanoparticles optical properties of responsive hybrid au@polymer nanoparticles the effect of the water on the curcumin tautomerism: a quantitative approach fluorescence study of the curcumin-casein micelle complexation and its application as a drug nanocarrier to cancer cells organic additive, -methylsalicylic acid induces spontaneous structural transformation of aqueous pluronic triblock copolymer solution: a spectroscopic investigation of interaction of curcumin with pluronic micellar and vesicular aggregates synthesis of a self organizable curcumin derivative and investigation of its interaction with metals in % aqueous media bodipy-conjugated thermo-sensitive fluorescent polymers based on -( -methoxyethoxy)ethyl methacrylate the authors gratefully acknowledge the financial support provided by the spanish ministerio de ciencia, innovación y universidades (grant mat - -r and pgc - -b- ). a.s.-q. acknowledges conacyt for the grant epe - . the authors would like to thank david gómez vargas assistance with the sem measurements. key: cord- -la svzml authors: strohl, william r. title: current progress in innovative engineered antibodies date: - - journal: protein cell doi: . /s - - - sha: doc_id: cord_uid: la svzml as of may , , antibody-based molecules have been approved by a regulatory authority in a major market. additionally, there are and antibody-based molecules in phase iii and phase i/ii clinical trials, respectively. these total antibody-based clinical stage molecules include naked iggs, antibody-drug conjugates, bispecific antibodies, total fc fusion proteins, radioimmunoglobulins, antibody fragments, and immunocytokines. new uses for these antibodies are being discovered each year. for oncology, many of the exciting new approaches involve antibody modulation of t-cells. there are over antibodies in clinical trials targeting t cell checkpoints, t-cell-redirected bispecific antibodies, and chimeric antigen receptor (car) cell-based candidates (all currently in phase i or ii clinical trials), totaling more than t cell interacting clinical stage antibody-based candidates. finally, significant progress has been made recently on routes of delivery, including delivery of proteins across the blood-brain barrier, oral delivery to the gut, delivery to the cellular cytosol, and gene- and viral-based delivery of antibodies. thus, there are currently at least antibody-based clinical stage molecules or cells, with incredible diversity in how they are constructed and what activities they impart. these are followed by a next wave of novel molecules, approaches, and new methods and routes of delivery, demonstrating that the field of antibody-based biologics is very innovative and diverse in its approaches to fulfill their promise to treat unmet medical needs. this year, , marks the th anniversary of the approval by the united states food and drug administration (us fda) of rituxan® (rituximab) and zenapax® (daclizumab), for treatment of b cell malignancies and for use to suppress organ rejection in renal transplants, respectively (table ) . while two antibodies had previously been approved by the fda (table ) , the approval of rituxan® and zenapax® in was a watershed moment in the history of monoclonal antibody (mab) therapeutics. the reasons are very different for each molecule. rituxan® has become both a huge medical and commercial success, with indications in b cell malignancies as well as in the treatment of rheumatoid arthritis (ra) (storz, ) . rituxan® is currently the fourth best-selling innovative drug of any kind with worldwide sales of $ , mm (table ) , about % of those sales coming in cancer indications and the other % from sales for treatment of ra (la merie publishing, ) . including rituxan®, seven of the top ten selling innovative drugs in the world in were proteins, six of which were antibodyrelated molecules (table ) . zenapax®, on the other hand, was the first humanized antibody to be fda approved but it never achieved significant commercial success and was eventually withdrawn from the market in . daclizumab, however, has been approved recently under the tradename zinbryta® for treatment of relapsing forms of multiple sclerosis (ms). to date, unique, innovative antibodies and fc fusion proteins have been approved for treatment of diseases in at least one major market (i.e., us, eu, japan) (table ) . of these, seven have been withdrawn from marketing either due to lack of efficacy, poor toxicity to efficacy profiles, or lack of market interest (table ) . of the approved antibody-based molecules, five contain completely murine (table ) . thus, as of may , , there are at least known antibody and fc fusion protein clinical-stage candidates (table ) . of these, are "naked" iggs, are "naked" antibody fragments (in both cases, "naked" refers to antibodies that are not antibody-drug conjugates [adcs], bispecific antibodies, radioimmunotherapeutics, or immunocytokines), are adcs, are bispecific antibodies, are fc fusion proteins, are conjugated with radioisotopes either as therapeutics or imaging agents, and are immunocytokines (table and fig. ). it is notable that, with the exception of fc fusion proteins, most of the non-"naked" antibodies are skewed towards the phase i/ii clinical stages, likely due to the more recent development of the various innovative technologies incorporated into those molecules (table ). in addition to these protein antibody-derived clinical stage molecules, there are documented phase i or ii clinical stage chimeric antigen receptor (car)-t cell or natural killer (nk) cell candidates that incorporate antibodies as their cars (table and fig. ) . thus, there are at least protein and cell based antibody-derived constructs either approved for medical use or being evaluated for their safety and efficacy in clinical trials. the unique antibody-based molecules/cells in development or approved for therapeutic use (table ) target unique antigens (table ). because several targets are important for multiple disease areas (e.g., vascular endothelial growth factor [vegf] as a significant target in both oncology and ophthalmology indications), there are more uses listed than antibodies. thus, unique molecules are used in different major therapeutic area indications (table ) , and the unique targets are distributed amongst major uses (table ) . about % of these protein and recombinant cell-based candidates are directed against targets in oncology (table ) . not surprisingly, all of the current car-t and car-nk candidates are in clinical evaluation for cancer indications. there are, however, preclinical efforts to generate car-t cells against viruses and virus-infected cell targets (sahu et al., ; liu et al., ; hale et al., ) , so this may change in the near future. another % of the clinical candidates are directed against targets in the immunology therapeutic area (including autoimmune and asthma, but excluding ms) ( antigen related cell adhesion molecule ], muc [mucin , cell surface associated]), are essentially "postal addresses" to which killing mechanisms can be targeted directly. these killing mechanisms can include, either individually or in combinations, antibody-dependent cellular cytotoxicity (adcc) (ochoa et al., ) , antibody-dependent cellular phagocytosis (adcp) (shi et al., ) , complement-dependent cytotoxicity (cdc) , antibody-drug conjugates (adc) (tsuchikama and an, ; beck et al., ) , antibody-induced apoptosis (sun et al., ; wang et al., ) , antibody-induced, non-apoptotic programmed cell death (alduaij et al., ) , bispecific antibody-redirected killer t or nk cells (lum and thakur, ; satta et al., ; suzuki et al., ) , or car-t/car-nk cells (ruella and gill, ; ruella and june, ; smith et al., ) . the second group, which overlaps with the first group, are receptors which may be targeted to block ligand binding and signal transduction (esparis-ogando et al., ; zhang and zhang, ) . the final category are checkpoint modulators, either to block t cell inhibitory pathways or to directly stimulate t or nk cells or macrophages. there are about t-cell related oncology targets in this category. of the unique targets for antibody-based drug candidates, the most widely targeted antigen is cd , which is recognized by clinical candidates, of which are cars ( table ). the second most targeted protein is cd e, found in clinical stage or approved molecules, of which are t cell-redirecting bispecific antibody candidates (table ) . thus, the two top targets, cd and cd e, are responsible for the engineered retargeting of t cells, either as car-t cells (ruella and gill, ; ruella and june, ; smith et al., ) or t-cell redirecting bispecific antibodies (lum and thakur, ; satta et al., ; suzuki et al., ) , to kill cancer cells. of the non-t-cell related targets, the proteins currently most widely targeted are erbb (her ), egfr, ms a (cd ), cd , pdcd (pd- ), msln (mesothelin), and erbb (her ), all for cancer indications. the th cytokine, il a, to which antibody-related biologics are directed, is currently the top non-oncology target (table ). there are unique molecules or recombinant cars directed against the top targets shown in table , representing about % of all of the clinical stage or approved antibody-based molecules/cells; the remaining (∼ %) candidates target the remaining unique targets. the approved mabs and fc fusion proteins are directed against unique targets, with tnf (tnf-α) and ms a (cd ) being the most widely targeted, with five antibody-based molecules each (table ). the five most valuable targets for approved mabs and fc fusion proteins are tnf (tnf-α), vegf, erbb (her ), ms a (cd ), and pdcd (pd- ) ( table ) . antibodies against the first four of these targets were approved more than ten years ago, so the market value has built up over time. remarkably, however, the anti-pd- antibodies, keytruda® and opdivo®, were approved , making pdcd (pd- ) a very fast rising target of value (table ) . the top ten antibody-based therapeutic targets (table ) comprise % of the value of the total targets, with the anti-tnf molecules leading the way with a market share of % (table ) . based on sales figures, recombinant proteins comprised seven of the top best selling drugs worldwide (table ) . of these seven proteins, five (humira®, rituxan®, remicade®, avastin®, herceptin®) are mabs and one (enbrel®) is an fc fusion protein (table ) . finally, since january (the past . years), antibodies and fc fusion proteins have comprised % ( / ) of innovative united states food and drug administration (us fda) drug approvals (fig. ) . this represents the greatest percentage ever since the beginning of the antibody era. thus, it is clear that mabs and fc fusion proteins are making an enormous impact on the pharmaceutical industry, both as novel approaches to treat difficult diseases and meet unmet medical needs, as well as providing an exciting new growth area for the industry. human iggs have been engineered in a multitude of ways to generate different effects (strohl and strohl, ) , as shown in table . in the earlier days of antibody engineering, the focus was on manipulation of the variable regions to humanize and affinity-mature antibodies, or to generate different types of antibody fragments such as scfvs (bird et al., ; huston et al., ) , diabodies (holliger et al., ) , pegylated fabs (choy et al., ) , tandabs (kipriyanov et al., ) , and domain antibodies (ward et al., ) . the next wave of antibody engineering focused more on the generation and application of "fit for purpose" antibodies (strohl, ) with tuned fc functions such as increased adcc, adcp, and cdc (strohl and strohl, ; brezski and georgiou, ; sondermann and szymkowski, ; barnhart and quigley, ) , or muted or silenced fc functions (labrijn et al., ; vafa et al., ; schlothauer et al., ; lo et al., ; borrok et al., ) . these functions have been approached using both glyco-engineering strategies such as low or no fucosylation for higher fcγriiia binding and increased adcc (shields et al., ; ferrara et al., ; malphettes et al., ; golay et al., ) , higher levels of sialylation for dampened immune responses (anthony and ravetch, ) , or non- abbreviations: fc, fragment crystallizable; ms, multiple sclerosis; gpcr, g-protein coupled receptor. * these numbers add up to more than the unique targets noted in table because several targets have major indications in multiple therapeutic areas (e.g., anti-vascular endothelial growth factor [vegf] antibodies with major indications in both oncology and ophthalmology). ** mostly single-pass membrane targets, either as monomeric cell-bound proteins, homodimeric receptors, or heterodimeric receptors. glycosylated antibodies for partly subdued fc effector function (walker et al., ; nesspor et al., ) . these and more fc effector modulations can also be generated with amino acid changes in the lower hinge and fc (shields et al., ; lazar et al., ; strohl, ; strohl and strohl, ; vafa et al., ; brezski and georgiou, ; sondermann and szymkowski, ; barnhart and quigley, ) . there are currently three approved antibody-based molecules with modified fc functionality. these include the ctla -fc fusion proteins, abatacept (orencia®) and belatacept (nujolix®), both of which have modified hinges to reduce fc functionality (davis et al., ) , and the humanized anti-c mab, eculizumab (soliris®), which has an igg /igg hybrid fc to reduce fc functionality (rother et al., ) . all three of these molecules bind to immune (gray et al., ) followed by commonly used names in parentheses. system components and the muted fc design was intended to increase the safety margin. additionally, two glyco-engineered antibodies with improved adcc activities have been approved in at least one major market. the first, mogamulizumab (poteligeo®), is an afucosylated anti-ccr mab approved in japan for adult t-cell leukemia/lymphoma that is produced by a cell line with a mutation in the fut (α- , -fucosyltransferase) gene (potelligent® technology from biowa; yamane-ohnuki et al., ; kanda et al., ; malphettes et al., ) . the second, obinutuzumab (gazyva®), an anti-ms a (cd ) mab with low fucose content, has been approved for treatment of chronic lymphocytic leukemia (cll) (golay et al, ) . the low fucose of obinutuzumab is due to the addition to the producing cell line of a β- , -n-acetylglucosaminyltransferase iii (gntiii) gene which adds the bisecting n-acetylglucosamine (glcnac) that interferes with fucosylation (glycart glycomab® technology acquired by roche in ) (ferrara et al., ) . moreover, there are four glycoengineered (low or no fucose) and one aglycosyl-igg phase iii clinical candidates in the late stage clinical pipeline. currently there are no approved iggs with amino acidmodified increased fc effector function, although there are two such fc-modified, increased fc effector function iggs in late stage clinical trials, the anti-cd mab, mor (morphosys, xencor), in phase ii/iii clinical trials for treatment of b cell malignancies (nct ), and the anti-erbb (her ) mab, margetuximab (merck, macrogenics), in phase iii clinical trials for breast cancer (nct ). moreover, there have been many efforts to modulate aspects of igg biology beyond just increasing or decreasing fc effector function (table ). the first of these is modulation of half-life via modified interaction of the fc with the recycling receptor fcrn (roopenian and akilesh, ; abbreviations: cd, cluster of differentiation; egfr, epidermal growth factor receptor; her , human epidermal growth factor receptor- ; ig, immunoglobulin; il, interleukin; pd- , programmed cell death protein- ; rank, receptor activator of nuclear factor kappa-b; tnf, tissue necrosis factor; vegf, vascular endothelial growth factor. * names given as hugo gene nomenclature committee (hgnc) names (gray et al., ) followed by commonly used names in parentheses. ** rounded to one decimal point. data abstracted from la merie, . *** $ . b of $ . b is % of total mab and fc fusion protein value in ( of total actively marketed antibody-based products). baker et al., ) . the most important of these modifications has been the "yte" mutation (dall' aqua et al., ; dall'aqua et al., ) from medimmune (astrazeneca), which has been incorporated into a few early stage candidates at this point (robbie et al., ) . other half-life extension mutations of the fc also have been made, including xencor's xtend technology (zalevsky et al., ) , which has been incorporated into at least two early stage clinical candidates, alexion's anti-c mab, alxn , and the national institutes of health's (nih's) anti-cd mab, vrc ls. several other igg engineering technologies have been reported that have the potential to modulate the capabilities of existing and future clinical candidates (table ). these include protease activated "probody" iggs for tumor-localized activity (erster et al., ; desnoyers et al., ; polu and lowman, ) , protease-resistant iggs that are stable in the tumor micro-environment (kinder et al., ) , hexameric antibodies with high c q binding and concomitant cdc effector function (cook et al., ; de jong et al., ) , ph-dependent binding (igawa et al., a; chaparro-rogers et al., ; devanaboyina et al., ) and sweeping antibodies ) that improve upon the elimination profile for soluble antigens (igawa et al., ) , engineering variable regions for improved solubility and developability (clark et al., ; seeliger et al., ) , modulation of the pi or charge of the antibody variable sequences for improved half-life (igawa et al., b; li et al., ; datta-mannan et al., ) and/or separation and purification (sampei et al., ) , and mutation of protein a binding site for improved purification of a heterobispecific igg (tustian et al., ) . another area of antibody engineering that is starting to see significant activity is the engineering of igms as therapeutics, especially where high avidity effects are desired (chromikova et al., ; wang et al., b) . in a recent example, an anti-tnfrsf b (dr ) igm demonstrated -fold greater avidity and -fold greater killing effect than a similar igg (wang et al., b) . adcs target a cytotoxic drug to a tumor to kill cancer cells while lowering the systemic exposure of the active moiety, with the goal of increasing the size of the efficacy/toxicity window of highly toxic anti-tumor drugs (strohl and strohl, ; tsuchikama and an, ; beck et al., ) . adcs morrison et al., humanization mouse cdrs in human frameworks jones et al., ; queen et al., generation of scfvs fv domains fused with linker bird et al., ; huston et al., fc fusion proteins igg fc fused with peptides or proteins capon et al., affinity maturation improvement in binding to target hawkins et al., isotype switching for modified fc functionality fc functionality - change in fc activity greenwood et al., aglycosyl igg n x mutation to generate aglycosylated igg to reduce fcγr activity bolt et al., ; nesspor et al., heterodimeric consist of three components, the targeting antibody, the cytotoxic payload, and the linker that couples those two components together (fig. ) . with those three components come five considerations for the design and construction of an adc: first, the targeting antibody must bind to a protein that is found either exclusively on cancer cells or significantly overexpressed on cancer cells as compared with expression on normal tissues. the best targets for adcs may be oncofetal antigens or targets that may be overexpressed in cancer cells but present in normal tissues at low copy number or in tissues in which the toxicity is tolerable. the cell surface proteins most widely targeted with clinical stage (or approved) adcs currently are her (five adcs targeting), cd (four adcs targeting), cd (three adcs targeting), and mesothelin ( adcs targeting). cea-cam , egfr (wild-type), egfr (variant iii), cd , and cd each have two clinical stage adcs targeting them. the properties of good adc targets, as well as descriptions of candidate adc targets, have been reviewed (teicher, ; strohl and strohl, ). an interesting strategy being employed by cytomx to increase the tumor specificity of their adcs is the use of pro-antibodies that possess a peptide sequence covering the paratope, preventing binding to their target until it reaches the tumor microenvironment (tme). once in the tme, the paratope-shielding peptide is cleaved by matrix metalloproteinases (mmps), which are in high concentrations in most tmes, allowing the antibody to bind to targets in that local environment (desnoyers et al., ; polu and lowman, ) . second, the adc-directing antibody must be rapidly internalized upon ligation to its targeted receptor. antibodies that bind cell surface receptors may or may not internalize rapidly, so when isolating the antibody, incorporation of internalization screens into the discovery process is critical (poul et al., ; zhou et al., ) . third, the identity, number, and type of linker attachment sites is a critical issue. in first generation adcs, the linkers were typically attached to the ɛ-amine of lysine residues (tsuchikama and an, ; beck et al., ) . given that there are about lysine residues in a typical igg, ten of which can be accessed for chemical coupling (tsuchikama and an, ) , the results of such conjugations are highly heterogeneous. even with optimization, conjugation to lysines results in a drug to antibody ratio (dar) of about - , with a range of - (lazar et al., ; tsuchikama and an, ; beck et al., ) . there are multiple challenges with heterogeneous adcs including analytical challenges, batch-to-batch consistencies, the stability of the adc, and the potential for variable pharmacokinetics if conjugation sites in some antibodies interfere with normal fcrn-mediated recycling (beck et al., ) . site specific conjugation, which has been achieved through a variety of methods and can result in very tight dars and increased homogeneity (junutula et al., ; panowski et al., ; perez et al., ; beerli et al., ; ihospice et al., ; siegmund et al., ; thompson et al., ; tsuchikama and an, ; beck et al., ) , appears to be a significant advancement. new approaches using extension sequences, such as developed by mersana, can achieve a drug/antibody ratio of (yurkovetskiy et al., ) . fourth, the stability of the linker can have a huge influence on the efficacy and toxicity of the adc. in theory, a more stable linker which is only degraded within the lysosome should have the best safety profile. unfortunately, it is not that simple, as there are cases in which highly stable linkers resulted in safety issues. some of these may be due to mannose receptor, or potentially also fcγr-mediated binding and internalization of adcs, which could result in "off-target" toxicity issues (gorovits and krinos-kiorotti, ; beck et al., ) . finally, not all cancer cells within a tumor are target antigen-positive (singh et al., ) , thus allowing potential abbreviations: bbb, blood brain barrier; cdc, complement-dependent cytotoxicity; cdrs, complementarity determining regions; cns, central nervous system; ert, enzyme replacement therapy. innovative antibodies review escape of the antigen-negative cells from targeted therapies. it has been demonstrated that membrane permeability of the cytotoxin is a critical factor for potential bystander activity . thus, design of future adcs will need to take the chemistry of the resultant adc into account to optimize bystander effect and efficacy. there currently are clinical stage adcs, including three approved adcs, nine in phase iii development, and another in phase i/ii clinical development. the three approved adcs include mylotarg® ( , withdrawn in ), the cd -targeting adcetris®, and the erbb (her )-targeting kadcyla®. these clinical stage adc molecules are directed against at least different known targets, although a few have not been disclosed, so the actual number may be higher. the most targeted cell surface receptors currently are erbb and cd ( adcs against each), and cd , cd , and msln (mesothelin) ( adcs against each). there are known different classes of drugs incorporated into clinical stage adcs, of which are small molecule classes and five of which are protein-based. the most widely used drug class incorporated into clinical stage adcs are the auristatins (employed times), followed by the maytansanoids (in adcs), and benzodiazepines (used in adcs) (table ) . of the biologics, pseudomonas exotoxin pe is incorporated into four adcs (table ) . even though three adcs have been approved for therapeutic use, this technology is still relatively early in the developmental cycle and many of the "rules" for optimized adcs are still being sorted out (drake and rabuka, ; beck et al., ) . more details on the design and construction of adcs can be found in tsuchikama and an ( ) and in beck et al. ( ) . fc fusions are fusions of the igg fc domain with either a protein or peptide. in theory, the fusion can be to either the c-or n-terminus of the fc, but most fc fusions on the market and in clinical development today are n-terminal fusions. the primary reason for generating fc fusions is to extend the half-life of pharmacologically relevant protein or peptide by using the fcrn-mediated recycling of the fc (strohl and strohl, ; strohl, ) . currently, fc fusion proteins (strohl, ) , with the pharmacologically active "head groups" being blood factors, such as f (factor ix) and f (factor viii), peptides such as gcg (glp- ) and a thpo (thrombopoietin) analogue, and an enzyme, such as the tissue non-specific alkaline phosphatase (tnsalp; millan et al., ) in asfotase alfa (strensiq®) (hofman et al., ) . certain human cytokines such as il have been approved (marketed name, proleukin®) for systemic delivery and use in severe diseases such as metastatic melanoma and metastatic renal cell carcinoma (dutcher, ) . systemic delivery of the t cell-activating cytokine, il , however, brings with it the potential for adverse events. the concept of using antibodies to target cytokines to either tumors or to specific tissues came into fruition around the turn of the century (penichet and morrison, ; halin et al., ) . since that time, there has been an effort to target il , or other cytokines such as il and tnf, to the tumor microenvironment, where the desired activity can take place with reduced adverse systemic effects (neri and sondel, ) . this approach has been actualized by the fusion of cytokines to antibodies to make immunocytokines that may target vasculature associated with tumors (pasche et al., ; hemmerli and neri, ) , tumor cell surface antigens (klein et al., ) , or targets that would assist in accumulation in inflamed joints (hughes et al., ) . immunocytokines come in two major formats, cytokine-scfv (or other fragment) fusions which have a short circulating half-life and cytokine-igg fusions, which retain a long half-life (neri and sondel ) . antibody-directed modulation of immune cell checkpoint receptors has become one of the most exciting and important new areas in antibody therapeutics over the past few years. most efforts have been focused on t cell checkpoint modulation, but there is increasing interest in b cell, nk cell, and myeloid cell checkpoint modulation as well. t cell activation is regulated by a series of three signals. the first signal is provided by the interaction of the t cell receptor (tcr) with major histocompatibility complex (mhc, hla) class i (for cd t cells) or mhc (hla) class ii (for cd t cells) on antigen presenting cells (apcs). the secondary signal is provided through one of several checkpoint receptors (table ) , which can either provide a costimulatory signal to activate the t cells, or a blocking signal to dampen t cell response (topalian et al., ) . the third signal comes from the production of either pro-inflammatory, t cellactivating cytokines or anti-inflammatory cytokines that would act to reduce t cell response (chikuma et al., ; schirdewahn et al., ) . cancer cells can express ligands for t cell inhibitory receptors such as pdcd (pd- ) (ligand is cd [pd-l ]), ctla- (ligands are cd and cd ), and havcr (aka tim ) (ligand reported to be gal ) to inhibit t cell activation and cytolytic t cell responses. ligation of these receptors can lead to t cell anergy or exhaustion, resulting in the inability of the immune system to kill cancer cells. inhibition of the blocking responses to t cell activation using anti-pdcd , anti-ctla , or anti-cd antibodies has proven clinically to result in improved responses for a subset of patients with metastatic melanoma, nsclc, and potentially other forms of cancer (achkar and tarhini, ; kim et al., ) . additionally, efforts are ongoing to use combinations of anti-pd and anti-ctla antibodies to increase the percentage of patients experiencing durable responses, i.e., "raising the tail of the survival curve" (harris et al., ) . alternatively, several clinical candidates are agonists of t-cell activating receptors such as tnfrsf (ox ), cd , tnfrsf (cd , - bb), tnfrsf (gitr), icos (cd ), cd , or cd to stimulate t cell responses (antonia et al., ; table ) . additionally, t cell checkpoint pathways are potentially important in infectious diseases, in which t cell exhaustion halts t cells from eliminating viral and bacterial pathogens (dyck and mills, ) . finally, antibody intervention in t cell checkpoint pathways may play a role in autoimmune diseases, where blocking the activating signals or increasing the blocking signals may result in lowering the t cell activation response (van der vlist et al., ) . five mabs and two fc fusion proteins that target t cell/ apc checkpoints have been approved (table ) . two more t cell checkpoint inhibitor antibodies are currently in phase iii clinical trials and are in phase i/ii clinical trials, covering different t cell checkpoint targets. some of these checkpoint targets are being tested in both immune and oncology related diseases. for example, cd , cd , and tnfrsf (ox ) antagonists are in early stage clinical trials for treatment of various immune disorders, whereas cd , cd , and tnfrsf (ox ) agonists are in early stage clinical trials for various cancer indications (table ) . checkpoint ligands expressed on cancer cells also are potentially excellent targets, both because they can block the inhibitory checkpoint interaction as well as targeting the ligand-expressing cancer cells with fc-active antibodies. for this approach, there are now three approved anti-pd-l antibodies and another seven in clinical trials, as well as three clinical stage anti-cd (cd ligand) mabs and one cd -targeting car-t cell product in phase i clinical trials., as well as four anti-cd (b h ) antibodies are currently in phase i clinical trials. b cell transitional checkpoints are centered around b cell homeostasis and the choice of whether the b cell should mature or proceed to apoptosis. this process ensures that b cells expressing autoreactive immunoglobulins are purged (cancro et al., ) . key regulators of b cell maturation that function in b cell checkpoints are tnfsf b (soluble blys, ligands b lymphocyte stimulator; also known as b cell activating factor [baff] ) and tnfsf (april, a proliferation- * abbreviations: imm, immunology; onc, oncology. ** known preclinical programs that should progress to clinical trials by end of . *** names given as hugo gene nomenclature committee (hgnc) names (gray et al., ) followed by commonly used names in parentheses. inducing ligand). tnfsf b can bind the tnfsf b receptor (br ; also known as baff-r) to promote b cell survival, and both tnfsf b and tnfsf can bind tnfrsf b (transmembrane activator- and calcium modulator and cyclophilin ligand-interactor, taci) and tnfrsf (b cell maturation antigen, bcma), both of which result in ig class switching and t cell-dependent responses (cancro et al., ). overexpression of tnfsf b can lead to autoimmune consequences, such as system lupus erythematosus (sle) or sjögren's syndrome (cancro et al., ). one b cell checkpoint inhibitor (anti-tnfsf b mab, benlysta®) is approved, two more are currently in phase iii clinical trials, and three are in phase i/ii clinical trials, all targeting the b cell activating factor regulatory pathway. another approach that has gained interest in very recent years is the immunomodulation of nk cells. nk cells, as well as cd t cells, express a series of inhibitory receptors including klrc -form a (nkg a), tigit, cd , and kir family members (carotta, ) . as an immune defense mechanism, tumor cells express ligands to bind to these receptors to inhibit unwanted activation of nk cells. currently there are six antibodies in phase i/ii clinical trials binding these targets to remove the brake on nk cell activation. finally, another checkpoint that regulates the activity of macrophages and their phagocytosis of target cells is the cd /sirpa (signal regulatory protein alpha) and calr (calreticulin)/lrp pathway. the cd /sirpa ligation is often referred to as the "don't eat me" signal, whereas calr/ lrp ligation is known as the "eat me" signal (mccracken et al., ) . blocking of cd by antibodies or fc fusion proteins can lead to an imbalance and a pro-"eat me" response (mccracken et al., ) . currently, four anti-cd antibodies or fc fusion proteins are being evaluated in clinical trials for treatment of cancer (table ) . one approach that has gained interest in recent years is the combination or mixture of antibodies, usually against a single target, included into a single dosage (raju and strohl, ; carvalho et al., ) . thus far, antibody mixtures are being used mostly for oncology and infectious disease indications. the danish biotechnology company, symphogen, has led this space, with four antibody mixtures currently being tested in clinical trials. these include sym , a mixture of two anti-egfr mabs, sym , a mixture of six antibodies against the erbb (erb-b receptor tyrosine kinase) family of receptors (ellebaek et al., ) , sym , a mixture of two antibodies targeting met (cmet), and sym , an undisclosed mixture of antibodies partnered with genentech for an infectious disease target. at least nine other antibody mixtures are being evaluated in clinical trials, all of which are against infectious diseases targets such as ebola virus, botulinum toxin, and other viruses. one very interesting new approach in this area that could see significantly greater upside in the coming years is the generation of fully human antibody mixtures, or polyclonal mixtures, in transgenic (tg) cattle (matsushita et al., (matsushita et al., , . these may, if found safe and efficacious, at least partially replace "specific" intravenous immunoglobulin (ivig), which is igg purified from individuals who have been vaccinated or from convalescing patients who have produced iggs against a specific target (llewelyn et al., ; mire et al., ) . the upside of tg cattle-produced human iggs is supply, consistency across lots, and the ability to vaccinate the cows with antigens not available for human vaccination due to regulatory and safety considerations. one such polyclonal mixture from tg cattle already being evaluated in clinical trials is sab- (sab therapeutics), a polyclonal mixture of human iggs targeting middle east respiratory (mers) virus (nct ; luke et al., ) . bispecific antibodies, first conceptualized in (milstein and cuello, ) , are antibodies that can bind two different antigens simultaneously. there are five fundamental groups of bispecific antibody formats: (i) asymmetric bivalent, bispecific igg-like antibodies with heterodimeric heavy chains (hcs) (ridgeway et al., ; merchant et al., ; gunasekaran et al., ; strop et al., ; klein et al., ; labrijn et al., von kreudenstein et al., brinkmann and kontermann, ) ; (ii) tetravalent multispecific antibodies that are comprised of iggs, with additional binding domains, e.g., scfvs, fvs, vhh domains, or non-antibody binding scaffolds such as fynomers (brack et al., ; silacci et al., ) , fused to either the n-or c-termini of either the heavy or light chains (lcs) (coloma and morrison, ) ; (iii) engineered binding domains within the normal igg structure, such as the "two-in-one" bispecific approach from genentech (bostrom et al., ; eigenbrot and fuh, ) and the f-star approach of designing novel second binding sites within the c h domain , (iv) engineered antibody fragments linked by short peptide linkers which can be made into bivalent, trivalent, or tetravalent formats addressing two to three targets (mack et al., ; holliger and winter, ; kipriyanov et al., ; reusch et al., ; egan et al., ) . these may be fused to an fc domain or other half-life extending molecule ; and (v) iggs that are chemically coupled to generate igg-igg conjugates (e.g., brennan et al., ; garrido et al., ) . examples of these five basic formats are shown in fig. . many variations on these central themes have been reviewed multiple times (kontermann, ; spiess et al., ; kontermann and brinkmann, ; ha et al., ; brinkmann and kontermann, ) . over the past decade there has been a literal explosion of novel bispecific antibody technologies, approaches, and clinical candidates. today there are at least bispecific or bifunctional antibodies in clinical trials that are made from at least different bispecific platform technologies (table ) . these include ten asymmetrical igg-based platforms ( bispecific antibodies), five appended igg platforms ( bispecific antibodies), a single platform for chemically coupled iggs (four bispecific antibodies), eight fragment-based platforms ( bispecific antibodies), and one igg-based bispecific generated with an unknown platform (table ) . two bispecific antibodies have thus far been approved for medical use, both in the field of oncology. the first bispecific antibody of any kind to be approved was catumaxomab (removab®), a bivalent, trifunctional, hybrid mouse igg arat igg b antibody targeting cd e with one arm and epcam with the other. catumaxomab, approved in (only in the european union) for treatment of malignant ascites, was generated by the three-way fusion of a mouse b-cell, a rat b-cell, and a myeloma cell to form a quadroma cell line (triomab® technology) (zeidler et al., ) . the second bispecific antibody to be approved was the anti-cd e x anti-cd "bispecific t cell engager" (bite) mt- , constructed by linking two scfvs with a five residue (g s) linker (mack et al., ) . this bite®, now known as blinatumomab (trade name, blincyto®), was approved in for treatment of b-cell acute lymphoblastic leukemia (all). the current clinical stage bispecific antibodies are used for a variety of different purposes. for example, of them bind two soluble antigens such as il and il (e.g., sar ; nct ), nine bind two receptors on the , igg-scfv fusion, mabtyrin (igg with non-antibody binding scaffold "centyrin" fused to c-terminal end of heavy chains); (c) iggs to which additional antigen combining sites have been added within the structure (e.g., two-in-one antibodies, mat "modular antibody technology" platform from f-star); (d) engineered antibody fragments linked by short peptide linkers which can be made into bivalent, trivalent, or tetravalent formats addressing two to three targets (e.g., bispecific t-cell engager (bite), nanobody platform, dual-affinity re-targeting (dart) antibodies, "tandem antibody" structures (tandabs)); (e) chemically coupled iggs. brennan et al., ; garrido et al., innovative antibodies mack et al., ; schlereth et al., ; baeuerle et al., tandab antibody fragment- william r. strohl same cell surface such as egfr and met (e.g., jnj- ; nct ), and four bind a cell surface target such as delta like canonical notch ligand (dll ) with one combining site and a soluble ligand such as vegf with the other (e.g., navicixizumab; nct ). two current clinical stage bispecific antibodies are biparatopic, i.e., both arms bind to the same receptor, albeit at two different nonoverlapping epitopes (e.g., zymeworks zw , which binds two non-overlapping epitopes of erbb ; nct ). the most significant use of bispecific antibodies, however, is for t cell redirection, in which one combining site is directed toward a cell surface target on a cancer cell and the other combining site binds cd e on t cells to redirect those cells to the targeted cancer cell (see below). twenty-seven clinical stage bispecific antibodies are immune cell redirection bispecific antibodies. one of these targets fcgr a (cd a) for nk cell redirection, while the other bispecific antibodies target cd e on t cells to redirect the cytotoxic t cells (ctls) to kill and lyse cancer cells. of these, are constructed from antibody fragments, seven are asymmetric bispecific iggs, four are conjugated bispecific iggs that are used to activate t cells ex corporally (brennan et al., ; garrido et al., ) , and two are bispecific iggs with appended domains (table ). the two appended iggs also utilize an asymmetric fc format so that only one cd e-binding arm is present. it is generally accepted that the most potent t cell redirecting antibodies are fragments, with unmodified bites and darts (dualaffinity re-targeting antibodies) demonstrating sub-picomolar ic values for in vitro killing activities (moore et al., ) . of the two approved antibodies, blincyto® is a mouse bite, while removab® is an asymmetric rat/mouse igg. given that both are "first generation" t cell redirecting, fully mouse antibodies for very different indications, it is difficult to say today which type of platform (fragment vs. igg-based) will ultimately be the most efficacious for treatment of diseases. the larger igg-based forms appear to be significantly less potent based on in vitro activities and in vivo preclinical dosing than are fragments (unpublished data). thus, there is a balance between sheer potency, which can be achieved with small size, and long half-life, which typically brings with it greater size and less potency. additionally, both the size of the cell surface receptor of the target cells and the epitope to which the antibody binds appear to be critical factors in potency as well (bluemel et al., ) . moreover, the potency of t cell redirected bispecific antibodies depends on the affinity of the arms for each antigen. typically in the case of bispecific t cell redirection antibodies, the affinity for the cancer cell surface target is much higher (i.e., -fold or more) than the affinity for the cd e chain on t cells (zhukovsky et al., ) . in summary, factors that may influence potency in t cell redirected antibodies are size of the antibody, size of the target cell surface protein, epitope on that protein to which the antibody binds, and affinity. another area that has not yet been fully investigated with respect to t cell redirection is the role of fc functionality. the triomab® platform, on which removab® is designed, has a highly active fc domain that interacts with human fcγrs to increase the immune response (chelius et al., ; hess et al., ) . on the other hand, most of the current fragment-fc, asymmetric igg, or appended igg platforms have used muted or silenced fcs so as not to over stimulate the immune system via interactions with myeloid effector cells. even with the absence of fc activity, many treatments with t cell redirecting bispecific antibodies are accompanied by cytokine storms that need to be addressed as part of the therapeutic paradigm . thus, it seems likely that most t cell redirecting antibodies made in the future will continue to avoid fc activity in an effort to limit the release of pro-inflammatory cytokines by t cells and other effector cells in the tumor microenvironment. cars are anti-tumor targeted antibodies that have been fused genetically to a stalk or linker, a transmembrane domain, and intracellular t cell activation domains that have been borrowed from activation checkpoint receptors such as cd , tnfrsf (cd ), and/or tnfrsf (ox ) ( fig. m ; figueroa et al., ; van der stegen et al., ; ruella and gill, ; smith et al., ; ruella and june, ; lim and june, ) . while the concept of car-t cells has been around since the early s (eshhar et al., ) , the advancement of technologies required to turn this into a viable "manufacturable" process was only realized in recent years. thus, similar to bispecific antibody technology, while conceptually old, truly developable car-t technology is still relatively young and still developing (lim and june, ) . there are fundamentally two types of cars. the first is autologous, in which a patient's t cells are collected by a process known as apheresis, and then either as a whole pool, or a fractioned pool of cd t cells, cd t cells or possibly both cd and cd t cells, are transduced with the cars using either viral vectors such as lentivirus or transposons such as sleeping beauty or piggyback (figueroa et al., ; lim and june, ) . the recombinant t cells, now armed with cars targeting a tumor expressed on their surface, are activated and infused back into the patients from which they were derived to kill cancer cells bearing the antigen (figueroa et al., ) . the second major type of car is allogeneic, or universal. an "off-the-shelf" cell line is constructed, typically devoid of mhc class i molecules (ren et al., a ) and endogenous t cell receptors (macleod et al., ; ren et al., a) to decrease the risk of host vs. graft (rejection) and graft vs. host disease (gvhd), respectively. this universal t cell line also would express cars for treatment of cancer or possibly viral infections. thus far, the barriers to generate truly off the-shelf allogeneic cell lines are still quite high, with control of proliferation, continued activation of the cells once they are engrafted, and incorporation of kill switches for safety purposes as critical issues still to be worked out. nevertheless, significant progress has been made in just the past year suggesting that fully modified allogeneic car-t cell therapy is quickly becoming a reality (ren et al., a, b) . to date, there are four generations of autologous car-t cell constructs. the first generation typically consisted of the extracellular, cancer cell-targeting scfv fused to the cd stalk and transmembrane domain followed by cd (aka cd ζ), which provided the activation signal (park and brentjens, ; figueroa et al., ; lim and june, ) . the first generation cars possessed ample cytotoxicity but lacked proliferative and survival signals. the second-generation cars typically linked the exodomain scfv to the transmembrane domain of cd , tnfrsf (cd , - bb), or tnfrsf (ox ) to provide a proliferation signal, followed by cd (cd ζ) to provide the cytolytic activation signal. the third generation cars have typically linked the targeting scfv to the cd transmembrane domain, followed by either the tnfrsf (cd , - bb), or tnfrsf (ox ) activation domains, and then cd (cd ζ) (park and brentjens, ; figueroa et al., ; smith et al., ; lim and june, ) . these cars combined cytolytic activity with both proliferation and survival signals to enhance both their activity and their persistence in the patient's serum. fourth generation cars add new activities such as a suicide mechanism to kill off the cars in case they become overproliferative, or utilize t cells that have been conditioned to recognize viral antigens which can be used as "vaccines" to increase the persistence of the car-t construct (chmielewski et al., ; smith et al., ; lim and june, ) . there are currently different car constructs in clinical trials. as stated earlier, all of the car candidates are in phase i or ii clinical trials. almost half ( / ) of the current cars originated in china, with originating in the us, and originating in europe. cars have been generated against different targets, of which are cell-surface proteins on cancer cells and one, wt , an mhc-displayed peptide target derived from an intracellular antigen (rafiq et al., ) . fifty-three (∼ %) clinical car candidates are directed against cd . the next most targeted antigens are gd and msln (mesothelin) ( cars each), erbb (her ) and cd ( cars each), and gpc (glypican- ) and tnfrsf (cd ) ( cars each). most of the current clinical stage car constructs are autologous car-t constructs generated from αβ t cells (table ) , but there are a few examples of other formats, including early formats of allogeneic car-t cells, autologous car γδ t cells, both autologous and allogeneic car-nk cells, car-nkt cells, and cars made from tcrs (table ) . it is too early to judge the success of the car field, although it is clear that this area has generated an enormous amount of interest, as well as funding well exceeding $ b. it is noteworthy that novartis recently ( / / ) filed a biologics license application (bla) to the us fda for treatment of relapsed and refractory b-cell acute lymphoblastic anemia (b-all) with ctl (tisagenlecleucel-t), making it the first car construct to be submitted for regulatory approval (kingwell, ) . moreover, kite pharma announced shortly thereafter ( / / ) that they had completed their rolling bla submission for treatment of non-hodgkin lymphoma (nhl) using kte-c (axicabtagene ciloleucel). if either ctl or kte-c is, or both are, approved within the next year, it will mark a huge milestone in this exciting new field. an area that has been of interest for many years, but has proven challenging, is the targeting of antibodies to compartments into which they do not normally go. these include, for examples, targeting antibodies to the gut via an oral route, to the brain by crossing the blood-brain barrier, or to the cytosolic intracellular compartment. all of these compartments present significant challenges, but in the past few years, significant strides have been made for all of them. the most advanced tissue-targeted antibody-based product the bone-targeted enzyme replacement-fc fusion, asfotase alpha (strensiq®), which was approved by the us fda for treatment of hypophosphatasia (hofman et al., ) . asfotase alpha (tnsalp-fc-deca-aspartate fusion protein) is targeted to bone with a deca-aspartate peptide fused to the c-terminus of the fc (millan et al., ) . the second area of antibody targeting that is represented by clinical candidates is based on the route of delivery to get the antibodies to the desired compartment. at least three orally-delivered, antibody-related proteins targeted to the intestinal tract are currently being evaluated in clinical trials. these include prx- (protalix®), an anti-tnf plant cellexpressed and delivered fc fusion protein in phase ii clinical trials (nct ) for the treatment of ulcerative colitis biologics for certain types of cancer where the tumor is more accessible (zeltsman et al., ) . a novel approach for delivering mabs and/or fc fusion proteins is via delivery of the gene or genes that produce them, either as naked dna, rna, or by a viral-based vector. this is not an entirely new approach, since studies were done around the turn of the century showing that rna (giraud et al., ) and viral (lewis et al., ) delivery of igg genes could result in demonstration of in vivo igg activity. nevertheless, there was not much interest until the past few years, when it has become evident that vectored or nucleic acid delivery of igg could potentially be a significant new approach to deliver antibodies for therapeutic use. one of the more exciting forms of delivery is the intramuscular injection of adeno-associated viruses (aavs) encoding antibodies, followed by years of consistently high expression of those antibodies in non-human primates greig et al., ) . it is important to note that aavs exist in the muscle cells as extrachromosomal elements and do not integrate, which increases the safety of their use for long term expression of antibodies or other proteins (greig et al., ) . this suggests that such an approach might be appropriate for delivery of anti-hiv antibodies to help patients either to become cured or, minimally, less reliant on highly active anti-retroviral therapy (haart) (schnepp and johnson, a; . there are several very promising, potent anti-hiv antibodies in clinical trials currently, some of which have been expressed in vivo using gene-based delivery of antibodies for potential therapeutic use (schnepp and johnson, b; yang and wang, ; . similarly, but with a different twist, aav-delivered antibodies to the nasal passages of mice have demonstrated excellent prophylaxis against flu virus (limberis et al., ; balazs et al., ; adam et al., ) . since these aavs enter epithelial cells that are sloughed off over several months, this provides a potentially safe route for delivery of prophylactic anti-flu antibodies that would cover the entire flu season. the potential significance of this approach is that there are several ha-binding and neutralizing antibodies available now that are nearly universal influenza virus inhibitors. these could potentially be used in clinical trials to determine whether or not this prophylactic, pan-influenza nasal delivery approach might be feasible. finally, the concept of using oncolytic viruses to deliver anti-tumor or checkpoint modulating antibodies to a tumor is very exciting. oncolytic viruses have been engineered for years to deliver immune-modulating molecules such as csf (gm-csf) to the tme (bommareddy et al., ) , so it makes sense that they could be engineered to deliver tme modulating antibodies (du et al., ) . (adelfinger et al., ; liikanen et al., ; fajardo et al., ) . over the past decade there has been a significant shift from discovery and development of basic antibodies, e.g., naked igg isotype antibodies with no additional engineering other than perhaps humanization and affinity maturation, to more sophisticated forms of antibodies in all kinds of shapes and sizes. these newer forms include fc-modified, glyco-engineered, bispecific, drug-conjugated, and cell surface expressed antibodies (i.e., cars) as new weapons to fight difficult to treat diseases. we now see this dramatic shift in the types and numbers of modified antibodies now reaching clinical trial studies. this new phase of antibody drug discovery and development represents an exciting and bold new era that should see antibody-based therapeutics expanding their influence in many types of diseases. in the next few years we will likely see the first regulatory approvals of car-t based antibodies and immunocytokines, as well as approvals of additional new bispecific antibodies, new adcs, fc engineered antibodies, and glyco-engineered antibodies. additionally, we should see new advances in targeting antibodies to the cns and intracellular compartments, as well as nucleic acid or viral-vectored delivery. what an exciting time to be an antibody engineer! abbreviations aavs, adeno-associated viruses; adc, antibody-drug conjugates; adcc, antibody-dependent cellular cytotoxicity; adcp, antibodydependent cellular phagocytosis; adcs, antibody-drug conjugates; car, chimeric antigen receptor; ccr , c-c motif chemokine receptor; cdc, complement-dependent cytotoxicity; cxcr , c-x-c motif chemokine receptor ; egfr, epithelial growth factor receptor; epcam, epithelial cell adhesion molecule; erbb , erb-b receptor tyrosine kinase ; gpcrs, g-protein coupled receptors; hiv, human immunodeficiency virus; mab, monoclonal antibody; ms, multiple sclerosis; nk, natural killer; ra, rheumatoid arthritis; rsv, respiratory syncytial virus; tnf-α, tumor necrosis factor-alpha; vegf, vascular endothelial growth factor william r strohl declares that he has financial interest in johnson & johnson, for whom he was recently an employee, but no other potential conflicts of interest. this article does not contain any studies with human or animal subjects performed by the author. structure and function of the blood-brain barrier the blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells the use of immunotherapy in the treatment of melanoma adeno-associated virus -mediated airway expression of antibody protects old and immunodeficient mice against influenza virus preclinical testing oncolytic vaccinia virus strain glv- b expressing an anti-vegf singlechain antibody for canine cancer therapy novel type ii anti-cd monoclonal antibody (ga ) evokes homotypic adhesion and actin-dependent, lysosome-mediated cell death in b-cell malignancies a novel 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immunoglobulin fc heterodimer platform technology: from design to applications in therapeutic antibodies and proteins. front immunol engineering hiv-resistant, anti-hiv chimeric antigen receptor t cells enhancement of the antitumor activity of interleukin- by targeted delivery to neovasculature immuno-oncology combinations: raising the tail of the survival curve selection of phage antibodies by binding affinity. mimicking affinity maturation the antibody-based targeted delivery of interleukin- and to the tumor neovasculature eradicates tumors in three mouse models of cancer mor /es , a novel bispecific antibody targeting psma for the treatment of metastatic castration-resistant prostate cancer cancer therapy with trifunctional antibodies: linking innate and adaptive immunity asfotase alfa: enzyme replacement for the treatment of bone disease in hypophosphatasia diabodies: small bispecific antibody fragments diabodies": small bivalent and bispecific antibody fragments targeting of viral interleukin- with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain fv analogue produced in escherichia coli antibody recycling by engineered ph-dependent antigen binding improves the duration of antigen neutralization reduced elimination of igg antibodies by engineering the variable region engineered monoclonal antibody with novel antigen-sweeping activity in vivo sweeping antibody as a novel therapeutic antibody modality capable of eliminating soluble antigens from circulation sitespecific conjugation of monomethyl auristatin e to anti-cd antibodies improves their pharmacokinetics and therapeutic index in rodent models a plant cell-expressed recombinant anti-tnf fusion protein is biologically activity in the gut and alleviates immune-mediated hepatitis and colitis effector cell recruitment with novel fv-based dual-affinity re-targeting protein leads to potent tumor cytolysis and in vivo b-cell depletion replacing the complementarity-determining regions in a human antibody with those from a mouse site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index comparison of cell lines for stable production of fucose-negative antibodies with enhanced adcc endosomal acidic ph-induced conformational changes of a cytosol-penetrating antibody mediate endosomal escape survival benefit of immune checkpoint inhibitors according to the histology in non-small-cell lung cancer: a meta-analysis and review engineered proteaseresistant antibodies with selectable cell-killing functions car t therapies drives into new terrain bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics progress in overcoming the chain association issue in bispecific heterodimeric igg antibodies cergutuzumab amunaleukin (cea-il v), a cea-targeted il- variant-based immunocytokine for combination cancer immunotherapy: overcoming limitations of aldesleukin and conventional il- -based immunocytokines vectorization in an oncolytic vaccinia virus of an antibody, a fab and a scfv against programmed cell death- (pd- ) allows their intratumoral delivery and an improved tumor-growth inhibition medi : a pcsk ab-glp- fusion molecule that elicits robust antidiabetic and antihyperlipidaemic effects in rodents and non-human primates easd dual targeting strategies with bispecific antibodies bispecific antibodies sales of recombinant therapeutic antibodies & proteins. weikersheim efficient generation of stable bispecific igg by controlled fab-arm exchange oral administration of okt mab to patients with nash, promotes regulatory t-cell induction, and alleviates insulin resistance: results of a phase iia blinded placebo-controlled trial analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry engineered antibody fc variants with enhanced effector function clinical use of blinatumomab for b-cell acute lymphoblastic leukemia in adults a her -specific modified fc fragment (fcab) induces antitumor effects through degradation of her and apoptosis generation of neutralizing activity against human immunodeficiency virus type in serum by antibody gene transfer fusion protein from rgd peptide and fc fragment of mouse immunoglobulin g inhibits angiogenesis in tumor framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge intracellular released payload influences potency and bystander-killing effects of antibody-drug conjugates in preclinical models oncolytic adenovirus expressing monoclonal antibody trastuzumab for treatment of her -positive cancer the principles of engineering immune cells to treat cancer self-assembled protein nanocarrier for intracellular delivery of antibody intranasal antibody gene transfer in mice and ferrets elicits broad protection against pandemic influenza novel cd -based bispecific chimeric antigen receptor designed for enhanced anti-hiv potency and absence of hiv entry receptor activity mgd , a cd x cd dualaffinity retargeting bi-specific molecule incorporating extended circulating half-life for the treatment of b-cell malignancies discovery of antibodies effector attenuating substitutions that maintain antibody stability and reduce toxicity in mice enhancing endosomal escape for intracellular delivery of macromolecular biologic therapeutics human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo targeting t cells with bispecific antibodies for cancer therapy a small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity integration of a cd car into the tcr alpha chain locus streamlines production of allogeneic gene-edited car t cells conversion of ctla- from inhibitor to activator of t cells with a bispecific tandem single-chain fc ligand highly efficient deletion of fut in cho cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies delivery of antibodies to the cytosol triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production species-specific chromosome engineering greatly improves fully human polyclonal antibody production profile in cattle molecular pathways: activating t cells after cancer cell phagocytosis from blockade of cd "don't eat me" signals an efficient route to human bispecific igg enzyme replacement therapy for murine hypophosphatasia hybrid hybridomas and their use in immunohistochemistry passive immunotherapy: assessment of convalescent serum against ebola virus makona infection in nonhuman primates application of dual affinity retargeting molecules to achieve optimal redirected t-cell killing of b-cell lymphoma beat® the bispecific challenge: a novel and efficient platform for the expression of bispecific iggs chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains humanized porcine vcam-specific monoclonal antibodies with chimeric igg /g constant regions block human leukocyte binding to porcine endothelial cells immunocytokines for cancer treatment: past, present and future avidity confers fcγr binding and immune effector fuction to aglycosylated igg increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle antibody-dependent cell cytotoxicity: immunotherapy strategies enhancing effector nk cells sitespecific antibody drug conjugates for cancer therapy adoptive immunotherapy for b-cell malignancies with autologous chimeric antigen receptor modified tumor targeted t cells the antibody-based delivery of interleukin- to the tumor neovasculature eradicates murine models of cancer in combination with paclitaxel antibody-cytokine fusion proteins for the therapy of cancer antibody-drug conjugates: current status and future directions probody therapeutics for targeting antibodies to diseased tissue selection of tumor-specific internalizing antibodies form phage libraries a humanized antibody that binds to the interleukin receptor optimized t-cell receptormimic chimeric antigen receptor t cells directed toward the intracellular wilms tumor antigen potential therapeutic roles for antibody mixtures multiplex genome editing to generate universal car t cells resistant to pd- inhibition a versatile system for rapid multiplex genome-edited car t cell generation a tetravalent bispecific tandab (cd /cd ), afm , efficiently recruits t cells for the potent lysis of cd (+) tumor cells. mabs knobs-into-holes' engineering of antibody ch domains for heavy chain heterodimerization a novel investigational fc-modified humanized antibody, motavizumab-yte, has an extended halflife in healthy adults fcrn: the neonatal fc receptor comes of age development of pf- , a highly potent anti-p-cadherin/anti-cd bispecific dart molecule with extended half-life for the treatment of cancer discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria how to train your t cell: genetically engineered chimeric antigen receptor t cells versus bispecific t-cell engagers to target cd in b acute lymphoblastic leukemia chimeric antigen receptor t cells for b cell neoplasms: choose the right car for you anti-hiv designer t cells progressively eradicate a latently infected cell line by sequentially inducing hiv reactivation then killing the newly gp -positive cells identification and multidimensional optimization of an asymmetric bispecific igg antibody mimicking the function of factor viii cofactor activity redirection of t-cell effector functions for cancer therapy: bispecific antibodies and chimeric antigen receptors the third signal cytokine il- rather than immune checkpoint inhibitors contribute to the functional restoration of hepatitis d virus-specific t-cells eradication of tumors from a human colon cancer cell line and from ovarian cancer metastases in immunodeficient mice by a single-chain ep-cam-/cd -bispecific antibody construct novel human igg and igg fc-engineered antibodies with completely abolished immune effector functions adeno-associated virus delivery of broadly neutralizing antibodies vector-mediated in vivo antibody expression boosting antibody developability through rational sequence optimization improved cancer therapy and molecular imaging with multivalent, multispecific antibodies trastuzumab triggers phagocytic killing of high her cancer cells by interaction with fcγriv on immune cells high resolution mapping of the binding site on human igg for fc gamma ri, fc gamma rii, fc gamma riii, and fcrn and design of igg variants with improved binding to the fc gamma r lack of fructose on human igg n-linked oligosaccharide improves binding to human fcgamma rlll and antibody-dependent cellular toxicity spontaneous isopeptide bond formation as a powerful tool for engineering site-specific antibody-drug conjugates discovery and characterization of cova , a clinical stage bispecific tnf/il- a inhibitor for the treatment of inflammatory diseases quantitative characterization of in vitro bystander effect of antibody-drug conjugates a novel, native-format bispecific antibody triggering t-cell killing of b-cells is robustly active in mouse tumor models and cynomolgus monkeys chimeric antigen receptor (car) t cell therapy for malignant cancers: summary and perspective harnessing fc receptor biology in the design of therapeutic antibodies alternative molecular formats and therapeutic applications for bispecific antibodies in vitro and ex vivo strategies for intracellular delivery rituximab. how approval history is reflected by a corresponding patent filing strategy optimization of fc-mediated effector functions of monoclonal antibodies isotype selection and fc engineering: design and construction of fit-for-purpose therapeutic antibodies fusion proteins for half-life extension of biologics as a strategy to make biobetters therapeutic antibody engineering: current and future advances driving the strongest growth area in the pharma industry generating bispecific human igg and igg antibodies from any antibody pair nivolumab effectively inhibit platinum-resistant ovarian cancer cells via induction of cell apoptosis and inhibition of adam expression chimeric antigen receptos and bispecific antibodies to retarget t cells in pediatric oncology armed oncolytic adenovirus expressing pd-l mini-body enhances anti-tumor effects of chimeric antigen receptor t-cells in solid tumors cytotoxic mechanisms of immunotherapy: harnessing complement in the action of antitumor monoclonal antibodies antibody-drug conjugate targets straightforward glycoengineering approach to site-specific antibodypyrrolobenzodiazepine conjugates generation of binding molecules immune checkpoint blockade: a common denominator approach to cancer therapy antibody-drug conjugates: recent advances in conjugation and linker chemistries development of purification processes for fully human bispecific antibodies based upon modification of protein a binding avidity engineered glycoforms of an anti-neuroblastoma igg with optimized antibody-dependent cellular cytotoxic activity an engineered silent fc variant of an igg eliminates all immune effector functions via structural perturbations improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design aglycosylation of human igg and igg monoclonal antibodies can eliminate recognition by human cells expressing fcγri and/or fcγrii receptors study of natural antibodies against vascular endothelial growth factor receptor in hepatocarcinoma multimeric anti-dr igm antibody displays potent cytotoxicity in vitro and promotes tumor regression in vivo binding activities of a repertoire of single immunoglobulin variable domains secreted from escherichia coli brain penetration, target engagement, and disposition of the bloodbrain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin establishment of fut knockout chinese hamster ovary cells; an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity passive immunization against hiv/aids by antibody gene transfer boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates a polymer-based antibody-vinca drug conjugate platform: characterization and preclinical efficacy enhanced antibody half-life improves in vivo activity simultaneous activation of t cells and accessory cells by a new class of intact bispecific antibody results in efficient tumor cell killing surgical immune interventions for solid malignancies receptor tyrosine kinases in carcinogenesis (review) internalizing cancer antibodies from phage libraries selected on tumor cells and yeast displayed tumor antigens bispecific antibodies and cars: generalized immunotherapeutics harnessing t cell redirection (ilan et al., ) . the plant cells are thought to protect the fc fusion while traversing through the stomach. over the last decade, the mouse anti-cd e mab, okt , has been evaluated in clinical trials for oral delivery to the gut for treatment of nonalcoholic steatohepatitis (nash; nct ), with results suggestive of clinical activity (lalazar et al., ) . a second anti-cd e mab formulated for oral delivery is foralumab (ni- ; novimmune, tiziana life sciences), a fully human mab currently being prepared for phase ii clinical trials for oral delivery for the treatment of nash. getting antibodies to cross the blood-brain barrier has been a goal for well over two decades. igg levels in the human brain are approximately . % of the serum concentration of - mg/ml (abbott et al., ) . this differential is due to the blood-brain barrier (bbb) which effectively keeps antibodies out of the brain. considering the wealth of potential targets for biologics in the central nervous system (cns), there has been a great effort to find mechanisms to improve the ability to translocate biologics into the cns. in recent years, significant progress has been made in getting antibodies to traverse the bbb. yu et al. ( ) used a bivalent, bispecific antibody binding tfrc (transferrin receptor, cd ) with one arm and bace (β-secretase- ) with the other arm, to demonstrate that low affinity antibodies to tfrc were more efficient at transcytosis than high affinity antibodies. while they only achieved about -fold higher accumulations of antibody in the brain over controls, they clearly demonstrated anti-bace pharmacological activity of the antibody, proving that the antibody had accumulated within the brain (yu et al. ) . they also generated a bivalent, bispecific antibody targeting human and non-human primate (nhp) tfrc with one arm and human bace with the other arm . the best variants, which were low-to-moderate affinity antibodies to tfrc, were accumulated -fold higher in the brain than control antibodies and they demonstrated in vivo pharmacological activity in nhps .neiwoehner et al. ( ) compared the efficiency of transcytosis using a tetravalent, bispecific antibody with two arms each binding to tfrc and app (amyloid-beta, aβ) to a trivalent, bispecific antibody with only one arm binding tfrc. they found that monovalent binding to tfrc promoted efficient transcytosis whereas bivalent binding to tfrc resulted in shuttling the complex towards lysosomal degradation. they demonstrated a -fold improvement in target engagement over the control (neiwoehner et al. ) . in contrast to these studies in which monovalent targeting of the transcytotic receptor was optimal, the anti-tmem a (α( , )-sialoglycoprotein), llama single-domain antibody, fc (abulrob et al., ) , appeared to be transcytosed more efficiently as a dimer rather than a monomer (farrington et al., ) . recently, fc was fused in an scfv format to the n-terminus of the hc of an anti-grm (glutamate metabotropic receptor , mglur ) antagonist igg to shuttle it across the bbb (webster et al., ) , achieving pharmacological activity with a -fold enrichment of the antibody in the brain parenchyma (webster et al., ) . thus, it still appears that there is much to be learned about optimizing antibodies for transcytotic delivery of proteins to the cns.william pardridge and his colleagues have isolated an anti-human insr (insulin receptor) antibody that can be transcytosed by insr on endothelial cells lining the vasculature in the brain (boado et al., ) . they have used the anti-insr antibody as a transcytotic carrier to move enzymes across the bbb for cns enzyme replacement therapy (ert) (boado et al., (boado et al., , . these candidates are constructed by fusion of the enzymes to the c-terminus of the bbb-traversing anti-insr igg "hirmab" (boado et al., (boado et al., , . agt- , which is a tetravalent (two antibody arms and two enzymes) fusion of an anti-insr antibody and α-l-iduronidase (ali) (boado et al., ) , is being evaluated in phase i clinical trials (nct ) for the treatment of mucopolysaccharidosis i (mps i; hurler syndrome). agt- was recently demonstrated to be taken up by nonhuman primate brain at . % of injected dose as compared to % injected dose of α-l-iduronidase alone (boado and pardridge, ) , demonstrating the pharmacological relevance of the bbb-traversing bispecific antibody. agt- , comprised of a fusion of iduronate -sulfatase (ids) to the c-termini of the anti-insr hcs (boado et al., ) , is under phase i clinical testing (nct ) for the treatment of mucopolysaccharidosis ii (mps ii; hunter syndrome).the final delivery-related technology that has gotten very interesting in recent years is the delivery of mabs to the cytosol of cells via pinocytosis and endosomal escape (marschall et al., ; lönn et al., ; stewart et al., ; lim et al., ) . multiple approaches have been taken to get biologically active antibodies into the cytosol of cells, including the use of cell penetrating peptides (marschall et al., ; lönn et al., ; lim et al., ) . just recently, a unique antibody has been generated for the delivery of an igg to the cytosol of cells via endosomal escape (choi et al., ) . this antibody, which has a unique sequence in its light chain variable region, has been matured to increase the proportion of igg that enters the cytoplasm . this, and other cell penetration technologies (marschall et al., ; lönn et al., ; lim et al., ) bring hope that one day, antibodies will be used to target cytosolic antigens. traditional forms of delivery for mabs and fc fusion proteins has been via either intravenous (iv) or subcutaneous (sc) administration of formulated proteins. generally, high dose mabs for oncology indications are limited to iv dosing, whereas low dose antibodies such as adalimumab, golimumab, and ustekinumab can easily be delivered in sc doses. additionally, in recent years there has been increased interest in intratumoral dosing of antibodies and other key: cord- -hsu yo c authors: rosa, gonçalo m.; santos, nuno; grøndahl-rosado, ricardo; fonseca, francisco petrucci; tavares, luis; neto, isabel; cartaxeiro, clara; duarte, ana title: unveiling patterns of viral pathogen infection in free-ranging carnivores of northern portugal using a complementary methodological approach date: - - journal: comp immunol microbiol infect dis doi: . /j.cimid. . sha: doc_id: cord_uid: hsu yo c pathogen surveillance in free-ranging carnivores presents challenges due to their low densitie and secretive nature. we combined molecular and serological assays to investigate infections by viral pathogens (canine parvovirus (cpv), canine distemper virus (cdv) and canine coronavirus (ccov)) in portuguese carnivores (canis lupus, vulpes vulpes, lutra lutra, martes foina, m. martes, meles meles, and genetta genetta) over a period of years. additionally we explored spatio-temporal patterns of virus occurrence in canis lupus. our study identified cpv dna in all carnivore species with an overall prevalence of . %. cpv was detected in all sampled years and seasons in canis lupus, supporting its enzootic nature. cdv rna was mainly detected in the canidae family, with viral nucleic acid recorded between and with a peak prevalence of % among the wolf population, followed by a sharp decline, suggesting an epizootic behaviour of the virus. antibodies show that mustelids and viverrids were often exposed to cdv. ccov was first recorded by molecular methods in wolf samples in , remaining in the wolf populations with marked fluctuations over time. the dual serological and molecular approach provided important epidemiological data on pathogens of wild carnivores in portugal. these programmes should also include monitoring of other potential reservoir hosts such as domestic cats and dogs. there is a great diversity of known pathogens among free ranging populations of carnivores, which can be transmitted to both domestic and stray animals. thus, epidemiological surveillance and improvement of current methodologies for pathogen detection are crucial to understand the ecology, impact and dynamics of the diseases [ ] . general knowledge about disease dynamics in free-ranging carnivores is sparse, mainly due lack of epidemiological information concerning pathogen distribution among wild populations [ ] . viruses are important pathogens of wild carnivores that can affect populations through increased mortality and/or decreased general health [ ] . canine parvovirus (cpv) and canine distemper virus (cdv) are known pathogens of domestic and wild carnivores worldwide [ ] [ ] [ ] [ ] [ ] . although cpv infection is a relatively new disease first reported in the late s in domestic dogs [ ] , the virus has spread rapidly [ , ] . in europe, the presence of cpv has been documented in a variety of freeranging carnivore populations from canids to mustelids and viverrids, through serology or molecular methods [ , [ ] [ ] [ ] . additionally, duarte et al. [ ] reported high exposure to parvoviruses in mesocarnivores from portugal, but to date, no cases of mortality among wild carnivores have been reported in iberian peninsula [ , ] . canine distemper virus (cdv) is a highly contagious viral pathogen mainly transmitted by aerosols through respiratory secretions, and responsible for a lethal systemic disease in dogs and other carnivores [ ] . domestic dogs have largely been responsible for introducing cdv to previously unexposed wildlife [ , ] . in iberian peninsula, the virus has already affected wild carnivores, being responsible for death or disease of common genet (genetta genetta) [ ] , red fox (vulpes vulpes) [ ] and iberian lynx (lynx pardinus) [ ] . direct mortality due to canine distemper in wolves was documented in portugal [ ] , and several cases have been reported in other parts of europe (e.g. [ ] . in contrast, knowledge on coronaviruses (ccov) in free-ranging carnivores is very limited. serological surveys have been providing evidence that some of these species may serve as hosts for this viral pathogen [ ] . nevertheless, ccov seems to be enzootic worldwide in dogs [ , ] , which likely transmit the virus to wild carnivores [ ] . in europe, wolf population have been found positive for this virus in france and italy, although with prevalence < % [ ] . despite the sampling of several iberian populations of wild carnivores, only a single coronavirus-positive has been reported from a mongoose (herpestes ichneumon) [ ] [ ] [ ] . several carnivore species have suffered dramatic declines in europe during the last two centuries, with human activities leading to fragmented populations and consequently genetic isolation [ , ] . although infectious diseases are not usually the main factor affecting the survival of these species, virulent pathogens can act as a mortality source and cause epidemics. in fact, small fragmented populations, such as those of iberian wolves, are thought to become more vulnerable to disease outbreaks [ , ] . multi-host pathogens, such as cpv, cdv or ccov, can be maintained in the system through domestic dogs or other reservoir hosts, potentially impairing the viability of smaller more vulnerable wolf populations [ ] . despite all three viruses having already been reported in the portuguese territory in wild carnivores, either by detection of antibodies (abs) or nucleic acids (see [ , , ] , an assessment of the potential threat posed is hampered by the difficulty of overlaying the different methods for analyses. the available diagnose tools used (serology or molecular) or the target material (scats or body tissues) impair, to some extent, the interpretation and comparison between datasets. thus, with the present study we intend to investigate occurrence of infection and spatio-temporal patterns of selected viral pathogens (cdv, ccov and cpv) circulating in free-ranging populations of carnivores from northern portugal. the use of combined approaches that include serological (abs) and molecular detection as means for an enhanced perception of the results and inter study comparisons is discussed. the resulting information has critical implications for understanding of disease dynamics in these species and broader conservation policies. the study was conducted on species of free-ranging carnivores, in a total of individuals: canids -iberian wolf canis lupus signatus (n = ), and red fox vulpes vulpes silacea (n = ); mustelids -eurasian otter lutra lutra (n = ) and eurasian badger meles meles (n = ); viverrids -common genet genetta genetta (n = ), stone marten martes foina (n = ), and pine marten martes martes (n = ). samples were collected from animals opportunistically found dead between and in northern portugal, including four protected areas (peneda-gerês national park, montesinho natural park, douro internacional natural park and alvão natural park) and in beira interior region comprising the serra da estrela natural park (fig. ) . the main known cause of death was accidental road kills and shootings (table ) . samples were stored at - °c at the institute for the conservation of nature and forest (icnf) tissue depository (btvs/icnb) and in the monitoring system of dead wolves (smlm/icnf). the collection site, gender, age and the preservation status was recorded for each animal (table s ). sampled tissues from the iberian wolf included liver, spleen and lymph node; liver, spleen, small intestine/rectum and lungs for the red fox and for the remaining animal species small intestine/rectum and lungs. tissue homogenates were performed in phosphate buffered saline (pbs) solution and directly used for co-extraction of total dna and rna with the dneasy tissue and blood kit (qiagen, germany), according to the manufactures instructions. nucleic acids were extracted in a separate laboratory and after quantification in a nanodrop c (thermoscientific) were kept at − °c until analysed. according to the organ availability, lung, liver or spleen tissue extracts were prepared as previously described [ ] , stored at − °c and later used for specific ab detection. we conducted molecular screening with a taqman® quantitative pcr system already described [ , ] . cpv dna was amplified by quantitative pcr (qpcr) in a μl reaction with ng of template, using the taqman® gene expression × master mix (applied biosystems), . μm of primer forward, . μm of primer reverse and . μm of taqman probe. cdv and ccov rna were amplified by one step reverse transcription-qpcr (rt-qpcr) using the taqman® rna-to-ct (tm) step kit, . μm of primer forward, . μm of primer reverse and . μm of taqman probe in a μl reaction with ng of template. we performed the amplification in the applied instrument (applied biosystems) and the cycling conditions comprised an initial denaturation step at °c for min, followed by cycles at °c for s and min at °c. when the template was rna the amplification cycle included an initial reverse transcription step at °c for min. reagents assemble and template addition was performed in separate areas to prevent sample contamination. as positive control for cpv, we used tenfold dilutions of cpv- - cornell strain (tetradog®, merial). regarding cdv and ccov each recombinant plasmid was used as positive control as already described [ ] . water was used as negative control representing % of the total samples tested in each run. we used lung, liver or spleen tissue extracts to detect cdv and cpv abs by indirect enzyme-linked immunoassay commercial kits ingezim parvo canino . .cpv.k. and ingezim moquillo igg . .cdg.k. (ingenaza, spain), according to the manufacturer instructions. tissue extracts were tenfold diluted for cdv and cpv testing. the anti-dog conjugate provided in the kits was used for detection of the primary antigen (ag)/abs complex in the canid samples (wolves, red foxes); in the viverrid and mustelid samples it was replaced by the protein a-peroxidase from staphylococcus aureus/horseradish (sigma-aldrich) [ ] . for wolf and red fox we investigated the possible association between molecular findings and age (cub, juvenile, adult) and sex (male, female) of the animals with fisher's exact test. taking advantage of the larger sample size of wolf, we used the same test to assess the effect of season (winter/ spring vs. summer/ autumn) on the infection status by all the three viruses. to do so, we only considered the interval of years in which each virus was detected. both statistical analyses were computed using ibm spss statistics . software. a % confidence interval (ci) was calculated using the wilson score interval method without correction for continuity (the richard lowry's vassarstats online calculators: http://vassarstats.net/prop .html). lastly we tested if the samples preservation status would have an effect on abs detection also using fisher's exact test. we detected cpv dna in all the species with an overall prevalence of . % (table ) . particularly in c. lupus, cpv dna was detected in all sampled years (table ; fig. ) with prevalence of infection close to % across the four seasons. thus, time of the year had no significant effect on the infection status in wolves (fisher's exact test, p = . ). samples showed an average ct of . , ranging from . to . cts (standard deviation . ). c. lupus liver samples showed a higher frequency of viral dna positivity, but positive results were also obtained from the spleen and lymph node. vulpes vulpes were also highly positive ( . %; table ) with viral dna detected in the lungs, liver, spleen and small intestine. both mustelids tested positive for cpv with viral dna detected in the small intestine in l. lutra and lungs in m. meles (table ) . among the few screened viverrids, g. genetta yielded the lowest prevalence with out of animals positive (table ) in the lung. both marten species generated positives in lungs and small intestine. antibodies against cpv were found in . % of the samples, including canids where seroprevalence in wolves reached almost % (table ). nevertheless, evidence of a positive association was found between cpv seropositivity in wolves and the sample conservation status (fisher's exact test, p = . ), where better preserved samples had a higher likelihood of detecting ab. among mustelids, the two sampled individuals of m. foina had been exposed to cpv. cdv rna was detected in two families (canidae and mustelidae) with an overall low prevalence ( . %) ( table ). the organs with the higher yields were the liver and spleen in c. lupus, but the intestinal tract in v. vulpes and g. genetta. cdv rna was only recorded in and (table ; fig. ). the wolf populations reached a frequency of % (table : (table : %; / ). we found a higher prevalence during the summer/ autumn months and progressively decreasing towards spring (fig. ) . however, the small sample size did not allow for a statistically significant effect of seasonality (fisher's exact test, p = . ). cdv abs were detected from onwards ( table ) in . % of the samples where both canid species presented relatively high prevalences (c. lupus . %; v. vulpes . %) ( table ). we found seropositive samples in all species but g. genetta (table ). through molecular analyses, we obtained an overall prevalence of ccov of . %. ccov rna was detected in all three families. thirteen c. lupus ( . %; table ) tested positive in the spleen and four v. vulpes ( . %; table ) in the spleen and small intestine. positive results were also found in the two g. genetta and the single l. lutra, both in the rectum ( table ) . the virus was first detected in a single fox in . however, ccov rna was not recorded in our wolf samples until . we did not detect the virus also in and after (table ). although the data shows higher prevalence in the cooler months with a trending decrease in detection from autumn to summer (with no positive animals found in the warmer months; fig. ), the differences are not statistically significant (spring/ summer vs. autumn/ winter: fisher's exact test, p= . ). we recorded co-infections by multiple pathogens (two or three viruses) across the three families, representing . % ( / ) of the table distribution of viral nucleic acid and antibody detection, through the collection years. table ). unfortunately for most mixed infections, there was no information on the cause of death or any additional data that could help with the diagnosis. co-infection by the three viruses was detected in a single individual of c. lupus ( fig. ; table ). co-infection by cdv/ccov was also infrequent and detected in one juvenile of g. genetta (table ) . no significant associations were detected between age (fisher's exact test, p ≥ . ), or sex (fisher's exact test, p ≥ . ), and viral nucleic acid positive samples in any of the two canid species. diagnosis and detection of viruses and other pathogens in wildlife populations can be extremely difficult. long-term collection of samples allowed us to show that all three taxonomic families (canidae, viverridae and mustelidae) have been exposed to cpv, cdv and ccov in northern portugal. viral detection in free-ranging animals was confirmed by molecular and serological data. our study identified a high prevalence of cpv among all surveyed species. this seems to be particularly significant in the two species of canids, where wolves presented an infection rate close to %, although with a high variability of viral loads (ranging from . - . cts). cpv was detected in all sampled years and across all seasons in c. lupus, suggesting its enzootic nature in this study area. we believe that the social habits of iberian wolves [ ] may increase the frequency of intra-specific contacts and thus promoting the pathogen transmission. the findings mimic the results obtained by duarte et al. [ ] , where some southern populations of mesocarnivores in portugal also reached % prevalence, using the same methodological approach. in foxes, for example, cpv dna was detected in almost % of the tissue samples, not far from . % obtained in our study and within the confidence interval. contrastingly, other european studies described a much lower prevalence, ranging from . - . % in wolf scats in italy [ , ] , or . % obtained in france [ , ] , and even a total lack of viral dna detection in tissue samples from several species (germany [ ] . cpv infection is typically established by oronasal route by contact with contaminated faeces and after initial replication in lymphoid organs cpv localizes predominantly in the intestinal tract [ ] . whenever matched tissue samples were available, mustelids and viverrids tested positive in the lung but not in the small intestine, contrasting with the canid samples, particularly from wolves, in accordance with this pattern of virus dissemination. the pathogen's high stability in the environment and the ability to induce carrier states in susceptible hosts [ ] , support our findings. the high resistance of cpv to adverse environments [ ] , the higher sensitivity and specificity of qpcr, and the availability of target tissues for viral replication in vivo [ ] , may explain the higher detection of cpv dna, with variability of the viral load. nevertheless, there was a lower seroprevalence of cpv, as positive samples were approximately % lower compared to results obtained through molecular assays. this discrepancy may be explained by several factors including: the use of lung, liver or spleen tissue extracts as the biological matrix for ab detection; the use of the anti-dog conjugate in the elisa assay for canids samples, and the lower sensitivity of elisa assays for abs detection versus qpcr for nucleic acid detection [ , ] . additionally, we found a link between sample preservation status and seropositivity. yet, the seropositivity rate obtained suggests a high rate of cpv exposure, but a fig. . proportion of viral infections and co-infections in the two species of canids sampled from northern portugal. pathogen detection was assessed through nucleic acid detection. non pathogen detection represented by white proportion. viral infections and co-infections in carnivores of northern portugal evaluated through nucleic acid detection. low rate of cpv induced mortality [ ] , suggested by the perdominant low viral load detected in the samples. cdv was mainly detected in the canidae family with an overall prevalence below %. viral rna was only recorded between and with a prevalence peak in the first year among the wolf population ( %) followed by a drop down, suggesting an epizootic behaviour of the virus. similar infection pattern has been documented in several animal species worldwide [ ] , including in europe [ , , ] , with cdv often implicated in disease outbreaks and mortality events. the highly infectious nature of cdv [ ] may explain the sudden increase in prevalence. cdv serological data shows a much higher proportion of positive samples ( %) compared to nucleic acid detection. in addition to canids, we confirmed that mustelids and viverrids have also been exposed to cdv. the observed high seroprevalence suggests a high rate of circulation among the carnivore in northern portugal and confirms these species as being highly susceptible to cdv infections. considering cdv epidemiology, our finding may indicate cdv spill-over into wild populations, quite possibly from domestic and stray dogs acting as disease reservoirs [ , , , ] . ccov rna was first recorded in our wolf samples in , remaining with marked fluctuations over time. both canid species present similar overall prevalence values. in comparison, the incidence was much lower in italian and french wolf populations using faecal samples [ ] , which is not surprising given the low stability of viral particles in scats [ ] . ccov rna has been detected in dog tissues across europe [ ] , but despite previous efforts, only a single positive mongoose has been recorded in portugal [ ] . thus, our results represent the first record of viral infections in wild populations of c. lupus and v. vulpes in iberia, as well as for l. lutra and g. genetta. although our prevalence data are poorly informative about the impact of infection and disease in the populations, previous studies have shown that ccov infection seems not to be frequently associated with fatal disease; instead, it often causes mild, self-limiting enteritis followed by rapid recovery [ ] . nevertheless, mortality may occur as a consequence of mixed infections with any of the two studied virus, cpv and/ or cdv [ , ] . a proportion close to % of the carnivores was involved in mix infections where cpv dominated most of the combinations in all affected species. the latter is not surprising giving the high prevalence of cpv among the carnivore populations, as previously discussed. conversely, when compared to previous studies [ , ] , we found the overall proportion of co-infections we obtained to be relatively high. infections by ccov seem to be aggravated when occur simultaneously with cpv [ , ] and/or cdv [ ] , which could explain seven of the unknown deaths in wolves where we detected any of the two combinations and even one individual infected by the three viruses. yet, mech & goyal [ ] showed that the negative impact of cpv seems to be attenuated once the virus becomes enzootic in a population, which could explain the apparent lack of outbreaks in our system, even with such a high prevalence of co-infections. however, this suggestion should be interpreted with caution due to the absence of demographic data and survival of pups for the populations here considered. as our data does suggest that cdv is not enzootic in the northern portugal populations of wolf, individuals may be more vulnerable to cdv infection but as well as to co-infection with cdv and ccov. nevertheless, only a single individual was detected with this mixed infection but cause of death was unknown. lastly, dual infection by cdv/cpv seems to be overall less frequent, at least in wild carnivores in european as we only detected in three canids in our study. the lack of case reports makes it hard to predict impacts and consequences of infections. however, research on other species highlighted a case of fatal canine distemper infection in african wild dogs with concurrent infection by cpv in / individuals, leading the authors to believe that co-infections may have contributed to the fatal outcome in some of the dogs [ ] . free-ranging carnivores are of vital importance to the stability and integrity of most ecosystems, but some viral infectious diseases have shown the potential to negatively impact wild populations and cause declines [ , ] . almost half of the known infectious diseases of freeranging carnivores are of viral origin [ ] . although the three viruses here studied have been associated with mortality in wild and domestic canids worldwide, and some viruses could, theoretically, threaten the viability of small isolated populations (e.g. cpv [ ] ), their impact in wild carnivore populations is still largely unknown, particularly among the northern portuguese populations. despite high prevalence levels of cpv and cdv our populations, none of these pathogens seem to have been associated with declines in iberia, or listed among the major threats to the wild carnivores [ ] . this can be explained by a greater impact of fragmentation (road-kills) and illegal hunting, and/ or simple lack of robust data and regular disease surveillance. nevertheless, our results suggest a high and common exposure to cpv and cdv, with a higher seroprevalence of abs when compared to similar studies conducted in the iberian peninsula [ , ] . furthermore, we detected different antibody titers towards cpv and cdv, suggesting different contact timings between the animals and the viral pathogens. immune response towards cpv and cdv infection is considered lifelong, thus detection of seropositive animals implies exposure but also protection against the virus [ ] . considering the availability of recent data regarding cdv and cpv in portugal [ , ; this study], it is suggested an active viral circulation among the animal population studied. unvaccinated dogs, as well as wolf prey species, or scattered infectious faeces/urine, make the transmission of viral pathogens between populations a likely scenario [ ] . the control and widespread vaccination of domestic dogs and cats could reduce the potential spill-over of pathogens. additionally, constant evolution of virus through mutations in the genome [ , ] present a challenge for pathogen detection and consequently for adequate wild carnivore populations' management. this means that the results obtained in this and other studies may underestimate the infectious pressure, particularly when a single 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evolution of feline parvovirus (fpv) high rate of viral evolution associated with the emergence of carnivore parvovirus we acknowledge the financial support by portuguese national we would also like to acknowledge all the colleagues and collaborators who collected the samples in the field. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.cimid. . . key: cord- -ytbs wi authors: sabzpoushan, s. h. title: a system biology-based approach for designing combination therapy in cancer precision medicine date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: ytbs wi in this paper, we have used an agent-based stochastic tumor growth model and presented a mathematical and theoretical perspective to cancer therapy. this perspective can be used to theoretical study of precision medicine and combination therapy in individuals. we have conducted a series of in silico combination therapy experiments. based on cancer drugs and new findings of cancer biology, we hypothesize relationships between model parameters which in some cases represent individual genome characteristics and cancer drugs, i.e., in our approach, therapy players are delegated by biologically reasonable parameters. in silico experiments showed that combined therapies are more effective when players affect tumor via different mechanisms and have different physical dimensions. this research presents for the first time an algorithm as a theoretical viewpoint for the prediction of effectiveness and classification of therapy sets. study of cancer as the second leading cause of human mortality is essential. early diagnosis and appropriate therapies can be a significant help to the improvement of cancer survivals. although surgery in the case of solid tumors, antitumor drugs, radiation, and immunotherapy have been the treatment of choice in some instances, but ineffectiveness of treatments, drug resistance, side effects of therapies, and tailoring treatment to the individual characteristics of each patient are still major clinical problems. where precision medicine will allow researchers to predict more accurately which therapies will work better in which groups of people, combination therapy is a keystone of cancer therapy and potentially reduces drug resistance, while simultaneously providing therapeutic anticancer benefits, such as reducing tumor growth and metastatic potential, arresting mitotically active cells, reducing cancer stem cell populations, and inducing apoptosis. a high percent of oncology drugs and therapies fails in clinical trials [ ] . this imposes extra expenses to patients and causes the loss of time in cancer therapies. mathematical models, in silico experiments, and simulations can be a great help for evaluation of different therapies and examining diverse strategies of drug therapies. ten major characteristics of cancer, known as cancer hallmarks, have been universally recognized as ( ) unlimited multiplication, ( ) evasion from growth suppressors, ( ) promoting invasion and metastasis, ( ) resisting apoptosis, ( ) stimulating angiogenesis, ( ) maintaining proliferative signaling, ( ) elimination of cell energy limitation, ( ) evading immune destruction, ( ) genome instability and mutation, and ( ) tumor-enhanced inflammation [ ] . regarding above hallmarks, it is plausible that we attribute drug therapy efficiencies to individuals' genome, i.e., individual's heterogeneity should be taken into account in cancer therapies by some means [ ] . efforts have been devoted to determine how cellular and noncellular components of the tumor's surrounding environment may help it to acquire these characters. this environment and its cellular and noncellular components are called tumor microenvironment (tme) [ ] [ ] [ ] [ ] . the recognition that cancer cells need their microenvironment to efficiently display their phenotype has opened the door to hypothesize and implement new therapeutic strategies. today, the main tumor therapy strategies consist of surgery, radiological intervention, chemotherapy, and somatostatin analogs to control symptoms. however, it seems that tumor cells are particularly clever and elastic, and may adapt to treatments and environmental modifications quickly, i.e., once one component has been blocked, other mechanisms will rapidly follow. this may be one of the main factors that lead to poor cancer therapies [ ] . it is why different obstructing mechanisms at the same time might lead to the best results of tumor development prevention [ ] . the above facts illuminate the motivation for researches in the field of combination therapies. precision medicine refers to the tailoring of medical treatment to the individual characteristics of each patient. it often involves the application of system biology to analyze the cause of an individual patient's disease at the molecular level and then to utilize targeted treatments (often combinatorial) to address that individual patient's disease process. the branch of precision medicine that addresses cancer is referred to as "precision oncology" [ , [ ] [ ] [ ] . tumors are encircled by extracellular matrix (ecm) and stromal cells, and the physiological state of the tme is closely connected to every step of tumorigenesis. evidence suggests that the vital components of the tme are ( ) fibroblasts and myofibroblasts, ( ) neuroendocrine cells, ( ) adipose cells, ( ) immune and inflammatory cells, ( ) the blood and lymphatic vascular networks, and ( ) extracellular matrix (ecm) [ ] . the combinatorial complexity of possible combination therapies [ ] and the expense and risks of trial and error experiments, as well as the lack of time for cancer patients, are the main reasons for combination therapies fail in clinical trials. in this circumstance, appropriate biologically realizable models and bioinformatics can be a solution. mathematical models and computer simulations can be good alternatives for preestimations and evaluations of effectiveness of drug therapies strategies. computational oncology [ ] and in silico trials are good preclinical alternatives to predict the progress of the disease in individuals and suggest new diagnostic and therapeutic methods. patients with cancer are known to be at an increased risk for community-acquired respiratory viruses, such as sars cov- . there is high proportion of patients who acquired the infection while already in the hospital for cancer treatment affairs. using bioinformatics, mathematical and computational models and in silico analysis are very safe and cost-effective tools for design and analyzing therapy strategies. in silico trials as precision medicine simulators can reduce patient commuters to hospitals and high-risk health centers [ ] . cancerous system models can be categorized into three general groups: continuous, discrete, and hybrid, where each one may have deterministic or stochastic formalisms. continuous models describe the system by using ordinary differential equations (odes) or partial differential equations (pdes). several researchers have used odes to study the growth of tumors. to capture spatial structures of tumors, one should use pdes. pdes can better express the temporal and spatial properties of tumor growth at the same time [ ] [ ] [ ] . in the family of agent-based models (abms), cells are considered as discrete elements, and the interaction between them is defined by biological-based rules [ ] [ ] [ ] [ ] [ ] [ ] [ ] . abms can simulate emergent structures, i.e., structures that a number of not too complex components work together and form more complex behaviors as a group. it is noteworthy that in differential equation-(de-) based models, rules are applied to the whole system where in abms, the functional rules of each single agent can be specific and special to that agent. these make abms more appropriate for emergent behaviors like tumor growth and tme dynamics modeling. abm is also one of the most frequently used methods for modeling multiscale systems like cancers [ ] . today, cancer therapy has dramatically changed, i.e., surgery and radiotherapy are not the only effective ways to fight tumor. novel methods and approaches are emerging, where the molecular and agent features of tumors seem to be the keystone of any therapy. new antibodies, small molecules, antiangiogenics, viral therapy, and precision medicine methods are typical examples. because of the abovementioned new therapies use microscopic or molecular level agents explicitly, so system biology-based cancer models can be the most satisfactory candidates for in silico experiments and studies. it is notable to remember that system biology is a term to describe the study of the interactions between the components of the biological systems and how these interactions give rise to the function and behavior of that system. however, although system biology-based mathematical models of cancer are very useful, but they have also limitations, because the recognition of the mechanisms governing cancerous systems has practical limitations as well [ , ] . in this paper, we use our recently published agent-based stochastic tumor growth model (absm) as a cancer system. we design several combination therapies which can be hypothesized and regulated as precision medicine. we postulate five novel quantitative merits for comparing possible effectiveness of different combination therapies. we show how in silico experiments can help oncologists to conduct and design combination therapies, and test their ideas, biomed research international in this research, we have used our previously proposed absm model [ ] as a cancer system, i.e., we may fit it to a given patient and use it for demonstrating our system biology-based approach for designing combination therapy in cancer precision medicine. agent-based modeling is a stochastic approach used to describe a population of interacting agents, where agents behave according to a set of rules that represent the dynamic features of system. in this way, our absm has been established on four bases: ( ) biological assumptions, ( ) physical structure, ( ) agents and their states, and ( ) states transition rules. the multilayer structure of the absm is shown in figure . in absm, host tissue is assumed as a two-dimensional lattice composed of ncell × ncell squares as illustrated in figure . here, each square of the lattice is called a cell. in the absm, two types of agents (immune (ia) and nonimmune (na) cells) are presumed, where three types of nas (normal cell or empty space (na_ ), proliferating tumor cell (na_ ), nonproliferating or quiescent cell (na_ ), and necrotic cell (na_ )) are considered. in the absm, we assume two types of na_ cells with different division probabilities: in the first type denoted by na_ _ , the cell division is not influenced by its neighbors, while in the second symbolized by na_ _ , the cell division probability (ρ pt ) is affected by its healthy neighbors (na_ s). in the absm, it is assumed that each na_ _ may be divided into one na_ _ and one na_ _ daughter cells with the probability nmm or into two na_ _ daughter cells with the probability ( − nmm). it means the higher the value of nmm, the more susceptibility of cancerous cell division to its microenvironment. in table , the basic elements of absm and their brief descriptions are summarized. as it was stated beforehand, the absm is a system biology-oriented model in the sense that it is constructed from several agents and interaction rules between them, and as is shown in [ ] , these elements and interactions can give rise to the function and behavior of cancer growth system. with a conceptual and intuitive look at the elements of table , some of them can be assumed individual-dependent, those are indicated by a star mark. you see all but one of them has probability dimensions. we will discuss more this matter in following sections. more details and descriptions about the absm can be found in appendix a and [ ] as well. medicine. combination therapy, i.e., a treatment approach that combines two or more therapeutic agents, is a keystone of cancer therapy. the consolidation of anticancer drugs and therapies enhances efficiency compared to the monotherapy approach, because it targets key pathways in a characteristically synergistic or an additive manner. this approach potentially reduces drug resistance, while simultaneously providing therapeutic anticancer benefits, such as reducing tumor growth and metastatic potential, arresting mitotically active cells, reducing cancer stem cell populations, and inducing apoptosis [ , ] . in this section, we perform in silico experiments as preclinical tests to design and suggest combination therapies with the use of absm. based on cancer drugs and new findings of cancer biology, we hypothesize relations between model parameters and cancer drugs, and therapies, i.e., we assume each drug in a target group can impact related parameter(s) and show by controlling combination of drugs (controlling parameters); we may simulate combination therapy strategies and control tumor size. having in mind system biology approaches, a survey of cancer therapy literature shows that we may classify five possible groups of target agents in cancer therapies as (a) new vessel formation agents, (b) progrowth signal amplification agents, (c) progrowth signal transmission agents, (d) dna replication-related agents, and (e) cell cycle activation agents. these groups and their relative levels are schematically illustrated in figure . concerning the definitions and roles of the parameters of the absm, we have hypothesized the relations of four model parameters, p , p , age, and nmm, with above groups as is illustrated in table . in absm, the parameters p , p are base probabilities of division of na_ _ and na_ _ cells, respectively, and n mm is the probability of production of a na_ _ from the mitosis of a na_ _ . age is maximum allowable time duration to stay in na_ mode without proliferation. we see p , p , and nmm are probability numbers; thus, their values can be on [ , ] interval, where age is time or more precisely number of iterations [ ] . progrowth signal transmission, as is depicted in figure , sends division signals to the cell; after the first molecule in a pathway receives a signal, it activates another molecule; this process is repeated until the last molecule is activated and the cell function is carried out. therefore, the assumption that age may be considered as a control tool of the target group c is biologically plausible, i.e., "age" can control the delay of progrowth signal. as a drug in the group b, one may name "trastuzumab", an antibody drug conjugate (adc) consisting of the recom-binant antiepidermal growth factor receptor (her ) monoclonal antibody trastuzumab conjugated to the maytansinoid dm via a nonreducible thioether linkage (mcc) with potential antineoplastic activity. the trastuzumab moiety of this adc binds to her on tumor cell surfaces; upon internalization, the dm moiety is released and binds to tubulin, thereby disrupting microtubule assembly/disassembly dynamics and inhibiting cell division and the proliferation of cancer cells that overexpress her . all of these mean that it is reasonable to relate parameters such as p p = . ,p that are related to cell division (proliferation) by any means to drugs like trastuzumab and treat them as tools for controlling the target groups (b, d, and e). in absm, the parameter nmm is related to proliferative cell type and quality; this is why we have attributed it to the target group d. on the one hand, all parameters in absm have exact mathematical definitions and biological interpretations, and on the other hand, all the abovementioned drugs and many others which are used in cancer therapy [ ] are validated clinically [ ] [ ] [ ] [ ] [ ] , so we can treat table as a theoretical deduction which implicitly is supported clinically, i.e., the mapping from treatments to model parameters is implicitly validated. for simplicity and ease of graphical and qualitative analysis, in this research, we only consider combinations of two target groups for therapies, and because we have no delegated parameter in the group a, so the number of sets of two p = . table . biomed research international groups of four groups, b, c, d, and e, will be six as bc, bd, be, cd, ce, and de. reconsidering these six possible combinations and dismissing repeated parameter sets, i.e., combinations that have same players, we consider three distinct combinations of the groups, bc, bd, and cd, with the attributed players as are listed in table . as we stated earlier, p , p , and nmm are probability numbers, so they range on the [ , ] interval, and besides considering their definition in absm, they generally repre-sent the probability of (abnormal) proliferation of their respective agents (cells). therefore, a large value, i.e., near unit, means high probability of cell division where a small value (near zero) means low probability. generally speaking, anticancer drugs of the groups b, c, d, and e try to slow cell reproduction via their underlying mechanisms; therefore, it will be plausible if we assume a relation between drug dosage and its effect on cell division probability, i.e., the more the value of drug dosage, the less p = . table . biomed research international the value of expected probability value will be. because maximum allowable dosage of a drug differs from case to case, we consider qualitative measures high (h), medium (m), and low (l) and assume that these values act against h, m, and l proliferation (division) probability, respectively. in this situation, we are so lucky because probability number interval is known to be on [ , ] interval. in table , some typical values are listed, although these values are chosen randomly for simulation investigations, but we may attribute them to individuals' variabilities in genes or biological characteristics and may also attribute them to high, medium, and low dosages by some means. this matter will be discussed more in discussion. in this strategy of combination therapies, we assume that therapy agents disturb (target) progrowth signal amplification and progrowth transmission at the same time. we assume that the tumor growth system is the same as the one given in figure (in fact using medical records of a patient, a physician can use absm to simulate existing tumor growth in the patient, i.e., run a simulation like figure and draw a table like table , so she or he can have an estimate of parameters: p , p , nmm, and age). we see this (assumed) given patient has individual characteristics or personalized genetics as p = : , p = : , nmm = : , and age = ; we set up in silico combination therapy experiments and examine the tumor growth, with controlling the set (p , age) via dosages. the results are depicted in figures (a)- (i) . these figures show tumor structural details qualitatively in the day th. in all figures, the light grey represents normal tissue cells, the heavy grey represents outer region of tumor that is comprised of proliferating cells, the middle region of white color is nonproliferative (quiescent) cells, and the black region is necrotic cells. table compares the tumor structures quantitatively; as you see, we have chosen necrotic fraction (nf), pure tumor growth fraction (pgf), and growth fraction (gf) [ ] , as quantitative measures to judge the effectiveness of the therapies. as one sees, the size of tumor in this regime (pgf), on day th , is a number between and percent of the considered tissue depending on the doses values, where the harmfulness of it (gf) varies between and percent; in this circumstance, the nf measure, that can be assumed as a therapy effectiveness feature after days, is a number between and percent. as we will see later, the best result of therapy is seen in this set; it is highlighted in in figure (d) and table . another possible combination of players in the bc group is (p , age). we repeat our in silico study as above; the qualitative and quantitative results are depicted in figures (a)- (i) and table , respectively. the size of tumor in this regime (pgf), on day th, is around percent of the considered tissue, i.e., larger than the previous one. where the harmfulness of it (gf) varies between and percent, in this condition, nf, that can be assumed as a therapy effectiveness feature after days, is a number between and percent. it seems that this set of players (p , age) is weaker than its counterpart (p , age) of the bc group in the fight against cancer. in this tactic of combination therapy, we assume that therapies' players disturb progrowth signal amplification and dna replication process at the same time. here again, it is assumed that the tumor growth system is the one that considered in figure . tables - show the tumor structures quantitatively for each set, respectively. regarding table , the size of tumor in this regime (pgf), on day th , is around to percent of the considered tissue. the harmfulness measure of the tumor (gf) varies between and percent; in this condition, nf (the therapy effectiveness feature) is a number between and percent. this set of players has the worst results of fighting against cancer among the all considered sets, where it is highlighted in red color in figure (a) and table . figure and table illustrate results of therapies in the bd group, where (p , nmm) are players. although these players are a bit better than previous players of this group, but they should be categorized as weak players still. another set of players in the bd group is (p , nmm), where their play results against tumor growth are summarized in figure and table . results confirm that we may label these players as weak ones like the other players of the bd group. . . group cd combined therapies. in this approach of combination therapies, we assume that therapy actors disturb progrowth transmission and dna replication process at the same time. for investigating the behavior and properties of this kind of combination therapies, we do the same as the two previous groups. this strategy has three sets of two players: (age, p ), (age, p ), and (age, nmm). it is noteworthy that the effects of players (age, p ) and (age, p ) have been investigated as players (p , age) and (p , age) in the bc group beforehand; therefore, we only consider the player set (age, nmm) as the agents of the cd group. qualitative and quantitative measures of therapies, when (age, nmm) act against tumor growth, are summarized in figure and table , respectively. it is seen that although these agents are better in comparison with the bd group teams, but are not as well as bc teams. the tumor size of % can be reached after days of therapy, where the score p = . p = . p = . figure : snapshots of tumor structure where therapy regime hits progrowth signal amplification and dna replication process. all tumor structures of cancer system on day , but at different dosages (a-i) of a combination therapy. biomed research international of % of harmfulness will be realizable (the % of therapy effectiveness measure); nf can be accessible by this team. precision medicine and combination therapies can improve the life expectancy of most patients and diminish damages to the tissues surrounding the tumor [ ] [ ] [ ] . in this way, mathematical models and in silico experiments are of great help. in this research, we used absm and hypothesized some relations between the model parameters and anticancer drug (agents) groups; besides, some parameters were attributed implicitly to individual variability. we performed in silico experiments and investigated the effects of some combinations of these parameters as therapy players. here, we analyze and discuss the postulated combination (and precision) therapy strategies and evaluate the results against recent findings in cancer biology and therapies. as stated beforehand, one of the intents of the researchers in the field of oncology is to take into account individual variability for each person (e.g., in genes, environment, and lifestyle) and understand and examine potential role(s) of any combinations of therapies on cancer cells' growth and spread [ , , , , , ] ; in addition, with regard to cancer hallmarks named in introduction, we see that six out of ten hall-marks are related to cell division explicitly or implicitly (hallmark numbers: , , , , , ) , and generally speaking (and setting aside environment and lifestyle), cancer is basically a genetic disease of cells; all of these mean that cell division probability (and cell cycle as well) is a genetic, i.e., individual representative. a dose refers to a specified amount of medication (therapy) taken at one time, and dosage is the prescribed administration of a specific amount, number, and frequency of doses over a specific period of time, i.e., a dosage guides a drug regimen. based on therapy type, doses are expressed in different metrics like mass units (e.g., milligrams), drops, and radiations. however, to avoid a special kind of metric or therapy, we use more general metrics (high, medium, and low doses) as it is common in medicine. in this situation, a physician can have his or her own interpretation from high, medium, and low metrics according to underlying cases. in this way, we may attribute [ , ] interval as a mathematical representation (or normalized quantity) of [low, high] doses (and dosage as well). this issue is illustrated in figure . note that figure illustrates only the mapping between domains, and not the exact relations among variables of different domains; in fact, relationships may be complex and nonlinear. nevertheless, "high, medium, low" approach can be considered table . biomed research international as an alternative for avoiding engagements in complex relations among variables of different domains. to evaluate the best combination therapy strategy, we not only used qualitative graphical structures of the tumors on day th that are depicted in figures - but also used quantitative measures (pure tumor growth fraction (pgf), growth fraction (gf), and necrotic fraction (nf)) [ ] . pgf, that is the fraction of the whole number of tumorous cells to the whole number of all cells in tissue, ncell × ncell, can be treated as a quantitative measure of the tumor size; a less pgf on day th means a smaller tumor. gf, that is the fraction of the population value of tumor proliferative cells to the population value of all tumorous cells, can be treated as a measure of the aggression and invasion potential of tumor; therefore, if two tumors have the same size on day th , we should look at their malignancy and prognosis for their future growth; in this case, gf can be used as a measure; the more the value of gf, the more harmfulness and aggression can be expected. nf, i.e., necrotic fraction, is the ratio of the number of necrotic cells to the whole number of tumorous cells; therefore, in spite of gf, nf can be treated as a measure of table . biomed research international manageability and controllability of the tumor; a larger value of nf on day th means a less dangerous tumor. nf can be treated as a measure that shows how a therapy can prevent tumor cells to be proliferative. one of the main problems in the examining different combinations of drugs (agents) is the huge number of different alternatives [ ] , e.g., when trying to identify the best combination of drugs at doses (e.g., high, medium, and low doses), one will have to test combinations. in this research, by defining five new features, we introduce a general algorithm for prediction (or suggestion) of the number of possible more (less) effective combinations. the usefulness of this algorithm is that we can try the best expected combinations of therapies at first and consume the time and expenses in clinical trials. it is well established in oncology that different obstructing mechanisms at the same time (compound therapy) might lead to the best results of tumor development prevention [ ] . in this circumstance, we expect therapies with more diverse players, i.e., the sets with different players from different groups and parameters that have different interpretations from cancer biology point of view will create more successful pairs of fighters against cancer. in this way, because different physical dimensions usually represent different action mechanisms, we give a positive score to therapy teams which have players with different physical dimensions; to deal with this favorite score, we have introduced a novel metric (dpd) in our research. to introduce our algorithm and attribute the above biological findings to combination sets, we reorganize table as depicted in table and add the following feature numbers to it: the number of different players of the considered target groups (dtg) in a set, the number of same players of the considered target groups (stg) in a set, the number of different mechanisms of parameter (dmp) actions, the number of same mechanism of the parameter (smp) actions, and finally, let us give an additional positive score to the teams with diversity number of the physical dimension (dpd) of the players. as an example for better description of table , in the fourth row, the features of the combined group "cd" have been summarized, i.e., considered groups are c and d. reconsidering tables and , we see that parameters in each three sets, (age, p ), (age, p ), (age, nmm), are all from different target groups, so we attribute the number to dtg feature of each set. we also see that in all three sets, (age, p ), (age, p ), (age, nmm), there is no parameter in common between target groups c and d; therefore, we assign the number to stg feature of each set. regarding the definitions of each parameter in each three sets, (age, p ), (age, p ), (age, nmm), it is clear that they affect the tumor growth by different mechanisms; therefore, we assign the number to dmp feature of each set. as reasoned above, we should give the number to smp feature of each set. it is interesting to note that each parameter in each set of (age, p ), (age, p ), (age, nmm) has different physical dimensions (time or probability); so as you see in the last column of table , we may assign an additional positive score to each team, i.e., the number to dpd feature of each set for the cd group. considering definition of terms, dtg, stg, dmp, smp, and dpd, it is clear that in a considered therapy, the higher the value of a term that begins with letter d and the lower the value of a term that begins with letter s, the better feature that therapy has. inspecting tables - , we can find the best and the worst therapies as follows: the best therapy result belongs to the combined group bc, where p and age are treated as therapies agents; the tumor features of this therapy on day th are highlighted in table . the worst result can be assigned to the combined group bd, where p and p are therapies players; the tumor features of this therapy on day th are highlighted in table . the features of these two therapies are compared in table . it is clear that p and age are players from different target groups (dtg = , stg = ) and affect tumor growth with different mechanisms (smp = ) and having different physical dimensions (dpd = ). it is where p and p are players that are common between target groups b and d (dtg = , stg = ) and affect tumor growth with different mechanisms ( smp = ) and having same physical dimensions (dpd = ). in this research, the hypothesized algorithm has been discussed for the set of therapies with two players; nevertheless, it can be extended to three and more player sets in the future researches. in fact, the importance and meaning of presented quantitative merits (dtg, stg, dmp, smp, and dpd) are more realizable, when one faces the sets with three or more players. although the presented research can help one to broaden the conclusions drawn from existing medical data, suggest new experiments, test hypotheses, predict behavior in experimentally unobservable situations, but has some limitations that need further investigations. where the value of a parameter as a therapy player may be controlled by dose value, some of parameters may also be related to individuals' genetic contents. in this context, although table represents the best therapy results, but it is seen that the best of the best results is where we set a high dose value to control p and an intermediate value for age; in table , it is seen that the worst results are for high doses for controlling p and p when we deal with a given patient with personal characteristics like table . the implementation of the roles of dose values in our presented algorithm should be more investigated. biological systems are complex; they are composed of several agents and sophisticated interactions (which look to be stochastic) among them. this is where stochastic system biology-based models can help us. in fact, in this circumstance, models such as our absm seems to be more compatible with the biology of cancer than ode or pde ones; in addition, although personalization and relationship between dosages and parameter values in absm look like to be difficult and a challenging issue, but proposed "high, medium, low" approach which is pharmacologically (biologically and therapeutically) plausible seems to be satisfactory; however, this matter should be more investigated in the future researches. one of the current limitations of our model is that we have not considered angiogenesis in it, and therefore, it has no delegated parameters in the target group a. we are hoping to add angiogenesis to absm in our future work. in this research, because the number of model parameters that are attributed to players of different target groups is limited, therefore, we could not investigate all possible combination sets of group players, e.g., be, ce, and de. we think about making a training and teaching software tool, using our proposed model and method. the results of this work should be more explored from clinical applications' point of view, and our research colleagues in cancer institute of iran are looking for more biological validations of our findings by implementing in vitro experiments. an interesting direction for future research can be the investigation of side effects of each combined groups. a side effect is an important issue in cancer drug therapy [ ] . it can decrease the life quality of a patient. it seems reasonable that combination therapies that can employ lower doses of each therapy element have the potential to milder side effects. it means that for more precious deductions from tables - , we may introduce a new merit that involves dosedependent side effects of each combined groups. in addition, because side effect, i.e., damage to na_ agents due to drugs, is not involved in absm, we hope to implement the modified version of the model in the future. our colleagues in the cancer research institute of iran [ ] are working on the setting up of statistical studies to test the proposed combination precision cancer therapy. however, although the quality of a scientific field depends on how well the mathematical descriptions developed on the theoretical side agree with results of experiments, possible lack of agreement between theoretical mathematical explanations and experimental measurements often leads to important advances as better theories are developed. it should be noted that absm like other agent-based models may suffer from computational bottleneck for large numbers of cells [ ] . this matter should be investigated, and possible use of "hybrid" approaches can be considered in the modified version of absm. treatment schedule and therapies strategies can improve the life expectancy of cancer patients. although precision medicine along with consolidation of cancer therapies, combination therapy enhances efficiency compared to the monotherapy approach, but which combination for which person is the most effective one is an open question. in this study, we presented a method and novel quantitative merits-dtg, stg, dmp, smp, dpd-that an oncologist can use to estimate and predict effectiveness of therapies for each individual and make quantitative rankings among therapies. in silico experiments showed that combined therapies are more effective when players affect tumor via different mechanisms and have different physical dimensions. our absm is founded on four bases: ( ) biological assumptions, ( ) physical structure, ( ) agents and their states, and ( ) states transition rules. in absm, host tissue is assumed as a two-dimensional lattice composed of ncell × ncell squares as illustrated in figure . here, each square of the lattice is called a cell. as is shown in figure , we assume that a tumor consists of three layers: ( ) the outer layer of proliferative tumor cells (dotted region), ( ) the middle layer of nonproliferating or quiescent tumor cells [ ] (dashed grey region), and ( ) the inner layer of necrotic cells (dark grey core). in the absm, with the physical neighborhood, we mean moore neighborhood as is depicted in figure (b). in the absm, two types of agents (immune and nonimmune cells) are presumed. definitions of agents and their states are summarized in table . each agent represents a biological cell placed at (i, j) coordinate, where < i, j < n − cell. the ia is a moving agent and may accidentally collide with nas. in this paper with the total number of tumor cells, we mean the sum of na_ , na_ , and na_ . in the absm, it is supposed that each ia has the ability to call other immune cells. in absm, na_ cells play the role of empty places for na_ or ia cells. for example, the ia cells that are looking for cancer cells or recalled to a specific location may substitute the na_ cells, i.e., possess na_ sites. to our knowledge from cancer biology, if na_ cells are surrounded by more healthy cells, they will access more nutrients and oxygen; it means that the higher is the number of healthy cells, na_ (empty space) around a proliferative tumor cell (na_ ), the greater is the likelihood of division of the na_ cell. two types of pt cells with different division probabilities are considered. in the first type denoted by na_ _ , the cell division is assumed independent from the environmental conditions, i.e., the division probability (ρ pt is constant), while in the second type is symbolized by na_ _ , i.e., the cell division probability (ρ pt is a function of its healthy neighbors (na_ s)). in the absm, it is assumed that each na_ _ may be divided into one na_ _ and one na_ _ daughter cells with the probability nmm and into two na_ _ daughter cells with the probability ( − nmm). however, it is assumed that each na_ _ cell can only be divided into two na_ _ daughter cells. at every simulation time step, the chance of each na_ (either na_ _ or na_ _ ) for proliferation (division) is checked, i.e., the existence of at least one empty place in the neighborhood of that parent cell is checked. if there is at least one empty space (na_ ) in its neighborhood, the na_ cell will divide with the probability ρ pt into two na_ daughter cells, so that one of the daughter cells will stay in the position of her parent and the other daughter will place in that empty neighbor. if there is more than one empty place in the neighborhood, the second na_ daughter cell will pick one of the empty spaces randomly (with the same probability). if the na_ cell cannot find any empty place in its neighborhood or if it cannot proliferate (with the probability − ρ pt ), it would stay at na_ state. the maximum of time length at which na_ can stay without any division is limited to age. if na_ has no chance for proliferation for a time length (age), then its state will be changed to nonproliferating (quiescence) tumor (na_ ). researches indicate that there are many levels of quiescence. various signals such as serum starvation or loss of nutrients, loss of adhesion, and cell contact inhibition at high cell density can induce quiescence to a tumor cell [ ] [ ] [ ] . in the absm, the above fact has been considered by introducing the lifetime parameter age. if a na_ cell is placed at a radial distance less than the radius of the necrotic core (r n in figure ), then it will turn to na_ (necrotic cell) at the next time step. if a na_ cell is at a radial distance less than w p from the external edge of the tumor (see figure ), it may turn to a na_ cell due to accessing nutrient. the na_ cells accumulate in the inner zone of the tumor and form a necrotic layer. their state will not ever change to any other states since they are already dead. researchers have found that tumor cells release signals [ ] in the host tissue which can be detected by immune cells via specific receptors. the immune cells will receive these signals and search for the tumor cells. for more compatibility of the absm with cancer biology, we introduced pure tumor growth fraction ðpgfðmÞÞ and growth fraction ðgfðmÞÞ concepts. pgfðmÞ refers to the ratio of the total number of tumor cells at time m to the total number of cells in the studied tissue (equation (a. ) ). gfðmÞ denotes to the fraction of at the start of the tumor growth in a tissue, when pgfðmÞ is negligible, ias enter the tissue from one corner of the lattice figure , where they have long distance from the center of the tumor. in this circumstance, each ia is assumed to move directly towards the center of the tumor and will take the place of an empty space in its moore neighborhood. this empty space neighbor is one that has the shortest euclidean distance from the center of the tumor. figure shows a typical simulation result of the absm. it can be seen that at time m ≥ , the growth fraction factor (gfðmÞ) remains almost constant, which means that the necrotic layer is formed and is noticeable. after this time, the ias can walk randomly in any direction to meet tumor cells. in the absm, we implement short-range or contactdependent biological communication [ ] . it means that any interaction between ia and na agents takes place only when they are in physical contact, i.e., when they are neighbors. when an ia encounters an na, one of the following events may take place: ( ) antitumor action. in this case, the na will fail. if ia_ encounters the na_ , the na_ cell may die with the probability ρ i (equation (a. )) and will change to na_ state at the next iteration in equation (a. ), k dt is tumor death constant;ni i,j and npt i,j are the numbers of ia_ cells and na_ cells, respectively, in the neighborhood of the studied cell (cell located at position (i, j)). in other words, the ia_ will substitute the na_ which is located in its neighborhood with the probability k dt , and the na_ will be dismissed from the microenvironment. if there are more than one na_ in the neighborhood of the ia_ , the ia_ will kill all na_ cells and randomly replace one of them. the other na_ cells will change into na_ . if the collision agent is ia_ , the appropriate rules will apply only to one of the randomly chosen (with a uniform density function) na_ cells in its neighborhood. ( ) null action. in this case, nothing happens, i.e., both agents; na_ and ia will survive and remain in their states. meanwhile, the ia will continue its search to find another na_ ( ) protumor action. in this case, the ia will fail in killing na_ and die with probability ρ t (equation (a. ) ). the ia will become an empty space, i.e., the ia will be removed from the tissue and will be replaced by a na_ where ntðmÞ and nptðmÞ are the sum of the number of tumor cells and the total number of na_ cells, respectively, in the studied tissue at time m. in fact, the more immune cells are successful to fight against tumor cells (v − f > ), the more will be recalled. however, if v − f < , there will be no newborn immune cell in the studied tissue. the newborn immune cells will enter from the four corners of the lattice into the tissue randomly. the transition rules of ia will be applied to each of the newborn ias. the basic absm elements and their brief descriptions are summarized in table . as it is deduced from table and above discussions, the absm is a system biology oriented one, i.e., it is constructed from several agents and interactions rules between them and as we will see these elements and interactions can give rise to the function and behavior of cancer growth system. with a conceptual and intuitive look at the elements of table , some of them can be assumed individual-dependent; those are indicated by a star mark. for better realization of our model, we hypothesize possible connections of each parameter to cancer hallmarks and tme follows. the hypothesized relations that are based on biological findings and arguments are listed in table . in this table, the number of hallmarks or tmes is same as the number that was given to them in introduction. for example, in the first and second columns of the first row of table , we have listed the possible effect of the valve of parameter p in cancer hallmarks and possible tme players which are delegated by p ; here, the number means that we hypothesize that parameter p will influence the hallmark number (unlimited multiplication) and tme player number (fibroblasts and myofibroblasts) as well. here, our approach is based on our interpretations from oncology literature, following discussions, and in silico experiments. as we mentioned earlier, fibroblasts and myofibroblasts are the number one component of tme. researches have confirmed that only the activated fibroblasts are required to initiate and endorse tumor growth [ ] [ ] [ ] [ ] . when the fibroblasts remain activated after the initial insult has regressed, these activated fibroblasts may work with other molecular pathways to boost neoplasm initiation, where it has a significant impact on cancer progression through remodeling ecm, inducing angiogenesis, employing inflammatory cells, and directly stimulating cancer cell proliferation via the secretion of growth factors, immune suppressive cytokines, and mesenchymal-epithelial cell interactions [ , ] . experimental evidences prove that neuroendocrine cells, the number two component of tme, exhibit a combination of neuronal and endocrine features [ ] . neuroendocrine cells are the accomplices of tumor formation [ ] . it is proven that the neuroendocrine system strongly influences the function of the immune system. neuroendocrine cells could stimulate the proliferation of prostate carcinoma cells and increase their aggressiveness [ ] , while in the development of neuroendocrine cell tumors, they may play a principal role [ ] . some features of adipose tissue, the number three component of tme, are associated with cancer. adipose cells secrete more than different cytokines, chemokines, and hormone-like factors [ , ] . these factors, whose production may upregulate in obesity, may be assistants in tumor initiation. obese adipose tissue hypoxia establishes a highly proinflammatory microenvironment, which is more likely to breed tumors [ ] [ ] [ ] . adipose tissue reprogramming and the associated systemic secretion may have an effect on cancer growth and progression [ ] . excess adiposity leads to high circulating blood estrogen [ ] and chronic, lowgrade inflammation, which is involved in cancer development, a major energy source, relating with inflammation, recruiting immune cells, and supporting vasculogenesis [ , , ] . the mammalian immune system monitors tissue homeostasis to protect against invading infectious pathogens and to eliminate damaged cells [ ] . findings recommend that immune surveillance has significant roles in recognizing and eradicating a large part of emerging tumor cells [ ] . however, unlike normal functions, immune inflammatory cells, the number four component of tme, would persist in sites of chronic inflammation, linked to diverse tissue pathologies, including fibrosis, aberrant angiogenesis, and neoplasia [ ] . recent discoveries in immune system research illuminate that it is difficult to ignore the crucial issue that immune inflammatory cells may be the early support structure of cancer, having a double effect on tumor formation [ ] [ ] [ ] [ ] [ ] [ ] [ ] . blood and lymphatic vascular networks, the number five component of tme, help tumor cells escape immune surveillance in the process of tumor progression. escape measures are mainly divided into two categories. directly, the lymphatic microenvironment will weaken or eliminate the normal function of immune cells. for instance, the myeloidderived suppressor cells could restrict the normal operation of t cells [ ] [ ] [ ] . when the metastatic tumor enters a new environment, cd + and cd + t cells may help them to evade the host immune system [ , ] . indirectly, the remodeling of unusual endothelial venules can influence immune cells to move into the lymph nodes [ ] . ecm, the number six component of tme, is a dynamic and complicated environment. evidences show that ecm can help neoplasms to get away from immune surveillance [ ] . ecm contains all the cytokines, growth factors, and hormones secreted by stromal and tumor cells. a model indicated that ecm heterogeneity is crucial for controlling collective cell-invasive behaviors and determining metastasis efficiency [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . ecm may affect tumors through extracellular secretion. in addition, ecm may alter the phenotype type of stromal cells or tumor cells [ ] . the ecm of tumor will provide a hypoxic or acidic environment in which the tumor cells have greater survival advantages than normal cells. ecm will select survival cells to aid in tumor growth and invasion at the fastest rate. using matlab codes, the absm was implemented. in following simulations, at first, it is supposed that the entire tissue (lattice in figure ) is covered with na_ s. the state of one cell in the center of the tissue is changed into na_ at time m = . the . % of total number of cells (i.e., : × ncell × ncell), where ncell = , in the studied tissue, are entered as ias from a corner of the lattice randomly. figure shows the initial condition for a typical simulation, where table depicts typical value of parameters. the absm was evaluated qualitatively and quantitatively considering the following criteria: the average radius of the external edge of tumor at iteration mðr t ðmÞÞ, the average radius of the necrotic zone at iteration mðr n ðmÞÞ, growth fraction at iteration m ðgfðmÞÞ, necrotic fraction at iteration m ðnfðmÞÞ, and the number of na_ , na_ , na_ , and ia cells at iteration m. besides, the graphical illustration of the growth is used as qualitative evaluation in some cases. figure shows the number of na_ , na_ , na_ , and ia vs. iterations. it is seen that the number of ia enhances due to recruitment which is qualitatively consistent with cancer biology. in this simulation, although ia cells enter the tissue to fight against tumor cells, but the permanent presence of cells of na_ in tissue is observed. figure compares the size of the simulated tumor growth using 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vivo impairs tumor-specific immune responses special thanks are due to dr. alizadeh (cancer biology research center, cancer institute of iran, tehran university of medical sciences, tehran, iran) and dr. esmati (department of radiotherapy, imam khomeini hospital, tehran university of medical sciences) for their valuable guidance and advice. the author declares that there is no conflict of interest regarding the publication of this paper. key: cord- - tax ajw authors: bhopal, raj s. title: covid- zugzwang: potential public health moves towards population (herd) immunity date: - - journal: nan doi: . /j.puhip. . sha: doc_id: cord_uid: tax ajw summary covid- is pandemic, and likely to become endemic, possibly returning with greater virulence. outlining potential public health actions, including hygiene measures, social distancing and face masks, and realistic future advances, this paper focuses on the consequences of taking no public health action; the role of natural changes such as weather; the adverse public health consequences of lockdowns; testing for surveillance and research purposes; testing to identify cases and contacts, including the role of antibody tests; the public health value of treatments; mobilising people who have recovered; population (a synonym for herd) immunity through vaccination and through natural infection; involving the entire population; and the need for public debate. until there is a vaccine, population immunity is going to occur only from infection. allowing infection in those at very low risk while making it safer for them and wider society needs consideration but is currently taboo. about - % population immunity is sufficient to suppress an infection with a reproduction number of about or slightly more. importantly, in children and young people covid- is currently rarely fatal, roughly comparable with influenza. the balance between the damage caused by covid- and that caused by lockdowns needs quantifying. public debate, including on population immunity, informed by epidemiological data, is now urgent. in public health, as in chess, planning well-ahead is essential. many leaders, however, are hesitant about articulating long-term plans for tackling the covid- pandemic. discussing future options might distract from the stay-at-home and social distancing messages. leaders are focused on immediate tasks including 'the peak' of hospitalisations and deaths, but reaching it will mean negotiating treacherous downhill terrain . ( ) the covid- pandemic has placed us in zugwang-a position in chess where every move is disadvantageous where we must examine every plan, however unpalatable. curative treatments and vaccines may be long-delayed and lockdowns harm the public's physical and mental health and not just the economy. mass testing will not be feasible globally especially in many low and middle-income countries, as it is in the well organised, advanced economies like the us and germany. we need to think beyond these interventions. an overview of public health options is summarised in table , constructed around the classic public health triad of primary (stop it occurring), secondary (pick it up early) and tertiary prevention (minimise the consequences) and interventions on the viral agent, host/population and environment. ( ) this classification is normally applied to individuals but in this paper i also use it for populations. this analysis fits with the who strategy update published on april ( ) and subsequent updates on its website, and an outline of options by bedford et al. ( ) i focus on core public health issues considering their relevance to population immunity, commonly called herd immunity, and used here as a better synonym. this is the protection from a contagious disease that the community enjoys because a high proportion of people are immune, thereby impeding transmission of infection from person to person. it is normally invoked through vaccination but also occurs naturally. ( , ) many important matters are in the table that are not fully discussed in this paper e.g. that research on the transmission, infective dose and changing genetics, and especially virulence of the virus (agent), is essential (column , table ). pinpointing the causes of the high risk in older populations, males, and people with cardiovascular disorders and type- diabetes is paramount. i assume that interventions targeted on the host/population including hygiene measures, avoiding handshaking and social distancing will be prolonged, that lockdowns will be imposed perhaps intermittently and with variable intensity, and that the role of face masks and temperature checks in public life will become clearer (column , table ). ( , ) public health surveillance systems and epidemiological research must be strengthened with more accurate data on incidence, prevalence, and death rates examined by both demographic variables including age, sex, socio-economic status and ethnicity/race and clinical risk factors (column , table ). environmental changes, permitting behavioural changes including social distancing and hygiene, especially in overcrowded housing and working settings, will be needed (column three, table ) as will effective, comprehensive healthcare together with financial aid for industries (column , table ). early and even ongoing trivialisation of covid- as 'just like flu' was incorrect for it is somewhat less severe in children but much more severe in older people (table ) . ( ) the world health organisation (who) estimates that about % of those diagnosed become seriously ill, % critically , and % die.( ) (as the number of people infected is usually unknown the infection fatality rate is variably estimated at between . - . %.) if - % of the world's population was infected without interventions there could be about - billion people infected with covid- , one billion seriously sick and up to million dead prematurely. there will be indirect consequences from the infection, lockdown measures and economic damage e.g. starvation, homelessness, poverty and mental health and physical morbidity and death. the collateral damage will be especially high in low and middle income countries and in children. ( ) that happened with the spanish flu epidemic in / .( ) the covid- pandemic arises during one of the most prosperous periods in human history permitting a more vigourous response than in . allowing the pandemic to unfold uncontrolled would rapidly produce population immunity, but this is not a palatable public health response, hence is not in table . pandemics can fizzle out. we hope this will happen with covid- . this is not a public health intervention and not in table . in the spanish flu epidemic there was a respite before the virus returned more virulently .( ) summers might bring respite even if lockdowns are relaxed but the virus seems to survive and be contagious in most climates. infection in summer may be less common and less severe than in winter because other respiratory infections are less common and being outdoors presents lower risk of acquiring infection than indoors. in very hot climates, however, people go indoors to escape the heat. lockdowns are important in primary, secondary and tertiary prevention (table , column ). most nations are applying strict lockdowns for - weeks, but as the who emphasises, this merely provides time to reduce the reproduction rate of infection (r) from about three to less than one.( ) the lockdown in wuhan, china was severe and strict from january- april, about weeks. however, life there is not normal even now and the infection has recurred. hubei province, where wuhan is, has about million people in china i.e. less than % of the population. the province, unlike countries, had access to the resources of the nation to survive. some economies cannot sustain even - week lockdowns and they are being lifted or breaking down within weeks e.g. after three weeks in ghana, and some places are resisting them e.g. brazil.( ) prolonged lockdowns may cause more morbidity and mortality than covid- , especially in the poorest countries, where the populations are relatively young on average and at little risk of death. we must evaluate the health consequences of lockdowns, assessing the benefits and costs. ( ) lockdowns are likely to become variable and local, depending on circumstances. mixing amongst local populations is already occurring and widespread international travel is returning. a uk-based strategy has suggested easing lockdowns when widespread testing is in place and when the number of daily deaths is below ( , deaths annually), and relaxing most measures when deaths are fewer than ( , deaths annually). ( ) the uk government seems to be following this approach in england, with more cautious approaches in scotland, wales and northern ireland. during lockdowns population immunity is being acquired slowly, and those at highest risk of severe morbidity and death are being shielded. population immunity will be accelerated as lockdowns are eased. testing is vital for both primary, secondary and tertiary prevention, and helps identify places where the disease has not yet occurred (table , column ). who has emphasised testing as key until a vaccine or a cure are discovered( ) but the reasoning is seldom explicit to the public. testing in selected populations is essential for public health surveillance and medical research to establish the incidence, prevalence and outcomes of the disease (table , column ). ( , ) such data help adjust our plans through feedback including indicating the proportion of the population that has acquired the infection and is potentially immune. the benefit of detecting the virus or the viral antigen in suspected cases is accurate diagnosis for clinical management (including protection of frontline staff) and to permit isolation/quarantine of proven cases. tracing of contacts becomes possible so they can be isolated to minimise spread. this is better than asking everyone with respiratory symptoms to isolate without doing tests but it requires extensive public health infrastructures (in addition to apps) and access to laboratories and testing kits (column , table ). to work, both testing and feedback of results must be prompt. testing helps control the acquisition of population immunity. testing for virus and antigen is also useful to check whether people recovering from covid- can return to normal without infecting others or harming themselves by premature activity (tertiary prevention) (column , table ). people who have recovered will need to be careful as their immunity may be partial and some may continue to shed the virus.( ) some recovered people, especially in essential services who have already returned to work, may be shedding virus but whether these people are contagious needs research. immunity to respiratory viruses is complex but cellular responses by macrophages and lymphocytes, including t-cells, are critical.( ) antibody, whether igm or igg, is a marker of potential immunity but its absence does not necessarily imply lack of immunity. people who have recovered from proven covid- must be partially immune. ( ) adults with mild or even asymptomatic illnesses may not mount a strong antibody response but like children probably have a strong, innate defence system. ( , ) we need an accurate antibody test to identify, retrospectively, people who have been infected and several are available. when the population prevalence is about % as is the case in many countries, even at % sensitivity and specificity only about % of positive tests are correct (predictive power of a positive test). ( ) higher accuracy could be achieved by using more than one kind of test. people who have self-isolated because of typical covid- symptoms and have antibody have probably had covid- and are partially immune. some false positives and false negatives are still inevitable. ( , ) nonetheless, antibody tests are invaluable for measuring the prevalence of population immunity (table , column ) . surprisingly, the accuracy of a test required to measure prevalence is usually different from the accuracy required for clinical practice. ( ) mobilising people who have recovered we need to normalise recovered people, especially those delivering essential services. the concept of immunity passports has been discussed. immunity passport implies a guarantee that cannot be given but a certificate indicating that a person has had the infection, has recovered clinically and is likely to be partially or wholly immune is more accurate. ( ) the number of eligible people globally could soon be in the hundreds of millions so the clinical, ethical, legal, and practical issues arising need urgent consideration. ( ) ( ) ( ) i have called for public debate including a citizen's jury. ( ) the ideal treatment would be preventative i.e. it would stop the infection occurring (column , table ). such treatment would, however, need to be extremely safe, especially in young people without underlying disorders where covid- is rarely fatal. (table )( ) it would probably be unaffordable for low-and middle-income countries. more likely, treatments may attenuate the illness, reducing the duration and fatality of the infection. ( , ) they could be valuable in public health in reducing the transmission of disease, especially to healthcare workers (column , table ). this will slightly slow the acquisition of population immunity. people with comorbidities, who are usually in the older age groups, are most severely affected by covid- , and especially if they have cardiovascular disorders, type diabetes, hypertension, and chronic respiratory disorders. ( , ) clearly, public health interventions should promote control of risk factors that lead to these diseases e.g. smoking, physical inactivity, high levels of alcohol consumption, high levels of salt and exposure to air pollution (column , table ). people in these groups could minimise their risk of exposure to covid- , await effective vaccination and benefit indirectly as population immunity through natural infection increases (column , table ). ( , ) involving the entire population especially those most vulnerable as the who has emphasised the strategy for controlling covid- needs to be global, and reach out to everyone. (column , table ) the who has emphasised the needs of populations in crowded circumstances, including asylum seekers, refugees and migrants, where the infection can spread readily. ( , ) regulations and laws making it illegal or difficult to house, employ or provide health and other services to vulnerable people (e.g. undocumented migrants, who cannot access public funds), need to be reviewed, especially as international travel is problematic. ( ) the pandemic hits minorities and migrants hard, given their greater overcrowding in homes and workplaces, relative poverty, the difficulties of understanding and acting upon social distancing guidelines, and the propensity to cardiovascular diseases and type diabetes. ( ) ( ) ( ) large numbers of such populations are being infected but being relatively young, comparatively fewer will die from covid- , thereby contributing disproportionately to population immunity. vaccination is the acceptable way of gaining population immunity, and our main hope for controlling the pandemic (columns and , table ).( , , ) numerous trials to develop a vaccine are underway and the who has set up a vaccine task force.( ) we cannot, unfortunately, pin all hopes on vaccines as they may only work for a short time especially if the virus evolves new strains. a vaccine that is effective, proven to be safe, manufacturable in billions of doses and available globally is unlikely this year, and may take years, even decades. proven safety is essential especially in children or young people.( ) serious illnesses or deaths in young people following immunisation, whether coincidental or causal, could impede vaccination. in people over -years of age, or the immunosuppressed, where the vaccine is needed most, a strong immune response is unlikely. efficacy of vaccines needs to be demonstrated in older groups and in those with underlying disorders. the technical phrase is herd immunity, with connotations of animals, rather than humans. herd immunity provokes hostility and controversy as it is usually interpreted as allowing the pandemic to unfold without interventions. the concept needs revisiting. if safe and effective vaccines and life-saving preventative and therapeutic medications are not found, lengthy lockdowns prove impossible, and the pandemic does not disappear spontaneously, population immunity is the only, longterm solution (column , table ). everyone infected and achieving any degree of immunity contributes to population immunity, ( ) and this is likely to be through a combination of cellular and antibodybased (humoral) responses. the duration of such immunity is unknown although it is reasonable to assume it will last this season with some long-term benefits given exposure to the same or similar strains of the virus. through social distancing and lockdown measures most societies have brought the reproduction number from about three ( ) to about one or less. the proportion of the population required to be immune to control an infection is called the herd (population) immunity threshold. it is difficult to calculate this number exactly in real world circumstances. to control an infection with an r of about and even somewhat higher we need about % of the population to have immunity (unlike measles where over % is needed).( , ) currently, the prevalence of covid- infection is variably estimated from - % according to locality and work settings. however, if covid- becomes endemic, the proportion of the population with immunity will rise fast, especially where lockdowns have been lifted .( ) this immunity will be helpful, though not fully protective, as new strains of covid- will probably emerge, so people will be re-infected but probably less severely so, as is the case for influenza. opening up the economy, schools, colleges and social life is accepting that many people will become infected even with test, track and isolate strategies. most young people will probably acquire the infection, often without a diagnosis as they will be asymptomatic or mildly affected. given this, we need to minimise the already low risks of adverse effects in young people (table ) , especially by identifying the reasons why a few become seriously ill. ( , ) hygiene and some social distancing measures will continue to be required in homes as children and young people return to nurseries/school/colleges. ( , , ) some young people with immunity related disorders could be advised not to return to school or university presently, while awaiting the rise of population immunity in their classmates, which will protect them indirectly. young people present risks to transmitting covid- to people in their household, especially parents and grandparents who have underlying disorders or are in the oldest age groups. home school may be needed for children in these exceptional circumstances. teachers and others in close contact with children and young people, especially those in older age groups and with chronic disorders, need shielding and/or personal protection equipment. we need excellent facilities for diagnosis, isolation, quarantine, and treatment for these young people and their contacts as they return to normal life. the public will need to be informed frankly about the risks by comparing those of covid- with infections they are familiar with e.g. influenza (table ) . ( ) the idea of covid- 'parties' by young people has been met with shock. intermingling is inevitable as workplaces, schools, colleges and universities are reopened. young people will make decisions that are logical for them given their risks and life circumstances. could we consider allowing young people without underlying disorders to get covid- naturally while shielding those most at risk through continued social distancing and isolation? ( , , ) young people might prefer this route rather than remaining in lockdown or acquiring the infection in riskier circumstances e.g. while travelling abroad. such people could be given advice and lightly monitored to minimise adverse effects. this is not unprecedented. chickenpox parties were occurring even in the s even though such infections posed risk to pregnant women and the ethics have been considered. ( ) the acquisition of covid- naturally by the young and healthy is, arguably, the safest way towards the goal of about % population immunity while protecting those most at risk and maximising benefits for society, whether in terms of the economy or achieving the full potential of future generations. ( , ) this question poses ethical, legal, logistical and clinical challenges similar to those arising in the proposal to test covid- vaccines in healthy volunteers.( ) allowing the covid- pandemic to run its course uncontrolled must not be permitted. none of the responses of countries internationally are optimal as partly reflected in their variability.( , ) covid- has placed us in zugzwang so we need precise and detailed plans and well-calculated series of moves that minimise the harms, tailored for each country and region according to their context and resources. the pandemic needs to be prevented from returning year-on-year, potentially more severely, especially in young people and children, and mandating repeated lockdowns.( ) we urgently need to consider all reasonable public health actions and plans (table ) . hope in natural forces, effective and safe vaccines and curative treatments is important but, given uncertainty, we need to consider other, admittedly difficult, paths. adults should now reflect on and debate, together with their elected policymakers and scientific advisers, the balance of risks they accept for themselves, versus the risks imposed to wider society, and thus directly inform potential strategies. covid- is having a major impact on children and their voice needs to be heard. ( ) ageism must be avoided whether through shielding or workplace policies that might inadvertently cause harm. everyone has the right to balance risks and benefits in relation to their own quality of life. this pandemic is complex while the messages being given to the public are overly simplistic. we need global and national leadership, imagination, courage and honest public discussion to shape and influence our future.( , ) table the levels of prevention in relation to the causal triad of virus (agent), the human host and the environment (physical and social) and the control of covid- , applied at both individual and population levels (or both) covid- -a framework for decision making. edinburgh: the scottish government concepts of epidemiology : integrating the ideas, theories, principles and methods of epidemiology covid- : towards controlling of a pandemic individual variation in susceptibility or exposure to sars-cov- lowers the herd immunity threshold herd immunity": a rough guide case isolation, contact tracing, and physical distancing are pillars of covid- pandemic control, not optional choices. the lancet infectious diseases physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov- and covid- : a systematic review and meta-analysis. the lancet children's mortality from covid- compared with all-deaths and other relevant causes of death: epidemiological information for decisionmaking by parents, teachers, clinicians and policymakers early estimates of the indirect effects of the covid- pandemic on maternal and child mortality in low-income and middle-income countries: a modelling study. the lancet global health on the centenary of the spanish flu: being prepared for the next pandemic a sustainable exit strategy: managing uncertainty, minimising harm test, test, test for covid- antibodies: the importance of sensitivity, specificity and predictive powers. public health rapid roll out of sars-cov- antibody testing-a concern positive rt-pcr test results in patients recovered from covid- immunity to respiratory viruses the immune system of children: the key to understanding sars-cov- susceptibility? the lancet child & adolescent health patients who have recovered from covid- : issuing certificates and offering voluntary registration covid- immunity passports and vaccination certificates: scientific, equitable, and legal challenges. the lancet chile plans controversial covid- certificates. the lancet remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial. the lancet effect of dexamethasone in hospitalized patients with covid- : preliminary report covid- ) and cardiovascular disease global, regional, and national estimates of the population at increased risk of severe covid- due to underlying health conditions in : a modelling study. the lancet global health evaluation of "stratify and shield" as a policy option for ending the covid- lockdown in the uk rohingya refugees at high risk of covid- in bangladesh. the lancet global health covid- : immense necessity and challenges in meeting the needs of minorities, especially asylum seekers and undocumented migrants is ethnicity linked to incidence or outcomes of covid- ? covid- and african americans sharpening the global focus on ethnicity and race in the time of covid- . the lancet the dual epidemics of covid- and influenza: vaccine acceptance, coverage, and mandates safety, tolerability, and immunogenicity of a recombinant adenovirus type- vectored covid- vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial the reproductive number of covid- is higher compared to sars coronavirus hyperinflammatory shock in children during covid- pandemic. the lancet covid- in children and adolescents in europe: a multinational, multicentre cohort study. the lancet child & adolescent health how to hold an ethical pox party human challenge studies to accelerate coronavirus vaccine licensure. the journal of infectious diseases singapore: university of singapore national what's the way out? potential exit strategies from the covid- lockdown a future for the world's children? a who unicef commission. the lancet professor martin mckee posed the questions on april that initiated this paper on april . i thank him for his feedback at that time. i am grateful to professional colleagues will tapsfield, anand bhopal, sunil bhopal jay bagaria, viola priesmann, jason yap, sarah dalglish, david mccoy, and laurence gruer george davey smith, liam smeeth , joan barry, neil french, john teare, paul roderick, and paolo vineis for detailed critical scrutiny of earlier dtafts from a medical and public health perspective. roma bhopal, sanjoy das, mark wilson, and ulrike wilson provided viewpoints from the perspective of members of the general public. roma bhopal also helped find age-specific data and help with proofreading and editing. people acknowledged are not responsible for any of the viewpoints expressed here (and may not agree with me). i have no conflicts of interest to declare. there was no external funding for this work. deaths with confirmed or presumed covid- , coded to icd- code u . . influenza death counts include deaths with pneumonia or covid- also listed as a cause of death. population is based on postcensal estimates from the u.s. census bureau ( ) ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord- -xxvol t authors: amos, louella b. title: cough date: - - journal: nelson pediatric symptom-based diagnosis doi: . /b - - - - . - sha: doc_id: cord_uid: xxvol t nan cough is an important defense mechanism of the lungs and is a common symptom, particularly during winter months. in most patients, it is self-limited. however, cough can be ominous, indicating serious underlying disease, because of accompanying problems (hemoptysis) or because of serious consequences of the cough itself (e.g., syncope and hemorrhage). the cough reflex serves to prevent the entry of harmful substances into the tracheobronchial tree and to expel excess secretions and retained material from the tracheobronchial tree. cough begins with stimulation of cough receptors, located in the upper and lower airways, and in many other sites such as the ear canal, tympanic membrane, sinuses, nose, pericardium, pleura, and diaphragm. receptors send messages via vagal, phrenic, glossopharyngeal, or trigeminal nerves to the "cough center," which is in the medulla. because cough is not only an involuntary reflex activity but also one that can be initiated or suppressed voluntarily, "higher centers" must also be involved in the afferent limb of the responsible pathway. the neural impulses go from the medulla to the appropriate efferent pathways to the larynx, tracheobronchial tree, and expiratory muscles. the act of coughing ( fig. . ) begins with an inspiration, followed by expiration against a closed glottis (compressive phase), resulting in the buildup of impressive intrathoracic pressures ( - cm h o). these pressures may be transmitted to vascular, cerebrospinal, and intraocular spaces. finally, the glottis opens, allowing for explosive expiratory airflow ( m/sec) and expulsion of mucus, particularly from the larger, central airways. the inability to seal the upper airway (tracheostomy) impairs, but does not abolish, the effectiveness of cough. weak ventilatory muscles (muscular dystrophy) impair both the inspiratory and the compressive phase. the patient history often provides the most important body of information about a child's cough. a diagnosis can often be discerned with relative certainty from the family history, the environmental and exposure history, and the acute nature and characterization of the cough. the patient's age (table . ) helps to focus the diagnostic possibilities. congenital anatomic abnormalities may be symptomatic from birth, whereas toddlers, who may have incomplete neurologic control over swallowing and often put small objects in their mouths, are at risk for foreign body aspiration; adolescents may experiment with smoking or inhaled drugs. socioeconomic factors must be considered; a family that cannot afford central heating may use a smoky wood-burning stove; spending time at a daycare center may expose an infant to respiratory viruses; and several adult smokers in a small home expose children to a high concentration of respiratory irritants. the various cough characteristics can help determine the cause of cough. the causes of acute, recurrent, and chronic coughs may be quite different from each other ( fig. . ; see also table . ). a cough can be paroxysmal, brassy, productive, weak, volitional, and "throatclearing," and it may occur at different times of the day (tables . and . ). the previous response or lack of response to some therapies for recurrent and chronic cough can provide important information (see table . ). furthermore, some coughs may be caused or worsened by medications (table . ). a history of accompanying signs or symptoms, whether localized to the respiratory tract (wheeze, stridor) or elsewhere (failure to thrive, frequent malodorous stools) can give important clues (table . ; see also tables . and . ). it is essential to remember that the daily language of the physician is full of jargon that may be adopted by parents but with a different meaning from that understood by physicians. if a parent says that a child "wheezes" or "croups" or is "short of breath," it is important to find out what the parent means by that term. because many disorders of childhood have genetic or nongenetic familial components, the family history can provide helpful information: • are there older siblings with cystic fibrosis (cf) or asthma? • is there a coughing sibling whose kindergarten class has been closed because of pertussis? • is there an adolescent or adult with chronic cough (bronchitis) who may have pertussis or tuberculosis? • was the child premature, and, if so, did he or she spend a month on the ventilator, and does he or she now have chronic lung disease (bronchopulmonary dysplasia)? • did the toddler choke on a carrot or other food months ago? • did the child have rsv, bronchiolitis, or rhinovirus infection as an infant? • did the child receive a bone marrow transplant a year ago? • is the child immunized? • did the infant have a tracheoesophageal fistula repaired in the neonatal period? chest, abdomen, and spine. the shape of the chest gives information. is the anteroposterior (ap) diameter increased, which indicates hyperinflation of the lungs from obstruction of small airways (asthma, bronchiolitis, cf)? is this diameter small, as can be seen with some restrictive lung diseases with small lung volumes (muscular dystrophy, spinal muscular atrophy)? the normal infant has a "round" chest configuration, with the ap diameter of the chest about % of the transverse (lateral) diameter. with growth, the chest becomes more flattened in the ap dimension, and the ap-to-transverse ratio is between % and %. although obstetric calipers can be used to give an objective assessment of the ap diameter of the chest, most clinicians rely on their initial inspection often reveals the seriousness of an illness: • is the child struggling to breathe (dyspnea)? • does the child have an anxious look? • can the child be calmed or engaged in play? • is the child's skin blue (representing cyanosis) or ashen? • does the child appear wasted, with poor growth that may indicate a chronic illness? the respiratory rate is often elevated with parenchymal lung disease or extrathoracic obstruction. respiratory rates vary with the age of the child (fig. . ) and with pulmonary infection, airway obstruction, activity, wakefulness and sleep, fever, metabolic acidosis, and anxiety. odors may also give helpful clues. does the examining room or the clothing smell of stale cigarette smoke? is there a foul odor from a diaper with a fatty stool, which may suggest pancreatic insufficiency and cf? is the child's breath malodorous, as can be noticed in sinusitis, nasal foreign body, lung abscess, or bronchiectasis? fingers. cyanotic nail beds suggest hypoxemia, poor peripheral circulation, or both. the examiner looks for the presence of digital clubbing (fig. . ) , which makes asthma or acute pneumonia extremely unlikely. the absence of digital clubbing but a history of severe chronic cough in an older child makes cf unlikely. † anatomic abnormality includes tracheobronchomalacia, tracheoesophageal fistula, vascular ring, abnormal position or take-off of large bronchi. common; less common; much less common. cf, cystic fibrosis; ger, gastroesophageal reflux. less easy to notice than intercostal retractions is their bulging out with expiration in a child with expiratory obstruction (asthma). contraction of the abdominal muscles with expiration is easier to notice and is another indication that a child is working harder than normal to push air out through obstructed airways. inspection of the spine may reveal kyphosis or scoliosis. there is a risk of restrictive lung disease if the curvature is severe. palpating the trachea, particularly in infants, may reveal a shift to one side, which suggests loss of volume of the lung on that side or extrapulmonary gas (pneumothorax) on the other side. placing one hand on each side of the chest while the patient breathes may enable the examiner to detect asymmetry of chest wall movement, either in timing or in degree of expansion. the former indicates a partial bronchial obstruction, and the latter suggests a smaller lung volume, voluntary guarding, or diminished muscle function on one side. palpating the abdomen gently during expiration may allow the examiner to feel the contraction of the abdominal muscles in cases of expiratory obstruction. hyperinflation may push the liver down making it palpable below the costal margin. palpation for tactile fremitus, the transmitted vibrations of the spoken word ("ninety-nine" is the word often used to accentuate these subjective assessment of whether the diameter is increased: does the patient look "barrel-chested"? intercostal, subcostal, suprasternal, and supraclavicular retractions (inspiratory sinking in of the soft tissues) indicate increased effort of breathing and reflect both the contraction of the accessory muscles of respiration and the resulting difference between intrapleural and extrathoracic pressure. retractions occur most commonly with obstructed airways (upper or lower), but they may occur with any condition leading to the use of the accessory muscles. any retractions other than the mild normal depressions seen between an infant's lower ribs indicate a greater than normal work of breathing. because lung sounds tend to be higher-pitched than heart sounds, the diaphragm of the stethoscope is better suited to pulmonary auscultation than is the bell, whose target is primarily the lower-pitched heart sounds (table . ). the adult-sized stethoscope generally is superior to the smaller pediatric or neonatal diaphragms, even for listening to small chests, because its acoustics are better (figs. . and . ). adventitious sounds come in a few varieties, namely, stridor, crackles, rhonchi, and wheezes. other sounds should be described in clear, everyday language. • stridor is a continuous musical sound usually heard on inspiration and is caused by narrowing in the extrathoracic airway, as with croup or laryngomalacia. • crackles are discontinuous, representing the popping open of air-fluid menisci as the airways dilate with inspiration. fluid in larger airways causes crackles early in inspiration (congestive heart failure). crackles that tend to be a bit lower in pitch ("coarse" crackles) than the early, higher-pitched ("fine") crackles are associated with fluid in small airways (pneumonia). although crackles usually signal the presence of excess airway fluid (pneumonia, pulmonary edema), they may also be produced by the popping open of noninfected fibrotic or atelectatic airways. fine crackles are not audible at the mouth, whereas coarse crackles may be. crackles is the preferred term, rather than the previously popular "rales." • rhonchi, or "large airway sounds," are continuous gurgling or bubbling sounds typically heard during both inhalation and exhalation. these sounds are caused by movement of fluid and secretions in larger airways (asthma, viral uri). rhonchi, unlike other sounds, may clear with coughing. , or the phalangeal depth ratio, is normally less than but increases to more than with finger clubbing. the dpd/ipd ratio can be measured with calipers or, more accurately, with finger casts. the hyponychial angle is measured from lateral projections of the finger contour on a magnifying screen and is normally less than degrees but greater than degrees with finger clubbing. schamroth sign is useful for bedside assessment. the dorsal surfaces of the terminal phalanges of similar fingers are placed together. with clubbing, the normal diamond-shaped aperture or "window" at the bases of the nail beds disappears, and a prominent distal angle forms between the end of the nails. in normal subjects, this angle is minimal or nonexistent. ( • wheezes are continuous musical sounds (lasting longer than msec), caused by vibration of narrowed airway walls, as with asthma, and perhaps vibration of material within airway lumens. these sounds are much more commonly heard during expiration than inspiration. the chest radiograph is often the most useful diagnostic test in the evaluation of the child with cough. table . highlights some of the radiographic features of the most common causes of cough in pediatric patients. radiographic findings are often similar for a number of disorders, and thus these studies may not indicate a definitive diagnosis. chest radiographs are normal in children with psychogenic (habit) cough and in children with sinusitis or gastroesophageal reflux (ger) as the primary cause of cough. a normal chest radiograph indicates the unlikelihood of pneumonia caused by respiratory syncytial virus (rsv), influenza, parainfluenza, adenovirus, chlamydia species, or bacteria. although children with cough resulting from cystic fibrosis (cf), mycoplasma species, tuberculosis, aspiration, a bronchial foreign body, or an anatomic abnormality usually have abnormal chest radiographs, a normal radiograph does not exclude these diagnoses. hyperinflation of the lungs is commonly seen on chest radiographs of infants with rsv bronchiolitis or chlamydia pneumonia, and a lobar or round (coin lesion) infiltrate is the radiographic hallmark of bacterial pneumonia. the diagnosis of sinusitis cannot be sustained with normal sinuses on radiograph or computed tomography (ct) scan. the white blood cell (wbc) count may help exclude or include certain entities for a differential diagnosis. for example, a wbc count of infections are the most common cause of acute cough in all age groups and are responsible for some chronic coughs. the age of the patient has a large impact on the frequency of the type of infection. viral upper respiratory infections (common cold); croup (laryngotracheobronchitis); viral bronchiolitis, particularly with rsv or human metapneumovirus; and viral pneumonia are the most frequently encountered respiratory tract infections and hence the most common causes of cough in infancy. viral illness may predispose to bacterial superinfection (croup and staphylococcus aureus tracheitis or influenza and h. influenzae or s. aureus pneumonia). viral upper respiratory infections (uri). viral uri symptoms and signs usually include stuffy nose with nasal discharge, sore throat, and sneezing. there may be fever, constitutional signs (irritability, myalgias, and headache), or both. cough is common and may persist for - days. the mechanism by which uris cause cough in children is undetermined. in adults, it is generally thought that "postnasal drip"-that is, nasal or sinus secretions draining into the posterior nasopharynx-causes cough and, in fact, may be one of the most frequent causes of cough. indeed, sinus ct in older patients with uris often reveals unexpected involvement of the sinus mucosa. other authorities believe that cough in a child with a uri indicates involvement (inflammation or bronchospasm) of the lower respiratory tract. over-the-counter cough and cold medications are commonly used. evidence of efficacy of these medications for children with uri is lacking. because of the known risk for unintentional overdose from these medications, their use is not recommended in children under age years. , with % lymphocytes strongly suggests pertussis, but not every child with pertussis presents such a clear hematologic picture. the presence of a high number or large proportions of immature forms of wbcs suggests an acute process, such as a bacterial infection. immunoglobulins provide supportive evidence for a few diagnoses, such as chlamydial infection, which rarely occurs without elevated serum concentrations of immunoglobulins g and m. specific bacteriologic or virologic diagnoses can be made in a number of disorders causing cough, including rsv, influenza, parainfluenza, adenovirus, and chlamydia pneumonia. in most cases, the viruses can be rapidly identified with amplification of the viral genome through polymerase chain reaction (pcr). in bacterial pneumonia, the offending organism can be cultured from the blood in a small proportion ( %) of patients. a positive culture provides definitive diagnosis, but a negative culture specimen is not helpful. throat cultures are seldom helpful (except in cf) in identifying lower respiratory tract bacterial organisms. sputum cultures and gram stains may help guide initial empirical therapy in older children with pneumonia or purulent bronchitis, but their ability to identify specific causative organisms with certainty (with the exception of cf) has not been shown clearly. infants and young children usually do not expectorate but rather swallow their sputum. specimens obtained via bronchoscopy may be contaminated by mouth flora, but heavy growth of a single organism in the presence of polymorphonuclear neutrophils certainly supports the organism's role in disease. if pleural fluid or fluid obtained directly from the lung via needle aspiration is cultured, the same rules apply: positive cultures are definitive, but negative cultures are not. a number of specific tests can help to establish diagnoses in a child with cough (see table . ). these include a positive response to bronchodilators in a child with asthma; visualizing the red, swollen epiglottis in epiglottitis (to be done only under very controlled conditions); the bronchoscopic visualization of the peanut, plastic toy, or other offender in foreign body aspiration; a positive purified protein diaphragm, with an enlarged retrosternal air space in as many as % of patients, peribronchial thickening in approximately %, and consolidation and/or atelectasis in - %. the diagnosis is confirmed with demonstration of rsv by pcr of nasopharyngeal secretions. in most cases, no treatment is needed because the disease does not interfere with the infant's eating or breathing. apnea is a common complication of rsv bronchiolitis in neonates and may necessitate close monitoring. in severe cases, often those in which there is underlying chronic heart, lung, or immunodeficiency disease, rsv can be life-threatening. in severe cases, hospital care with supplemental oxygen and intravenous fluids is indicated. suctioning of secretions is an essential part of the treatment. many other treatment modalities have been tried for hospitalized infants with bronchiolitis. aerosolized bronchodilators and systemic glucocorticoids do not seem to alter clinical outcome and are not recommended in most patients. nebulized saline may reduce the length of hospitalization. use of high-flow nasal cannula may reduce the need for more invasive forms of respiratory support in infants with impending respiratory failure. viral pneumonia. viral pneumonia can be similar to bronchiolitis in its manifestation, with cough and tachypnea, after a few days of apparent uri. there can be variable degrees of fever and of overall illness. infants and children with viral pneumonia may appear relatively well or, particularly with adenovirus or influenza, may have a rapidly progressive course. frequent symptoms include poor feeding, cough, cyanosis, fever (some patients may be afebrile), apnea, and rhinorrhea. frequent signs include tachypnea, retractions, crackles, and cough. cyanosis is less common. the most common agents causing viral pneumonia in infancy and childhood are rsv, influenza, and parainfluenza. adenovirus is less common, but it is important because it can be severe and leave residua, including bronchiectasis and bronchiolitis obliterans. adenovirus pneumonia is often accompanied by conjunctivitis and pharyngitis, in addition to leukocytosis and an elevated erythrocyte sedimentation rate (esr); the esr and leukocyte count are usually not elevated in other types of viral pneumonia. additional viral agents include enteroviruses, human metapneumovirus and rhinovirus. radiographs most often reveal diffuse, bilateral peribronchial infiltrates, with a predilection for the perihilar regions, but occasionally lobar infiltrates are present. pleural effusions are not common. on occasion, if an infant is extremely ill, bronchoscopy with bronchoalveolar lavage may be indicated to isolate the virus responsible for the pneumonia. treatment is largely supportive, with oxygen and intravenous fluids. mechanical ventilation may be necessary in a small minority of infants. in young infants, the afebrile pneumonia syndrome may be caused by chlamydia, ureaplasma, or mycoplasma species; cytomegalovirus; or pneumocystis jiroveci. in this syndrome, cough and tachypnea are common. severe pneumonia may develop in neonates as a result of herpes simplex. pertussis (whooping cough). pertussis is a relatively common cause of lower respiratory tract infection in infants, children, adolescents, and adults, especially in those who are underimmunized or not immunized. the causative organism, bordetella pertussis, has a tropism for tracheal and bronchial ciliated epithelial cells; thus the disease is primarily bronchitis, but spread of the organism to alveoli, or secondary invasion by other bacteria, can cause pneumonia. the disease can occur at any age, from early infancy onward, although its manifestations in young infants and in those who have been partially immunized may be atypical. most commonly, pertussis has three stages: • catarrhal, in which symptoms are indistinguishable from a viral uri common viral pathogens include rhinovirus, rsv, coronaviruses, and parainfluenza viruses. the differential diagnosis includes allergic rhinitis, which often demonstrates clear nasal secretions with eosinophils and pale nasal mucosa, and sinusitis, which presents with mucopurulent nasal secretions containing neutrophils and erythematous mucosa. croup (laryngotracheobronchitis). infectious croup (see chapter ) is most common in the first years of life. its most dramatic components are the barking ("croupy") cough and inspiratory stridor, which appear a few days after the onset of a cold. in most cases, the patient has a low-grade fever, and the disease resolves within a day or two. in severe cases, the child can be extremely ill and is at risk for complete laryngeal obstruction. there may be marked intercostal and suprasternal retractions and cyanosis. stridor at rest signifies significant obstruction. diminishing stridor in a child who is becoming more comfortable is a good sign, but diminishing stridor in and of itself is not necessarily good: if the child becomes fatigued because of the tremendous work of breathing through an obstructed airway and can no longer breathe effectively, smaller-than-needed tidal volumes make less noise. it is important to distinguish croup from epiglottitis in the child with harsh, barking cough and inspiratory stridor because the natural histories of the two diseases are quite different (see table . ). epiglottitis occurs more commonly in unimmunized toddlers than in infants (see chapter ). treatment of mild croup is usually not needed. for decades, pediatricians have recommended putting a child with croup in a steamy bathroom or driving to the office or emergency department with the car windows rolled down. it is likely that these remedies are effective because of the heat exchange properties of the upper airway; air that is cooler or more humid than the airway mucosa will serve to cool the mucosa, thus causing local vasoconstriction and probably decreasing local edema. in a child who has stridor at rest, evaluation is indicated. symptomatic, often dramatic relief through decreased laryngeal edema can usually be achieved with aerosolized racemic epinephrine ( . % solution, . to . ml/dose). it is essential to remember that the effects of the epinephrine are transient, lasting only a few hours, although the course of the illness is often longer. the result is that when the racemic epinephrine's effect has worn off, the child's cough and stridor will probably be as bad or even worse than before the aerosol was administered. this is not a "rebound" effect: the symptoms are not worse because of the treatment but, rather, because of the natural progression of the viral illness. repeating the aerosol will probably again have a beneficial effect. a child who responds favorably to such an aerosol needs to be observed for several hours because further treatment may be needed. a single dose of dexamethasone ( . mg/kg orally, intramuscularly, or intravenously) reduces the severity and hastens recovery. aerosolized steroids (budesonide) may also be effective in patients with mild to moderately severe croup. bronchiolitis. bronchiolitis is a common and potentially serious lower respiratory tract disorder in infants (see chapter ). it is caused usually by rsv but on occasion by parainfluenza, influenza, human metapneumovirus, adenovirus, enterovirus, and human rhinovirus. it mostly occurs in the winter months, often in epidemics. rsv bronchiolitis is seen uncommonly in children older than years. typically, "cold-like" symptoms of rhinorrhea precede the harsh cough, increased respiratory rate, and retractions. respiratory distress and cyanosis can be severe. the child's temperature is seldom elevated above °c. the chest is hyperinflated, widespread crackles are audible on inspiration, and wheezing marks expiration. the chest radiograph invariably reveals hyperinflation, as depicted by a depressed .e (see nelson textbook of pediatrics, p. .) (see nelson textbook of pediatrics, p. .) colds and cough in infants (see previous discussion) apply to this older age group. the differential diagnosis of rhinorrhea is noted in table . . sinusitis. the sinuses may become the site for viral and subsequent secondary bacterial infection spreading from the nasopharynx (fig. . ) . the signs and symptoms are usually localized, including nasal congestion, a feeling of "fullness" or pain in the face (fig. . ) , headache, sinus tenderness, day or night cough, and fever. maxillary toothache, purulent nasal discharge for more than days, and a positive transillumination (opacification) are important clues. sinus radiographs or (more accurate) ct scan may facilitate the diagnosis of sinusitis by demonstrating opacification of the sinus with mucosal thickening. sinusitis is thought to be a cause of cough in adults and can probably be listed, with lower certainty, as a cause of cough in children. sinusitis is frequently seen in other conditions known to cause cough, especially cf, asthma, ciliary dyskinesia, and granulomatosis with polyangiitis with or without eosinophilia. it may be difficult to ascertain whether the cough is a direct result of the sinus infection or the underlying problem (purulent bronchitis in the child with cf or ciliary dyskinesia, exacerbation of asthma). in the first two situations, it may not matter because treatment is the same. in the case of the child with asthma, it is important to treat the asthma with bronchodilating and antiinflammatory agents, as well as to treat the infected sinuses with antibiotics. • paroxysmal, dominated by repeated forceful, paroxysmal coughing spells; spells may be punctuated by an inspiratory "whoop," posttussive emesis, or both • convalescent, in which the intensity and frequency of coughing spells gradually diminish each stage typically lasts - weeks, except the paroxysmal stage, which lasts many weeks. (pertussis is known as the " day cough" in china.) most children are entirely well between coughing spells, when physical findings are remarkably benign. infants younger than months of age are at highest risk for complications. the majority of infants with pertussis need to be hospitalized. diagnosis can be difficult because the definitive result-namely, culturing the organism from nasopharyngeal secretions-requires special culture medium (bordet-gengou, which must be prepared fresh for each collection). culture specimens are much less likely to be positive during the paroxysmal stage than during the catarrhal stage, when the diagnosis is not being considered. pcr assay of an adequate nasopharyngeal (np) specimen is the most commonly used test because of improved sensitivity and faster turnaround time compared to culture. an elevated wbc count, as high as , - , , with lymphocytes predominating is suggestive of pertussis in infants and children but often absent in adolescents. chest radiographic findings are nonspecific. infants with severe disease may require hospitalization. treatment is largely supportive, with oxygen, fluids, and small frequent feedings for patients who do not tolerate their normal feedings. treatment with azithromycin decreases infectivity and may ameliorate the course of the disease if given during the catarrhal stage. complications include those related to severe coughing (table . ) and those specific to pertussis, such as seizures and encephalopathy. the risk of acquiring pertussis is markedly reduced by immunizations (three primary immunizations and regular booster immunizations). neither pertussis infection nor immunization produces lifelong immunity. chlamydial infection. chlamydia trachomatis can cause pneumonia in young infants, particularly those aged - weeks. cough, nasal congestion, low-grade or no fever, and tachypnea are common. conjunctivitis is an important clue to chlamydial disease but is present in only % of infants with chlamydial pneumonia at the time of presentation. affected infants may have a paroxysmal cough similar to that of pertussis, but post-tussive emesis is less common. crackles are commonly heard on auscultation, but wheezing is much less common than the overinflated appearance of the lungs on radiographs would suggest. the organism may be recovered from the nasopharynx by culture or antigen testing. the complete blood cell count may reveal eosinophilia. chlamydial infection responds to oral erythromycin therapy. ureaplasmal infection. ureaplasma urealyticum pneumonia is difficult to diagnose but causes cough in some infants. there are no particularly outstanding features to distinguish this relatively uncommon infection from viral pneumonias. bacterial pneumonia. bacterial pneumonia is relatively less common in infants than is viral pneumonia but can cause severe illness, with cough, respiratory distress, and fever. chest radiographs are abnormal, and the wbc count is elevated. treatment is with antibiotics effective against pneumococci, group a streptococci, and, if illness is severe, s. aureus. viral uris. in early childhood, as children attend daycare and nursery schools, they are constantly exposed to respiratory viruses to which they have little or no immunity (e.g., rsv, rhinoviruses, adenoviruses, parainfluenza, and enteroviruses). young children may have as many as - or even more uris in a year. the remarks concerning the treatment of sinusitis involves the use of oral antibiotics active against the common pathogens (i.e., streptococcus pneumoniae, nontypable h. influenzae, moraxella catarrhalis, and, in rare cases, anaerobic bacteria or streptococcus pyogenes). treatment regimens include the use of amoxicillin, amoxicillin-clavulanate, cefuroxime, cefpodoxime, or cefdinir. amoxicillin is considered the initial agent of choice. oral (pseudoephedrine, phenylephrine) or topical (phenylephrine, oxymetazoline) decongestants may be of benefit by increasing the patency of the sinus ostia, which permits drainage of the infected and obstructed sinuses. oral antihistamines may benefit patients with an allergic history. treatment with antimicrobial agents should continue for at least days after the patient has responded. this may require - days of therapy. many patients with presumed sinusitis recover without antibiotic therapy. complications of acute sinusitis include orbital cellulitis, abscesses (orbital, cerebral), cranial (frontal) osteomyelitis (pott puffy tumor), empyema (subdural, epidural), and thrombosis (sagittal or cavernous sinus). pneumonia. the features discussed for viral pneumonia in infants are relevant for viral pneumonia in older children. the differentiation of viral or atypical pneumonia from classical bacterial pneumonia is noted in table . . adenovirus and influenza pneumonia may present similar to bacterial pneumonia in severity and acuteness. bacterial pneumonia is more common in toddlers and older children than in infants. the most common pathogen is s. pneumoniae. (table . ). cough may not be as prominent a presenting symptom or sign as tachypnea and grunting. raised respiratory rates (> in infants - months old, > in children - years old) plus retractions and grunting with or without hypoxia (oxygen saturation < %) have a high specificity and sensitivity for pneumonia. chest pain, abdominal pain, headache, or any combination of these symptoms may occur. upper lobe pneumonia may produce meningeal signs, and lower lobe involvement may cause abdominal pain and an ileus. examination of the chest shows tachypnea but may be otherwise surprisingly normal. in older children, there may be localized dullness to percussion, with crackles or amphoric (bronchial) breath sounds over a consolidated lobe. the chest radiograph may be normal in the first hours of the illness, inasmuch as the radiographic findings often lag behind the clinical manifestations. nonetheless, both anteriorposterior and lateral views are the main diagnostic tools; lobar consolidation is usual, with or without pleural effusion. in infants, the pattern may be more diffuse and extensive. *atypical pneumonia syndrome (see table . ); atypical in terms of extrapulmonary manifestations, low-grade fever, patchy diffuse infiltrates, poor response to penicillin-type antibiotics, and negative sputum gram stain. sars, severe acute respiratory syndrome. some clinical and radiographic features may be suggestive of the bacterial cause of pneumonia. children (especially infants) with staphylococcal pneumonia are more likely to have a rapid overwhelming course. staphylococcal pneumonia may be accompanied by more extensive radiographic abnormalities, including multilobar consolidation, pneumatocele formation, and extensive pleural (empyema) fluid. the presence of a pleural effusion is not helpful in indicating the specific bacterial diagnosis because other bacterial pneumonias may be accompanied by pleural effusion. pleural effusions may represent a reactive parapneumonic effusion or an empyema. pleural fluid may be characterized as transudate, exudate, or empyema (table . ). if the effusion is of sufficient size, as demonstrated by a lateral decubitus radiograph or ultrasonography, a thoracentesis may be indicated to differentiate the nature of the effusion and to identify possible pathogens. for young children who require sedation for thoracentesis and who have an effusion needing drainage, a primary chest tube placement is preferred over thoracentesis to decrease the risks from multiple procedures with sedation. differentiating among the causes of bacterial pneumonia can be done with certainty only with positive cultures from blood, pleural fluid, fluid obtained by direct lung tap, or, in rare cases, sputum. current or previous antibiotic treatment diminishes the yield of such cultures. bronchoscopy with or without lavage may yield helpful specimens from the progressively ill child or the child who has not responded promptly to empirical antibiotics. treatment of uncomplicated presumed bacterial pneumonia is with antibiotics. ampicillin is the drug of choice for the previously healthy child who requires hospitalization with lobar pneumonia who is fully immunized. if the child is not fully immunized, either cefotaxime or ceftriaxone is indicated. for the critically ill child, vancomycin and cefotaxime/ceftriaxone may be considered for possible drug-resistant s. pneumoniae and methicillin-resistant s aureus (mrsa). many children with pneumonia do well with oral antibiotics and respond within hours to the first dose. repeated or follow-up chest radiographs may remain abnormal for - weeks after appropriate treatment and are not indicated for a single episode of uncomplicated pneumonia (i.e., to an adult with active tuberculosis. nonetheless, tuberculosis is an infrequent cause of cough in children, even in those with active disease. the diagnosis is made primarily by skin testing (purified protein derivative [ppd]) or a positive quantiferon test; a history of contact with a person who has tuberculosis; and recovery of the organism from sputum, bronchoalveolar lavage, pleural fluid or biopsy, or morning gastric aspirates (table . ). the yield from these procedures is relatively low, even from children with active pulmonary tuberculosis. no effusion, no abscess, and good response to treatment). mycoplasma pneumoniae is a common cause of pneumonia among school-aged children. the disease often occurs in community outbreaks in the fall. the illness typically begins with extrapulmonary symptoms (i.e., sore throat, myalgias, headache, fever), which then progress to include cough, which can be paroxysmal at times. patients do not often appear acutely ill, but cough may persist for weeks. there may be no specific abnormalities on the chest examination, although a few crackles may be heard, and about one third of younger patients wheeze. the radiographic findings in mycoplasmal pneumonia can mimic almost any intrathoracic disease; scattered infiltrates with nonspecific "dirty" lung fields, predominantly perihilar or lower lobes, are common, and lobar infiltrates and pleural effusion are occasionally seen. laboratory data (complete blood cell count, esr, sputum culture) may not be helpful. a rise in antimycoplasma immunoglobulin g over - weeks may be demonstrated but is seldom helpful in guiding therapy. a positive immunoglobulin m response may be useful, although it can persist in serum for several months and, consequently, may not indicate current infection. pcr is helpful. the cold agglutinin test yields positive results in about % of patients with mycoplasmal pneumonia, but they are also positive in other conditions, including adenovirus infection. the more severe the illness is, the greater is the frequency of positive cold agglutinins. the diagnosis is often made from the history of an older child who has a lingering coughing illness in the setting of a community outbreak, unresponsive to most (nonerythromycin) antibiotic regimens. treatment with azithromycin, clarithromycin, or erythromycin in children < years old or tetracycline or doxycycline in children ≥ years old usually shortens the course of illness. extrapulmonary complications of mycoplasmal infection include aseptic meningitis, transverse myelitis, peripheral neuropathy, erythema multiforme, myocarditis, pericarditis, hemolytic anemia, and bullous otitis media (myringitis). in patients with sickle cell anemia, severe respiratory failure and acute chest syndrome may develop. infection with chlamydia pneumoniae mimics respiratory disease resulting from m. pneumoniae, inasmuch as it occurs in epidemics, is seen in older children, and produces an atypical pneumonia syndrome and pharyngitis. tuberculosis. tuberculosis is uncommon in developed countries; % of the disease burden worldwide is in developing countries. tuberculosis must be considered in the child with chest disease that is not easily explained by other diagnoses, especially if the child lives in or has migrated from an endemic area of the world or has been exposed cutaneous induration ≥ mm • close exposure to known or suspected active tb • chest radiograph consistent with tb (old or active) • clinical evidence of tb • children receiving immunosuppresive therapy or with immunosuppressive conditions cutaneous induration ≥ mm children at increased risk • age < yr of age • medical high risks (chronic renal failure, malnutrition, diabetes mellitus, lymphoma) children with likelihood of increased exposure • children born in high-prevalence regions of the world • children who travel to high-prevalence regions of the world • children frequently exposed to adults who are hiv infected, homeless, users of illicit drugs, residents of nursing homes, incarcerated, or institutionalized cutaneous induration ≥ mm • all children ≥ yr of age without any identifiable risk the patterns of disease in normal hosts include primary pulmonary tuberculosis, with subsequent inactivation usually noted in young children and reactivation pulmonary disease among adolescents. primary pulmonary disease is often noted as a lower or middle lobe infiltrate during the period of t lymphocyte reaction to the initial infection. before resolution, the mycobacterium tuberculosis infection may disseminate to the better oxygenated upper lobes and extrathoracic sites, such as bone, or the central nervous system. if the immune response contains the initial infection, the radiographic findings may be indistinguishable from those of any other pneumonic process. with altered immune function, however, there may be progressive local disease, dissemination to miliary pulmonary disease, or early reactivation (months to years) at distal sites, which produces tuberculous meningitis or osteomyelitis. reactivation of upper lobe pulmonary disease may produce cavities that are similar to the disease among adults. cavitary and endobronchial lymph node involvement are highly infectious, in contrast to the much less contagious nature of the hypersensitivity reaction noted in primary pulmonary disease. inhaling food, mouth or gastric secretions, or foreign bodies into the tracheobronchial tree causes acute, recurrent, or chronic cough. interference with normal swallowing disrupts the coordination of swallowing and breathing that prevents aspiration. structural causes of disordered swallowing include esophageal atresia (in neonates), strictures, webs, or congenital stenoses. mediastinal lesions (tumors, lymph nodes), including vascular rings, may compromise the esophageal lumen and esophageal peristalsis, increasing the likelihood of aspiration. functional disorders include central nervous system dysfunction or immaturity, dysautonomia, achalasia, and diffuse esophageal spasm. prior neck surgery, including tracheostomy, may alter normal swallowing. tracheoesophageal fistula and laryngeal clefts are congenital malformations with direct physical connections between the tracheobronchial tree and the upper gastrointestinal tract; thus oral contents enter the lungs directly. making the diagnosis of aspiration as the cause of cough may be difficult. barium contrast studies during swallowing may help characterize these disorders if barium enters the trachea. because most patients aspirate sporadically, a normal barium swallow does not rule out aspiration. radionuclide studies can be helpful if ingested radiolabeled milk or formula is demonstrated over the lung fields at severalhour intervals after the meal. bronchoscopy and bronchoalveolar lavage that recover large numbers of lipid-laden macrophages suggest that aspiration has taken place; however, the finding is neither sensitive nor specific for aspiration. treatment depends largely on the cause of aspiration. because many patients who aspirate do so because of lack of neurologic control of swallowing and breathing, it is often difficult to prevent. even gastrostomy feedings cannot prevent aspiration of oral secretions. in extreme cases, tracheostomy with ligation of the proximal trachea has been employed. this not only prevents aspiration but also prevents phonation, and it must be considered only in unusual situations. aspiration pneumonia is often treated with intravenous ampicillinsulbactam or clindamycin to cover mouth flora of predominant anaerobes. additional coverage against gram-negative organisms may be indicated if the aspiration is nosocomial. any child with cough of abrupt onset should be suspected of having inhaled a foreign body into the airway. toddlers, who by nature put all types of things into their mouths and who have incompletely matured swallowing and airway protective mechanisms, are at high risk. infants with toddlers or young children in the household who may "feed" the baby are also at risk. in older children, it is usually possible to obtain an accurate history of the aspiration event. these events are described as choking, gagging, and coughing while something (e.g., peanuts, popcorn, small toys, sunflower seeds) is in the mouth. the child may come to the physician with cough and wheeze immediately after the event, with a clear history and a straightforward diagnosis. in many children with a tracheobronchial foreign body, however, the initial episode is not recognized; these children may not come to medical attention for days, weeks, or even months. the initial episode may be followed by a relatively symptom-free period lasting days or even weeks, until infection develops behind an obstructed segmental or lobar bronchus. at this point, cough, perhaps with hemoptysis, with or without wheeze, recurs. on physical examination early after an aspiration episode, there is cough, wheeze, or both, often with asymmetry of auscultatory findings. there may be locally diminished breath sounds. later, localized wheeze or crackles may be detected. the triad of wheezing, coughing, and decreased breath sounds is present in fewer than % of patients. the presence of laryngotracheal foreign bodies often manifests with stridor, retractions, aphonia, cough, and normal radiographs. chest radiographs may be normal in % of patients with intrathoracic foreign bodies but should be obtained in both inspiration and expiration because in some cases the only abnormality is unilateral or unilobar air trapping, which is occasionally more clearly identified with an expiratory radiograph. in this view, an overdistended lung that had appeared normal on the inspiratory view does not empty, but the normal, unobstructed lung empties normally. this phenomenon causes a shift of the mediastinum toward the emptying lung, away from the side with the obstructing foreign body (fig. . ) . in other patients, localized infiltrate or atelectasis may be present behind the obstructing object. in a few patients, it may be possible to identify the foreign body itself; nonetheless, most inhaled food particles are not radiopaque and cannot be seen on radiographs. aspiration is usually unilateral ( %); - % of the objects are in the right lung (the lobe depends on body position-supine versus standing-but is often the right middle lobe). the definitive diagnostic and therapeutic maneuver is bronchoscopy; either the flexible or rigid open-tube bronchoscope enables direct visualization of the object; the rigid instrument also enables its removal. ger is a common cause of cough in all age groups (see chapter ). the typical patient is an infant in the first months of life who spits up small amounts of milk frequently after feedings. this "regurgitant reflux" most commonly resolves by year of age. however, many toddlers and children continue to have reflux, although it may be "silent" or nonregurgitant (without spitting up). in most people with ger, it is merely a nuisance or not noticed. in some there are sequelae, and this condition is designated gastroesophageal reflux disease (gerd). one manifestation is cough; the mechanisms for the cough are not fully understood. aspiration of refluxed material is one mechanism for cough but is probably not very common in neurologically intact children. a major mechanism for gerd with cough is mediated by vagal esophagobronchial reflexes (bronchoconstriction), stimulated by acid in the esophagus. whether acid in the esophagus is sufficient stimulus to cause bronchoconstriction by itself or whether it merely heightens bronchial reactivity to other stimuli is not yet clear. many children with reactive airways disease have cough or wheeze that is difficult to control until their concurrent ger is also treated. many episodes of cough caused by gerd occur in children with asthma that is difficult to control. (cftr), which acts as a chloride channel and affects other aspects of membrane transport of ions and water. not all the consequences of the defective gene and protein have been determined. in general, however, the defective gene product results in the long-observed clinical manifestations of the disease, including thick, viscid mucus in the tracheobronchial tree, leading to purulent bronchiolitis and bronchitis with subsequent bronchiectasis, pulmonary fibrosis, and respiratory failure; pancreatic duct obstruction, leading to pancreatic insufficiency with steatorrhea and failure to thrive; and abnormally high sweat chloride and sodium concentrations. the airway disease in cf is characterized by infection, inflammation, and endobronchial obstruction. the infection begins with s. aureus, h. influenzae, escherichia coli, klebsiella species, or combinations of these organisms but eventually is dominated by nonmucoid or mucoid pseudomonas aeruginosa. other organisms, such as burkholderia cepacia, stenotrophomonas maltophilia, alcaligenes xylosoxidans, aspergillus fumigatus, or nontuberculous mycobacteria may also appear; their significance remains undetermined. in some patients, b. cepacia has been associated with rapid deterioration and death, and in others, aspergillus species has caused allergic bronchopulmonary aspergillosis (abpa). the airway inflammation in all patients with cf appears to be the result of toxic substances, including elastase, released by neutrophils as they respond fig. . .) a b the diagnosis of gerd must also be considered in the child with chronic or recurrent cough with no other obvious explanation. the child who coughs after meals or at night, when the supine position may provoke ger, should be evaluated for ger. if ger is confirmed, the next step is a therapeutic trial of antireflux therapy. treatment in a child whose cough is related to ger may be accomplished by treating the reflux (see chapter ) or by a combination of antireflux and antiasthma treatment (see chapter ). on occasion, the cough may be abolished by stopping all antiasthma medications. in such cases, the cough was a manifestation of reactive airways with esophageal acidification as the trigger for bronchospasm; the esophageal acidification was caused by the bronchodilator effects on the lower esophageal sphincter. cough is frequently the sole or most prominent manifestation of asthma; wheezing may be entirely absent. in fact, asthma is almost certainly the most common cause of recurrent and chronic cough in childhood (see chapter ). some of the features that characterize the cough of a child with asthma are listed in table . . treatment for asthma manifesting as cough is the same as the treatment for asthma. cystic fibrosis (cf) is a common cause of recurrent or chronic cough in infancy and childhood. cf occurs in in - live births among white persons, is far less common among african americans ( in , ), and is rare among native americans and asians. early diagnosis improves the prognosis for untreated cf; if untreated, many patients die in infancy or early childhood. with current state-of-the-art care, median length of survival is upper s. cf is a genetic disorder, inherited as an autosomal recessive trait. the cf gene is on the long arm of chromosome ; more than mutations have been identified at the cf locus. of these mutations, one (Δf , indicating a deletion, Δ, of a single phenylalanine, f, at position of the protein product) is the most common, responsible for - % of all cf chromosomes. the mutation affects the gene's protein product, termed cystic fibrosis transmembrane regulator any age (even infants) coexistence of allergy increases likelihood, but absence of allergy does not decrease likelihood wheeze need not be present ↑cough with upper respiratory infections ↑cough with (and especially after) exercise ↑cough with hard laughing or crying ↑cough with exposure to cold ↑cough with exposure to cigarette smoke usually a history of dramatic response to inhaled β-agonists cf may manifest at birth with meconium ileus ( - % of patients), or later, with steatorrhea and failure to thrive despite a voracious appetite, in an apparent effort to make up for the calories that are lost in the stool (see chapter ). the most common presenting symptom is cough, which may appear within the first weeks of life or may be delayed for decades. the cough can be dry, productive, or paroxysmal. cough may respond to antibiotics or perhaps steroids, but it is less likely to improve with bronchodilators (see tables . and . ). although cf is a genetic disease, there is often no family history. furthermore, in atypical cases, patients may not have pancreatic insufficiency (~ % of patients) and thus may not demonstrate steatorrhea and failure to thrive. in addition, malabsorption may not be evident in the neonatal period. there is no such thing as a child who looks "too good" to have cf; common abnormalities found on physical examination are noted in table . . one of the most important physical findings is digital clubbing. in most patients with cf, clubbing develops within the first few years of life. although the list of conditions associated with digital clubbing (table . ) is long, they are less common than cf, or the incidence of digital clubbing with these conditions is low. there is some relationship between the degree of pulmonary disease severity and the degree of digital clubbing. a child who has had years of severe respiratory symptoms without digital clubbing is not likely to have cf. the diagnosis is confirmed by a positive sweat test or confirming the presence of two cf mutations on chromosome . the sweat test, if not performed correctly in a laboratory with extensive experience with the technique (as, for example, in an accredited cf center), yields many false-positive and false-negative results. the proper technique is to use quantitative analysis of the concentration of chloride in the sweat produced after pilocarpine iontophoretic stimulation. chloride concentrations higher than mmol/l are considered positive, and those lower than mmol/l are negative (normal). healthy adults have slightly higher sweat chloride concentrations than do children, but the same guidelines hold for positive tests in adults. the non-cf conditions yielding elevated sweat chloride concentrations are listed in table . . false-negative results of sweat tests can be seen in cf children presenting with edema or hypoproteinemia and in samples from children with an inadequate sweat rate. sweat testing can be performed at any age. newborns within the first few weeks of life may not produce a large enough volume of sweat to analyze ( mg minimum), but in those who do (the majority), the results are accurate. indications for sweat testing are noted in table . . in patients for whom sweat testing is difficult (e.g., because of distance from an experienced laboratory, small infants who have not produced enough sweat, patients with extreme dermatitis, or patients with intermediate-range sweat chloride concentrations), dna mutation testing can be useful. demonstration of two known cf mutations confirms the diagnosis. finding one or no known mutation makes the diagnosis less likely but is not exclusive, inasmuch as there are patients all states are using a neonatal screen for cf. the cf screen assays include measuring serum immunoreactive trypsinogen (irt) levels, which are elevated in most infants with cf for the first several weeks of life, and dna analysis for cftr mutations. the main drawback of the irt assay is that it has relatively poor specificity; as many as % of the positive results on the initial screen are false-positive results. if an infant's irt screen is positive, the test should be repeated, or dna analysis for the most common cftr mutations should be performed. at - weeks of age, which is when the irt is repeated, the false-positive rate has fallen dramatically but is still quite high ( %). definitive testing with the sweat chloride test needs to be carried out on infants with positive screening results. laboratory data that may support the diagnosis of cf include low levels of fecal elastase. this suggests pancreatic insufficiency, which occurs most commonly in cf but can be seen in other diseases. the test is not perfect for confirming cf as some cf patients have sufficient pancreatic function. pulmonary function test findings with an obstructive pattern, incompletely responsive to bronchodilators, are consistent with cf but, of course, can be seen in other conditions. conversely, some patients with cf also have asthma and may show a marked response to a bronchodilator. complications of cf that should suggest the diagnosis are noted in table . . the treatment of patients with cf requires a comprehensive approach, best performed in, or in conjunction with, an approved cf center. several studies have shown survival to be significantly better in center-based care than in non-center-based care. table . lists the main anatomic abnormalities that cause cough. vascular rings and slings. vascular rings and slings are often associated with inspiratory stridor because the abnormal vessels compress central airways, most commonly the trachea (see chapter ). the patient may also have difficulty swallowing if the esophagus is compressed. the diagnosis may be suspected from plain radiographs of the chest, especially those showing tracheal deviation and a right-sided aortic arch. further support for the diagnosis can be found at bronchoscopy (which shows extrinsic compression of the trachea or a main stem bronchus), barium esophagram (which shows esophageal compression), or both. the definitive diagnosis is made with computed tomographic angiography or magnetic resonance angiography. treatment is surgical. pulmonary sequestration. pulmonary sequestration is relatively unusual, occurring in in , children. it occurs most commonly in the left lower lobe and can manifest in several ways (fig. . ; see also table . ). the chest radiograph usually shows a density in the left lower lobe; this density often appears to contain cysts (fig. . ) . the feature distinguishing a sequestered lobe from a complicated pneumonia is that the blood supply arises from the aorta and not the pulmonary circulation. doppler ultrasonography and ct angiography provide the definitive diagnosis. the treatment is surgical removal. congenital pulmonary airway malformation (cpam). congenital pulmonary airway malformations (formerly known as congenital cystadenomatoid malformations or ccams) are rare. they manifest in infancy with respiratory distress in nearly % of cases; the other half may manifest as cough with recurrent infection later in childhood or even adulthood. the chest radiograph reveals multiple cysts, separated by dense areas. chest ct scans can help make the diagnosis with near certainty. surgical removal is the treatment of choice if the lesion is symptomatic. congenital lobar emphysema. congenital lobar emphysema occurs in one of , live births. it can manifest dramatically with with not-yet-characterized mutations. furthermore, commercial laboratories do not identify all of the mutations. recovery of mucoid pseudomonas aeruginosa from respiratory tract secretions is strongly suggestive of cf. similarly pansinusitis is nearly universal among cf patients but is quite uncommon in other children. respiratory distress in the neonatal period or later (fig. . ) , with cough or wheeze, or as an incidental finding on a chest radiograph. radiography shows localized overinflation, often dramatic, with compression of adjacent lung tissue and occasionally atelectasis of the contralateral lung because of mediastinal shift away from the involved side. the appearance on chest ct scan is typical, with widely spaced blood vessels (as opposed to congenital cysts, for example, which have no blood vessels within the overinflated area). bronchoscopy can document patent bronchi and should probably be performed in older children in whom congenital lobar emphysema can be confused with acquired overinflation of a lobe as the result of bronchial obstruction, as with a foreign body. if the disease is symptomatic, treatment is surgical. tracheoesophageal fistula. tracheoesophageal fistula is common, with an incidence of about one in live births. of these fistulas, the large majority ( %) are associated with esophageal atresia; only % are the isolated, h-type fistula (a patent esophagus with fistulous tract connecting the esophagus and trachea). a neonate with esophageal atresia experiences respiratory distress, excessive drooling, and choking and gagging with feeding. the h-type fistula causes more subtle signs and may be undiagnosed for months or even years. the child may have only intermittent feeding trouble, especially with liquids. there may be recurrent lower respiratory tract infections. the diagnosis is not challenging in the infant with esophageal atresia; a nasogastric tube cannot be passed, and swallowed barium outlines the trachea. in the older child with h-type fistula, a barium esophagogram may or may not reveal the fistula. bronchoscopy and esophagoscopy should permit direct visualization of the fistula; however, the opening may be hidden in mucosal folds. treatment is surgical. many children born with tracheoesophageal fistula have recurrent cough and lower respiratory tract infection for many years, even after successful surgical correction. the cough is characteristically the harsh cough of tracheomalacia, which is present at the site of the fistula. the infections result from several causes, including ger, with or without aspiration, and altered mucociliary transport. treatment involves regular chest physiotherapy and early and aggressive use of antibiotics whenever there is evidence of increased pulmonary symptoms. hemangiomas. hemangiomas may be present within the airway and can cause cough, rarely with hemoptysis. stridor (if the hemangioma is high in the airway) and respiratory distress (if the hemangioma is large) may also occur. in rare cases, with very large airway hemangiomas, there may even be dysphagia from extrinsic compression. children with cutaneous hemangiomas in the mandibular or neck region ("beard" distribution) are at risk for an airway hemangioma. the diagnosis is made with bronchoscopy. these lesions may resolve spontaneously over the first year or so. however, if they cause symptoms, it may not be advisable or possible to wait for them to resolve. many airway hemangiomas regress with steroid treatment; however, due to the side effect profile, propranolol is considered the treatment of choice. asthma is a contraindication for propranolol treatment due to its beta-blocking effect and potential to worsen asthma. laser ablation may be indicated in some refractory cases that do not respond to first-line treatment. in the case of a large subglottic hemangioma, a tracheostomy is performed and maintained until the mass regresses. enlarged lymph nodes. enlarged mediastinal lymph nodes, such as those resulting from tuberculosis, leukemia, other hematologic malignancies, or other infections, are occasionally a cause of cough in children ( plain radiographs of the chest. the x-ray study or bronchoscopy may show extrinsic compression of the trachea. treatment is directed at the underlying cause. bronchial stenosis. occasionally bronchial stenosis, either congenital or acquired, may cause cough. the diagnosis is made with bronchoscopy, after suspicion has been raised by the child having recurrent infiltrates in the same lobe, especially with localized wheeze. treatment may be difficult. in some cases, endoscopic balloon dilatation or airway stent placement is successful; in others, surgical resection of stenotic areas may be necessary. bronchogenic cysts. bronchogenic cysts are uncommon, but they can cause cough, wheeze, stridor, or any combination of these. they may also cause recurrent or persistent pneumonia if they block a bronchus sufficiently to interfere with normal drainage of the segment or lobe. radiography may show localized overinflation if the cyst causes a ball-valve-type obstruction. the cyst itself may or may not be seen on plain radiographs. bronchoscopy reveals extrinsic compression of the airway. ct studies often definitively show the lesion. surgical removal is indicated. on occasion, a school-aged child may develop a cough that lasts for weeks, often after a fairly typical cold. this cough occurs only during wakefulness, never during sleep. in many cases, the cough is harsh and foghorn-like. it often disrupts the classroom, and the child is asked to leave. the child is otherwise well and may seem rather unbothered by the spectacle created. there is no response to medications. it seems that this type of cough, previously termed "psychogenic," or "psychogenic cough tic," but now called habit cough, has given the child valuable attention. this attention then serves as the sustaining force, and the cough persists beyond the original airway inflammation. in the small minority of cases, there may be deep-seated emotional problems of which the cough is the physical expression. during the history or physical examination, the child appears completely well and may cough when attention is drawn to the child or when the word "cough" is uttered. the physical examination findings are otherwise completely normal, as are laboratory values. because this may occur in any child, evidence of mild reactive airways disease (history or pulmonary function testing) does not rule out the diagnosis. once a physician has seen a child with this problem, it is usually possible to make the diagnosis with certainty on entering the examining room or, indeed, from the hallway outside the room. treatment can prove more difficult. the child and family should be reassured that the child is well. suggestion therapy empowers and encourages the patient to suppress the cough for short increments of time. the goal is for them to gradually lengthen the cough-free intervals. speech therapy may be helpful ( infiltrates, and there is invariably iron deficiency anemia. the diagnosis is based on lung biopsy findings. tumors. tumors causing cough are rare in childhood. cough occurs because of bronchial blockage, either extrinsic or endobronchial (see table . ). the diagnosis is usually made from bronchoscopy, chest ct, or both. treatment depends on the cell type, but it usually involves at least some surgical removal. chemotherapy or radiation may be used in some cases. tracheomalacia and bronchomalacia. isolated tracheomalacia or bronchomalacia is uncommon but can cause cough in some children. the cough of tracheomalacia is typically harsh and brassy. treatment is difficult but, fortunately, is seldom needed. spasmodic croup. some children, usually preschoolers, may episodically awaken at night with stridor and a harsh, barking cough indistinguishable from that of viral croup. this entity is termed spasmodic croup and is of unclear origin. viral and allergic causes have been postulated. ger may be the cause in some patients. treatment with cool mist or racemic epinephrine is effective in most patients. if ger is the underlying cause, antireflux treatment is beneficial. obliterative bronchiolitis. obliterative bronchiolitis is very rare except in lung transplant recipients. in other instances, it may arise after adenovirus, measles, or influenza pneumonia; after exposure to certain toxins; or in other rare circumstances. children may exhibit cough, respiratory distress, and exercise intolerance. the diagnosis is suggested by the pulmonary function test or radiographic evidence of small airways obstruction; however, these findings are not always present. not all chest radiographs show overinflated lungs, and not all pulmonary function tests show decreased small airways function. the definitive diagnosis is histologic via open or transbronchial biopsy. no specific treatment is available. most children with obliterative bronchiolitis recover, but many progress to chronic disability or death. the child who coughs out blood or bloody mucus presents special diagnostic and therapeutic challenges. although hemoptysis is relatively uncommon in children, particularly among those without cf, many conditions can cause it (table . ). it is important (and not always easy) to distinguish cases in which blood has originated in the tracheobronchial tree (true hemoptysis), the nose (epistaxis), and the gastrointestinal tract (hematemesis). table . gives some guidelines to help localize sites of origin of blood that has been reported or suspected as hemoptysis. none of these guidelines is foolproof, partly because blood that has originated in one of these sites might well end up in another before being expelled from the body; for instance, blood from the nose can be swallowed and vomited or aspirated and expectorated. infection is among the most common causes of hemoptysis. lung abscess and tuberculosis need to be considered. bronchiectasis can readily cause erosion into bronchial vessels, often made tortuous by years of local inflammation, and produce hemoptysis. other infectious causes are less common and include necrotizing pneumonias and fungal and parasitic lung invasion. foreign bodies in the airway can cause hemoptysis by direct irritation, by erosion of airway mucosa, or by secondary infection. pulmonary embolus is uncommon in children and adolescents, but it needs to be considered in the differential diagnosis of an adolescent with hemoptysis of unclear origin. clues to the diagnosis of pulmonary embolus include a positive family history, severe dyspnea, chest pain, hypoxia, a normal chest radiograph, an accentuated second bronchiectasis. bronchiectasis is defined as an abnormal dilation of the subsegmental bronchi and is usually associated with chronic cough and purulent sputum production. it occasionally occurs after severe pneumonias (bacterial or viral); it eventually develops in nearly all patients with cf. diagnosis may, on occasion, be made with plainchest radiographs, but high-resolution ct scanning is the diagnostic procedure of choice. treatment of bronchiectasis consists of airway clearance with chest physiotherapy with postural drainage or highfrequency chest wall oscillation, occasionally bronchodilators and mucolytic agents, and antibiotic therapy during exacerbations. surgical resection may be indicated in cases that are progressive and localized when medical therapy has failed. the prognosis of bronchiectasis depends on the underlying cause. cf-associated bronchiectasis is a major cause of cf-related morbidity and mortality; whereas, non-cf bronchiectasis may remain stable or even regress with therapy. ciliary dyskinesia. conditions in which the cilia do not function properly (immotile cilia or ciliary dyskinesia) lead to cough, usually because infection (and bronchiectasis) occurs in the absence of normal mucociliary transport. treatment is similar to that for cf, with regular chest physiotherapy and frequent and aggressive use of antibiotics at the first sign of airways infection, most commonly increased cough. interstitial lung disease. interstitial lung diseases are now classified based on those that occur during the neonatal period and those that are not as prevalent in infancy. interstitial lung diseases that manifest with cough include aspiration (chronic and recurrent) pneumonitis, hypersensitivity pneumonitis, bronchiolitis obliterans, and cryptogenic organizing pneumonia (formerly known as bronchiolitis obliterans organizing pneumonia or boop). lung biopsy may be required for a diagnosis. pulmonary hemosiderosis. pulmonary hemosiderosis is a rare, and often fatal, condition of bleeding into the lung that can manifest with cough. if sputum is produced, it is often frothy and blood-tinged. there may be frank hemoptysis. however, the cough may be nonproductive, or the sputum may be swallowed. some cases are associated with milk hypersensitivity (heiner syndrome), and affected children may have upper airway obstruction. some cases are associated with collagen vascular disorders. radiographs usually show diffuse fluffy postnasal drip (?) bronchiectasis ciliary dyskinesia interstitial lung disease heart failure/pulmonary edema pulmonary hemosiderosis drug-induced (see table bronchoscopy can sometimes localize a bleeding site, identify a cause (e.g., a foreign body or endobronchial tumor), or recover an offending bacterial, fungal, or parasitic pathogen. in many instances, bronchoscopy does not help except by excluding some possibilities, because either no blood or blood throughout the tracheobronchial tree is found. bronchial artery angiography may help to identify the involved vessel or vessels. treatment of hemoptysis depends on the underlying cause. it can be a terrifying symptom to children and their parents, and a calm, reassuring approach is essential. because hemoptysis is seldom fatal in children, reassurance is usually warranted. furthermore, hemoptysis most often resolves, and treatment of the bleeding itself is not often needed. what is required is treatment of the underlying cause of the hemoptysis, such as therapy for infection, removal of a foreign body, or control of collagen vascular disease. when death occurs from hemoptysis, it is more likely to be from suffocation than from exsanguination. in cases of massive bleeding, the rigid open-tube bronchoscope may help suction large amounts of blood while ventilating and keeping unaffected portions of lung clear of blood. interventional radiologists treat as well as localize a bleeding site by injecting the offending vessel with occlusive substances (embolization). in extremely rare instances, emergency lobectomy may be indicated. heart sound, an abnormal compression ultrasonographic study of the leg veins, a positive homans sign, a positive helical ct scan, and a high-probability lung ventilation-perfusion scan. the diagnosis of several causes of hemoptysis is straightforward. for example, hemoptysis that occurs immediately after a surgical or invasive diagnostic procedure in the chest should suggest an iatrogenic problem. the chest radiograph can help suggest lung abscess, pulmonary sequestration, bronchogenic cyst, or tumor. chest ct can help with cases of arteriovenous malformations, and additional laboratory values can support the diagnosis of collagen vascular disease. cough that may produce significant complications (see table . ). for most diseases, suppressing the cough offers no advantage. disadvantages include narcotic addiction and loss of the protective cough reflex with subsequent mucous retention and possible superinfection. demulcent preparations (sugar-containing, bland soothing agents or honey) temporarily suppress the cough response from pharyngeal sources, and decongestant-antihistamine combinations may reduce postnasal drip. cough itself seldom necessitates specific treatment. nonetheless, cough is not always completely benign (see table . ). most complications are uncommon, and most accompany only very severe cough, but some are serious enough to justify treatment of the cough itself. cough suppressants include codeine and hydrocodone (two narcotics) and dextromethorphan (a nonnarcotic d-isomer of the codeine analog of levorphanol). such agents should be used only for severe cough is important because it is a symptom and sign of underlying disease that frequently merits treatment. in the acute setting, severe disease, including massive hemoptysis or profound dyspnea or hypoxemia, warrants immediate attention, rapid diagnosis, and rapid management. certain chronic conditions, including those that suggest cf and those in which symptoms have persisted and interfere with a child's daily activities and quality of life, warrant further evaluation and treatment. finally, a child whose cough fails to respond to what should have been reasonable treatment should be referred to a pulmonary specialist (table . ). evaluation of chronic or recurrent cough guidelines for evaluating chronic cough in pediatrics recommendations for the assessment and management of cough in children upper respiratory infection bacterial tracheitis: report of eight new cases and review the common cold adverse events from cough and cold medication in children does this patient have sinusitis? diagnosing acute sinusitis by history and physical examination pertussis (whooping cough) report of the committee on infectious diseases bts guidelines for the management of pleural infection in children chlamydial respiratory infections the management of communityacquired pneumonia in infants and children older than months of age: clinical practice guidelines by the pediatric infectious diseases society and the infectious diseases society of america infant pneumonitis associated with cytomegalovirus, chlamydia, pneumocystis, and ureaplasma: follow-up value of radiological follow-up of childhood pneumonia british thoracic society guidelines for the management of community acquired pneumonia in children: update tuberculosis (myconbacterium tuberculosis) etiology and treatment of pneumonia pulmonary manifestations of acquired immunodeficiency syndrome standardized diagnosis of pneumonia in developing countries hypoxaemia in young kenyan children with acute lower respiratory infection tuberculosis in children clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis the evaluation of the child with recurrent chest infections nebulised hypertonic saline solution for acute bronchiolitis in infants bronchoscopic removal of aspirated foreign bodies in children tracheobronchial foreign bodies subglottic foreign bodies in pediatric patients tracheobronchial foreign bodies cystic fibrosis the cystic fibrosis genotype-phenotype consortium. correlation between genotype and phenotype in patients with cystic fibrosis cystic fibrosis therapeutics. the road ahead wheezing in infants with cystic fibrosis: clinical course, pulmonary function, and survival analysis cystic fibrosis physiotherapy in cystic fibrosis efficacy and safety of short-term administration of aerosolised recombinant human dnase i in adults with stable stage cystic fibrosis the diagnosis of cystic fibrosis: a consensus statement emerging therapies for cystic fibrosis pseudomonas cepacia: pulmonary infection in patients with cystic fibrosis effect of long-term treatment with inhaled budesonide on adult height in children with asthma a clinical index to define risk of asthma in young children with recurrent wheezing airway responsiveness in early infancy predicts asthma, lung function, and respiratory symptoms by school age statement: asthma control and exacerbations. standardizing endpoints for clinical asthma trials and clinical practice low-dose inhaled corticosteroids and the prevention of death from asthma venous thromboembolic complications in children clinical assessment and management of massive hemoptysis massive haemoptysis: medical management will usually arrest the bleeding pathogenesis and management of hemoptysis in children key: cord- - oti zg authors: panlilio, adelisa l; gerberding, julie louise title: occupational infectious diseases date: - - journal: textbook of clinical occupational and environmental medicine doi: . /b - - - - . - sha: doc_id: cord_uid: oti zg nan infections acquired in the work setting represent an eclectic group that is seldom, if ever, considered together as a single category. occupational infections involve several organ systems, respiratory, enteric, and skin infections being particularly common. transmission involves not only casual person-to-person contact, but also a variety of other routes in special work environments. it is thus important to consider what unique features characterize the infectious diseases that can be considered occupational. perhaps one useful way of thinking about these diseases is that, as a group, they tend to be transmitted during work schedules or practices that are systematized. therefore, they can be anticipated, and to the extent that unsafe infectionprone practices can be identified and modified, they can be systematically prevented. another common feature is that many of the occupational infectious diseases can be regarded as behavioral. to the extent that unsafe practices have been defined, and practice policies modified to reduce infection risk, continued transmission often represents failure to follow accepted standards. although certain occupational infections can be prevented by vaccines (e.g., hepatitis b), prevention often depends on simple behavioral changes, such as hand hygiene, use of gloves, and not working while ill. finally, the anthrax cases during the fall of in the united states demonstrate the possibility of intentional (and criminal) exposure to infectious agents in the workplace, in this instance among those handling mail. these intentional exposures, fortunately, are rare events, but should be considered in assessing sources of exposure for unexpected illnesses. prevention depends primarily on defining risky occupational practices or environments, clearly articulating policies for preventing communicable disease acquisition; removing structural barriers to compliance with policies (e.g., providing soap, hand cleansers, and gloves; allowing time away from work during periods of illness); and promoting healthy practices through behavioral change. because infectious diseases may represent the most common cause of time lost from work, it is important for the clinician concerned with occupational medicine to understand the relationship of specific infections to specific work environments and practices, and to give at least as much attention to prevention as to diagnosis and treatment. occupationally acquired infections have historically been associated with animal exposures and unsanitary work environments. modernization of agrarian techniques and improvement in sanitation have markedly decreased the incidence of these infections in the developed world, though they remain problematic in developing countries. while nearly all infectious diseases could conceivably be transmitted in the workplace, the emphasis here is on those that can be transmitted by casual contact or by specific workrelated exposures, with emphasis on diseases that are most common, most serious, or most readily prevented. healthcare settings pose a unique challenge because of the proximity of infectious patients, susceptible patients, and healthcare personnel. infections transmitted from personnel may have devastating effects on certain groups of patients, particularly the immunosuppressed. likewise, certain infections transmitted to personnel, such as multidrug-resistant tuberculosis, may have serious or even fatal, consequences. table . summarizes the microbial etiology, sources, routes of infection, categories of workers at risk, and clinical manifestations of selected infectious diseases that have occupational predilections. a detailed discussion of waterborne microbial diseases is also provided in chapter . because the treatment of occupationally acquired infections does not differ from that of infections acquired non-occupationally, the emphasis of this chapter is on the recognition and prevention of these infections. etiologic category, as well as the potential for recurrences, account for the high incidence of the common cold, even among healthy adults. colds are more common in the fall, winter, and early spring, perhaps because of increased crowding among children during the colder seasons. workers are most apt to acquire colds from exposure to young children in the home. secondary cases among coworkers may then develop. adults experience two to four colds each year, although the incidence among adult women exceeds that of men by a small margin, and smokers have a substantially increased risk. the modes of transmission of cold viruses are not entirely elucidated. for rhinoviruses, transmission among experimental subjects occurs most readily by direct handto-hand contact, with a case followed by autoinoculation of the mucous membranes of the eye or nose. such finger-tomucous membrane contact is ubiquitous and unavoidable. other viruses are transmissible by aerosolized droplets. the importance of fomites (such as drinking glasses, telephone receivers, and shared office equipment) as vectors of transmission has not been determined. clinical manifestations typical cold symptoms include nasal congestion, coryza, non-productive cough, sneezing, pharyngitis, and laryngeal irritation. fever is often low grade, or may be absent. viral upper respiratory infections usually resolve within - days, but longer durations are not uncommon. treatment, prevention, and control treatment for uncomplicated infections is symptomatic. decongestants are more useful in relieving symptoms than are antihistamines. expectorants, saline gargles, and other nonprescription remedies are useful in some cases. antibiotics should not be prescribed for the treatment of colds. colds are difficult to prevent. a policy of work restriction until symptoms improve may prevent the spread of colds but is likely to be impractical (table . ). the cost-benefit analysis of such an approach could be useful, especially in childcare and healthcare settings. hand washing after contact with nasal secretions may be helpful. care should be taken to use tissues when coughing or sneezing and to dispose of soiled tissues after use. epidemiology influenza is a self-limited respiratory illness caused by types a and b influenza virus. epidemics of influenza occur annually in the winter months. adults remain susceptible to the illness despite prior episodes of infection because the antigenic structure of influenza viruses changes frequently, leading to new epidemics. influenza is spread from person to person, primarily by the coughing and sneezing of infected persons or sometimes by direct contact, either with infected persons or a contaminated surface. the disease is easily transmitted, and a single index case may transmit to a large number of susceptible persons in a short period of time. adults and children typically are infectious from - days before through - days after the onset of symptoms. clinical syndromes an attack of influenza starts abruptly with fever, malaise, myalgia, and headache. respiratory symptoms mimicking those of the common cold and lower respiratory symptoms including dry cough also are frequent. fever resolves in uncomplicated cases in - hours, but other symptoms may persist for days to weeks. influenza pneumonia, associated with hypoxemia, cough, and interstitial infiltrates, is not common in healthy adults. elderly patients and those with underlying immunodeficiencies and chronic pulmonary diseases are at high risk for secondary bacterial pneumonias, often caused by streptococcus pneumoniae and less often by haemophilus influenzae and staphylococcus aureus. the diagnosis of influenza frequently is made on the basis of clinical symptoms and signs. however, influenza is very difficult to differentiate from respiratory illnesses caused by other pathogens on the basis of clinical symptoms alone. other pathogens that can cause similar symptoms include, but are not limited to, mycoplasma pneumoniae, adenovirus, respiratory syncytial virus (rsv), rhinovirus, parainfluenza viruses, and legionella species. many pathogens, including influenza, rsv, and parainfluenza, cause outbreaks in a seasonal pattern. laboratory confirmatory tests can be performed to differentiate influenza from other illnesses. appropriate patient samples to collect for laboratory testing can include a nasopharyngeal or throat swab from adults, or nasal wash or nasal aspirates, depending on which rapid test is used. samples should be collected within the first days of illness. rapid influenza tests provide results within hours; viral culture provides results in - days. most of the rapid tests are more than % sensitive for detecting influenza and more than % specific. because as many as % of samples that would be positive for influenza by viral culture may give a negative rapid test result, negative rapid tests should be followed by viral culture in a sample of the swabs collected. viral culture can also identify other causes of influenza-like illness when influenza is not the cause. serum samples can be tested for influenza antibody to diagnose acute infections. two samples should be collected per person: one sample within the first week of illness and a second sample - weeks later. if antibody levels increase from the first to the second sample, influenza infection likely occurred. because of the length of time needed for a diagnosis of influenza by serologic testing, other diagnostic testing should be used for rapid detection of possible outbreaks. during community outbreaks, specific virologic or serologic diagnosis is not necessary once the type(s) of influenza virus causing the outbreak have been identified. treatment, prevention, and control persons at high risk for serious morbidity (persons aged and older, persons with chronic underlying diseases) should receive influenza vaccine annually. immunization also is recommended for healthcare personnel and others at risk for transmitting influenza to high-risk patients ( had been associated with reduced work absenteeism and fewer deaths among nursing home patients. , most employers do not provide influenza prevention programs for workers outside the healthcare field. amantadine and rimantadine can reduce the duration of uncomplicated influenza a illness when administered within days of onset of illness in otherwise healthy adults. zanamavir and oseltamivir can reduce the duration of uncomplicated influenza a and b illness by approximately day compared with placebo. none of these antiviral agents has been shown to be effective in preventing serious influenza-related complications. to reduce the emergence of antiviral drug-resistant viruses, the duration of therapy should typically be no longer than days. both amantadine and rimantadine are indicated as prophylaxis for influenza a, but not for influenza b infection. oseltamivir has been approved as prophylaxis for influenza a and b. zanamivir has not been approved for prophylaxis, but has been shown to be as effective as oseltamivir in preventing febrile, laboratory-confirmed influenza illness. they are approximately - % effective in preventing illness from influenza a. chemoprophylaxis can be a component of influenza outbreak control programs. epidemiology the incidence of measles (rubeola) has steadily declined in the united states during the last decade and is no longer considered endemic. measles is a major cause of morbidity and mortality worldwide. the majority of cases in the united states in recent years have been imported or secondary cases epidemiologically linked to imported cases. infected persons are highly contagious by the air-borne route during the viral prodrome, and when cough and coryza are prominent, until about days after the rash appears. infection confers lifelong immunity. although most adults born prior to experienced childhood infection and are no longer susceptible, up to % may lack natural immunity. in recent epidemics, cases occurred among unimmunized children, as well as children and young adults who had received a single vaccination with live virus, and among older adults. clinical syndromes measles progresses in several phases. initial virus replication occurs in the respiratory tract and leads to a primary viremic phase, which usually is asymptomatic. release of virus from infected reticuloendothelial cells produces secondary viremia and infection of the entire respiratory system, accompanied by symptoms of coryza, cough, and in some, bronchiolitis or pneumonia. koplik's spots, a bluish-gray enanthem most prominent on the buccal mucosa, precede development of the rash. in a typical case of measles, the rash begins on the face, then progresses to the trunk and distal extremities, and disappears in the same sequence after - days. treatment, prevention, and control live-attenuated vaccine for prevention of measles became available in the early s. all healthy children should receive the vaccine at age months. because % do not respond to a single dose of vaccine, a second dose is now recommended to improve vaccine efficacy. all healthy adults born after who have not received two doses of live virus vaccine or have not experienced measles also are advised to receive vaccine (table . ). persons who received killed vaccine have a risk of developing atypical measles, and require re-immunization with live virus vaccine. live virus vaccine is contraindicated in infants, pregnant women, and immunosuppressed persons. passive immunization with γ globulin is available for unimmunized persons exposed to infected individuals, but is not routinely recommended for adults. measles rarely can exacerbate tuberculosis and cause a temporary inhibition of delayed hypersensitivity. vaccine administration should be delayed for month after tuberculin testing, and until treatment is under way in persons with active tuberculosis. epidemiology mumps is a viral illness transmitted by the oral or respiratory route during contact with contaminated fomites or aerosolized droplet secretions. mumps is less contagious than measles or rubella but produces significant morbidity, especially among adults. the incubation period ranges from to weeks. virus is detectable for days prior to and days after the appearance of symptoms. most adults are immune to mumps, but - % of unimmunized adults have no serologic evidence of prior infection and are considered susceptible. mumps incidence is now very low in all areas of the united states. the substantial reduction in mumps incidence during the past few years likely reflects the change in the recommendations for use of measles mumps rubella (mmr*) vaccine. clinical syndromes parotitis typically is bilateral, but unilateral disease and involvement of other salivary glands occurs in some persons. localized parotid tenderness and swelling, fever, and painful swallowing suggest the diagnosis. aseptic meningitis is common but benign. encephalitis is a rare but serious manifestation. about % of affected postpubescent men develop orchitis, epididymitis, or both, which is bilateral in % of cases and may be the sole manifestation of mumps infection. about % of cases of mumps orchitis result in testicular atrophy, but neither sterility nor impotence are common sequelae. oophoritis occurs in about % of women with mumps. vaccine separated by at least month (i.e., a minimum of days), and administered on or after the first birthday, are recommended for all children and for certain highrisk groups of adolescents and adults. adult men and healthcare personnel with no history of mumps or mumps immunization should be screened for immunity and vaccinated if they are susceptible. immunization is contraindicated in persons with immunosuppression and in pregnant women (table . ). passive immunization with mumps immune globulin decreases the incidence of orchitis and is recommended for mumps in adult men with a single testis. individuals with active mumps should be excluded from work until days after the onset of parotitis to avoid transmission to others in the workplace (table . ). epidemiology fifth disease, also called erythema infectiosum or 'slapped cheek disease', is an infection caused by parvovirus b . it is a common rash illness that is usually acquired in childhood, but can be an occupational risk for school and childcare personnel. it has been transmitted to personnel in healthcare settings. clinical syndromes symptoms begin with mild fever and symptoms of fatigue. after a few days, the cheeks take on a flushed 'slapped' appearance. there may also be a lacy rash on the trunk, arms, and legs. not all infected persons develop a rash. the child is usually not very ill, and the rash resolves in - days. most persons who get fifth disease are not very ill and recover without any serious consequences. an adult who is not immune can be infected with parvovirus b and either have no symptoms or develop the typical rash of fifth disease, joint pain or swelling, or both. usually, joints on both sides of the body are affected. the joints most frequently affected are the hands, wrists, and knees. the joint pain and swelling usually resolve in a week or two, but they may last several months. about % of adults, however, have been previously infected with parvovirus b , have developed immunity to the virus, and cannot get fifth disease. fifth disease is believed to be spread through direct contact, fomites, or large droplets. the period of infectivity is before the onset of the rash. once the rash appears, a person is no longer contagious. the incubation period is - days but may be as long as days. treatment, prevention, and control symptomatic treatment for fever, pain, or itching is usually all that is needed for fifth disease. adults with joint pain and swelling may need to rest, restrict their activities, and take anti-inflammatory medications to relieve symptoms. transmission can be prevented by careful attention to hygiene, especially hand washing. no special precautions are necessary. excluding persons with fifth disease from work, childcare centers, or schools is not likely to prevent the spread of the virus, since people are contagious before they develop the rash. epidemiology pertussis, or whooping cough, is an acute infectious disease caused by the bacterium bordetella pertussis. pertussis continues to be an important cause of mortality in the united states. a dramatic decline in the incidence followed the widespread use of whole-cell pertussis vaccines in the mid- s. however, since the early s, the reported pertussis incidence has increased cyclically with peaks occurring every - years. , contributing to this increase in incidence is the waning of immunity over time following vaccination, particularly in older age groups. transmission most commonly occurs by contact with respiratory secretions or large aerosol droplets from the respiratory tracts of infected persons and less frequently by contact with freshly contaminated articles of an infected person. analysis of national surveillance data for pertussis during - indicates that pertussis incidence continues to increase in infants too young to receive three doses of pertussis-containing vaccine and in adolescents and adults. clinical syndromes the incubation period of pertussis is commonly - days. pertussis begins insidiously with non-specific upper respiratory symptoms including coryza, sneezing, low-grade fever, and a mild, occasional cough, similar to the common cold. the cough gradually becomes more severe, and after - weeks, the second, or paroxysmal stage, begins. characteristically, the patient has paroxysms of numerous, rapid coughs generally with a characteristic high-pitched whoop, commonly followed by vomiting and exhaustion. the patient usually appears normal between attacks. older persons (i.e., adolescents and adults), and those partially protected by the vaccine may become infected with b. pertussis, but usually have milder atypical disease. pertussis in these persons may present as a more persistent cough of greater than days duration, and may be indistinguishable from other upper respiratory infections. inspiratory whoop is uncommon. b. pertussis is estimated to account for up to % of cough illnesses per year in older persons. even though the disease may be milder in older persons, these infected persons may transmit the disease to other susceptible persons, including unimmunized or underimmunized infants. the medical management of pertussis cases is primarily supportive, although antibiotics are of some value, with erythromycin being the drug of choice. this therapy eradicates the organism from secretions, thereby decreasing communicability and, if initiated early, may modify the course of the illness. there is no pertussis-containing vaccine (including dtap) currently licensed for persons years of age or older, and vaccination with dtap currently is not recommended after the th birthday. vaccine reactions are thought to be more frequent in older age groups, and pertussis-associated morbidity and mortality decrease with increasing age. studies are currently under way to determine if a booster dose of acellular pertussis vaccine administered to older children or adults may reduce the risk of infection with b. pertussis. this may in turn reduce the risk of transmission of b. pertussis to infants and young children who may be incompletely vaccinated. studies among older children, adolescents, and adults examining pertussis disease burden and transmission of disease to infants might guide future policy decisions on the use of acellular pertussis vaccines among persons more than seven years of age. currently, vaccination of children more than years of age, adolescents, and adults is not recommended either routinely or as an outbreak control measure. in the future, licensure of pertussis vaccines for adolescents or adults may lead to new recommendations for the use of vaccines in outbreaks. epidemiology most epidemics of bacterial pneumonia in the workforce are due to community-acquired infections. however, legionellosis is one type of pneumonia which can be transmitted in the workplace. legionella pneumophila is an important cause of both epidemic and endemic adult pneumonia, and it can be associated with outbreaks in the workplace. this organism colonizes aquatic ecosystems and potable water, and it is transmitted to humans by the air-borne route. contaminated air conditioners, humidifiers, and shower heads have been implicated in outbreaks among workers and hospital patients. outbreaks of legionellosis have occurred after persons have breathed mists that come from a water source (e.g., air conditioning cooling towers, whirlpool spas, showers) contaminated with legionella bacteria. persons may be exposed to these mists in homes, workplaces, hospitals, or public places. legionellosis is not passed from person to person. a careful occupational history should be obtained from all adults who present with pneumonia, because occupational exposures cause many otherwise rare pneumonias. public health authorities should be notified if an occupational source is suspected so that an epidemiologic investigation to identify transmission routes and other susceptible individuals may commence. clinical pneumonia syndromes community-acquired bacterial pneumonia usually is exhibited acutely, with fever, chills, productive cough, and often, pleurisy. chest examination demonstrates signs of consolidation that may be confirmed radiologically. sputum examination may aid implementation of empiric therapy by suggesting the etiologic pathogen. blood cultures should be obtained when invasive disease is suspected, and lumbar puncture to evaluate meningeal fluid is indicated when symptoms or signs of meningitis are present. patients with legionnaire's disease usually have fever, chills, and a cough, which may be dry or productive. some patients also have muscle aches, headache, tiredness, loss of appetite, and, occasionally, diarrhea. chest x-rays often show pneumonia but are not pathognomonic. it is difficult to distinguish legionnaire's disease from other types of pneumonia by symptoms alone; other tests are required for diagnosis. the definitive test is culture isolation of the organism in sputum, bronchoalveolar fluid, or pleural fluid. other useful diagnostic tests detect the bacteria in sputum by specialized stains, identify legionella antigens in urine samples, or compare antibody levels to legionella in two blood samples obtained - weeks apart. the time between the patient's exposure to the bacterium and the onset of illness for legionnaire's disease is - days. treatment, prevention, and control empiric ambulatory therapy of acute community-acquired bacterial pneumonia, not requiring hospitalization, should include coverage for pneumococcus and h. influenzae, if the patient has a history of chronic obstructive lung disease. amoxicillin, trimethoprim-sulfamethoxazole and cefixime are reasonable choices, unless atypical pneumonia caused by m. pneumoniae or c. pneumoniae is suspected, in which case erythromycin is preferred. erythromycin is the antibiotic currently recommended for treating persons with legionnaire's disease. in severe cases, a second drug, rifampin, may be added. preventing bacterial pneumonia is a difficult challenge. workers at risk for pneumococcal and haemophilus infections should be immunized, although the efficacy of this approach among patients at highest risk is debated (table . ). influenza immunization could eliminate a major risk factor for both primary and secondary bacterial pneumonias. occupational exposures to potential pathogens should be minimized with proper ventilation. prevention of legionellosis is achieved by maintaining an environment that is not conducive to survival or multiplication of legionella. the necessary preventive measures may involve water treatment or modification of air conditioning and ventilation systems. these preventive steps, which may be costly, should be directed at healthcare facilities, and occupational settings where cases have been identified. epidemiology rubella (german measles) virus is transmitted person to person by mucosal exposure to infected droplets of respiratory secretions. since , children in the united states have been routinely immunized against rubella at age months, so that the majority of recognized cases today occur in adults and unimmunized children. since , reported indigenous rubella has continued to occur at a low but relatively constant endemic level with an annual average of less than rubella cases. recent data indicate that the rate of rubella susceptibility and risk for rubella infection are highest among young adults. no large epidemics have occurred since the vaccine was licensed for use in . however, outbreaks continue to occur among groups of susceptible persons who congregate in locations that increase their exposure, and among persons with religious and philosophic beliefs against vaccination. several recent outbreaks have occurred in workplaces where most employees are foreign born, particularly from latin america. reinfection can occur following natural or acquired immunity, but it is usually asymptomatic and only rarely accompanied by viremia. rubella virus is shed from the respiratory tract of infected persons beginning days before the development of rash and for several days after the rash appears. the onset of the rash coincides with the period of maximal contagiousness, and infected persons are not considered infectious for more than days after the rash appears. infected infants shed virus for several months despite the presence of antibody. clinical syndromes adult rubella is often asymptomatic. symptoms occur - days after exposure. following a prodrome of fever and malaise, adults exhibit a maculopapular rash that begins on the face and extends downward, persists for - days, and often is accompanied by regional lymphadenopathy of the head and neck, which persists for days to weeks. one-third of adult women may develop arthritis in the fingers, knees, and wrists during the exanthematous phase of illness. children develop hemorrhagic complications more often than adults. in contrast, encephalitis, albeit rare, is more common in adults and is fatal in - % of cases. maternal rubella infection acquired in the first weeks of gestation frequently results in congenital rubella. the earlier in pregnancy rubella occurs, the more severe the fetal consequences. infection in the first trimester results in deafness, congenital heart disease, cataracts or glaucoma, endocrine abnormalities, and mental retardation in up to % of newborns. spontaneous abortion also occurs commonly. treatment, prevention, and control immunization of children and susceptible adults with live attenuated rubella virus effectively prevents rubella and accounts for the dramatic decline in the incidence of this disease in the united states. however, many adult women of childbearing age remain susceptible to rubella and require immunization prior to conception to prevent congenital rubella. the hemagglutination-inhibition serologic assay detects natural or acquired immunity. the advisory committee on immunization practices (acip) recommends screening of healthcare personnel who have not been vaccinated, and immunization of susceptible individuals. complications of rubella vaccine occur among adults and include lowgrade fever, symmetric polyarthralgias, distal paresthesias, lymphadenopathy, and rash. vaccine is contraindicated in immunosuppressed persons and pregnant women. pregnancy should be avoided for months after vaccination (table . ). susceptible household contacts of infected adults and children pose a transmission risk in the workplace during the period of virus shedding, beginning about days before the development of rash (about week after exposure) until days after rash appears. therefore, susceptible individuals should not report to work during this time interval (table . ). epidemiology tuberculosis (tb) is caused by mycobacterium tuberculosis and, rarely today, by m. bovis. the incidence of tuberculosis (tb) in the united states declined steadily until the mid- s, but then sharply increased, especially in urban areas. the resurgence of tb in the united states in the late s and early s was associated with the emergence of multidrug-resistant tb (mdr-tb) and the hiv/aids epidemic. with this resurgence of tb in the united states came several high-profile nosocomial outbreaks associated with lapses in infection control practices and delays in diagnosis and treatment of persons with infectious tb, as well as the appearance and transmission of mdr-tb strains. since , the declines in the overall number of reported tb cases, including the level of mdr-tb, appear to reflect successful efforts to strengthen tb control following the resurgence of tb and the emergence of mdr-tb. activities emphasizing the first priority of tb control (i.e., promptly identifying persons with tb, initiating appropriate therapy, and ensuring completion of therapy) have been the most important factors in achieving this improvement. such activities reduced community transmission of m. tuberculosis, particularly in areas with a high incidence of aids. improvements in implementation of infection control measures in healthcare settings, concurrent with mobilization of the nation's tb control programs, succeeded in reversing the upsurge in reported cases of tb, and case rates have declined to their lowest levels to date. the threat of mdr-tb is decreasing, and the transmission of tb in healthcare facilities continues to abate due to implementation of infection controls and reductions in community rates of tb. nevertheless, some healthcare personnel are at risk for acquiring tb. pulmonary tb is most commonly transmitted in healthcare settings by inhalation of aerosolized droplet nuclei derived from the respiratory secretions of patients with active respiratory tb. close contact usually is required. most other categories of workers generally are not at risk without close and sustained workplace contact with a person who has active untreated disease. ingestion of unpasteurized milk from cows infected with m. bovis is no longer an important source of tb in most industrialized countries. clinical syndromes primary infection usually is asymptomatic in adults. teenagers and young adults are at higher risk for rapid progression to active disease, usually characterized by apical cavitary disease, than are older adults. primary infection in the elderly usually is exhibited as lower lobe consolidation with hilar adenopathy. primary tuberculosis in persons with advanced hiv infection is commonly symptomatic and progressive. once infection occurs, the organism may disseminate from the lungs to other sites, including the gastrointestinal and genitourinary tracts, and bone. normally, the infection is contained by the host's immune response at this stage. the risk for reactivation is highest in the first year after exposure and declines thereafter. however, aging and stressors such as immunosuppression, intercurrent illness, and chronic malnutrition may increase the risk for reactivation or dissemination of the disease later in life. clinically, reactivation tuberculosis usually is exhibited as upper lobe pulmonary cavitary disease, but virtually any organ system may be involved. treatment, prevention, and control tuberculin skin testing allows determination of prior exposure to tb in immunologically healthy adults, by assessing delayed hypersensitivity to tuberculin antigens using purified protein derivative. the tuberculin skin test (tst) is the only proven method for identifying infection with m. tuberculosis in persons who do not have tb disease. although the available tst antigens are neither % sensitive nor specific for detection of infection with m. tuberculosis, no better diagnostic methods have yet been devised. the preferred skin test for diagnosing m. tuberculosis infection is the mantoux test. it is administered by injecting . ml of tuberculin units (tu) ppd intradermally into the dorsal or volar surface of the forearm. tests should be read - h after test administration, and the transverse diameter of induration should be recorded in millimeters. there are three cut-off levels recommended for interpretation of the tst results. in hiv-infected persons, any reaction resulting in an induration larger than mm is read as positive. among others, the presence of mm or more of induration always indicates a positive test, - mm indicates a positive result in persons at risk for tb, and less than mm is negative. a positive tst means an individual has been exposed to tb in the past and is at risk for reactivation. a baseline chest radiogram should be performed on all persons with newly diagnosed tst positivity. if the x-ray study suggests active disease, sputum samples should be obtained, stained for acid-fast bacilli, and cultured for mycobacteria. treatment should be implemented immediately if the index of suspicion is high. public health officials should be notified to institute case management and evaluation of contacts in the home and work environment. if the x-ray study is negative, treatment with isoniazid to suppress or eradicate latent organisms may be recommended, especially in persons younger than age and for those who have recently converted to positive tsts. although bcg vaccine is the most widely administered of all vaccines in the world, and has the highest coverage of any vaccine in the who expanded programme on immunization, it appears to have had little epidemiologic impact on tb. despite its shortcomings, and because of its beneficial effect in children and against leprosy, bcg vaccine likely will remain a component of childhood vaccination strategies in developing countries. however, because of questions about the vaccine's efficacy, and because it induces dermal hypersensitivity to purified protein derivative (ppd) tuberculin in most recipients, bcg has never been recommended for programmatic use in the united states. healthcare providers should follow appropriate infection control procedures, including use of isolation rooms and respiratory protection, when caring for patients with active tuberculosis. , varicella/zoster epidemiology varicella virus, the causative agent of chickenpox and zoster, is a highly contagious herpes virus spread by the respiratory route from person to person. the incubation period is about days, and the period of infectivity begins a few days prior to the onset of the rash to about days after the first crop of vesicles appears. immunosuppression usually prolongs the period of infectivity, especially if varicella zoster immune globulin (vzig) has been administered. zoster represents reactivation of varicella virus that is latent in sensory nerve ganglia, and it is not a manifestation of primary infection except in newborns infected in utero. the incidence of zoster increases with age and immunosuppression. susceptible persons in direct contact with zoster lesions risk developing primary varicella. in the prevaccine era, varicella was endemic in the united states, and virtually all persons acquired varicella by adulthood. as a result, the number of cases occurring annually was estimated to approximate the birth cohort, or approximately million per year. this incidence has likely decreased since licensure of the vaccine in . varicella is not a nationally notifiable disease, and surveillance data are limited. clinical syndromes varicella in otherwise healthy children usually is a benign, self-limited disease characterized by low-grade fever and vesicular rash, often preceded by a viral prodrome. varicella vesicles of primary infection appear first on the scalp and trunk and disseminate in crops showing various stages of development over the next - days. healing results in crusting accompanied by intense pruritus. manifestations of varicella are more severe in adults than in children. about % of adults with varicella show radiographic evidence of pulmonary involvement, but this is rarely clinically significant. however, cough, tachypnea, and impaired gas exchange can occur and persist for months after infection. varicella during pregnancy can produce congenital varicella. in its most severe form, this infection can result in mental retardation, blindness, growth retardation, deafness, chorioretinitis, and a peculiar dermatomal lesion of the upper or lower extremity associated with limb atrophy. zoster, the most common manifestation of varicella infection among adults, characteristically produces unilateral vesicular eruptions preceded by pain in one to three dermatomes. disseminated zoster, which is more likely in immunosuppressed patients, probably poses the same risk of infection transmission as primary varicella infection. the major complication of zoster is postherpetic neuralgia, which is especially common in the elderly and may be extremely debilitating. zoster frequently produces cerebrospinal fluid pleocytosis and occasionally encephalitis. immunologically healthy persons may experience recurrences of zoster, usually in the same dermatome as the initial outbreak. zoster is a marker of deteriorating cellmediated immunity among hiv-infected patients, and it may disseminate. treatment, prevention, and control passive immunization with vzig is recommended for immunosuppressed susceptible persons, children with leukemia and other malignancies, and neonates exposed in utero within days before delivery. several antiviral drugs are active against varicella zoster virus, including acyclovir, valacyclovir, famciclovir, and foscarnet. famciclovir and valacyclovir are approved for use only in adults. clinical studies indicate that these drugs may be beneficial if given within hours of onset of rash, resulting in a reduction in the number of days new lesions appeared, in the duration of fever, and in the severity of cutaneous and systemic signs and symptoms. antiviral drugs have not been shown to decrease transmission of varicella, reduce the duration of absence from school, or reduce complications. oral acyclovir can be considered in otherwise healthy adolescents and adults or secondary cases in the household, because of the increased risk of severe illness in these groups. antiviral therapy may also be considered for persons with chronic cutaneous or pulmonary disorders, persons receiving long-term salicylate therapy, and for children receiving short, intermittent or aerosolized courses of corticosteroids. antiviral drugs are not recommended for routine postexposure prophylaxis. systemic steroids in older adults (more than years old) may reduce the incidence and severity of postherpetic neuropathy if started early (within days of skin manifestations). varicella has been difficult to prevent because of the high degree of contagion in households, schools, and healthcare settings. live attenuated virus vaccines have demonstrated efficacy in preventing primary infection, and one was licensed for use in the united states in . routine immunization is now recommended for children less than months of age. varicella vaccination should be given to susceptible adolescents and adults who are at high risk of exposure to varicella. this group includes persons who live or work in environments in which there is a high likelihood of transmission of varicella, such as teachers of young children, residents and staff in institutional settings, and military personnel. varicella vaccination is also recommended for susceptible adolescents and adults who will have close contact with persons at high risk for serious complications of acquired varicella, including healthcare personnel and susceptible family contacts of immunocompromised individuals. the acip recommends that all healthcare personnel be immune to varicella, either from a reliable history of prior varicella infection or vaccination, to reduce the risk of infection and its complications, and to decrease the possibility of transmission of varicella zoster virus to patients (table . ). susceptible adults exposed to children with varicella or with disseminated zoster pose a risk of transmitting varicella to non-immune coworkers, and they should not work until the incubation period is over or, if they become ill, until all lesions are crusted (table . ). dermatomal zoster is not spread efficiently by the air-borne route and otherwise healthy adults afflicted with this illness may be allowed to work if they can avoid touching the lesions and contaminating the work environment. the role of vaccine in the postexposure management of susceptible employees needs to be elucidated. data from the united states and japan in a variety of settings indicate that varicella vaccine is effective in preventing illness or modifying the severity of illness if used within days, and possibly up to days, of exposure. acip recommends vaccine be used in susceptible persons following exposure to varicella. personnel should be excluded from work who have onset of varicella until all lesions have dried and crusted (table . ). following exposure to varicella, personnel who are not known to be immune to varicella (by history or serology) should be excluded from duty beginning on the th day after the first exposure until the st day after the last exposure ( th day if vzig was given). epidemiology acute gastrointestinal infection follows upper respiratory illness as the next leading category of infectious diseases causing absenteeism among adult workers. a wide array of pathogens, including viruses, bacteria, and protozoa, can result in acute infections of the stomach, small bowel, or colon. a comprehensive discussion of enteric pathogens is provided in chapter (waterborne microbial diseases). most of the etiologic agents are acquired by the fecal-oral route; produce mild, self-limited diseases; and resolve without specific therapy. agents of dysentery (e.g., shigella spp.) often are highly transmissible through low-inoculum exposures. occupational transmission of food-borne or water-borne illnesses occurs; person-to-person transmission has propagated outbreaks of many of these illnesses in healthcare settings, daycare and nursery schools, and institutions where sanitation is poor. instances of such transmission have generally involved food handlers, who are often poorly trained and short-term employees, serving as sources of transmission to others. occupations requiring travel to countries with poor sanitation present a major risk for gastrointestinal infections. poultry workers are frequently exposed to salmonella infections. avoidance of oral contact with sources of fecal contamination is the most important strategy for preventing transmission of pathogens associated with intestinal infections. maintaining good personal hygiene, including careful hand hygiene after using restrooms and before food preparation; proper cooking and storage of foods; and avoidance of contaminated foods and water when traveling are essential prevention strategies. food handlers with diarrheal illnesses should not work until symptoms have resolved, and cure of bacterial infections should be documented by obtaining negative stool cultures more than hours after antimicrobial therapy is completed (table . ). the only vaccines for any of the etiologic agents for acute enteric infections are for typhoid and hepatitis a, which are recommended for personnel in laboratories who frequently work with salmonella typhi or hepatitis a virus (table . ). epidemiology cytomegalovirus (cmv) is a ubiquitous herpes virus transmitted by direct inoculation with infected body fluids (including blood, blood products, respiratory secretions, saliva, and urine) and through sexual contact with infected partners. at least % of healthy adults have serologic evidence of prior cmv infection. infection can be acquired perinatally, in utero during maternal primary infection, during birth by passage through infected vaginal secretions, or through ingestion of infected breast milk. cmv is known to be highly transmissible in daycare centers and nursery schools. sexually active adults and recipients of blood products are also at high risk for infection. infants and young children excrete cmv in their urine, saliva, and respiratory secretions for several months after infection. virus is much less readily detected in healthy adults, but intermittent shedding has been documented. like all herpes viruses, cmv remains latent in the host after initial infection. previously infected persons may be reinfected with new strains of cmv. occupational transmission of cmv has been documented in childcare settings, where person-to-person spread through exposure to infected secretions and urine is believed to provide an efficient mode of transmission. up to % of seronegative workers in preschool daycare centers have acquired cmv infection in some studies, indicating a potentially serious risk to women of child-bearing age, because of the adverse effects of primary maternal cmv infection on the fetus. at one time, employment in healthcare settings also was believed to pose a high risk for cmv acquisition. however, epidemiologic investigations suggest that most infections in healthcare personnel are acquired sexually, or from exposure to young children in the home, and not from work-related contact. is asymptomatic in healthy persons. a self-limited mononucleosis-like illness occurs in a minority, which may be complicated by hepatitis, pneumonitis, hematologic abnormalities, and myocarditis. immunosuppressed children and adults with primary cmv infection, reactivation, or reinfection may develop severe sequelae. organ transplant recipients, hiv-infected patients, and persons with malignancies have a risk of developing cmv viremia, pneumonia, hepatitis, pancreatitis, enteritis, and retinitis. primary cmv infection at any stage of pregnancy carries a greater risk to the fetus than does recurrent cmv infection during pregnancy. symptoms of congenital cmv infection may be present at birth, and are due to the consequences of active virus replication and resultant end-organ damage. congenital cytomegalic inclusion disease, the most severe form of this entity, includes central nervous system disease, respiratory distress, hepatitis, hepatosplenomegaly, rash, and multi-system failure. infection acquired from exposure to cervical cmv during birth usually is asymptomatic and detected by the onset of virus shedding - weeks postpartum. with ganciclovir or foscarnet for cmv infection is reserved for immunosuppressed persons at high risk for severe complications. the safety and effectiveness of these agents in preventing congenital cmv have not been established. avoidance of mucosal contact with infected body fluids is the best strategy for preventing cmv transmission. hand washing after contact with secretions and fomites is essential, especially in nurseries and daycare settings. the presence of persons at risk for cmv shedding in the workplace does not pose a hazard to other employees unless direct contact with infected secretions is anticipated. isolation of infected neonates or children is not essential if hand washing is performed after contact with secretions, blood, and urine. pregnant healthcare providers compliant with hand washing protocols can generally safely care for patients with cmv infection. [ ] [ ] [ ] no work restriction is necessary for individuals with cmv infection (table . ). direct exposure to blood and other infected body fluids. children born to infected mothers are at high risk for hbv infection. persons parenterally exposed to blood, including multi-transfused patients, hemophiliacs, dialysis patients, and injection drug users, also are at significant risk. sexual contact with infected partners is another efficient mode of hbv spread. in most industrialized countries, adult infections usually are acquired sexually or by injection drug use. hbv is a relatively hardy virus capable of surviving on environmental surfaces and fomites. transmission in households is well documented and may, in part, be attributable to mucosal contact with fomites contaminated with secretions or blood from infected persons. healthcare personnel and others at risk for occupational blood exposure through percutaneous, mucosal, or dermal routes can acquire hbv infection. the risk associated with accidental needle-stick inoculation of infected blood to susceptible healthcare personnel varies between % to %, depending on the hepatitis b e antigen (hbeag) status, and hence the viral titer of the source. in up to % of occupational infections, a discrete exposure cannot be identified. hepatitis b has an incubation period of - days. the period of infectivity precedes the development of jaundice by - weeks and correlates with the presence of hepatitis b surface antigen (hbsag) in the serum; - % of persons with acute (but often clinically silent) infection develop chronic antigenemia. in the united states, up to % of adults are carriers of hbv, and provide a reservoir for maintenance of the disease in the population. apparent hepatitis in about one-third of acutely infected adults. clinical hepatitis may be preceded by a prodrome of fever, malaise, urticarial or maculopapular rash, and arthralgias for several days. fever usually resolves before the onset of jaundice. jaundice, dark urine, and scleral icterus usually are present by the time patients seek medical attention. right upper quadrant tenderness, mild hepatic enlargement, and occasionally, splenomegaly are signs that should suggest the diagnosis. the most striking laboratory abnormality is the finding of extreme elevations in the aminotransferase enzymes. alanine aminotransferase (alt) and aspartate aminotransferase (ast) may be elevated to more than times the normal levels, whereas the bilirubin and alkaline phosphatase levels are increased to a much lesser extent. fulminant liver involvement occurs in about % of adults and may be complicated by more serious abnormalities, including hypoglycemia, coagulopathy, and hypoalbuminemia. hepatic encephalopathy, hepatorenal syndrome, and bleeding diatheses are life-threatening complications seen in these patients. about % of adults with clinically apparent acute hbv infection proceed to chronic hbs-antigenemia, and are at risk for chronic hepatitis, postnecrotic cirrhosis, and primary hepatocellular carcinoma. patients with asymptomatic primary hbv infection are at higher risk for chronic infection than those with symptomatic infection. while chronic persistent hepatitis, a benign illness of little clinical consequence except for the potential for hbv transmission to susceptible individuals, may occur, the major health concern is chronic active hepatitis, which eventually may produce cirrhosis, liver failure, and hepatoma. hepatitis b is differentiated from other causes of hepatitis by serologic assays. a positive hepatitis b surface antigen (hbsag) test identifies patients with current infection and correlates with infectivity during acute and chronic infection. titers of hbsag in the chronic phase of illness may wax and wane and occasionally fall below the limits of laboratory detection, so sequential testing should be performed if chronic hbv is suspected. the presence of hbeag correlates with active virus replication and is a marker of high infectivity and high titer of hbv in the liver and blood. antibody to hepatitis b surface antigen (hbsab) appears when hbsag is cleared and is positive in individuals with immunity after recent or prior infection or immunization. persons with hbsab are not susceptible to acute infection or chronic hepatitis b, except in the very rare case in which reinfection occurs with a strain of hepatitis b against which the normal antibody response does not provide cross-protection. hepatitis b core antibody (hbcab) appears before hbsab and just after hbsag is cleared from the serum, and this is a useful test for diagnosing acute hepatitis b in the window period before hbsab appears. high titers of hbcab persist in chronically infected persons and obviously do not predict immunity from further liver disease. there currently is no treatment for acute hepatitis b. alpha interferon and lamivudine have been licensed for the treatment of persons with chronic hepatitis b. these drugs are effective in up to % of cases. hbv infection is largely preventable. inoculation with recombinant vaccines containing hbsag components is safe and highly immunogenic, and appears to confer protection from infection for at least years (table . ). postvaccination testing should be done - months after completion of the three-dose series to document an appropriate response (i.e., > miu/ml). more than % of persons immunized with three properly timed doses (e.g., , , and months) of vaccine administered intramuscularly in the deltoid region develop protective hbsab levels. factors associated with a lack of response include improper vaccination (improperly stored vaccine, gluteal inoculation, subcutaneous injection), obesity, older age, and smoking. persons who do not respond to the primary vaccine series should receive a second three-dose series or be evaluated for hbsag positivity. since , substantial progress has been made toward eliminating hbv transmission in children and reducing the risk for hbv infection in adults. recommendations of acip have evolved from universal childhood vaccination, to prevention of perinatal hbv transmission, vaccination of adolescents and adults in high-risk groups, and catch-up vaccinations for susceptible children in high-risk populations. , the acip vaccination strategies for children and adolescents have been implemented successfully in the united states, and routine immunization of all children is now recommended. the occupational safety and health administration's (osha's) blood-borne pathogen standard mandates provision of vaccine at no cost to all healthcare employees and others at occupational risk for blood exposure. substantial declines in the incidence of acute hepatitis b have occurred among highly vaccinated populations, such as young children and healthcare personnel. vaccine should also be provided to susceptible individuals before sexual maturity, particularly to teenagers in those settings (e.g., inner cities, concentration of poverty) where hbv is highly prevalent, and to all adults at risk for sexual or occupational exposure. preimmunization screening for evidence of prior or persistent infection usually is not cost effective. however, postimmunization testing for antibody response is recommended - months after the rd dose to detect nonresponders among persons at high risk for exposure. titers of hbsab fall over time and may be undetectable after - years. the duration of vaccine protection is under investigation. most data suggest that protection persists even when hbsab titers fall below the level of detection, and routine screening and boosting are not recommended. the need for prophylaxis for persons sustaining accidental percutaneous or mucosal exposures to blood should be based on several factors, including the hbsag status of the source, and the hepatitis b vaccination and vaccineresponse status of the exposed person. such exposures usually involve persons for whom hepatitis b vaccination is recommended. any blood or body fluid exposure to an unvaccinated person should lead to initiation of the hepatitis b vaccine series. a summary of prophylaxis recommendations for percutaneous or mucosal exposure to blood according to the hbsag status of the exposure source and the vaccination and vaccine-response status of the exposed person is shown in table . . when hepatitis b immune globulin (hbig) is indicated, it should be administered as soon as possible after exposure (preferably within hours). the effectiveness of hbig when administered more than days after exposure is unknown. when hepatitis b vaccine is indicated, it § hepatitis b immune globulin; dose is . ml/kg intramuscularly. ¶ a responder is a person with adequate levels of serum antibody to hbsag (i.e., anti-hbs ≥ miu/ml). ** a non-responder is a person with inadequate response to vaccination (i.e., serum anti-hbs < miu/ml). † † the option of giving one dose of hbig and reinitiating the vaccine series is preferred for non-responders who have not completed a second three-dose vaccine series. for persons who previously completed a second vaccine series but failed to respond, two doses of hbig are preferred. § § antibody to hbsag. should also be administered as soon as possible (preferably within hours) and can be administered simultaneously with hbig at a separate site (vaccine should always be administered in the deltoid muscle). for exposed persons who are in the process of being vaccinated but have not completed the vaccination series, vaccination should be completed as scheduled, and hbig should be added as indicated (table . ). persons exposed to hbsag-positive blood or body fluids who are known not to have responded to a primary vaccine series should receive a single dose of hbig and reinitiate the hepatitis b vaccine series with the first dose of the hepatitis b vaccine as soon as possible after exposure. alternatively, they can receive two doses of hbig, one dose as soon as possible after exposure, and the second dose month later. the option of administering one dose of hbig and reinitiating the vaccine series is preferred for non-responders who did not complete a second three-dose vaccine series. for persons who previously completed a second vaccine series but failed to respond, two doses of hbig are preferred. states. it is estimated that . % of americans have been infected with hcv. hcv-associated end-stage liver disease is the most frequent indication for liver transplantation among us adults. the incubation period for acute hcv infection ranges from to weeks (averaging - weeks). hcv transmission occurs primarily through exposure to infected blood, such as through injection drug use, blood transfusion, solid organ transplantation from infected donors, unsafe medical practices, occupational exposure to infected blood, and birth to an infected mother (i.e., vertical transmission). hcv may also be acquired through sexual contact, but the importance of this mode of transmission in the united states is not well characterized. hcv is not transmitted efficiently through occupational exposures to blood. healthcare personnel who are parenterally exposed to infected blood through needlestick injuries may acquire hcv infection, but the magnitude of risk (approximately in hcv needlesticks) is less than that associated with hbv exposure. one epidemiologic study indicated that transmission occurred only from hollow-bore needles compared with other sharps. transmission rarely occurs from mucous membrane exposures to blood, and no transmission in hcv has been documented from skin exposures to blood. data are limited on survival of hcv in the environment. in contrast to hbv, the epidemiologic data for hcv suggest that environmental contamination with blood containing hcv is not a significant risk for transmission in the healthcare setting, with the possible exception of the hemodialysis setting where hcv transmission related to environmental contamination and poor infection control practices have been implicated. the risk for transmission from exposure to fluids or tissues other than hcv-infected blood also has not been quantified but is expected to be low. hcv is not known to be transmissible through the airborne route, through casual contact in the workplace, or by fomites. clinical syndromes hepatitis c virus infection produces a spectrum of clinical illness similar to hbv and is indistinguishable from other forms of viral hepatitis based on clinical symptoms alone. serologic tests are necessary to establish a specific diagnosis of hepatitis c. most adults acutely infected with hcv are asymptomatic. after acute infection, - % of persons appear to resolve their infection without sequelae as defined by sustained absence of hcv rna in serum and normalization of alt levels. chronic hcv infection develops in most persons ( - %), with persistent or fluctuating alt elevations indicating active liver disease developing in - % of chronically infected persons. no clinical or epidemiologic features among patients with acute infection have been found to be predictive of either persistent infection or chronic liver disease. moreover, various alt patterns have been observed in these patients during follow-up, and patients might have prolonged periods (greater than or equal to months) of normal alt activity even though they have histologically confirmed chronic hepatitis. thus, a single alt determination cannot be used to exclude ongoing hepatic injury, and long-term follow-up of patients with hcv infection is required to determine their clinical status and prognosis. the course of chronic liver disease is usually insidious, and progresses slowly without symptoms or physical signs in the majority of patients during the first two or more decades after infection. chronic hepatitis c frequently is not recognized until asymptomatic persons are identified as hcv positive during blood donor screening, or elevated alt levels are detected during routine physical examinations. most studies have reported that cirrhosis develops in - % of persons with chronic hepatitis c over a period of - years, and hcc in - %, with striking geographic variations in rates of this disease. however, when cirrhosis is established, the rate of development of hcc might be as high as - % per year. longer follow-up studies are needed to assess lifetime consequences of chronic hepatitis c, particularly among those who acquired infection at young ages. although factors predicting severity of liver disease have not been well defined, recent data indicate that increased alcohol intake, being aged greater than years at infection, and being male are associated with more severe liver disease. in particular, among persons with alcoholic liver disease and hcv infection, liver disease progresses more rapidly; among those with cirrhosis, a higher risk for development of hcc exists. in addition, persons who have chronic liver disease are at increased risk for fulminant hepatitis a. screening enzyme immunoassay (eia) and supplemental confirmatory immunoblot tests are licensed and commercially available to detect antibodies to hcv (anti-hcv). anti-hcv may be detected within - weeks after the onset of infection but a single anti-hcv test cannot distinguish between acute, chronic, or past infection. hcv rna can be detected within - weeks of exposure to the virus and several weeks before elevations of alt and detection of anti-hcv. testing for anti-hcv by eia is recommended - months after an exposure to detect infection; testing for hcv rna may be performed - weeks after exposure if earlier detection of infection is desired. treatment, prevention, and control hcv-positive patients should be evaluated for the presence and severity of chronic liver disease. initial evaluation for presence of disease should include multiple measurements of alt at regular intervals, because alt activity fluctuates in persons with chronic hepatitis c. patients with chronic hepatitis c should be evaluated for severity of their liver disease and for possible treatment. alpha interferon (with or without ribavirin) treatment of hcv appears to prevent hcv replication, decrease hepatic inflammation, and improve symptoms among chronically infected persons. persons with chronic hcv have undergone successful liver transplantation, although recurrences have been documented in this setting. antiviral therapy is recommended for patients with chronic hepatitis c who are at greatest risk for progression to cirrhosis. these persons include anti-hcv-positive patients with persistently elevated alt levels, detectable hcv rna, and a liver biopsy that indicates either portal or bridging fibrosis or at least moderate degrees of inflammation and necrosis. therapy for hepatitis c is a rapidly changing area of clinical practice and consultation with a knowledge specialist (e.g., hepatologist) is recommended. no clinical trials have been conducted to assess postexposure use of antiviral agents (e.g., interferon with or without ribavirin) to prevent hcv infection, and antivirals are not fda approved for this indication. available data suggest that an established infection might need to be present before interferon can be an effective treatment. , because there is currently no postexposure prophylaxis (pep) for hcv, the intent of recommendations for postexposure management is to achieve early identification of infection and, if present, referral for evaluation of treatment options. in addition, no guidelines exist for administration of therapy during the acute phase of hcv infection. however, limited data indicate that antiviral therapy might be beneficial when started early in the course of hcv infection. when hcv infection is identified early, the person should be referred for medical management to a specialist knowledgeable in this area. at present, avoidance of parenteral exposure to blood is the only available strategy for preventing hcv infection. epidemiology it is estimated that more than million persons worldwide had been infected by hiv and that million were living with hiv/aids by the end of the year . in the united states, almost million persons are living with hiv. most individuals with hiv infection are active adults employed in the workforce. the primary means of acquiring infection among adults is either through behaviors such as unprotected homosexual or heterosexual intercourse with an infected partner, involving the exchange of body fluids, or injecting drug use involving shared needles and syringes. the virus also is perinatally transmitted to approximately - % of children born to infected mothers, (e.g., vertical transmission). breastfeeding is a bidirectional mode of transmission, to nursing infants of infected mothers and, rarely, to mothers of nursing infants when nipple maceration and biting occur. since , all donated blood in the united states has been screened for hiv infection. the risk of hiv infection due to transfusion of blood products screened by current methods is estimated to be in , , units transfused. screening does not completely eliminate the potential for a seronegative but infected unit from a recently infected donor to escape detection. hiv is not transmitted by the air-borne route, by household or workplace contact with infected persons, by exposure to contaminated environmental surfaces, or by insect vectors. the virus is easily inactivated by most common disinfectants, including household bleach (diluted : ). commercial sex workers are at the greatest risk of acquiring hiv infection occupationally. the other group of workers at risk for acquiring hiv infection occupationally is healthcare personnel. healthcare providers and other workers in contact with blood or other body fluids who sustain accidental percutaneous or mucosal inoculations with virus-infected material are at risk for infection. the magnitude of risk depends on the severity of exposure, but on the average, about in hiv needlesticks results in infection. the risk for infection following mucosal exposures is estimated to be lower at approximately . %. in the absence of direct exposure, healthcare providers are not at occupational risk for hiv infection. in the united states, through december , there have been cases of occupationally acquired hiv infection reported with an additional possible cases. clinical syndromes the clinical course of hiv infection is variable and changing with the advent of antiretroviral therapy, as well as treatment and prophylaxis for infectious complications. early after infection, within a few weeks to months, an acute febrile illness characterized by malaise, pharyngitis, lymphadenopathy, maculopapular rash, and headache may occur. the frequency of this mononucleosis-like illness has varied widely in reports of seroconverting individuals. at initial presentation of such patients, hiv antibody screening tests (enzyme immunoassay (eia)) may be negative, but viral antigen (p antigen) and serologic reactivity to one or more viral components (western blot test) allows the diagnosis to be established at this stage. hiv infection should be suspected in any person with a mononucleosis syndrome lacking a positive heterophil antibody response (monospot test). following initial infection, most persons have generalized asymptomatic lymphadenopathy and appear well. however, laboratory tests document a gradual decline in the number of circulating t-helper lymphocytes (cd cells), beginning soon after infection and continuing over the next several years. t-helper cells are essential components of the immune system and mediate aspects of both cellular and humoral immunity. symptoms, signs, and illness suggestive of mild to moderate immunodeficiency appear after about years, when cd cells decrease by about %, to less than cells/dl. intermittent fever, oral thrush, bacterial pneumonia, enteric infections, and reactivated tb are typically diagnosed at this time. signs suggestive of more rapid deterioration include oral hairy leukoplakia (a wart-like white growth in the oral cavity), shrinking lymphadenopathy, fever, weight loss, and elevated erythrocyte sedimentation rate. when cd cell counts fall below , serious opportunistic infections can be anticipated. pneumocystis pneumonia (pcp) was the most common index diagnosis in the first years of the epidemic, but the advent of effective pcp prophylaxis has altered the picture. other opportunistic infections and malignancies, including kaposi's sarcoma, lymphoma, disseminated tb, toxoplasmosis, and cryptococcal meningitis, now account for the majority of index hiv diagnoses. with the exception of tb, the infectious complications of hiv infection generally are not transmissible to healthy individuals and pose no risk in the workplace. indeed, the causative organisms are ubiquitous and most adults have already been exposed. opportunistic infections in hivinfected patients usually represent reactivation of dormant organisms when the immune system can no longer keep them inactive. be offered to all patients with symptoms ascribed to hiv infection. recommendations for offering antiretroviral therapy in asymptomatic patients require analysis of many real and potential risks and benefits. information regarding treatment of acute hiv infection from clinical trials is very limited. ongoing clinical trials are addressing the question of the long-term clinical benefit of potent treatment regimens for primary infection. in general, treatment should be offered to individuals with fewer than cd t cells/mm or plasma hiv rna levels exceeding , copies/rnl (by rt-pcr or bdna assay). once the decision has been made to initiate antiretroviral therapy, the goals should be maximal and durable suppression of viral load, restoration and/or preservation of immunologic function, improvement of quality of life, and reduction of hivrelated morbidity and mortality. hiv-infected individuals found to have latent tb infection should be treated with antituberculous therapy to prevent activation of disease. persons at risk for direct contact with blood and other potentially infected materials should receive specific instruction in universal/standard precautions for infection control, as recommended by the centers for disease control and prevention , and mandated by the occupational safety and health administration. for most environments outside healthcare settings, common sense and attention to personal hygiene are adequate to protect workers. gloves should be worn to clean up visible sites of blood contamination. environmental surfaces can then be decontaminated with disinfectant solutions or household bleach (diluted : ). , individuals sustaining accidental parenteral exposures to hiv should be counseled to undergo baseline and followup testing for months after exposure (e.g., weeks, months, and months) to diagnose occupational infection. postexposure chemoprophylaxis with antiretroviral agents has been recommended by the us public health service since after certain exposures to hiv-infected sources which pose a risk of infection transmission, such as needlesticks, mucous membrane, and non-intact skin exposures (tables . and . ). data from animal models of prophylaxis with these agents suggest that antiviral activity is diminished when treatment is delayed for more than hours. for this reason, immediate reporting and access to chemoprophylaxis is recommended. occupational exposure is a frightening experience. consultation with clinicians knowledgeable about hiv transmission risks who can provide supportive counseling to the worker is essential during the follow-up interval. cdc recommends that occupationally exposed workers refrain from unsafe sexual practices, pregnancy, and blood and organ donation for months after exposure to minimize the risk of transmission. zoonoses are infections that are maintained in nature by transmission between vertebrate animals, and they can be transmitted from other vertebrates to humans or from humans to other vertebrates. zoonotic pathogens can be divided into two major groups: ( ) those transmitted primarily among wild animals (e.g., yersinia pestis, rabies), and ( ) those transmitted primarily among domestic animals (e.g., sporothrix schenkii, non-typhoid salmonella spp.). other infections not properly classified as zoonoses can result from working directly with animals (e.g., infected wounds resulting from animal bites) or with animal products (e.g., anthrax in carpet weavers). many zoonotic infections present occupational risks, not only to those who work with live or dead vertebrate animals or animal products but also to workers exposed to certain environments contaminated by animals or animal products. thus, workers in veterinary medicine, animal husbandry, and animal research are at risk for acquiring a host of zoonotic infections specific to the type of live animal exposure, just as those involved in healthcare work with humans are at risk for infections acquired from humans. examples of such zoonotic infections include q fever in veterinarians, psittacosis (caused by chlamydia psittaci) in duck farmers, orf (contagious ecthyma) in shepherds, lymphocytic choriomeningitis (e.g., leptospirosis) in laboratory workers who handle rodents, fatal herpes virus simiae infection in primate handlers, and more recently, monkeypox in veterinarians and pet store owners. , influenza a (h nl) (avian flu) infection was shown to have been transmitted from ducks and chickens to poultry workers in hong kong and has become an important source of epidemic infection in various international settings; lyssavirus (related to rabies virus) infections have been transmitted from bats to humans in australia, and a large outbreak of febrile encephalitic and respiratory illnesses among workers who had exposure to pigs was shown to be due to infection with a previously unrecognized paramyxovirus (formerly known as hendra-like virus, now called nipah virus). brucellosis is an example of a zoonotic infection in abattoir workers exposed to live or dead animals or animal products. examples of zoonotic infections acquired by workers exposed to environments harboring or contaminated by contagious animals include leptospirosis in rice field workers, and argentine hemorrhagic fever typically acquired by adult males harvesting corn in cornfields inhabited by rodents, which serve as the reservoir for junin or machupo virus. it is beyond the scope of this chapter to review the large number of zoonoses (about have been described) that could pose a risk to workers in unique jobs that involve contact with various animals or environments. for each type of occupation that involves regular animal contact, it is important to recognize the types of infectious disease risks involved, consider baseline studies and storage of serum for future serologic tests if risks are high, plan preventive measures when possible, and prepare for early diagnosis and treatment of such infections when illness occurs. some of the zoonoses, the occupational groups they affect, and their clinical presentations are included in table . . infectious diseases continue to emerge, posing threats to the health of workers in numerous settings. a prime example of such a threat is severe acute respiratory syndrome or sars, first identified in early and responsible for illness and death primarily among exposed healthcare personnel. emerging infectious issues which may prove to be challenges for occupational health include those posed by bioterrorism, biotechnology, and emerging and reemerging infections. these emerging infections emphasize the need for continued vigilance and for careful history taking about occupational exposures when evaluating individuals for illnesses that could possibly be occupationally acquired. timeliness in identification and reporting of cases assists in the accurate estimation of the magnitude of the infectious disease problem and in the development of additional preventive and therapeutic measures. screening of employees for infection with or susceptibility to infectious diseases is an important part of healthcare maintenance, especially when the occupational setting poses a significant risk of transmitting or acquiring infections. screening also is warranted if specific interventions are available to prevent disease transmission among workers. assessment of behaviors, such as smoking, that increase the risk of acquiring infections also is valuable so that employees can be provided educational and other interventions to modify risks. preventing infectious diseases in workers can decrease absenteeism and financial costs associated with disability, sick leave, and health insurance, even if the primary source of infection is non-occupational. attending to these issues at the time of employment obviates the need for ongoing surveillance of many infections and simplifies outbreak investigations by documenting the pre-exposure immune status of contacts. tst screening for active disease identifies those persons who would benefit from prophylaxis (tables . and . ). tb vaccination with bacillus of calmette-guerin (bcg) vaccine, a live attenuated strain of m. bovis, is provided for children and some workers in most european countries, but it is not recommended in the united states because of its unproven efficacy when used in adults and because it induces dermal hypersensitivity to purified protein derivative (ppd) tuberculin in most recipients, impeding the usefulness of tst as a screening tool. persons age years and older, persons with chronic diseases or pulmonary disorders, and healthcare personnel should be offered pneumococcal vaccine and annual influenza vaccine (table . ). rubella immune status should be ascertained and men and women immunized in settings where women of childbearing age are employed (table . ). even though rubella is not often transmitted in the workplace, outbreaks can occur among susceptible individuals and vaccination is an important public health intervention. medical personnel should demonstrate proof of rubella immunity or vaccination prior to patient contact. measles vaccine should be provided to all workers born after with no documented history of measles who have not received two injections of live virus vaccine (table . ). screening for immunity to varicella and mumps is not routinely recommended, except for healthcare providers and adults with no history of infection with these agents who are exposed to young children. all adults require tetanus immunization. tetanus diphtheria toxoid (td) boosters should be administered every years to adults who have completed primary immunization (table . ). employees with no prior history of tetanus diphtheria immunization or with uncertain histories should receive a series of three primary vaccine injections. similarly, adults with no history of polio immunization should undergo primary immunization with inactivated polio vaccine, especially if they are employed in healthcare settings or when travel to endemic areas is anticipated. persons employed in occupations that pose a risk for parenteral contact with blood and other body fluids should be offered hepatitis b immunization (table . ). healthcare personnel, laboratory workers, animal handlers, first responders, and personal service workers such as barbers, tattooists, and cosmetologists are included in this category. adults with multiple sexual partners also should be encouraged to undergo immunization. serum banking to allow documentation of baseline serostatus is useful for laboratory and healthcare personnel at risk for other bloodborne infections such as hiv or more exotic infections. laboratory workers and animal handlers may be at risk for unusual infectious diseases. q fever, a rickettsial disease transmitted by the air-borne route, is a special risk encountered by handlers of sheep and similar animals. serologic testing for q fever titers prior to occupational exposure is important to document baseline status and to detect seroconversion at follow-up testing. although smallpox vaccine is no longer recommended routinely, genetically engineered vaccines prepared from vaccinia may pose a risk to researchers and clinicians treating patients enrolled in vaccine trials. laboratory workers who handle vaccinia or recombinant vaccinia preparations in culture or in animals should receive vaccinia vaccine. healthcare personnel caring for patients immunized with vaccinia or other orthopoxviruses or tissues and specimens from patients with these infections also should be immunized. a program for smallpox vaccination for selected individuals who may be in the frontline for responding to a bioterrorist attack has recently been initiated in the united states. consultation should be obtained to determine the need for screening, immunization, and testing for other exotic infections. some animal handlers are at risk of acquiring rabies through bites or exposure to infected secretions and tissues. immunization with human diploid cell vaccine (three -ml intradermal doses on day , , and or ) should be provided to workers at risk for rabies and for persons traveling for more than month to areas where rabies is endemic (table . ). booster injections should be provided every years for those with continuing exposure. surveillance of infectious diseases is conducted to detect increased occurrence of disease so that preventive interventions can be initiated. surveillance can be passive (based on employee health consultations or reports from contractual providers or supervisors) or active (actual monitoring of disease occurrence). active surveillance for infectious diseases is not required in most occupational settings. in work environments where exposure to m. tuberculosis may occur -such as healthcare settings, residential care facilities, shelters, and correctional facilities -active tst surveillance among susceptible individuals is indicated. periodic tsts are especially important in the wake of several recent outbreaks associated with drug-resistant strains of m. tuberculosis in hospitals, adult care settings, and home healthcare settings. skin testing should be performed at least annually in these settings, and perhaps as often as every months, for personnel at high risk for exposure to active tb. surveillance of teachers, travelers to endemic areas, and employees in other institutional settings where close contact with infected individuals is possible also may be warranted, depending on the local prevalence of tb. surveillance for infections among laboratory workers and animal handlers exposed to specific pathogens should be individualized in accordance with standard guidelines for biosafety in microbiologic and biomedical laboratories. maintaining standardized records of reportable infectious diseases is an important component of passive surveillance in the workplace. centralized collection and assessment of these records at regular intervals may allow early detection of outbreaks of occupational infections amenable to specific control interventions. geographic or temporal clusters of cases or clustering among persons with similar attributes or occupational tasks suggest a common source of exposure and infection and warrant investigation. local public health officials and regulatory agencies should be consulted promptly when an outbreak is initially suspected. reporting of occupationally acquired infections permits public health agencies to identify clusters of old and emerging illnesses and ultimately prevent them. these events should be reported as mandated by state and local regulations. , return-to-work criteria employees diagnosed with communicable infectious diseases should not return to work until the period of infectivity is past. specific guidelines should be consistent with local public health regulations. some workers, for example food handlers with certain diarrheal illnesses, cannot resume their duties until culture evidence of cure is obtained. employees should be advised of the return-towork policies at the time of employment and when illness is diagnosed. a table for length of work restriction for healthcare personnel can be used to guide return-to-work policies for the workplace (table . ). common sense dictates attention to personal hygiene among all workers. hand washing after using the bathroom and before handling food is essential. the mouth should be covered while sneezing or coughing, and soiled tissues and dressings should be disposed of in trash containers. employers have a responsibility to minimize crowding in the work setting. facilities for hand washing should be available in bathrooms and food preparation areas. proper ventilation also is important. trash should be emptied at regular intervals, and work areas should be clean and free of pests. smoking should be prohibited in common work areas. spills of blood, body fluids, and other potentially infectious substances should be removed with disposable paper towels or other suitable procedures. contaminated areas should then be disinfected with commercial products or with a solution of household bleach (diluted : ). , infection control in healthcare settings infection control programs in healthcare settings are necessary to prevent transmission of healthcare-related infections to patients and healthcare personnel. the cdc has established a two-tiered system of infection control precautions. the first tier consists of 'standard precautions' which are precautions recommended for delivery of care to all patients regardless of diagnosis or presumed infection status. they are designed to limit exposure to blood or other body substances and include elements such as hand hygiene and use of appropriate protective barriers, e.g., masks, eye protection, and gloves, as needed to prevent direct contact. the second tier of precautions recommended by cdc are 'transmission-based precautions', designed for the management of patients known or suspected to be infected with pathogens whose transmission can be limited by the adoption of additional measures beyond those which are part of standard precautions. they apply to pathogens transmitted by the air-borne or aerosol routes, droplets, and by direct and indirect contact. respiratory precautions are employed for patients with infections communicable by the air-borne route. such patients are housed in private rooms with special ventilation and should wear surgical masks when leaving their rooms. respiratory protection (i.e., n- respirators) also are advised for providers in close contact with patients on respiratory precautions. however, the re-emergence of epidemic and mdr-tb has led to a re-emphasis of other fundamentals for prevention of transmission of tuberculosis in healthcare and other settings. early identification of tb allows early indication for therapy, and requires alertness in considering tb in high-risk patients with pulmonary symptoms, especially those with hiv infection. special ventilation measures and respiratory protection are especially important for cough-inducing procedures, such as sputum induction and aerosolized pentamidine administration. healthcare personnel who have the potential for being exposed to m. tuberculosis should be screened on employment and at least annually thereafter by ppd skin testing, comparing previous test results to current results to identify those who have converted to skin test positivity. procedures for disposal of infectious wastes have been developed by the cdc. needles and other sharp objects should be sterilized prior to disposal. liquid and laboratory wastes may be dumped into sewage systems. materials heavily contaminated with bacteria or blood should be placed in special bags that are specifically labeled for infectious waste and should be disposed of according to community standards for such materials. employers have a responsibility to educate employees about infection control. barriers to prevent exposure, including masks, gowns, eye protection, and gloves, should be readily available to workers at risk. hand washing facilities and hand hygiene supplies are essential. where access to sinks or running water is not feasible, alcohol hand rubs or packaged towels containing disinfectants should be provided. impervious containers for the disposal of needles and other sharp objects are essential. such containers should be made available on ambulances and provided to home health aides and other visiting healthcare personnel. personal service workers who use needles, razors, and other sharp objects also should have access to safe disposal units. persons receiving care at home who require injections or other procedures that demand the use of needles also should be provided with impervious disposal containers and instructed in proper disposal methods to protect sanitation workers and others in contact with waste. despite improvements in engineering controls, work practices, and personal protective equipment, laboratory personnel are nevertheless at risk for occupationally acquired infections. laboratory personnel may acquire infection by aerosolization of specimens, mouth pipetting, or percutaneous injury or mucocutaneous contact. methods of infection control applicable to laboratory settings are described in the cdc document entitled 'biosafety in microbiological and biomedical laboratories'. by , it was estimated that . million children were attending out-of-home daycare in a variety of settings including licensed child daycare centers, regulated daycare homes, and unregulated family daycare homes. many serious infections occur as endemic or microepidemic problems in the daycare setting. these include h. influenzae type b, hepatitis a, cytomegalovirus, parvovirus b , and enteric infections (shigella, giardia, rotavirus, clostridium difficile, campylobacter, cryptosporidium, calcivirus, salmonella, enteric adenovirus, astrovirus, and several types of e. coli infections). in addition, the high rate of acute respiratory infections leads to early onset of otitis media, frequent antibiotic use, and emergence of multidrug-resistant enteric pathogens. thus, workers in close contact with children risk exposure to a wide variety of communicable pathogens contained in secretions, urine, and stool. all such personnel, as well as all children in schools and daycare centers, should be screened for immunity to common childhood infections and vaccinated if immunity is not present (table . ). regulation of childcare facilities is essential to reduce risks to children and workers. national standards for infection control in childcare facilities were promulgated in . hand washing facilities and policies are the most important component of disease prevention in school and daycare settings. hands should be washed after contact with mucous membranes and potentially infected body fluids. older children should be instructed in personal hygiene. children with fever or diagnosed infections should be excluded from attending daycare or school until transmission risk is no longer present, and policies for such exclusion should be in place. prompt reporting of disease outbreaks and prompt involvement of public health authorities are essential. employees should be instructed in common-sense first aid procedures for handling wounds, bites, and other situations in which exposure to infected blood or tissues is possible. barrier protection is rarely required in schools, but gloves should generally be available for emergencies requiring first aid. infection with a variety of agents during pregnancy has the potential to cause fetal damage, especially when primary infection occurs. while a number of these infections can be community acquired, the likelihood of exposure to certain of these pathogens can be greater in healthcare settings. infections with as rubella, cmv, and parvovirus are among the infectious agents which may be of special concern to pregnant healthcare personnel. in general, adherence to standard precautions as well as preexposure immunizations when available and appropriate are the best way of preventing the devastating effects of such infections (table . ). , immunodeficient workers are at increased risk of devastating infections, particularly with opportunistic agents. the greatest risk for such workers is likely in the healthcare setting where there can be ample opportunity for exposure to these agents. many immunocompromising illnesses would be viewed by the us legal system as disabilities and therefore, individuals with those conditions would be covered under the provisions of the americans with disabilities act of (see chapter . ). such persons should be informed about their risks and furthermore, their employers should make reasonable accommodations to allow their employees to continue to perform their jobs, taking into consideration the provisions of applicable federal, state, and local regulations. occupational infectious diseases encompass a large variety of infections which can involve many organ systems. they include some common infections, such as influenza, that pose a special problem in the workplace because of close interpersonal contact and crowding, and that taken together account for a large proportion of time lost from work. many of these infections are preventable by policies that promote hygiene and provide exclusion from work during periods of contagion. in addition, a variety of less common, but sometimes serious, infections are particularly associated with specific occupations. recognition of the types of infection risk associated with specific occupations can, in most cases, lead to effective, often simple steps for primary prevention, as well as opportunities for early diagnosis and treatment. bioterrorismrelated inhalational anthrax: the first cases reported in the united states principles and practice of infectious diseases centers for disease control and prevention. prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) influenza vaccination in long-term-care hospitals reduces the mortality of elderly patients measles, mumps, and rubella -vaccine use and strategies for elimination of measles, rubella, and congenital rubella syndrome and control of mumps: recommendations of the advisory committee on immunization practices (acip) changing epidemiology of pertussis in the united states: increasing reported incidence among adolescents and adults guidelines for the control of pertussis outbreaks ashrae guideline - : minimizing the risk of legionellosis associated with building water systems guideline for prevention of nosocomial pneumonia. the hospital infection control practices advisory committee progressing toward tuberculosis elimination in low-incidence areas of the united states: recommendations of the advisory council for the elimination of tuberculosis centers for disease control and prevention. targeted tuberculin testing and treatment of latent tuberculosis infection the role of bcg vaccine in the prevention and control of tuberculosis in the united states: a joint statement by the advisory council for the elimination of tuberculosis and the advisory committee on immunization practices guidelines for preventing the transmission of tuberculosis in health-care facilities guideline for isolation precautions in hospitals. the hospital infection control practices advisory committee prevention of varicella: update recommendations of the advisory committee on immunization practices (acip) epidemiology and prevention of vaccine-preventable diseases guideline for infection control in healthcare personnel guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force infection control precautions for the pregnant healthcare worker. bailliere's risk and management of bloodborne infections in healthcare workers updated us public health service guidelines for the management of occupational exposures to hbv, hcv, and hiv and recommendations for postexposure prophylaxis achievements in public health: hepatitis b vaccination -united states hepatitis b virus: a comprehensive strategy for eliminating transmission in the united states through universal childhood vaccination: recommendation of the immunization practices advisory committee (acip) protection against viral hepatitis: recommendations of the immunization practices advisory committee (acip) occupational safety and health administration, department of labor. cfr part . , occupational exposure to bloodborne pathogens; final rule recommendations for prevention and control of hepatitis c virus (hcv) infection and hcv-related chronic disease national institutes of health consensus development conference panel statement. management of hepatitis c an unequal epidemic in an unequal world the risk of transfusion-transmitted viral infections surveillance of healthcare personnel with hiv/aids, as of panel on clinical practices for the treatment of hiv infection. guidelines for the use of antiretroviral agents in hiv-infected adults and adolescents. us department of health and human services recommendations for prevention of hiv transmission in healthcare settings update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis b virus, and other bloodborne pathogens in health-care settings guideline for environmental control in healthcare facilities centers for disease control and prevention. multistate outbreak of monkeypox -illinois risk of influenza a (h n ) infection among poultry workers, hong kong, - emerging viral diseases: an australian perspective outbreak of hendra-like virus -malaysia and singapore, - risks and prevention of nosocomial transmission of rare zoonotic diseases sars -looking back over the first days occupational standards for the protection of employees in biotechnology centers for disease control and prevention. general recommendations on immunization: recommendations of the advisory committee on immunization practices (acip) and the american academy of family physicians (aafp) recommendations for using smallpox vaccine in a pre-event vaccination program: supplemental recommendations of the advisory committee on immunization practices (acip) and the healthcare infection control practices advisory committee (hicpac) us department of health and human services centers for disease control. case definitions for public health surveillance mandatory reporting of infectious diseases by clinicians. mandatory reporting of occupational diseases by clinicians selecting, evaluating, and using sharps disposal containers. dhhs (niosh) publication no. - child care arrangements for preschoolers by family characteristics: fall national standards for infection control in out-ofhome child care americans with disabilities act of , stat. , u.s.c. sec. et seq immunization of health-care workers: recommendations of the advisory committee on immunization practices (acip) and the hospital infections control practices advisory committee (hicpac) known responder ¶ known non-responder** antibody response unknown hbig § × and initiate hepatitis b vaccine series no treatment hbig x and initiate revaccination or hbig × † † test exposed person for anti-hbs: § § if drug resistance is a concern, obtain expert consultation. initiation of postexposure prophylaxis (pep) should not be delayed pending expert consultation, and, because expert consultation alone cannot substitute for face-to-face counseling, resources should be available to provide immediate evaluation and follow-up care for all exposures. § source of unknown hiv status (e.g., deceased source person with no samples available for hiv testing). ¶ unknown source (e.g., splash from inappropriately disposed blood). ** small volume (i.e., a few drops). † † the designation 'consider pep' indicates that pep is optional and should be based on an individualized decision between the exposed person and the treating clinician. § § if pep is offered and taken, and the source is later determined to be hiv negative, pep should be discontinued. ¶ ¶ large volume (i.e., major blood splash). key: cord- - dwqqgv authors: christian, michael d. title: biowarfare and bioterrorism date: - - journal: crit care clin doi: . /j.ccc. . . sha: doc_id: cord_uid: dwqqgv bioterrorism is not only a reality of the times in which we live but bioweapons have been used for centuries. critical care physicians play a major role in the recognition of and response to a bioterrorism attack. critical care clinicians must be familiar with the diagnosis and management of the most likely bioterrorism agents, and also be adequately prepared to manage a mass casualty situation. this article reviews the epidemiology, diagnosis, and treatment of the most likely agents of biowarfare and bioterrorism. weapons and their potential use by states with the consideration of use by nonstate actors (eg, terrorists). more recent definitions of bioterrorism include both a broader range of potential biological agents as well as considering more diverse groups of potential targets and impacts of biological weapons. spencer defines bioterrorism as "the use of micro-organisms as weapons of catastrophic effect which can be described as: the category or method of use of a weapon system that results in a significant negative impact on a nation's physical, psychological or economic well-being, thereby causing a major modification of routine activity." this definition highlights several key points. first, it highlights that a wide range of microorganisms must be considered and that their impact is not merely physical but may also include psychological and economic factors. spencer further elaborates on his definition of bioterrorism by stating "bioterrorism is best described as the use of micro-organisms (pathogens) or the products of living organisms (toxins) to inflict harm on a wider population, including animals and crops." the elaboration on his original definition highlights that not only are humans directly vulnerable to bioterrorism but we are vulnerable through indirect attacks on our livestock or crops, which has also been termed agroterrorism. , other investigators broaden the definition further to not only include microorganisms and biotoxins but also larger organisms, specifically insects. as with many other medical issues that intensivists face in their busy clinical and academic practices, pressured by ever-increasing time and budgetary restraints, there is a necessity to prioritize efforts and resources toward the most common and higherimpact concerns. it is difficult to provide a clear-cut answer as to where bioterrorism should be prioritized on this list. although some experts state that the risk of a largescale bioterrorist attack is low, in a more recent analysis, us senators graham and talent quote their conclusion form the commission on the prevention of weapons of mass destruction proliferation and terrorism in , which stated "unless the world community acts decisively and with great urgency, it is more likely than not that a [biologic] weapon of mass destruction will be used in a terrorist attack somewhere in the world by the end of ." anthrax in particular remains such a concern, because of both the lethality of the agent and also the potential availability given the number of governments that produced weaponized anthrax in the past. the later issue is of concern because of both the availability of expertise as well as the risk of residual caches of anthrax in failed states that are vulnerable for misappropriation. in addition, it may require less expertise to develop aerosolizable anthrax then previously believed. , box lists the capabilities required of any organization, whether state or nonstate, to conduct and deliver a bioterrorist attack. even a small or moderate bioterrorism event has the potential to overwhelm local medical resources and cause significant civil and economic disruption as a result of the psychological impact of such an attack. given the potential risk for a bioterrorism event and the major impact that could occur, these factors alone provide a strong argument as to why an understanding of bioterrorism is warranted for critical care physicians. however, if these arguments are not persuasive enough, the knowledge that critical care physicians will play a key role in a bioterrorist event, and that their effectiveness in responding to the event is dependent on their medical knowledge regarding bioterrorism agents, should compel one to take the time to read this review. agents used for bioterrorism typically cause critical illness and therefore are of clinical relevance to the intensivist. several of the category a and b organisms (see later discussion) also produce human infections in nature, and therefore, knowledge of their presentation and treatment can also be applied in nonbioterrorism settings. the us centers for disease control and prevention (cdc) has identified and categorized a list of potential bioterrorism agents ( table ). the agents identified by the cdc have been accepted, by most authorities globally, as the highest priority for preparedness and research. those agents from category a form the primary focus of this article. however, relman reminds us that it is important not to solely focus on the agents from the cdc list because they were largely driven by past military programs and do not include agents that were not of particular interest or relevance in the past but may be in the future given technological advances. in addition, military programs focused on weaponizing agents, whereas terrorists could seek to manipulate the natural spread of existing organisms or the development of novel strains. agents this section provides a basic overview and description of the commonly considered bioterrorism threats. specific details about the epidemiology, diagnosis, treatment, and outcomes associated with each of the organisms follow in the subsequent sections. anthrax the bacteria causing anthrax is bacillus anthracis, an encapsulated, grampositive, spore-forming bacilli. , , , when seen on a gram stain, it is often described as box cars given its appearance as a series of railway boxcars in a train viewed from above ( fig. ) . bacillus anthracis is a soil-borne organism and can be found in the environment globally. the organism grows quickly on standard culture media ( - h) and its spores are highly resistant, potentially being viable for decades. in naturally occurring infections, the organism may infect humans by transcutaneous inoculation (cutaneous anthrax), ingestion (gastrointestinal [gi] anthrax, including oropharyngeal), or inhalation (thoracic anthrax). in terms of use as an organism of bioterrorism, it is most likely to be delivered in its spore form as an inhaled agent. once inhaled, bacillus anthracis spores enter alveolar macrophages by phagocytosis and are transported to regional lymph nodes, where they germinate, typically within to days but they may be delayed up to days. , , symptoms start after germination and bacterial replication begins to occur. what causes the variability in incubation period associated with the time from infection with spores to germination to the vegetative bacillus is unknown. bacillus anthracis produces exotoxins: edema and lethal, comprised of components: ( ) edema factor (ef), which impairs neutrophil function and disrupts cell water hemostasis, resulting in massive edema; ( ) lethal factor (lf), which causes release of tumor necrosis factor a and interleukin b, which are believed to mediate the severity of illness produced; and ( ) protective antigen, found in combination with both ef and lf, which allows binding and transportation of the other toxins to and across cell membranes. in addition to the toxins, there are several other virulence factors, including an antiphagocytic capsule. anthrax is not transmitted person to person, but patients should be isolated with droplet precautions as part of standard febrile respiratory infection precautions until the cause of their illness is confirmed. patients with cutaneous anthrax require contact precautions. decontamination should include removing clothing and jewelry and washing skin with soap and water. caution must be exercised not to generate secondary aerosols when handling contaminated clothing items. environmental contamination from patients exposed to an aerosol of bacillus anthracis spore can be performed with a . % hypochlorite bleach solution. this procedure is not sufficient for site decontamination after the release of bacillus anthracis spores, because much more extensive decontamination is required. [ ] [ ] [ ] plague plague is caused by yersinia pestis, a nonmotile, gram-negative, bipolar coccobacillus that can be found worldwide. , , human infections occur in nature regularly, and plague is endemic in regions such as the southwest united states. yersinia pestis virulence factors include: v and w antigens, lipopolysaccharide endotoxin, capsular envelope (antiphagocytic), coagulase, and fibrinolysin. the natural reservoir for yersinia pestis is rodents and the vector to humans is the oriental rat flea (xenopsylla cheopis). plague is highly communicable in the pneumonic form and may present as any of the following clinical syndromes: bubonic plague, primary pneumonic plague, primary septicemic plague, plague meningitis, plague pharyngitis, pestis minor, and subclinical infection. aerosolized yersinia pestis likely produces a clinical presentation identical to pneumonic plague. however, yersinia pestis does not produce spores and is susceptible to destruction by drying, heat, and ultraviolet light, therefore making it significantly more challenging to weaponize it then anthrax. it is more readily transmitted via an infected vector, such as the oriental rat flea, or by person-to-person transmission. the incubation period depends on the clinical presentation; bubonic plague takes to days and pneumonic plague as short as hours to days. isolation for plague requires droplet precautions until hours of effective antibiotic therapy. decontamination is not required specifically for plague. health care institutions should follow their usual procedures for cleaning after patient discharge from a room. tularemia francisella tularensis is the organism responsible for causing tularemia; it has often been associated with rabbits and various rodents and is occasionally referred to as rabbit fever and deer fly fever. , [ ] [ ] [ ] francisella tularensis is a fastidious, small gram-negative, facultative intracellular coccobacillus able to live in soil, water, and decomposing carcasses for long periods. francisella tularensis can be transmitted by direct contact with mucous membranes, cutaneous inoculation through broken skin or bites from infected ticks (or other arthropods), ingestion and inhalation (although human-to-human transmission has not been reported ) . as discussed further in the epidemiology section, tularemia has been previously used as a biological weapon. the mode of deployment as a biological weapon in the past has often been through infected vectors ; however, a modern bioterrorist would most like deploy the agent via aerosolization and it could present as: primary pneumonic tularemia (inhalation), oculoglandular tularemia (eye contact), ulceroglandular (broken skin contact), or oropharyngeal (mucous membrane contact without deep inhalation). tularemia is not communicable between humans, so specific isolation is not required; only standard universal precautions should be used. decontamination for aerosolized exposure should involve remove clothing and jewelry and washing the skin with soap and water. q fever the only category b bacterial organism to receive any extensive discussion in this article is the zoonotic rickettsial organism coxiella burnetii, the causative agent of q fever in humans. [ ] [ ] [ ] coxiella burnetii is a small, obligate intracellular, gramnegative highly pleomorphic coccobacillus. it has a typical gram-negative cell wall structure, but does not stain well with gram stain although it can be seen with a gimenez stain. coxiella burnetii has morphologic forms: large and small cell variants. the small cell form is the extracellular form, which is metabolically inactive and resistant to chemical agents as well as environmental and physical conditions. the large cell variant is the metabolically active and pathogenic intracellular form. the disease is called q fever for query fever, because the causative organism had not been identified at the time of the first documented large outbreaks. q fever occurs worldwide and is associated with contact with sheep, goats, or cattle, particularly during birthing when placental exposure occurs. human infection is through the inhalation of aerosolized organisms from infected animals. coxiella burnetii is highly infectious to humans, and its sporelike small cell variant form makes it a potentially viable biological weapon. most human cases of disease are zoonoses, with human-to-human infection only rarely reported; therefore, only standard infection control precautions are required in the clinical setting. however, coxiella burnetii is highly infectious from culture and therefore should always be handled under biosafety level conditions. environmental decontamination should a culture spill or bioterrorism release occur is difficult given the highly resistant features of the small cell variant. other bacterial agents in addition to the bacterial agents already discussed, there are several other potential organisms that could be used as bioweapons (see table ). [ ] [ ] [ ] rickettsia prowazekii is the causative agent of louse-borne typhus and, similar to q fever, efforts have been made in the past to develop it into a bioweapon. brucella sp are small, aerobic, intracellular gram-negative coccobacilli, which cause primarily zoonotic infections in sheep, cattle, goats, and other animals. brucellosis can manifest as either christian systemic or localized infections and is acquired by contact with or ingestion of fluids from infected animals, particularly by the consumption of unpasteurized milk or cheese. brucella suis was reportedly weaponized by the united states, and possibly other countries, in the past. the organism was to have been aerosolized from a bomb. burkholderia pseudomallei is a gram-negative, facultative anaerobic, motile bacillus that causes melioidosis. there are naturally occurring cases of melioidosis in southeast asia and australia yearly. the disseminated form causes an acute illness, with high mortality. the disease is also known to cause abscesses, which, in some cases, do not present until many years after the initial exposure. burkholderia mallei is a gram-negative, aerobic, nonmotile bacillus that causes glanders. it exists only in living organisms and cannot survive in the external environment. glanders usually presents in humans as a nodular disease with regional lymphangitis; however, systemic dissemination of the organism does occur on occasion, producing septic shock, potentially leading to death. control measures have led to the eradication of glanders from most countries in the world, with the exception of ongoing zoonotic endemics in parts of the middle east, asia, africa, and south america. both burkholderia sp tend to be transmitted to humans through inoculation via a break in the skin, although inhalation is occasionally also a means of human infection. both organisms have been studied, and reportedly developed, as biological weapons in the past by the germans and russians. given the possibility of human-to-human transmission, droplet precautions should at least initially be used for patients with systemic or pulmonary involvement. the more significant transmission risk is from cultured organism, and therefore, any cultures should be managed in a biosafety level laboratory. smallpox once believed to be a disease of the past, smallpox is now one of the most significant bioterrorism threats to the world. , , [ ] [ ] [ ] there are no animal reservoirs for smallpox and the last naturally occurring case was in somalia in . smallpox is caused by the dna variola virus, a member of the genus orthopoxvirus (family poxviridae), which also includes molluscum contagiosum, vaccinia (virus used in the smallpox vaccine), and monkeypox. a large vulnerable population has existed since smallpox vaccination was stopped in the late s and early s. variola is highly communicable, and can be transmitted by airborne, droplet, and fomite (bed linen and clothing) transmission in addition to direct contact. infection starts in mucosa of airway, then the virus replicates in regional lymph nodes before subsequently resulting in an asymptomatic primary viremia to days after infection with spread to the bone marrow, spleen, and lymph nodes. secondary viremia occurs (day [ ] [ ] [ ] [ ] [ ] , in which the virus localizes in the dermis and oropharyngeal mucosa and is marked by the onset of symptoms and infectivity. five clinical syndromes of variola infection are seen: classic (unvaccinated only), modified (vaccinated or unvaccinated), flat (vaccinated or unvaccinated) hemorrhagic, and variola sine eruptione (vaccinated only). variola has several features that make it a potentially good bioweapon, including the ability to be made into a lyophilized form, which can be aerosolized and is heat resistant (box ). however, the virus is easily killed, even in the lyophilized from, by ultraviolet light and disinfectants. the primary concern related to smallpox as a bioterrorism agent is that it was produced in large quantities as a bioweapon by the soviet union and the security of its viral stock remains uncertain after the collapse of the superpower. mathematical models suggest that even a moderate-sized attack infecting to people would lead to a massive global pandemic that would require significant interventions to control, which in turn would carry with them significant economic and civic ramifications. airborne and contract precautions should be used to manage patients with smallpox. clearly, in a large-scale attack or outbreak, isolation of all patients in negative pressure rooms would not be feasible. all fomites (bed linen and patient clothing) should be transported in sealed biohazard bags and autoclaved before washing or incineration. standard hospital procedures for room cleaning can be followed once a patient has been discharged from the room. viral hemorrhagic fevers viral hemorrhagic fevers (vhf) ( table ) are a group of infections caused by families of rna viruses (arenaviridae, bunyaviridae, filoviridae, and flaviviridae), which are believed to primarily exist within animal or arthropod reservoirs, but occasionally infect humans. , , each virus is generally contained to a specific geographic region. all of the viruses have in common that they attack the vascular endothelium, leading to vascular leak and potentially producing shock or coagulopathies. with the exception of hantavirus, natural transmission is primarily by respiratory droplets and body fluids but not by airborne aerosol. aerosol transmission of hantaviruses from the urine of rodents has been well documented. most secondary (humanto-human) transmission of vhf is to family members or health care workers caring for patients with vhf. the primary mode of transmission in these cases is direct contact with infected body fluids and percutaneous exposures. however, questions still remain regarding the possibility of aerosol transmission, particularly for lassa fever or from aerosol-generating procedures. several of the vhf viruses have reportedly been weaponized by the russian and the us militaries. isolation for all vhf is strict droplet and contact precautions. patients who are end stage or have significant pulmonary involvement may warrant airborne isolation in a negative pressure room, if available. for specific details regarding isolation and environmental decontamination procedures for vhf, see the cdc's interim guidance for managing patients with suspected viral hemorrhagic fever in us hospitals, available online at http://www.cdc. gov/hai/pdfs/bbp/vhfinterimguidance _ _ .pdf (accessed january, ). in addition to clinical infection control precautions, it is important to minimize risk to box why smallpox is a good candidate as a bioterrorism agent . it is transmissible by the aerosol route, both in a weaponized from and from infected to susceptible persons. . it is heat stable in a lyophilized form. . the populations of most countries contain a high proportion of susceptible persons. . smallpox is associated with high morbidity and about % mortality. . there is a significant psychological fear of smallpox among both the public and health care workers. . initial diagnosis of cases of the disease may be delayed, given that it has not been seen clinically for more than years. . other than the vaccine, which may be effective only in the first few days after infection, there is no proven drug treatment for clinical smallpox. the laboratory and other health care workers by strictly limiting the number of samples and blood work collected. no fungal organisms are currently on the cdc list of category a or b agents, although it could be argued that some should be considered as potential category c agents. coccidioidomycosis is caused by the soil-borne spore-forming organisms coccidioides immitis and coccidioides posadasii found in the americas. these organisms typically cause a self-limited respiratory illness in immunocompetent individuals. coccidioidomycosis is typically transmitted by inhalation, particularly after events such as earthquakes or dust storms, which generate airborne particulate mater from the soil. human-to-human transmission does not occur. overall, the long incubation period and mild clinical presentations in most individuals make coccidioides an unattractive agent as a bioweapon. however, given the ease of access to the organism and its natural propensity for aerosol transmission, its potential use should not be ignored. keeping an open mind about possible future bioterrorism agents is essential if we are truly going to be prepared. biological toxins technically toxins are chemicals but because of their source (living organisms), biotoxins are typically classified as biological weapons. , , , the cdc list of potential bioterrorism agents (see table ) includes several toxins. in addition, there are several other toxins that also have the potential to be used as agents of bioterrorism (box ). toxins can be deployed via aerosol dispersal devices or by contaminating food sources and have been used as bioweapons in the past ( table ) . to be effectively deployed as an airborne agent, the toxin must be - mm for optimal aerosolization. ricin ricin is a protein toxin obtained from the organism ricinus communis (castor beans). castor beans are available worldwide and ricin is a natural by-product produced in the processing of castor beans to castor oil. the waste products resulting from the processing of castor beans contain approximately % ricin. in its pure form, ricin is a white powder that is soluble in water and stable over a wide range of ph. chemically, ricin is a glycoprotein lectin with a and b chains joined by a disulfide bond. the b chain binds to galactose-containing proteins and lipids on cell surfaces, causing direct membrane damage and release of cytokines, whereas the a chain inhibits eukaryotic ribosomes by removal of adenine from the rrna loop in the subunit, therefore stopping protein synthesis, leading to cell death. ricin is less toxic by weight compared with botulinum toxin or staphylococcal enterotoxin type b (seb), but is more readily available and easy to produce in large quantities than either of these agents. ricin can be deployed as a bioweapon via several routes, including inhalation, ingestion, or injection. clearly, as a bioterrorism agent with the intent to cause mass casualties, inhalation or ingestion is more likely than injection. after exposure, decontamination should include removal of clothing and jewelry, washing with soap and water, followed by rinsing with copious amounts of water. clostridium botulinum toxin (botulism) botulinum toxin is the most poisonous substance known to man with a dose lethal to half those exposed (ld ) of ng/kg. clostridium botulinum is a gram-positive, spore-forming, anaerobic bacillus found in soil and water globally. rarely, the toxin is also produced by clostridium butyricum and clostridium baratii. botulinum toxin is produced in pharmaceutical grade and is commercially available for cosmetic and other medical purposes. seven types of toxins exist and are identified as types a to g, with a, b, and e causing most cases of human disease. the toxin contains chains (heavy, light, nontoxic hemaglutinin). the heavy chain permits the toxin to bind with the cell, and the light chain contains zinc-dependent endopeptidase, which stops calcium release. botulinum toxin binds to peripheral presynaptic cholinergic terminals (including muscarinic and nicotinic) and blocks the calcium-dependent exocytosis process. the central nervous system is not affected by the toxin. botulism is most often considered a food-borne illness, given that most of the naturally occurring cases are the result of ingesting contaminated food products. other naturally occurring forms of toxicity are wound botulism (soil-contaminated wounds) and infantile botulism (ingestion of spores). the spores themselves are heat resistant, tolerating temperatures up to c, but the toxin itself denatures and becomes inactivated with even brief exposures to temperatures higher then c. use as a potential bioweapon includes contaminating food sources or via aerosolization. however, the toxin quickly degrades when exposed to environmental conditions, thus limiting its usefulness as a bioterrorism agent. botulinum toxin cannot cross the skin but can be absorbed by mucous membranes. should exposure to the toxin occur via aerosolization, decontamination should include removing clothing and jewelry, washing with soap and water, and rinsing with copious amounts of water. wash contaminated surfaces with a . % hypochlorite bleach solution to destroy the toxin. micotoxins several hundred varieties of toxins are derived from fungi, with the trichothecenes and aflatoxins being most concerning. aflatoxins are produced by aspergillus flavus or aspergillus parasiticus and are common contaminants in harvested food. aflatoxins bind to and damage dna and cellular proteins. trichothecenes are produced by a large number of fungi including: fusarium, stachybotrys, trichoderma, myrothecium, and cephalosporium. the most likely toxins from the class trichothecenes to be used as bioweapons are: t (trichothecene) and deoxynivalenol (vomitoxin). t causes skin irritation/pain and can be absorbed through the skin or inhaled, and then disseminated systemically, binding to peptidyltransferase inhibiting protein synthesis. it also interferes with dna polymerase and monoamine oxidase. the organs affected first are those are with rapidly reproducing cell lines (gi tract, bone marrow, and skin) as well as impairing proteins involved in coagulation and the krebs cycle. in addition, the breakdown of serotonin, epinephrine, and norepinephrine is impaired. t is a yellow droplet in appearance. should exposure occur, decontamination includes removal of clothing and jewelry, scrubbing the skin with soap and water, and isolating clothing or other contaminated objects. staphylococcal enterotoxins staphylococcal enterotoxins are heat-stable toxins produced by the common staphylococcus aureus bacteria. there are more than types of enterotoxins, with type a commonly known for causing food poisoning and type f causing toxic shock syndrome. the genes for staphylococcal enterotoxins are found on plasmids and bacteriophages, allowing transfer between different strains. seb can be aerosolized and has been studied as a potential incapacitating biological agent. seb is a superantigen, which produces massive stimulation of t cells and cytokine storm, which is rarely fatal but is significantly incapacitating to the victim. if there is exposure, decontamination requires removing clothing and jewelry, followed by washing skin with soap and water. clostridium perfringens epsilon toxin clostridium perfringens is an anaerobic, grampositive, spore-forming bacillus that is found in soil in all parts of the world. clostridium sp produce several toxins, one of which is the epsilon toxin, which commonly causes food poisoning. similar to seb, epsilon toxin could be another potential incapacitating bioweapon. insects were some of the earliest bioweapons ever used against an enemy (see table ). , there are potential ways to use insects in bioterrorism: direct attacks, agents of agroterrorism, or as vectors of disease. direct attacks most often use stinging insects and have been used in the past to defend fortifications and rout enemies from entrenched positions (the bee hive in the log, as seen in cartoons). another insect that has been studied by the indian military as a potential bioweapon is the paederus beetle, found in the middle east, which produces the toxin pederin. pederin is potent, causing intense pain, festering lesions, and blindness if it contacts the eyes. ingestion of the beetle can be lethal, as is injecting pederin into the bloodstream. agroterrorism is "the deliberate introduction of an animal or plant disease as well as damage to crops and livestock with the goal of generating fear, causing economic losses and/or undermining social stability." an example of insects used as an agent of agroterrorism is the medfly (ceratitis capitata), a fruit fly found in parts of the world but not the united states. their larvae eat many plants, causing significant crop destruction. in , a group threatened to release medflies in california if the government did not stop a pesticide-spraying program. if the insects had been released, the estimated damage to the economy would have been $ . billion. insects can be used in bioterrorism as disease vectors. table lists potential vector-borne diseases of concern. epidemiology it is challenging to describe the epidemiology of agents of bioterrorism in the traditional manner for several reasons. first, as discussed in the introduction, theoretic risks are largely being discussed, with many unknown factors involved. further, given that most of the work done in the area of bioweapons has been conducted by military or state organizations, only a small percentage of the overall activities have been publically reported. to provide the best possible overall perspective on the epidemiology of bioterrorism agents, table offers a comprehensive overview of past use of bioweapons as compiled from several sources. , , , , , , , , [ ] [ ] [ ] in addition to the general risks discussed in the introduction, academic health centers potentially play a unique role in the epidemiology of bioterrorism. any public area where mass gatherings occur or any infrastructure critical for the smooth functioning of society are potential targets for bioterrorism attacks. not only do hospitals in general, and academic health centers specifically, fit this profile, but academic health centers may also be at an increased risk because they are also potential sources of agents of opportunity (ao). the term ao is used to connote the use of a routine and unregulated chemical, biological, or radiologic agent by terrorists. table potential bioterrorism vector-borne diseases of concern for humans vector disease two key features of an ao are ( ) its availability, and ( ) a practical means of dissemination. box lists potential biological aos available in many academic health centers. an ao does not have to cause significant morbidity or mortality to be effective; the psychological effects on the public and impact on society/critical infrastructure functioning are sufficient to have a serious effect. aos are of significant concern to law enforcement officials because potential barriers to the development of a highly effective biological weapon are the cost and expertise required to develop them, whereas aos mitigate both of these factors. in the past, the development of bioweapons has required significant funding and labor, which typically only states can mobilize. it has been said that the us military bioweapon program employed more than staff and the ussr bioweapons sections employed more than , staff, both at costs of millions of dollars. although presenting a significant obstacle in bioweapon development, cost does not always prevent nonstate actors from pursuing bioweapons; the aum shunrikyo cult may have spent more than $ million dollars in its failed attempt to develop weapon-grade anthrax. the diagnosis, or identification, of a bioterrorism event is likely the most challenging step in the response. recognition of a bioterrorism event has components: ( ) identifying that an intentional rather then natural phenomenon has produced several cases of illness, and ( ) diagnosing the specific organism or agent causing the illness. they may occur in either order depending on the circumstances. bedside clinicians, specifically critical care physicians, play a crucial role in both aspects of recognizing that a bioterrorism event has occurred. , , recently, a great deal of attention and money has been directed toward bioterrorism detection technologies, such as environment sampling and syndromic surveillance. however, these efforts are highly unlikely to replace the bedside clinician in recognizing the event. , in a simulated anthrax attack designed to test the performance of a syndromic surveillance system currently in use, nordin and colleagues found that performance of syndromic surveillance varies with the infection rate. based on their analysis in a metropolitan area if there was a release of anthrax at a large public venue, a syndromic surveillance system that monitored approximately % of the local population would detect the event most of the time if the infection rate was % and all of the time if the infection rate was more than %, but it would take to days in both situations. therefore, these investigators concluded that "a suspicious clinician may detect the first case of anthrax before a syndromic surveillance system sounds an alarm and public health determines it is an anthrax release." identifying the important role that the beside clinician plays in recognizing a bioterrorism event is not intended to diminish or exclude the role of others in the process, especially not public health. the rapid recognition, and effective response, to a bioterrorism event requires that clinicians and public health officials work together. [ ] [ ] [ ] in some circumstances, such as smallpox, the diagnosis of a single case triggers the alarm that a bioterrorism event has occurred. however, for any of the diseases with a naturally occurring incidence, as well as with a novel organism or illness that has not yet been identified, it is the existence of a cluster of cases in a geographic area that triggers suspicion of a possible bioterrorism attack or emerging epidemic ( table ) . recognition of a cluster of patients typically requires a level of situational awareness that is beyond what an individual clinician can acquire and is usually attainable only by a regional, state, or federal public health agency depending on how widely distributed the cases are. for example, if an anthrax attack took place in a busy international airport, the cases would be widely distributed across a nation or internationally (eg severe acute respiratory syndrome [sars]). in this case, to the individual clinicians receiving the cases of inhalational anthrax, each would be an unusual, but not necessarily alarming, occurrence. however, the reporting of several cases of inhalational anthrax to a federal or international public health agency simultaneously would certainly trigger an alarm. however, we are often left with trying to distinguish between a naturally occurring event (epidemic) and an intentional event (bioterrorism). it can be difficult to distinguish between an artificial incident (terrorism) and a natural occurrence (epidemic). , box lists some of the characteristics that might help christian table case number triggering criteria for considering a potential bioterrorism event disease(s) a a this factor is in part based on the background incidence of disease in a geographic area and therefore varies from location to location as well as based on seasonal variations. for example, a viral encephalitis that is naturally transmitted by an endemic vector occurring in the summer when mosquitoes are prevalent would not necessarily raise concerns; however, a single case occurring in the winter or in a geographic area where that infection is not typically seen may raise concerns immediately. features that suggest a bioterrorism event rather than an epidemic . an epidemic curve that suggests a point source (common source) outbreak or extended source rather then a naturally propagated (transmitted) source (fig. ) . identification of a cluster of cases (large numbers of patients from a similar geographic area with similar symptoms) distinguish between the events based on epidemiologic features. recently, radosavljevic and belojevic have developed a scoring system to help distinguish between a bioterrorism incident and an epidemic ( table ) . these investigators use an interesting approach, which scores several qualitative and quantitative features of the incident categorized under groupings: person (cases), place (spatial distribution), and time. a score of or higher on this system is said to suggest that an artificial (intentional or accidental) event is more likely then a naturally occurring epidemic. the questions considered in this score have to be answered at the public health level, not the individual clinician level, because they require a level of situational awareness not attainable by a clinician in a hospital. the importance of clinicians working effectively with public health agencies is further reinforced by computer modeling work by hupert and colleagues, which suggests that even minor delays in detecting (diagnosing) and initiating the response to a large-scale anthrax attack would make even perfectly executed prophylaxis campaigns ineffective in preventing the health care system from being overwhelmed by cases. as noted in the earlier discussion, identifying the disease or organism involved can be key to identifying the event as a bioterrorism incident. this section provides an overview of the clinical and investigational findings that assist in diagnosing the specific diseases. key to any biological diagnosis are laboratory, particularly microbiological, tests. table summarizes the appropriate specimens and tests for diagnosing potential agents of bioterrorism. however, as with many aspects in critical care, it is important not to be overly reliant on technology. the college of american pathologists regularly tests the ability of laboratories to accurately diagnose, or appropriately refer to a reference laboratory, clinical potential agents of bioterrorism. in their recent testing of laboratories, the college of american pathologists found rates of acceptable identification responses were as follows: bacillus anthracis, % ( ) although the rates of appropriate diagnosis are improving, there still remains a delay in notification of the reference laboratory and a not insignificant failure rate for diagnosing category a organisms other then anthrax. anthrax as discussed earlier, anthrax presents as typically distinct clinical syndrome: cutaneous, gi, and thoracic. , , , diagnosis of anthrax often involves a combination of clinical, radiographic, and microbiological data. the clinical presentation of the anthrax syndromes is outlined in table . cutaneous anthrax most often affects the hands, arms, and face. one of the main differential diagnoses for cutaneous anthrax are spider bites, and table presents clues to help distinguish between the . the diagnosis of cutaneous anthrax can often be made clinically followed by laboratory confirmation. other laboratory and radiologic investigations are of limited use in cutaneous anthrax. conversely, in the cases of gi and thoracic anthrax, the clinical presentation is relatively nonspecific, and radiologic investigations, especially for inhalational anthrax, along with laboratory and microbiological testing are essential for making the diagnosis. with thoracic anthrax, sputum cultures are rarely positive but first blood cultures are almost always positive. with the relatively nonspecific findings associated with thoracic anthrax, it can be challenging for clinicians to differentiate it from the more commonly seen community-acquired pneumonia (cap). kyriacou and colleagues conducted a retrospective review of cases of bioterrorism-acquired thoracic anthrax and compared them with cases of cap or influenza. these investigators found that the most accurate predictor of anthrax was mediastinal widening or pleural effusions on chest radiograph, which were % sensitive, . % specific compared with cap, and . % specific compared with influenza (see fig. ). other features more common in anthrax were: nausea, vomiting, pallor, cyanosis, diaphoresis, altered level of consciousness, and increased hematocrit level. two groups , have developed screening criteria to help diagnose anthrax after a known bioterrorism incident. however, a costeffectiveness analysis showed that although both criteria would have identified cases abbreviations: ct, computed tomography scan; cxr, chest radiograph; mri, magnetic resonance imaging. a for microbiological findings, refer to table . data from refs. , , , , of anthrax, the mayer criteria would have screened only patients (cost $ usd), whereas the hupert criteria would have screened patients ($ , usd). although it is rare with cutaneous anthrax, any case of anthrax has the potential to develop hemorrhagic meningitis. therefore, any evidence to suggest meningitis should prompt appropriate investigations such as neuroimaging and a lumbar puncture. plague as with anthrax, the diagnosis of plague varies significantly based on the clinical syndrome as which it presents. , , plague may present as any of the following clinical syndromes: bubonic plague, primary pneumonic plague, primary septicemic plague, plague meningitis, plague pharyngitis, pestis minor, and subclinical infection. the first syndromes are the most common presentations and their diagnostic features are outlined in table . bubonic plagues is characterized by initial lymphadenitis, most commonly inguinal, followed by the development of a systemic illness, which may include secondary plague pneumonia. primary septicemic plague is similar to bubonic plague, without the initial appearance of buboes. it remains unclear if it is the same entity with subclinical lymphadenitis or a distinct pathophysiologic process. pneumonic plague may be primary, which presents as an acute respiratory infection, or may occur as a result of hematologic spread (secondary) from bubonic or primary septicemic plague. hemoptysis is frequently seen with pneumonic plague and can help differentiate it from anthrax or tularemia. general laboratory and radiologic investigations are nonspecific and the diagnosis is primarily made via microbiology testing (see table ). automated culture detection systems may not detect or can misidentify yersinia pestis, and therefore, if there is a high level of suspicion for plague, the microbiology laboratory should be made aware of this when the cultures are ordered. tularemia tularemia is another disease with several distinct presentations that are variable and dependent on the mode of transmission. , the clinical spectrum of disease includes: typhoidal, ulceroglandular, glandular, oculoglandular, oropharyngeal, and pneumonic tularemia, with the last being the most likely presentation from a bioterrorism incident. the average incubation period is to days, but ranges from to days. the pneumonic form typically presents with: fever, chills, headache, malaise, coryza, cough, chest pain, pharyngitis, abdominal pain, arthralgia, septic shock, respiratory failure, and acute respiratory distress syndrome (ards). the ulceroglandular form presents with cutaneous ulcers . to cm with heaped-up edges and regional lymphadenopathy. ulceroglandular infections may progress to systemic disease similar to the pneumonic form. the typhoidal form does not involve lymphadenopathy. the typhoidal form usually involves the lung primarily, whereas the ulceroglandular form first affects the mediastinal lymph nodes, then progresses to parenchymal involvement. microbiological testing is required to make the diagnosis, although cultures are generally low yield given the difficulty growing the organism. therefore, alternative techniques for identification such as immunofluorescence and polymerase chain reaction (pcr) are preferred. radiologic findings are generally nonspecific, although approximately % of patients with tularemia present with bronchopneumonia, typically bilateral, and may develop cavities. only approximately % have lymphadenopathy and pleural effusions, which is significantly lower than would be expected with thoracic anthrax. q fever q fever, when seen in naturally occurring infections in humans, causes a spectrum of disease from asymptomatic fevers and fevers of unknown origin through to life-threatening infections, typically associated with endocarditis or pneumonia. , the primary concern in a bioterrorism setting is inhaled coxiella burnetii, which presents with fever and cough alone in cases of lower inoculation through to severe pneumonia and respiratory distress with high inoculums. the diagnosis of q fever requires microbiological testing. the chest radiograph and computed tomography (ct) finding are nonspecific and consistent with any other type of pneumonia. there is no significant adenopathy seen on the chest radiograph or ct, which may help to rule out anthrax. other bacteria the other bacterial agents that are discussed are brucella sp and burkholderia sp. brucellosis has multiple clinical presentations and can be difficult to diagnose. typically brucellosis infects organs (lung, liver, spleen), bone marrow, bones, or central nervous system and is an insidious infection, presenting primarily with constitutional symptoms and organomegaly. the incubation period ranges from to weeks. diagnosis of brucellosis is best made by bone marrow cultures or blood cultures, although they are less sensitive. burkholderia mallei causes the clinical disease glanders, which presents as skin nodules and lymphadenopathy after inoculation through the skin, but in a bioterrorism event with inhalation exposure, the most likely presentation would be a nonspecific systemic febrile illness, which may or may not be accompanied by a pustular rash. burkholderia pseudomallei causes the clinical syndrome of melioidosis, a disease with a broad spectrum of presentations, including skin ulcers, pneumonia, acute fulminate sepsis, and chronic abscesses. if used in a bioterrorism setting with inhalational exposure, the acute presentation would most likely be either pneumonia or sepsis. smallpox smallpox can present as of clinical syndromes (table ) : classic, modified, flat, hemorrhagic, and variola sine eruptione. , , the primary challenge in the diagnosis of smallpox is the lack of clinicians who have experience with the disease. the cdc has published a case definition to aid in the diagnosis of cases (box ). classic smallpox begins with a prodrome of fever and constitutional symptoms. after to days, the rash begins to emerge, initially as small red spots in the mouth and throat, which develop into sores that erupt and discharge virus, making the patient highly infectious at this point. at about the same time that the sores in the mouth begin to open, a rash develops on the skin. the rash is said to spread in a centrifugal pattern, starting on the face, then spreading to the extremities. the lesions progress from macules, to papules (day of rash), umbilicated vesicles (day [ ] [ ] , and pustules (day ) before crusting over (fig. ) . in contrast to chickenpox (varicella), in smallpox, all the lesions progress at the same stage and the lesions are more densely concentrated on the face and extremities (figs. and ) . a single suspected case of smallpox is a public health emergency and the local public health agency should be notified immediately if there is a suspected case. imaging does not play a major role, because the respiratory symptoms tend to develop after the skin lesions. a few individuals who care for smallpox victims, or who have been vaccinated, develop a pulmonary form of smallpox without skin lesions. in these cases, chest radiography or ct may be helpful. the chest radiograph shows ill-defined nodular opacities in the upper lung fields, which may persist for months before calcifying. vhf clinical presentation of vhf depends on several factors, including specific virus, virulence of the strain, route of exposure, dose, and host factors (table ) . , the general feature that all vhf have in common is that they cause microvascular damage, leading to symptoms associated with increased vascular permeability, potentially resulting in hypovolemic shock or frank hemorrhage. medical imaging is not helpful in diagnosing these conditions, and the general laboratory findings are nonspecific, but often reveal evidence of hepatitis and hypovolemia. the specific diagnosis depends on microbiological testing (see table ). the diagnosis of a bioterrorism event secondary to the use of biological toxins depends on the presentation of unusual clusters of cases. identifying the specific toxin can be challenging but the clinical presentation of some toxidromes can be useful in suggesting the agent involved. the clinical syndromes associated with ricin poisoning vary based on the mode of entry into the body. inhalation of ricin leads to symptoms within hours, which include dyspnea, fever, cough, pulmonary edema, and chest tightness, whereas ingestion of ricin results in vomiting and diarrhea, leading to dehydration, shock, hallucinations, seizures, and hematuria. the typical onset of symptoms after ricin poisoning averages from to hours up to hours. laboratory findings associated with ricin poisoning are nonspecific but include metabolic acidosis, increased results for liver function tests, anemia, increased creatinine level, leukocytosis, and hematuria. aerosolized botulinum toxin, likely to be experienced in a bioterrorism setting, produces disease that mimics food-borne, infant, and wound botulism clinically. the clinical features that are anticipated with the inhalation of botulinum toxin include descending paralysis starting with diplopia, ptosis, fixed dilated pupils, dysphagia, respiratory failure, urinary retention, and constipation. with the ingestion of botulinum toxin, the clinical features begin with gi symptoms, including nausea, vomiting, diarrhea, bloating, and pain, before the development of the paralysis. onset of symptoms after exposure to botulinum toxin occurs as early as hours after inhalation, but if ingested typically there is a -hour to -hour delay before symptoms begin. however, the onset may be delayed as much as days. the diagnosis of botulinum intoxication is based on laboratory detection of the toxin in blood, food, or stool. the clinical presentations of mycotoxins vary based on the specific toxin. aflatoxin ingestion produces clinical features acutely, including hemorrhagic liver necrosis and pulmonary edema with chronic features, including the development of hepatic cancer. the effects and clinical presentation of inhaled aflatoxin are not known. t can be absorbed through the skin or inhaled. the clinical presentation in the acute phase includes pain on contact with skin or eyes, conjunctivitis, blurred vision, rhinorrhea, epistaxis, dyspnea, wheezing, tachycardia, shock, vomiting, diarrhea, erythema, and blistering. delayed symptoms present approximately week after exposure and include thrombocytopenia, neutropenia, anemia, and coagulopathy. seb is a superantigen, which causes massive stimulation of t cells and cytokine storm, which produces the associated clinical features. ocular exposure results in conjunctivitis and eye swelling, whereas inhalation causes fever, chest pain, gi symptoms, pulmonary edema/ards, and shock. the respiratory effects may not be seen for up to hours after exposure. as with most other infectious diseases, considerations for the management of bioweapons should include consideration of ( ) vaccination to prevent infection/illness, ( ) treatment of infection/illness, and ( ) prophylaxis after exposure to prevent clinical infection/illness. also, as is common with most other infectious diseases, there are often several antimicrobial regimens that can effectively treat a specific bioterrorism agent, and for others there are no effective treatments. table presents the generally accepted international recommendations for vaccination, treatment, and prophylaxis of bioterrorism agents. however, clinicians should always consult the most recent guidelines published by their public health authority when making clinical management decisions regarding bioterrorism agents. in addition to the current therapies, new drugs and vaccines are being developed specifically to address the threat of bioterrorism. with the exception of possibly cutaneous anthrax, there are no specific indications for surgical management of any bioterrorism organisms. even in the case of anthrax, surgical debridement has primarily been used for injection anthrax. , supportive care for victims of a bioterrorism event involves components: the care of the individual patient and the response to a mass casualty event. despite the heightened awareness of the potential for bioterrorist events since , many hospitals remain unprepared for biological threats. a study of uk emergency departments (eds) revealed that % did not have isolation facilities, and only % had departments with independent ventilations systems that would allow for the department to be isolated from the rest of the hospital. in addition, the survey found that isolation procedures in many eds were poor; for example, % would not have isolated a patient with potential sars and % would not isolate a patient with chickenpox. the task force for emergency mass critical care provides guidelines for hospitals to prepare for and manage mass critically ill casualties from events such as a bioterrorism attack. critical care physicians should be familiar with these guidelines and prepared to respond in such circumstances, because if they are not, all of their skills and knowledge for treating individual patients are of little use as the system becomes overwhelmed. supportive care of individual patients can generally be grouped into categories based on the class of agent involved: bacterial, viral, or toxin. bacterial and viral agents typically produce a sepsis syndrome with vascular leak, particularly in the case of vhf, as well as variable degrees of a systemic inflammatory response. supportive management is similar to the standard best practices for managing septic patients (box ). particular attention needs to be directed toward ventilatory support, with lung protective ventilatory strategies to minimize the development or exacerbation of adult respiratory distress syndrome. significant fluid shifts are to be expected given the vascular leak, requiring judicious volume resuscitation and cardiovascular support with vasopressors and inotropes based on goal-directed therapy. supportive therapy for patients involved in bioterrorism attacks with biotoxins varies based on the specific toxin. in the case of exposure to botulinum toxin, the primary support required is mechanical ventilation, hydration, and nutritional support until the paralysis resolves. toxins such as seb produce a response similar to sepsis and are supported by the sepsis protocols described earlier. ricin and mycotoxins both act at the cellular level, and although supportive therapies should be attempted, they may be of little benefit in altering the outcomes for patients. for mycotoxins specifically, there may be a role for the addition of steroids as a component of supportive care. data regarding patient outcomes after exposure to bioweapons or bioterrorism agents are, for the most part, lacking, given the paucity of cases and that most research has been carried out by government or military organizations and the results cannot be shared publically. table summarizes the published outcomes after exposure to bioterrorism agents when data are available. however, the outcomes from previous incidents, and in particular from naturally occurring illness, may not predict future outcomes, because bioterrorism agents may undergo genetic manipulation to increase their virulence or introduce antibiotic resistance, which decreases the efficacy of treatments. although to the average clinician, bioterrorism may seem to be only a remote possibility, it is not only a reality of the times in which we live but also bioweapons have been used for centuries. critical care physicians play a critical role in the response to a bioterrorism attack, and even more importantly, in identifying that an attack has occurred in the first place. an effective response to a bioterrorism incident requires a coordinated effort between clinicians and public health. critical care clinicians must be familiar with the diagnosis and management of the most likely bioterrorism agents, and also be adequately prepared to manage a mass casualty situation. index case of fatal inhalational anthrax due to bioterrorism in the united states the anthrax attacks years later biological weapons control. prospects and implications for the future potential bio-terror agents entomological terrorism: a tactic in asymmetrical warfare bioterrorism: preparing for the impossible or the improbable the threat of bioterrorism anthrax as a biological weapon, : updated recommendations for management bioterrorism-preparing to fight the next war anthrax infection an overview of anthrax infection including the recently identified form of disease in injection drug users the sverdlovsk anthrax outbreak of bioterrorism-related inhalational anthrax: the first cases reported in the united states a biological decontamination process for small, privately owned buildings comparative sporicidal effects of disinfectants after release of a biological agent decontamination after a release of b. anthracis spores threats in bioterrorism. i: cdc category a agents tularemia as a biological weapon: medical and public health management tularemia: the disease and the weapon insects as weapons of war, terror, and torture q fever: the neglected biothreat agent threats in bioterrorism. ii: cdc category b and c agents pathogenic rickettsiae as bioterrorism agents pathogenesis of burkholderia pseudomallei and burkholderia mallei rethinking smallpox an overview on the use of a viral pathogen as a bioterrorism agent: why smallpox? modeling potential responses to smallpox as a bioterrorist weapon the economic impact of a bioterrorist attack: are prevention and postattack intervention programs justifiable? viruses of the bunya-and togaviridae families: potential as bioterrorism agents and means of control interim guidance for managing patients with suspected viral hemorrhagic fever in us hospitals coccidioides species as potential agents of bioterrorism bioterrorism: toxins as weapons developing a consensus framework and risk profile for agents of opportunity in academic medical centers: implications for public health preparedness the history and threat of biological warfare and terrorism unusual epidemic events: a new method of early orientation and differentiation between natural and deliberate epidemics bioterrorism preparedness and response: clinicians and public health agencies as essential partners biologic and chemical weapons of mass destruction simulated anthrax attacks and syndromic surveillance late recognition of sars in nosocomial outbreak critical care and biological disasters: lessons learned from sars and pandemic influenza planning definition and functions of health unified command and emergency operations centers for large-scale bioevent disasters within the existing 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anthrax due to bioterrorism: evaluating proposed screening protocols the office of the surgeon general at tmm publications state-of-the-art therapeutic medical countermeasures for viral threat agents injection anthrax causing compartment syndrome and necrotising fasciitis the surgical management of injectional anthrax emergency departments (eds) in the united kingdom (uk) are not prepared for emerging biological threats and bioterrorism summary of suggestions from the task force for mass critical care summit prevention of laboratory-acquired brucellosis comparison of efficacy of ciprofloxacin and doxycycline against experimental melioidosis and glanders key: cord- -jm qwxhz authors: ouidir, marion; mendola, pauline; buck louis, germaine m.; kannan, kurunthachalam; zhang, cuilin; tekola-ayele, fasil title: concentrations of persistent organic pollutants in maternal plasma and epigenome-wide placental dna methylation date: - - journal: clin epigenetics doi: . /s - - - sha: doc_id: cord_uid: jm qwxhz background: prenatal maternal plasma persistent organic pollutant (pop) concentrations have been associated with neonatal outcomes. however, the underlying mechanisms remain unknown. placental epigenetic mechanisms may be involved, but no prior epigenome-wide studies have investigated the impact of maternal pops on placental dna methylation. we studied the association between maternal plasma pop concentration in early pregnancy and epigenome-wide placental dna methylation among pregnant women from the nichd fetal growth studies. results: our analysis focused on pops with more than % plasma concentrations above the limit of quantification, including organochlorine pesticides (hexachlorobenzene, trans-nonachlor, p,p’-dichlorodiphenyldichloroethylene), polybrominated diphenyl ether (pbde ), polychlorinated biphenyls ( / , , ), and poly- and perfluorinated alkyl substances (pfass) (perfluorodecanoic acid, perfluorohexanesulfonic acid, perfluorononanoic acid, perfluorooctanesulfonic acid, perfluoroundecanoic acid (pfunda)). using % false discovery rate, pops were associated with a total of differentially methylated cpg sites (nominal p values ranging from . × (− ) to . × (− )). out of the cpg sites, ( %) were significantly correlated with placental expression of genes. notably, higher pfunda was associated with increased methylation at cpg sites (cg , cg , cg ) annotated to tusc , and increased methylation at those cpg sites was correlated with decreased expression of tusc in the placenta. increased methylation at cg (tusc ) and decreased expression of tusc were correlated with shorter birth length. out of the cpg sites, methylation at cpg sites was correlated (p value < . ) with at least one neonatal anthropometry measure (i.e., birth weight, birth length, and head circumference). seven cpg sites mediated (p value < . ) the association between pbde and neonatal anthropometry measures. genes annotating the top differentially methylated cpg sites were enriched in pathways related to differentiation of embryonic cells (pbde ) and in pathways related to brain size and brain morphology (pfass). conclusions: dna methylation changes in the placenta were significantly associated with maternal plasma pops concentration. the findings suggest that placental dna methylation and gene expression mechanism may be involved in the prenatal toxicity of pops and their association with neonatal anthropometry measures. persistent organic pollutants (pops) have been used for decades in a large variety of products. despite the international consensus to reduce or ban these chemicals, exposure persists, mainly through diet, with concentrations still detected in human serum, including in us pregnant women [ ] and newborns [ ] [ ] [ ] . pops are ubiquitous endocrine-disrupting compounds (edc) that interfere with maternal hormones and can impact fetal development and health in later life. studies have reported that maternal levels of pops during pregnancy are associated with decrements in fetal growth and birth weight [ ] [ ] [ ] [ ] [ ] , neurodevelopmental disorders [ ] , earlier age of menarche [ ] , and genitourinary conditions in offspring [ ] . the mechanisms underlying the relationships between maternal exposure to these chemicals and fetal outcomes are not yet fully understood. alterations in placental development have been reported in response to maternal edc exposure [ ] , such as modification of the size of the placenta in mice [ ] , and degeneration of placental trophoblast in rats [ ] . therefore, changes in dna methylation in the placenta may be one of the potential mechanisms that explain the impact of pops on human fetal outcomes [ ] . existing studies of pops and placental dna methylation were based on candidate gene-based approaches [ ] [ ] [ ] [ ] . two studies among pregnant women from the check (children's health and environmental chemicals in korea) cohort reported associations of β-hexachlorocyclohexane (β-hch) with decreased methylation in line- (a surrogate marker of global methylation) and p,p′-dichlorodiphenyltrichloroethane (p,p′-ddt) with increased methylation of igf (implicated in placental and fetal growth) [ ] and mct among boys [ ] . p,p′-dichlorodiphenyldichloroethylene (p,p′-dde) and polybrominated diphenyl ether- (pbde ) were significantly associated with increased methylation in dio among female infants [ ] . higher pbde in cord blood was associated with decreased placental methylation in line- and higher pbdes and with decreased placental methylation of igf among chinese [ ] while others did not find any associations [ ] . most studies have investigated individual pops, although pregnant women are exposed to a mixture of chemicals [ ] . in this study, we assessed placental dna methylation related to individual pops and to chemical classes (i.e., sum of pops in each chemical class). we performed an epigenome-wide association study (ewas) to identify placental dna methylation associated with maternal plasma concentration of pops in early gestation ( weeks days to weeks days) among pregnant women participating in the eunice kennedy shriver national institute of child health and human development's (nichd) fetal growth studies-singletons cohort (which comprised pregnant women from clinic sites within the usa). genes annotated to the differentially methylated cpg sites were tested for enrichment of molecular pathways. we assessed correlations between dna methylation at the pop-associated cpg sites and placental expression of the annotated genes. further, to evaluate the relevance of the differentially methylated cpg sites to fetal growth, we examined correlations of methylation and gene expression with birth weight, birth length, and head circumference. lastly, we investigated the potential mediating pathway from pops to neonatal anthropometry measures via placental dna methylation at the pop-associated cpg sites that were correlated with neonatal anthropometry measures (i.e., birth weight, birth length, and head circumference). characteristics of the women included in the analysis are presented in table . the mean (sd) age and prepregnancy bmi were . ( . ) years and . ( . ) kg/m , respectively, and ( . %) were nulliparous (table ) . there was no difference in characteristics of women included in our analytic sample and the full nichd fetal growth study cohort (supplementary table s ). our analysis included chemicals with more than % plasma pop concentrations above the limit of quantification (loq). these included persistent lipophilic chemicals: organochlorine pesticides (ocps: hexachlorobenzene (hcb), trans-nonachlor, p,p′-dde), polybrominated diphenyl ethers (pbde ) and polychlorinated biphenyls (pcb congeners / , , ), and persistent non-lipophilic chemicals: poly-and perfluorinated alkyl substances (pfass: perfluorodecanoic acid (pfda), perfluorohexanesulfonic acid (pfhxs), perfluorononanoic acid (pfna), perfluorooctanoic acid (pfoa), perfluorooctanesulfonic acid (pfos), perfluoroundecanoic acid (pfunda)) (supplementary table s ) . in total, maternal early pregnancy plasma concentrations of pops were significantly associated with placental dna methylation at cpg sites annotated to genes (bacon-corrected false discovery rate (fdr) p values < . , nominal p values ranging from . × − to . − , supplementary table s ). the majority of the differentially methylated cpg sites ( . %) were located in cpg island regions (supplementary figure s ). ocps (i.e., hcb, trans-nonachlor, and p,p′-dde) were associated with methylation at cpg sites. the smallest association p value and highest strength of association was between trans-nonachlor and cg (rbm , β = − . , % confidence interval (ci) − . to − . , fdr p value = . × − ; table , fig. ). pbde was associated with methylation at cpg sites, the smallest association p value being with cg (selk; fdr p value = . × − ; fig. ). the highest strength of association was with cg (ero lb, β = − . , % ci − . to − . ). all measured pcbs (congeners / , , and ) were associated with methylation at cpg sites with the smallest association p value being between pcb and cg (ndufa ; fdr p value = . × − ). the highest strength of association was between pcb / and cg (nhej , β = − . , % ci − . to − . ). pfass (i.e., pfda, pfhxs, pfna, pfos, and pfunda) were associated with cpg sites, of which the smallest association p value being between pfda and cg (spg ; fdr p value = . × − ; table ). the highest strength of association was between pfunda and cg (ilf , β = − . , % ci − . to − . ). notably, higher pfunda concentration was associated with increased methylation at differentially methylated cpg sites annotated to tusc (cg , cg , and cg ). analyses of chemical classes (i.e., sum of ocps, sum of pcbs, and sum of pfass) found that the sum of ocps was associated with cpg sites, the smallest association p value being with cg (tex ; fdr p value = . × − ; supplementary table s ). the highest strength of association was with cg (mgc , β = − . , % ci − . to − . ). the sum of pcbs was associated with cpg sites, the smallest association p value being with cg (fbxo ; fdr p value = . ), while the highest strength of association was with cg (nhej , β = − . , % ci − . to − . ). analysis of differentially methylated regions (dmr) found that pbde was associated with three dmrs, the smallest association p value being the dmr annotated to hla-dmb (fdr p value = . × − ), and highest strength of association being annotated to znf (β = − . , % ci − . to − . ); pfunda was associated with one dmr annotated to tusc (β = . , % ci . to . , fdr p value = . × − , supplementary table s ) . out of the differentially methylated cpg sites, cpg sites were correlated with placental gene expression of unique genes (p values < . , table , supplementary table s ). the strongest correlation was between cg and expression of tusc (r = − . , p value = . × − ). further examination of the correlations between individual pop concentrations and gene expression levels found that higher maternal transnonachlor concentration was marginally correlated (r = − . , p value = . ) with decreased expression of sh pxd b (supplementary table s ). this is consistent with our dna methylation analysis finding where higher maternal plasma concentration of trans-nonachlor was associated with decreased methylation at cg (sh pxd b) ( table ) , and lower methylation at cg was correlated with decreased expression of sh pxd b (table ). sh pxd b displayed the highest expression in female reproductive tissues (supplementary figure s ). the genes mapping the differentially methylated cpg sites were enriched in ingenuity pathway analysis (ipa) canonical disease and function pathways (supplementary table s ). genes annotating the cpg sites differentially methylated with increasing levels of pbde were enriched in disease and functional annotation pathways including differentiation of embryonic cells (p value = . × − ). genes annotating cpg sites associated with pfas exposure were enriched in disease and functional annotation pathways, including size of the brain (p value = . × − ) and morphologies of the central nervous system (p value = . × − ), brain (p value = . × − ), and head (p value = . × − ). the top ipa canonical pathways included estrogen receptor signaling table s ). to understand whether single nucleotide polymorphisms (snps) influence dna methylation of the top-associated cpg sites, we assessed methylation quantitative trait loci (cis-meqtls) within mega base distance from the differentially methylated cpg sites in published databases of meqtls in the placenta [ , ] and blood (including cord blood and peripheral blood samples from whole blood, buffy coats, white blood cells, and blood spots) [ ] . in the placenta, cg (gramd ) and cg (tusc ) that were associated with pfunda concentration in our study have been reported to be cis-meqtl targets for snps in gramd and tusc (supplementary table s ). both genes displayed the highest expression in the placenta (supplementary figure s ). in the blood, cpg sites that were associated with transnonachlor, pbde , pfda, and punda in our study have been reported to be cis-meqtl targets for unique snps located in/near genes (supplementary table s ), including sh pxd b and tusc . previously, we found significant associations between maternal plasma concentrations of specific pops and fetal growth and birth anthropometry measures [ , ] . to examine whether the change in methylation at the cpg sites significantly associated with pops in the present analysis are related to neonatal anthropometry, we tested the correlations of methylation levels at each of the cpg sites with neonatal anthropometry measures (birth weight, birth length, and head circumference). among them, cpg sites were correlated with at least one neonatal anthropometry measure (supplementary table s ). we conducted mediation analysis to investigate whether placental dna methylation at those cpg sites was in the pathway between pops and neonatal anthropometry. five cpg sites (cg (bcl a), cg (sema b), cg (stk l), cg (map k ), and cg (kiaa )) significantly mediated the inverse association between pbde and birthweight (table ). in addition, two cpg sites (cg (kiaa ) and cg (kiaa )) significantly mediated the inverse association between pbde and head circumference. pbde was negatively correlated with birth weight (r = − . , p value = . ) and head circumference (r = − . , p value = . ) (fig. ) . out of the genes for which expression levels were correlated with dna methylation at the top-significant cpg sites, higher expressions of tusc were positively correlated with increased neonatal length (r = . , p value = . ) (supplementary table s ). in this study, we assessed the relation between maternal plasma pop concentrations during early pregnancy and genome-wide dna methylation in the placenta. specific maternal plasma pop concentrations were associated with differentially methylated cpg sites. of the cpg sites, were correlated with placental expression of the annotated genes. we found strong evidence for association of maternal plasma pfunda concentration with tusc based on consistent findings from dna methylation, gene expression, and meqtl analyses. similarly, we found consistent evidence from dna methylation and gene expression data on the impact of maternal plasma concentration of trans-nonachlor on sh pxd b. the correlations between dna methylation at the pops-associated cpg sites and neonatal anthropometry suggest that placental epigenetic mechanisms may underlie the influence of specific maternal plasma pop concentrations on fetal growth. two differentially methylated cpg sites identified in the present study have been associated with exposure to chemicals in previous ewas analyses: cg (znf ) associated with pbde in our study has been associated with polybrominated biphenyl in adult blood [ ] , and cg (tusc ) associated with pfunda in our study has been associated with pcbs in peripheral blood leucocytes [ ] . furthermore, cpg sites table s ). for example, cux , fam a, and ftcd-annotated cpg sites were associated with specific pfass in our study as was pfas concentrations in cord blood [ ] . many genes identified as being associated with maternal plasma concentration of pbde in our study have been associated with polybrominated biphenyl in adult blood (i.e., hla-dm, cib , arhgef l, bcl a, cds , eno , hoxb , inhba, pcbp , sdk , spred , zbtb , zeb , zhx , znf , and znf ) [ ] . the sh pxd b gene found to have methylation at cg and gene expression signatures associated with trans-nonachlor in our study is a genome-wide association study (gwas) locus for waist-hip ratio [ , ] , body weight [ ] , and balding measurement [ , ] . methylation at cg (sh pxd b) in liver tissue biopsies has been previously associated with obesity [ ] . mediation analysis revealed a potential explanation of the association between pbde and smaller birth weight through placental dna methylation at map k . these findings support earlier studies that reported associations between pops and birth weight [ ] and a study in mice that highlighted activation of mitogen-activated protein kinases (mapk) in the placenta of mice treated with pbde [ ] . we also observed positive association between dna methylation at another mitogenactivated protein kinase gene (mapk ip ) and pfunda. both map k and mapk ip placental dna methylation levels were negatively correlated with birth weight in our study. furthermore, pbde was associated with increased dna methylation at cg (kiaa ), and higher methylation at cg was associated with smaller weight and head circumference at birth. pbde was also associated with increased dna methylation at cg (kiaa ), and higher dna methylation at cg was associated with decreased head circumference. differential methylation at cg has been previously associated with neurodevelopmental syndromes [ ] . the kiaa gene is known to play a role in brain calcification [ ] , abnormal cerebellum morphology, and functional neurological abnormalities related to dysfunction of the [ ] . moreover, our mediation analysis suggested relations between pbde and head circumference via placental dna methylation (cg (kiaa ) and cg (kiaa )). together, our study corroborates previous findings on the impact of maternal plasma pop concentrations and placental methylation, and adds to recent ewas evidence for novel placental methylation sites that could potentially impact placental function and fetal development [ ] . we found strong corroborative evidence of exposure to pfunda on methylation and gene expression of the tusc gene, which is highly expressed in the placenta. in addition, placental methylation at cg (tusc ) and expression of tusc gene was correlated with birth length. a published placental meqtl study [ ] showed that sequence variants regulate methylation of cg of tusc in the placenta. tusc gene is a protein coding gene associated with several biological functions including cellular magnesium uptake, protein glycosylation, and embryonic development. tusc is a gwas locus for mental health disorders and general cognitive ability including educational attainment and mathematical ability [ ] , obsessive-compulsive disorder [ ] , and schizophrenia [ ] . in previous ewas analyses, methylation at cg (tusc ) in cord blood has been associated with prenatal arsenic exposure [ ] and pcb exposure [ ] . pfunda was observed to be associated with decreased methylation at cg (kif b), and lower methylation at cg was associated with smaller birth length. kif b has been associated with body height in a published gwas. in the same cohort, maternal plasma pfunda and pfhxs concentrations have been significantly associated with decreased neonatal thigh length at birth [ ] . studies have shown associations between prenatal exposure to pfas and bone development [ , ] . our findings may point to potential corr. spearman correlation, acme average causal mediation effects, ade average direct effect, te total effect p-values: * < . ; **p < . ; ***p < . ; ****p < . adjusted for maternal self-reported race/ethnicity, maternal age in years, fetal sex, maternal pre-pregnancy bmi, cotinine level, total lipids (except pfass), methylation sample plate, first three methylation principal component (pcs), and the first genotype pcs pathway through placenta dna methylation; however, the mediation analysis results were not significant. our study had several potential limitations that need to be considered in weighing our results. we are not aware of data from similarly-designed cohort studies, limiting our ability to replicate our findings in other independent populations. however, we were able to enrich the interpretation of our results using previous ewas analyses involving biospecimens collected from adults or cord blood. there may be spatial and cell populationbased differences in gene expression in the placenta, which was beyond the scope of our study. our ewas was able to identify modest methylation changes associated with maternal plasma pops concentration, but we acknowledge that studies with larger sample sizes are needed to detect cpg sites with smaller methylation changes. the study was undertaken at clinical sites that could potentially be related to differential methylation. however, in a sensitivity analysis evaluating models using the akaike information criterion (aic), out of differentially methylated cpg sites ( . %) were better explained in models without than models with clinical sites, and all association β values were comparable (supplementary table s ). finally, as chemicals mixture may be more complex than the sum of chemicals, we encourage future studies to further investigate the potential implication of interaction and non-linear relationships of pops on placental epigenetic changes. our study had several strengths. we adjusted our analysis for genotype-based principal components (pcs) in addition to methylation-based pcs, effectively minimizing spurious associations due to population stratification [ ] . to date, there is no reference for cell type composition for placenta; therefore, we implemented a validated reference-free adjustment for cell type proportion variation implemented in sva [ ] and further corrected our analysis for genomic inflation using bacon, a method demonstrated to maximize study power while controlling for false discovery rate [ ] . we also integrated our ewas findings with placental gene expression and neonatal anthropometry measures to highlight potential mechanisms of prenatal pop toxicity through placental epigenetic changes. findings from the present study suggest that maternal plasma concentrations of specific pops may influence placental dna methylation resulting in differences in neonatal birth size. furthermore, we observed strong evidence consistently supporting a role for pfunda concentration on tusc including placental dna methylation, gene expression, and placental meqtl analyses. taken together, these findings shed light on potential placental epigenetic mechanisms that may explain associations between prenatal exposure to pops and birth outcomes. this analysis included pregnant women from the nichd fetal growth studies-singleton cohort-who provided placenta samples at delivery and had pops concentration measures. briefly, the nichd fetal growth studies-singleton cohort included pregnant women enrolled between weeks and days and weeks and days between july and january from clinic sites within the usa [ ] . eligible women could not have a past history of adverse pregnancy outcomes or self-reported behavioral risk factors such as use of cigarettes, illicit drugs, or alcohol in the months prior to pregnancy. the study was approved by institutional review boards at nichd, all participating clinical and laboratory sites, and the data coordinating center. for this ancillary study, all maternal blood samples used were collected at enrollment ( weeks days to weeks days) in phlebotomy equipment determined to be free of the pop contaminants under study. a total of persistent organic pollutants (pops) were measured, as described in a previous published paper [ ] . we excluded chemicals where more than % of the concentrations were below laboratory loq. to capture the effect of the total exposure of each chemical class (i.e., ocps, pcbs, pfass), we summed individual chemicals within a class assuming additivity. briefly, pfass were quantified using μl of plasma, and pbdes, pcbs, and ocps were quantified using ml of plasma. all samples were shipped in dry ice to the wadsworth center, new york state department of health [ ] . total plasma lipids were calculated using the short formula: total lipids (in ng/ml) = . * total cholesterol + triglycerides + . [ , ] , where total cholesterol and triglycerides (in nangrams per millimeters) were measured from non-fasting plasma stored plasma in − °c freezers using the roche cobas chemistry analyzer (roche diagnostics, indianapolis, in) [ ] . machine-measured pop concentrations were modeled without substituting concentrations below the loq with a constant to minimize bias introduced when assessing health outcomes [ ] . for analysis, pop concentration were log ( + chemical) transformed for all pops except for p,p′-dde, pcb # / , sum of ocps, and sum of pcbs where a log ( + chemical) transformation was used. all concentrations were then rescaled by their standard deviations to provide results in interpretable units, i.e., change per sd for each and summed pops. placental samples (n = ) were obtained within h of delivery. briefly, placental parenchymal biopsies measuring . cm × . cm × . cm were taken from the fetal side, placed in rnalater, and frozen for molecular analysis, as previously described [ ] . extracted dna was assayed using illumina's infinium human methylation beadchip (illumina inc., san diego, ca). standard illumina protocols were followed for background correction, normalization to internal control probes, and quantile normalization. quality control procedures were followed as previously described [ ] . of the pregnant women with placental dna methylation data that passed quality filters, women had pop concentrations available for analysis and represent the study cohort ( / , . %). for analyses, beta values were converted to the m value scale by using the formula: m value = log (beta/( -beta)). any resulting infinity or missing m values ( . %) were imputed by the k-nearest neighbors method, setting k = for inclusion in the analysis [ ] . the placental biopsies used for dna extraction were also used for extracting rna from placentas using trizol reagent (invitrogen, ma), and sequenced using the illumina hiseq system. the expression of the transcripts were quantified using salmon [ ] which account for experimental attributes and biases such as fragment gc-content bias that is commonly observed in rna-seq data. participants with rna-seq, dna methylation, and pops level at enrollment (n = ) were used to test correlation between dna methylation and gene expression levels. epigenome-wide analyses were performed for each pop and each summed chemical class as the predictor and placenta dna methylation at each cpg site as the outcome using the r/bioconductor package "limma" [ ] . placental genome-wide snp genotype data were used to estimate genotype-based pcs representing the population structure. the r package "prcomp" was used to calculate pcs on the samples' percent methylation profiles [ ] . the ewas analysis included robust linear regression models that were adjusted for self-reported maternal race/ethnicity (non-hispanic white, non-hispanic black, hispanic, asian), age (in years), offspring sex (male/female), pre-pregnancy bmi (kg/m ), total plasma lipid concentration (ng/ml, except pfass), logtransformed plasma cotinine level (ng/ml), methylation sample plate (n = ), the first three methylation pcs and the first genotype pcs to account for population structure [ ] , and putative cell-mixture estimated using surrogate variable analysis (sva) components (n = ) to account for latent source of noise such as batch effects and cell composition [ ] [ ] [ ] . to account for the inflation of statistical test in ewas, we implemented the bayesian method to obtain bacon-corrected inflation estimates (bλ) and bacon-corrected p values using the r/bioconductor package bacon [ ] . corrected p values from bacon were then controlled for fdr [ ] giving bacon-corrected fdr p values. quantilequantile plots of p values and the corresponding inflation estimates before (λ) and after (bλ) baconcorrection are reported in supplementary figure s . the inflation statistic was close to after bacon correction (supplementary figure s ) . genome-wide manhattan plots were used to report results from ewas. the residual-vs-fitted plots for the first top hits of each ewas are presented in supplementary figure s . to identify genomic regions that are differentially methylated with maternal plasma concentrations of pops, we implemented dmr analysis using the r package dmrff [ ] . significant dmr was defined based on three criteria: ( ) the dmr length can be at most base pairs; ( ) the region has ewas fdr p values < . , and ( ) ewas effect estimates for the cpgs have the same direction. we estimated the correlation between mrna levels of the genes mapping to pop-associated cpg sites in our study (bacon-corrected fdr p values < . ) and placenta dna methylation and corresponding maternal pops blood concentration using spearman correlations. genes annotating to the epigenome-wide significant cpg sites (bacon-corrected fdr p values < . ) were further explored. we identified canonical pathway, networks, and diseases and biological function involved using the "core analysis" function in ingenuity pathway analysis (ipa, qiagen redwood city, ca, usa, www. qiagen.com/ingenuity). diseases and function annotation with molecules or more were considered. statistical significance of overrepresented canonical pathways was determined using fisher's exact test after adjustment for multiple testing using the benjamini-hochberg method [ ] . to identify snps that may influence dna methylation at the cpg sites in cis (i.e., within megabase on either side of a gene's transcript start site (tss) [ ] ), we examined the significant cpg sites (bacon-corrected fdr p values < . ) in a list of snps that are known to be meqtl in the placenta from two previously published papers [ , ] . then the significant cpg sites (bacon-corrected fdr p values < . ) were queried in the mqtl database (http://www.mqtldb.org/) that documents methylation quantitative loci (meqtl) in human blood at serial time points across the life-course: birth, childhood, adolescence, middle age, and pregnancy [ ] . then, the cis-meqtl snps identified through the query were annotated using tools and databases including haploreg v . [ ] and genotype-tissue expression (gtex) [ ] . additional file : figure s . residual-vs-fitted plots for the top hits of each ewas. for each figure, one color represents one cpg site. additional file : supplementary tables: table s . characteristics of the study subsample and of the full nichd fetal growth studies -singletons. table s : description of the maternal plasma persistent organic pollutants concentrations. table s : top-significant adjusted difference (bacon-corrected fdr p-values < . ) in placenta dna methylation associated with maternal pop. table s : differentially methylated regions (dmr) analysis. table s : correlations between dna methylation at the top differentially methylated cpg sites and gene expression in placenta. table s : significant associations between maternal blood levels of pop and expression of genes near the top significant dna methylation cpg sites. table s : top pathways of diseases and biological function from ipa. table s : ipa canonical pathway. table s : network identified by ingenuity pathway analysis. table s : placental cis-eqtl analysis of top-significant (bacon-adjusted fdr p-value< . ) cpgs. table s : cis-meqtl analysis of top-significant (bacon-adjusted fdr p-value< . ) cpgs. table s : spearman correlation between top differentially methylated cpg sites and neonatal anthropometry. table s : spearman correlation between neonatal anthropometry and gene-expression that were significantly correlated with the methylation on the corresponding cpg sites. table s : comparison with previous ewas on chemicals. table s : sensibility analysis further adjusted for clinical sites for the cpg sites differentially methylated in the original model. environmental chemicals in pregnant women in the united states: nhanes prenatal exposure to endocrine disrupting chemicals and risk of being born small for gestational age: pooled analysis of seven european birth cohorts the transfer of polychlorinated biphenyls (pcbs) and polybrominated 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functional changes in gene expression in newborn cord blood and subsequent birth outcomes prenatal concentrations of perfluoroalkyl substances and bone health in british girls at age effects of developmental exposure to perfluorooctanoic acid (pfoa) on long bone morphology and bone cell differentiation accounting for population stratification in dna methylation studies an epigenome-wide association study of obesity-related traits controlling bias and inflation in epigenome-and transcriptome-wide association studies using the empirical null distribution cohort profile: nichd fetal growth studies-singletons and twins calculation of serum "total lipid" concentrations for the adjustment of persistent organohalogen toxicant measurements in human samples chlorinated hydrocarbon levels in human serum: effects of fasting and feeding plasma concentrations of lipids during pregnancy and the risk of gestational diabetes mellitus: a longitudinal study the limitations due to exposure detection limits for regression models sex differences in the associations of placental epigenetic aging with fetal growth dna methylation age of human tissues and cell types salmon provides fast and bias-aware quantification of transcript expression limma powers differential expression analyses for rna-sequencing and microarray studies methylkit: a comprehensive r package for the analysis of genomewide dna methylation profiles capturing heterogeneity in gene expression studies by surrogate variable analysis sva: surrogate variable analysis. r package version . . controlling the false discovery rate: a practical and powerful approach to multiple testing dmrff: identifying differentially methylated regions efficiently with power and control. biorxiv expression quantitative trait loci: present and future haploreg: a resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants the genotype-tissue expression (gtex) pilot analysis: multitissue gene regulation in humans mediation: r package for causal mediation analysis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we acknowledge the study participants of the nichd fetal growth studies. we we examined whether maternal pop concentration and the methylation levels and gene expression level of the annotated gene for the significant differentially methylated cpg sites were correlated with neonatal anthropometry at birth (i.e., birth weight, length, and head circumference) using spearman correlation coefficients. since maternal plasma pop concentrations were previously reported to be associated with neonatal anthropometry [ ] , we tested the possible mediation of the methylation at the cpg sites in the relation between the specific pop and neonatal anthropometry at birth using the r package "mediation" [ ] . mediation analyses were tested using the following criteria: ( ) pop concentrations were associated with placental dna methylation at the cpg site (bacon-corrected fdr p values < . ), ( ) correlations between the cpg site and neonatal anthropometry measures had a p value < . . the total effect represents the effect of the pops on the neonatal anthropometric outcome without adjusting for methylation at the cpg sites; the direct effect represents the effect of the pop on the neonatal anthropometry measure at a fixed methylation level; the indirect effect represents the effect of the pop through the placental dna methylation. analyses were adjusted for self-reported maternal race/ethnicity (non-hispanic white, non-hispanic black, hispanic, asian), age (in years), offspring sex (male/female), pre-pregnancy bmi (kg/m ), total plasma lipid concentration (ng/ml, expect pfass), log-transformed plasma cotinine level (ng/ml), methylation sample plate (n = ), the first three methylation pcs, and the first genotype pcs. supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file : figure s . cpg sites relation to island.additional file : figure s . manhattan plots of associations between dna methylation in placenta and maternal plasma concentrations of asum of ocps, b-sum of pcbs, and c-sum of pfass.additional file : figure s . gene expression from the human protein atlas, consensus dataset.additional file : figure s . quantile-quantile (qq) plots with raw pvalues and inflation estimates (λ) and with bacon-corrected p-values and bacon-corrected inflation estimates (bλ).authors' contributions mo, pm, and ft-a planned the analysis. mo conducted data analysis, drafted, and revised the manuscript. pm and ft-a contributed to the interpretation. pm, gbl, kk, cz, and ft-a provided critical intellectual content. gbl was the pi of the nichd fetal growth studies and along with co-investigator (cz) were responsible for implementation of the study protocol including data collection. kk was the pi on the subaward for toxicologic analysis of pops and their modelling assumptions and interpretations. all authors approved the final version of the manuscript. the placental genome-wide dna methylation, gene expression, and genotype data are available through dbgap with accession number phs .v .p . the maternal genotype data analyzed in the current study are available from the corresponding author upon request. summary statistics from the ewas analysis are available as supplementary data. the study was approved by institutional review boards at the nichd, all participating clinical and laboratory sites, and the data coordinating centers. all pregnant women participated after giving informed consent. the authors declare that they have no competing interests.author details key: cord- -obrq q authors: benghanem, sarah; mazeraud, aurélien; azabou, eric; chhor, vibol; shinotsuka, cassia righy; claassen, jan; rohaut, benjamin; sharshar, tarek title: brainstem dysfunction in critically ill patients date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: obrq q the brainstem conveys sensory and motor inputs between the spinal cord and the brain, and contains nuclei of the cranial nerves. it controls the sleep-wake cycle and vital functions via the ascending reticular activating system and the autonomic nuclei, respectively. brainstem dysfunction may lead to sensory and motor deficits, cranial nerve palsies, impairment of consciousness, dysautonomia, and respiratory failure. the brainstem is prone to various primary and secondary insults, resulting in acute or chronic dysfunction. of particular importance for characterizing brainstem dysfunction and identifying the underlying etiology are a detailed clinical examination, mri, neurophysiologic tests such as brainstem auditory evoked potentials, and an analysis of the cerebrospinal fluid. detection of brainstem dysfunction is challenging but of utmost importance in comatose and deeply sedated patients both to guide therapy and to support outcome prediction. in the present review, we summarize the neuroanatomy, clinical syndromes, and diagnostic techniques of critical illness-associated brainstem dysfunction for the critical care setting. the brainstem is the caudal portion of the brain that connects the diencephalon to the spinal cord and the cerebellum [ ] . the brainstem mediates sensory and motor pathways between the spinal cord and the brain and contains nuclei of the cranial nerves, the ascending reticular activating system (aras), and the autonomic nuclei. it controls the brainstem reflexes and the sleepwake cycle and is responsible for the autonomic control of the cardiocirculatory, respiratory, digestive, and immune systems. brainstem dysfunction may result from various acute or chronic insults, including stroke, infectious, tumors, inflammatory, and neurodegenerative diseases. in the context of critical illness, the brainstem can be susceptible to various insults that can be categorized as structural and non-structural origin. brainstem dysfunction can then contribute to impairment of consciousness, cardiocirculatory and respiratory failure, and thus increased mortality [ ] [ ] [ ] [ ] . in the present review, we describe brainstem functional neuroanatomy, clinical syndromes, and assessment methods before addressing the concept of critical illnessassociated brainstem dysfunction. the brainstem can be categorized into three major parts: midbrain, pons, and medulla oblongata ( figs. and ) . the brainstem contains both gray and white matter, with the basilar artery representing the vascular supply. the gray matter includes the nuclei of the cranial nerves (anterior part), the aras (posterior part), the extrapyramidal and the central autonomic nervous system (ans). this gray matter controls brainstem reflexes, arousal, automatic movements, and homeostasis, respectively. the white matter is composed of ascending sensory pathways and descending pyramidal and extrapyramidal pathways (table ) . brainstem pathology should be considered in cases of (a) sensory or motor deficits combined with cranial nerve palsy, (b) impairment of consciousness, (c) dysautonomia, or (d) neurological respiratory failure. the pyramidal and extrapyramidal tracts connect the upper motor neurons and the extrapyramidal nuclei with the lower motor neurons located in either the brainstem or the spinal cord [ ] . while the former controls voluntary movement, the latter is involved in reflexes, motion, complex movements, and postural control (tables and ). upper motor neuron damage can lead to symptoms, ranging from hemiparesis to the locked-in syndrome, which is typically characterized by intact awareness, quadriplegia, anarthria, and absence of eye movements except for preserved vertical gaze. it usually results from bilateral pontine white matter lesions [ ] . characteristic clinical features of brainstem lesions include ipsilateral cranial nerve palsies or cerebellar signs combined with contralateral motor deficits. brainstem lesions may present with abnormal movements, such as hemichorea, hemiballism, dystonia, tremor, asterixis, pseudo-athetosis, and non-epileptic myoclonus [ ] ( table ) . bilateral motor corticobulbar tract lesion may present with swallowing impairment, dysphagia, dysphonia, velo-pharyngo-laryngeal impairments, uncontrollable crying/laughing episodes, and emotional lability (i.e., pseudobulbar affect; table ). a brainstem lesion of the posterior column-medial lemniscus pathway and the spinothalamic tract results in a contralateral proprioceptive/touch and temperature/pain deficit, respectively. the testing of the cranial nerves and brainstem reflexes is described in table . abnormal spontaneous eye position and movements may be encountered in patients with brainstem lesions and can be seen in comatose patients. assessment of pupillary size allows the diagnosis of third nerve lesion (i.e., mydriasis) or horner's syndrome (i.e., myosis, ptosis, enophtalmia, and anhidrosis). pupillary light, corneal, oculocephalic, and gag reflexes are routinely assessed in the critical care setting. the oculovestibular responses and oculocardiac are less frequently tested, except to determine brain death. the absence of brainstem reflexes and spontaneous breathing is a prerequisite for the diagnosis of brain death [ ] . automated pupillometry could improve the assessment of the pupil light reflex and thereby its prognostic value [ ] . corneo-mandibular reflexes can be detected in acute brain injury, but its prognostic relevance remains controversial. finally, assessments of primitive reflexes are less relevant in the icu context but can (table ) . when suspecting brainstem lesions, mri will have the highest yield to further localize and characterize brainstem lesions [ ] (table ). evoked potentials may be also useful for detecting a brainstem lesion. eeg [ ] may be supportive in patients with abnormal movements and disorders of consciousness, and cerebrospinal fluid (csf) analysis for those with suspected inflammatory or infectious diseases. the aras controls the sleep-wake cycle and includes several nuclei mainly located in the pontine and midbrain tegmentum [ ] (table , figs. and ): the rostral raphe complex, the parabrachial nucleus, the laterodorsal tegmental nucleus, the locus coeruleus (lc), the nucleus pontis oralis, the basal forebrain, and the thalamus. monoaminergic neurons are directly linked to the cortex and are inhibited during deep sleep. cholinergic pedunculopontine and laterodorsal tegmental nuclei are indirectly connected to the cortex via the thalamus and remain active during rapid eye movement sleep. these pathways are modulated by hypothalamic neurons [ ] . disorders of consciousness can be organized between acute and subacute or chronic [ ] . acute impairments of consciousness include coma which is defined as a "state of unresponsiveness in which the patient lies with eyes closed and cannot be aroused to respond appropriately to stimuli even with vigorous stimulation" [ ] . the association of a prolonged non-responsive coma with a complete cessation of brainstem reflexes and functions suggests the diagnosis of brain death which is defined as an irreversible loss of all functions of the entire brain. delirium is defined as an acute and fluctuating disturbance of consciousness, including attention and impairment of cognition, associated with motor hyperactivity or hypoactivity [ , ] . delirium has been associated with long-term cognitive impairment, functional disability in icu survivors, and hospital mortality [ ] . brainstem dysfunction could account for some features of delirium, such as fluctuations in arousal and attentional impairment that could be related to aras and to ponto-mesencephalic tegmentum dysfunction, respectively. other states of acute impairment of consciousness include clouding of consciousness and stupor, but they are less frequently used [ ] . subacute or chronic disorders of consciousness include the vegetative state (vs, also called unresponsive wakefulness syndrome) defined as state of unresponsiveness in which the patient shows spontaneous eye opening without any behavioral evidence of self or environmental awareness [ ] . the minimally conscious state (mcs) is defined as state of severely impaired consciousness with minimal behavioral evidence of self or environmental awareness, characterized by the presence of non-reflexive behavior (visual pursuit, appropriate motor response to painful stimulus) or even intermittent command following indicating a cortical integration [ , ] . the vs and mcs are related to a preservation of brainstem arousal functions but with persistent impairment of supratentorial networks implicated in consciousness [ ] . stimulation of the aras may improve consciousness in vegetative or mcs patients [ ] . in addition to deep brain stimulation, vagal nerve stimulation, which probably modulates the activity of the nucleus of the tractus solitarius and the dorsal raphe, has shown promising results [ ] . in addition to these classical syndromes, other consciousness impairments have been described. peduncular lesions can cause hallucinations [ ] which may be encountered in icu patients. more generally speaking, it is likely that brainstem dysfunctions account for a portion of the sleep-wake cycle impairments experienced by icu patients. brainstem lesions can induce cognitive deficits including impaired attention, naming ability, executive functioning, and memory impairment [ ] , ascribed to a disruption of interconnection between the frontal- subcortical system and the brainstem [ ] . finally, deep sedation is a pharmacologically induced coma, and its mechanisms of action involve the brainstem gaba and n-methyl-d-aspartate (nmda) receptors [ ] . assessments of consciousness are based on neurological examination to confirm the diagnosis, determine the underlying cause, and evaluate the prognosis. in clinical practice, this assessment most commonly relies on the glasgow coma scale (gcs) [ ] . focusing on the brainstem in particular, the four (full outline of un-responsiveness) score is to be preferred as it includes the corneal, pupil light, and cough reflexes and respiratory patterns [ ] . in comatose patients, pupil sizes and reactivity can be suggestive of particular etiologies, such as drug overdose (myosis for opioids or mydriasis for tricyclic anti-depressants). in comatose brain-injured patients, brainstem reflex assessment is crucial to detect a uncal or downward cerebellar (tonsillar) herniation [ ] . while the absence of corneal and pupillary light reflexes is strongly associated with poor outcome in post-anoxia, their prognostic value is less validated in other causes [ ] . patients with severe critical illness may be comatose due to sedation, which in clinical practice can be assessed using the rass (richmond agitation sedation scale) [ ] . in deeply sedated patients (i.e., rass − or − ), the brainstem reflexes assessment sedation scale (brass) might be useful to assess the effect of sedatives on the brainstem and potentially detect a brainstem dysfunction [ ] ( table ). the cam-icu and icdsc are appropriate to monitor delirium [ , ] . finally, in vs and mcs patients, the coma recovery scale-revised has also been validated [ ] . coma due to structural brainstem lesions is predominantly related to pedunculo-pontine tegmental lesions, usually detected on mri [ ] (table ) . neurophysiological tests may be useful to assess the neurological prognosis in patients with impairment of consciousness. somatosensory evoked potentials (ssep) assess conduction from peripheral nerves (n ) to the somatosensory cortical (n ) regions passing through the brainstem (p ). brainstem auditory evoked potentials (baep) are described in table [ ] . interestingly, sedation increases latencies and decreases amplitudes of evoked potentials in a dose-dependent manner but does probably not change the amplitudes with low to moderate doses used in icu [ ] . the intracranial conduction time and intrapontine conduction time are assessed by measures of the p -n inter-peak latency on ssep and the iii-v interpeak latency on baep [ ] . the prognostic value of baep has been explored in various causes of coma [ ] [ ] [ ] . after cardiac arrest, the predictive value of baep for poor outcomes is limited [ ] . however, in traumatic brain injury, preserved baep are associated with a good outcome [ ] . wave i can disappear if the auditory nerve is injured (traumatic or hypoxic injuries) [ ] . reactivity on eeg to auditory, visual, or nociceptive stimuli is important to assess after cardiac arrest because its absence is associated with poor outcome [ , ] . absent reactivity can result from a thalamus-brainstem loops and aras dysfunction [ ] [ ] [ ] [ ] . the electrophysiological measurement of the blink reflex (table ) is a way to study the trigemino-facial loop [ ] , but its prognostic value in comatose patients remains insufficiently supported [ ] . the ans plays a key role in homeostasis and allostasis by controlling vital functions and the immune system [ ] and is composed of sympathetic (e.g., noradrenergic) and parasympathetic (e.g., cholinergic) systems. sympathetic effects originate from the spinal cord (d to l ), while parasympathetic neuronal cell bodies are present in the nuclei of cranial nerves iii (edinger westphal nuclei), vii, ix, and x and the sacral spinal cord (s to s ). activation of the parasympathetic nervous system results in a decrease in heart rate (hr) and blood pressure (bp), and an increase in gastrointestinal tonus, vesical detrusor contraction, and myosis. activation of the sympathetic system results in opposite effects. cortical input can modulate responses in the ans [ ] as well as various receptors throughout the body, including the baroreceptors [ ] . brainstem injury may cause dysautonomic symptoms, which can be life-threatening [ ] (table ) . cardiac arrhythmias frequently occur after brainstem stroke and are associated with increased mortality [ ] . an intracranial hypertension-induced midbrain insult can impair parasympathetic control and thereby induce adrenergic storm. in brain death, there is a disappearance of the vasomotor tone and an impairment of myocardial contractility [ ] . as exhaustive discussions of tests that allow testing of the ans are beyond the scope of this review, we will focus on cardiovascular tests degenerative/atrophic injury mri magnetic resonance imaging, tdm tomodensitometry, csf cerebrospinal fluid, ecg electrocardiogram mri results according to etiologies: vascular injury: diffusion and flair-weighted sequence hyperintensity restricted to a vascular territory hemorrhage: swi/t * sequence hypointensity inflammatory: diffuse or multifocal white matter lesions on t -and flairweighted sequences, with or without contrast enhancement inflammatory nmo (mri of optical nerve and medullary mri): extensive and confluent myelitis on more than three vertebrae and optical neuritis with possible contrast enhancement traumatic injury: hyperintensity on diffusion sequence, diffuse axonal injuries on dti (diffusion tensor imaging) sequence, hemorrhage lesions on t */swi metabolic: t hyperintensity specifically involves the central pons infectious: abscess/nodes with contrast enhancement paraneoplastic: limbic encephalitis with temporal diffusion and flair hyperintensity tumor: mass with possible necrosis, contrast enhancement and oedema revealed by a flair hyperintensity around tumor degenerative injury: brain and brainstem atrophy (colibri sign) absence of grimacing to pain and absence of ocr absence of grimacing to pain and presence of ocr ocr: oculocephalic reflex brass is a clinical score that has been developed for scoring brainstem dysfunction in deeply sedated, non-brain-injured, mechanically ventilated, critically ill patients and ranges from to the brass has prognostic value, as -day mortality proportionally increases with the brass score applicable to icu patients. standard monitoring allows for the detection of variations in hr and bp that can be suggestive of dysautonomia. however, the lack of apparent changes in cardiovascular signals does not rule out dysautonomia, which can be then assessed with the hr and bp spectral analysis. high frequency (hf) band (i.e., . to . hz) variability of the hr is thought to predominantly reflect parasympathetic tone, while low frequency (lf) variability (i.e., . to . hz) is primarily mediated by sympathetic activity. the lf/hf ratio reflects the sympathovagal balance. therefore, spectral analysis allows studying the sympathetic, parasympathetic, and baroreflex activities both at rest and during stimulation [ ] . if the valsalva maneuver, the cold pressure test, and the pharmacological tests (with yohimbine or clonidine) allow testing the ans, their use in icu is very limited. conversely, pupillometry is much more applicable for assessing dysautonomia in icu. thus, patients with dysautonomia present a pupil dilatation at resting state and a slow redilatation time [ ] . there are two types of muscles that play a major role in the respiratory system, dilatator muscles of the superior airway that are innervated by the brainstem via cranial nerves (motor neurons present in the v, vii, and xii nuclei) and contractor/pump muscles (diaphragm, intercostal, sternocleidomastoid, abdominal muscles) that are innervated by spinal motor neurons. they are controlled by bulbospinal (automatic command) and corticospinal (voluntary command) pathways. the respiratory drive originates from neurons of the latero-rostro-ventral medulla oblongata, which includes the pre-botzinger complex and the parafacial respiratory group that control inspiration and expiration, respectively [ ] ( table ) . this center receives various inputs to automatically adjust the respiratory drive to metabolic and mechanic changes [ ] . metabolic inputs are mediated by both peripheral (aortic and carotid) and central (medulla oblongata and lc) chemoreceptors [ ] . the mechanical inputs are mediated by mechanoreceptors localized in the pulmonary parenchyma, bronchial wall, and muscle. at the level of the pons, the pedunculopontine tegmentum, the lc, the lateral parabrachial and kölliker-fuse nuclei are involved in the automatic respiratory control [ ] ( table ) . automatic and voluntary control of respiratory motor neurons can be injured together or separately. for instance, automatic control is impaired in central congenital and acquired hypoventilation syndrome (i.e., ondine syndrome), while voluntary control is preserved [ ] . acquired hypoventilation syndrome can result from brainstem tumoral, traumatic, ischemic, and inflammatory injuries [ ] , which implies the need for long-term mechanical ventilation. ventilator management may be significantly affected by brainstem lesions, and importantly, clinical features of neurological respiratory dysfunction are related to the localization of brainstem injury. the more caudal the lesion is, the more it is associated with an impairment of the respiratory drive. midbrain injuries do not usually affect the respiratory rate (rr). injuries to the upper pons increase the tidal volume and decrease the rr, while injuries of the lower pons are associated with respiratory asynchrony (e.g., ponto-peduncular injury). ataxic breathing (irregular pauses and apnea periods) and central apnea are observed in rostro-ventral medulla oblongata injuries and associated with poor outcomes. central neurogenic hyperventilation results from activation of the medullary respiratory center. finally, yawning or refractory hiccups may be seen with lesions of the posterolateral medulla oblongata [ ] . swallowing impairment contributes also to the difficulty of weaning mechanical ventilation and can be an indication for a tracheostomy. there are various structural and non-structural causes of neurological respiratory dysfunction, including infratentorial lesions, drug toxicity, heart failure, and sepsis [ ] [ ] [ ] . diagnosis relies on standard assessments of brainstem lesions can result in absent or delayed peaks iii and v, prolonged iii-v and i-v inter-peak latency, or a reduced i/v amplitude ratio (< . ) delay or absence of r indicates a facial /trigeminal nerve injury. r can be delayed in comatose patient and is also bilaterally delayed or absent in wallenberg's syndrome (with a r preserved) respiratory function (e.g., ventilator curves, tidal volumes (vt), and rr in mechanically ventilated patients) but also on assessing the ventilatory response to hypercapnia (e.g., during a t-piece trial). an electromyogram of the respiratory muscles, notably the diaphragm, provides relevant information on the central drive. this technique may be helpful in patients that are impossible to wean from mechanical ventilation. as a caveat, it may be at times difficult to differentiate central respiratory dysfunction from critical illness neuropathy/myopathy. emg and nerve conduction studies may help with the distinction, but limited assessments of every respiratory muscle group and available at highly specialized units limit this approach [ ] . in mechanically ventilated patients, spirography can be performed (with the vt/inspiration duration (ti) ratio reflects the ventilatory command intensity) as well as the occlusion pressure measurement (i.e., p . ). the latter reflects the "unconscious"/central respiratory command, but variability of its measurements limits routine application. the leading causes of primary brainstem dysfunction are summarized in table and major differential diagnosis of brainstem dysfunction in table . in the following section, we will discuss evidence for brainstem dysfunction encountered in critically ill patients beyond primary brainstem dysfunction. the "brainstem dysfunction" hypothesis originates from our study on usefulness of neurological examination in non-brain-injured critically ill patients who required deep sedation. these patients have usually a severe critical illness and therefore a higher risk to develop severe secondary brain insult [ , ] . furthermore, protracted deep sedation is still required in more than % of critically ill patients [ ] and has been reported to be associated with increased mortality [ ] . we found that assessment of brainstem reflexes was reproducible in this population [ , ] . we also found that routinely used sedative and analgesic agents such as midazolam and fentanyl do not impair pupillary light, corneal, and cough reflexes in % of cases but depress oculocephalic response and grimacing to painful stimulation (absent in and %, respectively) [ , , ] . the cessation of brainstem reflexes results from the combining effects of critical illness (i.e., secondary brain insult), sedative, and analgesic agents. it is interesting to note that guedel observed more than years ago that sedative drugs abolish brainstem reflexes according to a sequential pattern (the loss of consciousness, followed by the cessation of brainstem reflexes in a rostro-caudal way until apnea) [ ] . in deeply sedated non-brain-injured critically ill patients, the cessation of brainstem responses follows two distinct patterns. the first is characterized by a depression of whole brainstem responses (similar to guedel's description), and the second is characterized by a preferential impairment of the corneal reflex, the pupillary light reflex, and to a lesser extent the cough reflex, with paradoxical preservation of the oculocephalic response. the latter profile is associated with the severity of critical illness and the depth of sedation. interestingly, this pattern cannot be ascribed to a unique focal brainstem lesion which most likely relies on a functional rather than a structural origin. this suggests that some neuroanatomical centers are more sensitive to deep sedation, critical illness, or both. opioids might also contribute to brainstem dysfunction, as they depress the aras, respiratory centers, and brainstem reflexes (notably pupillary light and cough reflexes). however, morphine infusion rates did not differ in our study between the two cessation patterns of brainstem reflexes [ ] . to assess brainstem reactivity in deeply sedated critically ill patients, we developed the brass [ ] ( table ). the principle of the brass development is not in agreement with the traditional paradigm of jackson, which states that the brainstem reflexes are abolished in a rostro-caudal way. it thus differs from the four score [ ] , which conditions the assessment of the cough reflex to the cessation of the pupillary light and corneal reflexes. besides improving the prediction of mortality in deeply sedated patients, the assessment of brainstem reflexes, with help of either the brass or the four score, might prompt the icu physician to perform a brainstem imaging. it is however likely that the processes involved in critical illness-related brainstem dysfunction are radiologically assessable. neurophysiological tests provide further arguments for brainstem dysfunction in critically ill patients without primary brainstem injury. for instance, eeg is not reactive in % of patients with sepsis [ , ] , knowing that absence of reactivity can result from a dysfunction of the aras [ ] [ ] [ ] [ ] . middle latency baep responses and ssep latencies were increased in % and % of deeply sedated non-brain-injured critically ill patients, respectively [ ] , indicating an impairment of the brainstem conduction. interestingly, mean values of these latencies did not differ from those recorded in deeply sedated brain-injured patients. critical illness is also associated with decreased variability in hr and bp, with an impaired sympathetic tone and baroreflex [ , ] and also with a reduced tidal volume variability [ ] that can correlate with weaning failure. since most of these findings concerned sedated patients, one may argue that sedative agents might be involved as a revealing or aggravating underlying insults. this hypothesis is further supported by the fact that increase in evoked potential latencies cannot be only ascribe to sedation since long-term swallowing disorders [ ] and aspiration pneumonia are more frequent in sepsis survivors [ ] . thus, a multimodal assessment of brainstem dysfunction in critical illness is warranted. the undergoing multicenter proretro study (clinicaltrials.gov: nct ) aims to evaluate a multimodal approach based on neurological examination and neurophysiological tests. neuroimaging and neuropathological studies show that the brainstem is prone to vascular, inflammatory, and excitotoxic insults [ ] . for instance, sepsis can be associated with impaired autoregulation of cerebral blood flow and microcirculatory dysfunction, which may compromise the brainstem perfusion. second, a multifocal necrotizing leukoencephalopathy involving the brainstem can be secondary to an intense systemic inflammatory response [ ] . finally, the neuro-inflammatory process can culminate in neuronal apoptosis, which is evidenced in brainstem autonomic nuclei in patients who died from septic shock or in experimental sepsis [ ] . interestingly, it has been shown that apoptosis of autonomic nuclei can induce hypotension in septic rat [ ] . both humoral and neural pathways can induce a neuroinflammatory process. the former involves the area postrema (fig. ) , which allows the diffusion of circulating inflammatory mediators into the brainstem; the latter involves mainly the vagal nerve, which mediates the transmission of peripheral inflammatory signals to the brainstem [ , ] . autonomic brainstem nuclei are regulated by these two pathways, which then play a major role in the control of systemic inflammatory response. finally, metabolic processes can be involved. it is well known that electrolyte disturbances but also renal and liver failure impair brainstem responses, as illustrated by centro-pontine myelinolysis or by usefulness of four score in hepatic encephalopathy [ ] . the predictive value of the neurological examination findings and neurophysiological responses has been assessed in critically ill patients. there is a proportional relationship between the brass value and mortality. interestingly, absence of a grimacing response associated with preserved oculocephalic responses is the most predictive of mortality [ ] , suggesting that prediction is better when first based on a combination of signs, and second, a decoupling process between the upper and lower part of the brainstem is involved [ ] . the absence of eeg reactivity and of ssep p response and increased p -n ssep latencies are associated with increased mortality [ , , ] . impaired hr variability and decreased sympathetic control are associated with mortality and organ failure [ ] . there are arguments for a relationship between delirium and brainstem dysfunction. the drugs currently used for treating delirium are involving brainstem receptors. thus, neuroleptics are antagonists of the dopamine d and serotoninergic ht a receptors that are prevalent in the brainstem [ ] . dexmedetomidine is a selective agonist of alpha- receptor, notably at the level of the lc [ ] . the role of the brainstem in patients with delirium is supported by these pharmacological data and further supported by neuropathological findings that demonstrate hypoxic and ischemic insults of the pons in delirious patients [ ] . absent oculocephalic responses and delayed middle-latency baep have been associated with delayed awakening or delirium after sedation discontinuation [ ] . in neuroanatomical point of view, it is likely that cessation of the oculocephalic response reflects a dysfunction of the aras while cessation of the cough reflex reflects a dysfunction of the cardiovascular and respiratory autonomic nuclei. finally, if conceivable, we do not know to what extent brainstem dysfunction contributes to long-term post-icu mortality and functional disability. another contributing factor of the brainstem dysfunction in critical illness course might be the impaired sympatho-vagal control of the inflammatory response. the vagus nerve first senses and modulates peripheral inflammation, constituting the so-called cholinergic reflex [ ] ; second, it senses the microbiota metabolites, being a major component of the gut-brain axis [ ] ( table ). the adrenergic system controls the immune system, with alpha and beta- receptors being proinflammatory and beta- receptors anti-inflammatory [ ] . it is therefore conceivable that a brainstem-related neuro-immune impairment can contribute to infection, organ failure, or death by facilitating a maladapted immune response. the modulation of the cholinergic reflex by α nachr agonists and by vagal nerve stimulation has been proposed in sepsis and critical illness to improve peripheral immune response and reduce organ dysfunction [ ] . in addition to its peripheral immune effects, cholinergic modulation and vagal stimulation can promote anti-inflammatory microglial polarization [ ] . however, we shall remind that rivastigmine, a cholinesterase inhibitor, is deleterious in critically ill patients. vagal nerve stimulation is also proposed in refractory status epilepticus [ ] and consciousness disorders [ ] , suggesting its potential but not yet demonstrated effect in critical illness-related encephalopathy. beta-blockers reduce the mortality in cardiac diseases by attenuating the deleterious effects of sympathetic hyperactivation and increasing the vagal tone [ ] . in sepsis, betablockers improve hr control, reduce systemic inflammation, and decrease mortality, acknowledging that their routine use is not yet warranted [ , ] . brainstem dysfunction can present with central sensory and motor deficits, cranial nerve palsies and 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medicine. vasc health risk manag systematic review of use of β-blockers in sepsis the effect of beta-adrenergic blockade on inflammatory and cardiovascular responses to acute mental stress publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the reviewers for their comments and suggestions to improve our manuscript. authors' contributions sb, am, br, and ea drafted the manuscript. jc, crs, vb, and ts critically revised the manuscript for important intellectual content. all authors read and approved the final manuscript. availability of data and materials not applicable ethics approval and consent to participate key: cord- -sl kredk authors: jiménez-ruiz, ernesto; hassanzadeh, oktie; efthymiou, vasilis; chen, jiaoyan; srinivas, kavitha title: semtab : resources to benchmark tabular data to knowledge graph matching systems date: - - journal: the semantic web doi: . / - - - - _ sha: doc_id: cord_uid: sl kredk tabular data to knowledge graph matching is the process of assigning semantic tags from knowledge graphs (e.g., wikidata or dbpedia) to the elements of a table. this task is a challenging problem for various reasons, including the lack of metadata (e.g., table and column names), the noisiness, heterogeneity, incompleteness and ambiguity in the data. the results of this task provide significant insights about potentially highly valuable tabular data, as recent works have shown, enabling a new family of data analytics and data science applications. despite significant amount of work on various flavors of this problem, there is a lack of a common framework to conduct a systematic evaluation of state-of-the-art systems. the creation of the semantic web challenge on tabular data to knowledge graph matching (semtab) aims at filling this gap. in this paper, we report about the datasets, infrastructure and lessons learned from the first edition of the semtab challenge. tabular data in the form of csv files is the common input format in a data analytics pipeline. however, a lack of understanding of the semantic structure and meaning of the content may hinder the data analytics process. thus, gaining this semantic understanding will be very valuable for data integration, data cleaning, data mining, machine learning and knowledge discovery tasks. for example, understanding what the data is can help assess what sorts of transformation are appropriate on the data. tables on the web may also be the source of highly valuable data. the addition of semantic information to web tables may enhance a wide range of applications, such as web search, question answering, and knowledge base construction. tabular data to knowledge graph (kg) matching is the process of assigning semantic tags from kgs (e.g., wikidata or dbpedia) to the elements of the table. this task however is often difficult in practice due to metadata (e.g., table and column names) being missing, noisy, incomplete or ambiguous. there exist several systems that address the tabular data to kg matching problem (e.g., [ , , ] ) and use state-of-the-art datasets with ground truths (e.g., [ , , ] ) or custom datasets. however, there does not exist a common framework to conduct a systematic evaluation of these systems, which leads to experimental results that are not easy to compare as they use different notions for true/false positives and performance measures. furthermore, available datasets are either small in size (e.g., [ , ] ) or low in quality and messy (e.g., [ ] ). the creation of the semantic web challenge on tabular data to knowledge graph matching (semtab) [ ] aims at filling this gap. the main contributions of this paper are summarized as follows: (i) we introduce an automated method for generating benchmark datasets for tabular data to kg matching. (ii) we release generated benchmark datasets (see zenodo repository [ ] ), and the code for evaluating the systems results (see github repository [ ] ). (iii) we report and analyze the results of the systems that participated in the first edition of the semtab challenge, using our benchmark datasets. the rest of the paper is organized as follows. section introduces the matching problems and its challenges. in sect. , we discuss related initiatives. the automatic dataset generator is described in sect. . section presents the semtab evaluation. finally, sect. provides the lessons learned and experiences from the semtab challenge and points to future lines. in this section, we provide some basic definitions about kgs and tabular data. we also introduce the selected matching tasks and their associated challenges. knowledge graph (kg). we consider rdf-based kgs which are represented as a set of rdf triples s, p, o , where s represents a subject (a class or an instance), p represents a predicate (a property) and o represents an object (a class, an instance or a data value, e.g., text, date and number). rdf entities (i.e., classes, properties and instances) are represented by uniform resource identifiers (uris). a kg consists of a terminological component (tbox) and an assertion component (abox). the tbox is often composed of rdf schema constructs like class subsumption (e.g., dbo:scientist rdfs:subclassof dbo:person) and property domains (e.g., dbo:doctoraladvisor rdfs:domain dbo:scientist). the abox contains relationships among entities and semantic type definitions (e.g., dbr:albert einstein rdf:type dbo:scientist). an owl ontology associated to the kg may provide more expressive constructors without a direct translation into triples, which will contribute to the inference of new triples via logical reasoning. a kg can typically be accessed via a sparql endpoint and via fuzzy matching based on an index of the lexical information associated to the kg entities. the latter is often referred to as kg lookup (e.g., spotlight for dbpedia [ ] and opentapioca for wikidata [ ] ). we assume that all columns and rows have the same size, with possibly cells with empty values. in arbitrary tabular data, unlike in relational tables, column names and row identifiers (i.e., primary keys) may be missing. in web tables and relational tables, rows typically characterize an entity, while in arbitrary tabular data (e.g., typical csv files in data science) there may not be a leading entity in each row (see for example table b) . matching tasks. we have selected the following tasks for the semantic annotation of tabular data: (i) column-type annotation (cta), (ii) cell-entity annotation (cea), and (iii) columns-property annotation (cpa). these matching tasks can be seen as subtasks that can serve the larger purpose of matching an entire table to a class, or matching a row of a table to an entity. the cta task expects the prediction of the semantic types (i.e., kg classes) for every given table column c k in a table t , i.e., ct a(t, c k , kg) = {st , . . . , st a }. the cea task requires the prediction of the entity or entities (i.e., instances) that a cell t i,j ∈ t represents, i.e., cea(t, t i,j , kg) = {e , . . . , e b }. finally, the cpa task expects as output a set of kg properties that represent the relationship between the elements of the input columns c k and c l , i.e., cp a(t, c k , c l , kg) = {p , . . . , p c }. note that cta (resp. cea) task focuses on categorical columns (resp. cells) that can be represented with a kg class (resp. kg entity). some numerical values may also represent entities if they play a foreign key role, but this would involve a different data wrangling task not considered in this work. challenges. the above matching tasks are challenging for various reasons including but not limited to: (i) lack of metadata or uninformative table and column names, a typical scenario in web tables and real-world tabular data. (ii) noisiness in the data (e.g., "virgen" in table b ). (iii) knowledge gap, cells without a correspondence to the kg (e.g., oxford start-ups in table c ). (iv) ambiguous cells pointing to more than one possible entity (e.g., "congo" in table a or "virgin" and "orange" in table b ). (v) missing data (i.e., cells without a value) increasing the effect of the knowledge gap (e.g., capital of "brazil" in table a ). (vi) short labels or acronyms, which typically bring more ambiguity to kg matching (e.g., "bt" in table b ). [ ] . imdb and musicbrainz are other popular benchmarks. imdb contains over , tables from imdb movie web pages, and musicbrainz contains some , tables from musicbrainz web pages [ ] . the entity mention cells are annotated with freebase topics. viznet [ ] contains million datasets mined from open data repositories and visualization data repositories. although viznet was initially derived for use in visualizations, it has been used in the context of column-to-type matching (cta task) of tables in a system called sherlock [ ] . sherlock provides a total of , crowdsourced annotations from human participants. however the annotations are not publicly available yet. numdb [ ] is a dataset of tables generated from dbpedia where the primary emphasis is on creating tables for identifying numerical columns. it allows the varying of the size of the table, as well as injection of different degrees of noise in the data, particularly in the textual data that can be used to match 'key' columns to test the robustness of any numerical matching approach. although these benchmarks are widely used in recent studies, they still suffer from a few shortcomings: (i) some benchmarks like limaye and t dv are quite small, with only limited contents; (ii) those large benchmarks like efthymiou are often in short of high quality ground truths, especially when all the three tasks need to be evaluated at the same time; (iii) large benchmarks often have a large number of rows but simple relations and contents (classes); (iv) most benchmarks have ground truth annotations from only one kg. meanwhile, using a fixed benchmark limits the evaluation of some special cases, such as the big knowledge gap when a large part of cells have no entity correspondences, while a system for generating benchmarks with an ad-hoc configuration enables researchers to evaluate the performance in face of these special cases. our efforts target this lacuna in benchmarks. benchmarks have been also developed for the related task of ontology matching, which is a well studied problem [ , ] . our benchmarking effort was inspired by the yearly ontology alignment evaluation initiative (oaei). the main difference between our benchmarks and the oaei benchmarks is the level of heterogeneity involved in the two data sources to be matched. instead of two semantically rich ontologies, as those in the oaei benchmarks, we consider one rich ontology corresponding to the kg, and one typically shallow table in terms of semantics. this heterogeneity creates an additional challenge, which ontology matching tools were not originally designed to cope with, but we believe that those tools can also benefit from our benchmarks. therefore, we also provide our benchmark data in rdf format and experiment with publicly available ontology matching tools (e.g., logmap [ ] ), to better evaluate their potential strengths and weaknesses from a different perspective than oaei (cf. sect. . ). to overcome the limitations of the existing benchmark datasets, and to create new benchmark datasets for each round of the challenge without extensive human annotation, we designed an automated data generator that creates tabular data given a sparql endpoint. the idea is to create tabular data similar to tables found on the web, but ensure a reasonable diversity in terms of size and coverage of classes and properties from various domains. in what follows, we describe each of the steps in the data generation pipeline summarized in fig. . although we used the english dbpedia as our source for this edition of the challenge, given that most state-of-the-art systems and the most widely used benchmarks use dbpedia mappings, our goal was to design a generic method of creating benchmark data that can go beyond dbpedia annotations. this way, dbpedia can be replaced with e.g. wikidata, or a domain-specific kg. we can also switch to other languages or create a multilingual collection. given this goal, the first step in data generation is a profiling step in which the list of classes, properties, and some basic statistics are extracted. the output of the profiling step is: ) a list of classes along with the number of instances per class; ) a list of properties for each class along with: (i) the number of instances that have a value for the property; (ii) the datatype for datatype properties and the range class for object properties. this information will be used in the next step to construct sparql queries. although our current profiling is simple, performing the necessary sparql queries over existing rdf stores could still be slow, and so a raw processing of rdf dumps may be required. another option is to use a profiling tool such as loupe [ ] . for table generation with numeric columns, refer to [ ] . in this step, we go through the list of classes from the output of the profiling, and generate a set of sparql queries for each class. this way, each table will have one class as the main topic with each row containing values from the properties of an instance of the class (or its subclasses, if any). in order to pick a set of properties for each class to turn into a set of columns in the output table, we use a simple randomized method. we use the gathered statistics only to avoid properties with very few instance values that could in turn result in sparql queries with empty or very small result set. for each class, we randomly select a number of properties within a predefined range. for the tables generated for the challenge, we select a minimum of and a maximum of columns for each table. we then create a query to retrieve the (primary) label of the instance along with labels of object properties and values of data type properties. when multiple values are present, we only select a single value for the corresponding cell. we also ensure in the query that the type of the object property matches the expected range in the ontology (if any) since, particularly in dbpedia, there might be objects of various types as property values of the same property. finally, we need to ensure a diversity of classes in the output and a balanced collection in terms of table size so that we avoid very small tables, and larger classes (e.g. person in dbpedia) do not end up dominating the collection. for small query result sets (less than rows for this edition), we drop the query and try selecting a new random subset and repeat the process until all properties are included or no new tables can be generated. to deal with larger classes, we break larger query results into randomly sized subsets, and ensure that we do not have more than a fixed number ( for this edition) of tables for the same query, and no more than a fixed number ( , for this edition) of rows across the collection for a given class. the final outcome is a collection of sparql queries, each resulting in tabular data with (i) columns that can be annotated with the expected type (class for the case of object properties), (ii) cell values that can be annotated with instance uris, and (iii) pairs of columns that can be annotated with a property. the outcome of the previous step is a collection of tables with all their contents completely based on values in the source (english dbpedia for this edition) which is somewhat unrealistic as real tables often have noise as well as columns/rows/values that cannot be matched with our knowledge source. for this edition, we implemented only a few simple refinement strategies to make the tables more realistic and so the matching task more difficult. we plan to significantly improve this refinement step to create more realistic collections and also collections geared towards particular features, e.g., the ability to handle certain kinds noise or the so-called "nil detection". the first simple refinement step includes adjusting some label values in a rule-based approach. for this edition, we do this only for person entities, by abbreviating first names. it is possible to do this string value manipulation based on introducing errors (e.g. typos, using the method used in the uis data generator [ ] ) or using sources of synonym terms and alternative labels. to further make the matching tasks more challenging, we have used another refinement process which is applied over a number of automatically generated collections (which differ due to the random creation of sparql queries described above). the goal of this refinement is to retain only a subset of benchmark tables from the generated collections, after discarding fairly easy matching cases. this process can be further divided into three sub-processes: (i) identifying tables in which the matching tasks is more challenging, (ii) identifying rows in a challenging table that are still fairly easy to match (cea task), and (iii) adapting the benchmark tables and the ground truths accordingly. for sub-process (i), we use the so-called refined lookup approach [ ] to identify more challenging tables. in summary, this two-step approach first looks up the contents of each table cell in a kb index, and for each top-ranked result, it stores its rdf:type. in the second step, it performs the same lookup operation, but this time, it restricts the results to only those belonging to the most frequent types per column, as retrieved from the first step. despite its simplicity, this approach provided decent effectiveness results compared to more sophisticated methods. we set an empirical threshold for f -score ( . ), and we report all the tables for which the simple lookup method returns an f -score lower than the threshold. the tables in the final benchmark dataset will only consist of tables that are reported in this step, i.e., easier tables are ignored. for sub-process (ii), we scan in depth the error logs of the previous subprocess, in which we report how many wrong results were reported per row and per column in a table. we remove the rows for which the simple baseline method provided only correct results ( errors), as long as the pruned table has more than rows. finally, for sub-process (iii), we adapt the ground truth files to reflect the refinement step. we first remove all the information about tables that were entirely discarded, and for the remaining tables, we adapt the row numbering to reflect the changes made in sub-process (ii). in order to allow ontology matching tools to use our benchmark datasets, we also provide our datasets in rdf format, as described by a simple owl ontology that we generate automatically from the tables [ ] . note that this process is currently only applicable when column headers are available in a table. in summary, we assume that each table corresponds to an owl class, with each row being an instance of this class. the table columns correspond to either data type or object properties, which have as domain the class corresponding to the table. we detect a special label column (using heuristics, as in [ , ] ), which we use as the rdfs:label property. based on the values of each column we define the range of each data property (e.g., xsd:integer, xsd:date, xsd:string) and object property. in the case of object properties, the range class is defined as a new class, named after the header of the corresponding column. this way, the values for the columns that describe object properties are treated as instances of the owl class, which is the range of this column. in the example of table a , assume that we have an additional row at the beginning, with the values: "country", "capital". in that example, we would create an owl ontology with the classes country and capital, and the object property hascapital. the owl class describing the table would be country, and this class would also be the domain of all the properties (in this case only hascapital ). the range of hascapital would be the class capital. finally, each row in the table corresponds to an instance of a country, with the rdfs:label of each instance defined from the value of the country column (which is determined as the label column). for example, the rdf triples generated for the first row would be: :china rdf:type :country, :china rdfs:label "china", :china :hascapital :beijing, and :beijing rdf:type :capital. the edition of the semtab challenge was collocated with the th international semantic web conference as a semantic web challenge and with the th ontology matching workshop as a special oaei evaluation track. the semtab challenge started in mid april and closed in mid october . it was organised into four evaluation rounds where we aimed at testing different datasets with increasing difficulty. evaluation framework. we relied on aicrowd as the platform to manage the semtab challenge tasks: cta, cea and cpa. aicrowd provides a number of useful functionalities such as challenge presentation, participant registration, automatic evaluation, ranking, submission limitation, and so on. for the (automatic) evaluation, an aicrowd python code template was provided, according to which the semtab evaluation interface and metrics were implemented and deployed [ ] . datasets and rounds. table provides a summary of the statistics of the datasets. for example, round dataset was composed of , tables; there were , target cells in cea, , target columns in cta, and , target column pairs in cpa. round was based on the t dv dataset [ ] and served as sandbox for the participating systems. as t dv provides only class annotations at table level, for cta, we extended the annotation of types for the other (entity) columns. we also manually revised the original and the new column types. round dataset was composed of (i) , relatively clean tables from the wikipedia tables presented in [ ] (i.e., not including tables with multiple column/row span, and large textual cell contents as in [ ] , and (ii) an automatically generated dataset of , tables as described in sect. . rounds and were composed of an automatically generated dataset with enhanced characteristics and a focus on non-trivial annotations. the ground truth for all four rounds was based on dbpedia. in this edition of the challenge, the ground truth was blind during the competition; but the target cells, columns and column pairs were provided to the participants. participants executed the matching tasks as defined in sect. for each of the given target table elements. the solutions for the cea task were expected in a file with lines having these fields: " table id" , "column id", "row id" and "dbpedia entity (only one)" (e.g., "table ", " ", " ", "dbr:norway"). similarly, cpa solutions had the following fields per line: " table id ", "head column id", "tail column id" and "dbpedia property (only one)" (e.g., "table ", " ", " ", "dbo:releasedate"). for cta, more than one type annotations, separated by a space, were accepted: ' table id" , "column id" and "dbpedia classes ( or more)" (e.g., "table ", " ", " ", "dbo:country dbo:place"). note that those annotations outside the targets were ignored. multiple annotations to one target cell or column pair, and multiple lines associated to the same target element returned an error. submission and schedule. participants had to submit their solutions for the three matching tasks via the aicrowd platform. the performance scores were automatically computed and systems were publicly ranked in the aicrowd webpages. in rounds and , the number of submissions was unlimited so that participants could fine-tune their systems. the number of submissions per day was limited in rounds and to avoid the effect of over-tuning. table shows the opening and closing dates for each round. the objective of round and the limited time also aimed at identifying potential over-tuning in the participating systems. evaluation metrics for cea and cpa. for cea and cpa, we compute precision p , recall r and f score (primary score) as follows: where target annotations refer to the target cells for cea and the target column pairs for cpa. note that it is possible that one target cell or column pair has multiple ground truths, as modern kgs often have duplicate components. one example is the wiki page redirected entities in dbpedia. an annotation is regarded as true if it is equal to one of the ground truths. the comparison for equality is case insensitive. recall that at most one annotation was submitted for each target cell or column pair. evaluation metrics for cta. for cta we used a different set of metrics to take into account the taxonomy (hierarchy) of classes in the kg, namely average hierarchical score (ah) and average perfect score (ap ): t denotes all the columns for annotation. we refer as perfect annotations (p ) the most fine-grained classes in the (ontology) hierarchy that also appear in the ground truth, while annotations involving the super-classes (excluding very generic top classes like owl:thing) of perfect classes are referred to as okay annotations (o). other annotations not in the ground truths are considered as wrong (w ). ah gives a full score to the perfect annotation, a half score to the okay annotations, and a negative score to wrong class annotation. ah is used as the primary score as it considers both correct and wrong annotations, while ap is used as secondary score as it only considers the rate of perfect annotations. table shows the participation per round. we had a total of systems participating in round . round had a reduction of participating systems (from to ), which helped us identify the core systems and groups actively working in tabular data to kg matching. round and round preserved the core participants across rounds and all three tasks. it is worth mentioning that logmap [ ] , a pure ontology alignment system, participated in round . logmap was given as input (i) the tabular data in rdf format as described in sect. . , and (ii) a relevant portion of the dbpedia kg. the obtained results were reasonable, but far from the specialised system in the challenge. this is expected as systems like logmap rely on the semantics of the input ontologies or kgs, which is missing in the input tabular data. next, we provide a brief description of the core participants, who also submitted a system paper to the challenge. mtab [ ] . mtab is a system that can jointly deal with the three tasks cta, cea and cpa. it is based on the joint probability distribution of multiple table to kg matching, following the probabilistic graph model by [ ] . however, the team improves the matching by using multiple services including dbpedia lookup, dbpedia endpoint, wikipedia and wikidata, as well as a cross-lingual matching strategy. idlab [ ] . the idlab team developed an iterative matching procedure named csv kg with the following steps: (i) gets crude entity matching with cells; (ii) determines the column types and column relations with these entities; (iii) corrects the cell to entity matching with the column types and column relations; (iv) corrects the remaining cells with the head cells; and (v) calculates the column type again with all the corrected cell to entity matching. adog [ ] . this systems utilizes a nosql database named arangodb to load dbpedia and index its components. adog then matches tabular data with the entities of dbpedia using levenshtein distance, a string similarity metric. tabularisi [ ] . the team developed a system with two steps: candidate generation and selection. the former uses the wikidata api and a search index based on dbpedia labels to obtain a list of entities for each cell, while the later scores the candidates with lexical features which are based on lexical similarity metrics, and semantic features which capture the cell coherence of each column. dagobah [ ] . this participant system proposes an embedding approach which assumes that entities in the same column should be closed in the embedding space. it gets candidate entities by kg lookup, and uses pre-trained wikidata embeddings for entity clustering and cluster type scoring. the challenge of this method lies in the setting of hyper parameters such as the cluster number. team sti [ ] . this team developed a tool named mantistable that can automatically annotate, manage and make the semantics of tables accessible to humans and machines. the tool has some built in functions for the three matching tasks, including a sparql query for entity matching, a relation annotator based on maximum frequency and a class annotator based on voting by entities. note that this system also provides a web interface for manual annotation. lod all [ ] . this system implements a pipeline for the three tasks with five steps: (i) extracts ranked candidate entities of cells with direct search by ask sparql queries and keywords; (ii) gets the type of each entity; (iii) determines the type of each column with a weighted combination of ratio score and a normalized class score; (iv) determines the entity of each cell with the type constraint; and (v) extracts the relation of entities in each row and select the inter-column relation by frequency. in this section, we report the results of the challenge rounds - for the systems participating in at least two rounds, which include the above core participants and a system called saggu that only participated in cea. complete evaluation results are available from the challenge website [ ] . the results for all three matching tasks are presented in fig. . mtab and idlab were the clear dominants in all three tasks. tabularisi was in a clear overall rd position in cta and cpa. the overall rd position in cea was shared among tabularisi and adog. special mention requires team sti which had an outstanding performance in round of cea. in terms of average scores, round was the most challenging one, although it is not comparable to rounds and as it includes a different source dataset. rounds and completely rely on the dataset generator. round aimed at being more challenging than round by only including non-trivial cases. this was partially achieved for cea, with the exception of mtab and team sti. the relative performance of systems across rounds is similar in cea and cta with the exception of team sti in cea, where there is an important improvement in round , and lod all that decreased performance in round of cta. according to the results, complementing dbpedia with additional resources like wikidata (e.g., mtab and tabularisi) brings an important value. in general, the use of elaborated lexical techniques seems to be the key for a good performance. other approaches based on more sophisticated methods like semantic embeddings (e.g., dagobah) do not seem to bring the expected value to the final performance, but they may suffer a lighter impact with respect to changes in the datasets and the kg. another factor that may impact their performance is the long time spent for learning or fine tuning the embeddings of a large kg like wikidata and dbpedia. sponsorship and awards. sirius and ibm research sponsored the prizes for the challenge. this sponsorship was important not only for the challenge awards, but also because it shows a strong interest from industry. figure d shows the average ranking of the participating systems in each task. mtab, idlab and tabularisi obtained the st, nd and rd prize, respectively, across the three matching task. adog shared the rd prize in cea with tabularisi. finally, team sti obtained the outstanding improvement prize in cea. in this paper, we have presented the datasets and the results of the first edition of the semtab challenge. the experience has been successful and has served to start creating a community interested in the semantic enrichment of tabular data. both from the organization side and the participation side, we aim at preparing a new edition of the semtab challenge in . next, we summarize the issues we encountered during the different evaluation rounds, the lessons learned, and some ideas for the future editions of the challenge. importance of the challenge. we received very positive feedback from the participants with respect to the necessity of a challenge like semtab to conduct a systematic evaluation of their systems. our challenge was also well-received from industry via the sponsorship of ibm research and sirius. minor issues. we faced a few minor issues during the evaluation rounds, which will help us improve the future editions of the challenge. next, we summarise some of them: (i) explicit reference to the version of the kg used; (ii) incompatible encodings when merging different datasets; (iii) low quality of the dbpedia wikiredirects; (iv) wikipedia disambiguation pages as annotations; (v) property hierarchy was not considered; (vi) the average hierarchical score (ah) was not easy to interpret for participants as, in the way it is currently defined, it does not have a clear upper bound. nevertheless, we believe these issues affected all participants in a similar way and they did not have an important impact in the relative comparison among systems. evaluation platform. on the one hand, the aicrowd platform makes the management of submissions, evaluation and ranking very easy. on the other hand, it has no interface for automatic deployment of the evaluation codes and data, which makes it inconvenient to deal with online errors or changes, as challenge organisers depend on the aicrowd team. it was also hard to communicate with participants not using the aicrowd discussion forum. for next editions, we may consider alternative solutions. the limitation of number of submission per day was not welcomed by all participants. however, we find that unlimited submissions may lead to over-tuning the matching model that will have limited generalization performance. in future editions, we will try to better split the datasets for finetuning from the ones for testing. instance matching. we produced an rdf version of the dataset in round , but we did not attract the expected attention in the oaei community and the participation of (ontology) instance matching or link discovery systems was limited to logmap. in future editions of the challenge, we aim at facilitating the participation of oaei systems. real-world datasets. several participants highlighted the necessity of more realistic datasets, however manually annotated datasets are limited in quantity and size. a possible solution is to create a consensual ground truth by combining the output of several systems. this solution has already been used in several evaluation tracks of the oaei campaign [ ] . reproducibility. as semtab was the first edition of the challenge, our priority was to facilitate participation and allow participants to directly submit their solutions for each matching task. this plays a negative role in terms of reproducibility of the results. in future editions, we are considering to require from participants (i) the submission of a running system as in the oaei campaign, or (ii) the publication of their system as a (web) service. matching targets. in semtab we advocated to provide this information to the users to make the matching and the evaluation easier. in future editions we may hide this information to the participants. participants will have less guidance which will especially be reflected in the cpa task. evaluation will also be more challenging as incompleteness of the ground truth should not penalize potentially correct predictions. improved data generator. as outlined in sect. , there are a number of ways to improve our data generator to create more realistic datasets. in particular, much work needs to be done in creating tables that are more challenging to match, and contain more variety of representations and contents that cannot be matched to the source kg. also, our data generator has a number of parameters which can be adjusted to create different benchmarks each suitable for a different use case. we intend to work on these extensions, create more diverse and realistic collections, and make our data generator publicly available which will allow us to seek contributions from the community. results of the ontology alignment evaluation initiative dagobah: an end-to-end context-free tabular data semantic annotation system aicrowd evaluation codes learning semantic annotations for tabular data colnet: embedding the semantics of web tables for column type prediction mantistable: an automatic approach for the semantic table interpretation opentapioca: lightweight entity linking for wikidata matching web tables with knowledge base entities: from entity lookups to entity embeddings annotating web tables through ontology matching ontology matching benchmarks: generation, stability, and discriminability ontology matching semantic web challenge on tabular data to knowledge graph matching semtab : semantic web challenge on tabular data to knowledge graph matching - data sets the merge/purge problem for large databases viznet: towards a large-scale visualization learning and benchmarking repository sherlock: a deep learning approach to semantic data type detection logmap: logic-based and scalable ontology matching making sense of numerical data -semantic labelling of web tables a large public corpus of web tables containing time and context metadata annotating and searching web tables using entities, types and relationships dbpedia spotlight: shedding light on the web of documents loupe -an online tool for inspecting datasets in the linked data cloud semantic table interpretation using lod all mtab: matching tabular data to knowledge graph using probability models adog -anotating data with ontologies and graphs matching html tables to dbpedia csv kg: transforming tabular data into semantic knowledge entity linking to knowledge graphs to infer column types and properties effective and efficient semantic table interpretation using tableminer+ we would like to thank the challenge participants, the iswc & om organisers, the aicrowd team, and our sponsors (sirius and ibm research) that played a key role in the success of semtab. this work was also supported by the aida project (alan turing institute), the sirius centre for scalable data access (research council of norway), samsung research uk, siemens ag, and the epsrc projects analog, oasis and uk fires. key: cord- -fbe bgi authors: russo, alice g; kelly, andrew g; enosi tuipulotu, daniel; tanaka, mark m; white, peter a title: novel insights into endogenous rna viral elements in ixodes scapularis and other arbovirus vector genomes date: - - journal: virus evol doi: . /ve/vez sha: doc_id: cord_uid: fbe bgi many emerging arboviruses are not transmitted by traditional mosquito vectors, but by lesser-studied arthropods such as ticks, midges, and sand flies. small rna (srna) silencing pathways are the main antiviral defence mechanism for arthropods, which lack adaptive immunity. non-retroviral integrated rna virus sequences (nirvs) are one potential source of srnas which comprise these pathways. nirvs are remnants of past germline rna viral infections, where viral cdna integrates into the host genome and is vertically transmitted. in aedes mosquitoes, nirvs are widespread and produce piwi-interacting rnas (pirnas). these are hypothesised to target incoming viral transcripts to modulate viral titre, perhaps rendering the organism a more efficient arbovirus vector. to explore the nirvs landscape in alternative arbovirus vectors, we validated the nirvs landscape in aedes spp. and then identified novel nirvs in six medically relevant arthropods and also in drosophila melanogaster. we identified novel nirvs in phlebotomus papatasi, culicoides sonorensis, rhipicephalus microplus, anopheles gambiae, culex quinquefasciatus, and ixodes scapularis. due to their unexpected abundance, we further characterised nirvs in the blacklegged tick i. scapularis (n = ). interestingly, nirvs are not enriched in r. microplus, another hard tick, suggesting this is an ixodes-specific adaptation. i. scapularis nirvs are enriched in bunya- and orthomyxo-like sequences, reflecting that ticks are a dominant host for these virus groups. unlike in mosquitoes, i. scapularis nirvs are more commonly derived from the non-structural region (replicase) of negative-sense viruses, as opposed to structural regions (e.g. glycoprotein). like other arthropods, i. scapularis nirvs preferentially integrate into genomic pirna clusters, and serve as a template for primary pirna production in the commonly used embryonic i. scapularis ise cell line. interestingly, we identified a two-fold enrichment of non-long terminal repeat (non-ltr) retrotransposons, in genomic proximity to nirvs, contrasting with studeis in ae. aegypti, where ltr retrotransposons are instead associated with nirvs formation. we characterised nirvs phylogeny and integration patterns in the important vector, i. scapularis, revealing they are distinct from those in aedes spp. future studies will explore the possible antiviral mechanism conferred by nirvs to i. scapularis,which may help the transmission of pathogenic arboviruses. finally, this study explored nirvs as an untapped wealth of viral diversity in arthropods. over million people contract arthropod-borne viruses (arboviruses) each year (who ) . many arboviruses are classified as emerging diseases, meaning their incidence is rapidly increasing; additionally, a large proportion lack preventative measures or treatment (jones et al. ; labeaud, bashir, and v c the author(s) . published by oxford university press. this is an open access article distributed under the terms of the creative commons attribution non-commercial license (http://creativecommons.org/ licenses/by-nc/ . /), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. for commercial re-use, please contact journals.permissions@oup.com king ) . consequently, managing these diseases is essential to limit the morbidity and mortality they cause in human and animal populations. two classes of arthropods transmit arboviruses to mammals: insects and arachnids (mellor ) . all insect vectors are classified into the order diptera (true flies). of these, mosquitoes (culicidae) within the genera aedes and culex are the most significant arbovirus vectors, transmitting, e.g. flaviviruses (e.g. dengue virus (denv), yellow fever virus (yfv) and zika virus (zikv)) and togaviruses (e.g. chikungunya virus (chikv) and ross river virus (rrv)) ( table ) . collectively, these viruses cause over million human infections per year and over , deaths (labeaud, bashir, and king ; bhatt et al. ; who ) . sand flies (psychodidae) and midges (ceratopogonidae) also transmit pathogenic viruses of humans and livestock, mostly in southern europe (table ) (mellor, boorman, and baylis ; depaquit et al. ) . of the arachnids, ticks (order ixodida) are the only group known to transmit arboviruses and are second only to mosquitoes in the diversity of viruses they can transmit (table ) (mansfield et al. ). since , several emerging tick-borne viruses have caused human outbreaks in europe, china, north america, india, and the middle east (mansfield et al. ) . compared with mosquitoes, tick-borne viral infections are under-sampled and understudied. in addition, no specific treatments are available, and vaccines exist only for three tick-borne viruses (louping-ill virus, tick-borne encephalitis virus, and kyasanur forest disease virus) but are not widely distributed (mansfield et al. ) . in contrast to vertebrates, arthropods tolerate arboviral infections well; they do not usually develop disease once infected and maintain the infection for life. this suggests an innate ability to control viral titre, while still permitting arboviral transmission to the next host (mellor ; blair ). however, not all haematophagous arthropods are efficient vectors for the same virus. the ability of a particular virus to replicate within a host is referred to as vector competence (hardy et al. ) . understanding the factors that influence vector competence is of practical interest, as modifying these can foster the development of targeted arboviral control strategies (weiss and aksoy ) . vector competence is driven by the complex interaction between the virus and the host immune system. arthropods lack an adaptive immune response and rely entirely on innate immunity to control viral infections (keene et al. ; sanchez-vargas et al. ). small rna (srna) silencing pathways (srna sps), such as rna interference (rnai), are a major component of arthropod immunity (bronkhorst and van rij ) . rnai is a gene-silencing mechanism modulated by srnas, the bestdescribed family of which are small interfering rnas (sirnas, - nt in length). the sirna pathway is triggered by the accumulation of dsrna which is cleaved to generate sirnas. the interaction of sirnas with proteins of the argonaute family triggers the silencing of target rnas (e.g. viral transcripts) mediated by the rna-induced silencing complex. in mosquitoes, suppression of rnai enhances the accumulation of viral rna in anopheles gambiae (o'nyong nyong virus) and ae. aegypti (sindbis virus) and in some cases causes increased mortality (keene et al. ; campbell et al. ; myles et al. ) . in ticks, knockdown of key proteins involved in rnai results in a significant increase of tick-borne encephalitis virus rna in ixodes cells (weisheit et al. ) , suggesting a similar defence mechanism. piwi-interacting rnas (pirnas) ( - nt) were discovered in drosophila, where they defend against the deleterious effects of transposable elements (tes) in the germ-line . pirnas are distinct from other srna classes as they interact with a distinct set of proteins (piwi proteins), and they arise from a ssrna, not a dsrna, precursor (siomi et al. ). there are two pirna biogenesis pathways. primary pirnas are generated from host transcripts (usually arising from transposon-rich genomic pirna clusters) that are cleaved to generate - nt pirnas. these are amplified via the pingpong cycle, leading to the cleavage of target rna (e.g. active transposons), producing secondary pirnas. while pirna sequences are not conserved between organisms, primary pirnas unequivocally have a uridine at their end ( u bias), and secondary pirnas have an adenine bias at the tenth nt from their end ( a bias) (aravin, hannon, and brennecke ) . unlike in drosophila, mosquitoes can use pirnas to mount an antiviral defence in cooperation with other srna sps in both somatic and germ cells (morazzani et al. ) . mosquito pirnas can be directly produced from exogenous viral rna, indicating that pirnas may be derived from both endogenous and exogenous sources, and may play an antiviral role (vodovar et al. ; miesen, girardi, and van rij ) . although recent studies indicate widespread conservation of the pirna pathway among arthropods (lewis et al. ) , less is known about whether they could play an antiviral role in non-mosquito arthropods. one source of endogenous pirnas is non-retroviral integrated rna virus sequences (nirvs) (vijayendran et al. ) . nirvs arise from the reverse transcription of viral rna into cdna and its integration into the genome of a host germ cell, followed by vertical transmission to offspring (katzourakis and gifford ) . rna viruses do not integrate into the host genome as part of their lifecycle, so it was initially thought that integration of non-retrotranscribing viruses was serendipitous-especially in vertebrate genomes which contain relatively few insertions (katzourakis and gifford ). yet recent evidence has highlighted the abundance of nirvs in some arthropod genomes-e.g., ae. aegypti and ae. albopictus contain > nirvs from over eight rna virus families encompassing þssrna, Àssrna, and dsrna viral groups (palatini et al. ; whitfield et al. ) . additionally, large-scale sequence analysis has shown that many of these nirvs cluster within pirna producing genomic regions and produce primary pirnas (whitfield et al. ; ter horst et al. ) . this suggests that nirvs have a functional role, perhaps involving the pirna pathway. nirvs are also a rich source of information about long-term viral evolution, diversity, and host range. in its exogenous state, an rna virus will typically evolve at À substitutions/site/year (jenkins et al. ) , but when integrated into a eukaryotic genome, will evolve several orders of magnitude more slowly (aiewsakun and katzourakis ) . nirvs represent near-intact ancient viral sequences, and so can be useful to estimate the timescale of long-term viral evolution, e.g., dating the emergence of modern viral families (belyi, levine, and skalka ) . nirvs are also a useful tool to expand the known host range of viral families, as finding a particular nirvs family in a genome indicates an ancestral infection event of that host lineage (aiewsakun and katzourakis ) . the true spectrum of arthropod-infecting rna viruses is only recently coming to light (shi et al. ) and further information on host range and viral diversity can be assisted by the identification and analysis of nirvs in arthropod genomes. most studies thus far have focussed on the abundance and diversity of nirvs in aedes genomes, due to the high quality of available genomes and the clinical significance of these vectors cpipj /johannesburg scaffold (hammon et al. ; sudia et al. ; monath and tsai ; turell et al. ; guo et al. ) togaviridae ( chromosome (hoskins et al. ) (crochu et al. ; roiz et al. ; palatini et al. ; whitfield et al. ) . other studies have also indicated the presence of nirvs in non-dipteran lineages (shi et al. ; ter horst et al. ) . as the incidence of disease caused by arboviruses rises it will become important to generate a deeper understanding of nirvs diversity among non-mosquito vectors. we aimed to examine the spectrum of nirvs in arbovirus vectors other than aedes mosquitoes, which represent a threat in the context of emerging viruses. using the well-studied genomes of aedes mosquitoes to validate our analysis, we employed a bioinformatic pipeline to characterise nirvs in seven non-aedes arbovirus vectors that have representative genomic sequences (table and fig. ). to identify nirvs in arthropod genomes, a list of query viral protein sequences was assembled. type species for each viral family as defined by the international committee for virus taxonomy (ictv) master species list were used (ictv ). these included all proteins from reference viral genomes of dsrna viruses, þssrna viruses and Àssrna viruses. protein sequences from viruses recently identified in arthropods through deep rna sequencing studies were also included to increase the likelihood of finding sequences from divergent viruses (cook et al. ; chandler, liu, and bennett ; li et al. ; shi et al. ). this dataset comprised , protein sequences representing , distinct viruses (supplementary table s ). the arthropod genomes used for analysis are detailed in table . the genome of drosophila melanogaster (release ) (adams et al. ; hoskins et al. ) was also searched for nirvs. this is the best-assembled and annotated arthropod genome and contains no known nirvs. therefore, this allowed us to assess whether our pipeline was likely to generate false positives. identification of nirvs was based on methods outlined previously (katzourakis and gifford ) which used basic local alignment search tool (blast). a tblastn search was undertaken separately for each organism in geneious (v . . ) (kearse et al. ) , with all viral protein sequences queried against each arthropod genome. blast hits were filtered by evalue ( e À ), and duplicate blast hits (covering the same region of a host genome) were removed manually. for blast hits representing the same nirvs, the hit containing the maximum genomic coverage was retained. to verify that the blast hits were truly viral-derived sequences, a reciprocal search was performed by using translated blast hits as query sequences in a tblastn search against the ncbi nonredundant (nr) database. matches to retroviruses, viral cloning vectors, and non-specific matches to host loci or other arthropod loci were discarded. to visualise which region of the viral genome nirvs were derived from, nirvs were aligned individually to the genome of a closely related extant virus using clustalw (v . ), which does not penalise end-gaps in the nucleotide alignment (larkin et al. ). alignments were concatenated, and duplicates of the reference sequence were removed. alignments were visualised in graphpad prism (v . ) based on the numerical mapping position of the nirvs to the reference sequence. for alignments and trees containing mononegaviruses and mononega-like viruses, the chuviridae was included. the chuviridae is not technically part of this order but shares a close phylogenetic relationship with mononegaviruses (li et al. ) . to calculate which proportion of nirvs had significant identity to other nirvs within the same genome, indicating that they were probably duplicated sequences, a clustalw (v . ) alignment of all nirvs within each genome was performed in geneious (v . . ). the distance matrix from this alignment was used to identify nirvs with > per cent nt identity to any other nirvs. to create phylogenetic trees broadly representing each group of nirvs in this study, a collection of representative nucleotide sequences corresponding to the respective rna-dependent rna polymerase (rdrp) region of each group was downloaded from ncbi (supplementary table s ). translated nt sequences, which were of similar sequence lengths, were aligned using mafft (v . ) (katoh et al. ) and trimmed using trimal (v . . ) (capella-gutierrez, silla-martinez, and gabaldon ). maximum-likelihood (ml) phylogenetic trees were created in raxml (v . . ) (stamatakis ) . to perform phylogenetic analysis of specific nirvs from i. scapularis, twenty-five closely related viral sequences were identified by a tblastn search against the nr database. nt sequences of the same gene from which the nirvs was derived (e.g. replicase) were downloaded for these viruses, as well as a more distantly-related outgroup sequence. sequences were aligned and trimmed as above. ml phylogenetic trees were created in raxml (v . . ) (stamatakis ) , with an automated aa substitution model and rapid bootstrapping with nonparametric replicates. eukaryotic hosts of each viral species included in the phylogenetic analyses were deduced from study metadata and manually indicated on the tree. . pirna cluster prediction in the i. scapularis genome analysis of a publicly available srna dataset from i. scapularis was performed on the university of queensland galaxy server (https://usegalaxy.org.au) (afgan et al. ) . raw sequencing reads were downloaded from ncbi [bioproject accession prjna ], read quality was assessed with fastqc (andrews ) , and illumina srna adapters were trimmed with trim galore! (galaxy v . . . ). this dataset, along with the i. scapularis iscaw genome assembly (gulia-nuss et al. ) , was used to predict pirna-producing loci with protrac (v . . ) (rosenkranz and zischler ) applying custom parameters as previously defined (ter horst et al. ) . nirvs residing completely within predicted pirna clusters were identified using a megablast search implemented in geneious (v . . ), with blast matches exhibiting both per cent nt identity and query cover considered to reside within the cluster. the probability of integration into a pirna cluster was defined as the percentage of the genome occupied by pirna clusters. a cumulative binomial distribution was used to estimate whether nirvs had preferentially integrated into pirna clusters, as opposed to randomly within the genome. to judge te enrichment in regions around nirvs, genomic sequence lying kb either side of each nirvs was extracted. for nirvs that did not contain more than kb flanking sequence due to the size of the scaffold, the sequence up until the end of the genomic scaffold was retained. repeatmasker (v . . ) (smit, hubley, and green ) was used to estimate the te composition of these regions, with a list of predicted tes identified in the iscaw genome assembly as a reference. these proportions were then compared with the genome-wide te composition (gulia-nuss et al. ) . where two annotations encompassed more than nt of overlapping genomic region, the annotation with the highest smith-waterman score was retained. we also analysed an nirvs 'hotspot', where multiple nirvs from different families were present in nearby loci. this was scaffold ds , nt position , - , , which contained nirvs from the orthomyxoviridae (n = ), mononegavirales (n = ), and bunyavirales (n = ) in close proximity (supplementary table s ). the above srna dataset [prjna ] was used to further characterise nirvs-derived srnas. for evaluation of the nirvs sequence content of total srna, the forward reads from all four sequencing runs were merged and hisat (galaxy v . . ) (kim, langmead, and salzberg ) was used to align the reads to a reference fasta file containing all i. scapularis nirvs. aligned reads were extracted and quantified with salmon (v . . ) with custom parameters: (-kmerlen ; -unmatedreads; incompatprior . ) (patro et al. ) . read counts were then grouped by the nirvs' viral family of origin, and the proportion of total reads corresponding to each family was calculated. to assess nirvs-derived srna abundance by sequence length, the aligned reads (forward and reverse strand) for each k-mer ( - nt) were quantified with salmon (v . . ) and then divided by the total number of sequences comprising that dataset to calculate the relative number of nirvs-derived srnas for each k-mer. to assess nt bias at each position of the srna sequences, mapped srna reads ( - nt) were trimmed to nt from the end and used as input in weblogo (v . ) (crooks et al. ) . the srna dataset used contained two replicates each of mock infection, and two of infection with anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis. although this was not a viral infection, we examined whether nirvs transcription was upregulated upon general immune responses to bacterial infection. forward reads from the two infection datasets (sra id srr -srr ) were mapped to all nirvs and quantified with salmon (v . . ). this was repeated for the mock datasets (sra id srr -srr ). differential expression counts of the srnas were calculated with deseq (galaxy v . . ) (love, huber, and anders ). recent literature has described a large number of aedes nirvs, so we initially analysed the genomes of ae. aegypti and ae. albopictus to confirm the robustness of our pipeline. both species had a similar abundance of nirvs (ae. aegypti, n = , ae. albopictus, n = ). we then detected nirvs in a further six arthropods that are known to transmit pathogenic vertebrate viruses. the i. scapularis genome harboured the next largest number of nirvs (n = ), followed by cx. quinquefasciatus (n = ), an. gambiae (n = ), c. sonorensis (n = ), p. papatasi (n = ), and r. microplus (n = ) (supplementary table s ). integrations from negative-sense viruses dominated in most organisms: ae. aegypti, n = ( %), ae. albopictus, n = ( %), i. scapularis n = ( %), cx. quinquefasciatus n = ( %), an. gambiae n = ( %), and c. sonorensis n = ( %) ( fig. a ). in p. papatasi and r. microplus there was one Àssrna derived nirvs ( fig. a ; supplementary table s ). all other nirvs were related to either þssrna or dsrna viral sequences (figs and ). no nirvs were identified in the negative control genome of d. melanogaster. many arthropods contain a high proportion of tes in their genomes; ae. albopictus and ae. aegypti contain about and per cent tes, respectively (nene et al. ; chen et al. ) . due to the important role of repeat sequences in arthropod evolution (peccoud et al. ), we suspected that many of the nirvs identified were generated due to post-insertion duplication and not by distinct integration events. using a clustalw alignment and distance matrix, we calculated whether nirvs shared high sequence identity with other nirvs within the genome. of nirvs in ae. aegypti, ( %) shared at least per cent nt identity with at least one other nirvs. in ae. albopictus, this number was ( %), in i. scapularis it was ( %); in cx. quinquefasciatus ( %), and in an. gambiae ( %) (supplementary table s ). there was no evidence of duplicated nirvs in other organisms with multiple nirvs (c. sonorensis and p. papatasi) (supplementary table s ). in ae. aegypti and i. scapularis, we visually represented duplicated nirvs within the groups mononegavirales, bunyavirales, and orthomyxoviridae (fig. ). a similar pattern was observed in i. scapularis where most Àssrna nirvs were classified in the mononegavirales in either the rhabdoviridae (n = [ % of Àssrna nirvs]) or among unclassified mononegaviruses (n = , %) (fig. ) . nirvs related to bunyaviruses (n = , %) were notably more abundant than in dipterans, and evenly distributed among the phenuiviridae (n = , %) and nairoviridae (n = , %) (fig. ) . orthomyxo-like nirvs (n = , %) were the next most prevalent, followed by the chuviridae (n = , %) (fig. ) . in cx. quinquefasciatus and an. gambiae, the Àssrna nirvs were related to unclassified mononegaviruses (n = and n = [ and % Àssrna nirvs, respectively]), chuviruses (both n = [ and %, respectively]), or rhabdoviruses (cx. quinquefasciatus n = , %) (fig. ). an. gambiae also contained both one phasma-and one phenui-like insertion (supplementary table s table s ). this pattern was consistent for cx. quinquefasciatus (n = / , %) and an. gambiae (n = / , %) ( fig. a) . notably, i. scapularis possessed more nonstructural [l] region integrations from mononega-like viruses (n = / , %), with the remainder from structural regions (n = / , %) ( fig. a) . to visually demonstrate this difference, a comparative alignment between negative-sense nirvs in ae. aegypti and i. scapularis was created, demonstrating the skew towards either structural or non-structural integration in each organism (fig. a) . in terms of specific structural integrations from mononegalike viruses, gp-derived nirvs were overrepresented in ae. albopictus (n = / mononega-like insertions, %), cx. quinquefasciatus (n = / , %), and an. gambiae (n = / , %), with either fewer or no np-derived nirvs in these organisms (n = / [ %], n = / [ %], and n = / [ %], respectively). alternatively, np-derived nirvs were more abundant in ae. aegypti (n = / , %) with fewer gp-derived integrations (n = / , %), and a single integration was derived from the m gene (fig. a ) and another from a protein of unknown function (supplementary table s ). similarly, structural proteinderived mononega-like nirvs in i. scapularis were mostly derived from np sequences (n = / , %), with only two gprelated nirvs ( %) (fig. a ). the hepe-virga clade and the flaviviridae were the two major lineages comprising nirvs from þssrna viruses ( fig. ; supplementary fig. s ). þssrna nirvs related to the flaviviridae were the most abundant, but were only observed in aedes mosquitoes (ae. aegypti, n = [ % þssrna nirvs], ae. albopictus, n = , %) (fig. ) . this was also the case for negelike viruses (ae. aegypti, n = [ %], ae. albopictus, n = [ %]), and hepe-virga-like viruses (ae. aegypti, n = [ %], ae. albopictus, n = [ %]) ( fig. ; supplementary fig. s ). no þssrna virusderived nirvs were detected in p. papatasi, r. microplus, or d. melanogaster. putative nido-like nirvs were initially detected in ae. albopictus (n = ), and once each in ae. aegypti, cx. quinquefasciatus, an. gambiae, c. sonorensis, and d. melanogaster; however, the identity of this as a true nirvs was disputed (see further discussion below, section . ). . . dsrna virus derived nirvs dsrna virus-derived nirvs were the least abundant across all organisms (n = total; supplementary fig. s ). dsrna nirvs could be classified as either partiti-like or toti-like, i.e. relatives of the fungal and protozoan-infecting families partitiviridae and totiviridae, respectively (fig. ) . in ae. aegypti and ae. albopictus, all dsrna-like nirvs (n = and n = , respectively) were toti-like (fig. ) when searching for nirvs, false positive hits can arise from viral query proteins that have chance homology to eukaryotic proteins (e.g. heat shock proteins). these can usually be eliminated with a reverse tblastn search against the nr database, where no viral hits are produced, as hits to eukaryotes are much stronger. we observed an unusual blast hit in ae. aegypti, ae. albopictus, an. gambiae, cx. quinquefasciatus, c. sonorensis, and d. melanogaster with homology to the orf a (replicase) protein of mosquito-specific viruses in the mesoniviridae (order nidovirales) ( rdrp sequences, as listed in supplementary table s ). shapes correspond to the abundance of nirvs from that group as shown in the legend, and colour represents the organism analysed (right-hand panels). viral orders are encircled with dotted lines, and families are shaded grey. the scale bar represents aa substitutions per site. database generated a list of close matches to hypothetical proteins from related insect species, in addition to more distantly related viral hits, so we did not fully eliminate this as a false positive hit. we next analysed the flanking regions of this putative nirvs, to see whether it was part of a functional protein coding sequence. the putative nirvs was part of an open reading frame (orf) spanning approximately , nt in all surveyed organisms, which encoded an annotated hypothetical protein in ae. aegypti, ae. albopictus, an. gambiae, cx. quinquefasciatus, and d. melanogaster (table ) . therefore, it seemed that this hit represented a conserved protein containing a 'nido-like' domain ($ nt identity, ranging from . to . %) (supplementary table s ). we performed a blastp search of this conserved protein to find potential homologues but generated a list of results similar to the initial tblastn search (supplementary table s ). owing to the emerging threat of tick-borne viruses, it is crucial to sample viral diversity in ticks. nirvs can represent novel phylogenetic lineages which have not been sampled in the exogenous form. therefore, we performed phylogenetic analysis on six i. scapularis nirvs from different taxonomic groups. this encompassed a wide range of genetic diversity, while using the longest sequence from each group ensured high phylogenetic resolution (table and fig. ). the single partiti-like nirvs ( nt) was derived from the conserved rdrp region, and clusters monophyletically with norway partiti-like virus [mf ] that was isolated from i. ricinus (fig. a) ). overall, these arthropod-specific partiti-like viruses form a clade related to, but distinct from, the partitiviridae family (fig. a ). however, due to the short sequence length there is low bootstrap support (< %) for most tree nodes (fig. a) . the longest chuvirus-like nirvs ( nt, genomic scaffold ds ) was related to the chuviral glycoprotein (m) gene. it sits basally to a larger cluster of arthropod-specific chuviruses; which includes a tick-specific cluster ( (fig. b) . orthomyxo-like nirvs in i. scapularis were more abundant than in any dipteran, and the longest ( nt, derived from the pb protein coding region), clustered in a group of . . this cluster is distinct from sandfly borne human pathogens (e.g. rift valley fever phlebovirus), and from the well-described tick-specific uukuniemi virus clade (fig. d) . the np-derived nairo-like nirvs falls into an ixodes-specific cluster within the nairoviridae, distinct from several clusters infecting either hard ticks (ixodidae) or soft ticks (argasidae) (fig. e) . lastly, the rhabdolike nirvs which was particularly long ( , nt) can be classified into a tick-specific rhabdo-like cluster, where its closest relative is norway mononegavirus (fig. f ). this classification notably did not align with its closest blast result which was the equine-infecting vesicular stomatitis indiana virus (table ) . using protrac (v . . ), , pirna clusters were predicted in i. scapularis which comprised a total of , , bp ( . % of the genome). thirty-two nirvs ( %) resided totally within these clusters, with the remaining either partially present or absent from the clusters. the cumulative binomial probability that at least nirvs integrated into pirna clusters was p < . , indicating a strong preference for integration into these sites. in ae. aegypti, ltr retrotransposons (pao_bel/ty _gypsy) are associated with nirvs integration (palatini et al. ). in i. scapularis, genomic regions flanking nirvs were associated with a two-fold enrichment of fragments of non-long-terminal repeat (non-ltr) class i retrotransposons ( . % in the whole genome vs. . % in nirvs flanking sites), but not of ltr retrotransposons ( . vs. . %) or dna transposons ( . vs. . %) ( table ). among the non-ltr retrotransposon fragments, the most relatively enriched were r elements ( . vs. . %), i elements ( . vs. . %), and l elements ( . vs. . %) ( table ). the nirvs 'hotspot' on scaffold ds also contained a high proportion of non-ltr retrotransposons ( . %), but not as many i elements ( . %) or r elements ( . %) ( table ) . it has been shown that pirnas are produced by nirvs in a variety of arthropods (palatini et al. ; ter horst et al. ) . to determine whether this was the case in our own dataset, we analysed a publicly available i. scapularis srna dataset for the presence of pirnas derived from nirvs loci (bioproject accession prjna ). out of , , srna reads originating from four independent sequencing runs, , ( . %) mapped exactly to at least one nirvs. these originated primarily from nirvs related to rhabdoviruses ( . %), unassigned mononegaviruses ( . %), orthomyxoviruses ( . %), phenuiviruses ( . %), nairoviruses ( . %), and chuviruses ( . %); these proportions are very similar to the abundance of each nirvs family within the genome (fig. a) . the most abundant nirvs-derived srnas were nt in length ( . % total -mers), followed by nt ( . %), nt ( . %), nt ( . %), nt ( . %), nt ( . %), nt ( . %), nt ( . %), nt ( . %), nt ( . %), nt ( . %), and nt ( . %) (fig. b) . additionally, nirvsderived srnas were predominantly antisense ( , reads, . %) as opposed to sense ( , reads, . %) (fig. b) . the length of the nirvs-derived srnas (primarily - nt srnas) their predominantly antisense nature, and the strong bias for a uridine at their position make them strongly reminiscent of primary pirnas (fig. b and c) . in the range of - nt, the most abundantly expressed nirvs were ds (rhabdo-like, l protein, , reads), ds _ (rhabdo-like, l protein, , total reads), ds _ (phenui-like, l protein, , reads), ds _ (nairo-like, n protein, , reads), and ds _ n protein, , reads) (supplementary table s ). no srnas were significantly up-or down-regulated (adjusted p-value < . ) upon infection with a. phagocytophilum. arthropods that transmit viruses to vertebrate hosts maintain a fine balance between carrying a viral infection for an extended figure . ml phylogenetic trees of six nirvs from the blacklegged tick i. scapularis. following identification of nirvs within the i. scapularis genome, the longest from each family of negative-sense viruses was selected for phylogenetic analysis (n ¼ ). a dsrna nirvs was also selected as it was the unique nirvs of this type identified (a). first, a tblastn search was performed using the nirvs against the ncbi nr database to generate a list of close matches. the top most closely related sequences were chosen, as well a more distantly related outgroup sequence. sequences were aligned with mafft (v . ) and trimmed with trimal (v . . ). a raxml period and avoiding the deleterious effects of viral infection. nirvs are proposed to be a heritable antiviral defence mechanism, interacting with srna sps to attenuate transcripts of an infecting virus (palatini et al. ) . it is now well-established that nirvs accumulation is a feature of multiple arthropod classes (ter horst et al. ). yet besides aedes spp., an in-depth analysis of nirvs in emerging arbovirus vectors is lacking. to address this gap, we selected six non-aedes arbovirus vectors for nirvs analysis. we uncovered more nirvs in the mosquitoes cx. quinquefasciatus and an. gambiae than reported previously, and we characterised novel nirvs in the non-mosquito dipterans c. sonorensis and p. papatasi, and in the asian blue tick r. microplus. the focus of our study, however, became the preponderance of nirvs in the deer tick i. scapularis, which exhibit unusual characteristics and are likely to be associated with the pirna pathway. ae. aegypti and ae. albopictus are the best-studied arthropods in the context of nirvs and were therefore used as validation organisms for our nirvs identification pipeline. our count in ae. aegypti (n = ) (figs - ) was considerably more than palatini et al. ( ) (n = ) who used a similar blast-based approach, and a recent version of the same genome (aaegl . ) (palatini et al. ). our results may reflect a more extensive range of query viruses (n = , vs. n = ). alternatively, whitfield et al. ( ) reported nirvs in ae. aegypti, but used a long-read based genome assembly for nirvs identification. since the current aaegl . assembly is based on short-read technology, highly repetitive nirvs-rich regions may be masked (dudchenko et al. ; whitfield et al. ) . these variable figures suggest that the number of nirvs reported is not definitive but is contingent on filtering parameters and the quality of host genome assembly. in ae. albopictus, we reported nirvs (figs - ) , also higher than palatini et al. (n = ) , yet they used a genome from a different source (foshan strain as opposed to c / cell line used in our study). however, the true number of nirvs in ae. albopictus is probably as high as or higher than ae. aegypti, as it contains an even higher proportion of tes (chen et al. ) , including ltr retrotransposons which may be responsible for nirvs formation. despite the discrepancies, both studies report similar nirvs characteristics. these nirvs were dominated by insertions from the structural gene-coding regions (n and g) of negative-sense viruses (predominantly mononegaviruses), with fewer insertions from þssrna and dsrna viruses. this indicates that the methodology of the current study, which identified nirvs without generating excessive false positive hits, is robust. we observed that cx. quinquefasciatus and an. gambiae harboured approximately twenty nirvs each (figs - ; supplementary table s ) , which is inconsistent with palatini et al. ( ) who identified no nirvs in an. gambiae, and only one nirvs in cx. quinquefasciatus. these mosquitoes therefore possess more nirvs than previously thought, with similar characteristics to aedes nirvs, i.e. dominated by integrations from structural regions of negative-sense viruses ( fig. a ; supplementary table s ) . in this study, we included two non-mosquito dipterans which transmit emerging mammalian viruses (table ) . analysis revealed nirvs in p. papatasi (n = ) (bunya-and partiti-like) and c. sonorensis (n = ) (chu-and bunya-like) (figs table . transposable element (te) occupancy in the entire i. scapularis genome, in regions with nirvs, and in a region containing multiple nirvs from three different viral groups (located on scaffold ds ). iscaw genome assembly ( , , , table s ), demonstrating that these nirvs probably originated from highly divergent viruses. the virome of these organisms has not been extensively sampled, yet our results imply the presence of circulating, divergent, and arthropod-specific viruses in these species, which may be important for future studies. overall, our results suggest that nirvs accumulation is primarily a feature of mosquitoes, and not other dipterans. nirvs are not usually present as protein coding regions, but as fragmented sequences containing stop codons. however, viral proteins can be repurposed by their hosts (exapted) in remarkable instances of horizontal evolution (koonin and krupovic ) . a blast hit in six dipteran genomes revealed the presence of a hypothetical protein containing a nido-like domain ( table s ). although hits to nidoviruses still arose, homology to these was generally weaker than to other insect proteins (e.g. in ae. aegypti), the hypothetical protein loc exhibited matches to uncharacterised proteins from other insects (e-values ranging from to . e À ) before a viral hit occured (e-value . e À ) (supplementary table s ). the pp a nidovirus protein, to which blast matches were generated, is a multifunctional replicase enzyme (ziebuhr ) . we speculated that this region of similarity could signify a conserved enzymatic function between virus and eukaryote, e.g. helicase; however, no functional protein motifs were observed in this viral region. due to the nature of the putative nirvs it could represent an exapted viral protein which integrated prior to the divergence of the hosts, now serving a novel function in present-day lineages. this possibility is especially interesting as arthropods are predicted to be the origin of some present-day viral groups (marklewitz et al. ) . alternatively, it could represent a case of host-to-virus exaptation, or protein mimicry by the virus (alcami ) , although these possibilities are unlikely in the case of an rna virus. further analysis of these nirvs are beyond the scope of this manuscript, but these possibilities present an intriguing opportunity for future analysis. we revealed an abundance of nirvs in i. scapularis (n = ), which is broadly consistent with other studies of ixodes spp. who report > nirvs (ter horst et al. ) . the majority of our nirvs generated a blast match to viruses isolated from hard ticks (supplementary table s ) , and phylogenetic analysis of six nirvs supported their classification into tick-specific viral clades (fig. ) . originating from both the sense (green) and antisense (purple) genomic strand. (c) sequence logo depicting ribonucleotide preference (probability) among the first ten nucleotides of nirvs-derived srnas, from both the sense and antisense genomic strands. we found both similarities and differences between nirvs in i. scapularis and dipterans. like the dipterans, i. scapularis nirvs were dominated by negative-sense viruses (n = , %) which consisted primarily of insertions from non-segmented negative-sense (nns) viruses (i.e. mononegaviruses) (n = , %) (figs and ; supplementary table s ). yet among the nns viral insertions, we observed a bias towards non-structural region integration (l protein) as opposed to structural regions (g and n), which are conversely more abundant in mosquitoes (fig. a ). this bias towards nirvs from the structural region of negative-sense viruses is thought to reflect relative mrna abundance during infection, where the np is the most abundant and l the least (holmes ) . here, the unexpected abundance of l integrations could reflect an unusual template preference for the endogenous reverse transcriptases (rts) and integrases in i. scapularis which catalyse nirvs formation. another unusual aspect of nirvs in i. scapularis was the overrepresentation of integrations from segmented negativesense (sns) rna viruses (orthomyxo-like and bunya-like) (n = , %) (figs and ). in contrast, all other surveyed organisms contained few sns virus-like nirvs (ae. aegypti n = , ae. albopictus n = , an. gambiae n = , c. sonorensis n = , r. microplus n = ) ( fig. ; supplementary table s ). specifically, the orthomyxo-like nirvs in i. scapularis were exclusively related to tick-borne viruses in the genus quaranjavirus (presti et al. ). orthomyxoviruses replicate in the nucleus (ruigrok et al. ) , where proximity to the host genome may predispose them to nirvs formation. however, studies on orthomyxovirus replication are heavily biased towards influenza viruses, and mechanisms of quaranjavirus replication are little explored. similar observations were made for the bunya-like insertions, again abundant in i. scapularis (n = ) despite being infrequent in other surveyed organisms. these could be classified as phenuilike or nairo-like (related to the phenuiviridae or nairoviridae). in contrast, phasma-like (phasmaviridae-related) insertions were confined to ae. aegypti, ae. albopictus, p. papatasi, and an. gambiae (supplementary table s ). this mirrors the known host range of these families, whereby the nairoviridae is tick-borne, the phenuiviridae infects insects and arachnids, and the phasmaviridae is insect-borne (lasecka and baron ) . overall, the detection of multiple sns viral insertions here suggests that ticks are the dominant hosts of these viral groups. curiously, i. scapularis lacked nirvs from positive-sense rna viruses, which comprise about one tenth of nirvs in aedes mosquitoes ( fig. ; supplementary table s ). in particular, flavivirus-derived nirvs are the most abundant positive-sense group in mosquitoes, yet despite the existence of a tick-borne flavivirus clade, we did not observe any flavi-like nirvs in i. scapularis. this is surprising, as tick-borne flaviviruses are vertically transmitted in nature (brackney and armstrong ) . their absence could reflect that positive-sense viral genomes are unfavourable templates for ixodid nirvs formation. alternatively, since most tick-borne arboviruses are confined to certain groups (bunyavirales, orthomyxoviridae, flaviviridae), tick viruses outside these groups are significantly under-sampled (junglen ) . accordingly, identification of tick-specific viruses in other groups may reveal positive-sense nirvs which are too divergent to be detected here. analysis of the r. microplus genome revealed only one nirvs, a much lower figure than i. scapularis (n = ) ( fig. ; supplementary table s ). this is an interesting disparity, both ticks contain a high proportion of repetitive elements ($ %) (barrero et al. ) . our data suggest that nirvs accumulation is perhaps confined to ixodes ticks, an observation validated by another study which also identified numerous nirvs in i. ricinus (ter horst et al. ) . this difference could reflect factors such as inefficient vertical viral transmission among rhipicephalus spp., or an alternative te landscape which does not favour nirvs formation. importantly, genome sequencing of more hard and soft ticks will help to further characterise tick nirvs. a requirement for the formation of an nirvs is infection of a germ cell, permitting transmission of the integrated viral sequence to offspring (aiewsakun and katzourakis ) . each nirvs is the footprint of a germline viral infection, suggesting vertical transmission of all the viral nirvs groups identified here. although vertical transmission of tick viruses is proposed to occur in nature (labuda and nuttall ) , information is limited. our data suggest that in i. scapularis, vertical transmission of mononegaviruses, bunyaviruses, and orthomyxoviruses may occur regularly, giving them ample opportunity to integrate. most of the nirvs here appeared to originate from tickspecific viral groups (fig. ). an exception was nirvs from the genus quaranjavirus (orthomyxoviridae) (fig. c) . quaranjaviruses appear to have a dual vertebrate-arthropod host range; e.g., wellfleet bay virus can cause mass mortality in wild bird populations (allison et al. ) . the presence of nirvs derived from this group therefore implies the potential for vertical transmission of quaranjaviruses, which has implications for the management of these viruses in wild tick populations. . nirvs are associated with the pirna pathway in i. scapularis srna sps are the key antiviral response in insects (blair and olson ) , yet current research is heavily biased towards mosquitoes. several studies indicate involvement of an srna response in tick flaviviral infection (schnettler et al. ; weisheit et al. ), yet the area is understudied. in particular, the observation that nirvs are proximal to pirna clusters for a variety of arthropods suggests that nirvs either ( ) integrate into these clusters preferentially or ( ) are positively selected for post-integration (palatini et al. ; whitfield et al. ; ter horst et al. ) . interestingly, despite the identification of multiple nirvs in i. scapularis in another study, no association with pirna clusters was noted (ter horst et al. ). repetition of this analysis for our own dataset uncovered a strong preference for integration into predicted pirna sites in the i. scapularis genome (p ( . ). despite this discrepancy, our observation of many nirvs-derived pirnas, as discussed below, suggests that our clustering analysis was robust. the second line of evidence for association with the pirna pathway is the production of nirvs-specific pirnas. to investigate this phenomenon in the blacklegged tick, we analysed datasets from i. scapularis ise cells (n = ) for the presence of nirvs-derived srnas. we found that nirvs-derived srnas from all families (except a partiti-like insertion) were produced proportionally to their abundance within the genome (fig. a ). further analysis revealed that these srnas exhibited characteristics of primary pirnas; specifically; they were primarily - nt in length, antisense, and had the canonical u pirna bias ( fig. b and c) . this strengthens the hypothesis that the nirvs-pirna association exists in i. scapularis as it does in other arthropods. further studies can elucidate whether nirvsderived pirnas are upregulated upon viral infection, and whether suppressing the pirna pathway confers an antiviral effect. additionally, virus-specific pirna production is known to differ between germ-line (the origin of the ise cell line) and somatic tissues in most arthropods (lewis et al. ) . therefore, it would be interesting to determine whether these nirvs-derived pirnas are also produced in somatic tissues where an infecting virus encounters the tick's main antiviral defences. . non-ltr retrotransposons are the predominant te class associated with nirvs integration in i. scapularis the production of cdna from viral rna is critical to the formation of an nirvs. this requires rt activity, which is likely provided by the retroelements common in eukaryotic genomes (holmes ) . although the association between nirvs and the pirna pathway is clear for a variety of arthropods, analysis of the te classes associated with nirvs only exists for ae. aegypti, where ltr retrotransposons are increased two-fold in nirvs-rich regions (palatini et al. ). our analysis indicated that non-ltr retrotransposons were instead two-fold enriched ( . vs. . %) in nirvs-associated regions in the i. scapularis genome, while ltr retrotransposon sequences were not increased ( . vs. . %) ( table ). this association corresponds to the diminished proportion of ltr retrotransposons in ticks ( . % of the genome) compared with mosquitoes (about % in the genomes of ae. albopictus, ae. aegypti, and cx. quinquefasciatus) (chen et al. ; gulia-nuss et al. ) . our data suggest that the rt activity of non-ltr retrotransposons, particularly i, l , and r elements, which were increased in nirvs-rich regions (table ) , might be responsible for nirvs formation in ixodid ticks. however, further studies of the reverse transcription of viral rna in tick cells is warranted to support this hypothesis. here, we have provided novel insights into nirvs in the genomes of important arbovirus vectors. our data suggest that nirvs in i. scapularis are distinct in both phylogeny and viral region of origin. furthermore, our data indicate a link to the pirna pathway in i. scapularis and an association with non-ltr retrotransposons. due to the increased incidence of tick-borne viruses, further studies into how this affects tick antiviral immunity are warranted. supplementary tables accompanying this text are available at https://arusso .github.io/nirvs/. supplementary data are 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brief on vector-borne diseases the ecology, genetic diversity, and phylogeny of huaiyangshan virus in china the coronavirus replicase: insights into a sophisticated enzyme machinery this research includes computations using the linux computational cluster katana supported by the faculty of science, unsw, australia. a.g.r. and d.e.t. acknowledge support through australian government research training program scholarships. all photographic images in fig. a.g.r., a.g.k., d.e.t., m.m.t., and p.a.w. conceived project and designed experiments. a.g.r., and a.g.k. performed experiments and analysed data. a.g.r. and p.a.w. wrote the manuscript. all authors read, edited, and approved the manuscript before submission.conflict of interest: none declared. key: cord- -a w nnos authors: yang, jiwen; tian, gang; chen, daiwen; zheng, ping; yu, jie; mao, xiangbing; he, jun; luo, yuheng; luo, junqiu; huang, zhiqing; wu, aimin; yu, bing title: dietary -hydroxyvitamin d( ) supplementation alleviates porcine epidemic diarrhea virus infection by improving intestinal structure and immune response in weaned pigs date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: a w nnos simple summary: porcine epidemic diarrhea is one of the major problems in current swine husbandry worldwide, and effective measures for prevention and treatment are scarce. we found that high dose -hydroxyvitamin d( ) supplementation could ease intestinal injury and inhibit intestinal immune response induced by porcine epidemic diarrhea virus (pedv), suggesting that feeding a high dose of -hydroxyvitamin d( ) could be used as an approach against pedv infection. abstract: we conducted this experiment to determine if feeding -hydroxyvitamin d( ) ( (oh)d( )) to weaned pigs would alleviate porcine epidemic diarrhea virus (pedv) infection and immune response. forty-two weaned pigs were allotted to of dietary (oh)d( ) treatments ( . , . , . , . , . , . μg (oh)d( )/kg diet) for days. on day of the trial, all the treatments were orally administrated with pedv except for one of the . μg (oh)d( )/kg treatments, which was challenged with the same volume of sterile saline and served as control. another . μg (oh)d( )/kg group for pedv challenge was named con-pedv. average daily gain (p < . ) was reduced by pedv infection. pedv administration also induced severe diarrhea (p < . ), reduction of villous height and the ratio of villous height to crypt depth, and increase of crypt depth and serum diamine oxidase activity (p < . ). serum igm and complement component levels were increased by pedv challenge. however, . μg (oh)d( )/kg supplementation alleviated intestinal damage (p < . ) compared with con-pedv. furthermore, . μg (oh)d( )/kg supplementation downregulated the mrna abundance of inflammatory cytokines and interferon signal pathway-related genes (p < . ) compared with con-pedv. these results suggested that dietary supplementation of . μg (oh)d( )/kg could alleviate intestinal damage and protect against pedv-induced inflammatory status. porcine epidemic diarrhea virus (pedv) infection causes severe damage to the intestinal function and barrier integrity of pigs [ ] , leading to diarrhea, vomiting, dehydration, and high mortality in piglets [ ] . recently, it was shown that the villus height and transepithelial resistance were decreased in pedv-infected pigs [ ] . pedv also induced intestinal mucosa and systemic proinflammatory cytokine responses in pigs [ , ] . currently, vaccination is the main means for preventing pedv infection. however, vaccines are poorly effective because of genetic variants of the viruses [ , ] . vitamin d not only enhances calcium and phosphate absorption but also regulates immune function [ ] . previous studies have shown that vitamin d can inhibit rotavirus replication and alleviate infection symptoms in piglets [ , ] . pedv infection has shown similar symptoms to rotavirus and, therefore, we speculated that (oh)d might ease the infection of pedv in pigs. however, studies on the feasibility of vitamin d as anti-pedv infection agent in piglets are scarce. it is generally believed that the bioavailability of (oh)d is higher than vitamin d [ ] [ ] [ ] . in this study, (oh)d was used to investigate whether it could alleviate pevd-infected diarrhea and intestinal injury. the experimental protocol involved in the present study was approved by the animal care advisory committee of sichuan agricultural university (animal ethics committee approval number is cd-syxk- - ). forty-two crossbred healthy weaned pigs (duroc × landrace × yorkshire, days old) with an initially body weight (bw) of . ± . kg were used in the days trial. on the first day of the trial, all pigs were allotted on the basis of bw to six groups and each fed diets supplemented with either . , . , . , . , . , or . µg (oh)d /kg. each treatment consisted of four gilts and three barrows. at day of this study, all the treatments were orally administrated with ml of pedv ( . × tcid /ml) except for one of the . µg (oh)d /kg ( iu vitamin d/kg equivalent) treatment, which was served as control (con) and administrated with the same volume of sterile saline. another . µg (oh)d /kg treatment for pedv challenge was named by con-pedv. in order to prevent infection, the con groups were housed in the next room of challenge groups. the settings for the two rooms were the same. pigs in the con group were negative for pedv throughout the trial period. as shown in table , the basal diet was formulated to meet or exceed nutrient requirement for weaned piglets [ ] , except for vitamin d , which was not prepared in the vitamin premix. each treatment was formed by supplementing with indicated (oh)d levels in the basal diet. all pigs had ad libitum access to water and experimental diets throughout the trial. (oh)d (hy-d) was kindly provided by dsm nutritional products ltd. shanghai, china. the pedv was kindly presented by professor zhiwen xu, college of veterinary medicine, sichuan agricultural university. average daily gain (adg) and average daily feed intake (adfi) were determined via weighing to determine body weight and recording of feed intake. after the pedv challenge, the fecal consistency and diarrhea incidence were assessed every day according to hu et al. [ ] : = hard feces, = firm well formed, = soft and partially formed feces, = loose, semi-liquid feces, and = watery feces. diarrhea rate (%) = (a/ d) × , in which a = total days per pig with diarrhea after pedv challenge. mean cumulative score = a/b, in which a = the sum of daily scores, and b = pigs per treatment. all pigs were bled via anterior vein on day. the blood was used for extracting serum via centrifugation at g for min, and the serum samples were stored at − • c until analysis. all pigs were then euthanized by intramuscular injection of shumianning (comprised of ketamine, xylazine, and midazolam, nanjing agricultural university, . ml/kg body weight). about cm jejunal tissue sample was stored in % paraformaldehyde solution for histological analysis. mucosal samples from the middle jejunum were scraped and rapidly frozen in liquid nitrogen, and then stored at − • c for further analysis. the concentration of igg, igm, and complement component (c ) and c (sichuan maker biotechnology co. ltd. chengdu, china) in serum were detected by automatic biochemical analyzer (model ; hitachi, tokyo, japan). immunology multiple control were performed before sample determination to ensure the outcomes were correct. after being embedded in paraffin, the jejunal samples were stained with hematoxylin and eosin for intestinal morphology measurement. a minimum of well-orientated villi and crypts from each intestinal sample of pigs were measured using image-pro plus . software. as a measurement of intestinal permeability, serum diamine oxidase activity (dao) was detected using commercial assay kits (nanjing jiancheng institute of bioengineering, jiangsu, china) following the protocols of the manufacturer. total rna was extracted from the mucosa of jejunum tissue using trizol reagent (invitrogen, shanghai, china). reverse transcription was performed with rna using a primescript rt reagent kit (takara, dalian, china). the mrna expression of genes of interest were quantified using an abi ht detection system (applied biosystems, foster, ca, usa) and the sybr premix ex taq ii with rox reagents (takara, dalian, china). the primer sequences used for rt-pcr are listed in table . all primer pairs were designed to have melting temperatures of approximately • c. cycling conditions were as follows: • c for s, followed by cycles of • c for s and • c for s. the relative mrna expression of each gene was calculated according to a previous publication [ ] . expression levels were normalized to β-actin. protein sample processing was performed according to zhang et al. [ ] . briefly, jejunal mucosa protein was extracted with lysis buffer (beyotime, shanghai, china). after centrifugation for min at , rpm, the supernatants were harvested for bicinchoninic acid assay to detect protein concentration. then, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, and incubated with the corresponding antibodies: anti-sucrase-isomaltase (si), anti-siga, anti-occludin (abcam, shanghai, china), and anti-β-actin (santa cruz, shanghai, china). following washing, the samples were incubated with secondary antibodies, and then proteins were incubated with electro-chemi-luminescence reagent for chemiluminescence. the protein expression was analyzed by image lab and normalized to β-actin. in this study, each pig was used as the experimental unit. the differences between con and con-pedv group were assayed by student's t-test. on the condition of pedv challenge, the data were performed using the proc mixed sas . procedure according to the following model: where y = dependent variable; µ = mean; α = effect of treatment; β = block effect of bw, and ε = error. orthogonal comparisons were also applied for linear and quadratic responses of increasing dietary (oh)d levels ( . , . , . , . , . µg (oh)d /kg). data of the innate immune gene expression were analyzed by tukey's test for post hoc comparisons. the significance was declared at p < . and trends at p < . . as shown in table , pedv challenge (con-pedv) decreased adg (p < . ) and adfi (p = . ) compared with con. however, the performance was not influenced among the different (oh)d supplementation groups. pedv infection induced severe diarrhea in piglets (table , p < . ). however, the dietary supplementation of (oh)d decrease diarrhea scores (p < . ) and diarrhea rate ( table , p < . ). table . growth performance and diarrhea parameter of weaned piglets fed (oh)d as indicated with porcine epidemic diarrhea virus (pedv) challenge. pedv infection increased serum igm and c concentrations compared with con (p < . , table ). however, different levels of (oh)d supplementation had no effect on serum igm, igg, and c levels on the condition of pedv challenge. in addition, dietary supplementation of . µg (oh)d /kg decreased serum c levels compared with the . µg (oh)d /kg group. compared with con, pedv challenge decreased villous height and the ratio of villous height to crypt depth (vcr), and increased the crypt depth of jejunum in the con-pedv group ( table , p < . ). however, . and . µg (oh)d /kg treatments significantly increased villous height and vcr and decreased crypt depth compared with the con-pedv group ( table , p < . ). furthermore, dietary supplementation of (oh)d increased villous height and vcr and decreased crypt depth in a linear way (p < . ). in addition, serum dao activity of pigs in the con-pedv group was significantly increased compared with con ( figure , p < . ). however, the dietary supplementation of (oh)d tended to decrease serum dao activity (p < . ). animals , , x of in addition, serum dao activity of pigs in the con-pedv group was significantly increased compared with con ( figure , p < . ). however, the dietary supplementation of (oh)d tended to decrease serum dao activity (p < . ). compared with con, the gene expression of intestinal barrier-related genes was not influenced by pedv (table ) . upon pedv challenge, the dietary supplementation of (oh)d increased jejunal claudin- gene expression significantly and this treatment effect was linear (p < . ). (oh)d also tended to increase muc and mrna expression (p < . ). interestingly, pedv increased sucraseisomaltase (si) and occludin protein levels, but high doses (oh)d ( . μg (oh)d /kg) compared with con, the gene expression of intestinal barrier-related genes was not influenced by pedv (table ) . upon pedv challenge, the dietary supplementation of (oh)d increased jejunal claudin- gene expression significantly and this treatment effect was linear (p < . ). (oh)d also tended to increase muc and mrna expression (p < . ). interestingly, pedv increased sucrase-isomaltase (si) and occludin protein levels, but high doses (oh)d ( . µg (oh)d /kg) inhibited these increase (figure ). pedv induced a significant increase in the mrna expression of rigi, tlr , myd , il , il , ifnλ , and mxa compared to con (table , p < . ). however, a high dose of dietary (oh)d supplementation decreased tlr , tlr , myd , il , il , ifnλ , stat , mxa, ifnar , and trif mrna expression compared with con-pedv (table , p < . ). means not sharing the same superscript differ at p < . . the p means t-test between con and con-pedv, and p value means multiple comparisons among the pedv challenge groups. early studies suggested that vitamin d exerts broad-spectrum antiviral effects, including inhibiting the replication of dengue virus [ ] , hepatitis c virus [ ] , rotavirus [ ] , and others. due to the similarity in symptoms after rotavirus and pedv infection in pigs, we carried out this study to investigate whether (oh)d could alleviate pedv infection. we have previously demonstrated that increasing dietary (oh)d levels linearly increased serum (oh)d concentrations, but no treatment effects were observed in the growth performance of weaned pigs [ ] . in the current study, pedv challenge decreased adg and adfi, and resulted in severe diarrhea of the piglets. previous studies have reported that pedv infection reduced growth performance and resulted in severe diarrhea [ ] [ ] [ ] . we have analyzed the pedv-n gene in jejunum mucosa by pcr, and the results revealed that pedv was prevalent in the pedv-inoculated pigs, whereas con treatments were negative for pedv (supplementary materials). this suggested that the pedv infection model was successfully established. in the current study, dietary (oh)d supplementation decreased diarrhea scores, and . µg (oh)d /kg treatment showed the minimum diarrhea scores and diarrhea rate, which indicated that supplementation with a high dose of (oh)d might alleviate the symptoms of pedv infection. the complement system is a part of the innate immune system, and not only participates in inflammation but also enhances the adaptive immune response [ ] . in this study, serum c level was increased with pedv challenge as compared with con. we also found that serum igm levels were increased by pedv challenge. immunoglobulins are the major secretory products of humoral immunity [ ] . these results indicated that the pedv infection activated the innate and humoral immune response. however, dietary (oh)d supplementation had no effect on serum c and igm levels under the conditions of pedv challenge. we inferred that (oh)d might exert an antiviral effect on intestinal mucosa, which is the main target of pedv replication [ ] . pedv infection always leads to morphological changes of the small intestine with a reduction in villus height and damage to intestinal integrity [ , ] . in this study, we also found that villus height and vcr were decreased, and crypt depth was increased with pedv challenge. in addition, serum dao activity was also increased by pedv challenge. serum dao activity is a marker of mucosal integrity [ ] . these results indicated that pedv induced morphological changes of jejunum and an increase in intestinal permeability. the . µg (oh)d /kg (oh)d treatment was shown to be optimal in alleviating intestinal injury induced by pedv. therefore, this may be the reason why the . µg (oh)d /kg treatment showed the minimum diarrhea scores and diarrhea rate among the treatments. moreover, we found that claudin gene expression showed a linear response with increasing dietary (oh)d concentration, and (oh)d tended to increase muc expression under the conditions of pedv challenge. it suggested that high dose (oh)d supplementation could improve tight junction protein expression to maintain intestinal barrier integrity. interestingly, levels of si, an intestinal absorptive cell marker, were significantly increased by pedv challenge. a previous study has shown that pedv infection decreased goblet cells in intestinal villous [ ] . hence, we infer that pedv infection could cause disorder differentiation of intestinal cells, which promotes the differentiation of intestinal stem cells to intestinal absorption cells, while decreasing differentiation to intestinal secretion cells. however, high dose (oh)d supplementation significantly reduced the si expression induced by pedv, which might be beneficial for maintaining normal intestinal function. we also found occludin expression was increased with pedv challenge compared with con, but . and . µg (oh)d /kg groups inhibited this increase. luo et al. [ ] demonstrated that overexpression of occludin in target cells makes them more susceptible to pedv infection, which indicated that occludin plays an essential role in pedv infection. in the present study, we speculated that . µg (oh)d /kg supplementation might ease pedv infection through decreasing occludin expression. innate immune response plays an important role in defense against viral infections in mammalian cells. during viral infection, the virus is recognized by pattern-recognition receptors (prrs) including toll-like receptors (tlrs) and retinoic acid-inducible gene i (rigi) or melanoma differentiation gene (mda ), then infs and proinflammatory cytokines are produced for initiation of the inherent antiviral immune response [ , ] . different types of ifns bind to different receptors. type i ifns (ifn-α and ifn-β) signal through ifnar and ifnar to activate the jak-stat signaling pathway. and type iii ifns (ifn-λs) signal through ifnlr and il r to activate jak-stat signaling pathway to induce the expression of hundreds of interferon stimulating genes [ ] . unlike type i interferon receptors, which are seemingly ubiquitous, type iii ifn receptors are confined to the mucosal epithelium [ ] . thus, ifn-λs mainly play an antiviral role in mucosal epithelial cells. in the current study, pedv increased the prr, inflammatory cytokine, and ifnλ expression in the jejunum mucosa. this indicates that the ifns signaling pathway was activated by pedv in the intestine of piglets. since the high dose of (oh)d supplementation showed a better protective effect than the low dose groups, we investigated whether high dose supplementation could alleviate pedv infection by regulating immunity. we found that a high dose of (oh)d inhibited the prr, ifnλ, stat , and mxa expression. it was suggested that dietary (oh)d supplementation inhibited the activation of intestinal immunity induced by pedv. previous studies have shown that vitamin d attenuated rotavirus infection and reduced the viability of mycobacterium tuberculosis through regulating autophagy and cathelicidin [ , ] . therefore, we speculated that the suppression of the ifn signaling pathway from high dose supplementation of (oh)d might be due to decreased pedv replication. in addition, we also found that high doses of (oh)d inhibited jejunal mucosa il and il mrna expression compared with con-pedv. this suggested that high dose (oh)d supplementation might inhibit intestinal inflammation induced by pedv. reducing the expression of intestinal inflammatory cytokines is also beneficial in maintaining normal intestinal function. in summary, the results of the current study indicate that dietary supplementation of . µg/kg (oh)d alleviated the severity of diarrhea of piglets infected with pedv by improving the intestinal structure and immune response, and maintaining regular intestinal function. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . figure s . porcine epidemic diarrhea virus induces caspase-independent apoptosis through activation of mitochondrial apoptosis-inducing factor porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis effects of porcine epidemic diarrhea virus infection on nursery pig intestinal function and barrier integrity immunohistochemical detection of the vomiting-inducing monoamine neurotransmitter serotonin and enterochromaffin cells in the intestines of conventional or gnotobiotic (gn) pigs infected with porcine epidemic diarrhea virus (pedv) and serum cytokine responses of gn pigs to acute pedv infection differential gene modulation of pattern-recognition receptor tlr and rig-i-like and downstream mediators on intestinal mucosa of pigs infected with pedv non s-indel and pedv s-indel strains outbreak of porcine epidemic diarrhea in suckling piglets porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus vitamin d and immune function vitamin d supplementation alleviates rotavirus infection in pigs and ipec-j cells via regulating the autophagy signaling pathway dietary vitamin d supplementation attenuates immune responses of pigs challenged with rotavirus potentially through the retinoic acid-inducible gene i signalling pathway biological activity of -hydroxycholecalciferol, a metabolite of vitamin d feeding -hydroxycholecalciferol improves gilt reproductive performance and fetal vitamin d status evaluation of relative bioavailability of -hydroxycholecalciferol to cholecalciferol for broiler chickens nutrient requirements of swine: eleventh revised edition effects of montmorillonite-zinc oxide hybrid on performance, diarrhea, intestinal permeability and morphology of weanling pigs a new mathematical model for relative quantification in real-time rt-pcr butyrate promotes slow-twitch myofiber formation and mitochondrial biogenesis in finishing pigs via inducing specific micrornas and pgc- α expression the alpha, -dihydroxy-vitamin d reduces dengue virus infection in human myelomonocyte (u ) and hepatic (huh- ) cell lines and cytokine production in the infected monocytes vitamin d: an innate antiviral agent suppressing hepatitis c virus in human hepatocytes effects of dietary -hydroxyvitamin d supplementation on growth performance, immune function and antioxidative capacity in weaned piglets n-acetylcysteine supplementation alleviates intestinal injury in piglets infected by porcine epidemic diarrhea virus porcine epidemic diarrhea virus reduces feed efficiency in nursery pigs the effect of porcine reproductive and respiratory syndrome virus and porcine epidemic diarrhea virus challenge on growing pigs i: growth performance and digestibility the complement system in regulation of adaptive immunity structure and function of immunoglobulins in situ hybridization for the detection and localization of porcine epidemic diarrhea virus in the intestinal tissues from naturally infected piglets the effect of porcine reproductive and respiratory syndrome virus and porcine epidemic diarrhea virus challenge on growing pigs ii: intestinal integrity and function diamine oxidase (histaminase). a circulating marker for rat intestinal mucosal maturation and integrity goblet cell depletion in small intestinal villous and crypt epithelium of conventional nursing and weaned pigs infected with porcine epidemic diarrhea virus tight junction protein occludin is a porcine epidemic diarrhea virus entry factor immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling distinct poly (i-c) and virus-activated signaling pathways leading to interferon-β production in hepatocytes ifn-lambda (ifn-λ) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo vitamin d induces autophagy in human monocytes/macrophages via cathelicidin this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we sincerely acknowledge the assistance of runqi fu, huifen wang and linyan jia for their assistance during the animal experiments and laboratory analyses. the authors declare no conflict of interest. the funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -xvc wx authors: wink, michael title: chapter allelochemical properties or the raison d'être of alkaloids date: - - journal: nan doi: . /s - ( ) - sha: doc_id: cord_uid: xvc wx this chapter provides evidence that alkaloids are not waste products or functionless molecules as formerly assumed, but rather defense compounds employed by plants for survival against herbivores and against microorganisms and competing plants. these molecules were developed during evolution through natural selection in that they fit many important molecular targets, often receptors, of cells, which are seen in molecules that mimic endogenous neurotransmitters. the chapter discusses that microorganisms and herbivores rely on plants as a food source. since both have survived, there must be mechanisms of adaptations toward the defensive chemistry of plants. many herbivores have evolved strategies to avoid the extremely toxic plants and prefer the less toxic ones. many herbivores have potent mechanisms to detoxify xenobiotics, which allow the exploitation of at least the less toxic plants. in insects, many specialists evolved that are adapted to the defense chemicals of their host plant, in that they accumulate these compounds and exploit them for their own defense. alkaloids function as defense molecules against insect predators in the examples studied, and this is further support for the hypothesis that the same compound also serves for chemical defense in the host plant. it needs more experimental data to understand fully the intricate interconnections between plants, their alkaloids, and herbivores, microorganisms, and other plants. organisms. we must also consider that plants compete with other plants (of the same or different species) for light, water, and nutrients. how do plants defend themselves against microorganisms (including bacteria, fungi, and viruses), herbivores, and plants? because plants do rather well in nature, this question has often been overlooked. we are well aware of the defensive strategies of higher animals against microbes and predators ( , , , , , , ) . the complex immune system with its cellular and humoral components is a well-studied area in the context of vertebrate-microbe interactions. against predating animals, nature evolved weapons, armor, crypsis, thanatosis, deimatic behavior, aposematism, flight, or defense chemicals (usually called "poisons") ( ). it is evident that most of these possibilities are not available for plants with their sessile and "passive" life-style. what then is their evolutionary solution? we can distinguish the following defense mechanisms in plants ( , , , , ) ; the mechanisms are not independent and may act cooperatively and synergistically. we should be aware that many species have additionally evolved specialized traits in this context. . mechanical protection is provided by thorns, spikes, trichomes, glandular hairs, and stinging hairs (which are often supported by defense chemicals). . formation of a thick bark on roots and stems can be considered as a sort of armor, and the presence of hydrophobic cuticular layers as a penetration barrier directed against microbes. b. cell walls are biochemically rather inert with reduced digestibility to many organisms because of their complex cellulose, pectin, and lignin molecules. callose and lignin are often accumulated at the site of infection or wounding ( , ) and form a penetration barrier. c. synthesis of inhibitory proteins (e.g., lectins, protease inhibitors) or enzymes (e.g., chitinase, lysozyme, hydrolases, nucleases) that could degrade microbial cell walls or other microbial constituents would be protective, as well as synthesis of peroxidase and phenolase, which could help inactivate phytotoxins produced by many bacteria and fungi. these proteins are either stored in the vacuole . allelochemical properties of alkaloids or are secreted as exoenzymes into the cell wall or the extracellular space ( , ) . these compounds are thus positioned at an "advanced and strategically important defense position." in addition, storage proteins (of cereals and legumes) are often deficient in particular essential amino acids, such as lysine or methionine. d. as a widely distributed and important trait, secondary metabolites with deterrenthepellent or toxic properties against microorganisms, viruses, and/or herbivores may be produced ( - , - ) . these allelochemicals can be constitutively expressed, they may be activated by wounding (e .g., cyanogenic glycosides, glucosinolates, coumaryl glycosides, alliin, ranunculin), or their de ~o u o synthesis may be induced by elicitors (so-called phytoalexins), infection, or herbivory ( , , [ ] [ ] [ ] . these products are often synthesized and stored at strategically important sites [epidermal tissues or in cells adjacent to an infection ( , )] or in plant parts that are especially important for reproduction and survival [flowers, fruits, seeds, bark, roots ( , , ) ]. in animals, we can observe the analogous situation in that many insects and other invertebrates (especially those which are sessile and unprotected by armor), but also some vertebrates, store secondary metabolites for their defense which are often similar in structure to plant allelochemicals ( , , , , , - , [ ] [ ] [ ] ) . in many instances, the animals have obtained the toxins from their host plants ( , , , , - ). hardly any zoologist or ecologist doubts that the principal function of these secondary metabolites (which are often termed ''toxins" in this context) in animals is that of defense against predators or microorganisms ( , , , [ ] [ ] [ ] . these defense compounds are better known as natural products or secondary metabolites. the latter expression originally meant compounds which are not essential for life, and thus distinct from primary metabolites ( , , ) . unfortunately the term "secondary" has also a pejorative meaning, indicating perhaps that the compounds have no importance for the plant. as discussed in this chapter, just the opposite is true. more than , natural products have been reported from plants so far ( , , ) . owing to the sophistication in phytochemical methods, such as chromatography (hplc, glc) and spectroscopy (nmr, ms) , new products are reported at rapid intervals. because only - % of all higher plants, which consist of over , species, have been analyzed phytochemically in some detail, the overall real number of secondary products is certainly very large. it is a common theme that an individual plant does not produce a single natural product, but usually a moderate number of major metabolites and a larger number of minor derivatives. within a taxon secondary metabolites often share a common distribution pattern and are therefore of some importance for phytochemical systematics. classic taxonomy, however, has taken little account of alkaloid distribution: if the same alkaloid is present in two plants of the same taxon, this is interpreted as evidence for a relationship, but its occurrence in two plants of nonrelated taxa is taken as evidence of independent evolution. because secondary metabolites are also derived characters that were selected during evolution, their general value for taxonomy and systematics is certainly smaller than formerly anticipated ( ). for many years, secondary metabolites were considered as waste products or otherwise functionless molecules, merely illustrating the biochemical virtuosity of nature ( , ) . in and , errera and stahl ( , , ) published the idea that natural products are used by plants for chemical defense against herbivores. since the leading plant physiologists of that time were mostly anti-darwinian, they were not willing to accept the defense argument, which was too much in line with the darwinian concept. therefore, this early defense concept was negated and remained forgotten for nearly years. in , fraenkel( ) reopened the debate in a review article and presented new data supporting the view that secondary metabolites serve as chemical defense compounds against herbivores. during the next three decades this concept was improved experimentally, and we can summarize the present situation as follows although the biological function of many plant-derived secondary metabolites has not been studied experimentally, it is now generally assumed that these compounds are important for the survival and fitness of a plant and that they are not useless waste products, as was suggested earlier in the twentieth century ( , ) . in many instances, there remains a need to analyze whether a given compound is active against microorganisms (viruses, bacteria, fungi), against herbivores (molluscs, arthropods, vertebrates), or against competing plants (so-called allelopathy). in some instances, additional functions are the attraction of pollinating or seed-dispersing animals, for example, by colored compounds such as betalains (within the centrospermae), anthocyanins, carotenoids, and flavonoids or by fragrances such as terpenes, amines, and aldehydes ( , ) . physiological roles, such as uv protection [by flavonoids or coumarins ( , )], nitrogen transport or storage ( , , ) , or photosynthesis (carotenoids), may be an additional function. allelochemicals are often not directed against a single organism, but generally against a variety of potential enemies, or they may combine the roles of both deterrents and attractants (e.g., anthocyanins and many essential oils can be attractants in flowers but are also insecticidal and antimicrobial). thus, many natural products have multiple functions, a fact which is easily overlooked since most scientists usually specialize on a narrow range of organisms (i.e., a microbiologist will usually not check whether an antibiotic alkaloid also deters the feeding of caterpillars). to understand all the interactions we need to adopt a holistic, that is, interdisciplinary, approach. it might be argued that the defense hypothesis cannot be valid since most plants, even those with extremely poisonous metabolites (from the human point of view), are nevertheless attacked by pathogens and herbivores. however, we have to understand and accept that chemical defense is not an absolute process. rather, it constitutes a general barrier which will be effective in most circumstances, that is, most potential enemies are repelled or deterred. plants with allelochemicals at the same time represent an ecological niche for potential pathogens and herbivores. during evolution a few organisms have generally been successful in specializing toward that niche (i.e., in a particular toxic plant) in that they found a way to sequester the toxins or become immune to them ( , , ) . this is especially apparent in the largest class of animals, the insects (probably with several million species on earth), which are often highly host plant specific. the number of these "specialists" is exceedingly small for a given plant species as compared to the number of potential enemies that are present in the ecosystem. we can compare this situation with our immune system: it works against the majority of microorganisms but fails toward a few viruses, bacteria, fungi, and protozoa, which have overcome this defense barrier by clever strategies. nobody would call the immune system and antibodies useless because of these few adapted specialists! we should adopt the same argument when we consider plants' defenses by secondary metabolites ( ) . since secondary metabolites have evolved in nature as biologically active compounds with particular properties in other organisms, many of them are useful to mankind as pharmaceuticals, fragrances, flavors, colors, stimulants, or pesticides. in addition, many allelochemicals provide interesting lead structures that organic medicinal chemists can develop into new and more active compounds. about - % of higher plants accumulate alkaloids ( , ). the incidence of alkaloid production varies between taxa to some degree; for example, about - % of species of the solanaceae and apocynaceae are michael wink alkaloidal, whereas other families contain few alkaloid-producing species. some alkaloids have a wide distribution in nature: caffeine occurs in the largest number of families, lycorine in the largest number of genera and berberine in the largest number of species. alkaloids are not restricted to higher plants (although they are here most numerous); they are also present in club mosses (lycopodium), horsetails (equisetum), fungi, and animals such as marine worms (e.g., nereidae), bryozoans, insects (e.g., coccinellidae, solenopsidae), amphibians (toads, frogs, salamanders), and fishes. alkaloids thus represent one of the largest groups of natural products, with over , known compounds at present, and they display an enormous variety of structures, which is due to the fact that several different precursors find their way into alkaloid skeletons, such as ornithine, lysine, phenylalanine, tyrosine, and tryptophan ( ) ( ) ( ) . in addition, part of the alkaloid molecule can be derived from other pathways, such as the terpenoid pathway, or from carbohydrates ( [ ] [ ] [ ] . whereas the structure elucidation of alkaloids and the exploration of alkaloid biosynthetic pathways have always commanded much attention, there are relatively few experimental data on the ecological function of alkaloids. this is the more surprising since alkaloids are known for their toxic and pharmacological properties and many are potent pharmaceuticals. alkaloids were long considered to be waste products [even by eminent alkaloid researchers such as w. . james and kurt mothes ( , , )l. because nitrogen is a limiting nutrient for most plants, a nitrogenous waste product would be a priori unlikely. the waste product argument probably came from animal physiology: carnivorous animals take up relative large amounts of proteins and nucleic acids, containing more nitrogen than needed for metabolism, which is consequently eliminated as uric acid or urea. a similar situation or need, however, is not applicable for plants. in fact, many plants remobilize their nitrogenous natural products (including alkaloids) from senescing organs such as old leaves ( , , ). if alkaloids were waste products, we would expect the opposite, namely, accumulation in old organs which are shed. on the other hand, the alkaloids produced by animals were never considered to be waste products by zoologists, but rather regarded as defense chemicals ( , , ) . thus, the more plausible hypothesis is that alkaloids of plants, microorganisms, and animals, like other allelochemicals, serve as defense compounds. this idea is intuitively straightforward, because many alkaloids are known as strong poisons for animals and homo sapiens. as a prerequisite for an alkaloid to serve as a chemical defense compound we should demand the following criteria. ( ) the alkaloid should have significant effects against microbes and/or animals in bioassays. ( ) the compounds should be present in the plant at concentrations that are of the same order (or, better, even higher) as those determined in the bioassays. ( ) the compound should be present in the plant at the right time and the right place. ( ) evidence should be provided that a particular compound is indeed important for the fitness of a plant. although more than , alkaloids are known, only few (- - %) have been analyzed for biochemical properties, and even fewer for their ecophysiological roles. in most phytochemical studies only the structures of alkaloids have been elucidated, so that often no information is available on their concentrations in the different parts and through the ontogenetic development of a plant, or on their biological activities. furthermore, the corresponding studies were usually designed to find useful medicinal or sometimes agricultural applications of alkaloids, not to elucidate their evolutionary or ecological functions. these objections have to be kept in mind, because an alkaloid is sometimes termed "inactive" in the literature, which usually means less active than a standard compound already established as a medicinal compound (such as penicillins in antimicrobial screenings). in many medicinal experiments relatively low doses are applied because of the toxic properties of many alkaloids. if the same compound would have been tested at relevant (which normally means elevated) concentrations that are present in the plant, an ecologically relevant activity might have been detected. another restriction is that the activities of alkaloids have been tested with organisms that are sometimes irrelevant for plants but medicinally important. however, if a compound is active against escherichia coli, it is likely that is is also active against other gram-negative and plant-relevant bacteria. nevertheless, most of the data obtained in these studies (tables i-viii ) provide important information which at present permits extrapolation to the function of alkaloids in plants. in this chapter the focus is on the biological activity of alkaloids (the information available on the pharmacological properties of alkaloids is mostly excluded), and we try to discuss these data from an ecological perspective. in the following, the possible functions of alkaloids in plant-animal, plant-plant, and plant-microbe interactions are discussed in more detail. it is nearly impossible to cover the literature exhaustively. therefore, an overview of the allelochemical properties of alkaloids is presented. because of the large amount of data (literature up to is included), the selection of examples must remain subjective to some degree. nevertheless, the author would be grateful to receive information or publications about relevant omissions. because homo supiens and domestic animals are to some degree herbivores, a large body of empirical knowledge has accumulated on the toxic properties of alkaloids (tables i through v) and alkaloid-containing plants. previously, the toxic properties of alkaloids in vertebrates was part of the definition (as a common denominator) for this group of natural products ( , ) . in the following, the toxic or adverse effects of alkaloids are separately discussed for invertebrates (mainly insects) and vertebrates. among the invertebrates, insects have been extremely successful from the evolutionary point of view, and they form the largest class of organisms on our planet as far as the number of both individuals and species is concerned. entomologists estimate that the number of insects is at least million, but tropical rain forests may harbor up to - million species, many of which are still unknown and, owing to the fast extinction of this ecosystem, will probably also disappear without having been discovered and studied by scientists. most insects are herbivores, and adaptation to host plants and their chemistry is often very close and complex ( i , , , , , - , , ) . whereas insects rely on plants for food, many plants need insects for pollination and seed dispersal. in the latter context we often find that plants attract insects by chemical means (colors, fragrances, sugars, amino acids). at the same time, other secondary metabolites are employed to discourage the feeding on flowers and seeds. the close association between plants, especially the angiosperms, and insects evolved during the last million years. some scientists have called this phenomenon a "coevolutionary" process, but it has to be recalled that the associations seen today are not necessarily those in which the chemical interactions originally evolved ( , , ). applications of synthetic insecticides have shown that resistance to these new compounds can occur rapidly, sometimes encompassing only a dozen generations. times can also be much longer. if plant species are introduced to a new continent or island, it usually takes a long time before new pathogens or herbivores become adapted and specialized to this new species. for example, lupinus polyphyllus from north america has a number of specialized herbivores, but is rarely attacked by herbivores in europe. this lupine left its enemies behind when it was transferred to europe three centuries ago. about years ago, however, the north american lupine aphid (macrosiphum albifrons) was introduced to europe accidentally. this aphid is specialized to alkaloid-rich lupines with lupanine as a major alkaloid. at present, this aphid has spread over most of europe and is now colonizing its former host, l. polyphyllus ( , ) . insect herbivores can be divided into two large groups whose strategies with respect to the plant's defense chemistry differ substantially ( ). the polyphagous species can exploit a wide range of host plants, whereas the mono-/oligophagous insects are often specialized on one or a small number of (often systematically related) hosts. polyphagous insects, namely, species which feed on a wide variety of food plants, are usually endowed with fantastic and powerful olfactory receptors ( ) that allow the distinction between plants with high or low amounts of "toxins." the receptors also allow insects to ascertain the quality of the essential products present, such as lipids, proteins, or carbohydrates ( ). these "generalists," as we can also call this subgroup of herbivores, are usually deterred from feeding on plants which store especially noxious metabolites and select those with less active ones (such as our crop species, where man has bred away many of the secondary metabolites that were originally present; see table xi ). alternatively, they change host plants rapidly and thus avoid intoxication. in addition, most polyphagous species have evolved active detoxification mechanisms, such as microsomal oxidases and glutathione peroxidase, which lead to the rapid detoxification and elimination of dietary secondary products ( , , , ). in contrast, mono-and oligophagous species often select their host plants with respect to the composition of the nutrients and secondary metabolites present. for these "specialists" the originally noxious defense compounds are often attractive feeding and oviposition stimulants. these insects either tolerate the natural products or, more often, actively sequester and exploit them for their own defense against predators or for other purposes ( , , - , , , , , - ). these observations seem to contradict the first statement, that secondary metabolites are primarily defense compounds, and a number of renowned authors have fallen into this logical pit, such as mothes ( ) and robinson ( ). however, these specialized insects are exceptions to the general rule. for these specialists, the defense chemistry of the host plant is usually not toxic, but they are susceptible to the toxicity of natural toxins from non-host plants ( ) . as compared to the enormous number of potential herbivores, the number of adapted monophagous species is usually very small for a particular plant species. quite a number of alkaloids have been tested toward herbivorous insects (table i ). in general it is observed that many alkaloids can act as feeding deterrents at higher concentrations (>i%, w/w). given the choice, insects tend to select a diet with no or only a small dose of alkaloids. also, specialists avoid most "toxins" except those of their host plants. these data indicate that under natural conditions plants with a high content of alkaloids should be safe from most herbivorous insects, with the exception of particular monophagous species or a few very potent polyphagous ones. if insects have no choice or if they are very hungry, the deterrency threshold value is much reduced, and they often feed on a diet with alkaloids that they would normally avoid ( , ). in this case we have the chance to test the toxicity of an ingested alkaloid. if insects do not take up alkaloid-containing food, alkaloid toxicity can be assessed to some degree by topical application or by injection ( table i) . as can be seen from table i a substantial number of alkaloids display significant insect toxicity, including nicotine, piperine, lupine alkaloids, caffeine, gramine, strychnine, berberine, ephedrine, and steroidal alkaloids. only the specialists can tolerate the respective alkaloids. the tobacco hornworm (manduca sexta), for example, can grow on a diet with more than % nicotine without any adverse effects. most of the nicotine is either degraded or directly eliminated via the malpighian tubules and in feces ( ). because nicotine binds to the acetylcholine (ach) receptor, it is likely that in manduca this receptor has been modified in such a way that ach can still bind, but not nicotine (so-called target site modification). the toxic effects of alkaloids in insects (table i) can be caused by their interference with diverse cellular and intracellular targets. since most mechanisms have not yet been elucidated for insects, this issue is discussed below in the section on vertebrate toxicity (see table iv ). with some caution we can extrapolate to insect toxicity. because homo sapiens and domestic animals are largely herbivores, a voluminous body of information on the adverse effects of secondary metabolites has accumulated over the centuries. many allelochemicals and alkaloids are feeding deterrents for vertebrates, owing to their bitter or pungent taste or bad smell, and instinctively a foul-smelling, bitter, or pungent diet is normally avoided. examples of bitter alkaloids (at least for man) are quinine, strychnine, brucine, and sparteine, and for pungent alkaloids are capsaicin, and piperine. it should be recalled that these taste properties are not identical for all animals. for example, geese, which are obligate herbivores, hardly avoid food with alkaloids or smelly compounds (amines, mercaptoethanol) that man would hardly touch ( ). conversely, fragrances that are attractive to us are highly repellent to geese ( ). even within a given population taste can differ significantly. it has been observed that a substantial proportion of homo sapiens cannot detect the smell of hcn, whereas others are highly sensitive. furthermore, olfactory sensitivity can differ with age, sex, and hormonal cycles. bitterness varies with the chemical structure of an alkaloid. with the quinolizidine alkaloids (qas) the following scale was assessed for man: mean detection levels are . % for sparteine, . % for lupanine, and . % for hydroxylupanine ( ). whereas we know a few parameters of olfactory qualities in homo sapiens, often much less or hardly anything is known for most other vertebrates. alkaloids are famous for their toxic properties in vertebrates, and plants that produce alkaloids are often classified by man as poisonous or toxic plants. for a number of alkaloids the respective ld,, values have been determined with laboratory animals, especially mice, but also rats, guinea pigs, cats, rabbits, dogs, or pigeons. table i presents an overview for alkaloids, including the very poisonous alkaloids aconitine, coniine, atropine, brucine, curarine, ergocornine, physostigmine, strychnine, colchicine, germerine, veratridine, cytisine, delphinidine, and nicotine. toxicity is usually highest if the alkaloids are applied parenterally [intravenously (i.v.), intraperitoneally (i.p.), and subcutaneously (s.c.)] as compared to oral application [per s (p.o.)]. also, some of the alkaloids which are made or stored by animals are strong vertebrate poisons, including batrachotoxin, batrachotoxinin a, anabasine, glomerine, maitotoxin, nereistoxin, palytoxin, saxitoxin, and tetrodotoxin ( , , , ) . although the general toxicity of alkaloids differs from species to species, the data in table i generally show that many alkaloids are more or less toxic to vertebrates. the toxic effects observed with intact animals has its counterpart in the cytotoxic effect, which has been recorded for nearly alkaloids (table ). these data have been obtained by screening many natural products for anticancer activity. however, an alkaloid that can kill a cancer cell is usually also toxic for "normal" cells. therefore, the data shown in table i are another indication of the general toxicity of alkaloids toward animals. because this toxicity applies also for herbivores, the production of alkaloids by plants can certainly be interpreted as a potent antiherbivore mechanism. for a number of alkaloids the mechanisms underlying the toxic effects have already been elucidated in some detail. we can distinguish molecular targets and processes that are important for all cells, such as synthesis of dna, rna, and proteins, replication, transcription, translation, membrane assembly and stability, electron chains, or metabolically important enzymes or proteins including receptors, hormones, and signal compounds (table iv ). in the following we discuss some of these toxic effects. a. cellular targets nucleic acids. dna, the macromolecule which holds all the genetic information for the life and development of an organism, is a highly vulnerable target. it is not surprising that a number of secondary metabolites have been selected during evolution which interact with dna or dnaprocessing enzymes. some alkaloids bind to or intercalate with dna/rna (table iv) and thus affect replication or transcription, or cause mutations, leading to malformations or cancer (table v) : -methoxyellipticine, dictamnine, ellipticine, harmane alkaloids, melinone f, quinine and related alkaloids, skimmianine, avicine, berberine, chelerythrine, coptisine, coralyne, fagaronine, nitidine, sanguinarine, pyrrolizidine alkaloids (pas), cycasin, olivacine, etc. many of the intercalating molecules are planar, hydrophobic molecules that fit within the stacks of at and gc base pairs. other alkaloids act at the level of dna and rna polymerases, such as vincristine, vinblastine, avicine, chelilutine, coralyne, fagaronine, nitidine, amanitine, hippeastrine, and lycorine, thus impairing the processes of replication and transcription. whereas these toxins usually cause a rapid reaction, some alkaloids cause long-term effects in vertebrates in that they are mutagenic or carcinogenic (table v) . besides basic data obtained in salmonella or drosophila, there are a few reports which illustrate the potent mutagenic effect of alkaloids on vertebrates. anagyrine, anabasine, and coniine cause "crooked calf disease" if pregnant cows or sheep feed on these alkaloids during the first period of gestation ( , , , , , ) . the offspring born show strong malformation of the legs. some of the steroid alkaloids (e.g., cyclopamine, jervine, and veratrosine), which are produced by veratrum species, cause the formation of a central large cyclopean eye ( - , an observation that was probably made by the ancient greeks and thus led to the mythical figure of the cyclops. it is likely that any herbivore which regularly feeds on plants containing these alkaloids will suffer from reduced productivity and reduced fitness in the long term. in effect, the plants which contain these alkaloids are usually avoided by vertebrate herbivores. another long-term effect caused by alkaloids with carcinogenic properties has been discovered only recently (tables iv and v) . the alkaloid aristolochic acid, which is produced by plants of the genus aristolochia, is carcinogenic. the mechanism of action of this alkaloid is believed to be similar to the well-known carcinogen nitrosamine ( , ) , because of its no, group. pyrrolizidine alkaloids and their n-oxides, which are abundantly produced by members of the asteraceae and boraginaceae but also occur in the families apocynaceae, celestraceae, elaeocarpaceae, euphorbiaceae, fabaceae, orchidaceae, poaceae, ranunculaceae, rhizo- esterified ( , ) . after oral intake, the n-oxides are reduced by bacteria in the gut. the lipophilic alkaloid base is resorbed and transported to the liver, where it is "detoxified" by microsomal enzymes. as a result, a reactive alkylating agent is generated, which can be considered as a pyrrolopyrrolidine. the alkaloid can then cross-link dna and rna and thus cause mutagenic or carcinogenic effects (especially in the liver) ( ). thus, pyrrolizidine alkaloids represent highly evolved and sophisticated antiherbivore compounds, which utilize the widespread and active detoxification system of the vertebrate liver. the pa story is very intriguing, since it shows how ingenious nature was in the "arms race." the herbivores invented detoxifying enzymes, and nature the compound which is activated by this process. a herbivore feeding on pa-containing plants will eventually die, usually without reproducing properly. only those individuals which carefully avoid the respective bitter-tasting plants maintain their fitnes and thus survive. the protection due to pa can easily be seen on meadows, where senecio and other pa-containing plants are usually not taken by cows and sheep, at least as long other food is available. protein biosynthesis is essential for all cells and thus another important target. indeed, a number of alkaloids have already been detected (although few have been studied in this context) that inhibit protein biosynthesis in uitro (table iv) , such as vincristine, vinblastine, emetine, tubulosine, tyramine, sparteine, lupanine and other quinolizidine alkaloids, cryptopleurine, haningtonine, homohamngtonine, haemanthamine, isohamngtonine, lycorine, narciclasine, pretazettine, pseudolycorine, tylocrebrine, tylophorine, and tylocrepine. for lupine alkaloids, it was determined that the steps which are inhibited are the loading of acyl-trna with amino acids, as well as the elongation step. the inhibitory activity was strongly expressed in heterologous systems, that is, protein biosynthesis in the producing plants, such as lupines, was not affected ( ). electron chains. the respiratory chain and atp synthesis in mitochondria demand the controlled flux of electrons. this target seems to be attacked by ellipticine, pseudane, pseudene, alpinigenine, sanguinarine, tetrahydropalmatine, ch,-(ch ),,- , -methyl-piperidines, capsaicin, the hydroxamic acid dimboa, and solenopsine. as mentioned before, however, only a few alkaloids have been evaluated in this context (table v) . biomembranes and transport processes. a cell can operate only when it is enclosed by an intact biomembrane and by a complex compartmenta- tion that provides separated reaction chambers. because biomembranes are impermeable for ions and polar molecules, cells can prevent the uncontrolled efflux of essential metabolites. the controlled flux of these compounds across biomembranes is achieved by specific transport proteins, which can be ion channels, pores, or carrier systems. these complex systems are also targets of many natural products (table iv) . disturbance of membrane stability is achieved by -methoxyellipticine, ellipticine, berbamine, cepharanthine, tetrandrine, steroidal alkaloids, irehdiamine, and malouetine. steroidal alkaloids, such as solanine and tomatine, which are present in many members of the solanaceae, can complex with cholesterol and other lipids of biomembranes; cells are thus rendered leaky. cells carefully control the homeostasis of their ion concentrations by the action of ion channels (na+,k+, ca + channels) and through na+,k+-atpase and ca +-atpase. these channels and pumps are involved in signal transduction, active transport processes, and neuronal and neuromuscular signaling. inhibition of transport processes (ion channels, carriers) is achieved by (table iv) acronycine, ervatamine, harmaline, quinine, reserpine, colchicine, nitidine, salsolinol, sanguinarine, stepholidine, caffeine, sparteine, monocrotaline, steroidal alkaloids, aconitine, capsaicine, cassaine, maitoxin, ochratoxin, palytoxin, pumiliotoxin, saxitoxin, solenopsine, and tetrodotoxin. a special class of ion channels in the central nervous system and involved in neuromuscular signal transfer are coupled with receptors of neurotransmitters such as noradrenaline (na), serotonin, dopamine, glycine, and acetylcholine (ach). we can distinguish two types. type is a ligand-gated channel (i.e., a receptor), which is part of an ion-channel complex, such as the nicotinergic ach-receptor. in type the receptor is an integral protein. when a neurotransmitter binds, the receptor changes its conformation and induces a conformational change in an adjacent gprotein molecule, which consists of three subunits. the a subunit then activates the enzyme adenylate cyclase, which in turn produces camp from atp. the camp molecule is a second messenger which activates protein kinases or ion channels directly, which in turn open for milliseconds (e.g., the muscarinergic ach receptor). a number of alkaloids are known whose structures are more or less similar to those of endogenous neurotransmitters. targets can be the receptor itself, the enzymes which deactivate neurotransmitters, or transport processes, which are important for the storage of the neurotransmitters in synaptic vesicles. alkaloids relevant here include (table iv) brucine, ergot alkaloids, eseridine, serotonin, physostigmine, gelsemine, p-carboline alkaloids, strychnine, yohimbine, berberine, bicuculline, bulbocapnine, columbamine, coptisine, coralyne, corlumine, ephedrine, ga- lanthamine, laudanosine, nuciferine, palmatine, papaverine, thebaine, cytisine and other quinolizidine alkaloids, heliotrine, chaconine and other steroidal alkaloids, cocaine, atropine, scopolamine, anabaseine, arecoline, dendrobine, gephyrotoxin, histrionicotoxin, methyllycaconitine, muscarine, nicotine, pilocarpine, psilocin, psilocybin, morphine, mescaline, and reserpine. a number of these alkaloids are known hallucinogens, which certainly decrease the fitness of an herbivore feeding on them regularly. cytoskeleton. many cellular activities, such as motility, endocytosis, exocytosis, and cell division, rely on microfilaments and microtubules. a number of alkaloids have been detected which can interfere with the assembly or disassembly of microtubules (table iv) , namely, vincristine, vinblastine, colchicine, maytansine, maytansinine, and taxol. colchicine, the major alkaloid of colchicum autumnale (liliaceae), inhibits the assembly of microtubules and the mitotic spindle apparatus. as a consequence, chromosomes are no longer separated, leading to polyploidy . whereas animal cells die under these conditions, plant cells maintain their polyploidy, a trait often used in plant breeding because polyploidy leads to bigger plants. because of this antimitotic activity, colchicine has been tested as an anticancer drug; however, it was abandoned because of its general toxicity. the derivative colcemide is less toxic and can be employed in the treatment of certain cancers ( ). also, cellular motility is impaired by colchicine; this property is exploited in medicine in the treatment of acute gout, in order to prevent the migration of macrophages to the joints. for normal cells, and thus for herbivores, the negative effects can easily be anticipated, and colchicine is indeed a very toxic alkaloid which is easily resorbed because of its lipophilicity . another group of alkaloids with antimitotic properties are the bisindole alkaloids, such as vinblastine and vincristine, which have been isolated from catharanthus roseus (apocynaceae). these alkaloids also bind to tubulin ( ). both alkaloids are very toxic, but are nevertheless important drugs for the treatment of some leukemias. from taxus baccata (taxaceae) the alkaloid taxol has been isolated. taxol also affects the architecture of microtubules in inhibiting their disassembly ( ). nonalkaloidal compounds to be mentioned in this context include the lignan podophyllotoxin ( ). in conclusion, any alkaloid which impairs the function of microtubules is likely to be toxic, because of their importance for a cell, and, from the point of view of defense, a wellworking and well-shaped molecule. enzyme inhibition. the inhibition of metabolically important enzymes is a wide field that cannot be discussed in full here (see table iv ). briefly, inhibition of camp metabolism (which is important for signal transduction and amplifications in cells), namely, inhibition of adenylate cyclase by anonaine, isoboldine, tetrahydroberberine and inhibition of phosphodiesterase by -ethyl-p-carboline, p-carboline- -propionic acid, papaverine, caffeine, theophylline, and theobromine are some examples. inhibition of hydrolases, such as glucosidase, mannosidase, trehalase, and amylase, is specifically achieved by some alkaloids (table iv) b. action at organ level. whereas the activities mentioned before are more or less directed to molecular targets present in or on cells, there are also some activities that function at the level of organ systems or complete organisms, although, ultimately, they have molecular targets, too. central nervous system and neuromuscular junction. a remarkable number of alkaloids interfere with the metabolism and activity of neurotransmitters in the brain and nerve cells, a fact known to man for a thousand years (table iv) . the cellular interactions have been discussed above. disturbance of neurotransmitter metabolism impairs sensory faculties, smell, vision, or hearing, or they may produce euphoric or hallucinogenic effects. a herbivore that is no longer able to control its movements and senses properly has only a small chance of survival in nature, because it will have accidents (falling from trees, or rocks, or into water) and be killed by predators. thus euphoric and hallucinogenic compounds, which are present in a number of plants, and also in fungi and the skin of certain toads, can be regarded as defense compounds. some individuals of homo sapiens use these drugs just because of their hallucinogenic properties, but here also it is evident that long-term use reduces survival and fitness dramatically. the activity of muscles is controlled by ach and na. it is plausible that an inhibition or activation of neurotransmitter-regulated ion channels will severely influence muscular reactivity and thus the mobility or organ function (heart, blood vessels, lungs, gut) of an animal. in the case of inhibition, muscles will relax; in the case of overstimulation, muscles will be tense or in tetanus, leading to a general paralysis. alkaloids which activate neuromuscular action (so-called parasympathomimetics) include nicotine, arecoline, physostigmine, coniine, cytisine, and sparteine. inhibitory (or parasympatholytic) alkaloids include hyoscyamine and scopolamine, (see above) ( ) . skeletal muscles as well as muscle-containing organs, such as lungs, heart, circulatory system, and gut, and the nervous system are certainly very critical targets. the compounds are usually considered to be strong poisons, and it is obvious that they serve as chemical defense compounds against herbivores, since a paralyzed animal is easy prey for predators or, if higher doses are ingested, will die directly (compare ld,, values in table ). inhibition of digestive processes. food uptake can be reduced by a pungent or bitter taste in the first instance, as mentioned earlier. the next step may be the induction of vomiting, diarrhea, or the opposite, constipation, which negatively influences digestion in animals. the ingestion of a number of allelochemicals such as emetine, lobeline, morphine, and many other alkaloids causes these symptoms ( ). another mode of interference would be the inhibition of carriers for amino acids, sugars, or lipids, or of digestive enzymes. relevant alkaloids are the polyhydroxyalkaloids, such as swainsonine, deoxynojirimycin, and castanospermine, that inhibit hydrolytic enzymes, such as glucosidase, galactosidase, trehalase (trehalose is a sugar in insects which is hydrolyzed by trehalase), and mannosidase selectively (table iv) . nutrients and xenobiotics (such as secondary metabolites) are transported to the liver after resorption in the intestine. in the liver, the metabolism of carbohydrates, amino acids, and lipids takes place with the subsequent synthesis of proteins and glycogen. the liver is also the main site for detoxification of xenobiotics. lipophilic compounds, which are easily resorbed from the diet, are often hydroxylated and then conjugated with a polar, hydrophilic molecule, such as glucuronic acid, sulfate, or amino acids ( ). these conjugates, which are more water soluble, are exported via the blood to the kidney, where they are transported into the urine for elimination. both liver and kidney systems are affected by a variety of secondary metabolites, and the pyrrolizidine alkaloids have been discussed earlier (tables iv and v) . the alkaloids are activated during the detoxification process, and this can lead to liver cancer. also, many other enzyme or metabolic inhibitors (e.g., amanitine), discussed previously, are liver toxins. many alkaloids and other allelochemicals are known for their diuretic activity ( ). for an herbivore, an increased diuresis would also mean an augmented elimination of water and essential ions. since na' is already limited in plant food (an antiherbivore device?), long-term exposure to diuretic compounds would reduce the fitness of an herbivore substantially. disturbance of reproduction. quite a number of allelochemicals are known to influence the reproductive system of animals, which ultimately reduces their fitness and numbers. antihormonal effects could be achieved by mimicking the structure of sexual hormones. these effects are not known for alkaloids yet, but have been confirmed for other natural products. estrogenic properties have been reported for coumarins, which di-merize to dicoumarols, and isoflavones ( , ) . insect molting hormones, such as ecdysone, are mimicked by many plant sterols, which include ecdysone itself, such as in the fern polypodium uulgare, or azadirachtin from the neem tree ( , ) . juvenile hormone is mimicked by a number of terpenes, present in some coniferae. spermatogenesis is reduced by gossypol from cottonseed oil ( ) . the next target is the gestation process itself. as outlined above, a number of alkaloids are mutagenic and lead to malformation of the offspring or directly to the death of the embryo ( table v) . the last step would be the premature abortion of the embryo. this dramatic activity has been reported for a number of allelochemicals, such as mono-and sesquiterpenes and alkaloids. some alkaloids achieve this by the induction of uterine contraction, such as the ergot and lupine alkaloids ( ) . the antireproductive effects are certainly widely distributed, but they often remain unnoticed under natural conditions. nevertheless, they are defense strategies with long-term consequences. blood and circulatory system. all animals need to transport nutrients, hormones, ions, signal compounds, and gas between the different organs of the body, which is achieved by higher animals through blood in the circulatory system. inhibitors of the driving force for this process, the heart muscle, have already been discussed. however, the synthesis of red blood cells is also vulnerable and can be inhibited by antimitotic alkaloids such as vinblastine or colchicine ( ) . some allelochemicals have hemolytic properties, such as saponins. if resorbed, these compounds complex membrane sterols and make the cells leaky. steroidal alkaloids from solanum or veratrum species display this sort of activity as well as influencing ion channels (table iv) . allergenic effects. a number of secondary metabolites influence the immune system of animals, such as coumarins, furanocoumarins, hypericin, and helenalin. common to these compounds is a strong allergenic effect on those parts of the skin or mucosa that have come into contact with the compounds ( , , ) . activation or repression of the immune response is certainly a target that was selected during evolution as an antiherbivore strategy. the function of alkaloids in this context is hardly known. this selection of alkaloid activities, though far from complete, clearly shows that many alkaloids inhibit central processes at the cellular, organ, or organismal level, an important requisite for a chemical defense compound. however, most of the potential targets for the , alkaloids known at present remain to be established. if no activity has been reported, it often means that nobody looked into this question scientifically, and not that a particular alkaloid is without a certain biological property. summarizing this section, it is safe to assume that most alkaloids can affect animals and thus herbivores significantly. dead plants easily rot due to the action of bacteria and fungi, whereas metabolically active, intact plants are usually healthy and do not decay ( ) . how is this achieved? the aerial organs of terrestrial plants have epidermal cells that are covered by a more or less thick cuticle, which consists of waxes, alkanes, and other lipophilic natural products ( , ) . this cuticle layer is water repellent and chemically rather inert, and it thus constitutes an important penetration barrier for most bacteria and fungi. in perennial plants and in roots we find another variation of this principle in that plants often form resistant bark tissues. the only way for microbes to enter a healthy plant is via the stomata or at sites of injury, inflicted by herbivory, wind, or other accidents. at the site of wounding, plants often accumulate suberin, lignin, callose, gums, or other resinous substances which close off the respective areas ( , ) . in addition, antimicrobial agents are produced such as lysozyme and chitinase, lytic enzymes stored in the vacuole which can degrade bacterial and fungal cell walls, protease inhibitors which can inhibit microbial proteases, or secondary metabolites with antimicrobial activity. secondary metabolites have been routinely screened for antimicrobial activities by many researchers, since the corresponding assays are relatively easy to perform. these studies have usually been directed toward a pharmaceutical application, and they often employ the routine methods for screening microbial or fungal antibiotics. it may happen that these tests do not detect an antibacterial activity of a compound because the wrong test species or a nonrelevant concentration was assayed. in the pharmaceutical context we search for very active compounds which can be employed at low concentrations. therefore, the higher concentrations, which would be more meaningful ecologically, are often not tested. these precautions have to be kept in mind when screening the literature for data on the antimicrobial activity of alkaloids. secondary compounds known for their antimicrobial activity include many phenolics (e.g., flavonoids, isoflavones, and simple phenolics), glucosinolates, nonproteinogenic amino acids, cyanogenic glycosides, acids, aldehydes, saponins, triterpenes, mono-and disesquiterpenes, and last but not least, alkaloids ( , , , , ) . in table vi alkaloids are tabulated for which antibacterial activities have been detected. the alkaloids usually affect more gram-positive than gram-negative bacteria. especially well represented are alkaloids which '-hydroxytabernamine hydroxytetrahydrosecamine tetrandrine thalicarpine thalicerbine thalidasine thalidezine thaliglucinone thalistine thalistyline thalmelatine thalmirabine thalphenine thalrugosaminine thalrugosidine thalrugosine tubocurarine ( i +. active; -, no activity observed in the concentration range tested (many alkaloids were only assayed in low concentrations as microbial antibiotics); ad, agar diffusion, al, agar dilution; bg, biogram; ld, liquid culture; mic, minimal inhibitory concentration; pd, paper disk; sp, suspension; tlc, tlc disk test according to wolters and eilert ( ) . if more than one value is given, the data refer to different bacterial species tested. derive from tryptophan (indole alkaloids) and phenylalaninehyrosine, which may be due to the fact that these alkaloids have obtained considerable scientific attention since the discovery of many medicinally important compounds within these groups ( , , , , , , - ) . some of these alkaloids are highly antibiotic, with similar activities as fungal antibiotics, namely, cinchophylline ( ), dictamnine ( , fagarine ( ), stemmadine ( ), yuehchukene ( ), liriodenine ( , lysicamine ( ), oxonantenine ( ), sanguinarine ( ), solacasine ( , ) , rutacridone epoxide ( ), tryptanthrine (i@#), and tuberin ( , ) (table vi) . in many instances, when alkaloids are assessed for their antibacterial activity, they are often also tested for antifungal properties. usually yeasts and candidu are used as test organisms (table vii) . table vii lists ( i , ), thaliglucinone ( ), demissidine ( , ), solacasine ( ), soladulcidine ( , ), solasodine ( , )tidine ( , ), tomatine ( , ) , verazine ( ), cryptopleurine ( ) hydroxyrutacridone epoxide ( , tryptanthrine ( ), and tuberin ( ) . whereas the mode of action and targets of antibiotics of fungal and bacterial origin have been elucidated in many instances (see table iv ), relevant information for plant-derived compounds is scant. however, the molecular targets of some alkaloids have been determined at the general level, but not specifically for bacterial or fungal systems (table iv) that may be responsible for the antibiotic effects observed. the following interactions of alkaloids having antimicrobial properties with molecular targets of bacterial or fungal cells are likely (compare tables vi and vii with tables iv and v) . protein biosynthesis in ribosomes is affected by sparteine ( , , lupanine, angustifoline, -tigloyloxylupanine, and hydroxylupanine ( , , , , , ) . intercalation or binding to dna is influenced by fagaronine, dictamnine ( ), harman alkaloids ( , ) [binding to dna is light dependent ( )], berberine ( - , chelerythrine ( ), and sanguinarine ( , ) ; these compounds may thus inhibit important processes such as dna replication and rna transcription that are also vital for microorganisms. the stability of biomembranes may be disturbed by cepharanthine, tetrandrine, and steroidal alkaloids such as solamargine ( , solanine ( , , ), and solasonine ( ) , thus leading to an uncontrolled flux of metabolites and ions into microbial cells. inhibition of metabolically important enzymes is affected by berber- ine ( ), chelerythrine ( , ), chelidonine ( ), palmatine ( ), sanguinarine ( , ), solacongestidine ( ) , and papaverine. in contrast to antibiotics of microbial origin that could be classified as alkaloids from a chemical point of view in many instances, and which often interfere with the biosynthesis or maintenance of the cell wall (murein) (table iv) , such an interaction has not been described for plantderived compounds. since this topic has not been studied in detail it remains open whether this complex is another target for alkaloids. we can distinguish between secondary metabolites that are already present prior to an attack or wounding, so-called constitutive compounds, and others that are induced by these processes and made de now. inducing agents, which have been termed "elicitors" by phytopathologists, can be cell wall fragments of microbes, the plant itself, or many other chemical constituents ( , , - ) . the induced compounds are called "phytoalexins," which is merely a functional term, since these compounds often do not differ in structure from constitutive natural products. in another way this term is misleading, since it implies that the induced compound is only active in plant-microbe interactions, whereas in reality it often has multiple functions that include antimicrobial and antiherbivoral properties (see below). many of the antimicrobial alkaloids found are constitutively expressed and accumulated, that is, they are already present before an infection. using plant cell cultures, it was observed that some cultures start to produce new secondary metabolites when challenged with bacterial or fungal cell walls, culture fluids, or other chemical factors ( , , - ) . among the compounds found to be inducible are alkaloids such as sanguinarine and hydroxyrutacridone epoxide (see table xi ). quinolizidine alkaloids display some antimicrobial properties, besides their main role in antiherbivore defense ( ) (see table i ). on wounding, qa production is enhanced, thus increasing the already high alkaloid concentration in the plant; in other words, the antimicrobial and herbivoral effect is further amplified (table xi) ( , , ) . the reactions leading to the induction and accumulation of phytoalexins with phenolic structures have been studied in molecular detail ( , , - ) . these studies revealed that plants can detect and react rapidly to environmental problems, such as wounding or infection: within min of elicitation, mrnas coding for enzymes that catalyze the reactions leading to the respective defense compounds are increasingly generated, leading to the accumulation of the respective enzymes and consequently the production of the secondary metabolites ( , , - ) . similar processes are likely for alkaloids, but so far the mechanisms have not been elucidated. we assume that a substantial number of the , alkaloids have antimicrobial properties (which remain to be tested in most cases) that are directed against the ubiquitous and generalist microbes which have not table vi . if a range is given, the first value gives a % inhibition, the second value a % inhibition. specialized on a particular host plant. however, alkaloid production does not necessarily have to be involved with antimicrobial defense. for example, phytophthora or fusarium will attack alkaloid-rich plants of nicotiana, solanum esculentum, and s . tuberosum. cladosporium and fusarium can develop in nutrient-containing media enriched with alkaloids, and aspergillus niger can utilize alkaloids as a nitrogen source ( ). in addition, most plant species are known to be parasitized or infected by at least a few specialized bacteria or fungi which form close, often symbiotic, associations. in these circumstances an antimicrobial effect expected from the secondary metabolites present in the plant can often no longer be observed. we suggest that these specialists have adapted to the chemistry of their host plants. mechanisms may include inhibition of biosynthesis of the respective compounds, degradation of the products, or alteration of the target sites, which are then no longer sensitive toward a given compound (so-called target site modification). these mechanisms need to be established for most of the microbial specialists living on alkaloid-producing plants. some associations between plants and fungi are symbiotic in nature, such as rhizobia in root nodules of legumes or microrhizal fungi in many species. in lupines, nitrogen-fixing rhizobia are present both in alkaloid-rich and alkaloid-free plants. they must therefore be able to tolerate the alkaloids, which are also present in the root. alkaloid production in lupines is more or less unaffected whether or not the plants harbor rhizobia ( , ) . an ecologically important symbiosis between plants and fungi can be observed in fungal species that produce ergot alkaloids. graminaceous species that are infected by ergot suffer much less from herbivory because of the strong antiherbivoral alkaloids produced by the fungi ( ). a similar relationship may occur for other fungal species of plants, many of which produce secondary metabolites possessing animal toxicity. from the pharmaceutical point of view, few alkaloids are interesting as antibiotics, because many are highly toxic to vertebrates (tables i and ). since many alkaloids are antibacterial and antifungal (tables vi and vii) and are present in plants at relatively high concentrations (section iila), it seems likely that from an ecological perspective alkaloids, besides their prominant role in antiherbivore strategies, may play an important role also in the defense against microbial infections. it should be recalled that even alkaloid-producing plants synthesize antimicrobial proteins, such as chitinase and lysozyme, and other antimicrobial secondary products, such as simple phenolics, flavonoids, anthocyanins, saponins, and terpenes ( - , ) . a cooperative, or even synergistic, process could thus be operating. c. antiviral properties plants, like animals, are hosts for a substantial number of viruses, which are often transmitted by sucking insects such as aphids and bugs (heteroptera). resistance to viral infection can be achieved either by biochemical mechanisms that inhibit viral development and multiplication or by warding off vectors such as aphids in the first place. the assessment of antiviral activity is relatively difficult. as a result, only a few investigators have studied the influence of alkaloids on virus multiplication. nevertheless, at least alkaloids have been reported with antiviral properties (table viii) . only sparteine ( ) and cinchonidine ( ) have been tested for antiviral activities against a plant virus, the potato x virus. all other evidence for antiviral activities (table viii) table viii are difficult to interpret at present. polyhydroxy alkaloids, such as swainsonine, can block the action of endoplasmic reticulum-and golgi-localized glucosidases and mannosidases, which are important for the posttranslational trimming of viral envelope proteins. because alkaloids often deter the feeding of insects, such as aphids and bugs (table i ), viral infection rates may be reduced in alkaloid-rich plants. such a correlation exists for alkaloid-rich lupines (so-called bitter lupines) and low-alkaloid varieties (the so-called sweet lupines) (see table xii) . plants often compete with other plants, of either the same or different species, for space, light, water, and nutrients. this phenomenon can be intuitively understood when the flora of deserts or semideserts is analyzed, where resources are limited and thus competition intense ( , , - ) . a number of biological mechanisms have been described, such as temporal spacing of the vegetation period in which some species flower at an earlier season, when others are still dormant or ungerminated. it was observed by molisch in ( ) that plants can also influence each other by their constituent natural products, and he coined the term "allelopathy" for this process. secondary products are often excreted by the root or rhizosphere to the surrounding soil, or they are leached from the surface of intact leaves or from decaying dead leaves by rain ( , ) . both processes will increase the concentration of allelochemicals in the soil surrounding a plant, where the germination of a potential competitor may occur. allelopathy, namely, the inhibition of germination or of the growth of a seedling or plant by natural products, is well documented at the level of controlled in v i m experiments ( , , , - ) , but how it operates in ecosystems is still often a matter of controversy. it is argued, for example, that soil contains a wide variety of microorganisms which can degrade most organic compounds. thus allelochemicals might never reach concentrations high enough to be allelopathic. allelopathic natural products have been recorded in all classes of secondary metabolites. few research groups have studied the effect of alkaloids in this context, but at least alkaloids have been reported with allelopathic properties (table ix) . as can be seen from table ix , allelopathic activities can be found within nearly all structural types of alkaloids. at higher alkaloid concentrations, a marked reduction in the germination rate can be recorded regularly. more sensitive, however, is the growth of the radicle and hypocotyl. they respond to alkaloids at a much lower level, and usually a reduction in growth can be observed but sometimes also the opposite, either of which reduces the fitness of a seedling. in species which produce the compounds, the inhibitory effects can be absent, as was reported for quinolizidine alkaloids in lupines and colchicine in colchicum autumnale ( , ) . it is likely that autotoxicity is prevented either by a special modification of cellular target sites or by other mechanisms. alkaloids ( , , ), berberine ( - ), sanguinarine ( , ) and veratrum alkaloids]; inhibition of protein biosynthesis [e.g., emetine ( ) and quinolizidine alkaloids ( , , - , ) tables iv and ix) . the inhibitory action of quinolizidine alkaloids should be explained in this context ( , ) . they are very abundant in lupine seeds (up to - % dry weight). during germination, -hydroxylupanine is converted to ester alkaloids, such as -tigloyloxylupanine. the latter compound is predominantly excreted via the roots of young seedlings and in germination assays proved to be the most allelopathic qa. these alkaloids influence only heterologous systems, not the germination of lupine seeds themselves. when lupine and lepidium seeds were grown together in the same pot, growth of the lepidium seedlings was much reduced and inhibited, indicating that qas may also be relevant in the ecological context ( ) . although the number of alkaloids with known allelopathic properties is not large, owing to the limited number of studies conducted, it is clear from table ix that alkaloids can be toxic to plants, probably by interfering with basic metabolic or molecular processes. although comparably few alkaloids have been studied for their biological activities in detail, and considering that our data collection (tables i-ix) is far from complete, we can safely state that alkaloids have potent deterrent or poisonous properties in herbivorous animals, and also affect bacteria, fungi, viruses, and plants. the next question will be whether all the adverse activities of alkaloids, which are often assayed in in uitro systems only, are meaningful in nature. because most of the allelochemical activities are dose dependent (others may be synergistic, additive, etc.), the question is whether the amounts of alkaloids produced and stored in plants are high enough to be ecologically meaningful. it is difficult, and also dangerous, to make a general statement concerning alkaloid levels in plants. we must remember that alkaloid composition and levels are often tissue or organ specific ( , , ) . they may vary during the day [a diurnal cycle has been observed for qas and tropane alkaloids ( , , )l or during the vegetation period ( . , ) . furthermore, as in all biological systems, there are differences at the level of individual plants and between populations and subspecies. unfortunately, many phytochemical reports do not contain any quantitative information, or these data are given for the whole plant without realizing the above-mentioned variables. in addition, concentrations are usually given on a dry weight basis, which is appropriate in the chemical or pharmaceutical context. however, herbivores or pathogens do not feed on the dry plant in general, but on the "wet" fresh material. in the context of chemical ecology we urgently need data on a fresh weight basis. as an approximation, in this chapter we use a conversion factor of to convert dry weight to fresh weight data if only the dry weight data are available. summarizing the relevant phytochemical literature, we find that alkaloid levels are between . and % (dry weight), which is equivalent to o.oi-ls%fresh weight, or . - mg/gfresh weight. for plantscontaining quinolizidine alkaloids, actual alkaloid contents are given for a number organs or parts (table x ) , which fall in the range deduced before. we have evaluated the situation for quinolizidine alkaloids and found that the actual concentrations of alkaloids in the plant are usually much higher than the concentrations needed to inhibit, deter, or poison a microorganism or herbivore ( , , , ) . this means that plants obviously play safe and have stored more defense chemicals than actually needed. if we look at the ed,, and ld,, values given in tables through ix, it is likely that the situation is similar for other alkaloid-producing plants, but these correlations need to be experimentally established in most instances. it seems trivial that plants not only synthesize but also store their secondary products, which makes sense only in view of their ecological functions as defense compounds, since they can fulfil these functions only if the amounts stored are appropriate. achieving and maintaining the high levels of a defense compound are very demanding from the point of view of physiology and biochemistry. most allelochemicals would probably interfere with the metabolism of the producing plant if they would accumulate in the compartments where they are made ( ). whereas biosynthesis takes place in the cytoplasm, or in vesicles (berberine) or organelles such as chloroplasts (qas, coniine), the site of accumulation of water-soluble alkaloids is the central vacuole, and that of lipophilic compounds includes latex, resin ducts, or glandular hairs (e.g., nicotine) ( , ) . in this context it should be recalled that many alkaloids are charged molecules at cellular ph and do not diffuse across biomembranes easily. during recent years, evidence has been obtained that at least some alkaloids pass the tonoplast with the aid of a carrier system. the next problem is determining how the uphill transport, that is, the accumulation against a concentration gradient, is achieved. proton-alkaloid antiport mechanisms and ion trap and chemical trap mechanisms have been postulated and partially proved experimentally ( , , ) . thus, the sequestration of high amounts of alkaloids in the vacuole is a complex and energy-requiring task, which would certainly have been lost during evolution were it not important for fitness. as a rule of thumb, we can assume that all parts of an alkaloidal plant contain alkaloids, although the site of synthesis is often restricted to a particular organ, such as the roots or leaves. translocation via the phloem, xylem, or apoplastically must have therefore occurred. phloem transport has been demonstrated for quinolizidine, pyrrolizidine, and indolizidine alkaloids, and xylem transport for nicotine and tropane alkaloids ( , , ) . if the plant relies on alkaloids as a defense compound, these molecules have to be present at the right place and at the right time. alkaloids are often stored in specific cell layers, which can differ from the site of biosynthesis ( , , ) . in lupines, but also in other species ( , , alkaloids are preferentially accumulated in epidermal and subepidermal cell layers, reaching local concentrations between and mm (table x) , which seems advantageous from the point of view of chemical ecology, since a pathogen or small herbivore encounters a high alkaloid barrier when trying to invade a lupine. the accumulation of many alkaloids in the root or stem bark, such as berberine, cinchonine, and quinine, can be interpreted in a similar way. a number of plants produce laticifers filled with latex. for example, isoquinoline alkaloids in the family papaveraceae are abundant in the latex ( ), where they are sequestered in many small latex vesicles. in latex vesicles of chelidonium mujus the concentration of protoberberine and benzophenanthridine alkaloids can be in the range of . - . m, which is achieved by their complexation with equal amounts of chelidonic acid ( ). if a herbivore wounds such a plant, the latex spills out immediately. besides gluing the mandibles of an insect, the high concentration of deterrent and toxic alkaloids will usually do the rest, and, indeed, chelidonium plants are hardly attacked by herbivores. in addition, as these alkaloids are also highly antimicrobial (table iv) , the site of wounding is quickly sealed and impregnated with natural antibiotics. other well-known plants that have biologically active alkaloids in their latex belong to the families papaveraceae (genera papauer, macleya, and sanguinaria) and campanulaceae (genus lobelia) ( ) . it is intuitively plausible that a valuable plant organ must be more protected than others. alkaloid levels are usually highest during the time of flowering and fruit/seed formation. in annual species actively growing young tissue, leaves, flowers, and seeds are often alkaloid-rich, whereas in perennial ones, like shrubs and trees, we find alkaloid-rich stem and root barks in addition. all these plant parts and organs have in common that they are important for the actual fitness or for the reproduction and thus the long-term survival of the species. spiny species, which invest in mechanical defense, accumulate fewer alkaloids than soft-bodied ones ( ); examples are isoquinoline alkaloids in cacti or qas in legumes ( ) . if a plant produces few and large seeds, their alkaloid levels tend to be higher than in species with many and small seeds ( , ); thus. a plant with few and big seeds is generally a rich source of alkaloids, which makes sense in view of the defense hypothesis. these few examples show that accumulation and storage of alkaloids have been optimized in such a way that they are present at strategically important sites where they can ward off an intruder at the first instance of attack. thus, specialized locations must be regarded as adaptive. alkaloid concentrations can fluctuate during the vegetation period, or even during a day ( , ). but in biochemical terms their biosynthesis and accumulation are constitutive processes. this ensures that a certain level of defensive compounds is present at any time. furthermore, continuous turnover is a common theme for molecules of the cells whose integrity is important, such as proteins, nucleic acids, and signal molecules. the same seems to be true for a defense compound. an alkaloid which mimics a neurotransmitter, such as hyoscyamine, nicotine, or sparteine, could be oxidized or hydrolyzed in the cell by chance, and thus would be automatically inactivated. only by replacing these molecules continuously can the presence of the active compounds be guaranteed. for example, it was suggested that nicotine has a half-life of hr in nicotiana plants, and that more than % of the co, fixed passes through this alkaloid ( ). in other groups of natural products it was possible to show that plants can react to infection by microbes or to wounding by herbivores by inducing the production of new defense compounds. these compounds are termed "phytoalexins" in phytopathology ( ) ( ) ( ) . classic examples of phytoalexins include isoflavones, phenolics, terpenes. protease inhibitors, coumarins, and furanocoumarins. using plant cell cultures it could be shown that a similar process can be observed with some alkaloidal plants, which start to produce alkaloids with antimicrobial properties (e.g., sanguinarine, canthin- -one, rutacridone alkaloids) when challenged with elicitors from bacterial or fungal cell walls (table xi) . but what is the situation after herbivory? when plants are eaten by large herbivores, a de nouo synthesis would be almost useless for a plant (except maybe trees), since this would not be quick enough. the situation is different, however for small herbivores such as insects or worms, which may feed on a particular plant for days or weeks. here the de nouo production of an allelochemical would be worthwhile. there are indeed some preliminary experimental data that support this view. in liriodendron rirlipifera several aporphine alkaloids accumulate after wounding, which are otherwise not present ( ). in tobacco the produc- " cc, cell culture; pl, plant tion of nicotine, in lupines that of qas, and in atropci belleidonnu that of hyoscyamine are induced by wounding, thus increasing the already high levels of alkaloids by up to a factor of . whereas the response was seen after - hr in lupines, it took days in nicotiunu and in atropei (table xi) . we suggest that the wound-induced stimulation of alkaloid formation is not an isolated phenomenon, but rather an integral part of the chemical defense system. the induced antimicrobial and antiherbivoral responses show that plants can detect environmental stress and that secondary metabolism is flexible and incorporated in the overall defense reactions. many details on how a plant perceives and transmits information remain to be disclosed, but this will surely be a stimulating area of research in the future. although the physiology and metabolism of most alkaloids are extremely intricate ( ) and often not known, the available data suggest that they are organized and regulated in such a way that alkaloids can fulfill their ecological defense function. in other words, the alkaloids are present at the right time, the right place, and the right concentration. the aforementioned arguments strongly support the hypothesis that alkaloids serve as defense compounds for plants. besides circumstantial evidence, we would welcome critical experiments which clearly prove that alkaloids are indeed important for the fitness and survival of the plants producing them. we suggest that if a plant species which normally produces alkaloids is rendered alkaloid-free, it should have a reduced fitness because it is much more molested by microorganims and herbivores than its alkaloid-producing counterpart. for one group of alkaloids, the quinolizidine alkaloids, these experiments have already been performed ( , , , , ) . as mentioned before, qas constitute the main secondary products of many members of the leguminosae, especially in the genera lirpinus, genistu, cyfisiis, bccptisiu, thrrmopsis, sophoru, ormosici, and others ( ). lupines have relatively large seeds which contain up to - % protein, up to % lipids, and - % alkaloids. to use lupine seed for animal or human nutrition, homo scipiens, for several thousand years, used to cook the seeds and leach out the alkaloids in running water. this habit has been reported for the egyptians and greeks in the old world, and for the indians and incas of the new world. the resulting seeds taste sweet, in contrast to the alkaloid-rich ones which are very bitter. in mediterranean countries people still process lupines in the old way, and sometimes the seeds are salted afterward and served as an appetizer, comparable to peanuts. at the turn of the twentieth century, german plant breeders set out to grow alkaloid-free lupines, the so-called sweet lupines. although sweet lupines are extremely rare in nature ( in > . ), the efforts were largely successful, and at present, sweet varieties with an alkaloid content lower than . % exist for lupinus albus, l. mutabilis, l . luteus, l. angustifolius, and l . polyphyllus. as far as we know, the sweet varieties differ from the original bitter wild forms only in the degree of alkaloid accumulation. this offers the chance to test experimentally whether bitter lupines have a higher fitness than sweet ones with regard to microorganisms and herbivores. the results of these experiments were clearcut ( , , , ) (table xu). in the greenhouse, where plants are protected from herbivores or pathogens, no clear advantage was seen. when lupines were planted in the field, without being fenced in and without man-made chemical protection, however, a dramatic effect was regularly encountered, especially with regard to herbivores ( , , , ) . rabbits (cuniculus europaeus) and hares (lepus europaeus) clearly prefer the sweet plants and leave the bitter plants almost untouched, at least as long as there was an alternative food source. before dying rabbits will certainly try to eat bitter lupines. a similar picture was seen for a number of insect species, such as aphids, beetles, thrips, and leaf-mining flies (table xii) , namely, the sweet forms were attacked, whereas the alkaloid-rich ones were largely protected. the alkaloid-poor variety of l . luteus also became a host of acyrthosiphon pisii ( ). in poland, where the sweet yellow lupine is one of the more important fodder plants, the invasion of the aphids became a serious problem not only because the aphid enfeebles the plants by sucking its phloem sap, but also because it transfers a viral disease. the disease, known as lupine narrow leafness, decreases seed production in infected plants, and the infection takes place early, that is, prior to the plants' blossoming. thus, a mixed population of sweet and bitter lupines can, after a few generations, lose all sweet forms. infestation by the aphid and the following viral infection accelerate the elimination of alkaloid-poor plants, which, even without infection, are already inferior in seed production ( ). this observation again stresses the importance of alkaloids for the fitness of lupines. plant breeders have also observed that bacterial, fungal, and viral diseases are more abundant in the sweet forms, but this effect has not been documented in necessary detail. these experiments and observations clearly prove the importance of qas for lupines, but it should not be forgotten that other secondary metabolites, such as phenolics, isoflavones, terpenes, saponins, stachyose, erucic acid, and phytic acid, are also present in lupines and may exert additional or even synergistic effects. the lupine example also tells us about the standard philosophy and problems of plant breeding. with our present knowledge on the ecological importance of qas for the fitness of lupines, it seems doubtful whether the selection of sweet lupines was a wise decision. in order to grow them we have had to build fences and, worse, to employ man-made chemical pesticides, which have a number of well-documented disadvantages. it can be assumed that similar strategies, namely, breeding away unwanted chemical traits, have been followed with our other agricultural crops, with the consequence that the overall fitness was much reduced ( ). we can easily observe the reduced fitness by trying to leave crop species to themselves in the wild: they will quickly disappear and not colonize new habitats. there are, however, alternatives. taking lupines as an example, we could devise large-scale technological procedures to remove alkaloids from the seeds after harvest (similar to sugar raffination from sugar beets). at present a few companies are actively exploring these possibilities. one idea is to produce pure protein, lipids, dietary fibers from bitter seeds. a spin-off product would be alkaloids, which could be used either in medicine (sparteine is exploited as a drug to treat heart arrhythmia) or in agriculture as a natural plant protective, that is, as an insecticide ( , ) . it is evident, however, that each plant has developed its own strategy for survival. if all plants would follow the same strategy, it would be an easy life for herbivores and pathogens, since being adapted to one species would mean adapted to all species. this specialization becomes evident if we analyze the qualitative patterns of secondary metabolite profiles present in the plant. we regularly see one to five main alkaloids in a plant, but also several (up to ) minor alkaloids. this qualitative pattern is not constant, but differs among organs, developmental stages, individuals, populations, and species. normally, we classify the compounds as belonging to one or two chemical groups. this does not mean, however, that their biological activities are identical. on the contrary, the addition of a lipophilic side chain to a molecule seems to be a small and insignificant variation from the chemical point of view, but this may render the compound more lipophilic, and thus more resorbable. in consequence, its toxicity may be higher (see qas in table i ). thus, a herbivore or pathogen has to adapt not only to one group of chemicals but to the individual compounds present. as the composition of these chemicals changes, it is even more difficult for them to cope. therefore, we suggest that structural diversity and continuous variation are means by which nature counteracts the adaptation of specialists. in medicine, we do a similar thing if we want to control microbial diseases. to overcome or to prevent resistance of bacteria toward a particular antibiotic, very often mixtures of structurally different antibiotics are applied, whose molecular targets often differ. if only one antibiotic were given to all patients, the development of resistance would be much favored. it has been argued that alkaloids cannot have a significant role in plants because not all plant species produce alkaloids (only % of all plants do). these authors, such as robinson ( , have overlooked the fact that if all plants would produce one single alkaloid, even a very toxic alkaloid such as colchicine, it could be certain that nearly all herbivores would have developed a resistance toward this alkaloid. only the variation of secondary metabolites, and thus of the targets which they affect, provides a means to develop efficient defense compounds. the arguments of robinson would be correct if there were higher plants without any secondary metabolites, which, nevertheless, would thrive in nature; however, these plants are not known. from an evolutionary perspective it is not important whether the defense chemical is an alkaloid or a terpene; it is only essential that it affect certain and important targets in herbivores or pathogens. although the biological activities of many alkaloids have not yet been studied and their ecological functions remain to be elucidated or proved, we can nevertheless safely say that alkaloids are neither waste nor functionless molecules, but rather they are important fitness factors, probably mostly antiherbivore compounds. since nature obviously favored multitasking, additional activities, such as allelopathic or antimicrobial activities, are plausible. for quinolizidine and pyrrolizidine alkaloids, these multiple functions are already well documented (tables i-x) . plants that defend themselves effectively constitute an ecological niche almost devoid of herbivores and pathogens. it is not surprising that during evolution a number of organisms evolved which have specialized on a particular host plant species and found ways to tolerate, or even to exploit, the defense chemistry of their hosts ( , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as compared to the huge number of potential enemies, the number of adapted specialists is usually small, and in general a "status quo" or equilibrium can be observed between the specialists (or parasites) and their hosts. a specialist is not well advised to kill its host, since this would destroy its own resources; a mutualism is more productive for survival. host plant-specific specialists occur within bacteria, fungi, and herbivores. the interaction of the former two groups is a central topic for plant pathologists. they often find that susceptible and nonsusceptible microbe strains exist. in most cases, it is not known how these microbial specialists achieved a relationship with the host plant chemistry, for example, whether they degrade secondary metabolites or whether they simply toler-ate them. many phytopathogenic bacteria and fungi produce their own secondary metabolites, which are often toxic to plants. it is assumed that these phytotoxins serve to weaken the host plants' defense, but may be this is not the whole story. many grasses are infected with fungi that produce ergot alkaloids. it has been assumed that these fungi (e.g., clauiceps) are proper parasites. in recent years, however, experimental evidence suggests that the relationship between grasses and ergot may be of a symbiotic nature ( ). ergot alkaloids are strong vertebrate toxins (tables i-iv) ; they mimic the activity of several neurotransmitters, such as dopamine, serotonin, and noradrenaline (table iv) . in fact, the impact of herbivores on populations which were highly infected by fungi was more reduced than those without. this means that the fungi exploit the nutrients of their host plants and supply them with strong poisons, which are not produced by the plants themselves. since the fungi do not kill their hosts, this close interrelationship seems to be of mutual interest. we expect that similar relationships are likely to be detected in the future. as mentioned earlier, a large number of mono-and oligophagous insects exist which have adapted to their host plants and the respective defense chemistry in complex fashions. in general, we can see the following main schemes ( , , , , , ) . in type adaptations, a species "learns" (or, as we should say, during evolution variants have been selected by natural selection which can tolerate a noxious defense compound) (a) by finding a way to avoid its resorption in the gut; (b) if resorption cannot prevented, by eliminating the toxin quickly via the malpighian tubules or degrading it by detoxifying microsomal and other enzymes; and (c) by developing a target site that is resistant to the toxin, such as a receptor which no longer bind the exogenous ligand. alternatively, in type strategies a species not only tolerates a plants' defense compound, but exploits it for its own defense or for other purposes, such as pheromones i , , ) . examples of type include manduca sexra, whose larvae live on nicoriana and other solanaceous plants. the alkaloids present in these plants, such as nicotine or hyoscyamine, are not stored but are degraded or directly eliminated with the feces ( ). in addition, it has been postulated that nicotine may either not diffuse into nerve cells or that the acetylcholine recpetor no longer binds nicotine as in "normal" animals ( ). the potato beetle (leptinotarsa decernlineata) lives on solanurn species containing steroid alkaloids, which are tolerated, but not stored, by this species, the bruchid beetle callosohruchus fasciarus predates seeds of qa-rich plants, such as laburnum anagyroides; this beetle eliminates most of the dietary cytisine with the feces ( ). examples of type are to some degree more interesting. in a number of plants alkaloids are translocated via the phloem ( ). when aphids live on these plants they are in direct contact with the alkaloids present. a number of examples are known at present which show that adapted aphids can store the dietary alkaloids. examples are the quinolizidines in aphis cytisorum, a. genistae, and macrosiphum albifrons, the pyrrolizidines in aphis jacobaea, a . cacaliaster, and aconitine in aphis aconiti ( , ) . for alkaloid-storing m . albifrons it was shown experimentally that the qas stored provide protection against carnivorous beetles, such as carabus problematicus or coccinella septempunctata ( , ) . acyrthosiphon spartii prefers sparteine-rich cytisus scoparius plants ( ); although it is likely that this species also stores qas, it has not been demonstrated to do so. larvae of the pyralid moth uresiphita reversalis live on qa-producing plants, such as teline monspessulana. the larvae store some of the dietary alkaloids, especially in the integument and also the silk glands. the uptake is both specific and selective and is achieved by a carrier mechanism. whereas alkaloids of the -oxosparteine type dominate in the plant, it is the more toxic cytisine that is accumulated by the larvae, with the oxosparteines being eliminated with the feces ( , ) . the larvae gain some protection from storing qas, as was shown in experiments with predatory ants and wasps. when the larvae pupate, most of the alkaloids stored are used to impregnate the silk of the cocoon, thereby providing defense for this critical developmental stage ( , ). the emerging moth lives cryptically, has no aposematic coloring, and does not contain alkaloids. in contrast the alkaloid-rich larvae are aposematically colored and live openly on the plants ( , ) . the larvae of the blue butterfly (plebejus icaroides) feed only on lupines, rich in alkaloids. as far as we know, the larvae do not sequester or store the dietary alkaloids ( ). helopeltis feeds on cinchona bark, which is rich in cinchonine-like alkaloids; it stores and uses them for its own defense ( ). larvae of the butterflies pachlioptera aristolochiae, zerynthia polyxena, ornithoptera priamus, and battus philenor live on arisrolochia plants and were shown to take up and sequester aristolochic acid, a carcinogenic alkaloid discussed earlier, as an effective defense compound ( , , ) . the best-studied group of acquired alkaloids are the pyrrolizidines, which are produced by plants, especially in the families asteraceae and boraginaceae ( ). some arctiid larvae of tyria jacobaea, cycnia mendica, amphicallia bellafrix, arginia cribaria, and arctia caja were shown to store the dietary pas and exploit them for their own defense ( , , , , - , ) . in tyria jacobaea, arctia caja, diacrisia sannio, phragmatobia fuligonosa, and callimorpha dominula pas are taken up and stored in the integument ( ). monarch butterflies (e.g., danaus plexipus) combine two sets of natural compounds. larvae feed on plants rich in cardiac glycosides and use them as chemical defense compounds. adult butterflies visit plants with pas, where they collect pas that are converted to pheromones or transferred to their eggs ( ,f , , , f, ) . a similar pa utilization scheme was observed with larvae of the moth utetheisa ornatrix ( , ) , where the compounds were shown to be deterrent for spiders and birds ( , ) . the chrysomelid beetle oreina feeds on pa-containing plants, such as adenostyles, and stores the dietary pas in the defense fluid ( , ). in the arctiid creatonotos transiens was observed an advanced exploitation of pas ( , , , - ) . the alkaloids are phagostimulants for larvae, which are endowed with specific alkaloid receptors. dietary pyrrolizidine n-oxides are resorbed by carrier-mediated transport. after resorption, free pas are converted to the respective n-oxides and ( s)-heliotrine to ( r)-heliotrine. the latter form is later converted to a male pheromone, ( r)-hydroxydanaidal. pas are stored in the integument, where they serve as defense compounds and are not lost during metamorphosis. in the adult moth, however, the pas are mobilized. in the female adult, pas are translocated into the ovary and subsequently into the eggs. in the male, pas are necessary for the induction of abdominal scent organs and concomitantly for the biosynthesis of pa-derived pheromones, which are dissipated from these coremata. in addition, pas are transferred into the spermatophore and thus donated to the female. a significant amount of pas is further transferred to the eggs, which thus obtain chemical protection from the pas previously acquired by both male and female larvae. marine dinoflagellates produce a number of toxins, such as saxitoxin, surugatoxin, tetrodotoxin, and gonyautoxin, that affect ion channels (table iv). these algae are eaten by some copepods, fish, and molluscs that also store these neurotoxins ( , , , , , ) . as a consequence, these animals have acquired chemical defense compounds, which they can use against predators. this discussion is not meant to be complete, but should illustrate that a number of insect herbivores exploit the chemistry of their food plants. these insects are adapted and have evolved a number of molecular and biochemical traits that can be considered as prerequisites. however, many of the respective plant-insect interactions have not yet been studied, and it is therefore likely that the acquisition of dietary defense compounds is even more widely distributed in nature than anticipated. whereas insect herbivores are often highly host plant specific, vertebrate herbivores tend to be more of the polyphagous type, although some specialization may occur. for example, grouse (lagopus lagopus) or capercaillies (tetra urogallus) prefer plants of the families of ericaceae or coniferae, and crossbills seeds of picea and abies species, which are rich in terpenes. the australian koala is oligophagous and prefers terpene-rich species of the genus eucalyptus. for approximately million years, the only true herbivorous vertebrates have been the mammals. the mesozoic reptiles disappeared following the mesophytic flora. birds, though a few species feed on seeds and berries, seldom eat leaves (except geese and grouse), and they frequently use insects, in addition to plant parts, as a food source ( ). although a single plant can be a host for hundreds of insect larvae, hundreds of plants comprise a daily menu for a larger mammal. the strategies of the polyphagous species include the following. avoidance of plants with very toxic vertebrate poisons (these species are usually labeled toxic or poisonous by man) by olfaction or taste discrimination. often such compounds may be described as bitter, pungent, bad smelling, or in some other way repellent. . sampling of food from a wide variety of sources and thus minimizing the ingestion of high amounts of a single toxin. . detoxification of dietary alleochemicals, which can be achieved by symbiotic bacteria or protozoa living in the rumen or intestines, or by liver enzymes which are specialized for the chemical modification of xenobiotics. this evolutionary trait is very helpful for homo sapiens, since it endowed us with a means to cope with our man-made chemicals which pollute the environment. carnivorous animals, such as cats, are known to be much more sensitive toward plant poisons ( ). it was suggested that these animals, which d o not face the problem of toxic food normally, are thus not adapted to the handling of allelochemicals. some animals, such as monkeys, parrots, or geese, ingest soil. for geese ( ) it was shown that the ingested soil binds dietary allelochemicals, especially alkaloids ( ). this procedure would reduce the allelochemical content available for resorption. . animals are intelligent and can learn. the role of learning in food and toxin avoidance should not be underestimated, but it has not been studied in most species. for most vertebrate herbivores, the ways they manage to avoid, tolerate, or detoxify their dietary allelochemicals have not been explored. sometimes, only domesticated animals were used in experiments, but they tend to make more mistakes in food choice than the wild animals. more evidence on this subject is available for homo sapiens, who has evolved a number of "tricks," some of them obviously not anticipated by evolution. first, man tends to avoid food with bitter, pungent, or strongly scented ingredients. as a prerequisite he needs corresponding receptors in the nose or on the tongue which evolved during the long run of evolution as a means to avoid intoxication. second, our liver still contains a set of detoxifying enzymes which can handle most xenobiotics. furthermore, some of these enzymes, such as cytochrome p. oxidase, is inducible by dietary xenobiotics. third, besides these biological adaptations, man has also used his brain to avoid plant allelochemicals. (a) many fruits or vegetables are peeled. as many alkaloids and other compounds are stored in the epidermis, for example, steroid alkaloids in potato tubers or cucurbitacins in cucurbits, peeling eliminates some of these compounds from consumption. (b) most food is boiled in water. this leads to the thermal destruction of a number of toxic allelochemicals, such as phytohaemagglutinins, protease inhibitors, and some esters and glycosides. many watersoluble compounds are leached out into the cooking water and are discarded after cooking (e.g., lupines or potatoes). (c) south american indians ingest clay when alkaloid-rich potato tubers are on the menu. since clay binds steroidal alkaloids, geophagy is thus an ingenious way to detoxify potential toxins in the diet ( ) . (d) man has modified the composition of allelochemicals in his crop plants, in that unpleasant taste components have been reduced by plant breeding. from the point of view of avoidance, this strategy is plausible, but, as was discussed earlier, it is deleterious from the point of view of chemical ecology. these plants often lose their resistance against herbivores and pathogens, which then has to be replaced by man-made pesticides. in general, only a few plants are exploited by man as food, as compared to the , species present on our planet. this means that even homo sapiens with all his ingenuity has achieved only a rather small success, indicating the importance and power of chemical plant defenses. in this context, it is worth recalling that a number of animals are able to synthesize their own defense compounds, among them several alkaloids ( , , , - ) . these animals have the common feature that they are usually slow-moving, soft-bodied organisms. marine animals, such as mol-luscs, sponges, zooanthids, and fishes, have been shown to contain a variety of alkaloids, such as acrylcholine, neosaxitoxin, murexin, pahutoxin, palytoxin, petrosine, and tetramine, that are toxic to other animals ( . , , , , , , , , , ) . a number of nemertine worms, such as amphiporus or nereis, produce alkaloids such as , -bipyridyl, anabaseine, nemertelline, or nereistoxin, which are toxic to predators such as crayfish ( , , , ,) . arthropod-made alkaloids include glomerine and homoglomerine in glomerus ( ) , adaline in adalia ( ), coccinelline, euphococcinine, and derivatives in coccinella, epilachna, and other coccinellid beetles ( , , , ) , and stenusine in stenus ( ) , which are considered to be antipredatory compounds ( , , , - ) . solenopsis ants produce piperidine alkaloids which resemble the plant alkaloid coniine. these alkaloids are strong deterrents and inhibit several cellular processes, such as electron transport chains (table iv) ( , ) . many insects indicate the content of toxic natural products by warning colors (aposematism) or by the production of malodorous pyrazines ( , , , ) . not only are lower animals able to synthesize alkaloids, but also vertebrates, especially in the class amphibia. tree frogs of the genus dendrobates accumulate steroidal alkaloids, such as batrachotoxin, pumiliotoxins a-c, gephyrotoxin, and histrionicotoxin, in their skin, which are strong neurotoxins (table iv) ( , , ) . natives have used the alkaloids as arrow poisons. similar alkaloids (i.e., homobatrachotoxin) have recently been detected in passerine birds of the genus pitohui ( ) . salamanders, salamandra maculosa, which are aposematically colored, produce the toxic salamandrine and derivatives, alkaloids of the steroidal group ( , , ). salamandrine is both an animal toxic (paralytic) and an antibiotic. toads (bufonidae) produce in their skin cardiac glycosides of the bufadienolide type, but also a set of alkaloids, such adrenaline, noradrenaline, adenine, bufotenine, or bufotoxin ( , , ). except for bufotoxin, the other chemicals are, or mimic, neurotransmitters. these examples show that alkaloids found in animals can either be derived from dietary sources (see section ,d, ) or be made endogenously. common to both origins is their use as chemical defense compounds, analogous to the situation found in plants. in animals we can observe the trend that sessile species, such as sponges and bryozoans, or slow-moving species without armor, such as worms, nudibranchs, frogs, toads, and salamanders, produce active allelochemicals ( , , , ) , but not so those with weapons, armor, or the possibility for an immediate flight. plants merely developed a similar strategy as these "unprotected" animal species. in this context it seems amazing that hardly anybody has doubted the defensive role of alkaloids in animals, whereas people did, and still do, where alkaloids in plants are concerned. evidence is presented in this overview that alkaloids are not waste products or functionless molecules as formerly assumed ( , ), but rather defense compounds employed by plants for survival against herbivores and against microorganisms and competing plants. these molecules were obviously developed during evolution through natural selection in that they fit many important molecular targets, often receptors, of cells (i.e. they are specific inhibitors or modulators), which can clearly be seen in molecules that mimic endogenous neurotransmitters (table iv; section ii,a, ,a). on the other hand, microorganisms and herbivores rely on plants as a food source. since both have survived, there must be mechanisms of adaptations toward the defensive chemistry of plants. many herbivores have evolved strategies to avoid the extremely toxic plants and prefer the less toxic ones. in addition, many herbivores have potent mechanisms to detoxify xenobiotics, which allows the exploitation of at least the less toxic plants. in insects, many specialists evolved that are adapted to the defense chemicals of their host plant, in that they accumulate these compounds and exploit them for their own defense. alkaloids obviously function as defense molecules against insect predators in the examples studied, and this is further support for the hypothesis that the same compound also serves for chemical defense in the host plant. the overall picture of alkaloids and their function in plants and animals seems to be clear, but we need substantially more experimental data to understand fully the intricate interconnections between plants, their alkaloids, and herbivores, microorganisms, and other plants. defense in animals introduction to ecological biochemistry defense mechanisms in plants herbivores: their interaction with secondary okologische biochemie allelochemicals: role in agriculture and forestry cell culture and somatic cell genetics of plants" (f. constabel and . m. wink gifttiere und ihre waften perspectives in chemoreception behavior biochemie und physiologie der sekundaren pflanzenstoffe plant metabolites biosynthese der alkaloide biochemistry of alkaloids the alkaloids: the fundamental chemistry antiseptika baerheim svendsen lloydia . n. m. rojas hernandez vallejos and . a. roveri the merck index proc. rd inf. lupin conf insect biology in the future micromolecular evolution, systematics and ecology phytochernical ecology: allelochernicals, mycotoxins and insect pheromones phytochem biogene gifte rozniki nauk rolnikzych die gift-und arzneipflanzen in mitteleuropa antibiotics: mechanism of action of antimicrobial and antitumour agents antimicrob. agents chemo pharmazeutische biologie , biogene arzneistoffe pharmazeutische biologie drug use in pregnancy the alkaloids handbook of enzyme inhibitions cold spring harbor conf primary and secondary metabolism of plant cell cultures chemical defenses of arthropods der einfluss einer pflanze auf die andere-allelopathie the science of allelopathy allelopathy lectures on insect olfaction focus on insect-plant interactions alkaloid biology and metabolism in plants molecular aspects of insect plant associations methods of plant biochemistry secondary products in plant tissue culture proc. natl the alkaloids the work of the author was supported by the deutsche forschungsgemeinschaft. i thank dr. th. twardowski for reading an earlier draft of the manuscript. key: cord- - gtnsyts authors: wolf, michael s.; serper, marina; opsasnick, lauren; o'conor, rachel m.; curtis, laura m.; benavente, julia yoshino; wismer, guisselle; batio, stephanie; eifler, morgan; zheng, pauline; russell, andrea; arvanitis, marina; ladner, daniela; kwasny, mary; persell, stephen d.; rowe, theresa; linder, jeffrey a.; bailey, stacy c. title: awareness, attitudes, and actions related to covid- among adults with chronic conditions at the onset of the u.s. outbreak: a cross-sectional survey date: - - journal: ann intern med doi: . /m - sha: doc_id: cord_uid: gtnsyts background: the evolving outbreak of coronavirus disease (covid- ) is requiring social distancing and other measures to protect public health. however, messaging has been inconsistent and unclear. objective: to determine covid- awareness, knowledge, attitudes, and related behaviors among u.s. adults who are more vulnerable to complications of infection because of age and comorbid conditions. design: cross-sectional survey linked to active clinical trials and cohort study. setting: academic internal medicine practices and federally qualified health centers. patients: adults aged to years living with or more chronic conditions. measurements: self-reported knowledge, attitudes, and behaviors related to covid- . results: a fourth ( . %) of participants were “very worried” about getting the coronavirus. nearly a third could not correctly identify symptoms ( . %) or ways to prevent infection ( . %). one in adults ( . %) believed that they were “not at all likely” to get the virus, and . % reported that covid- had little or no effect on their daily routine. one in respondents was very confident that the federal government could prevent a nationwide outbreak. in multivariable analyses, participants who were black, were living below the poverty level, and had low health literacy were more likely to be less worried about covid- , to not believe that they would become infected, and to feel less prepared for an outbreak. those with low health literacy had greater confidence in the federal government response. limitation: cross-sectional study of adults with underlying health conditions in city during the initial week of the covid- u.s. outbreak. conclusion: many adults with comorbid conditions lacked critical knowledge about covid- and, despite concern, were not changing routines or plans. noted disparities suggest that greater public health efforts may be needed to mobilize the most vulnerable communities. primary funding source: national institutes of health. t he severe acute respiratory syndrome coronavirus (sars-cov- ) and resultant coronavirus disease (covid- ) have evolved into a pandemic, requiring persons around the world to attend to rapidly changing messages about public health and take immediate actions to minimize their risk for infection and the spread of the virus ( ). this unprecedented global crisis has also been marked by miscommunication regarding the imminent threat of covid- , leading to public confusion and inaction ( ) . older adults and those with underlying health conditions are at greatest risk for severe infection and death due to covid- ( ) . the same factors that make individuals more vulnerable are also associated with reduced ability to access and understand health information, make well-informed decisions, and take optimal health-promoting actions-a skill set commonly called "health literacy" ( , ) . this is especially true when the health information itself is not timely, trusted, consistent, or actionable. health literacy has emerged over the past decades as one of the strongest psychosocial determinants of health, and it has also been shown to explain a range of health disparities by age, race/ ethnicity, and socioeconomic status ( ) . thus, in uncertain times like this, when the interpretation of critical and ever-changing public health messages is paramount, many vulnerable populations may be further marginalized by inadequate health communication, posing substantial risks to themselves and their communities. we did a time-sensitive study among higher-risk, older adults living with or more chronic conditions to determine their current awareness of covid- , their perception of the seriousness of its threat, their level of worry and concern related to contracting the virus, whether it is affecting their daily routine or existing plans, how prepared they feel to handle an outbreak, and their confidence in the federal government response. this took place from through march during the initial outbreak of the virus in the united states. we focused on the role of health literacy and other psychosocial health determinants in understanding risks for covid- and the initiation of preventive behaviors. health services, are sponsored by the national institutes of health, and are taking place among primary care sites ( academic internal medicine clinics and federally qualified health centers) across the greater chicago area ( table ) . health literacy and cognitive function among older adults (r ag ) is a cohort study examining cognitive and psychosocial factors associated with self-management and outcomes of chronic disease over time among predominately older adults. three randomized controlled trials-ehr-based universal medication schedule to improve adherence to complex regimens (r nr ), a universal medication schedule to promote adherence to complex drug regimens (r ag ), and transplant regimen adherence for kidney recipients by engaging information technologies: the take it trial (r dk )-evaluate health system strategies that leverage electronic health records and available consumer technologies to improve patient adherence and safe use of complex drug regimens. these studies were selected because they enroll mostly middle-aged or older adults (range, to years) with or more chronic conditions who therefore would be at greater risk for covid- . the studies use common assessments, allowing for uniform measurement of many patient characteristics. the northwestern university institutional review board approved study procedures, and all patients included in this telephone survey had provided prior consent to be contacted for future research opportunities. data were collected from through march . inclusion criteria varied across studies by age, presence of specific chronic conditions, having been prescribed complex regimens (≥ medications), and being an active patient at specified primary care sites; table provides study-specific eligibility criteria. methods of these studies have also been described in prior publi-cations ( ) ( ) ( ) . in brief, recruitment procedures included identifying potentially eligible participants via electronic health record queries; sending them a letter describing the study; then telephoning any patients who did not opt out of being contacted to introduce the study, screen for eligibility, and schedule an in-person baseline interview. common exclusion criteria for all studies include the presence of a severe and uncorrectable cognitive, visual, or hearing impairment that would preclude a participant's ability to complete interviews. for this survey, we targeted participants whose last interview was done between and the present. this time frame was selected to ensure that previously collected data from each parent study-which were merged with data from this survey-were most current; participants with the most recently collected prior data were prioritized for recruitment. trained research interviewers contacted participants outside their normally scheduled research interviews to invite them to answer a short set of questions pertaining to covid- by telephone. participant responses were recorded by interviewers using redcap web-based survey software. on average, surveys took less than minutes, and participants who completed the survey were told that they would be mailed a $ gift certificate for their time. in total, adults were enrolled in the parent studies and were eligible for the survey; were contacted during the week under investigation. of these, declined participation and could not be reached or asked to be contacted at a later date. in all, completed the study, for an overall cooperation rate of . %. across all studies, there was prior, uniform collection of patient demographics (age, sex, race, and ethnicity), socioeconomic status (household income, num- awareness, attitudes, and actions related to covid- awareness, attitudes, and actions related to covid- annals.org annals of internal medicine ber in household, educational attainment, employment status, and health insurance), and self-reported chronic conditions. all included the newest vital sign to assess health literacy ( ) . the consumer health activation index was used to determine patient activation across studies ( ) . in addition, a single item was used to capture self-reported overall health (excellent, very good, good, fair, or poor). for r ag , both englishand spanish-speaking patients participated; limited english proficiency (lep) was determined by patients self-reporting how well they spoke english. survey items were adapted from questionnaires used to study prior outbreaks ( ) . awareness of covid- was assessed using items that asked whether participants had heard of the novel coronavirus, if they had been told they had it or believed they did, or if someone they knew had been told they had it or believed they did ( table lists the items). perceived concern for covid- (more plainly called "coronavirus") was evaluated by first asking participants to rate, on a scale of to ( being no threat at all and being very serious), how serious a public health threat they believed the coronavirus is or might become. in addition, a single question asked participants to rate their level of worry about getting the coronavirus (very worried, somewhat worried, a little worried, or not worried at all). to provide context, this same question was asked with regard to influenza, and participants were also asked whether they had received an influenza vaccine in the past year. demonstrated knowledge of covid- was assessed through open-ended questions asking participants to name symptoms of the coronavirus and actions they could take to avoid becoming infected. five trained expert clinician raters (m.s., j.a.l., t.r., d.l., and m.a.) documented and independently coded verbatim responses, which were then thematically analyzed by members of the research team. in addition, participants were asked to estimate the percentage of persons who acquire the coronavirus who will die of it and the percentage who will have only mild symptoms. participants were asked whether they were currently making changes to their daily routines as a result of the coronavirus and whether they had changed any of their plans. verbatim responses were documented for participants stating that they had changed plans, and responses were also independently coded by trained raters and then thematically analyzed. respondents were asked about the likelihood of themselves or someone they know getting the coronavirus (very likely, somewhat likely, not that likely, or not at all likely). they also answered questions about their sources for information about the coronavirus, confidence that the federal government could prevent a national outbreak (very confident, somewhat confident, not very confident, or not confident at all), and perceived preparedness if a widespread outbreak were to occur (very prepared, somewhat prepared, a little prepared, or not prepared at all). descriptive statistics (means with sds and percentage frequencies) were calculated for all patient characteristics and survey responses. associations between patient characteristics and responses to covid- awareness, perceived concern, knowledge, and related awareness, attitudes, and actions related to covid- behavior items were then examined in bivariate analyses using tests, t tests, or analysis of variance, as appropriate. multivariable linear regression models were used to estimate least-squares means (with % cis) for the continuous outcome of perceived concern. for dichotomous outcomes, a multivariable poisson distribution was used rather than odds ratios for the relative risk estimates ( ) . all models included health literacy as a primary covariate of interest, additional variables affecting knowledge and behavior (age, gen-der, race, and income), day the survey was done, and parent study. statistical analyses were performed using stata/se, version (statacorp). the study was supported by national institutes of health projects. the funding sources had no role in the design, conduct, or analysis of the study or the decision to submit the manuscript for publication. table summarizes respondent characteristics. participants were older overall, and . % were female. the sample was racially and ethnically diverse, and many participants were socioeconomically disadvantaged: nearly a third ( . %) were living below the poverty level. about half of adults had low or marginal health literacy, all had at least chronic condition, and two thirds ( . %) were living with or more chronic conditions. all participants had heard of the coronavirus (covid- ) , and most considered the potential threat to be high ( table ) . one in ( . %) said that they were "very worried" about getting the coronavirus, and . % were not worried at all ( table ); in contrast, . % said that they were "very worried" about getting influenza, and . % were not worried at all. half ( . %) rated their worry about covid- and influenza the same, whereas . % were more worried about getting covid- . very few participants ( . %) believed that they would definitely or probably get the coronavirus. the threat of a covid- outbreak was rated to be more serious by adults aged years or older and by women before adjustment; those with or more chronic conditions rated the threat as less serious than those with fewer conditions ( table ) . black participants were more likely than white participants to report that they were "not worried at all" about getting the coronavirus; this was also true for those reporting poorer health. women, black and hispanic persons, those with lep, those living below the poverty level, those with lower health literacy, and unmarried persons were significantly more likely to respond that it was "not at all likely" that they would get covid- . in multivariable analyses, women remained more likely than men to rate the seriousness of the covid- threat as high, whereas adults living below the poverty level rated it as less serious than those with higher incomes ( table ). respondents' ratings of the seriousness of covid- also significantly increased by day of awareness, attitudes, and actions related to covid- interview, with higher ratings at the end of the survey period than at the beginning. blacks were more likely than whites to be only "a little worried" or "not worried at all" about getting the coronavirus, and black race, living below the poverty level, and low health literacy all remained independently associated with participants' belief that it was "not at all likely" that they would get sick with covid- . on average, respondents estimated that more than half ( . %) of infected persons will have only mild symptoms and . % will die of covid- ( table ) . most participants correctly identified symptoms ( . %) and ways to prevent infection ( . %). women estimated fewer mild cases and more deaths than men (table ). this was also true for blacks relative to whites, for those living below the poverty level, and for those with lower health literacy. participants who were older, black, unmarried, unemployed, or retired; had poorer health; or had lower health literacy showed poorer knowledge of covid- ( table ). those who identified as being hispanic and having lep demonstrated greater covid- knowledge. after multivariable adjustment, patient characteristics were no longer associated with knowledge of covid- symptoms or means of prevention ( table ) . more than half of patients ( . %) reported that the coronavirus had caused them to change their daily routine "a lot," whereas . % said that they had changed existing plans as a result ( table ). men; black persons; those with lep, lower health literacy, or or more chronic conditions; those living below the poverty level; and persons who were unmarried, unemployed, or retired were less likely to makes changes because of the coronavirus ( table ) . after multivariable adjustment, these patient factors were no longer associated with changes to either daily routine or existing plans. in contrast, respondents who were interviewed later in the -week survey period were more likely to report that their daily routine had changed "a lot" ( table ) . one in respondents ( . %) reported that they were "very prepared" for a widespread outbreak. nearly a third ( . %) had no confidence that the federal government could prevent a nationwide outbreak; . % were very confident ( table ) . black and hispanic adults; those with lep, lower health literacy, lower health activation, or poorer health; those living below the poverty level; and those who were unmarried, unemployed, or retired were more likely to con- awareness, attitudes, and actions related to covid- annals.org annals of internal medicine sider themselves either "a little prepared" or "not prepared at all" ( table ). in multivariable analyses, black race and low health literacy were both independently associated with a greater likelihood of feeling only "a little prepared" or "not prepared at all" ( table ) . hispanic persons, those with lep, those living below the poverty level, and those with lower health literacy were also more likely to be "somewhat" or "very" confident in the federal government. in multivariable analyses, only low health literacy remained associated with feeling "somewhat" or "very" confident in the federal government's ability to prevent a nationwide outbreak ( table ). in a survey of more than sociodemographically diverse adults with chronic health conditions living in chicago, we found that most respondents perceived the threat of a covid- outbreak to be serious, although the level of worry varied; half equated the threat with that of influenza, and only a few reported being more worried about getting influenza than covid- . nearly one third could not identify symptoms or proper measures to prevent infection. most respondents reported that the virus was affecting their daily routine and leading to changes in already made plans, yet in adults believed that it had little or no effect on their lives or plans. nearly in participants believed that they were only a little or not at all prepared for a covid- outbreak, whereas just in believed that they were very prepared. only in respondents was very confident that the federal government could prevent a nationwide outbreak of this virus. at the time of writing, illinois ranks seventh in the united states with more than covid- cases, and state residents have died. when our c survey started on march , there were only cases and no deaths; by the end of the survey on march, there were cases and deaths. across the united states and worldwide, the outbreak was increasing at a rate of % to % more new cases daily during the week of the interviews. at the same time, several measures were announced in succession: schools began closing across illinois, employers were sending staff home to work remotely, various public restrictions were implemented (bar and restaurant closures and limitations on gatherings), and ultimately a "shelter at home" order was announced. thus, our findings provide a rare snapshot of how a cohort of mostly middle-aged and older adults with underlying health conditions adapted to this unprecedented time and took action, or not. our study identified concerning demographic and socioeconomic differences in how individuals perceived the threat of covid- and, perhaps, their own ability to take actions to prevent illness. specifically, those who were black, were living below the poverty level, and had low health literacy were less likely to believe that they might become infected, and black respondents were less worried about the pandemic. black adults also felt less prepared for an outbreak than white adults, and individuals with low health literacy reported not only being less prepared but also having more confidence in the federal government response. although the reasons for these findings are not clear, similar results were reported during the h n influenza pandemic in ( ) . trust in public health officials, information-seeking behaviors, sources of information, frequency of media exposure, knowledge, and worry related to the outbreak were all highlighted determinants of documented disparities in uptake of recommended behaviors. in our study, disparities by race, socioeconomic status, and health literacy were not reflected in ratings of the seriousness of the covid- threat, demonstrated knowledge of its symptom presentation or general means to prevent it, or reported changes to daily routines and plans. prior research has documented racial differences pertaining to trust in the health care system ( ) ( ) ( ) . for those who are living below the poverty level or have low health literacy, perceptions of personal risk and the ability to prevent infection may be limited. this may be due to feeling less able to change one's social circumstance, or lack of public health communications that are explicit and actionable and provide clear, efficacious messaging pertaining to recommended protective behaviors ( , ) . a previous report found socioeconomic and literacy disparities in mortality associated with the influenza pandemic; likewise, our findings should raise caution ( ) . although the current public health infrastructure is different, existing efforts may not be adequately reaching these vulnerable populations. our study, working to quickly capture the opportunity to understand how the most vulnerable are processing current events, clearly has limitations. first, this survey was done among a selected group of patients who were all active participants in cohort studies or clinical trials sponsored by the national institutes of health in large u.s. city. thus, these findings may have limited generalizability, especially for younger adults and those without underlying health conditions. however, our study samples purposefully include men and women who are socioeconomically, racially, and ethnically diverse and are at greatest risk for covid- because of age and underlying conditions. second, to rapidly implement our investigation and quickly recruit as large a sample as possible during the first of multiple waves of interviews, we were limited in the depth of our survey and number of items to use. prior research on virus outbreaks guided our selection and creation of survey items ( ), but we lacked the time or opportunity to validate all questions, particularly in the midst of a public health crisis. however, items followed best practices for the design of assessments for use among persons with lower literacy ( ) . third, our outcomes capture only initial awareness of covid- , degree of worry, fundamental knowledge, attitudes, and a limited set of behaviors. understanding of the virus has since evolved, and we could not expand on those developments. items included in planned follow-up waves of original research awareness, attitudes, and actions related to covid- the survey will adapt accordingly and expand data capture on behaviors, among other just-in-time topics. finally, as a time-sensitive study, what we have learned in this initial, critical week, when covid- most fully took hold in the united states, is that public health messaging has dramatically changed: new policies, state restrictions, and information are being shared not just daily but hourly. it is likely that all of what we report in this -week glimpse has considerably altered. regardless, our findings depict the initial lack of clarity in understanding, perceived susceptibility, and personal efficacy regarding the pandemic among those at greatest risk. that is why we intend to continue to follow this cohort as part of an ongoing c initiative. this first wave of the c study revealed profound gaps in awareness, knowledge, concern, and preemptive public health action. the potential for the covid- pandemic to exacerbate health disparitiespotentially through mechanisms related to inadequate or conflicting public health messaging among those who are socioeconomically disadvantaged, belong to racial minority groups, or have more limited health literacy-may be exceptionally high. actions are needed now to ensure that as the pandemic unfolds, all citizens are adequately made aware of the gravity of the threat; with great clarity and attention to health literacy best practices, we need to explain specific steps that must be taken to avoid harm. grant support: by grants r ag , r ag , r dk , and r nr from the national institutes of health (nih). disclosures: dr. wolf reports grants from the nih during the conduct of the study; grants from merck, the gordon and betty moore foundation, the nih, and eli lilly outside the submitted work; and personal fees from sanofi, pfizer, and luto outside the submitted work. dr. serper reports personal fees from biovie outside the submitted work. ms. batio reports grants from the nih during the conduct of the study. dr. ladner reports grants from the national institute of diabetes and digestive and kidney diseases during the conduct of the study. dr. persell reports grants from omron healthcare and pfizer outside the submitted work. dr. bailey reports grants from the nih during the conduct of the study; grants from merck, the nih, and eli lilly outside the submitted work; grants and personal fees from the gordon and betty moore foundation outside the submitted work; and personal fees from sanofi, pfizer, and luto outside the submitted work. authors not named here have disclosed no conflicts of interest. disclosures can also be viewed at www.acponline.org/authors /icmje/conflictofinterestforms.do?msnum=m - . study protocol and statistical code: available from dr. wolf (e-mail, mswolf@northwestern.edu). data set: available to those who meet prespecified criteria; access allowed to deidentified data only. available from dr. wolf (e-mail, mswolf@northwestern.edu). corresponding author: michael s. wolf, phd, mph, ma, feinberg school of medicine, northwestern university, north lake shore drive, th floor, chicago, il ; e-mail, mswolf@northwestern.edu. current author addresses and author contributions are available at annals.org. covid- coronavirus outbreak. accessed at www .worldometers.info/coronavirus on coronavirus disease : the harms of exaggerated information and non-evidence-based measures prevalence of comorbidities in the novel wuhan coronavirus (covid- ) infection: a systematic review and meta-analysis health literacy and functional health status among older adults the prevalence of limited health literacy the relationship between health literacy and health disparities: a systematic review rationale and design of the regimen education and messaging in diabetes (remind) trial literacy, cognitive function, and health: results of the litcog study development and rationale for a multifactorial, randomized controlled trial to test strategies to promote adherence to complex drug regimens among older adults quick assessment of literacy in primary care: the newest vital sign development and validation of the consumer health activation index perceptions and plans for prevention of ebola: results from a national survey a modified poisson regression approach to prospective studies with binary data what have we learned about communication inequalities during the h n pandemic: a systematic review of the literature the role of risk perception in flu vaccine behavior among african-american and white adults in the united states determinants of influenza vaccination among high-risk black and white adults. vaccine association of patient perceptions of cardiovascular risk and beliefs on statin drugs with racial differences in statin use: insights from the patient and provider assessment of lipid management registry how does education lead to healthier behaviours? testing the mediational roles of perceived control, health literacy and social support disparities in influenza mortality and transmission related to sociodemographic factors within chicago in the pandemic of development of the patient education materials assessment tool (pemat): a new measure of understandability and actionability for print and audiovisual patient information original research awareness, attitudes, and actions related to covid- current author addresses: drs. wolf, o'conor, arvanitis, persell feinberg school of medicine, northwestern university, north lake shore drive, th floor dr. ladner: feinberg school of medicine, northwestern university, north saint clair street critical revision of the article for important intellectual content key: cord- -rnjnxymc authors: kariithi, henry m.; İnce, İkbal agah; boeren, sjef; murungi, edwin k.; meki, irene k.; otieno, everlyne a.; nyanjom, steven r. g.; van oers, monique m.; vlak, just m.; abd-alla, adly m. m. title: comparative analysis of salivary gland proteomes of two glossina species that exhibit differential hytrosavirus pathologies date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: rnjnxymc glossina pallidipes salivary gland hypertrophy virus (gpsghv; family hytrosaviridae) is a dsdna virus exclusively pathogenic to tsetse flies (diptera; glossinidae). the kb gpsghv genome contains open reading frames and encodes more than confirmed proteins. the asymptomatic gpsghv infection in flies can convert to symptomatic infection that is characterized by overt salivary gland hypertrophy (sgh). flies with sgh show reduced general fitness and reproductive dysfunction. although the occurrence of sgh is an exception rather than the rule, g. pallidipes is thought to be the most susceptible to expression of overt sgh symptoms compared to other glossina species that are largely asymptomatic. although glossina salivary glands (sgs) play an essential role in gpsghv transmission, the functions of the salivary components during the virus infection are poorly understood. in this study, we used mass spectrometry to study sg proteomes of g. pallidipes and g. m. morsitans, two glossina model species that exhibit differential gpsghv pathologies (high and low incidence of sgh, respectively). a total of host proteins were identified, of which and proteins were significantly up- and down-regulated, respectively, in g. pallidipes compared to g. m. morsitans. whereas gpsghv proteins were detected in g. pallidipes f( ) progenies, only viral proteins were detected in g. m. morsitans. unlike in g. pallidipes, qpcr assay did not show any significant increase in virus titers in g. m. morsitans f( ) progenies, confirming that g. m. morsitans is less susceptible to gpsghv infection and replication compared to g. pallidipes. based on our results, we speculate that in the case of g. pallidipes, gpsghv employs a repertoire of host intracellular signaling pathways for successful infection. in the case of g. m. morsitans, antiviral responses appeared to be dominant. these results are useful for designing additional tools to investigate the glossina-gpsghv interactions. glossina pallidipes salivary gland hypertrophy virus (gpsghv; family hytrosaviridae) is a dsdna virus whose kb genome encodes more than confirmed proteins (abd-alla et al., , b kariithi et al., a) . the hytrosaviridae family consists of only one other member, the housefly musca domestica (diptera; muscidae) hytrosavirus (mdsghv; coler et al., ) . however, detection of hytrosavirus-like infection symptoms, i.e., the salivary gland hypertrophy syndrome (sgh) in the narcissus bulb fly merodon equestris (diptera; syrphidae; amargier et al., ) and in male accessory gland filaments of the parasitic wasp diachasmimorpha longicuadata (hymenoptera; braconidae; luo and zeng, ) implies that the hytrosaviridae potentially contains other members. the intrinsic properties of hytrosaviruses, i.e., covert chronic infection of adult stages without expression of detectable sgh symptoms, have probably hindered the discovery of other hytrosaviridae family members up until now. gpsghv is exclusively pathogenic to the tsetse fly (diptera; glossinidae), the vector of a group of neglected tropical diseases called the african trypanosomiases (mattioli et al., ) . research on gpsghv pathobiology has been hindered by a lack of an in vitro cell culture system to support the virus replication (abd-alla et al., a) . attempts to multiply gpsghv in alternative insect hosts such as m. domestica have so far been unsuccessful. the only available method to multiply gpsghv is via intra-hemocoelic injections of virus suspension in g. pallidipes (kariithi et al., b) . a mature gpsghv virion contains four distinct structural components (nucleocapsid core, tegument, envelope, and helical surface projections) composed of virally-encoded proteins . the gpsghv virion also contains hostderived cellular proteins: some are incorporated into the virus particles and may play roles in virus replication and transmission (kariithi et al., a,b) . in g. pallidipes, gpsghv is transmitted horizontally via saliva during feeding (abd-alla et al., ) and vertically (transovarial) via the fat body tracheal system and milk gland secretions (boucias et al., ) . gpsghv infection in laboratory colonies of g. pallidipes can either be asymptomatic or symptomatic with the former being the most rampant in laboratory colonies of this tsetse species (abd-alla et al., ) . however, the asymptomatic infection state can convert to a symptomatic state, leading to reproductive dysfunction and reduced fecundity in addition to sgh symptoms (abd-alla et al., ; lietze et al., ; boucias et al., ) . more than % of salivary gland (sg) proteins appear to be specifically expressed in g. pallidipes flies with overt sgh symptoms but not in asymptomatic flies (kariithi et al., ) . unlike in the laboratory tsetse fly colonies, gpsghv infection is mainly covert (latent) in wild g. pallidipes populations. occurrence of sgh symptoms have been reported in other glossina species such as g. m. morsitans (jura et al., ) and g. m. centralis (sang et al., ) . however, sgh symptoms are rare especially in species other than g. pallidipes. notably, even in g. pallidipes the occurrence of sgh symptoms is an exception rather than the rule (boucias et al., ) . the pathobiology of gpsghv in species other than g. pallidipes has not been so far investigated. whether naturally or artificially infected, the gpsghv infection rate is low, but males are more susceptible to infections compared to females (abd-alla et al., ; boucias et al., ) . after acquisition through a blood meal, gpsghv translocates to the sgs where it primarily replicates (garcia-maruniak et al., ) . in g. pallidipes, intra-hemocoelic gpsghv injection leads to significant increase in the viral titters in the whole fly, but the injected virus is not released via saliva during feeding and there is no development of overt sgh symptoms (boucias et al., ) . rather, sgh symptoms are overt in the f progenies of the infected mothers. it is yet to be confirmed in which host tissues gpsghv replicates after artificial injection. however, the current school of thought is that in naturally-infected g. pallidipes, the virus replicates in the male reproductive accessory glands (sang et al., ) and the gut (sang et al., ) but without any teratogenic effects. the pathological, morphological and ultrastructural effects of gpsghv infection in g. pallidipes sgs have been studied to considerable length (kariithi et al., (kariithi et al., , a guerra et al., ) . however, no such studies have been performed in other glossina species. further, the molecular basis for the differential gpsghv pathology in different glossina species is still unclear. here, we investigated gpsghv-induced modulation of total protein expression in the sgs of g. pallidipes and g. m. morsitans, with special emphasis on the host pathways that are potentially employed by the virus during infection. we hypothesized that gpsghv infection in glossina is under the control of host-and/or virus-encoded factors (proteins/peptides) whose interactions influence the expression or lack of overt sgh symptoms. we tested the hypothesis by comparing the sg proteomes of gpsghv-infected vs. mock-infected g. pallidipes and g. m. morsitans flies. the host (and viral) proteins identified in this study are potential targets for control of gpsghv infections in tsetse fly mass production facilities. for instance, antiviral strategies could be developed to block virus replication and egress (esfandiarei et al., ; cheshenko et al., ; chen et al., ) , prevent the establishment of virus replication complexes (saxena et al., ) and prevent development of cellular proliferation (guergnon et al., ) . such antiviral approaches are applicable in the control of virus infections in mass production of other insects. the g. m. morsitans and g. pallidipes flies used in this study were obtained from a colony maintained at the joint fao/iaea insect pest control laboratories (ipcl), seibersdorf, austria. for each treatment described below, groups of experimental flies were kept in holding cages (diameter of cm and height of cm) at a density of flies per cage and a mating ratio of : (male: female). the holding cages had netting on top and bottom for fly feeding and pupae collection, respectively. the experimental flies were reared at ± • c, - % relative humidity, h scotophase and fed on defibrinated bovine blood meals ( - min; times per week; feldmann, ) . the pupae from the sequential larviposition cycles were collected and incubated at • c until eclosion of the adult f progenies. for further analyses, male f progenies were selected from the fourth larviposition cycle (g ) based on available data that the incidence of sgh symptoms reaches % at the g (boucias et al., ) . it should be noted that males were used because they are significantly more susceptible to expression of sgh symptoms than the females (abd-alla et al., ) . to allow for development of sgh symptoms, the selected f male progenies were reared for weeks (equivalent to blood meals) under the same insectaria conditions and handled as the parents. all the treatments described here were replicated at least three times. to prepare the gpsghv inoculum, one intact pair of sgs displaying overt sgh symptoms were dissected from an adult ( -day old) male g. pallidipes fly and stored in ml of icecold sterile saline (ph . ). the sgs were then homogenized and clarified by brief centrifugation ( × g; min; • ) to remove tissue debris. the supernatants were sterilized by passing through a . -µm filter unit and the virus titters present in the filtrate were estimated by a quantitative polymerase chain reaction (qpcr) as described by abd-alla et al. ( a) . by this qpcr method, an average of × virus copies were estimated to be present in a µl aliquot of the virus preparation and was used for tsetse fly injections. for infections, teneral (newly eclosed; nonfed) female g. m. morsitans and g. pallidipes flies were artificially (intra-hemocoelic) injected with the virus preparations using a protocol described by boucias et al. ( ) . briefly, the female flies selected from the colony as described above were inoculated with either µl of the virus inoculum or µl of filter-sterilized pbs (mock infections). following the injections, the females were mated with asymptomatic males; these females were then separated from the males and subsequently maintained in the insectary until they produced the f progenies as described above. detection of viral dna in infected g. pallidipes and g. m. morsitans flies to confirm gpsghv infections, the week-old f male progenies produced by the mock-and virus-infected mothers were screened using a diagnostic pcr protocol described by abd-alla et al. ( ) . for this, genomic dna was extracted from one intermediate excised leg of individual flies using dneasy tissue kit (qiagen inc., valencia, ca). pcr amplifications were performed using primers and conditions previously described (abd-alla et al., , b , and the pcr products analyzed on % agarose gels. flies were considered to be non-infected, moderately-infected or highly infected if there were no visible bands or showed faint bands or thick bands, respectively, on agarose gels as previously described (abd-alla et al., ) . the actual virus copy numbers and the virus density levels were determined using qpcr essentially as described by abd-alla et al. ( b) . for virus density levels, the qpcr data were normalized against the tsetse β-tubulin gene . the samples with high virus infections (based on the agarose gels) were subsequently used for mass spectrometry measurements as virus-infected samples. this cut off was especially used in the case of g. m. morsitans flies, which do not usually show overt sgh symptoms. for g. pallidipes, samples with overt sgh symptoms (corresponding to samples with high infections from the agarose gels) were selected for mass spectrometry measurements. for negative controls, samples from -week-old male flies were selected from the mock-infected fly groups (confirmed to be pcr-negative). ten flies from each of the replicated treatments described above were selected for subsequent sg dissections. to prepare protein extracts, sgs from the f progenies described above were dissected days after the flies had their last blood meals to allow for full digestions (abd-alla et al., a) . from each of the selected flies, intact pairs of sgs were individually dissected, during which the occurrence of sgh symptoms was assessed. the dissected sgs were preserved (at • c) in µl sterile saline complemented with protease inhibitors (roche diagnostics, germany). then, each of the pool of pairs of sgs were homogenized using a glass/teflon homogenizer and ultrasonicated (sonifier cell disruptor, branson, ct, usa) as described by kariithi et al. ( a) . the homogenates were freeze-thawed and clarified three times by centrifugation ( × g; min; • c). the supernatants were pooled and the proteins quantified using bca protein assay (bio-rad) according to manufacturer's instructions. then, equal quantities ( ng) of the proteins (from each of the pooled sgs per each of the three replicates) were electrophoresed using % sds-page gels (invitrogen) as described by green and sambrook ( ) . the gels were stained with colloidal cbb stain (nupage novex; invitrogen). the middle sections of entire gel lanes were excised, and each of the gel lanes was divided into eight slices (equal portions) each of which was cut into approximately mm pieces. to prepare peptides for mass spectrometry measurements, the gel slices containing the sg proteins were subjected to in-gel trypsin digestions as previously described (kariithi et al., a) . briefly, after washing the gel pieces with mm ammonium bicarbonate (abc) buffer and abc buffer/ % (vol/vol) acetonitrile (acn), proteins were reduced and alkylated using dithiothreitol and iodoacetamide, followed by washing with abc/abc-acn buffers and trypsin digestions. tryptic peptides were then analyzed by liquid chromatography-tandem mass spectrometry (lc-ms/ms; lu et al., ) . to identify the sg proteins, the ms/ms spectra obtained from the lc-ms/ms measurements were searched against a tsetse fly database, a gpsghv database, a contaminant database containing sequences of common contaminants, and a decoy database constructed by reversing all protein sequences downloaded from uniprot. the ms searches were performed using maxquant v . . . (cox and mann, ) and andromeda as the database search engine (cox et al., ) . a maximum false discovery rate (fdr) of less than . was set at the peptide and protein levels. maxquant search parameters included variable oxidation of m, fixed carboxamidomethylation of c, and extra variable modifications for de-amidation of n and q. "label-free quantification" (lfq) and "match between runs" (set to min) options were enabled. de-amidated peptides were allowed to be used for protein quantification. to quantify the sg proteins, the resulting maxquant protein list was filtered to show only those proteins with a minimum of two peptides matching the same protein, of which at least one peptide was unique and unmodified. all other quantification settings were set at default. any hits to the decoy sequences and hits with modified peptides only were deleted from the list of protein/peptides groups. to easily compare abundances of the same proteins between the controls and virus-infected samples, logarithms (log ) of normalized lfqs were used. logarithms of the total intensity corrected for the number of measurable peptides (i.e., intensity based absolute quantization; ibaq) were used to compare the levels of different proteins from the same sample (mocks vs. gpsghv-infected; schwanhausser et al., ) . proteins were considered to be up-or down-regulated when their log protein abundance ratios were larger or smaller than zero, respectively. proteins were considered significantly upregulated when their log protein abundance ratios were larger than six. gene ontology annotation of the identified proteins were created using blast go v . . (conesa et al., ) . analyses of the protein motif/domain were performed using various bioinformatics softwares, including smart (schultz et al., ) and interproscan (zdobnov and apweiler, ) . we first analyzed gpsghv replication in the f progenies from virus-infected mothers. we detected varying levels of gpsghv infections in g. m. morsitans flies: non-infected, moderately infected and highly infected as evidenced by the absence of bands, faint bands and thick bands, respectively, in the agarose gels presented in the figure a . the different gpsghv infection levels we obtained for g. m. morsitans in figure were comparable to the results obtained for g. pallidipes, as well as results from our previous studies in g. pallidipes (compare with figure in abd-alla et al., ) . qpcr analysis of the samples in the "highly infected" category revealed high virus genome copies in g. m. morsitans ( figure b) . when dissected however, none of these f progenies from gpsghv-infected g. m. morsitans mothers showed any sgh symptoms. this is unlike in the g. pallidipes f progenies, which revealed % prevalence of sgh symptoms (data not shown), a result which was in agreement with our recent report (boucias et al., ) . notably, the gpsghv density levels in f progenies of g. pallidipes were significantly higher (p = . ) compared to the virus-infected g. m. morsitans f progenies ( figure c) . importantly, the virus density levels in both the mock-and the virus-infected g. m. morsitans f progenies were comparable to those of the mock-infected g. pallidipes (see figure c ). we then performed mass spectrometry on the sg protein extracts from the f progenies of g. m. morsitans flies with high viral titters and the g. pallidipes flies with overt sgh symptoms. analyses of the gpsghv-infected g. m. morsitans and g. pallidipes proteomes compared to their mock-infected counterparts resulted in unique peptides that mapped to non-redundant proteins. of these proteins, . % (n = ) were host (glossina)-specific, while . % (n = ) and . % (n = ) were from gpsghv and bacterial endosymbionts (wigglesworthia glossinidia and sodalis glossinidius), respectively. we then filtered out proteins with single and modified peptides, which resulted in proteins, of which , , and proteins were from the host, gpsghv and w. glossinidia, respectively. all the identified proteins are detailed in supplementary material (tables s -s ). when we compared the lc-ms/ms data sets obtained from the gpsghv-infected to mock-infected sgs, we found clear differential protein expression patterns in response to the virus infections in g. pallidipes and g. m. morsitans vs. their respective mock-infected controls (compare figure and figure ). from these two figures, gpsghv infection had more drastic effects in the protein expression in the sgs of g. pallidipes than that of the g. m. morsitans. in the figure , gpsghv infection in g. m. morsitans had little overall effects on the host's sg protein expression patterns, i.e., majority of the proteins (confidently identified by ≥ unique peptides per protein) were aligned around the y-axis (circled). on the other hand, a cohort of sg proteins were detectable in the proteome of gpsghv infected g. pallidipes, but were hardly detectable in the proteome of the mock-infected flies (see circled proteins in figure ) . notably, in contrast to the g. m. morsitans sg proteins (figure ), only few proteins were not significantly affected by gpsghv-infection in g. pallidipes (see proteins along the y-axis in figure ). overall, comparing the gpsghv-infected flies to their mockinfected counterparts, the majority of the host's sg proteins in g. m. morsitans had less than -fold up-or down-regulation compared to the proteome of g. pallidipes (see dotted red lines in figures , ) . we then made a more comprehensive comparison of the gpsghv-induced protein modulation by generating a log-log plot of the abundance distribution ratios of g. m. morsitans and g. pallidipes sg proteins. by combining the proteomics datasets obtained from the g. m. morsitans and g. pallidipes frontiers in microbiology | www.frontiersin.org , ) . shown are non-infected samples (no bands), moderately-infected samples (faint bands) and highly infected samples (thick bands). m is molecular marker. the pcr amplifications were performed using genomic dna extracted from single intermediate legs excised from -week old male f progenies produced by the control (mock), or virus-infected (gpsghv) flies. determination of the gpsghv copy numbers and the virus density levels by qpcr are shown in (b,c), respectively. for determination of virus copy numbers (b), -fold serially diluted viral dna (targeting odv-e gene) were used as internal standards as described by abd-alla et al., a . for determination of the virus expression levels, qpcr data were normalized using a tsetse fly housekeeping gene (β-tubulin). viral density levels in the virus-infected g. pallidipes progenies were significantly higher (p = . ) than the levels in the virus-infected g. m. morsitans flies. the values in the parentheses (c) indicate the virus density levels. letters a and b represent significant differences between the samples (i.e., there was no significant difference between samples labeled a, while a and b were significantly different). sg proteomes (figures , , respectively), the identified host proteins fell into two broad categories. the first category consisted of proteins that were down-regulated or up-regulated in either or both g. m. morsitans and g. pallidipes proteomes. the second category consisted of the proteins that were detectable in one of the two glossina species and in not the other. these categories are presented in the figure . proteins in the first category consisted of four groups: first, compared to mock-infected controls, a total of proteins were up-regulated in the gpsghv-infected g. pallidipes sg proteome but were down-regulated in g. m. morsitans ( figure a ; table s ). of the proteins, showed more than -fold upregulation in g. pallidipes proteome and they were all downregulated in virus-infected g. m. morsitans (table ) . second, proteins were up-regulated in both gpsghv-infected g. m. morsitans and g. pallidipes sg proteomes compared to their mock-infected counterparts ( figure b ; table s ). third, compared to the mock-infected controls, proteins were downregulated in the virus-infected g. pallidipes but up-regulated g. m. morsitans ( figure c ; table s ), nine of which were upregulated ≥ -fold in g. m. morsitans compared to the g. pallidipes sg proteomes (table ) . lastly, nine proteins were down-regulated in both virus-infected g. m. morsitans and g. pallidipes as measured from their proteomes ( figure d ; table s ). similarly, the proteins that were detectable in the proteome of one of the two gpsghv-infected tsetse species and not in the other fell into two main groups. first, compared to mockinfected flies, proteins were detectable in the virus-infected g. m. morsitans sg proteome but not in that of g. pallidipes (figure , x-axis), . % (n = ) of which were up-regulated (table s ) . second, proteins were detectable in g. pallidipes but not in g. m. morsitans (figure , y-axis), . % of which were up-regulated (table s ) . a closer look at the proteomics data indicate that majority of the heavily modulated proteins in the sg proteome of g. pallidipes appear to be spread over a wide range of host pathways. these pathways included, among others: atp/ubiquitin-dependent and s proteasome (ups) pathways, integrin-liked kinase pathway, transketolase pathway, hippo signaling pathway and diverse signaling pathways ( table ). in the case of g. m. morsitans, proteins potentially involved in the host's antiviral defenses appear to be quite dominant. some of the antiviral defense-related systems included induction of innate immune response via reactive oxygen species (virus degradation), the ubiquitin/ s proteasome system, v-atpase system (virus degradation via acidification of endosomes and or/lysosomes), the phagocytic engulfment system (to clear virus infection) and adaptive mitochondrial-mediated immune responses (interfere with production of progeny virus) ( table ) . frontiers in microbiology | www.frontiersin.org whereas only five gpsghv proteins (encoded by orfs sghv , sghv , sghv , sghv and sghv ) were detectable in g. m. morsitans, a total of proteins were detected in the sg proteome of g. pallidipes (see figures - ; table s ). these proteomics results reflects the findings we obtained from the qpcr assays described above. it should be noted that sghv is the most abundant of all gpsghv proteins; sghv is a high-molecular weight ( kda) viral protein, while sghv is a one of the major viral envelop proteins (kariithi et al., a) . the high abundance and large size, respectively, of these virion proteins possibly explains their detection in g. m. morsitans. further, proteins encoded by the orfs sghv and sghv have not been detected in our previous proteomic studies in g. pallidipes, and were in the current study only detectable in the g. m. morsitans proteome but not in the g. pallidipes proteome (figure ) . the failure to detect other viral proteins in g. m. morsitans does not necessarily imply complete absence of these proteins. rather, their abundances could have been too low or their detection could have been masked by the highly abundant host proteins. all the nine wigglesworthia proteins were in the detected in the sg proteome of g. m. morsitans (table s ) , probably because unlike the g. m. morsitans, the genome of g. pallidipes is not yet available. whereas four of the nine wigglesworthia proteins were up-regulated in the virus-infected compared to mock-infected flies, five were down-regulated (figure ) . the up-regulated proteins included transcription repair coupling factor, chaperone protein dnak, chaperone protein htpg, and imp-cyclohydrolase. down-regulated proteins included protein tola, dna polymerase i, exonuclease v, bifunctional enzyme glmu and a potassium transporter protein. since wigglesworthia is housed within the host's bacteriocytes (pais et al., ) , the detection of wigglesworthia proteins in the g. m. morsitans sgs suggest that these proteins likely "leak" into the hemolymph, potentially during bacteriocytes turnover, and eventually move from the hemocoel to the sgs. hytrosaviruses replicate primarily in the sg tissue of their insects hosts (garcia-maruniak et al., ) . as such, one would expect that the virus-induced modulation of the sg microenvironment (i.e., morphological and biochemical/functional features of the tissue) results in the expression of various proteins/peptides specifically to the advantage of viral replication and dissemination. whereas gpsghv in g. pallidipes occurs in both asymptomatic and symptomatic infection states, the virus infection in other glossina species is almost always asymptomatic. we have previously demonstrated the dynamics of the development of virus-induced sgh symptoms (abd-alla et al., and trans-generational transmission of the virus in g. pallidipes (boucias et al., ) . however, the case of gpsghv infection in other glossina species has not been investigated. importantly, we have so far not observed any overt sgh symptoms in the g. morsitans colony maintained at the ipcl seibersdorf, which was used as the source of the experimental flies we have described here. in the current study, the effects of gpsghv infections in g. m. morsitans were remarkably different from previous studies. for instance, virus injection into newly larvipositioned third-instar larvae of two morsitans groups resulted in varying prevalence of sgh-like symptoms in developed adults, which ranged from . % (jura et al., ) to . % (kokwaro et al., ) in g. m. morsitans, and up to % in g. m. centralis (sang et al., ) . in our case, we did not observe any sgh symptoms in g. m. morsitans, potentially because whereas we injected the virus into newly eclosed adults, the researchers in the previous studies injected the virus suspensions into larvae. we therefore conclude that the injected virus is capable of infecting and replicating during ontogeny on the sgs during pupation (as evidenced from the previous studies). further, the injected virus appears figure | abundance distribution ratios of g. pallidipes sg proteins. the figure depicts the distribution of proteins detected in the sg proteome of g. pallidipes infected by gpsghv compared to the mock-infected controls. the host proteins detected by two or more peptides per protein are shown in blue and red, respectively, while the gpsghv proteins are shown in green. the proteins that were up-regulated and down-regulated in gpsghv-infected sg are shown on the right and left sides of the y-axis, respectively. the large blue circle depicts proteins that were detectable in the sg proteome of gpsghv-infected but not in the proteome of mock-infected g. pallidipes. proteins which were not significantly modulated are depicted along the y-axis. the dotted red lines represent -fold protein regulation. ibaq denotes intensity-based absolute quantification. incapable of infecting and inducing overt sgh symptoms in fully differentiated sg cells in adults (as evidenced from our study). notably, the observed high virus titters in g. m. morsitans could represent dna replication but may not represent production of infectious viral particles. the comparable virus titters between the virus-infected g. m. morsitans and the mockinfected g. pallidipes suggest that the virus may be undergoing only partial replication in adult cells to maintain steady-state titters throughout the adulthood. this is in agreement with previous studies in g. pallidipes whereby, utilizing a diagnostic pcr, abd-alla et al. ( ) detected gpsghv pcr positives in % of colonized g. pallidipes that did not exhibit overt sgh symptoms. taken together, the analyses of gpsghv loads (by agarose gels), virus density levels (by qpcr), and protein expression (by lc-ms/ms) imply that either g. m. morsitans, at least in the sgs, is far less permissive to virus replication or that the virus undergoes limited replication in g. m. morsitans (whereby only a subset of genes are expressed) compared to g. pallidipes. potentially, unlike in g. pallidipes where sgh symptoms can be overt, we are of the opinion that gpsghv infection is entirely latent in g. m. morsitans as previously proposed by kariithi et al. ( b) . potentially, the difference in gpsghv replication, i.e., partial replication or latency in g. m. morsitans vs. active replication in g. pallidipes, explains the differences in the repertoire of proteins detected in the sg proteomes of the two glossina species. the clear gpsghv-induced differential modulation of sg protein expression in glossina raises the question of what host pathways are potentially globally regulated to facilitate successful virus infection. it is well known that for cellular entry and induction of pathogenesis, many viruses manipulate key host signaling pathways that globally regulate many cellular processes (diehl and schaal, ) . so far, we have not been able to elucidate the precise mechanism(s) through which gpsghv induces overt sgh symptoms, mainly because of a lack of cell culture system to support the virus multiplication (arif and pavlik, ) . therefore, the precise mechanisms of gpsghv infection (cellular attachment, entry, intracellular trafficking, replication, maturation, and egress) in glossina remains elusive. in an attempt to unravel the pathobiology of gpsghv, we draw inferences from other virus-host systems that have been studied so far. of the proteins we identified in this study, proteins that showed significant differential expression patterns in virusinfected flies are particularly interesting since they potentially reflect involvement in gpsghv pathogenesis. in this regard, the proteins that were significantly up-regulated in the sg proteome of g. pallidipes but down-regulated in that of g. morsitans (table ) are interesting to focus on. also important were the nine proteins found to be up-regulated in proteome of g. morsitans but down-regulated in the proteome of g. pallidipes (table ) . notably, our annotation of the nine proteins revealed that these proteins may be involved in pathways related to the host's antiviral responses to virus infection (see table and the references therein). in the following sections, we briefly discuss the potential roles of the proteins stipulated in tables , with regard to viral entry into host cells, intracellular trafficking, and evasion of host's immune response, replication/translation and cellular proliferation. for entry, some viruses attach to host cell receptors thus inducing conformational changes that cause fusion of the viral envelope with the host's plasma membrane (thorley et al., ) . this is followed by delivery of the viral nucleocapsids into the cellular cytoplasm and uncoating of the viral genome. integrinlinked kinases (ilks), which were up-regulated in g. pallidipes (d tmu ; table ), have been implicated in viral cellular entry. for instance, kaposi's sarcoma-associated herpesvirus (kshv) envelop glycoprotein b (gb) hijacks ilks to induce the fak-src-pi k-rhogtpase signaling pathway (naranatt et al., ; sharma-walia et al., ) . similar to the kshv gb envelope protein, the gpsghv sghv protein, which was detected in the current study (table s ) , contains an arginyl-glycyl-aspartic acid (rgd) motif that may interact with the host's ilks. pending experimental validations, gpsghv potentially employs an entry mechanism similar to kshv . following cellular entry, a critical phase in viral pathogenesis is intracellular trafficking of viral nucleocapsids, a process that requires intricate signaling. one of the key pathway components targeted by several viruses is the gtpase rab protein. notably, this protein was found to be up-regulated in g. pallidipes (d tma ; table ) , unlike in g. m. morsitans. gtpases regulate membrane trafficking, particularly in the formation, motility and docking of vesicles (zerial and mcbride, ) . some viruses activate gtpase-mediated pathways to facilitate their intracellular trafficking (chien et al., ) . for instance, in the absence of rab a/b, herpes simplex virus (hsv- ) was unable to traffic from the er to cytoplasmic viral assembly complexes, leading to a build-up of un-enveloped viral particles in the cell cytoplasm (zenner et al., ) . gtpases were indeed found to be up-regulated in shrimps infected with whispovirus (wu and zhang, ) , another large invertebrate dsdna virus like gpsghv. it is tempting to postulate that gpsghv up-regulates gtpases for intracellular trafficking in g. pallidipes, especially because the virus genome shares at least putative homologs with the above-mentioned large dsdna viruses, including hsv and whispoviruses (abd-alla et al., ) . apart from the knowledge that the host's sg is the primary replication organ for gpsghv (garcia-maruniak et al., ) , and that the virus is transmitted from the infected mother to the progeny via the milk gland secretions (boucias et al., ) , the precise virus replication and dissemination mechanisms are unknown. by comparing our data with the data available from other virus-host systems, it is possible to postulate theories on gpsghv replication and dissemination in glossina. some viruses modulate the mitochondrial transport machinery to provide energy necessary for replication, especially for the viruses whose genomes are a+t-rich (ohta and nishiyama, ; anand and tikoo, ) . the gpsghv genome is a+t-rich ( %; abd-alla et al., ) , implying that virus-modulation of glossina mitochondrial transport machinery is a good possibility. the mitochondrial oxoglutarate/malate carrier (ogc) protein is important for the tricarboxylic acid cycle (tca), gluconeogenesis and nitrogen metabolism (cappello et al., ) . ogc is reportedly upregulated as an adaptive response to prevent mitochondrial injury (ripoli et al., ) . thus, the up-regulation of ogc (d tm ) in g. pallidipes (table ) may be gpsghv-induced when robust virus replication occurs, especially in the event of overt sgh symptoms. another host protein targeted by viruses to facilitate replication is the transketolase (tktl)- (d tm ). we found tktl protein to be up-regulated more than -fold in g. pallidipes (see table ) unlike in g. m. morsitans. tktl provides a link between the glycolytic, pentose-phosphate, and nucleotide synthesis pathways (brault et al., ) . during active virus replication when rapid dna synthesis is required, carbohydrate molecules are channeled to the dna synthesis machinery through the tktl pathway, a process of utmost importance in proliferating tissues (chen et al., ) . this is of particular interest in this case of induction of the sgh symptoms in g. pallidipes, especially because sgh is mainly due to cell proliferation (guerra et al., ) . since the tktl pathway allows synthesis of ribose without the need of oxygen, gpsghv may highjack the tktl pathway to circumvent the need for oxygen (noch and khalili, ) , thus allowing rapid gpsghv genome replication. another host protein involved in viral replication is proteasome α- (d tp ; table ), a key protein in the atp/ubiquitin-dependent non-lysosomal proteolytic pathway. for instance, the interaction of proteasome α-subunit psma with hepatitis c virus (hcv) led to an inhibition of host protease activity and thus stimulated transcription trans-activation by hcv (krüger et al., ) . several other host proteins involved in viral replication that were detected in the current study included eif m (d tmn ; cheshenko et al., ) , molecular chaperones (e.g., hsp ; d ts ; kariithi et al., ) and s proteasome regulatory complex proteins (d tnj ; verchot, ; see table ). the hypothetical conserved protein (d trx ; table ) is % identical to the m. domestica anoxia up-regulated like protein and its expression in our case is virus-induced. mutuel et al. ( ) reported a significant induction of reactive oxygen species (ros) in the tracheal and fat body systems of lepidopteran insects early in infection with junonia coenia densovirus. the authors made this observation prior to viral replication before any detectable disease symptoms. interestingly, decrease of ros induction positively correlated with exponential phase of viral infection. it has been proposed that gpsghv may replicate in the host's fat bodies, and that the host's tracheal system provides a conduit for the virus transmission (kariithi, ) . it is likely that this and perhaps other similar proteins play roles during gpsghv replication. taken together, our data provide potential targets for future investigations of how gpsghv replicates and is disseminated in the host. upon successful cellular entry, viruses must evade the host's immune responses, a process for which the host's ubiquitin/proteasome system (ups) has significant roles. in the current study, we detected the main components of the ups, i.e., e ligase (d tnj ) and s proteasome (d trz ) ( table ) . the ups is essential for persistent infection of some viruses. for instance, plant rna viruses in the family luteoviridae (genera poleroviruses and enamoviruses) encode viral suppressors of rna silencing (vsrs) that hijack the ups components to promote degradation of key components of the host's rnainterference (rnai) system, thereby promoting virus replication (verchot, ) . dna viruses are known to be under host rnai surveillance. these include invertebrate iridoviruses (bronkhorst et al., ; kemp et al., ) , baculoviruses (jayachandran et al., ) , densoviruses (ma et al., ) , whispoviruses (huang and zhang, ) , and plant viruses (blevins et al., ) . as stated above, gpsghv infections are frequently observed, but remain asymptomatic and seldom result in sgh symptoms both in nature and in laboratory-bred glossina species. although the questions of how gpsghv infection progresses from a covert asymptomatic infection to an overt symptomatic infection are yet to be answered, we speculate that the virus is under host rnai surveillance, hence components of the host's ups systems form ideal candidates for further studies. another group of host antiviral defense proteins detected in this study were the v-atpases (d tli ; d tsc ; d tlr ; and d tlb ; table ), whose activity leads to acidification of intracellular compartments, necessary for multiple cellular processes (jefferies et al., ) . recently, lu et al. ( ) reported that over-expression of v-atpase in bombyx mori nucleopolyhedrovirus (bmnpv)-infected cells significantly inhibited viral proliferation. potentially, the acidification of endosomes and lysosomes by v-atpase renders these organelles competent for viral degradation. it is therefore not surprising that in the current study, v-atpase were up-regulated in the sg proteome of g. m. morsitans as opposed to that of g. pallidipes ( table ) as the former appear to be less permissive to gpsghv replication compared to the latter. similar to the v-atpase, mitochondrial atp synthase was down-regulated during white spot syndrome virus (wssv) infection in shrimps (wang et al., ) . the ubiquinol-cytochrome c reductase iron-sulfur subunit (rieske subunit/bc ) detected in the current study (d tr ; table ) was demonstrated to be up-regulated during the infection of anopheles gambiae by plasmodium falciparum (marie et al., ) . the up-regulation of bc in these two cases could have been due to the presence of the parasites in the mosquito, which could be a response involved in parasite resistance. the α-and β-subunits of mitochondrial processing peptidase (mpp; d trb ; table ) are homologous to the core and core proteins of the bc complex (braun and schmitz, ) . mpp and bc complex appear to have similar pathogen-induced modulation patterns, and their up-regulation in g. m. morsitans may be an adaptive antiviral host resistant response. it is still not clear how gpsghv induces cellular proliferation in the host's sg tissue. however, other studies have demonstrated that some viruses induce cellular proliferation via modulation of specific signaling pathways. protein phosphatase a (pp a; d tln ; table ) is critical in the regulation of cell proliferation, signal transduction, cytoskeletal dynamics, and apoptosis (seshacharyulu et al., ) . some viral proteins such as the small t antigen of sv specifically target and directly interact with and displace pp a's scaffolding b subunit thereby inducing cellular proliferation (guergnon et al., ) . to activate intracellular signaling pathways, some viruses use various approaches to hijack the g-protein-coupled receptors (gpcrs; d tpg ; table ), leading to enhancement of viral pathogenesis (sodhi et al., ; lin et al., ) . other viruses such as kshv encode potent and constitutively active gpcr homologs that modulate cellular proliferation (kirshner et al., ) . hypothetically, some gpsghv envelop proteins (table s ) could interact with host proteins to trigger signaling pathways resulting in hyperplasia as has been reported in other viruses such as the fowl poxvirus (afonso et al., ) . it is interesting to experimentally validate whether these virus and/or host proteins are actually involved in the development of sgh in glossina. an important step during assembly of viruses is processing of viral mrnas, which in some cases involve the host transacting splicing factors such as serine/arginine-rich proteins (srps; akopian et al., ) . to ensure production of their own protein diversity, adenoviruses, hsv- , influenza a viruses (iav) and hiv manipulate mrna splicing by phosphorylating srps (estmer-nilsson et al., ; sciabica et al., ; fukuhara et al., ; dubois et al., ) . therefore, it not surprising that in the current study, srp (d tn ) was up-regulated in gpsghv-infected g. pallidipes (active viral replication), but down-regulated in g. m. morsitans (less permissive to viral replication) ( table ) . however, it is currently unknown how gpsghv mrnas are processed. another host protein involved in viral assembly is tailless-complex polypeptide protein- (tcp- ; d tp and d tmk ; table ). for instance, tcp- has been implicated in the assembly of hepatitis b/c virus capsids (lingappa et al., ; inoue et al., ) , while annexins (d trs and d tme ; table ) are involved in the hiv- assembly in lipid rafts (harrist et al., ; saxena et al., ) . other proteins that may be involved in viral assembly include the s proteasome non-atpase regulatory subunit (atpase ; d tn ), which was down-regulated as seen in the sg proteome of g. pallidipes (table ) . potentially, this protein may regulate (by blocking) degradation of viral proteins. a gene similar to atpase was found to be down-regulated more than -fold in the rice stripe virus (rsv) infected small brown plant hopper, laodelphax striatellus (lee et al., ) . induction of the sgh symptoms is possibly a reflection of active production of viable progeny virus particles. during active virus progeny production, enveloped viruses are known to depend on the endoplasmic reticulum (er) for maturation of viral envelope glycoproteins, and proteins involved in the formation of replication complexes, assembly, envelopment and genome packaging (medigeshi et al., ; scheel and rice, ) . this imposes a tremendous protein load in the er, leading to er stress. consequently, er stress results in the induction of the unfolded protein response (upr), an evolutionary conserved prosurvival pathway that signals the nucleus to induce the expression of various chaperones (walter and ron, ) . in some cases, interaction between the induced chaperones and viral proteins is critical for processing of viral proteins and assembly of mature virions. during prolonged and overwhelming er stress, upr switches from being prosurvival to proaptotic (szegezdi et al., ) . prolonged virus-induced er stress/upr responses modulate a variety of signaling pathways that contribute to viral pathogenesis (fung and liu, ) , and may lead to cellular proliferation and hypertrophy. the up-regulation of upr-associated proteins and several molecular chaperones in flies with sgh symptoms (see table ) implicates the upr/er stress machinery in the development of overt sgh symptoms in g. pallidipes. as discussed above, the tktl pathway may also be involved in the expression of overt sgh symptoms in g. pallidipes. the current data provide potential targets for development of rationally designed antiviral strategies in large tsetse fly rearings for sterile insect technique. the data presented in this study provide hints as to why g. m. morsitans is much less susceptible host to gpsghv infection compared to g. pallidipes. the known and/or putative functions inferred from sequence similarity analyses revealed that the differentially modulated proteins we have identified are potentially involved in various aspects of gpsghv pathogenesis. specifically, and like in many other viruses, gpsghv appears to deploy a repertoire of strategies to exploit the host intracellular signaling pathways for replication, especially in g. pallidipes. in the case of g. m. morsitans, host proteins involved in antiviral defense systems appeared to be dominant. some of the pathways that appear to be targets of the virus include the upr and tktl pathways, implicating their involvement in expression of overt sgh symptoms in g. pallidipes. the proteins involved in these pathways deserve further functional (experimental) validations to understand the relevance of the differences in their expression patterns. the current study is a critical baseline data in a new coordinated research project (crp) initiated by iaea, aimed at gaining a deeper knowledge of the glossina/symbiont/gpsghv tripartite interactions and how these interactions affect trypanosoma parasite transmission (van den abbeele et al., ) . we have designed rnai bioassays to further investigate how asymptomatic gpsghv infection is maintained in glossina. candidate proteins experimentally validated as essential for efficient gpsghv pathogenesis are ideal targets for developing rationally designed antiviral strategies to control the virus infections in tsetse mass rearing facilities. in a larger view, our data are important for future studies on molecular and biochemical routes employed by members of the new entrants into the family of insect viruses, the hytrosaviridae. hk,İİ, jv, mv, and aa participated in the design of the study. aa and im set up the bioassays. hk andİİ processed and quantified the salivary gland proteins. sb performed the lc-ms/ms measurements. sb and hk analyzed the proteomics data sets. hk and em annotated/characterized the proteins. hk wrote the manuscript. jv, mv, aa,İİ, em, eo, and sn contributed in writing the paper, providing critical comments and suggestions. all the authors read and approved the final manuscript. the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fmicb. . development of a non-destructive pcr method for detection of the salivary gland hypertrophy virus (sghv) in tsetse flies quantitative pcr analysis of the salivary gland hypertrophy virus (gpsghv) in a laboratory colony of glossina pallidipes genome analysis of a glossina pallidipes salivary gland hypertrophy virus reveals a novel large double-stranded circular dna virus managing hytrosavirus infections in glossina pallidipes colonies: feeding regime affects the prevalence of salivary gland hypertrophy syndrome hytrosaviridae: a proposal for classification and nomenclature of a new insect virus family dynamics of the salivary gland hypertrophy virus in laboratory colonies of glossina pallidipes (diptera: glossinidae) tsetse salivary gland hypertrophy virus: hope or hindrance for tsetse control? universal primers for rapid detection of hytrosaviruses the genome of fowlpox virus signal recognition particle: an essential protein-targeting machine ubiquitin and plant viruses, let's play together! plant physiol mise en evidence et purification d'un virus dans la proliferation monstrueuse glandulaire d'insectes. 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regulation discovery of the genes in response to white spot syndrome virus (wssv) infection in fenneropenaeus chinensis through cdna microarray intercommunity effects on microbiome and gpsghv density regulation in tsetse flies characterization of a rab gtpase up-regulated in the shrimp peneaus japonicus by virus infection interproscan-an integration platform for the signature-recognition methods in interpro analysis of rab gtpase-activating proteins indicates that rab a/b and rab are important for herpes simplex virus secondary envelopment rab proteins as membrane organizers the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © kariithi, ince, boeren, murungi, meki, otieno, nyanjom, van oers, vlak and abd-alla. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - op m pu authors: wu, zhiqiang; yang, li; ren, xianwen; he, guimei; zhang, junpeng; yang, jian; qian, zhaohui; dong, jie; sun, lilian; zhu, yafang; du, jiang; yang, fan; zhang, shuyi; jin, qi title: deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases date: - - journal: the isme journal doi: . /ismej. . sha: doc_id: cord_uid: op m pu studies have demonstrated that ~ %– % of emerging infectious diseases (eids) in humans originated from wild life. bats are natural reservoirs of a large variety of viruses, including many important zoonotic viruses that cause severe diseases in humans and domestic animals. however, the understanding of the viral population and the ecological diversity residing in bat populations is unclear, which complicates the determination of the origins of certain eids. here, using bats as a typical wildlife reservoir model, virome analysis was conducted based on pharyngeal and anal swab samples of bat individuals of major bat species throughout china. the purpose of this study was to survey the ecological and biological diversities of viruses residing in these bat species, to investigate the presence of potential bat-borne zoonotic viruses and to evaluate the impacts of these viruses on public health. the data obtained in this study revealed an overview of the viral community present in these bat samples. many novel bat viruses were reported for the first time and some bat viruses closely related to known human or animal pathogens were identified. this genetic evidence provides new clues in the search for the origin or evolution pattern of certain viruses, such as coronaviruses and noroviruses. these data offer meaningful ecological information for predicting and tracing wildlife-originated eids. emerging infectious diseases (eids) pose a great threat to global public health. approximately %- % of human eids originate from wildlife, as shown by the typical examples of hemorrhagic fever, avian influenza and henipavirus-related lethal neurologic and respiratory diseases that originated from rodents, wild birds and bats (jones et al., ; wolfe et al., ; lloyd-smith et al., ; marsh and wang, ; smith and wang, ) . the primary issues associated with the prevention and control of eids are how to quickly identify the pathogen, determine where it originated and control the chain of transmission. these issues, along with the limited knowledge of the viral population and ecological diversity of wildlife, complicate the study of eids. therefore, meaningful information afforded by the understanding of the viral community present in wildlife, as well as the prevalence, genetic diversity and geographical distribution of these viruses, is very important for the prevention and control of wildlife-borne eids daszak et al., ) . bats are mammals with a wide geographical distribution, extensive species diversity, unique behaviors (characteristic flight patterns, long life spans and gregarious roosting and mobility behaviors) and intimate interactions with humans and livestock (calisher et al., ) . they are natural reservoirs of a large variety of viruses, including many important zoonotic viruses that cause severe diseases in humans and domestic animals, including henipaviruses, marburg virus and ebola virus (luis et al., ; quan et al., ; o'shea et al., ) . the severe acute respiratory syndrome (sars) outbreak in , which resulted in nearly cases and deaths worldwide, was suspected to have originated in bats and then spread to humans (li et al., ) . all of these examples reveal that zoonotic viruses carried by bats can be transmitted directly or via certain intermediate hosts from bats to humans or domestic animals with high virulence. as most bat-borne pathogens are transmitted by four routes (airborne, droplet, oral-fecal and contact transmission) from the respiratory tracts, oral cavities or enteric canals of bats to other species, it is particularly important to determine the viral communities present at these locations. in this study, bat individuals of representative species across china were sampled by pharyngeal and anal swabbing, to assess the variety of viruses residing in bat species. metagenomic analysis was then conducted to screen the viromes of these samples. here we outline the viral spectrum within these bat samples and the basic ecological and genetic characteristics of these novel bat viruses. the identification of novel bat viruses in this study also provides genetic evidence for cross-species transmission between bats or between bats and other mammals. these data offer new clues for tracing the sources of important viral pathogens such as sars coronavirus (sars-cov) and middle east respiratory syndrome cov (mers-cov). pharyngeal and anal swab samples were collected separately and then immersed in virus sampling tubes (yocon, beijing, china) containing maintenance medium and were temporarily stored at − °c. the samples were then transported to the laboratory and stored at − °c. viral nucleic acid library construction, next-generation sequencing and taxonomic assignments a tube with either a pharyngeal or anal swab sample in maintenance medium was vigorously vortexed to re-suspend the sample in solution. samples from the same bat species and from the same site were then pooled. the pooled samples were processed with a viral particle-protected nucleic acid purification method and then amplified by sequenceindependent reverse transcriptase-pcr as described previously wu et al., ) . briefly, the samples were centrifuged at g for min at °c. supernatant from each sample was filtered through a . -μm polyvinylidene difluoride filter (millipore, darmstadt, germany), to remove eukaryotic and bacterial-sized particles. the filtered samples were then centrifuged at g for h at °c. the pellets were re-suspended in hank's balanced salt solution. to remove naked dna and rna, the re-suspended pellet was digested in a cocktail of dnase and rnase enzymes, including u of turbo dnase (ambion, austin, tx, usa), u of benzonase (novagen, darmstadt, germany) , and u of rnase one (promega, madison, wi, usa) at °c for h in × dnase buffer (ambion). the viral nucleic acids were then isolated using a qiamp minelute virus spin kit (qiagen, valencia, ca, usa). viral first-strand cdna was synthesized using the primer k- n ( ′-gaccatctagcgacct ccac-nnnnnnnn- ′) and a superscript iii system (invitrogen, carlsbad, ca, usa). to convert firststrand cdna into double-stranded dna, the cdna was incubated at °c for h in the presence of u of klenow fragment (neb, ipswich, ma, usa) in × neb buffer (final volume of μl). sequenceindependent pcr amplification was conducted using μm primer k ( ′-gaccatctagcgacct ccac- ′) and . u phusion dna polymerase (neb). the pcr products were analyzed by agarose gel electrophoresis. a dna smear of larger than bp was excised and extracted with a minelute gel extraction kit (qiagen). the amplified viral nucleic acid libraries were then analyzed using an illumina ga ii sequencer (illumina, sandiego, ca, usa) for a single read of bp in length. the raw sequence reads were filtered using previously described criteria , to obtain valid sequences. each read was evaluated for viral origin by conducting alignments with the ncbi nonredundant nucleotide (nt) database (nt) and protein database (nr) using blastn and blastx (with parameters -e e- -f t). reads with no hits in nt or nr were further assembled using the velvet software (v . . , pittsburgh supercomputing center, pittsburgh, pa, usa) and the contigs were again aligned with nt and nr to identify any viruses that were present. the taxonomies of the aligned reads with the best blast scores (e-value o − ) from all lanes were parsed and exported with megan -metagenome analyzer . besides the alignment-first analytical strategy, we also tested assembly-first strategy to analyze the sequence data. the reads were firstly assembled by metagenomics assemblers (for example, metavelvet and idba-ud) (namiki et al., ; peng et al., ) and the output contigs were then aligned to nt and nr. as the percentage of viral reads was small in the whole data sets and most of the assembled contigs were thus of bacterial and host origins, the assembly-first strategy generally had lower sensitivity than the alignmentfirst strategy in this study. identification of the prevalence and positive rate of each virus according to the molecular clues provided by metagenomic analyses, the sequence reads classified into the same virus family or genus by megan were extracted and then assembled with seqman program (lasergene, dnastar, madison, wi, usa). a draft genome with several or a large number of single-nt polymorphisms of each virus was obtained. based on the partial genomic sequences of the viruses obtained by the assembly, we designed specific nested primers for pcr or reverse trancriptase-pcr to screen for each virus in individual samples from each bat species (the primer sequences for each virus are available in supplementary table s ). genome sequencing of each virus in positive samples by pcr the accurate locations of the reads and the relative distances between reads of the same virus were determined based on the alignment results exported using megan . representative positive samples for each virus were selected for genome sequencing as viral quasi-species. the reads with accurate genomic locations were then used for reads-based pcr to identify partial genomes. based on the partial genomic sequences obtained by specific nested pcr, the remaining genomic sequences were determined using inverse pcr, genome walking, and ′-and ′ rapid amplification of cdna ends. genome sequences and phylogenetic and evolutionary analyses all genome sequences have been submitted to genbank. the accession numbers for all sequences are listed in supplementary table s . the ga ii sequence data have been deposited into the ncbi sequence reads archive under the accession number sra . routine sequence alignments were performed using megalign program (lasergene) or t-coffee with manual curation. mega . (phoenix, az, usa) was used to align the nt and amino acid (aa) sequences using muscle package with the default parameters. the best substitution model was then evaluated using model selection package. finally, a maximum-likelihood method with an appropriate model was used to conduct phylogenetic analyses with bootstrap replicates. supplementary table s . bat viromes in china z wu et al main bat species, the most common genera and families of both frugivorous and insectivorous bats that reside in urban, rural and wild areas throughout china. all pharyngeal and anal swab samples were classified and combined into pools and then subjected to virome analysis . finally, a total of gb of nt data ( valid reads, bp in length) was obtained. in total, reads were best matched with viral proteins available in the ncbi nr database (~ . % of the total sequence reads). the number of virusassociated reads in each lane varied between and . in total, families of phages, insect viruses, fungal viruses, mammalian viruses and plant viruses were parsed. after excluding bat habit-related non-mammalian viruses, including insect viruses (mainly of the families baculoviridae, iflaviridae, dicistroviridae and tetraviridae; subfamily densovirinae), fungal viruses (mainly of the families chrysoviridae, hypoviridae, partitiviridae and totiviridae), phages (the order caudovirales and families inoviridae and microviridae) and plant viruses (matched reads of the non-mammalian virus families are provided in supplementary table s ), overview of the reads of families of mammalian viruses in each pooled sample is shown in figure a and supplementary table s . in addition, an overview of the classification from family to genus of the identified bat viruses is shown in figure b . the reads related to the family parvoviridae comprised the largest proportion of viruses as shown in figure , most of which were classified into the subfamily densovirinae. the dominating abundance of insect densoviruses was associated with the insectivorous habits of bats. the existence and prevalence of virus strains in of the families were confirmed. in total, positive results were confirmed by pcr screening (positive rates are shown in supplementary table s ) and viruses of representative positive samples were selected for genomic sequencing as quasi-species of these viruses (supplementary table s ; the full-length sequenced viruses are labeled in red). the most widely distributed families of mammalian viruses were herpesviridae, papillomaviridae, retroviridae, adenoviridae and astroviridae. the diverse reads related to these families occupied~ % of the total viral sequence reads (supplementary table s ). the assessment of diverse reads of bat herpesviruses, bat papillomaviruses, bat retroviruses, bat astroviruses and bat adenoviruses (supplementary figure s ) , and full-length sequenced representative strains of these viruses revealed that most of these viruses were distinct from each other within each family ( %- % nt identities). in addition to the above families, many reads related to the families circoviridae, paramyxoviridae, coronaviridae, caliciviridae, polyomaviridae, rhabdoviridae, hepeviridae, bunyaviridae, reoviridae, flaviviridae and picornaviridae, and the subfamily parvovirinae, exhibited low nt or aa sequence identities with known viruses. although sequence reads related to the families orthomyxoviridae and hepadnaviridae were occasionally present in some of the samples, we failed to amplify any sequences of viruses in these families, which may have been a result of very low viral loads. although samples from frugivorous bats of two species (rousettus leschenaultia and cynopterus sphinx) were collected for virome analysis, only a few reads related to herpesvirus, papillomavirus and retrovirus were found. this finding revealed that the virome of frugivorous bats is far less abundant than that of insectivorous bats in this study. bat main single-stranded rna viruses (coronaviridae, paramyxoviridae and picornaviridae) the family coronaviridae contains two subfamilies, coronavirinae and torovirinae. the subfamily coronavirinae includes four approved genera, alphacoronavirus, betacoronavirus, deltacoronavirus and gammacoronavirus. it is a group of large enveloped viruses with a positive single-stranded rna genome (~ to kb in length). six αand β-covs (hku , oc , nl , e, sars-cov and mers-cov) are human pathogens that cause mild-tosevere disease (corman et al., b; li et al., ; cui et al., ; o'shea et al., ) . here, novel bat covs (btcovs) were identified separately in bat species of genera from provinces (supplementary table s and supplementary figure s ). covs from seven bat species are reported for the first time. phylogenetic trees were constructed based on the deduced rna-dependent rna polymerase (nsp ) (figure a spike (s) and figure b proteins). the sequence identities of these btcovs are shown in supplementary table s . eleven btcovs were assigned to a group with lineage-b beta-covs and three btcovs were assigned to a group with lineage-c beta-covs. the btvs-betacov/sc identified in vespertilio superans bats had a closer genetic relationship with mers-cov than with other btcovs (supplementary figure s ) , as well as with neocov identified in african neoromicia capensis bats (corman et al., a) . one btcov, bthp-betacov/zj , represented a separate clade related to lineage b. the overall nt identity of this cov genome with lineage-b covs was only % and the rna-dependent rna polymerase and s proteins shared only % and % aa identities with those of lineage-b covs. this cov contained an unusual putative s-related orf between orf ab and the s gene (supplementary figure s and supplementary table s ) (quan et al., ) . a signal peptide ( - aa) and a transmembrane region ( - aa) were identified in the s-related orf , suggesting that this protein might be a surface protein. the remaining btcovs were all assigned to the genus alphacoronavirus and formed many novel separate clades. for btmr-alphacov/sax and btnv-alphacov/sc , although the phylogenetic tree constructed based on rna-dependent rna polymerase indicated that these two viruses were clustered with hku , the phylogenetic tree based on the s protein indicated that these two viruses represented two separate clades far from other covs, suggesting that recombination may occur in their genomes. although the orf ab, e, m and n genes of btrf-alphacov/hub and btms-alphacov/ z wu et al gs shared very high sequence identities (higher than %), the s genes of these two viruses shared only % nt identity. a similar phenomenon was observed between btrf-alphacov/yn and hku . the results of sample-by-sample screening of bat samples from the same gathering place revealed that btcov strains of the same species identified in the same cave had significantly diverse features. some key gene segments, such as the s and orf genes, presented great diversity with low sequence identities (supplementary figure s ) , indicating that these two genes are hypervariable regions within the btcov genome. similar to recently reported sars-like covs (sl-covs) (wiv , rs and lyra ) (ge et al., ; he et al., ) , two btcovs, btrs-betacov/ yn and btrs-betacov/gx , identified in rhinolophus sinicus from the yunnan and guangxi provinces shared the highest similarities with sars-covs in the backbone (including orf ab, e, m and n genes), orf , orf a, orf b and orf genes compared with other bat lineage-b β-covs (supplementary tables s -s and supplementary figures s a and b and s ) . furthermore, the s genes of lineage-b β-covs from r. sinicus had much higher genetic diversity and were scattered among the phylogenetic clades of sars-covs and lineage-b covs from other bat species (supplementary figure s c) . co-infections of cov strains of sublineages and of group in miniopterus fuliginosus were detected in two anal specimens collected in guangdong and henan. the covs of sublineage with highly similar backbone sequences presented differing degrees of variation in the s region. recombination was confirmed by similarity plots, bootscan analysis and detection of putative breakpoints around the s regions in the genomes of lineage covs in m. fuliginosus and m. pusillus. (supplementary table s and supplementary figure s ). the family paramyxoviridae is a group of large enveloped viruses with negative-sense singlestranded rna genomes (~ to kb in length) that are responsible for a variety of mild-to-severe human and animal diseases (mayo, ; smith and wang, ) . bat paramyxoviruses (btparavs) were separately identified in bat species from provinces of china (supplementary table s and supplementary figure s ). the full-length sequence of viruses (btmf-parav/ah , btml-parav/ qh and btha-parav/gd ) were almost completely determined (supplementary figure s and supplementary table s ) and other viruses were partially or completely sequenced in the l gene. twelve of the novel btparavs identified in the different bat species could be clustered together and formed a separate phylogenetic clade. alternatively, these btparavs could be classified into a potential separate genus, shaanvirus, with btmf-parav/ah and btml-parav/qh as prototypes. the genomic organizations of these two viruses were similar to those of three previously reported members of the genus jeilongvirus. however, the genomes of btms-parav/anhui and btml-parav/qh were shorter in length than those of the three rodent viruses and the sequence identities were low (supplementary table s ). the remaining two novel btparavs could be clustered together with rubulaviruses as new species (figure c . the central domain of the n protein contained three conserved motifs common to all paramyxoviruses, and the six conserved domains within the l proteins of the order mononegavirales (lau et al., ) could be found in all three fulllength sequenced viruses. picornaviruses (picovs) of the family picornaviridae are small, non-enveloped, positive single-stranded rna viruses with a genome of - kb in size. the members of the family picornaviridae cause mucocutaneous, encephalic, cardiac, hepatic, neurological and respiratory diseases in a wide variety of vertebrate hosts (tracy et al., ; wang et al., ) . nineteen bat picovs (btpicovs) were identified in bat species from provinces (supplementary table s and supplementary figure s ). phylogenetic analysis of the rnadependent rna polymerase genes was conducted (figure d . seven btpicovs could be clustered with three previously reported btpicovs (clade ) and could then be divided into separate sub-clades according to their host genera. clade contained two sub-clades formed by four btpicovs of two bat genera. clade was formed by two btpicovs identified in different bat genera and clustered in a sister relationship with the genus sapelovirus. clade contained two btpicovs identified in the same bat genus that were closely related to the genus kobuvirus. the previously reported miniopterus schreibersii picov- was closely related to the genera cardiovirus and senecavirus. two btpicovs, btrf-picov- /yn and btmf-picov- /sax , showed lower aa identities with other known picovs. different btpicovs identified from the same bat genus, such as rhinolophus or miniopterus, in different locations showed very close genetic relationships. the aa identities of the predicted rnadependent rna polymerase proteins of these novel btpicovs were low compared with known picovs (supplementary table s ). the predicted p , p and p regions and cleavage sites of these viruses showed typical features of picovs (supplementary table s ). the family circoviridae is a group of viruses with small, non-enveloped, circular single-strand dna genomes of . - kb in length (fauquet and fargette, ) . porcine circovirus (cv)- is the main swine pathogen (chae, ) . thirty-four novel bat cvs (btcvs) were identified in bat species table s and supplementary figure s ). the genome sizes of these viruses varied from to nts. seven new clades of btcvs in the genera circovirus and cyclovirus were found. clade was formed by two viruses clustered in a sister relationship with two pathogenic viruses, porcine and dog cvs (chae, ; li et al., ) , at the same root with a short branch length. three btcvs were closely related to human cycloviruses. seven btcvs were assigned to the proposed genus cyclovirus and formed four separate clades, three of which were closely related to human cycloviruses. a separate clade, clade , was constructed from two btcvs and was closely related to the genus cyclovirus. in addition to these viruses, novel btcvs branched out of the root of cvs and cycloviruses revealed the presence of new genera different from the known genera (figure a) . viruses of the family parvoviridae comprise a group of small, non-enveloped viruses with linear positive-sense single-stranded dna (~ kb genomes) that infect vertebrate animals and cause mild-tosevere diseases (brown, ) . bat parvoviruses (btpvs) and bat bocaviruses were identified in nine bat species from eight provinces (supplementary table s and supplementary figure s ). in addition to viruses in the genera bocavirus, parvovirus and amdovirus, four btpvs were most similar to the recently reported human bufavirus members and the bufavirus-related wuharv parvovirus, with similar ns and vp proteins (higher than % aa identities) (phan et al., ; yahiro et al., ) . bthp-pv/gd , in the genus parvovirus, was very closely related to a recently identified rat pv, with % aa identity (figure b) . the aa identities among the predicted vp proteins of these btpvs and bat bocaviruses, and other known members of the subfamily parvovirinae, are shown in supplementary table s . bat adeno-associated viruses have been described previously (li et al., b) ; however, considering the nonpathogenic nature of adeno-associated viruses, we did not perform further verification of these viruses. other rare bat viruses six bat caliciviruses (btcalvs), four bat polyomaviruses, one bat hepatitis e virus, one bat rhabdovirus, one bat bunyavirus, one bat orthoreovirus and one bat rotavirus were identified. phylogenetic in previous studies, zoonotic viruses in more than virus families have been identified in bats around the world (chen et al., ; o'shea et al., ) . two bat virome analyses conducted by li et al. ( a) and donaldson et al. ( ) have revealed the presence of covs, herpesviruses, picovs, cvs, adenoviruses, adeno-associated viruses and astroviruses in some bat species of north america. one bat virome analysis conducted by ge et al. ( ) mainly described insect viruses in some bat species of china. one bat virome analysis conducted by ng et al. ( ) has revealed the presence of a novel rhabdovirus in big brown bats, and one bat virome analysis conducted by he et al. ( ) has described the spectrum of viruses harbored by several bat species in myanmar. different from these previous reports, this study was the first to characterize the pharyngeal and anal virome of representative bat samples in china. we did not perform additional verification of non-mammalian viruses because of the association of the abundance of these viruses (not initially harbored in bats) with their life habits. this report suggests that bats harbor a large spectrum of mammalian viruses. except for a few viruses, such as btcalvs, btcvs and btpvs, which are closely related to known viruses, most of the bat viruses identified here that were widely distributed . these findings reveal that these three bat genera may act as major reservoirs for diverse mammalian viruses in china. notably, all bats collected in this study were considered to be apparently healthy and showed no overt signs of disease, further confirming that bats can tolerate diverse viruses through their unique metabolic and immune systems (o'shea et al., ) . this study extends the host range for members of each viral family and reveals unique ecological and evolutionary characteristics of bat-borne viruses. the diverse btcovs were grouped into several novel evolutionary clades that significantly differed from those of all known αand β-covs, providing additional evidence to support investigations of the evolution of bat-originated covs. with regard to btparavs, a previous study has revealed that bats host major mammalian paravs in the genera rubulavirus, morbillivirus, henipavirus and the subfamily pneumovirinae (drexler et al., ) . however, in this study, except for viruses assigned to the genus rubulavirus, the remaining viruses formed a new genus distant from the known genera and the identified btparavs showed no direct relationship with the known human or animal pathogens of the family paramyxoviridae. these results suggest an entirely different distribution of btparavs in china than previously reported. although the classifications of bat herpesviruses, bat papillomaviruses, bat retroviruses, bat astroviruses, bat adenoviruses, btpicovs, btcvs, btpvs and bat bocaviruses were extended according to the current virus taxonomy file released by the international committee on taxonomy of viruses, the large number of novel viruses grouped into the various evolutionary clades identified in this study further expand the taxa to include many new viral genera and species. many new clades formed by btcvs distinct from all known members of the genera circovirus and cyclovirus could be candidates for many new genera. viruses related to henipaviruses, ebola virus, rabies virus and pathogenic bunyaviruses were not detected in the chinese bat species examined in this study. diverse herpesviruses and papillomaviruses identified countrywide support the hypothesis that these dna viruses from different bat species are located in different phylogenetic positions within each family without strict host or geographic specificity (garcia-perez et al., ) ; however, many other dna or rna viruses, such as btcovs, btparavs, btpicovs and btpvs or bat bocaviruses, identified from the same or different bat species from different locations shared high sequence identities and close genetic relationships. these phenomena indicate that certain bat-originated dna and rna viruses have the potential for intra-or cross-species transmission concomitant with the migration, co-roosting and intra-or inter-species contact of their bat hosts. the identification of some viruses, such as certain rat pv-related btpv, norovirus-related btcalvs, human or swine cv-related btcvs and bat rotavirus in the rotavirus a group, also provides a new understanding of the evolution of these viruses in different mammalian hosts and possible transmission events that occur between bats and other hosts. furthermore, btcovs had more distinctive features than the other bat viruses. highly diverse s genes or orf s were present in particular covs carried by bats of the same species from different locations or even the same gathering place. considering the diversity of covs, co-infections may create opportunities for recombination and the emergence of new covs that are able to adapt to new hosts. these findings may explain why tracing the potential cov-related eids in insectivorous bats is often complicated by the presence of diverse key genomic segments and no virus with an identical genome sequence related to the pathogens causing human or animal eids has been identified in insectivorous bats. instead, the origin of the ebola virus and henipaviruses could be relatively easily confirmed by the identification of identical viruses in frugivorous bats (calisher et al., ; leroy et al., ; smith and wang, ) . only lineage-b β-covs of six bat species (r. ferrumequinum, r. sinicus, r. pusillus, r. macrotis, r. affinis and chaerephon plicata) from china (sl-covs) are closely related to sars-covs, as 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camels metavelvet: an extension of velvet assembler to de novo metagenome assembly from short sequence reads distinct lineage of vesiculovirus from big brown bats, united states bat flight and zoonotic viruses idba-ud: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth acute diarrhea in west african children: diverse enteric viruses and a novel parvovirus genus identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in nigeria bats are a major natural reservoir for hepaciviruses and pegiviruses bats and their virome: an important source of emerging viruses capable of infecting humans evolution of virulence in picornaviruses hepatitis a virus and the origins of picornaviruses origins of major human infectious diseases coronavirus diversity, phylogeny and interspecies jumping discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus virome analysis for identification of novel mammalian viruses in bat species from chinese provinces novel henipa-like virus, mojiang paramyxovirus, in rats novel human bufavirus genotype in children with severe diarrhea, bhutan unbiased parallel detection of viral pathogens in clinical samples by use of a metagenomic approach novel sars-like betacoronaviruses in bats mers-related betacoronavirus in vespertilio superans bats receptor usage and cell entry of bat coronavirus hku provide insight into bat-to-human transmission of mers coronavirus covs, and the highly diverse s genes present in only r. sinicus imply that frequent recombination events occur among sl-covs of r. sinicus and other hosts, suggesting that the transmission of sl-covs from r. sinicus to other mammals is a result of the viruses obtaining novel s genes. these data indicate that human sars-covs most likely originated through zoonotic transfer, either directly or indirectly, from chinese horseshoe bats via a complicated adaptation process and a series of rare recombination events.although the transmission of mers-cov has been confirmed by the detection of identical mers-cov sequences in dromedary camels and humans (azhar et al., ; chu et al., ; meyer et al., ) , the zoonotic transmission of this virus from dromedaries to humans is still considered to be rare (hemida et al., ) and the wildlife source of mers-covs remains unknown. the data obtained here and a recently reported lineage-c btcov, neocov, identified by another group (corman et al., a; yang et al., a) provide new clues about the sources and pathways of human-and camel-derived mers-covs in bats of the family vespertilionidae. bat species of this family may have important roles in mers-cov evolution (yang et al., b) . furthermore, the diverse s genes of these mersrelated covs may provide an opportunity for their recombination to ultimately generate new covs.in conclusion, the understanding of the viral community characteristics, genetics and ecological distribution of bat viruses could enable the rapid identification of novel viruses with variant genomes and could thus facilitate the tracing of eids in bats. furthermore, this strategy could be extended to other wildlife or livestock worldwide, ultimately increasing knowledge of the viral population and ecological community, thus minimizing the impact of potential wildlife-originated eids on public health by providing meaningful basic data. the authors declare no conflict of interest. key: cord- - h rl authors: nan title: full issue pdf date: - - journal: jacc: cardiovascular imaging doi: . /s - x( ) - sha: doc_id: cord_uid: h rl nan studies in patients with mitral valve disease have identified atrial fibrillation (af) as a risk factor ( , ) , others have found pulmonary artery systolic pressures (pasps) to be the most powerful predictor of tr progression ( ) . in a recent study ( ) , neither increased pasp nor af were associated with the rate of tr progression. cross-sectional studies suggest that tr is frequent, with a prevalence similar to that of aortic stenosis, and may represent a significant public health problem ( ) . however, there are no studies that have explicitly measured the population burden of hemodynamically significant tr attributable to specific risk factors. population burden measurements, for example, populationattributable fractions (pafs) ( , ) , are directly relevant to prevention and research prioritization because they estimate the number of events that may be preventable if the prevalence of a modifiable risk factor was reduced. therefore, this study sought to identify risk factors for the development of significant functional tr and their relative importance in a large cohort of patients with trivial or mild tr at baseline. to gain additional insight into the effect of various risk factors, the burden of tr that could be attributed to each risk factor also was estimated. a separate analysis was performed among subjects without valvular heart disease (vhd). a retrospective cohort study was conducted using the presence of an rv pacemaker lead; and ) additional valvular heart disease. patients with organic tricuspid valve disease and in whom pasp was not adequately estimated were excluded. tr was quantified by an integrated approach ( ) as described in previous studies ( , , ( ) ( ) ( ) . first, tr was graded qualitatively by using color doppler flow mapping. when more than mild tr was present, severity was determined by integrating data from the following parameters: the vena contracta width when feasible ($ mm, denoting severe tr); the presence and degree of malcoaptation of the tricuspid leaflets; tenting distance; the presence of mid-to-late systolic flow reversal in the hepatic veins; and evaluation of right heart chambers ( , , , ) . lv ejection fraction (lvef) and la size were estimated by a combination of visual assessments and standard chamber measurements. the cutoff values for lvef and la size values were based on american society of echocardiography guidelines at the time the echocardiographic studies were performed ( ) . rv function was assessed qualitatively by integrating visual assessment of the contractility of the rv walls from different views and classified using an ordinal scale (normal or mildly, moderately, or severely reduced) ( , , ) . the final rv chamber size and function were qualitatively graded as a binary variable (< or $ moderate). pasp was estimated from a clearly defined tr signal by continuous-wave doppler and inferior vena cava size and respiratory variation ( ) . isolated significant tr was defined as previously described ( ) . study endpoints. the primary endpoint of the study was the progression of tr severity from trivial/ mild to moderate/severe. statistical analysis. baseline characteristics of the groups were compared using an unpaired t-test for continuous variables and by the chi-square statistic for categorical variables. because the exact time of tr progression between the first and second echocardiographic examination was unknown, multivariate stepwise binary logistic regression models were fitted to determine independent predictors of progression to significant tr. were performed to provide additional information for the most predictive variables for tr progression ( ) . to estimate the potential contribution of various baseline risk factors to the burden of moderate or severe tr in the population, the pafs were computed, expressed as a percentage for each primary risk exposure ( , ) . generally, the pafs are the fractions of outcomes that would be prevented if the risk factors under consideration were removed, comparing current risk factor exposure to a counterfactual zero exposure. pafs associated with independent risk factors for the development of moderate or severe tr were calculated using the stata command punaf ( ) . the pafs presented are adjusted for confounders in a similar manner to the corresponding logistic regression. continuous risk factors (age and pasp) were dichotomized at their median values for the pafs calculations. in step , the logistic model was specified using paired echocardiographic data to evaluate the combined effects of baseline predictors and the appearance of new predictors between the echocardiograms. logistic regression with robust clustered standard errors was used to adjust for within-subject repeated measures ( ) . in this model, a total of , consecutive patients who met the study inclusion criteria were screened. of these, patients were excluded due to moderate or severe tr. of the remaining patients, , patients had at least additional echocardiographic study (supplemental baseline demographic and echocardiographic characteristics of the study cohort are shown in table . patients who progressed to moderate or severe tr were older, more likely to be female, and have more history of heart failure, af, and a pacing lead. in addition, there was a higher frequency of all echocardiographic variables denoting left-sided myocardial or valvular disease among individuals with tr progression. the frequency of rv enlargement or dysfunction was low, albeit higher in subjects with tr progression ( table ) . table ). the c-index of the model was . ( % ci: . to . ), indicating good model discrimination. the incidence rates of progression to moderate or severe tr as a function of age are shown in figure . similar results were obtained accounting for time in a competing risk regression model (supplemental table ). the predictive model for tr progression in patients without significant valvular disease at baseline the cart also demonstrated that, without the presence of these parameters, the risk of developing significant tr was small, regardless of other risk factors. for example, a young person with a pacing lead but with normal pap and normal la size and no af has a w % probability of developing significant tr. pafs for significant tr. the proportion of incident moderate or severe tr that could be attributed to each of the risk factors is shown in figure . age associated with a more rapid progression to significant tr. however, in that study, there was no control group of patients without tr progression. in addition, important predictors found in previous studies ( , , ) , ph, af, and left atrial size were not considered in that study ( ) . to the authors' knowledge, this is the largest study pertaining to tr progression. the present study provides a comprehensive assessment of all potential risk factors for tr progression in a large cohort of patients, providing insight into the pathogenesis of hemodynamically significant functional tr, both in terms of individual risk prediction and from the population perspective. ph was the strongest risk factor to antedate the development of significant tr. the mechanisms by which ph promotes the development of tr have been previously reviewed ( , ) . importantly, however, the causes of ph in the contemporary population are primarily aging ( ) and diastolic heart failure ( ) , rather than vhd. the present study explicitly calculated the population burden of tr attributable to various risk factors. in the overall study cohort, aging and ph had the highest paf. by contrast, the paf was substantially lower for pacing and even vhd. the low paf of pacing and vhd may be partly due to the low prevalence of these risk factors but also due to the stronger effects of other risk factors. for example, left-sided vhd may lead to tr primarily through ph and af, and pacing may depend on other concomitant risk factors to produce significant tr (e.g., tricuspid valve [tv] annulus that is already dilated). these results indicate that, even if left-sided vhd was eliminated, the incidence of moderate or severe tr would not be substantially reduced, given the strength of association and high prevalence of aging, ph, and af in the population. similarly, from the population perspective, strategies to mitigate tr post-pacemaker or -implantable cardioverterdefibrillator insertion are likely to have limited impact on the prevalence of significant tr. the cart analysis also ranks covariates in terms of their importance in the tree ( , ) . the analysis demonstrated that variables (ph, la size, age, and af) provided prognostic information that was almost identical to the full independent variables logistic regression model. interestingly, the cart analysis also provided a sensible interpretation of the most powerful predictors of tr progression that was consistent with our current mechanistic understanding (central illustration) ( ) . elevated pap represent established lhd, where pulmonary pressures are already elevated due to a more advanced lv systolic or diastolic dysfunction or vhd, with or without af. in turn, ph results in maladaptive rv remodeling, with further tricuspid valve tenting and tricuspid annular dilation ( , , ) . in the absence of elevated pap, la enlargement represents the same underlying conditions but at an earlier stage. an increase in la size most commonly reflects increased la pressure secondary to increased lv stiffness and diastolic dysfunction and is a marker of both the severity and the chronicity of diastolic dysfunction and magnitude of la pressure elevation ( ) . over time, however, an enlarged la portends pulmonary venous hypertension ( ) and a higher incidence of af ( ) . af with right atrial dilation and increased tv annular area appears to be sufficient to cause significant functional tr ( ) . however, this condition occurs infrequently in the absence of ph and is often categorized under "idiopathic" tr ( , ) . note that the cart analysis is consistent with this concept, as a small number of patients with af and normal pap remain at substantial risk for tr progression. the cart analysis is also useful in determining the need and intensity of follow-up in individuals with mild tr. in the absence of any of the major risk factors (central illustration), progression to moderate or severe tr occurred in w % of patients and therefore these patients can be followed at longer intervals. in patient with or more of the major risk factors, closer follow-up is justified and is often also dictated by the presence of significant lhd. furthermore, based on the present study results and current mechanistic concepts ( , ) , the best strategy to mitigate the progression of tr is to aggressively treat ph and af and their causes. tr is linked to lhd progression. previous studies of tr progression have generally studied risk variables at baseline data ( , ) . the present results demonstrate that tr progression entails a striking increase in the associated left-sided cardiac comorbidities such that, at the time of the final echocardiographic study, lhd was far more advanced. thus, in addition to a more severe lhd at baseline, patients whose tr progress represents a subset characterized by an accelerated course of lhd, including a more rapid increase in la size, pulmonary pressures, and higher rates of new atrial fibrillation, reduction in lvef, worsening valvular disease, and need for pacemaker or device therapy. this finding further establishes the inextricable link between functional tr progression and lhd. ( ) . nonquantitative methods are accepted ( ) and have been used in multiple previous studies ( , , , , , ) but may be less accurate. individual susceptibility to tr progression is deter- limited ability of conventional imaging modalities to assess its severity and define its pathophysiology ( , ) . left-sided heart diseases and pulmonary hypertension (ph) (either pre-or post-capillary) are the most frequent causes of ftr ( ) . less common (although its prevalence increases with age) is ftr due to isolated tricuspid annular dilatation in the setting of aging or long-standing atrial fibrillation (af) resulting in right atrial enlargement and tricuspid annular remodeling, whereas right ventricular (rv) shape and function remain normal ( ) . another uncommon etiology of ftr is primary rv disease, including cardiomyopathies or rv infarction. however, in a sizable number of cases, the etiology is uncertain, and the term "idiopathic" or "isolated" ftr has been suggested ( that have been associated with increased risk for faster ftr progression ( ) ( ) ( ) . in this issue of ijacc, [ ] ) in a sizable number of patients. rv abnormalities were visually graded as a binary variable (less than moderate or moderate or greater), and the sizes of tricuspid annulus and right atrium were not contemplated, despite evidence suggesting that both can play a role in ftr development and progression ( ) . retrospective studies based on clinically indicated tests suffer from obvious selection biases of the initial population, and subsequent follow-up tests are more likely in sicker or highly symptomatic patients. because patients with moderate ftr, and even a significant proportion of those with severe ftr, are completely asymptomatic, we cannot exclude that patients who underwent repeated echocardiography were those who had to follow-up their left heart disease or those who were symptomatic. in the reported study, only , patients ( %) of the initial cohort of , underwent repeated echocardiography. although mutlak et al. ( ) used an integrative approach to assess the severity of ftr, patients were selected on the basis of regurgitant jet area on color doppler: trivial (jet area < . cm ), mild (jet area to cm ), moderate (jet area to cm ), and severe (jet area > cm ) ( ) . however, these are the criteria used to grade the severity of mitral regurgitation. using this metric to assess the severity of ftr is a rtificial intelligence broadly refers to algorithms that iteratively learn from plethora of clinical, imaging, and other diagnostic information to uncover patterns and make decisions from data ( ) ( ) ( ) . these techniques have empowered new discoveries to be made via modern bioinformatics approaches developed to achieve improved representations of complex disease processes ( ) ( ) ( ) . an exemplar application is the "multiomics" approach for precision medicine, wherein patients are clustered or classified based on similarities in various clinical features to enable improved individual-level risk stratification ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . specifically, the "phenomics" approach in precision medicine allows for systematic dis- ( ) . although these methods have already been claimed to be useful in genomics and system biology ( , , ) , there has been limited enthusiasm to address their application in clinical cardiovascular medicine. the growth of cardiac imaging techniques has offered an ever-increasing array of measurements for understanding cardiac structure and function. for example, continuous technical developments and the evolution of novel parameters in echocardiography, ranging from -dimensional and doppler-based measurements ( ) to speckle tracking echocardiography (ste) ( ) , vector flow mapping (vfm) ( ) , and contrast particle imaging velocimetry-derived ( , ) parameters of left ventricular (lv) muscle and blood vector flow mapping. images were analyzed using commercially available off-line software (das-rs , hitachi aloka medical ltd., tokyo, japan). the lv endocardial border was manually traced at the onset of the qrs complex, which the software automatically tracked. we included a total of parameters obtained from vfm to be consistently measured in most patients (> %). these parameters have been previously described, and although validation studies are limited ( ) ( ) ( ) , the published data suggest that these parameters may carry clinically useful information. they included: ) indexes of vortex, such as vortex area, vortex core diameter, circulation, maximum vorticity, and boundary linear velocity from the flow-velocity curve ( ) ; ) energy loss (el) ( , ) ; and ) the intraventricular pressure gradient (ivpg) ( ) . the vortex was automatically tracked and analyzed throughout the cardiac cycle ( ) . the els were calculated as the mean and peak values in systole and diastole ( ) . ivpg was measured on a line that went through the center of the lv from the base to the apex ( the illustration shows the steps involved in our study in which the data was extracted from the patient's electronic medical record. first, -dimensional and doppler echocardiography, vector flow mapping of left ventricular (lv) muscle and blood flow mechanics, and lv and left atrial speckle-tracking parameters were obtained from patients. in the second step, unsupervised machine learning was used to generate patient similarity network and the subsequent clusters were identified and validated across clinical parameters and outcomes. finally, the data was used to train supervised learning models with cluster membership as target variable. three separate models, namely, decision trees, ensemble, and deepnets, were trained and tested to classify the patients to the correct group. la ¼ left atrium. appendix. the ste and vfm analyses were performed by a single observer blinded to the patients' clinical information (j.s. cho). machine learning framework. we first used an unsupervised learning method that analyzes unlabeled points in multidimensional feature space to identify patient phenotypic clusters with similar features along a network (patient similarity network). the clusters are then used as target for the supervised learning method to predict the patient groups. unsupervised clustering prior to classification has been shown to improve the predictive capabilities of supervised learning models ( ) ( ) ( ) . patient details of the baseline characteristics are described in table . patient-patient similarity network. we integrated complex echocardiographic variables (table , figure ) to create a patient-patient similarity network. the network and the shape of the data that materialized was a loop with a side branch on the left of the loop ( figure ) using an automated platform with unsupervised machine learning. cluster i was found in the upper region of the loop. the patients in cluster i were younger (age ae years; p < . vs. others) and had a lower prevalence of comorbidity than was found in the rest of the patients ( table ) . typically, healthy volunteers ( , %) were grouped in this cluster. clusters ii and iii were in the right and bottom regions of the loop. cluster ii contained older patients (age ae years; p < . ) with a lower prevalence of comorbidities than those found in other clusters. compared with patients in clusters i and ii, those in cluster iii (age ae years; p < . ) presented a higher prevalence of comorbidities ( table ) . cluster iv was a side branch that included patients. the patients in cluster iv were older (age ae years), were predominantly women ( %), and had a significantly higher prevalence of comorbidities than those found in other clusters (hypertension: %, atrial fibrillation: %, significant valvular heart disease: %, and cardiomyopathy: %; all p < . vs. others) ( table ) . conventional echocardiographic parameters. figures, cluster i had the most preserved conventional echocardiographic features, including lvef, eʹ, e/eʹ, and the la volume index and lv mass index, each of which progressively worsened from cluster i to iv (p < . for all). features such as e/a and lv volume showed a bimodal pattern in which the values decreased from cluster i to ii and increased from cluster ii to iv (p < . for both) ( table ) . speckle-tracking parameters. the ste analysis showed that most parameters (i.e., lv gls, lv srs, lv sre, pals, and la srs) progressively decreased in magnitude from clusters i to iv (p < . for all) ( table ) . however, in contrast to lv gls, pals showed a bimodal pattern in which it increased in magnitude from cluster i to ii and then decreased in magnitude from cluster iii to iv (p < . ) ( figures c and d) . vector flow mapping. the majority of the vfm parameters, especially the early diastolic vortex area, core diameter, and circulation, showed a bimodal pattern in which they decreased from clusters i to ii and subsequently increased toward clusters iii and iv (p < . for all). however, maximum vorticity and boundary linear velocities decreased from clusters i to ii and remained decreased in clusters iii and iv, whereas the late diastolic vortex core diameter continuously increased from clusters i to iv (p < . ). the late diastolic vortex area, circulation, figure and supplemental table a lge extent was visually defined as the number of segments that manifested lge. interobserver and intraobserver variability for all cmr indexes used in the present registry are shown in supplemental tables a and b. endpoint and follow-up. the only clinical endpoint of this registry was all-cause mortality. follow-up was centrally carried out by cardiologists using the unified electronic regional health system registry. adjudication of events was made by consensus of the cardiologists authorized by the ethics committee for this purpose. the specific objectives were to explore the potential of the extent of ischemic burden associated with the following: ) long-term all-cause mortality; and ) the effect of revascularization procedures prompted by the index vasodilator stress cmr study on longterm, all-cause mortality. ischemic burden (segments with pd post-stress) lge segments) values are mean ae sd, n (%), and n (range figure ). although lge was not included in the multivariate analysis, its presence and extent were also associated with all-cause mortality in different ischemic burden populations (supplemental table . variables used to obtain the propensity-matched population (n ¼ , , treated and not treated with a cmr-related revascularization procedure) are displayed in table . all-cause mortality occurred in of the , patients included in this population ( %)- ( %) of non-revascularized cases and ( %) of revascularized cases (p ¼ . ) ( figure b ). baseline and cmr characteristics of patients (treated and not treated) with a cmr-related revascularization procedure are listed in table . the all-cause mortality rate in patients with and without cmr-related revascularization differed according to the extent of the ischemic burden. in non-revascularized patients, the all-cause mortality rate increased as the ischemic burden rose. however, this tendency did not occur in patients who underwent a cmr-related revascularization procedure ( figure , central illustration). figure illustrates the divergent effect of the ischemic burden on the allcause mortality risk depending on the use or not of cmr-related revascularization. we used a cutoff point of segments with pd poststress to stratify patients into categories of extensive and nonextensive ischemic burden. this was the best cutoff point, which was derived from the youden index, and applied to the receiver-operating curve analysis that explored the association of the extent of ischemic burden with all-cause mortality. in addition, this selection coincided with the point where the effects of the ischemic burden on all-cause mortality in revascularized and non-revascularized patients intersected ( figure ). in the nonextensive ischemic burden group (# ischemic segments), patients who underwent a cmrrelated revascularization procedure displayed a significantly higher risk of all-cause mortality than those who did not ( % vs. %; p ¼ . ). in contrast, patients in the extensive ischemic burden group (> ischemic segments) treated with a cmr-related revascularization procedure displayed a lower risk of all-cause mortality than those who had cmrrelated revascularization was not done ( % vs. (figures and lv end-systolic volume index was removed of multivariable analysis due to excessive co-linearity (supplemental table ) and correlation (supplemental table the present registry demonstrated that in unselected patients with known or suspected sihd who under- the propensity score, aimed at predicting the use of cmr-related revascularization, was obtained using a non-parsimonious model that included in model , those co-factors that were independently related to the occurrence of all-cause mortality (age, male sex, dm, current smoker, lvef, and ischemic burden). the c-statistics of the resulting model was . , indicating good discrimination. then, the rest of variables associated with the use of cmr-related revascularization were also tested in model . none of these variables significantly improved the c-statistics of model . however, lge extent was also used for selecting the : propensity scoreÀmatched population (final model) because of its potential relevance in indicating the use of cmr-related revascularization. abbreviations as in table all-cause mortality rates based on the extent of the ischemic burden in revascularized and non-revascularized patients in the (a) entire registry and in the (b) matched patients. hrs (with the respective % confidence intervals) of all-cause mortality increased in parallel with the extent of the ischemic burden in non-revascularized patients, but the opposite tendency occurred in patients who underwent cmr-related revascularization (p for interaction < . ), both in the (c) entire registry and in the (d) matched patients. cofactors independently associated with all-cause mortality in multivariable analysis (age, male, dm, current smoker, lvef, and ischemic burden) and the propensity score to undergo cmr-related revascularization were used for adjustment in the (c) entire registry. abbreviations as in figures and . a physiology-based strategy (pci only in lesions with fractional flow reserve < . ) was superior to an anatomy-based strategy (pci in lesions > % by angiography) in the fame trial ( ) . in the fame- (fractional flow reserve versus angiography for multivessel evaluation) trial ( ) , it was also more effective than optimal medical therapy alone in reducing the risk of a combined endpoint (but the benefit was restricted to the rate of unplanned revascularization lge (no. of segments) values are mean ae sd, n (%), and n (range). all covariates had standardized mean difference (smd) of < . , which was considered to be a sign of covariate balance. other abbreviations as in table . the currently recommended % threshold of ischemic myocardium is mainly derived from studies that used exercise combined with nuclear imaging ( , ) . we based the analyses in our series on the extent (number of segments) of inducible pd. this is probably the most widely used and easily interpreted in summary, all these observations seem to suggest that to achieve a significant risk reduction in such a potent endpoint as all-cause mortality, an extensive ischemic burden by any of the available all-cause mortality was selected as the only endpoint. this is a verifiable and worrisome event, and the retrospective strategy for the revision of data within a unified electronic regional health system guaranteed the quality of information obtained. other events such as the cause of death, relief of symptoms, unplanned revascularization procedures, or re-infarctions could have contributed a more holistic vision of the course of patients. a trial fibrillation (af) is often asymptomatic, and even more frequently asymptomatic with increasing age ( , ) . in patients with stroke risk factors, continuous implantable monitoring reveals subclinical af in w % ( ), whereas cohort studies have found subclinical af in pacemaker patients to be associated with increased risk of stroke ( ) . systematic screening for af is currently not supported, but reliable predictors of af and possibly stroke may help in selecting patients for screening. assessment of left atrial late gadolinium enhancement (la lge) with cardiovascular magnetic resonance (cmr) imaging has primarily been used before or after af ablations, and it is well established that the extent of la lge before ablation is inversely associated with procedural success ( ) . furthermore, stroke is associated with increased la lge ( , ), but only a few studies of la lge in patients without af or stroke have been published ( ) ( ) ( ) . increased la lge has been found to be associated with future diagnosis of atrial arrhythmias in patients with preexisting cardiac disease ( ) but no study has investigated the prognostic value of la lge in a population-based cohort or by use of continuous monitoring. the aim of this study was to investigate the extent of la lge in a population with stroke risk factors but no history of af and to investigate whether la lge was associated with incident af as detected by continuous monitoring using an implantable loop recorder (ilr). the study is part of the loop study (nct ). a detailed description of the study design has previously been published ( ) . in short, participants from the general population were randomly selected from civil registries and invited by mail. total of study participants died during follow-up, all due to noncardiac causes. in a total of patients ( %), adjudicated incident af episodes lasting $ min were detected, and experienced at least af episode lasting $ . h. among patients with af, the mean af burden was . ae . %, median . % (iqr: . % to . %). when asked about symptoms, only patients ( %) reported symptoms at af debut and/or during af. baseline characteristics are summarized in table , showing no differences between participants with and without af. cmr parameters are summarized in table ). there was no difference in baseline demographic risk factors between men and women, but lv remodeling was more pronounced in men compared with women (supplemental table ). correlations between cmr variables figure ). in this prospective study, we performed la lge scans in patients followed by continuous monitoring for af for more than years. we found that, in addition to la volumes and function, increased la lge was significantly associated with risk of af. the long continuous monitoring enabled us to show that la lge is associated not only with short episodes of af ($ min), but also with long episodes ($ . h), as well as high af burden. importantly, most of the patients ( %) were completely asymptomatic. la remodeling and af. markers of la remodeling such as la size and function have been linked in previous studies to the development of symptomatic af ( , ) . these studies all depend on a clinical diagnosis of af and hence asymptomatic patients are not represented. our study found that la volume and ejection fraction were associated with incident af, with similar hazard ratios as previously reported ( , ) asymptomatic af arguably represents a phenotype with less atrial remodeling. although af has been shown to induce atrial fibrosis in animal models ( ) , many of the participants in the current study with no detected af displayed surprisingly large amounts of la lge ( table ) . a few other population-based studies have also found la lge in patients without af ( , ) , although these studies were not able to rule out subclinical af. hence, it appears that even relatively severe la lge is not strictly linked to af, which speaks for atrial enhancement as an independent phenomenon from known risk factors for af as part of a specific fibrotic atrial cardiomyopathy ( ) , possibly with a bidirectional causality between af and fibrosis ( ) . in this context, it is important to remember that la lge may not be specific to atrial fibrosis. as lge reflects accumulation of contrast, other causes of increased interstitial space, for example, inflammation, or blood stasis may also result in enhancement. prediction of af is important because af is often asymptomatic, even with long af episodes ( , ) . although subclinical af is associated with an increased risk of stroke compared with no af ( ), it appears that shorter episodes are less hazardous ( ) . we found that la lge was strongly associated with longer episodes of af ($ . h) with a higher hazard ratio than for af episodes of any duration ( figure ) . similarly, we found that la lge was significantly higher in patients with a high af burden ( figure ). our findings indicate that la lge may serve as a predictor of longer af episodes and higher af burden. we thus speculate that more widespread atrial fibrosis can sustain af once it occurs. excessive atrial ectopic activity is strongly associated with incident stroke independently of manifest af ( ) and increased la lge has been found in patients with stroke of undetermined cause ( ) . although la lge was significantly higher in the patients with incident af during follow-up, some of the participants without af also displayed extensive la lge. as strokes are often temporarily discordant from af ( ) , it may very well be that remodeling of the left atria increases the risk of af but also in itself constitutes an increased risk of stroke. as an alternative method of measuring fibrosis, ventricular native t relaxation times and extracellular volume have been associated with af recurrence after ablation ( ) . we were not able to confirm these findings with regard to incident screening-detected af. this may be because the degree of diffuse fibrosis is lower before development of subclinical af compared with the af phenotype seen in patients undergoing ablation procedures. previous cmr studies have found correlations between la lge and age ( , ) , hypertension ( ), and female sex ( , , ) . similar to other previous cmr studies and studies on autopsy findings on atrial fibrosis ( , ), we could not confirm the associations with age and comorbidities. this was possibly because included participants were $ years of age and had at least of hypertension, heart failure, diabetes mellitus, and/or previous stroke, meaning that the healthiest among the participants were arguably those with only risk factor (e.g., well-managed hypertension), whereas no participants were young or free of risk factors. as described, men displayed significantly more la lge than women in non-af participants, although this was not the case in patients with af. in general, men also displayed more lv remodeling as well as lv lge (supplemental table ) compared with women, which may be linked to the increased la lge in male participants. finally, a u g u s t : - increased la lge in women compared with men has earlier been found to reverse with age ( ) . study limitations. first, our study is limited by a modest cohort size. out of ethical considerations, participants were to have an estimated glomerular filtration rate of at least ml/min per . m to be included in the study. second, la lge has well-known limitations. however, as in a recent study ( ) , we found good inter-and intraobserver reproducibility and the scan-rescan variability for la lge has recently been shown to be acceptable ( ) . the upper iir threshold used for la lge is well-described with data from healthy controls ( ) and with reference to electroanatomic mapping ( ) . because most of the patients were asymptomatic, we cannot exclude the presence of existing asymptomatic paroxysmal af at inclusion. nevertheless, the increasing incidence of af throughout the entire follow-up period suggests that a substantial number of af episodes were truly new-onset. in this prospective cohort-based study, we found that lge measured by cmr is predictive of future subclinical af. translational outlook: future studies are needed to assess whether la lge is valuable in selecting patients for screening for af or for stroke risk stratification. if reduction of la lge is possible, this could potentially reduce af burden and progression. cally, animal models support the relevance of clustered fibrosis in leading to re-entrant activations that sustain af ( ) . additionally, it is known that af itself can lead to tissue fibrosis, as illustrated by the fact that rapid atrial pacing induces fibrosis and af ( ) , and that atrial fibrosis and electric remodeling underlie the transition from paroxysmal to persistent af and contribute to af perpetuation ( ) . therefore, the relationship between af and fibrosis is clear. what is less clear is the direction of this relationship. af and fibrosis go hand-in-hand, but which is the proverbial "chicken" and which is the "egg"? is af a consequence of fibrosis, is fibrosis the consequence of af, or is the answer a combination of the ? another clinical conundrum revolves around the mechanisms, determinants, and predictors of stroke in af. the mechanistic paradigm of thrombus formation in the left atrial appendage ( ) is undisputed and seems to be overwhelmingly predominant. however, recent data seems to question its exclusiveness: the lack of temporal correlation between strokes and af episodes-within the preceding month ( )-seems to suggest that additional mechanisms are operative, or that af may be not only be a proximate risk factor for stroke, but also a marker of stroke risk from other mechanisms. and yet, the longer the af episodes, the higher the risk of stroke ( ) . how can this make sense? how can the presence of af be associated with strokes even at times when af does not happen? one fascinating hypothesis is that there is an underlying disease process that can both be a mechanism of stroke susceptibility and a mechanism of af, and that both can occur independent of one published retrospective study identified an association between la lge and incident new-onset af ( ) . in this issue of ijacc, bertelsen et al. ( ) patients with rfs were not enrolled in the study. coronary cta advanced plaque assessment was performed. outcome measures were combined endpoints: acute coronary syndrome (acs), cardiac death þ acs, and cardiac death þ acs þ late revascularization. results among the patients enrolled in the capire study, in patients, a mean follow-up of ae months was obtained ( patients were excluded due to < segment involvement score < at core lab coronary cta analysis and patients were lost at follow-up). higher atherosclerotic burden was found in patients with higher clinical risk, but prevalence of elevated noncalcified plaque volume did not significantly differ between low-versus high-risk patients. quantitative plaque parameters by coronary cta were associated with composite endpoints at multivariable analysis when corrected for univariate predictors. elevated noncalcified plaque volume, expressed as dichotomic variable, was associated with all combined endpoints. even if the low absolute number of events represents a limitation to the present study, patients with low noncalcified plaque volume had similar risk of cardiac events independently from the presence of multivessel disease, while patients with high noncalcified plaque volume had higher rates of cardiac events. continuous variables normally distributed were compared using the student's t-test for independent . ae . ; p ¼ . ) ( table ) . medical therapy was more prevalent at baseline among patients with a higher clinical risk profile. plaque volume was analyzed both as continuous and as discrete dichotomic variables, using the hq ( th percentile) among the entire population as cutoff values that resulted to be > mm for total plaque volume, > mm for noncalcified plaque volume, and > mm for low-attenuation plaque. the same approach was used for sis, sss, and ct-adjusted leaman score and the hq cutoff resulted to be > for sis, > for sss, and > for the ct-adjusted leaman score. the majority of coronary cta data suggested higher atherosclerotic burden in patients with higher clinical risk; however, the prevalence of elevated noncalcified plaque volume when considered as a dichotomic variable did not significantly differ between low-risk versus high-risk patients ( table ) . concerning the advance ct plaque analysis, all qualitative high-risk plaque features were associated with all different combined endpoints (table ) . finally, the quantitative plaque parameters were associated to all the combined endpoints, both when values are mean ae sd or n (%). ace ¼ angiotensin-converting enzyme; at -blockers ¼ angiotensin receptor blockers; bmi ¼ body mass index; cad ¼ coronary artery disease; hb ¼ hemoglobin; hdl-c ¼ high-density lipoprotein cholesterol; hstnt ¼ highsensitivity troponin t; ldl-c ¼ low-density lipoprotein cholesterol; ptx ¼ pentraxin- ; scd ¼ sudden cardiac death; total-c ¼ total cholesterol. considered as continuous or as dichotomic variables ( table ) . separate multivariable analysis in low-versus highrisk patients and excluding patients with diabetes confirmed plaque volume < hu as an independent predictor of events in both groups of patients (supplemental tables and ). akaike information criterion was calculated and is reported in supplemental table . similarly, patients with low noncalcified plaque volume were almost free of events at follow-up even if the -year risk of heart attack (frs) was more than % ( % and % acs-free survival probability in patients with or without frs > %, respectively). on the contrary, the subgroup of patients judged to values are n (%) or mean ae sd. cad ¼ coronary artery disease; ct ¼ computed tomography; cta ¼ computed tomography angiography; hpf ¼ high-risk plaque feature; hq ¼ highest quartile; hu ¼ hounsfield units; lap ¼ low-attenuation plaque; lm ¼ left main disease; nrs ¼ napkin ring sign; pl vol ¼ plaque volume; ri ¼ remodeling index; sc ¼ spotty calcification; sis ¼ segment involvement score; sss ¼ segment stenosis score; other abbreviations as in table . have a low or moderate clinical risk, but with coronary cta showing high noncalcified plaque volume, has been reclassified by coronary cta avoiding underestimation of their cardiovascular risk ( % mace-free survival probability in patients with low frs but with high noncalcified plaque volume; log-rank p < . ). similar results were obtained when atherosclerotic cardiovascular disease risk score was used instead of frs (supplemental figure ). the main finding of this analysis of capire study supports quantitative parameter of coronary cta plaque assessment, more specifically the coronary plaque volume and particularly the noncalcific plaque volume, as the most powerful predictor of cardiovascular events at follow-up over lumen stenosis and clinical risk profile. excluding patients with known heart disease enabled to specifically address the association between coronary atherosclerosis evaluated by coronary cta and future acute coronary events; moreover, the peculiar study design, leading to a dichotomic distribution of traditional risk factors among our population, enabled us to demonstrate that the prevalence of elevated noncalcific plaque volume was not differently distributed in low-versus high-risk patients. the traditional approach to cad is based on primary prevention for risk factor control in asymptomatic subjects and on myocardial values are mean ae sd or n (%), unless otherwise indicated. acs ¼ acute coronary syndrome; ci ¼ confidence interval; hr: ¼ hazard ratio; other abbreviations as in table . values are n (%), unless otherwise indicated. abbreviations as in tables to . notably, the multivariable analysis to predict cardiac events showed that the addition of mvd to the a u g u s t : - editorial comment body of published data evaluating whole-heart plaque quantification using coronary cta. however, much work remains to be done before its routine clinical use ( figure ) during the administration of mg of adenosine triphosphate (atp) at mg/kg/min for > min ( ) twenty-three patients were lost to follow-up; therefore, the final study population comprised patients with suspected or known coronary artery disease. ct ¼ computed tomography. table shows baseline patient characteristics in all patients and those with suspected or known cad. the dose-length products for cta, ctp, and ctde ( ) ( ) ( ) family history of cad ( ) ( ) ( ) body mass index > kg/m ( ) ( ) ( ) echocardiography lvef < % ( ) ( ) ( ) wall motion abnormality ( ) ( ) ( ) symptom typical ( ) ( ) ( ) atypical ( ) ( ) ( ) nonanginal ( ) ( ) ( ) dyspnea ( ) ( ) ( ) history of cad ( ) old myocardial infarction ( ) ( ) ( ) values are n (%) or mean ae sd. cabg ¼ coronary artery bypass grafting; cad ¼ coronary artery disease; lvef ¼ left ventricular ejection fraction; pci ¼ percutaneous coronary intervention. obstructive cad ( ) ( ) ( ) severe stenosis ( ) ( ) ( ) multivessel disease ( ) ( ) ( ) proximal lad stenosis ( ) ( ) ( ) stenosis score $ ( ) ( ) ( ) dynamic ct perfusion ischemic score $ * ( ) ( ) ( ) ischemic score $ ( ) ( ) ( ) ct delayed enhancement myocardial scar ( ) ( ) ( ) $ segments with scarring ( ) ( ) ( ) scar score $ ( ) ( ) ( ) scar score $ ( ) ( ) ( ) values are n (%). *ischemic perfusion defect was defined as ischemic score $ . cad ¼ coronary artery disease; ct ¼ computed tomography; lad ¼ left anterior descending artery. ischemic score $ was the strongest predictor of hard events (hr: . ; % ci: . to ; p ¼ . ). regression analysis. models for multivariate analysis were created to evaluate whether ipd (ischemic score $ ) was an independent predictor when adjusted for each of the predictors ( table ). in all patients, ipd was an independent predictor when adjusted for obstructive cad, severe stenosis, proximal lad stenosis, scar score $ , and wall motion abnormality. in these models, obstructive cad, severe stenosis, proximal lad stenosis, and myocardial scarring were also independent predictors against ipd, but wall motion abnormality was not. in patients with suspected cad, ipd remained an independent predictor when adjusted for obstructive cad, severe stenosis, proximal lad stenosis, scar score $ , and ccs $ . in these models, obstructive cad, severe stenosis, proximal lad stenosis, scar score $ , and ccs $ were independent predictors against ipd. in patients with known cad, ipd was an independent predictor when adjusted for obstructive cad, severe stenosis, proximal lad stenosis, multivessel disease, and scar score $ . in these models, multivessel disease and scar score $ were independent predictors against ipd, whereas obstructive cad, severe stenosis, and proximal lad stenosis were not. multivariate analysis of predictors for hard events are listed in supplemental table . ipd remained an independent predictor of hard events in all patients when adjusted for current smoker, wall motion abnormality, severe stenosis, and scar score $ . in these analysis, current smoker was an independent predictor against ipd, whereas wall motion abnormality, severe stenosis, and scar score $ were not. kaplan-meier curves by ipd ( figure ) showed that annualized event rates for maces were significantly different between patients with and without ipd among all patients ( . % vs. . %; p < . ) ( figure a) and those with suspected ( . % vs. . %; p < . ) nakamura et al. annualized event rates for maces in patients with ischemic scores of # , to , and $ were . %, . % and . % respectively, in all patients (p < . ); . %, . %, and . % in patients with suspected cad respectively (p < . ); and . %, . %, and . % in patients with known cad, respectively (p < . ) (supplemental figure ). obstructive cad. to evaluate the incremental prognostic value of ipd over obstructive cad, global chi-square scores and concordance index were calculated ( figure ) . figure a shows that, by adding ipd to obstructive cad, global chi-square scores increased significantly from . to . in all patients (p < . ), from . to . in patients with suspected cad (p < . ), and from . to . in patients with known cad (p ¼ . ). roc curve analysis for prediction of maces ( figure b ) showed that the concordance index after adding ipd to obstructive cad was significantly higher than that in obstructive cad alone in all patients and in those with suspected and known cad. figure a shows that, among patients with stents who had no cabg, a significant difference in annualized event rates was apparent between patients with and without ipd ( . % vs. . %; p < . ). in patients with heavy calcification, among those with suspected cad, ipd had a significant association with poor prognosis. annualized event rates for maces were significantly higher in patients with ipd than in those without among patients with a calcium score ( . % vs. . %; p < . ) ( figure b ). degree of stenosis. kaplan-meier curves by ipd according to cta results ( figure ) showed that patients with ipd had a worse prognosis than those without ipd among those with moderate ( % to %) stenosis but no severe ($ %) stenosis (annualized event rate: . % vs. . %; p < . ) tables and . who had undergone bypass grafting were excluded. abbreviations as in figure . prognostic value of stress dynamic ctp with ctde in patients with ipd (þ) and scar score $ , with ipd (þ) and scar score < , with ipd(-) and scar score $ , and with ipd(-) and scar score < , respectively (p < . ). in figure b , global chisquare tests showed that ipd and scar score $ had incremental prognostic value over each other (p < . ). details of events in patients with ipd or scarring are given in supplemental table . there was no significant difference in the number of each event between ipd and scar score $ . the main findings of the present study were that: ) figure . nakamura et al. fibrosis associated with abnormally increased extracellular volume (ecv). in the left anterior descending coronary artery (lad), a stent is represented in its midportion, characterized as patent by cta, without significant pressure drop by ct ffr (negative), but associated with a previous distal myocardial infarct, characterized by ct de. moreover, the stented lesion is also associated with a distal fixed perfusion defect due to reduced capillary density in the scar, which commonly extends to its peri-infarct border caused by lower capillary density (mixed normal and scarred myocardium) combined or not with microvascular dysfunction. ct ecv is greatly increased in the scar, as expected, and possibly increased in the peri-infarcted region as well because of tissue mixing or repeated ischemia and apoptosis. the heart cartoon also shows a first large lad diagonal affected by diffuse atherosclerosis but with no significant morphological stenosis, as demonstrated by cta. ct ffr can be abnormal if too many lesions in series lead to an overall pressure drop across the entire epicardial vessel length. ctp may be normal or abnormal because of microvascular disease and dysfunction. finally, although ct de is most often negative for myocardial scar, ct ecv can be increased because of chronic ischemia from microvascular disease with interstitial fibrogenesis. importantly, the cartoon illustrates that multiple scenarios due to different combinations of macrovascular and microvascular disease can occur, and very frequently do, in the same heart in a patient with cad, particularly in those with advanced ischemic heart disease. in this regard, although computed tomographic replacement fibrosis phenotyping through delayed enhancement is more than a decade old ( ), the recently demonstrated ability to quantify interstitial myocardial fibrosis by ct ( ) could provide additional insight into the pathogenesis of heart failure in patients with preserved ejection fraction ( ) . given that heart failure in general and heart failure with preserved ejection fraction specifically are frequently associated with macrovascular atherosclerosis and microvascular ischemic heart disease ( , ) , such avenues for further investigation may prove uniquely important to patients with heart disease in the future. in summary, the work by nakamura et al. ( ) demonstrates an important option to physicians who seek to detail the prognostic paths their patients with table . overall, baseline characteristics in the two groups were mostly balanced. the majority of patients ( %) had pacemakers, % had icds, % underwent cardiac resynchronization therapy (crt), either with a pacemaker or a defibrillator, and % had an epicardial lead. implanted prosthetic heart valves were present in . % of the study population. overall, the most prevalent pathogens causing cdrie were staphylococci organisms ( %). values are n (%). *cox proportional hazards analyses were applied to calculate hazard ratios (hrs) and % confidence intervals (cis); for the in-hospital mortality, and new heart rhythm and conduction disturbance odds ratios (ors) with % ci were calculated instead. values in bold indicate statistical significance. the in the analysis patients with positive m tc-hmpao-spect/ct scans for cdrie had higher in-hospital mortality rates than those with negative mtc-hmpao-spect/ct results ( . % vs. %, respectively; p ¼ . ). p < . were considered statistically significant. abbreviations are as in figure . and log-rank test results. p < . were considered statistically significant. abbreviations are as in figure . holcman et al. the total incremental admission mortality rate for patients with cdrie varies from . % to . %, depending on the cied type and the patient's status ( uncontrolled infection, renal replacement therapy, reoperation due to cdrie, new heart rhythm, and conduction disturbances. p < . were considered statistically significant. abbreviations are as in figure . abbreviations are as in table . holcman et al. abbreviations as in table . table ) . holcman and colleagues in this issue ( ) this has already been part of clinical routine in cancer subsequently, endocardial and epicardial contours of the short-axis lge images were traced manually. myocardial scar core size was automatically quantified using the full-width at half-maximum method, which defines scar as myocardium with signal intensity > % of the maximum signal intensity in the hyperenhanced area. in addition, scar border zone was defined as myocardium with signal intensity > %, but < % of the maximum signal intensity in the hyperenhanced area, as described previously ( ) . total scar size, scar core size, and border zone size were expressed as grams and percentages of the total lv mass. in addition, transmurality of scar core was evaluated, and segments were characterized as % to %, % to %, % to %, and % to % transmurality. ace ¼ angiotensin converting enzyme; arb ¼ angiotensin-ii-receptor blockers; ca ¼ calcium; cabg ¼ coronary arterial bypass graft; crt-d ¼ resynchronization therapy; nt-probnp ¼ n-terminal prohormone of brain natriuretic peptide; nyha ¼ new york heart association; pci ¼ percutaneous coronary intervention; va ¼ ventricular arrhythmia. for [ c]hed image analysis, plasma parent fractions, and ratios of plasma to whole-blood concentrations derived from manual arterial blood samples were fitted to a sigmoid function ( ) . subsequently, the arterial whole-blood tac was multiplied by the there were no significant differences in clinical characteristics between patients with or without va, but patients who showed va tended to be younger ( ae vs. ae years; p ¼ . ). figure shows an example of cmr and pet imaging in a patient who remained free of va. characteristics. the results of cmr imaging are presented in table and figure . the current study indicated that the amount of scar border zone was associated with va, whereas scar core was not. previous studies demonstrated that myocardial scar is strongly associated with scd in patients with ischemic cardiomyopathy ( , ) . studies evaluating both scar core and border zone in patients with coronary artery disease often indicate that scar border zone might be a better predictor for va than scar core mass alone ( , , , ) . our results are consistent with findings of roes et al. ( ), who showed that scar border zone was the strongest predictor for va, whereas scar core and total infarct zone were not associated with va ( ). the infarct border zone often comprises a mixture of viable and nonviable tissue with heterogeneous conduction times, which may give rise to re-entry pathways and provide substrate for va ( ) . however, a recent study of acosta et al. ( ) evaluated patients with ischemic and nonischemic cardiomyopathy and concluded that both scar mass and border zone were independently associated with va. these inconsistent findings may partly be explained by sample-size differences and the use of different methods for defining scar core area ( , ) . in the current study, the full width at half maximum method was used to define scar areas, as this method has been shown to provide the most reproducible results ( ) . this study failed to show an association between quantitatively assessed myocardial perfusion using pet and the occurrence of va. in this same patient cohort, however, an independent relationship between impaired global hyperemic mbf and inducibility of va was demonstrated using an electrophysiological study ( ) . previous studies that a u g u s t : - been linked to electric instability, as areas of denervated myocardium show prolonged refractory periods and supersensitivity to cathecholamines ( ) ( ) ( ) . in a previous study, the size of sympathetic denervated myocardium was found to be larger in patients with inducible va ( ) . in addition, the positron emission tomography) study revealed that total sympathetic denervation size independently predicts appropriate icd therapy for fast va ( ace ¼ angiotensin converting enzyme; arb ¼ angiotensin-ii-receptor blocker; cfr ¼ coronary flow reserve; ci ¼ confidence interval; hed ¼ hydroxyephedrine; hr ¼ hazard ratio; lvedvi ¼ leftventricular end-diastolic volume index; lvef ¼ left-ventricular ejection fraction; mbf ¼ myocardial blood flow; nt-probnp ¼ n-terminal prohormone of brain natriuretic peptide; nyha ¼ new york heart association; ri ¼ retention index. may be hemodynamically tolerated or self-limiting ( ) . it is therefore possible that the role of our imaging parameters on the effect of scd was overemphasized. icds for primary prevention of scd, increased current problem: primary prevention icd patients experience a low incidence of appropriate icd therapy. improved risk stratification of scd is important to identify patients who could benefit of icd implantation. compromising the high frame rate ( ) . consequently, blood and tissue motion in addition to tissue structure may now be explored using high frame rate ultrasound imaging (central illustration). ultrafast doppler imaging. conventionally, blood flow is detected with doppler ultrasound by discriminating signals according to their temporal characteristics based on doppler frequency ( ) . because red blood cells tend to move faster than the surrounding tissue, their doppler frequency is generally higher, and the signals can be separated by applying a temporal filter. in cardiac imaging, myocardial tissue also moves, which can make blood flow detection more challenging. in ultrafast ultrasound, all the pixels (or voxels) in a frame (or in a volume) are analyzed simultaneously, and both the temporal and spatial characteristics of the incoming signals can be exploited ( ) . tissue is known to be more spatially coherent than blood and is far less deformable than blood. this results in the signals from neighboring pixels or voxels inside a tissue area to be more likely correlated to one another. this additional spatial information allows the use of spatiotemporal filters, which dramatically improve blood detection ( ) . hence, ultrafast ultrasound not only brings the possibility of imaging high velocity flow but also the ability to efficiently discriminate blood flow from tissue movement (video ). coronary ultrafast doppler angiography. flow detection in the coronary circulation requires distinguishing the blood signal from myocardial movement. this becomes even more challenging for imaging the coronary microvasculature (with a diameter < mm). the use of specific and adaptive spatiotemporal filters assessment of myocardial stiffness. assessment of myocardial stiffness using acoustic radiation force. link between mechanical activation and electrical stimulation. speckle tracking of intraventricular blood flow. wall shear stress (wss). standard doppler techniques using in high frame rate. tissue structure backscatter tensor imaging (bti) elastic tensor imaging (eti) villemain here, the shear wave is generated by an internal stimulus (valve closure) and the ultrafast imaging allows to see its propagation. on m-mode acquisition along the midline of the left ventricular septum, a spatiotemporal representation of the local tissue acceleration is obtained and allows to estimate the shear wave speed. adapted with permission from petrescu ( ) . ca ¼ cardiac amyloidosis; hv ¼ healthy volunteer; other abbreviation as in figure . villemain et al. ( ). from a practical point of view, the variation of frame rate (from conventional to ultrafast) does not change the clinical constraints of pediatric cardiology. indeed, the transmission/reception frequency, allowing optimization of the spatial resolution and thus adaptation of the anatomy explored, will be generally the same whatever the frame rate. in addition, the type of probe (phased array or linear) and angular dependency remains the same. in the future, the manufacturer (and not the practitioner) will need to adapt its systems and "probe to system" transmissions to allow the application of ultrafast imaging in pediatrics (as in adult cardiology). for this reason, few details will be discussed in this section on the type of equipment, type of probe, or emission/reception frequencies because these parameters will be broadly comparable to daily clinical practice. in the case of an exception (e.g., for coronary ultrafast doppler angiography [cuda] only demonstrated with a linear probe for technical reasons), precision will be provided. in this paper, the applications will therefore be grouped by type of disease and not by age group (as traditionally used in pediatrics) because all the techniques presented do not have specific limits according to the patient's age or weight. in addition, they showed, using eti (previously compared with histology [ ] ), that the fractional anisotropy was different between healthy volunteers ultimately, bti could alter our comprehension of the the importance of quantitative cvp assessment is well described for prognostication of pah patients ( ) . however, in pediatrics, because of a lack of reliable tools, this parameter is not widely used to evaluate pah. likewise, the assessment of filling pressures in the functionally univentricular heart is limited, but important because of the preload dependence of the fontan circulation. in addition, the significant risks of long-term hepatic impairment in patients with a functionally univentricular heart is emerging as an increasingly important prognostic factor ( , ) . therefore, better diagnostic tools to characterize liver morphology, stiffness, and their relationship to the cardiac status and pulmonary arterial pressures in the fontan circulation is needed. noninvasive quantitative evaluation of right heart filling pressures was very limited before the advent of ls ( ) . therefore, quantitative assessment of cvp by swe in this population deserves further study. nevertheless, the parameters that can influence ls (notably fibrosis, inflammation, or cholestasis) can complicate its interpretation at the patient's bedside. therefore, myocardial stiffness must be analyzed in terms of contractile stress properties. espvr is a linear parameter ( figure ) and can be characterized by a slope (e es , end-systolic elastance) and a volume axis intercept (v o ), so that: where p es and v es are end-systolic pressure and volume, respectively. importantly, this relationship was initially shown to be independent of afterload ( ) . ultimately, the espvr characterizes properties of the ventricular chamber when the myocardium is at maximal activation at a given contractile state. the stakes will be the same for ultrafast imaging. the development of a d multiplane analysis, or more generally a d ultrafast imaging approach, will probably be necessary to analyze the heart as a whole. initial preclinical studies are beginning to open this possibility ( , ) , clinical proof of concept is now the next step. last, and more specifically, concerning natural shear waves generated by the mitral valve, the electrical conduction through the myocardium leads to ultrafast ultrasound imaging could be a central noninvasive imaging tool, particularly in congenital and pediatric cardiology. myocardial stiffness assessment by ultrafast ultrasound imaging has the potential to become a cornerstone of ultrasound imaging in cardiology, particularly for the noninvasive assessment of systolic and diastolic physiology. further clinical developments could potentially reduce the need for cardiac magnetic resonance or imaging techniques requiring radiation. the pathogenesis of covid- is characterized by distinctive but synergistic mechanisms, the first ( ) . the extent to which these finding may also apply to sars-cov- is unknown. to date, no cases of sars-cov- nucleic acid isolation from myocardial specimens have been described. however, several cases have reported on the occurrence of severe myocarditis during laboratory-proven covid- ( ) ( ) ( ) ( ) ( ) . in all these cases, myocarditis a u g u s t : - procoagulant state with increased risk for arterial and venous acute thrombotic events, including type myocardial infarction (mi) and pulmonary embolism (pe). indeed, there is increasing concern that patients with covid- are more prone to develop thromboembolic venous events and disseminated intravascular coagulation ( , ) . secondary cardiac involvement may also be the consequence of covid- pneumonia is characterized by initial interstitial damage with a bilateral, peripheral, and posterior distribution followed by parenchymal involvement ( ) . lus effectively detects the areas pleural effusion is rare *the term "ground-glass opacities" is also used in cxr to refer to areas of blurred opacities. lus score, validated with chest ct comparison, provides a numeric assessment of regional loss of aeration that can be used to assess the response to treatments ( ) (figure ) . table . values are n/n (%) or median (interquartile range). *rv dilatation has been defined as rv mid diameter > mm. †rv dysfunction has been defined as either tricuspid annular plane systolic excursion < mm or doppler tissue imaging s wave (s wave) < . cm/s. ‡ph has been defined as spap > mm hg. cvp ¼ central venous pressure; lvef ¼ left ventricular ejection fraction; ph ¼ pulmonary hypertension; rv ¼ right ventricular; rwma ¼ regional wall motion abnormality; spap ¼ systolic pulmonary arterial pressure. and were assigned if parenchymal opacification involved %, < %, and $ %, respectively, of each region (severity score range to ). the individual scores for each lung as well as the total score were significantly higher in patients with clinically severe covid- compared with mild cases. a severity score < . was highly effective in ruling out severe covid- pneumonia, with a negative predictive value of . % ( ). in the same way lus could be effective in evaluating covid- pneumonia severity and monitoring its modifications over time. for this purpose the numeric assessment of regional loss of aeration measured by global lus score could represent a useful tool ( ) . the global lus score can be calculated as the sum of regional aeration scores attributed to each lung region during a standard -zone examination: involving # % of the pleura, ¼ b-lines becoming coalescent or involving > % of the pleura, and ¼ tissue-like pattern ( ) (figure ) . the global lus score showed a good correlation with lung density as assessed on ct and has been applied in the icu setting to quantify and monitor lung aeration in weaning from mechanical ventilation and in patients with ards on extracorporeal membrane oxygenation ( ) . so far, the implementation of the global lus score to monitor disease evolution and to guide decision making in patients with covid- has not been systematically investigated. cardiac involvement is present in up to % of patients with covid- . multimodality imaging is essential in different clinical settings in covid- . multimodality imaging is useful in diagnosis, risk stratification, and management. strategies for preventing viral transmission during examinations must be adopted. the t he highly anticipated ischemia (international study of comparative health effectiveness with medical and invasive approaches) trial, the largest study to-date conducted to assess for the incremental clinical benefits of an initial invasive management strategy over an initial medical therapy strategy in patients with stable ischemic heart disease (sihd), was just published ( ). this study has important implications for the field of imaging as well as coronary interventions-how the trial will change these fields and each modality will be debated fiercely over the next many months. we want to pro- summary. stress testing remains a vital diagnostic option for patients with ischemic heart disease. in particular, with its extensive evidence-base clear, focused, and clinically relevant study elegant study design: large, randomized, multicenter, international recruitment robust outcome measures well-powered study (> % power to detect an . % relative reduction in the primary endpoint assuming an aggregate -yr cumulative event rate of approximately %) excellent follow-up: > % complete follow-up outstanding optimal medical therapy (w % statin use, % angiotensin-converting enzyme or angiotensin-converting enzyme inhibitor use, % aspirin use) excellent risk factor control (nearly % nonsmokers, and % with well controlled systolic blood pressure < mm hg). a lack of equipoise; published data was conflicting, and physicians were not willing to randomize all patients with more than moderate ischemia. we do not know the percentage of patients screened who were eventually enrolled. some sites report this to be % of patients who were identified as having moderate to severe ischemia, where there was a negative bias toward enrolling patients in the trial particularly when ischemia was more severe. thus, the generalizability may be difficult to ascertain. smaller numbers of women, nonwhite, and hispanic participants. only % ( , of , ) of the randomized cohort comprised women. women more often manifested symptoms, and ischemic changes in the absence of obstructive cad (women comprised % of the cohort excluded for nonobstructive cad). smaller cohorts of nonwhite ( . %) and hispanic ( . %) patients were included ( ) . heterogeneity in patients enrolled, revascularization practices, and the definition of ischemia. b sites ( randomizing sites) from countries recruited subjects into this study. this has added heterogeneity in patient characteristics ( ) , and practice patterns, as free stents and free medicines were provided to some of the international sites, which may have affected subject selection for study enrollment, revascularization method selection in the invasive arm, as well as cross over from the conservative to invasive arm. b ischemia tests included electrocardiographic ischemia, radionuclide/cardiac magnetic resonance myocardial perfusion abnormalities, and ischemic wall motion abnormalities, which may have different implications for burden of ischemia and prognosis. due to slow recruitment, study inclusion criteria were amended to include patients based on exercise stress test evidence of ischemia, the study primary endpoint was amended as pre-specified into the study, and follow-up was reduced in . inclusion of exercise stress alone cohort is problematic as the magnitude of electrocardiography changes do not correlate to the extent and severity of ischemia. it added heterogeneity to the patient cohort and may have diminished the study power for drawing conclusions for the imaging-based ischemia alone. whether this exercise stress-only cohort underwent fewer revascularizations is not clear. the exercise stress-only cohort had a stricter cutoff of % stenosis in a coronary artery serving a large myocardial region before randomization ( ) . it is not surprising then that the exercise stress-only cohort had greater frequency of severe ischemia ( % vs. . % for imaging stress test) ( ), greater frequency of -vessel cad, lad, and proximal lad stenosis than participants undergoing stress imaging. on the other hand, they were also younger, less often white, with lower prevalence of hypertension and prior pci or cabg, stroke, or mi ( ) . exercise stress-only cohort made up % of all patients enrolled. all eligible stress-only patients were assigned to the severe category, thus accounting for % of severe ischemia, a disproportionate portion of patients that may have diluted the severe ischemia cohort defined on imaging. it is well-recognized that high-risk ischemic features on nuclear imaging (transient ischemic dilation, heart to lung ratio, right ventricular tracer uptake, left ventricular ejection fraction, and myocardial blood flow values by positron emission tomography) portend greater risk. it is not clear if these were considered and we await the subanalyses. unblinded study without sham procedure for revascularization. this may have potentially increased hospitalization for unstable angina or heart failure and cross over from conservative to invasive arm ( ) . completeness of revascularization has not been reported yet, but chronic totally occluded coronary arteries were common and frequently not revascularized. at this time, we do not know if the revascularization was directed to the coronary stenoses that subtended ischemic territories. the study was underpowered to evaluate cabg vs. pci separately. invasive patients may have been treated according to local practices. this may have made the selection of all invasive modalities (pci and cabg) suboptimal and, as such, appear no better than medical therapy (paraphrased from communication with m. ruel, md) (pending citation). cabg ¼ coronary artery bypass surgery; cad ¼ coronary artery disease; lad ¼ left anterior descending; mi ¼ myocardial infarction; pci ¼ percutaneous coronary intervention. substantiating its utility and versatility and continued refinements in its diagnostic precision, stress echocardiography will retain a prominent role as a robust tool for assessment of patients with ischemic heart disease. the ischemia study has several strengths and weaknesses ( table ) . do results interpretation and concern of extrapolating from ischemia. as with many trials, there are differences in the interpretation of the ischemia results, and various attempts will be made at extrapolating beyond the data provided and questions asked in the trial. a problematic interpretation by some is that the given the astronomical financial losses faced by our health systems, will our careers face major hurdles in turn affecting our lives? these are but a few of the stressors that we continue to face in these challenging times. across our hospitals, nearly all elective procedures, including a large volume of patients with planned stress tests, were deferred, and most were rescheduled into june or later (figure ) . interactions often left us feeling that many questions were unanswered and with residual concern about the patient's state and need for intensified cardiovascular care, which we were unprepared to provide. at its onset, covid- was deemed primarily a respiratory disease, but we rapidly recognized multiorgan involvement with distinct and devastating cardiovascular manifestations. for the sickest patients, acute cardiac decompensation was observed, whereas others had elevated troponin levels, raising concern for an initial diagnosis of acute myocardial what has happened over the past few months is unimaginable, and its emotional toll on our lives has yet to be fully realized. there are so many ways that these challenges have added stress to our day, for example, anxiety related to the potential shortages of personal protective equipment, concern over cut- other studies, which reported that a worse baseline lvgls was associated with negative outcomes ( - ). a total of studies generated receiver-operating characteristic curves ( , , , ) . in studies, the cutoff value of lvgls was found to be À %, À . %, and À . %, which could be used to predict worse outcomes in patients with chronic primary mr undergoing mitral valve surgeries ( , , ) . stress single-photon emission computed tomography (spect) incurred higher -months cost for ischemic heart disease testing versus stress cardiac magnetic resonance (cmr) across a range of duke activity status indexes (dasi) (p < . ). (table) vt optimal cutoff points. we studied subjects ( % were men; age . ae table ; intraclass correlation coefficient values for all measurements were good to excellent ( . to . ). with to our knowledge, this was the largest prospective rtof cmr study of high-risk patients with significant pr. our multinational cohort consisted of a balanced representation of children, young adults, middleaged adults, and older adults across the age quartiles, which enhanced generalizability (as opposed to previous data reported from a single country with younger subjects) ( ). our results confirmed that male and female hearts with rtof were distinct, and adaptive response of cardiac dimensions and systolic function to chronic volume overload appeared to vary according to age and by sex. at more advanced ages, women displayed more extensive change within the right heart. older adult women had larger rv volumes, whereas the extent of rv dilation did not differ significantly in older males versus younger males, which suggested that age might be a more important consideration for timing of pv intervention in females to mitigate irreversible rv remodeling in older women with larger rv volumes. approach. with the knowledge that females with rtof have smaller hearts at baseline but may be more susceptible to rv enlargement and rv systolic dysfunction with advancing age, one must question whether referral for intervention should incorporate sex and age variables. we suggest that our data may carry the potential to further inform risk stratification and timing of pv intervention in rtof. interclass correlation coefficients (icc) with % confidence intervals (cis) were performed to test intrareader and inter-reader consistency of agreement, and mean differences (and % ci) were estimated. lge was quantified using a threshold of sds above the mean signal intensity of the reference myocardium. the median duration of follow-up was months year; % ci: . % to . %; p ¼ . ). t decreased over time in men not on ert (À . ms per year; % ci: À . to À . ; p ¼ . ), although there was a tendency to a smaller reduction on ert (À . ms; % ci: À . to À . ; p ¼ . ; between groups: p ¼ . ). however, in women, t decreased over time in those not on ert (À . ms per year; % ci: À . to À . ; p < . ) but increased in the ert group (þ . ms per year; % ci: . to . ; p ¼ . ; between groups: p < . ). this longitudinal cmr study was consistent with a sex-specific myocardial response in fd. men had more advanced cardiac involvement at baseline and progressed at a greater rate than women, despite use figures h and i a plaque in the right coronary artery (rca) without high-risk plaque (hrp) features progressed into an obstructive lesion at follow-up, whereas a plaque in the left anterior descending (lad) artery with hrp features at baseline remained nonobstructive at follow-up. the diameter stenosis was similar between the lesions, but total percent atheroma value (pav) was greater in the rca lesion 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in the lateral wall was calculated for patients with known lv lead position. †values of cw and ww of the septum and lateral wall were compared in patients (crt responders n ¼ , %) in which assessment of mw indices was feasible immediately after crt implantation mwt ¼ -min walk test gfr ¼ glomerular filtration rate lbbb ¼ left bundle branch block lvedv ¼ left ventricular enddiastolic volume lvef ¼ left ventricular ejection fraction lvesv ¼ left ventricular end-systolic volume pi ¼ postimplantation ww ¼ wasted work a novel clinical method for quantification of regional left ventricular pressure-strain loop area: a non-invasive index of myocardial work left ventricular contractile reserve by stress echocardiography as a predictor of response to cardiac resynchronization therapy in heart failure: a systematic review and meta-analysis role of myocardial constructive work in the identification of responders to crt critical care utilization for the covid- outbreak in lombardy, italy: early experience and forecast during an emergency response cardiac ct with delayed enhancement in the characterization of ventricular tachycardia structural substrate: relationship between ct-segmented scar and electro-anatomic mapping management of covid- respiratory distress acute respiratory distress syndrome: the heart side of the moon surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease (covid- ) changes in arterial pressure during mechanical ventilation echocardiographic findings in covid- pneumonia the spectrum of cardiac manifestations in coronavirus disease (covid- )-a systematic echocardiographic study prognostic value of right ventricular longitudinal strain in patients with covid- risk stratification in patients with aortic stenosis: pay more attention to the right-side unit! right ventricle to pulmonary artery coupling in patients undergoing transcatheter aortic valve implantation rv contractile function and its coupling to pulmonary circulation in heart failure with preserved ejection fraction: stratification of clinical phenotypes and outcomes prognostic value of right ventricular longitudinal strain in patients with covid- moderate and severe acute respiratory distress syndrome: hemodynamic and cardiac effects of an open lung strategy with recruitment maneuver analyzed using echocardiography prognostic value of right ventricular longitudinal strain in patients with covid- abnormal right ventricularpulmonary artery coupling with exercise in heart failure with preserved ejection fraction lack of a tricuspid regurgitation doppler signal and pulmonary hypertension by invasive measurement diagnosis of pulmonary embolism with use of computed tomographic angiography pulmonary embolism and coexisting deep vein thrombosis: a detrimental association? hoyt have filed for a patent for the ai algorithm. all other authors have reported that they have no relationships relevant to the contents of this paper to disclose. the authors attest they are in compliance with human studies committees and animal welfare regulations of the authors' institutions and food and drug administration guidelines, including patient consent where appropriate. for more information, visit the jacc: cardiovascular imaging author instructions page. we thank drs. tsolaki and zakynthinos for their in- we appreciate the comments by dr. fukui and colleagues about our paper ( ), and we fully agree with some of their views. however, there are some issues that need to be clarified and discussed.we acknowledge and agree that right ventricular (rv) function is closely linked with the afterload. in among them, the majority was mild or trivial tr, which may affect the accuracy of pasp assessment. as the right heart catheterization was not available in our designated treatment hospital, pasp derived from key: cord- -yw jvf authors: li, w. d.; hou, j. l.; wang, w. q.; tang, x. m.; liu, c. l.; xing, d. title: effect of water deficit on biomass production and accumulation of secondary metabolites in roots of glycyrrhiza uralensis date: - - journal: russ j plant physiol doi: . /s sha: doc_id: cord_uid: yw jvf two-year-old seedlings of licorice plant (glycyrrhiza uralensis fisch) were exposed to three degrees of water deficit, namely weak ( – %), moderate ( – %), and strong ( – %) relative water content in soil, whereas control plants were grown in soil with – % water content. moderate and strong water deficit decreased the net photosynthetic rate, stomatal conductance, and biomass production. water use efficiency and the root-to-shoot ratio increased significantly in response to water deficit, indicating a high tolerance to drought. weak water deficit did not decrease root biomass production, but significantly increased the production of glycyrrhizic acid (by %) and liquiritin (by %) in the roots. therefore, a weak water deficit can increase the yield of root medical compounds without negative effect on root growth. licorice (glycyrrhiza uralensis fisch.) is a very popular medicinal plant, which roots contain glycyr rhizic acid and liquiritin mainly accumulated in the root and rhizome tissues [ , ] . recently, glycyrrhizic acid has been found to be highly active in inhibiting the replication of the severe acute respiratory syn drome (sars) associated virus and has been sug gested as a potential therapeutic agent for chronic hepatitis and acquired immunodeficiency syndrome (aids) [ ] . licorice plants appear to be highly drought tolerant, being a favorable plant to restore degraded desert, arid and semiarid ecosystems of northwest china [ ] . however, data on physiological processes, such as biomass production and secondary metabolite yield, in response to environmental condi tions are lacking [ ] . water deficit usually inhibits plant growth and pro ductivity by affecting gas exchange and especially pho tosynthesis [ , ] . water use efficiency (wue) can be traditionally defined as the ratio of net photosynthesis to transpiration over a period of seconds or minutes [ ] . the higher wue has been mentioned as a strategy this text was submitted by the authors in english. to improve crop performance under water limited conditions [ ] . however, in licorice plants photosyn thesis and biomass production as well as wue in response to water deficit were not studied. water deficit can induce the biosynthesis of some secondary metabolites [ ] [ ] [ ] , resulting in their accumulation in medicinal plants [ , , ] . for example, the concentration of rutin and chlorogenic acid increased with drought severity in tomato plants [ ] . although the responses of the metabolites to drought have been investigated in some medicinal plants [ , ] , no reference concerning the effect of various water deficit levels on their production by lic orice roots is available. the present study aims to determine the effect of water deficit on gas exchange, biomass and secondary metabolites production in licorice plants. it was hypothesized that a suitable water deficit, in addition to saving water, can also increase the amount of root secondary metabolites without negative effect on root growth. water treatments were carried out from may until the end of october in . four levels of soil rel ative water content (wc), - , - , - , and - %, represented control plants, weak, moderate, and strong water deficit, respectively. each pot was weighed and water was added to reach the target level at : p.m. every day. there were four replications per treatment arranged in a completely randomized block design. leaf gas exchange. the newly developed leaves from the middle part of the shoot were chosen for gas exchange measurement using a li portable pho water use efficiency (wue) was calculated as the ratio of the net photosynthetic rate to transpiration rate. biomass determination. biomass determination was carried out by the end of october. licorice plants were separated into the roots and shoots. dry weights were determined after drying for h at °c in an oven. root to shoot ratio = root dry weight: shoot dry weight. then the dried roots were used for glycyrrhizic acid and liquiritin analyses. glycyrrhizic acid and liquiritin analyses. glycyr rhizic acid and liquiritin were extracted as described in [ ] . dry roots were extracted with a tenfold volume of . % ammonia for min under ultrasonication ( w, khz). glycyrrhizic acid and liquiritin con centrations were determined with a hp high per formance liquid chromatography system (agilent technologies, united states) consisting of a g a pump, a g a degasser, and a g a autoinjector connected to a g b diode array detector (dad). the separation was performed on a dikma diamon sil tm c column ( mm × . mm × µm) with a mobile phase consisting of . % h po (solvent a) and acetonitrile (solvent b). the sample ( µl) was eluted with a gradient profile, and the column was maintained at °c [ , ] . statistical analysis. statistical treatment was per formed using a spss statistical package (version , spss, chicago, united states). the difference between the mean values of each treatment was deter mined using duncan's multiple range test and consid ered significant at p < . . the net photosynthetic rate, stomatal conduc tance, and transpiration rate decreased with increas ing water deficit (table ). compared to the control, - % wc had no effecton the net photosynthetic rate and stomatal conductance, but at - and - % wc, photosynthesis and transpiration were sig nificantly reduced. however, water deficit increased wue and the highest value was observed at the - % wc. dry weight of the plant and its organs decreased with increasing water deficit, but no effect was exerted at - % wc ( table ) . root dry weight decreased by . , . , and . % with increasing water deficit, while shoot dry weight decreased by . , . , and . %, respectively (table ). root to shoot ratio increased as water deficit progressed (table ) . the gain in the content of glycyrrhizic acid and liq uiritin in the roots under - % wc had the highest ( . and . %, respectively), followed by the mod erate treatment ( . and . %, respectively) as com pared to the control plants. there was no significant difference between the strong wc treatment and con trol (figs. a, b) . at - % wc, the amounts of glycyrrhizic acid and liquiritin in plant roots were the highest among the studied four levels of soil water con ditions: the gain increased by . and . %, respectively (figs. c, d) . at the lowest wc, glycyr rhizic acid amount in plant roots decreased, although no difference in liquiritin amount between these and control plants was found (figs. c, d ). it is well known that water deficit is one of the major factors limiting plant growth and yield [ , ] . in this study, licorice plants were able to grow and pro duce biomass even at - % wc (table ) , suggest ing that this plant can acclimate in response to unfa vorable environment and exhibit high drought resis tance. water deficit induces partial closing of stomata and both transpiration and photosynthesis decrease, thereby slightly increasing wue [ ] , especially at - % wc ( table ). the increase in the efficiency of water use under drought occurs at the expense of absolute biomass production [ ] . in order to dimin ish metabolism consumption and increase uptake of water under dry conditions, plants often decrease their growth rate and biomass production, and contribute more synthesized biomass to roots, so that they could maintain a higher root to shoot ratio ( table ) . parti tioning more assimilate to the underground parts and maintaining the higher root to shoot ratio may con tribute to enhanced water uptake [ ] . thus, these responses allow licorice plants to survive and even to continue to grow under conditions of water shortage, i.e., to develop drought tolerance. water deficit decreases plant photosynthesis and thereby reduces plant growth and biomass production ( tables , ). however, the dry weight of the roots decreased less than that of shoots (table ) . no signif icant difference was found between the dry weights of roots and the shoots at - and - % wc, while other treatments significantly decreased biomass pro duction (table ). our findings indicate that weak water deficit did not affect growth and biomass pro duction of licorice roots. it has been well documented that water deficit can also affect the production of secondary metabolites in some medicinal plants [ , , ] . biosynthesis of secondary metabolites is known to be affected by drought, indicating influence by environmental stim uli [ , ] . for example, the content of several alka loids increased in response to drought in tabernae montana pachysiphon [ ] . liu [ ] also found that camptothecin concentrations briefly rose when camp totheca acuminata seedlings experienced drought. glycyrrhizic acid and liquiritin, the major bioactive components of g. uralensis, are accumulated in the underground parts of licorice plants [ , ] . thus, soil wc plays a key role in their biosynthesis. weak water deficit significantly increased not only glycyrrhizic acid and liquiritin concentrations per gram dry weight (figs. a, b) , but also total amount of these com pounds in plant roots (figs. c, d) . the synthesis of secondary metabolites was stimulated under weak drought conditions. zhu et al. [ ] also found that mild water deficit significantly increased saikosaponin a and d contents in bupleurum chinense roots. our results confirm that weak water deficit can increase secondary metabolite contents and thereby increase the quality of medical row material. development of a monoclonal antibody based enzyme linked immu nosorbent assay for the analysis of glycyrrhizic acid separation of glycyrrhizic acid and liquiritin from glycyrrhiza uralensis fisch extract by three liquid phase extraction systems glycyrrhizin, an active component of liquorice roots, and replication of sars associated coronavirus effect of salt and drought stress on antioxidant enzymes activities and sod isoenzymes of liquorice (glycyrrhiza uralensis fisch), plant growth regul effect of low light intensity on growth and accumulation of secondary metabo lites in roots of glycyrrhiza uralensis fisch photosynthetic response to water stress and changes in metabolites in jasminum sambac, photo synthetica photosynthetic responses to chromosome doubling in relation to leaf anatomy in lonicera japonica subjected to water stress physiological and growth responses of populus davidiana ecotypes to different soil water contents impact of water supply on photosynthesis, water use and carbon isotope discrimination of sugar beet genotypes drought induced in vivo synthesis of camptothecin in camptotheca acuminata seedlings anti oxidant potential and indole alkaloid profile variations with water deficits along different parts of two variet ies of catharanthus roseus impact of fertilization on drought response in the medicinal herb bupleurum chinense dc growth and alkaloid contents in leaves of tabernaemontana pach ysiphon stapf (apocynaceae) as influenced by light intensity, water and nutrient supply drought stress in tomatoes: change in plant chemis try and potential nonlinear consequences for insect herbivores simultaneous determina tion of glycyrrhizic acid, liquiritin and fingerprint of licorice by rp hplc effects of drought stress and n supply on the growth, biomass partitioning and water use efficiency of sophora davi dii seedlings we would like to thank dr. e.s.j. harris (medical school, harvard university, united states) and dr. h. xu (institute of botany, chinese academy of sciences, china) for critical reviews of the manuscript.the work was supported by natural science foun dation of china (nos. , ). key: cord- -ln qfg authors: bertolesi, gabriel e.; zhang, john zhijia; mcfarlane, sarah title: plasticity for colour adaptation in vertebrates explained by the evolution of the genes pomc, pmch and pmchl date: - - journal: pigment cell melanoma res doi: . /pcmr. sha: doc_id: cord_uid: ln qfg different camouflages work best with some background matching colour. our understanding of the evolution of skin colour is based mainly on the genetics of pigmentation (“background matching”), with little known about the evolution of the neuroendocrine systems that facilitate “background adaptation” through colour phenotypic plasticity. to address the latter, we studied the evolution in vertebrates of three genes, pomc, pmch and pmchl, that code for α‐msh and two melanin‐concentrating hormones (mch and mchl). these hormones induce either dispersion/aggregation or the synthesis of pigments. we find that α‐msh is highly conserved during evolution, as is its role in dispersing/synthesizing pigments. also conserved is the three‐exon pmch gene that encodes mch, which participates in feeding behaviours. in contrast, pmchl (known previously as pmch), is a teleost‐specific intron‐less gene. our data indicate that in zebrafish, pmchl‐expressing neurons extend axons to the pituitary, supportive of an mchl hormonal role, whereas zebrafish and xenopus pmch+ neurons send axons dorsally in the brain. the evolution of these genes and acquisition of hormonal status for mchl explain different mechanisms used by vertebrates to background‐adapt. different types of camouflage (i.e., masquerade, disruptive and distractive coloration, surface disruption and countershading) are generated by a combination of skin patterns and colours selected through evolution in many prey and predator species as a means to reduce the likelihood of detection (crypsis) (skelhorn & rowe, ) . in general, they are not mutually exclusive and work best when several of their components match the background colour (eterovick et al., ) . the matching of skin pigmentation to background colour can be genetically determined (defined here as "background matching"), or induced by physiological plasticity through differential secretion of hormones by neuroendocrine systems ("background adaptation") (baker & bird, ; hoekstra, ). an example of genetically based background matching are mutations that produced a hyperactive melanocortin receptor (mc r), the main receptor for α-melanocyte-stimulating hormone (α-msh) (robbins et al., ) . the emergence of genetic polymorphisms in mc r expressed by pigment cells, and the selection of standing genetic variations, resulted in adaptive evolution of background matching in populations of reptiles, birds and mammals (linnen et al., ; price & bontrager, ; rosenblum, hoekstra, & nachman, ) . in contrast, background adaptation is plastic, with colour change mediated by pigment cell receptors activated by hormones secreted after eye photoperception of surface colour. although background adaptation occurs in many species, differences exist in the mechanisms between species that are not well understood. here, the evolutionary analysis of three critical genes involved in background increase in organisms with a black background de rijk, jenks, & wendelaar bonga, ; jenks, overbeeke, & mcstay, ) . the existence of a second hormone secreted from the pars nervosa of the pituitary, with opposite effects on background adaptation to α-msh, was suggested initially by hogben and slome ( ) in their "dual-hormonal control" theory. mch is processed from the c-terminal end of the preprohormone pmch ( figure ). in fish, an "mch" peptide is produced in the hypothalamus and transported down axons to be secreted from the neurohypophysis upon stimulation (baker & bird, ; enami, ; kawauchi, ; kawauchi, kawazoe, tsubokawa, kishida, & baker, ) (figure ). the ability to adapt to a white background by aggregating melanosomes associates with the synthesis and secretion of an "mch" in several teleost species (gröneveld, balm, martens, & wendelaar bonga, a; kishida, baker, & eberle, ; mizusawa et al., ; sugimoto, f i g u r e zebrafish and xenopus both background-adapt but only in zebrafish do mchl+ neurons project axons to the pituitary. (a) schematic of the genomic structure of the three-exon pmch and one-exon pmchl genes, the mrnas, the prehormones and the aligned a.a. sequence of mch and mchl. the two cysteines involved in the disulphide bonds in mch and mchl are indicated in red, and a.a. identity in green, while the predicted dibasic peptide at the cleavage site (rr) is underlined. (b) quantification of the pigmentation index (left) and dorsal pictures of the head (right) from zebrafish and frog larvae developed for days with a white (wb) or a black (bb) background. bars indicate average (normalized to wb) from two independent experiments (n = ; n = ; ±se; *p < . , anova). bottom right panels are enlargements of the red squares. (c) fish against pmch and pmchl followed by immunohistochemistry against mch/mchl. transverse brain sections ( µm) processed for fish (red; white arrows) and with a polyclonal anti-human mch antibody (green) that recognizes both mch and mchl. note mch immunoreactivity surrounding the pituitary (p) in zebrafish but not xenopus. pmchl+ neurons project their axons to the pituitary in fish (yellow arrows), while the axons of pmch+ neurons from both species project dorsally and are restricted to the brain (hypothalamus areas) nagamori, yasui, & oshima, ) . thus, a dual-hormonal control of regulation by α-msh and an mch with "hormonal status" may exist in fish. mch can also act as a neurotransmitter or neuromodulator to regulate physiological processes associated with feeding and energy homoeostasis (amano & takahashi, ; ludwig et al., ; matsuda et al., ) . the fish mch system differs from that in mammals with regard to the hormonal status of mch and the presence of two genes, pmch and pmchl, with distinct genetic structures (berman, skariah, maro, mignot, & mourrain, ). the single pmch gene of mammals and birds contains three exons and encodes a prohormone that generates an mch that acts as a neurotransmitter (breton, presse, hervieu, & nahon, ; cui et al., ; nahon, presse, bittencourt, sawchenko, & vale, ) . zebrafish have this gene, named originally pmch (renamed here pcmh), but also possess a one-exon gene that ultimately produces a peptide with "melaninconcentrating activity" (berman et al., ; ono, wada, oikawa, kawazoe, & kawauchi, ; takayama, wada, kawauchi, & ono, ) (figure ). the mammalian mch, encoded by the three-exon gene, is almost identical to the -a.a. fish "mch," encoded by the single-exon gene (berman et al., ; kawauchi, ; kawauchi et al., ; nahon et al., ; ono et al., ) . because rat mch was characterized after the fish peptide, both received the same name (nahon et al., ) . synteny analysis indicates, however, that the mammalian and zebrafish three-exon genes are linked evolutionarily, and higher similarity exists between the mch peptides encoded by the three-exon genes than that by the one-exon gene (named hereafter pmchl) (figure a ) (berman et al., ) . a few studies have compared the expression of the pmch genes in fish adapted to different backgrounds (berman et al., ; kang & kim, ; mizusawa et al., ; zhang et al., ) , but it is unclear whether it is mch or mchl that mediates teleost background adaptation. moreover, an evolutionary analysis of pmch and pmchl genes is lacking. our data indicate that pmchl-expressing cells extend "mchl"positive axons to the zebrafish pituitary, while the pmch-expressing neurons extend "mch"-positive axons dorsally in the brain, suggesting that mchl rather than mch has the hormonal role. we used the phylogeny of chordates to analyse the evolution of pomc, pmch and pmchl genes. we suggest an explanation for the differences in the mechanisms that evolved to regulate background adaptation by inferring ancestral relationships, and assessing associations with plastic colour change, the ability of α-msh, mch and mchl peptides to be synthesized and secreted and to work as neuroendocrine hormones, as well as their effects on skin pigmentation. we propose that the evolution of background adaptation relates mainly to key changes in the pmch genes, with the topological structure of the pomc gene and the α-msh a.a sequence both conserved during evolution. we show that the intron-less pmchl gene appears only in the teleostei lineage, possibly as a result of an ancestral retroposition or during the teleost-specific genome duplication (tsd). based on our analyses, we suggest renaming the intron-less gene, originally named pmch in several fish (kawauchi, ; ono et al., ; takayama et al., ) , as pmchl, which is in concordance with the current zebrafish nomenclature convention and reflects its evolutionary origin. pmchl gained "hormonal status" and is found only in teleosts, while the three-exon pmch peptide product was conserved as a brain neurotransmitter that regulates feeding behaviour. to be consistent with the evolutionary origin of both genes, in this paper we call the mammalian and fish three-exon gene, and the prehormone and the peptide generated, as pmch, pmch and mch, respectively, and the fish intron-less gene, pmchl, and its corresponding peptides pcmhl and mchl. of note, special care should be taken with the literature, as a different nomenclature was used. physiological skin pigmentation indices were measured on -dayold embryos at the middle of the light cycle period ( hr on), as described previously (bertolesi, hehr, & mcfarlane, ) . briefly, pictures of the dorsal head of tadpoles were taken with identical conditions of light, time exposure and diaphragm aperture, and converted to binary white/black images using nih imagej (u. s. national institutes of health, bethesda, md) public domain software. the areas of ten dorsal head melanophores/fish were measured (n = fish; n = experiments), while in xenopus (n = ; n = ) the pigmentation present within an area of the dorsal head between the eyes was used for quantification, since melanophore processes overlap. statistical differences were determined by anova (*p < . ). pmch and pmchl from zebrafish were amplified by reverse transcriptase pcr (rt-pcr) from single-strand cdna generated from zebrafish embryos at days post-fertilization (dpf) using superscript™ ii rnase h reverse transcriptase (invitrogen) according to the manufacturer's instructions. pcr amplifications were carried out in a total volume of μl using fermentas pcr mix (thermo fisher scientific inc., ottawa, ontario). pcr amplification products were cloned into the pcrii-topo vector and sequenced at the dna services facility, u. calgary. pcrii-topo-pmch-zf and pcrii-topo-pmchl-zf amplified by pcr produced sequences with % identity to those deposited previously in the genbank database [fj . between nucleotides and (pmch) and fj . between nucleotides and (pmchl), respectively). a pcmv sport plasmid, containing the full-length cdna encoding x. laevis pmch obtained from an image clone (open biosystems image clone number ic ; pcmv-sport -pmch-xl), was used to generate pmch probes. antisense and sense riboprobes were synthesized from linearized plasmid using sp or t rna polymerases (roche) and digoxigenin (dig) (roche, montreal, qc, canada)-labelled nucleotides and stored at − °c. fish was performed on -µm cryostat sections obtained from embryos at dpf. hybridized probes were detected using anti-dig antibody ( : ; roche) conjugated to horseradish peroxidase. the staining process was carried out with a tsa cyanine (red) system kit (perkin elmer, usa), according to the manufacturer's instructions. immunohistochemistry was performed on sections after fish using standard procedures. briefly, the fish reaction was stopped with several washes of tn-t ( mm tris ph . , mm nacl, . % bovine serum albumin (sigma), and . % tween- (bdh)) before addition of primary antibody (anti-mch rabbit polyclonal m ; / dilution; sigma-aldrich) in blocking buffer ( . % blocking f i g u r e schematic diagram of related taxa (classes or orders) of vertebrates with a similar state of evolution and/or mechanism for background adaptation. extant species divided (see materials and methods) into groups corresponding with taxonomic classes (top rectangles) or taxonomic orders. evolutionary years (millions) dividing different era are indicated on the left. whole-genome duplication events ( r; teleost-specific duplication [tsd r] and salmonid-specific duplication [ss r]) are indicated in yellow, while blue circles denote the age of the oldest preserved fossils with fur or feathers with insulation capacity. homoeotherms (birds and mammals) are in grey boxes. background adaptation is indicated with a white/black circle. group : cyclostome (agnathans: hagfish and lamprey); group : chondrichthyes (cartilaginous fish; sharks); group : cladistei (polypteriformes; bichir); group : chondrostei (sturgeon); group : holostei (bowfin; gar); group : teleostei; group : euteleostei and neoteleostei; group : acanthomorpha (see taxonomic orders for groups , and in table s ); group : coelacanthi and dipnoi (lungfish); group : anura (frogs); group : caudata (salamanders); group : squamata (lizard and snakes); group : testudine (turtles); group : crocodilia (crocodiles, alligators); group : aves (birds); and group : mammalian (mammals). not included in the analysis are the taxonomic orders from amphibian gymnophiona (caecilians) and from reptile rhynchocephalia (tuatara) (grey circle) reagent fp in tn-t; perkin elmer). alexa fluor-tagged secondary antibodies (green; nm emission, anti-rabbit) diluted : , were used to detect the primary antibody. a variety of organisms from agnathans and gnathostomes with readily available protein reference sequence were used to probe for pmch, pmchl and pomc. homologues were identified using zebrafish pmch, pmchl and pomc as queries with a cut-off expect (e) value for positive candidates set at . . the organisms selected are listed in tables s and s (pmch and pmchl) and (pomc). the genetic-structural analysis of pmch and pmchl was performed from organisms listed in the ncbi gene database that contained the exonintron structure and gene identification number, as referred to in the organism list. although organisms with fully sequenced genomes were chosen wherever possible, some organisms in key lineages with unpublished or incomplete genomes were also included. in a few cases, for example, for organisms that are used as model systems for background adaptation (i.e., oncorhynchus keta and verasper moseri), we included nucleotide sequence obtained from cdna rather than derived from genomic sequences. different species were chosen to represent a broad range of organisms in sixteen clades (taxonomic classes or orders) as referred to in figure . vertebrates were divided into sixteen clades. group contains species in the infraphylum agnathan (cyclostomes, represented by two taxonomic classes, myxini and cephalaspidomorphi, which includes the hagfish and lamprey, respectively). groups - correspond to extant gnathostomes. group contains the cartilaginous fish (sharks; taxonomic class, chondrichthyes), while the ray-fin fish (actinopterygii) were divided into six groups representing taxonomic classes or orders (groups - ). the taxonomic classes cladistei, chondrostei and holostei were maintained as independent groups (groups , and , respectively), while the clade teleostei was divided into three groups based on the taxonomic orders and differences in their background adaptation response (see results and table s ). the taxonomic orders in teleostei were divided as follows: group (teleostei) contains taxa, group (euteleostei plus neoteleostei) contains taxa, and group (acanthomorpha plus percomorpha) contains taxa (table s ) . lobe-fin fish (sarcopterygii) were group . the amphibians were grouped into their three taxonomic orders: anura (frogs; group ), caudata (salamanders; group ) and gymnophiona (caecilians, not analysed because of lack of information on pomc, pmch and pmchl). reptiles were also grouped according to their taxonomic order: squamata (lizards and snakes; group ); testudine (turtles; group ); crocodilia (crocodiles; group ); and rhynchocephalia (tuataras), with the latter not considered because only one living species (sphenodon punctatus) exists and genetic information is lacking. finally, groups and correspond to the clade aves (birds) and mammals, respectively. validated sequences were aligned using muscle (multiple sequence alignment) and used to build a hidden markov model (hmm) by using a maximum-likelihood architecture construction algorithm. all phylogenetic analysis and alignments were performed by using the public domain mega x software (www.megasoftware.net) (kumar, stecher, li, knyaz, & tamura, ) . detailed descriptions and statistics are indicated in the figure legends. mch and mchl show a similar a.a. sequence (figure a ), and both bind cultured mammalian hek cells that overexpress mch r and mch r (mizusawa et al., ) . the data are more convincing, however, for mchl having "hormonal status." at physiological concentrations (< nm), mchl induces greater melanosome aggregation in cultured skin melanophores and xanthophores than mch (mizusawa et al., ) . further, mchl is the "factor" with melanin-concentrating capacity that was shown initially to be secreted by the pituitary of catfish (parasilurus asotus) (enami, ) , and was then purified from the pituitary of chum salmon (o. keta) ( figure a ) (enami, ; kawauchi et al., ) . finally, pmchl mrna is upregulated in organisms with a white background (berman et al., ; kang & kim, ; mizusawa et al., ; zhang et al., ) , and pmchl overexpression in medaka (oryzias latipes) increases mchl plasma levels and lightens the skin, but leaves feeding behaviours unchanged (kinoshita et al., ) . these data argue that mchl has hormonal status. whether the related mch is also secreted into the circulation blood to induce melanosome aggregation is unclear. in order to answer this question, we used two experimental models: zebrafish (danio rerio) and the african clawed frog (x. laevis), which possess mch/mchl and only mch, respectively ( figure a ). of note, studies have defined the hormonal status of an mch in fish based on the immunohistochemical detection of mch+ axon fibres that project to the neurohypophysis (baker, ; baker & bird, ; powell & baker, ) and/or the biochemical detection of mch peptides in blood kishida et al., ) . those studies, however, were performed before it was known that two pmch genes exist, and used polyclonal antibodies raised against either mammalian mch (human or rat) or salmonid mchl (coho salmon), which likely cross-react in that mch and mchl a.a. sequences are almost identical ( figure a ). in order to compare the background adaptation response between the two model systems, we grew three-dayold zebrafish and xenopus larvae for days with a white or a black background, and then measured skin pigmentation (bertolesi et al., ) . both species showed melanosome aggregation and a light skin colour with a white background ( figure b ). next, we asked whether the pmch+ and pmchl+ neurons in zebrafish and the pmch+ neurons of xenopus are present in the hypothalamus, and project axons to the neurohypophysis. of note, in zebrafish pmch and pmchl are expressed by different neurons (berman et al., ) our experimental data suggest mchl possesses hormonal status in zebrafish, while mch may act as a brain neurotransmitter. together with the presence of two pmch genes in fish and one in mammals, these observations led us to hypothesize that the uni-hormonal or dual-hormonal theories of background adaptation in vertebrates might be explained by the evolution of the pomc, pmch and pmchl genes. to address this possibility, we assessed in species across evolution first the presence of a physiological background adaptation response, via an analysis of the literature, and, second, the structure and sequences of the pomc, pmch and pmchl genes and their corresponding peptides. thus, the evolution of the physiological background adaptation response was inferred by comparison of extant organisms grouped in clades (taxonomic classes or orders) as referred to by the integrated taxonomic information system (https://www.itis.gov/) (figure ). we focused our evolutionary analysis on two assemblages in the phylum chordata that emerged over the past million years: the agnathans (bilateral organisms with an internal support system but that lack a hinged jaw; cyclostome; group ) and the gnathostomes (bilateral organisms with an internal cartilaginous or bony skeleton, and a hinged jaw; groups - ). the protochordata (organisms that lack an internal cartilaginous or bony skeleton) were not considered in this study, since the mechanisms to change skin colour differ from those in chordata (umbers, fabricant, gawryszewski, seago, & herberstein, ) , and the pomc gene is not detected (sundström, dreborg, & larhammar, ) . cyclostomes (group ) change skin colour in response to environmental light (dark/light) through melatonin secreted by the pineal gland (joss, ) ; however, we are unaware of reports of quick physiological skin adaptation prompted by changing the surface colour (figure , circle with a red cross). background adaptation does occur in extant gnathostomes starting in group (cartilaginous fish; chondrichthyes) with sharks altering their skin pigmentation to adapt to the "background" colour generated by deep and shallow water (lowe & goodman-lowe, ) , and continuing through to mammals (beltran, burns, & breed, ; zimova et al., ) (figure , black and white circles; see additional references in table s ). the ray-finned bony fish (groups - ; actinopterygii) constitute the largest clade of living vertebrates and distribute over cladistei, chondrostei, holostei and teleostei taxa ( figure ). here, we maintained three of the four taxonomic classes (cladistei, chondrostei and holostei, with , and living species, respectively) as independent groups (groups , and , respectively), with all containing species that background-adapt ( figure and table s ). the fourth actinopterygii taxonomic class, teleostei, contains more than , species. we divided this clade into three groups that reflect different evolutionary lineages based on recent molecular and genomic data (groups - ) (betancur-r et al., ; betancur-r et al., ; near et al., ) . cypriniforme zebrafish (d. rerio) and the anguilliforme eel (anguilla anguilla) with a dual-hormonal mechanism (figure , tables s and s ). group contains the euteleostei plus neoteleostei ( figure and table s ). background adaptation occurs in the esociform esox lucius, as well as in several species of salmonids, including oncorhynchus mykiss, and the chum salmon (o. keta) from which an mch peptide (we call mchl) was originally purified (kawauchi et al., ) . studies in salmon helped reveal the dual-hormonal mechanism, in that circulating mch/mchl and α-msh levels increase with a white and black background, respectively kishida et al., ) (figure and table s ). finally, group , acanthomorpha, are collectively known as the spiny-rayed fish ( figure ) . percomorphas, the clade that encompasses most of the diversity of modern acanthomorpha, is the major evolutionary lineage at the top of the teleost tree ( figure and table s ). organisms of this group backgroundadapt and include the important models beloniforme medaka (oryzias latipes), tetraodontiforme fugu (takifugu rubripes) and the pleuronectiforme flounder (verasper moseri) (klovins et al., ; mizusawa, kobayashi, yamanome, saito, & takahashi, ; sugimoto et al., ) . interestingly, several species in this group lack the dual-hormonal mechanism, in that mch/mchl plasma levels but not α-msh vary with background adaptation, including flounders (verasper moseri and platichthys flesus) (gilham & baker, ; kang & kim, ; mizusawa et al., ) and tilapias from the genus oreochromis (nile and mozambique) (gröneveld, balm, martens et al., a; gröneveld, balm, & wendelaar bonga, b; van der salm, metz, wendelaar bonga, & flik, ) . group consists of the coelacanthi and dipnoi lobe-finned fish (lung fish), with only eight living species (figure ) . captive dipnoi adapt their skin colour to the background (smith & coates, ) , while no information is available for coelacanths. to best characterize evolutionary changes in pomc and pmch, we grouped the species in the taxonomic classes amphibia and reptilia according to their taxonomic orders (figure ): the amphibian frogs (anura; group ), salamanders (caudata; group ) and caecilians (gymnophiona; not analysed, grey circle), and the reptilia squamata (lizards and snakes; group ), testudine (turtles; group ), crocodilia (crocodiles; group ) and rhynchocephalia (tuataras not analysed, grey circle) (figure ) . although colour skin pigmentation of several species of caecilians mimics the above-ground soil where they move (wollenberg & measey, ) , is not clear if it is a genetically mediated process (background matching) or driven by physiological colour plasticity. therefore, caecilians were not grouped for further analysis. in contrast, background adaptation in amphibian frogs and salamanders is well documented (table s ) and appears to be driven by a uni-humoral mechanism, since mch/mchl do not trigger melanosome aggregation (ferroni & castrucci, ; wilkes et al., ) . background adaptation is also present in species in the clade reptilia ( figure and table s ). finally, the homoeotherms of the taxonomic classes aves (birds; group ) and mammals (group ) we next performed an evolutionary analysis of the mch and mchl peptides and genes. we focused first on the gene structure of pmch, analysing mainly organisms whose genome was fully sequenced and listed in the ncbi gene database with the exon-intron structure and gene identification number (table s ). the three-exon gene appears conserved during evolution, as it is present in all groups in which we were able to find the gene, from chondrichthyes (group ) to mammals (group ). in contrast, the intron-less gene is present only in teleost fish (groups - ; figure and table s ). teleosts possess ~ % more pigmentation-related genes than tetrapods, most of which were duplicated and retained during the tsd and are involved in specification and differentiation of pigment cells (braasch, brunet, volff, & schartl, ; lorin, brunet, laudet, & volff, ) . thus, the idea that pmchl was retained in teleostei by evolutionary pressure selection on a gene with a key role in pigmentation regulation (e.g., background adaptation) is tempting. (minth, qiu, akil, watson, & dixon, ) and o. kisutch (mch ) (nahon, presset, schoepfer, & vale, ) . we find four variants in the arctic char (salvelinus alpinus) and the atlantic salmon (salmo salar), which express the mrnas (leong et al., ) ( figure and table s ). phylogenetic analysis of the predicted proteins of the group salmonids and the esociform esox lucius shows that pmcha and b, and pmchla and pmchlb are related to pmch and pmchl from esox lucius, respectively, but appeared more recently in evolution ( figure ). this result, together with the ab- (courseaux & nahon, ) . we next analysed pmch and pmchl at the a.a. level. both pmch and pmchl contain a predicted mch and mchl sequence able to generate a cyclic ring with a.a. between two internal cysteines that mediate a disulphide bond essential for receptor binding (audinot et al., ; matsunaga, hruby, lebl, castrucci, & hadley, ) , as well as two consecutive basic residues (e.g., arg -arg in mammals and rodents) critical for cleavage and release (viale et al., ) ( figures a and ) . mch peptides are at least two a.a. longer ( - a.a. / species) than mchl ( a.a. / species in groups - ). the latter is highly conserved (> % identity, / a.a.) in all species of groups - (figure and table s ). also, highly conserved in f i g u r e carboxy-end amino acid sequence of the evolutionary conserved pmch and the teleost-specific pmchl. structure (top) and alignment of the carboxy-end region of pmch and pmchl of several species grouped as indicated in figure . the putative peptides processed from pmch include mch and neuropeptide glutamic acid-isoleucine (nei), and from pmchl include mchl, neuropeptide glutamic acid-valine (nev) and mch gene-related peptide (mgrp). the length of the a.a. sequences and the number of exons (ex; red) that contribute to the open reading frame are indicated on the right. note three (or more) exons and one exon contribute to pmch and pmchl, respectively. multiple protein sequence alignments were performed by using muscle and correspond to pmch from group mchl is a valine inside the ring ( / species from groups - ). in mch, the a.a. at this site is isoleucine or leucine, except for in two group species, d. rerio and paramormyrops kingsleyae, in which valine is present (figure ) . we next analysed the presence of other putative peptides in the pmch and pmchl prohormones. in mammals, pmch is processed to mch and neuropeptide glutamic acid-isoleucine (nei) (viale et al., ) , a -a.a. peptide that affects the hypothalamic-gonadal axis (bittencourt & celis, ; fujimoto, fukuda, sakamoto, takata, & sawamura, ) . conserved sequence for the mammalian nei is present in amphibians and reptiles (groups - ), while several a.a. are absent from group species and missing completely from group and species (figure ) . these data suggest that nei has no biological function in teleosts. similarly, in birds (group ), the predicted nei peptide is highly modified, and the loss of the k/r r site for cleavage in chicken and japanese quail (figure ) suggests that the peptide is not processed and released. thus, nei is conserved in land organisms, but not aquatic organisms or birds. interestingly, a arginine-valine (nev) neuropeptide from the rainbow trout sequence was suggested as a homologue of the mammalian nei, based mainly on the topological position and the presence of a dibasic rr motif nahon et al., ) . our analysis shows that nev is predicted to be processed from pmchl but not pmch, and in species from groups and , but not group (figure ). an additional putative peptide, named mch gene-related peptide (mgrp) (figure ) , is present in nile tilapia, but does not participate in background adaptation (gröneveld, balm, & wendelaar bonga, c) . (hu et al., ; takahashi et al., ) . thus, the three-exon gene is translated to pmch, which then releases mch after processing. in mammals, and probably in fish (berman et al., ; mizusawa et al., ) , mch acts as a neurotransmitter to regulate feeding. the paralogue pmchl intron-less gene (originally named pmch), and the mchl peptide to which it gives rise, appears only in teleostei. its existence, coupled with the potential of mchl to reach the circulation, associates with the dualhormonal mechanism for background adaptation in teleosts. with α-msh as a key regulator of background adaptation, and data which suggest that pomc and mcr in chordates are linked evolutionarily (dores & baron, ; dores et al., ) , we next analysed the a.a. sequences of pomc, α-msh and acth. acth and α-msh arise from processing of pomc in the anterior pituitary and pars intermedia pituitary, respectively, and are utilized in distinct neuroendocrine circuits (zhou, bloomquist, & mains, ) (figure a ). β-, γ-and δ-msh are also processed from pomc, but have no known role in f i g u r e topological organization of pomc and amino acid sequence alignment of acth/α-msh. (a) schematic representation of pomc with the positioning of the different processed peptides. γ-msh, β-msh, acth (green) and α-msh (red), which is at the amino-end of acth, are indicated and positioned based on the presence of the conserved hfrw sequence. β-endorphin is located at the carboxy-end. the a.a. length and the position of the first a.a. of hfrw are indicated on the right and inside the acth/α-msh domain, respectively. note that δ-msh is exclusive to group (sharks) and γ-msh is not present in teleosts (groups , and ), cyclostomes (group ) and the lineage from which arose the snakes (group ), but is present in lizards (half blue rectangle). also indicated are mutations in hfrw in groups and species. in lamprey, acth and α-msh are encoded by different genes, named pom and poc, respectively (heinig et al., ; takahashi et al., ) . (b) aligned sequences of acth and α-msh (green and red box, respectively). amino acids in α-msh are coloured. note similarity between groups to . topological organization and sequence alignment were performed by using the following pomc prohormones (species, genbank acc. background adaptation (gilham & baker, ; sumpter & lowry, ) . for group cyclostomes, we analysed the peptides that would arise from two genes with homology to pomc, pom (pro-opiomelanotropin) and poc (pro-opiocortin), which contain the acth and the α-msh sequences, respectively (heinig, keeley, robson, sower, & youson, ; takahashi et al., ) . common to acth and all three msh peptides is the presence of a his-phe-arg-trp (hfrw) sequence, critical for binding and activation of mcrs (dores et al., ; schwyzer, ) . we used this motif to align the various pomc peptides of the different taxonomic groups (figure a ). the comparison of msh peptides between groups supported our taxonomic grouping of phylogenetic clusters. first, in agreement with previously described differences (dores, cameron, lecaude, & danielson, ; takahashi, amemiya, nozaki, sower, & kawauchi, ) , the δ-msh sequence is detected only in species of group ( / species) (figure a and table s ). second, γ-msh exists in cartilaginous fish and tetrapods, but is absent in the agnathan poc and pom (amemiya, takahashi, suzuki, sasayama, & kawauchi, ) ( figure a and table s ). in agreement with previous studies (dores & baron, ; kitahara et al., ) , we found γ-msh is also absent from groups , and . novel is that γ-msh in squamata is missing in the lineage giving rise to snakes ( / species), but is present in lineages from which lizards and geckos arose ( / species) ( figure a and table s ). further, we find mutations in the functionally relevant hfrw motif of γ-msh in species from groups and [e.g., hfhw ( / species) and hfrs ( / species), respectively) ( figure a and table s ), which may result in non-functional peptides. together, our analysis suggests that while γ-msh was lost in teleosts and snakes, it may already have become non-functional earlier in evolution (chondrostei and holostei lineages). the analysis of acth/α-msh sequences between the agnathan poc/pom and gnathostome pomc indicates an association with the ability to background-adapt. the α-msh sequence is similar in length ( a.a.) and homology (identity between % and %; / and / a.a., respectively) only between groups that background-adapt (groups to ), but is remarkably different in cyclostomes (figure b ). these differences in cyclostome α-msh may explain the lack of background adaptation in lamprey. first, while the α-msh peptide predicted from the pom gene is identical to the msh-b peptide isolated from the pituitary glands of the sea lamprey petromyzon marinus , it is longer than gnathostome α-msh, with additional a.a. located near two amino terminal basic a.a. essential for endopeptidase cleavage ( figure b) . second, the homology in the region surrounding hfrw is low in agnathans ( %; / a.a. as compared to more than % in gnathostomes) (figure b) . finally, the affinity of α-msh to the lamprey mcr is , times lower than to the human receptor (haitina et al., ; schiöth, petersson, muceniece, szardenings, & wikberg, ) . the fact that α-msh and acth bind to the lamprey receptor with similar affinities, while the affinity of α-msh for the human mcr is times higher than that of acth (haitina et al., ; schiöth et al., ) , suggests that acth may be the biological and the "ancestral" ligand for the mcr in agnathans. interestingly, the localization of α-msh and acth to the intermedia and anterior pituitary, respectively, is nonetheless conserved between agnathans and gnathostomes , as is the tetrapeptide hfrw required to bind mcr (figure b ) (costa et al., ; schwyzer, ) . as mentioned, species of groups to all show background adaptation. the literature supports the idea that this pigmentation response associates with the conservation of α-msh sequence between these groups. particularly relevant in this regard, given their evolutionary separation from mammals, are group species, which also lack mchl. the dogfish squalus acanthias α-msh (figure b ) (lowry, bennett, & mcmartin, ) is almost identical to that of human (identity / a.a; figure b ). interestingly, in sharks, background adaptation is reversed by intravenous administration of α-msh antibodies (rodrigues & sumpter, ) , and the marine elasmobranch and mammalian α-msh both induce melanosome dispersion after intravenous injection (sumpter & lowry, ) . indeed, α-msh regulates pigmentation in species from groups (potamotrygon reticulatus) (visconti & de l. castrucci, ) to group (abdel-malek et al., ) , suggesting a conserved role (supporting references in table s ). of note, while α-msh functions in background adaptation in homoeotherms (groups - ), the peptide source may differ in key ways from that in groups - . in birds, α-msh is likely produced in a paracrine manner in the integument, and not from the pars intermedia pituitary, which birds lack (boswell & takeuchi, ) . in mammals, α-msh levels change seasonally, rising during spring/summer and decreasing in autumn/winter (altmeyer, stöhr, & holzmann, ; lincoln & baker, ; mcfarlane et al., ) , but the mechanisms and the source of α-msh are unknown. a possible explanation for the differences with groups - is that species therein require rapid colour plasticity for thermoregulation (fan, stuart-fox, & cadena, ; tattersall, eterovick, & andrade, ) , a need minimized in birds and mammals with an "insulated" integument. in summary, our analysis reveals that α-msh (and β-msh) was conserved during evolution, while γ-msh and δ-msh were not. interestingly, the differences in α-msh a.a. sequence between agnathans and gnathostomes associate with the capacity to background-adapt. in ectotherms, background adaptation (and thermoregulation) is regulated by α-msh secreted from the pars intermedia pituitary. in homoeotherms, however, a role of a pituitary-derived α-msh in background adaptation is unclear. we used both the structure and presence of pomc, pmch and pmchl as a "genetic basis," alongside mch/mchl "hormonal status," to explain differences in the physiological mechanisms for background adaptation over evolution. the genetic basis includes a conserved α-msh sequence and function between groups and , but not group (figure ) . a conserved three-exon pmch gene, whose function appears conserved and relates to feeding behaviours and energy homoeostasis, is present in species of all groups. a pmchl gene appears in teleosts (groups - ) and associates with hormonal status for mchl ( figure ) and axon projections from pmchl+, but not pmch+, neurons to the hypophysis. therefore, we suggest mchl arose evolutionarily in the teleostei lineage, acquiring hormonal status and a new role in skin pigmentation. finally, the "genetic basis" and the "hormonal status" together explain the following differences in the mechanisms that regulate background adaptation in vertebrates (figure ; conclusions): . group : no significant role for α-msh and mch. the absence of a background adaptation response in cyclostomes likely reflects the inability of mch and α-msh to regulate skin pigment dispersion or aggregation, with a distinct α-msh sequence and affinity to mcr from that in gnathostomes. lamprey skin does not respond to synthetic salmon mchl (baker & bird, ) , and a "melanophore-aggregating factor" is detected in hypothalamic but not pituitary extracts (baker & rance, ) , suggesting that mch does not possess hormonal status. additionally, mchl is not present in cyclostomes (figure ). groups - : α-msh but not mch regulates background adaptation. mchl is not detected in these groups, and mch does not appear to have hormonal status. for instance, a shark mch antibody detects cells in the hypothalamus, but not the pituitary gland, of sphyrna lewini (group ) (mizusawa et al., ) . additionally, neither human mch ( % identity to shark mch; / a.a.), nor synthetic mchl, produce aggregation in triakis scyllium and potamotrygon reticulatus (group ) (mizusawa et al., ; visconti & de l. castrucci, ). yet, pituitary-derived α-msh regulates background adaptation in several shark species (visconti & de l. castrucci, ) (figure ; supporting references in table s ). interestingly, skin melanophores from erpetoichthys calabaricus (group ), and acipenser stellate and polyodon spathula (group ), do not respond to synthetic mch and mchl, and mch antibodies fail to label fibres in the posterior pituitary (baker & bird, ; sherbrooke & hadley, ) , arguing against hormonal status for mch in groups and . background adaptation in species from these groups, however, requires a functional pituitary and involves α-msh-induced dispersion of melanosomes, pointing to α-msh, but not mch, as the key regulator of skin pigmentation (figure and additional references in table s ). group : hormonal status and skin response to mch appears. in holosteans, mch+ fibres extend to the pituitary in lepisosteus osseus and amia calva, and mchl causes melanosome aggregation (baker & bird, ; sherbrooke & hadley, ) . therefore, a dual hormonal role of α-msh and mch in regulating skin pigmentation is possible. whether mchl is responsible is unclear, in that we were unable to find pcmhl in any holostean genome, including that of the fully sequenced lepisosteus oculatus (figure ). thus, it is possible that the dualhormonal system was present in the common ancestor of holosteans and teleosts. a key question that needs to be addressed is what selective advantage, if any, was provided by the duplicated genes of teleosts? evolutionary selection of pmchl may be explained as a case of subfunctionalization, with essentially no functional change before and after the duplication in holosteans. groups - : a dual control mechanism at the bottom of the teleostei lineage (groups and ), and a single main regulator, likely mchl, at the top (group ). we suggest pcmhl appears early in the teleost lineage by retroposition or during the tsd, with mchl-expressing neurons then selected over evolution to extend f i g u r e genetics and the hormonal status of mchl explain the different mechanisms for background adaptation. groups able to background-adapt are indicated with a white/black circle. the genetic basis corresponds to the presence of a highly conserved -a.a. α-msh (red square), a three-exon pmch gene (three yellow bars) or a one-exon pmchl gene (yellow square), while hormonal status indicates the capacity of an mch to be released into the bloodstream. a teleost-specific pmchl gene encodes mchl, which acts as a hormone in background adaptation, while the conserved pmch gene encodes mch, which we suggest acts as a neurotransmitter to regulate energy homoeostasis and food intake. the six different mechanisms for background adaptation include the following: (a) group : no significant role for α-msh and mch; (b) groups to : α-msh but not mch participates; (c) group : hormonal status appears with a dual role of α-msh and mch in regulating skin pigmentation; (d) groups - : a "dual control theory" mechanism for background adaptation at the bottom of teleostei lineage (groups and ) and a single main regulator, likely mchl, at the top (group ); (e) group : α-msh but not mch regulates background adaptation; (f) groups - : a uni-humoral α-msh system that also is involved in thermoregulation; and (g) groups and : α-msh and mch roles changed in homoeotherms as insulation systems (fur and feathers) appear during the evolution of endothermy their axons to the pituitary. in this manner, function was split between neurotransmitter (mch) and hormonal (mchl) roles. in support of the dual control theory in groups and , physiological changes in both α-msh (black background) and mchl (white background) are produced when background is altered. for example, in the eel (group ), mch induces melanosome aggregation, and a salmon mchl antibody labels secretory granules in neurons with fibres that terminate on neurohypophysial blood vessels, whose density varies between white and black adapted organisms (gilham & baker, ; powell & baker, ) . plasma α-msh levels increase in eels with a black background . in zebrafish, in addition to the hormonal status for mchl suggested by our expression analysis (figure ) , a white background increases pmchl mrna levels, while fasting increases the expression of pmch (berman et al., ) . a black background in zebrafish triggers melanosome dispersion via α-msh interaction with mcr expressed by melanophores (richardson et al., ) . in two group species, starry flounder (platichthys stellatus (kang & kim, ) ) and barfin flounder (verasper moseri (mizusawa et al., ) ), pmch and pmchl mrnas increase in white backgroundadapted organisms, although most robustly for pmchl. because mchl, but not pomc mrna/α-msh, levels change with background in several group species at the top of the teleostei lineage, it is possible that during evolution the balance between α-msh and mch changed from α-msh»mch in groups - , to a dual mechanism in groups and (α-msh = mch ), to α-msh«mchl in group species (figure ) . group : α-msh but not mch regulates background adaptation. mch+ nerve fibres are absent from the neural lobe of the pituitary of protopterus annectens, and synthetic salmonid mchl does not influence skin cells of lepidosiren paradoxa (dipnoi) (baker, ; visconti & de l. castrucci, ) , suggesting that mch does not have hormonal status and plays no role in skin pigmentation ( figure ). . groups - : a uni-humoral α-msh control system for background adaptation and thermoregulation. mchl is not present in amphibians and reptiles, and mch appears to lack hormonal status ( figure ) . indeed, while the hypothalamus contains mch-expressing cells in frogs (rana esculenta; group ), squamata (podarcis muralis and natrix natrix; group ) and turtles (trachemys scripta; group ), their axons do not project to the posterior pituitary (cardot et al., ; lázár et al., ) . thus, α-msh is the main regulator of background adaptation and may also be important for temperature regulation of ectotherms though colour plasticity (fernandez & bagnara, ) . groups and : new roles for α-msh and mch. in birds and mammals, mchl does not exist, mch+ cells do not extend axons to the pituitary (cardot et al., ; cortés et al., ) , and the pars intermedia pituitary disappears in birds, arguing against a hormonal status for mch (figure ) . birds rely on carotenoid ingestion for background adaptation, while in mammals α-msh possibly associates with seasonal colour moulting. thus, while colour plasticity is important for thermoregulation in ectotherms, in feather/hair covered homoeotherms, skin pigment cells changed with regard to cell type (only melanocytes in birds and mammals), pigment type (eumelanin and pheomelanin) and properties (pigment secretion vs. dispersion/aggregation). together, our experimental data and comparison of evolutionary studies suggest that mchl but not mch has hormonal status in teleosts. a conserved α-msh peptide and the presence of mch or mchl constitute the "genetic basis" for background adaptation that together with the "hormonal status" for mchl explains the different background adaptation mechanisms observed in vertebrates. the authors declare no conflict of interest. geb designed the experiments. geb and jzz performed the experiments and analysed the data. geb and sm wrote the paper. https://orcid.org/ - - - mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides seasonal rhythm of the plasma level of alpha-melanocyte stimulating hormone melanin-concentrating hormone: a neuropeptide hormone affecting the relationship between photic environment and fish with special reference to background color and food intake regulation a newly characterized melanotropin in 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the background-adapted medaka, oryzias latipes: change in the intracellular signaling system the involvement of melanotrophins in physiological in the dogfish scyliorhinus canicula colour change concomitant duplications of opioid peptide and receptor genes before the origin of jawed vertebrates desalle r isolation and characterization of melanotropins from lamprey pituitary glands evolutionary significance of proopiomelanocortin in agnatha and chondrichthyes possible involvement of melanin-concentrating hormone in food intake in a teleost fish, barfin flounder structures of two genes coding for melanin-concentrating hormone of chum salmon tribute to r. g. boutilier: skin colour and body temperature changes in basking bokermannohyla alvarengai (bokermann ) reversible colour change in arthropoda alpha-msh, the melanocortin- receptor and background adaptation in the mozambique tilapia cellular localization and role of prohormone convertases in the processing of pro-melanin concentrating hormone in mammals melanotropin receptors in the cartilaginous fish, potamotrygon reticulatus and in the lungfish, lepidosiren paradoxa synthesis of a cyclic melanotropic peptide exhibiting both melaninconcentrating and -dispersing activities why colour in subterranean vertebrates? exploring the evolution of colour patterns in caecilian amphibians pineal-specific agouti protein regulates teleost background adaptation the prohormone convertases pc and pc mediate distinct endoproteolytic cleavages in a strict temporal order during proopiomelanocortin biosynthetic processing function and underlying mechanisms of seasonal colour moulting in mammals and birds: what keeps them changing in a warming world key: cord- -v wnmg authors: flanagan, katie l.; best, emma; crawford, nigel w.; giles, michelle; koirala, archana; macartney, kristine; russell, fiona; teh, benjamin w.; wen, sophie ch title: progress and pitfalls in the quest for effective sars-cov- (covid- ) vaccines date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: v wnmg there are currently around sars-cov- candidate vaccines in preclinical and clinical trials throughout the world. the various candidates employ a range of vaccine strategies including some novel approaches. currently, the goal is to prove that they are safe and immunogenic in humans (phase / studies) with several now advancing into phase and trials to demonstrate efficacy and gather comprehensive data on safety. it is highly likely that many vaccines will be shown to stimulate antibody and t cell responses in healthy individuals and have an acceptable safety profile, but the key will be to confirm that they protect against covid- . there is much hope that sars-cov- vaccines will be rolled out to the entire world to contain the pandemic and avert its most damaging impacts. however, in all likelihood this will initially require a targeted approach toward key vulnerable groups. collaborative efforts are underway to ensure manufacturing can occur at the unprecedented scale and speed required to immunize billions of people. ensuring deployment also occurs equitably across the globe will be critical. careful evaluation and ongoing surveillance for safety will be required to address theoretical concerns regarding immune enhancement seen in previous contexts. herein, we review the current knowledge about the immune response to this novel virus as it pertains to the design of effective and safe sars-cov- vaccines and the range of novel and established approaches to vaccine development being taken. we provide details of some of the frontrunner vaccines and discuss potential issues including adverse effects, scale-up and delivery. there are currently around sars-cov- candidate vaccines in preclinical and clinical trials throughout the world. the various candidates employ a range of vaccine strategies including some novel approaches. currently, the goal is to prove that they are safe and immunogenic in humans (phase / studies) with several now advancing into phase and trials to demonstrate efficacy and gather comprehensive data on safety. it is highly likely that many vaccines will be shown to stimulate antibody and t cell responses in healthy individuals and have an acceptable safety profile, but the key will be to confirm that they protect against covid- . there is much hope that sars-cov- vaccines will be rolled out to the entire world to contain the pandemic and avert its most damaging impacts. however, in all likelihood this will initially require a targeted approach toward key vulnerable groups. collaborative efforts are underway to ensure manufacturing can occur at the unprecedented scale and speed required to immunize billions of people. ensuring deployment also occurs equitably across the globe will be critical. careful evaluation and ongoing surveillance for safety will be required to address theoretical concerns regarding immune enhancement seen in previous contexts. herein, we review the current knowledge about the immune response to this novel virus as it pertains to the design of effective and safe sars-cov- vaccines and the range of novel and established approaches to vaccine development being taken. we provide details of some of the frontrunner vaccines and discuss potential issues including adverse effects, scale-up and delivery. the recent emergence of severe acute respiratory syndrome coronavirus- (sars-cov- ), the cause of coronavirus disease , is wreaking havoc due to widespread dissemination throughout the world. on march , who formally declared that a global pandemic and by the end of august , almost million cases and over , deaths had been reported worldwide involving all continents, except antarctica ( ) . strategies to identify cases and limit spread by widespread testing and physical distancing have been challenging to implement, healthcare and public health systems have been overwhelmed, resulting in continued escalation in many countries and profound effects on lives and livelihoods. while the majority of people are either asymptomatic or experience a mild respiratory infection, ∼ % of cases are more severe and require hospital admission ( ) . reported mortality rates vary by geographic region, ranging from almost % in france to < % in many other countries ( ) . this wide discrepancy suggests selection bias due to differences in local testing strategies and capacity, consistent with differences in health system capacity, population demographics and other health determinants. certain groups such as the elderly and those with particular comorbidities are more likely to die of covid- ( ) . healthcare workers in particular have experienced significant morbidity and mortality from covid- ( ), causing clear psychological impacts and threatening delivery of healthcare services ( ) . there is no known effective treatment for this virus and currently no available vaccine, with the result being that sars-cov- continues to spread throughout the virus naïve population of the world. the urgent need for effective sars-cov- vaccines cannot be overstated. immunization can not only protect individuals but also, if provided to enough people in a timely way with (even) partially protective vaccines, induce sufficient herd immunity to curtail the spread of this virus and reduce morbidity and mortality across the globe. the race to develop safe and effective vaccines has seen sars-cov- candidate vaccines developed at a scale and pace never imagined before: currently almost potential vaccines are in various stages of development ( ) ( ) ( ) . a range of vaccine design approaches and platforms have been employed. however, since > % of candidate vaccines typically fail it is expected that the eventual number of successful vaccines may be only be a handful. they may also become available in different time frames and suitable for use in different populations. most vaccine candidates are currently in preclinical trials, but a number have entered phase or phase / studies, with plans to rapidly scale up to phase and . trials are being conducted at "pandemic speed" ( ) and using novel designs. this early success has already seen cooperation and collaboration as well as significant funding across the globe. for example, the coalition for epidemic preparedness innovations (cepi), a notfor-profit global coalition launched in to deal with the worldwide threat of epidemic outbreaks, is playing a pivotal role in supporting many of the frontrunner vaccines ( ) . herein, we review what is currently known about the immune response to sars-cov- and the various vaccine platforms being used to develop the sars-cov- vaccines. understanding the mechanism of action of the various candidate vaccines is the key focus of this review. we also discuss potential challenges at the immunological level, assessment of vaccine safety and scale-up and delivery. coronaviruses are enveloped positive-sense single stranded rna viruses belonging to the coronaviridae family. they infect birds and mammals causing a range of symptoms from respiratory to gastrointestinal disease ( ) . a number of relatively common seasonal coronaviruses are known to infect humans ( ) , causing mild respiratory illness ("the common cold"). two previous lethal human coronavirus diseases, namely severe acute respiratory syndrome (sars caused by sars-cov- ) ( ) and middle east respiratory syndrome (mers caused by mers-cov) ( ) arose in and , respectively. they have a high mortality rate of ∼ and %, respectively, but fortunately, neither reached pandemic levels. sars-cov- was able to be contained by public health measures to prevent human-to human transmission and then disappeared before vaccine development had progressed significantly. the genome of sars-cov- encodes for the structural proteins spike (s), envelope (e), membrane (m) and nucleocapsid (n) as well as a number of accessory and non-structural proteins (figure ) ( ) . the m and n proteins give the virion its shape, and the s proteins appear as spikes on the viral surface giving it a solar corona appearance. the s glycoprotein (spike) exists in trimeric form and is the structure by which binding to the host cells occurs. the virus receptor, angiotensin-converting enzyme (ace ), expressed on the figure | structure of sars-cov- and key antigenic components. illustration of sars-cov- which is a single stranded rna virus. the key antigenic components being targeted in vaccine design are shown on the right, consisting of the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. the main emphasis for human vaccines is based on the spike (s) protein, consisting of an s binding region and s fusion and cell entry region. the s domain contains the receptor binding domain (rbd) responsible for binding to the ace receptor on the surface of host cells. following fusion, the s protein sheds the s region and undergoes a dramatic structural change to its post-fusional state in order for the virus to enter the host cells. surface of multiple human cells is engaged via the receptor binding domain (rbd), which is part of the s subunit of the s glycoprotein ( ) (figure ) . the s subunit consists of two heptad repeat fusion regions, hr and hr , responsible for membrane fusion and cell entry. the rbd domain can either be down and buried or rotated up and primed for ace binding ( ) . the hidden down state is the predominant state of the s protein trimer in sars-cov- , and likely a mechanism the virus uses to hide the entry epitopes and evade the host immune response ( ) . two s trimers can concurrently bind to an ace dimer. following s protein binding in its prefusion state, the s subunit is cleaved and shed permitting the s dramatic conformational changes which constitute the post-fusion state required for viral entry ( , ) . innate immunity to covid- : protection or hyper-activation? sars-cov- stimulates an innate immune response via pattern associated molecular patterns (pamps) expressed by the virus, which in the case of sars-cov- consists of viral rna and its intermediates produced during replication ( ) . these conserved pamps stimulate multiple immune response pathways via pattern recognition receptors (prrs), including sensing by endosomal toll-like receptors (tlrs) and , cytosolic retinoic acid-inducible gene (rig- ) and melanoma differentiationassociated protein (mda ). local tissue damage in the lungs also releases damage-associated molecular patterns (damps) further contributing to local inflammation. the resultant inflammatory response provides immediate antiviral immunity via activation of antiviral type and interferon (ifn) pathways leading to an upregulation of inflammatory cytokines such as interleukin (il- ) and il- β, further recruiting neutrophils, other innate immune cells and stimulating anti-sars-cov- adaptive memory t cells and b cells ( ) (figure ) . there is still much to understand about the immune response to sars-cov- and the immunological differences in those with mild as compared to severe infection. emerging data suggest that the innate response to sars-cov- is aberrant ( , ) . for example, the early type i and iii interferon responses are relatively suppressed by sars-cov- , an immune evasion strategy employed by the virus, leading to early failure to control the virus. furthermore, uncontrolled local inflammation, or what has been described as "cytokine storm, " leading to tissue damage with inflammatory cell infiltration and the acute respiratory distress syndrome (ards) is thought to characterize late stage, severe manifestations of covid- ( ) ( ) ( ) . for example, patients with severe covid- had higher levels of il- , il- r, il- , and tumor necrosis factor alpha (tnf-α) than those with moderate disease, the former of which correlated with clinical severity and death ( , ) . those covid- patients requiring icu admission demonstrated greater plasma levels of il- , il- , il- , granulocyte colony-stimulating factor (gcsf), interferoninducible protein (ip- ), monocyte chemoattractant protein- (mcp- ), macrophage inflammatory protein- α (mip- α) and tnf-α than the non-icu covid- patients, all supporting enhanced innate immunity in the sicker patients ( ) . of note, the il- levels described in covid- patients are a log-fold lower than those described in classic cytokine storm ( ) . furthermore, il- is an immunosuppressive cytokine suggesting dysregulated immune homeostasis rather than pure inflammation. upregulated chemoattractant chemokines further recruit inflammatory cells including neutrophils, macrophages, natural killer (nk) cells and t cells resulting in further immunopathology ( ) (figure ). antibodies (abs) produced by activated b cells play a key role in anti-viral immunity via several mechanisms including viral neutralization, antibody-dependent cellular cytotoxicity (adcc), antibody-dependent cellular phagocytosis (adcp), and antibody-dependent complement activation (adca) (figure ) . it is thought that generation of high levels of neutralizing antibodies (nabs) against sars-cov- are required for a successful human vaccine ( ) . however, some patients recover without producing high levels of nabs and those with severe disease may experience an early rise in nabs ( ) . nevertheless, most vaccine efforts are focused on the induction of nabs to the s protein in order to block attachment of rbd to the ace receptor on host cells ( ) . as mentioned before, this domain is hidden in the s protein's prefusion state, presenting challenges to the success of rbd-based vaccines ( ) . for this figure | key components of the innate immune response to sars-cov- . antigen presenting cells (apcs), such as monocytes, macrophages, and dendritic cells (dcs), recognize pattern associated molecular patterns (pamps) expressed by sars-cov- via their pattern recognition receptors (prrs), such as toll-like receptor (tlr) and . this activates intracellular signaling pathways leading to the expression of type and interferons (ifns), which in turn activate innate immune cells to produce pro-inflammatory cytokines and chemokines. this leads to an influx and activation of neutrophils, further apcs and other innate immune cells, such as natural killer (nk) cells. reason, some groups have focused on eliciting nabs to the less immunogenic s subunit of the spike protein ( ) and sars-cov- vaccines based on other antigens, including the n protein, are also being developed. a recent report from the us describes outcomes among , covid- patients, many of whom had critical illness, transfused with the plasma from people who have recovered from covid- (convalescent plasma [cp]) ( ) . early cp infusion within days of illness had a lower -day mortality than those treated after or more days ( . % vs. . %). in this uncontrolled study, those who received higher levels of igg abs in the transfused plasma had a better mortality outcome. these data, alongside results from another uncontrolled study showing recovery in severely unwell covid- patients treated with cp, lends support to the notion that naturally acquired abs can be protective ( ) . however, other plasma factors such as cytokines, defensins and other non-specific abs may also play a protective role in these studies ( ) . sars-cov- -specific nabs recovered from infected humans also protected syrian hamsters and rhesus macaques in challenge studies ( , ) . ideally, a sars-cov- vaccine would induce long-lasting abs, but it is not yet known how long specific abs persist in sars-cov- infected individuals or how long they would persist after vaccination. indeed, several recent studies indicate rapid waning of sars-cov- nabs in some individuals following natural infection, although others maintained high levels to - days ( , ) , raising concerns about the persistence of nabs post-immunization; whether nabs plateau or continue to decline over time is yet to be determined. by contrast, nabs to sars and mers have been detected up to - years after infection in human survivors ( ) and a recent study reports nabs years after sars infection, suggesting that long-lasting coronavirus-specific nabs can be induced in some instances ( ) so hopefully ab mediated immunity to sars-cov- will be equally long-lasting. importantly, class-switched igg memory b cells to s and rbd have been demonstrated in covid- patients confirming the generation of b cell memory which can provide a rapid recall response on subsequent sars-cov- exposure ( ) . furthermore, we do not yet know what level of nabs are required for protection ( ) . the standardization of a range of assays to support vaccine studies, such as viral neutralization assays, to enable comparison of different vaccine candidates in different populations will be key to facilitating vaccine development, an issue which represents a current focus of the who ( ). t cell adaptive immunity: protection, immune suppression or disease enhancement? processing and presentation of sars-cov- epitopes by antigen presenting cells (apcs) via human leukocyte antigen (hla) class i leads to activation of naïve cd + t cells which differentiate into cytolytic effectors (cytotoxic t lymphocytes [ctl]) that kill virus-infected cells. activation of t helper (th ) cd + t cells via viral peptides presented on hla class ii alleles further enhances the cd + t cell response, while hla class figure | key components of the adaptive immune response to sars-cov- . the adaptive immune response is activated following viral uptake and antigen processing by a range of apcs. the apcs present viral antigen to b cells which then differentiate into antibody producing plasma cells. the neutralizing antibodies (nabs) then bind to key viral proteins, such as the spike protein, and neutralize their activity. other ab-mediated antiviral functions include antibody dependent cellular cytotoxicity (adcc), antibody dependent cellular phagocytosis (adcp), and antibody dependent complement activation (adca). cytotoxic cd + t cells kill virally infected cells via the production of granzymes and perforin and the expression of fas ligand (fasl), all of which mediate cellular apoptosis. a series of cd + t cell populations are involved in the adaptive cellular response to sars-cov- . follicular helper t cells (t fh ) and th cd + t cells both provide help for b cell antibody production. th and th cd + t cells are also thought to play a role in the inflammatory response and viral killing. cd + regulatory t cells have been implicated with an immunoregulatory role in sars-cov- infection via the production of anti-inflammatory cytokines and contact-mediated cellular suppression. whether cd + tregs and bregs play a role is not currently known. ii-restricted follicular helper (t fh ) and th cells enhance virusspecific antibody production (figure ) . effective viral clearance therefore requires a combination of cd + ctl and cd + t cell mediated enhancement of b cell and cd + t cell responses ( ) . pro-inflammatory th also play a role as evidenced by their high frequency in lung biopsy specimens of covid- patients ( ) (figure ) . severe viral disease, associated with an over exuberant t cell response, leads to local inflammation and toxicity, thus activation of immune modulatory factors or checkpoints is crucial in limiting the damage. indeed, regulatory t cells (tregs) play a key role in the control of inflammation and immunopathology in other coronavirus infections ( ) , but this has yet to be investigated for sars-cov- (figure ) . most activated t cells subsequently apoptose and die, but some persist and provide long-term virus-specific t cell immunity. natural infection with sars-cov- induced long-lasting memory t cells up to years after infection ( ) and memory t cells persisted to years after mers infection ( ) , supporting the potential for long-term persistence in sars-cov- infection. these should provide protective immunity on subsequent exposure to the pathogen, the ultimate goal of an effective vaccine. severe covid- patients exhibit immunological features associated with t cell anergy or exhaustion. for example, the lungs of severe covid- patients contain high levels of cd + t cells expressing classic markers and genes of exhaustion ( ) , while patients who have recovered from moderate or severe covid- seem to have robust memory t cell formation ( ) . these infiltrating cells also express high levels of markers of activation and cytotoxicity. other studies have similarly demonstrated that more activated and, in some cases, more exhausted t cells develop in symptomatic covid- patients compared to levels during the prodromal stage ( ) . those who experience mild-moderate disease maintain their lymphocyte counts and have more polyfunctional t cells; while studies in those with severe disease are contradicting, reporting both lower and higher cd + t cell cytotoxicity ( ) . severe disease is associated with profound lymphopaenia lending support to the theory that the disease also causes immunosuppression, although lymphocyte trafficking to the site of infection may also be responsible ( ) . a biphasic t cell response has therefore been proposed, characterized by an early cd + cytotoxic t cell response to control the virus followed by t cell exhaustion and depletion as a result of viral persistence over many weeks in some patients. this model could account for poorer outcomes in the elderly who have diminished t cell repertoires and better outcomes in children who have a diverse and abundant naïve t cell pool ( ) . nevertheless, the likely early protective role for cd + and cd + t cells in sars-cov- clearance suggests that their induction should be exploited in sars-cov- vaccine development strategies. no vaccine is licensed or available for sars or mers, however, considerable preclinical development studies have been performed. a range of vaccine development approaches were explored, including live attenuated vaccines and inactivated vaccines (such as inactivated whole virus), soluble protein vaccines, viral vectors, nanoparticles, and dna vaccines; most of these platforms are also being utilized for sars-cov- , as discussed below ( , ) . most of the sars and mers subunit candidate vaccines have targeted the s glycoprotein and been examined in studies conducted in animal models, although n, m, and e protein-based vaccines have also been tested ( ) . however, the fact that animal models, including mice and nonhuman primates, display only limited clinical manifestations of sars and mers, that it is not usually lethal, severely limit the translation of results into humans. to overcome this, animal adapted strains and transgenic animal models of severe disease have been developed ( , ) . in virus challenge studies in animals, high titers of nabs to both sars-cov- and mers virus correlated with protection ( , ) . relatively few studies have investigated the protective role of t cells, and while some correlate cd + and cd + t cell responses with protection, adoptive transfer and t cell depletion assays in mice suggest they are not necessary for protection in mice immunized with a dna-based sars vaccine ( ) . furthermore, vaccination of mice with sars and mers candidate vaccines has been shown to result in th lung pathology with eosinophilic infiltration following wild-type virus challenge ( ) ( ) ( ) ( ) . this immunopathological effect has been associated with whole virus vaccines with and without adjuvant and linked with responses to the n protein in particular; s protein-based vaccines may have a lesser effect. an n protein-based dna sars vaccine also elicited a delayed type hypersensitivity reaction in mice, further cautioning against using this protein for sars-cov- vaccines ( ). furthermore, s-based vaccines appear more immunogenic and protective in these studies and the duration of protection has been shown in challenge studies to last - months in mice immunized with a vesicular stomatitis virus (vsv)-based sars vaccine and animals immunized with dna and/or protein-based mers vaccines ( , ) . of note, in the former study, protection against viral challenge was complete in younger mice but limited in senescent mice, highlighting potential issues with successful vaccination in older individuals ( ) . the only sars vaccines to reach human phase clinical trials were based on inactivated virus, soluble s glycoprotein and dna approaches ( ) . mers vaccines tested in human phase clinical trials include a dna-based vaccine alone ( ) ; dna in combination with adenovirus or modified vaccinia ankara (mva) viral vectors as a prime-boost strategy ( , ) ; and a chimp adenovirus vaccine ( ) . efficacy against disease has not been tested in humans since it has not been deemed ethical to perform challenge studies with lethal viruses for which there is no effective treatment. the first sars-cov- genome was isolated from patients with pneumonia from wuhan, china in early and identified as a novel human coronavirus ( , ) . since that time, multiple viruses have been isolated and sequenced from patients throughout the world providing an understanding of the evolutionary capacity and diversity of this pathogen ( ) . structural genomics and molecular interaction analysis (interactomics) can be used to rapidly identify putative functional sites, facilitating rational vaccine design by identifying potential b and t cell epitope regions of key viral proteins ( ) . several groups have used computational programs and immunoinformatics to predict cd + and cd + t cell and b cell epitopes across multiple sars-cov- antigens from pathogen sequence data to design novel sars-cov- vaccines ( , ) . the genetic diversity and stability of these regions over time can then be characterized, and cross-reactivity predicted and examined to ensure a vaccine provides cross-strain immunity. the s gene of human coronaviruses is well-known to undergo genetic drift which could compromise sars-cov- vaccines based on this region ( , ) . reassuringly, comparisons between the sequences of known sars-cov- b and t cell epitopes from the n and s proteins have been shown that some of the epitopes map identically to proteins from sars-cov- ( ). importantly, these epitopes showed no mutations among available sars-cov- sequences suggesting that they reside in stable parts of the s protein. the t cell epitopes also covered a broad range of hla alleles and could therefore be future targets for vaccine design. one issue with this approach is that the epitopes will be specific for human hla alleles, making it difficult to test them for protective efficacy in animal models. one way to overcome this problem is to use humanized mouse models which have functional human immune systems ( ) , bearing in mind that mice can also be modified to express the ace receptor ( ) . also, there are often animal homologs of human cd and cd t cell epitopes so it may still be possible to screen for efficacy. however, it should be borne in mind that due to the urgent need to develop an effective sars-cov- vaccine, animal challenge data are not a mandatory pre-requisite to progression to clinical trials; nevertheless, ideally these should be conducted as part of the vaccine development strategy. the recent development of a laboratory-adapted sars-cov- strain that is pathogenic in mice, provides an ideal animal challenge model for testing the efficacy of sars-cov- candidate vaccines in mice ( ) . vaccine technology has been advancing rapidly and there is a plethora of approaches to constructing sars-cov- vaccines. these range from the original technique employed centuries ago in the smallpox vaccine development of using modified or killed whole organism, to protein and peptide-based vaccines, nucleic acid dna and rna vaccines and nanoparticle constructs. each approach has unique advantages and disadvantages including varying time to development and scalability, cost, stability, as well as anticipated safety and immunogenicity profiles; all approaches are represented among preclinical and clinical trials ( - ) (figure ). some vaccine candidates, particularly protein-based vaccines, require an adjuvant to boost their immunogenicity ( ) . to date, very few adjuvants have been licensed for human use since the original adoption of aluminum salts (alum) from the early s, which bias immunity toward a th response ( ) . several of the newer adjuvants consist of toll-like receptor agonists to stimulate innate immunity combined with alum, such as as which is based on the tlr ligand monophosphoryl lipid a (mpl). oil-in-water emulsion adjuvants including mf and as are stronger adjuvants than alum and have been adopted in several licensed influenza vaccines. the adjuvant as , present in the malaria vaccine rts,s, combines mpl and a saponin qs- ( ) . a wide variety of adjuvant approaches are being taken with the various sars-cov- vaccines in development, including those mentioned above, some of which are described in the relevant sections below ( ) . many vaccines are given via the intramuscular route, including the majority of the current covid- vaccines in development ( table ) . however, since sars-cov- causes infection via the respiratory tract, a vaccine targeting the mucosal immune system might be preferable ( ) . mucosal vaccines have the advantage of being needle-free and thus practical for mass-administration, but immune tolerance induction can be an issue. the formulation, including the use of mucosal adjuvants, is therefore important to ensure stimulation of adequate local and systemic immunity ( ) . several groups have developed mucosal sars-cov- vaccines. a deoptimized live attenuated whole virus vaccine is being developed for intranasal (i.n.) administration, and several adenovirus-based vaccines will also be administered via the i.n. route ( table ). an oral probiotic pill-based sars-cov- dna vaccine (bactrl) is also planned for clinical trials ( table ) . the sublingual route is also considered an attractive route for inducing mucosal immunity ( ) . an alternative approach is to combine parenteral vaccines with adjuvants such as retinoic acid and caf which are known to induce protective iga mucosal responses ( , ) . researchers are also investigating self-administered mechanisms for sars-cov- vaccines to overcome the need for a healthcare worker to administer the vaccine. for example, inovio tm have developed a novel hand-held cellectra r intradermal delivery device which uses a brief electrical pulse to reversibly open skin cell pores to allow the entry of their ino- dna plasmid vaccine ( ) ( table ). the university of pittsburgh school of medicine has developed an intradermal microneedle patch that can deliver sars-cov- protein antigens, either s or rbd (pittcovacc), through the skin which will enter human trials soon ( ) ( table ) . this approach delivers antigen and danger signals to the high-density dermal apcs stimulating a highly effective immune response, even in the absence of adjuvant. it requires only low doses of antigen thus reducing production costs. indeed, this method induced more potent s -specific nabs in mice than the traditional needle injections, with or without the inclusion of tlr ligand sequences ( ) . the development of live attenuated vaccines requires the organism to be modified via passage multiple times under unique conditions in the laboratory until it loses enough key virulent protein and peptide subunit vaccines are usually combined with an adjuvant in order to enhance immunogenicity. the main emphasis in sars-cov- vaccine development has been on using the whole spike protein in its trimeric form or components of it, such as the rbd region. multiple non-replicating viral vector vaccines have been developed, particularly focused on adenovirus; while there has been less emphasis on the replicating viral vector constructs. nucleic acid-based approaches include dna and mrna vaccines, often packaged into nanocarriers such as virus-like particles (vlps) and lipid nanoparticles (lnps). nanoparticle and vlp vaccines can also have antigen attached to their surface or combined in their core. the immune cell therapy approach uses genetically modified sars-cov- -specific cytotoxic t cells and dendritic cells expressing viral antigens to protect against sars-cov- infection. each of these vaccine approaches has benefits and disadvantages in terms of cost and ease of production, safety profile and immunogenicity, and it remains to be seen which of the many candidates in development protect against factors so as to not cause disease, but retains its ability to replicate in the vaccine recipient, and stimulate a robust immune response that protects against infection ( ) ( ) ( ) . these vaccines tend to be highly immunogenic, do not require an adjuvant and provide long-lasting immunity. however, they are usually contraindicated in those immunocompromised or pregnant. it is difficult to develop a live attenuated sars-cov- vaccine quickly because of the time and knowledge required to ensure that it is suitably attenuated and all virulence factors removed. reverse genetics can be employed for the rational design of the ideal attenuated sars-cov- vaccine virus, for example by determining key virulence factors, as has been done for other coronaviruses ( ). long-term maintenance of consistent live vaccine stocks is also problematic ( , ) . despite the challenges, three codon-pair deoptimized live attenuated vaccines are currently undergoing pre-clinical testing, but none have yet progressed to clinical trials ( ) ( table ) . these three candidates are being developed by codegenix/serum institute of india, indian immunologicals ltd/griffith university and mehment ali aydinlar university in turkey, respectively ( ). this approach relies on using a single virus strain which may not cross-protect against other strains, particularly as the virus continues to spread worldwide and mutates as selection pressure increases once immunity becomes more widespread. inactivated vaccines are produced by growing the virus and then killing or "deactivating" it so that it can no longer replicate. the whole virus can be used, although alternative approaches include splitting the virus with a detergent to disrupt it or purifying antigenic components to create a subunit vaccine ( ) . these vaccines are safer than live-attenuated vaccines but less immunogenic and often require an adjuvant. there are currently four inactivated vaccines in human clinical trials and a further nine in the preclinical stages of development (tables , ). an alum adjuvanted purified formaldehyde inactivated whole virus vaccine developed by sinovac biotech., called picovacc, has been shown to be immunogenic in mice, rats and non-human primates ( ) . it induced nabs to both vaccine and non-vaccine strains and provided partial or complete protection of macaques in challenge studies and has now entered human clinical trials in china ( pre-clinical data confirming induction of high levels of nabs in several animal models and protection against intra-tracheal sars-cov- challenge in rhesus macaques after doses of vaccine ( ) . the institute of medical biology/chinese academy of medical science has also developed an inactivated whole virus construct that is in a phase / trial; and bharat biotech international ltd also has one in a phase / trial ( table ) . rather than using the whole or a split fragment of the virus as the vaccine, antigenic proteins (or peptides) of the virus can be generated using recombinant technology in the laboratory instead ( ) . this is the most popular approach in the sars-cov- vaccine development field, with protein/peptide constructs in preclinical trials and that have entered clinical trials ( table ) . they are relatively simple vaccines to make and thus cheap to produce. a major advantage over whole virus vaccines is their relative safety since unnecessary reactogenic components such as lipopolysaccharides and toxins can be excluded. however, protein/peptide vaccines often require adjuvants and multiple doses in order to stimulate an adequate immune response. peptide vaccines usually consist of - amino acid b cell and t cell epitope regions of key antigens allowing for a precise and targeted immune response. peptidebased vaccines have been developed as candidates against several viral pathogens, but few have been licensed except for use in veterinary practice ( ) . this is in part due to their inherent limitations including a narrow breadth of immune response, potential for incorrect confirmation for epitope recognition, lack of b cell receptor cross-linking for b cell epitopes, hla restriction for t cell recognition, failure to induce cross-reactive immunity against different viral strains and failure to elicit longlasting immunity. the use of whole proteins in vaccines broadens their immunogenic potential, however the protein can also lose its native structure and thereby lose immunogenicity. in addition, as knowledge of the immune response to sars-cov- is still in its early stages, so too is the understanding of precisely what may be the most conformationally optimal, immunogenic and safe protein or peptide to utilize; this will be discovered as vaccine candidates using this approach are examined in clinical trials. the majority of sars-cov- candidate vaccines in development are based on the s protein or its rbd region (figure ) ( ) . indeed, five of the current protein/peptide vaccine candidates in clinical trials target the full s protein and the other two target rbd only ( table ) . some novel technologies have been employed to overcome the issue of the s glycoprotein losing its immunogenic conformational form. the university of queensland has developed a molecular clamp technique that uses proteins to stabilize the s protein in its coiled shape. this seeks to ensure that functional nabs are produced that will tightly bind to virus in its native form and thus prevent cell entry ( ) ( table ) . other groups have focused on using powerful adjuvants to ensure immunogenicity, for example novavax's recombinant s protein sars-cov- nanoparticle vaccine, nvx-cov , combined with an adjuvant called matrix-m tm . this is a saponin-based adjuvant that enhances apc recruitment and action and stimulates high levels of nabs and cell-mediated immune responses ( ) . the s protein in nvx-cov is present its native trimeric form and has been shown to stimulate high levels of nabs in mice and baboons in preclinical studies. primary phase clinical trial results have just been reported from a randomized, observer-blind, placebo-controlled trial of nvx-cov conducted in healthy adults ( ) ( table ) . safety and immunogenicity were assessed for doses of vaccine ( and µg) with (n = ) or without (n = ) the matrix-m tm adjuvant (a µg dose). a strong correlation was observed between nab titers and anti-spike igg to s protein with the adjuvanted, but not the unadjuvanted, vaccine. levels were comparable to those in convalescent sera (r = . ). both and µg adjuvanted doses elicited responses of similar magnitude and every participant seroconverted by both assays. t cell responses measured in participants showed that nvx-cov /matrix-m induces antigen-specific polyfunctional cd + t cell responses (ifn-γ, il- , and tnfα) in response to spike protein stimulation; there was a strong bias toward this th phenotype. the safety data are discussed in section comparative safety and tolerability results for current sars-cov- vaccine candidates in clinical trials and this vaccine is now progressing to phase trials. clover biopharmaceuticals's protein trimer tag© vaccine is combined with as adjuvant; the uq vaccine uses mf adjuvant; the vaxine pty ltd. s protein vaccine has a special advax tm adjuvant; and the medigen s based construct is combined with the adjuvant cpg ( table ) . generex biotechnology, in partnership with epivax, have developed a sars-cov- vaccine (epv-cov ) based on li-key technology with undisclosed sars-cov- peptides ( ). this uses synthetic peptides chemically linked to the -amino acid ii-key to ensure robust immune activation of th and th cd + t cells in particular, which in turn facilitate antibody production ( , ) . multiple viral vectors have been used for vaccine delivery due to their ability to infect cells and deliver the gene product encoding antigenic proteins for production inside the host cell following administration, usually via the intramuscular (i.m.) route ( ) . vector viruses are usually genetically modified to reduce their virulence and render them replication defective. adenoviruses and poxviruses are the most widely used non-replicating viral vectors, but many other viruses can also be adapted for vaccine delivery. replicating viral vectors, such as measles virus and vesicular stomatitis virus (vsv), can also be used for sars-cov- vaccine design; they mimic natural infection, rendering them self-adjuvanting and therefore more potent, and can be used in lower doses ( ) . while subunit vaccines are more focused on induction of humoral immunity, viral vector vaccines are able to induce robust cell mediated immunity (cmi) in addition to abs and have been shown in animal models to protect against challenge with pathogenic coronaviruses ( ) . a modified vaccinia ankara (mva) vector expressing a mers-cov recombinant protein induced t cell responses and nabs in mice, while a rabies virus-based sars-cov- vaccine protected mice against sars-cov- challenge ( ). human adenoviruses are the most common non-replicating viral vectors being used for sars-cov- vaccine development; constituting of the in clinical trials and of the in preclinical development ( table ) . the three in clinical trials include cansino's ad -ncov, janssen's ad covs ( ) and gamelaya's ad -based gam-covid-vac lyo vaccine ( table ) . all three encode the s protein. while adenovirus vectors are well-tolerated and highly immunogenic in most people, preexisting immunity to the viral vector can hamper the induction of immunity, particularly after repeated doses. animal adenoviruses can be used as vectors instead of human ones to overcome this problem, which is the approach used for the oxford university chimpanzee adenovirus-based candidate sars-cov- vaccine chadox ncov- (also called azd ) and the simian adenovirus-based reithera vaccine grad, both of which are in clinical trials ( table ) . anti-vector immunity can still develop after the first dose of vaccine even if a simian adenovirus vector is used. despite the concern about anti-vector immunity, of the adenovirus-based sars-cov- vaccines in clinical trials are planned to be given as a single dose, whereas almost all the other vaccines in clinical trials require at least doses ( table ) . phase and trials have now been completed and published for the cansino (ad -ncov) and oxford (chadox ncov- ) , the safety aspects of which will be discussed later in this article. the ad -ncov randomized, double-blind, placebo-controlled phase trial results involving volunteers ≥ years of age showed > % seroconversion by rbd-elisa, but only % seroconversion of nab responses in the higher dose group and % in the lower dose group after a single immunization ( ) . fifty two percent of volunteers had pre-existing anti-ad nabs, and those with higher anti-ad immunity had approximately half the rbd-specific elisa and sars-cov- nabs as compared to those with low pre-existing anti-vector immunity, supporting interference with vaccine immunogenicity. preliminary findings from a phase / , single-blind, randomized controlled chadox ncov- vaccine trial conducted in , healthy volunteers aged - years induced nabs in % of participants after a single dose of vaccine, rising to % after a booster dose ( ). neutralization titers were comparable to those observed in convalescent plasma from people who had recovered from covid- . both vaccine constructs elicited t cell responses which peaked at days post-immunization, as determined by interferongamma (ifn-γ) enzyme linked immunospot (elispot) assay ( , ). day responses appeared higher with chadox ncov- (median per million peripheral blood mononuclear cells [pbmc] ) as compared to the highest dose used of ad -ncov (mean per million pbmc) in the phase trial ( ); ad -ncov elicited a median of - sfu/million pbmc at day in the phase trial ( ). intracellular staining confirmed production of ifn-γ, tumor necrosis factor (tnf) and interleukin- (il- ) by both cd + and cd + t cells following ad -ncov immunization, with more cytokine detected in cd + t cells as compared to cd + t cells ( ) . institute pasteur replicating viral vector vaccine candidate tmv- , based on measles virus, is the only viral vector vaccine that has entered phase clinical trials to date ( table ) , but there are more at the pre-clinical stage of development. these include each based on influenza virus or vsv, more measles virusbased candidates, and each based on avian paramyxovirus, horsepox (called tnx- ), newcastle disease virus, and yellow fever virus ( table ) . the majority target the s protein or rbd, although one measles virus-based candidate also targets the n protein. two of the influenza-based candidates are designed for intranasal administration. a live attenuated yellow fever vector-based vaccine (yfs ) expressing the prefusion form of the s protein recently showed that a single dose protected most hamsters in a sars-cov- challenge model ( ) . nucleic acid-based vaccines (dna or mrna) offer a costefficient and scalable approach to sars-cov- vaccine development ( ) . the major nucleic acid-based approaches are described below. dna-based vaccines are stable and safe to handle. however, while there are multiple dna vaccine constructs targeting numerous viral infections in animal and human studies, to date none have been licensed for human use. naked dna can be injected and taken up by apc or apc can be directly transfected with plasmid dna encoding the target antigen. subsequent expression and presentation of the target antigen leads to the induction of antigen-specific cd + and cd + t cells and antibody production. however, dna vaccines tend to be poorly immunogenic, necessitating various strategies to improve immunogenicity such as the use of viral promotors, immunostimulatory sequences and adjuvants. nanocarriers such a virus like particles (vlps) can also be used to improve dna delivery, avoid its degradation and be immunostimulatory. there are safety concerns about potential integration into the host dna causing dysregulated gene expression although the risk of this is thought to be very low. a series of animal studies with sars-cov- nucleic acidbased vaccines have shown induction of nabs and cellular immunity, with partial protection against turkey cov challenge ( ) and vaccine-induced ab-mediated protection to the s protein in mice ( ) . twelve dna-based sars-cov- vaccines are currently in preclinical development and more have entered phase / clinical trials ( table ) . two dna-based candidates in clinical trials are administered i.m., whereas the other are administered via the intradermal (i.d.) route ( table ) . inovio pharmaceuticals is testing their cellectra r electroporation i.d. delivery device to administer their s protein-based dna plasmid vaccine ino- which induces nabs and t cells in mice and guinea pigs ( ) ( table ). an alternative approach currently in pre-clinical trials is being taken by symvivo with their dna b. longum vaccine bactrl which is administered as an oral pill ( ) . messenger rna (mrna) vaccines consist of an rna strand that codes for a target antigen ( , ) . the two main types are those packaged and delivered in non-replicating form or those packaged with other rna as an in vivo replicating vaccine. they offer a promising alternative to conventional vaccine approaches due to their high potency, capacity for rapid development and potential for low-cost manufacture, which could prove crucial for global accessibility for a covid pandemic vaccine. they also have a good safety profile, since they are not made with pathogen and do not integrate into host dna. several challenges encountered by mrna vaccines include the need for packaging, for example into particles or liposomes, as naked rna is otherwise rapidly broken down and the need for freezing or refrigeration for wide scale delivery. there are currently rna-based vaccines in pre-clinical trials and have entered clinical trials ( table ) . the clinical trial candidates include two lipid nanoparticle encapsulated rna sars-cov- vaccines that have already progressed to phase trials ( table ) . moderna/niaid have published preliminary results from their phase dose-ranging, safety and immunogenicity trial of doses of mrna- encoding the s protein conducted in healthy adults aged - years ( ) . the s protein in mrna- is stabilized in its prefusion state by proline substitutions in the s subunit; and the lipid nanoparticle coat consists of lipids. all participants developed rbd abs by elisa and sars-cov- -specific nabs by neutralization assay after doses in the upper range of those who have recovered from covid- (convalescent serum). t cell responses measured by intracellular staining for th (tnf, il- , ifn-γ) and th (il- , il- ) cytokines following stimulation with an s protein peptide pool showed a th cytokine bias and low-level cd t cell responses. an interim report from the phase / dose-ranging study of doses of biontech's mrna s protein vaccine candidate bnt administered to adults aged - years elicited rbdbinding igg and sars-cov- nabs in the order of . - . fold that of convalescent human plasma. t cell responses were not reported in this paper. this vaccine encodes trimerized rbd which is modified by adding a "foldon" trimerization domain to increase immunogenicity ( ) . both mrna- and bnt are now in phase clinical trials in adults ≥ years ( table ). the safety data for both will be reviewed in a later safety section. the remaining rna-based candidates in clinical trials include three in phase and one in phase / ( table ) . the imperial college london lnp-encapsulated rna vaccine consists of an s protein-based self-amplifying rna construct (lnp-ncovsarna); designed because sarna vaccines induce a more stable dna product and are more immunogenic than conventional nucleic acid vaccines ( ) ( table ) . nanoparticle-based vaccine approaches have received increasing interest in recent years due to their good safety profile and high immunogenic potential, with an ability to efficiently target dendritic cells (dcs) for processing and presentation, providing a clear advantage over less immunogenic dna and rna vaccines ( ) . materials used to construct nanoparticle vaccines include self-assembling viruses (virus like particles [vlps]), lipids (as liposomes), proteins, metals (e.g., gold) and polymers which also act as their own adjuvant ( ) . many nanoparticles are highly stable and less prone to degradation than other constructs such as "naked" protein, peptide, dna and rna vaccines ( , ) . selected antigens, which may be proteins or nucleic acid, can either be attached to the surface of the nanoparticles or combined in the particle core. they can be synthesized in a variety of shapes and sizes to induce robust innate as well as adaptive immunity. other modifications include altering the nanoparticle surface to target certain cells or enhance immunogenicity and packaging with tlr ligands and other immune modulators. one of the most popular approaches for viral vaccine development is engineering vlps consisting of self-assembled viral membrane in a monomeric complex which display the viral epitopes but lack multiple key viral components, ensuring they have no replication capacity ( ) . the widely used human papillomavirus (hpv) vaccines are an example of this approach. a nanoparticle polypeptide vaccine displaying sars-specific b cell epitope repeats from the c terminal heptad repeat region of the s protein in its native coiled formation was shown to elicit nabs in mice ( ) . a number of the protein and nucleic acid-based sars-cov- vaccine candidates use nanocarriers to package the vaccine product to improve stability and ensure efficient antigen processing ( ) . as mentioned above, novavax's full length s protein construct nvx-cov is a nanoparticle vaccine and the majority of mrna-based sars-cov- vaccines are packaged lnps, including mrna- and bnt . there are sars-cov- vlp vaccines in preclinical development and just one in a phase clinical trial ( table ) . the latter vaccine developed by mendicago inc. consists of plant-derived recombinant vlps made by transfecting viral genes into the cell nuclei of leaves permitting transient expression of viral proteins which form into vlps which are extracted and purified for clinical use ( ) ( table ) . sars-cov- vaccine approaches based on car-t cell concepts are also being tested in clinical trials. the shenzhen genoimmune medical institute in china is using a lentiviral vector system to create viral minigenes which express viral proteins (s, m, e and n proteins) and immune modulatory genes (p polyprotein protease) to modify dendritic cells (dcs). the lv-dc presenting sars-cov- specific antigens will activate cytotoxic t cells. novel dc (lv-smenp dc) and antigenspecific cytotoxic t cell vaccines are currently being tested by s.c. injection or i.v. infusion in a phase / multicenter therapeutic clinical trial in participants ranging from months to years of age ( ) ( table ) . the same company has also developed artificial dcs expressing sars-cov- mini-genes for various viral proteins which are being tested in a phase clinical trial as a s.c. injection in sars-cov- infected individuals in the same age range ( table ) . autologous dcs expressing sars-cov- antigens have also been developed by aivita biomed inc. in usa and are being tested as a s.c. administered vaccine in phase clinical trials in healthy adults > years of age ( ) ( table ) . whilst not suitable for large-scale delivery and use, immune cell therapy might be used in the context of pre-emptive therapy in high-risk patients such as the elderly and immunocompromised. there is increasing evidence that immunization with live vaccines can improve survival against non-vaccine targeted infections, a phenomenon termed "non-specific, " "off-target, " or "heterologous" effects of vaccines ( , ) . some of the best evidence comes from studies of immunization with the tuberculosis vaccine bacillus calmette-guérin (bcg); for example, bcg has been shown in three randomized trials to markedly reduce all-cause mortality in low birthweight neonates ( ) . bcg causes epigenetic modification of innate immune cells (monocytes and natural killer (nk) cells) thereby enhancing innate responses in a process called innate immune training ( , ) . experiments in both murine models and humans have further shown that bcg protects against certain viral pathogens ( ) . reports that people living in countries where childhood bcg is routinely given have lower covid- mortality have been posited as further evidence for protective effects of bcg ( ). however, these observations are fraught with confounders, including difficulty ascertaining covid- infection rates in these countries, the time the virus first entered the country and differences in national control strategies ( ) . furthermore, other studies contradict these findings, such as a geographic regression discontinuity analysis along the east and west german border showing that the covid- infection rates do not vary according to the bcg vaccination strategies employed during the cold war ( ) ; and a study from israel showing that sars-cov- infection rates in adults did not differ by bcg at birth status ( ) . even if childhood bcg immunization does not protect against covid- , a bcg dose as an adult may provide some short-term protection. indeed, it has been widely postulated that the nonspecific beneficial effects of bcg might protect against covid- , resulting in an explosion of interest in this vaccine as a protective strategy in recent months ( , ) . this has led to a number of clinical trials being established in north and south america, australasia, africa and europe, all testing the ability of bcg vaccine to protect against sars-cov- infection, mostly in high-risk exposed healthcare workers ( ) ( table ) . these trials collectively aim to recruit > , participants, with the largest trial of , healthcare workers currently recruiting across multiple sites in australia and europe. non-specific protective effects of live vaccines have also been postulated for oral polio vaccine (opv) ( ) and measles vaccine ( ) . as a result, a study of the protective effects of opv against covid- is due to commence in usa in june ( ) , and a clinical trial of measles-mumps-rubella (mmr) immunization and covid- protection is being conducted in egypt ( table ) . a consistent feature of the off-target effects of live vaccines has been their sex-differential nature; females often showing greater susceptibility to these immunomodulatory effects ( , , ( ) ( ) ( ) . indeed, sex differences in targeted vaccine immunogenicity and adverse events have also been widely described ( , ) . this raises the possibility that any off-target protective effect of live vaccines may differ between the sexes and that the sars-cov- vaccine candidates may show sex differences in immunogenicity and reactogenicity. it is possible, but uncertain, that strategies to use these already licensed vaccines may provide a modest level of protection against covid- , including those at highest risk, such as healthcare workers. it is important to recognize, however, that data from these studies will still need to accrue whilst we are waiting for sars-cov- targeted vaccines to come through the pipeline. given the unprecedented speed at which targeted vaccines are being developed, this approach may not be necessary or required; furthermore, these existing live vaccines are not currently recommended for this indication, so should only be utilized in this context in the setting of a clinical trial. even if an efficacious vaccine is developed there are a number of immunological challenges that need to be considered. furthermore, there is the enormous challenge of mass production and rapid and equitable delivery ( table ) . it is extremely unlikely that any of the sars-cov- vaccines will be % effective; while they may not prevent becoming infected, it is hoped that they will prevent progression to severe disease. indeed, the seasonal influenza vaccine is generally about % protective against infection but does decrease disease severity and hospitalization rates ( ) . a recent study in which macaques were vaccinated with the oxford university and astrazeneca adenovirus vaccine, chadox ncov- , found that the primates were protected from sars-cov- -induced pneumonia ( ) . however, the macaques still had high levels of virus replicating in their upper respiratory tract. it is hoped that even if the vaccines do not prevent infection in the upper airways, they may reduce viral load and disease severity and in turn, the amount of virus a vaccinated person transmits to others. even a modestly efficacious sars-cov- vaccine could mitigate the severity of this pandemic and be highly beneficial in a world struggling to contain this novel virus and its devastating social and economic effects. most vaccines are tested in healthy young adult males and non-pregnant women and, if safe, they are then tested in healthy children prior to licensure. this therefore raises the issue that any vaccines may initially have less empiric data available on use in certain key vulnerable populations such as the elderly, immunocompromised groups and pregnant women. it is plausible that vaccines may be considerably less immunogenic in older and frail elderly who experience the most severe outcomes from covid- , hence the importance of adjuvants in many of the vaccine candidates, both for dose sparing and enhancing immunogenicity ( ) . efficacy in this population is also likely to vary by the type of vaccine construct and adjuvant used. this has been seen for other diseases such as influenza, where the adjuvanted and high dose vaccines are more immunogenic than the standard inactivated vaccines in the elderly ( , ) . similarly, for herpes zoster, the efficacy of the live-attenuated vaccine in the elderly is approximately % compared with > % for a subunit adjuvanted vaccine (glycoprotein e and liposomebased as b adjuvant) ( ) . thus, ensuring studies can be conducted in these priority target groups, either pre-or earlypost deployment, will be critical and special formulations may be required to ensure adequate immunogenicity. throughout life, humans are repeatedly exposed to the endemic human seasonal coronaviruses and develop human cov (hcov) antibody repertoires that can potentially cross-react with sars-cov- -specific antigens ( ) . this early immune imprinting leads to preferential expansion of cross-reactive abs when a related antigen is encountered, in this case sars-cov- ( ). this long-recognized process is called original antigenic sin (oas) ( ) and is known to occur with several common viruses, such as influenza and respiratory syncytial virus ( , ) . oas can either lead to a less effective immune response or cause enhanced immunity and immunopathology ( , , ) . oas is one of the mechanisms responsible for the phenomenon of antibody dependent enhancement (ade) of immunity which is thought to occur in covid- , whereby non-neutralizing cross-reactive abs against other coronaviruses enhance host cell entry and viral infectivity and worsen disease severity ( , , ) . this was seen to occur in cats vaccinated against feline infectious peritonitis coronavirus ( ) ( ) ( ) and certain sars and mers vaccine platforms also appear to have shown worsened immunopathology in animal challenge studies compared with placebo ( ) . recently, it was shown in vitro using human cells that nabs to coronavirus s protein can also trigger ade by causing a conformational change of the s protein ( ) . antigen-experienced older individuals are more susceptible to the phenomenon of ade, while less antigen-experienced younger individuals mount more targeted antibody responses to viral neoantigens. this may in part account for the greater immunopathology of covid- in older individuals. indeed, it has been shown recently that covid- -naïve children and elderly have quite different cross-reactive sars-cov- -specific antibody signatures, which would play differing roles upon challenge with the wild-type virus ( ) . theoretically, a sars-cov- vaccine could skew the immune system toward production of less effective or even harmful cross-reactive hcov abs via these processes of oas and ade, rather than induce highly-targeted sars-cov- -specific abs as intended ( ) . indeed, anti-spike abs taken from critically unwell covid- patients with severe lung injury skewed macrophages in vitro into a pro-inflammatory phenotype, implicating the abs in driving the surge in lung-resident proinflammatory macrophages and subsequent pro-inflammatory cytokine release in the lungs ( ) . the authors attribute an aberrant igg glycosylation pattern in severe covid- patients with their more pro-inflammatory properties so hopefully the iggs induced by vaccination will not have this problem. indeed, none of the current sars-cov- vaccines in human clinical trials have reportedly caused ade to date, although the number of human subjects studied to date is still relatively small. since animal challenge data are not required to progress to clinical sars-cov- vaccine trials, the opportunity to screen for potential adverse events including ade at the pre-clinical stage of development may be missed. having said that, many of the sars-cov- vaccine candidates have undergone considerable pre-clinical testing, including challenge studies. there was no evidence of ade in pre-clinical sars-cov- challenge studies in rhesus macaques following immunization with dose of chadox ncov- ( ) or doses of the inactivated whole virus vaccine candidate bbibp-corv ( ), both of which were protective in these studies. it is postulated that basing the vaccine on the s rbd terminal subunit of the s glycoprotein should overcome this problem by inducing nabs only, although this has not been proven and, notably, only a few vaccine candidates have taken this approach. furthermore, the in vitro conformational changes causing ade described above occur in the rbd domain, so this approach may not be successful. aberrant manifestations of t cell responses were observed in the s for other viral vaccine candidates, such as inactivated vaccines against measles and respiratory syncytial virus (rsv), resulting in vaccine associated enhanced respiratory disease (vaerd) upon subsequent wild-type pathogen exposure ( , ) . a major driver of this was accentuation of th cytokines, with resultant allergic (eosinophilic) and airway hyperesponsiveness. this was compounded by another mechanism related to the conformation of the vaccine antigen, resulting in the generation of excessive non-neutralizing ab. having a high amount of binding, but not neutralizing, ab caused immune complex deposition and complement activation with local inflammation in the presence of a high viral load. it will therefore be important for vaccine candidates to mitigate the risk of vaerd by considering the t cell profile induced by vaccination, avoiding th biased cd + t cell immunity and biasing toward cd + cytotoxic t cell responses. the alum adjuvanted inactivated vaccine candidate picovacc could theoretically drive a th skewed immune response. while this was not observed in macaque studies when analyzing th and th cytokine profiles, it is not clear from the paper what samples were used to measure these cytokines, clarification of which will be crucial in vaccine safety assessment ( ) . many of the other vaccine constructs favor th immunity, as demonstrated in the published human trial results for several of the viral vector ( - ) and mrna vaccines ( ) and the novavax nanoparticle matrix m adjuvant vaccine ( ) . original antigenic sin also applies to t cell responses and therefore pre-existing cov-specific t cell memory may be enhanced by immunization with a sars-cov- vaccine leading to either suboptimal t cell responses or immunopathology ( , ) . vaccine safety assessment is integrated into all stages of the clinical development pipeline for candidate vaccines and continues once vaccines are deployed into the population. randomized controlled clinical trials examine safety, as well as immunogenicity and efficacy. decisions on the participant numbers to be included in phase studies will likely be powered on efficacy endpoints, but these studies also need to be of sufficient size and planned duration to ensure comparison of injection site, systemic and unanticipated or "unsolicited" adverse events between groups. comprehensive safety studies are particularly critical because some candidate vaccines use platform technologies that have not been examined extensively in human subjects to date, including some of the viral vectors, mrna and nanoparticle constructs, and because of the potential for enhanced disease and adverse events related to aberrant immune responses to be seen upon infection pre-and post-licensure. a list of adverse events of special interest (known as aesi) across all body systems, including immunological, cardiovascular, neurological, musculoskeletal and dermatological manifestations, have been agreed by the brighton collaboration in conjunction with cepi and the who with input from regulatory agencies and other experts, as have the associated case definitions and surveillance strategies ( , ) . listed conditions include anaphylaxis, vasculitides, myocarditis, generalized convulsions and meningoencephalitis, among many others. comprehensive data on safety are essential to ensure that the benefit:risk ratio of vaccination is favorable, to support decision-making by policy makers on wide-scale program implementation among healthy people, and to ensure that individuals are sufficiently confident to accept vaccination. some of these aesi will require post marketing (phase ) studies but are also undergoing evaluation in some of the preclinical and early phase evaluations, particularly the larger phase studies. interim phase / safety results have recently been published for two leading adenovirus-based vaccines, chadox ncov- ( ) and ad -ncov ( , ) ; two mrna based vaccine candidates, mrna- ( ) and bnt ( ) ; an aluminum adjuvanted inactivated whole virus vaccine ( ) ; and an adjuvanted recombinant protein nanoparticle vaccine, nvx-cov /matrix-m ( ) . all candidates exhibited acceptable safety profiles, and while local and systemic reactogenicity was common for the mrna, adenovirus and adjuvanted protein nanoparticle constructs, the inactivated whole virus vaccine was considerably less reactogenic ( table ) . reactions were generally mild to moderate and resolved within days. importantly, there were no serious adverse events reported for any of these vaccine candidates. pain at the injection site was a particularly common feature with the mrna vaccines (approaching % in some groups), and while it was the commonest reaction to the inactivated vaccine, it was still lower for this candidate than for the other vaccines (table ) . headache, myalgia and fatigue were the other most commonly reported symptoms for the mrna, adenovirus and adjuvanted protein nanoparticle candidate vaccines ( table ) . documented fever was relatively common for mrna and adenovirus candidates but ≤ % for the inactivated virus and adjuvanted protein nanoparticle candidates ( table ). the chadox ncov- trial underwent a protocol amendment during the trial allowing several sites to administer paracetamol prior to vaccination and hourly for h. this slightly reduced reports of pain, feeling feverish, chills, mylagia, headache and malaise but had no effect on confirmed fever. pre-existing adenovirus immunity, increased age and male sex were all associated with decreased fever following immunization with ad -ncov ( ) . in terms of blood parameters, immunization with chadox ncov- was associated with transient neutropaenia and bnt b with lymphopaenia. in the ad -ncov phase and trials reactogenicity was dose-dependent for pain, headache and fever; while it was dosedependent for almost all parameters for the mrna vaccines ( table ) . as a result of the high reactogenicity after a single dose of bnt b , it was decided not to give a second dose. interestingly, the ten participants given a booster dose of the chadox ncov- vaccine had less reactogenicity after the second dose. the opposite was found for the bnt- b mrna and nvx-cov /matrix-m vaccines for which reactogenicity was greater after the second dose ( , ) . these favorable safety results (alongside the good immunogenicity reported earlier in this article) have allowed the progression of the adenovirus and mrna candidate vaccines to phase clinical trials. whilst the sars-cov- vaccine pipeline is progressing at unprecedented speed, there is a concern that suppression of viral transmission in many countries will make evaluation of vaccine efficacy (ve) difficult, as phase studies need sufficient infection rates to compare disease incidence in vaccinated with control individuals. reassuringly, planned studies are currently expanding to higher burden countries where infrastructure to conduct complex adaptive clinical trials exists. nevertheless, the time needed to accrue sufficient data on efficacy will be a critical determinant impacting vaccine availability. in the setting of this uncertainty "human challenge models" for sars-cov- have been proposed. this methodology involves the intentional infection of research participants, providing safety data and pointing to immune correlates of protection. this approach has recently advanced vaccine development for infections like salmonella typhi (typhoid vaccine) and group a streptococcus ( , ) . the difference between these bacterial human challenge models and sars-cov- is that these pathogens have been researched for decades and have an effective antibiotic "rescue" treatment available. infecting a human with sars-cov- to test vaccine efficacy raises numerous ethical issues ( ) , including those around informed consent of the healthy volunteer, noting that while severe covid- is less common in young adults, deaths have still occurred in this age group. strict infection control and personal protective equipment (ppe) measures would also be required to limit "third-party" risk to staff co-ordinating any studies. while it is uncertain if this approach will be required, the who is progressing a framework, including key criteria, that will be required by ethical review boards in order to facilitate "human challenge" trials ( ). as noted by stanley plotkin, "extraordinary diseases require extraordinary solutions" ( ) . it normally takes decades for a vaccine to be developed and licensed for use. however, the race is on to develop a sars-cov- vaccine for worldwide distribution in an unparalleled timeframe to vaccinate the world's population and provide widespread herd immunity. this is already being facilitated by rapid progression through the normally slow bureaucratic regulatory and approval processes and unprecedented worldwide collaboration between governments, universities, pharmaceutical companies and philanthropic organizations; such efforts must continue. in addition, ensuring good public communication regarding the safety and effectiveness of the vaccine will be key to gaining public trust and acceptance of a vaccine that could be seen as rushed. additionally, having carefully designed post-marketing (phase ) surveillance is vital to detect rare or unexpected safety signals and aesi which may only be detected once the vaccine is rolled out on a mass scale. a key player in global access to a sars-cov- vaccine is the not-for-profit coalition for epidemic preparedness innovations (cepi) which is working with global health authorities and vaccine developers worldwide to support sars-cov- vaccine development ( , ) . cepi was founded in with the consensus that new and sustainable models of partnership are needed to respond to worldwide infectious diseases threats. founding members include the bill & melinda gates foundation (bmgf), wellcome trust uk, world economic forum, india department of biotechnology and the government of norway. cepi's goal is not just to advance development, but to also advance licensing, manufacturing, delivery and stockpiling of vaccines once an effective vaccine has been developed ( ). it has raised approximately $ . billion for sars-cov- vaccine development and initiated a series of partnerships in a bid to advance frontrunner candidate sars-cov- vaccines. cepi is investing across a range of vaccine technologies including novavax's nanoparticle vaccine (nvx-cov and matrix-m tm ), clover biopharma's s-trimer protein-based vaccine (scb- ), university of queensland's molecular clamp s protein vaccine, university of oxford's adenovirus vector vaccine (chadox ncov- ), inovio's dna plasmid vaccine (ino- ) and moderna's and curevac's mrna vaccines ( ) . many other government and philanthropic organizations are injecting large sums of money into the effort to develop effective sars-cov- vaccines. the european commission has registered an impressive $ billion in donations toward the development and deployment of vaccines, treatments and diagnostics against sars-cov- ( ). the us government agency biomedical advanced research and development authority (barda) has pledged almost $ million to accelerate the development of moderna's mrna- vaccine, with phase trials expected to begin soon ( ) . barda has also funded janssens's adenovirus (ad ) i.n. vaccine, astra zeneca's azd vaccine (formerly chadox ncov ), merck and iavi's rvsvg-cov and sanofi's recombinant sars-cov- protein vaccine and novavax's nanoparticle matrix m adjuvant vaccine ( ) , demonstrating the range of vaccine strategies being supported. importantly, these phase / studies are being conducted in countries with high disease burden (e.g., north and south america and south africa), so the vaccine efficacy (ve) and safety results can be obtained as rapidly as possible whilst meeting the vaccine regulators' requirements. the logistics of scaling up to produce the billions of doses required to immunize the world's population is an onerous task. it is not yet clear how long immunity will last and therefore whether booster doses or annual immunization will be required. furthermore, multiple initial doses may be needed and the vaccine may have to change as the virus evolves naturally. barda have pledged that they will scale up to million annual sars-cov- vaccine doses in the us each year under operation warp speed, while bmgf have recently awarded $ million to inovio's dna based vaccine ino- with the intention of providing over million doses by the end of . novavax has completed phase studies and is progressing rapidly to phase studies, with the aim of producing up to million doses by the end of and entering large-scale manufacturing of billions of doses in ( ) . curevac has a gmp-certified production facility that can produce million doses of their mrna vaccine in a single production run and million doses of azd (chadox ncov- ) will be available by july ( ) . even if sufficient sars-cov- vaccine doses can be manufactured, worldwide delivery presents another major logistic and financial hurdle. storage requirements will be enormous and the vaccine may need to be either frozen or refrigerated, presenting cold-chain issues. the decision about whether to prepare the vaccine in single-dose or multi-dose vials will impact manufacturing, storage, delivery and potential infection risks ( ) . infrastructure and manpower will be required to distribute and administer the vaccine. equity will be a major issue since richer countries may procure the vaccine for their citizens while poorer countries may not be able to afford it. cepi is negotiating global access upfront in order to ensure equitable access, hopefully averting "vaccine nationalism" ( ) . in early may, who launched the access to covid- tools (act) accelerator, which aims to handle the logistics of vaccine procurement and allocation ( ) . covax, co-led by cepi, gavi and who, is the vaccine pillar of act charged with accelerating vaccine development and manufacture and guaranteeing fair and equitable worldwide access. in an open letter, more than world leaders have united and called for a sars-cov- vaccine to be freely available to all people in all countries of the world in what they call "the people's vaccine, " which is surely the fairest way to tackle this unprecedented global pandemic ( ) . by contrast, pneumococcal conjugate vaccine which has been available for years, is still not included in the immunization programs of many countries due to lack of affordability, many children are left unprotected and about half a million deaths from pneumococcal disease each year. initially, as vaccine production commences, it will not be possible to deliver a sars-cov- vaccine to the entire world population and it is anticipated that key vulnerable populations will need to be targeted. these would likely include healthcare workers, the elderly and those with significant risk factors such as those with co-morbidities or the immunocompromised. it might even be used as a travel vaccine as borders re-open across the world, but is unlikely to be incorporated into infant schedules for some time given the very low risk of severe disease in that age group and time needed to conduct pediatric studies. while the world eagerly awaits effective sars-cov- vaccines as the solution to ending this pandemic, it should be borne in mind that there are many caveats even if robust sars-cov- specific nabs, cd + and cd + t cell responses can be induced by vaccination. the number of doses and dosing frequency will need to be determined, and, particularly if repeated or annual vaccination is required, the financial burden and logistics of delivery will have to be supported. a better understanding of what level of nabs correlate with protection and development of standardized viral neutralization along with other assays to compare vaccine candidates is eagerly awaited. potential disease enhancement and other theoretical safety concerns related to each type of vaccine need to be understood and carefully monitored for, while potential suboptimal immunity may need to be overcome. certain vulnerable populations may respond poorly to vaccination, or the vaccine may predominantly protect against severe disease, rather than infection. if t cells prove critical for protection, it will be necessary to ensure that the included t cell epitopes are recognized by enough hla types to ensure worldwide coverage. scale-up of production to billions of doses and delivery to all regions of the world is a massive logistic challenge. in the short-term the strategy will likely need to be targeted vaccination toward those most at risk (e.g., healthcare workers) with the strategy reviewed as vaccine production increases. on the positive side, given the multiple candidates being developed, there is considerable optimism that some of the vaccines currently in trials will prove effective and be available for use in with delivery scaled-up thereafter. kf proposed the project and coordinated the study group. kf, nc, and fr wrote the first draft. eb, mg, ak, km, bt, and sw reviewed and edited the manuscript, provided comments, and suggested references substantially contributing to the content. all authors approved the final submitted version of the manuscript. this project was supported by the australasian society for infectious diseases (asid) vaccination special interest group (vacsig). covid- in the usa epidemiology of covid- : a systematic review and meta-analysis of clinical characteristics, risk factors, and outcomes global covid- case fatality rates deaths in healthcare workers due to covid- : the need for robust data and analysis factors associated with mental health outcomes among health care workers exposed to coronavirus disease the milken institute. covid- treatment and vaccine trackers. 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( ) available online at logistical challenges for potential sars-cov- vaccine and a call to research institutions, developers and manufacturers access to covid- tools (act) accelerator. ( ) available online at world leaders unite in call for a people's vaccine against covid- we would like to thank claudio rosa (www.claudiorosa.com) for medical illustration. key: cord- - yy y n authors: freye, enno; levy, joseph victor title: mechanism of action of opioids and clinical effects date: journal: opioids in medicine doi: . / - - - - _ sha: doc_id: cord_uid: yy y n nan the opium poppy probably reached china about the seventh century a.d. through the efforts of arab traders who advocated its use for medicinal purposes. in chinese literature, however, there are earlier references to its use. the noted chinese surgeon hua to of the three kingdoms ( - a.d.) used opium preparations and cannabis indica for his patients to swallow before undergoing major surgery. the opium poppy, papaver somniferum, is an annual plant, i.e., the plant matures one time, and does not regenerate itself. new seed must be planted each season. from a small seed, it grows, flowers, and bears fruit (a pod) only once. the entire growth cycle for most varieties of this plant takes about days. the tiny seeds (like the seeds on a poppy seed roll) germinate quickly in warm air and sufficient soil moisture. in less than weeks, the young plant emerges from the soil, grows a set of four leaves, and resembles a small cabbage in appearance. the lobed, dentate (jagged-edged) leaves are bluish-green with a dull gray or blue tint. the major legal opium production areas in the world today are in governmentregulated opium farms in india, turkey, and tasmania (australia). the major illegal growing areas are in southwest asia (afghanistan, pakistan, and iran) and in the highlands of mainland southeast asia (burma, laos, vietnam, and thailand)popularly known as the golden triangle ( figure ii- ) . opium poppy is also grown in colombia, mexico, and lebanon. opium poppies containing small amounts of opium alkaloids were, at one time, widely grown as an ornamental plant and for seeds in the united states. the opium poppy control act of declared the possession of this plant illegal. from the cut capsule latex is exuded, which is collected and further processed in order to gain the different ingredients ( figure ii- ) . within the secreted latex collectors will find the major constituents of opium poppy, which are as follows: . morphine ( %- %), the most important alkaloid, which was discovered by the pharmacist sertürner in a small town of einbeck, located in lower saxonia in germany in . he decided to name the extract from the opium poppy morphine ( figure . thebaine ( . %- %) a precursor of many of semi-synthetic opioid agonists (i.e. etorphine, oxymorphone) and antagonists (naloxone, naltrexone, diprenorphine, cyprenorphine), mixed agonist/antagonists (nalbuphine) as well as the partial agonist buprenorphine ( figure c ). . benzylisoquinolines are a group of agents, which do not interact with the opioid receptor. the most important one is papaverine ( . %- %) a phosphodiestrase inhibitor, which relaxes the smooth muscle, and noscapine ( %- %), which is used as a cough suppressant ( figure d) . raw or cooked opium contains more than different alkaloids, including morphine, codeine, and thebaine ( figure ii- ) . in mainland southeast asia, the morphine alkaloid alone accounts for approximately % of the total weight of opium. heroin manufacturers must first extract the morphine from the opium, before converting the morphine to heroin. the extraction is a simple process, requiring only a few chemicals and a supply of water. morphine sometimes is extracted from opium in small clandestine laboratories, which are typically set up near the opium poppy fields. since the morphine base is about one-tenth the weight and volume of raw opium, it is desirable to reduce the opium to morphine before transporting the product from the field to a heroin laboratory. the following is a step-by-step description of morphine extraction in a typical mainland southeast asian laboratory. an empty -gallon oil drum is placed on bricks about a foot above the ground and a fire is built under the drum. thirty gallons of water are added to the drum and brought to a boil. ten to fifteen kilograms of raw opium are added to the boiling water. with stirring, the raw opium eventually dissolves in the boiling water, while soil, leaves, twigs, and other non-soluble materials float in the solution. most of these materials are scooped out of the clear, dark brown liquid opium solution. slaked lime (calcium hydroxide) or, more often, a readily available chemical fertilizer with a high content of lime, is added to the solution. lime will convert the water-insoluble morphine alkaloid into water-soluble calcium morphenate. (other opium alkaloids do not react with lime to form water-soluble calcium salts, as does morphine.) codeine is an opium alkaloid that is slightly water-soluble and some codeine will be carried over with the calcium morphenate in the liquid. otherwise, for the most part, the other alkaloids will become a part of the sludge. as the solution cools, the morphine solution is scooped from the drum and poured through a filter. cloth rice sacks are often used as filters and can then be squeezed in a press to remove most of the solution from the wet sacks. liquid saponated cresol (lysol) is commonly added to the solution to facilitate filtering. the morphine-rich solution is then poured into large cooking pots and reheated but, this time, not boiled. ammonium chloride (a powder) is added to the heated calcium morphenate solution to adjust the alkalinity to a ph of to , and the solution is then allowed to cool. within or h, morphine base precipitates (crashes) out of the solution and settles to the bottom of the cooking pot. the solution is then poured off through cloth filters. any solid morphine base chunks in the solution will remain on the cloth. the morphine base is removed from both the cooking pot and from the filter cloths, wrapped and squeezed in cloth, and then dried in the sun. when dry, the crude morphine base is a coffee-colored coarse powder. this form of morphine is commonly known by the chinese term pi-tzu in mainland southeast asia. if morphine base is to be stored or transported to another location, it may be pressed into blocks. crude morphine base is generally %- % morphine, and is an intermediate product in the heroin process. addicts do generally not use this morphine base. this crude morphine base may be further purified (and changed to morphine hydrochloride) by dissolution in hot water and hydrochloric acid, then adding activated charcoal, reheating, and filtering. the solution is filtered several times before being allowed to cool. as the solution cools, morphine hydrochloride precipitates out of the solution and settles to the bottom. the precipitate is trapped (or captured) by filtration. if the morphine hydrochloride is to be stored or transported to another location, it may be pressed into bricks. morphine hydrochloride (often tainted with codeine hydrochloride) is usually pressed into brick-sized blocks in a press and wrapped in paper or cloth. the most common block size is in. by in. by in., and weighs about lb ( . kg). it takes a full day to extract morphine from opium. as described in the preceding paragraphs, the chemicals used to isolate morphine from opium (known as extraction) include calcium hydroxide (slaked lime) and ammonium chloride. the precursor chemical normally used in the conversion of morphine to heroin (known as acetylation) is acetic anhydride. chemical reagents used in the conversion process include sodium carbonate and activated charcoal. chemical solvents needed are chloroform, ethyl alcohol (ethanol), and ethyl ether. other chemicals may be substituted for these preferred chemicals, but most or all of these preferred chemicals are readily available from smugglers and suppliers. laboratory equipment includes large chinese cooking woks, measuring cups, funnels, filter paper, litmus paper, and enamel (or stainless steel) pots. only the most sophisticated heroin laboratories use glass flasks, propane gas ovens, vacuum pumps, autoclaves, electric blenders, venting hoods, centrifuges, reflux condensers, electric drying ovens, and elaborate exhaust systems. it is common to find portable, gasoline-powered generators at clandestine heroin conversion laboratories. generators are used to power various electrical devices. heroin synthesis from morphine (either morphine base or morphine hydrochloride) is a two-step process that requires between and h to complete . heroin base is the intermediate product. typically, morphine hydrochloride bricks are pulverized and the dried powder is then placed in an enamel pot. acetic anhydride is added, which then reacts with the morphine to form heroin acetate. (this acetylation process will work either with morphine hydrochloride or morphine base.) the pot lid is tied or clamped on, using a damp towel for a gasket. the pot is carefully heated for about h, below boiling, at a constant temperature of c ( f). it is never allowed to boil or to become so hot as to vent fumes into the room. tilting and rotation agitate the mixture until all of the morphine has dissolved. when cooking is completed, the pot is cooled and opened. during this step, morphine and the anhydride become chemically bonded, creating an impure form of diacetylmorphine (heroin). water is added to the thick, soupy mixture and the mixture is stirred as the heroin dissolves in the solution. sodium carbonate (a crystalline powder) is dissolved in hot water and then added slowly to the heroin solution until effervescence stops. this precipitates heroin base, which is then filtered and dried by heating in a steam bath. for each kilogram of morphine, g- g of crude heroin base is formed, depending on the quality of morphine. the tan-colored heroin base (about % pure heroin) may be dried, packed, and transported to a heroin-refining laboratory, or it may be purified further before conversion to heroin hydrochloride (a water-soluble salt form of heroin) at the same site. mainland southeast asian heroin base is an intermediate product that can be further converted to either smoking heroin (heroin no. ) or injectable heroin (heroin no. ). to make heroin no. , the crude base is mixed with hydrochloric acid, resulting in heroin hydrochloride (hcl). adulterants, including caffeine, are added after this conversion. for each kilogram of crude heroin base, about kg of caffeine is used. various flavorings such as quinine hydrochloride or strychnine hydrochloride are sometimes added to heroin no. . next, the wet paste mix is stirred to dryness over a steam bath. the resulting dry heroin no. will be in the form of coarse lumps. the lumps are crushed and passed through a mesh sieve, and the grains (pieces) are then packaged for sale. the entire process takes about h and requires only minimal skill. while extra attention to stirring is required to assure dryness, one person can prepare kg of heroin no. during this time. the reaction of morphine with acetic anhydride produces heroin acetate. to the heroin acetate mixture in the pot, water is added and mixed by stirring. a small amount of chloroform is added. the mixture is stirred and then allowed to stand for min. doing so dissolves highly colored impurities and a red, greasy liquid is formed at the bottom of the container. the water layer is carefully poured off and saved in a clean pot, leaving the red grease in the pot. in a clean pot, activated charcoal is stirred into the aqueous solution and is filtered to remove solid impurities. the decolorizing effects of the charcoal, combined with the chloroform treatment, will leave a light yellow solution. the use of charcoal is repeated one or more times, until the solution is colorless. sodium carbonate (a crystalline powder) is dissolved in hot water and then added slowly to the heroin solution until effervescence stops. this precipitates the heroin base, which is then filtered and dried by heating on a steam bath. the heroin base is heated until dried. the powder should be very white at this stage. if not white, the base is redissolved in diluted acid, treated repeatedly with activated charcoal, re-precipitated, and dried. the ultimate purity and color of the resulting heroin hcl will depend largely on the quality of the heroin base. the heroin base is then dissolved in ethyl ether. conversion to the hydrochloride salt is achieved by adding hydrochloric acid in ethanol to the heroin mixture. the heroin then precipitates. the process of extracting morphine from opium involves dissolving opium in boiling water, adding lime (calcium oxide), or slaked lime (calcium hydroxide), or limestone (calcium carbonate) to precipitate non-morphine alkaloids, and then pouring off the morphine in solution. ammonium chloride is then added to the solution to precipitate morphine from the solution. the chemicals used to process opium to morphine have a number of legitimate purposes and are widely available on the open market. an empty oil drum, some cooking pots, and filter cloths or filter paper are needed. in the united states, opium preparations became widely available in the nineteenth century and morphine was used extensively as a painkiller for wounded soldiers during the civil war. the inevitable result was opium addiction, contemporarily called the army disease or soldier's disease. these opium and morphine abuse problems prompted a scientific search for potent, but nonaddictive, painkillers. in the s, chemists developed an opium-based and supposedly nonaddictive substitute for morphine. the bayer pharmaceutical company of germany was the first to produce the new drug in large quantities under the brand name heroin. this product was obtained by acetylation of morphine. soon thereafter studies showed heroin to have narcotic and addictive properties far exceeding those of morphine. although heroin has been used in the united kingdom in the treatment of the terminally ill, its medical value is a subject of intense controversy. among the commonly known classes of opioids/opiates being used in practice are morphine, codeine, heroin, and the antagonist naloxone ( figure ii- ). morphine by itself is still made from opium and although there is a major first-pass effect (i.e. degradation by liver enzymes), oral administration is still possible, but requires substantial dosage increase. codeine, which is also taken orally, has a strong ability to inhibit coughing, but it induces less analgesia. among the phenylpiperidines a number of synthetic compounds have entered the market. the most known is meperidine/pethidine (demerol®), which is very similar to morphine, but is more efficacious when given orally for the control of pain. another derivative is loperamide (imodium®), an agent being used as a common antidiarrheal, which does not enter the brain, as it is incapable of crossing the blood-brain barrier. hence it is not abused and therefore is sold as a doc (drug over the counter). contrary, fentanyl (sublimaze®) is an opioid, which is at least times as potent as morphine. this agent is used with nitrous oxide or droperidol (a neuroleptic) in intravenous anesthesia (neuroleptanesthesia), but it is also a used in a transdermal patch for the control of chronic pain. another known opioid is methadone, which has a good oral efficacy, a much longer half-life than morphine ( - h) , and in regard to its effect much like morphine. it is used for treatment of heroin addiction and for the control of chronic pain. a methadone congener, which is being used solely in the methadone substitution programs is laam ( -levoacetylmethadol), only needs to be taken once every h. the opioid propoxyphene (darvon®) has figure ii- . molecular structure of different opioid ligands with agonistic or antagonistic properties the lowest analgesic potency ( . times morphine). it is almost always given together with aspirin for the control of mild to moderate pain. it is very popular clinically due to misplaced concerns about the abuse potential of codeine. it is interesting to note that all commonly used opioids have a similar structure in regard to their terminal morphine ring and the distance between the ring and the n-substitution. such common traits suggests that opioids must have a common structure in order to interact with a specific receptor site ( figure ii- ) . thus, central analgesics mediate their action by means of an interaction with specific opioid receptor sites located within specific areas of the central nervous system, which are engaged in the transmission of nociceptive afferences and the identification of pain. there, opioids act as agonists at highly definite receptor sites, and there is general agreement on the existence of at least three types of opioid receptor sites (table ii- ). . the morphine mu receptor ( ) at which the prototype morphine binds, . the kappa receptor ( ) at which the prototype agonist is ketocyclazocine, and . the delta receptor ( ) at which the prototype endogenous opioid ligand enkephalin binds. opioid receptors are distributed widely in brain and found in spinal cord and peripheral sensory and autonomic nerves. there are the three well-characterized members of the opioid receptor family, designated by the greek symbols , and . the more recently discovered orl receptor is placed with this family due to its high degree of structural homology. these receptors were renamed op , op , op and op , respectively, by an international union of pharmacology (iuphar) nomenclature committee in [ ] . this nomenclature has proved unpopular. the nomenclature (x-opioid peptide receptor) has been proposed giving , mu or mop; , delta or dop; , kappa or kop and orl or nop receptors. in order to keep matters straightforward the original nomenclature is used in the following chapters. the products of endogenous opioid peptide genes activate opioid receptors physiologically: proenkephalin (giving methionine-and leucine-enkephalin; metenk and leu-enk, respectively; figure ii- ), prodynorphin (dynorphins a and b and -neo-endorphin) pro-opiomelanocortin ( -endorphin) and pronociceptin (nociceptin, also known as orphanin fq). met-enk and leu-enk have highest affinity for -receptors, less affinity for , and very low affinity for -receptors; the dynorphins have preferential affinity for -receptors, but bind to the and types with high affinity; -endorphin binds with high affinity to and receptors, but has little affinity for receptors. all the peptides are full agonists at their cognate receptors. endomorphin- and - , derived from an unknown precursor, are endogenous peptides with high selectivity for -receptors. these peptides are unusual in that they are partial agonists. none of the proenkephalin, prodynorphin or pro-opiomelanocortin peptide products or the endomorphins displays affinity for the orl receptor. similarly, the orl receptor agonist nociceptin has no appreciable affinity for , or receptors. the four receptor types have been cloned and shown to be -transmembrane receptors activating g proteins of the pertussis-toxin insensitive g i/o family, but including g z. evidence for subtypes of , and opioid receptors exists, but the molecular basis for the observed functional and pharmacological differences is unclear. putative and receptors are differentiated by several agonist and antagonist ligands. however, there is only one receptor gene, the protein product of which has properties of the putative receptor. the distinction between the proposed and receptors is based largely on the apparent preferential blockade of the type by the antagonist, naloxonazine [ ] . there is only one cloned receptor gene, corresponding to the putative receptor, but several forms of the -receptor mrna arising from alternative splicing have been reported. the receptors these encode differ at the end of the c-terminal tail and show subtle differences in the binding profile of opioid ligands; a role for the variants is not known. the cloned -receptor, with high affinity for u is the subtype. the proposed and subtypes are poorly defined in both molecular and pharmacological terms (table ii- ) . a recent explanation for subtypes has evolved with the identification of opioid receptor heterodimers or hetero-oligomers that appear to have properties different from the monomeric receptors. an interesting addition to ligands that bind to the receptor is the hallucinogen salvinorin-a. this is a highly efficacious and potent agonist, but is most unusual in that it has no nitrogen atom. endogenous opioid systems have a functional role in modulating pain perception; opioid agonists are therefore potent analgesics. opioid receptors are also present in hypothalamus (figure ii- ), where they influence temperature regulation and control of hormonal secretion. in the forebrain, endogenous opioids are involved in behavioral reinforcement and appear to play a role in anxiety and in the expression of emotions. in addition, opioids influence gastrointestinal and autonomic nervous system function. originally, a fifth binding site, the sigma receptor, was included in this group. however, actions mediated through this receptor are not reversed by naloxone so it is not a true opioid receptor. the -receptors have been further sub-classified into two distinct subtypes ( and ), as have the -receptors. kappa receptors have been divided into , , and sub-types. recently, several of these receptors have been cloned successfully. in animal models, some laboratories have cloned up to -receptor subtypes [ ] . however, the functional significance of these "spliced variants" remains unclear at present. originally suggested by martin and coworkers [ ] , all three opioid receptor types mediate different opiate effects as they figure ii- . difference in topographic density of the three opioid receptor sites within the central nervous system. adapted from [ ] normally serve endogenous opiates (the endorphins, dynorphins, and enkephalins; table ii- ): . activation of the mu receptors ( ) results in analgesia, euphoria, respiratory depression, nausea, gi slowdown, and miosis. receptors of this type are mostly located in periaquaductal gray (pag), spinal trigeminal nucleus, caudate and geniculate nuclei, thalamus, and spinal cord. . binding at the kappa receptors ( ) induces modest analgesia, dysphoria, feelings of depersonalization and disorientation, miosis, and mild respiratory depression. these receptors are mainly found in basal ganglia, nucleus accumbens, ventral tegmentum, cortex, hypothalamus, periaqueductal grey, the spinal cord, and in the periphery. . occupation of the delta receptors ( ) results in anxiolysis and central pain relief, although its overall significance is not all that well understood. they are mainly found in the nucleus accumbens and the limbic system (table ii- ) . molecular biology techniques have enabled the primary amino acid sequence of the human -, -, and -opioid receptors to be determined. the pharmacological and functional properties of the cloned receptors, the development of "knockout" animals, which are deficient in a receptor or part of a receptor, and the manipulation and substitution of various amino acids in critical domains of the various opioid receptors have provided new insights in opioid action. in this regard, the three opioid receptor genes, encoding mu (mor), delta (dor), and kappa (kor) have been cloned. the binding affinities of a range of opioids to the -, -, and -opioid receptors and also to the cloned orphinan receptor have been examined in animals. the animal data indicate that while the commonly prescribed opioids (agonists and antagonists) bind preferentially to the -receptor, they also interact with all three receptor types. morphine and normorphine (a minor metabolite of morphine) show the greatest relative preference for the -receptor. methadone (which also has some nmda-receptor blocking activity) shows significant binding to -receptors, while buprenorphine, and to a lesser extent naloxone, avidly binds to all three receptor types. there is evidence (albeit inconsistent) that the d-enantiomer of methadone blocks the nmda receptor [ ] . the binding affinity of buprenorphine to the receptor is much greater than that of naloxone, which explains why the latter only partially reverses buprenorphine overdose. animal data also indicate that codeine and diamorphine have very poor binding to opioid receptors, which reinforces the possibility that both are prodrugs where the pharmacologically active species are morphine [ ] and -monoacetyl morphine, respectively [ ] . oxycodone may also act through an active metabolite, though there are some data, which suggest that this is not the case [ ] . pethidine is considered to be a potent -receptor agonist, but it does bind weakly to all three opioid receptors (table ii- ) . ketobemidone has a lower affinity for the -receptor than does morphine, but it shows greater discrimination for this receptor compared to -receptors. the binding of both of these opioids to the -receptor is similar [ ] . this difference in opioid action is also mirrored in the difference in affinity of various narcotic ligands interacting with the three relevant opioid receptor sites (table ii- ) . it should be noted that some of those ligands, either pure antagonists, mixed agonist/antagonists or partial agonists, are characterized by displacement potency at a specific receptor site. from the above binding and displacement values it can be seen, that opioid practically bind to all three receptor sites, however with different affinity. the preference in binding to one receptor site manifests itself in the visible clinical effect, which may either be of agonistic or of antagonistic nature. the binding of morphine, methadone, buprenorphine, and naloxone to the cloned human -receptor shows excellent congruence with the animal data [ ] . fentanyl shows a similar binding affinity, while codeine demonstrates greater binding affinity to the cloned human receptor (table ii- ; figure ii - ). thus, for these commonly administered opioids, there is no great variability in their affinity for the humanreceptor. the clinical relevance of these data is that different opioids act in different ways. from anecdotal clinical experience there is considerable interindividual adapted from [ , , , , ] variability in response to each opioid and this reinforces the need to assess an individual's response to opioid analgesia carefully. it also is premature to extrapolate from laboratory data, which in many instances have not yet been replicated, to the clinic. however, data increasingly inform the clinical use of these drugs and will be particularly relevant to new approaches to their use such as "opioid switching". figure ii- shows the putative analgesic effect mediated by the main -opioid receptor depicting that higher affinity also correlates closely with analgesic potency. but aside from -receptor interaction, analgesia can also be mediated through a -receptor and a -receptor site. the classification of different opioid receptor types is based on the original description by martin and coworkers from [ ] . the effects presumed to be mediated at -receptors have been defined as a result of both human and animal studies, while the effects mediated at -receptors derive predominantly from animal models. receptors mediate analgesia that persists in animals made tolerant to -agonists. the -agonists produce less respiratory depression and miosis than -agonists. it is assumed that opioid receptors mediate opioid receptors are found in several areas of the brain, particularly in the periaqueductal grey matter, and throughout the spinal cord ( figure ii- ). supraspinal systems have been described for -, -, and -receptors, whereas -and -receptors modulate pain at the spinal level [ , , ] . the different distribution of the various opioid subsites suggests different mechanisms of action in the mediation of analgesia. thus, -selective opioids like morphine, fentanyl and sufentanil, due to the high density of binding sites, mediate their main action within the brain stem and the midbrain. due to their close vicinity to respiratory and cardiovascular regulating centers in the brain stem, selective -opioids accordingly induce a marked depression of respiration and blood pressure. on the other hand, due to the main distribution of the -receptors within the cortex (lamina v, vi) [ ] it is conceivable that these ligands induce a lesser respiratory and cardiovascular depressive effect. as a consequence and contrary to -ligands, -ligands induce a marked sedative appearance. in addition, there is a lesser addiction liability with -ligands, which is easily derived from the fact that the relevant areas in the limbic system show a low concentration of -binding sites. also, the lesser analgesic potency of -ligands is enlightened by the fact that most of the -selective receptors can be found in the deep lamina vi of the cortex. since their dendrites retrograde descend to the thalamus, all ascending nociceptive input is modified, resulting in a depression of nociceptive afferences and a reduction in arousal. certain dendrites of petrosal cells of the cortex also descend down to the brain stem, whereby the activating, ascending reticular system (ars) is affected resulting in a reduction of vigilance [ ] . in summary, due to the dissimilarity of distribution of the three opioid receptor subtypes with the spinal cord and the supraspinal areas of the cns, a functional differentiation can be expected. this effect is reflected in difference of binding affinities with the brain where % of all receptor sites are referred to the -, % to the -and % to the -opioid receptor [ , ] . the present understanding of the effect profiles of opioid receptors, however, remains incomplete, as new advances make it clear that their disposition and structure are extremely complex. opioids inhibit pain signals by different mode of actions: • inhibition of ca ++ -influx into the buttons of the presynaptic cell (e.g. the one releasing substance p; figure ii - ) . this is because ca ++ -influx is necessary for neurotransmitter release, whereby opioids reduce or prevent substance p from being released. • acting as an inhibitory neurotransmitter, since the opioid hyperpolarizes the postsynaptic cell by enhancing k + -flow out of the neuron, which makes it more difficult for all incoming nociceptive afferences to stimulate the next neuron, and thus more difficult to send painful information. • moderation of central perception of painful information in the limbic system so as to make it less aversive when it is perceived. several facts have led to the assumption that opioids interact with specific binding sites in the cns. a slight molecular substitution at the side chain of the morphine molecule structure results in considerable changes of potency (table ii- ) . whereas pethidine (meperidine, usp), a piperidine derivative, may be considered a weak analgesic, fentanyl, a piperidine derivative, is about - times more potent than morphine. the opioid antagonists levallorphan and naloxone are noted for a low and a analgesic effect, respectively. furthermore, only the levorotator (levo-) isomers of opioids, which appear in their natural form (i.e. compounds which, when in solution, rotate plane-polarised light to the left) are pharmacologically active (e.g. levorphanol). their dextrorotatory (dextro-) isomers, which can be synthesized in the laboratory (e.g. dextrophane), shows a negligible pharmacological effect. both substances are structurally the mirror image of each other ( figure ii- ) . in this context it is important to note that only the levo-stereoisomer of the racemic mixture is the pharmacologically active ingredient. this observation supports the part ii figure ii- . mechanism of action of opioids at the central nervous system. by binding at the same receptor site as the endogenous opioids (i.e. enkephalins, endorphins), the release of excitatory neurotransmitters such as acetylcholine and glutamate is decreased thereby reducing the receiving cells excitatory input. the degree of opiate receptor binding is proportionally to the net release of excitatory transmitters and the reduction of depolarization produced by the arriving nociceptive nerve impulse. this enkephalin inhibitory system normally modulates the activity of the ascending pain pathways within the spinal cord and the brain. opioid agents act by binding to unoccupied enkephalin receptors, thereby potentiating the analgesic effects of the system notion that stereroselectivity of an opioid analgesic is a prerequisite in order to bind to the opiate receptor site, thus inducing analgesia. based on their interactions with the various receptor subtypes, opioid compounds can be divided into agonist, agonist/antagonist, and antagonist classes (table ii- ) . by definition an agonist is a drug that has affinity for and binds to cell receptors to induce changes in the cell that stimulate physiological activity. the agonist opioid drugs have no clinically relevant ceiling effect to analgesia. as the dose is raised, analgesic effects increase in a log linear function, until either analgesia is achieved or dose-limiting adverse effects supervene. efficacy is defined by the generally opioids exist in optical isomers, which are a mirror image in the molecular form. only the levorotatory (levo)-isomer, which in solution rotates plane-polarized light to the left, produces the characteristic analgesic effect of an agent. the dextrorotatory isomer is totally inactive. this sterospecificity of opiate action supports the concept of selective receptor binding to a site, which is able to distinguish in "handedness or goodness of fit" of an opioid molecule maximal response induced by administration of the active agent. in practice, this is determined by the degree of analgesia produced following dose escalation through a range limited by the development of adverse effects. potency, in contrast, reflects the dose-response relationship. potency is influenced by pharmacokinetic factors (i.e. how much of the drug enters the body systemic circulation and then reaches the receptors) and by affinity to drug receptors. the concepts of efficacy and potency are illustrated in the following figure, which shows the dose-response curves for two drugs with a full agonistic and a partial agonistic action. if the logarithm of dose is plotted against response an agonist will produce an s-shaped or sigmoid curve. the efficacy of the two drugs, defined by maximum response is the same. the full agonist produces the same response as a partial agonist but at a lower dose, and therefore is described as more potent ( figure ii- ). an antagonist by definition is an agent that has no intrinsic pharmacological action but can interfere with the action of an agonist. competitive antagonists bind to the same receptor and compete for receptor sites, whereas non-competitive antagonists block the effects of the agonist in some other way. contrary the mixed agonist/antagonists analgesics can, in turn, be subdivided into the mixed agonist/antagonists and the partial agonists, a distinction also based on specific patterns of drug-receptor interaction. both the partial agonist and the agonist/antagonist drugs have a ceiling effect for analgesia, and although they produce analgesia in the opioid-naive patient, in theory they can precipitate withdrawal in patients who are physically dependent on morphine-like drugs. for these reasons, they have been considered generally to have a limited role in the management of patients with cancer pain. the mixed agonist/antagonist drugs produce agonist effects at one receptor and antagonist effect at another. pentazocine is the prototype agonist/antagonist: it has agonist effects at the -receptors and weak to medium antagonistic action at the figure ii- . typical dose-response curves of a full agonist, a partial agonist and an antagonist on opioid-related effects -receptor thus in addition to analgesia, pentazocine may produce -mediated psychotomimetic effects not seen with full or partial agonists. when a mixed agonist/antagonist is administered together with an agonist, the antagonist effect at the -receptor can generate an acute withdrawal syndrome. a partial agonist has low intrinsic activity (efficacy) so that its dose-response curve exhibits a ceiling effect at less than the maximum effect produced by a full agonist ( figure ii- ) . buprenorphine is the main example of a partial agonist opioid. increasing the dose of such a drug above its ceiling does not result in any further increase in response. this phenomenon is illustrated in the figure in which buprenorphine is the partial agonist. the full agonist is more potent than the partial agonist (in the lower part of the curve it will produce the same response at a lower dose), but is less effective than both coadministered ligands because of its ceiling effect. when a partial agonist is administered together with an agonist, displacement of the agonist can cause a net reduction in pharmacological action, which may be sufficient to generate an acute withdrawal syndrome. while this is a theoretical possibility with morphine and buprenorphine, no such interaction has been reported clinically. similarly, it has been suggested that the effects of morphine may be blocked in a patient switched from buprenorphine, because of the prolonged action of buprenorphine and the assumption that it will "antagonize" the effect of morphine. this has been one of the reasons, why buprenorphine has not been in cancer pain management. however, the recent development of a transdermal formulation of buprenorphine may encourage its use in chronic cancer pain (and chronic noncancer pain). an analgesic ceiling with buprenorphine is only reached at doses of - mg or more in h [ , ] . when used in usual recommended doses (e.g., two patches of g/h of transdermal buprenorphine, equivalent to - mg per h) buprenorphine can be considered a full -agonist since at these doses its effect will lie on the linear part of the dose-response curve [ ] . relative potency is the ratio of the doses of two analgesics required to produce the same analgesic effect. by convention the relative potency of each of the commonly used opioids is based upon a comparison with mg of parenteral morphine. data from single-and repeated-dose studies in patients with acute or chronic pain have been used to develop an equianalgesic dose table (table ii- ) that provides guidelines for dose selection when the drug or route of administration is changed. the information contained in the equianalgesic dose table does not represent standard doses, nor is it intended as an absolute guideline for dose selection. many variables may influence the appropriate dose for an individual patient, including intensity of pain, prior opioid exposure in terms of drug, duration, and dose (and the degree of cross-tolerance that this confers), age, route of administration, level of consciousness, metabolic abnormalities (see below), and genetic polymorphism in the expression of relevant enzymes or receptors. in addition, a substitution at the side chain, for example the substitution of a methyl-group by an allyl-group or the substitution by a cyclopropyl-group results in the new opioid antagonist naloxone, diprenorphine and naltrexone respectably, or mixed agonists/antagonists (nalorphine, levallorphane), which have the capability of antagonizing the effect of the parent compound ( figure ii- ) . similarly, when the n-methyl group of the highly potent opioid oxymorphone or the pure agonist etorphine ( times of morphine) is replaced by a cyclopropylmethyl group, the highly potent antagonists naltrexone and diprenorphine are derived. these compounds are . times as potent as naloxone and while the former is used as an oral preparation in the rehabilitation of the earlier opiate addict, the latter is used in veterinary medicine for the reversal of immobilization of wild animals. in addition, diprenorphine is also the original substance of buprenorphine where additional three methyl groups are incorporated in the molecule ( figure ii- ). such minor changes in the molecular structure and their resultant major pharmacological effect suggest, that similar to hormones and catecholamines, opioids bind with specific receptor sites which results in the characteristic effects such as analgesia. various research groups corroborated this hypothesis almost simultaneously. pert and snyder [ ] , terenius [ ] and kosterlitz [ ] were the first research group to identify selective binding sites in the cns using radioactive labeled opioids. these so-called opiate binding sites were found mainly in neuronal structures and nervous pathways involved in the transmission of nociceptive signals such as the first relay station of pain transmission, the substantia gelatinosa of the spinal column. in the posterior horn the impulse is passed over to the second neuron while, simultaneously, descending nerve fibers from the reticular system (the cortico-and reticulo-spinal tract) induce either a facilitation or an attenuation of pain transmission, which results in a modulation of pain impulses at the spinal level ( figure ii- ). the course of pain transmission is to the contralateral side of the spinal cord where impulses have already undergone a distinct separation. it is the paleospinothalamic pathway, consisting of nonmyelinated c-fibers, which mediate the excruciating, dull pain component, which is difficult to localize as it ends in the nonspecific nuclei of the medial thalamus [ ] . en route, the paleospinothalamic tract sends off afferent fibers to the midbrain area, such as the periaqueductal grey matter and the reticular formation [ ] . the pathway ends in intralaminar nuclei of the thalamus and the nucleus limitans, a patch of pigmented nerve cells border the mesencephalon ( figure ii- ) . from there, subcortical pain pathways link with the pallidum, the alleged psychomotoric center that sends fibers to all areas of the brain hemisphere. the neospinothalamic pathway, in contrast, consists of myelinated a -fibres, which transfer impulses to the nucleus ventrocaudalis-parvocellularis (n.v-c parvocellularis). from there pain sensations are projected to the postcentral gyrus, which enables the patient to localize the source of pain ( figure ii- ) . both, the central grey matter and the pallidum are characterized by a dense accumulation of opiate binding sites [ , ] . it is worth noting that nervous pathways transmitting the dull, chronic and less pinpointed pain components are more affected by opioids, while , is an important relay station in the mediation of nociceptive afferents to higher pain modulating and discriminating centers of the cns, which is necessary for the nonspecific feeling of pain and is closely coupled with emotions the neospinothalamic pathway conveys the sharp and well localized pain components which accompany any injury and are always the first to be perceived. the indefinable, dull, emotional component is perceived later, giving pain its negative character. this separation in pain pathways is of special importance. impulses from the fast pathway usually antagonize the mediation of slow afferent impulses on all levels in the cns: substantia gelatinosa and reticular formation, as well as the specific and the nonspecific projecting nuclei of the thalamus [ ] . opioid binding sites, as they are visualized with receptor-binding techniques, strikingly map the paleospinothalamic pain pathway ( figure ii- ) . furthermore, there is a high density of opioid binding sites in various other parts of the brain [ , , , ]: . the corpus striatum, being part of the limbic and the extrapyramidal motor system, is responsible for triggering opioid-induced muscular rigidity. it is not only the regulatory center for locomotion but it is also the center for the regulation of attention and perception. . the nucleus solitary tract in the brain stern, which is the origin of the noradrenergic dorsal pathway bundle, which is in command of vigilance and the cough reflex. . the nuleus amygdala, being part of the limbic system, is in charge of the mediation of euphoria (or "kick") when opioids are used for other purposes than pain. . the locus coeruleus being the origin of the neurosympathetic system in the brain stem, regulates peripheral vasodilatation. . lastly, a dense accumulation of opioid binding sites is found in the substantia gelatinosa at the dorsal horn of the spinal cord. -current thinking is that effective opioid analgesics work through stimulating mu ( ) receptors, which also produce euphoria. -euphoria is mediated by the actions of opiates at a cluster of brain areas that include the nucleus accumbens and ventral segmental area. dopamine influx seems to cause subjective pleasure, or euphoria. -opioids may have a "disinhibiting" (inhibition of inhibitory neurons) effect that allows greater dopamine influx. because the main property of opioids is the blockade of nociceptive transmission in the mesencephalon (i.e. the nucleus limitans and the limbic system) the following effects can be observed: . no pain (analgesia), since any sensation is not identified as painful. . a lack of the negative emotional component of pain. on the contrary, euphoria may result and pain is no longer experienced as an emotional distress, even though pain impulses are transmitted via the ventrocaudal-parvocellular nucleus to the postcentral cortex. . during the opioid-induced pain-free state, the site of pain, however, still can be localized. as a consequence pain has lost its negative character and is no longer experienced or perceived as uncomfortable and distressing. in contrast to the analgesics that have a peripheral site of action (e.g. acetylsalicylic acid; asa), opioids act at the relay station of nociceptive-propagating pathways at the synapse of nerve conduction. within the nerve, pain impulses are transmitted as a change in electric conduction. and in order to guarantee maintenance of the nociceptive impulse, the excitatory impulse releases a neurotransmitter at the terminal nerve. due to its chemical configuration, the transmitter fits exactly into a binding site at the opposite nerve ending resulting in an increase of excitability and a change in the electrical nerve conduction. opioids have the property of binding to specific receptor sites at pre-and post terminal nerve endings resulting in an inhibition of a release of the excitatory neurotransmitter. the continuity of the impulse is interrupted, the nociceptive signal is no longer transmitted and thus can no longer be perceived as such ( figure ii- ). due to the difference in stereoconfiguration, opioids differ in their affinity (i.e. goodness of fit) at these binding sites ( figure ii- ). this explains why different opioids are characterized by a large variety in potency. in addition, opioids also differ in their intrinsic activity (i.e. the degree of conformational change of the receptor site) resulting in different intracellular effects. taken together affinity and intrinsic activity results in the efficacy of a drug within the system ( figure ii- ) . thus, binding properties are reflected in varying analgesic potencies. contrary, the intensity of binding with the receptor site (i.e. the intensity with which the opioid adheres to the binding site) is reflected in the duration of effects (table ii- and figure ii- ) [ , , ] . for instance opioid analgesics such as sufentanil or lofentanil have an exceptional goodness of fit to the opioid receptor site, which results in high potency. on the other hand, the low dissociation coefficient from the receptor of buprenorphine or lofentanil is characterized by a long duration of action, while the high association coefficient demonstrates increase of affinity to the binding site. contrary to agonists, antagonists are able to displace an opioid from its receptor binding site and take up his position. displacement is only possible because the antagonist has a greater affinity to the binding site. therefore, affinity of an opioid antagonist is expressed in its antagonistic potency. naloxone or naltrexone have a very high affinity to the receptor and easily displace an opioid whereas levallorphan is five times weaker ( figure ii- ) . in order to induce a similar antagonistic effect, a higher dose of levallorphan is necessary. in order to induce increasing effects with opioids, the goodness of fit not only is a prerequisite. of additional importance is the conformational change the receptor undergoes after binding, which is expressed in the "the intrinsic activity". an opioid must, therefore, not only fit to the receptor; it must also induce a chain reaction in the transmembrane receptor domain resulting in a net effect ( figure ii- ) . the reaction after opioid binding seems to depend on the side chain of the molecule. thus it appears that one portion of the opioid molecule provides the binding to the receptor whereas another portion is responsible for the induction of a conformational change (i.e. intrinsic activity), which will either be of agonistic or antagonistic nature. in a sensitive and specific opiate-receptor assay, the guinea pig ileum with its dense accumulation of receptor binding sites, it was possible to demonstrate receptor affinity and pharmacological efficacy ( figure ii- ) . this assumption is underlined by the effects induced by "pure" opioid antagonists such as naloxone or naltrexone, which also have a good fit with the receptor site, however when given on their own do not induce an analgesic effect. for instance, if naloxone is given by itself, the compound does not induce effects similar to its parent compound oxymorphone ( figure ii- ) . also, in contrast to a potent opioid like fentanyl, the antagonist naloxone has a lower dissociation coefficient resulting in a shorter duration of action, which may result in a reoccurrence of an opioid-like effects such as respiratory depression. however, due to its high source: adapted from [ , , ] part ii figure ii- . difference in affinity and intrinsic activity of various opioids. note, that codeine has a similar intrinsic activity as sufentanil. however, due to the higher affinity of the latter the net analgesic potency is much larger association coefficient (i.e. affinity), it induces a rapid displacement of the agonist and a reversal of all opioid effects. on the other hand mixed agonist/antagonists, such as pentazocine, nalorphine, levallorphan, nalbuphine and butorphanol, demonstrate characteristics, which enable them to displace a pure agonist at the receptor site (antagonistic effect), but at the same time when administered by themselves, they induce opioid related effects such as analgesia and respiratory depression (agonistic effects; table ii- ) . such dual activity is only possible by means of their intrinsic activity at two distinct and different receptor sites: one the antagonistic activity at the -and its agonistic action art the -receptor site. and lastly, partial agonists like meptazinol and buprenorphine induce their analgesic potency via the -opioid receptor. although having a high affinity, their analgesic ceiling effect at the higher dose range is due to a lesser intrinsic activity, resulting in a lesser net analgesic appearance than pure agonists. such difference in the characteristic traits of opioids can be summarized as follows: . the affinity to the receptor (displacement properties or extrinsic activity) . the intensity of binding to the receptor (duration of effect) . the ability to change the conformation of the receptor (intrinsic activity) . the competitive potency (antagonism) . the degree of metabolism (duration of effect) note the relatively high antagonistic potency of buprenorphine, however, is due to its high affinity to the receptor site resulting in the displacement of a ligand at the preoccupied receptor site. similarly like a hormone or other extracellular "first messengers" that bind to its receptor on a cell surface, a signal is transmitted or "transduced" to the cells interior, thus setting a series of events that produce a biological response. such "events" include both chemical reactions and physical reactions like a conformational change in the protein molecules. the biological responses include cell differentiation, altered metabolism and cell growth and division. there are three signaling pathways that share many of the same intracellular events. each pathway is characterized by its receptor and by the cascade of intracellular events that lead to a biological response. each receptor has an extracellular, transmembrane, and intracellular component and the binding of a ligand to the receptor represents the "primary message". the term "secondary messenger" is used for those mediators that diffuse from one part of the intracellular space to its spatially removed target. among these secondary messengers are adenosine- ,- -cyclic phosphate (c-amp). many integral membrane glycoprotein membranes share a seven transmembrane alpha-helix motif ( figure ii- ) . the ß-adrenergic receptor, whose natural ligands are epinephrine and norepinephrine, is an example of such receptors. similarly in the opiate receptor, binding of a ligand presumably initiates a conformational change in the membrane protein that is transmitted to the cell interior. this physical reaction can then facilitate other physical or chemical reactions, which are conveyed to ion channels, resulting in a change of transmembrane ion flow. the transduction of the signals from external messengers, including opiate ligands involves intracellular heterotrimeric g-proteins, which are bound to the inner cell (plasma) membrane, a secondary messenger system, involving cyclic amp, and a target response. as the name implies, these proteins are trimers, consisting of an , , and subunit. they are bound to the inner membrane and the subunit can bind the guanine nucleotides, gtp and gdp. g-proteins are involved in vision, smell, cognition, hormone secretion and muscle contraction in humans, and in mating in yeast. there are more than receptors (not including odor receptors) that utilize g-proteins, and there are at least members of the g-protein family, with each member having its characteristic , , and subunits. while the subunit is different for each g-protein, the ß/ pair can be the same. however, all of the g-proteins share a similar structure. in regard to the opioid receptor, specifically the g-proteins transmit the signal from the intracellular part of the receptor to the effector. adenylyl cyclase (ac), which is an inner membrane-bound enzyme, regulates the production of the secondary messenger, adenylyl cyclase. other effectors that are g-proteindependent include additional enzymes, like cyclic gmp phosphodiesterase, and transmembrane ion channels ( figure ii in its resting conformation, the g-protein consists of a complex of the three subunit chains and a gdp molecule bound to the alpha subunit. the alpha subunit is in close proximity to the intracellular part of the transmembrane receptor and, when a ligand binds to the receptor, the change in its conformation causes it to bind to the g-protein at the alpha subunit. this results in an exchange of bound gdp for gtp, which is more abundant in the cell than gdp. gtp causes a conformational change in the alpha subunit, thus "activating" it so that the alpha subunit dissociates from the -pair. the alpha subunit diffuses along the membrane until it binds to an effector, thereby activating it. the alpha subunit is also a gtpase, so the signal transduction is regulated at this level by hydrolysis of gtp to gdp and inorganic phosphate. such hydrolysis can occur spontaneously or upon interaction with a gtpase activating protein, "gap". the gdp-alpha subunit complex then binds to the ß/ complex to reform the original trimeric protein. since the stimulation of the external receptor can activate a number of g-proteins, signal amplification can occur. while this is a desired response in many instances, control at this level is needed to modulate it. g-proteins, then, are nano-switches when they turn on the effector by binding of the alpha subunit and turning it off when the gtp is hydrolyzed. the duration of production of secondary messenger, like cyclic amp, is determined by the rate of hydrolysis. in this sense, the g-protein acts as a nano-timer. although there is controversy over the role of the ß/ subunits in modulation of signals, it is likely that there are both inhibitory and stimulatory effects. if different receptors act on the same g-protein, or if different g-proteins act on the same effector, the potential exists for a "graded" response to an extracellular signal. if the same receptor acts on many g-proteins, or if one g-protein acts on many effectors, then there may be many simultaneous responses to the primary messenger. following binding the g-proteins activates the membrane-bound effector, adenylyl cyclase (ac). this enzyme catalyzes the synthesis of cyclic amp resulting in the formation of atp, camp and pyrophosphate. because this molecule is freely diffusing through the cytoplasm, it is a "secondary messenger" (figure ii- ). the reverse reaction, the formation of atp from camp and pyrophosphate, is catalyzed by a specific phosphodiesterase. camp is involved in a number of physiologic processes. for the breakdown of glycogen, stimulation of the ß-adrenergic receptor involves activation of adenylyl cyclase and synthesis of cyclic amp. the activity of camp-dependent protein kinase (capk) requires camp in order to phosphorylate ser and thr residues on other cellular proteins. glycogen phosphorylase is activated by capk, making glucose- -phosphate available for glycolysis. adenylyl cyclase activity is regulated at a number of levels, including modulation of gtpase activity of ga, phosphodiesterase activity, and protein phosphatases. inhibitory g proteins, gi, are analogous to the stimulatory g proteins, gs, except for the exchange of gtp by gdp by the -subunit and the subsequent inhibitory action of gia on adenylyl cyclase. rather, it has been determined that ligand induced dimerization is the mechanism through which the receptor ptks are activated. this dimerization brings the tyrosine kinase catalytic domain on each receptor into close enough arrangement so that each kinase can phosphorylate tyr residues in the other's tyrosine kinase domain. such activated catalytic domains can then phosphorylate tyrosines outside of the catalytic domains, which can then modify other intracytoplasmic proteins, either by phosphorylation or by other means. all these changes are reversed with an overexpression of activation when an opioid is antagonized by a specific antagonist such as naloxone with activation of the excitatory nmda-(n-methyl-d-aspartate) receptor, resulting in a rebound with an increase in transmission of stimuli ( figure ii- ) . the next step in the signaling pathway involves activation of an inner membranebound monomeric g protein known as ras, which initiates a series of kinase reactions that ultimately carry the signal to the transcriptional apparatus of the nucleus. ras, being a g protein, is activated when its bound gdp in the resting state is replaced by gtp. it, too, has gtpase activity, but the half-life is too slow to allow for effective regulation of a signal. another gtpase activating protein, gap, increases the rate of gtp hydrolysis by ras. a "kinase cascade" ensues, involving raf (a ser/thr kinase), map kinase (also known as mek, which is both a tyr kinase and ser/thr kinase, and a family of proteins known as mapks or erks. opioids induce a variety of clinical relevant effects, which can be subdivided in being advantageous and/or even detrimental. one of the major consequences following opioid administration is that of analgesia, or antinociception. and while nsaids induce their antiniociceptive effect via cyclooxygenase (cox) inhibition, local anesthetics selectively block ion-channels, thus inhibiting the transmission of nociceptive efferent to the higher pain modulating centers in the cns. contrary, opioids bind to those areas, which not only are involved in transduction but also in the modulation and identification of painful afferences. although the majority of opioids are able to induce a maximal analgesic effect, the dosages necessary to induce such a result differ significantly. for instance, an opioid like sufentanil part ii needs a much lower dosage than the less potent opioid morphine. this is due to the higher affinity and intrinsic activity of sufentanil, suggesting that only a lesser portion of receptors needs to be occupied in order to induce the desired effect. however, a high analgesic potency necessarily does not reflect a better efficacy. this is because in certain painful conditions, some opioids are more efficacious than others. on the other hand, not all painful conditions, as the patient expresses them, can be treated successfully with an opioid. therefore, before starting an opioid therapy it is mandatory to evaluate the kind of painful condition the patient has, use the specific opioids as indicated, and avoid those painful states where opioids are contraindicated or result in a lesser therapeutic effect. however, there is the general position: in intense to severe, excruciating pain, opioids are the sole agents, which are able to induce sufficient analgesia -pain from muscular dysfunction. in patients who present pain of myofacial nature, opioids are contraindicated since they will not result in an alleviation of nociception. due to muscle spasm or an increase in tension physical therapy presents the first defense line in the therapeutic approach. this is accompanied by the administration of a benzodiazepine, which induces a muscle relaxant effect and/or the injection of a corticoid together with a local anesthetic ( . % bupivacaine) in so-called trigger points ( figure ii- ) . trigger points are typical points which are sore and from which the pain radiates to referred areas. such points can be felt as knots or bumps under the palpating finger, which can be moved over the underlying musculature. following the in injection of the local anesthetic the circulus vituosus of increased muscle tension and myofacial pain is interrupted. local ischemia is alleviated and local accumulation of pain mediating substances is flushed out. a typical example is tension-type headache, which is the moist common type of headache. originating from increased stress, it is accompanied by emotional factors and fear. thus the painful condition can be considered of psychosomatic nature. -pain of neurogenic or deafferentiation origin, also termed as complex regional pain syndrome (crps), this type of pain is mostly seen after injury of peripheral nerves leading to spontaneous and paroxysmal discharges. such pain typically is seen as post-herpetic pain, central pain after stroke, diabetic peripheral neuropathy, phantom limb pain, traumatic nerve avulsion, trigeminal neuralgia, lumbosacral plexopathy, all being circumscribed as neuropathic pain. it originates proximal of the peripheral nociceptor ( figure ii- ) , and characteristically is due to a dysfunction or lesion of the peripheral nerve fibers and/or centrally located nervous structures. typically this type of pain is accompanied by a sensory deficit: it is of a burning, shooting, stabbing, piercing, tearing or electricshock-like, paroxysmal and vice-like nature. this pain is of paresthetic, hypoor hyperesthetic quality often is refractory to any opioid therapy. the causes for such painful conditions may be quite different: compared to a normal situation (top), there is spontaneous ectopic firing from increased sodium-channel activity after peripheral nerve injury (middle), which eventually results in central sensitization with stimulus independent pain (bottom). modified from [ ] ectopic spontaneous discharges originating from a lesioned or a cut nerve, this results in an upregulation of sodium-channels being the source of spontaneous discharge. continuous nociceptive barrage later results in central sensitisation and "wind-up" (figure ii- ). partial denervation with spontaneous discharge of nerve activity is followed by an induced release of a nerve growth factor (ngf). such release induces sprouting of fibers into adjacent afferent somatic nerve fibers resulting in an enlargement of the receptive field and an increased conduction of nociceptive impulses to higher pain centers ( figure ii- other topical formulations with capscaicin or emla cream present additional therapeutic options for treatment of neuropathic pain. in addition, transcutaneous electrical stimulation (tens) or even spinal cord stimulation (scs) may present an alternative and effective strategy, resulting in the attenuation of pain. in the latter technique, analgesia is induced by the electrically induced release of endogenous opioids (enkephalins, endorphins, dynorphin) in the spinal cord and within the hypothalamus activating the descending serotoninergic and noradrengergic pathways. -opioids in visceral painful condition. another type of pain, which cannot be treated sufficiently with an opioid, is visceral pain. such a painful condition may arise from the intestine (e.g. the irritable bowel syndrome or ibs) or pain emerging from other internal organs such as the gall bladder, the urinary tract or pain following an appendectomy, cholecystectomy or hysterectomy ( figure ii- ). due to the participation of smooth muscles in such a condition a peripheral analgesic with a muscle relaxant effect can be of advantage. because the sympathetic nervous system to a major part is involved in such a condition, therapeutic implications incorporate a ganglionic blocker, surgical sympathectomy, or intravenous conduction anesthesia with guanethidine. -psychosomatic painful conditions, if treated with an opioid, in the long run are bound to end in opioid resistance. such a painful condition is mainly seen in the depressive patient or it may even be a premonitoring sign of schizophrenia. however, pain can also be part of a conversion-neurotic syndrome [ , ] , where aside from pain fear, phobia, and obsessive-compulsive symptoms are the dominant elements. painful conditions being sensitive to opioids all agonizing painful states, which are due to • posttraumatic • postoperative • ischemic, or of • tumor origin, can be treated sufficiently with an opioid. the rational for the therapy with a central analgesic is related to the fact, that the nociceptive impulses are transmitted via specific pain afferents reaching supraspinal areas. by blockade of specific receptor sites within the brain, opioids induce a reduction and/or result in a total blockade of nociceptive impulses. opioids therefore present the main line of defense in all medium to severe painful conditions. when using opioids one has to realize that this group of ligands, besides their beneficial analgesic effect at the same time also induces a detrimental respiratory depression. this is a mayor drawback when using opioids in acute pain and is directly proportional to their analgesic potency. for instance a potent opioid such as fentanyl already is able to induce respiratory depression in the lower dose range. however, a less potent opioid like codeine or tramadol, even when given in dosages higher than their therapeutic margin, will not induce a clinically relevant respiratory depressive effect ( figure ii- ) . because opioids given for alleviation of chronic pain are given orally and in a controlled release formulation, there is no acute rise in opioid plasma level, which otherwise would induce respiratory impairment. in addition, chronic pain patients cannot be considered as being opioid naïve. their respiratory center already has developed some degree of habituation, being less sensitive to the opioid agent. those opioid ligands, which inherit a lesser respiratory depressive effect, however, are characterized by a comparable reduced analgesic potency. also, a pure -type ligand such as morphine, fentanyl or sufentanil is characterized by a doserelated decrease in respiration until total apnea becomes apparent. contrary, the potent partial agonist buprenorphine with increasing doses demonstrates a ceiling effect, which is seen at a dose of g/kg ( figure ii- ) . typically, when administering high dosages of potent -type ligands such as fentanyl or alfentanil, a time related sequence of effects on respiration can be observed. the progression of respiratory depression is a characteristic trait, which develops within seconds to minutes: . a reduction in respiratory rate (bradypnea) with a partial compensation of tidal volume. . a respiration, which is only kicked off by external stimuli, such as noise or pain. changes in tidal volume (liter/min) in subjects being exposed to increasing dosages of the pure -type ligand fentanyl and the partial agonist buprenorphine. note, the ceiling effect in respiratory depression at higher doses of buprenorphine. adapted from [ ] . a short period where respiration is forgotten, originally termed in europe as "oublie respiratoire", as it was observed in the early times of neuroleptanesthesia when using fentanyl together with a neuroleptic agent for the induction and maintenance of anesthesia. at this stage, however, the patient can be ordered to take deep breaths. . the total apnea, where in spite of any external stimuli or the command to breathe the patient spontaneously will not be able to take a deep breath. he needs immediate respiratory assistance. this centrally-induced opioid-related respiratory depression is due to a blockade of the respiratory regulating centers within the brain stem (pons and medulla), resulting in a lesser sensitivity to an increase in arterial pco and/or a reduction of arterial po [ , ] . in addition the activating reticular system (ars), which descends down into the brain stem, acts as a regulatory pacemaker for the inspiratory center, by which respiratory depressive effect of opioids are affected. this is reflected in the clinics when in addition to an opioid a benzodiazepine is added, which by depressing vigilance, results in an immediate cessation of respiration [ ] . any opioid-induced respiratory depression instantaneously and effectively can be reversed by the administration of a specific opioid antagonist such as naloxone. because of the higher affinity of the antagonist, naloxone displaces the agonist from the receptor site (competitive antagonism), and after binding respiratory depression is reversed and normal ventilation is instigated ( figure ii- ) . clinically, an opioid-related respiratory depression is reversed by titrating the dose of naloxone necessary to • initiate a sufficient spontaneous respiration, however • avoiding an acute abstinence syndrome with tachycardia and hypertension, and at the same time • remaining a sufficient level of analgesia during reversal one should consider the half-life of naloxone, which is between and min [ , ] . therefore "remorphinisation" with a reoccurrence of respiratory depression may appear if the half-life of the agonist is longer than the antagonist, or if high concentrations of the agonist are still circulating in the blood plasma [ ] . following successful reversal it therefore is mandatory to administer an additional dose of naloxone intramuscularly, which acts like a depot, or hook the patient up to a continuous intravenous drip of a naloxone solution, sufficient for long-term receptor occupation by the antagonist. all these procedures, however, do not replace the need for a continuous surveillance, which is necessary in order to detect any possible gradual development of respiratory impairment. respiratory depression can also be reversed by a mixed agonist/antagonist such as nalbuphine. although being less potent than naloxone, it however is one of the mixed ligands having a sufficient antagonistic potency (table ii- ) . at the same time the ligand has a -fold longer duration of action than [ , ] , while the lesser antagonistic potency results in a more gradual and not in abrupt displacement, resulting in lesser sympathetic overdrive [ ] ( figure ii- ) . another pure antagonist, nalmefene shows the longest duration of antagonism with up to h of action [ ] . because of its high antagonistic potency ( . -fold of naloxone) an acute abstinence syndrome can be induced if the necessary dose is not titrated to patients need [ ] . it had been proposed that different receptor sites in the cns mediate opioid-related analgesia and respiratory impairment [ ] . such difference has also been demonstrated for fentanyl-analogues in the animal [ ] , suggesting a clinical relevance for reversal of respiratory impairment, however at the same time maintaining antinociception. such connotation was further corroborated by experimental data where the selective antagonist naloxonazine was able to reverse opioid induced analgesia, but not respiratory impairment. this led to the assumption that -opioid subreceptors are involved in the mediation of opioid-induced respiratory depression (i.e. and ) [ , ] . clinical data seem to underline this assumption, as low doses of sufentanil demonstrated a lesser respiratory depressive effect and a higher analgesic potency when compared to fentanyl. such difference in action reportedly is due to a disparity in receptor affinity to -subsites, with a higher affinity to the -and a lesser affinity to the -receptor [ ] . other experiments, however, suggest that the co-binding of -selective ligands to the -receptor results in respiratory impairment. subanalgesic doses of the -selective ligand d-ala -d-leu-enkephalin, when co-administered with morphine, induced a potentiation of analgesia, while another -ligand d-ala -met-enkephalinamid produced a reduction in analgesia [ ] . such -related differentiation in efficacy was also seen with the potent ligand sufentanil. there respiratory impairment was reversed while at the same time maintaining antinociception using the highly selective -antagonists naltrindol and naltribene respectively [ ] ( figure ii- ) . the implication of / -receptor interaction is further supported by receptor binding studies, where sufentanil demonstrates higher -selectivity than fentanyl (table ii- ) . such putative interaction between -and -receptors is further corroborated when co-administering intrathecally a -and a -selective ligand resulted in a potentiation of effects [ ] . from such data it can be concluded that a coupling mechanism between the -and -opioid receptor not only seems to result in an increase in analgesia, but at the same time also seems to cause respiratory depression. such coupling mechanisms may result in a modulation to potentiation of effects whereby it is still uncertain whether both sites independently operate from each other or whether the -receptor only accentuates the effects induced by -binding. besides a direct action of opioids on the sensitivity of the respiratory center to changes of arterial po and pco , also centrally-induced sedative effects very likely influence respiration. such sedative effects can be derived with the aid of the electroencephalogram where clinically different potent opioids qualitatively induce a different in the eeg pattern. since such eeg changes are dose-related, one is able to derive a dose-relationship. at the same time such eeg-changes reflect the bioavailability of centrally active agents acting on nervous structures of the cns, depicting the effect-concentration site [ , ] . thus, following intravenous administration of an agent, it is not the plasma concentration, which is responsible for a centrally induced effect. more importantly, it is the actual concentration of the opioid at the receptor site, which is affected significantly by issues such as distribution of an agent, its lipophilicity, or the present brain perfusion. therefore vigilance changes can be considered as important aspects in an opioidrelated respiratory impairment being derived in two relevant experiments: . wakefulness by itself already is a fact resulting in sufficient respiration. this could be demonstrated nicely in volunteers where hyperventilation and the resultant hypocapnia resulted in a rhythmic respiratory pattern. if however, the same volunteers were asleep or in anesthesia, hypocapnia was followed by apnea [ ] . . in the animal laryngeal stimulation during anesthesia resulted in apnae, without, however, initiating a cough reflex. being awake, a cough reflex without apnae was induced following laryngeal stimulation [ ] . . there is a close exponential correlation of the physiologic regulatory mechanism affecting respiration. this had been demonstrated after sufentanil application in the canine, whereby increasing dosages of a selective antagonist not only dose-related reversal of sufentanil-induced hypercarbia and hypoxia with the two selective -antagonists naltrindol (nti) and naltribene (ntb) respectively, in the canine. due to the higher lipophilicity of naltribene being able to pass through the blood-brain barrier, there is a superior reversal effect. in spite of increasing doses of the antagonist there is a blockade of response to the electrically induced evoked potential, which is only reversed by the highly specific -antagonist cyprodime. adapted from [ ] reversed the depressed respiratory drive but at the same time induced an increase of power in the high frequency beta band ( - hz) of the eeg ( figure ii- ) , reflecting increase in vigilance [ ] . . clinically such sedative related respiratory depression can also be derived in patients, when cumulative dosages of an opioid reach a point where the respiratory center "forgets" to respond adequately by initiating deep breaths (oublie respiratoire). this is seen in classical neuroleptanalgsia where the patient's vigilance can be increased to a point by external stimuli (e.g. pain, auditory stimuli) resulting in the initiation of an inspiratory effort [ ] . from all these data it can be derived that the simultaneous binding of opioid within the activating reticular system (ars) in the brain stem, vigilance is depressed, which secondarily affects the response of the respiratory center following hypercapnia. at such instances the overall mesencephalic reticular control mechanism is no longer able to adequately respond to a stimulus and only with an increase in vigilance there is an accelerated reactivity, being able to sufficiently respond to an increase in arterial pco . since the reticular mechanism is coupled with reticulo-cortical afferences, such changes can be derived from cortical changes in the eeg. such a "forgotten" reaction to sufficiently respond to a given stimulus [ ] is also seen in the clinical environment when a benzodiazepine is given on-top of an opioid resulting in a further deterioration of respiratory drive. this is because a benzodiazepine depresses the reaction of the ars, and the concomittant reduction in vigilance results in a lessened reaction to external stimuli, producing a clinically relevant suppression of respiration. in general prolongation of respiratory depression after opioid administration has to be expected when the following agents are coadministered: . all agents that inhibit the biotransformation of opioids such as contraceptives, anti-tumor agents, anti-arrhythmics, antidepressants, systemically administered antimycotics, neuroleptic drugs, and volatile anesthetics [ , , , , ] . by inhibition of conjugation of glucoronide and oxidative dealkylation, the necessary metabolic pathways for degradation and termination of activity of most agents, a prolongation of action has to be expected. . all agents, which are able to displace the opioid from protein binding within the plasma, resulting in a higher portion of the pharmacologically active agent. preparations such as cumarine derivatives, and phenylbutazone, which when coadminstered are prone to result in a prolongation of effects [ , , , ] . . in addition, hypoproteinemia and acidosis of the blood, both of which result in lesser protein binding, cause a higher concentration of non-bound opioid in the blood plasma. such increase in plasma concentration now is able to bind to the receptor site with an increase of efficacy and a longer duration of action [ ] . following opioid-based anesthesia, several factors cause an overhang of opioid action, which may even result in a "re-morphinisation" and the re-occurrence of respiratory impairment: . the excessive intramuscular premedication with an opioid, which may act like a depot. . the premedication with a long-acting benzodiazepine, which is able to induce a reduction in vigilance lasting into the postoperative period. . the uncritical intraoperative use of high concentrations of a volatile anesthetic, which results in a lesser biodegradation of the opioid. . the intraoperative administration of fractional doses of an opioid, which results in an accumulation. due to the fact that a portion of each dose of an intravenously administered opioid is also taken up by peripheral sites (e.g. fatty tissue, musculature, skin, internal organs) there is an accumulation of the agent, which act like a depot. from there the drug later diffuses into the blood-stream, resulting in a prolongation of effects ( figure ii- ). . an insufficient loading dose of the opioid, which may result in the necessity of re-administration of small amounts of the drug intraoperatively with consequent peripheral accumulation. . long-term intravenous administration of an opioid by drip, resulting in the increase of the agent in the peripheral compartment with later recirculation into the blood stream. . the combination of opioids with different half-lifes, which may result in an unforeseen potentiation of effects. . uncritical administration of bicarbonate resulting in alkalosis of the blood, which induces a faster release of the opioid from the peripheral compartment. . a non-corrected hypovolemia, which coincides with lesser protein binding of the agent and a higher portion of the free active compound. . uncritical use of a selective antagonist such as naloxone, not considering that its half-life is shorter than the agonist, resulting in a later reoccurrence of respiratory impairment. the sedative effect of opioids goes in hand with their capability to induce sleep (lat. hypnos). such an effect is mostly seen with the mixed agonist/antagonists, while morphine takes a medium position ( figure ii- ) . the hypno-sedative effect of opioids is useful in premedication and during postoperative analgesia, where a sedated status of the patient is advantageous. in contrast to a potent sedative nature of mixed agonist/antagonists, the pure -type ligand fentanyl is characterized by a very low sedative potency. when in the beginning of use of neuroleptic analgesia for anesthesia, fentanyl was used together with the neuroleptic agent droperidol, often patients reported of intraoperative "awareness". although being an obligatory part of anesthesia, sleep, was not sufficiently maintained throughout the whole procedure. therefore in order to guarantee a sufficient level of sleep in patients receiving a fentanyl-based anesthetic technique, an additional hypnotic (propofol), a benzodiazepine (midazolam), a neuroleptic agent (i.e. dehydrobenzperidol), or a volatile anesthetic (sevoflurane, desflurane, or enflurane) has to be given on-top the opioid. nowadays the problem of awareness again has gained much attention [ ] , since the technique of total intravenous anesthesia (tiva) with remifentanil and [ ] propofol, completely omitting nitrous oxide (n o), often results in an insufficient level of sleep with awareness. typically pure -ligands such as bremacozine and tifluadom (table ii- ) , which in comparison to morphine have a -fold analgesic potency [ , ] , do not induce a respiratory depressive effect [ ] . their lack in respiratory impairment is due to the selective binding in deep layers of the cortex [ , ] , where in comparison to the brain-stem, a more than % higher concentration of -binding sites is found [ , ] . their predominant sedative effect is due to centripetal fibers descending down from deep layers of the cortex to the thalamus, thus decreasing the nociceptive input [ ] . although having the advantage of an increased sedative effect combined with the lack in respiratory impairment, clinically, the use of -ligands had to be abandoned. this is because of their intense dysphoric side effects, which lasts for several hours. in addition, their analgesic potency, in comparison to pure -ligands is much lower. therefore such agents cannot be regarded as suitable for intraoperative use, where an intense nociceptive barrage can only be blocked by a potent -opioid. only the mixed agonist/antagonists (e.g. nalbuphine, butorphanol), which exert their analgesic action through binding at the -site, currently are in clinical use mainly for postoperative analgesia [ , ] . this is because cumulative dosages, contrary to a typical -ligand like morphine, result in a ceiling effect for respiratory depression ( figure ii- ). in addition, because of their wide margin of safety, high dosages part ii figure ii- . ceiling effect of respiratory depression following cumulative doses of the mixed agonist/antagonist nalbuphine. in contrast to morphine, at a certain dose there is no further increase in the degree of respiratory impairment. adapted from [ ] have been advocated in balanced anesthesia where the opioid resulted in an up to % reduction in mac (minimal alveolar concentration) of the volatile agent [ , ] . opioids in general induce a dose-related hyposedative component, which is mirrored in the electroencephalogram by an increase of activity in the slow -with concomitant decrease of power in the fast -domain. however, when giving a large bolus dose of fentanyl ( - g/kg body weight) alfentanil ( g/kg body weight) morphine ( - mg/kg body weight) or sufentanil ( - g/kg body weight) an immediate dominance of delta-waves in the eeg becomes evident, being accompanied by sleep. for instance, such effects clinically are seen when high-dose opioid anesthesia is used in cardiac patients for the induction of anesthesia. such a sleepinducing effect is due to a short-term blockade of all afferences being switched in the activating reticular system (ars) of the mesencephalon. aside from a blockade within the nucleus limitans a deep level of analgesia is initiated [ ] . such a "narcotic component", with dominance of delta-activity in the eeg, and contrary to equi-analgesic doses of fentanyl, it is more apparent after sufentanil [ ] , which makes this agent more suitable for the induction of cardiac patients ( figure ii- ) . following induction with a potent -ligand such as fentanyl or sufentanil the initial "narcotic component" later transforms into a "pure analgesic component". this is because the opioid is redistributed, which results in a lesser concentration within the cns and a lesser binding in areas within the ars. at this stage there is a note, a pronounced delta activation after injection and a lesser arousal reaction induced by laryngoscopy and intubation, which in the sufentanil group reflected in a lesser decline of power in the slow delta-domain of the eeg. adapted from [ ] dominance in the -band ( - hz) of the eeg, which is stable, not being affected by any nociceptive stimuli [ , ] . clinically, such an effect has been described for the precursor of fentanyl, the opioid phenoperidine [ ] and for fentanyl [ ] . after a period of - min the deep narcotic component changes into a sedative state, which is stable and cannot be reversed to desynchronization by any nociceptive stimulus. during such "analgesic state" the patient again is able to respond to verbal commands, while at the same time having a deep analgesic level ( figure ii- ) . without the addition of nitrous oxide, such patients are awake, however, nociceptive afferents are not able to modulate the ars, the endotracheal tube is tolerated while at the same time nociception is only sensed as a touch. such phenomena are due to afferents ascending along the spinothalamic tract, which directly ascend to the postcentral cortical area by which the impulse can be localized. collaterals, which ascend through the nucleus limitans within the limbic system and convey nociception, are sufficiently blocked by the opioid and the patient does not perceive pain ( figure ii- ) . the opioid receptor system bordering the fourth cerebral ventricle and the underlying activating reticular system is the relevant anatomical structure in mediating sedation. selective perfusion of increasing concentrations of the opioid fentanyl in the awake canine induced a dose-related enlargement of slow-wave high amplitude delta-activity within the eeg, characterized by a sleep-like behavior ( figure ii- ) . this effect was reversed by the levo-isomer of naloxone inducing an arousal reaction. it, however, was not reversible by the dextro-isomer of the antagonist [ ] . the physiological significance of opioid receptors in the control of vigilance is also reflected in the high density of opioid binding sites in the mesencephalon [ ] . physiologically this is mirrored by an arousal reaction following intense acoustic or a nociceptive stimulus, both of which induce a reversal from the low frequency delta-to high frequency beta-activity in the eeg ( figure ii- ) . in summary, it is concluded that opioids primarily affect the limbic system, the specific site for inducing the negative component of nociception. lastly such assumption is underlined by the result from mc kenzie and coworkers in the animal where the opioids morphine and pethidine were not able to sufficiently block any pain related nervous transmission from the mesencephalon to the higher cortical areas [ ] . in contrast, both ligands were able to block nociceptive transmission from the mesencephalon to hippocampal areas of the limbic system, the part of the cns, which is responsible for the identification of pain, causing the negative, grief, stinging and an intense emotional feeling associated with pain ( figure ii- ) . such differences in pain modulation were corroborated in patients undergoing stereotactic, painful stimulation within specific areas of the cns [ ] . nociceptive afferents of the spinothalamic tract end in the nucleus ventrocaudalis parvo-cellularis part ii figure ii- . selective perfusion of increasing doses of fentanyl through the fourth cerebral ventricle of the awake canine. note, the direct sedative (delta-synchronisation in the eeg) effect via the underlying ars. this effect is mediated through opioid receptors located on the floor of the ventricle, since it was reversible with naloxone (desynchronisation with beta activation in the eeg). adapted from [ ] thalami, from where they further ascend to different cortical areas. since these nuclei reflect a specific somatotopic differentiation, electrical stimulation within this area induced painful sensations in different parts of the body. decoding of painful afferents was only possible when the stimulating electrode was placed within the nucleus limitans, where collaterals of the spinothalamic tract switch to the limbic system. there stimulation induced a less well-localized, however, intense unspecific displeasure [ ] . following the administration of high dosages of opioids with different potency, epileptogenic activities in the eeg with tonic-clonic seizures can be induced in the animal. pethidine (meperidine), morphine, alfentanil, fentanyl and sufentanil when administered in doses above , , , and mg/kg body weight respectively, induced epileptogenic discharges [ ] . because such massive dosages are never used in anesthesia or for analgesia in acute or chronic pain, epileptogenic effects, although being cited in the literature after fentanyl [ , ] and sufentanil [ ] are of insignificant nature. this is because the clinical picture resembles tonicclonic seizures, however, in the eeg no such discharges could be derived [ ] . therefore, those high doses of opioids, which induced epileptogenic activity in the rat [ ] or the canine [ ] are far off from therapeutic range. thus, in general, an epileptogenic activity of opioids can be canceled out. one exception is the use of high dosages of pethidine (meperidine), where the metabolic product norpethidine is a potent epileptogenic compound, which especially in the newborn is able to induce epileptogenic activity [ ] . the cause for the few observations of a pseudoepileptogenic activity of opioids when being administered within the therapeutic range very likely is due to a desinhibition of the cortical motor center within the cns, as this phenomenon was observed during the induction of anesthesia or following a decline in plasma concentration. such assumption is underlined by part ii figure ii- . two main components of painful afferents: localization of nociception (cortical area) and the initiation of the negative component (i.e. the limbic system within the mesencephalon). opioids mainly modify the latter area "epileptogenic activity" during the induction of anesthesia with the pure hypnotic etomidate, where desinhibition of the motor cortex activity was the cause of cortical discharges [ ] . each cough involves a complex reflex arc beginning with the stimulation of sensory nerves that function as cough receptors. there is evidence, primarily clinical, that the sensory limb of the reflex exists in and outside of the lower respiratory tract. although myelinated, rapidly adapting pulmonary stretch receptors (rars), also known as irritant receptors, are the most likely type of sensory nerve that stimulates the cough center in the brain, afferent c-fibers and slowly adapting pulmonary stretch receptors (sars) also may modulate cough. rars, c-fibers, and sars have been identified in the distal esophageal mucosa; however, studies have not been performed to determine whether they can participate in the cough reflex. although gastroesophageal reflux disease can potentially stimulate the afferent limb of the cough reflex by irritating the upper respiratory tract without aspiration and by irritating the lower respiratory tract by micro-or macroaspiration, there is evidence that strongly suggests that reflux commonly provokes cough by stimulating an esophageal-bronchial reflex. each involuntary cough involves a complex reflex arch beginning with the stimulation of sensory nerves in the airway epithelium that function as "cough receptors." efferent impulses from these receptors are conducted by means of the vagus nerve to the "cough center" in the brain stem. because cough can be voluntarily suppressed, controlled, or initiated, there also can be afferent input from the cerebral cortex. the function of this "cough center" is to receive these impulses and produce a cough by activating efferent nervous pathways to the diaphragm and laryngeal, thoracic, and abdominal musculature. the possibility that there might be afferent input other than the vagus nerve and cerebral cortex was based on clinical observations described in case reports and a few animal studies [ ] . histologic studies of the respiratory tract in both animals and humans have revealed sensory nerve endings within the basal layer of the epithelium and between epithelial cells of the larynx, trachea, and bronchi [ ] . these nerve endings are thought to be cough receptors. they contain neuropeptides, such as substance p and calcitonin-gene related peptide (cgrp), which mediate neurogenic inflammatory events in the airways. these sensory nerve endings have been found to be most numerous in the posterior wall of the trachea, at branching points of large airways, and less numerous in the more distal, smaller airways. none have been found beyond terminal bronchioles. it is not known for certain which type of afferent nerve mediates cough. a model that summarizes the current understanding of cough is schematically depicted in the figure. it shows the myelinated, rapidly adapting pulmonary stretch receptors (rars), also referred to as irritant receptors, as the most likely type of sensory nerve that stimulates the cough center in the brain. both mechanical and chemical stimulation of rars have been shown experimentally to cause cough. another type of sensory nerve in the airways, c-fibers, may also participate in regulating cough. they are unmyelinated, vagal afferent fibers that may be activated by the same triggers as rars. their activation releases neuropeptides locally that may secondarily stimulate cough by activating rar nerves. however; impulses transmitted by c-fibers alone probably do not stimulate cough, because experimental evidence has shown that they inhibit cough centrally in the brain. a third type of sensory nerve, the slowly adapting pulmonary stretch receptors (sars), may modulate cough. although these nerves do not directly respond to chemical and mechanical triggers, they do appear to be activated by the deep breath of a cough and may enhance cough by making the expiratory effort more forceful. in addition to mechanical and chemical stimuli, cough has been caused in animals by thermal and electrical provocation. the sites most sensitive to all stimuli are the larynx, trachea, and cannulae of the larger airways. outside of the lower respiratory tract, cough receptors have been demonstrated histologically only in the hypopharynx [ ] . however, it has been inferred from clinical studies that sensory nerve endings subserving the cough reflex via the vagus nerve probably exist in the extemal auditory canals and eardrums, hypopharynx, pericardium, stomach, and esophagus, because stimulation of these sites has been reported to cause cough [ ] . based on the fact that cough can be voluntarily initiated, postponed, and/or suppressed, this provides evidence that there also can be afferent input from the cerebral cortex. in addition to directly stimulating cough by carrying impulses from cough receptors to the cough center, vagal afferents may indirectly provoke cough by another mechanism. they may stimulate neurotransmitter release or mucus secretion from airway submucosal glands that, in turn, stimulate the cough reflex [ ] . the existence of a discrete central cough center is controversial. what is known is that afferent pathways first relay impulses to an area in or near the nuc eus tractus solitarius. these impulses then are integrated into a coordinated cough response in the medulla oblongata of the brain stem, probably separate from the medullary centers, which control breathing. although electrical stimulation studies of different areas in the medulla have evoked cough in animals, suggesting that the cough center is diffusely located [ ] a discrete cough center still may exist, because these electrical stimulations may have activated afferent pathways of the cough reflex. the motor outputs trom the cough center are in the ventral respiratory group, with the nucleus retro-ambigualis sending impulses via motoneurons to the respiratory skeletal muscles and the nucleus ambiguus sending impulses to the larynx and bronchial tree. more specifically, the efferent impulses of the cough reflex are transmitted from the medulla to the expiratory musculature, through the phrenic nerve and other spinal motor nerves, and to the larynx through the recurrent laryngeal branches of the vagus nerves ( figure ii- ) . vagal efferents also innervate the tracheobronchial tree and mediate bronchoconstriction [ ] . although stimulation of cough and bronchoconstriction can be experimentally separated using nonpermeant anions to stimulate cough without bronchoconstriction, these two phenomena normally are activated simultaneously to facilitate the most effective cough. bronchoconstriction may improve clearance of secretions by narrowing the cross-sectional area of the airways, thereby increasing the velocity of air leaving the patients lower respiratory tract during the expiratory phase of coughing [ ] . experimentally, it has been shown in animals that the efferent pathways of the cough reflex are anatomically distinct and separate from the efferent pathways of normal spontaneous ventilation. blockade of the cough reflex arch by means of opioids, known as the antitussive action, refers to the fact that they suppress this protective reflex. this is of benefit during anesthesia and/or in patients being artificially ventilated in the intensive care unit (icu) because it results in the tolerance of an endotracheal tube. however, the antitussive potency differs significantly among the various opioids ( figure ii- ) . the action is not related to a specific receptor site, because a stereoselective action of opioids in regard to their antitussive effect could not be demonstrated. in addition, reversal with the selective antagonist naloxone is less selective [ ] . the mode of action is a blockade of the cough center within the brainstem. three of the most commonly used suppressors of the cough reflex are hydrocodone, codeine and hydromorphone. all of them are characterized by low analgesic potency, they model for afferent limb of cough reflex in airways. myelinated. rapidly adapting pulmonary stretch receptors (rars) and unmyelinated c-fibers are sensory nerves that participate in the afferent limb of the cough reflex. mechanical and chemical stimuli activate sensory nerve enelings in the epithelial layer. rars appear to be the main type of sensory nerve stimulating cough centrally. although c-fibers may inhibit cough centrally, neuropeptides released in the periphery upon stimulation of c-fibers may indirectly stimulate cough by activating rars. slowly adapting pulmonary stretch receptors (sars) do not respond to irritant stimuli that initiate cough but may enhance cough centrally by making expiratory muscular effort more forceful demonstrate a negligible dependence liability, and they are common components in doc (drugs over the counter) cough medicine. a similar antitussive potency, however, is also seen with the more potent opioids such as diamorphine, fentanyl or sufentanil. the latter are used in an opioid-based anesthetic regimen or in icu patients who are in need of ventilatory support. when a potent opioid such as sufentanil is used, the patient is adapted much easier to the respiratory cycle of the ventilator resulting in lesser doses of additional sedative agents. morphine in this regard has a much weaker antitussive activity while pethidine (meperidine) and all mixed agonist/antagonists are characterized by a negligible antitussive action ( figure ii- ) . it can be summarized that potent opioids also inherit a marked antitussive effect, while weak opioids and especially the mixed opioid analgesics are unable to sufficiently suppress the cough reflex. during the induction of anesthesia, while injecting an intravenous bolus dose of a potent opioid such as fentanyl or sufentanil, often a cough reflex is initiated. such part ii dependence or addictive liability (how addictive a drug is likely to be) depends upon how quickly the drug enters/leaves the brain. also, it is directly proportional to the analgesic potency and it depends on the opioid receptor sites with which the ligand interacts. for instance, members of mixed agonist/antagonists (i.e. nalbuphine, pentazocine, butorphanbol) demonstrate a predominant interaction with the -opioid receptor, which is characterized by a low dependence liability ( figure ii- ). in addition, development of dependence also is related to the speed for instance, an opioid like buprenorphine is characterized by a low dissociation constant reflecting long binding to the receptor, a long duration of action and a slow separation from the receptor. the definition of addiction is based in two different terms: . physiological dependence produced by repeated drug-taking that is characterized by a withdrawal syndrome, when drug is removed (e.g. alcohol, opiates). . psychological dependence produced by repeated drug taking that is characterized by obsessions and compulsive drug-seeking behaviors; results in a detrimental impairment in physical, mental or social functioning. there are five classes of abused psychoactive drugs . opioids produce a dream-like state; effects include: analgesic (reduction in pain), hypnotic (sleep inducing), euphoria (sense of happiness or ecstasy) using morphine, heroin, or the cough suppressant codeine. . depressants produce feelings of relaxation/sedation and a dream-like state, anxiolytic (anxiety-reducing) and hypnotic effects; reduce central nervous system activity. members of the class are alcohol, barbiturates, and benzodiazepines. . stimulants increase alertness, arousal, and elevated mood; activate central nervous system (sympathomimetic = mimic the activation of the sympathetic nervous system). members of this class are cocaine, amphetamines, nicotine, caffeine. . psychedelics produce distortions of perception and an altered sense of reality. typical representatives are lsd, psilocybine, mescaline, mdma (ecstasy), and pcp (phencyclidine). . marijuana with one of its major active ingredient thc produces feelings of well-being and sense of acuity (sharpness). it also can produce feelings of relaxation. also, it is recognized, that opioids, which demonstrate a fast onset of action or due to their galenical preparation (injection, smoking) result in an immediate high plasma concentration, followed by an instantaneous receptor binding, results in a "kick" with an euphoric feeling. such preparations therefore are more prone to induce a behavior pattern of abuse. therefore addictive liability or how addictive a drug is likely to be, depends upon how quickly the drug enters/leaves the brain (table ii- ) . most importantly, the tendency of opioids to result in an abuse is very much linked to the fact of why and when the opioid is ingested. for instance, an opioid taken only for the mere pleasure will rapidly result in the development of dependency. contrarily, if an opioid is taken for the attenuation of pain, the likelihood to develop an abuse behavior is very low. aberrant drug related behavior has to be suggested when the following signs of abuse are obvious: in physical examination. a routine physical examination can elucidate common complications of heroin use or assist in diagnosing opioid dependence. chronic intravenous use can be confirmed by the presence of "track" marks, which are callouses that follow the course of a subcutaneous vein. these are caused by repeated injections into adjacent sites over an accessible vein. tracks are often found in easily accessible body areas, such as the backs of the hands, antecubital fossae, on the legs, or in the neck. signs of recent injection may be found in unusual places in patients attempting to hide their sites of injection. a thorough examination for tracks or recent injection sites should include looking between the table ii- . factors that influence how quickly a drug will enter the brain . chemical structure: how fatty is the drug (i.e. its lipophilicity)? does the drug have nutrients that our brain uses? . how fast does the drug cross the blood brain barrier (bbb) lipophilic drugs (i.e. heroin, fentanyl) cross the bbb much faster than hydrophilic agents (i.e., morphine); they mimic nutrients our brain needs and can "slip" through transporters in the bbb. . route of administration: is the drug entering directly into the blood stream or is it entering first into the stomach? the routes of administration increase the likelihood that a drug enters the blood stream whereby it increases the addictive liability of the drug. intravenous injection > smoking/snorting > sublingual application > inhalation via nostrils and lungs because they have abundant blood capillaries and more blood supply under the tongue and the lung respectively. fingers and toes, under the fingernails and toenails, in the axillae, breast veins, and the dorsal vein of the penis. one complication of drug use that can be found on examination is nasal septal perforation from repeated intranasal insufflation (especially when cocaine is mixed with heroin and snorted). a heart murmur may indicate subacute bacterial endocarditis, a complication of intravenous injection without using good sterile technique. posterior cervical lymphadenopathy may suggest early viral infection, especially with hiv. hepatic enlargement may indicate acute hepatitis; a small, hard liver is consistent with chronic viral hepatitis due to hepatitis b or c virus, which are common among injection drug users who share needles. signs of opioid intoxication may include pinpoint pupils, drowsiness, slurred speech, and impaired cognition. signs of acute opioid withdrawal syndrome include watering eyes, runny nose, yawning, muscle twitching, hyperactive bowel sounds, and piloerection. on the other hand the following behaviors are more suggestive of an addiction disorder: • selling prescription drugs • prescription forgery • stealing or "borrowing" drugs from others • injecting oral formulations • obtaining prescription drugs from nonmedical sources • concurrent abuse of alcohol or illicit drugs • multiple dose escalations or other non-compliance with therapy despite warnings • multiple episodes of prescription "loss" • repeatedly seeking prescriptions from other clinicians or from emergency rooms without informing the prescriber or after warnings to desist • evidence of deterioration in the ability to function at work, in the family, or socially that appear to be related to drug use • repeated resistance to changes in therapy despite clear evidence of adverse physical or psychological effects from the drug the following behavior pattern is less suggestive of an addiction disorder. • aggressive complaining about the need for more drugs. • drug hoarding during periods of reduced symptoms. • requesting specific drugs. • openly acquiring similar drugs from other medical sources. • unsanctioned dose escalation or other noncompliance with therapy on one or two occasions. • unapproved use of the drug to treat another symptom. • reporting psychic effects not intended by the clinician. • resistance to a change in therapy associated with "tolerable" adverse effects with expressions of anxiety related to the return of severe symptoms. and while addiction is characterized by a compulsive drug-using and drug-seeking behavior that interferes with normal functioning and causes use of the drug despite increasingly damaging consequences, there are different mechanisms from dependence as addicts can experience intense cravings for drugs even years after sobriety (after body set-points, etc. should have adjusted back to normal). the nervous pathways involved in the development of dependence is the mesolimbic-dopaminergic reward system, where direct activation (i.e. cocaine, nicotine, alcohol) or inhibition of the inhibitory gabaergic neurons in the ventral tegmental area (vta) directly project to dopaminergic neurons in the nucleus accumbens (i.e. opioids such as heroin) results in an increased release of dopamine in the nucleus accumbens and stimulation of the prefrontal area resulting in euphoria. on the other hand, insufficient release of dopamine in this area results in dysphoria, which is seen in abstinence ( figure ii- ). in the acute phase, opioids inhibit adenylyl cyclase and camp. over time, the downstream transcription factor (creb) is activated which increases adenlyly cyclase production ( figure ii- ) . chronic opioid use leads to upregulation of camp and creb, directing to tolerance, dependence and withdrawal symptoms. the significance of intracellular changes in creb is supported by data in mice without creb, which are less likely to develop addiction. also, recent research has also implicated an opioid peptide, dynorphin, in this pathway. besides initial changes in creb, chronic administration of an opioid results in the induced increased formation of peptides syntheses fosb within the nucleus accumbens. such overexpression of fosb increases the sensitivity to cocaine and opioids resulting in an increased likelihood of relapse. in general an opioid-related dependency has to be distinguished from a dependency of the barbiturate-, alcohol-or cocaine-type. this is because they all result in different psychopathological and withdrawal symptoms. the latter is a set of physiological reactions that occur in response to removal of a drug following repeated treatment; often (although not always), the reactions are opposite those produced by the drug itself. in the beginning it is the pleasure seeking behavior that results in repetitive drug administration until finally, the drugs of abuse that produce physical dependence (e.g., opiates or alcohol), results in the avoidance of the unpleasant withdrawal syndrome can contribute to repeated drug-taking ( figure ii- ). the main physiological consequence of nausea and emesis is the removal of toxins, which is an important protective reflex mechanism being induced during food intoxication. it however, is also seen after radiation or chemotherapy, after the administration of an opioid-based anesthetic regimen or during long-term therapy for alleviation of chronic pain. about % of all patients experience nausea and/or emesis after opioid anesthesia. the cause of such reaction is a stimulation of the chemoreceptor biochemical changes induced during addiction and the development of tolerance, which is followed by withdrawal when an antagonist is administered or by lack in maintenance dosages trigger zone (ctz), which lies in close vicinity to the emetic center, bordering the fourth cerebral ventricle, above the area postrema ( figure ii- ). this area is richly supplied with dopaminergic, histaminergic, serotonergic ( -ht ), and cholinergic receptor sites, being the origin of metabolic or drug induced vomiting [ ] . contrary to the other areas within the cns, the ctz is characterized by leaking capillaries (windowed capillaries), through which opioids as well as toxins can disseminate. such anatomical difference indicates that this area does not contain the usual blood-brain barrier (bbb). being located within the dorsal part of the activating reticular formation (ars), all visual, cortical and limbic efferences, as well as efferences of nearby nuclei of the vasomotor center and the center for salivation and respiratory control are switched, resulting in a controlled succession during vomiting. once the vomiting center is stimulated by any of the efferent stimuli, a coordinated sequence of events is commenced: • stop of rhythmical contractions of the stomach, followed by an accumulation of food in the abdomen, resulting in. • retroperistaltic action. • contraction of the cardia with increase of pressure in the stomach. • due to the coordinated contractions of diaphragm, intercostal muscles and the rectus abdominis muscle, food is being expelled forcefully via the opened orifice of the stomach, the dilated esophagus and the opened glottis. because the ctz shows a dense accumulation of serotonin receptors, the serotoninantagonist ondansetron (zofran®) is able to induce an antiemetic effect [ , ] . other agents, which are given for reversal of emesis, are metoclopramide, and/or the neuroleptic agents haloperidol, triflupromazine, or alizapride-hcl, all of which interact through direct binding with the dopaminergic d -receptor. another antiemetic is diphenhydramine, which exerts its action via binding at the cholinergic and histaminergic receptors ( figure ii- ) . postoperative nausea and emesis (ponv), however, still present a problem specifically related to anesthesia. in a large survey with over patients and using multivariance analysis, the following main risk factors for ponv were identified: • female sex • young age • history of ponv/motion sickness • nonsmoking status • long duration of anesthesia each min increase in duration increases ponv risk by %, and a baseline risk of % is increased to % after min [ ] . the type of operation, the addition of nitrous oxide (n o), high age and/or the addition of an opioid to the anesthetic regimen, in comparison to the above risk factors, had a lesser impact on the incidence on ponv [ , ] . commonly the key strategic antiemetic agents for reducing patients ponv are as follows (figure ii- ): the -ht -receptor antagonists are used for both the prevention and treatment of ponv and have a low side-effect profile. they are given toward the end of surgery for greatest efficacy, and are more effective in preventing vomiting than in preventing nausea. dolasetron, granisetron, and ondansetron all have favorable side-effect profiles. no evidence has revealed differences in efficacy and safety among the -ht -receptor antagonists used for the prophylaxis of ponv. a recent study demonstrated the equivalent efficacy and safety of granisetron and ondansetron when these agents were used in combination antiemetic therapy. in this study, low-dose granisetron ( . mg) plus dexamethasone mg was found to be not inferior to ondansetron mg plus dexamethasone mg in patients undergoing abdominal hysterectomy with general anesthesia. the combinations prevented vomiting in % and % of patients, respectively, in the first h after tracheal extubation, and in % and % of patients, respectively, in the h after extubation. dexamethasone has been found to be effective for the management of ponv and their proposed mechanism of action is that of membrane stabilization and inhibition of inflammation. use of this agent is controversial because of its alleged association with delayed wound healing. it has a slow onset but a prolonged duration of action, and therefore it is advised that dexamethasone be administered upon induction of anesthesia. the most commonly used dose for adults is - mg i.v. smaller doses of . - mg have also been used and found to be as effective. based on a quantitative, systematic review of the data, no adverse side effects, especially delayed wound healing, have been noted following a single antiemetic dose of dexamethasone [ ] . the neuroleptic drug droperidol, a butyrephenone derivative, is widely used for ponv prophylaxis and is comparable with ondansetron as a prophylactic antiemetic. similar to haloperidol it acts as a dopamine antgonist at the ctz and the area postrema. for greatest efficacy, droperidol is administered at the end of surgery or concomitantly with morphine via patient-controlled analgesia systems. the use of low doses ( . - . mg) of droperidol has not been associated with the typical side effects of higher doses of this drug (hypotension, extrapyramidal symptoms, sedation, akathisia, dysphoria). in , the food and drug administration began requiring that droperidol labeling include a "black box" warning stating that the drug may cause death or life-threatening events resulting from qtc prolongation and the possibility of life-threatening torsades de pointes. the labeling requirement was based on reported cases associated with droperidol use (at doses of . mg) during its approximately years on the market [ ] . however, no case reports in peer-reviewed journals have linked droperidol with qtc prolongation, cardiac arrhythmias, or death at the doses used for the management of ponv. also, in a randomized, doubleblind, placebo controlled trial, droperidol was not associated with a significant increase in the qtc interval in comparison with saline solution [ ] . in another recent study, droperidol did not increase the qtc interval any more than did ondansetron [ ] . transdermal scopolamine (transderm scop® . mg), an antimuscarinic ganet, works by blocking the cholinergic receptor. it has an antiemetic effect when applied the evening before surgery or h before the end of anesthesia preventing the patient from post-discharge nausea, vomiting and retching. the phenothiazines, promethazine, and prochlorperazine act both as d -and the h -receptor antagonist. they also inhibit histamine receptors and possibly cholinergic receptors in the gut. both have been shown to be effective antiemetics when administered intravenously at the end of surgery. all three drugs may cause sedation, dry mouth, and dizziness. metoclopramide is benzamide that blocks d -receptors both centrally and peripherally in the gastrointestinal tract increasing gastric emptying. the antihistamines, especially diphenhydramine act on both the ctz and the vestibular pathways of the inner ear. at higher doses however, they can prolong general anesthesia and recovery times. consensus guidelines agree that patients at high or moderate risk for ponv are most likely to benefit from prophylaxis. patients at low risk for ponv are usually not candidates for prophylaxis unless their condition may be compromised by the medical sequelae or vomiting. those at moderate risk for ponv should receive antiemetic monotherapy or combination therapy. those at high risk should receive combination therapy with two or three antiemetics from different classes. drugs with different mechanisms of action can be combined for optimal efficacy. for example, the -ht -receptor antagonists (more effective against vomiting) can be combined with droperidol (more effective against nausea). a multimodal approach that incorporates both baseline risk reduction and antiemetic therapy should be adopted for ponv prophylaxis. a recent prospective, double blind, randomized, controlled trial compared three strategies for the prevention of ponv in patients undergoing laparoscopic cholecystectomy: ( ) a multimodal approach using ondansetron, droperidol, and total intravenous anesthesia (tiva) with propofol; ( ) a combination of ondansetron and droperidol, with isoflurane and nitrous oxide-based anesthesia; and ( ) tiva with propofol alone. the complete response rate was higher in the multimodal group ( %) than in the combination group ( %) or tiva-only group ( %), as was the degree of patient satisfaction. nausea and vomiting may persist in some patients after they leave the postanesthesia care unit (pacu). after medication and mechanical causes of ponv have been excluded, rescue therapy with antiemetics can be initiated. for patients who received no prophylaxis, low-dose therapy with -ht -receptor antagonists may be initiated. consensus guidelines also recommend low-dose therapy with a -ht -receptor antagonist for patients in whom dexamethasone prophylaxis has failed. for patients in whom initial -ht -receptor antagonist prophylaxis has failed, a -ht -receptor antagonist rescue therapy should not be given within the first h after surgery. similarly, patients in whom prophylactic combination therapy with a -ht -receptor antagonist plus dexamethasone has failed should be treated with an antiemetic from a different class. as a general guideline, patients who experience ponv within h after surgery should be treated with an antiemetic other than the one used for prophylaxis. for the treatment of patients who experience ponv > h after surgery, drugs from the prophylactic antiemetic regimen may be repeated, except for dexamethasone and transdermal scopolamine, which have a longer duration of action. also, propofol may be used in small doses ( mg as needed) for the treatment of ponv in a supervised environment. the preliminary results of a recent analysis support the recommendation that a rescue antiemetic should be from a class other than that of the original antiemetic agent [ ] . this analysis of a previous trial reported that in patients who failed prophylaxis with ondansetron or droperidol, promethazine was significantly more effective in controlling ponv than the original agent. dimenhydrinate was also more effective than droperidol in patients who failed prophylaxis with droperidol. in summary, the first step in the management of ponv is to identify surgical patients at high or moderate risk for ponv, then reduce baseline risk factors in these patients. combination antiemetic therapy is recommended for patients at high risk for ponv for patients at moderate risk, monotherapy or combination therapy may be considered. a multi modal approach for the prevention of ponv including the use of antimetics with different mode of action, hydration and tiva with propofol has been shown to be most effective. patients who have not received prophylaxis and experience ponv can be treated with a low dose of a -ht -receptor antagonist. in patients who fail prophylaxis treatment with an antiemetic, another agent than the one used for prophylaxis is recommended. opioids can induce muscular rigidity, which is due to an increased tone of the striatal muscle. especially, the muscles of the thoracic cage and of the abdomen show this rigidity, a phenomenon, which is observed after the bolus injection of a potent opioid, such as the fentanyl series (i.e. fentanyl, sufentanil, alfentanil and remifentanil; figure ii- ) . increase in muscle tone is directly correlated to the -receptor interaction, because mixed agonist/antagonists and highly selective -opioid antagonists (i.e. ctap), but not -(e.g. nor-binaltorphine) nor -antagonists (e.g. naltrindole) were able to reverse such muscular rigidity [ , ] . in addition, administration of the selective antagonist methylnaltrexone in the nucleus raphe pontis was able to reverse increased muscle tone after alfentanil in the animal [ ] suggesting this nucleus is an additional important site of action of opioids to induce rigidity. clinically this rigidity is a disadvantage because it results in an insufficient ventilation of the patients and is characterized by the following features [ , ] : • it appears shortly after intravenous injection of a potent opioid. • it can be induced especially in the elderly patient population. • it is potentiated by nitrous oxide (n o). • it is more likely to develop in patients with parkinson's disease. the anatomical correlate by which opioids induce muscular rigidity is the striatum, and, being part of the basal ganglion system, it has the task to control locomotion ( figure ii- ) . within the striatum there is a dense accumulation of opioid binding sites, which interact with dopaminergic d -receptors. similar as in parkinson's disease, there is a reduction in the dopamine level with an ensuing imbalance of the cholinergic transmitter system, both of which are in balance with each other and a necessary prerequisite for the control of muscle tone [ ] . while in parkinson's disease, increased muscle tone is induced by decrease of dopaminergic neurons in the striatum, opioid-induced rigidity is due to an enhanced degradation of the transmitter dopamine resulting in a functional deficit of a sufficient level in the nigro-striatal pathway [ ] . the exact mode of action of opioids to reduce dopamine level within the nigrostriatal system and induce muscular rigidity, very likely is induced by inhibition of tyrosine hydroxylase, the necessary enzyme for the synthesis of dopamine [ ] . due to the interconnection with the inhibitory gabaminergic system, output of gaba in the pallidum declines ( figure ii- ) . this, in turn, causes an overactivity of cholinergic neurons projecting to thalamic neurons. from here the area a of the cortical premotor center is activated and the corticospinal tract leads efferents to the anterior horn of the spinal cord [ ] . although opioids do not directly affect muscle tone, rigidity rapidly can be reversed by the injection of a competitive or non-competitive muscle relaxant [ ] . although the increased efferent output at the neuromuscular junction is not reduced, muscle relaxants induce their action by inhibiting the binding of acetylcholine at the motor endplate ( figure ii- ) . because the gabaminergic system in the putamen is involved in the mediation of opioid-induced muscular rigidity, any increase in gabaminergic transmission can also ease this side effect. such a notion has been supported by results, where a benzodiazepine reduced the increased muscle tone, an effect that could be reversed by the specific benzodiazepine antagonist flumazenil [ ] . in addition, significance of the neurotransmitter dopamine in basal ganglia of the cns, which are involved in the regulation of muscle tone = pallidum externum; = putamen; = nucleus caudatus; = thalamus; = hypothalamus; = lobus parietalis; = central grey; = corticospinal tract; = inhibitory dopaminergic pathway; = thalamo-cortical neurons; = substantia nigra since neighboring -receptors interact with the substantia nigra, opioid-related rigidity could be attenuated by the additional administration of the -agonist dexmedetomidine [ ] . the miotic action of opioids on the pupil is an easily recognizable and quantifiable effect in man. the neural pathways responsible for regulating pupil size are reasonably well defined. yet, the mechanisms behind this and related effects of opioids on the eye in humans and laboratory animals have just begun to be explored. opioid-induced miosis in the human, dog and rabbit is thought to be mediated through the central nervous system. this action is a specific opioid effect as demonstrated by its antagonism by naloxone. theories have been advanced suggesting comparison between groups p < . p < . figure ii- . alfentanil-induced truncal rigidity in patients following induction of anesthesia. in comparison to the rapid bolus injection of the drug, slow injection over a period of min resulted in a significant lesser reduction of thoracic compliance. the increase in truncal rigidity was instantly reversed by a low dose ( mg/ kg body weight) of the fast acting muscle relaxant succinylcholine. adapted from [ ] that morphine produces its effects by direct stimulation of the edinger-westphal (preganglionic parasympathetic) nucleus [ ] . an alternative view has been postulated that morphine depresses cortical centers, which normally inhibit the edinger-westphal nucleus. others have suggested that miosis is caused by stimulation of opioid receptors located on the iris sphincter, although this opinion seems to be in the minority. the exact site, or sites, of action within the cns, which are responsible for opioid-induced miosis remain obscure. it is generally accepted, however, that sympathetic innervation is not essential, the miotic effect being entirely dependent on the integrity of the parasympathetic system. for example, lee and wang [ ] have shown that dogs with a sectioned oculomotor nerve fail to show miosis even with a fold increase in the dose of morphine. in contrast, dogs show normal responses following sympathectomy. while local application of a muscarinic antagonist (scopolamine) that blocks the pupil sphincter completely abolishes the pupillary effects of morphine in the rabbit, application of a sympathetic neuronal blocker (guanethidine) or of an alpha-adrenergic antagonist (phentolamine) that block the pupil dilator had no effect in those experiments. thus, pharmacologic dissection of the autonomic innervation of the pupil suggests that opioid-induced miosis is mediated solely through the parasympathetic system. other cns structures may also be involved in opioid-induced miosis. lee and wang [ ] have shown that removal of the cerebral hemispheres potentiates the miotic action of morphine in the dog. they interpreted this effect as being a reflection of the loss of tonic inhibition originating in the occipital lobes. the latter are known to play a regulatory role in pupillary function, particularly with respect to the near-response (accommodation, convergence and miosis) and, hence, their removal might be expected to alter the pupillary response to drugs. the same authors also observed that acute or chronic optic nerve section did not alter the miotic response to morphine in dogs. in humans, it was shown that morphine produced a dose-related miosis under conditions of low ambient light. taken together, these findings suggest that morphine may cause miosis through more than one mechanism. the main neural structures, which are thought to regulate pupillary size are found in the midbrain, mainly the pretectal area and the edinger-westphal nucleus of the oculomotor complex. because neuronal unit activity in the edinger-westphal nucleus has been shown to correlate with light-induced pupillary constriction. opioids therefore depress or abolish spontaneous and light-induced firing of pupilloconstrictor neurons in the pretectal area, while the opposite effect is observed in the edinger-westphal nucleus where a marked increase in spontaneous firing rate resulting in madriasis. it is because of this increase in activity that certain animal species (rat, cat, monkey) demonstrate an opioid-induced mydriasis. in addition, the brain stem region regulating pupil size is known to have multiple inputs, including the cortex and midbrain, and several others can be assumed to exist. depression by morphine of tonic inhibitory input trom the cortex may partially account for the miosis observed by lee and wang [ ] . these findings suggest that opioids may act directly on the neurons subserving the parasympathetic light reflex. also, in contrast to other workers, morphine has no local action on the iris. for example, lee and wang [ ] could not produce miosis by injecting % of an effective systemic dose of morphine ( mg) directly into the anterior chamber of the eye in dogs. although opioid binding sites have been found in the retina of the rat, cow, toad and skate, opioids injected into the anterior chamber may stimulate retinal receptors in some species, causing miosis via reflex parasympathetic output. following oral ingestion, but also after the systemic administration, opioids also bind to selective receptors located within the intestinal tract. the physiological significance of peripherally located opioid binding sites within the intestine is that of regulation of the propulsive transit. the intestine with a total surface of nearly m is an underestimated important anatomical site as it has a high accumulation of neuronal tissue, which has been termed the enteral nervous system (ens), which acts like a second brain. since there is a close interconnection of the ens with the cns via the vagus nerve, regular impulses to and from the ens are being exchanged. anatomically the intestine is surrounded by two separate syncytial, netlike nervous structures. one is the myentericus plexus (auerbach) located between the longitudinal and the circulatory muscle fibers ( figure ii- ) . the second is the submucosal meissner plexus, located between circulatory and the submucosal muscle fibers. within the auerbach plexus of the intestinal tract, there is a balance between the cholinergic and enkephalinergic neurons: binding of systemically applied opioids to enkaphalinergic receptor sites results in an inhibition of transit followed by constipation. contrarily, cholinesterase inhibitors induce an accumulation of acetylcholine at ach-receptors with an increase in motility and an enhancement of transit. presently, however, not very much is known of the long-term effect of central analgesics on opioid-receptors within the myenteric plexus, and if opioid ligands induce only a constipating effect, whether they also depress the immune system in the intestine or result in a distress the neuroregulatory and endocrine function. while analgesia, respiratory depression, bradycardia, antitussive action and miosis all are centrally induced opioid effects, the most relevant peripheral opiod action is that of constipation [ ] [ ] . this is most relevant in patients with chronic pain taking an opioid for its attenuation. being one of the major side effects, it often results in the necessity to take a laxative on a routine basis. the cause for such constipation is the constriction of the pylorus resulting in a delay in emptying of the stomach [ ] . however, most important, opioids induce a constriction of the small intestine resulting in a delay of the propulsive transit. because selective opioid binding sites are mainly located in the small intestine, opioids inhibit the release of local acetylcholine [ , , ] , followed by a concomitant loss of coordinated propulsive movements of the gut. a constipating effect of opioids on the large intestine is of significantly lesser degree, because this part of the intestinal tract contributes to a much lesser extent to the overall constipating effect. this is because continuous propulsive movements are not seen on this area, and contrary to the small intestine, the percentage of enkephalinergic neurons is significantly lower [ , ] . in addition, enkephalin derivatives are able to inhibit transit in the small while at the same time increasing contractions in the large intestine [ ] . systemicallyselective applied opioids therefore primarily interact with enkephalinergic neurons in the antrum, the duodenum, and the small intestine, all of which results in a delay of transit [ , , , ] . the constipating effect of an opioid can be reversed by a selective peripheral acting antagonists such as methylnaltrexone [ , ] or alvimopane [ ] . opioids, which interact primarily with the -opioid receptor induce a lesser constipating effect [ , ] , while -selective ligands induce no effect on gastrointestinal transit [ ] . comparable to a ketamine-or a volatile anesthetic based regimen, an opioid-based anesthesia results in a longer delay of gastrointestinal emptying in the postoperative period [ , ] (figure ii- ) . this, however, is clinically of little significance, as the potential constipating effect does not last longer than h after anesthesia. contrarily to many other anesthetics, opioids in general do not depress the cardiovascular system. this is also reflected in the higher therapeutic range (ld /ed ) being derived in the animal (table ii- ) . such data can also be conveyed to the human, since a wide therapeutic margin of safety is directly correlated with a lack in cardiovascular impairment. while carfentanil, with a potency twice that of sufentanil, is solely used in veterinary medicine for the immobilization of wild animals [ ] , lofentanil ( fold potency of fentanyl), due to its intensive receptor binding, is characterized by a duration of action of h [ ] . both fentanyl derivates are not in clinical use, because the high potency and the intense receptor binding would be difficult to handle in patients. from the table, however, it is obvious that the higher the selectivity to the receptor site, and the higher the potency, the lesser the amount of cardiovascular depression [ , , , , , ] . following the injection of potent opioids, bradycardia is the most prominent cardiovascular effect seen in patients. this is due to a direct central stimulation of the nucleus nervi vagi and a typical effect of -ligands. thereafter, a reduction of the sympathetic drive is initiated resulting in an overexpression of parasympathetic activity. also, a direct peripheral negative inotropic activity with a potentiation of acetylcholine release at the sinus node of the heart is discussed [ ] . the increase in vagal tone and the reduction of sympathetic drive on the peripheral vasculature results in a decline of mean arterial pressure. a reduction of sympathetic tone on vessel tone and a reduction of resistance is also termed as "pooling" of circulating blood volume. such a reduction of peripheral resistance in certain cases may be of benefit for the patient, as it is accompanied by a reduction in afterload of the heart [ , ] . bradycardia, the reduced peripheral resistance (i.e. afterload of heart) as well as the pooling effect with a reduction of preload of the heart, can be of benefit for a patient with myocardial infarction. this is because those three variables are major determinants in myocardial oxygen consumption (mvo ) [ , ] . it, however, should be noted that the sympatholytic action with pooling of blood volume induced by potent opioids might demask a previously compensated hypovolemic condition in a patient resulting in significant hypotension. for instance, in patients with multiple trauma a reduced dose of the opioid should be given, either diluted or slowly injected while measuring blood pressure continuously. in general, however, especially in polytraumatized patients, opioids are of benefit, as they reduce the stress-related release of hormones and particularly of angiotensin ii, maintaining the effect of circulating catecholamines on the vasculature. opioid-related bradycardia with an accompanying hypotension can rapidly be reversed with increasing doses of the vagolytic agent atropine ( . - - . mg/kg body weight). the incidence and the severity of such a drop in blood pressure cannot be foreseen. it is related to the autonomic basal tone of the patient and the dose of the injected potent -ligand ( figure ii- ) . depending on the product, the autonomic basal tone of, and the applied dosages to the patient, either parasympathetic (inhibitory) and/or a sympathetic (excitatory) symptoms are induced (table ii- ) . such clinical effects can be diminished by atropine, an -blocker (e.g. phenoxybenzamine), a ß-blocker (e.g. propranolol), and a ganglionic blocker (e.g. hexamethonium) respectively [ ] . the stimulatory effects of opioids can also be explained in the laboratory where stimulation of cyclic amp formation, phosphinoside hydrolysis, and the elevation of intracellular calcium, resulting from mobilization of calcium stores and by stimulating influx, which leads to an increased neurotransmitter release and neurotransmission [ ] . thus, at the cellular level these changes may underlie the opioid stimulatory effect. in addition, such stimulation is also discussed as playing a part in the development of tolerance to opioid drugs [ ] . the effect of increasing doses (mg/kg body weight) of potent opioids on the cardiovascular system of the canine, where low amounts result in parasympathetic activation, and high to massive doses induce an increase of sympathetic drive. adapted from [ ] in opioid-based anesthesia, vagal-or sympathetic-induced side effects can be reduced or eliminated by the following techniques: . the preliminary administration of atropine (up to mg/kg body weight). . the simultaneous administration of a volatile anesthetic (n o, enflurane, desflurane, sevoflurane). . the simultaneous use of a neuroleptic agent (e.g. droperidol, haloperidol). . the simultaneous use of a benzodiazepine (e.g. diazepam, midazolam, lorazepam). . the simultaneous use of a hypnotic (e.g. barbiturate, etomidate, propofol). adapted from [ , ] all these agents induce a depression of cns activity in different areas of the central nervous system, which results in equilibrium of the autonomic nervous system discharge, thus, reducing the overshoot of sympathetic and/or parasympathetic tone ( figure ii- ) . mixed agonist/antagonists, when given in dosages above the therapeutic range, induce a cardiostimulatory sympathomimetic effect, which purportedly is induced via stimulation of -receptor sites [ ] . as a result, tachycardia, an increase in peripheral vascular resistance, and an increase in pulmonary artery pressure are induced (table ii- ) , all of which increase myocardial oxygen consumption (mvo ). therefore agonist/antagonists should not be given above their therapeutic range in patients with mi or with a preexistent cardiovascular disease [ ] . a malfunction at the atrio-ventricular node in the myocardium, followed by prolongation of the p-q interval is a phenomenon, which can be induced in patients demonstrating a preexisting abnormal conduction system in the heart. such prolongation manifests itself especially when potent opioids are being administered (fentanyl, sufentanil), whereby the opioid-induced acetylcholine release induces a stimulation of vagal activity. thus, patients already having a prolongation of p-q time or who present a sick-sinus syndrome, extreme bradycardia has to be anticipated, which could result in concomitant cardiac arrest. in order to prevent such a scenario, the opioid should not be given as a bolus, but rather as a diluted solution. in addition, the solution should be injected slowly over a long period of time neuroleptics block afferents from entering the ascending reticular formation, which increase vigilance; tranquillizers protect the hippocampus from an excitatory activation, while barbiturates, hypnotics and volatile anesthetics primarily block the cerebral cortex from arousal table ii- . different cardiovascular effects of -ligands, mixed agonist/antagonists, and partial agonists resulting in a decrease (⇓) or an increase (⇑) blood pressure heart rate pulmonary artery pressure adapted from [ , , ] of at least min. if, however, extreme bradycardia is recognized on the monitor, atropine is the agent of choice ( . - . mg/kg body weight) for rapid reversal. in very extreme cases, the antiarrythmic agent metaproterenol may become necessary, as it is able to increase atrio-ventricular conduction. high doses of methadone or its derivative -levoacetylmethadol (laam) may result in life threatening torsades de points with the potential of ensuing ventricular fibrillation. predisposing factors for the development of such a situation are a prolongation of atrio-ventricular conduction time, hypopotassemia, and/or the simultaneous intake of agents, which inhibit metabolism of the opioid (e.g. tricyclic antidepressants, imidazol derivatives, antimalaria agents, or antihistaminics). a direct negative inotropic effect on the myocardium has been demonstrated in the isolated papillary muscle and in the langendorff preparation of the heart for a variety of opioids [ , ] . such direct effects, however, are not of clinical significance, because such a depression is only evident in concentrations above the therapeutic range. in addition, compensatory cardiovascular and the autonomic regulatory mechanisms come into play when an opioid is given to a subject. following the intravenous injection of pethidine (meperidine, usp), hypotonia and syncope may result. because of the atropine-like molecular structure of this agent, tachycardia, as well as reflex bradycardia can be observed [ ] . for the reason of these potential side effects pethidine should not be given to patients with myocardial infarction [ ] . in addition, it is observed that in a shock-like situation, due to the release of endogenous opioids (enkephalins, endorphins), the additional administration of an exogenous opioid results in an additional occupation of opioid binding sites within the myocardium. this aspect is followed by a negative inotropic effect with an unfavorable consequence on hemodynamics [ ] . some experimental work has postulated a putative direct negative inotropic effect of n o in an opioid-based anesthetic regimen [ ] . since this is mainly seen when n o is given in concentrations above % with a resultant drop in fio , this very both agents dose-dependently reduce adrenaline-induced ventricular extrasystoles. adapted from [ ] pvc-premature ventricular countraction likely is due to an insufficient myocardial oxygen supply. in addition, high concentrations of n o have a direct vasodilatory effect, resulting in a reduction of venous return to the heart and a drop in blood pressure [ ] . it therefore is advocated that in patients receiving opioid anesthesia with a preexisting cardiovascular disease, the optimal concentration in fio should be around . . opioids also have been demonstrated to induce an anti-arrhythmic effect. this has been shown in the animal for meptazinol [ ] and in experimental coronary artery occlusion, using fentanyl, sufentanil and carfentanil respectively [ , , ] (figure ii- ). the reason for such an antifibrillatory effect seems to be due to the increase in vagal tone [ ] . international union of pharmacology. xii. classification of opioid receptors separation of opioid analgesia from respiratory depression: evidence of different receptor mechanism opiate receptors: different ligand affinity in various brain regions identification and characterization of three new alternative spliced mu-opioid receptors the effects of morphine and nalorphine-like drugs in the nondependant and morphine-dependant chronic spinal dog the d-and the l-isomers of methadone bind to the non-competitive site on the nmda receptor in the rat forebrain and spinal cord plasma concentrations of codeine and its metabolite morphine, after single and repeated oral administration novel receptor mechanisms for heroin and morphine- ß-glucoronide analgesia the intrinsic antinociceptive effects of oxycodone appear to be k-opioid receptor mediated ketobemidone, methadone and pethidine are non-comptitve n-methyl-d-aspartate (nmda) antagonists in the rat cortex and spinal cord the binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties selectivity of ligands for opioid receptors, in opioids i. handbook of experimental pharmacology comparison between epidural fentanyl, sufentanil, carfentanil, lofentanil and alfentanil in rats: analgesia and other in vivo effects interaction of -opioid receptor agonists and antagonists with the analgesic effect of buprenorphine in mice european federation of the international association for the study of pain chapters opiate receptor: demonstration in nervous tissue multiple opiate receptors: support for unique mu, delta and kappa sites autoradiographic localization of opiate k-receptors in the guinea pig brain autoradiographic localization of kappa opiate receptors to deep layers of the cerebral cortex may explain unique sedative and analgesic effects characterization of opioid receptors in nervous tissue buprenorphine causes ceiling effect in respiratory depression but not in analgesic effect the clinical pharmacology of buprenorphine: extrapolating from the laboratory to the clinic clinical pharmacology of buprenorphine: ceiling effects at high doses specific uptake of narcotic analgesics by subcellular fractions of the guinea pig ileum in vitro models in the study of structure-activity relationships of narcotic analgesics Über die antagonistischen systeme der schmerzempfindung und des schmerzgefühls im peripheren und zentralen nervensystem a morphine-like factor "enkephalin" in rat brain: subcellular localization determination of methionine enkephalin in discrete regions of rat brain pain mechanisms: a new theory neurotransmitter receptor binding in the brain interaction of naloxone with mu-and delta-opioid agonists on respiration of rats h-sufentanil, a superior ligand for the mu-opiate receptor: binding properties and regional distribution in rat brain and spinal cord alfentanil (r ) -a particularly shortacting narcotic analgesic in rats n- -substituted -( arylethyl)- -piperidinyl-nphenylpropanamides, a novel series of extremely potenet analgesics with unusually high safety margin neuropathic pain: aetiology, symptoms, mechanisms, and management current views on the role of psychiatrists in the management of the chronic pain psychodynamics and psychotherapy in the treatment of patients with chronic pain the effects of morphine and meperidine on the central respiratory mechanisms in the cat: the action of levallorphan in antagonizing these effects respiratory and cardiovascular effects of met-enkephalin applied to the ventral surface of the brain stem fentanyl in the fourth cerebral ventricle causes respiratory depression in the anesthetized but not in the awake dog pharmacokinetics of naloxone in rats and man. basis for its potency and short duration of action prevention of late fentanyl-induced respiratory depression after the injection of the opiate antagonists naltrexone and s- as compared to naloxone pharmacokinetics of fentanyl as a possible explanation for recurrence of respiratory depression analgesic effectiveness of the narcotic agonist-antagonists opioid agonists, antagonists and mixed narcotic analgesics: their use in postoperative and chronic pain management reversal of narcotic-induced respiratory depression with nalbuphine hydrochloride reversal of fentanyl-related respiratory depression with nalbuphine; effects on the co -response curve of man antagonism of postoperative opioid-induced respiratory depression: nalbuphine versus naloxone nalbuphine reverses fentanyl-related eeg changes in man prolonged antagonism of opioid action with intravenous nalmefene in man antagonism of fentanyl-induced respiratory depression with nalmefene enkephalins: isolation, distribution and function multiple opioid receptors mediate the respiratory depressant effect of fentanyl-like drugs in the rat minireview: multiple mu opiate receptors multiple morphine and enkephalin receptors and the relief of pain differences in magnitude and duration of opioid induced respiratory depression and analgesia with fentanyl and sufentanil mu and delta receptors: their role in analgesia and in the differential effects of opioid peptides on analgesia opioid-induced respiratory depression and analgesia may be mediated by different subreceptors the delta receptor is envolved in sufentanilinduced respiratory depression delta opid receptor enhancement of mu opioid receptor-induced antinociception in spinal cord the pharmacokinetics and pharmacodynamics of gi b. anesthesiology context-sensitive half-time in multicompartment pharmacokinetic models for intravenous anesthetic drugs influence of cerebral activity in wakefulness on regulation of breathing waking and ventilatory responses to laryngeal stimulation in sleeping dogs respiratory safety association des analgésiques centraux et des neuroleptiques en cours d'intervention part ii drug interactions of clinical significance with opioid analgesics drug interaction and side effects index™ biotransformation von fentanyl. ii. akute arzneimittelinteraktion -untersuchungen bei ratte und mensch the magnitude and the duration of respiratory depression produced by fentanyl and fentanyl plus droperidol in man a quick guide to common drug interaction, in patient care plasma protein binding of phenytoin after cholecystectomy and neurosurgical operations intravenous fentanyl kinetics morphine and phenytoin binding to human plasma protein in renal and hepatic failure plasma concentrations of fentanyl in normal surgical patients and those with severe renal and hepatic disease reduction in the incidence of awareness using bis monitoring bremazocine: a potent, long-acting opiate kappa-agonist tifluadom (kc- ) induces suppression and latency changes of somatosensory-evoked potentials which are reversed by opioid antagonists bremazocine: an opiate which induces sedation and analgesia but no respiratory depression cellular distribution of the mrna for the k-opiod receptor in the human neocortex: a non-isotopic in situ hybridization study opiate receptor binding sites in human brain utilisation pratique des analgesiques centraux en anesthesie et reanimation patient-controlled analgesia using butorphanol for postoperative pain control: an open label study nalbuphine versus pentazocine in postoperative pain after orthopedic surgery the enflurane sparing effect of morphine, butorphanol, and nalbuphine mac reduction of enflurane and isoflurane and postoperative findings with nalbuphine hcl and fentanyl: a retrospective study ceiling effect for respiratory depression by nalbuphine the "narcotic" component of fentanyl. l'anesthese vigile et subvigile die hypnotische wirkung von fentanyl und sufentanil kardiovaskuläre und zentralnervöse effekte unter fentanyl versus sufentanil bei der intubation herzchirurgischer patienten eeg findings in neuroleptanalgesia central nervous effects of neuroleptanalgesia as induced by haloperidol and phenoperidine exzitatorische und inhibitorische phänomene am zentralnervensystem, verursacht durch fentanyl perfusion of the fourth cerebral ventricle with fentanyl induces naloxone reversible hypotension, bradycardia, baroreflex depression and sleep in unanaesthetized dogs regional distribution of opiate receptor binding in monkey and human brain the effects of morphine and pethidine on somatic evoked responses in the midbrain of the cat, and their relevance to analgesia wechselwirkungen zwischen dem system der schnellen schmerzempfindung und dem des langsamen Über die zweiteilung der schmerzempfindung und des schmerzgefühl, in schmerz comparative study of cardiovascular, neurological, and metabolic side effects of eight narcotics in dogs genralized grand mal seizure after recovery from uncomplicated fentanyl-etomidate anesthesia another case of grand mal seizure after fentanyl aministration tonic-clonic activity after sufentanil seizure-like movements during fentanyl infusion with absence of seizure activity in a simultaneous eeg recording the effects of high-dose fentanyl on cerebral circulation and metabolism in rats opioid analgesics and antagonists, in the pharmacological basis of therapeutics diagnose and treatment of symptoms of the respiratory tract h-codeine binding in the guinea pig lower brain stem physiology and pharmacology of vomiting treatment of postoperative nausea and vomiting after outpatient surgery with the -ht antagonist ondansetron comparison of ondansetron versus placebo to prevent postoperative nausea and vomiting in women undergoing ambulatory gynecologic surgery coinsensus guidelines for managing postoperative nausea and vomiting postoperatives erbrechen -ein score zur voraussage des a meta-analysis of nausea and vomiting following maintenance of anaesthesia with propofol or inhational agents dexamethasone for the prevention of postoperative nausea and emesis-a quantitative systemic review food and drug admistration black box warning on the perioperative use of deoperidol: a review of the cases effect of low-dose droperidol on the qt interval during and after general anesthesia: a placebo-controlled study prolongation of qtc interval after postoperative nausea and vomiting treatment by droperidol or ondansetron the effectiveness of rescue antiemetics after failure of prophylaxis with ondansetron or droperidol: a preliminary report role of opioid receptors in the substantia nigra in morphine-induced muscular rigidity antagonism of dermorphin-induced catalepsy with naloxone, trh-analog cg and the benzodiazepine antagonist, ro - catatonia" produced by alfentanil is reversed by methylnaloxonium microinjections into the brain abdominal muscular rigidity induced by morphine and nitrous oxide studies in muscular rigidity, nitrous oxide and narcotic analgesic agents the effect of fentanyl and droperidol on the dopamine metabolism of the rat striatum morphine katalepsy in the rat: relation to striatal dopamine metabolism tyrosine hydroxylation in the rat striatum after fentanyl and droperidol in vivo decrease of neocortical choline acetyltransferase after lesion of the globus pallidum in the rat attenuation of fentanyl-induced truncal rigidity die lungencompliance wird durch die rasche injektion von alfentanil beeinträchtigt dexemedetomidine, acting through central alpha -adrenoceptors, prevents opiate-induced muscle rigidity in the rat gastrointestinal effects of opioids naloxone and morphine inhibit gastric emptying of solids effect of synaptic transmission blockade on morphine action in the guinea pig myenteric plexus enkephalin-like immunoreactivity in the human gastrointestinal tract autoradiographic localisation of opiate receptors in rat small intestine influence of opiates on colonic motility relative involvement of mu, kappa, and delta receptor mechanisms of opiate-mediated antinociception in mice inhibition of gastrointestinal transit by morphine in rats results primarely from direct drug action on gut opioid sites effect of morphine on gastric emptying a comparison of the effect of oral controlled release morphine and intramuscular morphine on gastric emptying methylnaltrexone prevents morphine-induced delay on oral-cecal transit time without affecting analgesia: a double-blind randomized placebo-controlled trial oral methylnaltrexone for opioid-induced constipation effects of adl - , a peripherally restricted mu opioid anntagost, on gut motility in methadone and laam-dependent patients with opioidinduced constipation. a dose-ranging study the effects of morphine and nalbuphine on intestinal transit in mice peptide opioid antagonist seperates peripheral and central opioid antitransit effects die gastro-coekale transitzeit nach fentanyl/midazolam-im vergleich zur enfluran-und ketamin/midazolam-narkose keine hemmung der intestinalen motilität nach ketamin-/midazolamnarkose immobilization of free-ranging wild animals using a new drug carfentanil and lofentanil sufentanil, a very potent and extremely safe intravenous morphine-like compound in mice, rats and dogs the development of new synthetic narcotics pharmacotherapy of opioids: present and future developments hämodynamische wirkungen hoher dosen von fenanyl, meperidine und naloxon beim hund cardiovascular effects of high doses of fentanyl, meperidine and naloxone in dogs filling pressures of the heart and pulmonary circulation of the patient with coronary artery disease after large doses of morphine control of myocardial oxygen consumption utilisation pratique des analgésiques centraux en anesthésie et réanimation determinants of the stimulatory opioid effect on transmitter release and possible cellular mechanisms: overview and original results stimulatory effects of opioids sigma receptor ligands: function and activity. neurotransmissions comparative effects and analgesic efficacy of the agonist-antagonist opioids a review of its pharmacological properties and therapeutical uses, in new drug series contractile responses to morphine, piritramid and fentanyl: a comparative study of effects on the isolated myocardium myocardial opiate receptor activity is stereospecific, independent of muscarinic receptor antagonism, and may play a role in depressing myocardial function utilisation de la pentazocine comme analgesique pour le traitement des douleurs post-operatoires. etude comparative entre le pethidine, la piritramide et la pentazocine, in utilisation de la pentazocine en anesthesie et reanimation hemodynamic changes following corticosteroid and naloxone infusion in dogs subjected to hypovolemic shock without resuscitation nitrous oxide as an adjunct to narcotic anesthesia does nitrous oxide or a reduced fi alter the hemodanymic function during high dose sufentanil anesthesia? the antiarrhythmic effect of meptazinol effects of high doses of fentanyl on myocardial infarction and cardiogenic shock in the dog the antifibrillatory effect of fentanyl, sufentanil, and carfentanbil in the acute phase of local myocardial ischemia in the dog antifibrillatory action of the narcotic agent fentanyl protective effect of the vagotonic action of morphine sulfate on ventricular vulnerability les effets anti-arrythmiques des opiaces. comparison avec un beta-bloqueur chez le chien. cah d'anesthesiol opposite pupillary effects in the cat and the dog after microinjection of morphine, normorphine and clonioline in the edinger-westphal nucleus mechanism of morphine -induced miosis in the dog key: cord- -x r rlm authors: yokhana, j. s.; parkinson, g.; frankel, t. l. title: effect of insoluble fiber supplementation applied at different ages on digestive organ weight and digestive enzymes of layer-strain poultry date: - - journal: poult sci doi: . /ps/pev sha: doc_id: cord_uid: x r rlm two experiments were conducted to study effects of dietary insoluble fiber (if) on digestive enzyme function in layer poultry. in experiment , wk old pullets were fed a control diet (group c) or a diet (group if) supplemented with % if (arbocel rc). after wk, pullets per group were killed and organ samples collected. the remaining pullets in group c were divided into two groups: half were fed the control diet (group c) and half were given the if diet (group c-if). similarly, half the pullets in group if continued on the if diet (group if) and half on the control diet (group if-c). at wk, organ samples were collected. bw at wk (if, . g; c, . g) and wk (if, . g; if-c, . g; c-if, . g; c, . g) were not different. at wk , the relative proventricular weight ( . g/ g bw) and activities of pepsin ( . pepsin units/g proventriculus/min) and pancreatic general proteolytic activity (gp) ( . μmol tyrosine produced/g tissue) were greater (p < . ) than those of group c (proventricular relative weight, . ; pepsin activity, . ; gp activity, . ). at wk , relative weights of liver and gizzard of group if were heavier (p < . ) than other treatments; activities of pepsin, gp, trypsin and chymotrypsin of if pullets were significantly greater than other treatments as was mrna expression for pepsinogens a ( . vs. . ) and c ( . vs. . ). in experiment , wk old hens were fed a control diet or a diet containing . % if (arbocel rc) for wk. final bw after wk was not different (if, . g; c, . g). pancreatic gp activity was greater (p < . ) in group if hens than group c at wk ( . vs. . μmol tyrosine released/min/g tissue)) as was relative gizzard weight ( . vs . g/ g bw). the significantly improved digestive organ weights and enzyme activities in if pullets may contribute to an improvement in feed utilization. growth and development in layer pullets during rearing and early lay are important to the commercial poultry industry because of their influence on long-term production efficiency (summers and leeson, ) . incharoen and maneechote ( ) showed that dietary insoluble fiber (if) such as whole rice hulls (wrh) at g/kg diet can be used to enhance growth and uniformity of pullets and improve egg production of laying hens. the inclusion in the diet of if from oat hulls, at levels between to % improves the growth performance of broilers fed low-fiber diets and dietary inclusion of % inulin or cellulose (arbocel fd ) as an if source has been shown to reduce feed intake and improved egg production of broiler breeder hens from to wk of age (mohiti-asli et al., the potential benefits of fiber are dependent to a great extent on the physicochemical characteristics of the fiber source. the inclusion of various if products as feed additives in the diets of growing or productive broiler-and layer-strains of poultry have been shown to induce positive changes on digestive physiology (e.g., hetland et al., hetland et al., , mateos et al., ; lim et al., ) . transit time (retention time) of ingested food from the crop to the gizzard and gizzard activity can be increased by if and these physiological effects can improve mixing of feed particles with digestive secretions and hence improve digestion and absorption of nutrients . digestive enzymes secreted by specific organs and regions of the digestive tract such as proventriculus, pancreas, and small intestines are responsible for the hydrolysis of dietary nutrients and play vital roles in the efficient absorption of the products of digestion. adding if in the form of oat hulls and wood shavings to broiler and layer diets has been shown to benefit nutrient digestion (hetland et al., (hetland et al., , possibly as a result of an increased amylase concentration in the chyme of the jejunum (hetland et al., ) . the majority of experiments examining the effects of if on the functioning of digestive enzymes have examined the effects on pancreatic or intestinal lipases, amylases and peptidases (hetland et al., ; taylor and jones, ; sarikhan et al., ; svihus, ) . there has been very little systematic work on the effects of if on proteolytic enzymes of the different supply organs of the digestive tract (proventriculus, pancreas and small intestine) of layer-strain poultry. enhanced activity of such enzymes may improve the supply of amino acids for body and reproductive organ growth, which continue after supply organs have developed, and may thus contribute to improve egg production. despite pepsin digestion being the first stage in the enzymatic digestion of protein, there has been very little investigation into dietary stimulation of pepsin function or enhancement of synthesis of pepsinogens in poultry. pepsinogens are the zymogen precursors of the active enzyme pepsin, and they are synthesized in poultry in the oxyntic cells of the proventriculus that also secrete hydrochloric acid (hirschowitz, ) . in adult poultry, two pepsinogens have been detected, pepsinogen a (cpga) and pepsinogen c (cpgc). pepsinogen a is secreted in greater amounts than cpgc (donta and van vunakis, ; sakamoto et al., ) . the first experiment was designed to determine whether the addition of a commercial if product (arbocel rc, jrs co. inc., rosenberg, germany) in the diets of young layer-strain pullets between the ages of and wk influenced supply organ growth and proteolytic enzyme activities in the proventriculus, pancreas, and small intestine. in addition, the experiment was designed to determine whether there was a residual effect of if on the supply organs and enzyme activities wk after it was removed from the diet. the effect of starting the if feed at a later age, wk, was also determined. concentration of pepsin in proventricular tissue can be affected by hormones such as neurotensin and food in the gut (degolier et al., ) . on the other hand, pepsinogen mrna activity is unlikely to be affected by short-term effects on pepsinogen secretion from oxyntic cells. we therefore wished to determine whether there was a correlation between pepsin activity and mrna in proventricular tissue so as to provide an alternate means of measuring the effects of dietary changes on digestive function. the second experiment was to determine the effect of the commercial if product arbocel rc on enzyme activities of layer hens kept under commercial conditions from point of lay at to wk of age through early egg production to wk of age. in experiment , -week-old hy-line brown pullets were obtained from a commercial supplier in melbourne, victoria. all pullets were kept in the one house containing pens ( . × . × . m, w × l × h) with slatted floors, two experimental diets were used: a control diet (table , ridley agriproducts pty ltd. , pakenham, , australia) without additives (diet c) and the if diet consisting of the control diet plus % if (arbocel rc fine, jrs co. inc., rosenberg, germany which contains (manufacturer's analyses) % crude fiber and % acid detergent lignin). the analyzed concentrations of crude fiber in the two diets were . % and . % for diet c and diet if, respectively (feedtest, agrifood technology pty ltd, werribee, vic., australia). feed and water were provided ad libitum. in experiment , hy-line brown hens between the ages of and wk old were obtained from a commercial egg producer (country lane poultry farm, tynong north, victoria australia). the company was testing, for their own information, whether addition of . % arbocel rc to the feed they normally used, would increase egg productivity, decrease cannibalism and decrease water content of the manure. they agreed to let us collect hens from their feeding trial. two diets were formulated for the company by ridley agriproducts pty ltd. the control diet (table ) was fed to half the hens in the layer house (group c) and the other diet containing . % arbocel was fed to hens in the other half of the layer house (group if). the concentrations of crude fiber in the two treatments diets were analyzed by the company and were . % for the control diet and . % for the if diet. the layer house held , hens and had fully automated temperature and humidity control with temperature sensors placed throughout the house to monitor conditions and adjust ventilation accordingly. there were cages per tier of the -tier a-frame units; hens were housed five per cage and cage floor area was cm per bird. hens were placed in the house at to wk of age. the control diet was fed to hens in one half of the house and the if diet was fed to hens in the other half of the house. feed and water was provided at all times. experiment on the day of arrival leg-bands were placed on all pullets and they were weighed. fortyeight pullets were allocated randomly to pens, four birds per pen. pullets in six pens were fed the control diet (group c) for the first wk and those in the remaining six pens were fed the % if diet (group if). pen treatments were randomized within the house. after wk, when the pullets were wk of age, six pullets (one per pen) were randomly selected from each treatment and killed with an intravenous overdose of pentobarbitone sodium (lethabarb, virbac animal health, milperra, nsw, australia). within min of the absence of a detectable palpebral reflex, the liver, gizzard, proventriculus, pancreas, small intestine and ceca were removed. the gizzard was cleaned of contents and both it and the liver weighed. whole proventriculus and pancreas were rinsed with cold . % saline, blotted dry, weighed and stored at - • c for later analysis of enzyme activities. the contents of the small intestines (jejunum and ileum) were washed out with . % saline then the tract was divided into three equal lengths (proximal, medial and distal sections) and cm segments from the middle of each section were collected, weighed separately and stored at - • c for later analysis of enzyme activities. the contents of the ceca were washed out with saline. at wk the remaining pullets in group c were divided so that eight pullets continued on the control diet (group c) and eight were given the if diet (group c-if). eight of the pullets from group if continued to be fed the if diet (group if) and eight were given the control diet (group if-c). the pullets were housed in three pens per treatment with two pens of three and one of two pullets. after a further wk ( wk after the start of the experiment) when the pullets were wk of age, they were killed and samples collected as before. in addition, tissues samples from the proventriculi of pullets in groups c and if were taken and stored in rnalater r (cat. no. r , sigma-aldrich, castle hill, nsw, australia) at - • c for later analysis of pepsinogen gene expression. the experiment was conducted in accordance with the principles and specific guidelines of la trobe university animal ethics committee (ltu-aec) project number aec - . experiment because we had no control of the experimental layout of the layer house with hens in one half of the house being fed the control ration (group c), and hens in the other half of the house being fed the ration containing . % if (group if), it was decided that hens from the middle tiers of the two central rows in the house would be taken for sample collection. by doing so, samples would be taken from hens that were kept under as similar environmental conditions (but not feed and water delivery) as possible. at , , , and wk after the start of the feeding trial, eight hens from group c and eight from group if were randomly collected, one hen per cage. the hens were collected from cages along the length of a row of cages. the hens were then transported by road (approximately h) to the university campus and weighed, killed, and tissue samples (excluding small intestines) were collected as in experiment . the experiment was conducted in accordance with the principles and specific guidelines of ltu-aec, project number aec - . proventricular pepsin activity the assay for pepsin activity was based on the method of anson ( ) as modified by susbilla et al. ( ) and delia ( ) . the whole proventriculus was thawed and homogenized on ice with a polytron homogenizer (kinematica ag, lucerne, switzerland) in a . m sodium buffer, ph . the homogenized samples were centrifuged and an aliquot of diluted supernatant was added to a test tube containing . % acidified bovine hemoglobin (cat. no. h , sigma-aldrich) substrate, ph . . the samples were incubated at • c in a shaking water bath (julabo sw, west germany) for min and the reaction stopped with % trichloroacetic acid (tca) solution. the sample was filtered and neutralized with . m sodium bicarbonate solution. free tyrosine released through hydrolysis of hemoglobin was measured with folin-ciocalteu's phenol reagent (cat. no. f , sigma-aldrich) and absorbance measured at nm in a spectrophotometer (model u- spectrophotometer, hitachi, japan). a tyrosine standard curve (cat. no. t , sigma-aldrich) was prepared from standard solutions ranging in concentration from to mm. porcine stomach mucosal pepsin (pepsin a, ec . . . , cat. no. p , sigma-aldrich) was used to convert tyrosine released to pepsin units. pepsin was dissolved in . m sodium phosphate buffer, ph and standards of to μg/ml produced. tyrosine released from hemoglobin by pepsin was measured as for the proventricular tissue. one unit of pepsin activity was defined as μmol of tyrosine released/min/g proventricular tissue. pancreatic general proteolytic (gp) activity the method of susbilla et al. ( ) with slight modification by delia ( ) was used to determine the general proteolytic activity of the pancreas. whole pancreas was homogenized in ringer's solution, ph . . the tissue extract was activated for h on ice, with enterokinase (from porcine intestine, cat. no. e , sigma-aldrich) in tris-hcl solution, ph . . the substrate ( % casein, cat. no. c , sigma-aldrich in a . m sodium phosphate buffer, ph ) was then added to a test tube containing tissue extract. after incubating in a shaking water bath at • c for min, the reaction was stopped with % tca (wt/vol) and the solution filtered through whatman no. filter paper. after addition of . m na co , free tyrosine in the solution was measured with folin-ciocalteu's solution. the absorbance was read at nm and amount of tyrosine released from casein was determined from a tyrosine standard curve in . m sodium phosphate buffer, ph . the gp activity was expressed as μmol of free tyrosine released from casein/min/g pancreatic tissue. pancreatic trypsin and chymotrypsin activities these were measured by the method of erlanger et al. ( ) as modified by caviedes-vidal and karasov ( ) . pancreatic homogenate plus enterokinase solution was incubated for h in a shaking water bath at • c. trypsin activity was then determined with a mm nα-benzoyl-dl-arginine -nitroanilide hydrochloride (dl-bapna, cat. no. b , sigma-aldrich) solution, ph . and chymotrypsin activity with mm n-glutaryl-l-phenylalanine -nitroanilide (gpna, cat. no. g , sigma-aldrich) solution, ph . . samples for trypsin and chymotrypsin determinations were incubated in a shaking water bath at • c for min. the reaction was stopped with % acetic acid and the absorbance was read at nm. the amount of -nitroaniline released from a substrate was obtained from a standard curve of -nitroaniline (cat. no. n , sigma-aldrich) in . m tris-hcl buffer, ph . , for trypsin activity and ph . for chymotrypsin activity. trypsin (ec . . . ) from bovine pancreas, (t , sigma-aldrich) was used to convert -nitroanaline concentration to trypsin units (delia, ) . standard concentrations of trypsin were incubated with mm dl-bapna solution, ph . and -nitroaniline measured as for the pancreatic tissue. one unit of trypsin activity was defined as μmol of -nitroanaline released/min/g pancreatic tissue. to convert -nitroanaline concentration to chymotrypsin units, standards of αchymotrypsin (ec . . . , type ii from bovine pancreas, cat. no. c , sigma-aldrich) were prepared in a mm hcl and incubated with mm gpna solution, ph . , as for pancreatic tissue. one unit of chymotrypsin activity was defined as μmol of nitroanaline released/min/g pancreatic tissue. small intestinal dipeptidase and aminopeptidase activities the dipeptidase activity in each of the three intestinal segments (proximal, medial, and distal) was measured according to the method described by delia ( ) . homogenates of each segment were incubated for min with the substrate, mm glycyl-lleucine (ec . . , cat. no. g , sigma-aldrich) in ringer's solution, ph . , in a shaking water bath at • c. after filtering through whatman no. paper, the absorbance was measured at nm. the amount of glycyl-l-leucine hydrolyzed by the activity of dipeptidase in the small intestine was determined by calculating the decrease in absorbance. dipeptidase activity per g intestinal tissue (nmol of glycyl-l-leucine hydrolyzed per g of jejunal plus ileal tissue/min) was calculated from the sum of the average activities determined for proximal, medial and distal sections. the method of caviedes-vidal and karasov ( ) was used to determine aminopeptidase-n activity. homogenates of each segment were incubated with mm l-alanine- -nitroanilide hydrochloride (cat. no. a , sigma-aldrich) in a . m sodium phosphate buffer, ph , at • c for min in a shaking water bath. the absorbance was read at nm and concentration of -nitroaniline formed was calculated from a nitroaniline standard curve in . m sodium phosphate buffer, ph , at concentrations ranging from to . mm. the activity of aminopeptidase in the small intestine (jejunum and ileum) was calculated as for dipeptidase activity and was expressed as μmol of free nitroaniline released from l-alanine- -nitroanilide per g jejunal plus ileal tissue/min. the protein concentrations in the homogenate extracts of the tissues were measured using the method of lowry et al. ( ) . bovine serum albumen (cat. no. a , sigma-aldrich) was used as standard. for the proventriculus, albumen was prepared in . m phosphate buffer, ph . , while for pancreas and small intestine it was prepared in ringer's solution, ph . . after incubation, tyrosine concentration was measured using folin-ciocalteu's solution. total mrna was extracted from proventricular tissues ( mg) using ml of trizol reagent (invitrogen, life technologies, australia) according to the manufacturer's instructions. a nano drop nd- spectrophotometer (thermofisher scientific, scoresby vic., australia) was used to quantify the extracted mrna. only samples with an absorbance a / ratio of . to . were used. extracted mrna was further purified with ambion x turbo dnase buffer (cat. no. am , life technologies) according to the manufacture's instructions and stored at - • c. the synthesis of cdna from extracted mrna was carried out with a dynamo tm cdna synthesis kit (cat. no. f- l, thermofisher scientific, australia) as recommended by the manufacturer. a thermal cycler (stratagene mx p qpcr system, agilent technologies, santa clara, ca), was used to convert the mrna into cdna. primers for target and reference genes were, pepsinogen a (ab ) forward gggtgccctctatc-tattgc, reverse cagtgtcatagcccaggatg; pepsinogen c (ab ) forward ggtgtcctact-gtgcctgtg, reverse cctggtttgtgatgg agatg; β-actin (nm˙ ) forward atggctc-cggtatgtgcaa, reverse tgtctttctggcc-cataccaa (grommen et al., ) ; glyceraldehyde- -phosphate dehydrogenase (gapdh) forward gccatcacagccacacaga and reverse tttcc-ccacagccttagca (azzam et al., ) . simplex real-time qpcr analysis (rt-qpcr) was conducted using sensimix sybr low-rox kit (cat. no. qt - , bioline australia pty ltd) in a stratagene mx p qpcr system (agilent technologies, santa clara, ca). each well of a -well plate contained the same volume of both forward and reverse primer of either a target or a housekeeping gene along with template cdna (or dnase-free water for no template controls) and reagents according to the manufacturer's instructions for the kit. for each sample a mixture of all reagents was added to a sterile . -ml microcentrifuge tube and mixed well. the first two wells of a -well plate contained buffer instead of the cdna template and were used as a control. from each . -ml microcentrifuge tube that contained a -μl reaction mixture a -μl was aliquot added to each well ( replicates per sample). agarose gel ( % agarose powder (cat. no. bio- , bioline (aust) pty. ltd., alexandria, nsw, australia) electrophoresis was carried out to confirm the purity and size of the bands of the pcr products. the pcr reaction composed of taq buffer, dntps, forward and reverse primers, taq dna polymerase, cdna template and nuclease-free water (nfw) according to the manufacturer's instructions (qiagen-australia, doncaster, australia). calculation of gene expression primer efficiency of the target genes was tested using five dilutions of cdna in duplicate. complementary deoxyribonucleic acid (cdna) was used and diluted as neat (no dilution), : , : , : and : in nfw. efficiency of pcr was determined for both the target and endogenous control genes from the standard curves generated with different dilutions of the cdna synthesized from the chicken proventriculus using the formula pcr efficiency = (− /slope). the slope values were calculated from standard curves and used in the following formula (harrison et al., ; gopinath et al., ) to obtain the corrected cycle time (ct): where ct is the mean cycle time of the target gene ct, nt is the mean ct of housekeeping genes of all the experimental samples, ct is the mean ct of housekeeping gene of the particular sample, s is the target gene slope, andŚ is the endogenous control slope. homogeneity of variances and normality of data were all test using ibm spss statistics version . for windows (ibm corporation, armonk, ny) before analysis of significance between treatments was carried out. for experiment , significance between treatments of pullets sampled wk after the start of the experiment were analyzed with an independent t-test (spss . for windows) with six pen replicates. for samples collected wk after the start, the three pen replicates per treatment were analyzed by one-way anova; when a significant difference between means was observed, post hoc significance between treatment means was determined using the bonferroni test. linear regression of pepsinogen a and pepsin activity was determined with spss. for experiment , each hen was an experimental unit as no cage of five hens was sampled more than once. differences between means were analyzed with an independent t-test. a value of p < . was considered statistically significant. table . experiment live body weights (g) and relative weights (g/ g live-weight) of internal organs of wk old hy-line brown layer strain pullets given a control diet † (c) or a diet supplemented with %, insoluble fiber † (if) for wk then either diet c or diet if for a further wk (mean, pooled sem). ‡ weight ranges for hy-line brown layer-strain, wk - g; wk - g (hy-line international, ) n = (pen replicate) at wk, n = (pen replicate) at wk. * means at wk within the same row are significantly different (p < . ). a,b means at wk within the same row with different superscript letters differ, p < . . c = control diet (no added if) from to wk. if = insoluble fiber (if) diet containing % arbocel rc fine, from to wk. c-if = control diet from to wk; if diet from to wk. if-co = if diet from to wk; diet c from to wk. inc., rosenberg, germany. ‡ normal weight ranges for hy-line brown layer-strain, wk , - , g; wk , - , g; wk , - , g; wk , - , g (hy-line international, ) . * means within the same row at same time point differ (p < . ). the presence of % if in the diet of pullets for five (groups c-if and if-c) or wk (group if) resulted in heavier bw than controls (group c, table ) but there was no significant difference among the treatments. in experiment , the bw of hens in group if were also not significantly different from control hens ( table ). the inclusion of if in the diets had no detrimental effect on bw of the pullets or laying hens as, in all treatments in experiment (table ) , mean bw was heavier than the normal ranges for hy-line brown pullets and in experiment the bw of both treatment groups (table ) was within the normal ranges (hy-line international, ) . others have shown that if can improve bw gain in both layer-strain and broiler birds, and it is possible that weight gain may be affected by age or developmental age at which if is given or by the strain of poultry used. incharoen and manee-chote ( ) showed that if supplied as whole rice hulls to younger pullets and of a different strain than used here, -to -week-old hn nick brown layer-strain pullets, resulted in a significant increase in bw. gonzález-alvarado et al. ( ) showed that bw gain in broiler chicks was significantly improved by increased fiber content of the diet from -to -day-old but was not affected in younger birds and bogus lawska-tryk ( ) showed that feeding broiler chickens from to d of age with arbocel bww- at four different levels (the final amounts added to the finisher ration being (control), . %, . %, and . %) led to significantly increased bw at the lowest and highest levels when compared with controls. feeding the if diet for or wk in experiment did result in significant increases in the relative weights of some organs (table ) . after being fed the if diet for wk the relative weight of the proventriculus in wk old pullets (experiment , table ) was significantly greater than that of the pullets on the control diet. feeding the if diet for a further wk to wk of age resulted in significantly greater relative weights of the liver and gizzard (but not proventriculus) compared to the weights in the control (group c) and other two treatments in which if was fed for a shorter period (table ) . a significant difference among relative weights of the small intestines was seen between those of group if and group c-if pullets (table ) although those from group if were heavier than small intestines in the other two groups (groups c and if-c). no other significant differences were found among relative weights of organs in experiment . insoluble fiber has been shown to affect the weights of supply organs and can affect functioning of the git, digestibility of feed and feed conversion of poultry (e.g., hetland et al., hetland et al., , mateos et al., ) . addition of pea fiber, wheat bran and oat bran either at low ( g/kg) or high levels ( g/kg) in broiler diets was shown by jørgensen et al. ( ) to cause a significant increase in the weight of the git tract which is similar to the results of experiment in pullets given the if diet for wk. the effects of if on gizzard and proventricular weights shown here are similar to those in an experiment by gonzález-alvarado et al. ( ) , in which if was included in the diets of broiler chicks from to d of age: the if was included either as % oat hulls or % soy hulls, in low-fiber diets (crude fiber . %). oat hulls caused a significant increase in gizzard weight: proventricular weight was only significantly increased with soy hulls (gonzález-alvarado et al., ) . thus source of if (and therefore proportions of the different fiber components) can influence organ development differently. similar to the experiment reported here using arbocel rc, bogus lawska-tryk ( ) also reported no effect of the if source, arbocel bww- , on pancreas weight. adding if to poultry feeds has been shown to change the weight and length of the git (siri, ; viveros et al., ; jørgensen et al., ; smits et al., ) which supports the results obtained in experiment . taylor and jones ( ) suggested that increased gizzard size could be a reason for the reduction in weight of the small intestine they found and may reflect an adaptation of the gut to increased availability of nutrients as a result of feeding a diet containing increased fiber concentration. a welldeveloped gizzard can positively enhance digestion as food is retained for a longer time in the upper digestive tract (proventriculus and gizzard) and this may activate digestive secretions from specific organs and result in more efficient nutrient assimilation (jones and taylor, ) . gonzález-alvarado et al. ( ) showed that fiber improved total tract apparent retention of feed and feed conversion and it also caused a decrease in the ph in the gizzard. fewer experiments using if have been carried out on layer-strain poultry than broilers; however, freitas et al. ( ) showed that wk old hy-line brown pullets given increased dietary neutral detergent fiber (ndf) for wk had significantly increased liver and intestinal weights but gizzard weight, unlike that in experiment , was reduced. in the adult hens in experiment only the gizzard at wk and liver at , , and wk after the start of the trial showed significantly higher relative weights. others have reported on the effects of if on organ weights in adult hens: the weights of gizzard and pancreas in -week-old leghorn hens (hetland et al., ) were similar to those of the hy-line brown hens in experiment and the authors reported an increase in relative gizzard weight with increased if intake. the authors (hetland et al., ) also showed there to be a significant increase in the relative weights of the gizzards in -week-old leghorn pullets supplemented with if as wood shavings for wk when compared with unsupplemented controls. the effect of if on concentrations of protein in proventriculus and pancreas differed between the two experiments reported here. the only significant difference in protein contents of the proventriculus were seen between groups if and c after wk in experiment (table ). however in the laying hens in experiment (table ) significantly higher protein contents were seen in pancreatic tissue at , , and wk after the start of the experiment in group if hens compared with those in group c. overall, the results of the experiments reported here show that organ weights can be increased by the addition of if to diets of growing pullets and in hens after the start of egg-laying. such effects may help to contribute to improved feed utilization as has been reported by others. besides the effects of if on live-weights and supply organ weights the experiments reported here show significant effects of if supplementation on the activities of proteases required at the different stages for digestion of dietary protein. five wk after the start of experiment , proventricular pepsin and pancreatic gp and chymotrypsin activities (table ) , were higher (p < . ) in group if pullets than those in group c. after wk on those diets (table ) , activities of pepsin, pancreatic gp, trypsin and chymotrypsin were significantly higher in group if than group c pullets. this may suggest that a longer time of feeding if is required to induce a significant response. feeding the if diet to the older pullets ( to wk of age) for wk (group c-if) had no significant effect (compared to controls) on activities of pepsin or the three pancreatic enzymes measured (table ). although activities of pepsin and pancreatic gp, trypsin and chymotrypsin in pullets fed first the if and then the control diets from to wk of age (group if-c) were always higher than those of controls (table ) significant differences in enzyme activities in the pancreatic enzymes were only table . experiment enzyme activities (per min/ g tissue) and protein content (mg/ g tissue) of wk old hy-line brown layer-strain pullets given a control diet † (c) and the control diet supplemented with % insoluble fiber † (if) for wk then either diet c or diet if for a further wk (mean, pooled sem). n = (pen replicate) at wk, n = (pen replicate) at wk. * means after wk on experimental diets within the same row are significantly different (p < . ). a-c means at wk within the same row with different superscript letters differ p < . . for definitions of c, if, c-if and if-c, see table . pepsin = pepsin activity in proventriculus, pepsin units μmol tyrosine produced/ min/g proventricular tissue. gp-pancreas = general proteolytic activity, μmol free tyrosine released from casein substrate/ min/g pancreas. chymotrypsin = chymotrypsin units of -nitroaniline released from n-glutaryl-l-phenylalanine -nitroanilide (gpna)/ min/g pancreas. trypsin = trypsin units of -nitroaniline released from nα-benzoyl-dl-arginine -nitroanilide (dl-bapna)/min/g pancreas. dipeptidase = μmol glycyl-l-leucine hydrolyzed/min/g tissue. aminopeptidase = nmol -nitroaniline released l alanine-p-nitroanalide/min/g tissue. for units of activity and abbreviations see table . seen for gp activity when compared with that of group c but trypsin activity was significantly greater than activities in groups c and c-if (table ). for small intestinal dipeptidase and aminopeptidase activities in experiment no significant differences were observed among treatments after or wk on the experimental diets (table ). in the laying hens (experiment ), the significant effects of if were less than in the younger pullets in experiment , and this could have been due the lower concentration of if used in the second experiment ( . versus . %). pepsin activities (table ) increased gradually with age from to wk of age in both treatment groups however pepsin activity in group if hens was significantly higher after wk compared with those in the control group. pancreatic gp activity in group c hens (table ) appeared to decrease with age while at all collection times ( , , , and wk) gp activities were significantly higher in in group if hens than those of hens in group c. for trypsin activities of the pancreas (table ) the only significant difference between treatments was seen at wk, with group if having higher activities. chymotrypsin activities (table ) in pancreatic tissues were significantly higher in group if compared with those of the control group at nine and wk after the start of the feeding trial. the mrna expression in proventricular tissues of week-old pullets was greater for cpga than for cpgc in both treatment groups. pullets in group if had significantly greater expression of both pepsinogen a (corrected ct . ± . ) and c (corrected ct . ± . ) compared to those in group c where corrected ct for cpga mrna was . ± . and that for cpgc, . ± . . there was a positive correlation (p < . ) between cpga mrna activity and pepsin activity (y = . x + . , r = . , n = ). because of the significant increase of both proventricular pepsin activity and mrna pepsinogen expression in if pullets (experiment ), it is unlikely that the higher levels of pepsin activity that were measured here were due to reduced secretion of pepsin (gabriel et al., ) into the proventricular lumen. decreased pepsinogen release would contribute to a higher retention of pepsinogen in the tissues (gabriel et al., ) . the significantly greater mrna expression of pepsinogens a and c in the pullets given if reflects the higher pepsin activities determined in proventricular homogenates and probably reflects an increase in production of the zymogens of pepsin enzymes. the similarity in increase between mrna of pepsinogen a and that of pepsinogen c suggests that the mechanism of stimulation of the two zymogens was similar and could have been caused by physical effects on the oxyntic (or oxynticopeptic) cells of the proventriculus or by one or more of the following that have been shown to influence pepsinogen production: increased production of cholecystokinin (cck), decreased ph in the proventriculus and increased reflux of bile from the small intestine via the gizzard (sommer and kasper, ; duke, ; li and owyang, ; sacranie et al., ; jiménez-moreno et al., ; svihus, ; sacranie et al., ) . pepsin activity is stimulated by low ph and possibly through an increase in solubility of mineral salts (incharoen and maneechote, ) and others have reported on the decrease in proventricular and gizzard ph caused by if (e.g., hetland et al., ; svihus, ) . the results obtained here on increased trypsin and chymotrypsin activities in the group if pullets after wk on if (experiment ), confirm those of bogus lawska-tryk ( ) who showed that trypsin and chymotrypsin activities were increased relative to controls in -day-old broilers fed if as arbocel bww- . regulation of the production of pancreatic enzymes is through hormones such as cck, secretin and gastrin produced in the digestive tract (sommer and kasper, ; li and owyang, ; owyang and logsdon, ; wang and cui, ) . hormone production in the intestinal mucosa of pullets fed if could have been increased through increased intestinal tissue production (table ) , and this in turn could lead to increased production of zymogens of the pancreatic enzymes and thus to measurement of an increased activity of the enzymes. significant increases in pancreatic gp and chymotrypsin activities were seen in both experiments reported here when if was added to diets of layer-strain pullets or hens but trypsin activity was only significantly increased in pullets in experiment . these different responses could have been due to the stage of maturity of the birds or to the differences in fiber concentration of the diets fed to the poultry in the two different experiments. the effects of increased enzyme activity in if supplemented poultry may have contributed to the observed increase in weights of supply organs in both experiments but it is not possible to identify the specific components of the if that caused such effects. the commercial product, arbocel rc, used in these experiments contained % crude fiber with % lignin (manufacturer's data) and is probably very different in composition from sources of if used by other research workers. the results of these experiments showing increased proteolytic digestive enzyme activities in the proventriculus and pancreas and in growth of supply organs may explain in part the improvements in protein digestibility reported by others following if supplementation in poultry (e.g., jaroni et al., ; jiménez-moreno et al., ) . however, in order to be able to understand mechanisms by which if can alter enzyme activities and or organ growth and to be able to understand why different if sources produce different effects, it will be necessary to identify more accurately the different types and proportions of the constituents making up the if sources used in poultry feeds. the estimation of pepsin, trypsin, papain, and cathepsin with haemoglobin effect of supplemental l-threonine on mucin gene expression and intestine mucosal immune and digestive enzymes activities of laying hens in environments with high temperature and humidity effect of different levels of cellulose in the diet on the proteolytic activity of the pancreas in broiler chickens developmental changes in digestive physiology of nestling house sparrows, passer domesticus neurotensin decreases pepsin output and gastrointestinal motility in chickens protein requirements, digestion and taste preferences of two lorikeet species chicken pepsinogens and pepsins. their isolation and properties gastrointestinal motility and its regulation the preparation and properties of two new chromogenic substrates of trypsin fiber level for laying hens during the growing phase differences in the digestive tract characteristics of broiler chickens fed on complete pelleted diet or on whole wheat added to pelleted protein concentrate effect of type of cereal, heat processing of the cereal, and inclusion of fiber in the diet on productive performance and digestive traits of broilers rapid detection of newcastle disease virus replication in embryonated chicken eggs using quantitative real time polymerase chain reaction identification of unique thyrotropin receptor (tshr) splice variants in the chicken: the chicken tshr gene revisited lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture effects of oat hulls and wood shavings on digestion in broilers and layers fed diets based on whole or ground wheat role of insoluble fiber on gizzard activity in layers role of insoluble nonstarch polysaccharides in poultry nutrition hy-line brown management guide -commercial layers control of pepsinogen secretion the effects of dietary whole rice hull as insoluble fiber on the flock uniformity of pullets and on the egg performance and intestinal mucosa of laying hens the effect of dietary wheat middlings and enzyme supplementation ii: apparent nutrient digestibility, digestive tract size, gut viscosity, and gut morphology in two strains of leghorn hens effects of source of fibre on the development and ph of the gastrointestinal tract of broilers the incorporation of whole grain into pelleted broiler chicken diets: production and physiological responses the influence of dietary fiber source and level on the development of the gastrointestinal tract, digestibility and energy metabolism in broiler chickens vagal afferent pathway mediates physiological action of cholecystokinin on pancreatic enzyme secretion beneficial effects of insoluble raw fiber concentrate addition to layer diet protein measurement with the folin phenol reagent poultry response to high levels of dietary fiber sources varying in physical and chemical characteristics effects of feeding regimen, fiber inclusion, and crude protein content of the diet on performance and egg quality and hatchability of eggs of broiler breeder hens new insights into neurohormonal regulation of pancreatic secretion the effect of insoluble fiber and intermittent feeding on gizzard development, gut motility, and performance of broiler chickens reflux of digesta and its implications for nutrient digestion and bird health analysis of temporal expression pattern and cis-regulatory sequences of chicken pepsinogen a and c effects of insoluble fiber on serum biochemical characteristics in broiler effects of dietary cellulose level on nutrient utilization in chickens. asian-aus dietary carboxymethylcellulose with high instead of low viscosity reduces macronutrient digestion in broiler chickens effect of acetylcholine, gastrin, and glucagon alone and in combination with secretin and cholecystokinin on the secretion of the isolated perfused rat pancreas laying hen performance as influenced by protein intake to sixteen weeks of age and body weight at point of lay quantitative feed restriction or meal-feeding of broiler chicks alter functional development of enzymes for protein digestion the gizzard: function, influence of diet structure and effects on nutrient availability the incorporation of whole grain into pelleted broiler chicken diets. ii. gastrointestinal and digesta characteristics effect of enzyme supplementation of a diet based on barley, and autoclave treatment, on apparent digestibility, growth performance and gut morphology of broilers how does cholecystokinin stimulate exocrine pancreatic secretion? from birds, rodents, to humans the ministry of higher education and scientific research-iraq (moher) provided scholarship for johnny yokhana. chris rowell from hy-line company and peter cransberg from ridley agriproducts pty ltd. and also to farm pride at tynong north, victoria provided assistance in acquiring birds and diet formulations. colleagues, diana delia, samuel armoo and vignesh rathinasamy provided information and assisted with lab work. the central animal house staff and in particular, rob evans and eva suric, helped with care of pullets. key: cord- - obi r authors: bretscher, lorenzo; hsu, alex; simasek, peter; tamoni, andrea title: covid- and the cross-section of equity returns: impact and transmission date: - - journal: rev asset pricing stud doi: . /rapstu/raaa sha: doc_id: cord_uid: obi r using the first reported case of covid- in a given u.s. county as the event day, we find that firms headquartered in an affected county experience, on average, a -bps lower return in the -day post-event window. this negative effect nearly doubles in magnitude for firms in counties with a higher infection rate (- bps). we test a number of transmission channels. firms belonging to labor-intensive industries and those located in counties with a large mobility decline have worse stock performance. firms sensitive to covid- -induced uncertainty also exhibit more negative returns. finally, more negative stock returns are associated with downward revisions in earnings forecasts. deepening economic depression started to set in as grim gross domestic product (gdp) growth predictions for the second quarter are updated. in this paper, we document that the domestic spread of in the united states has a negative and significant impact on firm equity valuations. in particular, we show that the pandemic negatively affects firms through its impact on labor supply and the uncertainty it generates. furthermore, the shock propagates through the equity market by putting downward pressure on analyst earnings forecasts. we attempt to address two questions: ( ) how does the sudden collapse in economic activity caused by affect equity valuation in the cross-section of firms? and ( ) what are the channels through which the covid- -induced economic downturn propagates and affects firm-level outcomes? the first question has important implications for investors' portfolio choices, banks' lending policies, and managers' investment decisions. the second question is key to understanding the effectiveness of policy interventions to stabilize the economy. using geographical dispersion of firm headquarter locations and the fact that the pandemic spread through the united states with variable timing, we perform a difference-in-differences estimation to understand the effect of the pandemic on equity returns. we find that, on average, daily returns of public firms are basis points (bps) lower in the day window right after the first case of is recorded (event day) in the county where the firm is headquartered. when we consider counties with high infection rates ( or more cases reached in less than days), the negative effect of the shock nearly doubles to bps. that is, returns are lower relative to days before the event day as well as to returns of firms residing in counties that have never experienced a covid- case during our sample period. the transmission of from a health care crisis to a corporate slump may be driven by several different economic mechanisms. to investigate the transmission mechanism, we focus on four potential, and not mutually exclusive, channels: labor supply, uncertainty, government spending and monetary policies, and cash flow expectations. in particular, the stay-at-home orders resulting from the spread of the virus may depress workers' ability to travel to work, while the largely unknown factors surrounding the disease generated considerable uncertainty about future economic activity. some firms may be less affected, thanks to existing government contracts providing stable demand. ultimately, given the widespread and rapid advance of , we anticipate analysts to frequently revise their earnings expectations. various recent studies and many economists predominantly describe the impact of the pandemic on the economy as a demand shock. indeed, because of social distancing measures and closure of public gatherings, consumer demand dropped significantly during march . the resultant momentous rise in unemployment further suggests that demand will be depressed for the foreseeable future. however, this does not rule out the pandemic as a supply shock. in fact, guerrieri, lorenzoni, straub, and werning ( ) suggest that negative supply shocks can turn into negative demand shocks. using a multisector model in incomplete markets, they show that a negative supply shock can trigger changes in aggregate demand as firms exit and jobs disappear. we believe that our natural experiment combining the geographical dispersion of firms with the staggered impact of provides a unique setting to examine the relative strength of various demand and supply channels through which the shock affects firm valuations. in our empirical setting, we rely on large public firms which sell their goods and services across the entire continental united states, and in many instances, internationally. hence, our county-level valuation effect due to the presence of confirmed virus cases is unlikely to be a pure demand-side phenomenon. in fact, our baseline regression results suggest that the crisis negatively affects the supply-side and labor productivity. specifically, to study the labor supply channel of the propagation, we estimate difference-in-differences regressions on a subsample of "labor-intensive" firms. we classify as labor-intensive firms within the mining, construction, and manufacturing sectors. the regression results show that the average daily return of a labor-intensive firm residing in a high intensity county is % lower in the -day post-event window. this suggests that the negative effect of shocks on equity returns is considerably stronger for laborintensive firms. in other words, the pandemic negatively affects economic output through its impact on labor supply. to further tie return dynamics to labor supply, we use the university of maryland mobility data to document that the countylevel percentage of people staying at home increased following the first reported case of coronavirus in the county. the decline in mobility due to the virus spread suggests that the pandemic negatively affects labor supply. next, we sort counties according to changes in the percentage of people staying at home before and after the first reported case. we find that firms with headquarters located in those counties with large increases in the percentage of people staying at home have more negative stock returns in the days following the first reported case compared to firms residing in counties with small changes in the percentage of people staying at home. taken together, results using mobility data provides evidence that the virus spread causes the population to stay at home more, which worsens the financial performance of local firms. using the date of the first reported case as opposed to another threshold allows us to separate out the effect stemming from the second moment (uncertainty) of the shock from its first moment (level). for example, the productivity level may possibly not fall immediately after the first case is reported, but business activities still can be negatively affected in expectation of a fall due to the uncertainty. to tease out the impact of covid- -induced uncertainty on equity returns, we employ data from hassan, hollander, van lent, and tahoun ( ) . these authors use textual analysis of earnings call transcripts to obtain firmlevel measures of risk exposure. the risk measure is related to the mentioning of words synonymous to "risk" or "uncertainty" in transcripts, and by construction it captures the management's attitude toward we find that firms concerned with the elevated uncertainty induced by exhibit particularly negative returns after the event day. moreover, we examine whether firm-level exposure to government spending and monetary policies can produce the heterogeneous return reaction we observe in the data. for government spending, goldman ( ) shows that government contractor firms weathered the financial crisis relatively better compared with noncontractor firms. we apply the goldman ( ) definition of government contractors to our sample of firms and check whether their stock outperformed the noncontractors after the first case of the coronavirus was reported in the county the firm is headquartered. in contrast to goldman ( ), we do not find this to be the case, which highlights a fundamental difference between the pandemic and the financial crisis. large government contractors are primarily in labor-intensive manufacturing industries leaving them more susceptible to the negative labor supply shock induced by the pandemic. focusing on a subsample of labor-intensive firms, we find that government contractors' stock returns are relatively more negative than noncontractors following the first reported case. hence, contractors enjoy stable demand due to contractual relationships with the government, but their stocks perform worse. this result suggests that government contractors might be particularly reliant on the presence of the labor force they employ. furthermore, we do not find evidence that heterogeneous sensitivity to monetary policy drives the cross-sectional return dynamics following the covid- news. the last channel of transmission we investigate concerns how the spread of covid- affects expected corporate earnings. to this end, we use analysts' forecast data from the i/b/e/s database and document that the first reported coronavirus case results in downward revision of earnings estimates of firms located in the same county. moreover, we find that firms with negative earnings revisions experience greater stock return declines relative to firms with no revisions or positive revisions in the -day window after the first case of is reported in a county. taken together, we deduce that the spread of covid- leads to financial market fragility partially by depressing the expected cash flows of affected firms. our work is related to a fast-growing literature on the asset price response to the pandemic in the first quarter of . gormsen and koijen ( ) look at the aggregate equity market and dividend futures during the outbreak to infer bounds on future gdp growth. baker et al. ( b) use text-based methods to study the u.s. stock market reaction to the covid- pandemic. cejnek, randl, and zechner ( ) provide evidence that index-level and singlestock near-maturity dividend futures reflect expectations of dividend cuts. pettenuzzo et al. ( ) analyze the signaling role of dividend announcements during the pandemic by looking at the stock market reaction of firms that changed their dividend policy. alfaro, chari, greenland, and schott ( ) show that aggregate equity market returns respond to daily unanticipated changes in predicted cases. toda ( ) uses a sir model to study the impact of the epidemic on the stock market. in a cross-country setting, ru, yang, and zou ( ) find that stock markets reacted more quickly and strongly in countries that had a sars outbreak, whereas gerding, martin, and nagler ( ) show that stock price reactions were stronger in countries with higher debt-to-gdp ratios. in a cross-country and cross-asset-class setting, croce, farroni, and wolfskeil ( ) study how epidemic news identified from twitter are reflected in asset prices. they conclude that the market price of contagion risk is very high. distinct from these studies, we do not focus on the aggregate u.s. equity market, but rather we rely on geographical heterogeneity of firm headquarters to directly a large number of papers investigate the effect of pandemics on economic growth, rather than asset prices. see barro, ursua, and weng ( ) , correia, luck, and verner ( ) , and ludvigson, ma, and ng ( ) . several papers also model the interaction between economic activity, economic decisions, and epidemic dynamics (see, e.g., alvarez et al., ; eichenbaum et al., ; jones et al., ; van binsbergen and opp, ) . more generally, various streams of papers extend the canonical epidemiology sir model to an exploration of the covid- epidemic (see, e.g., atkeson, ; favero, ) . identify the consequences of the pandemic in the cross-section of u.s. equity returns. in this respect, our work contributes to a large number of contemporaneous papers that investigate the firm-and industry-level price responses to covid- . albuquerque, koskinen, yang, and zhang ( ) focus on the performance of firms with high environmental and social ratings during the covid- outbreak. ramelli and wagner ( ) focus on the importance of trade (e.g., china-oriented stocks) and financial policies for firm value. these authors document that firms with more leverage and minimal cash holdings suffered severely in the period from february through march , even if these firms did not have international activities. they interpret this finding as evidence that business uncertainty caused by is amplified through certain financial channels. interestingly, hassan, hollander, van lent, and tahoun ( ) document that financing concerns are mentioned relatively rarely in earnings conference transcripts as covid- spreads globally. on the contrary, these authors find evidence for decreasing demand, disruption of the supply chain and closure of production facilities, and increased uncertainty as major concerns for firms. papanikolaou and schmidt ( ) construct an industry-level measure of exposure to work disruptions, and investigate whether the cross-sectional differences are predictive of differential economic outcomes during the pandemic. pagano, wagner, and zechner ( ) investigate how companies' exposure to social distancing not only during the outbreak but also prior to and after the outbreak affects asset prices. despite sharing a similar interest on the cross-sectional response of stock prices to the pandemic, we differ from all these papers in two respects. first, our econometric analysis combines the geographical dispersion of firms with the staggered impact of and is akin to the classical difference-in-differences approach commonly used in corporate finance. second, and related, a unique advantage of our natural experiment is that it allows us to examine the relative strength of various channels (specifically, the labor supply channel, the uncertainty channel, the government policy channel, and the cash flow news channel) through which the covid- shock affects firm valuations. we also contribute to broader streams of literature in finance and economics. first, our analysis of the labor supply channel speaks to the literature studying how labor-induced operating leverage affects asset prices. for example, donangelo, gourio, kehrig, and palacios ( ) show that high labor-share firms have operating profits that are more sensitive to shocks and have higher expected asset returns. moreover, donangelo (forthcoming) shows that operating leverage due to labor is important in explaining the value premium. belo, lin, li, and zhao ( ) , belo, lin, and bazdresch ( ) , and belo, donangelo, lin, and ding ( ) study how labor market frictions affect asset prices, and favilukis and lin ( a,b) study the consequences of rigid wages on asset prices and return predictability. our analysis of the labor supply channel also contributes to the recent theoretical contributions. for example, in the eichenbaum, rebelo, and trabandt ( ) model, an epidemic has both aggregate demand and aggregate supply effects. guerrieri, lorenzoni, straub, and werning ( ) develop a theory of keynesian supply shocks that trigger changes in aggregate demand larger than the shocks themselves. these authors argue that the economic shocks associated to the covid- epidemic may have this feature. second, our analysis of uncertainty as a possible propagation channel through which the news of the virus's spread onto asset prices speaks to a large literature on second moment shocks. starting with bloom ( ) , an increasing body of research studies how uncertainty shocks influence economic activity and asset prices. veronesi ( , ) examine the response of the equity risk premium to government-induced (political) uncertainty. croce, nguyen, and schmid ( ) show that a reduction of model uncertainty can come at the cost of depressing growth for the long-run. finally, bianchi, kung, and tirskikh ( ) evaluate the effect of different sources of uncertainty (i.e., demand and supply uncertainty) on macroeconomic and financial outcomes via structural estimation. third, our analysis of the possible effects of fiscal and monetary policies during the pandemic complements the literature that has investigated the effectiveness of various policy measures during the great recession (e.g., adelino et al., ; chodorow-reich et al., ; goldman, ; wilson, ) . lastly, our investigation of the reaction of earning forecasts of analysts and their relation to returns during the pandemic contributes to a vast literature that analyzes the roles of cash flow expectations and discount rates for asset prices. gormsen and koijen ( ) and landier and thesmar ( ) are two contemporaneous studies that look at these two channels during the covid- outbreak exploiting aggregate s&p dividend strips and ibes forecasts, respectively. similar to landier and thesmar ( ) , we also use ibes forecasts and focus on the crosssection of returns. we believe that our staggered difference-in-differences setup, and our analysis of returns reaction over a tight window around the event day, complements their analysis of cumulative returns response to the covid- crisis. we obtain public firm ticker symbols from compustat at the end of . using these tickers, we download daily closing prices from bloomberg from december , , to march , . we stop the sample on march th to minimize the effect of the cares act on stock prices. the cares act was passed on march , , and, by design, affects specific firms and industries in a heterogeneous manner. we then calculate daily returns for all firms in the sample using the end of day prices. we then drop firms that have at least one missing price or if the price dips below $ a share on any given trading day since the beginning of . the number of headquarter firms in our data set located in a particular u.s. county firm headquarter locations are also obtained from compustat. we convert zip codes to geographic identifiers (geoid) using a crosswalk from the u.s. census bureau. geoid's are unique to u.s. counties, which allows us to merge firm headquarter locations with the case data from the new york times. figure reports the density of firms in our data set headquartered in a particular county. the completed data set matches trading day-firm observations (firm characteristics as of the end of ) and the calendar day on which the first reported case appears in the county where the firm is headquartered. table illustrates the geographical distribution of firms in our sample by state. although california, new york, and texas dominate in having the highest number of firm headquarters, in that together they account for roughly one-third of the sample, substantial heterogeneity in headquarters locations across the united states is evident. in the appendix, as a robustness check, we repeat our baseline tests excluding the three populous states and document similar findings. using the day on which the first covid- case is reported as the event day (covid- ( ) dummy), we examine firm-level returns before and after the event day. we truncate our data at calendar days postevent day such that our post-event window is from day to day (post- . this choice aims to minimize the confounding effect due to other news arrivals. figure displays the evolution of cases by county. the figure reports time windows that contain the first reported case by county. the first window spans a longer time period as testing was initially sparse. table presents summary statistics of firms in our study. statistics (in millions of dollars) of total assets (compustat variable: at), capital expenditure (capx), sales (sale), property, plants, and equipment (ppent), and operating income (oibdp) for the baseline sample are shown in panel a. the sample comprises a total of , trading day-firm observations. these are large firms on average with mean total assets of $ . billion and median of $ . billion. they have substantial sales with mean of $ . billion and median of $ million. these firms are also highly profitable with average operating income of more than $ billion. taken together, table suggests the firms in our sample represent the largest companies in our economy. this matters for our identification strategy for the supply channel of the shocks. because these companies are so large, it is unlikely their sales are highly concentrated in the same county where the firm is headquartered. therefore, if the first reported case of in the county where the headquarters resides affects stock returns, the effect is more likely to be transmitted through the supply channel. table panels b, c, d, and e are firm summary statistics broken down by industry, defined by their fama-french (ff ) industry classification. for labor-intensive firms (ff industry to ) firms in panel b, the average total assets is $ . billion and the average sales is $ . billion. for retail (ff industry ) firms in panel c, the average total assets is $ . billion and the average sales is $ . billion. for financial (ff industry ) firms in panel d, the average total assets is $ . billion and the average sales is $ . billion. for services (ff industry ) firms in panel e, the average total assets is $ . billion and the average sales is $ . billion. the average operating income across all major industries is well over $ billion. we conclude that large firms with high sales volume are well distributed throughout various industries in our sample. in this section, we examine the impact of the covid- pandemic on firm-level equity returns. we devise a natural experiment by combining the geographical heterogeneity of firm headquarter location with the staggered spread of the virus to identify the causal relationship between and firm performance. the date on which the first case of is reported in a given county in the united states defines our event. the differential timing of the first reported case across counties allows us to pin down the treatment effect of covid- on the crosssection of firms. employing difference-in-differences estimations and spline regressions, we compare stock performance of firms before and after the first reported case as well as between firms located in and non- (no reported covid- case during our sample period) counties. to establish our baseline result, we perform a diff-in-diff estimation of firm-level returns in the event window around the first confirmed case of , or event day, in the county a given firm headquarter is located. specifically, we regress the panel of daily log returns on the dummy and the post- dummy as shown in equation ( ): ( ) coefficient loadings β and γ are meant to capture the difference in returns on the day of the event and in the -day post-event window, respectively, compared with returns prior to the event day. returns of firms residing in a county which never reported a covid- case in the sample period also serve as part of the control group. to control for unobserved heterogeneity along several dimensions, we include firm (ψ i ), industry (ρ j ), county (δ c ), and day (φ t ) fixed effects in the analysis. standard errors are double clustered at the county and trading day levels. the full sample comprises , trading day-firm observations. column of table presents the results for the baseline diff-in-diff regression. although the estimated coefficient for the ( ) dummy is insignificant, the estimated coefficient for the post- dummy is negative and statistically significant at the % level. the point estimate of - . suggests that returns are on average bps lower in the -day post-event window relative to returns in the control group. in column , instead of using raw returns, we use total returns as the dependent variable. total returns are obtained from bloomberg, and adjust for near-term cash dividends. the point estimate on the post-covid- dummy is - . , very similar to the counterpart in column . using excess return over the effective fed funds rate leaves the results unchanged. this also holds true for all results discussed below. given the baseline finding showing a localized impact of covid- on firm-level returns, we postulate that this effect should be stronger in counties where the spread of the virus is more intense. we then construct a growth intensity measure for each affected county using the number of days for reported cases to go over (see figure ). employing only observations in high growth intensity counties ( days or less), we repeat the diff-in-diff regression in equation ( ) and tabulate results in columns and of table . for raw returns, the γ coefficient is still negative and significant at the % level; more importantly, the magnitude has increased to - . , almost doubling relative to the estimate in column . for total returns, the coefficient loading strengthened from - . in column to - . in column . see figure for a graphical representation. the divergence between cumulative returns in high growth versus low growth intensity counties widens dramatically after the first reported case. these findings validate our hypothesis that in locations considered "hot zones" (likely more densely populated), returns are more negatively affected by the spread of coronavirus. table difference-in-differences regression results for firm-level returns on the shock this table presents difference-in-differences regression results. the sample period is between january , , and march , . the regression equation is log(return)ijct = α+βcovid- ( ) ct +γpost- +ψi +ρj +δc +φt +εijct, where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients β and γ are shown. both raw returns and total returns (including dividends) are used as dependent variables. the dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the post- dummy encompasses the days after the event day. the high ( growth subsample includes only firms residing in counties where the growth between and reported cases took less than days. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. high growth ( ) ( ) ( ) ( ) raw return total return raw return total return ( ) - . - . - . - . (- . ) (- . ) (- . ) (- . ) post- . ** - . * - . ** - . ** (- . ) (- . ) (- . ) (- . ) - . *** - . *** - . *** - . *** (- . ) (- . ) (- . ) (- growth intensity of covid- cases by county. growth intensity is defined as the number of days before the county reaches reported cases measured from the day the initial case was reported in a county. to further dissect the response of firm-level returns to the shocks, we utilize spline regressions around the day the first coronavirus case is reported in a county. in particular, we regress firm level returns on dummies assigned to each day around the event. we assign individual dummies to each of the days in the post-event window, to . in the pre-event window, we assign individual dummies to each of the calendar days leading up to ( ) ( to ), as well as one dummy to capture all the days prior to the pre-event window (covid- < (- ) ). the regression specification is shown here: where the dummies capture days leading up to the event day, the covid- ( ) dummy represents the day of the first reported case, and the dummies are assigned to individual days following the event. the and dummies are dropped so the returns on those days serve as the benchmark. as mentioned previously, all trading days from the beginning of the sample up to days before the event day are designated by the covid- < (- ) dummy. we also truncate the data at days post-event. similar to equation ( ), firm, industry, county, and day fixed effects are included to control for unobserved heterogeneity. table documents spline regression results. column represents the full sample using raw returns; column represents the full sample using total returns; column represents the high covid- growth intensity sample using raw returns; and column represents the high covid- growth intensity sample using total returns. across the four columns, none of the coefficients for the dummies denoting days immediately before the event day, to , is statistically significant. this suggests there is no pre-trend leading up to the event and thus satisfies the parallel trend assumption of the diffin-diff estimation. the second item of interest is that the ( ) dummy is not significant across the four columns of table . there does not appear to be an immediate impact of reported virus cases to stock returns on the same day. in the post-event window, estimated coefficients for the dummies are generally negative. in table , columns and , for the full sample, these coefficients are not statistically significant until . although the coefficient loadings on the post-covid- dummy in table , columns and , are negative and significant, it takes about a week for the stock valuation impact to be realized. however, when the sample is narrowed to high growth intensity counties, results shown in columns and of table demonstrate that the impact of the shock is concentrated on day and after from the initial reported case. the estimated coefficient of - . and - . , in columns and , respectively, are significant at the %. this implies the average return is roughly bps lower days after the event day relative to the days immediately prior to the event day. we conclude from the spline exercise that the first reported case of in the county where a firm is headquartered has a significant negative effect on its stock return, but there is a delay between the reporting date and the stock reaction date. this delay is minimized in counties which have high rates of virus spread. table spline regression results for firm-level returns on the this table presents spline regression results. the sample period is between january , , and march , . the regression specification is shown in equation ( ). both raw returns and total returns (including dividends) are used as dependent variables. the dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the ) dummy encompasses all trading days prior to the -day window leading up to the event day. the high ( growth subsample includes only firms residing in counties where the growth between and reported cases took less than days. all regressions include firm, industry, county, and day fixed effects. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. high growth ( ) ( ) ( ) ( ) raw return total return raw return total return covid- < (- (- . ) (- . ) constant - . *** - . *** - . *** - . ** (- . ) (- . ) (- . ) (- . how does affect the cross-section of returns? the pandemic is all encompassing. for the financial markets, other than generating unprecedented uncertainty (or risk), it also changes investors' expectations about the future level of earnings and dividends. in this section, we examine a number of potential channels through which the pandemic potentially affects firms. in what follows, we focus on four aspects of the covid- crisis: the labor supply channel, the uncertainty channel, the government spending and monetary policies channel, and the cash flow expectations channel. we start by investigating whether our evidence of localized negative firm returns is consistent with the pandemic having a negative effect on local labor productivity. if this is indeed the case, we would expect firms relying more on labor input in the production function to be asymmetrically affected. to test this hypothesis, we focus on "laborintensive" firms and repeat the diff-in-diff analysis. labor-intensive firms are defined to encompass fama-french industry codes between and , which excludes firms in utilities, retail, financial, and services industries. table shows the results. +ψi +ρj +δc +φt +εijct, where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients β and γ are shown. both raw returns and total returns (including dividends) are used as dependent variables. the dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the post-covid- dummy encompasses days after the event day. the labor-intensive subsample includes only firms in the fama-french industries to (excluding utilities, retail, financial, and services). robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. high growth and labor intensive ( ) ( ) ( ) ( ) raw return total return raw return total return ( ) - . . - . - . (- . ) ( . ) (- . ) (- . ) post- . * - . * - . ** - . ** (- . ) (- . ) (- . ) (- . ) - . *** - . *** - . *** - . *** (- . ) (- . ) (- . ) (- for both raw returns and total returns, coefficient loadings on the post- dummy in the labor-intensive subsample are negative and statistically significant at the % level in columns and . in terms of magnitude, − . and − . respectively, these estimates are larger than the full sample estimates in tables , columns and . furthermore, when we focus on labor-intensive firms headquartered in high growth intensity counties, the γ coefficients are significant at the % level. this can be seen from columns and in table . when compared with columns and in table , the point estimates suggest that labor-intensive firms located in high growth intensity counties experience a much larger drop in equity value. to further examine how labor supply is affected by the outbreak of the pandemic, we use data from the university of maryland covid- impact analysis platform. specifically, we obtain the county-level variable "% staying home" to investigate how the first reported case of coronavirus in a given county affects the population's ability to go to work. similar to our analysis of firm-level returns, we perform a difference-in-differences estimation and a spline regression at the county level employing "% staying home" as the dependent variable. table shows the results. the sample period is from january , , to march , . in column , the difference-in-differences estimates on the covid- ( ) dummy and the post-covid- dummy are both positive and significant at the % level. for example, after the first case of is reported in a given county, the percentage of people staying home goes up by . % in the -day post-window relative to all days prior to the news. from the spline regression results displayed in column of table , we see that there is no parallel trend violation as all dummies before are insignificant, and all indicator dummies from up to + are positive and highly statistically significant, which validates the difference-in-differences finding in column . thus, we conclude that the first reported case of coronavirus in a county has a noticeably negative effect on labor supply in the same county as the population stay at home more. next, we link the results documented in table to firm-level returns. to this end, we conjecture that firms located in counties with a large increase in the percentage staying home should have worse stock performance in comparison to firms residing in counties with small changes in percent staying home. that is, if labor supply is indeed a factor in the transmission of the news to the financial market, firms located in areas in which labor is most negatively affected by the pandemic should be penalized more by investors. to test this hypothesis, we sort counties into bins according to the change in percent staying table percentage of people staying home after first reported this table regresses the percentage of people staying home by county on date dummy variables around the event day. data for the percentage of each county staying home come from the maryland transportation institute. the sample period is between january , , and march , . the dummy denotes the day on which the first case of is reported in the county. the post-covid- dummy encompasses days after the event day. the covid- < (- ) dummy encompasses all trading days prior to the -day window leading up to the event day. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. ( ) ( ) % staying home % staying home covid- < (- ) - . (- . ) - . (- . ) (- ) - . (- . ) (- ) - . (- . ) ( ) home in a window around the first reported case of coronavirus. then we assign a staying home dummy to all firms headquartered in counties with large increases in percent staying home. in the return regression, we interact this staying home dummy with the covid- ( ) dummy and the post-covid- dummy, as defined previously. the full regression specification is where i sh is the staying home dummy. β and γ are estimated coefficients for firms located in counties experiencing large staying at home increases following the covid- news. table reports the results. table columns and report regression results when the countylevel change in percent staying home is measured from days prior to the first reported case to days after, ∆ % stay home (- ,+ ), whereas columns and report results when the change is measured from days before the first reported case to days after, ∆ % stay home (- ,+ ) . in all scenarios, we find that the post-covid- dummy by itself is no longer statistically significant. on the other hand, all coefficient loadings on the interaction between the post-covid- dummy and the staying home dummy are negative and statistically significant at the % level. firms headquartered in counties experiencing large rises in percent staying home earn bps lower returns relative to those firms residing outside of these counties in days following the first reported case of coronavirus. overall, the findings documented in this section verify our conjecture that the labor supply channel plays an important role in the transmission of the covid- shock to firm performance. with no known cure or vaccine as of may , the economic outlook around the world is highly variable. citizens are left wondering when state-by-state shelter-in-place orders will be lifted, when social distancing measures will be relaxed, and when eating and drinking establishments will be allowed to open up again. medical experts have also warned about the risk of a second wave of contagion as the lockdown measures are gradually relaxed (see, e.g., xu and li, ) . at the same time, because of the lack of historical precedence of comparable events, economists and market participants are perplexed by the magnitude and length with which output, demand, employment, earnings, etc., are expected to decline. in a recent paper, baker, bloom, davis, and the largely unanticipated nature of the current pandemic is confirmed by the widely read global risk report: the world economic forum did not report a global pandemic among the most likely risks. the first five most likely risks were related to the environment, with climate change listed as the main threat to the planet in (cf. wef global risks perception survey - ). however, we cannot rule out that alert investors may have table interaction results for firm-level returns on shock and percentage of people staying home this table presents interaction regression results. the sample period is between january , , and march , . we interact and dummies with indicators for whether a county has experienced an above-median change in the percentage of the population staying home. we use windows of both and days prior to the event day to a period of days after the event day. where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients β, γ, β, and γ are shown. both raw returns and total returns (including dividends) are used as dependent variables. the covid- dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the post-covid- dummy encompasses days after the event day. i sh c is a dummy variable that equals one if a county is designated as having an above-median change in the percentage of people staying home. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. ∆ % stay home (- ,+ ) ∆ % stay home (- - . *** - . *** - . *** - . *** (- . ) (- . ) (- . ) (- terry ( a) use three different forward-looking uncertainty measures to document the enormous rise in economic uncertainty as of march of . further, their economic model attributes % of the forecasted output contraction by q of (mean of %) to covid- induced uncertainty. figure plots the quarter-on-quarter gdp forecast taken into account pandemic concerns in their portfolio choices in advance of the current covid- event. see, for example, pagano et al. ( ) for an analysis of firm returns before the disaster. dispersion from professional forecasters between and . the spike at the end of this series corresponds to a dramatic increase in uncertainty unlike anything we have experienced in the postwar period. we attempt to tease out the impact of covid- on equity returns due to the uncertainty it produces. to that end, we employ data from the work of hassan, hollander, van lent, and tahoun ( ) (hhvlt herein). these authors use textual analysis of earning call transcripts to obtain firm-level measures of covid- exposure as well as sentiment and risk associated with that exposure. the sentiment measure is related to the conditional mean of the shock, whereas the risk measure is related to the variance of the shock. by construction, the risk measure captures the management's attitude toward we merge our return data set with the hhvlt data set from q . the two variables of interest are risk. net sentiment can be positive or negative depending on the according to bloomberg data, as of april , . historical data from philadelphia fed. the text classification approach used to construct the risk and sentiment measures has been validated in recent work by hassan et al. ( ) in the context of measuring a firm's exposure to, for example, political risk. firm's outlook due to the shock. risk can be zero or a positive value if the firm expresses an uncertain outlook following the shock. after merging the data, we construct a risk dummy i risk , which we define as any firm in our sample that has a nonzero risk measure in the first quarter of . we then eliminate all firms with a positive net sentiment value since these firms expect to benefit from the pandemic. to see whether uncertainty is associated with the decline in equity following the first reported case of coronavirus in the same county where the firm is headquartered, we extend the diff-in-diff regression from section . to include interaction terms with i risk . in particular, the regression specification is where β and γ are coefficient loadings on the interaction between the covid- dummy and the post- dummy, respectively, with the covid- risk dummy. similar to the baseline diff-in-diff regressions, firm, industry, county, and day fixed effects are used as controls. table presents the results. table shows results for the full sample and the high covid- growth intensity sample. notice the γ coefficients in the fourth row are negative and statistically significant across the columns for both raw and total returns. this means that firms affected by risk > ) experience even lower returns after the event day (covid- ) relative to those firms not affected by this uncertainty (covid- risk = ). overall, we find confirmation in these interaction regressions that covid- -induced uncertainty drives firm-level equity returns after the shock is realized. policies in this section, we investigate whether the stability of recurrent government purchases makes firms more resilient to the economic shock induced by the pandemic. indeed, in the context of the [ ] [ ] financial crisis, goldman ( ) shows that government contractorsdefined as firms deriving more than % of sales from the federal government-experienced smaller declines in sales, profitability, and market values than otherwise similar firms. to assess the (direct) effect of government purchases at the firm level, we compare the stock performance of government contractors table interaction regression results for firm-level returns on the this table presents interaction regression results. the sample period is between january , , and march , . we merge our sample with the hassan, hollander, van lent, and tahoun ( ) data set from q . their data using textual analysis contains firm-level exposure to . in particular, we use variables net sentiment and risk to measure the first (level) and second (uncertainty) moment, respectively, of the covid- shock to each firm. all firms with a positive net sentiment with respect to the shock are dropped. the regression equation is log(return)ijct = α+βcovid- ct +γpost- +β ct ×i risk i +γ post- +ψi +ρj +δc +φt +εijct, where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients β, γ, β , and γ are shown. both raw returns and total returns (including dividends) are used as dependent variables. the covid- dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the dummy encompasses days after the event day. i risk i is a dummy variable that equals one if a firm has a covid- risk value greater than zero in q . the high ( growth subsample includes only firms residing in counties where the growth between and reported cases took less than days. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. high growth ( ) ( ) (- . ) (- . ) (- . ) . *** - . *** - . *** - . *** (- . ) (- . ) (- . ) (- (gc) and other firms (non-gc) during the pandemic. the regression government purchases directly affect the performance of government contractors that derive a large portion of their revenues from those purchases; in turn, government contractors' performance may also spill over onto other firms, for example, along the supply chains. we study only the direct effect. specification is is a dummy variable that flags government contractor firm i. the parameters of interest are β and γ -the difference-in-differences estimate of the effect of being a government contractor during the pandemic. table presents the regression results. columns and summarize findings in the full sample. we observe that none of the coefficient loadings on the interaction terms is statistically significant. this implies that following the first reported case of coronavirus in a county, government contractors' financial performance is not better compared to noncontractor firms. this result suggests that the current economic contraction and the one observed in the financial crisis are fundamentally different phenomena. indeed, while the stability of government purchases provided government contractors with a hedge during the [ ] [ ] financial crisis (goldman, ) , this effect is largely absent in the current pandemic. most large government contractors belong to the manufacturing industry (defined as belonging to fama-french industry codes to ). hence, it is possible, that they are particularly susceptible to negative labor supply shocks. as a result, despite the fact that gc's benefit from the stability of having government contracts during economic downturns, the covid- pandemic adversely affects these firms' productivity through the labor supply channel, as discussed in section . . to test this hypothesis, we focus on the subsample of labor-intensive firms and reestimate the regressions including the gc dummy. results are presented in columns and of table . within the labor-intensive subsample, estimated coefficients for the covid- × i gc and the post-covid- × i gc terms are all negative and statistically significant. in particular, the interaction between the post-covid- dummy and the gc dummy exhibits high statistical significance at the % level in columns and . gc's have lower equity returns than non-gc's in labor-intensive industries in the aftermath of the first reported covid- case. hence, this evidence table interaction regression results for firm-level returns on the shock and government contractors this table presents interaction regression results. the sample period is between january , , and march , . we merge our sample with the goldman ( ) data set, where the author identifies government contractors from compustat. these are firms deriving more than % of sales from the federal government. the regression equation is log(return)ijct = α+βcovid- ct +γpost- where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients β, γ, β, and γ are shown. both raw returns and total returns (including dividends) are used as dependent variables. the covid- dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the post-covid- dummy encompasses the days after the event day. i gc i is a dummy variable that equals one if a firm is a designated government contractor. the labor-intensive subsample includes only firms in the fama-french industries to (excluding utilities, retail, financial, and services). robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. labor intensive ( ) ( ) (- . ) (- . ) (- . ) post-covid- × i gc . - . - . *** - . *** ( . ) (- . ) (- . ) (- . ) - . *** - . *** - . *** - . *** (- . ) (- . ) (- . ) (- suggests that the strain on labor supply due to the pandemic appears to outweigh the benefits of stable demand for the gc firms. next, we investigate whether heterogeneous exposure to monetary policy can generate the return reaction to news. to the extent that the pandemic causes the federal reserve to implement accommodative monetary policy for the foreseeable future, it is possible that some firms benefit from this (expected) monetary policy easing more than others. for a simple measure of monetary policy exposure at the firm level, we use the financial constraint index developed by whited and wu ( ) . chava and hsu ( ) show that financially constrained firms are especially sensitive to monetary policy shocks around fomc announcements. firms for which it is more difficult to obtain external financing, as proxied by a high value of the whited and wu ( ) index, respond strongly to monetary policy surprises. in table b . in the appendix, we find this not to be the case. the interaction term between the post-covid- dummy and the financial constraint dummy is insignificant in the full sample as well as in the labor-intensive subsample. our analysis suggests that the pandemic effects are not confined to financially constrained firms, as these firms do not display differential returns following the first reported case relative to unconstrained firms. we thus conjecture that monetary policy is unlikely to be a primary driver of stock market reaction to the coronavirus spread. this is in line with the findings by ramelli and wagner ( ) . these authors show that the liquidity facilities announced by the fed-the "primary market corporate credit facility" and the "secondary market corporate credit facility"-had only a temporarily effect, which was largely reversed within a -day window. in this section we evaluate the effect of the covid- crisis on earnings revisions, and link these revisions back to the cross-sectional variation in firm returns documented in section . . we use firm-level earnings per share (eps) forecasts for various forecast horizons ( , , and years, corresponding to fiscal years , , and ) from the i/b/e/s database. to construct a firm-level eps forecast at a daily frequency, we follow landier and thesmar ( ) and construct the daily forecast as the average of the analysts' individual forecasts for a given firm. we then calculate daily forecast revisions that are either equal to the average across the three forecast horizons (combined revisions) or horizon specific. that is, the combined revision is defined as and the horizon-specific revisions are defined as where h ∈ { , , } and f t ep s ih is the eps forecast at time t for a firm i and forecast horizon h. in an attempt to capture the evolution of firm-level eps forecasts, we then construct the cumulative revisions for a window from days before to days after the first case is reported in a given county. cumulative revisions are defined as follows: where x ∈ {combined, , , }, d corresponds to the day on which the first covid- case is reported and t ∈ [d − ,d + ]. we start by performing an analysis similar to that reported in table . specifically, we regress the panel of cumulative earnings revisions on the covid- dummy and the post-covid- dummy. table presents the results for revisions averaged across the forecast horizons (column ), as well as for revisions occurring at the individual -(column ), -(column ), and -year (column ) horizons. focusing on column , we observe that the estimated coefficient γ on the post- dummy is negative and statistically significant at the % level. the point estimate of − . suggests that there is on average a negative . % change in analysts' revisions in the -day post-event window relative to firms in the control group. the remaining columns shows that the coefficient stays flat across forecast horizons at about − . (although it misses statistical significance at the shortest -year horizon). tables and together provide evidence that both returns and cumulative eps revisions are significantly lower in the -day post-event window relative to before. motivated by these findings, we investigate to what extent eps forecast revision can account for decreases in returns. to this end, we interact the post- we are implicitly assuming that there is no revision in earnings beyond . the data indeed support this idea. see the discussion in landier and thesmar ( ) . our analysis is motivated by the classical campbell and shiller ( ) decomposition (and its manipulations), where unexpected stock returns are related to changes in expectations of future dividend growth and future stock returns: +ψi +ρj +δc +φt +εijct, where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients β and γ are shown. cumulative eps revisions are measured over the time window starting days before and ending days after the first case is reported in a county. moreover, the revisions are either averaged across the -, -, and -year forecast horizons (x = combined) or horizon specific (i.e., x = , x = , and x = , respectively). the dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the dummy encompasses days after the event day. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. ( ) (- . ) (- . ) (- . ) post- . ** - . - . ** - . *** (- . ) (- . ) (- . ) (- revision on a given day. the full specification is given by log(return) ijct = α+βcovid- ct +γ post- ct ×i n eg.rev. i +γ post- ct ×i n orev. i +γ post-covid- ct ×i p os.rev. i +ψ i +ρ j +δ c +φ t +ε ijct . ( ) table reports the results. interestingly, only those firms whose earnings forecasts have been revised downward experience returns that are lower (relative to returns in the control group) in the -day postevent window. despite it being possible for firms with no earnings revisions to experience lower returns (relative to the control group) due to an increase in discount rates, we find no evidence in our event window supporting this channel. relative to the baseline estimate of − bps reported in table , the effect in table is substantially larger in absolute terms. that is, returns are on average bps lower in the -day post-event window relative to returns in the control group. the remaining columns highlight that the relationship between firm returns and revisions in earnings forecasts is similarly strong for shortand medium-term expectations (i.e., for and earnings), but weakens substantially, both statistically and economically, at the -year forecast horizon. table firm-level returns and earnings per share forecast revisions this table presents interaction regression results. the sample period is between january , , and march , . we interact covid and post-covid- dummies with indicators for whether a firm has experienced a negative, positive, or no earning forecast revisions. we use windows of both and days prior to the event day to a period of days after the event day. log(return)ijct = α+βcovid- ct +γ post- +γ post- +γ post- +ψi +ρj +δc +φt +εijct, where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients β, γ , γ , and γ are shown. raw returns are used as dependent variable. the covid- dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the post- dummy encompasses days after the event day. i n eg.rev. i (i n orev. i and i p os.rev. i ) is a dummy variable that equals one if a firm experiences a positive (no and positive, respectively) eps forecast revision on a given day. moreover, these revisions can be calculated across a forecast horizon or be horizon specific. that is, columns to present results for daily eps forecast revisions averaged across the -, -, and -year forecast horizons (column ), -(column ), -(column ), and -(column ) horizons. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. . ** - . *** - . ** - . (- . ) (- . ) (- . ) (- . ) post- × no revision - . - . - . - . (- . ) (- . ) (- . ) (- . ) post-covid- × pos. revision . - . . - . ( . ) (- . ) ( . ) (- . ) - . *** - . *** - . *** - . *** (- . ) (- . ) (- . ) (- in all, our evidence suggests that, over a tight window around the event day, downward revisions in earnings expectations constitute an important driver of the firm returns response. in this paper, we document how the cross-section of equity returns is differentially affected by the pandemic in the united states. specifically, we exploit the staggered geographical dissemination of the virus at the county level and take advantage of the headquarter location of u.s. publicly traded firms. through our natural experiment, we find that firms headquartered in an affected county experience on average bps lower daily returns in the -day post-event window relative to returns before the event and compared with firms headquartered in non- counties. further, we show that this negative relationship is stronger for firms in counties where the virus spread is more intense (- bps) . we also shed new light on the economic channels through which the outbreak of covid- affects firm-level returns. we focus on four nonmutually exclusive transmission channels: labor supply, uncertainty, government spending and monetary policies, and cashflow expectations. concerning labor supply, firms in labor-intensive industries exhibit more negative returns (- bps) relative to the full sample after the first reported case. we further show that the county-level percentage of people staying at home increases in the aftermath of the news. related, firms residing in counties experiencing large rise in percent staying home have worse return performance than those located outside. regarding uncertainty, firms with idiosycratic exposure to covid-induced risk are proven to have more negative returns. on government policy, heterogeneous sensitivity to government spending and monetary policies turn out to be ineffective in explaining the crosssection of return dynamics due to the virus spread. however, in the labor-intensive subsample, government contractors actually experience larger return declines than noncontractors. this is suggestive evidence that the negative impact on labor productivity from the pandemic outweighs the benefit of stable demand provided by government contracts. lastly, the first reported case of covid- , on average, puts downward pressure on analyst earnings forecasts of firms headquartered in the same county. as a result, firms with negative earnings revisions display more negative returns in the post-first-case news window. this study is an early examination into the wide-ranging impact of the covid- pandemic on the u.s. economy. by looking at the cross-section of equity returns, our empirical design allows us to better understand the propagation mechanism behind the crisis, which hopefully will inform the policy makers' discussion about stabilizing the economy. we perform a number of robustness tests. first, we examine firm-level returns in a tight window around the event day. that is, we employ a -day event window ( days before and days after) and drop all return observations before the event window. furthermore, we also delete returns belonging to untreated firms headquartered in non- counties. we use the following spline regression specification: Φkcovid (-k) ct +βcovid ct + k= Ψkcovid (+k) st +ψi +ρj +δc +φt +εijct, where the and dummies are not used so those returns serve as the benchmark. table . however, similar to before, the coefficient loading on the dummy remains significant at the % level. the magnitude is slightly smaller at - . and - . in the tight event window. in columns and of table a . , we repeat the exercise for labor-intensive firms located in high covid- growth intensity counties and present the results. again, estimated coefficients for the to dummies are negative and statistically significant. importantly, none of the columns of table a . contains parallel trend violations. overall, our main findings remain in the narrow window around the covid- ( ) event day. next, we examine the subset of nonfinancial firms in the sample. it is common practice in the finance literature to leave banks and insurance companies out of the analysis due to their large asset size and balance sheet structure. we drop firms belonging to ff industry code . spline regression results are shown in table a . . regression specification follows equation ( ). comparing table a. with table , two items are worth noting. first, none of the estimated coefficients are statistically significant in the pre-event period. second, coefficient loadings on the covid dummy are also insignificant. unlike the loadings in table , however, those on the covid (+ ) dummy are statistically significant across the four columns in table a . . in columns and , the baseline sample, average nonfinancial firm return is . bps lower using raw returns and . bps lower using total returns than the benchmark return calculated from the two days immediately before the event day. the magnitude of these coefficients increase in columns and . for nonfinancial firms residing in high covid- growth intensity counties, the average raw return days after the event day is . bps lower than the benchmark return in column , and the average total return is . bps lower in column . we do not report results for the subsample of labor-intensive firms in this robustness check since they are by definition nonfinancial firms. findings documented in table a . suggests that our results actually strengthen in the subsample of nonfinancial firms. to ease the concern that our results are driven by a few firms located in large counties or population centers, we drop firms headquartered in the states of california, new york, and texas in the next robustness test. spline regression specified in equation ( ) is then applied to the reduced sample. we report the results in tables a. . in the baseline sample, shown in columns and of table a . , regression results are stronger when we exclude firms in california, new york, and texas. estimated coefficients of raw returns and total returns on covid (+ ) and covid (+ ) are all statistically significant, at the % and % level, respectively. in columns and of table a . , for firms located in high covid growth intensity counties, results are similar. generally speaking, findings from this robustness test verify that our baseline results are not driven by a few firms located in large counties and population centers. counties are excluded. all regressions include firm, industry, county, and day fixed effects. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. high growth and labor intensive ( ) ( ) (- . ) (- . ) (- . ) (- . ) . . - . - . ( . ) ( . ) (- . ) (- . ) constant - . *** - . *** - . ** - . ** (- . ) (- . ) (- . ) (- french industry ) . the sample period is between january , , and march , . equation ( ) presents the regression specification. both raw returns and total returns (including dividends) are used as dependent variables. the dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the ) dummy encompasses all trading days prior to the -day window leading up to the event day. the high ( growth subsample includes only firms residing in counties where the growth between and reported cases took less than days. all regressions include firm, industry, county, and day fixed effects. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. full sample high growth ( ) ( ) ( ) ( ) raw return total return raw return total return covid- < (- ) - . - . - . - . this table presents spline regression results for firms headquartered outside of the states of california, new york, and texas. the sample period is between january , , and march , . equation ( ) reports the regression specification. both raw returns and total returns (including dividends) are used as dependent variables. the dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the ) dummy encompasses all trading days prior to the -day window leading up to the event day. the high ( growth subsample includes only firms residing in counties where the growth between and reported cases took less than days. all regressions include firm, industry, county, and day fixed effects. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. full sample high growth ( ) ( ) ( ) ( ) raw return total return raw return total return covid- < (- ) - . - . - . - . appendix b. financial constraint as a proxy for monetary policy sensitivity where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients β, γ, β, and γ are shown. both raw returns and total returns (including dividends) are used as dependent variables. the dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the post-covid- dummy encompasses days after the event day. i f c i is a dummy variable that equals one if a firm falls in the top half according to the ww index. the labor-intensive subsample includes only firms in the fama-french industries to (excluding utilities, retail, financial, and services). robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. labor intensive ( ) ( ) ( ) ( ) raw return total return raw return total return ( ) - . - . - . - . (- . ) (- . ) (- . ) (- . ) post- . ** - . ** - . - . (- . ) (- . ) (- . ) (- . ) ( ) - . *** - . *** - . *** - . *** (- . ) (- . ) (- . ) (- this table presents interaction regression results. the sample period is between january , , and march , . we interact and dummies with indicators for whether a firm has experienced a negative, positive, or no earning forecast revisions. we use windows of both and days prior to the event day to a period of days after the event day. log(return)ijct = α+βcovid- ( ) ct +γ post- +γ post- +γ post- +ψi +ρj +δc +φt +εijct, where i,j,c and t represent firm, industry, county, and day, respectively. estimated coefficients β, γ , γ , and γ are shown. total returns including dividends are used as dependent variable. the dummy denotes the day on which the first case of is reported in the same county where the firm is headquartered in. the post-covid- dummy encompasses days after the event day. i n eg.rev. i (i n orev. i and i p os.rev. i ) is a dummy variable that equals one if a firm experiences a negative (zero, and positive, respectively) eps forecast revision on a given day. moreover, these revisions can be calculated across forecast horizon or be horizon specific. that is, columns to present results for daily eps forecast revisions averaged across the -, -, and -year forecast horizons (column ), -(column ), -(column ), and -year (column ) horizons. robust standard errors with double clustering at the county and day levels are used in reporting the t-statistics in parentheses. 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evidence from global stock markets susceptible-infected-recovered (sir) dynamics of covid- and economic impact the effectiveness of life-preserving investments in times of covid- fiscal spending jobs multipliers: evidence from the american recovery and reinvestment act beware of the second wave of covid- key: cord- -xddikzxs authors: wiseman, a. title: avoidance of oxidative‐stress perturbation in yeast bioprocesses by proteomic and genomic biostrategies? date: - - journal: lett appl microbiol doi: . /j. - x. . .x sha: doc_id: cord_uid: xddikzxs aims: bioprocess oxidative stress caused by many reactive oxygen species (ros) can lead to largely irreversible perturbation of yeast bioprocesses. these include the production of proteins derived from recombinant dna yeast technology (aerobically grown saccharomyces cerevisiae). these proteins include rennin, amyloglucosidases (glucamylases), interferons, interleukins, insulin, monoclonal antibodies, tissue plasminogen activators (t‐pa), sexually transmitted disease antigens, and measles, mumps and rubella antigens, growth hormones, somatotropin, blood clotting factors viii and xiii. in addition, there may be a demand for severe acute respiratory syndrome–coronavirus antigens, hepatitis a, b and c viral‐selected antigens, hiv retroviral antigens, influenza antigens, trypanosomal antigens, and foot and mouth disease antigens. prevention of oxidative stress has been achieved by application of antioxidant redox metalloenzymes such as superoxide dismutases (containing cu/zn cytosolic, mn mitochondrial and fe bacterial) glutathione peroxidases (and other se‐containing proteins and enzymes such as the thioredoxins), catalases (fe‐containing), cytochrome c peroxidases (fe‐containing), ceruloplasmins (cu‐containing), metallothionines (these cysteine thiol‐rich proteins bind ions of cadmium and mercury) and tyrosinases(cu‐containing). methods and results: ros are generated inadvertently by single metal valency couples such as feii/feiii and by feiii/fev present in (including human) isoforms in cytochromes p mixed‐function oxidases (ec · · · ; o( ) : mono‐oxygenase nadph/nadh requiring). in addition, mixed‐metal couples such as valency unmatched forms in cui/feii and feiii/mniv can recycle electrons. moreover, proteins/protein chaperone couples can recycle electrons, often where futile‐recycling systems have been instigated. furthermore, oxidized membrane phospholipids (r) can form rooh (lipid hydroperoxides) and roh (lipid alkoxides) that can generate ros through fenton chemistry (iron‐catalysed) chain reactions. utilization of chain‐breaking antioxidants such as vitamin e (α‐tocopherol) in the lipid phase and vitamin c (ascorbate) in the aqueous phase can terminate these ros‐producing reactions. conclusions: the main significance of the study is that proteomic strategies of relief from bioprocess perturbation by ros of yeast fermentations (used to manufacture proteins required in the food and therapeutic bioindustries) may become possible through addition of selected proteins (including metalloenzymes). the main impact of the study is that the utilization of genetically modified (gm) yeast produced by recombinant dna technology genomic strategies could circumvent the bioprocessing problems that otherwise result from the bioprocess perturbations: this is as a result of oxidative stress caused by ros, which is avoidable by deployment of appropriate antioxidants such as vitamins e, c and d (and antioxidant proteins and enzymes often of microbial origin via recombinant dna technology). where biomolecular injury is caused by reactive-oxidant molecular species, such as reactive oxygen species (ros and toxic metals, see tables and ), redox chemistry plays a dominant role in pathogenesis, through the onset of outcomes resulting from biomolecular injury (see . in relation to this, many metals are able to perform futile one-electron recycling between couples formed between appropriate valency states (wiseman and woods ) . furthermore, hydrogen radical abstractions can occur that can trigger a chain reaction in unsaturated phospholipids of oxidized membranes (both periplasmic membranes and those of cytoplasmic organelles) that releases breakdown products (when heated with feii/ascorbate) such as malondialdehyde that are appropriate for bioassay by the thiobarbituric acid (tbars) procedure (halliwell and gutteridge ) . furthermore, the likelihood of superoxide anion-free radical (aeo ) generation produces an enhanced risk of biomolecular injury as a manifestation of the biohazard of particular metal contamination, both of the environment and in the body. the essential role of oxygen gas, which has built up in the atmosphere as a result of the photosynthetic activity of green plants over a ae -billion-year period is paramount in ros production. the toxic nature of oxygen (abele ) , although essential for aerobic life on earth, has been subject to amelioration of toxicity through evolution of antioxidant enzymes that have developed over the last million years. moreover these antioxidant enzymes in the body, such as superoxide dismutase, glutathione peroxidase and catalase, augment the role of dietary antioxidants such as vitamins e and c (wiseman et al. ; wiseman and woods ) . furthermore, the use of dna recombinant forms may confer unexpected technological advantage in bioprocesses in cases where novel proteins, such as biocatalyst enzymes and cells, are introduced into previously unfavourable environments. where these introductions are associated table toxic and nontoxic metals that may be detectable in yeastbioprocess feedstocks (see table with the biometabolism because of gut microflora of animals, dietary idiosyncrasy considerations are involved in the prediction of harmful outcomes. these predictions therefore need to be evaluated as part of the risk/benefit analysis associated with the necessary toxic hazard/risk recommendation by studies in silico and in real-lab (in petri). there is a major interest in the possibility that bioactive compounds in foods from agriculture, particularly those in plant and microbially fermented foods, can protect against a wide range of diseases . furthermore, bioactive compounds can have gender-differentiated effects that are dependent also on a number of other factors including age, genomics and stage of reproductive development. complacency may be the result of overconfidence in the prediction of 'nontoxicity', to bioprocess and to consumers, but failure to predict the likelihood of a disaster outcome with foodstuffs, has been manifest in the past. examples include turkey x-disease caused by aflatoxin (a hepatotoxin and carcinogen) in groundnuts contaminated in storage by aspergillus flavus (late s africa; see tables and ) ; spanish cooking oil syndrome caused by contamination of cooking oil with aniline ( s); syndromes caused by forms of organic mercury produced by micro-organisms in waterways ( s). natural foodstuffs may also contain toxins (see tables and ) and protectants (see tables and ). molecular toxicology has provided a good mechanistic basis for prediction of future problems, and this approach is supported by the power of computer modelling on a wide range of unusual microbially produced chemicals that reach the body via methods of bioprocessing of foodstuffs that include solid-state fermentation. injury to macromolecules, table prevention of biomolecular damage in bioprocessing through addition of antioxidant biochemicals, enzymes and chaperone proteins (see table ) antioxidant chemicals vitamins such as e and c are free radical production chain breakers phytoestrogens (wiseman et al. free radical scavengers such as other free radicals reducing agents (low redox potentials) some proteins (thiol-containing for example) xenoestrogens such as bisphenol a, alkylphenols (octylphenols and nonylphenols) antioxidant enzymes catalases ( table ) in yeasts and other micro-organisms (antioxidants such as vitamins e and c chain terminate the formation of membrane-degrading ros) relates to more than just structural damage that is sometimes obvious and predictable: functional damage may also follow. moreover, functional damage can be predicted in silico: injury therefore to catalytic proteins (enzymes) and honorary enzymes (many other proteins) may result in functional problems with nucleic acids and other biologically active biomolecules. further investigation is needed on the interactions of macromolecules especially in micro-organisms prior to harnessing these into food bioprocessing. prevention of toxicity by safe choice (or gm control) of micro-organisms is to be recommended. unfortunately, prevention is based upon prediction of toxic responses via risk-prediction analysis usually in silico. cure or at least the 'successful treatment' of manifest bioprocess toxic responses may become the easiest option: these will then become a strategy to ameliorate the undesirable damage caused by biomolecular injury in yeast bioprocesses such as brewing (wiseman ; wiseman and woods a,b) . the recognition of characteristic responses of classes of cellular biomolecules, such as nucleic acids, proteins and phospholipids to injury was developed in the twentieth century. nevertheless, a more holistic approach can advance further our understanding of the behaviour, of these macromolecules under oxidative stress. a web of interactions is evident amongst biomolecules in micro-organisms, disturbance of such biomolecular webs by toxic substances is often the basis of the toxic manifestations seen as loss of homeostasis in pure cultures of micro-organisms. furthermore, stability of microbial species in mixed cultures has been attributed to relatively few species in a web of interaction: perturbation to the web by external forces may be subjected to compensatory restoration of the 'equilibrium position' amongst the microbial participants. moreover, supply of metabolites is attributable to enzyme action within the living micro-organisms. for example, atmospheric nitrogen-fixing (using the nitrogenase system) by soil organisms such as azotobacter vinelandii (aerobic) or clostridium pasteuranum (anaerobic) are controlled by metabolite supply. in the latter case, supply of pyruvate allows a chemostat interpretation of growth kinetics, mediated by the coa pyruvate ferredoxin oxidoreductase-catalysed cleavage of pyruvate to acetyl phosphate (this allows a subsequent production of atp) plus carbon dioxide. such microorganisms occupy an ecological niche that can be subjected to perturbation by the introduction of the end-product of nitrogen fixation (ammonia). linking species webs, are therefore a reflection of the underlying enzyme-catalysed webs of biochemical metabolites that make up both the cellular interior milieu of organisms and the associated biochemical mixtures in the growth and maintenance media, especially in scale-up fermentation bioreactors. immobilized enzyme utilization complicates the biochemical mixes present, and make reductionist approaches in prediction of expected biochemical balance more risky, especially if an unidentified microbial mix is employed as the bioindicator in pilot plant studies. toxicity to one species-web, may however favour stability of a competing species web: the presence of known (or unknown) yeast- phytoestrogen isoflavones include the soya protein-derived glucones daidzin and genistin. these are degraded to the aglycone forms daidzein and genistein by gut microflora: and these initial products can be degraded further by other bacteria in the gut for example to equol derivatives and equol conjugates with glucuronic acid (excreted through the kidney). fermented soya foods, such as miso or tempeh, contain mostly the unconjugated isoflavone aglycones . bioprocess antitoxicants (these are often antioxidants against ros), however, lower the accurate prediction of final outcome in the balance equilibria observed with mixed cultures of micro-organisms including bioprocess yeasts, and their enzymes used in bioprocessing (tucker and woods ) . the dilemma faced by molecular toxicologists for biohazard prediction lies, therefore, in an identification of chemostasis based upon a relatively few named species webs. resilience as opposed to growth perturbation may be predicted wrongly in silico for some mixed cultures of micro-organisms: it is essential, therefore, that real-lab (in petri) studies are also undertaken. species bioindicators and molecular biomarkers should both be subject to continuous assessment by critical biomonitoring to predict such yeast-bioprocess perturbation as opposed to resilience outcomes. harmful free radicals (halliwell and gutteridge ) , such as superoxide anion are produced in micro-organisms during aerobic respiration because of only partial reduction of some oxygen molecules in the electron transport chain. this is because of one-electron reduction of each atom of oxygen, instead of two-electron reduction to form water. similarly, in many micro-organisms the electron transfer chain from nadh to water (with insertion of one oxygen atom into xenobiotic substrates) that uses cytochromes p- may display futile cycling, in the absence of substrate, to produce the superoxide anion (aeo ). reduction of ros, and also of reactive nitrogen species (rns), is a priority for avoidance of production in particular microbial cultures susceptible to oxidative stress. for example, damage to biomolecules can occur by attack on phospholipid cytoplasmic and internal membranes, and also notably on dna: and on the recombinant dna of gm yeasts employed in manufacture of proteins such as insulin and interferon, intended for human therapy. all redox biochemicals, existing in their oxidized and reduced forms, constitute an oxidation/reduction couple, for instance, with polyphenols and some nutraceuticals (ridgway and tucker ) . each of such redox couples displays an experimentally determined redox potential (reduction oxidation potential) of a particular voltage under standard (defined) conditions of temperature and pressure (usually °c, ph ae , atm). the standard redox potential scale (e o ) has good oxidizing agents (pro-oxidants) such as molecular oxygen at the top with the high value of + ae v, and the reducing agents (antioxidants) at the bottom. for example, nadh is at ) ae v (the scale passes through zero) and a voltage difference of ae v therefore is associated with electron flow from nadh to o (in oxygen-utilizing respiratory chains). this potential difference is normally sufficient to allow the production of a total of at least three atp, each one at a particular step in the respiratory chain via known carriers by proton pumping through the inner membrane of yeast mitochondria. for instance, in interconversion of fe(iii) : fe(ii) the latter loses one electron during its oxidation to the feiii ( +) form and thus these two forms of iron are pro-oxidant (fe + ) and antioxidant (fe + ), respectively, but with the extra involvement of perferryl fe + in the cytochrome p mechanism via [feo] + the oxonium cation (lewis ; williams et al. ) . fe + /ascorbate is commonly employed to generate fe + , ostensibly the antioxidant form of the iron iii : iron ii couple to catalyse the breakdown of phospholipid liposomal membrane model systems. fe + reacts through fenton chemistry (halliwell and gutteridge ) with lipid hydroperoxides (rooh) or lipid alkoxides (roh) in the phospholipid mixture after storage in an atmosphere that contains oxygen, to form these peroxyl (roo • ) and alkoxy (ro • ) free radical derivatives of phospholipids. these free radicals initiate a chain reaction of further attack of the phospholipids via degradation reactions that produce large amounts of these free radical species (wiseman et al. ; see figs and ) . avoidance biostrategies against ros and rss in many oxidatively stressed bioprocesses that utilise yeasts (and other micro-organisms) are essential where human therapeutic proteins are expected to be produced in high yield. these proteins intended for therapy include insulin, inter ferons, interleukins, growth hormone and blood clotting factors (viii and xiii). moreover, the renewed demand for antigens of viruses such as hepatitis, severe acute respiratory syndrome (coronaviruses), foot and mouth disease, and hiv retroviruses, has established a greater demand for nonperturbation of yeast bioprocesses by ros (wiseman ) by development of stable phenotype despite variation in gene deletion (stearns ) . moreover, antioxidants, natural and synthetic, are being developed to supply the correct level of yeast-bioprocess resilience (wiseman ) (see tables - ) through a range of genomic and proteomic strategies. furthermore, the coupling in yeast bioprocess of thioredoxin (se) as an electron donor for peroxiredoxins (these use catalytic cysteine-cysteine residues to remove peroxide) can be viewed as an useful extension to other antioxidant enzymes which could overload the yeast bioprocess with unwanted metals such as cu and zn (see table ). nevertheless, se-supplementation could be viewed as health benefiting where soils are deficient in this metalloid antioxidant component of glutathione peroxidases (rayman ) . isoflavone phytoestrogens as functional ingredients in soya products: physiological importance of metabolism and possible indications for their rapid absorption following consumption of textured vegetable protein. functional foods ' th uk-nl nitrogen cycle meeting quantitative analysis of the flavonoid content of commercial tomatoes, onions, lettuce and celery free radicals in biology and medicine guide to cytochromes p structure and function se brought to earth procedure for the partial purification of apple leaf polyphenoloxidase suitable for commercial application safeguards and spurs enzymes in food processing crystal structure of human cytochromes p c with bound warfarin phytochemicals epidemiological factors biosustainability-limit to industrial biocatalysts if no supersedence of bioprocess perturbation by resilience? an enhanced multifunctional food -beer? the brewer international welcome praise for copper deleterious outcome of bioprocesses: unless ecobuffering can be effluent-incorporated by the utilisation of immobilised enzymes safe choice of metals in food bioprocess enzyme mimicry biomolecular free radical toxicity: causes and prevention isoflavone aglycone and glucoconjugate content of high-and low-soy uk foods use in nutritional studies key: cord- -tycd i authors: severino, amie l.; mittal, nitish; hakimian, joshua k.; velarde, nathanial; minasyan, ani; albert, ralph; torres, carlos; romaneschi, nicole; johnston, camille; tiwari, suchi; lee, alex s.; taylor, anna m.; gavériaux-ruff, claire; kieffer, brigitte l.; evans, christopher j.; cahill, catherine m.; walwyn, wendy m. title: μ-opioid receptors on distinct neuronal populations mediate different aspects of opioid reward-related behaviors date: - - journal: eneuro doi: . /eneuro. - . sha: doc_id: cord_uid: tycd i μ-opioid receptors (mors) are densely expressed in different brain regions known to mediate reward. one such region is the striatum where mors are densely expressed, yet the role of these mor populations in modulating reward is relatively unknown. we have begun to address this question by using a series of genetically engineered mice based on the cre recombinase/loxp system to selectively delete mors from specific neurons enriched in the striatum: dopamine (d ) receptors, d receptors, adenosine a (a a) receptors, and choline acetyltransferase (chat). we first determined the effects of each deletion on opioid-induced locomotion, a striatal and dopamine-dependent behavior. we show that mor deletion from d neurons reduced opioid (morphine and oxycodone)-induced hyperlocomotion, whereas deleting mors from a a neurons resulted in enhanced opioid-induced locomotion, and deleting mors from d or chat neurons had no effect. we also present the effect of each deletion on opioid intravenous self-administration. we first assessed the acquisition of this behavior using remifentanil as the reinforcing opioid and found no effect of genotype. mice were then transitioned to oxycodone as the reinforcer and maintained here for d. again, no genotype effect was found. however, when mice underwent d of extinction training, during which the drug was not delivered, but all cues remained as during the maintenance phase, drug-seeking behavior was enhanced when mors were deleted from a a or chat neurons. these findings show that these selective mor populations play specific roles in reward-associated behaviors. introduction m-opioid receptors (mors), the principal target of addictive analgesics are widely expressed in diverse brain regions associated with reward (for review, see le merrer et al., ) . mors are expressed on the gabaergic neurons that innervate the dopaminergic neurons of the ventral tegmental area (vta) so are poised to enable dopamine release an important mediator of rewarding behavior. mors are also expressed in the striatum which controls movement and the formation of behavioral habits associated with reward. these two behaviors, reward and locomotion, are mediated by different dopaminergic signaling profiles in distinct neurons (howe and dombeck, ) and are often used to generate a profile of reward behavior in mice (mitchell et al., ; zhang and kong, ) . mors are widely expressed in the different neuronal populations and subregions of the striatum (wang et al., (wang et al., , wang and pickel, ; miura et al., ; cui et al., ) . they are expressed on dopamine (d ) receptor, d , and adenosine a (a a) subpopulations of medium spiny neurons (cui et al., ; oude ophuis et al., ) . they are also expressed on cholinergic interneurons (ponterio et al., ) and on cortical or thalamic glutamatergic neurons innervating medium spiny neurons (for review, see miura et al., ) . within these different neuronal populations, mors are differentially expressed in striatal subregions. for example, they are expressed in the patches or striasomes where they colocalize with dynorphin-expressing d medium spiny neurons (brimblecombe and cragg, ) but also in the matrix where their expression is less and on d or d medium spiny neurons (cui et al., ) . although we do not fully understand the functional role of each of the striatal neuronal populations, we do have insight as to their function from their cellular expression patterns and electrophysiology studies. mors are expressed presynaptically on glutamatergic afferents projecting to the striatum and postsynaptically on striatal dendrites and dendritic spines (wang et al., ) . activation of these receptors inhibits both glutamatergic afferent activity and that of gabaergic collaterals from medium spiny neurons (blomeley and bracci, ; ma et al., ; james et al., ) . in addition, mors inhibit cholinergic interneurons so regulating local spontaneous dopamine release (sandor et al., ; ponterio et al., ; ponterio et al., ) . presynaptic mors are also found on low threshold spike interneurons and so modulate their spontaneous activity (elghaba and bracci, ) . at the behavioral level, several studies point toward a role of striatal mor populations in reward behaviors. earlier studies showed that ablating mor-enriched striosomes of the dorsal striatum produces deficits in motorskill learning (lawhorn et al., ) . forebrain mors are known to play a role in alcohol, food and heroin reward behaviors charbogne et al., ) and in the hedonic reward value of food reward (boulos et al., ) . in addition, mor re-expression on dynorphin expressing medium spiny neurons in an otherwise null background are sufficient to reinstate some, but not all, opioid reward behaviors (cui et al., ) . given the broad but diverse distribution of mors on different neuronal subtypes throughout the striatum, we set out to determine the contribution of these mors to opioid reward behaviors. in order to do this, we bred flmor mice, in which exons two and three of the mor gene (oprm ) are flanked by loxp, with four different cre-recombinase mice (d cre, d cre, a acre, chatcre). we first verified these deletions using rnascope in situ mrna hybridization and quantitative pcr. we then assessed opioid-induced hyperlocomotor, sensitization of this effect, and intravenous opioid self-administration (ivsa). from these studies, we conclude that each of these mor-expressing populations are required for distinct aspects of opioid reward-related behaviors. all procedures were authorized by the institutional animal care and use committee (iacuc) and are in compliance with the policies on the use of animals in research as outlined by this journal. all transgenic mice used in this study were bred by the animal breeding colony. d flmors, d flmors, a aflmors and choline acetyltransferase (chat) flmors were generated by breeding flmor mice (loxp sites flanking exons - of the oprm gene on a : c bl/ j: sv background, stock # , the jackson laboratory) with four cre driver lines to obtain cre recombinase on one (d cre; stock # -ucd, d cre; -ucd, a acre; -ucd, mmrrc, nih, dhhs, % c bl/ j) or two (chat-ires-cre; stock # , the jackson laboratory, % c bl/ j) alleles and flmor on both alleles. control flmor mice of the same background were generated as littermates from the breeding strategies used. mice lacking all mors (stock # , % c bl/ j, the jackson laboratory) were bred as heterozygous pairs to generate knock-out (ko) and wild-type (wt) littermates. male and female transgenic mice were used between age - weeks and - g of body weight. animals were maintained on a / h light/dark cycle with ad libitum access to food and water, and experiments were conducted at zt -zt (zeitgeber time). all mice were group housed for the duration of the experiment except for the ivsa experiments during which mice were singly housed in an enriched environment after surgery. all schedule ii drugs, remifentanil, oxycodone, cocaine, and morphine, were obtained from the nida drug supply program (rti). mice were euthanized, their brains removed and flash frozen. all equipment and surfaces were cleaned with rnase inhibitor solution and ish (advanced cell diagnostics) performed as previously described (severino et al., ) . to characterize mor knock-down in the d -, d -, a a-, and -flmor mice, the following riboprobes were used; oprm (catalog # , atto ), drd (catalog # -c , alexa fluor ), and drd a (catalog # -c , atto ). to characterize mor knock-down in chatflmor mice, the same oprm and drd a riboprobes were used as well as a chat riboprobe (catalog # -c , alexa fluor ). rna in situ hybridization was imaged using a  oil immersion objective on a leica sp stimulated emission depletion microscope (sted, leica microsystems) at the advanced light microscopy core. the images were compiled in adobe illustrator and brightness and contrast and the tonal adjustments feature uniformly applied across the entire composite image. to determine the extent to which mor was deleted from specific neuronal types within each of the mouse lines generated, we counted the number of drd a, drd , or chat-positive cells and then determined the number of these cells that were mor positive (having a minimum of three grains). the data are expressed as the percentage of mor-positive cells within each of the subgroups (drd a, drd , or chat). quantitative reverse transcription-pcr (qpcr) qpcr was performed in flmor, d -, d -, a a-, and chat-flmor mice to define the relative expression levels of oprm , drd a, and drd using the primers shown in table and methodology as previously described . relative ratios comparing conditional kos to flmor expression for each gene of interest were calculated by using b -actin as reference gene and the -ddct method to evaluate differential expression levels. fiberglass open field boxes (   cm) were placed on a horizontal glass pane cm above an infrared camera (aca - gm basler ace camera) at lux. after d of habituation, mice were placed in the chamber for min followed by a subcutaneous injection of saline or drug and placed back in the chamber for min and their locomotion activity recorded (ethovision xt , noldus). this was repeated at the same time of day for three consecutive days. an intravenous catheter ( . mm i.d., . mm o.d., norfolk access) was inserted into the right jugular vein of mice under sterile conditions as previously described (james et al., ; storey et al., ; mittal et al., ) . after d of recovery, the mice began daily self-administration in operant chambers (med-associates) for h or reinforcers, whichever came sooner. a two-lever design was used in which the active cue and drug-paired lever, or the inactive lever, was randomly assigned. an active lever press resulted in an intravenous drug infusion ( . ml/g body weight) and the presentation of a -s tone and visual light cue. each reinforcer was followed by a -s "timeout" period during which no reinforcers could be delivered but presses could be made on either lever. on the first d of this protocol, mouse exploration of the levers was facilitated by placing a drop of % sweetened condensed milk on both the active and inactive levers (  per session). the mice initially underwent - d of acquisition training using remifentanil ( . mg/kg/infusion) at a fixed ratio of one (one lever press resulted in one infusion, fr ). oxycodone ( . mg/kg/infusion) was then used as the reinforcer for nine consecutive days, the maintenance phase, on the same fr schedule. this was followed by extinction training over d during which the mice underwent the same fr schedule to a maximum of reinforcers or h, but saline was delivered through the catheter. catheter patency was tested using an infusion of propofol ( ml of % propofol w/v in saline) every d. power analyses of prior data indicate that power is . or greater with cell means of n = (n = used for experiments where animal drop-out rates are expected because of jugular cannula failure, etc.). for experiments where we lacked sufficient prior data for an a priori power analysis, we used prior experience with similar methods to guide us. although we did use male and female mice, we did not analyze sex as a biological factor as we did not have sufficient power to do so. all experiments included both genotypes with males and females representing % and %, respectively, of the total number of mice used. several analytical methods were used anova one-way or two-way anova was used to analyze data obtained from the rna ish, qpcr, total locomotion and the intrasession ivsa datasets using prizm v (graphpad) with further details provided in the results and statistical tables. lmm were used to analyze the intrasession locomotion data so as to examine the slope and so rate of change over time of this dataset. we also used lmm with coefficients accounting for random slope or intercept within subjects to define and interpret the intersession ivsa datasets. we used the lmertest (kuznetsova et al., ) package in r to run lmm. the linear models were used to assess the effect of time, treatment group, or an interaction of these factors on each variable. the resulting model is a regression equation where the intercept or the slope is allowed to vary for each subject: where y characteristic is the characteristic being modeled (e.g., distance traveled, lever presses, etc.), each predictor variable is represented by its subscripted x, u subject represents the random intercept or slope associated with each individual subject. the coefficients (b ) are estimated and assessed for significance. whenever a significant effect was observed, an anova against a reduced null model was used to assess the impact of the respective factor. we first defined the selectivity of the loxp/cre recombinase system by rna in situ hybridization to examine cellspecific knock-down of the mor encoding gene (oprm ) in the dorsolateral striatum. we found that, for cells labeled with the drd probe, oprm and drd colocalization was reduced in d flmors (representative image, fig. ai ; quantified expression, fig. bi ; p , . , table , item a) and enhanced in d flmors ( fig. bi ; p , . , table , item a). for cells labeled by the drd probe, oprm and drd colocalization was reduced in d flmors (representative image, fig. ai ; quantified expression, fig. bii ; p , . , table , item b). a aflmors showed oprm expression in drd cells and a deletion from some, but not all drd cells (representative image, fig. ai ; quantified expression, fig. bii ; n.s, table , item b). in assessing oprm expression in chat cells, we found a loss of oprm expression in chatflmors compared with flmors but no change in drd expression, a positive control (representative image, fig. aii ; quantified expression, fig. biii ; p , . , table , item c). qpcr was performed to determine overall striatal expression levels of oprm , drd , and drd in flmor in the conditional knock-down strains. we found a loss of oprm cdna in d flmors (p = . ) and d flmors (p = . ; fig. ci ; table , item d) but no other line. there was no compensatory effect of these mor deletions on drd ( fig. cii ; table , item e) or drd ( fig. ciii ; table , item f) expression in the different lines. selective mor deletions define specific roles of d and a a mor populations in opioid-induced hyperlocomotion oxycodone as the analgesic effects of oxycodone may be non-specific (yang et al., ) , we first examined the locomotor effect of oxycodone ( mg/kg, s.c.) in mice lacking mors in all cells, a global mor ko, and their wt littermates, ( fig. a ) over three consecutive days. on day , we found no effect of oxycodone in mor kos compared with wts (p , . ), a lack of effect that did not differ from wts injected with saline (p = . , table , item a). by the third day, the oxycodone locomotor response had sensitized in wts (p , . ) but no change was observed in kos (p = . , table , item b). the -min timebins of the intrasession data further show oxycodone-induced hyperlocomotion in wt but not kos and sensitization of this response in only wts over time (fig. b , p , . ; table , item c). we then examined the dose-response relationship of oxycodone using (saline), , , and mg/kg subcutaneously in each of the genotypes (fig. a) . we found no effect of genotype following saline suggesting no effect of these deletions on basal locomotion (table , item a). however, a significant dose by genotype interaction was found following oxycodone (p , . , table , item b). ( ) dose. when compared with the saline group of the same genotype, the mg/kg dose of oxycodone had no effect, but and mg/kg of oxycodone induced hyperlocomotion in control flmors (p = . and p = . , respectively), a aflmors (p , . for both doses), and following mg/ kg in d flmors (p , . ) and chatflmors (p = . ). however, there was no effect of oxycodone in the d flmors (table , item b). ( ) genotype. d flmors showed a decreased response compared with flmors at mg/kg (p = . ) whereas a aflmors showed a greater locomotor response than flmors (p , . for both doses). neither chatflmors nor d flmors differed from flmors (table , item c). our first experiments examined the dose-dependent locomotor effects of morphine using (saline), , , and table for statistical analyses. all data are shown as mean sem, and the individual datapoints are shown in extended data figure - , for which this legend also applies. mg/kg (subcutaneously; fig. b ). we observed a dose  genotype interaction (p , . , table , item d) as follows. ( ) dose. when compared with the group receiving saline of the same genotype, we found that mg/kg morphine, but not any lower doses, induced hyperlocomotion in flmors (p = . ) and d flmors (p , . ). d flmors and chatflmors showed no response at any dose (table , item d) whereas a aflmors showed hyperlocomotion after both and mg/kg (p , . for both doses), but not mg/kg. ( ) genotype. between genotype analysis (table , item e) showed a similar effect of genotype following morphine as oxycodone treatment in that, when compared with flmors, a aflmors showed an enhanced response at the higher doses used, (p = . ) and (p = . ) mg/kg, whereas d flmors showed a reduced response at mg/kg (p = . ), but not mg/kg. both d -and chat-flmors were not different from flmors. to assess whether the changes in opioid-induced locomotor responses were generalizable to other drug classes, we determined the effect of genotype on cocaine-induced locomotion ( mg/kg, s.c.; fig. c ). we found cocaine-induced locomotion in all genotypes (fig. c , p . ; table , item f) but this effect was enhanced in chatflmors (p , . , table , item g). repeated opioid exposure is well known to induce a sensitization of the initial hyperlocomotor response (tao et al., ) . this occurs concurrently with an increase in the incentive motivational properties of a drug and has been considered as a window into this property of drugseeking behavior (robinson and berridge, ) . to assess the role of each of these mor populations in this phenomenon, we examined sensitization to oxycodone ( mg/kg, s.c.), morphine ( mg/kg, s.c.), and saline, over three consecutive days of drug exposure in all genotypes. the data were analyzed by two-way anova to assess the effect of day and drug on the first and last days of the test. the flmors showed a genotype  day interaction as both oxycodone (p = . ) and morphine (p = . ), but not saline, induced sensitization ( fig. d ; table , item h). the d flmors showed no sensitization effect following oxycodone or morphine and this response was not different from saline ( fig. e ; table , item i). the d flmors were similar to flmors as they sensitized to both oxycodone (p , . ) and morphine (p , . ) but not saline ( fig. f ; table , item j). the a aflmors sensitized to oxycodone (p , . ) but not to morphine or saline. (fig. g ; table , item k). the chatflmors similarly sensitized to oxycodone (p , . ), but not morphine or saline ( fig. h ; table , item l). we then defined the locomotion profile induced by each drug with each session using linear mixed model analysis to assess the effect of time and genotype. this was done using -min timebins on day and day of mg/kg oxycodone or mg/kg morphine. ( ) there was a genotype  time interaction on day of oxycodone ( fig. i , p , . ; table , item m). the d flmors (table , item n, p = . ) and a aflmors (p , . ), but not flmors, d -or chat-flmors showed a change in locomotor activity within the session. ( ) we did not find a timebin  genotype interaction (table , item o) on day of oxycodone. however, the d flmors showed decreased activity over time (fig. j , p , . ; table , item p), but no other change in activity over time was observed in other lines. ( ) there was a genotype  time interaction on day of morphine (fig. k , p , . ; table , item q), with d flmors showing a different locomotor profile than flmors (p = . ). further post hoc analyses showed that flmors (table , item r, p , . ), d flmors (p , . ), a aflmors (p , . ), and chatflmors (p = . ), but not d flmors, increased their locomotor activity during the session. ( ) we also observed a significant genotype  time interaction on day (fig. l , p , . ; table , item s), with both d flmor (p , . ) and chatflmor (p , . ) showing less activity during the session than (table , item t, p , . ), d flmors (p , . ), a aflmors (p , . ), and chatflmors (p = . ), but not d flmors, increased their locomotor activity during this session. selective mor deletions define specific roles of a a and chat mor populations in opioid ivsa although opioid-induced locomotion and sensitization of this response have been used as an index of reward behaviors (robinson and berridge, ; stewart and badiani, ) , ivsa is considered as a more direct measure of reward seeking and addiction (everitt et al., ) . we therefore examined whether deleting mors from these neurons altered opioid ivsa through an indwelling jugular catheter under a short-access fr schedule. each of the phases of the ivsa protocol (remifentanil acquisition, oxycodone maintenance and extinction) were analyzed separately and results presented for each of the following four parameters; active and inactive lever presses, reinforcers earned and lever choice as shown by the percent of active lever/total lever presses made. remifentanil, a fast-acting opioid, was used to establish the association of an active lever press with an opioid infusion and associated cues. during this short acquisition phase, we did not find a genotype  day interaction or any main effect of genotype on any of the four parameters measured; (fig. a-d, respectively) . however, we found a main effect of day on active lever presses made (p , . , x = . ; table , item a), reinforcers earned (p , . , x = . ; table , item b), and percentage active lever presses (p , . , x = . ; table , item c), but not inactive lever presses, showing that all lines acquired this self-administration behavior but there was no effect of genotype. the mice were then transitioned to oxycodone selfadministration for d. compared with those on saline, mice receiving oxycodone made more active lever presses (fig. e , p , . ; table , item d), earned more reinforcers (fig. g , p , . ; table , item e), and had a higher percentage active lever presses (fig. h , p , . ; table , item f). there was no difference in the inactive lever presses made between the saline and . wts demonstrated a sensitization of this locomotor response (p , . ) from day to day that was absent in kos. refer to table for statistical analyses. all data are shown as mean sem, and the individual datapoints are shown in extended data figure - for which this legend also applies. oxycodone groups (fig. f) . there was no effect of genotype on any parameter. extinction has been shown to increase drug-seeking behavior following oxycodone self-administration (hakimian et al., ) . we similarly found that, when compared with saline, all genotypes showed a treatment  day interaction in the number of active lever presses made (p , . ; fig. e ; table , item g) and reinforcers earned (p , . ; fig. g ; table , item h), but not inactive lever presses (fig. f ) or percentage active lever presses (fig. d ) between the last day of oxycodone maintenance and the first day of extinction. post hoc analyses showed an effect of oxycodone in that mice receiving oxycodone made more active lever presses (p , . ; table , item i), inactive lever presses (p , . , table , item j) and earned more reinforcers (p , . ; table , item k) on the first day of extinction there was no effect of genotype following the vehicle ( ) injection showing no effect of any of these deletions on basal locomotor activity. b, morphine ( , , , mg/kg) also induced a dose-dependent increase in locomotor activity in flmors, d flmors, and a aflmors but not in d flmors or chatflmors (a: p , . vs , b: p , . vs ). compared with control flmors, this effect was enhanced in a aflmors (ppp , . and pppp , . vs flmor of the same dose). c, cocaine ( , mg/kg) induced hyperlocomotion in all lines when compared with saline ( ; pppp . ), an effect that was enhanced in chatflmors (a: p , . vs flmors). d-h, sensitization. after three consecutive days of repeated opioid injections, flmors (d) and d flmors (f) showed an enhanced, or sensitized, response to both oxycodone and morphine. d flmors (e) did not show this enhanced effect to either opioid whereas a aflmors (g) and chatflmors (h) sensitized to oxycodone but not morphine (pp , . and ppp , . , respectively, vs day ). i-l, intrasession locomotor analysis. this analysis assessed the locomotor response to oxycodone or morphine during each -min session on day and day . i, a single injection of oxycodone ( mg/kg) on day increased locomotor activity in d flmors (p , . ) and a aflmors (p , . ), whereas flmors, d flmors, and chatflmors showed no change in activity during the session. j, after d of repeated oxycodone administration, the locomotor activity of d flmors (p , . ) declined through the session and all other genotypes showed no change across time. k, a single injection of morphine ( mg/kg) on day resulted in a within-session increase in locomotor activity in flmors (p , . ), d flmors (p , . ), a aflmors (p , . ), and chatflmors (p , . ), but not d flmors. l, after d of repeated morphine administration, a similar pattern emerged as on day with flmors (p , . ), d flmors (p , . ), a aflmors (p , . ), and chatflmors (p , . ), but not d flmors, showing a within session increase in locomotor activity. refer to table for statistical analyses. all data are shown as mean sem, and individual datapoints are shown in extended data figure - for which this legend also applies. research article: new research versus the last day of maintenance. no such transition effect was observed across any parameter in the saline group. we then assessed the change in drug-seeking behavior over the d of extinction in mice that had received oxycodone using lmm analysis. we found no genotype  day interaction, however there was a main effect of genotype on reinforcers earned (fig. g , p , . ; table , item n) with chatflmors (p , . ) and a aflmors (p , . ) earning more reinforcers than flmor mice over these d. there was a trend toward a main effect of genotype for acquisition. during this short acquisition phase (days - ) during which remifentanil was self-administered there was no effect of genotype and no interaction or a main effect of genotype on any of the four parameters measured; active lever presses (a), inactive lever presses (b), reinforcers earned (c), or the percent active lever presses made (d). e-h, maintenance and extinction. mice were then transitioned to oxycodone self-administration for d followed by d of extinction. when compared with mice self-administering saline, those that self-administered oxycodone made more active lever presses (p , . ), earned more reinforcers (p , . ), and showed a preference for the active over inactive lever (p , . ) during the maintenance and extinction session. e, during the extinction but not maintenance phases, a aflmors made more active lever presses than flmors (p , . ). f, there was no effect of genotype on the number of inactive lever presses at any stage. g, similar to the number of active lever presses made, a aflmors and chatflmors earned more reinforcers than flmors during extinction (a: p , . ). h, there was no effect of genotype on active lever preference as shown by the percent active lever/total lever presses. i-n, within session analysis of the cumulative number of active lever presses made and reinforcers earned during the -h session was assessed on three specific days; the last day of oxycodone (day ) and the first (day ) and third (day ) days of extinction. this shows no effect of genotype on the last day of oxycodone for either the cumulative active lever presses (i) or reinforcers (j) earned. k, however, on the first day of extinction, a aflmors made more active lever presses than flmors (a: p , . vs flmor at and and - min). i, a similar effect was seen in the reinforcers earned during this session when a aflmors earned more reinforcers (a: p , . vs flmor at - and min) as did chatflmors (b: p , . vs flmor at - min). n, on the third day of extinction, there was no further effect of genotype on the number of active lever presses made. m, however, the chatflmors showed an increase in reinforcers earned on the third day of extinction (a and c: p , . and p , . , respectively, vs flmor at - min). refer to table for statistical analyses. all data are shown as mean sem. active lever presses (fig. e , p = . ; table , item l) and percentage active lever presses (fig. h , p = . ; table , item m), with a aflmors showing increased active lever presses (p , . ) and percentage active lever presses (p , . ) made over these d than the flmors. we also observed a main effect of day on reinforcers earned ( fig. g , p , . ; table , item o) with all mice showing a decrease in reinforcers earned over the d of extinction with no effect of genotype. no other effects were found for active lever presses, inactive lever presses and percentage active lever presses across these d. we also analyzed the cumulative frequency of active lever presses and reinforcers earned during the -h test on three specific days of the ivsa protocol ( fig. i-n) . the first of these days, day of the maintenance phase and the last day of oxycodone self-administration, showed a lack of genotype effect on either the cumulative active lever presses ( fig. i ; table , item p) or reinforcers earned ( fig. j ; table , item q). however, on the next day assessed, extinction day , a aflmors showed an increase in cumulative active lever presses (fig. k , p , . ; table , item r) and reinforcers earned (fig. l , p , . ; table , item s). chatflmors also earned more reinforcers than flmors on this day (fig. l , p , . ; table , item s). by the third day of extinction, there was no effect of genotype on cumulative active lever presses ( fig. m ; table , item t), but there was an effect of genotype on cumulative reinforcers earned with chatflmors earning more reinforcers than flmors during the last min of the test (fig. n ; table , item u). these findings outline distinct roles for mors on neuronal populations in behaviors associated with opioid-induced locomotion and reward behaviors. these are that selective ablation of mors from d receptor-expressing neurons prevents opioid-induced locomotor hyperactivity as well as locomotor sensitization but has no effect on opioid ivsa. second, removal of mors from a a neurons enhances opioid-induced hyperlocomotion, locomotor sensitization and drug-seeking behaviors during extinction following opioid ivsa. third, ablation of mors from chat neurons results in an agonist-dependent hyperlocomotor effect whereby morphine fails to elicit dose-dependent locomotor hyperactivity or sensitization yet oxycodone-induced effects are similar to control flmor mice. these mice also show an increase in drug-seeking behavior during extinction. fourth, despite the common theory that a a receptor expression is equivalent to d receptor expression in medium spiny neurons, our data suggests that the a a cre deletes mors from only a subset of d medium spiny neurons and, that, in stark contrast to mor deletion from a a neurons, mor deletion from d neurons results in no discernible change in these reward-based behaviors (fig. a ). our study shows that mors on d neurons are required for the initial locomotor and sensitization response to morphine and oxycodone. the effect of morphine is in line with a previous study in which the expression of mors in only d neurons in striatal patches in an otherwise null background reinstated morphine-induced locomotion (cui et al., ) . together these findings demonstrate both the requirement and necessity of this mor population for this striatal-mediated output. this may be a result of mors on d recurrent collaterals inhibiting d neurons to reduce striatal output and attenuate the motor effect of opioids, as modeled in figure b . another possibility is that these receptors are required for the release of dopamine in the vta (cui et al., ) , which is required for this response (steidl et al., ) . in regards our ivsa findings, the lack of effect of the d mor deletion in the acquisition of oxycodone ivsa is in contrast with previous work (cui et al., ) , perhaps as other mor populations such as those within the matrix, are also involved in the acquisition phase of this behavior. it is also possible that this is an example of an opioid-specific effect in which the faster-acting opioid, remifentanil, used in (cui et al., ) , results in greater lever pressing behavior than oxycodone. as d receptors are expressed on cholinergic interneurons (weiner et al., ) , the a a cre line has been used to selectively target d medium spiny neurons (fink et al., ; rosin et al., ; wang et al., ) . our findings show that this a a-mor population is an apparent subset of d medium spiny neurons that controls the locomotor sensitivity to oxycodone and morphine and drug-seeking behavior during extinction. these inhibitory receptors may be on some d -d collaterals (taverna et al., ) , where their deletion allows an earlier threshold to be reached to increase striatal motor output, as modeled in figure b . as mors on cholinergic interneurons remains intact and, surprisingly, mors are also present on some d striatal neurons, their deletion displays a remarkably different and striking phenotype from d flmors. this could reflect a role of this striatal population or an extrastriatal neuronal population that expresses both a a and m opioid but not necessarily d receptors. as regards mors on d neurons, we find that these receptors influence neither opioid-induced locomotion nor opioid ivsa. while deleting mors from d , d and a a neurons was performed to identify their role in gabaergic striatal neurons, deleting mors from cholinergic interneurons examines the role of these receptors in altering cholinergic neuronal activity. these neurons form - % of the striatal population yet they are remarkably influential in controlling striatal circuits (gritton et al., ) and output, and both mors and d -opioid receptors strongly inhibit their rhythmic activity to affect behavior (bertran-gonzalez et al., ; ponterio et al., ) . activation of mors could affect glutamate or acetylcholine release and subsequent dopamine release from nearby terminals (yorgason et al., ) to alter the activity of local circuits (for review, see clarke and adermark, ; berke, ) . omission of an expected reward induces a dip in dopamine release, a negative reward prediction error (rpe) accompanied by a pause in cholinergic interneuron activity (hart et al., ) to affect local d and d medium spiny neuron activity (mamaligas and ford, ) . deleting mors from these neurons may prevent the encoding of an rpe and facilitate drug-seeking, as shown by an increase in cue-induced reinforcers earned, but not active lever presses, during extinction (fig. m,n) . the rapid increase in hyperlocomotion following oxycodone and the sustained, gradual increase in hyperlocomotion following morphine (fig. i -l) is likely because of the different plasma-kinetic (pk) profiles of these two drugs. oxycodone has a higher percentage of unbound drug in the blood and a -fold greater influx rate than morphine (boström et al., ) . this results in a -fold higher ratio of unbound oxycodone in the brain: blood and a higher unbound steady state in the brain (boström et al., (boström et al., , likely explaining the larger increase in dopamine release following intravenous oxycodone than intravenous morphine (vander weele et al., ) . the ligand-dependent and genotype-dependent effect of morphine but not oxycodone in chatflmors further suggests that this receptor population is more sensitive to the pk profile of each ligand. this could be because of a time-dependent effect of these receptors in modulating intrinsic cholinergic interneuron activity and the control of local circuitry. there are several limitations of this study. one is that we have used the loxp-cre recombinase system to achieve developmental deletion of mors from various neuronal populations (gong et al., ) . for the most part these populations are striatal where the co-expression of mors with d or d receptors can be used to define different medium spiny neuron populations (gerfen et al., ; weiner et al., ) . however, dopamine neurons project to various brain regions in addition to the striatum, the hippocampus, amygdala, and prefrontal cortex. the behavioral outcomes in this study may therefore be influenced by mor expression on dopamine circuits outside the striatum. for example, mor expression on the intercalated neurons of the amygdala (gregoriou et al., ) , and in the globus pallidus (weiner et al., ; delfs et al., ) ablating mors from d expressing neurons removes mor inhibition and increases a a msn activity greater reduction in motor output. removes mor inhibition and increases d msn activity greater increase in motor output. gpe decreased motor output (increased with cocaine) figure . a, summary of our findings. deleting mors from d neurons reduces oxycodone-induced hyperlocomotion and sensitization but does not alter the ivsa profile. deleting mors from d neurons alters neither the locomotor effects of oxycodone nor the ivsa profile whereas deleting mors from a a neurons increases oxycodone-induced hyperlocomotion and sensitization and also drug-seeking behaviors following opioid ivsa. deleting mors from chat neurons does not alter oxycodone-induced hyperlocomotion and sensitization but does increase the locomotor effect of cocaine and drug-seeking behaviors following opioid ivsa. b, a possible mechanism by which mors on d or a a neurons alter striatal-mediated motor output. removing mors from d medium spiny neurons and so d -a a recurrent collateral increases a a neuronal activity to reduce striatal motor output. conversely removing mors from a a medium spiny neurons and so a a-d recurrent collaterals increases d neuronal activity to increase striatal motor output. reward behaviors (boulos et al., ) and mors and chat co-expression in secretomotor neurons of the colon suggests gut function may be altered in chatflmors (galligan and akbarali, ) . further studies could also assess the role of mors in different striatal subregions such as in patches or matrix, dorsal ventral striatum and co-expression with both d and d receptors (soares-cunha et al., ) . an additional limitation is that we did not assess the effect of the cre insertion alone as this would have required further back-crossing of all lines. striatal d and d neurons are traditionally considered to have opposing effects on striatal motor patterns resulting in a coordinated motor activity. in this simple model, activating d neurons of the direct pathway increases striatal output to facilitate movement whereas activating d neurons of the indirect pathway inhibits competing motor patterns and inhibits movement (kravitz et al., ) . this model has been expanded and developed to include several interacting factors that influence the threshold of these outputs by recurrent collaterals between d and d neurons (bahuguna et al., ) , regulation by different interneurons (taverna et al., ) , and the regional and compartmental expression patterns of d and d (cui et al., ; oude ophuis et al., ) . nevertheless, the opposing and complimentary effects of medium spiny neuron activation remains a central component of their activity. we show that the effect of deleting mors from d and a a neurons resembles such complementation, albeit the inverse, as it is the absence of mors from d or a a neurons that reduces or facilitates motor output, respectively. we propose that this can be explained by the presence of these g io -coupled receptors on recurrent medium spiny neuron collaterals, as shown by the schematic model in figure b . the roles of d and d medium spiny neurons in mediating reward are also seen as divergent yet complementary in that d neurons mediate drug reinforcement and positive reward behaviors, whereas the d s mediate aversion or ambivalence and are active during withdrawal (koo et al., ; cole et al., ) . in addition, d and d receptors also play complementary but opposing roles in learning value-based and motivated behaviors, an important component of the change in reward value during extinction (verharen et al., ) . in regards the roles of mors on these neurons, we show that rather than mediating positive reinforcement during the initial stages of opioid reward, that it is mors on a a or chat neurons that are important in controlling drug seeking during extinction, a period of increased anxiety and negative affect (carmack et al., ) . additional studies to further define the effect of these deletions on a a or chat neurons under different physiological 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mesopontine and rostromedial tegmental neurons tolerance and sensitization to the behavioral effects of drugs nicotine modifies corticostriatal plasticity and amphetamine rewarding behaviors in mice ( , , ) heteromers of m opioid and dopamine d receptors modulate opioid-induced locomotor sensitization in a dopamine-independent manner recurrent collateral connections of striatal medium spiny neurons are disrupted in models of parkinson's disease rapid dopamine transmission within the nucleus accumbens: dramatic difference between morphine and oxycodone delivery differential contributions of striatal dopamine d and d receptors to component processes of value-based decision making dendritic spines containing m-opioid receptors in rat striatal patches receive asymmetric synapses from prefrontal corticostriatal afferents ultrastructural immunocytochemical localization of mu opioid receptors and leu -enkephalin in the patch compartment of the rat caudate-putamen nucleus ultrastructural immunocytochemical localization of mu-opioid receptors in dendritic targets of dopaminergic terminals in the rat caudate-putamen nucleus medium spiny neurons of the anterior dorsomedial striatum research article: new research mediate reversal learning in a cell-type-dependent manner d and d dopamine receptor mrna in rat brain activation of delta-opioid receptor contributes to the antinociceptive effect of oxycodone in mice cholinergic interneurons underlie spontaneous dopamine release in nucleus accumbens locomotor activity: a distinctive index in morphine self-administration in rats key: cord- -jkg qkj authors: skowronski, danuta m.; hamelin, marie-eve; de serres, gaston; janjua, naveed z.; li, guiyun; sabaiduc, suzana; bouhy, xavier; couture, christian; leung, anders; kobasa, darwyn; embury-hyatt, carissa; de bruin, erwin; balshaw, robert; lavigne, sophie; petric, martin; koopmans, marion; boivin, guy title: randomized controlled ferret study to assess the direct impact of – trivalent inactivated influenza vaccine on a(h n )pdm disease risk date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: jkg qkj during spring-summer , several observational studies from canada showed increased risk of medically-attended, laboratory-confirmed a(h n )pdm illness among prior recipients of – trivalent inactivated influenza vaccine (tiv). explanatory hypotheses included direct and indirect vaccine effects. in a randomized placebo-controlled ferret study, we tested whether prior receipt of – tiv may have directly influenced a(h n )pdm illness. thirty-two ferrets ( /group) received . ml intra-muscular injections of the canadian-manufactured, commercially-available, non-adjuvanted, split – fluviral or pbs placebo on days and . on day all animals were challenged (ch ) with a(h n )pdm . four ferrets per group were randomly selected for sacrifice at day post-challenge (ch+ ) and the rest followed until ch+ . sera were tested for antibody to vaccine antigens and a(h n )pdm by hemagglutination inhibition (hi), microneutralization (mn), nucleoprotein-based elisa and ha -based microarray assays. clinical characteristics and nasal virus titers were recorded pre-challenge then post-challenge until sacrifice when lung virus titers, cytokines and inflammatory scores were determined. baseline characteristics were similar between the two groups of influenza-naïve animals. antibody rise to vaccine antigens was evident by elisa and ha -based microarray but not by hi or mn assays; virus challenge raised antibody to a(h n )pdm by all assays in both groups. beginning at ch+ , vaccinated animals experienced greater loss of appetite and weight than placebo animals, reaching the greatest between-group difference in weight loss relative to baseline at ch+ ( . % vs. . %; p = . ). at ch+ vaccinated animals had higher lung virus titers (log-mean . vs. . pfu/ml, respectively; p = . ), lung inflammatory scores ( . vs. . , respectively; p = . ) and cytokine levels (p> . ). at ch+ , both groups had recovered. findings in influenza-naïve, systematically-infected ferrets may not replicate the human experience. while they cannot be considered conclusive to explain human observations, these ferret findings are consistent with direct, adverse effect of prior – tiv receipt on a(h n )pdm illness. as such, they warrant further in-depth investigation and search for possible mechanistic explanations. during spring-summer , several observational studies from canada reported that prior receipt of the - trivalent inactivated influenza vaccine (tiv) was associated with increased risk of medically-attended, laboratory-confirmed a(h n )pdm illness, with estimated risk or odds ratios of . - . compared to those unvaccinated [ ] . this increased risk was not apparent among vaccinated people when comparing hospitalized to community cases [ ] , and observational studies in other settings showed contradictory results, including increased [ ] [ ] [ ] [ ] , null [ ] [ ] [ ] [ ] or protective [ , ] effects from vaccination. hypotheses to explain findings from canada initially focused on methodologic (observational designs) or product-specific (domestically-manufactured vaccine) considerations. however, a randomized-controlled trial (rct) in hong kong spanning november to october also showed significantly increased relative risk ( . ) among children who had received a different manufacturer's - tiv product (vaxigrip, sanofi pasteur, lyon, france) [ , ] . previous ferret studies have also shown mixed results although none have demonstrated - tiv to have been protective against a(h n )pdm [ ] [ ] [ ] [ ] [ ] [ ] . two small ferret studies reported no tiv effect on virus replication in nasal or lung specimens [ , ] but, where clinical outcomes have been assessed, several studies have shown consistent albeit non-significant trend toward greater weight loss and worsening of severity indicators in vaccinated ferrets [ ] [ ] [ ] [ ] . all of these ferret studies to date, however, have suffered from small sample size, typically comparing # animals per group in total. mechanistic hypotheses to explain increased a(h n )pdm risk among prior tiv recipients have included both direct and indirect vaccine effects [ ] . the direct effect hypothesis postulates that seasonal vaccine may directly influence host resistance to pandemic virus infection and/or replication whereas the indirect hypothesis proposes that seasonal vaccine may block the more robust, complex and cross-protective immunity otherwise afforded by seasonal virus infection thereby indirectly increasing the risk of pandemic illness. here we report on a randomized, blinded, placebo-controlled ferret study to test whether the commerciallyavailable tiv predominantly used in canada in - may have directly influenced a(h n )pdm disease risk. overview experimental procedures were conducted at the laval university animal care facility in québec between april and july , ( figure ). animals were housed two per cage in the same room and permitted food and water ad libitum. the primary outcome of this study was weight loss. sample size was assigned to test differences in proportionate weight loss from baseline following infection with pandemic h n virus. based on % power and -sided alpha of . to detect mean difference in weight loss relative to baseline of % in the placebo group and % in the vaccine group, and given standard deviation (sd) of - %, - ferrets per group would be required. we used ferrets per group. thirty-two male ferrets were randomly assigned to receive . ml intra-muscular injection of either - tiv (''vaccine'') or pbs (''placebo'') on days and . study personnel and investigators remained blinded to vaccine/placebo assignment throughout experimental procedures and assays. glaxosmithkline (gsk fluviral; manufactured in laval, québec, canada) and sanofi pasteur (vaxigrip; manufactured in lyon, france) supplied approximately % and %, respectively, of the seasonal split tiv distributed in canada during the - season. in this experiment we therefore used the dominant canadian manufactured, commercially-available, non-adjuvanted gsk sodium deoxycholate split-antigen fluviral containing the three who- the potency of all three vaccine strains of the post-expiry but cold-chain maintained - fluviral lot that was used was confirmed by single radial immuno-diffusion (srid) testing by the center for biologics evaluation and research, united states (us) food and drug administration (fda) [ ] . us specifications require $ mg/ml hemagglutinin (ha) for each antigen (dose . ml) based on the mean of three tests, with additional requirements around the standard deviation (sd). as last assessed in february , for each antigen, mean ha content for the tiv lot used remained $ mg/ml with sds within required specifications. on day , animals were lightly anesthetized and challenged (ch ) intra-nasally with ml ( . logtcid /ml) [ , ] (table s ) , administered half-volume per nostril. four randomly-selected ferrets per group were sacrificed on day (i.e. days post-challenge: ch+ ) and the remainder sacrificed on day (i.e. days post-challenge: ch+ ). pre-shipment sera collected between march and , and serum samples from anesthetized animals collected on days (may , ), , (ch ), (ch+ ) and (ch+ ) were assessed for antibody to tiv components and a(h n )pdm by haemagglutination inhibition (hi), microneutralization (mn), nucleoprotein (np)-based elisa and ha -based protein microarray assays. hemagglutination inhibition and microneutralizationantigens and analyses. reference viruses used in the hi and mn assays were obtained from canada's influenza reference laboratory, the national microbiology laboratory (nml), winnipeg, and passaged in mdck cells two to three times (table s ) . mdck-passaged viruses were tested by both hi and mn against reference ferret anti-serum provided by the nml, confirming antigenic integrity was maintained relative to the who-recommended reference viruses. viral hemagglutinin (ha) and neuraminidase (na) were also sequenced to assess amino acid (aa) identity relative to who reference viruses and the actual vaccine components selected by the manufacturer (table s and table s ). compared to respective vaccine strains, passaged assay viruses showed complete ha and na identity for seasonal h n and $ % for h n . compared to challenge virus, passaged a(h n )pdm assay virus also showed $ % ha and na identity. percent identity was reduced as expected when comparing seasonal to pandemic h strains ( - % in the ha protein). hi and mn assays were conducted in duplicate according to established protocols [ ] as detailed below and the individual result assigned as the geometric mean titer (gmt) of duplicate values. summary hi and mn serologic statistics were compared including the number meeting threshold titers of and of . changes in titers from baseline (day ) to days , (ch ), and (ch+ ) or (ch+ ) were assessed through sero-conversion, group gmts with % confidence intervals (ci) and group gmt ratios (gmtr). haemagglutination inhibition methods. in preparation for the hi assay, sera were treated with receptor destroying enzyme (accurate chemical & scientific, ny) to remove nonspecific inhibitors of agglutination, and further hemadsorbed with % turkey erythrocytes (lampire biologic laboratories, pennsylvania). sera were serially diluted beginning at : with phosphate buffered saline and ml of each serum dilution was reacted with ml of antigen (infected mdck cell lysate supernatant) containing ha units of virus for minutes. to each mixture ml of . % turkey erythrocytes were added, and after mixing, the preparations were incubated for minutes. results were recorded by photography. the hi titer was designated as the inverse of the highest dilution at which detectable hi activity was still present. influenza b virus was used in the hi assay in its ether-treated, inactivated form. briefly, the clarified influenza b virus cell lysates were each mixed by agitation with an equal volume of diethyl ether. the mixture was kept at uc for minutes to allow for the phases to separate. the top ether layer was aspirated and nitrogen gas bubbled through the virus preparation to remove residual ether, and the preparation was then used in the hi assay. microneutralization methods. for mn assay, % tissue culture infectious dose (tcid ) viral titers were determined on mdck cells. the sera were treated with receptor destroying enzyme (accurate chemical & scientific, ny) and serially diluted in serum-free medium (megavir, hyclone, utah) beginning at : . to each ml dilution, infectious units of virus were added. the plates were incubated for hours at uc to allow for virus antibody interaction. the contents of each well were then transferred onto microtiter plates with confluent monolayers of mdck cells. after hours of further incubation at uc, the medium in each well was removed and replaced with fresh megavir medium containing mg/ml l- -tosylamido- -phenylethyl chloromethyl ketone (tpck)-treated trypsin. the plates were further incubated at uc and monitored for cytopathic effects on days and . the mn titer was defined as the inverse of the serum dilution immediately preceding the wells with cytopathic effects. and ha -based protein microarray methods. competitive elisa was conducted on sera using a commercially-available np-based influenza a antibody test kit from idexx laboratories, inc. (switzerland) [ ] . competitive elisa antibody values were calculated as sample optical density (od) divided by negative control od with ratios , . considered positive and mean values with %ci displayed; smaller elisa ratios denote higher antibody levels. an ha -based protein microarray serological assay was conducted for study and non-study antigens listed in table s , performed as previously described [ ] with adaptation for detection of ferret antibodies. serum samples were tested in a single : dilution in blotto containing . % surfact ampt (both thermo fisher scientific inc., rockford, usa). all incubation steps were one hour at uc. recombinant ha proteins were spotted in duplicate and incubated with ml blotto followed by ml of diluted ferret serum and then two-step conjugation by incubation with ml mouse anti-mustelid igg (antibodiesonline, aachen, germany) followed by ml dylight conjugated goat anti-mouse igg (jackson immunoresearch, baltimore pike, usa) both in blotto containing . % surfact ampt. after washing and drying, the slides were scanned using a powerscanner (tecan, mä nnedorf, switzerland) and signals quantified using a scanarray scanner (perkin elmer, waltham, usa). individual spot signals were valued with correction by subtraction for per spot background fluorescence, truncated at zero where signal was below background. final individual per ferret values were assigned as the average of duplicates, log transformed after imputing a value of . for any zero values and compared for each antigen by study group and day. activity, rectal temperature, appetite and weight were recorded daily from days through and then from challenge (day ; ch ) until sacrifice. baseline reference weight per ferret was computed as the average of day to and ch weights. activity was scored - : one being the most alert and playful; two, alert but playful only if induced; three, alert but not playful (stays in hiding place); four, neither alert nor playful and five was the humane endpoint. appetite was scored as usual, diminished or no appetite. a(h n )pdm virus titers were assessed in nasal wash prechallenge day and daily post-challenge (ch+ ) until sacrifice and in whole right lung homogenates at each scheduled sacrifice. virus titers were determined by standard plaque assays using st gali-expressing mdck cells [ , ] , expressed as log transformed plaque-forming units (pfu) per ml. paraffin tissue sections were prepared from whole left lung of ferrets for histo-pathology assessment at each scheduled sacrifice. inflammation was graded on six indicators (bronchial/endobronchial, peribronchial, perivascular, interstitial, pleural and intraalveolar), each scored as: (normal), (mild), (moderate) or (marked) for a maximum combined score of [ ] . vascular congestion and pulmonary edema were similarly scored. lung immuno-histochemistry was undertaken to identify cells with virus antigen. paraffin tissue sections were quenched for minutes in aqueous % h o then pre-treated with proteinase k for minutes. a : , dilution of mouse monoclonal antibody to influenza a nucleoprotein (f np ; in-house) was applied for one hour. sections were visualized using horseradish peroxidaselabelled polymer, envisionh+system (anti-mouse) (dako, usa), reacted with the chromogen, diaminobenzidine and counterstained with gill's hematoxylin. change in lung cytokine mrna gene expression was assessed by relative quantitative pcr (qpcr) with rna extracted from ml of right lung homogenate using the qiaamp viral rna mini-kit. reverse transcription was performed using the high capacity rna-to-cdna kit (applied biosystems) on . mg of the total rna following manufacturer's protocol. qpcr was performed using the taqmanh gene expression master mix (abi) with primers designed to target published cytokine sequences [ ] [ ] [ ] from mustela putorius furo mrna sequences at a final concentration of . mm. assays were run on the stepone plus (abi) with the following conditions: uc- minutes; uc- minutes; followed by cycles of uc- seconds; and uc- minute. fold-change was calculated using the delta-delta ct method [ ] with uninfected ferrets as reference and gapdh as endogenous control. chi-square or fisher's exact test were used to compare categorical variables and t-test or wilcoxon methods for continuous variables. weight, nasal virus titers, and ha protein microarray values were analysed via contrasts in a mixed-effects linear model (group, visit and their interaction, with repeated measurements on each animal). for protein microarray values, analyses were repeated after excluding statistical outliers without substantively affecting main conclusions. inflammatory scores by individual ferret are presented and combined inflammatory scores and cytokine values at ch+ versus ch+ were compared via contrasts in a two-way anova model (group, scheduled sacrifice day and their interaction). observed p, . are described as significant; no attempt is made to correct for multiple inference. individual hi, mn and elisa assay results are displayed concurrently for pre-shipment, day , , and / time points, for the four vaccinated and four placebo animals sacrificed at ch+ in table s , and for the animals per group sacrificed at day in table s for vaccinated animals and table s for placebo animals. summary elisa results are shown in table and summary microarray results for study antigens in figure with study and non-study antigen cross-reactive responses shown in figure s . summary hi and mn statistics are shown in table s and table s , respectively. all animals were confirmed by np-based elisa to be influenza a naïve at pre-shipment and day . by day there was significant elisa antibody rise following immunization in the vaccine group, with further antibody rise evident by day (ch+ ) after a(h n )pdm challenge in vaccinated animals but not until day (ch+ ) in placebo ferrets ( table , table s , table s , table s ). ultimately, total influenza a elisa antibody was significantly higher among vaccine compared to placebo animals at both scheduled sacrifices ( table ) . protein microarray results were consistent with elisa but in addition showed vaccine-induced ha antibody to the seasonal h antigen, for which values were significantly higher in vaccinated animals relative to pre-immunization and compared to placebo from day , most pronounced from day after the first tiv dose (i.e. three weeks after two-dose vaccine series completion) ( figure ). there was significant rise in antibody relative to pre-immunization for the h n vaccine component at day , but only relative to the placebo group at day (i.e. five weeks after vaccine series completion). slight but significant decrease in a(h n )pdm antibody was evident at day postimmunization compared to baseline but not thereafter or relative to placebo, calling into question its clinical relevance. significant rise in antibody to a(h n )pdm relative to pre-immunization was evident at day in both the vaccine and placebo groups. of interest, infection with a(h n )pdm induced antibody by day not only to itself but also to closely related h antigen in both study groups (significantly higher for the latter in the vaccinated) ( figure s ). furthermore, at day among vaccinated but not among placebo animals, a(h n )pdm challenge induced significantly greater cross-reactivity to nonstudy ( , ) h variants to which the animals had never been exposed, evident relative to pre-immunization, to the placebo group and to pre-challenge at day ( figure s ). increase following a(h n )pdm challenge among the vaccinated was evident at day for other non-h , non-vaccine antigens (h , h ) relative to pre-immunization but not relative to placebo. all animals were also shown by hi and mn assays to be influenza naïve at pre-shipment (table s , table s , table s ). more variability in hi than mn antibody titers was evident thereafter. overall, however, the significant rise in vaccineinduced antibody shown by elisa and ha protein microarray by day was not evident by hi or mn assays except among a few of the vaccinated ferrets ( table s , table s ). conversely, ferrets in both groups showed substantial neutralizing antibody to a(h n )pdm at day (table s , table s ) with very high gmts and gmtrs relative to baseline, slightly (but nonsignificantly) higher in the placebo compared to the vaccine group by both hi and mn (table s , table s ). average baseline weight was comparable between the vaccine ( . kg) and placebo ( . kg) groups (p = . ). beginning at ch+ , more vaccinated animals showed diminished or no appetite compared to placebo recipients ( / versus / ; p = . ) with the greatest between-group difference at ch+ ( / versus / ; p = . ), that resolved by ch+ ( / vs. / ; p = . ). the greatest between-group difference for no appetite was at ch+ ( / vs. / ; p = . ). weight loss from baseline was greater in the vaccine than placebo group, evident beginning at ch+ and marginally significant across the full study period (% weight loss p = . ; absolute loss p = . ) ( figure a) . the greatest between-group difference in percentage weight loss from baseline was at ch+ ( . % vs. . %; p = . ) ( figure a ) on which day / ( %) vaccinated versus / ( %) placebo animals had lost % or more of their body weight compared to baseline (p = . ) ( table s , table s , table s ). consistent with random selection, the same pattern was evident regardless of sacrifice day, with mean percentage weight loss from baseline at ch+ of . % for the vaccine group and . % for the placebo group among animals selected for sacrifice at ch+ and . % and . %, respectively, among animals sacrificed instead at ch+ . animals had comparable scores of one for alertness/playfulness at all time points except from ch+ - for which activity levels were marginally worse (scored as two) in all animals in both groups. temperature patterns did not differ between vaccine and placebo groups with a peak in mean/median temperatures at ch+ of . uc and . uc, respectively. nasal a(h n )pdm titers did not differ significantly between groups over time (p = . ). nasal virus titers rose more steeply between ch+ - in the vaccine versus placebo group (mean difference in log-titres . versus . pfu/ml; p = . ) and then fell more steeply at ch+ - (mean difference in log-titres . versus . pfu/ml; p = . ) ( figure b ). lung a(h n )pdm titers at ch+ were significantly higher in the vaccine versus placebo group (log-mean . versus . pfu/ ml; p = . ) ( figure c ). neither group had detectable virus in the lung at ch+ . at ch+ , animals sacrificed from the vaccine group had higher combined mean/median lung inflammatory scores than placebo animals ( . / . versus . / . ), a difference that did not reach statistical significance (p = . ) ( table s ) . two of four vaccinated animals showed a combined lung inflammatory score of $ at ch+ compared to none of the four placebo animals (maximum score = ) ( table s ). salient histologic features are shown in figure for animals of both groups with the highest and lowest combined ch+ inflammatory scores, illustrating the severe bronchopneumonia that was evident in half of the vaccinated but none of the placebo animals. the increased micrometric scale in panel d reflects that the pathologic changes are marked and diffuse, best rendered at low magnification, whereas the mild to moderate and more focal changes in panels a to c necessitate photomicrographs at higher magnification. compared to ch+ , combined mean lung inflammatory score was significantly lower in the vaccine group among animals sacrificed at ch+ ( . vs. . ; p = . ) whereas this showed little change over time in the placebo group ( . vs. . ; p = . ) with significant interaction between scheduled sacrifice day and group (p = . ). combined mean/median lung inflammatory scores were not significantly different between the vaccine versus placebo group at ch+ ( . / . versus . / . ). in both the vaccine and placebo groups at ch+ , influenza antigen was detected by immuno-staining in bronchial and bronchiolar epithelium. within alveolar walls, most of the positive cells were identified as pneumocytes using double immunolabelling [not shown]. occasional cells had the morphology of macrophages. no viral antigen was detected in animals at ch+ . ifn gamma was below the limits of detection in both groups at both scheduled sacrifices. other lung cytokines were consistently but non-significantly higher in the vaccine versus placebo group at ch+ . all cytokine values were lower in both groups at ch+ , consistently but non-significantly lower in the vaccine animals. the ch+ versus ch+ difference was statistically significant for all vaccine group cytokines (all p# . ) except ifn alpha (p. . ) whereas differences were not significant in the placebo group, except for il (p = . ). overall, the interaction between scheduled sacrifice day and study group was not significant for any cytokine (figures a and b ). during spring-summer , several observational studies from canada reported that prior recipients of - tiv experienced approximately two-fold increased risk of medically-attended, laboratory-confirmed a(h n )pdm illness [ ] . recognizing that all observational designs are susceptible to methodological bias, gold standard rct analysis would typically be considered essential in clarifying such unexpected findings. in hong kong, an rct of the - tiv (vaxigrip) already underway had shown similar association among vaccinated children with significant relative risk of . (increased to . with adjustment for seasonal infection) [ , ] ; however, during follow-up rct using - vaxigrip and spanning august to december , the same investigators instead reported significant protective effects [ ] . both rcts lacked sufficient power for analysis based on virologically-confirmed infection so that conclusions were drawn instead from less reliable serologically-defined outcomes [ , ] . further rct test of the association in humans has now become practically impossible given that, since fall , all seasonal tiv routinely contains protective, homologous a(h n )pdm antigen [ ] . we therefore undertook rct evaluation of the possible direct effects of prior heterologous tiv receipt on a(h n )pdm disease risk in ferrets as the ideal alternate model of human influenza infection [ ] . although none of the animals became moribund or so severely ill as to require euthanasia, ferrets immunized with two doses of - tiv did show significantly worse clinical, virologic and pathological features following pandemic h n infection compared to placebo recipients. as originally powered to show, vaccinated animals experienced significantly greater weight loss relative to baseline following infection, maximally different from placebo at ch+ . nasal wash titers did not differ, but vaccinated animals showed significantly higher lung virus titers. consistent with lung virus findings, lung inflammation was also increased more than . -fold in vaccinated compared to placebo animals at ch+ although with fewer animals sacrificed on that day the difference fell just short of statistical significance. inflammatory indicators were, however, significantly higher at ch+ compared to ch+ in the vaccinated animals and showed no change across that period in placebo recipients. lung cytokines showed a similar pattern. although illness activity levels appeared similar between groups based on categories of induced playfulness, these may have been of insufficient resolution to reflect clinical differences in lung disease, manifest otherwise through significant loss of appetite and weight. by day post-challenge animals in both groups had recovered. in human studies, a doubling of the risk of medically-attended pandemic h n illness was observed among - tiv recipients, but an increase in the risk of hospitalization was not shown [ ] . this was broadly interpreted to suggest increased risk of acquiring infection per se whereas here we report increased disease severity among influenza-naïve, systematically-infected ferrets. as such, our findings in ferrets may not replicate the experience in humans. it is worth noting, however, that the source population in the human observational studies was patients seeking medical care [ ] . although illness severity among outpatient visits was not specifically compared between vaccinated and unvaccinated participants, subjects had experienced influenza-like illness severe enough to prompt medical consultation within one week of illness onset, and that outcome was significantly increased among the vaccinated. in that regard, the pattern of acute worsening of a(h n )pdm illness during the first week of infection in vaccinated ferrets, followed by subsequent recovery by day , is consistent with increased outpatient but not hospitalization risk observed in vaccinated humans. as such, this ferret rct suggests that earlier findings from observational studies in humans cannot be dismissed on the basis of methodological bias alone and that direct mechanistic explanations should be sought. whether these findings may be product-specific or may also apply to other tiv products has yet to be separately assessed in follow-up studies. to date, hypotheses about biological mechanisms to explain increased a(h n )pdm risk among prior tiv recipients have included both direct and indirect vaccine effects [ ] . indirect mechanisms include the infection block hypothesis whereby effective seasonal vaccine may prevent the more robust and complex cross-protective immunity against heterologous viruses afforded by seasonal infection, such as through cell-mediated responses to conserved internal virus components [ , , ] . other epidemiological investigators have favoured this hypothesis, or related variations (such as temporary immunity hypothesis) [ , ] , but in appendix g of our original publication [ ] we demonstrated these indirect hypotheses to be insufficient and implausible to fully explain a doubling of risk, requiring as they do unreasonably high estimates of infection attack rates, infectioninduced cross-protection, and tiv effectiveness [ , ] . to test whether increased risk in vaccinees may have occurred without invoking infection block mechanisms we specifically designed the current ferret experiment without the intermediary of heterologous seasonal influenza infection. as such, we cannot rule out an additive role for indirect vaccine effects mediated through infection block mechanisms, but show that direct vaccine effects are likely to have at least contributed to our previous findings. possible direct vaccine effects include antibody-dependent enhancement (ade) whereby virus uptake by cells is enhanced in the presence of low-level, cross-reactive, non-neutralizing antibodies, best described for dengue [ , , ] . a possible role for cross-reactive antibodies in explaining severe a(h n )pdm manifestations in otherwise healthy adults, and in archived lung sections from fatal adult cases during the h pandemic has previously been suggested [ ] . another recent study has reported an association between higher ratios of cross-reactive elisa versus neutralizing antibody titers early during a(h n )pdm infection and more severe illness [ ] . enhanced respiratory disease in vaccinated swine has also been reported following challenge with a(h n )pdm or other heterologous, homosubtypic h viruses that do not share cross-reactive neutralizing antibodies [ ] [ ] [ ] [ ] . as in our ferret experiment, clinical worsening in vaccinated swine was evident at two to five days post-challenge [ , ] and was correlated with elevated pro-inflammatory cytokine responses in the lung [ ] . unlike the current ferret or prior human studies, however, these swine studies used adjuvanted whole virion vaccine that was additionally heterosubtypic for the neuraminidase surface protein (i.e. n versus n ), the relevance of which is uncertain. ade is classically associated with enhanced virus uptake in macrophages or other fc-receptor-bearing cells, demonstrated in vitro for influenza [ ] [ ] [ ] [ ] and more recently also specifically for a(h n )pdm in the presence of heterologous human anti-sera [ ] . in our vaccinated ferrets, higher lung virus titers were observed, but immuno-histochemistry could not distinguish affected cells of the lung in vaccine versus placebo animals, and macrophages were not predominant in either group. more recently in swine, however, heterologous antibody has been shown to enhance a(h n )pdm infection of other mammalian (mdck) cells, described in the context of fusion-enhancing crossreactive anti-ha stalk antibodies and absent neutralizing antibodies targeting the ha globular head [ , ] . our experiment was unable to further elucidate these putative immunologic mechanisms. we identified significant vaccineinduced influenza a antibody rise by elisa and confirmed this to include anti-ha antibody response to the seasonal h n tiv component. there was also rapid and significant increase in influenza a elisa antibody following a(h n )pdm challenge among vaccinated but not placebo ferrets sacrificed at ch+ but ha -based and neutralizing antibodies to a(h n )pdm were not evident until ch+ in either group. lower neutralizing antibody to a(h n )pdm even at ch+ among vaccinated versus placebo ferrets, although not statistically significant, is consistent with human immunogenicity trials showing blunting of pandemic h n vaccine-induced responses in association with prior seasonal vaccine receipt [ ] [ ] [ ] [ ] . conversely, pandemic h n immunization has been observed to boost pre-existing heterosubtypic antibody which may also be consistent with our ch+ findings for the h n tiv component and the broad boosting of cross-reactive antibodies to other antigenically-distant h variants observed by protein microarray in vaccinated but not placebo animals [ ] . although protein microarray did not show cross-reactive a(h n )pdm antibody prior to ch+ , it was not designed to detect the sort of anti-ha stalk antibodies highlighted above in association with severe disease in vaccinated swine [ ] . ultimately, therefore, we are unable to discern whether the rise in ch+ elisa antibody in vaccinated animals suggests early crossreactive, non-neutralizing antibody to a(h n )pdm or further antibody increase to tiv antigens weeks after their second dose (or both) although microarray indicates the latter certainly contributed. t-cell hypo-responsiveness may be an alternate explanation compatible with a hypothesis of direct vaccine effect. this phenomenon has been reported in same-season influenza vaccine booster-dose studies [ ] with parallels also in the allergy literature suggesting peptide-induced t-cell hypo-responsiveness beginning at - weeks and lasting up to weeks [ ] . however, while ferret # (placebo; inflammatory score . ) indicating very mild/minimal peri-bronchial inflammation; (b) ferret # (vaccinated; inflammatory score . ) indicating very mild/minimal peri-vascular inflammation; (c) ferret # (placebo; inflammatory score . ) indicating moderate bronchial and mild peri-bronchial/peri-vascular inflammation; (d) ferret # (vaccinated; inflammatory score . ) indicating severe bronchopneumonia. the increased micrometric scale in panel d reflects that the pathologic changes are marked and diffuse, best rendered at low magnification, whereas the mild to moderate and more focal changes in panels a to c necessitate photomicrographs at higher magnification. corresponding histopathology scores are shown in table s . doi: . /journal.pone. .g interferon-gamma was below detectable limits in both groups, lung cytokines in vaccinated ferrets were otherwise consistently (but non-significantly) higher compared to placebo animals at ch+ , notably including the th il [ ] , pro-inflammatory il [ , ] and regulatory il [ ] cytokines. il has been implicated as super-inducer of neutrophil infiltration and acute lung immuno-pathology following influenza infection [ ] , with counteractive dampening interactions by il [ ] . in an earlier canadian ferret experiment in which animals administered a single dose of - tiv also experienced worse a(h n )pdm illness, il in nasal wash was substantially raised in the fluviral group and il significantly in the flumist group, with disease enhancement suggested in both vaccine groups compared to controls [ ] . we did not assess nasal wash cytokines or flumist and were not statistically powered to explore cytokine differences, but lung il and il were also both non-significantly raised at ch+ in our fluviral versus placebo ferrets. all cytokine values were then lower at ch+ in the absence of lung pathology. however, none of the between-group cytokine differences at either time point were statistically significant. there are limitations to this study. although ferrets are considered the ideal animal model for human influenza infection, there are anticipated differences in immunologic and clinical aspects of immunization, infection and illness responses (timing, dosing and intensity) across species. overall patterns may be compared but ferret studies do not support precise quantification of actual risk in humans. the greater likelihood of more severe disease based on several clinical indicators (weight loss, lung virus titers) among vaccinated compared to unvaccinated ferrets may not replicate the greater likelihood of medically-attended a(h n )pdm illness we previously reported in vaccinated humans. in using influenza-naïve, systematically infected ferrets there are clear differences from the human experience with respect to pre-conditions (influenza exposure history and immunologic context), process (infection acquisition), and other relevant parameters (vaccine immunogenicity, clinical outcomes and monitoring). clinical relevance of the differences we report between vaccinated and placebo ferrets is ultimately best interpreted in the context of our study objectives assigned in follow up to the prior human observations we reported. the main objective of the ferret study was to assess through randomized, controlled design whether prior receipt of - tiv may have had direct, adverse effects on a(h n )pdm illness, specifically powered related to weight loss. although we cannot more precisely elucidate the underlying mechanisms involved, the current ferret study supports the hypothesis of direct vaccine effect. taken together with prior human and swine studies, these findings represent a signal that warrant further investigation and better understanding though they cannot be considered conclusive. the most prominent concern in this ferret study may relate to our failure to show neutralizing antibody response to vaccine, an issue we therefore consider in detail. we observed some greater albeit low-level variability in antibody titers by hi, a nonfunctional assay, than by microneutralization or np-based elisa. intra-and inter-laboratory variability in antibody assay results is well-recognized [ , ] and we thus interpret findings in the context of combined hi, mn, and elisa results, overall indicating our animals were naïve at pre-shipment and baseline. vaccine-induced hi and mn responses were not evident thereafter but significant vaccine-induced antibody rise was shown by both np-based elisa and ha -based protein microarray assays, the latter also shown only at high serum/low antibody concentrations (i.e. testing dilution of : ). although srid testing of the expired - vaccine lot that we used still met standard ha potency requirements for annual commercial vaccine approval by the fda [ ] , we cannot rule out other unrecognized vaccine changes with time that may have been influential. mdck-passaged viruses used in our hi and mn assays were antigenically equivalent to reference strains and amino acid sequencing showed they were also identical in their ha and na to the - h n vaccine component. this argues against such linear differences to explain the suboptimal neutralizing responses we measured; however, we cannot rule out other conformational changes to protein structure in the original vaccine comprised of (sodium-deoxycholate) disrupted and inactivated virus. the h n virus used in our assays was also antigenically equivalent to the who-recommended reference virus with which it shared $ % ha antigenic site amino acid identity. however, we did not conduct antigenic testing in relation to the actual vaccine component used by manufacturers and cannot rule out differences between assay and vaccine viruses in the suboptimal vaccine responses we measured (table s and s ). protein micro-array also showed variable low-level response to the h n component among immunized ferrets at day , significantly greater than placebo and compared to pre-immunization only at day (i.e. five weeks after the second vaccine dose). whether the latter was due to further antibody rise with time following immunization or cross-reactive response following a(h n )pdm challenge is unknown; however, similar effect was not observed relative to placebo for other non-vaccine h subtype viruses included in the microarray, suggesting h n vaccine response. thresholds for defining antibody response are also anticipated to vary across species and assays; these have not been crosscorrelated/ validated in ferrets for the various antigens and assays we used. in fact, sero-protective thresholds have not yet been established for ferrets by any assay. however, failure to induce a robust hi or mn antibody response to inactivated influenza vaccine in naïve ferrets has long been recognized, after single or several doses, in the absence of prior infection or adjuvant for seasonal, novel or pandemic vaccines [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in order to replicate more closely the observations in humans, we used a commercially available, non-adjuvanted - fluviral lot that had been administered in canada. it is of note that in human studies conducted with the same product in - (pediatric) [ ] , - (elderly) [ ] and in a mouse study conducted in - [ ] , fluviral also induced suboptimal hi and/or mn responses to the same seasonal a/brisbane/ / -like h n vaccine antigen. for example, in the pediatric trial including infants and toddlers - months of age similarly naïve to influenza as were our ferrets, the same schedule of two . ml doses of a thimerosal-free version of the - fluviral induced significantly lower h n antibody responses than even half that volume ( . ml) per dose of vaxigrip [ ] . [ ] . a similar pattern, though less pronounced, was also observed with the h n component [ ] . the reasons for diminished immunogenicity of the canadian vaccine are unknown although authors of the pediatric trial proposed more complete clearance of intact virus among other possible explanations. in human observational studies, the - tiv was still shown to be protective overall against homologous seasonal influenza [ ] . spring-summer observations of increased risk of heterologous a(h n ) illness were identified six or more months after tiv receipt. in that regard, lower vaccine-induced antibody titers in ferrets at our three-week post-immunization a(h n )pdm challenge time point may better replicate end-ofseason antibody conditions when vaccinated humans were exposed to a(h n )pdm virus. a prior ferret study to assess the same - fluviral also suggested disease enhancement but was able to induce homologous hi antibody response to the h n component with mean antibody titre exceeding within two weeks of a single . ml vaccine dose [ ] , higher even than induced in the pediatric study population cited above. such variability in serologic responses may reflect lot-to-lot or laboratory differences. not knowing the precise mechanisms involved in vaccine-associated enhanced respiratory disease, and unable to exactly know or replicate the human immunologic context in spring-summer , we did not adjust the human vaccine formulation, dose or schedule to force higher ferret vaccine responses. instead we focused on clinical parameters, recording the observed effects according to standard immunization practice. in general, ferret studies to date have suffered from small sample size and insufficient power [ ] [ ] [ ] [ ] [ ] [ ] ] . our study was powered for clinical (percentage weight loss) comparison and follow-up to days post-challenge. failure to reach statistical significance for other consistent indicators should not prompt their dismissal but should stimulate further investigation. it may be argued that lung findings at ch+ were chance occurrences among few ferrets poorly-representative of the full group experience. however, animals in both groups were randomly and blindly selected for ch+ sacrifice, the comparison of baseline and ch+ characteristics showed no significant within-group differences according to scheduled endpoint, and ch+ lung findings in vaccinated animals (higher virus titers and lung inflammation) were consistent with overall clinical patterns (greater loss of appetite and weight). nevertheless, future experiments should be powered with more animals to specifically examine these early acute clinical, immunologic, and pathologic findings and explore their possible mechanisms in greater detail. we assessed only influenza-naïve animals whereas most humans, other than young children, will have prior potentially cross-attenuating influenza infection history. the use of influenzanaïve animals in previous swine [ ] [ ] [ ] [ ] , ] and the current ferret studies may be relevant to the increased severity highlighted in vaccinated animals but to a lesser extent noted with the association in people. further experiments are needed to explore nuances related to infection and/or immunization history which additionally and variously complicate the human experience. in a recent publication, disease enhancement was included among possible hypotheses to explain greater pandemic h morbidity in the americas compared to australia, new zealand or europe, with reference to findings in vaccinated swine interpreted ecologically in the context of regional differences in prior heterologous seasonal h n virus circulation; given findings in vaccinated ferrets and swine, however, regional differences in prior heterologous seasonal h n vaccine (i.e. tiv) coverage may also be relevant to consider [ ] . of note, mechanisms such as ade, if explanatory, require a precise balance of low-level, crossreactive, non-neutralizing antibody to be manifest [ , , ] , a particular but sliding immunologic scale that may not have been captured in all animals or humans at the time of a(h n )pdm exposure. a spectrum of illness is anticipated with any infection process and a greater likelihood of severity does not require that all exposed individuals experience that outcome. however, this additional immunologic complexity related to ade, if involved, may have contributed to the variability in clinical outcomes we observed among vaccinated ferrets and to the variability in reporting the association in humans. our experiment assessed the unique context of heterologous but homosubtypic pandemic h n challenge. it has been suggested that original antigenic sin as an aspect of the cross-reactive, non-neutralizing antibody required for ade applies when antigenic differences of less than - % exist across related but distinct prime-boost strains [ ] ; amino acid differences in the ha between the - seasonal and pandemic h antigens were within this range ( - % similarity; table s ) with much closer homology ( %) across the ha ( amino acid differences across residues). however, without better understanding of the underlying mechanisms or specific virologic interactions we cannot speculate whether the same association could apply to other emerging heterologous or hetero-subtypic variants; the antigenic distance and other criteria required to define or forecast that likelihood remain unknown. in summary, although these ferret findings cannot be considered conclusive in explaining earlier human observations from canada, they support the hypothesis that prior receipt of - tiv may have had direct, adverse effects on a(h n )pdm illness. both human and ferret findings from canada are consistent with observations elsewhere of enhanced disease following heterologous influenza challenge in vaccinated swine. given the potential implications for informing influenza immuno-epidemiology and public health response to other emerging viruses, these signals warrant further in-depth evaluation and a search for possible mechanistic explanations. figure s ha microarray values for study and nonstudy antigens by group and study day. table s amino acid sequence substitutions in hemagglutinin (ha) and neuraminidase (na) of mdck-passaged assay and challenge viruses. (pdf) table s pairwise identity (% (number of amino acid mutations)) in influenza a hemagglutinin (ha ) peptide and antigenic sites across viruses used in antibody assays and a(h n )pdm challenge. (pdf) association between the - seasonal influenza vaccine and pandemic h n illness during spring-summer : four observational studies from canada seasonal influenza vaccine and increased risk of pandemic a/h n -related illness: first detection of the association in british columbia clinical and epidemiologic characteristics of an outbreak of novel h n (swine origin) influenza a virus among united states military beneficiaries association between seasonal influenza vaccination in - and pandemic influenza a (h n ) infection among school students from kobe effectiveness of pandemic h n vaccine against influenza-related hospitalization in children effectiveness of - trivalent influenza vaccine against pandemic influenza a (h n ) -united states notes from the field: outbreak of pandemic influenza a (h n ) virus at a large public university in delaware pandemic influenza h n infection in victoria, australia: no evidence for harm or benefit following receipt of seasonal influenza vaccine in 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infections with seasonal influenza a virus induce cross-protective immunity against a(h n ) pandemic influenza virus in a ferret model control of pandemic (h n ) influenza virus infection of ferret lungs by nonadjuvant-containing pandemic and seasonal vaccines immunogenicity and safety of dose levels of a thimerosal-free trivalent seasonal influenza vaccine in children aged - months: a randomized, controlled trial seasonal trivalent inactivated influenza vaccine does not protect against newly emerging variants of influenza a (h n v) virus in ferrets global mortality estimates for the influenza pandemic from the glamor project: a modeling study authors wish to acknowledge ms. lisan kwindt for assistance with literature retrieval, manuscript preparation and formatting, and ms. catharine chambers for assistance with literature review. we also wish to thank dr. yan li of the national microbiology laboratory for providing the reference viruses and anti-sera used in the serologic assays, as well as the authors and originating and submitting laboratories of the reference who and vaccine virus sequences obtained from gisaid's epiflu database. contributed reagents/materials/analysis tools: ss xb cc al ceh mk edb rb. wrote the paper: dms meh gds nzj gl ss xb cc al dk ceh sl mp gb mk edb rb. key: cord- - nhvbvy authors: kase, samuel m.; gribben, jeanie l.; waldman, elisha d.; weintraub, andrea s. title: a pilot study exploring interventions for physician distress in pediatric subspecialists date: - - journal: pediatr res doi: . /s - - -x sha: doc_id: cord_uid: nhvbvy background: while institution-sponsored wellness programs may be effective, little is known about their availability and utilization in pediatric subspecialists, and about programs physicians wish were available. methods: a survey of perceptions about, and availability and utilization of institutional wellness activities, was distributed electronically to pediatric subspecialists nationally. bivariate analyses were performed using χ( ) tests or independent t tests. multivariable logistic regression models for categories of institution-sponsored programming as a function of potential predictors of program utilization were performed. qualitative content analysis was performed for free-text survey answers. results: approximately % of respondents participated in institution-sponsored wellness opportunities. debriefs, schwartz center rounds, mental health services, and team building events were the most available institution-sponsored wellness activities, whereas debriefs, team building, schwartz center rounds, and pet therapy were most frequently utilized. respondents desired greater social/emotional support, improved leadership, enhanced organizational support, and modifications to the physical work environment, with no significant differences across subspecialties for “wish list” items. conclusions: physician wellness requires more than a “one-size-fits-all” initiative. our data highlight the importance of encouraging and normalizing self-care practices, and of listening to what physicians articulate about their needs. pre-implementation needs assessment allows a “bottom-up” approach where physician voices can be heard. the importance of physician well-being has moved to the forefront of national dialogue about healthcare. [ ] [ ] [ ] [ ] the impact of burnout, secondary traumatic stress, compassion fatigue, and moral distress on the personal and professional lives of us physicians can no longer be ignored. [ ] [ ] [ ] [ ] institutions and healthcare systems have begun implementing physician "wellness initiatives" for students, trainees, and faculty. , a working definition of physician wellness has been suggested to include not merely the "absence of ill-being," but "physical and mental well-being that allows physicians to develop their full potentials across personal and work-life domains." to that end, the triple aim (enhanced patient experiences, population health, and health cost reduction) has expanded to a quadruple aim that incorporates the importance of collective wellbeing of the patient care team to achieve desired healthcare objectives. , despite the inherent difficulties, the most stressful and emotionally challenging elements in physicians' lives may also be the activities from which the greatest satisfaction is derived. therefore, it is critical to better understand what drives work-related distress and satisfaction and whether there are ways to support one while minimizing the other. although personal self-care strategies and institution-sponsored programs are both essential for physician wellbeing, workplace initiatives likely have greater impact in reducing burnout. , although some institution-sponsored programs may be effective, little is known about who uses them and how likely they are to be utilized. of particular importance is understanding which programs/services physicians wish they had available, as this may help target interventions to specific physician audiences and suggest new possibilities to explore. to date, academic pediatric subspecialists' opinions on existing and desired workplace wellness interventions have not been examined. in our current study, we conducted a cross-sectional national survey of physicians in five pediatric subspecialties (neonatology, critical care, palliative care, emergency medicine, and hematology-oncology) to: ( ) characterize existing institution -sponsored wellness offerings; ( ) determine utilization of available institution -sponsored wellness activities; and ( ) identify interventions physicians wish were available to them. we hypothesize that desired interventions will be influenced by subspecialty as well as by individual personality characteristics, and that there is a disconnect between wellness programming that is offered to pediatric subspecialists and what they themselves truly desire. email addresses for pediatric subspecialists in neonatology (nicu), palliative care (pc), critical care (cc), emergency medicine (em), and hematology-oncology (ho) acquired as part of our prior studies of compassion fatigue, burnout, and compassion satisfaction [ ] [ ] [ ] [ ] [ ] were utilized as sources of potential subjects for the current study. this project was designated as exempt human research by the institutional review board at the icahn school of medicine at mount sinai. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. instrument a -item questionnaire of personal and professional characteristics (including the "big five personality factors" , ) as well as perceptions about, and availability and personal utilization of institutional "wellness" activities, was distributed electronically and anonymously to pediatric subspecialty providers nationally. a brief description of the study, with a hyperlink to the questionnaire, was distributed via surveymonkey ® to all potential study participants. the invitation to participate was resent to non-responders at -week intervals for a total of three attempts. individual survey responses were downloaded, coded, and entered into spss statistics version (ibm, armonk, ny). qualitative content analysis was performed for free-text answers to "what programs/support systems would you create to help you cope with compassion fatigue and burnout?" descriptive statistics were calculated for all other survey responses. bivariate analyses were performed using χ tests or independent t tests as appropriate. multivariable logistic regression models for categories of institutional programming as a function of potential predictors of program utilization were constructed. of the surveys delivered by surveymonkey ® , responses were returned ( . % response rate). of these, two individuals declined to participate and participants returned a blank survey. eighteen surveys were excluded because the participant had not completed availability and personal utilization questions, and thus the study questions could not be addressed. twenty-six surveys were excluded because the participant was not currently a nicu, pc, cc, em, or ho provider. this left a final study population of individuals (fig. ) . characteristics of the study population are shown in table , supplementary table s , and supplementary fig. s . of the five subspecialties included in the study, the largest number of responses came from cc and em physicians ( . % and . % of participants, respectively). the majority of participants were female, were mid-career or senior level practitioners, and had worked at their present institution for more than years. nicu and pc respondents had been in practice and employed at their current institutions longer than cc, em, and ho participants (p < . ). while almost two-thirds of respondents had some education about burnout, less than half had education about compassion fatigue, and % were entirely unfamiliar with the concept. nicu and ho physicians were less likely than pc and em providers to have had education about compassion fatigue (p < . ) and were also less likely to have received education about burnout when compared with em (p < . ). personal self-care virtually all participants engaged in one or more self-care activities outside the workplace. time spent with family/friends/pets, conversations about work-related distress, and exercise were the most frequently reported personal wellness activities (table ) . em and cc physicians were significantly more likely to exercise than ho (p < . and p < . , respectively). em physicians were significantly more likely to talk about work-related distress than cc, ho, or nicu physicians (supplementary table s ; p < . , p < . , and p < . , respectively). pc physicians were significantly more likely to socialize, explore creative arts as a therapeutic outlet, participate in holistic practices, and utilize mental healthcare services than the other subspecialists (supplementary table s ), and were the least likely to engage in "negative behaviors" (e.g., self-designated excessive food or alcohol consumption, recreation drug abuse; supplementary table s ). nicu physicians were the least likely to talk about work-related distress, socialize with friends/family, engage in creative arts, or utilize mental healthcare as part of personal wellness regimens (supplementary table s ). female study participants were significantly more likely than male participants to use social connections and holistic practices as self-care (p < . ). in addition, participants who self-identified with the personality trait "extroversion" were significantly more likely to exercise (p < . ) and to socialize (p < . ) as part of personal self-care, whereas individuals who identified with the trait "openness" were more likely to utilize creative arts (p < . ), and subjects who identified with "neuroticism" were more likely to cope using "negative habits" (p < . ) and mental health services (p < . ). institution-sponsored self-care approximately % of respondents reported that they participated in institutionally offered wellness opportunities ( table ) . the most frequently cited barriers to participation were the (in) convenience of scheduling ( . %), time constraints ( . %), the preference of participants to "handle things on my own" ( . %), and the sentiment that partaking in the activity would not be helpful ( . %). sixteen percent of participants had "no idea" what programs their institution offered for physician wellness, with ho physicians the least aware and pc providers the most aware of available activities (supplementary table s , p < . ). impediments to participation were independent of subspecialty (supplementary table s ). while female study participants were significantly more likely than male participants to report "inconvenience" of institutionally sponsored wellness programs as a barrier ( . % vs. %, p < . ), male physicians were more likely to prefer to "handle things on their own" ( % vs. . %, p < . ) and to assume that wellness interventions would not be helpful ( % vs. %, p < . ). participants who self-identified with "extroversion" or "agreeableness" were less skeptical of the potential personal value of attending institution-sponsored wellness activities (p < . ), while those who identified as "conscientious"' were more likely to find events "too time-consuming" (p < . ). scheduled debriefs/critical incident management, schwartz center rounds, and professional counseling/support groups were the institutionally sponsored wellness activities that were most available to study participants ( table ) . availability of debriefs/critical incident management, schwartz center rounds, pet therapy, and organized team building events were significantly different across subspecialties (supplementary there were significant differences in utilization of almost all institution-sponsored wellness activities across pediatric subspecialties (supplementary table s ). pc providers were more likely to take advantage of all institution-sponsored wellness programs (with the exception of organized team building activities) than any of the other subspecialists surveyed (p < . ). nicu physicians were least likely to utilize pet therapy or institution-sponsored mental health services, while em physicians were least likely to attend schwartz rounds or holistic programs, and cc physicians were least likely to participate in debriefs/critical incident management or partake of peer-to-peer support programs. utilization of institution-sponsored wellness activities was not affected by sex nor by personal selfcare activities. individuals who self-identified with the personality trait "openness" were significantly more likely to attend institutionsponsored holistic programs (p < . ) and those who self-identified with "agreeableness" were more likely to attend schwartz rounds (p < . ). respondents' free-text responses (fig. ) for "wish lists" for specific institutional supports to combat compassion fatigue and burnout were coded into four main categories: social/emotional support, improvements to leadership, organizational support, and modifications to the physical work environment. the categories of social/emotional support and organizational support were further parsed as shown in table and fig. . there were no significant differences across subspecialties for any "wish list" items (supplementary table s ). for the four main categories of "wish lists," there were no significant differences between male and female physicians; however, female physicians were significantly more likely to desire greater social/emotional support as it related specifically to enhanced opportunities for debriefing and patient connection ( % vs. %, p < . ). self-identified "extroverts" were significantly more likely to desire modifications of their physical work environments (p < . ), while those who identified with "conscientiousness" were more likely to want organizational support (p < . ) and less likely to want social/emotional support in the form of enhanced team building (p < . ). individuals who identified with "agreeableness" were also significantly less likely to request greater social/emotional support in the form of enhanced team building (p < . ), as well as less likely to want increased general administrative support (p < . ). compassion fatigue, moral distress, and burnout, alone or in combination, take their toll on the physical and emotional health of physicians. [ ] [ ] [ ] [ ] in this cross-sectional national survey of pediatric subspecialists, we present the first comprehensive evaluation of availability and utilization of institution-sponsored physician wellness activities. we also present the first investigation into the workplace wellness interventions that pediatric subspecialists wish were available to them. because compassion fatigue, moral distress, and burnout are interrelated phenomena that negatively impact well-being and professional performance, physicians may be unable to pinpoint a specific cause of their emotional distress. , being able to "name it to tame it" may well apply to physician distress. in our study population, nearly two-thirds of participants had some education about burnout, whereas less than half had received education about compassion fatigue. greater knowledge about the prevalence of these phenomena, as well as their signs and sequelae, may help pediatric subspecialists recognize the importance of integrating selfcare routines into their daily lives. we identified differences in personal self-care practices based on subspecialty, sex, and personality characteristics. while it is perhaps not surprising that pc providers were the most engaged in self-care outside of the workplace, it was unexpected and concerning that nicu physicians in our sample were less involved than the other subspecialists in activities that strengthen social/emotional connections in private life. that female study participants were more likely to utilize social/ emotional liaisons for self-care is also not surprising, given what is understood about differences in relational styles between men and women. similarly, that "extroverts" participated in activities with greater social/emotional connection and that "neurotics" were more likely to engage in "negative" habits and utilize mental health services was notable but not entirely unexpected. , in recent years, many institutions and healthcare organizations around the country have embarked on wellness initiatives to address the widespread problem of physician distress and burnout. , the cost of investing in institutional programs to promote physician well-being and retention is far less that the cost of replacing a "burned-out" physician. in our study population, the range of available institution-sponsored physician wellness activities differed across pediatric subspecialties. this raises important questions about how institutions determine which wellness interventions to provide. are pre-implementation needs assessments being performed? or are less systematic smatterings of activities simply put in place to "check the box" of providing physician wellness opportunities? availability of an institution-sponsored wellness intervention in no way guarantees that the target audience of physicians knows it exists, uses it, or finds it effective in easing compassion fatigue or burnout. in our cohort, nearly % of participants reported having "no idea" what institution-sponsored self-care activities were available to them. because it can be extremely difficult for a distressed or "burnedout" physician to seek support, it is disheartening that dissemination of this information may be so variable and/or inadequate. that barriers to participation differed by sex and personality traits speaks to the importance of providing more than a "one-size-fitsall" approach to what is offered. in our population, while there were significant differences in utilization of institution-sponsored wellness activities across subspecialties, the overall utilization rate was~ %. while this level of involvement in self-care at work is somewhat encouraging, it does not in any way speak to the effectiveness of these interventions. given the current alarming levels of burnout and dissatisfaction among physicians nationally, some institution-sponsored wellness activities may not be as beneficial as intended. in our study cohort, scheduled team debriefings, schwartz center rounds, mental health services, and organized team building events were the most available institution-sponsored wellness activities, whereas debriefs, organized team building, schwartz center rounds, and pet therapy were the most frequently utilized programs. interestingly, while professional counseling/support groups were available to % of participants, only % utilized the intervention. this is in contrast to team building events and pet therapy, which while less available ( % and %, respectively), had greater participation ( % and %, respectively). it is possible that participants perceive less stigma associated with these activities. this underscores the importance of an institutional culture that views a physician's receipt of mental healthcare as self-care rather than frailty. when our study population was queried about specific institutionsponsored wellness programs they lacked but desired, the majority sought greater social/emotional support. free-text responses (fig. ) included desire for "more social events within the group to deepen relationships and build camaraderie" and "social gathering to share difficult situations and debrief about them." this is aligned with the most commonly utilized institutional offerings. coworker relationships impact overall job satisfaction, and work-related distress arising from coworker conflict has been associated with burnout in some pediatric subspecialists. [ ] [ ] [ ] [ ] [ ] in specific, participants sought increased opportunities for "real-time debriefing sessions for difficult cases… either after a death or even during care of a critically ill patient." debriefs/critical incident management has been shown to provide a sense of support and community, and may improve resilience. the knowledge that peers face similar struggles can serve as a source of relief. however, successful critical incident management requires skill and sensitivity in order to avoid causing additional traumatic stress. there are several limitations to our study. our survey response rate was low, which raises reasonable concerns about nonresponse bias. response rates to physician surveys have declined steeply in recent years, with job demands and email/survey fatigue as the most commonly cited reasons. , there is no scientifically validated, minimally acceptable survey response rate, and a low response rate, in and of itself, is not an indicator of response bias. survey response rates are not strongly associated with survey quality. that said, we acknowledge that our response rate (at < % in this pilot study) may introduce a greater degree of non-response bias. it is not possible to compare variables between those who responded to the survey and those who did not. however, because pediatric academic subspecialists share similar attitudes and behaviors, it is possible that responders and non-responders have comparable characteristics and thus provide similar responses. contact information for some study participants may have been outdated; thus, our response rate may actually have been higher than our calculation. we did not query social/emotional support, improvements to leadership, organizational support, and modifications to the physical work environment. the categories of social/emotional support and organizational support were further parsed as shown. nicu neonatology, pc palliative care, cc critical care, em emergency medicine, ho, hematology-oncology. a pilot study exploring interventions for physician distress in pediatric. . . sm kase et al. participants about feelings of distress or well-being on the day of survey, but either of these sentiments may have impacted participation and/or responses. the generalizability of our findings may be limited by the sample size and by the fact that participants were largely female and mid-career and senior level faculty. our study was not designed to evaluate the impact or effectiveness of any interventions described by participants. it is encouraging that physician wellness is now a benchmark worthy of consideration alongside improvements in patient care experiences and population health, and diminution in healthcare costs. however, actualization of physician wellness requires more than simply implementing a "one-size-fits-all" wellness initiative. institutional leadership must continue to work diligently and thoughtfully to change the current pervasive culture that contributes to physician distress. our data highlight the importance of providing better education to pediatric subspecialists about sources and sequelae of work-related distress, of encouraging and normalizing physician selfcare practices both at home and in the workplace, and of listening and responding to what physicians articulate about their own needs. thus, pre-implementation needs assessment allows a "bottom-up" approach where physician voices can be heard and, hopefully, answered. the well-being of physicians physician wellness: a missing quality indicator what do we mean by physician wellness? a systematic review of its definition and measurement physician burnout-a serious symptom, but of what? the personal and professional consequences of physician burnout: a systematic review of the literature physicians aren't 'burning out' they're suffering from moral injury a crisis in health care: a call to action on physician burnout interventions to prevent and reduce physician burnout: a systematic review and meta-analysis evidence-based interventions for medical student, trainee, and practicing physician well-being: a charm annotated bibliography for the collaborative for healing and renewal in medicine (charm) best practices subgroup physician well-being: expanding the triple aim from triple to quadruple aim: care of the patient requires care of the provider exploring the rewards and challenges of paediatric palliative care work not all coping strategies are created equal: a mixed methods study exploring physicians' self-reported coping strategies compassion fatigue, burnout, and compassion satisfaction in neonatologists in the u a cross-sectional analysis of compassion fatigue, burnout, and compassion satisfaction in pediatric palliative care providers in the united states a cross-sectional analysis of compassion fatigue, burnout, and compassion satisfaction in pediatric critical care physicians in the united states a crosssectional analysis of compassion fatigue, burnout, and compassion satisfaction in pediatric emergency medicine physicians in the united states a cross-sectional analysis of compassion fatigue, burnout, and compassion satisfaction in pediatric hematology-oncology physicians in the united states handbook of personality: theory and research the mini-ipip scales: tinyyet-effective measures of the big five factors of personality compassion fatigue: coping with secondary traumatic stress disorder in those who treat the traumatized moral distress and its contribution to the development of burnout syndrome among critical care providers the new science of personal transformation caring for oneself to care for others: physicians and their self-care language and social identity how physicians identify with predetermined personalities and links to perceived performance and wellness outcomes: a crosssectional study the relationship between the big five personality factors and burnout: a study among volunteer counselors estimating the attributable cost of physician burnout in the united states building a program on well-being: key design considerations to meet the unique needs of each organization the impact of stigma and personal experiences on the helpseeking behaviors of medical students with burnout implementing emotional debriefing in pediatric clinical education a primer on critical incident stress management: what's really in a name? response burden and questionnaire length: is shorter better? a review and meta-analysis surveys and response rates nonresponse bias in a mail survey of physicians. eval. health prof response rates and nonresponse errors in surveys exploring physician specialist response rates to webbased surveys healing the professional culture of medicine we thank ni-ka ford, academic medical illustrator at the icahn school of medicine at mount sinai, for her assistance with fig. . the online version of this article (https://doi.org/ . /s - - -x) contains supplementary material, which is available to authorized users. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -yehnlabq authors: barh, debmalya; tiwari, sandeep; weener, marianna e.; azevedo, vasco; góes-neto, aristóteles; gromiha, m. michael; ghosh, preetam title: multi-omics-based identification of sars-cov- infection biology and candidate drugs against covid- date: - - journal: comput biol med doi: . /j.compbiomed. . sha: doc_id: cord_uid: yehnlabq sars-cov- has ushered a global pandemic with no effective drug being available at present. although several fda-approved drugs are currently under clinical trials for drug repositioning, there is an on-going global effort for new drug identification. in this paper, using multi-omics (interactome, proteome, transcriptome, and bibliome) data and subsequent integrated analysis, we present the biological events associated with sars-cov- infection and identify several candidate drugs against this viral disease. we found that: (i) interactome-based infection pathways differ from the other three omics-based profiles. (ii) viral process, mrna splicing, cytokine and interferon signaling, and ubiquitin mediated proteolysis are important pathways in sars-cov- infection. (iii) sars-cov- infection also shares pathways with influenza a, epstein-barr virus, htlv-i, measles, and hepatitis virus. (iv) further, bacterial, parasitic, and protozoan infection pathways such as tuberculosis, malaria, and leishmaniasis are also shared by this virus. (v) a total of candidate drugs including the prophylaxis agents and pathway specific inhibitors are identified against covid- . (vi) betamethasone, estrogen, simvastatin, hydrocortisone, tositumomab, cyclosporin a etc. are among the important drugs. (vii) ozone, nitric oxide, and photosensitizer drugs are also identified as possible therapeutic candidates. (viii) curcumin, retinoic acids, vitamin d, arsenic, copper, and zinc may be the candidate prophylaxis agents. nearly % of our identified agents are previously suggested to have anti-covid- effects or under clinical trials. among our identified drugs, the ones that are not yet tested, need validation with caution while an appropriate drug combination from these candidate drugs along with a sars-cov- specific antiviral agent is needed for effective covid- management. the first case of covid- caused by sars-cov- infection was reported between december , to december , from huanan, hubei province, china [ ] [ ] [ ] and as of september , , the virus has infected , , people with , , deaths globally [ ] . currently, the usa is the most affected country by the covid- pandemic with an estimated , , infections and , , deaths, followed by india ( , , infected, , deaths), and brazil ( , , infected, , , deaths) [ ] . although, the "person-to-person transmission" of sars-cov- was reported on january , [ ] and several quarantine and management strategies were adopted in various countries, the infection could still not be controlled and neither the morbidity. one of the major causes for this uncontrolled number of infection and deaths is the unavailability of sars-cov- specific vaccines and antiviral drugs. to overcome the crisis and to identify effective anti-sars-cov- drugs, repurposing of the existing fda approved antiviral drugs are given high priority [ ] . although, individual host omics such as interactome [ ] , proteome [ ] , and transcriptome [ , ] based pathways of sars-cov- infected host cell or subject have been described and potential drugs targeting such pathways were reported, so far, no integrative omics based approaches have been employed to study the sars-cov- infected host biology and pathways in order to predict the potential drugs. in this paper, we have used an integrative omics approach considering the sars-cov- infected host interactome, proteome, transcriptome, and bibliome datasets and analysed the covid- associated host genetic information to identify common host pathways that are deregulated during sars-cov- infection and potential drugs targeting those pathways. we have also reported the sars-cov- infected host biology/ pathways and potential drugs from each omics-based approach separately. in this work, we analysed five different peer-reviewed published omics data sets to identify the infection biology and candidate drugs against sars-cov- . the overall approach applied in our work is presented in fig- . j o u r n a l p r e -p r o o f we have used five omics data sets in this analysis. the first data set, designated as the interactome, outlines sars-cov- interaction with human (hek t/ cell line) proteins as described by gordon et al., [ ] . the second data set used in this study is the transcriptome (designated as transcriptome- ) of lung epithelial cells upon viral infection. we have considered up-regulated genes from this transcriptome profile [ ] . the third data set comprise of up-regulated genes from the peripheral blood of covid- patients (designated as transcriptome- ) as described by xiong for identification of potential drugs from various gene sets, we used two additional tools enrichr (https://amp.pharm.mssm.edu/enrichr/) [ ] and web-based gene set analysis toolkit (webgestalt, www.webgestalt.org) [ ] apart from the toppfun module of toppgene suite [ ] . enrichr uses dsigdb (a drug signatures database) [ ] and drugmatrix (a comprehensive toxicogenomic database) for gene set analysis based candidate drug enrichment. all default parameters were used in enrichr and toppfun. in webgestalt [ ] , the over-representation analysis was performed using human genome as reference set and drugbank [ ] as the functional database. default parameters for all these tools were used and a p-value < . was considered significant and used to generate the results. to understand the network-based biological processes, pathways, and protein-drug interactions for each data set and also the combined data set, networkanalyst . (www.networkanalyst.ca) [ ] was used. since sars-cov- predominantly infects the lung cells, in our network analysis the human species was used, and to generate the lung tissuespecific protein-protein interaction (ppi) network, from the tissue-specific ppi module of networkanalyst, the lung tissue type was selected. for generic ppi, the imex interactome database (innatedb) [ ] was used. the top ten genes, based on their degree and j o u r n a l p r e -p r o o f betweenness, were considered. for network-based pathways, the kyoto encyclopedia of genes and genomes (kegg) database [ ] and for biological processes, the panther:bp [ ] modules that were integrated with networkanalyst was used. the common pathways and bps under top ten hits were selected for further analysis. the genes associated with the selected common bps and pathways were collected and analysed using toppfun [ ] to identify drugs for these gene sets. additionally, the protein-drug interactions module of networkanalyst . [ ] was also used to identify drugs for each omics sample-based network using the drugbank database [ ] and filtered with minimum-network. in this approach, we combined the top ten pathways from each of our analysis (individual and combined omics analysis). based on their cumulative number of appearances, the common pathways were ranked. pathways enriched with at least omics-based analyses are presented in the results. we followed a similar approach for selecting bps also. to identify the most frequent drugs enriched across all our analysis, we also adopted a similar strategy. additionally, in this case, we classified all the drugs following the drug categories as suggested by the drug information portal of the u.s. national library of medicine (https://druginfo.nlm.nih.gov/drugportal/) and listed the most common drug categories across all the analysis. further, we also presented the most frequent drugs under each category for better identification of the important drugs. this drug based enrichment was performed for both omics-based and pathway-based drugs that were identified in our analysis. in our analysis, several common and unique biological processes and drugs are enriched depending on the omic data sets or the analysis tools used. here, we report the important pathways, biological process, and drugs. the detailed outcomes of each analysis can be found in the supplementary materials. first, we used the toppgene suite [ ] for candidate gene prioritization. as the bibliome based gene set is a combination of all kinds of sars-cov- infection associated human genetic data, we used the bibliome based gene set collected through disgenet [ ] as the training set for this toppgene based analysis. (table s ). however, while we combined the top genes from all the candidate gene-based analysis and table s ) . gordon et al., [ ] in their ppi study showed that % of the interactome are related to endomembrane and er/golgi/ mitochondrial trafficking. sars-cov- predominantly interacts with innate immune pathways, host translation machinery, and cullin ubiquitin ligase complex. in our interactome set enrichment, we also observed that, the interactome is mostly associated with organelle membranes and involved in intracellular protein targeting or transport. additionally, we observed that the sars-cov- interacting human proteins are mostly involved in cell cycle, nuclear pore complex (npc) disassembly, mitochondrial protein import, and regulation of glucokinase (table s (i)). in our lung-specific interactome analysis, this interactome was also found to be involved in viral carcinogenesis, mrna splicing, negative regulation of apoptosis, ubiquitin mediated proteolysis, protein processing in endoplasmic reticulum, and spliceosome pathways (table s (i)) . in their analysis, gordon et al., [ ] suggested that protein biogenesis inhibitors, sigma and sigma receptor inhibitors, eif a inhibitors, sec inhibitors, anti-cancer compounds, and antipsychotic antihistamines could be potential drugs against sars-cov- . in our analysis, we also observed enrichment of anticancer or antipsychotic drugs. our identified potential drugs are antineoplastic antibiotics (idarubicin, romidepsin, daunorubicin, epirubicin), antineoplastic agent (irinotecan), anti-depressive and anti-psychotic agents (fluoxetine, fluphenazine), estrogens (coumestrol, conjugated estrogen), anti-bacterial agents (tunicamycin, latamoxef), enzyme inhibitor (thapsigargin), central nervous system agent (chlorzoxazone), dermatologic agent (verteporfin), adrenergic agent (dobutamine), and metal/growth substance (polaprezinc). apart from these, flavin adenine dinucleotide, guanosine- '-diphosphate, myristic acid, and kappadione are also enriched in our analysis (table s (i), table s (i)). anticoagulants (citric acid) are also found to be good candidates (table s (p), table s (p)). blanco-melo et al., [ ] in their in vitro transcriptome (transcriptome- ) study using normal human bronchial epithelial (nhbe) cells have shown that sars-cov- induces a low ifn-i and -iii and high chemokine signalling. this unique and inflammatory response was not observed in sars-cov- , mers-cov, iav, and rsv [ ] . in our analysis, we also found that this up-regulated transcriptome is associated with interferon and cytokine signaling pathways. further, the transcriptome- is also enriched for influenza a, herpes, and hepatitis c infections (table s (t- )). the network-based analysis enriched negative regulation of apoptotic process, mrna splicing, viral carcinogenesis, hepatitis b, epstein-barr virus infection, nod-like receptor signaling pathway, ubiquitin mediated proteolysis, il- signaling pathway, measles, hepatitis c, and influenza a (table s (t- )). the results are mostly common to the cellular proteome-based analysis, except the ubiquitin mediated proteolysis, which is not found in case of the proteome data set (table s (p)). for the up-regulated cellular transcriptome (transcriptome- ), we identified several potential drugs. these drugs belongs to several drug categories such as-adrenal cortex hormones (table s (t- ), table s (t- )) xiong et al., [ ] reported that, upon infection with sars-cov- , the patient's blood transcriptome (transcriptome- ) profile shows increased complement activation, humoral immune response, immunoglobulin mediated response, acute inflammatory response, and lymphocyte apoptosis. we also observed a similar profile for transcriptome- , based on our gene set enrichment analysis. however, in addition to these biological processes and pathways, we found that the malaria pathway is also shared by sars-cov- (table s (t- ) ). in the network-based lung-specific ppi analysis, we found a profile that is similar to the bronchial epithelial cell specific transcriptome-based (transcriptome- ) findings by blanco-melo et al., [ ] . we found viral carcinogenesis, negative regulation of apoptotic process, endocytosis, mrna splicing, epstein-barr virus infection, hepatitis b, htlv-i infection, measles, and ubiquitin mediated proteolysis pathways in our analysis (table s (t- )). the candidate drugs for this up-regulated patient's blood transcriptome are identified as: antihypertensive agent (hydrochlorothiazide), antirheumatic agent (leflunomide), antiasthmatic agent (theophylline), and anesthetic (procaine). we also found that anthelmintic drug lucanthone is enriched in our analysis (table s (t- ), table s (t- )). the bibliome-based gene set analysis showed mostly similar biological events that we had (table s (b), table s (b)). our combined omics associated gene set enrichment analysis (proteome and transcriptomes) revealed a similar biology and pathways of sars-cov- as we found in each individual transcriptome analysis. the key bps and pathways involved are complement activation, humoral immune response, and cytokine and interferon signaling ( (table s (p+t), table s (p+t)). for the combination of all the five omics data sets, we identified antineoplastic agents (table s (p+t+i+b), table s (p+t+i+b)). in our analysis, we observed sars-cov- infection shares other viral pathways such as to identify pathway specific drugs, we used the genes involved in the five most important common pathways (viral processes including all the individual virus pathways, mrna splicing, ubiquitin mediated proteolysis, cytokine signaling in immune system, and protein processing in endoplasmic reticulum). for each pathway, genes from all the five omics data sets (identified from our network based analysis and toppfun) were combined and toppfun [ ] , webgestalt [ ] , and enrichr [ ] were used to identify gene set specific drugs. (table s ) . additionally, several candidate drugs are also identified for each pathway. glibenclamide (table s ) . since we identified several pathways, biological process, and drugs in our various analyses, to identify the most important ones, we combined all the results and then ranked them according to their number of appearances. we selected the pathways, biological process, and drugs that are commonly enriched in at least of our omics-based analysis. while we combined biological processes from all our analysis and performed the ranking, we found that rna splicing, viral process, mrna processing, mrna splicing via spliceosome, negative regulation of apoptotic process, regulation of cell cycle, rhythmic process, cell proliferation, defense response, immune system process, protein phosphorylation, and complement activation are the top pathways involved in the sars-cov- infection process ( fig- a , table s ). similarly, epstein-barr virus infection, htlv-i infection, pathways in cancer, cell cycle, cytokine signaling in immune system, focal adhesion, hepatitis b, influenza a, leishmaniasis, ubiquitin mediated proteolysis, viral carcinogenesis, and mrna splicing/spliceosome are found as top pathways (fig- b , table s ). apart from these pathways, measles, tuberculosis, toxoplasmosis, and malaria pathways are also found to be common in at least three of our analyses (fig- b , table s ). (fig- ) . purvalanol, resveratrol, sorafenib, tanespimycin, and vorinostat (fig- a , table s ). the identified top drug categories are antineoplastic agents, analgesics, anti-infective agents, antibacterial agents, enzyme inhibitors, metal/ growth substances, alkylating agents, antihemophilic factors, and vitamin d that have at least drugs in each category (fig- b , (fig- ) . among the anticancer drugs identified in our analysis, a combination of topoisomerase inhibitors (irinotecan and etoposide) is reported to be an effective treatment strategy to counter cytokine storms in critically ill covid- patients [ ] . our identified doxorubicin was previously suggested for repurposing to treat covid- patients [ ] . another identified anticancer drug dasatinib is found to be safer to treat chronic myeloid leukemia (cml) patients infected with sars-cov- [ ] . binimetinib, brigatinib , seliciclib, sorafenib, and vorinostat were also previously predicted to be effective against sars-cov- infection [ ] [ ] [ ] [ ] . therefore, the additional anticancer drugs identified by our analyses, such as phenethyl isothiocyanate, abciximab, lucanthone, cisplatin, tositumomab, procarbazine, and noscapine may also be tested against sars-cov- . our identified antidepressant drug fluoxetine (prozac) was previously reported to inhibit the replication of sars-cov- [ ] and therefore, may be tested for potential covid- therapy. the antipsychotic medicine fluphenazine is also enriched in our analysis that has previously been tested on sars-cov- [ ] . similarly, sertraline (a serotonin ( -ht) inhibitor), that is identified in our study, has recently been found to block sars cov endocytosis and suggested for drug repurposing against covid- [ ] . thus, the additional j o u r n a l p r e -p r o o f antidepressant/ antipsychotic drugs identified by our analyses, such as droperidol (antidopaminergic), may also be tested to understand their effectiveness against sars-cov- . in our analysis, several cyclooxygenase (cox) inhibitors and anti-histamine compounds found potential against covid- . among these drugs, indomethacin (cox inhibitor) is shown to exert its anti-sars-cov- activity in canine coronavirus model [ ] . further, indomethacin in combination with resveratrol (also identified in our analysis) has been proposed to be used for treating covid [ ] . two other cox inhibitors (loxoprofen and acetaminophen) identified in our analysis were also previously tested in silico for their potential antiviral activity against sars-cov- [ ] . niflumic acid which is also a cox inhibitor as well as a ca +-activated clchannel blocker is also enriched in our analysis and also been previously identified as a potential drug against sars-cov- [ ] . other antiinflammatory drugs such as andrographolide, apremilast (phosphodiesterase type inhibitor) that are identified in our analysis were also predicted by previous studies as potential covid- therapeutics [ , ] . in our analysis, we also found the rheumatoid arthritis drug tofacitinib as a good candidate for covid- management. tofacitinib is a jak inhibitor and reduces immune system inflammation and is currently under clinical trials for covid- treatment (nct , nct ), therefore, the additional anti-allergic and antiinflammatory drugs identified by our analyses including sulindac (cox inhibitor) and terfenadine (h -receptor binding anti-histamine drug) need attention to be tested against sars-cov- . similarly, other channel inhibitors enriched in our analysis such as acetohexamide (atp-dependent k + channel inhibitor), suloctidil (calcium antagonist), glibenclamide (atp-sensitive potassium channels inhibitor), and thapsigargin (a sarco/endoplasmic reticulum ca²⁺ atpase inhibitor) may also be tested. tissue plasminogen activators (tpa), antihemophilic factors, immune globulin, and antithymocyte globulin are found to be good candidates in covid- treatment as per our various omics-based analyses. reports suggest that, tpa with targeted anti-inflammatory treatment may be a potential therapy against covid- [ ] . tenecteplase, a tpa, that is identified in our analysis, is considered in managing acute coronary syndromes associated with covid- [ ] and is currently under clinical trial to manage covid- patients (nct ). lanoteplase, a third generation recombinant plasminogen derived from alteplase, is also identified in our analysis. alteplase is currently under clinical trial to j o u r n a l p r e -p r o o f evaluate its efficacy against covid- (nct ). we also found antihemophilic factors could be also effective against covid- . the factor viii which is an antihemophilic factor is elevated in covid- patients [ ] . therefore, factor viii inhibitors may be a potential therapeutic strategy. immune globulin is also enriched in our analysis. immune globulin is present in plasma and convalescent plasma therapy is effective in treating covid- patients [ ] . our identified human serum albumin may be a good delivery vehicle to maximize the cellular internalization and enhancement of other drugs used in covid- treatment [ ] . since most of our identified blood based components are suggested to be effective in covid- , the anti-thymocyte globulin which is also identified in our analysis may also be tested for its efficacy in the management of sars-cov- infection. estrogen or estrogen analogues are enriched in almost all of our omics data set-based analyses. dimorphism of covid- infection has been recently reported. men are found to be more prone to sars-cov- infection or covid- associated deaths as compared to women [ ] . the disease severity of covid- is also found to be dependent on erα:erβ expression ratio and e level [ ] . reports also suggest that the expression of human ace , which is the receptor for sars-cov- , is regulated by estrogen [ ] . therefore, estradiol agonist may have a protective role against covid- [ ] . recently, estrogen patch is under clinical trial to evaluate its efficacy against covid- (nct ). in our analysis, we observed that phytoestrogens including coumestrol could also be a potential therapeutic against covid- . we observed that, steroids could play an important role in fighting against covid- . the corticosteroid dexamethasone and methylprednisolone which we identified in our analysis, were previously found to significantly reduce mortality rates from covid- [ , ] and currently under clinical trials (nct , nct ). the other corticosteroid that is predominantly enriched in our integrative omics approach is betamethasone. betamethasone is found to be safe for pregnant subjects infected with sars-cov- [ ] and is currently also under clinical trial at sites where dexamethasone is not easily available (nct ). therefore, other corticosteroids identified in our analysis such as fludrocortisone acetate may also be tested against covid- . among our identified glucocorticoids, hydrocortisone has been tested on covid- patients [ ] and prednisone and procaine may be tested in the coming days. j o u r n a l p r e -p r o o f no known targeted antiviral drug is enriched in our analysis. however, we identified several antibiotics. among these antibiotics, clindamycin was previously suggested by a computational approach [ ] and is recommended to treat sars-cov- infection [ ] . we identified two additional antitumor antibiotics, geldanamycin and tanespimycin, which are also heat shock protein (hsp ) inhibitors. while geldanamycin is suggested for covid- treatment [ ] , tanespimycin was previously identified by an in silico method [ ] . apart from sharing various viral pathways, we also found that sars-cov- shares pathways associated with tuberculosis, leishmaniasis, and malaria. the antimalarial drug hydroxychloroquine alone or in combination with remdesivir effectively inhibits sars-cov- infection [ , ] . hydroxychloroquine is under clinical trial (nct ). in our analysis, we found that artenimol or artesunate, an alternative of chloroquine could also be effective in treating covid- . moreover, artesunate was previously predicted to be a good candidate against sars-cov- [ , ] and is currently under clinical trial (nct ). in our analysis, we found immunosuppressant drugs may also be effective against covid- . among the identified immunosuppressants, cyclosporin a is enriched in our multiple analyses. a recent in vitro study indicates that cyclosporin a is effective against sars-cov- [ ] and therefore, can be a potential drug to be further tested in covid- patients [ ] . cyclosporin a is currently under clinical trials (nct , nct ) . similarly, the other immunosuppressant leflunomide identified in our analyses is also under clinical trial (nct ). in our integrative omics approach, we found that ozone could be a potential therapy against ozone-based management for early control of covid- are under various stages of clinical trials (nct , nct ). nitric oxide, which is known to provide innate antiviral protection [ ] , is also enriched in our analysis. nitric oxide inhalation is recently reported to be beneficial to manage the acute respiratory distress syndrome due to sars-cov- infection [ ] and currently nitric oxide inhalation therapy is under clinical trials to understand its efficacy in managing covid- patients (nct , nct , nct ). verteporfin, a photosensitizer drug for photodynamic therapy is found to be an effective drug in our analysis. verteporfin is also previously reported to be a potential antiviral therapy against sars-cov- [ ] . in our multi-omics-based analysis, acetylcysteine is enriched. acetylcysteine is a mucolytic agent that can prevent the severity of sars- ). therefore, our results suggest that our analytic approach in this study is highly accurate and curcumin, retinoic acids, and ergocalciferol could be good prophylaxis candidates against covid- . arsenenous acid and arsenic trioxide are identified both in our candidate gene-based integrative omics analysis as well as viral pathway-based analysis. recent in silico analysis suggests that arsenic based drug darinaparsin may inhibit sars-cov- rna polymerase and proteases, and therefore, may inhibit viral replication [ ] . in homeopathy medicine arsenicum album , prepared from arsenic trioxide, is recommended as a prophylactic medicine against sars-cov- infection [ ] . in our analysis, copper is found to inhibit all three important pathways: mrna splicing, ubiquitin mediated proteolysis, and protein processing in endoplasmic reticulum pathways. copper irreversibly disintegrates coronavirus genomes, envelope, and spike [ ] . report also suggests that enrichment of copper levels in j o u r n a l p r e -p r o o f plasma could boost both the innate and adaptive immunity [ ] and therefore, copper could also be a good prophylactic agent against sars-cov- infection. similarly, polaprezinc is identified as a potential molecule to block protein processing in the endoplasmic reticulum pathway. zinc supplementation is recommended as a good prophylaxis and therapeutic strategy against covid- [ ] . currently, several clinical trials are on-going to evaluate the anti-covid- efficacy of zinc in combination with other drugs (nct , nct ). in this work, we have used four different host specific omics data sets that were recently published along with a bibliome based gene set to identify the sars-cov- infection biology, potential drugs, and prophylaxis agents against this virus. based on our individual omics-based or their combination-based analysis, it is evident that the sars-cov- infection shares various virus, bacteria, and protozoon infection pathways. although, we identified several candidate drugs and prophylaxis candidates, we were not able to identify any sars-cov- specific antiviral agent. nearly ~ % of our identified agents were previously reported to have anti-covid- activity, and a number of these agents are currently under various stages of clinical trials. since the most observed effective drugs/agents are among those that were used (or introduced) previously, our used integrative omics-based methodology is highly credible and warrants its wide-spread adoption and application. this method would also be highly applicable for drug repositioning research in future waves of covid- infection and also other possible pandemics. however, as sars-cov- shares multiple pathogens' infection pathways, individual drugs targeting a single pathway may not be effective and therefore, a combination of drugs needs to be formulated to block the multiple infection pathways of this virus. further, the prophylaxis agents identified here also need an effective combination. all these identified drugs need multiple validations, large scale clinical trials, and caution before their use in covid- management. db: conceived and designed the experiment, data collection and analysis, result interpretation, and wrote the paper; st: preformed re-analysis; pg: data interpretation and edited the article, mei, agn, mmg and va: provided technical inputs. authors declare no competing interest. j o u r n a l p r e -p r o o f supplementary table legends table s : candidate gene based analysis of multi-omics data. table s : gene set enrichment based analysis of multi-omics data. table s : network analysis based lung-specific multi-omics data. epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study a new coronavirus associated with human respiratory disease in china a novel coronavirus from patients with pneumonia in china coronavirus disease (covid- ) a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster a review of sars-cov- and the ongoing clinical trials a sars-cov- protein interaction map reveals targets for drug repurposing imbalanced host response to sars-cov- drives development of covid- transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid- patients the disgenet knowledge platform for disease genomics: update toppgene suite for gene list enrichment analysis and candidate gene prioritization enrichr: a comprehensive gene set enrichment analysis web server update dsigdb: drug signatures database for gene set analysis webgestalt : gene set analysis toolkit with revamped uis and apis drugbank: a comprehensive resource for in silico drug discovery and exploration networkanalyst . : a visual analytics platform for comprehensive gene expression profiling and meta-analysis innatedb: systems biology of innate immunity and beyond--recent updates and continuing curation kegg: new perspectives on genomes, pathways, diseases and drugs panther version : more genomes, a new panther go-slim and improvements in enrichment analysis tools the ai-discovered aetiology of covid- and rationale of the irinotecan+etoposide combination therapy for critically ill covid- patients vulnerabilities of the sars-cov- virus to proteotoxicityopportunity for repurposed chemotherapy of covid- infection, biorxiv sars-cov- (covid- ) and chronic myeloid leukemia (cml): a case report and review of abl kinase involvement in viral infection morphological cell profiling of sars-cov- infection identifies drug repurposing candidates for covid- , biorxiv silico discovery of candidate drugs against covid- , viruses prediction of repurposed drugs for treating lung injury in covid- artificial intelligence approach fighting covid- with repurposing drugs covid- : drug targets and potential treatments the serotonin reuptake inhibitor fluoxetine inhibits sars-cov- , biorxiv broad anti-coronaviral activity of fda approved drugs against sars-cov- in vitro and sars-cov in vivo, biorxiv understanding sars cov endocytosis for covid drug repurposing indomethacin has a potent antiviral activity against sars cov- in vitro and canine coronavirus in vivo, biorxiv indomethacin and resveratrol as potential treatment adjuncts for sars cov /covid identification of novel compounds against three targets of sars cov- coronavirus by combined virtual screening and supervised machine learning repurposing drugs for covid- based on transcriptional response of host cells to sars-cov- , arxiv andrographolide as a potential inhibitor of sars-cov- main protease: an in silico approach apremilast as a potential treatment option for covid : no symptoms of infection in a psoriatic patient combination of thrombolytic and immunosuppressive therapy for coronavirus disease : a case report considerations for management of acute coronary syndromes during the sars-cov- (covid- ) pandemic factor viii and functional protein c activity in critically ill patients with coronavirus disease : a case series, a a pract use of convalescent plasma therapy in two covid- patients with acute respiratory distress syndrome in korea serum albumin-mediated strategy for the effective targeting of sars-cov- estrogen and the disease severity of sars-cov- infection estrogen regulates the expression of sars-cov- receptor ace in differentiated airway epithelial cells beta-estradiol, a potential ally to alleviate sars-cov- infection use of corticosteroids in coronavirus disease pneumonia: a systematic review of the literature effectiveness and safety of available treatments for covid- during pregnancy: a critical review effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods peripheral blood examination findings in sars-cov- infection drug repositioning suggests a role for the heat shock protein inhibitor geldanamycin in treating covid- infection hypothesis: mpges- -derived prostaglandin e , a so far missing link in covid- pathophysiology? remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro artesunate: could be an alternative drug to chloroquine in covid- treatment? in-silico studies of antimalarial-agent artemisinin and derivatives portray more potent binding to lys and lys -binding hotspots of sars-cov- spike protein than hydroxychloroquine: potential repurposing of artenimol for covid- , chemrxiv drug repurposing screens reveal fda approved drugs active against sars-cov- , biorxiv cyclosporine a: a valid candidate to treat covid- patients with acute respiratory failure? potential cytoprotective activity of ozone therapy in sars-cov- /covid- the nitric oxide pathway provides innate antiviral protection in conjunction with the type i interferon pathway in fibroblasts nitric oxide inhalation as an interventional rescue therapy for covid- -induced acute respiratory distress syndrome potent antiviral effect of protoporphyrin ix and verteporfin on sars-cov- infection, biorxiv catechin and curcumin interact with corona ( -ncov/sars-cov ) viral s protein and ace of human cell membrane: insights from computational study and implication for intervention a novel combination of vitamin c, curcumin and glycyrrhizic acid potentially regulates immune and inflammatory response associated with coronavirus infections: a perspective from system biology analysis inhibitory effects of resveratrol on the epstein-barr virus lytic cycle resveratrol as a novel anti-herpes simplex virus nutraceutical agent: an overview effective inhibition of mers-cov infection by resveratrol natural compounds as potential inhibitors of novel coronavirus (covid- ) main protease: an in silico study vitamin d deficiency and co-morbidities in covid- patients -a fatal relationship? letter: covid- , and vitamin d in silico identification of a potent arsenic based approved drug darinaparsin against sars-cov- : inhibitor of rna dependent rna polymerase (rdrp) and necessary proteases advisory for corona virus homoeopathy for prevention of corona virus infections unani medicines useful in symptomatic management of corona virus infection is copper beneficial for covid- patients? potential role of zinc supplementation in prophylaxis and treatment of covid- htlv-i, measles, and hepatitis virus infection pathways. • sars-cov- also shares tuberculosis, malaria, and leishmaniasis infection pathways. • mrna splicing, cytokine and ifn signaling, and ubiquitin are important pathways. • anticancer, antipsychotic, anti-inflammatory, antibiotic, immunosuppressant, corticosteroid cyclosporin a are top candidate drugs nitric oxide, and photosensitizer drugs are also important against covid- retinoic acids, vit-d, arsenic, copper, and zinc are candidate prophylaxis agents • ~ % of our identified drugs are previously suggested or under clinical trial for covid- . • our newly identified candidate drugs need validation with caution none key: cord- -xn cf a authors: tanz, robert r.; shulman, stanford t. title: sore throat date: - - journal: practical strategies in pediatric diagnosis and therapy doi: . /b - - - - . - sha: doc_id: cord_uid: xn cf a nan sore throat is a common chief complaint. each year approximately million patients in the united states visit physicians because of throat complaints. the majority of these illnesses are nonbacterial and neither necessitate nor are alleviated by antibiotic therapy (tables - to - ). acute streptococcal pharyngitis, however, warrants accurate diagnosis and therapy to prevent serious suppurative and nonsuppurative complications. furthermore, life-threatening infectious complications of streptococcal and nonstreptococcal oropharyngeal infections may manifest with mouth pain, pharyngitis, parapharyngeal space infectious extension, and airway obstruction (tables - and - ). most episodes of pharyngitis are caused by viruses (see tables - and - ). it is difficult to clinically distinguish between viral and bacterial pharyngitis with a very high degree of precision, but certain clues may help the physician. accompanying symptoms of conjunctivitis, rhinitis, croup, or laryngitis are common with viral infection but rare in bacterial pharyngitis. many viral agents can produce pharyngitis (see tables - and - ). some cause distinct clinical syndromes that are readily diagnosed without laboratory testing (see tables - , - , and - ). in pharyngitis caused by parainfluenza and influenza viruses, rhinoviruses, coronaviruses, and respiratory syncytial virus (rsv), the symptoms of coryza and cough often overshadow sore throat, which is generally mild. influenza virus may cause high fever, cough, headache, malaise, myalgias, and cervical adenopathy in addition to pharyngitis. in young children, croup or bronchiolitis may develop. rsv is associated with bronchiolitis, pneumonia, and croup in young children. rsv infection in older children is usually indistinguishable from a simple upper respiratory tract infection. pharyngitis is not a prominent finding of rsv infection in either age group. parainfluenza viruses are associated with croup and bronchiolitis; minor sore throat and signs of pharyngitis are common at the outset but rapidly resolve. infections caused by parainfluenza, influenza, and rsv are often seen in seasonal (winter) epidemics. adenoviruses can cause upper and lower respiratory tract disease, ranging from ordinary colds to severe pneumonia. the incubation period of adenovirus infection is to days. upper respiratory tract infection typically produces fever, erythema of the pharynx, and follicular hyperplasia of the tonsils, together with exudate. enlargement of the cervical lymph nodes occurs frequently. when conjunctivitis occurs in association with adenoviral pharyngitis, the resulting syndrome is called pharyngoconjunctival fever. pharyngitis may last as long as days and does not respond to antibiotics. there are many adenovirus serotypes; adenovirus infections may therefore develop in children more than once. laboratory studies may reveal a leukocytosis and an elevated erythrocyte sedimentation rate. outbreaks have been associated with swimming pools and contamination in health care workers. the enteroviruses (coxsackievirus and echovirus) can cause sore throat, especially in the summer. high fever is common, and the throat is slightly red; tonsillar exudate and cervical adenopathy are unusual. symptoms resolve within a few days. enteroviruses can primary infection caused by herpes simplex virus (hsv) usually produces high fever with acute gingivostomatitis, involving vesicles (which become ulcers) throughout the anterior portion of the mouth, including the lips. there is sparing of the posterior pharynx in herpes gingivostomatitis; the infection usually occurs in young children. high fever is common, pain is intense, and intake of oral fluids is often impaired, which may lead to dehydration. in addition, hsv may manifest in adolescents with pharyngitis. approximately % of new-onset hsv-positive adolescent patients have herpetic lesions; most patients with hsv pharyngitis cannot be distinguished from patients with other causes of pharyngitis. the classic syndrome of herpetic gingivostomatitis in infants and toddlers lasts up to weeks; data on the course of more benign hsv pharyngitis are lacking. the differential diagnosis of vesicular-ulcerating oral lesions is noted in table - . a common cause of a local and large lesion of unknown etiology is aphthous stomatitis ( fig. - ) . some children have a combination of periodic fever (recurrent at predictable fixed times), aphthous stomatitis, pharyngitis, and cervical adenitis (pfapa); this syndrome is idiopathic and may respond to oral prednisone or cimetidine. pfapa usually begins before the age of years and is characterized by high fever lasting to days, occurring every to weeks, and resolving spontaneously. infants and toddlers with measles often have prominent oral findings early in the course of the disease. in addition to high fever, cough, coryza, and conjunctivitis, the pharynx may be intensely and diffusely erythematous, without tonsillar enlargement or exudate. the presence of koplik spots, the pathognomonic white or bluewhite enanthem of measles, on the buccal mucosa near the mandibular molars provides evidence of the correct diagnosis before the rash develops. acute exudative pharyngitis commonly occurs with infectious mononucleosis caused by primary infection with epstein-barr virus (ebv) ( table - ) . mononucleosis is a febrile, systemic, self-limited lymphoproliferative disorder that is usually associated with hepatosplenomegaly and generalized lymphadenopathy. the pharyngitis may be mild or severe, with significant tonsillar hypertrophy (possibly producing airway obstruction), erythema, and impressive tonsillar exudates. regional lymph nodes may be particularly enlarged and slightly tender. infectious mononucleosis usually occurs in adolescents and young adults; ebv infection is generally milder or subclinical in preadolescent children. in united states high school and college students, attack rates are to per , population per year. ebv is transmitted primarily by saliva. after a -to -week incubation period, patients with infectious mononucleosis usually experience an abrupt onset of malaise, fatigue, fever, and headache, followed closely by pharyngitis. the tonsils are enlarged with exudates and cervical adenopathy. more generalized adenopathy with hepatosplenomegaly often follows. fever and pharyngitis typically last to weeks, while lymphadenopathy and hepatosplenomegaly subside over to weeks. malaise and lethargy can persist for several months, possibly leading to impaired school or work performance. laboratory studies of diagnostic value include atypical lymphocytosis; these lymphocytes are primarily ebv-specific, cytotoxic t lymphocytes that represent a reactive response to ebv-infected b lymphocytes. a modest elevation of serum transaminase levels, reflecting ebv hepatitis, is common. tests useful for diagnosis include detection of heterophile antibodies that react with bovine erythrocytes (most often detected by the monospot test) and specific antibody against ebv viral capsid antigen (vca), early antigen (ea), and nuclear antigen (ebna). acute infectious mononucleosis is usually associated with a positive heterophile test result and antibody to vca and ea ( fig. - ) . the findings of acute exudative pharyngitis together with hepatomegaly, splenomegaly, and generalized lymphadenopathy suggest infectious mononucleosis. early in the disease and in cases without liver or spleen enlargement, differentiation from other causes of pharyngitis, including streptococcal pharyngitis, is difficult. indeed, a small number of patients with infectious mononucleosis have a throat culture positive for group a streptococci. serologic evidence of mononucleosis should be sought when splenomegaly or other features are present or if symptoms persist beyond days. patients with infectious mononucleosis require supportive treatment. corticosteroids may be indicated for acute life-threatening conditions, such as airway obstruction caused by enlarged tonsils. in the evaluation of a patient with sore throat, the primary concern is usually accurate diagnosis and treatment of pharyngitis caused by group a streptococci, which accounts for about % of all episodes of pharyngitis. the sequelae of group a streptococcal pharyngitis, especially acute rheumatic fever and acute glomerulonephritis, at one time resulted in considerable morbidity and mortality in the united states and continue to do so in other parts of the world. prevention of acute rheumatic fever in particular depends on timely diagnosis of streptococcal pharyngitis and prompt antibiotic treatment. group a streptococci are characterized by the presence of group a carbohydrate in the cell wall, and they are further distinguished by several kinds of cell wall protein antigens (m, r, t). these protein antigens are useful for studies of epidemiology and pathogenesis. group a streptococcal pharyngitis has been endemic in the united states; epidemics occur sporadically. episodes peak in the late winter and early spring; rates of group a streptococcal pharyngitis are highest among children aged to years old. spread of group a streptococci in classrooms and among family members, especially in crowded living conditions, is common. transmission occurs primarily by inhalation of organisms in large droplets or by direct contact with respiratory secretions. pets do not appear to be a frequent reservoir. untreated streptococcal pharyngitis is particularly contagious early in the acute illness and for the first weeks after the organism has been acquired. antibiotic therapy effectively prevents disease transmission. within hours of institution of therapy with penicillin, it is difficult to isolate group a streptococci from patients with acute streptococcal pharyngitis, and infected children can return to school. molecular epidemiology studies of streptococcal pharyngitis have shown that numerous distinct strains of group a streptococci circulate simultaneously in the community during the peak season. "dna-fingerprinting" techniques further demonstrate that children with streptococcal pharyngitis serve as a community reservoir for strains that cause invasive disease (e.g., sepsis, streptococcal toxic shock syndrome, cellulitis, necrotizing fasciitis) in the same geographic area and season. the classic patient with acute streptococcal pharyngitis has a sudden onset of fever and sore throat. headache, malaise, abdominal pain, nausea, and vomiting occur frequently. cough, rhinorrhea, conjunctivitis, stridor, diarrhea, and hoarseness are distinctly unusual and suggest a viral etiology. examination of the patient reveals marked pharyngeal erythema. petechiae may be noted on the palate, but they can also occur in viral pharyngitis (see table - ). tonsils are enlarged, symmetric, and red, with patchy exudates on their surfaces. the papillae of the tongue may be red and swollen; hence the designation "strawberry tongue." anterior cervical lymph nodes are often tender and enlarged. combinations of these signs can be used to assist in diagnosis; in particular, tonsillar exudates in association with fever, palatal petechiae, and tender anterior cervical adenitis strongly suggest infection with group a streptococci. however, other diseases can produce this constellation of findings. some or all of these classic characteristics may be absent in patients with streptococcal pharyngitis. younger children often have coryza with crusting below the nares, more generalized adenopathy, and a more chronic course, a syndrome called streptococcosis. when rash accompanies the illness, accurate clinical diagnosis is easier. scarlet fever, so-called because of the characteristic fine, diffuse red rash, is essentially pathognomonic for infection with group a streptococci. scarlet fever is rarely seen in children younger than years old or in adults. the rash of scarlet fever is caused by infection with a strain of group a streptococci that contains a bacteriophage encoding for production of an erythrogenic (redness-producing) toxin, usually erythrogenic (or pyrogenic) exotoxin a. scarlet fever is simply group a streptococcal pharyngitis with a rash and should be explained as such to patients and their families. although patients with the streptococcal section one respiratory disorders streptococcal toxic shock syndrome is usually associated with a primary cutaneous rather than a pharyngeal focus of infection. the rash of scarlet fever has a texture like sandpaper and blanches with pressure. it usually begins on the face, but after hours it becomes generalized. the face, especially the cheeks, is red, and the area around the mouth often appears pale in comparison (circumoral pallor). accentuation of erythema occurs in flexor skin creases, especially in the antecubital fossae (pastia's lines). the erythema begins to fade within a few days. desquamation begins within a week of onset on the face and progresses downward, often resembling that seen after a mild sunburn. on occasion, sheetlike desquamation occurs around the free margins of the fingernails and is usually more coarse than the desquamation seen with kawasaki disease. the differential diagnosis of scarlet fever includes kawasaki disease, measles, and staphylococcal toxic shock syndrome (table - ). although signs and symptoms may strongly suggest acute streptococcal pharyngitis, laboratory diagnosis is highly recommended, even for patients with scarlet fever (fig. - ) . scoring systems for diagnosing acute group a streptococcal pharyngitis on clinical grounds have not proved very useful. using clinical criteria alone, physicians overestimate the likelihood that patients have streptococcal infection. the throat culture has traditionally been used to diagnose streptococcal pharyngitis. plating a swab of the posterior pharynx and tonsils on sheep blood agar, identifying β−hemolytic colonies, and testing them for the presence of sensitivity to a bacitracinimpregnated disk is the "gold standard" diagnostic test, but it takes to hours to obtain results. there are a number of rapid diagnostic tests that take less than minutes. these "rapid strep" tests detect the presence of the cell wall group a carbohydrate antigen after acid extraction of organisms obtained by throat swab. rapid strep tests are highly specific (generally > %), with the throat culture used as the standard. unfortunately, the sensitivity of most of these rapid tests can be considerably lower. in comparison to hospital or reference laboratory throat culture results, the sensitivities of these tests are generally % to % and can be lower. however, when both throat cultures and rapid tests performed in physicians' offices are compared with cultures performed in reference laboratories, the sensitivities, specificities, and overall accuracy of the office culture and the office rapid test are quite similar; the latter often performs better than the culture. the low sensitivity of these tests, coupled with their excellent specificity, has led to the recommendation that two swabs be obtained from patients with suspected streptococcal pharyngitis. one swab is used for a rapid test. when the rapid antigen detection test result is positive, it is highly likely that the patient has group a streptococcal infection, and the extra swab can be discarded. when the rapid test result is negative, group a streptococci may nonetheless be present; thus, the extra swab should be processed for culture. physician offices that have demonstrated that their rapid test and throat culture results are comparable may be able to rely on the rapid test result even when it is negative, without performing a backup culture. in general, patients with a negative result of the rapid test do not require treatment before culture verification unless there is a particularly high suspicion group a streptococcal infection (e.g., scarlet fever, peritonsillar abscess, or tonsillar exudates in addition to tender cervical adenopathy, palatal petechiae, fever, and recent exposure to a person with group a streptococcal pharyngitis). testing patients for serologic evidence of an antibody response to extracellular products of group a streptococci is not useful for diagnosing acute pharyngitis. because it generally takes several weeks for antibody levels to rise, streptococcal antibody tests are valid only for determining past infection. specific antibodies include antistreptolysin o (aso), anti-dnase b, and antihyaluronidase (aht). when antibody testing is desired in order to evaluate a possible post-streptococcal illness, more than one of these tests should be performed to improve sensitivity. treatment begun within days of the onset of group a streptococcal pharyngitis is effective in preventing acute rheumatic fever. therapy does not appear to affect the risk of the other nonsuppurative sequela, acute post-streptococcal glomerulonephritis. antibiotic therapy also reduces the incidence of suppurative sequelae of group a streptococcal pharyngitis, such as peritonsillar abscess and cervical adenitis. in addition, treatment produces a more rapid resolution of signs and symptoms and terminates contagiousness within hours. for these reasons, antibiotics should be instituted as soon as the diagnosis is supported by laboratory studies. there are numerous antibiotics available for treating streptococcal pharyngitis (table - ). the drug of choice is penicillin. despite the widespread use of penicillin to treat streptococcal and other infections, penicillin resistance among group a streptococci has not developed. penicillin can be given by mouth for days or intramuscularly as a single injection of benzathine penicillin. intramuscular benzathine penicillin alleviates concern with patient compliance. a less painful alternative is benzathine penicillin in combination with procaine penicillin. intramuscular procaine penicillin alone is inadequate for prevention of acute rheumatic fever because adequate levels of penicillin are not present in blood and tissues for a sufficient time. other β-lactams, including semisynthetic derivatives of penicillin and the cephalosporins, are at least as effective as penicillin for treating group a streptococcal pharyngitis. their broader spectrum, their higher cost, and the lack of formal data concerning prevention of acute rheumatic fever relegate them to second-line status. the decreased frequency of dose administration of some of these agents may improve patient compliance and makes their use attractive in selected circumstances. patients who are allergic to penicillin should receive erythromycin or another non-β-lactam antibiotic, such as clarithromycin, azithromycin, or clindamycin. resistance of group a streptococci to erythromycin has increased dramatically in areas such as japan, france, spain, taiwan, and finland, where erythromycin has been widely used. this has not yet emerged as a major problem in the united states, where the rate of macrolide resistance is about %. sulfa drugs (including sulfamethoxazole combined with trimethoprim), tetracyclines, and chloramphenicol should not be used for treatment of acute streptococcal pharyngitis because they do not eradicate group a streptococci. antibiotic therapy has greatly reduced the likelihood of developing suppurative complications caused by spread of group a streptococci from the pharynx or middle ear to adjacent structures. peritonsillar abscess ("quinsy") manifests with fever, severe throat pain, dysphagia, "hot potato voice," pain referred to the ear, and bulging of the peritonsillar area with asymmetry of the tonsils and sometimes displacement of the uvula (fig. - ; see table - ). on occasion, there is peritonsillar cellulitis without a well-defined abscess cavity. trismus may be present. when an abscess is found clinically or by an imaging study such as a computed tomographic scan, surgical drainage is indicated. peritonsillar abscess occurs most commonly in older children and adolescents. sore throat necrotizing fasciitis diarrhea *case definition of streptococcal toxic shock syndrome requires (i) isolation of group a streptococci from (a) a normally sterile site (blood, synovial or peritoneal fluid) or (b) a nonsterile site (throat, wound). (ii) severity is defined by (a) hypotension and (b) two or more of renal impairment, coagulopathy, liver involvement, adult respiratory distress syndrome, a generalized erythematous macular rash (with or without later desquamation), and soft tissue necrosis (necrotizing fasciitis, myositis, gangrene). the definitive diagnosis requires criteria ia and iia plus b. criteria ib and iia plus b are considered probable if no other identifiable cause is present. alt, alanine aminotransferase; ast, aspartate aminotransferase; bun, blood urea nitrogen. retropharyngeal abscess represents extension of infection from the pharynx or peritonsillar region into the retropharyngeal (prevertebral) space, which is rich in lymphoid structures (figs. - and - ; see table - ). children younger than years old are most often affected. fever, dysphagia, drooling, stridor, extension of the neck, and a mass in the posterior pharyngeal wall may be noted. surgical drainage is often required if frank suppuration has occurred. spread of group a streptococci via pharyngeal lymphatic vessels to regional nodes can cause cervical lymphadenitis. the markedly swollen and tender anterior cervical nodes that result can suppurate. otitis media, mastoiditis, and sinusitis also may occur as complications of group a streptococcal pharyngitis. additional parapharyngeal suppurative infections that may mimic streptococcal disease are noted in table - . furthermore, any pharyngeal infectious process may produce torticollis if there is inflammation that extends to the paraspinal muscles and ligaments, producing pain, spasm, and, on occasion, rotary subluxation of the cervical spine. the differential diagnosis of torticollis is presented in table - . oropharyngeal torticollis lasts less than weeks and is not associated with abnormal neurologic signs or pain over the spinous process. nonsuppurative complications include acute rheumatic fever (see chapters and ), acute post-streptococcal glomerulonephritis (see chapter ), and possibly reactive arthritis/synovitis. in addition, an association between streptococcal infection and neuropsychiatric disorders such as obsessive-compulsive disorder and tourette syndrome has been postulated. this possible association has been called pandas (pediatric autoimmune neuropsychiatric disorders associated with streptococci). therapy with an appropriate antibiotic within days of onset of symptoms is highly effective in preventing rheumatic fever, but acute glomerulonephritis is not prevented by treatment of the antecedent streptococcal infection. pharyngitis caused by one of the nephritogenic strains of group a streptococci precedes the glomerulonephritis by about days. unlike acute rheumatic fever, which occurs only after group a streptococcal pharyngitis, acute glomerulonephritis also can follow group a streptococcal skin infection. treatment with penicillin cures group a streptococcal pharyngitis but is unable to eradicate group a streptococci from the pharynx in approximately % of patients ( fig. - ) . this causes considerable consternation among such patients and their families. penicillin resistance is not the cause of treatment failure. a small proportion of these patients are symptomatic and are thus characterized as having clinical treatment failure. reinfection with the same strain or a different strain is possible, as is intercurrent viral pharyngitis. some of these patients may be chronic pharyngeal carriers of group a streptococci who are suffering from a new superimposed viral infection; others may be noncompliant with regard to therapy. many patients who do not respond to antimicrobial treatment are asymptomatic and are identified when follow-up culture specimens are obtained, a practice that is usually unnecessary. patients who are compliant with regard to therapy are at minimal risk for acute rheumatic fever. one explanation for asymptomatic persistence of group a streptococci after treatment is that these patients were chronic carriers of group a streptococci who were initially symptomatic because of a concurrent viral pharyngitis and who did not truly have acute streptococcal pharyngitis. patients who are chronically colonized with group a streptococci are called chronic carriers. carriers do not appear to be at risk for acute section one respiratory disorders headache pyuria (sterile); gallbladder hydrops rheumatic fever or for development of suppurative complications, and they are rarely sources of spread of group a streptococci in the community. there is no reason to exclude these carriers from school. there is no easy way to identify chronic carriers prospectively among patients with symptoms of acute pharyngitis. the clinician should consider the possibility of chronic group a streptococcal carriage when a patient or a family member has multiple culturepositive episodes of pharyngitis, especially when symptoms are mild or atypical. a culture specimen is usually positive for group a streptococci when the suspected carrier is symptom-free or is receiving treatment with penicillin (intramuscular benzathine penicillin is recommended in order to eliminate concern about compliance). carriers often receive multiple unsuccessful courses of antibiotic therapy in attempts to eliminate group a streptococci. physician and patient anxiety is common and can develop into "streptophobia." unproven and ineffective therapies include tonsillectomy, prolonged administration of antibiotics, use of β-lactamase-resistant antibiotics, and culture or treatment of pets. available treatment options for the physician faced with a chronic streptococcal carrier include the following: . obtaining a rapid test, throat culture, or both each time the patient has pharyngitis with features that suggest streptococcal pharyngitis, and treating with penicillin each time a test is positive. . treating with one of the regimens effective for terminating chronic carriage. the first option is simple, as safe as penicillin, and appropriate for many patients. the second option should be reserved for particularly anxious patients; those with a history of acute rheumatic fever or living with someone who had it; or those living or working in nursing homes, chronic care facilities, hospitals, and perhaps schools. the two antibiotic treatment regimens that have been effective for eradication of the carrier state are: • intramuscular benzathine penicillin plus oral rifampin ( mg/kg/ dose up to mg, given twice daily for days beginning on the day of the penicillin injection) • oral clindamycin, given for days ( mg/kg/day up to mg, divided into three equal doses) clindamycin may be preferred because it is easier to use than intramuscular penicillin plus oral rifampin and may be somewhat more effective. in controlled, comparative trials, no other antibiotic regimens have been demonstrated to reliably terminate the chronic streptococcal carrier state. successful eradication of the carrier state makes evaluation of subsequent episodes of pharyngitis much easier, although chronic carriage can recur upon reexposure to group a streptococci. some patients seem remarkably susceptible to group a streptococci. the reasons for frequent bona fide acute group a streptococcal pharyngitis are obscure, but appropriate antibiotic treatment results in resolution of symptoms and eradication of group a streptococci. the role of tonsillectomy in the management of patients with multiple episodes of streptococcal pharyngitis is controversial. fewer episodes of sore throat have been reported among patients treated with tonsillectomy (in contrast to patients treated without surgery) during the first years after operation. the patients enrolled in that study had experienced numerous episodes of pharyngitis, but it appears that not all episodes were caused by group a streptococci. of particular concern is the reported tonsillectomy complication rate of % and the improvement over time noted among the nontonsillectomy patients. in addition, the presence of tonsils is not necessary for group a streptococci to infect the throat. tonsillectomy cannot be recommended except in unusual circumstances. it seems preferable to treat most patients with penicillin whenever symptomatic group a streptococcal pharyngitis occurs. obtaining follow-up throat specimens for culture helps distinguish recurrent pharyngitis from chronic carriage. certain β−hemolytic streptococci of serogroups other than group a cause acute pharyngitis. well-documented epidemics of food-borne group c and group g streptococcal pharyngitis have been reported in young adults. in these situations, a high percentage of individuals who have ingested the contaminated food promptly developed acute pharyngitis, and throat cultures yielded virtually pure growth of the epidemiologically linked organism. there have been outbreaks of group g streptococcal pharyngitis among children. however, the role of these non-group a streptococcal organisms as etiologic agents of acute pharyngitis in endemic circumstances has been difficult to establish. group c and group g β streptococci may be responsible for acute pharyngitis, particularly in adolescents. however, the exact role of these agents, most of which are carried asymptomatically in the pharynx of some children and young adults, remains to be fully characterized. when they are implicated as agents of acute pharyngitis, groups c and g organisms do not appear to necessitate treatment, inasmuch as they cause self-limited infections. acute rheumatic fever is not a sequela to these infections, although post-streptococcal acute glomerulonephritis has been documented in rare cases after epidemic group c and group g streptococcal pharyngitis. arcanobacterium (formerly corynebacterium) haemolyticum is a gram-positive rod that has been reported to cause a scarlet fever-like illness with acute pharyngitis and scarlatinal rash, particularly in teenagers and young adults. detecting this agent requires special methods for culture, and it has not routinely been sought in patients with scarlet fever or pharyngitis. the clinical features of a. haemolyticum pharyngitis are indistinguishable from group a streptococcal pharyngitis; pharyngeal erythema is present in almost all patients, patchy white to gray exudates in about %, cervical adenitis in about %, and moderate fever in %. palatal petechiae and strawberry tongue may also occur. the scarlatiniform rash usually spares the face, palms, or soles. it is erythematous and blanches; it may be pruritic and demonstrate minimal desquamation. erythromycin appears to be the treatment of choice. section one respiratory disorders ( to ) , and infection has been documented in several travelers from western europe. the pathogenesis of diphtheria involves nasopharyngeal mucosal colonization by c. diphtheriae and toxin elaboration after an incubation period of to days. toxin leads to local tissue inflammation and necrosis (producing an adherent grayish membrane made up of fibrin, blood, inflammatory cells, and epithelial cells) and it is absorbed into the blood stream. fragment b of the polypeptide toxin binds particularly well to cardiac, neural, and renal cells, and the smaller fragment a enters cells and interferes with protein synthesis. toxin fixation by tissues may lead to fatal myocarditis (with arrhythmias) within to days and to peripheral neuritis within to weeks. acute tonsillar and pharyngeal diphtheria is characterized by anorexia, malaise, low-grade fever, and sore throat. the grayish membrane forms within to days over the tonsils and pharyngeal walls and occasionally extends into the larynx and trachea. cervical adenopathy varies but may be associated with development of a "bull neck." in mild cases, the membrane sloughs after to days and the patient recovers. in severe cases, an increasingly toxic appearance can lead to prostration, stupor, coma, and death within to days. distinctive features include palatal paralysis, laryngeal paralysis, ocular palsies, diaphragmatic palsy, and myocarditis. airway obstruction (from membrane formation) may complicate the toxigenic manifestations. accurate diagnosis requires isolation of c. diphtheriae on culture of material from beneath the membrane, with confirmation of toxin production by the organism isolated. laboratories must be forewarned that diphtheria is suspected. other tests are of little value. treatment includes equine antitoxin to neutralize circulating toxin, as well as systemic penicillin or erythromycin. in a teenager, the retropharyngeal space normally does not exceed mm when measured from the anterior aspect of the c vertebral body to the posterior pharynx. in infants, the retropharyngeal space is usually less than one width of the adjacent vertebral body. however, during crying, this distance may be three widths of the vertebral body. also, under normal circumstances, the retrotracheal space does not exceed mm in teenagers when measured from the anterior aspect of c- to the trachea. dotted lines depict the "thumbprint" sign, noted on a lateral neck radiograph, made by a swollen epiglottis. (from reilly bm: sore throat. in practical strategies in outpatient medicine, nd ed. philadelphia: wb saunders, .) acute symptomatic pharyngitis caused by neisseria gonorrhoeae occurs occasionally in sexually active individuals as a consequence of oral-genital contact. in cases involving young children, sexual abuse must be suspected. the infection usually manifests as an ulcerative, exudative tonsillopharyngitis but may be asymptomatic and resolve spontaneously. gonococcal pharyngitis occurs in homosexual men and heterosexual women after fellatio and is less readily acquired after cunnilingus. gonorrhea rarely is transmitted from the pharynx to a sex partner, but pharyngitis can serve as a source for gonococcemia. diagnosis requires culture on appropriate selective media (e.g., thayer-martin medium). recommended therapeutic regimens include a single intramuscular dose of mg of ceftriaxone or a single oral -mg dose of ciprofloxacin. spectinomycin is ineffective performance of a predictive model for streptococcal pharyngitis in children group a streptococcal infections and acute rheumatic fever acute pharyngitis optical immunoassay test for group a ß-hemolytic streptococcal pharyngitis: an office-based, multicenter investigation potential mechanisms for failure to eradicate group a streptococci from the pharynx the changing epidemiology of invasive group a streptococcal infections and the emergence of streptococcal toxic shock-like syndrome: a retrospective populationbased study persistence of group a streptococci in eukaryotic cells-a safe place? what is a throat culture streptococcal pharyngitis: the carrier state, definition and management streptococcus associated toxic shock persistence of acute rheumatic fever in the intermountain area of the united states outbreak of group a streptococcus septicemia in children: clinical, epidemiologic, and microbiological correlates working group on severe streptococcal infections: defining the group a streptococcal toxic shock syndrome: rationale and consensus definition other pathogens community-wide outbreak of group g streptococcal pharyngitis periodic fever, aphthous stomatitis, pharyngitis, adenitis: a clinical review of a new syndrome arcanobacterium haemolyticum in children with presumed streptococcal pharyngotonsillitis or scarlet fever chlamydial pharyngitis mononucleosis caused by epstein-barr virus and cytomegalovirus in children: a comparative study of cases pharyngitis associated with herpes simplex virus in college students pharyngoconjunctival fever caused by adenovirus type epstein-barr virus infections: biology, pathogenesis, and management epstein-barr virus infectious mononucleosis in children: i. clinical and general laboratory findings epstein-barr virus infectious mononucleosis in children: ii. heterophil antibody and viral-specific responses arcanobacterium haemolyticum: biology of the organism and diseases in man life-threatening infections of the head and neck clinical infections and nonsurgical treatment of parapharyngeal space infections complicating throat infection pharyngitis followed by hypoxia and sepsis: lemierre syndrome peritonsillar abscess: clinical and microbiologic aspects and treatment regimens upper respiratory tract infections in young children: duration of and frequency of complications deep neck infections and respiratory distress in children new approaches to the treatment of group a streptococcal pharyngitis treatment of streptococcal pharyngotonsillitis: reports of penicillin's demise are premature penicillin and the marked decrease in morbidity and mortality from rheumatic fever in the united states efficacy of tonsillectomy for recurrent throat infection in severely affected children: results of parallel randomized and nonrandomized clinical trials effect of antibiotic therapy on the clinical course of streptococcal pharyngitis resistance to erythromycin in group a streptococci streptococcal pharyngitis: the case for penicillin therapy duration of positive throat cultures for group a streptococci after initiation of antibiotic therapy clindamycin treatment of chronic pharyngeal carriers of group a streptococci in gonococcal pharyngitis. examination and testing for other sexually transmitted diseases and pregnancy are recommended. chlamydia species and mycoplasma pneumoniae may cause pharyngitis, although the frequency of these infections is disputed. chlamydia trachomatis has been implicated serologically as a cause of pharyngitis in as many as % of adults with pharyngitis, but isolation of the organism from the pharynx has proved more difficult. chlamydia pneumoniae has also been identified as a cause of pharyngitis. because antibodies to this organism show some crossreaction with c. trachomatis, it is possible that infections formerly attributed to c. trachomatis were really caused by c. pneumoniae. diagnosis of chlamydial pharyngitis is difficult, whether by culture or serologically, and neither method is readily available to the clinician.m. pneumoniae most likely causes pharyngitis. serologic (positive mycoplasma immunoglobulin m [igm]) or, less often, culture methods can be used to identify this agent, which was found in % of college students with pharyngitis in one study. polymerase chain reaction (pcr) is diagnostic.there is no need to seek evidence of these organisms routinely in pharyngitis patients in the absence of ongoing research studies of nonstreptococcal pharyngitis. the efficacy of antibiotic treatment for m. pneumoniae and chlamydial pharyngitis is not known, but these illnesses appear to be self-limited. treatment of more complicated m. pneumoniae infections, such as pneumonia (table - ), is indicated with erythromycin, azithromycin, or clarithromycin; doxycycline may be used if the patient is older than years. key: cord- -acwkh ed authors: kallio-kokko, hannimari; uzcategui, nathalie; vapalahti, olli; vaheri, antti title: viral zoonoses in europe date: - - journal: fems microbiol rev doi: . /j.femsre. . . sha: doc_id: cord_uid: acwkh ed a number of new virus infections have emerged or re-emerged during the past years. some viruses are spreading to new areas along with climate and environmental changes. the majority of these infections are transmitted from animals to humans, and thus called zoonoses. zoonotic viruses are, as compared to human-only viruses, much more difficult to eradicate. infections by several of these viruses may lead to high mortality and also attract attention because they are potential bioweapons. this review will focus on zoonotic virus infections occurring in europe. during the past years a number of new virus infections have emerged or re-emerged. most of them, such as sin nombre and andes hantaviruses, sars coronavirus, avian influenza, nipah and hendra viruses, have appeared in subtropical or tropical regions. dengue is spreading to new areas and west nile virus has reached the new world. infections by several of these viruses may lead to high mortality and also attract attention because they are potential bioweapons. some viruses such as tick-borne encephalitis virus are spreading to new areas along with climate and environmental changes. most of these infections are zoonoses and clearly viruses shared by animals and humans are, unlike human-only viruses, much more difficult to eradicate. here, we review zoonotic virus infections occurring in europe. the infections like lassa fever and dengue that are imported to europe but are not indigenous to european nature will not be discussed in detail in the review. we have divided the virus infections into two categories, those that are transmitted to humans directly from vertebrate animals (like rodents, foxes, bats and birds) and those that are primarily transmitted by arthropods (mosquitoes, ticks, sandflies). the latter class is formed by arboviruses but notably they have vertebrate hosts in nature. hantaviruses are enveloped viruses with a tri-segmented negative-stranded genome and belong to the family bunyaviridae [ , ] . the . kb l (large) segment rna encodes the kda rna polymerase, the . kb m (medium) segment the two glycoproteins - kda gn and - kda gc, -formerly known as g and g ; and the . kb s (small) segment the - kda nucleocapsid protein (n) ( table , fig. ). in addition, the s segment of some hantaviruses has another open reading frame named ns but its product or function remains to be discovered. viral messenger rnas of the members of the bunyaviridae are not polyadenylated and are truncated relative to the genome rnas at the termini. messenger rnas have -methylated caps and - nontemplated nucleotides which are derived from host cell mrnas. the termini of all three segments are conserved and complementary to each other, a feature that has assisted in cloning and discovery of new hantaviruses. unlike most other bunyaviridae, hantaviruses are not arthropod-borne (arboviruses), but are rodent-borne, roboviruses. each hantavirus is primarily carried by a distinct rodent/insectivore species although a few host switches seem to have occurred during the tens of millions of years of their co-evolution with their carrier animals [ ] . we now know that the genetic diversity of hantaviruses is generated partly by (i) genetic drift (accumulation of point mutations and insertions/deletions) leading to quasispecies [ , ] , (ii) genetic shifts (reassortments of genome fragments within the same virus genotype/species), and (iii) according to recent findings [ , ] , by homologous recombination, a mechanism not previously observed for negative-strand rna viruses. hantaviruses, which cause hemorrhagic fevers with renal syndrome (hfrs) in eurasia and hantavirus cardiopulmonary syndrome (hcps) in the americas, are prime examples of emerging and re-emerging infectious agents. like most of these infections hantaviral diseases are zoonoses. with the exception of the south-american andes virus, which can be transmitted directly from human to human, hantavirus infections are thought to be transmitted to humans primarily from aerosols of rodent excreta. only some [ , ] . topografov hantavirus isolated from siberian lemmings (lemmus sibiricus) has not been detected in north european lemmings (lemmus lemmus) although it can grow in them [ ] . infections in rodents are mainly asymptomatic and persistent. hantavirus infections are quite common in europe [ ] (table ) , puumala virus is common in northern europe, european russia and parts of central-western europe (fig. ) . dobrava virus is found mainly in the balkans (fig. ) . saaremaa virus has been detected in eastern and central europe but its epidemiology is not well defined (fig. ) . apart from laboratory infections [ , ] seoul virus has been detected in wild rats, only in france [ ] . it is also apparent that many parts of europe, such as britain, poland and byelorussia, remain ''white'' on the european hantavirus map [ ] . this means either that hfrs is rare or nonexistent in these regions or is not widely recognized and diagnosed by the biomedical community. in northern europe hfrs as well as the carrier rodents exhibit peaks in - year cycles [ ] while in central europe the hfrs incidence follows the fluctuations of ''mast years'', i.e. the availability of beech and oak seeds for the hantavirus-carrying rodents. in central europe hfrs peaks in the summer while in northern europe most cases occur in late autumn and early winter, from november to january. risk factors to catch hantavirus infections and hfrs include professions such as forestry, farming, and military, or activities such as camping, and the use of summer cottages. males are more likely to be exposed than females [ , ] . puumala and dobrava viruses both cause hfrs but the infections differ considerably in severity [ ] : both are characterized by acute-onset fever, headache, abdominal pains, backache, temporary renal insufficiency -first oliguria, proteinuria and increase in serum creatinine and then polyuria -and thrombocytopenia but the extent of hemorrhages (hematuria, petecchiae, internal hemorrhages), requirement for dialysis treatment, hypotension and mortality are much higher in [ ] . this is important since the infection is so common in many areas of europe [ ] (table , fig. ). in addition, in some patients puumala virus infection may invade the pituitary gland and lead to mortality or at least hypophyseal insufficiency requiring hormone-replacement therapy [ ] . the pathogenesis of hfrs is poorly understood [ , ] . however, it is known that b integrins can mediate the entry of pathogenic hantaviruses [ ] and that hantaviruses can regulate apoptosis [ ] [ ] [ ] [ ] ] . also there is evidence [ , ] that increased capillary permeability is an essential component in the pathogenesis of both hfrs and hcps, although different target tissues, kidneys and lungs are affected in the two diseases. hfrs patients show locally increased levels of tnf-a in the plasma and kidneys [ , ] and high levels of urinary secretion of the proinflammatory cytokine il- [ ] . studies with a monkey model mimicking human puumala virus infection [ ] may assist in elucidating the mechanism of pathogenesis. of the four structural proteins, both in humoral and cellular immunity, the n protein appears to be the principal immunogen [ ] . cytotoxic t-lymphocyte (ctl) responses are seen [ ] and may be important both for protective immunity and pathogenesis of hantavirus infections [ ] . the diagnosis of acute hfrs is primarily based on serology, since viral rna cannot be regularly detected in the blood or urine of patients [ , ] . both immunofluorescence tests and enzyme immunoassays are widely used for detection of specific igm or low-avidity igg antibodies, characteristic of acute infection [ ] [ ] [ ] . in addition, immunochromatographic -min igm-antibody tests [ , ] have been developed. vaccines against hantavirus infections have been used for years in china and korea, but not in europe or the americas [ ] . no specific therapy is used in europe, although both ribavirin and interferon-a have been successfully used in trials in china [ , ] . a major problem is that at the time hfrs patients are hospitalized, virus replication is already disappearing. members of the genus lyssavirus within the family rhabdoviridae are bullet-shaped, enveloped viruses approximately nm in diameter and nm in length. the kb non-segmented negative-strand genome encodes five proteins (starting from the end): the - kda nucleoprotein (n), the - kda phosphoprotein (p), the - kda matrix protein (m), the trimeric - kda glycoprotein (g) and the kda polymerase protein (l) ( table , fig. ). proteins are separately transcribed in cascade by a special mechanism from a single -end promoter which results in a decreasing transcription and expression gradient for proteins encoded from the to end. the major antigen with neutralizing epitopes and pathogenetic determinants is the glycoprotein, which is responsible for receptor recognition and membrane fusion. after endocytosis the viral envelope fuses with endosomal membranes provoking the release of the internal viral nucleocapsid in the cytoplasm where transcription and replication takes place. the helically wound nucleocapsid results from the intimate association of the nucleocapsid protein and the rna genome. it serves as template for the polymerase (l protein and p cofactor) for transcription and replication. it buds to intracytoplamic membranes in infected neurons, but on plasma membranes in salivary gland epithelial cells. the lyssaviruses currently consist of established genotypes, or lineages, of rabies(-like) viruses [ ] of which the classical rabies virus, found throughout the world and associated with terrestrial mammalian hosts and american bats, forms genotype ( table , fig. ). other lineages, or genotypes, are found in bats, of which mokola and lagos bat viruses seem to be less pathogenic, and towards which the vaccines based on the classical genotype rabies virus are not protective [ a, b] . in addition to the genotypes, new bat lyssa- virus genotypes have been recently found, e.g. from russia [ c]. during the past years or more in europe, classical rabies virus has made two major host shifts, firstly from the dog to red foxes, and then to racoon dogs brought from east asia to be raised for fur; this species then became widely established in the wild (fig. ) . phylogenetic data also suggest that westand southward spread of rabies virus occurred during the last century [ a] . although in europe the most important carriers of classical rabies virus are foxes and racoon dogs, the virus can be transmitted to secondary hosts such as domestic animals (dog, cat, cattle, horse, sheep) or e.g. deer -practically any mammal species could be a potential carrier. bats are a special case; lyssaviruses are maintained in bats, even in the absence of classical rabies in carnivores; thus in countries where classical rabies has been eliminated, bat rabies has become the dominating or only source of rabies virus and retains the potential for host-switching into the carnivore reservoir which constitutes a more direct threat for public health [ b]. as many bats are protected species, the detection or ''absence'' of bat rabies is dependent also on the intensity of screening efforts [ , ] . as of the beginning of , the following countries in europe were declared ''rabies-free'' by who (meaning no indigenous cases occurred during the last two years): belgium, cyprus, finland, greece, iceland, ireland, italy, luxembourg, norway, portugal and sweden (fig. ) . in , rabies virus was detected in europe in wild animals (excluding bats), domestic animals; bat and human rabies cases were diagnosed (see table ). the countries where rabies was circulating include (in diminishing order of cases) russia, ukraine, lithuania, belarus, latvia, estonia, croatia, poland, slovakia, serbia-montenegro and turkey with hundred(s) to thousands of (animal) cases, romania, bosnia-herzegovina, germany, moldova and bulgaria with dozens of cases, and individual cases were registered (some imported and not affecting the rabies-free status) in slovenia, the netherlands, denmark, france, albania, finland, austria and switzerland ( members of out of the lyssavirus genotypes (except lagos bat virus, i.e. genotype ) have caused rabies disease in man. the infection is inevitably fatal in humans or other mammals unless immune intervention (vaccination and administration of antibodies) is used. transmission may occur though the bite of an animal delivering the virus deep in to striated muscle or connective tissue, but infection may also occur after abrasion of the skin or licking of mucous membranes. the bite of a bat with small teeth may go unnoticed; on the other hand the bat lyssaviruses may infect human skin more easily than has been recognised. the incubation period is - days, but has been shown in some cases to be months, even years. the classical picture of rabies includes prodromal illness with fever and non-specific symptoms, as well as itching and local paresthesia. this is followed by neurological signs, consisting of either encephalitic ''furious rabies'' or paralytic ''dumb rabies''. in the former, episodic hyperactivity and excitation of the cns manifest as, e.g. hydrophobia, hypersalivation and convulsions. the patient finally develops paralysis, coma and cardiorespiratory failure. in the paralytic form, no excitation is seen, but paralytic disease develops to coma and death [ , a, b] . after entry of the virus to the body, the virus must gain access to peripheral nerves and to be transported towards the cns. rabies virus components are attached either directly or by encapsulated vesicles to the dynein motor carrying the ''cargo'' along the axonal microtubular system towards the cell stroma, approximately at a speed of mm/day [ , , ] . it is transmitted after replication on neuronal membranes transsynaptically to adjacent neurons and finally invades the cns where it first disturbs the limbic system associated with the excitation, and later neocortex, with little histopathological changes in neurons. centrifugal spread of the virus from the cns to many tissues through somatic and autonomic nerves also occurs, where the salivary glands are especially important for the spread of the virus to the next victim. immunological responses do not occur before cns involvement. all the viruses in phylogroup i are also pathogenic to mice by i.m. injection, and can cross-neutralize each other. this has practical implications, as fortunately the vaccine strains (of genotype ) also appear to protect against the ebvl viruses [ ] . a pathogenic determinant common to phylogroup i viruses seems to be amino acid r in the glycoprotein [ ] ; substitution of this amino acid also abolishes the retrograde transport [ ] . when symptoms develop, no cure is available. however, after exposure to a bite or scratch of a potentially rabid animal, rabies must and can effectively be prevented by post-exposure prophylaxis, originally developed by louis pasteur. the treatment includes in a non-vaccinated person (a) washing (with water and detergent) and disinfecting the wound to minimize the amount of cell-free virus (this alone can increase survival %), (b) starting a post-exposure vaccine regimen which includes (in europe) five doses of cell-culture derived vaccine intramuscularly into the deltoid muscle on days , , , , and (c) in case of severe or deep injury additional passive anti-rabies immunoglobulin, which should be administered principally to the wound area. if the person was pre-vaccinated or the animal can be caught and studied for the presence of rabies, the protocol can be adjusted accordingly [ , a] . clinical suspicion of rabies in a case of encephalitis of unknown origin is the starting point. ante mortem diagnostics can be achieved most easily with rt-pcr from saliva. in addition, rabies antigen can be detected in brain or nuchal skin biopsies, and in some cases antibodies in serum or csf may be found. post-mortem diagnostics is most rapid with antigen detection or rt-pcr from the brain, virus isolation is also possible. in addition to post-exposure prophylaxis, vaccines in humans can be used for pre-exposure prophylaxis ( doses) in risk groups (veterinarians, wildlife workers, travellers to endemic areas; especially small children who may be unable to explain a potential exposure to a rabid animal) [ , a, ] . the primary method for control of rabies is vaccination of dogs (and cats). this practice was established at the beginning of the last century in most of europe, but has not yet been achieved in many developing countries, where annually people still die of rabies. in most of europe (excluding eastern europe) rabies has been eradicated from terrestrial wildlife species (carnivores) by aerial distribution of vaccine baits across the countryside. the vaccines used to protect wildlife species include attenuated vaccines as well as a recombinant vaccinia virus carrying the rabies virus glycoprotein (the latter has in one case caused a skin infection in man). although rabies in terrestrial animals can be controlled efficiently, eradication from the bat reservoir is not currently feasible. arenaviruses are the only members of the rna virus family arenaviridae. these viruses are enveloped, lipid solvent-sensitive, pleomorphic particles with a mean diameter of nm (ranging between and nm). host cell-derived ribosomes are present in the virions, and give the virus particles a ''sandy'' appearance under the electron microscope, hence the name arenavirus (arena: sand in latin). the virion structure of arenaviruses is quite simple, virus particles contain two rna segments (s and l) linked to nucleocapsid proteins and viral polymerase molecules, and these nucleocapsidpolymerase complexes are surrounded by a lipid envelope into which two glycoproteins (g and g ) are linked protruding on the outside of the virion. arenaviral rna segments have an ambisense coding arrangement, the s segment ( . kb) encoding a -kda nucleocapsid protein (n) in the viral complementary sequence, and in the viral-sense -end sequence a -kda glycoprotein precursor (gpc) which is posttranslationally cleaved to two glycosylated proteins - kda g and g ; and the l segment ( . kb) encoding a -kda viral polymerase (l) in the viral complementary sequence, and in the viral-sense -end of the sequence a - kda ring finger z protein, which has been shown to have a role in arenavirus budding [ a] (table , fig. ). initiation of transcription may involve cap-snatching, although the transcription mechanism is not yet fully elucidated. arenaviruses include viruses all carried by different rodent hosts (except tacaribe virus which has been isolated only from fruit bats) [ b]. arenaviruses are capable of causing chronic infections in their rodent hosts, and infectious virus is present in the blood and is also secreted into body fluids (saliva, urine, semen), which is presumably the route of transmission to humans. the appearance and incidence of arenaviral infections are closely associated with the distribution of the rodent host species and the rodent population dynamics. arenaviruses have co-evolved with their specific host species during millions of years, and have been divided into old world and new world groups first on a serological bass, and later into evolutionary lineages (new world group) using genetic analysis [ a, b, c]. both groups contain viruses that are included in the category a pathogen list (defined by cdc, usa), which means that the propagation of these agents is allowed only in biosafety level laboratories. these highly pathogenic arenaviruses include the south american junin, machupo, guanarito, and sabia viruses from the new world group, and the african lassa virus from the old world group. all these viruses can cause hemorrhagic fevers in humans, and are considered potential bioterrorism agents being thus included in biohazard preparedness programs. in europe, these viruses occur only rarely as imported cases. the only arenavirus endemic in europe is lymphocytic choriomeningitis virus (lcmv), shown to circulate in mus musculus populations (table ) , and associated with pet hamster derived epidemics [ b, a]. relatively few epidemiological data are available concerning the actual distribution of lcmv in europe, but in addition to serological evidence from mus sp. material from spain [ b], antibodies against lcmv have been detected in rodent species other than mus musculus (our unpublished data), which indicates that other yet unknown arenaviruses may circulate in europe. at least ten arenaviruses have been reported to be able to cause disease in humans. as mentioned above, five arenaviruses (lassa, junin, machupo, guanarito, and sabia viruses) are even capable of causing a lifethreatening viral hemorrhagic fever [ a, b] . none of these five viruses are endemic in europe, but a few imported lassa virus infections have been diagnosed in germany and great britain during the last few years with no secondary infections detected [ ] [ ] [ ] . increased travelling increases also the risk for transmission of exotic arenaviruses to non-endemic areas such as europe. lcmv is thus far the only known endemic arenavirus in europe [ b]. in humans lcmv infections are mostly either asymptomatic or influenza-like diseases. in some cases aseptic meningitis or meningoencephalomyelitis is seen. lcmv is also capable of causing congenital infections manifested by hydrocephalus, microcephalus, chorioretinitis, and mental or psychomotor retardation [ , a, b] . lcmv infections are rarely fatal for humans. the diagnosis of arenaviral infections is based on serology and/or direct detection of the virus [ ] . for serodiagnosis methods using immunofluorescence assay (ifa) as well as enzyme immunoassay (eia) have been described. either a four-fold rise in igg antibody titers or presence of igm antibodies is considered indicative of acute infection. the antibodies that appear first in the acute phase of infection are directed against the nucleocapsid protein; neutralizing antibodies against the glycoproteins appear later in the convalescent phase (if at all). this means that typing of the causative agent is difficult on a serological basis at the early stage of infection, and is actually possible only in the convalescent phase due to the slow rise of virus typespecific neutralizing antibodies. for direct detection of the virus, antigen detection assays are useful in the early diagnosis of lassa fever especially. also reverse transcriptase (rt)-pcr tests have been developed to detect arenaviral rna in patient samples [ a, b, a, b, c]. virus isolation attempts can also be successful. the supportive treatment of arenaviral infections includes ensuring the fluid, electrolyte and osmotic balance. in severe hemorrhagic fever-cases early diagnosis is important because the use of ribavirin has been found effective if the treatment commences within the first six days after onset of symptoms [ ] . immune plasma containing neutralizing antibodies has also been useful in some cases. for prevention of arenaviral diseases, several attempts to develop vaccines have been made [ a] . one vaccine, candid , has been successfully used in the prevention of argentine hemorrhagic fever caused by junin virus with a clear reduction in the number of infections observed in humans [ b, ]. the genus orthopoxvirus in the family poxviridae consists of large - nm brick-shaped viruses, with a double-stranded dna genome ( - kb) ( table ) , that are serologically cross-reactive and -protective. the middle part of the genome is very conserved among orthopoxviruses encoding structural proteins and replication machinery whereas the ends are more variable comprising genes involved with host-specificity and counteracting the immune response [ ] . replication of orthopoxviruses occurs in the cytoplasm and includes translation of early mrnas (such as dna polymerases and immune defense molecules), dna replication, translation of intermediate mrnas (for late transcription factors), and late mrnas encoding structural proteins and late enzymes, respectively. altogether, e.g. the cowpox virus genome encodes nearly open reading frames. intracellular non-enveloped virions are first formed, comprising the majority of the infectious viral progeny; some particles develop in er/golgi into enveloped, either cell-attached or extracellular viral particles, often motile due to attached actin tails [ ] . homologous recombination occurs readily between orthopoxvirus sequences which has raised some concerns about the use of vaccinia virus-based vaccines in wild animals [ ] . some orthopoxviruses are host-specific, whereas some are more promiscuous but have a distinct reservoir. their nomenclature may be misleading; e.g. monkeypox is not carried by monkeys, neither is cowpox carried by cows. smallpox or variola virus, now eradicated and historically the cause of one of the most feared human diseases, was specific to man. many other orthopoxviruses circulate in wildlife species and are often zoonotic. examples include the vaccinia virus, the modern smallpox vaccine, the origins of which are unclear but was originally described as cowpox by edward jenner [ ] in late th century england, and which later has also re-escaped to nature in other parts of the world [ ] ; monkeypox virus, pathogenic to primates, including humans, causing a smallpox-like disease with secondary transmission and with a likely reservoir in small rodents in central africa; cowpox virus, which is the main orthopoxvirus in europe and may be transmitted to man either directly from rodents or from a secondary carrier, typically cat. cowpox virus has been detected in western eurasia and in europe, voles of clethrionomys and microtus species and apodemus mice are the main reservoir hosts [ ] ( table ) . shedding of the virus from rodents is apparently transient. infection of cattle is rare; domestic cats relatively frequently present with clinical disease, but infection of zoo animals, e.g. elephants, has also been reported [ ] . following the cessation of smallpox vaccination, more than years ago, the number of humans susceptible to cowpox has increased. more than human cowpox cases have been reported in the literature since [ , ] . all age groups may acquire cowpox, but most cases have been in girls under years of age, who have had a cat or e.g. a field mouse as a pet. infection probably occurs through abrasions in the skin; persons with atopic eczema are more prone to the infection. [ ] [ ] [ ] [ ] . the incubation period is - days, after which papules develop into lesions - cm in diameter which proceed through pustular, ulceral and eschar stages over a period of a about two weeks. they may be painful and vary in number, size and severity. local lymphadenopathy, pyrexia and nausea may occur; secondary bacterial infections are common. typically, solitary lesions are found, located mainly in fingers, hands or face (e.g. eyelid) [ , ] . in - weeks the lesions heal gradually, some residual scars may remain. in some cases severe generalized skin infection occurs [ ] , especially in atopic and in immunocompromised individuals and may in extreme cases lead to death [ ] . cidofovir (a phosphorylated nucleoside analog of cytosine) may have potential as an antiviral against cowpox virus [ ] . man-to-man transmission of cowpox virus (unlike for monkeypox) has not been reported. it is usually possible to detect orthopoxvirus particles directly from the skin lesions by electron microscopy. the virus can also be readily isolated in e.g. vero cells or chorioallantoic membrane of chicken embryos from the lesions and subsequently characterised [ ] . several sensitive pcr approaches have been described, some related to the bioterrorism (smallpox) preparedness [ ] [ ] [ ] ; in each case further typing at the species level is needed. in addition, during acute cowpox infection, igm antibodies and low-avidity igg antibodies have been detected [ ] . following the cessation of smallpox vaccination approximately years ago, the younger age groups are the most susceptible population, both to smallpox and to cowpox, which is more closely related to vaccinia virus. recent estimates indicate as low as % protection levels among europeans. for instance in finland, in the age group over , everybody had orthopoxvirus antibodies as measured by immunofluorescence assay. the seroprevalence decreased gradually towards younger age groups reflecting the gradual cessation of smallpox vaccination, with the last vaccinations in finland occurring in [ ] . smallpox was finally declared to be eradicated from the world in [ ] after which few people in europe have received the vaccine. in addition to wild rodents carrying cowpox, import of exotic pets may also pose a risk for orthropoxvirus transmission, as was seen in a recent outbreak of monkeypox virus in the usa [ ] . orthomyxoviruses are enveloped, negative-strand rna viruses with - genome segments, of which avian influenza, (i.e. influenza a) viruses may cause severe disease in domestic poultry and cause zoonotic infections. the influenza viruses in wild aquatic birds are the source of these epidemics in chickens as well as providing a gene pool for reassortants with human influenza a viruses which then may become established in humanto-human transmission resulting in influenza pandemics. in addition, influenza a viruses are known pathogens of pigs, horses, mink, seals and whales [ , ] . influenza a virus is - nm in diameter and has genome segments varying in size from . to . kb. the three largest segments - encode the polymerase subunits pa ( kda), pb ( kda) and pb ( kda), respectively; whereas the segments - each encode one viral protein, namely the kda hemagglutinin (ha), the kda nucleoprotein (np), and the kda neuraminidase (na), respectively (table , fig. ). the two smallest segments, and , encode each two proteins, the kda matrix protein (m ) and the kda membrane protein (m ), and the kda ns and the kda ns proteins, respectively (table , fig. ). in common with the bunyaviridae, the genomic rna is packed in the nucleoprotein which carries polymerase subunits, and the and -ends are conserved and complementary to each other and thus able to form panhandle structures in which the promoter regions reside. ''cap-snatching'' from cellular mrnas and an oligo-u motif are used to create viral mrnas starting with a cap structure and ending in a poly-a region. the virus enters the cells after binding to the sialic acid receptors by endocytosis, which is followed by acidification of the endocytic vesicle and, mediated by the ion channel forming m protein, of the interior of the virus, which leads then to fusion of the viral and endosomal membrane and release of the viral nucleocapsids to the cytoplasm, respectively. however, untypically for an rna virus, the replication, transcription and nucleprotein assembly occur in the nucleus. the envelope proteins are processed to the plasma membrane, where budding of the virions finally occurs. the envelope proteins are also the most important antigenic determinants. the homotrimeric hemagglutinin (ha), defines the ''h type'' which is responsible for the binding to the sialic-acid containing host cell receptors and membrane fusion properties. the neuraminidase (na) defines the ''n type'', and cleaves terminal sialic acid residues from glycoconjugates enabling the virus to reach target cells in the mucin-rich epithelium and facilitating release of the virus from the cells [ , ] . the catalytic site of the neuraminidase is a target for antivirals oseltamivir and zanamivir, and the m protein is a target for amantadine and rimantadine. to become active, the hemagglutinin protein needs to be cleaved by trypsin-like proteases found in respiratory and gastrointestinal epithelia. human-adapted influenza viruses replicate in the respiratory tract, whereas in avians the virus replicates primarily in the gut. when transmitted to and within poultry, the normal hemagglutinin of influenza a virus h or h of wild aquatic birds of the ''low pathogenic type (lpai) may be mutated. accumulation of basic residues at the cleavage site makes the ha cleavable by most proteases of cells, such as furin, and results in ''highly-pathogenic avian influenza'' (hpai) virus able to replicate in most tissues killing rapidly up to % of chickens [ ] [ ] [ ] . hpai, also known as ''fowl plague'' was first described in and the virus was first isolated in suggesting that humans have been directly exposed to avian influenza a viruses for centuries [ ] . influenza a virus gene pools reside in wild aquatic birds where at least ha types and na types are found, as compared to ha (h -h ) and na (n , n ) types circulating in man. also, the genes in aquatic birds are in evolutionary stasis without undergoing changes due to selective pressures. influenza a viruses in man are under constant selective pressure imposed by population immunity causing the hemagglutinin of influenza a virus to change its antigenic properties by accumulation of mutations (genetic drift). however, through double infection and reassortment (genetic shift), novel (e.g. hemagglutinin) genes from aquatic birds may become established in human influenza viruses giving rise to pandemics due to lack of adaptation to and immunity in, humans. previously, it was thought that the different receptor specificity -favoring different side chains of sialic acid -of human and avian influenza viruses would make direct transmission of avian viruses to humans unlikely, but both could be expected to infect swine, which carry receptors for both. thus pigs are considered to be potential reservoirs for generating new influenza virus variants. it has only recently been discovered that viruses considered unique to avian species may also infect man, although this may involve change in receptor specificities [ ] . in conjunctivitis in uk caused by avian influenza viruses (and previously, by seal influenza a viruses) was reported [ , ] (table ) . direct zoonotic transmission of avian influenza viruses to man resulting in human respiratory illness, was not known to occur or had not been diagnosed before the outbreak of h n avian influenza virus in hong kong in where / patients died of lower respiratory tract infection [ ] . after this, in europe, an outbreak of h n hpai avian influenza was encountered in italy ( ) ( ) without reported transmission to humans [ ] . in , a major h n hpai avian influenza outbreak occurred in the netherlands [ , ] . during this epidemic, veterinarians and people who culled infected poultry were at greatest risk of infection. it was noted by active surveillance that human h infections (and simultaneous h infections) were occurring, and consequently prophylactic treatment with oseltamivir was started. the h virus was confirmed to be transmitted to humans of which the majority had conjunctivitis, seven had an influenza-like illness; one veterinarian (who did not receive prophylactic oseltamivir) died. his symptoms started with high fever and headache two days after visiting a farm with infected chickens. one week later, he was admitted to hospital with pneumonia, where his status deteriorated to multi-organ failure, with death due to respiratory insufficiency weeks after onset of symptoms. in addition, in three cases household primary contacts were shown to have the disease by human-to-human transmission [ ] . both na inhibitors, zanamivir and oseltamivir inhibited virus obtained from humans during this outbreak and a significant difference was found in avian influenza virus detection between oseltamivir users ( / ) and those who had not taken prophylactic medication ( / ) [ ] . outbreaks of avian influenza have continued to occur in other parts of the world, especially the devastating ongoing h n epidemic in south-east asia since late with as of june a total of deaths/ cases in viet nam, thailand and cambodia. the epidemic has had a dramatic impact on poultry farming and industry with million birds dying or being destroyed to reduce virus dispersal. this ongoing epidemic clearly has ''pandemic potential''. avian influenza viruses may be detected by virus isolation (in cell culture or embryonated eggs) or rt-pcr which may be targeted at the specific ha subtype or may be generic to influenza a viruses (e.g. targeting the matrix protein gene) [ ] . also, serological tests such as. hemagglutination inhibition may be used. typing may be based on serological methods (such as hemagglutination inhibition), specific primers/probes or sequencing. many commercial influenza a antigen tests detect the nucleoprotein or na activity and should be applicable to the avian viruses, although this is poorly studied and documented. furthermore, it should be noted that rt-pcr from throat swabs of the lethal case in the netherlands were negative [ ] . avian influenza, or ''fowl plague'' outbreaks usually arise following contact with wild aquatic birds, such as mallards and ducks, in which the virus replicates as a rule without causing symptoms. the virus that is excreted in the gut can typically be isolated (or detected by rt-pcr) from cloacal swabs. hinshaw et al. [ ] studied over nine thousand birds and detected % prevalence in young and % prevalence in adult birds. influenza virus is readily transmitted in cold environments and is stable for - days at + °c, or in wet faeces (as shown for h n virus) for at least days at °c and more than days at + °c [ , ] . the main preventive and control measures include proofing the chicken breeding facilities against wild birds. raising chickens and turkeys in the open is a risk, minimizing secondary spread of outbreaks by stamping out the infected poultry, followed by cleaning, disinfection and controlling movements of humans and animals, trade embargoes and reporting the outbreaks (''fowl plague'' is in the top priority ''list a'' of the international animal health code of the office international des epizooties). selling poultry live, a common practice in south-east asia, is a definite risk factor. vaccination of poultry has been used as an additional control measure but may lead to undetected shedding and transmission of mutant virus selected under the pressure imposed by the vaccine [ ] . to prevent further transmission to humans, rapid measures are needed due to the short incubation period. the dutch experience suggests that results can be achieved by personal protection (e.g. protective eye glasses, masks) for all workers who screen and cull poultry, vaccination with regular inactivated influenza virus vaccine and prophylactic oseltamivir for those handling potentially infected poultry, to be continued for days after last exposure [ ] . for humans, specific vaccines containing h and h antigens would be welcome. the only known zoonotic agent of the togaviridae family to cause human disease in europe is the mosquito-borne sindbis virus (sinv), in the genus alphavirus. sinv is distributed throughout the old world and australia and causes rashes and polyarthritis outbreaks in northern europe, similar to chikungunya, oÕnyong-nyong virus and ross river virus in far east asia, africa and australia, respectively, whereas other alphaviruses causing encephalitic infections in man (venezuelan, western and eastern equine encephalitis virus) are found in the new world. alphaviruses are enveloped, positive-strand rna viruses with an kb genome. the non-structural proteins (nsp - ) are encoded from the -terminus of the genome, and a separate subgenomic s rna from the -end is used as a messenger for the structural proteins: the - kda capsid protein (c), and the - kda envelope glycoproteins (e and e ) [ ] (table , fig. ). sinv was first isolated in the nile delta in the s from a pool of mosquitoes without knowledge of any disease association. it is now known to be the causative agent of mosquito-borne epidemic polyarthritis with accompanying rashes and when described in northern europe it was given the names ockelbo disease, pogosta disease, or karelian fever when found in sweden, finland and russia, respectively. most infections occur during august-september, and larger outbreaks tend to appear with a seven-year interval (e.g. in finland, serologically verified cases occurred in , cases in ; and in sweden - cases are reported during peak years) [ , ] , with a peak incidence ( %) in -year old females. the association of sinv with pogosta disease was first discovered in the early s and it is believed that the virus may have been distributed throughout northern europe around this time. this conclusion is based on the evidence that thousands of human and bird sera collected during the early s in finland, were negative for sinv antibodies, whereas in the s - % of the population were sinvantibody positive [ ] . antibodies to sinv without polyarthritis outbreaks have been recorded in italy, romania, greece and the former yugoslavia [ ] . however, human disease due to sinv has been recorded in south africa, from where it may have originated. sinv was isolated from mosquitoes in sweden, norway and russia in the early s [ ] [ ] [ ] and sinv antibodies are found in wild tetraonic and migratory birds, most commonly (in sweden) from passeriformes such as redwing (turdus iliacus), fieldfare (turdus pilaris), blue tit (parus caeluleus), chaffinch (frinigilla coelebs), songthrush (turdus philomelos); thrushes have been suggested to be a major candidate as an amplifying host [ , ] . in addition, antibodies are commonly found in galliformes [ , ] . phylogenetically, sinv strains are similar in northern europe and south africa (in the north to south dimension), but differ considerably from strains circulating in asia and australia, where sinv-associated rash-arthritis is not recorded. this is consistent with the notion of a north-south dispersal of sinv strains by migratory birds [ ] . several bird species can be infected experimentally [ ] . it is evident that sinv cycles between birds and ornithophilic (culex and culiseta) mosquitoes (table ) . however, it may spill over to other hosts (evidence from many vertebrates from a frog to a bear) and vectors (including ticks and aedes mosquitoes) [ ] . recently, during an outbreak in finland in , the causative agent of pogosta disease was isolated for the first time in europe from skin biopsies and a blood sample of patients [ ] ; the virus strains were most closely related to sinv strains isolated from mosquitoes in sweden and russia years previously. the incubation period for the disease is about one week and the onset is accompanied by arthritis/arthralgia and itchy rash as the dominant symptoms, and also fatigue, mild fever, headache and muscle pain. hematological laboratory parameters are within the normal range and levels of c-reactive protein (crp) are not elevated. the rash is usually located on the trunk and thighs and lasts for a couple of days. one third to a half of patients, suffer from joint pains for more than months [ , ] . usually several joints are affected, the most common being the ankle, wrists, knee, and finger joints ( % or more of patients), as well as hip, shoulder and elbow joints [ , a, b ]. diagnosis is based on serology using enzyme immunoassay, immunofluorescence assay or hemagglutinationinhibition, and detection of seroconversion or a -fold rise in titre between two samples, or positive igm in a single sample. the first sample is usually taken during the first week after onset of illness, another sample is required to diagnose or exclude sinv infection; igm antibodies are detectable until approximately days post-onset, and igg antibodies can be detected approximately days after onset of illness [ b, ] . in some cases persisting igm antibody can be detected years after infection [ ] . for research purposes, the virus can be detected by ] or isolated from skin biopsies [ ] . no specific preventive measures are available, apart from avoiding mosquito bites. flaviviruses comprise a diverse group of pathogens that have been traditionally classified as arthropodborne viruses. they are linear positive single-stranded rna viruses with a monopartite genome ( - kb) that encodes structural proteins: the kda capsid protein (c), the - kda major envelope protein (e) and the . kda glycoprotein m (in mature virions); and non-structural proteins (ns , ns a, ns b, ns , ns a, ns b and ns ) (table , fig. ). noncoding or untranslated regions flank the infectious rna genome. when seen in the electron microscope flaviviruses appear as uniform spherical particles, - nm in diameter. the virus particles consist of a lipid envelope that has a surface covered by protrusions that contain envelope (e) and membrane (m) structural proteins, organized as dimers. this envelope surrounds an isometric capsid protein of approximately nm in diameter [ ] [ ] [ ] . there are currently about members in the family flaviviridae [ ] which have been found infecting a wide variety of organisms including mammals, arthropods, avian and amphibians. many of these viruses are major pathogens of humans, domestic and farmed animals as well as wildlife species. with the possible exception of the dengue viruses, the flaviviruses are zoonotic, depending almost entirely for their existence on wildlife vertebrate and in many cases, invertebrate species. the type species of the genus is yellow fever virus (yfv), hence the term ''flavi'' from the latin word flavus, which in turn describes the yellowish color of the skin in yellow fever infections [ ] . the classification, and serological and phylogenetic studies of flaviviruses reflect the importance of the vector on the biology and evolution of this genus. there are essentially three groups of flaviviruses: tick-borne, mosquito-borne and non-vectored flaviviruses, although this grouping is, to some extent, arbitrary since some mosquito-borne viruses are also transmitted by ticks and vice versa [ ] (table ). phylogenetic analysis also shows very strong correlations between genetic relationships, epidemiology and ecology of these viruses [ , ] . some flaviviruses are responsible for a significant proportion of the morbidity and mortality that is registered annually worldwide. they cause epidemic outbreaks that involve encephalitis and/or haemorrhagic fever, often fatal and involving millions of humans or in some case birds or mammals. important flaviviruses affecting humans are the dengue viruses, yellow fever virus, west nile virus (wnv), tick-borne encephalitis virus (tbev), japanese encephalitis virus, saint louis encephalitis virus, and murray valley encephalitis virus among others. dengue virus alone causes more than - million cases worldwide each year and some million people are now at risk from dengue infections [ ] . the most important flavivirus in europe is tbev, which is endemic in many european countries, and also in russia, ( table , fig. ) northern china and northern japan [ , a] . it affects thousands of people annually and has a significant impact on public health. the virus is transmitted to humans mainly through a tick bite, however, the infection has also been reported to occur by drinking unpasteurised goat milk from viraemic animals [ b, c]. the virus is maintained in nature in a cycle involving ticks and wild vertebrate hosts and also by transovarial and transstadial transmission in its vector [ , ] . serological evidence and viral isolations, as well as sequence similarities have suggested that migratory birds could also play a role in the transmission of tbev from central europe to scandinavian countries; moreover, endemic areas of tbev are regions of high migratory bird activity [ ] . tbev is classified taxonomically into three subtypes: european subtype, far eastern subtype, and siberian subtype. the first subtype is transmitted mainly by ixodes ricinus, and the last two by ixodes persulcatus [ , ] . the distribution of tbev is well coordinated with the distribution of its vector, furthermore, different genotypes are located in distinct geographical areas and associated with specific vector hosts. recent data have shown the co-circulation of all three subtypes of tbev in the same geographical region, specifically in latvia, where the two vector ixodes species habitats meet [ ] . however, the endemic region in europe is patchy and covers only part of the geographical range of e.g. ixodes ricinus. there has been an increase of the incidence of tbev in many of its endemic areas but not in austria where a countrywide successful vaccination campaign was established reducing the disease incidence to lower levels [ c, d, a]. tbev affects principally the nervous system and can cause several clinical features of different severity table tick-borne enchephalitis viral infections in europe per country through time year year year year austria belarus -a croatia czech r. denmark -- estonia finland france germany - including meningitis, meningoencephalitis, meningoencephalomyelitis and meningoradiculoneuritis. hospitalization varies from days to months and in some cases years of treatment and rehabilitation are necessary as sequelae occur in approximately / of the patients. the incubation period of tbe is between and days. the disease is characteristically biphasic. the first phase (day - of onset of symptoms) is viraemic and can be either asymptomatic or fever, malaise, headache, anorexia, nausea, and muscle pains may be present. the second phase occurs in up to % of the patients after about days lag period (approximately days after the tick-bite) as a neurological disease of which about . % has a fatal outcome [ b, c, d]. the neurological symptoms and severity vary: the clinical picture includes meningitis (in about half of the patients), meningoencephalitis, meningoencephalomyelitis and meningoradiculoneuritis. hospitalization varies between days and months and in some cases years of treatment and rehabilitation are necessary in case of e.g. paresis. altogether, neuropsychiatric sequelae occur in approximately / of the patients. louping ill virus (liv) is endemic in ireland, in the northern region of great britain and in norway (fig. ) affecting principally sheep with a disease that is known as ovine encephalomyelitis, infectious encephalomyelitis of sheep, or trembling-ill. liv is transmitted to sheep by the tick vector ixodes ricinus. the natural life cycle of liv resembles that of tbev, it can be sustained in the natural environment through non-systemic transmission of virus between ticks cofeeding on rodents and other wild animals, which in turn infect grazing animals such as sheep and goat as a zoonotic disease. louping ill virus has also been observed in a bird-tick-bird cycle involving the red grouse (lagopus lagopus scoticus), ptarmigan bird species and the ixodes tick [ ] . infection with liv in sheep is characterised by a biphasic fever, depression, ataxia, muscular in-coordination, tremors, posterior paralysis, coma, and death. there is evidence for infection by liv in other domestic species and wildlife, i.e. cattle, horses, pigs, dogs, deer, shrews, wood-mice, voles, and hares [ , a] . it is generally believed that the majority of avian infections occur through a tick bite, however laboratory experiments and field observations have demonstrated that the red grouse can also become infected by feeding on infected ticks indicating that the vector bite is not the only route of infection [ b] . humans are also susceptible to infection with liv. however, the majority of the cases reported are accidentally acquired infections in laboratory workers. the second most frequent infection results from handling infected animal carcasses. infection with liv in humans can cause a neurological disease resembling the clinical picture observed for tbev infections, i.e. biphasic encephalitis, influenza-type illness, fever, articular pain, meningitis, myagia and poliomyelitis-like illness [ ] . other possible transmission routes for liv infection to humans include drinking contaminated milk from goat or sheep in an acute phase of infection [ ] , tick-bite, exposure to infective material, or through skin abrasions or wounds. antigenic and phylogenetic studies have shown that liv is most closely related to strains of the western european subtype of tbev and it is estimated to have emerged from this lineage approximately years ago [ ] . west nile virus (wnv) was first discovered in in uganda [ ] . it occurs throughout africa, the middle east, europe, russia, india and indonesia and was recently introduced into north america (new york) [ , ] . in the old world wnv is primarily considered to be an african virus which annually disperses northwards out of africa when birds migrate to europe, the middle east and asia [ ] . in humans, the majority of wnv infections cause a nonsymptomatic or mild febrile illness; however some infections can cause encephalitis and in most severe cases can lead to death, particularly in elderly patients. the incubation period is between and days after a mosquito bite (http://www.cdc.gov/ncidod/ dvbid/westnile/wnv_factsheet.htm). west nile virus is an illustrative example of the human impact on the dispersal and evolution of flaviviruses. the virus appeared for the first time in the usa, in new york in causing sixty-two confirmed human infections and seven deaths [ ] . the virus successfully over-wintered and during the next years dispersed widely throughout north america and now more recently also to central america and the caribbean. in north america the virus infects a very wide range of mosquito and animal species. the exact mechanism of introduction into north america is not known [ - a] . to date, more than human cases and deaths from wnv have been reported in the united sates of america (http:// www.cdc.gov/ncidod/dvbid/westnile). in europe, outbreaks caused by wnv have been recorded since the early s, especially in mediterranean countries, romania and southern russia (fig. ) . larger outbreaks (over cases) have been reported since the mid s in urban settings, especially a large outbreak in bucharest, romania in [ a, b, c] . in southern russia, specifically in volgograd, astrakhan and krasnodar regions, wnv has caused large epidemics of approximately human cases, with % mortality rate of reported cases. molecular epidemiological studies have shown that the latest large outbreaks in volgograd were caused by strains genetically similar to that of romania- , kenya- and newyork- reflecting the widespread distribution capacity of these epidemic viral strains [ b, b]. the incidence of wnv in europe is largely unknown. phylogenetic studies have showed that wnv has diverged into two main lineages, which form internal clusters or clades [ c, a, b, c]. more recently, a novel virus (rabensburg virus), antigenically and genetically closely related to wnv was isolated from a culex pipens mosquito pool in czech republic. this new virus could represent a third lineage of wnv, however more studies are needed to confirm this hypothesis or to determine whether or not this could represent a new member of the flaviviridae family [ a] . usutu virus (usuv) was first isolated from a mosquito in africa in [ c]. it was characterised serologically and classified within the japanese encephalitis virus serocomplex. usutu virus had rarely been isolated since then, with only one reported human case and being present only in two regions of africa. however, in the late summer of , usuv was found in vienna in austria during a study of an avian epidemic characterized by fatal encephalitis that was thought to be caused by wnv. the virus caused die-off among the bird population, especially blackbirds, in vienna (fig. ) and when isolated from birds and mosquitoes it showed % genetic identity to the african usuv [ b]. this is the first time usuv has been observed outside africa and also the first time it has been associated with fatal disease in animals. the virus re-appeared in the late summer of in the same region of vienna [ a] . most recently, in the virus has been isolated from humans (associated with rash in one patient) and from birds in austria and evidence exists as to the virus being able to overwinter establishing itself in this geographical area (nowotny, n. second european congress of virology, eurovirology ). as mentioned above the distribution of flaviviruses is worldwide, and we have described the zoonotic flaviviruses endemic to europe. however, it is important to note the increasing incidence of imported flaviviruses totaling over cases mainly due to changes in human behavior, such as increased travel to non-european destinations (http://www.eurosurveillance.org). the dengue viruses (denv) are a world-wide public health problem. they are transmitted to man by mosquitoes, particularly aedes aegypti and cause a wide range of different clinical outcomes [ c, a] . it is estimated that million cases of dengue fever (df) and of dengue hemorrhagic fever (dhf) cases occur annually [ ] . in a study based on the epidemiology and clinical course of travellers with symptoms of dengue infection it was found that the incidence of df among european travellers is underestimated, since diagnostic procedures, in general, for febrile patients often do not include tests for tropical arthropod-borne diseases, such as dengue virus [ b] . there have also been some imported cases of yfv to europe mainly by tourists travelling from endemic areas. however, the incidence of yfv as an imported disease is much lower than that of dengue infections (http://www.eurosurveillance.org) presumably because of the different nature of these diseases, denv having spread worldwide throughout the tropics and yfv occurring only in its endemic regions in africa and south america [ c]. traditionally, diagnosis of flaviviruses in europe has been restricted to detecting tbev infections through serological assays; igm-capture enzyme immunoassay, hemagglutination inhibition-, and immunofluorescence assay. igm antibodies in serum, and in some cases in cerebrospinal fluid during the neurological disease usually provide the diagnosis. rt-pcr is rarely positive at the second phase of the disease [ a]. because of the close antigenic relatedness of flaviviruses, laboratory findings can easily lead to misdiagnosis [ b]. in a study conducted in hungary, tbev-positive serum panels were tested retrospectively against wnv and some sera were found to show higher titres to wnv than to tbev when compared in serological assays (e. ferenczi, personal communication). as mentioned above, a study including travellers returning to europe from tropical destinations proved that many cases remain undetected. there is a need to improve the diagnosis for flaviviruses in order to determine the true incidence and prevalence of these infectious diseases in europe, and to study the ecology of imported viruses to determine which viruses have been able to establish themselves in the continent and which are still continuously being imported from africa or asia. meanwhile anti-vector campaigns and vaccination for travellers to endemic areas are the only measures to prevent and control these infectious diseases [ ] . there are effective vaccines for tbev and yfv but a protective immunogen does not exist for denv or wnv. tbev vaccines from two commercial manufacturers (baxter and chiron) are available and widely used, especially in austria and germany, both are based on formalin-inactivated virions, three injections are needed for full protection. booster immunizations are recommended every - years for people living or spending holidays in endemic areas. . . nairoviruses: crimean-congo hemorrhagic fever virus the genus nairovirus (family bunyaviridae) is composed of predominantly tick-borne viruses that have been divided into seven serogroups [ ] including several associated with severe human and livestock diseases (especially crimean-congo hemorrhagic fever virus (cchfv) and nairobi sheep disease virus). of the pathogenic viruses only cchfv causes significant human morbidity and mortality and is found in europe. like other members of the bunyaviridae family nairoviruses possess a tripartite single-stranded rna genome of negative polarity consisting of large (l), medium (m) and small (s) segments. the kb l segment encodes an rna-dependent rna polymerase (deduced size kda in cchfv), the . kb m segment encodes the precursor for the two envelope glycoproteins gn ( kda in cchfv) and gc ( kda in cchfv), and the . kb s segment the viral nucleocapsid n ( kda) ( table , fig. ). like with other members of the bunyaviridae viral messenger rnas (mrna) are not polyadenylated and are truncated relative to the genome rnas at the termini. messenger rnas have -methylated caps and - nontemplated nucleotides which are derived from host cell mrnas. the nairovirus l segment encoding the rna polymerase is conspicuously large, almost twice the size of many other members within the family bunyaviridae, and may thus provide other functions such as viral helicase and a papain-like cysteine protease activity predicted from the nucleotide sequence [ , ] . viral protein analysis has suggested that gn and gc may be derived from and kda precursors, of which the latter contains an n-terminal region with a highly o-glycosylated mucin-like domain [ ] . the cchfv nucleocapsid protein colocalizes and interacts with human mxa protein; this interaction may explain the antiviral effect of interferons on cchfv [ ] . similar to the hantavirus-rodent association, the high genetic variation of viruses of the genus nairovirus reflects the diversity of their predominant tick hosts [ ] . within cchfv isolates, comparison between m and s segment phylogenetic groupings suggests that reassortment events have occurred in some virus lineages [ ] . reverse genetics, recently established for cchfv [ ] , provides a unique opportunity to study the biology of nairoviruses and tailor optimal therapeutic and prophylactic measures against cchfv infections. cchfv is transmitted most efficiently by hyalomma ticks, followed by rhipicephalus, dermatocentor spp and many other species of ixodid (hard) and some argasid (soft) ticks (table ) . among ticks cchfv is capable of transmitting infection both transovarially and transstadially. the life cycle of cchfv also includes a tick-vertebrate host cycle involving both wild and domestic animals. the virus or antibodies against it have been detected in rodents, hares, hedgehogs and some birds but human infections seem to be principally from contacts with livestock (mainly cattle, sheep, goats) including in africa farmed ostriches and less often ticks [ , ] . thus cchfv is more commonly seen in persons exposed to blood and tissues of infected animals during occupational activities, such as farming, herding, veterinary examination and abattoir work. nosocomial infection is common and often results in small outbreaks. yet, outdoor and recreational activities also represent a risk factor. in addition, horizontal transmission of cchfv from mother to child may occur [ ] . the global distribution of cchf follows closely that of hyalomma spp. in the middle east, asia, africa and southeast europe [ , ] . sequence analysis of cchfv s rna segments gives patterns following links between different geographic locations, in some cases suggesting links originating from trade in livestock and long-distance carriage of virus or infected ticks during bird migration [ ] . in europe, cchf occurs in the balkan peninsula (albania, bulgaria, greece, turkey, yugoslavia) but also in southern russia, hungary, france and portugal [ , , ] . the symptoms and signs of crimean-congo hemorrhagic fever are similar to those of other viral hemorrhagic fevers [ , ] . the incubation period is generally short ranging usually from to days. there is typically a very sudden onset of illness with fever, rigors, chills, intense headache and backache or leg pains, myalgia, nausea, and vomiting. patients may also present with photophobia, somnolence and menigism with confusion or aggression. hemorrhages in the form of petecchiae, ecchymoses, epistaxis, melena and bleeding from various organs usually begin a few days later. tachycardia is common and lymphadenopathy is seen occasionally. the case-fatality rate is - %. the pathology consists of hemorrhagic and necrotic lesions in various organs as well as fibrin deposits. the methods used for detection of cchfv infection in humans or livestock include indirect immunofluorescence on virus-infected or n-expressing transfected cells. both igm and igg antibodies reactive for cchf appear within a week in patients [ , ] . alternatively a recombinant nucleocapsid protein-based enzyme immunoassay [ ] [ ] [ ] may be used. rapid detection and quantification of cchfv rna is possible by real-time reverse transcription pcr [ ] . ribavirin is clearly effective in treatment of crimean-congo hemorrhagic fever [ , ] but supportive treatment (blood, thrombocytes, coagulation factors) is equally important. immune plasma from recovered patients has also been used to treat patients but not in controlled studies with a follow-up of virus-neutralizing activities [ ] . the most effective method of preventing infections is to take measure to avoid exposure to ticks (protective clothing, tick repellent, frequent body searches to remove ticks). inactivated virus prepared in mouse brains has been used on a limited scale as a vaccine in southeastern europe and the former ussr but the sporadic and unpredictable occurrence of the disease renders it difficult to identify target populations and has slowed development of a safe and modern vaccine [ ] . orthobunyaviruses belong to the family bunyaviridae. these enveloped viruses have a three-segmented negative-strand rna genome. the . kb s segment encodes the - kda nucleocapsid protein (n) and a - kda nonstructural protein (ns); the . kb m segment encodes the polyprotein precursor of two glycoproteins, g and g ( - kda and - kda, respectively), and a nonstructural protein nsm ( ) ( ) ( ) ( ) ; and the . kb l segment encodes the rna polymerase ( kda) ( table , fig. ). viral messenger rnas are not polyadenylated and are truncated relative to the genome rnas at the termini. messenger rnas have -methylated caps and some nontemplated nucleotides which are derived from host cell mrnas. orthobunyaviruses are transmitted to humans by a variety of mosquito species, and in europe two known human pathogenic representatives of the genus circulate, tahyna virus and inkoo virus (mostly transmitted by aedes spp.) [ , , ] (table ). these viruses belong to the california serogroup, and their incidence is associated with the distribution of the carrier mosquitoes. the natural cycle of these viruses involve also mammal hosts. tahyna virus circulates throughout most of europe, whereas inkoo virus is mainly reported from northern europe [ , a] . tahyna virus causes an influenza-like disease which may also present as a meningitis. the infection caused by inkoo virus is mostly sub-clinical or a mild disease, although encephalitis has been reported. the most typical symptoms associated with encephalitis or meningitis caused by californian serogroup viruses include fever, headache, nausea, vomiting, convulsions and confusion. in europe the seroprevalence for californian serogroup viruses varies from % to %, depending on the geographic area [ ] . in finland the seroprevalence against inkoo virus varies from % to % (Å land islands) to - % (finnish lapland) [ , putkuri et al, unpublished results] . in disease-endemic areas the seropositivity of the population increases with age [ b]. the diagnosis of orthobunyaviruses is based on serology, either as a rise in igg-antibody titers, or the presence of igm antibodies. also rt-pcr methods are under development to detect viral rna in cerebrospinal fluid samples of patients with encephalitis. phleboviruses are one of the five groups within the family bunyaviridae. they have a negative singlestranded rna tripartite genome, namely: l ( . kb), m ( . - . kb) and s ( . - . kb). the l segment encodes a single protein of kda (rna polymerase) in the complementary sense. the m segment encodes a protein precursor of kda in the complementary sense which is cleaved into two glycoproteins g ( kda) and g ( kda). the s segment has an ambisense coding arrangement, and it encodes the nucleocapsid protein (n) of kda in the complementary sense and a non-structural protein (nss) of kda in the virion sense [ , ] (table , fig. ). viral messenger rnas (mrna) are not polyadenylated and are truncated relative to the genome rnas at the termini. mrnas have -methylated caps and some nontemplated nucleotides which are derived from host cell mrnas. when seen under the electron microscope the virions are spherical and enveloped and approximately - nm in diameter. glycoprotein projections of - nm are also seen embedded in the membrane. there are between and spikes per virion [ , ] . phleboviruses are arthropod-borne viruses and are transmitted mainly by phlebotomine sandflies, hence the derivation of its name ''phlebo'' (table ) . however, mosquitoes and ticks can also transmit phleboviruses. phleboviruses are distributed throughout most of the world but have not been reported in australia [ ] . the first documented record of a disease resembling those caused by phleboviruses dates back to the s in the mediterranean countries of europe [ ] . however, the first virus isolation occurred for rift valley fever virus in in kenya [ ] . retrospective serological studies have indicated that outbreaks caused by phleboviruses have occurred since in the sub-saharan africa region [ ] . phleboviruses are major causal agents of encephalitis in humans, especially among children, in mediterranean countries. they also have an important impact in livestock especially in african endemic areas. infection of livestock is characterised by a high rate of abortions and pathologies associated with hemorrhagic illness (leukopenia, thrombocytopenia, fibrin thrombi, intravascular coagulopathy, etc.) [ ] [ ] [ ] . the distribution of the disease follows that of its vector. however the natural history of phleboviruses is largely unknown and for many species the amplifying host has not been identified [ ] [ ] [ ] . in general, phlebovirus infections cause a - days illness characterised by an incubation period of - days followed by fever, general malaise, headache, photophobia, and back and joint pain [ ] . the illness occurs during the summer months when the activity of phlebotomine flies is high [ ] . there are three recognised members of the genus phlebovirus, associated with disease in humans. rift valley fever virus (rvfv), which is the type species of the genus and is transmitted by mosquitoes, causing an influenza-like disease that affects domestic animals and humans. the majority of human infections are asymptomatic; however, in . % of cases it can cause severe haemorrhagic fever, encephalitis and death. the geographical distribution of rvfv includes africa, egypt, saudi arabia and yemen and occurs as epizootics involving aedes spp. mosquitoes and domestic animals [ ] . toscana virus (tsv) causes a mild influenza-like disease that can occasionally develop into an acute neurological disease, such as meningitis or meningoencephalitis in italy and possibly other mediterranean countries. a similar virus (granada virus) was recently isolated in spain [ ] . tsv is the number one cause of encephalitis among children in italy. the distribution of the virus follows that of its vector, phlebotomus perniciosus, found in italy, spain, portugal, and cyprus. human cases have also been reported from southern france and greece. the virus replicates in the sandfly vector population; however, experimental evidence suggests that an amplifying vertebrate host is needed to sustain the virus in the arthropod population [ ] [ ] [ ] [ ] [ ] [ ] . sandfly fever virus (sfv) sicilian and naples viruses can cause a mild-influenza like disease. the distribution of sfv also follows that of its vector, phlebotomus papatasii, found in the mediterranean basin extending to the middle east and arabian peninsula, the caucasus mountains, pakistan and india [ ] [ ] [ ] . diagnostics of phlebovirus is performed mainly by igm-capture eia and ifa. the virus can be isolated using intracranial inoculation in suckling mice or using a susceptible tissue culture cell line (vero, llc-mk , bhk- ) [ , a] . toscana virus and naples sandfly fever virus group together and are distinct antigenically and phylogenetically from sicilian sandfly fever virus [ b] . prevention of infections by phleboviruses varies depending on the type species. immunising livestock with the formalin-inactivated or live-attenuated vaccines can prevent rvfv by avoiding epizootics and hence human infections [ , ] . in the case of tsv and sfv, prevention is limited to controlling the vector population through insecticides and repellents [ , ] . what are the reasons for the appearance of all these emerging and re-emerging viruses? have changes taken place in the viruses themselves? apparently not. the principal reasons are changes in the environment and human activity that have created new attacks with nature. factors such as climate change, increasing intensity of agriculture, the building of dams and introduction of other irrigation measures which generate new breeding sites for mosquitoes, crises and wars which bring rodents and arthropods closer to humans, population growth and especially in suburban slums, and global air traffic are important factors generating such new contacts, or may affect the ecological cycles of these viruses. many epidemics may be unexpected outcomes of such changes. moreover, the new molecular biology techniques facilitate detection of new viral agents. many prevention and control measures, such as the anti-aedes 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to explore how different diets may affect human brain development and if genetic and environmental factors play a part. design: cohort study. setting: uk biobank data were collected from centres across the uk. participants: only white british individuals free of alzheimer's or dementia diseases were included in the study, where participants had quality-controlled genetic data, and participants had qualified brain mri data. main outcome measures: grey matter volume, intake of cereal and coffee, body mass index and blood cholesterol level. results: we investigated diet effects in the uk biobank data and discovered anti-correlated brain-wide grey matter volume (gmv)-association patterns between coffee and cereal intake, coincidence with their anti-correlated genetic constructs. these genetic factors may further affect people's lifestyle habits and body/blood fat levels through the mediation of cereal/coffee intake, and the brain-wide expression pattern of gene cplx , a dedicated marker of subplate neurons that regulate cortical development and plasticity, may underlie the shared gmv-association patterns among the coffee/cereal intake and cognitive functions. conclusions: our findings revealed that high-cereal and low-coffee diets shared similar brain and genetic constructs, leading to long-term beneficial associations regarding cognitive, bmi and other metabolic measures. this study has important implications for public health, especially during the pandemic, given the poorer outcomes of covid- patients with greater bmis. increases in human brain volume, due to growth, begin at an early stage of embryonic development and continue until late adolescence . after this, the brain experiences a persistent but slow decrease in size throughout adulthood . generally, development is tissue-specific but systematically organized across the brain and may be susceptible to both genetic and environmental influences - , as well as their interactions, e.g. through epigenetic modifications . diet is a common environmental factor that can influence the trajectory of brain size. for example, a lack of nutrients over an extended period of time causes both structural and functional damage to the brain , and improved diet quality is associated with larger brain volumes . furthermore, evidence suggested that ingested substances (both food and drink) in well-fed and healthy adults may also cause changes in brain size. for example, in a small-scale study, an increase in the size of the hippocampus was inferred to have occurred as an effect of both low and high coffee consumption . while there are extensive studies of the degree to which different diets affect the body - , there is an absence of systematic investigation into how different diets may affect the human brain in both the short and long term. thus, it is not known if impacts of different diets on brain structures follow similar patterns, or whether different brain regions exhibit differential sensitivity to diet and other environmental factors. in addition, there is a lack of knowledge about whether genetic factors play any role in the sensitivity of the brain to environmental factors. in the present work, we provide a detailed analysis of brain-size changes that occur in healthy adults due to the ingestion of different common foods and drinks to investigate whether these influences from diets were . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint systematically organized across the brain, whether these diets influences have underlying genetic factors, and whether these genetic factors have further implications in people's daily activities, metabolism and cognitive functions. study samples were from the uk biobank study, a prospective epidemiological study that involves over , individuals in centres across the uk . between and , approximately . million mailed invitations to participate in the survey were sent to people in the national health service registry who were aged - years and living < miles from a study centre. participants were recruited to collect a range of questionnaires about detailed phenotypic information including diet, lifestyle, anthropometric and cognitive function assessments, biological samples, including blood and medical records obtained from the nhs registries. since , a subsample of the original population has been invited back to collect magnetic resonance imaging of body and brain, and questionnaires about diet, lifestyle, and cognitive function assessments. in the current study, we used data collected at both recruitment and mri scan. the original sample comprised individuals ( . ± . years; . % women). we included white british individuals and then excluded individuals who were diagnosed with alzheimer's or dementia defined by codes g /f in the th edition of the international classification of diseases (icd- ). of the individuals, individuals had quality-controlled genetic data, and individuals had available brain mri data. table s summarized relevant demographic information. behavioural and neuroimaging data collection and protocol are publicly available on . all participants provided written informed consent to uk biobank. data access permission was granted under ukb application (pi jianfeng feng). dietary information was obtained from the touchscreen questionnaire at the baseline and the mri scan appointment. cereal intake was the number of bowls of cereal the participants consumed per week. the types of cereal included bran cereal, biscuit cereal, oat cereal, muesli, and other types (e.g., cornflakes, frosties). coffee intake was the number of cups of coffee the participants drank per day. the types of coffee included decaffeinated coffee, instant coffee, ground coffee, other types of coffee. detailed information can be found in supplementary materials. lifestyle phenotypes included physical activity, sleep, smoking and alcohol and were obtained from the touchscreen questionnaire at the baseline appointment. physical activities were assessed using met (metabolic equivalent task) scores derived based on international physical activity questionnaire) of total physical activity (including walking, moderate, and vigorous activity) and usual walking pace. the time spent watching television was also included to reflect physical activity. sleep data included information for sleep duration, morningness or eveningness type, insomnia symptoms, daytime dozing, getting up in morning, and nap during day. smoking status included smoking history and the number of cigarettes currently smoked daily. alcohol intake was examined using frequency and amounts of alcohol drinking. detailed description can be found in supplementary materials. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint cognitive function performances were examined at the baseline and the mri scan appointment. the cognitive tests included fluid intelligence score, reaction time, numeric memory, pairs matching, prospective memory, matrix pattern completion, symbol digit substitution and trail making. detailed descriptions of procedures can be found in supplementary materials. body mass index was calculated from the participant's measured weight (kg)/height (m ). cholesterol, high-density lipoprotein (hdl) cholesterol, low-density lipoprotein (ldl) cholesterol, and triglycerides were measured in the blood sample collected at recruitment. we used a proxy phenotype for alzheimer's disease (ad) case-control status derived from the genetic risk index for ad based on parents' diagnoses as suggested in a previous study . the proxy phenotype ranged approximately from to , with values near zero when both parents were unaffected (lower for older parents and possible values below zero if both parents were over age ) and values of two when both parents were affected. covid- test results data are linked to uk biobank by public health england (phe). data were available for the period th march to rd august . data provided included specimen origin (hospital inpatient indicating severe covid- vs. other settings). detailed information is available on the website (http://biobank.ndph.ox.ac.uk/ukb/exinfo.cgi?src=covid _tests). to focus on the covid- , we excluded individuals passed away except for those who had positive test results. there were unique test results available, of which ( . %) were positive; ( . %) tests were conducted on inpatients; ( had available data on bmi and diet) were inpatients and positive. detailed structural mri data collection and acquisition procedures can be found in supplementary materials. all uk biobank structural mri data were preprocessed in the statistical parametric mapping package (spm ) using the vbm toolbox with default settings, including the usage of high-dimensional spatial normalization with an already integrated dartel template in montreal neurological institute (mni) space. all images were subjected to nonlinear modulations and corrected for each individual head size. images were then smoothed with a mm full-width at half-maximum gaussian kernel with the resulting voxel size . mm . the estimated total intracranial volume (tiv) covariate, were calculated as the summation of the grey matter, white matter, and csf volumes in native space. the automated anatomical labelling (aal ) atlas , which partitioned the brain into regions of interest, was employed to obtain the total brain grey matter volume and region-wise grey matter volume. t images were successfully preprocessed, and the grey matter volumes of the aal atlas of the discovery sample were extracted. the majority of participants were assessed in the cheadle mri site ( . %) and the rest in the newcastle site ( . %). detailed genotyping and quality control procedures of the uk biobank can be found in supplementary materials or http://biobank.ctsu.ox.ac.uk/. in this study, we performed stringent qc standards by plink . . single-nucleotide polymorphisms (snps) with call rates < %, minor . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint allele frequency < . %, deviation from the hardy-weinberg equilibrium with p< e- were excluded from the analysis. in addition, we selected subjects that were estimated to have recent british ancestry and have no more than ten putative third-degree relatives in the kinship table using the sample quality control information provided by ukb. for more details, we refer to the official document for genetic data of the ukb (http://www.ukbiobank.ac.uk/scientists- /genetic-data/). after the quality control procedures, we obtained a total of , snps and participants. we followed the ahba preprocessing pipeline suggested by arnatkevic̆iūtė et al. and using the same pipeline as shen et al. , including probe-to-gene re-annotation, data filtering, probe selection. in the next step, we separated the samples into the areas based on their mni coordinates, using the automated anatomical labelling (aal ) atlas and excluding the samples located outside of the grey matter defined by this atlas. to control for the inter-individual differences, we conducted two within-donor normalizations. the expression data were first normalized within-sample and acrossgene and then normalized across samples. one gene failed the normalization and therefore was deleted, resulting in , genes. we used the mean expression of samples located in the brain region and the mean expression in the brain region of all subjects as the gene expression in each brain region defined by aal atlas . we conducted linear regression analysis to test the pairwise associations between the diet phenotypes and the total and regional grey matter volume (gmv), respectively. the covariate variables included were age at imaging scan, sex, imaging sites (dummy variable), and total intracranial volume (tiv). to further understand the biological insights of the shared variants of cereal intake and coffee intake, we performed linear regression analysis to examine the pairwise associations between the independent lead snps and other diets, lifestyle, and body/blood fat covarying age, sex, the top genetic principal components. we also performed linear regression analysis to examine the pairwise associations between the cereal/coffee intakes, other diets, lifestyle, and cholesterol covarying age, sex. we performed genome-wide association analysis adjusting for age, sex, and the top ancestry principal components using plink . to assess the association between phenotype and genotype on cereal intake and coffee intake separately. after association analysis, we employed the fuma online platform (version . . , http://fuma.ctglab.nl/) to define genomic risk loci. the gwas summary statistics was submitted as input. fuma identifies significant variants with p value less than e- that were largely independent of each other (r < . ). based on the clumping of the independent significant variants (r < . ), independent lead variants were obtained. shared lead snps of cereal and coffee were mapped to genes based on cis-eqtl (p value≤ . ) in the brain using database gtex v with fuma . the eqtl mapping assigned snps to genes up to mb apart. the ldsc software (https://github.com/bulik/ldsc) was employed to estimate the heritability of cereal intake and coffee intake as well as their genome-wide genetic correlation . we used the . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint pre-calculated ld scores using genomes european data. we used the overlap of summary statistics variants and hapmap variants as recommended . mediation effects were examined using baron and kenny's ( ) causal steps approach. the causal steps approach involved four steps to establishing mediation. firstly, a significant relation of the independent variable to the dependent variable is required in = ! + + ! ( " : = ). secondly, a significant relation of the independent variable to the hypothesized mediating variable is required in = # + + # ( " : = ). thirdly, the mediating variable must be significantly related to the dependent variable when both the independent variable and mediating variable are predictors of the dependent variable in = $ + ′ + + $ ( " : = ) fourthly, the coefficient relating the independent variable to the dependent variable must be larger (in absolute value) than the coefficient relating the independent variable to the dependent variable in the regression model with both the independent variable and the mediating variable predicting the dependent variable ( . . | | > | ′|). to further evaluate the p-value of the significant mediation identified by the above process, we performed times bootstrap of the individuals to obtain the distribution of the proportion of the mediation, i.e., = (| | − | % |)/| |, under the alternative hypothesis. thus, the pm was expected to be positive by definition, and the corresponding p-value could be calculated as the doubled chance of observing the pm less than zero during the bootstrap procedure. as no priory assumption about whether diet or lifestyle/ blood and body fat levels should serve as the mediator for their associations with the lead snps, we, therefore, identified the most likely mediator with an excess pm, i.e., the model showing higher pm, of which the significance level was again evaluated through a -bootstrap process. we examined the similarity among the brain-wide gmv-association patterns of cereal/coffee intake and cognitive functions. specifically, we first performed association analyses between region-wide gmv and each phenotype. then, we calculated the pearson correlation coefficient (similarity) between the gmv-association patterns of a pair of phenotypes of interest, of which the significance level was evaluated through times permutation that shuffled the individual's ids of the gmv data at each iteration. the similarity between brain-wide gmv-association pattern of a given phenotype and the brainwide gene-expression pattern was also examined through their pattern correlation, of which the null distribution was established through times permutation that at each iteration, the pattern correlation was re-calculated with the gmv-association patterns been regenerated with shuffled ids of the gmv data. the corresponding p-values were hence calculated as the chance of randomly getting a higher pattern correlation than the observed one in terms of their absolute value based on the established null distribution. the above permutation process was employed to ensure that the potential oversampling of brain regions will not inflate the false positive rate. we used anonymised data collected by uk biobank study. no patients were involved in setting the research questions or the outcome measures. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint we first investigated the relationship between grey matter volume (gmv) and different diet phenotypes, which were both measured at the second visit (i.e. at follow-up) of participants to a research center . we found that the total grey matter volume of the brain (tgmv) is affected by diet. some dietary items were negatively correlated with the tgmv, thus decreasing consumption of these items had the tendency to increase the tgmv, while other items were positively correlated, and had the opposite tendency on the tgmv. with a statistically significant correlation (p< . bonferroni corrected), intake of coffee, water, processed meat, beef, lamb/mutton and pork were found to be negatively correlated with tgmv, while intake of cereal and dried fruit were positively correlated with tgmv (see table s ). we note that predated measurements (i.e., baseline measurements) of cereal and coffee intake were also related to the follow-up values of tgmv (table s ) , and these remained significant even after controlling for the corresponding follow-up intakes (table s ). this indicates a persistent, rather than a short-term connection between diet and gmv. table s and s , based on an alternative way of measuring tgmv, obtained similar results, confirming the methodological stability of the above findings. correlations were further investigated between diet phenotypes and the volumes of brain regions defined by the automated anatomical labelling (aal ) atlas . a total of statistically significant correlations (bonferroni correction: p< . / / ) were found, again mainly between gmv and intake of cereal, coffee, water, dried fruit, processed meat, beef, pork and lamb/mutton (fig. .a and table s ). it is interesting to note that the gmv-association pattern of cereal intake highly resembles, although in the opposite direction, the gmv-association pattern of coffee intake (pattern correlation across the whole brain: r=- . , pperm< e- based on -permutation; fig. .b). we conducted genome-wide association studies (gwas) for the intake of both cereal (n= ) and coffee (n= ) at baseline and identified and independent lead genome-wide significant variants with p< e- (i.e., the lead snps) respectively (fig. , table s &s and fig.s ). a linkage disequilibrium (ld) score regression analysis indicates that both findings were free from systematically inflated false-positive rates, e.g., due to population stratification, with intercepts of . (cereal intake) and . (coffee intake), and the corresponding snp-based heritabilities were estimated as . (se= . ) and . (se= . ) respectively. furthermore, we observed a significant negative genetic correlation between intake of cereal and coffee (rg=- . , se= . , z-score=- . , p= . e- ), i.e., the alleles associated with higher cereal intake were likely to be in association with reduced coffee intake, which is in line with the above gwas findings, where the three shared lead snps, i.e. rs , rs and rs , were found in associations with both cereal and coffee intake, again in opposite directions (table s ) . the minor c-allele at rs was associated with a higher intake of cereal (regression coefficient . , % confidence interval . to . , tdf= = . , p= . e- ) and a lower intake of coffee (regression coefficient - . , % confidence interval - . to - . , tdf= =- . , p= . e- ). the minor c-allele at rs was associated with lower intake of cereal (regression coefficient - . , % confidence interval - . to - . , tdf= =- . , p= . e- ) and higher intake of coffee (regression coefficient . , % confidence interval . to . , tdf= = . , p= . e- ). the minor t-allele, at rs , was associated . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint with lower intake of cereal (regression coefficient - . , % confidence interval - . to - . , tdf= =- . , p= . e- ) and higher intake of coffee (regression coefficient . , % confidence interval . to . , tdf= = . , p= . e- ). while rs and rs have both been previously associated with coffee/caffeine consumption - , caffeine metabolism , and alcohol consumption , this is the first study to identify an association with cereal intake. it is notable that snps rs (the c-allele) and rs (the t-allele) were also strongly associated with higher intake of tea (regression coefficient . , % confidence interval . to . , tdf= = . , p= . e- for rs ; regression coefficient . , % confidence interval . to . , tdf= = . , p= . e- for rs , respectively) and lower intake of water (regression coefficient - . , % confidence interval - . to - . , tdf= =- . , p= . e- ; regression coefficient - . , % confidence interval - . to - . , tdf= =- . , p= . e- , respectively) (fig. .a and table s ), although both intakes were not observed with significant long term impacts on the tgmv (table s ). this result is remarkable because there is a median to large anti-correlation between the intake of coffee and tea (r=- . , regression coefficient - . , % confidence interval - . to - . , tdf= =- . , p< . e- ), which is likely due to the seesaw effect given the limited amount of beverages one may consume each day. thus, individuals with both snps (i.e., c-allele of rs and t-allele of rs ) might generally prefer flavoured beverages to the water. as both cereal and coffee intake, as well as their shared lead snps, were associated with different table s & s ), we then investigated possible mediation roles of diet or/and lifestyles on their associations with snps. as no prior assumptions about whether diet or lifestyle should serve as the mediator for their associations with the lead snps, we evaluated the most likely mediator, based on the corresponding proportion of mediation (pm) that they are responsible for (see the supplementary material for more details). we found the following: ) both intake of cereal and coffee were likely to mediate the positive association of the frequency of alcohol intake with the t-allele of rs (pm= . %, pbootstrap= . e- and pm= . %, pbootstrap= . e- respectively; table s ); these were superior to alternative mediation models with the frequency of alcohol intake as the mediator (excess pm> % with pbootstrap< . for both alternative models, fig. .b and table s ; see supplementary materials for detailed analyses); ) the association between higher t-alleles of rs and less daytime sleeping was mediated by cereal intake (pm= . %, pbootstrap= . e- ; fig. .b and table s ), which was superior to the alternative mediation model with daytime sleeping as the mediator (excess pm= . % with pbootstrap= . , table s ; see supplementary materials for detailed analyses); ) both difficult in rising and less daytime sleeping were found to mediate the negative association of cereal intake with the c-allele of rs , so did the alternative mediation models with the cereal intake as the mediator. however, neither group of mediation models was superior to the other (table s ) ; ) interestingly, while individuals with rs (the c-allele) were more likely to be an 'evening person' and experience difficulties in rising, both lifestyle traits did not mediate the associations of . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint the snp with higher cereal intake or lower coffee intake (nor did the alternative mediation models), which was mainly due to nonconcordant correlations, e.g., a positive correlation was observed between ease in rising and higher cereal intake while a negative one was expected (fig. table s , s & s ). in addition to lifestyle, both cereal and coffee intake, as well as their shared lead snps, were also associated with blood (for example with total cholesterol, r=- . , p< . e- for cereal and r= . , p= . e- for coffee) and body fat levels (for example with the body mass index (bmi), r=- . , p< . e- for cereal and r= . , p= . e- for coffee) (table s , table s ). therefore, we further explored possible mediator roles of fat levels and the intake of cereal and coffee. we found the following: ) associations between rs (c-allele) and: an increased body mass index (bmi), triglycerides and decreased hdl cholesterol, were mediated by increased coffee intake (pm= . %, pbootstrap= . e- , pm= . %, pbootstrap= . e- , and pm= . %, pbootstrap= . e- , respectively), which were superior to the alternative mediation models with corresponding fat levels as mediators (excess pms= . %, . % and . %, respectively; all corresponding pbootstrap< . ) (table s ) ; ) associations between rs (t-allele) and higher body mass index (bmi), total cholesterol, and ldl cholesterol, were mediated by higher coffee intake (pm= . %, pbootstrap= . e- , pm= . %, pbootstrap= . e- and pm= . %, pbootstrap= . e- , respectively), as well as by lower cereal intake, to a lesser extent (pm= . % for total cholesterol, pbootstrap= . e- and pm= . % for ldl cholesterol, pbootstrap= . e- ). the above models were superior to alternative mediation models with corresponding fat levels as mediators (for the coffee intake: excess pms= . %, . % and . %, respectively, with all corresponding pbootstrap< . ; for the cereal intake: excess pms= . % and . %, respectively, with all corresponding pbootstrap< . ) (table s ) . related to the current covid- pandemic, using the uk biobank data we found that individuals who tested positive of covid- (n= , inpatients only) had higher bmis (cohen's d= . , t= . , p= . e- ) and lower cereal intake (cohen's d=- . , t=- . , p= . ) than the rest population (n= , either tested negative or not tested). this further highlights the importance of our finding for public health that cereal intake is associated with lower bmis. to further characterize the negatively correlated brain-wide gmv-association patterns for cereal and coffee intakes, we further investigated if such similarities have any implications for cognitive functions, and we found that brain-wide gmv-association patterns of most cognitive functions were significantly correlated with those of both cereal and coffee intake, although in opposite directions, at both baseline and follow-up (gmv were measured at follow-up only). in particular, performance in tasks of matrix pattern completion, symbol digit substitution, and numeric and alphabet-numeric trail making showed similar brain-wide gmv-association patterns with both cereal (in positive correlation) and coffee (in negative correlation) intake at both baseline and follow-up (|r|min= . , all pfdr< . ; fig. and table s &s ), while the fluid intelligence score only showed a similar brain-wide gmv association pattern with the cereal intake (at both . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint baseline and follow-up; rmin= . , all pfdr< . ; table s &s ). in line with the above findings, higher risk of alzheimer's disease (estimated as the proxy-ad ), characterized by reduced cognitive functions, was associated with reduced cereal intake (r=- . , p= . e- ), as well as increased coffee intake to a much lesser extent (r= . , p= . ), in contrast to previous findings of either protective or non-significant effect of high coffee intake on alzheimer's disease. we further investigated if the identified putative genetic variants may also contribute to observed similarities of brain-wide gmv association patterns between diet and cognitive function, through the expression of candidate genes. we first performed eqtl mapping of the lead snps using software fuma and identified candidate protein-coding genes (table s ) that also have brain-wise gene expression information from the allen institute for brain science (aibs) . after also mapping to the aal atlas, the brain-wide expression pattern for each candidate gene (i.e. the mean expression level across all aibs individuals for each brain region) was then correlated with the brain-wide gmv association patterns for cereal and coffee intakes. while multiple candidate genes had their brain-wide expression pattern in significant correlation with brain-wide gmv associations patterns for the coffee intake (table s ) , only gene cplx showed significant 'gene-expression vs gmv-association' pattern similarity with both intakes of cereal (r= . , pperm= . e- , pfdr-corrected= . ) and coffee (r=- . , pperm= . e- , pfdr-corrected= . ). it is of particular interest that the gene-expression of cplx (a known prominent marker that is specific for subplate neurons that regulate cortical development and plasticity across the brain - and also respond to both light and electrical stimuli in retinal neurons ) also showed significant pattern correlations with almost all cognitive functions (i.e., r= . for fluid intelligence, r= . for numerical memory, r= . for prospective memory, r= . for matrix pattern completion, r= . for symbol digit substitution, and r= . /r= . for both trail making tasks; all corresponding pfdr-corrected< . ; table s ). in the large-scale imaging/genetics analysis presented in this work, we have: (i) gained insights into long-term associations between brain-wide gmv and diets, especially the anti-correlated impacts from cereal and coffee intake, (ii) identified shared genetic constructs for both higher cereal and lower coffee intake, and explored the complex relationship among cereal/coffee intake, their genetics constructs, lifestyle, and body/blood fat level, (iii) revealed shared brain-wide gmvassociation patterns between cognitive function and the intake of cereal and coffee and further showed that such similarity might be underlaid by the brain-wide expression patterns of gene cplx , a shared genetic determinant identified for the intake of cereal and coffee. these novel findings hence suggest the existence of a brain-wide systematic organization of gmv that is susceptible to both genetic and environmental influences, which may have further impacts on people's lifestyles, cognitive functions, and metabolic measures (e.g. bmi and blood cholesterol level). two lead snps shared by the intake of coffee and cereal, i.e. rs and rs , have been previously associated with coffee/caffeine consumption - , caffeine metabolism , and alcohol consumption . however, this is the first study to identify their associations with cereal intake. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint moreover, while cplx , within the ld complexity around the lead snp rs along with other genes (based on r package "biomart" ), has previously been proposed as a candidate gene for both coffee consumption and blood pressure , it is the first time that this gene has been linked with multiple cognitive functions, such as intelligence, as well as with cereal intake. remarkably, the expression of cplx is a highly specific marker of subplate neurons that regulate cortical development and neuronal plasticity across the brain . specifically, while most subplate neurons were short-lived during the development of the brain, previous studies have shown that the cplx -positive subplate neurons could survive into adulthood in mice . therefore, our findings were not only congruent with the role of subplate neurons in the cortical development, but with further implications that these cplx -positive subplate neurons might mark the dynamic system of gmv in the brain that is susceptible to environmental factors. such a hypothesis could be supported by previous findings that cplx protein's regulation of exocytosis in mice retinal neurons could be altered by both light and electrical stimuli . uk biobank only includes participants aged and above. therefore, some of our results may not necessarily reflect the situation in the younger population, although the gwas findings are highly consistent with previous literature across different age bands, which may help to alleviate such a concern. in addition, further studies are still needed to fully understand the molecular and metabolic pathways involved in the systematic alternation of gmvs in the brain, as well as its influences on cognitive function and metabolism. since high cereal diets, but low coffee diets, have long-term beneficial associations regarding the brain, cognition, bmi and other metabolic measures, this study has significant implications for public health. our findings highlight the importance of a 'cereal' breakfast across the life span, but perhaps especially for children and adolescents whose brains are still in development and for all uk biobank data used in this work were obtained under data access application and are available to eligible researchers through the uk biobank (www.biobank.ac.uk). gene expression data from the allen institute for brain science are freely available at https://human.brain-map.org/static/download. custom code that supports the findings of this study is available from the corresponding author upon request. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint tables s -s . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint fig. . correlations between grey matter volume (gmv) and different daily diets. (a) circular heatmap of correlations between gmvs of brain regions from aal (the outer layer) and different diets (along radius). as indicated by the colour bar, positive correlations were highlighted in red while negative correlations were highlighted in blue. the inner layer indicates the lobes that brain regions belong to. (b) brain regions with significant correlations between their gmvs and the intake of cereal (upper) and coffee (middle), as well as the overlapped significant regions (bottom). sm: sensorimotor. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint fig. . manhattan plots of the genome-wide association results for the intake of cereal (upper) and coffee (bottom). the gray line indicates the genome-wide significance level (i.e. p-value= e- ). variants with significant associations with both cereal and coffee intake were highlighted with red dots. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint fig. . scatter plots of brain-wide gmv-association patterns of cognitive functions and the intake of cereal (upper) and coffee (bottom). each dot represents one of aal brain regions, where the colours indicate at which lobes the brain regions were located. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint cognitive development and aging brain structural trajectories over the adult lifespan morphometry and development: changes in brain structure from birth to adult age development and aging of cortical thickness correspond to genetic organization patterns genetic architecture of subcortical brain structures in , individuals heritability of regional brain volumes in large-scale neuroimaging and genetic studies epigenome-wide meta-analysis of blood dna methylation and its association with subcortical volumes: findings from the enigma epigenetics working group coffee consumption and health: umbrella review of meta-analyses of multiple health outcomes uk biobank: an open access resource for identifying the causes of a wide range of complex diseases of middle and old age multimodal population brain imaging in the uk biobank prospective epidemiological study genome-wide meta-analysis identifies new loci and functional pathways influencing alzheimer's disease risk a new spm toolbox for combining probabilistic cytoarchitectonic maps and functional imaging data automated anatomical labelling atlas plink: a tool set for whole-genome association and population-based linkage analyses a practical guide to linking brain-wide gene expression and neuroimaging data what is the link between attention-deficit/hyperactivity disorder and sleep disturbance? a multimodal examination of longitudinal relationships and brain structure using large-scale population-based cohorts functional mapping and annotation of genetic associations with fuma the genotype-tissue expression (gtex) pilot analysis: multitissue gene regulation in humans an atlas of genetic correlations across human diseases and traits association studies of up to . million individuals yield new insights into the genetic etiology of tobacco and alcohol use coffee consumption and health: umbrella review of meta-analyses of multiple health outcomes coffee consumption and risk of dementia and alzheimer's disease: a dose-response meta-analysis of prospective studies an anatomically comprehensive atlas of the adult human brain transcriptome molecularly defined subplate neurons project both to thalamocortical recipient layers and thalamus subplate neurons: crucial regulators of cortical development and plasticity subplate neurons regulate maturation of cortical inhibition and outcome of ocular dominance plasticity complexin increases the fidelity of signaling in a retinal circuit by regulating exocytosis at ribbon synapses functional roles of complexin and complexin at mouse photoreceptor ribbon synapses mapping identifiers for the integration of genomic datasets with the r/bioconductor package biomart genome-wide association analysis of coffee drinking suggests association with cyp a /cyp a and nrcam genetic analysis of over million people identifies new loci associated with blood pressure traits high prevalence for obesity in severe covid- : possible links and perspectives towards patient stratification all authors have completed the icmje uniform disclosure form and declare: no support from any organisation for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years, no other relationships or activities that could appear to have influenced the submitted work. key: cord- -mf pn authors: weber, stefanie; ramirez, christina; doerfler, walter title: signal hotspot mutations in sars-cov- genomes evolve as the virus spreads and actively replicates in different parts of the world date: - - journal: virus res doi: . /j.virusres. . sha: doc_id: cord_uid: mf pn severe acute respiratory syndrome coronavirus- (sars-cov- ) was first identified in wuhan, china late in . nine months later (sept. , ), the virus has infected > million people world-wide and caused > . ( . %) fatalities in countries and territories. research on the genetics of the sars-cov- genome, its mutants and their penetrance can aid future defense strategies. by analyzing sequence data deposited between december and end of may , we have compared nucleotide sequences of sars-cov- genomes from china, europe, the us, and india to the sequence of the wuhan isolate. during world-wide spreading among human populations, at least distinct hotspot mutations had been selected and found in up to > % of viral genomes. many of these mutations led to amino acid exchanges in replication-relevant viral proteins. mutations in the sars-cov- genome would also impinge upon the secondary structure of the viral rna molecule and its repertoire of interactions with essential cellular and viral proteins. the increasing frequency of sars-cov- mutation hotspots might select for dangerous viral pathogens. alternatively, in a . nucleotide-genome, there might be a limit to the number of mutable and selectable sites which, when exhausted, could prove disadvantageous to viral survival. the speed, at which novel sars-cov- mutants are selected and dispersed around the world, could pose problems for the development of vaccines and therapeutics. the coronavirus disease pandemic has presented unusual challenges to genetic and virological analyses [fauci et al. ; na zhu et al. ; qun et al. ]. one of the major scientific problems confronted with by the sars-cov- pandemic lies in our limited understanding of the interactions between the viral and the human host genomes and the latter's defense mechanisms against this pathogen. the frequency of new mutations in viral genomes depends on a multitude of viral and host factors which determine mutant selection. nucleic acid sequence and the presence of repair mechanisms in the viral genome, the secondary and tertiary structures of the viral genome; the intensity of viral replication, the host's genetically determined defense mechanisms, environmental factors, like ambient temperature, uv radiation, among many unidentified factors, all contribute to the stability or instability of viral genomes. sars-cov- , like sars- cov- ( / ), and mers-cov ( , which were also responsible for human severe acute lung diseases, belongs to the group of beta-coronaviruses. human beta-coronaviruses oc or hku cause less severe seasonal upper respiratory tract infections. coronaviruses carry plus-strand rna genomes of between , and , nucleotides in length, the largest genomes among rna viruses [helmy et al. ; coronavirus disease pandemic, ] . during viral replication, a virus-encoded exonuclease and additional non-structural proteins form a replication complex with the viral rna-dependent-rna polymerase (rdrp) to generate new virion-packaged genomes [hartenian et al. ; subissi et al. ; wang et al. ]. this complex functions in proof-reading and corrects copying errors by the viral rdrp [subissi et al. ] . for sars-cov- , this proof reading mechanism is still under study. the debate that during sars-cov- rna replication the generation of mutants remains low in comparison to other plus-strand rna viruses, has not been resolved. however, for our understanding of this epidemic, it will be more relevant to research how efficiently sars-cov- rna mutants are selected and become dominant in the course of the covid- pandemic. mutations affecting the secondary and tertiary structures of sars-cov- rna might be the most relevant ones when considering selection for viral survival among human populations. there are unique aspects of sars-cov- genetics and mutagenesis in that the virus managed to jump very recently from mammalian to human hosts and thereafter expanded with unprecedented speed among a world population of almost billion who live under vastly different geographic and socioeconomic conditions. the growing distance from that critical animal-to-human transition point and the chance for rapid propagation under an ensemble of environmental factors and human genetic backgrounds has enabled sars-cov- genomes to select for replication-efficient mutations. the plasticity of the sars-cov- genome has enabled us to study the frequency of occurrence, the types of nucleotide exchanges and the selection of hotspot mutations in sars-cov- genomes from isolates that were collected on different continents between december and the end of may . [ncbi: https://www.ncbi.nlm.nih.gov/genbank/sars-cov- -seqs/]. most observed mutations were point mutations and were found once or a few times among a large number of isolates and may not be significant. there arose, however, strongly selected mutations which show predominant representation and increase in frequency as sars-cov- explosively replicates in human populations with different geographic, socioeconomic, climatic, and genetic backgrounds. the sars-cov- genome sequences, which were analyzed for sequence variations, were randomly selected from the ncbi databank "sars-cov- (severe acute respiratory j o u r n a l p r e -p r o o f syndrome coronavirus- ) sequences"(https://www.ncbi.nlm.nih.gov/genbank/sars-cov- seqs/). the nucleotide sequences included in the comparisons shown in tables s to s had been deposited in the data bank as follows: table s -china: december , to march , table s -europe: january to may , table s -germany: february to march , table s -usa i: february to april , table s -usa ii: june to july , table s -india: january to may , nucleotide sequences from china, europe, the us and india were compared to the reference genome of the sars-cov- isolate form wuhan-hu- , ncbi reference sequence: nc_ . . the program vector nti advance™ (invitrogen™), tool align x was used for the alignment of sequences from the us and germany. nucleotide sequences of isolates from china, india, and europe were analyzed with the program snapgene (gsl biotech) by using the algorithm muscle (multiple sequence comparison by log-expectation). amino acid sequences were also analyzed with the program snapgene. dna sequence analyses of reverse-transcripts of an rna genome will have to be considered with oversight. errors could have been introduced at several steps, e.g., by preferred reading mistakes of the reverse transcriptase due to specific sequence or structural properties of sars-cov- rna. we have tried to overcome this obvious complication by analyzing a large number of genomes. more specifically, the absence of the distinct hotspot mutations in the majority of sequences from samples isolated in china, convincingly argues against the possibility of technical problems during the generation of sars-cov- nucleotide sequences. in some of the statistical investigations on the data presented, notably comparisons of mutation frequencies between sample collections usa-i and usa-ii, the permutation test as well as the kolmogorov-smirnov test were applied. all sequence alignments performed in our laboratory have been stored and are available to inspection on request. the alignments are also accessible via "google drive" [https://drive.google.com/drive/folders/ fuoqjv_cd_gceaiwh iiq gwwsjfoqoi?usp=sha ring]. we have investigated whether and to what extent mutants of the sars-cov- rna sequence of , nucleotides are selected as the virus spreads around the world. and predominate in human populations with different geographic, societal, and genetic backgrounds at the time of beginning our analyses, about . nucleotide sequences of sars-cov- had been published of which were randomly selected and compared to the reference sequence of the wuhan isolate from late (ncbi reference sequence: nc_ . ). figure was reproduced here as an example of part of the sars-cov- nucleotide sequence at around position and displays viral isolates from different geographic regions (lines - ). this sequence was aligned for comparison with the sequence of the wuhan sars-cov- isolate (line ). in of selected sequences, the ggg sequence of the wuhan sequence (in blue) was mutated to aac (in white). additionally, in sequence positions and a few c → t point mutations were apparent. as in this example, all mutations described in this report were identified by inspection and comparison of individual sequences to the wuhan reference. the results of all sequence comparisons were presented as supplemental materials in tables s to s and summarized in table . we hypothesize that signal hotspot mutations, in particular those identically noted in different populations, have functional significance and have been selected for advantages during active viral replication. the possible impact of these mutations on heightened pathogenicity will require further study. according to the best information available, the world-wide spread of sars-cov- originated from wuhan, china, officially in late [fauci et al. ; helmy et al. ; na zhu et al. ; qun et al. ]. in table s (supplemental materials), the comparison of nucleotide sequences from sars-cov- isolates from china with the wuhan standard sequence revealed a total of deviations from the wuhan reference. most notably, in sequence positions and , the point mutations cc to tc and ta to ca, respectively, were observed in isolates out of the sequences examined. single cytidine (c) to thymine (t) transitions, which were often seen only / , occasionally to times in the sequences, were noted in many different sequence locations. the frequent cytidine-to-uracil (c → t) transitions, as demonstrated here, might be caused by one of the cellular apobec (apolipoprotein b mrna editing enzyme) cytidine deaminases attempting to restrict viral propagation [digiorgio et al. ]. the high frequency of c to t transitions and its evolutionary implications had been pointed out earlier by simmonds ( ) . among these c to t transitions, originated from cg dinucleotides in sequence positions , , , and . such mutations can possibly be attributed to the oxidative deamination of a presumptive -methyl-c nucleotide ( -mc) to uracil (u). it is unknown whether -mc occurs in sars-cov- rna. single nucleotide mutations, other than c to t transitions, which also occurred only in / isolates or were present to times were also documented among the isolates. the functional meaning of these very rare sequence alterations can be considered questionable, unless the frequency of sequence alterations in a certain spot expands with time and/or at certain geographic locations. some of the hotspot mutations discovered in isolates from locations other than china, like in positions , , , and , were only seen each in / isolates from china (see table ). thus, the frequency of mutations in these locations strongly increased when the virus massively replicated in regions outside china (see below). this was also true for the presumably important ggg → aac mutation in position which was found expanded particularly in isolates from europe (see below and discussion section). table s presents analyses of nucleotide sequences in sars-cov- isolates from european countries (czech republic, finland, france, greece, italy, netherlands, poland, serbia, spain, and sweden). compared to the wuhan reference sequence, five signal hotspot mutations were ( / ), ( / ), ( / ), ( / ), ( / ), ( / ), ( / ), ( / ) and others, mutations were also found, though much less frequently. the mutation in position comprised three nucleotides, ggg to aac in / of the analyzed sequences. this mutation affected the open reading frame for the nucleocapsid phosphoprotein n, altered the original sequence agg gga to aaa cga (see table ), and led to a change in amino acid sequence positions and from arg-gly (rg) to lys-arg (kr) and thus juxtaposed two very polar basic amino acids. possibly due to the strictly implemented lockdown early in the pandemic and by good luck, this country has so-far been spared excessive fatalities, although more recently (september ) there has been an increasing number of covid- cases. it is solely for this reason that search results from different isolates from germany table s have been listed separately from samples from other european countries. some of the previously identified mutation hotspots were confirmed at nucleotide numbers ( / ), ( / ), ( / ), ( / ), and ( / ). there were two additional hotspot mutations in positions (gc → ac, / ) and (gc → ac, / ). in positions and , the high mutation rates in samples from europe (table s ) were not observed in the samples from germany. in this context, it will be interesting to compare the results from the published munich study [böhmer et al. ] , in which sequences of isolates from patients were described. they had contracted covid- in january , and their sars-cov- infection could reportedly be tracked to a single conference attendee from china in the munich area. in the munich patient cohort, only sequence positions ( / ), ( / ), and ( / ) showed high mutation frequencies, whereas others failed to be represented (data included in synopsis table ). moreover, position presented with a mutation frequency of / which did not register in samples from china or any other country. in the samples from china chosen for our analyses (table s ), the high frequency mutations from the munich study were altogether absent or (at nucleotide ) present at a low frequency of / . notably, the mutation in position was absent from the munich list, although it was found at / in the samples from china (table s ). of course, there might have been mutations in chinese sars-cov- isolates not represented in the sequences we analyzed (table s ). the data on the analyses of isolates from the us confirmed the steady rise in mutation frequencies as sars-cov- spread to different parts of the world (table s ). these samples from the us were collected before the more recent (june/july ) recurrence of covid- in several parts of the us ( table s ) . some of the mutations annotated in tables s (china) and s (europe) were found even more frequently in this us cohort, like in sars-cov- the usa, unfortunately, has become the most severely sars-cov- -hit country in the world with . million covid- cases and has tallied about , deaths (census september ). we therefore analyzed an additional sars-cov- rna sequences from the most severely affected states arizona (az), california (ca), florida (fl) and texas (tx) (tables and s ) . the inspected sequences (table s ) had been deposited between june and july , . the sequences discussed here had not been included in table s . in table , the usa-i and usa-ii analyses were juxtaposed and demonstrate that the previously identified hotspot mutations were still represented at about the same frequencies as shown in table s . the mutations at intermediate frequencies ( / ) in positions , , and registered at frequencies between / and / at slightly lower frequencies in table s . we tested the hypothesis of differential distribution of mutation frequencies in the sequences from time point (usa-i) and time point (usa-ii) using a permutation test as well as kolmogorov-smirnov test and failed to reject the null hypothesis of equivalent distributions, p > . , suggesting that there is no significant difference in hotspot mutation frequencies between the two different time periods of sequence analyses from the us. in table s , mutation analyses in nucleotide sequences of isolates from india have been summarized. some of the hotspot mutations documented in isolates from the us were found in rna samples from india as well, partly at increased frequencies, at sequence in the samples studied here, the sars-cov- sequences from india showed the largest number ( ) of hotspot mutations, which also had the highest occurrence (up to / , i.e. %) of mutation at a given nucleotide position ( table s ) . as sars-cov- infections find susceptible populations around the world, and as the rate of viral replication shoots up in these populations, the number of new hotspots of mutation and the frequencies of mutations in individual hotspots were found increased. permitting the virus to replicate with growing efficiency might render its genome better adapted and increasingly dangerous to human health. alternatively, the possibility has to be considered that the lack of j o u r n a l p r e -p r o o f deleterious effects on the host and its tolerance might have favored the selection of mutants. moreover, repeated bottlenecks in the replication of viruses have been shown in the case of vesicular stomatitis virus to reduce its fitness (so called muller's ratchet) [duarte et al. ] . lastly, accumulation of mutants during the world-wide spread of sars-cov- might eventually decrease its virulence. hence, the consequences of an increase in the number of viral mutations on its pathogenicity will be very difficult to predict. eventually, a possible decrease in virulence might turn out to set a limit to sars-cov- 's pandemic potential. so far, we have inspected the available seven sars-cov- sequences from russia and found mutation hotspots identical to the ones predominant in europe (data not shown) as yet, not enough sars-cov- rna sequences have been deposited to allow a meaningful analysis. table juxtaposes all hotspot mutations in isolates from different geographic regions. the majority of de novo mutation hotspots arose after sars-cov- had been transmitted to regions outside china and been allowed active replication in different environments (tables s to s ). the mutations in positions and with frequencies / in sequences from china (table s ) were found outside china only in the us i and ii samples, though with moderate frequencies ( / or / ) and at even lower frequencies in the indian samples ( / ) ( table ) . hence most of the world-wide mutation hotspots described here (tables s to s ) must have originated and been selected in the course of massive replication of sars-cov- in its world-wide expansion. a challenging aspect arose from the identification of sars-cov- mutants in covid- patients from the munich, germany area in january [böhmer et al. ]. this report explained that a manufacturer of automobile parts in the vicinity of munich was visited by a collaborator from china in january . after the visitor had returned to china, she fell ill with covid- , and her contacts in the munich area also came down with the disease. a comparison of the occurrence and frequencies of the mutants in positions , and in all patients in the munich report [böhmer et al. ] to those in all other parts of the world (table ) , does not render the chinese traveler the most plausible source for sars-cov- in the munich area. these very mutations, however, are frequent hotspots in europe, germany, the us (i and ii) and india. of course, it will have to be investigated whether the hotspot mutations apparent in the isolates from the munich cohort might have been present, j o u r n a l p r e -p r o o f though infrequently, among chinese isolates, in particular in the munich visitor (patient ) from china. mutations in sequence positions - - or occurred at low frequency or were absent in samples from china, but expanded to levels up to / ( %) and / ( %) in the us and in india, respectively ( table ) . increases in the number of hotspot mutations and the heightened percentage of sequences altered in a given hotspot in samples from europe, and even more so from the us and india, suggested that these increases were tied to the intense replication of sars-cov- in these countries upon its spreading from china in the course of just a few months. it will be interesting to investigate whether sars-cov- spreading to and massively replicating in constantly new, immunologically naïve populations furthers the selection of viral mutants, in particular of those with heightened pathogenicity. the comparison of the number and frequency of hotspot mutations between samples from the usa i ( states) ( table s ) and usa ii (az, ca, fl, tx only) cohorts (table s ) revealed no differences ( table , see above), as corroborated by statistical analyses (see above). in an earlier report [pachetti et al. ] , the authors analyzed genomic sequences derived from patients infected by sars-cov- between december and mid-march and found eight mutations of sars-cov- , located at positions , , , , , , and . these authors report mutations in positions , , , and to have been observed mainly in europe, those at positions , and in north america. their results are interesting in that the mutations in sequence positions , , , and did not show up in our analyses, whereas mutations in positions , , , and were found in our collection as well ( table ) . since their sequence selection anteceded ours by several months, it is conceivable that the non-common mutations might have been counter-selected as the pandemic spread throughout the world. of course, the less likely possibility existed that by chance their group and ours worked on non-overlapping sequences in the data bank. furthermore, in a preprint deposit [laamarti et al. ], the authors analyzed , sars-cov- genomes isolated from countries during the first three months of the pandemic and found site mutations distributed in six genes of the sars-cov- genome. these mutations belonged to certain genotypes which appeared to be specific to certain geographic regions. table summarizes the amino acid exchanges due to sequence alterations in hotspot mutations identified in this study (table ) . mutations in sequence positions , , , , , , and did not cause amino acid exchanges and were not included in table . aside from the possible functional and structural alterations in proteins altered by either codon-neutral or codon-affecting mutations, any mutation in sars-cov- rna can impinge upon the structure of the single-stranded viral rna molecule itself with consequences for important viral rna-protein interactions. in sequence position , the mutation agg gga → aaa cga changes the amino acid sequence of the nucleoprotein n in positions and from arg-gly (rg) to lys-arg (kr) ( table ) . the frequency of this mutation was found in isolates from different countries to be / (china), / (europe), / (germany), / (us i), / (us ii) and / (india). the mutant amino acid sequence in nucleocapsid protein n between positions to (kkprqkr) and to (rkrpeq) ( table ) raises questions about the possible generation of a nuclear localization signal [kalderon et al. ] and strong dna-binding motifs in protein n. for sars-cov- very little is known about activities in the nuclei of infected cells. one publication reports cell cycle-dependent nucleolar, not nuclear, localization of protein n, although for non-sars coronaviruses [cawood et al. ]. it is presently unknown whether a mutated n protein might fundamentally alter the biology and pathogenicity of this sars-cov- mutant. it is also interesting to ponder how the wuhan sequence in position ggg was altered to aac. could rna-rna recombination have played a role? several of the mutants affect virus-encoded proteins with functions in the replication of sars-cov- , like non-structural proteins (nsp , nsp ) and the helicase. the important surface glycoprotein and the nucleocapsid phosphoprotein were also affected by codon changes. evidence has been adduced that the d g mutation of the sars-cov- spike protein bestows advantages on viral replication both in patients and in cell culture (korber et al. ) . in a preprint deposit [isabel et al. ], a sars-cov- this d g spike protein mutation has also been described. among the mutants listed in table , were transitions, transversions, led to amino acid changes in the proteins they were coding for, were synonymous mutations. of the transitions, were c to t (u) mutations. so far, we have not detected any insertions or deletions in the sequences analyzed. a deletion in the sars-cov- genome with evolutionary implications has been recently described [su et al. ]. it will now be important to correlate the identified hotspot mutations with the course and outcome of individual infections in humans. this demanding problem has not yet been tackled. hopefully, the results of our study will provide a platform for those in sars-cov- research who take care of patients with sars-cov- infections. sars-cov- has the ability to mutate and, in its course of world-wide dissemination, to select for distinct signal hotspot mutations depending on high rates of genome replication and complex environmental and genetic conditions in newly invaded territories of the world. during its intercontinental journey, the exposure of sars-cov- to the st century's repertoire of medical resources may have been an additional selective force. the impact of an increase in hotspot sars-cov- mutations on immunogenesis and the prospects for vaccine development [jackson et al. ] might be experienced and will have to be examined in the future. s.w. carried out all work involving sequence selection and formal analyses, was involved in the conceptualization of the project and in the analysis and interpretation of data. c.r. analyzed data for their statistical significance. w.d. initiated the project, was involved in the conceptualization of the project and in the analysis and interpretation of data and wrote the manuscript. yes, i will be a co-author on the paper. tables s to s -presentation of all data in detail. in these tables all alterations of sars-cov- rna/dna reverse transcript nucleotide sequences in comparison to the wuhan sequence have been compiled. sequence positions between to , , nucleotide alteration, and frequency of mutations in individual positions were listed. table s -china ( sequences analyzed -guangzho, hongkong, beijing, wuhan, fuyang, hangzhou, shanghai, yunnan). table s -europe ( sequences analyzed -czech republic, finland, france, greece, italy, netherlands, poland, serbia, spain, and sweden). table s -germany ( sequences analyzed düsseldorf, -heinsberg). table s investigation of a covid- outbreak in germany resulting from a single travel-associated primary case: a case series cell cycle dependent nucleolar localization of the coronavirus nucleocapsid protein covid- ) pandemic. geneva: world health organization evidence for host-dependent rna editing in the transcriptome of sars-cov- rapid fitness losses in mammalian rna virus clones due to muller's ratchet covid- -navigating the uncharted the molecular virology of coronaviruses the covid- pandemic: a comprehensive review of taxonomy, genetics, epidemiology, diagnosis, treatment, and control evolutionary and structural analyses of sars-cov- d g spike protein mutation now documented worldwide an mrna vaccine against sars-cov- -preliminary report a short amino acid sequence able to specify nuclear location tracking changes in sars-cov- spike: evidence that d g increases infectivity of the covid- virus large scale genomic analysis of sars-cov- genomes reveal a clonal geo-distribution and a rich genetic variations of hotspot mutations a novel coronavirus from patients with pneumonia in china emerging sars-cov- mutation hot spots include a novel rnadependent-rna polymerase variant early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia rampant c→u hypermutation in the genomes of sars-cov- and other coronaviruses: causes and consequences for their short-and long-term evolutionary trajectories discovery and genomic characterization of a -nucleotide deletion on orf b and orf during the early evolution of sars-cov- one severe acute respiratory syndrome coronavirus protein complex integrates processive rna polymerase and exonuclease activities structural basis for rna replication by the sars-cov- polymerase we are grateful to esteban domingo, universidad autónoma de madrid for alerting us to the duarte et al. the authors declare no competing interests. key: cord- -azpg yrh authors: mead, dylan j.t.; lunagomez, simón; gatherer, derek title: visualization of protein sequence space with force-directed graphs, and their application to the choice of target-template pairs for homology modelling date: - - journal: j mol graph model doi: . /j.jmgm. . . sha: doc_id: cord_uid: azpg yrh the protein sequence-structure gap results from the contrast between rapid, low-cost deep sequencing, and slow, expensive experimental structure determination techniques. comparative homology modelling may have the potential to close this gap by predicting protein structure in target sequences using existing experimentally solved structures as templates. this paper presents the first use of force-directed graphs for the visualization of sequence space in two dimensions, and applies them to the choice of suitable rna-dependent rna polymerase (rdrp) target-template pairs within human-infective rna virus genera. measures of centrality in protein sequence space for each genus were also derived and used to identify centroid nearest-neighbour sequences (cnns) potentially useful for production of homology models most representative of their genera. homology modelling was then carried out for target-template pairs in different species, different genera and different families, and model quality assessed using several metrics. reconstructed ancestral rdrp sequences for individual genera were also used as templates for the production of ancestral rdrp homology models. high quality ancestral rdrp models were consistently produced, as were good quality models for target-template pairs in the same genus. homology modelling between genera in the same family produced mixed results and inter-family modelling was unreliable. we present a protocol for the production of optimal rdrp homology models for use in further experiments, e.g. docking to discover novel anti-viral compounds. ( words) since high-throughput sequencing technologies entered mainstream use towards the end of the first decade of the st century, there has been an explosion in available protein sequences. by contrast, there has been no corresponding high-throughput revolution in structural biology. obtaining solved structures of proteins at adequate resolution remains a painstaking task. x-ray crystallography is still the gold standard for structure determination more than years after its first use in determining myoglobin structure [ ] . the result of this discrepancy between the rate of protein sequence determination and the rate of protein structure determination is the protein sequence-structure gap [ ] . homology modelling is a rapid computational technique for prediction of a protein's structure from (a) the protein's sequence, and (b) a solved structure of a related protein, referred to as the target and the template, respectively. since structural similarity often exists even where sequence similarity is low [ , ] , homology modelling has the potential to reduce massively the size of the protein sequence-structure gap, provided the models produced can be considered reliable enough for use in further research. the rna-dependent rna polymerase (rdrp) of rna viruses presents an opportunity to test and expand this approach. rdrps are the best conserved proteins throughout the rna viruses, being essential for their replication [ ] . conservation is particularly high in structural regions that are involved in the replication process, for instance the indispensable rna-binding pocket [ ] . rdrps are also of immense medical importance as the principal targets for antiviral drugs. evolution of resistance against anti-viral drugs is a major concern for the future, and the design of novel anti-viral compounds is a highly active research area. solved structures of rdrps are of great assistance to these efforts, as they enable the use of docking protocols against large libraries of pharmaceutical candidate compounds [e.g. refs. [ , ] ]. although some human-infective rna viruses have solved rdrp structures, there are still large areas within the virus taxonomy that lack any. this paper will first identify where the protein sequencestructure gap is at its widest in rdrps. because of the sequencestructure gap, it is therefore impossible in many genera to perform docking protocols against solved structures of rdrp for discovery of novel anti-viral compounds. under these circumstances, replacement of real solved structures with homology models for docking experiments requires that the homology models used should be both high quality and also optimally representative of their respective genera. our second task is to present several similarity metrics in sequence space that assist in the identification of the virus species having the rdrp sequence that is most representative of its genus as a whole. we then present the first use of force-directed graphs to produce an intuitive visualization of sequence space, and select target rdrps without solved structures for homology modelling. these are then used to perform homology modelling using template-target pairs within the same genus, between sister genera and between sister families, monitoring the quality of the models produced as the template becomes progressively more genetically distant to the target sequence being modelled. finally, we produce homology models for reconstructed common ancestral rdrp sequences. in the light of our results, we comment on the strengths and weakness of homology modelling to reduce the size of the protein sequence-structure gap for rdrps, and produce a flowchart of recommendations for docking experiments on rdrp proteins lacking a solved structure. we chose rdrps from human-infective viruses based on the list provided by woolhouse & brierley [ ] . given the global medical importance of aids, we also included lentivirus reverse transcriptases (rts) for analysis. solved structures for these proteins, where available, were downloaded from the rcsb protein data bank (pdb) [ ] . table presents our criteria for selecting suitable homology modelling candidates. rdrp and rt amino acid sequences for all virus species satisfying the criteria of table were downloaded from genbank [ ] . alignment of sequence sets for each genus, was performed using mafft [ ] . alignments were refined in mega [ ] using muscle [ ] where necessary, and the best substitution model determined. alignment of target sequences onto their solved structure templates for homology modelling was carried out using the molecular operating environment (moe v. . , chemical computing group, montreal h a r , canada). we define sequence space as a theoretical multi-dimensional space within which protein sequences may be represented by points. for an alignment of n related proteins, the necessary dimensionality of this sequence space is n- , with the hyperspatial co-ordinates in each dimension for any protein determined by its genetic distance to the n- other proteins. for n ¼ , direct visualization of all dimensions of sequence space is impractical at best, since a -dimensional space must be simulated in three dimensions, and is effectively impossible for n ! . the following methods were used to reduce sequence space to two and three dimensions for ease of visualization. to simplify calculations, we allow an extra dimension defined by the distance from each sequence to itself. the value of the co-ordinate in that dimension is always zero and our sequence space has n dimensions rather than n- . the pairwise distance matrix (m d ) for each genus, calculated from the sequence alignment in mega, consists of entries m d (i,j) giving the genetic distance between each pair of sequences i and j where {i, j} { , …. n} and i s j, for a set of n sequences. in our data set n ranges (see supplementary table) the similarity matrix was then used as input for r package qgraph [ ] . the "spring" layout option was chosen, which uses the fruchterman-reingold algorithm to produce a two-dimensional undirected graph in which edge thickness is proportional to absolute distance in n dimensions and node proximity in two dimensions is optimized for ease of viewing while attempting to ensure that those nodes closely related in the n-dimensional input are also close in the two-dimensional output [ ] . iterations were performed, or until convergence was achieved. for each alignment, the pairwise distance matrix (m d ) was used as input for r package cmdscale, which uses multi-dimensional scaling to produce a three-dimensional graph from the n-dimensional input, with node proximity again reflecting relative similarity [ ] . spotfire analyst (tibco spotfire analyst, v. . . , ) was used to visualize the output of cmdscale. we define the centroid as a hypothetical protein sequence located at the centre point of the sequence space of an alignment. the real sequence closest to the hypothetical centroid is termed the centroid nearest neighbour (cnn). we calculate the position of the cnn in three ways. table list of criteria used to select rna-dependent rna polymerases (rdrps) for homology modelling. human-infective virus importance to human health ncbi refseq annotated genome easy retrieval of high quality rdrp sequence rdrp located at the end of polyprotein or on its own segment eliminates unconventional rdrps at least one solved rdrp at a range of different taxonomic levels, e.g. in same species, same genus, same family, same order to be used as the templates in homology modelling at different levels of genetic distance . . . shortest-path centroid nearest neighbour for a sequence i { , …. n} in an alignment of n sequences, its total path length d(i) to the other n- sequences may be calculated from the distance matrix m d as follows: is zero. this may be omitted to enforce a strict n- dimensions for n input sequences, but we leave it in to simplify subsequent calculations. we define i* as the index that minimizes d(i). the shortest path cnn is therefore sequence i*. for alignments where clusters of closely related sequences exist, giving many values of m d (i,j) close to zero, this method will tend to place the cnn within a cluster. to overcome this problem, the arithmetic mean and median, respectively, were used to determine the mean cnn and the median cnn. the values of d (equation ( )) may be averaged to produce mean total path distance d: where again n is the total number of sequences in the alignment. we now re-define i* as the index that minimizes d(i) -d. in the event of equation ( ) returning zero, the mean cnn and the true centroid are identical. as with all variables using means, the mean cnn is liable to skewing by outliers. we generate a vector d over i { , …. n}, in which each entry d(i) represents the total path length for sequence i (equation ( )). the values of vector d are then ranked in ascending order x s( ) to x s(n) to produce vector d s . the median cnn is the sequence with value d(i) situated in the middle of the array d s , at d(m), where d(m) is either d (m odd ) or d (m even ) for alignments with odd or even numbers of sequences respectively. we now re-define i* as the index that minimizes d(i) -d(m). again, in the event of equation ( ) returning zero, the median cnn and the true centroid are identical. as with all variables using medians, the median cnn is liable to skewing by the presence in the alignment of multiple sequences with the same value of d(i). the choice of solved structures as templates for homology modelling, and the choice of targets to be modelled, within each genus was governed by the following rules: ( ) for each genus the solved structure that covered the highest proportion of the rdrp or rt sequence was chosen as the template for that genus. ( ) if more than one candidate template structure was found at this sequence length, the structure with the lowest resolution in angstroms was selected. see table for the templates satisfying these two criteria. ( ) within each genus, the sequence with the greatest genetic distance from the template, was chosen as the target for homology modelling. see table for the template-target pairs satisfying this criterion. ( ) criterion was applied to find template-target pairs in different genera (see table ) and different families (see table ), thus testing the limits of homology modelling at high genetic distances. homology modelling was carried out using the molecular operating environment (moe v. . , chemical computing group, montreal h a r , canada). ten intermediate models were produced using the amber :eht forcefield under medium refinement. the model that scored best under the generalised born/ volume integral (gb/vi) was selected to undergo further energy minimisation using protonate d, which predicts the location of hydrogen atoms using the model's d coordinates [ , ] . to assess the stereochemical quality of the homology models produced, ramachandran plots were derived in moe, and used to calculate the proportion of bad outlier f-j angles in the model, after subtraction of the number of outlier f-j angles in the template. generally, outlier angle percentage below . % indicates a very high quality model, and a percentage below % indicates a good quality model [ ] . models were superposed with their templates in moe and rootmean-square deviation (rmsd) value derived for the alpha carbons (ca) in the two structures. generally, an rmsd below Å indicates a good quality model [ ] . qualitative model energy analysis (qmean) was used to analyse models using both statistical and predictive methods [ ] . the qmean z-score is an overall measure of the quality of the model when compared to similar models from a pdb reference set of x-ray crystallography-solved structures. a z-score of would indicate a model of the same quality as a similar high quality x-ray crystallographic structure, while a z-score below À . indicates a low quality model [ ] . maximum likelihood (ml) trees [ ] were produced for each genus in mega. the ml tree and the corresponding multiple sequence alignment were input into the ancestral reconstruction server, fastml [ ] . the reconstructed sequence for the root of the tree, i.e. the putative common ancestor rdrp or rt sequence for the genus was used as the target for homology modelling in moe, using the template chosen according to the rules in section . . the reconstructed ancestral sequence was added to the alignment and the force-directed graph re-drawn. fig. b , showing the targettemplate pairs for homology modelling may be compared with fig. c , showing the ancestor-template pairs. our first observation is that there are still large areas of the viral taxonomy where no solved rdrp structures exist. no suitable templates for homology modelling were found within the entire nidovirales order of rna viruses. this order contains several coronaviruses important to human health including severe acute respiratory syndrome-related coronavirus (sars-cov) and middle east respiratory syndrome-related coronavirus (mers-cov) [ ] . in the order mononegavirales, vesiculovirus was the only genus with a solved rdrp structure suitable for homology modelling. however, this order contains many medically important viruses such as zaire ebolavirus, hendra henipavirus, measles morbillivirus, and mumps rubulavirus [ ] . in the order bunyavirales, phenuiviridae stands out as an important family lacking a solved rdrp, despite it containing various human-infective arboviruses such as rift valley fever phlebovirus and sandfly fever naples phlebovirus [ ] . (table ) . fig. shows two-dimensional force-directed graphs of similarity for each genus with more than four rdrp reference sequences (or rt sequences in the case of lentivirus). in principle, it would be possible to draw force-directed graphs for entire families and even orders. however, the input to qgraph is the similarity matrix calculated from the distance matrix, and the distance matrix is calculated in mega from an alignment. once taxonomic distance begin to extend beyond genera, alignment becomes progressively less reliable, with all the downstream statistics tending to degrade as a consequence. we therefore confine our construction of forcedirected graphs to intra-genus comparisons. it is evident from fig. that sequences are not necessarily evenly distributed in sequence space. clustering is noticeable in the genus flavivirus, with two sub-groups and an outlier sequence evident. mammarenavirus also shows division into two sub-groups. by contrast, picobirnavirus has only five relatively equidistant reference sequences, thus producing a highly regular pentagram. similarly, rotavirus has eight reference sequences, with four at each end of a fairly regular cuboid. fig. a also shows how the various methods equations ( )e( ) for determining the cnn of sequence space for each genus, are in poor agreement. only in rotavirus and table solved structures of rdrps and reverse transcriptase (for hiv- ) selected as templates for homology modelling. all are derived by x-ray crystallography except a which is a cryo-electron microscopy structure. for protein coverage, indicates that the template covers more than % of the sequence, indicates less. for f-j outliers and qmean z-score, indicates good-quality, indicates poor-quality, determined by the following thresholds: f-j ¼ %, qmean z-score ¼ À . . table homology modelling at intra-genus, inter-species level. templates are as given in table . targets are the rdrp (or reverse transcriptase for lentivirus) sequences from the reference genome accession numbers given. rmsd: root mean square deviation in angstroms between template and model when superposed in moe. indicates good quality, indicates poor quality, determined by the following thresholds: f-j < %; qmean z-score > À . ; rmsd < Å. indicates good quality, but using a partial template (see table ) *imjin thottimvirus was reclassified in by the international committee on taxonomy of viruses (ictv) in a new genus thottimvirus. table homology modelling at intra-family, inter-genus level. templates are as given in table . targets are the rdrp (or reverse transcriptase for spumavirus) sequences from the reference genome accession numbers given. rmsd: root mean square deviation in angstroms between template and model when superposed in moe. indicates goodquality, indicates poor-quality, determined by the following thresholds: f-j < %; qmean z-score > À . ; rmsd < Å. picobirnavirus are mean and median cnns found in the same sequence. fig. a also shows that the best solved structure for the purposes of template choice in homology modelling is rarely close to the centre of sequence space. only in lentivirus is the optimal template also the mean cnn, and only in vesiculovirus is the optimal template a shortest-path cnn. fig. b shows the relations of the template-target pairs in sequence space, illustrating how intra-genus homology modelling template-target selection attempts to traverse the largest genetic distance available within the genus. figs. and compare, for genera orthohantavirus and mammarenavirus respectively, the force-directed graphs of fig. with the three-dimensional equivalent output of multidimensional scaling. fig. shows a sequence clustering within orthohantavirus that is not readily apparent in the force-directed graph. the cnns are distributed among four clusters, as there is no sequence close to the geometrical centre of the three-dimensional space, where the notional centroid is located. the solved structure has other sequences in its proximity in the three-dimensional space, roughly table homology modelling at intra-order, inter-family level. templates are as given in table . targets are the rdrp (or reverse transcriptase for lentivirus) sequences from the reference genome accession numbers given. rmsd: root mean square deviation in angstroms between template and model when superposed in moe. indicates goodquality, indicates poor-quality, determined by the following thresholds: f-j < %; qmean z-score > À . ; rmsd < Å. fig. . force-directed graph visualisations of similarity of rdrps (or reverse transcriptase for lentivirus) within genera. the genetic distance matrix for each alignment was converted into a similarity matrix equations ( ) and ( ). the fruchterman-reingold algorithm ( minimisation iterations) was implemented in r module qgraph to produce a force-directed graph. relative similarity is represented by node proximity, and absolute similarity is proportional to edge thickness. the solved structure and the three types of centroid nearest neighbour (cnn) sequences are highlighted. the species names corresponding to the numbered nodes are listed in the supplementary table. cardiovirus has less than four reference sequences and is omitted. a: location of solved structure and the three cnns in sequence space equations ( )e( ). some genera have two median cnns. equivalent to the lower right quadrant of the two-dimensional force-directed graph. similarly, the shortest-path cnn and mean cnn are both located within another three-dimensional cluster also containing sequences, which is roughly equivalent to the upper right quadrant of the two-dimensional force-directed graph. fig. presents a similar picture for mammarenavirus. the forcedirected graph for mammarenavirus has more obvious clustering that for orthohantavirus, showing a lower-left to top-right split. in the three-dimensional representation, these are equivalent, respectively, to the three clusters on the right and two clusters on the left. as with orthohantavirus, there is no cnn near the geometrical centre of the three-dimensional space, but the cnns are distributed around two clusters. three dimensional representations of all the genera in fig. are available from the link in the raw data section. homology modelling was carried out as follows: ( ) intra-genus, inter-species ( models, table ) ( ) intra-family, inter-genus ( models, table ) ( ) intra-order, inter-family ( models, table ) ( ) intra-genus, on reconstructed common ancestor ( models, table ) table shows that homology modelling with template and target within the same genus, produced good quality models in most cases, as judged by percentage of f-j outliers and rmsd within the high quality range. only the models for american bat vesiculovirus and tamana bat virus have percentages of f-j outliers outside of the high quality range. qmean, however, is rather more critical of the output with only the model for porcine picobirnavirus falling within the high quality range. the model for imjin thottimvirus scores eighth best on percentage of f-j outliers and second best on rmsd, despite the re-classification (occurring after the completion of our experimental work) by the ictv of this virus, originally in genus orthohantavirus into a new thottimvirus genus [ ] . it should be noted that the models for imjin thottimvirus, burana orthonairovirus and brazilian mammarenavirus were based on very short template structures (see table ). table shows that homology modelling with template and target within the same family but different genera, still produced good quality models in most cases, as judged by percentage of f-j outliers and rmsd within the high quality range. only the models for lleida bat lyssavirus and macaque simian foamy virus have percentages of f-j outliers outside of the high quality range. however, once again, qmean assesses all models as outside the high quality range. table shows that homology modelling with template and target within the same order but in different families, is a far more difficult proposition than at the lower taxonomic levels. the model for mammalian orthobornavirus fails all three quality tests and only the model for rift valley fever phlebovirus manages to pass two out of three. table shows that modelling the structure of the reconstructed sequence of the common ancestor of each genus, produces models of the same standard as intra-genus modelling (compare tables and ). by contrast with almost all the other models, the qmean scores are within the high quality range, with only two exceptions, table. the common ancestors of genera rotavirus and vesiculovirus. fig. c shows the force-directed graphs with the locations of the ancestral sequences added. table summarises the results of tables e inclusive. as the taxonomical distance increases, production of high quality homology models becomes more difficult. however, modelling the reconstructed ancestral sequence of each genus is typically productive of a better scoring model even than the real sequence targets chosen for intra-genus modelling. fig. shows representative examples of homology models of high and low quality superimposed with their template solved structure along with their corresponding ramachandran plots and qmean quality scores. all homology models in tables e are available from the link in the raw data section. the first objective of this study was to identify viral taxa which are comparatively lacking in solved structures for rna-dependent rna polymerase (rdrp). we observed that the entire order nidovirales, the families bornaviridae, filoviridae and paramyxoviridae within the order mononegavirales, and the family phenuiviridae within the order bunyavirales, fall into this category. additionally, within the genera orthohantavirus, orthonairovirus and mammarenavirus, all within the order bunyavirales, the solved structure available for rdrp covers less than % of the protein sequence. given the medical importance of many viruses within these taxa, and the number of anti-viral drugs that target rdrps, we suggest that they are prioritized for x-ray crystallography to close the "sequence-structure gap". our second objective was to assess how well homology modelling could provide models that might serve for computerassisted drug discovery of novel anti-viral compounds. to assist in the visualization of sequence space, we produced the first application of force-directed graphs to protein sequences (fig. ) . we also applied multidimensional scaling for comparative purposes (figs. and ) . force-directed graphs enable the visualization of complex data in two dimensions. the three dimensional visualization produced from multidimensional scaling is visually richer, but this benefit can only be appreciated when a viewing application such as spotfire is available so that the three-dimensional image can be rotated. force-directed graphs convey much of the information in a single image which may be printed on a page or viewed on screen. this two-dimensional collapsing of sequence space also allows for easy simultaneous comparison of multiple datasets, in the present case multiple genera, which cannot readily be performed if separate three-dimensional viewers require to be open. the most common method of visualizing sequence space is the phylogenetic tree. for instance, starting from a distance matrix, agglomerative hierarchical clustering, such as the upgma method [ ] , can be performed to generate a tree. slightly more sophisticated methods, such as neighbour-joining [ ] can generate trees where the branch lengths are proportional to genetic distance. force-directed graphs do not represent genetic distance as accurately as phylogenetic trees, since the distances between nodes, table. although optimized to reflect relatedness, are constrained by the fruchterman-reingold algorithm to the best representation in two dimensions. however, force-directed graphs again allow easier simultaneous comparison of several data sets than phylogenetic trees. fig. would be impossible to create on a single page if trees were used instead of force-directed graphs. trees represent ancestral sequences as nodes on the tree, with only existing taxa as leaves. force-directed graphs, by contrast, allow ancestral sequences to be represented in the same way as existing ones. fig. c shows that ancestral sequences do not necessarily appear as outliers in force-directed graphs. indeed, for genera flavivirus, hepacivirus, orthobunyavirus and orthohantavirus in particular, the insertion of the reconstructed ancestral sequence into the forcedirected graph in fig. c does not overly distort its original shape in fig. aeb . the reason for this becomes apparent when one considers a phylogenetic tree represented in unrooted "star" format. the ancestral sequence is then at the centre of the star topology and it can be seen that the genetic distance from the root to any particular leaf sequence may often be less than for many pairwise leaf sequence combinations. we did not perform calculation of centroid nearest neighbours (cnns) for alignments incorporating reconstructed ancestral sequences, but we are tempted to speculate that many of the ancestral sequences would have been cnns, had they been included. table homology modelling the common ancestor for each genus. templates are as given in table . targets are the reconstructed ancestral rdrp (or reverse transcriptase for lentivirus) sequences. rmsd: root mean square deviation in angstroms between template and model when superposed in moe. indicates good-quality, indicates poorquality, determined by the following thresholds: f-j < %; qmean z-score > À . ; rmsd < Å. table mean model (or structure) quality. the top line shows the mean quality scores for the solved structures used. the other lines show the mean quality scores for the models produced at various levels of taxonomic distance between template and target. indicates good-quality, indicates poor-quality, determined by the following thresholds: f-j < %; qmean z-score > À . ; rmsd < Å. numbers in brackets indicate the revised scores if the model for imjin thottimvirus is moved out of the intra-genus category and into the intra-family category in the light of its subsequent transfer into the new genus thottimvirus. ), and outliers ( cross, text). the z-score graphics show model quality on a sliding scale: low-quality ( ), high-quality ( ). qmean shows the overall z-score, "all atom" shows the average z-score for all of the atoms in the model, "cbeta" the z-score for all cb carbons, "solvation" is a measure of how accessible the residues are to solvents, and "torsion" is a measure of torsion angle for each residue compared to adjacent residues. it is important to remember that homology models are theoretical constructions and caution must be exercised in treating them as input material for further experiments. among the various statistics for assessment of model quality, f-j outlier percentage is a measure of the proportion of implausible dihedral angles in the model, and indicate where parts of the model backbone are likely to be incorrectly predicted. nevertheless, it is also important not to become too dependent on statistics such as f-j outlier percentage, as "bad" angles do occasionally occur in solved structures. for instance in the present study, the thresholds of < . % for a very high quality model, and < % for a good quality model given by lovell et al. [ ] would suggest that six of the twelve template solved structures used here ( table ) would not have been assessed as "very high quality" had they been models rather than solved structures. indeed the templates from indiana vesiculovirus and rotavirus a have more than . % f-j outliers, and also have the poor quality scores for qmean. these two structures also have the poorest resolution of any of our templates, at > Å. the poor quality scoring may therefore simply be a consequence of uncertainties in positioning of atoms in these structures. one might reasonably posit that the use of template solved structures having such issues might influence the resulting models to contain the same outliers. however, the model for rotavirus i has a lower level of f-j outliers than its rotavirus a template ( table ) . as might be expected, production of high quality models becomes more difficult as the genetic distance between target and template increases, as show in tables e nevertheless, even at the level of template-target pairs in separate genera (table ) , the average performance is acceptable, as summarized in table . we therefore suggest that homology modelling may be used to produce rdrp models for research use even for genera where no solved structure exists, provided a template structure exists within the same family. here, we provide examples (table ) of such successful inter-genus, intra-family, models for genera coltivirus and parechovirus. our inter-genus models for lyssavirus and spumavirus are slightly less successful. moving to the next taxonomic level, models with template-target pairs in separate families (table ) are generally less successful. one exception is our model for family phenuiviridae, which is better than some of the intra-family models. this is encouraging, since phenuiviridae is a family without any solved rdrp structure. homology models have been produced at much larger taxonomic distances than those dealt with here, for instance from bacteria to eukaryotes [ ] , so it should be stressed that we make no claim for the generality of our findings outside of the viral orders under consideration, or for proteins other than rdrp. multi-domain proteins in particular, may produce higher quality models for some domains than others. one surprising result was the high quality of the models of reconstructed ancestral sequences (table , summarized in table ). as previously discussed, this may be due to the fact that the ancestral sequence is, assuming a regular molecular clock, potentially equally related to all descendent members of its genus. in this paper, we calculated centroid nearest neighbours (cnns) as the central points in sequence space for each genus (fig. ) . a reconstructed ancestral sequence may also be considered as a candidate central point. the value of central points is that they may serve as targets that could be used to make models representative of their genus as a whole. for instance, the shortest-path, mean and median cnns of genus orthohantavirus are sequences , and (see supplementary table for a list of sequences for each genus) , representing sin nombre orthohantavirus, rockport orthohantavirus and cao bang orthohantavirus respectively. the partial solved structure used as the template for modelling in the genus orthohantavirus in the present paper is from hantaan orthohantavirus ( ize, see table ) and the target used, imjin thottimvirus (sequence in orthohantavirus panel of fig. ) , is now classified as belonging to a new genus thottimvirus (table ) . the three cnns, sin nombre orthohantavirus, rockport orthohantavirus and cao bang orthohantavirus are %, % and % identical to ize respectively, whereas imjin thottimvirus is only % identical. the latter was of course chosen to test the effectiveness of intra-genus homology modelling over as wide a genetic distance as possible (see section . ). for the performance of subsequent experimental procedures on orthohantavirus rdrps, for instance docking to discover novel anti-viral compounds, a homology model corresponding to one of the three cnns mentioned above or to the reconstructed ancestor (table ) would be the preferred target, along with the existing solved structure. where a solved rdrp structure exists in a genus, it should be used. however, if that solved structure is not a cnn, a homology model of a cnn or ancestral sequence should be produced for comparative purposes. where no solved rdrp structure exists in a genus, a structure from another genus in the same family may be used. on the basis of our investigations, we recommend a procedural flowchart for selection of an rdrp structure for further study, for instance docking to discover novel anti-viral compounds, in any rna virus genus of interest (fig. ) . where a solved structure exists within a genus, it is the obvious choice for further experiments. however, where that solved structure is far from any of the cnn sequences of the genus, as judged by the force-directed graph, a cnn may also be homology modelled for comparative purposes, using the existing solved structure as a template. any differential performance of the solved structure and the homology model in, for instance, a docking experiment, may give clues as to the generality of conclusions derived from the solved structure alone. a reconstructed ancestral rdrp may also be used as an alternative to, or in addition to, a cnn. the limits of homology modelling would appear, on the basis of the results presented here, to be at the intrafamily, inter-genus level. template-target pairs in different viral families are unlikely to be of practical use, as the predicted quality of the resulting models is low. our models were produced using moe, and we have not performed comparisons using other modelling tools, such as swiss-model [ ] or modeller [ ] . we feel that it is unlikely that significant differences in output would be produced, but when the object of the exercise is drug-discovery, we recommend that the protocol in fig. be implemented using several alternative modelling softwares. crystallographic structural genome projects are badly needed to close the sequence-structure gap. in the meantime, systematic attempts to fill the gaps via homology modelling may be useful. however, for many taxa e all of the order nidovirales and much of mononegavirales -the paucity of solved structures to act as templates remains a serious obstacle. all code, inputs and outputs are available from: https://doi.org/ . /lancaster/researchdata/ . a three-dimensional model of the myoglobin molecule obtained by x-ray analysis protein modeling: what happened to the "protein structure gap the high throughput sequence annotation service (ht-sas) -the shortcut from sequence to true medline words the evolution and emergence of rna viruses crystal structure of the full-length japanese encephalitis virus ns reveals a conserved methyltransferase-polymerase interface molecular docking revealed the binding of nucleotide/ side inhibitors to zika viral polymerase solved structures using bioinformatics tools for the discovery of dengue rna-dependent rna polymerase inhibitors epidemiological characteristics of humaninfective rna viruses the rcsb protein data bank: integrative view of protein, gene and d structural information reference sequence (refseq) database at ncbi: current status, taxonomic expansion, and functional annotation mafft: iterative refinement and additional methods mega : molecular evolutionary genetics analysis version . for bigger datasets muscle: multiple sequence alignment with high accuracy and high throughput network visualizations of of relationships in psychometric data graph drawing by force-directed placement some properties of classical multidimensional scaling protonate d: assignment of ionization states and hydrogen coordinates to macromolecular structures the generalized born/volume integral implicit solvent model: estimation of the free energy of hydration using london dispersion instead of atomic surface area structure validation by calpha geometry: phi,psi and cbeta deviation on the accuracy of homology modeling and sequence alignment methods applied to membrane proteins qmean: a comprehensive scoring function for model quality assessment toward the estimation of the absolute quality of individual protein structure models evolutionary trees from dna sequences: a maximum likelihood approach fastml: a web server for probabilistic reconstruction of ancestral sequences sars and mers: recent insights into emerging coronaviruses taxonomy of the order mononegavirales: second update emerging phleboviruses taxonomy of the order bunyavirales: second update construction of phylogenetic trees for proteins and nucleic acids: empirical evaluation of alternative matrix methods the neighbor-joining method: a new method for reconstructing phylogenetic trees swiss-model and the swiss-pdbviewer: an environment for comparative protein modeling modeller: generation and refinement of homology-based protein structure models supplementary data to this article can be found online at https://doi.org/ . /j.jmgm. . . . key: cord- - y vjhux authors: wang, qihui; yan, jinghua title: isolation of monoclonal antibodies from zika virus-infected patient samples date: - - journal: zika virus doi: . / - - - - _ sha: doc_id: cord_uid: y vjhux the combination of sorting antigen-specific memory b cells with determining immunoglobulin (ig) genes at the single-cell level enables the isolation of monoclonal antibodies (mabs) in individuals. this method requires a small amount of blood (usually ml) and is rapid (less than weeks to isolate antigen-specific mabs). due to the application of antigens as the bait to capture the specific memory b cells, the majority of isolated mabs are true binders to the antigen, which increases the isolation efficiency. here, applying this approach, we describe the characterization of mabs against zika virus from a convalescent patient sample. from ml whole blood, we sorted zika envelope (e) protein-interacting single memory b cells. the ig genes from cells were determined, and mabs were found that bind to zika e protein with varied binding affinities. zika virus has caused global concern due to the accumulating evidence suggesting that infection is associated with microcephaly and neurological complications, such as guillain-barré syndrome [ ] [ ] [ ] [ ] . however, there are currently no approved antivirals against zika infection [ ] . administration of polyclonal or purified neutralizing monoclonal antibodies (mabs) to pregnant mice helps to clear the virus and alleviates the neurological disorders in their fetuses [ , ] , providing proof of concept that neutralizing mabs can be used to treat zika virus infections. currently, multiple strategies have been reported to generate human neutralizing mabs against zika infection, including sequencing antigen-specific memory b cells [ , ] or generating epstein-barr virus-immortalized memory b cells from zika patient samples [ ] , and identifying functional mabs from phage display naïve antibody libraries [ ] . in addition, murine mabs against zika virus have also been reported [ ] . here, we introduce a method to apply zika envelope (e) glycoproteins, which play pivotal roles in virus entry and contain important neutralizing epitopes, to sort single memory b cells from a convalescent zika patient. subsequently, the immunoglobulin (ig) genes encoded by the sorted cells were determined at the single-cell level. the approach to determine the ig genes from single cells was first reported by tiller et al. [ ] . later, scheid et al. modified this approach to sequence single gp -binding memory b cells, which were isolated from human immunodeficiency virus (hiv) patients [ ] . then the mabs against human papillomavirus (hpv) or respiratory syncytial virus (rsv) were isolated from immunized or naturally infected human donors, respectively [ , ] . during the epidemic of zika virus, we and other groups also applied this strategy to isolate mabs targeting zika virus from zika patients [ , ] . from ml whole blood, we were able to pair mabs from collected single cells. as detected by surface plasmon resonance (spr), of the mabs were true binders to zika e protein [ ] . although the protocol depicted in this chapter focuses on the isolation of zika mabs from a zika convalescent patient, it could also be expanded to other viruses, and we recently reported the isolation of human neutralizing mabs against rift valley fever virus (rvfv) from a convalescent rvf patient, applying the same strategy [ ] . . primers for reverse transcription (rt) are listed in table . . primers for the first round pcr (pcra) to amplify v h are listed in table . . primers for the pcra to amplify v κ are listed in table . . primers for the pcra to amplify v λ are listed in table . . primers for the second round pcr (pcrb) to amplify v h are listed in table . . primers for the pcrb to amplify v κ are listed in table . . primers for the pcrb to amplify v λ are listed in table . . other primers used in this study are listed in table . table primers for the pcra to amplify v κ forward primer cκ-ext gag gca gtt cca gat ttc aa table primers for the pcra to amplify v λ forward primer . lb agar plates containing ampicillin: dissolve g of tryptone, . g of yeast extract, . g of nacl, and . g of agar in l of ddh o. the agar will not dissolve until autoclaved. autoclave for min and then allow it to cool until the bottle can be held with bare hands. add ml of ampicillin ( mg/ ml). invert to mix thoroughly. carefully pour out into sterile . trypsin-edta ( . %) and phenol red. table other primers used in this study . transfer buffer: dissolve . g of tris base and . g of glycine together in . l of ddh o. then add ml of methanol and mix. at last, add ddh o to a final volume of l. characterization for the mabs . biacore t system. . biacore t evaluation software, version . . . collect ml of whole blood, using a k edta tube, from a convalescent zika patient or a healthy donor with informed consent (see note ). . dilute the anticoagulated blood with ml pbs (one volume of the original blood). . invert the density gradient medium bottle several times to ensure thorough mixing. . add ml of density gradient medium to a ml centrifuge tube with a conical bottom. . carefully layer the diluted blood sample onto the density gradient medium solution (see note ). . centrifuge at  g for min at room temperature (see notes and ). . draw off the upper layer containing plasma and platelets using a sterile pipette, leaving the pbmc layer undisturbed at the interface. aliquot ml of the upper layer per vial, which contains the plasma, and store them at À c ( fig. ). . transfer the layer of pbmcs to a sterile ml centrifuge tube using a sterile pipette (see note ). . add pbs until the total volume reaches ml ( . volumes of the original blood). resuspend the cells by gently drawing them in and out of a pipette. . centrifuge at  g for min at room temperature, and discard the supernatant. . add ml pbs to resuspend the cells. mix thoroughly and transfer μl of cell suspension in a . ml tube for cell counting. . load μl of cell suspension into a cell counting chamber. . insert the chamber into an automated cell counter and run the counting. . repeat step in this section. . quickly resuspend the cell pellet by adding freezing medium to a cell density of  /ml (see note ). . aliquot ml of the cell-medium mixture per vial, and place in a freezing container. . freeze the cells overnight at À c. . transfer vials to a liquid n tank for storage (see note ). cytometry ( . day) . samples used for staining are designed as follows: (a) zika patient pbmcs stained with zika e protein, as well as a panel of memory b-cell markers (see note ). (c) healthy donor pbmcs stained with mers-rbd protein (positive control for anti-his antibody). . transfer a vial containing the frozen pbmcs of either a convalescent zika patient or a healthy donor from the liquid n tank, and place them into a c water bath (see note ). . gently swirl the vial in the c water bath, and quickly thaw the cells (< min) until there is just a small bit of ice left in the vial. . transfer the vials into a hood. before opening, wipe the outside of the vials with % ethanol. . transfer the thawing cells into a centrifuge tube ( ml) containing ml of pre-warmed culture medium. . centrifuge the cell suspension at  g for min at room temperature. . decant the supernatant without disturbing the cell pellet. . gently resuspend the cells in facs buffer, μl per cells. . keep the mixture on ice for h. . add ml of facs buffer to the cells and pellet the cells by centrifuging at  g for min at c. decant the supernatant without disturbing the cell pellet (see note ) . . resuspend the cells with μl of facs buffer per cells (see note ). . prepare staining master mix in a . ml microcentrifuge tube (see table ) and store them at c in the dark before use. . add μl of the staining master mix to cells that prepared in step in this section. incubate them on ice for min. . add ml of facs buffer to the mixture of cells and antibodies in the last step and pellet the cells by centrifugation at  g for min at c. decant the supernatant without disturbing the pellet. . resuspend the cells with . ml facs buffer. place the tube on ice and avoid light before cell sorting. controls ( h) . label a facs tube for each of the six fluorochromes that will be used in the cell sorting. add . ml of facs buffer into each tube. . mix compensation beads by vigorously inverting at least ten times. . add μl of compensation beads into each tube. . load the zika pbmcs sample stained with zika e protein and memory b-cell markers. gate the target cells as in fig. d. . adjust the flow rate so that the event rate is approximately events/s. . mix and briefly centrifuge each component in the rt-pcr synthesis system for the first-strand cdna before use. table in a . ml tube. table ) into each well containing the collected single cells prepared in step in subheading . . . incubate the plate at c for min and then place it on ice for at least min. . prepare the following cdna synthesis mix in a . ml tube, adding each component in table in the indicated order. table ) to each well, mix gently, and collect by centrifugation at ,  g at c for s. . the rt reaction is performed at c for min and terminated at c for min. chill on ice and then store at À c until use. . prepare primers for the pcra (see tables - ). the amount for reactions is listed in table . . perform the pcra to amplify the ig genes. prepare the reaction system as indicated in table (see note ) . the cycling conditions for the pcra are shown in table . store the pcra products at À c until further use. . prepare primers for the pcrb (see tables - ). the amount listed in table is for reactions. . perform the pcrb to amplify the ig genes. prepare reaction system as indicated in table (see notes and ). the cycling conditions for the pcrb are indicated in table . . load the samples onto an agarose gel ( . %), and separate the dnas by electrophoresis. . a typical band size for v h , v κ , and v λ is approximately bp, as displayed in fig. . . perform the pcrc. prepare reaction system as indicated in table . . load the samples onto an agarose gel ( . %), and separate them by electrophoresis. . cut out the~ bp bands and extract the dna segments using a gel extraction kit according to the manufacturer's instructions. table ) is incubated at c for min, followed by cycles of c for s, c for s, and c for s, with a final incubation at c for min. lf new ( μm)  pcr buffer table reaction system for amplification of the constant regions  pcr buffer high-fidelity dna polymerase . cut out the~ bp bands for c h and~ bp for c κ and c λ . extract the dna segments using a gel extraction kit according to the manufacturer's instructions. . perform overlapping pcr to generate the expression cassette (see table ). the pcr reaction is incubated at c for min, followed by cycles of c for s, c for s, and c for min, with a final incubation at c for min. . digest the pcr products from the last step with ecori and xhoi for both the heavy and lambda chains. digest the kappa chain with kpni and xhoi (see table ). mix gently and spin down for a few seconds. incubate at c overnight. . purify the digested pcr segment using a universal dna purification kit according to the manufacturer's instruction. . ligate the expression cassette to the pcaggs vector (see table ). for both heavy and lambda chains, the vector was  pcr buffer high-fidelity dna polymerase pcaggs-z h encodes the heavy chain of the rd sorted cell). . the generation of vectors for mab expression is displayed in schematic diagram in fig. . fig. strategy to clone and express zika e-specific human mabs. the ig genes from the sorted cells were determined and cloned into the expression vectors by a reported approach with some modifications [ , ] . the ig transcripts in the collected zika e-interacting memory b cells were first reverse-transcribed into cdna using the ig gene-specific primer mix at the single-cell level. then the variable regions for heavy, kappa, and lambda chains were amplified by nested rt-pcr. the first round pcrs were performed with the forward primer mix specific for the leader region and reverse primers specific for the constant regions of heavy, kappa, and lambda chain, respectively. the second round pcrs were performed with the forward primer mix specific for framework segment fr and respective nested reverse primers specific for the heavy, kappa, and lambda constant regions. then the pcr products were separated by electrophoresis and sent for sequencing. in terms of the correct segments, another round of pcr (pcrc) was performed with the forward primer containing the signal peptide of mouse ig κ and reverse primer paired with the framework segment fr . then the resultant pcr segments were overlapped with the respective constant region to get the full expression cassettes for each chain, which were then ligated to the linearized pcaggs vector. all expression plasmids were sequenced and aligned with those in pcrb . load the samples onto a precast sds-page gel ( - %), and separate the proteins by electrophoresis. . transfer the proteins onto a nitrocellulose membrane. . perform western blotting to assess the expression of each mab using anti-higg/hpr (diluted by : ). as indicated in fig. . etbr stains dna by intercalating between the bases of dna. it will also intercalate into human dna, so wear gloves to prevent contact with it. a separate space is also recommended for performing all experiments using etbr-containing materials. do not pull the comb out too soon, as it causes the wells to collapse. it will take - min to gel. if the gel cannot be used in h, it is recommended to transfer the gel without comb into a tank containing  tae buffer. . the primers used for the amplification of ig genes are the same as reported (see tables - ) [ ] . however, after sequencing, cloning of the expression vectors was designed based on the pcaggs vector. . all primers are stored in small aliquots to avoid repeated freezing and thawing. . the blood sample of the convalescent zika patient was collected days post onset of fever, headache, and dizziness. it is reported that after immunization, antigen-specific b cells with a memory phenotype could be detected in the blood within week [ ] . in the presence of antigens, memory b cells undergo affinity maturation, and their b-cell receptors have increased affinities for the antigen. studies on memory b cells after smallpox vaccination in humans indicated that antigenspecific memory b cells initially declined postimmunization ( year) but then reached a plateau~tenfold lower than peak and were stably maintained for > years after vaccination [ ] . thus, considering the time that needed for affinity maturation, we recommend collecting the blood month- year post onset of symptoms to isolate specific mabs. . when layering the sample, do not mix the density gradient medium solution and the diluted blood sample. keep them in separate layers. . the break should be turned off at this step. for other steps using centrifuging, set the break on. . if the blood has been collected for > h, extend the centrifuging time to min. . usually, - ml of solutions containing pbmcs will be pipetted out from ml of the blood. . for example, if the cell density is determined to be  / ml at step in subheading . , the total cell number is  /ml  ml ¼ .  . thus, we need to add . ml freezing medium to resuspend the cells. . freshly prepared pbmcs exert higher efficiencies for ig gene amplification than frozen cells. thus, freshly prepared pbmcs are recommended for the isolation of mabs. in case the following cell sorting cannot be performed immediately, the methods using the freezing cells are provided in subheading . . . memory b cells express cd and cd , but not cd . in this study, we focus on the igg + memory b cells. . if fresh pbmcs are used here, please skip to step . if using frozen cells, please follow step . . before turning the tube right side up, it is recommended to aspirate the liquid left around the tube orifice. . it is difficult to decant the entire buffer. usually,~ μl of buffer will be left. thus, we add μl facs buffer for cells or μl for  cells. . there are three antibodies conjugated with pe-cy ® . they are anti-human cd /pe-cy ® , anti-human cd /pe-cy ® , and anti-human cd a/pe-cy ® . any of the three are suitable for preparation of the compensation beads for pe-cy ® . . the steps in this section are specific to, but not limited to, a bd facsaria iii cytometer. . here, we use the markers for t cells, nk cells, and platelets for negative selection, to exclude their disturbance. . here, we include fsc-a and fsc-h to exclude cell aggregates. we are not sure about the proportion of memory b cells that bind to zika e protein. thus, it is difficult to set the threshold to distinguish antigen-specific memory b cells from those targeting other antigens in the sample of zika patient pbmcs. in previous work, we studied the interaction between the receptor-binding domain (rbd) of middle east respiratory syndrome coronavirus (mers-cov) and its receptor cd , which is widely expressed on lymphocytes, including t cells. here, we used the mers-rbd, which is also tagged at its c-terminus with  his like the zika e protein, to stain pbmcs from a healthy donor. then, anti-his/pe was applied to bind to the his tag. through comparison between the results in fig. b and c, we could determine the threshold to gate the zika e-specific memory b cells, as indicated in fig. d . . the pcr reactions are performed in -well plates. . the combined primers are used to amplify ig genes in the pcrb. however, different primers in a single tube might disturb each other and reduce their annealing efficiency. thus, in terms of the single cells that yield typical bands for the kappa or lambda chain but not for heavy chain in gel electroporation, we usually repeat pcrb using the separate primers. in addition, mgcl exerts effects on the activities of dna polymerase (for sing-cell pcr). varied concentrations of mgcl , ranging from . to . mm, can also be applied to amplify the variable region of the heavy chain from single cell, whose light chains' variable regions have been sequenced. . from pmbcs isolated from ml whole blood of a convalescent patient, we finally sequenced paired mabs, which have been published previously [ ] . qihui wang and jinghua yan . here, we introduce the traditional method to double digest the pcr segments with two restriction enzymes and clone them into the same sites in the linearized pcaggs vector using t ligase. however, other methods (e.g., in-fusion reaction [ ] ) can be used. . here we displayed the results for z , z , and z . . the steps in this section are specific to, but not limited to, a biacore t system. . in addition to spr, enzyme-linked immunosorbent assay is another typical method to assess the interaction between mabs and antigens. however, for zika e-specific mabs, we found that some of them displayed relatively low response to the antigens coated on the plate (data not shown here). it is possible that zika e proteins undergo some conformational changes when adsorbing to the plate, which results in the decreased binding to certain mabs. in terms of spr experiments, the mabs were captured on the chip through interactions with protein a, which binds to the fc region. thus, the cdrs of mabs orient to the buffer flowing over the chip surface. in addition, the zika e proteins in the buffer of hbs-ep are label-free and more prone to be in its native conformation than those coated on the plate. thus, we chose spr assay to detect the interaction between mabs and antigens. . due to the different binding kinetics between an antigen and its mab, we set varied dissociation time. for example, mab z dissociates with zika e with very low rate; thus we set s for their dissociation. however, for both z and z , they are ready to dissociate with the antigen, and s was used for their dissociation (fig. s g, j, and l in the paper [ ] ). . due to the different binding kinetics between mabs and zika e protein, the data could be fit by either a steady state affinity model or : (langmuir) binding model. guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study zika virus associated with meningoencephalitis zika virus associated with microcephaly zika virus in brazil and macular atrophy in a child with microcephaly monoclonal antibodies against zika virus: therapeutics and their implications for vaccine design transfer of convalescent serum to pregnant mice prevents zika virus infection and microcephaly in offspring a single injection of human neutralizing antibody protects against zika virus infection and microcephaly in developing mouse embryos molecular determinants of human neutralizing antibodies isolated from a patient infected with zika virus recurrent potent human neutralizing antibodies to zika virus in brazil and mexico specificity, cross-reactivity and function of antibodies elicited by zika virus infection neutralization of zika virus by germline-like human monoclonal antibodies targeting cryptic epitopes on envelope domain iii structural basis of zika virus-specific antibody protection efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning broad diversity of neutralizing antibodies isolated from memory b cells in hiv-infected individuals rapid profiling of rsv antibody repertoires from the memory b cells of naturally infected adult donors characteristics of memory b cells elicited by a highly efficacious hpv vaccine in subjects with no pre-existing immunity neutralization mechanism of human monoclonal antibodies against rift valley fever virus high-throughput isolation of immunoglobulin genes from single human b cells and expression as monoclonal antibodies early appearance of germinal center-derived memory b cells and plasma cells in blood after primary immunization cutting edge: long-term b cell memory in humans after smallpox vaccination sitespecific recombinational cloning using gateway and in-fusion cloning schemes key: cord- -f qdr authors: may, valerie; larkum, a. w. d. title: a subtidal transect in jervis bay, new south wales date: - - journal: austral ecol doi: . /j. - . .tb .x sha: doc_id: cord_uid: f qdr long‐term variations in the intertidal algal flora of the entire rock platform at plantation point, jervis bay, are described by may ( ). the study now reported presents similar long‐term changes in the flora of a subtidal region of the same headland, observed within the same period of time. this first detailed report of subtidal macroalgal communities in new south wales describes a several‐year study of the benthic communities along a transect in the upper sublittoral region of a rocky headland at plantation point, jervis bay. eighty‐nine species of algae were recorded, five of which were previously unrecorded for new south wales. the area studied is dominated by the large brown algae ecklonia radiata and phyllospora comosa, large areas of which were cleared periodically by storms. turf, shade and crustose coralline algal communities also were present. storms, seasonal variation and longer term changes all affected the abundance and distribution of the algal species growing along the transect and hence the floristic composition of the area. changes in the intertidal algal flora of the entire rock platform at plantation point, jervis bay, are described by may ( ) . it was considered that additional information on changes in the flora of a subtidal region of the same headland, observed within the same period, could yield valuable additional information. hence serial observations were begun concurrently on an underwater transect. the transect was chosen, on practical grounds, to include a variety of communities and to exhibit gradual changes in such factors as slope, depth, light, exposure to wave action and kind of substrate. it was also in the general region of a planned sewer outfall. this paper gives the results of a study of part of the benthic algal flora of jervis bay and presents the first report for new south wales of a wholly subtidal transect. the only other work published on subtidal communities of australia is that of shepherd & womersley ( , , which relates solely to south australia. the present work was begun in september as part of a systematic survey of the intertidal and subtidal algae of plantation point, jervis bay, in response to plans to site a sewer outfall there. the outfall was placed in close proximity to the study site in august , months after the beginning of the study. sampling continued until . the present paper deals entirely with the subtidal algae. the intertidal algae, including a separate study of those occurring near the sewerage outfall, are the subject of separate communications. collett ) . the point is exposed to a certain amount of wave action, but this is much less than on the adjacent open ocean coast. a transect was located near the eastern tip of plantation point and ran seaward in a southerly direction for some m (fig. ) . seven sampling stations (a-g) were marked along this transect and were chosen so that they would include the major vegetational and topographic zones along the transect. station a was at an angle to the other transect stations and was a gully connecting directly to the site of the sewer outfall. the sampling region was approximately m wide. collecting was carried out by scuba diving. samples of representative algae were collected by hand and placed in plastic bags. small chips of rock were also taken for identification of smaller algae. to avoid disturbance to the vegetation of the stations, collecting was carried out over a wide but prescribed area, which was chosen according to the size and characteristics of each station. it is considered that sampling by this technique had a negligible effect on the flora, especially in comparison with the effects of storms and seasonal changes. the algae were identified in the laboratory by one of us (v.m.). the study area was first investigated in june and on several subsequent occasions, but collecting along the transect did not begin until september . collections were made on separate occasions from to (see table a ). the collection of april was lost and only a photographic record of forty coloured slides remained so that the list of species compiled from the photographs for that date is likely to be incomplete, particularly for smaller species. however both this (with twenty species) and the next collection in july, in which twentynine species were recorded, followed closely upon storms which denuded many rock surfaces in the transect area of their algae. for comparison with other collections see table a . the sublittoral algal communities of plantation point have been described briefly by larkum ( ) . during the period of the present study both ecklonia radiata and phyllospora comosa occurred, sometimes intermixed, on rock surfaces around the point. however, in the shallower region west of the point where the exposed rock platform grades into a shallowly shelving bottom of shale, sand and shells these two algae were replaced by smaller brown algae (such as sargassum spp. and caulocystis cephalornithos) and many turf algae; caulerpa cactoides also occurred frequently there. in the deeper water ( - m) of this area there were beds of the seagrass posidonia australis growing in sand. however, these beds did not occur on the southern side of the point. on the southern side ofthe point the ecklonia radiata and fhyllospora comosa were patchily distributed. they were interspersed with communities of crustose coralline algae, turf algae and, on overhanging rocks, of shade algae and sponges. the transect site crossed algal communities representative of those found in the sublittoral region on the eastern and southern sides of the rock platform. eighty-nine species of algae were found during this study (see appendix). five species of algae in the study have not previously been recorded for new south wales although they are known for adjoining states. these species are as follows: ( ) cladophora feredayi harv., known from victoria (womersley ). ( ) glossophora nigricans (j. ag.) wom., known from victoria (womersley ) . ( ) lophosiphonia prostrata (harv.) falk., known from queensland (cribb ) . as recorded previously, this prostrate species occurred epiphytic on members of the zonarieae, here mostly on lobophora variegata and zonaria turneriana. ( ) lophosiphonia reptabunda (suhr) cribb, previously known from queensland (cribb ). ( ) stypopodium zonale (lamour.) pap., reported from queensland (bailey ) . specimens of these and other species of algae recorded from plantation point, including the transect, are stored at the national herbarium of new south wales, royal botanic gardens, sydney (nsw). table lists the occurrences and distribution of the species which were common in the transect during this study and table gives similar information for the rare species. the flora of the gully (station a) was very heterogeneous, often with mixed ecklonia radiata and phyllospora comosa on the sides and e. radiata and turf algae on rock slabs on the floor. the rock slabs on the floor, some measuring up to several cubic metres in volume, were overturned on several occasions by severe storms, which kept the vegetation at a young stage of growth. the next station of the transect (station b), a horizontal shelf, had a uniform turf flora ( fig. ) with no large plants of e. radiata, p. comosa or sargassum spp.; breaking wave action at this site seems to have been too severe for large algae. this station and station d were probably subject to the heaviest wave action occurring in the transect. station c consisted of the sides of a deeper channel flanked by submerged overhanging rocks. there was no visible growth in the channel bottom which was composed of loose shells and boulders. there was some water surge. this station appears to be distinctive from other stations of the transect because of its low light intensity, as shown in table . it had a uniform flora of shade algae, crustose or small creeping reds and occasional small brown and green algae ( table ) . station d was towards the outer (weather) edge of a submerged rock outcrop exposed to heavy breaking wave-action. the vegetation on this outcrop changed in a distance of m from crustose coralline algae through turf algae (on the seaward side) to a mixed stand of ecklonia radiata and particularly phyllospora comosa (on the shoreward side). the latter species sometimes overlaid the adjacent turf of small algae as shown in fig. . station e was a small site at the base of the rock outcrop of station d, on a subhorizontal ledge. it was a deep site ( m-see fig. ) and was shaded by the rock outcrop so that the irradiance was much less than at other sites, apart from stations c and f. wave action was usually less than in preceding stations, but the site was subject to storm-generated abrasive action of small rocks and gravel from the adjacent site f. the commonest algae at this site were filamentous or small terete or foliose algae. station f included the deepest site ( to . m) and consisted of a fairly flat seabed covered with sand, gravel, shells and semi-mobile rocks. many hairy mussels (trichomya hirsute) were present and these consolidated a large part of substrate. a number of crustose and articulate coralline algae were also common and these too helped to consolidate the substrate. nevertheless the severe storms that took place during the study mobilized much of the area and the resultant abrasion had a severe detrimental effect on the mussels and the algal flora. large plants of ecklonia radiata and phyllospora comosa were absent from this area except on some large boulders near the outer extremity. sargassum spp., cystophora moniliformis and cladostephus verticillatus were common. delisea pulchra was also common at this station and at station g but was absent or very rare elsewhere. station g was situated on a large shallow submerged rock outcrop some m offshore. this station was the least disturbed by water movement. extensive patches of large plants of ecklonia radiata occurred in this area before the great storm of june (see below) and these had largely become re-established by late . however, there were many large patches with crustose coralline algae, turf algae or flrgfls u/ri spp. caulerpa cactoides, common in , was noted to be covered with crustose coralline algae in april following storms in february-march. after further storms from june - , plants of c. cactoides at station g were observed in july to be almost white and this was the last time that this species was observed in the transect or near it, although a specimen was observed in a protected gully in deeper water near to station g in november . o pl. n, , jan. dec. most of the algal species occurring in the transect stayed relatively constant in distribution or varied seasonally. some algae were of rare occurrence (table ). presumably these were present more abundantly elsewhere; for example, on the sheltered northern side of the headland, or intertidally, and invaded the transect area for short periods of favourable conditions. the most prominent algal associations of the transect area were those dominated by ecklonia radiata and phyllospora comosa. these two associations occurred as a mosaic, intermixed throughout the transect area, including the gully (station a). they were both unevenly distributed and intermixed with patches of other large algae such as sargassum spp. and cystophora moniliformis, small areas of turf algae (dominated by zonaria turneriana, lobophora variegata and various red algae), and patches of rock covered by encrusting calcareous algae. the most abundant algae beneath large plants of ecklonia radiata and phyllospora comosa in stations a, d and g were codium lucasii, lobophora variegata, zonaria turneriana, amphiroa anceps and corallina officinalis. light measurements indicated that the submarine irradiance reaching the understorey vegetation varied between and % of that incident on the canopy. thus the understorey algae were strongly shaded; they included some of the same species as at shaded station c, but in different relative abundance. at station c the most abundant species were (?) nitophyllum sp. peysonnelia capensis and heterosiphonia australis. the following algae were to be found at most stations throughout the transect (table ) : cladophora repens, enteromorpha intestinalis, lobophora variegata, sphacelaria tribuloides, stypopodium zonale, zonaria turneriana, acrosorium uncinatum, fosliella farinosa, gelidium pusillum, heterosiphonia australis, laurencia brongniartii, plocamium cartilagineum, polysiphonia sp. and a red encrusting calcareous alga. however, their abundance often varied markedly from station to station and from season to season. by contrast a few species occurred only at a single station e.g. caulerpa cactoides at station g and zonaria sinclairii at station b ( table ) . the stations at which the commoner species occurred most frequently are shown in table . if two stations had identically high numbers, both are given. from this table it appears that stations a, d and g had a greater number of species occurring with high frequency ( , and respectively). it is noteworthy that these stations as a group, were not distinct from all other stations by virtue of their area, distance from shore, depth, light intensity or degree of water movement. at the other extreme. station c had only five frequently occurring species and thirty species found elsewhere on the transect never occurred there. the distinctive feature of this station appeared to be its low light intensity. a number of species showed seasonal changes in distribution and abundance. the total number of species occurring on any one collection date are seen in table a . pooled data for the periods august-february (early spring to summer) and march-july (autumn to winter) showed that more species were widespread in spring/summer than in autumn/winter (table b) . summer - had somewhat fewer species, and the following winter somewhat more species, than in other years. table lists the season at which many species were most widely distributed. twenty-nine species showed seasonal (mainly summer) maxima although the time spans were not necessarily the same. four species (indicated) showed an erratic upswing in distribution in july, presumably due to increased space being available, after the heavy storms in the previous month. reproductive tissue was not found sufficiently frequently in the collected material to obtain good seasonal information on reproduction. in most species reproductive plants appeared to occur throughout the year. however, sargassum spp. were more fertile in october-february (summer) and heterosiphonia australis was fertile (tctrasporic) from may to october (autumn-spring). - - -- - --- ------- ------ -- -- - -- - (b) . pooled data on the total no. of species (including all sampling stations) and average number of species (all stations) per collection for the periods aug. -feb. and march-july. - - - - the many storms that occurred during the study period caused much damage to the larger algae. the very severe storm of june almost completely denuded the area of e. radiata and phyllospora comosa. subsequent storms in may , february-march , june , may , and june caused much damage to all large algae. however, in the period - , a gradual re-establishment of e. radiata and p. comosa occurred, bringing their densities back by late to those prior to june (see fig. ). (field observations by a.w.d.l.). apart from the storm-induced changes in abundance of certain species there were few dramatic changes in the flora of the transect from to . however floristic changes did occur. these are seen by comparing the two years of detailed observations ( and ) , there being eight collections in each year. considering first the rare species: during the study period twenty-one species were rare (defined here as up to three station-occurrences during the whole survey)-see table . it is seen that only one of these, platysiphonia miniata, occurred in both years, despite the fact that these years had eleven and nine occurrences of rare species, respectively. thus there appears to be a relatively constant number of these plants of rare occurrence, but they consist of species which differ from time to time. other differences between the floras of and were apparent when the number of occurrences of certain common species at various stations was totalled. although this method does not take the species abundance into account, it does indicate changes in distribution of species. those species showing greatest numerical differences in frequency of occurrence between the two years are listed in table , groups and . group lists some less common (but not rare species as listed in table ) algae, six of which disappeared in or and did not reappear, and one species which was seen for the first time in . when the numbers of species in each of these groups are totalled (table ) it appears that ten species were more prevalent in , while another eleven species were more prevalent in . these twenty-one species should be considered in terms of the total transect flora of eighty-nine species. as with the species of rare occurrence, the total number of species occur- november-march september-november (also peak in july, ) october-march (only) october-december (also peaks in july) august-january (only) september-december (also peak in july. ) august-november july-january october-december september-march july-november august-october february-april november-january july-november september-november august-november july-december july-december 'sargassum species are not considered since young plants cannot be determined to species. tsee footnote in appendix, ring stayed relatively constant, but some of the component species changed in their frequency of occurrence between the two years. another means of comparing the flora in and is to calculate similarity coefficients on the basis of species presence or absence (hruby ) . for this comparison the eight observations of each of these years were pooled and the total number of species occurring in them used to calculate the s(^rensen similarity coefficient (table -complete similarity is attained with a coefficient of unity). each station showed some change, with a coefficient between . and . for the total number of species. station e showed a very low coefficient ( . ) for the green algae; that is, a very significant difference between and . however, the number of green algae involved (ten species) was small, so that large random changes might be expected. shepherd & womersley ( , , have published the only previous work for australia, on subtidal communities in south australia. they suggest that three subtidal zones exist: ( ) an upper sublittoral zone, the depth of which is dependent on wave action, in which only turf algae occur, ( ) a mid-sublittoral zone dominated by ecklonia radiata and other large brown algae, and ( ) a lower sublittoral zone at depths greater than m where light is severely limiting. in contrast to shepherd & womersley's studies, no distinct upper sublittoral zone could be discerned in our studies. however, the somewhat sheltered site at plantation point may well have produced a merging of the two upper zones. womersley ( ) also reports that in calmer water the second zone of larger brown algae may extend almost to low tide level. station b taken at a depth beginning at m, had only small plants of the larger species of brown algae together with a complete covering of turf algae and may well have been representative of this merged zone and . on the open coast adjacent to jervis bay, ecklonia radiata and other large grown algae were restricted to considerably deeper waters and an upper turf or crustose coralline zone existed. the transect site clearly contained a heterogeneous assortment of communities ranging from areas dominated by shade algae (station c) to those containing fairly large plants of ecklonia radiata (e.g. station g). the e. radiata stands never achieved the density and consistency found at some other deeper sites in new south wales. this could clearly be ascribed to the effect of storms during the -year study period. although was exceptional for severe storm damage it seems probable that fairly regular cycles of denudation of larger algae and regrowth would occur at i\ times. therefore we believe that storms were the major factor controlling the size and density of these particular communities and causing the patchy heterogeneous distribution. it appears likely (see table b ) that storms had a severe effect on the algal populations of the transect in the first six months of but that most species recovered within a few months-an obvious exception (see table , group ) being caulerpa cactoides, which never reappeared. further, the erratic july increase in the distribution of certain species (marked in table ) is likely to be due to the increased availability of denuded sites following storm damage. wave action at times other than during storms october. november, april, october, october, maximum another major physical factor affecting the communities was underwater irradianee, the limitation of which under overhanging rocks, as in station c, and possibly under the canopy ole. radiata and p. comosa, gave rise to distinct shade communities. the influence of biotic factors on the communities was not investigated. a number of herbivores was present, such as sea urchins (heliocidaris erythogramme and centrotus rodgasii), limpets and the large turban-shell (ninella torquata). therefore herbivorous activity may have been an additional controlling factor at some sites. any assessment of long-term algal population changes is very difficult but there does seem to be a definite change in the composition of the whole transect when the two years and are considered. the distribution changes (table ) , the differences in composition of the rare species flora (table ) and the similarity coefficients (table ) , all do suggest a change in the algal populations between these years. possible causes of such a change during the study period (if it is not part of a long-term cycle) are the effect of the particularly severe storm damage of or the release of sewage effluent some m from station a from august onwards. if the latter were the cause, a gradient of change might be shown with the stations furthest from the effluent outlet showing the least change. no such gradient was apparent in the analysis of the similarity coefficients (table ) but, since the stations are adjoining each other, the gradient might be expected to be slight at best except perhaps in station a, which was nearer to the outfall site. it is possible that the changes under discussion result from the denudation of ecklonia radiata and phyllospora comosa in the storms of , and its gradual re-establishment throughout the study period. certainly, large changes in occupied space, available irradiance and water turbulence attended the re-establishment of large plants of e, radiata and p, comosa. however, this re-establishment of large plants occurred particularly only at stations a and g, whereas the similarity coefficients (table ) indicated changes of similar magnitude at all stations throughout the transect. we are therefore unable to determine the cause of the changes that occurred in species composition and frequency; we merely demonstrate its occurrence. although the cause of this drift over a matter of years is as yet undetermined, its * occurrence needs to be considered in such studies as environmental impact surveys. a comparison of marine floras based on observations taken over only two years is obviously inadequate. may ( ) demonstrated the occurrence of long term variation in algal intertidal floras, extending over several years. the present study indicates that such changes occur also in subtidal regions. algae. in; comprehensive calatogue of queensland plants records of marine algae from south-eastern queensland polysiphonia & lophosiphonia seasonal changes in two algal populations from the coastal waters of washington state the marine plants of jen-is bay long-term variation in algal intertidal floras a comparative study of epiphytic algal communities on two common genera of seagrasses in eastern australia the sublittoral ecology of west island, south australia. . environtnental features and the algal ecology pearson island expedition . . the subtidal ecology of benthic algae the subtidal algal and seagrass ecology of a critical survey ofthe marine algae of southem australia. i chlorophyta a critical survey ofthe marine algae of southem australia. ii phaeophyta aspects on the distribution and ecology of subtidal marine algae on southem australian coasts acrosorium uncinatum (j. ag.) kylin amphiroa anceps (lamour.) decne. anotrichium tenue (ag.) naeg. (formerly grifftthsia tenuis c. ag.) audouinella daviesii (dillw gerloff bangia fuscopurpurea (dillw.) lyngb. bryopsis sp. (as recorded in may etal., ) callithammion sp. -^polysporangia found but insufficient material for definite identification*-caulerpa cactoides centroceras clavulatum (c. ag.)mont. ceramium sp. chaetomorpha aerea (dillw.) kuetz. champia sp. (as recorded in may etal., ) chondria dasyphylla (woodw cystophora moniliformis (esper.) wom. & niz. delisea pulchra (grev.)mont. dictyopteris acrosdchoides ectocarpales complex (as recorded in may etal compacta cribb lobophora variegata (lamour.) wom. lomentaria australis (kuetz.) levr. lophosiphonia prostrata (harv.) falk suhr.)cribb martensia elegans hering crouan (?) nitophyllum sp. padina fraseri (grev.) grev. petalonia fascia (muell.) kuntze petrospongium rugosum polysiphonia sp. (inci. both p. mollis h. & h. & p. macrocarpa harv. as recorded in may et al., ) porphyra columbina mont. pterosiphoniapennata (ag.) falk. ralfsia sp. rhabdonia nigrescens harv. (as recorded in may etal., ) rhodymenia australis (sond.) harv. sargassum flavicans (mert.) c.ag subtidal transect in jervis bay sphacelaria tribuloides menegh. spyridiafilamentosa (wulf.) harv. stypopodium zonale (lamour.) pap. symphyocladia marchantioides (harv.) falk. viva lactuca. l. young siphonous alga; possibly vaucheria sp. (as recorded in may er a/., ) wrangelia plumosa harv these genera have not so far been recorded from new south wales, and the present inadequate collections are considered insufficient for a determination and location record. hence the collections are here reported merely as caluihamnion sp we are grateful to members of the nsw state fisheries for making transport and diving facilities available to us. in particular we are grateful to dr l. collett, now at the department of conservation, victoria, for much help and encouragement. dr bruce allender, monash university, kindly identified the stypopodiwn zonale, mr r. west, mr k. black and dr m. greenway provided underwater assistance on several occasions. key: cord- -abs rvjk authors: liu, ming; kong, jian-qiang title: the enzymatic biosynthesis of acylated steroidal glycosides and their cytotoxic activity date: - - journal: acta pharm sin b doi: . /j.apsb. . . sha: doc_id: cord_uid: abs rvjk herein we describe the discovery and functional characterization of a steroidal glycosyltransferase (sgt) from ornithogalum saundersiae and a steroidal glycoside acyltransferase (sga) from escherichia coli and their application in the biosynthesis of acylated steroidal glycosides (asgs). initially, an sgt gene, designated as ossgt , was isolated from o. saundersiae. ossgt -containing cell free extract was then used as the biocatalyst to react with structurally diverse drug-like compounds. the recombinant ossgt was shown to be active against both β- and β-hydroxyl steroids. unexpectedly, in an effort to identify ossgt , we found the bacteria laca gene in lac operon actually encoded an sga, specifically catalyzing the acetylations of sugar moieties of steroid β-glucosides. finally, a novel enzymatic two-step synthesis of two asgs, acetylated testosterone- -o-β-glucosides (at- β-gs) and acetylated estradiol- -o-β-glucosides (ae- β-gs), from the abundantly available free steroids using ossgt and ecsga as the biocatalysts was developed. the two-step process is characterized by ecsga -catalyzed regioselective acylations of all hydroxyl groups on the sugar unit of unprotected steroidal glycosides (sgs) in the late stage, thereby significantly streamlining the synthetic route towards asgs and thus forming four monoacylates. the improved cytotoxic activities of ′-acetylated testosterone -o-β-glucoside towards seven human tumor cell lines were thus observable. steroidal glycosides (sgs) are characterized by a steroidal skeleton glycosidically linked to sugar moieties, which can be further acylated with aliphatic and aromatic acids thus forming complex acylated steroidal glycosides (asgs) . the resulting steroidal glycoside esters (sges) exhibit a wide variety of biological activities, like cholesterol-lowering effect , anti-diabetic properties , anti-complementary activity , immunoregulatory functions , and anti-cancer actions [ ] [ ] [ ] , which made asgs promising compounds with pharmaceutical potential. numerous methods, including direct extraction , chemical synthesis , and biosynthesis , have been developed to synthesize these acylated steroidal glycosides. direct extraction from varied organisms is one of the main methods to obtain asgs [ ] [ ] [ ] . however, the content of asgs was usually low in natural sources [ ] [ ] [ ] . moreover, the extraction routes were highly time-consuming and required laborious purification procedures [ ] [ ] [ ] , resulting in poor yields and/or low purity of the final products. the production of asgs was also achieved by chemical synthesis previously [ ] [ ] [ ] [ ] [ ] . however, these efforts often encounter a fundamental challenge, namely, regioselective acylation of single hydroxyl group of unprotected sgs in the late stage of the chemical synthesis of asgs. sgs generally possess multiple hydroxyl groups with similar reactivity. regioselective acylation of a particular one of multiple hydroxyl groups generally requires multi-step protection/deprotection procedures, which makes the synthetic pathway of these sges costly, wasteful, long and timeconsuming, and results in low yield in the end. the biosynthesis of asgs from free steroids based on enzymatic catalysis was deemed to reduce the number of protection/ deprotection steps due to the high selectivity of enzymes. theoretically, the biosynthesis of asgs includes two steps. in the first reaction, the sugar moiety from nucleotide-activated glycosyl donors was attached to steroids at different positions, most commonly at the c- hydroxyl group (oh), under the action of nucleotide dependent sgts , . the glycosylation of a hydroxyl group at the c- position of steroids was well characterized and a few of steroidal β-glucosyltransferases were isolated from diverse species , . however, the reports of sgts specific for positions other than c- of steroids are limited. the sugar moieties of the resultant sgs can further be acylated by sgas to form asgs in the next step , . compared to sgts, surprisingly little is known about sgas. up to date, no sga genes has yet been cloned, which in turn limit the enzyme-mediated biosynthesis of asgs. hence, the successful gene isolation and functional characterization of sgas have become a prominent challenge for bioproduction of asgs. herein, the functional characterizations of a plant-derived sgt with activity against both β-and β-hydroxyl steroids, and a bacterial sga, as well as their application in the biosynthesis of asgs are reported. initially, a steroidal glycosyltransferase ossgt was isolated from medicinal plant o. saundersiae and showed activities for β-and βhydroxyl steroids. unexpectedly, in an effort to identify the function of ossgt , we characterized laca protein (designated as essga ) from e. coli as a sga, catalyzing testosterone- -oβ-glucoside (t- β-g) and estradiol- -o-β-glucoside (e- β-g) to form corresponding acylates. further, under the synergistic actions of ossgt and essga , the biosynthetic preparation of two acylated steroidal glycosides (asgs), namely acetylated t- β-gs (at- β-gs) and e- β-gs (ae- β-gs), was first achieved, thereby yielding four monoacetylated steroidal glucosides, namely -o, -o, -o and -o-acylates (scheme ). the cytotoxic activities of these monoacylates were evaluated against seven human tumor cell lines (hct , bel , mgc , capan , nci-h , nci-h and a ) and -acetylated testosterone -o-β-glucoside was observed to display improved cytotoxic activity against these seven cell lines (scheme ). the species o. saundersiae is a monocotyledonous plant rich in steroidal glycosides, suggesting that it may contain sgts responsible for the glycosylation of steroidal aglycons [ ] [ ] [ ] . o. saundersiae is thus selected as the candidate plant for sgts isolation. the transcriptome of o. saundersiae was thus sequenced with the aim of isolating genes encoding sgts. a total of , , raw reads were generated after the transcriptome sequencing of o. saundersiae. after removal of dirty reads with adapters, unknown or low quality bases, a total of , , clean reads were retained. these clean reads were combined by assembling soft trinity to form longer unigenes. finally, an rna-seq database containing , unigenes with mean length of bp was obtained. next, these unigenes were aligned to publicly available protein databases for functional annotations, retrieving unigenes displaying the highest sequence similarity with sgts. unigene with bp in length was thus retrieved from the unigene database for its high similarity with sgts ( supplementary information fig. s ). moreover, orf finder result showed that this unigene contained a complete open reading frame (orf) of bp, starting at nucleotide with an atg start codon and ending at position with a tga stop codon. the unigene contained bp of -utr (untranslated region) and bp of -utr. therefore, unigene was selected for further investigation. to verify the identity of unigene , a nested pcr assay was therefore carried out to amplify the cdna corresponding to the orf of unigene using gene-specific primers (supplementary information table s ). an expected band with approximately . kb was obtained, as observed in agarose gel electrophoresis ( supplementary information fig. s a ). the amplicon was then inserted into peasy tm -blunt plasmid (supplementary information table s ) to form a recombinant vector for sequencing. results indicated that the amplified product was % identity with that of unigene , confirming unigene was a bona fide gene in o. saundersiae genome. the bp orf encoded a polypeptide of amino acids (aa) with a predicted molecular mass of . kda and pi of . . blast analysis of the deduced protein revealed its predominant homology with sterol β-glucosyltransferase from elaeis guineensis (xp_ . , %), musa acuminata subsp. malaccensis (xp_ . , %) and anthurium amnicola (jat . , %). the cdna was therefore designated as ossgt and submitted to genbank library with an accession number of mf . the sequence analyses of ossgt were first assessed with the aim to direct its expression and functional verification. no putative trans-membrane domain was observed in ossgt based on the prediction results by tmhmm (http://www.cbs.dtu.dk/services/ tmhmm/), suggesting ossgt is a cytoplasmic sgt and may be expressed heterologously in e. coli in a soluble form. multiple alignment of ossgt and other plant sgts indicated that the middle and c-terminal parts of these sgts were more conservative than the n-terminal region ( supplementary information fig. s ), consistent with previous notion . moreover, two conservative motifs, namely a putative steroid-binding domain (psbd) and a plant secondary product glycosyltranferase box (pspg), were observed in ossgt ( supplementary information fig. s ). the region named psbd located in the middle part of ossgt and was thought to be involved in the binding of steroidal substrates . pspg box is about aa in length and close to the carboxy-terminus. this box is a characteristic "signature sequence" of udp glycosyltransferase and deduced to be responsible for the binding of the udp moiety of the nucleotide sugar . the presence of psbd and pspg boxes suggests that ossgt may be involved in secondary metabolism, catalyzing the transfer of udp-sugars to steroidal substrates thereby forming steroidal glycosides. the phylogenetic tree based on deduced amino acid sequences of ossgt and other sgt was generated by mega . . as scheme an enzymatic two-step synthesis of at- β-g ( b- e) and ae- β-g ( b- e) from the free steroids testosterone ( ) and estradiol ( ) . firstly, two sgs, t- β-g ( a) and e- β-g ( a), were prepared from their corresponding steroidal substrates testosterone ( ) and estradiol ( ) in the presence of a steroidal glycosyltransferase ossgt from o. saundersiae. the resulting t- β-g ( a) was further regioselectively acetylated under the action of an acyltransferase ecsga from e. coli, thereby yielding four monoacetylated steroidal glucosides ( b- e) with the yield ratio of : : : . likewise, e- β-g ( a) was acetylated by ecsga to form monoacetylated products b- e in a ratio of : : : . shown in supplementary information fig. s , all selected sgts were clusted into four clades, mon, di, ba and fun clades. the four clades included sgts from monocots, dicots, bacteria and fungi, respectively. ossgt belonged to mon clade, suggesting that ossgt was most similar to sgts from monocots. ossgt was then inserted into pet- a(þ) to yield a recombinant pet a-ossgt (supplementary information table s ) , which was transformed into transetta(de ) (transgen, beijing, china) for heterologous expression. sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) indicated that most of the expressed ossgt protein was present in the form of insoluble inclusion body, which was regarded to be devoid of bioactivity. it was well known that chaperone proteins were able to assist protein folding and thus increase production of active protein . therefore, a chaperone plasmid pgro (takara biotechnology co., ltd., dalian, china) was applied to be co-expressed with pet aossgt in bl (de ) (transgen, beijing, china), facilitating the soluble expression of ossgt . as shown in supplementary information table s , the plasmid pgro contains two genes encoding chaperone proteins groes and groel. under the synergistic action of chaperones groes and groel, an intense band with an apparent molecular mass of kda was present in the crude extract of bl (de )[pet aossgt þpgro ], but not in the crude proteins of the control strain bl (de )[pet- a (þ)þpgro ] ( supplementary information fig. s b ). the immunoblot analysis with an anti-polyhistidine tag antibody showed a bound band, but the control extract did not cross-react with the antibody ( supplementary information fig. s c ). these data collectively indicated that ossgt was successfully expressed in e. coli in a soluble form (supplementary information figs. s b and c) in accord with the predicted result of soluble expression of ossgt . to identify the activity of ossgt , the ossgt -containing crude protein was used as the biocatalyst for glycosylation reactions. each member of the acceptor library ( - , fig. b and supplementary information fig. s ) was first assessed as sugar table s ). of the substrates, only steroids were observed to be glucosylated by ossgt , forming corresponding monoglucosides (figs. - and supplementary information figs. s - ) . the ten steroids included seven β-hydroxysteroids ( - , supplementary information figs. s - ), two β-hydroxylsteroids ( - , figs. and ) and one β, β-dihydroxysteroid ( , supplementary information fig. s ). the reaction activities of ossgt towards the ten substrates indicated that ossgt was an sgt showing activities towards both β-and β-hydroxysteroids, consistent with the predicted result by bioinformatics analyses (supplementary information figs. s and s ). in fact, the reports of sgts with glycosylation activity against steroids at positions other than c- were limited and only three sgts from yeast were verified to exhibit selectivity towards both β-and β-hydroxylsteroids . ossgt was therefore viewed as the first plant sgt with selectivity towards both β-and β-hydroxylsteroids (figs. and and supplementary information figs. s - ) . however, the glycosylation activity of ossgt towards βhydroxyl group would be lost if additional hydroxyl group at β-( β-oh-testosterone, ), β-( β-oh-testosterone, ), β-( β-oh-testosterone, ), or α-position ( α-oh-testosterone, ) , even a methyl group at c -position (methyltestosterone and its derivatives, - ) was attached to testosterone ( ), generating not any glycosylated products. moreover, ossgt has no activity towards other compounds, including steroids without β-and β-hydroxyl groups ( - and - ) , flavonoids ( - ), alkaloids ( ) ( ) ( ) ( ) ( ) ( ) ( ) , triterpenoids ( - ), phenolic acids ( - ) and coumarins ( - ) as shown in supplementary information fig. s . among reactive β-and β-hydroxylsteroids, dehydroepiandrosterone ( ) had a maximum conversion approaching %, followed by diosgenin ( ) with % conversion and the other compounds having conversion below % (fig. a) . to produce sufficient glucosylated products for structural characterization, scale-up of ossgt -mediated reactions to preparative scale ( ml) was conducted. the resultant glucosides ( ) glycosylation. hplc chromatogram of reaction product of estradiol ( ) incubated with ossgt protein (a) or without ossgt (b). uv spectra of and enzymatic product a are shown in upper panels. the hplc conditions are available in supplementary information table s . were prepared by hplc and subjected to nmr analysis for structural elucidation. to determine the glycosylation sites of a- a, a and a, the c nmr analyses of the corresponding aglycons - , and were also performed (supplementary information figs. s - and table s ). the c nmr glycosylation shifts (Δδ, δ glucoside -δ aglycon ) of these glycosides were thus examined to ascertain the glycosylation position (table , and supplementary information tables s - ). the steroidal glycosides were observed to have significant glycosylation shift Δδs for c- (glycosides - ) or c- position (compounds - ), showing their -or -glycosides. for a and a, the location of glucose group was determined to be at c- based on their hmbc correlations between h- and c- (supplementary information figs. s and ). the β-anomeric configuration of the d-glucose unit in these ten glucosides ( a- a) was determined from the large anomeric proton-coupling constants of h- (j ¼ . hz) ( table and supplementary information tables s - ). the structures of these glucosides were thus assigned to βglucosides ( a- a and a) or β-glucosides ( a and a) of steroids based on h nmr ( a- a) and c nmr ( a- a) signals, hsqc ( a- a, a- a, a- a), hmbc ( a- a, a) and dept ( a, a) spectra (table , supplementary information figs. s - and tables s - ). these data collectively showed that ossgt was an inverting-type glycosyltransferase. in the preparation of t- β-g ( a), when the concentrated reaction mixture was separated by reversed phase highperformance liquid chromatography (rp-hplc), we accidentally discovered that in addition to the major peak representing t- β-g ( a), a minor peak ( b) was also present in the hplc profile ( supplementary information fig. s a ). the minor product with a t r of . min was also subjected to lc-ms analysis. surprisingly, the [m þ h] þ value of the minor product was assigned to . , more than that of monoglycosylated testosterone ( supplementary information fig. s b ). this finding hints that the minor product may be an acetylated testosterone glucoside. to characterize the exact structure of b, the minor product was prepared in bulk for nmr experiment (supplementary information figs. s - ). details of h and c nmr spectra were tabulated in table . the minor product was thus identified as -acetylated testosterone -o-β-glucoside ( -at- β-g, b). to test if the acetylated product b was from glucoside a, the purified glucoside a was used as the substrate to incubate with crude extract of e. coli expressing pet- a(þ) or pet ossgt , respectively. in both conditions, we observed the presence of b ( supplementary information fig. s ). on the contrary, no acetylated product b was detected in the e. coli lysate without the addition of substrate a ( supplementary information fig. s ). we therefore inferred that testosterone ( ) was first glycosylated at the β-hydroxyl group by ossgt to form t- β-g ( a), which was then selectively acetylated at c- of sugar moiety to yield the -at- β-g ( b) by a soluble bacterial acetyltransferase ( supplementary information fig. s ) . likewise, two metabolites, e- β-g ( a) and -acetylated estradiol -o-β-glucoside ( -ae- β-g, b), were detected in the concentrated ossgt -catalyzed reaction mixture of estradiol ( ) as shown in supplementary information figs. s - , tables s and s . these data collectively revealed that e. coli cell contained at least one sgas specific for the acetylation of steroidal β-glycosides. moreover, the sugar donor promiscuity of ossgt -catalyzed glycosylation reactions was also investigated. β-sitosterol ( ) and testosterone ( ) were chosen as the sugar acceptors to react with varied sugar donors listed in the supplementary experimental section, respectively. results demonstrated that both β-sitosterol ( ) and testosterone ( ) had no reactive activity towards other udp-activated nucleotides except udp-glc under the action of ossgt , indicating ossgt was specific for udp-glc. to characterize the genes encoding sgas, the first task was to analyze the genome sequence of bl (de ), which was public in ncbi database (accession no. cp . ). this bacterial strain contains at least putative acetyltransferase genes, in which laca, maa and wech, were predicted to encode o-acetyltransferase (supplementary information table s ). as shown in supplementary information fig. s , further sequence analyses revealed wech protein was a membrane-bound protein with a total of membrane-spanning helixes, inconsistent with the above results, in which the candidate acetyltransferase was determined to be a soluble protein in bacterial lysate. laca and maa proteins were predicted to have no transmembrane helixes, suggesting their soluble form in bacterial. thus, the remaining two genes, laca and maa, were further investigated. first, the entire orfs of the two genes were isolated from bacterial genome using gene-specific primers (supplementary information fig. s a and table s ). the orfs of laca and maa genes were and bp encoding polypeptides of and aa, respectively. the predicted molecular weights of the two proteins were . and . kda. the two genes were then inserted into pet- a(þ) to generate two recombinant vectors, which were introduced into bl (de ) for heterologous expression. after isopropyl-β-d-thiogalactoside (iptg) induction, the accumulation of approximately or kda was observed in the lysate of bacterial strain harboring pet a-laca or pet a-maa (supplementary information fig. s b ). moreover, the presence of bacterially-expressed his-laca or his-maa fusion protein in the bacterial lysate was verified by western-blot with anti-his antibody ( supplementary information fig. s c ). the expressed laca or maa protein was then purified to near homogeneity by affinity chromatography ( supplementary information fig. s b ). the purified his-laca or his-maa fusion protein was used as the biocatalyst to react with t- β-g ( a) and acetyl-coa. the reactions were monitored by hplc-uv/ms analysis using the method e (supplementary information table s ). as shown in fig. , -at- β-g ( b) was detected in laca-catalyzed bioconversion of t- β-g ( a), attesting laca encoded a sga (fig. , upper panel). on the contrary, there were not any new products in maa-mediated reaction. laca was thus designated as ecsga (e. coli steroidal glucoside acetyltransferase) for convenience hereinafter and submitted to genbank with an accession number of mf . it is generally accepted that hydrolases and acyltransferases are two classes of enzymes responsible for acylation reactions of sgs . the enzymatic acylations reported now are largely performed by hydrolases like lipases . on the other hand, not any genes encoding sgas are isolated up to date , . ecsga is therefore regarded as the first steroidal glycoside acyltransferase catalyzing the attachment of acyl groups into the hydroxyl groups of steroidal β-glycosides, to our knowledge. also, ecsga was observed to catalyze another steroidal βglycoside, e- β-g ( a), to form corresponding acylate ( b, fig. , upper panel) . on the other hand, the other glucosides listed in supplementary information fig. s could not be acetylated by ecsga , testifying ecsga was specific to steroidal β-glycosides. moreover, the acyl donor promiscuity of ecsga was investigated. t- β-g ( a) or e- β-g ( a) was used as the acyl acceptor to react with different acyl donors (acetyl-coa, succinyl-coa, arachidonoyl-coa, palmitoyl-coa and acetoacetyl-coa) under the action of the purified ecsga . results manifested that neither t- β-g ( a) nor e- β-g ( a) could react with these acyl donors except acetyl-coa, indicating that ecsga had strict donor selectivity. after careful check of ecsga -catalyzed reaction mixture in hplc profile, we have found several other minor peaks adjacent to the major product b (fig. , upper panel) . these minor peaks are so close that we could not distinguish. therefore, an efficient hplc method, namely method i (supplementary information table s ), was developed to separate these peaks. as shown in fig. (lower panel) , besides the major product b (t r ¼ . min), we observed three other minor peaks at t r ¼ . , . table s ). lower panel, hplc profile of acetylated products of a separated by chromatogramic method i (supplementary information table s ). and . min, respectively. the lc-ms measurement of these minor peaks showed that all of them have an [m þ na] þ value of . , thus suggesting their monoacetylated testosterone glucosides ( supplementary information fig. s ) . likewise, e- β-g ( a, t r ¼ . min) was observed to form four acetylated glucosides using purified ecsga as the biocatalyst (fig. ) . besides the well-characterized -ae- β-g ( b, t r ¼ . min), the other three products were determined to be monoacetylated estradiol glucosides based on their ms data ( supplementary information fig. s ). it was assumed that ecsga could introduce an acyl group into different hydroxyl groups of steroidal β-glycosides, generating varied monoacetylated products (figs. and ). to obtain sufficient amount of monoacetylated testosterone glucosides for structural characterization and further cytotoxicity assay, an enzymatic two-step process for at- β-gs ( b- e) was developed (scheme ). firstly, the whole cell biotransformation for the formation of at- β-gs ( b- e) was exploited due to its simple catalyst preparation. when testosterone ( ) was incubated with the engineered strain bl (de )[pet a-ossgt þpgro ], not any new products were detected. on the other hand, when t- β-g ( a) was added into the same whole-cell system, -at- β-g ( b) was present in the reaction mixture ( supplementary information fig. s ). these data indicated that testosterone ( ) could not be transported into the cell while the glycosylation of testosterone ( ) significantly improved the intercellular transport. thus, the formation of at- β-gs ( b- e) from testosterone ( ) using the single whole-cell biocatalyst is infeasible. a two-step process is therefore established to address this limitation. specifically, ossgt -catalyzed reaction was performed in the membrane-free crude cell extract of bl (de )[pet a-ossgt þpgro ], while ecsga -mediated acetylation was conducted in the whole-cell system of bl (de )[pet a-essga ]. the optimal ph and temperature of ossgt -catalyzed reaction using the cell-free extract of bl (de )[pet a-ossgt þp-gro ] as the biocatalyst were first determined to be alkaline ph value of and c, respectively (supplementary information fig. s ). next, the μl screening scale of ossgt -catalyzed glycosylation reaction was scaled to ml scale, in which mg testosterone ( ) were glycosylated to form mg t- β-g ( a) under optimized conditions (scheme ). the resultant t- β-g ( a) was subsequently used as the substrate applied in the scale-up of the whole-cell system of bl (de )[pet a-essga ] ( ml) under optimized ph . and c. the resulting reaction mixture was subjected to high-performance liquid chromatography-solid phase extraction-nmr spectroscopy (hplc-spe-nmr) measurement. comparison of the h and c nmr spectra of c- e with those of b suggested that compounds c- e had the same framework as b and the structural difference might be the position of the acetyl group. the location of acetyl group was determined to be at c- based on the hmbc correlations between h- (δ . ) and c- " (δ . ) as shown in supplementary information fig. s . thus, compound c was assigned as -at- β-g. the isolated glucose proton at δ . (h- ) of compound d exhibited long-range correlations with carbonyl carbons at δ . ( supplementary information fig. s ). moreover, h- (δ . ) of compound e showed long-range table s ). lower panel, hplc profile of acetylated products of a separated by chromatogramic method (supplementary information table s ). correlations with c- " (δ . ), as revealed by the hmbc spectrum ( supplementary information fig. s ). these data supported that the structure of d and e was elucidated as -at- β-g and -at- β-g, respectively. hence, the three trace products at t r ¼ . , . and . min were thus assigned to be -( d), -( e) and -at- β-g ( c) based on their respective nmr data (tables - and supplementary information figs. s - ) . these data indicate that ecsga can effectively introduce the acetyl group into the primary hydroxyl group and each secondary hydroxyl group of t- β-g ( a), yielding four monoacylates without the formation of diacylates ( fig. and scheme ). because the primary c( )-oh was the most reactive of the four hydroxyl groups in t- β-g ( a), acetylation of t- β-g ( a) took place preferentially at the c ( )-oh, giving -o-acylate predominantly in % yield ( fig. and scheme ). also, ecsga can regioselectively acetylate each secondary hydroxyl of t- β-g ( a) in the presence of the primary hydroxyl group, giving -( c), -( d) and -at- β-g ( e) in %, % and % yield, respectively. these data revealed the reactivity trend of hydroxyls is -oh -oh -oh -oh. likewise, the formation of four monoacylates was also present in ecsga -catalyzed acylation of e- β-g ( a, t r ¼ . min, fig. ). in addition to the well-characterized major product b, there are three trace products c- e. because of their trace amount, we did not further enrich these monoacetylated estradiol glucosides for nmr analysis. however, according to the catalytic behavior of ecsga towards t- β-g ( a), it was easy to infer that these products were most likely -( c, t r ¼ . min), -( d, t r ¼ . min) and -ae- β-g ( e, t r ¼ . min, fig. ). the order of reactivity of the hydroxyls was determined as -oh -oh -oh -oh with a yield ratio of : : : (fig. ) . regioselective acylation of one of the multiple hydroxyl groups in sgs is the major obstacle to the synthesis of sges and direct methods for site-selective acylation of unprotected sgs have rarely been documented. in this contribution, we successfully achieved the regioselective acylation of fully unprotected sgs using ecsga as the biocatalyst, thereby leading to an extremely short-step synthesis of asgs. acetylated steroidal glucosides, namely b, c, d and e, together with b were tested for their in vitro cytotoxicity against seven human cancer cell lines including hct , bel , mgc , capan , nci-h , nci-h and a . the results indicated that -at- β-g ( d) exhibited a wide spectrum of cytotoxic activities against the tested cell lines (table ). -at- β-g ( b) displayed much less cytotoxicity than -at- β-g ( d) but showed a mild cytotoxicity against human non-small cell lung carcinoma cell line nci-h with ic values of . μmol/l ( table ) . on the contrary, the control t- β-g ( a) did not display significant cytotoxicity towards these tested cell lines (ic . μmol/l). these evidences revealed that the acyl groups of sges are of importance to their cytotoxicity and direct regioselective acylation of sgs is thus believed as a powerful tool for the discovery of drug candidates. acylated steroidal glycosides have attracted our attentions primarily due to their biological and pharmacological significances , , , . there are two enzymes, namely sgts and sgas, responsible for the biosynthesis of asgs. to synthesize asgs, the primary premise is to obtain glycosyltransferases capable of catalyzing the formation of sgs from the abundantly available free steroids. o. saundersiae is thus selected as the candidate plant for sgts isolation. o. saundersiae is a monocotyledonous plant rich in acylated steroidal glycosides, suggesting that it may contain sgts and sgas responsible for the biosynthesis of asgs . thus, the transcriptome of o. saundersiae was sequenced with the aim to facilitate the genes discovery. ossgt was then isolated from o. saundersiae based on the rna-seq data. subsequently, ossgt -containing cell-free extract was used as the biocatalyst for glycosylations of structurally diverse drug-like scaffolds. the use of cell-free extract offers a number of advantages. unlike the ambitious purification procedures, the preparation of cell-free extract was simple and timesaving. moreover, compared the purified enzymes, the recombinant proteins used in crude extract-based system were more stable. steroidal glycosides are one of the main sources of innovative drugs . sgt-catalyzed glycodiversification of steroids could expand the molecular diversification, thereby facilitating the discovery of pharmacological leads. thus, the search of sgts with catalytic promiscuity may provide potent biocatalysts for glycodiversification. therefore, a library containing structural diverse drug-like molecules was utilized to react with the recombinant ossgt with the aim to explore the substrate flexibility of ossgt . in vitro enzymatic analyses revealed that ossgt was active against various steroids, including physterols ( - , supplementary information figs. s - ) , steroid hormones ( , - , figs. and , and supplementary information figs. s , s and s ), steroidal sapogenin ( , supplementary information fig. s ) and cardiac aglycon ( , supplementary information fig. s ) , exhibiting a wider substrate range than that of previously identified sgts from plant , . to investigate the regioselectivity of ossgt , diversified steroids were selected as the sugar acceptors for ossgt -catalyzed glycosylations. as illustrated in figs. - and supplementary information s - , ossgt specifically attacked the hydroxyl groups at c- and c- positions, but no activities towards hydroxyl groups at c- ( , ), c- ( ), c- ( ), c- ( ) , c- ( ), c- ( ) , c- ( , and ) and c- ( and ) . when steroids having two potentially reactive hydroxyl groups, like α-hydroxypregnenolone ( ) or androstenediol ( ), were used as the substrate for ossgt -assisted glycosylation, only glycosides with a glycosyl substituent in c- position were detected in the reaction mixture, suggesting ossgt exhibited prominent regioselectivity towards the -oh of both substrates ( supplementary information figs. s and s ) . also, ossgt the stereoselectivity of ossgt was also assessed in this study. estradiol ( ) and α-estradiol ( ) differ for the configuration of the hydroxyl group at c- position. when each of the two compounds was used to react with ossgt , only β-configurated glycosides were generated (fig. ) . likewise, ossgt showed βselective glycosylation towards the hydroxyl group at c- position. cumulatively, these evidences revealed that ossgt catalyzed glycosylations were conducted in a region-and stereoselective fashion. one of the most striking findings of this study is the characterization of bacterial laca protein as a steroidal glycoside acyltransferase. it is well known that laca is one of three structural genes (lacz, lacy and laca) in lac operon , . the function of lacz and lacy is well-characterized , . lacz encodes a βgalactosidase, catalyzing the cleavage of lactose into glucose and galactose. lacy encodes a lactose permease responsible for lactose uptake , . the third structural protein encoded by laca gene in lac operon was initially inferred to be an acetyltransferase. the exact action of this protein, however, remains in doubt until now. in this investigation, in an effort to identify the function of ossgt , we unexpectedly characterized laca protein from e. coli as a sga. in vitro enzymatic analyses revealed that laca protein could specifically catalyze the attachment of acyl groups into the hydroxyl groups of sugar moieties of steroidal β- glycosides (figs. and ) . although we have no evidences for the role of laca protein in vivo, these findings in the present work may provide a starting point for identifying the exact activity of laca protein in lactose metabolism. the bottleneck in enzymatic synthesis of asgs is the lack of well-characterized sgas. the successful characterization of laca made it to be the first sga and laca protein was thus designated as ecsga . a novel enzymatic two-step synthesis of at- β-gs and ae- β-gs from the abundantly available free steroids under the sequential actions of a steroidal glycosyltransferase ossgt from o. saundersiae and ecsga was achieved. the two-step process is characterized by acyltransferase-catalyzed regioselective acylations of all hydroxyl groups of unprotected sgs in the late stage, thereby significantly streamlining the synthetic route towards asgs and thus forming four monoacylates. regioselective acylation could expand molecular diversity, thereby facilitating the discovery of pharmaceutical leads. in this investigation, ecsga -catalyzed acetylation of two steroid βglucosides (t- β-g and e- β-g) leaded to the production of eight new monoacylates. furthermore, the cytotoxic activities of these monoacylates were tested and -at- β-g was observed to display improved activities towards seven human tumor cell lines, suggesting this compound had promisingly pharmacological potential. this study therefore reports for the first time a novel synthetic process for the green preparation of acylated steroidal glycosides with medicinal interest. a steroidal glycosyltransferase ossgt from o. saundersiae was identified to be the first plant sgt with selectivity towards both β-and β-hydroxylsteroids. one of the most striking findings of this study is the characterization of bacterial laca protein as a steroidal glycoside acyltransferase, catalyzing the attachment of acyl groups into the hydroxyl groups of steroidal β-glycosides. a novel enzymatic two-step synthesis of at- β-gs and ae- β-gs from the abundantly available free steroids under the sequential actions of ossgt and ecsga was achieved. the two-step process is characterized by acyltransferase-catalyzed regioselective acylations of all hydroxyl groups of unprotected sgs in the late stage, thereby significantly streamlining the synthetic route towards asgs and thus forming four monoacylates. moreover, the cytotoxic activities of these monoacylates were tested and -at- β-g was observed to display improved activities towards seven human tumor cell lines. this study therefore reports for the first time a novel synthetic process for the green preparation of acylated steroidal glycosides with medicinal interest. in this contribution, four compound libraries, namely sugar acceptor, sugar donor, acyl acceptor and acyl donor libraries, were provided for enzyme-mediated reactions. the compounds listed in fig. and supplementary information fig. s include diverse structures like steroids , flavonoids ( ) ( ) ( ) ( ) ( ) ( ) ( ) , alkaloids ( ) ( ) ( ) ( ) ( ) ( ) ( ) , triterpenoids ( - ), phenolic acids ( - ) and coumarins ( - ) are used as the sugar acceptors for ossgt -catalyzed glycosylation reactions ( fig. and supplementary information fig. s ). the sugar donors consist of seven udp-activated nucleotides, among which, udp-dglucose (udp-glc), udp-d-galactose (udp-gal), udp-d-glucuronic acid (udp-glca) and udp-n-acetylglucosamine (udp-glcnac) were obtained from sigma-aldrich co., llc. (st. louis, mo, usa). udp-d-xylose (udp-xyl), udp-l-arabinose (udp-ara) and udp-d-galacturonic acid (udp-gala) was synthesized by enzymemediated reactions in our laboratory [ ] [ ] [ ] . the acyl acceptor library is made up of steroidal glucosides ( a- a) and other glucosides ( - ) listed in supplementary information fig. s . the acyl donor library includes acetyl-coa, succinyl-coa, arachidonoyl-coa, palmitoyl-coa and acetoacetyl-coa, all of which were purchased from sigma-aldrich co., llc. the other chemicals were either reagent or analytic grade when available. the resulting raw reads from sequence library of o. saundersiae was firstly filtered to obtain clean reads, discarding dirty reads with adaptors, unknown or low quality bases. these clean reads were subsequently combined to form longer unigenes by assembling program trinity. these unigenes obtained by de novo assembly cannot be extended on either end. next, these unigenes were aligned by blast x algorithm to protein databases, such as nr, swiss-prot, kegg and cog (e-valueo . ) for functional annotation. the unigenes displaying similarity to sgts were retrieved for further orf analysis. in a word, those unigenes with a complete orf and displaying high similarity to sgts were selected as the candidate for further investigation. to verify the authenticity of the candidate unigene, cdna isolation was performed using gene-specific primers by a nested pcr assay as previously described (supplementary information table s ) [ ] [ ] [ ] [ ] . the obtained amplicon was inserted into peasy tm -blunt plasmid (transgen co., ltd., beijing, china) and then transformed into e. coli trans -t competent cells for recombinant plasmid selection (supplementary information table s ). the resultant recombinant plasmid was isolated and subjected to nucleotide sequencing. the obtained cdna was thus designated as ossgt for convenience. the bioinformatics analyses of ossgt , like prediction of physiochemical properties, multiple sequence alignment and phylogenetic analysis, were performed as detailed in our previous reports [ ] [ ] [ ] [ ] . ossgt was amplified using gene-specific primers (supplementary information table s ) and the resulting pcr product was ligated into ecori and hind iii sites within the pet- a(þ) vector (novagen, madison, usa) using seamless assembly cloning kit (clonesmarter technologies inc., houston, tx, usa) as described previously . the generated construct pet a-ossgt was transformed into e. coli strain transetta (de ) for expression as described previously . also, to improve heterologous expression of ossgt , pet a-ossgt was cotransformed into e. coli bl (de ) strain with a chaperone plasmid pgro (takara biotechnology co., ltd., dalian, china) as introduced by yin et al. the expression of ossgt was induced by iptg at a final concentration of . mmol/l. the expressed ossgt was checked by sds-page and western-blot analyses as described by guo et al. next, the bl (de ) [pet a-ossgt þpgro ] suspension cells were disrupted in a high-pressure homogeniser (apv- , albertslund, denmark) operated at bar. disrupted cells were centrifuged at , rpm for min to discard the pellet. the resultant supernatant, namely the membrane-free crude extract, was used as the biocatalyst for steroidal glycosylation. after verification of heterologous expression of ossgt , the crude extract containing the recombinant ossgt was applied as the biocatalyst to react with various sugar acceptors and donors ( fig. and supplementary information fig. s ). the total reaction mixture was μl contained mmol/l phosphate buffer (ph . ), a sugar acceptor ( mmol/l), a sugar donor ( mmol/l) and μl crude ossgt proteins. the reaction mixture was incubated at c for h. the formation of glycosylated products was table the cytotoxic activities of monoacylates against human tumor cell lines. unambiguously determined by a combination of hplc-uv, hplc-ms and nmr as described previously , . the determination conditions for hplc-uv were summarized in supplementary information table s . the genome dna was extracted from e. coli strain bl (de ) using bacteriagen dna kit (cwbio co., ltd., beijing, china) according to the supplier recommendation. the resulting genome dna was then used as the template of pcr amplification to isolate these candidate sga genes using gene-specific primers (supplementary information table s ). the amplified pcr products were inserted into peasy tm -blunt plasmid to generate recombinant vectors for sequencing verification. next, these oacetyltransferase-encoding genes were heterologously expressed in bl (de ) as described above. sds-page and western-blot of these recombinant proteins were conducted as that of ossgt (see above). the recombinant o-acetyltransferase proteins were subjected to purification with ni-nta agarose columns according to the manufacture's protocol. purified protein concentrations were determined using bradford protein assay (bio-rad, hercules, ca, usa). the enzymatic activities of ecsgas were determined in μl citrate buffer solution (ph . ) containing an acyl acceptor ( mmol/l) listed in supplementary information fig. s , an acyl donor ( mmol/l) summarized in chemicals section and the purified protein ( . μg). the reactions were incubated at c for h. then μl methanol was added to terminate the reaction. the reaction mixture was monitored by hplc-uv (supplementary information table s ) and the structure of the generated product was determined by a combination of hplc-ms and nmr as reported by liu et al. . . biotransformation of testosterone and testosterone- -oβ-glucoside using the whole cell system after iptg induction, the engineered bl (de )[pet a-ossgt þpgro ] cells were harvested by centrifugation at , rpm for min and then resuspended in m medium with cell density of od value of . . the substrate testosterone ( ) or testosterone- -o-β-glucoside (t- -o-β-g, a) with the final concentration of . mmol/l was added into the m medium and continued to incubate at c overnight. the formation of products was monitored by hplc analysis as mentioned above. the effects of ph and temperature on ossgt -and ecsga catalyzed reactions were investigated. the crude extract of bl (de )[pet a-ossgt þpgro ] and the purified ecsga protein were applied as the biocatalyst in their respective reactions. the effects of ph on both reactions were determined at varied buffers including citric acid/sodium citrate buffer ( . mol/l, ph . - . ), na hpo /nah po ( . mol/l, ph . - . ), na hpo / naoh buffer ( . mol/l, ph . - ). the influences of temperature were explored in a range of to c with intervals of c (ossgt -mediated glycosylation) or c (ecsga -catalyzed acylation) in the standard reaction mixture as described above. scale-up of ossgt -and ecsga -catalyzed reactions was performed to obtain sufficient at- β-gs for structural characterization and further cytotoxicity assay. initially, the μl ossgt catalyzed reaction was directly scaled to ml, in which mg testosterone ( ) were added into and then incubated with crude cell extract at optimal ph and temperature for h. the resultant reaction mixture was applied to preparative hplc to isolate pure t- β-g, which was then used as the substrate in ml ecsga -catalyzed reaction for at- β-gs production. structure characterization of at- β-gs was performed using hplc-spe-nmr technique as described by liu et al. except some modifications on chromatographic conditions. hplc separation was carried out on an ymc-pack ph column ( μm, nm, mm  . mm) with an isocratic elution of % water-trifluoroacetic acid (a, . %: . %, v/v) and % methanol (b) at a flow rate of ml/min. seven human cancer cell lines, hct- (human colon cancer cell line), bel (human hepatocellular carcinoma cell line), mgc (human gastric carcinoma cell line), capan (human pancreatic cancer cell line), nci-h , nci-h and a (human lung cancer cell lines) were used in the cytotoxicity assay. the viability of the cells after treated with various chemicals was evaluated using the mtt ( -( , -dimethylthiazol- -yl)- , -diphenyl tetrazolium bromide) assay performed as previously reported , . the inhibitory effects of these tested compounds on the proliferation of cancer cells were reflected by their respective ic ( % inhibitory concentration). steryl glycosides and acylated steryl glycosides in plant foods reflect unique sterol patterns rice bran extract containing acylated steryl glucoside fraction decreases elevated blood ldl cholesterol level in obese japanese men structural analysis of novel bioactive acylated steryl glucosides in pre-germinated brown rice bran a potent anticomplementary acylated sterol glucoside from orostachys japonicus therapeutic potential of cholesteryl o-acyl alpha-glucoside found in helicobacter pylori immunological functions of steryl glycosides cholestane glycosides with potent cytostatic activities on various tumor cells from ornithogalum saundersiae bulbs a new rearranged cholestane 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gymnema sylvestre r.br. catalyzing biosynthesis of steryl glucosides cloning and functional expression of ugt genes encoding sterol glucosyltransferases from saccharomyces cerevisiae, candida albicans, pichia pastoris, and dictyostelium discoideum glycosyltransferases in plant natural product synthesis: characterization of a supergene family chaperone coexpression plasmids: differential and synergistic roles of dnak-dnaj-grpe and groel-groes in assisting folding of an allergen of japanese cedar pollen, cryj , in escherichia coli regioselective enzymatic acylation of complex natural products: expanding molecular diversity structure, bioactivity, and chemical synthesis of osw- and other steroidal glycosides in the genus ornithogalum functional diversification of two ugt enzymes required for steryl glucoside synthesis in arabidopsis lac operon induction in escherichia coli: systematic comparison of iptg and tmg induction and influence of the transacetylase laca the lac operon galactoside acetyltransferase cdna isolation and functional characterization of udp-d-glucuronic acid -epimerase family from ornithogalum caudatum transcriptome-guided discovery and functional characterization of two udp-sugar -epimerase families involved in the biosynthesis of anti-tumor polysaccharides in ornithogalum caudatum transcriptome-guided gene isolation and functional characterization of udp-xylose synthase and udp-d-apiose/udp-dxylose synthase families from ornithogalum caudatum ait transcriptome-wide identification of sucrose synthase genes in ornithogalum caudatum transcriptomeenabled discovery and functional characterization of enzymes related to ( s)-pinocembrin biosynthesis from ornithogalum caudatum and their application for metabolic engineering functional analyses of ocrhs and ocuer involved in udp-l-rhamnose biosynthesis in ornithogalum caudatum interactions among sars-cov accessory proteins revealed by bimolecular fluorescence complementation assay cdna isolation and functional characterization of squalene synthase gene from ornithogalum caudatum probing steroidal substrate specificity of cytochrome p bm variants steroids hydroxylation catalyzed by the monooxygenase mutant - from bacillus megaterium bm metabolic engineering of escherichia coli for -butanol production cytotoxic cholestane glycosides from the bulbs of ornithogalum saundersiae supplementary data associated with this article can be found in the online version at doi: . /j.apsb. . . . key: cord- -fwsicgv authors: thomas, v. j.; bliemel, martin; shippam-brett, cynthia; maine, elicia title: endowing university spin-offs pre-formation: entrepreneurial capabilities for scientist-entrepreneurs date: - - journal: nan doi: . /j.technovation. . sha: doc_id: cord_uid: fwsicgv abstract university spin-offs are important mechanisms for creating and capturing value from scientific inventions. academic scientists are uniquely positioned to shape such opportunities long before the university spin-off is founded. to better understand how science-based university spin-offs can be endowed for success, the pre-formation stage of ventures co-founded over a year period by a star-scientist-entrepreneur is analysed by matching his co-invented us patents granted to co-authored publications and these ventures. employing the extended case method, including the analysis of extensive archival data, iterative interviews, and this unique, longitudinal, multi-level dataset, existing dynamic capabilities theory is confronted and extended with evidence as to how a star-scientist-entrepreneur senses and shapes and seizes opportunities to endow university spin-offs pre-formation. a process model is developed depicting four pre-formation entrepreneurial capabilities with which these science-based university spin-offs are endowed for success. recommendations are made for scientist-entrepreneurs, investors, university leadership, and for innovation policymakers. universities generate a large and growing proportion of scientific inventions (edwards et al., ; leih and teece, ; martin and tang, ; roberts et al., ) . university spin-offs are important mechanisms for creating and capturing value from these inventions (leih and teece, ; maine and seegopaul, ; shane, ) . academic scientists are uniquely positioned to shape such opportunities long before the university spin-off is founded (clarysse and moray, ; clarysse et al., ; maine and thomas, ; murray, ; rasmussen et al., ) . while scholars have noted that the location and growth of science-based ventures can be linked to the presence of highly productive academic scientists (maine et al., a; zucker et al., ) , the process by which scientists endow university spin-offs remains unknown. and, though understudied, the entrepreneurial capabilities of scientists are much maligned. in fact some scholars have cast doubts on whether scientists should play a leading role in the commercialization of science through spin-off formation (gurdon and samsom, ; stuart and ding, ; vohora et al., ) . this study is motivated by the research question: how can scientist-entrepreneurs endow university spin-offs pre-formation? the extended case method (burawoy, ) is employed to confront and extend dynamic capabilities theory by elucidating the manner in which an exemplar star-scientist-entrepreneur (sse) senses, shapes and seizes opportunities to endow university spin-offs pre-formation. a process model of entrepreneurial capabilities leading to the emergence of science-based university spin-offs is developed. an exemplar sse was identified as an outlier (fig. ) based on the science-based university spin-offs he had co-founded, their level of success, and after initial data collection showed that his career patenting output exceeded most firms in the emerging nanobiotechnology industry. the research leading to the formation of a science-based university spin-off may precede the founding of the venture by a decade or more. thus, although the first spin-off co-founded by the focal scientist-entrepreneur was formed in , extensive data including papers, patents, and ventures with the scientist as a co-author, co-inventor, and cofounder was gathered for the period - , allowing for a longitudinal examination of the progression of science from research laboratory to science-based university spin-off. a sensing and shaping capability is linked to seizing capabilities pre-formation through matching patents and papers to ventures. the patent-paper-venture matching provided objective data on the timeline from invention to spin-off formation, through analysing and linking patents to their associated papers and spin-off ventures. interviews and secondary sources provided evidence on technology-market matching -a key sensing and shaping capability -which was then anchored in time via patent-paper-venture matching to the corresponding seizing capabilities. a method was developed to identify platform technologies from papers, along with a technique to identify matched patents which were broad, blocking, and relevant (maine and thomas, ) . data on the founding and financing of ventures was sourced from the us securities and exchange commission (sec) filings, archived documents, company reports, and press releases (see appendix ). the entrepreneurial capabilities leading to well-endowed science-based university spin-off emergence were verified and refined through interviews with the scientist-entrepreneur, senior personnel at the mit technology licensing office (tlo), a lab alumni and academic co-founder, a business co-founder identified and nurtured by the scientist-entrepreneur, a venture capitalist who was also ceo of a co-founded spin-off, and an ip counsel of a co-founded spin-off (see appendix ). this study contributes to the academic entrepreneurship and dynamic capabilities literatures in several important ways. first, through the extended case method, detailed evidence of the commercialization activities of a star-scientist-entrepreneur is used to confront and extend dynamic capabilities theory to the individual level pre-formation (table & fig. ) . consistent with existing dynamic capabilities theory (eisenhardt and martin, ; helfat and petraf, ; teece, ; teece et al., ) both a sensing and shaping capability as well as seizing capabilities are observed at the firm-level. extending dynamic capabilities theory, at the individual level the key pre-formation sensing and shaping capability of technology-market matching and the pre-formation seizing capabilities of claiming and protecting ip, attracting and mentoring the founding team, and strategic timing are identified and elucidated. a process model of these four entrepreneurial capabilities which lead to well-endowed science-based university spin-offs is developed, identifying the role of the scientist-entrepreneur, his academic collaborators, the university's technology licensing office, and the external environment. second, the novel method of patent-paper-venture matching is developed which enables a detailed longitudinal examination of the processes of science commercialization from the flow of research outputs and personnel from the lab to the emergence of science-based university spinoffs. through this method, we reveal how the coordination, sequencing, and timing of commercialization decisions by the sse (along with his collaborators and the tlo), helps prepare the nascent venture in the pre-formation and early post-formation stages of venture emergence. third, this research adds to the growing literature on science-based entrepreneurship by providing empirical evidence and longitudinal analysis of the emergence and performance of science-based university spin-offs. in doing so, a nuanced perspective on a crucial and understudied period in the lifecycle of science-based university spin-offs (druilhe and garnsey, ; phan, ; rasmussen, ) is provided. from this analysis, we offer recommendations for scientist-entrepreneurs, investors, university leadership, and policymakers to further facilitate the commercialization of university science. despite an extensive literature on academic entrepreneurship, the pre-formation stage of sciencebased university spin-offs remains something of a black-box. this is problematic because constraints to the commercialization of public science -and the capabilities required to overcome them -are poorly understood (maine et al., a; pisano, ) . enabling further commercialization of science from universities requires a deeper understanding of the capabilities demonstrated by highly successful scientist-entrepreneurs, and the ecosystems within which they operate. in this section, relevant literature on academic entrepreneurship, scientistentrepreneurs, dynamic capabilities, and entrepreneurial capabilities is reviewed. universities contribute to economic growth through academic entrepreneurship, and more specifically, through university spin-off emergence (roberts et al., ; rothaermel et al., ; shane and stuart, ; siegel and wright, ) . this mechanism is particularly important for the commercialization of breakthrough technologies which have the potential to create new industries or transform existing ones. though the importance of this phenomena of university spin-off emergence is broadly recognized, extant studies have concentrated at the macro/ institutional level leading to the critique that the micro-level remains understudied (fuller and rothaermel, ; siegel and wright, ) . moreover, the vast majority of the academic entrepreneurship literature examines university spin-offs post-formation (mustar et al., ; rothaermel et al. ). yet much remains unknown. for example, though it is well recognized that academic scientists are the key decision makers developing the technology and shaping the commercialization strategy in the very early stages with the technology having been developed in their scientific lab (jain et al., ; krabel and mueller, ; perkmann et al., ) , further elucidation is needed to understand how scientists can endow university spin-offs for success. science-based academic entrepreneurship draws into sharp distinction the need for microlevel (early stage) evidence in the academic entrepreneurship literature (rasmussen, ) . science-based university spin-offs face challenges that are well recognized -in particular, high uncertainty and high commercialization costs coupled with long timelines from invention to revenue generation (agrawal, ; maine and seegopaul, ; pisano, ; shane, ) . although some potential strategies to overcome these challenges have been identified, all remain insufficiently understood. in particular, little is known about the pre-formation stage, and the role that a scientist-entrepreneur may play in endowing a university spin-off with the resources required for a higher likelihood of success. this gap has been noted by other academic entrepreneurship scholars, who call for qualitative research on sector-based entrepreneurial capabilities, and specifically ask "how do star scientists and technology gatekeepers influence the development of sector-based entrepreneurial capabilities?" de massis et al. ( , pp. ) . science-based university spin-offs require significant resources and capabilities in their preformation and early post-formation stages. the lack of such endowments result in most university spin-offs failing within a decade of founding (dimov and de clerq, ; timmons, ) . few science-based university spinoffs succeed in raising substantial vc financing or reaching an initial pubic offering (ipo) (fini et al., ; maine and thomas, ) . highly productive scientists are thus at an advantage in being able to attract much needed resources to these spin-offs through their reputation and signalling effects (stuart and ding, ) . highly productive scientists have also been labelled "elite" or "stars" and several scholars have shown that such scientists contribute disproportionately to the discovery of scientific inventions from universities (baba et al., ; lotka, ; zucker et al., ) . this productivity has led them to be identified in several ways: having an above average level of productivity in generating scientific publications and patents (baba et al., ; lawson and sterzi, ; schiffauerova and beaudry, ; subramanian et al., ) , being nobel prize winners (higgins et al., ), or having identified and characterized specific dna sequences (zucker et al., (zucker et al., & . beyond the underlying theme of productivity in patents and papers, these scientists are often actively involved in commercializing their discoveries (fuller and rothaermel, ; rothaermel et al., ; stuart and ding, ) , possibly because of their ability to signal the quality of research and to attract resources towards nascent science-based spin-offs. in fact, the founding of firms in emerging science-based industries is disproportionately co-located with star scientists (maine et al., a; zucker et al., ) , and spin-offs co-founded by star scientists are more likely to reach an ipo (fuller and rothaermel, ) . thus, highly productive scientists contribute disproportionately to academic entrepreneurship in scientific fields. dynamic capabilities theory seeks to explain why some firms are able to show better performance in a changing environment (eisenhardt and martin, ; helfat and petraf, ; teece, ; teece et al., ) . while researchers have made significant progress in identifying the antecedents, moderators and mechanisms leading to firm performance, much work remains (schilke et al., ) . the predominant focus of most studies on dynamic capabilities has been at the firm-level. while this focus is essential, valuable insights can also be gained by examining individual-level capabilities (felin et al., ; helfat and petraf, ) . this emphasis on individual-level capabilities is particularly important in the pre-formation stage of new ventures because "it is entrepreneurs who bring agency to opportunity", shane ( ) , by sensing, shaping and seizing opportunities. the pre-formation stage is a crucial and understudied period in the lifecycle of sciencebased university spin-offs (druilhe and garnsey, ; phan, ; rasmussen, ) . pathdependent decisions on key elements of science commercialization such as intellectual property (ip), founding team, and target markets are taken during this stage. for example, the quality of patent protection achieved pre-formation and the manner in which the patents are licensed out by the inventors and their institutions impact the ability of the licensee science-based venture to commercialize the technology. academic scientists from whose research labs these inventions emerge, are important stakeholders in the pre-formation stage. while extant researchers have suggested that most academic scientists are neither well-suited nor trained for science commercialization (gurdon and samsom, ) , a few outlier star-scientist-entrepreneurs have emerged. their unusual success in co-founding a large number of science-based university spinoffs can shed light on the entrepreneurial capabilities they possess and enrich the dynamic capabilities framework. productive streams of research have investigated the influence of dynamic capabilities in firms post-formation (eisenhardt and martin, ; teece, pisano, and shuen, ) . moving from the level of the firm to the individual, research on entrepreneurial capabilities has also focussed predominantly on the post-formation stage (alvarez and barney, ; shane, ; shane and venkataraman, ) . however, the pre-formation stage is where critical decisions which affect the future success of the spin-off may be taken by the scientist and his or her academic collaborators (druilhe and garnsey, ; rasmussen, ; rasmussen et al., ; shane, ) . the entrepreneurial capabilities of a founder and of a founding team can impact venture success (eesley et al, ; gruber et al., ; maine et al., ) , and we argue that this is particularly true for scientist-entrepreneurs, given the critical decisions they take pre-formation. technology-market matching is a crucial capability for science-based businesses (freeman, ; maine and garnsey, ; schmookler, ) . while early stage market selection is important for any innovating firm (gruber et al., ) , it takes on far more importance for science-based ventures commercializing technologies with broad applicability (maine and seegopaul, ; maine et al., a) . technology-market matching has predominantly happened post the formation of science-based ventures, and not in the labs of academic scientists (maine and garnsey, ; maine et al., a ). yet, given the long timelines from invention to innovation and the large sums of capital involved, early-stage entrepreneurial capability in technology-market matching could be enormously beneficial. makes sense to select an "extreme" case when the phenomenon of interest is "transparently observable". the study of an exemplar is appropriate and valuable to develop or expand theories, particularly in contexts with evolving, complex processes and little primary data elucidating them (eisenhardt and graebner, ; yin, ) . science commercialization is such a context (fini et al., ) . the relationships uncovered through such in-depth, longitudinal and multilevel analyses of single case studies (i.e. eisenhardt, & ; murray, ) have proved invaluable in informing the metrics of subsequent quantitative studies. the extended case method is a technique developed to link the macro and the micro levels through pre-existing theory (burawoy, ) . it is particularly useful when dealing with complex, multi-layered and unstructured phenomena (bjerregard, ; matthyssens and vandenbempt, ) . researchers identify a case which is used to confront pre-existing theory, with the aim of using the anomalies from the case to identify the ways in which existing theory can be refined. this method emphasizes the importance of context, focusing on the specific characteristics of the single case that can illuminate the wider processes that can enable the focal organizations to survive and thrive. this study uses the extended case method to confront dynamic capabilities theory with the specific case of a star-scientist-entrepreneur who has cofounded over science-based ventures ( table ) . star-scientist-entrepreneurs are defined here as academic scientists with an above average level of productivity in generating scientific publications and patents and who have cofounded at least one science-based university spin-off (bubbles in figure ) . interestingly, there is a skewed distribution, with the greatest impact (in terms of number of spin-offs founded, vc financing raised, number of ipos, significant social issues addressed) coming from outliers (fuller and rothaermel, ; nightingale and coad, ) . thus, rather than studying a sample of sses, who may or may not have put any systematic thought into guiding spin-off emergence, the careful study of multiple spin-offs co-founded by an exemplar sse (large bubble on top right of figure ) can reveal entrepreneurial capabilities honed through the formation of multiple spinoffs over several decades. the star-scientist-entrepreneur investigated in our study was identified based on insights from an earlier study (maine et al., b) and selected for further examination based on his extensive productivity in generating scientific papers, patents and ventures. our multi-level, longitudinal analysis encompasses the sse's capabilities, the backgrounds of his academic and business co-founders for each spin-off, temporal and strategic patterns revealed through patentpaper-venture matching, and venture success measured through financing raised and reaching an ipo. in studying the sse, his lab and his co-founded spin-offs over decades ( table ) , data on all of the papers and granted us patents with the star scientist as a co-author and co-inventor until st december are first gathered and then these patents are matched to the academic papers through a combination of extensive automated and manual matching. papers which advanced a platform technology were identified, and the journal impact factors of publications were gathered from the journal citation report . patents which were broad, blocking and relevant, were identified following the method outlined in section . . co-authored papers were identified from the web of science and also compared with the publication list on the lab website of the sse (http://langer-lab.mit.edu/publications). after eliminating dual entries and errors, and accounting for any inconsistencies in the coverage of the web of science dataset, the total number of papers was . all us patents issued between july and december with robert s. langer or robert s. langer jr. as a co-inventor residing in massachusetts were identified from the uspto. in all, us patents were identified and analysed through our patent-paper-matching technique (section . ). acknowledging the importance of context as required by the extended case method, the researchers conducted several interviews with the scientist-entrepreneur, the mit ip counsel, a scientific co-founder, and a co-founded venture ceo among others (details in appendix and section . ). extensive secondary data on each co-founded venture which included information on their patents, papers, initial leadership team, and scientific alumni from the scientist entrepreneur's lab, were collected from a variety of sources ranging from the us patent office, the mit tlo, the sec, web of science, google scholar, individual scientist cvs and webpages, university and firm webpages, and firm press releases, to published interviews of the scientist-entrepreneur and co-founded venture leadership team in periodicals and online sources (details in appendix ). the scientist-entrepreneur was also invited to confirm patent-paper matches in a number of instances. in doing so, the researchers collaborated with the focal subject and confirmed their analysis through multiple follow-up interactions with the scientistentrepreneur and the mit tlo as suggested by the extended case method. to enable the mapping of sensing and shaping capabilities to seizing capabilities in the preformation stage, the patent-paper matching technique (murray and stern, ) was extended to include ventures. matching patents and papers to ventures allows for a nuanced understanding of the progression of science from research laboratory to science-based university spin-off. patents were matched to papers by first creating a list of the top matched papers for each patent. to do so, for each co-authored patent, the year of patent filing was identified, a list of inventors from each patent was compiled, and co-authored papers that had been published in the +/-five year period from the patent filing date were identified. next, the patent inventors were matched with the co-authors of the papers in this period. in many instances, multiple inventors were matched as co-authors on papers submitted and published within this period. the number of overlapping words between the titles and abstracts of the focal patent and the papers were also identified. once the list of top possible matched papers was generated through this technique, an overlap in the text and/or the figures in both sets of documents were manually verified, so that each patent could be accurately matched to the papers that inform it (murray and stern, ) . the patent-paper matches thus identified are a combination of extensive automated and manual matching (bubela et al., ) which, to the knowledge of the authors, has not previously been attempted on a dataset of this scale. once the patent-paper matches were complete, the core technology of each firm cofounded by the star-scientist-entrepreneur was identified. from descriptions on the company website, annual reports, press releases, sec filings, published ceo interviews and firm media reports, the patent-paper sets were matched to the firms. in some instances firms had listed the papers and/ or patents on which the firm's technology was based. this also helped in the matching exercise. the resultant patent-paper-venture matching enables the tracking of the commercialization of public science. a patent is a property right granted by a government to an inventor, with the aim of protecting intellectual endeavours and supporting technological progress. a patent confers the right to exclude others from making, using, offering for sale, selling, or importing an invention into a particular jurisdiction for a specified period of time. inventions or discoveries of a new and useful process, machine, manufacture, or composition of matter, or new and useful improvements in these categories, may be patented. the breadth of protection is determined through the patent claims, which define the scope of protection or the legal boundaries of the invention. each patent can have two types of claims, independent and dependent. independent claims stand alone and do not reference other claims within the patent. dependent claims reference other claims, and can be considered as subsets of the claims on which they depend. the first claim in a patent is independent, defines the broadest scope of the patent protection (huys et al., ) and is least restrictive (uspto, : mpep § . (m)). patents are more valuable to science-based university spin-offs and to their investors when they are broad, blocking and relevant, as argued in maine and thomas ( ) : "a broad patent is one which enables a wide range of applications (more value creation): filing a broad patent requires forethought of how widely a patent can be applied in the future. a blocking patent enables a spin-off to appropriate that value, as competitors have difficulty inventing around such a patent. a relevant patent is one which is deemed promising and useful, (for example, because it meets an unmet market need, has a large potential social impact, and/or is in an emerging area of scientific discovery), stimulating significant follow-on activity both by the firm and by others." (maine and thomas, ) science-based spin-offs which emerge from university labs with broad, blocking and relevant patents are thus better resourced to enable the translation of breakthrough technologies. identifying broad, blocking and relevant patents, particularly in the us, is not straightforward. unlike in europe, where the blocking nature of a patent can be inferred by the x and y classification in the search reports of forward citing patents (torrisi et al., ) , in the us no equivalent classification exists. existing proxies for patent breadth, such as the number of ipc classes (lerner, ) and the number of patent claims (lanjouw and schankerman, ) , also have drawbacks (maine and thomas, ; reitzig, ) . to address this, the method described in maine and thomas ( ) was followed to identify broad, blocking and relevant patents in the us. first, the first characters of the patent claims (the independent claim) were searched for the presence of the word "comprising," which has been identified as an indicator of the broad nature of a patent (radack, ) . this indicator was then combined with data on patent forward citations, as these citations are indicative of the cumulative development of the technology by the scientist, his collaborators, and competitors. the criteria used to identify broad, blocking, relevant patents was those patents which have the word "comprising" in the first characters of the patent claims and have more than forward citations within years from patent issue date or more than forward patent citations within years of patent issue date. our proxy for broad, blocking, relevant patents combines text from patent claims with forward patent citations, and enables large scale empirical studies. this approach responds to calls for using combinations of procedural and text based indicators of patent value (reitzig, ) . the argument has been made that forward citations are inconsistent with the blocking nature of a patent -because by definition a blocking patent cannot be cited, as it prevents other inventors from entering the space (blind et al., ). however, other research suggests that self-citations are a greater indicator of market value than external forward citations (hall et al., ) . maine and thomas ( ) argue that the star scientist's collaborators build on his blocking patents, extending the technology in differing directions, resulting in high citations. content analysis was employed on the star scientist's co-authored papers to determine platform technologies (hsieh & shannon, ) . the top frequently occurring words in the titles and abstracts of all these papers were identified. three of the authors then independently assessed this list and eliminated commonly used words in english and in the biomedical domain. in the few cases where there was a difference in perception, the authors discussed and resolved their differences. co-occurring words such as "tissue engineering" and "controlled release polymers" were combined as key phrases and included in the list. the refined list consisted of words such as nanoparticles, aptamers, and polymers, which are highly indicative of platform technologies as they are broadly applicable across multiple domains. using this selective, validated list, papers were identified which had used these words in the titles and abstracts and thus classified as describing platform technologies in the biomedical domain. the list generated by this automated and manual verification exercise is available on request. the patterns revealed by the patent-paper-venture matching, the identification of broad, blocking patents, and the identification of platform technologies were compared and contrasted, with particular attention paid to paper characteristics, patent characteristics, the selection of markets, co-authors, co-inventors, co-founders, and timing of spin-off formation. a process model of four key entrepreneurial capabilities which lead to well-endowed university spin-offs (figure ) was iteratively developed. these capabilities were then further explored at the level of the university spin-off and their impact on success assessed. ten interviews were conducted and three additional video interviews with key individuals were also used to inform and refine the process model (appendix ). consistent with the extended case study method, following initial interviews with the star-scientist-entrepreneur and initial patent-paper-venture matching, the analysis was iteratively refined with direct involvement of the sse in categorizing patents and in confirming co-inventor and co-founder involvement. three interviews were conducted with the mit tlo ip counsel who leads the patent portfolio of the sse. a scientific co-founder, a business co-founder, a venture capitalist who was also ceo of a langer lab spin-off, and an ip counsel of a co-founded spin-off were also interviewed. interviews with these important stakeholders in the innovation ecosystem informed and validated analysis of the entrepreneurial capabilities of the focal sse. further data was gathered on all spin-offs to demonstrate the four entrepreneurial capabilities identified through our patent-paper-venture matching and through our interviews and archival data. for the spin-offs co-founded by the sse, technology-market matching was observed and validated at the project formulation stage ( table ) , as well as at the platform technology and the firm-level (figure ) . claiming and protecting the invention was demonstrated for all spin-offs by observing the presence or absence of elite publications, blocking patents, and platform technologies in the emergence of each venture ( table ) . strategic timing was measured as the time from the issuing of the first blocking patent associated with the spin-off to the time of firm founding ( table ) . attracting and mentoring the founding team was observed through the founding team composition, including documenting the involvement of collaborating labs, academic cofounders, and his lab alumni ( table ). the tenure of the initial ceo -sometimes a founder and sometimes recruited up to years after the firm was founded -was documented, and their prior education was coded as phd scientist or non-scientist. the prior business experience of the founding ceo was coded as biopharma executive, biopharma, medtech, vc, serial entrepreneur, and cosmetics mnc executive ( table ) . the sse's formal roles at founding, as documented in sec filings, were coded as board member, scientific advisory board member, beneficial owner and/or promoter. informal roles carried out by the sse in attracting and mentoring the founding team were observed and coded as one or more of: mentoring scientist-entrepreneurs, identifying and nurturing business talent, attracting experienced ceos, and attracting vcs ( table ) . mentoring scientist-entrepreneurs may happen both pre-and post-formation. it can occur in the star scientist's lab or it may occur when the star scientist continues to mentor former lab alumni through his membership in the scientific advisory boards of co-founded university spin-offs. identifying and nurturing business talent occurs when the eventual ceo of a venture was identified and mentored by the sse prior to firm formation. attracting vcs is observed when a vc is a co-founder along with the sse and in the case of documentation of a vc investing (sometimes in multiple ventures) because of the sse's reputation. science-based university spin-offs endowed with valuable ip, skilled people, and technology-market orientation enjoy enhanced chances of success. this measure of "wellendowed university spin-off" is proxied with ten-year survival status and with total financing raised / year since founding. the total amount of financing raised by each university spin-off was gathered from sec filings, press releases, and other secondary sources, and includes seed and venture capital financing raised, money raised through an ipo, and money received upon the acquisition of the firm. this measure includes all non-government finance raised from firm founding until firm exit or until february , and thus may understate the total financing received by the spin-offs. to control for the wide range of age of the firms, the success metric of millions of us$ raised per year was calculated for each of the spin-offs which emerged from the sse's lab. this measure aims to be a proxy for well-endowed university spin-offs. the impact on success of founding team characteristics and the sse's role in attracting and mentoring the founding team are assessed. the science-based university spin-offs co-founded by the exemplar star-scientist-entrepreneur are well-endowed compared to an average science-based spin-off (table ) . his stats are compelling on every dimension (for comparison in the biomedical sector, see holley and watson, ) . first, as a prolific academic, he has published over papers, and is the most highly cited chemical engineer in history (> , citations). with over patents issued or pending worldwide, including over issued us patents, he exceeds the patenting output of all but the most established biopharmaceutical firms. technologies developed in his lab have improved the lives of millions of people. and as the co-founder of well-endowed sciencebased university spin-offs, he has refined commercialization processes which help translate his laboratory inventions to ventures with a higher likelihood for success. the focal sse did not begin his career as a star scientist, nor was he identifiable as such for over a decade. when he graduated with a phd in chemical engineering from mit in , at the height of the oil crisis, he turned down four lucrative job offers from oil companies, preferring to work as an engineer in the clinical research laboratory of dr. judah folkman at the harvard medical school. his interdisciplinary research was fruitful, but not recognized by much of the establishment, and was met with scepticism from leading researchers, granting agencies and the us patent office. his first nih grants were rejected, and his first patent filings failed the "non-obviousness test". he was granted his first patent only after presenting the patent office with signed affidavits from established scientific leaders in his field attesting to the highly unconventional nature of his work. from this inauspicious start, spin-offs from the lab of this star scientist have raised over us$ billion cumulatively. these spin-offs have achieved success by several measures including meeting the -year survival threshold, raising multiple rounds of financing, and, for some of the more mature ventures, getting products to market ( table ) . all ventures spun out of the lab of the sse meet or are in the process of meeting the -year survival threshold, if acquisitions are included as survival ( table ). in contrast, most science-based university spin-offs are likely to fail within the first decade of founding (dimov and de clerq, ; timmons, ) . as to financing, all but two of the ventures spun out of the lab of the sse raised at least us$ million in financing (table ) . zhang ( , table ) finds that, of those university spin-offs which raise venture capital, average total vc money raised is us$ . million, giving a comparison level for our data. this provides further evidence that spin-offs co-founded by the star scientist entrepreneur are well-endowed pre-formation. the evidence of entrepreneurial capabilities deployed by the sse is analysed with a focus on the capacity to sense and shape opportunities and to seize opportunities pre-formation. one pre-formation entrepreneurial capability central to sensing and shaping opportunities was observed and validated through further interviews. three pre-formation entrepreneurial capabilities involved in seizing opportunities were revealed through patent-paper-venture matching, further data analysis, and additional interviews. the four entrepreneurial capabilities are depicted in a process model (figure ) , which begins with technology-market matching at the project formulation stage in the research lab. the process of the emergence of the university spin-off was observed to proceed sequentially onwards through claiming and protecting the invention (influenced by the university tlo and the uspto), to attracting and mentoring the founding team (influenced by vcs, academic collaborators and experienced entrepreneurs), and finally to the founding of science-based university spin-offs (with strategic timing influenced by the scientist, his academic collaborators and vcs). the cycle resumes as new lab members are attracted to the star scientist's lab and directed towards solving unmet market needs, often leveraging existing platform technologies. these four entrepreneurial capabilities leading to well-resourced science-based university spinoff emergence are described next, along with the evidence which supports them. the star-scientist entrepreneur sensed and shaped opportunities through technology-market matching and this capability was passed on to his graduate students and through them to his cofounded ventures. strong technology-market matching capability was demonstrated within the research lab in formulating research projects targeting unmet market needs (table ), in cofounding ventures based on technologies outside his lab ( table ) , and in co-founding multiple spin-offs from the same platform technology (figure ) . the star scientist has built an unusual lab culture around identifying and prioritizing research ideas. he purposefully steers his lab members and academic collaborators in the selection and development of research projects that address significant unmet market needs. as depicted in table , we observed pre-formation technology-market matching at the project formulation stage in out of spin-offs from the lab of the sse. the focal sse practiced pre-formation technology-market matching in both directions: leading from the market, and leading from the technology through formation of multiple ventures from a single platform technology. leading from the market is less common for scientistentrepreneurs but can be clearly demonstrated in the genesis of the star scientist's co-founded ventures applied inhalation research (air) and microchips. in the case of air, when applied mathematician david edwards came to him to get advice, the star scientist steered him towards a known problem which would utilize both edwards' mathematical modelling expertise and the star scientist's most renowned platform technology of controlled release polymers. this marketfocused research sought to design therapeutic particles which could be inhaled deeply and dispersed widely throughout the lungs. edwards and the star scientist went on to develop large porous particles for pulmonary drug delivery, filing a broad, blocking patent in and publishing the key research in science in , and co-founding air that same year ( table ) . in another instance, the star scientist was inspired by conventional microchip fabrication and envisaged another solution to a health problem, that of people forgetting to take medications and contraceptives at the prescribed times. as he had no previous experience with microchip fabrication technology, the star scientist discussed his ideas for a programmable, implanted biomedical device which would enable the controlled release of therapeutics over years or even decades, with an mit colleague in materials engineering, prof. michael cima. this matching of technology and market in the star scientist's mind led to an exploratory research collaboration and the eventual co-founding of microchips (table ) . co-founder michael cima credits the star scientist with being "gifted at 'connecting technologies to true medical needs.'" (schaffer, ) . the sse's capability in technology-market matching was also observed in the ventures he co-founded where the technology did not come from his lab ( table , shaded rows). in these instances, scientists from other labs and institutions approached him because of his known entrepreneurial capabilities. derrick rossi, a researcher at boston children's hospital and cofounder of moderna therapeutics, approached him with a stem cell invention, and was redirected to commercialize a breakthrough platform technology: rossi's first breakthrough was to create a disguise for the mrna so that it could slip into the cell unnoticed. as he explained to langer, he did this by modifying two of the mrna's nucleotides, or building blocks. once they breached the cells' defense mechanism, the mrna reprogrammed the cells into ips cells. that was the feat that got rossi so much acclaim. but what most struck langer as he listened to rossi was the first part: the technique that rossi had developed to modify the mrna. "this is a much bigger discovery than something that affects stem cell behavior," langer told rossi, already imagining the potential. "you could apply it to make anything." (elton, ) the formation of multiple ventures from a single platform technology is another form of technology-market matching, in this case leading from the technology, although the process is still iterative. as depicted in figure , the spin-offs co-founded by this star scientist stem from platform technologies. most notably, he has co-founded spin-offs over years which draw on his platform technology of controlled release polymers. this technology is so broad that one firm has virtually no chance of tackling the entire breadth of value creation, and multiple ventures are formed even from sub-platforms. for example, following the development of controlled release nanoparticles (gref et al., ) , the star scientist and his post-doctoral fellow, omid farokhzad, developed stealth nanoparticles which could bind to targeted sites to treat cancer. this led to the formation of bind therapeutics, selecta biosciences, and blend therapeutics over the following years (fig. ) figure ). once an opportunity is sensed and shaped, additional capabilities are required to seize that opportunity (teece, ) . we observed this seizing of opportunities through three entrepreneurial capabilities demonstrated by the star-scientist-entrepreneur pre-formation (figures and ) . notably these pre-formation entrepreneurial capabilities imprint upon the nascent firm through the mentoring of lab members who go on to leadership roles in the emerging science-based spin-off. a second entrepreneurial capability demonstrated was through claiming and protecting lab inventions in order to create and capture value. the sse and his academic co-founders repeatedly invented a platform technology, published the results in an elite journal, and obtained at least one broad, blocking, and relevant patent. such patents send a strong signal to potential investors (maine and thomas, ) . this strategy was consistent in spin-offs formed from to , demonstrated by of spin-offs from the lab of the star scientist ( table ) . this method of claiming and protecting scientific inventions enhances the probability of raising external financing for the spin-off. through patent-paper-venture matching, the prevalence and importance of broad, blocking, relevant patents on a platform technology is revealed (table ) . surprisingly, the timing of filing such patents is also distinctive in the approach of this star scientist ( table , far right column). the conventional wisdom for biomedical firms is to patent narrowly first and patent more broadly later in commercialization (hegde et al., ) . in contrast, the star scientist, his lab members, collaborating labs, and the mit tlo, file broad patents as early as possible and in such a way as to give his collaborators protected room to take the technology in different directions through patent continuations-in-part (cips) sharing the same priority date as their parent document. the broad, blocking patents are filed early ( table ) , and broad protection is also generated through multiple patents on alternative scientific mechanisms. it was clear from primary interviews (appendix , interviews and ), as well as previous studies (hsu and bernstein, ; nelsen, ; shane and stuart, ) , that the mit tlo plays a critical role throughout the patenting and licensing process for all mit scientists, by selecting the invention disclosures to take forward into patent application and prosecution. in cases where the inventors are from multiple organizations, the mit tlo often plays the role of the lead organization, coordinating negotiations with licensees and carefully licensing out pieces of the intellectual property by fields of use, with clauses indicating that the licence has to be returned in case commercialization milestones are not met within a specified timeframe (appendix , interview ). by ensuring that the technologies developed in the star scientist's lab and across mit are protected by patents before public dissemination, the tlo is able to satisfy both the need for sharing new knowledge in scientific journals while at the same time maintaining strong property rights to incentivize commercialization. unusually, we see evidence of the sse and the mit tlo coordinating the manner in which claiming and protecting the ip is done to enable broader technology-market matching from platform technologies. in several instances, the star scientist's patents have been licensed by field of use and even by type of therapeutic payload to allow multiple opportunities for value creation. this strategic management of ip is particularly evident with the star scientist's platform technologies of controlled release polymers and biodegradable polymer devices (figure ) . such field of use licensing is practiced both by the mit tlo and by the spin-offs themselves, once they have in-licensed and built their own ip portfolio. the star-scientist-entrepreneur demonstrated another entrepreneurial capability in attracting and mentoring the founding team. he limited operational involvement in his spin-offs, primarily acting in a scientific advisory role and as a board member ( table ) . this created a greater need for scientific leadership within the spin-off, given the need to refine inventions from the sse's lab into replicable, viable products. as shown in table , in of the spin-offs from his lab, a lab alumnus is a co-founder (the sse co-founded more spin-offs with faculty at other boston institutions and was the sole academic founder of acusphere). these lab co-founders, along with other lab alumni and earlier lab spin-offs, co-developed the platform technology underlying the spin-off and are exceptionally dedicated to the cause (langer, ) . in most instances, these lab co-founders take on a scientific leadership role, and are paired with experienced managers taking on the role of ceo, although, interestingly, there is sometimes a lag between firm founding and the hiring of the first ceo. thus the university spin-off benefits from tight linkages to the sse's lab through lab alumni in scientific roles permitting easier flow of tacit knowledge. past the sse's formal role at founding (such as being on the board of directors and/or the scientific advisory board), three ways in which he attracted and mentored the founding team were observed and were coded as mentoring scientist-entrepreneurs, identifying and nurturing business talent, and attracting vcs (table ) . notably, direct evidence of the star scientist attracting experienced ceos was not observed. in the most successful ventures, evidence of mentoring scientist-entrepreneurs and attracting vcs was found ( table ) . this capability was identified more frequently in the latter half of his career ( table ) . the mentoring of scientist-entrepreneurs can happen in the university lab and also -in the case of repeat ventures -in the spin-off when the star-scientist-entrepreneur provides mentoring through the scientific advisory board. as identified in table , evidence of such mentoring was found in of ventures spun out of the lab of the focal sse. lab alumni who co-founded a spin-off with the sse had been mentored both as a scientist and with an entrepreneurial mindset pre-formation. in the same way that the sse was mentored in solving important problems in the folkman lab (cooke, ) , he mentored lab members in his own lab. a pattern began in with sontra medical, the first of these ventures which commercialized the research of a student supervised in the lab of the sse. in the case of repeat ventures with the same alumni, mentoring continued to occur through the scientific advisory board. out of the ventures for which this informal role was not observed, were co-founded with experienced scientist-entrepreneurs and the remaining were at the beginning of the star scientist's entrepreneurial career. a less frequent but unusual role observed was that of the star-scientist-entrepreneur identifying and nurturing business talent. clear evidence of this was found in ventures ( (elton, ) . alternatively, venture capitalists may join the founding team themselves as placeholder ceos until a suitable ceo is found, as observed in living proof (table ) , or later joining as ceo when pursuing an ipo, as observed in bind. the business co-founders (and the initial ceo) chosen brought experience as a serial entrepreneur, a venture capitalist, or as a senior executive in a biotech, pharmaceutical or chemical multinational corporation. lab alumni rarely took on business leadership roles, more typically co-founding and/or joining in scientific leadership roles. a vc who served as ceo in one of the spin-offs co-founded by the star scientist made the case that the leadership of university spin-offs was as much or more important to their success as the technology: [a science-based university spin-off] "want[s] foundational patents that can define game changing technology" ... [and] ... "you want great science, some patent protection and a great team around it." .... a "great team can be successful with a not so great technology" [but you can ruin the commercialization of a great technology with a poor team]. (appendix , interview ) spin-offs whose initial ceo had executive experience at a larger biopharma company were more likely to be successful ( table ) . there are two notable exceptions: in the case of living proofa spin-off with no clinical trial hurdles and a competitive commercialization environment, the executive experience of the initial ceo comes from a cosmetics multinational corporation; in the case of air -it was acquired after only years, and focused on scientific development during those first years. thus, the university spin-off benefits from both the networks and experience of attracted venture capitalists and the initial ceos, who facilitate alliance partnerships and investment. the patent-paper-venture matching methodology enabled the identification of the entrepreneurial capability of strategic timing, which contributes to the emergence of well-endowed university spin-offs. the star-scientist-entrepreneur and his collaborators identify critical unmet needs and formulate projects in an attempt to solve them. for projects which lead to breakthrough solutions, they patent, publish, and continue to refine these ideas (sometimes in stealth mode) until they are ready for commercialization through spin-off formation. the sse guides technology-market matching during project formulation and mentors lab members in understanding, evaluating, and mitigating the gap between successful experiments in the research lab and commercial viability. pre-formation, technology-market matching begins unusually early -at the project formulation stage ( table ) . next, the sse repeatedly files broad, blocking, relevant patents at the earliest opportunity. these are coordinated in timing and context with elite publications ( table ). in contrast, the sse co-founds the venture far later than might be expected ( table ) . thus, the spin-offs from the lab of the sse often have a prolonged gestation period within the university. during this gestation time, further technical goals are set and achieved, with additional journal publications and follow-on patent protection. as a tangible example, in the case of bind therapeutics, the unmet market need was targeted drug delivery through the bloodstream and the controlled release of that drug. ruxandra timing his publishing and patenting and, in particular, spin-off formation strategically, the sse was able to build a strong case for multiple rounds of spin-off funding. early focus on an unmet market need guided the development of the breakthrough research. early timing of the initial elite paper and broad, blocking relevant patent were essential to prevent either scientific or commercial pre-empting of the innovation idea. but a conscious choice was made to delay venture formation until the technology was assessed to be viable, until scientific leadership of the venture was mentored, and until venture capitalist timelines and perception of value was met. venture capitalist and co-founder terry mcguire endorses this strategy but notes how unusual it is for scientist-entrepreneurs to delay venture formation (arnaud, ). yet, given the typical vc investment window of - years, ventures with a longer time to commercial viability postformation, will be less likely to raise vc financing (maine and thomas, ; pisano, ) . our broader observations suggest that, since , founding of the sse's lab spin-offs have been purposively delayed until the technology is closer to commercial viability. gestation times are presented for all of the spin-offs formed from technologies co-developed in the focal star scientist's lab and with him as a co-inventor ( this timing has grown to an average of . years in the latter half (table ) . thus, stategic timing is observed in the coordination and sequencing of the other three pre-formation entrepreneurial capabilities and is most notable in the timing of firm formation ( table and figure ). most science-based university spin-offs emerge through long, complex pathways (roberts, ) . some entrepreneurship scholars suggest that these spin-offs are less efficient at developing inventions than incumbents and make predictable mistakes: "for instance, they may hire the wrong people, develop the product for the wrong market, or try to develop it for too many markets and succeed at none, or simply run out of money." (dalay and fosfuri, , pp. ; arora, fosfuri, and ronde, ) . linking science-based university spin-offs to the patentpaper pairs informing them reveals how early decisions taken by the star scientist entrepreneur (and his academic collaborators, with the support of the tlo), along with their coordination and sequencing, have laid a strong foundation for the emergence of these spin-offs pre-formation. the role of the sse in four key entrepreneurial capabilities, which endow these science-based university spin-offs for success is depicted in figure and discussed in this section. teece ( ) has identified three key dynamic capabilities which impact firm performance: sensing and shaping opportunities, seizing opportunities, and transforming opportunities. while these capabilities are at the firm-level, there are growing calls for focusing on the "entrepreneurial function embedded in dynamic capabilities" protogerou et al., p. . this is particularly relevant as the pre-formation stage of venture formation is seen as a neglected issue in the spin-off literature (druilhe and garnsey, ) and in entrepreneurship theory (phan, ; rasmussen, ) . in focusing on entrepreneurial capabilities pre-formation, this study follows the emerging trend of focusing on individual-level skills and capabilities (augier and teece, ; helfat and petraf, ) to further enrich the dynamic capabilities framework. our study also responds to calls for more process-oriented studies to extend dynamic capabilities theory (schilke et al., ). at the firm-level, our study confirms dynamic capabilities of sensing and shaping capabilities, and seizing opportunities. the sensing and shaping capability is observed through technology-market matching at the firm-level (fig. ) . the seizing opportunities dynamic capability is also observed at the firm-level through claiming and protecting the invention, attracting and mentoring the founding team, and strategic timing ( table ) . these firm-level capabilities enable the well-endowed science-based spin-offs to continue to outperform ( table ) . extending dynamic capabilities theory, we employ the patent-paper-venture matching technique developed in this paper to elucidate four entrepreneurial capabilities which the starscientist-entrepreneur uses to better endow co-founded spin-offs pre-formation (fig. ) . whereas all spin-offs shown in fig. are co-founded by the sse, those shown in bold font also have the sse as a co-inventor or co-author, indicating his direct involvement from guiding the science from concept through potential applications and on to its commercialization through application specific ventures. this individual-level entrepreneurial capability termed technology-market matching (table and figure ) is an essential component of the sensing and shaping dynamic capability at the firm-level (figure ) . the firm-level dynamic capability of seizing opportunities can be linked to the individual-level entrepreneurial capabilities of claiming and protecting the invention, attracting and mentoring the founding team, and strategic timing (fig. ) . the entrepreneurial capability of claiming and protecting the invention at the individual level can be observed through the role of the sse as a co-inventor on broad, blocking and relevant patents ( table ). the entrepreneurial capability of attracting and mentoring the founding team by the sse can be observed in table and through founding team members who are langer lab alumni ( table ) . the individual level entrepreneurial capability of strategic timing can be observed through table . technologymarket matching happenes very early on during project formulation. after technology market matching, successful breakthrough research is protected through broad, blocking, relevant patents which are then paired with elite publications. in constrast, venture formation is frequently delayed. as teece ( teece ( , p. notes, "opportunity creation and/or discovery by individuals require both access to information and the ability to recognize, sense, and shape developments." for science-based ventures, the entrepreneurial capability central to sensing and shaping opportunities -and linked to venture success -is technology-market matching (gruber et al., ; maine and garnsey, ; maine et al., a) . this entrepreneurial capability has rarely been observed pre-formation in a research laboratory. a notable exception is rasmussen's study of university spin-offs where he notes the early shaping of research projects according to perceived market needs (rasmussen, , pp. ). in the star-scientist-entrepreneur's research lab, we observed this capability practiced in the project formulation stage -both leading with the market opportunity and leading with the technology opportunity. in the first approach to technology-market matching, large unmet needs trigger the search for new technologies to solve these problems. star scientists can do this at the project formulation stage if they have an extensive awareness of multiple scientific domains and the ability to bring together an interdisciplinary team of other faculty members, postdoctoral fellows or graduate students. reputation, trust, and past commercialization experience might underpin the ability of a star scientist to form such interdisciplinary teams. in this way the human capital and social capital of the star scientist and the human capital embodied in his network of collaborators (murray, ) , help in selecting promising avenues of research and in forming interdisciplinary project teams which can then target large unmet needs. the second approach to technology-market matching is the ability to match existing technologies to viable market applications. although sometime derided as "technology-driven," effective technology-market matching can happen from either direction (maine and garnsey, ) . in fact, a star scientist may choose to use her in-depth domain expertise to carve out multiple university spin-offs from the same or similar platform technologies, each targeting a different unmet market need (fig. ) . in this manner, the sse is able to leverage the learning from one set of experiments and use it to form another spin-off targeted at a different market. this approach also serves to diversify the risk of failure among multiple university spin-offs: the success of one can benefit other related spin-offs, but the failure of one spin-off may not mean that the other spin-offs based on similar technology will fail. this risk diversification argument is consistent with shane's ( , pp. - ) arguments for general purpose technology (gpt) commercialization by university spin-offs. however, whereas shane ( ) anticipated this risk diversification to yield benefit to a single venture, the funding environment of science-based ventures seldom allows a single venture to focus on the full breadth of opportunities afforded by the platform technology. the additional benefit of multiple spin-offs from one platform, coordinated by the sse and the tlo, is observed in this study. in introducing his second category of dynamic capability -seizing opportunities - teece ( teece ( , p. ) notes "once a new (technological or market) opportunity is sensed, it must be addressed through new products, processes, or services. this almost always requires investments in development and commercialization activity." teece ( ) also elucidates the potential competitive advantage of locking up assets and of timing commercialization decisions. for a science-based university spin-off, the individual-level entrepreneurial capabilities pre-formation central to seizing opportunities are claiming and protecting the invention, attracting and mentoring a founding team to commercialize the invention, and strategic timing. each entrepreneurial capability is discussed below in the context of related literature. patenting and publishing are central to the commercialization of breakthrough scientific inventions, constituting the currency of the venture (hsu and ziedonis, ; maine and thomas, ; pisano, ) . consistent with murray ( ) the star-scientist-entrepreneur investigated in this study played a central role in claiming and protecting his inventions through publishing the results of his research in elite scientific journals while also protecting it with one or more broad, blocking patents prior to spin-off founding (table , figure ) . in doing so, the star scientist and tlo create currency for the university spin-off in their subsequent quest to raise financing (hsu and ziedonis, ; maine and thomas, ) . further, broad, blocking patents on platform technologies enable different spin-offs to simultaneously develop the technology for specific market applications, as one spin-off may not be able to commercialize all applications of a platform technology. this coordination enables broader value creation from platform technologies (arora et al., ; gambardella and mcgahan, ) . such practices reduce the holdup problem, particularly problematic with large incumbent firms, which can lose interest in in-licensed technologies (langer, ) . supporting the arguments of al-aali and teece ( ), this case evidence demonstrates how the strategic management of ip can influence venture success, and suggests strategies which may be followed by other scientist-entrepreneurs and their university tlos. the evidence summarized in table suggests that the pre-formation entrepreneurial capability of the star-scientist-entrepreneur in attracting and mentoring the founding team impacts university spin-off success. this finding is consistent with founder imprinting (beckman and burton, ) , but -critically -this is imprinting pre-formation. in line with the perspective of mathias et al. ( ) that "certain sources of imprint have an enduring effect on how entrepreneurs think about themselves, their opportunities, and their ventures," graduate school can be considered a formative stage in the life of a scientist, and the imprinting from an sse during this sensitive stage can manifest itself in the decision of the lab alumni to undertake science commercialization through science-based university spin-off formation. more broadly, the sse influences the founding team of the university spin-off (and thus the human capital endowment of the firm) through mentoring scientist-entrepreneurs, identifying and nurturing business talent, attracting vcs, and through their networks, experienced ceos. there is substantial evidence of the sse mentoring lab members, both in their scientific approach and in their entrepreneurial role (table ). in line with founder imprinting effects (beckman and burton, ) , we argue that the graduate students and postdoctoral fellows who are part of the team inventing the underlying technology can embody the characteristics of the star scientist founder. we propose that founder imprinting may not only occur between founder and firm post-formation, but can also occur between the sse and his or her lab members preformation. thus, lab alumni who go on to be scientist-entrepreneurs may imprint the values and characteristics of the sse on their spin-off, whether co-founded with the sse or not. mentoring lab members to become co-founders in a scientific role brings four advantages to university spin-offs: one, a direct link between the lab and the spin-off is created, facilitating tacit knowledge flow. two, the sse is freed to explore additional avenues to extend the platform technology within the academic research lab, which may directly or indirectly benefit the spinoff. three, the level of passion and commitment for the commercialization of this technology will be much higher than with a licensor scientist, especially if the underlying technology has been based on the thesis/ project of the lab member. four, the entrepreneurial mindset and practices of the sse will have been imprinted on the lab alumni pre-formation. our findings also concur with and elucidate findings by eesley et al. ( ) , who argue that the impact of founding team diversity is contingent on technology innovativeness. consistent with their study, we find that science-based university spin-offs with purely scientific founding teams, where no ceo is hired for several years after firm formation, do not appear to be penalized while raising financing ( table ) . the most commonly observed pattern seen in our sample, however, is the sse pairing his lab alumni with experienced business co-founders, generally identified through the networks of vc investors. notably, spin-offs were more successful at raising financing when their initial ceos had executive experience at a biopharma company ( table ) , suggesting that such managerial experience and social capital enable success in this sector. such executives are not typically within the networks of academic scientists. thus, the role of the sse in attracting vcs and, through their networks, experienced business co-founders, also contributes to the success of the venture. venture capitalists provide much needed capital resources to science-based university spin-offs (maine and thomas, ; shane and stuart, ) . they also leverage their network in the search for professionals with appropriate management experience who can lead the fledging university spin-off (hellmann and puri, ) . beyond their financial capital and social capital, association with a reputed venture capitalist is in itself a signalling mechanism to other potential investors (hsu, ) . such an association with a venture capitalist can help the university spin-off raise multiple rounds of financing, which is the most important predictor of an ipo (shane and stuart, ) . during the uncertainty-filled pre-formation stage, significant efforts are needed to attract high quality human capital and sufficient ongoing funding. the timing of key decisions in the early stages of venture emergence is attracting growing attention. scholars have noted that nascent entrepreneurs take important decisions during this period, and that the coordination and sequencing of such decisions can impact venture viability (dimov, ; hopp and greene, ; rasmussen, ) . related research on the importance of timing to commercialization has looked at this problem from the view of firm entry and exit strategies (arora, fosfuri and ronde, ; suarez et al., ) , the impact of timing patents on future licensing (kim et al., ) , and signalling the quality of the intellectual property to potential investors (hsu and ziedonis, ; maine and thomas, ) . this study contributes to the timing literature by identifying the pre-formation coordination, sequencing, and timing of technology-market matching, elite publications and broad, blocking, relevant patents, and firm formation, demonstrating how a star scientist endows science-based university spin-offs. teece ( teece ( , pp. argues that well positioned firms can afford to wait to exploit opportunities until the timing is most advantageous. we demonstrate the same is true in the case of nascent science-based ventures. the early timing of technology-market matching and of publications and patents, coupled with delayed spin-off formation by the star scientist and his collaborators, can give a positive ex ante signal to better align venture timelines and expectations with those of venture capital investors. this timing is important as few science-based spin-offs are able to raise venture capital or reach an ipo (fini et al., , maine and thomas, ) and yet science-based spin-off ventures require vast amounts of risk capital over extended time periods to commercialize their inventions (maine and seegopaul, ; pisano, ) . teece ( , pp. ) argues for the importance of "getting the timing right" and that "the capacity to make high-quality, unbiased but interrelated investment decisions in the context of network externalities, innovation, and change…", is the dynamic capability of seizing opportunities. we argue such an entrepreneurial capability is also relevant pre-formation (figure ) , in that the coordination, sequencing, and timing of commercialization decisions made by the scientist entrepreneur enable opportunities to be seized. the relative timing of patenting to firm formation allows insight into this dynamic capability around strategic timing. table depicts the gestation period (timing in years from grant of first blocking patent until firm formation) of each venture. this period is notably long as the sse times patents and key papers as early as possible, and yet delays the formation of the venture. post patenting, the assessment of when a science-based university spin-off is sufficiently endowed for the biomedical commercialization challenge involves dynamic assessment of technology and commercial viability (arnaud, ; gruber and tal, ; maine, probert, and ashby, ; teece, ) , the readiness of a scientific-entrepreneur to lead further technological development within the firm (clarysse and moray, ) , and the fit of the remaining developmental timeline with venture capitalists' institutional logic and window for investment (maine and seegopaul, ; maine and thomas, ; pahnke, katila, and eisenhardt, ) . during the longer gestation times, the sse further develops the technology towards commercial viability, mentors scientific leadership for the eventual venture, and helps attract vcs. these are all aspects of the pre-formation entrepreneurial capability of strategic timing. for those ventures spun out of the lab of the sse which had longer gestation times, we observed evidence that the technology was not initially considered to be viable in the chosen market opportunity, and that, during the gestation time, the sse mentored scientist-entrepreneurs and was involved in attracting vcs (tables and ) , as well as further developing the technology towards commercial viability. such strategy has systematically led to multiple rounds of financing from venture capitalists and the public market (table ) . these levels of financing are far higher than typical vc financing for university spin-offs (zhang, , table ) much basic research and certainly generic platform technologies allow for a broad range of applications over time (maine and garnsey, ) . as projects that draw on this platform technology are formulated, iterative technology-market matching done at the project formulation stage allows the star scientist and lab members additional time to design experiments to establish commercial viability prior to firm formation. as depicted in figure , ventures were spun out of the platform technology of controlled release polymers over nearly years: noteably, for of these spin-offs, technology-market matching occurred in the project formulation stage ( table ). the strategic importance of early technology-market matching and of delaying venture formation is likely to have increased during the past two decades. innovation scholars argue that managing and rewarding science commercialization has become increasingly difficult during this period of time (arora et al., ; arora, fosfuri, and ronde, ; pisano, ) . our data is consistent with the increasing difficulty of taking a breakthrough technology to market, with the gestation period of science-based university spin-off ventures lengthening from an average of . years for the first ventures co-founded by the star scientist entrepreneur to an average of . years for the latter ventures co-founded by the star scientist entrepreneur. beyond deciding when a venture is commercially viable, the importance of coordination and sequencing in the timing of key commercialization decisions is a contribution to the literature. although each commercialization decision may be recognized as important individually, our study reveals the importance of the coordination and sequencing of these key decisions in leading to well-endowed university spin-offs pre-formation. repeatedly, we observed technology-market matching during the project formulation stage, the broad, blocking, relevant patents linked to elite publication and the delayed firm formation. such coordination, sequencing, and timing of decisions by the sse (with his collaborators and the tlo), mostly in stealth mode, helps prepare the nascent venture in the pre-formation and early post-formation stages of venture emergence. the holistic nature of this extended case method study, and the nuanced empirical evidence anchored in time by the patent-paper-venture matching method represent a significant contribution to the strategic timing literature, as well as to the broader literatures on academic entrepreneurship and dynamic capabilities. investors might also benefit from the insights provided by our study. our measure of broad, blocking, relevant patents might be a useful metric for them to utilize in identifying high potential ventures ex-ante. investors -particularly those with domain-specific expertise -may also choose to invest in long term relationships with scientist-entrepreneurs, helping them develop technology-market matching capabilities and introducing them to their networks of potential venture ceos. there are several implications of this research for university leadership and innovation policymakers. the need for strategic leadership in the role played by universities in our knowledge-based economy has never been greater (fini et al., ; leih and teece, ) . creating an entrepreneurial culture and facilitating interdisciplinary collaboration, while acknowledged as important, require significant change (leih and teece, ; mathias et al., ; sharp, ) . at mit, both have been purposefully nurtured over the past decades (roberts et al., ; sharp, ) . the importance of strategic management of intellectual property to science-based university spin-offs is demonstrated. all too often, claiming, protecting and commercializing ip is unintentionally constrained by university tlos and national innovation policies (bubela and caulfield, ; hall et al., ; huang-saad et al., ) . success metrics would ideally align the incentives of university tlos with long term objectives for the university as well as with regional and national systems of innovation (bubela and caulfield ; christini, ; langford et al., ) . for example, technology licensing offices, if better resourced, could follow mit in deciding to file patents on invention disclosures with potential impact (nelsen, ; appendix , interview ) rather than rationing their resources based on short term revenue or cost recovery considerations. entrepreneurial education for potential scientistentrepreneurs can lead to better utilization of tlo resources by reducing basic mistakes and by making faculty/tlo interactions more productive (bienkowska et al., ; council of canadian academies, ; huang-saad et al., ) . there are also implications from our findings for national and regional innovation policymakers. consistent with fini et al. ( ) , this study suggests innovation policies which support "bottom up" initiatives to enable the emergence of higher quality university spin-offs are needed. we show that the entrepreneurial capabilities of a star-scientist-entrepreneur can enable well endowed (in other words, high quality) university spin-offs. yet policies focused on importing star-scientist-entrepreneurs are both impractical and insufficient. rather than importing star scientists, policymakers can create the conditions to nurture their emergence through innovation policies aimed at developing and supporting pre-formation entrepreneurial capabilities. the dynamic capability of science-based ventures to rapidly respond to opportunities or crises such as the covid- pandemic, originates pre-formation in the purposeful building of the four key entrepreneurial capabilities described in this study. thus, the firm-level dynamic capability of technology-market matching at moderna therapeutics was developed in a path dependent manner from the entrepreneurial capabilities practised and imprinted pre-formation. entrepreneurship programs aimed at scientists and engineers can develop entrepreneurial capabilities in technology-market matching, claiming and protecting ip, attracting and mentoring the founding team, and strategic timing. the us nsf training program i-corps has demonstrated some success in educating scientist-entrepreneurs in later stage technology-market matching, and also broadening their entrepreneurial networks (huang-saad et al., ) . however, harms et al. ( ) argues that existing programs are ill-suited to sectors with high technology uncertainty and long gestation times. additional or alternative educational programs, focused on building pre-formation entrepreneurial capabilities in university scientists, could unlock substantial additional value from university inventions. policies which lower barriers to claiming and protecting ip and to strategic timing by scientist-entrepreneurs -such as by better resourcing tlos, making patenting an eligible cost by granting agencies, and not requiring venture formation by faculty in order to be eligible for early commercialization grants -when combined with committed university leadership, will support the emergence of well-endowed sciencebased university spin-offs. this study has two main limitations. first, by concentrating on the career of an unusually prolific biomedical star-scientist-entrepreneur at mit, the generalizability of the results may be questioned. clearly, entrepreneurial outcomes at elite universities located in leading technology clusters are not representative of the average university or region (nightingale and coad, ; siegel and wright, ) . nevertheless, we contend that such universities are where the phenomenon of science-based university spin-off emergence is most prevalent. stuart and ding ( ) find that elite universities form the most science-based spin-offs, with scientists at the top us universities being times more likely to become a scientist-entrepreneur, and that scientists involved in the founding of a university spin-off published at a rate of . times that of their matched "pure" scientist. there is also evidence of an increasing prevalence of serial scientist-entrepreneurs (lawson and sterzi, ; stuart and ding, ) . the nuanced evidence provided in this study about the role of this exemplar scientist-entrepreneur in university spin-off formation can inform and enhance commercialization activities by academic-entrepreneurs at a broad range of institutions. for example, technology-market matching at the project formulation stage is a learned capability which can be developed by academic scientists in any region. second, the entrepreneurial capability of claiming and protecting ip may not be easily replicable in less munificent environments. some universities may not be able to provide funds to support extensive patenting activities. government grants may also not support patenting. while acknowledging this limitation, we contend that limited funds at university tlos mean that even more care needs to be taken to ensure that the fewer patents being filed are broad and blocking in nature. thus even if the inventor decides to license their technology to incumbents, having broad, blocking, patent protection (along with elite journal publications) can increase the chances that their inventions will reach society. a caveat here is that securing a broad, blocking, relevant patent is not always under the control of the scientist or the tlo, and can be characterized as a protracted give-and-take affair with the patent office. utilizing the extended case method, existing dynamic capabilities theory is confronted and extended with evidence as to how an exemplar scientist-entrepreneur senses, shapes, and seizes opportunities to endow university spin-offs pre-formation. responding to calls for processoriented, individual-level studies to extend dynamic capabilities theory (protogerou et al., , pp. ; schilke et al., ) , this study contributes to the academic entrepreneurship literature by extending dynamic capabilities theory to the individual-level during the pre-formation stage of science-based university spin-offs. methods were developed to identify biomedical platform technologies and match the inventions (embodied in granted us patents and papers) of a biomedical star-scientist-entrepreneur with the university spin-offs he co-founded over years of his career, allowing for a longitudinal examination of the progression of science from research laboratory to science-based university spin-off. a process model was developed, depicting the key role played by the sse in four pre-formation entrepreneurial capabilities which endow these science-based university spin-offs for success: technology-market matching, claiming and protecting the invention, attracting and mentoring the founding team, and strategic timing. this paper demonstrates how these entrepreneurial capabilities can be developed and deployed by scientist-entrepreneurs. we propose that such entrepreneurial capabilities can also be taught more broadly to university scientists. our study suggests that innovation policies aimed at innovative start-ups should focus on supporting scientist-entrepreneurs in the pre-formation stage of university spin-off emergence. the recommendations drawn from this research can guide academic scientists, investors, university leadership, and policy makers in fostering the commercialization of scientific inventions through university spin-offs. fig. ) can be disaggregated into the capacity ( ) to sense and shape opportunities ( ) to seize opportunities ( ) to maintain competitiveness through enhancing, combining, protecting, and when necessary, reconfiguring the enterprises. (teece, ) a call for more processoriented studies to extend dynamic capabilities theory (schilke et al., ) . a call for "focusing on the entrepreneurial function embedded in dynamic capabilities i.e. managerial capabilities for sensing and seizing opportunities." (protogerou et al., , pp. ) , and "how do star scientists and technology gatekeepers influence the development of sectorbased entrepreneurial capabilities?" de massis et al. 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biotechnology internal human capital and the birth of u.s. biotechnology enterprises the authors thank l. foster, r. langer • the pre-formation stage of ventures co-founded by a star scientist-entrepreneur is analysed. • co-invented us patents granted are matched to co-authored publications and these ventures.• existing dynamic capabilities theory is extended to show how a star scientistentrepreneur senses and shapes and seizes opportunities. • a process model is developed depicting four pre-formation entrepreneurial capabilities. key: cord- - o g q authors: polychronakis, ioannis; riza, elena; karnaki, pania; linos, athena title: workplace health promotion interventions concerningwomenworkers' occupational hazards date: journal: promoting health for working women doi: . / - - - - _ sha: doc_id: cord_uid: o g q nan in the european labor market, women today constitute an increasing part of the working population, equaling about percent of the european workforce (european agency for safety and health at work a) as a result of their dynamic entrance in the labor market during the last few decades. while women have occupied posts even in professions that so far have been considered as "traditionally male," the european labor market retains a high degree of segregation regarding women's participation rates in certain occupational sectors (european agency for safety and health at work a; ) . the european union (eu) has so far applied a gender-neutral approach (european agency for safety and health at work a; ) to policies and legislation concerning occupational safety and health (osh) to comply with world health organization (who) guidelines for equality in health standards and access to health service. however, this approach does not seem to suffice for effectively meeting gender-specific issues of occupational hygiene and safety that have emerged concerning female workers in particular. the female working population carries certain characteristics that have to be taken into consideration through the process of design and implementation of osh policies, because their interaction with the occupational environment may produce additional hazardous effects for women employees: women's workday concerns arising from their roles as mothers, spouses, or carers for the elderly, add an extra load on the mental and physical fatigue they sustain in their workplace (artazcoz et al. ; artazcoz, borrell & benach ) . everyday household tasks amount to hours of unpaid overtime on top of the -hour working day, increasing their total physical and psychological strain. as a consequence, women workers are more easily affected by burnout effect or suffer more frequently from work-related stress than their male colleagues, who continue to participate significantly less than women in house tasks. working conditions in terms of ergonomics, working pace, managing heavy workloads, and using tools or personal protective equipment (ppe) (tapp ; murphy, patton, mello, bidwell, & harp ) are often designed according to the size and the physical strength of an average male worker. this is a consequence of the fact that many occupational sectors were, until recently, almost exclusively staffed by men, and even today employ an overwhelming majority of male workers. despite the increase in the participation of female workers in many professional fields, the high cost of adequate interventions still constitutes a forceful barrier to adjusting the modern workplace to female employee's needs for health and safety. because women of child-bearing age constitute a significant part of the female workforce, the protection of women's reproductive health is an issue of great concern for eu policymakers, in terms of legislation. this applies to factors and working conditions that both directly and indirectly influence the female reproductive system, including fertility (biological, physical, or chemical hazards-e.g., endocrine disruptors that affect women's ability to conceive), pregnancy (detrimental factors for the foetus during intrauterine development), and lactation. one also has to underline the fact that pregnant women are in need of specially designed ergonomic workplaces (niedhammer, saurel-cubizolles, piciotti & bonenfant ) , that consider changing physical and biological conditions and needs throughout the gestation and post-partum period. biological predisposition determines that women employees have reduced physical strength in comparison with their male colleagues (hooftman, van der beek, bongers, & van mechelen ) . this fact creates a comparatively higher burden for female workers who perform the same tasks as men, and creates a greater risk for musculoskeletal strain. furthermore, women's reduced average muscle force places them in an unfavorable position in cases of bullying and physical violence at their workplace, both from co-workers or the public (e.g., psychiatric ward nurses). women workers are still a minority group in certain professional fields (e.g., construction, mineral extraction, heavy industry), and in most cases they remain in lower managerial positions in comparison with men. under these circumstances, women employees have limited control over administrative decisions (european agency for safety and health at work ) concerning occupational health and safety, and often lack access to the appropriate communication channels to report cases of bullying, mobbing, or even sexual harassment-especially when superiors are involved. in certain areas of industrial production (e.g., the textile industry), the female working population consists predominantly of immigrant workers with poor literacy skills, or difficulty communicating. this language barrier may, in some situations, cause work-related accidents, as well as expose workers to occupational hazards due to misconception or ignorance of safety instructions or warning labels and signs. women in europe present higher percentages of part-time employment than men, as shown in figures . and . . in many occupational sectors (e.g., cleaning industry, cashiers), the overwhelming majority of women work part time. in addition, female employees show a higher turnover rate during their career and seem to spend shorter periods, on average, in the same position (mcdiarmid & gucer ) . because of this effect, women's occupational diseases are, in many cases, significantly underreported, introducing a systematic bias in many studies on occupational hazards and creating the misperception that female workers generally occupy safer jobs. to make matters even worse, women in this kind of unstable employment pattern have, in most cases, only limited access to occupational health services and workplace health promotion activities, even though they constitute a high-priority group for similar interventions. it should be underlined, however, that under no circumstance does this genderspecific approach lead to the false conclusion that women workers constitute a (jouhette & romans ) high-risk group requiring preferential treatment over issues of occupational safety and health in comparison to male workers. such a misinterpretation could cause unacceptable discrimination against women and, in some cases, their exclusion from occupational sectors where female workers have for a long time now proven their worthiness as employees. even though available research data in the literature may suggest that certain traits or characteristics connected with gender could possibly influence the occupational risk of female employees, they fail to identify occupational hazards that are selectively or exclusively harmful to women. at this point, it is useful to categorize all gender-related parameters that have been identified as distinguishing occupational health and safety issues between male and female workers. according to previous studies, three fields of possible gender influence (kennedy & koehoorn ) on estimated occupational risk can be identified. because of job and task segregation observed among the european workforce, osh studies based on occupational categorization alone have been insufficient in assessing potential health risks for women, because their tasks may vary significantly from men's even if they carry the same professional title (Östlin ) . women follow different time patterns of exposure through part-time or shift work, and usually carry out tasks requiring more precise, repetitive movements than male workers (hooftman et al. ; stellman ) . • female workers have smaller (on average) body dimensions (hooftman et al. ) , which differentiates their occupational exposure: a. in professions involving manual handling, greater physical workload may be required by women to perform the same tasks as men. b. in cases of chemical exposure through the skin, the female body provides smaller available surface for absorption. c. protective equipment is often ineffective for women employees (protective clothing, gloves, masks, and respirators) (han dh ) . protective equipment originally designed for male workers does not fit appropriately to the shape and size of the female body and does not fully prevent exposure to hazardous agents. • under normal conditions, women present lower alveolar ventilation rate and cardiac output (brown, shelley & fisher ) , which reduces the input rate of volatile chemicals into their body • in the case of benzene (a proven carcinogen) and other volatile organic compounds (vocs), it has been experimentally demonstrated that women present higher blood/air partition coefficients (brown et al. ) (greater blood / air concentration fraction), increasing the amount of chemicals diffused from alveoli to the blood compartment • concerning the metabolism of chemical compounds, potential gender-related disparities in enzymic activity (gandhi, aweeka, greenblatt & blaschke ) (e.g., cytochromes p , transporting enzymes) have been reported, although research results are contradictory • in the case of exposure to metals, women appear to absorb greater amounts of cadmium through digestion, possibly due to a common absorption pathway for iron and cadmium (vahter, berglund, Åkesson & lidén ) (especially for menstruating women with low body-iron storage) • chemicals absorbed into the bodies of women workers are distributed in a relatively smaller body mass than men, because their body mass index (bmi) is lower (gandhi et al. ) . as a further consequence: a. women present a relatively higher organ blood flow, which increases the rate at which chemical substances circulating in blood compartment are delivered to the tissues. b. women's renal clearance (gandhi et al. ) (a parameter that is directly related to body weight) is slower in comparison with men's, and therefore their capacity to excrete toxic compounds, as well as their metabolites, through daily production of urine is low. • bodily distribution of chemicals in women also differs in regard to their concentration in plasma. experiments on gender influence on the distribution of certain drugs, indicate that (gandhi et al. ): a. plasma volume is generally lower in females (the same total-body chemical burden may produce more toxic plasma concentrations in women). a. the concentration of certain binding proteins for drug metabolites or other chemicals in plasma depends heavily on hormonal status-especially estrogens (e.g., pregnancy, menstrual phase, and menopause). • the female body carries a greater proportion of adipose tissue than that of males (brown et al. ; gandhi et al. ) , and as a result it demonstrates a different pharmacokinetic response to lipophilic metabolites (e.g., prolonged retain and increased metabolism of benzene). • in professions involving exposure to inorganic lead, blood concentrations do not provide a reliable criterion of chronic exposure in the case of female employees. as the metal gradually accumulates in the bone tissue, demineralization of women's skeleton during periods of increased bone turnover (as in pregnancy or menopause) releases significant quantities of lead into their bloodstream (vahter m et al. ) . • women present different social and dietary habits, such as smoking (e.g., cadmium absorption) (vahter m et al. ) and alcohol or coffee consumption (mcgovern ) , which may act as modifiers to environmental exposures • the use of chemical substances for household tasks (e.g., cleaning products), hobbies (e.g., fertilizers in gardening), or other activities involving application of potentially harmful agents (including cosmetics and artificial hair dyes) may subject women to further exposure outside their daily work hours • wearing jewelery is an additional nonoccupational source of skin exposure to metals for women (e.g., nickel) (vahter m et al. ) , increasing the burden of metal-induced occupational dermatitis for women employees • female employees in occupations involving manual tasks may also have to sustain additional workloads arising from family demands, especially in large families with children under years old, or elderly persons over years old (artazcoz et al. ) , which may contribute to producing symptoms of physical fatigue or musculoskeletal strain. • increased family demands of female workers, combined with strenuous job tasks may also have a serious impact on women's mental health (disturbed work-life balance, inadequate leisure time, lack of personal life) (artazcoz et al. ) • besides the immediate toxic effects of certain metals such as cadmium on humans (affecting both men and women), there is ongoing research on possible estrogenlike activity as well as its potential association with breast cancer through the activation of estrogenic receptors (brama m et al. ) • the manifestation of certain gender-specific cancers (e.g., breast cancer, which occurs almost exclusively in women) seems to involve among others, interaction between genetic expression (e.g. atm tumor suppressors) (mcgovern ) and environmental exposures • the manifestation of autoimmune diseases (highly frequent among the female population) might be triggered or accelerated by substances or agents commonly used in certain professions (as in the case of lupus erythematosus and mercury exposure) (mcgovern ) • the varying composition of the labor force in different occupational sectors may have introduced a significant bias in epidemiological studies concerning occupational hazards for women: a. especially in the heavy industry and construction sectors, which employ almost exclusively male employees, the small minority of women who work alongside their male co-workers in various positions may have been overlooked (niedhammer et al. ) in osh studies, introducing exclusion bias (concerning women workers) because of the difficulties researchers had in finding women employees to participate in their studies. b. on the other hand, women-focused osh research has concentrated on the relatively small number of professions that master the majority of the female work-force. this fact probably explains the relatively large volume of studies on health-care professions (which are easily accessible to research), while women workers remain heavily underrepresented in osh studies in other sectors (messing & stellman mager ) c. the majority of studies that focus exclusively on women workers deal with mental health issues and psychological parameters (messing & stellman mager ; niedhammer et al. ) , while other work-related hazards such as exposure to chemicals, radioactive material, biological factors, electromagnetic fields, noise, or ergonomic factors are either indirectly examined by surveys on mixed working populations (where results are adjusted for gender), or even worse, by generalizing epidemiological evidence of osh conducted among male employees. • the segregation of tasks performed within the same job department or even under the same occupational title, may introduce misclassification bias when the influence of gender on occupational risk is under study. any observed excess risk among women workers (e.g. musculoskeletal injuries) in comparison with men, should not necessarily be attributed to the role of gender, especially when such results are based only on job title (hooftman et al. ). in such cases, further quantification of exposure (job exposure matrices, stratification according to tasks) is essential in determining whether the declination in study results arises from differences in performed tasks, or is truly related to gender-e.g., the excess risk for developing carpal tunnel syndrome in female workers seems to be eliminated in professions with strictly defined tasks (mcdiarmid, oliver, ruser & gucer ) . • other forms of bias related to gender have been identified in the design of clinical, as well as osh, studies: a. an observer error due to adopting "male perspective and way of thinking" (pinn ) in interpreting epidemiological data. b. the "male norm" bias, arising from the use of male workers as standard (pinn ) , even for occupational health and safety issues where both sexes are affected (e.g., occupational cancer). • there are indications that many of the existing studies on women workersespecially those concerning occupational musculoskeletal injury-may suffer from perceptual bias (the increased likelihood of employees to report injuries), or overrating the severity of related symptoms in questionnaire surveys according to the way they perceive their working environment or their degree of job satisfaction (strazdins & bammer ) . taking into account that female workers are generally occupied in less satisfactory, underpaid jobs with repetitivemonotonous tasks (hooftman et al. ) , over-reporting may contribute significantly to the excess risk found by many relevant studies for female employees. • for the majority of female workers employed outside the dangerous industrial or construction sectors, there is little public awareness of the occupational exposures they sustain from their working environment because they usually do not face immediate danger of acute toxic effects or death. this fact may introduce a significant recall bias in relative studies because women workers are either unable to identify potentially harmful agents they have been exposed to, or tend to underestimate the extent of such exposures (e.g., unawareness of types of agents involved in their tasks that may constitute reproductive hazards) (bauer, romitti & reynolds ) . • in mixed working populations, the healthy worker effect appears stronger for male than female employees , which is possibly attributable to the fact that men are hired to perform more physically demanding tasks than women and are therefore subjected to more rigorous selection during the hiring process. the existing research evidence indicates a widely accepted false sense of safety in many of the professional sectors employing predominantly women, which has been recognized in earlier occupational health and safety studies in the united states as the so-called generally recognized as safe (gras) status (mcdiarmid & gucer ) for most of the female professions. this is partially due to the fact that male workers, especially in heavy industry (construction workers, miners, welders, heavy machinery operators), are expected to face a higher number of severe or even fatal incidents or occupational diseases (niedhammer et al. ) , than those in the safe tertiary sector. gras reflects the commonly held belief that certain drugs and chemicals are safe if empirical knowledge obtained by their wide use over a period of years does not indicate they are detrimental to the population. as a consequence, this approach is also adopted in occupational sectors, where such materials have been widely used-the majority of which involve femaledominated professions where, until recently, osh research has been considered nonessential. contemporary evidence-based medicine, however, requires more solid epidemiological data to conclude whether this group of occupations is as safe as is currently presumed. in addition, there is an increasing need to study the possible sideeffects on health from exposure to thousands of chemical compounds present in jobs generally considered as nonhazardous (cleaning agents, drugs, cosmetics, food preservatives). the latter translates as a need to expand the field of occupational health and safety research and place the so-called female professions under a more thorough and systematic investigation. according to official statistics of the european agency for safety and health at work, certain occupational sectors (health professionals, education workers) employ mostly females while the percentage of women in other professions (construction workers, heavy industry) (european agency for safety and health at work ) remains relatively low. figure . presents the distribution of the female working population in different occupational activities, in the european union. for many of the professions where women are highly represented, research has explored specific occupational hazards. in tables . in the health services sector, women are employed in various positions (e.g., nurses, laboratory technicians, emergency room technicians) and face a multitude of occupational risks, some of which are cited in table . . women are also often employed in the education sector, especially in nursery and primary education, and therefore face diverse occupational risks, some of which are specific to the profession (e.g., voice disorders). table . presents some of the related occupational hazards for this category of workers. while affected by many occupational hazards, some of which are cited in table . , women working in the cleaning industry are also disadvantaged due to the fact that (gavana, tsoukana, giannakopoulos, smyrnakis, & benos, ; gyorkos et al., ; nakazono, nii-no, & ishi, ; skillen, olson, & gilbert, ; valeur-jensen et al., ) • vascular problems (kovess-masfety, sevilla-dedieu, rios-seidel, nerriere, & chee, ) of the lower extremities due to extended standing (sandmark, wiktorin, hogstedt, klenell-hatschek, & vingard, ) in upright position • voice disorders due to overuse of vocal chords duff, proctor, & yairi, ; kooijman et al., ; kosztyla-hojna, rogowski, ruczaj, pepinski, & lobaczuk-sitnik, ; roy, ; sliwinska-kowalska et al., ; sulkowski & kowalska, ; thibeault, merrill, roy, gray, & smith, ; williams, ) • exposure to increased levels of noise (behar et al., ) • musculoskeletal problems (fjellman-wiklund, brulin, & sundelin, ; sandmark, ; yamamoto, saeki, & kurumatani, ) (handling and lifting small children in day care centres, physical education teachers, inadequate body posture) • work-related stress (fjellman-wiklund et al., ; zidkova & martinkova, ) • children's or adolescent's violent behavior (lawrence & green, ) • exposure to infectious agents • dermatitis due to direct skin contact with irritating substances (weisshaar et al., ) • dermal infections (staphylococcus, fungi) (mcbryde, bradley, whitby, & mcelwain, ) • inhalation of irritating vapours and airborne micro-particles containing dust or other allergens (j. j. jaakkola & jaakkola, ) • musculoskeletal disorders due to handling or lifting heavy objects, inadequate body posture (balogh et al., ; mondelli et al., ) • fall injuries (stairs, slippery floors) (kines, hannerz, mikkelsen, & tuchsen, ) • workplace violence (chen & skillen, ) • sexual harassment table . food production industry workers workplace hazards • inhalation of airborne allergens emitted from food processing (e.g., artificial dyes, flour, animal proteins) • dermal infections (staphylococcus, b-haemolytic streptococcus, bacillus anthracis, fungi) • dermatitis (allergic or irritating) from skin contact to foods themselves or substances used for their processing (jappe, bonnekoh, hausen, & gollnick, ; kanerva, estlander, & jolanki, ) • exposure to zoonoses (processing animal products) • musculoskeletal disorders (handling and lifting excessive loads, inappropriate body postures, poor ergonomic design of workstations, repetitive strain) (chyuan, du, yeh, & li, ) • injuries (falls due to slippery floors, burns, lacerations from knives or used tools) (courtney et al., ) • exposure to extreme temperatures (cold in refrigerators, excessive heat in kitchens) their occupation is often unregulated, and thus no occupational safety and health services are available to them. the food production sector involves various types of work, from food preparation to packaging, storing, and more, involving mainly biological and chemical hazards due to immediate contact with food. table . presents a non-exhaustive lists of related occupational hazards. a large number of women are employed in the sector of hospitality services (e.g., waitresses, cooks, bar attendants) and are subject to a number of risks, some of which are listed in table . . the textile sector is heavily industrialized, and women working in this sector face many and serious risks, some of which are cited in table . . laundry workers are also faced with heavy tasks such as long hours on their feet, exposure to extreme temperatures, and lifting heavy loads, as can be seen in table . . table . hospitality services industry-restaurant workers' workplace hazards • exposure to extreme temperature conditions (excessive heat in cookers) • musculoskeletal injury due to handling or lifting heavy objects-repetitive movementsstrenuous workload (chyuan et al., ; dempsey & filiaggi, ) • dermatitis induced by skin contact with foods or cleaning agents (jappe et al., ; kanerva et al., ) • dermal infections (skin contact to infected food surfaces, development of fungal infections due to extended exposure to humidity) • injuries (falls due to slippery floors, falling objects, skin lacerations from sharp objects, burns from heat-emitting objects or appliances) (courtney et al., ; horwitz & mccall, ) • inhalation of micro-particles (food-cooking, passive smoking, poor ventilation) (svendsen, jensen, sivertsen, & sjaastad, ) • workplace violence (graham, bernards, osgood, & wells, ) • sexual harassment • work-related stress (low levels of job satisfaction, stressful working conditions) table . textile industry-clothing manufacture workplace hazards • exposure to increased levels of noise (weaving machines) (bedi, ; cardoso, oliveira, silva, aguas, & pereira, ) • increased concentration of fibres, micro-particles and organic solvents (artificial dyes, chemicals used in textile processing) in workplace environment (bakirci et al., ; ghio et al., ) • musculoskeletal injury (poor ergonomic design (choobineh, lahmi, hosseini, shahnavaz, & jazani, ) of the production line, repetitive movements (bjorksten, boquist, talback, & edling, ) , lifting and handling heavy objects) • visual fatigue • injuries (entanglement in moving parts of equipment, skin lacerations by sharp objects) • intense work-related stress (strenuous workload, intense work pace in production lines, low level of job satisfaction) ceramic and pottery workers face a series of specific occupational risks connected with the nature of their profession, as presented in table . . light manufacturing includes many types of industries, employing mainly nonspecialized workers and therefore involving diverse types of exposure. table . presents some of the hazards involved in these occupational activities. (dorevitch & babin, ) • musculoskeletal injury due to poor ergonomic design, handling heavy loads, repetitive muscle strain, vibrations (martinelli & carri, ) • stressful working conditions -strenuous work pace in production lines table . light manufacture workers' workplace hazards • musculoskeletal injury due to poor ergonomic design (equipment, tools and workstations that don't fit the physical dimensions of female workers), handling and lifting heavy loads, repetitive movements (bjorksten et al., ; roquelaure et al., ) • visual fatigue (untimanon et al., ) • exposure to chemical agents (e.g., metals & solvents in electronic circuits manufacture, drug by-products in the pharmaceutical industry) (clapp, ; ladou, ) • stressful working conditions in production lines call center work is a newly developed sector that employs mostly women who are faced with risks such as visual fatigue, musculoskeletal disorders, and other hazards as presented in table . . hairdressing is a female-dominated sector that, until recently, has been regarded as a safe occupation. however, current literature associates this profession with various hazards, some of which are included in table . . the tertiary sector-especially office workers-are faced with hazards arising mainly from poor ergonomic design and poor indoor air quality, as shown in table . . hazards in agriculture are linked mainly to a high risk of injuries and to the use of chemical substances such as pesticides, herbicides, and others, as shown in table . . (best et al., ) (inadequate body postures (osteras, ljunggren, gould, waersted, & bo veiersted, ) , poor ergonomic design (boyles, yearout, & rys, ) ) • vascular problems of the lower extremities due to prolonged standing in upright position • dermal infections (ballas, psarras, rafailidis, konstantinidis, & sakadamis, ; schroder, merk, & frank, ) (skin lacerations from scissors or other sharp tools (moghadam, mazloomy, & ehrampoush, ) , dermal fungi from continuous exposure to humidity) • dermatitis (khrenova, john, pfahlberg, gefeller, & uter, ; perkins & farrow, ) (irritating or allergic) (cavallo et al., ; doutre, ) induced by contact to cosmetics (amado & taylor, ; iorizzo, parente, vincenzi, pazzaglia, & tosti, ; katugampola et al., ; sosted, hesse, menne, andersen, & johansen, ) , artificial hair dyes (belinda thielen, ; rastogi, sosted, johansen, menne, & bossi, ) or even protective gloves (foti et al., ) • allergic asthma (akpinar-elci, cimrin, & elci, ; allmers, nickau, skudlik, & john, ; macchioni et al., ; moscato et al., ) induced by exposure to volatile substances (baur, ; berges & kleine, ; gala ortiz et al., ; hoerauf, funk, harth, & hobbhahn, ; hollund & moen, ; labreche, forest, trottier, lalonde, & simard, ; piipari & keskinen, ) and particles (cosmetics, hair sprays (albin et al., ; montomoli, cioni, sisinni, romeo, & sartorelli, ) , dryers • job-related stress (strenuous working conditions, low job satisfaction) (mcbride, firth, & herbison, ; perry & may, ) • exposure to zoonoses due to close contact with animals or animal products (bacillus anthracis, mycobacterium, brucellosis, viral infections e.g. avian influenza) • exposure to chemical compounds during transportation, storage, mixing or application of fertilizers, pesticides or herbicides (buranatrevedh & roy, ; garcia, ) • exposure to allergens through inhalation or direct skin contact (pollen, animal proteins, fungi) (linaker & smedley, ) • exposure to natural phenomena (extreme heat, frost, thunderstorms, floods) • job related stress (stressful working conditions, job insecurity, low income, low job satisfaction) • violence at the workplace (verbal or physical abuse) • sexual harassment this section will focus on how theories and models of health promotion can be put into practice for the design and implementation of workplace interventions concerning osh issues targeted at female workers. the example that will be used is work-related reproductive disorders. the specific health topic has been selected as an example for three primary reasons: • reproductive disorders have been associated with a wide range of occupational hazards (e.g. physical, chemical, biological agents) • a large number of professions employing women involve exposure to hazards such as those mentioned in the above point • further research is needed on this topic because many of the traditionally female professions considered generally safe may involve unidentified risks for women's reproductive health reproductive hazards constitute a field of increasing interest for occupational hygienists and health professionals across the world. there is little or no information at all about the possible effects on female reproductive health of the vast majority of chemical substances introduced by the thousands every year in industrial production (lawson et al. ) . even in cases of widely used chemicals, the existing literature of their possible detrimental effects on women's reproductive physiology is relatively poor. for most of the agents considered as hazardous for the reproductive system, their causal relationship to problems in human reproduction has not been adequately documented and gender differences in exposure or toxicity have not been thoroughly examined. because female workers constitute a nonhomogenous population of diverse occupational categories, various physical, chemical, and biological exposures are under examination concerning their potential risks on the reproductive health of women. a non exhaustive list of factors under investigation concerning their potential harmful effects on female reproductive health is presented in table . . the precede-proceed model of planning will be used as a framework to guide the diagnostic phase of the suggested intervention (gielen & mcdonald ; green & kreuter ; green, kreuter, deeds & partridge ; national cancer institute b; ransdell. ) . the outline of this theoretical model is presented in figure . . • precede provides the methodological framework for the design of tailored educational interventions targeting specific populations. it is based on the medical model, involving an initial diagnostic approach to the needs of a patient, before prescribing a specific treatment. as an analogy, precede constitutes a tool to design a specific educational plan, according to the identified needs of the target group. • proceed has been an addition to the original model, to further include environmental determinants (e.g., policies, managerial and economic issues) that influence human attitudes towards specific health behaviors. this model follows a reverse course, towards the origin of certain health behaviors to target interventions for the causal factors themselves, rather than just the symptoms. the outline of the process that takes place in nine stages is presented in figure . . for our example, only the diagnostic part of the model will be analyzed. despite the fact that we have already chosen occupational reproductive hazards as our intervention subject in this case, the stage of social diagnosis is supposed to have taken place before making our choice. for any workplace health promotion effort to be effective, the key issue must be tailored according to the needs of the predefined target population. even though women's reproductive health may seem like a scientifically important field of intervention, our target group of women employees may not consider it to be a highpriority issue-either because they consider having more important health problems or because they are not adequately informed on the possible impact of similar disorders on their personal health status. the main focus of health professionals at this stage is to investigate: • the target group's perception of their quality of life • the most important determinants of their quality of life (e.g., career, family, health) • their expectations and concerns about their health status • whether reproductive health issues are considered an important enough factor for women that an intervention through a whp program is valuable the focus of health professionals during this phase is to identify-through analyzing epidemiological evidence-the impact of the specific problem on the predefined target group (e.g., female workers in a factory, women employed in a specific profession). furthermore, this procedure aims at prioritizing the specific subgroups that face the highest risk of exposure to reproductive hazards and need more immediate preventive measures. this stage includes: • identification of work-related parameters, as well as individual behaviors that may influence the reproductive health status of women employees • evaluation of specific indicators of reproductive health disorders in our target population. some of these indicators are listed in table . one of the specific interests of health professionals at this stage is to locate groupings of reproductive disorder indicators in certain subgroups (specific job tasks, worksite-specific reproductive hazards) of the population, to prioritize them as intervention groups (e.g., focusing a whp program for the prevention of reproductive disorders on oncology unit nurses in case they present higher incidence of congenital defects compared to the rest of health care personnel) table . potential indicators of reproductive disorders (lawson et al. ) . increased infertility rates among women of a specific industry . a prolonged conception period among female workers . frequent reports of menstrual disorders and early menopause by female employees in the company's medical files . male/female ratio of births . reported pregnancy complications among employed pregnant workers (e.g., diabetes, hypertension, pre-eclampsia, etc.) . reduced (or increased) average birth-weight of infants . increased rates of pre-term deliveries (and miscarriages) . number of sick-leave days among pregnant employees (for problems related to pregnancy) . increased rates of congenital defects among infants of female workers . increased incidence of neoplasms of reproductive organs among employed women it is imperative during the initial design of a tailored whp intervention program for the prevention of occupation-induced female reproductive disorders, to incorporate a set of behavioral and environmental change indicators that serve as general objectives for the program. whp professionals, prior to the development of an intervention plan, should conduct behavioral and environmental diagnosis to identify existing key issues concerning osh attitudes and beliefs and practices in the organization (employees, executives, and company administration) and the safety status of facilities, procedures, and equipment. these key issues may include (state of alaska ): • personal accountability: this parameter is crucial for the success or failure of any prevention program, both on worksites and in the general population. it is important to adjust the program's aims and methods according to women's perceptions of its personal influence on their health status. female employees should be able to recognize their personal responsibility and contribution to the effective implementation of the preventive measures and practices by the completion of the whp intervention. • attitude towards change: a key component for the design of an effective intervention prevention program is taking into account the degree to which women agree with the proposed changes (safety behavior, practices, osh regulatory environment), so that invention methods can be modified accordingly. at this stage, therefore, health professionals should evaluate the awareness status and the ability of female employees to adopt the desired safety practices introduced by the whp program on both personal and collective levels to determine the kind of messages and strategies appropriate for the specific population. • participation: one of the primary targets of the whp intervention is to achieve a high degree of participation in the program's activities, as it is one of the key elements that significantly influences results. it is important at this stage to recognize and alleviate barriers that are driving women to abstain from similar programs. furthermore, there is a need to identify the subgroups of female workers where the focus of the intervention needs to be to promote their involvement. • occupational hazard identification: apart from recognizing that female workers are at a high risk for reproductive disorders, the whp program should also concentrate on specific protective measures and proposals for these groups of employees. it is essential, therefore, to identify and record existing working conditions of women in the specific organization in detail, to determine their possible detrimental effect on the reproductive physiology of those same women, and assess the existent osh status of their job tasks. this process includes recording: . procedures (production line, manual tasks, strenuous work pace, extreme climate conditions, emission of fumes/particles, and stressful conditions) . hazardous agents (physical, chemical, and biological) involved in female workers' tasks or working environment . equipment used in specific tasks (radiation sources, vibrating parts, electromagnetic fields) . existing protective measures (ventilation systems, separate mixing chambers for chemicals, lifting devices for manual handling, radiation shields, ppe, rotation of night shift workers, etc.) for women employees . potential for osh improvement (substitution of procedures or agents, automatization of tasks, amelioration of working conditions, change of job post, or rotation of workers) this part of the diagnostic process involves the identification of the educational needs of female employees, as well as the structural changes that are needed in the specific organization to effectively introduce the whp interventions for the protection of women worker's reproductive health. this process will be used to shape our strategic approach towards the target population, through the analysis of determinants of compliance with safety practices at individual, collective, and organizational levels. three categories of such factors may be identified-namely, predisposing, enabling, and reinforcing factors, that will be further analyzed: predisposing factors: health professionals may recognize multiple potential fields of intervention on which to focus the whp program: the whp program may introduce certain interventions to promote the desirable change to compliance in osh practices. • personalized information on female reproductive system and occupational risks involved • health awareness building on reproductive health issues and their importance • wide dissemination of existing scientific evidence on reproductive hazards for women employees (population awareness) • creation of peer support systems among groups of women workers to promote compliance with safety procedures • detailed recording of job tasks for female employees and identification of sources of exposures to known or potential reproductive hazards • introduction of specific safety guidelines and policies for the prevention of reproductive disorders • establishment of clear communication channels between employees and administration to report their concerns or personal experience on relative issues • improvement of the existing surveillance system for reporting suspicious cases among women workers reinforcing factors whp program officials may utilize numerous tactics to support the desired prevention strategy at this field. • provision of access to supplementary information resources on reproductive health issues and available prevention methods to the population of women employees • application of periodic follow-up sessions and use of frequent reminders (letters, telephone calls, e-mail messages) to retain an increased awareness level among female workers • dispensation of easily accessible screening services for exposure of employees to reproductive hazards • building a support network for the compliance of individuals with occupational safety practices by appointing safety committees that include female workers at risk for reproductive hazards • organization of group discussions among workers of specific occupational categories to share common experiences and concerns on related issues • projection of specific employees as models of good conduct in osh issues involving reproductive hazards prevention • presentation of statistics on results of exposure level reduction, or outcomes, if available (e.g., reduction on rate of miscarriages) organizational level- • active participation of women employees in the decision-making process concerning applied safety policies in the company • representation of female workers from different occupational sectors within the organization in administrative issues regarding the design of workstations and job tasks, and the introduction of new technologies, materials, and procedures • introduction of incentives for the compliance of employees with safety policies whp professionals should conduct this final diagnostic procedure before the implementation of the prevention program, to determine whether the program's scope and activities are compatible with the administrative and policy framework of the organization. the main issues to be identified at this stage are: • whether the policies and safety regulations related to potential reproductive hazards are in accordance with the program's requirements and the existence of requisite modifications or complementary arrangements • whether the program introduces any interventions that are in conflict with the organization's operational framework • whether the selected form of intervention (information campaign, skill building sessions, group activities) is appropriate for the existing company culture in osh issues • which of the existing structures and activities in the organization are useful to the program's strategic planning. some examples of similar structures and activities are presented in table . . • whether the company's administration is sufficiently flexible to adopt the participatory decision model proposed by the program for the resolution of osh issues • whether the organization's field of activities and operational status allows for alternative practices, procedures, and materials. table . sites an indicative list of similar practices and procedures. . systematic record of occupational medical history of workers . safety committees appointed by company's employees . trade-union department specialized in osh issues for female employees . registry of recognized occupational reproductive disorders . official forms for reporting employees remarks on working conditions and related hazards . detailed registry of materials, substances and processes utilized in each department of the organization (toxicity, carcinogenicity, potential for endocrine disrupting activity) . regular group meetings among workers and administration representatives . periodic screening of working population for hazardous occupational exposures the primary concern of whp professionals in the design of an educative intervention for women workers is to provide a tailored program according to the specific target population and its educational needs. the selection of a specific approach for this educational needs assessment depends heavily on the available resources (staff, time, expenditure limitation) of the program. listed below are some of the available techniques, and the form in which they may be employed, to obtain related information from the female workers' population (national cancer institute a; pfizer ; younger, wittet, hooks & lasher ) : women employees can be accessed individually, either at their worksite or through telephone or internet surveys, to fill in specifically designed questionnaires. some of the questions that may be included in such a questionnaire are listed in table . . this approach involves two-hour sessions of small work groups of six to ten women employees who testify their individual concerns, experiences, and percep- tions on work-related reproductive health issues. the activity takes place under the continuous supervision of an expert facilitator (health professional). the workgroup is selected on the basis of common socioeconomic and ethnic characteristics (e.g., representation of low literate immigrant female workers) as well as their specific job tasks. the application of this technique offers the whp program a more comprehensive insight into the target population profile (younger et al. ) , as well as the specific needs of certain special subgroups of womens workers (e.g., effective approach and training techniques, use of appropriate educative material). women employees are interviewed in the form of open-ended questions, where they are encouraged to identify themselves and their educational needs by trained professionals (instead of being guided by specific queries). even though this technique is the most time-consuming, it offers the most in-depth needs identification (younger et al. ) . these committees are formed by women employee representatives of specific at-risk populations, and consult whp professionals on specific issues related to reproductive health disorders among certain categories of workers, contributing their own experiences and concerns. the ecological model (mcleroy, bideau, steckler & glanz ) that was presented in the first chapter of this book offers the opportunity to identify the determinants of individual behavior within the wider context of social groups or organizations to which a person belongs. this perspective can therefore be useful for implementing comprehensive whp programs addressing specific health issues. analyzing the profile of a specific organization according to the five individual levels of the ecological perspective model-intrapersonal, interpersonal, institutional, community, and policy-one can identify multiple and multi-component potential interventions for the protection of female workers from the main categories of reproductive hazards recognized in the existing literature. at the intrapersonal level, workplace health promotion activities focus on individual skill building for female workers in the form of personal counseling on issues of reproductive health. the general scope of these interventions, some of which are presented in table . , is the introduction of a number of issues including: • identification of occupational reproductive hazards, personal risk factors, and related symptoms • requested behavioral changes towards prevention • skill-building in the correct use of equipment, materials, and safety practices • access to scientific resources and specialized health services workplace health promotion interventions at this level appeal to groups of working women instead of individuals. this stage of whp involves skill-building sessions, team collaboration and support activities, and health education, which may vary in group size or duration according to the educational needs of a specific working population. these groups can be selected according to common epidemiological or social characteristics of the workers (e.g., age, education, ethnicity) to adequately tailor any intervention. table . presents some of the group activities that may take place within the context of whp: cited in table . is a list of available interventions at the institutional (or company) level concerning organizational measures, practices, and policies that may be implemented for the protection of women worker's reproductive health. to implement effective workplace health promotion programs for the protection of female reproductive health, health professionals should not neglect the fact that the female working population in a specific worksite acquires certain characteristics that • group training sessions on radioactive material safe handling, and protective measures against ionizing radiation • group skill-building on stress management techniques, workplace design for the protection of pregnant women employees, safe manual handling methods • group education on infectious agents hazardous for female reproductive health, their transmission pathways, methods for prevention generic: • creation of employee's groups to constitute models of "best practice" and provide a supportive environment for the adoption of safety techniques (or "healthy behaviour") among women workers • creation of idea-exchanging groups, for identification of specific workplace reproductive hazards and special issues of concern for women workers define a community. the main focus of a successful whp project at this level is to incorporate the issue of reproductive health in the safety culture of female workers. this involves organizing targeted group activities for female workers that addresses the problem of reproductive hazard prevention through the community's system of "group norms, inner rules and beliefs" (edlich, winters, hudson, britt & long ) , and create a climate of awareness on issues related to female employees. at this level, workplace health promotion programs involve contacts with stakeholders (trade union members, employee representatives, company executives) and policymakers to propose measures, policies, and legal provisions for the protection of female workers' reproductive health, some of which are listed in table . . whp programs may utilize multiple channels of communication to deliver messages related to women's reproductive health protection. the approach may take various forms (prevention ): • proposals (by health professionals) for specific changes in the production line -substitution of chemical factors which are detrimental for female reproductive health with "safer" compounds • proposals for specific design changes in facilities, to isolate chemical procedures • frequent measurements by health technicians in the workplace to record concentration of chemicals, and identification of high risk population • training of occupational physicians and nurses in: a) identification of chemical reproductive hazards b) available preventive measures c) early diagnosis of reproductive disorders • re-positioning of pregnant employees to reduce potential exposure to chemicals • provision to all staff members of specific company's guidelines on occupational safety and reproductive hazards prevention (official forms, leaflets, electronic mail), as well as standard operating procedures for using chemicals • rotation of workers to reduce potential exposure • definition of a strict company's policy on health protection for non-smokers • placement of warning signs to prohibit smoking in the company • provision of adequate outdoor spaces, and timebreaks for smokers • funding of campaigns and incentives on smoking cessation for employees • proposals for specific design changes (e.g. armoring improvement, isolation of radioactive material) to avoid exposure of personnel to radiation • frequent radiation measurements in the workplace environment to identify potential sources of exposure and high risk employees • provision (to all staff) of specific directives on safe use of radiation emitting equipment (instruction sheets, official forms, leaflets, electronic mail) • re-positioning of pregnant employees to reduce potential exposure. rotation of workers to reduce potential exposure • proposals to administration to conduct adequate modifications in ergonomic design and provision of special equipment table . (continued) potential hazard intervention opportunities • proposals to administration for the introduction of specific safety regulations concerning female employees (especially during pregnancy): a) time-schedule modification, b) maximum working hours, c) mandatory time-breaks, d) provision of sick-leave days, e) maximum allowed weight for manual handling, f) re-positioning of pregnant employees, g) zero-tolerance policy on aggressive behavior against employees • providing staff with the organization's safety guidelines for preventing physical strain (instruction sheets, official forms, leaflets, electronic mail). rotation of workers to reduce physical strain • provision to all staff of educative material on potential effects of infectious agents to female reproductive health • introduction of collective safety guidelines for all health-care personnel • provision of adequate safety equipment to prevent accidental transmission of infectious agents (safety syringe mechanisms, syringe disposal vessels, goggles, masques, gloves) • extensive vaccination program for child-bearing age personnel (health care workers, teachers, nursery workers) lectures constitute single courses or one-shot education interventions (prevention ) providing general information on the topic of occupational reproductive hazards for women (risk factors, generic preventive measures). presentations and lectures may be used as well to carry health messages among the staff in an effort to establish general acceptance and support for the company's pertinent safety regulations and policies. (lawson et al., ) • systematic occupational exposure assessment to identify high risk female employees • proposal for legislative regulation to substitute hazardous agents with "safer" chemical compounds • promotion of scientific research on gender specific reproductive issues for female workers • obligatory reproductive health surveillance of women workers in high risk for reproductive disorders • systematic registry of birth defects according to maternal occupational exposures • production of specifically designed protective equipment for female workers this form of communication is able to provide wide-scale access to the population of working women (e.g., access to agricultural workers in distant areas, low-literacy skill employees) through billboards, magazines, and newspapers published by the company, or items of special interest to certain professional sectors, such as leaflets referring to reproductive safety issues. health professionals may utilize this channel to address generic guidelines for prevention on issues of reproductive health either to female workers themselves or to their coworkers, which may effect women employees through their tasks or behavior (e.g., men employees smoking indoors, safe storage or handling of hazardous chemicals in the workplace). this form of health promotion is based on establishing an interactive information service for the prevention of occupational reproductive disorders (prevention ) . this service should incorporate a telephone center with the ability to provide personal telephone counseling to women employees, as well as a hotline for answering women's questions concerning reproductive health issues. furthermore, this service should develop an electronic library, accessible through the internet, for all female workers and the general public, providing official safety guidelines for reproductive hazards and answers to related questions. finally, it enables continuous sensitization of high-risk female employees through frequent electronic reminders and their activation in safeguarding their reproductive health. media has proven to be one of the most effective communication channels for many health issues and health promotion activities. it can be utilized to address messages on a larger scale, mostly by health professionals who work in workplace health promotion programs on community, national, or european level. it uses public announcements, short messages, and commercials to address reproductive health hazard prevention issues through radio, television, and newspapers. in some cases, this form of health promotion may be useful for approaching female workers who are not easily accessible by other workplace health promotion programs, such as occupations in small enterprises, part-time employment, and female agricultural workers in remote areas. • violent behavior intense psychological stress (artazcoz lazcano, cruz i cubells, moncada i lluis, & sanchez miguel exposure to ionizing radiation (x-ray & computed tomography departments, nuclear laboratories, patients treated with radioactive drugs) anaesthetic gases, cytotoxic drugs • infectious diseases airborne, blood borne • dermatitis (allergic, irritative) • disturbance of the regular circadian -metabolic rhythm gimeno, felknor, burau, & delclos, ) and micro particles indoor air quality and health in offices and other non-industrial working environments prevalence and risk factors of occupational asthma among hairdressers in turkey incidence of asthma in female swedish hairdressers current findings about the development of obstructive airway diseases in hairdressers (bk numbers: / ) women's occupational dermatologic issues associations of self estimated workloads with musculoskeletal symptoms among hospital nurses combining job and family demands and being healthy: what are the differences between men and women? gender inequalities in health among workers: the relation with family demands stress and work-related tension in the nurses and clinical aides of a hospital predictors of early leaving from the cotton spinning mill environment in newly hired workers interdigital pilonidal sinus in a hairdresser self-assessed and directly measured occupational physical activities-influence of musculoskeletal complaints, age and gender evaluation of reports of periconceptual occupational exposure: maternal-assessed versus industrial hygienist-assessed exposure work-related obstructed airway diseases by irritative noxae in the low concentration range evaluation of occupational environment in two textile plants in northern india with specific reference to noise noise exposure of music teachers laundry workers: cleaning up an industry. we can do it: protecting women workers hazardous substances in the air at hairdressers' workplaces work-related musculoskeletal disorders in hairdressers the hazards of surgical smoke. not to be sniffed at! british journal of perioperative nursing : the journal of the neck and 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and challenges. aaohn journal : official journal of the american association of occupational health nurses workstation design in carpet hand-weaving operation: guidelines for prevention of musculoskeletal disorders nurses confronting sexual harassment in the medical environment musculoskeletal disorders in hotel restaurant workers mortality among us employees of a large computer manufacturing company factors influencing restaurant worker perception of floor slipperiness handle with care: the american nurses association's campaign to address work-related musculoskeletal disorders prioritizing safe patient handling: the american nurses association's handle with care campaign epidemiology of voice problems in dutch teachers cross-sectional investigation of task demands and musculoskeletal discomfort among restaurant wait staff work-related violence and the ohs of home health care workers working in clients' homes: the impact on the mental health and well-being of visiting home care workers. home health care services quarterly health hazards of ceramic artists occupational contact urticaria and protein contact dermatitis are health care providers who work with cancer drugs at an increased risk for toxic events? a systematic review and meta-analysis of the literature prevalence of voice disorders in african american and european american preschoolers prevention of disabling back injuries in nurses by the use of mechanical patient lift systems gender issues in safety and health at work -a review. luxembourg: office for official publications of the european communities european agency for safety and health at work ( b) including gender issues in risk assessment [electronic version mainstreaming gender into occupational safety and health. luxembourg: office for official publications of the european communities eurostat ( ) european social statistics: labour force survey results : office for official publications of the european communities fjellman-wiklund a, brulin c, and sundelin g ( ) physical and psychosocial work-related risk factors associated with neck-shoulder discomfort in male and female music teachers prevalence of sensitivity to rubber additives and latex in hairdressers with hand and/or forearm contact dermatitis diagnostic approach and management of occupational asthma by persulfate salts in a hairdresser. allergy and asthma proceedings : the official journal of regional and state allergy societies sex differences in pharmacokinetics and pharmacodynamics pesticide exposure and women's health adequacy of vaccination coverage at school-entry: cross-sectional study in schoolchildren of an urban population pulmonary fibrosis and ferruginous bodies associated with exposure to synthetic fibers the precede-proceed planning model organisational and occupational risk factors associated with work related injuries among public hospital employees in costa rica bad nights or bad bars? multi-level analysis of environmental predictors of aggression in late-night large-capacity bars and clubs health promotion planning: an educational and environmental approach knowledge, attitude and practices among health care workers on needle-stick injuries high rubella seronegativity in daycare educators fit factors for quarter masks and facial size categories the risk of adverse reproductive and developmental disorders due to occupational pesticide exposure: an overview of current epidemiological evidence traumatic injuries in agriculture haz-map ( b) cadmium and compounds. occupational exposures to hazardous agents retrieved glycol-ethers. occupational exposure to hazardous agents occupational exposure to sevoflurane, halothane and nitrous oxide during paediatric anaesthesia. waste gas exposure during paediatric anaesthesia chemical exposure in hairdresser salons: effect of local exhaust ventilation gender differences in self-reported physical and psychosocial exposures in jobs with both female and male workers quantification and risk analysis of occupational burns: oregon workers' compensation claims, to environmental factors in infertility risk factors for neck pain in office workers: a prospective study allergic contact dermatitis in hairdressers: frequency and source of sensitisation professional cleaning and asthma office work, sbs and respiratory and sick building syndrome symptoms garlic-related dermatoses: case report and review of the literature principal results occupational allergic contact dermatitis from spices a multicentre review of the hairdressing allergens tested in the uk occupational risk for hepatitis a: a literature-based analysis exposure assessment in epidemiology: does gender matter? development of hand eczema in hairdressers, to years after the start of training -a follow-up study of "posh-studie" participants perilous work: nurses' experiences in psychiatric units with high risks of assault industrial sectors with high risk of women's hospital-treated injuries risk factors for voice problems in teachers an analysis of occupational dysphonia diagnosed in the north-east of poland do teachers have more health problems? results from a french cross-sectional survey work practice and some adverse health effects in nurses handling antineoplastic drugs case report: occupationally related recurrent varicella (chickenpox) in a hospital nurse characterization of chemical exposures in hairdressing salons printed circuit board industry perceiving classroom aggression: the influence of setting, intervention style and group perceptions workgroup report: implementing a national occupational reproductive research agenda-decade one and beyond gender differences in the healthy worker effect among synthetic vitreous fiber workers voice symptoms of call-centre customer service advisers experienced during a work-day and effects of a short vocal training course respiratory illness in agricultural workers making manufacturing a safe work environment for women workers. we can do it: protecting women workers evaluation of the exposure to biomechanical overload of the upper limbs and clinical investigation in a female population employed in the manual loading of production lines in ceramics factories noise exposure and hearing loss in agriculture: a survey of farmers and farm workers in the southland region of new zealand an investigation of contact transmission of methicillin-resistant staphylococcus aureus women in agriculture: risks for occupational injury within the context of gendered role male and female rate differences in carpal tunnel syndrome injuries: personal attributes or job tasks? the "gras" status of women's work sex matters: exploring differences in responses to exposures an ecological perspective of health promotion programs workplace violence in health care: recognized but not regulated sex, gender and women's occupational health:the importance of considering mechanism ovarian intrafollicular processes as a target for cigarette smoke components and selected environmental reproductive disruptors the effect of health education in promoting health of hairdressers about hepatitis b based on health belief model: a field trial in yazd carpal tunnel syndrome and ulnar neuropathy at the elbow in floor cleaners occupational asthma among hairdressers occupational asthma and occupational rhinitis in hairdressers energy cost of physical task performance in men and women wearing personal protective clothing rubella infections of the school teachers in sapporo municipal schools after their employment adapting osha ergonomic guidelines to the rehabilitation setting theory at a glance: a guide for health promotion practice theory at a glance" a guide for health promotion practice how is sex considered in recent epidemiological publications on occupational risks? muscle pain, physical activity, self-efficacy and relaxation ability in adolescents gender inequalities in occupational health: harvard school of public health accidental exposure to biological risk among health care workers of a sanitary area prevalence of occupational hand dermatitis in u.k. hairdressers noise and chemical induced hearing loss: special considerations for farm youth pfizer principles for clear health communication nd edition agents causing occupational asthma in finland in - : cow epithelium bypassed by moulds from moisture-damaged buildings sex and gender factors in medical studies: implications for health and clinical practice lik sprava safe handling of hazardous drugs needlestick injuries among health care workers. a literature review designing and evaluating intervention plans evaluating cdc-funded health department hiv prevention programs occupational risky business: injury prevention behaviors of farm women and children using the precede-proceed model to increase productivity in health education faculty [electronic version unconsumed precursors and couplers after formation of oxidative hair dyes labour market latest trends - rd quarter data active epidemiological surveillance of musculoskeletal disorders in a shoe factory histology and pathology laboratories. chemical hazard prevention and medical/health surveillance musculoskeletal dysfunction in physical education teachers physical work load in physical education teachers barber's hair sinus in a female hairdresser: uncommon manifestation of an occupational dermatosis framing personal risk in public health nursing the prevalence and risk factors for occupational voice disorders in teachers glutaraldehyde exposure and its occupational impact in the health care environment contact dermatitis to hair dyes in a danish adult population: an interview-based study division of labor standards and safety, occupational safety and health ( ) the manager's handbook: a reference for developing a basic occupational safety and health program for small businesses women, work and musculoskeletal health occupational voice disorders: an analysis of diagnoses made and certificates issued in - exposure to cooking fumes in restaurant kitchens in norway effects of glutaraldehyde exposure on human health occupational risk factors associated with voice disorders among teachers health risks of occupational exposure to anticancer (antineoplastic) drugs in health care workers assessment of dna damage in nurses handling antineoplastic drugs by the alkaline comet assay assessment of dna damage in nurses handling antineoplastic drugs by the alkaline comet assay visual problems among electronic and jewelry workers in thailand association between occupational asthenopia and psycho-physiological indicators of visual strain in workers using video display terminals please put this in the proper format and move this reference to its alphabetical place in the "p's" weaver vm secondary individual prevention of occupational skin diseases in health care workers, cleaners and kitchen employees: aims, experiences and descriptive results. int arch occup environ health williams nr ( ) occupational groups at risk of voice disorders: a review of the literature chemical occupational risks identified by nurses in a hospital environment work-related musculoskeletal disorders and associated factors in teachers of physically and intellectually disabled pupils: a self-administered questionnaire study seroprevalence of varicella, measles and hepatitis b among female health care workers of childbearing age immunization and child health materials development guide zidkova z and martinkova j ( ) psychic load in teachers of elementary schools key: cord- - ewi dka authors: nan title: primary immunodeficiencies date: journal: pediatric allergy, asthma and immunology doi: . / - - - - _ sha: doc_id: cord_uid: ewi dka primary immunodeficiencies (pids), once considered to be very rare, are now increasingly recognized because of growing knowledge in the immunological field and the availability of more sophisticated diagnostic techniques and therapeutic modalities [ ]. however in a database of > , inpatients of a general hospital for conditions suggestive of id patients were tested, and an undiagnosed pid was found in ( %) of the subjects tested [ ]. the publication of the first case of agammaglobulinemia by bruton in [ ] demonstrated that the pid diagnosis is first done in the laboratory. however, pids require specialized immunological centers for diagnosis and management [ ]. a large body of epidemiological evidence supports the hypothesis of the existence of a close etiopathogenetic relation between pid and atopy [ ]. in particular, an elevated frequency of asthma, food allergy (fa), atopic dermatitis and enteric pathologies can be found in various pids. in addition we will discuss another subject that is certainly of interest: the pseudo-immunodepressed child with recurrent respiratory infections (rris), an event that often requires medical intervention and that very often leads to the suspicion that it involves antibody deficiencies [ ]. structural genes, and also perhaps on the lack of / c genes [ ] . mbl deficiency is due to one of three point mutations in the gene for mbl, each of which reduces levels of the lectin by interfering with the protein oligomerization [ ] . in children with this kind of deficiency, the level of mbl is . mg/l compared to the mg/l in controls [ ] . regardless of whether the children are homozygote (hz) or heterozygote (het) in relation to a given mutation, the defect appears to be more consistent in small babies aged - months [ ] , who show an immaturity in providing immune response to capsular bacteria and in whom low levels of opsonin are incapable of compensating for this [ ] . the risk of contracting infections is similar in hzs [ ] and hets [ ] , though it persists throughout life in hzs because of an abnormal allele, while it exhausts itself in the hets, where the frequency of abnormality is similar to that of the general population [ ] . anomalies in immunoglobulins (ig) and in opsonization have been observed, respectively in %- % of children suffering from frequent asthma and igg subclass deficiencies. children suffering from cystic fibrosis also present an elevated prevalence of immediate cutaneous reactions to aeroallergens, and although without primary defects of adoptive immunity, they are susceptible to severe rris; therefore it may be possible that they suffer from the mucosal antigenic exclusion [ ] . unlike asthmatic children, in whom a relatively high concentration of ige for respiratory viruses was observed [ , , ] , positive skin prick tests (spts) are more common for aspergillus fumigatus [ ] . the hypothesis suggested by these observations is that atopy derives from an unbalanced immune response to foreign antigens, with a consequent lack of their early identification or the capacity to neutralize or eliminate them. this hypothesis is based on the evidence that id precedes the development of atopy: in the taylor et al studies, newborn babies, the children and/or siblings of atopic patients, presented a significant reduction in serum concentrations of iga when aged months: this was transient hypogammaglobulinemia (hgg) of infancy (thi). the association of very low iga levels with atopy has been proposed again in the classic prospective study on the association of viral respiratory infections (vri) and the onset of allergic manifestations, which proved serum iga levels at the lowest normal levels in the children studied [ ] . this data has been confirmed within primary immunodeficiencies (pids), once considered to be very rare, are now increasingly recognized because of growing knowledge in the immunological field and the availability of more sophisticated diagnostic techniques and therapeutic modalities [ ] . however in a database of > , inpatients of a general hospital for conditions suggestive of id patients were tested, and an undiagnosed pid was found in ( %) of the subjects tested [ ] . the publication of the first case of agammaglobulinemia by bruton in [ ] demonstrated that the pid diagnosis is first done in the laboratory. however, pids require specialized immunological centers for diagnosis and management [ ] . a large body of epidemiological evidence supports the hypothesis of the existence of a close etiopathogenetic relation between pid and atopy [ ] . in particular, an elevated frequency of asthma, food allergy (fa), atopic dermatitis and enteric pathologies can be found in various pids. in addition we will discuss another subject that is certainly of interest: the pseudo-immunodepressed child with recurrent respiratory infections (rris), an event that often requires medical intervention and that very often leads to the suspicion that it involves antibody deficiencies [ ] . in several pids (table . ) [ , , , , , , , , , ] , atopic symptoms are present: gastroenteric and rhinitis in selective iga deficiency (sigad), severe ad in wiskott-aldrich syndrome (was) and hyper-ige syndrome (higes), in which it spreads over the entire body, and to which other allergic manifestations can also be associated such as asthma, rhinitis and angioedema. various acquisitions indicate that pid is also an opsonization deficiency, observed in % of the normal population [ ] . in this disease, microorganism phagocytosis by polymorphonuclear (pmn) leukocytes appears annulled, and the patient is subject to severe infections supported by capsular bacteria: the deficiency, described in association with severe and recurrent infantile infections [ , , ] , depends on the lack of mannose-binding lectin (mbl) [ ] , its primary immunodeficiencies a possible atopy dependence on iga underproduction rather than on ige hyperproduction ( fig. . ): in children with levels of iga at the minimum normal level, and followed from birth until the age of - months, a greater severity of atopic manifestations and an increased cumulative incidence of asthma, ad and otitis media with effusion (ome) were observed compared to controls. the close links between id and atopy are confirmed by symptoms similar to ad present in some forms of was ( %), higes ( %), xla (x-linked agammaglobulinemia) or autosomal recessive (ar), ataxia-telang- chapter primary immunodeficiencies proposed that in these patients cd -th levels are sufficient for modulating ige synthesis, but cd t-cell levels are inadequate for inhibiting ige synthesis, which results in increased ige synthesis. this hypothesis is supported by the observation that omenn syndrome, was and especially higes, with an immunological phenotype characterized by a quantitative and qualitative reduction of cd t cells, are accompanied by extremely high levels of serum ige [ , , ] . lymphocytes in subjects with normal levels of ige are incapable of producing them, not even after stimulation with polyclonal activators such as pwm (pokeweed mitogen) or ebv (epstein-barr virus), while patients with high antibody levels spontaneously synthesize in culture sige (specific) levels between and , pg/ml, also releasing factors capable of increasing ige secretion (ige-pf) [ ] . supernatant derivatives from the t cells of patients with higes are in fact capable of inducing in vitro the pre-b cells to increase ige production; furthermore, when the t lymphocytes in these patients are isolated on the basis of receptors for the ige fc fragment, the remaining cells release ige-pf [ ] . considering the suppressive activity of human lymphocytes with cd phenotype on sige, it has been observed that these lymphocytes are able to suppress sige synthesis in patients with high antibody levels; similarly cd + cells from a bone marrow transplant (bmt) can suppress ige production in the hla-compatible recipient [ ] . the study of patients with id associated with hyper-ige has supplied useful information concerning ige system biology, although the immune defect essentially responsible for ige increased production and for severe atopic iectasia (ata), thymic alymphoplasia, scid (severe combined id) ( %) [ ] and, occasionally, by digeorge syndrome (dgs), id with hyper-igm (higms) now cd /cd l deficiency, selective igm deficiency, biotin-dependent carboxylase deficiency, cgd (chronic granulomatous disease), primary neutropenia, and in netherton, nezelof, omenn and shwachman syndromes [ ] . other forms, in addition to those discussed, are associated with gastrointestinal symptoms: diarrhea and malabsorption of xla and thi, diarrhea in was and dgs, food-related allergies ( %) in sigad and also an elevated frequency of asthma [ ] .among secondary id, only aids is associated with ad (chap. ). the association between a deficiency of t cells and high levels of ige, observed in patients with higes, nezelof syndrome, ata, was and other diseases, has been known for some time (table . ) [ ] . experimental studies on animals indicate that there may be an inverse correlation between serum ige levels and t-cell functions: this could be attributed to a t-lymphocyte deficiency in atopics, genetically determined, which makes them more vulnerable to the camp inhibiting activity, and consequently causing an imbalance between the two subclasses of t cells, which could lead to ige hyperproduction and atopy development; however, in no case is there evidence of a relationship between cd deficiency, ige levels and allergic symptoms. it has been serum ige concentrations chronic granulomatous disease m- y < - , hyper -ige syndrome - y - , , nezelof syndrome m- y - , non-x-linked agammaglobulinemia - y - other variable immunodeficiency normal infants and adults - y - data from reference [ ] . m months, y years, gm geometric mean. manifestations has not yet been identified. the most interesting syndromes from this point of view are the three syndromes analyzed above, characterized by common clinical indications such as early ad onset, increased susceptibility to all varieties of pathogens, as well as an exceptionally high ige serum level [ , ] ( table . ). several pids have autoimmune features, including chédiak-higashi syndrome, cgd, complement deficiencies c q, c r, c s, c , c , griscelli syndrome, higms (cd deficiency), lad (leukocyte adhesion deficiency), hla class i deficiency, hla class ii deficiency, omenn syndrome, was, and xlp ( ) , which will be dealt with subsequently. in children with a mean age of months, autoimmunity was chronic and severe requiring prolonged immunosuppression, however with no spontaneous remission of such manifestations [ ] . definition pids ( fig. . ) [ ] consist of a heterogeneous spectrum of congenital, individual and combined anomalies of the immune system (humoral deficiencies, combined deficiency of b and t cells, the complement, phagocytes, neutrophils, etc.), as well as syndromes and diseases associated with id that are traditionally classified as pids. the updated classification (table . ) has divided ª pids into six main groups, also including secondary id with infections (first among them all aids) that cause deficiency and immunosuppression [ ] . the classifications of pids is based on characteristic clinical features and specific alterations in immune status. advances in molecular genetics now make it possible to complete the table according to the types of genetically altered molecules involved [ ] . to complete this data, see table . and table . [ , , , ] showing the behavior of antibodies and circulating b and t cells. pids occur infrequently and are highly heterogeneous in nature, relatively few centers gain extensive experience in the diagnosis, so it is difficult to estimate the prevalence of these disorders from routinely collected health statistics [ ] . studies in countries on all continents have included , patients: tables . and data concerning incidence has increased considerably thanks to a greater availability of specific tests and more widespread knowledge in the medical profession related to these pids, including ata [ ] . however, because primary immunodeficiencies [ , ] ; other data from [ ] (omenn syndrome) and [ ] (jak ). ada adenosine deaminase, id immunodeficiencies, jak janus-family kinase, pnp purine nucleoside phosphorylase, Ø decreased, ØØ markedly decreased, ≠ increased, -absent, + present, n normal. a progressive. b not functional. predominantly t-cell defects the total may not correspond to the sum of the cases because it may include some pid with very low incidences. the figures should be divided into the years that were considered. a incidence × live births; the thi figure includes probable cases. thi selective igg subclass deficiency autosomal hyper-igm syndrome selective antibody deficiency with normal igs cellular and antibody id syndromes associated with other major defects ata wasp syndrome digeorge anomaly hie nijmegen anomaly immunodeficiency associated with or secondary granulocyte dysfunctions defects of phagocyte number and function cgd cyclic neutropenia kostmann's syndrome schwachman syndrome complement deficiency -undefined total pid , time period years years / - / latin america includes eight countries. xla x-linked agammaglobulinemia, cvid common variable immune deficiency, thi transient hypogammaglobulinemia of infancy, scid severe combined id, was wiskott-aldrich syndrome, ata ataxia-telangiectasia, cgd chronic granulomatous disease. to be reliable and can also be used for other recessive x-linked pids to identify cell lines with genetic defects, as is the case of was [ ] . one must, however, properly consider the phenomenon of mutations, that can render useless the inactivation method, as has been proved in was, in xla and also in scid, in which the mutation is not in the maternal t cells but in the germlines [ ] . the main clinical aspects of humoral and cellular pid are schematized in table . [ ] ; further in numerous pids there is a deficiency of chemotaxis (table . ) as in cgd [ ] . in antibody deficiencies, current treatment, while waiting for genetic treatment to become available, complicated in xla by several btk mutations, is based on the prophylactic administration of ivig, combined with quick antibiotic treatment during infectious episodes. the genes responsible for id linked to chromosome x have been recently mapped on the respective chromosome bands (fig. . ): the bands on the short limb are designated "p" and those on the long limb "q" (table . ). this was possible thanks to the refinement of dna recombinant technology (rdna), including dna probes (sequences of radio-marked dna) and restriction fragment length polymorphism (rflp). the closer the gene segregates to rflp, the lower the chance that they might be separated by recombination phenomena when meiosis occurs: the identification of deficient genes allows early diagnosis, even prenatal, and if necessary gene therapy or bmts [ ] . furthermore, the observation that numerous pids are transmitted with an x-linked modality allows a relatively simple diagnosis of males with a positive family history (fh); if fh is negative ( %- % of xla cases) or there are females presenting a clinical pattern of pid, or when sporadic cases are caused by a new mutation, carrier identification is based on the study of immunologically normal female carriers, with two populations of b precursors, using x-chromosome inactivation analysis. this test does not take into account the existence of possible gene mutations and the availability of already affected relatives, and it is also relatively simple and fast [ ] . molecular studies follow the hypothesis that, at an early stage during embryogenesis, one of the two x chromosomes is randomly inactivated in the cells of all tissues of female embryos (persisting as barr's chromatin) [ ] . therefore in normal conditions, one has a cell mosaic that actively expresses for % the paternal x chromosome and for the remaining % the maternal x chromosome (lionization) [ ] (fig. . a) [ ] . in female carriers of xla, the cell mosaic expresses % for an x chromosome with btk in an active form and the remaining % for an x with a mutated btk (bruton tyrosine kinase). this means that in the carrier mother it is inactivated in preference to the x chromosome carrier of the defective gene in b, which matures therefore in an unbalanced manner (not randomly), while in all other cells activation occurs randomly. it follows that in fixed carriers only the b lymphocytes that have the x carrier of a normal gene complete the differentiating route, while the precursors that express the x chromosome with a mutated btk do not mature into b cells, but remain blocked [ , ] (fig. . b) . in x-scid, the study of fixed carriers follows the corrected lyon hypothesis, because the cells with a normal active x develop into normal t lymphocytes; however, when t precursors with a mutant x reach the stage where the x is needed, they do not find it and consequently do not develop: thus female carriers have only one normal and active x, instead of the random mixture of cells with one of the two active x [ ] . the inactivation test appears predominantly b-cell immunodeficiency inherited in an x-linked trait, only % of males have a fh positive for pid; female cases are also known, supporting an ar trait [ ] . classically affected subjects present levels of igg at < mg/dl, with very low circulating iga, igm and b cells (table . ), in which are found, in addition to bm, pre-b lymphocytes in an almost normal quantity [ ] . xla is characterized by a blocking of b-cell differentiation that results in an arrest of the evolution of pre-b a cells: low levels of cytoplasmic igm and high levels of surrogate light (l) chains (cd b) into later-stage b cells [ ] .the b-cell differentiation arrest in the majority of xla patients appears to be homogeneous, with approximately % of the pro b-cell compartment being negative for cytoplasmic igm expression [ ] . the size and nature of the residual more mature b-cell population (leakiness) varied among patients, independent of the type of btk mutation. further, it appears that the pro b-cell compartment composition in bone marrow (bm) of some xla patients can be influenced by low levels of wild-type btk mrna [ ] . on the contrary, t cells are normal both in function and in number, as is thymus architecture, including hassall's corpuscles and thymus-dependent areas of spleen and lymph nodes. b lymphocyte zones are typically depleted, with an absence of gcs, plasma cells, and cortical and medullar differentiation compared to normal (figs. . and . ) and an absence of adenoidal tissue (fig. . ). the intestinal lamina shows a similar deficiency [ ] , even if both b and t cells use the same recombination (chap. ). in the bm, increased pre-b lymphocytes without cd and cd can be observed. the pre-b cells are capable of transcribing and translating microgram intracytoplasmic (ic) h chains, but not the l chains [ ] , thus pre-b only form microgram chains not associated with v h , while only % of normal cells produce incomplete chains [ ] . experimental data currently indicate that the defect lies in the xla gene mutations that codify for btk [ , ] . the xla gene is expressed by b cells during differentiation, but is not transcribed in the t cells, thereby explaining the b lymphocyte maturative block at the pre-b level [ ] (fig. . ), immediately after their appearance in the bm [ ] . xla, however, presents a genetic heterogeneity, explained by mutations in the btk domain (ph, th, sh , sh , kinase), with a frequency proportional to the pertinent domain dimensions [ ] . the mutation size was ascertained by finding mutations in patients ( fig. . ) . equally, genes codifying for marker proteins and receptors that are essential for b-cell maturation and development are also involved: in fact many of these proteins, including btk, hm chains and surface proteins are crucial for b-cell differentiation [ ] . studying chil-dren of both sexes with xla, various mutations of hm germline have been identified, in addition to deletions affecting the d, j h and cm genes and other gene alterations capable of blocking h chain synthesis on b lymphocytes [ ] . an equivalent molecular defect was observed in an infant girl with xla, with differentiation block preceding the ig gene rearrangements by early pre-b cells [ ] . there are also the so-called leaky forms, with absent or few b cells and various antibody deficiencies [ ] , which can be attributed to individual mutations of btk [ ] , for example in the non-kinase domain, which permits the expression of normal btk levels [ ] . however, btk mutations can be even more detrimental for b lymphocyte proliferation compared to the total kinase absence [ ] . xla is clinically characterized from its onset in male babies, at - months of life (but also at the end of the st year), when the maternal igg passive protection ceases. it usually attracts attention due to delayed growth and mostly recurrent and severe bacterial infections dominate (sinusitis, otitis, bronchitis, [ ] . although representing the phenotypic picture of humoral id, confirmed as a separate deficiency [ ] , xla associated with ghd is mapped in the same region as the x chromosome of the isolated xla. the observation of hz deletion of one or more c h genes for the h chains of ig in %- % of normal patients has led to the identification of various polygenic deletions concerning the genes of one or more isotypes and subclasses [ , ] . some subjects are lacking in genes of all or some igg subclasses, associated with iga and ige deficiency, with no clinical symptoms in % of cases [ , ] . in italy these deletions have a frequency of . % and the expected frequency in hzs is of : , [ ] . only some of the families that produce l chains and not the k chains are known. in one family the molecular bases of the deficiency were ascribable to two different punctiform mutations, one in each cκ allele that prevented the formation of -s-s bridges between the k and h chains. the k:l ratio in human ig is : , and the relative alterations can be observed in numerous primary or secondary ids [ ] . only one patient is known with an l chain deficiency, hgg and rri (upper and lower respiratory tract) [ ] . [ , , ] . rotavirus and echo viruses also cause severe meningoencephalitis in %- % of patients [ ] . phenotypic variability may occasionally be present, as in a family spanning three generations [ ] . in patients with a median age of . years the median age at the xla onset was months and the median age of diagnosis was years, with a median diagnosis delay of months. the common infectious diseases were pneumonia, otitis, diarrhea, sinusitis, and arthritis. the most common chronic infections were seen in . % of the patients: in the respiratory tract in . %, in the gastrointestinal tract in . %, in the central nervous system (cns) in . %, and in the musculoskeletal system in . % of patients [ ] . bronchiectasis, malabsorption, arthritis, autoimmune and tumor-related diseases are the most common complications, as well as edema, contractures, etc. (fig. . ). one must predict the onset of bronchiectasis and intervene quickly with specific physiotherapy, because forms that are initially localized later spread, causing respiratory failure in older children and adolescents. one-third of all cases start with mono-or rheumatoid arthritis (ra) caused by ureaplasma urealyticum with a sterile exudate, which usually regresses following treatment with ivig [ ] .anti-polio vaccinations with live attenuated viruses should be forbidden, because they can cause very severe pneumonia [ ] . about ten cases of xla associated with ghd are known. in addition to reduced growth, clinical symptoms are typical of xla, though it is not a variant, sporadic cases have been described, occasionally associated with sigad ( %- %) and more often with ata ( %) and susceptibility to infections [ ] or without rris [ ] , differentiating patients with probable pid from those with low levels of igg (table . ) [ ] , in whom it may represent delayed maturation [ ] . selective deficiency of igg subclasses presents three different aspects: ∑ total lack of a subclass ∑ two sd levels below average ∑ inability to produce antibodies relative to the subclass in question, even when hematic concentrations are normal [ ] the following selective deficiencies are present [ , ] : ∑ isolated igg : deficiency in only a few cases has been described, also because this subclass represents %- % of all iggs (the others account for % [igg ], % [igg ] and % [igg ]), its absence is very probably an indication of an evident hgg; these patients usually have a reduced level of total iggs and react normally to antigens with a polysaccharide capsule. ∑ other combined id (cid): for example igg +igg , igg +igg , igg +igg +igg , igg +igg , igg +igg + igg , some of which are associated with a deficiency of iga or its subclasses [ , ] . the association of sigad and defects of igg subclasses is explainable in view of ig production ontogenesis by b cells: one starts with igm, moving on to igd, and then to igg ending up with iga passing by ige [ ] ; therefore the deficiency could originate with an immunological defect involving the t lymphocyte regulating work or b cells secreting ig, with an effect on the final stages of their production. in rare cases, more or less extended deletions in chromosome have been observed, in the region that codifies the h chains; in most cases the genome is instead intact, confirming a possible defect in b lymphocyte switching [ ] . very rarely this deficiency depends on gene hz deletions [ ] . this pid is probably the most common of all (tables . , . ), especially in nonselected populations of caucasian origin [ ] . it is defined by the presence of a serum level of iga < mg/dl and the absence of siga in the total deficiency; in the partial sigad levels are < mg/dl but < sd compared to normal levels for age, with measurable siga [ ] . in total deficiencies, igm and igg levels can be normal [ ] (table . ), but igg and igg levels are low [ ] . in several cases, the partial deficiency is transient [ ] , returning to normal levels of iga in % of cases by the age of and in % by ( fig. . ) [ ] . the sigad is transmitted sporadically; however, cases of multifactorial and dominant ar, with a variable or incomplete expression [ , ] transmitted within the same family have been reported. functional alterations reflect on the final maturing process of b lymphocytes, given that about % of b iga + lymphocytes show an igm+igd+iga+ membrane phenotype, a normal aspect only in newborn babies [ ] . studies on chromosome have not led to conclusive results, because deletions in children with sigad are associated with mental retardation, facial dysmorphisms, failure to thrive, etc. an association with hla haplotypes situated on chromosome is instead more consistent, and common in patients with cvid. interesting indications for understanding the pathogenesis come from molecular genetic studies that have allowed the formulation of a hypothesis of multifactorial origin, given that the combinations of more widely involved hla haplotypes and extended haplotypes involve the class i-iii genes, to the extent that they are more often encountered in the general population [ ] .among the class iii alleles the most studied is the gene that dictates the c a, among class i and ii the most common are a , a , b , b , b , cw , dr , dr , dr , dqw , associated with haplotypes such as a , b , dr ; b , dr ; a , b or a , b . other haplotypes are extended, such as b /sco /dr , bw /sc [ , ] /dr , bw /sc /dr and b f/fc /dr , the first of which is increased in patients with a combined iga, igg and ige deficiency [ , , ] . the association with dr gives sigad a risk factor of (table . ). one hla supertype is also found in deficiencies of -hydroxylase with a late onset, suggesting an important locus for iga differentiation close to class iii hla genes [ ] . it has been thought that to induce sigad a non-hla gene or an environmental penetration factor might be necessary, due to the possible sigad discordant expression in hla-identical twins [ ] . however, the analyzed sequence of involved alleles showed a significant sigad correlation with some alleles belonging to hla-dq locus, composed of a protective allele with aspartic acid and a susceptible one with valine or alanine in the b chain in position [ ] . we have observed that the presence of aspartic acid ensures protection in dm. furthermore, the immunological deregulation extends to the typical formation of auto-antibodies and characterized by the presence of igg anti-iga [ , ] . in these subjects, there is a wide symptom range, mostly represented by rris, allergic and autoimmune diseases (aids), among which is diabetes. allergic diseases are twice as common in partial deficiencies, unlike aids [ ] . from a clinical point of view, the frequency of chronic diarrhea and malabsorption, associated with celiac disorders and giardia lamblia infestation are not surprising, considering siga's prominent iga , igg , igg and ige due to deletion of ig h chain constant region genes were associated with undue susceptibility to infection [ ] (see "rri"). a few cases of selective igm deficiency are known, associated with rris and various other symptoms [ ] . igm deficiency was detected in four children with rris. isolated igm defect was present in two children, and two more children had an associated igg subclass deficiency [ ] . cvid includes a heterogeneous group of unhealthy conditions that have in common hgg and rris; it has an incidence of between : , and : , [ ] . the inheritance of two susceptibility genes within the hla on the short arm of chromosome : one located near the class ii region and the other near the junction between the class iii and class i regions is a serious risk for the development of cvid [ ] . there are autosomal dominant or ar forms also linked to sex; sporadic cases are the most common [ ] . the molecular bases are not totally clarified as yet: the pathogenetic mechanisms may depend on b lymphocyte ( % of patients) and t lymphocyte ( %) defects [ ] . the b-cell intrinsic defect is attributable to an alteration of the differentiating line at different stages of maturation, resulting in a poor formation of antibodies, with hgg of variable degrees, while in patients with xla the circulating b lymphocytes are virtually absent. the iggs are < mg/dl (with a reduction in all the subclasses: a normal phenotype is observed in only % of patients) [ ] . more often there is a hierarchical order in the shortage: igg < igg < igg < igg [ ] . iga and igm antibodies are < - mg/dl [ ] , reflecting the potential cd underexpression, implying an activation deficiency [ ] or a t-b cooperation defect [ ] . a study of t lymphocyte subpopulations indicates various subgroups of patients: % have t cells with scarce il , il and il levels, while % have a reduced cd :cd ratio, with an increase in cd bearing the cd marker, which suppresses igg production, elaborates normal il levels and increases ifn-g production [ ] . it can also accompany a deficiency of interleukins (ils: il , ifn-g), suggesting a defect in the signaling mechanisms based on the tcr/cd [ ] . a t-lymphocyte deficiency is therefore difficult to evaluate [ ] , also because this could be a vri effect [ ] . cvid can also be observed following congenital rubella or ebv infections; it can also be induced by some drugs such as phenytoin [ ] . in / cvid families, subjects were identified with identical large mutations in the icos (inducible costimulator) gene, expressed on the surface role in the formation of a barrier against the penetration of polypeptidic macromolecules through the intestinal mucosa. sigad therefore facilitates the penetration of food antigens through the mucosa followed by the formation of specific antibodies. for example, % of patients present cics and precipitins to cm, % to bovine anti-serum and % to anti-serum of calf fetus [ ] . symptoms affecting the respiratory tract are also caused by the absence of siga, as in / children aged - with increased susceptibility to rris [ ] . patients balance the siga deficiency with the sigm, but in some cases the compensation is insufficient for exempting them from rris and asthma [ ] . sigad should be diagnosed on the basis of both serum and secretory iga, because normal levels in adults are achieved at different times (table . ) . some drugs such as phenytoin (an anticonvulsant) can determine sigad, sometimes persisting in time after the drug has been discontinued. the clinical symptoms in these cases are not different from those of patients with sigad [ ] . there is no random treatment; these patients do not benefit from therapy with ivig, even when enriched in iga. there are no counter-indications for obligatory and optional vaccinations [ ] . whole blood or plasma transfusions containing iga can sensitize patients or cause anaphylactic shock in those already sensitized [ ] . life expectancy is excellent; however, the random discovery of sigad in asymptomatic children should not be underestimated. they should in fact undergo periodic clinical and laboratory controls so as to identify as early as possible any possible pertinent symptoms. at the same time, there is the need to ensure a good life quality with adequate prevention of rris in those patients whose respiratory tract is affected [ ] . sadni translates into the inability to respond to certain antigens, especially if polysaccharide. while some individuals are normal, others contract sinopulmonary infections. the reduction of igg levels is more of an associative relationship than a random one; igg levels, on the other hand, do not predict antibody responses. subjects who do not respond to anti-hepatitis vaccination may fall into this category [ ] . in one retrospective survey at a pediatric tertiary care center, sadni was the most frequent diagnosis, accounting for % of id diagnoses [ ] . there are cases of patients in good health, without ige due to gene deletion [ ] . in two siblings, deficiency of of activated t cells, which interacts with the icos ligand gene expressed on b cells. an additional patients with sporadic cvid were examined, and no mutations were found. only in patients with cvid screened thus far (< %) have been found to have icos mutations. one unexplained feature of cvid is that the onset of clinical symptoms does not occur until late childhood or adulthood [ ] . pid is variable either in the clinical and immunological pattern, or in the onset period, more common during the school years or in adults, but also between the ages of and [ ] . the acute bacterial recurrent and/or severe lower respiratory tract infections (lrti) are characteristic: sinusitis ( % of pediatric cases), otitis media ( %), bronchitis, pneumonia ( %) and/or digestive tract infections (diarrhea %) [ ] . the prevalence of infections caused by mycetes has increased as well as cases of pneumonia caused by pneumocystis carinii, a signal for cell-mediated immunity (cmi) [ ] . the gastroenteric tract is dominated by symptoms similar to those seen in celiac disease, with generalized malabsorption, steatorrhea, lactose intolerance, protein-losing enteropathy, inflammatory bowel disease (ibd), saccharidase deficiency and malabsorption of vitamin b and folic acid, supported also in this case by intestinal infestation caused by giardia lamblia [ ] . the tumor necrosis factor receptor family (tnfr) member taci (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) mediates isotype switching in b cells. in / unrelated subjects with cvid and / subjects with sigad there was a missense mutation in one allele of tnfrsf b (encoding taci). none of these mutations were present in healthy subjects. tnfrsf b mutations cosegregated with the phenotype of cvid or sigad in family members of the index subjects. b cells from subjects with taci mutations expressed taci but did not produce igg and iga in response to the taci ligand april (a proliferation-inducing ligand), probably reflecting impaired isotype switching [ ] . other characteristics are hemopathies, hepatosplenomegaly, autoimmune hemolytic anemia (aiha) and x-linked lymphoproliferative disease (xlp), and cutaneous and internal organ granulomas (which differentiate it from xla), in particular ra, thrombocytopenia, and neutropenia [ ] . offspring of cvid patients are at risk throughout their lives for cvid development and should be monitored with a high index of suspicion [ ] . based on experimental evidence, it has been hypothesized that iga-and cvid-associated deficiencies may be the extreme opposites of one clinical spectrum: there is a block of b-cell differentiation, different only in the isotype involved. both defects often appear in different members of the same family groups and more or less the same alleles are present [ ] . the most accredited hypothesis is that a number of extended haplotypes of the hla system are shared, to which gene duplications, deletions and polymorphisms codifying for some class ii and iii alleles correspond [ ] . in fact, a number of common hla haplotypes, especially belonging to class iii, are observed in patients, and at least two haplotypes in % of cases [ ] , such as hla-dqb * , hla-dr , c b-sf, c a, g - , bf- . , c a, hsp- - . , tnfa- , hla-b and hla-a , postulating therefore the existence of a common genetic basis [ ] , with a susceptible gene ( p . ) possibly the association marker [ ] . for example in five members of a large family with one of the two pids, duplications of the c genes were associated with a selected group of hla class ii and iii genes [ ] . the fact that four members without pid also had these haplotypes indicates that their presence alone is not sufficient for expressing pid, leaving room therefore for other factors [ ] such as overlapping relations with celiac disease. the analysis of linked genes has confirmed a strong association with locus a, suggesting that an important role in both pid is played by the gene codifying c a or an adjacent one [ ] . see "x-linked hyper-igm (or hyper-igd) or cd deficiency (xhigms)" for further discussion. unlike transient hgg that occurs when the maternal iggs gradually disappear from circulation (table . ), in the original study by taylor et al iga levels had fallen, becoming regular in newborn babies with thi after year, corresponding to the nonatopic levels [ ] . it consists therefore of a pathological delay in the normal antibody production maturing process. walker et al have calculated that the prevalence of thi is × in children, equal to the prevalence of symptomatic sigad ( × ) [ ] . in all children the igg and in / ( %) the iga were < th percentile, / ( %) had igm levels < th percentile resolved around the nd month; further confirmation consisted in the fact that the children had symptoms either of ad or of fa or food intolerance [ ] . from table . the mean incidence is from to × . during years children aged - months were diagnosed with thi and an incidence of . /year [ ] . in other studies the main defect was in the igg: in one it had normalized between and months [ ] , in another trial / babies ( . %) exhibited at the age of months an absence of serum igg levels and of specific antibodies to viral agents, which in eight children were detected before the serum igg levels returned to normal, whereas in basis is an il r deficiency [ ] , more precisely of the g receptor mapped on chromosome xq [ ] . the sole deficiency of il r is not sufficient for producing an immunological phenotype as devastating as scid [ ] . because the g chain of il r (il rg), a shared component of il r, il r, il r, il r, il r, il r and il r [ ] , gc mutations interfering with its link to the ils deprive the lymphoid progenitor cells of the crucial signals for normal lymphocyte intrathymic development [ ] . mutations in any of the genes: il rg, il ra, jak , artemis, rag , rag , cd , ada, cd cause scid [ , , , , , , , , , , ] . a total of il rγ gene mutations have been sequenced, of which are unique [ ] . each of these mutations has resulted in γc deficiency with varying degrees of id. the mutations are distributed throughout the eight exons of the gene, as well as in the regions necessary for proper transcription and translation. the penetrance of each of the above il rg mutations is unknown. exons and have mutation hot spots. the types of mutations identified include missense, nonsense, insertions, deletions, splice mutations, and mutations that affect rna processing and translation [ ]. among mutations in patients, the most numerous ( %) are punctiform mutations (fig. . ) plus one missense related to amino acid residues [ ] , with a lack of jak and gc interactions [ ] . in an atypical form, the substitution with residual cysteine of the arginine at position appears to be decisive for gc chain expression, probably a mutation reversion at the basis of the molecular defect, with a numeric and functional t-cell normalization [ ] . the mutations, by inactivating the common g chain, render the t cells of boys with scid-x unresponsive to several ils. the result is a block in t-cell development and a severe deficiency of mature t cells. b cells, although pre-two children normal igg levels were detected even before the appearance of specific viral antibodies. igg levels usually normalize at between and months [ ] , at the age of years (fig. . ) or before months of age in / children; however, / still had low ig levels at - months of age [ ] . at was in / children ig levels were still < sd for age and in / various igg subclass deficiencies were detected [ ] . a prospective study with an -year follow-up found that igg and iga deficiency is normalized by the age of , but in a minority of cases this may be a prodrome of sigad or another humoral deficiency [ ] . a study with a -year follow-up of children with igg deficiency as well as iga deficiency in % of the cases, observed multiform clinical symptoms. since thi can gradually normalize, some children have low antibody titers, and others low igg levels. however, both groups experienced significant infections [ ] . in some cases, thi is asymptomatic; in others infections, especially of the respiratory tract, are present. the designation of thi may be a misnomer, and an alternative designation could be added to thi such as "with recovery" or "with development of other dysgammaglobulinemia" [ ] . general characteristics of combined t-cell/b-cell immunodeficiency are summarized in table . [ ] . t -b + scid is a heterogeneous group with an incidence of between : , and : , livebirths [ ] . xlinked fh is positive in % of cases [ ] . the genetic sent in normal or even increased numbers than in other forms of scid, are dysfunctional [ ] . b cells do not mature or produce antibodies due to a complete b-cell differentiation arrest at the pre-bcr checkpoint, showing the absence of complete vdj recombination [ ] . other forms are also known with an attenuated phenotype and a partial t-cell function [ ] . typical scid-x represents the most common form, with . % of cases [ ] ( . % in tables . , . ) . in the thymus, there is a severe hypocellularity, without lymphocytes and hassall's bodies where thymic epithelial cells predominate without grossly evident corticomedullary differentiation (fig. . ). severe lymphopenia is often associated with eosinophilia; nk cells are within the norm or rare. the majority of in-fants with scid-x lack both t and nk cells (t -b + nkphenotype) [ , ] . cd t cells, if present, are of maternal origin, because the block, as also in scid ar, occurs at the level of cd -, cd -, cd -, cd + and cd a + ; developing t cells and cd + thymic dc are reduced > -fold when compared to age-and gendermatched control thymus [ ] (fig. . , pre-t, tn), thus scid t -b + . the study of other subpopulations distinguishes the scid subtypes: t cells are reduced in all variants, the absolute cord blood (cb) number is - , lymphocytes/mm (tables . , . , so that any count below , /mm is lymphopenic). moreover, in ada deficiency (adenosine-deaminase) there is a maximum reduction in total lymphocytes, in scid-x and in jak defi- chapter primary immunodeficiencies alies) and against a common t and nk cell drop [ ] . furthermore, in two cohorts [ , ] the affected females had the same phenotype, indicating a possible complex molecular defect [ ] . there is no response to delayed spts, and there is an absence of lymphocyte proliferative responses to mitogens and to a specific antigen such as tetanic toxoid [ , , ] . the average age at diagnosis was . months in children [ ] , similar to the ages reported in > children with scid from all causes [ , ] . the male:female ratio is : and diagnosis is often missed or occurs too late to save the lives of those infected infants who may manifest gvhd early on with morbilliform eruption in the first few days of life due to the transplacentally acquired maternal t cells, intractable diarrhea resulting in a severe malabsorption ( fig. . ), severe interstitial pneumonia (fig. . ), or giant cells caused by anti-measles vaccination or bcg (bacillus calmette guérin), with death caused by chickenpox or infections caused by pneumocystis carinii, herpes, adenovirus, cmv (cytomegalovirus), etc. [ ] . the absence of tonsils is observed and also lymphoid tissue [ ] and thymus [ ] hypoplasia. these children must be transferred urgently to a specialized center and be placed in a sterile room to receive a bmt [ ] . on rare occasions, il rγ mutations have caused an atypical mild scid that presented beyond infancy [ ] . up-regulation of bcl- by an il r lacking il rb tyrosine residues leads to increased cell survival after il deprivation; astonishingly, this survival signal does not occur when gc tyrosine residues are absent. thus, if ciency, the number of b cells is the highest and that of nk cells is the lowest (b + >t ->nk -); nk cells on the contrary reach their highest levels in the ar form [ ] . in scid there can also be b alymphocytosis [ ] . this divergent data is, however, characteristic of t -b + nkmolecular defects (gc, jak defects), b + t -nk -(ada deficiency), or t and b (possible recombination anom- gc-dependent signals are revealed only in the absence of il rb tyrosine, il r engages at least two distinct signaling pathways to regulate apoptosis and ccl- expression [ ] . in two clinical series, patients with mutations in il rg represent %- % of all scid cases [ , ] . children with il rg mutations have lymphopenia in % of cases, with total lymphocyte counts < , /mm (normal levels, , - , /mm ), based on clinical case series [ , ] . all patients have very low or absent t cells, and approximately % have low or absent nk cells [ ] . ar mutated genes on autosomal chromosomes have been identified in ada deficiency, jak deficiency, and rag or rag deficiency [ ] . the existence of b-and t-lymphocyte lymphoid precursor differentiated defect is particular, in some cases a rag and rag mutation, the two genes that activate vdj recombination [ ] was observed; however, this rag gene function has been questioned [ ] since a rag defect is more present in t -b -scid and the omenn syndrome [ ] . in babies suffering from scid, there is a marked reduction of t and b lymphocytes (table . ) and all in vivo and in vitro responses are absent. onset and clinical and histological pattern is similar to that of x-scid. the jak gene mutation (tables . - . ) variant has a frequency of . %- . % [ , ] among babies affected by scid and in the absence of t ( ± %) and nk cells ( ± %) [ ] . the molecular base is the mutation affecting the jak , which prevents it from associating with the gc chain and from sending signals to the abovementioned ils [ ] and to other marker proteins belonging to the jak-stat complex [ ] . at the origin is a lack of t lymphocytes that transform into the scid phenotype [ ] . these patients present b + t -nk -: the b ( ± %) with iga equal to ± % [ ] , and those with x-scid present a defective differentiation, but are capable of producing elevated levels of ige in the absence of other isotypes [ ] . this data indicates that gc and jak are essential for t-and nk-cell development [ ] . the clinical characteristics are identical to those in x-scid, with the difference that the scid-jak phenotype is also observed in females ( %) [ ] . furthermore, a jak deficiency could be an important cause of scid ar and should be considered in all patients with the b + t -nkphenotype, without an x-recessive heritage [ ] . in a -month scid-x infant presenting with a history of recurrent infection and failure to thrive, a novel splice mutation, gc-dependent, was described, characterized by near-normal count of functionally deficient nk cells (b + t -nkcell phenotype). cell surface gc expression was undetectable on nk cells and in trace amounts in the minority of b cells. t cells were absent, igg and iga undetectable, and igm were within the normal range [ ] . bmt is not a perfect therapy, because b-cell function developed in / children, and nk functions normalized in / children after bmt [ ] . the family pedigree shows an inbred family with consanguinity across five generations. two brothers were diagnosed with scid. one, at the age of months, presented with persistent oral thrush, oral ulcers, and failure to thrive. he had no palpable lymph nodes and no thymus shadow on a chest x-ray film. the second was diagnosed soon after birth and the third brother has always been healthy. three other male cousins died in infancy from severe infections consistent with scid; a th cousin presented with oral candidiasis at the age of weeks and failure to thrive. no thymic shadow was detected on chest x-ray film and peripheral blood lymphocytes showed persistent lymphopenia. he had no lymph nodes, failed to reject a skin allograft and did not show an increase in the blood igg and igm antibodies for dtp after three vaccinations. the three affected patients were hz for a caet transition at nucleotide in exon , leading to a proline to serine substitution (p s) in the extracellular domain of il r. the cousins and their parents harbored both wild and mutant alleles. this partial deficiency is sufficient to block t-cell development and lead to a scid phenotype. the fh of severe pid with multiple affected male infants strongly suggested an x-linked inheritance. nevertheless, this family consanguinity is in favor of an ar inheritance [ ] . defective il r expression caused in three patients a t -b + nk + scid, indicating that the t-cell defect in scid-x resulted from inactivation of il ra signaling. thus il r-mediated signaling is required for t cells but not for nk ontogenes. mutations in the gene for the il r chain on chromosome p were found in all three patients [ ] . these infants resemble those with other types of scid with respect to their susceptibility to infection and the absence of functional t cells and b cells. however, they differ in that their circulating lymphocytes are primarily nk cells. rag and rag are required for the rearrangement of tcr and bcr genes [ ] . half of the patients with t -b -scid had mutations in their rag or rag genes, thus highlighting the crucial role of these genes in normal v(d)j recombination machinery [ ] . rag-thymocytes lack a functional pre-tcr and hence arrest at the cd -/cd + stage of differentation [ ] : without rag and rag , mature ig and tcr genes cannot be assembled, and lymphocyte development is arrested at very early stages [ ] . abnormalities of the chest, scapula and iliac bones and short and stumpy limbs [ , ] . x-ray abnormalities are documented in fig. . : the absent thymic shadow and a notable cupping and flaring of the ribs' ends (arrows) can be observed, while histological studies of the chondrocostal junctions document their total cellular disorganization (fig. . ). this deficiency is the object of a great deal of attention because it was the first to be treated using gene therapy [ ] . the lack of ada is observed in . % of patients with scid [ ] . the ada enzyme catalyzes the conversion of adenosine and deoxyadenosine into inosine and deoxynosine; although ada is found in all cells (cd anchors ada to the lymphocyte cell surface (table . ), the deficiency damages above all the immune system [ ] . table . [ ] summarizes the biochemical foundations of this pid. more than ada mutations are known, including > amino acid substitutions, deletions and punctiform mutations or anomalies of the gene itself, such as exon deletion and exons , , and - mutations with a total of , nine of these in patients with ada-scid and six in those with a partial deficiency [ ] . an additional mutant alleles have been found ( missense and single-codon deletion) [ ] . adenosine and deoxyadenosine are also apparent suicide inactivators of the enzyme s-adenosylhomocysteine (sah) hydrolase, with consequent accumulation of sah, a powerful inhibitor of virtually all cellular methylation reactions [ ] . the accumulation of metabolites, including camp, deoxy-atp and '-o-methyladenosine, has a toxic effect on the cells by blocking dna synthesis and dividing and resting t lymphocyte proliferation [ ] (fig. . ). four different clinical phenotypes have been described for ada-deficient subjects (table . ) [ ] , which cover a broad spectrum of immunological aberrations, from the complete absence of b and t immunity, indicating scid ( %- % of patients) (the thymus in fig. . ) to forms with a delayed onset or partial deficiency ( %- %) [ ] . in children, the delay between onset of symptoms and diagnosis has been estimated to average months [ ] . if clinical symptoms indicate an early onset, in addition to typical scid symptoms, there are also x-ray pathognomonic skeletal abnormalities of the chondrodysplasia type, especially this very rare ar type of scid was observed for the first time in in identical twin male infants who exhibited a total lack of both lymphocytes and granulocytes in their peripheral blood and bone marrow. it has a frequency of % in cases of scid [ ] . the children are symptomatic in % of cases within the first days after birth [ ] and is usually fatal within the rd month of life without a bmt [ ] . due to the common stem cell (sc) non-maturation [ ] , it is characterized by total block in lymphoid and myeloid precursor differentiation, therefore not only by an extraordinary lymphopenia, but also by a marked cytopenia in all sections (table . ) [ ] , in the spleen, in the lymph nodes and in the gastroenteric tract, and a high frequency of severe successive infections [ ] . the thymus is always much reduced in volume, no hassall's bodies are seen [ ] . seven of the eight infants reported by who with this defect died between and days of age from overwhelming infections; the eighth underwent complete immunological reconstitution from a bmt [ ] . an additional three of five children who required two hscts (hematopoietic sct stem-cell transplantation) and received intensive conditioning therapy before haploidentical hsct (matched for of the hla loci) are alive and well an activated phenotype and poor functional capacity [ ] . studies involving hla typification and dna polymorphism show that t cells belong to the host, ruling out, therefore, the etiology of maternal cell engraftment [ ] , unlike other types of scid [ ] . the absence of circulating b is also characteristic [ ] , equal to . %- . % of normal levels [ ] , reaching % [ ] , high ige levels ( ui/l), hypereosinophilia reaching , ¥ cells/l (normal, - . cells/l), and low ig levels at the beginning [ ] then declining to the point of agammaglobulinemia [ ] , comparable to that in reticular digenesis (table . ) [ ] . the marked b-cell depletion can also bear rag and rag gene missense mutations that decrease the efficiency of vdj recombination, which results in impaired but not absent rearrangement of both bcr and tcr. four missense mutations were detected in the rag- in / patients [ ] . in / patients ( %) the mutations affected the rag gene, and in / ( %) the rag gene [ ] . increased ige is linked to th primary infiltration, with spontaneous production of il ifn-g, il , il and il , which is down-regulated by ifn-g therapy [ ] . clonal expansion of vb + cd + , cd -cd secreting high il levels and low il and ifn-g levels [ ] could indicate an analogy with the fas (cd ) defect. t lymphocytes show an activated phenotype and a spontaneous apoptosis associated with reduced expression of bcl- gene product, and a higher cell death of cd + cd r + cells [ ] . given that high cd levels in the lymph nodes, skin and serum of three children generated th lymphocytes [ ] , a th -mediated pathogenesis is possible: the cd are th markers (chap. ). as in human scid, b and t cells are found in mice with scid, but with a final repertoire that is decidedly oligoclonal and lacks the heterogeneity characteristic of a normal immune system, so lymphopenic scid and omenn syndrome could be two aspects of the same disease with different clinical expressions, especially of time [ ] . clinically, young babies soon after birth show a generalized exudative erythroderma and desquamation, often mistaken as ad, alopecia, widespread lymphadenopathy, hepatosplenomegaly, persistent and profuse diarrhea, failure to thrive with malnutrition ( fig. . ) , aiha, recurrent infections caused by common and opportunistic germs ( fig. . ), and markedly elevated serum ige levels [ , , , ] . this outline included four babies from the same family with the same symptoms until death occurred at - months, but who did not present hypereosinophilia and were diagnosed as dm [ ] . differential diagnosis may be challenging since omenn syndrome and gvhd show dyskeratosis and basal vacuolation, but the first always shows acanthosis and usually parakeratosis. gvhd shows a flat epidermis and rarely parakeratosis. both can be distinguished after immunohistochemical staining for cd and cd , which shows predominantly lymphocytes in the dermal infiltrate in omenn syndrome, and relatively more macrophages in gvhd [ ] . with myeloid and t-and b-cell lymphoid reconstitution [ ] ; another child is alive and well after months [ ] . b-scid, characterized by increased cell sensitivity to radiation secondary to mutations of the artemis gene, could carry a poorer prognosis because of defective repair of dna breaks [ ] , occurring around the time of bmt, from the effects of chemotherapy, infections, and gvhd [ , , ] . one group of patients with scid with an additional sensitivity to radiation was found to harbor large deletions or truncation mutations in the artemis gene mapped on chromosome p [ ] , implying a role for artemis in dna double-strand break repair, which is mutated in human scid [ ] . omenn syndrome is classified as a scid because newborn babies exhibit symptoms similar to a gvhd, due to a antigen expression and cd a absence [ ] , and because it can coexist in families with alymphocytosis [ ] . this is an ar syndrome with an unknown pathogenesis, sharing characteristic clinical and immunological abnormalities with t + b -scid [ ] . severe cutaneous lesions with hyperkeratosis, apoptotic malpighian necrosis and basal membrane destruction can be associated [ ] . no lymphoid cells or hassall bodies are found in the thymus [ ] . the immunological structure reveals histiocyte infiltration of the skin, bm and lymph nodes, with proliferation of t infiltrating the epidermis and the enteric mucosa, increased t cells with combined t-cell and b-cell deficiency igg (g/l) . - . igm (g/l) neutrophils (cells/ml) , - , data from [ ] . in a child with scid and circulating t cells within the norm, a gene transcription deficiency was ascertained [ ] . a male infant of first cousin parentage presented at the age of months with cmv pneumonia, persistent oral and esophageal candidiasis, adenovirus gastro-enteritis, and failure to thrive. he developed lymphadenopathy, hepatosplenomegaly, iron deficiency anemia with no evidence of hemolytic anemia, and chronic inflammation of his lungs and mandible. biopsies showed extensive lymphocytic infiltration of his lung, liver, gut, and bone. serum igg and igm were elevated, but iga was low. he had t-cell lymphocytopenia, with an abnormal cd :cd ratio of : . the t cells responded poorly to anti-cd , phytohemagglutinin and other mitogens, and to il . he was found to have a truncated mutation of the il ra chain (cd ). he was given a successful allogeneic bmt after cytoreduction [ ] . about cases have been reported, [ , , ] , % [ ] or % [ ] of which have the x-linked form of cd deficiency [ ] . patients are generally male, but there can be a non-x-linked form [ ] , in which % of patients are females [ ] . an estimated minimal incidence was calculated of in , , live births. over half of patients developed id symptoms and were diagnosed by year of age, and over % by years of age [ ] .although carriers of xhigms are considered to be asymptomatic, an extreme lyonization of the normal x can lead to a mild expression of the xhigms which is similar to cvid [ ] . it can be secondarily caused by environmental factors and also stem from congenital rubella [ ] : this indicates its heterogeneity. mutations in the tnfrsf encoding cd in xhigm patients result in a lack of b-cell signaling by activated t cells [ ] . however, boys out of failed to express cd , and tnfrsf mutations were found in of these boys, whereas no tnfrsf mutations were found in boys with weak expression of cd [ ] . as a result, xmigm b cells fail to undergo isotype switching and produce only igm due to a defect in the rna editing enzyme, activation-induced cytidine deaminase (aicda), an enzyme expressed only in b cells and required for the processes of class-switching and somatic hypermutation of ig genes [ ] . the marked reduction of igg (< mg/dl), ige and iga is accompanied by a sharp increase in mature igm and circulating igd, but b cells do not express other ig [ ] . interestingly, % of patients with confirmed xhigms who had tnfrsf mutations had low concentrations of igg, iga, and igm. most of the remaining patients with xhigms had the classic pattern of normal or raised igm with low concentrations of iga and igg [ ] . the cd gene defect is usually expressed on the membrane by activated t lymphocytes, which therefore cannot bind b-cell cd [ , ] . figure . shows cd localizations and mutation frequencies, in . % of cases mistakenly. for example, a sense codon substitutes for a missense one, creates a premature stop signal: therefore specific pertinent mutations, such as g e, tunistic infections. this disorder is related to mutations in the gene that encodes the nuclear factor kb (nf-kb), which is required for activation of the transcription factor nf-kb, or nemo (nf-kb essential modifier), also known as ikk (inhibitor of b kinase). the phenotype observed in x-higms-eda patients shows that the putative zinc-finger domain of nemo has a regulatory function and demonstrates the definite requirement of cd -mediated nf-kb activation for b cell ig classswitching [ ] . three other genes, expressed by b cells, have been associated with the higm phenotype giving place to higm - . mutations of activation-induced cytidine deaminase (aicda) (higm ) and uracil glycosylase (ung) (higm ), both expressed by follicular b lymphocytes, lead to defective class switch recombination and somatic hypermutation. mutations of cd , the cd receptor, cause a rare autosomal form with a clinical phenotype similar to cd deficiency (higm ). these rare pids may shed light on the complex events leading to the production of high-affinity, antigen-specific antibodies of different isotypes [ , ] . early treatment with ivig associated with antibiotic prophylaxis have reduced the incidence of life-threatening infections and improved the growth of children with higms [ ] . cycles of g-csf (granulocytecolony stimulating factor) in the presence of severe neutropenia are advised [ ] . substitute therapies with soluble forms of recombinant or gene type cd [ ] are being studied. a recent review of cd -deficient patients showed that % develop liver disease and only % survive into the third decade of life [ ] . bmt has a successful outcome in young children ( %); older patients with more ad-can interfere directly with the link site for cd ( fig. . ). consequently the signal which indicates that b cells should begin isotype switching, limited to the production of low-affinity igm, is missing [ ] . without isotype switching, gc formation is minimal [ ] (fig. . ) and follicular dendritic cells (fdcs) are reduced in number, also having an abnormal phenotype [ ] . as shown by figs. . - . , the lack of cross-linking of cd by cd results in b-cell failure to up-regulate cd and cd , important costimulatory molecules that interact with immunoregulatory molecules on t cells such as cd and ctla- . two patients with normal levels of cd have also been described [ ] . as in males with xla, infections start during the th month, those most often observed are otitis, pneumonia or sepsis cased by pyogenic bacteria, opportunistic infections, in particular caused by pneumocystis carinii [ ] , and also ulcerative stomatitis, ra, neutropenia, aiha, lymphoproliferating complications and type b gastroenteric lymphomas with igm [ , ] . the most prominent clinical infections were pneumonia ( % of patients), upper respiratory infections (urti) ( %- %) including sinusitis ( %) and recurrent otitis ( %), lrti ( . %) recurrent/protracted diarrhea ( %- . %), cns infections ( . %- %), sepsis ( %- . %), cellulitis ( %), hepatitis ( %- . %), and osteomyelitis ( %) [ , ] . lymphoid tissues are normal or hyperplastic [ ] . recently, a rare form of higms associated with hypohydrotic ectodermal dysplasia (eda) characterized by the absence or hypoplasia of hair, teeth, and sweat glands has been described. unlike patients with higms, these patients failed to have a history of oppor- purine-nucleoside phosphorylase (pnp) deficiency, ar, for which patients have been reported [ ] , is characterized by the absence of an enzyme necessary for the catabolism of purines, which converts inosine, deoxynosine, guanosine and deoxyguanosine into hypoxanthine and guanine ( fig. . ); the responsible gene has been mapped to chromosome q at position . [ ] . this has also been observed in patients with nezelof syndrome [ ] . a variety of mutations have been found in the pnp gene in patients with pnp deficiency [ ] . although ada and pnp are both purine salvage pathway enzymes, pnp deficiency does not lead to as severe an id as ada deficiency. patients have considerably reduced concentrations of serum and urinary uric acid. numbers of t cells fall progressively, more than that of b cells (table . ), just like the proliferating responses to mitogens and antigens, especially because pnp deficiency causes an intracellular accumulation of deoxy-gtp (guanosine triphosphate) inhibiting ribonucleotide-reductase and t-and b-lymphocyte proliferation, so combined t and b defects are critical. pnpdeficient patients are as profoundly lymphopenic as those with ada deficiency, with absolute lymphocyte counts usually < /mm . ig levels and production of specific antibodies are all normal [ ] . onset may be early, as for scid, but also delayed until the age of - years. the clinical pattern is dominated by recurrent bacterial, viral and fungal infections, with an abnormal susceptibility to opportunisic germs. two-thirds vanced liver disease may die because cryptosporidia infection that has progressed rapidly following pretransplantation cytotoxic conditioning therapy [ ] . therefore, a patient with end-stage liver disease related to cd deficiency first received a liver graft, and as soon as liver-graft function was satisfactory, bmt was performed with a nonmyeloablative conditioning protocol of fludarabine and melphalan [ ] . the screening for cd deficiency should include children with severe rri, and with dysgammaglobulinemia with a normal or increased igm level [ ] . conventional allogeneic hsct from an hla-matched or a matched unrelated donor (mud) is curative and feasible, if performed before significant infections and organ damage occur [ ] . an approach for high-risk patients including nonmyeloablative hsct was workable in a retrospective analysis of european patients undergoing hsct for cd deficiency in eight european countries between and . the donor sc source included hla-identical siblings, muds, and two phenotypically matched parental stem cells (scs) ( tcd [t-cell depleted]). of these patients, ( %) died from infection-related complications, with a positive result in . % of patients [ ] . carriers can be detected, and this is useful for making a prenatal diagnosis [ ] . of patients suffer from neurological alterations, ranging from spastic symptoms and alterations, etc., to mental retardation and one-third from aids, the most common of which is aiha. the consequence of severe infections, generalized vaccination, severe chickenpox, lymphosarcoma and gvhd caused by blood transfusions in the first decade of life is death [ , ] unless bmt is successful [ , , , , ] . however, poor neurodevelopmental progression may result [ ] or may not [ ] . since the biochemical bases of pnp and ada deficiencies are similar, it is hoped that genetic treatment will also be effective in children with this pid [ ] . this deficiency of hla molecule expression occurs in the more severe forms of pid if they are class ii: about cases [ , ] are known of this ar syndrome [ ] , heterogeneous for the numerous complementation groups the patients are divided into [ ] . hla class ii molecules are absent in all tissues [ ] , to the extent that the cells of patients maintained in cultures for years preserve the negative phenotype [ ] . there is a deficiency of class ii gene transactivator (ciita) codified by chromosome , the expression of which plays an important role in t-cell activation: its absence makes class ii gene expression impossible [ ] . this function is shared with another protein mapped on chromosome , rfx , with a binding site in the promoter region of genes codifying class ii chains [ ] . two additional class ii-specific transcription factors are rfxap and rfxank [ ] . these act on the class ii promoter region and are essential and also nonreplaceable, to the extent that alternative routes cannot compensate for their absence [ ] . furthermore inactivation, or the deficiency of these factors, has a specific effect on the genes dictating hla class ii, the li chain and hla-dm, because there is no indication that other regulating systems may be involved [ ] . the absence of hla class ii is associated with a cd lymphopenia. hla class i expression is normal in the patients tested and cd lymphocyte numbers are not reduced [ ] . interestingly, in a twin study, despite the deficiency, there were antibody responses and class ii-dependent t cells; hence the authors envisage that this represented a hla class ii residual expression below the test sensitivity [ ] . the clinical outline is dominated very early on, before the age of months (range, weeks to months) [ ] , by severe and recurrent gastroenteric and pulmonary infections, with a severe and prolonged course, associated with malabsorption and failure to thrive [ ] . bacterial and viral infections, bronchopneumonia, hepatitis, cholangitis, viral meningoencephalitis and various autoimmune manifestations are common complications [ ] . even though an hla class ii deficiency is clinical-ly less severe than scid, the result is uniformly fatal during the first or second decade of life [ ] . the most evident immune defect consists in the complete lack of reactivity to exogenous antigens, which in vivo reflects an anergy to spts, as well as the complete lack of hla class ii expression and absence of cellular and antibody responses to antigen stimulation [ ] , which are instead positive to mitogens (table . ; fig. . ) [ ] . laboratory investigations show a normal b lymphocyte number, but children may be agammaglobulinemic [ ] . the thymus and other lymphoid organs are remarkably hypoplastic, with a severe cd lymphocyte depletion, while cd and b-cell levels are normal. the syndrome involving a deficiency of hla antigens confirms an important hla biological role in the complex system of t-b cooperation [ , ] . some studies suggest that there are more types of deficiency. when placed together in a culture, the b lymphocytes of these patients, previously transformed by ebv, the lymphocytes correct each other so as to allow hla class ii molecule expression. this has led to the identification of so-called complementary groups [ ] . the specification that the gene is mapped on chromosome p . can lead to an earlier prenatal diagnosis [ ] . longterm survival seems to depend primarily on hla-identical and hla-haploidentical bmt performed in the first years of life, before the acquisition of chronic virus carriage and sequelae of infections [ , ] . a child recently received a transplant [ ] with a novel protocol [ ] : the cd count increased up to cells/ml [ ] . a direct correction of the genetic defect is based on the transduction of cells from patients with lentiviral vectors encoding ciita, rfxank, rfx , or rfxap. the rfxank vector restored class ii expression in a t-cell line from one patient. the rfxap vector corrected primary cells from a second patient [ ] . the study of the common association of hla class i molecule deficiency, already known as bare lymphocyte syndrome, has led to the identification of various patients with an isolated deficiency and of one patient with a deficiency associated with class ii, the most severe [ ] . the deficiency is caused by tap- -tap- mutation, accompanied by severe and chronic bacterial rris [ ] . in two brothers with the ar hla class i defect, the onset of rris took place between the ages of and ; the poor expression of nk cells was so severe that it led to the development of bronchiectasis [ ] . the immunological structure is characterized by few cd : the deficient expression of hla class i molecules is diagnostic [ ] (tables . , . ). mutations with no symptoms referable to an id, therefore integrating a genetic heterogeneity. two other brothers and both parents were healthy [ ] . while the ε chain deficiency produces modest clinical symptoms, the other two are severe also from an immunological point of view: in the γ chain deficiency resulting from the profound cd and cd ra decrease caused by altered thymic activity that leaves the cd ro unaffected [ ] , and in those of the ζ chains due to the severe thymic atrophy [ ] and thymocytes falling to % of normal levels, with limits at between % and % [ ] . the cd δ deficiency due to a heritable mutation of the cd gene that prevents the synthesis of the cd protein has been reported in cases hz for the cd mutation. two cousins died at - months of age because of overwhelming infection. the thymus shadow is clearly visible on chest x-rays. the thymus becomes populated with developing thymocytes, with an arrest of differentiation at the cd -cd stage of t-cell development. a girl ( rd patient) survives after a bmt [ ] . this rare deficiency transmitted as an ar trait is caused by mutations of the zap- gene, a non-src family protein tyrosine kinase (ptc) important in t-cell signaling (tables . - . ). zap- , known to be crucial for t cell activation, is a key player in tcr down-modulation and z degradation [ ] . zap- has an essential role in the cd g chain deficiency due to g or e gene mutations [ ] determines a lack of cd and the absence of cd ra [ ] . in the first two cases described, one brother died at months because of viral pneumonia after a clinical history indicating scid with severe aiha, while the other was asymptomatic at the age of years, although with the same molecular defect [ ] . the study of these brothers proved that, in spite of the absence of functioning g chains and % of the expressive levels of the cd /tcr complex, the lymphocytes were normal. according to the authors, other chains may act in the place of missing ones; however, the correlated scarcity of cd may have negatively interfered with the mechanisms discriminating between self and non-self, while g chain deficiency could have modulated the onset of the deceased brother's severe autoimmune disease (aid) [ ] . a cd e deficiency was found in a -year-old child with mild rri symptoms and otitis media; the expression of the cd /tcr complex was only %, but the stimulation with anti-cd induced a normal proliferating response. in fact, despite the ongoing mutation, a northern blot analysis showed production of a low amount of transcribed rna, corresponding to a small quantity of e normal chains, even though their dimensions were smaller than normal ones [ , ] . the z chain deficiency found in the two brothers is similar to the deficient expression of cd /tcr [ ] . in the younger brother, the thymus, markedly reduced, showed no hassall bodies; the elder brother had similar chain [ ] . in several babies (most of mennonite origin) with scid [ , , , ] , the nonfunctional cd t cells (table . ) were either normal or increased (cd + cd + , %). cd absence in the thymus and in circulation (cd + cd + , %- %) [ , ] suggests that the selective process is arrested during the transition from double-positive (dp) to mono-positive (mp) t cells [ , , ] .arrested thymocytes had terminated rag gene expression and up-regulated tcr and bcl- expression, but failed to differentiate into mature cd or cd mp thymocytes, to be rescued from death by neglect or to sustain il ra expression [ ] . zap- deficiency results in an impairment of transendothelial migration that can be rescued by the transfection of zap- because cross-talk between the zap- signaling pathway and the chemokine receptor cxcr is required for t-cell migration [ ] . although the thymic architecture is normal with presence of hassall bodies [ ] and cd seem normal in the cortex, very few migrate to the medulla [ ] . the near absence of cd + cells and an increased cd :cd ratio dominate [ ] . the few cd coexpress cd + , the nk-cell marker; b cells appear normal and functional, cd -cd + is at a level of %- % [ , ] , and serum ig values are normal [ ] . the same phenotype was found in the brothers [ ] ; other relatives were het [ , ] . the absence of cd expression was shown to correlate with a missense mutation in both ig alleles of the cd a gene domain in a -year-old man and his sister, whereas high percentages of cd -cd -tcrab + t cells were found in the three siblings [ ] . the proliferative responses in vitro to phorbol myristate acetate (pma) and ionomycin, pkc activators (protein kinase c), were normal, unlike pha (phytohemagglutinin), pwm, tetanic toxoid, anti-cd , etc. [ , ] . the positives operate below the tcr, while the negatives react directly with the cd /tcr complex [ , , ] , confirming the zap- deficiency [ ] . the cd are present despite the deficiency because syk, the other member of the family, ensures a compensatory role in the infrathymic cd selection, although with a limited efficacy [ ] . seven months after bmt, a child was clinically well and immunologically recovered [ ] . studies in two siblings hz for a stop mutation in the tap- gene suggest that nk cells express still unknown inhibitory receptor(s) (the missing receptor, discussed in chap. ) capable of down-regulating the nk cell cytotoxicity on binding to surface ligand(s) expressed by t cell blasts. functional analyses were consistent with the concept that this putative inhibitory receptor is expressed by virtually all tap- /nk cells, whereas it is present only in rare nk cells from healthy persons. another prospect would be that tap- /nk cells are actually missing this still unidentified triggering receptor involved in nk cell-mediated killing of pha blasts. since cells derived from patients displaying defective expression of either of the tap subunits are characterized by a strong reduction of mature hla class i molecules at the cell surface, a tap deficiency is connected with hla class i deficiency [ ] . as discussed in chap. , nfat (nuclear factor of the activated t cells) is a transcription factor that forms a powerful transcriptional activating complex and, by linking with specific dna-regulating sites, plays a critical role in the synthesis of various t-cell ils which, due to the deficiency or excessive migratory mobility of nfat, although normal in number and in distribution, are incapable of activating and/or secreting the genes of il , il and ifn-g [ ] . a -year-old girl with scid presented during infancy with severe recurrent infections and failure to thrive; her mrna was not produced for il - and ifn-g due to poor t-cell proliferation, although these were normal in number and in distribution, to initiate the transcription of the relative genes, regulated by nfat, with a binding site in the proximity in the ¢ region. this severe clinical picture is accompanied by evident hgg [ , ] . nk-cell deficiency is found in scid, cvid, reticular dysgenesis, chédiak-higashi syndrome, xlp, lad in tap- deficiency and in cfs (chronic fatigue syndrome), in particular cid such as scid, suggesting an association between nk-and t-cell deficiencies [ ] . there is one known case of an adolescent with an isolated numerical and functional deficiency of nk cells and of precursors, recurrent neutropenia, severe and recurrent ebv, cmv, herpes simplex virus (hsv) infections and life-threatening chickenpox. another child, diagnosed at the age of . years with a cd deficiency, suffers from severe viral and bacterial infections although he has antibodies to various viruses [ ] . the growing list of human genetic defects that impair nk-cell function has been recently joined by nemo-id [ ] which occurs in a group of patients with antibody deficiency combined with exquisite susceptibility to infection with nontuberculous mycobacteria. infectious susceptibilities common to these disorders stress the important role for nk cells in host defense [ ] . the natural history of three boys with nemo mutations outside of the th exon has been described. including these boys, there have been families described as having nemo-id. the resulting estimated incidence of nemo-id is : , live male births, making this disorder significantly less common [ ] . cd ra cells and a marked clinical improvement. these data indicate that the thymus is differentially required in the maintenance of the tcr repertoire complexity [ ] . known also as idiopathic lymphocytopenia, primary cd t-cell deficiency is revealed by a profound and persistent reduction in circulating cd and with a cmi deficiency. it is documented in patients suffering from infections caused by opportunistic germs such as cryptococcus-induced meningitis and oral candidosis, also including ten children and a number of adolescents, for whom the following minimum levels of cd per age have been established: < , cells/mm from to months and < /mm from to years, or a total lymphocyte count of < % on two separate occasions without being hiv-infected [ ] . a family has been reported involving two brothers aged and with t counts between and /mm , recurrent respiratory, intestinal and cutaneous infections, and failure to thrive. the mother showed a low cd :cd ratio [ ] , while the entire family showed normal levels of ig and subclasses and hla molecules [ ] . other symptoms included mental retardation, pansinusitis, bronchiectasis [ ] , but no infections caused by opportunistic germs such as those reported by the who scientific group [ ] . one case of primary cd deficiency is known of a child with scid without genetic transmission of the deficiency. t-cell proliferative responses to mitogens were defective and il r expression was deficient on his t lymphocytes, and b cells did not differentiate into antibodysecreting cells when provided with the help of normal t cells [ ] . the index patient for primary cd deficiency was the first child of consanguineous kurdish parents. she presented aged months with a rash, pyrexia, hepatosplenomegaly, lymphadenopathy, pneumonitis, pancytopenia, and disseminated cmv infection. laboratory analysis showed absolute lymphopenia, low t cell numbers, with markedly low cd + and low cd + and normal b cell numbers. she responded well to anti-cmv treatment and at months underwent a mud bmt. t-cell engraftment was demonstrated weeks after bmt. despite continuous anti-cmv treatment, her undifferentiated scid human p lck deficiency p lck deficiency is an ar scid due to a defect of an src kinase critical for the generation of mature thymocytes in adult mice. p lck is important in tcr signaling and phosphorylation of the itams of the cd /tcr complex proteins. mutant mice lacking p lck have pronounced thymic atrophy, a critical reduction in dp (cd + cd + ) thymocytes, no detectable mp thymocytes, and only a few peripheral t cells. both proliferation and development of a given defined cell subpopulation depend on meuse age. the absolute numbers and proliferation of dn and isp (immature single positive) thymocytes only proliferate during fetal and early postnatal life up to days after birth, whereas the proliferation is significantly decreased beyond that age, thus lck may have differential roles in the proliferation and maintenance of dn, isp, and mp/dp thymocyte populations [ ] . the first demonstration of a human scid patient with an abnormal expression of p lck is an scid infant hospitalized at months for dehydration, failure to thrive, and sepsis. the immune phenotype included hgg, selective cd lymphopenia, lack of cd expression on cd + t cells and poor t cell blastogenic responses to various mitogens and il . p lck protein expression was only minimal with an unusual mrna splicing pattern of the lck gene. the levels of p fyn were normal and it is therefore possible that p fyn played a role, albeit incomplete, in the development of his mature t cells. the child has since undergone an allogeneic bmt (at months) from a matched unrelated donor (mud) [ ] . unfortunately the boy died months later due to cmv infection and gvhd (fd goldman, pers. comm., nov. ). whn (winged-helix-nude) encodes for a transcription factor that is crucial for maturation of the thymus microenvironment [ ] . nu/nu mice fail to develop a thymus and mature t cells due to a defect in the whn gene encoding a transcription factor necessary for terminal epithelial cell differentiation. a defective whn gene could lead to the disrupted early t cell development in the bm. t cell progenitors were associated with a lack of pta gene expression and a failure to give rise to mature t cells in adoptive euthymic hosts. wild-type hscs rapidly matured into functional t cell progenitors in the marrow of euthymic or thymectomized but not nu/nu hosts. therefore defects in bm prethymic t cell development can contribute to t cell deficiency in nu/nu mice [ ] . in two sisters a severe scid caused by mutation of the whn gene was associated with complete alopecia. hla-identical bmt in one of the two girls resulted in a clear reconstitution of cd + and cd + cmv reactivated, and she died days after bmt [ ] . a -bp deletion in the gene encoding cd resulted in the loss of glutamic acid and tyrosine in the first fibronectin type iii module of the extracellular domain of cd , identifying a region important for cd structural integrity and lack of surface cd expression. this was almost certainly responsible for the id in this girl [ ] . a second child presented at months of age with severe cid, showing similar t-cell defects. despite normal b-lymphocyte numbers, serum ig levels decreased with age [ ] . introduction of a functional cd minigene was sufficient to overcome the main scid-associated defects and represents a potential route to a gene therapy for human cd -deficient scid [ ] . two male infants born to consanguineous parents had scid despite phenotypically normal blood lymphocytes. their t cells were unable to produce il , ifn-g, il and tnf-a [ ] . another child with scid had defective transcription of il genes encoding il -il [ ] . dna binding of activation protein (ap- ), oct, creb, sp , and nf-k b was normal, but the binding of nfat to its il promoter response element [ ] , or the ability of nuclear factors from the child's t lymphocytes to bind response elements present in the il regulatory region [ ] was barely detectable [ , ] both before and after t-cell stimulation [ ] . these results indicate that the nfat abnormality may underlie the multiple il deficiency in these boys. nezelof syndrome, also known as cellular id with ig, or combined with a predominant t-cell defect, or as a scid variant, clinically less severe compared to the previous ones, is characterized by a form of ad, concentrations of ige that may also be extremely elevated (table . ), and normal or increased serum levels of other ig classes [ ] . the cmi study emphasized the mature t-cell reduction or absence, various expressions of immature cells, with cutaneous anergy to spts and a reduced or absent in vitro lymphocyte response to mitogens. from infancy, patients present recurrent or chronic pulmonary infections, pondostatural retardation, oral and/or cutaneous candidosis, chronic diarrhea, recurrent cutaneous and urinary tract infections, gram-negative bacterial sepsis and a particularly severe form of chickenpox [ ] . differential diagnosis must include pediatric aids, also marked by proportionably increased ig and a lack of antibody and t-cell function [ ] . inherited through ar modalities, cd deficiency has been observed in children, two of whom were brothers, with mutations of the fas gene, one hz and het [ , ] , as well as in unrelated children [ ] . these mutations most often arise as a result of mutations in the gene encoding the lymphocyte apoptosis receptor fas/apo-l/cd . a novel mutation has been identified in the intracellular apoptosis signaling domain of fas in members of a family, with several members monitored for up to years [ ] . thus, the deficiency is inherited in an autosomal dominant fashion but with a high degree of variability in clinical expression [ ] , but also in an ar fashion [ ] . the clinical picture is dominated by imposing hepatosplenomegaly with an early onset, even neonatal, accompanied by t-cell hyperproliferation, chronic and persistent lymphadenopathy, and failure to thrive [ ] . an extensive lymphocyte infiltration of lymph nodes, spleen and liver is observed, with t cells reaching , /ml [cd + , cd -cd -(dn) equal to - cells/ml compared to - in controls], as in omenn syndrome, also in the bloodstream, with possible oligoclonality of t cellularity. dn t cells expressed the a/b tcr [ ] . immune dysregulation is associated with g and a hgg (hypergammaglobulinemia, auto-antibodies and aids, especially of the hematological type, such as aiha, and with a severe and recurrent thrombocytopenia [ , ] . autoimmune features are discussed in chap. . an overlapping mechanism could belong to the etiopathogenesis of xlp and omenn syndrome. was has a prevalence of approximately × live births [ ] . it is transmitted as a recessive hereditary trait linked to the chromosome x, localized in a pericentrometric position on the short limb of chromosome x (xp . -p . ) [ ] . it is therefore possible to identify the female carriers and to provide prenatal diagnosis [ ] . the gene that codifies the was defective protein (wasp) has been isolated [ ] and has mutations distributed among all exons of the entire gene, of which are unique and familiar, with two large deletions, one embracing exons - and one intron [ , ] (fig. . ). six novel mutations have been identified that involve nonsense mutations, or small deletions, all of which result in predicted truncation of wasp synthesis [ ] . a new, recurrent mutation is v m, due to a cpg island was found in a hz girl, who showed microthrombocytopenia and infections to the same degree as her hemizygous father and brother. the amount of was protein was about % in platelets and % in mononucleated white cells [ ] . involved in ensuring the t lymphocyte functional polyvalence, also explaining why microvilli and platelet defects are absent [ ] .wasp and several related proteins (the wasp family) are all involved in the organization of the actin cytoskeleton. to carry out vital functions, cells have to rearrange their actin cytoskeletons [ ] . the characteristics peculiar to wasp as a meeting point for the marking pathways is illustrated in fig. . [ ] . the wasp function is absent in cases of was, in ten with attenuated was and in with xlt [ ] . in normal subjects, it is found in the cyto-molecular biology has proven that wasp found only in blood cells binds the small gtpase cdc h in the gtp but not in the gdp [ , , ] . cdc h plays a critical role in the assembly of actin filaments [ ] and in t-cell polarization when they encounter a b-lymphocyte apc [ ] . wasp activity is regulated by several proteins acting in concert to control wasp configuration. the wasp-interacting protein, when phosphorylated, releases wasp from its grip, allowing wasp to be activated by rho-family gtpases [ ] . experimental data also indicate that cdc , wasp and actin might be plasm but not in the nucleus of various cells such as platelets, t and b lymphocytes and monocytes [ ] . the xlt gene is located on the same locus as the was and could therefore be a variant [ ] ; the main immunological anomalies are summarized in table . [ , ] . children with was have significantly elevated levels of il and ige (table . ) and decreased levels of ifn-γ [ ] . the pathogenetic mechanism unifying the symptom triad is not clear; the glycosylation defect has been proved, primarily concerning sialidation, therefore resulting in an instability on the membranes of platelets, neutrophils and lymphocytes expressing a glycoprotein sialopherin (cd ) [ ] , localized on chromosome , which makes it an improbable candidate, even though cd is indeed the binding agent of cd and could therefore play a role in t-cell maturation, differentiation and activation, thereby acquiring marking capacities that are independent of tcr/cd [ ] . however, the tcr-mediated signaling defect is characteristic of was [ ] , in addition to the reduced expression of cd [ ] , which can explain immune and hematological defects. in the lymph nodes, there is a shortage of lymphatic follicles and the thymus-dependent and -independent areas are depleted, moderately at the age of years ( fig. . ) and to a greater extent at years ( fig. . ). the predominant immunological outline is constituted by elevated iga and ige levels, low igm and all igg levels, as well as the absence of a response to polysaccharide antigens, which is why the children's serum lacks isohemagglutinin [ , ] . in unweaned babies, the most striking finding is the cd :cd ratio = [ ] , compared to . in normal children aged . - . (tables . - . ). was usually starts at . months (range, - ) [ ] with hemorrhagic manifestations, petechiae and prolonged bleeding from the umbilical scar or the circumcision site, observed in newborn babies [ ] . the clinical triad is characterized by cutaneous lesions that are practically indistinguishable from rather severe ad ( %), congenital thrombocytopenia ( %), a marked susceptibility to rris ( %) [ ] (figs. . , . ) and gastroenteric symptoms such as hematemesis, melena and chronic diarrhea [ ] . other complications may include neutropenia ( %), arthritis ( %), skin vasculitis ( %), cerebral vasculitis ( %), inflammatory bowel disease ( %), and renal disease ( %) [ ] . a reduced thrombopoiesis (level < , /ml), with microthrombocytes and an accelerated turnover in boys must allow for a suspected diagnosis [ ] . it has recently been proven that the classic presentation is more common in children aged . months than in those aged . months ( % compared to %), unlike platelet counts [ ] . however, only % of unselected children with persistent thrombocytopenia, positive fh, small platelets and defects associated with t and/or b lines had the classic triad and % only thrombocytopenia before diagnosis [ ] . primary immunodeficiencies infections, appearing during the first months of life, are often marked by otitis media, pneumonia, meningitis and sepsis, caused by viruses (cmv and herpesvirus) and by bacteria (pneumococci or other capsular polysaccharide). these are followed by more common infections caused by opportunistic germs, pneumocystis carinii and mycetes such as candida albicans. differential diagnosis should also include a rare ar syndrome similar to was, also reported in female patients, characterized by ad, rris and thrombocytopenia with microthrombocytes [ ] . when caring for these children one must monitor the platelet count, the immunological structure (ig, lymphocyte and subpopulation counts) and the potential onset of autoimmunity and tumors [ ] .aiha may be found in % of children [ ] . prophylactic treatment for infections is done with ivig; mg/kg every weeks) and sulfamethoxazole ( mg/ kg/ days) after diagnosis [ ] . splenectomy may decrease the bleeding tendency [ ] , but early relapse of thrombocytopenia after splenectomy is predictive of a poor prognosis [ ] . on average death occurs around the age of ( in untreated children), but can occur between . and . [ ] , with survival also > . death is caused by massive hemorrhages ( %), tumors ( %) and severe infections ( %) [ ] . the second largest group of patients with id given bmts since are those with was, with . % of children aged < years [ ] . fourteen out of patients underwent phenoidentical (n= ) or haploidentical (n= ) hscts; the other four died before hsct could be undertaken [ ] . boys who had received a mud hsct transplant < years had survival rates similar to those receiving hla-identical sibling transplants, but the success rate decreases dramatically at the age of - [ ].wasassociated t-cell signaling defects can be improved upon retrovirally transduced hscts [ ] . recently, correcting the t-cell defects has been proposed. the potential for correction of the t-cell defects has recently been demonstrated by transduction with an oncoretroviral vector encoding the wasp, which resulted in correction of the deficient proliferative response to tcr stimulation characteristic of was [ ] . ata is a complex ar inherited syndrome, associated with neurological, immunological, endocrinological, hepatic and cutaneous abnormalities, characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, and increased susceptibility to rris [ ] with an incidence estimated at : , - , , live births [ ] . in italy the frequency on the general population, is of . × , with an increase in hets from . % to . % [ ] . it is characterized by a genetic heterogeneity, which is reflected in the division into four main groups of complementation, to which one must add the nijmegen and at-fresno variants, perhaps caused by other well-defined id syndromes the same gene, also localized on the long arm of chromosome q . [ ] . the -kb gene, called atm (at mutated) because of its mutations by defective splicing in all patients with ata, permits het identification [ ] . a dna clone complementary to atm shows considerable affinity to factors responsible for signals involved in regulating the cell cycle and codifying a protein similar to phosphatidylinositol- -kinase (pi k) [ ] , involved in mitotic signal transduction, meiotic recombination, and cell cycle control. a result could be a recombination defect which interferes with b and t lymphocyte gene rearrangement, involving tcr and isotype switching, consequent to a damaged dna triplication and therefore accounting for ig deficiencies [ ] . cells from these patients progress too rapidly from the g phase, in which they receive ionizing radiations, to the s phase, then continuing irradiation, to the g /m phase with further delay, evolving in apoptosis [ ] . this hypothesis has received further credit after observing that the p gene expression does not increase in human cells exposed to radiations [ ] . the p gene is part of the normal cell cycle and during the s phase provides time for the dna physiological repair after exposure to radiation that may also be cosmic [ ] . the thymic tissue is either absent or degenerated with a fetal appearance (fig. . ) : some follicles, also with b cells, are visible at the age of (fig. . ) , at there is complete cellular depletion (fig. . ). immune deficiencies are humoral and cellular (cutaneous anergy and depressed proliferative responses) [ ] . the karyogram shows that the lymphocytes have common rupture points at the chromosomal level with inversions and translocations involving precisely the tcr and ig genes [ ] . most chromosomal translocations involve the genes encoding tcr on chromosome and the ig h chains on chromosome : most breakpoints occur at the loci that encode ig and tcr for antigen (regions q , p , q , q ) [ ] , in areas typical for cod-ification of molecules of immunological importance (chap. ). an important role is played by genes belonging to ig gene superfamily (igsf) (table . ) . possibly the progressive id of ata, like its apparently unlinked manifestations, is at least in part linked to the accumulation of clonal anomalies affecting the tcr and the igsf: this suggests the intervention of "illegitimate" recombinations damaging above all the t cells [ ] . t-cell immunological deficiency is completed with lymphopenia, a decreased cd :cd ratio due to the drop in cytotoxic cd , and a rise of immature forms with tcrgd [ ] . another consequence is the isotype deficiency: about % of patients present sigad; > % are also affected by an igg -igg deficiency with igm becoming monoclonal, and % by serum igg deficiency [ , , ] . fig. . ,the degree of depletion of lymphocytes is extensive in both thymus-dependent and thymus-independent areas chromosome into cells, the chromosomal aberrations induced by x-rays were suppressed [ ] . the rare ar nijmegen breakage syndrome, so called because it was initially seen in two brothers of secondcousin parents living in that city, and at the moment observed in approximately patients, has various characteristics of ata but without ataxia, telangiectasia, or high concentrations of afp. clinical characteristics are singular: short stature and microcephaly with prenatal onset, bird-like profile, prominent midface, a long nose, low-set ears, cutaneous depigmentation with caféau-lait spots, an almost normal intelligence, and also rris and bronchiectasis. humoral and cellular id includes reduction of antibodies and lymphoproliferative responses [ , ] . during an -year period of observation, the id was found to be profound, highly variable, and with a tendency to progress over time in / children [ ] . there is a high proclivity to expressing rearrangements of chromosomes and as in ata [ , ] . dgs is usually sporadic, with known cases of positive fh [ ] . it is caused by a defective development of the rd and th branchial pouches which takes place before the th week of gestation, with consequent thymic hypoplasia or aplasia and parathyroid hypoplasia; the th and th pouches and branchial arches can also be affected [ ] . the cause can be found in the neural crest cell incapacity to migrate and interact appropriately with endothermic cells of the brachial pouches and arches [ ] . deletions (often microdeletions) at the pericentrometric region of chromosome q -pter have been described in %- % of cases [ ] . a microdeletion q . was recorded in children aged - months, % of whom had developmental delays, mild hypotonia, as well as language and speech delays [ ] . another children had deficits in the areas of attention, story and visuospatial memory, arithmetic performance relative to other areas of achievement, psychosocial functioning [ ] , and mental retardation in % of children [ ] , thus indicating the need for early intervention beginning in infancy [ ] . overlapping alterations are present in the syndrome complex known as catch , which in turn includes the charge association. other cases of dgs can derive from microdeleted chromosome p (fetal-alcoholic syndrome, retinoic embryopathy, maternal diabetes) [ ] . this variable phenotype is reliably referred to microdeletion q . ; the greater it is the more complex is the associated phenotype [ ] . another difference depends on the variable spectrum of t-cell abnormalities in individuals with dgs who might have normal t-cell numbers and function, low t-cell numbers but fairly normal t-cell proliferative function [ ] or no t cells purkinje cells (pcs) and degenerated granular cells. that the number of basket cells, so called because they form with the axons bunches of fibrils distributed so as to form a nest in which the nucleus of each pc settles, is almost normal, proving that pcs are probably normal at birth and degenerate only later [ ] . typical clinical manifestations are ataxia, telangiectasia of both auricular lobes and sclera, rris and an elevated incidence of neoplasia [ ] . from a review of patients [ ] , the percentages of symptoms are as follows: progressive ataxia ( %), typically cerebellar, becomes evident when children start walking or a little later, affecting intentional movement and becoming complicated by dysarthria ( %) and involuntary choreic movements ( %), causing the majority to be unable to walk by about the age of - [ ] . at a later stage it is possible to observe nystagmus ( %), strabismus, oculomotor apraxia ( %), reduction or absence of reflexes ( %), and dyslalia, increasingly amplified and, in some patients, also mental retardation [ ] . telangiectasias develop between the ages of and on the bulbar conjunctiva ( %) (fig. . ) , on the flexor surfaces of the limbs and areas exposed to sun rays ( %). height and weight are < th percentile ( %) and the appearance is progeric ( %). severe rris are common ( %), encouraged by antibody deficiencies. the pathogens involved can be bacterial or viral, often resulting in lung bronchiectasis, all starting after the onset of neurological manifestations [ , ] . associated neoplasia ( %) is usually lymphoreticular, less common than adenocarcinoma, with an eightfold increased trend for all kinds of tumors [ ] . in cultures the fibroblasts of these patients are three times as sensitive, compared to controls, to ionizing radiations and to radiomimetic chemical substances, but not to uv rays, unlike what is observed in the cells of subjects affected by xeroderma pigmentosum [ ] . in addition, the persistence of elevated serum a- -fetoprotein (afp) levels was observed in all patients. an interesting in vitro study has reported that by introducing a normal human other well-defined id syndromes fig. . . conjunctival telangiectasia in a girl with ata [ ] . a second group is referred to as having partial dgs (dgsp) or transient forms (dgst), with mild symptoms. the designation "complete digeorge syndrome" (dgsc) is reserved for the third group of infants who have absence of thymic function in addition to other defects of the rd and th pharyngeal pouches, < % of patients with dgs, although they can have high t-cell numbers that respond to mitogens [ , ] . these patients have profound id, with its associated clinical findings [ ] . dgst includes cases with a spontaneous quantitative and qualitative t lymphocyte recovery [ ] . the thymus can also be ectopic: in dgsc the t zones are depleted, the cd /cd markedly reduced both in number and in function with spt anergy, and b cells appear unaffected or increased [ ] . in dgsp, the most common type, t-cell number and function are instead usually normal, as are the cd /cd cells with a nk phenotype, or they may be moderately reduced [ ] . the proliferative response to mitogens can be pathologically reduced [ ] and the response to polysaccharide antigens may be absent [ ] . from neonatal age, there are malformations of other structures that form during the first weeks of embryogenesis, presenting a suggestive but not pathognomonic picture (table . ) [ ] . diagnosis is usually suspected within the first days after birth, due to the presence of hypocalcemic tetany caused by hypoparathyroidism and cardiac malformation. the facial dysmorphism is also characterized by a small mouth with thin lips described as fish-like [ , ] (fig. . ) . the two rare cardiopathies indicated in table . depend on neural crest nonintegration, as mentioned, which accounts for > % of the alterations alone [ ] . others can be observed affecting the right heart, such as fallot tetralogy, pulmonary athresia with an interventricular septum defect, and pulmonary infundibular stenosis [ ] . babies surviving the neonatal period manifest from the very first months an increased susceptibility to infections, particularly those of the respiratory and digestive tract, viral and/or fungal, but also caused by pneumocystis carinii, which can be fatal in dgsc [ ] . other findings include gastroesophageal reflux, speech delay, laryngomalacia, absent kidney, conductive or sensorineural deafness, th cranial nerve palsy, and hypothyroidism [ ] . treatment with high doses of vitamin d and diets enriched with ca gluconate are needed immediately, also ensuring that calcemia remains at the lower limit of normal values so as to avoid snc and renal damage. subsequently the possible correction of cardiac malformations should be evaluated. id may be severe, but can regress spontaneously with reconstitution of cmi and t functions; compensating hyperplasia of the residual parathyroid tissue can make it possible to discontinue ca and vitamin d treatment [ ] . dgs natural history is, however, complicated by mental retardation and the difficulties encountered in correcting cardiac malformations and in controlling hypoparathyroidism [ ] . because of variability in the id severity, it is difficult to evaluate claimed benefits of bmt: in two cohorts of [ ] and transplanted infants [ ] , the survivors were out of ( . %). recently, / and / infants underwent postnatal transplantation with cultured unrelated thymic tissue, with immunosuppression, with positive results [ ] . del q . syndrome, characterized by a -mb deletion on chromosome q . is the most frequent known chromosomal microdeletion syndrome, with an incidence of in , - , livebirths. patients show chapter primary immunodeficiencies clinical outlines not unlike gvhd [ ] (fig. . ) [ ] , suggesting a possible connection to a fas deficiency (cd ) or apoptosis syndrome. the sh d a gene was found altered in two families, thus indicating that xlp must be considered when more than one male patient with cvid is encountered in the same family, and sh d a must be analyzed in all male patients with cvid [ ] . recently a critical revisitation of this experiment in nature has allowed the identification of links between id and allergy [ , , , ] . the rare higes is associated with bacterial rris, chronic ad, coarse facial features and very elevated ige levels [ , ] ( table . ), up to , iu/ml [ ] . linkage to a region on chromosome q has been demonstrated in several affected families; however, neither the fundamental host defect nor the defective gene has yet been identified [ ] . fh is frequently positive for atopic disease, at times higes is combined with an unusual predisposition to staphylococcus aureus infections [ , , , ] . in buckley's study, it was present in . % of cases, of both sexes, indicating an autosomal dominant transmission with incomplete penetrance [ ] . onset occurs in the pediatric age in % of cases [ ] . clinical presentation is unusual: there are no complaints during the first months of life, toward the rd- th month a severe form of chronic ad appears all over the body, which can be associated with other allergic manifestations, including asthma in . % of cases [ ] . skin biopsy specimens reveal spongiosis and perivascular dermatitis and/or folliculitis with a predominance of eosinophils [ ] . there is an excessive predisposition to cutaneous and respiratory tract infections (deep and superficial abscesses, otitis, pneumonia, sepsis) ( fig. . ), also encouraged by neutrophil chemotactic deficiency caused by defective cellular functions (table . ), which, if present, is so pronounced that it becomes a characteristic, unlike ad where it is secondary [ ] . the subcutaneous abscesses, described as cold, not covered by warm and reddened skin, are pathognomonic to higes but not essential to the diagnosis [ ] . the abscess is filled with pus that always grows staphylococcus aureus; in some cases mucocutaneous candidosis and chronic herpetic keratitis are associated [ ] . infections appear within the first months [ ] . the face shows coarse and dysmorphic features, midline facial defects such as a prominent nose and a high, arched palate, and disproportionate cheekbones and mandible; pondostatural growth notably retarded [ ] , pneumatocele (fig. . ) and osteoporosis caused by reduced bone density with a tendency to fracture [ ] complete the picture. six consanguineous families have been reported with an ar form of higes, including cardiac abnormalities, t-cell deficits, cleft palate facial anomalies, and hypocalcaemia. at least genes have been mapped to the deleted region. recently, in / patients with del q . syndrome without q deletion mutations were found in t-box that is a major genetic determinant of the del q . syndrome [ ] . xlp is caused by a defect in the sh d a gene (table . ), which binds to the cytoplasmic domains of cd slam (signaling lymphocyte activation molecule) and b , and may regulate signals transmitted by these receptors in t and nk cells, respectively [ ] . xlp has been reported in > males from > families [ , ] , and in an other males [ ] , it is inherited with the x-linked model. it is set off in males aged - by an ebv infection that became manifest with a very polymorphous pattern, often with unusually severe or fatal infections mononucleosis caused by the immune system incapacity to respond to ebv, or evolving into a hgg with iga and igg deficiency and higms, or medullar aplasia and/or a burkitt type lymphoma [ ] . the disease has been reported in female subjects [ ] . xlp polymorphism could be explained by the fact that the ebv receptor is expressed on differentiating b lymphocytes starting with the preceding isotypic conversion stage [ ] . it has recently been verified that before ebv infection, males already suffer from dys-or pan-hgg, incapable of regulating the expression of ig and/or containing b or t lymphoproliferation. even after ebv infection, the immune system is unable to provide adequate th responses, and therefore releases cytotoxic alloreactive cd and th -like t cell ils, causing extensive damage to the entire parenchyma, exemplified by fulminating hepatitis, cellular infiltrations and tissular necrosis. the lymphoid tissues with an altered structure are also affected by necrosis, with a high incidence of mostly nonlocalized lymphomas [ ] . the thymus is also affected by thymocyte rarification, with other well-defined id syndromes fig. . . bone marrow biopsy specimen in a -year-old boy: numerous histiocytes in erythrophagocytosis affected children aged months to . years, with ar-higes presenting with the classic immunological findings, including rri, eczema, elevated serum ige, hypereosinophilia, and severe recurrent fungal and viral infections [ ] . notably, patients with ar-higes did not have skeletal or dental abnormalities and did not develop pneumatoceles, as seen in autosomal dominant-higes [ ] . among the immunological characteristics (table . ) [ , ] , cutaneous anergy to several antigens such as candida and tetanic toxoid is characteristic, which is associated with the anomaly of proliferative responses by the t cells to antigens and mitogens, in contrast with the integrity of other functions tested in vitro [ ] . t subpopulations appear to be normal [ ] . however, the lymphocyte proliferation to anti-cd /cd monoclonal antibodies can be impaired [ ] . as noted, ifn-γ deficiency associated with a pathological th prevalence has a fundamental impact on ige hyper-production [ , ] . in higes, some studies have confirmed ifn-g deficiency compared to controls [ , ] , also due to an impaired response to il [ ] , while others have not [ , ] ; however, compared to ad, normal levels of t producers of il are characteristic [ ] . considering the ifn-g/il + correlation of ad, in higes no specific t-cell anomalies are noted, nor does the hyper-ige explain this pediatric abnormal susceptibility to infections: high ige levels are also seen in children with ad, who do not, however, have an unusual predisposition to abscess formation [ ] . one typical characteristic is sige directed against microbial antigens: the anti-staphylococcal sige rise to . % compared to normal levels of . %- . %. another constant finding is the increase in % of cases of eosinophil concentrations, which make up %- % of leukocytes [ ] , reaching %- % [ ] . by expressing the cd -cd duo, they stimulate the isotype b-cell switching to ige.we studied children affected by severe ad, chronic fa-induced diarrhea and asthma. the allergens responsible were cm and der p [ ] . in case of higes caused by fa, atopic manifestations can clearly improve following an exclusion diet, reducing the frequency of infections and partially correcting the immune defect [ ] , revealing how fa can induce several immunological anomalies. diagnosis is made on the basis of the data in table . ; differential diagnosis with ad is schematized in table . [ ] . treatment with cromolyn is extremely effective, anti-staphylococcal antibiotic treatment [ , ] and if necessary antifungal therapy provide good results [ ] . griscelli disease, mapping to chromosome q [ ] , is an ar syndrome caused by mutations in the myo a (gs ), rab a (gs ), or mlph (gs ) genes, all of which lead to a similar pigmentary dilution [ , ] . the disease is also characterized by partial oculocutaneous albinism, predisposition to pyogenic infections and in most patients by abnormal regulation of the immune system, which results in a syndrome of macrophage hyperactivation, known as hemophagocytic lymophohistiocytosis [ ] . mutations in the gtp-binding protein rab a (gs ), which appears to be involved in an uncontrolled t lymphocyte and macrophage activation syndrome, leading to death in absence of bmt, occur in this syndrome [ ] . a mutation was found in the myo a gene (gs ) associated primarily with neurological impairment [ ] . two identical twin boys aged months were reported with persisting fever, mouth ulcers, hepatosplenomegaly, pancytopenia and failure to thrive [ ] , as was an -month-old infant [ ] . both infants had silvery-gray hair and pigment clumps on the hair shafts, and skin biopsy showed accumulation of melanocytes on melanosomes. their parents were first cousins and a sibling with similar manifestations had already died, as did the twins. a genetic study revealed a -bp deletion in the rab a gene ( del aagcc in exon ) [ ] . in a -year-old child with hemophagocytic syndrome, id, and secondary neurological disorders, typical melanosome accumulation was found in skin melanocytes and pigment clumps were observed in hair shafts. two heterozygous mutant alleles of the rab a gene, a c-t transition (c t) leading to q stop and a g-c transversion on the exon splicing donor site (g + c) were found [ ] . the finding of gray strands of hair, gray eyebrows, and eyelids in childhood should alert pediatricians to considering griscelli syndrome since an early diagnosis is life-and health-saving [ ] . the phagocyte system with the biochemical basis of cgd is analyzed within the framework of innate immunity. chronic granulomatous disease (cgd) has an overall prevalence of : , to : , although this could be underrated (table . ), considering that some subjects may have a very mild clinical phenotype that escapes diagnosis [ ] . a us registry of birth rates found a prevalence of : , to : , live births for the period - [ ] . the youngest patient was days old [ ] and in children with cgd the mean age at the onset of infections was months, with a median delay in diagnosis of . years [ ] . otherwise the the clinical features of this rare ar disease include oculocutaneous albinism and susceptibility to especially s. aureus and b-hemolytic streptococcus [ ] . approximately % of patients develop an accelerated phase of the disease, with deposition of lymphohistiocytes in the liver, spleen, lymph nodes and bm, resulting in hepatosplenomegaly, lymphadenopathy, bm infiltration hemophagocytosis, pancytopenia as well as fever, jaundice, prolonged bleeding, easy bruisability, neurological changes (nystagmus and neuropathy), mild mental retardation, and partial ocular and cutaneous albinism [ , , ] . the cellular hallmarks of the disease include large lysosomal granules in leukocytes, giant melanosomes in melanocytes and affecting other cells of the body such as neural schwann cells, renal tubular cells, gastric mucosa, pneumocytes, hepatocytes, langerhans cells of the skin, and adrenal cells [ , ] . the fundamental defect in this disorder was found to be caused by mutations in a gene mapped to chromosome q -q [ ] encoding a cytosolic protein on chromosome named lysosomal-trafficking (lyst) regulator, encoding a -kd protein whose function remains unknown [ ] . bmt is resolutive in these children [ ] . normal protein expression (x + form), but with a total absence of oxidase due to incorrect binding [ ] . ar-cgd is caused by a mutation in the genes encoding the remaining oxidases of kd (p phox ) (phox, phagocytic oxidase) [ ] (ncf- ), p phox of kd (cyba), and p phox of kd (ncf- ) [ , ] . the ar-cgd forms ( . %- % of cases) [ , ] are identified using the immunoblotting technique, depending on whether they affect the p phox , the p phox or the p phox [ , , , ] , with greater prevalence in an american study [ ] . patients with the x-cgd appear to have a more serious clinical phenotype than patients with the ar-cgd, based on the fact that they are diagnosed significantly earlier (mean, . years of age vs . years of age, respectively), have a significantly higher prevalence of infections and a higher mortality ( . % vs . %) [ ] . mutations in any of the structural molecules (table . ) lead to cgd. mutation of rac (see lad), the predominant g protein in neutrophils, leads to defects in so production, as well as in chemotaxis [ ] . activation of the nadph oxidase requires complex rearrangements between the protein median age at onset was . months, and the median age at diagnosis was . years [ ] . the deficiency appears in two forms ( [ , ] , with an h-chain deficiency, is divided into four x subtypes (table . [ , , , , , ] ), also identified on the basis of nbt results, depending on whether the x is absent (the most common form), reduced or present but inactive; subtype x is divided into two variants: in one of them the nbt is slightly positive in %- % of cells ( % of patients), in the other in %- % ( % of patients) [ , , , ] . more precisely, the four subtypes are caused by mutations in the four gp phox regions, many of which depend on cybb gene mutations, causing the x form, while mutations depend on the nadphoxidase activity (x form) and eight others lead to a phagocyte deficiency subunits, which are in part mediated by noncovalent binding between src-homology domains (sh domains) and proline-rich motifs [ ] . cgd is a hereditary disease (table . ) characterized by severe recurrent pyogenic infections. this marked susceptibility is caused by the phagocytes' incapacity to kill in particular the catalase-positive bacteria, because of a genetic defect of the nadph-oxidase enzymatic system situated in the wall of the phagocytic vacuole. in cgd, phagocytosis occurs normally, but the nadph-oxidase is unable to markedly produce anion superoxide (o •-), h o and other o free radicals, thereby permitting the survival of microorganisms within the cells, where they are protected from the antibodies and from most antibiotics [ ] . another consequence of the lack of o radicals is the development with countless inflammatory episodes, which then result in typical granulomas [ ] . the nitroblue tetrazolium (nbt) reduction test is based on the chemical characteristics: in fact, the phagocytes without o •are unable to reduce the yellow nbt of products activated by pha aspecifically stimulated phagocyte o , or specifically with corpuscle particles such as preopsonized yeasts (fig. . ) . the result was % in children [ ] . at a molecular level, the genes that codify the two subunits of flavocytochrome b , gp phox and p phox have been cloned: respectively the cytochrome, h (b) and l (a) chains situated on the phagosome vacuole membrane, and also the cytosolic factors p phox , p phox and p phox , deriving from the nadph-oxidase activation, all proteins placed inside the cytoplasm and that belong to innate immunity. it has therefore been possible to identify molecular lesions at the cgd origin, with the exception of the p rac [ , , ] . the clinical pattern is severe in the x form and variable in the other two x forms; onset occurs within the st year of life in / of cases, and in others within the nd year [ ] , although it can appear also at the age of [ ] . purulent recurrent infections, with a granulomatous evolution, predominantly affect the epithelial surfaces normally colonized by bacteria, such as cutaneous, subcutaneous, mucous membranes, the respiratory tract and the intestine: cutaneous and mucosal infections, and lymphadenitis lead to suppuration and fistulation (fig. . ) , pneumonia or lung abscesses (fig. . ) are more frequently characterized by persistent fever and diarrhea [ ] (table . ) [ , ] . pneumonia was the most prevalent infection in patients ( %) (mostly by aspergillus), followed by suppurative adenitis ( %), subcutaneous abscess ( %) and liver abscess ( %); mostly by staphylococcus, osteomyelitis ( %) mostly by serratia, and sepsis ( %), and by salmonella [ ] . in a long-term trial, pneumonitis was the most prevalent infection ( %) followed by lymphadenitis ( %), aphthous stomatitis ( %), liver abscesses ( %) and chronic lung disease the rates (%) consist of a first [ ] and of a second number related to the european study [ ] ; us data regarding ar cgd are in parentheses [ ] . data from [ , , , , ] . x x-linked, ar autosomal recessive, ad autosomal dominant inheritance, nd not done. a the superscript symbols indicate the level of immunoreactive proteins: undetected,diminished, + normal protein levels. granulomas in the entire lung parenchyma, which are formed by mononucleates (fig. . a) with giant cells (fig. . b) . the chronology of infection onset is summarized in table . [ ] : lymphadenitis is the earliest. osteomyelitis is usually a worrying complication: ( %) [ ] . lymphadenitis, lung infections, enteral infections, and hepatic abscesses were the most frequent infections in a cohort of children [ ] . staphylococcal liver abscesses are almost pathognomonic of cgd [ , ] . because the infections develop in areas drained by lymphatics, they tend to diffuse via the lymphohematogen route, thus causing arthritis and osteomyelitis and abscess formation, especially affecting the bones, which are the most severe manifestation, and hepatitis with common upsurge of hepatosplenomegaly. lung infections are almost the rule: those initially segmented and parallel tend to gradually spread over the entire lobe [ , ] . histological examination shows widespread gastric outlet obstruction urinary obstruction extensive bone destruction involves various segments, for example the vertebra, the metacarpus and the metatarsus, causing widespread damage, which is difficult to treat and is also irreversible [ , ] . aspergillus, pulmonary, bone (fig. . c) or encephalic infections constitute a severe therapeutic problem and are threatening events, with a mortality rate of %, but with specific treatment the prognosis is good as far as recovery is concerned [ ] . the treatment includes prophylaxis with trimethoprim-sulfamethoxazole (tmp/smx) ( mg/day given in two divided doses), and ifn-g ( mg/m subcutaneously thrice weekly) in all patients with cgd, regardless of genotype [ , ] . itraconazole therapy ( and then mg/kg/day) has an excellent tolerance in all cases and was effective in of children ( . %) [ ] . survival until the age of and beyond is achieved by % of patients with cgd xl and % of those with cgd ar [ ] . because the prognosis is uncertain, as observed, the only possibility for a definite resolution is with a bmt, from family donors who are x-cgd or x-cgd-identical [ ] . bmt was successful in children out of ( . %) (see table . ), including a -year-old boy with x-cgd who underwent successful hla-identical peripheral blood sc transplantation during invasive pulmonary aspergillosis and osteomyelitis, which was unresponsive to antifungal treatment [ ] . lad is due to mutations in the gene on chromosome at position q . encoding cd (table . ). it is divided into five types: lad type i to lad type v [ , , , ] . the three subunits of the cd /cd complex are involved in pid (lad type i syndrome), ar, linked to the lack of a m b equal to cd b/cd (table . ) surface expression on all leukocyte populations caused by different mutations in the cd encoding gene, often severe in infancy [ , ] . children with a deficiency of these integrins have a defect above all in phagocyte action, suffer from severe infections from the neonatal period [ ] due to absent b -integrin activity, which impairs neutrophil ability to exit the circulation and travel to sites of infection. on the contrary, leukocyte movements are not prevented, indicating the normal involvement of cd and cd (table . ) . the clinical basis for defining this disease, described in over cases [ ] , dates back to a study at the soothill school in [ ] . there are two forms of lad type i [ ] : if the deficiency is full blown (no detectable cd ), the clinical symptoms (table . ) [ , ] are dominated by severe and recurrent infections with a negative prognosis in the first years of life unless corrected by an allogenic bmt, the only resolutive treatment [ ] . if instead it is a partial deficiency with residual cd expression, the clinical outline is less severe and some patients, with appropriate treatment, can live to adult age [ ] . lad type ii, ar (cd levels are %- % of the normal levels), with a molecular base represented by an slex ligand (cd s) is a defect common to cd e and p, which mediate neutrophil rolling. in the absence of a gdp-fucose transporter, the slex is not made. lad type results from mutations in this transporter that takes fucose into the golgi apparatus for posttranslational fucosylation of newly synthesized proteins. this is the ligand for cd e; without it, leukocytes cannot make initial attachment to vascular endothelium [ ] . lad has been described in two children aged and with mental retardation, from different families, but both with parents who were blood relatives [ ] . it has a lower mortality rate [ ] . mice with a deficiency of both selectins show a lad-like syndrome, providing a useful model for studying these syndromes [ ] . lad type iii shows defective tethering and adhesion and bleeding diathesis. this is a new syndrome where in vitro leukocytes showed normal rolling along endothelial cell cultures but defective tethering and tight adhesion. thus this is a defect in the capability of vascular integrins on circulating leukocytes to rearrange with their endothelial ligands at adhesive contacts and rapidly arrest on target vascular endothelium in response to endothelial-displayed chemoattractants. however, the expression levels of the major integrins on lymphocytes and neutrophils were largely conserved in the patient cells, ruling out a lad-i syndrome. patient leukocytes showed no lad-ii like fucosylation defect, since they expressed normal levels of the fucosylated marker cd s, comprising the slex carbohydrate selectin ligand [ ] . defects in both leukocyte and platelet functions that are biochemically and molecularly distinct from the adhesion disorders previously described suggest a mutation in an early myeloid pathway. the defect is associated with regulation of the gtpase activating protein rap , as demonstrated by the intact rap expression and activation by phorbol esters, thus ruling out an lad defect in rap gtp loading [ ] . lad type iv manifests defective cd e expression or tethering. a girl developed pseudomonas omphalitis at weeks of age, recurrent ear and urinary tract infections, and had clinical evidence of impaired pus formation reminiscent of a lad syndrome, but her neutrophils were functionally normal and expressed normal levels of cd , cd e, and slex. however, the patient showed an absence of cd e from the endothelium, although e-selectin mrna was present. in contrast to patients with lad , she had mild chronic neutropenia but appropriate leukocyte increases in response to infections or gm-csf. a bm biopsy performed during a period of health showed normal cellularity for her age. her fh is remarkable only for a previous sibling who had died at weeks of gestation of a staphylococcal infection of the fetus, amniotic fluid, and phagocyte deficiency placenta. she also has two half-sisters who are completely well. the fh is negative for recurrent infections in either parent or more distant relatives [ ] . lad type v caused by rac deficiency. a -week-old boy born to unrelated parents had delayed uc separation, perirectal abscesses, poor wound healing, and absent pus at sites of infection in the setting of neutrophilia, suggesting a neutrophil defect. his neutrophils exhibited decreased chemotaxis, polarization, azurophilic granule secretion, as well as significantly reduced stimulated superoxide production but had normal expression and up-regulation of cd b. rac constitutes more than % of the rac in neutrophils [ ] . a -yearold boy who had multiple recurrent, life-threatening infections characterized by leukocytosis and notable for the absence of pus in the inflamed tissues was reported. the presence and density of cd b, cd c, and cd were normal. the expression of cd p and cd l were also normal. a bmt was curative. the boy shared a phenotype that closely mimicked that of a mouse mutant deficient in the rho gtpase, rac [ ] . the disease was shown to be attributable to an ad mutation in the rho gtpase rac at an amino acid needed for proper interaction with other intracellular proteins. rac comprises > % of the critically important g protein rac in neutrophils. each member of the family appears to control a distinct function of the actin cytoskeleton (chemotaxis and degranulation) and nadph oxidase (superoxide production) function [ ] . a male child from the mother's first pregnancy was born at term from parents of arab ethnic origin who were first cousins. he had a severe genetic disorder associated with functional defects in multiple leukocyte integrins, reflected in recurrent infections, profound leukocytosis and a bleeding diathesis. platelet transfusions and antibiotic courses reduced the symptoms, which remained a significant clinical problem. at age years, he died from disseminated fungal infection after a mismatched bmt. a younger brother presented with the same clinical and hematological phenotypes at birth and died at age week from sepsis. g pd converts g p to -phosphogluconolactone, generating nadph and a h + ion from nadp + . nadph oxidase catalyzes the monovalent reduction of o to o •-, with the subsequent conversion to h o by superoxide dismutase [ ] . in the form of a partial deficiency, known as the cause of hemolytic anemia or favism, the enzyme's residual activity ( %- %) permits nor-mal bactericidal activity. the g pd variants have been classified by the level of residual enzyme activity and propensity for hemolysis and grouped into five classes: class i, severely deficient with chronic hemolytic anemia; class ii, severely deficient with occasional hemolytic anemia (< % residual activity); class iii, moderately deficient ( %- % residual activity); class iv, normal activity ( %- %); class v, increased activity [ ] . in a trial on g pd-deficient subjects originating from different parts of italy, a greater molecular heterogeneity than described by others was observed, especially in sardinia [ ] . in a complete deficiency, sexually transmitted, whose gene is localized on the chromosome x at position p and characterized by several mutations and their variants, with a consequent deficiency of bactericidal activity, the neutrophils are unable to kill s. aureus, e. coli and serratia, and therefore there is an increased susceptibility to infections, rather like cgd [ ] . the diagnostic work-up of children may reveal a child with recurrent infections who initially received the diagnosis of g pd deficiency, subsequently shown to have the phenotype of x-linked cgd [ ] . the disorder has a higher incidence in mediterranean countries and asia, in japan ( . %) than in indonesia ( . %), as ascertained with a novel screening kit [ ] , and is low in newborns in tehran, iran ( . %) [ ] . within the framework of oxygen-dependent killing defects, hereditary myeloperoxidase deficiency (mpo) is the most common neutrophil biochemical defect and plays an important role in the host defense mechanism against microbial diseases. the neutrophil disorder characterized by the lack of mpo activity is speculated to be associated with a decreased level of immunity. mpo is unusually accompanied by a specific pathology. ar transmitted, it appears far more common than previously suspected ( : , for the partial deficiency to : , for the total deficiency). it is a disorder that is prevalently recorded in entirely healthy patients and therefore, in most cases, a random laboratory finding. in addition to three already-known mutations, the genetic characterization of an italian population showed the presence of six novel mutations: four missense mutations, a deletion of an adenine within exon (c. dela) and a mutation within the ¢ splice site of intron (c. - a>c). the c. dela deletion causes a shift in the reading frame with the occurrence of a premature stop codon within the pro-peptide. the activation of a cryptic ¢ splice site located nt upstream of the authentic ¢ splice site causes a shift in the reading frame that may lead to the generation of an abnormal mpo precursor lacking the enzymatic activity [ ] . in a japanese patient with complete mpo deficiency, neutrophil function analysis revealed that mpo activity was alkaline phosphatase [ ] . monocyte functional alterations in the second individual suggest that c/ebph plays a critical role in monocyte/macrophage development of humans and implicates abnormalities in monocytes/macrophages and neutrophils in the onset and development of the disorder [ ] . severe congenital neutropenia (scn) and cyclic neutropenia are disorders of neutrophil production predisposing patients to recurrent bacterial infections. recently, mutations of the gene encoding neutrophil elastase (ela ) have been indicated as the most common cause for scn as well as the cause for autosomal dominant cyclic neutropenia [ ] . deficiency of ela leads to regularly fluctuating levels of neutrophils [ ] . linkage analysis on affected pedigrees have shown that cyclic neutropenia and sporadic cases of this disease are due to a mutation in the gene for ela , located at p . [ ] . this enzyme is synthesized in neutrophil precursors early in the process of primary granule formation [ ] . a mutation in the ela gene was detected in one of three apparently autosomal dominant kindreds with familial scn. no mutations were identified in the apparently ar families [ ] . these results fit those showing that mutations were found in all five scn families [ ] , but they suggest that not all cases of autosomal dominant scn caused by mutations in ela [ ] . however, the high frequency of het mutations in the neutrophil elastase gene in sporadic scn confirms a previous report [ ] . considering that four novel mutations and a low-frequency polymorphism were detected, nearly all cases of sporadic scn may result from de novo het mutations in ela [ ] . in recurrent scn, an absolute neutrophil count of < cells/mm (or < . ¥ /l) [ ] oscillates with an approximate -day periodicity. circulating neutrophils vary between almost normal numbers and zero [ ] . in about % of patients with cyclic neutropenia, however, the cycles range from to days [ ] . in children referred during a -year period pids were as follows: cyclic neutropenia ( . %), shwachman-diamond syndrome ( . %), kostmann syndrome ( %), and chédiak-higashi syndrome ( . %). the mean absolute neutrophil count of children was . ± . cells/mm (range, - , /mm) at the first visit. the children first experienced symptoms of infection suggesting neutropenia at a median age of . months (range month to years), also suffering from oral ulcer, otitis, pneumonia, diarrhea, cutaneous abscess, and oral candidiasis [ ] . fever, stomatitis, and periodontitis and skin infections occur during periods when the neutrophil count is low. significantly diminished with slightly elevated superoxide production. mutational analysis of the patient revealed a glycine to serine substitution (g s) in the exon region [ ] . because the granulocytes without mpo cannot kill candida, some subjects, presumably carriers of a more extensive mutation and in association with other diseases, present severe and recurrent candida infections. the mpo defect can be diagnosed via a cytochemical investigation or a quantitative count of enzyme levels [ ] . neutrophil-specific granule deficiency is a rare autosomal dominant disorder characterized by recurrent pyogenic infections, defective neutrophil chemotaxis and bactericidal activity, and lack of neutrophil secondary granule proteins [ ] . the markedly decreased level of mrna expression for the bactericidal/permeability-increasing (bpi) protein, the activation factor pu- and defensins in these patients suggests a role for ccaat/enhancer binding protein (c/ebpη) gene in earlier phases of the myeloid differentiation program [ ] . c/ebpη is a member of the leucine zipper family of transcription factors, expressed primarily in myeloid cells [ ] . recessive mutations in the c/ebpη gene were described in one patient; analyses of the c/ebpη locus indicated that the disorder could have resulted from hz recessive inheritance of the mutant allele from an ancestor shared by both parents [ ] . loss of c/ebph function is the primary genetic defect in this disease [ ] . in a second individual lacking functional c/ebph, analysis of peripheral blood leukocytes revealed aberrant expression of cd , cd b, cd , cd , and cd on the proband cells [ ] . a male patient lacking neutrophilspecific granules died from complications of pneumonia at age [ ] . neutrophil-specific granules contain important microbicidal components (table . ) . among other deficiencies of oxygen-independent killing, this ar defect is characterized by severe recurrent bacterial deep-tissue skin infections without patients showing an increased susceptibility to a particular pathogen. they have defects in chemotaxis, disaggregation, and receptor up-regulation. deficiencies of the oxidoreduction and microorganism-killing mechanisms have also been described. the markedly decreased level of mrna expression for the bactericidal/ permeability-increasing (bpi) protein, the activation factor pu- and defensins in these patients suggests a role for c/ebph in earlier phases of the myeloid differentiation program [ ] . the defect is identified through a blood test colored with a wright reactive in which polymorphonucleates do not present the specific granules that normally contain lactoferrin. from a morphological point of view, the nuclei appear bilobated and the nuclear membrane may show intro-and extroversions. it is also possible to identify the membrane's lack of cyclic neutropenia is an autosomal dominant disorder in which cyclic hematopoiesis causes intervals of neutropenia and susceptibility to opportunistic infection. in nine families whose children displayed typical blood patterns, pedigrees confirmed dominant inheritance without evidence of heterogeneity or decreased penetrance; three pedigrees suggested new mutations [ ] . a wide spectrum of symptom severity, ranging from asymptomatic to life-threatening illness, was observed within the nine families. the phenotype changed with age. children displayed typical neutrophil cycles with symptoms of mucosal ulceration, lymphadenopathy, and infections [ ] . patients are usually asymptomatic, but during the period of severe neutropenia, recurrent overwhelming infections, inflammation, and ulcers occur in about % of patients and can lead to significant chronic morbidity [ ] . severe neutropenia was shown by children, moderate by , and mild by : of these children had leukopenia, anemia, thrombocytopenia, and monocytosis. during follow-up, respiratory infections developed in , oral manifestations in children. the most common infections, in descending order of frequency, were otitis media, abscesses, pneumonia, oral ulcers, acute diarrhea, cutaneous infections, oral candidiasis, and periodontits. sinusitis, cystitis, conjunctivitis, meningitis, and osteomyelitis were less frequently observed. hepatomegaly was also detected in children and splenomegaly in one; children died of recurrent infections. therefore, recurrent infections always deserve further evaluation for detecting such disorders [ ] . abdominal pain must be assessed aggressively because of the high frequency of clostridium infections during the period of severe neutropenia [ ] . during the course of scn, bm shows lack of maturation of granulocyte precursors beyond myelocytes, and there is myeloid hyperplasia during the remainder of the cycle. occasionally, there is a reduction in the severity of neutropenia and the accompanying infections over time [ ] . a complete clearing of symptoms and a significant increase in quality of life is noteworthy in children [ ] . however, while the disease is commonly described as benign, four children in three of the nine families died of clostridium or e. coli colitis, documenting the need for urgent evaluation of abdominal pain [ ] . pediatric cyclic neutropenia is effectively treated with rhug-csf (recombinant human g-csf), usually at doses of - mg/kg/day (median dose, . mg/kg/day) [ ] or twice weekly, or once a month. typically, children are noted in early infancy to have persistent scn with absolute neutrophil counts < . ¥ /l lasting for months or years [ ] . in children aged days to months, the initial and lowest median absolute neutrophil counts were . ¥ /l and . ¥ /l, respectively [ ] . usually, children suffer from long-term recurrent bacterial infections, and maturation arrest of myelopoiesis at the promyelocytemyelocyte stage of bm development [ ] . the disease begins during the st year of life, and its infectious complications include cellulitis, perirectal abscess, peritonitis, stomatitis, and meningitis, commonly as a result of infections with s. aureus, e. coli and pseudomonas aeruginosa [ ] . the numbers of circulating monocytes and eosinophils are often increased [ ] . missing the most important cells in the defense against bacterial infections, the neutrophil granulocytes, children suffer from episodes of severe, often life-threatening bacterial infections [ ] . they spend many days in hospital, requiring iv antibiotic treatment. recurrence of bacterial infections leads to irreversible tissue damage, for example in the lungs, requiring often disabling surgical interventions. a high incidence of significant bone mineral loss was seen in children with scn [ ] . the presence of qualitative and quantitative abnormalities of primitive myeloid progenitor cells expressing g-csfr may play an important role in the impairment of granulopoiesis in these patients, thus nearly all patients have a response to pharmacological doses of rhug-csf: neutrophil counts rise, infection rates fall, and mortality is reduced [ ] . since the introduction of rhug-csf, most children enjoy a normal life span and a greatly improved quality of life, although they still have problems with infections, especially chronic gingivitis and periodontitis [ ] . it is more likely that the bone loss was caused by the pathophysiological features of the underlying disease, but it is possible that rhug-csf accelerates bone mineral loss [ ] . prolonged administration of rhug-csf at a dose of u/kg bw twice daily may be associated with increased bone resorption, mediated by osteoclast activation and leading to bone loss. in children, the resulting osteopenia can be successfully managed with antiresorptive bisphosphonate therapy with significant improvement in bone density [ ] . a child maintained on long-term rhug-csf therapy developed acute myelogenous leukemia associated with a g-csfr mutation.after having undergone successful allogeneic bmt, both ela- mutation and g-csfr mutation became undetectable by pcr [ ] . shwachman syndrome, a rare ar condition, characterized by pancreatic insufficiency, reduced mobility and neutrophil chemotaxis, cyclic neutropenia, thrombocytopenia, metaphyseal dysostosis, delayed growth and recurrent pyogenic infections, in two cases was associated with isolated gh deficiency [ , ] . in addition to metaphyseal chondrodysplasia, neutropenia, and pan-poor to absent granuloma formation [ ] . salmonella and certain viral infections [hsv, cmv, parainfluenza, and respiratory syncytial virus (rsv)] are also seen [ ] . most patients bearing an ifn-gr deficiency present gross mutations that truncate the protein and prevent its expression, giving rise to severe mycobacterial infections and, frequently, a fatal outcome [ ] . mortality in these children is high, and infections are severe and recurrent [ ] , as in an -year-old girl before receiving a bmt [ ] . a point mutation may be fatal: an individual, probably hz for the mutation, died from meningitis due to mycobacterium bovis [ ] .a hz missense ifn-gr mutation was identified in two siblings who did not respond to low or intermediate concentrations, yet responded to high ifn-g concentrations, probably for a reduced affinity of ifn-gr for its ligand [ ] . otherwise the mutation results in normal surface expression of ifn-gr that do not bind ifn-g [ ] . a dominant deletion in the ifn-gr gene has been reported in a female patient hz for a -bp deletion in exon of ifn-gr who developed postvaccinal disseminated bcg infection [ ] . the ar form of partial ifn-gr deficiency was reported in patients of unrelated kindred with susceptibility to mycobacterial infection [ ] . an -year-old girl with ifn-gr deficiency, also with recurrent mycobacterial infections and liver cirrhosis with portal hypertension, received red cell-depleted bmt from her hla-identical sister. the transplantation course was uneventful and years later the child remains in excellent clinical condition and free of mycobacterial infections [ ] . a complete ifn-gr deficiency was found in a child due to a hz dinucleotide deletion resulting in a premature stop codon in the protein extracellular domain. this gene defect emphasizes the critical role that ifn-g plays in host defense against mycobacteria [ ] . a girl with bcg and salmonella enteritidis infection and a hz recessive deletion in the p subunit of il leading to a complete il p deficiency has been reported. a large hz deletion within the il p subunit gene was found, precluding il p (composed of p and p subunits) functional expression by activated dcs and phagocytes. the net result was a markedly impaired ifn-g production by lymphocytes. however, addition of recombinant exogenous il p in the assay was able to restore normal ifn-g production in vitro [ ] . the girl suffered from well-organized granulomas, possibly due to residual il -independent ifn-g production [ ] . another kindred [ ] and two siblings and one unrelated patient [ ] carried the same large deletion, also accompanied by disseminated infections. a -year-old female was repeatedly hospitalized since the age of weeks for recurrent episodes of pneumococcal pneumonia with sepsis and other infections in the absence of creatic exocrine insufficiency, the findings in children are noted as variable extremity shortening, cup deformation of the ribs, metaphyseal widening and hypoplasia of the iliac bones, and increased echogenicity of the pancreas with no change in size [ ] . recurrent infections begin during the st year of life and commonly involve the sinuses, lungs, bones, skin, and urinary tract [ ] . neutropenia, either cyclic or intermittent, occurs in all patients, and %- % of patients also have pancytopenia [ ] . immune functions may be involved in this syndrome, including marked pan-hgg, especially of the iga, normal/increased cellular immunity, but depressed humoral and nk cell immunity [ ] . in patients diagnosed in infancy, a significant growth improvement and a decreasing frequency of infections were observed over time, in addition to improvement or normalization of exocrine pancreatic function [ ] . a continuous spectrum from systemic bcg infection to local recurrent nontuberculous mycobacterial infection covered by the clinical features of affected children has recently helped to identify several genetic defects in the monocyte-macrophage-th t-cell pathway [ ] . different types of mutations in four genes (ifn-gr , ifn-gr , il p , il rb ) forming the ifn-γ/il axis [ ] have revealed both allelic and nonallelic heterogeneity and result in different disorders whose common pathogenic pathway is impaired ifn-g-mediated immunity [ , ] . several children have been reported who presented a new kind of hereditary id with severe and/or recurrent infections caused by only one microorganism family, in opposition to other patients with classic pid. five new syndromes may encompass these children with a genetic predisposition to infectious diseases. if the ifn-g/il axis is impaired, the host becomes highly susceptible to infection with organisms that replicate intracellularly (susceptibility to mycobacterial disease). stat- (signal transducer and activator of transcription- ) deficiency predisposes to viral disease, nemo and irak- (il r-activating kinase- ) deficiencies predispose to infections caused by pyogenic bacteria [ ] . this pid encompasses several defects: complete, partial, and ar ifn-gr deficiency, and complete, partial, and ad ifn-gr deficiency [ ] . ifn-g and the cellular responses induced by it are essential for controlling mycobacterial infections. patients with ar mutations leading to complete loss of ifn-gr or ifn-gr expression have the most severe phenotypes, and they present early in life with disseminated severe infections, especially if they have received bcg vaccination, and have fever. she exhibited il deficiency that was associated with an abnormality of the il p gene. although present, ifn-g was reduced [ ] . a genetic lack of il rb surface expression predisposes to severe infections by pathogenic mycobacteria or salmonella and causes strongly decreased, but not completely abrogated ifn-g production [ ] . the deficiency may be complete as well as partial [ ] . several patients with these features have been reported [ ] . three unrelated individuals with severe, idiopathic mycobacterial and salmonella infections were found to lack il rβ chain expression. il rβ sequence analysis revealed genetic mutations that resulted in premature stop codons in the extracellular domain [ ] . a patient with severe infections as above and multiple adverse drug reactions had t cells unable to produce ifn-g or proliferate in response to il , despite the expression of wild-type il rβ and il rβ [ ] . defective il r signaling leads to low t-cell and nk-cell ifn-g production [ ] . il rb and il rb chains are associated in an ar deficiency with susceptibility to mycobacteria and salmonella infections [ ] . the stat ( forms, ad and ar [ ] ) s mutant failed to restore ifn-g production in stat -deficient il rb transgenic cells [ ] . stat , - , and - activation by il was lost, an impairment specific for il ; nor is activation of stat alone sufficient for il -induced ifn-g production and proliferation [ ] . two unrelated infants hz with respect to mutated stat suffered from mycobacterial disease, but unlike patients with ifn-gr deficiency both died of viral disease [ ] the complement is an integral part of the humoral defense system against infections and also for promoting inflammatory process (figs. . , . ). complement deficiency was found in / dutch patients ( . %) over a -year period ( . % × year) [ ] . from the study of blood donors the prevalence may be of . % in the general population [ ] . congenital deficiencies have been described for most of the proteins it is composed of (tables . and . f [ , , , ] , usually following the ar model. properdin deficiency is the only complement deficiency that is x-linked [ ] . hets can be easily identified because their relevant component is present in the serum with a % concentration. the lack of one component at the hz level serologically involves the blockage of enzyme release below and the absence of hemolytic activity, while that of controlling proteins causes its uncontrolled activation, consuming the factor that is the object of control and, in various ways, also of successive components [ ] . nonfunctional c q variants have been observed, c r and c s deficiencies are often associated, probably because they are mapped on contiguous genes of chromosome (c q on ) [ ] . the b, c and c genes, situ-ated on the short limb of chromosome , constitute along with others the hla class iii (chap. ). c and c are codified on chromosome p and have a similar structure; c shows a different structure, because the molecule consists in three a, b and g chains, united to form two subunits, a-g and b dictated by different genes [ ] . alternative pathway deficiencies are extremely rare [ ] . complement deficiencies are accompanied by an increased frequency of infectious pathologies [ ] , although it is not rare to come across them in individuals who are apparently in good health, as in the case of c hereditary deficiency [ ] . also frequent are teens and young adults with autoimmune manifestations (chap. ). classic pathway deficiencies are often associated with sle-like diseases (systemic lupus erythematosus), id of the early components of complement (c -c ) are associated with risks of infections caused by encapsulated bacteria such as streptococcus pneumoniae, haemophilus influenzae type b, as well as by meningococci [ ] . the incidence of sle in patients with c q, c , or c deficiency is %, %, and %, respectively [ ] . partial c deficiency is also associated with sle; % of patients with sle exhibit c a deficiency [ ] . several components are associated with development of membranoproliferative glomerulonephritis (table . [ , , , ] ). in alternative pathway id, the infections recognize pyogens as the most common etiological agents, while final common pathway id (c -c ) or properdin (p) have been associated with recurrent or invasive infections by neisseria (n) gonorrhoeae or n. meningitidis, gramnegative bacteria, and asplenia, agammaglobulinemia [ , , , ] . it is estimated that the frequency of meningitis in subjects with hz deficiency of the final c -c pathway is %, , -fold higher than in non-id individuals [ , ] . some characteristics appear to associate the patients with complement deficiency and meningococcal disease: frequent recurrent episodes, an older age at the first onset, lower mortality compared to patients with a normal complement, and a prevalence of males [ ] . over patients with c q, c r and c s deficiencies have been described, c s deficiency only in two cases [ ] . a selective and complete c s deficiency in a -year-old girl with complex aids including sle-like syndrome, hashimoto's thyroiditis, and autoimmune hepatitis has been reported. exon-specific amplification of genomic dna by pcr followed by direct sequence analysis revealed a mz nonsense mutation in the c s gene exon xii at codon . both parents were het for this mutation [ ] . a deficiency in one of these proteins is sufficient to block the classic pathway activation; deficiency results as a consequence of non-synthesis, which in the unlike c , hz c deficiency is very rare and is caused by the non-expression of all alleles ( of c a and of c b, maternal and paternal alleles), which can occur due to punctiform mutations, gene deletions, or other gene alterations that prevent gene transcription [ ] . the two a and b genes are polymorphous, just like c , c , c , c a and c b and the b factor (bf); polymorphic variants of the other proteins are rare, with different alleles for c a and for c b identified at the moment. moreover, two loci c a and c b null alleles q (quantity ), do not codify for any phenotype, although often present in the general population [ ] . in a -year-old caucasian child who suffered from several bouts of pneumonia caused by respiratory viruses, eight episodes of acute otitis media, prolonged respiratory and urinary tract infections, molecular studies of the c gene region revealed hz deletion of hla class iii cyp a-tnxa-rp -c b, generating total deficiency of case of c q amounts to % of cases, while in the remaining % the molecules are malfunctioning but cross-reacting with the native molecule [ ] . c r and c s deficiencies are usually combined, due to the contiguity of the two genes; typically in these patients c r is absent and c s levels are reduced ( %- %) [ ] . affected patients suffered from a sle-like syndrome and sporadically from an extended predisposition to infections [ ] . associated symptoms in c q deficiency are sle-like syndrome, rheumatic disease, and infection. several children suffered from meningitis, recurrent septicemia, recurrent otitis media, pneumonia, and stomatitis; two died from meningitis septicemia [ ] . the incidence is based on the data from [ ] ; the inheritance is always ar, with the exception of c -inh deficiency (autosomal dominant) and factor p deficiency (autosomal recessive or x-linked). data from [ , , , ] . gn glomerulonephritis, jra juvenile rheumatoid arthritis. c b and the flanking ¢ region up to c a [ ] . moreover, in / cases the c a*q alleles were related to a c a/cyp p gene deletion within the hla-b c c bfs c aq b dr haplotype. in / cases, the c b*q allele was related to a c b/cyp p gene deletion within the hla-b c c bff c a bq dr haplotype [ ] . the c null allele incidence is so elevated that % of the population expresses all c genes, while % lacks - alleles [ ] . the elevated number of q probably derives from the marked similitude of the two genes, which facilitates the unequal crossover, but this crossover in the hla can modulate the expression of three c a alleles and one c b or vice versa; the c aq allele spreading amplifies the risk of contracting sle and juvenile ra (jra). hz c deficiency, the most common in the caucasian population, has an incidence that varies between : , and : , , whereas the het carrier rate is . % [ ] . it is usually found in the a , b , dr , bfs, c q , c a, c b haplotype context which, due to its considerable rarity, could assume a predictive value [ ] . two different types of c deficiency are known: in type i the synthesis is deficient due to the protein nontranslation, in type ii there is a selective absence of secretion but not of synthesis, therefore c levels are . %- % of normal values [ ] . hets have a nonfunctioning gene, the complement profile is characterized by serum c concentrations equal to % of normal values; about % are asymptomatic, while the other half exhibit frequent infections and quite a few suffer from sle and correlated syndromes [ ] . het c deficiency was associated with a -bp deletion in the c gene (type i), mainly within the hla-a b c q bfs c a b dr haplotype [ ] . in a certain number of cases, a c deficiency is accompanied by a partial bf malfunctioning, genetically close to it. hzs can also have a deficient function of the alternative pathway [ ] . possibly this deficiency, like other quite common ones, may not always be reported in the literature: the total number of cases therefore underestimates the real prevalence, as is also found in children with c deficiency [ ] . c deficiency must be suspected in all patients presenting pneumococcal infections after the age of years [ ] . both c and c predispose to sle, but this is not the expression of a particular genetic association caused by the same gene localization, because c q, r, s, deficiencies, which also cause sle, are situated as mentioned outside the hla system [ ] . the molecular bases of this pid appear heterogeneous. the c gene exists in different allelic forms, some of which have reduced functionality. one must remember the c important role in immune responses, also as far as apc and b cells are concerned, as well as the defensive role played in innate immunity along with c . since both pathways converge in the cleavage and activation of c , there is no way that this defect can be corrected, and furthermore the opsonic power is greatly deficient, as is the c chemotaxis; therefore patients affected by hz deficiency mostly present clinical symptoms totally similar to a congenital hgg with severe recurrent infections and at times the symptoms of cic disease [ ] . although id is severe, some patients apparently remain in good health and the syndrome may also in time become less severe, probably due to the higher number of immune experiences that allow a better effector function to antibody reactions mediated by the fc receptor [ ] . c protein was defective in noninfected nigerian children with protein-energy malnutrition (pem), but rose significantly in the presence of bacterial infection, thus sharing the values found in healthy controls [ ] . in its clinical expressions, c deficiency does not differ from the other deficiencies discussed here; the clinical consequences of absent c a anaphylotoxin are unclear [ ] . one-fourth of all patients are asymptomatic. in caucasians incidence is : , [ ] . deficiencies associated with c -c are rare but reflect the close genetic proximity of their pertinent genes; in c -c deficiencies, % of patients lacking one component experience at least one severe episode of neisseria infection and . % one aid [ ] . rare in europe, c deficiency is the second most common complement deficiency in the japanese ( . %) [ ] . in italian children it has a prevalence of % and has been identified also in healthy siblings [ ] . in japan c deficiency is more associated with meningococcal meningitis than with neisseria infections [ ] . in a highly inbred arab population, a c deficiency was associated with a mutation (g c) that is also prevalent among israeli jews of moroccan ancestry [ ] . a total deficiency of this -kd protein, inherited as an ar trait, has been described in a young patient with a hemolytic-uremic syndrome and in one case in italy whose parents were first cousins [ ] . among relatives of the proband studied, encompassing generations, ten had low factor h levels, including her two children, indicating a het factor h deficiency [ ] . h deficiency results in uncontrolled breakdown of c , and in depletion of bf, p and c [ ] . c and c components are decreased in varying degrees, while c and c are found in plasma in traces and only as activated molecules [ ] . inheritance of factor d deficiency is for the moment uncertain [ ] (table . ): a partial deficiency ( %- % of normal concentrations) has been described in two mz twins, and a total deficiency in one male. this deficiency, serologically characterized by the non-functionality of the alternative pathway, is clinically accompanied by an increased susceptibility to neisseria infections [ ] , leading us once more to emphasize the alternative pathway significance as a substantial means of defense for a broad spectrum of damaging actions caused by bacterial infections. properdin deficiency is the only one inherited as a characteristic linked to the chromosome x and only affects males [ ] . at the moment, > cases have been described. the deficiency can be materialized by a total p absence, with levels reduced to %, with normal levels, however, showing an altered functional activity [ ] . specific research has shown a remarkable reduction of c a and b titers, which represent the c -convertase proteins, with a consequent heightened consumption due to the alternative pathway spontaneous activation. males are affected by septic episodes caused by neisseria, sometime fulminating, with onset even occurring during the st year of life [ ] . there is no evidence of increased susceptibility to cic diseases or infections caused by other organisms [ ] , thus implying that a functioning alternative pathway is particularly important for a defense against infections [ ] . correctly identifying children with complement abnormalities is important and worthwhile if any of the following factors are present: id (such as repeated or unusual infections with other organisms, fh, unusual course of the illness, etc.), repeated neisserial infections, infection with an unusual serogroup, fulminant disease in males (p deficiency), coexisting angioedema, autoimmune, or connective tissue disorders [ ] . in the two forms of c deficiency (c a + c g and c b mapped on chromosome ), the subunit not involved is present in the serum, also with reduced levels, and accompanied by altered functionality of the one involved. c deficiency has different characteristics due to the diverse associations of the b and a-g chains; therefore caucasians with this deficiency lack the b chain ( %), while colored patients lack the a and g chains ( %) [ ] . c deficiencies are poorly considered because they are often asymptomatic [ ] ; however, in japan there is an incidence of . % [ ] and between % [ ] and % of cases [ ] present meningococcal meningitis. congenital c deficiencies are very common among the japanese ( . %- . %) and represent . % of all deficiencies (table . ). there have been > cases of congenital c -c deficiencies reported, distributed unevenly between the various ethnic groups: c and c deficiencies are prevalent in colored patients and the c deficiencies appear to be more common in caucasians [ ] . an analysis of published studies shows that % of patients with sporadic meningococcal infections may have a c -c deficiency [ ] . clinical patterns are overlapping. the most common complement deficiency is c -inh deficiency (c inhibitor), responsible for hereditary angioedema that causes symptoms in hets (chap. ). at least cases of factor i deficiency are known [ ] , with autosomal co-dominant transmission because parents show normal complement levels at % of factor i [ ] . as in an h deficiency, serologically one has the alternative pathway activation, due to c b non-catabolization that continuously forms c conversion with severe c deficiency, the levels of which do not exceed % of normal values [ ] . in hzs, in addition to subsequent pyogenic infections, as in c deficiency [ ] , cutaneous rashes and urticaria caused by massive release of histamine and pro-inflammatory cellular products by anaphylotoxic fragments are reported [ ] . the fact that a child during the initial period of life should experience a certain number of urtis or lrtis is within the norm: rris are mainly caused by immunological immaturity or inexperience, both transient. a typical symptom outline is difficult to define, and prevalence is also little known. in id children, a basic pathology is present instead, which encourages the recurrence of infections. although a distinction between infection and associated disease is important, childhood infections might have a key role in stimulating the maturation of the immune system, and the microbial burden in early life has been invoked as a protective factor against wheezing and asthma (chap. ). preschool-age children have an especially high frequency of vris, with most having three to eight infections per year and %- % have ≥ vris per year. the rate in children aged - has been about in children compared with about in for children aged - , and about in , for those aged - [ ] . rris may be defined as >six episodes of urti and/or > lrtis in the previous year, or based on age and ≥ episodes per year if aged < or ≥ episodes per year if aged ≥ . several risk factors can influence the onset and recurrence of infections [ ] . it is clear that the younger the child is, the more he/she may fall ill: this is also related to serum ig levels (table . ). the dogma of primary and secondary responses also may not apply to infections, at least those caused by rotavirus, which confers considerable protection only after various infectious events and in children aged [ ] . environmental factors in the absence of a basic pathology are important. it is also obvious that the more crowded the environment the child lives in, the more probable that infection becomes. in addition to the number of siblings, other factors such as socioeconomic status, age (preschool children), contact with outside persons, especially babysitter, early social contacts, exposure to passive smoke, indoor and outdoor pollution may be found to be related to the hygiene hypothesis (chap. ). however, daycare attendance, which was considered to be an indicator of exposure to respiratory pathogens, and the presence of siblings, increased the risk of urti in preschool children aged - [ ] , and in the st year of life for children with fha [ ] . among children with fha, the protective effect of day care attendance in early life against the development of atopy only begins by years, and against wheezing this may not be observed until after years [ ] . the particular ease of smoking parents and/or relatives who fall ill with influenza has been known for some time, to the same extent that the children who live with them are affected by rris (table . ). the impact on rri incidence as it is related to children in kindergartens, has been reflected by a significantly increased morbidity observed in babies aged months to years who go to daycare, who show a number of more severe and longer lasting infections per year [ ] , with an incidence of . % in children who stay at home compared to . % of those in kindergartens [ ] and an increase of % of ome persistence (chap. ). in children exposed to cigarette smoke, the risk increased -fold for lrti [ ] or by . -fold, equal to ≤ episodes of respiratory infections each year [ ] . studies on environmental pollution have identified the most damaging agents: conclusive data on fine particles in suspension and polluting derivatives is available, proving a significantly increased risk of infantile rris: table . indicates that no reduces immune defenses against rtis, provoking alterations of the epithelium and of the lymph node cells, with negative effects on mucociliary clearance and macrophages. the biological role played by no in the domestic pollution derived from the home has been ascertained to be related to cooking and the smoke released by combustion [ ] . using wood for heating leads to so development, while radiators cause the air to dry, which in turn causes potentially infected particles to remain in suspension. pollutants are increasingly responsible for indoor pollution (chap. ). although levels of micro-pollution are not easily ascertained, significant associations with acute rris and conditions such as polypnea and dyspnea have been reported, especially in < year-old children [ ] . these children's capacity to evoke adequate responses is genetically controlled; however, it is commonly known that their parents or siblings have suffered similar illness as children. in subjects with physiological immaturity of the immune system, vris more easily cause infectious episodes, which are important factors to be considered only in the presence of recurrent or incompletely cleared conditions [ ] . predisposing factors related to a basic pathology derive from perinatal factors, more common in premature babies, which can lead to respiratory tract alterations and consequently to bronchopulmonary dysplasia; anatomical anomalies; cystic fibrosis, which can become recurrent pneumonia; adenoiditis causing otitis and ome; congenital ciliary dyskinesia; humoral deficiency and pid characterized by recurrent sinopulmonary infections [ ] (table . ) [ ] . viruses are the principal etiological agents, and over a novel member of the coronavirus family has been characterized, which is associated with cases of sars (severe acute respiratory syndrome). phylogenetic analyses and sequence comparisons showed that sarscoronavirus is not closely related to any of the previously characterized coronaviruses [ ] . as investigated by a -year study the overall prevalence is age-related, and different between children aged - ( fig. . ) [ ] and those aged - ( fig. . ) [ ] : rsv and rhinovirus have a different impact on the first group ( % vs %) and the influenza viruses on the second group ( %- %). incidence in young children was . -fold higher than in those aged - [ ] . rsv causes bronchiolitis in breast-fed babies, with a higher frequency the younger the child is (tables . , . ) , and rhinitis in older siblings. even an infectious agent neglected for some time, such as ureaplasma urealyticum, causes a lung pathology in younger children while sparing those > years old [ ] . the bacteria most commonly involved are: streptococcus pyogenes (pharynx and larynx); haemophilus in- complement deficiency fluenzae (middle ear and larynx reaching the epiglottis); and streptococcus pneumoniae (middle ear). there are often secondary bacterial infections such as complications caused by vris, which certainly contribute to recurrent infections and/or the onset of chronicity [ ] . in chap. we reported several studies which concluded that early infections may protect from atopy development (hygiene hypothesis). we must distinguish pid outlines and pseudo-id in children with rris. summarizing the aforementioned, severe and recurrent lrti and sinusitis are the principal clinical manifestations in children affected by deficiencies prevalently involving humoral immunity (table . ). these children fall ill during the first weeks of their lives and often contract infections caused by opportunistic agents, fungi, protozoa and viruses, and as months go by are also affected by malnutrition and failure to thrive. episodes affecting the airways, particularly common in cellular and combined id, tend to become longer or severe, especially if complicated by pneumonia [ ] . chronic disorders such as sinusitis and bronchiectasis (sinobronchial syndrome) are not rare; interstitial pneumonia is in most cases caused by pneumocystis carinii and results in tachypnea and lung hyperinflation [ ] . infections supported by the herpes simplex, ebv and cmv are also common: males with xlp exhibit a deficient response to ebv [ ] . severe and recurrent sinopulmonary, was, higes and ata infections as well as severe rris in cgd, complement deficiencies and lad to v must also be borne in mind. cases of recurrent pneumonia should be warning signals to rule in . % pediatric cases of pids [ ] . the second most important manifestation is chronic diarrhea: in some cases the infections are caused by rotavirus and enterovirus among which the echo: giardia lamblia, salmonella and campylobacter can also cause chronic enteric infection; malabsorption resistant to treatment can be ascribed to cryptosporidium [ ] . rris are common in children. they reflect the immaturity of the immune system in its encounter with environmental antigens; this developmental delay during the first years of life fosters the development of rris. thus, rris are part of the growing-up process of any child [ ] . the consequences of rris can be of a profound and sometimes protracted alteration of the different immune defense mechanisms, which place the child in an undefended position, similar to the condition observed in children with pids, compromising the phagocytes, lymphocytes, nk cells, antibody production, and ils at every occasion [ ] . the responsible viruses for these infections have a development limited to surface mucous cells, spreading from cell to cell due to contiguity, while the viremic stage is absent or remains marginal. the incubation period is therefore brief, normally < days; consequently the immune response may not be capable of ensuring a protective function, or it only intervenes partially, clarifying the potentially unlimited number of infectious episodes [ ] . from a pathogenetic point of view, the virus works by triggering the development of ige and allergic sensitization and/or damaging the immune structures [ , ] . in the first case, it is known that experimental infection in mice with rsv is capable of significantly increasing the absorption of ovalbumin (ova) administered by aerosol, of igg, ige and anti-ova siga (fig. . ) and of increasing the synthesis of ige and specific igg to ragweed also administered by aerosol. the result is that the majority of infants become infected with rsv, although lrtis develop in only about % [ ] . approximately %- % of those subsequently experience recurrent acute asthma from vri [ ] . the mechanism by which vris induce atopic sensitization in experimental models is identified with antigen penetration and sige synthesis [ ] . studies show that viruses increase mucosal permeability, by modulating antigen uptake and altering antigen processing by the mucosa, which results in the ige-suppressor t-cell depression, while ifn-g-modulated histamine release further increases mucosal permeability [ ] . it is probable that the immune deficiency is secondary to vris, because many viruses are capable of inducing transient [ ] . the lack of nk cells, which are in the front line of defense against viral infections, and with which the altered production of il and factors activating the phagocytes are associated, appears significant, but it is unclear whether the deficiency is primary or secondary to viral infections [ ] . the basic question with potential therapeutic consequences therefore remains unanswered, and is probably destined to remain so until more sophisticated tests are available to clarify this issue, although the nk-cell reduction in these children corroborates the first hypothesis. recent data emphasizes the important defensive activity of cytotoxic cd t cells and by different ils, including il , il and il active in antiviral defense: if the infected cells express on the surface the viral antigen processed in association with hla class i molecules, cytotoxic cd take care of killing the cell. normally the cd are activated by the virus itself or by soluble factors released by the infected cells and mediate the cellular lyses after recognizing hla class i antigens on the same cell. however, if the apc is a macrophage, it can be parasitized by the virus, with consequently reduced chemotaxis and microbicidal activity and in particular the capacity to cooperate with t cells [ ] . considering that macrophages may have evolved specific mechanisms for directing t-cell development toward the th , since cmi can solve some infections, it is clear that their dysfunctions appear in the insufficient nk cell activation and inadequate th development in response to infections. ifn-g, produced not as a direct consequence of infection, but probably by il and/or il , stimulates the nearby cells to block the nucleic acid transcription and therefore the viral replication, preventing the infection of those cells to which virus spreads due to contiguity. therefore the ifn-g species-specific antiviral activity takes place at the very first stages of the infection, preceding the antibodies [ ] . the response to germs' capsular polysaccharides, particularly deficient until the age of , although encounters with these germs are abundant during this period of time, still remains to be evaluated [ ] . however, selected children, although normal from an immunological point of view, may have a deficient antibody response even aged - [ ] with a percentage of modifications of both humoral and cmi, therefore not only of antibody synthesis or phagocyte and neutrophil functions, but also of t lymphocytes and related ils [ ] . it is also possible to hypothesize that persistent vris are capable of inducing th activation by antigens or super-antigens: th t cells with il help induce virus-specific cd + to produce il , which recruits eosinophils in the respiratory tract, thus reducing ifn-g secretion. thus there may be an increased interaction of ige mast cells in these subjects with immunoregulatory alterations, due to a marked lymphoproliferative response and the elaboration of other ils, which consequently amplifies ige production in the respiratory tract. the ige response is thought to lead to a greater production of bronchoconstrictor mediators by effector cells; viral infections themselves may induce these cells to release histamine. it is also known that humoral deficiency, especially of iga, very often opens the way to gram-positive germs causing viral and bacterial infections, thereby completing the circle [ ] . interestingly, only / children have at least a significant production of antigen-specific salivary iga against klebsiella pneumoniae [ ] . even if the alterations are transient and aspecific in children with rris, their occurrence and persistence for a number of months, for a year and even longer, leads to believe that the immunosuppressive mechanisms set off by the first episode occasion more severe, profound and lasting consequences for the immune functions than those occurring in their normal peers [ , ] . having at least one physician-diagnosed lrti in the st year of life was significantly associated with recurrent wheezing (or, . ) and asthma (or, . ) [ ] . at the base of this exclusive predisposition in children for contracting rris, there is a nk cell reduction [ ] and immune deficiencies related to the global lymphocyte population, the cd and the cd :cd ratio, unbalanced toward cd t cells, prevalent in children expressing coughing compared to those with bronchial hyperreactivity (bhr) [ ] . other t-cell deficiencies in children with humoral anomalies include a considerable spontaneous production of il and il , both generated by th phenotypes, or alternatively by the th , which express both ils [ ] . it is similarly feasible that virus-specific cd deprived of cytolytic activity are converted into th -like t cells when il is present [ ] . cmi in these children consists above all in the transient t-cell numeric and functional depression, coinciding with a deficiency of ils necessary for their activation, proliferation and differentiation. the virus toxic effect also acts directly on the t cells, inciting rough structural modifications including giant polynucleate cells, which jeopardize homing and recirculation capacities, to the point of immunosuppression, affecting the specific lymphocytes for that particular virus, thus favoring the attacking organism [ ] . a condition of immunosuppression occurs also as a result of superantigen (sa) orchestration, which, stimulating a large num-nonreactive children of %- % [ , ] , rising to %- % if with an iga and/or igg subclass deficiency [ , , ] . while % of children have low antipneumococcus igg , antibody responses are totally absent in % of dysimmunoglobulinemics with a virtual absence of iga and igg [ ] , confirming previous data [ ] . iga and igg -deficient children show a clinical pattern with elevated susceptibility to s. pneumoniae infections [ ] . von waldeyer ring (nalt) is a significant constituent of immune response, and of resistance to nalt-dependent infections. among the consequences of chronic adenotonsillitis (table . ) the statistically significant decrease in ig levels, including siga, must be evaluated eventually in relation to rri complications (chap. ). igg subclass deficiencies can be present in children with chronic and/or severe asthma, associated or not with sigad, in children affected or not by asthma with severe rris or with chronic nonallergic respiratory clinical symptoms, in children with sigad, ata, was, cvid, scid and in healthy subjects [ ] . the anti-virus antibodies generally belong to the igg and igg isotypes while the igg protect from microbes with polysaccharide antigens, such as s. pneumoniae and group a and type b h. influenzae [ ] . table . shows normal values for igg subclasses in subjects aged - . unlike total igg concentrations, igg and igg reach normal levels within the st year, igg mature more slowly, ensuring an effective antibody response only after the nd year, and igg develop even more slowly. various authors believe that the role played by igg subclasses is unique and vital in defending from infections: igg deficiency is associated with an increased susceptibility to infections by bacteria expressing capsular polysaccharides, such as pneumococci, meningococci, h. influenzae, bordetella pertussis, etc., as well as other factors capable of setting off an inflammation [ , ] . igg deficiency instead seems associated with a marked predisposition for rris [ ] . however, undetectable igg subclass levels are a common finding in normal individuals and an accurate detection of very low levels of igg is technically difficult to achieve [ , ] . babies aged ≥ month have levels of circulating lymphocytes secreting all four subclasses in a higher number than adults; therefore the capacity to produce antibodies exists well before a full humoral response is developed. subnormal igg subclass concentrations, especially of igg , are also observed in healthy children who do not present an increased susceptibility to infections. low subclass levels do not necessarily indicate that the subject will experience immune disorders exclusively linked to these, nor do normal concentrations guarantee that the child will be spared complications. sub-jects have been observed both with normal igg levels and with recurrent infections and with a subclass deficiency, which often do not form other types of antibodies [ ] . igg subclass determination does not indicate what the humoral level restricted to that molecule is: this is a characteristic in children, unlike adults, even though research was carried out using the same methods in the two studies [ ] . interesting hints come from studies involving children with rris: of children aged - , with a confirmed diagnosis of susceptibility to infections, / ( . %) had a igg subclass deficiency, almost all concerning igg , with several associations [ ] . in other children, the selective igg deficiency was statistically significant compared to atopic controls without rri, associated not so much with a well-defined state of id as to a respiratory tract defense mechanism deficiency, in view of the fact that the relative prevalence of this subclass in secretions may indicate a role in the mucosal defense [ ] . other children with typical symptoms of recurrent infections, lymphadenopathies, failure to thrive and hgg exhibited low igg levels, confirming that normal levels of total igg do not exclude a subclass deficiency [ ] . in a cohort of young babies, igg deficiency was only present in / subjects ( %) [ ] ; however, the absence of a control group makes the results incomparable. an igg subclass deficiency is therefore able to induce or worsen chronic respiratory symptoms in allergic and nonallergic children, especially if predisposed to developing these affections [ ] , or in subjects with sigad [ ] . with time these deficiencies, and eventually also those associated with iga, can normalize [ ] . in conclusion, transient and persistent iga and/or igg deficiencies have been reported in a small percentage of asymptomatic children [ ] , but even if iga and igg subclasses are not always required as such for a normal immune response, their deficiency may predispose to rris [ ] . as previously illustrated in chap. , the close links between atopy and rris are known, and this is confirmed by the observation that asthmatic children have a higher incidence of rris than their nonasthmatic siblings. it has been known that rris during the early periods of life can play a role in the development of bhr and atopy: in the classic study by frick et al [ ] , in out of allergic children sensitization was propitiated by rris. with continued observation, the authors noted the presence of high ige levels, positive rast and histamine released by leukocytes after infections [ ] . in a cohort of asthmatic children aged . - . , the affected by th week of pregnancy is possible, so as to evaluate the immune phenotype in the fetal blood [ ] . wccs (white-cell counts) in cb and differential counts can be used to detect the lymphopenia that is commonly present in infants with scid. however, subset analysis by flow cytometry is necessary to enumerate t, b and nk cells. subsequently, scid diagnosis will be suspected when overwhelming opportunistic infections occur [ ] . depending on whether one suspects a humoral, cellular or innate immune deficiency, we begin with the algorithm in table . [ ] , positive if infants or children have ≥ of these signs, then , ] on laboratory tests can be consulted. however, children with variable levels of antibody id may end up with different diagnosis [ ] . children with higms presented initially with a history of an increased susceptibility to infection including pneumocystis carinii pneumonia [ ] in % of children [ ] . in pids affecting phagocytes, because of the relatively narrow spectrum of disease-specific infections (such as aspergillosis in cgd), careful attention to the microbiology laboratory early in the course of evaluation of a patient suspected of having a pid is crucial to orient the work-up in the appropriate direction [ ] . in a male newborn referred to hospital at days of age for fever, hemodynamic failure and an inflammation syndrome caused by pulmonary infection, culture of tracheal, bronchoalveolar lavage samples and lung biopsy grew positive for a. fumigatus, enabling the diagnosis of cgd [ ] . to investigate whether patients with undiagnosed id could be identified in diverse inpatient hospital populations, a scoring algorithm and computer screening method was updated [ ] on the basis of icd- codes to survey the discharge diagnoses of all hospitalized patients over periods of time. thus id patients were identified, eight of whom were children aged - ( %), two with neutropenia, two with igg deficiency, one with lad, one with dgs, etc. [ ] . we also suggest including congenital phagocytic defects in the differential diagnosis of recurrent bacterial or fungal infections in a child [ ] . a congenital complement deficiency should be suspected if levels of even one component are reduced [ ] . early diagnosis is essential for choosing the necessary treatment [ ] . the differential diagnosis of pid will emphasize the different characteristics schematized in table . , to which one must add objective rarity, while fh and child gender become important [ ] . in subjects suffering from omenn syndrome, was, severe combined and cellular ids, the screening of clinical symptoms may be useful at birth and during the first few months after birth (table . ) [ , , ] . the localization of infections is multiple, the id child usually appears to be ill, and the peripheral lymph nodes and lymphatic rris had a higher incidence of fh positivity (p= . ), increased ige (p= . ), as well as a combined iga (p= . ) and igg (p= . ) deficiency [ ] . ige hyperproduction could be the result, not only of the wellknown association between igg subclasses and ige and their coregulation of il expression [ ] , but also of a virus-caused unbalanced cd :cd ratio [ ] . these results link atopy to rris, confirming that the state of chronic inflammation and bhr induced by allergic sensitization is an ideal substratum for the adhesion and chronic evolution of bacterial and/or viral infections. there are no specific clinical outlines for rris. on the contrary, symptoms are extremely varied, with, as previously mentioned, infections caused by bacteria and viruses. urtis are common at age . during the last months, . % of the children experienced more than one bout of acute otitis media, . % had more than one pharyngotonsillitis episode, . % contracted > common colds, and . % had rhinitis weekly or monthly [ ] . there are children who, during the period of maximum exposure due to biological immaturity and immunological inexperience, suffer from one episode each month affecting different organ systems, as well as lymphadenopathies and failure to thrive. the capacity for inducing bhr in normal subjects and worsening the symptoms in those already ill are precisely caused by vri, also facilitating greater penetration of inhaled viral allergens [ ] (table . ); rris in turn predispose to sinusitis. lower ifn-g levels produced by of children at months of age were even greater if the comparison was made between children with rris and those with no or maximally one rri during the follow-up period [ ] . rhinovirus-induced infections (table . ) take the appearance of common rhinitis, but stimulate mastocytes to release histamine, contributing to bhr development and the perspective of delayed reactions. a differentiating feature is the respiratory infections in the id child that may also result from opportunistic pathogens [ ] (table . ). respiratory infections should be under control. a screening of humoral immunity revealed low ig levels in . %, low iga levels in . %, and sigad in . % of children [ ] . during the last few years, increasingly sophisticated diagnostic techniques have permitted prenatal diagnosis in many cases (table . ) [ , , , , , ] : in forms supported by rag- and/or rag- mutations a diagnosis even at the th- th or at the pharyngeal tissue are almost imperceptible [ , ] . the seriously undernourished appearance should be noted, more often observed in children with scid [ ] . rris can be observed in other cmi forms [ ] : deficiencies of cd g and e chains [ , , ] , zap- [ , ] and hla class ii [ , ] . finally, children with aids will seem to be in severe general condition and this disease is a paradigmatic example of how hiv can overturn the t lymphocyte immune defense with regards to opportunistic infections [ ] . in some cases of pediatric aids there is hgg that is indistinguishable from pid and that belongs to the differential diagnosis of severe recurrent infections during the first few months after birth [ ] . as far as tih is concerned, the confirmation of a normal presence of the b lymphocytes and low levels of intrinsically produced ig is resolutive, compared to agammaglobulinemia [ ] . an articulate case history often identifies the familiarity of rris, usually with an absent basic pathology and the frequent predisposing environmental factors (table . ), among which passive cigarette smoking stands out. maximum prevalence occurs during the first years of life or during first contacts with school, the disease is limited in time, and there is usually a single location. in most cases the pseudo-immunodepressed child is clinically normal in all other respects [ ] . in ten reported sars-infected children from hong kong, fever, cough, and runny nose were complement deficiency lateral pharyngeal x-ray to visualize adenoidal tissue cytokine production (il , il , il , il , ifn-g, il ) chromosome fragility (ataxia-telangiectasia, bloom's syndrome, etc.) data from [ , , ] . ada adenosine-deaminase, adcc antibody dependent cellmediated cytotoxicity, ctl cytotoxic t lymphocytes, ltt lymphocyte transformation test, pha phytohemagglutinin, pma phorbol myristate acetate, pnp purine nucleoside phosphorylase, ppd purified protein derivative. sars seems to have a less aggressive clinical course in younger children [ ] . when in doubt, a broad spectrum of laboratory tests are available: cbc, proteinemia and protidogram, serum ig levels, or in secretions and igg subclasses (table . ), immunoelectrophoresis (homogeneous components, k/l), dosage of isohemagglutinin, five other natural antibodies, the sweat test [ ] , in strictly selected cases also a lymphocyte population and subpopulation count (tables . - . ), and x-ray of paranasal sinuses. analysis of the lymphocyte profile sometimes shows a number of deficiencies, statistically differentiated from those found in other children affected by an asthmatic pathology; however, none of the immunological deficiencies indicated (table . ) are characteristic in pseudo-immunodepressed children. common diagnostic methods may not be capable of revealing a deficiency of igg subclasses or of selective igg : the chance that there may be abnormal igg or igg levels is not excluded by the normality of igg serum concentrations [ ] . furthermore, the distribu-tion of igg into four subclasses makes it difficult to identify these deficiencies simply by measuring total serum igg levels [ ] ; only for the past few years have there been highly specific reagents for measuring individual subclass levels and methods such as radial immunodiffusion (rid) [ , ] . the aaaai has recommended not relying on subclass levels [ ] , especially igg levels, which seem to be unmeasurable in % of the population [ ] . rid, which has proven to be more sensitive than the elisa used by the cdc in atlanta, has shown that % of normal children have values below normal for at least one subclass [ ] ; a similar deficiency was present in % of children with rris [ ] . a recent study measuring the igg with both methods, has proved that the rid can show higher values of igg and igg in low serum levels of both ig [ ] , data with an unquestionable negative effect in pediatrics. in conclusion, at the moment our knowledge suggests that we should also carefully interpret low levels of one or more subclasses, because on the one hand this might indicate a transient or paraphysiological condition, on [ ] . igg levels in all patients also rose considerably compared to previous treatment: the average levels in different determinations was > mg [ ] . finally, all children grew normally; the height achieved by each child is between the rd and th percentile, within the limits of theoretical values calculated on the height of their parents. substituting therapy with ivig has allowed patients to return to their normal activities, with a considerable improvement in quality of life. in all these years, we have never come across substantial unwelcome reactions or infectious complications [ ] , as also found by other authors [ , ] . ivig could also be effective for reducing the allergic symptoms discussed thus far: presuppositions are not lacking, such as the blocking of allergens and mastocyte fcr thanks to the modest quantities of igg present in the preparations [ ] . in addition, the increased understanding of the igg transplacental passage (chap. ) can absolve the function of timing their transfusion in the case of mothers with antibody id, so that the fetal defenses can be complete and quantitatively adequate. in sigad common ivig preparations cannot be used, even if with a low content of iga, nor enriched, both because of the extremely short iga life-span, which would therefore suggest iga administration every - days, and because the infused iga do not reach the secretions [ ] . should ivig be indicated for a deficiency associated with igg , or should transfusions of blood derivatives become necessary, one must first investigate serum antibody anti-iga levels (igg and ige) and, should these be positive, avoid infusions or administer them in a hospital under strict medical supervision, or use washed red blood cells [ ] . the same precaution must be taken for subjects with ata for whom ivig, if is appropriately administered, also ensure beneficial effects on quality of life, while there are no known therapies for contrasting neurological symptoms. in patients with humoral deficiency, alongside ivig, if appropriate, an antibiotic prophylaxis is suitable with monthly cycles, alternating amoxicillin, cephalosporin, co-trimoxazole, etc., bearing in mind family compliance. in cvid recurrent infections caused by giardia lamblia should be treated using furazolidone ( mg/kg/day) or methronidazole ( mg/kg/day) for days, if necessary to be repeated. cvid treatment in specialized centers involves recombinant il , il and cimetidine [ ] . some t-cell pids represent a severe clinical emergency, such as omenn syndrome, in which hypovolemic shock and reticular dysgenesis are immanent in the battle for survival. although precise figures are unavailable, thousands of patients worldwide with different forms of the other a modest deficiency can result in clear hgg [ ] . subnormal igg levels can indeed be associated with various manifestations of immune dysfunctions; it is therefore advisable in this case to proceed with specific investigations, measuring the response to polysaccharide antigens and studying the lymphocyte activity in vitro [ ] . in children with normal serum levels, who are instead lacking in antibody responses to polysaccharide antigens [ , ] , this is a conclusive investigation [ ] (fig. . ) . a study of children aged > , half atopic and half not, has confirmed this thesis, concluding that the answers were similar in both groups, therefore excluding a greater rri predisposition in the atopic children [ ] . many patients with high ige levels do not present atopic manifestations: it is thought that an increased concentration is related to a reduced inhibiting activity of the thymus in ige synthesis [ ] . recurrent sinopulmonary infections must make one also consider cystic fibrosis and immotile cilia syndrome [ ] . children with malnutrition (chap. ) suffer from numerous ids, prevalently concerning cmi; their vulnerability makes them succumb to severe bacterial me infections and urtis, often also risking death. obese subjects may also be affected by rris due to a possible adipose tissue hypovascularization or to a defect in the granulocyte microbicidal activity [ ] . in the presence of antibody deficiency, antibiotic treatment is chosen as a preventive therapy in less severe cases, otherwise the preferable therapy consists in ivig (fig. . ). this treatment is restricted to a limited number of diseases, including some forms of id, secondary or cytopenic id, in which effectiveness has been proved in dbpc studies [ , , ] , like other positive forms of intervention described, while it appears to be of no use in uncomplicated thi [ , ] . two children aged . and with higes and kawasaki disease were administered mg/die of ivig for days and one . -year-old with higes received only one dose, with ige levels falling from , - , to - , ui/ml on the th day. hence there was almost a normalization of ige production with symptom relapse after months; similar results using a single dose were also obtained in two children with higes and severe ad [ ] . the following data represent a number of clinical and immunological parameters in children suffering from humoral pid and ata, with igg levels < mg/dl. treatment with ivig, also at a higher dosage, was very well tolerated by patients: all children presented a clear- verely affected by several factors [ ] . either hla-identical marrow or t-cell-depleted (tcd) haploidentical parental marrow is the standard of care for scid ( fig. . ) . when histocompatible related donor bmt is unavailable, a bmt either with hla-identical unfractionated or tcd haploidentical parental marrow is the standard of care for scid [ ] . all but one ( %) of scid infants who received tcd identical or haploiden-genetically determined id have been given bmt in attempts to correct their underlying id [ ] , including a recent series [ ] . specific treatment for cellular pid consists in a bmt from a hla-compatible donor [ ] . the ideal sc donor is normally a sibling who shares identical hla class i and class ii loci. without such a donor, these transplantations usually resulted in fatal gvhd. if death did not occur, event-free survival was se- treatment tical bmt in the first days of life are currently alive, with the period of survival ranging from months to > . years after transplantation. this compares favorably with a % survival rate of infants receiving transplants at a median age of days (range, - days) [ ] .a girl with t -b -scid received a full matched bmt from her sister at age weeks [ ] . a worldwide survey conducted by buckley from through , with subsequent additions of published cases from the literature, revealed that of ( %) patients with pid transplanted with hla-identical marrow during a period of years were alive [ ] . there are > patients worldwide who have survived scid as a result of successful transplantation of hla-identical or haploidentical bm [ ] . most importantly, of infants ( %) undergoing transplantation in the first . months of life are currently alive [ , ] , compared with a cut-off at months ( % vs %, children younger vs those aged > months) receiving bmt (or . [ ] ). we stress that neonates developed higher lymphocyte responses to phytohemagglutinin and higher numbers of cd + and cd ra + t cells in the first years of life than those receiving bmts late. t-cell antigens peaked earlier and with higher values in the neonatal bmts ( days to year) than in the late bmts ( - years) [ ] . over the past years % of all scid patients ( / ) receiving bmt at duke university medical center survive to varying ages up to ≥ years after bmt. only had an hla-identical donor. all others received rigorously tcd haploidentical bm from a parent, most often the mother. the soy lectin, srbc rosetting technique was used (r. buckley pers. comm. november th, and april th, ). an uncommon bmt to treat ar scid was undertaken in a -month-old girl. the donor was her hla-mismatched -year-old sister, who had previously received a bmt from her father [ ] : presently, they are aged and and are affected by molloscum contagiosum infection [ ] . bmt, both hla identical unfractionated and tcd hla aploidentical [ ] .a recent trial found that because only %- % of affected children have a familial hlaidentical donor (rid), the alternative therapeutic options are bmt from a mud or a haploidentical bmt or from hla-mismatched related donors (mmrds). only % of these children may find a matched donor; therefore, the remaining pid-affected children are candidates for a tcd haploidentical bmt [ ] . mud hsct is successful in young children [ ] , but the success rate decreases dramatically above the age of - years [ ] (table . ) [ , , , , , , , , , , , , , , , , , , , , , , , , , - , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ] . bmt survived. compared with mmrd bmt, survival was significantly higher with rid or with mud ( / = . %) [ ] . when an hla-identical sibling as the donor is unavailable, a phenotypic hla-matched unrelated bmt is needed, also used in cd deficiency [ , ] , with a clinical and immune outline normalization [ ] and a variable effect on igg subclasses treatment [ ] . c median ( . - years after bmt). p, at the time of publication. [ ] . possibly because of earlier diagnosis before untreatable opportunistic infections develop, the results have improved considerably during the last two decades [ ] . bmts have been successful when applied within the first - days of life in infants with scid, ( %) of those still alive range from months to years, not justifying in utero transplants. a completely normal t-cell function was obtained within - days [ ] and in an other patients was still present after - years [ ] . of the children who received a mismatched parental bmt from to , ( %) remain alive with t-cell immune reconstitution, a median of . (range, - . ) months after bmt [ ] . of children out of who received allogeneic bmt after the first days of life, ( %) are alive [ ] . three out of five children who received a hsct are alive and well after - months [ ] . breast feeding appeared correlated to an earlier reconstitution when the donor was the mother [ ] . intra-amniotic gene transfer has been successfully carried out on a laboratory animal, registering in a dose-dependent manner the fetal gastroenteric and respiratory effects [ ] . if confirmed in human beings, this method of treatment will certainly prove useful for prenatal correction of pid. bmt/hsct should be completed by conditioning regimens with busulfan and cyclophosphamide, less toxic than total lymphoid irradiation or a combination of nucleoside analogs and anti-lymphocyte antibody preparations [ ] . for example, busulfan ( mg/kg), melphalan ( mg/m ) and anti-thymocyte globulin ( mg/kg) [ ] . to enhance the engraftment rate in haploidentical bmt in pid, it was recently suggested to add donor peripheral scs after mobilization with g-csf ( mg/kg for days) and bm cells. with this procedure the cell load is increased, which allows intensification of the conditioning regimen for induction of faster engraftment [ ] . in utero bmt suggested advantages include the sterile environment in utero, and immaturity of the fetal immune system enabling the prevention of clinical manifestations of the disease in the neonate, and the engraftment without the use of cytotoxic conditioning regimens: a child thus treated was well at age months [ ] . two series of six and four patients [ , ] and two additional case reports [ , ] of in utero transplants have been published, yet failure of b-cell engraftment and function may result in long-term dependence on ivig replacement [ ] . better results than those published for in utero bmt for scid were implicit in infants admitted and diagnosed at a median age of days because of a fh of a previously affected infant. bmt was successful and all children are alive and well with follow-up to . years [ ] or < days vs approximately months [ ] . however, it is suggested that in utero transplants may carry the risks associated with injecting the fetus and the inability to detect gvhd during gestation [ ] . umbilical cb transplantation (ucbt) was done in two children affected by a zap- deficiency and an omenn-like syndrome. both are alive and well at . and . years after ucbt [ ] . unrelated ucbt in eight children with severe t-cell id [ ] and in three with was [ ] resulted in consistent and stable t -, b -, and nk cell development [ , ] . faster availability of ucbts is a meaningful advantage for patients requiring urgent transplantation: a median of days more rapidly than did those receiving bone marrow [ ] . in a large report [ ] , patients with scid, seven with was, and other unspecified pid received an unrelated ucbt. ucb was evaluated as a sc source for immune reconstitution in children with severe primary t-cell ids such as scid, reticular dysgenesis, thymic dysplasia, cid, dgs, and was when a matched sibling donor was unavailable, and has been used to date in more than , patients [ ] . three infants who rejected a tcd-mismatched parental bmt without prior cytoreduction engrafted after infusion of ucbt [ ] . a . -year-old girl with hla class ii deficiency had a successful related ucbt for graft failure following tcd nonidentical bmt [ ] . a girl with reticular dysgenesis failed to engraft following her first transplant, but fully engrafted after a second unrelated ucbt. five of six patients showed grade i gvhd, although one child experienced grade iv skin and gut gvhd. immunological ucbt resulted in consistent and stable t-cell, b-cell, and nk-cell development [ ] . long-term event-free survival (≥ months) with recovery of antigen-specific responses was reported following an unrelated ucbt in a child with omenn's syndrome [ ] . gene therapy, which has revolutionized and could revolutionize even more pid treatment in the near future, is analyzed in table . [ , ] . the requisite for applying this form of genetic engineering treatment is that the responsible gene must be cloned; the most common techniques involve knock-out mice and inactivating a particle [ ] , or employing retroviral vectors (table . ) [ ] , totally deprived of their genomic factors except for the normal copy of the gene to be inserted. this is indispensable for allowing the vector to reach the human nucleus, where it will integrate with the cellular genome [ ] . in this case, pbls are collected through leukapheresis and cultivated in vitro with retroviral particles containing a normal gene rna copy: thus the healthy gene is introduced into the cell genome using a vector and the manipulated cells are reinfused, so as to restore normal immune functions. this system has been used to treat two children aged months suffering from scid [ ] and from ada deficiency by employing autologous pbls [ ] , bm cells [ ] , and cb cells [ ] . three reports [ , , ] of successful gene therapy in infants with x-linked scid [ , ] and in t -b -scid [ ] are a major step forward among repeated efforts to achieve better immune reconstitution in ada-scid with gene therapy than with bmt/sct [ ] . immediately after the diagnosis had been made in two capacity of wild-type, replication-competent retroviruses to cause leukemia in immunologically immature neonatal mice [ ] , and in humans (two children out of ) [ , , ] . retroviruses can cause insertional oncogenesis, a long-known potential complication of retroviral gene transfer attempts, because gene integration occurs at random in the genome, thus deregulating the expression of cellular oncogenes [ ] . this complication has been thought to be unlikely with such vectors, because they are capable of inserting only once into the cell's chromosomes and cannot repeatedly reproduce and integrate. lentiviruses may be more effective than murine retroviruses for gene transfer into human hematopoietic scs and t lymphocytes [ ] . in ada-scid, the safety and efficacy of hsc gene therapy combined with nonmyeloablative conditioning for the treatment of scid has allowed two children to live at home and clinically well, with normal growth and development [ ] . on january , , fda placed on "clinical hold" all active gene therapy trials using retroviral vectors to insert genes into blood scs after having learned that a second child treated in the french gene therapy trial developed a leukemia-like condition. gene therapy is on hold despite enormous promise for certain scid/ cid variants. survival. in scid, the european experience with unfractionated hla-identical and tcd or non-tcd haploidentical or mud bmts in patients with scid reported that between and , a -year survival with sustained engraftment was significantly better after hla-identical than after mismatched transplantation ( % vs %).within the hla-identical group, survival after bmt from genotypically or phenotypically identical related or muds was %, %, and %, respectively [ ] . in non-scid, -year survival after genotypically hla-matched, phenotypically hla-matched, mmrd, and mud bmt was %, %, %, and %, respectively [ ] . in a retrospective analysis of bmts performed between and at european centers in children as young as month with different pids (excluding scid), the overall survival among recipients of hla genetically identical bmt was %, . % in patients who received closely matched bmt, and % in recipients of bmt with two or three mismatched hla antigens. a significant improvement in survival has been achieved in most pids (overall survival, . % vs . %, primarily because of a decrease in the frequency of infectious complications [ ] . in the similar analysis performed in children with scid at european centers between and , out of ( . %) patients were alive with evidence of engraftment months after bmt. however, patients died > months post-bmt, mainly due to cgvhd and/ or viral infection. thus gvhd months after bmt and b -scid vs b + scid were the main factors associated with a poor outcome [ ] . the disease-free survival was significantly better for patients with b + scid ( . %) than for those with b -scid ( . %) [ ] . in a children aged and months including a novel splice imitation in the common gc chain [ ] , haploidentical cd + peripheral progenitor cells mobilized with gm-csf were isolated to a purity of more than %. these cells were infused with no prior chemoablation and no prophylaxis against gvhd. both children showed signs of t-cell reconstitution beginning weeks after the cd + infusion and were weaned from continuous cures. they are in excellent health, without gvhd, and months after transplantation. one child does not need replacement ig. the other received a booster infusion of cd + scs from the original donor year later to improve b-cell function and now receives ig every months. both were followed for months after gene transfer [ ] . however, retroviral vectors have the treatment data from [ , , ] . ciita class ii transactivator, cgd chronic granulomatous disease, xla x linked agammaglobulinemia; for other abbreviations see table . . a done with success, see text for details. b in utero transplant of maternal stem cells. trial on children with pid receiving bm from hla-nonidentical related donors or from hla-identical unrelated donors at european centers between august and june , out of children ( . %) survived - months. bm was tcd by use of either erythrocyte rosetting or monoclonal antibodies to prevent gvhd [ ] . additional survival rates were reported previously (table . ). in a series of consecutive ud bmts / children with scid and non-scid pids who received a bmt with reduced-intensity conditioning (ric) regimen between and survived after a . -year follow-up, as well as / children who received a bmt with myeloablative conditioning (mat) between and and survived after an . -year follow-up. therefore a ric regimen results in improved survival and reduced bmt-related mortality compared with mat in hr children undergoing an ud bmt [ ] . in transplanted patients with was, the -year probability of survival differed according to donor type: % with hla-identical sibling donors, % with other related donors, and % with mud. significantly, boys who had received a mud transplant before years of age had survival rates similar to those receiving hlaidentical sibling transplants [ ] . however, the time required to develop immune function after haploidentical scts is quite different from that after unfractionated hla-identical bm. lymphocytes with mature t-cell phenotypes and functions fail to rise significantly until - months after bmt; normal t-cell function is reached between and months [ ] . b-cell function develops much more slowly, averaging - . years for normalization; many do not have b-cell function, despite normal t-cell function. [ ] . ex vivo rigorous depletion of post-thymic t cells from donor marrow that cause gvhd is efficient and feasible, even in haploidentical settings [ ] , presumably because of more effective infection-control measures and better transplantation strategy [ ] . for non-scid, sct can provide a cure, and grafts from unrelated donors are almost as beneficial as those from genetically hla-identical relatives [ ] . in most patients, deficient b-cell function persists after transplantation and requires lifelong ivig therapy [ , ] , which is necessary to prevent bacterial and common viral infections [ , ] . some patients also have persistent deficiencies of t-cell function after sct [ , ] . in children with rris, depending on the nature of the infection, the pediatrician will prescribe the most appropriate symptomatic and/or antibiotic therapy. in the presence of persistent inflammation, or during the winter, when the risk of close acute recurrent episodes is higher, anti-inflammatory preparations will be prescribed via aerosol, chromones, ketotifen, b -adrenergic and if necessary steroids for topical use, strictly depend-ing on the need. we suggest monitoring measures, such as keeping a clinical diary, in which each acute episode should be briefly noted, continuing registration until clinical symptoms have not regressed for at least days and returning to keep notes in the diary each time there is a cough and/or nasal and/or bronchial inflammation, completing this with pef as well as some respiratory parameters right at the beginning and then every months. it is obvious that if medical intervention is not resolutive a center specialized in infantile respiratory physiopathology should be contacted [ ] . children with sars were treated with high-dose ribavirin, oral prednisolone, or iv methylprednisolone, with no shortterm adverse effects [ ] . antibiotics must be used very carefully in these children because they can influence positively or negatively the innate, cellular or humoral immunity (chap. ), interaction with ils and growth factors are not known, repeated use often causes phenomena involving allergy/ intolerance [ ] , and most infection-prone children suffering from vris are given antibiotics unnecessarily. in italian children ( . % of males) aged months to years (median, years) with a history of rris, macrolide therapy of acute respiratory infections influenced the natural history of rris, probably because of their elective activity on atypical bacteria [ ] . considering the emergence of antibiotic-resistant bacterial stock, as for example s. pneumoniae, immunotherapy has been proposed as a means of preventing rris by providing children with small doses of inactive bacterial antigens liable to trigger specific and protective immune responses (table . ) [ ] . for example, om- bv significantly reduces the urti rate, particularly in a dbpc study in children aged - with a history of acute urtis [ ] , is active in preventing rri episodes [ ] with a meaningful reduction in the number of days of suffering acute urtis [ ] . bacterial ribosomal and membrane proteoglycans of s. pneumoniae, which stimulate b cells with secretory responses, as well as memory cells, may be used for responding to future infections [ ] . ribosomal immunotherapy appears to be not only well tolerated, but also ideally targeted to induce mucosal responses [ ] . among the preparations reserved for specific use, a study of pidotimod in dbpc trials proved its effectiveness in a sample of children with rris, also showing increased cd , absent in placebo-treated children [ ] . the use of immunostimulants should be limited to children with proven high susceptibility to acute urti, or overexposed children attending daycare facilities, or attending kindergarten or elementary school [ ] . however, according to a meta-analysis, immunostimulants are an effective treatment for the prevention of acute urti in children [ ] . furthermore the indiscriminate and purely empiric use of ivig must be discouraged in every child with rri, whereas in a prospective, dbpc study of ivig and co-trimoxazole, of children < years referred for recurrent bacterial rris became infection-free over a -month obser-if caused by pneumococci. other kinds of vaccines have provided disappointing results: fig. . [ ] indicates the immune bases of a specific immunization and the possibility of specific interventions. in children with pid, one should bear in mind all the aforementioned facts. until the past few years, there was a busy motion into the fundamental problems underlying a majority of these conditions. many have now been mapped to specific chromosomal locations, and an impressive number vation period [ ] , in addition to having an extremely unfavorable cost-benefit ratio [ ] immunization children with rris and deficient antibody responses to germs expressing a capsular polysaccharide can be successfully vaccinated, but avoiding the administration of live virus vaccines and integrating this if necessary with an igg replacement therapy [ ] . furthermore, in view of the availability of conjugated vaccines, it will be possible to induce antipneumococcus-igg , providing an effective treatment for children with rris, especially treatment of the fundamental biological errors have been identified. the pediatrician is entrusted with a more difficult job, that of identifying as early as possible the possible existence of pid, remembering the suggestions for case history in chap. , with the exception of clinical emergencies such as omenn syndrome and reticular dysgenesis. this specific research becomes a necessity thanks to the new diagnostic and therapeutic advances that have been conceived over the past few years: the earlier one acts, on the one hand with a prenatal diagnosis and on the other with a bmt or sct therapy, the greater the chance to increase life expectancy for these children, in addition to ensuring better quality of life. the discovery and cloning of the genes for these diseases have obvious implications for the potential of gene therapy. the rapidity of these advances suggests that there will soon be many more to come. one of the most common differential diagnoses will occur with a child affected by rris, for whom we believe the number of infections 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shwachman-diamond syndrome igg subclasses t cell depleted haploidentical bone marrow transplantation for the treatment of children with severe combined immunodeficiency a family of wasps clinical, immunologic, and genetic features of an autoimmune lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis defective opsonization. a common immunity deficiency immunology in the pediatrician's office independent mutations of the human cd e gene resulting in a t cell receptor/cd complex immunodeficiency genetically determined immunodeficiency disease and malignancy report from immunodeficiency cancer registry complementation cloning of an mhc class ii transactivator mutated in hereditary mhc class ii deficiency (or bare lymphocyte syndrome) severe combined immunodeficiency: a retrospective single-center study of clinical presentation and outcome in patients atypical x-linked severe combined immunodeficiency due to a possible spontaneous reversion of the genetic defect in t 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the pluripotent molecule of the innate immune system consensus conference: il bambino con deficit di iga: le caratteristiche cliniche, immunologiche e genetiche per una corretta gestione il bambino immunodepresso th t-cell and monocyte defects autoimmune lymphoproliferative syndrome (alps) in a child from consanguineous parents: a dominant or recessive disease? rotavirus infection in infants as protection against subsequent infections regulation of immunoglobulin (ig)e synthesis in the hyper ige syndrome the gene involved in x-linked agammaglobulinemia is a member of the src family of protein-kinases primary immunodeficiency mutation databases v(d)j recombination defects in lymphocytes due to rag mutations: severe immunodeficiency with a spectrum of clinical presentations a cd minigene restores regulated isoform expression and immune function in cd -deficient mice: therapeutic implications for human cd -null severe combined immunodeficiency analysis of natural killer cells in tap 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of patient with hyperimmunoglobulinaemia e syndrome (letter) primary immunodeficiency diseases in latin america: first report from eight countries participating in the lagid. latin american group for primary immunodeficiency diseases bcl activates the nf-kappab pathway through ubiquitination of nemo virally induced immunosuppression the xlinked hyper-igm syndrome: clinical and immunologic features of patients brief report: twin boys with major histocompatibility complex class ii deficiency but inducible immune responses neuropsychological profile of children and adolescents with the q . microdeletion case definition for surveillance of severe acute respiratory syndrome sars primary immunodeficiency diseases role of tbx in human del q . syndrome high incidence of significant bone loss in patients with severe congenital neutropenia (kostmann's syndrome) key: cord- -yfuuirnw authors: severin, paul n.; jacobson, phillip a. title: types of disasters date: - - journal: nursing management of pediatric disaster doi: . / - - - - _ sha: doc_id: cord_uid: yfuuirnw disasters are increasing around the world. children are greatly impacted by both natural disasters (forces of nature) and man-made (intentional, accidental) disasters. their unique anatomical, physiological, behavioral, developmental, and psychological vulnerabilities must be considered when planning and preparing for disasters. the nurse or health care provider (hcp) must be able to rapidly identify acutely ill children during a disaster. whether it is during a natural or man-made event, the nurse or hcp must intervene effectively to improve survival and outcomes. it is extremely vital to understand the medical management of these children during disasters, especially the use of appropriate medical countermeasures such as medications, antidotes, supplies, and equipment. skeleton as a result of incomplete calcification and active bone growth centers. protected organs, such as the lungs and heart, may be injured due to overlying fractures. cervical spine injuries can also be pronounced, as in patients with head trauma. in fact, spinal cord injury may be present without any radiographic abnormalities of the spine. finally, vital signs will vary based on the pediatric patient's age. this may be a pitfall during rapid evaluation by any nurse or hcp not accustomed to the care of children. younger pediatric patients have higher metabolic rates and, therefore, higher respiratory rates and heart rates. this can be a distinct disadvantage versus older pediatric patients when encountering similar diseases. an example is inhaled toxins (e.g., nerve agents and lung-damaging agents). infants and children will suffer greater toxicity since they inhale at a faster rate due to higher metabolic demands and thus, distribute the toxin more rapidly to various end-organs. understanding respiratory differences is essential to the management of the acutely ill pediatric patient. the most common etiology for cardiorespiratory arrest in children is respiratory pathology, typically of the upper airway. most of the airway resistance in children occurs in the upper airway. nasal obstruction can lead to severe respiratory distress due to infants being obligate nose breathers. their relatively large tongue and small mouth can lead to airway obstruction quickly, especially when the neuromuscular tone is abnormal such as during sedation or encephalopathy. in infants, physiologic (i.e., copious secretions) and pathologic (i.e., edema, vomitus, blood, and foreign body) factors will exaggerate this obstruction. securing the airway in such events can be quite challenging. typically, the glottis is located more anterior and cephalad. appropriate visualization during laryngoscopy can be further hampered by the prominent occiput that causes neck flexion and, therefore, reduces the alignment of visual axes. the omega or horseshoeshaped epiglottis in young infants and children is quite susceptible to inflammation and swelling. as in epiglottitis, the glottis becomes strangulated in a circumferential manner leading to dangerous supraglottic obstruction. children also have a natural tendency to laryngospasm and bronchospasm. finally, due to weaker cartilage in infants, dynamic airway collapse can occur especially in states of increased resistance and high expiratory flow. along with altered pulmonary compensation and compliance, a child may rapidly progress to respiratory failure and possibly arrest. cardiovascular differences are critical in the pediatric patient. typical physiological responses tend to allow compensation with seemingly normal homeostasis. with tachycardia and elevated systemic vascular resistance, younger pediatric patients can maintain normal blood pressure despite decreased cardiac output and poor perfusion (compensated shock). since children have less blood and volume reserve, they progress to this state quickly. in pediatric patients with multiorgan injury or severe gastrointestinal losses, these compensatory mechanisms are pushed to their limits. the unaccustomed hcp may be lulled into complacency since the blood pressure is normal. all the while, the pediatric patient's organs are being poorly perfused. once these compensatory mechanisms are exhausted, the patient rapidly progresses to hypotension and, therefore, hypotensive shock. if not reversed expeditiously, this may lead to irreversible shock, ischemia, multiorgan dysfunction, and death. pediatric patients with altered mental status pose significant problems. the differential diagnosis will be very broad in the comatose patient based on development alone. for example, younger pediatric patients can present with nonconvulsive status epilepticus (ncse) instead of generalized convulsive status epilepticus (gcse). in fact, ncse is more common among younger pediatric patients than gcse, especially in those from to months of age. furthermore, many of them are previously well without preexisting diseases such as epilepsy. other disease states may include poisoning, inborn errors of metabolism, meningitis, and other etiologies of encephalopathy. using the modified pediatric glasgow coma scale (gcs) is the cornerstone when evaluating the young pediatric patient when they are preverbal. pupillary response, external ocular movements, and gross motor response may be challenging to evaluate in a developmentally young or delayed pediatric patient. pediatric traumatic brain injury is extremely devastating. whether considered accidental (motor vehicle crash) or nonaccidental (abusive head trauma), evaluation of the neurological status of the acutely injured pediatric patient can be problematic, especially the gcs. some prefer to use the avpu system (alert, responds to verbal, responds to pain, and unresponsive). due to the disproportionately larger head and weaker neck muscles, there is more risk of acceleration-deceleration injuries (fall from a significant height, vehicular ejection, and abusive head trauma). furthermore, the softer skull, dural structural differences, and vessel supply will place the pediatric patient at risk for brain injury and intracranial hemorrhage. finally, due to pediatric brain composition, the risk of diffuse axonal injury and cerebral edema is much higher. although spinal cord injury is rare in young pediatric patients, morbidity and mortality are significant. in pediatric patients less than years of age, the most commonly seen injuries are in the atlas, axis, and upper cervical vertebrae. in young pediatric patients, spinal injuries tend to be anatomically higher (cervical) versus adolescents (thoracolumbar). furthermore, congenital abnormalities, such as atlantoaxial abnormalities (trisomy ), may exaggerate the process. the clinical presentation of spinal cord injury varies in young pediatric patients due to ongoing development. laxity of ligaments, wedge-shaped vertebrae, and incomplete ossification centers contribute to specific patterns of injuries. finally, spinal cord injury without radiographic abnormality (sciwora) may result. because of the disproportionately larger head, weaker neck muscles, and elasticity of the spine, significant distraction and flexion injury of the spinal cord may occur without apparent ligament or bony disruption (hilmas et al. ; jacobson and severin ; severin ). motor skills develop from birth. gross and fine motor milestones are achieved in a predictable manner and must be assessed during each hcp encounter. cognitive development will follow a similar pattern of maturation. the development of these skills can often predict injuries and their extent. for example, consider a house fire. a young infant, preschooler, and adolescent are sleeping upstairs in house when a fire breaks out in the middle of the night. the smoke detectors begin to alarm. each child is awoken by the ensuing noise and chaos. based on the development, the adolescent will most likely make it out of the house alive. he will comprehend the threat, run down the stairs, and exit the house without delay. smoke inhalation may be minimal. if it is a middle adolescent, an attempt may be made to jump out of the window leading to multiple blunt trauma with or without traumatic brain injury. the preschooler most likely will be too scared and not understand how to escape. tragically, he may hide under a bed or in a closet. when the firefighters arrive and search the house, the preschooler may remain silent because of fear, especially of strangers in the house. he will most likely succumb to thermal injuries along with the effects of carbon monoxide and die. as far as the infant, he cannot walk, climb, crawl, or run. furthermore, he cannot scream for help or know how to escape. as the smoke engulfs the room, he will most likely suffer severe smoke inhalation injury including extensive carbon monoxide toxicity along with thermal injuries and die. this example also points out another important difference in pediatric patients: their dependence on caregivers. when considering neonates, for example, their entire existence depends on the caregiver, including feeding, changing of diapers, nurturing, and environmental safety. these dynamics are essential to the pediatric patient's health and survival, especially during a disaster. another aspect of development is the attainment of language skills. this, too, develops over time in a predictable fashion. one of the biggest challenges in pediatrics is the lack of the patient's ability to verbally convey complaints. as described above, verbal milestones vary among the different age ranges of the pediatric patient. hcps are often faced with a caregiver's subjective assessment of the problem. although it can be revealing and informative, this may not be available in an acute crisis situation. it will take the astute hcp to determine, for example, if an inconsolably crying infant is in pain from a corneal abrasion or something more life-threatening such as meningitis. this can also be a challenging task in a teenager, especially during middle adolescence. an hcp will have to determine, for example, if the seemingly lethargic middle adolescent is intoxicated with illicit drugs or has diabetic ketoacidosis. finally, the hcp will have to address developmental variances among their pediatric patients and any comorbid features. young pediatric patients can regress developmentally during any illness or injury. this is especially seen in patients with chronic medical conditions (cancer) or during prolonged hospitalization with rehabilitation (multisystem trauma). furthermore, those pediatric patients with developmental and intellectual disabilities, for example, will be difficult to evaluate based on the effects of their underlying pathology. these pediatric patients typically have unique variances in their physical exams (jacobson and severin ; severin ) . please refer to chap. for more detailed information on pediatric development. pediatric patients will often reflect the emotional state of their caregiver. they take verbal and physical cues from their caregiver. at times, this may also occur in the presence of a nurse or hcp. the psychological impact of illness will vary greatly with the child's development and experience. children tend to have a greater vulnerability to post-traumatic stress disorder especially with disaster events. furthermore, they are highly prone to becoming psychiatric casualties despite the absence of physical injury to themselves. and as any pediatric hcp can tell you, the younger pediatric patients tend to also have greater levels of anxiety, especially while preparing for invasive procedures such as phlebotomy and intravenous line placement (hilmas et al. ; jacobson and severin ; severin ) . please refer to chap. for more detailed content on mental health. the world health organization and the pan american health organization define a disaster as "an event that occurs in most cases suddenly and unexpectedly, causing severe disturbances to people or objects affected by it, resulting in the loss of life and harm to the health of the population, the destruction or loss of community property, and/or severe damage to the environment. such a situation leads to disruption in the normal pattern of life, resulting in misfortune, helplessness, and suffering, with adverse effects on the socioeconomic structure of a region or a country and/or modifications of the environment to such an extent that there is a need for assistance and immediate outside intervention" (lynch and berman ). types of disasters usually fall into two broad categories: natural and man-made. natural disasters are generally associated with weather and geological events, including extremes of temperature, floods, hurricanes, earthquakes, tsunamis, volcanic eruptions, landslides, and drought. naturally occurring epidemics, such as the h n , ebola, and novel coronavirus outbreaks, are often included in this category. man-made disasters are usually associated with a criminal attack such as an active shooter incident, or a terrorist attack using weapons such as explosive, biological, or chemical agents. however, man-made disasters can also refer to human-based technological incidents, such as a building or bridge collapse, or events related to the manufacture, transportation, storage, and use of hazardous materials, such as the chernobyl radiation leak and the bhopal toxic gas leak. even though disasters can be primarily placed into any of these two categories, they can often impact each other and compound the magnitude of any disaster incident (united states department of homeland security, office of inspector general ). a prime example is the march tohoku earthquake leading to a tsunami (natural) that triggered the fukushima daiichi nuclear disaster (man-made). disasters can also be characterized by the location of such an event. internal disasters are those incidents that occur within the health care facility or system. employees, physical plant, workflow and operations of the clinic, hospital, or system can be disrupted. external disasters are those incidents that occur outside of the health care facility or system. this impacts the community surrounding the facility, proximally or distally, but does not directly threaten the facility or its employees. as with natural and man-made disasters, internal and external disasters can impact each other. for example, an overflow of patients during a high census period may lead to the shutdown of the hospital to any new patients (internal disaster). this will place the hospital on bypass and possibly stress other hospitals in the community beyond their means (external disaster). a terrorist event, such as the release of sarin in a subway system during a busy morning commute, can lead to massive disruption in the community (external disaster). all the victims of the attack will seek medical care at nearby hospitals, possibly overwhelming the health care staff and depleting critical resources (internal disaster). characterization of disasters by geography (local, state, national, and international) can also be used. again, no matter the site of the incident, a disaster in one area could easily create a disaster in another geographical region. for example, a factory and its community could be ravaged by a hurricane (local disaster). if this is the only factory in the world to produce a certain medication, this could lead to critical shortages to hospitals all around the world (international disaster). the term "disaster preparedness" has been used over the years as a way to describe efforts to manage any disaster event. however, preparedness is only one aspect of the process. the use of the term disaster planning is more appropriate. it considers all aspects needed for an effective effort and is dependent on additional phases, not just preparedness. national preparedness efforts, including planning, are now informed by the presidential policy directive (ppd) that was signed by the president in march and describes the nation's approach to preparedness (united states department of education, office of elementary and secondary education, office of safe and healthy students ; united states department of homeland security b). a recommended method for disaster preparedness efforts is the utilization of an "all-hazards" model of emergency management (adini et al. ; waugh ) . the four overlapping phases of the model include mitigation, preparedness, response, and recovery. the mitigation phase involves "activities designed to prevent or reduce losses from a disaster" (waugh ) . examples include land use planning in flood plains, structural integrity measures in earthquake zones, and deployment of security cameras. the preparedness phase includes the "planning of how to respond in an emergency or a disaster, and developing capabilities for more effective response" (waugh ) . examples include training programs for emergency responders, drills and exercises, early warning systems, contingency planning, and development of equipment and supply caches. up to this point, all planning efforts are proactive and not reactive. often times, a hazard analysis is conducted to delineate areas of strengths and identify potential risks. it helps in "the identification of hazards, assessment of the probability of a disaster, and the probable intensity and location; assessment of its potential impact on a community; the property, persons, and geographic areas that may be at risk; and the determination of agency priorities based on the probability level of a disaster and the potential losses" (waugh ) . after a disaster or emergency incident occurs, the response phase, or "immediate reaction to a disaster", (waugh ) begins. examples include mass evacuations, sandbagging buildings and other structures, providing emergency medical services, firefighting, and restoration of public order. in some situations, the response period may be a short (e.g., house fire), intermediate (e.g., bomb detonation), or extended (e.g., pandemic influenza) duration. after a period of time, the recovery phase follows. these are "activities that continue beyond the emergency period to restore lifelines" (waugh ) . examples include the provision of temporary shelter, restoration of utilities such as power, critical stress debriefing for responders, and victims, job assistance and small business loans, and debris clearance. recovery always seems to be the most unpredictable; it may take days to months to years. as demonstrated with recent hurricanes harvey, irma, and maria in , the most affected regions are still in the phase of recovery and may be along a prolonged track as hurricane katrina in . as mentioned, the early phases of planning (mitigation and preparedness) truly hinge upon the environment or community surrounding the health care site (e.g., clinic, hospital, or long-term care facility). identification of potential hazards and risks is a key step in disaster planning. using a hazard vulnerability assessment (hva) or a threat and hazard identification and risk assessment (thira) can provide a basis for mitigation and prevention tasks. an hva/thira emphasizes which types of natural or man-made disasters are likely to occur in a community (e.g., tornado, flood, chemical release, or terrorist event). they further highlight the impact those disasters may have on the community and any capabilities that are in place that may lessen the effects of the disaster (illinois emergency medical services for children ). a basic principle of the hva methodology is to determine the risk of such an event or attack occurring at a given hospital or hospital system. simply, the risk is a product of the probability of an event and the severity of such an event if it occurs (risk = probability × severity). however, there are many complexities in quantifying terrorism risk (waugh ; woo ) . it is important to note that in some circumstances, exposure may need to be included in the equation (risk = probability × severity × exposure), but usually for operational risk management applications (mitchell and decker ) . at any rate, issues to consider for the probability of an event occurring include, but are not limited to, geographic location and topography, proximity to hazards, degree of accessibility, known risks, historical data, and statistics of various manufacturer/vendor products. severity, on the other hand, is dependent on the gap between the magnitude of an event and mitigation for the given event (severity = magnitude -mitigation). magnitude varies upon the impact of the event to humans, property, and/or business. mitigation varies upon the development of internal and external readiness before a disaster strikes. as one can surmise, if the magnitude of the event outstrips the mitigation, the event is considered a threatening hazard. once the hva is completed, the health care site should immediately prioritize planning efforts for the top - hazards and develop plans accordingly. all other identified hazards must also be addressed to ensure a broad and robust disaster plan. it is important to realize that local and regional entities also perform comprehensive hvas. a concerted analysis among a hospital and key community stakeholders is optimal for a coordinated plan. an hva/thira contains both quantitative and qualitative components. specific tools have been developed through private and public organizations (e.g, fema) that can help in the analysis (united states department of homeland security, federal emergency management agency ). using these tools as a guide, the entity can determine what types of hazards have a high, medium, or low probability of occurring within specific geographic boundaries. typically, these tools do not have components specific to children or other at-risk populations. however, the tools can be adapted either directly by adding children to specific hazards or ensuring considerations specific to children are incorporated into the hva/thira calculations. the hva/thira should be reviewed and updated minimally on an annual basis to identify changing or external circumstances. this includes conducting a pediatric-specific disaster risk assessment to identify where children congregate and their risks (e.g., schools, popular field trip designations, summer camps, houses of worship, and juvenile justice facilities) (illinois emergency medical services for children ). of note, hva techniques have been utilized for pediatric-specific disaster plans. having a separate pediatric hva (phva) is crucial to a well-rounded and robust health care disaster plan. first, it demonstrates the extent of the pediatric population in the community. it is estimated that % of the population fits within the age range of pediatric patients. in some situations, it may be more. during the performance of a phva, it was demonstrated that % of the community was less than years of age (jacobson and severin ) . second, a phva increases the situational awareness of those tasked to plan for disasters that involve children and adolescents. often times, children and adolescents are excluded from local and regional disaster plans. the unique vulnerabilities of pediatric patients will demand appropriate drills, exercises, equipment, medications, and expertise. thirdly, identifying pediatric risks in a community will help prioritize efforts of planning, especially in those hospitals not accustomed to caring for pediatric patients. finally, a phva helps to develop a framework for global pediatric disaster planning. this can extend beyond a local community and actually advance city, state, regional, and national disaster planning efforts. there has been a development of web-based tools to simplify and enhance the phva process (jacobson and severin ) . after an hva/thira has been completed, the results should be used to help direct and plan drills/exercises based on high impact and high probability threats. it is advised to conduct an hva/thira on an annual basis to assess specific threats unique to your organization's physical structure as well as the surrounding geographic environment. it will also provide insight into whether there is an improvement in previous planning efforts. completion of a population assessment that provides a demographic overview of the community with a breakdown of the childhood population is strongly recommended in conjunction with the hva/ thira. collaborating with other community partners, such as local health departments and emergency management agencies, can assist an organization with the conduction of a comprehensive hva/thira (illinois emergency medical services for children ). please see chap. for further information on hospital planning. pediatric supplies, equipment, and medications will be scarce during a disaster. it will become more of an issue if the health care facility is not accustomed to caring for acutely ill pediatric patients. this will be further exacerbated by a massive surge of acutely ill pediatric patients, a widespread or prolonged disaster, and supply line disruptions. to protect the health security of children and families during a public health emergency, the assistant secretary for preparedness and response (aspr) manages and maintains the strategic national stockpile (sns), a cache of medical countermeasures for rapid deployment and use in response to a public health emergency or disaster (fagbuyi et al. ) . various pediatric-specific supplies and countermeasures are included in the sns. maintaining a supply of medications and medical supplies for specific health threats allows the stockpile to respond with the right product when a specific disease or agent is known. if a community experiences a large-scale public health incident in which the disease or agent is unknown, the first line of support from the stockpile is to send a broad-range of pharmaceuticals and medical supplies. place and martin ) . the emergency equipment and supply lists can easily be adapted for any pediatric disaster emergency (place and martin ) or incident requiring pediatric mass critical care (desmond et al. ) . ageappropriate nutrition, hygiene, bedding, and toys/distraction devices should also be available (illinois emergency medical services for children ) (tables . and . ). endotracheal tubes • uncuffed: . and . mm • cuffed or uncuffed: . , . , . , . , and . mm • cuffed: . , . , . , . , and . mm feeding tubes ( f and f) laryngoscope blades curved: and ; straight: , , , and laryngoscope handle magill forceps (pediatric and adult) nasopharyngeal airways (infant, child, and adult) oropharyngeal airways (sizes - ) stylets for endotracheal tubes (pediatric and adult) suction catheters (infant, child, and adult) tracheostomy tubes (sizes . , . , . , . , . , . , and . mm) yankauer suction tip bag-mask device (manual resuscitator), self-inflating (infant size: ml; adult size: ml) clear oxygen masks (standard and nonrebreathing) for an infant, child, and adult masks to fit bag-mask device adaptor (neonatal, infant, child, and adult sizes) nasal cannulas (infant, child, and adult) nasogastric tubes (sump tubes): infant ( f), child ( f), and adult ( f- f) laryngeal mask airway a vascular access arm boards (infant, child, and adult sizes) catheter over-the-needle device ( - gauge) intraosseous needles or device (pediatric and adult sizes) intravenous catheter-administration sets with calibrated chambers and extension tubing and/or infusion devices with ability to regulate rate and volume of infusate umbilical vein catheters ( . f and . f) b central venous catheters ( . f- . f) intravenous solutions to include normal saline, dextrose % in normal saline, and dextrose % in water fracturemanagement devices extremity splints, including femur splints (pediatric and adult sizes) spine-stabilization method/devices appropriate for children of all ages c (continued) laryngeal mask airways could be shared with anesthesia but must be immediately accessible to the ed b feeding tubes (size f) may be used as umbilical venous catheters but are not ideal. a method for securing the umbilical catheter, such as an umbilical tie, should also be available c a spinal stabilization device is one that can stabilize the neck of an infant, child, or adolescent in a neutral position when a pediatric disaster victim presents acutely ill to the hospital, various emergency interventions will be needed to stabilize the patient. evaluation of the pediatric patient should include a primary survey (abcde), secondary survey (focused sample history and focused physical examination), and diagnostic assessments (laboratory, radiological, and other advanced tests). this will guide further therapeutic interventions. particular attention should be given to the identification of respiratory and/or circulatory derangements of the child, including airway obstruction, respiratory failure, shock, and cardiopulmonary failure. interventions will be based on physiologic derangements of the pediatric patient and determined by the scope of practice and protocols, such as standard resuscitation algorithms for neonatal (american academy of pediatrics and american heart association et al. ) and pediatric (american heart association ) victims. the hcp must be knowledgeable of various emergency medications (table . ) used for children, the appropriate dosages and their mechanism of action, any potential side effects, and drug/drug interactions. other medications, such as antibiotics, antidotes, or countermeasures, may be needed as well. pharmacologic therapy should be initiated immediately based on clinical suspicion and not delayed due to pending laboratory tests (e.g., antibiotics for presumed infection/sepsis or antidotes for suspected nerve agents). dosages should be based on the patient's weight or a length-based weight system. (montello et al. ) or hard copy countermeasure manuals may be more practical, especially during a disaster incident when computer service or internet access may be unreliable. in , the centre for research on the epidemiology of disasters (cred) launched the emergency events database (em-dat). em-dat was created with the initial support of the world health organization (who) and the belgian government. the main objective of the database is to serve the purposes of humanitarian action at national and international levels. the initiative aims to rationalize decision-making for disaster preparedness as well as provide an objective base for vulnerability assessment and priority setting. em-dat contains essential core data on the occurrence and effects of over , mass disasters in the world from to the present day. the database is compiled from various sources, including united nation agencies, nongovernmental organizations (ngos), insurance companies, research institutes, and press agencies (cred ). as described in the cred report entitled natural disasters : lower mortality, higher cost, a disaster is entered into the database if at least one of the following criteria is fulfilled: or more people reported killed; or more people reported affected; declaration of a state of emergency; and/or call for international assistance (cred ). in economic losses, poverty and disasters - : cred/unisdr report, the cred defines a disaster as "a situation or event which overwhelms local capacity, necessitating a request at national or international level for external assistance; an unforeseen and often sudden event that causes great damage, destruction and human suffering" (cred ). the cred em-dat classifies disasters according to the type of hazard that triggers them. the two main disaster groups are natural and technological disasters. there are six natural disaster subgroups. geophysical disasters originate from the solid earth and include earthquake (ground movement and tsunami), dry mass movement (rock fall and landslides), and volcanic activity (ash fall, lahar, pyroclastic flow, and lava flow). lahar is a hot or cold mixture of earthen material flowing on the slope of a volcano either during or between volcanic eruptions. meteorological disasters are caused by short-lived, micro-to meso-scale extreme weather and atmospheric conditions that last from minutes to days and include extreme temperatures (cold wave, heat wave, and severe winter conditions such as snow/ice or frost/ freeze), fog, and storms. storms can be extra-tropical, tropical, or convective. convective storms include derecho, hail, lightning/thunderstorm, rain, tornado, sand/dust storm, winter storm/blizzard, storm/surge, and wind. derecho is a widespread and usually fast-moving windstorm associated with convection/convective storm and includes downburst and straight-line winds. hydrological disasters are caused by the occurrence, movement, and distribution of surface/subsurface freshwater and saltwater and include floods, landslides (an avalanche of snow, debris, mudflow, and rockfall), and wave action (rogue wave and seiche). flood types can be coastal, riverine, flash, or ice jam. climatological disasters are caused by longlived, meso-to macro-scale atmospheric processes ranging from intraseasonal to multidecadal climate variability and include drought, glacial lake outburst, and wildfire (forest fire, land fire: brush, bush, or pasture). biological disasters are caused by the exposure to living organisms and their toxic substances or vectorborne diseases that they may carry and include epidemics (viral, bacterial, parasitic, fungal, and prion), insect infestation (grasshopper and locust), and animal accidents. extraterrestrial disasters are caused by asteroids, meteoroids, and comets as they pass near-earth, enter earth's atmosphere, and/or strike the earth, and by changes in the interplanetary conditions that affect the earth's magnetosphere, ionosphere, and thermosphere. types include impact (airbursts) and space weather (energetic particles, geomagnetic storm, and shockwave) events (cred ). there are three technological disaster subgroups. industrial accidents include chemical spills, collapse, explosion, fire, gas leak, poisoning, radiation, and oil spills. a chemical spill is an accidental release occurring during the production, transportation, or handling of hazardous chemical substances. transport accidents include disasters in the air (airplanes, helicopters, airships, and balloons), on the road (moving vehicles on roads or tracks), on the rail system (train), and on the water (sailing boats, ferries, cruise ships, and other boats). miscellaneous accidents vary from collapse to explosions to fires. collapse is an accident involving the collapse of a building or structure and can either involve industrial structures or domestic/nonindustrial structures (cred ). technological disasters are considered man-made, but as suggested by their subgroup, they are accidental and not intentional. the united nations office for disaster risk reduction (unisdr) and cred report, economic losses, poverty, and disasters - , reviews global natural disasters during that time period, their economic impact, and the relationship with poverty. between and , climate-related and geophysical disasters killed . million people and left a further . billion injured, homeless, displaced, or in need of emergency assistance. although the majority of fatalities were due to geophysical events, mostly earthquakes and tsunamis, % of all disasters was caused by floods, storms, droughts, heatwaves, and other extreme weather events. the financial impact was staggering. in - , disaster-hit countries reported direct economic losses valued at us$ billion, of which climate-related disasters caused us$ billion or % of the total. this was up from % (us$ billion) of losses (us$ billion) reported between and . overall, reported losses from extreme weather events rose by % between these two -year periods. in absolute monetary terms, over the last -years, the usa recorded the biggest losses (us$ billion), reflecting high asset values as well as frequent events. china, by comparison, suffered a significantly higher number of disasters than the usa ( vs. ) but lower total losses (us$ billion) (cred ) (figs. . , . , . , . , . , . , . , . and . in , climate-related and geophysical incidents in the world were estimated with , deaths and over million people impacted. indonesia recorded approximately half of the deaths with india accounting for half of those impacted by disasters. notable features of were intense seismic activity in indonesia, a series of disasters in japan, floods in india, and an eventful year for both volcanic activity and wildfires. however, an ongoing trend of lower death tolls from previous years continued into (centre for research on the epidemiology of disasters (cred) and united nations office for disaster risk reduction (unisdr) ) (tables . , . , . , . , . , . and . there are no specific deviations when medically managing children after a natural disaster. according to sirbaugh and dirocco ( ) "small-scale mass casualty incidents occur daily in the united states. few present unusual challenges to the local medical systems other than in the number of patients that must be treated at one time. except in earthquakes, explosions, building collapses, and some types of terrorist attacks, the same holds true for large-scale disasters. sudden violent disaster mechanisms can produce major trauma cases, including patients needing field amputations or management of crush syndrome. for the most part, medicine after a disaster is much the same as it was before the disaster, with more minor injuries, more people with exacerbations of their chronic illnesses, and number of patients seeking what is ordinarily considered primary care. this is true for children and adults." it should be noted, however, that children have a predisposition to illness and injury after natural disasters. the hcp must be able to identify any health problems and treat the child effectively and efficiently while utilizing standard resuscitation protocols as indicated. traumatic injuries may be seen after any natural disaster. the injuries can range from minor scrapes and bruises to major blunt trauma or traumatic brain injury. children are at increased risk for injury since adults are distracted by recovery efforts and may not be able to supervise them closely. the environment may not be safe due to environmental hazards, such as collapsed buildings, sinkholes, and high water levels. dangerous equipment used during relief efforts may be present, such as heavy earth moving equipment, chainsaws, and power generators. hazardous chemicals, such as gasoline and other volatile hydrocarbons, may be readily accessible or taint the environment. without suitable shelter, children are also exposed to weather, animals, and insects (sirbaugh and dirocco ) . infectious diseases may also pose a problem to children after a natural disaster. infectious patterns will persist during a disaster based on the season and time of year. there may be outbreaks or epidemics of highly contagious infections (e.g., influenza, respiratory syncytial virus, streptococcus pyogenes) due to mass sheltering of children and families. poor nutrition or decreased availability of food may lower their resistance against infections. various water-borne or food-borne diseases may cause illnesses in children. poor hygiene and mass shelter environments may exacerbate these illnesses. immunized children should be protected against common preventable diseases after a natural disaster but still could be a problem in mass groups that are not completely or appropriately immunized. after the haiti earthquake, there were increased cases of diarrhea, cholera, measles, and tetanus in children months after the earthquake despite some level of vaccination (sirbaugh and dirocco ) . children are at risk for various environmental emergencies. austere environments will impact children greatly. heat exposure coupled with minimal access to drinkable water may lead to severe dehydration. exposure to the cold may lead to frostbite or hypothermia. children are at risk for carbon monoxide toxicity due to generator use or natural gas poisoning due to disrupted gas lines. there is always a risk for thermal injury due to the use of candles and other flame sources. exposure to animals (snakes) and insects (spiders) may increase the risk of envenomation. submersion injury and drowning incidents may escalate. this will be due to lack of supervision of children around storm drains, newly formed bodies of water, or rushing waters of storm diversion systems (sirbaugh and dirocco ) . mental health issues are often seen in children after natural disasters. even though a child may not be injured, they may become "psychiatric casualties" due to the horrific sights they have seen during or after the disaster. children and adolescents with behavioral or psychiatric problems may experience worsening symptoms and signs due to stress, trauma, disruption of routines, or availability of medications. this is often exacerbated if the parent, guardian, caregiver, or hcp is also having difficulty coping with the stress of the disaster. in general, the most common mental health problem in children is a post-traumatic stress disorder. however, separation anxiety, obsessive-compulsive symptoms, and severe stranger anxiety can also be seen in children after a traumatic event (sirbaugh and dirocco ) . see chap. for more detailed information. terrorism impacts children and families all around the world (tables . and . ). after the events of / , much attention has been given to the possibility of another mass casualty act of terrorism, especially with weapons of mass destruction, that include chemical, biological, nuclear, radiological, and explosive devices (cbnre), or other forms of violence such as active shooter incidents and mass shootings (jacobson and severin ) . since then, other incidents, both foreign and domestic, have involved children and complicates the concept of and the response to terrorism. johnston ( ) said it best in his review of terrorist and criminal attacks targeting children: "one of the more accepted defining characteristics of terrorism is that it targets noncombatants including men, women, and children. however, terrorist attacks specifically targeting children over other noncombatants are uncommon. this is for the same reason that most terrorists have historically avoided mass casualty terrorism: the shock value is so great that such attacks erode support for the terrorists' political objectives. the / attacks represent an increasing trend in mass casualty terrorism. at the same time, policymakers are examining this evolving threat, they must increasingly consider the threat of terrorist attacks targeting children." based on historical events, it is clear infants, toddlers, children, and adolescents have been victims of terrorism. this global trend of terrorists targeting children seems to be escalating (johnston ) . therefore, it is imperative to understand terrorism and ways it impacts the children and families served by the health care community. combs ( ) defines terrorism as "an act of violence perpetrated on innocent civilian noncombatants in order to evoke fear in an audience". however, she goes on to argue that to become an operational definition, there must also be the addition of a "political purpose" of the violent act. therefore, "terrorism, then, is an act composed of at least four crucial elements: ) it is an act of violence, ) it has a political motive or goal, ) it is perpetrated against civilian noncombatants, and ) it is staged to be played before an audience whose reaction of fear and terror is the desired result." (combs ) . there are different typologies of terrorism. at least five types of terror violence have been suggested by feliks gross: "mass terror is terror by a state, where the regime coerces the opposition in the population, whether organized or unorganized, sometimes in an institutionalized manner. dynastic assassination is an attack on a head of state or a ruling elite. random terror involves the placing of explosives where people gather (such as post offices, railroads, and cafes) to destroy whoever happens to be there. focused random terror restricts the placing of explosives, for example to where significant agents of oppression are likely to gather. finally, tactical terror is directed solely against the ruling government as a part of a 'broad revolutionary strategic plan'" (combs ). an additional typology offered is "lone wolf terror which involves someone who commits violent acts in support of some group, movement, or ideology, but who does stand alone, outside of any command structure and without material assistance from any group" (combs ) . martin ( ) reviews eight different terrorism typologies in the ever shifting, multifaceted world of modern terrorism. the new terrorism "is characterized by the threat of mass casualty attacks from dissident terrorist organizations, new and creative configurations, transnational religious solidarity, and redefined moral justifications for political violence" (martin ) . state terrorism is "committed by governments against perceived enemies and can be directed externally against adversaries in the international domain or internally against domestic enemies" (martin ) . dissident terrorism is "committed by nonstate movements and groups against governments, ethno-national groups, religious groups, and other perceived enemies" (martin ) . religious terrorism is "motivated by an absolute belief that an otherworldly power has sanctioned and commanded the application of terrorist violence for the greater glory of the faith…[it] is usually conducted in defense of what believers consider to be the one true faith" (martin ) . ideological terrorism is "motivated by political systems of belief (ideologies), which champion the self-perceived inherent rights of a particular group or interest in opposition to another group or interest. the system of belief incorporates theoretical and philosophical justifications for violently asserting the rights of the championed group or interest" (martin ) . international terrorism "spills over onto the world's stage. targets are selected because of their value as symbols of international interests, either within the home country or across state boundaries" (martin ) . criminal dissident terrorism "is solely profit-driven, and can be some combination of profit and politics. for instance, traditional organized criminals accrue profits to fund their criminal activity and for personal interests, while criminalpolitical enterprises acquire profits to sustain their movement" (martin ) . gender-selective terrorism "is directed against an enemy population's men or women because of their gender. systematic violence is directed against men because of the perceived threat posed by males as potential soldiers or sources of opposition. systematic violence is directed against women to destroy an enemy group's cultural identity or terrorize the group into submission" (martin ) . the all-hazards national planning scenarios are an integral component of dhs's capabilities-based approach to implementing homeland security presidential directive : national preparedness (hspd- ). the national planning scenarios are planning tools and are representative of the range of potential terrorist and natural disasters and the related impacts that face the nation. the federal interagency community has developed all-hazards planning scenarios for use in national, federal, state, and local homeland security preparedness activities. the objective was to develop a minimum number of credible scenarios to establish the range of response requirements to facilitate disaster planning (dhs ) (table . ). twelve of the scenarios represent terrorist attacks while three represent natural disasters or naturally occurring epidemics. this ratio reflects the fact that the nation has recurring experience with natural disasters but faces newfound dangers, including the increasing potential for use of weapons of mass destruction by terrorists. the scenarios form the basis for coordinated federal planning, training, exercises, and grant investments needed to prepare for all hazards. dhs employed the scenarios as the basis for a rigorous task analysis of prevention, protection, response, and recovery missions and identification of key tasks that supported the development of essential all-hazards capabilities (united states department of homeland security, federal emergency management agency ) (table . ). each of the scenarios follows the same outline to include a detailed scenario description, planning considerations, and implications. for each of the terrorismrelated scenarios, fema national preparedness directorate (npd) partnered with dhs office of intelligence and analysis (i&a) and other intelligence community and law enforcement experts to develop and validate prevention prequels. the prequels provide an understanding of terrorists' motivation, capability, intent, tactics, techniques and procedures, and technical weapons data. the prequels also provide a credible adversary based on known threats to test the homeland security community's ability to understand and respond to indications and warnings of possible terrorist attacks (united states department of homeland security, federal emergency management agency ). a chemical agent of terrorism is defined as any chemical substance intended for use in military operations to kill, seriously injure, or incapacitate humans (or animals) through its toxicological effects. chemicals excluded from this list are riot-control agents, chemical herbicides, and smoke/flame materials. chemical agents are classified as toxic agents (producing injury or death) or incapacitating agents (producing temporary effects). toxic agents are further described as nerve agents (anticholinesterases), blood agents (cyanogens), blister agents (vesicants), and lung-damaging agents (choking agents). incapacitating agents include stimulants, depressants, psychedelics, and deliriants (banks ; departments of the army, the navy, and the air force, and commandant, marine corps ). nerve agents are organophosphate anticholinesterase compounds. they are used in various insecticide, industrial, and military applications. military-grade agents include tabun (ga), sarin (gb), soman (gd), cyclosarin (gf), venom x (vx), and the novichok series. these are all major military threats. the only known battlefield use of nerve agents was the iraq-iran war. however, other nerve agent incidents, such as the tokyo subway attack (sarin), the chemical attacks in syria (chlorine, sarin, mustard), and the attempted assassination of sergei skripal in salisbury, uk (novichok), support that civilian threats also exist. nerve agents are volatile chemicals and can be released in liquid or vapor form. however, the liquid form can become vapor depending upon its level of volatility (e.g, g-agents are more volatile than vx). the level of toxicity depends on the agent, concentration of the agent, physical form, route and length of exposure, and environmental factors (temperature and wind) (tables . and . ). nerve agents exert their effects by the inhibition of esterase enzymes. acetylcholinesterase inhibition prevents the hydrolysis of acetylcholine. the clinical result is a cholinergic crisis and subsequent overstimulation of muscarinic and nicotinic receptors throughout the body including the central nervous system. clinical muscarinic responses include sludge (salivation, lacrimation, urination, defecation, gastrointestinal distress, and emesis) and dumbels (diarrhea, urinary incontinence, miosis/muscle fasciculation, bronchorrhea/bronchospasm/bradycardia, emesis, lacrimation, and salivation). nicotinic responses vary by site. preganglionic sympathetic nerve stimulation produces mydriasis, tachycardia, hypertension, and pallor. however, stimulation at the neuromuscular junction leads to muscular fasciculation and cramping, weakness, paralysis, and diaphragmatic weakness. central nervous system presentations range from anxiety and restlessness to seizures, coma, and death (banks ; rotenberg and newmark ; rotenberg b ). pediatric manifestations (table . ) may vary from the classic clinical responses due to their unique vulnerabilities (hilmas et al. ): • children may manifest symptoms earliest and possibly more severe presentations. • could be hospitalized for similarly related illnesses and diseases. • smaller mass. • lower baseline cholinesterase activity. • tendency to bronchospasm. • pediatric airway and respiratory differences. • altered pulmonary compensation. • lower reserves of cardiovascular system and fluids. • isolated central nervous system signs (stupor, coma). • less miosis. • vulnerability to seizures and neurotransmitter imbalances (excitability). • immature metabolic systems. differential diagnoses include upper or lower airway obstruction, bronchiolitis, status asthmaticus, cardiogenic shock, acute gastroenteritis, seizures, and poisonings (carbon monoxide, organophosphates, and cyanide). diagnostic tests include acetylcholinesterase levels, red blood cell cholinesterase levels, and an arterial blood gas. treatment (tables . and . ) includes decontamination (reactive skin decontamination lotion ® [potassium , -butanedione monoximate], soap and water, and . % hypochlorite solution), supportive care, and administration of nerve agent antidotes (atropine, pralidoxime chloride, and diazepam). atropine is a competitive antagonist of acetylcholine muscarinic receptors and reverses peripheral muscarinic symptoms. it does not restore function at the neuromuscular junction nicotinic receptors. it does, however, treat early phases of convulsions. pralidoxime chloride separates the nerve agent from acetylcholinesterase and restores enzymatic function. it also binds free nerve agent. the major goal is to prevent "aging" of the enzyme (e.g., gd). diazepam provides treatment of nerve agent-induced seizures and prevents secondary neurologic injury. typically, associated seizures are refractory to other antiepileptic drugs. the antiseizure effect of diazepam is enhanced by atropine (banks ; cieslak and henretig ; messele et al. ) . potential medical countermeasures include trimedoxime (tmb ), hi- (an h-series oxime), obidoxime, "bioscavengers" (butyrylcholinesterase, carboxylesterase, organophosphorus acid anhydride hydrolase, and human serum paraoxonase), novel anticonvulsant drugs, n-methyl-d-aspartate (nmda) receptor antagonists (ketamine, dexanabinol), and common immunosuppressants such as cyclosporine a (jokanovic ; merrill et al. ; national institutes of health ; united states department of health and human services ). all patients should be observed closely for electroencephalographic changes and neuropsychiatric pathologies. polyneuropathy, reported after organophosphate insecticide poisoning, has not been reported in humans exposed to nerve agents and has been produced in animals only at unsurvivable doses. the intermediate syndrome has not been reported in humans after nerve agent exposure, nor has it been produced in animals. muscular necrosis has occurred in animals after high-dose nerve agent exposure but reversed within weeks; it has not been reported in humans (banks ). on march , , sergei skripal, a former russian double agent, and his daughter, yulia skripal, were found unresponsive on a park bench in salisbury, uk. they were brought to a nearby hospital and treated for signs consistent with a cholinergic crisis due to a nerve agent exposure. analysis of the skripals found the presence of a secret nerve agent called novichok. further testing found high concentrations of the agent on the front-door handle of his home. one of the investigating police officers, detective sergeant nick bailey, unknowingly touched the door-handle and also became ill. all three survived due to rapid recognition of the nerve agent exposure by hospital personnel. four months later, two other people, dawn sturgess and charlie rowley, became ill with identical symptoms in the town of amesbury, miles from salisbury. they were later confirmed to have high concentrations of novichok on their hands from a perfume bottle found in a recycling bin. both were immediately treated, but dawn sturgess later died. charlie rowley survived. it was believed the discarded perfume bottle contained novichok and was discarded by the assailants after the attempt on sergei skripal. on september , , the uk government revealed that their investigation uncovered two suspects from closed circuit television (cctv) footage near the skripal's home. the suspects entered the uk on russian passports using the names alexander petrov and ruslan boshirov, stayed in a london hotel for days, visited salisbury briefly, and then returned to moscow. minute traces of novichok were also found in the london hotel where they had stayed. the uk prime minister, teresa may, said that the suspects are thought to be officers from russia's military intelligence service the glavnoye razvedyvatel'noye upravleniye (gru), and that this showed that the poisoning was "not a rogue operation" and was "almost certainly" approved at a senior level of the russian state. the two suspects later appeared on russian tv denying the accusations and saying they were just "tourists" who had traveled all the way from moscow to salisbury just to see the "famous cathedral". however, cctv of the cathedral area found no evidence of the two men visiting the cathedral, although they were captured on cctv near the skripal's home. in a development in september , one of the men was revealed as actually being a russian intelligence officer named colonel anatoliy chepiga and was a decorated veteran of russian campaigns in chechnya and ukraine. and later in october, the second man was named as dr. alexander mishkin, a naval medical doctor allegedly recruited by the gru (chai et al. ; may ) . novichok (Новичоќ: russian for "newcomer") is a highly potent nerve agent developed from the russian classified nerve agent program known as foliant. almost everything known about these agents is due to a russian defector, vil mirzayanov ( ) who was an analytical chemist at the russian state research institute of organic chemistry and technology (gosniiokht). he has described the details of the novichok program in his book "state secrets: an insider's chronicle of the russian chemical weapons program". the first three nerve agents of the novichok series developed in the program were substance- , a- , and a- (table . ). they were synthesized as unitary agents, like vx, tabun, soman, and sarin. unitary means that the chemical structure was produced at its maximum potency. however, the novichok agents were developed as binary agents: maximum potency when two inert substances are combined together prior to deployment to create the active nerve agent (cieslak and henretig ) . very little is known about the chemistry of these weaponized organophosphate agents. however, they appear to be more potent than current nerve agents. for example, the ld of novichok agents is reported . μg/kg similar to -(dimethylamino)ethyl n,n-dimethylphosphoramidofluoridate (vg), a novel fourth generation nerve agent. furthermore, novichok- is × more effective than vx and novichock- is × more effective than soman (cieslak and henretig ; hoenig ) . clinically, they behave like other organophosphates by binding to acetylcholinesterase preventing the breakdown of acetylcholine thereby leading to a cholinergic crisis. there appears to be a similar "aging" process as seen with other nerve agents. in addition, the novichok agents binding to peripheral sensory nerves distinguishes this class of organophosphates. prolonged or high-dose exposure results in debilitating peripheral neuropathy. exposure to these agents is fatal unless aggressively managed (cieslak and henretig ) . decontamination is essential to prevent ongoing exposure to the patient and medical personnel. clothing should be removed and quickly placed in a sealed bag (prevents ongoing exposure to the emission of vapors) followed by thorough washing with soap and water. application of dry bleach powder should be avoided as it may hydrolyze nerve agents into toxic metabolites that can produce ongoing cholinergic effects. supportive care is essential. antidote therapy should be given as usual for nerve agents, including atropine, diazepam, and pralidoxime chloride (united states department of health and human services, office of the assistant secretary for preparedness and response, national library of medicine ; united states department of health and human services, chemical hazards emergency medical management (chemm) ). of note, the toxicity of the novichok agents may not rely on anticholinesterase inhibition. some have suggested that reactive oximes like potassium , -butanedione monoximate are preferred oximes for antidotal therapy (cieslak and henretig ) . cyanide is a naturally occurring chemical. it can be found in plants and seeds. it is also used in many industrial applications and is a common product of combustion of synthetic materials. typical cyanogens include hydrogen cyanide (ac) and cyanogen chloride (ck). low levels of cyanide are detoxified by a natural reaction in the human body using the rhodanese system. there is reversible metabolism with vitamin b a to vitamin b (cyanocobalamin). an irreversible reaction occurs with sulfanes to produce thiocyanates and sulfates. the former is excreted via the urinary tract. when cyanide overwhelms this natural process, cyanide binds to ( ) a vx = venom x (cieslak and henretig ) cytochrome oxidase within the mitochondria and disrupts cellular respiration. cyanide has an affinity for fe+ in the cytochrome a complex and oxidative phosphorylation is interrupted. cells can no longer use oxygen to produce atp and lactic acidosis ensues from resultant anaerobic metabolism. when inhaled, cyanide produces rapid onset of clinical signs. findings include transient tachypnea and kussmaul breathing (from hypoxia of carotid and aortic bodies), hypertension and tachycardia (from hypoxia of aortic body), and neurologic findings such as seizures, muscle rigidity (trismus), opisthotonus, and decerebrate posturing. other findings include cherry red flush, acute respiratory failure/ arrest, bradycardia, dissociative shock, and cardiac arrest. venous blood samples exhibit a bright red color. arterial blood gas may demonstrate a metabolic acidosis with an increased anion gap due to lactic acid (banks ; cieslak and henretig ; rotenberg a) . pediatric manifestations (table . ) may vary from the classic clinical responses due to their unique vulnerabilities (hilmas et al. ): • thinner integument leading to shorter time from exposure to symptom development. • higher vapor density (ck) and concentration accumulation in living zone of children, • higher minute ventilation and metabolism. • abdominal pain, nausea, restlessness, and giddiness are common early findings. • cyanosis mostly noted other than classic cherry red flushing of the skin. • resilient with recovery even when just using supportive measures alone. differential diagnoses include meningitis, encephalitis, gastroenteritis, ischemic stroke, methemoglobinemia, and poisonings (nerve agents, organophosphates, methanol, hydrogen sulfide, and carbon monoxide). diagnostic tests include arterial blood gas, lactic acid, and thiocyanate levels. treatment (tables . and . ) includes decontamination, supportive care, and administration of cyanide antidote kit (nitrites and thiosulfate). the nitrites facilitate the production of methemoglobinemia (fe+ ) which attracts cyanide molecules forming cyanmethemoglobin. amyl nitrite pearls are crushed into gauze and placed over the mouth/nose or in a mask used for bag/mask ventilation. sodium nitrite is given parenterally and dosed according to the patient's estimated hemoglobin so as to prevent severe methemoglobinemia. since the formation of cyanmethemoglobin is a reversible reaction, and sodium thiosulfate is given to extract the cyanide. dosing is also dependent upon estimated hemoglobin. along with the naturally occurring rhodanese enzymatic system, the irreversible reaction forms thiocyanate. thiocyanate is water soluble and is excreted harmlessly via the kidneys (banks ; cieslak and henretig ). potential medical countermeasures (national institutes of health ; united states army medical research institute of infectious diseases (usamriid) ) include hydroxocobalamin, cobinamide (a cobalamin precursor), dicobalt edetate, cyanohydrin-forming compounds (alpha-ketoglutarate and pyruvate), s-substituted crystallized rhodanese, sulfur-containing drugs (n-acetylcysteine), and methemoglobin inducers ( -dimethylaminophenol and others). blistering agents, or vesicants, promote the production of blisters. typical examples include sulfur mustard (hd), nitrogen mustard (hn), and lewisite (l). these agents, especially sulfur mustard, are considered capable chemical weapons since illness may not occur until hours or days later. vesicants are alkylating agents that affect rapidly reproducing and poorly differentiated cells in the body. however, they can also produce cellular oxidative stress, deplete glutathione stores, and promote immature cognitive function unable to flee emergency immature coping mechanisms inability to discern threat, follow directions, and protect self high risk for developing ptsd bbb blood-brain barrier, bsa body surface area, cns central nervous system, ptsd post-traumatic stress disorder (hilmas et al. ) intense inflammatory responses. clinical findings are initially cutaneous (erythema, pruritus, yellow blisters, ulcers, and sloughing), respiratory (hoarseness, cough, voice changes, pneumonia, respiratory failure, acute lung injury, and acute respiratory distress syndrome), and ophthalmologic (pain, irritation, blepharospasm, photophobia, conjunctivitis, corneal ulceration, and globe perforation) in nature. after exposure through these primary portals of entry, other sites are affected, including the gastrointestinal tract (nausea, vomiting, and mucosal injury), the hematopoietic system (bone marrow suppression), the cardiovascular system (l), reproductive system (hd, hn) , and the central nervous system (lethargy, headache, malaise, and depression) (banks ; yu et al. ) . pediatric manifestations (table . ) may vary from the classic clinical responses due to their unique vulnerabilities (hilmas et al. ): • thinner integument leading to shorter time from exposure to symptom development. • higher vapor density and concentration accumulation in the living zone. • higher minute ventilation and metabolism. • greater pulmonary injury. • ocular findings more frequent (less self-protection and more hand/eye contact). • gastrointestinal manifestations more prominent. • unable to escape and decontaminate. • unable to verbalize complaints (i.e., pain). treatment (tables . and . ) includes decontamination and supportive care. currently, there are no antidotes for mustard toxicity (cieslak and henretig ) . agents under investigation include antioxidants (vitamin e), anti-inflammatory drugs (corticosteroids), mustard scavengers (glutathione, n-acetylcysteine), and nitric oxide synthase inhibitors (l-nitroarginine methyl ester). other therapeutics under investigation include the use of british anti-lewisite (bal), reactive skin protectants, and ocular therapies (national institutes of health ; usamriid ). lung-damaging agents are toxic inhalants and potentially can affect the entire respiratory tract. typical examples include chlorine (cl ), phosgene (carbonyl chloride), oxides of nitrogen, organofluoride polymers, hydrogen fluoride, and zinc oxide. since many of these chemicals are readily available and have multiple industrial applications, they are considered terrorist weapons of opportunity. toxicity is dependent upon agent particle size, solubility, and method of release. large particles produce injury in the nasopharynx (sneezing, pain, and erythema). midsize particles affect the central airways (painful swelling, cough, stridor, wheezing, and rhonchi). small particles cause injury at the level of the alveoli (dyspnea, chest tightness, and rales). highly soluble agents, such as chlorine, dissolve with mucosal moisture and immediately produce strong upper airway reactions. less soluble agents, such as phosgene, travel to the lower airway before dissolving and subsequently causing toxicity. it is important, however, to realize that very few lungdamaging agents affect only the upper or lower airway (e.g., cl ). if the agent is aerosolized, solid or liquid droplets suspend in the air and distribute by size. if it is a gas or vapor release, there is uniform distribution throughout the lungs and toxicity will be based on solubility and reactivity of the agent (banks ; burklow et al. ; cieslak and henretig ) . pediatric manifestations (table . ) may vary from the classic clinical responses due to their unique vulnerabilities (hilmas et al. ): • pediatric airway and respiratory tract issues (obligate nose breathers, relatively small mouth/large tongue, copious secretions, anterior/cephalad vocal cords, omega or horseshoe-shaped epiglottis, tendency of laryngospasm and bronchospasm, and anatomically small, "floppy" airways). • high vapor density and concentration accumulation in the living zone. • unable to verbalize or localize physical complaints. • rapid dehydration and shock secondary to pulmonary edema. • increased minute ventilation and metabolism. differential diagnoses include smoke inhalation injury, cardiogenic shock, heart failure, traumatic injury, asthma, bronchiolitis, and poisoning (cyanide). treatment (tables . and . ) includes decontamination and supportive care. currently, there are no antidotes for lung-damaging agent toxicity (cieslak and henretig ) . potential countermeasures include novel positive-pressure devices, drugs to prevent lung inflammation, and treatments for chemically induced pulmonary edema (beta agonists, dopamine, insulin, allopurinol, and ibuprofen). in addition, drugs are being investigated that act at complex molecular pathways of the lung the centers for disease control and prevention (cdc) has delineated bioterrorism agents and diseases into three categories based on priority. category a agents include organisms with the highest risk because the ease of dissemination or transmission from person-to-person, result in high mortality rates, have the potential for major public health impact, promote public panic and social disruption, and require special action of public health preparedness. these agents/diseases include smallpox (variola major), anthrax (bacillus anthracis), plague (yersinia pestis), viral hemorrhagic fevers (filoviruses [ebola, marburg] and arenaviruses [lassa, macupo]), botulinum toxin (from clostridium botulinum), and tularemia (francisella tularensis). category b agents, the second highest priority, include those that are moderately easy to disseminate, result in moderate morbidity and low mortality rates, and require specific enhancements of diagnostic capacity and enhanced disease surveillance. these agents/diseases include ricin toxin (ricinus communis), brucellosis (brucella species), epsilon toxin of clostridium perfringens, food safety threats (salmonella species, escherichia coli o :h , shigella), glanders (burkholderia mallei), meliodosis (burkholderia pseudomallei), psitticosis (chlamydia psittaci), typhus fever (rickettsia prowazekii), q fever (coxiella burnetii), staphylococcal enterotoxin b, trichothecenes mycotoxin, viral encephalitis (alphaviruses, such as eastern equine encephalitis, venezuelan equine encephalitis, and western equine encephalitis), and water safety threats (vibrio cholera, cryptosporidium parvum). category c agents have the next priority and include emerging pathogens that could be engineered for mass dissemination because of availability, ease of production and dissemination, and have the potential for high morbidity and mortality rates and major health impact. recognition of a biologic attack is essential. there are various epidemiologic clues to consider when determining whether the outbreak is natural or man-made (markenson et al. ; cieslak ; usamriid ) : • the appearance of a large outbreak of cases of a similar disease or syndrome, or especially in a discrete population. • many cases of unexplained diseases or deaths. • more severe disease than is usually expected for a specific pathogen or failure to respond to standard therapy. • unusual routes of exposure for a pathogen, such as the inhalational route for disease that normally occur through other exposures. • a disease case or cases that are unusual for a given geographic area or transmission season. • disease normally transmitted by a vector that is not present in the local area. • multiple simultaneous or serial epidemics of different diseases in the same population. • a single case of disease by an uncommon agent (smallpox, some viral hemorrhagic fevers, inhalational anthrax, pneumonic plague). • a disease that is unusual for an age group. • unusual strains or variants of organisms or antimicrobial resistance patterns different from those known to be circulating. • a similar or identical genetic type among agents isolated from distinct sources at different times and/or locations. • higher attack rates among those exposed in certain areas, such as inside a building if released indoors, or lower rates in those inside a sealed building if released outside. • outbreaks of the same disease occurring in noncontiguous areas. • zoonotic disease outbreaks. • intelligence of a potential attack, claims by a terrorist or aggressor of a release, and discovery of munitions, tampering, or other potential vehicle of spread (spray device, contaminated letter). one should know the cellular, physiological, and clinical manifestations of each biologic agent. furthermore, knowledge of distinct presentation patterns of children will be helpful to diagnosis. in any event, the ten steps in the management of biologic attack victims, pediatric, or otherwise, should be applied (cieslak and henretig ; cieslak ; usamriid smallpox is caused by the orthopoxvirus variola and was declared globally eradicated in . the disease is highly communicable from person-to-person and remains a threat due to its potential for weaponization. the only stockpiles are at the cdc and at the russian state centre for research on virology and biotechnology. however, clandestine stockpiles in other parts of the world are unknown. since the cessation of smallpox vaccination, the general population has little or no immunity. the three clinical forms of smallpox include ordinary, flat, and hemorrhagic. another form, modified type, occurred in those previously vaccinated who were no longer protected. the asymptomatic incubation period is from to days (average days) after exposure. a prodrome follows that lasts for - days and is marked by fever, malaise, and myalgia. lesions start on the buccal and pharyngeal mucosa. the rash then spreads in a centrifugal fashion, and the lesions are synchronous. initially, there are macules followed by papules, pustules, and scabs in - weeks. other clinical features include extensive fluid loss and hypovolemic shock, nausea, vomiting, diarrhea, bacterial superinfections, viral bronchitis and pneumonitis, corneal ulceration with or without keratitis, and encephalitis. death, if it occurs, is typically during the second week of clinical disease. variola minor caused a mortality of % in unvaccinated individuals. however, the variola major type caused death in % and % in those vaccinated and unvaccinated, respectively. flat (mostly children) and hemorrhagic (pregnant women and immunocompromised) types caused severe mortality in those populations infected. the differential diagnoses for smallpox include chickenpox (varicella), herpes, erythema multiforme with bullae, or allergic contact dermatitis. varicella typically has a longer incubation period ( - days) and minimal or no prodrome. furthermore, the rash distributes in a centripetal fashion and the progression is asynchronous (images . and . ). diagnosis of smallpox is mostly clinical (centers for disease control and prevention a). if considered, contact public health immediately. laboratory confirmation (cdc or who) can be done by dna sequencing, polymerase chain reaction (pcr), restriction fragment-length polymorphism (rflp), real-time pcr, and microarrays. these are more sensitive and specific than the conventional virological and immunological approaches (goff et al. ) . generally, treatment is largely supportive (table . ). fluid losses and hypovolemic shock must be addressed. also, due to electrolyte and protein loss, replacement therapy will be required. bacterial superinfections must be aggressively treated with appropriate antibiotics. biologic countermeasures and antivirals against smallpox are under investigation, including cidofovir, brincidovir (cmx- ), and tecovirimat (st- ). these agents have shown efficacy in orthopoxvirus animal models and have been used to treat disseminated vaccinia infection under emergency use. cidofovir has activity against poxviruses in animal studies (in vitro and in vivo) and some humans (eczema vaccinatum and molluscum contagiosum). brincidovir is an oral formulation of cidofovir with less nephrotoxicity and has recently been announced as an addition to the strategic national stockpile (sns) for patients with smallpox. tecovirimat is a potent and specific inhibitor of orthopoxvirus replication. a recent study found that treatment with tecovirimat resulted in % survival of cynomolgus macaques challenged with intravenous variola virus. the disease was milder in tecovirimat-treated survivors and viral shedding was reduced compared to placebo-treated survivors. prophylaxis comes in the form of the smallpox vaccine (vaccinia virus), acam ® , which replaced wyeth dryvax™ in . safety profile of the two vaccines appears to be similar. side effects of vaccination range from low-grade fever and axillary lymphadenopathy to inadvertent inoculation of the virus to other body sites to generalized vaccinia and cardiac events (myopericarditis). rare, but typically fatal complications include progressive vaccinia, eczema vaccinatum, postvaccination encephalomyelitis, and fetal vaccinia. modified vaccinia ankara (mva) smallpox vaccine (bavarian nordic's imvamune ® ) is a live, highly attenuated, viral vaccine that is under development as a future nonreplicating smallpox vaccine (greenberg et al. ; kennedy and greenberg ). passive immunoprophylaxis exists in the form of vaccinia immune globulin (vig) and is used for primarily treating complications from smallpox vaccine. limited information suggests that vig may be of use in postexposure prophylaxis of smallpox if given the first week after exposure and with vaccination. monoclonal antibodies may represent another form of immunoprophylaxis. postexposure administration of human monoclonal antibodies has protected rabbits from a lethal dose of an orthopoxvirus. as mentioned, smallpox is highly communicable person-to-person (table . ). contact precautions with full personal protective equipment (ppe) are required. airborne isolation with the use of an n- mask is needed for baseline protection. an n- mask or powered airpurifying respirator (papr) is recommended for protection during high risk procedures (beigel and sandrock ; goff et al. ; rotz et al. ; pittman et al. ; usamriid ). anthrax is caused by the aerobic, spore-forming, nonmotile, encapsulated gram-positive rod bacillus anthracis. it is a naturally occurring disease in herbivores. humans contract the illness by handling contaminated portions of infected animals, especially hides and wool. infection is introduced by scratches or abrasions on the skin. there is concern for potential aerosol dispersal leading to intentional infection through inhalation: it is fairly easy to obtain, capable of large quantity production, stable in aerosol form, and highly lethal. anthrax spores enter the body via skin, ingestion, or inhalation. the spores germinate inside macrophages and become vegetative bacteria. the vegetative form is released, replicates in the lymphatic system, and produces intense bacteremia. the production of virulence factors leads to overwhelming sepsis. the main virulence factors are encoded on two plasmids. one produces an antiphagocytic polypeptide capsule. the other contains genes for the synthesis of three proteins it secretes: protective antigen, edema factor, and lethal factor. the combination of protective antigen with lethal factor or edema factor forms binary cytotoxins, lethal toxin, and edema toxin. the anthrax capsule, lethal toxin, and edema toxin act in concert to drive the disease. three clinical syndromes occur with anthrax: cutaneous, gastrointestinal, and inhalational. cutaneous anthrax is the most common naturally occurring form. after an individual is exposed to infected material or the agent itself, there is a - day (average days) incubation period. a painless or pruritic papule forms at the site of exposure. the papule enlarges and forms a central vesicle, which is followed by erosion into a coal-black but painless eschar. edema surrounds the area and regional lymphadenopathy may occur. gastrointestinal anthrax is rare. typically, it develops after ingestion of viable vegetative organisms found in undercooked meats of infected animals. the two forms of gastrointestinal anthrax, oropharyngeal and intestinal, have incubation periods of - days. the oropharyngeal form is marked by fever and severe pharyngitis followed by ulcers and pseudomembrane formation. other findings include dysphagia, regional lymphadenopathy, unilateral neck swelling, airway compromise, and sepsis. the intestinal form begins with fever, nausea, vomiting, and abdominal pain. bowel edema develops which leads to mesenteric lymphadenitis with necrosis, shock, and death. endemic inhalational anthrax (woolsorters' disease) is also extremely rare and is due to inhaling spores. therefore, any case of inhalational anthrax should be assumed to be due to intentional exposure until proven otherwise. the incubation period is - days but can be up to days. there is a prodrome of - days consisting of fever, malaise, and cough. within h, the disease rapidly progresses to respiratory failure, hemorrhagic mediastinitis (wide mediastinum), septic shock, multiorgan failure, and death. patients with inhalational anthrax may also have hemorrhagic meningitis. mortality is greater than % in - h despite aggressive treatment of inhalational anthrax. the differential diagnoses of ulceroglandular lesions include antiphospholipid antibody syndrome, brown recluse spider bite, coumadin/heparin necrosis, cutaneous leishmaniasis, cutaneous tuberculosis, ecthyma gangrenosum, glanders, leprosy, mucormycosis, orf, plague, rat bite fever, rickettsial pox, staphylococcal/ streptococcal ecthyma, tropical ulcer, tularemia, and typhus. the differential diagnoses of ulceroglandular syndromes include cat scratch fever, chancroid, glanders, herpes, lymphogranuloma venereum, melioidosis, plague, staphylococcal and streptococcal adenitis, tuberculosis, and tularemia. the differential diagnoses for inhalational anthrax include influenza and influenza-like illnesses from other causes. the differential diagnoses of mediastinal widening include normal variant, aneurysm, histoplasmosis, sarcoidosis, tuberculosis, and lymphoma. the diagnosis of anthrax is by culture and gram stain of the blood, sputum, pleural fluid, cerebrospinal fluid, or skin. specimens must be handled carefully, especially by lab personnel and those performing autopsies. elisa and pcr are available at some reference laboratories. the chest radiograph of inhalational anthrax shows the classic widening of the mediastinum. additional findings include hemorrhagic pleural effusions, air bronchograms, and/or consolidation (purcell et al. ). supportive treatment is indicated, including mechanical ventilation, pleural effusion drainage, fluid and electrolyte support, and vasopressor administration. for inhalational anthrax, antibiotic treatment is unlikely to be effective unless started before respiratory symptoms develop. treatment (table . ) includes ciprofloxacin (or levofloxacin or doxycycline), clindamycin, and penicillin g. raxibacumab, a monoclonal antibody, was approved by the fda in for the treatment of inhalational anthrax in combination with recommended antibiotic regimens and prophylaxis for inhalational anthrax when other therapies are unavailable or inappropriate. it works by inhibiting anthrax antigen binding to cells and, therefore, prevents toxins from entering cells (kummerfeldt ) . the adult dose is mg/kg given iv over h and min. the dose for children is weight based; ≤ kg: mg/kg; > - kg: mg/kg; > kg: mg/kg. premedication with diphenhydramine iv or po is recommended h before the infusion. it can also be used as postexposure prophylaxis in high risk spore exposure cases (cieslak and henretig ; migone et al. ; the medical letter ). obiltoxaximab (anthim) is a recently approved monoclonal antibody treatment for inhalational anthrax in combination with recommended antibiotic regimens and prophylaxis for inhalational anthrax when other therapies are unavailable or inappropriate. adults and children > kg should receive a single obiltoxaximab dose of mg/kg. the recommended dose is mg/kg for children > - kg and mg/kg for those weighing ≤ kg. premedication with diphenhydramine is recommended to reduce risk of hypersensitivity reactions (the medical letter ). in patients with inhalational anthrax, intravenous anthrax immune globulin (anthrasil) should be considered in addition to appropriate antibiotic therapy (mytle et al. ; the medical letter ; usamriid ). postexposure prophylaxis includes ciprofloxacin (or levofloxacin or doxycycline) for days plus administration of vaccine; since spores can persist in human in addition to appropriate antibiotic regimen, monoclonal antibody therapy (see text for dosing) and intravenous anthrax immune globulin should be administered for inhalational anthrax c levofloxacin or ofloxacin may be an acceptable alternative to ciprofloxacin d rifampin or clarithromycin may be acceptable alternatives to clindamycin as a drug that targets bacterial protein synthesis. if ciprofloxacin or another quinolone is employed, doxycycline may be used as a second agent because it also targets protein synthesis e ampicillin, imipenem, meropenem, or chloramphenicol may be acceptable alternatives to penicillin as drugs with good cns penetration f assuming the organism is sensitive, children may be switched to oral amoxicillin ( - mg/kg/d divided q h) to complete a -day course. the first days of therapy of postexposure prophylaxis, however, should include ciprofloxacin or levofloxacin and/or doxycycline regardless of age. vaccination should also be provided; if not, antibiotic course will need to be longer g according to most experts, ciprofloxacin is the preferred agent for oral prophylaxis h ten days of therapy may be adequate for endemic cutaneous disease. a full -day course is recommended in the setting of terrorism, however, because of the possibility of concomitant inhalational exposure tissues for a long time, antibiotics must be given for a longer period if vaccine is not also given. the anthrax vaccine adsorbed (ava biothrax™) is derived from sterile culture fluid supernatant taken from an attenuated strain of bacillus anthracis and does not contain any live or dead organisms. the vaccine is given . ml intramuscularly at and weeks then at , , and months followed by yearly boosters (pittman et al. ; usamriid ) . consult with cdc for current pediatric recommendations. anthrax is not contagious in the vegetative form during clinical illness (table . ). contact with infected animals increases likelihood of spread. therefore, contact should be limited and the use of appropriate ppe in endemic areas is indicated (beigel and sandrock ; purcell et al. ; usamriid ) . plague is caused by yersinia pestis, a nonmotile, nonsporulating gram-negative bacterium. it is a zoonotic disease of rodents. it is typically found worldwide and is endemic in western and southwestern states. humans develop the disease after contact with infected rodents, or being bitten by their fleas. after a rodent population dies off, the fleas search for other sources of blood, namely humans. this is when large outbreaks of human plague occur. pneumonic plague is a very rare disease and when it is present in a patient, it may be highly suspicious for intentional dispersal of this deadly agent. three clinical syndromes occur with plague: bubonic plague ( %), septicemic plague ( %), and primary pneumonic plague ( - %). bubonic plague occurs after an infected flea bites a human. after an incubation period of - days, there is onset of high fever, severe malaise, headache, myalgias, and nausea with vomiting. almost % have abdominal pain. around the same time, a characteristic bubo forms which is tender, erythematous, and edematous without fluctuation. buboes typically form in the femoral or inguinal lymph nodes, but other areas can be involved as well (axillary, intraabdominal). the spleen and liver can be tender and palpable. the disease disseminates without therapy. severe complications can ensue, including pneumonia, meningitis, sepsis, and multiorgan failure. pneumonia is particularly concerning since these patients are extremely contagious. mortality of untreated bubonic plague is %, but % with efficient and effective treatment. septicemic plague is characterized by acute fever followed by sepsis without bubo formation. the clinical syndrome is very similar to other forms of gram-negative sepsis: chills, malaise, tachycardia, tachypnea, hypotension, nausea, vomiting, and diarrhea. in addition to sepsis, disseminated intravascular coagulation can ensue leading to thrombosis, necrosis, gangrene, and the formation of black appendages. multiorgan failure can quickly follow. untreated septicemic plague is almost % fatal versus - % in those treated. pneumonic plague is very rare and should be considered due to an intentional aerosol release until proven otherwise. the incubation period is relatively short at - days. sudden fever, cough, and respiratory failure quickly follow. this form produces a fulminant pneumonia with watery sputum that usually progresses to bloody. within a short period of time, septic shock and disseminated intravascular coagulation develop. ards and death may occur. mortality rate of pneumonic plague is very high but may respond to early treatment. plague meningitis is a rare complication of plague. it can occur in % of patients with septicemia and pneumonic forms and is more common in children. usually occurring a few weeks into the illness, it affects those receiving subtherapeutic doses of antibiotics or bacteriostatic antibiotics that do not cross the blood-brain barrier (tetracyclines). fever, meningismus, and other meningeal signs occur. plague meningitis is virtually indistinguishable from meningococcemia. the differential diagnoses of bubonic plague include tularemia, cat scratch fever, lymphogranuloma venereum, chancroid, scrub typhus, and other staphylococcal and streptococcal infections. the differential diagnoses of septicemic plague should include meningococcemia, other forms of gram-negative sepsis, and rickettsial diseases. the differential diagnosis of pneumonic plague is very broad. however, sudden appearance of previously healthy individuals with rapidly progressive gram-negative pneumonia with hemoptysis should strongly suggest pneumonic plague due to intentional release. diagnosis can be made clinically as previously described. demonstration of yersinia pestis in blood or sputum is paramount. methylene blue or wright's stain of exudates may reveal the classic safety-pin appearance of yersinia pestis. culture on sheep blood or macconkey agar demonstrates beaten-copper colonies ( h) followed by fried-egg colonies ( h). detection of yersinia pestis f -antigen by specific immunoassay is available, but the result is available retrospectively. chest radiograph of patients will demonstrate patchy infiltrates (centers for disease control and prevention a; worsham et al. ) . treatment includes mechanical ventilation strategies for ards, hemodynamic support (fluid and vasopressor administration), and antimicrobial agents (table . ). gentamicin or streptomycin is the preferred antimicrobial treatment. alternatives include doxycycline or ciprofloxacin or levofloxacin or chloramphenicol. in cases of meningitis, chloramphenicol is recommended due to its ability to effectively cross the blood-brain barrier. streptomycin is in limited supply and is available for compassionate use. it should be avoided in pregnant women. postexposure prophylaxis includes doxycycline or ciprofloxacin. no licensed plague vaccine is currently in production. a previous licensed vaccine was used in the past. it only offered protection against bubonic plague but not aerosolized yersinia pestis. the plague bacterium secretes several virulence factors (fraction (f ) and v (virulence) proteins) that as subunit proteins are immunogenic and possess protective properties. recently, an f -v antigen (fusion protein) vaccine developed by usamriid provided % protection in monkeys against high-dose aerosol challenge. there is no passive immunoprophylaxis (i.e., immune globulin) available for pre-or postexposure of plague (usamriid ). use of standard precautions for patients with bubonic and septicemic plague is indicated. suspected pneumonic plague will require strict isolation with respiratory droplet precautions for at least h after initiation of effective antimicrobial therapy, or until sputum cultures are negative in confirmed cases. an n- respirator should be used for baseline protection (table . ). it is also recommended to use an n- respirator or papr for high risk procedures (beigel and sandrock ; ; centers for disease control and prevention ; centers for disease control and prevention b; pittman et al. ; usamriid ) . in a mass casualty setting, parenteral therapy might not be possible. in such cases, oral therapy (with analogous agents) may need to be used b ofloxacin (and possibly other quinolones) may be acceptable alternatives to ciprofloxacin or levofloxacin; however, they are not approved for use in children c concentration should be maintained between and μg/ml. some experts have recommended that chloramphenicol be used to treat patients with plague meningitis, because chloramphenicol penetrates the blood-brain barrier. use in children younger than may be associated with adverse reactions but might be warranted for serious infections d ribavirin is recommended for arenavirus or bunyavirus infections and may be indicated for a viral hemorrhagic fever of an unknown etiology although not fda approved for these indications. for intravenous therapy use a loading dose: kg iv once (max dose, g), then mg/kg iv q h for days (max dose, g), and then mg/kg iv q h for days (max dose, mg). in a mass casualty setting, it may be necessary to use oral therapy. for oral therapy, use a loading dose of mg/kg po once, then mg/kg/day po in divided doses for days viral hemorrhagic fever has a variety of causative agents. however, the syndromes they produce are characterized by fever and bleeding diathesis. the etiologies include rna viruses from four distinct families: arenaviridae, bunyaviridae, filoviridae, and flaviviridae. the filoviridae (includes ebola and marburg) and arenaviridae (includes lassa fever and new world viruses) are category a agents. based on multiple identified characteristics, there is strong concern for the weaponization potential of the viral hemorrhagic fevers. specifically, there has been demonstration of high contagiousness in aerosolized primate models. there are five identified ebola species, but only four are known to cause disease in humans. the natural reservoir host of ebola virus remains unknown. however, on the basis of evidence and the nature of similar viruses, researchers believe that the virus is animal-borne and that bats are the most likely reservoir. four of the five virus strains occur in an animal host native to africa. marburg virus has a single species. geographic distribution of ebola and marburg is africa (fitzgerald et al. ). both diseases are very similar clinically. incubation period is typically - days with a range of - days. symptoms may include fever, chills, headache, myalgia, nausea, and vomiting. there is rapid progression to prostration, stupor, and hypotension. the onset of a maculopapular rash on the arms and trunk is classic. disseminated intravascular coagulation and thrombocytopenia develops with conjunctival injection, petechiae, hemorrhage, and soft tissue bleeding. there is a possible central nervous system and hepatic involvement. bleeding, uncompensated shock, and multiorgan failure are seen. high viral load early in course is associated with poor prognosis. death usually occurs during the second week of illness. mortality rate of marburg is - % and for ebola - %. in a retrospective cohort study of children during the / ebola outbreak in liberia and sierra leone (all less than years with a median age of years with one-third less than years of age), the most common features upon presentation were fever, weakness, anorexia, and diarrhea. about % were initially afebrile. bleeding was rare upon initial presentation. the overall case fatality rate was %. factors associated with death included children less than years of age, bleeding at any time during hospitalization, and high viral load (smit et al. ) . in another retrospective cohort study of children at two ebola centers in sierra leone in (all less than years of age), presenting symptoms included weakness, fever, anorexia, diarrhea, and cough. about % were afebrile on presentation. the case fatality rate was higher in children less than years ( %) versus - years of age ( %) and times more likely to die if child had a higher viral load. signs associated with death included fever, emesis, and diarrhea. interestingly, hiccups, bleeding, and confusion were only observed in children who died (shah et al. ) . lassa virus and new world viruses (junin, machupo, sabia, and guanarito) are transmitted from person-to-person. the vector in nature is the rodent. the incubation period is from to days. the geographical distribution is west africa and south america, respectively. the south american hemorrhagic fevers are quite similar but differ from lassa fever. the onset of the south american viruses is insidious and results in high fever and constitutional symptoms. petechiae or vesicular enanthem with conjunctival injection is common. these fevers are associated with neurologic disease (hyporeflexia, gait abnormalities, and cerebellar dysfunction). seizures portend a poor prognosis. mortality ranges from % to over %. on the contrary, lassa viruses are mild. less than % of infections result in severe disease. signs include chest pain, sore throat, and proteinuria. hemorrhagic disease is uncommon. other features include neurologic disease such as encephalitis, meningitis, cerebellar disease, and cranial nerve viii deafness (common feature). mortality can be as high as %. differential diagnoses include malaria, meningococcemia, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and typhoid fever. diagnosis is through detection of the viral antigen testing by elisa or viral isolation by culture at the cdc. no specific therapy is present and generally involves supportive care, especially mechanical ventilation strategies for ards, hemodynamic support, and renal replacement therapy. for the arenaviridae and bunyaviridae groups, ribavirin may be indicated ( (pittman et al. ) . there is no current vaccine for ebola that is licensed by the fda. an experimental vaccine called rvsv-zebov was found to be highly protective against ebola virus in a trial conducted by the world health organization (who) and other international partners in guinea in . fda licensure for the vaccine is expected in . until then, , doses have been committed for an emergency use stockpile under the appropriate regulatory mechanism in the event and an outbreak occurs before fda approval is received (centers for disease control and prevention b; henao-restrepo et al. ) . another ebola vaccine candidate, the recombinant adenovirus type- ebola vaccine, was evaluated in a phase trial in sierra leone in . an immune response was stimulated by this vaccine within days of vaccination and strict contact precautions (hand hygiene, double gloves, gowns, shoe and leg coverings, and face shield or goggles) and droplet precautions (private room or cohorting, surgical mask within ft) are mandatory for viral hemorrhagic fevers. airborne precautions (negative-pressure isolation room with - air exchanges per h) should also be instituted to the maximum extent possible and especially for procedures that induce aerosols (e.g., bronchoscopy). at a minimum, a fit-tested, hepa filter-equipped respirator (e.g., an n- mask) should be used, but a battery-powered papr or a positive pressure-supplied air respirator should be considered for personnel sharing an enclosed space with, or coming within ft of, the patient. multiple patients should be cohorted in a separate ward or building with a dedicated airhandling system when feasible (table . ). environmental decontamination is accomplished with hypochlorite or phenolic disinfectants (beigel and sandrock ; radoshitzky et al. ; usamriid ; won and carbone ) . francisella tularensis, a small aerobic, nonmotile gram-negative coccobacillus, causes tularemia (rabbit fever). clinical disease is caused by two isolates, biovars jellison type a and b. this organism can be stabilized for weaponization and delivered in a wet or dry form. the incubation period is usually - days (range - days). initial symptoms are nonspecific and mimic the flu-like symptoms or other upper respiratory tract infections. there is acute onset of fever with chills, myalgias, cough, fatigue, and sore throat. the two clinical forms of tularemia are typhoidal and ulceroglandular diseases. typhoidal tularemia ( - %) occurs after inhalational exposure and sometimes intradermal or gastrointestinal exposures. there is abrupt onset of fever, headache, malaise, myalgias, and prostration. it presents without lymphadenopathy. nausea, vomiting, and abdominal pain are sometimes present. untreated, there is a % mortality rate in naturally acquired cases (vs. - % in those treated). it is higher if pneumonia is present. this form would be most likely seen during an aerosol release of the agent. ulceroglandular tularemia ( - %) occurs through skin or mucus membrane inoculation. there is abrupt onset of fever, chills, headache, cough, and myalgias along with a painful papule at the site of exposure. the papule becomes a painful ulcer with tender regional lymph nodes. skin ulcers have heaped up edges. in - %, there is focal lymphadenopathy without an apparent ulcer. lymph nodes may become fluctuant and drain when receiving antibiotics. without treatment, they may persist for months or even years. in some cases ( - %), the primary entry port is the eye leading to oculoglandular tularemia. patients have unilateral, painful, and purulent conjunctivitis with local lymphadenopathy. chemosis, periorbital edema, and small nodular granulomatous lesions or ulceration may be found. oropharyngeal tularemia with pharyngitis may occur in % of patients. findings include exudative pharyngitis/tonsillitis, ulceration, and painful cervical lymphadenopathy. the differential diagnosis is antibiotic unresponsive pharyngitis, infectious mononucleosis, and viral pharyngitis. pulmonary involvement ( - %) is seen in naturally occurring disease. it ranges from mild to fulminant. various processes include pneumonia, bronchiolitis, cavitary lesions, bronchopleural fistulas, and chronic granulomatous diseases. left untreated, % will die. differential diagnoses include those for typhoidal (typhoid fever, rickettsia, and malaria) or pneumonic (plague, mycoplasma, influenza, q-fever, and staphylococcal enterotoxin b) tularemia. diagnosis should be considered when there is a cluster of nonspecific, febrile, systemically ill patients who rapidly progress to fulminant pneumonitis. tularemia can be diagnosed by recovering the organism from sputum (pcr or dfa) or serology at a state health laboratory. chest radiograph is nonspecific with possible hilar adenopathy. treatment is streptomycin or gentamicin (table . ). alternatives include doxycycline, ciprofloxacin, or chloramphenicol. a live-attenuated vaccine (ndbr ) exists and typically used for laboratory personnel working with francisella tularensis. there is no passive immunoprophylaxis. ciprofloxacin or doxycycline can be given as pre-and postexposure prophylaxis (beigel and sandrock ; hepburn et al. ; pittman et al. ; usamriid ) . botulinum neurotoxins (bont) are produced from the spore-forming, gram-positive, obligate anaerobe clostridium botulinum. it is the most potent toxin known to man. a lethal dose is ng per kilogram. it is , times more toxic than sarin (gb). there are seven serotypes of botulinum toxin (a through g). a new serotype (h) has been tentatively identified in a case of infant botulism but has not been fully investigated. most common are serotypes a, b, and e. the toxin acts on the presynaptic nerve terminal of the neuromuscular junction and cholinergic autonomic synapses. this disrupts neurotransmission and leads to clinical findings. there are three forms of botulism: foodborne, wound, and intestinal (infant or adult intestinal). botulinum toxin can also be released as an act of bioterrorism via ingestion or aerosol forms. incubation can be from h after exposure to several days later. clinical findings of botulism include cranial nerve palsies such as ptosis, diplopia, and dysphagia. this is followed by symmetric descending flaccid paralysis. however, the victim remains afebrile, alert, and oriented. death is typically due to respiratory failure. prolonged respiratory support is often required ( - months). differential diagnoses include guillain-barre syndrome, myasthenia gravis, tick paralysis, stroke, other intoxications (nerve gas, organophosphates), inflammatory myopathy, congenital and hereditary myopathies, and hypothyroidism. diagnosis is mostly clinical. laboratory confirmation can be obtained by bioassay of patient's serum. other assays include immunoassays for bacterial antigen, pcr for bacterial dna, and reverse transcriptase-pcr for mrna to detect active synthesis of toxin. cerebrospinal fluid demonstrates normal protein (unlike guillain-barre syndrome). emg reveals augmentation of muscle action potential with repetitive nerve stimulation at - hz. treatment (table . ) is mainly supportive including intubation and ventilator support. tracheostomy may be required due to prolonged respiratory weakness and failure. antibiotics do not play a role in treatment. botulism antitoxin heptavalent [a, b, c, d, e, f, g]-equine (bat) was approved by the fda in . bat was developed at usamriid as one of two equine-derived heptavalent bont antitoxins. bat is approved to treat individuals with symptoms of botulism following a known or suspected exposure. it has the potential to cause hypersensitivity reactions in those sensitive to equine proteins. the safety of bat in pregnant and lactating women is unknown. evidence regarding safety and efficacy in the pediatric population is limited. in , the fda approved botulinum immune globulin intravenous (babybig), a human botulism immune globulin derived from pooled plasma of adults immunized with pentavalent botulinum toxoid. it is indicated for the treatment of infants with botulism from toxin serotypes a and b. immediately after clinical diagnosis of botulism, adults (including pregnant women) and children should receive a single intravenous infusion of antitoxin (bat or, for infants with botulism from serotypes a or b, babybig) to prevent further disease progression. the administration of antitoxin should not be delayed for laboratory testing to confirm the diagnosis. the pentavalent toxoid vaccine (previously for protection against a, b, c, d, and e; but not f or g) is no longer available as of . no replacement vaccine is currently available. standard isolation precautions (table . ) should be followed (beigel and sandrock ; dembek et al. ; pittman et al. ; timmons and carbone ; usamriid ). ricin is a potent cytotoxin derived from the castor bean plant ricinus communis. it is related in structure and function to shiga toxins and shiga-like toxin of shigella dysenteriae and escherichia coli, respectively. it consists of two glycoprotein subunits, a and b, connected by a disulfide bond. the b-chain allows the toxin to bind to cell receptors and gain entrance into the cell. once ricin enters the cell, the disulfide chemical linkage is broken. the free a chain then acts as an enzyme and inactivates ribosomes thereby disrupting normal cell function. cells are incapable of survival and soon die. ricin has a high terrorist potential due to it characteristics: readily available, ease of extraction, and notoriety (maman and yehezkelli ) . three modes of exposure exist: oral, inhalation, and injection. four to eight hours after inhalation exposure, the victim develops fever, chest tightness, cough, dyspnea, nausea, and arthralgias. airway necrosis and pulmonary capillary leak ensues within - h. this is followed quickly by severe respiratory distress, ards, and death due to hypoxemia within - h. injection may cause minimal pulmonary vascular leak. pain at the site and local lymphadenopathy may occur. however, it may be followed by nausea, vomiting, and gastrointestinal hemorrhage. ingestion leads to necrosis of the gastrointestinal mucosa, hemorrhage, and organ necrosis (spleen, liver, and kidney). diagnosis is suspected when multiple cases of acute lung injury occur in a geographic cluster. serum and respiratory secretions can be checked for antigen using elisa. pulmonary intoxication is managed by mechanical ventilation. gastrointestinal toxicity is managed by gastric lavage and use of cathartics. activated charcoal has little value due to the size of ricin molecules. supportive care is indicated for injection exposure. in general, treatment is largely supportive, especially for pulmonary edema that can result from the capillary leak. there is no vaccine available or prophylactic antitoxin for human use. however, there are two ricin vaccines in the development that focus on the ricin toxin a (rta) chain subunit. a mutant recombinant rta chain vaccine, rivax, has been shown to be safe and immunogenic in humans. the other vaccine is another recombinant rta chain vaccine, rvec . it has shown effectiveness in animal models by producing protective immunity against aerosol challenge with ricin in animal models. standard precautions are advised for health care workers (pittman et al. ; roxas-duncan et al. ; traub ; usamriid ). recent events which include the nuclear reactor meltdown at fukushima and international tension between nuclear powers, spark concern over potential devastation from nuclear catastrophes. there are numerous examples of radiation disasters in history. sixty-six thousand people were killed in hiroshima and thirty-nine thousand people were killed in nagasaki from nuclear bombs detonated over these cities in (avalon project-documents in law, history and diplomacy n.d.). many other people suffered from long-term consequences of radiation poisoning. in , , square kilometers of land in russia, ukraine, and belarus were contaminated with radiation from a meltdown at a nuclear power plant in chernobyl, ukraine. one hundred and thirty-five thousand people were permanently evacuated from their homes (likhtarev et al. ) . long-term health consequences included many children who developed thyroid cancer several years later. many of these children died. a tsunami pummeled the east coast of japan in march of . the power outage that ensued at the fukushima power plant led to a failure of the cooling system of the fuel rods, leading to a meltdown of four of the reactors at the plant. a massive quantity of radiation was released into the atmosphere, forcing people to evacuate their homes indefinitely. creative thinking and heroic actions by the tokyo fire department prevented entire populations of cities from being poisoned with radiation. terrorism experts are concerned that terrorist organizations will produce and detonate a radiological dispersion device (rdd), sometimes referred to as a dirty bomb. this is a conventional explosive, loaded with radioactive material which would be dispersed upon detonation. this would likely involve only one radioisotope. fewer people would be exposed and a smaller area would be contaminated than what would transpire with the detonation of a nuclear weapon. spreading fear and panic would be the primary purpose of such a device (mettler jr and voelz ) . radiation is the emission and propagation of energy through space or through a medium in the form of waves. radiation can be ionizing or nonionizing depending on the amount of energy released. most radiation that people encounter is low energy and, therefore, nonionizing with no biological effects. ionizing radiation emits enough energy to strip electrons from an atom, which provokes cellular changes and thereby, results in biological effects. radiation emitted from nuclear decay is always ionizing (radiation emergency assistance center/training site (react/s-cdc) ). atomic nuclei are held together by a very powerful binding energy despite positively charged protons repelling each other. this energy is released from unstable nuclei in the form of electromagnetic waves or particles. when ionizing radiation reaches biological tissue, chemical bonds are disrupted, free radicals are produced, and dna is broken. electromagnetic waves are of two types, x-rays and gamma rays. x-rays are relatively low energy and less penetrating. gamma rays have a shorter wavelength and contain relatively higher energy, making them more penetrating of biological tissue. ionizing radiation in the form of particles consists of alpha particles, beta particles, and neutrons. alpha particles are the largest of the forms of particulate radiation. they are composed of two neutrons and two protons. they do not easily penetrate solid surfaces, including clothes and skin. however, they can cause severe damage to an organism if internalized. in , in the united kingdom, alexander litvienko, an ex kgb agent was poisoned with a radioactive element called polonium (mcphee and leikin ). a small amount of polonium was sprinkled into his food. polonium releases alpha particles when it decays. it was relatively safe for the assassin to carry this element with him because of the relatively poor ability of alpha particles to penetrate clothing and skin. once it is ingested, however, alpha particles have profound biological effects. mr. litvienko became very ill, and ultimately died. beta particles are high energy electrons discharged from the nucleus and are highly penetrating. neutrons emitted from a nucleus are also highly penetrating. in general, neutrons are only released by the detonation of a nuclear weapon. ionizing radiation of any form cannot be detected by our senses. it is not smelled, felt by touch, tasted, or seen. it is possible to be exposed to a lethal dose of radiation without realizing it. in goiania, brazil, in , children found a canister of radioactive cesium ( cs) that had been looted from a medical center and left in the street. the children liked the appearance of the substance but were not able to sense any abnormalities or danger with it. they began to rub it on their bodies because they liked the way it made them glow in the dark. the children all became ill. ultimately, people were exposed to this radioisotope. it took days before physicians recognized that the people had radiation poisoning. four people died of acute radiation syndrome. four factors determine the severity of exposure to ionizing radiation: time, distance, dose, and shielding. time is the time of exposure to the radiation source. distance is the distance from the radiation source. based on the inverse square law, exposure is reduced exponentially with increasing distance from the radiation source. dose is measured by the amount of energy released by the source and is numerically described by how many disintegrations per second occur, in curies (ci) or becquerels (bq). shielding is the efficacy of the barrier to the radioactive source. lead is well-known to be a very effective shield to x-rays. in a radiation exposure, injury to skin from trauma or burns may cause a greater degree of contamination because of loss of the shielding of the skin. there are four important principles for the nurse or hcp to understand with regard to exposure to ionizing radiation: external exposure, external contamination, internal contamination, and incorporation. external contamination occurs when radioactive material is carried on a person after exposure. this person can then contaminate others. removing contaminated clothing eliminates % of the toxin. others are then less vulnerable to exposure. internal contamination is when a radioactive substance enters the body through inhalation, ingestion, or translocation through open skin. incorporation is internalization of the toxin into body organs. incorporation is dependent on the chemical and not the radiological properties of the radioactive toxin. radioactive iodine, i, is taken up by the thyroid gland because iodine enters the gland as part of normal physiology (advanced hazmat life support (ahls) ). ionizing radiation can damage chromosomes directly and indirectly, causing ravaging biological effects. indirect damage comes from the production of h + and oh − . free radical formation upsets biochemical processes and causes inflammation. these effects can take anywhere from seconds to hours to be expressed. clinical changes can take from hours to years to be realized (zajtchuk et al. ). immediately after a major radiation exposure, the clinical matters of most concern are those related to trauma from blast and thermal injuries. these injuries may be life-threatening and must be addressed first. after thermal and traumatic injuries are addressed, attention should be paid to the severity of radiation exposure. severe exposure can cause acute radiation syndrome. "the acute radiation syndrome is a broad term used to describe a range of signs and symptoms that reflect severe damage to specific organ systems and that can lead to death within hours or up to several months after exposure" (national council on radiation protection (ncrp) and measurements ; national council on radiation protection (ncrp) and measurements ). the mechanism of cell death from toxic radiation exposure is related to the inhibition of mitosis. organs with the most rapidly dividing cells are the most susceptible. the gastrointestinal and the hematopoietic are the organ systems most notably affected. the organs of pediatric patient have a higher mitotic index, in general, to those of adults and are more vulnerable to injury from radiation poisoning. the time of onset and the severity of acute radiation syndrome are controlled by the total radiation dose, the dose rate, percent of total body exposed, and associated thermal and traumatic injuries. there is a % death rate (ld ) within days for people exposed to a dose of radiation of . - . gy. the ld is lower for the pediatric population. the acute radiation syndrome is composed of four phases: prodromal, latent, manifest illness, and death or recovery. inflammatory mediator release during the prodromal phase causes damage to cell membranes. this phase occurs during the first h after exposure to radiation. nausea and vomiting and fever can occur during this time. if these symptoms occur during the first h after exposure, there is a poor prognosis. the onset of the latent phase is usually in the first days post exposure but can ensue anytime during the first days thereafter. all cell lines of the hematopoietic system are affected. lymphocytes and platelets, the most rapidly dividing cells of the bone marrow, are most severely affected. the illness phase manifests after days since radiation exposure. infection, impaired wound healing, anemia, and bleeding occur during this time of illness. the hematopoietic, gastrointestinal, central nervous, and integumentary are the organ systems affected. there is a marked reduction of cells from all cell lines of the bone marrow. there is a direct correlation with the drop in absolute lymphocyte count with the dose of radiation received. the absolute lymphocyte count is commonly used to estimate the dose of radiation received. the gastrointestinal (gi) epithelial lining, one of the most rapidly dividing cell lines of the body is the second most vulnerable to radiation poisoning. the radiation dose required to affect the gi system is gy. vomiting, diarrhea, and a capillary leak syndrome for gi tract are common manifestations. hypovolemia and electrolyte instability ensue. translocation of bacteria into the bloodstream, combined with the diminished immunity caused by the decimation of the hematopoietic system, place victims at high risk for septic shock. another organ system affected by the acute radiation syndrome is the central nervous system. this requires a large dose of at least gy. manifestations include cerebral edema, disorientation, hyperthermia, seizures, and coma. acute radiation syndrome that involves the central nervous system is always fatal. the integumentary system is frequently affected by the acute radiation syndrome, especially if the skin is in direct contact with a radioisotope. epilation, erythema, dry desquamation, wet desquamation, and necrosis occur respectively with increasing severity associated with increasing doses of radiation. radiation burns can be distinguished from thermal or chemical burns by their delayed onset. it can take days to weeks for radiation burns to affect victims. thermal and chemical burns cause signs and symptoms more acutely. hospitals that anticipate victims of radiation should prepare areas of triage with decontamination supplies and techniques ready to be deployed. an emergency department (ed) should be divided into "clean" and "dirty" areas. the dirty area is created for the purpose of decontamination to prevent the spread of radioisotopes. all health care personnel should wear ppe including surgical scrubs and gowns, face shields, shoe covers, caps, and two pairs of gloves. the inner pair of gloves is taped to the sleeves of the gown. each health care worker should be monitored for the exposure of the radiation and its dose with a dosimeter worn underneath the gown. the radiation safety officer of the hospital should take a leadership role in health care worker protection and decontamination procedures. consultation from the radiation emergency, assistance center (react/ts) is imperative. react/ts is a subsidiary of the u.s. department of energy. its contact information is as follows: phone number during business hours is - - . the phone number is - - after business hours. the react/ts website is http://orise.orau. gov/reac/ts/. as victims arrive, triage protocols of mass casualty scenarios should be implemented. it should be noted that radiation exposure is not "immediately" lifethreatening. initial clinical management should focus on the abcde (airway, breathing, circulation, disability, and exposure) of basic trauma protocol. the "d" in the above acronym can also be a symbol for decontamination. after airway, breathing, and circulation are addressed, initial phase of decontamination entails careful removal of potentially contaminated clothing. caution should be exercised to remove the clothing gently, while rolling garments outward to prevent the release of dust of radioactive material that could contaminate people in the treatment area. further decontamination procedures take place after initial stabilization. skin decontamination procedures are identical to those of toxic chemical exposure with the following exceptions: • ppe are slightly different as described above. • gentle skin rubbing is done to prevent provocation of an inflammatory response and further absorption of the radioactive toxin. • only soap and water are used. rubbing alcohol and bleach should be avoided. it is advisable to shampoo the hair first, because it is usually the site of the highest level of contamination of the body, and runoff onto the body can then be cleansed during skin decontamination (radiation event medical management (remm) of the u.s. dept. of health and human services n.d.). it should be noted that health care workers are not at risk for contamination if they wear proper ppe during the resuscitation and decontamination process. the lack of knowledge of this point may lead to reluctance to treat patients and increase morbidity and mortality for victims. "no hcp has ever received a significant dose of radiation from handling, treating, and managing patients with radiation injuries and/or contamination."(react/s-cdc ). when initial resuscitation and decontamination have been completed, attention should be paid to ongoing support of ventilation, oxygenation, the management of fluid and electrolytes, and treatment of traumatic and burn injuries. infection control procedures are important due to the impending immunocompromised state of the victims. it is important to ascertain the details of the catastrophic event. data on the nature and size of the exposure and the types of radioactive agents involved are vital for ongoing management and decontamination. after the details of the nature of the exposure are uncovered, diagnostic tests should be done, including serial cbc and cytogenetic analysis of lymphocytes, otherwise known as cytogenic dosimetry (react/s-cdc ). measurements of change in lymphocyte counts and cytogenetic dosimetry are sensitive markers for the dose of radiation received by a victim. measurements of internal decontamination are done by the sampling and analysis of nasal and throat swabs, stool, and h urine. wound samples and irrigation fluid should also be sampled. after initial stabilization, external decontamination, and diagnostic testing, internal decontamination is performed. external decontamination involves removal of clothes and cleaning the skin and hair. internal decontamination removes radioisotopes that are internalized via inhalation, ingestion, and entry into open wounds. because ionizing radiation is being released inside the body, internal decontamination must be performed promptly after initial resuscitation. since radioisotopes behave identically to their nonradioactive counterparts, antidotes are chosen based on the chemical, and not the radiological properties of the element. basic strategies of internal decontamination include chelation, competitive inhibition, enhanced gastrointestinal elimination, and enhanced renal elimination. specific agents are used for chelation of different radioisotopes. dtpa (diethyenetriaminepentaacetic acid) is administered for the elimination of heavy metals such as americium, californium, curium, and plutonium. dtpa comes in two forms, calcium dtpa (ca-dtpa) and zinc-dtpa (zn-dtpa). ca-dtpa is ten times more effective than zn-dtpa. for adults and adolescents, administration is as follows: • g of ca-dtpa iv initially in the first h, followed by g zn-dtpa iv daily for maintenance. • for children less than years of age administer: • fourteen mg/kg ca-dtpa iv initially, followed by fourteen mg/kg of zn-dtpa iv daily thereafter (national council on radiation protection (ncrp) and measurements ). • the initial dose of dtpa may be administered via inhalation to adolescents and adults if the contamination occurred via inhalation. this method of administration is not approved for pediatric use. chelation with dimercaprol (bal) is used to eliminate polonium. bal is a highly toxic drug and should be administered with caution. the dose is . mg per kg im four times a day for days, then twice a day on the third day and once a day for - days, thereafter (national council on radiation protection (ncrp) and measurements ). alkalinization of the urine is renal protective during administration. a less toxic alternative to bal, dimercaptosuccinic acid (dmsa), otherwise known as chemet ® is also available. the dose of dmsa is ten mg per kg po every h for days. the same dose is given every h for days, thereafter (national council on radiation protection (ncrp) and measurements ). another mechanism for internal decontamination is competitive inhibition. the radioisotope, i, is released during a meltdown of a reactor at a nuclear power plant. potassium iodide (ki) is widely recognized as a competitive inhibitor to its radioactive counterpart, i, from being incorporated into the thyroid gland. ki blocks % of i uptake into the thyroid gland if ki is given within the first hour of exposure. it will block % of incorporation if given within h of exposure. its protective effect lasts for h. with administration of this drug, thyroid function should be monitored closely. dosing guidelines (table . ) are included in the table below (u.s. food and drug administration n.d.). gastrointestinal elimination is another mechanism of internal decontamination (table . ). an ion exchanger, prussian blue, (ferric ferrocyanide), binds elements that circulate through the enterohepatic cycle. since it is not absorbed through the gastrointestinal tract, prussian blue carries the toxins into the stool. it is highly effective in the elimination of cs or thallium and was used during the cs incident in goiania, brazil. the dosing of prussian blue is as follows: • infants: . - . mg per kg po three times a day (not fda approved). • children - years of age: g po three times a day. • children ≥ years of age: g po three times a day. • prussian blue is administered for at least days, and can be adjusted based on the degree of poisoning (national council on radiation protection (ncrp) and measurements ). urinary elimination is another useful method of internal decontamination. tritium can be eliminated with excess fluid administration. uranium is eliminated by alkalinizing the urine to a ph of - . sodium bicarbonate is given at a dose of meq/kg iv every - h and is titrated to effect. if renal injury occurs, dialysis may be required. the basic approach to treating acute radiation syndrome is supportive therapy. gi losses from gastrointestinal difficulties are treated with iv fluids and electrolyte replacement. -ht antagonists can be used to suppress vomiting and benzodiazepines for anxiety. a patient suffering from acute radiation syndrome may be severely immunocompromised and requires a room with positive pressure isolation. colony stimulating agents for granulocytes and erythrocytes can be used for bone anemia and leukopenia. bone marrow transplant may be required for severe cases. a patient with skin contamination with radiation should be decontaminated with soap and water. a geiger counter can be helpful to identify areas of contamination. scrubbing is performed in a concentric matter, beginning at the outer layers of contamination and moving into the center since the area of greatest contamination is in the center. in this way, the area of contamination remains contained. attention should be paid to good nutrition and pain control. burn and plastic surgery service should also be consulted. more details on decontamination can be found in chap. . the psychological impact of a radiation catastrophe on the pediatric victims is likely to be devastating (american academy of pediatrics (aap) ). sleep disturbances, social withdrawal, altered play, chronic fear and anxiety, and developmental regression can occur. a correlation between the parent's psychological response and that of the child would occur as with other types of disaster. mental health professionals should be consulted in the event of this type of situation. please refer to chap. for more information. a lot of concern has been expressed over the possibility of terrorist attacks involving explosive devices in recent years (depalma et al. ) . explosive devices are relatively simple to manufacture and easy to detonate. they can injure and kill many people and spread fear over large populations. victims of bomb blasts sustain more body regions injured, have more body injury severity scores, and require more surgeries than victims of nonexplosive trauma incidents. victims of explosives also have a higher mortality (kluger et al. ) . these observations are also true of pediatric victims (daniel-aharonson et al. ) . many factors influence the number of people injured and the severity of the injuries in an explosion. the magnitude of the explosion and its proximity to people and the number of people in the area affect the severity and number of injuries. other factors include the collapse of building or structure from the blast, promptness of the rescue operation, and the caliber and proximity of medical resources in the vicinity. victims who experience explosions in closed spaces are especially vulnerable to more severe injuries. twenty-nine case reports of injuries from terrorist bombings were reviewed (arnold et al. a) . the investigators compared the injury severity of victims of explosions who sustained injuries from structural collapse, closed space explosions without structural collapse, and open space explosions. the mortality rate for these victims was %, %, and %, respectively. hospitalization rates were %, %, and %, respectively. ed visits were %, %, and %, respectively. victims of closed space explosions without structural collapse experienced greater hospitalizations rates than those involved in a structural collapse, because many of the victims involved in the structural collapse experienced immediate death. an explosion is defined as a rapid chemical conversion of a liquid or solid into a gas with energy release. substances that are chemically predisposed to explosion, called explosives, are characterized as low or high order, depending on the speed and magnitude of energy release. low-order explosives release energy at a relatively slow pace and explosions from these substances tend not to produce large air pressure changes or a "blast." the energy release is caused by combustion, producing heat. the involved material "goes up in flames." gunpowder, liquid fuel, and molotov cocktails are examples of low-order explosives (centers for disease control and prevention ). explosions from high-order materials cause a blast with a pressure wave in addition to causing the release of heat and light. the blast pressure wave causes compression of the surrounding medium which is physically transformed in all directions from the exact point of explosion. when an explosion occurs on land, air is the surrounding medium compressed. in bodies of water, the surrounding medium is water. the degree of medium compression and the distance that the energy wave travels is determined by the magnitude of the explosion. the power of the blast is measured in pounds per square inch (psi). the pressure blast wave has distinctive characteristics. the amplitude of the wave reaches its highest point immediately after the blast. the blast wave then rapidly decays as it travels through space. as the blast wave propagates, and compresses the surrounding medium, it leaves a vacuum because of displaced molecules in the surrounding medium and a negative phase of the wave ensues. in a land explosion, air molecules are displaced by the initial positive pressure, after which a negative pressure occurs in the vacated space. a wave that propagates through a confined space rebounds off of the wall and reverberates. it may interact with victims in the confined space many times, causing more severe injuries (stuhmiller et al. ) (fig. . ). four kinds of injury occur in high energy explosions. primary blast injuries occur directly from the pressure wave of the blast. secondary injuries occur from being struck by flying objects from the blast. these injuries can be blunt or penetrating. tertiary injuries occur when victims are displaced from a location and strike other objects or surfaces. all other injuries related to the blast are called quarternary. they include burns, inhalational injuries, toxic exposures, and traumatic injuries from structural collapse. primary injuries from blast waves affect bodily tissues with a tissue gas interface. when a pressure wave enters the body, tissue of gas filled organs compress slower than the air inside the tissue, causing stress in the tissue, possibly damaging it. this baseline positive phase originally described by friedlander, a blast wave consists of a short, high-amplitude overpressure peak followed by a longer depression phase. injury potential depends on the wave's amplitude as well as the slopes of its increase and decrease in pressure. x-axis refers to time and y-axis refers to pressure. (jacobson and severin ) also known as the "spalling effect." as the negative pressure phase of the blast wave propagates through, it causes more stress on the tissue and further damage. in addition to damaging tissues with an air tissue interface, pressure blasts can cause injury to the brain and can lead to limb detachments. despite the fact that primary blast injuries can be ravaging, they are less common than other types of injury from blasts. the tympanic membranes, lungs, and gastrointestinal tract are the most common organs sustaining injury from pressure waves. the tympanic membrane is the most vulnerable of these three organ systems (depalma et al. ; garth ) . five psi, which is considered a weak blast, will rupture % of tympanic membranes. to put this in perspective, c , a commonly used explosive generates a pressure of four million psi. otoscopy can reveal ruptured tympanic membranes. neuropraxia, deafness, tinnitus, and vertigo are symptoms that can be experienced. severe blast injuries of the ear can result in damage to the organ of corti, resulting in permanent hearing loss. the second most common organ injured from a blast wave is the lung. fifteen psi are required to cause injury to this organ. lung injuries are more likely to occur from a blast within a closed space, or when victims sustain burns (burns commonly cause acute lung injury from release of inflammatory mediators). direct alveolar damage, blood vessel with bleeding, and inflammation are the three different manifestations of lung injury from blasts. alveolar damage can cause pneumothorax and pulmonary interstitial emphysema. when air dissects along the bronchovascular sheath, pneumomediastinum, pneumopericardium, and subcutaneous emphysema can occur. air that enters the pulmonary venous system can result in a systemic arterial air embolism, and possibly, a stroke. inflammation of the lungs from direct pressure damage to the tissue, cause acute lung injury and possibly, disseminated intravascular coagulation. clinical signs of lung injury include tachypnea, chest pain, hypoxia, rales, and dyspnea. if there is vascular disruption, hemoptysis can occur. air leaks from alveolar injury can result in diminished breath sounds, subcutaneous crepitance, increased resonance, and tracheal deviation. hemodynamic compromise will occur with tracheal deviation. alveolar damage, leading to air in the pulmonary venous system, can lead to a systemic arterial air embolism. air in the coronary arteries can lead to coronary ischemia with st and/or t waves changes on ecg. air embolism to cerebral arteries leads to cerebral vascular accidents (strokes) with focal neurological deficits. other manifestations of systemic air embolism include mottling of the skin, demarcated tongue blanching, and/or air in the retinal vessels (the most common sign of arterial air embolus). rapid death after initial survival is most often caused by arterial air embolus. initiation of positive pressure ventilation may trigger this event (ho and ling ) . a lung injury from a blast can also precipitate a vagal reflex resulting in bradycardia and hypotension. it is postulated that this occurs from the stimulation of c fibers in the lungs (guy et al. ). the gastrointestinal system is the third most common organ system affected by primary blast injury. physical stress and/or mesenteric infarct leads to weakening of the bowel wall with possible rupture. hemorrhage can also occur (paran et al. ; sharpnack et al. ) . the most common site of injury is the colon. injury to the bowel can be delayed and occur up to several days after the inciting incident. solid organs are spared because of their homogeneity and lack of air tissue interface. brain injury is becoming increasingly recognized as a result of primary blast. shearing injuries of the brain occur as a result of wave reverberation in the skull. hippocampal injury causing cognitive impairment has been shown in animal studies (cernak ; cernak et al. ; singer et al. ) . observations in humans have revealed electroencephalographic abnormalities and attention deficit disorder (born ) . human autopsies have revealed punctate hemorrhages and disintegration of nissl substance in victims who sustained blast injury without direct head trauma (guy et al. ) . research involving yucatan minipigs revealed that the brain sustains neuronal loss in the hippocampus after being subjected to primary blast injury. brain injury also occurred from the inflammation that ensued post blast (goodrich et al. ) . novel therapeutic approaches may be on the horizon for treatment of traumatic brain injury, including that caused by primary blast. intranasal insulin administered to rats subjected to traumatic brain injury resulted in enhanced neuronal glucose uptake and utilization, and subsequently improved motor function and memory. decreased neuroinflammation and preservation of the hippocampus were also noted (brabazon et al. ) . in a different investigation, a neuroprotective nucleotide, guanosine, was administered to rats subjected to traumatic brain injury. the treatment group of rats had better locomotor and cognitive outcomes than did the placebo group. programmed cell death and inflammation were also attenuated in the treatment group (gerbatin et al. ) . the leading cause of death from blast is from flying objects striking victims (secondary blast injury). eyes are particularly vulnerable. injuries resulting from displacement of the victims who strike objects are known as tertiary injuries. lighter weight children are particularly susceptible to this type of injury. burns, toxic exposures, and crush injuries constitute quaternary injuries. crush injuries commonly occur in explosions with structural collapse. the "crush syndrome" can occur when a trapped limb sustains prolonged compromise to the circulation, leading to rhabdomyolysis. tissue destruction and inflammatory response then occur. lifethreatening electrolyte abnormalities including hyperkalemia, renal failure, hyperuricemia, metabolic acidosis, acute respiratory distress syndrome, disseminated intravascular coagulation, and shock can result from crush syndrome (gonzalez ) . the crush syndrome is commonly seen in natural disasters that result in a lot of structural collapse. structural collapse and fires can cause the release of toxic materials such as carbon monoxide and cyanide. knowledge of the details of a blast can greatly enhance the ability of nurses and hcps to care for victims of a blast in a hospital setting. knowledge of whether a blast occurred in a closed or open space, whether structural collapse occurred, or if a victim was rescued from a collapsed area are details that can alert nurses and hcps as to what kind of injuries that they may anticipate. if toxic substances are released with a blast, nurses and hcps can prepare for decontamination techniques and antidote therapies. it would be advantageous for a hospital to be aware of the number of victims that are arriving for care. a mass casualty incident will stress the resources of the institution. hospital personnel should take stock of the resources that are available. the number of available ventilators and o-blood are examples of finite resources that should be considered. advanced trauma life support (atls) principles should be applied to all blast injury victims. abcd of initial resuscitation is applied. the "d" stands for disability as well as decontamination. decontamination techniques should be deployed if there is uncertainty about toxic exposure as described elsewhere in this chapter. on completion of abcd of initial resuscitation a secondary survey is performed, as described by atls protocol. attention should be paid to potential injuries that occur with blast injuries. ruptured tympanic membranes should alert the nurse or hcp of problems from primary blast injury. impaled objects should remain in place and removed in the operating room by surgical staff so that bleeding may be controlled. a thoracoscopy tube should be placed with an open three point seal over a wound on the side of the chest with an open pneumothorax. a hemothorax is also treated with a thoracoscopy tube. an autotransfusion setup can be applied to recirculate the blood from the pleural cavity of a hemothorax (wightman and gladish ) that would help preserve donor blood for other victims. for severe respiratory distress and/or impending respiratory failure, endotracheal intubation should be performed and positive pressure ventilation should be instituted. because lung tissue could be weakened from primary blast injury, caution should be exercised because of a high risk of pneumothorax, hemorrhage, or arterial air embolus. gentle application of positive pressure ventilation should be applied to avoid these complications. if only one lung is injured unilateral lung ventilation can be considered for larger children and adults. this technique is not suitable for babies and small children. supplemental oxygen with an fio of % should be administered to patients suspected of having an arterial air embolus. hyperbaric oxygen therapy could even be considered to help accelerate the removal of air from the arteries. placement of the patient in the left lateral recumbent position may reduce the likelihood of the air lodging in the coronary arteries. victims of blast injuries should be treated identically to those of other types of trauma after initial resuscitation is completed. if primary blast injury occurred, frequent chest and abdominal x-rays should be performed in consideration of the possibility of lung or gastrointestinal injuries. limbs with open fractures should be immobilized and covered with sterile dressings. systemic, broad spectrum antibiotics should be administered to patients with open limb injuries. eyes that sustained chemical injury should be irrigated with water for an hour. all injured eyes should be covered. most ruptured tympanic membranes will heal spontaneously. victims with tympanic membrane injury should be advised to avoid swimming for some time. topical antibiotics are prescribed if dirt or debris is seen in the ear canal. oral prednisone is prescribed for hearing loss. victims with crush injuries should be treated with large volumes of iv fluids to treat inflammatory shock and possibly rhabdomyolysis. electrolytes should be monitored carefully as these patients are at risk for hyperkalemia, hyperphosphatemia, hyperuricemia, hypocalcemia, and acidosis. smoke inhalation, burns, and toxic exposures should be treated according to guidelines of burn, trauma, and toxicology protocols. mass casualty incidents (i.e. mass shootings, active shooter events, bombings, and other multifatality crimes) often attract extensive media coverage as well as the attention of policy makers. many agencies and organizations record and publish data on these incidents. the measurement and reporting does vary based on the absence of a common definition. however, it is clearly evident that mass casualty incidents (mcis) continue to increase in both number and scope (federal bureau of investigation ; office for victims of crime, office of justice programs, u.s. department of justice ). in the u.s., mass shootings are the most common and most closely tracked. the congressional research service (crs) defines mass shootings as events where more than four people are killed with a firearm "within one event, and in one or more locations in close proximity." congress uses the term mass killings and describes these events as "three or more killings in a single incident." the federal bureau of investigation (fbi) uses the term active shooter, which it defines as "an individual actively engaged in killing or attempting to kill people in a populated area." it is important to realize that nongovernmental ( ranking third of all locations for and , seven of the incidents occurred in educational environments resulting in five killed and wounded. two incidents occurred in elementary schools, resulting in two killed (including a firstgrade student) and eight wounded (one teacher shot, three students shot, and four wounded from shrapnel). one incident occurred in a junior/senior high school, resulting in none killed and four wounded (two from shrapnel, all students). four incidents occurred at high schools (one outside a school during prom), resulting in three killed (all students) and seven wounded (all students). fortunately, no incident occurred at institutions of higher learning during or (advanced law enforcement rapid response training (alerrt) center, texas state university and federal bureau of investigation, u.s. department of justice ). notably, two of the incidents occurred in houses of worship, resulting in killed and wounded. one of these incidents occurred at the first baptist church in sutherland springs, texas, and had the third highest number of casualties ( killed and wounded) in . the dead included women, men, children ( girls and boy), and an unborn child (goldman et al. ) . a summary report has also been developed for all active shooter incidents from to , including incidents per year (fig. . ), casualties per year (fig. . ) , and location ( fig. . ) categories (federal bureau of investigation ; federal bureau of investigation ). overall, there was an increase in number of active shooter incidents and casualties per year. location categories with number of incidents and statistics of their contribution were provided: areas of educational environments account for a large portion of locations for active shooter incidents, ranking only second to commercial areas. of the incidents ( . %) occurring at schools, one took place at a nursery (pre-k) school and one incident occurred during a school board meeting that was being hosted on school property but no students were involved (neither perpetrator or victim). the remainder ( incidents) were perpetrated by or against students, faculty, and/or staff at k- schools (federal bureau of investigation ). finally, active shooter incidents ( %) did occur at institutions of higher learning. as a reminder, no incident occurred at institutions of higher learning during or . table . provides a detailed summary of educational environment incidents from to . since the beginning of , other tragic active shooter attacks have occurred in the u.s. and greatly impacted children and adolescents. two of these such events have occurred in educational environments (united states secret service national threat assessment center ). on february , , a gunman opened fire at marjory stoneman douglas high school. fourteen students and three staff members were killed while fourteen others were injured (follman et al. ) . twelve victims died inside the building, three died just outside the building on school premises, and two died in the hospital. the shooter was a former student of the school. another active shooter event occurred on may , at santa fe high school in santa fe, texas. the shooter killed ten individuals including eight students and two teachers while injuring others. the shooter was an enrolled student at the school (follman et al. ) . based on the statistics of active shooter incidents, casualties, and locations, it is vital to prepare schools and plan for such events. national preparedness efforts, including planning, are now informed by the presidential policy directive (ppd) that was signed by the president in march and describes the nation's approach to preparedness. this directive represents an evolution in our collective understanding of national preparedness based on the lessons learned from terrorist attacks, hurricanes, school incidents, and other experiences. ppd- defines preparedness around five mission areas and can be applied to school active shooter incidents. on march , , at : p.m., jeffery james weise, , armed with a shotgun and two handguns, began shooting at red lake high school in red lake, minnesota. before the incident at the school, the shooter fatally shot his grandfather, who was a police officer, and another individual at their home. he then took his grandfather's police equipment, including guns and body armor, to the school. a total of nine people were killed, including an unarmed security guard, a teacher, and five students; six students were wounded. the shooter committed suicide during an exchange of gunfire with police campbell county comprehensive high school (education) on november , , at : p.m., kenneth s. bartley, , armed with a handgun, began shooting in campbell county comprehensive high school in jacksboro, tennessee. before the shooting, he had been called to the office when administrators received a report that he had a gun. when confronted, he shot and killed an assistant principal and wounded the principal and another assistant principal. the shooter was restrained by students and administrators until police arrived and took him into custody pine middle school (education) on march , , at : a.m., james scott newman, , armed with a handgun, began shooting outside the cafeteria at pine middle school in reno, nevada. no one was killed; two were wounded. the shooter was restrained by a teacher until police arrived and took him into custody essex elementary school and two residences (education) on august , , at : p.m., christopher williams, , armed with a handgun, shot at various locations in essex, vermont. he began by fatally shooting his ex-girlfriend's mother at her home and then drove to essex elementary school, where his ex-girlfriend was a teacher. he did not find her, but as he searched, he killed one teacher and wounded another. he then fled to a friend's home, where he wounded one person. a total of two people were killed; two were wounded. the shooter also shot himself twice but survived and was apprehended when police arrived at the scene orange high school and residence (education) on august , , at : p.m., alvaro castillo, , armed with two pipe bombs, two rifles, a shotgun, and a smoke grenade, began shooting a rifle from his vehicle at his former high school, orange high school in hillsborough, north carolina. he had fatally shot his father in his home that morning. one person was killed; two were wounded. the shooter was apprehended by police weston high school (education) on september , , at : a.m., eric jordan hainstock, , armed with a handgun and a rifle, began shooting in weston high school in cazenovia, wisconsin. one person was killed; no one was wounded. the shooter was restrained by school employees until police arrived and took him into custody west nickel mines school (education) on october , , at : a.m., charles carl roberts, iv, , armed with a rifle, a shotgun, and a handgun, began shooting at the west nickel mines school in bart township, pennsylvania. after the shooter entered the building, he ordered all males and adults out of the room. after a -min standoff, he began firing. the shooter committed suicide as the police began to breach the school through a window. five people were killed; five were wounded on april , , at : a.m., su nam ko, aka one l. goh, , armed with a handgun, began shooting inside oikos university in oakland, california. he then killed a woman to steal her car. seven people were killed; three were wounded. the shooter was arrested by police later that day on august , , at : a.m., robert wayne gladden jr., , armed with a shotgun, shot a classmate in the cafeteria of perry hall high school in baltimore, maryland. the shooter had an altercation with another student before the shooting began. he left the cafeteria and returned with a gun. no one was killed; one person was wounded. the shooter was restrained by a guidance counselor before being taken into custody by the school's resource officer sandy hook elementary school and residence (education) on december , , at : a.m., adam lanza, , armed with two handguns and a rifle, shot through the secured front door to enter sandy hook elementary school in newtown, connecticut. he killed students and six adults, and wounded two adults inside the school. prior to the shooting, the shooter killed his mother at their home. in total, people were killed; two were wounded. the shooter committed suicide after police arrived taft union high school (education) on january , , at : a.m., bryan oliver, , armed with a shotgun, allegedly began shooting in a science class at taft union high school in taft, california. no one was killed; two people were wounded. an administrator persuaded the shooter to put the gun down before police arrived and took him into custody new river community college, satellite campus (education) on april , , at : p.m., neil allen macinnis, , armed with a shotgun, began shooting in the new river community college satellite campus in the new river valley mall in christiansburg, virginia. no one was killed; two were wounded. the shooter was apprehended by police after being detained by an off-duty mall security officer as he attempted to flee santa monica college and residence (education) on june , , at : a.m., john zawahri, , armed with a handgun, fatally shot his father and brother in their home in santa monica, california. he then carjacked a vehicle and forced the driver to take him to the santa monica college campus. he allowed the driver to leave her vehicle unharmed but continued shooting until he was killed in an exchange of gunfire with police. five people were killed; four were wounded sparks middle school (education) on october , , at : a.m., jose reyes, , armed with a handgun, began shooting outside sparks middle school in sparks, nevada. a teacher was killed when he confronted the shooter; two people were wounded. the shooter committed suicide before police arrived arapahoe high school (education) on december , , at : p.m., karl halverson pierson, , armed with a shotgun, machete, and three molotov cocktails, began shooting in the hallways of arapahoe high school in centennial, colorado. as he moved through the school and into the library, he fired one additional round and lit a molotov cocktail, throwing it into a bookcase and causing minor damage. one person was killed; no one was wounded. the shooter committed suicide as a school resource officer approached him berrendo middle school (education) on january , , at : a.m., mason andrew campbell, , armed with a shotgun, began shooting in berrendo middle school in roswell, new mexico. a teacher at the school confronted and ordered him to place his gun on the ground. the shooter complied. no one was killed; were wounded: students and an unarmed security guard. the shooter was taken into custody (continued) on june , , at : p.m., aaron rey ybarra, , armed with a shotgun, allegedly began shooting in otto miller hall at seattle pacific university in seattle, washington. he was confronted and pepper sprayed by a student as he was reloading. one person was killed; were wounded. students restrained the shooter until law enforcement arrived reynolds high school (education) on june , , at : a.m., jared michael padgett, , armed with a handgun and a rifle, began shooting inside the boy's locker room at reynolds high school in portland, oregon. one student was killed; teacher was wounded. the shooter committed suicide in a bathroom stall after law enforcement arrived marysville-pilchuck high school (education) on october , , at : a.m., jaylen ray fryberg, , armed with a handgun, began shooting in the cafeteria of marysville-pilchuck high school in marysville, washington. four students were killed, including the shooter's cousin; students were wounded, including one who injured himself while fleeing the scene. the shooter, when confronted by a teacher, committed suicide before law enforcement arrived florida state university (education) on november , , at : a.m., myron may, , armed with a handgun, began shooting in strozier library at florida state university in tallahassee, florida. he was an alumnus of the university. no one was killed; were wounded. the shooter was killed during an exchange of gunfire with campus law enforcement. umpqua community college (education) on october , , at : a.m., christopher sean harper-mercer, , armed with several handguns and a rifle, began shooting classmates in a classroom on the campus of umpqua community college in roseburg, oregon. nine people were killed; were wounded. the shooter committed suicide after being wounded during an exchange of gunfire with law enforcement. madison junior/ senior high school (education) on february , , at : a.m., james austin hancock, , armed with a handgun, allegedly began shooting in the cafeteria of madison junior/senior high school in middletown, ohio. he shot two students before fleeing the building. no one was killed; four students were wounded (two from shrapnel). the shooter was apprehended near the school by law enforcement officers antigo high school (education) on april , , at : p.m., jakob edward wagner, , armed with a rifle, began shooting outside a prom being held at his former school, antigo high school in antigo, wisconsin. two law enforcement officers, who were on the premises, heard the shots and responded immediately. no one was killed; two students were wounded. the shooter was wounded in an exchange of gunfire with law enforcement officers and later died at the hospital townville elementary school (education) on september , , at : p.m., jesse dewitt osborne, , armed with a handgun, allegedly began shooting at the townville elementary school playground in townville, south carolina. prior to the shooting, the shooter, a former student, killed his father at their home. two people were killed, including one student; three were wounded, one teacher and two students. a volunteer firefighter, who possessed a valid firearms permit, restrained the shooter until law enforcement officers arrived and apprehended him on january , , at : a.m., ely ray serna, , armed with a shotgun, allegedly began shooting inside west liberty salem high school, in west liberty, ohio, where he was a student. after assembling the weapon in a bathroom, the shooter shot a student who entered, then shot at a teacher who heard the commotion. the shooter shot classroom door windows before returning to the bathroom and surrendering to school administrators. no one was killed; two students were wounded. school staff members subdued the shooter until law enforcement arrived and took the shooter into custody freeman high school (education) on september , , at : a.m., caleb sharpe, , armed with a rifle and a pistol, allegedly began shooting at freeman high school in rockford, washington, where he was a student. one student was killed; three students were wounded. a school employee confronted the shooter, ordered him to the ground, and held him there until law enforcement arrived and took him into custody rancho tehama elementary school and multiple locations in tehama county, california (education) on november , , at : a.m., kevin janson neal, , armed with a rifle and two handguns, began shooting at his neighbors, the first in a series of shootings occurring in rancho tehama reserve, tehama county, california. after killing three neighbors, he stole a car and began firing randomly at vehicles and pedestrians as he drove around the community. after deliberately bumping into another car, the shooter fired into the car and wounded the driver and three passengers. the shooter then drove into the gate of a nearby elementary school. he was prevented from entering the school due to a lockdown, so he fired at the windows and doors of the building, wounding five children. upon fleeing the school, the shooter continued to shoot at people as he drove around rancho tehama reserve. law enforcement pursued the shooter; they rammed his vehicle, forced him off the road, and exchanged gunfire. the shooter's wife's body was later discovered at the shooter's home; the shooter apparently had shot and killed her the previous day. in total, five people were killed; were wounded, eight from gunshot injuries (including one student) and six from shrapnel injuries (including four students). the shooter committed suicide after being shot and wounded by law enforcement during the pursuit aztec high school (education) on december , , at approximately : a.m., william edward atchison, , armed with a handgun, began shooting inside aztec high school in aztec, new mexico. the shooter was a former student. two students were killed; no one was wounded. the shooter committed suicide at the scene, before police arrived a in a study of active shooter incidents in the united states between and , the fbi identified locations where the public was most at risk during an incident. these location categories include commercial areas (divided into business open to pedestrian traffic, businesses closed to pedestrian traffic, and malls), education environments (divided into schools [prekindergarten through th grade] and institutions of higher learning), open spaces, government properties (divided into military and other government properties), residences, houses of worship, and health care facilities. in , the fbi added a new location category, other location, to capture incidents that occurred in venues not included in the previously identified locations (federal bureau of investigation ). this table only includes educational environments. an entire list of all incidents from to at all locations can be found at https://www.fbi.gov/file-repository/activeshooter-incidents- .pdf/view (federal bureau of investigation prevention means the capabilities necessary to avoid, deter, or stop an imminent crime or threatened/actual mass casualty incident. prevention is the action schools take to prevent a threatened or actual incident from occurring. protection means the capabilities to secure schools against acts of violence and man-made or natural disasters. protection focuses on ongoing actions that protect students, teachers, staff, visitors, districts, networks, and property from a threat or hazard. mitigation means the capabilities necessary to eliminate or reduce the loss of life and property damage by lessening the impact of an event or emergency at the school. it also means reducing the likelihood that threats and hazards will happen. response means the school's or school district's capabilities necessary to stabilize an emergency once it has already happened or is certain to happen in an unpreventable way, establish a safe and secure environment, save lives and property, and facilitate the transition to recovery. recovery means the capabilities necessary to assist schools affected by an event or emergency in restoring the learning environment. it also means teaming with community partners to restore educational programming, the physical environment, business operations, and social, emotional, and behavioral health. the majority of prevention, protection, and mitigation activities generally occur before an incident, although these three mission areas do have ongoing activities that can occur throughout an active shooter incident. response activities occur during an incident, and recovery activities can begin during an incident and occur after an incident (united states department of education, office of elementary and secondary education, office of safe and healthy students ; united states department of homeland security b; united states department of homeland security ). in the k- school security guide, the u.s. department of homeland security (dhs) focuses on prevention and protection since the activities and measures associated with them occur prior to an incident ( ). effective preventative and protective actions decrease the probability that schools (or other facilities) will encounter incidents of gun violence or should an incident occur, it reduces the impact of that incident. the guide emphasizes that the level of security at a facility will be based on hazards relevant to the facility, people, or groups associated with it. it also warns that as new or different threats become apparent, the perception of the relative security changes and insecurity should drive change to reflect the level of confidence of the people of groups associated with the facility. the dhs utilizes a hometown security approach that emphasizes the process of connect, plan, train, and report (cptr) with the objective to realize effective, collaborative outcomes (united states department of homeland security b). the initial phase is connect and occurs by a school or district reaching out and developing relationships in the community, including local law enforcement. having these relationships before an incident or event can help speed up the response when something happens. each school must begin with identification or development of a security team, group, or organization. this phase also emphasizes outreach, collaboration, and building of a coalition. there should be coalition members from within a school and may include district/school administrators, teachers, aides, facility operations personnel, human resources, administrative, counseling, and student groups. external groups directly related to the school might include boards of education, parent organizations, mental health groups/agencies, and teacher and bus driver unions. external groups indirectly related to the school include all responder organizations such as police and fire departments, sheriff's office, emergency medical services, emergency management, and the local dhs protective security advisor (psa). other tangential groups such as volunteer organizations, utility providers, and facilities in close geographic proximity should also be considered. core and advisory members of the coalition are established. a coalition champion is also identified and is the person who owns the majority of the responsibility for achieving a school's security goals. the champion organizes the coalition as it grows and matures (united states department of homeland security b). the next phase is plan. this will bring the coalition together. the guide for developing high quality school emergency operations plans (united states department of education, office of elementary and secondary education, office of safe and healthy students ) is an excellent resource for the coalition. a school security survey for gun violence can be completed and the coalition or user can quickly and effectively determine a facility's security proficiency (united states department of homeland security. ). specific portions of or topics within a school plan should be assigned to individuals, committees, or working groups most qualified to address them. the planning process must be sustainable. the amount of time spent in the planning phase should be commensurate with the amount of effort expended on the other phases (united states department of homeland security b). the next phase of the process is to train on the plan developed by the coalition. determining who is responsible for what and how it should be done is the basic function of planning. in fact, telling various members of the team what is expected of them and when to do that activity is the function of training. it is vital to utilize the curricula development expertise possessed by the k- community. school administrators should take advantage of this skill set and find creative ways to address difficult topics, such as gun violence. it should be carried out in an effective and nontraumatic way. presenting the training in pieces or steps allows for a more comprehensive learning experience. it is important to validate training through exercises and drills, all of which should include the students. the training event should be followed by the completion and implementation of an after-action improvement plan with adjustment of the cptr as indicated (united states department of homeland security b). the final phase in the process is report. the reporting phase is arguably the most important of all the phases. reporting principles underlie the other three phases and have profound prevention and protection impacts by driving forward information. dhs models the reporting phase using the "if you see something, say something ® " campaign (u.s. dhs, ) and the nationwide suspicious activity reporting (sar) initiative (nationwide suspicious activity reporting initiative (nsi) ). "if you see something, say something ® " focuses on empowering anyone who sees suspicious activity to do something about it by contacting local law enforcement, or if an emergency to call - - (united states department of homeland security a). this is a compelling capability when well organized and managed. a good plan for reporting, especially for a k- school, involves training staff and students on what is considered suspicious. there are many methods in which schools can employ to facilitate this, such as dedicated telephone numbers, websites for anonymous reporting, email or text messaging, and mobile phone applications. conducting simple drills for reporters and receivers keeps skills sharp and reinforces the importance of the effort with the goal to save lives. if the plan includes sharing all suspicious activity calls with the local fusion center then the probability of higher fidelity reporting increases (united states department of homeland security b). when making changes to a school's plans, procedures, and protective measures, it is imperative the needs of individuals with special health care needs be addressed throughout the process. planning, training, and execution should always consider accessible alert systems for those who are deaf or hard of hearing; students, faculty, and staff who have visual impairments or are blind; individuals with limited mobility; alternative notification measures; people with temporary disabilities; visitors; people with limited english proficiency; sign cards with text-and picture-based emergency messages/symbols; and involving people with disabilities in all planning (united states department of homeland security, interagency security committee ). it is important to understand that no "profile" exists for an active shooter (united states department of education, office of elementary and secondary education, office of safe and healthy students ). however, research indicates there may be signs or indicators. o'toole ( ) presents an in depth, systematic procedure for school shooter threat assessment and intervention. the model was designed to be used by educators, mental health professionals, and law enforcement agencies. its fundamental building blocks are the threat assessment standards, which provide a framework for evaluating a spoken, written, and symbolic threat, and the fourpronged assessment approach which provides a logical, methodical process to examine the threatener and assess the risk that the threat will be carried out. schools should learn the signs of a potentially volatile situation that may develop into an active shooter situation and proactively seek ways to prevent an incident with internal resources, or additional external assistance (united states department of education, office of elementary and secondary education, office of safe and healthy students ). potential warning signs of a school shooter may include increasingly erratic, unsafe, or aggressive behaviors; hostile feelings of injustice or perceived wrongdoing; drug and alcohol abuse; marginalization or distancing from friends and colleagues; changes in performance at work or school; sudden and dramatic changes in home life or in personality; pending civil or criminal litigation; and observable grievances with threats and plans of retribution (united states department of homeland security b). at a minimum, schools should establish and enforce policies that prohibit, limit, or determine unacceptable behaviors and consequences of weapons possession/use, drug possession/use, alcohol/tobacco possession/use, bullying/harassment, hazing, cyber-bullying/harassment/stalking, sexual assault/misconduct/harassment, bias crimes, social media abuse, and any criminal acts (united states department of homeland security b). in addition to policies and positive school climates, school districts and administrators should establish dedicated teams to evaluate threats, such as a threat assessment team (tat). the team should include mental health professionals (e.g., forensic psychologist, clinical psychologist, and school psychologist) to contribute to the threat assessment process (united states department of homeland security b). it is the responsibility of the tat to investigate and analyze communications and behaviors to make a determination on whether or not an individual poses a threat to him/herself or others (united states department of education, office of elementary and secondary education, office of safe and healthy students ). as well as tats, some schools have even opted to establish social media monitoring teams which look for keywords that may indicate bullying or other concerning statements. if a school opts to create such a team, it should work very closely with the tat to ensure that applicable privacy, civil rights and civil liberties, other federal, state and local laws, and information sharing protocols are followed. please refer to chap. for further information. after an active shooter incident, field triage (e.g., jumpstart) must commence and the patient must be evaluated by an experienced emergency medicine or trauma surgeon, preferably by a pediatric specialist in those disciplines. if an active shooter incident is coupled with detonation of an explosive device, the child must be screened and decontaminated for radiation exposure ("dirty bomb"). triage tags are extremely helpful when multiple victims present in a short period of time. medical response to an active shooter event will focus on control of external hemorrhage along with circulatory stabilization. operative emergencies will be common and receive the highest priority. severe extremity injuries may be controlled with tourniquet application or other forms of hemorrhage control. re-evaluation is paramount to prevent ischemia to distal regions. however, thoracic or abdominal (truncal) injuries will need immediate surgical exploration and intervention. penetrating trauma will cause more vascular injuries than blunt trauma, and vascular surgical trays may be in short supply at a hospital. major procedure or surgical trays may become short in supply based on the increased operative demand. resuscitative blood transfusion therapy may utilize a massive blood transfusion protocol. since whole blood may be short in supply, some will simply use the : : rule (administer one unit of packed cells: one unit of fresh frozen plasma: one unit of platelets). a unit for children may be substituted as an aliquot based on size of the patient (e.g., administer ml/kg of packed cells: ml/kg of fresh frozen plasma: ml/kg of platelets). calcium must also be replaced when there is a large volume transfusion. due to extensive blood product utilization, there may be a heavy impact on institutional or regional blood supplies. plans should be in place to address these problems, including the implementation of allocation of scarce resources. mental health support and staff debriefs are essential and should be included after an active shooter event (hick et al. ). in conclusion, all forms of disasters, whether man-made or natural, impact infants, children, and adolescents throughout the world. effective and efficient interventions remain the cornerstone of sustaining a child's well-being while reducing untoward complications due to all forms of disasters. having a deep understanding of pediatric physiology and pathophysiology is crucial to all levels of disaster diagnostics and therapeutics. all nurses and hcps have an obligation to understand these principles and deliver excellent, compassionate care to the pediatric disaster victim. advanced law enforcement rapid response training evidence-based support for the all-hazards approach to emergency preparedness ahls advanced hazmat life support provider manual active shooter incidents in the united states in and radiation disasters and children-committee on environmental health apls: the pediatric emergency medicine resource textbook 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ravcheev, dmitry a.; hu, dan; zhang, fengyu; gong, xiufang; hao, lina; cao, min; rodionov, dmitry a.; wang, changjun; feng, youjun title: two novel regulators of n‐acetyl‐galactosamine utilization pathway and distinct roles in bacterial infections date: - - journal: microbiologyopen doi: . /mbo . sha: doc_id: cord_uid: kbiwze z bacterial pathogens can exploit metabolic pathways to facilitate their successful infection cycles, but little is known about roles of d‐galactosamine (galn)/n‐acetyl‐d‐galactosamine (galnac) catabolism pathway in bacterial pathogenesis. here, we report the genomic reconstruction of galn/galnac utilization pathway in streptococci and the diversified aga regulons. we delineated two new paralogous agar regulators for the galn/galnac catabolism pathway. the electrophoretic mobility shift assays experiment demonstrated that agar (agar ) binds the predicted palindromes, and the combined in vivo data from reverse transcription quantitative polymerase chain reaction and rna‐seq suggested that agar (not agar ) can effectively repress the transcription of the target genes. removal of agar (not agar ) from streptococcus suis zyh augments significantly the abilities of both adherence to hep‐ cells and anti‐phagocytosis against raw . macrophage. as anticipated, the dysfunction in agar ‐mediated regulation of s. suis impairs its pathogenicity in experimental models of both mice and piglets. our finding discovered two novel regulators specific for galn/galnac catabolism and assigned them distinct roles into bacterial infections. to the best of our knowledge, it might represent a first paradigm that links the galn/galnac catabolism pathway to bacterial pathogenesis. amino sugars are referred to a variety of diversified/complex monosaccharides in which a hydroxyl group is chemically replaced with the amine group. most of current knowledge on the metabolism of amino sugars comes from studies with escherichia coli (reizer et al. ) , bacillus subtilis (freymond et al. ; gaugue et al. ; plumbridge ) and streptomycetes coelicolor (rigali et al. ) . relative to the best-known examples of amino sugars, glucosamine (glcn) and n-acetylglucosamine (glcnac), the investigations on the other two amino sugar derivatives of galactose, n-acetyl-d-galactosamine (galnac) and d-galactosamine (galn), are relatively limited, but increasingly accumulated (abu-qarn et al. ; leyn et al. ) . it seemed very likely that galn/galnac amino sugars as common components participate in the formation of various cell structures/constitutes in three domains of life (abu-qarn et al. ; plumbridge ) . in general, not only does galnac act as an element of lipopolysaccharide displayed on bacterial cell wall (bernatchez et al. ; leyn et al. ) , and but also connects carbohydrate chains in mammalian mucins (carraway and hull ) . additionally, it functions as the substrate in n-acetyl βgalactosidation, a new type of post-translational modification of protein in organisms, including bacterial pathogens (sadler et al. ; barr and nordin ; davis et al. ; abu-qarn et al. ) . given the multiple roles played by galn/galnac, we therefore anticipated a hypothesis that galn/galnac metabolism might be linked to bacterial infectivity. the paradigm pathway for galn/galnac utilization/ catabolism, which was initially proposed for e. coli in (reizer et al. ) , contained the following five steps ( fig. ): ( ) the transport and phosphorylation of galn/galnac substrates (catalyzed by phosphortransferase system [pts] systems agabcd and agavwef, respectively) (brinkkotter et al. ) , ( ) the agaa-mediated deacetylation of galnac- -p (reizer et al. ) , ( ) the deamination/isomerization of galn- -p by the bi-functional enzyme agas into tag- -p (reizer et al. ) , ( ) the agaz kinase-aided phosphorylation of tag- , -p from tag- -p (reizer et al. ) , and ( ) cleavage of tag- , -pp by the class ii aldolase, agay (brinkkotter et al. ) , to produce glyceraldehyde -phospahte and glycerone phosphate (pep). interestingly, a recent comparative genomics-based study suggested an extensive diversity in galn/galnac utilization pathways of proteobacteria such as shewanella (leyn et al. ) . in particular note the first two steps of this pathway exhibit of high variability, while the latter three steps are largely conserved (leyn et al. ) . in e. coli, not only does agar regulator that belongs to the deor family of transcriptional factors, act as an autoregulator, but also negatively controls the expression of two aga genes (agaz and agas) of galn/ galnac catabolism pathway via direct binding of the specific palindromes in front of these target genes (ray and larson ) . although the fact that both galn and galnac can induce activities of these aga genes-encoding protein products is aware, the physiological ligands for agar repressor remain unclear (leyn et al. ) . streptococcus suis, a gram-positive bacterium, is a zoonotic agent with the ability to infect both its natural host swine and human individuals with close contact with swine/porkrelated products . according to the differentiation in their bacterial capsule structure, this streptococcus species is categorized into serotypes (feng et al. , b . among them, serotype (ss ) is generally believed to be most virulent, in that it is frequently isolated from clinical diseased swine (ma et al. ; feng et al. b ) and human sporadic cases (feng et al. b) and/or big-scale outbreaks (tang et al. ; chen et al. ) . we are aware that ss has spread to more than countries/regions and claims nearly cases of human infections worldwide (feng et al. b ). the molecular mechanism underlying bacterial pathogenicity has been partially delineated thus far (feng et al. b) , and these identified virulence determinants include the previouslyknown virulence factors exemplified with capsule (benga et al. ; seitz et al. ) and suilysin (jacobs et al. ; lun et al. ; takeuchi et al. ). in addition to the regulatory networks amongst the newly identified factors (e.g., the two-component systems salk/salr , nisk/nisr [xu et al. ] , ciarh [li et al. ], etc.) , it would be of particular interest to note the contributions of enzymes from central metabolisms (enolase [eno] [esgleas et al. ; feng et al. a; zhang et al. a; lu et al. ] , glutamine synthase [glna] [si et al. ], inosine -monophosphate dehydrogenase [impdh] zhou et al. ], etc.) to bacterial pathogenesis (feng et al. b) . however, nothing is aware regarding the potential relevance of bacterial galn/galnac metabolism and/or its regulation to s. suis infection. in this work, we employed the integrative approaches combining the bioinformatics and comparative genomics to conduct the genomic reconstruction of the galn/galnac utilization pathway in lactobacillaceae, including the zoonotic pathogen s. suis. also, we are the first to report the functional definition of two new gntr-type transcription factors (referred to agar and/or agar ) with involvement of galn/galnac catabolism. more intriguingly, we observed that agar -dependent regulation of galn/galnac utilization pathway is required for bacterial virulence of s. suis serotype . to the best of our knowledge, it represents the first example that the genetic control of galn/galnac catabolism is linked to bacterial infectivity of streptococcus. suis are highlighted with yellow and light-blue background arrows, respectively. galnac, n-acetyld-galactosamine; galn, galactosamine; pts, phosphotransferase system; bgac, βgalactosidase; agaa, galnac- -p deacetylase; agas, galn- -p deaminase/isomerase; tag, tagtose; agaz, tag- -p kinase; agay, tag- , -pp aldolase. here corresponded to the human laryngeal epithelial cell hep- (cctcc gdc ) and the mouse macrophagocyte raw . (atcc tib- , rockville, md), respectively (table s ) (hu et al. ) , and cultivated at °c in the presence of % co in dulbecco's modified eagle's medium with % fetal bovine serum (roche, indianapolis, in, usa), μg/ml gentamycin, and μg/ml penicillin g . the s. suis agar (ssu _ ) gene was amplified using polymerase chain reaction (pcr) with primers ssu _ -f plus ssu _ -r (table s ) and ligated into the bamhi and xhoi sites of pet a(+) expression vector , resulting in the recombinant plasmid pet - (table s ) . similarly, the other expression plasmid pet - was given through direct cloning of s. suis agar (ssu _ ) gene carrying bamhi and sali sites introduced by primers (ssu _ -f plus ssu _ -r) into the expression vector pet a(+) with the same cuts (tables s , s ). the above two plasmids (pet - and pet - ) are designed to prepare in vitro proteins of agar and agar , respectively. for functional complementation, the two genes (ssu _ and ssu _ ) were separately inserted into the low-copy shuttle vector pva (romero et al. ) , giving the plasmids pva - and pva - , respectively (table s ) . all the recombinant plasmids involved in this study were confirmed with both pcr assays and direct dna sequencing. figure . discovery of the genome-wide regulons encoding the galnac/galn utilization pathways in lactobacillales arrows represent the galnac/ galn catabolism-related genes, and circles denote the predicted agar-recognizable sites. the genes are variably colored according to differential functions assigned in figure and labeled with the last letter of the corresponding protein. genes from the same genetic loci that are not adjacent/ neighbored each other are separated by a slash. similarly, genes from different genetic loci are separated by a double slash. given the two types of putative agar-binding palindromes shown on the top (details in table s ), agar and agar sites are accordingly colored with red and blue. sequence logos were given using the weblogo package (http://weblogo.berkeley.edu/logo.cgi). the detailed information on the displayed loci is listed in table s and figure s . galnac, n-acetyld-galactosamine; galn, galactosamine. the two recombinant plasmids (pet a- and pet a- ) were separately transformed into bl (de ), giving the engineered strains fyj , and fyj , respectively (table s ). the two versions of hexahistidine-tagged agar protein (referred to agar [ssu _ ] and agar [ssu _ ]) were produced using the above two engineered strains fyj and fyj , respectively. in brief, when bacterial optical density at wave-length of nm (od ) reached . - . , the bacterial cultures with an appropriate plasmid (table s ) were induced with . mmol/l isopropyl-βdthiogalactopyranoside (iptg) at °c for - h. as we described before (feng and cronan ) , two rounds of french pressure-based lysis were conducted for release of the recombinant protein agar (and/or agar ), and subsequent procedures of protein purification included the nickel column-based affinity purification followed by fast phase liquid chromatography (fplc) feng and cronan ) . consequently, the protein of interest was concentrated via ultrafiltration , and the purity was judged by % sodiumdodecyl sulfate polyacrylamide gel electrophoresis (sds-page). western blot was performed routinely to further verify the hexahistidine-tagged agar (and/or agar ) protein. the protein samples were separated with % sds-page and thereafter transferred to a nitrocellulose membrane (amersham, ge healthcare, piscataway, nj, usa). the primary antibody was an anti-hexahistidine mouse monoclonal antibody, and the secondary antibody was a peroxidase-conjugated goat anti-mouse immunoglobulin g. the signal of target protein band was captured by an exposure to the high-performance chemiluminescence ecl film (amersham, ge healthcare, piscataway, nj, usa). to verify the identity of the recombinant agar (and/ or agar ), the resultant peptides by digestion with sequencing grade trypsin (g-biosciences, st. louis, mo, . ng μl − in mmol/l ammonium bicarbonate) were subjected for analyses of a waters q-tof api-us quad-tof mass spectrometer linked to a waters nano acquity uplc (feng et al. b ). the mass data acquired were assayed through waters protein lynx global server . . , mascot (matrix science, boston, ma, usa) combined with blast against ncbi nr database (feng et al. a ). the nickel column-based purified agar (and/or agar ) protein was further assayed by gel filtration chromatography using a superdex column (amersham, ge healthcare, piscataway, nj, usa) run on an Äkta fast protein liquid chromatography system (ge healthcare) as described figure . maximum-likelihood phylogenetic tree of agar proteins. agar (ssu _ ) is given in red, and agar (ssu _ ) is highlighted in blue. the nagr proteins from bacillus subtilis, streptococcus suis, and lactobacillus plantarum are used as outtree. the logos for the predicted palindromes recognized by agar (agar ) is presented on the right hand. all the homologs of agar (agar ) is accessed to table s . nagr proteins from b. subtilis (bertram et al. ) , l. plantarum wcfs , and s. suis zyh were used as an outgroup. nagr was selected because, as like agar and agar , this protein is a member of the hutc subfamily of the gntr family and a regulator of aminosugar metabolism. previously cronan , ) . the column effluent was evaluated at a flow rate of . ml/min in pbs buffer ( mmol/l na hpo , mmol/l kh po , mmol/l tris-hcl, mmol/l nacl, . mmol/l kcl, ph . ). the protein peak at the position of expected elution volume was sampled and verified by % sds-page. to further elucidate the solution structure of agar (and/or agar ) protein from s. suis, we conducted chemical cross-linking experiments in which ethylene glycol bis-succinimidylsuccinate (egs) (pierce, rockford, il, usa) was added . in each reaction system ( μl in total), the interested protein (~ mg/ ml) was separately incubated with the egs cross-linker (at different levels [ . , . , . , . , . , , , μmol/l for agar protein; , . , , , μmol/l, for agar protein]) for h at room temperature. finally, the reaction products were visualized via % sds-page followed by commassiee brilliant blue staining. the function of the predicted agar and agar binding sites were proved using electrophoretic mobility shift assays (emsa) as we established earlier (feng and cronan ; feng et al. a,b) with minor modifications. all the doublestrand dna probes were produced in vitro by annealing two complementary oligonucleotides in ten buffer ( mmol/l tris-hcl, mmol/l ethylene diamine tetraacetic acid (edta), mmol/l nacl; ph . ), and labeled with dig-ddutp (roche, indianapolis, in, usa) by the terminal transferase cronan , ) . three agar specific dna probes used here included ssu _ / site probe ( bp), ef site probe ( bp), and ef site probe ( bp). in addition to ssu _ probe ( bp) as the negative control, the other four tested agar recognizable sites corresponded to ssu _ probe ( bp), ssu _ probe ( bp), ssu _ / site ( bp), and ef site probe ( bp) (table s ). after min of incubation of the dig-labeled dna probes ( . pmol) with or without agar (and/or agar ) protein in the binding buffer (roche) at room temperature, the dna-protein complexes were separated by the native % page gel and transferred onto an equilibrated, positively charged nylon membrane (roche) by contact blotting followed by uv cross-linking ( mj for sec) (feng and cronan ; feng et al. b ). finally, the signals were captured by exposure to the high-performance chemiluminescence film (amersham hyperfilm ecl) cronan , ) . mid-log phase cultures of s. suis strains (wild type [wt], Δagar , Δagar , cΔagar and cΔagar ) grown in thb media (with/without galnac- p) were collected to prepare the total bacterial rna using an rneasy bacterial rna isolation kit (qiagen, hilden, germany) li et al. ). as we performed before cronan , ) , rna integrity/quality was validated by separation of . % agarose gel electrophoresis. the possible contamination of trace genomic dna in the rna samples was ruled out by pcr-based detections, using the total rna as the template (feng and cronan . using the qualified rna preparations, complementary dnas (cdnas) were synthesized by reverse transcription (rt). then, the real-time quantitative pcr (qpcr) combined with the sybr green method feng and cronan ) was carried out to probe possible relevance of agar (and/or agar ) to the altered expression profile of genes encoding galnac utilization pathway. the method of −ΔΔct ( ) was applied to determine the relative level of the target genes associated with galnac utilization in which the s rrnaencoding gene s rdna as the internal reference (table s ) . target genes tested here are agas (ssu _ ), figure . binding of agar to the predicted cognate sites. alignment of the agar -binding sites from species of lactobacillales (a) and the resulting sequence logo (b). in (a) the identical residues are white letters in red background, similar residues are black letters in yellow background, and varied residues are in black letters. names and locus tags for genes from streptococcus suis are shown by red font. in (b) the sequence logo is generated using weblogo (http://weblogo.berkeley.edu/logo.cgi). the detailed information of the cognate sites is seen in table s . (c) agar does not bind its own promoter. (d) binding of agas (ssu _ ) promoter to agar proteininteraction of agar protein with the promoter regions of both agaa (ssu _ ) (in e) and bgac (ssu _ ) (in f) agar protein from streptococcus suis cannot bind to the agar site of ef of enterococcus faecalis v (in g), whereas it binds to its agar site (in h). the minus sign denotes no protein of agaa added. the protein levels of agar (in the right hand four lanes of each panel [left to right]) were . , , and pmol. the protein samples were incubated with . pmol of dig-labeled probe in a total volume of μl. a representative result from three independent gel shift assays ( % native page) is given. agaay (ssu _ / ), bgac (ssu _ ), and gad-vwef (ssu _ , ssu _ , ssu _ and ssu _ ), respectively. the agar (or agar ) gene from the s. suis strain zyh was replaced with the spectinomycin resistance (spc r ) cassette by homologous recombination hu et al. ) . briefly, the spc r cassette from pset (takamatsu et al. ) was cloned into the puc vector (invitrogen) to give the intermediate plasmid puc -spc (table s ) , and then the two dna fragments adjacent to the agar (or agar ) gene were separately inserted into the puc -spc vector, giving the knockout plasmid puc:: and puc:: , respectively (table s ). as we did before li et al. ) , the plasmid of puc:: (or puc:: ) was electroporated into the competent cells of s. suis zyh to acquire positive transformants with spc r . the multiplex-pcr techniques were adopted to screen the Δagar (and/or Δagar ) mutant (table s ) . consequently, the mutants we acquired for functional experiments were further proved by direct dna sequencing. the two plasmids of pva - and pva - were separately transformed into Δagar and Δagar mutants to give the complemented strains cΔagar and cΔagar , respectively (table s ) . as hytönen et al. (hytonen et al. ) reported, s. suis bacteria (wt, Δagar and cΔagar ) grown in the midlog phase were subjected to cell lines-based analyses. the two cell lines are hep- (human laryngeal epithelial cell line) and murine macrophage raw . cells hu et al. ). bacterial adherence was tested with hep- cell line, and the evaluation for ability of antiphagocytosis was based on the raw . cells. to reveal the role of agar (and/or agar ) in bacterial pathogenesis/virulence, two different kinds of experimental animals were employed, including balb/c ( -week old, female) mice and spf-piglets. in the infection experiment of mice, totally animals were challenged that are classified into six groups ( mice/group). except that thb acted as a negative control, the other five groups infected with s. suis (at a dose of × cfu per mouse) corresponded to wt, Δagar , cΔagar , Δagar , and cΔagar , respectively. the result obtained from the experiment of mice infection was further checked using the infection test of piglets, its natural host of s. suis. given the fact that only agar (not agar ) plays a role in bacterial infectivity, three groups of piglets (six piglets/group) were rechallenged by wt, Δagar , and cΔagar , respectively. clinical syndromes of the infected mice/piglets were monitored for h. of particular note, deaths were recorded and moribund animals were humanely killed. all experiments on live vertebrates in this study were approved by the ethics figure . galnac/galn utilization pathway is repressed by agar and induced by addition of galnac. (a) schematic diagram for agar and its cognate target genes.circles denote the agar -recognizable sites, and the arrows represent the genes of the galnac utilization pathway. note: ssu _ and ssu _ constitutes an operon agaay, whereas the four ordered genes (ssu _ , ssu _ , ssu _ , and ssu _ ) might form an operon of bgac-gadvwef. (b) real-time qpcr assays for effects of agar on expression profile of genes of galnac/galn utilization pathway in streptococcus suis. (c) addition of galnac increases expression level of galnac/galn utilization pathway-encoding genes in s. suis.the data are expressed as averages ± sd, and error bars mean sd. no less than five independent experiments were carried out here. the minus sign denotes no addition of galnac into growth media, whereas the plus sign denotes addition of galnac in vitro. note: p < . . galnac, n-acetyld-galactosamine; galn, galactosamine; qpcr, quantitative pcr; sd, standard deviations. command and performed in accordance with the relevant guidelines and regulations (cao et al. ; hu et al. ) . genome sequences were downloaded from the microbesonline genomic data base (dehal et al. ). identification of orthologs was performed using the blastp search in the non-redundant database (altschul et al. ) and microbesonline tree browser. for functional protein annotations by distant homology to characterize proteins, blast search in the swissprot/uniprot database was used. analysis of chromosomal gene clustering was performed by microbesonline and seed web resources (dehal et al. ; disz et al. ). the galnac utilization subsystem curation and analysis were conducted, using the seed platform (disz et al. ) . protein domains were determined by protein similarity search tools in the pfam database (sonnhammer et al. ) . multiple sequence alignments were constructed by either clustalw (http://www.ebi.ac.uk/tools/clustalw /index.html) (thompson et al. ) or muscle (edgar ) . phylogenetic trees were constructed using the maximum likelihood algorithm implemented in the phylip package (felsenstein ) and visualized via the dendroscope tool (huson et al. ). sequences logos were constructed using weblogo package (crooks et al. ) . for genomic reconstruction of the regulons, we used the well-established comparative genomics approach (rodionov ) . the approach includes inference of transcriptional factor-binding sites (tfbss), construction of nucleotide positional weight matrices (pwms) for tfbss motifs, and reconstruction of regulons in complete genomes on the basis of prediction of putative tfbss in the promoter gene regions. first, in the studied lactobacillales genomes, we revealed orthologs of previously known genes for galnac utilization (table s ). second, we predicted possible transcriptional regulators for the uncovered galnac utilization. candidate regulators were attributed to the regulons by using a genomic colocalization and co-occurence of a putative regulator with the identified galnac utilization genes. such analysis defined two groups of transcriptional regulators belonging to the hutc subfamily of the gntr family (fig. ) . for each group of agar proteins, we identified putative binding motifs analyzing upstream regions of presumably regulated gene by the discover profile tool implemented in the regpredict web server (novichkov et al. ) . in this approach, putative tfbss were determined as overrepresented words in upstream regions of putatively coregulated genes. based on the genomic co-occurrence and co-presence of genes, it has been proposed that agar binding sites should be located upstream hydrolase and pts genes while agar sites ought to be upstream of deacetylase, isomerase, and aldolase genes. for the prediction of putative tfbss, we analyzed upstream regions of probably related operons, expanding − to + bp relative to start codon of the first gene of the operon. both agar and agar belong to hutc subfamily. tfbss for hutc subfamily proteins have structure of an even palindrome (rigali et al. ; suvorova et al. ) . thus, for prediction of the agar and agar tfbss motifs, we searched for even palindromic dna motifs of - bp. among all motifs found for each set of upstream regions, we selected the longest motif with the highest information content. the selected motif was quality controlled by two approaches, ( ) consistency check and ( ) phylogenetic footprinting. consistency check (mironov et al. ; rodionov ) was done, that is, motif was checked for the presence in multiple number of genomes. high quality motif should find predicted tfbss in upstream regions of predicted regulated operons in genomes having the analyzed regulator, but not in genomes lacking the regulator. phylogenetic footprinting technique is based on analysis of multiple alignments for upstream regions of orthologous genes (shelton et al. ). high quality motif should find predicted tfbss that are located inside conserved islands of multiple alignments. when the high quality of the motif was confirmed by both consistency check and phylogenetic footprinting, the identified regulatory motifs were used for the construction of the pwms (profiles) and the obtained matrices were used to determine additional candidate regulatory sites in the analyzed genomes, using the run profile tool in the regpredict web server. the scores of sites were calculates as a sum of nucleotide weights for each position. the data used here were expressed as mean ± sd. unless specified, data were analyzed by two-tailed, unpaired t test, and all assays were repeated no less than three times. the threshold for significance refers to the p < . . the galnac utilization pathway has been previously reconstructed by the comparative genomics approach in a large number of proteobacteria species (leyn et al. ) and experimentally analyzed in e. coli (brinkkotter et al. ) and shewanella sp. ana- (leyn et al. ). here we used the same approach for reconstruction of the galnac utilization pathway in the zoonotic agent, s. suis ( fig. and table s ). for integrated genomic reconstruction of galnac utilization pathways and concordant transcriptional regulation, we searched orthologs of known galnac utilization genes (leyn et al. ) in complete lactobacillales genomes. as a result, the genes encoding, this metabolic pathway was identified in genomes representing four families, streptococcaceae, lactobacillaceae, enterobacteraceae, and carnobacteriaceae (table s ). the minimal number of genes in the reconstructed regulons was detected in lactobacillus helveticus and streptococcus pyogenes. in these organisms, only one or two genes from the galnac utilization pathway were found because these genes were insufficient for galnac utilization. most probably, these organisms cannot utilize galnac. the minimal gene set allowing utilization of galnac is present in a group of closely related genomes including streptococcus gordonii, streptococcus mitis, and streptococcus pneumonia. this gene set contains genes for regulator (agar), galactosamine- -phosphate deaminase/isomerase (agas), glycoside hydrolase (bgac), and pts (gadvwef). the other well-studied streptococcus genomes also contain genes for the tagatose- , -diphosphate aldolase (agay). in contrast, all the analyzed lactobacillus lack agay gene but contain the gene for n-acetylgalactosamine-phosphate deacetylase (agaa). amongst streptococcaceae, agaa gene was identified only in the s. suis genome. the gene for tagatose- -phosphate kinase (agaz) was found only in enterococcus faecalis and carnobacterium sp. - . previously agay gene was shown to be not obligatory for the galnac utilization and function of agaz was proposed to be actualized by other genes, such as lacc (ec . . . ) or pfk (ec . . . ). orthologs of these genes were found in the studied genomes, for example in s. suis lacc is encoded by gene with locus tag ssu _ and pfk is encoded by ssu _ . the presence or absence of agaa gene is the crucial point for the ability or disability to utilize galnac. thus, the absence of this gene in the aga regulons points to the ability of the organism to utilize only galn, but not galnac utilization. thus, organisms lacking agaa gene should have galn-specific transporters whereas organism having agaa gene should be able to transport galnac (leyn et al. ) . to predict the specificity of the lactobacillaceae transport systems, we compared all the identified ptss to the previously described ones. the phylogenetic analysis revealed that all the lactobacillaceae ptss are orthologous to the transport system from haemophilus parasuis (fig. s ). this pts was previously proposed to be specific to galn-containing oligosaccharides. co-occurrence and co-localization of this pts with the hydrolase genes in the most studied genomes confirms its specificity to the oligosaccharides. on the other hand, pts genes are co-localized also with the agaa gene in the large number of studied genomes (table s ) that designate to the possibility of this system to transport galnac or both galnac and galn containing oligomers. however, we cannot have success in predicting the precise specificity of the gad-vwef encoded pts, using only the comparative genomics approach. for the integrated genomic reconstruction of galnac utilization pathways and the concordant transcriptional regulation, we searched orthologs of known galnac utilization genes (leyn et al. ) in complete lactobacillales genomes. as a result, the genes encoding this metabolic pathway were identified in genomes representing four families, streptococcaceae, lactobacillaceae, enterobacteraceae, and carnobacteriaceae (table s ). analysis of conserved chromosomal loci with the galnac utilization genes detected the presence of one or two suggested transcriptional regulators per genome ( fig. and table s ). all predicted regulators belong to the gntr family of transcription factors (hoskisson and rigali ). phylogenetic analysis revealed that the predicted regulators form two separate orthologous groups that we named agar and agar (fig. ) . a strong tendency of agar and agar genes to cluster onto the chromosome with the galnac utilization genes suggests conservation of their function (fig. ) . two lactobacillaceae and four streptococcaceae genomes have both agar and agar genes, whereas in other genomes only one of the regulator genes was present ( fig. and table s ). to infer the agar /agar regulons in lactobacillales, we used the comparative genomics approach applied in the regpredict web server. this approach combines prediction of candidate regulator-binding sites with crossgenomics comparison of regulons. since agar and agar protein orthologous groups are distantly related to each other ( % identity), we propose that their binding motifs should also be different. in most analyzed genomes, agar is co-localized with genes for glycoside hydrolase and pts, whereas agar tends to be clustered onto the chromosome with the genes for deacetylase, isomerase, and aldolase ( fig. and table s ). thus, we proposed that agar -binding sites should be located upstream hydrolase and pts genes while agar sites ought to be upstream of deacetylase, isomerase, and aldolase genes. upstream regions of genes presumably regulated by each transcriptional factor were analyzed using discover profile tool of the regpredict web resource. after the identification of putative-binding motif, we searched for additional regulatory sites in the analyzed genomes and finally reconstructed agar and agar regulons. the candidate motifs for both the studied regulators have an even palindrome structure (figs. , a and table s ) that is in good agreement with previous observations on binding motifs for proteins of the hutc subfamily of gntr family (hoskisson and rigali ). predicted agar -binding sites have a length bp, whereas the predicted-binding sites for agar are bp sequences ( fig. a and table s ). both agar and agar -binding motifs have a palindrome at-rich central part. the agar -binding motif is at-rich at all times and quite degenerated, that is, has moderate information content. on the other hand, the agar -binding motif is more cg-rich and conserved and has a higher information content than agar -binding motif. additionally, agar -binding motif demonstrate similarities with the motifs of some regulators, previously characterized experimentally or in silico, such as gnbr of lactobacillus casei bl (bidart et al. ) and nagr proteins of b. subtilis (bertram et al. ; leyn et al. ) and various lactoacillales (ravcheev et al. ) . composition of the agar and agar regulons varies between species (fig. ) . in lactobacillus rhamnosus (l. rhamnosus), enterococcus faecalis (e. faecalis), and carnobacterium sp. - , all genes for galnac utilization are organized in a single operon that is regulated by a single regulator, agar in the first two genomes and agar in the last one ( fig. and table s ). in the most part of the streptococcaceae and lactobaciilaceae genomes, we observed a strong "division of labor" between the two regulators (fig. ) . thus, agar -binding sites were found upstream of hydrolase/pts genes, whereas agar looks to regulate genes for deacetylase/isomerase/aldolase (fig. ) . in the genomes of s. suis, lactobacillus gasseri (l. gasseri) and lactobacillus johnsonii (l. johnsonii), overlapping of regulons was detected (fig. ) . in all these genomes, genes for the transport system seemed likely to be under double regulation by agar and agar . also, in the vast majority of the analyzed genomes autoregulation was identified for both agar and agar genes (table s ) . to probe the putative function of the two new members (s. suis agar and agar ) of the gntr family of transcription factors, we employed bl (de )/pet (a) a prokaryotic expression system to prepare the above two proteins in vitro. consequently, the n-terminal hexahistidine tagged s. suis agar protein was purified to homogeneity and gave a single-protein band of appropriate molecular mass (~ kda for monomer) (fig. a) . the xhis tagged version of this recombinant s. suis agar protein was also determined by western blot using the anti- xhis tag primary antibody (fig. b) . chemical cross-linking assays with the egs cross-linker visualized clearly the egs dose-dependent dimerization of agar , indicating its predominant solution structure of this protein is a dimer (fig. c) . liquid chromatography mass spectrometry-based determination of tryptic peptides of the recombinant agar protein band excised from an sds-page gel (fig. a ) verified its identity, in that the peptides matched s. suis ssu _ protein with % coverage of the expected peptides (fig. d) . somehow different from the agar protein, two forms of agar protein (monomer, the predominant form, and trace amount of dimer) still can be detected by the separation with % sds-page (fig. s a) . given that two possibilities are present (either the contaminated protein with the molecular mass at the dimeric position, or the dimeric form of agar ), western blotting with the anti- xhis tag primary antibody was conducted. as anticipated, it clearly showed that two protein bands with an appropriate molecular mass (~ kda for monomer and ~ kda for dimer), ruling out the possibility of protein contamination (fig. s b ). this observation is unexpected, but not without precedent. in fact we recently encountered a similar scenario in the case of the brucella bior regulator that is also a member of the gntr family transcription factor (feng et al. a ). the essence of bior forming a dimer was further proved by chemical cross-linking assays (fig. s c) . ms-based analyses of two protein bands of agar (one is cut from a dimer, the other is collected from a monomer) validated exactly the identity wherein the two forms of peptides covered s. suis ssu _ protein at the level of no less than % ( fig. s d and e). it seemed very likely that the genome of s. suis zyh (accession no.: cp . ) encodes a fully functional galnac utilization machinery and most of the genes encoding this pathway are regulated by agar (and/or agar ) (fig. ) . thereby, we employed emsa to probe the binding of the agar (and/or agar ) protein to the cognate palindromes (table s , fig. a and b) . on the s. suis chromosome, totally three putative agar -binding sites (ssu _ probe, ssu _ probe and ssu _ / site probe) were localized, whereas only one possible agar -recognizable site (ssu _ / site probe) was detected. because of the absence of the predicted agar binding site upstream of the agar gene, we expected that this gene is not autoregulated. indeed, emsa experiments suggested that agar protein cannot bind to its own promoter region (ssu _ probe), ruling out the possibility of autoregulation by agar regulator (fig. c ). by contrast, emsa tests revealed clearly that agar protein bound the other three agar -binding palindromes in a dose-dependent manner (fig. d-f) . additionally, we also used s. suis agar protein to evaluate the function of the two agar-binding sites in front of the ef locus of e. faecalis v , a close relative of s. suis. as a result, agar physically interacted with the ef site , ttgtggttataaccagtt (fig. h) , but not the ef site , tttattgacaaaataaaaaa (fig. g) further validated the specificity of agar binding. different from the scenario with agar , s. suis agar was only found to have the ability to interact with the predicted ssu _ / site , tctattaatatactaacact (fig. s a ). as anticipated from the comparative genomic analysis of the regulons, no interplay was observed between the agar -specific ssu _ / site , ttgtggttatgtccagta and s. suis agar protein (fig. s b) , ruling out the possibility of cross-regulation by agar and agar . more importantly, the two putative agar recognizable sites from e. faecalis v (referred to ef site , aaattcaatatattaagata and ef site ) were demonstrated to be functional, using gel shift assays with the s. suis agar protein ( fig. s c and d) . given the observation that agar efficiently binds to the promoter regions of the three genes/operons (agas (ssu _ ), agay (ssu _ ), and bgac (ssu _ )) encoding galnac utilization pathway in s. suis (figs. , a), we therefore attempted to elucidate its possible function in modulating streptococcus galnac utilization machinery. the Δagar (Δ ) isogenic mutant was constructed, using an approach of homologous recombination, and its complementary strain cΔagar (cΔ ) was generated through the low-copy plasmid pva -borne expression of agar gene (table s ). we noted that the deletion of agar does not affect its growth (fig. s a) . additionally, no obvious alteration of capsule was observed in the Δ agar mutant in comparison with the wt strain zyh (fig. s b and c) . the qpcr-based transcriptional analyses showed that removal of agar gene gave at least five-fold increment of agas (agaay and bgac-gadvwef operon) expression (fig. b) . whereas the transcription of the above target genes was restored to the level seen in its parental strain s. suis zyh (fig. b) . similar results were also observed in altered expression profile of the Δagar mutant revealed by the rna-seq (e.g., expression of the three genes ssu _ (agas), ssu _ (agaa), and ssu _ (agay) are elevated upon removal of agar , table ). therefore, it seemed true that agar is a functional repressor for the streptococcus galnac utilization pathway. in contrast, we failed to visualize any significant alteration of the bgac (ssu _ ) expression level in the Δagar mutant in comparison with the wt strain and its complementary strain cagar (not shown). the suggested possibility that agar might play an uncovered role or be an evolutional relic with loss of physiological demand and/or advantage, in that ( ) there is no in vivo regulatory role attributed to its protein product agar although it retains dna-binding activity to its own promoter, ( ) the fact that all the other galnac metabolism-related genes are without control by agar is due to the lack of the predicted agar -recognizable palindromes, ( ) no physiological function can be present in galnac metabolism, even agar can autoregulate itself. thus, our subsequent interests focused on agar regulator. considering the direct relevance of agar to galnac utilization in s. suis, it is reasonable to probe the possibility whether this regulator acts to be responsive to the presence of galnac. we thereby grew wt strain s. suis zyh in the medium with/without the supplementation of galnac, and compared the expression profile of relevant target genes like agas. as anticipated, the expression level of all the tested galnac catabolism pathway-encoding genes (including agas and bgac) was given a -to -fold increment upon the addition of mmol/l galnac into growth conditions (fig. c) . together, we concluded that the galnac/galn utilization pathway is repressed mainly by the agar regulator, whereas induced by an addition of galnac. contribution of agar (agar ) to bacterial pathogenesis as we knew that amino sugars (galn and/or galnac) constitute common residues for surface structure of various bacterial cell walls (bernatchez et al. ; freymond et al. ) , we thus ambitiously reasoned that ( ) such a bacterial surface structure might be involved in mutual communication/crosstalk between bacterial pathogens and the inhabited/infected hosts, ( ) the maintenance and regulation of galn/galnac catabolism is probably implicated into successful infections of some bacterial pathogens. to probe possible interference between galn/galnac catabolism and the formation of virulence-associated surface structures, we systemically employed two lines of approaches including cell lines-based tests and infections of experimental animals. first, hep- cell line was used to evaluate the ability of bacterial adherence ( fig. a and b) , and raw . macrophage was subjected to dissecting its capability of anti-phagocytosis ( fig. c and d) . fluorescence-activated cell sorting (facs)-based experiments elucidated that around threefold increment of bacterial adherence potential to hep- cells was given in the Δagar mutant relative to the wt zyh strain (fig. a and b) . furthermore, the deletion of agar gene was found to give nearly fivefold increment of anti-phagocytosis ability against raw . macrophage ( fig. c and d) . this finding was in much similarity to scenarios seen with both Δneub and Δcps b mutants of s. suis (note: the two genes are involved in bacterial surface architecture) . thereby, we anticipated that the enhancement in abilities of bacterial attachment and anti-phagocytosis might be partially due to the altered carbohydrates with galn/ galnac residues on bacterial cell wall surface and this kind of alteration could be attributed to the dysfunction in agar -mediated regulation of galn/galnac utilization pathway. in contrast, no obvious difference between the Δagar mutant and its parental strain zyh was observed on either bacterial adherence to hep- cell line or bacterial anti-phagocytosis against raw . macrophage (not shown). in fact, such a different effect exerted by agar (and agar ) is not very surprising, in that only aga (not agar ) evolved to possess a regulatory role in controlling galnac catabolism in s. suis. given the fact that not only the two mutants (Δneub and Δcps b) of s. suis feature the potential of both increased adherence/anti-phagocytosis, but also exhibit dramatically reduced virulence in infection models of both mice and piglets , it is of much interest to further probe a possible role of agar in bacterial virulence. in the mice infection assays, all the balb/c ( week old, female) mice infected with the wt strain zyh were sick shortly, and most of the rats died within h (fig. e ). by contrast, mice of the negative control group inoculated with thb survived (not shown). in particular note, nearly all the mice infected with the Δagar mutant survived, while most of animals injected with the complementary strain cΔagar died within days. to verify the results obtained from the above model of mice, we repeated the infection tests using the model of spf-piglets, natural hosts for s. suis. as a result, we observed that the six spf-piglets inoculated with wt virulent strain developed most of the typical disease symptoms (high fever, limping, swollen joints, etc.), and most of them died on day (fig. f) . as expected, all the piglets infected with the Δagar mutant survived during the period of entire experiment. however, the re-introduction of agar gene into the Δagar mutant restored fully its strong virulence (fig. f) . collectively, we believed that the prevalent regulator agar for galn/galnac utilization pathway contributes to bacterial infectivity of s. suis, although we failed to note an apparent role of the other secondary regulator agar in bacterial pathogenicity (not shown). the data shown here represents a first report that illustrated the genomic reconstruction of galn/galnac catabolism pathway in streptococci and firmicutes. different from the scenarios described in e. coli (reizer et al. ) and shewanella (leyn et al. ), a significant variation of this pathway was proposed for streptococci ( fig. and table s ). as you can see from the working model, agaz is absent in s. suis. it seemed likely that our continued genomic analyses pointed out that s. suis actually lacks any ortholog of this gene. thereby, we speculated that the loss of agaz function in s. suis might be compensated by two other genes that are not in aga regulon: ( ) ssu _ (lacc ; tagatose -phosphate kinase, ec . . . ), ( ) ssu _ (pfk, -phosphofructokinase, ec . . . ). this hypothesis required further experimental verification. through extensive analyses for genomic contexts from firmicutes, we dissected two galn/galnac pathwayspecific regulators namely agar and agar . the agar gene is divergently transcribed into genes forming an operon and encoding a βgalactosidase (bgac) (hu et al. ) and a pts sugar uptake system (named agabcde that could be a galn uptake system). the agar gene in many streptococcus species is located in front of the genes encoding the predicted galactosamine- -phosphate isomerase and tagatose , -diphosphate aldolase, two key enzymes in the aga catablic pathway. in s. suis, the locus ssu _ is annotated to be agar , whereas ssu _ is referred to agar . somewhat evolutionally distinct from the paradigm regulator (the e. coli agar protein product) that belongs to the deor family of transcription factor, the two newly identified regulators agar and agar are classified into the gntr family of transcription repressors (figs. , s ) . additionally, it seemed likely that the two gntr-like regulators (agar and agar ) themselves fit into two different subgroups and might possess the varied preference in their dnabinding sites (figs. , s ) . the fact that agar -recognizable palindrome is predicted to be present in front of multiple target genes whereas agar has only one site located in its own promoter region (fig. ) raised the possibility that agar could have been evolved into a prevalent/ major regulator for galn/galnac utilization pathway (agar might be a minor/and even a cryptic regulator). our in vitro gel shift assays validated the direct interaction between the agar (and/or agar ) protein and its binding sites (figs. , s ). given the facts that a significant regulated expression of galn/galnac utilization-related genes (such as agas, agaay , and bgac) by agar was observed, however, no in vivo role could be attributed to agar , we came to be more confident that agar relative to agar acts as a prevalent/leading player in modulating expression of genes encoding bacterial galn/ galnac catabolism in most firmicutes. however, we did not obtain evidence for physiological ligand/molecular effector of agar repressor, although we observed that galnac does induce the expression of galn/galnac catabolism-related genes such as agas (fig. c) . the essence that amino sugars (galn and/or galnac) are common components on the surface of bacterial cell walls (bernatchez et al. ; freymond et al. ) has driven us to test possible roles for the regulation of galn/ galnac catabolism in the infectivity of bacterial pathogens. as expected, an interference of galn/galnac catabolism/ utilization by disrupting agar encoding a prevalent regulator, significantly altered abilities of both bacterial adherence to hep- cells and anti-phagocytosis against raw . macrophage (fig. ) . given the fact that in this alteration we observed a similar scenario seen with the two virulence-associated determinants (neub and cps b) with biological roles in constitution/development of bacterial surface architecture , we reasoned that regulation/maintenance of galn/galnac catabolism pathway is essential for full virulence of s. suis. in fact, this proposal was subsequently proved in that the dysfunction in agar -mediated regulation impairs bacterial infectivity of s. suis in both mice and piglets (fig. ) . also, we are not surprised to establish the relevance of galn/galnac catabolism to bacterial pathogenicity, in that the alteration of carbohydrates with galn/galnac residues on bacterial cell surface interferes with the bacterial pathogen-binding host cells. taken together, we are first to report the genomic reconstruction of the galn/galnac utilization pathway in s. suis and its novel regulatory network with variations. more intriguingly, we revealed that the interference of galn/galnac utilization pathway by the inactivation of the agar regulator attenuates greatly the bacterial infectivity of the zoonotic pathogen s. suis. our finding might provide a metabolic basis for design of small molecule drugs (inhibitors)-based therapeutics against s. suis infection through targeting the regulatory network of galn/ galnac utilization pathway. additional supporting information may be found in the online version of this article: table s . strains and plasmids used in this study. table s . dna primers used in this study. table s . agar regulons in firmicutes. table s . agar (agar ) binding sites. figure s . multiple sequence alignments of ssu _ (agar ) with two other bacterial homologs. the three homologous proteins used here included bacillus subtilus nagr (nc_ . ), ssu _ (agar ) of streptococcus suis zyh (nc_ . ), and escherichia coli agar (nc_ . ). the program of clustalw (http://www. ebi.ac.uk/tools/clustalw /index.html) was applied to conduct the multiple alignment of protein sequences, and the final output is generated by the espript . program (http:// espript.ibcp.fr/espript/cgi-bin/espript.cgi). identical residues are in white letters with a red background, similar residues are in red letters with a white background, varied residues are in black letters, and dots represent gaps. the predicted protein secondary structure is given on the top. designations: nagr, n-acetylglucosamine repressor; agar, acetyl-galactosamine repressor; bs, bacillus subtilus; ec, e. coli, α, αhelix; β, βsheet; t, βturns/coils. figure s . purification, verification, and characterization of the agar (ssu _ ) protein. (a) % sds-page profile of the purified agar (ssu _ ) protein from streptococcus suis. (b) western blot analyses for the nterminal x his tagged agar protein, using the anti- xhis tag primary antibody. the monomeric protein with expected size of ~ kda is indicated with an arrow, whereas the dimer form (~ kda) is highlighted with an asterisk. designations: m, protein standard marker; wb, western blot. (c) determination for the solution structure of the agar protein, using chemical cross-linking assays. the chemical cross-linker used here is ethylene glycol bis-succinimidylsuccinate (egs). the triangle on the top represents the addition of the egs cross-linker in varied concentrations ( . , . , . , . , . , , , μmol/l in the right-hand eight lanes [left to right]). the minus sign denotes no addition of egs. the protein sample was separated with % sds-page. the ms-based identification of the purified agar protein with the solution structure of both monomer (in d) and dimer (in e). the tryptic peptides that match the agar protein are given in bold and under-lined type. figure s . binding of agar to the predicted palindromes. the predicted agar -binding site (in a) of ssu _ / gene bound agar (ssu _ ) protein, whereas the agar -binding site (in b) of this locus does not interact with agar protein. (c) streptococcus suis agar protein bound to the predicted agar site of ef gene from enterococcus faecalis v . (d) s. suis agar protein bound to the putative agar site of ef gene from e. faecalis v . the minus sign denotes no addition of agaa protein. the protein levels of agar (on the right-hand four lanes of each panel [left to right]) were . , , , and pmol. the protein samples were incubated with . pmol of the dig-labeled probe in a total volume of μl. a representative result from three independent gel shift assays ( % native page) is given. figure s . phylogenetic analyses of phosphotransferase system (pts). in total, the pts system is classified into five sub-groups, one of which is pts-v (highlighted in blue). locus tag of pts system is showed here for agac, ssu _ is indicated in red. not just for eukarya anymore: protein glycosylation in bacteria and archaea gapped blast and psi-blast: a new generation of protein database search programs biosynthesis of glycoproteins by membranes of acer pseudoplatanus. incorporation of mannose and n-acetylglucosamine polysaccharide capsule and suilysin 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serotype this work was supported by the start-up package of zhejiang university (youjun feng), the zhejiang provincial natural science foundation for distinguished young scholars (grant no. lr h ), the national natural science foundation of china (grant no. , , , , , and ), the russian academy of sciences (program "molecular and cellular biology".), the national research fund (# ), luxembourg, and was co-funded under the marie curie actions of the european commission (fp -cofund). dr. feng is a recipient of the "young talents" award. none declared. key: cord- -s l yxc authors: ranga, vipin; niemelä, erik; tamirat, mahlet z.; eriksson, john e.; airenne, tomi t.; johnson, mark s. title: immunogenic sars-cov- epitopes: in silico study towards better understanding of covid- disease—paving the way for vaccine development date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: s l yxc the emergence of the covid- outbreak at the end of , caused by the novel coronavirus sars-cov- , has, to date, led to over . million infections and nearly , deaths. consequently, there is an urgent need to better understand the molecular factors triggering immune defense against the virus and to develop countermeasures to hinder its spread. using in silico analyses, we showed that human major histocompatibility complex (mhc) class i cell-surface molecules vary in their capacity for binding different sars-cov- -derived epitopes, i.e., short sequences of - amino acids, and pinpointed five specific sars-cov- epitopes that are likely to be presented to cytotoxic t-cells and hence activate immune responses. the identified epitopes, each one of nine amino acids, have high sequence similarity to the equivalent epitopes of sars-cov virus, which are known to elicit an effective t cell response in vitro. moreover, we give a structural explanation for the binding of sars-cov- -epitopes to mhc molecules. our data can help us to better understand the differences in outcomes of covid- patients and may aid the development of vaccines against sars-cov- and possible future outbreaks of novel coronaviruses. the ongoing pandemic outbreak of covid- has resulted in the declaration of a global health emergency around the world on january by the world health organization (who) [ ] . the first reported case was on december from the chinese city of wuhan, from which the virus quickly spread to other countries and territories and, as of july , resulted in at least . million infections and over , deaths [ ] . based on the early epidemiology of the covid- disease statistics, the who estimated that the fatality rate of the novel coronavirus is around %, significantly higher than the mortality rate caused by common human coronaviruses [ ] . there are currently numerous scientific, clinical, and socio-economical efforts aimed at combating the covid- disease and its ramifications around the world [ , ] . peptides are loaded on mhc receptors using a peptide loading complex consisting of tap and several other proteins [ ] . in order to narrow down the specific epitopes that could elicit an effective mhc class-i-mediated t cell response, we predicted linear -mer immunogenic sars-cov- peptides and their prominent interacting hla allotypes using the immune epitope database and analysis resource (iedb) and netctl . web servers. the identified peptides were then analyzed in conjunction with the available experimental data for sars-cov-derived linear t-cell epitopes. the three-dimensional structural models of selected ternary complexes of sars-cov- epitope-hla allotype-t cell receptor were created to assess interactions at the structural level. our results can at least partially explain individual differences in the covid- severity and could potentially be used for vaccine development against sars-cov- . all protein sequences encoded by the most up-to-date sars-cov- genomic sequence (refseq: nc_ . ) were retrieved from ncbi refseq database [ ] on march (full accession identifiers in table ). the iedb (http://tools.iedb.org/mhci/) [ ] and netctl . (http://www.cbs.dtu.dk/services/ netctl/) [ ] web servers were used with default parameters to predict sars-cov- epitopes and their binding affinities (expressed as ic ) to different hla allotypes. the iedb server sorts the predicted mhc-i-binding viral epitopes based on the percentile score, which is calculated by comparing the predicted binding affinities of sars-cov- peptides and affinities calculated for a large set of peptides, randomly selected from the swissprot database [ ] ; the iedb server integrates an artificial neural network (ann), stabilized matrix method (smm) and combinatorial library (comblib). the iedb method is highly accurate in classifying mhc class i epitopes, having an auc for the roc curve greater than . [ ] . the netctl . server integrates prediction of proteasomal cleavage, tap transport and peptide-binding to mhc-i supertypes (see table s a for a list of hla-a and hla-b allotypes that belong to these supertypes, with data extracted from the published scientific literature [ , ] ). the netctl . method allows for the identification of class i mhc epitopes with a sensitivity of . and specificity of . based on the default filtering threshold score of . [ ] . since the netctl . and iedb servers use different multistep approaches to predict the binding of sars-cov- peptides to hlas, we used both servers. mhc-i allotypes are known to bind epitopes with lengths of to amino acids. the optimal epitope length was determined by potting the ic values of the predicted (iedb) top percentile epitopes against -to -mers. moreover, the immunogenicity of the top percentile epitopes were predicted using the mhc-i immunogenicity server of iedb (http://tools.iedb.org/immunogenicity/) [ ] . the epitopes with an immunogenicity score greater than . were considered for comparison with epitopes predicted using the netctl . server. in order to identify experimental epitopes matching the predicted sars-cov- epitopes, the data of experimentally known sars-cov epitopes and their interacting mhc allotypes (validated using t-cell assays and mhc ligand assays) were downloaded from the iedb database on april (https://www.iedb.org/database_export_v .php) [ ] . the characteristics of each "match" (protein name, sequence, mapped start-end, mhc-i allotype, etc.) were tabulated and are presented in table s . the grand average of hydropathicity index (gravy) was calculated using the kyte-doolittle hydropathy index scale [ ] . tmhmm . was used to search potential transmembrane helices [ ] . the jpred web server was used to predict secondary structures in non-transmembrane proteins [ ] . the half-lives of the predicted emhc-i complexes was predicted using the netmhcstabpan . web server [ ] . data were plotted using graphpad prism (graphpad software, version . . ; https://www.graphpad.com). the crystal structures of an influenza a virus epitope in complex with the hla-a* : allotype (pdb id: tez, chain a and c; . Å) [ ] and hepatitis b core antigen with hla-a* : (pdb id: oxr, chain a and c; . Å) [ ] were downloaded from the protein data bank (pdb) [ ] (see table s b for the pdb codes of mhc-i allotype structures indicated in table s a ). for docking to hla allotypes, the rosetta flexpepdock web server [ ] was used, after the -mer influenza a epitope was mutated to match selected sars-cov- epitopes using pymol (the pymol molecular graphics system, schrödinger, llc). for ternary complex analysis, the t-cell receptor (tcr)-hla-influenza a epitope complex (pdb id: tez) was used as a template. coordinates of the tcr α and β chains (pdb id: tez; chain i and j, respectively) and the docked epitope-hla-a* : complex were saved using pymol, and interacting residues visually inspected at the interface. molecular dynamics (md) simulations followed the general protocol detailed in [ ] . briefly, the ternary composite structure of hla-a* : in complex with the sars-cov- s protein epitope fiagliaiv and tcr was used as a starting structure to perform molecular dynamics simulation. the structure was initially prepared using maestro (schrödinger release - : maestro, version, schrödinger, llc) by adding hydrogen atoms, determining the protonation states of ionizable side-chains and energy minimizing the structure to remove bad contacts. three independent simulations were carried out using the amber program (version ) [ ] and the ff sb protein forcefield [ ] . the ternary structure was solvated with tip p water molecules [ ] in an octahedral box, keeping a Å distance between solute atoms and the surface of the box. the simulation system was neutralized by adding na + ions, with additional na + /cl − ions incorporated to bring the system salt concentration to . m. the system was then energy-minimized for cycles using the steepest descent and conjugate gradient methods. the minimization was carried out in six-stages, where a restraint on solute atoms was gradually lowered from to kcal mol − Å − . subsequently, the system was heated to k during ps with a kcal mol − Å − restraint on solute atoms. next, equilibration was performed for ns in four steps by systematically reducing the restraint force to kcal mol − Å − . finally, a restraint-free ns production simulation was carried out at constant temperature ( k) and pressure ( bar). coordinates were saved every ps and the sampled conformations were analyzed using vmd [ ] , cpptraj [ ] and chimera [ ] programs. root-mean-square fluctuation (rmsf) calculation was computed using the cα atoms of the initial structure as a reference. hydrogen bonds were defined with a bond length ≤ . Å and a bond angle ≥ • . in order to estimate the potential antiviral cytotoxic t-cell response linked to specific hla allotypes, we predicted the binding affinity of all possible linear -to -mer peptides derived from the proteins (table ) of the sars-cov- proteome (n = , n = , n = and n = ) to hla-a and hla-b supertypes using the iedb web server [ ] . the hla-c supertype-an extremely good ligand for killer-cell immunoglobulin-like receptor (kir) on natural killer (nk) cells-was not selected for this analysis because it is known to be less effective in presenting antigens to cytotoxic t-cells than either hla-a or hla-b [ ] . the class i mhc-epitope (emhc-i) complexes were classified into three different groups based on the predicted epitope-to-mhc binding affinity scores [ ] : strong binders (ic ≤ nm), weak binders ( nm < ic ≤ nm) and non-binders (ic > nm). out of the highest scoring sars-cov- epitopes (top percentile), the -and -mers had, on average, a higher binding affinity to mhc class i supertypes than either the -or -mer epitopes ( figure a ). moreover, there were % more -mer peptides ( ) predicted to bind to class i mhc receptors (ic ≤ nm) in comparison with -mer peptides ( ) ( figure b) . consequently, the top one percentile -mer peptides were selected for further analysis. the predicted (iedb) sars-cov- -mer epitopes (top one percentile) for different hla allotypes (table s a) with an immunogenicity score ≥ . were compared to those predicted using the netctl . server [ ] . the mhc class i epitopes identified from this consensus/combined approach were classified using the binding affinity values from iedb prediction method as strong mhc binders (table s ) , weak binders (table s ) or non-binders (table s ). based on this analysis, many peptides derived from nonstructural proteins (nsp), surface glycoproteins (s) and membrane proteins (m) of sars-cov- are likely to be presented by mhc class i receptors ( figure c , table s ), and hence have a high potential to activate an immune response or the destruction of infected host cells, with many epitopes being derived from the s, e, c-like proteinase ( clpro) and -to- exonuclease ( exo) proteins. in order to predict the most potent epitopes, including those without experimental data, we screened in silico the proteins of sars-cov- . we ranked the epitopes based on predicted binding to mhc-i molecules (affinity values; iedb) and "combined score" (netctl . ), predicting the potency of an epitope to be presented by mhc-i (tables s and s ). the top-ranked, common epitopes from both prediction methods (table ) were selected for further analysis; all five of these predicted epitopes are unique to sars-cov- and not identical in sars-cov. this comparison, we identified common epitopes of sars-cov/sars-cov- (table s ) and hla allotypes hla-a* : and hla-a* : molecules as the top antigen presenters; both allotypes having strong binding affinities (ic ≤ nm) to six peptide epitopes (table ) : residues fiagliaiv from the s protein, vllflafvv and flafvvfll to the e protein, vlawlyaav to clpro, llsagifga to nsp and vlwahgfel to exo of sars-cov- . in order to obtain experimental proof that the mhc-i-binding sars-cov- epitopes predicted in this study are presented by the mhc class i antigen processing pathway in vivo, we compared the in-silico-identified -mer epitopes (n = , table s ) to the equivalent, experimentally identified epitopes of sars-cov strains (n = ; mhc ligand assays data from the iedb database) [ ] . in this comparison, we identified common epitopes of sars-cov/sars-cov- (table s ) and hla allotypes hla-a* : and hla-a* : molecules as the top antigen presenters; both allotypes having strong binding affinities (ic ≤ nm) to six peptide epitopes (table ) : residues fiagliaiv from the s protein, vllflafvv and flafvvfll to the e protein, vlawlyaav to clpro, llsagifga to nsp and vlwahgfel to exo of sars-cov- . table . sars-cov- -derived hla-a* supertype-binding epitopes that are identical to the epitopes of sars-cov strains experimentally known to activate cytotoxic t-cells. to examine the evolutionary conservation or "sequence stability" of the six common epitopes of sars-cov/sars-cov- binding strongly to hla-a* : and hla-a* : , we performed a blastp (ncbi) search against the non-redundant database of sars-cov- [ ] . only one epitope- llsagifga from nsp -was found to have heterogeneity in its sequence (table ) , whereas the other five epitopes were fully conserved, i.e., not yet having changed during the evolution of sars-cov- , suggesting an important role for these peptide sequences for the virus and, at the same time, making these peptides top-candidate antigens for activating the cytotoxic t-cell response against sars-cov- itself. the five conserved and experimentally proven epitopes we selected for further analyses. since mhc class i molecules must retain bound epitopes long enough at the cell surface to successfully induce t-cell-specific immune responses [ ] , we estimated the half-life in hours for each of the experimentally identified epitope-hla complexes (table ) using the netmhcstabpan . web server [ ] . this analysis revealed that the hla-a* : and hla-a* : allotypes have longer predicted half-lives than hla-a* : when in complex with the immunogenic epitopes (table ): all five hla-a* : -epitope complexes and three out of five hla-a* : -epitope complexes had a predicted half-life longer than h, whereas the single hla-a* : -epitope complex had the lowest half-life of less than one hour. table . predicted half-lives of complexes of the conserved sars-cov- -derived most immunogenic experimentally identified epitopes and hla-a* allotypes shown in table . secondary structures, localization within sars-cov- and gravy (grand average of hydropathicity index) scores of the epitopes. allotypes in order to examine the distribution of the hla-a* -antigen complex half-lives in general, we analyzed more than experimentally known complexes of hla-a* supertypes (see table s a for a list of allotypes) bound to bacterial-and viral-pathogen-derived epitopes, which were extracted from the iedb database [ ] . this revealed that immunogenic (ic ≤ nm) hla-a* : -epitope complexes with predicted half-lives of more than three hours were almost double in number in comparison to the hla-a* : -epitope complexes, whereas no immunogenic hla-a* : -epitope complexes were found that would have a similar half-life ( figure s a ). interestingly, the amino acid sequences of the α chains of hla-a* : and hla-a* : are . % identical; the only difference in the sequences-a phenylalanine to tyrosine substitution-is located at the epitope-binding site and is the likely reason for the difference in the epitope binding affinities and half-lives. out of the five most stable epitopes (table ) , three- fiagliaiv (s protein), vllflafvv (e protein) and flafvvfll (e protein)-were derived from transmembrane proteins and were, as expected, more hydrophobic (gravy score > ) than the vlawlyaav ( clpro) and vlwahgfel ( exo) epitopes originating from intravirion proteins ( table ). the epitopes derived from the s and e proteins respectively map to transmembrane helical segments wyiwlgfiagliaivmvtimlcc and livnsvllflafvvfllvtlail that, based on analysis using the tmhmm . web server [ ] , are bitopic in nature, i.e., the predicted transmembrane helices span the lipid bilayer only once. in fact, our predicted epitopes share features, such as being membrane associated, with the well-studied and clinically important epitopes in hiv [ ] and tuberculosis [ ] . this supports the idea that the transmembrane helical epitopes of sars-cov- could potentially stimulate cytotoxic t-cell-mediated immune responses. in order to assess whether hydrophobic residues are enriched in the immunogenic epitopes, we compared the gravy score distribution of the immunogenic (n = , ic ≤ nm) and non-immunogenic (n = ; ic > nm) hla-a* -bound epitopes of bacterial and viral pathogens (retrieved from the iedb database) ( figure s b ). we found that, for epitopes with a gravy score greater than one (having at least seven non-charged residues in a -mer epitope), the immunogenic epitopes were more enriched in hydrophobic residues ( %) in comparison to the non-immunogenic epitopes ( %). thus, our analysis suggests that hla-a* supertype molecules prefer binding to hydrophobic epitopes (ic ≤ nm), and this agrees with published results [ ] . moreover, we obtained similar results from our in-silico-identified sars-cov- -derived novel epitope-mhc-i complexes ( table ) : the most potent identified epitopes had long half-lives and were derived from either hydrophobic, transmembrane regions of sars-cov- or from intravirion proteins that were also found to be mutated among sars-cov- sequences (table ) . table . predicted half-lives of the novel sars-cov- -derived, most immunogenic in-silico-identified epitopes in complex with the allotypes shown in table . secondary structures, localization within sars-cov- , gravy scores and known mutations in the epitopes. in order to compare the interaction patterns adopted by the predicted top five immunogenic epitopes (table ) of mhc-i molecules, we docked the epitopes to the cleft between the α and α helices of hla-a* : (pdb id: tez, chain a) and hla-a* : (pdb id: oxr, chain a). this docking analysis agrees with our other prediction data and suggests that the immunogenic epitopes bind to both the hla-a* : and hla-a* : allotypes by adopting a similar backbone conformation, as has been observed for the canonical epitope gilgfvftl of the influenza a virus (pdb id: tez, chain c) ( figure s c,d) . in more detail, we observed that the residues at position (pos) , , and are fully buried within the antigen-binding cleft of hlas and act as anchoring residues, providing steric constraints to the nand c-terminus of the epitopes (figure a,b) . comparison of the root-mean-square deviation (rmsd) of the superposed cα atoms of the epitopes revealed a maximum deviation for the vlwahgfel epitope ( exo), possibly due to the bulky, aromatic side chain of w buried at pos and two charged residues: h at pos and e at pos (table ) . interactions between the partially solvent-exposed hydrophobic residues at pos -pos of the docked epitope fiagliaiv (s protein) and the solvent-exposed residues a , k , v , t , k , v and q of the α and α helices of hla-a* : , complement what is seen in the x-ray crystal structure of the influenza a virus epitope-hla-a* : complex (pdb id: tez, chain a and c), and suggest that the sars-cov- epitope-hla complex interacts with tcr ( figure c ). and cdr β of the tcr-β chain, recognize the hla-a* : -sars-cov- fiagliaiv epitope ( figure d ); the cooperative interacting nature of residues in these loops could provide specificity towards class i mhc molecules. moreover, the residues l and w within the cdr β loop and i within the cdr α loop could be important for antigen-hla complex recognition due to their likely direct contacts with residues l , i and i of the fiagliaiv epitope and residues a , v and q of hla-a* : ( figure e ). to understand the molecular basis of tcr binding to the sars-cov- antigen-loaded mhc-i cell surface molecules, hla-a* : , in complex with the s protein epitope fiagliaiv , was superimposed with the hla-a* : allotype of the influenza a virus ternary complex structure (pdb id: tez, chain a), and the atomic coordinates of tcr (tcr-α and tcr-β chain; pdb id: tez, chain i and j, respectively) were then utilized for visual analysis of binding. this visualization suggests that the loops cdr α, cdr α and cdr α of the tcr-α chain, and loops cdr β, cdr β and cdr β of the tcr-β chain, recognize the hla-a* : -sars-cov- fiagliaiv epitope ( figure d ); the cooperative interacting nature of residues in these loops could provide specificity towards class i mhc molecules. moreover, the residues l and w within the cdr β loop and i within the cdr α loop could be important for antigen-hla complex recognition due to their likely direct contacts with residues l , i and i of the fiagliaiv epitope and residues a , v and q of hla-a* : ( figure e) . to assess the conformational and intermolecular interaction dynamics of the hla-a* : - fiagliaiv s protein epitope-tcr complex, a ns simulation was carried out on the ternary structure in triplicate. the global conformational dynamics was assessed by computing the rmsf over the cα atoms ( figure a ), which shows a stable tcr and epitope, with an average rmsf of . ± . Å and . ± . Å, respectively. the α and α domains of hla-a* : were also stable ( . ± . Å, . ± . Å), unlike the α domain, which exhibits a higher fluctuation ( . ± . Å) likely arising from the flexibility of the loop between domains α and α and the lack of stabilizing β -microglobulin. figure b compares the complex at , and ns during the simulation. these observations were consistent among the three independent simulations ( figure s ). the intermolecular interactions taking place in the complex structure were also examined by visually inspecting the trajectory and calculating the number of hydrogen bonds formed during the simulation (table s ) . for example, in simulation , the highest number of hydrogen bond interactions recorded were between hla-a* : and backbone atoms of the epitope peptide, with t -v and w -i interactions topping the list, as they were respectively formed during % and % of the simulation time. on the other hand, hydrogen bond interactions between the tcr and the epitope were almost exclusively from interactions between w (β chain)-i ( %) and q (α chain)-i ( %). the hla-a* : -tcr hydrogen-bonding interactions were mostly between residues from the cdr α, cdr α, cdr α, cdr β and cdr β loops of the tcr and the α and α helices of hla-a* : . visual analysis also revealed that hydrophobic interactions are integral to the interaction the epitope is making, both with hla-a* : and the tcr, as the hydrophobic epitope peptide was tightly enclosed by hydrophobic clusters from the proteins throughout the simulation, reflecting observations made on the basis of the original docked complex. thus, our structural analysis suggests that the s protein epitope fiagliaiv of sars-cov- (and sars-cov), and the other epitopes listed in table , could form strong complexes with hla-a* : and hla-a* : allotypes, and that the epitope-hla complexes can also be recognized by tcrs to initiate cytotoxic t-cell-mediated immune responses. the intermolecular interactions taking place in the complex structure were also examined by visually inspecting the trajectory and calculating the number of hydrogen bonds formed during the simulation (table s ) . for example, in simulation , the highest number of hydrogen bond interactions recorded were between hla-a* : and backbone atoms of the epitope peptide, with t -v and w -i interactions topping the list, as they were respectively formed during % and % of the simulation time. on the other hand, hydrogen bond interactions between the tcr and the epitope were almost exclusively from interactions between w (β chain)-i ( %) and q (α chain)-i ( %). the hla-a* : -tcr hydrogen-bonding interactions were mostly between residues from the cdr α, cdr α, cdr α, cdr β and cdr β loops of the tcr and the α similar to the docking results obtained from the experimentally known epitopes with the hla-a* supertype, we observed that residues at pos - and pos (a , e , w and l ) of the novel epitope aewflayil -derived from a transmembrane segment of the nsp protein of sars-cov- -are fully buried within the antigen-binding cleft of hla-b* : (pdb id: iex, chain a). moreover, the location of the partially solvent-exposed residues at pos -pos (l , a , y and i ) of the docked epitope along with solvent-exposed residues r , t , t , e , q , y , e and w of the α and α helices suggest an interaction of the aewflayil -hla-b* : complex with tcr ( figure f ). in order to tackle the current covid- pandemic, it is critically important to better understand the underlying mechanism that gives rise to the individual differences in disease severity as well as to aid the vaccine development against the causative virus, sars-cov- . effective vaccinations are needed to eradicate the virus from populations all over the world and knowledge regarding the immunological response should have a significant impact on understanding disease progression. however, due to the limited experimental and clinical data currently available on the specific immune responses against sars-cov- , the development of an effective vaccine against covid- will be a challenge. this study sought to better understand the individual differences in the viral antigen presentation pathway and to aid the development of vaccines against covid- by predicting in silico sars-cov- immunogenic epitopes. based solely on in silico predictions, the most potent sars-cov- -derived mhc class i binding epitopes are aewflayil and lvaewflay in terms of binding affinity, hydrophobicity and stability. however, for these "in silico epitopes", only limited experimental data are available to correlate with. therefore, in this study we mainly focused on potential sars-cov- epitopes that were conserved with sars-cov epitopes experimentally known to activate cytotoxic t-cells, and hence could be used in vaccine development. the s glycoprotein-derived epitope fiagliaiv binds to the hla-a* : and hla-a* : allotypes with experimental ic values lower than nm (table ). our docking analysis supports these predictions, i.e., that epitope fiagliaiv could bind tightly to these allotypes and that ternary complexes with tcrs could form. moreover, recent data demonstrate that patients with a severe form of covid- have a stronger t-cell response after stimulation with the sars-cov- s-protein peptide pool compared to those with a mild manifestation of the disease [ , ] . the disease progression of covid- is also associated with a higher magnitude of inflammatory cytokine-producing cd + t cells [ ] . whether these immune responses are due to strong binding of sars-cov- epitopes, including fiagliaiv , to certain hla allotypes, such as hla-a* : and hla-a* : , or whether tight virus epitope-hla interaction in general can actually be harmful for covid- patients by causing, e.g., an immunological over-reaction, is not yet fully understood [ , ] . furthermore, both cd + and cd + t-cells have been shown to be stimulated by overlapping peptides ( -mers overlapping by amino acids) of the entire s glycoprotein sequence [ ] . does this mean that the s protein might function as a double-edged sword-that is, being crucial for viral entry into the host cell, but also important for overstimulating the immune responses, causing severe inflammation that aids the spread of the virus to surrounding cells? this still needs to be answered. the latter "sword" is known to be avoided at least by hiv, which has a sophisticated mechanism to limit the infection rate in order to better avoid immune surveillance [ , ] . a recent in vivo study shows that epitopes derived from the c-terminus of the s protein had a significantly stronger cd + t helper cell response in healthy donors in comparison to those infected with sars-cov- [ ] . the cd + t cells' cross-reactivity to the s protein might represent the key for understanding the different disease manifestations of covid- , particularly in the asymptomatic infections in children and adolescents. our predicted epitope fiagliaiv overlaps with the c-terminal sequence of the s protein containing the s subunit, which is internalized after tmprss cleavage. therefore, this particular amino acid sequence may also be important for inducing a protective immunological response towards immunity in covid- . however, canonically t helper cells recognize mhc class ii molecules, whereas our prediction is based on the mhc class i molecules' ability to present viral antigens for a possible cytotoxic t-cell response [ ] . indeed, we found four -mer epitopes that include the intact -mer fiagliaiv epitope sequence and showed binding affinities < nm with drb * : allotype of mhc class ii (http://tools.iedb.org/mhcii/) [ ] , an allotype that is common in caucasoid and oriental ethnic backgrounds (https://www.ebi.ac.uk/ipd/imgt/hla/ethnicity.html) [ ] . nevertheless, there are a few reports regarding mhc class i-reactive cd + t helper cells, including the study where co-cultures of highly purified cd + t cells, together with a stimulatory mhc class ii-negative cell line transfected with mhc class i molecules, were used to show the direct interaction of t helper cells with mhc class i molecules [ ] . however, whether or not the fiagliaiv epitope is presented to both cytotoxic and helper t cells needs to be experimentally verified; a recent study that appeared while the current work was under review suggested that cross-reactivity could affect disease progression in covid- [ ] . furthermore, the latest experimental reports suggest that the s glycoprotein of sars-cov- is both o-and n-glycosylated, especially on the rdb domain, which could mask immunogenic epitopes and may play an important role in sars-cov- immune evasion [ ] [ ] [ ] . fortunately, the predicted epitope fiagliaiv is part of a transmembrane helix, and consequently is neither o-nor n-glycosylated; the closest glycosylation site is at pos , rendering this particular amino acid sequence potentially suitable for vaccine development. intriguingly, the sars-cov- -derived, membrane glycoprotein epitope hlriaghhl , which has low binding affinity (ic = . nm; table s ) towards hla-b* : , is % identical in sequence with the intravirion sars-cov epitope hlrmaghsl also from a membrane glycoprotein and shown to elicit a strong t-cell response in patients with the hla-b* : allotype [ ] . furthermore, hla-b* : has been shown to have a protective role against the severe forms of sars-cov [ ] . this inspired us to do a separate in silico binding affinity analysis of the sars-cov hlrmaghsl epitope to the hla-b* : allotype: a high binding affinity (ic = . nm) of the hlrmaghsl epitope with the hla-b* : was predicted and agrees with the reported [ ] protective immune response against sars-cov. thus, there seems to be a difference between the highly conserved sars-cov-derived hlrmaghsl and sars-cov- -derived hlriaghhl epitopes in their potency towards the hla-b* : allotype. moreover, the predicted low binding affinity of the sars-cov- epitopes with the hla-b* : allotype (table s ) might not be sufficient to induce immune responses, thus rendering the potential of this specific epitope unfavorable for vaccination. based on our predictions, hla-b* : is one of the worst allotypes for presenting sars-cov- -derived epitopes with an average binding score (ic ) of nm for the four epitopes predicted to bind (table s ). this is in line with similar results from the predictions for sars-cov- and previous clinical data from sars-cov patients, demonstrating that this particular hla allotype gives susceptibility to a more severe form of the viral disease [ ] . furthermore, our prediction shows that hla-b* : is not binding to either the s or m protein-derived peptides, strengthening the reported general view that hla-b* : is not optimal for eliciting an immune response in covid- patients [ ] . taken together, identification of the predicted most immunogenic epitopes of sars-cov- could aid vaccine development. since the sequences of the "top" epitopes of sars-cov- and sars-cov are highly conserved and the sars epitopes are known to elicit an immunological response based on a previous study [ ] , a common vaccine protecting against both viruses and potential future strains is possible. moreover, the selected experimentally known epitopes have been shown not to evoke an unwanted t cell cross-reactivity in vitro, further validating the potential use of the conserved sars-cov/cov- peptides in vaccine development without disrupting self-tolerance [ ] . however, there are many hurdles that need to be addressed; for example, due to the hydrophobic nature of these epitopes, they probably would need to be loaded in a liposome or nanocarrier for efficient vaccine delivery [ ] . moreover, as these conserved epitopes are presented by only a few hla allotypes, i.e., a* : , a* : , a* : , which are common in american indian, caucasoid, hispanic and oriental ethnic backgrounds [ ] , the estimated world population coverage using these epitopes would only be around . % (http://tools.iedb.org/population/) [ ] . therefore, developing a globally effective sars-cov- vaccine will probably require a pool of both novel and conserved epitopes, making a globally effective sars-cov- vaccine development a challenging task. in the present study, we identified sars-cov- epitopes that were predicted to be presented by the mhc class i antigen processing pathway to the cytotoxic t cells. we report five purely in-silico-predicted most potent epitopes unique to sars-cov- and five potent sars-cov- epitope peptides identical to and experimentally determined for sars-cov. the novel sars-cov- epitopes were analyzed for their interaction with hla allotypes using the iedb and netctl . web servers and three-dimensional structural models of selected, molecular dynamics simulation proven ternary complexes of sars-cov- phla-tcrs were created to assess interactions at structural level. hla-a* : and hla-a* : were found to have the greatest potential to present the selected epitopes, which are hydrophobic in nature and originated mainly from the transmembrane region of sars-cov- proteins. our results could assist in the understanding of the individual and varying disease progression of covid- , as well as paving the way towards vaccine development against sars-cov- . supplementary materials: the following material are available online at http://www.mdpi.com/ - x/ / / / s , figure s : (a): distribution of the predicted half-lives (log scale) of predicted (iedb) -mer epitope-hla-a* supertype complexes. the complexes are classified as immunogenic (ic ≤ nm) and non-immunogenic (ic > nm) based on the epitope binding affinity with the mhc molecule, (b): distribution of the gravy scores of immunogenic (ic ≤ nm) and non-immunogenic (ic > nm) epitopes, figure s c and s d: comparison of the backbone conformation of the five epitopes docked into the cleft of hla-a* : (c) and hla-a* : (d) molecules against the canonical epitope gilgfvftl of the influenza a virus (pdb id: tez, chain c), figure s : structural dynamics of the hla-a* : - fiagliaiv s protein epitope-tcr complex during three replicate ns simulations. (a) cα atom rmsf of the ternary complex. (b) average cα atom rmsf. table s a : mhc class i allotypes association with supertypes described in published scientific literature [ , ] . superscript "x" indicates availability of x-ray crystal structure in protein data bank (pdb) [ ] , table s b : pdb codes for the x-ray crystal structures of mhc class i allotypes, table s : sars-cov- -derived mhc class i binding epitopes identified with iedb and netctl . prediction methods as having strong binding affinity (ic ≤ nm) with mhc molecules, table s : sars-cov- -derived mhc class i binding epitopes identified with iedb and netctl . prediction methods as having weak binding affinity ( nm < ic ≤ nm) with mhc molecules, table s : sars-cov- -derived mhc class i binding epitopes identified with iedb and netctl . prediction methods as having non-binding affinity (ic > nm) with mhc molecules, table s : sars-cov- -derived epitopes (immunogenicity score ≥ . ) and their prominent interacting hla allotypes identified with iedb and netctl . prediction methods, table s : most potent sars-cov- -derived epitopes having immunogenicity score ≥ . and predicted binding affinity (ic ) ≤ nm with their prominent interacting hla allotypes identified with iedb prediction method, table s : most potent sars-cov- -derived mhc class i binding epitopes identified with netctl . (combined score ≥ ) prediction method, table s : most potent sars-cov- -derived mhc class i binding epitopes that are identical to the experimentally known epitopes (mhc ligand assays data from the iedb database) of sars-cov strains to activate cytotoxic t-cells. predicted half-lives of the epitope-mhc-i complexes, gravy scores and mutations in the epitopes are shown. table s world health organization declares global emergency: a review of the novel coronavirus (covid- an interactive web-based dashboard to track covid- in real time preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies data sharing and outbreaks: best practice exemplified potential inhibitors against -ncov coronavirus m protease from clinically approved medicines a review of coronavirus disease- (covid- ) acute respiratory distress syndrome advances in diagnosis and treatment an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov- epidemic: an observational cohort study middle east respiratory syndrome coronavirus (mers-cov) in oman: current situation and going forward emerging coronaviruses: genome structure, replication, and pathogenesis structural genomics of sars-cov- indicates evolutionary conserved functional regions of viral proteins universal three-dimensional construction of eleven amino acids near the catalytic nucleophile and base in the superfamily of (chymo)trypsin-like serine fold proteases sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor a pneumonia outbreak associated with a new coronavirus of probable bat origin structural basis of receptor recognition by sars-cov- functional assessment of cell entry and receptor usage for sars-cov- and other lineage b betacoronaviruses human leukocyte antigen (hla) and immune regulation: how do classical and non-classical hla alleles modulate immune response to human immunodeficiency virus and hepatitis c virus infections? front association of hla class i with severe acute respiratory syndrome coronavirus infection memory t cell responses targeting the sars coronavirus persist up to years post-infection hla-a* t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus nucleocapsid and spike proteins efficient induction of cytotoxic t lymphocytes specific for severe acute respiratory syndrome (sars)-associated coronavirus by immunization with surface-linked liposomal peptides derived from a non-structural polyprotein a presence of sars-cov- -reactive t cells in covid- patients and healthy donors pathways of antigen processing ncbi viral genomes resource the immune epitope database (iedb): update large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction the immune epitope database and analysis resource in epitope discovery and synthetic vaccine design definition of supertypes for hla molecules using clustering of specificity matrices hla class i supertypes: a revised and updated classification a simple method for displaying the hydropathic character of a protein predicting transmembrane protein topology with a hidden markov model: application to complete genomes jpred : a protein secondary structure prediction server pan-specific prediction of peptide-mhc class i complex stability, a correlate of t cell immunogenicity structural basis for clonal diversity of the human t-cell response to a dominant influenza virus epitope structural insights into the binding of hepatitis b virus core peptide to hla-a alleles: towards designing better vaccines the rcsb protein data bank: new resources for research and education rosetta flexpepdock web server-high resolution modeling of peptide-protein interactions deciphering the structural effects of activating egfr somatic mutations with molecular dynamics simulation improving the accuracy of protein side chain and backbone parameters from ff sb comparison of simple potential functions for simulating liquid water visual molecular dynamics software for processing and analysis of molecular dynamics trajectory data ucsf chimera-a visualization system for exploratory research and analysis friends or foes? retrovirology systematically benchmarking peptide-mhc binding predictors: from synthetic to naturally processed epitopes ncbi blast: a better web interface peptide-mhc class i stability is a better predictor than peptide affinity of ctl immunogenicity primary human immunodeficiency virus type -specific cd + t-cell responses induced by myeloid dendritic cells identification of antigens presented by mhc for vaccines against tuberculosis tcr contact residue hydrophobicity is a hallmark of immunogenic cd + t cell epitopes a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov- targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals phenotype of sars-cov- -specific t-cells in covid- patients with acute respiratory distress syndrome key words natural killer cells in hiv- infection: a double-edged sword structural basis of transmembrane coupling of the hiv- envelope glycoprotein human leukocyte antigen susceptibility map for sars-cov- major histocompatibility complex class i-restricted alloreactive cd + t cells deducing the n-and o-glycosylation profile of the spike protein of novel coronavirus sars-cov- site-specific analysis of the sars-cov- glycan shield site-specific n-glycosylation characterization of recombinant immunogenetics in sars: a casecontrol study hla studies in the context of coronavirus outbreaks a epitopes described in-immune epitope database (iedb) quantitating t cell cross-reactivity for unrelated peptide antigens harnessing self-assembled peptide nanoparticles in epitope vaccine design conflicts of interest: e.n. has filed a patent application regarding a method for preventing the spreading and lowering the infection rate of pathogens, finnish patent application number , filing date . . . all other authors declare that they have no competing interests. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.vaccines , , key: cord- -ph eji authors: mostajo, nelly f; lataretu, marie; krautwurst, sebastian; mock, florian; desirò, daniel; lamkiewicz, kevin; collatz, maximilian; schoen, andreas; weber, friedemann; marz, manja; hölzer, martin title: a comprehensive annotation and differential expression analysis of short and long non-coding rnas in bat genomes date: - - journal: nar genom bioinform doi: . /nargab/lqz sha: doc_id: cord_uid: ph eji although bats are increasingly becoming the focus of scientific studies due to their unique properties, these exceptional animals are still among the least studied mammals. assembly quality and completeness of bat genomes vary a lot and especially non-coding rna (ncrna) annotations are incomplete or simply missing. accordingly, standard bioinformatics pipelines for gene expression analysis often ignore ncrnas such as micrornas or long antisense rnas. the main cause of this problem is the use of incomplete genome annotations. we present a complete screening for ncrnas within bat genomes. ncrnas affect a remarkable variety of vital biological functions, including gene expression regulation, rna processing, rna interference and, as recently described, regulatory processes in viral infections. within all investigated bat assemblies, we annotated ncrna families including snornas and mirnas as well as rrnas, trnas, several snrnas and lncrnas, and other structural ncrna elements. we validated our ncrna candidates by six rna-seq data sets and show significant expression patterns that have never been described before in a bat species on such a large scale. our annotations will be usable as a resource (rna.uni-jena.de/supplements/bats) for deeper studying of bat evolution, ncrnas repertoire, gene expression and regulation, ecology and important host–virus interactions. bats (chiroptera) are the most abundant, ecologically diverse and globally distributed animals within all vertebrates ( ), but representative genome arrangements and corresponding coding and non-coding gene annotations are still incomplete ( ) . except for the extreme polar regions, bats can be found throughout the globe, feeding on diverse sources such as insects, blood, nectar, fruits and pollen ( ) . their origin has been dated in the cretaceous period, with a diversification explosion process dating back to the eocene ( ). the bat families known to date are classified into the suborders yinpterochiroptera and yangochiroptera ( , ) ( figure ). although they account for > % of the total living mammalian diversity ( ) , the genomes of only bat species of the estimated > species ( ) have been sequenced with adequate coverage to date and are publicly available ( figure ) . bats have developed a variety of unique biological features that are the rarest among all mammalian, including laryngeal echolocation ( , ) , vocal learning ( ) and the ability to fly ( ) . they occupy a broad range of different ecological niches ( ) , have an exceptional longevity ( - ) and a natural and unique resilience against various pathogenic viruses ( , ) . for example, bats are the suspected reservoirs for some of the deadliest viral diseases such as ebola and sars ( ) ( ) ( ) , but appear to be asymptomatic and survive the infection. possibly, the solution to better understand and fight these pathogens lies in the uniquely developed immune system of bats ( , ) . studying bats and their genomes is likely to have high impacts on various sci- figure . we used available genomes of bat species from eight out of families for non-coding rna annotation in this study. the tree shows their phylogenetic relationship and is based on a molecular consensus on family relationships of bats ( ), further adapted and extended from ( ) . bat families and species with published genomes currently available in the ncbi are shown (details see table ). bat families still lacking a published genome assembly are written in gray color. rna-seq data sets were selected from species marked with an asterisk and additionally obtained from a myotis daubentonii cell line (see table ). bat silhouettes were adapted from artworks created by fiona reid. entific fields, including healthy ageing, immune and ecosystem functioning, the evolution of sensory perception and human communication, and mammalian genome architecture (see the recent bat k review for further details ( ) ). despite the unique biological characteristics of these flying mammals and their important role as natural reservoirs for viruses, bats are one of the least studied taxa of all mammalian ( ) . accordingly, there is little knowledge about the non-protein-coding transcriptome of bats, which plays a crucial role in an extensive number of cellular and regular functions and comprises a very diverse family of untranslated rna molecules ( , ) . in addition, it is believed that due to the early evolutionary radiation of bats (compared to other mammals) their innate and acquired immune responses have a different set of molecules ( ) . genome assemblies and annotations are essential starting points for many molecular-driven and comparative studies ( ) . especially, studies of non-model organisms play important roles in many investigations ( ) . in most cases, however, these organisms lack well-annotated genomes ( ) , which severely limit our ability to gain a deeper understanding of these species and may further impede biomedical research ( ) . in this study, we comprehensively annotated non-coding rnas in available bat genome assemblies (table ) . for each bat species, we provide final annotations that are compatible with current ensembl and ncbi (national center for biotechnology information) standards (gtf format) and that can be directly used in other studies, for example for differential gene expression analysis. we compare our new annotations with the currently available annotations for bats and show that a large number of non-coding genes are simply not annotated and are therefore overlooked by other studies. we used six rna-seq data sets comprising different conditions (table ) to validate our annotations and to determine the expression levels of our newly annotated ncrnas. exemplarily, we show that our novel annotations can be used to identify ncrnas that are significantly differential expressed during viral infections and were missed by previous studies. we downloaded the last recent genome versions for bat species in september from the ncbi genome database (table ) . within the order of yinpterochiroptera, nine genomic sequences were obtained covering the bat families pteropodidae, hipposideridae, rhinolophidae and megadermatidae whereas for the order of yangochiroptera another seven genome assemblies were available for the bat families phyllostomidae, mormoopidae, vespertilionidae and miniopteridae ( figure ). we introduced a unique three-letter abbreviation code (table ) to easily distinguish between the bat species in the manuscript and intermediate annotation files provided in the electronic supplement. we used quast (v . . ) ( ) to calculate several assembly statistics for all genomes, shown in supplementary table s . at the end of , two new bat genomes were presented by the bat k project (http://bat k.ucd.ie) ( ), comprising a newer version of the greater horseshoe bat genome (rhinolophus ferrumequinum; rhinolophidae) and the genome of the pale spear-nose bat (phyllostomus discolor; phyllostomidae). however, these two bat genomes were not included in our present study due to the data use policy of the bat k consortium and to support a fair and productive use of these data. to validate our novel ncrna predictions, we selected six rna-seq data sets ( , ( ) ( ) ( ) ( ) comprising all together samples gathered from four different bat species. we have labeled each published rna-seq data set based on the first authors last name and the year of data set publication ( table and supplementary table s ). all samples were quality trimmed using trimmomatic ( ) (v . ) with a nt-step sliding-window approach (q ) and a minimum remaining read length of nt. for the field- data set ( ) , we additionally removed the three leading ' nucleotides from the reads of each sample because of a generally low quality observed by fastqc (www.bioinformatics.babraham.ac.uk/projects/ table . we have annotated ncrnas within bat genomes of different assembly quality. we introduced three-letter abbreviations for each bat species used throughout the manuscript and in supplemental files and annotations. genome sizes were estimated (est.) by using c-values (dna content per pg) from the animal genome size database (http://genomesize.com) and by applying the following formula: genome size = ( . · ) · c. if multiple entries for one species were available, an average over all c-values was calculated and used to estimate the genome size. if one species could not be found, an average c-value for the corresponding genus was used. supplementary table s provides additional assembly statistics calculated by quast (v . . ) ( ) . ncbi acc. -genbank assembly accession without the prefix gca fastqc/) (v . . ). the remaining reads of all processed samples were individually mapped to all bat assemblies using hisat ( ) (v . . ) and transcript abundances were subsequently calculated from all resulting mappings by featurecounts ( ) (v . . ). if suitable, the appropriate strand-specific counting mode was applied for each data set (see table for information about the strand specificity). for each bat genome assembly, the merged annotation of already known (ncbi) and newly identified (this study) ncr-nas was used (supplementary files s ). due to the size of the annotations and the huge amount of overlaps, long ncr-nas were counted and analyzed for differential expression separately. to enable a better investigation of small rnas (sr-nas), we included a data set of the targeted sequencing of srnas (especially mirnas) from m. daubentonii cells (weber- ). to obtain this srna sequencing data, total rna of samples, which was obtained using the same procedure like explained for the rrna-depleted m. daubentonii data ( ) , was preprocessed using the illumina truseq smallrna protocol, sequenced on one hiseq lane, and finally uploaded in the course of this study under geo accession gse . the reads of these srna samples were additionally preprocessed by removing potential adapter sequences with cutadapt ( ) (v . . ) followed by a quality (q ) trimming using again a window-size of and a minimum length of nt by prinseq ( ) (v . . ). the processed srna samples were either individually mapped to the bat genomes for differential expression analysis or combined and mapped on each bat genome to predict known and novel mirnas with mirdeep ( ). only uniquely mapped reads were counted and used for the differential gene expression analyses with deseq ( ) (v . . ). annotated rrna genes were removed prior de-seq and tpm (transcripts per million) analysis. all raw read counts from samples of one data set were combined and normalized together using the built-in functionality of deseq , followed by pairwise comparisons to detect significant (adjusted p-value < . ; absolute log fold change > ) differential expressed ncrnas. besides the deseq normalization, we calculated tpm values for each ncrna in each sample as previously described ( ) : where c i is the raw read count of ncrna i, l i is the length of ncrna i (and the cumulative exon length in the case of lncrnas) and n is the number of all ncrnas in the given annotation. to this end, we obtained for each rna-seq sample, each bat annotation, and each ncrna one tpm value representing the normalized expression level of this ncrna. if available, we calculated all tpm values in relation to the expression of all already known coding and non-coding genes and not only based on our novel ncrna annotation. although we performed mappings, read countings, and normalization for all samples, bat genome assemblies and all six data sets ( table ; overall mappings), we only selected one comparison per data set to exemplarily show novel and significantly differential expressed ncrnas (supplementary files s . -s . ; divided by data set and input annotation). for each data set, we chose the bat species that was also used in the corresponding study. for the hölzer- and weber- data sets, we used the closely related m. lucifugus genome assembly and annotation as a refer- table . six rna-seq data sets comprising all together samples derived from four different bat species were used to evaluate our novel ncrna annotations. all samples were quality trimmed and individually mapped to all bat assemblies using hisat ( ) and transcript abundances were subsequently calculated from all mappings by featurecounts ( ) . we labeled each rna-seq data set based on the first authors last name and the year of data set publication. raw read data of the enriched sequencing of small rnas (especially mirnas) of a m. daubentonii cell line (accompanying gse ( ) ) have been uploaded in the course of this publication under geo accession gse (weber- ). polya+ -library preparation with mrna selection; rrna--library preparation with rrna depletion and size selection (> nt); srna -library preparation with size selection (< nt); se/pe -single-/paired-end sequencing; ss/not-ss -strand-specific/unstranded sequencing; ss s /ss a -strand-specific in sense orientation/in antisense orientation figure . please note that for m. daubentonii currently no genome assembly is available, so the genome assembly of m. lucifugus was used as a close relative. all of our annotations follow the general transfer format (gtf) as described and defined in the ensembl ( ) database (https://ensembl.org/info/website/upload/gff. html). therefore, each row of each annotation file is either defined as a gene, transcript, or exon (by the feature column) and strictly following a hierarchical structure, even if only one exon (as for most ncrnas) is reported. by adhering to this annotation format, our novel annotations can be easily merged with existing ones as derived from ensembl or ncbi and are directly usable as input for computational tools such as hisat for mapping or featurecounts for transcript abundance estimation. we defined gene, transcript, and exon ids following the ensembl pattern: <species><feature><ncrna>< -digit-number>. in general, we used specialized computational tools for the annotation of specific ncrna classes (supplementary tables s -s ). if not otherwise stated, the main ncrna-discovery is based on homology searches against the rfam database ( - ) (v . ). we used the gorap pipeline (https://github.com/koriege/gorap), a specially developed software suite for genome-wide ncrna screening. gorap screens genomic sequences for all ncrnas present in the rfam database using a generalized strategy by applying multiple filters and specialized software tools. to this end, gorap takes huge advantage of infernal ( , ) (v . . ) to annotate ncrnas based on input alignment files conserved in sequence and secondary structure (so-called stockholm alignment files; stk). all resulting alignment files were automatically pre-filtered by gorap based on different ncrna class-specific parameters including taxonomy, secondary structure and primary sequence comparisons. due to repeats, pseudogenes, undiscriminable un-/functional genes and overlapping results from the different assembly methods, we defined a ncrna set per species for annotation that includes filtered sequences, but allows for variants and multiple copies. this final annotation set is defined by hand-curating the resulting stk alignments of gorap with the help of emacs ralee mode ( ) . due to the removal of sequences in the stockholm alignments, the remaining sequences were extracted and again aligned into stockholm format using cmalign --noprob from infernal. the rfam-derived ncrna alignments were further split into snornas (supplementary table s ), mirnas (supplementary table s ) and other ncrnas including snrnas, lncrnas and other structural rnas (supplementary table s ). in general, our annotation results give an overview about the amount of different ncrnas in bat species and intentionally can include false positive hits and duplicates. all ncrna hits are placed as stk (if available), gtf and fasta-files in the electronic supplement and osf (doi. org/ . /osf.io/ cmdn). rrnas. we used the prediction tool rnammer ( ) (v . ) to identify . s, s and s rrna genes using hidden markov models. the tools output was transformed into regular gtf file format. all output files can be found in supplementary table s . trnas. for the annotation of trnas, we applied trnascan-se ( ) (v . . ) to the bat contigs using default parameters. the results were filtered by removing any trnas of type 'undet' or 'pseudo' and the tabular output was transformed into the gtf file format. additional information about the anticodon and the coverage score were added to the description column. we provide the raw trnascan-se files in supplementary table s . snornas. we annotated snornas based on available alignments from the rfam database using gorap and additionally marked and classified them into box c/d and box h/aca when intersecting with the set of snornas from http://www.bioinf.uni-leipzig.de/publications/supplements/ - ( ) (supplementary table s ). mirnas. additionally to the rfam-screening (supplementary table s ), mirnas were predicted by the mird-eep pipeline ( ) (v . . . ) using default parameters and the combined smallrna-seq data set (weber- ; samples) mapped to each individual bat assembly as an input for mirdeep (supplementary table s ). to validate the accuracy of our approach, we compared our mirdeep annotations of m. lucifugus/p. alecto (based on the transcriptomic data derived from m. daubentonii; weber- ) with annotations of mirnas for transcriptomic data of myotis myotis ( ) and p. alecto ( ) . for reference mapping, huang et al. also used the m. lucifugus genome, so we were theoretically able to directly compare our annotations with the annotations of both studies. unfortunately, no positional information (annotation file) of the identified mirnas derived from the transcriptomic data of m. myotis were given in the manuscript or supplement ( ) . therefore, we blasted the precursor mirna sequences identified with the help of the m. myotis transcriptome against the m. lucifugus genome and retained only hits with a sequence identity of %. the so obtained positional information was further used to calculate the overlap between our predicted mirnas in m. lucifugus. we used the same approach for the p. alecto comparison. if the annotated location of an mirna and one of our identified mirna locations in m. lucifugus/p.alecto were overlapping by at least %, we counted this location as a common prediction. lncrnas. long ncrnas were annotated using a high confidence data set h from the lncipedia ( ) (v . ) database comprising transcripts of potential human lncrnas. the transcripts were used as input for a blastn ( . . +, e − ) search against each of the bat assemblies (compiled as blast databases). the blastn result for each bat assembly was further processed to group single hits into potential transcripts as follows: first, for each query sequence q ∈ h, hits of q found on the same contig c and strand s were selected (hits c, s, q ) and the longest one, q , was chosen as a starting point so that trscp c, s, q = (q ). second, all hits q i ∈ hits c, s, q with q i ∈trscp c, s, q , which overlap neither in the query q nor in the target sequence and do not exceed a maximum range of nt from the most upstream to the most down-stream target sequence position of all q j ∈ trscp c, s, q ∪q i , were added iteratively to trscp c, s, q . to this end, we introduced a simple model of exon-intron structures, naturally occurring when using transcript sequences as queries against a target genome assembly. we defined the nt search range based on an estimation of lncrna gene sizes derived from the human ensembl ( ) annotation. if the sum of the lengths of all q i ∈ trscp c, s, q covers the query transcript length length q at least for %, trscp c, s, q was considered as a transcript and its elements q i as exons, otherwise all q i ∈ trscp c, s, q were withdrawn. this procedure is repeated until all hits ∈ hits c, s, q were used or withdrawn. therefore, each so-defined group of non-overlapping hits derived from the same query sequence and found on the same contig and strand should represent a lncrna transcript with its (rough) exon structure. the defined transcripts were saved as blast-like output and transformed into gtf file format. to follow the gtf annotation format and to harmonize our lncrna annotations with the other ncrna annotations, each lncrna transcript was also saved as a gene annotation and consists of at least one exon. as we observed a lot of different sequences from lncipedia aligning to the same positions in the genomes, we decided to condense exons at the same sequence positions, considering transcripts with one or multiple exons separately. for each contig and strand, starting from the ' end, exons with a minimum overlap of nt were grouped together. in the case of multiple exons, groups of exons were merged, if they shared any transcript origin. if all exons in the group originated from the same lncipedia gene, the group was considered as one gene with several transcripts and its associated exon(s). otherwise, we defined a lncrna hot spot on gene level with several transcripts and their associated exon(s). the lncipedia names of the gathered transcripts of a lncrna hot spot, as well as start and end positions of all exons, are listed in the gtf gene attribute field (supplementary table s ). the scripts used for the identification of lncrnas can be found at https://github.com/rnajena/bats ncrna. table ) . for the other six species we used blastn ( . . +, e − ) with the mlu and pva mitochondrial genomes as queries against the remaining bat genomes. for ehe, we found a possible mtdna contig in full-length ( nt; awhc ) in the genome assembly. due to the circularization of mtdna, we rearranged the sequence of this contig to start with the gene coding for the phenylalanin trna and to match the gene order of the other mitochondrial genomes. only for esp, rsi, mly, efu and mna, we were not able to detect any possible contigs of mtdna (table ). all mitochondrial genomes were annotated with mitos ( ). the ncrna results were filtered by e-value (threshold . ), thus one of two small rrnas in mbr and rfe and one of two large rrnas in ehe were discarded as false positive hits (supplementary table s ). for the five bat assemblies directly including mtdnas (table ) , the mitos annotations were added to the final merged ncrna annotation. all other mtdnas and annotations can be found in supplementary table s . as we annotated all bat assemblies by using different tools, we needed to merge the resulting gtf files to resolve overlapping annotations and to receive a final annotation of ncrnas for each bat species. furthermore, we extended the available ncbi annotations (including protein-and noncoding genes) by integrating our novel ncrna annotations. the scripts used to merge the different annotation files and to calculate overlaps between annotations can be found at https://github.com/rnajena/bats ncrna. due to their size, we have not included the lncrna annotations based on lncipedia. these can be downloaded and used separately (supplementary table s ). of novel non-coding annotations. for each bat species, we merged the ncrna annotations (except for lncrnas) using a custom script (merge gtf global ids.py). after reading in all features and asserting correct file structure, overlaps were resolved in the following manner: (i) exons are considered overlapping if > % of the shorter one is covered by table . mitochondrial bat genomes (mtdna) publicly available and used for annotation with mitos ( ). for out of the bat species investigated in this study, mtdna could be found in the ncbi. for four bat species, the mtdna is also part of the genome assembly as determined using blastn. for e. helvum, no mtdna could be found in the ncbi, but we were able to identify a single contig that is part of the genome assembly as mtdna using blastn and the mitochondrial genomes of the other bats as query. the contig was rearranged to match the gene order of the other mtdnas. r -found via blastn and rearranged merge of ncbi and novel annotations. we first converted and filtered the ncbi annotations to a compatible format with a custom script (format ncbi.py) and then combined the results with our merged novel ncrna annotations using the same strategy to resolve overlaps as above, but imposing less strict format rules (merge gtf ncbi. py). at best, a genome assembly represents the full genetic content of a species at chromosome level. whereas the first complex eukaryotic genomes were generated using sanger chemistry, today's technologies such as illumina short-read sequencing and pacbio or oxford nanopore long-read approaches are increasingly used ( ) . the currently available bat genomes vary widely regarding their assembly quality and completeness (table and figure ; supplementary table s ) and were predominantly assembled by using short illumina-derived reads and low (∼ x) ( ) up to moderate/higher coverage ( - x) approaches ( ) ( ) ( ) ( ) ( ) ) . a new assembly of the cave nectar bat (eonycteris spelaea) ( ) was exclusively generated from long-read data derived from the pacbio platform, and the genome of the egyptian fruit bat (rousettus aegyptiacus) ( ) was assembled by using a hybrid-approach of illumina and pacbio table and supplementary table s ) supplementary table s . data. these two genomes from the pteropodidae family are of a generally higher quality (figure and supplementary table s ). regardless of their assembly quality, these genomes need to be annotated to identify regions of interest, for example, encoding for protein-and non-coding genes or other regulatory elements. current genome annotations, mostly generated by automatic annotation pipelines provided by databases such as the ncbi ( ) or ensembl ( ), are predominantly focusing on protein-coding genes and well-studied ncrnas such as trnas and rrnas. accordingly, the available bat genome annotations vary a lot regarding their quality, ranging from more comprehensive annotations for longstanding bat genomes such as m. lucifugus or p. vampyrus to annotations on region level, completely missing any coding or non-coding gene annotations at the current ncbi version ( figure and table ). furthermore, by using strandspecific rna-seq data, we could show that some genes (e.g. ifna /ifnw in the ensembl annotation of m. lucifugus ( ) ) are annotated on the wrong strand and are therefore entirely missed by differential expression studies when relying on a strand-specific read quantification. for all publicly available bat genomes, ncrnas are generally annotated on low levels and are highly incomplete, mostly only comprising some trnas, rrnas, snrnas, snornas and lncrnas ( figure and table ). therefore, many ncrnas, especially mirnas, are simply overlooked by current molecular studies, for example from rna-seq studies that aim to call differential expressed genes based on such in-complete genome annotation files. studies that have made additional effort on annotating ncrnas in bats ( , , , ( ) ( ) are not reporting their results on a level that can be directly used for further computational assessment (e.g. as a direct input for rna-seq abundance estimation). currently, in the ncbi database, five bat assemblies are entirely lacking any coding/non-coding annotations and mirnas are not annotated at all ( table ). the rfam database ( ) contains mainly for m. lucifugus and p. vampyrus ncrna families. other ncrnas are currently unknown from bat genomes or not well documented. the genome assembly status of different bat species varies widely: ranging for example from contigs and an n of nt (d. rotundus) to contigs and an n of only nt (m. lyra), see table and supplementary table s . accordingly, the annotation status also varies a lot (table ) . within this work, we give an overview of potential ncrna annotations in bats. however, the precise number of ncrnas remains unclear, because of ncrnas being present several times in the assemblies, and others still remaining undiscovered. to give a better estimation of transcribed and potentially functional ncrnas, we used six illumina short-read rna-seq data sets derived from four bat species (table ) to estimate the expression levels of our novel annotations. note that we refer throughout this paper to an rna-seq data set by the first author's name and the year of the respective data set publication. the only included data set derived from a species of the yinpterochiroptera suborder (r. aegyptiacus) was obtained from a study dealing with the differential transcriptional responses of ebola and marburg virus infections in human and bat cells ( ) (data set: hölzer- ) . in this study, total rna of nine samples of r e-j cells, either challenged by the ebola or marburg virus or left un-infected, were harvested at , or h post infection (poi) and sequenced. unfortunately, no biological replicates could be generated for this study. therefore, we did not use this data set for the differential ex-pression analysis, but also calculated normalized expression values (tpm; transcripts per million) as done for all rna-seq data sets. the other five data sets comprise yangochiroptera species of the vespertilionidae (m. lucifugus, m. daubentonii) and miniopteridae (m. natalensis) families (table ). field et al. conducted two transcriptomic studies ( , ) (field- , field- using wing tissue of the hibernating little brown myotis bat. they were especially interested in transcriptional changes between un-infected wing tissue and adjacent tissue infected with pseudogymnoascus destructans, the fungal pathogen that causes the white-nose syndrome. two other data sets were obtained from virus-(rvfv clone ) and interferon (ifn) alphainduced transcriptomes of a myotis daubentonii kidney cell line (mydauni/ c). rna of mock, ifn and clone samples were gathered at two time points, and h poi ( ) . from the same samples, rrna-depleted (hölzer- ) and smallrna-concentrated (weber- ; see methods section for details) libraries were generated and sequenced. finally, we used data of the long-fingered bat m. natalensis initially obtained to characterize the developing bat wing ( ) . here, total rna was extracted from paired forelimbs and hindlimbs from three individuals at three developmental stage. we have mapped each sample to each bat genome, regardless of the origin of the rna. expectedly, the mapping rate decreases in bat species that are evolutionary more far away from the original species from which the rna was sequenced. however, we were interested to find out which ncrnas are consistently transcribed in all investigated bat species or only in certain bat families and sub-groups. over all bat assemblies, we annotated ncrna families for in total trnas, rrnas, mirnas ( predicted by mirdeep ), snornas, mitochondrial (mt-)trnas and mt-rrnas as well as other ncrnas additionally derived from the rfam database ( ) (selected ncrnas are shown in table ). with a broad approach, we have identified potential lncrnas and defined between (m. lyra) and (d. rotundus) lncrna hot spots. all annotations, separated for each ncrna class and summarized for each bat species, can be found in the open science framework (doi.org/ . /osf.io/ cmdn) and in our electronic supplement (rna.uni-jena. de/supplements/bats) and are compatible with the genome assembly versions listed in table . thus, our annotations together with the bat genome assemblies obtained from the ncbi can be directly used for subsequent analysis such as differential gene expression detection. we detected s and s rrna for the majority of investigated bat species (supplementary table s ). the varying number of rrnas is in line with all currently available metazoan genomes, lacking the correct composition of rrnas due to misassemblies. however, the number of . s rrna varies a lot between for p. vampyrus and for m. natalensis (table and supplementary table s ). interestingly, of pteropodidae show a higher number of . s rrnas compared to the other species (∼ - fold). only the p. alecto assembly is in line with the other bat assemblies in regard to the amount of . s rrna copies. for all bat species, we observed various numbers of trnas (table and supplementary table s ). we could detect full sets of trnas for e. fuscus and m. davidii, whereas between one and four trnas are missing from the assemblies of h. armiger, m. brandtii, m. lucifugus, m. lyra, m. natalensis, p. parnellii, r. ferrumequinum, r. sinicus and d. rotundus, and between nine and twelve are missing for e. helvum, p. alecto, p. vampyrus, r. aegyptiacus and e. spelaea (supplementary table s ). interestingly, we identified a large number of trnas ( ) for m. lyra in comparison to all other bat species (supplementary table s ), likely a result of the low assembly quality (figure and supplementary table s ). the lowest amount of trnas was annotated for p. parnellii and d. rotundus with only and copies, respectively. in all other bat genomes, we found between and trna genes (supplementary table s ). the trna encoding for valine (val) with the anticodon structure tac had high copy numbers (over ) in all genome assemblies of the pteropodidae family (table ) . similar copy numbers were achieved by the r. ferrumequinum and r. sinicus assemblies regarding the trna encoding for isoleucine (ile) with the anticodon aat (supplementary table s ). for trna(ile) and the anticodon gat, we also observed high copy numbers in h. armiger. interestingly, all species with high trna(val) and trna(ile) copy numbers had rather low counts (between and ) of trna(sec) with the anticodon tca, while this trna was found with higher copy numbers in p. parnelli ( ) and in d. rotundus ( ) and with even higher counts (between and ) in all other bat species (supplementary table s ). however, high copy numbers might be also occur due to assembly quality and false positive predictions of trnascan-se. in supplementary table s we list all detected snornas, divided into box c/d and box h/aca types. overall, we found snorna families within the investigated bat species, comprising box c/d, box h/aca and unclassified snornas. many snornas were found with exactly one copy present in each bat genome assembly (e.g. scarna ), whereas others were found in multiple copies for each bat species (e.g. snord ) or completely absent from certain bat families (e.g. aca ), see table . exactly one copy of the small nucleolar rna aca was found within the genomes of the pteropodidae family and multiple copies for d. rotundus, p. parnellii, m. natalensis and members of the vespertilionidae family; however, this snorna seems to be completely absent from bat species of the megadermatidae and rhinolophidae families ( table ). the h/aca box snorna aca is predicted to guide the pseudouridylation of s rrna u ( ) . interestingly and as another example, snora was found in higher copy numbers in the genomes of the vespertilionidae family. among others, this h/aca box snorna was described to be commonly altered in human disease ( ) . over all bat assemblies, we detected mirna families based on rfam alignments (supplementary table s ) and predicted between (e. helvum) and (m. davidii) mirnas based on the combined small rna-seq data sets (weber- ) using mirdeep ( ) (supplementary table s ). the higher number of mirnas predicted for myotis species can be explained because the small rna-seq data set is derived from a myotis daubentonii cell line. similar to other ncrna classes, we observe various differences in mirna copy numbers between the bat families. for example, mir- and mir- are absent in all vespertilionidae and m. natalensis (table ) , whereas mir- is present in all yangochiroptera (except d. rotundus) but absent from all pteropodidae (supplementary table s ). the mirna is absent from all yangochiroptera except d. rotundus. there are many other examples of absent/present mirnas in certain bat species/families such as mir- (absent from pteropodidae), mir- (absent from yinpterochiroptera) and mir- (absent from rhinolophidae and megaderma lyra), see supplementary table s . with mirdeep , we detected hundreds of potential mirnas for all investigated bat species (supplementary table s ). generally, all bat species can be divided into two groups. for the majority of the bat assemblies ( out of ) about mirnas were predicted. for the other species of the vespertilionidae family about times as many mir-nas could be found. this is in concordance with the small rna-seq data used for the prediction, that was obtained from myotis daubentonii kidney cells (see methods). nevertheless, ∼ conserved mirnas can be predicted in the evolutionary more distant bat species. table . general genome information for each of the investigated bat assemblies and selected ncrna examples annotated in this study. we selected ncrnas with interesting copy number distributions among the investigated bat species. for snora , pvt and hottip and we additionally found sophisticated differential expression patterns in at least one of the used rna-seq data sets (absolute log fold-change greater , tpm > ). full tables and detailed information for each ncrna class (fasta, stk, gtf files) can be found in the electronic supplement online (supplementary tables s -s ) lncrnas for the annotation of lncrnas, we have deliberately chosen a broad blast-based approach, using , transcripts of potential human lncrnas obtained from the lncipedia database ( ) . we have consciously chosen this approach, because lncr-nas have diverse genomic contexts, reveal various functions and act in different biological mechanisms ( ) ( ) ( ) . from lncipedia transcripts, we annotated genes and lncrna hot spots. we defined regions in a genome as a lncrna hot spot, if different lncipedia transcripts derived from different genes map to the same region (see methods section for detailed description). overall, we found between and potential lncrna genes in m. lucifugus and r. sinicus, respectively, and between and lncrna hot spots in m. lucifugus and d. rotundus, respectively. we annotated the previously described lncrnas tbx -as and hottip ( ) in all bat genomes, except tbx -as in m. lucifugus, presumably due to the lower genome assembly quality (table ) . besides evolutionarily explainable differences in the presence of lncrnas, we have observed that the number of lncrnas and lncrna hot spots increases with increasing assembly quality (e.g. with a higher n ; see supplementary figure s ). we have not observed such a clear correlation between assembly quality and annotation results for short ncrnas. based on the rfam alignments, we were able to detect other ncrna families in addition to the rrnas, trnas, snornas and mirnas described before (supplementary table s ). overlaps with annotated lncrnas (supplementary table s ) are intentional, because the rfam includes only highly structured parts of long ncrnas. the highest number of ncrna copies ( ) was detected for the u spliceosomal rna in m. brandtii, already known to have a lot of pseudogenes ( ) . for ncrnas such as caesar (rf ), g-csf slide (rf ), nron (rf ), tusc (rf ), xist exon (rf ), linc (rf ), and hammerhead hh and hh (rf , rf ), we found exactly one copy in each investigated bat genome assembly (supplementary table s ). again, we also observed ncrna families that are lost for some species or entire families, for example the ribozyme cotc (rf ) that seems to be absent from all vespertilionidae members and m. natalensis. for each investigated bat species except e. spelaea, r. sinicus, m. lyra, e.fuscus and m. natalensis (where no mitochondrial contigs could be identified; table ) mitochondrial protein-coding genes and ncrnas were annotated (see methods). in total, mitochondrial genes comprising trnas, two rrnas ( s and s) and protein-coding genes were detected for each bat species as known for other metazoans ( ) (supplementary table s ). the mitochondrial genome lengths range from nt in e. helvum to nt in m. davidii. for the five bat species, where the mitochondrial genome could be identified as a part of the ncbi genome assembly, we appended the mtdna annotation to the final annotation of ncrnas. exemplarily, we investigated known and novel differentially expressed (de) ncrnas found in the genome of m. lucifugus in more detail. to this end, we used the rna-seq data sets field- , field- , hölzer- and weber- (table ) as a basis to identify de ncrnas that were newly discovered in this study and were not part of the current ncbi or ensembl genome annotations for this bat species. more detailed de results can be found in supplementary file s . we filtered for novel m. lucifugus de ncrnas by (i) an absolute log fold change (fc) > , (ii) an adjusted pvalue < . , and (iii) a tpm > . we further manually investigated the expression patterns with the igv ( ) and discarded de ncrnas overlapping with the current ncbi (myoluc . annotation release ) or ensembl (myoluc . . ) annotations. based on the small rna-seq comparison of mock and virus-infected (clone ) samples h post infection (weber- ), we found several mirnas (rfam-and mirdeep -based) and snornas to be differentially expressed ( figure and details in supplementary files s . ). in general, replicates of virus-infected and ifn-treated samples h post infection tend to cluster together only based on the expression profiles of small ncrnas (mainly mirnas) ( figure a and b) . most differences can be observed between the h virus-infected and all other samples, which seem to show no clearly distinguishable expression pattern. interestingly, at h post infection, we see replicates clustering together regardless of their treatment (mock, ifn, clone ). thus, after only h, few mirnas are differentially expressed and therefore the samples of each replicate (mock, ifn, clone ) cluster together, because they have the same passaging history but the passaging history in between the replicates differ ( ) . after h, more and more mirnas are significantly differentially expressed and the samples can be better distinguished based on their treatment ( figure a and b). we observed that, in general, mirnas tend to be down-regulated ( figure b ; upper half), while snornas tend to be up-regulated (lower half) after h of clone infection compared to mock. for example, we found a novel mirna (mlugd in our annotation; predicted by mirdeep ; supplementary table s ) located in an intron of the protein-coding gene sema g, significantly down-regulated (log fc = - . ) during clone infection ( figure c and d) . based on rfam alignments we further found a histone '-utr stemloop (rf ), an rna element involved in nucleocytoplasmic transport of the histone mrnas, significantly down-regulated during infection. for the same comparison of mock and virus-infected samples at h, the rrna-depleted data set (hölzer- ) revealed several de lncrnas. for example, we found a lncrna potentially transcribed in an intron of mx (mlugl ) up-regulated (log fc = . ) during infection. another lncrna (mlugl ), we found potentially transcribed as a part of two exons of the plat protein-coding gene and down-regulated (log fc = − . ) during viral infection (see details in supplementary files s . ). interestingly, based on the field- rna-seq data, we found two internal ribosomal entry site (ires) in the genes vegfa and odc with rfam ids rf and rf , respectively, to be -fold up-regulated during p. destructans infection. in this study, we comprehensively annotated ncrnas in readily available bat genomes obtained from the ncbi database (table ) . we provide novel annotations in the common gtf format, following a hierarchical structure of gene, transcript and exon features to allow direct integration of our annotations into already available ones (supplementary tables s -s ). finally, we provide for each bat genome assembly an extended annotation file merged with the protein-and non-coding gene annotations that were already available by the ncbi database (supplementary files s ; leaving out potential lncrnas that can be downloaded separately, see supplementary table s ) . we used six rna-seq data sets derived from the transcriptomic sequencing of four bat species (table ) to calculate normalized expression values for our newly annotated ncrnas and exemplarily show significantly differential expressed ncrnas (figure ) , which were never before described on such a large scale for any bat species. in addition to the evolutionarily explainable differences in the pure existence and the amount of annotated ncrnas in bats, we have observed that the assembly quality can also influence the annotation results. while the effects on the annotation of short ncrnas seem to be small (with some exceptions), the number of identified lncrnas and lncrna hot spots increases with increasing assembly quality (e.g. with a higher n ; see supplementary figure s ). however, this observation may be true for our data and analyses, but it also depends strongly on the annotation method used. recently, the bat k project (http://bat k.ucd.ie/) was announced as a global effort to sequence, assemble and annotate high-quality genomes of all living bat species ( ) . we aim to extend our annotation of ncrnas regularly and whenever new bat genomes become publicly available. in mid january , new low-coverage bat genomes of nine families were submitted by the broad institute to the ncbi genome database. unfortunately, our timeconsuming and computationally extensive analyses were already completed at this time point. we want to further automate our ncrna annotation workflow, to easily include these and any new bat (or other mammalian) genomes that will be sequenced and assembled in the future. our current identification of ncrnas in bat species will be usable as a resource (electronic supplement) for deeper studying of bat evolution, ncrnas repertoire, gene expression and regulation, ecology, and important host-virus interactions. detailed information about the bat genomes used in this study, their assembly quality and all ncrna candidates (in fasta, stk and gtf format) can be found in the electronic supplement (rna.uni-jena.de/supplements/ bats). the final extended annotations for each investigated bat species can be found in supplementary files s and the lncrna annotations in supplementary table s . to allow full reproducibility of our study, all final and intermediate data files (such as used genome files and mapping files in bam format) were uploaded to the open science framework under accession doi.org/ . /osf.io/ cmdn. python scripts used to filter and merge our annotations were deposited at github (github.com/rnajena/ bats ncrna). the virus-infected and ifn-stimulated small rna-seq data of the m. daubentonii kidney cell line was uploaded to geo (gse ). bats: important reservoir hosts of emerging viruses bat biology, genomes, and the bat k project: to generate chromosome-level genomes for all 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pacbio long-read sequencing the egyptian rousette genome reveals unexpected features of bat antiviral immunity reference sequence (refseq) database at [ncbi: current status, taxonomic expansion, and functional annotation sequencing and annotation for the jamaican fruit bat (artibeus jamaicensis) down but not out: the role of micrornas in hibernating bats a computational screen for mammalian pseudouridylation guide h/aca rnas small rnas with big implications: new insights into h/aca snorna function and their role in human disease long noncoding rnas: past, present, and future incredible rna: dual functions of coding and noncoding cncrnas: bi-functional rnas with protein coding and non-coding functions evolution of spliceosomal snrna genes in metazoan animals animal mitochondrial genomes integrative genomics viewer (igv): high-performance genomics data visualization and exploration icarus: visualizer for de novo assembly evaluation we thank ivonne görlich key: cord- - yu yl authors: bailey, stacy cooper; serper, marina; opsasnick, lauren; persell, stephen d.; o’conor, rachel; curtis, laura m.; benavente, julia yoshino; wismer, guisselle; batio, stephanie; eifler, morgan; zheng, pauline; russell, andrea; arvanitis, marina; ladner, daniela p.; kwasny, mary j.; rowe, theresa; linder, jeffrey a.; wolf, michael s. title: changes in covid- knowledge, beliefs, behaviors, and preparedness among high-risk adults from the onset to the acceleration phase of the us outbreak date: - - journal: j gen intern med doi: . /s - - - sha: doc_id: cord_uid: yu yl background: the us outbreak of coronavirus disease (covid- ) accelerated rapidly over a short time to become a public health crisis. objective: to assess how high-risk adults’ covid- knowledge, beliefs, behaviors, and sense of preparedness changed from the onset of the us outbreak (march – , ) to the acceleration phase (march –april , ). design: longitudinal, two-wave telephone survey. participants: predominately older adults with ≥ chronic condition recruited from active, federally funded studies in chicago. main measures: self-reported knowledge of covid- symptoms and prevention, related beliefs, behaviors, and sense of preparedness. key results: from the onset to the acceleration phase, participants increasingly perceived covid- to be a serious public health threat, reported more changes to their daily routine and plans, and reported greater preparedness. the proportion of respondents who believed they were “not at all likely” to get the virus decreased slightly ( . to . %; p = . ), but there was no significant change in the proportion of those who were unable to accurately identify ways to prevent infection ( . to . %; p . ). in multivariable analyses, black adults and those with lower health literacy were more likely to report less perceived susceptibility to covid- (black adults: relative risk (rr) . , % confidence interval (ci) . – . , p = . ; marginal health literacy: rr . , % ci . – . , p < . ). individuals with low health literacy remained more likely to feel unprepared for the outbreak (rr . , % ci . – . , p = . ) and to express confidence in the federal government response (rr . , % ci . – . , p < . ) conclusions: adults at higher risk for covid- continue to lack critical knowledge about prevention. while participants reported greater changes to daily routines and plans, disparities continued to exist in perceived susceptibility to covid- and in preparedness. public health messaging to date may not be effectively reaching vulnerable communities. the global outbreak of severe acute respiratory syndrome coronavirus (sars-cov- ), the virus that causes coronavirus disease (covid- ) , has rapidly evolved into a worldwide public health crisis. in the uncertainty of this pandemic, effective public health messaging is needed to inform the us population of risks posed by covid- and to motivate individuals, communities, and those in power to take action to minimize infection and prevent further spread of the virus. this need is particularly salient as recent data have revealed striking racial and ethnic disparities with covid- infection and mortality. , in chicago, where black residents represent less than a third of the total population, they account for half of those who have tested positive for covid- and two-thirds of those who have died. the success of the us response to this pandemic will depend largely upon the actions taken to protect and support the most vulnerable as well as how effectively public health information is conveyed to all americans, particularly those at greatest risk of severe infection and death. , , evidence has shown that how individuals obtain, interpret, and react to public health messages is influenced by socioeconomic, cultural, and health-related factors. [ ] [ ] [ ] to examine these influences within the context of the emerging coronavirus pandemic, our team previously conducted a telephone survey from march to , among high-risk, predominately older adults living with one or more chronic conditions. we investigated participants' level of awareness and knowledge of covid- , their impressions of the seriousness of the pandemic, their perceived susceptibility to the virus, whether the outbreak was affecting their daily routine and plans, and how prepared they felt for the pandemic. at the onset of the us outbreak, we found that almost a third of respondents lacked critical knowledge of covid- symptoms and methods of prevention. there were also marked disparities: individuals who were black, living in poverty, and/or with low health literacy were significantly less likely to believe they would become infected with covid- ; they also reported feeling less prepared for an outbreak. from march to april , , more than , americans tested positive for the coronavirus and over , died. by april , states had issued orders to close non-essential businesses and schools, restrict individual movement, and limit gatherings. an estimated million americans, % of the us population, were living under "shelter in place" or similar orders to reduce transmission of the virus. given this monumental shift in americans' daily lives, the purpose of this investigation was to longitudinally examine how adults' covid- knowledge, beliefs, behaviors, and sense of preparedness changed over time, from the onset to the acceleration phase of the us outbreak. we also explored psychosocial and sociodemographic factors influencing adults' beliefs surrounding covid- as well as individuals' adoption of preventive behaviors. the chicago covid- comorbidities (c ) survey is a longitudinal, telephone-based study conducted among predominately older adults in chicago, il. we administered wave of the survey at the onset of the us outbreak (march - , ) and wave during the acceleration phase of the pandemic (march -april , ). the northwestern institutional review board (irb) approved the study procedures. c participants were active enrollees in one of four ongoing, federally funded studies led by our team. parent studies have been described in depth previously. , [ ] [ ] [ ] in brief, these studies included a longitudinal cohort study examining the cognitive function and aging among older adults and three randomized trials evaluating technology-based strategies to improve patient adherence to complex drug regimens. , - participants were recruited into parent studies after receiving care from one of academic or community health centers in chicago, il. the eligibility criteria for each study varied and have been described in detail. overall, the target populations for these studies were older, english-speaking patients and those diagnosed with ≥ chronic condition; one trial also recruited spanish-speaking adults. only participants who provided consent to be contacted for future research opportunities and who completed a parent study interview from to were eligible to participate in wave . a description of wave methods has been published. all participants who completed wave were eligible to participate in wave . data collection was standardized across waves. research staff contacted participants via telephone and invited them to complete a survey about covid- . responses were recorded using redcap. surveys took < min to complete and participants were offered a $ gift certificate per wave. participants completed standardized assessments of personal attributes as part of their participation in the nih parent studies. this included items assessing sociodemographic (e.g., age, sex, race/ethnicity, income) and health characteristics (self-reported chronic conditions, overall health) as well as health literacy (the newest vital sign) and health activation (consumer health activation index). , all assessments were administered, and results analyzed, according to published guidelines. , covid- awareness, perceived seriousness, and perceived susceptibility. awareness of covid- was measured by asking participants whether they had heard of the coronavirus and, if so, if they knew someone who had or thought they had the virus. perceived seriousness of covid- was assessed by asking participants to rate, on a scale of to ( being no threat at all and being very serious), how serious a public health threat they believed the coronavirus is or might become. for perceived susceptibility, respondents were asked to rate the likelihood that they would get sick from the coronavirus (definitely will, probably will, it's possible, or not at all). assessed through two open-ended questions which asked participants to name symptoms of the coronavirus and actions they could take to prevent infection. clinician raters independently coded verbatim responses as correct or incorrect. participants were also asked to estimate the percentage of people who would die from the coronavirus once infected and the percentage of those who would have mild symptoms. covid- reported behaviors, perceived preparedness, and confidence in government. participants were asked how much the coronavirus had changed their daily routine (a lot, some, a little, or not at all) and whether they had changed plans due to the virus. they also rated their confidence in the federal government's ability to prevent further covid- outbreak (very confident, somewhat confident, not very confident, or not confident at all) and perceived preparedness for a widespread outbreak (very prepared, somewhat prepared, a little prepared, or not prepared at all). descriptive statistics (means with standard deviations and percentage frequencies) were calculated for all patient characteristics and survey responses. paired t tests, ranked tests, and mcnemar's tests were conducted to determine whether patient responses at waves and differed significantly. associations between patient characteristics and responses to covid- knowledge, beliefs, reported behaviors, and perceived preparedness items were evaluated in bivariate analyses using chi-square tests. multivariable regression models were used to examine differences in outcomes; a poisson distribution was used rather than odds ratios for the relative risk estimates. all models included health literacy, age, sex, race, poverty, number of days between waves and and parent study; this mirrored the models conducted for wave . in order to assess change between waves and , generalized linear mixed models with a repeated effect were used to model interactions between wave and relevant covariates for each outcome. least square means were then calculated for all covariates at wave and , and post hoc paired tests were conducted within each level of the covariate. all p values from the paired tests were bonferroni adjusted. analyses were performed using sas, version . (cary, nc). of the participants in wave , declined to participate in wave , could not be reached or asked to be contacted later, had incomplete or missing data, and completed the survey, for an overall cooperation rate of . %. the adults who did not complete wave were more likely to be black, to live below the poverty line, and to have low health literacy than those who participated. table summarizes participant characteristics for wave . overall, patients were socioeconomically diverse, at an average of . years old, and . % had ≥ chronic conditions. all participants had heard of the coronavirus and % said they knew of someone who had or thought they had the virus. participants rated the coronavirus as a more serious public health threat in wave than wave (mean (sd) . ( . ) vs. . ( . ); p < . ; table ). almost a quarter ( . %) of participants at wave believed they were "not at all" likely to get sick from the coronavirus ( table ). in bivariate analyses, individuals who were female, black, living below the poverty level, unemployed, and with low or marginal health literacy were more likely to believe that they were "not at all" likely to become infected (table ) . after multivariable adjustment, individuals who were black or had marginal health literacy skills were significantly more likely to state that it was "not likely" they would become sick (table ). this was similar to wave , which found that adults who were black, living below the poverty line, and with low health literacy believed it was "not likely" they would become sick. while overall perceptions of susceptibility increased over time (table ), in multivariate analyses investigating change from wave to wave , no significant interaction between survey wave and any participant characteristic was found. this suggests there was no change in how certain groups, particularly by race, income, or health literacy, at wave , participants estimated significantly higher fatality rates from covid- than in wave (mean (sd) . ( . ) vs. . ( . ), p = . ; table ). most participants ( . %) were able to correctly identify symptoms and . % could name methods of preventing infection. knowledge of symptoms increased significantly from wave to while knowledge of prevention did not (table ). in bivariate analyses, men, individuals with low or marginal health literacy, englishspeaking adults, and those with multiple chronic conditions had less knowledge of coronavirus symptoms ( table ) . as in wave , hispanic and lep adults had significantly greater knowledge of methods to prevent coronavirus (table ). after multivariable adjustment, no participant characteristics were significantly associated with knowledge of coronavirus symptoms or prevention. this was consistent with wave findings. when examining change from wave to wave , individuals who were older, female, black, or with low health literacy skills were more likely questions re-worded at wave to account for acceleration in the outbreak. at wave , questions were "how confident are you that the federal government can prevent a nationwide outbreak of the coronavirus?" and "how prepared do you think you are if there were to be a widespread coronavirus outbreak?" covid- coronavirus disease *values are percentages unless otherwise stated † participants did not respond at wave ; did not respond at wave ‡ participants did not respond at wave ; did not respond at wave to increase their knowledge of coronavirus symptoms; those who lived below the poverty level as well as those above the poverty line also experienced increases in symptom knowledge (table ) . at wave , . % of participants reported that coronavirus had changed their daily routine "a lot" and . % stated that they had changed plans due to coronavirus. there was a significant increase in both of these behaviors from waves to ( table ) . participants who were older, unemployed, and with low health literacy were less likely to report changing their daily routine "a lot" at wave while men, individuals with low health literacy, and adults with three or more chronic conditions were less likely to report changing plans (table ) . after multivariable adjustment, there were no participant characteristics that were associated with changes in daily routine or plans at wave . this was consistent with wave . in repeated effect interaction models assessing changes between waves and , increased changes in behavior were noted across a number of participant characteristics. specifically, participants under the age of , both men and women, individuals of both black and white race, those living below and above the poverty line, and those with low, marginal and adequate health literacy experienced either more changes to daily routines or plans or both (table ) . confidence in the federal government's ability to prevent further outbreak did not change significantly between waves and . % of participants at wave reported that they were "very confident" in the government's response. in multivariate analyses, individuals with low or marginal health literacy were more likely to express that they were "somewhat" or "very" confident in the government (low: relative risk . , % ci . - . ; p < . ; marginal: relative risk . , % ci . - . ; p = . ); a similar relationship with low health literacy was found in wave . more than a quarter ( . %) of participants at wave believed they were "very prepared" for the coronavirus outbreak and most ( . %) thought they were "somewhat prepared" while . % stated they were "a little" or "not prepared at all." there were significant differences in the level of perceived preparedness between waves and , with more participants at wave feeling prepared (table ). however, similar to wave , individuals who were hispanic, those with limited english proficiency, individuals living below poverty level, the unemployed, those with low or marginal health literacy, and participants with low or moderate health activation were more likely to state that they were "not at all prepared" in bivariate analyses (table ) . in multivariable analyses, only individuals with low health literacy were more likely to be "a little" or "not prepared at all" (table ). in wave , black participants and individuals with low health literacy were more likely to report feeling low preparedness. in regard to change, adults living below the poverty line and those who were black, male, and ages - were less likely to report being "a little" or "not at all" prepared during the acceleration phase of the us outbreak in comparison to the onset. findings from this longitudinal study reveal key changes in adults' knowledge, beliefs, behaviors, and preparedness from the onset of the us outbreak to the acceleration phase. our results indicate that adults increasingly perceive covid- as a very serious public health threat and made more changes to their daily routine and plans as the pandemic progressed. importantly, most individuals' perceptions of their own preparedness for the outbreak also increased. these changes are particularly notable among black adults and those living below the poverty level, who at the onset of the pandemic were disproportionately more likely to feel unprepared. participants' ability to identify covid- symptoms improved over time. despite these positive developments, serious public health concerns remain. participants with low health literacy continue to be more likely to report feeling unprepared for the outbreak and in of these high-risk adults was unable to accurately identify methods of preventing covid- , a proportion that was unchanged since the onset of the pandemic. additionally, almost a quarter of participants-who are predominately older, with multiple underlying health conditions-reported believing that it was "not at all likely" that they would become sick from the coronavirus. black adults and those with lower health literacy continued to be more likely to have lower perceived susceptibility. the c study has provided a unique opportunity to understand how high-risk adults' knowledge, beliefs, behaviors, and preparedness changed over the initial stages of a pandemic. wave was conducted at the onset of the us outbreak, when the number of cases of covid- in illinois ranged from to and no "stay at home" order had been issued. in contrast, during wave , there were between and , cases in illinois and all state residents had been ordered to stay home. unsurprisingly, there was a marked shift in participants' daily routines between waves and and increased recognition of the public health threat posed by covid- . yet, our findings also reveal clear differences in how adults perceive their own individual risk for covid- , their sense of preparedness for the outbreak, and their knowledge of what can be done to prevent infection. these findings suggest that public health messages on risk factors for coronavirus, its potential spread, and methods of preventing covid- are not reaching all americans, particularly the most vulnerable. misinformation on effective methods of prevention are widespread and may be contributing to poorer understanding of how to effectively reduce transmission of the virus. , individuals with low health literacy, who are likely to struggle to obtain, process, and understand public health messages related to covid- , are more likely to feel unprepared for the pandemic and to perceive themselves as less susceptible to covid- , which may place them at greater risk. , this study has limitations. participants were predominately older, most had three or more chronic conditions, and all lived in metro chicago, il. as such, findings may not be generalizable to younger or healthier adults or to individuals in other geographic locations. findings indicate that adults' knowledge, beliefs, behaviors, and preparedness changed over time with the pandemic; as such, results from this wave may not be reflective of current norms by the time of publication. however, our team is mobilizing to conduct additional waves of this study at future dates. finally, this study relied upon self-report measures. it is possible that social desirability bias may have led participants to over-report changes to their behaviors. however, given the restrictions currently in place in illinois, it is likely changes in daily routines and plans occurred for most participants. our first longitudinal assessment of the c study revealed that participants increasingly perceived covid- to be a serious public health threat, reported more changes to their daily routine and plans, and had a greater sense of preparedness from the onset to the acceleration phase of the outbreak. yet, knowledge deficits remain and many high-risk participants still perceived themselves as being "not at all likely" to become sick even after cases had greatly increased in the region. accurate, easy-to-understand, and consistent public health messaging that can reach all americans, including the most at risk and vulnerable, is needed to reduce the threat posed by the covid- outbreak and to ensure that everyone is prepared and aware of the actions that can be taken to protect themselves, their families, and their communities from this rapidly evolving public health crisis. data availability: the datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request. corresponding author: stacy cooper bailey, phd mph; division of general internal medicine & geriatrics, feinberg school of medicine at northwestern university, n. lake shore drive, th floor, chicago, il , usa (e-mail: stacy-bailey@northwestern.edu). funding information this work was supported by grants r ag , r ag , r dk , and r nr from the national institutes of health (nih). dr. linder is supported by a contract from the agency for healthcare research and quality (hhsp i) and by grants from the national institute on aging (r ag , r ag , p ag ), the agency for healthcare research and quality (r hs ), and the peterson center on healthcare. the northwestern institutional review board (irb) approved the study procedures. effective health risk communication about pandemic influenza for vulnerable populations hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease -covid-net, states latest data: covid- . city of chicago effective communication during an influenza pandemic: the value of using a crisis and emergency risk communication framework protecting vulnerable populations from pandemic influenza in the united states: a strategic imperative public response to community mitigation measures for pandemic influenza health literacy: a prescription to end confusion demographic and attitudinal determinants of protective behaviours during a pandemic: a review awareness, attitudes, and actions related to covid- among adults with chronic conditions at the onset of the u.s. outbreak: a cross-sectional survey see which states and cities have told residents to stay at home rationale and design of the regimen education and messaging in diabetes (remind) trial development and rationale for a multifactorial, randomized controlled trial to test strategies to promote adherence to complex drug regimens among older adults literacy, cognitive function, and health: results of the litcog study quick assessment of literacy in primary care: the newest vital sign development and validation of the consumer health activation index springfield: illinois department of public health. accessed at: www.dph.illinois.gov/covid on why misinformation and distrust are making covid- more dangerous for black america. national public radio (npr) united kingdon: reuters institute for the study of journalism. accessed at reutersinstitute.politics.ox.ac.uk/types-sources-and-claims-covid- -misinformation on public health literacy in america: an ethical imperative conflict of interest: dr. bailey reports grants from the nih, merck, pfizer, the gordon and betty moore foundation, and eli lilly and personal fees from the gordon and betty moore foundation, sanofi, pfizer, and luto outside the submitted work. dr. serper reports personal fees from biovie outside the submitted work. ms. batio reports grants from the nih during the conduct of the study. dr. ladner reports grants from the national institute of diabetes and digestive and kidney diseases during the conduct of the study. dr. persell reports grants from omron healthcare and pfizer outside the submitted work. dr. wolf reports grants from the nih during the conduct of the study; grants from merck, the gordon and betty moore foundation, the nih, and eli lilly outside the submitted work; and personal fees from sanofi, pfizer, and luto outside the submitted work. authors not named here have disclosed no conflicts of interest. key: cord- - w kwzu authors: timmermans, ans; melendrez, melanie c.; se, youry; chuang, ilin; samon, nou; uthaimongkol, nichapat; klungthong, chonticha; manasatienkij, wudtichai; thaisomboonsuk, butsaya; tyner, stuart d.; rith, sareth; horm, viseth srey; jarman, richard g.; bethell, delia; chanarat, nitima; pavlin, julie; wongstitwilairoong, tippa; saingam, piyaporn; el, but sam; fukuda, mark m.; touch, sok; sovann, ly; fernandez, stefan; buchy, philippe; chanthap, lon; saunders, david title: human sentinel surveillance of influenza and other respiratory viral pathogens in border areas of western cambodia date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: w kwzu little is known about circulation of influenza and other respiratory viruses in remote populations along the thai-cambodia border in western cambodia. we screened outpatients (median age , range – ) presenting with influenza-like-illness (ili) at sentinel sites in western cambodia between may and december . real-time reverse transcriptase (rrt) pcr for influenza was performed on combined nasal and throat specimens followed by viral culture, antigenic analysis, antiviral susceptibility testing and full genome sequencing for phylogenetic analysis. ili-specimens negative for influenza were cultured, followed by rrt-pcr for enterovirus and rhinovirus (ev/rv) and ev . influenza was found in cases ( %) and occurred almost exclusively in the rainy season from june to november. isolated influenza strains had close antigenic and phylogenetic relationships, matching vaccine and circulating strains found elsewhere in cambodia. influenza vaccination coverage was low (< %). western cambodian h n ( ) isolate genomes were more closely related to earlier cambodia isolates ( . % genome conservation) than to thai isolates ( . % genome conservation), despite sharing the majority of the amino acid changes with the thai references. most genes showed signatures of purifying selection. viral culture detected only adenovirus ( . %) and parainfluenza virus ( . %), while non-polio enteroviruses ( . %) were detected among culture-negative samples including coxsackievirus a , a , a , a , a , b , b and echovirus e and e using nested rt-pcr methods. a single specimen of ev was found. despite proximity to thailand, influenza epidemiology of these western cambodian isolates followed patterns observed elsewhere in cambodia, continuing to support current vaccine and treatment recommendations from the cambodian national influenza center. amino acid mutations at non-epitope sites, particularly hemagglutinin genes, require further investigation in light of an increasingly important role of permissive mutations in influenza virus evolution. further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in cambodia is also needed. acute respiratory infection (ari) is the leading cause of morbidity and mortality in cambodia [ ] . previous studies have attributed the etiology of acute viral respiratory infections in cambodia to rhinovirus, respiratory syncytial virus (rsv), parainfluenza virus (piv), influenza virus a and b, human metapneumovirus (hmpv), bocavirus, adenovirus, enterovirus, and coronavirus [ ] . influenza has been described to be one of the most important causes for hospitalization of children with an ari [ ] . it was suggested that some southeast asian countries, where influenza a infection is present year round, play an important role in the global spread of influenza that could trigger annual epidemics in temperate regions [ , ] . until the establishment of the national influenza center (nic) at institut pasteur du cambodge (ipc) and a national influenza-like-illness (ili)-sentinel surveillance system in , little was known about influenza circulation in cambodia. since then, the sentinel surveillance system has, together with event-based surveillance, demonstrated evidence of seasonal, pandemic and avian influenza. previously published data revealed that thailand and cambodia, which are positioned geographically in the northern hemisphere, demonstrate differing influenza epidemiology with regard to seasonality, with thailand exhibiting a northern hemisphere transmission pattern while transmission in cambodia typically occurs from june to december, similar to the southern hemisphere [ ] . the rainy season in western cambodia is june to november and the dry season is december to may. the more recent data suggests that influenza seasonality is more similar in thailand and cambodia than previously thought. [ ] . depending on the type of genes and the specific residues, phenotypic effects of random or neutral mutation of the virus can be positive, purifying or neutral [ ] . influenza virus efficiently escapes from host antibodies through an accumulation of mutations/single amino acid changes (antigenic drift) at the antigenic sites (epitopes) in surface glycoproteins of the hemagglutinin (ha) gene, and to a lesser extent, neuraminidase (na) genes [ ] . the antigenic sites are five somewhat overlapping regions (designated a to e for the h strains [ ] [ ] [ ] and ca , ca , cb, sa, and sb for the h strains [ ] ). antigenic mapping by smith et al. [ ] showed that antigenic clusters of viruses emerged during the -year period following the introduction of the a/h n virus in humans in and that major jumps (or "cluster transitions") occur between antigenically distinct clusters of viral sequences roughly every years (antigenic shift) [ ] . until , the cambodian national sentinel surveillance system only covered out of provinces based on a network of urban sentinel-sites at referral hospitals. no data existed about circulating influenza strains and other respiratory pathogens in western-cambodia along the thai-cambodia border, which is a poor and rural area with large volume of crossborder traffic. between and , the armed forces research institute of medical sciences (afrims) established four additional influenza sentinel surveillance sites in four border provinces in western cambodia, in collaboration with the cambodian communicable disease control (ccdc) department and the ipc. this study describes the circulation of influenza and non-influenza respiratory viruses and the genetic diversity of influenza viruses in western cambodia along the thai-cambodia border which has provided information for treatment, prevention, and control strategies for these populations. the ili and event-based surveillance systems are public health activities organized by the ministry of health in cambodia and as such have a standing authorization from the national ethics committee. samples were all anonymized for the purpose of this study. the study was approved by scientific review committee at afrims in thailand, the institutional review boards at the walter reed army institute of research (wrair) in the united states and the national ethics committee for health research (nehcr) in cambodia. written informed consent was obtained from volunteers or parents or legal guardians of children enrolled in the study. between may and december , we collected specimens and surveillance data for influenza and other viral respiratory pathogens from a subset of outpatients presenting with influenza-like-illness (ili) at four sentinel sites-located in five health centers and hospitals in battambang, oddar meanchey, pailin and banteay meanchey provinces in cambodia (fig ) . fig was created using arcview gis version . (http://arcview.software.informer.com [ , ] ) sentinel health centers and hospitals were selected based on sufficient overall patient volume and patients with upper respiratory infections (uri), geographical representativeness and ease of specimen transport to the study laboratory. sites were established sequentially: battambang in ( sites), oddar meanchey and pailin in , and banteay meanchey in . due to the cross-sectional design of our study in a region for which no previous baseline respiratory surveillance existed, no minimal sample size was calculated. nevertheless, and in accordance with national and world health organization influenza surveillance guidelines [ ], we aimed to collect a cross-section of data distributed over time and space. for every site -and pending on local epidemiology and health facility workload, to ili specimens were collected each week (monday to friday) for weeks per year and largely from the first two subjects meeting the predefined inclusion criteria. demographic, clinical data and respiratory specimens were collected from patients who met the case definition for ili defined as persons of year of age or older, arriving at the health center or hospital within days of fever onset with a fever (axillary . °c or oral . °c) and cough or sore throat in the absence of other diagnoses. patients younger than year, who could not provide consent or those with nasal lesions or epistaxis were excluded. demographic data included gender, occupation, presenting signs and symptoms, self-reported influenza vaccination history, medication use and travel within the last -days. parents reported for children, which in our study are defined as all study volunteers younger than years old. descriptive statistics of demographic and clinical data including frequencies and cross-tabulations were entered in a study database by double data-entry using microsoft excel and analyzed using ibm spss statistics . (spss, ibm inc.) up to three respiratory swabs per volunteer were collected by trained staff with standard procedures. one nasal swab was tested on-site with quickvue influenza a+b (quidel, san diego, calif.) using monoclonal antibodies specific for influenza a and b virus. a combined nasal and throat specimen was placed in a universal transport medium (utm, copan diagnostics, corona, ca, usa), stored between - °c or in liquid nitrogen and shipped to the afrims-cnm (cambodia national malaria center) laboratory at the battambang referral hospital for testing for influenza virus. all ili-specimens were typed and subtyped for influenza a and b at the afrims-cnm laboratory in battambang. influenza-positive specimens were forwarded to the virology unit at institut pasteur du cambodge (ipc) in phnom penh and the who collaborating center (whocc) for reference and research on influenza in melbourne, australia. nucleotide sequence and phylogenetic analysis of influenza viruses was conducted at the virology branch at the walter reed army institute of research (wrair), silver spring, maryland, u.s. influenza-negative specimens were sent to the department of virology at afrims in bangkok, thailand for viral culture and further characterization of other respiratory viruses. rna was extracted from an aliquot of μl of each combined nasal and throat specimen using qiagen viral rna mini kits (qiagen, hilden, germany). the influenza genome was amplified and detected using a standardized the real-time reverse transcription (rrt)-pcr assay on a smartcycler ii platform (cepheid, sunnyvale, ca), to test for influenza a and/or b types. samples positive for influenza a and/or b were further tested to confirm influenza a and/or b positivity and to determine influenza a subtypes. the hemagglutinin-gene (ha) specific primers and probes were used for subtyping of human influenza a subtypes h , h , h and pandemic h (ph , previously known as swh ). nucleoprotein-gene (np) specific primers and probe for universal swine influenza was also used to confirm influenza a subtype ph detection in some samples. the μl reaction mixture consisted of μl of rna eluate and μl of the master mixture which included . μl enzyme mixture (superscript iii rt/platinum taq dna polymerase-life technologies), . μl rnaseout™ recombinant ribonuclease inhibitor (invitrogen™, carlsbad, ca), . μl x reaction mix, . μl of both primers and . μl of probe, and μl rnase-free water (see s table for primer sequences). thermocycling reaction conditions for influenza typing were as follows: initial reverse transcription at °c for min, followed by denaturation and pcr activation by taq inhibitor activator at °c for min and cycles of pcr amplification (< °c for sec then °c for sec). for influenza a subtyping: reverse transcription at °c for min, pcr activation by taq inhibitor activator at °c for min and cycles of pcr amplification (< °c for sec then °c for sec). after influenza typing by rrt-pcr, two μl aliquots of every influenza-positive specimen was transferred into cryogenic vials for transport in liquid nitrogen to ipc in phnom penh. specimens testing positive for influenza by rrt-pcr were inoculated onto madin-darby canine kidney (mdck) cells for isolation of influenza strains at the virology unit of ipc. the influenza strains were analyzed by a hemagglutination inhibition test using reference antigens and anti-sera provided by the whocc for reference and research on influenza in melbourne, australia. the na-star kit (life technologies, carlsbad, ca, usa), a chemiluminescent neuraminidase (na) inhibition assay which utilizes a , -dioxetane derivative of sialic acid as substrates, was used for na inhibitor susceptibility testing on a subset of isolates, randomly selected across specimen collection sites and dates. the concentration of drug required to inhibit % of the na activity (ic ) was calculated using the non-linear curve-fitting function in the graphpad prism package (graphpad software, inc., la jolla, ca, usa). the average ic (nm) (± standard deviation) of two independent determinations was calculated for each virus. outliers of more than standard deviations from the overall mean were retested twice. a subset of isolates were forwarded to whocc in melbourne for confirmation of strain analysis by ha inhibition test and na inhibitor susceptibility using an na enzyme inhibition assay with a fluorescent substrate munana [ '-( -methylumbelliferyl)-α-d-n-acetylneuraminic acid sodium salt hydrate]. the typical range of ic was calculated as the mean ic ± standard deviations using a panel of well-characterized reference strains kindly provided by the who collaborative center for reference and research on influenza, melbourne, australia. isolates with ic values within or close to the typical ic range were considered to be sensitive isolates. ic values outside of the typical range and between and nm were isolates with mildly reduced sensitivity and ic values well outside the typical range and greater than nm were considered as isolates with highly reduced sensitivity. viral rna segments, extracted from mdck supernatant were sequenced at ipc in phnom penh from samples collected in and from samples collected in for both pandemic influenza (ph n ) and influenza h n (h n ), representing our four study sites with varying success (s table) . cleaned nucleotide sequence data was sent in fasta-format to the bioinformatics section at the virology branch of the wrair in silver spring, maryland, usa. sequences for ph n and h n sequence datasets respectively were combined with references available from the influenza viral resource at genbank [ ] and the gisaid epiflu database [ ] representing global diversity of ph n from - . sequences were aligned using muscle version . . [ ] implemented in seaview version . . [ ] and manually inspected for accuracy. best models were determined from alignments using jmodeltest version . . [ ] . maximum likelihood (ml) phylogenetic trees were generated using phyml version . as implemented in seaview version . . [ ] . see table for a detailed description of datasets and models used in analysis. ml phylogenies were annotated using mega version and figtree version . . [ ] . all influenza sequences used in analysis have been deposited into genbank under accessions ku -ku . alignments were analyzed for diversity, percent nucleotide similarity, antigenic variation and selection using mega version [ ] and the hyphy package [ ] implemented through the datamonkey webserver [ ] [ ] [ ] . annotation was conducted using genbank's annotation tool in the influenza viral resource [ ] and a literature review. all specimens tested negative by influenza pcr were cultured for isolation of respiratory virus and detection by cytopathic effect (cpe) and/or with either immunofluorescence assay (ifa), or direct fluorescence assay (dfa). a subset of culture-negative specimens (collected between may and april ), where we found a higher proportion ( . %) of non-polio enteroviruses in children less than years old as compared with previous studies ( %) in cambodia [ ] , were tested for enterovirus and rhinovirus by two separate nested rt-pcr methods adapted from coiras et al., and singh et al., [ , ] , one for simultaneous detection of pan-enteroviruses and rhinoviruses, and the other specific for enterovirus (ev ). pcrproducts positive for enterovirus or rhinovirus were sequenced for nucleotide analysis (fig ; s table) . cell culture of influenza-negative specimens enterovirus ev detection included single-step first-round rt-pcr and semi-nested pcr (primers are in s table) . the ev type specific primers were modified from the previous study by singh et al. [ ] . detection of pan-enteroviruses and rhinoviruses (pan-ev/rv nested rt-pcr) is similar to the procedure for ev including rounds of pcr as reported before [ ] . the primers for simultaneous detection of enteroviruses and rhinoviruses were designed in the polyprotein gene, between non-coding region ( ncr) and vp /vp regions that was previously described by coiras et al. [ ] . ev/rv- n was modified from primer -ev/rv [ ] for using in nested pcr reaction. single-step first-round rt-pcr reaction was the same as described above except that . pmol of each forward and reverse primer ( -ev/rv and -ev/rv) was used (s table) . the rt step was performed at °c for min, followed by cycles of thermocycling °c for seconds, °c for min, and °c for min. the reaction was further incubated at °c for min. five micro-liter of : diluted first round pcr products were added to the nested pcr reaction with the same reagents as in the first-round but without dtt and amv-rt and the primers were . pmol of each forward and reverse primer, -ev/rv and ev/rv- n. the reaction was incubated at °c for min followed by cycles of thermocycling °c for seconds, °c for min, and °c for min. the pcr products ( μl each) were subjected to electrophoresis in agarose gels, with -bp dna ladder serving as a molecular marker. a negative control of rna-free water and a positive control of cdna template from the ev reference strain (atcc no. vr- tm ) were included in each experiment. the pcr products from the positive samples were sequenced on both strands with the pcr primers (s table) . the specific bands from the first or second rounds pcr were purified using qiaquick gel extraction kit (qiagen, germany) before sending for direct sequencing. sequencing service was performed by ait biotech (singapore). the sequences from both strands were combined for analysis and edited with sequencher (gene code corp., usa). homology searches were through nucleotide blast program [ ] along with the percentage of sequence identity of the two given sequences. ili-patients (median age year, range - ) were enrolled from five sites from may to december ; among these, ( %) tested positive for influenza by rrt-pcr (table ) . most frequent symptoms reported were fever ( %, inclusion criterion), cough ( %,), runny nose ( %), congestion ( %), sore throat ( %,) and headache ( %). although gender distribution was similar in ili-patients, more females were positive for enterovirus ( %) or rhinovirus ( %). patients with influenza were slightly older (median age years old) and presented more often with a sore throat ( %). compared with other ili-cases, patients testing positive for enterovirus reported body pain ( % vs. %) and headache ( % vs. %) less frequently, although chills ( % vs. %), vomiting ( % vs. %) and abdominal pain ( % vs. %) were more common. dyspnea was uncommon, yet most often seen in patients positive for adenovirus ( %). diarrhea was observed in ili-patients ( %- %) except those infected with enterovirus ( / ). influenza vaccination coverage in and approximated % in response to the influenza a/ph n ( ) pandemic yet decreased to zero in by subjects' reports. five (table and figs and a). vaccination coverage during the entire study period was % ( / ). the probability of developing influenza among ili-patients who had been vaccinated ( / ) and who had not ( / ) was similar and not significantly different ( . vs. . , p = . ). all ic -values of influenza a and b isolates tested from were within the susceptible range for both oseltamivir and zanamivir (table ) . overall, at least respiratory virus was detected in out of ( %) ili-specimens collected between may and december (fig ) ; of which most were influenza ( , % of ili-cases), followed by rhinovirus ( , %), adenovirus ( , %), non-polio enterovirus ( , %) and parainfluenza virus (piv) ( , %). all of the ili specimens that tested negative for influenza a or b by rrt-pcr were sent for viral culture. forty ( %) were culture positive; adenovirus, piv , piv , piv were isolated (figs and b ). no rsv, hmpv or coronaviruses were identified. pan-ev/rv and ev nested rt-pcr (s table) detected samples positive for enterovirus, which included coxsackievirus a (n = , a , a , a , a , a ), echovirus (n = , e , e ) and coxsackievirus b (n = , b , b ). a single specimen was found to be negative during the first round of rt-pcr but positive by second round pan-ev/rv and ev nested rt-pcr. the base pairs pcr product fragment obtained from pan-ev/rv nested rt-pcr was sequenced using the rv/ev inner primers. the bases obtained from sequencing were found to be % identical to the enterovirus a /homo sapiens/vnm/ / strain (accession: kj ). when the bp pcr product obtained from ev nested rt-pcr was sequenced using the ev inner primers, bases was obtained. this sequence was found to be % identical to enterovirus a /cambodia: banteay meanchey strains (accessions: kp , kp , kp , kp , kp , kp , kp , kp ). out of the specimens positive with rhinoviruses, only could be serotyped. of these , most were rhinovirus a (n = ), followed by rhinovirus b (n = ) and rhinovirus c (n = ) (fig b) . amplicons were sequenced and sequences were analyzed using genbank's blast tool to identify the virus species. pandemic influenza a (ph n ). all sequences were highly related with no more than . % divergence between the strains detected in the study and respective seed viruses included in the vaccine composition (a/california/ / , a/brisbane/ / , and a/christchurch/ / ; s table, fig ) for any of the segments analyzed. when compared to the a/california/ / vaccine strain, all cambodian viruses on average had amino acid changes within the ha segment. amino acid changes found in the ha gene can be found in table . no evidence for amino acid substitutions leading to changes in glycosylation was seen in the ha dataset; however, several changes among sequenced samples occurred in antigenic sites, polymorphic sites or had a recorded effect in in vitro analyses per the literature (s table) . for the na gene, several amino acid substitutions resulted in addition/loss of glycosylation sites, or table) . for the mp gene, all samples contained the s n mutation that may confer amantadine resistance as determined by the genbank influenza viral resource annotation tool [ ] . no features or changes of interest were seen within the np or ns segment. the ha and na segments contained the most amino acid substitutions when compared to the reference (a/california/ / ) as well as to the yearly defined groups ( and ). the mp and np segments showed the fewest substitutions (table ). there were no samples within the ha amino acid analysis that had unique substitutions relative to the global sequences. however, the na gene had unique amino acid substitutions not found with the global references; the function of these substitutions is unknown (s table) . selection pressure was determined using the dn/ds statistic. values of dn/ds > are indicative of positive or adaptive selection, values < are indicative of purifying or negative selection and a value = indicates neutral or no selection ongoing in the dataset. all samples were under purifying selection for all segments with exception to the ns segment of the cambodian samples, which had a dn/ds value of . suggesting neutral selection (s table) . for ph n there was no evidence of geographic clustering of sequences within sampling site (colored branches in fig ) ; nor was there evidence of reassortment among the sequences sampled. sequences (references and samples) from cambodia distributed throughout the trees; however, the majority of sequences, including those corresponding to strains detected in this study appeared within a specific cambodian clade. however, this clade was only evident in ha and na phylogenies and did not appear in np, mp or ns phylogenies (s -s figs). influenza a h n (h n ). all sequences were highly related with no more than . % divergence between the samples and the furthest related respective vaccine (s table, fig ) for any of the segment. the majority of segments were more closely related to the a/victoria/ / ( ) table) . there were several amino acid changes among the sequenced samples occurring in antigenic sites, polymorphic sites or which resulted in a change of glycosylation (table ). for the na gene, several amino acid substitutions resulted in addition/loss of glycosylation sites, or were found in polymorphic or antigenic/catalytic sites (epitopes) ( table ). for the mp gene, it was noted that all samples contained the s n mutation that may confer adamantane resistance as determined by the genbank influenza viral resource annotation tool [ ] . no features or changes of interest were seen within the ns segment for any sample. the ha and na segments contained the most amino acid substitutions when compared to the reference (a/victoria/ / ) as well as to the yearly defined groups ( and ). the mp and ns segments showed the fewest substitutions (s table) . there were several amino acid substitution within the ha amino acid analysis that were unique relative to the global sequences (and vaccine references); two of which occurred in antigenic regions (table ) and five additional changes of unknown effect or location significance (s table) . one amino acid substitution in the na gene was found to be unique in antigenic site a (table ) and unique substitutions of unknown effect or location significance were also found (s table) . all 'unique' aa substitutions were not found with the global sequences or vaccine references. all samples were under purifying selection for all segments with the exception to the ns segment. when samples were separated based on the respective years of collection, the ns gene showed evidence of neutral selection (dn/ds~ ). however, when all samples were combined to increase sample size, it showed overall evidence of purifying selection (dn/ds < ; s table) . overall, for h n , there was no evidence of geographic clustering of sequences within the same sampling site except samples from oddar meanchey in which all but one sample clustered into the ohio vaccine cluster (green branches in fig ) . this clustering was visual only, not statistically significant due to small sample size, though the cluster was well supported (cluster node value , value obtained using associated likelihood ratio test (alrt); analogous to bootstrap analysis). there was no evidence of reassortment among the samples analyzed and all samples were consistently grouping with either the a/ohio/ / or a/victoria/ / vaccines. there was one exception; sample a/cambodia/v / consistently fell outside of both the ohio and victoria vaccine clusters (fig ) for all segments analyzed. this is to the best of our knowledge the first influenza surveillance study in remote border areas in western cambodia. over the time course of months, spanning influenza seasons including the last few months of pandemic a/ph n ( ) influenza circulation, seasonal influenza virus was the most commonly detected respiratory virus in this predominantly pediatric population of subjects who presented with ili (fig ) . despite detection of human highly pathogenic avian influenza (hpai) h n virus in western cambodia since early [ ] [ ] [ ] , h n , typically detected in south-central cambodia, was not found at our sentinel sites. influenza subtypes varied by year with a/ph n ( ) being predominate in , influenza b in , and influenza a/h n in . we did not expect to detect any h n in our laboratory surveillance system as most suspected h n cases would have been screened out by the health centers based on reported exposure to dead or diseased poultry. the specimen of one patient with reported h n exposure in was investigated in our laboratory and tested negative for h n . table . changes in glycosylation, antigenic and polymorphic sites linked to amino acid (aa) substitutions and sub-antigenic/catalytic sites (epitopes) where aa substitutions were found for hemagglutinin (ha) and neuraminidase (na) segments of h n . [ ] . our data revealed that the influenza b did not match well with strains included in vaccine composition of trivalent vaccines; therefore, if vaccination is to be implemented, the quadrivalent influenza vaccine that contains the two influenza b lineages, rather than the trivalent vaccine, may be more suitable. aside from a limited quantity of oseltamivir, stockpiled by the government for management of severe disease by pandemic or avian influenza [ ] , use of antivirals for influenza is very low in cambodia [ ] . as a result, all strains we detected were susceptible to neuraminidase inhibitors (nis) such as oseltamivir and zanamivir, which can be partially explained by the absence of these medications in cambodia. due to widespread adamantane resistance, we did not conduct susceptibility testing on this class of antiviral drugs; however, we did find the s n mutation conferring adamantane resistance in all of our influenza isolates tested. phylogenetically, all our samples clustered together within each of the two subtypes (a/ ph n or a/h n ), which is not unusual given the location and specimen collection dates compared with the reference sequences used. our western cambodian ph n ( ) isolates were more closely related, based on full genome analysis, to earlier isolates from cambodia ( . % genome conservation) than to the thai isolates ( . % genome conservation) or the california -vaccine reference. however, it was also noted that amino acid changes were shared with the thai references suggesting a possibility of mixing between thai and cambodian influenza as expected. isolates from battambang, being western cambodia's major transportation hub, showed the highest diversity of amino acid changes. analysis of sequence data from our influenza patients revealed a random or neutral mutation of the influenza genomes as expected. koel et al. [ ] discovered that periodic major antigenic change in influenza a/h n virus was caused mainly by single amino acid substitutions, which occurred at only seven out of possible amino acid positions in ha at antigenic sites immediately adjacent to the receptor binding site (rbs). these substitutions were located in antigenic sites a (position ) and b (positions , , , , , and ) , with none in sites c, d, or e. we did not see these changes within antigenic sites a or b in our dataset. the ha and na genes in our h and h isolates showed signatures of purifying selection meaning that the virus keeps these genes conserved by removing random mutations as they occur. these single amino acid mutations were found at non-epitope sites of ha [ ] that have not been associated with major antigenic changes on their own. however, it is possible that some of these mutations may constitute permissive or compensatory mutations that would be important in enabling co-mutations that could affect viral fitness, and as such, have an incentive to remain fixed under purifying selection. permissive mutations have been described in the spread of oseltamivir resistant influenza a/h n virus that carry the h y mutation and are increasingly being recognized as a major force in evolution [ , ] . glycosylation (addition of oligosaccharide chain to the surface protein) is another common form of protein modification. alteration of glycosylation sites can affect folding and conformation changes in the surface glycoprotein, hereby impacting virus survival and transmissibility. in addition, glycosylation can affect interaction with receptors and cause a virus to be more [or less] recognizable by the innate host immune system and antibodies [ ] . none of the changes in glycosylation in our isolates have been reported in the literature as having an effect on viral structure or function; but more data may be needed. mapping sites of mutation and glycosylation on epitopes provides a better understanding of antigenic drift and is important for improving vaccine strategies [ ] . overall, and despite adequate storage and transportation of our specimens, culture yield was low in our dataset (~ %) with isolation of only adenovirus and parainfluenza. this may be due to an exclusion of children younger than year, the outpatient setting with less severe disease and possibly a later presentation to the health facility (which was possible up to days after fever onset) resulting in a lower viremia. previous work among hospitalized patients has shown presence of human coronavirus, human bocavirus, hmpv and rsv in cambodia [ ] but these were not detected by our culture techniques. therefore, the number of non-influenza viruses in our population is possibly underestimated. additional testing is ongoing at afrims in order to elucidate true disease burden of non-influenza viruses on the population. in comparison with previous work in cambodia [ ] , we found a relatively higher proportion ( . % versus . %) of ili-patients testing positive for adenovirus over dry seasons in and which may be explained by our younger study population who may be disproportionately affected by this virus. piv- ( . %), which in previous dry seasons was described as the most common parainfluenza-virus in cambodia [ ] , was the least common type during our surveillance period, whereas piv- ( . %) was most common, particularly during november to march . this is not consistent with previous reports for cambodia [ ] . the lack of presence of piv- in our study population may be due to an outbreak of piv- during the surveillance time frame and the fact that we did not include children under year old, an age when % of piv- is usually detected [ ] . by employing a highly sensitive and specific nested rt-pcr assay for enteroviruses and rhinoviruses, we found a higher proportion ( . %) of non-polio enteroviruses in children less than years old compared with previous studies ( %) in cambodia [ ] which could be partially explained by the predominantly outpatient population in our study sample. this is likely an underestimation of the true prevalence in this population since we excluded children less than one year of age, an age group more likely infected by enteroviruses at rates exceeding those of older children and adults by several fold [ ] [ ] [ ] . the most common enteroviruses found among children under years old in our population were coxsackieviruses ( . %) and echoviruses ( %). although we did not find coxsackievirus a , the most common etiologic agent for hand-foot-mouth-disease (hfmd), we found other coxsackieviruses that can cause hfmd such as coxsackievirus a (n = ) and a (n = ). coxsackievirus a is known to cause either an atypical rash resembling eczema herpeticum or chickenpox (united states - and in europe since ) or nail loss one to two months after onset of symptoms (finland , taiwan , japan ). coxsackievirus a was one of the enteroviruses implicated in hfmd outbreaks in china in and [ ] . while these patients reported symptoms of fever ( . - °c), runny nose, chills, cough, congestion and abdominal pain, they did not report symptoms specific to hfmd such as herpangina and skin rash. we did not detect in our study ev virus genotype c , which was the principal etiologic agent causing the hfmd outbreak across southeast-asia that manifested as a life-threatening neuro-respiratory syndrome in children under years old from provinces in cambodia [ ] . although we did not have ev test results from specimens collected during the peak of the outbreak, our data suggests that ev virus was not present yet in the border areas of western cambodia before april , the time that the first cases were found elsewhere in cambodia. regarding testing for non-influenza viruses, the use of the less sensitive viral culture as the first-line test supplemented by ifa on only a subset of influenza-negative ili-specimens underestimates overall extent of transmission as well as co-infections. exclusion of children under year of age may also contribute to underestimation. our data was derived from a passive surveillance system with convenience sampling. while we attempted to select surveillance sites representative of the nearby geographic locations, we had no accurate figures of the catchment populations or denominators at our respective sentinel sites. our staggered method of adding sentinel sites suggests that not all sentinel sites enrolled patients evenly over influenza seasons. similarly, with a small sample size and limited number of sampling locations, no inferences could be made with regard to temporal or spatial flow of influenza strains in our phylogenetic analysis. now with a more established system in place, our data may provide a useful baseline for future molecular evolution studies of influenza in cambodia and in the region. despite proximity to thailand, influenza activity, seasonality, antigenicity and anti-viral susceptibility in western cambodian isolates followed patterns observed elsewhere in cambodia rather than thailand. this supports earlier recommendations from the cambodian nic to use the northern hemisphere influenza vaccine on a southern hemisphere vaccination schedule. additionally, use of the quadrivalent versus the trivalent vaccine should improve coverage of influenza b strains circulating in cambodia. neuraminidase inhibitors may still be used for treatment and chemoprophylaxis for seasonal influenza given little evidence for resistance. the amino acid mutations at non-epitope sites, in particular of ha, require further investigation in light of the increasingly important role of permissive mutations in the evolution of influenza virus. further research to clarify the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in cambodia is needed. table. samples collected for genetic analysis in this study including virus subtype, sample origin, specimen type, sampling date and segments successfully sequenced. (docx) s table. primers for rt-pcr and nucleotide sequencing for ev/rv and ev : the enterovirus type specific primers were modified from the previous study by [ ] . the modifications were made by following the alignments of vp sequences of different ev strains collected during - from thailand, taiwan, philippines, vietnam, and china available in genbank including the sequences with the accession no. jn - , fj , fj , jq , jq , am - , jq , and jx . the primers for simultaneous detection of enteroviruses and rhinoviruses were designed in the polyprotein gene, between non-coding region ( ncr) and vp /vp regions that was previously described by coiras et al. [ ] . ev/rv- n was modified from primer -ev/rv [ ] for using in nested pcr reaction. (docx) s table. total number of viruses identified by pan-enteroviruses and rhinoviruses (ev/ rv) pcr and nucleotide sequencing † (docx) s table. ph n average percent nucleotide sequence identity between vaccine strains and cambodia isolates gene segments. (docx) s table. changes in glycosylation, antigenic and polymorphic sites linked to aa substitutions and sub-antigenic/catalytic sites (epitopes) where amino acid (aa) substitutions were found for for the ha and na segments of ph n . aa substitution nomenclature is as follows; reference amino acid (a/california/ / ), amino acid site, sample amino acid. amino acids are numbered from the start codon of the segment (atg:methionine). (docx) s table. unique ph n amino acid (aa) changes of unknown function in specific samples for the na gene as compared to a/california/ / . aa substitution nomenclature is as follows; reference amino acid (a/california/ / ), amino acid site, sample amino acid. amino acids are numbered from the start codon of the segment (atg:methionine). (docx) s table. ph n sample amino acid substitution summary and selection analysis by group per segment analyzed. (docx) s table. h n average percent nucleotide sequence identity between vaccine strains and cambodia isolates gene segments. (docx) s table. h n sample amino acid substitution summary and selection analysis by group per segment analyzed. (docx) s table. unique h n amino acid changes of unknown function to specific to samples for the ha gene as compared to a/victoria/ / . aa substitution nomenclature is as follows; reference amino acid (a/victoria/ / ), amino acid site, sample amino acid. amino acids are numbered from the start codon of the segment (atg:methionine). (docx) s table. unique h n amino acid changes of unknown function to specific to samples for the na gene as compared to a/victoria/ / . aa substitution nomenclature is as follows; reference amino acid (a/victoria/ / ), amino acid site, sample amino acid. amino acids are numbered from the start codon of the segment (atg:methionine). 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ministry of health avian influenza-situation in cambodia-update influenza vaccination guidelines and vaccine sales in southeast asia responding to pandemic influenza in cambodia and lao pdr: challenges in moving from strategy to operation substitutions near the receptor binding site determine major antigenic change during influenza virus evolution estimating the fitness advantage conferred by permissive neuraminidase mutations in recent oseltamivir-resistant a(h n )pdm influenza viruses permissive secondary mutations enable the evolution of influenza oseltamivir resistance virus glycosylation: role in virulence and immune interactions predicting the evolution of human influenza a use of a multiplex pcr/rt-pcr approach to assess the viral causes of influenza-like illnesses in cambodia during three consecutive dry seasons parainfluenza viruses a continuing surveillance of enterovirus infections in healthy children in six united states cities. i. viruses isolated during and coxsackievirus b infection and aseptic meningitis in neonates and children an epidemic of echovirus meningitis aedes hensilli as a potential vector of chikungunya and zika viruses complete sequence analyses of enterovirus strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in we gratefully thank all the volunteers for their study participation and study site and provincial health department staff in battambang, oddar meanchey, banteay meanchey and pailin for collecting the data and specimens. we would like to acknowledge the thai-cambodian afrims field team for their dedication and commitment: mr key: cord- -ywaefpe authors: rodon, jordi; muñoz-basagoiti, jordana; perez-zsolt, daniel; noguera-julian, marc; paredes, roger; mateu, lourdes; quiñones, carles; erkizia, itziar; blanco, ignacio; valencia, alfonso; guallar, víctor; carrillo, jorge; blanco, julià; segalés, joaquim; clotet, bonaventura; vergara-alert, júlia; izquierdo-useros, nuria title: pre-clinical search of sars-cov- inhibitors and their combinations in approved drugs to tackle covid- pandemic date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: ywaefpe there is an urgent need to identify novel drugs against the new coronavirus. although different antivirals are given for the clinical management of sars-cov- infection, their efficacy is still under evaluation. here, we have screened existing drugs approved for human use in a variety of diseases, to compare how they counteract sars-cov- -induced cytopathic effect and viral replication in vitro. among the potential antivirals tested herein that were previously proposed to inhibit sars-cov- infection, only % had in vitro antiviral activity. moreover, only eight families had an ic below µm or iu/ml. these include chloroquine derivatives and remdesivir, along with plitidepsin, cathepsin inhibitors, nelfinavir mesylate hydrate, interferon -alpha, interferon-gamma, fenofibrate and camostat. plitidepsin was the only clinically approved drug displaying nanomolar efficacy. four of these families, including novel cathepsin inhibitors, blocked viral entry in a cell-type specific manner. since the most effective antivirals usually combine therapies that tackle the virus at different steps of infection, we also assessed several drug combinations. although no particular synergy was found, inhibitory combinations did not reduce their antiviral activity. thus, these combinations could decrease the potential emergence of resistant viruses. antivirals prioritized herein identify novel compounds and their mode of action, while independently replicating the activity of a reduced proportion of drugs which are mostly approved for clinical use. combinations of these drugs should be tested in animal models to inform the design of fast track clinical trials. there is an urgent need to identify novel drugs against the new coronavirus. although different antivirals are given for the clinical management of sars-cov- infection, their efficacy is still under evaluation. here, we have screened existing drugs approved for human use in a variety of diseases, to compare how they counteract sars-cov- -induced cytopathic effect and viral replication in vitro. among the potential antivirals tested herein that were previously proposed to inhibit sars-cov- infection, only % had in vitro antiviral activity. moreover, only eight families had an ic below µm or iu/ml. these include chloroquine derivatives and remdesivir, along with plitidepsin, cathepsin inhibitors, nelfinavir mesylate hydrate, interferon -alpha, interferon-gamma, fenofibrate and camostat. plitidepsin was the only clinically approved drug displaying nanomolar efficacy. four of these families, including novel cathepsin inhibitors, blocked viral entry in a cell-type specific manner. since the most effective antivirals usually combine therapies that tackle the virus at different steps of infection, we also assessed several drug combinations. although no particular synergy was found, inhibitory combinations did not reduce their antiviral activity. thus, these combinations could decrease the potential emergence of resistant viruses. antivirals prioritized herein identify novel compounds and their mode of action, while independently replicating the activity of a reduced proportion of drugs which are mostly approved for clinical use. combinations of these drugs should be tested in animal models to inform the design of fast track clinical trials. a novel betacoronavirus, the severe acute respiratory syndrome coronavirus (sars-cov- ), is causing a respiratory disease pandemic that began in wuhan, china, in november , and has now spread across the world (chen et al., ) . to date, remdesivir is the only approved antiviral drug for the specific treatment of this coronavirus infectious disease or covid- (beigel et al., ; grein et al., ) . however, several drugs are being used in the frontline of clinical management of sars-cov- -infected individuals in hospitals all around the world, to try to avoid the development of the covid- associated pneumonia, which can be fatal. by the end of september , almost a million people had died from covid- , and over million people have been infected (who situation report). although different drug regimens are being applied to hospitalized patients, no clinical study has evidenced their efficacy yet. under this scenario, initiatives launched by the world health organization (who), such as the solidarity study that has compared remdesivir, hydroxychloroquine, ritonavir/lopinavir and ritonavir/lopinavir plus ßinterferon regimes, have been of critical importance to prioritize the use of the most active compounds (who, ) . unfortunately, although remdesivir has proven efficacy in randomized controlled trials (beigel et al., ; grein et al., ) , a recent update of the who clinical trial has failed to detect any effect on overall mortality, initiation of ventilation and duration of hospital stay with any of the antivirals tested (pan et al., ) . thus, there is an urgent need to identify novel therapeutic approaches for individuals with covid- developing severe disease and fatal outcomes. in this report we present a prioritized list of effective compounds with proven antiviral efficacy in vitro to halt sars-cov- replication. compounds were analyzed depending on their expected mechanism of action, to identify candidates tackling diverse steps of the viral life cycle. sars-cov- entry requires viral binding and spike protein activation via interaction with the cellular receptor ace and the cellular protease tmprss (hoffmann et al., ) , a mechanism favored by viral internalization via endocytosis. interference with either of these processes has proven to decrease sars-cov- infectivity (hoffmann et al., ; monteil et al. ) , and therefore, inhibitors targeting viral entry may prove valuable. in addition, sars-cov- enters into the cells via endocytosis and accumulates in endosomes where cellular cathepsins can also prime the spike protein and favor viral fusion upon cleavage (hoffmann et al., ; mingo et al., ; simmons et al., ) , providing additional targets for antiviral activity. once sars-cov- fuses with cellular membranes, it triggers viral rna release into the cytoplasm, where polyproteins are translated and cleaved by proteases (song et al., ) . this leads to the formation of an rna replicase-transcriptase complex driving the production of negative-stranded rna via both replication and transcription (song et al., ) . negative-stranded rna transcribes into positive rna genomes, allowing for the translation of viral nucleoproteins, which assemble in viral capsids at the cytoplasm (song et al., ) . these capsids then bud into the lumen of endoplasmic reticulum (er)-golgi compartments, where viruses are finally released into the extracellular space by exocytosis. potentially, any of these viral cycle steps could be targeted with antivirals, so we have thus searched for these compounds as well. finally, as the most effective antiviral treatments are usually based on combined therapies that tackle distinct steps of the viral life cycle, we also tested the active compounds in combination. these combinations may be critical to abrogate the potential emergence of resistant viruses and to increase antiviral activity, enhancing the chances to improve clinical outcome. we have tested the antiviral activity of different clinically available compounds and their combinations by assessing their ability to inhibit viral induced cytopathic effect in vitro. our strategy was circumscribed mostly to compounds approved for clinical use, since they are ideal candidates for entering into fast track clinical trials. drug selection criteria first focused on compounds already being tested in clinical trials, along with well-known human immunodeficiency virus- (hiv- ) and hepatitis c virus (hcv) protease inhibitors, as well as other compounds suggested to have potential activity against sars-cov- in molecular docking analysis or in vitro assays. we first assessed the activity of compounds with hypothetical capacity to inhibit viral entry, and then we focused on drugs thought to block viral replication upon sars-cov- fusion. molecular docking studies provided an additional candidates, which were predicted to inhibit the sars-cov- main protease. finally, compounds with unknown mechanism of action were also assessed. by these means, we have compared drugs and of their combinations for their capacity to counteract sars-cov- induced cytopathic effect in vitro. we first tested compounds that could have an effect before viral entry by impairing viruscell fusion (supp . table ) . hydroxychloroquine is an anti-malarial drug that exerts its activity by disrupting the endosome pathway, and that has been proposed as an anti-sars-cov- agent wang et al., ) . we confirmed the inhibitory effect of hydroxychloroquine against sars-cov- -induced cellular cytotoxicity on vero e cells . a constant concentration of a clinical isolate of sars-cov- (id epi_isl_ ) was mixed with increasing concentrations of hydroxychloroquine and added to vero e cells. to control for drug-induced cytotoxicity, vero e were also cultured with increasing concentrations of hydroxychloroquine in the absence of sars-cov- . by these means we calculated the concentration at which hydroxychloroquine achieved a % maximal inhibitory capacity (ic ). as shown in fig. a , this drug was able to inhibit viral-induced cytopathic effects at concentrations where no cytotoxic effects of the drug were observed. the mean ic value of this drug in repeated experiments was always below µm (supp . table ) . these results aligned with previous reports highlighting the in vitro inhibitory capacity of chloroquine derivatives wang et al., ) , but contrasted with data on animal models (maisonnasse et al., ; rosenke et al., ) or currently ongoing clinical trials that have failed to detect any associated benefit of hydroxychloroquine treatment (boulware et al., ; cavalcanti et al., ) . since hydroxychloroquine was first administered in combination with the antibiotic azithromycin (gautret et al., ) , which induces antiviral responses in bronchial epithelial cells (gielen et al., ) , we further tested the activity of this compound in our assay. however, in the vero e model, azithromycin did not show any antiviral effect (fig. a) , and the combination of hydroxychloroquine with azithromycin had a similar activity to that of the chloroquine derivative alone (fig. a) . indeed, this was also the case when we tested hydroxychloroquine in combination with different hiv- protease inhibitors and other relevant compounds currently being tested in clinical trials (supp. table ). additional food and drug administration (fda)-approved compounds previously used to abrogate viral entry via clathrin-mediated endocytosis were also tested in this sars-cov- -induced cytotoxicity assay (supp . table ) . indeed, interference with clathrinmediated endocytosis is one of the potential mechanisms by which hydroxychloroquine may exert its therapeutic effect against sars-cov- . one of these compounds was amantadine, which blocks coated pit invaginations at the plasma membrane (phonphok and rosenthal, ) and is licensed against influenza a virus infections and as a treatment for parkinson's disease. in addition, we also tested chlorpromazine, an antipsychotic drug that inhibits clathrin-mediated endocytosis by preventing the assembly and disassembly of clathrin networks on cellular membranes or endosomes (wang et al., ) . when we assessed the antiviral efficacy of these clathrin inhibitors against sars-cov- , we did not find any prominent effect; only a partial inhibition at µm for amantadine (fig. b) . the broad cathepsin b/l inhibitor e -d also showed partial inhibitory activity (fig. b) . e -d exerts activity against viruses cleaved by cellular cathepsins upon endosomal internalization, as previously described using pseudotyped sars-cov- (hoffmann et al., ) . while these results could not be confirmed using the specific cathepsin b inhibitor ca- -me due to drug-associated toxicity (supp . table ) , it is important to highlight that none of these cathepsin inhibitors is approved for clinical use. these data suggest that sars-cov- entry in vero e partially relies on clathrin-mediated endocytosis and cellular cathepsins, which cleave the viral spike protein and allow viral fusion once sars-cov- is internalized in endosomes. however, hydroxychloroquine antiviral activity was much more potent than that exerted by amantadine or e- d ( fig. a-b) . recently, it has also been suggested that hydroxychloroquine could block sars-cov- spike interaction with gm gangliosides (fantini et al., ) . gm gangliosides are enriched in cholesterol domains of the plasma membrane and have been previously shown to bind to sars-cov spike protein (lu et al., ) . this mode of viral interaction is aligned with the capacity of methyl-beta cyclodextrin, which depletes cholesterol from the plasma membrane to abrogate sars-cov- induced cytopathic effect (fig. b) , as previously reported for sars-cov (lu et al., ) . removal of cholesterol redirected ace receptor to other domains, but did not alter the expression of the viral receptor (lu et al., ) . moreover, nb-dnj, an inhibitor of ganglioside biosynthesis pathway, also decreased sars-cov- cytopathic effect (fig. b) . these results highlight the possible role of gangliosides in viral binding, although the polar head group of gm ganglioside ( ' sialyllactose) was not able to reduce viral-induced cytopathic effect (supp. table ). agents involved in autophagy, such as the becn -stabilizing compounds niclosamide or ciclesonide, inhibit the release of infectious sars-cov- to the supernatant (gassen et al., ) or reduce the expression of viral nucleoprotein h post-infection (jeon et al., ) . however, these autophagy inhibitors were highly toxic in our three-day assay (supp . table ). arbidol is a compound that intercalates into membrane lipids leading to the inhibition of membrane fusion between viruses and cells, and between viruses and endosomal membranes (haviernik et al., ) . although arbidol has exhibited in vitro efficacy against sars-cov- (li, ) it showed drug associated cytotoxicity in our assay (supp. table ). we also tested the antiviral activity of two jak inhibitors: baricitinib and tofacitinib. baricitinib was previously suggested to reduce viral entry by interfering with ap -associated protein kinase (aak ) necessary for clathrin mediated endocytosis stebbing et al., ) . however, neither this compound or tofacitinib protected vero e cells from sars-cov- induced cytopathic effect (supp. table ). while we did not detect an antiviral effect for jak inhibitors, these compounds may still be useful to control hyperinflammation and cytokine storm at later stages of infection ). finally, we tested camostat, a serine protease inhibitor with capacity to abrogate sars-cov- spike priming on the plasma membrane of human pulmonary cells and avoid viral fusion (hoffmann et al., ) . camostat showed no antiviral effect on vero e cells (fig. c) , what indicates that the alternative viral endocytic route is the most prominent entry route in this renal cell type. of note, a broader cellular protease inhibitor such as the alfa -antitrypsin (att), used to treat severe att human deficiency, was able to exert an antiviral effect on vero e . however, it required high concentrations that will most likely rely on the activity of these proteases in the endosomal route (fig. c) . in order to confirm that previously identified compounds listed in supp. table specifically inhibit the viral entry step, we next employed a luciferase-based assay using pseudotyped lentivirus expressing the spike protein of sars-cov- , which allows to detect viral fusion on hek- t cells transfected with ace . as a control, we used the same lentiviruses pseudotyped with a vsv glycoprotein, where no entry inhibition above % was detected for any of the drugs tested (data not shown). in sharp contrast, sars-cov- pseudoviruses were effectively blocked by most of the drugs previously tested on vero e with wild-type virus (fig. d) . the main differences were observed with ca- -me, ciclesonide and arbidol. these compounds showed a partial blocking effect on ace hek- t cells that was not obvious when using replication competent sars-cov- on vero e (supp. fig. ). in addition, nb-dnj failed to block viral entry ( fig. d ), suggesting that ganglioside dependence may be reduced in ace overexpressing cells or, alternatively, that this drug requires a longer exposure time to effectively reduce the content of gangliosides via biosynthesis blocking. overall, using alternative sars-cov- viral systems, we could identify chloroquine derivatives, cathepsin inhibitors and cholesterol depleting agents as the most promising candidates to block sars-cov- endocytosis in vero e and hek- t cells transfected with ace . however, chloroquine derivatives were the only ones that displayed an ic below µm (supp . table ) , and were also active abrogating pseudoviral entry into hek- t cells expressing ace (fig. e) . although camostat failed to inhibit viral fusion on ace hek- t cells (fig. e) , its activity was rescued when these cells were transfected with tmprss . the opposite effect was observed for chloroquine, which reduced its inhibitory activity on tmprss transfected cells (fig. e ). thus, the expression of cellular proteases on the plasma membrane facilitates the fusion with viral membranes, decreasing the likelihood of viral entry through the endosomal route. these data concur with previous findings in pulmonary cells, where viral entry via endosomal route was not active since chloroquine failed to abrogate viral fusion (maisonnasse et al., ) ; however, camostat effectively blocked this entry (hoffmann et al., ) . our results highlight that alternative routes govern sars-cov- viral entry and these pathways vary depending on the cellular target. thus, effective treatments may need to block both plasma membrane fusion and endosomal routes to fully achieve viral suppression. in our search for antivirals inhibiting post-viral entry steps, we first focused on remdesivir, which has in vitro activity against sars-cov- after viral entry and has already been approved for the treatment of covid- by the fda and ema. we further confirmed the in vitro capacity of remdesivir to inhibit sars-cov- induced cytopathic effect on vero e ( fig. a) . the mean ic value of this drug in repeated experiments was always below µm (supp. table ). in combination with hydroxychloroquine, however, remdesivir did not significantly modified its own antiviral effect ( fig. b) , either when hydroxychloroquine was added at increasing concentrations or at different fixed concentrations of the drug. this was also the case for other antivirals tested in combination (supp. table ). of note, other rna polymerase inhibitors such as galdesivir, which was proposed to tightly bind to sars-cov- rna-dependent rna polymerase (elfiky, ) , showed no antiviral effect (supp . table ). favipiravir, approved by the national medical products administration of china as the first anti-covid- drug in china (tu et al., ) , showed only partial inhibitory activity at the non-toxic concentration of µm (supp . table ) . we also assessed clinically approved protease inhibitors with potent activity against hiv- . however, none of the hiv- protease inhibitors detailed in supp. table showed remarkable protective antiviral activity against sars-cov- infection on vero e cells, with the exception of nelfinavir mesylate hydrate, which showed an ic value below µm (supp . table and fig. c ). lopinavir and tipranavir inhibited sars-cov- induced cytopathic effect at the non-toxic concentration of µm, and amprenavir exhibited activity at the non-toxic concentration of µm (fig. c) . darunavir, which is currently being tested in ongoing clinical trials, showed partial inhibitory activity at µm, although this concentration had . ± . % of cytotoxicity associated (fig. c) . of note, we tested hiv- reverse transcriptase inhibitors such as tenofovir disoproxil fumarate, emtricitabin, tenofovir alafenamide, and their combinations, but they also failed to show any antiviral effect against sars-cov- (supp. fig. ) . these results indicate that future clinical trials should contemplate the limited antiviral effect displayed by these anti-hiv- inhibitors against sars-cov- in vitro. we also assessed the inhibitory capacity of hcv protease inhibitors, but none showed any antiviral activity (supp. table ). of note, exogenous interferons alpha and gamma displayed antiviral activity against sars-cov- (supp. table ). in light of these results, we tested the inhibitory effect of the tlr agonist vesatolimod that triggers interferon production. although this agonist was not able to protect from the viralinduced cytopathic effect on vero e (supp . table ) , as expected since it is an interferon-producer deficient cell line (emeny and morgan, ) , it could still be useful in other competent cellular targets. since severe covid- patients display impaired interferon responses (hadjadj et al., ) , these strategies may be valuable to avoid disease complication. in addition, we also assessed several compounds with the best computational docking scores among approved drugs against the cl protease of sars-cov- , but none of them were effective to protect vero e from viral induced cytopathic effect (supp. table ) . the most potent antiviral tested was plitidepsin (fig. d) , which targets the eukaryotic elongation factor a (eef a ) and has been previously used for the treatment of multiple myeloma. the mean ic value of this drug in repeated experiments was always in nm concentrations (supp . table ). in combination with other active antivirals, we did not observe a reduction on ic values (supp. table ). this result indicates no significant synergy, but also highlights the possibility of using plitidepsin without reducing its antiviral activity in combined therapies (fig. d) , what could be relevant to avoid possible selection of resistant viruses. overall, plitidepsin showed the lowest ic values of all the compounds tested in this in vitro screening ( table ) . we also assessed the inhibitory capacity of several inhibitors and broad anti-bacterial, anti-parasitic, anti-malarial, anti-influenza and anti-fungal compounds, along with other pharmacological agents previously suggested to interfere with sars-cov- infection (supp . table ). such was the case of ivermectin, an fda-approved broad spectrum anti-parasitic agent previously reported to inhibit the replication of sars-cov- in vitro as measured by rna accumulation (caly et al., ) . however, among these potential antivirals, only three types of molecules exerted detectable antiviral activity in our assay: itraconazole, fenofibrate, and calpain and cathepsin inhibitors such as mdl and npo compounds. itraconazole, an antifungal that may interfere with internal sars-cov- budding within infected cells (wu et al., ) , displayed an ic value of µm ( fig. a and supp. table ). fenofibrate is clinically used to treat dyslipidemia via activation of ppara, and also inhibited the cytopathic effect exerted by sars-cov- on vero e at µm ( fig. b and supp. table ). as fenofibrate is a regulator of cellular lipid metabolism, we made use of the luciferase-based viral entry assay to try to elucidate its mode of action. when lentiviruses pseudotyped with the spike protein of sars-cov- were added to ace- expressing hek- t cells in the presence of fenofibrate, viral entry was abrogated (fig. c) . the most potent agent found was mdl , a calpain iii inhibitor in a pre-clinical stage of development that displayed activity in the nanomolar range ( fig. d and supp. (riva et al., ) . moreover, three out of four different calpain and cathepsin inhibitors named npo showed potent antiviral activity too (supp. figure ) . of note, in combination with other active antivirals, we did not observe a reduction on ic values of mdl (supp. table ) . inhibitors of calpains, which are cysteine proteases, might impair the activity of viral proteases like cl (main protease) and plpro (papain-like protease) (riva et al., ; schneider et al., ) . however, calpain inhibitors may also inhibit cathepsin bmediated processing of viral spike proteins or glycoproteins, including sars-cov and ebola (schneider et al., ; zhou and simmons, ) . to understand the mechanisms of action of calpain and cathepsin inhibitors such as mdl , we added lentiviruses pseudotyped with the spike protein of sars-cov- to ace- -expressing hek- t cells and the same cells also expressing tmprss in the presence of this drug. importantly, mdl only blocked viral entry in ace- -expressing cells (fig. e) . this result indicates that mdl blocks cathepsins that are implicated in sars-cov- entry via the alternative endosomal pathway, as described for chloroquine derivatives and e- d (fig. e) , which are all active when tmprss is not present and their inhibitor camostat displays no activity (fig. e) . in conclusion, among the compounds and their combinations tested herein for their potential capacity to abrogate sars-cov- cytopathic effect, we only found compounds with antiviral activity, and only eight types of these drugs had an ic below µm or iu/ml ( table ) . these eight families of compounds were able to abrogate sars-cov- release to the supernatant in a dose dependent manner (fig. ) , indicating that the reduction in the cytopathic effect that we had measured in cells correlates with viral production. as these eight families of compounds tackle different steps of the viral life cycle, they could be tested in combined therapies to abrogate the potential emergence of resistant viruses. we have assessed the anti-sars-cov- activity of clinically approved compounds that may exert antiviral effect alone or in combination. although we were not able to detect any remarkable synergy in vitro, combined therapies are key to tackle viral infections and to reduce the appearance of viral resistance. we have tested more than seventy compounds and their combinations, and verified a potent antiviral effect of hydroxychloroquine and remdesivir, along with plitidepsin, cathepsin and calpain inhibitors mdl and npo, nelfinavir mesylate hydrate, interferon a, interferon-g and fenofibrate. these are therefore the most promising agents found herein that were able to protect cells from viral-induced cytopathic effect by preventing viral replication. our findings highlight the utility of using hydroxychloroquine and mdl or other cathepsin inhibitors to block viral entry via the endosomal pathway in kidney cell lines such as vero e or hek- t. however, the endosomal viral entry route is absent in pulmonary cells and, therefore, camostat should be considered as the primary inhibitor to limit sars-cov- entry in pulmonary tissues or in cells expressing tmprss . these findings can explain why randomized clinical trials using hydroxychloroquine have failed to show a significant protective effect (boulware et al., ; cavalcanti et al., ) . nonetheless, in combined therapies, it should be noted that agents targeting the alternative endosomal sars-cov- entry route such as hydroxychloroquine or mdl could be key to stop viral dissemination in other extrapulmonary tissues where viral replication has been already detected , and viral entry could take place through this endosomal pathway. this could partially explain why in a retrospective observational study including more than patients, hydroxychloroquine treatment showed a significant reduction of in-hospital mortality (arshad et al., ) . thus, since alternative routes govern sars-cov- viral entry depending on the cellular target (ou et al., a) , effective treatments might be needed to block both plasma membrane fusion and endosomal entry to broadly achieve viral suppression. sars-cov- replication could be effectively blocked using nelfinavir mesylate hydrate, remdesivir and plitidepsin. while nelfinavir showed lower potency, remdesivir and plitidepsin were the most potent agents identified. however, remdesivir and plitidepsin are not yet suitable for oral delivery and require intravenous injection, complicating their clinical use for prophylaxis. finally, we also confirmed the antiviral effect of type i and ii interferons as well as fenofibrate, which have been extensively used in the clinic for many years and may therefore prove valuable for therapeutic use. the data presented herein should be interpreted with caution, as the ic values of drugs obtained in vitro may not reflect what could happen in vivo upon sars-cov- infection. the best antiviral compounds found in the present study need to be tested in adequate animal models. this strategy already helped to confirm the activity of remdesivir against sars-cov- , while also questioning the use of hydroxychloroquine in monotherapy (maisonnasse et al., ) . thus, assessing antiviral activity and safety in animal models is key to identify and advance those compounds with the highest potential to succeed in upcoming clinical trials. in turn, in vitro results confirmed in animal models will provide a rational basis to perform future clinical trials not only for treatment of sars-cov- -infected individuals, but also for pre-exposure prophylaxis strategies that could avoid novel infections. prophylaxis could be envisioned at a population level or to protect the most vulnerable groups, and should be implemented until an effective vaccine is developed. in particular, orally available compounds with proven safety profiles, such as fenofibrate, could represent promising agents. germans trias i pujol (hugtip) approved this study. the individual who provided the sample to isolate virus gave a written informed consent to participate. eagle medium, (dmem; lonza) supplemented with % fetal calf serum (fcs; euroclone), u/ml penicillin, µg/ml streptomycin, and mm glutamine (all thermofisher scientific). hek- t (atcc repository) were maintained in dmem with % fetal bovine serum, iu/ml penicillin and µg/ml streptomycin (all from invitrogen). hek- t overexpressing the human ace were kindly provided by integral molecular company and maintained in dmem (invitrogen) with % fetal bovine serum, iu/ml penicillin and µg/ml streptomycin, and µg/ml of puromycin (all from invitrogen). tmprss human plasmid (origene) was transfected using x-tremegene hp transfection reagent (merck) on hek- t overexpressing the human ace and maintained in the previously described media containing mg/ml of geneticin (invitrogen) to obtain tmprss /ace hek- t cells. virus isolation, titration and sequencing. sars-cov- was isolated from a nasopharyngeal swab collected from an -year-old male patient giving informed consent and treated with betaferon and hydroxychloroquine for days before sample collection. the swab was collected in ml medium (deltaswab vicum) to reduce viscosity and stored at - ºc until use. vero e cells were cultured on a cell culture flask ( cm ) at . x cells overnight prior to inoculation with ml of the processed sample, for h at ºc and % co . afterwards, ml of % fcs-supplemented dmem were supplied and cells were incubated for h. supernatant was harvested, centrifuged at x g for min to remove cell debris and stored at - ºc. cells were assessed daily for cytopathic effect and the supernatant was subjected to viral rna extraction and specific rt-qpcr using the sars-cov- upe, rdrp and n assays (corman et al., ) . the virus was propagated for two passages and a virus stock was prepared collecting the supernatant from vero e . viral rna was extracted directly from the virus stock using the indimag pathogen kit (indical biosciences) and transcribed to cdna using the primescript™ rt reagent kit (takara) using oligo-dt and random hexamers, according to the manufacturer's protocol. dna library preparation was performed using swift amplicon sars-cov- panel (swift biosciences). sequencing ready libraries where then loaded onto illumina miseq platform and a bp paired-end sequencing kit. sequence reads were quality filtered and adapter primer sequences were trimmed using trimmomatic. amplification primer sequences were removed using cutadapt (martin, ) . sequencing reads were then mapped against coronavirus reference (nc_ . ) using bowtie tool (langmead, b. and salzberg, s, ) . consensus genomic sequence was called from the resulting alignment at a x x average coverage using samtools (li et al., ) . genomic sequence was deposited at gisaid repository (http://gisaid.org) with accession id epi_isl_ . pseudovirus production. hiv- reporter pseudoviruses expressing sars-cov- spike protein and luciferase were generated using two plasmids. pnl - .luc.r-.e-was obtained from the nih aids repository. sars-cov- .sctΔ was generated (geneart) from the full protein sequence of sars-cov- spike with a deletion of the last amino acids in c-terminal, human-codon optimized and inserted into pcdna . -topo (ou et al., b) . spike plasmid was transfected with x-tremegene hp transfection reagent µm to . nm at ⅕ serial dilutions. plitidepsin was also assayed at concentrations ranging from µm to . nm at / dilutions. interferons were assayed at concentrations ranging from to . iu/ml at ⅕ serial dilutions. when two drugs were combined, each one was added at a : molar ratio at concentrations ranging from µm to . nm at ⅕ serial dilutions. in combination with other drugs, plitidepsin was also assayed at concentrations ranging from µm to . nm at / dilutions. e cells together with . tcid /ml of sars-cov- , a concentration that achieves a % of cytopathic effect. untreated non-infected cells and untreated virus-infected cells were used as negative and positive controls of infection, respectively. to detect any drugassociated cytotoxic effect, vero e cells were equally cultured in the presence of increasing drug concentrations, but in the absence of virus. cytopathic or cytotoxic effects of the virus or drugs were measured days after infection, using the celltiter-glo luminescent cell viability assay (promega). luminescence was measured in a fluoroskan ascent fl luminometer (thermofisher scientific). were adjusted to a non-linear fit regression model, calculated with a four-parameter logistic curve with variable slope. cells not exposed to the virus were used as negative controls of infection, and were set as % of viability to normalize data and calculate the percentage of cytopathic effect. statistical differences from % were assessed with a one sample t test. all analyses and figures were generated with the graphpad prism v . b software. in silico drug modeling. we performed glide docking using an in-house library of all approved drug molecules on the cl protease of sars-cov- . for this, two different receptors were used, the lu pdb structure, after removing the covalently bound inhibitor, and a combination of two crystals from the diamond collection (https://www.diamond.ac.uk/covid- ). receptors were prepared with the schrodinger's protein wizard and glide sp docking was performed with two different hydrogen bond constraints: glu and his (with epsilon protonation); we enforced single constraints and also attempted the combination of both. the best molecules, based on glides's docking score were selected. top docking scores, however, did not exceed - , indicating poor potential binding. pseudovirus assay. hek- t overexpressing the human ace and tmprss were used to test antivirals at the concentrations found to be effective for sars-cov- without toxicity, which were the following: µm for niclosamide; µm for chloroquine, chlorpromazine, ciclesonide, mdl and fenofibrate; µm for hydroxychloroquine, ca- -me and arbidol hcl; µm for e- d; µm for baricitinib; µm for amantadine, nb-dnj, ' sialyl-lactose na salt, tofacitinib, and camostat mesylate; µm for methyl-b-cyclodextrin, and , mg/ml for att. a constant pseudoviral titer was used to pulse cells in the presence of the drugs. h postinoculation, cells were lysed with the glo luciferase system (promega). luminescence was measured with an ensight multimode plate reader (perkin elmer). table . compounds with antiviral activity grouped in colors depending on their ic values, expressed in µm unless otherwise indicated. activity of hydroxychloroquine and azithromycin. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of hydroxychloroquine, azithromycin, and their combination. drugs were used at a concentration ranging from . nm to µm. when combined, each drug was added at the same concentration. non-linear fit to a variable response curve from one representative experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. the particular ic value of this graph is indicated. cytotoxic effect on vero e cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). b. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of amantadine, a clathrin-mediated endocytosis inhibitor, e- d, a pancathepsin inhibitor acting downstream once viruses are internalized in endosomes, nb-dnj, an inhibitor of ganglioside biosynthesis and methyl-b-cyclodextrin, a cholesteroldepleting agent. all drugs were used at a concentration ranging from . nm to µm aside from methyl-b-cyclodextrin, which was used times more concentrated. nonlinear fit to a variable response curve from one experiment with two replicates is shown (red lines). cytotoxic effect on vero e cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). c. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of camostat, a tmprss inhibitor, and att, an alfa- antyitrypsin, a broad cellular protease inhibitor, as described in a. d. effect of entry inhibitors on luciferase expression of reporter lentiviruses pseudotyped with sars-cov- spike in ace expressing hek- t cells. values are normalized to luciferase expression by mock-treated cells set at %. mean and s.e.m. from two experiments with two replicates. cells were exposed to fixed amounts of sars-cov- spike lentiviruses in the presence of a non-toxic constant concentration of the drugs tested on vero e . statistical deviations from % were assessed with a one sample t test. e. comparison of entry inhibitors blocking viral endocytosis, such as chloroquine, with inhibitors blocking serine protease tmprss expressed on the cellular membrane, such as camostat, on different cell lines. ace expressing hek- t cells transfected or not with tmprss were exposed to sars-cov- spike lentiviruses as described in b. values are normalized to luciferase expression by mock-treated cells set at %. mean and s.e.m. from at least two representative experiments with two replicates. statistical deviations from % were assessed with a one sample t test. a. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of remdesivir. drug was used at a concentration ranging from . nm to µm. non-linear fit to a variable response curve from one representative experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. the particular ic value of this graph is indicated. cytotoxic effect on vero e cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). b. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of remdesivir and its combination with hydroxychloroquine, as detailed in a. drugs in combination were used at a concentration ranging from . nm to µm (left panel). alternatively, remdesivir was used at a concentration ranging from . nm to µm at the fixed indicated concentrations of hydroxychloroquine (right panel). c. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of protease inhibitors against hiv- . nelfinavir mesylate hydrate was the only drug with activity. inhibitors were used at a concentration ranging from . nm to µm. the particular ic value of this graph is indicated d. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of plitidepsin and its combinations with hydroxychloroquine and remdesivir. when combined, each drug was added at the same concentration. drugs were used at a concentration ranging from . nm to µm. the particular ic value of these graphs is indicated. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of itraconazole. drug was used at a concentration ranging from . nm to µm . non-linear fit to a variable response curve from one representative experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. the particular ic value of this graph is indicated. cytotoxic effect on vero e cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). b. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence were assessed with a one sample t test. supp. table . antiviral activity of potential entry inhibitors tested against sars-cov- . na; not active. ic values are reported in µm unless otherwise indicated. supp. table . antiviral activity of potential inhibitors against sars-cov- tested in combination. na; not active. table . antiviral activity of potential post-entry inhibitors against sars-cov- . na; not active. ic values are reported in µm unless otherwise indicated. supp. table . antiviral activity of potential inhibitors against sars-cov- with predicted capacity to block sars-cov- viral protease. na; not active. supp. table . antiviral activity of potential inhibitors against sars-cov- with unknown mechanism of action. na; not active. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of hiv- reverse transcriptase inhibitors. drugs were used at a concentration ranging from . nm to µm. non-linear fit to a variable response curve from one experiment with two replicates is shown (red lines). cytotoxic effect on vero e cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of calpain and cathepsin inhibitors npo. drugs were used at a concentration ranging from . nm to µm. non-linear fit to a variable response curve from one experiment with two replicates is shown (red lines). cytotoxic effect on vero e cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). the research of cbig consortium (constituted by irta-cresa, bsc, & irsicaixa) is supported by grifols pharmaceutical. the authors also acknowledge the crowdfunding initiative #yomecorono (https://www.yomecorono.com). js, jva and niu have nonrestrictive funding from pharma mar to study the antiviral effect of plitidepsin. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. a patent application based on this work has been filed (ep . ). the authors declare that no other competing financial interests exist. treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with covid- remdesivir for the treatment of covid- -preliminary report a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- the fdaapproved drug ivermectin inhibits the replication of sars-cov- in vitro hydroxychloroquine with or without azithromycin in mild-to-moderate covid- epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study detection of novel coronavirus ( -ncov) by real-time rt-pcr ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study regulation of the interferon system: evidence that vero cells have a genetic defect in interferon production structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov- infection analysis of sars-cov- -controlled autophagy reveals spermidine hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial azithromycin induces anti-viral responses in bronchial epithelial cells compassionate use of remdesivir for patients with severe covid- impaired type i interferon activity and inflammatory responses in severe covid- patients histopathological findings and viral tropism in uk patients with severe fatal covid- : a post-mortem study arbidol (umifenovir): a broad-spectrum antiviral drug that inhibits medically important arthropod-borne flaviviruses the novel coronavirus ( -ncov) uses the sarscoronavirus receptor ace and the cellular protease tmprss for entry into target cells insights from nanomedicine into chloroquine efficacy against covid- identification of antiviral drug candidates against sars-cov- from fda-approved drugs (microbiology) fast gapped-read alignment with bowtie an exploratory randomized controlled study on the efficacy and safety of lopinavir/ritonavir or arbidol treating adult patients hospitalized with mild/moderate covid- (elacoi) the sequence alignment/map format and samtools hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro lipid rafts are involved in sars-cov entry into vero e cells hydroxychloroquine use against sars-cov- infection in non-human primates cutadapt removes adapter sequences from high-throughput sequencing reads ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to npc + endolysosomes is a rate-defining step inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace . hydroxychloroquine-mediated inhibition of sars-cov- entry is attenuated by tmprss (microbiology) characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov repurposed antiviral drugs for covid- -interim who solidarity trial results stabilization of clathrin coated vesicles by amantadine, tromantadine and other hydrophobic amines baricitinib as potential treatment for -ncov acute respiratory disease discovery of sars-cov- antiviral drugs through large-scale compound repurposing hydroxychloroquine proves ineffective in hamsters and macaques infected with sars-cov- (pharmacology and toxicology) severe acute respiratory syndrome coronavirus replication is severely impaired by mg due to proteasome-independent inhibition of m-calpain inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry from sars to mers, thrusting coronaviruses into the spotlight covid- : combining antiviral and anti-inflammatory treatments a review of sars-cov- and the ongoing clinical trials mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro solidarity" clinical trial for covid- treatments clinical benefit of remdesivir in rhesus macaques infected with sars-cov- (microbiology) analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods development of novel entry inhibitors targeting emerging viruses we are grateful to patients at the hospital germans trias i pujol that donated their samples for research. for his excellent assistance and advice, we thank jordi puig from fundació lluita contra la sida. we are most grateful to lidia ruiz and the clinical sample management team of irsicaixa for their outstanding sample processing and management, and to m. pilar armengol and the translational genomics platform team at key: cord- -qu mm r authors: xu, zhonglin title: sampling theory date: - - journal: fundamentals of air cleaning technology and its application in cleanrooms doi: . / - - - - _ sha: doc_id: cord_uid: qu mm r in order to ensure the reliability of the measurement results of particle concentration, except for the reasonable detecting method and the sophisticated detection equipment, the correct sampling principle to minimize the sampling errors must be followed, which requires that people who carry out the measurement should master the correct sampling theory. in order to ensure the reliability of the measurement results of particle concentration, except for the reasonable detecting method and the sophisticated detection equipment, the correct sampling principle to minimize the sampling errors must be followed, which requires that people who carry out the measurement should master the correct sampling theory. sampling is generally divided into the planktonic method (the airborne state of sampled particles remains the same) and the capture method (airborne particles are captured). in the capture method sampling system, there are a few instruments: the sampler (as the clamp of a filter material, it is preferably made of stainless steel with the commonly effective diameter mm, as shown in fig. . ), the sampling tube (usually plastic pipe), flowmeter (the float flowmeter is commonly used; the flow rate is - l/min), vacuum pump (vacuum degree ! mmhg; the flow rate ! l/min), and valve (the needle valve or other fine-tuned valve). in the planktonic method sampling system, it is mainly composed of the dust analyzer (such as a particle counter) and the sampling tube. when the sampling system is designed, there are two problems should be paid attention to. one is the orientation of the sampling probe, and the other is the position of the flowmeter. onto the inner wall of the tube or the mouth edge, while some cannot enter the sampler or the sampling tube, so that the resultant collected particle number is less than the actual value. figure . shows the curve about the effect of the inclination angle between the sampling mouth and the direction of the flow [ ] . when the angle is less than , for particles with diameter less than mm, the sampling error is less than %. figure . provides a curve which can be used to calculate this kind of error more accurately [ ] . one accurate method is to obtain the direction with the maximum dynamic pressure, i.e., the airflow direction, when the pitot tube is used. when sampling is performed outside, the filter material should be perpendicular to the ground to avoid the deposition of large particles or debris on the filter material. the essence of this problem is the influence of additional resistance on the flowmeter. the calibration of flowmeter is carried out in the environment with certain resistance (i.e., the additional resistance at the entrance of the flowmeter), pressure, and temperature, but in the actual application period, all these conditions changed, so it will certainly affect the original calibration value. in particular, the additional resistance caused by sampler, filter, and valve is often neglected, so it is necessary to emphasize this point. figure . shows the experimental system to investigate the influence of additional resistance caused by different installation positions of the flowmeter [ ] . for the completely same flowmeters and (after calibration), they are connected in series and k is placed in between to create resistance. its value is indicated by . flowmeter reflects the standard flow, and flowmeter reflects the flow under different resistances. when the vacuum pump is turned on, it can be found that the flow rates showed by the flowmeter and are different. the former is less than the latter. the larger the resistance is, the larger the difference is. if k and k are adjusted in the way that the value indicated by the flowmeter is fixed while the resistance of k varies, the variation of the values indicated by flowmeter is shown in fig. . . the main reason for this result is that air pressures inside the two flowmeters are different due to the additional resistance caused by spiral clamp k . when air enters from the bottom of the flowmeter , there is almost no additional resistance (flowmeter calibration is often carried out in this case). when air goes through k , the resultant air pressure in the flowmeter decreases. according to the principle for orifice flowmeter and float flowmeter, when the same gas under different conditions is measured with a flowmeter, the following relationship about flow rates can be obtained: where q and q are the indicated flow rates under two kinds of working conditions; ρ and ρ are gas densities under two kinds of working conditions. it is known that the gas density ρ is proportional to the pressure p and is inversely proportional to temperature t. the above expression can be written as where p and p are the pressures upstream of the flowmeters under two different working conditions (the air pressure in calibration condition is generally one atmospheric pressure or mmhg. the air pressure in measurement condition is the subtraction of the absolute value of the readings of the pressure gauge upstream of the flowmeter from the local atmospheric pressure b or the subtraction between b and the known additional total resistance before the flowmeter. only in the circumstance with the sampler, the total resistance becomes the resistance of the filter media under the sampling velocity.); t and t are air temperatures in two different working conditions, which is generally c in calibration condition. there are two kinds of consideration methods for the application of this expression. the first consideration method. let the indication value q represent the scale value in calibration condition, q the indication value to be measured in the actual working condition, p one atmospheric pressure in calibration conditions, and p the pressure in the measurement condition; it is obvious that p < p , and q > q (for the convenience of discussion, it is assumed that there is no difference for the temperature), namely, the actual flow through the flowmeter is greater than the indication value. this is because of the reduced density and the decreased air pressure in the flowmeter. the second consideration method. let the indication value q represent the actual measured indication value, p the actual measured pressure, and q the indication value with the calibration pressure p ; it is obvious that p < p and q < q , i.e., the actual indication value q in actual condition should be reduced to q in the calibration condition, which is always easily neglected by people. the round dots in fig. . are the indication values in the calibration condition after calculation (the temperature difference is ignored), and the value agrees well with the experimental data. if the above two calculation results should be converted to other conditions, the following equation can be used: where the superscript "'" in the equation represents the conversion state, such as the calibration state, standard state, sampling state, and special state in the flowmeter. the subscript " " means the adopted symbol during the application of eq. ( . ) with one of the two consideration methods. as for what kind of the destination state of conversion, it depends on the need. for example, in inspection process of product quality or quality accident, the actual local particle concentration should be known, so it should be converted into the sampling state. for the general measurement, for the purpose of comparison, it should be converted to calibration state or standard state. so for the general measurement, the second method to correct the flow rate should be used, because after correction the result under calibration state is obtained, which no longer needs state conversion. according to the influence of the additional resistance on the flow rate, the following points in the sampling should be paid attention to: . when the particle concentration is measured with the filter method, under the premise of the particle collecting efficiency, the filter material with small resistance should be used, and the resistance of the pipeline should be reduced as much as possible. . when the flowmeter is installed at the suction end of a vacuum pump, the valve used to regulate the flow must be installed behind the flowmeter, as shown in fig. . . it should not be installed between the sampler and the flowmeter. if the flowmeter is installed at the exhaust end of the vacuum pump, there should be a buffer bottle between the flowmeter and the vacuum pump. particularly for removing large oil drops during the use of mechanical pump, apparatus such as valve and filter cannot be placed outside of the buffer bottle, as shown in fig. . . if sampling is performed in the cleanroom, the vacuum pump can only be placed indoors. because the exhaust of vacuum pump must pass hepa filter, it is not suitable to place the flowmeter at the exhaust end. . when filter paper is used as the media, because the resistance during sampling is not more than mmhg, so its influence can be neglected. when microporous membrane filter is used for sampling, because its resistance during sampling is often more than mmhg, the influence of this additional resistance on the indication value of flow rate will be more than %. therefore the result must be corrected. now examples for calculation will be given as follows: example . . during the indoor sampling in a cleanroom, the indication value of the flowmeter is l/min, and the sampling time is min. the resistance of sampling filter material (microporous filter membrane) under the sampling flow rate is mmhg. after calculation, the total number of particles is , under the air temperature c and the air pressure mmhg during measurement. what kind of air cleanliness level can be achieved in the room? ans: . the flow without any corrections total sampling flow rate is q ¼  ¼ l the average particle concentration is n ¼ , / ¼ #/l so the air cleanliness in this room reaches class . with the first consideration method: (a). the actual flow rate through the flowmeter. the reading value of the flowmeter l/min is the indication value in the calibration condition, so the actual flow rate q through the flowmeter should be according to the actual collected particle concentration . l/min after correction, the calculated indoor particle concentration is . #/l, so the indoor air cleanliness level does not reach class . from the above calculation result, it is found that the influence of additional resistance cannot be overlooked. when sampling is taken in the flowing air (especially when in the pipeline), the plane of the sampling mouth should be vertical to the direction of the air flow (namely, the axis of the sampling pipe and the direction of flow are the same), and the sampling velocity must be equal to the air flow velocity. otherwise, the sampled concentration (i.e., the measured concentration) will be greater than or less than the true concentration, which will cause an error. this is the principle of the isokinetic sampling. on the left of fig. . , the airflow velocity u is greater than the sampling velocity u. in this case, the boundary limit of flow inhaled into the sampling tube is less than the diameter d of the sampling tube. if there are large particles outside the boundary limit of the inhaled flow or outside of the streamline with diameter d , it will not follow the streamline to flow along the tube wall when it approaches to the tube mouth; instead it will enter the tube due to the inertial effect. small particles on this boundary limit will not enter the tube. in this way, in the same sampling air volume, particles outside this air volume will be carried to enter the tube, especially the large particles. so the sampled concentration of large particles increases, which means the whole sampled concentration will be higher than the actual concentration. on the right in fig. . , the airflow velocity u is less than the sampling velocity u. in this case, the boundary limit of the flow inhaled into the sampling tube is bigger than the diameter d of the sampling tube. even if there are big particles within the boundary limit of the inhaled flow or within the streamline range with diameter d , when it approaches the tube mouth, it will not enter into the tube but instead flow away along the tube wall, and only small particles in the streamline can enter into the tube. so in the same volume of airflow entering into the tube, particles especially big particles will be lost. so the total sampled concentration will be less than the actual one. therefore, factors that affect the sampling concentrations mainly include the difference between the air flow velocity u and the sampling velocity u (i.e., the velocity at the sampling mouth), the dimensionless inertial parameter describing the inertial effect (stokes parameter) st. the form of the expression is where n is the sampled concentration; n is the real concentration. as for the error caused by non-isokinetic sampling, the following semitheoretical formula [ ] [ ] [ ] is now widely used: in refs. [ , ] it provides: in ref. [ ] it provides: it is shown that the relationship between n n and u u is more obviously demonstrated directly in eq. ( . ) (published in ) than eq. ( . ) (published in ), so eq. ( . ) was referred by e. the difference of the results with the two formulas is little, and the latter is slightly larger. from eq. ( . ) , it is shown that the general characteristic is: for the situation when particles are very big, and the flow rate is large, and the diameter of the sampling tube is very small); st ¼ const (the larger the difference between u and u is, the greater the concentration difference is) u /u ¼ const (the larger st is, the bigger the concentration difference is) when both eqs. ( . ) and ( . ) are plotted in fig. . [ ] , the above feature can be vividly demonstrated. it is shown that: . in the conditions of non-isokinetic sampling in the wide range u / u ¼ . - . , as long as st ≯ . , or u /u ¼ . - and st ≯ . , the sampling error≯ %. because large particles are the sampled object in the field of dust removal technology, the requirement for the velocity deviation is less strict when error is ≯ %. for example, in japanese standard jis z , the sampling error for non-isokinetic sampling is within ae % when the error of non-isokinetic velocity is between À %~+ %. . the influence at the situation u /u > which has slower sampling velocity (as on the right of fig. . ) is much larger than that at the situation u /u < which has faster sampling velocity (as on the left of fig. . ). this is also the reason why the negative deviation is less than the positive deviation specified in jis z - . at c, μ ¼ .  À pa · s. from table . , it is found that c ¼ . ( . μm) or . ( μm). the following results can be obtained with eq. it should be emphasized that, as pointed out in chap. and relevant literatures [ ] , the unit of μ during calculation of st must be paid attention to. if the engineering unit in the past is used, the value of μ decreases by . times, which will increase the particle loss rate by . times and will draw different conclusions. the above results show that the error with non-isokinetic sampling for μm particles is less than % even when particle counter with the sampling flow rate . l/min is used. but it reaches % for ! μm particles. if the requirement of sampling error is not only for particles with a certain size, but also for whole particles with diameter larger than a certain size such as ! . μm, it should be calculated according to the standard distribution of particle size. from chaps. , , and , when suppose particles with diameter ! . μm occupy %, particles with diameter . μm occupy %~ %, so: it can be calculated that the error is - . % for all the particles ! . μm with large sampling flow rate, and the average is . %. apparently the error is much smaller with the small sampling flow rate. this result is much smaller than the experiment data in the wind tunnel, but the error of the experiment data is . % on average [ ] , which is also less than %. for sampling of large particles in the flow field with large velocity fluctuation, such as the sampling in the field of dust removal technology, the error will be % by the velocity deviation %. the application of isokinetic sampling is mentioned in all the monographs about this aspect, where the allowable error is also mentioned to be more than % [ ] [ ] [ ] [ ] . the velocity is required to be controlled strictly in these literatures. one method is the prediction of velocity. when a micromanometer is connected to both inside and outside of the sampling probe, the sampling velocity is adjusted so that the indication value is . of course, when the sampling flow rate changes, it is necessary to use the cumulative flowmeter, and the rotameter is only for the purpose of surveillance and control. in addition, there is another method called isodynamic pressure method. chinese academy of preventive medicine has developed a specialized isokinetic sampling probe. in cleanroom, it is obvious that the velocity field is stable. but it is not uncommon that the velocities at many positions are different from the average velocity by one time, or even much larger (e.g., in the blinder area below the shadowless lamp holder in the center of the operating table, where the air cleanliness must be measured). the transient change rate of velocity at each position can also be more than one time (as shown in fig. . ). because of the time difference at each sampling position, the variation of velocity may be offset with each other. literatures have emphasized that the isokinetic sampling procedure must be followed. but the feasibility of isokinetic sampling at various measurement positions in large space is much less than that in the pipeline. in unidirectional flow cleanroom, the sampling velocity can be chosen to be the average velocity. but in turbulent flow cleanroom, it is meaningless to mention the average velocity. so for the measurement of the concentration field in space, the allowable error should be considered. all test results have errors. if the error is within the allowable range, the test results can be accepted. e proposed that the error less than % is acceptable which is caused by non-isokinetic sampling. it also points out that when u:u is between . : and : , the error is less than %. the diagram to make correction was also given as appendix c in e, which is shown in fig. . . the example of the application of fig. . is shown as follows: when the particle counter with small sampling volume . l/min is used to detect particles with diameter μm (only for this particle size) from airflow with velocity . m/s, should any correction be made? ans: we know that u/u ¼ . / . ¼ . , and st ¼ . for μm particles, so point a can be found on fig. . , which falls in the area where correction is not needed. so there is no need for correction. the specification of isokinetic sampling is listed below from the main standards, which is shown in table it is shown that no matter what the particle size and the allowable error are, d required isokinetic sampling for unidirectional flow. but for e, isokinetic sampling is needed only when the error is > % for particles ! μm, where the flow pattern is not specified. in fact it is turbulence flow. because particle counter with small sampling flow is used, the sampling error is smaller and influence exists only for large size particles. so e clearly pointed out: "although isokinetic sampling is good, if it is not feasible, the sampling deviation should be estimated with the method in appendix c," "it is only meaningful for particles with diameter equal to or larger than μm in clean area by non-isokinetic sampling." in iso - , it only kept the specification about the minimum sampling volume and the probe orientation which were given in e, while the requirement of isokinetic sampling is not explicitly mentioned. this is identical with the general opinion of e. although the sampling error is big for particles ! μm with non-isokinetic sampling, a lot of practice shows that μm particles do not exist in cleanroom, and μm particles do not exist in high-level cleanroom either, and only a few exist in low-level cleanroom. so the influence on the particle number is small, which can be used for the understanding of iso specifications. the next chapter will illustrate that the sampling error including the non-isokinetic sampling by particle counter will generally not exceed the standard estimation range. based on the above analysis, we can suggest that (sampling error ≯ % is used as the standard): . under the condition of u /u ¼ . - . , as long as st ≯ . , isokinetic sampling is not required. . for sampling in cleanroom according to the current domestic and international standards, isokinetic sampling is not required for particles ! μm. . if particle counter with large sampling flow rate is used in cleanroom, isokinetic sampling is required for particles ! μm. (for particles less than μm, calculation should be performed to determine whether isokinetic sampling is needed. generally isokinetic sampling is needed for μm particles.) . when isokinetic sampling is needed but it is impossible, correction of the result should be made with the estimation error on fig. . . of course, it is absolutely impossible to obtain motionless air, and this is also the case even in some experimental device. here the so-called quiescent state means that the air velocity is very low, and it is completely in natural state. there are two kinds of errors when the sampling probe is facing upward in the still air: when the sampling rate is very low and the sampling mouth is upward, due to the settlement of particles, some particles outside the sampling volume will "fall" into the sampling probe. in the extreme case, the sampling flow rate is zero; sampling error will be infinite because of the natural sedimentation of particles. this is similar to the sampling in flowing air. with the larger sampling flow rate and the larger particle size, these particles may not be collected. in order to reduce the first kind of error, sampling with adequate sampling velocity must be performed. in order to reduce the second kind of error, sampling probe with large diameter should be used. davies provided a mathematical expression with the above two conditions [ ] : for the first kind of condition μ ! ν s ( . ) where v s is the settling velocity. therefore the following expression can be obtained (where the slip boundary correction can be omitted) where d is the diameter of the sampling probe; q is the sampling flow rate; d p is the particle diameter when ρ ¼ . for the second kind of condition (where the slip boundary correction can be omitted): with the mathematical expressions, figs. . and . can be obtained [ ] , which facilitates the usage. the sampling conditions below the dotted line in fig. . show that the requirement for both the minimum and maximum allowable sizes of sampling probe cannot be simultaneously satisfied. when isokinetic sampling is required, it does not necessarily change the diameter of the whole sampling tube. it is fine as long as sampling probes with different diameters are placed at the head to the sampling tube, as shown in fig. . . if air velocity u is expressed with "m/s," the sampling flow rate q with "l/min," and the sampling probe diameter d with "mm," we can obtain: it is a matter of concern about the measurement error caused by particles loss in the sample tube. it is serious when the sampling loss of microbiology reaches . % for a . m long sampling tube [ ] , which is not impossible. except for the electrostatic effect during sampling and filtration processes because of the electrostatic charge, there are also other reasons for the particle loss, which mainly include the deposition of particles in the tube caused by diffusion, deposition, collision, and coagulation effect. they will be introduced separately as follows. particles loss by diffusional deposition will occur since they will attach onto the tube wall by diffusional movement. expressions to calculate the particle loss by diffusional deposition are different according to the different flow states inside the tube. for the common particle counters with large, medium, and small sampling flow rates at home and abroad, reynolds numbers re in the sampling tube are shown in table . . it is laminar flow when re < , . it is turbulent flow when re > , . so for the particle counter with large sampling flow rate, it should be considered as the turbulent flow, while the laminar flow should be considered for the particle counter with medium or less sampling flow rate. according to the summary of the conclusions by fuchs in [ ] , the following formula can be obtained. let when α > . when α < . where n/n is the penetration of particles through the sampling tube; n is the particle concentration at the entrance of the sampling tube, i.e., the sampling concentration, #/l; n is the particle concentration at the distance x from the entrance of the sampling tube, #/l; d is the diffusional coefficient of particles, m /s; r is the radius of the sampling tube, m; x is the length of the sampling tube, m; u is the air velocity inside the sampling tube, m/s. with the above formula, the dotted line shown in fig. . can be obtained [ ] . the relationship can be found between particle loss rates, particle size, and flow rate. but it is inconvenient to determine the required length of the tube from the theoretical curve. fuchs gave a slightly different equation in the published monograph in new york in [ ] (in the past this equation was referred by author in relevant publications). when α > . , when α < . , the α value above may also be . in some literatures [ ] . by comparison, the result from eq. ( . ) is slightly smaller than that from eq. ( . ) , and the result from eq. ( . ) is slightly larger than that from eq. ( . ) . but the differences for these two situations are very small. for particle counters with medium and low sampling flow rates, the value of α of the latter is greater than the former. the smaller the particles are, the larger the diffusion is, which is more disadvantageous. the most disadvantageous situation corresponds with d p ¼ . μm, when d ¼  À m /s. we can obtain so for calculating the diffusional loss of particles for particle counters with medium and small sampling flow rates, α < . . eq. ( . ) can be used to obtain the relationship diagram between α and À n/ n , as shown in fig. . [ ] . in comparison of fig. . , this figure directly presents the relationship between α and the particle loss rate which is described in eq. ( . ) . it is convenient to calculate the particle loss rate and the pipe length with the known particle loss rate. it is shown in fig. . that as long as α . , the diffusional loss rate will be %. suppose the allowable maximum loss rate is %, we know so the following requirement must be satisfied: for particle counter with medium and low sampling flow rates, calculation results are shown in table . . since the diffusional coefficient is independent of the particle density, the allowable tube length in table . is also not related to the particle density. from the calculated results, the diffusional loss for ! . μm particles is extremely small, which can be completely ignored. in the literature from b.y.h. liu [ ] , similar equation as eq. . was used to obtain the left part of fig. . (only specified that α < . ). although it was pointed out that the formula was valid for laminar flow, it did not indicate in the figure that it is not suitable for the application in non-laminar flow such as the particle counter with large sampling flow rate. and example was given for this kind of particle counter (e.g., diameter d t ¼ cm, and sampling velocity u ¼ m/s). that means for a sampling tube with d t cm, when pass-through time of the flow is s and particle loss rate by diffusion is %, the allowable tube length is s  m/s ¼ m. if eq. ( . ) is used for calculation, when the particle loss rate is %, we can obtain there is little difference between these two results. so it is acceptable to consider that results are consistent with fig. . and eq. ( . ) for calculation of the turbulent flow. but as mentioned before, the above formula and figure should not be used for the particle counter with large sampling flow rate where turbulent flow is inside. so it is incorrect to choose either m or m. it is difficult to obtain the particle loss in the particle counter with medium sampling flow rate from fig. . . but it is convenient to use eq. ( . ) . the allowable pass-through time t for the flow inside the tube can also be calculated with eq. ( . ). because because the particle loss by diffusion is known to be extremely small, the allowable tube length, i.e., the acceptable value t, is very big. the specific number of t will not be calculated. of course, the value of t can be obtained with the known tube length and flow velocity. the diffusional deposition onto the surface of tube wall by turbulent flow is much complex than that by laminar flow, and diffusional deposition onto the indoor wall which is introduced in sect. . . as pointed out in sect. . , there is a very thin diffusional boundary layer close to the wall. particle concentrations beyond the boundary layer will become uniform by turbulent flow. the boundary layer thickness is δ, which is difficult to determine. in hinds' monograph [ ] , it is pointed out that davis had already discussed the problem, and fuchs provided the diffusional boundary layer thickness is δ inside the tube. so the whole penetration p through the tube with length x can be obtained with the following formula after the diffusional loss by the turbulent flow is considered: where v d is the diffusional velocity (see sect. . ) (m/s); where ρ is the air density. let log-log paper can be used to plot the linear relationship between the loss rate -p and y. so the value of y can be obtained from the figure according to the required loss rate, and then the value of x can be calculated. suppose it is required: substituting it into the eq. ( . ), we can obtain: substituting μ ¼ .  À pa · s and ρ ¼ . kg/m into the above expressions, we obtain: with these above conditions, the loss rate %. if the value of y is obtained under a certain loss rate, we obtain: x yd t u d for the particle counter with large sampling flow rate where turbulent flow exists inside the sampling tube, the allowable lengths are specified in table . . as shown in table . , the allowable tube length calculated according to the turbulent diffusion situation for .l μm is much less than that with laminar flow diffusion. it is obvious that it's inappropriate to calculate by the latter situation. and the allowable pass-through time t of the turbulent flow can also be obtained with eq. ( . ), which is: for the sampling in turbulent flow by particle counter with large sampling flow rate, the allowable pass-through time through the sampling tube is shown in table . . of course, the allowable pass-through time can also be calculated with the division between the allowable tube length and the airflow velocity. as mentioned before, b.y.h. liu just quoted the formula for diffusion loss in laminar flow [ ] . he thought that the diffusional loss in the turbulent flow can be calculated according to the laminar flow at first, and then an additional item considering the vortex deposition is added. but the calculation method is very inconvenient for use, so it will not be introduced here. for laminar flow, fuchs quoted the same formula of Г. Натансол in two versions of his monographs [ , ] : for turbulence flow, fuchs gave the following expression in his latter version [ ] : it has been pointed out that the difference is small between the calculation results when n/n ! . in two conditions. here calculation for μm particles is taken as an example: the allowable tube length is as follows when n/n ¼ . : deviations for these two kinds of flow regime are both within %, so they can be calculated by laminar flow. the theoretical curve made by eq. ( . ) has been shown in fig. . [ ] , which is the solid line. it is also not convenient to determine the tube length by this curve. the relationship diagram between β and À n/n is shown in fig. . , which is very convenient for use to calculate the loss rate. [ ] . for the sampling tube with d t ¼ cm, taking μm, for example, when the particle penetration is . , we obtain t d t % s cm the allowable tube length for the particle counter with large sampling flow rate u ¼ m/s is the deviation is % with the value . m in table . . since interpolation is needed during the application of the figure, it is inconvenient to calculate specifically. in addition to vertical and horizontal tubes, the sampling tube generally has a certain degree of bending. figure . shows a real photo. it forms a bend in the extreme case, as shown in fig. . [ ] . the minimum bending radius is the calculation results of collision loss are listed in table . . it is shown from the table that the larger the particle is, the greater the collisional loss is; the bigger the value st is, the bigger the collisional loss is; the smaller the value r is, the greater the collisional loss is. for particles with diameter equal to and less than μm (this is the maximum controlling particle size in various recent standards), the collisional loss can be ignored. but for l μm particles, it should be taken into consideration. equation ( . ) has given the method to calculate the coagulation concentration of monodisperse particles. if it is used to estimate the coagulation concentration of polydisperse particles, separate calculation should be performed with different particle size. and further correction can be made according to the geometric standard deviation σ g (see chap. ) of the group particles. the correction values from literature [ ] are listed in table . . take the unfavorable . μm particles as an example: when t ¼ s, and n ¼ #/cm , the coagulation coefficient is found from table . that k ¼ .  À cm /s. so even when polydispersity is taken into consideration with σ g ¼ , k increases by . times. the result shows that the coagulation loss is very small, so it can be completely ignored. from the above theoretical calculation, the particle loss by collision, eddy, and coagulation for particles with diameter equal to and smaller than μm inside the sampling tube is much smaller than that by diffusion and deposition, which generally can be ignored. it should be considered only for particles with diameter more than μm and in special sampling conditions. for the particle loss by diffusion and deposition, the deposition loss is much important. the diffusional loss of large particles can also be negligible. comparison is performed between three experimental data and theoretical calculation results: . for the horizontal sampling tube with diameter . cm and length cm (a) and cm (b), respectively, particle counter with small sampling flow rate was used to measure the concentration of standard particles after steady state reached. the sampling velocities were both cm/s. the test result is shown in table . [ ] . for tube a with length only cm, even for μm particles, the theoretical deposition loss rate is much lower than %, and the diffusional loss is even smaller. so the average concentration in tube a can be considered as the standard value without any particle loss, which can be used to compare with that in tube b (table . ). . for a horizontal sampling tube with diameter d t . cm and length cm, measurement was performed to test the concentration of the standard particles when the sampling velocity was . m/s. the result is shown in fig. . [ ] . by comparison of the particle loss rate in the figure with the theoretical particle loss rate, results are shown in table . . in the two experiments, small spherical psl particles were used as the standard particles, whose density is . g/cm . from comparison, the actual particle loss rate is close to the theoretical value for particles with diameter less than μm. but the actual loss rate is % of the calculated value for particles with diameter larger than μm. this is because the resuspension phenomenon is not considered in the formula after particles deposit. however, it cannot be ignored for big particles. because the smaller the particles are, the larger the molecular force is. the corresponding suspension velocity is bigger, which needs bigger force to blow them up, and it is hard to resuspend again. as shown in fig. . , the suspension velocity suddenly decreases from about - μm. it showed that it is hard for particles with diameter more than μm to resuspend, while it is quite easy for particles with diameter more than μm to resuspend again. it shows that the deposition loss rate is smaller than that of small particles. . for the horizontal sampling tube with diameter . cm and length cm, when the sampling velocity is . m- . m/s, the actual loss rate for polydisperse dop particles in the tube is shown in fig. . [ ] . taking u ¼ . m/s, for example, comparison with theoretical results is shown in table . (the particle density is . g/cm ). in the experimental results with six grades of velocity channels, most of the actual tested results are closed to the theoretical value, and both have the same trend. for example, the loss rate becomes bigger when the sampling velocity is small, and the loss rate increases quickly with the increase of the particle size. with the same sampling velocity, for most particle sizes, the particle loss rate in pipes with small diameter is bigger than that in big pipes. through the comparison of three examples, it is reasonable to consider that the theoretical calculation is closer to the reality. correct on the actual test data will be performed according to the theoretical calculation if necessary. what should be emphasized is that, the theoretical calculation results are all obtained from the formula and the given parameters in this section, instead of directly in the fig. . . the values from this figure are a little smaller. since the values of some parameters are unknown during the plot of the figure, the reason why it is smaller cannot be confirmed (table . ). through the above analysis of particle loss inside the sampling tube, the following conclusions can be obtained: . for particles with diameter less than μm, the length of sampling tube should be considered just according to the particle loss by diffusion and deposition. . for . - μm particles, the deposition loss rate is gradually bigger than that by diffusion, so the length of the sampling tube should be considered according to the deposition loss. . for particles with diameter less than . μm, the length of the sampling tube should be considered according to the diffusional loss when only particle counter with large flow rate is used. when particle counters with medium and small sampling flows, the length should be considered according to the deposition loss. in the appendix b . of fs e, the sampling tube in the "air sampling system" for particle counters is required that "the size of the sampling tube should be determined in such a way that the flow time in the tube should not be more than s." and in the b . . , further suggestion was given in the section about "the consideration for particle delivery." "for . - μm particles, the maximum sampling tube could be m. for - μm particles, the length of the sampling tube should not be more than m. in this condition, the particle loss rate can be less than % for small particles by diffusion and large particles by deposition and inertia (see appendix c)." in the above references, the reynolds number of sampling flow is required to be - , , and the diameter of the sampling tube is not specified. so in this re conditions, when calculation is performed with the turbulent formula, if the diameter is bigger than that the diameter cm for the particle counter with large sampling flow rate by %, the allowable length could be m for . - μm particles. if the diameter is cm, the maximum length could not be more than m according to table . . the second channel should not be - μm, but instead it should be - μm as the interval, because μm is the prescribed maximum control particle size in e. therefore from table . , it is suitable that the length of the horizontal sampling tube is not more than m ( e only mentioned it as the sampling tube, which is not accurate). if the diameter of control particles is - μm, it cannot be more than m. for the special needs with the - μm particles as the control object, it should not be more than m for the horizontal sampling tube. so for e, it is reconcilable that - μm is changed to - μm. for the particle counter with so-called medium sampling flow rate . l/min in china, the control particle size generally begins from . μm. so the lower limit . - . μm can be classified as one channel (corresponding to . - μm in e). based on the results from table . and the calculated results from ref. [ ] cited in e, the length of the allowable sampling tube can be long in terms of the diffusional loss. therefore only the deposition loss should be considered. from table . , when the atmospheric particle concentration is large ( ), it is safe to set the length of the sampling tube within m in theory. for cleanroom where the maximum control size is μm commonly, it is more likely that only the deposition loss is considered. from table . , it is known that the horizontal sampling tube should be less than . m, which means that in order to ensure the loss rate less than % for μm particles, it is better that the length of the horizontal sampling tube is less than . m. as shown in fig. . , it is allowable that the length of the horizontal sampling tube is not more than the instrument length, and it is not suitable if the tube is longer. ref. [ ] gives the suggested length value of the sampling tube for domestic situation, as listed in table . . for the environment with different air cleanliness levels, the sampling flow rates should be different. during the measurement with the balance meter, it is relevant to the sensitivity of the balance. during the dust sampling with membrane filter, the sampling flow rate is related to the background value of the membrane filter. an appropriate proportion must be kept between the captured particle number and the background value of the membrane filter. for both cases, appropriate sampling flow rates are needed. the minimum sampling volume put forward here is for the particle counter [ ] . in the s, particle counters with medium and big sampling flow rates had appeared. due to the difference of the measurement results between large sampling flow rate and small sampling flow rate, this problem is naturally proposed. for measurement in cleanroom with air cleanliness level higher than class , what is the requirement for the sampling flow rate of the particle counter? if requested, how to determine the minimum sampling volume? this is the new problem that is put forward by the development of air cleaning technology. in us standards before c in , including that from federal government, nasa, and air pollution control association, and in related standards from britain and germany, the problem of the minimum sampling volume, the minimum sampling times, and the necessary number of measuring points were not provided. only in , b generally put forward that for turbulent flow cleanroom, it is suitable to set the sampling flow rates . , . , and . ft /min, and for laminar (now unidirectional flow) cleanroom, it is suitable to set the sampling flow rates . , . , and . ft /min. in , german standard vdi- -iii only put forward that it is better to use particle counter with sampling flow rate ft /min for cleanroom with air cleanliness level above class . in the standard ies-rp-cc- made by institute of environmental sciences and technology in usa pointed out the number of measuring points for the first time, and the method to determine the number of measuring points by the area of the cleanroom was proposed. for turbulence and laminar flow cleanroom, the area (ft ) corresponding to each measuring point is ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi air cleanliness level p , but the minimum sampling volume was also not mentioned. in october of , sato [ ] proposed to perform short-period sampling on several positions with small sampling volume particle counter for the first time. for class cleanroom, it is reasonable to doubt the accuracy of the measured results. but how to determine the sampling volume was not discussed. it was the first time in china that the concept of the minimum sampling volume was officially put forward also in october of . the nonzero principle to determine this minimum sampling volume was given, and the calculation method was also provided [ ] . in , the concept of necessary number of measuring points and the corresponding calculation method were put forward [ ] . the minimum sampling volumes obtained by this method and by data (data from japan jis standard in adopted from us standard) from c later (in ) belong to the same class, which will be described in detail later. particle counter can only display numbers which are positive integers such as , , , and . if the particle concentration of the measured space is extremely low, and the sampled air volume for each reading number every time, namely, the sampling volume is very small, the average concentration in the sampling volume may be a number that is less than . in this case, each reading may appear many times with the value of " ," or the majority is " ." for the occasion that the particle concentration is extremely low, when the average reading at each sampling point is " " and there are few sampling points such as only three points, the average concentration should be " ." if there are errors, for example, there are two times of " " and one time of " ," the average concentration becomes " . ," so the difference between two concentrations reaches . times. in this case, the real particle concentration cannot be really reflected. if the sampling volume is big, each reading from every sampling volume may be more than , such as and , then the frequency with number " " in each sampling volume is few. even if there are some particles, the average concentration is only affected in the magnitude of . - . , so the difference from true concentration will be smaller. the above situation mainly appears in the place with high air cleanliness requirement (such as higher than class ). therefore, the minimum sampling volume of the particle counter should have such characteristics. the possibility that particles fall into the minimum sampling volume is large, which means there is no possibility for the appearance of zero particle inside the sampling volume. this method can be called "nonzero inspection principle." as pointed out in chap. , if the average concentration in the sampling volume is set as λ, the particle distribution in the space can be described by the poisson distribution when λ . eq. ( . ) has given the probability p for the appearance of k particles at most in the sampling volume. it is rewritten here: so the probability for " " particle is: the curve in fig. . can be made with the two above formulas. the " particle" curve corresponds to the probability for the appearance of " particle." the " particle" corresponds with the sum of appearance of " " and " " particle. the probability of appearance " " particle is the difference between " " and " ," which is analogous for other cases. from fig. . , if the probability of nonzero reading is required to reach %, namely, the probability of the appearance of " " particle is only %, λ must reach . the sampling volume obtained is the minimum sampling volume. the calculation example of the minimum sampling volume will be given below. now the lower limit of the particle concentration for each air cleanliness level is used to represent the concentration for this level (which is safer than that with the upper limit of the concentration). we can obtain: the minimum sampling volume ¼ particles the lower limit of concentration at this level ( . ) the calculated results are shown in table . . from table . , we can obtain: . in china, the legal unit system is used, while in air cleaning technology, "l" is used as the basic unit. so it is appropriate to take . l. . for the environment with the particle concentration less than #/l, when ordinary particle counter with small sampling flow rate is used, the sampling time should be extended to ensure adequate sampling volume and larger possibility of the appearance of nonzero particle. if the sampling time is not prolonged and the sampling volume is not increased, most of the measured results will be zero, which will reduce the reliability of the measurement results. . for the measurement in the space with high air cleanliness requirement with ordinary small sampling flow rate particle counter, if the extension of the required time is not long, the extension of time is feasible. if the extension of the time is too long, it is not only uneconomic, but also likely to cause other errors. so it is necessary to use the particle counter with large sampling flow rate in this case. but the effect of large sampling volume on the concentration field must be considered, which may be also likely to produce false phenomena. so in the above circumstances, it may not be necessary to use large sampling flow rate particle counter. as already mentioned before, the poisson distribution may not be suitable to describe the particle concentration distribution in the cleanroom. although this aspect has been explained in chap. , which believed that the poisson distribution can still be used as the basic means to predict the particle distribution in space with high air cleanliness, if local serious leakage occurs in unidirectional flow cleanroom, it will have effects. but this kind of influence also exists in the turbulent flow cleanroom, especially when the sampling points are close to the leakage airstream. so code for construction and acceptance of cleanroom (gb - ) has required that air filters installed in the cleanroom must undergo the leakage detection one by one. after installation, leakage scanning must be performed along the frame of the air supply surface. when all the situations are confirmed, the statistical result is effective. otherwise, if the frequency of a particle size is more than their due value, it is apparent that there are abnormal reasons, which means the data is not believable or the possible reason must be found. it does not mean that the poisson distribution cannot be used to predict the particle distribution. for example, during the measurement with times (the sampling volume is l), there are times with " " particle and one time with " " particle. please judge if this is a normal situation. with eq. ( . ), we can calculate λ ¼ / ¼ . , which means the probability of the appearance of " " particle should be %, namely, it should be times, which is consistent with the actual condition. so although the sampling volume is not enough according to the value of λ, the result can be considered as normal. when the first measured value is " " in the above example, please judge if this is a normal situation. after calculation, we obtain λ ¼ . , so the frequency of the appearance of " " particle should be times. there are obvious differences. from eq. ( . ) we know: where k is the total times of measurement; x is the times of the appearance with " " particle. if there are times of the appearance of " " particle in total times, λ ¼ . , which means the average particle concentration in theory should be . #/l, instead of / ¼ . #/l. since the average concentration in the sampling volume l is . , the probability for the appearance of three particles is determined by eq. ( . ) in this sampling volume, which is: so the probability of the appearance of three particles is extremely small, which is impossible. if it appears, it is an illusion, and it cannot be used to calculate the average concentration. it is very likely that the occurrence of three particles is caused by leakage. but as illustrated in e, the statistics method is not aimed to changing multipoint leakage positions during sampling (the possibility of leakage at one place is very small), which means the application of statistical methods or the minimum sampling volume cannot modify the random abnormal error caused by the leakage. in other words, the abnormal situation of measurement results should not deny the particle distribution characteristic and the application of statistical methods. instrument performance may also become one of the abnormal reasons. table . listed foreign measurement data (the data at each point is the average value of many sampling times), where air cleanliness class was realized in the cleanroom when instrument was used, while the conclusion was opposite when instrument was used [ ] . the performances of these two instruments were compared, and it was found that the function to reset zero cannot be performed in instrument . so it is inappropriate to conclude that the above particle distribution characteristic is not correct and the minimum sampling volume is not applicable when two instruments with different performances were used for measurement and the measured results do not follow the above distribution characteristics. in table . , an example was presented where two instruments with identical performance after calibration were used to determine the particle distribution in three rooms, and the results are completely consistent. when statistical analysis is made on these data, it is more likely to conform to the distribution characteristic law. the monograph air cleaning technical measures was published before the concept of "nonzero inspection principle." so the data of table . were not adopted, but the empirical data were used. at that time the concept of the minimum sampling volume was not established, and instead the concept of the sampling volume at every time was used. that is: when particle concentration < #/l (equivalent to class ), sampling volume ! . l; when particle concentration - #/l (equivalent to class ), sampling volume ! . l; when particle concentration ! #/l (equivalent to class ), sampling volume ! . l. the code for design of clean room approved in and implemented in also adopted the similar data as the monograph "air cleaning technical measures," which was later than the "nonzero inspection principle." the data are: the concept of the minimum sampling volume was also not given. it did not mention that particle distribution in cleanroom belongs to poisson distribution, so the method with "nonzero inspection principle" was not cited to obtain the minimum sampling volume. as for the sampling volume specified in code for construction and acceptance of cleanroom (jgj - ) published in and code for design of clean room (gb - ) published in , it is consistent with related international standards ( d, iso - ). the exact data will be presented later. the above discussion is aimed to measure accurately, so the concept of the minimum sampling volume must be used. but it is still not enough only to use the minimum sampling volume; there must be enough sampling points, which have already pointed out in the past [ ] . but the specification in this aspect in related foreign standards is not enough. the problem will be discussed in the next chapter. number [ ] [ ] [ ] because sampling process is the collection of the counting data at every sampling point, it can be treated as the poisson process. according to the poisson distribution, the interval estimation of the average value for the test data can be made. from eq. ( . ), if the total number n obtained from the actual measurement is used to replace k, the distribution function of the estimation value λ for λ is where λ is the upper confidence limit of λ , as shown in fig. . . when ζ is the probability (i.e., the confidence level) for the given value of Þand integration can be performed for the right expression of the above equation, the upper limit of λ of the estimation parameter λ can be obtained with the value of n for different ζ. results are shown in table . . the random absolute error of this sampling is: the random relative error is: l' l l as shown in fig. . , for the given confidence level ζ, the more the sampled total particle number is, the bigger the upper confidence limit λ is. from eq. ( . ), the relative error r decreases. as for the minimum sampling volume in us c standard, it is based on the principle that the total sampled particle number is for the corresponding air cleanliness level according to the explanation of b revision attendant. with this principle, when the confidence level is %, the maximum concentration is particles and the minimum is particles. that means there is % probability that the total particle number sampled is between and . for example, when the upper concentration limit is calculated, it could be . % when table . is used with the confidence level . %, which is consistent with the above explanation by b revision proposals. from the table, the sampling error is r ¼ . %. if the confidence level reduces to %, λ ¼ particles. although the increased total sampled particle number can reduce error, the sampling time will be prolonged. it is completely artificial to set particles. so we obtain: minimum sampling volume ¼ upper concentration limit with the air cleanliness ( . ) for class , the upper concentration limit is #/ft , / ¼ . ft . for class , the upper concentration limit is , #/ft , / , ¼ . ft . according to the same principle, iso - uses "l" to represent the sampling volume, so: minimum sampling volume ¼ upper concentration limit with the air cleanliness=m  ; l ( . ) in code for design of clean room (gb - ), the value , in the above equation was mistaken as . the above results are shown in table . . the mantissa of l in e and iso are different slightly. at the same time, iso specified that the minimum value should not be less than l. from the comparison of tables . and . , we can know: . the above two principles to determine the minimum sampling volume are based on the collection of the point data. so all of them applied the poisson distribution, but with different methods. the method with λ ¼ is aimed to guarantee that the probability of nonzero data is not less than %. the method with total particle number ¼ is slightly optional. so although eq. ( . ) and eq. ( . ) have similar shapes, the nature is different. only american literature [ ] has not pointed out that the test statistics is based on the counting points' data when it comes to poisson distribution. so when the statistical analysis about the average concentration (it will be introduced in detail in next chapter) was mentioned in this literature, it concluded that "both experience and theory have proved that poisson distribution is seldom applicable for the actual concentration in the cleanroom," and t distribution was recommended, which will cause misunderstanding. it seems that t distribution may fit the particle concentration in the cleanroom. in fact, different principles were used to treat the statistical problems with two different features between the counting points' data and the average value. from statistics theory, it is known that this is the problem with two different natures, and it is not a problem that which distribution is more suitable to describe the particle distribution in cleanroom. one is the density distribution, where the poisson distribution is applicable. the other is the particle number distribution according to the particle size, which is aimed to calculate the error of the average value, namely, the so-called treatment of "actual measured concentration data from the cleanroom," so t distribution method reflects the average distribution characteristics from small subsamples. the method will be discussed in the next chapter. . the us federal standard c adopted the english unit, which chooses ft as the basic unit. it is understandable that . of this unit, namely, . ft , is taken. . the minimum sampling volume specified in c is calculated according to the upper concentration limit. although the concentration increased by times, λ only improves . times. so in contrast, the minimum sampling volume of each air cleanliness level should be less than that in table . . because the difference of the specific value represented by . unit for air cleanliness level lower than class , the minimum sampling volume (l) is bigger than that in table . . but in all, the two values of the minimum sampling volume are very close. like the measurement of particles, there is also the problem of the minimum sampling volume for airborne bacteria sampling. now the minimum sampling volumes are calculated with "nonzero inspection principle" and the airborne bacterial concentration, which is shown in table . [ ] . as pointed out in chap. , deposition of particles on surface follows the poisson distribution. therefore like the minimum sampling volume mentioned in the above section, there is also the minimum sampling volume problem during the measurement of particle settlement. if the deposition area is too small, it will reduce the reliability of the whole measurement because the probability of the appearance of " " particle is extremely large. this problem is especially important during measuring the colony in biological cleanroom with the deposition method. as the minimum sampling volume analyzed in the previous section, in order to ensure % of readings are nonzero, the particle deposition density must also reach or exceed . from table . , the allowable colony in the specified period in each petri dish is known, which is the deposition density (take one petri dish as the base). so the minimum deposition area needed for measuring the bacterial concentration with the deposition method in the environment with different air cleanliness levels can be obtained, which corresponds with the minimum number of petri dishes. for example, when it is . #/l from table . , the probable maximum settlement in each petri dish is . cfu. then in order to obtain three cfu, it needs . dishes, approximately dishes. results are listed in table . . if it is #/l, the number of petri dishes is and . that is the upper concentration limit corresponding with the air cleanliness level in fig. . or the value used by author in the past. the minimum number of petri dishes should be adapt to the necessary number of sampling points or the number of sampling points specified in the regulation, which will be introduced later. if the minimum number of petri dishes is bigger than the number of sampling points, the former value is adopted, and both the requirement of the number of sampling points and the minimum deposition area should be satisfied. if the minimum number of petri dishes is less than the number of sampling points, the number of petri dishes should choose the number of measurement points. if the sampling space is small and petri dishes cannot be placed, the deposition period could be appropriately prolonged (not more than h), and the number of the petri dishes can decrease proportionally. measurement method of particle concentration in exhaust air aerosol technology (trans: sun yufeng). heilongjiang science and technology press influence of additional resistance on the flowrate calibration of the flow meter aerosol technology (trans: sun yufeng). heilongjiang science and technology press aerosol technology: properties, behavior and measurement of airborne particles handbook of air cleaning on the sampling error due to non-isokinetic sampling analysis of sampling problem in laminar flow cleanroom theory and practice of dust removal and collection (trans: chan wenchang). scientific and technical documents publishing house dust removal in the workshop of crushing and screening industry ventilation dust removing technology heilongjiang science and technology press aerosol technology (trans: sun yufeng). heilongjiang science and technology press the fifth institute of military medical college, development of jwl- type sampler the mechanics of aerosols (trans: gu zhenchao) particle loss in sampler the mechanice of aerosols electrostatic effects in aerosol sampling and filtration discussion of the sampling tube length of the particle counter aerosol technology (trans: sun yufeng). heilongjiang science and technology press heilongjiang science and technology press study on property of atmospheric aerosol (the st report) dust meter -light scattering particle counter calculation of the minimum detection volume of the particle counter with different air cleanliness levels status of industrial cleanroom calculation of the necessary sampling points in cleanroom certifying a class cleanroom using federal standard c. (trans: zhang yaping, checked by feng peiming) rationale for proposed revisions to federal standard b (cleanrooms) (trans: liu xianzhe, zhan yingmin) interval estimation of particle concentration -process of measured data measurement of low concentration particles and evaluation of air cleanliness level in clean area measurement of bacterial concentration. handouts for the seminar on the specification of construction and acceptance inspection of cleanroom key: cord- - atidai authors: abo-elkhier, mervat m.; abd elwahaab, marwa a.; abo el maaty, moheb i. title: measuring similarity among protein sequences using a new descriptor date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: atidai the comparison of protein sequences according to similarity is a fundamental aspect of today's biomedical research. with the developments of sequencing technologies, a large number of protein sequences increase exponentially in the public databases. famous sequences' comparison methods are alignment based. they generally give excellent results when the sequences under study are closely related and they are time consuming. herein, a new alignment-free method is introduced. our technique depends on a new graphical representation and descriptor. the graphical representation of protein sequence is a simple way to visualize protein sequences. the descriptor compresses the primary sequence into a single vector composed of only two values. our approach gives good results with both short and long sequences within a little computation time. it is applied on nine beta globin, nine nd (nadh dehydrogenase subunit ), and spike protein sequences. correlation and significance analyses are also introduced to compare our similarity/dissimilarity results with others' approaches, results, and sequence homology. information encoded in the genome of any organism plays a central role in defining the life of that organism. e nucleotide sequence that forms any gene is translated into its corresponding amino acid sequence. is sequence of amino acids becomes functional only when it adopts its tertiary structure. experimental methods such as x-ray diffraction and nuclear magnetic resonance are considered authoritative ways for obtaining proteins' structure and function. ese experimental methods are very expensive and time consuming. erefore, computational methods for predicting protein structure have become very useful. proteins with similar sequences are usually homologous, typically displaying similar d structure and function. sequence alignment is the first step of d structure prediction for protein sequences. alignment approaches are classified into alignment-based and alignment-free methods. blast (basic local alignment search tool) and clustalw are the most widely used computer programs for alignmentbased approaches [ ] [ ] [ ] . results of these programs provide an approximate solution to the protein alignment problem. on the other hand, many alignment-free approaches are proposed for sequence comparison. most biological sequence analysis methods still have weaknesses, including having low precision and being time consuming [ , ] . similarity/dissimilarity analysis of biological sequences is used to extract information stored in the protein sequence. many mathematical schemes have been proposed to this end. graphical representations of biological sequences identify the information content of any sequence to help biologists choose another complex theoretical or experimental method. graphical representation provides not only visual qualitative inspection of gene data but also mathematical characterizations through objects such as matrices. some d and d graphical representations are created by selecting a geometrical object that is used to describe nucleic acid bases or residues [ ] [ ] [ ] [ ] [ ] . others are based on assigning vectors of two or three components to nucleic acid bases or amino acids [ ] [ ] [ ] [ ] [ ] [ ] [ ] . adjacency matrices are also introduced in some articles [ ] [ ] [ ] [ ] , where an exact solution is obtained to the protein alignment problem. additional methods use discrete fourier transform (dft) in which dna sequences are mapped into four binary indicator sequences, followed by the application of dft on these indicator sequences to transform them into a frequency domain [ , ] . dynamic representation is used to remove degeneracies in the previously mentioned approaches [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . another method is based on the simplified pulsecoupled neural network (s-pcnn) and huffman coding where the triplet code was used as a code bit to transform dna sequence into numerical sequence [ ] . in this study, we introduce a new alignment-free method for protein sequences. each amino acid in the protein sequence is represented by a number, and a new d graphical representation is suggested. a new descriptor is introduced, comprising a vector composed of the mean and standard deviation of the total numbers of each protein sequence (a t , sa t ). our graphical representation eliminates degeneracy and has no loss of information. it is suitable for both short and long sequences. as a proof of concept, our approach is applied on nine beta globin protein sequences and nine nd (nadh dehydrogenase subunit ) protein sequences. it can be applied on any sequence length with the same efficiency. correlation and significance analyses are introduced among our results, along with pid% [ ] and clustalw [ ] to demonstrate the utility of our approach. all the protein sequences used in this study were downloaded from e national center for biotechnology a new d graphical representation is introduced. each amino acid in any protein sequence is represented by the suggested intensity y x (i) and intensity level a x (i). e intensity (y x (i)) of each amino in the sequence depends on its abundance and location in the different sequences. it is calculated using where f x is the frequency of amino acid x in the sequence, number of times of x/n. n is the protein sequence length, number of residues in protein sequence. i is the position of each amino acid x in a sequence. en, the intensity level a x (i) of each amino acid (x) in the sequence is calculated by using the natural logarithm function as in erefore, each amino acid has its own intensity level which is a vector of n elements according to equation ( ) . finally, the combined intensity level of the protein sequence a t (i) is obtained by the summation of the intensity levels' vectors a x (i) of the protein sequence by using equation ( ) . e combined intensity level a t (i) is also a vector of n elements: each amino acid has its own graph. now, twenty graphs are obtained for each sequence of the different amino acids. e combined graph is obtained by combining these graphs within a single graph. is combined intensity level is our new d graphical representation. our approach is first applied on two short segments of protein from "yeast saccharomyces cerevisiae": protein i: "wtfesrndpakdpvilwlnggpgcs-sltgl" protein ii: "wffesrndpandpiilwlnggpgcs-sftgl" ese two short proteins consist of amino acids each. e two sequences are different in amino acids at positions , , , and . e values y x (i) and a x (i) for each amino acid in the two sequences are calculated. for protein i, the g amino acid is repeated four times in the protein sequence. ese four repeats occur in positions , , , and . e frequency, f g , equals ( / ). by substituting in equations ( ) and ( ), the results of y g (i) and a g (i) are presented in table . by summing the values of a x (i) for all amino acids in protein i, the total value of a t (i) is obtained, as shown in figure (a). e position i of each amino acid is located on the x-axis, and the total intensity level a t (i) is located on the y-axis. we next apply our approach on nine beta globin and nine nd (nadh dehydrogenase subunit ) protein sequences, which are illustrated in tables and . e d graphical representation for human, chimpanzee, and opossum beta globin protein sequences is illustrated in representations for fin whale and rat nd protein sequences are illustrated in figures (a) and (b), respectively. we finally apply our approach on coronaviruses protein sequences which are illustrated in table . e d graphical representation of tgevg from class i and gd t from sars_cov protein sequences is illustrated in figures (a) and (b) respectively. mathematical descriptors help in recognizing major differences among similar protein sequences quantitatively. a new descriptor for protein sequences is suggested, which is a vector composed of the arithmetic mean a t and standard deviation sa t of the combined intensity level value a t (i) of the protein sequence. ey are evaluated according to the following equations: is descriptor compresses the information from primary protein sequences into a single vector composed of only two values. e beta globin, nd , and coronaviruses protein sequence descriptors are illustrated in tables - , respectively. table shows that the mean of all coronaviruses is around . and with a range from . to . while their standard deviation varies according to their class. ey are divided into four classes. e first four viruses belong to class i. e fifth to the ninth coronaviruses belong to class ii. class iii contains the tenth and eleventh viruses. e rest viruses from the th to the th belong to sars-cov. according to our approach, the standard deviation of class i ranges from . to . . class ii's standard deviation ranges from . to . . class iii's standard deviation has values from . to . . sars-cov's standard deviation almost equals . . e resulting standard deviation values of the coronaviruses classify them correctly to the four classes. e coronaviruses classes' ranges according to our approach are shown in figure . to compare the species' protein sequences, the euclidean distance among species' descriptors is evaluated. for example, the human beta globin protein sequence's descriptor is ( . , . ) and the chimpanzee beta globin protein sequence's descriptor is ( . , . ) . to measure the degree of similarity between human and chimpanzee, the euclidean distance between these vectors is evaluated. e similarity/dissimilarity matrices of beta globin and nd protein sequences are illustrated in tables and , respectively. table results show that human and chimpanzee sequences are similar. ere is also striking similarity between mouse and rat sequences, while human and opossum sequences are obviously dissimilar. species id length gorilla caa chimp caa human aaa rat caa mouse caa gutta ach duck caa gallus caa opossum aaa pigmy chimpanzee, common chimpanzee, human, and gorilla nd protein sequences are similar, while the blue whale is similar to the fin whale, and mouse is similar to rat. similar to the other sequence, human and opossum are still dissimilar. however, our algorithm cannot measure the degree of similarity very well for pigmy chimpanzee. e distance between human and pigmy chimpanzee is . , while the distance between human and gorilla is . , as shown in table . e results of both tables and are approximately comparable to previous reports [ , , , - ]. we got the phylogenetic trees of beta globin and nd protein sequences by applying the upgma (unweighted pair group method with arithmetic mean). e phylogenetic tree based on tables and of our method is presented in figures and , respectively. figure proves the utility of our similarity/dissimilarity analysis for beta globin protein sequences. figure shows our analysis of similarity/dissimilarity of nd . it is mentioned that our algorithm cannot measure the degree of similarity very well for pigmy chimpanzee with human. is appears of course in figure . e p. chimp branch should be close to c. chimp. despite this error, the tree shows that human, common chimpanzee, pigmy chimpanzee, and gorilla belong to the same cluster. to check the effect of this error on our algorithm, the results of our algorithm are compared to sequence homology. a correlation and significance analysis is also provided. e results of our algorithm are compared to the sequence homology by two methods. first, we use the smith waterman algorithm to calculate the number of identical residues in each pair of protein sequences [ ] . e results of the pid% of nine beta globin sequences are illustrated as a similarity/dissimilarity matrix in table . e larger pid% represents the more similar protein sequences. a correlation and significance analysis is provided to compare our approach in table with pid% in table . e correlation of the two sets of data is sufficiently strong when the correlation coefficient (r) is greater than . . e negative sign of (r) indicates that when the first data set increases, the second data set decreases. we then assess statistical significance for correlation coefficient values greater than . to ensure that they likely do not occur by chance. our sample set is composed of nine protein sequences. erefore, we use degrees of freedom. a t-value of . or greater indicates that a less than . chance of the results occurred by coincidence. e results for correlation coefficients and t-values for our approach are illustrated in table . second, clustalw is a widely used system for aligning any number of homologous nucleotides or protein sequences [ ] . e clustalw program's distance matrix of nine nd protein sequences is illustrated in table . correlation and significance analyses are also provided to compare our approach in table with clustalw results in table . e results of the correlation and significance analyses of our approach and other approaches [ , ] are illustrated in table . our sample set of nd is also composed of nine protein sequences. erefore, we use degrees of freedom and a t-value of . or greater. despite the unusual result for pigmy chimpanzee that appeared in table : e correlation and significance analysis between our similarity analysis results of beta globin protein sequences in table and pid% similarity matrix in table . tables and in [ ] and table in [ ] and clustalw similarity matrix in table . correlation coeff. (r) of our approach t-value of our approach correlation coeff. (r) of [ ] t-value of [ ] correlation coeff. (r) of [ ] (table ) t-value of [ ] ( table , the correlation coefficient of pigmy chimpanzee in our similarity matrix and clustalw matrix is . . is value likely does not occur by chance, as the t-value equals . , as illustrated in table . e comparison between our results and both pid% and clustalw and other approaches' results indicate the utility of our approach. a new graphical representation of protein sequences is introduced. it is the combined intensity level of the amino acids composing any protein sequence. each amino acid in a given protein sequence has its own intensity and intensity level. ey are vectors of n elements as n is the protein sequence length. e combined intensity level is then computed and graphed to represent any protein sequence graphically. our d graphical representation effectively displays differences between protein sequences without degeneracies. e graph does not overlap or intersect with itself. our new descriptor suggested a vector of two elements, which are the mean and standard deviation of the combined intensity level (a t and sa t ). a similarity/dissimilarity analysis is evaluated by computing euclidean distance between each two species' descriptors. examination of similarity/dissimilarity among nine beta globin, nine nd , and coronaviruses protein sequences provided good results compared to previous approaches. e suggested approach is effective for both short and long sequences, and the computations are very simple. furthermore, loss of sequence information is avoided. correlation and significance analyses with pid% and clustalw are also introduced to show the utility of our approach. basic local alignment search tool gapped blast and psi-blast: a new generation of protein database search programs clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice graphical representation of proteins similarity/dissimilarity calculation methods of dna sequences: a survey highly compact d graphical representation of dna sequences, sar and qsar unique graphical representation of protein sequences based on nucleotide triplet codons novel -d graphical representation of proteins representation of protein sequences on latitude-like circles and longitude-like semi-circles on a geometry-based approach to protein sequence alignment dna sequence comparison by a novel probabilistic method -d graphical representation of proteins based on physico-chemical properties of amino acids a d graphical representation of protein sequence and its numerical characterization -d maps and coupling numbers for protein sequences d graphical representation of protein sequences and their statistical characterization dna sequence representation without degeneracy protein map: an alignment-free sequence comparison method based on various properties of amino acids on novel representation of proteins based on amino acid adjacency matrix protein alignment: exact versus approximate. an illustration very efficient search for protein alignment-vespa similarity analysis of protein sequences based on d and d amino acid adjacency matrices a measure of dna sequence similarity by fourier transform with applications on hierarchical clustering a new method to cluster dna sequences using fourier power spectrum descriptors of d-dynamic graphs as a classification tool of dna sequences distribution moments of d-graphs as descriptors of dna sequences similarity studies of dna sequences using genetic methods d-dynamic representation of dna sequences d-dynamic representation of dna sequences spectral-dynamic representation of dna sequences four-component spectral representation of dna sequences d-dynamic representation of protein sequences a novel dna sequence similarity calculation based on simplified pulse-coupled neural network and huffman coding f-curve, a graphical representation of protein sequences for similarity analysis based on physicochemical properties of amino acids a novel descriptor for protein similarity analysis adld: a novel graphical representation of protein sequences and its application comparative analysis of protein primary sequences with graph energy e graphical representation of protein sequences based on the physicochemical properties and its applications a novel method of d graphical representation for proteins and its application novel numerical characterization of protein sequences based on individual amino acid and its application all data are mentioned clearly in the manuscript in section under the title "dataset, technology, and tools." in this section, we illustrate the data in three tables: tables , , and . we also mention that data are downloaded from "gene bank." all data files are with extension", fasta". e authors declare that they have no conflicts of interest. key: cord- -l f gp authors: nan title: oral and poster manuscripts date: - - journal: influenza other respir viruses doi: . /j. - . . .x sha: doc_id: cord_uid: l f gp nan pandemic influenza h n (h n pdm) virus of swine-origin causes mild disease, but occasionally is associated with acute respiratory distress syndrome and death. , it is important to understand the pathogenesis of this new disease. previously we showed a comparable virus tropism and host innate immune responses between h n pdm and seasonal h n influenza virus in the human respiratory tract, however h n pdm virus differed from seasonal h n influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection. we now compare the tropism and host responses elicited by pandemic h n with that of related swine influenza viruses and a pandemic-swine reassortant virus in ex vivo and in vitro cultures of the human respiratory tract and conjunctiva. we have used recombinant virus to investigate the role of the hemagglutinin (ha) and neuraminidase (na) of h n pdm virus in its conjunctival tropism. these findings are relevant for understanding transmission and therapy. fragments of human conjunctiva, bronchi, and lung tissues were cut into - mm fragments within h of collection and infected with influenza a viruses at a titer of tcid ⁄ ml. viruses investigated included h n pdm (a ⁄ hk ⁄ ⁄ ), swine h n virus (a ⁄ swine ⁄ hk ⁄ ⁄ ), which shares a common derivation for seven genes with h n pdm, a natural swine reassortant h n (a ⁄ swine ⁄ hk ⁄ ⁄ ), which has acquired the na gene from h n pdm and other swine influenza h n viruses. reverse genetics derived recombinant viruses with ha and na gene segments of seasonal h n and pandemic h n swapped were also studied. lung fragments were cultured at °c in culture plates; conjunctival and bronchial biopsies were cultured in air-liquid interface at and °c respectively. tissue fragments were infected for h and incubated for , , and h post infection. infectious viral yield was assessed by titration in mdck cells. the infected tissues were fixed with formalin and analyzed by immunohistochemistry for influenza antigen. cytokines profiles induced by influenza virus infected respiratory epithelial cells in vitro were measured by quantitative rt-pcr and elisa. we found comparable replication in seasonal and pandemic h n viruses in human respiratory tract, while the swine influenza a ⁄ swine ⁄ hk ⁄ ⁄ (h n ) virus and a ⁄ swine ⁄ hk ⁄ ⁄ (h n ) virus failed to infect and replicate in human lung ex vivo culture, but it replicated productively in human bronchus ex vivo. interestingly, the swine reassortant influenza h n (a ⁄ swine ⁄ hk ⁄ ⁄ ) virus (with the na from h n pdm) infected and productivity replicated in lung ex vivo and in vitro. pandemic h n pdm virus, but not seasonal h n virus, was able to infect ex vivo cultures of human conjunctiva, suggesting subtle differences in receptor binding profile in h n pdm, seasonal viruses, and the swine related h n viruses. using reverse genetics derived recombinant viruses, we were able to demonstrate that the ha and na segments of h n pdm, but not the polymerase genes, were required for the conjunctival tropism of h n pdm ( figure ). in contrast with highly pathogenic influenza h n virus, which induced high cytokine and chemokine decretion, the related swine viruses, a ⁄ swine ⁄ hk ⁄ ⁄ (h n ), as well as the swine pandemic reassortant virus, a ⁄ swine ⁄ hk ⁄ ⁄ (h n ) we studied were similar to h n pdm and seasonal influenza viruses in their intrinsic capacity for cytokine dysregulation. collectively, our results suggest that pandemic h n pdm virus differs in modest but subtle ways from seasonal h n virus in its intrinsic virulence for humans, findings that are in accord with the epidemiology of the pandemic to date. the ha and na gene segments are key to the conjunctival tropism manifested by the h n pdm virus. the pandemic reassortant influenza h n (a ⁄ swine ⁄ hk ⁄ ⁄ ) virus isolated from swine with the na from h n pdm shares with h n pdm the capacity for productive replication in lung ex vivo and in vitro. these findings are relevant for understanding transmission and therapy. isolation of influenza viruses from specimens is traditionally performed in two classical systems: embryonated chicken eggs and mdck cell culture. nevertheless, several publications are dedicated to the theme of alternative cell culture systems, which may be used for influenza virus isolation and cultivation. [ ] [ ] [ ] this is in part because mdck cells are of animal origin, which means that they cannot be used as a proper model for estimating interactions between a human virus and a human cell culture as a host. a variety of human monolayer and suspension cell cultures have been tested on their capability to support influenza virus replication. among them, some support influenza a virus growth as well as mdck cells do, others support replication of a virus, but do not enable the formation of mature viral particles, whereas others show only a weak level of replication or are not permissive at all. caco- cells, for example, represent a good substitute for mdck cells, because it has been shown that the rate of viral isolation in caco- cells is as effective as in mdck, and sometimes is even better. the success of viral replication is determined not only by the cell culture type, but also by the virus itself. despite the accepted view that it is the type of receptor that defines the interaction between the virus and the host cell, there is evidence that it is not the only factor that predetermines the fate of the cell. the fate of the infected cell can also differ. a series of articles show that apoptosis is the most probable mechanism of cell killing by influenza viruses. , influenza a viruses of different subtypes induce apoptosis to a different extent (e.g. h viruses provoke more strong apoptotic response than h viruses do ). nevertheless, it has been demonstrated that caco- cells do not follow the apoptotic pathway and die through necrosis. the sjpl cell line also dies through necrotic pathway and not apoptosis. the aim of our work was to compare growth characteristics of different flu viruses (e.g. avian, swine, and human) in various human and animal cell cultures and to evaluate their influence on cell culture growth. the parameters measured in the study were as follows: cytopathic changes of cell cultures following virus infection, hemagglutinin production, np synthesis, the dose-dependent effect of infection on cell proliferation, and the ability of viruses to induce apoptosis. influenza viruses used included: highly pathogenic avian h n a ⁄ kurgan ⁄ ⁄ , low pathogenic avian h n a ⁄ gull ⁄ kostanai ⁄ ⁄ , swine h n a ⁄ swine ⁄ ⁄ , human h n v a ⁄ california ⁄ ⁄ , human h n v a ⁄ saint-petersburg ⁄ ⁄ , human h n a ⁄ brisbane ⁄ ⁄ , and human h n a ⁄ brisbane ⁄ ⁄ . the viruses were propagated in -days embryonated chicken eggs, the allantoic fluid was collected, the aliquots were made and stored at ) °c for further use. to evaluate tcid for each virus on all cell cultures, -well plates were used. the cells were seeded ae ml per well (concentration of - ae · cells ⁄ ml). the confluent -h old monolayer was used for viral inoculation. the cells were washed twice with serum-free medium, then ae ml of tenfold viral dilutions from viral aliquots were added and left for min for contact at °c. the cells were then washed to remove the non-attached particles, and the wells were filled with tpck-trypsin ( lg ⁄ ml)-containing medium without bovine fetal serum. the plates were observed daily for cytopathic effect, and the results were evaluated at h after infection for cytopathic effect and by reaction of hemagglutination with suspension of chicken erythrocytes ( ae %). infection of suspension cell cultures was done in centrifuge tubes. cells (concentration - · ) were inoculated with viral dilutions (moi = - ). after min of contact, cells were washed, resuspended in rpmi with trypsin and fetal serum, and seeded in -well plates ( ml in each well). the results were fixed after h, calculating the number of cells grown and estimating the rate of apoptosis by hoechst- staining. cells were grown in -well plates with seeding concentration · cells ⁄ ml. one millilitre of cell suspension was placed in each well, inoculated with viral dilution (moi = - ) and left for h. after, the cells were detached from plastic with versene and calculated in fuks-rosental camera to evaluate the number of cells. the monoclonal antibodies obtained in research institute of influenza towards viral nucleoprotein np were used following the standard protocol described in. for all viruses tested, mdck turned out to be more permissive than sp cell culture. avian viruses, independently of their pathogenicity, replicated efficiently on both animal cultures tested. human h n and h n viruses demonstrated weaker replication in sp cells. the most significant differences were seen for swine influenza and pandemic h n v viruses which replicated in mdck cells at the rates comparable with other viruses, but showed poorer growth in sp cell line (see table ). human cell lines displayed clear differences in their susceptibility to viruses of various origins. avian influenza viruses replicated in all cell lines except girardi heart, and the most intense replication rate was observed for ecv- , l- , and rd lines. a- and a- were poorly infected, as well as all suspension cell lines tested. seasonal human h n , as well as h n viruses, replicated in all cell cultures tested, but the rate of infectivity was rather low in practically all cultures tested with the exception of rd and t- g cell lines. strikingly, swine influenza virus and human pandemic h n v viruses didn't replicate well in any of human lines tested. a weak replication rate was observed in ecv- , rd, and t- g, but in general, human cell lines were the titers produced by swine and pandemic influenza viruses are shaded in grey. *low-pathogenic avian influenza virus; **highly-pathogenic avian influenza virus poorly susceptible to pandemic h n v. swine influenza virus differed because it infected weakly a- and girardi heart cell cultures, which was not the case for h n v viruses. our study has shown that all influenza viruses were able to induce apoptosis in the cell cultures tested. the degradation of chromatin found in the nucleus with hoechst- staining was seen before the first symptoms of cytopathic effect (cpe) in monolayer of cells. in cell cultures where the cpe was not visible, high doses of virus still induced apoptotic response. the process of apoptosis is rather well studied in mdck cells and some other cell types, so we've focused on three human monolayer cell cultures that are relatively poorly studied: a- , ecv- , and flech. these cell cultures are less susceptible to viral infection, and besides, it was interesting to find out whether the viruses that do not cause any cpe do infect these cultures. a- turned out to be most sensitive to apoptotic response, while flech turned out to demonstrate weak reaction. time needed for apoptosis induction by different flu viruses also varied. the earliest apoptosis was noted for h n and h n viruses and h n viruses induced apoptosis at about h postinfection. it is well-known that apoptosis can be induced only by a reproducing virus, and that uv-kills viruses that are not capable of it. we tested whether swine and pandemic h n v viruses (that do not show cpe in these cultures) do replicate in them and induce apoptosis with the help of monoclonal antibodies against viral np. the obtained data show that they indeed do replicate in these cell cultures, as we observed np fluorescence, and that they also induce apoptosis (see table ). we've shown earlier thus, we've tested the ability of swine and pandemic h n v viruses in this aspect. it was shown that these viruses were comparable with the effect seen for seasonal h n virus. moreover, swine influenza virus induced stronger apoptotic response in hemablastoid cell lines in comparison with pandemic h n v viruses, which also have a swine origin. we also checked the ability of flu viruses to influence monolayer cell cultures growth. the data clearly indicated that only ecv- endothelial line and t- g glioblastoma line displayed cell proliferation in response to low moi. apoptosis wasn't registered in these stimulated cultures, apparently because the moi was very low. all the other monolayer cultures didn't respond to low moi by stimulation of their proliferation. interaction between an influenza virus particle and a host cell can follow several scenarios. cpe seen in infected cells is accompanied with high rates of viral particles production and leads to cell death. the death itself may be through apoptotic or necrotic pathways. , also, infection process in low doses can stimulate cell proliferation -the effect seen for hemablastoid lines, histiocytes, peripheral blood cell lines, , and in glioblastoma and endothelial cell lines as it was described here. considering the origin of ecv line, these cells bear all the antigenic, biochemical, and physiological traits of umbilical cord and are actively used in pharmacological tests as well as glioblastoma cells; they also are of special interest for oncogenesis studies. table . replication, apoptosis induction, and np synthesis of influenza viruses in a- , ecv- and flech cell cultures. the numbers represent the log tcid ⁄ ae ml calculated by reed-muench method as described in. the ()) symbol means that no cpe could be observed in any dilution and no hemagglutination could be registered. the (+) symbol means that apoptosis was observed with hoechst- staining though the productive replication and production of progeny viruses in human cell lines was generally low, it is evident that viral infection does occur in these cells, even for swine and h n v viruses. it can be demonstrated by the presence of np de novo synthesis and by stimulation of virus-induced apoptosis. in fact, we observe a contradiction: avian influenza viruses actively reproduce in human cell lines, but we do not see their vast spreading in human population, while h n v viruses that hardly replicate in all human cultures tested have caused the latest pandemic. influenza viruses continue to cause problems globally in humans and their livestock, particularly poultry and pigs, as a consequence of antigenic drift and shift, resulting frequently and unpredictably in novel mutant and reassortant strains, some of which acquire the ability to cross species barriers and become pathogenic in their new hosts. long-term surveillance of influenza in migratory waterfowl in north america and europe have established the importance of anseriformes (waterfowl) and charadriiformes (gull and shorebird) in the perpetuation of all known subtypes of influenza a viruses. the available evidence suggests that each of the hemagglutinin (ha) and nine neuraminidase (na) subtype combinations exist in harmony with their natural hosts, cause no overt disease, and are shed predominantly in the feces. , in this study we determined the subtypes and prevalence of low-pathogenic influenza a viruses present on the territory of kazakhstan in - and further analysed the ha and na genes of these isolates in order to obtain a more detailed knowledge about the genetic variation of influenza a virus in their natural hosts. (institute for biological safety problems, gvardeiskiy, zhambyl oblast, kazakhstan)). samples that were identified as influenza a virus positive by matrix rrt-pcr were thawed, mixed with an equal volume of phosphate buffered saline containing antibiotics (penicillin u ⁄ ml, streptomycin mg ⁄ ml, and gentamicin lg ⁄ ml), incubated for minutes at room temperature, and centrifuged at g for minutes. the supernatant ( ae ml ⁄ egg) was inoculated into the allantoic cavity of four -day old embryonated hens' eggs as described in european union council directive ⁄ ⁄ eec. embryonic death within the first hour of incubation was considered as non-specific, and these eggs were discarded. after incubation at °c for days the allantoic fluid was harvested and tested by haemagglutination (ha) assay as describe in european union council directive ⁄ ⁄ eec. in the cases where no influenza a virus was detected on the initial virus isolation attempt, the allantoic fluid was passaged twice in embryonated hens eggs. the number of virus passages in embryonated eggs was limited to the maximum two to limit laboratory manipulation. a sample was considered negative when the second passage ha test was negative. the subtypes of the virus isolates were determined by conventional haemagglutination inhibition (hi) test and neuraminidase inhibition (ni) test, as describe in european union council directive ⁄ ⁄ eec. rna extraction and pcr with specific primers rna was extracted from infective allantoic fluid using rneasy mini kit (qiagene, gmbh, germany) according to the manufacturer's instructions. the rna was converted to full-length cdna using reverse transcriptase. the rt mix comprised ae ll of dmpc water, ll of · first strand buffer (invitrogen), ae ll of mm dntp mix (amersham biosciences), ll of mm uni primer, u of rnaguard (amersham biosciences), u of mmlv reverse transcriptase (invitrogen) and ll rna solution in total volume of ll. the reactions were incubated at °c for minutes followed by inactivation of the enzyme at °c for min. pcr amplification with ha and na gene specific primers was performed to amplify the product containing the full length ns gene. twenty-five microliter pcr-mix contained · platinum taq buffer (invitrogen), lm dntp, ae mm mgcl , nm each of fw primer and rw primer, u platinum taq dna polymerase (invitrogen) and ll cdna. reactions were placed in a thermal cycler at °c for min, then cycled times between °c seconds, annealing at °c for seconds, and elongation at °c for seconds and were finally kept at °c until later use. sequences of the purified pcr products were determined using gene specific primers and bigdye terminator version ae chemistry (applied biosystems, foster city, ca), according to the manufacturer's instructions. reactions were run on a abi tm dna analyzer (applied biosystems). sequencing was performed at least twice in each direction. after sequencing, assembly of sequences, removal of low quality sequence data, nucleotide sequence translation into protein sequence, additional multiple sequence alignments, and processing were performed with the bioedit software version ae ae ae with an engine based on the custal w algorithm. the phylogenetic analysis, based on complete gene nucleotide sequences were conducted using molecular evolutionary genetics analysis (mega, version ae ) software using neighbor joining tree inference analysis with the tamura-nei c-model, with bootstrap replications to assign confidence levels to branches. [ ] [ ] [ ] [ ] ha and na sequences obtained from genbank the ha and na gene was analyzed both with selected number of influenza isolates and in comparison with virus genes obtained from genbank were used in phylogenetic studies [ ] . the nucleotide sequence data obtained in this study has been submitted to the genbank database and is available under accession numbers fj , fj ae , fj , fj , gu -gu for ha and fj , fj ae , fj , fj , gu -gu for na. avian influenza prevalence in our study h , h , and h influenza a virus subtypes were found to circulate at the same time, in the same geographic region in the kazakhstan. this finding most likely indicates the existence of a large reservoir of different influenza a viruses in kazakhstan. we analyzed the ha and na gene sequences of the eight influenza a viruses isolated in kazakhstan together with selected number of isolates, reported between year to , and previously published in the genbank. phylogenetic analysis of the h ha gene showed that all viruses separated into the american and eurasian lineages ( figure ). an evolutionary tree suggests that north american isolates have diverged extensively from those circulating in other parts of the world. geographic barriers which determine flyway outlay may prevent the gene pools from extensive mixing. the lack of correlation between date of isolation and evolutionary distance suggests that different h ha genes co circulate in a fashion similar to avian h ha genes and influenza c genes, implying the absence of selective pressure by antibody that would give a significant advantage to antigenic variants. analysis of phylogenetic relationships among the ha ha genes reported in this study clearly shows that viruses belong to the western pacific flyway, one of the major migratory flyways in this region that have subsequently spread throughout eurasia. these findings provide further evidence of the dynamic influenza virus gene pool in this region. along the western pacific migratory flyway, the influenza virus gene pool in the domestic waterfowl of southern china has 'mixed' longitudinally with viruses isolated from japan, mongolia, and siberia. however, it appears that there has also been 'mixing' latitudinally through overlapping migratory flyways, thereby facilitating interaction between the influenza virus gene pool in domestic waterfowl in the eastern and western extremities of the eurasian continent. this helps to explain the latitudinal spread of the qinghai-like (clade ae ) h n virus in the last years, while h n outbreaks in korea and japan may represent the longitudinally transmitting pathway. ha of subtype h so far has been found exclusively in shorebirds, such as gulls, and in a pilot whale (potentially a spillover from shorebirds), but not in other avian species that are natural hosts of influenza a virus, such as ducks and geese; therefore the study of the evolution of these viruses is very interesting. phylogenetic analysis h ha gene revealed three significantly different evolutionary lines: an american line, a european line, and a line comprising the isolates from america and eurasia. further we analyzed na genes of influenza viruses (figure ) . the na gene is important both because of its functional role in promoting the dissemination of the virus during infection, and because, like ha, it is a principal target of the immune system. it was shown that phylogeny of na genes of influenza have the same properties as hemagglutinin. na genes of kazakhstanian viruses belong to eurasian lineage of virus evolution. obtained data are important for surveillance and diagnostics because some of the lpai viruses examined in this study can infect and be shed by chickens and turkeys and may have epidemiology potential during further recombination with other influenza viruses. influenza virus is divided into different subtypes based on hemagglutinin (ha) and neuraminidase (na) on the virus surface. within each subtype, ha continues to mutate and produce immunologically distinct strains, as antigenic drift. the continuous mutation of influenza virus (iv) is important for annual epidemics and occasional pandemics of disease in humans. antigenic drift requires vaccines to be updated to correspond with the dominant epidemic strains. in humans, ivs show both antigenic drift frequently. in contrast, ivs from birds are in evolutionary stasis, and they show little amino acid changes. , the reason is that ivs in bird intestine are not subjected to strong immune selection. hemagglutinin (ha) gene of influenza a virus encodes the major surface antigen, which is the target for the protective neutralizing antibody response that is generated by infection or vaccination. in humans, influenza a viruses show antigenic drift with amino acid changes in the globular head of the ha so as to evade herd immunity of the population. on the contrary, avian influenza a viruses show evolutionary stasis in wild birds. h aivs have occurred frequently in chicken farms in the world. although vaccination is not permitted, h n aivs have circulated in taiwan for a time. the seroprevalence in chicken flocks reaches about % in the field. h n aivs invades internal organs, such as kidney and lung. thus, viruses in chicken flocks are pressured into antibody selection. here, we report that h n aivs in the field have showed evolutional changes instead of evolutional stasis. in response to requests from poultry farmers for diagnostic investigations of illness in poultry flocks, the authors did necropsy at the pen-site. after careful examination, tracheae were taken and kept in cold for virus isolation in the laboratory. for avian influenza virus isolation, trachea was homogenized : in tpb with antibiotics. the homogenate was frozen and thawed three times and then centrifuged at g for minutes. the supernatant was passed through a ae lm filter. the homogenate was examined for the presence of virus by inoculation into five -to -day-old specific-pathogen-free (spf) chicken eggs for two passages. thirteen h n aivs were isolated in this laboratory during and from different parts of taiwan. besides the viruses isolated in this laboratory, the ha sequences of chicken h n aivs were from the genbank. the accession numbers of hemagglutinin of aiv reference strains included in this study were as the following: g ⁄ , dq ; g ⁄ , dq ; ⁄ , dq ; na ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ns ⁄ , dq ; sp ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; pf ⁄ , dq ; pf ⁄ , dq ; pf ⁄ , dq ; a ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ⁄ , dq ; ch ⁄ , dq ; ⁄ , dq ; a ⁄ , dq and ⁄ , dq . the viruses isolated were propagated in the allantoic cavities of -day-old embryonated spf eggs for hour. the virus rna was extracted using qiaamp viral rna miniprep kit (qiagen) . six-week-old balb ⁄ c mice were injected emulsion intraperitoneally with lg of purified and concentrated a ⁄ chicken ⁄ taiwan ⁄ v ⁄ (h n ) virion with complete freund's adjuvant. every two weeks, the mice were boosted supplementary five times with lg of virion in incomplete freund's adjuvant. when the mice were boosted, blood was collected from tail vein and tested by the western blot assay to check the antibody titers. the mice were then injected intraperitoneally with lg of virion at week . five days after the last injection, the splenocytes in the mice were fused with myeloma cells (sp ⁄ -ag ). one week before fusion, the myeloma cell line was expended in dmem medium (hyclone laboratories, logan, ut) with % fetal bovine serum at °c to ensure they were in the exponential growth phase. the spleen cells from immunized mice were washed, harvested, and mixed with the previously prepared myeloma cells and fused by gradually adding % polyethylene glycol- . the resulting pellet was plated into well tissue culture plates. only the fused cells grew in medium with hypoxanthine-aminopterin-thymidine (hat). with fresh medium replacement over weeks, the hybridomas were ready for screening. hightiter monoclonal antibody (mab) preparations were obtained from the ascetic fluid of mice injected with the selected hybridoma clones. the antibody from mouse ascetic fluids was purified by precipitation with ammonium sulfate, then aliquoted and frozen at ) °c, avoiding repeated freezing and thawing. eventually, six mabs were obtained and named ch -d , eb -b , eb -e , eb -f , ff -f , and ff -f , respectively. the hi test was performed following a standard method. all the viruses were diluted twofold and reacted with % chicken erythrocytes in the v-bottomed microtiter plate by the hemagglutination test. after agglutination, four hemagglutinating units of a ⁄ chicken ⁄ taiwan ⁄ v ⁄ (h n ) and ascetic fluids from the immunized mice of the six mabs were prepared for hi test. hi titers of or more were regarded as positive. the cases submitted for diagnosis from chicken farms had respiratory signs, increase in mortality, or drop in egg production (e.g. egg production dropped from % to %). the extent of drop in egg production depended on the chicken ages. for example, the age of case was weeks, a stage of increasing egg production. however, after h n aiv infection, the egg production decreased % instead of increasing and then stayed at % for a week. the infected chickens showed signs of decreasing activity, anorexia from g per bird to g per bird, and respiratory signs. case showed infection in the second floor first and then transmitted to third and fourth floor, indicating that the virus transmitted by air or human movement. however, most cases showed air borne transmission from one flock to another in spite of enforcing restrictions of persons entering the poultry pens and changing clothes and booths. in most cases, males' mortality was higher than that of female pen mates. by comparing the sequences of ha of those h n viruses, we found that amino acid changes in ha were higher than those in ha , showing that antigenic changes on the globular head of ha molecule rather than randomly on the whole ha protein, indicating that h n viruses in taiwan had been selected in the presence of antibody pressure. the aa residues and changes that showed yearly trends were the followings: a- s, i s, v i, n s, e k, l m, e d, q k, a v, or t, s n, s r, k n, y d, n t, s i, g d, l v, i v, g e, t n, g s, a v, k e, d n, i m, and m i. however, their significance on antigenic variation was previously unknown. by hemagglutinination inhibition (hi) assays, except mab ch -d , all other monoclonal antibodies elicited from v ⁄ showed different hi titers with the different h n viruses (table ). however, those mabs showed negative hi to and , the early h n strains. this indicated that the epitopes recognized by those mabs were undergoing antigenic drift. introduction aquatic birds are recognized as the natural reservoirs of the influenza a virus as all known subtypes (h -h , n -n ) have been found in them. phylogenetic analyses of influenza viruses found in other animals revealed that all were directly or indirectly derived from viruses resident in aquatic birds. however, the prevalence, movement, and evolutionary dynamics of influenza viruses in these avian hosts have not been well defined. southern china was hypothesized to be an 'epicenter' for the generation of human pandemic influenza viruses as all major influenza pandemic viruses in the th century emerged from this region. the ecological background that facilitates the occurrence of these pandemic influenza strains has not been fully explored. in the past two decades, four lineages, belonging to h n , h n , and h n viruses, have become established and long-term endemic in different types of poultry in this region. [ ] [ ] [ ] some of these viruses were disseminated to many countries in eurasia and africa and have continued to cause sporadic human infection, posing a persistent pandemic threat to the world. in the mean time, the endemic influenza lineages have undergone extensive genetic reassortment events giving rise to many variants, dramatically increasing the genetic diversity of the influenza virus in this region. questions remain as to how and where these viruses emerged, and what were the sources of the gene segments incorporated within the novel reassortant variants of the h n , h n , and h n virus lineages. to address these questions, surveillance of influenza in migratory and domestic (sentinel) ducks has been conducted since at poyang lake, the biggest fresh-water lake and the major migratory bird aggregation site in southern china. the aim of this study is to identify the prevalence, seasonality, and movement of virus between migratory and domestic ducks. migratory ducks were captured during over-wintering, from november to march. cloacal swabs and blood samples were collected from each individual bird. all birds were released after sampling. to observe the interaction between migratory ducks and domestic birds, we also sampled domestic ducks from two duck farms (designated as sentinel ducks) surrounded by rice fields and inaccessible to other types of poultry, but accessible to migratory birds. that is, the sentinel ducks share the same water body with migratory ducks and have the chance to spread viruses to each other. for sentinel ducks, sampling was conducted fortnightly, all year-round, on the two farms from august onwards. cloacal swabs and fresh fecal droppings were taken. about birds were randomly sampled fortnightly from these farmed ducks. all swabs were soaked in vials containing ae ml transport medium with antibiotics and kept on ice-packs during sampling and immediately stored in ) °c freezers for further use. blood samples from migratory ducks were treated according to methods previously described. serological survey and virus subtyping in migratory and sentinel ducks used hemagglutination inhibition (hi) and neuraminidase inhibition (ni) tests as previously described. for isolates that were not identified by reference antisera, subtypes were determined by rt-pcr using subtype specific ha and na diagnostic primers. prevalence and seasonal patterns of influenza virus in migratory and sentinel ducks during during - a total of cloacal swabs from migratory ducks and cloacal or fecal swabs from sentinel ducks were collected at poyang lake. from these specimens, influenza isolates were obtained from migra- tory ducks and from sentinel ducks; isolation rates of ae % and ae %, respectively (table ) . it was noted in sentinel ducks that virus occurrence formed a seasonal peak from november to february, which completely overlapped the over-wintering months of migratory ducks. this suggests that virus movement or transmission between migratory and sentinel ducks occurred during this period at poyang lake. thirty positive samples (hi titer ‡ ) were identified from blood samples collected during november and december in . among these, samples were positive to h , were positive to h , were positive to h , and were positive to h . one serum sample was positive to both h and h , which suggested co-infection of influenza virus in migratory ducks might occur in natural conditions. poyang lake, which is located in the northeastern part of jiangxi province, is the largest freshwater lake in china and is part of the eastern asia-australia migration route. every year, hundreds of thousands of migratory ducks congregate at poyang lake during the migration season. recent farming practice involves raising domestic waterfowl in dense populations in the poyang lake region. farmraised domestic waterfowl are allowed to feed in and share the same water body with migratory birds, thereby facilitating direct interactions between domestic waterfowl and freeranging migratory birds. this makes poyang lake an ideal site to observe the dynamics of influenza virus interactions between migratory and sentinel ducks in southern china. in our longitudinal surveillance during [ ] [ ] [ ] [ ] [ ] [ ] , the overall virus isolation rate from migratory ducks was less than %, which suggests a low prevalence of viral infection during the birds' southern migration. similar results have been observed in taiwan, which is also an important stopover site for migratory birds along the eastern asia-australia migration route during years of surveillance. the overlap in seasonal patterns of virus infection between migratory and sentinel ducks found in our study suggests that virus movement or transmission between migratory and sentinel ducks occurred during the period of time migratory birds were at poyang lake. the ha subtypes harbored in migratory and sentinel ducks were similar in our study. for migratory ducks, h , h , h were the predominant subtypes, while h , h , and h were the major subtypes in sentinel ducks. hpai h n was only detected from migratory ducks in early on two sampling occasions. from phylogenetic analyses the h n viruses isolated from migratory ducks were closely related to the viruses endemic in domestic poultry in southern china. therefore, it appears that h n viruses endemic in domestic poultry could be transmitted to migratory ducks via close contact in southern china. only lp h viruses were detected from sentinel ducks at poyang lake during this period. whether h n virus infection was absent from sentinel ducks at poyang lake needs further investigation. serological surveys provided further evidence for the prevalence of aiv in migratory ducks at poyang lake. the serological results in did not match well with the epidemiological results during [ ] [ ] [ ] [ ] [ ] [ ] , which suggests that influenza virus infection in migratory birds could be influenced by multiple factors, such as host immune status, population size, spatial and temporal variations, and migration routes. southern china has the biggest domestic duck population in the world. our study demonstrates that dynamic interactions between migratory ducks and sentinel ducks occurred frequently throughout the surveillance period. thus, sentinel ducks could be treated as intermediate hosts between the ''real gene pool'' from migratory ducks and domestic poultry in the whole influenza virus ecosystem. a sentinel duck sampling system may be a feasible method to represent the viruses in the natural gene pool and a baseline for virus or gene interactions between migratory and domestic ducks. further investigations and surveillance are required to better understand the role of the domestic duck population in facilitating virus interactions and the generation of genetic diversity. two distinct lineages of h n influenza viruses represented by a ⁄ chicken ⁄ beijing ⁄ ⁄ (ck ⁄ bei-like) and a ⁄ quail ⁄ hong kong ⁄ g ⁄ (g -like) have become established and endemic in poultry in southern china. these established h n lineages continue evolving to generate many different reassortant variants (or genotypes) , and are causing sporadic cases of human infection. , studies of h n viruses isolated from pigs in hong kong and shandong province have also raised the possibility of reassortment with human-like viruses from pigs. , in addition, h n viruses isolated beyond the late s had preferential binding with a- , -neuacgal human-like receptors. these observations suggest that the h n influenza viruses still have pandemic potential. unlike highly pathogenic h n influenza viruses that have been rarely detected in the live-poultry markets in hong kong since , h n viruses are still frequently isolated in our surveillance program. therefore, we try to understand the continuing evolution of h n viruses through genetic characterization and phylogenetic analyses of the viruses isolated in hong kong live-poultry markets from to . a total of terrestrial poultry were sampled at different live-poultry markets in the hong kong sar between january and december . of those samples, were from chickens and the others were from minor poultry species including chukar, pheasant, guinea fowl, silky chicken, and pigeon. fecal droppings, cloacal and tracheal swabs, drinking water, and environmental samples from cages were collected into transport medium. viruses were isolated in -to -day old embryonated eggs as described previously. virus isolates from positive sampling occasions were selected for sequence analysis. rna extraction, cdna synthesis, and pcr were carried out as described previously. dna sequencing was performed using bigdye terminator v ae cycle sequencing kit on an abi dna analyzer (applied biosystems) following manufacturer's instructions. all sequences were assembled and edited with lasergene ae (dnastar, madison, wi) software. sequence alignment and residue analysis were performed with the bioedit sequence alignment editor, version ae . all eight gene segments of sequenced viruses were characterized and analyzed phylogenetically together with virus sequence data available in public databases. maximum-likelihood trees were constructed using garli ae . estimates of the phylogenies were calculated by performing neighbor-joining bootstrap replicates using paup* ae . systematic surveillance of live-poultry in hong kong from to resulted in h n isolates from samples (overall isolation rate, ae %) ( table ). there were strains isolated from chicken samples (isolation rate, ae %). of these viruses, four were isolated from tracheal swabs (isolation rate, ae %), while isolates were isolated from cloacal or fecal swabs (isolation rate, ae %). an additional isolates were collected from drinking water samples (isolation rate, ae %). there were strains of h n viruses isolated from minor poultry samples (isolation rate, ae %) ( table ) . of these viruses, only one was isolated from tracheal swabs (isolation rate, ae %), whereas strains of viruses were isolated from cloacal or fecal swabs (isolation rate, ae %). the isolation rate in drinking water in minor poultry was again higher when compared with other sampling methods with strains isolated from drinking water samples (isolation rate, %). taken together, these findings suggest that the h n viruses mainly replicated in the intestinal tract of chickens and minor poultry species. also, the high isolation rate in drinking water samples could be a sensitive indicator for monitoring the prevalence of h n viruses in the field. to better understand the evolutionary pathway of h n viruses in southern china, representative viruses, isolated from hong kong live-poultry markets from to , were sequenced and genetically characterized. phylogenetic analysis of the h ha gene revealed that ck ⁄ bei-like viruses were predominant and one chicken isolate had a g -like ha gene ( figure ). this is the first time the g like h ha gene has been detected in chickens from livepoultry markets in hong kong. the ck ⁄ bei-like lineage is further divided into two subgroups as previously described. subgroup is represented by qa ⁄ st ⁄ ⁄ and subgroup is represented by dk ⁄ hk ⁄ y ⁄ . all h n viruses in this study belonged to subgroup of the ck ⁄ bei-like lineage except for the virus with the g -like ha gene. phylogenetic analysis of the na gene also showed a similar evolutionary pattern to the ha gene with all viruses clustered within the ck ⁄ bei-like lineage. these results revealed that ck ⁄ bei-like viruses are predominant in both chickens and minor poultry. all of the pb , pa, np, ns and m genes clustered with those of h n lineage viruses previously prevailing in ter- restrial poultry in southern china. phylogenetic analysis of the pb gene revealed three different lineages; g -like (n = ), ck ⁄ sh ⁄ f ⁄ -like (n = ), and unknown avian (n = ). the sh ⁄ f ⁄ -like lineage (or f ⁄ -like) was previously reported in eastern china and was used previously for vaccine production in an intensive vaccination program. this pb gene lineage was also distinguishable from the ck ⁄ bei-like lineage and its presence in the viral genome may be due to reassortment between the vaccine strain and field isolates, followed by selective establishment in terrestrial poultry. gene constellation analyses of the viruses revealed six genotypes. thirty-four of the viruses analyzed belonged to two genotypes, b and b , which were also the prevailing reassortants found in other provinces in southern china since . the remaining sixteen viruses belonged to four novel genotypes that have not been identified before in this region. characterization of h n influenza viruses isolated from live poultry in hong kong markets from a year surveillance program revealed that ck ⁄ bei-like viruses were predominant in southern china and were continuing to evolve. two recognized and four novel genotypes were identified in this study. one characterized virus, ck ⁄ hk ⁄ nt ⁄ , had a g like ha gene (the first time this has been detected in hong kong poultry markets) that showed a close relationship with two human h n strains isolated in . g -like viruses were usually detected and caused outbreaks in chickens of middle eastern and european countries, [ ] [ ] [ ] and minor poultry, mainly quail, in southern china. whether the g -like virus was transmitted from china to middle eastern and european countries, as the highly pathogenic h n virus did in the last five years, or vice versa, is still unknown. since the ck ⁄ hk ⁄ nt ⁄ strain clustered with other g -like strains isolated previously in minor poultry in southern china, the g -like viruses in chicken may be due to interspecies transmission from minor poultry species. genetic studies demonstrated that reassortants with genotypes b and b persistently occurred in either chickens or other minor poultry species from to . other genotypes that were prevalent in southern china might be being gradually replaced and four novel genotypes were identified in this study. these novel genotypes were generated through reassortment of viruses with different lineages. a newly emerged f ⁄ -like lineage originating from eastern china is responsible for generation of some of the novel genotypes found in this study. the ck ⁄ bei-like lineage is gradually being replaced by f ⁄ -like lineages which are becoming dominant in northern and eastern china. , animal experiments have also demonstrated that f ⁄ -like viruses are more effective in replication and transmission in chickens compared with ck ⁄ bei-like viruses. since the f ⁄ -like lineage of the pb gene has been introduced into southern china, this newly emerged lineage may have a higher tendency to replace the rnp genes in the circulating ck ⁄ bei-like viruses and subsequently become the endemic virus in terrestrial poultry. in vietnam, the modelling of the pandemic h n progression estimates that ( - ) pigs might be exposed to the virus on the basis of cases among swine owners ( - ). a poor level of biosecurity, high animal densities, and a mix of species could increase the risk of influenza virus flow, persistence, and emergence on swine and poultry farms. this study was set up in the red river delta, where a third of the national pig husbandry is produced. the aims are to give preliminary information of the epidemiological state of swine influenza and in order to further assess the risk of infection of swiv, through cross-species transmissions from poultry to pigs. this paper will present the preliminary results on swiv and the risk factors of pig seropositivity in vietnam. a cross-sectional study was conducted in two provinces of the red river delta in april . pig farms were randomly selected from nine communes representative of at risk area of avian h n . in each farm, pig and poultry were sampled and collected to virological and serological analyses. interviews were conducted in all farms by trained interviewees. questionnaires included closed and open questions on ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - livestock husbandry ⁄ management and household characteristics, such as herd size and structure, health history and vaccination, pig housing, watering and feeding system, reproduction, purchasing of animals, biosecurity measures, pig contact with poultry, and environmental factors. the virological detection assay was performed on pools of nasal swab specimens from pigs. we investigated whether real-time rt-pcr assay could detect gene m on pools of nasal swab specimens before attempting virus isolation from individual nasal swab specimens. the poultry and pig sera were tested against influenza type a with an enzyme-like immunosorbant assay (elisa) competition test idvetª. this commercial kit is designed to specifically detect antibodies directed against the np protein antigen of influenza type a viruses. the positive serum samples were examined in hemagglutination inhibition (hi) to determine antibody titers and subtypes. the hi test was tailored for h , h , and h subtypes in pigs and h and h subtypes in poultry. seroneutralization tests by pseudo particles were used to test the presence of antibodies directed against h subtype. we analysed the data for relationships between influenza a serological status (the outcome variable) and possible risk factors using r version ae ae (r development core team). the statistical unit was the individual. initially, the quantitative variables were encoded into categorical variables according to the quartiles or median. descriptive statistics (e.g., means or medians, proportions, standard deviations) were calculated for all herd-level and commune level predictors to assist in the subsequent modeling process. we also performed the independence test among all variables to determine if variables were dependant. then, univariate analysis of potential risk factors for the pigs being positive for swiv and estimation of odds ratios were performed using generalised linear mixed models with binary outcome and logit link function for each herd-level and commune-level variable to determine which variables were individually associated with influenza a seropositivity at a significance level of p < ae . herd and commune of residence were included as a random effect to account for the correlation of observations at the herd level. the third stage of the analyses included the four herdlevel variables found to be significantly (p < ae ) associated with influenza a seropositivity. an automatic process using all possible associations between the selected variables was computed into a mixed logistic regression models, with random effects. when two variables were collinear, as determined before, only one variable was likely to enter the multivariable model, and therefore, the selection of which collinear variable to enter the model was guided by biological plausibility and statistical significance. all of the pools of nasal swabs were rt-pcr negative. the maximal possible prevalence considering perfect diagnostic tests would be of ae % at a confidence level of %, in an infinite population within these regions (win-episcope ae ). six hundred-and-nine pig sera were tested in nonvaccinating farms. the herd seroprevalence of swine influenza in the commune previously infected by the avian h n in the red river delta raised by ae % [ ae ; ae ] in april . but among seropositive farms, only four had at least two seropositive pigs. the within-herd seroprevalence is very low, and no seropositivity was detected in the majority of farms. estimates had large confidence intervals due to small sample sizes. the individual seroprevalence raised ae % [ ae ; ae ]. the subtyping of seropositive sera is still in process. descriptive statistical analyses on five major risk factors of swiv: farm size, breeding vs. fattening, purchasing, percentage of family income, and poultry production, were conducted. based on this analysis, three types of farming systems were identified and included in mixed models ( table ) . percentage of family income by pig production and poultry production were not differentiating factors for this typology. whereas types and seem to be specialized in fattening, the type produces and might sell piglets on the farm site. the exploration of the different variance components indicated that the random effect variances were mainly associated with the herd, while the commune did not seem to have any effect. therefore we included in all models only the herd as a random effect. the random effect term for herd was modelled, assuming a normal distribution with a table . typology of farming system type : large fattening farms largest scale production, with more than pigs per year specialized in fattening, and purchase more than pigs per year type : small fattening farms small scale of production, with less than pigs per year specialized in fattening, and purchase less than pigs per year type : medium breeding-fattening farms medium scale of production, with less than pigs per year breeding and fattening piglets, with rare purchase common variance [$n( ,r herd)]. the univariate analyses were conducted on variables and typology variables, with herd as random effect. some coefficient or confidence intervals were inconsistent because of small effectives, especially for the percentage of self-product culture or the pig freegrazing because of the lack of positive results in the dataset. the only one significant (p value < ae ) parameter was the percentage of pig sales in the familial annual income. surprisingly, common risk factors of swine influenza infection, such as farm size, animal movements, and sanitary parameters got low odds ratio individually (without being significant); the typology provides the hypothesis of complex interactions effects that increase the risk of infection. as shown in table , the farming system type got a higher seroprevalence of ae % [ ae - ae ] and a higher risk indicator, with or = ae (p-value = ae ) in comparison with type . this finding was not significant. in the multivariate mixed model, the percentage of familial income provided by pig production was the only one significant variable, with or = ae [ ae - ae ]. the focus on diseased animals in the winter-time is usually required in order to increase the likelihood to isolate the virus, although the isolation rate on healthy or clinical samples never exceed %. the season and the lack of disease reports might explain the difficulties to detect influenza viruses. additionally, the pooling method tends to decrease the isolation rate because of a dilution effect, potential presence of pcr assay inhibitors, or uneven distribution of virus in the sample. our seroprevalence results must be confirmed and the subtypes identified, especially because we found only one positive animal in a few farms that could be attributed to false positive results of the elisa test (performances are not known). these preliminary results are in favor of a virus circulation at low level in the spring, but must be completed by further surveys in the winter and before the new year (têt celebration) when pig production, trade, and movement increase at their maximum. no clear prior information on the expected prevalence of swine influenza in vietnam, tests sensitivity, and speci-ficity could be obtained from literature or reliable sources. bayesian methods will be carried out in the future in order to compute prevalence and ⁄ or to estimate the probabilities of freedom. the risk factors analysis was limited by the lack of positive results. further studies are necessary to identify the at-risk season and type of farming systems at risk of swine influenza infection. however, this investigation of risk factors leads to the hypothesis that medium size breeding-fattening farms had a higher risk than large or small size fattening farms. further investigation are needed to precise this typology. the risk of swiv infection increases with a combination of three major factors. poultry production does not seem to play any role on swine infection. the generalized linear mixed model afforded to take into account all the non investigated parameters at the herd level. although we investigated the most common risk factors of swine influenza infection covering different kind of fields, the herd random effect might explain risk variations. mixed models have become a frequently used tool in epidemiology. due to software limitations, random effects are often assumed to be normally distributed. since random effects are not observed, the accuracy of this assumption is difficult to check. further studies, such as case-control or cohort studies could help to identify more precisely risk factors of swine influenza seropositivity, as these study designs are more adapted than cross-sectional studies. the concept that swine are a mixing-vessel for the reassortment of influenza viruses and for the emergence of pandemic influenza viruses has been re-enforced by the emergence of the recent pandemic. the pandemic h n virus of (h n pdm) is believed to have emerged through the reassortment of north american triple reassortant and eurasian avian-like swine influenza viruses. since the immediate precursor of this pandemic virus has not yet been identified, it is not possible to be definite whether the reassortment leading to the pandemic occurred in swine, but swine influenza viruses are the nearest known ancestors of each gene segment of h n pdm. , the mechanisms of pandemic emergence are not clear. it is believed that the pandemics of and arose through reassortment of the pre-existing human seasonal influenza virus with avian influenza viruses, and swine have been proposed to be a possible intermediate host where such reassortment between human and avian viruses may take place. the pandemic was the first to arise for over years and the first to occur after the understanding that pandemics arise from animal influenza viruses. systematic studies of influenza virus ecology and evolution in swine are, therefore, important in order to understand the dynamics of pandemic emergence. furthermore, since swine are the likely host within which h n pdm virus originated, it was predicted that this virus would readily infect swine and may reassort with endemic swine influenza viruses. these predictions have now been confirmed with reports of h n pdm being detected in pigs in many countries and reassortment with endemic swine influenza virus being confirmed. while h n pdm has been genetically and antigenically stable in humans, reassortment between h n pdm, which is well adapted to transmission in humans, and other avian or swine viruses may lead to the origin of novel viruses posing a threat to public health. in addition to endemic swine virus lineages, avian influenza viruses such as h n and highly pathogenic avian influenza (hpai) h n have also been occasionally identified in pigs in parts of asia. , it has been shown that h n pdm readily reassorts with h n to generate viable progeny in vitro. it is therefore essential to monitor the ecology, evolution, and biological characteristics of swine influenza viruses so that their continued evolution and zoonotic and pandemic potential can be monitored. there is however, a paucity of surveillance data on swine influenza viruses worldwide. this is in part related to the negative commercial consequences that may arise from detection of influenza in a swine herd leading to a major economic loss to the producer. here we outline a surveillance system that has been in place in hong kong for the last decade, based on sampling animals arriving at an abattoir in hong kong. we demonstrate the feasibility of such surveillance in an abattoir setting and compare methods used for detection influenza viruses in swine. virus isolation was carried out by inoculation into mdck cells and by allantoic inoculation in embryonated eggs as previously described. virus isolates were subtyped by haemagglutination inhibition tests using specific antisera and genetically characterized by sequencing and phylogenetic analysis of the haemagglutin gene. , virus detection by rt-pcr a subset of recent specimens was tested in parallel by real time pcr using the biorobot universal system (qiagen) that enables fully-automated viral nucleic acid extraction and downstream reaction setup in a -well plate format. total viral nucleic acids were extracted in a -well plate format with the qiaamp virus biorobot mdx kit (qiagen) on the biorobot universal system (qiagen) according to the manufacturer's instructions. briefly, ll of sample was lysed in ll buffer al, supplemented with ae lg carrier rna in a s block (qiagen), which placed the samples into a well plate format. after protease digestion, samples were transferred to silica based membrane in well plate format for binding. following two washing steps, rna was eluted in ll of elution buffer (buffer ave) into a well elution microplate cl (qiagen) . for the synthesis of cdna, ll of purified rna was used in a ll reaction containing ll of · buffer, ae nm of each deoxynucleotide triphosphate (dntp), mm dithiothreitol, lg random primer, u of rnaseout recombinant ribonuclease inhibitor, and u of superscript iii reverse transcriptase (all from invitrogen). reactions were performed in the geneamp thermocycler (applied biosystems) with the following parameters: minutes at °c, minutes at °c, and soak at °c. subsequent to the reactions, ll of cdna was diluted ⁄ by adding ll of ae buffer (qiagen) . real-time pcr was performed using the power sybrÒ green pcr master mix (applied biosystems) according to the manufacturer's instructions. briefly, ll of ⁄ diluted cdna was amplified in a ll reaction containing ae ll of · power sybr green pcr master mix, nm of forward primer m c ( ¢-ctt cta acc gag gtc gaa acg- ¢) and nm of reverse primer m r ( ¢-agg gca ttt tgg aca aag ⁄ t cgt cta- ¢). the primers have been designed to amplify the sequences in the conserved region of influenza a virus matrix gene, thereby detecting viruses from different species including swine influenza viruses. real-time pcr was performed in the abi fast system (applied biosystems) with the following cycling conditions: minutes at °c once, seconds at °c, and minutes at °c for cycles, followed by melting curve analysis with seconds at °c, minutes at °c, and seconds at °c. in each assay, serially diluted plasmids containing the full length m gene cloned from a ⁄ vietnam ⁄ ⁄ (h n ) were included as standards to perform absolute quantification. a manual baseline was set from cycles - and a manual cycle threshold (ct) was set at ae . samples that were positive or unequivocal results from the real-time pcr were confirmed by performing gel electrophoresis on the pcr products. positive visual identification was made in the presence of the target pcr product at bp in length. a total of tracheal and nasal swabs were processed during the years january -april and yielded influenza virus isolates, an overall virus isolation rate of ae %. of these, were subtype h (classical swine, eurasian avian-like swine, and triple-reassortant), were human-like h viruses, and were eurasian avianlike swine h n viruses. culture in mdck cells yielded % of h subtype viruses, % of the human seasonal-like h n viruses, and ae % of the avian-like eurasian swine h n viruses. culture in embryonated eggs yielded ae % of the h subtype viruses, % of the human seasonal-like h n viruses, and ae % of eurasian avian-like swine h n viruses ( figure ). tracheal and nasal swabs each gave comparable overall virus isolation rates ( ae %). however, isolation rates for human-like h n viruses were ae fold higher in nasal swabs ( ae % versus ae % respectively; p = ae ) ( figure ) . a parallel evaluation of rt-pcr and culture was carried out in specimens. rt-pcr detected ⁄ ( %) of the culture positive specimens. rt-pcr was also positive in ⁄ ( ae %) culture negative specimens, but all these specimens had very low virus load in the rt pcr tests. virus could not be cultured from these culture negative specimens even by attempts at virus re-isolation from the frozen specimen. surveillance in an abattoir setting provides an acceptable yield of influenza viruses and is a feasible method of swine influenza surveillance. sampling in a large abattoir setting allows surveillance to be carried out anonymously with no negative consequences to the supplier. the supply-chain of pigs to the hong kong abattoir involves pigs being trucked in over long distances and may provide opportunity for virus amplification during transport. thus, virus isolation rates may be lower in more vertically integrated and homogenous production and slaughter systems where less mixing of pigs occurs. our results indicate that mdck cell culture is essential for optimizing virus isolation during swine influenza surveillance. allantoic inoculation of embryonated eggs by itself is sub-optimal for isolation of swine influenza viruses. it is however possible that inoculation of embryonated eggs by the amniotic route may lead to better isolation rates than allantoic inoculation. rt-pcr detection is an alternative method for virus detection. but the additional specimens detected by rt-pcr did not yield culturable virus, even following attempts at re-isolation and sequential passage. the rt-pcr positive ⁄ virus isolation negative specimens had very low virus load, and this may be the explanation for the inability to isolate such viruses. in addition, rt-pcr did not detect all viruses isolated by culture. tracheal and nasal swabs gave comparable isolation rates with the exception of human-like h n viruses which were more frequently isolated from nasal swabs. this may suggest that, in contrast to endemic swine influenza virus lineages, these human-like h n viruses are less adapted to replication in the lower respiratory tract. in summary, collection of nasal or tracheal swabs in an abattoir setting together with virus isolation in mdck cells provides a feasible approach to surveillance of swine influenza viruses. kong, kong, - introduction wild waterfowl are the natural reservoir of influenza a viruses (aiv), and they play an important role in the genesis of pandemic influenza. it is suggested that the pandemic virus was purely derived from avian virus, which adapted to humans and caused efficient human-to-human transmission, while the pandemics of and had acquired the viral haemagglutinin, pb polymerase, and in , the neuraminidase gene segments from the avian gene pool. the major regional outbreaks of highly pathogenic avian influenza (hpai) h n in asia, europe, and africa highlight the potential role played by migratory waterfowl in disseminating highly pathogenic influenza viruses. therefore defining the influenza virus gene-pool in wild birds is of vital importance. surveillance was carried out - times weekly from to during the winter months of october to april in the hong kong mai po nature reserve and lok ma chau, hong kong. the hong kong mai po nature reserve and lok ma chau are along the east asia-australian flyway where a peak of more than ducks and grebes congregate every winter. fecal droppings were collected and transported in vials containing ae ml of vtm, which was prepared from m ( ae g ⁄ l), penicillin g ( · u ⁄ l), polymyxin b ( · u ⁄ l), gentamicin ( mg ⁄ l), nystatin ( ae · u ⁄ l), ofloxacin hcl ( mg ⁄ l), and sulfamethoxazole ( g ⁄ l). an aliquot of ll from each swab sample was inoculated into the allantoic cavity of a -to -day-old chicken embryonated egg, and incubated for days at °c. positive ha isolates were subtyped using standard antisera , and rt-pcr was performed with the used of one-step rt-pcr assay (invitrogen) described earlier, followed by sequencing on abi prism xl dna analyzer. the determination of species of origin was performed by dna barcoding of the mitochondrial cyto-chrome oxidase i gene from dna extracted from the fecal droppings. during the -year surveillance period, a total of influenza viruses were isolated from samples collected, an overall isolation rate of ae %. a total of isolates were obtained from specimens collected during the winter period coinciding with the southern migration of waterfowl along the east asian flyway and one isolate obtained from samples collected in spring during the period when northern migration of waterfowl took place along the east asian-australasian flyway. the isolation in hong kong was slightly lower than a similar study conducted in south korea in which the isolation rate of migratory birds was ae % in - . this suggested a slightly lower prevalence of influenza virus present in hong kong as the birds migrated southwards. the viruses isolated in hong kong, representing hemagglutinin (ha) subtypes of h -h and neuramidinase (na) subtypes of n -n , were all from wild waterfowl ( table ) . out of the twelve ha subtypes isolated, h and h were the two subtypes that were isolated frequently every year for h and in six out of seven years for h , respectively. h and h viruses accounted for ae % and ae % of all virus isolated, respectively. on the other hand, h , h , and h were the least prevalence ( ae %) and were only isolated once in years. of the na subtypes, n and n were isolated most often ( ae % and ae % of all isolates, respectively) and n was the least ( ae %). november was the month that had the highest prevalence of influenza virus ( ae % of samples being positive) compared to only ae % in march. the subtype's variation was the most diverse in december during our years of surveillance. this suggested that more of these wild migratory birds may be carrying influenza virus when they arrive in hong kong. however the continued isolation of viruses suggests continued circulation of these viruses in the vicinity of mai po. the study of dna barcoding for the mitochondrial cytochrome oxidase i gene retrieved from fecal droppings revealed that the isolates originated mainly ( ae %) from birds of the order anseriform, family anatidae including eurasian wigeon, northern shoveler, northern pintail, common teal, and garganey. non-anseriformes which were found to have shed aiv viruses were cormorant, grey heron, and stint. none of the water samples collected from the ponds where these birds congregate were found to be positive for the virus. phylogenetic analyses of the ha gene of the lpai h viruses isolated in this study clustered with that of the other lpai h viruses isolated from hokkaido, mongolia, and siberia and were not closely related to the hpai h n . satellite tracking of eurasian wigeons and northern pintails in dec and revealed their flyway from hong kong to as far north as eastern russia, eastern mongolia, and northern china. no hpai h n viruses were isolated in this study from apparently healthy birds. however, as part of the surveillance of dead wild birds carried out by the department of agricultural, fisheries and conservation of the government of hong kong during this same period, over dead wild birds were tested positive for hpai h n and has been reported elsewhere. our influenza surveillance in hong kong has revealed a diversity of influenza virus subtypes the migratory waterfowl infected within the region. the result of the phylogenetic analysis correlated with the findings from satellite tracking that viruses isolated in hong kong were closely related to those isolated in areas along the migratory route. no healthy bird was isolated with hpai h n, although dead wild birds have been regularly found to have hpai h n virus, suggesting that infected birds might not live for a long period. introduction a novel swine-origin h n influenza virus emerged in mexico in april and rapidly spread worldwide, causing the first influenza pandemic of the st century. most confirmed human cases of h n ⁄ influenza have been uncomplicated and mild, but the increasing number of cases and affected persons worldwide warrant optimal prevention and treatment measures. today, almost all of the pandemic h n ⁄ viruses tested are resistant to m blockers. therefore, only the neuraminidase (na) inhibitors are currently recommended for treatment of this pandemic influenza. for the control of influenza infection, the clinical use of oseltamivir has increased substantially during the pandemic. to date, the majority of tested clinical isolates have remained susceptible to na inhibitors, oseltamivir and zanamivir, but oseltamivir-resistant variants with h y na mutation (n numbering) have been isolated from individuals taking prophylaxis, from immunocompromised patients, and from a few community clusters. , in view of the high prevalence of oseltamivirresistant seasonal h n influenza viruses in - , the isolation of resistant h n ⁄ viruses without known oseltamivir exposure raised great concern about the transmissibility and fitness of these resistant viruses. here we studied the transmissibility of a closely matched pair of pandemic h n ⁄ clinical isolates, one oseltamivir-sensitive and one resistant, in both direct contact and respiratory droplets routes among ferrets. viral fitness was evaluated by co-infecting a ferret with both the oseltamivir-sensitive and -resistant viruses. the viruses were also characterized by full genome sequencing, susceptibility to na inhibitors, and growth in mdck and mdck-siat cells. oseltamivir-resistant influenza a ⁄ denmark ⁄ ⁄ (h n ) virus (a ⁄ dm ⁄ ⁄ ) was isolated from the throat swab of a patient who had influenza-like symptoms and received post-exposure oseltamivir prophylaxis ( mg once daily). wild-type influenza a ⁄ denmark ⁄ ⁄ (h n ) virus (a ⁄ dm ⁄ ⁄ ) was isolated from a patient in the same cluster of infection as the a ⁄ dm ⁄ ⁄ virus. to assess growth kinetics of viruses, confluent mdck or mdck siat cell monolayers were infected with viruses at a multiplicity of infection (moi) of approximately ae pfu ⁄ cell (single-step) or ae pfu ⁄ cell (multi-step). supernatants were collected every h or h p.i. for time points. a modified fluorometric assay using the fluorogenic substrate ¢-( -methylumbelliferyl)a-d-n-acetylneuraminic acid (munana) was used to determine viral na activity. the drug concentration required to inhibit % of the na enzymatic activity (ic ) was determined by plotting the percent inhibition of na activity as a function of compound concentration calculated in the graphpad prism (la jolla, ca) software from the inhibitor-response curve. na enzyme kinetics were determined by measuring na activity every seconds for minutes under the same conditions as above, when all viruses were standardized to an equivalent dose of ae pfu ⁄ ml. the k m and v max were calculated by fitting the data to the appropriate michaelis-menten equations using nonlinear regression in the graphpad prism software. young adult ferrets ( - months of age) were obtained from the ferret breeding program at st. jude children's research hospital. all ferrets were seronegative for influenza a h n and h n viruses and for influenza b viruses. for transmission studies, the donor ferrets were lightly anesthetized with isoflurane and inoculated intranasally with tcid virus in ae ml sterile pbs . after the donor ferrets were confirmed to shed virus on day p.i., each donor was then housed in the same cage with two naïve direct-contact ferrets. two additional recipient ferrets were placed in an adjacent cage isolated from the donor's cage by a two layers of wire mesh (approximately cm apart) that prevented physical contact but allowed the passage of respiratory droplets. ferret weight and temperature were recorded daily for days. nasal washes were collected from donors and recipients on day , , , , , , , and p.i. by flushing both nostrils with ae ml pbs, and tcid titers were determined in mdck cells. serum samples were collected weeks after virus inoculation, and were tested for seroconvention by hi assay. full genome sequencing revealed that the pair of h n ⁄ viruses differed only at na amino acid position , where the pandemic a ⁄ dm ⁄ ⁄ virus had an h y amino acid mutation caused by a single t-to-c nucleotide substitution at codon . the wild-type a ⁄ dm ⁄ ⁄ was susceptible to oseltamivir carboxylate (mean ic : ae nm), but the a ⁄ dm ⁄ ⁄ carrying the h y na mutation had ic values approximately - times of the wild-type viruses (mean ic : nm). the ic of zanamivir was comparable for both viruses and were uniformly low (mean ic £ ae nm). the h y na mutation confers resistance to oseltamivir carboxylate but did not alter susceptibility to zanamivir. to understand the impact of the h y mutation on the na enzymatic properties, na enzyme kinetics was determined. the na of the oseltamivir-resistant virus had a slightly higher k m (mean = lm) and lower v max (mean = u ⁄ sec) than na of the sensitive virus (k m , mean = lm; vmax, mean = u ⁄ sec). the results suggested that the h y na mutation reduced na affinity for substrate and na catalytic activity, although the function of na was not severely impaired. to further evaluate the impact of the h y na mutation on virus growth in vitro, single-and multi-cycle growth studies of both viruses were performed in mdck and mdck-siat cells. in the both single-and multiple-cycle growth curves, the two viruses reached comparable levels eventually, but the initial growth of the resistant virus was significantly delayed by at least - logs in comparison to that of wild-type virus (p < ae ). the donor ferrets inoculated with wild-type a ⁄ dm ⁄ ⁄ or oseltamivir-resistant virus shed virus productively until day or day p.i., with a peak virus titer comparable to that of a ⁄ dm ⁄ ⁄ virus (table ). in a ⁄ dm ⁄ ⁄ virus group, two of direct-contact ferrets the weight loss in ferrets is the maximum percentage loss compared with the initial weight. virus shedding is indicated as number of virus-shedding animals ⁄ total number; mean peak virus titer (log tcid ⁄ ml) in nasal wash samples is indicated in parentheses. serum hemagglutination inhibition (hi) titer to homologous virus in ferret serum was determined on day p.i. duan et al. and of respiratory droplet-contact ferrets were infected through virus transmission, as indicated by the virus titers and inflammatory cell counts in their nasal washes and also by sero-conversion. under identical conditions, in a ⁄ dm ⁄ ⁄ group, only of direct-contact ferrets were infected through virus transmission, but neither respiratory droplet-contact ferrets was infected, as confirmed by the absence of sero-conversion (table ) . virus shedding in the direct-contact ferrets was lower and peaked after a longer interval in this group than in the oseltamivir-sensitive a ⁄ dm ⁄ ⁄ group (table ) , but the resistant viruses appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. these results showed that an oseltamivir-resistant h y mutant of pandemic h n virus, a ⁄ dm ⁄ ⁄ virus could be only transmitted efficiently by direct contact. to compare the relative fitness, growth capability, and transmissibility of the sensitive and resistant h n ⁄ viruses within host, a donor ferret was co-inoculated with a : ratio of the sensitive and resistant viruses, and another two naive ferrets were housed with the donor to test direct contact. during co-infection, the pattern of virus shedding and the clinical signs were similar to those in ferrets inoculated with either a ⁄ dm ⁄ ⁄ or a ⁄ dm ⁄ ⁄ virus (table ). in the inoculated donor ferret, the virus population in the nasal washes remained mixed but wild-type viruses outgrew the resistant virus progressively ( figure ). two of direct-contact ferrets were infected through virus transmission, but only wild-type virus was detected in both direct-contact ferrets ( figure ). in summary, oseltamivir-sensitive a ⁄ dm ⁄ ⁄ virus possessed better growth capability in the upper respiratory tract than did resistant a ⁄ dm ⁄ ⁄ virus, and thus had an advantage in directcontact transmission. our study determined the comparative transmissibility of two naturally circulating oseltamivir-sensitive and -resistant pandemic h n ⁄ viruses; we demonstrated inefficient respiratory-droplet transmission of an oseltamivir-resistant h y mutant of pandemic h n virus among ferrets, although it retained efficient direct-contact transmission. we suggest that the lower fitness of resistant virus within the host along with its reduced na function and delayed growth in vitro may in part explain its less efficient transmission. notably, the h y mutant of h n ⁄ used in this study was the first oseltamivir-resistant h n ⁄ isolate from a patient on oseltamivir prophylaxis to be characterized for transmissibility. our observation in the animal model is consistent with the epidemiological data collected from humans, which showed no evidence of predominant or continued circulation of oseltamivir-resistant viruses. as this study was undertaken, additional h y mutants of h n ⁄ viruses have emerged in the absence of oseltamivir use. , the emergence of these viruses should raise concerns as to whether resistant h n ⁄ viruses will acquire greater fitness and spread worldwide as the naturally resistant h n viruses did during the - season. two independent studies have evaluated the pathogenecity and transmission of other oseltamivir-resistant pandemic h n ⁄ clinical isolates in the animal models. , one of the studies, which also used an oseltamivir-resistant virus isolated from a patient under oseltamivir prophylaxis, observed similar results as ours: although the respiratory-droplet route of transmission was not investigated, it was shown that the resistant isolate was transmitted though direct-contact route and was as virulent as wild-type virus in ferrets. in another study, two oseltamivir-resistant isolates were transmitted through the respiratory-droplet route in ferrets, and the dynamics of transmission were different between the two isolates. apparently, these two oseltamivir-resistant isolates were still unequal in their transmissibility and were disparate from the resistant isolate in our study. the isolation history of the two resistant isolates was unclear in this study, and this would be an important factor to understand the fitness of drug-resistant viruses. further studies with more clinical isolates of diverse isolation background are warranted to identify how these novel h y mutants of pandemic h n ⁄ virus have changed to retain their full transmissibility. taken together, all these related studies underline the necessity of continuous monitoring of drug resistance and characterization of potential evolving viral proteins. this study was supported by contract hhsn c from the national institute of allergy and infectious diseases, national institutes of pigs have been considered as hypothetical ''mixing vessels'' facilitating the genesis of pandemic influenza viruses. , the pandemic h n ⁄ virus (ph n ⁄ ) contained a very unique genetic combination and was thought to be of swine origin, as each of its eight gene segments had been found to be circulating in pig populations for more than a decade. however, such a gene constellation had not been found previously in pig herds all around the world. only after its initial emergence in humans has this virus been repeatedly detected in pigs, and found to further reassort with other swine influenza virus. [ ] [ ] [ ] a primary question remaining to be answered is whether the ph n ⁄ -like and their genetically related viruses could become established in pig populations, thereby posing novel threats to public health. despite the fact that ph n ⁄ first appeared in mexico and the united states, and six of its eight gene segments were derived from the established north american triple reassortant swine influenza virus (trig), its neuraminidase (na) and matrix protein (m) genes belonged to the eurasian avian-like swine lineage (ea), which had never been detected in north america previously. , likewise, the trig-like viruses were never reported in europe. in contrast, both lineages of virus were frequently detected in asia, and reassortants between them have also been documented in recent years. , this has given rise to a complicated ecological situation, i.e. the simultaneous prevalence of multiple genotypes of h n and h n viruses in pigs. , among them, two representative reassortants showed the most similar genotypic characterization to the ph n ⁄ virus, the sw ⁄ hk ⁄ ⁄ (h n ) and sw ⁄ hk ⁄ ⁄ (h n ), which respectively harbor seven and six gene segments closely related to the pandemic strains. , to understand their in vivo characteristics and zoonotic potential, these two viruses, together with a human prototype strain and a swine ph n ⁄ -like isolate, were chosen for a study of their pathogenicity and transmissibility in domestic pigs, ferrets, and mice. the prototype ph n ⁄ virus, a ⁄ california ⁄ ⁄ (ca ), was provided by the world health organization collaborating centers for reference and research on influenza (atlanta, ga, usa). three ph n ⁄ -related swine influenza viruses were isolated through our surveillance program in south china as previously described. , the a ⁄ swine ⁄ guangdong ⁄ ⁄ (h n , gd ) virus was a ph n ⁄ -like swine isolate. a ⁄ swine ⁄ hong kong ⁄ ⁄ (h n , hk ), the closest pandemic ancestor known to date, possesses an m gene derived from the ea lineage, with the other gene segments from trig viruses. a ⁄ swine ⁄ hong kong ⁄ ⁄ (h n , hk ), a recent pandemic reassortant progeny, had a ph n ⁄ like na gene (also belonging to the ea lineage), an ea-like hemagglutinin (ha) gene, and six trig-like internal genes. all viruses were propagated in madin-darby canine kidney (mdck) cells for three passages, and their titers were determined by plaque assays. all experiments with live viruses were conducted in biosafety level (bsl- ) containment laboratories. pigs ( - week old, n = - ) and ferrets ( month old, male, n = ) were intranasally infected with pfu of each virus, and mice ( ) ( ) week old, female balb ⁄ c, n = ) with a dose of pfu. naïve uninfected pigs (n = ) were co-housed in the same cage with the inoculated ones from each group. body weights and clinical signs were recorded daily. virus replication was determined by titration of the virus in nasal and rectal swabs (pigs), nasal washes (ferrets), as well as from lungs and other organs (pigs and mice). seroconversion was tested by hemagglutination inhibition (hi) assays. histopathological and immunohistochemical analysis were performed as previously described. statistical analysis was performed by mean analysis with pasw statistics (spss inc., chicago, il, usa). the probability of a significant difference was computed using anova (analysis of variance). results were considered significant at p < ae . the pathogenicity of the four viruses tested differed significantly in inoculated mice. animals infected with pfu of hk experienced the most severe body weight loss ( ae ± ae %) but started to recover after days post-infection (dpi). hk caused similar peak body weight loss ( ae ± ae % on dpi) in mice as did ca ( ae ± ae %, on dpi), but the onset of clinical signs and weight loss (on dpi) was day later than those caused by the other three viruses. the gd -infected group suffered the least body weight loss ( ae ± ae %, dpi) and was the earliest to recover. although all four viruses were detected in the lungs with comparable virus titers on dpi (p > ae ), mice inoculated with gd consistently showed the lowest lung index (lung weight ⁄ body weight, %) on , , and dpi (p < ae ), suggesting the slightest injury and consolidation of the lungs. in concordance with the body weight change, the lung index from the hk group was higher than that from any other groups on and dpi, indicating the marked virulence of hk in mice. notably, virus titer of hk in the nasal turbinate was lower than the other groups both on and dpi (p < ae ), but virus replication in the lower respiratory tract was either higher (in the trachea) or similar (in the lungs). observations of the body weight changes caused by infection of ph n ⁄ or its genetically related swine viruses in ferrets have come to a similar conclusion as that for the mouse experiment. after nasal inoculation with pfu of each virus, all groups of ferrets experienced transient body weight loss for - days, except for those infected with gd , which showed no significant weight loss (p > ae ). although ferrets from the ca -infected group reached their peak weight loss ( ae ± ae %, dpi) one day earlier than those from the hk and hk groups, they began to regain body weight quickly thereafter. hk -infected ferrets also recovered rapidly and their body weights reached the same level as those of the gd -infected group at dpi. comparatively, ferrets inoculated with hk had the most retarded body weight recovery, which did not get back to the baseline level until dpi. hk was only detectable in the nasal wash on dpi, whereas the duration of virus shedding for gd , hk , and ca was - days. by combining the data obtained from the virus titration in the mouse turbinate and ferret nasal washes, a possible conclusion can be made that hk may have lower transmissibility than the other three viruses. after inoculation or exposure by direct contact (physical contact) with the ph n ⁄ virus and its close relatives, most pigs experience no or mild symptoms, such as slight loss of appetite and inactivity. body weight loss was only recorded in pigs inoculated with hk during the second week post-inoculation, but not in their contact pigs or in the other groups. diarrhea was observed intermittently in each of the inoculated or contact groups throughout the experiment, and viruses could be recovered in the rectal swabs, saliva, drinking water, and environmental swabs (inner cage walls accessible to the pigs) at various time points. however, virus titers in the positive rectal swabs were just slightly above the detection limit, while those from the environment sometimes could be higher. whether these viruses can replicate in the digestive tract or were just carried-over by contaminated foods and water requires further investigation. although virus could be detected in the nasal swabs of all infected or contact animals, the lowest peak titer was from pigs inoculated or in contact with hk ( ae - ae log tcid ⁄ ml lower than the other groups), suggesting unfavorable replication in the nasal cavity for this virus. postmortem examination on and dpi revealed that pigs infected with hk had the most extensive gross lesions in the lungs, and histochemical staining of viral nucleoprotein (np) in lung tissues on dpi also suggested the best replication for hk in the lower respiratory tract. on days post-contact (dpc), all pigs exposed to the inoculated animals developed sero-conversions (hi = - ) except for one from the gd contact group. however, on dpc, its hi titer reached , indicating slower seroconversion. this study revealed that both the pandemic h n and its genetically related swine viruses could readily infect mice, ferrets, and pigs causing mild to moderate clinical symptoms. they could also transmit efficiently between pigs. when compared with the pandemic stains and its reassortant progeny (hk ), the hk (h n ) virus containing the ea-like m gene in the genetic context of the trig virus showed consistently higher virulence in all three mammalian models tested, but it is still unknown what might happen if such a virus further reassorts to obtain the pandemic-like or ea-like na gene. however, our findings suggest that pigs could likely maintain the prevalence of different genotypes of pandemic-related influenza viruses, and highlight the zoonotic potential of multiple strains of swine influenza virus. pandemic influenza viruses emerge from the animal reservoirs. among the three pandemics that occurred in the last century, we learned that the h n and the h n pandemic viruses emerged by reassortment between circulating human virus and avian-origin influenza virus(es). studies on the emergence of the catastrophic spanish h n virus suggest that the virus may have obtained all of its eight gene segments from the avian reservoir, , or alternatively is a reassortant between mammalian and a previously circulating human influenza virus. over years since the last pandemic, the first pandemic in the st century arose in and was caused by a swine-origin influenza virus containing a unique gene combination, with gene segments derived from the circulating north america ''triple reassortant'' (pb , pb , pa, ha, np, and ns) and the ''eurasian'' (na and m) swine influenza viruses. , analysis of the pandemic h n viruses failed to identify known molecular markers predictive of adaptation to humans. the ''triple reassortant'' swine influenza viruses emerged in late s in north america is a reassortant between classical swine (descendent of the virus after adaptation in swine population), avian, and human influenza viruses. the eurasian influenza virus was originally an avian influenza virus that was introduced into the european swine population in the late s. , while incidents of zoonotic infection with triple reassortant or eurasian influenza in humans have been reported, , sustained human-to-human transmission has never been established. these results suggest that the unique gene combination seen with the pandemic h n viruses may confer its transmissibility among humans. we have carried out systematic prospective surveillance of swine influenza in southern china over that last years through samples routinely collected at an abattoir in hong kong. during this time, the surveillance results suggest co-circulation of classical swine h n , triple reassortant h n , eurasian swine h n , and a range of reassortants between these three virus lineages. , ferrets have been reported as a suitable model for the study of influenza transmission as they are naturally susceptible to influenza infection, exhibit similar clinical signs (including sneezing), and possess receptor distribution in the airway similar to that of humans. [ ] [ ] [ ] to identify molecular determinants that enable sustained human-to-human transmission, we compared the pandemic virus with genetically related swine influenza viruses obtained from this surveillance program for their ability to transmit from ferret to ferret by direct contact or aerosol transmission. viruses human h n influenza virus [a ⁄ wuhan ⁄ ⁄ (wuhan )] and pandemic h n influenza viruses [a ⁄ california ⁄ ⁄ (ca )] were included for the study. swine influenza viruses that are genetically related with the pandemic h n virus were selected from our surveillance system, including classical swine-like influenza virus a ⁄ sw ⁄ hk ⁄ ⁄ (h n ) (swhk ), triple reassortant-like a ⁄ sw ⁄ arkansas ⁄ ⁄ (h n ) (swar ), and one reassortant between triple reassortant and eurasia swine influenza viruses [a ⁄ sw ⁄ hk ⁄ ⁄ (h n ) (swhk )]. swhk contains seven gene segments (pb ,pb ,pa,ha,np,m,ns) closely related to the pandemic h n viruses. transmissibility was tested in -to -month-old male ferrets obtained from triple f farm (sayre, pa); all ferrets were tested to have hi titer £ against human seasonal influenza h n (a ⁄ tennessee ⁄ ⁄ ), h n (a ⁄ brisbane ⁄ ⁄ ), and influenza b (b ⁄ florida ⁄ ⁄ ) prior the experiments. in each virus group, three ferrets were inoculated with tcid of the virus. at day postinoculation (dpi), we introduced one naïve direct contact ferret to share the cage with inoculated ferret, and one naïve aerosol contact ferret into the adjacent compartment of the cage separated by a double-layered perforated divider. nasal washes were collected every other day and tested for influenza virus antigen and to determine viral titers (tcid ). weight changes, temperature, and clinical signs were monitored daily. transmission is defined by detection of virus from nasal washes and ⁄ or by seroconversion (> fold rise in the post-sera collected after - days post contact). experiments were performed in the p + laboratory at st. jude children's research hospital. all studies were conducted under applicable laws and guidelines and after approval from the st. jude children's research hospital animal care and use committee. at tcid inoculation dose, all viruses replicated efficiently in the ferret upper respiratory tract with peak titers detected from inoculated ferrets at dpi. lower peak titers were detected from swhk and swhk inoculated ferrets, however, the differences were not statistically significant (table ) . tissues collected from inoculated ferrets at dpi showed that pandemic h n and swine influenza viruses replicated both in the upper and lower respiratory tract of the ferrets, while the replication of human seasonal influenza wuhan was restricted in the upper respiratory tract. direct contact transmission from inoculated donor ferrets to their cage-mates was observed for all viruses studied, albeit at different efficiency. human seasonal influenza (wuhan ) and pandemic h n viruses (ca ) transmitted most efficiently via direct contact route as the virus can be detected on dpi from direct contact ferrets, and the peak titers were detected on dpi from direct contacts. moderate direct contact transmission efficiency was detected from swar and swhk viruses as the virus can be detected from direct contact ferrets at dpi, with peak titers detected at dpi or dpi. classical swine-like swhk showed least efficient contact transmission as virus could be detected from all direct contacts only at dpi, and the peak titer detected on dpi. aerosol transmission was detected in groups of human seasonal influenza virus wuhan ( ⁄ ), pandemic h n influenza virus ca ( ⁄ ), as well as swine precursor virus swhk ( ⁄ ). transmission of wuhan and ca to aerosol contacts was detected at dpi or dpi, while transmission of swhk was detected later at dpi, suggesting that the swhk virus possessed aerosol transmission potential, but may require further adaptation to acquire efficient aerosol transmissibility. in addition to viral detection from nasal washes, we also detected viruses from the rectal swabs of ferrets inoculated or infected with pandemic h n viruses (ca ) or classical swine-like virus (swhk ), which share the common origin for the ha, np, and ns gene segments. while many of the swine influenza viruses studied were able to transmit via the direct contact route, swhk , which shares a common genetic derivation for seven genes with h n pdm, possessed capacity for aerosol transmission, albeit of moderate efficiency. swhk differed from swine triple reassortant viruses in the origins of its m gene. it is possible that the m gene derived from eurasian avian- like swine viruses also contributes to the transmissibility of h n pdm influenza viruses. outbreaks of highly pathogenic avian influenza (hpai) of the h n subtype are of extreme concern to global health organisations as human infection can result in severe acute respiratory distress syndrome, multi-organ failure, and coma. hpai viruses of either h or h subtypes contain a characteristic multi-basic cleavage site in the hemagglutinin glycoprotein as well as other virulence factors that expand the viral tropism beyond the respiratory tract of poultry. there is also emerging evidence of viral rna or antigen in multiple organs and the cns of humans infected with h n that is consistent with systemic infection , and raises the question of the role of the cleavage site in dissemination of the virus in this species. the majority of human cases with h n have involved contact with sick or contaminated poultry and exposure to respiratory secretions of birds that can be inhaled and ingested. particular risk factors for h n infection include bathing with sick birds, improper hand washing after handling sick birds, or slaughtering poultry. viral inoculum may also be consumed directly during a variety of religious and cultural practices, such as drinking contaminated duck blood and kissing of merit release birds. h n infection is lethal in % of human cases, and the pathogenetic mechanisms leading to this level of mortality are unclear. to date cases have been reported to the who, although many more people have potentially been exposed to h n through contact with infected bird populations. some studies have suggested that genetic factors may predispose an individual to severe h n disease, but little is known about the influence of route of virus exposure on morbidity and mortality. in ferrets, an animal model frequently used to study influenza because of its similar disease profile to humans, swayne et al. observed that exposure to a virulent h n strain a ⁄ vietnam ⁄ ⁄ by intra-gastric gavage did not lead to disease and did not generate an antibody response, whereas ferrets that experienced a more natural exposure by being fed contaminated meat developed severe signs of infection. in this study we further assessed the disease profile of h n following a natural oral exposure in the ferret model. to achieve this inoculation condition, conscious ferrets voluntarily consumed a liquid inoculum of h n hpai strain a ⁄ vietnam ⁄ ⁄ . as a comparison anesthetised ferrets were exposed by intranasal administration of inoculum and the ensuing disease profiles of the different routes of infection were compared. eight ferrets per group were inoculated with egg infectious dose of a ⁄ vietnam ⁄ ⁄ in a volume of ll that was given to the nares of anaesthetized ferrets to establish a total respiratory tract (trt) infection or voluntarily consumed by conscious ferrets to establish an oral infection. ferrets were culled at a predetermined humane endpoint that was defined as either a > % weight loss and ⁄ or evidence of neurological signs, discussed in ; animals that did not reach the humane endpoint were euthanased on day after challenge. nasal washes and oral swabs collected during the course of infection and organ homogenates were assessed for the presence of replicating virus by growth in embryonated-chicken eggs; viral loads were determined by titration on vero cells and expressed as tcid . tissue samples were fixed with formalin and embedded in paraffin for sectioning. viral lesions were identified by hematoxylin and eosin staining of the sections and the presence of viral antigen in the sections was determined by staining with antibody to influenza a nucleoprotein. pre-and post-exposure antibody responses were assessed by hemagglutination-inhibition assays using irradiated a ⁄ vietnam ⁄ ⁄ virus. the majority ( %) of ferrets infected by the trt route rapidly became inactive, developed severe disease, and were euthanased at the humane endpoint following infection ( figure ). ferrets infected orally had an improved chance of survival, as only % of animals developed severe disease (figure ), and the surviving ferrets were more active than ferrets infected by the trt throughout the stage of acute infection (data not shown). the improved survival rate and wellbeing of ferrets infected orally was not a result of poor infection rates by this route, as of surviving ferrets developed h specific antibodies by day post-infection, and they did not have pre-existing antibodies to h n (data not shown). the two ferrets that developed severe disease after oral infection had similar disease profiles to ferrets infected by the trt route; they both progressed to a > % weight loss and exhibited neurological signs (data not shown). viral loads in organs of these two ferrets confirmed dissemination to extra-pulmonary sites (table ) : replicating virus was detected at high titres in the spleen, pancreas, liver, and brain. similar findings were recorded in ferrets with trt infections in this study (not shown) and elsewhere. viral load in nasal washes and oral swabs taken at days , , and post-infection by the oral route did not correlate with the development of severe disease, and virus was isolated only sporadically and at low titre from the nasopharynx of these animals (data not shown). interestingly, the two ferrets with severe disease after being infected orally had no detectable viral antigen or lesions in the olfactory epithelium and bulb (table ) , whereas of ferrets culled after infection by the trt route had lesions and viral antigen in both the olfactory epithelium and bulb (data not shown). trt oral figure . percentage of ferrets that survived infection after oral or trt infection. ferrets were exposed to a ⁄ vietnam ⁄ ⁄ by the total respiratory tract (trt) route (circles) or the oral route (triangles). the percentages of ferrets that survived infection are indicated at each day following challenge. ferrets exposed orally were more likely to survive h n infection than ferrets exposed to the same dose of virus by the trt. the improved survival rates that were observed after an oral infection could be a consequence of low-level viral replication in the upper respiratory tract in combination with delivery of a substantial portion of the inoculum directly to the stomach where it may have been inactivated by the harsh environment of the gastro-intestinal tract. most ferrets infected orally developed an h -specific antibody response which differs from the studies of swayne et al. in which ferrets gavaged with a liquid inoculum neither developed signs of disease nor an antibody response. however swayne et al. administered virus to anaesthetized ferrets by gastric gavage that would have bypassed the oropharynx. in our study virus was administered to the oral cavity directly and would have had access to the oropharynx. low level of replication at this site may have been sufficient to trigger an antibody response. the two ferrets that developed severe disease following oral infection had a similar profile of viral dissemination as ferrets infected by the trt route. differences were seen in the olfactory epithelium and bulb as lesions, and viral antigen did not occur in these sites following oral infection, although cerebral involvement was identified. one route of dissemination of h n into the cns may be by transport within nerves through the olfactory bulb into the cerebrum. due to the absence of lesions and antigen in these sites following oral infection the spread of virus into the brain in these two animals may be occurring through involvement of other cranial nerves or the hematagenous routes. nasal turbinates ) ae ) ) ) ) pharyngeal lymph node interactions of oseltamivir-sensitive and -resistant highly pathogenic h n influenza viruses in a ferret model < ae b ) + + + + olfactory epithelium nd a nd ) ) ) ) olfactory bulb nd nd ) ) ) ) trachea < ae ) ) nd ) nd lung ) < ae + + ) + spleen ae ) + + ) + small intestine ) ) ) ) ) + pancreas ) ae + ) + + the pandemic potential of highly pathogenic h n influenza viruses remains a serious public health concern. while the neuraminidase (na) inhibitors are currently our first treatment option, the possibility of the emergence of virulent and transmissible drug-resistant h n variants has important implications. clinically derived drug-resistant viruses have carried mutations that are na subtype-specific and differ with the na inhibitor used. the most commonly observed mutations are h y and n s in the influenza a n na subtype (n numbering here and throughout the text); e a ⁄ g ⁄ d ⁄ v and r k in the n na subtype; and r k and d n in influenza b viruses. h n influenza viruses isolated from untreated patients are susceptible to the na inhibitors oseltamivir and zanamivir, although oseltamivir-resistant variants with the h y na mutation have been reported in five patients after , or before drug treatment; and the isolation of two oseltamivir-resistant h n viruses with n s na mutation from an egyptian girl and her uncle after oseltamivir treatment were described. the impact of drug resistance would depend on the fitness (i.e., infectivity in vitro, virulence, and transmissibility in vivo) of the drug-resistant virus. if the resistance mutation only modestly reduces the virus' biological fitness and does not impair its replication efficiency and transmissibility, the effectiveness of antiviral treatment can be significantly impaired. the recombinant wild-type h n influenza a ⁄ vietnam ⁄ ⁄ (vn-wt), a ⁄ turkey ⁄ ⁄ (tk-wt) viruses, and oseltamivir-resistant viruses with h y na mutation (vn-h y and tk-h y) were generated by using the -plasmid reverse genetics system. susceptibility to na inhibitors was tested by using a fluorescence-based na enzyme inhibition assay with munana substrate at a final concentration of lm. viral fitness was studied in vivo in a ferret model: groups of three ferrets were lightly anesthetized with isoflurane and inoculated intranasally with vn-wt, vn-h y, or mixtures of the two at a different ratios at a dose of pfu in ae ml pbs; they were inoculated with tk-wt, tk-h y, or mixtures of the two at a different ratios at a dose of pfu in ae ml pbs. respiratory signs (labored breezing, sneezing, wheezing, and nasal discharge), neurologic signs (hind-limb paresis, ataxia, torticollis, and tremor), relative inactivity index, weight, and body temperature were recorded daily. virus replication in the upper respiratory tract (urt) was determined on days , , and p.i. the competitive fitness (i.e., co-inoculation of ferrets with different ratios of oseltamivir-resistant and -sensitive h n viruses) was evaluated by the proportion of clones in day- nasal washes that contained the h y na mutation. na mutations were analyzed by sequence analysis of individual clones ($ clones ⁄ sample) created by ligation of purified pcr products extracted from nasal wash samples into a topo vector. introduction of the h y na mutation conferred high resistance to oseltamivir carboxylate in vitro; the mean ic of the vn-h y and tk-h y viruses was and times, respectively, that of the corresponding wildtype viruses. the oseltamivir ic of the tk-wt virus was $ times that of the vn-wt virus. all four recombinant h n viruses were susceptible to zanamivir. introduction of the h y na mutation reduced $ % and % of the na activity of vn-h y and tk-h y viruses, respectively, as compared to the wild-type virus activity (p < ae ; two-tailed t-test). all ferrets inoculated with either vn-wt or vn-h y virus exhibited acute disease signs (high fever, marked weight loss, anorexia, extreme lethargy), rapid progression, and death by day - p.i., and no differences in clinical signs and replication in the urt of ferrets were observed between wild-type and oseltamivir-resistant viruses ( table ) . both of the tk viruses caused milder illness than did the vn viruses, despite a much higher dose ( pfu ⁄ ferret), and the tk-h y virus caused less weight loss and fever than the tk-wt virus (table ) . however, competitive fitness experiments revealed a disparity in the growth capacity of vn-h y and tk-h y viruses as compared to their wild-type counterparts: clonal analysis established the uncompromised fitness of vn-h y virus and the impaired fitness of tk-h y virus (table ) . although, the trend towards an increase ⁄decrease in the frequency of the h y na mutation relative to the wild-type was statistically significant (p > ae ) for two studied groups only. mutations within the na catalytic (r k) and framework (e a ⁄ k, i l, h l, n s) sites or near the na active enzyme site (v i, i t ⁄ v, q h, k n, a t) emerged spontaneously (without drug pressure) in both pairs of viruses (results not shown). the na substitutions i v and e a could exert compensatory effect on the fitness of vn-h y and tk-h y viruses. the lethality and continuing circulation of h n influenza viruses warrants an urgent search for an optimal therapy. our results showed that the h y na mutation affects the fitness of two h n influenza viruses differently: the oseltamivir-resistant a ⁄ vietnam ⁄ ⁄ -like virus outgrew its wild-type counterpart, while the oseltamivir-resistant a ⁄ turkey ⁄ ⁄ -like virus showed less fitness than its wild-type counterpart. we used a novel approach to compare the fitness of oseltamivir-sensitive and -resistant influenza viruses that included analysis of virus-virus interactions within the host (competitive fitness) during co-infection with these viruses. although mixed populations were present in the urt of ferrets on day p.i., the fitness of vn-h y virus was uncompromised as compared to that of its drug-sensitive counterpart, while that of tk-h y virus was impaired. a minor population of na inhibitor-resistant variants may gain a replication advantage under suboptimal therapy in two ways: (i) preexisting variants less sensitive to the drug are selected from the quasispecies population, leading to an increase of the number of resistant clones, and (ii) outgrowing variants may acquire additional compensatory mutations that enhance their fitness. it is possible that use of antiviral drugs (particularly at suboptimal concentration) against mixtures of oseltamivir-resistant and sensitive viruses will promote the spread of drug-resistant variants * ferrets in all groups inoculated with a ⁄ vietnam ⁄ ⁄ virus died by day - p.i. and were observed once daily for days. ** results obtained from one ferret. *** by inhibiting drug-sensitive variants that are competing with them for the dominance in the infected host. the influence of multiple genes on the fitness of viruses carrying h na mutation cannot be excluded. in our study we focused on additional na mutations, and sequence analysis of individual na clones was done to identify potential host-dependent and compensatory na mutations. we found that the na mutations e a and n s, which confer cross-resistance to oseltamivir and zanamivir, , can emerge spontaneously in clade . h n influenza virus in ferrets. further, we observed that mutations at na catalytic (r k) and framework (i l and n s) sites and in close proximity to the na enzyme active site (v i, i t ⁄ v, q h, k n, a t) emerged without drug pressure in both pairs of h n viruses. compensatory mutations in na or other genes may mitigate any fitness cost imposed by resistance mutations. our study identified six potential compensatory na changes (d v, f s, i v, e a, h l, and f s) that may affect the fitness of viruses with the h y na mutation. we suggest that na mutations at residues i v and e a are of importance. interestingly, we observed differences in predominance of i v and e a na mutations in different genetic backgrounds: i v mutation was identified in a ⁄ vietnam ⁄ ⁄ (h n )-like and e a in a ⁄ turkey ⁄ ⁄ (h n )-like genetic background. moreover, i v na mutation was identified only when ferrets were inoculated with the mixtures of vn-wt and vn-h y viruses, but not in ferrets inoculated with vn-h y virus. none of the potential compensatory na mutations was identified in the original inoculum used to infect ferrets. the h y na mutation causes a large shift in the position of the side chain of the neighboring e residue, which must form a salt bridge with r to accommodate the large hydrophobic pentyl ether group of oseltamivir. residue i is located near the na active site, and although it does not alter polarity, it results in a shorter side-chain and, thus, may indirectly affect the residues in the na active site. we suggest that antigenic and genetic diversity, virulence, the degree of na functional loss, and differences in host immune response and genetic background can contribute to the observed differences in the fitness of h n influenza viruses. therefore, the risk of emergence of drugresistant influenza viruses with uncompromised fitness should be monitored closely and considered in pandemic planning. this study was supported by contract hhsn c from the national institute of allergy and infectious diseases, national institutes of health, and by the american lebanese syrian associated charities (alsac). the data presented in the manuscript have been published at: govorkova ea, ilyushina na, marathe bm, mcclaren laninamivir (r- ) is a strong na inhibitor against various influenza viruses, including oseltamivir-resistant viruses. [ ] [ ] [ ] [ ] [ ] [ ] we discovered a single intranasal administration of laninamivir octanoate (cs- ), a prodrug of laninamivir, showed a superior anti-virus efficacy in mouse and ferret infection models compared to repeated administra-tion of oseltamivir and zanamivir. [ ] [ ] [ ] this suggested that cs- works as a novel long-acting na inhibitor of influenza virus in vivo. a single inhalation of cs- proved noninferiority in adult patients and significantly superior in child patients, compared to an approved dosage regimen of oseltamivir for treatment. cs- has been commercially available as an inhaled drug, inavir Ò , for the treatment of influenza in japan since october . the long-acting characteristics of cs- are explained by several reasons. first, cs- was quickly hydrolyzed to an active metabolite, laninamivir, after an intranasal administration to mice, and was retained for a long time as laninamivir in target organs, such as lung and trachea. however, with an intranasal administration of laninamivir, it disappeared quickly and did not demonstrate its longlasting characteristics. another reason is a strong binding of laninamivir to nas of seasonal influenza viruses compared to other three na inhibitors, oseltamivir carboxylate, zanamivir, and peramivir. in the following, the tight-binding ability of laninamivir to pandemic (h n ) na, as well as to the seasonal influenza virus nas, was demonstrated. in addition, we present a hypothesis of the mechanism of the long-lasting property of cs- in mouse based on a localization of an enzyme that hydrolyzes cs- to laninamivir. the influenza viruses, pandemic(h n ) (inf ), a ⁄ new caledonia ⁄ ⁄ (h n ), a ⁄ panama ⁄ ⁄ (h n ), and b ⁄ mie ⁄ ⁄ were treated with excess na inhibitors, such as oseltamivir carboxylate, zanamivir, peramivir, and laninamivir, and then unbound na inhibitors were removed from the mixtures with a bio-spin column bio-gel p- (bio-rad laboratories, hercules, ca, usa). the na substrate, -methylumbelliferyl-n-acetyl-a-d-neuraminic acid (nacalai tesque, japan) was added to the virus-na inhibitor complex, and the na activities were followed for hours at room temperature by measuring the fluorescence at an excitation wavelength of nm and an emission wavelength of nm. the enzyme which hydrolyzes cs- to laninamivir was partially purified from rat lungs using ion exchange column chromatography, and almost all bands separated by an sdspolyacrylamide gel electrophoresis were identified by mass spectrometry. the gene expression profiles of the enzyme were investigated by the bioexpress database (genelogic inc., gaithersburg, md, usa). the enzyme gene cloned from mouse lung mrna was transiently expressed in cos cells. antiserum to the esterase was prepared by immunizing rabbits, and immunostaining was done using histomouse-tm-max kit (invitorgen corp., carlsbad, ca, usa) according to the manufacturer's manual. binding stability of na inhibitors to the four viruses are shown in figure the enzyme that hydrolyzes cs- to laninamivir in rat lungs was identified as carboxyesterase. this esterase was shown to be expressed in epithelial cells of rat lung by in situ hybridization. the mouse homolog of the rat esterase was carboxylesterase (ces ). the mrna of the mouse ces was shown to be highly expressed in lung and liver by the gene expression profile, and ces was also found to contain signal sequences for retention in endoplasmic reticulum (er) and golgi at the c-terminus. the cloned ces gene and the ces gene lacking the signal sequence were exogenously expressed in the cos cells. the cs- -hydrolyzing activity associated with the cos cells expressing ces was recovered from the culture sup of the cos cells expressing ces lacking the retention signal sequence. localization of ces was immunohistologically confirmed inside the airway epithelium cells of mice, which are the target cells for influenza virus infection. the long acting property of intranasal administration of cs- in mice can be explained both by the long retention of laninamivir in the respiratory tract and by the stable binding of laninamivir to influenza virus na. again, stable binding of laninamivir to na of pandemic (h n ) virus was also observed similar to that of seasonal h n virus. the following are speculated as the mechanisms for the long-lasting characteristics of cs- in mice. we explain the mechanism by clarifying a cs- hydrolyzing enzyme and its localization inside cells. the hypothesis of the mechanism is presented in figure . briefly, hydrophilic laninamivir may not enter easily inside cells, whereas hydrophobic cs- may enter inside cells. ces with er ⁄ golgi retention signal hydrolyzes octanoate of cs- figure . difference of binding stabilities of various na inhibitors to influenza virus neuraminidases. the na substrate was added to the influenza virus-na inhibitor complex (oseltamivir carboxylate, n; zanamivir, h; peramivir, s; laninamivir, •; distilled water, ¤), and the na reaction was followed for minutes. the background (only the na substrate [d] ) is also shown. a part of data from. to generate the hydrophilic drug, laninamivir, and then it is trapped inside er ⁄ golgi because of its high hydrophilicity. the glycoprotein, na, which matures in er ⁄ golgi, meets laninamivir there and efficiently makes a stable complex with it. there are some questions that remain. how does cs- move from the cell membrane to er ⁄ golgi? is laninamivir indeed trapped inside er ⁄ golgi, and does it make a complex with na in mice? we are now making an attempt to clarify these concerns. in our study, we have explored the antiviral potential of two newly synthesized compounds to provide protection against the novel pandemic influenza virus h n ( ) strain. the compounds were reconstituted in dimethylsulphoxide (dmso), and so the initial studies began with cytotoxicity determination of solvent on uninfected and untreated madin-darby canine kidney (mdck) cells. on obtaining an upper limit for dmso, the compounds were tested for estimation of their maximum non-toxic dose to the mdck cells. thereafter, the effective dose of the compounds was evaluated and validated by a number of assays and gene expression profiling at both nucleic acid and protein level. we found that these newly synthesized compounds possess potent inhibitory activity towards the novel pandemic influenza h n ( ) virus. these findings are being evaluated in vivo for a better understanding of their inhibitory capabilities and also their effect on the host metabolism. this will be required in the course of development of new drugs for use in the prophylaxis and treatment against the influenza virus. the mdck cell line (from nccs, pune) was maintained in · dmem media (sigma, st. louis, mo, usa) supplemented with % fetal calf serum and antibiotics viz. unit ⁄ ml penicillin and lg ⁄ ml streptomycin at °c ⁄ % co . the synthesized compounds used in this study were kindly provided by the department of chemistry, university of delhi, delhi, india. the pandemic influenza h n ( ) virus was isolated and propagated in the allantoic cavities of embryonated chicken eggs during the pandemic period. the virus stocks were prepared and stored at ) °c. plaque assay was performed as previously described by hui et al., . briefly, ae · mdck cells ⁄ ml were seeded in six-well plates and maintained in dmem for hours at °c ⁄ %co . the monolayer of the cells was inoculated with serially diluted virus samples for minutes at °c ⁄ %co . subsequently, a mixture of agar overlay was added, and the plates were incubated at °c for days or until formation of plaques. the plaques were visualized after removal of the agar plug and staining with ae % crystal violet or neutral red solution. the virus titre was expressed as plaque forming unit (pfu) per milliliter. the in vitro cytotoxicity analysis was performed to determine the % cytotoxic concentration (cc ) of the compounds on mdck cells. the compounds were dissolved in dimethylsulfoxide (dmso), and so a prior cytotoxicity analysis was performed to determine the toxic concentration of dmso on the cells. various concentrations of compounds were mixed with dmem containing % fcs before addition to the preformed monolayer of mdck cells in -well plates. a series of suitable controls for in vitro cc determination was included in every plate, and the plates were incubated in the optimum environment for mdck cell culture. the cc of test compounds was analyzed by estimation of percentage cell viability of the compound-and mocktreated mdck cells by performing a colorimetric assay using tetrazolium salt -( , -dimethylthiazol- -yl)- , diphenyl tetrazolium bromide (mtt) at end-point of hours post-incubation. the assay was performed as described by mosman . briefly, mtt stock at a concentration of ae mg ⁄ ml was prepared in · pbs. the media was aspirated from the wells and ll of mtt dye from the stock was added to each well. following incubation at °c ⁄ % co for - hours, the dye was very carefully removed from the wells, and the cells were incubated with ll of stop solution (dmso) per well at °c ⁄ % co for hour. the absorbance of the supernatants from each well was measured at nm, and the percentage cell viability was calculated. madin-darby canine kidney cells were maintained overnight in a -well tissue culture plate at °c ⁄ % co . the cells were inoculated with various virus dilutions at °c ⁄ % co for minutes and observed for cytopathic effect (cpe). the media from the experimental wells were aspirated after - hours of infection and were subjected to plaque assay. the percentage cell viability was determined by performing mtt assay. the results of both these tests were used to assess tcid of the virus. the pre-formed monolayer of mdck cells was inoculated with the -fold dilution corresponding to tcid of the virus for hour at °c ⁄ co . the experimental setup included control wells for the cells, virus, and compound. meanwhile, the concentrated stocks of the synthesized compounds were diluted with dmem (with % fcs) to various concentrations within their respective cc ranges. one hour post-infection, the cells were incubated with these diluted solutions. the cells were observed at various time intervals post-inoculation for cpe, and ll media was collected from each experimental well for performing hemagglutination test. after h, the media was collected for plaque assay and the cells were subjected to mtt cell viability assay. preformed monolayers of mdck cells were infected with virus and treated with the respective inhibitory concentration of the compounds. forty-eight to hours post-incubation, total cellular rna was isolated using ribozol (amresco, solon, oh, usa) and treated with lg ⁄ ml of dnase (promega, madison, usa). the concentration and quality of the rna from each well were determined by measuring their absorbance at and nm. one microgram of the cdna synthesized from each rna sample was used for sybr green-based real-time pcr detection of the ha gene of pandemic influenza h n ( ) virus. as a control, human glyceraldehyde- -phosphate dehydrogenase (hgapdh) was also amplified using gene specific primers. , immunoblotting immunoblotting was performed to further validate the antiviral potential of the compounds. the experimental protocol was the same as for real time rt-pcr analysis. the cells were harvested hours post-treatment with the compounds to prepare whole cell lysates in mammalian cell lysis buffer [ ae m nacl, ae m tris cl (ph ae ), ae m edta (ph ae ), m m protease inhibitor cocktail, lg ⁄ ml pmsf]. the protein concentration was determined by bca protein assay. the cell lysates were fractionated on % polyacrylamide for western blotting. the blot was developed using sheep monoclonal antibody (santa cruz biotechnology, ca, usa) against ha protein of influenza virus and horseradish peroxide conjugated rabbit-anti sheep igg ( : dilutions) as secondary antibody. the median cytotoxic concentration for compound meuh came out to be lm, and that for flh was lm. compounds showing potent antiviral effect on the pandemic influenza h n ( ) virus propagation in madindarby canine kidney cells ( figure ). the viral titres remained constant in cells treated with the compounds, while they increased in the untreated virus infected cells. ed for the compounds meuh and flh were and lm, respectively. fifty-two percent (meuh) and % (flh) inhibition against the pandemic influenza h n ( ) virus was achieved using ed of the test compounds. both the compounds were able to reduce the rna levels of the ha gene by approximately - %, whereas approximately % inhibition was seen when both the compounds were used in combination. similar results were obtained by the immunoblotting analysis ( figure ). antiviral therapy has shown to be a promising tool in the management of various respiratory diseases, including those caused by influenza viruses. we have already shown inhibition of influenza virus replication in our earlier studies using catalytic nucleic acids, which can be used as an approach in the development of new therapeutic strategy. these therapies are very useful as the influenza virus vaccines need annual renewals due to frequent genetic drifts in the viral surface proteins. in pandemic situations the existing vaccines do not provide complete protection against the novel virus as the population generally remains naïve for the newly mutated surface antigens. the antiviral drugs play an important role in the control of novel viral strains for which there are no vaccines available. however, the key obstruction in the extensive use of antiviral drugs is their cost and relative therapeutic efficacy provided. two classes of drugs were being used for treatment and control of the influenza virus infection in humans, the m ionchannel blockers , (amantadine and rimantadine), which prevent viral uncoating, and the neuraminidase inhibitors , (zanamivir and oseltmivir), which prevent the release of influenza virions from the cytoplasmic membrane. but widespread resistance to these antiviral drugs , has limited their use. thus, novel drugs are required for the effective therapy against the emerging strains of influenza virus. the novel chemical compounds used in our study were tested for their antiviral efficacy against the pandemic influenza h n ( ) virus. a reduction in the cpe in compound treated virus infected mdck cells indicated presence of antiviral activity in chemical compounds. the persistence of constant viral titers in the compound treated cells provided evidence for the interference posed by the compounds in the replication of influenza virus. inhibition in the ha gene expression further validated our hypothesis for the antiviral effect of compounds. the efficacy of these compounds in animal models is currently being validated in our laboratory. further, molecular studies are required to ameliorate the awareness regarding the mode of action of these chemical compounds against the viruses. and is now licensed in japan, while another, laninamivir, is being developed as an inhaled prodrug. resistance to nais among circulating influenza viruses was previously low (< % worldwide). [ ] [ ] [ ] however, the - influenza season was marked by a worldwide emergence of oseltamivir-resistant seasonal influenza a (h n ) viruses with the h y (h y in n numbering) in the na. [ ] [ ] [ ] [ ] [ ] [ ] the prevalence of oseltamivir resistance was even higher in the subsequent - influenza season with many countries reporting up to % oseltamivir resistance, seasonal and pandemic influenza viruses collected globally between october , and september , were submitted to the who collaborating center for surveillance, epidemiology and control of influenza at the centers for disease control and prevention (cdc) in atlanta, ga, usa, and propagated in madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa). reference viruses representative of oseltamivir-sensitive and -resistant seasonal and pandemic viruses were also propagated in mdck cells. susceptibilities of virus isolates to the nais oseltamivir carboxylate (hoffman-la roche, basel, switzerland) and zanamivir (glaxosmithkline, uxbridge, uk) were assessed in the chemiluminescent ni assay using the na-star tm kit (applied biosystems, foster city, ca, usa) as previously described. additionally, subsets of virus isolates were tested for susceptibility to peramivir (biocryst pharmaceuticals, birmingham, al, usa). fifty percent inhibitory concentration (ic ) values were calculated using jaspr curve fitting software, an in-house program developed at cdc. curve fitting in jaspr was done using the equation: v = vmax · ( ) ([i] ⁄ (ki + [i]))), where vmax is the maximum rate of metabolism, [i] is the inhibitor concentration, v is the response being inhibited, and ki is the ic for the inhibition curve. box-and-whisker plot analyses of log-transformed ic s were performed for each virus type ⁄ subtype and nai using sas . software (sas institute, cary, nc, usa) to identify viruses with extreme ic values (outliers). outliers were characterized based on a statistical cutoff of ic greater than three interquartile ranges from the th percentile. outliers were subjected to genetic analysis by pyrosequencing and ⁄ or conventional sequencing to detect known or novel markers of nai resistance. those harboring previously characterized mutations in the na associated with nai resistance were considered drug-resistant; their descriptive statistics were determined separately from naisusceptible viruses. descriptive statistics to compute the mean, median, and standard deviation (sd), and a one-way analysis of variance were performed on original scale ic data, using sas . software (sas institute) for each nai and virus among seasonal influenza a (h n ) viruses tested for oseltamivir susceptibility (n = ), ( ae %) were outliers for the drug (table ) and harbored the oseltamivir-resistance conferring h y mutation in the na. by contrast, only a small proportion ( ae %) of tested h n pdm viruses (n = ) were resistant to oseltamivir. all influenza a (h n ) viruses (n = ) were sensitive to oseltamivir except for one outlier, a ⁄ ontario ⁄ rv ⁄ with d v mutation in the na, whose ic of ae nm was beyond the statistical cut-value off and > -fold the mean ic for the drug ( ae nm). all influenza b viruses (n = ) were sensitive to oseltamivir with exception of an outlier b ⁄ texas ⁄ ⁄ , with d e (d e in n numbering) mutation in the na, whose ic was beyond the cut-off, but only fourfold greater than the mean ic for the drug. all virus types ⁄ subtypes tested for zanamivir were sensitive to the drug (table ) , except for some outliers among seasonal influenza a (h n ) and a (h n ) outliers. the seasonal influenza a (h n ) outliers included a ⁄ thailand ⁄ ⁄ (h n ) and a ⁄ hawaii ⁄ ⁄ (h n ), both with combined h y and d d ⁄ g mutations in their na. the presence of concurrent mutations at na residues h and d in seasonal influenza a (h n ) virus isolates substantially enhances resistance to oseltamivir and peramivir and ⁄ or zanamivir, however, the changes at d are typically cell-derived and not present in clinical specimens. influenza a (h n ) outliers for zanamivir included a ⁄ ontario ⁄ rv ⁄ with d v mutation in the na, as well as a ⁄ maryland ⁄ ⁄ and a ⁄ vladivostok ⁄ ⁄ with d g and mixed d d ⁄ g mutations, respectively. some mild outliers for zanamivir among a (h n ) viruses with ic beyond the statistical cutoff but < -fold mean ic for the drug were also identified; their genetic analysis revealed presence of wildtype and mutant sequences at residue namely, d d ⁄ g, d d ⁄ n, or d d ⁄ a. mutations at residue d of the na are associated with reduced susceptibility to zanamivir in a (h n ) viruses, but were reported to be cell-culture derived in recent h n viruses. all virus isolates tested for peramivir (n = ) were sensitive to the drug, except for h y variants among seasonal influenza a (h n ) and h n pdm viruses, which exhibited reduced susceptibility to the drug. in addition, one influenza a (h n ) isolate, a ⁄ ontario ⁄ rv ⁄ with d v mutation in the na, showed reduced susceptibility to peramivir. the ic values determined in functional ni assays provide valuable information for detection of resistant viruses, but should not be used to draw direct correlations with drug concentrations needed to inhibit virus replication in the infected human host, as clinical data to support such inferences are inadequate. nevertheless, combining elevated ic values with the presence of established molecular markers of resistance in the na of virus isolates and their matching clinical specimens provides a reliable and reasonably comprehensive approach of identifying nai-resistant isolates for surveillance purposes. in this study, outliers with elevated ic values for oseltamivir among seasonal influenza a (h n ) and h n pdm viruses were confirmed to be oseltamivir-resistant based on the presence of the h y mutation in the na. outliers for oseltamivir and ⁄ or zanamivir among influenza a (h n ) viruses in this study were shown to harbor mutations at d , which were earlier associated with reduced susceptibility to zanamivir, and were cell-culture derived. the effects of d mutations on nai susceptibility appear to be strain-specific; however, there are no conclusive supporting data and further investigations are required. outliers among the influenza a viruses in this study exhibited changes in the na, derived naturally or through cell-culture, which altered their susceptibility to nais. however, mild outliers for oseltamivir and ⁄ or zanamivir among influenza a viruses with slightly elevated ic s, but without apparent changes in the na are sometimes identified. in such instances it is imperative to exclude the potential presence of influenza b among such outliers, using conclusive genetic tests such as real time pcr, since influenza b viruses exhibit higher ic values for oseltamivir and zanamivir than influenza a viruses. viruses exhibiting such mixes are typically excluded from statistical analyses of ic s for respective drugs and virus type ⁄ subtype. establishment of a clinically relevant ic cutoff value which could be used to differentiate statistical outliers from truly resistant viruses is imperative. global surveillance for nai susceptibility of influenza viruses circulating globally should be sustained to reflect the impact of seasonal and pandemic of influenza, given the limited pharmaceutical options available for control of influenza infections. nasopharyngeal swab specimens from patients with acute respiratory infection were collected at influenza sentinel surveillance units (outpatient and hospital-based) all over mongolia. specimens were transported to the virology laboratory, nccd, ulaanbaatar, and rt-rt pcr positive samples were grown in a mdck cell culture according to the protocol developed by cdc. and influenza virus gene segment (m genes) sequencing ( strains-genbank accession numbers: cy , cy , cy , cy , cy , cy , and cy ) and influenza virus gene segment (na gene) sequencing ( strains genbank accession numbers: cy and cy ) by the standard methods with applied biosystems xl genetic analyzer using primers supplied by who collaboration centers. a chemiluminescent na inhibition assay was performed with veritas microplate luminometer using the commercially available kit, na-star (applied biosystems, foster city, ca, usa), according to the manufacturers protocol. the na inhibitor susceptibility of influenza virus isolates was expressed at the concentration of na inhibitor needed to reduce na enzyme activity by % (ic ). oseltamivir carboxylate, was provided by f. hoffman-la roche ltd (basel, switzerland). na inhibition assay data were analyzed using robosage software comparing test data with the data produced by the reference na inhibitor sensitive and resistance strains, which were provided by the who influenza collaboration center, melbourne, australia. all viruses tested were sensitive to oseltamivir with two exceptions: a seasonal influenza virus a ⁄ ulaanbaatar ⁄ ⁄ (h n ) with ae nm ic value and a pandemic influenza virus a ⁄ dundgovi ⁄ ⁄ (h n ) with ae nm ic value ( figure ). there was oseltamivir resistance detected in ae % ( ⁄ ) of seasonal a (h n ) and in ae % ( ⁄ ) of a (h n ) pdm viruses. the oseltamivirresistant viruses were collected from untreated patients. in total, influenza b viruses were analyzed by na inhibition assay and all were sensitive to oseltamivir. the na of both oseltamivir-resistant strains contained h y mutation based on the sequencing analysis. the difference in the na amino-acid sequences between the mongolian oseltamivir-resistant viruses and the respective oseltamivir-sensitive reference viruses is shown in table all a(h n ) viruses analyzed for m channel inhibitor resistance by pyrosequencing contained the s n mutation and, thus, were resistant to this class of anti-influenza drugs. the segment sequencing revealed that seasonal a(h n ) viruses possess the common s n mutation. of note, a single strain a ⁄ zavkhan ⁄ ⁄ (h n ) contained an unusual s d change in the m protein. our study shows that the same prevalence [ ae % ( ⁄ )] of seasonal a(h n ) viruses with h y mutation in ⁄ season in mongolia with the published data for ⁄ season from japan. , however the prevalence of oseltamivir resistance in japan has dramatically increased in ⁄ season to % ( ⁄ ). the observed double mutations: h y and d g in a ⁄ ulaanbaatar ⁄ ⁄ (h n ) strain, which have been also found in japan in ⁄ season. the patient from whom the oseltamivir resistant seasonal influenza h n virus has been isolated was a -year-old boy, living in ulaanbaatar, the capital city, without history of using oseltamivir. the patient from whom the oseltamivir resistant a(h n )pdm virus was isolated was a year-old man, residing in the dundgovi, the southern province, also without history of antiviral treatment. according to the who data, isolation of the pandemic viruses carrying h y change from untreated patients has been uncommon. circulation of amantadine-resistant seasonal a (h n ) viruses has been increasing in mongolia since ⁄ influenza season. all pandemic influenza a(h n ) strains ( ) tested were resistant to m channel inhibitors due to the presence of the s n mutation in the m protein. among seasonal a(h n ) viruses, one contained a s d change whereas the others had s n, the well established marker of resistance to both amantadine and rimantadine. this is the first report of detecting the s d change in the seasonal a(h n ) viruses. according to the cdc data (unpublished), the s d change conferred the drug resistance in the a(h n ) viruses according to the virus yield reduction assay. it is essential to continue the antiviral resistance surveillance of influenza virus strains circulating in mongolia to ensure the efficiency of a proper clinical management of influenza patients. (conferred by the s n mutation). of note, the genotype and genotype dual resistant viruses from asia appear to be genetically similar to those previously reported dual resistant viruses from hong kong, sar. , the genotype virus was the only dual resistant virus with a nearly complete c genome. oseltamivir-resistance for this virus appears to be the result of a reassortment as demonstrated by the presence of the oseltamivir-resistant clade b na gene. although the detection of dual resistant seasonal influenza a (h n ) viruses is still rare, there has been an increased prevalence of dual resistance viruses during the last three seasons: . % ( of tested in - ), . % ( of in - ) , and % ( of in - ) (v p < . ). while the continued circulation or co-circulation of seasonal a (h n ) viruses is uncertain, the emergence of dual resistant influenza viruses in five countries does present a public health concern, especially since dual resistant viruses would limit the options for antiviral treatment to a single licensed antiviral drug: zanamivir. moreover, the markers of resistance seen in seasonal a (h n ) viruses also confer resistance in the more widely circulating pandemic a (h n ) virus. and, since the acquisition of mutations in influenza a viruses typically occur through drug selection, spontaneous mutation, or genetic reassortment with another drug resistant influenza a viruses, the detection of influenza a (h n ) viruses that are resistant to both adamantanes and oseltamivir warrants close monitoring, even if only detected at low frequency. new antiviral agents and strategies for antiviral therapy are likely to be necessary in the future. heightening concern that drug resistance will likewise become prominent in pandemic viral strains and highlighting the need for antiviral drug resistance surveillance. the h y mutation in h n neuraminidase is the most common mutation conferring resistance. however, due to the high mutation rates of viruses, new mutations can be expected that will also render viral neuraminidase less sensitive to antiviral drugs. pcr methods can be used to detect previously identified mutations; however, functional neuraminidase enzyme activity inhibition testing is necessary for detecting drug resistance that results from novel mutations. the two neuraminidase enzyme inhibition assays using either the fluorescent munana or chemiluminescent na-star Ò substrate are robust tools for ni susceptibility testing. the munan-a-based assay is broadly used by many groups, including many regional health organizations for ni susceptibility testing, yet no standardized protocol or dedicated kit has been in place for this assay, making comparison of data generated between different laboratories difficult. borrowing from multiple neuraminidase inhibitor susceptibility network (nisn)-published munana-based neuraminidase assay protocols, we have developed a kit-based fluorescent neuraminidase assay that offers both standardization and off-the-shelf quality-controlled reagents for ni susceptibility testing and other neuraminidase assay applications. the na-fluor tm influenza neuraminidase assay reagents and protocols were optimized in comparison to published nisn protocols according to the criteria of assay performance, ease-of-use, consideration of historically used assay conditions, reagent storage stability, and environmental impact. our optimized assay conditions consists of lm munana, ae mm mes, mm cacl , and ph ae in a ll assay volume, and performing the assay for minutes at °c following a minutes preincubation of drug with the virus. these conditions are consistent with the majority of published influenza ni screening data in publication. the standard na-fluor tm assay workflow for screening viral isolates for sensitivity to nis includes first titering the viral sample by neuraminidase activity to determine optimal virus concentration to be used in subsequent ic determination assays. the na-fluor tm assay is an ideal tool for titering virus based on neuraminidase activity in the viral coat. titering of viral samples prior to running the ic determination assays insured that assays would be performed within the fluorescence detection dynamic range of both the assay and the fluorometric instrument being used. viral titers giving rfus in the range of - were used for subsequent assays. comparison to traditional munana assays a primary goal of developing a standardized munana assay was to provide a standardized protocol and set of reagents that would allow for comparison of ni surveillance data between laboratories and over time. in addition, the assay should provide data comparable to historical data sets based on traditional munana-based protocols. to insure that our newly developed na-fluor tm assay met these criteria we performed side-by-side comparisons of the na-fluor tm assay to munana-based nisn protocols, as well as our na-xtd tm and na-star Ò chemiluminescent neuraminidase assays to compare assay sensitivity and dynamic range and for ni ic determination with multiple viral isolates. for all assay comparisons, assays were performed according to respective published protocols. for direct comparison of results, an equivalent amount of virus (and concomitant neuraminidase activity) was used for each assay. the na-fluor tm assay provides low-end sensitivity (by signal to noise ratio) and dynamic range similar to nisnpublished, munana-based protocols (data not shown). these assays all show a low-end detection of approximately ae u ⁄ well and dynamic range of - orders of magnitude when performed simultaneously side-by-side using serial dilutions of bacterial (clostridium perfringes) neuraminidase. these assays show approximately onefold less dynamic range and approximately fivefold less low-end sensitivity than chemiluminescent assays under these conditions. given the large amount of archived ni inhibition data for viral isolates over the past decade, it is very important for a standardized assay to generate data similar to established protocols so that data can be compared in relative terms. when run side-by-side, na-fluor tm assay provided oseltamivir carboxylate and zanamivir ic values similar to nisn-published, munana-based protocols. ic values vary somewhat for munana assays versus chemiluminescent assays depending on the viral isolate, as previously described. the na-fluor tm assay also exhibited similar sensitivity for detecting ni sensitive virus compared to nisn-published fluorescent assays as shown in figure . the large shift in ic values between oseltamivir-sensitive and resistant virus using the na-fluor tm assay enables detection of mutant virus in mixed viral samples ( figure ). this capability is critical for identifying resistant virus in clinical isolates presenting mixed populations of resistant and sensitive virus during ni susceptibility surveillance. several characteristics of the na-fluor tm assay make it an ideal assay for processing large numbers of viral isolates for ni sensitivity surveillance or for using the assay for high throughput screening for lead discovery of new antiviral reagents. the na-fluor tm assay signal was found to remain stable for up to hours after stop solution addition when stored at room temperature and for several days when stored at °c (data not shown). ic values did not change over these times, indicating that the assay is compatible with processing many samples in a short time frame. the na-fluor tm assay was also found to be highly reproducible giving a z' of ae or above indicating that the assay can be used confidently to identify nis in high throughput screening mode. the assay can tolerate up to % dmso, a common compound delivery reagent used in high throughput screens (data not shown). we have developed a standardized na-fluor tm assay suggested protocol that gives data similar to established mun-ana protocols. however, we have also found that several protocol adaptations can be made that generate comparable data while allowing the user more flexibility in assay mode, use of additional reagents, and to meet user-specified assay time requirements. the na-fluor tm assay can be run in either the standard minutes ⁄ °c endpoint mode described above or as real-time kinetic assay with repeated reads taken over time without the addition of stop solution, which both serves to terminate neuraminidase activity and to enhance the fluorescence of the product. for typical ni-sensitive viral strains, the rate of munana substrate turnover at °c is linear for at least hours (data not shown). as would be expected, rates of substrate turnover decrease in the presence of nis reflected in a decreased slope exhibited by real-time kinetic reads. real-time acquired rfus are typically - fold lower than rfus acquired after addition of stop solution at the same time point. ic values obtained using slope analysis for real-time assays are similar to values obtained by endpoint analysis. whether run in real-time or end-point mode, the linear rate of substrate turnover allows the user to run the assay for shorter or longer assay times than the standard protocol without compromise to assay performance. the na-fluor tm assay is also compatible with standard methods used in many laboratories to inactivate virus. we have shown that ni ic values for multiple viral strains remain unchanged when the assay is performed in the presence of ae % np- or % triton x- (data not shown). similar results are also obtained by adjusting the na-fluor tm stop solution to % ethanol prior to addition for assay termination. the assay is unaffected by phenol red concentrations present in cell culture media. we have developed a standardized munana-based fluorescent neuraminidase assay, the na-fluor tm influenza neuraminidase assay kit, which has been optimized for ni susceptibility screening. the assay provides data that can be compared to data generated using traditional munanabased protocols. the assay is economical, highly reproducible, easy to use, and environmentally friendly. the assay is flexible and amendable to user-specific adaptations including assay mode, assay timing, and reagent compatibility. trademarks ⁄ licensing ª life technologies corporation. all rights reserved. relenza is a registered trademark of glaxo- to test the prophylactic potency of h -vhhb, mice were treated intranasally with pbs, lg of h -vhhb, or negative control rsv-vhhb at , , or hours before infection with one ld of nibrg- ma virus. body weight loss was monitored daily, and on day mice were sacrificed to determine the viral load in the lungs. all mice that received h -vhhb retained their original body weight, whereas those receiving pbs or rsv-vhhb gradually lost weight (data not shown). intranasal administration of h -vhhb at or hours before challenge resulted in undetectable lung virus titers. when animals were treated with h -vhhb hours before challenge, virus titers were fold lower compared to pbs and rsv-vhhb treated mice, and three out of seven animals still had undetectable virus titers ( figure ). we next determined if h -vhhb nanobody Ò could be also be used therapeutically. we administered lg of this nanobody Ò intranasally to mice up to hours after chal-lenge with ld of nibrg- ma virus. four days after challenge, animals that received h -vhhb , , or hours after challenge had significantly higher body weight (data not shown) and lower lung virus loads than control mice. although mice treated with h -vhhb nanobody Ò hours after challenge were not clinically protected compared to control mice, they had significantly lower lung virus titers (figure ). to identify the ha amino acid residues that are potentially involved in h -vhh binding, escape viruses were selected by growth and plaque purification of nibrg- ma virus in the presence of h -vhhm or h -vhhb nanobodies Ò . the ha sequences of six independently isolated h -vhhm escape viruses revealed substitution of a lysine by a glutamic acid residue at position in ha (h numbering). in addition, two h -vhhm escape mutants carried an n d and four carried an n s substitution. the three-dimensional structure of nibrg- ha shows that n d ⁄ s and k e are close to each other as part of the corresponding antigenic site b in h ha. , interestingly, the n d ⁄ s mutations remove an n-glycosylation site, which is surmised to have evolved in h n ha as a strategy to mask an antigenic site. escape viruses selected in the presence of h -vhhb carried k n (n = ) or k e (n = ) substitutions. these results indicate that residues in antigenic site b, at the top of ha and very close to the receptor binding domain (rbd), are essential for neutralization of the virus by h -vhhm ⁄ b nanobodies Ò (figure ). the virus titer was measured in lung homogenates prepared on day after challenge. the x axis refers to the time points in hours relative to the challenge (time = hours) when ha-specific nanobodies (h -vhhb), control nanobodies (rsv-vhhb) or pbs was administered to the mice. # below detection limit, n not determined [n = - mice per condition: p values < ae (*)]. here we demonstrated that prophylactic and therapeutic treatment with llama-derived immunoglobulin single variable domain fragments is effective to control infection with h n influenza virus in a mouse model. we demonstrate that pulmonary delivery is a highly effective route of administration to treat or prevent influenza virus infection. in addition, we demonstrate that a homobivalent h -vhhb has powerful h n -neutralizing activity in vivo. it is important to note that we used a mouse-adapted derivative of the non-highly pathogenic nibrg- virus in our challenge model. nevertheless, this virus induces severe morbidity and lethality in mice. compared to conventional neutralizing monoclonal antibodies, vhhs offer the advantage that they are easy to produce in escherichia coli, typically with high yield. in addition, their small size ( kda for a monovalent vhh) and high folding capacity allow the generation of oligovalent vhh derivatives. in vitro escape selection revealed that a k e substitution in ha abolished the neutralizing effect of h -vhhm ⁄ b. a lys or arg residue at this position is conserved in all human h n virus isolates. of note, all selected escape mutants contained a glutamic acid or serine residue at position , which suggests that the conserved positively charged amino acid is important for neutralization by h -vhh nanobodies Ò . interestingly, escape mutants selected with h -vhhm also carried an n d ⁄ s co-mutation that removes an n-glycosylation site in this antigenic site of ha. the predicted n-glycosylation site at n in a ⁄ hong kong ⁄ ⁄ ha was shown to be glycosylated and may have evolved to mask an antigenic site near the rbd. , the selected amino acid changes are located near the receptor binding site of ha. therefore, it is possible that enhanced receptor binding properties of these escape viruses contribute to or are responsible for the loss of neutralizing activity of h -vhh nanobodies Ò . , we conclude that influenza virus neutralizing nanobodies Ò have considerable potential for the treatment of h n virus infections. although we focused on vhhs that presumably recognizes an epitope near the rbd, it is possible to select vhh molecules that bind to other epitopes in ha, including more conserved domains. more, a novel na (i m) substitution was discovered in a series of specimens from a patient. for the amantadine resistance, samples were tested, and all of them were confirmed to be resistant. we collected respiratory specimens from patients who had been clinically refractory to antiviral treatment since october upon ethical approval from the relevant institutions. to investigate the resistant pattern, sequence analysis to the na and matrix (m ) genes were conducted by reverse transcription (rt)-pcr and sequencing reaction. the obtained sequences were analyzed by the influenza sequences and epitopes database, which was developed in korea. eleven patients were found to be having oseltamivir-resistant pandemic (h n ) viruses with the h y substitution in the viral na genes (tables and ). some cases were associated with oseltamivir treatment on the basis of h y change from the oseltamivir-sensitive genotypes to oseltamivir-resistant genotypes in consecutive samples from the same patient. furthermore, a novel na (i m) substitution that may be associated with oseltamivir resistance was detected in specimens from one patient (patient g) who had myelodysplasia and received oseltamivir and peramivir (tables and ). in addition, we obtained viruses from clinical specimens (patients a and c) and evaluated antiviral susceptibility by measuring the dose of oseltamivir and zanamivir required for % inhibition (ic ) of na activity. these viruses (from patients a and c) were resistant only to oseltamivir (ic ae and ae nmol ⁄ l, respectively). susceptibility to zanamivir was not altered whether na contained y or h (ic ae and ae nmol ⁄ l, respectively). one isolate of pandemic (h n ) virus with an oseltamivir-sensitive genotype (h in its na) was susceptible to oseltamivir (ic ae nmol ⁄ l) and zanamivir (ic ae nmol ⁄ l). patients with oseltamivir-resistant pandemic (h n ) were treated during hospitalization with oseltamivir alone or with a combination of other antiviral drugs ( we found patients of oseltamivir resistance with h y mutation in the na gene of pandemic (h n ) virus through the surveillance of patient refractory to antiviral treatment. in addition, novel amino acid change (i to m) at position in the na gene, which might influence oseltamivir susceptibility, was detected in sequential specimens of a patient. these data showed that generation of oseltamivir resistance could be associated with oseltamivir treatment. therefore, it needs to strengthen the antiviral monitoring by supplementation of the clinical data including antiviral treatment. during the pandemic, oral oseltamivir was the primary antiviral medication used for treatment of hospitalized patients with ph n infection. many physicians worried that clinical deterioration or failure to respond to treatment with oseltamivir was due to either oseltamivir resistance or oseltamivir failure. in the united states, two investigational intravenous (iv) nais were available during - : peramivir through emergency use authorization and zanamivir by investigational new drug application. peramivir would be an option for patients with oseltamivir failure, but would not be appropriate for patients infected with h y oseltamivir resistant mutants. iv zanamivir was available in limited supply, but would be appropriate for severely ill patients infected with an oseltamivir-resistant ph n virus. during the pandemic, clinicians had few options for antiviral resistance testing in the united states. to respond to this need, the us centers for disease control and prevention (cdc) offered antiviral resistance testing for patients suspected to have clinical failure due to oseltamivir resistance. we describe the methods that cdc used to prioritize patients for testing during the pandemic and to detect markers for oseltamivir resistance, as well as the results from this testing. to facilitate decisions on which patients to test, we developed testing algorithms that were shared with state labora-tories, epidemiologists, and the emergency operation center at cdc. we prioritized patients who might benefit the most from antiviral testing given the inherent delay in providing antiviral results, e.g. patients who might have prolonged ph n shedding. patients that were critically ill [intensive care unit (icu) admission] or patients with severe immunocompromising conditions with clinical evidence for oseltamivir treatment failure (persistent detection of virus and clinical unresponsiveness to the drug) were prioritized. in addition, we tested specimens from patients that failed oseltamivir chemoprophylaxis. standard forms with information regarding specimen and minimal clinical information were collected on all patients. all protocols were validated and approved by clinical laboratory improvement amendments, e.g. quality standards to ensure accuracy, reliability, and timeliness of patient test results. information collected on patients was deemed public health response, not research, at cdc. clinical specimens, confirmed as pandemic influenza a (h n ), were tested for the h y mutation in the na using pyrosequencing. results were returned to sender within - hours of specimen receipt. from october until july , a total of specimens from patients were submitted for testing. viruses from ( %) of patients had h y mutation in the na in at least one submitted specimen. clinical information was available for patients (table ) . most patients had received oseltamivir for treatment prior to obtaining the specimen sent for antiviral testing. four patients received oseltamivir for chemoprophylaxis, all were immunosuppressed, and all had the h y mutant; duration of chemoprophylaxis until ph n infection was detected varied ( - days). among the patients with an h y mutant who were treated with oseltamivir, the median time on oseltamivir prior to collection of specimen with h y mutation was days (range - days). three patients were part of a hospital cluster of oseltamivir-resistant virus infections and were infected with h y mutants prior to oseltamivir treatment. patients with immunocompromising conditions accounted for almost half of all patient specimens tested, but they accounted for the majority of oseltamivir-resistant ph n virus infections (table ) ; among individuals with severe immunocompromising conditions and clinical failure while on oseltamivir therapy, ( %) had the h y mutant detected. among the immunosuppressed patients with an oseltamivir-resistant virus, ( %) had hematologic malignancies reported. in contrast, among the subset of icu patients without immunocompromising conditions and clinical failure while on oseltamivir therapy, we found little resistance: ( ae %) of icu patients had oseltamivir resistance detected. during the pandemic, we were able to provide timely and useful information to clinicians regarding suspected cases of oseltamivir resistance. our testing algorithm limited the number of specimens to specimens from the highest risk patients that would benefit the most from antiviral treatment. such an approach allowed us to offer this service without compromising our public health duties. in addition, the information we collected on patients from this service complimented our data on the national surveillance for antiviral resistance. we also performed national antiviral resistance surveillance from april to july . overall, resistant ph n viruses were identified from april to july in the united states among tested samples, including specimens described above, surveillance specimens, and resistant viruses reported in the literature. further studies to understand risk factors for oseltamivir-resistant ph n infection in patients with severe immunocompromising conditions are needed. while efforts to provide antiviral testing technology and materials to state laboratories are ongoing, clinicians still have limited options for such testing. rapid and inexpensive assays that could be performed by clinical laboratories, especially those caring for immunosuppressed patients, would be useful to inform patient care. the applied biosystems Ò na-xtd tm influenza neuraminidase assay kit provides the next-generation na-xtd tm , -dioxetane chemiluminescent neuraminidase (na) substrate, together with all necessary assay reagents and microplates, to quantitate sensitivity of influenza virus isolates to neuraminidase inhibitors. like the na-star Ò influenza neuraminidase inhibitor resistance detection kit, the na-xtd tm influenza neuraminidase assay provides highly sensitive detection of influenza neuraminidase activity. in addition, the na-xtd tm assay provides extended-glow light emission that eliminates the need for reagent injection and enables signal measurement either immediately or up to several hours after assay completion. the na-xtd tm assay is also used to quantitate influenza na activity directly in cellbased virus cultures to monitor viral growth or inhibition. global monitoring of influenza strains for resistance to neuraminidase inhibitors (nis) is essential for understanding their efficacy for seasonal, pandemic, or avian influenza, and studying the epidemiology of viral strains and resistance mutations. functional neuraminidase inhibition assays enable detection of any resistance mutation, making them extremely important for global monitoring of virus sensitivity to nis. the first-generation chemiluminescent na-star Ò influenza neuraminidase inhibitor resistance detection kit has been widely used for virus ni sensitivity assays, - including identification of a ⁄ h n pandemic virus resistant to oseltamivir. , in addition, this assay has been used for identification of new ni compounds, ni characterization, studies of virus transmission, drug delivery, na quantitation of virus-like particles, and cell-based virus quantitation. neuraminidase assays performed with chemiluminescent , -dioxetane substrates, including na-star Ò and na-xtd tm substrates, typically provide -to- -fold higher sensitivity by signal-to-noise ratio than assays performed with the fluorescent munana substrate. in addition, chemiluminescent assays provide linear results over - order of magnitude of neuraminidase concentration compared to - orders of magnitude with the fluorescent assay. the high assay sensitivity achieved with chemiluminescent assays enables use of lower concentrations of viral stocks, and the wide assay range minimizes the need to pre-titer virus stocks prior to ic determination. chemiluminescent reactions result in conversion of chemical energy to light energy, as light emission. the na-xtd tm substrate is a , -dioxetane structure bearing a sialic acid cleavable group. to perform the na-xtd assay, virus dilutions (from cell culture supernatant) are pre-incubated in the presence of neuraminidase inhibitor. then na-xtd substrate is added and incubated for minutes for substrate cleavage to proceed. finally, light emission is triggered upon addition of na-xtd accelerator, which provides a ph shift and a proprietary polymeric enhancer, both required for efficient light emission. chemiluminescent assays are performed in solid white microplates, and light emission is measured in a luminometer. the na-xtd tm substrate has a single structural difference from the na-star Ò substrate that provides a much longer-lasting chemiluminescent signal, with a signal half-life of approximately hours (not shown), compared to $ minutes with the na-star assay, eliminating the need for luminometer instruments equipped with reagent injectors and enabling more convenient batch-mode processing of assay plates. the na-xtd tm assay kit also provides a new accelerator solution, containing a next-generation polymer enhancer, and a triton Ò x- -containing sample prep buffer providing enhanced na activity. read-time flexibility is demonstrated by determination of oseltamivir ic values using data collected over hours after addition of na-xtd tm accelerator. although signal intensity slowly decreases over time, the ic curves and values are identical at each time point, shown using influenza b ⁄ lee ⁄ ( figure ) . triton x- detergent at % has been shown to inactivate flu virus while increasing neuraminidase activity. the addition of na sample prep buffer (containing % triton x- ) to virus stocks (at ⁄ volume, achieving a final concentration of %) provides increase in na activity up to fourfold, but is not consistently observed, and seems to be most effective with more concentrated virus stocks. ic values are unaffected by the addition of triton x- to the virus stock prior to virus dilution (not shown), so the assay is compatible with known virus inactivation reagents. assay sensitivity and ic values determined with the na-xtd assay have been compared to those obtained with both the chemiluminescent na-star assay and the fluorescent na-fluor assay (not shown). the chemiluminescent assays provide -to -fold higher sensitivity by signal-to-noise ratio, depending on the virus strain, wider assay dynamic range, and better low-end detection limit than the fluorescent assay. the wide assay range with the chemiluminescent assays enables determination of ic values over a range of virus concentrations, eliminating the need to titer virus prior to performing ic determination assays. ic values obtained with the na-xtd assay are nearly identical to those obtained with the na-star assay, with both oseltamivir and zanamivir neuraminidase inhibitors, and tend to be slightly lower than ic values obtained with the fluorescent assay. viral na quantitation provides a convenient read-out to measure viral growth or inhibition, including inhibition in the presence of inhibitory compounds or antibodies, described as accelerated viral inhibition with na as readout assay (avina). bation in the presence of varying concentrations of oseltamivir carboxylate. samples of culture media were assayed hours later. quantitation of na activity with the na-xtd tm assay demonstrates inhibition of viral growth by oseltamivir carboxylate in cell culture ( figure ). different volumes of culture media were assayed with the na-xtd assay, either in the culture plate or in a separate assay plate (not shown). performing the assay using the entire well contents ( ll) reduces assay sensitivity due to the high concentration of phenol red. assaying a smaller volume of culture medium (either in culture plate or a separate assay plate) provides higher sensitivity, and enables temporal monitoring or use of remaining culture medium for other assays. the applied biosystems Ò na-xtd tm influenza neuraminidase assay kit is a next-generation chemiluminescent neuraminidase assay providing high assay sensitivity and ''glow'' light emission kinetics for improved ease-ofuse. the applied biosystems Ò na-fluor tm influenza neuraminidase assay kit, based on the fluorescent mun-ana substrate, has also been developed to complement the na-xtd tm and na-star Ò chemiluminescence assays, for users lacking luminometer instrumentation or choosing to use fluorescence assay detection. together these kits offer: • standardized reagents and protocols • choice of detection technology • simple instrumentation requirements • high sensitivity for use with low virus concentrations • compatibility with batch-mode processing and largescale assay throughput • broad specificity of influenza detection • flexibility in assay format • additional na assay applications -cell-based viral assays, screening for new nis, detection of na from other organisms functional neuraminidase inhibition assays enable detection of any resistance mutation and are extremely important in conjunction with sequence-based screening assays for global monitoring of virus isolates for ni resistance mutations, including known and new mutations. together, these assays provide highly sensitive, convenient and versatile assay systems with standardized assay reagents, and simple assay protocols for influenza researchers. over hospitalizations and deaths in the us annually are attributable to seasonal influenza, primarily in chronically ill persons and the elderly. - following the emergence of pandemic h n influenza, severe illnesses have also been observed in children and young healthy adults. the occurrences of staphylococcal and pneumococcal pneumonia complicating influenza pandemics are well described. [ ] [ ] [ ] although temporal associations of bacterial pneumonia and influenza circulation have been reported, there is little precise data on rates of bacterial complications of seasonal or pandemic influenza. the study of bacterial lung infection has been hampered by insensitive tests for invasive disease and the difficulty of interpreting routinely obtained sputum culture results. , procalcitonin (proct), the prohormone of calcitonin, can discriminate viral and bacterial infections. this -aminoacid precursor protein normally produced by neuroendocrine cells of the lungs and thyroid gland was first shown to be elevated in bacterial infections in patients with pulmonary injury and pneumonitis. stimuli of proct include tnf-a, endotoxin, and other bacterial products. several studies indicate that bacterial infections commonly induce hyperprocalcitonemia, but that viral infections, including h n , are associated with only minimal increases. , , of note, proct induction is attenuated by viral-induced interferon-c. a meta-analysis of studies comparing proct and crp as markers for bacterial infection found that proct was more sensitive and specific than crp for differentiating bacterial from other causes of inflammation. , therefore, we measured proct levels in patients with seasonal and pandemic influenza and compared results with conventional methods for bacterial diagnosis. adults ‡ years of age admitted to rochester general hospital (rgh) from november st to june th for two winter seasons ( - ) with an admitting diagnosis compatible with acute respiratory tract infection were recruited for the study. patients were screened within hours of admission, and those with prior antibiotic use, immunosupression, or pregnancy were excluded. subjects or their legal guardian provided written informed consent. the study was approved by the university of rochester and rgh research subjects review board. at enrollment demographic, clinical and laboratory information was collected. influenza testing included nosethroat swabs (nts) for rapid antigen, viral culture, and reverse transcription-polymerase chain reaction (rt-pcr) and serology. testing for bacterial pathogens included blood cultures, sputum for culture and gram stain, nts for mycoplasma pneumoniae and chlamydophila pneumoniae pcr, s. pneumoniae antigen testing, and pneumococcal serology. if patients were unable to expectorate, sputum was induced with normal saline and bronchodilators. specimens were considered adequate by the standard criteria of > neutrophils (pmns) and < epithelial cells per high power field. serum was collected at admission and hospital day for proct measurements. influenza infection was defined a positive result for any of the following tests: . cloned proteins were coated on eia plates at ug ⁄ ml in bicarbonate buffer. after overnight incubation, plates were washed and two-fold dilutions of serum were incubated overnight at room temperature. plates were washed and incubated with alkaline phosphatase conjugate for hours, followed by substrate. a greater than or equal to fourfold rise in titer was considered evidence of infection with s. pneumoniae. urinary antigen for s. pneumoniae samples were assayed for antigen using the binax now kit. (binax inc, scarborough, me, usa). the proct was measured using time resolved amplified cryptate emission technology (kryptor pct; brahms, henningsdorf, germany). functional sensitivity is ae ng ⁄ ml (normal levels are ae ± ae ng ⁄ ml). mycoplasma and chlamydia pcr real-time pcr targeting the p adhesion gene for m. pneumoniae and the ompa gene for c. pneumoniae was used to detect atypical bacteria. results fifty-one of ( ae %) illnesses evaluated tested positive for influenza virus. of these, were due to ''seasonal influenza'' ( influenza a ⁄ h n and influenza b), and were identified as ''pandemic influenza'' ( h n ). demographics of both groups were similar: mean ages ± and ± years, respectively, and equivalent sex and racial characteristics. other than a higher incidence of underlying lung disease in the seasonal group ( % versus %, p = ae ), pre-existing medical conditions including obesity were similar. symptoms, physical findings, and discharge diagnoses did not differ, and chest radiographs (cxr) showed infiltrates in % and % of seasonal and pandemic subjects, respectively. two pandemic and one seasonal influenza patient developed respiratory failure, and none died. overall, bacterial infections were diagnosed in ( %) subjects ( -seasonal and -pandemic), and none were bacteremic. bacterial infections included: -s. pneumoniae, -m. pneumoniae, -s. aureus, and -h. influenzae. all seasonal patients were diagnosed with asthma or bronchitis, whereas three pandemic patients had pneumonia. mean serum proct (ng ⁄ ml) levels in seasonal versus pandemic patients on admission and day were: ae ± ae versus ae ± ae and ae ± ae versus ae ± ae , respectively, and were not significantly different (table ) . several patients in the pandemic group had high proct levels, and there was a trend toward more pandemic patients having admission proct values ‡ ae ng ⁄ ml than seasonal subjects [ ( %) versus ( %), p = ae ] ( figure a , b). of the four patients with proc-t > ae ng ⁄ ml, two had dense infiltrates on cxr, one had a peripheral wbc of ⁄ ml with a threefold increase in s. pneumoniae antibody, and one developed respiratory failure associated with copd exacerbation. reliable sputum samples (within hours of antibiotics) were collected in only ( %) subjects. of these, proct was ‡ ae ng ⁄ ml in two with influenza alone and three associated with bacterial infection, and < ae ng ⁄ ml in with influenza alone and five associated with bacterial infection. in the with reliable sputa and accepting the conventional bacterial diagnosis, sensitivity of a proc-t ‡ ae ng ⁄ ml for bacterial infection was %, specificity %, positive predictive value %, and negative predictive value %. notably, one patient considered to have influenza alone (proct - ae ng ⁄ ml) had group a streptococcus and s. aureus in a contaminated sputum and bilateral infiltrates on cxr. three of five patients with bacterial infections and proct < ae ng ⁄ ml had a clinical diagnosis of bronchitis. mean proct values were significantly higher in patients with infiltrates versus those with atelectasis or no acute disease on cxr ( ae ± ae ng ⁄ ml versus ae ± ae ng ⁄ ml, p = ae ). combining patients with proct values ‡ ae ng ⁄ ml with those having positive bacterial tests, rates of bacterial infection associated with seasonal and pandemic influenza were % and %, respectively. notably, antibiotics were administered to % of subjects despite % having no acute disease on cxr. in our study, bacterial infections were diagnosed in approximately % of adults hospitalized with influenza with no significant difference in rates noted between seasonal and pandemic influenza infected subjects. previous reports of bacterial infection rates of - % with seasonal influenza are difficult to compare with recent studies of pandemic influenza, because the latter tended to focus on more severely ill patients. [ ] [ ] [ ] bacterial pneumonia has been suspected or diagnosed in - % of patients in intensive care associated with h n infection and up to % of patients who died. , despite aggressive pursuit of specimens for bacterial testing, diagnoses could be confirmed in only ( ae %) of patients using conventional methodology. given the difficulty in establishing a diagnosis of bacterial infection, elevated proct values may be helpful to identify patients at high risk for invasive disease. in a study of patients with severe h n or bacterial infection necessitating intensive care, a threshold proct level of ae ng ⁄ ml, demonstrated % sensitivity and % specificity for bacterial infection. among patients with h n associated pneumonia, many of whom had respiratory failure, a threshold proct value of ae ng ⁄ ml provided a sensitivity of % and specificity of % for bacterial infection. access to samples from lower airways in ventilated patients in these studies may have improved recovery of bacteria and account for the different results we observed. it should be noted that none of our patients were bacteremic, which is a very strong stimulus for proct release. proct levels have been used successfully to guide therapy in community acquired pneumonia, and our data showing high proct levels in patients with infiltrates on cxr suggests proct may be most useful for excluding invasive disease. , elevated proct levels were not observed in patients with purulent sputum and clear cxr. it is notable that a proct level of < ae ng ⁄ ml did not exclude patients with bacterial bronchitis since proct has been used to guide antibiotic therapy in copd exacerbations. while it could be argued that healthy patients with bacterial bronchitis do not require antibiotic treatment, physician behavior in our study indicates antibiotics are frequently prescribed. combining patients with proct values ‡ ae ng ⁄ ml and those with a positive bacterial test, approximately % in patients in our study had bacterial complications associated with influenza infection. efforts should be made to curtail antibiotic use in hemodynamically stable patients with clear cxrs. given physician discomfort regarding discontinuing antibiotics, proct measurements in combination with routine bacterial cultures should be useful tools to guide therapy. influenza, mrsa, cytokines: diagnosis, treatment, prevention -a possible strategy for outpatient care we started the antiviral treatment of influenza in humans using neuraminidase inhibitors on january , in a successful attempt to cure a -year-old patient. since then, we have used the inhalant antiviral drug zanamivir, and later (october , ) changed to the use of oseltamivir with systemic bioavailability for treating patients with influenza. after years of experience with antiviral treatment of outpatients, we highlight the importance of early diagnosis and early treatment. the necessity of an earliest possible diagnosis was confirmed in the pandemic of . large hospitals reported that patients with an h n ⁄ infection had to be treated with extracorporeal membrane oxygenation. we are convinced this is due to delayed recognition of infection in most cases. valuable time is lost when the patient with a sudden onset has to be brought to a hospital for emergency treatment. the point at which the patient goes to the doctor is decisive, and this problem of timing and the delivery of early treatment is not specific to germany. in our medical office, we assessed patients with suspected influenza (to date seasonal infections, and in , h n ⁄ ) through clinical diagnosis, and then proven by point of care rapid test (quickvue; quidel, san diego, ca, usa) followed by pcr. all of the patients undergo concomitant lab tests: leukopenia, serum iron level, and the humoral inflammation status [sum of the c-reactive protein (crp) and fibrinogen levels]. because of the constant threat of a bacterial superinfection, a bacterial swab and antibiogram is carried out on every patient. in all cases positive for influenza, oseltamivir was given immediately. nowadays it is important that a double infection with influenza and mrsa must be recognized immediately and treatment started at once with antivirals and, when appropriate, with a suitable antibiotic. we pay particular attention to an extremely low iron level (signum mali ominis). in addition we monitor oxygen saturation and the course of the humoral inflammation status every - hours for every of our outpatients. among our patients with seasonal influenza, we saw within hours, within hours, and within hours after disease onset. for pandemic influenza, it was patients within hours, within hours, and two within hours. for all patients, we measured crp < ae mg and fibrinogen < mg ⁄ dl ( hours), crp < mg and fibrinogen < mg ⁄ dl ( hours), and crp > mg and fibrinogen < mg ⁄ dl ( hours, only seasonal cases). antibiotics were necessary in cases, heparin and oxygen administration in cases. one hundred forty-eight patients had a superinfection following influenza. the most common strains were haemophilus parainfluenzae and staphylococcus aureus. the subsequent use of a suitable antibiotic was only necessary in % of the patients. in all cases diagnosed, treatment (including heparin and oxygen administration) and monitoring were conducted in our medical office. none of our patients (seasonal and pandemic) had to be admitted to hospital. the early decision of whether or not antiviral and antibacterial treatment is taking effect is the only way the threat of a cytokine storm can be averted. not only does the primary care physician have to be aware of the pathophysiology involved, but also the necessary diagnostic and therapeutic options have to be made available to him. the result will lead to a saving of both lives and healthcare costs. this applies both in epidemic as well as in pandemic times. today we know that influenza leaves behind a defenceless immune system, and that the proteases of s. aureus contribute to influenza associated pneumonia. mark von itzstein, who discovered neuraminidase inhibitors, emphasized the synergistic cooperation of viruses and bacteria (personal communication, ). mrsa and influenza viruses are posing problems worldwide. the case of a -year-old boy with h n ⁄ infection demonstrates how fatal developments can be prevented. due to his constantly recurring colds, we had already detected the mrsa colonization years earlier and had always worked on boosting his general health and resistance. both the patient and his family were included in dealing with the problem. the patient was, and is, always vaccinated early with a virosomal vaccine (baxter). during the oktoberfest in munich in september , when h n infections were increasingly occurring, we learned that our patient had come down with an extremely acute feverish illness. with the help of the rapid test, we diagnosed an h n ⁄ virus infection and started treatment with oseltamivir immediately. the humoral inflammation status, which had increased very rapidly to more than ae mg ⁄ dl within hours, was treated with the effective cotrimoxazol from the antibiogram. at the same time, the patient was heparinized. the following day the patient had no fever and was symptom-free. it was only through our early knowledge of what could develop pathophysiologically that we were in a position to make the right decision at the right time. every doctor treating outpatients can follow this procedure if he is familiar with the pathophysiology of the disease and has the available tests on hand: virus rapid test, additional laboratory parameters (leukopenia, iron), and the humoral inflammation status. the decisive factor, however, is the constant clinical alertness towards the course of every acute feverish cold with acute onset. the patient has to remain in the care of the attending physician, and the chosen treatment has to be administered and monitored. this means constant spo measurements and checking the humoral inflammation status every hours. if a clinical worsening occurs during monitoring, the treatment regime has to be changed immediately, which means the administration of an appropriate antibiotic. this outpatient care on the part of the doctor has to be available days a week so that no time will be lost. reports from the netherlands and denmark show that, with the help of this preventive strategy under the motto 'search and destroy,' the dangerous, fatal course of infections reported in germany with at least four deaths a day, can be avoided. however, the doctor has to be adequately remunerated for the elaborate amount of time this intensive outpatient care requires. with our strategy, we have moved from divergence to convergence in the care of our patients. we reported on our years of clinical experience with this approach at the antivirals congress in peking. our main message was early diagnosis and early treatment. we were able to demonstrate this in outpatients with seasonal influenza and h n ⁄ outpatients. our creed is: as much outpatient care as possible and as little hospitalization as possible. virological and autopsy findings in suspected and confirmed fatal cases of h n pandemic influenza in the czech republic -preliminary results influenza viruses cause substantial morbidity and mortality. pandemic influenza may have a serious impact on certain (mainly younger) age groups in comparison with seasonal flu. influenza is one of few viral infections capable of causing a pneumonia that is difficult to cure and ⁄ or leads to sudden death. the aim of this study was to analyze and compare virological and autopsy findings in patients who died with suspected or confirmed h n pandemic influenza virus infection. there were virologically confirmed cases of pandemic influenza and deaths in the czech republic during pandemic wave. more than influenza strains belonging to the new pandemic variant were isolated in the national influenza reference laboratory. postmortem biological samples were collected from any patient who died with suspected influenza infection to test for respiratory viruses. the samples were screened for h n pandemic influenza virus by real-time pcr (rt pcr), and when rt pcr positive, by virus isolation assay. no immunohistochemical staining for influenza antigen was done on the rna pcr positive cases. other important respiratory viruses such as respiratory syncytial virus, parainfluenza viruses, and adenoviruses were detected by virus isolation assay in a suitable cell culture. epidemiological analysis of postmortem histopathologic findings in the airway tissue was carried out in of fatal cases. virological findings were subsequently correlated with histological changes and available demographic and clinical data. statistical analysis was performed by t-test using spss software. sixty-one deaths ( males, females) were analyzed. the rna of the h n pandemic influenza virus was detected by pcr in cases, while cases remained negative. five respiratory syncytial viruses and two adenoviruses were detected in the influenza negative group. the mean age of confirmed h n pandemic influenza victims was ae years, age range - years and median ae years. the mean age of influenza negative victims was ae years, age range - years and median ae years. the % ci for the difference in the age between the two groups is ) ae ; ae . the test is statistically significant at the % level. the obtained significance (p = ae ) can be explained by the relatively small size of the study group. the most common postmortem histopathologic finding in the lung tissue of the h n pandemic influenza virus-positive victims was diffuse alveolar damage (often bilateral) and ⁄ or hyaline membrane formation, possibly with signs of respiratory distress syndrome (in , i.e., ae %, of autopsied patients). in the h n pandemic influenza virus negatives, the most common finding was pneumonia or bronchopneumonia with the detection of various bacterial species (in , i.e., ae % of autopsied patients). the cause might be either primary bacterial infection or superinfection following primary infection with influenza virus that remained undetected. the h n pandemic influenza victims were younger than the patients who died with suspected but undetected h n pandemic influenza. the majority of deaths were primarily linked to rapidly developing respiratory failure. this result supports the previous reports of severe respiratory outcomes in younger age groups that are typically linked to the spread of a pandemic strain of influenza. due to limited amount of pandemic vaccine, especially at the beginning of pandemic, it is advisable to assess experiences with antiviral treatment, mainly dosing, and way of antiviral administration. primers specific for each of the eight genes of pandemic h n ⁄ were adopted from assays as described previously to discriminate against seasonal human h n and h n viral segments (table ) . the primers were allowed to cross-react specifically with the sister clade viral segments of pandemic h n ⁄ . the method we employed in this study was a -step singleplex sybr green-based real-time rt-pcr. this approach helped lower the running cost of the assays and facilitated downstream molecular analyses (e.g., sequencing) by using screened cdna samples. viral rna was extracted from viral cultures or clinical samples as described , and was converted to cdna in a universal rt-pcr. each ll rt reaction containing ae ll of purified rna, ll of · firststrand buffer (invitrogen), u of superscript ii reverse transcriptase (invitrogen), ae lg of uni ( ¢-ag-caaaagcagg- ¢), ae mm of deoxynucleoside triphosphates and mm of dithiothreitol was incubated at °c for minutes, followed by °c for minutes for heat inactivation. for each segment-specific real-time pcr, the ll reaction contained ll of a -fold diluted cdna samples, ll of fast sybr green master mix (applied biosystems), and ae lm of the corresponding primer pair. the thermocycling conditions of all eight segment-specific pcrs were optimized as °c for seconds, followed by cycles of °c for seconds and °c for seconds, and all eight assays were performed simultaneously in a sequence detection system (applied biosystems). at the end of the amplification step, pcr products went through a melting curve analysis to determine the specificity of the assay ( - °c; temperature increment: ae °c ⁄ seconds). cdna of a ⁄ california ⁄ ⁄ virus was used as a positive control. robust and specific amplification was achieved in all eight segment-specific real-time rt-pcr reactions. pcr product for each segment of pandemic h n ⁄ yielded unique melting curve pattern with distinctive melting temperature (tm), which was not observed in negative and water controls ( figure ). reactions with tm value within sds of the mean tm were determined as positive. we evaluated the assays with a number of serologically confirmed human clinical samples. all pandemic h n ⁄ samples (n = ) were positive in all eight assays, while all seasonal samples (h n = ; h n = ) were negative in all assays, as expected ( figure and data not shown). these results showed that no reassortant of pandemic h n and seasonal viruses was present in the tested human isolates. we applied these assays to our on-going influenza virus surveillance program in swine. nasal and tracheal swab samples were collected at an abattoir in hong kong and cultured in madin darby canine kidney cells or embryonated eggs as described. positive viral cultures in hemagglutination assays were tested with the established segmentspecific real-time rt-pcr assays. among swine viral isolates collected from to september , of them were recognized as pandemic h n ⁄ in all eight segments. they were confirmed to be of pandemic h n ⁄ origin by subsequent full genome sequencing analyses, showing that there were interspecies transmissions of the virus from humans to pigs. , the remaining viruses had one to seven gene segments positive in the segment-specific real-time rt-pcrs. thirty of them were selected as representative samples for full genome sequencing analyses based on the genotyping data generated in our assays. they were swine h n or h n viruses with their gene segments derived from tr or eurasian avian-like swine lineages. it should be highlighted that all of their positive gene segments in our assays belonged to the sister groups of pandemic h n ⁄ . their melting curve patterns were very similar to those derived from segments of pandemic h n ⁄ , except for ha of tr lineage. our results successfully demonstrated the use of these segment-specific real-time rt-pcrs to recognize gene segments of contemporary tr (pb , pb , pa, ha, np, and ns) and ea (na and m) swine viruses. the ha-specific assay was able to discriminate pandemic h n ⁄ from other contemporary swine viruses in the same lineage. nevertheless, to confirm the identity and to examine all the genetic variations in the viruses of interest, full genome sequencing analyses were necessary. in this study, the biggest obstacles in primer design were sequence similarity and diversity of influenza viruses. we attempted to use degenerated primers, but they were highly non-specific. the finalized non-degenerated primers crossreacted with genes from pandemic h n ⁄ and its sister clade tr (pb , pb , pa, ha, np, and ns) and ea (na and m) swine viruses with some minor sequence mismatches. three avian (h n , h n , and h n ) and classical swine (h n ) were also tested with our assays. all of these animal viruses were negative, except for ns gene of the classical swine virus. our segment-specific real-time rt-pcr assays might be used in high throughput genotyping. they detected pandemic h n ⁄ viruses and acted as a preliminary screen-ing tool to select virus reassortants of interesting genotypes for further sequencing analyses. in fact, we identified a novel reassortant in january during the course of this study. this sw ⁄ hk ⁄ ⁄ has a previously unidentified viral gene combination as shown in figure . it was confirmed to be a reassortant between pandemic h n ⁄ and other swine viruses in full genome sequencing characterization. it has a pandemic h n -like n gene, an ea-like h , and the other six internal genes derived from tr swine viruses. , the eight established real-time rt-pcrs can rapidly reveal the gene-origins of influenza viruses. we are currently using these assays in influenza surveillance in humans and other animals. it is believed that similar strategy might be applied to detect and genotype other influenza viruses and possible reassortants in the future. pandemic influenza a ⁄ h n ⁄ infects millions of people around the world. a significant fraction of the world's population may also already have been exposed to the virus and, although asymptomatic, may be at least partially immune to the disease. a precise assessment of the number of people exposed to the influenza a ⁄ h n ⁄ virus is epidemiologically relevant. however, assays typically used to estimate antibody titers against a particular influenza strain, namely hi and neutralization, require use of the actual virus. this seriously limits broad implementation, particularly in regions where high biosafety facilities are unavailable. we developed an elisa method for the evaluation of presence of specific h n influenza virus-antibodies in serum samples. mouse anti-histidine tagged antibodies ( ll; lg ⁄ ml; abd serotec Ò , uk) in pbs (ph ae ) were dispensed into standard -well plates and incubated for - hour at room temperature. excess antibody was removed by at least two successive alternate washings with pbs-tween ae % and pbs. commercial blocking solution ( ll, superblock Ò t ; pierce Ò , usa) was added and incubated for at least hour at room temperature. after successive washing steps with pbs-tween ae %, non-glycosylated histidine-tagged recombinant protein ( ll; lg ⁄ ml) was added to each well. this protein consisted of the receptor-binding domain of the hemagglutinin of the influenza a ⁄ h n virus. , after hour incubation, wells were washed for at least two alternating minutes cycles with pbs-tween and pbs. a : dilution of the serum or plasma sample to be assayed ( ll) was added to each well and incubated at room temperature for hour. after repeated alternating minutes pbs-tween ae % and pbs washes, anti-human igg antibody solution ( ll ⁄ well; : dilution in pbs-tween ae %) marked with horse radish peroxidase (pierce Ò , usa) was added and incubated for hour at room temperature. after repeated alternate washes with pbs-tween ae % and pbs), substrate solution ( ll; -step ultra tmb-elisa; pierce Ò ) was added to each well. after incubation for minutes at room temperature in darkness, the enzymatic reaction was stopped by addition of m h so ( ll ⁄ well). yellow color produced by the enzymatic reaction was evaluated by absorbance at nm in a biotek Ò microplate reader (usa). blank assays using albumin in place of human sera established the elisa background signal, which was subtracted from sample absorbance signals: abs serum sample ¼ abs serum sample before correction À abs albumin sample : absorbance values were normalized based on the average signal of non-exposed subjects (uninfected subjects), and expressed as normalized absorbance (abs norm ): where abs serum ample is the sample absorbance signal, abs albumin sample is the albumin control absorbance signal, abs non exposed subjects is the average absorbance signal of non-exposed subject samples. for ferret serum samples, the same basic protocol was followed, with minor modifications. an anti-igg anti-ferret polyclonal antibody preparation was used at a dilution of : in pbs-tween ae %. a recombinant receptor-binding domain of the ha of the influenza a ⁄ h n ⁄ virus, expressed in escherichia coli strains, was used as the elisa antigen. this kda protein, designated here as ha - -rbd, contained amino acids - of the influenza a ⁄ mexico ⁄ indre ⁄ (h n ) hemagglutinin. a sequence coding for a series of six histidines at the n-terminus of the protein was included in the genetic construct to allow purification using immobilized metal affinity chromatography (imac) and attachment to assay surfaces treated with anti-histidine antibodies (or alternatively co + or ni + ). a panel of four samples (kindly provided by st. jude from ferrets exposed to different influenza strains, namely h n , h n swine, and h n , was also tested by the elisa method using : dilutions. protein ha - -rbd specifically and selectively recognizes antibodies from serum samples from convalescent h n ⁄ influenza subjects. dubois et al. demonstrated that this protein, produced in e. coli, folds properly into a -d structure practically indistinguishable from the analogous region in the ha of the influenza a ⁄ h n ⁄ virus. ha - -rbd preserves three of the conformational immunogenic epitopes (sa, sb, and cb) described for influenza a ⁄ h n hemagglutinins. the recombinant protein was used as the antigen, attached through histidine tags to microplate surfaces treated with anti-histidine antibodies to discriminate between serum samples from subjects exposed and non-exposed to influenza a ⁄ h n ⁄ . samples collected before the pandemic onset, and therefore presumed to exhibit low specific antibody titers against influenza a ⁄ h n ⁄ , were analyzed by elisa using the antigen ha - -rbd. the histogram of normalized absorbance values from this sample set displayed a normal behavior with a standard deviation of ae units. only ae , ae , and ae % of these samples exhibited normalized absorbance values higher than ae , ae , and ae , respectively. no sample from non-exposed individuals presented an absorbance value higher than ae . variability among samples from non-exposed subjects was much lower than in samples with high specific serum antibody titers from convalescent h n ⁄ patients. exposure to the h n ⁄ influenza virus with this elisa method can be predicted by absorbance values normalized to those of abs norm ¼ ðabs serum ample À abs albumin sample Þ=ðabs non exposed subjects À abs albumin sample Þ ð Þ serum from uninfected subjects. consequently, for reliable results, inclusion of samples from non-exposed subjects on every assay microplate is necessary. figure shows the analysis of human serum samples, including samples from convalescent patients with positive diagnosis by rt-pcr. three positive (dark gray bars) and two negative controls (light gray bars) were included in the same microplate. all serum samples corresponding to convalescent subjects exhibited absorbance values ae - ae times higher than negative samples ( figure ). normalized absorbance values above ae suggested exposure to the virus, although, a more conservative threshold value of ae units is proposed for discrimination between exposed and non-exposed subjects. the elisa method described here yields adequate reproducibility and a high signal ⁄ noise ratio within determinations in the same microplate and among different microplates. using a normalized absorbance value of ae , the method was able to discriminate samples from convalescent patients, preferably after the third week of infection, and at least up to the twentyfourth week of exposure. assay sensibility was further validated against results from hi assays. a previously reported study showed that all members in a pool of fourteen samples diagnosed as positive by hi exhibited normalized absorbance values higher than ae , and % of them exhibited normalized absorbance values higher than ae . in general, high hi titers (> ) were correlated with normalized absorbance values higher than ae . figure a shows results using the ha-rbd elisa method and the hi assay on a pool of seventeen known positive serum samples corresponding to convalescent h n ⁄ patients. all samples determined as positive by hi ( samples) were also positive by elisa. while sensitivity of the hi assay was ⁄ = ae %, the elisa method recognized all samples correctly as positive ( % sensitivity) when a threshold of ae or ae was used. figure b shows that sera from ferrets infected with other influenza strains (h n , h n swine, and h n ) showed no cross-reactivity when analyzed by elisa. in summary, the ha-rbd elisa method presented here consistently distinguished influenza a ⁄ h n ⁄ infected and non-infected individuals, particularly after the third week of infection ⁄ exposure. since no actual viral particles are required, this assay can be readily implemented in any basic laboratory. in addition, should sufficient vaccine be unavailable, this elisa could determine the level of specific antibodies against the virus and presumably the extent of partial protection in a subject. therefore, the elisa protocol might allow better administration of vaccination programs during pandemic or seasonal influenza outbreaks. in april , a novel h n influenza virus emerged in north america and caused the first influenza pandemic of the st century. [ ] [ ] [ ] [ ] the pandemic h n (pdmh n ) has a unique gene constellation that was not previously identified in any species or elsewhere. it is genetically related to the triple reassortant swine h n influenza viruses currently circulating in north america, with the exception of the neuraminidase (na) and matrix (m) genes, which are derived from a eurasian swine influenza virus. swine h n influenza viruses were first isolated in and continued to circulate in north america with very little antigenic changes (classical swine h n ) until . since , however, the antigenic make up of swine h viruses has shown increased diversity due to multiple reassortment events and the introduction of h n genes from human influenza viruses. currently, four swine h clusters (a, b, c, d) are found endemic in the north american swine population. , these swine h viruses show substantial antigenic drift compared to the classical swine h viruses. cluster d swine h is derived from current human h viruses, and there is a substantial antigenic divergence between classical swine h and human seasonal h viruses. epidemiological evidence shows a two-way transmission of influenza viruses between swine and humans, and such events lead to the emergence of the pdmh n virus. , , phylogenetic analysis have suggested that possible ancestors of the eight genes of pdmh n were circulating in the swine population for at least years prior to the emergence of the pdmh n virus in humans, although the pdmh n virus itself was not isolated from pigs until after the pandemic. interestingly, pdmh n infections have been reported not only in humans and pigs, but also in other animal species such as turkeys, cats, ferrets, cheetahs, and dogs. [ ] [ ] [ ] after the first report of pdmh n infection in swine in canada, other countries, including argentina, australia, singapore, northern ireland, finland, iceland, england, united states, japan, and china reported outbreaks of pdmh n in swine as well. , [ ] [ ] [ ] the ample geographic range of pdmh n outbreaks in swine, its apparent broad host range, and the possibility of two-way transmission between swine and humans poses a tremendous challenge for controlling the virus. therefore, to differentiate pdmh n from other h strains, particularly in swine and human populations, is an important issue to ascertain the magnitude of the disease caused by the pdmh n . in this study, we developed an elisa assay to discriminate pdmh n strains from other swine and human h viruses. madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa) were maintained in modified eagle's medium (mem) containing % fbs. a ⁄ california ⁄ ⁄ ⁄ h n virus (ca ⁄ ) was kindly provided by the centers for disease control and prevention (cdc), atlanta, georgia. other viruses are listed in table . viruses were propagated in mdck cells and stored at ) °c until use. viruses were titrated by the reed and muench method to determine the median tissue culture infectious dose (tcid ). three monoclonal antibodies ( b , h , and f ) against ha of pandemic h n were prepared in our laboratory following previously described methods (shao and perez et al., unpublished). purification and labeling of mabs mab b , h and f were purified on a protein g-sepharose affinity column (upstate biotechnology, lake placid, ny, usa). biotinylation of the detection antibody in the elisa was performed using sulfo-nhs-lc-biotin (sulfosuccinimidyl- -(biotinamido)hexanoate; pierce, rockford, il, usa) according to the manufacturer's instructions. purified h and f were selected as the capture antibody, and biotin-conjugated b was selected as the detection antibody, and hrp-conjugated streptavidin (abcom, cambridge, ma, usa) was developed using the tmb substrate system (kpl, gaithersburg, md, usa). in brief, the mixture of the purified h and f ( ae and ae lg ⁄ ml respectively, in carbonate ⁄ bicarbonate buffer, ph ae ) was coated to -well plates (test well, t) for h at °c. at the same time, a control antibody was coated to -well plates (control well, c). after blocking the plates with % (w ⁄ v) non-fat milk in pbs for hour at °c, the samples were diluted in extract buffer ( %tween- , ae %bsa in pbs) and added to the wells ( ll ⁄ well, each sample was table . specificity assay of the sandwich elisa result (t ⁄ c) added to four wells-two for t wells and two for c wellsand the mixture was incubated at °c for hour. after four washes, ll biotin-conjugated b ( ae lg ⁄ ml) in dilution buffer ( ae % bsa in pbs) was added to the wells and the mixture was incubated for h at °c. following three washes, ll diluted hrp-conjugated streptavidin ( ae ng ⁄ ml) in dilution buffer was added to the plates. after incubation for h at °c, the plates were washed five times, and the binding developed using the tmb substrate system for minutes. the ratio of the average od value of the t wells to that of the c wells (t ⁄ c) of individual samples was calculated. t ⁄ c values > ae were considered positive in the sandwich elisa. we developed three monoclonal antibodies, b , h , and f , against a prototypical pdmh n strain, a ⁄ california ⁄ ⁄ (h n ) (ca ⁄ ). these monoclonals were used to develop a rapid sandwich elisa for specific diagnosis of pdmh n strains. purified h and f were used as capture antibodies, whereas the biotin-conjugated b was used as detection antibody. the sandwich elisa showed strong reaction with different pdmh n strains as described in in order to evaluate if the sandwich elisa could distinguish the pdmh n from other swine h clusters (a, b, c, d), swine influenza strains spanning these clusters were tested. these viruses were first diluted : in extract buffer, and then added to the coated plates. as shown in table , the t ⁄ c ratios of these viruses were < ae , and therefore showed negative elisa result. likewise, testing of human seasonal virus strains a ⁄ brisbane ⁄ ⁄ (h n ), a ⁄ malaya ⁄ ⁄ (h n ), a ⁄ wsn ⁄ (h n ), and a ⁄ brisbane ⁄ ⁄ (h n ) also showed negative elisa results. furthermore, the sandwich elisa showed no cross reaction with avian influenza viruses, including strains of the h , h , h , h , h , h , h , h , h , h , and h subtypes. more recently, the mutation d g in the ha of some pdmh n strains has been associated with exacerbated disease and altered receptor binding. [ ] [ ] [ ] [ ] [ ] to evaluate if such mutant could be detected in our sandwich elisa, we tested a mutant of a ⁄ netherland ⁄ ⁄ (h n ) carrying the d g mutation (engineered by reverse genetics). as described in table , our elisa could still capture the d g mutant virus and showed a positive reaction, which highlights the specificity of our assay for pdmh n strains, even those with mutations. to evaluate the sensitivity of the elisa, we used the serially diluted pdmh n viruses to determine the limit of detection (lod). as shown in table , in our elisa the highest positive dilutions of nl ⁄ and ca ⁄ were : and : , respectively. the lod of the sandwich elisa by tcid was ae · and ae · tcid ⁄ ml, for nl ⁄ and ca ⁄ , respectively. it is important to note that the t ⁄ c ratio from nl ⁄ and ca ⁄ viruses showed clearly a dose dependent effect, while the t ⁄ c ratio of a ⁄ swine ⁄ iowa ⁄ (h n ) did not show the same dependence and was always < ae , corroborating the high specificity of the sandwich elisa for pdmh n strains. although we did not compare our elisa with other current commercial rapid influenza detection kits, the lod of our elisa assay is similar to other commercial kits that detect human seasonal influenza virus. comparison of the sandwich elisa with the ''gold standard'' -virus isolation in order to further evaluate the feasibility of the application of the elisa to clinical samples, nasal wash samples ae · ^ )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) )( ae ) -from ferrets, of those previously infected with ca ⁄ and shown positive by virus isolation, were tested. the samples were diluted : in extract buffer and then tested using the sandwich elisa. result showed out of positive samples by virus isolation were positive also by the sandwich elisa (sensitivity ae %). the samples tested that were negative by virus isolation were also negative in the elisa, indicating % specificity for our assay. these results show not only that our elisa has high compatibility with the virus culture method, but also indicates this application can be used for clinical samples. although real time rt-pcr targeting the ha gene has been used for specific diagnosis of pdmh n with high sensitivity, [ ] [ ] [ ] [ ] [ ] [ ] it is a method that requires manipulation of the sample to extract viral rna, and it is prone to crosscontamination during the pcr steps. in this study, we described a convenient sandwich elisa based on three mabs developed against the pdmh n strain. the elisa not only shows high specificity for pdmh n strain, but also shows great sensitivity. the elisa could distinguish pdmh n strains from human seasonal h and h viruses and, more importantly, from other swine h viruses. we must note that current rapid diagnostic tests cannot be used to differentiate pdmh n from swine or human h viruses. it is also worth noting that the sensitivity of commercial rapid antigen-based diagnostic tests for detecting pdmh n is lower than that for human seasonal influenza viruses. , a study by kok et al. showed that sensitivity of the current rapid antigenic tests for pdmh n is only ae %, whereas that for seasonal influenza a is ae %. chen et al. developed a dot-elisa and increased the sensitivity for influenza rapid antigen detection. however, the dot-elisa developed by chen cannot distinguish among subtypes. the lod of our elisa is between ae · to ae · tcid ⁄ ml, comparable to the lod of rapid diagnostic tests for human seasonal influenza viruses. compared to the ''gold standard''-virus isolation-our sandwich elisa showed ae % sensitivity using ferret nasal washes. our results highlight the potential application of our sandwich elisa for the specific diagnosis of pdmh n viruses. the timely and reliable laboratory evidences are vital factors for field epidemiologists trying to control outbreaks of infectious diseases and for the practicing clinicians to properly manage disease cases. therefore, analysis of new detection methods in comparison to the routine ''classical'' methods is essential to select new methods to be introduced into health service practices, especially in developing countries. in this study we have compared rt-rt-pcr detection of influenza viruses and direct fluorescent-antibody assay using r-mix hybrid cells (a &mv lu) with the ''classical'' cell culture methods in developing country settings. in this study, we analyzed nasopharyngeal swabs col- the detection of influenza h , h , b, and pandemic influenza (h )pdm virus-specific nucleic acids was performed by rt-rt-pcr in abi fast real time pcr system using primers recommended by cdc, usa, and super-scriptÔ iii one-step rt-pcr and platinum Ò taq dna polymerase kits (invitrogen). the cycling protocol was: minutes at °c, minutes at °c, and cycles of seconds at °c, seconds at °c. rapid detection of influenza infected cells has been performed by dfa using the infected hybrid cells of r-mix within hours after inoculation, according to the manufacturers instruction (diagnostic hybrids, inc., usa). the isolation of influenza viruses was performed on mdck cell culture by the protocol recommended by cdc, usa. we detected ( ae %) influenza virus-specific nucleic acid fragments from all tested samples by rt-rt-pcr. among the positive samples, there were ae % a(h n ), ae % a(h n ), ae % influenza b, and ae % a(h n )pdm with different distributions by time series in different age-groups. inoculation of the cell lines by rt-rt-pcr positive samples selected randomly has detected influenza virus in ae % ( ⁄ ) on mdck cell culture and % ( ⁄ ) on r-mix hybrid cell culture with varying distribution for different strains. in other words, mdck cell culture technique was better for isolation for pandemic influenza viruses and dfa using r-mix hybrid cell culture technique for detection of seasonal influenza viruses (table ) . average times needed for the final results for different methods were: hours for rt-rt-pcr, hours for dfa on r-mix and days for mdck cell culture with two passages at least. the peak of the seasonal influenza a virus detection occurred in the - th weeks of , however the pandemic influenza detection peak was observed in the - th weeks of ( figure ). the outbreaks by seasonal influenza viruses was observed mostly among the children of - years of age, and pandemic influenza virus outbreak was observed mostly in the adults of - years of age. the results of this study indicate that rt-rt-pcr is the most suitable method for decision makers in epidemiological and clinical settings by sensitivity and timeliness. the final results show that r-mix dfa requires times longer, and by mdck cell culturing, times longer periods, than by rtrt-pcr. mdck cell culture technique has a higher isolation of pandemic influenza viruses, and r-mix dfa has a greater detection rate of seasonal influenza viruses by our results. according to our study, with rtrt-pcr, the isolation of positive samples by tissue culture of influenza a viruses was % and influenza b viruses was ae %, which is lower than in similar spanish study. however our study illustrates similar results with a canadian study where the sensitivity of dfa method and tissue culture technique was shown to be lower than rtrt-pcr sensitivity. as recorded by a study of american researchers, r-mix hybrid and conventional cell culture techniques have had similar sensitivity, which does not match the results of our study. however, the results of our study match with the results of italian and american scientists , where the r-mix hybrid method for seasonal influenza viruses is higher than mdck cell culture technique. background: viral kinetics is increasingly used to study influenza infectiousness. the choice of the study design, i.e. when and how many times nasal samples are to be collected in individuals depending on the sample size, is crucial to efficiently estimate the viral kinetics (vk) parameters. material and methods: we performed a model based optimal design analysis in order to determine the minimal number of nasal samples needed to be collected per subject and when to collect them in order to correctly estimate the vk parameters. the model used was a non linear mixed effect model developed with data collected from patients sampled nine times in days (initial design - samples collected), and we used d-optimization for design identification. we also computed the minimal number of participants necessary. results: considering that % of the influenza-like illness cases are not due to volunteer challenge studies have been used since the 's to provide data on virus shedding from the respiratory tract during influenza infection. recently, vk was studied in naturally acquired influenza infection. , these data are invaluable to describe the natural history of influenza-infection and to compute natural history parameters such as the latent period, generation time, or the duration of infectiousness. [ ] [ ] [ ] [ ] however, among the studies used in a meta-analysis about viral shedding kinetics, the designs varied greatly from one to another. these differences led to variable amount of available information concerning the vk. the lack of adequate sampling leads to imprecise estimates. on the other hand, intensive sampling or over-sampling, while associated with highly informative data, may lead to unnecessary discomfort for the patient and cost to the investigator. optimal design is increasingly used to conceive studies and provides cost-efficient designs. here we propose an optimised design to model vk in the case of influenza infection. we defined the number of participants, the number of samples to collect and their allocations. this design allows, at a minimum cost and discomfort, accurate vk curves and allows the natural history parameters to be well described. model a vk population model was proposed for influenza infection. this model describes with eight parameters the relations between free virus, uninfected target epithelial cells, infected epithelial cells, and early immune response. this model was built on a dataset of volunteers from which nasal samples were collected once a day over days. we call this dataset the ''original dataset''. three parameters, the induction of the early immune response, the virus production rate, and the virus clearance, did not show inter-individual variability and were precisely estimated (relative standard error below %). we considered them as fixed in this research work. five parameters were hence considered here: b the infection rate, d the infected cell mortality rate, w the effect of early immune response on virus production rate and v init the initial value of virus titre. in order to correctly estimate these parameters it is crucial to determine a design to collect informative data. optimal designs maximise the amount of information provided by the study. it involves the determination of the number and allocation of sample times per subject as well as the number of participants. d-optimization is based on the maximization of the determinant of the fisher information matrix and thus minimizes the variance of the parameters. we used the fedorov-wynn algorithm implemented in pfim . to maximize this determinant, which implies to pre-define a set of possible sample times. with the hypothesis that the inoculation occurred at : am, we chose three possible hours ( : , : , and : ) for each day with respect of the sleep-time. to validate the design, we simulated datasets of volunteers with the optimised design obtained. we then estimated the population parameters using monolix . for each of the datasets. we compared the estimated parameters obtained with the simulated datasets to the parameters used to build the optimal design. we computed the relative bias as: with n: number of successful estimations among the simulated datasets. h i : parameter value obtained with the ith dataset. h: parameter value obtained with the original dataset. we also compared the observed rse from these simulations with the rse predicted by pfim and the rse obtained with the original dataset. the rse is proportional to ffiffiffi n p , where n is the population size. we can hence deduce the smallest number of participants necessary to obtain rse below %. where rse predicted is the highest predicted rse (here rse for w) with participants and n predicted = and rse min is equal to ae . considering that % of the influenza-like illness cases are not due to influenza virus, the total number of participants should be multiplied by ae . we found that the best design was when all the participants are sampled five times: three times during the second day post-inoculation at : , : , and : hours and twice on the third day post-inoculation at : and : ( figure ). the comparison of the relative bias and rse predicted by pfim and those obtained after simulation and re-estimation of the parameters are shown in figure . v init and d in a lesser extent present bias. fixed effect parameters are precisely estimated and accordingly to pfim except for v init . we found that participants shedding virus or participants with ili symptoms are necessary if % of them are not infected with influenza virus. we propose an optimised design to accurately study the vk of influenza virus with the minimal number of samples. this design is well balanced between the amount of necessary information and the precision of estimation. we found that samples are necessary to precisely fit the vk curves, which is five times less than the number of samples collected in the original study. ??? the samples should be collected during the second and third days after inoculation. yet we showed in a previous work that the incubation period lasted ae days. ??? hence, the optimised sample times correspond to the two-first days of symptoms and this design could be applied to naturally acquired infections studies in which the inoculation time is unknown. an advantage of this design is its practicality and convenience. all samples are collected during the daytime and after the onset of symptoms. it can thus be used for studies with naturally acquired infections. the design was validated with several criteria concerning the accuracy of the estimation with the optimised design. the parameters estimates were generally satisfactory. the parameter describing the effect of the early immune response on the virus production rate was, however, less precisely estimated (predicted rse = %), and the initial value of the viral titre was very different of the one obtained with the original dataset (bias v init on figure ). this is probably due to the fact that it was measured at day post inoculation, and that the inter-individual variability is much higher than at day . furthermore, d (the infected cell mortality rate) seems also to be biased. this may be due to the fact that three parameters were fixed. the model used was developed from experimentally inoculated healthy volunteers with low serum haemagglutinin antibody titre and with virus inoculation time at : am. the applicability of the design to naturally acquired infection would depend on the pathogenicity of the virus as well as pre-existing immunity and the relevance of challenge method to natural influenza acquisition. our design could be directly used to accurately study vk during influenza infections and would reduce the discomfort of patients and the cost of the experimentation. usefulness of a self-blown nasal discharge specimen for use with immunochromatography based influenza rapid antigen test introduction influenza rapid antigen tests (irat) have become very popular and are widely used for confirming suspected clinical diagnosis of influenza in japan. most of the currently used irat that are based on immunochromatography (ic), nasopharyngeal swab, nasopharyngeal aspiration, and throat swab have been approved as specimens for japanese national health insurance purposes. but the specimen collection by these methods gives patients considerable discomfort, and sometimes appropriate specimens cannot be obtained due to patient resistance, especially by children. in the present studies, self-blown nasal discharge was used as the specimen for an irat, and the results were compared with the results of viral isolation and an identical kit primed with nasopharyngeal swab specimens for seasonal influenza viruses and pandemic (h n ) virus. patients who visited any of the clinics that belong to the influenza study group of the japan physicians association in the - and the - influenza seasons with influenza-like illnesses exhibiting findings were registered after providing informed consent. a square plastic sheet of · cm was handed to the patient. nasal discharge was collected by blowing the nose into the plastic sheet as a specimen for irat, i.e. self-blown specimen. two nasopharyngeal swab specimens were also obtained at the same time for irat and virus isolation. self-blown specimens were obtained successfully by ( ae %) of consecutive outpatients in the - season, as seen in table the sensitivity and specificity of various influenza rapid antigen tests have been reported in various settings. [ ] [ ] [ ] [ ] direct comparison of the results is difficult because of differences in patient or influenza virus, characteristics such as age, study designs, and other features. in this study of the - influenza season, the sensitivity, specificity, and accuracy of the ic kit primed with nasopharyngeal swab specimens were ae %, ae %, and ae %, respectively. these results were quite comparable to our results of the - season, in which the overall results of other ic kits were ae %, ae %, and ae %, respectively, indicating that the ic kit used is quite reliable. the sensitivity, specificity, and accuracy of an ic kit will vary by the method of specimen collection. in general, virus titer is considered to be highest with nasopharyngeal aspiration, lower with nasopharyngeal swabs, and lowest with throat swabs. practically, nasopharyngeal swab is the most popular. the sensitivity, specificity, and accuracy of the ic kit with self-blown discharge specimens compared well with those of an identical ic kit primed with nasopharyngeal swab specimens. for self-blown specimens, sensitivity and specificity were ae % and ae % for influenza a, ae % and ae % for influenza b, % and ae % for pandemic (h n ) . self-blown specimens display sensitivity, specificity, and accuracy comparable to that of conventional nasopharyngeal swab specimens. there was no significant difference in sensitivity, specificity, or accuracy between self-blown specimens and nasopharyngeal swab for influenza a, influenza b, and pandemic (h n ) . these results suggest that selfblown specimens are as useful as nasal cavity swab specimens for the diagnosis of influenza in the clinical settings. nasal discharge, obviously, cannot be collected from infants incapable of blowing their own nose or patients who do not develop a nasal discharge. in this study, self-blown specimens were obtained from ae % of the patients. the rate of successful collection was over % in the age groups of - and - years. these rates would seem to be sufficient for clinical use. the procedure of self-blown specimen collection using a plastic sheet is easy and causes no pain or discomfort. it seems to be more acceptable and safe than the other methods, especially for children. furthermore, this procedure reduces the risk of influenza transmission from patients to the medical staff members involved in sample collection. self-blown sample collection may be superior to other sample collection methods in these respects. we previously reported an inverse correlation between the amount of virus in a specimen and the time to a positive reaction. in this study, there was no significant difference in the mean time to a positive between self-blown self-blown specimens enough to be examined were obtained from consecutive outpatients, and specimens showed a tendency to be obtained large amount from children rather than the aged. there were no statistically significant differences between the ic kit results primed with self-blown discharge and nasopharyngeal swab specimens for influenza a, influenza b and pandemic (h n ) . and nasal swab specimens, suggesting that the self-blown specimens contained sufficient viral antigen for the ic kits. the influence of the presence or absence of nasal congestion on the results of the kit was assessed. the sensitivity of selfblown specimens from patients with nasal congestion was significantly lower than that from patients without nasal congestion. it is possible that insufficient capability to blow the nose due to nasal congestion might tend to lead to false negatives. the observation that the time to positive is longer for patients with nasal congestion than for patients without nasal congestion is concordant. application of self-blown specimen collection only to appropriate patients would increase the sensitivity, which would be important in a clinical setting. we tested only two commercial antigen detection kit, the quick vue rapid sp influ kit and quicknaviÔ-flu (denka-seiken co., ltd). the resulting sensitivity, specificity, and accuracy of the ic kit primed with self-blown specimens were considered adequate for clinical use. to confirm the usefulness of self-blown nasal discharge specimens, further investigation is necessary using other kits and in different settings. the usefulness of a self-blown nasal discharge specimen for an influenza rapid antigen test based on immunochroma-tography was evaluated in the - and - influenza season. results suggest that self-blown nasal discharge specimens are useful as specimens for influenza rapid antigen tests based on immunochromatography for not only seasonal influenza viruses, but also pandemic (h n ) virus. the specimen collection by the patients themselves will reduce the burden of other collection methods and the risk of infection to the medical staff. in april , a mixed-origin h n influenza virus was recognized as a new causative agent of influenza-like illnesses (ili) in humans. since its emergence, the virus has spread rapidly throughout the world and caused a pandemic. most commercial rapid antigen tests (rat) can detect influenza a or b viruses, but cannot specifically distinguish pandemic (h n ) virus with seasonal influenza. recent studies have indicated that the poor performance of the rat approach and nonspecific detec-tion of the pandemic (h n ) virus was the main obstacle to their widespread use in private clinics. , with the need for a new rapid kit with reasonable sensitivity and specificity for pandemic (h n ) virus, we developed a new rat kit in collaboration with company, standard diagnostics, inc., (yongin-si, gyonggi, korea). monoclonal antibody (mab) against haemagglutinin (ha) of the pandemic (h n ) virus was developed using korean isolate and applied to the new kit with the mab to seasonal influenza virus. we examined the detection limit of the kit using the serial dilution of korean pandemic virus isolate (a ⁄ korea ⁄ ⁄ ). during december , clinical specimens from patients with ili were collected at sentinel clinics of six provinces in korea. the specimens were tested by the new rat, and the results were compared with those of real-time reverse transcription polymerase chain reaction (rrt-pcr) by us cdc and virus isolation in mdck cell culture to determine the sensitivity and specificity for the diagnosis of pandemic (h n ) . the detection limit of the new kit against ha of a ⁄ korea ⁄ ⁄ virus was confirmed to be pfu ⁄ ml. by contrast, the detection limit against the np protein was pfu. however, when the kit was applied to clinical specimens, no difference between the two targets was found. using rrt-pcr and viral culture as the references, the performance of the ridt is shown in table . among specimens, were tested positive by rrt-pcr and were tested positive by viral culture. among the rrt-pcr confirmed cases, were positive, and among the viral culture confirmed cases, were positive with the new rat. using rrt-pcr as the reference standard, the overall sensitivity of rat was ae % ( % confidence interval (ci): ae - ae %) and specificity was ae % (ci: ae - ae %). with viral culture as the reference, the rat sensitivity and specificity was ae % (ci: ae - ae %) and ae % (ci: ae - ae %), respectively. when analyzed by the regions tested, the sensitivity ranged between ae % and ae % for rrt-pcr and between ae % and ae % for viral culture as a reference. among patients who had a record of their symptom onset and sample collection date, ( ae %) visited the clinic on the day of symptom onset, and ( ae %) visited day later. when the rat performance was evaluated by day of onset, the sensitivity was lower at three or more days after the onset of symptoms; however, the sensitivity was highest at days after onset and reasonable on the day of onset or at day after ( table ). we found that this new rat had reasonable sensitivity and high specificity compared with rrt-pcr and viral culture for detecting the pandemic (h n ) virus. in one recent study, the sensitivity and specificity of the new rat kit was % and %, respectively, and the ha protein for pandemic (h n ) was detected more sensitively than the np protein for influenza a virus. the sensitivity and specificity of our new rat were lower than those of that study. we found that the test performance varied depending on the clinics in which the tests were performed, and this might be attributable to the persons who collected the specimens. although the clinicians were trained well for *ci, confidence interval. **ppv, positive predictive value. ***npv, negative predictive value. collecting specimens, there might be some differences in performance. the new rat kit could detect pandemic (h n ) virus specifically. although the sensitivity was lower than those of rrt-pcr and virus culture, and negative rat results should be confirmed with more sensitive methods, this kit could be useful in sentinel clinics if used with caution. determination of infectious virus titres is central to many experiments designed to study the biology of influenza virus. assays based on the measurements of viral components, whether viral protein or nucleic acid, does not differentiate infectious virus from non-infectious or defective viral particles, which may have no infectivity or biological *three hundred and forty samples with a known date of onset and sample collection were analyzed. ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - activity. therefore the ''gold standard'' of virus measurement requires bioassays that examine the ability of viral particles to replicate and further infect other cells. titration on madin-darby canine kidney (mdck) cells in a well plate format is commonly used to measure influenza virus titre. this method is labour intensive, subjective in their read out of cytopathic effect, and takes several days to obtain a result. microneutralization tests that quantitate neutralizing antibody titres and assays of drugs for antiviral activity also require well based assays of residual virus infectivity. therefore, technologies that improve on the titration of infectious virus will be of great benefit. this study utilized the xcelligence system (roche applied science), which adopts microelectronic biosensor technology to monitor dynamic, real-time label free and non-invasive analysis of cellular events. the system measures electronic impedance using an array of microelectrodes located at the bottom of each culture well (e-plate ). adherent cells are attached to the sensor surface of electrode arrays, and changes in impedance can be detected and recorded. the xcelligence system can monitor cell events induced by viral infection, such as changes in cell number, adhesion, viability, morphology, and motility. measured electrode impedance is expressed as dimensionless cell index and is graphically represented using software to show the phenotypic changes of a cell population over time. the aim of this study is to demonstrate that using this platform to measure real-time cell index has potential to circumvent many of the limitations of the currently established procedures of end point titration of virus infectivity and for microneutralization assays. madin-darby canine kidney cells were propagated in growth medium consisting of minimum eagle's medium (invitrogen) supplemented with % fetal bovine serum (invitrogen), ae mg ⁄ l penicillin (invitrogen), and mg ⁄ l streptomycin (invitrogen), with incubation at °c in a % co humidified atmosphere. influenza a ⁄ hong kong ⁄ ⁄ (h n ), a seasonal influenza virus from a patient who suffered from a mild febrile illness, was propagated in mdck cells maintained in virus medium consisting of minimum eagle's medium (invitrogen) supplemented with ae mg ⁄ l penicillin (invitrogen), mg ⁄ l streptomycin (invitrogen), and mg ⁄ l np-tosyl-l-phenylalaninechloromethyl ketone-treated trypsin (sigma, st louis, mo, usa), with incubation at °c in a % co humidified atmosphere. virus stocks were aliquoted and stored at °c until use, and the % tissue culture infectious dose (tcid ) of the virus stock was determined by titration in mdck cells according to standard procedures, and the tcid of the stock virus was calculated by the method of reed and muench. to perform a microneutralization assay, mdck cells seeded at a density of cells ⁄ well in an e-plate was removed from the xcelligence system after approximately hour; growth medium was then removed, cells washed, and replaced with ll virus-medium. a human serum, which is known to contain high titre antibody against the h n virus was heat inactivated for min at °c, and twofold serial dilutions were performed in virus medium. the diluted serum was mixed with an equal volume of virus medium containing influenza virus at tcid ⁄ ll. after incubation for h at °c in a % co humidified atmosphere, ll of virus-antibody mixture was added to the mdck cells to give each well an equivalent virus dose of tcid . a back titration of the virus challenge dose was performed, and a cell control (free of virus) was performed in quadruplicates. after incubation at room temperature for minutes, the e-plate was then placed back onto the xcelligence system in the incubator and maintain at °c with % co , and the cell index values were measured every minutes for at least a further hour. the same procedures were performed with cells seeded in conventional well cell culture plates for parallel comparison with the currently used standard method. in this case, cells were examined for cytopathic effect under an inverted microscope after days of infection and the lowest virus dilution, which protected the cells from viral induced cytopathic effect taken as the neutralizing end point. after hour of seeding mdck cells at cells ⁄ well, standard microneutralization assay for influenza virus was performed. integral to this assay, a serial titration of the input virus at ae log increments was carried out. wells infected with the undiluted virus ( tcid ⁄ well), the cell index commenced dropping at a steeper gradient than the no-virus cell control after approximately hour of infection ( figure ). this drop in cell index continues at a consistent slope until it flattened out when approaching zero cell index. this steep decrease in cell index with constant gradient was also observed for virus dilutions up to and including log ( -folds), and the profile shifted with increased time in proportion to the dilution made to the virus. virus dilutions beyond log have cell index profiles similar to the no virus input control, and this corresponds to the absence of cytopathic effect as determined by microscopic observation at hour after infection. hence, there was a correlation between the amount of virus used for infection, the onset of the influenza virus-mediated cytopathic effect, and the steep decline in cell index. a human serum with known microneutralization antibody titre to h n virus was used in this study to investigate the real time cell index changes that occur during the assay ( figure ). using influenza virus treated with serum dilutions up to and including a dilution of : , the cell index profile remained essentially the same as the no virus cell control, which correlates with the lack of cytopathic effects under microscopic observation at hour of infection. at a serum dilution of : , the steep decrease in cell index, which is characteristic of cellular cytopathic effect induced by the virus, became evident at around hour post infection, and this was reduced to hour when serum dilution of : was used. in contrast, for the virus -no antibody control, the onset time for this steep decrease in cell index occurs at approximately hour. for both serum dilutions of : and : , full cytopathic effect was observed microscopically at hour of infection. from microscopic observation of cytopathic effect, according to the current standard procedures, the neutralizing titre of the human serum used in this study is at : as it is the last dilution of the serum that prevented cytopathic effect from being detected. an essential part of the microneutralization assay is to confirm the titre of the input virus (normally tcid ⁄well) by performing a titration assay with decreasing serial dilutions of the virus. under normal procedures, cells are examined microscopically after hour of infection for sign of cytopathic effects. in the case of mdck cells, the cytopathic effect is cell death, which is indicative of the presence of live influenza virus infecting and replicating in the cells. therefore, the titre of the virus is taken as the last dilution in which cytopathic effect is present. parallel realtime cell index measurements demonstrated that for wells with cytopathic effects, the profile exhibits a steep gradient linear decrease in cell index after infection with the virus, which can be termed the ''cpe plunge.'' the time in which the cpe plunge became evident appears to be inversely proportional to the amount of virus, therefore the opportunity exists to utilize this aspect to calculate or compare quantitatively different virus concentrations. for unequivocal assignment of cytopathic effect, it normally requires - days after infecting the cells, with days after infection being the standard time to read virus titration and microneutralization assays. using the real-time cell index monitoring, it is found that apparent cytopathic effect can only be observed microscopically when the cell index has dropped to near zero. as the time of onset of the ''cpe plunge'' becomes evident many hours prior to observable cytopathic effect, it is possible that the time to results can be drastically reduced after some formulation of the method. we compared the current standard method in perfoming a microneutralization assay with one utilizing the real-time cell index measurement to investigate whether this approach is able to offer better performance over the existing one. the current standard neutralization assay is the microscopic observation of antibody mediated protection from virus cytopathic effect in mdck cells. this study showed that this may also be achieved by examining the profile generated from the real-time measurements of the cell index. using real-time cell index monitoring, it is possible to detect inhibitory activity at higher dilutions of the anti-serum than can be detected by the standard microscopic observation of cytopathic effect. therefore, the realtime cell index monitoring could potentially be developed to be a more sensitive method for measuring anti-viral activity. as drug resistant strains of influenza a viruses including the pandemic h n are being reported, the real-time cell based monitoring system may also have the potential to be developed for use as a diagnostic platform for drug resistance assays. this study suggests that real-time cell index monitoring has the potential to substantially reduce human resources in reading results, as well as reducing time-to-result of these assays from days to two. the saving could be substantial for work involving bio-hazard level ⁄ pathogens such as h n viruses as personnel working with these organisms are require to be highly trained and experienced. in addition, the reduction in transferring plates to and from the microscope in reading cytopathic effect will substantially reduce the possibility of accidents from occurring. furthermore, the system provides objective digital data to an otherwise subjective assay method, which can improve standardization, data exchange, and hence collaboration between different laboratories. with more detailed validation and development, real-time cell index monitoring could transform the way we study and diagnose infection with pathogens such as influenza viruses. the emergence of a novel h n influenza a virus of swine origin, the pandemic a(h n ) , with transmissibility from human to human in april posed pandemic con-cern and required modifications to laboratory testing protocols. a new protocol for universal detection of influenza a and b viruses and simultaneous subtyping of influenza a (h n ) virus, composed of two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h n v (relab, italy), was evaluated and compared to the reference protocol recommended by who. fast set infa ⁄ infb was able to detect influenza a and b viruses circulating between and belonging to different subtypes and lineages, and no cross reactions were observed by either fast set infa ⁄ infb or fast set h n v. the who assay was found to have a slightly lower end-point detection limit ( ) dilution) in comparison to the new protocol ( ) ). specificity of the assays was % as assessed on a panel of stored clinical samples including adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, s. pneumoniae, n. meningitidis, h. influenza, and human influenza viruses. the new assay panel allows the detection, typing, and subtyping of influenza viruses as requested for diagnostic and surveillance purposes. the high sensitivity of the protocol is coupled with capacity to detect viruses presenting significant heterogeneity by fast set infa ⁄ infb and with high discriminatory ability by fast set h n v. a rapid and sensitive assay for the detection of influenza virus in clinical samples from subjects with ili or low respiratory tract infections is a fundamental tool for epidemiological and virological surveillance, management of hospitalized patients, and control of virus nosocomial transmission. the emergence, in april , of a novel h n influenza a virus of swine origin, the pandemic (a(h n ) ), with transmissibility from human to human poses pandemic concern and required modifications to the laboratory testing protocols. molecular diagnosis of influenza is generally achieved through a twophase process: a screening phase for the detection of virus, and the subsequent strain characterization performed by either sub-type-specific rt-pcr or entire ⁄ partial genome sequencing. during a pandemic, simultaneous implementation of both the detection of influenza a and b influenza viruses and identification of the new subtype is useful for clinical and epidemiological reasons. here, we describe a new protocol including two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h n v (relab, italy) that allows universal detection of all influenza a viruses and, simultaneously, all subtypes that are influenza a(h n ) . specificity and clinical sensitivity of the two-one-step rt-pcrs (fast set infa ⁄ infb and fast set h n v; relab, italy) were evaluated by testing selected specimens, including: • fifty samples collected from nasopharyngeal swabs representative of influenza viruses, belonging to differ-ent subtypes and lineages, and other respiratory viruses and bacteria circulating in italy between and . • six purified a(h n ), a(h n ), and a(h n ) strains, kindly supplied by alan hay, who influenza centre, london, uk. • two hundred-fifty influenza positive samples selected according to type, subtype, clade and viral concentration from > specimens received by the liguria influenza reference laboratory between january st and december st, . since , nasopharyngeal swabs sampled from patients suspected of having contracted the influenza virus have been collected in viral transport medium, and upon arrival into the laboratory, the samples were divided in ‡ aliquots. those not immediately processed were stored frozen at ) °c. stored samples were used for this evaluation, and all specimens were re-extracted for the study. samples collected between and included specimens positive for: no seasonal a(h n ) have been detected since january st, . furthermore, weak positive sample using fast set infa ⁄ infb, but negative at block pcr and typing ⁄ subtyping assays was tested. the analytical sensitivity of the test under investigation was determined testing ten-fold serial dilutions of seasonal influenza a(h n ), seasonal influenza a(h n ), new pandemic influenza a(h n ) , and b cell culture-grown viruses. the intra-assay reproducibility was measured by testing the same a(h n ) positive sample times in the same experiment, while the inter-assay reproducibility was confirmed by testing the same samples in independent experiments. to evaluate the performance of the protocol, all samples were tested using a block pcr confirmation test (seeplex Ò rv ace detection), and all specimens collected between january st and december st, and dilutions were also assayed using the recommended who ⁄ cdc protocol of real-time rtpcr for influenza a(h n ). typing and sub typing were performed using the who protocol and ⁄ or sequencing. viral rna was extracted from swabs using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's protocol. fast set infa ⁄ infb and fast set h n v are two multiplex one-step real time pcr assays developed and evaluated by the liguria regional reference centre for diagnosis and surveillance of influenza in collaboration with relab diagnostics. both assays contain primers and a dual-labelled hydrolysis probe that targets two regions of the matrix gene (table ) . amplification conditions were as follows: reverse-transcription °c for minutes, denaturation °c for minutes, then cycles of °c for seconds, °c for seconds. the entire amplification process extended for minutes. an internal control real-time assay was also incorporated in order to detect pcr inhibition, failed extraction ⁄ pcr and technical error. the cdc realtime rtpcr (rrtpcr) protocol for detection and characterization of swine influenza includes a panel of oligonucleotide primers and dual-labelled hydrolysis (taqman Ò ) probes to be used in real-time rtpcr assays for the in vitro qualitative detection and characterization of swine influenza viruses in respiratory specimens and viral cultures. this protocol recommends three primer-and-probe sets: infa, amplifying a conserved region of the matrix gene from all influenza a viruses; sw infa, designed to specifically detect the nucleoprotein (np) gene segment from all swine influenza viruses and sw h , designed to specifically detect the hemagglutinin gene segment from a(h n ) . the seeplex Ò rv ace detection for auto-capil-lary electrophoresis is a multiplex block rt-pcr that applies dpoÔ (dual priming oligonucleotide) technology and is designed to detect major respiratory viruses, respiratory rna (influenza a and b virus, parainfluenza virus type , and , respiratory syncytial virus a and b, rhinovirus a ⁄ b, coronavirus oc and e ⁄ nl ) viruses and dna (adenovirus) virus, from patients' samples including nasopharyngeal aspirates, nasopharyngeal swabs and bronchoalveolar lavage. conventional viral culture was performed inoculating ae ml of each specimen into mdck-siat seeded into -well plates for influenza isolation. virus detection was performed by the hemagglutination test using ae % guinea pig red blood cells (rbc). specificity and clinical sensitivity results of the new protocol are reported in table . fast set infa ⁄ infb was able to detect influenza a and b virus circulating between and belonging to different subtypes and lineages, and no cross-reactions were observed by either fast set infa ⁄ infb or fast set h n v. among specimens collected between january st and december st, , all fast set infa ⁄ infb and fast set h n v high titre positive samples resulted positive using the who ⁄ cdc assay and showing reactivity using infa and sw infa primer-andprobe sets. among low titre a(h n ) positive samples at fast set infa ⁄ infb, ( ae %) were not detected by the who ⁄ cdc assay, but were positive using seeplex Ò rv . the who ⁄ cdc sw h primer-and-probe set works in ae % ( ⁄ ) and ae % ( ⁄ ) of high and low titre a(h n ) positive samples, respectively. all a(h n ) strains collected during and initially detected by fast set infa ⁄ infb were confirmed after rna re-extraction by seeplex Ò rv and who ⁄ cdc assay showing reactivity using the infa primer-and-probe set. all infa ⁄ infb were confirmed after rna re-extraction by seeplex Ò rv . one influenza a case identified by the who ⁄ cdc kit (infa primer-and-probe set, ct values: ae , sw infa primerand-probe set: negative) and new protocol (a primer-andprobe set, ct values: ae , a(h n ) primer-and-probe set, ct values: ae ) was not detected by either seeplex Ò rv or by who subtyping protocol and ⁄ or sequencing, suggesting a very low viral load or unspecific results by real time assays. the analysis of serial dilutions of cell culturegrown a(h n ) showed that the detection limit of fast set infa ⁄ infb, fast set h n v, and seeplex Ò rv was identical ( ) ) and log lower than that using the who ⁄ cdc protocol ( ) ). a similar analysis with respect to a(h n ) and a(h n ) strains indicated that fast set infa ⁄ infb sensitivity ( ) and ) , respectively) was log lower than that showed by seeplex Ò rv ( ) and ) , respectively). in comparison with the new protocol, the who ⁄ cdc assays, considering infa primer-and-probe set, was found to have a slightly lower end-point detection, detecting the ) a(h n ) and a(h n ) dilution. also in detecting influenza b virus, fast set infa ⁄ infb sensitivity ( ) and ) , respectively) was log lower than that showed by seeplex Ò rv and the who ⁄ cdc protocol. data on intra-assay and inter-assay precision, measured as cv% of ct showed that the dispersion indices observed had values of less than %. since samples were detected using the new protocol that resulted negative using the who ⁄ cdc assays. the unfortunately low quantity of low titre a(h n ) samples collected during did not allow us to highlight differences between assays fast set infa ⁄ infb, and fast set h n v positivity was always confirmed by seeplex Ò rv , which demonstrated high sensitivity, showing a detection limit comparable or lower when compared with those observed using the who ⁄ cdc assays. the high analytical sensitivity of seeplex Ò rv is reported by kim who observed a detection limit of copies per reaction for each type ⁄ subtype of influenza viruses. the high sensitivity of the new protocol is coupled with its capacity to detect viruses presenting a significant heterogeneity by fast set infa ⁄ infb and high discriminatory ability by fast set h n v. fast set infa ⁄ infb was able to identify representative influenza viruses of circulating strains during the last decade belonging to different subtypes, lineages, and clusters, and fast set h n v primerand-probe set reacted selectively with a(h n ) target. a recent report demonstrated that the sw infa assay is not specific to a(h n ) and is able to detect both human and avian (h n ) influenza a viruses and so there is the potential for misidentification. high titre (ct ae and ae at fast set infa ⁄ infb) a(h n ) viruses did not react with fast set h n v primer-and-probe set (data not shown). available human a(h n ) sequences are similar within the h n v primer-and-probe regions, but having - mismatches in the forward primer and, more notably, two of the mismatches occurred within nucleotides of the end, an important determinant for primer specificity. in conclusion, this protocol can be a powerful tool in the diagnostic laboratory setting for specific simultaneous analysis of several samples in minimal time, showing enhanced sensitivity in detecting influenza viruses, and high discriminatory ability in identifying the new pandemic a(h n ) . a university-corporate partnership to enhance vaccination rates among the elderly: an example of a corporate public health care delivery public health campaigns usually rely on governmental infrastructure and finance for vaccine implementation programs. however, there are many financial and physical barriers which preclude widespread and effective vaccine administration, especially among the elderly. on an international scale, both government agencies and citizen groups have a vested interest in searching for more resourceful methods of attaining significant immunization levels (> % of the population). in fact, it seems to have become both a grassroots civic and governmental goal, especially among developing countries. we implemented the unique strategy of enlisting the assistance of a privately-owned food market chain to address the public health issue of mass vaccination for the elderly. in this context, publix pharmacy and the university of south florida (usf) recently developed both a handbook and a training program to facilitate the administration of vaccinations. between and , the publix-usf partnership resulted in administration of over thirty thousand influenza a (h n ) vaccinations, % of which were given to adults over years of age. consequently, vaccine administration costs were decreased by using corporate resources and bypassing overly strained municipal resources. this unique university-corporate partnership successfully delivered h n vaccine to a vulnerable cross-section of society at a lower cost and with minimal side effects and morbidity. it may be safely projected that university-corporate partnerships could result in an effective method for rendering a vital service to an aging and especially vulnerable segment of the population. government policy and funding are the foundation of immunization programs on an international scale. for example, in the united states, governmental programs account for over % of the monetary outlay used for immunization. until , the global alliance for vaccines and immunizations (gavi) acted as a catalyst for implementing vaccine and immunization programs in each targeted country. under the auspices of gavi-collaborations between governments, charitable organizations, and multinational health agencies (such as uncief and the who)-many countries have increased their spending for vaccination programs. however, development of financially sustainable immunization programs geared toward reaching the majority of the population are still at a nascent level of evolution. the development of more innovative and costeffective approaches has become imperative in order to reach a greater number of vaccination candidates. administering the influenza vaccine only to the subpopulation of over year olds would save an estimated quality-adjusted life years in a cohort of approximately half the world's population. widespread public vaccination programs are made more complex by the continuing development of newer vaccines, concomitant specialized administration costs, and the logistical challenge of conveying recipients to vaccination points of service. , in spite of the increasing complexity of mass vaccination, cost-benefit analyses clearly favor annual influenza vaccination in the elderly population on an international scale. , recently, in , influenza vaccine administration was reported to reach between % and % of the elderly population, which denotes varying degrees of success within each particular country. , however, there was also a report of a uniform plateau effect at around % of the population, beyond which additional vaccination coverage was difficult to achieve. physical limitations to vaccination seem to be more insurmountable for the elderly. unfortunately, this is the population segment which could experience the most significant vaccination-associated mortality reduction. we employed the unique strategy of involving the resources of publix supermarkets, a corporate food market chain, to address the public health issue of widespread vaccination for the elderly. we took advantage of recent changes in the florida statutes, which expanded the scope of pharmacists' practice to include administration of vaccines. subsequently, publix pharmacy and the university of south florida (usf) developed a handbook and training program to facilitate and enhance vaccine administration by publix pharmacists. by using proprietary pharmacists and more practical supply storage, we were able to decrease the costs of vaccine administration. the consumer was charged $ for administration costs plus the cost of the injection itself, regardless of insurance or eligibility for governmental subsidy. although patients were initially self-selected, they were ultimately excluded if they had demonstrated prior adverse effects to influenza vaccinations or to any of the components of such vaccinations. between and , the publix-usf partnership vaccinated people against influenza a (h n ), of which were florida residents. the age range was - years old with a median age of years old. seventysix percent of the participants were over years old (see figure ). within the population surveyed, the reported side effects of the vaccine in this study were not serious, but included: vertigo, cold sweats, chills, vomiting, syncope, rash, nausea, stomach pain, elevated blood pressure, injection site reaction, inflamed bursa, and bilateral thigh discomfort. participants from all socioeconomic classes were vaccinated. an income-by-zip code analysis revealed % of those vaccinated resided in zip code areas where the average household income was <$ per year. of those remaining, % had an average income of $ -$ per year, and % had an income of >$ per year. each person vaccinated was charged ten dollars for administration costs. this represents a decrease in the administration costs ranging from one dollar to ten dollars saved per vaccine. , conclusion this unique university-corporate partnership successfully delivered h n vaccine to a high-risk population with decreased vaccine administration costs. the influenza vaccine is well-tolerated, with minimal side effects when patients who have a history of adverse reactions are excluded. we can postulate that university-corporate partnerships may indeed be effective at reaching the aging population which is a challenge in most communities. this delivery model may prove to be another tool for improving the efficiency of mass immunization by facilitating accessibility, which results in wider coverage. this model also enhances delivery of healthcare by decreasing costs of immunization regardless of whether the payer is a government, insurance company, or self-pay consumer. the gavi initiative stressed three goals for accomplishing sustainability and independence in immunization programs. the goals were to: (i) mobilize additional resources from governmental and non-governmental sources; (ii) improve program efficiency to minimize additional administration resources needed; and (iii) increase the reliability of funding. empowering privately owned corporations within the community, such as food markets or pharmacies, to administer vaccines mobilizes additional resources to readily achieve the first goal of gavi. mobilizing resources of non-healthcare, corporate vaccination locations enhances accessibility due to travel convenience. in our study, participants came from all socioeconomic classes, suggesting that ease of access is independently hindering mass vaccination, and that people of all incomes are more likely engaged when access issues are eliminated. the second and third goals were also accomplished by recruiting a corporation's resources for vaccine administration (refrigeration, storage, and employees). this minimizes the money spent from vaccine program funds to support the infrastructure of immunizations, thus improving financial efficiency and sustainability. financial efficiency implies that money is spent to safely reach as large a portion of the population as possible. by using corporate storage facilities instead of paying for independent facilities, money can be spent elsewhere. more vaccines can be purchased and more money can be spent on media communications to encourage vaccination. sustainability requires the ability to fund annual vaccination programs which reach % of the population or greater. key to the control of pandemic influenza are surveillance systems that raise alarms rapidly and sensitively. in addition, they must minimise false alarms during a normal influenza season. we develop a method that uses historical syndromic influenza data from the existing surveillance system 'servis' monitoring seasonal ili activities in scotland. we develop an algorithm based on wcr of reported ili cases to generate an alarm for pandemic influenza. wcr is defined as the ratio of the number of reported cases in a week to the number of cases reported in the previous week. from the seasonal influenza data from scottish health boards, we estimate the joint probability distribution ( figure ) we compare our method, based on our simulation study, to the mov-avg cusum and ili rate threshold methods and find it to be more sensitive and rapid. the wcr method detects pandemics in larger fraction of total runs within the same early weeks of pandemic starting than does any of the other two methods ( figure ). as shown in the table, for % pandemic case reporting rate and detection specificity of %, our method is % sensitive and has mdt of weeks, while the mov-avg cusum and ili rate threshold methods are, respectively, % and % sensitive with mdt of weeks. at % specificity, our method remains % sensitive with mdt of weeks. although the threshold method maintains its sensitivity of % with mdt of weeks, sensitivity of mov-avg cusum declines to % with increased mdt of weeks. for a two-fold decrease in the case reporting rate ( ae %) and % specificity, the wcr and threshold methods, respectively, have mdt of and weeks with both having sensitivity close to %, while the mov-avg cusum method can only manage sensitivity of % with mdt of weeks. the first cases of the pandemic were reported in scotland in the th week of the season. the wcr algorithm as well as the mov-avg cusum method detects the pandemic weeks later in week . the ili threshold method detects it week later in week . both the wcr and mov-avg cusum methods therefore outperform the ili threshold method by week in the retrospective detection of the pandemic in scotland. while computationally and statistically very simple to implement, the wcr method is capable of raising alarms rapidly and sensitively for influenza pandemics against a background of seasonal influenza. although the algorithm has been developed using the servis data, it has the capacity to be used at large scale and for different disease systems where buying some early extra time is critical. more generally, we suggest that a combination of different statistical methods should be employed in generating alarms for infectious disease outbreaks. different detection methods would provide cross-checks on one another, boosting confidence in the outputs of the surveillance system as a whole. real-time evidence being created worldwide will greatly contribute to the full understanding of influenza pandemics. here we report the real-time epidemiology and virology findings of the influenza a(h n ) pandemics in mongolia. the epidemiological and virological data collected through isss of nic, nccd, mongolia (real-time information on registered ili cases and virological laboratory results are available from the weekly updates in the nic, mongolia website: http://www.flu.mn/eng/index.php?option=com_ content&task=category&sectionid= &id= &itemid= ) were used for analysis in relation to the previous seasonal influenza activities in the country. influenza viruses were detected in naso-pharyngeal samples from ili patients by rt-rt-pcr with applied biosystems fast real time pcr system , using primers and instructions supplied by cdc, usa. influenza viruses were isolated by inoculation of rt-rt-pcr-positive samples of mdck cell culture according to the standard protocol. ten representative strains of a(h n )pdm viruses were selected for sequencing of different gene segments, namely: a ⁄ ula- , and a ⁄ dundgovi ⁄ ⁄ . sequencing of influenza virus gene segments was performed in applied biosystems xl genetic analyzer using primers and instructions supplied by cdc, usa, and bioinformatic analysis was performed with abi ⁄ seqscape v. . and mega programs. the pandemic alert in mongolia was announced by the government on april , , just after the who announcement of the pandemic alert phase, and planned containment measures were intensified. despite intensive surveillance, no a(h n )pdm virus was detected in mongolia until the beginning of october . around suspected cases, mostly arriving from the a(h n )pdm epidemic countries, tested zero by rt-rt-pcr for a(h n )pdm virus. the first a(h n )pdm case detected by the routine surveillance system in ulaanbaatar city, the capital of mongolia, was confirmed by rt-rt-pcr on october , ( st week of ). the reported ili cases escalated rapidly, reached the peak in the - th week of , and gradually decreased thereafter ( figure ). week of . however, the registered ili cases increased again from the th week of , and peaked at the - th weeks of . the viruses isolated during this nd peak were influenza b strains ( figure and table ). for the genetic characterization of the mongolian pandemic isolates, gene segments i (pb ), gene segments ii (pb ), gene segments iii (pa), gene segments iv (ha), gene segments v (np), gene segments vi (na), gene segments vii (m), and gene segments viii (ns) of the representative a(h n )pdm mongolian strains were sequenced, and all sequences have been deposited in the genbank (accession numbers: cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy , cy ). all genes of mongolian strains were possessing ae - ae % similarity with the genbank deposited gene sequences of the original pandemic strain a ⁄ california ⁄ (h n ). the who declared the pandemic alert phase (phase iv) on april , , and was prompted to announce the pandemic phase (phase v) two days later. after days, the who declared the beginning of the pandemic peak period (phase vi) on june , . however, in mongolia, the pandemic alert period continued for days. mongolia was free of the pandemic virus during the whole first wave of the pandemics in the northern hemisphere. with the confirmation of the st influenza a(h n )pdm case on october , in ulaanbaatar, mongolia entered into the pandemic phase (phase v), and after just weeks, the registered ili cases peaked, confirming mongolia shifted into the pandemic peak period (phase vi), which i i i v i i v i v v v i i i i iii iv v vi vii viii worldwide by who in mongolia coincided with the nd wave of pandemics in many countries of the northern hemisphere (see, picture and table ). despite the relatively milder clinical manifestations, the disease burden for the health service was enormous, while the morbidity per population at the peak period was - times higher above the upper tolerant limit, and - times higher above the seasonal influenza outbreaks. in contrast to the seasonal influenza outbreaks where over % of the registered ili cases have been in the age group under , it has been observed that over % of the registered cases in this pandemic peak period were in the age group of - . on january , , we regarded the pandemic had entered into the post-peak period (phase vii) when the registered ili cases became lower than the upper tolerant limit, during which time mongolia experienced an influenza b outbreak. on may , we determined that mongolia entered the post-pandemic period (phase viii) as the influenza virus isolations were almost stopped, and after no pandemic virus detected for months. who announced pandemic vii and viii phases much later. , this first ever real-time laboratory confirmed influenza pandemics in mongolia and confirmed some variations of pandemic spread in different parts of the world. the comparison of deduced amino-acid sequence changes have shown that the mongolian strains belong to the clade , according to the classification of a(h n )pdm influenza strains suggested by m. nelson, which has circulated worldwide since july . this is also evidence that the st wave of the pandemics did not hit mongolia. the who public health research agenda for influenza is aimed to support the development of evidence needed to strengthen public health guidance and actions essential for limiting the impact of influenza on individuals and populations. each stream-specific group reviewed and discussed the proposed organization, content, rationale, and global health importance of their designated research stream. specific research recommendations were made for topics within each stream: background: a syndromic surveillance system using nonclassical data sources for detection and monitoring evolution of flu and flu-like illness (ili) in djibouti is reported here as part of the preliminary report of djibouti who-copanflu international study (wcis)**. methodology: clinical reports, over-the-counter drug sales, lab diagnosis report, and health communication trends were obtained for an integrated statistical analysis. results: transition to winter is concomitant with upsurge of ili cases and ili drug sales. in addition, more rural folks manage ili infections on self medicament than through clinical consultancy. inefficient and vague data collections were observed. a successful implementation of wcis will create a platform upon which challenges faced in djibouti health department in routine surveillance will be addressed to achieve a near-real time surveillance of flu pandemic. conclusion: innovations, prompt reporting, and instituting open source syndromic surveillance system software's in resource limited environment like djibouti will enhance early detection and evolution monitoring of pandemic flu. the spanish flu in ⁄ infected and killed millions of people, and threatened to wipe humanity off the face of planet. however, the recent scenarios of influenza h n ( ) pandemics' worldwide occurrence fell short of most scientific prediction on its magnitude and intensity. this dampened their confidence; they cannot state precisely as to when, how, where, and which of the spanish flu-like pandemic will occur in the future. in support of scientific community and governments, the who hasn't gone to slumber, but is reminding its member states to up their post pandemic surveillance and monitoring of influenza virus in circulation for advance preparedness in case of an outbreak. despite all uncertainty around the pandemic flu h n , there remains a common knowledge and understanding that this flu has shown a great potential to evolve and cause huge morbidity and mortality. although its future magnitude may be unpredictable, its recurring events have severe consequences on human health and the economic well being of everyone. and therefore, advance planning and preparedness is critical in protecting any population in the future, especially those located in resource limited environment without universal health cover and generous disaster emergency funds. . two collapsed sets of a weekly and monthly mean data (of four years period) were clustered in five categories of ili cases, drug sales, lab results, vaccine consumption, and health promotion. this was followed by a descriptive statistics analysis of cumulative weekly and monthly data to establish presence or absence of trend. time series analysis was not done due to data limitation. copanflu program: as at the time of going to press, the cohort study is at the household recruitment and inclusion phase and the study covers the djibouti city. it is in our intention to use the cohort study findings to validate or improve the niph ministry of health djibouti ili surveillance effort for better preparedness. clinical service: % of all health facilities are in djibouti city. of the ae % ( ) of the population that seeks medical care on influenza and influenza like illness each year, ae % ( ) and ae % ( ) of them are attended to at the city's public and private clinics, respectively ( figure ). the rest are attended from the regional health centers. the majority of ili incidence sharply rise with the onset of the winter season (october to april), affecting mostly the middle age group ( - years). pharmaco-surveillance: % ( ) of total prescriptions were antipyretic and antiflu drugs, ae % ( ) of which were consumed by peripheral regions, the non dji- lab diagnostics: the annual ili lab diagnosis was negligible ae % ( ), which can be attributed to less equipped virology laboratories to warrant routine service utility. documented cases were from previous bouts of avian influenza that had a human incidence from and . with support of egypt-based naval army medical research unit three (namru ), clinicians were motivated to sample all ili patients and submit to collaborating international reference influenza lab in cairo, egypt. vaccination: influenza vaccinations were undocumented, but at least ae % ( ) of population sought the service (for yellow fever and meningitis) as mandatory travel advisory or as childhood immunization need. at the time of going to the press, there were at least vaccine doses of h n ( ) virus donations yet to be administered. health promotion and hygiene: print and audiovisual risk communication remained favorite means of reaching out to urban dwellers ( ae %). while to the rural and nomadic population, person to person communications was the preferable means. to increasing public awareness that will encourage reporting of ili cases and entrench risk aversion health behavior that limits flu spread, who-copanflu international study djibouti has incorporated basic training on ili infection and personal hygiene by interviewers during household inclusion. improving national epidemic surveillance capacity and response under new international health regulation is important for any nation, including djibouti. our finding indicates the winter season predisposes one to ili infections; they therefore opt for medical services or self medication depending on their capability and ⁄ or understanding. in djibouti, almost no city dwellers favors self medication over clinical consultation, suggesting the presence of inhibitory factors like distance from the health centers and the cost of accessing consultancy. common in the absence of universal primary health care setting, it therefore calls for active innovativeness in outbreak detection, disease reporting, and preventive medicine on the part of health authority so as to achieve good population health. in respond to these, niph has turned resource limitation to a motivation instead and is working towards institutionalizing a near-real-time syndromic surveillance system as a core functional unit. it capitalizes on three major aspects within its reach: prompt accurate data generation for analysis, ehesp wcic-study input, and information technology use. prompt accurate data generation for analysis: data used in our analysis suffered from un-timeliness (weekly instead of daily basis), incompleteness (vague over-counter drug sales records), entry errors (incidence case reports), and poor collection format (most of data collection forms). use of satellite handset phones for regional health centers and mobile phones for city sentinel clinics will reduce unnecessary data delivery delays. in addition, creating awareness to data entry personnel on the importance of careful and completeness of entries is important, as is the need to reformat data collection forms to capture exact aspects of surveillance needs for relevant executable analysis. besides alerting for immediate impending epidemics, these data can also be adopted for projective predictive modeling of annual epidemics, including that for influenza. ehesp wcic-study input: djibouti wcic-study is complementary to the existing syndromic surveillance system, but with emphasis on flu and flu-like illness. various innovations as suggested above are used in seeking to overcome the prevailing challenges. while every attempt is made to realize its (wcic-study) objective and for global comparison, lessons learned from successful implementation will form a platform for future refined syndromic surveillance protocol as equally reported elsewhere in asian countries. , information technology: national institute of public health djibouti has an informatics department with sufficient working pcs and personnel to execute efficient data collection and management for epidemiological analysis. however, licensing cost of near-real time syndromic surveillance software is prohibitive, but the open access software with capacity to generate custom graphs, maps, plots, and temporal-spatial analysis output for specific syndromes should make implementation a lot easier. such output for conditions like flu (or gastroenteritis) will be essential to cause prompt response of the local public health office and international partners in saving lives and suffering of djibouti people. pandemic flu surveillance and preparedness requires multifaceted, interdisciplinary, and international approach whose efficiency and efficacy can only be refined over time. building on the health care system's swot for preparedness, the ehesp wcic-study promises to refine surveillance system operation and knowledge on individual's risk determinants to swine flu (h n ) virus infection at the household level in djibouti. these efforts are ultimately creating available control options at the time of need (pandemic occurrence), and at the same time exploring investment in quality data profiling and information technology, which will include syndrome surveillance software systems like essence, ewors, or other open sourced ones. the antibody efficacy -which compares the illness frequency between those with and those without a protective level of pre-epidemic hi antibodies ( ‡ : ) -has been proposed ; however, this index has rarely been used due to practical difficulties in confirming the strain-spe-cific disease corresponding to each of the vaccine-induced antibodies. we followed elderly individuals residing in a nursing home, whose serum specimens were obtained before and after undergoing trivalent influenza vaccination, in ⁄ influenza season (medium-scale mixed [a ⁄ h n and b] epidemic in study area, and a ⁄ h n was circulating at the nursing home). the serum antibody titre to each strain of influenza virus was measured by the hi method, using the same antigens as those in the vaccine. all participants' body temperatures, respiratory symptoms, other general symptoms, hospitalization, discharge, and death were recorded daily from november to april in a prospective manner. when the participants suffered any influenzalike symptoms, such as sudden fever ‡ ae °c, throat swabs were collected and tested using a rapid diagnosis kit for influenza, which utilizes an immunochromatographic method. the adjusted odds ratios (or adj ) for febrile illness and kit diagnosed influenza were evaluated using multiple logistic regression models adjusting for possible confounders (i.e., age, sex, coexisting conditions, and vaccine strains). after vaccination, the proportion of subjects achieving an hi antibody titre ‡ : (seroprotection level) were ae % ( ae - ae %) for a ⁄ h n , ae % ( ae - ae %) for a ⁄ h n , and ae % ( ae - ae %) for b. during the follow-up period, the a ⁄ h n strain was isolated therein, and subjects experienced sudden-onset fever ( ‡ ae °c), and eight subjects were positive for rapid diagnosis kit. patients with a seroprotection level of the hi antibody titre ( ‡ : ) had lower incidences of febrile illness (or adj , ae ; % ci, ae - ae ) and rapid kit diagnosed influenza (or adj , ae ; % ci, ae - ae ) than those with a lower titre. thus antibody efficacy ( ) or adj ) against fever related to a ⁄ h n and kit diagnosed influenza were both estimated to be %. although statistical significance was not detected due to limited sample size, these results lend support for the usefulness of antibody efficacy. some data presented within this manuscript was also published in hara et al. asia via a regional network from which epidemics in the temperate regions were seeded. the virus isolates obtained from nasopharyngeal swab specimens from outpatients were typed and subtyped by the hemagglutination (ha) inhibition assay. the emergence of a ⁄ fujian ⁄ ⁄ coincided with higher levels of influenza-like illness in korea than what is typically seen at the peak of a normal season. most of the intermediates and fujian-like strains were isolated from asian countries, and the mutational events associated with the fujian strains took place in asia. closely dated phylogeny from december , to august , showed that the antigenic evolution of the h n fujian strains had periods of rapid antigenic changes, equivalent to amino acid changes per year ( figure ). the fujian-like influenza strains were disseminated with rapid sequence variation across the antigenic sites of the ha domain. the antigenic evolution of the fujian strains was initiated by exceptionally rapid antigenic change that occurred in asia, which was then followed by relatively modest changes. some of the data presented in this manuscript was previously published in kang et al. we compared reactivity to the novel virus strain using haemagglutination inhibition (hi) assays performed on discarded plasma specimens left over from routine testing. samples were taken from healthy adult blood donors (> years) before and after the ph n influenza epidemic that occurred during the southern hemisphere winter of , and again prior to onset of the southern hemisphere influenza season. reactivity to the novel h n strain of influenza was relatively uncommon among the healthy adult population during the first australian winter wave, rising from a baseline of % to %. a further increase in the seropositive proportion from % to % was observed over the summer months, most likely attributable to immunisation. this level of immunity appears to have been sufficient to constrain the winter epidemic. together with a final serum collection, planned for late , these data will aid evaluation of the extent and severity of disease in this 'second wave' of ph n . assessment of the extent of disease due to novel influenza a(h n ) virus (ph n ) during the winter outbreaks in australia was made difficult by the generally mild nature of disease. the epidemic was experienced in a staggered fashion around the country, reflecting the considerable geographical distances between state and territory capital cities ( figure ). differences in the intensity of case-finding during the evolving pandemic response and between jurisdictions hindered comparisons of disease burden in distinct geographical regions. rates of reported hospitalisations and deaths appeared fairly similar across states but, without a consistent exposure denominator, assessment of relative severity was difficult. we conducted a national serosurvey of antibody to ph n using residual plasma from healthy blood donors collected before and after the epidemic to estimate ph n exposure. here we report the findings of that first collection, together with new data on seroprevalence of ph n antibody in specimens gathered in march-april . these latter samples were collected prior to onset of seasonal influenza activity to assess the impact of a national ph n vaccine program conducted in spring ⁄ summer ⁄ on the proportion of individuals with antibody titres deemed protective. findings informed estimates of population susceptibility to ph n prior to the influenza season and provided a baseline for a subsequent serosurvey that will be collected at the end of to assess the extent of exposure during the 'second wave.' tralian red cross blood service (the blood service) for dengue fever surveillance studies. these samples were used to provide a baseline estimate of prevalence of cross-reactive antibody to ph n in the australian population. discarded plasma specimens, taken for virologic testing from healthy adult blood service donors, were prospectively collected at two additional timepoints for measurement of antibody to ph n . collection periods were as follows: approximately plasma samples were randomly selected from donors in each of brisbane, hobart, melbourne, newcastle, perth, sydney, and townsville on each occasion. up to specimens were identified in each of the following age strata: - , - , - , - , - , and > years. at the last collection timepoint, there was deliberate over-sampling of the oldest and youngest age strata in which approximately specimens were collected (i.e., up to specimens per site). in accordance with the provisions of the national health and medical research council's national statement on ethical conduct in human research, individual consent was not required for use of these specimens, given the granting of institutional approval by the blood service human research ethics committee. reactivity of plasma against ph n was measured in haemagglutination inhibition (hi) assays using turkey red blood cells (rbc). egg-grown a ⁄ california ⁄ ⁄ virus was purified by sucrose gradient, concentrated and inactivated with b-propiolactone, to create an influenza zonal pool preparation (a gift from csl limited). plasma samples were pretreated with receptor destroying enzyme ii (denka seiken co. ltd), : (volume ⁄ volume) and tested as previously described. following hour incubation, ll % (volume ⁄ volume) of rbc was added to each well. hi was read after minutes. any samples that bound to the rbc in the absence of virus were adsorbed with rbc for hour and reassayed. samples in which background activity could not be eliminated by these means were excluded from the analysis. titres were expressed as the reciprocal of the highest dilution of plasma where haemagglutination was prevented. a panel of control sera and plasma samples was included in all assays. it comprised paired ferret sera pre-and postinfection with the pandemic virus or seasonal influenza a(h n ), a(h n ), or influenza b viruses and paired human plasma and sera collected from donors before april or after known infection with the pandemic virus or after immunisation with the australian monovalent pandemic vaccine. all assays were performed by the who collaborating centre for reference and research on influenza. for each of the three study timepoints and within each age group, the proportion of seropositive individuals (hi titres ‡ ) was calculated, with exact (clopper-pearson) confidence intervals. the contribution of individual variables (age, gender) and location to seropositive status was assessed in separate multivariate logistic regression models developed to assess the post-pandemic and pre-influenza season collections. all statistical analyses were conducted in stata . locations of specimen collection are shown in figure , together with the number of samples tested from each centre. samples with high background hi titres or discrepancies between assays were excluded at each timepoint as follows: at baseline, from the post-pandemic collection, and in early . pared with baseline was % overall, rising from % to % (table ). the only jurisdictions in which seropositive proportions were higher in october ⁄ november than in the baseline collection were hobart [ % ( % ci ae , ae )], perth [ % ( ae , ae )], and sydney [ % ( ae , ae )]. in the multivariate regression model, the only jurisdiction in which exposure appeared somewhat higher than the reference population of brisbane was hobart [or ae ( % ci ae , ae ), p = ae ]. a marked age effect on antibody status was observed at this timepoint, with an increase in the proportion of seropositive individuals in relation to the baseline collection only noted for those aged between and years (table ) . according to the multivariate model, the youngest and oldest cohorts had similar titres, with all other groups showing significantly lower seropositive proportions than the reference population of - years [e.g. - years or ae ( % ci ae , ae , p < ae )]. an overall increase in the seropositive proportion from % to % was observed between october and april , distributed throughout all jurisdictions ( ( , ) ]. antibody titres prior to the influenza season rose in all age groups, but remained significantly lower among [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] year olds than in the youngest age cohort (table ) . adjusted ors for the seropositive proportion in the multivariate model in these age groups were: - years [or ae ( % ci ae , ae )]; - years [or ae ( ae , ae )]. the relatively low titres observed in these groups reflected small incremental increases in the seropositive proportion across each of the time points studied, suggestive of both low rates of infection and vaccination. the rise in immunity observed across the population was most likely attributable to immunisation in the majority, given the absence of observed outbreaks and very few notified cases of ph n during the period between the two plasma collections. this study suggests that, while adult exposure to ph n during the southern hemisphere winter was uncommon at around %, vaccine uptake in the australian population over the period november -may was in the order of %. this latter estimate is in keeping with recently published figures for adult ph n vaccine coverage from a national immunisation survey conducted by the australian institute of health and welfare. in that survey, vaccine coverage was significantly higher in tasmania than in other states, but mostly in those over years of age, possibly in a subgroup whose health status may have differed from that of the donor population. no allowance has been made in this analysis for likely waning of natural or vaccine induced immunity, possibly resulting in lower estimates of natural and ⁄ or vaccine exposure than may have occurred over the period. regardless of such intervening processes, the seropositive proportion among australian adults at the start of the winter season appeared likely to be sufficient to constrain transmission of infection in the age groups tested. this assertion has been borne out in practice, with only modest levels of influenza reported during the late and protracted season. a final serum collection is planned for the end of the influenza season in australia from which to assess the level of exposure in relation to the baseline observed here. the need for epidemiologic studies such as this has been highlighted by groups such as the european centre for disease control to aid evaluation of the extent and severity of the 'second wave,' known to be variable from historical reports of past pandemics in disparate populations. in - , the first wave of the swine-origin novel h n flu (h n ) pandemic swept across the world, including japan. to examine the epidemiological nature of this novel infectious disease among school children within and among small regional communities, we have carried out a complete survey on the incidence of h n among school children using absentee reports provided by school health teachers in two small administrative districts (population: about in total) in japan. we then examined the epidemiological diversity on the inci-dence of h n within and among small regional communities. we investigated seventeen elementary and ten junior high schools in moroyama-town and sakado-city located in the central part of saitama prefecture. populations are: all ages, and ; elementary schools, and ; junior high schools, and , respectively. the number of school children in each school ranges from to . the surveillance system was built on an apache-and mysql-based web server using html, php, and java-script. school health teachers enter information on children absenteeism due to school infectious diseases via web browsers at each school infirmary on a daily basis. in addition to the trend graphs shown on the web browser, detailed analyses were reported to the schools and local educational boards weekly. the basic reproduction number (r ) of h n was estimated according to becker. agentbased modeling and simulations were also performed using a multi-paradigm simulator anylogic version . (xj technologies, st. petersburg, russia). by the end of march , cumulative incidence (ci) of h n among school children in moroyama and sakado reached % and %, respectively. the overall r among school children in this area was ae . vaccination rate of children in this area during the surveillance period was reported to be very low (< %). there was no considerable difference between the epidemic curves in this neighboring town and city. on the other hand, in the individual schools, the cis as of the end of march scattered from % to % ( figure ) even though the schools are closely located. to examine the cause of this diversity, we built an agent-based community model consisted of the same numbers of agents as those of children in the actual schools and people in moroyama and sakado to simulate the infection. the ratio of probability of infection in schools and the remaining places were assumed to be : or : . using a heuristic optimization scheme, we estimated the parameters for the simulations to give the overall ci of % (the ci as of the end of march ). we then performed simulations repeatedly. the cis obtained with the repetitive simulations with the assumption of higher probability of infection in schools scattered from % to %, indicating that the cis of the small population communities may vary considerably, even though all the agents were assumed to have the same susceptibility to infection at the beginning, and the other conditions were the same. the policies for surveillance ⁄ analyses ⁄ prevention of communicable diseases in local communities have generally been decided on governmental-and ⁄ or each local administrative district-basis (populations: several hundred thousands to several millions) in japan. we found the considerable variations in the cis of h n for children among much smaller areas, i.e., the school districts (populations: all ages, several thousands; school children, several hundreds). we thus conclude that the granularity of surveillance ⁄ analyses ⁄ prevention should be finer than in the past to achieve the most effective policies against influenza and similar communicable diseases in the local communities. the cause of this diversity can be explained in part by the stochastic nature of infection transmission processes in the small populations shown by the agent-based simulations. we have already conducted a complete questionnaire survey for the school children and their parents to clarify the relevance of the other issues including differences in environmental factors, preventive policies (e.g., vaccination, school closures), etc., in each school. the detailed analyses will be reported elsewhere. a www-based surveillance system for transmission of infectious diseases among school children within and among small regional communities. j epidemiol ; (s ):s . this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. the first cases of the a ⁄ h n v pandemic influenza were reported in mexico and the united states in april . given the context of this new influenza virus and considering the likelihood of its pandemic spread, the cohorts for pandemic influenza (copanflu) international consortium was created in order to study individual and collective determinants of pandemic a ⁄ h n v influenza across different countries by setting up prospective cohorts of households, followed during years. this study relies on the first available data from the copanflu-france project, which is part of the copanflu international consortium. we studied factors associated with elevated haemagglutination antibody titers against a/h n v at entry in the copanflu-france cohort. we focused in this primary analysis on the association between the titers and influenza vaccination (seasonal or pandemic) across age groups. the copanflu-france cohort was set up in fall . inclusions began on december , and ended on july , . households were sampled using a random telephonic design (mitofsky-waksberg method) in a stratified geographical sampling scheme, aimed at including a sample of subjects representative of french general population. all household members were eligible to the cohort, without any age limit. the inclusion of a household required the participation of all members: the refusal of one or more member(s) prevented the inclusion of other members. the protocol was approved by a research ethics committee and written informed consent was obtained for all subjects. this study requires several visits to the households by nurses who collect written data with questionnaires and biological samples. during the inclusion visits, nurses collected from all subjects detailed data regarding medical history, including vaccination and preventive measures against influenza. blood samples were collected at entry and centralized. a standard hai technique was adapted to the detection and quantification of antibodies to the a ⁄ h n v virus. the titration endpoint was the highest dilution that exhibited complete inhibition of haemagglutination in two independent readings. the lowest read dilution was ⁄ . geometric mean titers were calculated for hai assays with the use of generalized estimating equations for interval-censored data, , taking into account a within-household correlation. multivariate models were derived from this method to identify factors associated with elevated gmts. we defined the ''gmt ratio'' (gmtr) as the multiplicative factor applied to the gmt in presence of an explanatory variable. for qualitative explanatory variables, a gmtr of n means a predicted n-fold higher gmt for subjects exposed to the considered factor compared to others. for continuous explanatory variable, the same interpretation applies to a unit difference. the following variables were included in the multivariate models: age, history of pandemic or seasonal influenza vaccination, and history of ili. age was categorized in three groups: - years (reference group), - years and over years. the definition of ili was that used by the cdc : fever ‡ ae °c and cough and ⁄ or sore throat without another known cause. history of ili was defined as an ili reported by the subject between september , (beginning of the influenza epidemic in france) and the date of inclusion. this preliminary analysis included subjects belonging to households. results reported hereafter do not account for missing data. participating households were sized - subjects, mean size = ae . in comparison, the mean size of french households is ae according to the latest national census. the median age of subjects at entry was ae years [iqr: ae ; ae ] versus ae [ ae ; ae ] for french population. the proportion of subjects reporting a history of ili since the beginning of the epidemic varied from ae % for subjects over years to ae % for subjects below years (table ) . vaccination with the pandemic strain was the highest in subjects below ( %) whereas vaccination with the seasonal strain was the highest in subjects over ( ae %). detailed data regarding vaccination is given in table . this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. [ ] [ ] [ ] [ ] [ ] [ ] among non-vaccinated subjects, elevated gmt in the elderly may be the result of exposure to similar viruses in early life, whereas children and young adults with elevated gmt are likely to have been infected by the a ⁄ h n v virus. [ ] [ ] [ ] [ ] interestingly, a significant drop in the hai titer is observed during the months following vaccination with the pandemic strain. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. the reason for this association is not obvious: although we cannot discard the hypothesis of a higher incidence of a ⁄ h n v infections in seasonal vaccine recipients, as described by several other studies, - the main explanation may be a cross-reaction between pandemic and seasonal strains. , , further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. in april , the cdc alerted about the appearance of a new strain of ia h n with unknown virulence. infants under years old had higher risks of hospitalization, complications, and rate of death for sari. materials and methods: a cross-sectional study was executed from may to december in . the sources were: mandatory reporting form of the province surveillance system, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports. inclusion criteria: children under years old with diagnostic of ili or sari and confirmed cases with epidemiological nexus or laboratory confirmation (rrt-pcr, ifi). the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. results: the ili rate in infants under years old was ae ⁄ people ( % ci - ) being higher in infants of years old ( ⁄ people of years ( % ci - ) ( table ) . infants had less risk of getting sick in relation to the rest of the population (rr ae [ % ci ae - ae ]) (p < ae ). the chance of sari in infants was ae ( % ci ae - ae ) compared to the rest of the population. the lethality rate was higher in infants under year old ( ⁄ people [ ⁄ ]). discussion: the evidence suggests that the infants under years old had lower risk of getting sick than the rest of the population, but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under year old. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. in april , the cdc alerted about the appearance of a new strain of ia h n with unknown dissemination and virulence. in june, the world health organization declared the pandemic. , the ili often presents an unspecific clinical picture in infants under years old, from mild symptoms to sari, especially in the newborn babies. infants under years old have higher risks of hospitalization, complications, and rate of death for sari. , on may th, argentina declared the first imported case of ia h n , and by the end of the month, it announced the viral circulation in the country. the epidemiological surveillance system of the province arranged that all the patients with influenza diagnosis made by a doctor must be reported. from april th to november th, suspected cases of ili in the province of tucumán were reported. the ili rate was ⁄ people, and ia h n comprised ⁄ people. the lethal rate of sari ia h n was ae ⁄ people ( ⁄ ). the objective of this research was to determine the epidemiological characteristics of the pandemic ia h n in infants under years old in the province of tucumán between may and december in . the province of tucumán is placed in the center of the northwest of the republic of argentina. it has a population of inhabitants of which are infants under years old. the crude birth rate for was ae &. the infant mortality rate was ae &. respiratory pathologies in infants under years old were the third cause of death in the province ( %). the public health system of the province is composed by three sectors: public, private, and welfare. with health facilities as a total, the average of available beds is & per inhabitants and & per neonates. a cross-sectional study was executed from may to december in in the province of tucumán, argentina. the following sources were used: mandatory reporting form of the surveillance system of the province filled by a doctor, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports (patients with sari). inclusion criteria: • suspected case of ili: sudden appearance of fever higher than °c, cough, or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nausea or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea. ) were used for the analysis. the odds rations, risk ratio and % confidence interval were calculated to compare ambulatory with hospitalized patients, confirmed and dismissed, < years old and the rest of the population. it was considered significant a rate of p < ae . the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. the epidemiological surveillance system of the province received ili reports, ae % ( ⁄ ) were infants under years old. twenty seven percent were dismissed ( ⁄ ), and % ( ⁄ ) of suspected cases were confirmed. the first ia h n case was a child of years from the province of buenos aires, in th epidemiological week, and the last suspected case was reported in october , ( figure ). the ili rate in infants under years old was ae ⁄ people ( % ci - ), being higher in infants of years old ( ⁄ people of years, [ %ci - ]). the higher ili rates in confirmed the pandemic of ia h n ( ) was detected for the first time in the province of tucumán. the evidence suggests that infants under years old had lower risk of getting sick than the rest of the population (protective factor), but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under year old. towns with the highest demographic density had superior proportion of cases. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. outbreak of h n influenza - : behavior of influenza h n in school children in the province of tucumá n, argentina criteria: patients treated with antiviral medication for prophylaxis, respiratory pathologies which did not justify specific medication, and incomplete forms. results: from all notifications, were cases of ili in the group aged - years old; % were males. the incidence rate in this group was ae per thousands of inhabitants. the % of laboratory samples were influenza a h n , % were confirmed as unspecific influenza, and % were dismissed. the school aged children group had a high risks of getting sick (r.r. ae [ % c.i. ae - ae ]), especially males. it appeared that school aged children had a protective factor for presenting sari (or ae [ % c.i. ae - ae ], p < ae ). the lethality rate in this group was ae ⁄ thousands. headaches, myalgia, coryza, and sore throat were very common and significantly different (p < ae ) than the rest of the population. it was reported a decrease in the ew coinciding with winter holidays (ew ). the epidemic curve was different in males compared to females during the winter holidays. discussion: school aged children got sick more than the rest of the population, although they presented less proportions of sari. however, comorbidities were decisive in order to present sari or death. the epidemic curve was different in males compared to females. through its analysis, the beneficial effect of school closure was observed, as long as children meet the recommendation to stay home. in april , different countries reported cases of influenza a h n ; mexico reported a high mortality rate associates with this disease. the world health organization (who) declared the phase influenza pandemic alert on june . several reports from different countries describe the behavior of the pandemic in school aged children. this group plays an important role in the transmission of influenza. in germany, during the summer peak, pandemic hardly spread within this group. this might be explained by the timing of the summer school holidays, which started between ew and . since mid october, after the autumn holidays, the school-aged children began to be more affected, and the proportion increased from % in the initiation period to ae % in the acceleration period. in australia, % of h n cases were school aged children ( - years), with a median age of years ( % of cases were aged - years and, and % between - years). in canada, the infection rate was highest in this group. in chile, the incidence rate was ⁄ inhabitants, although in general they had mild desease. school closure can operate as a proactive measure, aimed at reducing transmission in the school and spread into the wider community, or reactive, when the high levels of absenteeism among students and staff make it impractical to continue classes. the main health benefit of proactive school closure comes from slowing down the spread of an outbreak within a given area and, thus, flattening the peak of infections. this benefit becomes especially important when the number of people requiring medical care threatens to saturate health care capacity. it has its greatest benefits when schools are closed very early in an outbreak, before % of the population falls ill. school closure can reduce the demand for health care by an estimated - % at the peak of the pandemic under ideal conditions, but too late in the course of a community-wide outbreak, the resulting reduction in transmission is likely to be very limited. policies for school closure need to include measures that limit contact among students when they are not in school. tucumán is placed in northwest argentina and has a total area of km . the population ( census, projection ) was inhabitants; of wich were - years old. the health system of the province is composed of sectors: public, private, and welfare. it has a total of health facilities with internement available and an average of & inhabitants. influenza-like illness (ili) has seasonal and endemic behavior in this province, as evidenced by past records from the national health surveillance system and influenza sentinel surveillance unit of the province. an increase of ili was reported in , with a peak in the ew . the objectives were: general objective to describe the behavior of the influenza a h n epidemic in school aged children from the province of tucumán, argentina. specific objectives • to explore the response to preventive measures by school aged population. • to assess the effect of the suspension of classes in this group. • to estimate the magnitude and severity of the disease. • to observe the effect of co-morbidities in this group. a cross-sectional study was executed from may to december . data were gathered through mandatory reporting forms, wich were collected from all public and private health centers. inclusion criteria: patients with compatible symptoms with influenza a; school aged children - years old. exclusion criteria: patients treated with antiv- iral medication as prophylaxis, respiratory pathologies which did not justify specific antiviral medication, and incomplete forms. • suspected case of ili: cases considered by clinical criteria (fever higher than °c, cough or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nauseas or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea). • confirmed case: person with positive laboratory results for influenza a h n or unspecificed influenza a (by laboratory results through rrt-pcr or immunofluorescence techniques). • dismissed case: by negative or different laboratory results, or different clinical evolution. • comorbidities: chronic illnesses like arterial hypertension, diabetes, asthma, recurrent obstructive bronchial syndrome (robs), smoking, chronic obstructive pulmonary disease (copd), immunosuppression, hiv ⁄ aids, cancer, nephropathy, obesity; pregnancy was also considered. data were analyzed using epi software (epi infoÔ cdc, atlanta, eeuu). rates were calculated and rr was estimated with their respective confidence interval (ci). population data were taken from national census projections. an estimation based on the same census was used for the group between and years old. to observe the effects of other co-variables, the or and their ci were calculated. logistic regression was used to evaluate the influence of the comorbidities. x was used to compare proportions. respiratory samples (nasopharyngeal and faryngeal swabs) were obtained. they were analyzed at influenza sentinel surveillance unit of tucumán, and ⁄ or sent to national reference laboratory dr. c. malbrán (rt-pcr). from all notifications ( ), were cases of ili in the group aged between and years old, % ( ⁄ ) of which were males. the incidence rate was ae , and it differed according to the sexes: ae males and ae females per thousands of inhabitants (p < ae ). of all laboratory samples ( ) % were confirmed as influenza h n , % were confirmed as unspecificied influenza, and % were dismissed. the remaining percentage corresponded to the isolation of other viruses (parainfluenza, respiratory syncytial virus, and adenovirus). the school aged group had higher risk of getting sick, in relation to the rest of the population (rr ae [ % ci ae - ae ]), especially males (rr ae ) compared with females (rr ae ). the highest attack rate was observed in the capital of tucumán ( ⁄ inhabitants). according to the rest of the population, it looked like being school aged children meant a protective factor for presenting sari (severe acute respiratory infection) (or ae [ % ci ae - ae ], p < ae ). the lethality rate was ae ⁄ thousand. the risk of dying was low compared to other ages. persons with comorbidities had significantly higher risk of presenting sari (or ae [ % ci ae - ae ], p < ae ) and of dying (or ae [ % ci ae - ae ], p < ae ). respiratory comorbidities were the most fre- quent: asthma ae % ( ⁄ ) and % rors ( ⁄ ). the symptoms headaches, myalgia, coryza, and sore throat were very common and significantly different (p < ae ) than the rest of the population. if we compared the group aged - years with - years old, the epidemic curve of the first group showed a decrease in the ew , coinciding with winter holidays (ew ) (figure ). there was a slight increase in the tendency when classes began, but it showed a clear declination afterwards. the analysis of rates in school aged children by ew showed a reduction of ae % in males and ae % in females (p < ae ) at ew . however, after the first week of winter holidays, the curve in males had a significant increased to ae % compared to ew , reaching the highest weekly rate of the epidemic ( ⁄ inhabitants). the reopening of classes coincided with a significant decrease of the rate ( ae %), from to ae ⁄ inhabitants in ew (p < ae ). in females, the school closure coincided with a plateau-shaped curve, and the reopening with a significant decrease of ae % of the rate, from ae to ae in ew ( figure ). the school children got sick a lot more than the rest of the population, although they presented less proportions of sari. however, comorbidities were determined in order to present sari or death. symptoms like headache, myalgia, coryza, and sore throat were considered more conducting for the definition of cases in this population in tucumán. the epidemic curve was different in males compared to females during the winter holidays. the beneficial effect of school closure was observed as long as persons met the recommendations. the difference between males compared to females during winter holidays could mean that women would have carried out social distance recommendations much better, for example, remained at home. the significant reduction after the opening of classes is a factor to be considered as an effective intervention in the declining stage of the curve. here, we report pdmh n infection attack rate (iar) during the first wave of the pandemic. we used our iar estimates to infer the severity of the pandemic strain, including the age-specific proportion of infections that led to laboratory confirmation, hospitalization, intensive care unit (icu) admission, and death. [ ] [ ] [ ] [ ] part of these results are now available in ref. subjects of a community study, - years old between november and october , we conducted a cohort study of pediatric seasonal influenza vaccination and household transmission of influenza. one hundred fifty-one children aged - were recruited and provided baseline sera in november and december . between september and december a further children aged - were recruited and provided baseline sera for the second phase of the study. for this serologic survey, we tested the sera collected before the first wave and the sera collected after the first pandemic wave. written informed consent was obtained from all participants. parental consent was obtained for participants aged or younger, and children between the ages of and gave written assent. all study protocols were approved by the institutional review board of the university of hong kong ⁄ hospital authority hong kong west cluster. age-stratified data on virologically confirmed outpatient consultations, hospitalizations, icu admissions, and deaths associated with pdmh n from april to november were provided by the hong kong hospital authority (the e-flu database). since may , patients admitted with acute respiratory illnesses routinely underwent laboratory testing for pdmh n virus by molecular methods. sera were tested for antibody responses to a ⁄ california ⁄ ⁄ by viral microneutralization (mn). most individuals infected with influenza develop antibody titers ‡ : by viral microneutralization after recovery. we defined the pdmh n seroprevalence rate as the proportion of individuals who had antibody titers ‡ : . while mn antibody titers of ‡ are not by themselves conclusive evidence for pdmh n infection, we have assumed that the increase in cross-sectional seroprevalence between the pre-and post-first wave time periods are evidence of recent pdmhn infection. the iar was defined as the proportion of individuals infected by pdmh n during the first wave. the case-confirmation rate (ccr), case-hospitalization rate (chr), case-icu-admission rate (cir), and case-fatality rate (cfr) were defined as the proportion of pdmh n infections that led to laboratory-confirmation, hospitalization, icu admission, and death. due to containment efforts until june , all laboratory-confirmed cases were required to be hospitalized for isolation regardless of disease severity. as such, only surveillance data from june onwards were used to estimate severity measures. we estimated the iar as the difference between the prefirst-wave and post-first-wave seroprevalence rate. we used the estimated iar as the denominator for calculating the ccr, chr, cir, and cfr. we used an age-structured sir model with age classes ( - , - , - , - , and ‡ ) to describe the transmission dynamics of pdmh n in hong kong between june and november . we assumed that the mean generation time was ae days. using the age-structured transmission model, we estimated the following transmission parameters from the serial cross-sectional serologic and hospitalization data: (i) r o , the basic reproductive number; (ii) p and p , the reduction in within-age-group transmission for - and - years old during summer vacation (compared to school days during september-december ); (iii) d r , the average time for neutralization antibodies titer to reach ‡ : after recovering from infection; (iv) h a , the age-specific relative susceptibility with - years old adults as the reference group. we assumed non-informative priors for all parameters and used monte carlo markov chain methods to obtain posterior distributions of the parameters. sources of specimens: [ ] pediatric cohort study ( - april virological surveillance data suggested that the first wave of pdmh n in hong kong occurred from august to october . most of the laboratory-confirmed infections in this first wave occurred in individuals aged below years old accounting for > % of the lab-confirmed cases and hospitalizations, % of icu admissions, and % of deaths. taking into account a delay of - weeks for antibody titers to appear during convalescence, we found that these virological surveillance data were consistent with our serial cross-sectional seroprevalence data, which indicated a sharp rise in seroprevalence among the - years old from september to november and a plateau thereafter (data not shown). among individuals aged - years, the seroprevalence rates were similar across time between pediatric outpatient subjects and pediatric cohort study subjects (data not shown). similarly, for older age groups, the seroprevalence rates were largely similar between blood donor subjects and hospital outpatient subjects (except for the - years old in november-december). this provided some evidence that despite biases in our convenience sampling scheme, the resulting serologic data provided a reasonably representative description of seroprevalence in the community. the estimated pre-and post-first-wave seroprevalence rates and the corresponding iar estimates are shown in table . the severity estimates (ccr, chr, cir, and cfr) are shown in table . in summary, we estimated the iar was ae % among - years old, ae % among - years old, ae % among - years old, ae % among - years old, ae % among - years old, and ae % among - years old. overall, we estimated a population-weighted iar of ae % ( - %) among individuals aged - years through the first wave in hong kong. ccr were around ae - ae % among the - years old. chr were around ae - ae % among the - years old. cir increased from ae ( ae - ae ) per infections in - years old to ( ae - ) per infections in - years old. cfr followed a similar trend with ae ( ae - ae ) death per infections in - years old to ae ( ae - ) deaths per infections in - years old. compared to children aged - , adults aged - were ae and times more likely to be admitted to icu and die if infected. the best-fit age-structured transmission model gave the following parameter estimates: . the basic reproductive number was ae ( %ci, ae - ae ). . it took an average of ( - ) days for recovered individuals to develop neutralization antibody titer ‡ : . table . estimated age-specific proportions of individuals with pdmh n infections that were laboratory-confirmed, were hospitalized, were admitted to icu, and died. case-icu and case-fatality rates are expressed as number of episodes per infections . compared to - years old, - years old children and - teenagers were ae ( ae - ae ) and ae ( ae - ) times more susceptible to pdmh n infection, respectively. . compared to - years old, - years old older adults and - years old elderly were only ae ( ae - ae ) and ae ( ae - ae ) times as susceptible as the - years old, respectively. . compared to the school period during september-december , summer vacation reduced within-agegroup transmission by % ( - %) among - years old, but only % ( - %) among - years old. using computer simulations, we estimated that if preexisting seroprevalence is zero, real-time serologic monitoring with about specimens per week would allow accurate estimates of iar and severity as soon as the true iar has reached % (data not shown). we estimated that during the first wave in hong kong, ae % of school-age children and ae % of individuals aged - were infected by pdmh n . a serologic survey in england found similar iars in london and the west midlands. both studies highlight the importance of including serologic surveys in pandemic surveillance. the geographically compact and well-mixed population in the urban environment of hong kong permits some degree of confidence in the validity of our iar and severity estimates. the completeness of the pdmh n surveillance system, welldefined population denominator, and our large-scale serologic survey provide accurate numerators and denominators for the severity measures. we based severity estimates for pdmh n on the iar as the denominator. in most previous studies of pdmh n severity, the denominator was clinical illness attack rate, which depends on the probability of symptoms as well as medical care seeking behavior of the population. , our estimated cirs and cfrs are broadly consistent with presanis et al.'s 'approach ' severity estimates, but around - times lower than their 'approach ' estimates. our estimates of chr are - times higher than their approach estimates of symptomatic chr. however, the hospitalization-death ratio was ⁄ = as of november in hong kong, but ⁄ = as of june in new york, suggesting that the clinical threshold for admission in terms of disease severity at presentation may have been lower in hong kong. our study has a number of limitations. first, we have used antibody titers of ‡ : by viral microneutralization as an indicator of recent infection, correcting for pre-existing seroprevalence levels, but this may lead to underestima-tion of the iar if some infections led to antibody titers < : , or if some individuals with baseline titers ‡ : were infected. second, our estimates of the iar would be biased upwards if infection with other circulating influenza viruses led to cross-reactive antibody responses resulting in antibody titers ‡ : . however between august and october , % of influenza a viruses detected in hong kong were pdmh n , and only % of isolated viruses were seasonal h n viruses. third, a minority of severe illnesses associated with pdmh n infection might not be identified by molecular detection methods, for example if admission occurred after viral shedding from the primary infection has ceased, in which case we may have underestimated the disease burden of pdmh n . finally, our analyses are primarily based on seroprevalence among blood donors to the hong kong red cross, who may not be representative of the whole population. we do not have detailed data on donors to compare their risk of infection with the general population, but we did observe very similar seroprevalence rates across the three groups of subjects in our study, i.e., blood donors, hospital outpatients and participants in a community cohort (data not shown). in conclusion, around ae % of the population aged - and half of all school-age children in hong kong were infected during the first wave of pandemic h n . compared to school-children aged - , older adults aged - , though less likely to acquire infection, had ae and times higher risk of icu-admission and death if infected. thus, although the iar of pdmh n is similar to that of a seasonal epidemic, the apparently low morbidity and mortality of pandemic influenza (h n ) appears to be due to low infection rates in older adults who had a much greater risk of severe illness if infected. the reasons why older adults appear relatively resistant to pdmh n infection even though they appear to lack neutralizing antibody remains unclear. if antigenic drift or other adaptation of the pdmh n virus allows these older age groups to be infected more efficiently, the morbidity and mortality of subsequent waves of the pandemic could yet become substantial. and the national institute of allergy and infectious diseases, national institutes of health (contract no. hhsn c; adb no. n -ai- ). the funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish. bjc reports receiving research funding from medimmune inc., a manufacturer of influenza vaccines. the authors report no other conflicts of interest. some data presented in this manuscript were previously published in wu et al. it is well known that a primary goal of vaccination is to generate immunological memory against the targeted antigen to prevent disease in a vaccinated person. this ensures an accelerated immune response in the event of future contact with the pathogenic agent, such as a virus. therefore, it is very important to develop criteria for the assessment of vaccine immunogenicity by measuring both t and b memory cell levels from the vaccinated host. in contrast to inactivated influenza vaccines, live attenuated influenza vaccines (laivs) have been shown to provide primarily cellular and local immune responses. - to date, however, the hemagglutination-inhibition (hai) test (i.e. detection of serum antibodies) remains the method widely accepted for evaluation of an influenza vaccine's immunogenicity. improved understanding of the role of cellular and mucosal immunity and their contribution to protecting against severe illness caused by influenza infection has emphasized the need to reconsider methodologies used to evaluate the immunogenic impact of various influenza vaccines. such new assays need to include methods to measure local antibodies and virus-specific lymphocytes, especially in the case of live attenuated influenza vaccines, because of their potential to induce such broad-based immune responses. the aim of this study was to assess the ability of new russian pandemic laivs a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) ('ultragrivak,' registered ae ae ) and a ⁄ ⁄ california ⁄ ⁄ (h n ) ('influvir,' registered ae ae ) to induce memory t-cells in naïve human subjects and to compare results to levels of hai antibodies from each subject. a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) laiv was generated by : genetic reassortment of low-pathogenic avian influenza virus a ⁄ duck ⁄ potsdam ⁄ - (h n ) and master donor strain a ⁄ leningrad ⁄ ⁄ ⁄ (h n ). , the vaccine strain contains ha gene from avian virus, as well as na and internal genes from the master donor virus. a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv was generated by classical ( : ) reassortment of a ⁄ california ⁄ ⁄ (h n ) with the master donor virus. the vaccine strain contains ha and na genes from a 'wild-type' h n strain and internal genes from the master donor virus. participants were aged to years and were without contra-indication of laiv vaccination. immunogenicity of a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) laiv was assessed in ten vaccinated persons and ten volunteers inoculated with a placebo (sterile physiological saline solution). immunogenicity of a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv was estimated in vaccinated volunteers and nine volunteers inoculated with placebo. viruses or placebo were administered intranasally twice with an interval period of days at a dosage of ae ml per nostril for each vaccination. physical examination, venous blood and nasal swab samples were collected at four time points during the study: (i) before vaccination (day ); (ii) days after first vaccination (day ); (iii) days after the second vaccination (day ); and (iv) weeks after the second vaccination (day ). serum hai antibodies were measured by standard hai assay using % human red blood cells. test antigens for the assay were a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) or a ⁄ ⁄ california ⁄ ⁄ (h n ) to match the appropriate vaccine antigen. local iga antibodies in nasal swabs were evaluated by elisa using whole purified a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) or a ⁄ ⁄ california ⁄ ⁄ (h n ) viruses at hau per ae ml for absorption to elisa plates. endpoint elisa titers were expressed as the highest dilution of sera that gave an optical density (od) greater than twice the mean od of six negative controls in the same assay. percentages of virus-specific cd + cd + ifn-c + and cd + cd + ifn-c + peripheral blood memory cells were determined using a flow cytometry iccs assay performed by the published method. pbmcs were prepared with standard histopaque- gradient centrifugation from heparinized whole blood. wilcoxon matched pair test, mann-whitney u test and the students t-test were used for statistical data analysis. prior to the first vaccination (day ), gmts of hai antibodies to a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) and a ⁄ ⁄ california ⁄ ⁄ (h n ) laivs were ⁄ ae and ⁄ ae , respectively. in addition, gmts of siga against these specific antigens from nasal swabs were ⁄ ae and ⁄ ae , respectively. no hai antibody titers greater than : were observed prior to vaccination. background levels of virusspecific t-cells varied significantly within groups. mean levels of virus-specific cd + ifnc + cells were ae % to a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) and ae % to a ⁄ ⁄ california ⁄ ⁄ (h n ). for cd + ifnc + cells, initial levels were ae % and ae %, respectively. thus, background levels of virus-specific antibodies were low, but prior vaccination or virus exposure in some volunteers produced some pre-existing levels of t cells, thus they were not absolutely immunologically naïve in this sense. preexistence of h n -crossreactive antibodies and t-cells has been observed previously. [ ] [ ] [ ] effect of vaccination antibody immune responses both influenza a (h n ) and influenza a (h n ) laivs stimulated production of serum hai antibodies and local iga antibodies in nasal swabs. following the first vaccination with influenza a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ (h n ) laiv, % percent of volunteers exhibited seroconversion of hai antibodies; after the second vaccination, % of volunteers exhibited seroconversion. after the first vaccination, a % conversion rate of siga was observed; after the second vaccination, % showed conversions in levels of siga. the first vaccination with a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv showed ae % of hai antibodies seroconversions vaccination, and % seroconversion after second vaccination. for local siga, those results were ae % and ae % following the first and second inoculation, respectively. figure summarizes cellular immune responses observed in the vaccinated versus the placebo group. after the influenza a (h n ) laiv inoculation, significant differences in both cd and cd ifnc-producing t-cells were observed at day after the second vaccination (d ). these data indicate that healthy young people who never received such avian influenza vaccines and were not exposed to h n wild-type viruses were able to respond to the live attenuated h n influenza vaccine. after the first influenza a (h n ) laiv vaccination, reliable increases were observed in cd + cells only. after the second vaccination, increases in both cd + and cd + fold changes were significantly higher in vaccinated volunteers compared to the placebo group. it is noteworthy that cellular immune responses (cd + and cd + cells) were more marked in the a ⁄ ⁄ california ⁄ ⁄ (h n ). considering the long-term circulation of h -subtype viruses among humans in contrast to the novelty of h viruses, such a result would be expected. similar data were also observed following vaccination with the h n laiv. after first vaccination, the percent of people with notable increases in virus-specific cd + and cd + t-cells was % and % to h n and % and % to h n , respectively. after the second vaccination, these results were % and % to h n and % and % to h n , respectively. importantly, a significant number of vaccinated volunteers without remarkable increases ( ‡ -fold) in hai antibodies had notable increases in cd + and ⁄ or cd + memory cells. the percent of people with notable increases in virus-specific t cells after the second vaccination among hai()) volunteers was % and % to h n and h n , respectively. these results indicate that laivs were able to induce broadly responsive, key antiviral immune responses that would not have been detected by the hai assay alone. thus, it can be deduced that hai data alone fails to reveal important broad and specific immune responses to laiv. consequently, the hai test alone is not suitable for assessment of laiv immunogenicity. furthermore, vaccination with h n laiv was able to induce cross-reactive memory t-cells to a seasonal vaccine strain, a ⁄ ⁄ solomon islands ⁄ ⁄ (h n ) ( table ) . reliable increases to a (h n ) were observed in up to % of volunteers. there was an inverse dependence between levels of memory t cells before and after vaccination. authors are thankful to path for the financial support of these studies. we are also thankful to jessica d'amico and dr. rick bright for their editorial review. options for the control of influenza vii background: increased susceptibility of older populations to secondary bacterial pneumonia-like infections following influenza infection has been well documented. recent evidence in mouse models suggests that this increased risk from secondary bacterial infection occurs through a desensitization of the innate immune response. this recent finding, however, does not account for potential differences in immune responsiveness due to age. materials and methods: to address this parameter, we used three age groups (aged, adult, and young mice) to evaluate the role of age in influenza-mediated vulnerability to secondary bacterial challenge with pseudomonas aeruginosa. all mice were evaluated for multiple parameters including: (i) survival; (ii) lung bacterial load; (iii) total lung protein content; (iv) immune cell infiltration; (v) cytokine ⁄ chemokine expression; and (vi) toll-like receptor (tlr) rna expression profiles. results: prior challenge with influenza contributed to aberrant cytokine ⁄ chemokine profiles and increased lung cellular infiltrate in response to secondary bacterial infection across all age groups, supporting a critical role for influenza infection in the alteration of immune responses to other pathogens. also similar to human influenza, these changes were exacerbated by age in mice as demonstrated by increased bacterial load, mortality, and total lung protein content (an indicator of lung damage) after p. aeruginosa challenge. conclusions: these data support a potential role for virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and the increased susceptibility of influenza virus infected mice to secondary bacterial infection. the understanding of the complex interaction of host and pathogen -and the role of age -in human influenza is critical in the development of novel therapeutics and improved vaccine approaches for influenza. our results support further examination of influenza-mediated alterations in innate immune responses in aged and non-aged animals to allow elucidation of the molecular mechanisms of influenza pathogenesis in humans. there is considerable evidence in the clinical literature to support the role of influenza infections with an enhanced risk for secondary bacterial pneumonias. [ ] [ ] [ ] given the increased pneumonia-related morbidity and mortality in both the young and elderly populations, there is rationale for gaining a deeper understanding as to the systemic changes in the pulmonary microenvironment. although there are some recent reports that account for some of the molecular mechanisms at work in this disease process, there is a paucity of experimental evidence that considers the potential effects of age. developmental changes in the immune system that occur in the aged environment have been well documented with regard to senescence of the adaptive immunity, global changes in myeloid cell function, and the establishment of a general pro-inflammatory state. , the aim of this work was to provide evidence for the contribution of the aged immune environment to the pathology of influenza mediated secondary bacterial infections. animals used in this study were housed under conditions approved by tulane university's institutional animal use and care committee. female balb ⁄ c mice used in these studies were divided into three age groups: aged ( months old), adult ( months old), and young ( months old). each age group was subdivided into two groups: influenza infected and naïve (control). mice were infected by the intranasal route with · pfu of mouse-adapted influenza a ⁄ pr ⁄ ⁄ . clinical disease was measured by body weight changes over a week period post influenza challenge, and recovery was determined as return to pre-infection weight. all mice were subsequently challenged intransally with · cfu pseudomonas aeruginosa strain pao . twenty-four hours post-pseudomonas challenge, bal with sterile pbs was performed on all mice in all groups. total rna from the cellular fraction was pooled from three experimental animals from each group. tlr mrna was detected by qrt-pcr, where expression levels were determined as relative to b-actin mrna levels. cdna was synthesized from total cellular rna from bal samples using iscript cdna synthesis kit (biorad). pcr reactions were composed of ae lg cdna forward and reverse primers according to optimized conditions and ae ll of · syber green icycler supermix (biorad), in a total vol-ume of ll and were run using a biorad icycler utilizing melting point determination. primers and concentrations used in this study included: mus_tlr f: tgctttcct-gctggagattt- nm, mus_tlr r: tgtaacgcaac agcttcagg- nm, mus_tlr f: atatgcgcttcaa tccgttc- nm, mus_tlr r: caggagcatactggt gctga- nm, mus_tlr f: ggcagcaggtggaattg tat- nm, mus_tlr r: aggccccagagttttgttc t- nm, mus_tlr f: ctggggacccagtatgctaa- nm, mus_tlr r: acagccgaagttccaagaga- nm, mus_tlr f: ggagctctgtccttgagtgg- nm, mus_tlr r: caaggcatgtcctaggtggt- nm, mus_ b-actinf: agccatgtacgtagccatcc- nm, mus_b-actinr: ctctcagctgtggtggtgaa- nm. as a measure of protein leakage into the alveolar space, total protein content in each bal was measured by bca assay of each supernatant fraction according to manufacturer's instructions (pierce). cytokine and chemokines levels were measured by multiplexed bead array (bioplex, biorad). immune cell characterization of bal was estimated by flow cytometry. lymphocyte populations were gated by forward versus side scatter and characterized as b cells (f ⁄ ) , cd + ) or t cells (cd b ) , cd + ). the myeloid population that is composed of macrophages, neutrophils, dendritic cells, and natural killer cells was enumerated by gating all but those found in the lymphocyte gate using forward versus side scatter plots. flow cytometry data was analyzed using flojo software (treestar). statistical analysis, where appropriate, was performed using a two-way analysis of variance (age versus influenza infection status) supported by bonferonni's correction for multiple comparisons. a recent finding by didierlaurent, et al., described an influenza mediated desensitization of tlr function as a primary contributor to an increase in bacterial burden when challenged after resolution of the primary influenza infection. this finding, however, was obtained using animals that were - weeks of age, where our study included two cohorts of older mice ( months and months). using whole protein content of the bal as an estimate of protein leakage into the lumen of the lung, we found elevated protein content in aged mice as compared to young and adult mice. in aged mice, a slightly lower total lung protein when comparing influenza infected to protein in the bal from influenza naïve mice challenged with p. aeruginosa (table ) . supporting previously published studies showing a generalized pro-inflammatory cytokine environment in the aged immune system, we provide evidence for significantly (p = ae ) and an increase in ifnc (p = ae ) was detected. the decrease in gm-csf correlates well with a previous report that gm-csf is less prevalent in influenza resolved animals (table ). we also report a noticeable change in the immune cell populations with respect to b-cells, cd + t-cells, and the myeloid cell populations. there is a trend of increased prevalence in cd t-cells in the post-influenza environment across all ages. b-cell numbers also trend toward increase in influenza treated animals in young and adult animals; however, there is a noticeable decrease in the bcells in aged animals. across all age groups, there is a general decrease in frequency of cells that would normally make up the myeloid cellular fraction of the bal (macrophages, neutrophils, dendritic cells, and natural killer cells) ( table ). our study also shows, as cited by others, that toll-like receptor (tlr) gene expression in the post-influenza environment is decreased in cells found in the bal after both influenza and pseudomonas infection. our data support the previous finding of a reduced expression of tlr mrna in influenza-cleared mice when we measured tlr , , , and . only tlr showed differences with respect to age with young mice showing little or no detectable change in tlr mrna expression. our results show an increase in the expression across all tlrs examined in the aged mice group (table ) irrespective of influenza infection status. these data support earlier studies performed with adult mice that showed reduced tlr mrna expression in the post-influenza environment. this study also expands the current understanding of the potential role of age in influenza mediated bacterial infection-induced mortality. the impact of these alterations in the immune microenvironment across age groups and infection status is highlighted by the ability of bacterially challenged animals to clear infection. assessment of bacterial load in the lungs of p. aeruginosa challenged mice indicated a difference in young and adult mice if previously infected with influenza virus. in aged mice, both influenza challenged and influenza-naïve mice had higher bacterial loads and less variability when comparing within the age group, supporting the risk of age alone in susceptibility to bacterial pneumonia (table , figure ). taken together, these data support the potential role for both virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and increased the susceptibility to secondary bacterial infection that results from influenza infection in mice. these findings highlight distinct differences in the immune environment between age groups and thus reveal necessity for further examination as to the mechanisms of immunity across age with respect to current infection status. garnering a clearer understanding as to the complex interaction of host and pathogen with respect to age in influenza infections is central to the development of increased efficacy in vaccine and therapeutic strategies. prospective estimation of the effective reproduction background pandemic influenza a (h n ) virus (ph n ) emerged in early and rapidly spread to every continent. an urgent priority for international and national public health authorities was to estimate the transmissibility of the pandemic strain for situational awareness and to permit calibration of mitigation strategies. the basic reproductive number, r , is defined as the average number of secondary cases that index case generates in a completely susceptible population, and is a common measure of transmissibility. however, it is difficult to estimate r without an understanding of the degree of any pre-existing immunity in the population. the effective reproductive number, r, is defined as the average number of secondary cases that index case generates, and can be estimated over time (i.e. r t ). wallinga and teunis described a method to estimate r t based on illness onset dates of the cases while assuming that all secondary cases would have been detected, and cauchemez et al. extended the method to permit prospective estimation by adjusting for secondary cases that have not yet experienced illness onset at the time of analysis. we describe how the method can further be extended to account for reporting delays, allowing true real-time estimation of r t during an epidemic, and we illustrate the methodology on notifications of ph n and associated hospitalizations in hong kong. we obtained data on all laboratory-confirmed ph n infections ('cases') reported between may and november , to the hospital authority and center for health protection in hong kong collated in the eflu database. a subset of the cases was hospitalised. the database also included information on age, sex, illness onset date, laboratory confirmation date, and contact history (for the early cases). laboratory-confirmed ph n infection was a notifiable condition throughout our study period. we extended existing methods for estimating r t over time to allow for reporting delays between illness onset and notification, and between illness onset, notification, and hospitalisation for those cases that were hospitalised, where the reporting delay distribution were estimated empirically from the data. we further extended the methodology to allow for imported cases (infected outside hong kong) contributing to the estimation of r t as infectors but not infectees. we used multiple imputation to allow for missing data on some symptom onset dates to make best use of all available data. we used a serial interval with mean (standard deviation) of ae ( ae ) days, and in sensitivity analyses, we used serial intervals with mean ae days and ae days. statistical analyses were performed in r version . . (r development core team, vienna, austria). in late april following the who global alert, hong kong initiated containment protocols to attempt to delay local transmission of ph n for as long as possible. these measures included screening at ports, airports, and border crossings, and enhanced surveillance for people with influenza-like illness, particularly for those who had recently returned from abroad. laboratory testing capacity was substantial due to heavy investment in local infrastructure following previous experiences with avian influenza a ⁄ h n in and severe acute respiratory syndrome in . laboratory-confirmed ph n cases were isolated until recovery, and their close contacts were placed under quarantine for days. imported cases were identified sporadically through may and early june . the first case of ph n not traceable to importation (i.e. a local case) was identified on june and triggered a change to mitigation phase measures. some containment measures, including isolation of cases, were continued until the end of june to allow a soft transition between containment and mitigation phases. as an immediate measure to try to reduce community transmission of ph n , all childcare centres, kindergartens, and primary schools were proactively closed for days (subsequently extended for another - days to summer vacation in early july). any secondary schools in which one or more confirmed ph n case was identified were reactively closed for days. on june the government opened eight designated flu clinics across the territory to provide free medical consultation for outpatients with influenza-like illness and free laboratory testing for ph n . these clinics resumed regular chronic disease services in mid-august, and laboratory testing and antiviral treatment was restricted to high risk groups in september. the various interventions are highlighted in figure (a), superimposed on the epidemic curve of laboratory-confirmed ph n cases and ph n -associated hospitalizations. around % of the cases were hospitalised, and this proportion increased somewhat towards the end of the epidemic. figure (b) shows the estimates of r t based on laboratory-confirmed ph n cases. the estimated r t peaked at ae on june , and fell below between june and july (which was within the school closure period). r t fluctuated between ae and ae through the school summer vacations in july and august, it subsequently increased to around ae - ae after schools reopened in september until the epidemic peaked in late september, and then fluctuated below as the epidemic declined. the trends in r t based on h n -associated hospitalizations were similar, although with wider confidence intervals due to the smaller number of events ( figure c ). the extension of the methods to allow for reporting delays avoided substantial bias in realtime estimates of r during the epidemic for the most recent days, and closely tracked the final estimates of r t . our results suggest that ph n may have had slightly lower transmissibility in hong kong than elsewhere. for example, estimates of r t were around ae - ae in new zealand and australia. lower transmissibility in hong kong has been associated with school closures in june and july followed by summer vacations from july through august. furthermore, in hong kong the influenza virus usually does not circulate after august, and therefore seasonality could also be a cause for the lower r t . on the other hand, the interventions applied during the mitigation phase, such as the widespread use of antiviral treatment in hong kong and the pre-existing immunity in the ageing population in hong kong, may also be associated with lower transmissibility. there are some limitations to our work. first, we only used aggregated data, and we did not consider the heterogeneity among the cases in terms of sex and age or other factors. therefore our estimates can only provide a snapshot of the overall trend, but limited information for any specific subset of population. secondly, we did not consider the possibility that cases might be infected in hong kong and exported to other countries, which could lead to slight underestimation of the transmissibility. one has to be careful in translating the estimated r t to the effectiveness of any specific interventions, as interventions may not be the only factor influencing the transmissibility; for example, a depletion of the susceptible population during an epidemic can also be a factor for the decline in r t . in conclusion, real-time monitoring of the effective reproduction number is feasible and can provide useful information to public health authorities for situational awareness and planning. in affected regions, laboratory capacity was typically focused on more severe cases, and changes in laboratory testing and notification rates meant that that case counts may not necessarily reflect the underlying epidemic. a useful alternative to case-based surveillance is surveillance of the subset of severe infections, for example hospital admissions, or icu admissions, and our results show that it was feasible to monitor ph n -associated admissions in real-time to estimate transmissibility. influenza antigenic cartography projects influenza antigens into a two or three dimensional map based on immunological datasets, such as hemagglutination inhibition and microneutralization assays. a robust antigenic cartography can facilitate influenza vaccine strain selection since the antigenic map can simplify data interpretation through intuitive antigenic map. however, antigenic cartography construction is not trivial due to the challenging features embedded in the immunological data, such as data incom-pleteness, high noises, and low reactors. to overcome these challenges, we developed a computational method, temporal matrix completion-multidimensional scaling (mc-mds), by adapting the low rank mc concept from the movie recommendation system in netflix and the mds method from geographic cartography construction. the application on h n and pandemic h n influenza a viruses demonstrates that temporal mc-mds is effective and efficient in constructing influenza antigenic cartography. the web sever is available at http://sysbio.cvm. msstate.edu/antigenmap. as a segmented, negative stranded rna virus, influenza virus is notorious for rapid mutations and reassortments. the mutations on the surface glycoproteins (ha and na) of influenza viruses are called antigenic drifts, and these antigenic drift events allow the virus to evade the accumulating immunity from previous infection or vaccination and lead to seasonal influenza epidemics. a reassortment event with a novel influenza antigen may result in antigenic shift and cause influenza pandemic. for instance, the h n pandemic virus is a reassortant with a swine origin ha antigen. vaccination is the primary option for reducing the effect of influenza, and identification of the right vaccine strains is the key to development of an effective vaccination program. the antigenicity of an optimal vaccine strain should match that of the epidemic strain. in influenza surveillance program, the influenza antigenic variants are generally identified by the immunological tests, such as hemagglutination inhibition (hi) assay, microneutralization (mn) assay, or elisa. these immunological assays measure the antigenic diversity between influenza viruses by comparing the reaction titers among the test antigens and reference antisera. however, data interpretation of the data from these assays is not trivial due to the embedded challenges such as data incompleteness, high noises, and low reactors. by mimicking geographic cartography, influenza antigenic cartography projects influenza antigens into a two or three dimensional map using immunological datasets. antigenic cartography can simplify the data interpretation, and thus, facilitate influenza antigenic variant identification. recently, we developed a novel computational method, temporal matrix completion-multidimensional scaling (mc-mds), in antigenic cartography construction. in this paper, we described the details of temporal mc-mds, especially the original concepts introduced in this method, and how they can achieve the robustness in antigenic cartography construction. our method included two integrative steps: it first reconstructs the hi matrices using low rank mc method, and then generates antigenic cartography using mds with a temporal regularization. the mc concept was adapted from the movie recommendation system in netflix and the cartography concept from geographic cartography. in , netflix, an online dvd and blu-ray disc rentalby-mail and video streaming company, held a -year netflix prize contest (http://www.netflixprize.com/) on computational methods for improving its recommendation system. in its recommendation system, netflix collected the rating data from the individuals. based on his or her renting history and the ratings in the systems (e.g., from evaluators and other renters), netflix recommendation system suggests certain movies to a renter. apparently, no individuals would be feasible to provide ratings for all of the movies, as it will take hundreds of years for a single person to rate over movies available from netflix. thus, the resulting rating data is an incomplete matrix, and it can be as sparse as less as %. the challenge in netflix recommendation system is a classic mc problem. [ ] [ ] [ ] [ ] [ ] as the inspiration of netflix prize contest, many efficient low rank mc algorithms were developed, for instance, opt-space, svt, cf, bellkor, pf, and fwls. eventually, the team bellkor's pragmatic chaos won this contest. their methods combines nonlinear probe blending and linear quiz blending to come up with a predictor bigchaos. matrix completion estimates the unobserved values based on the observed values. the users can refill the missing data without repeating the experiments. furthermore, mc will help reduce the noises in the data, for instance, those biases by different individuals performing experiments. in influenza antigenic characterization, hi assay is a commonly used assay for antigenic analysis, since hi assay is relatively economic and easy to perform. however, hi is labor intensive, and it is almost impossible for any individual lab to complete the hi assays for all pairs of antigens and antisera during influenza surveillance. in addition, both testing antigens and the reference antisera are dynamic. for instance, in seasonal influenza surveillance, generally only contemporary antisera are used in experiments. thus, we will have to integrate multiple hi tables in order to evaluate the overall antigenic changes for influenza vaccine strain selection. the resulting hi tables will be incomplete, and the observed entries in the integrated hi data can be as less as %. the completion of this matrix can be formulated as a typical mc. briefly, given the combination of hi matrix with m antigens and n antisera, the hi matrix can be represented as m m·n = (m ij ) m·n , where m ij denotes the hi values from the reaction between testing antigen i and antiserum j. the low rank mc assumes that both antigen and antiserum can be embedded into a low rank space. to be specific, the low rank mc method is to seek matrix u m·r , v n·r and a diagonal matrix r r·r , where m = u m·r r r·r (v n·r ) t . in order to achieve this goal, the optimization formulation has been employed, which can be represent as following, where e denotes the observed entries in hi matrix and g(x) is a regularization function. the eqn ( ) is the standard format of a low rank mc formulation. the geographic cartography is a common technique to display the cities and their geographic distances in a map. this cartography can be generated using mds based on a geographic distance matrix. figure (a) shows the antigenic cartography generated using a distance matrix with seven cities, and figure (b) is a map for comparison. as an analog of geographic cartography, the influenza antigenic cartography maps the influenza antigens into a two or three dimensional map based on the distance matrix generated using immunological data. this incomplete matrix can be filled through mc algorithm discussed in section mc and netflix. low reactors, non-random date incompleteness, and temporal model generally, three types of data are present in a combined hi matrix: high reactor, low reactor, and missing values. among these three data types, high reactors are the most reliable data points. the low reactors are those values present in the hi matrix as ''equal to or less than a threshold h'', where h can be , , , or . low reactors have similar values in the affinity dataset but could be from different binding settings. these low reactors are present due to the detection limits of biotechnology, and they are not reliable. both these missing values and low reactors make it very difficult to analyze and interpret antigenic correlations amongst tested antigens and reference antigens. to our best knowledge, none of the existing mc method can handle the threshold values. in addition, the non-random incompleteness of influenza immunological datasets generates an additional challenge in traditional mc methods, which are based on the assumption that the observed values are randomly distributed among the matrix. in a typical combined antigenic hi data, most of the off-diagonal entries are missing values or low reactor values. in order to overcome the above issues, we incorporated a regularization function into the eqn ( ), where this indicator function is only valid for those entries with low reactor values. an alternating gradient decent method is applied to solve the optimization problem in eqn ( ) . in addition, a temporal mds method is proposed to project the antigens into a or dimensional map. x where d ij is the average distance between virus i and virus j, t i is the isolation year of virus i, d ij is the distance between virus i and virus j in cartography, d ac i is the distance between virus a and center of group i, and d c i c j is the distance between the centers of group i and group j. all the parameters are tuned by cross validation. we named this method as temporal mc-mds. by applying temporal mc-mds method in an h n dataset, low reactors. figure (a) is a three-dimensional influenza antigenic map based on this data by using mc-mds method. the reported clusters (hk , en , vi , tx , bk , si , be , be , wu , sy , and fu ) were displayed in the core of a spiral s-shape, and bk and be are located at the turning point of this s-shape. however, the antigenic distances between some viruses are incorrect. for example, the distance between hk and fu in the projection is ae units, which is close to the distance between hk and bk ( ae units). the main reason leading to those inaccurate distances is the unique distribution of hi datasets described in section . . in comparison, with the temporal model, not only the viruses in clusters have been clearly separated, but also the antigenic distances between each cluster are proportional to their isolation time interval. in this updated cartography ( figure b ), the antigenic distance between hk and fu is ae units, where the distance between hk and fu is ae units. this result suggested that the temporal information is critical for antigenic cartography construction for immunological datasets spanning a long time period. the hi data from seasonal influenza surveillance belong to this category. for seasonal influenza virus ⁄ pandemic influenza viruses within a short time span, the temporal model is probably not necessary, as there is lack of long-term immunological pressure present in the population. figure (c) is an antigenic cartography generated using a hi dataset with h n influenza viruses spanning from april of to june of . this map demonstrates that there is lack of antigenic drifts during the first wave of this pandemic influenza as all of these viruses are mixed altogether. our limited studies on h and h avian influenza viruses suggested the temporal model is not needed for avian influenza viruses. however, extensive studies are required to investigate whether there is any special data structure present in this type of data. in this study, we described in details the concepts and applications of new computational method, temporal mc-mds for influenza antigenic cartography construction. we formulate the influenza cartography as two integrative steps: low rank mc problem from the concept of netflix movie recommendation system and mds from geographic cartography construction. in order to handle two additional challenges, including low reactor and non random distribution of antigenic data, a temporal model is incorporated into mc-mds as temporal mc-mds. our applications demonstrated that temporal mc-mds is effective in constructing influenza antigenic cartography. the three dimensional antigenic cartography for a ⁄ h n seasonal influenza virus without temporal model, and the antigenic clusters were defined in ref. [ ] ; (b) the three dimensional antigenic cartography for a ⁄ h n seasonal influenza virus with temporal model; (c) the two dimensional antigenic cartography for a ⁄ h n pandemic influenza without temporal model, and these viruses were labeled in shape by the corresponding month for them to be detected. one grid is corresponding to a twofold change in hemagglutination inhibition experiment. the mechanisms driving the three waves of infection and mortality in the uk in - are uncertain. although the circulation of three distinct viruses could have generated three waves of infection, the virological evidence required to prove or disprove this hypothesis is lacking. social distancing, an alternate mechanism for generating fluctuations in the effective susceptible pool and therefore explaining multiple waves of infection, , was not generally imposed in the uk as it was in the us and australia. we are therefore motivated to explore the possible role of continual population-level changes in the average protective response against the circulating virus in generating a multi-wave pandemic, within a biologically motivated deterministic model for influenza transmission. the nature and duration of protection against further infection following recovery from influenza is uncertain and depends on the mode and tempo of viral evolution, as well as the response of the cellular and humoral arms of the adaptive immune system. for a given seasonal ⁄ pandemic strain, memory b-cells may generate a specific antibody response in a portion of the adult ⁄ elderly population, depending on the exposure to related antigenic sub-types. however neutralising antibodies are unlikely to be a widespread immunological response to a novel (pandemic) strain. memory t-cells which recognise conserved internal viral proteins may be a more common mechanism for protection; the generation of very high levels of cytotoxic cd + t-cells potentially facilitates rapid viral clearance, , and lower levels of cd + t-cells perhaps provide partial protection. in this work we explore key drivers of multi-wave pandemics within phenomenological models that incorporate different immune response mechanisms building on existing models , incorporating the role of evolving population-level protection in multi-wave pandemics. we use weekly reports of influenza mortality rates for five administrative units in the uk (blackburn, leicester, newcastle, manchester and wigan) where records from block censuses instigated by local medical officers to record the cumulative incidence of reported symptoms in each wave in a sample of or more households are also available. the symptom reporting data allows us to estimate the case fatality rate and thus use the mortality time series to constrain our transmission model. furthermore, the incidence of individuals reporting symptoms in multiple waves provides information about the acquisition and loss of immunity. we extract the death rate and symptomatic (re)infection rates predicted by our model prevalence for a given set of parameters and estimate a likelihood-based on a comparison to all the death and cumulative reported incidence data assuming a negative binomial error distribution. we utilise monte carlo markov chain (mcmc) methods with parallel tempering algorithms to maximise this likelihood and obtain parameter estimates. parallel tempering -which concurrently searches for maximal likelihood parameter solutions on a set of scaled likelihood surfaces -allows for relatively rapid exploration of the parameter space. we use bayesian information criteria (combined with qualitative assessment of biological plausibility) to aid model selection. we have implemented a deterministic compartmental transmission model, which allows for a variety of phenomenological modes of protection against the pandemic virus. to facilitate this, we stratify the population into two groups; the 'experienced' population (stratum ) who have had been exposed to an influenza virus and the 'naive' population (stratum ) who have not. in each stratum, i hosts may be classified as either susceptible s i , exposed e i and e i , having (recovered from) a symptomatic i i (r i ), or asymptomatic a i (ra i ) infection. note that the states tq i , tq i , e i , t i , and t i are included so that the hosts move between the key epidemiological states with a peaked (rather than exponential) distribution of waiting times. hosts in the experienced stratum may exhibit reduced susceptibility, infectiousness, and symptomatic proportion compared to naive hosts, parameterised by e i , e s , and e a , respectively; however note that depending on the model parameters, there may be fully susceptible hosts within the experienced stratum. in addition, we assume homogeneous population mixing and a constant basic reproduction number r with the force of infection: modulated by a sinusoidal seasonal term with amplitude b with phase chosen to maximise transmission in the winter season. here n is the total population size, and x e is the initial fraction in the experienced strata. the proportion of symptomatic cases a and the case fatality rate l are permitted to vary from wave to wave (and given indices , or accordingly). the transmission dynamics is described by the following set of coupled ordinary differential equations. where s in, = p utq i and s in, = in order to divert recovered infectious hosts from the naive stratum into the experienced stratum. the probabilities of gaining permanent protection are q = q and q = . the latent exposed period is fixed to be c = ⁄ ae days, and the rate of recovery is parameterised by m = ⁄ t inf , where t inf is the infectious period. hosts with prior sterilising protection begin in q and move into s at rate u q = ⁄ t wq . recovered hosts (r i ) migrate back to s at a rate u = ⁄ t w . the state p contains hosts with permanent protection. the modes of protection captured in this model are: i. permanent prior protection (beginning in state p ), ii. waning prior protection (beginning in state q ), iii. permanent acquired protection with probability q (moving into state p ), iv. waning acquired protection with probability ) q, and, v. partial prior protection (beginning in state s ) resulting in reduced infectiousness (e i ), susceptibility (e s ), and symptomatic proportion (e a ). in the context of this model, 'permanent' protection refers to protection which lasts for the duration of the epidemic. here we explore the results of parameter fitting to two models which differ in the nature of the assumed pre-existing protection in the community at the beginning of the pandemic. protection hypothesis assumes that the prior protection is sterilising but temporary, whilst protection hypothesis assumes that the prior protection is partial but permanent and may act on susceptibility, infectiousness, and ⁄ or asymptomatic proportion. each model allows waning acquired protection and for a proportion q of the experienced population to gain permanent protection following infection. fitted parameters common to each model are t inf , b , q, t w , a, l and the proportion beginning in p x i . prior protection hypothesis : sterilising, waning prior protection we fix x e = and fit for q (t = ) ⁄ n and t wq so that protective modes i, ii, iii, and iv are enabled ( figure ). it is important to note that due to the slow convergence of the mcmc chains, we cannot guarantee that our parameter estimates correspond to the global minimum. furthermore, parameter estimates can only be meaningfully interpreted for good fits to the data. due to the prediction of a fourth (unobserved) wave for the model fit to blackburn, we do not report these parameter estimates here. the fits to the leicester data are generated with the parameter set r = ae , a = ae , a = ae , a = ae , t w = ae years, t wq = ae years, we fix q (t = ) ⁄ n = and fit for x e , e a , e i , and e s so that protective modes i, iii, iv, and v are enabled (figure ) . the parameters corresponding to the fit in figure for leicester are r = ae , a = ae , a = ae , a = ae , t w = ae years, p (t = ) ⁄ n = ae , s (t = ) ⁄ n = ae , b = ae , t inf = ae days, q = ae , e a = ae , e i = ae , and e s = ae . our model with protection hypothesis -which, similarly to the model discussed in ref. [ ] , assumes that a sub-population has waning sterilising prior protection -is able to generate multiple waves of infection via the continual replenishment of s from an initially large proportion (over %) of hosts with prior protection in q combined with the waning of acquired immunity in around % of cases on a time-scale of months. disease severity as measured by symptomatic proportion increases from % in the first wave to above % for the second and third waves. over a quarter of the population are initially permanently immune, and a large r value of ae drives transmission in the remaining population. protection hypothesis -which assumes that prior protection offers partial susceptibility and ⁄ or reduced infectiousness or symptomatic disease -performs slightly more poorly; the fit to the leicester data has an inferior likelihood (although the mortality data only likelihood is a little larger), despite the higher dimensionality of the model. nevertheless, the model fit still mirrors many characteristics of the data, particularly for leicester. we note that for this model, a is very near the lower limit, corresponding to ubiquitous exposure in the first wave. in this scenario, refuelling of the susceptible pool to generate secondary and tertiary waves is still possible due to a shorter waning time of acquired protection (well within months) and a lower probability of gaining permanent protection following infection, when compared with the parameter estimate for hypothesis . the parameter estimates suggest that approximately % of the population initially experiences reduced disease severity (e a $ ae ), but similar susceptibility and infectiousness. a larger value for r $ ae is required to drive transmission despite low numbers beginning in p , due to the large number of hosts who acquire temporary or permanent immunity early on in the pandemic. it is clear that, at least mathematically and perhaps biologically, there are multiple possibilities for the structure of population-level protection which are compatible with the generation of multiple pandemic waves. however, whilst the models considered here are able to explain the observed mortality and reinfection data for some patterns of infection and mortality (e.g. leicester), they are not consistently able to reproduce a pandemic which dies out after three waves across the connected populations we are studying (e.g. for blackburn). it is challenging to construct a deterministic model for the spread of disease within multiple locations in the uk in , which assumes homogeneous mixing without modulation of the transmission rate by social distancing. an improved model working with these assumptions likely requires a richer structure for the host protection response than the structures we have explored thus far. we are currently seeking improved fits to the data by implementing a number of biologically defensible exten-sions to our model, including incremental immunity whereby t w increases by a factor v after each exposure to the pandemic flu, and incremental loss of prior protection whereby a increases as hosts lose their sterilising prior protection. it is important to note that the mechanism(s) generating differences in the pandemic experience recorded in geographically connected locations is an open question; true differences in demography, varying degrees of reactive social distancing, inhomogeneities in the circulation (or circulation history, i.e. prior immunity) of viral strains, stochastic variations, and ⁄ or unique socio-cultural ⁄ behavioural conditions may all contribute to this effect. the h n experience in australia and elsewhere highlighted the difficulties faced by public health authorities in diagnosing infections and delivering antiviral agents (e.g. oseltamivir) as treatment for cases and prophylaxis for contacts in a timely manner. consequently, forecasts from mathematical models of the possible benefits of widespread antiviral interventions were largely unmet. we summarise results from a recently developed model that includes realworld constraints, such as finite diagnostic and antiviral distribution capacities. we find that use of antiviral agents might be capable of containing or substantially mitigating an epidemic in only a small proportion of epidemic scenarios given australia's existing public health capacities. we then introduce a statistical model that, based on just three characteristics of a hypothetical outbreak [(i) the basic reproduction number, (ii) the reduction in infectiousness of cases governments and public health agencies worldwide, spurred by outbreaks of sars and h n , have developed preparedness strategies to mitigate the impact of emerging infectious diseases, including pandemic influenza. pandemic response plans are presently being revised in light of the h n experience. [ ] [ ] [ ] many developed countries amassed large stockpiles of neuraminidase inhibitors (nais) with the expectation that they could be used to not only treat the most severely ill, but curb transmission in the community. without relevant field experience indicating how nais should be distributed, mathematical and computational modelling has been used to inform optimal deployment policy in a pandemic scenario. - models of population transmission were used to infer likely effects on epidemic dynamics, using data from human and animal studies of experimental infection and nai efficacy trials. in the australian (and wider) context, models indicated the potential for substantial benefit at the population level if nais were distributed in a liberal manner, targeting close contacts of indentified cases. furthermore, results indicated that use of limited nai resources in this way may improve the impact of case treatment due to the effects on epidemic dynamics. however, these models did not take into account logistic and other real-world constraints, such as finite diagnostic and antiviral distribution capacities, which were identified as limiting factors during the australian h n pandemic response. [ ] [ ] [ ] in particular, if using positive pcr diagnosis as a 'decision to treat' test, delays to confirmation of diagnosis, particularly once total laboratory capacity was exceeded, prevented timely delivery of nais to both cases and contacts of cases. in previous work, we have extended our existing models to examine how diagnostic strategies [e.g. using pcr confirmation versus syndromic influenza-like illness (ili) presentation as a decision to treat], diagnostic-capacity, and nai distribution capacity each impact on the ability to deliver an effective intervention. the model uses case severity (the proportion of infections deemed severe) to determine the overall presentation proportion, and so the ability to identify individuals eligible for nai treatment and contact prophylaxis. figure (a) shows a key result from the model. for each curve shown, we simulated thousands of epidemics, sam-pling across plausible ranges of parameters describing virus, population, and intervention characteristics using a latin hypercube sampling (lhs) approach. without intervention, the proportion of the population infected either symptomatically or subclinically by the end of the epidemic is around %. if a syndromic strategy (ili presentation) is used to determine provision of nais as treatment and prophylaxis, excessive distribution of drug to individuals who are not infected with influenza occurs early in the epidemic. early stockpile expiry accounts for a marginal impact of the antiviral intervention on the final outbreak size, in the order of a few percent. the second strategy modelled (pcr ⁄ syndromic) is one where pcr confirmation of diagnosis is required early in the epidemic to make treatment decisions until such time as laboratory capacity is exceeded. from this point, individuals are treated on the basis of symptoms alone -during an epidemic phase in which a substantial proportion of ili presentations will be attributable to influenza. under this strategy, the intervention is able to control the outbreak in approximately % of the simulated epidemics given the 'base case' constraints on diagnosis and delivery assumed in the model. the results highlight that a successful antiviral intervention requires a highly sensitive diagnostic strategy in the initial stages of the epidemic and comprehensive distribution of post-exposure prophylaxis. a pcr ⁄ syndromic strategy for decision to treat and provide contacts with prophylaxis is thus optimal. the surface in figure (b) shows the percentage of simulation runs for the pcr ⁄ syndromic strategy that have a final population attack rate of < % (a substantial reduction from the no intervention case of approximately %) as a function of pcr capacity and nai daily distribution capacity. as indicated by the arrow, the estimated australian pcr laboratory capacity appears to be sufficient, while significant benefits for the public health outcome may be achieved if logistical delivery constraints for nai distribution can be ameliorated. however, the probability that such an interventioneven with substantial increases in pcr and nai distribution capacity -would successfully mitigate an epidemic is low ( - %), and consequently it is difficult to universally recommend an antiviral intervention. in this study, we introduce a statistical model that predicts whether or not an nai distribution strategy based on a pcr ⁄ syndromic antiviral distribution policy will be successful in mitigating an epidemic. we thereby provide proof-of-principle for the design of a decision support tool that may be used by public health policy makers during an epidemic when faced with formulation of context specific nai distribution policy. synthetic data of hypothetical outbreaks and interventions were generated using the lhs simulations developed in ref. [ ] . we selected a random sample of outbreaks from a total of simulated epidemics ( % of model simulations). using these data, we identified independent model parameters that were most highly rank-correlated with the final attack rate. these parameters were included in a logistic regression model to assess their ability to predict whether an influenza epidemic would be successfully mitigated by an antiviral intervention (ar < %). model predictions were then validated against the full simulated dataset. full details of the simulation model, its structure, parameterisation and parameter distributions are available in ref. [ ] . use of the lhs simulation approach, and the method of model analysis and evaluation was similar to that previously described. matlab a (mathworks, natick, ma, usa) was used for the analysis and statistical model fitting. table shows results from our logistic regression model. key parameters sufficient to predict whether or not an outbreak may be controlled by the deployment of av agents are: . r , the basic reproductive number of the outbreak (assigned values between ae and ae for this example). as the value of r increases, the epidemic progresses more rapidly and is more difficult to control, explaining the negative correlation coefficient. . e t , the relative infectiousness of treated individuals (assigned values between ae and ae ). higher values for this parameter indicate only modest drug effects on transmission, explaining the negative correlation coefficient. . g, the proportion of infections that are severe (assigned values between ae and ae ), and which in turn determines the presenting proportion (derived values between ae and ae ). as the presenting proportion increases, the ability to identify and treat cases and deliver prophylaxis to contacts also rises, increasing the impact of the antiviral intervention. the roc curve ( -specificity versus sensitivity, not shown) for the logistic regression model specified in table has an area under the curve of ae , demonstrating that the model predicts the success of an antiviral intervention extremely well. for example, with a sensitivity of % we still have a specificity of approximately %. evaluation of the pandemic response has emphasised the need for early informed decision-making to implement proportionate disease control measures. our model identifies a low probability of successful epidemic mitigation using targeted antivirals alone (figure and ref. ), in distinction to results from models that fail to account for the diagnosis and delivery constraints inherent in any public health response. the decision support tool (table ) highlights key epidemic characteristics that are predictive of a high likelihood of effective mitigation. the reproduction number was one of the earliest parameters estimated from early outbreak data during the h n outbreak. , our findings reinforce the importance of characterising epidemic severity as early and as accurately as possible, in order to inform a proportionate pandemic response. critically, a typically mild pandemic (low g), such as that experienced in , is predictably difficult to contain using a targeted antiviral strategy due to the low proportion of infectious cases that present to health authorities. the relative infectiousness of treated individuals, e t , is strongly negatively correlated with successful mitigation, perhaps a surprising result given the model's underlying assumption (based on available epidemiological and human clinical trials data) that e t lies in the range [ ae , ]. that is, nais provided as treatment have a maximum impact of just a % reduction in infectiousness. however, our previous results show a strong synergistic effect of treatment when overlayed on a contact prophylaxis strategy, explaining the observation here that e t is critical in determining likely success of an intervention. despite the limited impact of treatment at the individual-level, the model outcomes are highly sensitive to the value of the relative infectiousness of treated cases. it follows that determination of e t is important for predicting the population-level outcome of a control effort. a 'small' reduction (of the order approximately %) may be extremely valuable in terms of success of a public health control strategy, and so should not be discounted. using a mathematical model which takes into account some of the key logistic constraints that are inherent to healthcare responses, we have derived a logistic regression model for estimating the probability that an antiviral intervention based on liberal distribution of nais as treatment and prophylaxis could successfully mitigate an influenza epidemic. the model demonstrates an excellent degree of accuracy when applied to synthetic data. the choice of parameters for the regression model was restricted to those that were both highly correlated with the success of the intervention and hopefully feasible to measure during the early stages of an emerging epidemic. the model could therefore be a useful near real-time decision support tool for public health policy in the face of an influenza epidemic, although further validation on a range of synthetic data (and real-world data where available) is required. influenza to seasonal flu status to avoid overstretching the demands on healthcare services. a great deal of information has emerged as the result of the pandemic response exercises conducted by affected countries. however, uncertainties remain regarding the effectiveness of intervention measures, as well as the feasibility and the timing of their implementation. mathematical and computational models [ ] [ ] [ ] have been used to project the outcomes of influenza outbreaks under various scenarios and epidemiological hypotheses. motivated by the events of and public health measures adopted by the taiwan cdc, we use a stochastic, individual-based simulation model to study the spatio-temporal transmission characteristics of the h n virus, so as to quantitatively assess the effects of early intervention strategies. our stochastic disease simulation model builds upon a highly connected network of individuals interacting with each other via social contact groups. to represent the daily interactions of approximately million people living in taiwan, we constructed a computer-generated mock population based on national demographic and employment statistics (to derive daily commute patterns) from the taiwan census (http://www.stat.gov.tw/). each individual is created with a set of attributes, including age, sex, residence, family structure, and social standing (employment status, etc.). based on their attributes and the time of day, each individual is assigned to miscellaneous contact groups, where the potential of interactions between any two individuals resulting in flu virus transmission occurs. such epidemiological properties are defined by empirically parameterized attributes such as basic reproduction number r , transmission probability, contact probability and associated probability distributions outlining the disease's natural history. additionally, intervention measures are implemented as scheduled events that could alter control parameters during the course of a simulation run. the targeted basic reproduction number (r ) in all our simulations is ae , following the suggested range by who of ae - ae . as the latent ⁄ incubation and infectious periods for h n have not yet been reliably ascertained, we adopt the natural history of the and pandemic influenza viruses. , here, the latent period ranged from to days, with a median value of ae days. the infectious periods begin day prior to symptom onset and can continue for - days, with a median value of ae days. twothirds of the infected individuals will develop clinical symptoms, and the asymptomatic cases will have half the infectious strength. the efficacy of antiviral drugs (oseltamivir) and vaccines are based on these studies. , for the source region of the infected cases, we use the north american continent (canada, mexico and united states) with an estimated total population of and an average hours of flight time to taiwan. the average daily passenger number is based on the annual statistical report on tourism, tourism bureau, taiwan (http://admin.taiwan.net.tw/english/statistics/year.asp? relno= ). each simulation lasts days and starts with a baseline simulation of r % ae h n pdm outbreak at the source region. the outbreak was adjusted to approximate clinical attack rate (car) in the united states, april -march , . we estimate the daily number of imported cases according to average daily passenger numbers and their probability of holding a disease status. we then apply airport exit ⁄ entry screening per corresponding success rates, by subtracting the number of identified symptomatic cases. we also consider latently infected passengers with inflight disease progression, by fitting a gamma distribution to the cumulative distribution of time to onset data with hours average flight-time, as presented by pitman et al. the daily imported cases are seeded according to the traveling patterns of foreign tourists and residents returning home. from the disease's natural history, we derive that roughly % of the infected travelers present no symptoms; the percentage increases if most symptomatic individuals elect not to travel in their condition, or are stopped by airport screening. we use the official epidemic data provided by the taiwan cdc to calibrate the simulation model and perform regression analysis on scenario parameters. this data is a close estimation of the weekly new clinical cases of h n pdm patients. it consists of weekly opd (outpatient department) icd- code (influenza) tallies collected by the bureau of national health insurance, taiwanadjusted to exclude seasonal flu patients and to account for uninsured patients. we formulate our scenario settings according to events in taiwan, and establish settings to approximate the actual events. with domestic events and intervention schedules fixed in time, the start date determines the simulation outcomes and the data range for selected indicators, such as the mean car, the epidemic peak, and several significant dates for the incoming index case events. we plot the taiwan weekly h n opd cases alongside the weekly new clinical cases from our simulation results in figure . our simulations not only capture the epidemic trend, but also pick out the most likely date, may , for identifying the first symptomatic case at airport screening based on practical assumptions. we further analyze the effectiveness of various mitigation measures with february , as the empirical start date for h n pdm in north america. the simulation result confirms that by the time we identified the first symptomatic case at the border screening, infected cases had already made their way to the public. by our calculation, roughly four such cases had passed in each of our scenario settings, with the first case happening as early as weeks before detection. figure also highlights the importance of the timing for the implementation of mitigation measures; for example, a -day-delay of the identical intervention plan results in nearly an additional % of the population being infected. therefore, the rule of thumb for healthcare officials is to implement intervention measures as early as possible. in our study, we have ignored the possibility of inflight transmission and any false positive results by airport screening procedures. to assess the effectiveness of each mitigation strategy of interest and their combinations, we take the calibrated simulation model and perform simulation realizations for groups of scenarios containing only those intended mitigation measures, and analyze the averaged results. for example, in the airport exit screening policy only scenario, the first imported symptomatic case can be delayed up to months, and the epidemic peak can be delayed up to days. as the data suggests, the exit screening policy alone has very little impact on car. combining various screening success rates for both exit and entry screening allows us to quantitatively assess their beneficial ramifications on the epidemic. for example, there is very little additional benefit between % and % suc-cess rates for entry screening policies when exit screening policies are adequate, as the enhanced border screening only delayed the epidemic peak by day, and reduced car by < ae %. base on this result, the government should not attempt to exhaust all its resources in securing the border during a pandemic event, because the return of such a policy will be disappointing. instead, a response plan with a shifting focus on health resource allocation and the capacity of adjusting intervention strategies in line with the developing epidemic will be most effective. based on the same principle, we perform experiments with assorted scenarios, including relaxing entry screening policies after identifying the first imported symptomatic case, mass vaccination based on the actual vaccination schedule of h n pdm in taiwan, and altering the start dates of the vaccination schedule. our results show that with a reasonable reduction in the airport entry screening success rate, we conserve valuable healthcare resources, but loose a few days for the strategic planning and preparation of subsequent response measures. in other simulation scenarios, a national vaccination campaign has very little impact on the outcome, due to the late start of the vaccination schedule. we then explore the effect of a national vaccination campaign with various starting dates. the simulation results are illustrated in figure , where the benefit of an early start date for mass vaccination is clearly demonstrated. considering a scenario with an % airport exit screening success rate, % airport entry screening success rate and % symptomatic case tracing success rate, the combined intervention strategy results in: a % reduction in car if the vaccination campaign starts in mid-november; % reduction if the campaign starts in mid-october; % reduction if the campaign starts in mid-september; and % reduction if the campaign starts in mid-august. in retrospect, the taiwanese government's response to h n pdm proved to be effective. first and foremost, it initiated enhanced border monitoring and on-board quarantine inspection as soon as the threat of a flu pandemic became clear. at the same time, the domestic preparations towards h n pdm were escalated, such as antiviral drug stockpiling and distribution, and vaccine acquisition. as the h n cases increased worldwide, various revised plans were adopted and implemented; such as the shift from labor-extensive on-board quarantine inspection to the notifiable infectious disease reporting system and realtime outbreak and disease surveillance system in order to effectively track down symptomatic and exposed passengers, apply prophylaxis treatment and mandatory in-home quarantine. as a result, all h n pdm related statistics are well below the international average. in modern society, countries rely heavily on the global economy for their own prosperity. shutting down the border for any length of time is not only costly, but could have disastrous economic effects that linger long after the event is over. moreover, with nearly % of the infected passengers presenting no symptoms whatsoever, they are not detectable by any port authority's screening procedures, and the importation of the novel flu virus is therefore inevitable. many studies conclude that entry screening is unlikely to be effective in preventing or delaying the importation of influenza, and has negligible impact on the course of subsequent epidemic. however, these studies are based on the assumption that effective exit screening is in place. our study shows that as the exit screening success rate decreases, the sensitivity of the entry screening policy becomes more pronounced. with the same methodology, we can also study the effects of varying the length of flight time, or the disease's incubation time. lastly, the benefit of entry screening is even more crucial for a small island country such as taiwan, since all incoming traffic must go through the port authority where entry screening can be enforced. in england and wales, three waves of the pandemic struck in summer, autumn, and winter seasons of - . although the proportion of people reporting symptoms was often greater in the first wave, - a puzzling feature was the much higher mortality in the second wave, in which . % of the population died, compared with . % in the out-of-season first wave and . % in the third wave. an obvious hypothesis to explain the changes in mortality from wave to wave would be that the virus mutated to higher virulence after the (lower mortality) first wave. although pandemic virus reconstituted from the high mortality waves has proven to have high virulence in animals, it has not been possible to recover virus from the first wave in for comparative purposes. indeed it is questionable whether virulence mutation(s) occurring between wave and wave could have spread to so many different populations in the time-frames observed. furthermore, in all three pandemic waves, there was the same agedistribution of mortality, with more deaths occurring amongst younger adults than older adults. [ ] [ ] [ ] this 'pandemic signature', arguably due to immune protection of older adults who were exposed to a similar virus in the years before , , suggests that the - viruses were at least immunologically similar in all three waves. a second hypothesis would be that the higher case fatality in the later waves was due to higher rates of complicating bacterial pneumonia, to increased transmission of influenza virus in the cooler months of the year, or to other seasonal effects. we have considered a third (immunological) hypothesis to explain the greatly increased mortality in waves and . the underlying idea is that the mortality rate in the first wave was lower than in later waves because most persons were protected by prior immunity in the first wave, and that the mortality was higher in later waves because of waning of that short-lived immunity. this hypothesis builds on our earlier modelling papers suggesting that even before the first wave in , military, school, and urban populations in england and wales apparently had (short-lived) immune protection, presumably induced by recent prior exposure to seasonal influenza. [ ] [ ] [ ] we suggest that this short-lived strain-transcending protection was in addition to the longer-lasting immunity, presumably induced by exposures to a similar virus circulating prior to , that arguably reduced pandemic mortality for older adults in - . , cumulative mortality rates attributed to pandemic influenza were available for each of the three waves in - for populations in england and wales. we have built immunological models to potentially explain the variation in mortality rates across waves and populations. to show proof of principle, we have fitted these models to mortality data from a randomly selected sub-set of twenty populations. our key assumption was that the risk of a fatal infection would be limited to persons with inadequate immunity who were being exposed to the pandemic virus for the first time. persons who were exposed and who survived an earlier wave were assumed to be protected against death in a later wave. model a and assumptions (see figure ) before the first wave, we assumed that people could be fully susceptible (s ), or partially protected (q ), or fully protected (p) by prior immunity which was not necessarily specific for the new virus. we assumed that exposure to the new pandemic virus would be fatal (m) in a proportion h of fully susceptible persons who were actually exposed (e) in the relevant wave. for those surviving that first exposure, it was assumed that they would be permanently protected against death in later waves by an immune assumed that viral exposure and multiplication would induce an immune response specific for the pandemic virus that would protect them against death in that wave and in subsequent waves. in contrast, for persons with strong prior immune protection, p, the virus would not be able to multiply to induce pandemic-specific immune protection. between waves, it is assumed that due to the waning of non-specific prior immunity, persons in the p state can move to the q state, and persons in the q state can move to an s state before the next wave. the proportion (e) of susceptible persons exposed to productive infection in each population was estimated by applying the following version of the final size equation to the proportion susceptible (s & q) in each wave, for each population: note: in both figures and , we have omitted the flows out of the q and e states that removed persons from the risk of death. parameters: s = proportion fully susceptible to infection and death before wave ; q = proportion susceptible to immunising infection, but not to death from exposure in wave ; p = proportion temporarily protected against both immunising infection and death from exposure in wave ; n = proportion even more protected against both immunising infection and death from exposure in wave (model b only); r = basic reproduction number (the average number of secondary cases for each primary case) in a fully susceptible population; f = proportion moving from q to s between waves; g = proportion moving from p to q between waves; d = proportion moving from n to p between waves (model b); h = proportion of e that actually move to m and die. model a could provide a very good fit for the summer, autumn, and winter waves of the - pandemic (results not shown). however, because of the replenishment of the pool of susceptible persons over time, model a also predicted a fourth wave of influenza in the spring season of . as no such wave was seen, and as we could not find parameters values for model a that did not predict a fourth wave, we must regard model a as inadequate. model b was similar to model a, but with an additional stage of prior immunity (n), which could wane to p. model b allowed us to not only fit the three observed waves, but also to fit the imputed data (zero cases) corresponding to the absent fourth wave. following earlier work, , we used a bayesian approach with markov chain monte carlo (mcmc) procedures to estimate model parameters, and we used hyper-parameters to allow for parameter variation between populations. the initial conditions were specified by the parameters: p , q , s and n . from these and the other parameters, it was possible to simulate the behaviour of model a over three waves, and of model b over four waves, and to estimate the expected numbers dying in each wave in each population. we calculated the log likelihood of the observed numbers of deaths given the parameter estimates, and we used mcmc simulation to generate the posterior distributions of parameters. although we obtained an excellent fit between observed and expected numbers of deaths in each of the three waves for the populations for model a, we could not find parameter values for model a that would fit the three observed waves without giving rise to a fourth wave in the spring of . accordingly, in the modified model b, we allowed for an additional stage of prior immunity (figure ) , and we fitted the model to the same data, plus imputed data corresponding to 'the absent fourth wave'. we obtained a very good fit to the three observed waves and the absent fourth wave in each population. the % credibility intervals for parameter estimates, derived from the posterior distributions of the hyper-parameters were: h = . - . , s = . - . , q = . - . ; n = . (fixed); p = ) s ) q ) n ; f = . - . ; g = . - . ; d = . - . and r = . - . . this analysis had allowed all parameters to vary from population to population under the constraints of the hyper-parameters. however, several of the biologically determined parameters might be expected to be more constant from population to population, whereas those dependent on mixing history and other social characteristics which vary more widely from population to population. to test this possibility, we fixed the mean values for the more biological parameters (f = . ; d = . ; g = . ) and estimated the % credibility intervals for the others as: h = . - . ; s = . - . , q = . - . ; and as before n = . (fixed); in a subsequent paper we will be able to provide more details of the method, the robustness of the assumptions, and the results from fitting to many more populations. this short report suggests that the observed patterns of mortality in england and wales over the three waves of the - influenza pandemic , can be explained by an immunological model. in particular, the lower mortality in wave one can be explained by the assumption of protective immunity antedating the first wave, arguably induced by prior exposure to seasonal influenza. , the much greater mortality in wave two can be explained by the waning, between wave one and wave two, of that short-lived and less-specific immune protection. the somewhat lesser mortality in wave three and the 'absent fourth wave' can be explained in terms of the progressive acquisition of immunity specific to the pandemic virus. the credibility estimates for parameters are of potential interest. for example, r estimates of . - . across different populations are consistent with our earlier findings. , if all persons had been susceptible, such r values imply that the virus would have infected most people in all populations. however, even in the first wave, the proportion susceptible, s + q , was < % in all populations, so that a considerable number of persons escaped productive infection in that wave; as their immunity waned, they became susceptible to infection in the later waves. it is likely that the variation in r between populations is due to different rates of population mixing. estimates for h indicate that between % and % of infections in the most susceptible persons were fatal; the higher values of h could reflect higher rates of secondary bacterial infection in the most socially disadvantaged and overcrowded populations. although we have shown the plausibility of an immunological explanation for wave to wave changes in pandemic mortality, we cannot assume that our particular model is even approximately correct. nor can we exclude the possibility that the higher mortality in the later pandemic waves in - was because of genetic change in the virus in later waves, or because of changing rates of secondary bacterial infection or seasonal effects. nevertheless, there is growing evidence that the population spread of pandemic influenza, whether in - , or in , , can be constrained by significant prior immunity, even for viruses that are ostensibly novel. previous reports, reviewed in ref. [ , ] , support the idea of strain-transcending immune protection, which can wane over periods of a few months. this form of protection, probably induced by recent exposure to seasonal influenza, may not be mediated by hi or neutralizing antibody. in contrast, strain-specific immunity, most often mediated by hi or neutralizing antibodies can be so long-lasting that after several decades it will still provide significant protection against any closely-related virus that re-appears in the population. it has not escaped our notice that although attack-rates in the h n pandemic were low in many countries, with generally mild symptoms, the virus did cause lifethreatening illness in a small proportion of younger affected persons. it seems likely that those who were most severely affected in were doubly unlucky: they had missed out on seasonal influenza infection or vaccination in the preceding season(s), and they were born too late to have been protected by the closely-related viruses that are thought to have circulated before . during the early phases of the influenza pandemic in italy, real-time modeling analysis were conducted in order to estimate the impact of the pandemic. in order to evaluate the results obtained by the model we compared simulated epidemics to the estimated number of influenza-like illness (ili) collected by the italian sentinel surveillance system (influnet), showing a good agreement with the timing of the observed epidemic. by assuming in the model mitigation measures implemented in italy, the peak was expected on week ( % ci: , ). results were consistent with the influnet data showing that the peak in italy was reached in week . these predictions have proved to be a valuable support for public health policy makers for planning interventions for mitigating the spread of the pandemic. mathematical models have recently become a useful tool to analyse disease dynamics of pandemic influenza virus can-didates. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] as of april , after the pandemic threat emerged worldwide, it was crucial for policy makers to have early predictions on the possible spread of the pandemic influenza virus in order to support, with quantitative insight into epidemic, policy decisions. thus, after the first pandemic alert was announced by the world health organization (who) in late april , a national crisis management committee headed by the minister of health was established in italy in order to provide weekly advice to the italian ministry of health. real-time analyses using an individual based model were undertaken. the transmission model was previously used for evaluating the effectiveness of the control measures adopted in the national pandemic preparedness plan and for assessing the age-prioritized distribution of antiviral doses during an influenza pandemic. to parameterize the transmission model, we used data derived from the national surveillance system until june and estimates of key epidemiological parameters as available at that time. in order to provide a preliminary assessment of the model predictions performed during the early stages of the epidemic, we compare model predictions with surveillance data of influenza-like illness (ili) available since august . after the first pandemic alert was announced by the who in late april , a national active surveillance system for the pandemic influenza was set up from april to july . however, over the period from april to october , surveillance systems, laboratory testing, and diagnostic strategies have varied considerably in italy. since end of july , following who recommendations, the focus of surveillance activities has changed in reporting requirements, as active case-finding became unsustainable and unnecessary. for this reason, the ministry of health (ministry of health, available in italian at the website: http://www.normativasanitaria.it) requested regional health authorities to report the weekly aggregated ili cases according to a new case definition (sudden onset of acute respiratory symptoms and fever > °c plus at least one of the following systemic symptoms: headache, malaise, chills, sweats, fatigue; plus at least one of the following respiratory symptoms: cough, sore throat, nasal obstruction). by october , following the increasing number of cases, the sentinel influenza surveillance system (influ-net available at: http://www.flu.iss.it) became the official surveillance system for ili cases in italy (ministry of health, available in italian at the website: http://www. normativasanitaria.it). since , influnet is routinely based on a nation-wide, voluntary sentinel network of sentinel community based physicians in the regions and autonomous provinces of the country. incidence rates are, therefore, not based on consultations, but on the served population of each reporting physician each week. influ-net usually consists of an average of (range - ) general practitioners (including physicians and pediatricians) per year, covering about ae - % of the general population, (representative for age, geographic distribution, and urbanization level) reporting ili cases (according with a specific case definition). italian influnet surveillance system is part of the european influenza surveillance scheme (eiss). a stochastic, spatially explicit, individual-based simulation model was used. individuals are explicitly represented and can transmit the infection to household members, to school ⁄ work colleagues, and in the general population (where the force of infection is assumed to depend explicitly on the geographic distance). the national transmission model was coupled with a global homogeneous mixing susceptible-exposed-infectious-removed (seir) model accounting for the worldwide epidemic, which is used for determining the number of cases imported over time. regarding the epidemiological assumptions (e.g., length and shape of the infectivity period, which lead to an effective generation time of ae days), this study is consistent with refs [ , , , ] , but for the proportion of symptomatic individuals, which is assumed to be ae %. the basic reproductive number of the national transmission model was set to ae , according to the early estimates as obtained during the initial phase of the epidemic in mexico in a community setting. , we initialized our simulations through the global homogeneous mixing model in such a way that imported cases were generated until june . this gives a reliable way for fixing the time in the simulations and thus determining the timing of school closure and vaccination in the simulations. the model accounts for school closure for both summer and christmas holidays: we assumed that in these periods contacts among students decrease, while contacts in the general community increase, as in ref. [ ] . we also considered scenarios accounting for partial immunity in the population. in order to investigate the effects of recommendations of the ministry of health (confirmed cases coming from affected areas were isolated for - days, either in hospital or at home) established in the early phase of the pandemic (april-july ), we assumed that a fraction of the imported symptomatic cases were isolated on the first day after the symptoms onset. this recommendation was in place until july . we also assumed, according to the italian school calendar, that schools were closed from june to september for the summer holidays, and from december to january for christmas holidays. the effects of prolonged school closure were also investigated. when considering vaccination, we assumed weeks for the logistical distribution of doses of pandemic vaccine. since at the time of simulation specific recommendations regarding the administration of a single dose of pandemic vaccine from ema were not available yet, we considered the administration of vaccine doses month apart). the pandemic vaccine was considered effective after the administration of the second dose with a vaccine efficacy of %. we assumed the vaccine to be administered by priority, vaccinating first the target population accounting for essential services workers (including health care workers and blood donors), pregnant women at the second or third trimester, and at risk patients (with chronic underlying conditions) younger than years old. the vaccination coverage was assumed %. regarding antiviral treatment and prophylaxis, recommendations of the ministry of health in the initial phase of the epidemic were to administer antivirals to all confirmed cases and to their close contacts. we assumed that the surveillance system would be able to detect % of symptomatic cases. after july , recommendations changed and antiviral treatment was considered only for cases with severe complications and in case of local clusters. since it was difficult to establish the proportion of treated cases, we considered different scenarios: antiviral treatment from % to % of the symptomatic cases. consistently with ref. [ ] , both treatment and prophylaxis were assumed to start day after the clinical onset of symptoms in the index case. treatment was assumed to reduce infectiousness by %, whereas antiviral prophylaxis was assumed to reduce susceptibility to infection by %, infectiousness by %, and the occurrence of symptomatic disease by %. as of july , approximately confirmed cases have been reported to the italian surveillance system for pandemic influenza. during july, the sudden increase of ili confirmed cases suggests for sustained autochthonous transmission in italy. by analyzing the number of ili cases reported to the surveillance during the weeks from to , we found that the exponential growth rate was ae ⁄ week and thus we estimated the national reproductive number to be r = ae . this estimate of the basic reproductive number supports the choice of the value adopted in the model simulations (r = ae ). in the absence of intervention measures, the predicted cumulative attack rate was ae % ( % ci: ae , ae ), and the peak was expected on week ( % ci: , ) with a peak day incidence of ae % ( % ci: ae %, ae %). by assuming case isolation, antiviral treatment, and prophylaxis to % of symptomatic cases until july , the peak was expected on week ( % ci: , ). when considering ae % of natural immunity in the population aged more than years, the peak was expected week later than in the previous scenario, i.e., on week ( % ci: , ). to validate the model, we compared model predictions (which are based only on the available information on the early phases of the epidemic) with ili data (figure ). based on model predictions, we estimated the underreporting factor of influnet ranging from ae to ae , considering different scenarios. by aligning the simulations with the ili data adjusted by the underreporting factor, we can observe that almost all the points in the increasing phase of the epidemic lie within the % ci of the model results (both considering or not natural immunity). the decay phase of the simulated epidemics shows a small delay with respect to the ili data. when introducing single and combined mitigation measures, such as case isolation, antiviral treatment, prophylaxis, and vaccination in the model, results showed that even a low proportion of symptomatic cases treated with antiviral drugs could have led to a relevant reduction in the epidemic size (table ) . we simulated the planned italian vaccination strategy (begun on october ), obtaining a limited but not negligible reduction in the attack rate with respect to the scenarios accounting only for antiviral treatment. moreover, the effect of vaccination would be higher if coupled with antiviral treatment; vaccination would have no effect on delaying the peak incidence. model predictions produced in italy during the early phase of the pandemic influenza are in excellent agreement with italian surveillance data on the beginning of the epidemic (when case isolation, antiviral treatment of index cases, and antiviral prophylaxis to close contacts were implemented by the italian regional public health authorities) and are basically consistent with the influ- net data during the course of the epidemic. the model has been useful for predicting the timing of the epidemic, while it has overestimated the impact of the influenza pandemic for adult and elderly individuals. however, the disalignment is probably due to the model parameterization. based on literature values, , we assumed a similar fraction of cases in the different social contexts considered in the model (namely ⁄ in households, ⁄ in schools ⁄ workplaces, and ⁄ in the general community), since analysis on the relative transmissibility of the virus was not carried out for any country yet. we were also able to estimate an underreporting factor for the influnet data in the range ae - ae . if we focus our attention on the reporting factor computed by considering the total number of cases (instead of symptomatic cases), the resulting value lies in the range - ae %, which is in excellent agreement with the range estimated in ref. [ ] on previous a ⁄ h n influenza seasons, namely ae %- ae %. moreover, based on our results showing that vaccinating % of the italian population was more than adequate to mitigate the pandemic, the ministry of health decided to stockpile a limited number of vaccines. we have also shown that starting the vaccination program in october (or later) could have had only a limited effect on reducing the impact of the epidemic, although it may have been useful to prevent a possible second wave and to protect essential workers and at-risk patients. finally, our results have shown that antiviral treatment would have been the most efficient strategy to reduce the impact of the influenza pandemic, even with a limited antiviral stockpile. a population-wide passive immunotherapy program in this paper, we assume that convalescent plasma (cp) is efficacious in treating severe cases of pandemic influenza. under this premise, we test the hypothesis that a population-wide passive immunotherapy program that collects plasma from a small percentage of convalescent individuals can harvest sufficient cp to treat a substantial percentage of severe cases during the first wave of the pandemic. the proposed program involves recruiting adults (individuals age - years) to donate blood if they have experienced influenza-like symptoms more than weeks ago (to account for the time needed for neutralizing antibodies to build up). the blood samples would be screened for infectious diseases (including hiv, hbv, hcv, htlv, and syphilis, etc., as in routine blood donation screening) and neutralizing antibodies against the pandemic virus. donors whose blood samples are free of known infectious agents and contain a sufficiently high titer of neutralizing antibodies would then be invited to donate plasma by plasmapheresis or routine whole blood donation. qualified donors with higher titers may be given higher priority for plasma donation. in this paper, we use the demographic and logistical parameters of hong kong as a case study. see figure for a schematic of the proposed passive immunotherapy program. we examine the following questions regarding the logistical feasibility and potential benefits of the proposed passive immunotherapy program: (i) what percentage of convalescent individuals (donor percentage) is needed in order for the program to significantly reduce pandemic mortality? (ii) how many severe cases can be offered passive immunotherapy? (iii) what are the ratelimiting factors in the supply of passive immunotherapy? (iv) what are the epidemiologic and logistical factors that determine the demand-supply balance of passive immunotherapy? a more detailed presentation of our results is now available in ref. [ ] . transmission and natural history model for pandemic influenza we use an age-structured disease transmission model to simulate the spread of pandemic influenza. the natural history model is similar to that used by basta et al. , the most important parameter in characterizing the growth of an epidemic is the basic reproductive number r , which is defined as the average number of secondary cases generated by a typically infectious individual in a completely susceptible population. we consider values of r between ae and , which is consistent with recent estimates. , [ ] [ ] [ ] logistical model for the passive immunotherapy program we assume that q d (%) of to year-old individuals who have recovered from symptomatic infections of pandemic influenza donate their blood for screening t r = days after cessation of symptoms. follow-ups of convalescent individuals infected with h n pdm in an ongoing clinical trial of passive immunotherapy suggested that neutralizing antibodies level reaches maximal level around - days after recovery and stays at that level for months after. we assume that q s (%) of these donors are qualified for plasma donation of which q r (%) are recurrent donors who return to donate plasma every t w = days. screening involves both detection of infectious agents and neutralizing antibodies against the pandemic virus. the latter is the rate-limiting step because neutralization tests of pandemic viruses can only be done in a bsl setting. we assume that five bsl -trained technicians are available to test the blood specimens, each running viral neutralization tests in days. therefore, the capacity and turnaround time of blood screening are u s = and t s = days, respectively. hong kong currently has nine plasmapheresis machines which allow a maximal throughput of plasma donations per day (assuming -hour daily operation with each donation taking minutes). therefore, the capacity and turnaround time of plasmapheresis are u p = and t p = ⁄ days, respectively. collected cp are ready for use in transfusion after final quality check, which takes t q = days. we assume that r t plasma donations are required to treat one severe case on average. the expert panel of the abovementioned study of passive immunotherapy for h n pdm in hong kong suggested that r t < . we assume that p h (%) of symptomatic cases will be severe cases for whom passive immunotherapy is suitable. although p h will be smaller than the case-hospitalization rate (passive immunotherapy may not be suitable for some hospitalized cases), we assume that the two have similar ranges and consider p h ranging from ae % to %. because each severe case requires r t plasma donations on average, demand for cp is simply r t p h times the number of symptomatic cases. therefore, r t p h can be regarded as a single parameter, which we refer to as the lumped demand parameter. we define the outcome as the percentage of severe cases that can be offered passive immunotherapy by the proposed program during the first wave of the local epidemic. we refer to this outcome as treatment coverage and denote it by q. we consider the base case scenarios assuming q r = % and q s = %. in general, the treatment coverage q increases sharply as the basic reproductive number r and the lumped demand parameter r t p h decrease (figure a ). in particular, when r is large and r t p h is small, q is very sensitive to r t p h , but insensitive to r . similarly, when r and r t p h are small, q is very sensitive to both. with a donor percentage of q d = %, the proposed program can supply passive immunotherapy to more than % of severe cases (q > %) if r < ae and r t p h < ae %, but < % if r > ae and r t p h > ae %. in general, the treatment coverage q increases sharply as the donor percentage q d rises from %, but with rapidly decreasing marginal increase ( figure b ). when r < ae and r t p h < ae %, q > % even if q d is as low as %, which is comparable to the current average blood donation rate of ae donations per population in developed countries. when q d is > %, q becomes largely insensitive to further increase in q d in most scenarios. the treatment coverage q for q d = % is more than % that for q d = % across all values of r and r t p h considered in the base case. therefore, increasing the donor percentage q d beyond % has a relatively small impact on cp supply. this is because increasing q d can boost supply only when plasmapheresis is not yet the supply bottleneck. for the same reason, once the donor percentage q d has reached %, the treatment coverage q is insensitive to further increase in q d even when the plasmapheresis and screening capacity are doubled ( figure b , lower panel). we conduct an extensive multivariate sensitivity analysis to test the robustness of our base case observations against uncertainties in parameter values. we generate epidemic scenarios by randomly selecting parameter values from their plausible ranges using latin-hypercube sampling. although there are numerous model parameters, the treatment coverage q is mainly determined by three lumped parameters: (i) r t p h , which indicates the magnitude of demand; (ii) q s q d , which indicates the magnitude of supply; (iii) the initial growth rate of the epidemic r (results not shown). while the dependence of q on r t p h and q s q d is readily comprehensible, it is not obvious a priori that q depends on the natural history and transmission dynamics of the disease via only the initial epidemic growth rate. when the plasmapheresis and screening capacity are very large, the supply-demand dynamics is further simplified: the treatment coverage q depends on lumped demand parameter r t p h and the lumped supply parameter q s q d only via their ratio. finally, q becomes insensitive to q s q d when the latter increases beyond - %, which is consistent with our base case observations. our results suggest that with plasmapheresis capacity similar to that in hong kong, the proposed passive immunotherapy program can supply cp transfusion to treat - % of severe cases in a moderate pandemic (basic reproductive number r < ae , lumped demand parameter r t p h < ae %) when the donor percentage is - %. increasing the donor percentage beyond % has little additional benefit because cp supply is constrained by the capacity of plasmapheresis during most stages of the epidemic. increasing plasmapheresis capacity could significantly boost cp supply, especially when there is a substantial pool of recurrent donors to alleviate the dependence of cp supply on donor percentage. in an ongoing clinical trial of passive immunotherapy for h n pdm virus infection in hong kong, % of convales- cent individuals agreed to donate their plasma for the study. therefore, the donor percentage required by the proposed passive immunotherapy program ( - %) is likely to be feasible. in view of the logistical feasibility of such program, we recommend that further clinical studies are conducted to evaluate the safety and efficacy of passive immunotherapy as a treatment for severe cases of pandemic influenza virus infection. our study is based on the premise that cp will be efficacious in reducing morbidity and mortality associated with pandemic influenza. in theory, the polyclonal nature of neutralizing antibodies in cp would lower the probability of an escape mutant emerging in treated patients. further, besides providing neutralizing antibodies against the pandemic virus, cp also might carry antibodies to other bacterial pathogens, which might decrease the severity of coexisting bacterial infections. as such, cp not only might reduce the case fatality rate but might also increase the recovery rate and shorten duration of hospitalization of severe cases. the proposed passive immunotherapy program can thus significantly reduce the burden on the healthcare system, especially the intensive care unit, which will likely be stressed, if not overloaded, at the peak of an influenza pandemic wave, hence benefiting the general public and not only those receiving passive immunotherapy. although the hypothesized efficacy of cp has yet to be proven in clinical trials, our modeling results show that a public health system similar to that in hong kong has the capacity to support a population-wide passive immunotherapy program that can supply cp treatment to a substantial percentage of the severe cases in a moderately severe pandemic. we estimate that compared to other developed countries, hong kong has a relatively low plasmapheresis capacity. our conclusions regarding donor percentage needed and rate-limiting factors remain valid for plasmapheresis capacity ranging from % to % of what we have assumed in the base case (results not shown). our conclusions are robust against uncertainties in the natural history and transmission dynamics of pandemic influenza. our sensitivity analysis shows that the outcome depends on these epidemiological characteristics only via the initial growth rate of the epidemic. as such, our results are applicable not only to pandemic influenza, but also to other emerging infectious diseases for which the time-scales of disease transmission and antibody response are similar to that for influenza virus. the three determinants of treatment coverage (the initial epidemic growth rate, the lumped demand parameter r t p h , and the lumped supply parameter q d q s ) are all readily measurable in real-time during an epidemic. therefore, our methods and results can be used as a general reference for estimating the treatment coverage of the proposed passive immunotherapy program for a given plasmapheresis capacity. background highly pathogenic h n virus continues to pose a serious threat to human health and appears to have the capacity to cause severe disease in previously healthy young children and adults. at present, antiviral therapy by oseltamivir remains the mainstay for managing h n patients. while early treatment improves survival, approximately % of patients treated within days of illness still succumb to the disease. in addition to the role of viral replication, there is good evidence that the host proinflammatory responses contributes to h n pathogenesis. this suggests that both antiviral and immune-modulatory drugs may have a role in therapy. we previously demonstrated that cyclooxygenase (cox- ) plays a regulatory role in h n hyperinduced pro-inflammatory responses, and its inhibitor has potent effects at modulating this host response. now we demonstrate that, in addition to its immune-modulatory effect, a selective cox- inhibitor, ns- has a direct antiviral effect against h n infection. materials and methods human primary monocytederived macrophages or alveolar epithelial cells (a ) were pre-treated with ns- or drug-vehicle for hour before h n virus infection. h n viruses at multipicity of infection (moi) of was used to infect the cells. following virus adsorption for mins, the virus inoculum was removed, and the cells were washed and incubated in corresponding medium with ns- or drug-vehicle as controls for , , , , and hours post-infection. cells were harvested for rna isolation at hours post-infection to study viral matrix (m) gene expression. supernatants were collected for % tissue culture infection dose (tcid ) assay to determine the virus titers at , , , and hours after h n infection. results ns- was found to suppress virus gene transcription and infectious virus yield in h n -infected human cells. conclusion we demonstrate that a selective cox- inhibitor, ns- , shows an inhibitory effect on h n viral replication in addition to its immune-modulatory effect that could counter the detrimental effects of excessive proinflammatory cytokine production. the findings suggest that selective cox- inhibitors may be a therapeutic target for treating h n disease in combination with appropriate antiviral therapy. the emergence and spread of the highly pathogenic avain influenza viruses (h n ) in poultry and wild birds with repeated zoonotic transmission to humans has raised pandemic concern. at the time of writing, human cases have been reported with fatalities, an overall case fatality rate of around % (cumulative number of confirmed human cases of avian influenza a ⁄ (h n ) reported to world health organization updated to october ). our previous data demonstrated that cox- was markedly up-regulated in h n -infected primary human macrophages, and that it played a regulatory role in the h n hyperinduced host pro-inflammatory responses. such cytokine dysregulation is proposed to be a major contributor to the pathogenesis of h n disease in humans. with the use of selective cox- inhibitors, we found that the h n -hyperinduced cytokine response was significantly suppressed by the drug in a dose-dependent manner. selective cox- inhibitor is a form of a non-steroidal anti-inflammatory drug that selectively targets cox- , and it is an inducible enzyme responsible for inflammatory process and immune response. here, we report a novel finding of a direct antiviral effect of a selective cox- inhibitor, ns- , against h n infection in human primary macrophages and alveolar epithelial cells. taken together with our previous findings that suggest an immuno-modulatory effect that can modulate virus driven cytokine dysregula-tion, these findings highlight a role for cox- and its downstream signaling as potential novel targets for adjunctive therapy of severe viral pneumonia, such as that caused by h n . such therapy may be combined with conventional antiviral drugs. the h n virus used was a ⁄ vietnam ⁄ ⁄ ( ⁄ ) (h n ), a virus from a patient with h n disease in vietnam during . the viruses were grown and titrated in madin-darby canine kidney cells cells as described elsewhere. virus infectivity was expressed as tcid . all experiments were performed in a biosafety level facility. monocyte-derived macrophages: peripheral-blood leucocytes were separated from buffy coats of healthy blood donors (provided by the hong kong red cross blood transfusion service) by centrifugation on a ficoll-paque density gradient (pharmacia biotech) and purified by adherence as reported previously. the research protocol was approved by the ethics committee of the university of hong kong. macrophages were seeded onto tissue culture plates in rpmi medium supplemented with % heat-inactivated autologous plasma. the cells were allowed to differentiate for days in vitro before use in the infectious experiments. alveolar epithelial cells: a cells were obtained from atcc and maintained in culture using dulbecco's modified eagle medium supplemented with % fetal calf serum, . mg ⁄ l penicillin, and mg ⁄ l streptomycin. differentiated macrophages or a cells were pre-treated with a selective cox- inhibitor, ns- (cayman), at concentrations as indicated or drug-vehicle for hour before infection. cells were infected with h n viruses at moi of . following virus adsorption for min, the virus inoculum was removed, the cells were washed and incubated in corresponding medium with ns- or drug-vehicle as controls throughout the experiments. cells were harvested for rna isolation at hours post-infection to study viral m gene expression. supernatants were collected for tcid assay to determine the virus titers at , , , and hours after h n infection. total rna was isolated using the rneasy mini kit (qiagen) according to the manufacturer's instructions. the cdna was synthesized from mrna with poly(dt) primers and superscript iii reverse transcriptase (invitrogen). transcript expression was monitored by real-time pcr using power sybr Ò green pcr master mix kit (applied biosystems) with specific primers. the fluorescence signals were measured using the real-time pcr system (applied biosystems). the specificity of the sybr Ò green pcr signal was confirmed by melting curve analysis. the threshold cycle (ct) was defined as the fractional cycle number at which the fluorescence reached times the standard deviation of the base-line (from cycle to ). the ratio change in target gene relative to the b-actin control gene was determined by the )ddct method as described elsewhere. ns- reduced the viral m gene expression in h n infected human macrophages in a dose-dependent manner ( figure ) . similarly, production of infectious virus yield in h n infected macrophages was found to be suppressed in the presence of ns- at lm compared to vehicletreated cells (figure a) . a comparable effect of ns- was observed in h n -infected human alveolar epithelial cells ( figure b ). we have previously demonstrated that cox- expression was dramatically upregulated following h n infection in human macrophages in vitro and in epithelial cells of lung tissue samples obtained from autopsy of patients who died of h n disease. this suggests that cox- may be an important host factor involved in h n pathogenesis and also provide a possible explanation on why h n virus replication is susceptible to a selective cox- inhibitor. cox- was previously reported to play an important role in the pathogenesis of other influenza a viruses. an in vivo study has highlighted the importance of cox- in h n - infected mice. findings showed that infection induced less severe illness and reduced mortality in cox- knock-out mice than in wild-type mice. on the other hand, cox- knock-out mice had enhanced inflammation and earlier appearance of proinflammatory cytokines in the bal fluid, whereas the inflammatory and cytokine responses were dampened in cox- knock-out mice. these data suggests that cox- and cox- may lead to opposite totally contrasting effects on influenza h n infected mice. cox- deficiency is detrimental, whereas cox- deficiency is beneficial to the host during influenza viral infection. therefore in the present study, instead of blocking cox enzymes in general as reported by others, we have chosen ns- that selectively block cox- but preserve cox- activity and showed that this drug significantly reduced h n virus replication in a dose-dependent manner. taken together with our previous report suggesting its immuno-modulatory effects, we believe that selective cox- inhibitors and cox- signaling pathways deserve investigation as a promising approach for targeting therapy in h n diseases. however, a few reports have suggested the importance of cox- in the late stage of inflammation for the resulution of inflammation, [ ] [ ] [ ] and this raises concern whether inhibition of cox- may be harmful in treating diseases related to dysregulation of host inflammatory response such as acute lung injury, which is a leading cause of death in h n patients. we previously looked at the autopsy samples of lung tissues from h n patients and found that cox- expression was markedly up-regulated compared with that from persons who died of non-respiratory causes. moreover, data also demonstrated that pro-inflammatory cytokines, such as tnf-a, was markedly elevated in the h n infected lung autopsies. taken together, with the histo-pathological findings, which showed predominant features of exudative inflammatory phase in autopsy lung samples from h n patients, , we may therefore speculate that people who had fatal h n infection died during acute inflammation phase, and before the resolution could occur, especially for the cases with a short disease duration (< - days). in conclusion, the roles of cox- in both pro-inflammation and pro-resolution phases deserves detailed investi-gation. the timing of selective cox- inhibitor therapy in h n infected patients may be extremely critical. therefore a time-dependent study using selective cox- inhibitors on h n -infected animal models will be particularly important in order to address the effectiveness of this drug in treating h n disease. avian antibodies to combat potential h n pandemic and seasonal influenza highly pathogenic avian influenza a virus (hpaiv) strain a ⁄ h n with unprecedented spread through much of asia and parts of europe in poultry remains a serious threat to human health. passive immunization (transfer of protective immunoglobulins) offers an alternative and ⁄ or additional strategy to prevent and cure influenza. here, we report that virus-specific immunoglobulin y (igy) isolated from eggs of immunized hens provide protection in mice against lethal h n virus infection by neutralization of the viruses in the lungs upon intranasal administration. importantly, chicken eggs obtained from randomly selected supermarkets and farms in vietnam, where mass poultry vaccination against a ⁄ h n is mandatory, contain high levels of igy specific for a ⁄ h n virus. when administered before or after the infection, igy prevented and significantly reduced replication and spread of hpaiv h n and related h n strains. thus, the consumable eggs readily available in markets of countries that impose poultry vaccination against a ⁄ h n could offer an enormous source of valuable biological material that provides protection against a ⁄ h n virus with pandemic potential. the approach could be used to control seasonal influenza. since , hpaiv of the h n subtype has resulted in more than cases of laboratory-confirmed human infection in countries with a death rate of more than % (http://www.who.int/csr/disease/avian_influenza/). h n influenza virus remains a global threat because of its continued transmission among domestic poultry and wild birds. passive immunization (the transfer of antigen-specific antibodies (abs) to a previously non-immune recipient host) offers an alternative and ⁄ or additional countermeasure against influenza. development of human monoclonal antibodies (mabs) against h n influenza haemagglutinin (ha) using epstein-barr virus (ebv) immortalization of b cells isolated from patients infected with h n , phage display, humanized mabs, and human recombinant abs has been attempted. chickens produce a unique immunoglobulin molecule called igy that is functionally equivalent to mammalian igg. igy is found in the sera of chickens and is passed from hens to the embryo via the egg yolk. egg igy has been used to prevent bacterial and viral infections (see review ) of the gastrointestinal tract and recently for protection against pseudomonas aeruginosa infection of the respiratory tract of patients with cystic fibrosis (cf). the epidemic of hpaiv h n virus has resulted in serious economic losses to the poultry industry, mostly in southeast asia. therefore, many countries including china, indonesia, thailand, and vietnam have introduced mass vaccination of poultry with h n virus vaccines that controls the h n epidemic to some extent. chickens immunized with recombinant h and ⁄ or inactivated h n reassortant vaccines produced a high level of virus-specific serum antibodies (abs) and were protected from h n virus challenge. theoretically, these abs could be found in egg yolk and separated for use in humans to prevent and cure h n hpaiv infection and disease, respectively. here, we examined the possibility that igy isolated from consumable eggs available in supermarkets in vietnam, where mandatory h n vaccination has been implemented, provide prophylaxis and therapy of hpaiv h n infection in mice. six-to -week-old female balb ⁄ canncrl (h- d) mice (charles river and jackson laboratory) and hy-line . igy abs were extracted from egg yolks as previously described. the % egg infectious dose (eid ) was determined by serial titration of virus stock in eggs, and eid ⁄ ml values were calculated according to the method of reed and muench. human virus stocks were grown in mdck cells as described previously , with viral titers determined by standard plaque assay. the % tissue culture infectious dose (tcid ) of virus was determined by titration in mdck cells. the standard elisa was performed for detection of anti-igy in the sera of igy-immunized mice. fifty percent lethal dose (ld ) titers were determined by inoculating groups of eight mice i.n. with serial -fold dilutions of virus as previously described. for infection, ketamine-anesthetized mice were inoculated intranasally with a lethal dose with pfu ( · ld ) of a ⁄ pr ⁄ ⁄ (h n ) virus as previously described, · ld of vn ⁄ (h n ) or · ld a ⁄ aquatic bird ⁄ korea ⁄ w ⁄ (h n ) resuspended in ll pbs per animal. ketamine-anesthetized mice were treated intranasally with ll of igy before or after infection. mice were observed for weight loss and mortality. subsets of animals were scarified for virus titre. we found comparable hai titers in the sera and egg yolks obtained from a farm in vietnam that was participating in a national mass vaccination program. furthermore we found % of eggs purchased in randomly selected supermarkets in hanoi, vietnam containing h -specific igy. the hai and vn titers of pooled egg yolk igy are comparable with those of sera obtained from hens selected randomly from the farm that underwent supervised h n vaccination. in contrast, igy separated from eggs purchased in korean markets where poultry are not vaccinated against avian influenza h n has no detectable h -specific hai or vn activity. we first treated naïve mice intranasally with h n -specific igy before infection with hpaiv h n strain, a ⁄ vietnam ⁄ ⁄ , isolated from a fatal case. such treated mice displayed mild weight loss and recovered completely by the end of the first week after inoculation ( figure a ). when animals were treated once with h n specific igy after h n inoculation they exhibited minimal weight loss during the first week after inoculation, and virus titers in the lungs were substantial reduced at day after infection; however, % of treated mice succumbed to infection during the second week after inoculation ( figure b) . it is possible that not all the hpaiv a ⁄ h n viruses were neutralized upon the single treatment with igy, and escaping viruses can spread systemically to organs outside of the lungs. these viruses may reappear in lung tissue later when specific igy is absent. indeed, vn ⁄ virus injected intravenously or into the brain can spread to the lungs. to circumvent the virus escape, we administered multiple treatments with h n specific igy after the infection. as a result, all infected mice recovered completely by the second week post-infection ( figure c) , and virus titers in the lungs were substantially reduced to the level that seen in protected mice that received single prior-infection treatment ( figure d) . similarly, the protective efficacy of h n -specific igy was observed in mice infected with lethal dose of mouseadapted avian influenza virus strain a ⁄ aquatic bird ⁄ korea ⁄ w ⁄ (h n ). this virus shares . % nucleotide sequence homology with ha (h ) but has different na (n ) from the one used for mass immunization in vietnam (reassortant avian h n influenza virus a ⁄ goose ⁄ gd ⁄ -derived, strain re- ). the results indicate that h n -specific igy isolated from eggs purchased in markets have preventive and therapeutic effects against infection with hpaiv h n and the related strain h n . the findings suggest that while a single treatment with igy prior to lethal infection was sufficient to protect the animals from the infection, multiple treatment is required for complete therapeutic effect after infection with hpaiv such as vn ⁄ strain. we further examined the protective efficacy of igy isolated from eggs laid by hens immunized in the laboratory with heat-inactivated human influenza a ⁄ h n virus, a ⁄ pr ⁄ ⁄ . we found substantial levels of hai and vn abs in the sera and yolks derived from immunized hens. when naïve mice were administered intranasally with such anti-pr ⁄ igy at - hours before or after infection with lethal dose of pr ⁄ virus, they were protected from the infection or lethal disease, respectively. the virus titers in the lungs of a ⁄ pr specific igy-treated mice at day after infection were also significantly lower than those seen in untreated mice or mice receiving normal igy. intranasal administration is the most effective route as compared to oral or peritoneal or intravenous administration for protection against lethal challenge, and the presence of virus-specific igy in bronchoalveolar lavage (bal) is required for the protection. the results provide a proof-of-concept that intranasal administration of virus-specific igy prevents influenza virus infection and cures the disease. the concept could be applied to control influenza outbreaks including seasonal and pandemic influenza. the protection was correlated with hai and vn activities of the igy and reduced virus titers in the lungs after treatments, suggesting that the protection is mediated by vn. we asked if administration of igy in the respiratory tract induces anti-igy ab response in mice. if this is the case, the next question is whether pre-existing anti-igy abs block igy-mediated protection. indeed, significant levels of anti-igy were observed in animals that received single or multiple administration of igy. when igy-immune mice were treated with virus-specific igy before or after lethal challenge, the results were identical to those obtained from treated naive mice, indicating that pre-existing anti-igy abs do not interfere with the protection mediated by virus-specific igy. consistently, incubation with anti-igy serum did not interfere with hai and vn activity of the virus-specific igy, indicating that anti-igy abs do not block virus binding by virus-specific igy (figure ). the finding suggests that the igy treatment could be applied to persons who have developed anti-igy during the individuals' life, and such treatment strategy could be repeated if multiple treatment is required and ⁄ or necessary later on to protect infections with other pathogens. the approach using specific igy for prevention and therapy of hpaiv h n infection offers a practical alternative to immunotherapy using convalescent plasma and an additional therapeutic option to antiviral drugs since widespread drug resistance has been recently reported among influenza virus strains. igy is relatively stable. we found no change in protective activity after at least months storage at °c, and lyophilization does not affect the activity, making production of igy practical. the use of igy immunotherapy has many advantages, since igy does not activate the human complement system or human fc-receptors, which all are well-known cell activators and mediators of inflammation. we chose the water dilution method for preparation of igy. the method is simple, efficient and does not require any toxic compounds or any additives. such igy preparations by this method have been used in other human study. , eggs are normal dietary components, so there is minimal risk of toxic side effects, except for those with egg allergy. thus, our study demonstrated that influenza virus-specific igy can be used in passive immunization that provides great help for immunocompromised patients and elderly who have weaken immune response to influenza vaccines. importantly, the consumable eggs readily available in the markets of countries that impose mandatory h n vaccination offer an enormous source of valuable, affordable, and safe biological material for prevention and protection against potential h n pandemic influenza. parts of the information and data presented in this manuscript were previously published in http://www.plosone.org/ article/info:doi% f . % fjournal.pone. . the polyphenol rich plant extract cystus is highly introduction the ⁄ h n influenza a virus pandemic clearly demonstrates that influenza is still a major risk for the public health. although the pandemic swine origin influenza a virus (soiv) caused only mild symptoms, the control of the outbreak still remains difficult. even as vaccine is available against this virus, the possibility of reassortment between the pandemic and a seasonal or avian a ⁄ h n influenza virus strain is indeed a frightening, but a likely event. this reassortant strain might be able to transmit easily between humans causing fatal infections, and the current soiv vaccine might no longer be sufficient to protect against the reassorted virus. in such a case, we can only rely on effective antiviral drugs. today, neuraminidaseinhibitors, such as oseltamivir, represent the most common clinically approved medication against influenza a viruses. unfortunately, the frequency of reports describing the appearance of drug-resistant seasonal h n and also h n influenza a viruses dramatically increased in the recent past. [ ] [ ] [ ] [ ] drug resistance to the known antivirals highlights the urgent need for alternative antiviral compounds with novel defense mechanisms. recently, we have reported that a polyphenol rich plant extract, cystus , which showed antiviral activity against influenza a viruses in cell culture and in mice. , moreover, the antiviral activity of cy-stus against seasonal influenza virus and common colds was also demonstrated in humans. however, the efficiency of cystus against soiv and a ⁄ h n isolates was unknown so far. therefore, we investigated cy-stus effectiveness against the pandemic strain and seven natural influenza a ⁄ h n isolates detected in several avian species during ⁄ avian influenza outbreak. additionally, the potency of the most common neuraminidase inhibitor oseltamivir was also investigated against these isolates. here, we show that cystus treatment was effective in in vitro studies against soiv and a ⁄ h n influenza virus. viruses avian h n isolates were originally obtained from the bavarian health and food safety authority, oberschleissheim, germany. the soiv a ⁄ hamburg ⁄ ⁄ was obtained from the robert-koch-institut, berlin, germany. all h n viruses were further propagated in embryonated chicken eggs or mdck ii (h n v) cells at the friedrich-loeffler-institut, tübingen, germany. for the cytopathological effect (cpe) inhibition screening, in accordance with sidwell, mdck ii cells were infected with different viruses at moi of ae . virus-infected cells were then treated with antiviral compounds cystus from ae to lg ⁄ ml or oseltamivir from ae nm to mm. after incubation for hours at °c and % co , cells were fixed, and viable cells were stained with crystal violet. after extraction of crystal violet from viable cells with % methanol, the extinction was measured with an elisa reader. immediately before infection, mdck ii cells ( · cells ⁄ well) were washed with pbs and subsequently incubated with virus diluted in pbs ⁄ ba ( ae % ba) mm mgcl , ae mm cacl , penicillin and streptomycin to a multiplicity of infection (moi) of ae for minutes at °c. cystus was added in a concentration of lg ⁄ ml directly to the virus-stock and on the cell monolayer simultaneously with the infection. after minutes incubation period, the inoculums were aspirated and cells were incubated with either mem or mem containing lm oseltamivir. at indicated time points, supernatants were collected. infectious particles (plaque titers) in the supernatants were assessed by a plaque assay under avicel as described previously. in order to investigate the antiviral potential of cy-stus , ec values based on the inhibition of the cpe on mdck ii cells were determined for cystus and in addition for oseltamivir. the ec values for cystus ranged from ae to ae lg ⁄ ml. cystus demonstrated the highest sensitivity against the soiv, sn and mb isolates with ec values below lg ⁄ ml. compared to these virus strains, cystus showed a slightly increased ec value for gsb ( ae lg ⁄ ml). in contrast the ec values for bb and bb were notably elevated ( ae and ae lg ⁄ ml). thus, the weakest antiviral effect of cystus was observed against these two isolates. the ec values evaluated for oseltamivir ranged from ae to ae lm ( table ), indicating that bb ( ae ) and gsb ( ae lm) can be considered resistant against oseltamivir. to confirm these results we investigated the ability of cystus to block virus replication as published before. as a control, virus infected cells were treated with oseltamivir as described earlier. in the absence of the drugs all influenza strains showed similar growth properties (figure , black squares) . first progeny viruses were detectable between and hours post infection (figure , black squares) . treatment with cystus resulted in reduction of virus titers of all influenza virus strains (fig. a-h, open triangles) . surprisingly, oseltamivir failed to inhibit the replication of two h n influenza virus strains (gsb and bb ), supporting the data of ec values ( figure d+h , grey rhombes). we assessed the antiviral activity of cystus against the newly emerged soiv and seven avian h n influenza viruses. cystus showed efficient antiviral activity against the pandemic h n v strain and was effective to a wide range of h n viruses. furthermore, cystus demonstrated a broader and more efficient antiviral potential than oseltamivir. cystus treatment leads to a stronger reduction of progeny virus titers, and more importantly, cystus was effective against all tested viruses, while oseltamivir was unresponsive against two of seven a ⁄ h n viruses. even though the pandemic strain in general is still sensitive to oseltamivir treatment, there are increasing numbers of reports of emerging resistant variants. the treatment with cystus does not result in the emergence of viral drug resistance since the mode of action is an unspecific physical binding of the virus particle that is also beneficial to reduce opportunistic bacterial infections. , , cystus is an extract from a special variety of the plant cistus incanus, and it is very rich in polymeric polyphenols. it is well known that polyphenols exhibit protein-binding capacity. however, cystus exhibited no neuraminidase inhibiting activity. therefore, ingredients of cystus may act in a rather unspecific physical manner by interfering with the viral hemagglutinin at the surface of the virus particle as demonstrated before. while this prevents binding of the virion to cellular receptors, it does not block accessibility and action of the viral neuraminidase. since, infections with influenza a viruses are still a major health burden and the options for control and treatment of the disease are limited, plant extracts such as cystus should be considered as a new candidate drug for a save prophylactic and therapeutic use against influenza viruses. attenuation of respiratory immune responses by antiviral neuraminidase inhibitor treatment and boost of mucosal immunoglobulin a response by co-administration of immuno-modulator clarithromycin in paediatric influenza the antiviral neuraminidase inhibitor osv and zanamivir are widely used treatment options for influenza infection and are being stockpiled in many countries. although mucosal immunity is the frontline of defense against pathogens, the effects of neuraminidase inhibitor treatment on airway mucosal immunity have not been reported. the suppression of viral rna replication and viral antigenic production by these drugs may result in a limited immune response against influenza virus. macrolides, such as cam and azithromycin, have anti-inflammatory and immunomodulatory properties that are separate from their antibacterial effects. [ ] [ ] [ ] this study examined the impact of osv treatment on immune responses in the airway mucosa and plasma in mice infected with iav and pediatric influenza patients. we also assessed the immuno-modulatory effects of cam in influenza patients who were treated with or without osv. female ae -week-old weanling balb ⁄ c mice were nasally inoculated with pfu of iav ⁄ pr ⁄ h n at day . immediately after infection, mice were given lg of osv orally or vehicle at -hours intervals for days. the levels of virus-specific siga in nws and bronchoalveolar fluids (balf) and igg in plasma were measured by elisa as reported previously. a retrospective clinical study was conducted. for the study, children with acute influenza were recruited and grouped according to the treatment received: days treatment with osv (n = ), cam (n = ), osv + cam (n = ), and untreated (n = ). since parents in japan are well aware of the adverse effects of osv especially the neuropsychiatric complications, the decision on whether to administer osv or not and to prescribe cam was made by the parents and the attending paediatricians, based on their anti-viral and immuno-modulatory activity. , comparisons were made of the levels of siga against iav ⁄ h n and iav ⁄ h n , total siga, in nws and disease symptoms before and after treatment. anti-ha siga and total siga in nws of patients were determined from the standard regression curves with human iga of known concentration in a human iga quantitation kit (bethy laboratories). because an affinity purified human anti-ha-specific siga standard of each influenza a subtype is not available, the relative value of anti-haspecific siga amount was expressed as unit (u). one unit was defined as the amount of one lg of human iga detected in the assay system as reported previously. the concentrations of siga in individual nws were normalized by the levels of total siga (lg ⁄ ml). oseltamivir suppresses viral rna replication and viral antigenic protein production. to investigate the influence of daily treatment with osv on ha-specific mucosal and systemic immune responses, we analyzed ha-specific siga levels in nws and balf as well as igg levels in plasma at days and post-infection in mice treated orally with osv or methylcellulose (mc) as vehicle. the osv treated mice showed lower antibody responses in nws and balf than control mice treated with mc solution (table ) . significantly reduced ha-specific siga responses were particularly noted in the osv group at day , the period of maximal mucosal siga induction. the airway secretions and plasma from mice at day did not contain detectable levels of ha-specific antibodies. these findings were supported by other data whereby mice treated with osv displayed significantly lower numbers of ha-specific iga antibody-forming cells (afcs) in the nasal lamina propria, mediastinal lymph nodes, and lungs compared with mc-treated mice. these results clearly indicate that oral administration of osv downregulates ha-specific siga responses in mucosa. on the other hand, there were no significant differences in the elevated levels of ha-specific plasma iga and igg antibodies or the increased numbers of ha-specific iga and igg afcs in the spleen between osv-and mc-treated mice. taken together, these results implicated the oral administration of osv in a suppressed induction of haspecific siga responses in respiratory lymphoid tissues, although systemic ha-specific antibody responses were not significantly affected by osv. since cam up-regulates il- , a mucosal adjuvant cytokine in the airways, and promotes the induction of siga and igg in the airway fluids of mice infected with iav, , we assessed the impact of treatment with osv and ⁄ or cam on the levels of anti-influenza siga in nws and clinical status of influenza patients. the concentration ratio of table . anti-ha-specific siga to total siga in nws was expressed as titer: anti-ha-specific siga (u ⁄ mg) ⁄ total siga (lg ⁄ mg) · . figure shows changes in the anti-ha(h n ) siga ratio (titer) and fold of increase in siga titer in each patient during the -days' treatment for the four different treatment groups. it is noteworthy that, upon admission to the hospital, the siga titers were < in % of patients. during the days of treatment, rapid increases in the titers were observed in almost all patients in cam, osv + cam, and no treatment groups. in contrast, in the osv group, the anti-ha-specific siga titers remained unchanged or decreased in the majority of patients. the finding of significant low induction of anti-viral siga in the osv group was supported by the results of animal experiments. however, the addition of cam to osv augmented siga production and restored mucosal siga levels; % of patients treated with osv + cam showed > -fold increase in the titers during treatment. these observations suggest that cam stimulated the local mucosal immunoresponse in the nasopharyngeal region of patients treated with osv. the prevalence of disease manifestations was also analyzed. among the symptoms listed, a significant decrease in the prevalence of cough was recorded between the no treatment group and the osv + cam group and between the osv group and the osv + cam group (**p < ae ), despite the limited number of patients in each group. the duration of the febrile period was significantly shorter in the osv and osv + cam groups than the no treatment group. however, no significant difference was observed between the osv group and osv + cam group. it has been reported that osv does not affect the cellular immune responses, such as cytotoxic t lymphocytes and natural killer cells. however, the effects of osv on mucosal immunity have not been studied so far. the present study showed that osv treatment of mice infected with iav induced insufficient protective mucosal siga responses in the respiratory tract, although treated mice showed the similar levels of systemic igg and iga antibody responses in plasma to those in mice treated with vehicle (table ) . the observed effect of osv on mucosal immunity was probably due to a suppression of viral replication and viral antigen production in the mucosal layer. these observations in mice are further supported by our clinical reports of siga in nws and balf of osv treated influenza patients. the membered-and membered-ring macrolides have been found to possess a wide range of anti-inflammatory and immuno-modulatory properties, , and to be effective in the treatment of respiratory syncytia and iav infection. , the efficacy of low doses administered on the long term against pathogens that are insensitive to macrolides indicates a mode of action that is separate from their antibacterial activity. , , , in the present study, we evaluated the immunomodulatory effects of cam on mucosal immune responses in pediatric influenza. a decrease in the proportion of total siga that was anti-ha-specific siga during treatment was observed in . % of patients in the osv group (those represented by the dotted lines and closed diamonds in figure ), whereas an increase in the proportion was observed in most patients of the other groups (except for one patient of the untreated group). despite the low or unchanged induction of anti-ha-specific siga in the majority of osv-treated patients, the additional use of cam with osv boosted the mucosal immune response and restored local mucosal siga levels. we are currently engaged in detailed immunological studies of the effects of cam and osv on the levels of mediators controlling iga class switching in nws of influenza patients and airway secretion of mice infected with iav. further studies should clarify the boost mechanisms of cam and the suppression mechanisms of osv in iga class switching. our findings suggest the risk of re-infection in patients showing a low mucosal response following osv treatment and cam effectively boosts the siga production for protection of re-infection. to date there is an urgent need to develop new antivirals against influenza. most of the molecules reported target influenza proteins that acquire rapid mutations of resistance. the development of new molecules that have a broad antiviral activity and are not subjected to influenza mutation is of particular interest. our laboratory and others recently showed that proteases can participate to the innate immune response in the airways through the activation of a family of receptors called par. in particular, through the release of interferon, par agonists curbed viral replication significantly in infected cells. in this study, since erk activation is crucial for virus replication, we investigated whether par could inhibit virus replication through inhibition of the erk pathway. results showed that while influenza a infection alone or par stimulation alone induced erk activation, par stimulation does not inhibit erk activation in influenza infected cells. thus, par agonists may be a potential new drug against influenza viruses that could be used in combination with other anti flu therapy such as the inhibition of the erk pathway. respiratory tract-resident proteases are key players during influenza virus type a infection. , in addition to their direct activating effect on surface viral proteins, lung mucosal proteases can regulate cellular processes by their ability to signal through protease-activated receptors (pars). after cleavage of the receptor by proteases, the new aminoterminal sequence of par binds and activates the receptor internally. these receptors are highly expressed at epithelial surfaces, in particular in the lung, where human influenza virus replicate in vivo. pars are thus directly exposed to proteases present in the airways. among the four different pars, par acts as an antiviral through an interferondependent pathway. , thus, agonists of par are potential new drugs against a broad range of influenza viruses, which is in accordance with the broad antiviral action of interferon. however, the signalling pathway induced by par agonists in influenza a infected cells has still to be investigated. in this manuscript, we showed that influenza infection or activation of par induced erk activation, a crucial step for efficient virus replication. , however, par agonists do not impaired erk activation in influenza a virus infected cells. since the pathway of par protection is likely to be erk-independent, the use of anti erk molecules in combination with par agonists maybe of potential interest in future anti-influenza therapy. influenza viruses a ⁄ wsn ⁄ (h n ) (a kind gift from nadia naffakh) was used in the present study. mdck (madin-darby canine kidney) and the human alveolar type ii a cell were obtained from atcc and grown as previously described. for western blot analysis, the following antibodies were used: monoclonal antibody for phospho-erk ⁄ (t ⁄ y ) and for erk ⁄ antibodies from cell signaling technology (beverly, ma), horseradish peroxydase (hrp)-coupled rabbit polyclonal antibodies against mouse or rabbit igg from paris (compiègne, france). a cells were infected with iav at an moi of in emem medium, as previously described. , at various time points post infection, cells were collected and proteins were analysed as previously described. , par stimulation was performed at °c in emem medium as previously described. after infection and ⁄ or stimulation, cells were lysed in ice-cold lysis buffer. lysates were centrifuged at g for min, and total proteins of the supernatants were analyzed by western blot analysis as previously described. , results since activation of the erk pathway is essential for efficient influenza replication, we first investigated the kinetics of erk activation after influenza infection in human a alveolar epithelial cells. for this purpose, a cells were infected with influenza viruses at a moi of at different time point post-infection, and activation of erk ⁄ pathway was assessed by western blot analysis using an anti-erk antibody. results showed that erk was phosphorylated after influenza infection in a time course depen-dent manner when compared to uninfected cells. in contrast, erk phosphorylation was not observed with heatinactivated viruses, suggesting that productive infection is needed for erk activation ( figure a ). antibodies against erk ⁄ were used as controls. since erk is activated after influenza infection, we then tested whether activation of par in uninfected cells also leads to activation of this pathway. for this purpose, a cells were stimulated with the selective human (h) or mouse (m) par agonist or a control peptide for the indicated time ( figure b ). when exposed to the par agonists and compared to controltreated cells, erk phosphorylation increased over the time course of stimulation. thus, influenza infection or stimulation of par without infection in a cells induced activation of the erk pathway at different time point post-infection. since influenza infection and par stimulation induced erk activation, we then investigated whether par could inhibit erk activation in influenza infected a cells. results in figure showed that in influenza infected cells, par activation for ten minutes does not inhibit erk activation after influenza infection. thus, erk activation is not inhibited by par activation in influenza stimulated cells. in this manuscript, we studied the activation of the erk pathway after par stimulation and or influenza infection. particularly interesting is the fact that either influenza infection or par stimulation alone induce erk phosphorylation in a epithelial cells, while erk activation is not inhibited in a infected cells compared to uninfected ones after par stimulation. proteases are key factor in the pathogenicity of influenza viruses. in addition to the cleavage of ha, necessary for iav replication, extracellular proteases also play a role in the modulation of the immune system against influenza viruses through the activation of pars. particularly par , activated by extracellular trypsin-like proteases, could inhibit virus replication through the release of interferon, , thus, strengthening the immune system via agonist peptides and providing new therapeutic potential against a broad range of influenza strains. in addition, targeting the host instead of the virus could provide a way to escape from virus resistance. thus, a better understanding of how virus escapes from immune surveillance may provide new therapeutic strategies to block iav. in addition, combinations of drugs that block virus replication via different pathways are of interest. the non classical molecules hla-g maybe an interesting new target as we recently showed that it is upregulated after influenza infection, and it is a well known immunotolerant molecule. indeed, it inhibits the innate immune response as well as the adaptive immune response. , also, as previously suggested, the erk signal transduction cascade is also of potential interest since it is crucial for virus replication and particularly influenza replication. , as shown here, it is unlikely that par protection occurs through an erkdependent pathway. thus strengthening the immune response with par agonists and blocking nuclear retention of the viral ribonucleoprotein complexes with inhibitors of the mek ⁄ erk pathway may be alternative combinatory approaches for influenza therapy. in addition, since those potential drugs target the host instead of the virus, this could help in the design of new antivirals molecules more resilient to iav mutations and thus to virus resistance. the initial waves of the first influenza pandemic of the st century have passed. in june , vaccine companies estimated they could produce in months almost . billion doses of pandemic vaccine. instead, they actually produced only million doses, of which % were non adjuvanted preparations. had these doses been produced with adjuvants (i.e., . lg instead of lg ha per dose), an additional billion doses could have been made available. yet there was public opposition to adjuvants in many countries, especially by regulatory officials in the united states. misperceptions about the safety of both adjuvanted and nonadjuvanted vaccines were widespread. added to this, shortfalls in vaccine production, delays in vaccine delivery, and the ''mildness'' of the pandemic itself meant that only a few countries achieved reasonable levels of vaccine coverage. millions of doses went unused and had to be destroyed. supplies of antiviral agents were even more limited. thus, despite the best efforts of influenza scientists, health officials, and companies, more than % of the world's people did not have timely access to affordable supplies of vaccines and antiviral agents. instead, they had to rely on th century public health ''technologies.'' given current understanding of biology in the early st century, they should have had -and probably could have had -something better. this report reviews evidence for an alternative approach to serious and pandemic influenza that could be used in all countries with basic health care systems. instead of confronting the influenza virus with vaccines and antiviral agents, it suggests that we might be able to modify the host response to influenza virus infection by using anti-inflammatory and immunomodulatory agents. this idea was introduced several years ago and has been reviewed in several publications. [ ] [ ] [ ] [ ] [ ] [ ] the central importance of the host response in the pandemic, young adults had high mortality rates. ever since, influenza virologists have sought to answer the question ''why did young adults die?'' by defining the molecular characteristics of the virus that were responsible for its virulence. in doing so, they have overlooked a crucial piece of clinical evidence from the pandemic: compared with young adults, children were infected more frequently with the same virus, yet they seldom died. consequently, the more important question is ''why did children live?'' this can only be explained by recognizing that children must have had a different host response to the influenza virus than adults. physicians have long recognized that for several other medical conditions, both infectious (e.g., pneumococcal bacteremia) and non-infectious (e.g., multiple trauma), children have a more benign clinical course than adults. , a corollary of this observation is that secondary bacterial pneumonia, although commonly found in young adults in , could not have been the primary cause of death. children must have had the same or higher rates of nasopharyngeal colonization with the same bacteria that were associated with pneumonia deaths in adults, yet children seldom died of secondary bacterial pneumonia. if young adults died with secondary bacterial pneumonia, underlying host factors must have made them more susceptible. few people who die of influenza do so during the first few days of illness when pro-inflammatory cytokine levels are high. instead, like patients with sepsis, they usually die in the second week, when anti-inflammatory cytokines and immunosuppression dominate. , , influenza deaths occur more frequently in older persons with cardiopulmonary conditions, diabetes, and renal disease, but as seen in the h n pandemic, they also occur in younger adults with obesity, asthma, and in women who are pregnant. regardless of age, people with all of these conditions share one characteristic in common: they have chronic low-grade inflammation. in effect, their ''innate immune rheostats'' have been set at different, and perhaps more precarious, levels that make them more vulnerable to influenza-related complications. laboratory studies of influenza virus infection confirm the importance of the host response. in several studies in mice in which the host response has been modified (e.g., cytokine knockout), survival has been improved without increasing virus replication in the lung. in fact, severe disease can be induced without any influenza virus replication. for example, fatal acute lung injury has been induced in mice by inactivated (not live) h n virus. in this model, antiviral agents would be useless; only the host response could be responsible for disease. these observations raise the following question: could the host response be modified so patients with severe seasonal and pandemic influenza might have a better chance of surviving? influenza is associated with acute coronary syndromes, and influenza vaccination and statins reduce their occurrence. these associations led to the suggestion in that statins might be used to treat pandemic influenza. other agents that might also be effective include ppara and pparc agonists (fibrates and glitazones, respectively) and ampk agonists (e.g., metformin). , these agents have been studied in laboratory models of inflammation, sepsis, acute lung injury, ischemia ⁄ reperfusion injury, energy metabolism, mitochondrial function, and programmed cell death. the results of these studies cannot be reviewed in detail here, but the major findings for cell signaling are summarized in the table . unfortunately, the results of experimental studies are not always clear cut. for example, in one study of influenza virus infected mice, il- was necessary for containing infection, but in another study il- appeared to be harmful. nonetheless, overall understand-ing of cell signaling pathways in influenza virus infections and the actions of statins, glitazones, fibrates, and ampk agonists strongly suggest that these agents could benefit patients with severe influenza. laboratory studies in mice infected with pr (h n ) h n and pandemic h n viruses show that resveratrol, fibrates, glitazones, and ampk agonists reduce mortality by - %, often when treatment is started - days following infection. - (resveratrol is a polyphenol found in red wine. it shares with these other agents many of the same cell signaling effects.) in h n -infected mice, treatment with celecoxib and mesalazine, together with zanamivir, showed better protection than zanamivir alone. remarkably, these immunomodulatory agents have not increased virus replication. even more remarkable, in another model of a highly inflammatory and frequently fatal conditionhepatic ischemia ⁄ reperfusion injury -glitazone treatment ''rolled back'' the host response of ''young adult'' mice ( - weeks old) to that of ''children'' ( - weeks old). this unique study suggests that immunomodulatory treatment might roll back the damaging and sometimes fatal host response of young adults with influenza to the more benign and rarely fatal response of children. several, but not all, observational studies have shown that outpatient statins decrease hospital admissions and mortality due to community-acquired pneumonia. for influenza itself, preliminary evidence presented in october suggests that immunomodulatory treatment of influ- table . cell signaling targets that might be affected by immunomodulatory treatment of severe seasonal and pandemic influenza* down regulate pro-inflammatory cytokines (e.g., nf-kappab, tnfa, il- , il- ) up regulate anti-inflammatory cytokines (il- , tgfb) up regulate pro-resolution factors (lipoxin a , resolvin e ) up regulate ho- and decrease tlr signaling by pamps and damps up regulate enos, downregulate inos, restore inos ⁄ enos balance and stabilize cardiovascular function decrease formation of reactive oxygen species and decrease oxidative stress improve mitochondrial function and restore mitochondrial biogenesis decrease tissue factor and its associated pro-thrombotic state stabilize the actin cytoskeleton in endothelial cells and intracellular adherins junctions, and thereby increase pulmonary barrier integrity and decrease vascular leak differentially modify caspase activation and apoptosis in epithelial and endothelial cells, macrophages, neutrophils and lymphocytes in the lung and other organs increase the bcl- ⁄ bax ratio in influenza virus-infected cells and prevent the apoptosis necessary for virus replication. *see references , , , for details. nf-kappab, nuclear factor kappab; tnfa, tumor necrosis factor alpha; tgfb, transforming growth factor beta; ho- , heme oxygenase - ; tlr, toll-like receptor; pamp, pathogen-associated molecular pattern; damp, damage associated molecular pattern; enos, endothelial nitric oxide synthase; inos, inducible nitric oxide synthase. enza patients with severe illness could be beneficial. in a study of almost patients hospitalized with laboratoryconfirmed seasonal influenza, inpatient statin treatment reduced hospital mortality by %. in these patients, the cell signaling effects of statin treatment, summarized in the table , probably acted to reduce pulmonary infiltrates, maintain oxygenation, stabilize myocardial contractility and the peripheral circulation, reverse immunosuppression, restore mitochondrial biogenesis, and prevent multi-organ failure. achieving these clinical effects led to a decrease in mortality. because of the molecular cross-talk between statins, fibrates, glitazones, and ampk agonists, , similar clinical benefits might be expected from other members of this ''family'' of immunomodulatory agents. simvastatin, pioglitazone, and metformin are produced as inexpensive generics in developing countries. they are used throughout the world in the daily treatment of millions of patients with cardiovascular diseases and diabetes. global supplies are huge. because most people with influenza recover without specific treatment (this was true in ), not all patients would require immunomodulatory agents. instead, only those at risk of ards, multi-organ failure, and death would need to be treated. importantly, the cost of treatment for an individual patient would be less than $ . (d.s. fedson, unpublished observations). moreover, unlike vaccines they could be used on the first pandemic day. thus far, influenza scientists and the institutions that support their work (e.g., nih and cdc, national health agencies in many countries, the bill and melinda gates foundation, the welcome trust, and the world health organization) have shown little interest in immunomodulatory treatment. nonetheless, when more than % of the world's people have no access to influenza vaccines and antiviral agents, their physicians must have access to an effective ''option,'' especially one that might be lifesaving. research on immunomodulatory agents for influenza must involve investigators in many fields outside influenza science -those with expertise in the molecular and cell biology of inflammation, immunity, sepsis, cardiopulmonary diseases, endocrinology and metabolism, ischemia ⁄ reperfusion injury, mitochondrial function, and cell death. laboratory studies needed to identify promising treatment agents would probably cost $ - million (d.s. the results of these studies would inform clinical trials that critical care physicians are already eager to undertake. , this work will be especially important for people in developing countries where critical care capacity is extremely limited and not likely to improve. like critical care physicians, influenza scientists too must recognize that they cannot afford not to undertake research to determine whether generic immunomodulatory agents might be useful in managing severe seasonal and pandemic influenza. the nf-kappab-inhibitor sc efficiently blocks h n influenza virus propagation in vitro and in vivo without the tendency to induce resistant virus variants introduction influenza is still one of the major plagues worldwide. the appearance of highly pathogenic avian influenza (hpai) h n viruses in humans and the emergence of resistant h n variants against neuraminidase inhibitors highlight the need for new and amply available antiviral drugs. we and others have demonstrated that influenza virus misuses the cellular ikk ⁄ nf-kappab signalling pathway for efficient replication, suggesting that this module may be a suitable target for antiviral intervention. here, we show that the novel nf-kappab inhibitor sc efficiently blocks replication of influenza a viruses, including avian and human a ⁄ h n isolates in vitro in concentrations that do not affect cell viability or metabolism. in a mouse infection model with hpai a ⁄ h n and a ⁄ h n viruses, we were able to demonstrate reduced clinical symptoms and survival of sc treated mice. moreover, influenza virus was reduced in the lung of drug-treated animals. besides this direct antiviral effect, the drug also suppresses h n -induced overproduction of cytokines and chemokines in the lung, suggesting that it might prevent hypercytokinemia we hypothesise to be associated with pathogenesis after infections with highly pathogenic influenza viruses, such as the a ⁄ h n strains. thus, a sc -based drug may serve as a broadly active nontoxic anti-influenza agent. to assess the number of infectious particles (plaque titers) in organs a plaque assay using avicel Ò was performed in -well plates as described by mastrosovich and colleagues. virus-infected cells were immunostained by incubating for hour with a monoclonal antibody specific for the influenza a virus nucleoprotein (serotec) followed by minutes incubation with peroxidase-labeled anti-mouse antibody (dianova) and minutes incubation with true blueÔ peroxidase substrate (kpl). stained plates were scanned on a flat bed scanner and the data were acquired using microsoft Ò paint software. the virus titer is given as the logarithm to the basis of the mean value. the detection limit for this test was < ae log pfu ⁄ ml. organs of infected and control mice were homogenized and incubated over night in ml trizol Ò reagent (invitrogen) at °c. total rna isolation was performed as specified by the manufacturer (invitrogen). rna was solubilised in ll rnase free water and diluted to a working concentration of ng rna ⁄ ll. reverse transcription real-time pcr was performed using quantifastÔ sybr Ò green rt-pcr kit and quantitect primer assays (qiagen) . all samples were normalized to gapdh and fold expression analyzed relative to uninfected controls. ct values were obtained with the smartcycler Ò (cepheid). to answer the question whether the nf-kappab inhibitor sc shows antiviral properties against influenza virus, h n infected mdck cells were treated with different concentrations of the inhibitor (figure ). already treatment with nm of sc led to a reduction of viral cpe of more than %. almost % protection of cells was achieved when cells were treated with lm sc . the results indicated that sc has antiviral properties at concentrations ranging from to nm. we next tested whether sc would also be effective in the mouse model of influenza virus infection. when h n mice were treated i.v. once daily for days with mg ⁄ kg sc , survival rate of the animals increased significantly (p < ae ). the same results were found when h n influenza virus infected mice were treated i.p. with mg ⁄ kg sc (data not shown). moreover, sc treatment was not only effective when the inhibitor was given prior to h n influenza virus infection, but also in a therapeutic setup when sc was applied to the animals days after infection (data not shown). since influenza virus infected mice showed increased survival after lethal infection, we next questioned whether the amount of influenza virus was reduced in the lung. therefore, we performed quantitative real-time (qrt) pcr to detect viral mrna. mice were treated with either sc or the solvent, and hour later the lungs were prepared to perform qrt-pcr. as shown in figure a the amount of viral mrna was reduced by % in sc treated mice compared to solvent treated controls, indicating that sc leads to a reduced expression of h n specific mrna in the lung of infected mice. since infection of mice with h n leads to hypercytekinemia, we also investigated the expression of cytokines in sc treated mice. as shown in figure b the amount of il- specific mrna was drastically reduced in sc treated mice compared to solvent treated controls. moreover, also the expression of ip- was altered in sc treated h n influenza virus infected mice. here, roughly % reduction of specific mrna was detectable ( figure c ). thus, sc leads to a reduced transcription of il- and ip- in h n infected mice. there is an urgent need for new concepts to develop antiviral drugs against influenza virus. targeting cellular factors is a promising but challenging approach, and the concerns about side effects are obvious. however, it should be considered that drugs targeting viral factors, such as amantadine or oseltamivir, also exhibit a wide range of side effects in patients. thus, drug safety has to be rigorously tested in clinical trials regardless whether a drug targets a cellular or a viral factor. moreover, resistance against human h n influenza viruses and highly pathogenic avian h n virus strains to oseltamivir and amantadine have been reported. in that respect, the strategy to target cellular factors , might be one way to ensure that new drugs against influenza virus will be useful and effective for a long time without causing the development of resistant virus variants. we were able to demonstrate that the nfkappab inhibitor sc is able to reduce influenza virus activity in cell culture. moreover, the compound was also effective against highly pathogenic avian influenza viruses of the h n and h n subtypes in the mouse model. next to the reduction of virus sc was also able to reduce h n -induced overproduction of cytokines and chemokines in the lung in the lung of mice after infection with h n . most importantly, the drug did not show any tendency to induce resistant virus variants (data not shown). thus, a sc based drug may serve as a broadly active non-toxic antiinfluenza agent. [ ] [ ] [ ] [ ] [ ] in hong kong, the first confirmed case was a tourist from mexico reported on may , . the local government made its first attempt to contain the spread of h n in the local community by closing the metropark hotel where that tourist was staying, and quarantining guests and staff for days. following identification of the first local case around weeks later on june , , the government closed all kindergartens and primary schools from june until early july. fever clinics were also opened, the alarm levels in hospitals were raised to the highest, and a public education campaign was implemented. previous studies of the community responses to severe acute respiratory syndrome (sars) and human-to-human h n avian flu identified the importance of understanding the background perceptions of risk and psychological impact on the community. [ ] [ ] [ ] [ ] [ ] in this study we investigated the psychological and behavioral responses of the general local community throughout the first wave of ph n , and we also examined the factors associated with greater use of preventive measures. a total of surveys were conducted between april and november , covering the entire first wave of the ph n pandemic. computer generated random-household telephone numbers from all land-based local telephone numbers covering over % of hong kong households were used to recruit a total of local adults. one cantonese-speaking adult (age ‡ ) was invited for interview in each selected household on the basis of a kish grid. the survey instrument was based on previous experience in sars and avian influenza projects. information, including knowledge on modes of transmission, psychological responses to pandemic influenza, preventive behaviors, attitudes towards the new vaccines and socio-demographics, was collected. informed consent was obtained prior to the interview. ethics approval was obtained from the institutional review board of the university of hong kong. descriptive statistics were weighted by sex and age based on the reference population data provided by the hong kong government census and statistics department. multivariable logistic regression analyses were used to examine the association between the use of preventive measures and knowledge, perceptions and behaviors, sociodemographic characteristics, and psychological responses to pandemic influenza. multiple imputation was used to cope with a small proportion of missing data and make the best use of all available data. statistical analyses were conducted in r version . . (r development core team, vienna, austria). twelve thousand and nine hundred and sixty-five local adults were recruited throughout the study period, with a total of telephone calls being made; the response rate among eligible participants was . %. hong kong entered the containment phase after the world health organization (who) announced a global alert, and policies including border screening, tracing, and quarantine of doi: . /j. - . . .x www.influenzajournal.com suspected cases were implemented. hong kong transitioned to the mitigation phase on june , when the first local case was reported. the chronology of these and other events plus the epidemic curve of laboratory-confirmed ph n cases are shown in figure (a) . the anxiety scores and risk perception of the respondents are shown in figure (b,c) . anxiety, measured by the state trait anxiety inventory, remained steady throughout the study period. in response to the announcement made by who and the unknown nature of the new virus, a higher proportion of the respondents expressed worry (more, much more, or extremely more worried than normal) if developed ili and perceived ph n severity (same, more, or much more serious than sars) initially in early may . fewer respondents reported worry if they developed ili as the pandemic proceeded, with a slight perturbation around the first deaths in july and a steady decline to . %, while perceived severity of ph n declined more dramatically after an early high. perceived risks of infection of respondents (absolute susceptibility) and risk relative to others (relative susceptibility) were also investigated and found to remain relatively stable throughout the first wave, with no indication of an increase during the period of peak ph n activity in september (figure c) . as the first wave of ph n progressed, knowledge on modes of transmission did not improve. on the contrary, later in the epidemic increasing proportions of respondents reported oral-fecal and cold weather as modes of transmission of ph n . around - % of the respondents did not recognize direct and indirect contact or touching infected persons and contaminated objects as transmission routes for ph n throughout the first wave ( figure d ). higher proportions of respondents avoided crowded places and rescheduled travel plans in the second half of june when local kindergartens and primary schools were closed and the first ph n -associated deaths were announced. social distancing measures such as avoiding crowded places and rescheduling travel plans remained stable with slightly decreasing trends thereafter. the use of hygiene measures and other social distancing strategies was relatively stable with slightly decreasing trends during the study period ( figure ). female sex and older age were generally associated with greater reported use of hand hygiene measures, home disinfection, avoidance of crowded places, and rescheduling of travel plans. female sex was also positively correlated with use of face masks and cough etiquette. we found a negative correlation between anxiety and use of all hand hygiene measures and cough etiquette, but a positive correlation between anxiety and use of home disinfection and (c) proportion of the respondents reporting higher worry if developed flu-like symptoms (more, much more, or extremely worried), higher perceived seriousness of h n compared to sars (much more or more severe), higher probability to contract h n over the next month (certain, much more, or more likely), higher probability to contract h n over the next month compared to others outside family (certain, much more, or more likely). (d) proportion of the respondents identifying possible modes of transmission as the actual modes of transmission of h n . social distancing measures. other significant factors contributing to greater use of preventive measures were worry and knowledge. greater worry was associated with higher probability of home disinfection, social distancing measures, and use of face masks. knowledge that h n could be spread by indirect contact was associated all the investigated preventive measures, and knowledge that h n could be spread by droplets was associated with cough etiquette, but not face masks. there were no consistent trends between all the investigated preventive measures and absolute and relative susceptibility. community transmission emerged in hong kong in mid-june , and prior to emergence of community transmission, perceived risk and perceived severity were high. as ph n spread in hong kong, risk perception declined, even at the same time as incidence was increasing. anxiety was low throughout, at around . on the -point scale, compared to a maximum of . during sars on the same scale. anxiety has been showed to be positively correlated to personal hygiene measures and social distancing in previous studies; , however, we found a negative correlation between anxiety and use of all hand hygiene measures, cough etiquette, and face masks, and a positive correlation between anxiety and home disinfection. the differences in findings may be due to the fact that our anxiety measure was not specific to h n , and the score could be affected by other factors including economics. unlike hygiene measures, higher anxiety level, greater worry, and higher risk of perception were all associated with more social distancing. , , , social distancing is the most direct strategy in avoiding infection from other people, and it is commonly observed in an outbreak that the general public avoids crowded places, travelling to other countries, and social gatherings, , but the economic impact could be substantial. as community incidence of h n peaked, we did not observe any increase in use of preventive measures (figure ) . we found that face mask use peaked at the early stage of the pandemic, while hand hygiene remained fairly constant, and the knowledge on the modes of transmission of ph n did not improve over time. the lack of substantial change in preventive measures or knowledge about the modes of ph n transmission in the general population suggests that community mitigation measures played little role in mitigating the impact of ph n in hong kong. on the other hand, knowledge that ph n could be spread by indirect contact was associated with all of the preventive measures studied. consistent with reports during the sars period, , this study also showed that females and those of older age were more likely than others to use hygiene measures, avoid crowded places, and reschedule travel plans. this study has some limitations. first, this was a crosssectional study that was carried out at different time points, rather than a longitudinal study following the same individuals over time, and so the inferences on changes in behavior may need to be interpreted more cautiously. second, we recruited samples from all land-based local telephone numbers that cover % of hong kong households, but the response rate was not high enough to guarantee a representative sample, and this could be a source of selection bias. third, the responses were self-reported, and this may lead to social desirability bias in estimating knowledge, attitudes, and preventive behaviors. fourth, since the hong kong population has previously gone through unique experiences from sars in and avian flu in , our results may not be comparable to other countries or settings. in conclusion, this study revealed that the ph n pandemic failed to generate an increase use of preventive measures in the local community. there was no association between anxiety level and the events of the pandemic. with a relatively low mortality and morbidity rates compared to sars, ph n was not a matter of concern in the hong kong community. the lack of substantial change in the use of preventive measures and improvement in knowledge on the modes of transmission of ph n suggested that public health campaigns during the pandemic may not have had substantial effects on the general public. london is a major tourist destination, the seat of government and finance in the uk, and in will host much of the olympic and paralympic games. along with the rest of the global community, in and early london faced the challenges of responding to the first pandemic of the st century. at the time, nhs in london was composed of organisations, including the london ambulance service, acute hospitals, mental health and primary care trusts, and the strategic health authority. while london's nhs is well practiced at responding to large, big bang incidents, the influenza a ⁄ h n v pandemic was a rising tide event that lasted many months. significant preparatory work had been undertaken prior to april , which meant that the nhs in london was ready to respond. nhs london (the strategic health authority for london) led the response in partnership with local managers in all nhs organisations. the first uk cases of influenza a ⁄ h n v were reported in scotland on april, with the first in london on april. cases continued to increase, and the first wave peaked in london in july. cases reduced over the school summer holidays, but increased again when children returned to school at the start of september, and a second, smaller wave occurred. it is essential that the nhs learns from the ⁄ influenza a ⁄ h n v pandemic to ensure it is prepared for future challenges. nhs london provided a standardised debriefing pack to all nhs organisations in the region to identify, capture, and learn lessons. each debrief event involved health and inter-agency partners to ensure all viewpoints were considered and brought together in a single local report. all local reports were compiled in an over-arching document, which brings together common themes to inform ongoing preparedness in the region. the debrief process identified a number of common themes, such as the need for clear and appropriate communication, the importance of working with partners, and the benefits of strong and early leadership. however, differences between and within organisations were also highlighted; for example, some wanted more freedom for local decision making, whereas others would have preferred more stringently applied central direction. the following paragraphs considers individual areas assessed in the debrief process. command and control was in the main effective, with clear direction delivered from the national centre through nhs london to local nhs organisations. effective leadership is essential; the identification of senior local individuals to lead the response with teams of people to support them was critical. appropriate use of technology to communicate messages and coordinate command and control processes greatly aided the response. this included the development of the nhs london noon brief, a daily digest and associated web portal, and regular teleconferencing. key points are: • operational management at all levels must be considered in pandemic planning. • appointing an executive lead in each organisation was invaluable in the response. • pandemic flu planning for london must continue to be regionally led. communication is an essential component of the response to any incident. it must be clear, timely, and accurate. in the main, communication was excellent and met these criteria. one of the most challenging aspects was when messages from partner organisations differed, which occasionally led to confusion, unnecessary work, or frustration. the use of technology greatly aided communication across the region and supported the response; this included secure web sites, bluetooth, and text messaging etc. key points are: • regular internal communications and staff briefings are critical in the response to emergencies. • regular teleconferencing should be incorporated into future plans. • organisations should consider proactive and innovative methods for communicating during emergencies. robust partnership working was an essential component of pandemic preparedness work; however in the event, the a ⁄ h n v pandemic had little impact on sectors in london other than health. resilient communication networks between organisations, a common understanding, and the ability to make decisions were essential to the response at local level. ipcs proved an excellent mechanism to maintain local working relationships and resolve problems. clarity on the seniority of those attending these meetings and whether multi-site organisations such as mental health trusts should attend every ipc should be considered on a local and regional basis. key points are: • pandemic planning must remain part of inter-agency working. • social care resilience and planning must be embedded and integrated in health planning. 'vulnerable groups' is a universal term that covers a large and fluid group of individuals with different needs. ensuring access to healthcare during the pandemic for those who became vulnerable due to the situation, or those identified as such prior to the event, was the role of the pct in partnership with the local authorities. work continues to ensure that communication with vulnerable people is appropriate and timely in all incidents, and that organisations work together to achieve this. key points are: • planning to support the breadth of vulnerable people must continue. • pandemic preparedness for the prison sector should be further developed. • red ⁄ amber ⁄ green ratings for assessing vulnerabilities of mental health service users in an emergency should be further developed across the region. correct and appropriate usage of ppe is an essential component of reducing influenza spread, particularly in healthcare settings. london's nhs had been working towards developing local stockpiles of ppe when the pandemic commenced; however, there was little in place. the unanticipated national stockpile, while providing ppe to all organisations, was accompanied with some challenges in that it was often unfamiliar stock. key points are: • work around local stockpiling of non-standard consumables should continue. • regular training and fit testing of respirators should be embedded in all organisations. antiviral treatment was a core component of the response to influenza a ⁄ h n v, and was provided free of charge from a national stockpile. npfs reduced pressure on frontline nhs services once it was activated; however, there were concerns that patients could 'cheat' the system and obtain the drugs prior their clinical need. information about storage requirements of countermeasures must be clearly explained when they are delivered to frontline services, and the potential for recall into national stockpiles should be planned for. key points are: • regular exercising of local mass countermeasures centres and antiviral collection points (acps) should continue. • the use of community pharmacies as acps should be further considered in the capital. pandemic influenza vaccine uptake by healthcare workers was better than usual seasonal influenza uptake in the majority of nhs organisations, but could have been even better. this was largely due to the second pandemic wave not being as significant as expected, lack of clarity around when the vaccine would be delivered, and limited amounts being available initially. • gp-led and mass vaccination models for pandemic vaccination should be considered in local plans. • local lessons from the pandemic vaccination campaign should be applied to seasonal flu vaccination. the ability to maintain or increase capacity in response to a surge in demand, no matter what the cause, must be planned for. any of a number of situations could result in reduced staff or more patients, such as industrial action, transport disruption, disease outbreak, major incident, or poor weather. the work undertaken during planning for and responding to the pandemic will stand organisations in good stead for future disruptions. the importance of robust business continuity planning locally cannot be overlooked, as this is a key component of maintaining and increasing capacity. key points are: • local gp 'buddy schemes' should be encouraged for response to extreme pressure events. • organisations should regularly run staff skills audits so as to be aware of their overall capability for managing emergencies. • less emphasis should be placed on the use of retired staff when planning service continuity. reporting is a necessary but onerous task, and is often one of the most time-demanding parts of any incident response. it is also the aspect least likely to be tested through exercising. nhs london worked with organisations to endeavour to reduce reporting pressures, but much of this was dictated by central government. it is essential that future reporting requirements are proportional, informative, and realistic. while recognising it is not possible to predict the detail of information that may be requested, some broad assumptions can be made. key points are: • organisations should consider how they would collect and collate data from disparate parts of their organisation, rather than focussing on the detail of what that might be. • national and regional planning should consider the need for information and how this is balanced with the demand this places on organisations. • the introduction of the concept of a daily dashboard to identify areas of pressure should be incorporated into pandemic flu planning. the winter and pandemic influenza resilience assurance process undertaken in autumn was a useful process to inform planning for the first winter when the pandemic virus would be circulating in the uk. this consisted of a regional inter-agency exercise and a comprehensive review of the winter and pandemic plans of all nhs organisations in london. • regular assurance of pandemic flu preparedness should be maintained. • future resilience assurance processes should be undertaken in a timely and measured manner. • local organisations should continue to undertake regular pandemic flu exercises. the recovery period is as important as the response, but often receives minimal attention and has the potential to suffer as staff return to their normal jobs. one of the aspects that was not anticipated during the pandemic was the amount of stock (ppe, antivirals, and vaccine consumables) that would be recalled into national stockpiles. this proved particularly challenging for pcts who had to coordinate the process across their local areas. key points are: • the recovery period of an emergency must be given the same status and importance as the response. • future pandemic flu planning must include the recovery of national stockpiles of equipment and medicines. it is essential the lessons from the ⁄ influenza a ⁄ h n v pandemic are learnt and embedded into business-as-usual and emergency response processes in preparation for the next pandemic and other incidents. even though the a ⁄ h n v pandemic was generally milder than previous pandemics, it still presented challenges to the nhs in london. the biggest challenge that remains is to ensure that the public and nhs staff are aware that a more virulent virus could cause significantly more illness, death, and disruption, and that we must maintain our preparedness should this happen. the influenza a ⁄ h n v pandemic has been a major stimulus to business continuity planning and emergency preparedness across health in london, and many of the experiences during the pandemic proved invaluable in the unusually severe weather in early . it is important that this impetus and focus is maintained. changes to the nhs landscape in london will be considered in ongoing pandemic and emergency preparedness to ensure we remain as well prepared as possible for future events, particularly as london approaches the olympic and paralympic games. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. the way national health systems operate during inter-pandemic and the pandemic alert periods and the methods they use to address potential threats posed by zoonotic viruses with pandemic potential, as well as sea-sonal influenza epidemics, can clearly indicate whether the countries have enough capacities to respond adequately to unexpected influenza outbreaks. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during a pandemic scenario, the group prioritized topics and questions relating to rapid action and response. five to key public health needs associated with a pandemic scenario have been identified for each of the research agenda streams: five priority public health topics were identified for a pandemic scenario as follows: • examination of host range and transmission dynamics of animal influenza viruses to guide surveillance, control strategies, and risk communication. • enhanced surveillance in animals and humans to monitor virus evolution: o early detection of novel reassortants or changes in genotype and ⁄ or phenotype related to virulence. o development of epidemiological and laboratory diagnostic tools and capacity building to optimize case finding. o develop a framework for surveillance in animals that address ethical, legal, and social barriers to intra-pandemic surveillance and reporting. • deconstruct the origins of the pandemic virus to identify factors that permitted efficient human transmission. • develop strategies to limit economic, social, and cultural disincentives of animal-based interventions to reduce intra-and inter-species transmission. • operational research to optimize risk communication in the early phases of the pandemic linked to animal husbandry and food safety. stream : limiting the spread of pandemic, zoonotic and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: transmissibility of influenza across the progression of infection and spectrum of disease: • relative contributions of the different modes of transmission for influenza. five priority public health topics were identified for a pandemic scenario as follows: • identification of groups at higher risk of infection and severe disease outcome through enhanced surveillance. • understanding disease severity and identification of predictors of severe outcomes. • investigation of vaccine effectiveness, especially in high risk groups in diverse geographic areas. • establishment ⁄ enhancement of pharmacovigilance, particularly for adverse events among at-risk groups. • optimization of strategies for rapid and targeted vaccine deployment. • rapid assessment to optimize acceptance of pandemic vaccine. six priority public health topics were identified for a pandemic scenario as follows: • collaboration and coordinated sharing of data, protocols, regulatory, and other implementation strategies and databases from different countries on all aspects of patient management and outcome to accelerate improvements in patient care. • development of best practices in patient management in different settings, including checklists and algorithms for clinical care and treatment, prognostic parameters, and tests to predict potential for the development of severe disease. • rapid, reliable, simple, low-cost point-of-care diagnostic tools for influenza. • best use of current antiviral drugs and optimal formulations in different target populations, such as parenteral and other routes of administration for severe infections. • use of combination therapies, including use of adjunctive therapies (e.g., use of convalescent serum and immunomodulators). • role of ongoing viral replication, host responses, and the effect of co-infections in the pathogenesis of severe disease. modern tools for early detection and monitoring of disease the group on surveillance tools concluded that the agreed topics of interest were equally applicable during a pandemic or inter-pandemic period: • studies to appraise and adapt modern technologies for early detection of influenza outbreaks in surveillance at the human-animal interface. • develop, integrate, and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems. • study efficient mechanisms on sharing data, clinical specimens, and viruses with consideration for local, ethical, legal, and research perspectives. • examine the timeliness and quality of data required for early detection from local to national and global levels for the respective stakeholders. five priority public health topics were identified for a pandemic scenario as follows: • identify environmental determinants of seasonal variation in influenza transmissibility in tropical and temperate regions. • estimate the transmission risk associated with types of contacts by comparing measured contact patterns with outbreak data. • incorporation of validated models of behavioral responses to risk and control measures in virus transmission. • development and implementation of novel technology for real-time sero-surveillance during a pandemic. • develop experimental and theoretical framework to assess host adaptation to study host receptor, antigenicity, and virulence. modern tools for strategic communication three priority public health topics were identified for a pandemic scenario as follows: • evaluate tools to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups to guide future communication efforts; develop tools and methods to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups, and thereby, guide future communication efforts. for communicating in different cultural settings, which engage and empower individuals and communities to practice and promote appropriate risk reduction measures. implementation of the identified research priorities is expected to underpin public health decision making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs and economic loss, and mitigate potential social disruption. complemented by an analogous research roadmap for a pandemic influenza scenario, the research recommendations for an interpandemic period represent a framework to provide evidence to guide public health policies on influenza control. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting public health needs for inter-pandemic periods according to its five key research streams: • stream . reducing the risk of emergence of pandemic influenza. • stream . limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza. • stream . minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza. • stream . optimizing the treatment of patients. • stream . promoting the development and application of modern public health tools. stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza inter-pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during an inter-pandemic phase, a more comprehensive approach was applied to establish research topics and prioritizing a range of questions that will build a solid foundation to guide research activities to support public health decision making. five to ten key public health needs associated with an inter-pandemic scenario have been identified for each of the research agenda streams: stream : limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: . transmissibility of influenza across the progression of infection and spectrum of disease . relative contributions of the different modes of transmission for influenza . biological, behavioral, and social host factors that influence the risk of transmission and infection . patterns, drivers, and mechanisms affecting the seasonality of transmission . viral and population factors that influence transmission and spread of different influenza types, subtypes, and strains . strategies to reduce the transmission of influenza in community, household, and health care settings, especially in less-resourced areas . impact and cost effectiveness of social measures, such as school closures, and the role of surveillance in assessing timing of these interventions . impact, effectiveness, and cost effectiveness of individual measures, such as isolation and quarantine . role of vaccination in limiting the spread of influenza and strategies for its use . impact of antiviral treatment and prophylaxis in reducing transmission of influenza stream : minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza . identify higher risk groups and severe disease through surveillance; disease severity and identification of predictors of severe outcomes . evaluate vaccination preventable disease burden and the potential impact of immunization programs through vaccine demonstration projects . enhancement of the properties of existing vaccines, including duration and breadth of protection, safety, immunogenicity, and dosesparing . development of new vaccines and vaccine platforms, especially suitable for under-resourced country settings . study the effectiveness of vaccine strategies to reduce disease burden in children and other high risk groups in a wide range of settings . improved uptake and acceptability of vaccines for both seasonal and pandemic influenza seven priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: inter-pandemic seasonal influenza scenario . research on the burden of severe disease with a focus on regionalspecific factors, such as the burden of tb and hiv and optimization of pandemic and management . development of new antiviral strategies and validation of surrogate endpoints which may aid in advancing understanding of disease progression . further clinical evaluation of current antiviral drugs, particularly in populations at risk . integration of seasonal influenza with pandemic preparedness; strengthen surveillance, health care systems, capacity, and preparedness planning . improving diagnostics (e.g., multiplex assays for viruses and bacteria), including antiviral resistance testing at point-of-care . dissemination of best practices, situation analysis, preparation for next epidemic (e.g., establish protocols for rotating stockpiles of antiviral drugs) . increased attention to basic science research such as studying immunomodulatory drugs five priority public health topics were identified for an inter-pandemic zoonotic influenza scenario as follows: inter-pandemic zoonotic influenza . antiviral susceptibility of circulating zoonotic viruses (e.g., h , h , h influenza viruses) . reassortment between zoonotic and human influenza viruses and the potential for inter sub-type spread of antiviral resistance and virulence modern tools for early detection and monitoring of disease the group focusing on surveillance tools concluded that the agreed topics of interest were equally applicable during both pandemic and inter-pandemic period: . identify modern technologies for early detection of influenza outbreaks as well as their application in surveillance at the human-animal interface . develop and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems . studies to address challenges on data, clinical specimens, and viruses sharing with consideration for local, ethical, legal, and research perspectives . examine the timeliness and quality of data required for early detection from local to regional, national, and global levels role of modeling in public health decision making five priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: . integration of genetic and epidemiological data to understand spatiotemporal spread to forecasts evolution for vaccine strain selection and to anticipate likely burden of disease . quantifying the relative contributions of different modes of transmission of human influenza and developing mechanistic modeling of transmission processes . research using data-capture technologies to characterize human contact and mobility patterns at local, regional, and global scales, and their correlation with transmission risk . integration of genetic, antigenic, and epidemiological analyses to optimize surveillance for newly emerging pathogens at the animal ⁄ human interface . identifying and quantifying human and environmental ecological, behavioral, and demographic determinants of the risk of cross-species transmission and pandemic emergence modern tools for strategic communication four priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: . review of evidence and experience related to health crisis communication from fields to organize knowledge and support evidencebased practice in strategic communication . identify and develop tools to rapidly and accurately monitor knowledge, attitudes, and practices in different population groups and guide future communication efforts . identify and develop communication tools and approaches for cultural settings and communities to practice and promote appropriate risk reduction measures . understand the potential ethical, social, economic, and political communication in crisis and develop strategies to work within constraints while maximizing opportunities complemented by an analogous research roadmap for a pandemic influenza scenario, the research topic recommendations for an inter-pandemic period represent an important outcome of joint international efforts by who, academicians, and public health experts. implementation of the identified research priorities is expected to underpin public health decision-making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs, and economic loss and mitigate potential social disruption over a medium-tolong term period. the impacts of school resumption on the incidence of pandemic (h n ) in school students introduction school closure is one non-pharmaceutical intervention that is often suggested in pandemic preparedness plans, and it was widely implemented in pandemic (h n ) to reduce transmission amongst school students. however, from past epidemiological studies, the effect of school closure in reducing respiratory disease transmission was inconclusive. given this public health intervention causes major disruption to the education system and potentially raises childcare issues to working parents, evaluating its effect in the recent pandemic is necessary to improve future pandemic planning. in hong kong, since school closure was implemented early in the pandemic and closure was effectively continued with the commencement of summer holiday, the lack of incidence data in the absence of school closure makes it difficult to analyse its effect directly. this has prompted us to analyse the situation indirectly from the angle of school resumption after summer holiday. in hong kong, public health surveillance on pandemic (h n ) was effective from th april- th september : healthcare professionals were advised to report suspected cases of infection to centre for health protection, department of health, hksar, for further laboratorial confirmation. demographics of reported cases were subsequently recorded into a computerised system (the ''e-flu'' database). following institutional approval, a dataset of all confirmed cases diagnosed from may to september was obtained, which included the age, gender, confirmation date, and notification date of each report. all cases were classified into four defined socio-economic classes by age: pre-schoolers ( - ), school students ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , adults ( - ), and retirees ( ‡ ). assuming cases had contracted infection on the earlier date between confirmation and notification, daily incidence in each age class was counted for epidemic curve construction. upon observing an unusual rise in the epidemic curve of school students when school season resumed in september, interrupted time series analysis (also known as intervention analysis) was applied to obtain the statistical significance of this observation. the analysis was applied to the incidence in school students from th july to th september , which covered the period from the start of summer holiday to the end of the th week of new school season. incidence in school students before summer holiday was deliberately dropped since not all schools were closed when the school closure policy was effective: all primary schools were closed proactively, whereas secondary schools were individually closed on a reactive basis if students were identified to have contracted the infection. school activity was formulated as a step function, which takes value from st september onwards (st = : t < st september, st = otherwise). a range of times series models were fitted by the maximum likelihood method and aic (akakine information criterion) was used to select the one with best fit. all computations were performed in sas version . . a total of ( ae %) pre-schoolers, ( ae %) school students, ( ae %) adults, and ( ae %) retirees were diagnosed with the infection in the surveillance period. the epidemic curves of preschoolers, school students, and adults showed a steady rise from th june onwards when local transmission of pandemic influenza was identified. an upsurge in the epidemic curve of school students can be observed in early september, coinciding with the commencement of the new school year (figure ) . interrupted time series analysis on the epidemic curve of school students returned an arima( , , ) model with equations: where st, yt, yt denote school activity, predicted and actual incidence in school students on day t, respectively. standard error and significance for model constants were: ae (se = ae , p = ae ), ae (se = ae , p = ae ). in short, the model can be interpreted as: the number of infected school students rose by ae per day on average during the entire study period, with a sharp increase by ae coming into effect when the new school year began. time series analysis showed, at the marginally significance level, that daily incidence in school students had a major increase when school season resumed. on the assumption that the increase was not caused by any change in health seeking behaviour, this result suggests that school resumption had facilitated transmission amongst school students. on the basis that school activity significantly increases incidence of pandemic influenza in school students, this study suggests closures of schools in the early phase of pandemic (h n ) and subsequently in the summer holiday probably had a major effect in mitigating transmission amongst school students. youngsters were postulated to be major vector for transmission in pandemic (h n ) . if this were true, it would be reasonable to expect the epidemic curves of the other age classes to show a similar upsurge when one is observed in school students. the absence of such observation in the epidemic curve of hong kong suggests school students were mostly disseminating the virus amongst themselves, but not to the other age groups. in november , gip convened the first global consultation on a public health research agenda for influenza to identify key research topics in each of the five main streams of public health research. during this meeting, the scientific working group (swg) of the sub-stream in ''modern tools for risk communication'' identified the requirements in research during influenza pandemics and inter-pandemic periods to provide clear, credible, and appropriate messages which meet the needs of diverse communities. the swg suggested that who hold a follow-up workshop to assess the use of modern tools related to strategic and risk communication and to further promote research in these areas. communication'' in may . one of the main objectives of the meeting was to generate a roadmap of public health research priorities related to strategic and risk communication. the research roadmap was developed by the group of invited experts on the basis of an analysis of available evidence and experience on public health and health crisis communication from relevant disciplines across global regions, as well as critical assessment of existing communication methods related to influenza control in different cultural, social, and ethnic settings. the workshop consisted of a series of presentations by experts in relation to experiences and lessons learned about communication during the sars, h n epidemic, and h n pandemic. there were also a series of group discussions on identifying research needs for pandemic and interpandemic periods in order to strengthen the research agenda. the expert group identified important public health needs in relation to communication during pandemics as well as in the inter-pandemic times. the main topics of discussion centered on communicating issues of influenza virus transmission, the use of influenza vaccines safety and efficacy, and use of antivirals as well as definition of the severity of the pandemic and the phase changes. in this context a number of research areas were identified, which can be broadly classified into four areas: understanding of communication principles and mechanisms is associated with an array of research topics covering different subject areas. one of the key questions here relates to the link between communication and ''behaviour change'' models and their application and appropriateness for different settings. the expert group defined the term ''behaviour change'' in this context as the modification of behaviour towards better health practices that are supported by clinical and scientific evidence for personal protection against infectious diseases and other adverse health risks. research topics related to these models require understanding and differentiating information and ''behaviour change'' needs of different audience segments, such as stakeholder mapping, target audience analysis, research into behaviour motivation, social norms, and the cultural, religious, social, legal, and political barriers and enablers of particular behaviors that are beneficial in influenza control. this research area also includes the analysis of media consumption among different audiences, role models, including ways to analyse how rumours and misinformation are spread, and ways to provide evidence-based information correctly. other important areas of investigation embrace methods to communicate uncertainty, learning how to build trust while communicating about a pandemic, and understanding what needs to be done before, during, and after a pandemic in order to create the best environment for influenza pandemic communication. critical key audiences identified for more intensive analysis were health workers, religious, public health, and societal (political and community) leaders. • investigation of the role of different communication channels and communication formats for different target audiences in a pandemic, particularly for groups that are ''hard-to-reach.'' • determining effects of perceptions related to pandemic influenza (severity, susceptibility, response efficacy, self efficacy, perceived social norms) on protective behaviours in different groups. • understanding audience in terms of their knowledge, preventive activities, and reasons why engaged ⁄ not engaged. • developing mechanisms to synergies between risk communication and behavior oriented approaches in the pandemic and inter-pandemic phases. • determining social, economic, cultural, and religious factors which support behaviours to limit spread and minimize impact in different settings. • identification of the key predictors ⁄ factors that influence people's behavior among different groups and populations vis-à -vis pandemic flu behaviors. • identification of elements that contribute to trust among populations and in different settings (country, public, professional, community), particularly where trust was previously compromised. • understanding psychology of different groups regarding their response to uncertainty, and finding the best way to communicate uncertainty. the research questions in this section relate to the planning, development, and evaluation of tools that can be quickly accessed and used in a pandemic situation. these may include communication materials and channels; the setting up of key stakeholder and champion communication networks; research protocols that are ready for rapid assessment during a pandemic or new communication tools. the use and understanding of terminology and language by both lay and professional groups and communities in planning for and ⁄ or reacting to a pandemic are important areas of research. acute examples, such as the naming of the viruses or the use of the word ''pandemic,'' illustrate this need well. the research focus of this area is to look at lessons learned from the a(h n ) pandemic and to document and evaluate case studies, both looking at best practices, challenges, and barriers that were experienced. different communication strategies need to be evaluated and models to be built not only in terms of reach, but also in terms of impact on thinking, emotional response, and behavioural modification. a key question was how to prepare communication for a pandemic and how can the pandemic communication contribute to longer term ''behavioural change.'' mathematical modelling on gauging outcomes of such ''behaviour change'' would provide strategic approaches in risk communication. this section aims to answer the question whether the modeling, mapping, and scenario planning are actually useful in the pandemic situation. the expert group agreed that the research on the above issues should use a variety of methods and engage a number of disciplines. this would include literature reviews, case studies, trials, ethnographic studies, modelling, surveys, network analysis, as well as any other useful methodology. in an inter-pandemic situation for actual behaviour under pandemic conditions. • study the synergies and develop priority research topics on strategic ⁄ risk communication for influenza under inter-pandemic situations that includes zoonotic and seasonal infections. the who public health research agenda for influenza initiated and facilitated a multi-disciplinary discussion for communication during pandemic and inter-pandemic situations. it focused on both theoretical and practical issues to improve practice and ensure the health of the public for influenza. critical areas for research were identified to build evidence in this field. it was recognized that there are extensive bodies of knowledge in a number of disciplines, , such as health promotion, behavioural psychology, social sciences, social and behaviour change communication, social marketing, and communication for development relating to these questions, and that these should be explored. outcomes of these research activities are expected to widen the evidence base which will support developing communication strategies for influenza by countries, institutions, and individuals and will, consequently, help to improve public health world-wide. abstract background: cytokine dysregulation contributes to the unusual severity of h n (reviewed in ). previously, we demonstrated that interferon regulatory factor (irf ) and p map kinase (p ) signaling pathways separately contribute to the induction of pro-inflammatory cytokines and chemokines in h n -infected cells. here we investigate the role of innate sensing receptors in the induction of these cytokines and chemokines in response to h n and seasonal h n infection. materials and methods: human macrophages derived from peripheral blood monocytes were infected with h n ( ⁄ ) or seasonal h n ( ⁄ ) viruses. the role of innate sensing receptors in cytokine and chemokine induction by h n virus was investigated using transient knock-down of these receptors with sirnas. the expression of innate sensing receptors in infected cells, and as a result of paracrine activation (by virus free supernatants of infected cells) of adjacent uninfected cells were also monitored by real-time pcr and ⁄ or western blotting. the involvement of janus kinase (jak) signaling pathways in these autocrine ⁄ paracrine cascades was investigated using a jak inhibitor. results: we previously showed that tnf-alpha, ifn-beta, and ifn-lambda are the key mediators directly induced by the h n virus in primary human macrophages with other cytokines and chemokines being induced as part of a secondary autocrine and paracrine cascade. here we demonstrated that retinoicacid-inducible gene i (rig-i) rather than toll-like receptor (tlr ) plays the predominant role in h n -induced cytokines and chemokines in human macrophages via the regulation of irf and nf-kb nuclear translocation. in addition to the effects on virus infected cells, paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the upregulation of sensing receptors. conclusions: h n directly induced tnf-alpha and ifnbeta mainly via rig-i signaling, and the subsequent activa-tion and nuclear translocation of irf and nf-kb in human macrophages. in addition to the effects on cytokine signaling, the innate immune sensing regulators themselves were also up-regulated by h n infection, much more so than by seasonal influenza infection, via jak signaling. the up-regulation of innate sensing receptors was not limited to the infected cells, but was also found in adjacent uninfected cells through paracrine feedback mechanisms. this may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. a more precise understanding of the signaling pathways triggered by h n virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h n influenza and also for treating other causes of acute respiratory disease syndrome. human h n infection is associated with a mortality rate of more than %. the basis for the unusual severity of h n disease has not been fully explained. cytokine dysregulation has been suggested to contribute to the disease severity of h n (reviewed in ). however, signaling pathways involved in the cytokine induction by h n virus are not fully understood. previously, we demonstrated that irf and p map kinase (p ) are separate signaling pathways which contribute to the induction of pro-inflammatory cytokines and chemokines in h n -infected cells. rig-i and melanoma differentiation-associated gene (mda ) are important cytosolic sensors of nucleic acid of pathogens, while tlr and tlr also recognize nucleic acid species of pathogens, but they are localized at the endosomal membrane. rig-i was found to be responsible for the recognition of influenza a virus infection, and the transfection of vrnps induces ifn-beta expression. while many studies have shown the role of rig-i in the induction of ifn-beta by influenza virus infection, the majority of these studies used either immortalized cell lines or mouse embryonic fibroblasts. there is a lack of data on the role of these innate sensing receptors in highly pathogenic avian influenza h n infection in primary human cells in vitro, which are more physiologically relevant. furthermore, there is little data on the autocrine and paracrine up-regulation of these innate immune sensors following virus infection. human macrophages were obtained from peripheral blood monocytes by adhesion and differentiation in vitro for days in rpmi medium supplemented with % autologous plasma. the cells were infected with h n ( ⁄ ) or seasonal h n ( ⁄ ) viruses at a moi of ae . a cells were obtained from atcc and cultured in mem medium supplemented with % fcs and % penicillin and streptomycin. the role of innate sensing receptors in cytokine induction by h n and h n viruses was investigated using transient knock down of these receptors with sirnas in human macrophages as previously described using specific sirnas purchased from qiagen. immunofluorescence staining assay of irf and nf-jb was employed to detect the nuclear translocation of these transcription factors after h n infection. rabbit polyclonal antibodies against human irf and and nf-kb were obtained from santa cruz biotechnology. goat anti-rabbit igg antibody conjugated with alexa fluor was a product of molecular probes. for investigation of paracrine effects on rig-i and tlr expression, culture supernatants collected from mock, ⁄ or ⁄ infected human macrophages were used to treat uninfected cells. the supernatants were first passed through a filter with -kda cut-off. virus particles as well as molecules with a molecular weight higher than kda were retained and removed, while the filtrate was collected for treatment of uninfected cells. the expression of innate sensing receptors in infected cells and in adjacent uninfected cells following paracrine activation by virus free supernatants of infected cells was monitored by real-time pcr. the involvement of jak signaling pathways in these paracrine cascades was investigated using a jak inhibitor (calbiochem). we previously showed that tnf-alpha, ifn-beta, and ifnlambda are the key mediators directly induced by the h n virus in primary human macrophages with others being induced as part of a secondary autocrine and paracrine cascade. in this study, we demonstrate that knockdown of rig-i or tlr led to the reduction of ifn-beta and tnf-alpha in human macrophages by both ⁄ (h n ) and ⁄ (h n ) infection. as shown in figure a , ⁄ virus induced higher level of ifn-beta mrna expression than ⁄ infection. cells transfected with rig-i or tlr sirna significantly reduced the expression of ifn-beta after ⁄ infection, by % and %, respectively. rig-i silencing also significantly reduced the ifn-beta expression in ⁄ infected cells by %. in contrast, silencing of mda or tlr did not suppress the induction of ifn-beta by either ⁄ or ⁄ infection; in fact, there was a slight ( %) increase of ifn-beta in cells transfected with mda sirna. based on these results we conclude that while both rig-i and tlr contribute to h n -induced interferon-beta induction in human macrophages, rig-i plays the dominant role. in order to investigate the relationship between these innate sensing receptors and the activation of transcription factors irf and nf-jb, we next measured the nuclear translocation of irf and nf-jb in cells with rig-i or tlr silencing after h n infection. immunofluorescence staining assay on irf and nf-jb was performed and the number of cells with nuclear translocation was quantitated. the percentages of cells with nuclear translocation were plotted in figure b . we demonstrated that rig-i knockdown led to a significant reduction of irf nuclear translocation after ⁄ infection, whereas the nuclear translocation of nf-jb after ⁄ infection was significantly suppressed by rig-i or tlr silencing. these results suggest that the involvement of rig-i and tlr in the cytokine induction by ⁄ was via the regulation of irf and nf-jb nuclear translocation. since rig-i and tlr are important in influenza a virus-induced cytokine expression, we next explored the expression of these innate receptors in neighboring uninfected human macrophages by treating the uninfected macrophages with the filtered culture supernatants collected from mock, ⁄ , or ⁄ infected macrophages. as shown in figure a , ⁄ supernatant differentially induced the mrna expression of rig-i, mda , and tlr compared to ⁄ supernatant treated human macrophages. the induction of rig-i was higher than the induction of mda and tlr . in the presence of lm of jak inhibitor, the up-regulation of all three innate sensing receptors was significantly reduced showing their induction was dependent on jak activity. human lung epithelial a cells were also treated with the supernatants collected from macrophages infected with mock, ⁄ , or ⁄ virus. differential induction of rig-i, mda and tlr by ⁄ supernatant compared to ⁄ supernatant treated cells was observed (figure b) . ⁄ supernatant dramatically induced all three innate sensing receptors, while ⁄ supernatant only marginally induced rig-i and mda , but not tlr . as in human macrophages, treatment with lm of jak inhibitor caused a significant suppression of ⁄ supernatantinduced rig-i, mda , and tlr expression in a cells. these results, taken together with the direct effects on virus infected cells, suggest that paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the up-regulation of innate sensing receptors. h n directly induced ifn-beta ( figure ) and tnf-alpha (data not shown) mainly via rig-i signaling and the consequent activation and nuclear translocation of irf and nf-kb in human macrophages. these results were consistent with a previous study using beas- b cells showing the essential role of rig-i in ifn-beta reporter activity by h n influenza virus infection. while tlr also played a role in induction of ifn-beta and the activation of irf and nf-kb, it plays a less important role compared to rig-i. the reduction of irf and nf-kb activation was also confirmed with the study by le goffic showing differential regulation of irf and nf-kb by rig-i and nf-kb can also be regulated by tlr . in addition to the direct role of rig-i and tlr in sensing and signaling the presence of influenza virus, the innate immune sensing regulators were themselves also highly upregulated in both infected (data not shown) and adjacent uninfected cells by influenza virus infection. compared with seasonal h n virus, the h n viruses had a much more dramatic effect on inducing innate sensing receptors via jak signaling pathways activated by autocrine and paracrine mediators. the up-regulation of rig-i, mda , and tlr was markedly induced by virus free culture supernatants from h n -infected macrophages, while supernatant from ⁄ -infected cells induced the expression of these receptors only to a lesser degree. the soluble mediators in the virus infected cell supernatant caused paracrine upregulation of rig-i, mda , and tlr in uninfected macrophages as well as human lung epithelial cells. these effects may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. taken together these results provide, at least, part of the explanation on the hyper-induction of cytokines in h n infection. a more precise identification of the signaling pathways triggered by h n virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h n influenza and also for treating other causes of acute respiratory disease syndrome. we generated mutants of y (h n ) and a ⁄ duck ⁄ hokkaido ⁄ vac generation and characterization of mutant viruses rgy sub (h n ), rgvac sub (h n ), and rgvac ins (h n ), which have a serial basic amino acid residues at their ha cleavage sites were generated by site-directedmutagenesis and reverse genetics. rgy sub (h n ) and rgvac ins (h n ) required trypsin to replicate in mdck cells, and showed similar levels of growth to their parental viruses (table ) . chickens intravenously inoculated with rgy sub (h n ) or rgvac ins (h n ) did not show any signs of disease. rgvac sub (h n ) replicated in mdck cells without exogenous trypsin, and one of the eight chickens inoculated with the virus showed slight depression at day post-infection. the h and h mutant viruses were serially passaged in the air sacs of chicks to assess their ability to acquire pathogenicity. plaque formation in mdck cells and pathogenicity in -day-old chicks and -week-old chickens are shown in table . rgy sub (h n ) replicated in mdck cells in the absence of trypsin and killed all of the chicks after six consecutive passages. two of the eight-four-weekold chickens inoculated intravenously with rgy sub-p (h n ) died within days. eventually, over % of the chickens intravenously infected with rgy sub-p (h n ) died by days post inoculation, and its pathogenicity was comparable to that of hpaivs. rgvac sub-p (h n ) was pathogenic to both chicks and -week-old chickens, and mortality increased after one more passage. rgvac ins-p (h n ) replicated in mdck cells in the absence of trypsin, killed all of the chicks, and caused % mortality among -week-old chickens. the lethal effect of rgvac ins-p (h n ) on chickens increased with one additional passage in the air sacs of chicks, as in the case of rgvac sub (h n ). to examine whether the pathogenicity of each virus via the natural route of infection correlated with that by intravenous infection or not, three -week-old chickens were challenged intranasally with the viruses at an eid of ae and observed for clinical signs until day post-infection (data not shown). all chickens inoculated with rgy sub-p (h n ) or its parental viruses survived without showing any clinical signs, and serum antibody responses were detected in the hi test. on the other hand, rgvac sub-p (h n ) and rgvac ins-p (h n ) were pathogenic as in the intravenous experiment, killing two of three chickens by day post-inoculation. one of three chickens were not infected with rgvac sub-p (h n ) or rgvac ins-p (h n ) via intranasal route (data not shown), indicating these p viruses had not been completely adapted to the host. to investigate the possibility of these p viruses to acquire further pathogenicity for chicken, rgvac sub-p (h n ) and rgvac ins-p (h n ) were obtained from the brain homogenates of the chickens that died on days post intranasal inoculation with the p viruses. although mortality rate of chickens inoculated with the p viruses was equal to that with p viruses, enhancement of pathogenicity was observed in intranasal inoculation study; all of the chickens inoculated with rgvac sub-p (h n ) were infected, and time to death was shortened to - days post inoculation in chickens with rgvac ins-p (h n ) (data not shown). to investigate whether tissue tropism of the viruses was involved in their pathogenicity, we determined viral titers in the tissue and blood samples from -week-old chickens intranasally inoculated with each virus on days post infection ( table ) . rgy (h n ) and rgvac (h n ) were scarcely recovered from the samples, and the mutant strains before passage showed broader tissue tropism than the parental viruses. none of the chickens inoculated with rgy sub-p (h n ) showed any signs of disease, and viruses were recovered from each of the samples except the brain and the blood. one chicken inoculated with rgvac sub-p (h n ) showed clinical signs such as depression, and viruses were recovered from virtually all of its organs and blood samples. two of three chickens inoculated with rgvac ins-p (h n ) showed disease signs, and one died days post inoculation. the viruses were recovered from almost all samples of the two chickens showing signs of disease. p viruses were efficiently replicated in systemic organs of the chickens as compared with p viruses. throughout the study, the viruses were recovered from the brains of all of the chickens showing clinical signs. here, we demonstrated that the h influenza virus acquired intravenous pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the ha and passaged in chicks. rgy sub-p (h n ) killed % of chickens infected intravenously, and its pathogenicity was comparable to that of hpaivs (table ) . however, chickens intranasally inoculated with rgy sub-p (h n ) did not show any clinical signs of disease (data not shown). these results are consistent with a previous study in chickens that found some h influenza viruses did not show intranasal pathogenicity although their intravenous pathogenicity index was over ae , classified as hpaiv according to the definition by european union. ohuchi et al. reported that the insertion of additional basic amino acids into the h ha cleavage site resulted in intracellular proteolytic cleavage. other groups reported that h and h has tolerated amino acid mutations into their cleavage sites, and the viruses with the mutated has replicated in mdck and ⁄ or qt cells in the absence of trypsin. , the results in the present study is in agreement with these, namely, cleavage-based activation by a ubiquitous protease is not restricted to the h and h has. the intranasal pathogenicity of the h and h mutants were different (data not shown), although these viruses similarly replicated in mdck cells in the absence of trypsin and killed chickens by intravenous inoculation ( table ) . the viruses were recovered from the brain and the blood of some chickens infected with rgvac mutants (h n ), and morbidity was closely associated with viral titers in the brain (table ) . on the other hand, no viruses were recovered from the brain of chickens infected with rgy mutants (h n ), explaining why rgy sub-p (h n ) did not show intranasal pathogenicity. all the viruses passaged in the air sacs of chicks killed chicken embryos by hours post allantoic inoculation (data not shown). rgvac sub-p (h n ) and rgvac ins-p (h n ) were more pathogenic to chicken embryos than rgy sub-p (h n ); the allantoic fluid obtained from the embryonated eggs inoculated with the h viruses passaged in air sacs was turbid. it has been reported that infection of a highly pathogenic h virus were strictly confined to endotherial cells in chicken embryos or chickens. , therefore, it is suggested that endotheliotropism differed between the h and h viruses passaged in air sacs and affected their intranasal pathogenicity. taken together, it is assumed that rgvac sub-p (h n ) and rgvac ins-p (h n ) showed marked intranasal pathogenicity with high levels of viremia caused by replication in vascular endothelial cells, leading to invasion of the brain. in the intravenous experiment, rgy sub-p (h n ) easily reached systemic organs, including the brain hematogenously, replicated through the cleavage of ha by a ubiquitous protease, and then exerted its pathogenicity. further study including a pathological analysis is currently underway to test this hypothesis. for all hpai viruses of subtypes h and h known to date, the cleavage of ha occurs at the c-terminal r residue in the consensus multibasic motifs, such as r-x-k ⁄ r-r with r at position p and k-k ⁄ r-k ⁄ t-r with k at p , and leads to a systemic infection. early studies demonstrated that the ubiquitously expressed furin and pcs are activating proteases of hpai viruses. furin and pcs cleave the consensus multi-basic motif r-x-k ⁄ r ⁄ x-r with r at position p . however, replacement of p r by k and a nonbasic amino acid significantly suppresses the processing activities of furin and pcs. most of the type ii transmembrane serine protease identified so far recognize a single r at position p , but the newly isolated mspl and its transcript variant tmprss preferentially recognize paired basic residue, particularly r and k at position p , at the cleavage site. [ ] [ ] [ ] thus, mspl and tmprss can activate various bioactive polypeptides with multibasic residue motifs, including fusogenic viral envelope glycoproteins. the present study was designed to characterize the proteolytic processing of the hpai virus ha by mspl and tmprss in comparison with furin. hpai virus a ⁄ crow ⁄ kyoto ⁄ ⁄ (h n ) was isolated from embryonated eggs inoculated with tracheal homogenates from dead crows. then, the mutant ha sequence was constructed by changing r residue to k residue (n'-rkkr-c' to n'-kkkr-c') at the ha cleavage site by sitedirected mutagenic pcr as described. we used human cell line ecv , which expresses mspl and tmprss at levels below detection, and established the cells stably expressing mspl and tmprss , such as ecv -mspl and ecv -tmprss . to determine the cleavage specificities of mspl ⁄ tmprss and furin, peptides ( lg each) were incubated with ae mu mspl ⁄ tmprss for hour and furin for hours at °c, respectively. after incubation, the samples were separated by reverse-phasehigh-performance liquid chromatography (rp-hplc) with the use of a c column. the elution samples were then identified by amino acid sequence analysis and by maldi-tof-ms. we analyzed the cleavability of -residue synthetic peptides derived from ha cleavage sites of hpai strains, such as a ⁄ chick ⁄ penn ⁄ ⁄ (h n ) and a ⁄ fpv ⁄ rostock ⁄ (h n ), and low pathogenic strain a ⁄ aich ⁄ ⁄ (h n ). after incubation with human mspl or human furin, the digested samples were separated by rp-hplc, and peptide fragments were characterized by mass-spectrometry and protein sequencing. in contrast to the low cleavage efficiencies of the h ha peptide with a single r at the cleavage site ( figure a) , both the h ha peptide with the k-k-k-r motif ( figure b ) and the h ha peptide with the r-k-k-r motif ( figure c) were fully processed at the correct positions by mspl within hour. in the case of h ha peptide with multiple basic residues, mspl cleaved two carboxyl-terminal sides of r in the cleavage site sequence of n'-k-k-rfl-k-k-rfl-g-c', while furin cleaved only at a single site of r with r at position p , n'-k-k-r-k-k-rfl-g-c' in the presence of mm cacl . these cleavage site specificities of furin were consistent with that reported for the h ha peptide of hpai virus a ⁄ hong kong ⁄ ⁄ (h n ) with r-k-k-r motif. however, the h ha peptide with k at position p ( figure b) was hardly cleaved by furin under the same experimental conditions. tmprss showed similar results (data not shown). these findings suggest that mspl and tmprss cover diverse cleavage specificities, including non-susceptible specificity to furin. full length recombinant ha of hpai virus with kkkr cleavage motif was converted to mature ha subunits with membrane-fused giant cell formation in mspl or tmprss transfectant cells. in addition, this conversion was suppressed by bowman-birk trypsin inhibitor, a membrane non-permeable highmolecular mass inhibitor against mspl ⁄ tmprss . to test for the generation of infective virus, the conditioned media of -day culture of ecv -wt and ecv -mspl cells infected with wt and mutant hpai h n viruses were inoculated into newly prepared cells and cultured for hours. although spreading of wt virus infection with ha cleavage motif of r-k-k-r was detected from the conditioned medium of both ecv -wt and ecv -mspl cells, that of mutant virus with ha cleavage motif of k-k-k-r was only detected from the condition medium of ecv -mspl cells. these results strongly suggest that the expression of mspl, but not furin, potentiates multicycles of hpai virus with k-k-k-r ha cleavage motif. seasonal human influenza a virus has have consensus monobasic cleavage site sequence, n'-q ⁄ e-x-rfl-g-c', and all hpai virus has have two types of cleavage site sequences with multiple basic amino acids, n'-r-k ⁄ r-k ⁄ r ⁄ x-rfl-g-c' with r at position p in a large number of hpai viruses and n'-k-k ⁄ r-k ⁄ t-rfl-g-c' with k at position p in a small number of hpai viruses. figure shows furin efficiently cleaved synthetic hpai a ⁄ hong kong ⁄ ⁄ (h n ) ha cleavage site peptide with the r-k-k-r motif, but hardly cleaved the hpai virus a ⁄ chick ⁄ penn ⁄ ⁄ ha cleavage site peptide with the k-k-k-r motif. furthermore, cleavage of the full-length ha of hpai virus with r-k-k-r motif was detected, but cleavage of hpai virus ha with k-k-k-r motif was hardly detected in ecv -wt cells containing furin ( figure ). these substrate specificities of furin suggest that proteases other than furin and pc ⁄ play a role in the processing of has of hpai virus with k-k ⁄ r-k ⁄ t-r cleavage motif. mspl and tmprss show unique cleavage site specificities of the double basic residues at the cleavage site, and r or k at position p greatly enhanced the efficiency, which none of the other ttsps have shown similar substrate specificities so far. furthermore, infectious and multicycle viral replication along with ha processing was also noted in genetically modified mutant recombinant live hpai virus a ⁄ crow ⁄ kyoto ⁄ ⁄ (h n ) with k-k-k-r cleavage motif in ecv -mspl cells (figure ) . these results were supported by the data of two cleaved peptides by mspl in figure c . these findings suggest that mspl has diverse cleavage specificities and may cleave ha at least two sites, although multiplicity of the mutant hpai virus was observed under the conditions. these results also suggest that mspl and tmprss in the membrane might potently activate the ha membrane fusion activity of hpai viruses and promote their spread. highly pathogenic avian influenza viruses replicate in various organs in birds, and the ha processing proteases might be widely distributed in these organs. indeed, tmprss and mspl are ubiquitously expressed in almost all human organs tested and are highly expressed in lungs, leukocytes, pancreas, spleen, and placenta. , in addition, mspl and tmprss are strictly localized in the plasma membranes, suggesting that proteolytic activation of hpai virus ha occurs not only through the trans-golgi network by furin and pc ⁄ , but also on the cell surface by mspl and tmprss . the pb -f protein, which is translated from the + reading frame of the pb gene segment, has been linked to the pathogenesis of both primary viral and secondary bacterial infections in a mouse model. - a mitochondrial targeting sequence is located in the c-terminal portion of the pb -f open reading frame, and expression of full length pb -f has been associated with mitochondrial targeting and apoptosis in a monocyte dependent manner. , it has been theorized that enhanced virulence could result from mitochondrial disruption with subsequent cell death mediated by pb -f . , a suggested second function of the pb -f protein is that it enhances immunopathology by triggering the inflammatory response. , in earlier studies from our group, the pro-inflammatory phenotype was markedly upregulated when the pb -f from the pandemic strain was expressed, arguing that this protein may be an important virulence factor for highly pathogenic pandemic viruses. , in this report we analyze the pb -f protein's contribution to pathogenesis in a mouse model, examining both inflammation and cell death. pb -f proteins from a variety of epidemiologically important iav strains including all pandemic strains from the th century, a highly pathogenic avian influenza virus of the h n subtype, and representative seasonal strains were utilized to determine the relevance to pandemic disease. we demonstrate that macrophage mediated immunopathology, but not apoptosis, are relevant functions of pb -f proteins from past or potential pandemic influenza viruses. using the predicted amino acid sequences of the pb -f proteins from pr , a ⁄ brevig mission ⁄ ⁄ , ⁄ singapore ⁄ ⁄ , a ⁄ hong kong ⁄ ⁄ , a ⁄ wuhan ⁄ ⁄ , and a ⁄ vietnam ⁄ ⁄ , peptides from the c-terminal end were synthesized as described. an additional n-terminal peptide was synthesized from the pr sequence as a positive control (mgqeqdtpwilstghistqk) as described. a panel of viruses were reverse engineered as described , and included laboratory strain pr , a virus unable to express pb -f (dpb -f ⁄ pr ), or expressing the pb -f of the pandemic strain ( pb -f ⁄ pr ) or the truncated h n strain (beij pb -f ⁄ pr ). , in addition, : reassortants encoding pb gene segments from a *current address: department of immunology and microbiology, university of melbourne, melbourne, vic., australia. highly pathogenic avian influenza of the h n subtype (h n pb ⁄ pr ), or from a human h n strain (h n pb ⁄ pr ) were utilized along with their isogenic deletion mutants for pb -f (h n dpb -f ⁄ pr and h n dpb -f ⁄ pr ). cell lines and cell death assays raw . cells were grown under conditions as described. cells were infected with one multiplicity of infection (moi) of virus for - hours, or exposed to lm (final concentration) of peptides derived from the c-terminal portion of pb -f for hour. cells from the supernatant and monolayers were harvested, washed, and stained with annexin (apc) and propidium iodide (pi) (becton dickinson, san jose, ca, usa), then analysed for cell death as described. six-to eight week old female balb ⁄ cj mice (jackson laboratory, bar harbor, me, usa) were maintained in a biosafety level facility in the animal resource center and procedures approved by the animal care and use committee at sjcrh. infectious agents and peptides were diluted in sterile pbs and administered intranasally to anesthetized mice (n = - ) in a volume of ll ( ll per nare) and monitored for overt signs of illness and weight loss daily. following euthanasia by co inhalation, the trachea was exposed and cannulated with a gauge plastic catheter (bd insyte; becton dickinson, sandy, ut, usa). bronchoalveolar lavage fluid (balf) was collected, red blood cell depleted, and cellular content analyzed via flow cytometry as described. one way analysis of variance (anova) was used for multiple comparisons of cell death and cellularity of balf. a p-value of < ae was considered significant for these comparisons. graphpad prism version . for windows (graphpad software, san diego, ca, usa) was utilized for all statistical analyses. to assess the contribution of pb -f to inflammation, we utilized a panel of previously described reverse engineered viruses in the mouse infection model. , the effect of pb -f expression was observed clearly in the inflammatory infiltrate in response to infection in the lungs. deleting pb -f from pr or expression of the c-terminally truncated beij pb -f had a significantly reduced influx of macrophages ( figure a) . expression of the pb -f caused similar inflammatory effects as the pr virus. disruption of pb -f expression the virus containing the h n pb gene segment in a pr background also significantly decreased the inflammatory response compared to the virus maintaining the ability to express full length pb -f ( figure a) . however, no differences were seen that could be attributed to the h n derived pb -f . the lungs of mice infected with the panel of pb -f variant viruses were examined at hours. pathologic changes typical of pr viral infection were observed in all lungs. these typical findings included perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris (figure ). in the lungs of mice infected with pr or pb -f ⁄ pr , however, significantly more perivascular cuffing was noted, with a prominent increase in numbers of macrophages (figure a, c) . the overall number of inflammatory cells throughout the lungs, including both airways and alveoli, was quantitatively greater in these mice than in mice infected with dpb -f ⁄ pr or beij pb -f ⁄ pr ( figure b, d) . as the function and influence of pb -f protein on normal viral function is not currently understood, and given the abrogation of enhanced inflammation induced by the truncated pb -f beij ⁄ pr virus, we sought to elucidate whether the c-terminal domain of pb -f could alone induce this inflammatory response. mice were exposed to a panel of peptides and were euthanized hours later for collection of balf. significant influxes of macrophages into the balf were seen following exposure to c-terminal pb -f peptides derived from pr , the pandemic strains from (h n ), (h n ), and (h n ), and the h n virus compared to controls ( figure b) . similar effects were not seen with the peptide derived from a more recent h n strain, a ⁄ wuhan ⁄ ⁄ . when peptide exposed mice were followed for morbidity for days, peptides proven to induce a heightened inflammatory response correlated strongly with overt clinical signs of illness (data not shown). thus, the ability to cause lung inflammation appears to be a property of pb -f proteins of viruses containing pb gene segments reassorted directly from the avian reservoir. the pb -f protein may contribute to virulence by rendering the host cellular immune response ineffective through inducing apoptosis. we sought to determine whether this was an epidemiologically important function for combating the host immune response to infection by testing the ability of pb -f proteins from several different iav strains to cause cell death. we therefore infected raw . cells with the panel of recombinant viruses at an moi of for - hours. as has been demonstrated previously, , , pr virus induces significant cell death compared to uninfected controls ( figure c ). when raw . cells were infected with pr virus, necrotic death peaked hours after infection. viruses lacking the c-terminal portion of pb -f , including the dpb -f ⁄ pr and the beij pb -f ⁄ pr were unable to cause cell death ( figure c ). in addition, expression of the pb -f also did not cause significant increases in cell death over controls. expression of pb -f or deletion of pb -f in either an h n or h n pb gene segment background similarly did not alter the cell death phenotype. to examine additional strains for which we did not have isogenic virus pairs, we next exposed the balbcj mouse derived macrophage cell line raw . to the panel of pb -f peptides derived from pr , the pandemic strains from (h n ), (h n ) and (h n ), and the h n for hours. cell death in raw . cells was caused only by the peptides derived from the laboratory strain pr and the peptide derived from the pandemic strain ( figure d ). viability was not affected by exposure of raw . to peptides derived from other virus strains. we conclude from these data that the mechanism by which pb -f contributes to the pathogenicity of pandemic influenza is unlikely to be through its reported ability to cause cell death. these data presented here demonstrate that the lung inflammatory response is enhanced by the influenza a virus pb -f protein in a mouse model. this inflammatory response was characterized by increased cellular infiltration of macrophages into the interstitial and alveolar spaces of the lungs, as well as enhanced perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris. this augmentation was shown to be induced by pb -f proteins only from those strains contributing to the formation of all pandemic strains of the th century and from the currently circulating, highly virulent h n strains that constitute an imminent pandemic threat. the iav h n strains circulating in humans since around code for a truncated pb -f . these viruses may lack the cterminal residues responsible for the inflammatory effects demonstrated in this publication. additionally, recently circulating h n strains, in contrast to their pandemic forbear from , have lost the capacity to cause pb -f mediated inflammation through mutation of the c-terminus of this protein. in a novel h n iav emerged from an animal reservoir and caused a human pandemic. disease burden from this strain has been considered mild in contrast to the three pandemics of the th century. the reasons for this disparity in pathogenesis are unclear. an examination of the origins of the three th century pandemics shows that only the hemagglutinin (ha) and pb gene segments were reassorted directly from the avian reservoir in every case, suggesting gene products of one or both of these may be important. the ha surface glycoprotein provided the antigenic novelty required for the each virus to achieve pandemic status. however, the significance of inclusion of a novel pb gene segment in each of the th century pandemics is not yet understood. we show here that the pb -f of these pandemic strains contributes to virulence through induction of inflammatory responses. thus pb -f may serve as a marker of the pathogenicity of pandemic strains. since the h n strain codes a truncated pb -f of only predicted amino acids, the lack of pb -f mediated inflammation may account in part for its relatively lower virulence. , of the panel of pb -f proteins studied, only that from the laboratory strain pr was capable of rendering responding host-immune cells ineffective by induction of cell death. we therefore hypothesize that molecular signatures specific to induction of apoptosis may have been lost through genetic mutation of the pb -f gene throughout the evolution of the iavs. our findings suggest that this apoptotic function is unlikely to be important for the virulence of any of the known pandemics. rather, the inflammatory phenotype appears to be the dominant contribution of pb -f to pandemic disease. influenza virus-cytokine-protease cycles are principal mechanisms of multi-organ failure in severe influenza and therapeutic approaches introduction influenza a virus is the most common infectious pathogen in humans, causing significant morbidity and mortality, particularly in infants and the elderly. mof with severe edema is observed in the advanced stage of influenza pneumonia. however, the relationships amongst factors that induce vascular hyper-permeability in severe influenza remain unclear. it is reported that significant increases in levels of pro-inflammatory cytokine levels, such as tnf-a, il- , and il- b, affect host survival both positively and negatively. the inflammatory response affects cell adhesion, permeability, apoptosis, and mitochondrial reactive oxygen species, potentially resulting in vascular dysfunction and mof. in addition, iav infection up-regulates several cellular proteases including ectopic trypsin and mmp- . up-regulated ectopic trypsin mediates the post-translational proteolytic cleavage of viral envelope hemagglutinin (ha), which is crucial for viral entry and replication and the subsequent tissue damage in various organs. the aim of the this study was to define the pathogenic impact of cytokine storm in iav infection and the molecular mechanisms by which pro-inflammatory cytokines and proteases cause vascular dysfunction in animal model. weanling female mice aged weeks (c bl ⁄ crslc) were infected with iav ⁄ wsn ⁄ ( pfu) with and without treatment of pdtc ( . mg ⁄ kg), nac ( mg ⁄ kg), and ndga ( mg ⁄ kg). these inhibitors were administrated once daily for days after infection. the levels of cytokines in tissue homogenates were measured by elisa kits. the effect of inhibitors on viral replications was determined by real-time pcr. gelatin zymography and western blotting were conducted as reported previously. host cellular responses in the airway after iav infection figure shows schematic view of typical biological responses in the airway of mice after iav infection. an initial response before viral proliferation is significant increases in pro-inflammatory cytokine levels. immediately after cytokine inductions, there is a marked up-regulation of ectopic trypsin along with an increase in virus titer in the airway, lung, and brain. ectopic trypsin mediates the post-translational proteolytic cleavage of iav ha, which is crucial for viral entry and replication and the subsequent tissue damage in various organs. we also found that iav infection markedly induces mmp- and matrix degradation. just after the peak of viral proliferation, the innate and adaptive immune responses of protective immunity are induced for defense and recovery, or oppositely on rare occasions, mof with vascular hyper-permeability is started into the advanced stage of influenza. the levels of tnf-a and il- in the lungs were increased persistently for days after iav wsn infection, and that of il- b peaked at days - post-infection (figure a ). since these cytokine responses are associated with activation of nf-jb and ap- , we treated mice once daily for days with anti-oxidant inhibitors: pdtc and nac against nf-jb activation, and ndga against ap- activation. pdtc and ndga significantly suppressed the up-regulation of tnf-a and il- b (p < . ), and nac suppressed tnf-a (p < . ), and il- (p < . ) at day post-infection. gelatin zymography showed up-regulation of ectopic trypsin and mmp- in mice lung, brain, and heart during infection for days ( figure b ). trypsin and mmp- induction was inhibited by treatment with pdtc, nac, and ndga, probably via blockade of nf-jb and ap- binding in the promoter region of the genes. viral rna replication in various organs at day post-infection was suppressed by more than one order of magnitude by pdtc, nac, and ndga ( figure c ). suppression of viral multiplication and induction of cellular factors by pdtc, nac, and ndga, significantly improved the survival of mice at day post-infection, the late stage of infection ( figure d ). to elucidate the mechanisms underlying brain vascular dysfunction of influenza-associated encephalopathy, changes in the levels of tight-junction proteins, intracellular zonula occludens- (zo- ) and transmembrane occludin, and the matrix protein laminin, were analyzed by western blotting. marked reductions in the expression levels of tight-junction constituents were detected at day post-infection, which were partly rescued by pdtc, nac, or ndga (figure e ). no other tight-junction protein, claudin- or matrix fibronectin and type iv collagen, were affected. the present study reports several new observations: (i) proinflammatory cytokines, tnf-a, il- b, and il- , when up-regulated by iav infection, induce trypsin and mmp- expression in various organs in mice; (ii) inhibitors of nf-jb and ap- effectively suppress the up-regulation of proinflammatory cytokines, trypsin, and mmp- and improve survival rates of infected mice. based on these results, we propose the 'influenza virus-cytokine-protease cycle' hypothesis as one of the mechanisms of vascular dysfunction in mof with cytokine storm in severe influenza and influenza-associated encephalopathy. the significance of pro-inflammatory hyper-cytokinemia, or 'cytokine storm,' in the pathogenesis of iav infection remains unclear. on the positive effects, cytokines promote lymphocyte activation and infiltration at the sites of infection and exert direct antiviral effects. however, on the negative effects of excess cytokines, the hyper inflammatory process evoked by viral infection may become harmful through intracellular activation of nf-jb, ap- , and the janus kinase-signal transducers and activators of transcription signaling pathways. , [ ] [ ] [ ] the in vivo experiments presented here showed that nf-jb and ap- inhibitors markedly suppress the expression of cytokines, trypsin, mmp- , and viral replication, resulting in a significant increase in the survival of infected mice. furthermore, cytokines interact with mitochondria to increase the production of reactive oxygen species, resulting in the production ⁄ activation of vasodilatory mediators such as nitric oxide and bradykinin, and subsequent endothelial dysfunction and edema in various organs. the molecular mechanisms underlying tight-junction disruption in endothelial cells and vascular hyper-permeability following the 'cytokine storm' remain unclear. tnfa up-regulation alters the cellular redox state, reduces the expression of four complex i subunits by increasing mitochondrial o ) production and depleting atp synthesis, decreases oxygen consumption thereby resulting in mitochondrial damage, , and increases [ca + ] i atp depletion dissociates zo- from the actin cytoskeleton and thereby increases junctional permeability. endothelial dysfunction induced by 'influenza virus-cytokine-protease cycle' in the early stage of severe influenza may further affect various circulating factors, coagulation factors and complement systems, and vascular interacting cells, such as neutrophils, macrophages and lymphocytes. mof is the final outcome of metabolic and mitochondrial fuel disorder, immunosuppression, endocrine disorder, and tissue injury followed by endothelial dysfunction in many organs. another key pathway of acute lung injury in the highly pathogenic avian influenza virus h n and acute respiratory syndrome-corona virus infection reported recently involves oxidative stress and formation of oxidized phospholipids, which induce lung injury via toll-like receptor signaling pathway. in addition to these data, up-regulated trypsin and pro-inflammatory cytokines may also affect tissue destruction and immunosuppression in the late stage of iav infection. further studies are required on the role of the 'influenza virus-cytokine-protease cycle' in the pathogenesis of mof, particularly in the late stage of viral infection. though influenza a virus replication kinetics and host responses have been previously studied in umbilical vein endothelial cell or transformed endothelial cell lines, the tropism of influenza a virus including h n and pandemic h n pdm for primary human lung microvascular endothelial cell has not been well defined. in this study we employed primary human lung microvascular endothelial cells, which are more physiologically relevant for understanding pathogenesis of influenza in the lung as to obtain a better understanding of the links of endothelial cell infection to systematic virus dissemination and multiple organ involvement in severe human influenza. supernatants of cells infected at moi of two were collected for cytokine protein assays, and total rna was extracted for gene expression analysis using qpcr. we found that seasonal influenza h n and h n viruses initiated viral gene transcription and viral protein expres- sion, but did not produce infectious progeny, while the highly pathogenic avian influenza h n and the pandemic influenza h n pdm virus could replicate even with the absence of exogenous protease (figure ) . furthermore, when compared to seasonal h n and h n , the h n virus was a more potent inducer of cytokine and chemokine including ifn-b, mcp- , rantes, ip- (figure ) , and il- , in virus infected endothelial cells, whereas h n pdm induced intermediate levels of cytokine and chemokine. avian influenza h n and pandemic h n pdm virus (but not the seasonal h n and h n virus) can productively replicate in human lung microvascular endothelial cells. this is likely to be of relevant to pathogenesis and provides a possible explanation for the extra-pulmonary infection seen in animal infection models. this extra-pulmonary spread may support the previous speculation and anecdotal evidence that h n and h n pdm virus can infect the gastrointestinal tract through the virus dissemination from the infected respiratory tract as the first target cells for influenza infection. [ ] [ ] [ ] in addition, the release of proinflammatory cytokine and chemokine induced by influenza h n and h n pdm virus infection in lung microvascular endothelial cells may be important contributors to the pathogenesis of severe human influenza disease leading to endothelial cell dysfunction that contributes to severe pulmonary disease symptoms. during its replication, influenza virus utilizes the host cellular machinery for many aspects of its life cycle. characterization of such virus-host protein-protein interactions is a must to identify determinants of pathogenesis. the m ion channel protein plays a crucial role during the entry and late stages of the viral life cycle where its c-terminal domain, well conserved among influenza a viruses, is accessible to cellular machinery after fusion with endosomal membrane and during its trafficking along the secretory pathway prior to assembly and budding. the aim of the study is to identify cellular interactants of m that play important regulatory roles during influenza infection. to identify cellular partners of m we performed a genome-wide yeast-two-hybrid (y h) screening approach using the cytosolic domain of m as bait and a human placenta random primed cdna library as prey and tested more than million interactions. from the y h screening, an interesting interaction with the human annexin a (anxa ) protein, a member of annexin family proteins that binds to phospholipds in a ca + -dependent manner, was identified. co-immunopre-cipitation of myc-tagged anxa and viral m proteins coexpressed in hek t cells after transfection and infection confirmed the direct interaction between anxa and m . we further investigated whether this interaction had any functional significance with regards to influenza life cycle. using a rna interference strategy to silence the anxa gene in human lung epithelial a cells, we observed increased progeny virus titers either in a single or multiple viral growth kinetics study, suggesting a negative regulatory role for anax during viral infection (figure ). a novel interaction between m and anxa was identified. more functional studies are in progress to define precisely the potential negative regulatory role of this interaction during viral infection. a systematic dissection of the viral life cycle will be performed to identify the step(s) affected by the anxa cellular factor using specific assays such as real-time quantitative rt-pcr in a single or multiple viral growth kinetics study, cell transduction with ha-and m -pseudotyped lentiviral particles, virion attachment and internalization assay, immunofluorescence staining of np protein as a marker of viral ribonucleoproteins localization, viral polymerase activity measurement, and viral budding observation by electron microscopy. rna extraction was achieved by qiagen biorobot ez prior to respiratory multiplex pcr analysis. what remained of the extracted material of each specimen was stored by refrigeration at °c. electronic patient records were searched for parameters, such as c-reactive protein (crp), white cell count (wcc), length of admission in days, and patient co-morbidities. patients were divided into three groups according to clinical severity: mild, moderate, and severe. the 'mild' group comprised of those admitted for three days or fewer, or not admitted at all. the 'moderate' group comprised those who required admission to hospital for more than days as a result of swine flu, but who did not require admission to an intensive care unit (itu). the 'severe' group comprised those who had required itu admission. invitrogen ' · reaction mix': ae mm of each dntp + mm magnesium sulphate. primer ⁄ probe mix recipe applied biosystems fast real-time pcr system, 'respiratory multiplex' program. well content ll; thermocycler initial stage ae °c for minutes, then °c for minutes. subsequent cycles of ae °c for seconds followed by °c for seconds for cycles. sequence detection software version . (applied biosystems). of clinical isolates analyzed, all samples produced amplification of pdh material; produced amplification of both swine flu and pdh material. human male dna (lot no. at ng ⁄ l, applied biosystems) at concentration calculated at ae cells ⁄ ll was diluted from ) to ) , yielding mean average ct values of respectively ae , ae , ae , and ae . plotting log of cell number versus ct gave a y = mx + c line from which ct could be interpolated into cell numbers. for swine flu quantification, a sample of swine flu ct ae was diluted through ) to ) . it must be noted that due to variability in resultant swine flu ct values, repetitions at these dilutions were done using an rna carrier ( lg ⁄ l, qiagen; cat no. ) in place of rnasefree water. the ) concentration was positive in nine out of assays; this fraction was used in the calculation described by simmonds to obtain a copy number of targets per reaction by the equation copy value = )ln(f), where f is decimal fraction of failure rate. here, f = ⁄ = ae ; )ln ae = ae copies. a control curve was generated with ct values of ae , ae , ae , and ae giving copy values of , ae , ae , and ae , respectively. using excel (microsoft office, ), these control series were adapted into formulae to convert swine flu and pdh ct values into copy numbers of these elements per reaction. simple division derived a value for swine flu copy per pdh copy, but this was chosen to be expressed as swine flu copy number per human cells. this will be referred to as the 'c' value. forty-two patients had known clinical details; average age was ae , female to male ratio : , and average admission length of days. of the mild group (n = ), nine cases were not admitted to hospital. of the remainder, the mean average admission length was ae days. mean average c value for all samples was ae · , with a standard deviation of ae · ; geometric mean was ae , and median average was ae . log(mean average c value) is shown for each severity group and for identified risk factors in the 'mild' severity group (figures a, b respectively) . in each case variation was too great to yield statistical significance. figure shows the range of c values observed in the 'moderate' severity group. > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > - · ; < - · > · ; < · > · ; < · > · ; < · > · ; < · > · ; < · > · ; < · in a study by duchamp et al., no significant correlation was observed between viral ct value and presence or absence of cardiaorespiratory disease, myalgia, digestive symptoms, or upper or lower respiratory tract infection (although a trend was observed towards patients presenting with signs of upper respiratory tract infection). to our knowledge, no other study has used a dual pcr for analysis of respiratory virus concentrations, and no study has attempted to correlate biochemical markers with respiratory virus concentration. the data exhibited a spectrum of c values, from values < · ) to over · . the three severity group standard deviations all overlapped with each other, preventing statistical significance. analysis of co-morbidities showed a high mean average c value when asthma was present ( ae · ), but again this was associated with an excessive standard deviation. whereas the median average c value in the presence of asthma was higher than the overall average c value ( ae versus ae ), it was significantly lower than the median c value when no co-morbidity was documented ( ae ). there are multiple caveats that may be the cause of such variety of c values obtained. the duration between initial rna extraction and study pcr had a range of to days, with mean average delay of days. the degradation of viral rna is an important contributor to assay variance and failure; rna degradation in clinical samples has been studied. [ ] [ ] [ ] degradation of human dna in clinical samples may have occurred. several studies have chartered degradation of stored human dna. , with regards to sampling, the clinical collection of throat swabs is naturally variable according to the method of the collector. a small number of bronchoalveolar lavage samples were analyzed, yet did not amplify, presumably due to rna degradation. the upper respiratory tract may be only a physical stepping stone for the virus, and take no further role in pathogenesis of severe disease (although undoubtedly is crucial for transmission). interestingly, a ferret study of pathogenesis observed that swine flu yields from the upper respiratory tract were greater than those given by ordinary seasonal h n , with consequently increased shedding. the review by mansfield cites significant findings regarding influenza pathogenesis, including the predilection of h n strains for type ii pneumocyte cells and alveolar macrophages. it also highlights the limitation of knowledge through dearth of human autopsy studies; an exception is the recognition of haematophagocytic syndrome in severe cases. it is known that specific immunoglobulin is effective against establishment of infection in the upper respiratory tract, whereas specific cytotoxic t lymphocytes (ctls) are necessary for clearance of the virus from the lower respiratory tract. it is also suggestive that a gap of two whole days transpires between initial infection and instigation of a specific immune response. it is plausible that in the healthy individual, virus progression is confounded by efficient natural mucosal immunity, in part through good secretory immunoglobulin levels. airway inflammation associated with asthma exacerbation is known to increase both risk of respiratory viral infection and poorer outcome. it is unproven but likely that the local inflammatory processes give rise to increased virion burdens in the upper airways; however, the same effect is conceivable for epithelial cell turnover. there will likely be variance within each clinical category due to patient circumstances and clinicians' judgment of required admission. unfortunately, the duration of symptoms prior to swab collection was often omitted in the clinical notes. finally, stratification of patient group by receipt of antiviral treatment was not studied. no correlations were observed with c values and crp, wcc or admission length. trends were observed towards higher c values in 'mild' cases, but without statistical significance. the relative small study size, coupled with the intrinsic variability of the parameters studied, warrants larger, better controlled, prospective studies to elucidate clinical use of the c value for influenza illness prediction and management. in mid-april a novel variant of a(h n ) influenza virus began to spread rapidly throughout the world, causing the first pandemic of the st century. the majority of the cases associated with this new virus show to be mild, but severe and fatal cases have been reported. molecular markers associated with severity have already been identified, as is the case of the mutation d g. resistant viruses to antiviral drugs have also been identified, highlighting the importance of rapid determination of the antiviral drug profile. global a(h n ) genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. the objective of this ongoing research study was, primarily, to thoroughly characterize the genetic profile and evolution of the emergent influenza a(h n ) virus circulating in portugal and its phenotypic expression on antiviral drugs susceptibility. the cases considered in this study were obtained from the community and from two collaborating hospitals in lisbon -a reference hospital for adults (hospital de curry cabral) and a reference hospital for children (hospital dona estefânia). the cdc real-time pcr protocol, recommended by world health organization (who), was the method used to confirmed all influenza a(h n ) cases. from a total of a(h n ) positive cases diagnosed and confirmed, were selected for this study, taking in consideration that they should cover the period of epidemic activity in portugal and include cases from persons belonging to risk groups and cases associated with more severe clinical features. ninety-six a(h n ) strains were isolated in mdck-siat cells, from combined naso-oropharyngeal swabs. for the evaluation of the genetic profile of a(h n ) virus circulating in portugal, of the isolates were characterized by genetic analysis of the ha, na, and mp genes. the remaining five gene segments (pb , pb , pa, ns, and np) were also sequenced for six of this isolates. briefly, sequencing was performed according to the protocol developed by cdc and recommended by who, using bigdye terminator v. . technology. nucleotide sequences were determined in a dna automatic sequencer abi prism xl genetic analyzer. for each genomic segment, genetic analysis was performed with lasergene v. . software (dnastar inc, usa) using an average of - overlapping readings, including sense and antisense, for precise nucleotide and amino acid sequence determination. genetic mutation and phylogenetic analysis were performed by neighbor-joining method, using mega . software, against published sequences from the vaccine strain (a ⁄ california ⁄ ⁄ ) and from selected a(h n ) strains available on gisaid epiflu database. all mutations were identified with reference to the vaccine strain genome sequence. antiviral drug susceptibility profile of a(h n ) influenza virus circulating in portugal was evaluated both phenotypically and genotypically for nais and genotypically for amantadine. phenotypic evaluation to nais, oseltamivir and zanamivir, was performed for all isolates by ic determination through munana fluorescence assays. genotypic evaluation was performed by searching for mutations associated with resistance to nais in all na gene sequences. amantadine susceptibility profile was performed for all isolates by searching on m sequence for the molecular markers associated with resistance to this antiviral drug (l f ⁄ i; v a ⁄ d; a t; s n; g e). genetic characterisation of the ha subunit of ha reveals point mutations in different strains. all analysed strains present p s and i v mutations, which distinguish them from the vaccine strain ( figure a ). thirty-three of the sequenced strains group in the s t branch. this mutation is referred in the literature as being associated with the putative antigenic site ca. most of these strains ( ) further subgroup in the d e branch, this mutation being associated with one loop of the receptor-binding site. from the early to the late epidemic period, an increased circulation of virus carrying the mutation s t was observed. this is in agreement with the association between this mutation and an enhanced viral fitness that is described in the literature. additional mutations were also observed in a small number of virus, of which we highlight: regarding the genetic characterisation of na, the majority of strains analysed ( of ) presents the mutations n d and v i ( figure b) . as mutation s t in ha gene, these two na mutations are described in the literature as associated with enhanced viral fitness. the few strains not carrying these mutations have circulated in the beginning of the epidemic period. fifteen of the analysed strains further subgroup in y h branch. additionally, mutation i v was identified in two strains. for the remaining gene segments available for the six analysed strains, the observations include: (i) no previously described virulence markers in pb , pb -f , and ns were detected; (ii) pb -f protein is present in the truncated form of amino acids; (iii) the presence of mutations i v and l q in ns and v i in np; (iv) the described association of mutation i v in ns and v i in np genes with viral fitness. phenotypic evaluation of nais susceptibility revealed the existence of three minor and two major outliers to oseltamivir ( figure ). the two minor outliers exhibited a reduction of approximately twofold in the susceptibility to this antiviral drug, comparing to the baseline level, while the reduction exhibited by the two major outliers was of approximately three-and fourfold. regarding zanamivir, two minor outliers were identified with a reduction of approximately twofold in the susceptibility, compared to the baseline level. these two minor outliers (a ⁄ portugal ⁄ ⁄ and a ⁄ portugal ⁄ ⁄ ) correspond to the two major outliers identified for oseltamivir. genetic analysis revealed the presence of the mutation i v in the na sequence of these two strains. the contribution of this mutation for the profile of reduced susceptibility identified for both nais is not known, but a mutation in the same na position (i r) has been referred to as being associated with a reduction in nais susceptibility. full genome sequence analysis of these strains shows that both strains also present the v i mutation in pb gene. however, no association of this mutation with antiviral drug susceptibility is referred in the literature. concerning genetic evaluation of susceptibility to amantadine, all analysed strains present a serine in position , which is a molecular marker of resistance to m inhibitors. these preliminary results allow us to discuss several points. however, the additional data that is being obtained through this ongoing study will be essential for a more complete analysis. for example, more information is needed to determine if the mutations found alter the biology and the fitness of the virus or if there are associated with an increased prevalence of the virus. the majority of the mutations identified in ha subunit have been detected in a(h n ) strains distributed throughout the epidemic curve, not evidencing a specific evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h n ) virus. the occurrence of mutations in the position of the ha subunit of a(h n ) virus have been described. however, more studies are needed to clarify the outcome of these mutations, as for example in patients with severe complications. it could also be relevant to investigate the presence of single and mixed variants in viruses and in clinical specimens and the possibility of these mutations affecting the binding specificity. regarding the susceptibility of a(h n ) pandemic viruses to antiviral drugs, all analysed strains were found to be resistant to amantadine. this resistant profile was not unexpected since the mp gene from this new variant had originated in the eurasian swine lineage, which is characterised by being resistant to this antiviral drug. the majority of the a(h n ) strains analysed revealed to be susceptible to both nais, with only five strains exhibiting a profile of reduced susceptibility, three to oseltamivir and two to both nais. for these last two, the presence of the i v mutation in the na sequence could explain the reduction observed, but a more complete analysis is needed to confirm this. the french national pandemic plan includes an early containment phase followed by a limitation phase. the efficacy of such a plan depends on pre-existing surveillance and laboratory networks. the grog community surveillance network and the hospital lab networks organized by the two french nics carried out the virological monitor- the efficacy of such plan depends on pre-existing influenza surveillance and laboratory networks. in france, the community surveillance is carried through the grog surveillance network. in addition, surveillance is also carried out in hospitals by the renal network. this renal network is divided in two sub-networks: the so-called h -labs network, activated during the containment phase and the extended renal lab network activated in the limitation phase. the h -labs have bsl- facilities that can be used for diagnosis purposes. as part of the national influenza surveillance system led by the french institute for public health surveillance (invs), the grog community surveillance network and the lab networks linked to the two french nics carried out the virological monitoring of the a(h n ) pandemic from the early containment phase up until the end of the pandemic phase. during the containment phase, all suspected cases were hospitalized and declared to invs. each patient was tested on the same day by specific virological diagnosis. hospital admission was not mandatory during the limitation phase, (i) the clustered cases were monitored to study transmission chains, and (ii) the circulation of the virus in the community was monitored through grog swabs collected by practitioners. the nics organized the influenza surveillance to fulfill several objectives according to the epidemiological situation. first, rt-pcr tools (influenza a m gene rt-pcr and a(h n ) specific h and n genes rt-pcrs) were developped and distributed to the lab networks on the th of may . from the early phase, the nics and the h -lab network analyzed all the samples collected from hospitalized and community patients. during the early phase of the limitation phase, an increasing number of labs were performing the specific assays. when the pandemic wave started, all hospital labs could do the testing. results were centralised by nic and reported on a weekly basis. in addition, nics carried out the monitoring of antiviral resistance emergence (na pyrosequencing, specific h y rt-pcr, and phenotypic assays), and real-time surveillance of genetic changes involved in virus adaptation (pb ) virulence factors or antigenic variations (ha). this sequencing was carried out by the pf sequencing platform of the institut pasteur. the first imported a(h n ) influenza cases were observed from the th of april . a limited number of cases have been reported in may. local transmission could be detected end of may. clusters were observed in schools in june and in summer camps during summer. as opposed to the epidemiology of the a(h n ) virus in other european countries, no summer wave was observed in france. only a limited number of sporadic cases were reported up until october. early september, a significant number of cases presenting with influenza-like illness was reported (figure ). the virological investigation of these cases showed high prevalence of rhinovirus infection. this circulation of rhinovirus was a counfounding factor of the pandemic. the pandemic wave lasted weeks between mid-october and the end of december (week to week , figure ). the pandemic wave started week - in the ile-de-france area, and only week - in the rest of france. the peak was recorded week ( figure ). the impact of the pandemic was mainly observed in the - years group of age. overall, severe cases have been admitted to the hospital, and deaths have been recorded by the end of the pandemic wave. the major impact was observed in the - years group of age ( % of deaths recorded). amongst the severe cases and the deceased cases, % and % of cases had no risk factor, respectively. these specimens, were positives for h n , representing ae % of total influenza virus detections. only nine brisbane-like h n , brisbane-like h n , and eight b viruses have been detected in the same period of time. the weekly positive rate ranged from % to %. phylogenetic and antigenic analyses of the viruses collected during the pandemic wave did not show any emerging genetic or antigenic variants (figure a,b) . eight patients, all among cases presenting with severe illness, were infected by a virus harbouring the d g mutation in the ha. amongst the virus tested for antiviral susceptibility or screened for the h y mutation by or specific rt-pcr, only oseltamivir-resistant viruses related to the na h y mutation have been detected. one of these cases also had an i r mutation associated to a reduced sensitivity to zanamivir. all but one resistant virus were detected in treated immunocompromised patients. overall, eight patients presented a virus with the d g mutation in the ha. all these patients had a severe infection; one of these had also a h y mutation in the na asociated to oseltamivir resistance. the pandemic started by the end of april . although the first cases recorded were as early as the th of april, the epidemic wave associated with a widespread spread of the virus was only recorded in october. the french population did not have to face a summer wave, as observed in north america and in numerous european countries. , it is difficult to speculate the reasons for the lack of summer wave; the specimens collected were negative for influenza. moreover, during september, it was anticipated that school openings would be the trigger for the beginning of the pandemic wave. as a matter of fact, a significant increase of influenza-like syndromes were observed at that time, but the virological investigation carried out by the laboratories showed thta is was related to a very large epidemic of rhinovirus. the epidemic circulation of other respiratory viruses can be counfounding factors for the surveillance of the influenza epidemic clinical when the survellance is only based on collection of clinical information. the starting of the pandemic wave was heterogeneous in france. the ilede-france region (paris and its suburbean area), where the population is dense, experienced an early start as compared to the rest of france. however, once the pandemic started in the rest of the county, the epidemic curves were quite similar. the peak was reached at identical times, although it may have been delayed in some remote places in france. overall, we estimate that % of the french population consulted for an ili presentation. the impact was mainly observed in the - years groupe of. however, this age groupe represented only a limited number of severe cases and deaths. on the other hand, the - years groupe of age, where the prevalence was not high, was the age group where the majority of severe cases and deaths was recorded ( % and %, respectively). this data is consistent with the observational data reported by numerous other countries. according to the profile of hospitalized cases, a(h n ) was more aggressive than seasonal viruses. the number of admission to the hospital was ten-fold that observed during a normal influenza epidemic. even if the mortality was limited ( cases), the age distribution of the deceased patients was different as compared to seasonal influenza ( % mortality in < years of age). the lack of recordeable excess mortality has been interpreted to be the consequence of a very mild pandemic, milder than some seasonal epidemics. however, the median age of the fatal cases was much younger than those observed during the seasonal flu, leading to a mis-interpretation of the real impact of the pandemic. when the impact is measurered in loss of years of life, the impact of this pandemic is larger than seen with seasonal influenza, and is quite comparable to these of the two last pandemics. the pandemic preparadness of numerous countries, the develoment of new intensive care techniques and equipment, and the large use of antivirals have reduced the overall impact of this pandemic. these are new factors that should be taken into account when evaluating the real impact of the h n virus. the virological monitoring of the pandemic was achieved by the community-based and hospital-based sea- sonal influenza networks, reminding the importance of maintining such networks. the diagnosis of influenza in most of the patients was carried out by molecular techniques. it has been clearly stated from the beginning of the pandemic that near-patient tests were lacking of susceptibility and could not be used for patient management. the distribution of a set of validated and comprehensive techniques by the two nic was very helpfull for the monitoring of the pandemic and the patients. however, this diagnostic procedure change should not preclude maintaining virus isolation that is necessary for whole genome analysis, monitoring of antigenic changes, and phenotypic testing for antiviral testing. some of the mutants that have been recorded, including viruses with antiviral resistance phenotype or genotype, could be analysed from grown virus strains. it is striking that despite a large antiviral usage, only a limited number of isolates had mutations associated to resistance. however, the frequent isolation of such resistant virus was observed in immunocompromised patients that presented severe infections and long virus shedding. the impact of the pandemic is still under evaluation. sero-epidemiological analysis will be performed to asses for the real attack rate of the pandemic virus. as in other countries, it has been recorded that asymptomatic infections could be observed frequently. it is quite unlikely that the impact of the pandemic was reduced by the vaccination campaign, although this vaccination started on the th of november, just when the pandemic started in france. it is estimated that millions received the vaccination. pandemic strains of the influenza virus sporadically emerge, deviating from the regular endemic strains of seasonal influenza. in april , a novel pandemic influenza virus a ⁄ h n emerged, swiftly spreading across the world. immediately, domestic and international public health agencies were forced to develop containment and mitiga-tion strategies in response to the pandemic. however, the dynamics and transmission patterns of this novel virus are yet to be fully understood. simultaneously, seasonal strains of influenza (a ⁄ h n , a ⁄ h n , and b) continued to circulate in many nations. both pandemic and seasonal variants of influenza are responsible for significant morbidity and mortality. to characterize the dynamics of this disease and the variation within strains, a more detailed understanding of the patterns in viral shedding during natural infection is required. the majority of data on the patterns of viral shedding during influenza infection are a result of volunteer challenge studies. in these studies, volunteers are commonly screened for pre-existing immunity against the challenge strain and are of a certain demographic and age. information on the patterns of viral shedding in natural influenza infections, pandemic or seasonal, is limited but should provide greater generalizability. we describe the trends of viral shedding and clinical illness in community acquired cases of pandemic and seasonal strains of influenza. in , a community-based study was conducted to analyse the effectiveness of non-pharmaceutical interventions to prevent the spread of influenza in households. in , a similar community-based study was initiated to collect comparative data from individuals infected with seasonal and pandemic influenza. both studies were conducted with very similar protocols, involving households in total. the specimens and symptom data required for this study all arise from secondary infections ascertained in these two community-based studies. the recruitment process in both studies was essentially identical. index cases were first recruited from their healthcare provider if they presented with influenza-like illness (ili). this individual would be included in the follow-up if he ⁄ she tested positive for influenza virus infection by rapid antigen test (quickvue) and was the first person in his ⁄ her household that showed signs of ili in the previous weeks. follow-up consisted of three home visits that spanned approximately - days. at each home visit, nasal and throat swab (nts) specimens were collected from all household members, regardless of the presence or absence of symptoms. symptoms were recorded in daily symptom diaries provided for every household member, and digital thermometers were provided to record daily tympanic temperature. the symptoms recorded were fever ‡ ae °c, headache, myalgia, cough, sore throat, runny nose, and phlegm. influenza virus infection and subtype was identified by reverse transcription polymerase chain reaction (rt-pcr) on the nts specimens. viral shedding was quantified from the same specimens by rt-pcr to determine viral loads, as well as by quantitative viral dilutions to determine median tissue culture infectious dose (tcid ). the details concerning laboratory methods have been described in a previous study. all analyses in this study focus exclusively on secondary cases; these are household contacts of recruited index cases who acquire influenza virus infection following the initial home visit. index cases generally presented with a certain threshold of illness severity requiring medical attention, whereas infections among household contacts can vary from asymptomatic to severe representing naturally acquired influenza infections. these secondary cases must be negative for influenza for their first nts specimen, and subsequently tested positive. we analysed mean viral loads measured by rt-pcr and quantitative culture by plotting by day since acute respiratory illness (ari) onset according to strain of influenza (pandemic a ⁄ h n , seasonal a ⁄ h n , seasonal a ⁄ h n , and seasonal b). ari is the reference time point, because the day of infection is unknown and is defined as the presence of ‡ of the symptoms mentioned above. average symptom scores were also plotted according to ari onset and grouped into upper respiratory symptoms (sore throat and runny nose), lower respiratory symptoms (cough and phlegm), and systemic signs and symptoms (fever ‡ ae °c, headache, and myalgia). mean daily tympanic temperatures were also plotted since date of ari onset and according to strain of influenza virus. all analyses were conducted using r software (version . . ; r development core team). a total of households and individuals were followed-up in the two studies. of household con-tacts tested by rt-pcr, were found to be influenza positive. among these influenza infections, ( ae %) were asymptomatic (rt-pcr positive plus symptoms recorded), were subclinical (rt-pcr positive plus symptom recorded), and presented with an onset of ari during the follow-up period. from the cases with ari onset, seven pandemic a ⁄ h n , seasonal a ⁄ h n , seasonal a ⁄ h n , and seasonal b influenza virus infections were identified. the age distribution among secondary cases was observed to be largely comparable across the four strains of interest (table ). there were a lower proportion of males who acquired pandemic a ⁄ h n compared to the seasonal strains of the virus. cough was the most commonly reported symptoms during follow-up in cases of pandemic a ⁄ h n and seasonal b, whereas runny nose was most common in seasonal a ⁄ h n and a ⁄ h n cases. cumulatively, fever ( ‡ ae °c) was reported in approximately half ( %) of the secondary cases. patterns of viral shedding were analysed in a subset of influenza positive individuals who recorded an onset of ari in their symptoms diaries (figure ). household contacts that were asymptomatic, subclinical, or did not have an ari onset were excluded from the analysis. viral shedding in all three influenza a strains were recorded to occur on the day of ari onset or day post-ari onset. follow- ing the peak, measured levels of viral shedding declined steadily to undetectable levels over - days. the trend of viral shedding in influenza b infected individuals rose days before ari onset, fluctuated for around days before eventually resolving. the patterns of viral shedding over time measured by quantitative viral culture were generally similar to the patterns measured by rt-pcr. the patterns of symptoms and signs were comparable in the four strains of influenza included in this study, peaking on the day or day post-ari onset, and gradually declining over a period of - days. in all strains, systemic symptoms and signs were observed to resolved faster than upper and lower respiratory symptoms. the trend of tympanic temperature in each influenza strain was comparable to the respective symptom pattern. patterns of viral shedding observed in influenza a strain infections (pandemic a ⁄ h n , seasonal a ⁄ h n , and seasonal a ⁄ h n ) were broadly similar. the pattern differed from the observed pattern of viral shedding in seasonal influenza b infections. the majority of viral shedding in influenza a strains occurred at and near ari onset, whereas there were variable amounts of viral shedding preand post-ari onset for those with influenza b. the biological reason for this difference is yet to be clarified. these differences are consistently observed regardless of laboratory method used to quantify the viral loads. it was observed that viral shedding measured by tcid resolved more quickly than when measured by rt-pcr, suggesting that rt-pcr is more sensitive, but it could be detecting inactivated fragments of rna instead of active virus. the trends observed for the seasonal strains of influenza in this study were similar to those reported in literature. the patterns of symptoms and signs as well as tympanic temperature in the four different strains of interest in this study were found to be comparable. these patterns closely resemble the patterns of viral shedding observed in the influenza a virus strains, but not in the influenza b virus strain. the trends of viral shedding, symptom scores, and tympanic temperature for pandemic a ⁄ h n were similar to trends observed for seasonal a ⁄ h n and seasonal a ⁄ h n infections, suggesting that the dynamics of these viruses are largely the same. the clinical course of infection with pandemic a ⁄ h n influenza virus appeared to be similar to the seasonal b influenza virus, but the patterns of viral shedding over time diverges. in general, our results suggest that the dynamics of the pandemic a ⁄ h n virus were similar to the seasonal a ⁄ h n and a ⁄ h n viruses, and clinically similar to the seasonal b virus. this study faced sample size limitations; very few cases of pandemic a ⁄ h n were detected and the secondary attack rate in general was low, though a total of households were followed up. this lack of power led to the inability to analyse the differences between adult and children and other characteristics that could be correlated with amount of viral shedding. there are also biases that must be factored in during recruitment. the eligibility criteria of only healthy households could select for households with higher innate immunity. on the other hand, recruitment at health care providers can be biased towards index cases that had more severe illness that required medical attention. the strength of the study is the broad generalizability of the results due to the strict classification of secondary cases. the infections reported in this study were all community-based and should represent true natural infections. pandemic potency of the influenza virus is largely determined by its transmissibility. the first objective of this study was to model the transmission of influenza h n and h n viruses. at present, vaccination with laiv has been used as a widespread, effective public health measure for influenza prophylaxis. some unsubstantiated concerns have been raised about a potential possibility of reassortment of circulating influenza viruses with laiv viruses following vaccination with laiv. thus, another objective of this study was to assess the probability of pig-to-pig transmission of cold-adapted viruses and their potential reassortment with wt influenza strains. female albino guinea pigs weighing - g were inoculated intranasally with eid of virus without anaesthesia. transmission studies were then performed hours after inoculation. inoculated animals were housed at % relative humidity and °c in the same cage with noninfected guinea pigs or in cages placed m away from non-infected pigs. virus replication was determined by virus isolation in hen eggs and by pcr. sera were collected at and days post inoculation. seroconversions were assessed by routine hai test. genome composition of reassortants was monitored by rflp analysis. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated, and virus growth properties were observed following virus titration in hen eggs. when infected pigs were co-caged with non-infected (naïve) individuals, vn , indo ⁄ , a ⁄ california ⁄ ⁄ , and nibrg- were isolated in %, % ae %, and % of contact animals, respectively. serological confirmation of virus transmission was higher than virological data ( %, %, %, and %, respectively). in addition, it was shown that when pigs inoculated with a ⁄ california ⁄ ⁄ were co-caged with animals inoculated with nibrg- , they got infected with both viruses ( table ) . the ability of direct transmission of cold-adapted viruses was also investigated. data show that the a ⁄ ⁄ california ⁄ ⁄ laiv candidate was detected in the upper respiratory tract of ae % vaccinated pigs. the mdv was identified in % of infected animals. however, neither group of contact pigs, co-housed with the vaccinate pigs, had evidence of infection with cold-adapted viruses. in addition, none of the contact pigs had any evidence of seroconversion to the coldadapted viruses as determined by hai assay. it was also most interesting to note that pig-to-pig transmission of the highly transmittable nibrg- reassortant virus was not seen when pigs, vaccinated with mdv, were co-caged with animals infected with nibrg- virus (table ) . this strongly implies a form of interference or protection from transmissibility that was provided by the cold-adapted virus. the results show that nibrg- and indo ⁄ viruses were able to spread between cages over the m distance ( % and % naïve animals were successfully infected, respectively). a ⁄ california ⁄ ⁄ influenza and vn viruses did not transmit between infected and non-infected guinea pigs housed in separated cages (table ) . pigs with confirmed a ⁄ california ⁄ ⁄ virus replication were also infected with nibrg- virus if h n -and h n -infected animals were separated by a space. thus, influenza virus transmission from h n -to h n -infected pigs has been shown, but the reverse pattern did not occur. transmission of nibrg- or a ⁄ california ⁄ ⁄ viruses was not observed when contact pigs were first vaccinated with the mdv and housed at a m distance ( table ) . it was also shown that efficiency of transmission of nibrg- was much higher than of other studied h n viruses; it can be transmitted between naïve guinea pigs separated from infected animals at a distance of - m (data not shown). five reassortants were isolated from animals which were infected with a ⁄ california ⁄ ⁄ virus and co-caged with pigs inoculated with nibrg- . two reassortants possessed different combinations of pr , nibrg- , and a ⁄ california ⁄ ⁄ genes and demonstrated the non-ca ⁄ non-ts phenotype typical of wt viruses. unexpectedly, two other reassortants inherited ha gene from nibrg- , na gene from a ⁄ california ⁄ ⁄ , and other genes from pr became ca and ts. : non-ts reassortant inherited pa gene from pr and seven other genes from a ⁄ california ⁄ ⁄ , gained ca properties. in spite of aforesaid experimental data, we cannot exclude the theoretical possibility of simultaneous infection of human host with cold-adapted and wt influenza viruses. to better understand possible consequences of such a reassortment event, we co-infected guinea pigs with a mixture of mdv and nibrg- viruses. nasal washes were collected and cloned by limited dilutions in hen eggs in the presence or absence of immune serum to the mdv. cloning of nasal washes without antiserum led to isolation of over clones, which were all identical to the mdv (data not shown). when nasal washes were cloned in the presence of antiserum, only nine clones were isolated. genome composition analysis showed that all isolates were triple reassortants, which had inherited pb and na genes from mdv, pa gene from pr , and ha gene from nibrg- . the origin of the other gene segments (pb , np, m, ns) in the genome of guinea pig-derived reassortants varied. reassuringly, all reassortants generated in vivo had the phenotype typical of the mdv. the severity of influenza outbreaks is partly determined by efficient spreading of the causative virus strain between human hosts. however, little is known about mechanisms underlying influenza virus transmission in humans. guinea pigs have been shown to be a suitable model for influenza transmission studies. our in vivo study showed that influenza a viruses vary in their transmissibility. nib-rg- and indo ⁄ viruses were able to transmit to naïve animals caged distantly from infected animals. in contrast, cold-adapted viruses, the same as those used for licensed laivs, showed no signs of transmission from one guinea pig to another. our study also provided evidence of a lower level of transmissibility of the novel pandemic h n virus compared to the nibrg- and indo ⁄ h n strains evaluated. benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. in our study, the mdv inoculated into guinea pigs appeared to interfere with and even offer protection from transmission of the highly transmissible nibrg- virus. the ability to immunize with the laiv and subsequently block the spread of a homologous h n subtype and a heterologous h n subtype influenza virus between guinea pigs has been shown. interference between cold-adapted and wildtype influenza virus infection was the most likely explanation for the data observed in our study. the mdv inoculated into guinea pigs might in some way interfere with transmission of highly transmissible influenza viruses. it is believed by some that widespread use of laiv could increase the potential risk of reassortment of the vaccine strain with circulating influenza viruses immediately following vaccination. however, it was shown that any such potential reassortments would most likely lead to yet attenuated viruses. our in vivo studies have shown that introduction of mdv genes into the genome of nib-rg- virus led to the generation of triple reassortants inherited pb and na genes of mdv and ha gene of h n virus. all isolates possessed phenotypical markers associated with attenuation of mdv. our data suggest that even if a reassortment event of such rare occurrence between a laiv strain and a circulating virus were to occur, it would most likely lead to a reassortant that would retain highly attenuated phenotypic properties of the vaccine strain. our data strongly support the safety of laivs, especially those developed against highly transmissible h n and h n pandemic influenza viruses. this information builds upon databases that have clearly shown the low likelihood of transmitting an laiv, as well as the high likelihood of any field reassortment of laiv with a circulating influenza virus to retain important properties of the cold-adapted, temperature-sensitive vaccine master composition. very interestingly, we also present data that show the potential of a laiv to prevent the transmission of highly infectious influenza viruses, perhaps identifying a broader role for laiv in the overall scheme of influenza virus prophylactic use. background: schlieren imaging is a non-invasive, real-time airflow visualization technique that relies on differences in air temperatures (and the resulting changes in the refractive index) to allow exhaled human airflows to be seen clearly against the background of more-stationary, ambient air. recently, this technique, well-known to engineers, has been applied to better understand and characterize airflow behaviors associated with everyday, as well as healthcarerelated, human respiratory activities. materials and methods: as a surrogate marker for the behavior of airborne infectious agents, schlieren imaging was used to visualize the airflow patterns produced by adult human volunteers of different ages while coughing with and without the wearing of standard surgical and n masks. results: the cough plumes were generally similar in shape and range for all the adult volunteers used in this study. although both the surgical and n masks decelerated and blocked some of the forward momentum of the coughed airflows, much of the cough plume was redirected and escaped around the top, bottom, and side edges of the masks to merge with the volunteer's natural, verticallymoving thermal plume. conclusions: schlieren imaging is a safe technique for visualizing exhaled airflows from human volunteers without the need for potentially-irritant or toxic particle tracers. findings from these schlieren imaging experiments will assist the development of more effective aerosol infection control guidelines in healthcare premises where patients infected with potentially airborne infectious agents (e.g., influenza and tuberculosis) are present. these infectious agents may be transmitted to healthcare workers, other patients, and their visitors by way of exhaled airflows. with the recent influenza pandemic , and the ongoing concerns about human cases of avian influenza h n infections, there is now a very real concern about the potential for the aerosol transmission of respiratory pathogens. such concerns amongst staff and patients in healthcare environments have led to a greater emphasis on the understanding and control of infectious airflows. , previous visualization techniques have used potentially-toxic or irritant gas or particulate tracers with hazardous laser light sources that have precluded the use of human volunteers as subjects. instead, various forms of lung models that simulate human respiratory patterns with such particulate tracers have been used. , schlieren imaging is a technique familiar to engineers and offers a non-invasive (i.e., no tracer required) airflow visualization method that depends only on differences in the refractive index of the warmer, human-exhaled air and the cooler ambient air. the use of a simple incandescent or light-emitting diode (i.e., non-laser) light source is safe and allows human volunteers to be used as experimental subjects, where their exhaled airflows are then observed using a large, precise spherical or parabolic telescopic mirror and a camera, and are recorded for later analysis and presentation. [ ] [ ] [ ] the analysis of these patterns of 'real-life' human airflows will be useful in optimizing aerosol infection control guidelines, which aim to reduce the transmission of airborne infectious agents to other healthcare personnel, patients, or their visitors. the images and analysis presented here have all been obtained from the large m diameter parabolic mirror (figure ) situated at the gas dynamics laboratory of penn state (directed by gary s. settles). this large schlie-ren imaging system has been in use for over years to obtain high quality schlieren images for various engineering applications. it has only recently been applied to clinically-relevant imaging. the objective of this paper is to augment and expand upon the details of the methods and results presented in an earlier study using this same schlieren imaging system. the aim of this series of studies is to visualize and capture a series of airflow images produced by coughing from adult human volunteers of different ages ( - years old). these included males (three of years, one of years of age) and females (one of years, one of - years, and one of - years of age). each volunteer was tested with and without wearing either a standard surgical mask or n mask. more specifically, the aim was to visualize the extent and direction of leakage around the mask whilst each subject was coughing. penn state institutional approval for experiments involving human subjects was also obtained. each volunteer was asked to stand approximately m in front of the schlieren mirror, facing across the surface of the mirror on one side, and to cough several times as the real-time, color image and video footage was recorded by the operator (using a nikon d camera; nikon inc. melville, ny, usa). this process was repeated whilst each volunteer was wearing a standard surgical mask then an n mask (supplied by mÔ, st paul, mn, usa). some of the schlieren images obtained from some of these volunteers have been published previously: for a -year old male, the year-old female and a -year old male, and the - year-old female. this article completes this series of schlieren images obtained from these experiments by including the images recorded for the older, year-old man. generally, it was found that the shape of the cough plumes (shown in the figure as darker shadows emanating from the subject's mouth) produced by adult humans of different ages was relatively similar. cough plumes are roughly conical in shape and very turbulent, usually passing beyond the extent of the m mirror (figure a) . a previous detailed study of one of these images measured a maximum airflow velocity of m ⁄ second for an adult cough. similarly, the effects of wearing surgical and n masks can be generalized across different ages. wearing a surgical mask allows leakage of the coughed air from the sides, top, and bottom of the mask ( figure b ). there is also some leakage through the mask, as indicated by the darker patches of air directly in front of the mask ( figure b, c) . the useful effect of the mask appears to be a deceleration and redirection of this coughed (and potentially infectious) air into the natural, upward-rising human thermal plume, which captures it and carries it upwards where it is diluted and less likely to transmit infection to others. the effects of the n mask are similar (i.e., deceleration and redirection), yet due to its tighter (mask-fitted) face seal, more of the coughed air appears to penetrate the front of the mask ( figure c ). this penetrating air is, however, also decelerated sufficiently to allow the wearer's natural thermal plume to carry it upwards. , discussion from these series of schlieren images presented in this and other related studies, [ ] [ ] [ ] it is clear that schlieren imaging offers a safe, non-invasive, real-time technique to visualize human exhaled airflows for all age groups. it is apparent that, at least where airflow patterns are an acceptable surrogate marker for airborne transmission risks, there are beneficial effects of wearing either type of mask, even when the mask fit is relatively poor. this is often the case when n -style masks are purchased and used by the general public -in contrast to the situation with healthcare workers, who are often accurately fit-tested for this type of mask. the immediate significance of this can be seen when masks are bought by parents for their children. often, these will not be of pediatric size and the mask-fit will be loose. children are well-known to be major sources of infection in the community because of their relatively poor immunity to many types of infectious agents due to their young age and, therefore, limited past-exposure history. these images allow infection control teams to literally see how far and how fast potentially-infectious human exhaled airflows can travel from an individual. this may have significant implications for guidance on the wearing of masks for infected staff and patients, on ward bed-spacing, as well as for the types of masks to be used in different situations. the important practical potential lies in the non-intrusive visualization of airflows associated with human volunteers, to assist in heightening the awareness amongst healthcare workers of the risks and potential for the airborne transmission of infectious agents, as well as the development of more effective aerosol infection control policies. schlieren images can be analysed more quantitatively, e.g., with the 'schlieren-piv' technique, , though this additional quantitative data is probably more of research interest than being of immediate practical use to everyday hospital infection control teams. these are the subtypes that we have studied. clearly, the question arises as to whether the changes in antigenicity are coupled with changes in germicide susceptibility. we have employed a modified log-reduction method in a cell culture system employing mdck cells in serum-free ex-cellÔ medium supplemented with trypsin. microscopic examination of cpe was the marker for infectivity together with plaque assay. we confirmed antiviral potency by using specific subtype influenza identification subtype technology, quidel quickvue Ò influenza a + b test. the log inactivation and percent inactivation by bac after a second contact time for the h , h , and h pandemic strains are as follows: a ⁄ swine ⁄ iowa ⁄ ⁄ h n , ae log ⁄ ae %; a ⁄ swine ⁄ cal ⁄ h n , ae logs ⁄ ae %; a ⁄ j ⁄ ⁄ h n , logs ⁄ ae %; and a ⁄ hong kong ⁄ h n , ae logs ⁄ ae % (table ). comparable results of antiviral efficacy are obtained with the tcid and plaque assays against all subtypes studied. when performing the plaque assay the sensitivity of virus recovery was better in the vessel with a larger surface area and overall recovery was in agreement with the potency determined by tcid assay. in our plaque assay, we inoculated a ⁄ hong kong ⁄ ⁄ virus dilutions into two different vessels with hours adsorption time: -well plate and t- flask, ml inoculum per replicate. virus titers obtained were: ae · pfu ⁄ ml from -well plate and ae · pfu ⁄ ml from t- flask ( table ). the discrepancy on virus potency can possibly be explained as: the binding of virus to host cell occurs only when virus gets a chance to interact with the cell on the monolayer during adsorption time. the percentage of virus population in the inoculum that has the opportunity to bind to the cell mainly depends on the surface area where this interaction takes place. therefore, in our experiment the plaque assay in the t- flask gave higher virus recovery ae versus ae · pfu ⁄ ml. the increased virus recovery can translate into better sensitivity of the test system for disinfectant and antiviral agents. the potency of the virus used in this study was determined by tcid was · tcid ⁄ ml. rapid diagnostic testing for influenza (quickvue Ò influenza a + b test, quidel) for aj versus bac was studied. the presence of influenza viral nucleoprotein a determined by quickvue kit correlated % with the viral infection based on by cpe in viral culture. interestingly, the inactivation of viral nucleoprotein was able to be revealed with diagnostic kit in the dilutions of virus ⁄ bac reaction mixture, which possessed prominent cytotoxic effect for the host cells in viral culture system. this type of molecular testing method is useful for interpreting antiviral efficacy against a background of cytotoxicity. these experiments are intended for the sponsor to substantiate to us fda that their antiviral substances are safe and effective. the data shows that the three hemagglutinin subtypes were highly susceptible to the quaternary ammonium compound in the short term in vitro experiment. the appearance of novel subtypes in the future can be met with the assurance that disinfectant and ⁄ or antiseptic resistance will be unlikely. certainly, from the above data, although genetic reassortment of human and swine viruses may modulate influenza pathogenesis and limit existing vaccine benefit, it is not likely be a factor in control of viruses on environmental surfaces by benzalkonium-type disinfectant ⁄ cleaning agents in community or health care environments. table . comparison of viral titer obtained in different vessels using quantal tcid and plaque assay methods plaque assay tcid assay t- ( cm ) -well plate ( cm ) tcid ⁄ ml tcid ⁄ ml ae · pfu ⁄ ml ae · pfu ⁄ ml · ae · options for the control of influenza vii outbreak influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. in this paper we share our experience of managing an outbreak of viral respiratory infection in an acf very early in the influenza pandemic and also describe some of the emerging issues relating to crossreacting antibodies to the pandemic (h n ) influenza virus in the very elderly. the outbreak investigation was conducted as part of an urgent public health intervention initiated by the new south wales (nsw) department of health during the early stages of the first southern hemisphere wave of the pandemic. nose and throat swabs for nucleic acid testing (nat) plus acute and convalescent serum samples ( weeks apart) were collected from all the residents of an acf where an influenza-like illness (ili) outbreak occurred. the investigation revealed dual outbreaks of pandemic (h n ) influenza and rhinovirus infection. out of residents, three had laboratory confirmed influenza [two with pandemic (h n ) ], and had rhinovirus infection on nat. testing of acute sera collected from every subject found elevated ( ‡ : ) pandemic (h n ) hai antibody in % ( ⁄ ) subjects aged years or more (born before and median age years; geometric mean titre-gmt ae ) compared with none of the residents aged under years (born after and median age years; gmt ae , p = ae ). the acf was closed to visi-tors for days. the symptomatic residents received treatment-dose oseltamivir, and all other residents were given oseltamivir prophylaxis. more than one virus may be circulating in an acf with an ili outbreak at any one time in winter. a significant proportion of elderly residents had pre-existing cross reacting antibody to the pandemic (h n ) , which may explain the minimal clinical impact of pandemic (h n ) in this elderly population. influenza is one of the leading causes of infectious death in elderly people, principally due to co-morbidities and declining immune competence with age. it is the most important agent in outbreaks of respiratory illness. influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. the clinical presentation of influenza in residents of acfs can be subtle, with a blunted febrile response and a non-specific decline in mental and functional status. residents commonly have underlying diseases that can be exacerbated by influenza infection, and in addition, they are at higher risk of serious influenza-related complications than community dwelling elderly people. people aged over years are also at higher risk of influenza-related death, and more than % of annual influenza-related mortality is usually confined to this high risk group. in australia, influenza and pneumonia have sub-stantial health impacts; recorded as being the underlying causes of death for persons in . since the world health organization declared an influenza pandemic in june , australia has suffered one of the highest rates of confirmed infection during the first southern hemisphere wave. by late october there were reported deaths due to pandemic influenza in australia, and to date there have been about deaths reported worldwide. although disproportionately far fewer elderly people developed clinical influenza during the current pandemic than occurs with seasonal influenza, their case-fatality rate remained substantial. early in the pandemic (june ), we investigated a suspected pandemic influenza outbreak in a rural acf in the state of nsw, australia. the epidemiology (including virulence and clinical outcome in the elderly) of the pandemic (h n ) virus was mostly unknown at the time of investigation, and as time passed, this investigation provided clarity on some important issues of the influenza epidemiology in the elderly population. in this paper we share our experience of managing a dual outbreak of viral respiratory infections early in the pandemic, and also describe some of the emerging issues relating to the cross-reacting antibodies to pandemic influenza in the very elderly. the outbreak investigation was conducted as part of urgent public health intervention initiated by the nsw department of heath in conjunction with the local public health unit, the national centre for immunisation research and surveillance (ncirs), and the institute of clinical pathology and medical research (a who national influenza centre). to determine the extent and cause of the outbreak, a public health research doctor (gk) was dispatched from sydney over a weekend to assist with outbreak investigation and control. on june th , the greater southern public health unit surveillance officer (bd) received a report of a possible pandemic (h n ) outbreak in a local acf. on investigation, it was discovered that days earlier a year old female resident had become generally unwell, but without specific symptoms of influenza like illness (ili). soon after, nine of the co-residents (but no staff) had developed symptoms suggestive of influenza. one other resident had returned from a melbourne (victoria) hospital (where pandemic (h n ) was known to be circulating) the previous week after surgery, but did not have ili symptoms. on june th, the symptomatic residents had nasal swabs taken by the local doctor for influenza [including pandemic (h n ) ] nucleic acid testing (nat). there was rising concern due to reports of widespread pandemic (h n ) influenza in a local army camp just over the border in nearby victoria, where pandemic (h n ) influenza was known to be circulating widely. on june th, the year old lady proved nat positive for pandemic (h n ) , but none of the other samples were pandemic (h n ) nat positive. concern arose that there might be an outbreak of pandemic (h n ) in the facility, and that some of the swabs from other residents might be false negatives. between and june, after consent was obtained, directly or through next of kin in demented residents, all submitted to venipuncture for serology, successfully, and the other as yet un-swabbed residents were swabbed. basic demographic data were collected from every resident with clinical information on co-morbidities and current medication use. convalescent blood samples were collected after weeks on th july from of the residents. swabs were sent to icpmr where nat for influenza a [including pandemic (h n ) ] and b was performed. the acute and convalescent serum samples were tested later (in december ), using haemagglutination inhibition assay (hai) to detect pandemic (h n ) antibody. , interventions the acf was closed to visitors from th until th june. treatment of the positive case and the nine symptomatic residents, with twice daily oseltamivir, was begun on saturday june th, and all other residents were started on once daily oseltamivir prophylaxis. the facility manager and local general practitioner (gp) monitored patient health on a daily basis, and none had to stop oseltamivir due to adverse events. one resident with ili who was known to have moderately impaired renal function was given once daily rather than twice daily oseltamivir treatment. the age range of the residents was - years with a median of years. all residents had underlying medical conditions, e.g., chronic cardiac and respiratory diseases ( table ) testing of acute sera collected from every subject found elevated ( ‡ : ) cross-reacting hai antibody to the pandemic (h n ) in % ( ⁄ ) of subjects aged years or more (born before and median age years; geometric mean titre-gmt ae ). however, the hai titre was consistently < : and significantly lower (gmt ae , p = ae ) in the residents aged under years (range - years, median years) (figure ). the index case (nat positive) did not show a significant raise in hai level in convalescence (going from to ). the pandemic (h n ) case that was determined by serology was pandemic (h n ) nat negative. to our surprise, seven of the other asymptomatic residents had rhinovirus detected on extended nat (reported on june th), despite being asymptomatic at time of swabbing and remaining so. the original nine influenza nat negative samples were then tested and three of these were also nat positive for rhinovirus; in total, ten proved nat positive for rhinovirus ( ae %). the serologically confirmed pandemic (h n ) case was also positive for rhinovirus infection. of interest was that only one resident had a documented fever. this investigation illustrates some of the difficulties in managing and investigating possible influenza outbreaks in real time in the context of an influenza pandemic. finding a nat positive case of pandemic (h n ) influenza among many other symptomatic cases raised the possibility (although not the probability) that pandemic (h n ) was the cause of the outbreak. rhinovirus infection, however, was confirmed by nat in ten residents. this outbreak illustrates that more than one virus (in this case and perhaps ) may be circulating in an acf at any one time in winter. in ili outbreaks in acfs, broad laboratory testing is recommended; nat is the most sensitive method of detecting influenza or other viruses in respiratory tract samples. studies have found that the pandemic (h n ) haemagglutinin (ha) gene is more closely related phylogenetically to the h n virus and classical swine influenza a ⁄ h n viruses than more recent seasonal human influenza a ⁄ h n viruses. it is antigenically similar to the h n pandemic virus in terms of the immunodominant antibody response to haemagglutinin. [ ] [ ] [ ] it is likely that individuals alive during the emergence and initial persistence of the pandemic virus would have higher levels of cross-reacting hai antibodies to the pandemic (h n ) , which would contribute towards better clinical protection. in our investigation, % of the residents born before (aged years or above in ) had pre-existing cross-reacting hai antibody to the pandemic (h n ) . in elderly populations, severe illness may be associated with organisms typically considered to be mild, such as rhinovirus. however, studies have shown that nursing home residents may be susceptible to outbreaks of rhinovirus that may cause mild to severe respiratory illness, particularly in those with a history of lung disease. one rhinovirus outbreak in a nursing home in the usa caused fatalities. another outbreak showed residents with underlying lung disease are more likely to have longer infection, require antibiotics, develop bronchospasm, and have difficulty breathing; two residents with underlying lung disease required emergency treatment and one died. a previous influenza outbreak in a nsw aged care facility in caused significant mortality and morbidity. that outbreak resulted in hospital admissions and six deaths. in our investigation we have found that % of the residents had chronic lung disease and % had chronic cardiac conditions both considered as high risk for severe complications of both rhinovirus and influenza infection. however, there were no hospitalisations or deaths in our outbreak investigation. indeed only one resident developed fever, indicating that non-specific signs of illness (such as in our index case) may be the only, or early, indication of an ili. our own experience with managing other ili outbreaks has also taught us that staff of acfs may not be vigilant enough to detect fevers. in this outbreak, the nursing home staff, local gp, public health unit and the outbreak investigation team and supporting laboratory staff acted quickly and in a coordinated way. pre-existing cross-reacting antibody in the very elderly (aged ‡ years) probably helped to limit the spread of the pandemic virus (compared to the circulation of rhinovirus) within the acf. exposure to the pandemic (or a close variant occurring before ) appears to be responsible for a high hai titre in the very elderly, which contributed towards better clinical protection. however, wider testing early on would have alerted us more quickly to the main cause of the outbreak. treatment and prophylactic use of oseltamivir may also have contributed to halting the spread of pandemic (h n ) and also to symptom relief. pandemic (h n ) influenza virus (ah pdm) has spread worldwide since march . in a paper of ah pdm, % of infected individuals have experienced gastrointestinal symptoms such as diarrhea and vomiting, which is higher than that of seasonal influenza. however, little is known whether viable virus shed from stool and replication of viruses are ongoing in the gastrointestinal tract. , viral load and isolation of ah pdm in cell culture in stool samples has been reported. stool specimens were collected from patients suspected to have pandemic (h n ) infection from november through may . virus isolation was conducted in cell culture by using madin-darby canine kidney (mdck) cells and taqman based rt-pcr from % (w ⁄ v) stool suspension in phosphate-buffered saline. taqman based rt-pcr was conducted by using primers, probes, and positive controls provided by niid (national institute of infectious diseases of japan). to confirm presence of ah pdm viral rna, lamp (loop-mediated isothermal amplification) was used as supplemental testing. of patients, one child (case ) submitted one nasal swab and four stool samples, another one nasal swab and two stool samples, and the other one stool sample. informed consent was obtained. strand specific rt-nested pcr was performed for only case by using only one primer at the rt reaction and also assayed neu aca - gal and neu aca - gal binding specificity about isolated strain derived from nasal swab and stool. receptor binding specificity was performed using a solid-phase binding assay with the sialylglycopolymers (poly a-l-glutamic acid backbones containing neu aca - galb - glcnacb-pap or neu aca - galb - glcnacb-pap bond as described. ) nucleotide sequences of the ha gene of ah pdm viruses isolated from stool sample and nasal swab were analysed. in order to exclude the possibility of contamination, the stool samples and nasal swabs were subjected to virus isolation separately. after getting the results on the nucleotide sequence, we also confirmed no strain harboring identical sequence was isolated in our laboratory before and after the day of sample collection. ah pdm viral rna was detected in nine ( %) of the subjects from stool samples. among nine subjects, one case (case no. ) was positive for viral isolation. case , a healthy -year-old girl, experienced fever and abdominal pain, and the others had gastrointestinal symptoms without upper respiratory symptoms. in case , influenza a virus was diagnosed by rapid antigen test on the day of symptom onset. viable ah pdm virus was isolated from the stool sample and nasal swab on the second day from onset using mdck cells (table ). viral load decreased gradually after symptom onset. however, viral shedding was still present days after symptom onset. positive stranded rna was detected days after symptom onset from the stool specimen ( figure ). above two ah pdm strains (isolated from nasal swab and stool specimen) bound exclusively to human type receptor, neu aca - gal. sequence analysis demonstrated that isolated virus from stool samples was identical with that from nasal swabs in comparison of ha gene ( bp). ah pdm influenza virus was isolated from the stool and nasal swab samples in the same patient simultaneously by using mdck cells. our results suggests the detection of viral rna and viable ah pdm influenza virus from stool samples may serve as a potential mode of transmission and has important implications in understanding the context of ah pdm influenza virus. strategies to prevent transmission of influenza include use of respirators. ffp and n respirators are certified to fil-ter at least % of particles ( ae lm in diameter), and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. [ ] [ ] [ ] we have developed a proprietary acid-polymer formulation to coat a standard ffp respirator with an antiviral layer. we aimed to test this coated respirator for antiviral efficacy against a range of influenza viruses. a series of tests compared the antiviral efficacy of coated and uncoated respirators in conditions designed to simulate real-life exposure to influenza by varying the route of inoculation, contact time, temperature, humidity, moisture, and contaminating substances. we also investigated whether infectious viruses could be transferred from contaminated respirator surfaces to gloves. we tested human, swine, and avian influenza viruses, including influenza a and b viruses. influenza a subtypes were the a ⁄ h n pandemic strain, seasonal h n , h n , h n , h n , and h n . in each test, suspensions of influenza viruses were prepared to - log tcid ⁄ ml in mem. in some tests, organic contaminants (yeast, bsa, and mucin) were added. one set of respirators was maintained at °c and % relative humidity for hours before the viral challenge, and repeatedly sprayed with he-pes buffer to simulate respiratory secretions. for each test, three coated (glaxosmithkline actiprotect) and three uncoated (sperian willson easy fit) ffp respirator samples were inoculated with ae ml of a viral suspension, which was applied with a pipette, sprayed, or aerosolised to create airborne droplets. after minute at room temperature (on a shaker), the respirator samples were assayed for the presence of infectious viruses using standard methods. in one test, after a minute contact time of the respirator with the virus, nitrile gloves were applied with light pressure to the outer surface of inoculated respirator samples and then assayed after minute. samples were put into test medium (mem, supplemented with antibiotics [penicillin, gentamycin, or streptomycin] and amphotericin b or l-glutamine). the supernatants were vortexed, extracted, and used to prepare serial -fold dilutions in mem. each dilution was used to inoculate four wells of rmk cells in a multi-well plate, and these cultures were incubated and scored over days for cytopathic effects, cytotoxicity, and viability. (some tests substituted mdck cells; others used inoculated embryonated chick eggs.) all tests included negative cell controls, cytotoxicity controls, and neutralisation controls. the spearman-karber formula was used to calculate viral loads as tcid or eid . antiviral efficacy was calculated from the difference between the geometric mean loads of influenza virus on the coated and uncoated respirators after minute of exposure. the viral loads applied to respirators in these experiments ranged from ae to ae log tcid , and were therefore high in comparison with respiratory secretions from infected patients at the peak of influenza symptoms (range - log tcid ). tables - show that the average viral loads detected on uncoated ffp respirator samples remained high in all conditions tested, ranging from ae to ae log tcid (or ae - ae log eid ). in contrast, the average viral load on coated respirators after minute of exposure ranged from below the limits of detection to £ ae log tcid ( ae log eid ). therefore, the relative antiviral efficacy of the coating ranged from ‡ ae to ae log . table shows that the relative antiviral efficacy of the coated mask remained high in simulated-use conditions such as organic contaminants and repeated saturation at high temperature and humidity. in the experiment to test transfer of viruses from respirators, the gloves applied to regular uncoated inoculated respirators had a viral load of ae log eid (table ) . by contrast, no viruses were detected on either the coated respirators or the gloves applied to them. the relative reduction in contamination was therefore ‡ ae log . ‡ ae log viral load with organic contaminants* ae ae ae log viral load after heat, moisture, and simulated secretions** ae ae ae log viral load transferred to glove** ae £ ae ‡ ae log eid *influenza subtype was a ⁄ h n , and strain was vnh n -pr ⁄ cdc-rg. **influenza subtype was a ⁄ h n , and the strain was hong kong ⁄ ⁄ . results are mean log tcid , unless specified otherwise. results are mean log tcid , unless specified otherwise, based on an infectivity assay in triplicate. limits of detection varied. * pandemic strains. **results are mean log eid , based on a haemagglutinin assay in duplicate. options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - strategies to prevent transmission of influenza include use of respirators, and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings for protection against pandemic influenza. - ffp respirators are certified in europe to filter at least % of nacl particles ( ae lm in diameter), and ffp and ffp respirators must filter at least % and % of these particles, respectively. influenza a viruses are typically ae lm, and can be carried in aerosolised droplets smaller than lm in diameter, which can disperse widely, remain airborne for hours, and be inhaled deeply into the respiratory tract. we have developed an acid-polymer formulation to coat the outer layer of a standard ffp respirator, in order to provide antiviral activity on the outer surface. we compared this coated respirator against standard ffp , ffp , and ffp respirators for filtration of aerosolised influenza viruses. the aim was to simulate protection against infectious viruses in droplets released when infected people cough and sneeze, and during aerosol-generating procedures in healthcare settings. the first assay compared three samples of coated ffp respirators (glaxosmithkline actiprotect) with three ffp controls (sperian willson easy fit). for each test, suspensions of influenza a (h n ) at ae log tcid ⁄ ml in ae · minimum essential medium (mem) were aerosolised with a nebulizer. the airborne droplets were introduced into a sterile chamber upstream of a respirator sample for minutes, at a flow rate of ae l ⁄ minute. constant airflow was maintained for another minutes after exposure to the virus. then the collection dish in the downstream sieve sampler (anderson) was assayed for infectious viruses using standard techniques. briefly, serial dilutions of the collection medium (mem with % fbs, % gelatine, and % hepes, supplemented with antibiotics and amphotericin b) in mem + trypsin were used to inoculate madin-darby canine kidney epithelial (mdck) cells in quadruplicate in a multi-well plate. these cultures were then incubated and scored over - days for cytopathic effects, cytotoxicity, and viability. negative cell controls and cytotoxicity and neutralisation controls were also performed. the spearman-karber formula was used to calculate tcid . the second assay compared five samples of coated respirators with five ffp controls ( m ) and five ffp controls ( m ). a suspension of influenza a (h n ), at ae tcid ⁄ ml, was nebulized for minute and seconds into the aerosol chamber, at a flow rate of ae l ⁄ minute, followed by constant airflow for minutes after exposure to the virus. then the collection medium in the downstream chamber (as before, with % nahco ) was assayed as described above. initial viral loads in the first and second assays were ae and ae log tcid , respectively, and were therefore high in comparison with respiratory secretions from infected patients at the peak of their influenza symptoms (range - log t-cid ). table shows that the average viral load that passed through the uncoated ffp respirators in the first assay was ae log tcid . the average viral load that passed through the coated respirators was ae log tcid . therefore, for active filtration of viruses, the relative efficacy of the respirator with antiviral coating was ae log greater than the uncoated respirator. for surface inactivation, the relative antiviral efficacy of the coated respirator was ae log . in the second study, table shows that the average viral load that passed through the uncoated ffp respirators was ae log tcid . in contrast, ae log tcid passed through the coated ffp respirators. by comparison with the viral load when no respirator was present ( ae log tcid ), the ffp respirators reduced the viral load by ae log , and the coated ffp by ae log . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load by ae log more than the ffp respirators. in this second study, the average viral load that passed through the uncoated ffp respirators was also ae log tcid . by comparison with the viral load when no respirator was present ( ae log tcid ), the ffp respirators reduced the viral load by ae log . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load passing through the mask by ae log more than the ffp respirators. table also shows that the coated respirators reduced the infectious viruses remaining on the mask surfaces by ae log more than the ffp respirators, and ae log more than the ffp respirators. even with a very high viral challenge, the coated respirators prevented passage of at least an additional ae log infectious viruses, compared with uncoated respirators. large numbers of infectious virions passed through all uncoated respirators tested. ffp respirators were no more effective than ffp respirators at blocking airborne influenza viruses. based on these in-vitro results, respirators with the antiviral coating could be expected to provide more protection than standard respirators from the risk of inhaling influenza viruses. strategies to prevent transmission of influenza include use of respiratory protection. ffp and n respirators are certified to filter at least % of nacl particles ( ae lm in diameter), and many guidelines have recommended that healthcare workers wear these respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. , we have developed a proprietary acid-polymer formulation, designed to coat a standard respirator and inactivate influenza viruses on contact. we tested this coated respirator for cytotoxicity, skin irritation, and sensitisation potential. the antiviral coating was also tested for stability and leaching under extreme environmental conditions, such as physical abrasion and simulated breathing at different temperatures, levels of humidity and co , and saturation with contaminants. eight coated respirators were tested at standard relative humidity ( % rh) for hours, and one at elevated humidity ( % rh) for hours. four coated masks were treated with synthetic blood or oral secretions, and then tested at % rh for hour. the sample respirators were sealed onto a mannequin head inside an airtight chamber, and air at °c and ppm co was pumped through the masks by a cyclic breathing machine at l ⁄ minute. a mm glass-fibre filter was placed behind the respirator, over the mannequin's mouth opening. at the end of all tests, these filters were eluted and analysed using high-performance liquid chromatography (hplc). standard in vitro methods were used to assess the cytotoxicity of the coated polyester and uncoated polypropylene layers of the respirator (glaxosmithkline actiprotect). samples were extracted in minimum essential medium (mem), supplemented with serum, penicillin, streptomycin, amphotericin b, and l-glutamine, at °c for hours. triplicate monolayers of mouse fibroblast cells (l- ) were dosed with each extract (including a reagent control and negative and positive controls), and incubated at °c in % co for hours. after hours of incubation with samples or controls, the monolayers of mouse fibroblast cells were examined microscopically for abnormal cell morphology or cellular degeneration. samples of the coated respirator (comprising four polypropylene layers bonded to the coated polyester outer layer) were applied under occlusive patch conditions to the skin of adults. controls, including individual layers, were applied in the same way. in a separate patch test, samples of the coated polyester outer layer and controls were applied under the same conditions to adults. after hours, test patches and controls were removed. sites were then scored for itching, erythema, oedema, epidermal damage, and papular response after and hours. the patches were applied three times a week for weeks. to evaluate sensitisation, test patches were applied - days later for hours at different sites to the original samples. after this challenge, skin was assessed and graded for sensitisation potential after and hours. table shows that no residues of the antiviral coating or degradation products were detected in the air that had passed through any of the eight respirators. cytotoxicity tests showed that the coated respirator material caused % cell lysis or toxicity, classified as slight reactivity (grade ), and that uncoated material caused no cell lysis or toxicity (grade ) ( table ) . results for positive and negative controls were severe reactivity and no reaction, respectively. from the results of the two human repeat-insult patch tests, neither the coated or uncoated layers nor the fullthickness respirator fabric caused irritation (including itching, erythema, edema, vesiculation, epidermal damage, papules, or reactions beyond the patch site) or sensitisation in any of the adult volunteers at any of the time points. based on these results, in conjunction with published data on acute and repeat-dose toxicity, mutagenicity, local irritation, dermal sensitisation, and inhalation safety for all components of the antiviral coating, the potential topical or inhalation exposure to the coated antiviral respirator does not pose a safety risk. the antiviral coating is durable and stable, and stays on the outer surface of the respirator, even in extreme environmental conditions. the coated respirator is non-irritating and non-sensitising. therefore, this respirator is considered to be well-tolerated and safe for its intended use. ies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. knowing how influenza virus is transmitted at home and in school is the key to preventing its spread. at the previous two meetings of this conference, , we introduced our study of household transmission of seasonal influenza and reported our conclusion that protracted survival of the virus even after treatment increases household transmission, and is a major factor in the transmission of the virus to infants. on the other hand, during the recent pandemic, many schoolchildren developed serious respiratory tract disorders, which again highlights the significance of schoolbased transmission of the disease. in this study, we compared transmission of a new influenza strain at home and in school with that of seasonal influenza and proposed countermeasures. the for the analysis of school-based transmission, the epidemic status of seasonal influenza in children at six elementary schools over the past two seasons ( - and - seasons) was compared with that of pdmh in children at two primary schools. using observational data of school-based transmission, we also constructed a model for influenza transmission , and evaluated the effects of factors that could affect influenza transmission (e.g., antibody prevalence, transmission rate, non-infectious latent period, infectious latent period, school closure) through the use of simulations. in this study, a diagnosis of influenza was confirmed by rapid influenza antigen detection kit. we previously reported the high sensitivity of the kits, - not only for seasonal influenza, but also for h n pandemic compared to virus isolation and pcr. serum antibody was not investigated. most of the index patients were treated with oseltamivir or zanamivir, and patients were treated with amatadine. no treatment was done for patients. no nai therapy was done as prophylaxis within the family. the incidence of households with an initial case patient who subsequently infected another member of the household was ae % ( of households) for seasonal influenza or ae % ( of households) for pdmh . thus, the household incidence of pdmh was lower than that of seasonal influenza. in addition, the percentage of family members in households who were infected by initial case patients (household transmission rate) was ae % ( of individuals) for seasonal influenza or ae % ( of individuals) for pdmh . thus, the household transmission rate was also lower for pdmh than that for seasonal influenza. effect of family size on household incidence and household transmission rate an analysis of the effect of family size on household incidence showed that, in families consisting of - individuals, the incidence of seasonal influenza in order of increasing family size was ae %, ae %, ae %, ae %, ae %, and ae %, respectively, and the incidence of pdmh was ae %, ae %, ae %, ae %, ae %, and ae %, respectively, indicating that household incidence tends to increase with increasing family size. in contrast, no definite relationship was noted between household transmission rate and family size. transmission rates for seasonal influenza in order of increasing family size were ae %, ae %, ae %, ae %, ae %, and ae %, respectively, or ae %, ae %, ae %, ae %, ae %, and ae %, respectively, for pdmh (shown in table ). effect of age cohort of initial case patient in household on household incidence and household transmission rate an analysis of the effect of the age cohort of the initial case patient in the household on household incidence and transmission rate showed that the household incidence of seasonal influenza in c , c , c , and c was ae % ( of households), ae % ( of households), ae % ( of households), ae % ( of households), and for m and f was ae % ( of households) and ae % ( of households), respectively. therefore, household incidence was the highest in c , followed by the parents. when the initial case patient was a child, the household incidence increased with decreasing patient age. in contrast, the household incidence of pdmh in c , c , c , and c was ae % ( of households), ae % ( of households), ae % ( of households), ae % ( of households), and for m and f was ae % ( of households) and ae % ( of households), respectively. therefore, household incidence was higher when the initial case patient was a parent, rather than a child. the household transmission rates for seasonal influenza from c to f were ae %, ae %, ae %, ae %, ae %, and ae %, respectively. therefore, as for household incidence, the highest rate ( ae %) was observed in c . the corresponding household transmission rates for pdmh were ae %, ae %, ae %, ae %, ae %, and ae %, respectively, with the highest transmission rates observed for infections from parents (shown in table ). if the rate of individuals with a secondary infection transmitted from the initial case patient in a household is presented as a percentage of the total number of affected individuals, the rates for seasonal influenza and pdmh were ae % ( of individuals) and ae % ( of individuals), respectively. therefore, the rate of individuals with a secondary infection was lower for pdmh than that for seasonal influenza. by age cohort, the corresponding rates of individuals for seasonal influenza in c , c , c , and c were ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), and for m and f was ae % ( of individuals) and ae % ( of individuals), respectively. for pdmh , the corresponding rates in c , c , c , and c were ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), ae % ( of individuals), and for m and f was ae % ( of individuals) and ae % ( of individuals), respectively. these findings indicate that, especially in the case of pdmh , most secondary infections in parents tend to be transmitted from another household member. the mean annual prevalence of seasonal influenza and the new influenza strain at the elementary schools for the two seasons was ae % and ae %, respectively, whereas the prevalence determined days after appearance of the first case in school was ae % and ae %, respectively. in the recent season at the same elementary schools, however, the prevalence was a high ae %. since the prevalence at days after the appearance of the first case in school was already ae %, these data show that the influenza virus spread quickly throughout the schools. at the schools with high transmission rates in the early period of the pandemic, new infections were confirmed even days after the school closure action was taken. these findings indicate that pdmh , the current influenza virus, has a long latent period during which it becomes infectious and spreads from infected individuals to numerous others in their vicinity. we constructed a model for influenza transmission in schools and estimated the time course of changes in the number of expected cases and the expected prevalence during the season. in this model, school children were divided into six groups depending on the stage of infection: uninfected period with no immunity, non-infectious latent period, infectious latent period, onset, post-onset infectious period, and immune period. it was assumed that schoolbased transmission occurred during the infectious latent period prior to onset and that no infections occurred during the post-onset infectious period because children were absent from school. due to the long latent period of pdmh , the distribution of the non-infectious latent period of pdmh was established as (day , day , day , day ) = ( %, %, %, %) and the distribution of the infectious period as (day , day , day ) = ( %, %, %). when simulations were performed under these conditions using the model for school-based transmission of influenza in which children from classes with an outbreak were kept at home for days, the time course of changes in the number of affected individuals actually observed and the time course of changes in the number of expected cases were determined. the expected prevalence under these conditions was %. to evaluate the effect of school closure, simulations were performed based on the assumption that children from affected classes were not kept at home for days. it was shown that there was an increase in the expected number of cases during the days corresponding to the period of actual school closure and that the expected prevalence increased to %. based on these findings, it was concluded that keeping children home from classes with an outbreak is an effective means of controlling the transmission of influenza in schools (shown in figure ). if the transmissibility of pdmh virus at home is estimated based on the speed of transmission and the degree to which pdmh is prevalent in schools, it would be expected that the household transmission of pdmh is also higher than that of seasonal influenza. in fact, the opposite is the case. this paradox can be explained in two ways. . the number of children aged or more and parents with pdmh influenza as a percentage of the total number of affected individuals is lower than those with seasonal influenza ( ae % versus ae %). further, although the number of parents with a secondary infection was high at home, the percentage of the total number of individuals with pdmh was a low ae % ( of individuals), compared to that for seasonal influenza ( ae % [ of individuals]). in other words, adults are less susceptible to pdmh infections and there was a correspondingly small number of affected individuals. therefore, it was considered that the transmission rate at home was lower than that at school for this reason. . the percentage of households with more than one affected individual within the same family was higher for pdmh at ae % ( of households) than for seasonal influenza at ae % ( of households). in the patients secondarily infected with pdmh , ae % of them showed symptoms of infection days or more after the onset in the first patient, suggesting that they were not infected at home, and the actual household transmission was ae % ( of households). therefore, although the prevalence was higher for pdmh , it seems that household transmission was lower because households with an affected individual implemented satisfactory control measures against infection. seasonal influenza differs greatly from pdmh influenza in its transmissibility at home and in school. in the household transmission of pdmh influenza, both the household incidence and household transmission rate of pdmh were low compared to those for seasonal influenza. although transmission of seasonal influenza from infants to parents was marked, in the case of pdmh , the reverse was true with transmission from parents to children being predomi-nant. it should be noted that household transmission in mothers was common in all eight seasons, suggesting the need to reconsider control measures against infection when nursing unwell family members. in the case of school-based transmission, pdmh was more prevalent than seasonal influenza, indicating that the virus spread quickly throughout the schools. this difference was attributed to the long infectious latent period when pdmh rapidly became rampant in the schools. an analysis of school-based transmission using a model for influenza transmission showed that, when % of the student population is infected, schools should be closed for five consecutive days in order to minimize the spread of the disease. the effectiveness of seasonal influenza vaccine in preventing pandemic and seasonal influenza infection: a randomized controlled trial introduction household transmission has been estimated to account for one-third of all influenza transmission, , and children are at high risk of spreading the disease. with reference to previous evidence, - some vaccine deployment strategies target children to prevent them from infection and transmitting influenza. nevertheless, few studies evaluated the effectiveness of vaccinating children in reducing household transmission. , during - , a pilot randomized controlled trial was conducted to investigate such effect by studying households with school age children randomized to receive trivalent inactivated seasonal influenza vaccine (tiv). the monovalent vaccine against pandemic influenza a (h n ) (ph n ) had yet been available until the end of the first wave. various conclusions have been made as to whether seasonal influenza vaccine might possibly protect against ph n . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] we report findings on the effectiveness of tiv against ph n observed in our cohort. households were screened if they expressed interest after receiving invitation letters distributed via their children's school or an existing pediatric cohort study. to be eligible, the household had to include at least one child aged - years who was not allergic or hypersensitive to any of the tiv components. children known to have immunosuppressive conditions or other contraindications against tiv were also excluded. written consent and assent were obtained from participants aged above years and those aged - years, respectively. proxy written consent was obtained from legal guardians or parents for participants younger than years. ethical approval was obtained from the institutional review board of the university of hong kong. consented households were allocated to the tiv and placebo group (in ratio : ) according a code generated by block randomization with random block sizes of , , and . an independent nurse prepared . one child (study subjects) from each household in the tiv group received a single dose of tiv with one child from each household in the placebo group receiving a single dose of saline placebo. parents and legal guardians were asked to report any adverse reactions days following vaccination. all participants, study nurses, and other research staff were blinded to the allocation and administration of vaccine or placebo. the vaccine allocation sequence was only disclosed to the investigators at completion of the study. serum specimens were collected from subjects shortly before (november-december ), one month after vaccination (december -january ), and after the winter (april ) and summer influenza seasons (august-october ). serum specimens were obtained from household contacts at baseline and after the winter and summer influenza seasons. all household members recorded any fever ‡ . °c, chills, headache, sore throat, cough, presence of phlegm, coryza, or myalgia daily on a symptom diary. they were also invited to report to the study hotline immediately if they experienced at least of the above signs or symptoms. as a response, the study nurse would visit the households with any sick members and collect nose and throat swab from all household members. the households were also telephoned monthly or increased to fortnightly during influenza seasons to monitor for signs and symptoms and remind them to report to the hotline. supermarket or book vouchers (for children) were given to the households including us$ for each serum specimen collected, us$ . for each home visit, and us$ for completion of the study. serologically-indicated influenza infection was the primary outcome of this study. it was define as a ‡ fold rise in antibody titer within each influenza season. other study outcomes included rt-pcr confirmed influenza virus infection, acute respiratory illness (ari) (two of any of the above listed signs or symptoms), and influenza-like illness (ili) (fever ‡ . °c with cough or sore throat). antibody titers against the vaccine strains were obtained by testing each serum specimens by haemagluttination inhibition (hai). viral microneutralization (vn) using standard methods was found to be more sensitive than hai in detecting antibody response against a ⁄ california ⁄ ⁄ (h n ) in another study conducted by our group and was, therefore, used in this study. the sera was initially diluted at ⁄ and further tested in serial doubling dilutions. nose and throat swabs were tested by reverse transcription polymerase chain reaction (rt-pcr) for influenza a and b viruses. technical details of the laboratory methods have been reported elsewhere. , fisher's exact test and chi-squared tests were used to compare count data including occurrence of side effects, laboratory confirmed, and clinically defined influenza infections. wilcoxon signed-rank test were used to compare the serum antibody titers between groups. exact binomial method or the wald approximation was used to estimate % confidence intervals where appropriate. all analyses were carried out in r version . . (r development core team, vienna, austria). twenty-five primary and secondary schools in the district of the study clinic were invited to participate. to parents of three schools that agreed to take part and another study cohort, invitation letters were sent and households were enrolled. personal referrals were made from these parents to enroll additional households. among enrolled households, subject with history of epileptic seizure was assessed to be contra-indicated against receiving the vaccine. blood taking failed in another subject, and both of them withdrew from the study. eleven households did not complete the study. table shows subject and household contacts of the tiv and placebo group were similar in demographics and prior influenza vaccination history. antibody titers before vaccination were comparable between groups (data not shown). most study subjects who received tiv showed antibody titer ‡ against the vaccine strains month after receiving tiv, and the proportion was significantly higher than those who received placebo (a ⁄ h n % in tiv versus % in placebo group, p < . ; a ⁄ h n % versus %, p < . ; b % versus %, p = . ). none of the study subjects had antibody titer ‡ against ph n following receipt of seasonal tiv. no serious adverse reactions were reported, and only pain at injection sites was slightly higher in tiv group (data not shown). subjects who received tiv had lower rates of serologically confirmed seasonal influenza a(h n ) ( % versus %, p = . ), a(h n ) ( % versus %, p = . ) and b infection ( % versus %, p = . , although the differences were not statistically significant (table ) . study subjects had higher rate of serologically confirmed ph n infection ( % versus %, p = . ), yet it was not statistically significant. after adjusting for potential cross reactive antibody response, % of subjects in tiv versus % in placebo groups showed ph n infection confirmed by either serology or rt-pcr (p = . ). little differences were observed for rt-pcr confirmed infection, ari, and ili in results combining the winter and summer influenza seasons. during winter season when seasonal influenza predominated, study subjects who had received tiv showed a lower tendency to develop ili ( % versus %, p = . ) or ari ( % versus %, p = . ). an opposite tendency was seen (ili % versus %, p = . ; ari % versus %, p = . ) during summer when ph n predominated. however, these differences were not statistically significant. rates of ili in subjects infected with ph n did not differ statistical significantly between subject who received tiv and placebo ( % versus %, p = . ). the study was not powered to detect indirect benefits to household contacts of vaccines resulting from reduced household transmission. attack rates were found to be similar between household contacts of subjects received tiv and placebo (data not shown). to examine potential factors that might affect risk of laboratory confirmed ph n infection, a multivariable logistic regression model was fitted to study all subjects and their household contacts. younger participants aged below years were found to have a higher risk (< years or = . , % ci . , . ; - years or = . , % ci . , . , > or = . ). after adjusting for age, sex, and date of study completion, receipt of tiv for the - influenza season was not found to affect risk of ph n infection. however, participants who had laboratory confirmed seasonal influenza infection during the study period had % lower risk of ph n infection (infected with seasonal influenza or = . , % ci . , . ; not infected with seasonal influenza or = . ). as (see table s for winter and summer results separately). influenza-like illness (ili) defined as temperature ‡ . °c plus cough or sore throat; acute respiratory illness (ari) defined at least any two of fever ‡ . °c, chills, headache, sore throat, cough, presence of phlegm, nasal congestion, runny nose, muscle or joint pain. limited by the sample size, we were not able to differentiate between the protective effect of seasonal a(h n ) and a(h n ) infection against ph n . other details of the results from the study were published elsewhere. discussion a non-significantly higher rate of ph n infection was observed in study subjects who received tiv compared to placebo. results from a multivariable logistic regression suggested that such a pattern might be explained by more common seasonal influenza infection in placebo group prior to the pandemic, protecting the placebo group against ph n . seasonal influenza infection within - months observed in our study might have conferred better cross protection than tiv against ph n . this resembles similar previous findings on cross protection between influenza infections in human and animal studies. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, the same phenomenon has not been observed in some studies on seasonal influenza vaccine against ph n . , , [ ] [ ] [ ] apart from differences in study design and vaccine used, we speculate that a short time interval between ph n and most recent seasonal influenza peak activities might be crucial for the phenomenon. hong kong is a subtropical area where the pandemic was preceded immediately by summer seasonal influenza circulation and a few months apart from the winter - influenza peak. if cross protection from seasonal influenza lasts for only a short period, it might have waned below partial cross protection from tiv over time from last seasonal influenza infection. the current study is limited by a small sample size, and further studies are required to confirm our hypothesis. while tiv is only effective against matching strains, a universal influenza vaccine could provide better protection against the ever evolving influenza viruses. introduction immunisation of healthy, as well as high risk, children has been the focus of much recent attention both in prevention of seasonal influenza and during the h n pandemic. detailed information on reactogenicity, particularly for newer vaccine formulations that include adjuvants, is limited. we recently reported results of a head-to-head comparison of two h n pandemic influenza vaccines in children in the uk. here we present new, detailed analyses of reactogenicity data from that study, which has important potential implications for future paediatric influenza vaccine development and use. we compared the safety, reactogenicity, and immunogenicity of two h n influenza vaccines, one as b (tocopherol based oil in water emulsion) adjuvanted egg culture derived split virion, the other non-adjuvanted cell culture derived whole virion, given as two dose schedules days apart, in a randomised, open label trial as previously reported. the study was age stratified ( months to under years & - years) to ensure adequate data in young children. age appropriate safety data (simplified for under year olds) were collected for days after each vaccine dose and serum was collected at enrolment & days after the second dose. nine hundred-thirty seven children received vaccines as per-protocol. when comparing the two vaccines, grade ( ‡ mm) local reactions were seen more frequently following the adjuvanted than the non-adjuvanted vaccine in both age groups, after both vaccine doses. in children over years old, ae % versus ae %, p < ae , after dose one; ae % versus ae %, p = ae , after dose two, in children under years old, ae % versus ae %, p = ae , after dose one (non significant, ns); ae % versus ae %, p < ae after dose two. fever ‡ °c (axillary measurement) was seen more frequently following the second dose of the adjuvanted vaccine compared to the non-adjuvanted vaccine in < year olds ( ae % versus ae %; p < ae ). looking specifically at the adjuvanted vaccine in under year olds, comparing the second dose with the first, there were significantly higher rates of fever ‡ °c (axillary measurement) ( ae % versus ae %, p < ae ), local grade ( ‡ mm) reactions ( ae % versus ae %, p = ae ), pain ( ae % versus ae , p = ae ), use of analgesia or antipyretic medication ( ae % versus ae %, p < ae ), and decreased activity ( ae % versus ae %, p < ae ). the adjuvanted vaccine was significantly more immunogenic, most notably in the younger children. in < year olds, haemagglutination inhibition (hi) seroconversion rates were ae % versus ae %, p < ae . among all general and local reactions measured, only the maximum temperature measured during the days after the second dose of the adjuvanted vaccine showed a significant (positive) association with post vaccination hi titres. for each °c rise in temperature there was a % increase in titre (p < ae ). these reactogenicity data demonstrate a step towards the future possibility of one-dose influenza immunisation programmes for young children associated with low rates of fever and other reactions. the occurrence of fever following adjuvanted vaccine, seen particularly after a second dose in younger children, was quantitatively associated with enhanced antibody titres. this association was not seen with unadjuvanted vaccine. this apparent difference between the relatedness of the pyrogenic and immunogenic effects of the two vaccines merits further investigation. novel adjuvants appear to have the potential to overcome the relatively poor immunogenicity previously experienced with inactivated influenza vaccines in infants and young children. however, careful adjustment may be needed to optimise the balance between high protection and acceptable reaction rates. tries causing sporadic human infections. vaccination has been used as an effective public health tool for influenza prophylaxis. the goal of this study was to evaluate live attenuated influenza vaccine (laiv) vaccine candidates for subtypes h and h . the attenuated phenotype of h and h laiv candidates has been proven in experiments in ovo and in vivo. in randomized clinical trials among adult volunteers, no significant adverse reactions attributable to the live vaccine occurred. our results indicate that pandemic laiv candidates were well tolerated and elicited serum, local, and cellular immune responses. the emergence and spread of highly pathogenic avian influenza h n viruses in avian populations and concurrent infections in humans since has prompted efforts to develop vaccines for use in the event of an influenza pandemic. in , the world faced a new h n pandemic. immunization with inactivated or live vaccines is the primary measure for preventing influenza. laivs appear to be safe and efficacious, and might possibly provide broader immune responses than inactivated vaccines. our study evaluated laiv pandemic candidates as part of the global influenza pandemic preparation project outlined by the who. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated by routine technique in embryonated hen eggs. laiv and placebo were supplied by microgen (irkutsk, russia). the monovalent laiv was produced from the pandemic vaccine candidates and formulated to contain and ae eid per dose ( ae ml) of a ⁄ ⁄ california ⁄ ⁄ and a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ , respectively. the vaccine or placebo was administered intranasally with a single-use dosing nasal sprayer. two doses were given at an interval of days. one hundred-ninety healthy adults aged - years were randomly divided into groups to receive either pandemic vaccine candidates ( ) or placebo ( ) . subjects were informed about purposes and methods of the study and potential risks associated with participation. all participants had an hai antibody titer of £ : to a ⁄ california ⁄ ⁄ (h n ) pandemic virus. in all there were and vaccines and and participants who received placebo, and were further tested for immune responses to h n or h n pandemic vaccine, respectively. another participants vaccinated with h n laiv were children between to years old. before the children were vaccinated, their parents were advised about study and their consent was required before any child was enrolled. on the advice of the national ethics committee, we did not include a placebo group in this study. individuals were not enrolled if they had an acute illness or fever at the beginning of the study or a history of egg allergy. immune responses of subjects were assessed by routine hai test (evaluation of serum igg antibodies), elisa (evaluation of iga antibodies eluted from the nasal swabs into steril pbs), and cytokine flow cytometry assay (evaluation of virus-specific cd + cd + ifnc + and cd + cd + ifnc + peripheral blood mononuclear cells). the results of phenotypic analysis in ovo showed that pandemic vaccine candidates retained the cold adapted-temperature sensitive (ca ⁄ ts) phenotype, typical of the coldadapted parental mdv. in contrast and as expected, a ⁄ california ⁄ ⁄ and a ⁄ duck ⁄ potsdam ⁄ - parental strains had the non-ts ⁄ non-ca phenotype typical of wt viruses. the h n pandemic vaccine candidate demonstrated an attenuated phenotype in mice and in java macaques and did not infect chickens. the vaccine attenuation study confirmed the attenuated phenotype of a a ⁄ ⁄ california ⁄ ⁄ pandemic laiv candidate in mouse, ferret, and guinea pig models. the phase i ⁄ ii randomized, controlled, double-blind clinical study safety evaluation of pandemic vaccine candidates in adults clinical examination of subjects who received two doses of pandemic vaccine candidates indicated that both vaccines were well tolerated. no fever reactions were observed after the first or second vaccination. after the first vaccination, ae % and ae % of reactogenicity events consisting of catarrhal symptoms, such as pharyngeal irritation or hyperemia, were observed for h n and h n vaccine candidates, respectively. after revaccination, subjects did not report local or systemic reactions. to determine whether a serological response occurred in the cohort of immunologically naïve subjects vaccinated with pandemic vaccine candidates, hai and elisa tests were used (table ) . post-vaccination geometrical mean titers (gmt) among subjects who received two doses of h n vaccine were significantly higher than pre-vaccination titers. the frequency of ‡ fold antibody rises was significantly higher ( ae %) after revaccination than after one dose ( ae %). the percentage of subjects with post-vaccination serum hai titers to h n ‡ : was ae % and for titers ‡ : , it was ae %. no seroconversions in the placebo group were detected. the virus-specific nasal iga antibody response to vaccination after two doses of the h n vaccine candidate demonstrated significant increases of ‡ fold rise iga antibodies ( %) compared to one dose. cumulative data of h n vaccination (all applied tests) showed % and % of conversions after the first and the second vaccination, respectively. increasing h n vaccine virus infectivity from ae to ae eid ⁄ dose lead to an enhancement of post-vaccination hai titers in vaccinees after the first vaccination to homologous h n antigen from ae % to ae % of ‡ fold antibody rises. values of post-vaccination serum hai antibody titers in subjects vaccinated with another pandemic vaccine candidate, a ⁄ ⁄ california ⁄ ⁄ , also proved to be rather low. after the primary vaccination, the percentage of subjects with hai protective antibody titers ‡ : were ae %. after revaccination, this parameter increased to ae %. four-fold increases in serum hai antibody titres were four-fold conversions after the first and the second vaccination was ae % and ae %, respectively. elisa antibodies in nasal swabs showed had an advantage in detecting induction of local iga as compared to serum hai antibodies. after revaccination four-fold serum hai antibody conversions were ae % vs. ae % of iga conversions in nasal swabs, respectively. taking into account cumulative data of h n vaccination (hai and elisa data), the obtained results were here and in the ae % and ae % of conversions after the first and the second vaccination, respectively. fourty-seven subjects were vaccinated with h n laiv, and who received a placebo were chosen for evaluation of cellular immune response by cytokine assays. after revaccination, the mean increases of both cd + and cd + memory cells were significantly higher in vaccinated subjects compared to the placebo group. interestingly, the same effect of vaccination was observed in vaccinees without detectable conversions of hai antibody titers. even after a single vaccination, the rate of subjects with significant increases of these cells in the blood was ae % (cd + ) and % (cd + ). after the revaccination, the percentage of subjects with significant increases in cd + and in cd + cells was ae %. immunogenicity of h n pandemic vaccine candidate in children hai antibody results among children aged to years proved to be significantly higher when compared to adult subjects: after the first vaccination, ae % of the children seroconverted; after revaccination, seroconversions reached ae % ( table ). the gmt rise to h n vaccine with primary vaccination was : ; after revaccination it increased to : . benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. many years of laiv seasonal trials have shown excellent tolerability and low reactogenicity. [ ] [ ] [ ] indeed, data showed that live influenza vaccines cause minimal systemic, local, and thermal reactions, generally from to %. a different situation was observed in the cohort of immunologically naïve volunteers vaccinated with pandemic vaccines. the rate of local reactions to a ⁄ ⁄ california ⁄ ⁄ and a ⁄ ⁄ duck ⁄ potsdam ⁄ ⁄ vaccine candidates increased to ae % and ae %, respectively. after revaccination no significant local and systemic reactions were observed. this confirms, indirectly, the development of a sufficiently high level of protection after the first vaccination with pandemic laiv. the most important criterion for assessing the quality of vaccines is their estimated safety, epidemic effectiveness, and immunogenicity. however, current regulatory documentation mandates that induction of serum antibodies, measured by hai, as the only criterion for a laiv immunogenicity evaluation. in addition to the standard hai assay, we determined serum (igg) and local (iga) antibodies in adult subjects vaccinated with an h n pandemic vaccine candidate. evaluation of overall results obtained in these additional serological tests, as well as those from the hai assay, showed an immune response to the vaccine in the majority of subjects ( ae % of ab seroconversions after the single vaccination and ae % after revaccination, respectively). these data show that methods used to routinely measure laiv immunogenicity should be revised to include a number of additional immunological methods such as igg and iga elisa, and cytokine assays consistent with the recently updated who recommendations on laiv monitoring. these clinical studies clearly demonstrated that pandemic laiv candidates are effective at generating pandemic specific influenza immunity. a key finding from this study is that it may be practical to give the vaccine as a single dose to both children and adults. evaluation of our laiv pandemic vaccine candidates was performed as part of the global influenza pandemic preparation project outlined by the who. it was considered that laiv could be produced in greater quantities and more rapidly than inactivated vaccines. together with the generation of herd immunity by laiv, this suggests that laiv implementation during the first wave of a pandemic may provide significant social, economic, and health benefits to the community. authors are thankful to path for the financial support of h n pandemic vaccine study. we are grateful for the the main evolutionary mechanism of influenza viruses during inter-pandemic period is the antigenic drift, but the epidemiological picture of circulating viruses is complicated by a high level of heterogeneity of strains, even though drift does not occur, due to co-circulation of drifted and old strains or to co-circulation of viruses belonging to the same type ⁄ subtype but with different antigenic patterns. [ ] [ ] [ ] [ ] [ ] [ ] lack of data exists on the impact of the wide heterogeneity of circulating strains on the seroprotection and on-field effectiveness of influenza vaccine: in particular, little is known about the ability of influenza vaccine to elicit an effective immune response against isolates with few amino acid mutations with respect to vaccine strains that represent the majority of circulating viruses. mf -adjuvanted vaccines, which are currently used for the prevention of seasonal influenza epidemics in elderly, are showed to confer higher seroprotection against homologous and drifted a(h n ) strains than non-adjuvanted vaccines. [ ] [ ] [ ] the broader immune response showed by mf -adjuvanted vaccine was measured using hi and nt assays against egg-grown drifted strains representing vaccine composition changes during the following seasons, but its ability to elicit a broader immune response against circulating viruses belonging to vaccine cluster and presenting amino acid mutations onto antigenic sites or against on-field isolates not-antigenically distant from vaccine strains has not yet been investigated. showing amino acid changes onto antigenic sites in position (n k), (n k), and (p s) with respect to a ⁄ california ⁄ ⁄ . in particular, a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ presents n d amino acid mutation detected in clade a ⁄ wyoming ⁄ ⁄ -like viruses. the ha sequences of a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ fell within the clade represented by the ha of a ⁄ califor-nia ⁄ ⁄ ; among these isolates, a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ showed antigenic site sequences very close to that of the ⁄ vaccine strain, whereas ha sequences of a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ posses amino changes onto antigenic site a(r k), c(g e) and d(r k), respectively. the ha sequences of more recent isolates fell within the clade represented by the ha of a ⁄ brisbane ⁄ ⁄ and characterized by the amino acid changes, relative to the ha of a a ⁄ california ⁄ ⁄ , g e and k i, with the exception of a ⁄ genoa ⁄ ⁄ , showing r g and l s amino acid changes present in viruses belonging to a ⁄ nepal ⁄ ⁄ clade. measure of genetic distance between vaccine and circulating strains was calculated as previously described by gupta. two blood samples were collected from each subject, just before and ± day post-vaccination. all sera were stored at ) °c. all samples were tested at the laboratory of health sciences department, university of genoa, by haemagglutination-inhibition (hi) and neutralization (nt) assays, performed following the who criteria and standardised method in our laboratory, respectively. [ ] [ ] [ ] guinea pig red blood cells were used for hi assay. all samples were assayed twice for hi and for nt. the obtained antibody titre was expressed as the reciprocal of the last sera haemagglutinating or inhibiting virus dilution. immunogenicity was determined by: geometric mean titre (gmt); mean-fold increase (mfi; ratio of post-to pre-vaccination titre); seroprotection rate (the percentage of subjects achieving an hi and nt titre ‡ iu); and seroconversion rate (percentage of subjects with a fourfold increase in hi or nt antibody titers, providing a minimal post vaccination titer of : ). post-vaccination gmt was reported as ratio, with the corresponding % confidence interval, of gmts after vaccination with mf -adjuvanted vaccine and with non-adjuvanted subunit vaccine. seroprotection and seroconversion rate % confidence interval was calculated using modified wald method. comparisons of seroconversion and seroprotection rates between subunit and mf -adjuvanted vaccine groups have been analyzed by fischer's exact test. the results were evaluated against the committee for medicinal products for human use (chmp) criteria for approval of influenza vaccines in the elderly, which require that at least one of the following criteria be met: mfi > ; seroprotection rate > %, or seroconversion rate > %. furthermore, hi titres were also transformed into binary logarithms, corrected for pre-vaccination status, as described by beyer et al. and were expressed as median titres, with the corresponding °- °i nter-quantile range. comparisons of corrected post-vaccination titers between subunit and mf -adjuvanted vaccine groups were analyzed by wilcoxon test. difference in immunogenicity profile between vaccine groups, expressed by ratio of different parameters, was correlated with genetic and antigenic distance between vaccine and viruses used in the study using spearman test. pre-vaccination titres were not significantly different between vaccine groups, for all strains (data not shown). post-vaccination gmt ratios between mf -adjuvanted and non-adjuvanted vaccine groups determined using hi and nt assays, with the corresponding % confidence interval, according to viral strain are shown in figure . both vaccines met chmp requirements for mfi (> ), seroconversion (> %), and seroprotection rate (> %) against a ⁄ wyoming ⁄ ⁄ -like, with the exception of a ⁄ genoa ⁄ ⁄ and a ⁄ california ⁄ ⁄ -like circulating viruses and against egg-grown a ⁄ wyoming ⁄ ⁄ , a ⁄ california ⁄ ⁄ , and a ⁄ wisconsin ⁄ ⁄ strains; the immune response against a ⁄ genoa ⁄ ⁄ met the requirements for mfi and seroprotection rate only in mf -adjuvanted vaccine group. requirements for mfi, seroconversion, and seroprotection rate against the a ⁄ brisbane ⁄ ⁄ -like virus a ⁄ genoa ⁄ ⁄ and the a ⁄ nepal ⁄ ⁄ -like genoa ⁄ ⁄ viruses and against egg-grown a ⁄ brisbane ⁄ ⁄ strain were reached only in subjects vaccinated with the mf adjuvanted vaccine. a similar pattern emerged from the analysis of mfi, seroconversion and seroprotection rates using nt assays. subjects vaccinated with the mf -adjuvanted vaccine showed significantly higher post-vaccination hi gmts against a ⁄ wyoming ⁄ ⁄ -like, a ⁄ california ⁄ ⁄ -like, a ⁄ nepal ⁄ ⁄ -like and a ⁄ brisbane ⁄ ⁄ like viruses, with the exception of a ⁄ genoa ⁄ ⁄ , and against egg-grown a ⁄ california ⁄ ⁄ , a ⁄ wisconsin ⁄ ⁄ , and a ⁄ brisbane ⁄ ⁄ strains, compared with individuals immunized with the non-adjuvanted vaccine ( figure ). the mf -adjuvanted vaccine also induced significantly higher seroconversion and seroprotection rates against following correction for pre-vaccination status, hi titres were significantly higher for the mf -adjuvanted vaccine group when evaluated against a ⁄ wyoming ⁄ ⁄ -like viruses, a ⁄ brisbane ⁄ ⁄ -like a ⁄ genoa ⁄ ⁄ , and a ⁄ nepal ⁄ ⁄ -like a ⁄ genoa ⁄ ⁄ strain ( figure ). pre-vaccination titre corrected response was higher in subjects vaccinated with mf adjuvanted vaccine also against egg-grown a ⁄ wyoming ⁄ ⁄ , a ⁄ california ⁄ ⁄ ⁄ , a ⁄ wisconsin ⁄ ⁄ , and a ⁄ brisbane ⁄ ⁄ . among viruses more closely related to a ⁄ california ⁄ ⁄ , subjects immunized with mf -adjuvanted vaccine showed a significantly higher corrected titres against a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ strains compared with the non-adjuvanted vaccine ( figure ) . spearman test showed a clear correlation between the distances and the advantage offered by mf expressed by ratio between mfi, post-vaccination gmts, corrected post-vaccination median, seroconversion, and seroprotection rates calculated using hi test in the two vaccine groups. similarly, ratio between mfi, seroconversion, and seroprotection rates calculated with nt test correlated with the genetic and antigenic distance between vaccine and viruses used for the study. the ability of mf to enhance the immunogenicity and to elicit a broader immune response against drifted strains than non-adjuvanted vaccine is consistent with other findings reported during the last decade. [ ] [ ] [ ] in subjects vaccinated with the mf -adjuvanted vaccine containing a ⁄ california ⁄ ⁄ , the immune response, expressed by a number of parameters, such as crude and corrected postvaccination titers, seroconversion, and seroprotection rates calculated using hi and nt assays, is higher than that observed in individuals immunized with subunit vaccine when it is evaluated against a drifted strains, such as a ⁄ brisbane ⁄ ⁄ -like and a ⁄ nepal ⁄ ⁄ -like strains, and against egg-grown a ⁄ brisbane ⁄ ⁄ virus. for the first time in this study, the impact of heterogeneity of circulating strains antigenically close to the vaccine on the antibody response elicited by mf -and non-adiuvanted vaccines is evaluated. immune response against viruses isolated during the ⁄ season, that appear more phylogenetically close to ⁄ vaccine strain a ⁄ wyoming ⁄ ⁄ , was higher in subjects vaccinated with mf -adiuvanted vaccine as demonstrated by higher crude and corrected post-vaccination hi titres and higher postvaccination nt titres, with the exception of a ⁄ genoa ⁄ ⁄ , against whom the nt post-vaccination gmt is identical in mf and subunit vaccine groups. furthermore, hi seroconversion and seroprotection rates were higher in mf vaccine group when evaluated against a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ . as far as the immune response against a ⁄ california ⁄ ⁄ -like viruses, the small number of enrolled subjects did not allow appreciating differences using qualitative response indicators, but crude post-vaccination hi titres were higher in mf vaccine group for all the strains. interestingly, a ⁄ california ⁄ ⁄ -like viruses with at least one amino acid change onto antigenic sites, i.e. a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ , showed a more marked difference in terms of response between the two vaccine groups. individuals immunized with mf -adiuvanted vaccine showed higher corrected post-vaccination hi titres and post-vaccination nt titres in comparison with subjects vaccinated with plain vaccine. these response indicators were similar in the two vaccine groups when the response was evaluated against a ⁄ genoa ⁄ ⁄ and a ⁄ genoa ⁄ ⁄ , which present no amino acid changes onto antigenic sites and identical hi titers respect with a ⁄ california ⁄ ⁄ at molecular and antigenic characterization, respectively. thus, the advantage offered by mf in terms of higher immunogenicity expressed by higher post-vaccination hi titres is observable also against viruses showing antigenic and molecular pattern undistinguishable from vaccine strain, but it became even more evident as the antigenic and molecular distance between vaccine and circulating strains grew. as emerged for a ⁄ genoa ⁄ ⁄ , a ⁄ genoa ⁄ ⁄ , and a ⁄ genoa ⁄ ⁄ , one amino acid was a sufficient change in antigenic sites for -fold decrease of hi titre against homologous vaccine strain to observe -fold higher post-vaccination nt titers (mf ⁄ subunit postvaccination gmt ratio range between ae and ae , figure ) and one-dilution higher corrected post-vaccination hi titers in mf vaccine group ( figure ) . finally, the correlation between the distance and the improvement offered by mf in terms of higher immunogenicity clearly emerged by spearman correlation analysis: it remains wellfounded both using a number of different response parameters obtained from hi and nt assays and calculating the distance by serological and genetic methods. outbreaks of h n pdm in pigs in commercial swine operations have been reported in several countries. in all incidents, epidemiological investigations have linked humans as the possible source of the infection to pigs. experimentally, it was established that the virus is pathogenic and transmits readily in pigs. the natural outbreaks of h n pdm and laboratory studies underscore the threat that the virus poses to the swine industry and highlight the need for developing effective control strategies. in the united states, a trivalent live attenuated influenza vaccine (flumistÒ) has been licensed for use in humans since . in swine medicine, however, temperature-sensitive laivs are not available. currently, only inactivated vaccines are available for pigs, but they provide limited protection against antigenically diverse influenza viruses. additionally, the use of inactivated vaccines has been associated with enhanced pneumonia when immunized pigs were challenged with divergent viruses. thus, the development of laivs has the potential to circumvent the drawbacks associated with commercial vaccines. with the aim of developing laiv temperature-sensitive influenza vaccines against the h n pdm virus, we have used reverse genetics to introduce attenuation markers in the polymerase genes of a swine-like tr h n influenza virus, a ⁄ turkey ⁄ ohio ⁄ ⁄ (h n ) (ty ⁄ ). we chose this isolate because it grows well in both eggs and cell culturebased substrates, displays a broad host range, and has internal genes similar to the h n pdm virus. safety and efficacy studies of the ty ⁄ att vaccine candidates in pigs demonstrated that this vaccine backbone is attenuated in swine and conferred sterilizing immunity upon an aggressive intratracheal challenge of pigs with the h n pandemic virus. thus, introduction of genetic signatures for att in the backbone of a swine-like tr influenza virus resulted in highly attenuated and efficacious live influenza vaccines with promising applications veterinary medicine. -t cells and mdck cells were maintained as previously described. a ⁄ turkey ⁄ ohio ⁄ ⁄ (h n ) (ty ⁄ ) has options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - been previously described and it was kindly provided by yehia saif, ohio state university. a ⁄ california ⁄ ⁄ (h n ) (ca ⁄ ) was kindly provided by the centers for disease control and prevention (cdc). generation of recombinant viruses by reverse genetics (rg) was done using a previously described method. the genetic signatures for attenuation were introduced into the pb and pb genes of ty ⁄ . ny : ty ⁄ att is a : reassortant with the surface genes from the a ⁄ new york ⁄ ⁄ (h n ) virus and the ty ⁄ att internal genes. all viruses were amplified in mdck cells to produce viral stocks. twenty-five pigs were divided into five groups (n = ) and intranasally inoculated with tcid ⁄ animal of either h n : ty ⁄ att or with ny(h n ) : ty ⁄ att vaccines diluted in ml of mem. two other groups were similarly inoculated with h n : ty ⁄ wt and h n : ty ⁄ rg and served as controls, whereas a fifth group was mockvaccinated with pbs alone. clinical observations were performed as previously described. , efficacy of h n ty ⁄ att vaccine in pigs fourty pigs were divided in four groups (n = )( table ) . group was vaccinated with tcid ⁄ animal of ny(h n ) : ty ⁄ att through intranasal route, whereas group was vaccinated intramuscularly with ml of an adjuvanted uv-inactivated ca ⁄ vaccine (uvadj-ca ⁄ ). group , non-vaccinated and challenged (nv+ca ⁄ ), and group , non-vaccinated, mock-challenged (nv+mock), were also included. pigs were boosted two weeks later. fourteen days post boost (dpb), pigs from groups - were challenged intratracheally with ml of · tcid of ca ⁄ . following challenge, pigs were monitored using methods as previously described. all statistical analyses were performed using graphpad prism software version ae (graphpad software inc., san diego, ca). the differences were considered statistically significant at p < ae . the ty ⁄ att-based vaccines are attenuated in swine pigs inoculated with wt ty ⁄ viruses developed fever (> °c) that peaked hpi ( figure a) and shed large amounts of in nasal secretions ( figure b) . similarly, viral titers in bronchoalveolar lavage fluid (balf) collected at dpi ranged from to tcid ⁄ ml ( figure c ). at necropsy, the lungs from animals inoculated with these viruses had severe pneumonia ( figure d ). in contrast, none of the animals inoculated with h n or h n ty ⁄ att viruses developed clinical signs following vaccination, indicating that the ty ⁄ att viruses were safe for administration to pigs ( figure a) . correspondingly, there was - fold less virus shedding from the nose of pigs vaccinated with ty ⁄ att viruses as compared to unmodified ty ⁄ viruses. in general, ny(h n ) : ty ⁄ att -vaccinated pigs shed less virus than h n : ty ⁄ att inoculated pigs ( figure b ). in addition, viral titers in balf were significantly reduced (p < ae ) in ty ⁄ attvaccinated pigs as compared to ty ⁄ wt-infected pigs ( figure c ). although both vaccines caused mild gross and microscopic lesions in the lungs, the percentage of lung ae ± ae * ± * ± * ± * balf, bronchoalveolar lavage fluid, uvadj-ca ⁄ , uv-inactivated ca ⁄ vaccine; nv+ca ⁄ , non-vaccinated, challenged positive control group; nv+mock, non-vaccinated, non-challenged negative control group. *significantly different from nv+ca ⁄ control group at p < ae . geometric mean hi titer against ca ⁄ at the day of challenge. à percentage of macroscopic lung lesions given as mean score ± sem. § average viral titer (log ) measure as tcid per ml. -average viral titer (log ) in balf at dpc. involvement was not significantly different from mock-vaccinated pigs, corroborating the clinical findings that these vaccines are sufficiently attenuated in pigs ( figure d, e) . histopathologically, nasal turbinates and trachea obtained from pigs immunized with either vaccine were similar to control animals, as opposed to the wt-inoculated pigs ( figure e ). vaccination with h n ty ⁄ att-based vaccines provides sterilizing immunity against h n pdm in pigs the clinical performance in pigs of the h n vaccines is summarized in table . nv+ca ⁄ animals had macroscopic pneumonia, viral replication in balf and shedding in the nose. uvadj-ca ⁄ vaccine provided satisfactory protection, but this protection was not sterilizing. remarkably, animals vaccinated with ny(h n ) : ty ⁄ att had sterilizing immunity. in both vaccine groups there was a significant reduction (p < ae ) in the percentage of macroscopic lung pathology compared to the nv+ca ⁄ group. control pigs had neither significant macroscopic nor microscopic lesions in the lungs. hi antibody titers measured at the day of challenge in both vaccine groups were approximately the same (table ). in the present study, we developed for the first time, temperature-sensitive laiv for use in pigs. data from our safety studies showed that both the h n and h n ty ⁄ att vaccines were attenuated in pigs. although the ty ⁄ att vaccines were detected in balf samples, the level of viral replication was significantly reduced in comparison to unmodified virus and, more importantly, caused no overt clinical signs. a minimal amount of replication is likely beneficial for eliciting t-cell responses to internal genes that may provide heterologous cross-protection. one of the most challenging tasks in producing effective live attenuated vaccines is to achieve an adequate balance between safety and efficacy. by introducing the att modifications into the polymerase genes of a swine-like tr strain, this desirable balance was achieved. the vaccines were histopathologic scores of nasal turbinates, trachea and lungs at dpi. ny(h n ) : ty ⁄ att (a virus that carries the surface genes of a ⁄ new york ⁄ ⁄ (h n ) and ty ⁄ att internal genes). all h n viruses have their surface genes derived from ty ⁄ . values are shown as the mean ± sem. * p < ae ; **p < ae ; *** p < ae . options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - attenuated in pigs and, more importantly, provided sterilizing immunity upon an aggressive challenge with pandemic h n as opposed to an experimental ca ⁄ inactivated vaccine, which elicited protective but not sterilizing immunity in all animals. in the face of influenza pandemics that have the ability to overcome the species barriers such as the h n , the supply of vaccines for use in agriculture could be jeopardized. our cell culture-based live att h n vaccines could be an attractive alternative for this possible pandemic vaccine shortage. because the ty ⁄ att live vaccines developed here are efficacious in swine, are easier to manufacture than inactivated vaccines, and do not require adjuvants, our study represents a major advance in vaccine development for the h n pandemic. in conclusion, our second generation of live att influenza vaccines based on modifications of the pb and pb genes of ty ⁄ retains its safety properties in vivo and can induce excellent protection against aggressive h n challenges in the swine host. influenza virus is one of the most important respiratory pathogens worldwide. , type a influenza causes an acute disease of the upper airways, and affects - million persons yearly. moreover, the threat of human influenza epidemic and pandemic has dramatically increased in recent years. vaccination is one of the crucial interventions for reducing the spread and impact of influenza. the generally used parenteral inactivated influenza vaccines induce mainly systemic antibody responses and only weak cell-mediated immunity and low levels if any mucosal immunity. on the other hand, intranasal immunization with live virus can induces a broad spectrum of both systemic and mucosal antibodies, and the immune response localized in the mucosa blocks the virus even during the first phase of infection. unfortunately, the use of live vaccines is always associated with a certain risk. the development of a crossprotective vaccine against potentially pandemic strains is an essential part of the strategy to control and prevent a pandemic outbreak. we induced intrasubtypic and intersubtypic cross-protection in balb ⁄ c mice by intratracheal (it) immunization with inactivated influenza viruses together with dead delipidated bacillus firmus (dbf) as an adjuvant. ten days after the nd immunization dose, the mice were infected with live influenza virus b ⁄ lee ⁄ lethal for mice (total infection dose corresponded to · ld ) or a⁄ pr ⁄ (total infection dose corresponded to ae - ld ). dbf adjuvant markedly increased both systemic and mucosal anti-viral antibody formation when applied together with inactivated influenza a or b viruses. protective significance was tested in vivo. mice were preimmunized with ) pbs (controls), ) dbf alone, ) virus alone, and ) vir-us+dbf. influenza b virus strains b ⁄ lee and b ⁄ yamanashi ⁄ ( years phylogenetically distant and antigenically substantially different, especially in terms of the main protective antigen -surface haemagglutinin) or two different influenza a subtypes -a ⁄ pr ⁄ (h n ) and a ⁄ california ⁄ (h n ) -were used (figures and ) . the mice were challenged with · ld of either b ⁄ lee ⁄ or a ⁄ pr ⁄ as appropriate. all controls died. the mice treated with dbf alone died with a delay or survived, which could be explained by stimulation of innate immunity. the animals immunized with virus alone were protected against homologous strains. adjuvant immunization was cross-protective: the mice immunized with a heterologous b strain (figure ) fell ill (pronounced body mass loss), but almost all survived and recovered. the mice immunized with a heterologous a subtype were excellently protected (negligible weight loss and zero mortality). intratracheal dbf ( lg per mouse) given to non-immunized mice hour before influenza infection eliminated the lethal effect in - % of infected animals depending on infection dose ( ae - ld ); in mice infected with lower than lethal doses ( ae ld ), weight loss was minimized or did not occur. the current mode of vaccination-induced immunity is mostly effective against a homologous strain of the virus used for vaccination. the attention is therefore focused on vaccines that are able to induce cross-protection and could be effective also in case of sudden appearance of a new virus variant. inactivated influenza viruses are known to be often insufficiently effective when used for mucosal immunization and for induction of cross-protection against drifted influenza viruses or novel subtypes. the drawback of vaccination with dead virus can be overcome by using a suitable adjuvant. mouse models were successfully immunized with vaccine containing inactivated virus in combination with cholera toxin or the escheria coli heat-labile toxin (lt). [ ] [ ] [ ] the use of cholera toxin in humans is precluded because of its high toxicity; a number of lt mutants that retain their adjuvant activity have been prepared; these mutants were likewise tested on the mouse model and should not cause any serious side effect in humans. for this reason, current studies aim at finding a suitable and safe mucosal and systemic immune response. dbf has been shown to be a very efficient adjuvant for mucosal immunization stimulating both innate and adaptive immunity. intratracheal immunization with inactivated influenza viruses and dbf as adjuvant induced efficient and even heterosubtypic cross-protection. dbf given hour before infection provided partial protection probably because of its strong stimulatory effect on the innate immunity. temperature-sensitive and cold-adapted candidates for live attenuated influenza vaccine with genomic composition of : based on highly pathogenic influenza a ⁄ h n viruses with pandemic potential were generated by the replacement of six internal genes from the influenza a ⁄ puerto rico ⁄ ⁄ (pr ) virus from pr -based rg-candidates for inactivated vaccine with appropriate internal genes of influenza a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) master donor virus (mdv) for russian laiv by methods of classical reassortment. all attempts to capture avian n neuraminidase into the genome of the mdv laiv production were ineffective. : reassortants were not generated. step by step co-infection of triple reassortants (h n -h n -h n ) with h n mdv in some cases was the only possibility to generate influenza a ⁄ h n cold-adapted vaccine reassortants. difficulties in generating : reassortants could be explained by a substantial gene constellation in the genome of pr based h n reassortant viruses. strong coupling of pb ⁄ pr and avian n genes in a ⁄ h n -pr -rg reassortants was revealed. annually updated laiv strains are generated by classical reassortment of circulating influenza viruses with well characterized, attenuated, ts ⁄ ca mdvs. resulting attenuated reassortants inherit the relevant ha and na of wild type parental virus and six internal genes of the mdv. candidates for inactivated influenza vaccines based upon avian influenza viruses with pandemic potential are generally generated by reverse genetics methods. in these cases, like with laiv, vaccine strains are : reassortants which possess the modified ha and na from potentially pandemic virus and six internal genes from the pr virus. the pr virus is considered to be of low virulence, i.e. attenuated, for humans, yet offers properties of high seed virus growth for influenza vaccine production. the ha of avian h influenza viruses with pandemic potential is engineered to remove four basic amino acid codons from the cleavage site of ha, resulting in a virus that is considered attenuated for natural hosts and safe for people. the objective of this study was to safely generate vaccine candidates for a laiv using highly pathogenic avian influenza viruses by the replacement of six internal pr genes in the genome of candidates for inactivated vaccine subtype h n (a ⁄ h n -pr -rg) with internal genes of the laiv mdv by methods of classical reassortment. len -mdv and a ⁄ h n -pr -rg virus were co-infected in embryonated chicken eggs. five rounds of selective propagation were performed, three of which were at low temperature ( °c). the production and selection of reassortants were carried out in the presence of rabbit antiserum to len -mdv. cloning by endpoint dilution was performed in each of the last three passages. a virus sample in an open petri dish was rocked gently for sec while being irradiated with a ge watt germicidal lamp at a distance of cm from the dish. the residual infection titer was measured by titration in embryonated chicken eggs. genome composition of reassortant viruses was monitored by rflp analysis. in addition, capacity of reassortant viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) for influenza viruses was determined by virus titration in chicken eggs. reassortment of the mdv with the vn-pr or indo-pr viruses either resulted in reassortants that contained six internal genes from len -mdv. however, all generated clones contained the na from the mdv. of ten such : reassortants based on vn-pr three reassortants had the pa gene from pr and one had ns gene from pr . : reassortants from the targeted h n composition were not generated. after repeated attempts, : temperature sensitive and cold adapted reassortants based on vn-pr and indo-pr viruses were obtained, but again, none had inherited the avian n neuraminidase (table ) . in contrast, nibrg- didn't reassort with the mdv at all. twelve unsucsessful attempts to develop : or : reassortants of nibrg- with mdv showed that the classical reassortment procedure (cloning by limited dilutions in the presence of anti-mdv serum, followed by co-infection of equal doses of two parental viruses in eggs and two selective passages at °c) did not work for this virus pair. to disharmonize the incredibly strong gene constellation of nibrg- , various modifications of the co-infection step were studied, such as: altering the nibrg- to mdv ratio (from : to : tions of anti-mdv serum alone or together with anti-pr serum. it was noted that even if the h n to mdv ratio was : , the clones obtained were presumably parental h n viruses without the transfer of any mdv-genes into genome of nibrg- . in all, clones were isolated, and of them were identical to nibrg- parental virus. in nine clones, only the pa gene from mdv was included, whereas in three clones only the 'cold' ns gene was included (data not shown). using uv inactivation of nibrg- prior co-infection was more encouraging. after the first round of co-infection of partially uv-inactivated nibrg- with mdv (at ratio : ), reassortants that inherited several internal genes of mdv were obtained in the context of the nibrg- background (b , c , c , d ) ( table ). some of them (c , c , d ) were chosen for the next round of co-infection. after the second round of co-infection, c , c , and d 'intermediate' reassortants with mdv (at ratio : or : ) : vaccine reassortants finally were obtained. live attenuated influenza vaccine is considered as one of the most promising pandemic vaccines. according to the who there is evidence that laiv might be more effective than inactivated vaccines. this study attempted the safe development of laiv for potential pandemic highly pathogenic avian a ⁄ h n viruses on the base of rg-reassortants for inactivated vaccine with modified h hemagglutinin and mdv for laiv. replacement of pr based internal genes into genome of vn-pr and indo-pr reassortants with appropriate genes of mdv was realized by the classical reassortment procedure. difficulties were encountered in obtaining : reassortants that contained both the ha and na from the wild type avian h n parental virus. in attempts to reassort the nibrg- with mdv, the classical reassortment procedure was unsuccessful. the challenge faced was to break an incredibly strong gene constellation of the nibrg- virus. partial uv-inactivation of nibrg- was encouraged in replacement of some pr internal genes with mdv genes in some cases avian-human reassortant viruses with gull h n and human influenza h n genes were difficult to generate, and reassortants with the desired genotype of six gull virus genes with human influenza a h and n genes were not isolated despite repeated attempts. the gull pb , np, and ns genes were not present in any of the gull-human h n reassortants generated. it is difficult to fully understand potential reasons for observed difficulties to reassort some avian viruses with human strains. unsuccessful attempts to develop : vaccine reassortants may be caused by an observed strong connection of pb and na genes in the genome of a ⁄ h n -pr -rg viruses. in our attempts, each reassortant that possessed avian n neuraminidase inheritied pb gene of pr as well. and vice versa, the 'cold' pb gene always appeared to be coupled with the n neuraminidase of the mdv. in some cases, step by step co-infection of triple reassortants (h n -h n -h n ) with h n mdv may be the only possibility to generate a cold-adapted vaccine reassortant. our studies demonstrate unique and significant challenges that are faced in the development of influenza vaccines for avian influenza viruses with pandemic potential. such challenges must be further studied to identify methodologies to allow for rapid development and response to emerging viruses in a crisis. it is imperative that these studies be continued and expanded to identify either mechanisms of such tight gene constellations in influenza viruses produced by rg-derived vaccine strains or inability some genes of human h n and avian h n viruses to cross. in addition, further studies to improve the efficiency of classical reassortment processes will be conducted. during the period from to , avian influenza outbreaks among humans have been registered in countries of asia, europe, and africa. morbidity and mortality of humans followed the global spread of avian influenza h n among wild and domestic birds, which caused great economic loss to the poultry industry in many regions including some highly developed countries. the global threat from avian influenza forced scientists to develop technologies for the production of a ⁄ h n human vaccine. the development of ai a ⁄ h n vaccines using strains isolated in kazakhstan and the organization of local production and creation of strategic stockpiles of effective vaccines is the an important issue for public health protection in the republic of kazakhstan. to address this, a scientific program 'influenza a ⁄ h n vaccine development for public health protection in kazakhstan' was approved and financed from to . in this article we give basic results of the development of a recombinant ai a ⁄ h n inactivated whole virion vaccine with aluminium hydroxide as adjuvant for public health protection in kazakhstan. [ ] [ ] [ ] the development of vaccine technology was conducted with the use of a ⁄ astanarg ⁄ : ⁄ (a ⁄ h n ) recombinant strain made of a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) and a ⁄ pr ⁄ ⁄ (h n ) strains by the reverse genetics. inactivation of virus containing allantoic fluid was carried out with the use of formalin in different concentrations. complete-ness of the virus inactivation was tested by -fold virus passaging in embryos. , purification and concentration of the inactivated viruscontaining allantoic fluid was conducted with the use of ultra filtration in tangential flow, which was followed by gel filtration. then we evaluated the content of total protein, hemagglutinin, and ovalbumin in purified and concentrated material. vaccine was composed of clarified and inactivated virus concentrate with the known ha dose containment, and ae % aluminum hydroxide was added in : proportions. composition components and quality control of finished vaccine was determined in the stages of semi-finished product and finished biopreparation. determination of quantitative ovalbumin content was conducted by elisa applying a strip test-system chicken egg ovalbumin elisa kit cat. n (alpha diagnostic international, usa). vaccine immunogenicity was evaluated by hai micro test in u-bottom -well plates produced by 'costar' (usa). vaccine apyrogenicity was evaluated after intravenous injection of the studied preparation to rabbits. , for confirmation of the results vaccine series were tested for bacterial endotoxins with the use of limulus amebocyte lysate produced by charles river laboratories, inc. usa. the vaccine toxicity was evaluated in white mice with body weight - gm and in rats with body weight - g both males and females according to glp principles. allergenic characteristics of the inactivated vaccine was determined in white outbred mice and guinea-pigs both males and females according to 'methodic guideline for evaluation of allergenic characteristics of pharmacological substances'. in the first series of experiments, we conducted work for obtaining influenza a(h n ) recombinant strain. bidirectional expression plasmid phw_b with full-length sequences of ha and na gene segments of the strain a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) isolated in kazakhstan were synthesized in geneart ag, (regensburg, germany). ha gene was modified by deleting the region encoding multiple basic amino acid rrrk motif in ha cleavage site. moreover, to prevent recovery of repeating basic amino acids motif due to polymerase slide, we inserted replacements g fi t and k fi t. thus the ha cleavage site consists of the following sequence ntpqgerrrkkrglfgai ntpqtetrglfgai. the basic amino acid motif of highly pathogenic strain a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) was replaced by the sequence tetr ⁄ glf, which is characteristic of low pathogenic strains of influenza h n . sequence of gene coding na in the strain a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) was cloned without modifications. the other segments pb , pb , pa, np, m and ns were obtained from influenza virus ivr- and synthesized and cloned in two-forked expression plasmid phw_b in geneart ag company, germany. the origin of genetic segments of vaccine strain a ⁄ astanarg ⁄ : ⁄ (h n ) is presented in table . vero cell culture ( passage) (who) was received from european cell culture collection (salisbury, wiltshire sp jg, great britain). the cell culture was grown in dmem ⁄ f medium with the addition of % of fetal bovine serum and mm l-glutamine. to obtain reassortant virus a ⁄ astana ⁄ ⁄ r- : , vero cells were infected with correlative plasmids by way of electroporation using nucleofector ii (amaxa) equipment. infected cells were placed in -well plates. after hour, dmem ⁄ f medium was changed into ml of opti-pro sfm (gibco) medium adding mm l-glutamine and lg ⁄ ml trypsin. two days after cytopathic effect appearance supernatant was collected and used for infection of spf-eggs. the virus a ⁄ astanarg ⁄ ⁄ - : was grown in chicken embryos, and then virus titer was determined in chicken embryos and madine-darby canine kidney (mdck) cell culture. the titer of two final a ⁄ astanarg ⁄ - : virus stocks was ae log eid ⁄ ml (chicken embryos); ae log tcid ⁄ ml (mdck cells); ha titer : . a ⁄ chicken ⁄ astana ⁄ ⁄ (h n ) virus contains motif of repeating basic amino acids in ha cleavage site. it is known that this sequence is the main determinant of ai virus pathogenicity. that is why this site was deleted in vaccine candidate strain. sequence results confirmed that influenza virus a ⁄ astanarg ⁄ ⁄ r- : strain ha gene sequence contains modified ha cleavage site and keeps mutations inserted for prevention of return to virus wild type. to confirm stability of modified ha gene sequence, five additional passages of recombinant strain a ⁄ astana rg ⁄ ⁄ - : were conducted in chicken embryos. sequencing and following phylogenetic analysis of the recombinant strain a ⁄ astana rg ⁄ ⁄ - : ha gene sequence proved the presence of modification in ha cleavage site. deletion of pathogenicity site of the obtained virus was confirmed by lethality test for chicken embryos, intravenous pathogenicity test in chicken, and in plaque-forming test with trypsin. pathogenicity test in chicken embryos showed that recombinant strain a ⁄ astanarg ⁄ - : is capable of growing up to high titers without causing embryos' death. a ⁄ astanarg ⁄ - : strain pathogenicity evaluation was conducted in - week-age white leghorns chicken, and this study proved that the strain a ⁄ astanarg ⁄ - : (h n ) is not virus pathogenicity inductor in chickens, which got intravenous injections of this virus (pathogenicity index is equal to ). h n strain ha cleavage site modification provides its cleavage capability only with tripsin-like proteases, which shows low level of pathogenicity. aiming at confirmation of ha cleavage site modification, we experimentally studied virus replication ability both with trypsin and without this enzyme. and we got the following results. in the plaque-forming test, a ⁄ astanarg ⁄ - : strain produced plaques in mdck cells only with trypsin, proving the trypsin-dependent phenotype characteristic of low pathogenic avian influenza viruses. to prove the ha subtype antigenic analyses of a ⁄ astana ⁄ ⁄ r- : strain was conducted by means of serological methods in hemagglutinin inghibition test with the use of postinfection antisera of rabbits and rats (influenza research institute swd rams), standard serum received from cdc, atlanta, usa. hai test proved that a ⁄ astana ⁄ ⁄ r- : strain belongs to h subtype. furthermore, toxicity of vaccine candidate strain was evaluated by way of subcutaneous injection of viral material to balb mice. the strain appeared to be non-toxic for white mice getting subcutaneous injection of ae ml of the preparation. the conducted research showed that according to all tested characteristics, a ⁄ astana ⁄ ⁄ r- : strain can be used for influenza a ⁄ h n inactivated vaccine production. according to its genetic characteristics, this strain belongs to the group of vaccine strains recommended by who for the development of influenza pre-pandemic inactivated vaccines. we determined basic cultivation parameters of the recombinant strain a ⁄ astanarg ⁄ - : in - day chicken embryos. the determined parameters are the following: infection dose, - eid ; cultivation period, hour; incubation temperature, °c. these cultivation parameters allow obtaining virus containing material with biological eid and hemagglutinating activity of ae - ae log eid ⁄ cm and : ha titre and even higher. in the next series of experiments, we conducted research on the determination of optimal sequence of technological stages of virus clarification, concentration, and inactivation in the order of vaccine production. samples of viral material were subjected to inactivation before and after clarification and concentration. the regimen of virus inactivation by formaldehyde with final concentration of ae %, period of inactivation of days, temperature of inactivation medium of - °c, ph of inactivation medium of - ae . on the basis of the conducted experiments we determined that the selected regimen of inactivation provides complete and irreversible inactivation of viral suspensions of the hpai strain irrespective of the kind of inactivated material. we did not observe reduction of ha activity in non-clarified viral suspensions. however, when we inactivated clarified and concentrated material, ha activity reduced by an order of magnitude. comparison of forms and sizes of virion structural elements in native (non-clarified) and formalin inactivated preparations did not reveal any significant differences. concentration of virus particles in the studied preparations was similar. the selected inactivation regimen provides obtaining completely avirulent viral suspension of the strain a ⁄ astanarg ⁄ - : , and it does not influence the structure of the virus. on the basis of the experiments results, we selected method of viral allantoic fluid inactivation without preliminary clarification. during further research, we tried to get highly clarified viral concentrate. this study resulted in the combined scheme, which includes clarification of inactivated viral allantoic fluid by low speed centrifugation at circulations per min for minutes, filtration through membrane filters with pore diameter of ae lm, ultrafilatration ⁄ diafiltration, gel filtration in b sepharose, and sterilization of viral suspension through membrane filters with pore diameter of ae lm. the experiments resulted in the development of production technology of embryonic inactivated vaccine based on recombinant strain a ⁄ astanarg ⁄ : ⁄ (h n ) contain-ing aluminium hydroxide as adjuvant. the developed influenza a ⁄ h n human vaccine has the trade name kazfluvacÒ. its composition components are presented in table . preclinical testing of the vaccine kazfluvacÒ was conducted according to the following parameters: general health condition of animals, change of body weight and temperature of immunised animals (for ferrets), presence of post vaccination antibodies response in sera, forming protective immune response against reassortant viruses of h subtype, study of acute and chronic toxicity of three experimental vaccine series in different doses and semi-finished vaccine product applying different ways of injection, study of allergic and immunotoxic characteristics of the vaccine, as well as study of pyrogenic reaction and analysis for bacterial endotoxins presence. [ ] [ ] [ ] [ ] preclinical tests of kazfluvacÒ vaccine safety showed that this vaccine does not have toxic effect on organisms of warm-blooded laboratory animals. double intramuscular injection of kazfluvacÒ vaccine in inoculative dose does not effect appearance, general health condition, behaviour of animals, their muscular strength and physical activity, does not have negative effect on biochemical parameters of blood and basic physical functions of animals organism, and does not cause pathomorphological changes. this shows the safety of the vaccine. local irritation action was not observed. the results of the vaccine allergic action study showed that the vaccine does not have allergic effect at the intravenous injection. the research also showed that the vaccine does not have negative effect on immune system of laboratory animals. research conducted on mice and ferrets showed high immunogenic activity of the vaccine at one-and two-dose regimen of injection. the research showed % of protective effect of kazfluvacÒ vaccine at two-dose injection regimen in ferrets infected by homological strain of influenza virus. the devised inactivated influenza a ⁄ h n vaccine kaz-fluvacÒ is a safe and immunogenic biopreparation that is not worse than the overseas analogues in its immunobiological characteristics. [ ] [ ] [ ] [ ] to date the whole-virion inactivated influenza a ⁄ h n vaccines of the producers such as omnivest (hungary), biken, denka seiken, kitasato institute, kaketsuken (japan), gsk biologicals (belgium), sinovac biotech (china) are registered. all of them are produced on the basis of chicken embryos and aluminum is used as an adjuvant. kazfluvacÒ differs from its analogues in the flowchart of the virus purification and concentration that makes possible to produce a safer preparation. , the results of the conducted research and preclinical testing allow starting work towards implementation of phase i preclinical tests on volunteers. it is planned to conduct a randomized blind placebo-controlled phase i study on double application of kazfluvacÒ vaccine in increasing doses. the preparation will be administered to volunteers aged - years for assessment of its safety and immunogenicity in doses of ae and ae lg of ha. when the world health organization (who) announced the sixth phase of a ⁄ h n v influenza pandemic, scientists all over the world started investigation to develop technology for production of prophylactic means against the disease. having taken into consideration the threat of a pandemic for kazakhstan, the ministry of education and science of the republic of kazakhstan launched the program ''monitoring, study, and development of diagnostic, prophylactic, and therapeutic means for influenza a ⁄ h n .'' this paper presents the experimental data obtained at the ribsp in the course of the studies towards the development of technology for production of an inactivated a ⁄ h n influenza vaccine, as well as the results of pre-clinical testing of the developed vaccine. the development of vaccine production technology was conducted with the use of who recommended vaccine strain nibrg- xp constructed by the method of reverse genetics in the national institute for biological standards and control (nibsc, great britain). the virus was inactivated with formalin at different final concentrations, and the extent of inactivation was evaluated via threefold virus passages in developing chicken embryos. the inactivated virus was purified and concentrated by the method of ultrafiltration in tangential flow followed by gel filtration. the purified and concentrated material was evaluated judging on the total protein, hemagglutinin (ha), and ovalbumin. the vaccine was prepared by pooling the purified and concentrated virus material with the certain weight content of ha and the work solution of aluminum hydroxide ( ae %) in the ratio : . the ovalbumin content was quantified in elisa with the use of the strip test system chicken egg ovalbumin elisa kit (cat. no. alpha diagnostic international, san antonio, texas, usa). weight content of the virus ha was determined according to sominina, burtseva. the content of the residual formaldehyde, aluminum (al + ) ions, and thiomersal in the vaccine was measured according to the operating instructions. the vaccine immunogenicity was assessed in the hemagglutination inhibition test, which was carried out as a microassay in -welled u-bottomed plates (''costar'', new york, usa). , apyrogenicity of the vaccine was assessed post intravenous administration of the tested preparation to rabbits. , to confirm the obtained results the vaccine batches were tested for bacterial endotoxins with use of the limulus amebocyte lysate (charles river laboratories, inc., wilmington, ma, usa). the toxicity of the vaccine was assayed in white mice weighing - g and in rats weighing - g (male and female) in compliance with the principles of good laboratory practice. allergenic properties of the inactivated vaccine were determined according to the ''operating instructions on assessment of allergenic properties of pharmaceutical substances'' in white outbred laboratory mice and guinea-pigs of both sexes. the first step in the course of developing technology for vaccine production was to determine the major conditions for influenza virus cultivation: usage of -days embryonated chicken eggs at the infectious dose within - eid , incubation temperature ( ± ae )°c, and duration of the incubation period hours. the established parameters for virus cultivation made it possible to produce virus-containing materials of infectious activity within ae - ae log eid ⁄ cm and hemagglutinating activity : and higher. in the subsequent experiments, an optimal method for virus inactivation was selected. on the basis of the experimental findings, the following conditions for inactivation of the native virus-containing material were elected: formalin of ae % final concentration as an inactivating agent; inactivation period of hours at temperature ( ± )°c. these conditions provide the complete inactivation of the virus (nibrg- xp strain) material, did not impact distinctly the structural organization of the virus, and did not reduce the antigenic activity. as it is well known, virus purification and concentration means very much in the development of technology for production of an inactivated whole-virion influenza vaccine. the investigation into optimization of the technological step of purification and concentration of the recombinant influenza virus nibrg- xp strain resulted in selection of an optimal pattern including such steps as clarification of the virus suspension by filtration through membranes with pore size ae lm, virus concentration by ultrafiltration in a tangential flow, dialysis filtration in a tangential flow, gel filtration on sepharose b, and sterilization of the viral suspension through membrane filters with pore size ae lm. the studies conducted by the ribsp specialists resulted in the development of technology for production of the first domestic whole-virion inactivated a ⁄ h n influenza vaccine with aluminum hydroxide as adjuvant and with the brand name refluvac Ò . the key processing characteristics of the whole-virion inactivated a ⁄ h n influenza vaccine vaccine refluvac Ò are shown in table . simultaneous with the performance of all process operations, the parameters such as sterility, inactivation extent, ph, vaccine specificity, total protein content, weight content of has, aluminum and formalin contents, content of thiomersal, and ovalbumin, pyrogenicity of the vaccine and its immunogenicity for mice, were optimized. the key qualitative characteristics of the designed influenza a ⁄ h n vaccine refluvac Ò are shown in table . before implementation of phase i clinical trials on volunteers, preclinical testing of three experimental batches of refluvac for immunogenic activity and safety was carried out. it was conducted in three laboratory bases of research institutions: the toxicology institute ⁄ federal medicobiological agency, russia (st petersburg), the research institute for biological safety problems (republic of kazakhstan), and the influenza research institute ⁄ north-western branch of the russian academy of medical sciences (st petersburg), with use of different animal models (mice, rats, chinchilla rabbits, guinea-pigs, ferrets). the results of the preclinical testing are as follows: • electron microscopy of the preparation has shown that the viral particles are well dispersed and do not aggregate. the portion of whole (intact) particles is over %, which is evidence of virion integrity; • assessment of polypeptide composition of the vaccine refluvac by electrophoresis in % polyacrylamide gel with sodium dodecyl sulfate has shown the vaccine to contain both surface antigens (ha, na) and highly purified inner virion proteins (np, m ) that are typespecific antigens, so the vaccine is a preparation of full immunological value; • judging on the parameters of acute and chronic toxicity for white mice and rats of both sexes, the vaccine is a non-toxic and safe preparation; • under conditions of a chronic experiment on white mice and rats, it was found that refluvac does not produce changes in behavior, somatic, or vegetative responses; • assay of hematological and biochemical blood characteristics of white mice and rats following vaccine administration did not reveal any significant differences as compared to the animals of the control group; • refluvac does not cause allergenic and immunotoxic impact; • the vaccine refluvac does not cause local irritative effect; • refluvac is apyrogenic for laboratory animals; • the pathomorphological and hystopathological analysis did not reveal any changes due to immunization in animal organs; • testing of immunogenic characteristics of the vaccine on mice and ferrets has shown formation of hemagglutinating antibodies in animals after single administration; • refluvac induces % protection in immunized ferrets at their challenge with the wild-type influenza virus a ⁄ california ⁄ ⁄ (h n v). the results of the performed preclinical testing have allowed concluding that refluvac, an inactivated whole-virion vaccine with aluminum hydroxide as adjuvant, is a safe and highly effective preparation against influenza a ⁄ h n v. the implemented study resulted in development of technology for production of the first domestic inactivated allantoic whole-virion influenza a ⁄ h n vaccine with aluminum hydroxide as an adjuvant under the brand name refluvac Ò based on the recombinant strain nibrg- xp. the devised pandemic vaccine meets who requirements as well as requirements concerning safety and immunogenicity of the national pharmacopeias of the republic of kazakhstan and russian federation. [ ] [ ] [ ] [ ] the devised technology for vaccine production differs from the previous technologies for production of allantoic whole-virion influenza a ⁄ h n vaccines in its processdependent parameters. presence of an adjuvant (aluminum hydroxide) increases significantly the vaccine immunogenicity and allows maximal reduction of the dose of the administered antigen that, in turn, results in diminished reactogenicity of the vaccine. aluminum hydroxide is an adjuvant that is most frequently used in clinical practice. to date the results of the double-centered randomized study of the europe-licensed vaccine fluval p [monovalent inactivated whole-virion influenza vaccine with aluminum phosphate based on strain a ⁄ california ⁄ ⁄ (h n ) nymc x- a (omninvest, pilisborosjeno, hungary)] that is similar to the refluvac preparation are published. the data of this research are an evidence of safety and high immunological effectiveness of the vaccine in dose lg ha at single administration both in adults and elderly persons. the results of the pre-clinical tests allow recommending carrying out phase clinical testing of the refluvac Ò vaccine for safety and immunogenicity. single immunization of volunteers with refluvac Ò in doses ae , ae , and ae lg of ha are planned. mid , respectively. the study results confirm that new h n laiv and h n laiv candidates are safe and immunogenic and confer protection from homologues influenza virus infection in mice. the recent emergence of a new pandemic h n virus and the threat of transmission of avian viruses to humans had stimulated research and development of live attenuated cold-adapted influenza vaccines against newly appeared influenza viruses. formulations of live attenuated influenza a vaccine (laiv) against pandemic influenza strains, including h n , h n , h n , and h n are currently being tested in preclinical and phase i clinical studies. the following paper describes the preclinical study of new h n and h n laiv candidates in mice. the study addressed the following three objectives: (i) to demonstrate that cold-adapted (ca) reassortant influenza a(h n ) and a(h n ) vaccine candidates are indistinguishable from the parental a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) master donor strain (mds) virus with regard to replication efficiency in upper and lower respiratory tract of mice; (ii) to demonstrate the immunogenicity of different doses of cold-adapted (ca) reassortant influenza a(h n ) and a(h n ) vaccine candidates in mice; and (iii) to demonstrate the protective efficacy of cold-adapted (ca) reassortant influenza a(h n ) and a(h n ) vaccine candidates in mice against a homologous wild-type virus challenge. the a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ) reassortant containing the ha and na genes from a ⁄ mallard ⁄ netherlands ⁄ (h n ) and six other genes from mds, the a ⁄ ⁄ california ⁄ ⁄ (h n ) reassortant containing the ha and na genes from a ⁄ california ⁄ ⁄ (h n ) and six other genes from a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) were generated by classical genetic reassortment in embryonated chicken eggs (ec). viruses were propagated in days old eggs ( °c, hours). fifty percent egg infectious dose (eid ) titers were determined by serial titration of viruses in eggs. titers were calculated by the method of reed and muench. female balb ⁄ c mice, - weeks of age were used in all experiments. mice were lightly anesthetized with ether and then inoculated intranasally (i.n.) with ll of infectious virus diluted in phosphate-buffered saline (pbs). mice were inoculated with mid ( % mouse infectious dose) of a ⁄ ⁄ california ⁄ ⁄ (h n ), a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ), and a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) mds. viral loads were measured in respiratory and brain tissues collected at and days post-infection (dpi). tissue homogenates prepared using a disruptor and clarified supernatants were titrated on eggs at permissive temperature to determine infectious concentrations. groups of animals were inoculated with mid or mid of either h n laiv or h n laiv intranasally after collecting a pre-immunization blood sample. a second blood sample was collected at dpi. on the same day, the animals received a second intranasal inoculation with the same virus that was used for priming at dpi. to assess protection, all animals were infected dpi with either mid of a ⁄ california ⁄ ⁄ (h n ) or mid a ⁄ mallard ⁄ netherlands ⁄ (h n ) virus by the intranasal route. four animals from each group were euthanized at dpi, and the respiratory and systemic organs were harvested for virus titration. a forth blood sample was collected at dpi from the remaining animals. hi antibody titers were determined for individual serum samples collected on days , , , and . body weights were taken daily following challenge through day postchallenge. sera were tested for hi against homologous h n and h n viruses. the h n laiv, h n laiv and h n mds influenza viruses replicate in mice lungs at level ae - ae lgeid ⁄ ml at dpi (figure ). at dpi, replication of the viruses in the lungs decreased to ae - ae lgeid ⁄ ml (data not shown). in contrast, the wild-type virus a ⁄ mallard ⁄ netherlands ⁄ (h n ) demonstrated high level replication in lungs - ae lgeid ⁄ ml. the levels of replication of studied viruses in nasal turbinates were ae - ae lg eid ⁄ ml at dpi (figure ) , and ae - ae lgeid ⁄ ml at dpi (data not shown). there were no significant differences between the viruses in regard to replication in upper respiratory tract of mice. thus, it was shown that a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ) and a ⁄ ⁄ california ⁄ ⁄ (h n ) vaccine candidates was indistinguishable from parental a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) in terms of replication in the lungs and noses of mice at and dpi. no virus was found in the brain tissue of immunized mice at and dpi (in undiluted samples tested). thus, it was shown that a ⁄ ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ), a ⁄ ⁄ cali-fornia ⁄ ⁄ (h n ) vaccine candidates are identical to a ⁄ leningrad ⁄ ⁄ ⁄ (h n ) in lacking neuroivasive capacity, and all three viruses similarly fail to replicate in the brain. it was shown that all immunized animals survived after challenge with wild-type a ⁄ mallard ⁄ netherlands ⁄ ⁄ (h n ) virus. the mice in vaccine groups showed no signs of morbidity. average weight changes were tracked from day to day in all study groups, but the changes did not exceed %. as shown in figure , the challenge virus actively replicated in respiratory tissue taken from mock immunized animals ( ae lgeid in the lung and ae lgeid in the nose), but failed to infect the brain and spleen. on the other hand, in both h n laiv vaccinated groups, all tested organs were free from presence of challenge virus. thus, immunization of mice with either mid or mid h n laiv protected the animals from the subsequent challenge infection with a homologous with wild-type h n virus. both h n and h n laiv candidates were found to be immunogenic. after one dose of mid of h n laiv, gmt of hi antibodies were ae . one dose of mid or mid h n laiv elicited hi antibody level with gmt of ae and ae , respectively. the second dose of h n laiv further stimulated serum hi antibody levels to gmt ae and ae , for mid or mid , respectively (data not shown). the mouse model is widely used to better understand the pathogenicity of avian influenza viruses for mammalian species, to be able to predict the pandemic potential of such viruses, and to develop improved methods for the prevention and control of the virus in a potential pandemic. a subset of the h viruses was evaluated for the ability to replicate and cause disease in balb ⁄ c mice following intranasal administration. h subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication. there is limited preclinical information available for laiv. thus, live monovalent vaccine against pandemic influenza virus h n (influvir) was tested for acute toxicity and its effect on the systems and organs of laboratory animals. according to toxicology and necroscopy results, the live monovalent influenza vaccine influvir, when applied intranasally, was safe and was well tolerated. in our current study we demonstrate that a(h n ) and a(h n ) laiv are indistinguishable from the parental mds virus with regards to replication kinetics in the upper and lower respiratory tract of mice. both h n and h n laiv candidates were immunogenic and protect mice against subsequent a challenge with the wild-type virus. live attenuated cold-adapted (ca) influenza vaccines are an effective means for the control of influenza, most likely due to their ability to induce both humoral and cellular immune responses. in our study we confirm that new h n laiv and h n laiv candidates are safe, immunogenic, and confer protection from influenza infection in mice. health organization (who) declared a pandemic by raising the worldwide pandemic alert level to phase . therefore, h n inactivated monovalent vaccine formulated with our proprietary oil-in-water emulsion based adjuvant was evaluated in ferrets for its potential to induce with low antigen dose efficient, robust, and rapid protective immunity against a wild type challenge virus (a ⁄ netherlands ⁄ ⁄ ). this adjuvant was also tested in ferrets in a h n avian influenza model for its ability to induce a cross-clade immunity and cross-protection. two independent studies (a&b) were carried out with male and female outbred ferrets (musleta putorius furo) in compliance with ''guide for the care and use of laboratory animals,'' ilar recommendations and aaalac standards. ferrets used in both studies were influenza seronegative by anti-nucleoprotein elisa and by hi assay against the pandemic and seasonal strains. in study a, four groups of seven ferrets aged approximately of months received one or two im vaccinations weeks apart of either af -adjuvanted ( ae lg of ha with af ) or unadjuvanted ( body weight loss was monitored as an indicator of disease and a mean body weight loss of % was recorded in the control group at day of necropsy. body weight loss was reduced to £ % and £ % in animals that had received and doses of either unadjuvanted or af -adjuvanted vaccine, respectively. viral lung titration showed high levels of virus replication ( ‡ ae tcid ⁄ g tissue) in the lungs of all control ferrets days after challenge. one or two administrations of unadjuvanted vaccine reduced lung viral load by and log , respectively. interestingly, ferrets that received either one or two doses of af -adjuvanted h n vaccine, showed significantly greater reduction of lung viral loads (> log ). no virus was detected in the lungs of ⁄ ( %) animals immunized with a single injection of the af -adjuvanted vaccine and in % of ferrets vaccinated twice. assessment of viral shedding from the upper respiratory tract showed that the af -adjuvanted a ⁄ h n monovalent vaccine was able to reduce the viral load in the nose and in the throat by ae and ae log , respectively, as compared to the control group. conversely, viral loads were only slightly reduced in the nose and mostly unchanged in the throat in ferrets immunized with either one or two doses of unadjuvanted a ⁄ h n monovalent vaccine. gross pathology and histology examinations revealed lung lesions consistent with influenza a ⁄ h n virus infec- however, a second dose of af -adjuvanted vaccine strongly increased hi and mn titers, which persisted for months (table ). antibody responses cross-reactive to heterologous clade . strain were elicited ferrets vaccinated with the af -adjuvanted clade . vaccine. hi antibody titers ‡ crossreactive to clade . and persistent up to d were observed in vaccinated animals. an inter-clade low crossreactive hi response to a clade strain was only detected in a few ferrets that had been vaccinated with the af -adjuvanted clade . . all af -adjuvanted clade . antigen vaccinated animals survived challenge either with the homologous or heterologous virus until euthanized day . after challenge, mean body temperature and mean body weights were monitored as indicators of disease. in the control ferrets, mean body temperature increased by - °c (depending on the challenge virus strain) h post challenge, with an accompanying mean body weight loss ranging from ae % to ae %. ferrets vaccinated with the af -adjuvanted clade . vaccine showed a lower and delayed fever compared to control ferrets that received the same viral challenge, whereas no significant differences were observed between vaccinated animals and their respective controls upon challenge with clade . or clade viruses. body weight loss was reduced in all vaccinated animals when compared to controls after challenge with either the homologous clade . strain or with one of the heterologous strains. lung virus titration showed high levels of virus replication in all control animals days after homologous challenge with the clade . virus. lung viral loads of all ferrets immunized with the af -adjuvanted clade . vaccine were reduced more than log . vaccination resulted in complete viral clearance from the lungs of % of animals assessed days after challenge. as compared to controls, a reduction of the mean viral load of about log was observed in ferrets vaccinated with the af -adjuvanted clade . vaccine after heterologous challenge with either the clade or clade virus. conversely, vaccination with af -adjuvanted clade . vaccine did not result in reduction of lung viral loads after challenge with the clade . heterologous virus strain. titration of pharyngeal swabs showed high levels of viral shedding in all control ferrets after challenge with clade . strain, whereas virus was not detected in any vaccinated animal. similarly, log reduction of viral shedding was seen in vaccinated versus control ferrets following clade heterologous challenge. lower reductions in viral shedding were observed after clade . challenge ( ae log ) and clade challenge ( ae log ). gross pathology and histology revealed lung lesions consistent with influenza a ⁄ h n virus infection all control animals challenged with the clade . , clade . or clade strains. mild to moderate lung lesions were observed in control animals following challenge with clade virus. macroscopic evaluation (percentage of affected lung parenchyma) and histopathological analysis (extent and severity of alveolitis, alveolar oedema and hemorrhage) showed that lung lesions were significantly reduced in af -adjuvanted clade . vaccinated animals after challenge with the homologous clade . virus strain as compared to controls. similarly, a reduction of the macroscopic and microscopic lung lesions was observed in vaccinated animals upon heterologous challenge with clade . and clade virus strains, whereas no differences were observed between control and vaccinated animals after challenge with clade virus. the results of these ferret challenge studies demonstrated that low doses of pandemic influenza vaccines formulated with an oil-in-water emulsion adjuvant, af , elicited strong antibody responses specific to the immunizing strain. importantly, these vaccines provided protection after homologous challenge with complete virus clearance in ferret lungs and reduced viral shedding from the upper respiratory tract suggesting an ability to reduce virus transmission. moreover, af -adjuvanted h n vaccine can provide cross-protection upon challenge with different h n clades by preventing mortality and reducing the viral burden in the lower and the upper respiratory tract. in conclusion, the results of these studies highlighted the ability of af -adjuvanted influenza vaccines to induce potent immune responses and full protection in ferrets against homologous challenge and suggested that protection may be mediated, at least in part, by antigenspecific humoral immunity. since , outbreaks of h n influenza virus infection in poultry have occurred in eurasian countries. phylogenetic and antigenic analysis of h n isolates revealed that there are three sublineages, consisting of g , g , and korean, among ha genes of the eurasian h n viruses. h n viruses do not cause severe disease in poultry, but co-infection of h n viruses with bacteria such as staphylococcus aureus, haemophilus paragallinarum, or attenuated coronavirus vaccine may exacerbate the disease. , h n viruses were isolated from domestic pigs in china and korea and from humans with febrile respiratory illness in hong kong in kong in , kong in , and it is, thus, postulated that in the present study, h virus strains were analyzed antigenically and phylogenetically to select a proper h n vaccine strain. inactivated whole virus particle vaccine was prepared, and its potency against h virus challenge was assessed in mice. viral rnas were extracted from the allantoic fluid of chicken embryos infected with viruses by using a commercial kit (trizol ls reagent; invitrogen, california, usa) and reverse-transcribed with the uni primer and m-mlv reverse transcriptase (invitrogen). the primers used for the ha gene amplification were h - f and h - r. for phylogenetic analysis, sequence data of the genes together with those from public database were analyzed by the neighbor-joining method. h influenza viruses were analyzed by hemagglutinationinhibition (hi) test. chicken hyperimmunized antisera against seven h viruses were prepared according to previous report. virus replication and pathogenicity against embryonated chicken eggs viruses were inoculated into -day-old embryonated chicken eggs and incubated for hours at °c. ha titers and % egg infectious dose (eid ) were measured every hours post-inoculation. pathogenicity of dk ⁄ hok ⁄ ⁄ against embryonated chicken eggs was evaluated by mean death time (mdt) as described previously. dk ⁄ hok ⁄ ⁄ was injected into the allantoic cavities of -day-old embryonated chicken eggs and propagated at °c for hours. the virus in the allantoic fluids ( ha) was purified by differential centrifugation and sedimentation through a sucrose gradient according to previous report. the concentration of protein was measured by od using ultrospec pro (amersham biosciences, tokyo, japan). the purified virus was inactivated with ae % formalin at °c for days. immunization of mice and challenge of immunized mice with hk ⁄ ⁄ four-week-old female balb ⁄ c mice were purchased from japan slc, inc. (shizuoka, japan). the mice were injected subcutaneously with , , ae , or ae lg proteins of inactivated dk ⁄ hok ⁄ ⁄ whole virus vaccine. two weeks later, the mice were boosted by subcutaneous injection with the same dose of the vaccine. control mice were injected with pbs. serum samples were tested by enzyme-linked immunosorbent assay (elisa) according to previous report. one week after the second vaccination, mice in each group were challenged intranasally with ll of ae eid of hk ⁄ ⁄ under anesthesia. on days postinfection, five mice in each group were sacrificed, and the lungs were separately homogenized to make a % (w ⁄ v) suspension with minimal essential medium (nissui, tokyo, japan). the virus titers of the supernatants of lung tissue homogenates were calculated in -day-old embryonated chicken eggs and expressed as the eid ⁄ gram of tissue. the other five mice in each group were monitored for body weight for days after challenge. the ha genes of h viruses were sequenced and analyzed by the neighbor-joining method. all of the h viruses were classified into the eurasian lineage ( figure ) . eleven, seven, and four strains were classified in the korean, g , and g sublineages, respectively. the h viruses of the korean and g sublineages were isolated from waterfowl, poultry, pigs, and humans in the east asian countries, and those of the g sublineage were isolated from poultry in the west asian countries. the cross-reactivity between these antisera and h n viruses were analyzed by hi test. the antisera against h viruses belonging to the korean sublineage were broadly cross-reacted to h viruses belonging to the g and g sublineages. h viruses belonging to the korean lineage were reacted to the antisera against h viruses belonging to the g and g sublineage compared with h viruses belonging to the other sublineage (data not shown). thus, it was suggested that h vaccine strain should be selected from the viruses of korean sublineage to prepare for the vaccine strain of h viruses. dk ⁄ hok ⁄ ⁄ replicated efficiently in -day-old embryonated chicken eggs (data not shown). pathogenicity of dk ⁄ hok ⁄ ⁄ against embryonated chicken eggs was determined by mdt. dk ⁄ hok ⁄ ⁄ was low pathogenic against embryonated chicken eggs (data not shown) and was selected as an h vaccine strain. to assess the potency of the vaccine against h virus infection, mice vaccinated subcutaneously with inactivated dk ⁄ hok ⁄ ⁄ were challenged intra-nasally with hk ⁄ ⁄ . immunogenicity of the inactivated vaccine was assessed by measuring the igg antibodies in mouse sera by elisa. antibody was detected in the group of mice injected lg protein after the first immunization and detected in the group of mice injected lg protein after the second immunization. thus, potency of the present inactivated whole virus vaccine was demonstrated in mice. next, to assess the protective immunity of the inactivated vaccine in mice, viral titers in the lungs was determined. the virus titers in the lungs were ae - ae eid ⁄ g in the groups of mice injected , and lg protein, and ae - ae eid ⁄ g in the other vaccinated groups. body weight reduction of mice were observed in the group of mice injected ae , ae lg protein, and control groups from dpi, and reached to % body weight loss from -to -day post-infection ( figure ). this result correlates with antibody titer in mouse sera and viral titers in the lungs. these results suggest that the test h inactivated whole vaccine confers prevent of weight loss and reduction of virus replication against h influenza virus infection in mice. recently, h n viruses of all of three sublineage have been isolated from wild birds and poultry in worldwide. h n viruses were isolated from pigs and humans in china and korea, suggesting that h n virus would be a potential for a pandemic influenza virus in human population. h n viruses were isolated from pigs in china and korea and were classified into the g and korean sublineage. in human cases, all h n virus isolated from humans in china was classified into the g sublineage. it was suggested that h n viruses isolated from pigs and humans vary in antigenicity of isolates between the korean, g , and g sublineages. therefore, it is important for the preparedness of influenza pandemic to develop h influenza virus vaccine, which could broadly cross-react to antisera of all sublineage viruses. so, we selected the vaccine candidate strain, dk ⁄ hok ⁄ ⁄ , which could broadly cross-react to antisera of all sublineage viruses, and which could replicate in this study, it was suggested that the test vaccine has potency to protect against challenge with h virus using mice for mammalian model. the challenge virus, hk ⁄ ⁄ , was isolated from human, replicates efficiently in mice, and shows pathogenicity in mice. the test vaccine inhibited viral replication and body weight loss in mice. whole inactivated vaccine produced protective immunity, supporting our approach of using whole virus particles for vaccine development. furthermore, whole particle virus vaccine could induce igg and mucosal iga levels after intranasal vaccination with whole particle vaccine. the present results may facilitate the studies of the vaccine for future pandemic caused by h influenza virus in humans. tants to attempt to improve growth. to determine whether wild type h n pdm grew better in the novartis mdck suspension cell line (mdck pf) than in eggs, isolations from h n pdm positive clinical samples were attempted in both substrates. the isolation rate of h n pdm viruses was higher in mdck pf cells ( %) ( ⁄ ) compared to allantoically inoculated eggs ( %) ( ⁄ ) . however the yields were lower than observed with seasonal viruses. little improvement in virus yield was seen with extra passaging or dilutions of h n pdm viruses isolated in mdck pf cells. with the emergence of the swine-origin pandemic h n (h n pdm) influenza in april , the need for efficient production of a suitable vaccine was a high priority. virus isolates were distributed by the who for the urgent development of suitable vaccine strains early in the pandemic. vaccine viruses can be grown in embryonated chicken eggs or in certified mammalian cells. , unfortunately wildtype h n pdm virus strains distributed by the who grew poorly in cell lines and eggs, requiring the generation of a series of conventional and reverse genetics derived reassortants to attempt to improve growth. from these reassortants, only the conventional egg derived reassortants nymc-x- a and nymc-x- (both based on one of the earliest known viruses a ⁄ california ⁄ ⁄ ) showed high enough growth and yield in eggs and cell culture to make them suitable for vaccine manufacture. these reassortants, while acceptable, still only gave haemagglutinin (ha) yields of approximately % that of seasonal h n reassortants. to determine if more recent wild type h n pdm viruses grew better in the novartis mdck suspension cell line (mdck pf), h n pdm positive clinical samples were cultured in mdck pf cells and also in embryonated hen's eggs. in addition, to improve virus yields from mdck pf isolates, extended passaging of three wild type h n pdm influenza viruses was performed using various virus dilutions at each passage level. the results were assessed using various serological and molecular biology techniques and compared to viruses isolated in eggs and conventional mdck cells. h n pdm viruses were received at the centre from who national influenza centres, who influenza collaborating centres and other regional laboratories and hospitals in australia, new zealand, and the asia ⁄ pacific region. viruses were received as original clinical specimens consisting of nasal swabs, throat swabs, nasopharyngeal aspirates, or nasal washes that had previously been shown to be h n pdm positive by real time rt-pcr. these specimens were then cultured in mdck pf cells with serum free medium containing trypzean (optaflu) and also independently inoculated into the allantoic cavity of day-old embryonated hen's eggs. virus cultures in mdck pf cells were sampled at and hour and evaluated by various means including ha titres. at hour, virus cultures were further passaged at varying dilutions ranging from ) to ) up to a total of passages. embryonated hen's eggs were incubated at °c for days and allantoic fluid was harvested and ha titres performed to determine whether a further passage was required in order to improve growth. the conventional reassortants were produced by a mixed infection of eggs or mdck pf cells with the wild type virus and a donor virus carrying the internal genes of the a ⁄ puerto rico ⁄ ⁄ virus. the reassortants were obtained by sequential passages using immuno-selective antisera against the surface antigen of the donor virus to remove virus populations carrying the ha and na protein of the donor strain. the reverse genetics viruses were rescued in vero cells using the plasmid system. both types of reassortants were generated and supplied by who collaborating centres and essential regulatory laboratories except the nvd-c- strain, which was produced by novartis. in this small study with recent h n pdm viruses, the isolation rate was higher in mdck pf cells ( %) ( ⁄ ) compared to allantoically inoculated eggs ( %) ( ⁄ ) . assessment of ha titres, however, showed higher ha titres in egg-isolated viruses compared to viruses isolated in mdck pf cells after two passages. egg generated or cell generated reassortant viruses gave higher ha titres compared to the homologous wild type viruses (table ) . no amino acid changes were observed in mdck pf isolated influenza viruses compared to original specimens or viruses isolated in conventional atcc derived mdck cells, unlike egg isolated viruses which showed a number of amino acid changes, many consistent with egg adaptation mutations (table ) . viruses isolated in mdck pf cells grouped phylogenetically with viruses isolated in conventional atcc derived mdck cells or viruses sequenced from original clinical samples, while egg isolated viruses grouped slightly differently (data not shown). as a result of the poor growth of h n pdm viruses in mdck pf cells, serial dilutions were performed over a number of passages ( figure ). based on the results obtained from the virus isolates, a ⁄ victoria ⁄ ⁄ , a ⁄ wellington ⁄ ⁄ , and a ⁄ darwin ⁄ ⁄ , a supplemental protocol was developed and used in the isolation of a ⁄ brisbane ⁄ ⁄ (figure ). only small differences in ha titer were seen between different dilutions, and copy number showed a similar trend to ha titer at each passage ( figure ). following the supplemental protocol for the isolation of a ⁄ brisbane ⁄ ⁄ results showed slightly higher ha titres with little variation between passages. the egg derived reassortants nymc-x- a and nymc-x- were also assessed for growth in mdck pf cells and were found to be superior by ha titer to other conventional reassortants (egg or cell derived), reverse genetics derived reassortants, or wild type viruses (table ) . two methods were used to determine the ratio of ha to other viral proteins: densiometric analysis using sds-page and reversed-phase hplc using a subtype specific standard. ha content in different vaccine seeds of influenza a subtypes demonstrated that the ha content per total virus protein from the nymc h n pdm reassortants was significantly different to the seasonal influenza a subtypes. for the seasonal h n the ratio of ha to p p p p p p p p p p p p p p p p n and m was ‡ %, for the h n the ratio of ha to n and m was £ %, while for the pandemic a ⁄ h n , the ratio of ha to n and m was much lower at £ % (data not shown). the results of this study has observed the growth of a series of - h n pdm viruses in vaccine suitable mdck pf cells to be generally lower than what has been seen with other seasonal influenza viruses. little improvement in virus yield was seen with extra passaging of h n pdm viruses isolated and passaged in mdck pf cells. passaging up to times in mdck pf cells using dilutions ranging from ) to ) resulted in supernatants with viral ha titres ranging from ha ⁄ ll to ha ⁄ ll. the isolation rate of h n pdm viruses was higher in mdck pf cells ( %) compared to allantoically inoculated (and passaged) eggs ( %), a trend also seen in previous work with seasonal influenza viruses. in contrast a study by hussain and colleagues found similar rates of isolation and replication of seasonal influenza viruses in mdck cells and eggs. the virus load as determined by matrix gene copy number showed a similar trend to ha titers. two of the isolates exhibited small rises and falls in ha titer during passaging, while a third, a ⁄ victoria ⁄ ⁄ gave consistently higher titers. interestingly this virus was unable to be isolated in eggs. the ha sequences of all strains were assessed at p , p , p , p , p and when available compared to the original clinical sample ha sequence. mdck pf-isolated viruses had few if any changes in their ha amino acid sequence, while the majority of egg isolates showed - amino acid changes compared to the clinical sample, with an egg adaption change (l i) evident in a number of them. the ha sequence of one of the better growing viruses, a ⁄ victoria ⁄ ⁄ , was found to have a g e change compared to the a ⁄ california ⁄ ⁄ reference virus. this change was also seen in the virus isolated in conventional, adherent mdck cells. these viruses with g e change when tested by hai have shown reduced reactivity with ferret antisera to a ⁄ california ⁄ ⁄ -like viruses, but normal reactivity with ferret antisera to h n pdm a ⁄ bayern ⁄ ⁄ -like viruses. despite this mutation all mdck pf derived viruses appeared to be a ⁄ california ⁄ ⁄ -like by hai. the h n pdm egg-derived reassortants (nymc x- a and nymc x- ) when grown in mdck pf cells were superior to wild type h n pdm viruses, reverse genetics derived reassortants, and other egg-derived reassortants. the yields of haemagglutinin from the nymc h n pdm reassortants were still below those seen with sea-sonal h n reassortants as was also seen in eggs. this trend has also been noted in other studies. in summary, attempts to improve growth and yield of the h n pdm wild types for mdck pf cells by extended passaging were not successful, and reassortants did not perform as well as seasonal h n reassortants have in the past. however, using higher dilutions for the passaging of h n pdm viruses in mdck pf cells did result in higher ha titres (a ⁄ brisbane ⁄ ⁄ ). further work is therefore required to generate pandemic h n seed viruses that grow well in a variety of cell culture and egg based vaccine production systems. the aim of this study is to evaluate antibody response to influenza virus neuraminidase (na) following immunization with live attenuated influenza vaccine (laiv). we adjusted the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. ( ) to assay neuraminidase inhibition (ni) antibody in sera taken from immunized mice and from human subjects in a clinical trial. for the assay, we prepared the a(h n ) reassortant virus containing the na of a ⁄ california ⁄ ⁄ (h n ) and the hemagglutinin (ha) of a ⁄ equine ⁄ prague ⁄ ⁄ (h n ). in addition, we used an na-specific igg elisa assay to test sera from immunized mice and volunteers. in mice, one dose of laiv induced ni antibody of a geometric mean titer (gmt) of ae , compared to ae in the control group. gmt of ni from human subjects who received two doses of pandemic a(h n ) were significantly higher than pre-vaccination titers. in unvaccinated human subjects, na-specific cross-reactive antibodies to pandemic a(h n ) were detected more often than cross-reactive antibodies to ha. antibody response to influenza virus na contributes to the overall immune response to influenza and may provide partial protection against influenza infection and reduce severity of disease in the host. a number of preclinical studies using purified or recombinant na have shown that various two-dose vaccine regimens in mice may significantly reduce pulmonary virus titers following viral challenge. [ ] [ ] [ ] a plasmid dna-vaccine model demonstrated cross-reactive antibodies to human n in mice could provide partial protection against a lethal challenge against h n or recombinant pr bearing the avian n . immunogenicity of current influenza vaccines, including laivs, is measured primarily as a level of strain-specific hemagglutination inhibition (hi) antibodies. however, the who meeting on the role of na in inducing protective immunity against influenza infection ( ) specified a need to develop suitable assays for anti-na antibody detection to enhance influenza vaccine evaluation in preclinical and clinical studies. the aim of the current study was to evaluate anti-na antibodies to pandemic a(h n ) influenza virus following laiv immunization. the rn ⁄ -swine a(h n ) reassortant influenza virus containing the na of a ⁄ california ⁄ ⁄ (h n ) and the ha of a ⁄ equine ⁄ prague ⁄ ⁄ (h n ) generated by classical genetic reassortment in embryonated chicken eggs (ce). parental a ⁄ equine ⁄ prague ⁄ ⁄ (h n ) influenza virus was obtained from the center for disease control and prevention, atlanta, ga, usa. viruses were propagated in day old ce and purified by sedimentation out of the allantoic fluid, followed by ultracentrifugation on - % sucrose step gradient. for the mouse studies, week old cba mice were inoculated intranasally with one dose eid ⁄ ae ml of a ⁄ ⁄ california ⁄ ⁄ (h n ) vaccine strain or received ae ml pbs. blood samples were collected on day post inoculation. healthy young adults were immunized twice, or days apart in the fall with a ⁄ ⁄ california ⁄ ⁄ (h n ) laiv manufactured by microgen, irkutsk, russia. for the human studies, peripheral blood specimens were collected from volunteers before vaccination, days after the first vaccination, and days after the second dose of vaccine. sera from five subjects diagnosed with influenza a(h n ) were collected in december , to weeks post infection and kindly provided by e. vo ıtsekhovskaia from biotechnology laboratory, institute of influenza, rams. also, sera obtained in from unvaccinated vol-unteers were tested for presence of cross-reactive antibodies to a ⁄ california ⁄ ⁄ (h n ). sera were treated with a receptor-destroying enzyme from vibrio cholera (denka-seiken, tokyo, japan) and then were tested in duplicates for hemagglutination-inhibition (hi) h specific antibodies by standard procedures using a ⁄ ⁄ california ⁄ ⁄ (h n ) test antigen. the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. was adjusted to assay ni antibody. briefly, -well plates (sarstedt, inc., nümbrecht, germany) were coated overnight with ll of lg ⁄ ml fetuin. the purified a(h n ) reassortant virus was diluted in pbs with % bsa and mm ca + to give a four times higher optical density at nm (od ) compared to control wells not containing virus. fifty-microliter volumes of serially diluted serum samples were incubated with an equal volume of prediluted virus for hour at °c. after incubation, the plates were washed and neuraminidase activity was measured by subsequently adding peroxidase-labeled lectin ( lg ⁄ ml; sigma, st. louis, mo, usa), incubating for hour at room temperature, washing the plates, and adding ll of peroxidase substrate (tmb). the reaction was stopped after minute by adding ll of n sulfuric acid. od values were measured at nm using the universal microplate reader (el x ; bio-tek instruments, inc., winooski, vt, usa). the ni titers were expressed as the reciprocal dilution that gave % od of positive control (virus, no serum control). in addition we used an igg elisa assay with ae lg ⁄ ml of purified na from a ⁄ california ⁄ ⁄ (h n ) to test sera from immunized mice and volunteers. data were analyzed with statistica software (version ae ) (statsoft, inc. tulsa, oklahoma, usa). geometric mean titers (gmt) were calculated and used to represent the antibody response. the comparisons were made within groups between pre-and postvaccinated titers (expressed as log ) after first and second vaccination using wilcoxon matched pairs test. to compare multiple independent groups we used a kruskal-wallis anova with subsequent multiple pairwise comparison based on kruskal-wallis' sums of ranks. a p-value of < ae was considered to be statistically significant. in mice, one dose of laiv induced antibody responses to both ha and na components of the a ⁄ california ⁄ ⁄ (h n ) influenza virus vaccine (table ) . geometric mean titers of ni antibody levels from vaccinated mice were ae and were significantly higher compared to those in unvaccinated control animals (p < ae ). elisa igg titers expressed as log were ae compared to ae in control group. there was good correlation between antibody rises obtained using ni or elisa tests (r = ae ). in a study during the fall of , % of examined unvaccinated subjects were negative to pandemic a(h n ) (hi titers £ : ). serum hi antibody titers to pandemic a(h n ) ‡ : were considered to be protective against *the postvaccination gmts of hi antibodies after revaccination were higher than respective prevaccination titers (p = ae ) **the postvaccination gmts of ni antibodies after revaccination were higher than respective prevaccination titers (p = ae ) serum hi and ni antibodies to a ⁄ california ⁄ ⁄ (h n ) after one or two doses of pandemic laiv were evaluated in subjects who had pre-vaccination hi titers £ : ( table ) . post-vaccination gmts of a(h n )-specific antibodies were significantly higher than pre-vaccination titers only among subjects who received two doses of laiv ( table ). the frequency of subjects with ‡ fourfold rises in hi antibody titers was higher after two doses ( ae %) compared to responses after one dose ( ae %) although the differences were not statistically significant ( table ). the highest antibody titers of hi and ni antibodies were achieved after natural infection (p < ae compared to all post-vaccination groups). all five subjects with confirmed influenza also had high levels of n -specific igg measured by elisa using purified na as the coating antigen (data not shown). influenza ha and na surface proteins are primary targets of neutralizing antibodies that provide protection against influenza infection. the correlation of strain-specific hi antibody titers ‡ : to protection of % of the subjects against influenza infection is based on a number of reports published in s. serum antibodies against viral na as result of influenza infection or vaccination also can neutralize the virus from infecting cells; however, little is known about protective levels of such antibodies. to evaluate ni antibodies directed against pandemic a(h n ) we used the reassortant a(h n ) influenza virus with mismatched ha to avoid non-specific inhibition. we demonstrated laiv immunization effectively increased levels of ni antibody, although in smaller amounts compared to influenza infection. our data suggest that an antibody to neuraminidase, resulting from an earlier infection of the circulating seasonal influenza a(h n ), evidently cross-reacted with the n of pandemic influenza virus, perhaps due to the previously reported % of conserved na epitopes in pandemic a(h n ). the peroxidase-linked lectin test using the reassortant a(h n ) influenza virus was shown to be a sensitive and time effective means of revealing homologous and cross-reactive anti-na antibodies after laiv immunization or influenza infection. this could be a useful method for influenza vaccine evaluation. significant levels of anti-na antibodies detected in peripheral serum from subjects infected with wildtype h n virus or with h n laiv. and the cross-antibody response to ph n . for calculation of geometric mean titer (gmt), a titer of < was assigned a value of . statistical significance was determined by paired t-test. cross-reactive antibody response to ph n in vaccinated populations of seasonal influenza virus table shows the antibody response to seasonal influenza viruses and ph n of participants. before vaccination, no or little antibody response to ph n had been detected in all age groups. vaccination with seasonal influenza vaccines resulted in seroresponse in over % of subjects, except children aged - years ( %) and subjects aged of - years ( %) vaccinated with - season influenza vaccine and adults aged ‡ years ( %) vaccinated with - season influenza vaccine. seroconversion was detected in over % of subjects of all ages. postvaccination to prevaccination gmt ratios for response to seasonal influenza viruses was more than ae -fold. in contrast, seroresponse to a ⁄ california ⁄ ⁄ after vaccination with - and - seasonal influenza vaccines were detected in only % and % of those aged - years, % of those aged - years, % and % of those aged - years, % and % of those aged ‡ years, respectively. seroconversion in all participants ranged from % to %, and postvaccination to prevaccination gmt ratios were < ae -fold. preexisting antibody response to ph n among subjects born before s in china according to a recent report, people who were born from to had a preexisting immunity to ph n . although only a very low level of cross-reactive antibody response to ph n had been observed among older subjects aged more than years old in china, we further analyzed these data by different age distribution of subjects, which can trace back to the previous infection that is genetically and antigenically more closely related to this new ph n influenza virus. the proportion of seroresponse to ph n with the titer of , , and (highest titer detected from participants of all ages in this study) and the value of gmt were analyzed according to the birth decade of subjects from . similarly, a peak of antibody response and the value of gmt occurred both in subjects born from to and sharply decreased afterward ( figure ). the seroresponse of subjects born in and before is significantly higher than subjects born afterward (p < ae ). similar to recent studies in some asia countries (guangxi province of china and singapore), limited antibody response to ph n had been detected in children and adults. , but, some other studies from european countries (finland, germany, the united kingdom) and the united states reported a high proportion of older individuals aged > years with pre-existing cross-reactive antibodies to ph n , which may possibly ba a result of previous exposure to antigenically related h n influenza viruses circulating in earlier decades or a lifetime of exposure to influenza a, which has resulted in broad heterosubtypic immunity among older individuals in those countries. previous infection and vaccination with a ⁄ new jersey ⁄ may also contribute to the high level of cross-reactive antibody response to ph n among adults older than years in the us. , the peak of the antibody response to ph n in subjects born between and , which is consistent with recent reports, may suggest the previous viral infections of spanish flu or closely related influenza viruses, which is before and little after the year of . recent antigenic report of new ph n viruses indicated that they are antigenically homogeneous among historical viruses, which are most similar to classical swine a(h n ) viruses. a number of reviews [ ] [ ] [ ] [ ] confirmed that the virus is the likely ancestor of all four of the human and swine h n and h n lineages, as well as the 'extinct' h n lineage. in , a(h n ) influenza viruses were first isolated from swine. they have been shown to be antigenically highly similar to the recently reconstructed human a(h n ) virus. the cellular responses may contribute to the sustaining and long term antibody response. probably, boosting by persisting antigenically related viruses in the early decades of the th century, may have contributed to the ability of these subjects to sustain memory b cells, and it is well established that a subset of plasma cells is long-lived, and these cells contribute to durable humoral immune responses, such as that observed after childhood smallpox vaccination. furthermore, t cells that recognize cross-reactive epitopes are preserved and might be enriched in the memory population; the course of each infection is influenced by the t-cell memory pool that has been laid down by a host's history of previous successive infections. our study indicated that wide transmission of this new virus or any antigenically close related influenza a(h n ) viruses may not have circulated among populations in china before the outbreak of ph n . our data also suggests the need for vaccination with ph n vaccine in all age groups. hypo-and agammaglobulinemia patients have an impaired immune system and are particularly susceptible to bacterial infections that are normally defended against by antibodies. therefore, patients routinely receive replacement therapy with immunoglobulins isolated from healthy blood donors. these patients are also prone to get viral infections, possibly due to defects in toll-like receptors and . because these patients lack an antigen specific humoral immune response, they are rarely vaccinated. the ability of hypogammaglobulinemic patients to produce a specific cell-mediated immune response upon vaccination has only been sparsely investigated. in contrast to local mucosal antibodies, vaccine-induced cell-mediated immunity is not believed to protect against pathogen entry per se, but may be sufficient to provide protection against severe disease and death following transmission of some microbes. , the aim of this pilot study was to investigate if influenza vaccination of hypogammaglobulinemic patients can induce an influenza-specific cell-mediated immune response. we therefore vaccinated hypogammaglobulinemic patients and healthy controls with pandemic h n virus vaccine and subsequently investigated the bcell and t-cell responses. the percentages of ifn-c, il- , and tnf-a cytokine producing cd + th -cells were determined, as these cytokines are important indicators of cell-mediated immunity. five a-or hypogammaglobulinemic patients were classified based on the freiburg classification : patient # is diagnosed with x-linked agammaglobulinemia, patient # and # are in group ia, patient # is in group ib and patient # is in group ii. the monovalent egg grown split virus vaccine adjuvanted with as was manufactured by glaxosmithkline (gsk), belgium. the vaccine strain was produced by reassortment between influenza a ⁄ california ⁄ ⁄ (h n ) and a ⁄ pr ⁄ ⁄ (h n ) to produce a ⁄ california ⁄ ⁄ -like virus (x a). the vaccine was mixed with adjuvant to contain ae lg haemagglutinin (ha) of a ⁄ california ⁄ ⁄ -like virus (h n ), squalene ( ae mg), dl-atocopherol ( ae mg), and polysorbate ( ae mg) per ml. healthy controls and hypogammaglobulinemia patients were vaccinated by intramuscular (im) injection. hypogammaglobulinemia patients received one or two vaccine doses days apart. the intention was to vaccinate the hypogammaglobulinemic patients with two doses of ae lg ha, but ae lg ha was inadvertently administered to the patients as the first dose. for patient # this was the second dose as he had received an initial dose of ae lg ha months prior to the study. patient # , # , and # received a second dose of ae lg ha. four healthy controls were immunised with one dose of ae lg ha according to norwegian national guidelines. peripheral blood mononuclear cells (pbmcs) were harvested and washed in pbs with % fbs. the pbmcs were resuspended in lymphocyte medium (rpmi with l-glutamine, ae mm non-essential amino acids, mm hepes ph ae , mm sodium pyruvate, iu ⁄ ml penicillin, lg ⁄ ml streptomycin, ae lg ⁄ ml fungizone and % fbs) prior to use in the enzyme-linked immunospot (elispot) and influenza-specific cd + t-cell assays. serum haemagglutination inhibition antibodies were tested by a standard method using ha units and ae % turkey erythrocytes. all samples were tested in duplicate and the test was repeated at least two times. titres < were assigned a value of for calculation purposes. for numeration of antibody-secreting cells (asc), an eli-spot assay was conducted as previously described with the following modifications. ninety-six well elispot plates were coated with lg ⁄ ml of a ⁄ california ⁄ ⁄ like (x a) h n virus diluted in pbs overnight at °c. after blocking with rpmi ( % fbs), pbmcs were added and incubated ( °c, % co ) for hour. secreted antibodies were detected with biotinylated goat anti-human igg, iga and igm specific antibody (southern biotech, birmingham, alabama, usa), incubated for hour at room temperature and developed with extravidin peroxidase and aec substrate. the numbers of spots were counted using an elispot reader (immunoscanÔ) and immunospot Ò software. the influenza-specific cd + th -cell response was measured by intracellular cytokine production of ifn-c, il- , and tnf-a. peripheral blood mononuclear cells ( per well) were incubated for hour ( °c, % co ) in ll lymphocyte medium containing lg ⁄ ml anti-cd , lg ⁄ ml anti-cd d, ae lg ⁄ ml monensin, lg ⁄ ml brefeldin a, (bd biosciences, franklin lakes, new jersey, usa), and the h n influenza split virus vaccine x a (either ae lg ⁄ ml or lg ⁄ ml ha). basal cytokine production was determined by incubating pbmcs in lymphocyte medium without influenza virus, and the percentage of cytokine positive cells without influenza stimulation were subtracted from influenza-stimulated cells. cells were stained for cd , cd , cd , ifn-c, il- , and tnf-a (bd biosciences) as previously described. finally, cells were resuspended in pbs containing % fbs and ae % sodium azide and analysed by bd facscanto flow cytometer ( - cells acquired). flowjo v ae ae (tree star, ashland, oregon, usa) was used for data analysis. five to six fold lower gmts were found in the patient group as compared to the healthy controls throughout the study ( figure a) . the lowest hi titres were obtained in patients # , # , and # , whilst patients # and # and all healthy controls fulfilled two of three european medicines agency committee for medicinal products for human use (chmp) seasonal influenza vaccine licensing criteria, by obtaining an hi titre > and a mean geometric increase of ae between pre-and post-vaccination. thus, the hi data indicate that two vaccine doses was sufficient to induce a protective hi antibody response in two out of five of the hypogammaglobulinemia patients tested in this study. the numbers of influenza-specific iga, igg, and igm asc were tested pre-vaccination and days post-vaccination with the h x a virus. few or no ascs were detected pre-vaccination (data not shown). at days post-vaccination the patient's iga, igg, and igm asc levels were significantly lower (p < ae ) compared to the healthy controls ( figure b) . but, the post-vaccination asc numbers in the patients were generally higher than at pre-vaccination stage ( - ascs). patient # had the highest iga and igg asc numbers, followed by patients # and # , whilst patient # and # had few or no asc's. these results confirm that the patients are indeed hypogammaglobulinemic and that some of the patients (# and # ) could be agammaglobulinemic in the context of producing influenza-specific antibodies. the asc levels of patients # , # , and # were lower than those of the healthy controls, but could possibly be adequate for reducing the severity of influenza disease. the influenza-specific th -cell response was evaluated by stimulating pbmcs with the influenza x a virus , , and days post-vaccination. stimulation of healthy control pbmcs with x a days after vaccination, induced ifn-c, il- , and tnf-a production by an average of ae %, ae %, and ae % cd + t-cells, respectively. patient # and # had higher responses than the healthy controls and stimulation with x a induced ae %, ae %, and ae % of t-cells from patient # to produce ifn-c, il- , and tnf-a, respectively (figure a) . the response of patient # was further boosted by a second vaccine dose, which resulted in ae %, ae %, and ae % cd t-cells producing ifn-c, il- , and tnf-a, respectively at day ( figure b ). these results show that the hypogammaglobulinemia patients studied here did not have a common impaired influenza-specific cd + th cytokine response. rather, there was a tendency towards increased responses, suggesting that the diminished antigen specific b-cell responses could induce a compensatory antigen specific th -cell response. the results from this pilot study suggest that some hypogammaglobulinemia patients may benefit from influenza vaccination. we found very different patient responses to influenza vaccination, but some of the patients (patient # and # ) did mount low influenza-specific asc responses. in addition, the vaccine-induced hi antibody titres above the protective level in patient # and # . these results are in accordance with previous publications, which described that polypeptide vaccines induce humoral responses in subgroups of common variable immunodeficiency patients. [ ] [ ] [ ] in this study, we also investigated cell-mediated immunity and found the percentages of homologous and cross-reactive influenza-specific cd + th -cells to be in the same range (for patient # , # , and # ) or higher (for patient # and # ) in the a-or hypogammaglobulinemic patients compared to the healthy controls. the higher response is probably due to the patients having received a vaccine dose of ae lg ha, whilst the controls received ae lg ha. in addition, the patients received a second booster dose, which influences the day and months responses. nonetheless, these results are the first to demonstrate that proliferation of pandemic influenza antigen specific th cells can be induced in hypogammaglobulinemic patients. in addition, vaccination induced influenza-specific asc's in some patients. the findings are promising and provide hope that hypogammaglobulineamic patients could be vaccinated against influenza and other diseases preventable by figure . peripheral blood mononuclear cells s from patients and healthy controls were isolated at day (a), (b), and day (c) and stimulated for hour with x a virus before staining and flow cytometric analysis. the figure shows the mean ± sd frequency of influenza-specific cd + cytokine producing cells (%) where the basal cytokine production from unstimulated cells has been subtracted. data for the hypogammaglobulinemia patients are additionally shown as a number for each patient. **significantly higher frequency of il- producing cd + t-cells in the patients compared to the healthy controls (students t-test p < ae ). titres are presented as the geometric mean titre ± % confidence interval. elispot data (b) are presented as the mean number of influenza-specific iga, igg, and igm ascs per peripheral blood mononuclear cells ± sem. data for the hypogammaglobulinemia patients are additionally presented by a number for each patient. *significantly higher numbers of ascs were detected in the healthy controls as compared with the hypogammaglobulinemia group (students t-test, p < ae ). vaccination. however, this hypothesis should be tested in larger clinical studies. the influenza virus undergoes antigenic evolution under intense immune selection pressure from herd immunity in humans through the process called antigenic drift and shift. , because of antigenic drift, yearly updating of vaccine strain is needed. a mismatch between the circulating strains and the vaccine strain in the subsequent season is often encountered, resulting in reduction of vaccine effectiveness and lack of protection from the circulating strain. in order to address this, a universal influenza vaccine based on a more conserved part of the influenza virus, which is not affected by antigenic change and that is conserved across all strains, remains the ultimate goal to afford cross-protection to drifted strains as well as to other subtypes of influenza which may arise from antigenic shift. , previous studies have investigated the potential of the m e. , m e has remained highly conserved since it was first isolated in . several studies have examined the use of m e as a vaccine component, using various approaches including proteins, peptides, dna vectors, and attenuated viral vectors. , [ ] [ ] [ ] [ ] [ ] [ ] although m e is a weak antigen, by linking the protein to a carrier hepatitis b virus core particle, protection against influenza has been achieved in mice particularly when administered with an adjuvant. some articles found that vaccination with m e coupled to hbc induces protective antibodies, whereas the contribution of t cells to protection was negligible. protection induced by vaccination with m e-hbc was weak overall and failed to prevent weight loss in vaccinated infected animals, and mice succumbed to high dose infection. we aimed to address the poor immunogenicity of m e-hbc by using igv as adjuvant. igv domain is common and conserved in the tim family. ligand binding sites of t cell immunoglobulin mucin (tim) located at igv domain. [ ] [ ] [ ] tim function is done by anti tim antibody which recognized the ligand binding sites of igv domain. tim family members share a common motif, including an igv domain. they are differentially expressed on th cells and th cells with the ability to regulate the immune system. , the igv domain of human b - is sufficient to co-stimulate t lymphocytes and induce cytokine secretion. soo hoo et al. vaccinated with tim- antibody and inactivated influenza and found enhanced vaccine-specific immune response. we report here for the first time the use of igv recombinant protein as adjuvant to immunize mice with influenza m e-hbc. results indicated that igv can induce the strong cellular immune response and cross reaction with different subtype influenza virus antigen. target igv may be used to develop the new method for vaccination strategies. expression and purification of recombinant igv protein rna was extracted from healthy human pbmc. one-step rt-pcr (qiagen, valencia, ca, usa) was done for the amplification igv gene. the pcr product was purified and cloned into pet a (novagen, madison, germany). the resultant construct pet a-igv has a histidine (his) tag ( his) at the n terminus. dna sequence of the insert was determined by sequencing. igv. recombinant protein was expressed in escherichia coli and was purified on a ni column (novagen). the purified protein was examined by sds-page and western blotting. six-eight weeks female balb ⁄ c mice (institute of zoology chinese academy of sciences, china) was used for the study. mice were immunized twice intradermally with ug m e-hbc (provided by cnic, china) combined with different doses of recombinant igv protein , , ug, respectively, or without igv as control. the area proximal to the tibialis anterior muscle was sterilized with % ethanol and different groups of mice were injected bilaterally with , , ug igv plus ug m e-hbc in ul phosphate buffer saline per mouse using a ml syringe with attached ⁄ ¢¢ g needle. the immunization was given at weeks intervals. four blood samples were obtained from every mouse: before immunization, after the first and second immunization, and after virus challenge by retro-orbital plexus puncture. after clotting and centrifugation, serum samples were collected and stored at ) °c prior to use for assays. mouse-adapted a ⁄ pr ⁄ ⁄ (h n ), a ⁄ brisbane ⁄ ⁄ (h n ), a ⁄ xinjiang ⁄ ⁄ (h n ), and a ⁄ guangzhou ⁄ ⁄ (h n ) were provided by chinese national influenza centre. nine to eleven days old embroynated specific pathogen free (spf) chicken eggs were inoculated with virus, and the eggs were incubated at °c for - days. the allantoic fluid was collected and purified by sucrose density gradient centrifugation, and the virus was inactivated by formaldehyde at °c overnight. to identify igg, igg , igg a against m e, elisa assays were used. in brief, -well (nunc, brunei, denmark) were coated with ul ⁄ well of m e recombinant protein (provided by gene lab of ivdc, xuanwu district, beijing, china) in carbonate buffer (ph ae ) overnight at °c. immediately before use, the coated plates were incubated with blocking solution ( % bsa in pbs) for h at °c and washed four times with pbs containing ae % tween (pbs-t). the serum samples were serially diluted and added in the plates. the detection color was developed by adding hrp-labeled goat anti-mouse igg, igg , or igg a ( figure ) . no cross-strain response was observed in the control group. the igv adjuvented groups show splenocytes stimulation with seasonal h n , h n , h n , and h n antigens. m e-hbc immunization without igv showed splenocyte stimulation, but the extent was lower than animals immunized in the presence of the igv adjuvent. these data suggested that igv had enhanced effect on priming against the conserved viral antigen matrix protein and generation cross-strain immune response. influenza is a respiratory disease causing epidemics every year. h n viruses and swine-origin h n have also infected humans in recent years. seasonal influenza vaccine cannot cope with significant antigenic drift or with the emergence of pandemic viruses of different subtypes not contained in the vaccine. the high extent of conservation of the m e makes it a promising immunogen. a vaccine based on coupling of the m e peptide to an appropriate carrier may provide a universal vaccine with effectiveness and safety. m e based vaccination induces protective antibodies not only in mice, but also in ferrets and monkeys. the carrier hepatitis b core as carrier with m e forms a virus like particle (vlp). vaccination with m e coupled to hbc induces protective antibody, whereas the contribution of t cell protection was negligible. protection induced by vaccination with m coupled to hbc was weak overall. in order to improve the vaccination effect of m e-hbc, new adjuvant igv was evaluated in combination with the m e-hbc. the tim molecules are a recently discovered class of proteins with the ability to regulate the immune system. crystal structures of the tim molecules has revealed a unique, conserved structure with ligand-binding sites in the igv domain. to determine the potential immunostimulatory molecular properties of igv, we have evaluated immune response of the igv in combination with m e-hbc vlp. previous papers reported that vlp immunized mice can induce the th and th immune response. different adjuvant combined vlp can produce biased immune response th ⁄ th mixed immune response, or th -preferred th ⁄ th profile. thus, the response following the use of igv as a new adjuvant combined with m e-hbc vlp needs to be evaluated. results indicated that igv combined groups showed th biased immune response and enhanced cross reactive t cell immune responses. this may show that igv immunized the mice and antiigv antibody can cross link the igv on t cells and enhance the cell figure . t cell proliferation assay. mice were immunized twice with , , , ug ⁄ ml igv plus m e-hbc, respectively, and naive group was immunized with pbs. three weeks after a boosting immunization, spleens were harvested from immunized and naive mice. different subtypes of inactivated virus antigen (a) h n , (b) h n , (c) h n , (d) h n were added and cocultured with different group splenocytes for h. quick cell proliferation assay kit was used to detect the cell proliferation. the - nm absorbance was read on a plate reader. data were showed were shown as mean values. the difference between naive group and different doses igv plus m e groups was determined using the student's t-test. all significance level is p < ae . response. we also evaluated the cross-protection produced by igv combined m e-hbc. we challenge with mouseadapted strain pr and prove the cross protection via reaction between the cells from the immunized animal and different subtypes of virus antigen. some subtypes of virus cannot infect the mice naturally, and therefore, virus challenge cannot be used to evaluate the effect. we co-cultured the t cells with inactivated antigen h , h , h , and h , and t cell proliferation was measured. results indicated that after immunization with igv plus m e-hbc, the t cells show cross-protection with other subtypes. this provides evidence that igv can enhance the cross protection across subtypes. the results of this study demonstrated that recombinant igv can be useful as an adjuvant and polarize the m e-hbc vlp immune response to a th profile. igv induced the m e-hbc vlp to induce t cell proliferation and cross-reactive responses to different influenza virus subtypes. this finding represents a new direction for the promotion of cell mediated immunity in m e based vaccine against influenza. a core european protocol, i-move, describing the methods to estimate influenza vaccine effectiveness (ive) was proposed by the european centre for disease prevention and control (ecdc) and epiconcept for the - season. it includes a case control method for pooled analysis based on a randomized ''systematic'' sample of swabs. , collection of swabs using a non randomized, i.e., ''ad hoc,'' sampling strategy, left at the appreciation of sentinel practitioners, provides a greater number of cases and con-trols for ive estimation more easily than using a systematic randomized sampling strategy. the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than specimens yearly from cases of acute respiratory illness (ari), using both sampling methods. , during the circulation of pandemic influenza viruses in france, it gave an opportunity to compare ive estimates using systematic randomized versus non systematic ''ad hoc'' sampling. influenza vaccine effectiveness was estimated by a casecontrol methodology according to ecdc i-move protocol, using on the one hand a systematic random sampling, on the other hand ''ad hoc'' non random sampling. the study was proposed to primary care practitioners of the grog network ( general practitioners and pediatricians) trained to collect data and swabs. the study population was patients from the community of all ages consulting a grog practitioner for an influenza like illness (ili) and having a nasal or throat swab taken within an interval of < days after symptom onset. ili was defined according to the european union (eu) case definition as sudden onset of symptoms with at least one of the following four systemic symptoms: fever or feverishness, malaise, headache, myalgia; and at least one of the following three respiratory symptoms: cough, sore throat, shortness of breath. swabs were performed through usual surveillance. no ethical approval was needed, but an oral informed consent was requested. cases were excluded if they refused to participate in the study or if they were unable to give informed consent or to follow the interview in native language because of aphasia, reduced consciousness, or other reasons. an individual was considered as vaccinated against pandemic influenza if he or she reported having received a pandemic influenza vaccination during the current season, and if at least one vaccine dose occurred more than days before ili onset. the study period started with the initiation of active influenza surveillance by the grog network, i.e., days after the beginning of the influenza vaccination campaign, and finished at the end of the influenza period defined as the last week with at least one swab positive for influenza within the grog network. ''ad hoc'' sampling patients from which swabs were taken were selected by the grog practitioners during the study period. systematic random sampling during the same period, patients were selected at random as follows. an age-group - years (gps and pediatricians); - years (gps and pediatricians); - years (gps); years or more (gps) was assigned to each practitioner, who was requested to swab the first patient of the week presenting with an ili within the pre-assigned age-group. swabs were collected in appropriate transport medium (virocult Ò , viralpack Ò , utm copan Ò ) and sent by post to the laboratory in triple packaging following the international guidelines for the transport of infectious substances (category b, classification un ). laboratory confirmation of influenza was by rt-pcr to detect currently circulating influenza a (subtypes h , seasonal and pandemic h ) and b viruses. an influenza case was defined as an ili case with a respiratory sample positive for influenza during the study period. controls were cases of ili having a swab negative for influenza during the study period. the outcome of interest is laboratory confirmed influenza. confounding factors and effects modifiers identified during the i-move preliminary study were registered: risk factors, chronic diseases, severity of underlying conditions, smoking history, former vaccinations, and functional status. data on cases and controls were collected by the practitioners using a standardized questionnaire adapted from the i-move study. questionnaires were sent by the practitioners with the swab to the virology laboratory, and sent to the grog national coordination. data entry and validation were ensured by open rome through the vircases computing tool. validation steps included control of exhaustiveness of centralization of questionnaires, comparison of data entered by the labs and the national grog coordination, coherence control, and identification of missing data. analysis was done for the two sampling groups (systematic and ad hoc) on cases ⁄ controls following the european method proposed by epiconcept, using excel ª (microsoft corp. redmond, washington, usa) and stata ª . baseline characteristics of cases and controls in unmatched studies were compared using the chi-square test, fisher's exact test, or the mann-whitney test (depending on the nature of the variable and the sample size). the association between vaccination status and baseline characteristics was assessed for both case and control groups. the vaccine effectiveness was computed as ive = )or (odds ratio). an exact % confidence interval (ci) was computed around the point estimate. analysis was stratified according to age groups, time (month of onset), presence or absence of chronic disease, and previous influenza vaccination. effect modification was assessed comparing the or across the strata of the baseline characteristics. confounding factors were assessed by comparing crude and adjusted or for each baseline characteristic. a multivariable logistic regression analysis was conducted to control for negative and positive confounding factors using a complete case analysis (with records with missing data dropped) and using multiple imputation with chained equations. the complete model included age group, number of gp visits, onset week, seasonal vaccination, previous seasonal influenza vaccination, presence of chronic disease and associated hospitalizations in the previous months, gender, and smoking status. variables were tested for multi-colinearity. interactions were tested using the likelihood ratio test (or wald test) and included in the model if significant at % level. a model with fewer variables (age group, number of gp visit, onset week, and seasonal vaccination) was also tested. several models were applied to both the ''ad hoc'' and systematic sampling groups of cases and controls. as shown in table , whatever the analysis method used, the ''ad hoc'' sampling strategy led to a slightly lower estimate of ive. the ci were extended when data were missing and reduced when using multiple imputations with chained equations. however, from a statistical point of view, comparison of ''ad hoc'' versus systematic strategies is not straightforward, because ''ad hoc'' sampling is not randomized and does not allow comparisons with statistical tests using statistical distribution laws. there are more missing data with the ad hoc sampling method. this is mainly due to our validation procedure: in the case of missing data in the systematic sampling group, as required by the i-move study protocol, queries were sent to sentinel practitioners using mail and phone calls. this specific heavy workload is not usually performed during routine surveillance and has not been achieved for the ''ad hoc'' sampling group given the great number of cases and controls ( ). within the framework of the i-move study, several items were added to the grog's usual clinical form accompanying swabs (hospitalizations, number of gp visits, smoking status, help needed for bathing or walking). in - , gps explained that this added workload was not compatible with their daily additional workload due to the pandemic situation. therefore, many of them refused to fill these new items systematically and threatened to leave the network. we thus obtained that the ''i-move items'' would be filled in for the clinical forms linked to systematic sampling, but were not in a position to obtain that for ''ad hoc'' sampling. the weekly distribution of systematic swabbing is not similar to that of ad hoc swabbing. the percentage of ad hoc swabs was higher than systematic swabs during the pandemic wave (mid-november to end of december) during which time the percentage of swabs positive for influenza was also higher ( figure ). this could explain the higher rate of positive swabs within the ''ad hoc'' samples. the vaccination campaign was launched by the ministry of health on october , , and vaccination coverage increased during the surveillance period. in february, the vaccination coverage was ae % in patients swabbed in the systematic group ( ae % on imputed data) and ae % [ ae - ae ] in the ad hoc group ( ae % on imputed data). at the national level, vaccine coverage is estimated at ae %. due to the over-mediatisation of pandemic vaccination and to rumors about its poor effectiveness, overconsultation of vaccinated patients and over-swabbing of vaccinated patients in the ad hoc group are not surprising. age distribution is significantly different between our two samples (p < ae ): the rate of - years old is lower in the systematic sampling group ( ae %) than in the ad hoc sampling group ( ae %). this can be explained by the fact that for the systematic sampling procedure, each grog practitioner had to swab the first ili patient in his assigned age group, whereas for ''ad hoc'' sampling, every grog practitioner could swab any ili patient irrespective of age. given the emphasis by health authorities and media on the burden of pandemic influenza among children and teenagers, one can hypothesize that when they were able to, sentinel practitioners focused on these age groups. gps in the ad hoc sampling scheme seem to have been more likely to select cases and further, to select vaccinated cases. those patients may have consulted earlier with specific symptoms (strong headache being more prevalent among cases). over-swabbing of patients having these symptoms in the ad hoc group is likely. the - pandemic influenza season was markedly different from previous ones: vaccination rate increased during and mainly after the pandemic peak; behaviors were strongly modified by unusual media hype; clinical features and risk factors might be different. it will be necessary to see if similar results are observed during a regular influenza season during which the vaccination rate increases before the epidemic peak with usual messages about vaccination and usual clinical influenza features. influenza early warning networks can estimate ive, taking into account many covariates. from a stakeholders and patients point of view, during the - influenza pandemic wave, there were no major discrepancies between ive estimated with an ad hoc sampling strategy, based on sentinel practitioners instinct, and ive estimated with a systematic random sampling strategy whatever the multivariable analysis methodology. although from a statistical point of view, comparison of the two strategies is not readily feasible because of the non random nature of ad hoc sampling. this latter strategy seems to result in slightly lower ive estimates, which could potentially be attributed to sentinel practitioners swabbing behavior. the ability to avoid missing data is a key point to decide which sampling method must be adopted, because ci extent depends greatly on the proportion of missing data among covariates. to match ive evaluation to surveillance networks practicality, selection of only those data essential for the study endpoint and easily collected by sentinel practitioners is paramount. it will be necessary to determine if results similar to those observed during the - pandemic season are found during a regular influenza season. influenza a viruses are important pathogens which remain a major cause of morbidity and mortality worldwide, and large numbers of the human population are affected every year. the first influenza pandemic in this century broke out in humans in march , and it was declared to be pandemic by mid-june. as of august jul , the pandemic virus had caused more than deaths worldwide, according to the world health organization (http:// www.who.int/csr/don/ _ _ /en/index.html). the infection and spread of the pandemic influenza was reduced in part due to the use of vaccines. however, the lack of h n pdm vaccine early in the pandemic illustrates the need to improve vaccine production and to generate vaccines that induce stronger cross-protection. inactivated split vaccines or live attenuated influenza virus vaccines (laivs) against h n pdm viruses were approved for human use by the united states food and drug administration. both the inactivated vaccines and laivs are produced by creating reassortant viruses that generally contain six vrnas (pb , pb , pa, np, m, and ns) from a master donor strain, plus the two glycoprotein vrnas (ha and na) from a virus that antigenically matches the strain predicted to circulate in upcoming influenza season (e.g. a ⁄ ca ⁄ ⁄ ). the reference viruses containing inactivated split virus vaccines are produced in embryonated chicken eggs, and primarily result in the production of antibodies that recognize the viral glycoproteins. both of these vaccine approaches require significant lead time for vaccine production, and modern approaches to speed preparation of vaccines and improve their efficacy is a global priority. , the ns protein of influenza a virus is a multifunctional protein that plays important roles in virus replication and as potent type i ifn antagonist. , mutations and ⁄ or deletions in ns typically induce stronger ifn responses by the host; those in turn suppress the replication of influenza virus - and can enhance immune recognition. [ ] [ ] [ ] [ ] in this study, we created a panel of experimental h n pdm ns-laiv candidates that have different deletions in the ns vrna and analyzed the vaccine potential of each ns-laiv in mice and ferrets to identify the best candidate(s). wt h n pdm influenza a virus a ⁄ new york ⁄ ⁄ (ny ) was created by reverse-genetics directly from a human swab specimen collected in new york state in april . deletions were introduced into the ny ns plasmid to create three mutant ns segments: ns - , ns - , and nsd . nucleotides - (cdna of ns segment) and - were replaced by stop codons to generate ns - and ns - ; nucleotides - were deleted to generate nsd , whose open reading frames for ns and nep were maintained. recombinant viruses were generated by co-transfection of eight reverse-genetics plasmids carrying the cdna of each gene segment into t ⁄ mdck cocultured monolayer adapted from hoffmann et al. , mouse studies experiments were performed in a biosafety level laboratories approved by the u.s. centers for disease control and prevention and the u.s. department of agriculture, and were conducted under approved animal care and use protocols. groups of -week-old female balb ⁄ cj (jackson laboratory, bar harbor, me, usa) were anesthetized with isoflurane and inoculated intranasally with tcid of each recombinant virus in ll of pbs diluent, or pbs as controls. body weights and clinical symptoms of the mice were monitored daily for days. nine mice in each group were euthanized on , , and days post inoculation (dpi), and nasal washes and lungs were collected for virus titration by tcid assay in mdck cells. at dpi, mice per group were challenged intranasally with · tcid ( ld in -week-old mice) of a mouse-adapted variant of ny (a ⁄ ny ⁄ ⁄ -ma ) (accepted, journal of virology). disease symptoms and weights of the vaccinated mice were monitored for days, and four mice from each virus group were euthanized at and days post challenge. lungs were removed and homogenized for virus titration by tcid assay. the mice that became moribund or lost > % of their starting body weight were euthanized for humane reasons. male fitch ferrets (triple f farms, sayre, pa, usa), - months of age and serologically negative by hemagglutination inhibition (hi) assay for currently circulating influenza viruses were used in this study. groups of or ferrets were inoculated intranasally with ae tcid of one of the viruses: ny wt (n = ), ns - (n = ), ns - (n = ), or nsd (n = ). ferrets were monitored for clinical signs through dpi as previously described. nasal washes were collected on , , , and dpi and were titrated in mdck cells by tcid assay. serum was isolated from blood collected ae weeks after immunization and used for neutralization assays. the ferrets were challenged with pfu of a ⁄ mexico ⁄ ⁄ ae weeks postimmunization and monitored for clinical signs of disease through dpi. nasal washes were collected on , , , and dpi, and were titrated in mdck cells by plaque assay. using reverse genetics, we created three laiv candidates weight loss of wt virus inoculated mice became evident at dpi, and the mice did not recover until dpi (figure a) . in contrast, mice inoculated with any one of the vaccine candidates had no clinical signs of disease and continued to gain weight at the same rate as did the mock- inoculated mice ( figure a ). viral titers in the lungs of ns - , and ns - infected mice were $ -fold lower than titers from wt virus-infected mice at all the time points analyzed ( , , and dpi) ( figure b) . notably, the nsd laiv was cleared from the mouse lungs very rapidly, and the mean titers were $ -fold and -fold lower than the titers of the wt virus at and dpi, respectively ( figure b) . the vaccinated mice were challenged with a mouseadapted variant of ny (accepted, journal of virology) on dpi. no disease symptoms were observed in the mice immunized by any of the ns-laiv candidates or the wt control. in contrast, disease symptoms including ruffled fur, hunched posture, and weight loss were observed in the mock-immunized mice as early as days post challenge (dpc); the symptoms progressed to severe disease, and the animals showed dramatic weight loss, became moribund, and succumbed to infection by dpc (figure c ). high titers of virus ($ tcid ⁄ ml) were present in the mock-immunized mice at dpc and at dpc ( figure d ). in contrast, virus was not detected in the lungs of immunized mice ( figure d ). this challenge data demonstrates that all of the ns-laiv candidates, including the highly attenuated nsd , induced sterilizing immunity that protected mice from a lethal ny h n pdm variant. groups of ferrets were intranasally immunized with ae tcid of each vaccine candidate or the wt virus. the titer of viruses recovered from nasal washes ranged from ae to ae tcid ⁄ ml through day in the wt virusinfected group, while the ns-laivs showed various degrees of attenuation (figure a) . the viral titer of all of the ns-laivs is at least -fold lower than that of wt in the nasal wash collected at dpi. the ns - laiv was the least attenuated in ferrets, and its replication was similar to that observed in mice. relative to the wt virus, the ns - laiv showed -fold reduction in titer, and the nsd laiv was below the limit of detection (at least fold reduction) at dpi. sera from blood collected ae weeks after immunization was analyzed for the presence of neutralizing antibodies by micro-neutralization assays. the ns-laiv candidates all induced very strong neutralizing antibody responses ( - ) that were similar to the titer elicited by wt virus infection ( figure b ). the ferrets were challenged with pfu of a ⁄ mexico ⁄ ⁄ (h n pdm) ae weeks post immunization. little disease or weight loss were observed in the naïve ferrets, and the ferrets immunized by infection with wt virus or the ns-laiv candidates didn't show any disease symptoms or weight loss. in contrast to the high titer of virus detected in the naïve ferrets through dpc, the ns-laiv immunized ferrets had very low levels of a ⁄ mexico ⁄ ⁄ in their nasal washes at dpc ( figure c ). the ferrets immunized with the ny ns-laivs had $ -to -fold lower viral titers than did the naïve animals ( figure d ). in summary, the ns-laiv candidates dramatically inhibited initial replication of the h n pdm virus under stringent challenge conditions ( pfu), and that the vaccinated animals rapidly cleared the infection (to below the limit of detection, by dpc). our results demonstrate that all of the ns-laiv candidates are attenuated compared to the wt h n pdm virus, and the degree of attenuation is dependent on the specific ns mutation. ns - was the least attenuated and does not represent a good vaccine candidate; whereas, nsd and ns - were highly attenuated in both the mouse and ferret models. although they were markedly attenuated, they elicited strong neutralizing antibody responses and protected mice and ferrets from subsequent challenge. nsd has a subtle in-frame deletion ( nt) that affects both the ns (residues - ) and nep (residues - ), and is analogous to a naturally attenuated variant of a normally highly pathogenic h n virus (a ⁄ sw ⁄ fj ⁄ ). the analogous ns deletion in a ⁄ sw ⁄ fj ⁄ (residues - ) was shown to reduce binding to host cleavage and polyadenylation specificity factor (cpsf), reduce ns protein stability, and enhance the type i ifn response of this h n virus. our study indicates that deletion of these nt in the ns vrna of the h n pdm also stimulates the host ifn response, specifically, ifn-ß, ifn-k , ip , and mxa (data not shown). the role of the deletion of residues - from nep has not been elucidated, but the induction of ifn and isgs by nsd was similar to, or slightly lower than, their induction by ns- , suggesting that the nep mutation also has an attenuating effect that warrants future investigation. in summary, we have generated a panel of laivs directly from a swab specimen containing a new pandemic virus and analyzed their attenuation and immunogenicity in two animal models. our study demonstrates that nsd is a novel ns-laiv that could be used to create laivs for diverse influenza a viruses. this study also validates the use of ns-laiv candidates, which are not only highly attenuated, but they also elicit strong innate and adaptive immune responses, resulting in protection of mice from subsequent challenge with a lethal mouse-adapted variant of ny , and ferrets from challenge with a ⁄ mexico ⁄ ⁄ (h n pdm). currently, a total of approximately million doses of inactivated influenza vaccine are being produced worldwide each year. one of the limitations in vaccine production is poor growth of human isolates in embryonated chicken eggs. this is essential to develop high yield seed viruses for large scale production of influenza vaccines. influenza a vaccine production utilizes high yield reassortants carrying ha and na genes from a wild type (wt) strain with generally - internal genes from the a ⁄ pr ⁄ ⁄ (pr ) strain, an highly egg adapted high growth donor strain. influenza b vaccines, however, have been produced directly from wt strains, partly because no high yield donor analogous to pr has been identified. in recent years, reverse genetics has been used as an alternative means of developing high growth vaccine viruses. , since in this plasmid-based technology, a : reassortant (six internal genes from a donor strain and two surface antigen genes from wild type strain) can be directly rescued, reverse genetics-derived reassortant viruses were expected to grow as efficiently as those derived from classical reassortment. however, reverse genetics reassortants have not produced the expected high growth for several reasons: (i) the : configuration is not always the best for virus yield, (ii) there is no process included for positive selection of adaptive mutants from quasispecies, and (iii) cell-derived viruses are not readily adapted to grow efficiently in eggs. our laboratory at new york medical college has been preparing b reassortants for several years by classical reassortment using b ⁄ lee ⁄ as a donor. it has been possible to develop b reassortants, which produce higher virus yields than wt strains in eggs, and it was found that the np gene of b ⁄ lee ⁄ was important in producing high yield b reassortants. however, b ⁄ lee ⁄ is inconsistent in providing high yield properties to b reassortants. in this study, in an attempt to find an alternative donor, we investigated the usefulness of b ⁄ panama ⁄ ⁄ for developing high yield b reassortants. as a wt strain, b ⁄ brisbane ⁄ ⁄ was used, which is one of the recommended influenza b virus vaccine strains for the ⁄ and ⁄ seasons. we found that b ⁄ panama ⁄ ⁄ is a useful donor, and some of the resultant reassortants were considered as vaccine candidates. b reassortant viruses were prepared by the classical reassortment method described by kilbourne. the antiserum to b ⁄ panama ⁄ ⁄ hemagglutinin and neuraminidase (hana) was raised in this study by immunizing rabbits with hana isolated from b ⁄ panama ⁄ ⁄ ; purified igg was used for antibody selection. the yields of the reassortants and their corresponding parent viruses were assessed by hemagglutination assay. viral rna was extracted directly from the allantoic fluid and amplified by rt-pcr to produce cdna for analyzing the gene composition. restriction fragment length polymorphism (rflp) analyses were performed to determine the origin of each gene segment of the high yield reassortants. restriction enzyme sets for each gene segment are available upon request. in this study we investigated the usefulness of b ⁄ panama ⁄ ⁄ as a donor for transferring high yield phenotype. b ⁄ panama ⁄ is a yamagata lineage strain with high growth phenotype (ha titer: - ). b ⁄ panama ⁄ ⁄ itself was a recommended b virus vaccine strain for ⁄ - ⁄ seasons. as a wt virus, a victoria lineage strain, b ⁄ brisbane ⁄ ⁄ , was used, which is a recommended b virus vaccine strain for use in the ⁄ and ⁄ seasons. reassortants were prepared according to classical reassortment protocol. after co-infection of b ⁄ panama ⁄ ⁄ and b ⁄ brisbane ⁄ ⁄ , progeny viruses carrying surface antigens (ha and na) of the vaccine strain were negatively selected by anti-b ⁄ panama hana antibodies, followed by passages without antibodies for positive selection of eggadapted viruses and finally limited dilution cloning. nymc bx- , bx- b, bx- d, and r- a are representative of resultant reassortants, which have significantly higher ha titers than the wt strain. the complete gene compositions of these reassortants were determined by rt-pcr ⁄ rflp analyses. as shown in table , all of these reassortants contained the pb of b ⁄ panama ⁄ ⁄ . other genes of b ⁄ panama ⁄ ⁄ (np of bx- , m of bx- b, and pb of bx- d) may not be involved in the high virus yield, since no significant growth difference among these reassortants in eggs was found as assayed by hemagglutination test. accordingly, the pb of b ⁄ panama ⁄ ⁄ is considered to be the sole factor involved in the high yield phenotype donated to the vaccine strain. we previously found that the b ⁄ lee ⁄ np gene was important in producing high yield b reassortants. it was of interest to examine whether b ⁄ lee ⁄ np and b ⁄ panama pb could work together to produce even higher yields. to test this possibility, bx- b ( : reassortant: pb and m genes from b ⁄ panama and the rest of the genes from b ⁄ brisbane) was selected and further reassorted with b ⁄ lee ⁄ . despite some difficulty in removing the na gene of b ⁄ lee ⁄ (r- c, b, b in table ), by monitoring ha and na genes of resultant viruses after each antibody selection passage with anti b ⁄ lee ⁄ hana antibodies, we were able to isolate and clone a triple reassortant, nymc bx- , which contains the np gene from b ⁄ lee ⁄ and pb and m genes from b ⁄ panama; the remaining genes are from b ⁄ brisbane ⁄ ⁄ (table ). in comparison with bx- b, no significant growth enhancement (nor reduction) in eggs was found for bx- over that seen for bx- b. nevertheless, bx- stably produces high virus yield and has been utilized as a seed virus for influenza b vaccine production for the - season by one or more vaccine manufacturers. there are contradictory reports - about the usefulness of reassortment for high yield influenza b viruses. however, we have been preparing b reassortants for several years by classical reassortment using b ⁄ lee ⁄ as a donor, and have been able to generate higher virus yield than wt strains. in this study, we found that b ⁄ panama ⁄ ⁄ serves as an efficient donor in providing the high growth capacity to b ⁄ brisbane ⁄ ⁄ (a recommended vaccine virus of victoria lineage for ⁄ - ⁄ seasons), and that the pb of b ⁄ panama ⁄ ⁄ is associated with the high yield phenotype. this particular strain from yamagata lineage might be useful to prepare high yield reassortants for other victoria lineage vaccine viruses. we noticed in this study that there may be segment incompatibilities between b ⁄ panama ⁄ ⁄ and b ⁄ brisbane ⁄ ⁄ . as shown in table , the pa and ns genes of all the high yield reassortants examined are derived from wt, b ⁄ brisbane ⁄ ⁄ , not from the donor, b ⁄ panama ⁄ ⁄ . this indicates that in this reassortment, the pa and ns genes are not replaceable with that of the donor to obtain high yield viruses. this degree of incompatibility might be common in b reassortment, resulting in low donor ⁄ wt reassortants, such as : and even : reassortants that we obtained in this study. if this is the case, reverse genetics based on : configuration may not result in generating high yield b reassortants unless a variety of donor ⁄ wt combinations are designed. one can speculate that in influenza b viruses, the surface glycoproteins (ha and na) and some of the internal proteins are functionally more closely related than in influenza a virus, as was seen in that pa and ns genes of b ⁄ brisbane ⁄ ⁄ reassort together with the ha and na genes of the same parent (table ). in our recent study on a reassortment between b ⁄ lee ⁄ and b ⁄ panama ⁄ ⁄ , it appeared that ha shapes overall gene constellations of the resultant reassortants, namely the reassortants tend to have more internal genes from the same parent of ha, no matter which parent's ha is selected by antibodies against the surface antigens of the other parent (data not shown). because of success in influenza a virus reassortment with pr , it is generally believed that reassortant with : or : configuration is optimal for virus yield. this may be the case in most instances of influenza a reassortment, but is not necessarily so in b reassortment. as shown in this study, only a single donor gene is capable of improving the yield of vaccine strain by reassortment. influenza a ⁄ h n v has spread rapidly in all parts of the world in as a true pandemic. epidemic events in russia occurred during the last week of september starting from far east region (yuzhno-sakhalinsk). kaliningrad (the western most russian city) was the second starting point of the epidemic. during october the epidemic spread over the whole russian territory. in a short period the new virus started to change genetically as it began to adapt to human populations during this pandemic (http://www.who.int; http://www.euroflu.org). in the period from may to december , clinical samples (nasopharyngeal swabs and postmortem materials) of patients with influenza-like illness from different regions of russian federation were analyzed to confirm the diagnosis using real-time reverse transcription pcr (rrt-pcr). clinical nasopharyngeal swabs and bronchoalveolar lavage and post mortal fragments of trachea, lungs, bronchi, spleen from saint petersburg hospitals and basic laboratories of federal influenza center were included in this study. all specimens were taken from patients with influenza-like illness or viral pneumonia. specimens were tested by rrt-pcr according to cdc protocols, i.e. using superscript iii platinum one-step qrt-pcr system (invitrogen) with primers and probes for infa, h seasonal, and h sw (biosearch technologies). in addition, the test-systems 'amplisense influenza virus a ⁄ b-fl' and 'influenza virus a ⁄ h -swine-fl' for pcr-detection, typing and subtyping of influenza viruses were also used. these test-systems are produced by central institute of epidemiology, moscow, russia and recommended by russian ministry of health as tests for influenza diagnosis. sequencing was carried out on an abi prism -avant genetic analyzer (applied biosystems, usa) with bigdye terminator cycle sequencing kit. phylogenetic analysis was performed using programs vector nti . (invitrogen) and mega . (psu, usa) by maximum likelihood with the tim+i+g model for ha, and -hky+i+g model for na. evolutionary model was selected by akaike information criterion (aic) in model-test (posada, crandall, ). statistical reliability of tree branches was evaluated by bootstrap test ( replications). immunohistochemical study was performed using novalink antibodies to ha and np with novocastra visualization system. influenza virus a ⁄ h n v rna was detected in patients with severe form of influenza-like illness and fatal cases. out of pcr-confirmed flu recovered cases % were patients under years of age, % were aged - years, and % were older than years. mean age of recovered patients was ae years (from month to years). viral rna in postmortem materials was detected mostly in lung tissue ( % of specimens) and trachea fragments ( %), and less commonly in spleen ( %). mean age of the deceased with confirmed flu (h n v) infection was ae years with age ranging from months to years. in % of fatal cases, influenza was complicated by viral or secondary bacterial pneumonia. median time from the onset of illness until death was days. according to our data, % of patients died had diabetes, ae % were obese, and % were pregnant women in the nd or rd trimester. ha and np were detected by immunohistochemical assay in lung tissue of dead patients with confirmed influenza virus a ⁄ h n v infection. ha and np was revealed in the endothelium of different sized blood vessels (capillaries and arterioles). these influenza virus proteins were also detected in some tissue macrophages apart from epithelium and endothelium. the localization of the two proteins was different: ha is mostly localized in cell membrane and cytoplasm, and np -mostly in the nucleus. here we present data on molecular genetic characteristics of strains of pandemic virus, strains obtained from clinical specimens, and from post mortal ones isolated in the research institute of influenza. all the strains studied contain the s n substitution in m protein, which indicates resistance to the adamantane antivirals, and have no h y substitution in the neuraminidase, which indicates resistance to oseltamivir. the phylogenetic analysis showed that russian viruses were similar to influenza viruses a ⁄ texas ⁄ ⁄ and a ⁄ california ⁄ ⁄ (ha similarity ae %). all russian viruses could be divided in two clusters: the first one includes viruses similar to the reference strain a ⁄ california ⁄ ⁄ , and the second one, which is the majority of viruses analyzed includes strains with substitutions ha s t, na n d, v i, and ns i v (figure ). bootstrap support was . the isolates with ha s t substitution can be classified in one of the five minor genome variants of a ⁄ h n v viruses found in the united states and mexico in . several viruses had strain-specific substitutions in antigenic sites sb and ca and the mutation d g in ha receptor-binding site. the substitution of amino acid residue asp to gly at position of ha was found in eight of eleven isolates ( %) from postmortem lung and trachea samples and two of forty isolates ( %) from nasopharyngeal swabs of patients with severe course of the disease. appearance of amino acid substitutions in the ha receptor-binding site (d e and d g ⁄ e) could be associated with influenza virus passaging on eggs. five strains that contained g at position of ha were isolated from post mortal specimens on mdck cells in this study, thereby excluding the possibility of substitution appearance hence to virus adaptation on eggs. in order to reveal genome changes in a(h n )v, strains isolated on the territory of russian federation during the pandemic, full genome sequences from genbank, and research institute of influenza database were analyzed comparing two groups of viruses (isolated before and after sept ). nine amino acid changes observed predominantly in late pandemic strains were found. five of them (s p, s n, d g, v i, v i) reside in ha, two in na (i v, n k), two in pb (k n, t i), and one in pa (f l). towards the end of the epidemic the viral population had demonstrated statistically certain rise in number of strains containing mutations in four genes. difference between groups was statistically significant (chisquare test, p = ae ). if v > ae , than difference between early and late strains is statistically significant. additionally fisher's test determined whether 'early strains' and 'late strains' differ significantly in the proportion of 'no mutation event' and 'mutation event' attributed to them in each particular position. all calculations were performed in fisher_tk freeware by vladimir belyaev similar to calcfisher (haseeb, ) fully described here (http://www.jstatsoft.org/v /i /paper). we have selected positions with statistically significant amino acid changes in late strains (p-value ae ). according to full genome analysis of influenza virus a ⁄ h n v strains, seven clades were distinguished, but the divergence between representatives of different clades remained small. (figure ). besides the strain a ⁄ perth ⁄ ⁄ also contains substitution s f in the same ha antigenic site. according to data obtained, the epidemic in russia was caused only by influenza virus a ⁄ h n v. unlike the previous epidemic periods when most severe influenza cases were registered among the children under years and among elderly people aged over years, the first wave of pandemic due to influenza virus a (h n )v resulted in increased level of mortality mainly among the people aged - years. though all pandemic viruses showed comparative genetic homogeneity, some evolutionary trends could be outlined. for clarification of the exact pathogenic role of mutation d g in ha receptor binding site, further studies are necessary. full-genome analysis of influenza virus a ⁄ h n v strains circulating in the southern hemisphere in the new epidemic season revealed the phylogenetic subgroup distinguished by seven substitutions in inner proteins (pb , pb , np, ns ) and sa antigenic site of ha (n d). the changes revealed could be caused by adaptation of the virus to an immunized human population. nasal and throat swabs (placed in ml mem and frozen at ) °c until use for viral rna extraction and tissue culture inoculation) were collected from patients with febrile illness, i.e., > ae °c. samples were received from clinics in us embassies and us military laboratories located throughout the world since the initial who declaration of novel h n outbreaks as a global pandemic on june , . viral isolates were obtained from inoculating cultures of mdck cells with ae - ae ml viral suspensions collected in mem originated from patients after - days incubation. [ ] [ ] [ ] [ ] due to low viral titers in normal clinical samples, most of full viral genome sequences were derived from viral stocks obtained by tissue culturing passages (mdck, - times). viral rna was extracted from clarified supernatant fluid of nasal ⁄ throat swabs or mdck cultures using the 'charge-switch' rna extraction system based on the user manual protocol from the manufacturer (invitrogen inc., ca, usa). total rna was eluted into volume equal to original sample volume, i.e., ll starting viral supernatant used to yield final ll rna in molecular grade water (invotrogen inc.) and stored at ) °c until tested. generating ⁄ preparing overlapped cdnas for full genome coverage of novel h n viruses by multiple rt-pcr amplifications the first step in the high-throughput sequencing pipeline for full influenza genome sequences was to establish a robust rt-pcr amplification scheme consisting different rt-pcr primer pairs covering all rna segments to ensure % amplification coverage of full viral genomes of all the incoming targeted viruses (houng, hs. , submitted for publication). extracted viral rna ( ll), derived from mostly mdck culturing stock or clinical sample containing sufficient viral load (> infectious units per ml) was added to primer-free rt-pcr total master mixture ( ae ll) for each virus followed by adding primer pair ( ll, pmole ⁄ ll per primer). rt-pcr was then performed: rt reaction through two hold-steps ( °c, minutes and °c, minutes); cycling amplifications ( °c for seconds, °c for seconds, °c for ae minutes). specific cdna amplicons corresponding to each individual primer pair were routinely monitored and visualized by agarose gel electrophoresis. pooled cdna products ( - lg) from each viral rt-pcr amplification run were used as sequencing substrates according to the roche flx user manual and bulletins by incorporating adaptors containing individually multiplex identifier [mid]-key assigned to each individually pooled viral cdna. up to different mid-keyed viral cdna were further pooled together to be clonally amplified on capture beads in water-in-oil emulsion micro-reactors (em amplifications), and pyrosequenced using one of two regions of a · mm picotiterplate. for each individual viral genome containing multiple assemblies ( rna segments), we obtained sff file(s) containing raw sequencing reads from which nucleotide sequence data and phredlike quality scores were extracted. on average, ae - ae % of - million mid-key specific nucleotides were extracted and mapped for consensus genome sequences. roche gsmapper (v. . and . ) software was used to assemble all sequencing raw data and sff files into consensus sequences. new reference mapping projects were created to assemble each individually mid-keyed viral cdna into consensus viral sequences. one of the earliest h n genomes of california origin, a ⁄ california ⁄ ⁄ (h n ), deposited in genbank, was routinely employed as a reference genome sequence for most of gsmapper projects. the resultant consensus sequences obtained were further verified and validated through the ncbi annotation utility check and ultimately deposited to the ncbi influenza database, genbank. nucleotide sequences specific to each individual rna gene were aligned by the geneious pro . . software (http://www.geneious.com). trees were built based on the tamura-nei genetic distance model using the neighbor-joining method with no outgroup used via geneious pro . . . phylogenetic trees of the h n genomes were constructed by importing fasta files containing specific concatenated target sequences of pb , pb , pa, ha, np, na, mp, and ns from each individual virus into the geneous pro software and going through the sequence assembling and tree building steps. high-throughput pyrosequencing of pooled novel h n cdnas by roche flx system up to viral cdnas could be routinely sequenced to completion for different full viral genomes from a single roche flx picotiter plate by utilizing the combination of pico-titer plate's two distinct regions as well as different mid-keyed adaptors. the 'shotgun' sequencing approach employed in this study is a feasible method to viral isolates (n) sequence multiple pooled h n viral genomes. for each pyrosequencing experiment, approximately - passed key reads (single fragment per bead) were obtained that yielded readable nucleic acid sequences. among those close to a million passed key reads, only - passed key reads had an average sequencing read length of > bps, defined as 'long reads' ( bps · reads = total of million bases of nucleic acid sequences) that were used to assemble into influenza genome sequences. mathematically, - million bases of raw sequencing data from each single roche flx experiment would provide sufficient sequencing bases to cover full genome sequences with approximate - · of sequencing depth coverage of influenza a with average genome size of bps for the total of eight segmented rnas. so far, more than full h n genomes sampled worldwide have been successfully sequenced and deposited in the ncbi database by division of viral diseases, walter reed army institute of research (wrair). the bioinformatics derived from unique viral genome sequences generated from this study based on constant rt-pcr amplification scheme and identical roche pyrosequencing protocols provide a reliable data set in predicting the evolutionary patterns of pandemic viruses. wrair received clinical samples from us embassies and military personnel throughout the world since the initial who announcement of novel h n outbreaks. nearly equal distributions of sequenced viruses derived from three broadly categorized geographic regions, north america, central ⁄ south america, and asia ⁄ europe ⁄ africa (data not shown). besides the geographic distribution pattern of viral isolates, figure displays the viral isolation time lines of all the sequenced viruses reflecting two peaks that coincided with two waves of pandemic infections, early-mid summer and fall of . phylogenetic trees of the eight influenza a segments of all sequenced viruses were tentatively generated. it was found that the substitution frequencies per site for the ha, na, and ns genes are at much higher rate than the other five genes, pb , pb , pa, np, and mp genes (data not shown). the observed higher genetic variations for ha and na genes of h n are consistent with the historical genomic and epidemiological dynamics data of human influenza a revealing higher temporal fluctuations in ha and na genes. [ ] [ ] [ ] [ ] analysis of full influenza genomes containing concatenated eight complete rna segments revealed the existence of two distinctive genetic clades in circulation since the beginning of pandemic, as shown in fig-ure . it is noteworthy that all viruses of mexico and california origins (clade shown at the top of figure ) were isolated at the beginning of pandemic prior to the isolation of all other viruses belonging to the second genetic clade . , discussion during the past decade, the advance of dna sequencing technology, such as development of ngs, in making full viral genome sequences readily available have enabled study of far broader and more detailed aspects of evolutionary change for any new emergent infectious pathogen. the massive sequencing capacity of roche flx system allows simultaneously process and sequence millions of individual cdna molecules, in contrast to processing and sequencing individual cdna fragments by conventional sanger sequencing method. within a short period of few months since the beginning of the pandemics, wrair accomplished large number of representative h n full genomes of worldwide origins via roche flx system. sequencing data derived from this study illustrates a much higher genetic variation rate for ha and na genes of h n that is compatible to the higher temporal fluctuation rate for ha and na genes of seasonal influenza a derived from decades of intensive monitoring and comparison studies and analyses. [ ] [ ] [ ] [ ] following the mexican and us reported cases, confirmed outbreaks of swine h n rapidly proliferated and spread throughout europe, asia, africa, and south america, most probably via global airline travel. , it seemed that new cases in the us and most cases throughout the world had been clinically mild relative to the initial reported cases in mexico. [ ] [ ] [ ] [ ] here we demonstrate through the phylogenetic relationship of sequenced h n full genomes that the clinical isolates could be divided into two different clades of viruses, i.e., the clade genetic group contains only viruses isolated at the beginning (march ⁄ april , mexico and california) of pandemics and the rest of other viruses all belong to the nd genetic group, clade . thus, it's likely that the currently circulating h n of clade causing worldwide infections is genetically different from the initial h n isolates that caused the early infections in mexico and california. , introduction a pandemic influenza virus ( h n ) was recently introduced into the human population. the hemagglutinin (ha) gene of h n is derived from 'classical swine h n ' virus, which likely shares a common progenitor strain with the human h n virus that caused the pandemic in . since antigenic changes of influenza virus ha occur more slowly in swine than in humans, we hypothesized that h n might still retain an antigenic structure similar to that of h n or the early isolates of its descendants. in this study, we compared ha antigenic structures of h n and human h n viruses by a molecular modeling approach to demonstrate the existence of shared epi-topes for neutralizing antibodies. we found that has of h n and the h n virus shared a significant number of amino acid residues in known antigenic regions. from this observation, we hypothesize that the h n ha antigenic sites will be targeted by antibody-mediated selection pressure in humans in the near future. we further discuss possible directions of antigenic changes in the evolutionary process of h n . sequence data of ha genes modeller v was used for homology modeling of ha structures. after models of the ha trimer were generated, the model was chosen by a combination of the mod-eller objective function value and the discrete optimized protein energy statistical potential score. after addition of hydrogen atoms, the model was refined by energy minimization with the minimization protocols in the accelrys discovery studio . software package using a charmm force field. steepest descent followed by conjugate gradient minimizations was carried out until the root mean square gradient was less than or equal to ae kcal ⁄ mol ⁄ a. the generalized born implicit solvent model was used to model the effects of solvation. the ha model was finally evaluated by using procheck, whatcheck, and verify- d. custom-made programs were developed with the ruby language and used for investigating the numbers of potential n-glycosylation sites and candidate codons (cand ) in ha sequences. it is known that the h ha molecules have four distinct antigenic sites: sa, sb, ca, and cb. , as a result, these sites consist of the most variable amino acids in the ha molecule of the seasonal human h n viruses that have been subjected to antibody-mediated immune pressure since its emergence in , although it was absent in humans from to . to investigate the structures of these antigenic sites of h n , d structures of the ha molecules of sc , the recent seasonal human h n virus (br ), and h n (ca ) were constructed by a homology modeling approach, and compared by mapping all the amino acid residues that were distinct from those of sc ha (data not shown). we found that most of these antigenic sites of br ha predominantly contained altered amino acid residues if compared with sc . by contrast, amino acid residues at these positions were relatively conserved in ca ha when compared with sc ha. notably, the sa and sb sites, which contain many amino acids involved in neutralizing epitopes near the receptor binding pockets, remain almost intact ( table ), suggesting that antibodies raised by natural infection with sc or its antigenically related descendant viruses play a role in specific immunity against ca . these observations lead us to hypothesize that such antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in the human population. based on this hypothesis, we speculated that h n would undergo patterns of amino acid substitutions in ha similar to those seen in seasonal human h n viruses during its epidemic period (i.e. those that have been substituted since ) (figure ) . we then predicted possible amino acid substitutions of h n from the sequence similarity of the antigenic sites. for example, both sc and ca had an asn residue at position in the sa site. for sc , the residue at this position has altered from asn to lys since . combining these two facts, it seems reasonable to hypothesize that ca will also undergo an amino acid substitution from asn to lys at position in the future. interestingly, we found that some of the recent variants of the h n virus have indeed undergone substitutions identical to those predicted in figure . it is important to monitor whether such variants will be selected and survive in sustained circulation in humans. next, we analyzed the acquisition of potential n-glycosylation sites associated with antigenic changes. previously, we reported that cand sites, a set of three codons that require single nucleotide substitution to produce n-glycosylation sequons, were important motifs to rapidly acquire n-glycosylation sequons. therefore, we investigated the number and location of potential n-glycosylation sites and cand sites in h n ha. we found that ca also had a single n-glycosylation sequon at the same position in the globular head region of ha, and lacked the multiple n-glycosylations that have been observed in the antigenic changes of the human h n virus during the early epidemic of this virus. we also found that ca ha possessed three cand sites that were present at the same position in sc ha (positions of the first asn residue, , , and ). of these, the cand sites with positions at and had actually become potential n-glycosylation sites in human h n viruses. this result suggests the likelihood of additional n-glycosylation at these sites during future antigenic changes of h n ha. notably, some of the recent h n variants (as of march , ) have an additional n-glycosylation sequon at position , where the h n virus readily acquired an n-glycosylation site during its circulation. the present study suggests that the antigenic structure of h n ha is similar, at least in part, to that of the h n ha. the and h n has share unique three-codon motifs that are important to readily acquire n-glycosylation sequons in their globular head region. based on these similarities, we predicted possible amino acid substitutions that might be associated with future antigenic changes of h n , and confirmed that such substitutions occurred in some of the recent variants of this virus. the present study provides an insight into likely future antigenic changes in the evolutionary process of h n in the human population. influenza viruses are classified into three types, a, b, and c, based upon the antigenic properties of nucleoproteins and matrix proteins. influenza a virus infects a wide range of hosts, including human, bird, swine, equine, and marine mammal species, while influenza b and c are less pathogenic than influenza a and are mainly found in humans, although there is evidence that they can also infect other species. influenza a has evolved in association with its various hosts on different continents for extended periods of time. to survive as a successful pathogen, the influenza viruses have developed a number of mechanisms, including antigenic mutation and genome reassortment, to continuously evolve and evade the surveillance of the host immune systems. antigenic and genetic analyses have provided important insights into the molecular dynamics of influenza virus evolution. however, a comprehensive understanding of influenza viral genetic divergence and diversity remains lacking. neuraminidase (na) is a major surface glycoprotein of influenza a and b, but is absent in influenza c. it plays a key role in virus replication through removing sialic acids from the surface of the host cell and releasing newly formed virions. influenza a viral na genes are classified into nine subtypes (na -na ) based upon their antigenic properties, while na genes of influenza b are not classified into subtypes. furthermore, most na subtypes of influenza a have evolved into distinct lineages and sub-lineages, which correspond to specific hosts or geographical locations. in this study, we conducted large-scale analyses of influenza na sequences in order to infer their evolution and to identify lineages (or sub-lineages) of influenza a viruses. a total of na sequences that excluded laboratory recombinant sequences were downloaded from genbank. sequences were aligned with muscle and mafft. the alignments were adjusted manually using translatorx, based upon corresponding protein sequences. phylogenetic analyses were conducted using the maximum-likelihood (ml) method in raxml. a set of perl scripts were written by us to facilitate this computational analysis. lineages and sub-lineages were determined based on the topology of the ml trees. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, but with the following modifications: a single digit is used to represent one of the nine subtypes and a letter is used to represent a lineage; a sub-lineage is also represented using a digit; a dot is used to separate a lineage and a sublineage. for example, a. means na subtype, lineage a, and sub-lineage . the time of most recent common ancestor (tmrca) was estimated using the bayesian mcmc method in beast. in all cases, we employed the gtr + u nucleotide substitution model, in which the first and second codon positions are allowed different rates relative to the third codon position. all data sets were analyzed under a relaxed molecular clock and the bayesian skyline population coalescent prior. the maximum clade credibility (mcc) tree across all plausible trees was computed from the beast trees using the treeannotator program, with the first % trees removed as burn-in. phylogenetic analysis based upon na sequences revealed two large groups corresponding to influenza a and b, respectively ( figure a ). within influenza a, two subgroups were found, one consisting of na , na , na , and na and the other consisting of the remaining five subtypes. subtype na was found to be a sister subtype of na , na being a sister subtype of na , and na a sister subtype of na . finally, each na subtype forms a distinct cluster, indicating its genetic uniqueness. influenza a and b viral na were estimated to have diverged around years ago ( figure b ). however, it had large % hpd values which ranged from years to years ago. the na subtypes of influenza a diverged from more than to several hundred years ago. the time of most recent common ancestor (tmrca) of each subtype of influenza a virus was generally recent and ranged from the calendar years to (figure b ). in addition, the tmrca for influenza b viral na was dated back to . a total of lineages were identified in influenza a (table ) . three lineages, a, b, and c, were identified for na based upon the tree topology. linage a originated from avian viruses and was further divided into sub-lineages: a. , a. , a. , a. , and a. . linage b consists of north american swine influenza viruses whereas c is a human lineage. two large lineages, a and b, were identified in na . lineage a is a human-specific lineage. interestingly, five major swine clades were observed within this lineage. lineage b is an avian-specific lineage, and consists of sub-lineages, b. , b. , and b. . three lineages were found in na . lineage a was found in north american avian, b in eurasian ⁄ oceanian avian, and c also in avian, but it does not show any geographical pattern. for na , na , and na , each was classified into lineages, one found in north american avian ( a, a, a) and the other in eurasian ⁄ oceanian avian ( b, b, b). three lineages identified respectively in na and na are north american avian ( a, a), equine ( b, b), and eurasian avian ( c, c). na was also found to have lineages: north american avian ( a), eurasian ⁄ oceanian avian i ( b), and eurassian ⁄ oceanian avian ii ( c), respectively. in this study, we conducted large-scale phylogeny and evolutionary analyses using influenza viral na sequences. the results showed that divergence between influenza a and b viruses occurred earlier than between any influenza a subtypes. this observation was consistent with previous findings based upon phylogenetic analysis of the ha gene, one of the most important genes related to host infection. within influenza a, two sub-groups were found, one consisting of na , , , and and the other consisting of the rest of five subtypes (na , , , , ) . this observation does not agree with the result described by liu et al., where na subtypes , , , , and formed one group and the remaining four subtypes (na , , , and ) formed the other group. this difference is apparently caused by the fact that an outgroup was not used in their phylogenetic analyses. in the present study, both influenza a and b viral na sequences were included in the analysis. high bootstrap values were obtained for major groups, indicating that the inferred evolutionary relationship should be highly reliable. classification and designation of the lineages and sublineages within the influenza a virus are essential for studies of viral evolution, ecology, and epidemiology. a total of lineages were identified within nine influenza a viral na subtypes and with the majority of the identified lineages found to be host or geographic specific or both. our results demonstrated a comprehensive view for the evolution of na genes and provided a framework for the inference of evolutionary history of pandemic viruses and for further exploring of viral circulations in multiple hosts. for example, the global pandemics of human h n in , h n in , the pandemic of human h n virus in , the crisis of h n hpai in hong kong in , and swine-origin h n influenza in , all can be mapped onto the lineages and sub-lineages identified in this study. such information will facilitate not only identification of known genetic origins but also early detection of novel influenza a viruses. influenza viruses constantly evolve to avoid the human immune pressure in the process of antigenic drift. through sequencing of viral genomes, the rates and direction of virus evolution can be observed. moreover, comparison of protein sequences allows us to determine amino acid substitutions that are related to immune pressure and antigenic drift. the creation of global influenza genetic databases, along with concurrent development of analytical tools, allows the comparison of multiple influenza virus strains. the main aim of this study was to perform antigenic and genetic comparison of pandemic influenza viruses (h n ) isolated during the - pandemic in ukraine and in other countries. nasopharyngeal swabs and autopsy materials collected from infected patients were received from the areas of ukraine. in addition, field isolates of influenza viruses from the ⁄ season and strain specific serum were used for identification by hemagglutinin inhibition assay. influenza viruses were identified and subtyped using real-time rt-pcr analyses using cdc primers and adopted protocols. sequencing was performed in two world health organization (who) influenza collaboration centers (centers for disease control and prevention, atlanta and national institute for medical research, london). hemagglutinin inhibition assay was conducted using chicken and guinea pig red blood cells following standard who protocols. the all ukrainian isolates of influenza viruses, which were isolated in ukraine during august-november , were identified as a ⁄ california the phylogenetic analyses confirmed the evolutionary relationship between ukrainian isolates and viruses from other countries, which were isolated during the first wave of the pandemic. high genetic and antigenic conservation of pandemic influenza viruses from ukraine and other countries also were demonstrated. considering that the emergence of the novel pandemic influenza strain occurred in countries of northern hemisphere during summer, it was very interesting and significant tracking the dynamics of genetic changes in influenza viruses, which were isolated at the beginning of epidemic and those isolated during the rise of the epidemic in ukraine. influenza a virus causes moderate to severe epidemics annually and catastrophic pandemics sporadically. due to the evasiveness of the influenza virus and the nature of its genome (eight single-stranded and negative-sense rna segments), it is essential to understand the evolution of this important pathogen. influenza virus evolves by two major mechanisms: mutation and reassortment. antigenic and genetic analyses have revealed partially the molecular dynamics of influenza virus evolution. , however, important questions, such as how many genotypes in the influenza a virus, remain unanswered. one of the major issues pertaining to this genotyping problem is how many lineages or sub-lineages can be determined for a subtype and according to what criteria. because of the unique structure of the influenza a viral genome, the computational genotyping methods developed for other viruses cannot be applied to the influenza virus. constructing phylogenetic trees is a powerful technique for the identification of evolutionary groupings (i.e., lineages ⁄ clades). however, for large trees, it is hard to determine how many lineages and the boundaries for each lineage. in this regard, multivariate analysis methods, such as multidimensional scaling (mds) and model-based hierarchical clustering, both taking advantage of dimension reduction and visualization, can complement conventional phylogenetic methods. hemagglutinin (ha), the fastest evolving segment, is recognized as the most important gene in the influenza virus that plays a key role in viral pathogenesis. however, we have only limited knowledge of lineages and sub-lineages occurring in the hemagglutinin (ha) gene of influenza a virus, although much effort has been made in assigning clades or sub-clades in highly pathogenic avian influenza (hpai) virus ha. in this study, both model-based hierarchical clustering and phylogenetic methods were used for sequence analysis. one objective for this study is to explore and develop a more accurate lineage approach for further comprehensive influenza lineage and genotype analyses. a total of hemagglutinin (ha) sequences (approximately nucleotides long), excluding laboratory recombinant sequences, were downloaded from genbank as of march, . sequences were aligned with muscle and mafft. the genetic distance matrix of all pairwise sequences was computed using the k p model under mega . . we then used the distance matrix as input to the cmdscale module in r . . for the mds analysis. the principle coordinates resulting from mds were used for the model-based hierarchical clustering analysis, again in r . . (the r foundation. available at: http://www.r-project.org/). the bayesian information criterion (bic) values were computed based upon ten different statistical data models -eii, vii, eei, evi, vei, vvi, eee, eev, vev, and vvv. the highest bic value was used to determine the number of clusters in the given sequence data. phylogenetic analysis was conducted using maximumlikelihood (ml) in raxml. raxml uses rapid algorithms for bootstrap and maximum likelihood searches and is considered one of the fastest and most accurate phylogeny programs for large-scale sequence analysis. all the analyses were conducted on the supercomputer cluster (holland computing center, http://hcc.unl.edu/main/index.php). the trees were visualized in figtree (version . . ) . lineages and sub-lineages were determined based on both the topology of the ml trees and model-based clustering results. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, with the following modifications: lineage analysis was conducted for each ha subtype, which agrees with the convention of influenza virologists that ha subtypes were identified in influenza a virus; ha lineages are represented with digits and letters, where the digit(s) represent one of the subtypes and a letter represents a lineage; here, we present sub-lineages or sub-sub-lineages also in digits, with smaller numbers representing earlier lineages or sub-lineages within the same subtype (e.g., lineage occurs earlier than lineage ); the digit is used to indicate inclusion of ancestral viruses in a lineage (or sub-lineage); a dot is used to separate lineages, sub-lineages, and sub-sub-lineages. for example, a. ae means ha subtype, lineage a, and sub-lineage , and sub-sub-lineage . the sub-lineage level can be extended as necessary. the model-based clustering method corroborates commonly used phylogenetic methods in lineage and sub-lineage assignment. here we use the h subtype as an example to show the lineage and sub-lineage assignment. the bayesian information criterion (bic) reaches its maximum when the number of clusters for h equals , regardless of which mode we choose ( figure a ). therefore, based on bic, the optimal number of clusters for the h subtype is . as a result, a total of clusters based upon the vvv model were identified ( figure b) . a significant correlation was found in lineage assignments by the phylogenetic method and the model-based hierarchical clustering method ( figure b,c) . lineages a and b were identified for h , which correspond to north american avian and eurasian avian, respectively. lineage a was further divided into sub-lineages, a. , a. , where a. is the ancestral sub-lineage in a. based on both model-based hierarchical clustering and phylogenetic analyses, a total of distinct lineages were identified among subtypes, averaging out to be ae lineages per subtype ( table ). the majority of the identified lineages were found to be host or geographic specific or both. for example, three lineages, a, b, and c, were identified for ha . lineage a was further divided into two sub-lineages, a. and a. . the a. is swine-specific, whereas a. is a human pandemic h n sub-line- how to accurately identify an evolutionary lineage of influenza a viruses is challenging. one commonly used approach is molecular phylogeny, where phylogenetic trees are constructed, and the tree topology is used for lineage determination. here, we used a bayesian model-based clustering method, along with phylogenetic methods, to decide lineages and sub-lineages of influenza a viruses based upon sequence data. the results demonstrated that the modelbased clustering method corroborates phylogenetic methods and increases the accuracy of lineage assignment. one salient feature of this study is its large-scale analysis of all available influenza a hemagglutinin sequences. a total of distinct lineages and sub-lineages were classified; the majority of them were found to be host or geographic specific. this observation agrees largely with previous findings. we are conducting further analyses of other influenza a segments and expect to identify their lineages and create a comprehensive genotypes database for all influenza a viruses. such information will allow us to detect the genetic origin of newly found viruses, track their genetic changes, and identify potential genome reassortments. a hierarchical nomenclature system has been proposed and adopted for hpai ha clades and sub-clades by who influenza surveillance centers. wan et al. also proposed a hierarchical approach for influenza a viral genotypes system. the work presented here is one of the first steps towards the development of a nomenclature system for influenza a virus lineages (at the segment level) and genotypes (at the genome level). whether the naming system will be accepted and used by the influenza research community is more challenging than the lineage analysis itself. identification of the genetic origins of influenza a viruses will enhance our understanding the evolution and adaptation mechanisms of influenza viruses. the phylogenetic analysis is the traditional approach to identify the influenza progenitor. first, the nucleotide sequences are aligned using multiple sequence alignment methods, such as clustalw, muscle, and t-coffee. second, phylogenetic analysis is performed on these aligned sequences to infer their evolutionary relationship using neighbor-joining (nj), likelihood, or bayesian inference. bootstrap analyses or computation of posterior probability are usually applied to estimate the phylogenetic uncertainty. however, this phylogenetic analysis is time consuming due to intensive computations in multiple sequence alignments and phylogenetic inferences. it is difficult to perform an analysis using this method on a large dataset, for instance, with more than taxa, as is the common case for influenza studies. alternatively, blast is applied to identify the prototype genes in the database. blast determines a similarity by identifying initial short matches and starting local alignments. since influenza viral sequences have very high similarities, especially for most conserved regions, blast usually generates a large number of outputs, which will not be helpful for progenitor identification. since blast is a local sequence alignment, the results from blast may not reflect the global evolutionary information between the sequences. the blast scores cannot be used to define the evolutionary relations between viruses, especially in the context of the entire genetic pool. recently, we have developed a distance measurement method, complete composition vector (ccv), that can calculate genetic distance between influenza a viruses without performing multiple sequence alignments. , we also adapted the minimum spanning tree (mst) clustering algorithm for influenza reassortment identification. the application of this approach in the analyses of pb genes of influenza a virus showed that the integration of ccv and mst allows us to identify the potential progenitor genes rapidly and effectively. based on these results, here we develop a webserver called ipminer for influenza progenitor identification. ipminer can identify potential progenitors for a query sequence against all public influenza datasets within a few minutes. in order to improve the computing efficiency, distance matrices were pre-computed by ccv, and they include for ha (h to ), for na (n to n ), and one for each of the internal gene segments (pb , pb , pa, np, ns, and mp). these pre-computed matrices will be updated weekly. ipminer just needs to compute the query matrices for a query sequence and sequences in the database. the standalone ccv program is also available at http://sysbio.cvm.msstate.edu/ipminer. in order to identify the influenza progenitor genes, ipminer first integrates the query matrix and a corresponding pre-computed matrix into a full distance matrix, which is then clustered by mst clustering algorithm. we adapted the threshold we measured previously in mst, u + nr, where u is the average distance and r is the standard deviation of a cluster. as a result, mst will generate a hierarchical structure for the clusters. in each cluster, we will randomly select viruses or % of the cluster size if this cluster has more than viruses. ipminer will return the viruses with the smallest distances when the search reaches to the lowest level (the largest n) in this hierarchical structure. our analyses have shown that the level has generally yielded good results for influenza a viruses. to visualize the overall mst structure, ipminer applies multi-dimensional scaling (mds) method to project all the viruses in the genetic pool onto a two dimensional graph, and the precursor viruses are marked in different shapes ( figure ). the users can select other prototype viruses from the graph for further phylogenetic analyses. a single job with one query sequence takes < min. the genbank identifiers and associated genetic distances and sequence identities are displayed. the users can download the sequences for the identified precursor viruses as well as those from the prototypes viruses. in addition, for the users' convenience, ipminer generates a phylogenetic tree using nj method implemented in phylip to illustrate the phylogenetic relationship among the query sequence(s), the identified progenitors, and the selected prototypes viruses. the programs in this solution package are written in java. the shell scripts are written in korn shell script in order to achieve high performance. cascading style sheets (css) are used for a consistent look across the pages. this also enables to change the overall design just by replacing the css definition file. php has been used as server side scripting and is written in java. in order to achieve high performance for computing in a genomic scale, we apply hash function or a binary tree, which enables that the precursor identification has a time complexity of o(n). for single queries, the users can visualize the results online. for batch queries of multiple sequences, the results will be sent to the users by e-mail. ipminer has been tested on microsoft internet explorer, mozilla firefox, and safari. the users need javascript to obtain full function of ipminer server. the webserver is available at http://sysbio.cvm.msstate.edu/ipminer. in summary, ipminer webserver has three major computational features for influenza progenitor identification: (i) it calculates the genetic distances through ccv and identifies the viruses with the shortest ccv distances against the query virus to be the progenitor genes; (ii) it projects influenza viruses onto a two dimensional map, which illustrates the global relationship between the progenitor genes and other viruses in the genetic pool; and (iii) it performs phylogenetic analyses between the query virus, the identified progenitor genes, and other selected prototype viruses. ipminer provides a user friendly web service for influenza progenitor identification in real time. the gisaid initiative offers an alternative to current public-domain database models in response to growing needs of the global influenza community for the sharing of genetic sequence and associated epidemiological and clinical data of all influenza strains. gisaid's publicly accessible epifluÔ database is governed by a unique sharing mechanism that protects the rights of the submitter, while permitting ongoing research as well as the development of medical interventions, such as drugs and vaccines. for the gisaid initiative, the max planck institute for informatics (mpii) saarbrücken, germany, has developed a web portal that is accessible at http://www.gisaid.org featuring the gisaid epifluÔ database that offers a unique collection of nucleotide sequence and other relevant data on influenza viruses. the database is based on software by oracle and the dante Ò system by a systems gmbh, germany. extensive metadata are also collected for most isolates. the database provides features for searching, filtering specific datasets for download, and user friendly upload functionality. to uphold gisaid's unique sharing mechanism, all users must positively identify themselves. while access is free of charge, all users agree that they will not attach any restrictions on the data, but will acknowledge both the originator of the specimen and the submitter of the data, and seek to undertake to collaborate with the submitter. all uploaded sequence data are submitted to rigorous curation by the friedrich-loeffler-institute for animal health (fli), germany. the database has been live since september , . among its contributors are all five who collaborating centers for influenza who routinely contribute data in addition to using the epifluÔ database for their semiannual vaccine strain selection. to provide a complete picture of data, all data available in the public domain is routinely imported. as of october , , the rapidly growing gisaid dataset comprises nucleotide sequences (from isolates) with (from isolates) uniquely submitted to this database. software development is underway to continually extend the spectrum of available data analysis tools. the intergovernmental process of the nd world health assembly specifically mentions gisaid as a publicly available database for depositing virus sequence data. starting in , germany's federal ministry of food, agriculture and consumer protection will be the long-term host of the gisaid platform. the mpii will continue to develop the portal and database software and enable gisaid to act as a catalyst for the development of advanced bioinformatics software connected directly to the database. gisaid has become an indispensible resource for the international scientific community on influenza. the consortium will expand its activities and offers to catalyze research and development on a wide variety of issues pertaining to risk analysis, drug development, and therapy of influenza. options for the control of influenza vii ª blackwell publishing ltd, influenza and other respiratory viruses, (suppl. ), - the pandemic h n virus emerged in and spread rapidly throughout the world, principally affecting children and young adults. as this virus is new to the human population, it is important to determine if these influenza infections are more commonly associated with other respiratory pathogens compared to previously circulating influenza strains. co-infecting respiratory viruses may cause increased morbidity in individuals with pandemic h n , and may also be unwanted contaminants in influenza vaccines if original clinical samples containing these adventitious viruses are used to directly inoculate certified cell lines for vaccine production. to examine this issue, stored rna from original clinical samples (nasal swabs, nasal aspirates, throat swabs) from australian and new zealand subjects that were collected in that were positive for pandemic h n and samples collected in that were positive for seasonal influenza by real time pcr assay (using the cdc, usa kits), were subjected to a resplex ii -panel version . (qiagen) pathogen screen. the resplex ii assay detects common respiratory viruses, such as respiratory syncytial viruses (rsv a, b), influenza a and b viruses, parainfluenza viruses (piv - ), human metapneumo-viruses (hmpv), coxsackieviruses ⁄ echovirus (cvev), rhinoviruses (rhv), adenoviruses (adv b, e), coronaviruses (nl , hku , e, oc ), and bocaviruses. resplex ii uses a combination of multiplex rt-pcr, hybridization of pcr onto target specific beads followed by detection using luminex-xmap technology. original clinical samples were received at the center from who national influenza centers, who influenza collaborating centers, and other regional laboratories and hospitals from australia, new zealand, and the asia ⁄ pacific region. most samples were from australia and new zealand. these samples consisted of nasal swabs, nasopharyngeal swabs, nasal washes, throat washes, and throat swabs. all samples were stored at ) °c until rna was extracted. rna was extracted from ll of clinical sample using either the magnapure extraction system (roche, australia) or the qiaxtractor system (qiagen, australia) according to the manufacturer's recommendations with an elution volume of ll and stored at ) °c until used. a ll aliquot of rna was used to amplify the selected influenza virus gene using specific primers and probes as supplied by cdc (atlanta, usa) along with super-script iii platinum one-step rt-pcr reagents (invitrogen, australia). real time pcr detection was performed on a fast system with sds software (applied biosystems, ca, usa). a cut off of a cycle threshold (c t ) of or below was considered positive. resplex ii panel ver . detection the qiagen molecular differential detection (mdd) system was used, which combines qiaplex amplification (multiplex rt-pcr) with detection on the liquichip workstation (luminex's xmap microsphere based multiplexing system) and qiaplex mdd software according to the manufacturer's instructions. a low level cutoff was used ( ) to obtain maximum sensitivity. from the clinical specimens that were positive for influenza from by real time pcr, there were ( %) a(h n ) seasonal influenza viruses, ( ae %) a(h n ) viruses, ( %) b viruses, and ( ae %) viruses which were influenza a positive, but could not be typed. clinical samples from selected to study were all influenza a(h n ) pandemic positive by real time pcr. detection of influenza virus in respiratory samples was much lower with the resplex ii assay (using a low cut off of units) for pandemic influenza a virus ( ⁄ ; sensitivity ae %) and to a lesser extent for seasonal influenza a ( ⁄ ; sensitivity of ae %) and b viruses ( ⁄ ; sensitivity of ae %) when compared to real time pcr. there were relatively few co-infecting respiratory viruses with either pandemic h infections in ( ae %) or seasonal influenza infections in ( ae %) ( table ). the most common dual infection seen with pandemic h n viruses and seasonal b viruses was with cvev ( ⁄ ; and ⁄ ; , respectively) while for a(h ) viruses there were no dominant co-infecting viruses ( table ). in one case was detected with three respira- tory pathogens in the same sample, a year old female who had pandemic h n , cvev, and rhv, and in a seasonal influenza sample, one case with a triple infection was detected (bocavirus, piv and influenza b). the median age of subjects with co-infections was younger for both pandemic h n with a median age of years (range: months to years), compared to the full sample set which had a median age of years (range: months to years), while for the patients from with seasonal influenza viruses with co-infections they had a median age of ae years (range: months to years) compared to all samples which had a median age of years (range: months to years). there was good concordance in detecting influenza a and b in respiratory samples collected in between real time rt-pcr and the resplex ii system ( % versus > ae % for seasonal influenza a and b respectively). this data compares well with other studies such as li et al. who found that resplex ii had ae % sensitivity and % specificity for seasonal influenza a viruses and ae % sensitivity and % specificity for influenza b viruses. in contrast, the present study found only ae % sensitivity for the resplex ii detection of influenza a with the samples that were positive for pandemic h n by real time rt-pcr. a recent study by rebbapragada et al. also showed lower sensitivity for pandemic h n viruses in nasopharyngeal samples with the resplex ii system ( % sensitivity and % specificity) compared to other commercial platforms seeplex rvp ( % sensitivity and % specificity) and luminex rvp ( % sensitivity and % specificity). interestingly the latest version of the resplex system offered by qiagen the resplex ii plus panel ruo now has a separate target for the pandemic h n virus (mexico ). in terms of detection of other respiratory viruses such as piv- , piv- , rsv and hmpv, high sensitivities ( ae %, ae %, ae %, and %, respectively) and specificities ( ae - %) compared to taqman rt-pcr have been reported from testing of nasal wash and nasopharyngeal clinical samples. in both the seasonal influenza positive and the pandemic h n positive (by real time rt-pcr) clinical specimens, few other respiratory viruses were detected. only of the samples had another virus detectable and one had two other viruses, while in out had another virus and one had two other viruses detected from a total of influenza virus positive samples collected in each year. enteroviruses, coronaviruses, and parainfluenza viruses were most often found with both seasonal and pandemic infections. younger age appeared to be associated with co-infections with those subjects in with dual infections having a median age of only years compared to the study groups years; and similarly for , the median age for subjects with dual infections was only ae years compared to the study groups' median age of years. a study by chong et al. on nasopharyngeal swabs collected during - using resplex ii and luminex xtag rvp fast, they found dual respiratory virus infections in ⁄ ( ae %) of samples and only ( ae %) with triple respiratory viral infections; however, these were from cases with any combination of multiple respiratory viruses not necessarily influenza, although influenza positive cases were the most common respiratory virus detected ( ae % of all positive samples). given the low level and variety of viral co-infections along with both seasonal and pandemic influenza seen in this study, it is unlikely that influenza infections predispose subjects to particular respiratory viruses, but may still allow bacterial colonization, such as has been seen with severe and fatal cases with pandemic h n with various bacteria including streptococcus pneumoniae, streptococcus pyogenes, staphylococcus aureus, or haemophilus influenzae. , low levels of other respiratory viruses along with the finding that certain cell lines (like the mdck -cells used in this study) do not propagate a number of these viruses (e.g. rsv a and b, rhinoviruses, coronaviruses), but do propagate others (e.g. parainfluenza ) should make testing for unwanted viruses that might be co-isolated with influenza viruses more focused and hence easier to detect and eliminate this isolate for future vaccine production. global influenza surveillance is one of the most important approaches to combat spread of disease. current laboratory methods for characterizing influenza are time-consuming and labor-intensive, and few viral strains undergo full characterization. even fewer strains from domestic poultry and swine or from wild aquatic birds are wellcharacterized. these strains are important for global surveillance since they are thought to be the precursors to pandemic influenza strains. we have designed a highthroughput global bio laboratory to address these surveillance needs. the goal of this project was to develop highspeed and high-volume laboratory capabilities for extensive surveillance and rapid and accurate detection and analysis of influenza. the workflow consists of surveillance, sample transportation, laboratory testing, data management and analysis. five robotic systems have been designed for this laboratory: sample accessioning, biobanking, screening, viral culture, and sequencing. sample accessioning logs barcodes, centrifuges, and aliquots samples are then sent to biobanking. the robotic biobank stores samples at ) °c and reformats tubes for screening. the screening system extracts rna and confirms the presence and subtype of influenza. aliquots of positive samples are sent to the viral culturing system for scale-up. finally, cultured samples are extracted and sent to the sequencing system for full genome sequencing. the sample accessioning, sequencing, and biobanking systems have been built, delivered, and validation processes are currently being completed. robotic screening and culturing systems have been fully designed and are ready to be built. a biosafety level -enhanced containment laboratory was built to enable the flow of samples containing highly pathogenic avian influenza viruses. in full operation, this approach to surveillance is designed to enable the sequencing of up to full virus genomes per year, more than the total of all full influenza genomes sequenced to date. the design of a robotic laboratory for influenza surveillance presents unique challenges and opportunities. before a robotic system is built, each assay is worked out on the bench top, each movement of the plates and reagents is defined, and the laboratory information management system (lims) must be able to address each step of the process. alternate assays are conceived for processes that are not automation-friendly. waste streams, worker safety, and space constraints are considered. each possibility is taken to reduce processes that have the potential to aerosolize or cross-contaminate influenza samples. instruments must be found that fit the capabilities needed. detailed specifications for each of the robotic systems were written including all the parameters listed above. once the systems are built, a long validation process takes place where the processes and instruments in each system are adjusted to function together properly. finally, a validation study is performed to ensure that the system is able to produce useful data for influenza research. the entire process takes months from start to finish for each robotic system and requires complete cooperation from a diverse team of researchers. the accessioning system logs initial sample information with the lims system. samples arrive in barcoded cryotubes. the liquid handler brings all samples up to a common volume and clarifies samples by centrifugation. samples are then transferred from screw-cap sample vials into storage plates containing individually punchable storage tubes. each tube ( ae ml) is individually identifiable with a d barcode on the bottom. six archive aliquots are made, and tubes are individually weld-sealed for storage. tips for aspiration are fixed and undergo a high-pressure plasma process between each use to sterilize tips and destroy nucleic acids. samples are stored at ) °c. each module has a capacity of remp plates or $ samples. the automated freezer system can assemble requested samples as -well plates while samples remain frozen. the screening system uses magnetic bead extraction chemistry, real-time pcr, and a liquid handling system to extract samples, confirm and quantify the presence of influenza, and reformat extracted samples for input into the sequencing system. serotype of human influenza samples will be performed by real-time pcr. many samples will not have enough material for further analysis and will need to be scaled up. the culturing system combines incubators, a liquid handling platform, plate reader, and real-time pcr to culture, monitor growth, harvest, and quantify influenza. when the system is not being used for culture and scale-up, it can be used to assay previously cultured influenza samples for drug resistance. a challenge to sequencing large numbers of influenza samples is the manpower required for sample preparation. the sequencing system has the capacity to prepare up to samples for sequencing per year for sanger sequencing. sanger sequencing was chosen because it is well-established for influenza surveillance, and automation-friendly. the system is designed to work with multiple primer sets ( , , ) . robotic systems all report to the lims. each process completion, plate movement, and data point are entered and checked by an online, web-based lims. status updates, notification, reporting, and data analysis can be achieved without entering the bsl containment facility. routine data analysis such as determining whether a cultured sample is ready to be harvested will be performed by the lims. complex data analysis, while still requiring significant human input, will be made easier by the data-acquisition functions of the lims. the implementation of a high-throughput influenza surveillance laboratory will provide an influenza research and response capacity that far exceeds what is available today. with the addition of each new system, we add a new capability to the influenza community and new opportunities to foster partnerships and collaborations with government, foundations, businesses, and academic institutions. this laboratory will not only enable cutting edge research, but will also enable a more effective response of near real-time surveillance during a pandemic outbreak. pandemics of and were believed to arise from avian influenza viruses. the tropism of avian and human seasonal influenza viruses for the human lower respiratory tract deserves investigation. the target cell types that support replication of avian influenza a viruses in the human respiratory tract in the early stages of clinical infection have not well defined. in a previous autopsy studies of human h n disease, influenza a virus were found to infect alveolar epithelial cells and macrophages. in this study, viral infectivity and replication competence of human and high and low pathogenic avian influenza viruses were systematically investigated in the human conducting and lower respiratory tract using ex vivo organ cultures. we compared the replication kinetics of human seasonal influenza viruses (h n and h n ), low pathogenic avian influenza viruses (h n , h n ) with that of the highly pathogenic h n viruses isolated from human h n disease. a range of human seasonal influenza a viruses of subtypes h n and h n viruses were included in this study from to . two isolates of low pathogenic avian influenza a (lpai) (h n ) viruses from different virus lineages isolated from poultry in hong kong in , a low pathogenic influenza a (h n ) virus isolate from wild ducks in hong kong in , and two virus isolates of highly pathogenic avian influenza (hpai) a subtype h n were included. fragments of human bronchi and lung were cut into multiple - mm fragments within hours of collection and infected in parallel with influenza a viruses at a titer of tcid ⁄ ml and as control cultures were infected with ultraviolet light inactivated virus. these tissues fragments were infected for hours and washed twice with pbs and incubated for , , and h at °c. the bronchial tissue was cultured in an air-liquid interface using sponge. viral yield was assessed by titration in mdck cells. one part of the infected tissue were fixed in formalin and processed for immunohistochemistry for influenza antigen. other part of infected tissue was homogenized and underwent rna extraction, and the expression of influenza virus matrix gene was measured by quantitative rt-pcr. human bronchus ex vivo cultures supported human seasonal influenza virus to replicate efficiently. avian influenza h n virus replicated, although less efficiently than that of seasonal influenza viruses, whereas hpai h n did not productively replicate in ex vivo cultures of human bronchus. this is in agreement with our previous finding in the well-differentiated bronchial epithelial cells in vitro. on the other hand, human lung ex vivo cultures supported prominent productive replication of human seasonal influenza h n ( figure a ) and hpai h n ( figure f ) viruses. lpai, such as h n ( figure c -d) and h n ( figure e ), also replicated productively, but with a lower viral yield. surprisingly, the replication of human influenza h n viruses ( figure b ) across the last three decades was greatly inhibited. there are clear differences in viral tropism of human seasonal and avian influenza viruses for replication in the human bronchus and lung. hpai h n virus can infect and productively replicate in the lower lung, which may account for the severity of human h n disease, but not in the conducting airways. surprisingly, there are marked differences in the replication competence of seasonal influenza viruses in ex vivo lung tissues, with influenza h n viruses being able to replicate efficiently while h n viruses do not. this may be related to the more strict siaa - gal binding preference of h n viruses. on the other hand, the efficient replication of influenza h n viruses in the alveolar spaces indicates factors other than tissues tropism alone play a role in the differences in disease severity between human seasonal h n and avian h n virus infections. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells, suggesting that viral protein(s) other than m gene-translational products facilitates the splicing of viral mrnas. the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns (c ⁄ ns ) and ns (c ⁄ ns ), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns and c ⁄ ns proteins, drastically reduced the splicing of ns mrna, raising the possibility that c ⁄ ns or c ⁄ ns enhances the splicing of viral mrnas. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns , whereas it was reduced by co-expression of influenza a virus ns protein (a ⁄ ns ). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns , whereas it was inhibited by that of a ⁄ ns . these results suggest that influenza c virus ns , but not a ⁄ ns , can up-regulate the splicing of viral mrnas. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. , the inefficient splicing of viral pre-mrnas can be understood partly by the fact that influenza a virus ns protein is associated with spliceosomes and inhibits pre-mrna splicing. , cis-acting sequences in the ns transcript also negatively regulate splicing. by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. the yamagata ⁄ ⁄ strain of influenza c virus was grown in the amniotic cavity of -day-old embryonated hen's eggs. cos- and t cells were cultured in dulbecco's modified eagle's medium containing % fetal calf serum. subconfluent monolayers of cos- cells were transfected with pme s containing influenza c virus m gene cdna using the lipofectamine procedure and then incubated at °c. total rna was extracted from both the transfected cells and cells infected with c ⁄ yamagata ⁄ ⁄ virus using the rneasy mini kit (qiagen). ribonuclease protection assay was performed using a ribonuclease protection assay kit rpa iii (ambion). briefly, a [ p]-labeled influenza c virus rna -specific rna probe (vrna sense) was synthesized by in vitro transcription and hybridized with the total rna at °c overnight. hybrids were digested with rnase a ( ae u) and rnase t ( u) at °c for minutes and then analyzed on a % polyacrylamide gel containing m urea. hmv-ii cells infected with c ⁄ yamagata ⁄ ⁄ and cos- cells transfected with pme s expressing influenza c virus ns were fixed with carbon tetrachloride at various times after infection and transfection, respectively. the cells were then stained by an indirect method using anti-gst ⁄ ns serum as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit igg (seikagaku kogyo) as the secondary antibody. the splicing efficiency of influenza c virus m gene-specific mrna (m mrna) in infected cells was higher than that in m gene-transfected cells the ratio of m encoded by a spliced m mrna to cm encoded by an unspliced m mrna in influenza c virusinfected cells was about times larger than that in m gene-transfected cells. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells (figure ). these data suggest that viral protein(s) other than m gene-translational products facilitates viral mrna splicing. the influenza c virus ns gene translational product may up-regulate the splicing of viral mrnas the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns (c ⁄ ns ) and ns (c ⁄ ns ), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns and c ⁄ ns proteins, drastically reduced the splicing of ns mrna, suggesting that c ⁄ ns or c ⁄ ns enhances viral mrna splicing. immunofluorescent staining showed that ns localized in the nucleus in the early phase of infection, and was distributed in both the nucleus and cytoplasm in the late phase of infection, raising the possibility that influenza c virus ns protein plays a role in viral mrna splicing that occurs in the nucleus. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns , whereas it was reduced by co-expression of influenza a virus ns protein (a ⁄ ns ) (figure a ). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns , though it was inhibited by that of a ⁄ ns ( figure b ). these results suggest that influenza c virus ns , but not a ⁄ ns , can up-regulate the splicing of viral mrnas. in influenza a virus-infected cells, splicing is controlled so that the steady-state amount of spliced mrnas is only - % of that of unspliced mrnas. , the mechanisms by which influenza a virus ns pre-mrnas are poorly spliced have been investigated and the following confirmed. influenza a virus ns protein associates with spliceosomes and inhibits pre-mrna splicing. , two cis-acting sequences in the ns transcript (positions - in the intron and positions - in the ¢ exon region) inhibit splicing. by contrast, influenza c virus m gene-specific mrna (m mrna) is efficiently spliced in influenza c virus-infected cells. in this study, we examined the mechanism by which influenza c virus m mrna is efficiently spliced and the regulatory mechanism of the splicing of ns gene-specific mrna (ns mrna). the introduction of a translational pre-mature termination into the influenza c virus ns gene, thereby blocking the synthesis of influenza c virus ns (c ⁄ ns ) and ns (c ⁄ ns ) proteins, drastically reduced the splicing rate of ns mrna. we further examined whether c ⁄ ns potentially facilitates viral mrna splicing. the splicing rate of m mrna of influenza c virus was increased by co-expression with c ⁄ ns , whereas it was reduced by co-expression with influenza a virus ns protein (a ⁄ ns ) (figure a ). the splicing of influenza a virus m gene-specific mrna was also increased by co-expression with c ⁄ ns , though it was inhibited by co-expression with a ⁄ ns ( figure b ). these results suggest that influenza c virus ns can facilitate viral mrna splicing, but in no way inhibit it, which is in striking contrast to the inhibitory effect of influenza a virus ns on pre-mrna splicing. , the mechanism for splicing enhancement by c ⁄ ns also remains to be determined. we speculate that c ⁄ ns may interact with some host proteins involved in splicing, thereby leading to an up-regulation in splicing, or that c ⁄ ns may bind to pre-mrna, increasing its accessibility to the spliceosome. the spliced mrna of the influenza c virus m gene encodes the m protein, which plays an important role in virus formation and determines virion morphology. , therefore, it is speculated that the mechanism for efficient splicing of m mrna, which provides the m protein necessary for virus assembly in a redundant amount, has been maintained in the influenza c virus. by contrast, unspliced mrna from the influenza c virus m gene encodes the cm ion channel, which is permeable to chloride ions, and also has ph-modulating activity. although the role of the influenza c virus cm ion channel in virus replication remains to be determined, it is conceivable that the over-expression of the cm protein has a deleterious effect on virus replication since the fact that a high level of influenza a virus m protein expression inhibits the rate of intracellular transport of the influenza a virus ha protein and other integral membrane glycoproteins has been demonstrated. if this is the case, efficient splicing of m mrna may control the amount of cm synthesized to optimize virus replication. therefore, we speculate that efficient splicing of m mrna leads to a high level of m expression and the reduced expression of cm , thereby creating conditions that are optimal for virus replication. in this study, we provided evidence that c ⁄ ns facilitates the splicing of m mrna. furthermore, c ⁄ ns may regulate the splicing efficiency of its own ns mrna during infection, controlling the amount of c ⁄ ns and c ⁄ ns proteins in infected cells. c ⁄ ns plays an important role in the nuclear export of vrnp, and is also associated with vrnp in the later stages of infection in virus-infected cells and is incorporated into virions, suggesting that c ⁄ ns is involved, not only in the sorting of vrnp into the assembly site, but also in virus assembly. therefore, it is likely that there is a mechanism by which an appropriate amount of c ⁄ ns is provided during infection to accomplish these functions. in conclusion, c ⁄ ns , which enhances the splicing of viral mrna, may regulate both the expression level of m gene-derived m and cm proteins, and that of ns gene-derived ns and ns proteins, thereby leading to optimal virus replication. propagation of the human influenza viruses in embryonated hen's eggs always results in a selection of variants with amino acid substitutions in the hemagglutinin (ha) that affect viral receptor-binding characteristics (reviewed ). brookes et al. recently studied infection in pigs using the egg-grown virus that contained a mixture of the original a ⁄ california ⁄ ⁄ (h n pdm) and its two egg-adaptation mutants with single amino acid substitutions d g and q r ( and in h numbering system). only the original virus and the variant with g were detected in the directly inoculated animals, indicating that the variant with r failed to infect. only the original virus was detected in nasal secretions of contact infected pigs, suggesting that the d g mutant failed to transmit. in contrast, there was an apparent selection of the d g mutant in the lower respiratory tract samples from directly inoculated pigs. the d g substitution is of a special interest as it can emerge during virus replication in humans and was associated with severe and fatal cases of pandemic influenza in - - and . here we compared phenotypic properties of the original clinical isolate of h n pdm virus a ⁄ hamburg ⁄ ⁄ and its d g and d r mutants to explain observed effects of these mutations on virus replication in swine and to predict their potential effects on virus replication in humans. a ⁄ hamburg ⁄ ⁄ (ham) was isolated from clinical material by two passages in mdck cells. the virus was passaged twice in -day-old embryonated hen's eggs and plaqued in mdck cells. the plaques were amplified in mdck cells and the sequences of the viral ha were determined. the variants with single mutation d g and q r were aliquoted and designated ham-e and ham-e , respectively. the receptor-binding specificity of the viruses was assessed by assaying their binding to desialylated-resialylated peroxidase-labeled fetuin containing either a - -linked sialic acid ( - -fet) or a - -linked sialic acid ( - -fet). in brief, viruses adsorbed in the wells of -well eia micro plates were incubated with serial dilutions of - -fet or - -fet, and the amount of bound fetuin probe was quantified by peroxidase activity. the binding data were converted to scatchard plots (a ⁄ c versus a ), and the association constants of the virus-fetuin complexes were determined from the slopes of these plots. viral cell tropism and replication efficiency in human airway epithelium were studied using fully differentiated cultures of human tracheo-bronchial epithelial cells (htbe). , to determine cell tropism, cultures were infected at a moi , fixed hours after infection, and double immuno-stained for virus antigen and cilia of ciliated cells. infected cells were counted under the microscope ( · objective with oil immersion) in the epithelial segment that included - consecutive microscopic fields containing between % and % ciliated cells relative to the total number of superficial cells. percentages of infected ciliated cells and infected non-ciliated cells relative to the total number of infected cells were calculated. ten segments per culture were analyzed and the results were averaged. to compare growth kinetics of ham and ham-e, replicate htbe cultures were infected with plaque-forming units of the viruses followed by incubation at °c under airliquid interface conditions. at , , and hours postinfection, we added dmem to the apical compartments of the cultures and incubated for minutes at °c. the apical washes were harvested, stored at ) °c, and analyzed simultaneously for the presence of infectious virus by titration in mdck cells as described previously. the non-egg-adapted h n pdm virus ham, similarly to the seasonal human virus a ⁄ memphis ⁄ ⁄ (h n ), bound to - -fet ( figure a ) and did not show any significant binding to - -fet. this result contrasted with the binding of h n pdm viruses to several - -specific probes in carbohydrate microarray analysis. reduced avidity of virus interactions with soluble glycoprotein in solution as compared to its binding to the probe clustered on the microarray surface could account for these differences in the assay results. the d g mutant ham-e differed from the parent virus by its ability to bind to -fet and by its reduced binding to -fet. the q r mutant only bound to - -fet, although less strongly than did the avian virus a ⁄ duck ⁄ alberta ⁄ ⁄ (h n ). the viral cell tropism in htbe cultures ( figure b ) correlated with receptor specificity. ham and mem ⁄ showed a typical human-virus-like tropism , with preferential infection of non-ciliated cells (< % of infected cells were ciliated). the mutant with r and control duck virus displayed a typical avian-virus-like tropism (preferential infection of ciliated cells). the d g mutant displayed a cell tropism that was intermediate between those of human and avian viruses; in particular, this mutant infected significantly higher proportion of ciliated cells than ham and mem ⁄ . observed alteration of receptor specificity and cell tropism ( figure ) suggested that egg-derived mutations can affect replication of the h n pdm virus in human airway epithelium. to test this, we first compared the capacity of the viruses to initiate infection in htbe cultures. replicate cultures were infected with identical doses of the viruses, fixed hours post-infection, and immuno-stained for viral antigen. under these conditions, ham and ham-e infected comparable numbers of cells, whereas ham-e infected at least times less cells (data not shown). this result indicated that the mutation q r markedly impaired the ability of ham-e to infect human airway epithelial cultures. we next compared two other viruses ham and ham-e for their multi-cycle replication in htbe cultures and found that the original virus reached threefold higher peak titers hours post infection than did the d g mutant ( figure ). the d g mutation in h n pdm virus facilitates virus binding to - -linked receptors and alters viral cell tropism in human airway epithelium. these changes could account for increased replication of the d g mutant in the lower respiratory tract in humans - and pigs and correlation of this mutation with severe pulmonary disease. [ ] [ ] [ ] [ ] [ ] the d g mutant replicates less efficiently in human airway cultures than the original virus. this finding correlates with an apparent lack of transmission of variants with g in humans and pigs. egg-derived mutation q r abolishes virus binding to - -linked receptors and strongly decreases infection in cultures of human airway epithelium. this result agrees with poor infectivity of the q r mutant in pigs and highlights potential pitfalls of using egg-adapted viruses with this mutation for the preparation of live influenza vaccines. nin-esterase-fusion (hef), nucleoprotein (np), matrix (m ) protein, cm , and the non-structural proteins ns and ns . , cm is the second membrane protein of the virus and is encoded by rna segment (m gene). [ ] [ ] [ ] [ ] [ ] [ ] it is composed of three distinct domains: a -residue n-terminal extracellular domain, a -residue transmembrane domain, and a -residue cytoplasmic domain. , , it is abundantly expressed at the plasma membranes of infected cells and is incorporated in a small amount into virions. , cm forms disulphide-linked dimers and tetramers, and is posttranslationally modified by n-glycosylation, palmitoylation, and phosphorylation. [ ] [ ] [ ] analyses of a number of cm mutants revealed the positions of the amino acids involved in the posttranslational modifications. , evidence was obtained that the n-glycosylation was not required for either the formation of disulfide-linked multimers or transport to the cell surface, and that none of dimer-or tetramer-formation, palmitoylation or phosphorylation was essential to the transport of cm to the cell surface. in the present study, in order to investigate the effect of cm palmitoylation on influenza c virus replication, we generated a cm palmitoylation-deficient influenza c virus, in which a cysteine at residue of cm was mutated to alanine, and examined the viral growth and viral protein synthesis in infected cells. t and hmv-ii cells were maintained as described previously. , llc-mk cells were maintained at °c in minimal essential medium with % foetal bovine serum and % calf serum. monoclonal antibodies (mabs) against the hef, np, and m proteins of c ⁄ ann arbor ⁄ ⁄ (aa ⁄ ), and antisera against the aa ⁄ virion and the cm protein were prepared as described previously. , [ ] [ ] [ ] the seven pol i plasmids for the expression of viral rnas of aa ⁄ , and the nine plasmid dnas for the expression of the influenza c viral proteins were reported previously. , plasmid dna, ppoli ⁄ cm -acy(-), in which -tgt- of the m gene was replaced with -gct- , was constructed based on ppoli ⁄ m. to generate a recombinant wild-type (rwt) virus, the above-mentioned plasmids were transfected into t cells as described previously. to rescue a mutant virus, rcm -c a, a recombinant influenza c virus lacking a cm palmitoylation site, the plasmid ppoli ⁄ cm -acy(-), instead of ppoli ⁄ m, was transfected together with the other plas-mids. at hours posttransfection (p.t.), the respective culture medium of the transfected- t cells was inoculated into the amniotic cavity of -day-old embryonated chicken eggs, and a stock of the recombinant virus was prepared. the infectious titres of the stocked recombinant viruses and the supernatants of recombinant-infected hmv-ii cells were determined according to the procedure reported previously. radioimmunoprecipitation hmv-ii cells infected with recombinants were labeled with [ s]methionine or [ h]palmitic acid. cells were then disrupted and subjected to immunoprecipitation with the indicated antibodies. the immunoprecipitates obtained were then analysed by sds-page on ae % gels containing m urea, and processed for fluorography. flotation analysis was performed according to the procedure described previously. to examine whether the cm protein without palmitoylation is synthesized in rcm -c a-infected cells, hmv-ii cells infected with the recombinants were subjected to , and the lysates of the cells were immunoprecipitated with anti-cm serum and analysed by sds-page. as shown in figure , the cm protein was synthesized both in the rwt-and rcm -c a-infected cells, but no incorporation of [ h]palmitic acid into the cm proteins synthesized in the rcm -c ainfected cells was observed, indicating that cm in the rcm -c a-infected cells was not palmitoylated. the rwt or rcm -c a viruses were infected to hmv-ii cells at an m.o.i. of and incubated at °c for up to hours. the infectious titres (p.f.u. ⁄ ml) of rwt were approximately -to -fold higher than those of rcm -c a at - hours p.i. (data not shown), indicating that rwt grew more efficiently than did rcm -c a. thus palmitoylation of cm appears to have some effect on the generation of infectious virions in cultured cells. to investigate the reason(s) for the difference in growth kinetics between the two recombinants, we analysed viral proteins synthesized in the infected hmv-ii cells. pulsechase experiments of hmv-ii cells revealed no significant differences in the synthesis and maturation of the hef, np, m , and cm proteins between the rwt-and rcm -c a-infected cells (data not shown). the infected cells pulse-labeled and chased were respectively immunoprecipitated with anti-cm serum in the presence of mm iodoacetamide and analysed by sds-page in non-reducing condition. in both populations of infected cells, several bands corresponding to cm a-monomer, -dimer, and -tetramer, as well as cm b-dimer and -tetramer were detected (data not shown). these results demonstrate an absence of any significant differences between palmitoylation-deficient cm and authentic cm in terms of conformational maturation and transport in infected cells. membrane flotation analysis revealed that no significant differences in the kinetics of the hef, m , and cm proteins were observed between rwt-and rcm -c ainfected cells (data not shown). in contrast, a slight difference in np kinetics was observed. the pulse-labeled np proteins were recovered in the bottom fractions in both rwt-and rcm -c a-infected cells. in the chase experiment, the amount of membrane-associated np proteins in fractions and was % of the total np in the rwt-infected cells, which was higher than that ( %) in the rcm -c a-infected cells (data not shown). this finding may suggest that the affinity of the np protein, presumably representing the viral ribonucleoprotein (vrnp) complex, to the plasma membrane in the rcm -c ainfected cells is lower than that in rwt-infected cells, leading to the less efficient generation of infectious virions. since cm is structurally similar to m , an influenza a virus membrane protein known to be involved in infectious virus production, [ ] [ ] [ ] [ ] [ ] it is possible that the cytoplasmic tail of cm participates in the genome packaging through interaction with vrnp. in the present study, we showed that the affinity of np to the plasma membrane of rcm -c a-infected cells was slightly lower than that to the plasma membrane of rwt-infected cells. this observation may suggest that palmitoylation of cm is involved in the viral ribonucleoprotein (vrnp) incorporation, leading to efficient infectious virion generation. we hypothesize that palmitoylation contributes to proper regional structure formation in the cm cytoplasmic tail, which is competent to recruit vrnp efficiently into virions. alternatively, the cm cytoplasmic tail without palmitoylation is not likely to reach the proper conformation, resulting in reduced interaction with vrnp and less efficient generation of infectious progeny virions. the questions of if and how the m protein is involved in the interaction between the cm cytoplasmic tail and np remains to be clarified. we showed that cm synthesized in rcm -c ainfected cells was oligomerized and transported to the cell surface. this finding is consistent with the previous observation that palmitoylation is not required for the transport of cm to the cell surface in cm -expressing cos- cells. however, the use of reverse-genetics system has enabled us to conclude that the palmitoylation of cm is required for efficient infectious virus production. this suggests that the significance of the other posttranslational modifications of cm during virus replication can be clarified using recombinant viruses lacking the respective modification sites. sialic acid (sia) linked glycoproteins are the classical influenza receptors for influenza virus haemagglutinin to bind. the distribution of sia on cell surfaces is one of the determinants of host tropism, and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. previous research has shown the differences in apical versus basolateral infection and release of different influenza virus from polarized epithelial cells and correlated this with sialic acid distribution in the human respiratory tract. moreover, mass spectrometric analysis was recently employed to elucidate the glycans present in the tissue in a higher resolution in human lung. the objective of this study was to examine in detail the distribution of these sia-linked glycans at the cellular level by the use of confocal microscopy. human primary type i-like and type ii pneumocytes were isolated from human non-tumor lung tissue by tissue fragmentation, percoll density gradient centrifugation, and magnetic cell sorting. the cells were seeded on coverslips and maintained in small airway growth medium. when confluence was reached, cell monolayers were fixed with % paraformaldehyde. we used the plant lectins, sambucus nigra glutinin (sna) from roche which binds to siaa - gal, maackia amurensis agglutinin (maa)i and maaii from vector lab, which bind the siaa - gal linked glycans using vector red as fluorescent chromogen. the cells were counter-stained with dapi or with fitc-conjugated antibody against endoplasmic recticulum (protein disulfideisomerase, pdi). the cells were imaged with multi-photon excitation laser scanning microscopy using zeiss lsm. the optical cross-section pictures were reconstructed by zeiss lsm meta. we found that there was more binding of maai and ma-aii to type ii pneumocytes than type i-like pneumocytes and more overall binding of these lectins than binding of sna ( figure ). in keeping with results from other polarized cells there was more binding to the apical than basolateral aspect, thus, explaining the previously published data on apical versus basolateral infection. as sialic acid has been implicated in the targeting of proteins to the surface, the relative lack of sialic acid on the basolateral aspect can explain why there is little seasonal influenza virus dissemination to the systemic circulation in human infections. furthermore, though there was little binding of sna to the figure . primary human type i-like and type ii pneumocytes stained with lecins (red), pdi (green), and dapi (blue) and imaged captured with confocal microscope. apical or basolateral aspects of the pneumocytes, the experimental findings of infection by influenza h n virus that has a strict siaa - gal tropism suggests that there are siaa - gal glycans present, which are not readily bound by the lectin sna. the in vitro model of primary human type i-like and type ii pneumocytes system formed a polarized epithelium that has a similar lectin distribution to human alveoli in vivo which demonstrated that it is a physiologically relevant model to study the tropism and pathogenesis of influenza a virus. human disease caused by highly pathogenic avian influenza (hpai) h n virus is associated with fulminant viral pneumonia and mortality rates in excess of %. cytokine dysregulation is thought to contribute to its pathogenesis. , we previously found delayed onset of apoptosis in h n infected human macrophages and, therefore, a longer survival time of the target cells for prolonged virus replication and cytokine and chemokine secretion, which may contribute to the pathogenesis of h n disease in humans. as bronchial and alveolar epithelial cells are target cells of influenza virus because of their proximal physiological location and interaction with macrophages, we further investigated if the differential onset of apoptosis could be found in influenza h n and seasonal influenza h n infected human respiratory epithelia. we dissected the apoptotic pathways triggered by influenza virus infection. seasonal influenza h n virus (a ⁄ hk ⁄ ⁄ ), a low pathogenic avian influenza h n lineage isolated from poultry (a ⁄ quail ⁄ hk ⁄ g ⁄ ), and two virus isolates of hpai a subtype (a ⁄ hk ⁄ ⁄ and a ⁄ vn ⁄ ⁄ ) were included. primary human bronchial and alveolar epithelial cells were infected with influenza viruses at moi of and the cell monolayer was collected at , , and hours post infection for tunel assay, and supernatant were collected for ldh assay. fragments of human lung tissues were cut into multiple - mm fragments within hours of collection and infected with influenza a viruses at a titer of tcid ⁄ ml. these tissues fragments were infected for hours and incubated for hours at °c. one part of the infected tissue was fixed in formalin and processed for immunohistochemistry for influenza antigen, and the other part was homogenized and underwent rna extraction. apoptosis cdna superarray platform (sabioscience) was employed to conduct apoptosis pathway analysis. in bronchial epithelial cells, seasonal influenza h n virus induced a high percentage of apoptotic cells by tunel assay at , , and hours post infection with a peak of (figure ) . a similar observation of delayed onset of apoptosis was found in influenza h n and h n infected alveolar epithelial cells. besides, cdna array data of ex vivo infected human lung showed that both influenza h n and h n virus induced trail expression compared with mock-infected tissue (approximately folds) at hours post infection, but influenza h n virus infected lung induced significantly more trail ( folds compared to mock infected cells), albeit with a limited viral replication ( figure ). influenza h n virus infected lung also elicited more tnf-alpha and fasr transcription than either h n or h n . these observations can account for the greater apoptotic response in influenza h n virus infected lung. as little impact on the expression of intrinsic pathway components was observed, it seems that the apoptotic response to influenza virus infection in lung was mainly through the extrinsic pathways. no significant changes in the expression of anti-apoptotic protein gene was found, except for a moderate induction of birc by influenza h n virus, which may act to modulate the apoptotic response. the delayed onset of apoptosis by hpai h n and low pathogenic avian influenza h n virus infected respiratory epithelial cells may be a mechanism for the influenza viruses to have more prolonged replication within the human respiratory tract, and this may contribute to the pathogenesis of human disease. hemagglutination (ha) assay % crbc suspension was treated by mu a , -specific sialidase at °c for minutes. complete elimination of a , -receptor on sialidase-treated crbcs was confirmed by receptor staining and flow cytometry. ha assay of live viruses with % crbc or % sialidase-treated crbc were performed in bsl- facility. synthetic ¢sln-paa-biotin(pa ), ¢sln-paa-biotin(pa ), ¢sln-ln-paa-biotin(pa ) was provided by the scripps research institute (tsri). as described elsewhere with some modifications, generally, serial dilutions of sialyglycopolymers were coated in -well-flat-bottom polystryrene plates, and hau live virus ⁄ well were added. alternatively, the plates were precoated with lg ⁄ ml sialyglycopolymers, and then , , , , hau live virus ⁄ well influenza viruses were added. rabbit antisera against a ⁄ ah ⁄ ⁄ diluted in pbs containing % bsa was added into the wells. bound antibody was detected by use of hrp-conjugated anti-rabbit igg antibody and tetramethylbenzidine substrate solution. each sample was determined in duplicates and the absorbance read at nm. a total of h n virus strains were obtained from to . the name and passage history of influenza viruses used in the study are listed in table . as the same sequences of eight rna segments were detected in a ⁄ js ⁄ ⁄ and a ⁄ js ⁄ ⁄ , only a ⁄ js ⁄ ⁄ was tested here. three amantadine-resistant variants with m mutation of screening of receptor-binding preference by ha assay representative results from three sets of independent experiments are shown in table . complete ha with sialidasetreated crbcs, which were only with a , -receptors, was detected in human influenza virus (a ⁄ brisbane ⁄ ⁄ , h n ) and two human h n virus strains, a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ . high binding of a , oligosaccharides to h n viruses was detected ( figure a -c). and enhanced a , -binding preference was also detected in a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ . the a , -binding was dose dependent for sialyglycopolymers and virus titer. notably, as compared with a ⁄ gd ⁄ ⁄ of both short-and long-a , recognition, a ⁄ gx ⁄ ⁄ prefers to bind to long-a , six oligosaccharides at low viral titer ( figure b,c) . however, both of them showed strong affinity to short-and long-a , oligosaccharides at high viral loads ( figure d ). sialoside-, galactoside-, mannoside-and sulfo-os-binding are the four types of carbohydrate-binding properties of influenza virus. binding of influenza virus to the a , -or a , -linked sialylated glycans on cell surface is important for host range restriction, and the preference to a , of h n virus limited its efficient infection in human. here, dual receptor-binding preferences were detected in a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ , which are of clade ae ae . although there is no direct evidence supporting the occurrence of human-to-human transmission in these infection events or the association between viral virulence and receptor-binding switching, viral systemic disseminations are found in the both fatal cases (data not shown). furthermore, with the introduction of clade ae ae into the adjacent countries of china, the finding of h n virus with - binding in human should be of concern. though h n virus with human-type receptor-binding was isolated from one patient treated by oseltamivir and those viruses were with ha and ⁄ or na substitutions, whether the substitutions responsible for receptor specificity switching is pre-existed or selected in human host remains unknown. our finding that three mutant viruses bearing m mutations of a s, a t, and s n cloned from one isolate a ⁄ hb ⁄ ⁄ suggested it is likely that the resistant viruses emerged in the host environment. no variation was found in their ha and na sequence, and all of them show high affinity to a - -binding. our data suggest that the binding-specificity was not affected by the mutations on viral envelope protein m . with the adaptation from wild aquatic birds to domestic poultry or even in human host environment, influenza virus may possess broader carbohydrate-binding spectrum or topology conformation. , we demonstrated differential a , -binding property of two human h n viruses, a ⁄ gd ⁄ ⁄ and a ⁄ gx ⁄ ⁄ . though minor effect of short-a , -binding was detected in viruses a ⁄ gx ⁄ ⁄ at low virus titer, both were of high affinity to long-a , glycans, even at the low titer which are rich on apical side of human upper respiratory epithelia. notably, no evident binding preference switching was detected in the viruses isolated from the sporadic human infection cases at the early of in china (table ) . however, higher affinity to the long-a , glycans was observed in bj ⁄ ⁄ , gz ⁄ ⁄ , and xj ⁄ ⁄ (data not shown). the discrepancy from the findings obtained by sialidase-treated crbc maybe associated with a limited abundance of n-linked a - with long branches on crbc, as demonstrated in a recent study. therefore, glycan dose-dependent binding assay is valuable and should be applied in flu surveillance. the underlying cause of the tendency is unknown, and further research on receptor-binding specificity of h n viruses is required. influenza a viruses of migrating wild aquatic birds in north america towards improved influenza a virus surveillance in migrating birds european union council directive ⁄ ⁄ eec the neighbor-joining method: a new method for reconstructing phylogenetic trees confidence limits on phylogenies: an approach using the bootstrap prospects for inferring very large phylogenies by using the neighbor-joining method mega : molecular evolutionary genetics analysis (mega) software version . the influenza virus resource at the national center for biotechnology information characterization of low-pathogenic h subtype influenza viruses from eurasia: implications for the origin of highly pathogenic h n viruses h n virus outbreak in migratory waterfowl a ⁄ h and a ⁄ h influenza viruses: different lines of one precursor evolution and ecology of influenza a viruses evolutionary processes in influenza viruses: divergence, rapid evolution, and stasis antigenic and genetic conservation of h influenza virus in wild ducks biologic characterization of chicken-derived h n low pathogenic avian influenza viruses in chickens and ducks genetic and pathogenic characterization of h n avian influenza viruses isolated in taiwan between and experimental selection of virus derivatives with variations in virulence from a single low-pathogenicity h n avian influenza virus field isolate evolution and ecology of influenza a viruses is china an influenza epicenter genesis of 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antigenic and genetic characterization of h n swine influenza in china cocirculation of avian h n and contemporary ''human'' h n influenza a viruses in pigs in southeastern china: potential for genetic reassortment? h n influenza a viruses from poultry in asia have human virus-like receptor specificity characterization of h subtype influenza viruses from the ducks of southern china: a candidate for the next influenza pandemic in humans? bioedit: a user-friendly biological sequence alignment editor and analysis program for window ⁄ ⁄ nt genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion phylogenetic analysis using parsimony (and other methods) . beta a novel genotype h n influenza virus possessing human h n internal genomes has been circulating in poultry in eastern china since characterization of h n influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation in eastern china during a year period characterization of avian h n influenza viruses from united arab emirates phylogenetic analysis of influenza a viruses of h haemagglutinin subtype h n subtype influenza a viruses in poultry in pakistan are closely related to the h n viruses responsible for human infection in hong kong diversified reassortants h n avian influenza viruses in chicken flocks in northern and eastern china genotypic evolution and antigenic drift of h n influenza viruses in china from the nucleoprotein as a possible major factor in determining host specificity of influenza h n viruses pigs as the ''mixing vessel'' for the creation of new pandemic influenza a viruses origins and evolutionary genomics of the swine-origin h n influenza a epidemic pandemic (h n ) outbreak on pig farm reassortment of pandemic h n ⁄ influenza a virus in swine from where did the 'swine-origin' influenza a virus (h n ) emerge? substitution of lysine at position in pb protein does not change virulence of the 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circulating worldwide from oseltamivir-resistant influenza viruses a (h n ), norway, - influenza activity -united states and worldwide, - season emergence of resistance to oseltamivir among influenza a(h n ) viruses in europe oseltamivir-resistant influenza virus a (h n ), europe, - season widespread oseltamivir resistance in influenza a viruses (h n ), south africa and composition of the - influenza vaccine emergence of h y oseltamivir-resistant a(h n ) influenza viruses in japan during the - season pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal influenza a viruses neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir host cell selection of influenza neuraminidase variants: implications for drug resistance monitoring in a(h n ) viruses neuraminidase receptor binding variants of human influenza a(h n ) viruses due to substitution of aspartic acid in the catalytic site -role in virus attachment? neuraminidase inhibitor susceptibility testing in human influenza viruses: a laboratory surveillance perspective update: drug susceptibility of swine-origin influenza a (h n ) viruses comprehensive assessment of pandemic influenza a (h n ) virus drug susceptibility in vitro detection of molecular markers of drug resistance in pandemic influenza a (h n ) viruses by pyrosequencing pandemic (h n ) and oseltamivir resistance in hematology/oncology patients fluview: a weekly influenza surveillance report prepared by the influenza division development of a sensitive chemiluminescent neuraminidase assay for the determination of influenza virus susceptibility to zanamivir evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network surveillance for neuraminidase inhibitor resistance among human influenza 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for complex multipathogen interactions in acute respiratory infections performance comparison of res-plex ii and xtag rvp fast for detecting respiratory viruses clinical virology symposium communityacquired respiratory co-infection (carc) in critically ill patients infected with pandemic influenza a (h n ) virus infection bacterial co-infections in lung tissue specimens from fatal cases of pandemic influenza a (h n ) -united states a quantitative risk assessment of exposure to adventitious agents in a cell culture-derived subunit influenza vaccine john's hopkins bloomberg school of public health design of an automated laboratory for high-throughput influenza surveillance human influenza surveillance: the demand to expand influenza: an emerging disease avian-to-human transmission of the pb gene of influenza a viruses in the and pandemics proinflammatory cytokine responses induced by influenza a (h n ) viruses in primary human alveolar and bronchial epithelial cells induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? influenza h n and h n virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation mapping of the two overlapping genes for polypepetides ns and ns on rna segment of influenza virus genome sequences of mrnas derived from genome rna segment of influenza virus: collinear and interrupted mrnas code for overlapping proteins influenza virus ns protein inhibits pre-mrna splicing and blocks mrna nucleocytoplasmic transport the influenza virus ns protein: a novel inhibitor of pre-mrna splicing identification of cis-acting intron and exon regions in influenza virus ns mrna that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes identification of a second protein encoded by influenza c virus rna segment influenza c virus ns protein upregulates the splicing of viral mrnas identification of an amino acid residue on influenza c virus m protein responsible for formation of the cord-like structures of the virus a mutation on influenza c virus m protein affects virion morphology by altering the membrane affinity of the protein detection of ion channel activity in xenopus laevis oocytes expressing influenza c virus cm protein evidence that the cm protein of influenza c virus can modify the ph of the exocytic pathway of transfected cells the ion channel activity of the influenza virus m protein affects transport through the golgi apparatus intracellular localization of influenza c virus ns protein (nep) in infected cells and its incorporation into virions receptor specificity, host range and pathogenicity of influenza viruses replication, pathogenesis and transmission of pandemic (h n ) virus in non-immune pigs world health organization. preliminary review of d g amino acid substitution in the haemagglutinin of pandemic influenza a (h n ) viruses 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specificity of pandemic influenza a (h n ) virus determined by carbohydrate microarray fields virology. philadelphia, pa: lippincott williams & wilkins the molecular virology and reverse genetics of influenza c virus identification of a second protein encoded by influenza c virus rna segment identification of a amino acid protein encoded by rna segment of influenza c virus influenza c virus cm protein is produced from a amino acid protein (p ) by signal peptidase cleavage a mutation on influenza c virus m protein affects virion morphology by altering the membrane affinity of the protein influenza c virus cm integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence evidence that the matrix protein of influenza c virus is coded for by a spliced mrna functional properties of the virus ion channels the cm protein of influenza c virus is an oligomeric integral membrane glycoprotein structurally analogous to influenza a virus m and influenza b virus nb proteins characterization of a second protein (cm ) encoded by rna segment of influenza c virus phosphorylation of influenza c virus cm protein the sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza c virus cm protein identification of an amino acid residue on influenza c virus m protein responsible for formation of the cord-like structures of the virus a human melanoma cell line highly susceptible to influenza c virus antigenic characterization of the nucleoprotein and matrix protein of influenza c virus with monoclonal antibodies construction of an antigenic map of the haemagglutinin-esterase protein of influenza c virus the synthesis of polypeptides in influenza c virus-infected cells new low-viscosity overlay medium for viral plaque assays the influenza virus m protein cytoplasmic tail interacts with the m protein and influences virus assembly at the site of virus budding the cytoplasmic tail of the influenza a virus m protein plays a role in viral assembly the influenza a virus m cytoplasmic tail is required for infectious virus production and efficient genome packaging distinct domains of the influenza a virus m protein cytoplasmic tail mediate binding to the m protein and facilitate infectious virus production influenza virus m ion channel protein is necessary for filamentous virion formation influenza h n virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells das inhibits h n influenza virus infection of human lung tissues receptor binding specificity of recent human h n influenza viruses differential onset of apoptosis in avian influenza h n and seasonal h n virus infected human bronchial and alveolar epithelial cells: an in vitro and ex vivo study human influenza virus a ⁄ hongkong ⁄ ⁄ (h n ) infection induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? proinflammatory cytokine responses induced by influenza a (h n ) viruses in primary human alveolar and bronchial epithelial cells differential onset of apoptosis in influenza a virus h n -and h n -infected human blood macrophages avian flu: influenza virus receptors in the human airway haemagglutinin mutations responsible for the binding of h n influenza a viruses to humantype receptors an avian influenza h n virus that binds to a human-type receptor evolution of highly pathogenic h n avian influenza viruses in vietnam between evolutionary dynamics and emergence of panzootic h n influenza viruses writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza a (h n ) virus recent avian h n viruses exhibit increased propensity for acquiring human receptor specificity a simple screening assay for receptor switching of avian influenza viruses glycan topology determines human adaptation of avian h n virus hemagglutinin h n chicken influenza viruses display a high binding affinity for neu acalpha - galbeta - ( -hso )glcnac-containing receptors a strain of human influenza a virus binds to extended but not short gangliosides as assayed by thin-layer chromatography overlay search for additional influenza virus to cell interactions avian flu: isolation of drug-resistant h n virus the surface glycoproteins of h influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties this study was supported by the li ka shing foundation, the national institutes of health (niaid contract hhsn c), and the area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn c, the li ka shing foundation, and we thank all french and vietnamese field staff involved in the data collection in viet nam for their enthusiasm and support and we are grateful to the pig farmers participating in the study for their cooperation and patience. this study was a part of the gripavi project and was funded by the french ministry of foreign affairs. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn c and the area of excellence scheme of the university grants commission (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. we acknowledge the food and environmental hygiene department of hong kong for facilitating the study. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn c, the li ka shing foundation, and the area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. we gratefully acknowledge our colleagues from iiii, shantou university and skleid, hku for their excellent technical assistance. the study was supported by the rfcid commissioned study (lab# ) from research fund secretariat, food and health bureau, hong kong sar; area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ), hong kong sar; and by niaid contract (sjceirs, hhsn c), nih, usa.ferrets in all groups inoculated with a ⁄ turkey ⁄ ⁄ virus survived the infection and were observed once daily for days. below lower limit of detection (< ae log eid ⁄ ml).statistical cutoff of ic values for nai susceptibility, determined by x ae + iqr. outliers with ic above this cutoff and > times the mean ic for each drug were characterized as extreme outliers; those with known drug-resistance mutations such as h y were classified as resistant and analyzed separately. h wildtype, oseltamivir-susceptible isolates. h y variants, oseltamivir-resistant virus isolates. iqr, interquartile ranges; nai, neuraminidase inhibitors. we wish to thank our collaborators in the who global influenza surveillance network and united states public health laboratories for the submission of virus isolates and clinical specimens. we also thank our colleagues from the virus reference team and the influenza sequence activity, influenza division, cdc, for their valuable technical assis-the findings and conclusions of this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention (cdc). we are indebted to yonas araya, theresa wolter, and ivan gomez-osorio for their excellent laboratory techniques and animal handling assistance. we would like to thank andrea ferrero for her laboratory managerial skills. this research was possible through funding by the cdc-hhs grant ( u ci ), niaid-nih grant, (r ai ), csrees-usda grant ( - ), and niaid-nih contract (hhsn c). we thank c bazzoli for advice. this work was supported by a grant from the european union fp project flu-modcont (no. ). we thank staff at seoul, incheon, daejeon, gwangju, gangwon, and jeonbuk provincial research institute of health and environments for their laboratory testing. additionally, we would like to acknowledge the contributions of participating sentinel doctors for evaluating the new rat kit. this study was supported by a grant from the korea cdc. we thank roche applied science for providing the materials and equipment for this evaluation. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn c and the area of excellence scheme of the university grants committee (grant aoe ⁄ m- ⁄ ) of the hong kong sar government. the authors would like express their sincere thanks to cdc, usa for supporting the routine surveillance of ili in we would like to acknowledge the australian red cross blood service (the blood service) and the australian government, which fully fund the blood service for the provision of blood products and services to the australian community. we also wish to thank the donors and staff of the blood service, who have assisted in provision of specimens for testing in this protocol, as well as the staff at the who we are grateful to liping long for her assistance in map generation. this project was supported by nih niaid rc ai . cz is supported partially by canadian nserc postdoc fellowship. the authors thank the national investigation team based at the national institute of health (istituto superiore di sanita'), italy (in particular antonino bella, maria cristina rota, stefania salmaso) for providing their support in data collection, and the european union this study was supported in part by a grant-in-aid ( ) and the special coordination funds for promoting science and technology of ministry of education, science, sports and culture of japan. this study was supported in part by a grant-in-aid from the ministry of education, science, and culture of japan ( ) and the special coordination funds for promoting science and technology of mext of japan. the work described here was supported by phs grant ai- (jam) and alsac. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. py, po, dw and kb are employees of glaxosmithkline. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the stud- authors are thankful to path for the financial support of this research. we would like to acknowledge jessica d'amico and dr. rick bright of path for their editorial review. this study was supported by path. the authors would like to thank rick bright, jessica d'amico, and vadim tsvetnitsky for editing assistance. the we thank dr. m. enami (kanazawa university) for generously providing plasmids containing cdnas to influenza a virus m and ns genes. we also gratefully thank dr. r. sho (department of public health, yamagata university faculty of medicine) for statistical analysis. some data shown in this study have also been presented in the reference paper. this work was supported in part by a grant-in-aid for scientific research from the ministry of education, culture, sports, science, and technology, japan, takeda science foundation, terumo life science foundation, and a grant-in-aid from the global coe program of the japan society for the promotion of science. we thank markus eickmann for his help in isolation and initial characterization of a ⁄ hamburg ⁄ ⁄ and for providing antisera against h n pdm. this study was supported by the european union fp global a(h n ) genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. in this study we analysed the genetic and antiviral drug susceptibility profiles of pandemic a(h n ) influenza virus circulating in portugal. genetic profile analysis was performed in isolates to the hemagglutinin (ha), neuraminidase (na) and mp genes, and in six of these isolates the pb , pb , pa, np and ns genes were also analysed. antiviral drug susceptibility profile was analysed for isolates, phenotypically and genotypically to neuraminidase inhibitors (nai) and genotypically to amantadine. the point mutations identified in ha, na, and mp genes of different strains do not seem to evidence an evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h n ) virus. all analysed strains were found to be resistant to amantadine, and five of these strains exhibited a reduced susceptibility profile to nai, three only for oseltamivir and two for both inhibitors. introduction: the dynamics of pandemic influenza a ⁄ h n compared to seasonal strains of influenza is not clearly understood. it is important to understand the patterns of viral shedding and symptoms over time in community-based infections.materials and methods: household infections were followed-up in two large community-based studies. patterns of viral shedding, symptoms and signs, and tympanic temperature were plotted over time and grouped according to strain for analysis.results: the patterns of viral shedding, symptoms and signs, and tympanic temperature in three influenza a strains (pandemic a ⁄ h n , seasonal a ⁄ h n , and seasonal a ⁄ h n ) were comparable. peak viral shedding occurred close to the onset of symptoms and resolved after - days. patterns of viral shedding in influenza b virus infections differed.discussion: the patterns of viral shedding and clinical course of pandemic influenza a ⁄ h n infections were broadly similar to seasonal influenza a ⁄ h n and a ⁄ h n . only the clinical course of seasonal influenza b infections was similar to pandemic influenza a ⁄ h n . the dynamics of pandemic influenza a ⁄ h n were observed to be largely alike to the dynamics of seasonal influenza a ⁄ h n and a ⁄ h n . the coated respirators inactivated a broad range of influenza strains within minute, including the pandemic strain and human, swine, and avian influenza viruses. antiviral effectiveness was not reduced by hot, humid conditions or repeated saturation, which might occur during prolonged use of respirators. in contrast, infectious virions were detected on the surfaces of all uncoated ffp respirators, and could be transferred to glove surfaces during handling of contaminated masks. growth of the viruses was monitored by ha titer using turkey red blood cells, by quantitative real time rt-pcr (qrt-pcr) to detect the influenza a matrix gene, and also by flow cytometry to detect virus positive cells using monoclonal antibodies (imagen influenza virus a and b). , matrix gene copy number was determined using qrt-pcr and analysed using the sequence detection software on a fast system sds (applied biosystems, california, usa). further characterisation was performed through sequence analysis and the ha inhibition (hai) assay. sequence analysis was performed using dnastar and all sequences obtained were compared with the sequence of either the original clinical specimen if available or the conventional atcc derived mdck cell isolate. the hai assay was used to characterize the viruses against a panel of known standard reference viruses and their homologous ferret antiserum.options for the control of influenza vii abstract background: we measured the cross-reactive antibody response to pandemic h n in children and adults before and after vaccination with [ ] [ ] [ ] [ ] influenza season vaccines as part of the rapid public health response to the emergence of ph n and to provide evidence for ph n vaccination policy development in mainland china. materials and methods: archived serum specimens from previous vaccine studies were detected by hemagglutination inhibition assay. results: limited crossreactive antibody response to ph n had been detected among participants of all age groups before and after they had been vaccinated with - , - influenza seasonal vaccines. vaccination with seasonal influenza viruses resulted in limited seroconversion to ph n in all age groups, compared with - % of seroconversion to seasonal influenza viruses. but similar to recent studies, a peak of cross-reactive antibody response to ph n was observed in % and % of participants born from to before and after vaccination. conclusions: in order to protect our populations in china, our study strongly suggests vaccination with ph n is required in all age groups and that older populations born before may be associated with a lower infection rate of ph n . on april and april , , cases of ph n were identified in specimens obtained from two epidemiologically unlinked patients in the united states and soon thereafter in texas and mexico. since that time, the virus has spread across the globe. assessment of cross-reactive antibody response to the ph n after vaccination with sea-sonal influenza vaccine was first reported from us centers of disease control and prevention (us cdc). according to their results, the seasonal influenza vaccines provided little or no protection against the ph n , but some degree of preexisting immunity to the virus existed, especially among adults aged ‡ years. in this study, using archived serum samples from previous vaccine studies, we measure the level of cross-reactive antibody response to ph n in children and adults vaccinated intramuscularly with trivalent inactivated vaccine developed for the northern hemi- serum specimens were collected and provided by provincial centers for disease control and prevention of china as a public health response to the emergence of ph n exempt from human-subjects review. a total of serum samples were collected from xinjiang uygur autonomous region, yunnan, and shandong provinces. all the serum specimens were grouped by the age of subjects ( - , - , - , ‡ years) and by different influenza seasons.hemagglutination inhibition assay was performed according to standard procedures in this study. [ ] [ ] [ ] as with h n components of the vaccine, the seasonal influenza viruses used in this study were a ⁄ solomon islands ⁄ ⁄ and a ⁄ brisbane ⁄ ⁄ . the ph n influenza virus used in this study was a ⁄ california ⁄ ⁄ provided by us cdc. all the viruses were propagated in specific pathogenfree embryonated chicken eggs and inactivated by & paraformaldehyde. the criteria recommended by the european agency for the evaluation of medical product was applied for the assessment of seasonal influenza vaccine gmt, geometric mean titer; hi, hemagglutination inhibition. three weeks after boosting immunization, spleens were harvested from immunized and control mice. splenocytes were prepared by lymphocyte separation media (ez-sepÔ, shen zhen, china). the cells were washed and resuspended in complete rpmi- containing fetal bovine serum (hyclone, logan, ut, usa), glutamax, um b-me. splenocytes were cultured in vitro in the presence of inactivated h n , h n , h n , and h n influenza virus antigen for h. quick cell proliferation assay kit (biovision, san francisco, ca, usa) was used to detect the cell proliferation. the - nm absorbance was read on a plate reader. all experiments have been repeated at least three times.results are presented as mean standard error of the mean (sem). comparison of the data was performed using the student's t-test. significance was defined as a p value of < ae . to evaluate the adjuvant effect of recombinant igv, the anti m e antibody subclasses was measured. igg and igg a were detected after the first and second immunization ( table ). the ratio of igg a ⁄ igg was calculated. immunization with only m e-hbc showed a lower igg a ⁄ igg ratio < ae . igv combined with m e-hbc led to a high igg a ⁄ igg ratio of up to - after first and second immunization. these igg subclass distributions indicated that igv can induce a th immune response. to determine whether the splenocytes were stimulated in vitro with different subtypes of inactivated influenza antigen after the igv plus m e-hbc antigen immunization, h n , h n , h n , h n inactivated antigen was used table . the serum igg, igg , igg a, and igg a ⁄ igg ratio were measured by elisa after first and second immunization. m e were coated on the wells plate overnight, and serial dilution sera of day , , after first and second immunization were added , ae , , , , ug ⁄ ml of igg, igg , igg a purified antibody were also added for obtaining the standard curve. hrp-labeled goat anti-mouse igg, igg , or igg a was then added, washed, and the optical density was read at nm. the results were showed at mean ± sem. day after first immunization days after second immunizationoptions for the control of influenza vii the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than specimens yearly from cases of acute respiratory illness (ari), using two sampling methods: systematic randomized and non systematic ''ad hoc'' sampling. although vaccines against influenza a virus are the most effective method by which to combat infection, it is clear that their production needs to be accelerated and their efficacy improved. a panel of recombinant live attenuated human influenza a vaccines (laivs), including ns - , ns - , nsd , were generated by rationally engineering mutations directly into the genome of a pandemic-h n virus. the vaccine potential of each laiv was determined through analysis of attenuation, immunogenicity, and their ability to protect mice and ferrets. the data indicate that the novel nsd -laiv was ideally attenuated and elicited strong protective immunity. this study also shows that attenuating mutations can be rapidly engineered into the genomes of emerging ⁄ circulating influenza a viruses in order to produce laivs. the influenza virus exhibits complicated evolutionary dynamics due to multiple reasons, such as diverse hosts, high mutation rates, and rapid replications. in this study, large-scale analyses of influenza neuraminidase (na) sequences revealed influenza a and b na genes diverged first around years ago, and subsequently the na subtypes of influenza a emerged around years ago. all nine na subtypes of influenza a were genetically distinct from each other, with a total of lineages identified. in addition, five and three sub-lineages were further identified in lineage a of na and lineage b of na , respectively. the majority of lineages and sub-lineages were found to be host or geographic specific. this study provides not only a better understanding of influenza na evolution, but also a database of lineages and sub-lineages that can be used for early detection of novel genetic changes for improved influenza surveillance. although phylogenetic approaches are commonly used and often found to be powerful, how to accurately identify lineages or sub-lineages of a gene segment of the influenza a virus remains a challenging issue. in this study, we address this issue by analyzing hemagglutinin (ha) sequences using a combination of statistical and phylogenetic methods. following a hierarchical nomenclature system that uses a letter to represent a lineage and a digit for a sub-lineage, we identified distinct lineages and sub-lineages in all ha subtypes through large-scale analyses of influenza a hemagglutinin sequences. the majority of the lineages or sub-lineages were host or geographic specific or both. further analysis of other segments will allow us to construct a comprehensive database for influenza a lineages and genotypes, facilitating early detection of new viral strains and genome reassortments and hence improve influenza surveillance. identification of the genetic origin of influenza a viruses will facilitate understanding of the genomic dynamics, evolutionary pathway, and viral fitness of influenza a viruses. the exponential increases of influenza sequences have expanded the coverage of influenza genetic pool, thus potentially reducing the biases for influenza progenitor identification. however, these large amounts of data generate a great challenge in progenitor identification. clinical (nasopharyngeal swabs) and post-mortem materials (fragments of trachea, bronchi, lungs, spleen) were obtained from clinics and ⁄ or out-patients from st. petersburg and from base virological laboratories (bvls) of the research institute of influenza in different regions of the country, which cover approximately ⁄ of the territory of russia. the informed consent for the bio-materials collection and studies was obtained from research subjects or from their relatives in cases of post-mortem materials. isolation of viruses was carried out in the mdck cell culture (cdc, atlanta, ga, usa) and in -day-old chicken embryos (e). isolation was done according the standard internationally accepted methods. the reaction of hemagglutination (ha) and the inhibition of hemagglutination (hai) were performed according the who recommended standard method. for the identification of epidemic isolates, we used the hyperimmune diagnostic bovine or ovine antisera annually obtained from the who reference center (cdc). for a detailed antigenic analysis we used the hyperimmune rat antisera against epidemic and reference influenza strains during the period from july , up to april , , we have obtained swabs from clinics and out-patients in st. petersburg and swabs from the bvls. in this period, rather high incidence of lethality from pneumonia was observed, which developed on the background of the pandemic flu h n v. thus, we received from bvls postmortem materials from deceased patients which manifested pcr+ influenza h n v-specific rna. all materials were tested for a possibility of isolation of influenza virus h n v both in eggs and in mdck cells. pcr-negative materials were discarded. we isolated strains of pandemic influenza from the materials collected in st. petersburg and region, which comprised ae % of the total number of analyzed samples. at the same time, we did not isolate any other sub-types of influenza in the season - except the pandemic flu. from the swabs purchased from bvls, strains were isolated, which compose ae % of the pcr+ samples, and strains from the post-mortem materials ( ae % of the pcr+ samples).altogether in the season - , we isolated, retrieved, and analyzed in hai influenza strains. ae % of them were pandemic strains a(h n )v, and only ae % influenza b viruses. these data together with the epidemiologic data and the results of pcr-diagnostic provide evidence in favor of nearly mono-etiological character of epidemic season - in russia for pandemic influenza a(h n )v.though the isolation of pandemic viruses was fulfilled in two traditional model systems, in the case of pandemic virus, we could observe the tendency of preferential multiplication in embryos compared to mdck, especially in cases of post-mortem material for which chicken embryos are the preferential system of isolation.h n v viruses, which were isolated and passaged in mdck, even with significant ha titers, quickly lost their ha activity provided they were kept at + °c. moreover, some other tested cell lines proved to be practically nonsensitive to the pandemic viruses h n v. we used hai reaction for the typing and antigenic characterization of isolated viruses. in the course of isolation of viruses in the reported period, we produced rat polyclonal antisera to the strains a ⁄ california ⁄ ⁄ and a ⁄ st. petersburg ⁄ ⁄ (h n )v and the antisera to the strains a ⁄ new jersey ⁄ ⁄ -the virus isolated during the epidemic in the united states and also of the swine origin -and to the 'swine' strains a ⁄ sw ⁄ ⁄ and a ⁄ iowa ⁄ ⁄ . the hai results of representative strains are given in figure . table shows that the isolated strains were homogenous in their antigenic properties and interacted with the diagnostic antiserum cdc for a(h n )v and also with the antisera to the strains a ⁄ california ⁄ ⁄ and a ⁄ st. petersburg ⁄ ⁄ up to - ⁄ homologous titer. viruses that were isolated from post-mortem materials did not differ by their antigenic characteristics from those isolated from swabs of live patients. only two strains could be attributed to the drift-variants of the strain a ⁄ california ⁄ ⁄ because they reacted with the appropriate antiserum up to ⁄ homologous titer; these strains were a ⁄ pskov ⁄ ⁄ and a ⁄ belgorod ⁄ ⁄ . it is interesting that the isolated strains reacted with the antisera to the strains a ⁄ new jersey ⁄ ⁄ and a ⁄ sw ⁄ ⁄ to ⁄ - ⁄ , and some particular strains even to ⁄ homologous titer. it is even more interesting that some pandemic isolates reacted with the antiserum to the strain iowa isolated in up to ⁄ - ⁄ homologous titer. despite of the fact that since the outbreak of 'swine flu' in the usa in new jersey years had gone (and for the strain iowa this period is nearly years) the ha of these viruses and of the pandemic influenza share some common antigenic determinants as was shown in hai.one more interesting feature of a considerable part of isolated strains is their capability to react with high titers with normal equine serum heated to and to °c, while all the strains of swine origin isolated earlier were inhibitor-resistant ( figure ). russian isolates of divided, in this respect, in two clear and approximately equal in number groups: one of them is similar to the reference strain amino acid substitutions, among them more than were disclosed in antigenic sites, so the degree of similarity to this strain is %. a new site of glycosylation was also discovered in the position of ha. essential distinctions of the aminoacid sequence of ha and antigenic properties of the h n v strains as compared with actual circulating and vaccine strains is one of the factors that determine the pandemic potential of this new influenza virus.according to the literature, the mutation in the ha gene d g could cause a broadening of the spectrum of receptor specificity of influenza virus by the acquisition of the capacity to bind both the residues a( fi ) and a( fi ) of the sialic acid of cellular receptors. both types of receptors are present at the human respiratory tract, but in different parts of it, and they exist in different proportions. according to the data of the european center of disease control and prevention (ecdc), the varieties g of the h n v virus were isolated in countries from subjects deceased of influenza or who suffered a severe form of illness, as well as from those who sustained only a light course of influenza. concerning the strains isolated in rii, this mutation was discovered in nine cases: four were isolated from live patients and five from post-mortem materials. thus, there are no convincing data at present that could prove a causal relationship of the given substitution and the aggravation of a disease course. this is in accordance with previous observations. concerning the resistance of studied strains to the widely used antiviral preparations, it was shown that all tested strains possessed the substitution s n in the m protein that determine the resistance to adamantanes. there was no substitution in the position of neuraminidase (na), which determines the resistance to oseltamivir (h y). these substitutions are the characteristic indices of the eurasian lineage of swine influenza viruses. thus all studied russian h n v isolates were resistant to adamantanes (rimantadine) and sensitive to oseltamivir. respiratory clinical samples taken in and that tested positive by real time reverse transcription (rt)-pcr for seasonal influenza viruses (a and b) and pandemic h n respectively were assessed for other respiratory viruses using the resplex ii panel ver . system distributed by qiagen. results showed that co-infections with another respiratory viruses were relatively rare, with a small number of samples having another co-infecting virus present, very few samples having two other viruses detectable in their samples, and none with further viruses. this low number of co-infecting viruses and the ability of certain cell lines not to support infection with particular viruses may make primary isolation of influenza viruses in cell lines easier than might have been thought previously. cm is the second membrane protein of influenza c virus and is posttranslationally modified by phosphorylation, palmitoylation, n-glycosylation, and dimer ⁄ tetramer formation. in the present study, we generated rcm -c a, a recombinant influenza c virus lacking cm palmitoylation site, and examined viral growth and viral protein synthesis in the recombinant-infected cells. the rcm -c a virus grew less efficiently than did the wild-type virus. membrane flotation analysis of the infected cells revealed that less np was recovered in the plasma membrane fractions of the rcm -c a-infected cells than that in the wild-type virus-infected cells, suggesting that palmitoylation of cm is involved in the affinity of the ribonucleoprotein complex to the plasma membrane, leading to the efficient generation of infectious viruses. influenza c virus has seven single-stranded rna segments of negative polarity, encoding pb , pb , p , haemaggluti- both the a , linkage and its topology on target cells were critical for human adaptation of influenza a viruses. the binding preference of avian flu virus h n ha to the a , -linked sialylated glycans is considered the major factor that limited its efficient infection in human. currently, the switch in binding-specificity of human h n viruses from a , to a , -glycans did naturally occur, and limited humanto-human transmission was found. to monitor their potential adaptation in the human population, receptor-binding specificity surveillance was made in china. here, the binding specificity of human h n virus strains isolated from to was demonstrated. dual binding preference to a , and a , -glycans were found in a ⁄ guangdong ⁄ ⁄ and a ⁄ guangxi ⁄ ⁄ . furthermore, both of them showed a high affinity to the long-branched a , -glycans, which predominate on the upper respiratory epithelial in human. our data suggests that the existence of h n virus with binding specificity to humans should be of concern.introduction via envelope glycoprotein hemagglutinin (ha), influenza viruses bind to cell-surface glycosylated oligosaccharides terminated by sialic acids (sa) where their linkage is celland species-specific. differential receptor binding preference is a host barrier for influenza virus transmission. although most h n viruses have low affinity to neu aca , gal (human-type) receptor, recent findings suggested that the adaptation of h n virus to human by mutations in the receptor-binding site (rbs) do indeed happen and resulted in enhanced affinity to human-type receptor. [ ] [ ] [ ] in contrast to its putative precursor, a ⁄ gs ⁄ gd ⁄ ⁄ , diverse genotypes were presented in currently circulating h n virus, accelerating evolution and widespread occurrence. , to date, distinct phylogenetic clades ( - ) were identified based on h n ha, and the confirmed human infections were caused by clade , , ae , ae , ae , and . in china, human h n disease was mostly caused by clade ae ae , which was identified in isolates from confirmed patients from provinces since . clade and clade ae are responsible for the case in and , respectively. two current cases of and were due to clade ae ae . now information on receptor property has been documented in some h n viruses of clade , ae , and ae . [ ] [ ] [ ] little is known about h n virus of clade ae ae , particularly from human.recently, a , -specific sialidase-treated red blood cell (rbc) agglutination assay was developed and used for receptor specificity screening of h n virus. , the a , or a , -binding preference can be distinguished by the change of hemagglutination titer reacted with rbcs and enzymatic rbcs. since fine receptor specificity existed in h n viruses, , the glycan array including sulfated-, fucosylated-, linear sialosides, di-sialosides, or direct binding assay with synthetic polyacrylamide (paa)-based sialylglycopolymers was also recommended for the receptor-specificity surveillance on h n viruses. furthermore, the long-branched a , sialylated glycans were currently identified to predominate on the upper respiratory epithelial in human and the recognition of this topology, ¢sln-ln is the key determinant for the human-adaptation of influenza a virus. here, we analyzed the receptor-binding specificity of human h n viruses isolated in china from to . since , a total of h n infection cases were confirmed in china from provinces. the pharyngeal swabs and lower airway aspirations from the patients were collected within days after disease onset, maintained in viral-transport medium, and tested within hours.options for the control of influenza vii key: cord- -gw mgg m authors: junter, guy-alain; lebrun, laurent title: cellulose-based virus-retentive filters: a review date: - - journal: rev environ sci biotechnol doi: . /s - - - sha: doc_id: cord_uid: gw mgg m viral filtration is a critical step in the purification of biologics and in the monitoring of microbiological water quality. viral filters are also essential protection elements against airborne viral particles. the present review first focuses on cellulose-based filter media currently used for size-exclusion and/or adsorptive filtration of viruses from biopharmaceutical and environmental water samples. data from spiking studies quantifying the viral filtration performance of cellulosic filters are detailed, i.e., first, the virus reduction capacity of regenerated cellulose hollow fiber filters in the manufacturing process of blood products and, second, the efficiency of virus recovery/concentration from water samples by the viradel (virus adsorption–elution) method using charge modified, electropositive cellulosic filters or conventional electronegative cellulose ester microfilters. viral analysis of field water samples by the viradel technique is also surveyed. this review then describes cellulose-based filter media used in individual protection equipment against airborne viral pathogens, presenting innovative filtration media with virucidal properties. some pros and cons of cellulosic viral filters and perspectives for cellulose-based materials in viral filtration are underlined in the review. abstract viral filtration is a critical step in the purification of biologics and in the monitoring of microbiological water quality. viral filters are also essential protection elements against airborne viral particles. the present review first focuses on cellulosebased filter media currently used for size-exclusion and/or adsorptive filtration of viruses from biopharmaceutical and environmental water samples. data from spiking studies quantifying the viral filtration performance of cellulosic filters are detailed, i.e., first, the virus reduction capacity of regenerated cellulose hollow fiber filters in the manufacturing process of blood products and, second, the efficiency of virus recovery/concentration from water samples by the viradel (virus adsorption-elution) method using charge modified, electropositive cellulosic filters or conventional electronegative cellulose ester microfilters. viral analysis of field water samples by the viradel technique is also surveyed. this review then describes cellulose-based filter media used in individual protection equipment against airborne viral pathogens, presenting innovative filtration media with virucidal properties. some pros and cons of cellulosic viral filters and perspectives for cellulose-based materials in viral filtration are underlined in the review. virus capture/purification/concentration is critical in a number of biopharmaceutical and clinical applications. validation of virus clearance is essential in the manufacture of therapeutic proteins, in particular blood products (bryant and klein ; klamroth et al. ; de mendoza et al. ; radosevich and burnouf ; shukla et al. ). on the other hand, large-scale, efficient purification schemes of viruses/ virus-like particles are required for the production of prophylactic vaccines and gene therapy vectors (rodrigues et al. ; segura et al. segura et al. , vicente et al. a, b) . virus inactivation technologies are commonly used to fulfil viral safety. they include physical (e.g., heat application, ultraviolet-and gamma irradiation) and chemical methods (e.g., solvent/detergent treatments) or their combination (e.g., exposure to photosensitizer plus uv light) (bryant and klein ; klamroth et al. ; klein and bryant ; pelletier et al. ; prowse ; radosevich and burnouf ; solheim ) . common methods for virus capture include filtration (charcosset ; grein et al. ; liu et al. ; van reis and zydney ) and chromatography in column or membrane configurations (charcosset ; gottschalk ; liu et al. ; orr et al. ; van reis and zydney ; segura et al. ). viral filtration is usually the final purification step in the downstream processing of biopharmaceutical products, e.g., monoclonal antibodies ( fig. ) , following one or more chromatography ''polishing'' steps that contribute to the overall virus removal efficiency of the process before concentration of the purified product. filtration technologies are also extensively used for the capture and concentration of waterborne viral pathogens (gibson ) from drinking, environmental, recreational or waste water samples (cashdollar and wymer ; ikner et al. ). in addition, particulate air filters are used in personal respiratory protective equipment (i.e., face masks and respirators) cohen and birkner ; rengasamy et al. ) to ensure shortrange protection of wearers against airborne pathogens-which include a number of viruses (tang et al. )-and in air purifiers/cleaners to limit longrange aerosol transmission of infection in healthcare settings (hyttinen et al. ; tang et al. ) . cellulosic membrane microfilters have been used routinely for ages in laboratories to perform the socalled ''sterile filtration'' (cold sterilization), i.e., the absolute removal of bacteria, yeasts and molds but not viruses (walsh and denyer ) from heat-sensitive liquid media. however, many virus filtration devices currently implemented are made from cellulose and its derivatives. some eighty years ago, gradocol, graded collodion (cellulose nitrate) membrane filters (elford ; bauer and hughes ) have been extensively used in ultrafiltration to estimate the size of several medically important viruses such as foot-and-mouth disease (galloway and elford ) , vaccinia (elford and andrewes ) , herpes (elford et al. ) , poliomyelitis (elford et al. ) and influenza (elford et al. ) (see also ferry ( ) for an exhaustive review). later on, commercial mixed cellulose ester filters with very low pore size, namely vf (virus fine), vm (virus medium) and vc (virus coarse) grade filters produced by millipore (billerica, ma, usa-a subsidiary of merck kgaa, darmstadt, germany), have also been applied to virus ultrafiltration for grouping by size assessment (hsiung ). as part of a series of papers surveying the antiviral applications of polysaccharide-based materials, the present review focuses on viral filtration using cellulosic filter media-most of which are commercially available products that have been extensively tested over the past twenty years for the removal/concentration/purification of viral particles from liquid samples, i.e., biopharmaceutical (namely blood products) and environmental (raw or treated) water samples. the application of cellulose-based filters in individual respiratory protective devices protecting the wearer against airborne viral pathogens is also detailed. polysaccharide-based chromatographic adsorbents for viral clearance or recovery/purification of viruses or virus-like particles will be presented elsewhere. in the processing of biological products, virus removal from the product stream while providing maximum product recovery is a critical task. it is more particularly difficult to eliminate small viral particles such as parvoviruses, which may contaminate blood products and mammalian cell cultures used in the production of recombinant proteins (charcosset ) . viral filters developed to answer this challenge are typically membrane (screen) filters ensuring a size-based rejection of viral particles via a sieving mechanism. given the size of viruses, ranging roughly from (parvoviridae) to nm (poxviridae) (segura et al. ) , viral filtration stands between microfiltration and ultrafiltration among pressure-driven filtration processes (fig. ) , though it is frequently but incorrectly (van reis and zydney ) classified as nanofiltration (burnouf et al. ). . regenerated cellulose hollow fiber (hf) membrane filters common virus filtration membranes are made from poly(ethersulfone) (pes), poly(vinylidene fluoride) (pvdf), and regenerated cellulose (burnouf and radosevich ; carter and lutz ; liu et al. ; van reis and zydney ) . among the latter, the commercial planova tm filters (asahi kasei medical, tokyo, japan) have been widely used to clear viruses from biologically produced pharmaceuticals, in particular blood products. planova filters are composed of cuprammonium regenerated cellulose hf (bemberg tm cupro fibers), prepared from cellulose cuprammonium spinning solution via microphase separation under precise spinning conditions (tsurumi et al. b, c) . the wall of each hf has a threedimensional web structure of pores consisting of large, bulky void pores interconnected by fine capillaries (tsurumi et al. a, b, c) (fig. ). during filtration, as the feed solution containing the product of interest is circulated through the hf bore, viruses accumulate in the large, bulky void pores of the fiber network while the product solution passes through the capillary pores. since the hf wall is several tens of micrometers thick (fig. ) , viruses are captured gradually inside the porous structure that can be considered multi-layered ( - layers) (hongo-hirasaki et al. ) . hence, planova filters behave like ''membrane depth filters'' (walsh and denyer ) operating on the basis of size exclusion. they offer a choice of nominal mean pore sizes, namely nm ( n), nm ( n), nm ( n) and nm ( n)-the large pore n model being essentially used as a prefilter to remove impurities or aggregates prior to final virus filtration-and can be operated in normal (dead-end/flow through) or tangential flow (cross flow) filtration mode (phillips et al. ). planova filters have emerged to answer a publichealth problem of worldwide magnitude, i.e., blood contamination by hiv (hamamoto et al. ; manabe et al. ) and hepatitis viruses (yuasa et al. ; sekiguchi et al. sekiguchi et al. , . table gives examples of viral clearance studies assessing the capacity of planova filters to remove viruses from blood products. in these viral clearance assays, product streams resulting from successive, scaleddown purification steps representative of the manufacturing process of the product (kundu and reindel ) , were artificially contaminated (''spiked'') with model viruses of different sizes (table ) (at much higher concentrations than what might be commonly found in the product intermediate) before being submitted to filtration. the virus removal efficiency of the filtration step was expressed as lrv (log reduction value, i.e., log ratio of the viral load in the spiked product feed stream to that recovered in the product filtrate), which implies that residual virus infectivity can never be reduced to zero but may be greatly reduced mathematically (ich ) . minimum lrv values were most frequently given. they were estimated when no viruses could be detected in filtered samples taking into account the detection limit of the assay (viral titer estimated as infectious unit per sample volume). from the measured lrvs, the filtration operation is usually classified as effective (lrv [ ), moderately effective ( \ lrv \ ) or makino et al. ( ) rev environ sci biotechnol ( ) ineffective (lrv \ ) (phillips et al. ). since filtration complements other viral clearance methods in the downstream processing of biotherapeutics (brorson ; klamroth et al. ; shukla et al. ) , several studies quoted in table also report lrvs for common virus reduction steps such as inactivation by pasteurization (gröner ; gröner et al. ; terpstra et al. ) or solvent/detergent treatment (dichtelmüller et al. ; terpstra et al. ) , and chromatographic capture (gröner ; gröner et al. ) . virus removal during the plasma fractionation process (bryant and klein ) leading from the crude plasma pool to the product (immunoglobulin here) stream has also been quantified (dichtelmüller et al. ; koenderman et al. ; terpstra et al. ) . overall virus reduction factors obtained by addition of successive lrvs are given in these works, as illustrated by table . in the virus spiking studies quoted in table , the actual viral filtration step was preceded by prefiltration to eliminate viral and/or protein aggregates that might be present in the spiked product solution. the presence of aggregated viruses may lead to overestimation of size exclusion effectiveness while contributing with protein aggregates to the fouling of viral filters, which reduces the membrane hydraulic permeability and may affect virus retention (phillips et al. ). pre-filtration was performed on the spikevirus stock suspension (caballero et al. ; gröner et al. ; roberts et al. ) on the product intermediate before virus inoculation (gröner et al. ; koenderman et al. ) , and/or on the virus furuya et al. ( ) and terpstra et al. ( ) cpv canine parvovirus b v gröner ( and terpstra et al. ( terpstra et al. ( , spiked material (chtourou et al. ; dichtelmüller et al. ; koenderman et al. ; terpstra et al. terpstra et al. , . for pre-filtration of the virus preparation, micro filters with a pore size adapted to the spike virus size, i.e., slightly lower than the virus size, were used to remove cell aggregates but not single viral particles-usual pore sizes ranging between . and . lm. micro filters with . - . lm pore size (koenderman et al. ; terpstra et al. ; , but also n (dichtelmüller et al. ) and n (chtourou et al. ) . planova viral filters were used to pre-filtrate the spiked product solution. in the latter work, the association in series of n and n filters led to efficient removal of small viruses such as ppv (chtourou et al. ). this was not the case when n was used as the main viral filter after pre-filtration with n (dichtelmüller et al. ) , which confirms previous date showing that the nm pore size was too large to retain the smallest viral particles (furuya et al. ; hongo-hirasaki et al. ). both n (caballero et al. ; furuya et al. ; gröner ) and n (caballero et al. ; roberts et al. ; terpstra et al. ; planova filters and their combination (gröner et al. ; koenderman et al. ) showed effective virus removal over a wide range of viral particle sizes (table ) . as a general rule, the removal efficiency of the filters increased with the ratio of pore size to virus size, as illustrated by gröner ( ) and terpstra et al. ( ) . this was also demonstrated by roberts et al. ( ) by filtrating poliovirus type (a picorvanirus) through planova filters manufactured with different pore sizes, ranging between (lrv = . ) and nm (lrv = . ). the two-filter combinations were globally more efficient than single filters at removing small viruses from spiked protein intermediates, though two n filters connected in parallel for tangential filtration (koenderman et al. ) yielded lrvs quite similar to those obtained using n alone (terpstra et al. ). care should be taken when comparing vertically the data collected in table , however. in addition to the sizes of viral particles and filter pores, the results of such virus-spiking studies are dependent on a number of process parameters such as the filtration operating conditions (filtration mode, transmembrane pressure, volume per filter area, tempera-ture…), the characteristics of the product feed stream (ph, conductivity, nature and concentration of the product…), or else the purity level and concentration of the virus spike. moreover, several studies include pre-filtration (dichtelmüller et al. ) or viral inactivation (koenderman et al. ; terpstra et al. ) in the calculation of lrvs. this variability in test conditions for filtration-based virus removal makes difficult any comparison between lrvs collected from multiple sources as in table , even though some results (caballero et al. ; hongo-hirasaki et al. ; roberts et al. ) contradict its influence on filtration performance, underlining the robustness of the filtration process. also, some variations in virus removal capacity may occur between commercial filter modules owing to their manufacture from different batches of hf, as illustrated by roberts et al. ( ) for n filters. like membrane microfiltration, depth filtration is widely used as a clarification step in biopharmaceutical purification to ensure removal of cell debris, large aggregates and contaminants from the product stream prior to purification processes such as column/membrane chromatography and viral filtration/inactivation steps (liu et al. ; van reis and zydney ; vicente et al. vicente et al. a, b) . commercially available depth filters currently employed in bioprocessing are composed of cellulose fibers with a porous inorganic filter aid and a resin binder, generally cationic. for instance, the zeta plus tm filter media ( m purification inc.-formerly cuno, meriden, ct, usa) is composed of a cellulose fiber depth matrix containing silica-based filter-aid material and positively charged by chemical bounding of a cationic charge modifier (ostreicher ) . varying retention ratings are available ( . - . lm). the triple layered a hc filter media from emd millipore include two depth filtration layers (cellulose ? diatomaceous earth) of different grades and a . lm-pore-size microporous membrane pre-filter made of mixed cellulose esters (van reis and zydney ). hence, these cellulosic depth filters rely on both size exclusion and electrostatic adsorptive binding to effect separation (liu et al. ) . in viral spiking studies (barnette et al. ; dichtelmüller et al. ; zhou et al. ) , they were found to retain viral particles whose surface is negatively charged over a wide range of ph as their isoelectric points (pi) range most frequently between . and . , with a mean value of . ± . (michen and graule ) . viruses are recognized as a major cause of waterrelated disease (bosch et al. ; fong and lipp ; gibson ; hamza et al. ) . enteric viruses, more particularly, which are associated primarily with gastrointestinal illness (bosch et al. ; fong and lipp ; gibson ; hamza et al. ) , are implied in most waterborne viral outbreaks (gibson ; hamza et al. ). all types of water, including waters used for drinking (surface or ground supplies), recreational waters (fresh, marine, and swimming pool), agricultural waters for irrigation (rivers and groundwater) and waste waters (sewage or industrial effluents), have been shown to be potential vehicles for virus transmission (bosch et al. ) . contamination is most frequently of fecal origin (bosch et al. ; wong et al. ) . although viral levels may be high at the contamination source, e.g., concentrations ranging between and virus particles per gram of feces are referred to in the literature (michen and graule ) , viral concentrations as low as - viral particles per liter may be found in environmental water (julian and schwab ) . since enteric viruses display high infectivity (fong and lipp ; julian and schwab ) , such titers may constitute a health risk and should be detected for reliable surveillance of viral pathogens in water. as a consequence, efficient concentration methods are needed to capture viruses in large-volume water samples and release the retained viral material in concentrated form. most common methods are based on filtration processes (cashdollar and wymer ; ikner et al. ). in particular, virus adsorptionelution (viradel) filter methods have been widely implemented since the s (cashdollar and wymer ) . briefly, in a viradel method, viruses from aqueous samples are reversibly adsorbed to microporous filters and then eluted from the filters in a small liquid volume (apha, awwa and wef ). adsorbent filters carry electrical charges and virus retention occurs via electrostatic interaction rather than by size exclusion. in other words, viradel filters act as depth filters rather than sieves. contrary to viral filters that retain viruses by a sieving mechanism to achieve complete viral clearance, their pore size lies in the microporous range, which allows the high flow filtration necessary for virus capture in large water samples. positively or negatively charged filters can be used in a viradel procedure, among which those made of cellulosic materials have been dominant over the last few decades. since their pi is below the ph of natural water (i.e., around neutrality) ( huang et al. ( ) table continued filter type a water nature no. [ tested earlier for concentration of viruses in water samples of different origins (table ). more recent studies made use of zeta plus tm mds microfilters, however (table ). in these works, pure (e.g., distilled), tap, environmental or sewage water samples were seeded with varying enteric viruses that are common waterborne pathogens, i.e., members of the families picornaviridae (polioviruses, coxsakieviruses, teschoviruses), adenoviridae (adenoviruses), caliciviridae (noroviruses, caliciviruses) and reoviridae (rotaviruses) (fong and lipp ) , or bacteriophages (enterobacteria phages) that are considered as alternative indicators of fecal contamination and as index organisms for the presence of enteric viruses in waters (goodridge and steiner ). naturally contaminated waters containing sufficient levels of indigenous viruses, more particularly bacteriophages, were also used in addition to spiking studies sensu stricto (table ) . following sample filtration, viral particles adsorbed to the filter were eluted in concentrated form using a variety of eluting solutions, the most common eluent consisting of a slightly alkaline (ph . - . ) protein solution (i.e., beef extract), frequently buffered with glycine-naoh or another amino acid solution (sometimes supplemented with salt to aid disruption of electrostatic interactions between viruses and filters (shields and farrah ) , a chaotropic agent (e.g., urea) or a surfactant (e.g., tween ) to affect virus-filter hydrophobic interactions . eluted viruses were quantified-using plaque titer assays, % tissue culture infectious dose (tcid ), quantitative realtime pcr (qpcr) and reverse-transcription pcr (rt-qpcr) (hamza et al. )-to assess the recovery efficiency of the filtration step by comparing to input titers. this efficiency depends on a number of process parameters including the filter type and filtration conditions (e.g., filtration rate and pressure), the elution buffer composition and eluting conditions, the nature, input titer and titration method of the tested virus-in addition to the water matrix characteristics that may affect virus quantification and filter performance (borchardt et al. ) . hence, tables and display a wide range of recovery yields. in practical tests aimed at the detection of naturallyoccurring viruses in field, large-volume water samples, the filtration (adsorption-elution) step is usually a primary concentration step which is followed by a secondary one to reach the virus detection threshold, e.g., organic flocculation or peg precipitation (lewis and metcalf ) . the recovery yield of such two-step concentration processes was evaluated in several studies using seeded or naturally contaminated water samples (tables , ) . as a general rule, the viral loss due to secondary concentration was balanced by a huge increase in concentration factor allowing virus detection in water concentrates from large-volume samples, as reported by chang et al. ( ) and raphael et al. ( ) for indigenous enteroviruses in wastewater and rotaviruses in sewage-polluted surface water, respectively. after determining the optimal elution conditions for mds disk-adsorbed noroviruses following filtration of spiked distilled water samples, lee et al. ( ) (table ) applied the optimized procedure to the detection of noroviruses in environmental water. they combined adsorptive filtration using mds cartridge with organic flocculation to enrich viruses from largevolume surface ( l) and ground ( l) water samples. this integrated two-step process led to high volume reduction factors, i.e., , and , for surface and ground water samples, respectively ( - for filtration, for organic flocculation), but its recovery efficiency was not evaluated by spiking studies. in the same way, beside spiking woo et al. ( ) . a ratio of the virus survival factor in the treated filter to that in the untreated filter, where the virus survival factor in a filter is the number of viruses recovered by elution from the filter divided by the number of viruses removed by the filtration process. b pf pp filter from commercial surgical mask (dupont tm n), ccf coarse pore cellulose filter paper (whatman tm grade , lm pore size), fcf fine pore cellulose filter paper (whatman tm grade , . lm pore size) studies like those detailed in table , mds filter cartridges have been widely applied to concentrate enteric viruses in water samples of different origins (table ) , ranging from large-volume samples of weakly contaminated water intended for drinking or drinking water production (borchardt et al. (borchardt et al. , lee et al. ; sedmak et al. ; verheyen et al. ) to wastewater samples with high viral content-to assess the virus removal efficiency of wastewater treatment plants (kuo et al. ; simmons et al. ) . in these tests of field water samples for enteric viruses, mds filtration was mainly combined with second-stage concentration by organic flocculation as detailed by fout et al. ( ) in the usepa (united states environmental protection agency) manual of methods for virology. the elution of filter-adsorbed viral particles was mostly performed with an alkaline beef extract solution supplemented or not with glycine (buffers eb and eb in table ). the work by verheyen et al. ( ) is an exception to these common procedures. to concentrate viruses in small-volume samples from drinking water sources, these authors used two filter cartridges in series with no additional concentration step and an alkaline powdered milk solution as elution buffer. they found that % ( / ) and % ( / ) of surface and ground (well) water sources, respectively, were contaminated with adenoviruses, with very few samples positive to rotavirus ( / surface water samples, / well water sources). despite the differences in virus concentration methods, these results compare with those obtained by xagoraraki et al. ( ) and cheong et al. ( ) for adenovirus detection in surface ( / samples, %) and ground water ( / samples, %), respectively. they also agree with data reported by borchardt et al. ( ) and cheong et al. ( ) showing the low contamination level of ground water by rotavirus. it is worth noting here, however, that the number of positive samples reported in these viral analyses of water (and the others quoted in table ) is dependent on the sensitivity of the detection techniques used to assess the presence of viruses, i.e., nucleic acid-based amplification methods such as conventional pcr, reverse-transcription pcr (rt-pcr), nested pcr, real-time pcr/rt-pcr or integrated cell culture pcr (icc/pcr) to determine virus infectivity (fong and lipp ; hamza et al. , mattison and bidawid ; watzinger et al. ) with different detection thresholds. it emerges from this glance at literature that cellulose-based electropositive filters are still commonly used for viral monitoring of water. these filters are expensive, however, and face competition with cheaper products, in particular nanoalumina fiber filters and glass wool filters (cashdollar and wymer ; wong et al. ) . the former, nanoceram tm filters manufactured by argonide corporation (sanford, fla., usa), are composed of nanosized ( nm diameter), alumina-based {mainly boehmite, c-alo(oh)} fibrilles dispersed in a microglass fiber matrix, resulting in an electropositive filter media with - lm average pore size kaledin , ) . the latter consist of commercial sodocalcic glass wool coated with mineral oil (type bourre qn/tech loose wool, isover saint-gobain, courbevois, france), hand packed into columns or filter holders in the laboratory (vilaginès et al. ) . these glass wool filters harbor electropositive sites while presenting hydrophobic surface characteristics. efficient enrichment of (seeded or/ and) indigenous viruses from various water samples using nanoceram (gibbons et al. ; ikner et al. ; pang et al. ) , and glass wool (deboosere et al. ; lambertini et al. ; wyn-jones et al. ) filters has been reported in recent years (see also cashdollar and wymer ; wong et al. ) . compared with mds filters (table ) , nanoceram filters showed similar , slightly lower (mcminn ) or higher (karim et al. ) virus concentration efficiency. commercially available under different pore sizes ( . - . lm), mixed cellulose ester membrane filters are negatively charged over a wide range of ph values, their overall negative charge increasing with ph (kessick and wagner ) . microporous filters in the . - . lm pore size range have been used for ages in laboratory and industry for size-based filtration of bacterial particles and cell debris (surface filters). these and larger pore size filters have been shown to retain enteroviruses, however, despite the much smaller size of viral particles compared to the nominal mean diameter of filter pores (cliver ). the presence of salts enhanced virus adsorption to cellulose ester membranes, this effect increasing with the cation valence (i.e., al ? [ mg ? [ na ? ) (wallis et al. ) . acidification of the viral suspension also improved virus adsorption efficiency, even in the absence of exogenously added salts (sobsey et al. ). these early results were later confirmed by lukasik et al. ( ) , who investigated the influence of mono-, di-, and trivalent salts (nacl, mgcl , and alcl ) on the adsorption of poliovirus and enterobacteria phages to the mf-millipore tm membrane filter type ha ( . lm pore size) and the electropositive m tm zeta plus tm mds microfilter at neutral or acidic ph. at ph , salts promoted virus adsorption to ha filter while affecting adsorption to mds filter. at ph . , more than % of the viruses tested adsorbed to ha filter with or without salt added to the viral suspension-the salts interfering again with viral adsorption to mds filter. furthermore, the addition of urea or tween to the salt solution affected virus adsorption to both filters at ph . and ha filter at ph . in agreement with previous studies by the same group (haramoto et al. (haramoto et al. , lukasik et al. ( ) explained these results by the antichaotropic effect of salts that increased hydrophobic interactions between filters and viruses and was impaired by the chaotropic agent or the detergent. at neutral ph, charge screeening by salt addition reduced electrostatic attractive and repulsive forces between viruses and mds or ha filter, respectively. cation-mediated cross-complexation between negative groups on virus and filter surfaces (kessick and wagner ) and, more particularly, strengthened hydrophobic virusfilter interactions contributed to improving the adsorption efficiency of ha filter. at acidic ph, viruses displayed a positive surface charge and their electrostatic interactions with ha filter switched from repulsive to attractive-and inversely for electropositive mds filter. the presence of salt probably affected these interactions but was balanced by the promoting effect of salt on hydrophobic interactions. hence, most tests reported so far for virus concentration from water samples using cellulose ester filters (essentially ha filters) have been performed after addition of multivalent cations (mainly mg ? ) to the water samples with or without ph adjustment to an acidic level. according to katayama et al. ( ) , the virus-loaded filters were rinsed with an acidic solution to eliminate remaining cations before elution with naoh or other alkaline buffers. haramoto et al. ( haramoto et al. ( , haramoto et al. ( , a proposed a variation in the method that consisted in pre-conditioning the filter with al ? ions. aluminum chloride was passed through the filter, making an electropositive ion coating, which avoided cation addition to the water sample. the recovery efficiency of both protocols has been evaluated by seeding water samples with enteric viruses and bacteriophages and quantifying eluted viruses (table ) . similarly to spiking studies (tables , ) and viral analyses of field water samples (table ) with electropositive filters, the ha-based filtration/elution process was frequently associated with a secondary concentration step to increase virus concentration factors. here, centrifugal ultrafiltration (cu) using commercial millipore (centriprep Ò , centricon Ò or amicon Ò ) concentrators was the elective concentration method. these cu units contain a low adsorptive regenerated cellulose membrane (ultracel Ò ) whose nominal molecular-weight cutoff ranges between and kda-a mwco value of kda being most frequently selected for secondary concentration of eluted viruses. they are routinely used in laboratories to purify and concentrate biomacromolecules such as peptides, proteins, and nucleic acids from small-volume biological samples (e.g., - ml for the centriprep filter unit). tested or not for virus recovery by spiking experiments, the combined concentration procedures have been extended to the detection of enteric viruses in field water samples including tap (haramoto et al. ), sea (katayama et al. ) , river (surface) (fong et al. ; hamza et al. ; haramoto et al. ) and waste (fong et al. ; katayama et al. ) water (table ) . samples with different volumes were collected according to the water source, i.e., sample volumes increased as the expected contamination level decreased. for instance, katayama et al. ( ) applied ha filtration, followed by acid rinse of the filter, viral elution with naoh and secondary concentration of the eluate by cu, to concentrate naturally occurring viruses (noroviruses, enteroviruses and hav-hepatitis a virus) in seawater samples. based on the volume reduction factor ( -l sample/ ml final concentrate, i.e., ) and the recovery yield of the two-step process (table ) , a virus concentration factor of was reached. only hav virus was not detected in any sample tested. later on, katayama et al. ( ) followed the same protocol to concentrate enteric viruses in the raw influent of a wastewater treatment plant. samples of -ml volume were collected and submitted to the two-step concentration process, yielding a volume reduction factor of c. . the four tested kinds of enteric viruses were detected in all wastewater samples but one lacking norovirus gi (nov gi). using peg precipitation as the secondary concentration step, hamza et al. ( ) tested river water samples for contamination by enteric viruses and bacteriophages. a volume reduction factor of ( -l sample/ ml final concentrate) and virus concentration factors ranging between (norovirus) and (adenovirus) (see table for recovery yields) were obtained. all analyzed samples were found positive for enteric viruses. human adenovirus and norovirus were detected in . and % of the samples, respectively. haramoto et al. ( ) illustrated the ability of al ? -coated filters to concentrate viruses from large-volume freshwater samples without salt addition by detecting noroviruses in tap water from tokyo university. tap water samples ( -l average volume) underwent two successive filtration/elution steps using -mm and -mm diameter ha filters prior to concentration by cu (final volume: . ml), which ensured a volume reduction factor higher than , . however, the virus recovery efficiency of the -step concentration process was not evaluated. ten of the tested samples were found positive for noroviruses. these results compare to those reported by lee et al. ( ) ( table ) using mds cartridge with organic flocculation to enrich noroviruses from large surface and ground water samples. despite these promising data, the two viradel methods based on electronegative filters (i.e., addition of mgcl to water samples or filter coating with al ? ions before filtration) have been essentially implemented for viral analysis of waters containing high amounts of indigenous viruses, requiring limited sample volumes (table )-electropositive filter cartridges being more adapted to virus concentration from large volumes of weakly contaminated water (table ). beside the above-mentioned work by haramoto et al. ( ) , only two studies among those detailed in table describe virus detection in environmental water samples with low viral content, namely drinking water sources after chlorination or without treatment (rigotto et al. ) and groundwater from artesian wells (chironna et al. ) . both studies used small-volume samples and cu as secondary concentration step, yielding a modest volume reduction factor of . very few from artesian well water samples were found positive to the tested enteric viruses: none for hav and enterovirus, for rotavirus and for norovirus (chironna et al. ) . a large proportion ( %) of samples from drinkingwater supplies was positive to adenovirus, however (rigotto et al. ) . these data are compatible with viral analyses of groundwater and water intended for drinking performed using mds filtration as the first virus concentration step (table ). it should be noted, however, that the same remark applies to data collected in table as to those in table concerning the numbers of virus-positive samples, i.e., their dependence on the virus detection method. this can be illustrated by the two following examples. using icc-pcr (measuring infectious viruses), katayama et al. ( ) detected enteroviruses in of seawater samples from bathing beach, but no sample was found positive by direct rt-pcr. de paula et al. ( ) found / river water samples positive for hav by quantitative real-time rt-pcr, but only / by nested rt-pcr. as attested by the large body of literature data quoted above, virus adsorption-elution methods using electropositive or electronegative filters are routinely applied to the primary concentration of waterborne viruses, each type of filter possessing its own advantages and drawbacks. ultrafiltration is considered another filtration-based option to concentrate viruses from water samples (cashdollar and wymer ; ikner et al. ). it has been indicated earlier that cu with microconcentrators based on cellulose ultrafiltration discs is commonly used as a secondary concentration step following the viradel process. hollow fiber ultrafiltration (hfuf), however, is considered a potential technique for primary concentration of viruses from large-volume water samples, yielding better virus recoveries than the viradel method performed with either electropositive or electronegative filters (cashdollar and wymer ) . commercial hfuf devices operated in cross-flow mode have been applied to the simultaneous concentration of biological particles, including viruses, spiked in tap (polaczyk et al. ) or reclaimed (liu et al. ) water samples, to virus recovery from seeded tap (rhodes et al. ) and estuarine (hernandez-morga et al. ) water samples, and also, more rarely, to virus recovery from field water samples (grassi et al. ; hernandez-morga et al. ). most of these devices are dialyzers that contain synthetic hf made from polysulfone. following the early work by belfort et al. ( ) showing that polysulfone hf membranes were superior to cellulose acetate ones for virus concentration, virus concentration experiments using hfuf dialyzers equipped with hf manufactured from cellulose are scarce. an example is given by liu et al. ( ) . they showed that the exeltra plus cellulose triacetate hf dialyzer (baxter healthcare corp., deerfield, il, usa) and the optiflux Ò f nr polysulfone dialyzer (fresenius medical care, walthamm, ma, usa) provided similar recovery efficiencies for ms and ux bacteriophages, escherichia coli, clostridium perfringens spores, and cryptosporidium parvum oocysts from spiked -l and -l samples of reclaimed water. an array of viral infections can be transmitted by the airborne route, in particular via aerosols (droplet nuclei) tang et al. ; tellier ; verreault et al. ). even though virus inactivation rates in the atmosphere are generally higher than those of bacterial and fungal contaminants, virus-containing aerosols can spread worldwide (després et al. ) . the threat of viral outbreaks and pandemics such as those caused by sars coronavirus (yu et al. ) and highly pathogenic strains of influenza a virus (tellier ) , allied with the fear of bioterrorism using viruses (e.g., smallpox and hemorrhagic fever viruses) (barras and greub ) as biological weapons, has encouraged the search for efficient protection equipment against aerosolized virus-containing particles. individual respiratory protective devices consist in respirators, i.e., air-filtering face masks designed to protect the wearer against inhalation of a hazardous atmosphere (here airborne infectious aerosols)-opposing to surgical masks designed to protect the environment from contaminants generated by the wearer's exhaled breath (i.e., prevention of surgical infections) . currently, most ''filtering facepiece'' (ffp) respirators have a multilayer composite structure with a central filtering layer displaying electret properties (gralton and mclaws ) . these electret filters (thakur et al. ) are produced by imparting an electrostatic charge to a nonwoven fibrous mat composed of synthetic polymer fibers such as poly(propylene) (pp) (mainly), poly(butylene terephthalate), poly(tetrafluoroethylene) and poly(carbonate) fibers. electrostatic charging of the filter media is commonly obtained by corona discharge, triboelectrification and electrostatic spinning (tsai et al. ) . electret filters collect particles through the combined action of mechanical and electrostatic forces (podgórski ; wang (cen ) should retain respectively at least and % of influent particles (nacl particles are used as nonbiological surrogate particles) (rengasamy et al. ). first worn by surgical teams a hundred years ago to prevent bacterial contamination of patient's open wounds, early surgical masks were constructed from layers of cellulose materials, more particularly cotton cellulose (gauze) (haller and colwell ) and derivatives (arnold ) (see also belkin ) . much more recently, face masks made from cotton fabrics have been tested as alternative respiratory protective equipment against pandemic outbreaks such as influenza (dato et al. ; davies et al. ; rengasamy et al. ) . like respirators, current commercially available surgical face masks include several layers of non-woven fabrics with, frequently, a cellulose inner layer in contact with the wearer's face to improve wearer's comfort. however, the filtering layer, usually made of meltblown fibers (ghosh ) , is devoid of electret properties. since particulate filtration is only mechanical, i.e., less efficient than that of respirators, most surgical masks are not certified for use as respiratory protective devices, standard tests to evaluate their filtration efficacy being less stringent (oberg and brosseau ) . while a number of studies have confirmed that surgical masks logically offer lower protection than respirators against aerosol particles, the effectiveness of both types of face masks at preventing viral respiratory infection, in particular influenza, is still a matter of controversy (bin-reza et al. ; cowling et al. ; gralton and mclaws ) . the improper facial fit of respirators coia et al. ; weiss et al. ) may affect their protection efficiency against infectious aerosols. it allows particulate flows outside the filtration area of the mask, resulting in face-seal leaks, i.e., the leakage of infectious particles around the edges of the mask (grinshpun et al. ; lei et al. ) . mask seal leakage can be minimized by applying existing guidelines for correct donning and fit checking of respirators . moreover, human face and head form models have been proposed as a tool for designing respirators with improved protection efficiency, simulating interactions between faces and facemasks and describing their fit (golshahi et al. ; lei et al. ) . the accumulation of viral particles at the surface and within the filtration media of respirators, where they can remain viable and infectious for extended periods of time (coulliette et al. ; sakaguchi et al. ) , is another significant problem with which respirator wearers are confronted in the prevention of viral transmission and spread. incorrect mask handling by the wearer may lead to accidental self-inoculation, cross-contaminations affecting both other healthcare workers and patients, and contamination of fomites (casanova et al. ) . furthermore, the risk of virus reaerosolization from respirators during extended use, if limited, cannot be ruled out (fisher et al. ) . recommendations for respirator doffing do exist but have proven insufficient to prevent virus transfer from respirator to healthcare employees' hands and clothing (casanova et al. ) . adding antiviral properties to the filtration process may contribute to limit the risk of viral transmission by improper handling of used respirators. a number of virucidal facemasks have been developed and patented over the past years (tiliket et al. ) , some of which are commercially available. in the spectrashield tm plus respirator masks designed by nexera medical inc. (fort lauderdale, fla.) (haas ), for instance, two layers of filter media are sandwiched between (inner and outer) antimicrobial layers where a silver-copper zeolite (carrier) antimicrobial agent (agion Ò antimicrobial, sciessent llc, wakefield, mass.) is embedded around the core of synthetic fibers (fosshield Ò , foss manufacturing, hampton, nh). the biofriend tm biomask tm ffp respirator, manufactured by filligent ltd. (hong kong) and distributed by medline industries, inc. (mundelein, ill.) , is another four-layered device in which the melt-blown pp filtration layer is inserted between an inner layer of spunbond pp and two antiviral layers, namely a cellulose/polyester layer containing copper and zinc ions and an outer layer of spunbond pp treated with a low ph (citric acidacidified) hydrophilic plastic coating (davison ; stewart et al. ) . the outer layer absorbs infectious aerosol droplets and viruses are denaturated by exposure to citric acid. in the second layer, inactivation of viruses with damaged structures is completed by the virucidal effect of divalent metal cations. contributing to the overall antiviral efficiency of the structure, the cellulosic component of this layer is a sulphated or sulfonated rayon fabric (stewart et al. (stewart et al. , to which a variety of viral human pathogens bind via the cationic sites of viral envelopes/capsids. beside the commercial biomask tm respirator, various composite structures designed for filtrationinactivation of airborne microbial contaminants, where cellulosic materials play an active antimicrobial role, have been patented in recent years bernard ; nakamura and nakamura ; tsutsumi ; zhang et al. ; zhong ) . several of them are based on bacterial cellulose layers/coatings provided with an antimicrobial component, i.e., silver (zhong ) or zeolite-supported silver (nakamura and nakamura ) nanoparticles, and chitosan (zhang et al. ) . the respiratory protective mask described by zhong ( ) comprises a three-layer bacterial cellulose membrane whose middle layer contains silver nanoparticles. the antimicrobial facemask invented by nakamura and nakamura ( ) includes two base cloth elements both of which are made of a woven textile (gauze), a nonwoven cellulose fabric (e.g., rayon), or a porous sheet (e.g., a . - . mm sliced sheet of urethane sponge). the first base cloth is filled with bacterial cellulose nanofibers that retain silver zeolite and a humectant (e.g., trehalose or , -butylene glycol) in their network structure. additional antimicrobial properties are provided to the mask by impregnating the second base cloth with a carboxylic acid (e.g., citric acid). zhang et al. ( ) also used bacterial cellulose in a composite antimicrobial material suitable for respiratory protective masks. a nonwoven polymer fabric rev environ sci biotechnol ( ) : - was coated with a film of bacterial cellulose mixed with poly(vinyl alcohol)-to improve the film-forming properties, mechanical strength and air permeability of the coating-and chitosan, a non-sulfated polysaccharide produced commercially by deacetylation of chitin that displays strong antibacterial potency (kong et al. ; rabea et al. ) and, to a lesser extent, antiviral activity (rabea et al. ; wang et al. ). in the antimicrobial air filtration device proposed by bernard ( ) , cellulose derivatives-more particularly, cellulose acetate phthalate (cap), a common pharmaceutical excipient for enteric coating of tablets and capsules with antiviral activity (pirrone et al. )-were used as ''biocidal prophylactic compounds'' to provide the classical filter media (e.g., a nonwoven pp web, electrostatically charged or not) with antimicrobial properties. cellulose derivatives could be incorporated as fibers or particles (micronized fibers) into the air filtration device either in a separate layer positioned before or after the filter media, or deposited onto the outer surface of the filter media relative to the air flow direction. to illustrate this invention, handsheets prepared from cap fibers intermixed or not with cap particles were successfully tested for virucidal efficacy by exposure to an aerosol challenge of enterobacteria phage Øx . by exploiting the antimicrobial properties of dialdehyde polysaccharides such as starch and cellulose dialdehydes, baney et al. ( ) proposed a new method for producing low-cost virucidal filters suitable for respiratory protective masks. the treatment of a standard cellulose filter paper (whatman tm grade filter paper) with sodium periodate improved its antiviral potency due to oxidation of some cellulose to dialdehyde cellulose inside the filter. challenged with aerosols of ms enterobacteria phage at high relative humidity ( % at °c), the treated filter showed higher filtration efficiency than a control, untreated one, i.e., better removal of viable viral particles (plaque forming units) joined to a lower resistance to air flow (pressure drop)-due to increased pore size distribution (woo et al. ) . higher inactivation of ms virus by the treated filter was also highlighted, confirming an improvement of its disinfection capability (woo et al. ). however, the filtration and inactivation of airborne viruses were less effective at lower moisture content of the filter, i.e., at air humidity levels commonly encountered in hospital settings, in particular operating rooms (balaras et al. ). furthermore, the inhalation resistance of the treated filter remained too high for application to respiratory protective masks. the same pros and cons apply to cellulose paper filters treated by immersion into aqueous suspensions of dialdehyde starch (das) (woo et al. ) . while this treatment did not affect the removal efficiency of viable ms viruses from aerosols at high relative humidity, virus survival on the treated filters was reduced (fig. ) and the pressure drop decreased as das concentration increased. the drop in air flow resistance remained insufficient for practical application to respirators. das treatment of a pp filter from a commercial ffp mask did not modify the filtration parameters (virus removal efficiency and pressure drop) of the filter but significantly improved its biocidal efficacy (fig. )-an opening of this treatment towards the development of virucidal facemasks. another example of cellulose filter material treated with cellulose derivatives to yield antiviral properties is given by tsurumi (tsutsumi ). this inventor devised a low-cost virucidal mask filter made of a standard cellulose nonwoven fabric impregnated with water extracts of sulfonated or aminated styrenegraft cellulose. to reinforce the water retention properties of the filter media, a superabsorbent resin such as poly(acrylic acid) was added to the graft cellulose extract before impregnation of the fabric or incorporated into a separate layer covering the impregnated fabric. low (hydrogen ions from sulfonic acid groups) or high (hydroxyl ions generated by aminated groups) ph conditions inside the hydrated material were claimed to make it virucidal against influenza a virus and caliciviruses. the patent gives no illustration of such antiviral properties, however. studies published by tiliket et al. ( ) and catel-ferreira et al. ( ) present another type of cellulosebased material for airborne virus filtration in which a low-cost nonwoven cellulose material, i.e., commercial kimwipes Ò (kw) wipes (kimberly-clark worldwide, inc., dallas, tex.), was chemically modified by coating with a synthetic polymer, i.e., poly(ethylenimine) (pei) (tiliket et al. ) or grafting of an antiviral agent, i.e., catechin (catel-ferreira et al. ) . the filtration efficiency of the modified filter media was first tested on aerosolized t d viruses (enterobacteria phage t , doermann's strain t d). then the treated filter was inserted inside a commercial medical mask in place of its cellulosic layer (kolmi m mask, kolmi-hopen, saint-barthélémy-d'anjou, france), and the reconstructed mask was challenged with td aerosols to evaluate its virus removal efficiency. both treatments significantly improved the virus capture factor (ratio of upstream to downstream pfu contents) of kw cellulose wipes and of reconstructed commercial masks compared to original masks and to masks reconstructed with untreated wipes. in these studies, the breathability of reconstructed masks was not quantified, nor was the virus survivability on the modified filter media. however, additional filtration experiments using aerosols of influenza a virus (low pathogenic h n strain) showed that challenged viral particles accumulated in pei functionalized kw layers with no loss in number and infectivity (tiliket et al. ) . thus these modified masks showed no virucidal effect, the peimodified layers behaving like an electret media owing to the polycationic character of pei. on the other hand, since catechin and catechin-grafted wipes showed antiviral activity against t d viruses in liquid media (catel-ferreira et al. ) , it may be assumed that part of viruses accumulated in excess in the treated wipes were inactivated by the polyphenol agent, i.e., the reconstructed masks were actually virucidal. despite these few examples, the use of cellulosic media in filtration devices aimed at the capture of airborne viral particles remains now very limited compared to filtration of virus-contaminated liquid samples. while patented, several cellulose-based, virucidal filter media designed for insertion in air filtering face masks have not been commercially developed yet. besides the wearing of personal respiratory protective equipment, another aspect of the fight against airborne infections lies in the decontamination of indoor air in healthcare facilities, and, more generally, of air processed through heating, ventilating and air conditioning (hvac) systems in the built environment. high efficiency particulate air (hepa) filters are the primary technology used for particulate removal in these collective protection devices-allied with uv irradiation, ozonation or air ionization to yield indoor air decontamination (bolashikov and melikov ; jacob et al. )-and may be complemented with a photytocatalyst such as tio to improve the inactivation of accumulated microorganisms pigeot-remy et al. ). since hepa filters are essentially made of glass fibers, they are beyond the scope of this review and their virus-retentive properties will not be detailed further. most frequently associated with other virus reduction (virus clearance of biopharmaceuticals) or concentration (viral analysis of water) steps, size-exclusion and/ or adsorptive (charge mediated) filtration is an essential tool to fight against viral contaminants in aqueous media. owing to inherent properties of cellulose, including mechanical strength and hydrophilicity (that opposes protein adsorption and biofouling), allied with widespread availability and biocompatibility, cellulose-based materials are still widely present in viral filters. developed commercially over the past thirty years, several cellulosic filter media have been and continue to be used for both routine analyses in laboratories and academic research studies aimed at improving the filtration performance, generating a wealth of data quantifying the efficiency of virus removal or recovery/concentration from biologic or water samples, respectively. as concerns biopharmaceutical compounds, these data arise from validation studies of the filtration step performed by spiking intermediate biologic solutions (mostly blood proteins) from multistage purification processes with known titers of viral particles. regenerated cellulose hf planova filters n -n , operated in the dead-end mode as a single filter or two units in series, were shown to provide lrv values ranging between and , on the average. increasingly tested since their launching in , these cellulosic filters compete with filter media made of synthetic polymers such as pvdf (e.g., viresolve Ò nfp-normal flow parvovirus-from emd millipore; ultipor Ò vf grade dv /dv from pall corporation, port washington, ny) or pes (e.g., millipore viresolve Ò pro; virosart Ò hs/hc/cpv from sartorius stedim biotech, aubagne, france). the hydrophilic nature of cellulose fibers is a definite advantage of planova filters over others for virus removal from protein solutions, minimizing flux decline due to proteindominated filter plugging (cake formation) at constant pressure (normal-flow) filtration (rathore et al. ). however, they are usually operated at lower flow rate than filters made of synthetic polymers whose operating pressure is higher. hydrophilic modified pvdf membrane filters designed for virus removal from high-concentration protein solutions at high flow rate are being developed by several manufacturers, including asahi-kasei (i.e., planova tm bioex filters, launched in ). many studies have also been reported to assess the virus concentration efficiency of the viradel method using spiked water samples, prior to the detection of waterborne viral contaminants in field water samples. in this method, cellulosic filters, whether positively or negatively charged, are used for adsorptive capture of viruses before virus recovery by elution with a lowvolume eluent. electropositive filters are more particularly suitable for virus capture in low-contaminated, high-volume water samples, e.g., samples of groundwater or source water for drinking water production ( table ) . though routinely used, these filters are expensive and face competition with nanoalumina fiber filters (e.g., the patented ahlstrom disruptor Ò electroadsorptive filter) (levi ) and glass wool filters, which are cheaper. on the other hand, efficient virus adsorption by electronegative filters-the standard, cost-effective mixed cellulose ester membrane microfilters-requires impractical sample pre-treatment: their use is limited to viral analysis of highlycontaminated waters, for which small-volume samples are sufficient (table ) . cellulose nanomaterials may represent a promising perspective for cellulosic materials in their application to viral filtration of liquid samples, yielding filtration membranes with higher mechanical strength, water permeability, surface hydrophilicicy and resistance to biofouling (carpenter et al. ) . ma et al. ( ) have presented a composite membrane consisting of an electrospun poly(acrylonitrile) nanofibrous scaffold deposited on a non-woven poly(ethylene terephthalate) support. this two-layered membrane was coated by a layer of cellulose nanofibers and tested for retention of ms- phages. at acidic ph, phage particles were adsorbed by the negatively charged cellulose nanofiber coating, yielding a lrv value [ . . later on, the same nanofibrous composite membrane was doped with cellulose nanofibers functionalized by grafting with poly(vinylamine) . ms- phages were adsorbed at neutral ph onto positively charged amino-modified nanofibers, with a lrv value of . metreveli et al. ( ) and asper et al. ( ) have presented a size-exclusionbased filter paper made of pure cellulose nanofibers for removal of swine influenza a and murine leukemia viruses, respectively. these non-woven materials could be tailored to ensure efficient virus retention (gustafsson and mihranyan a). self assembled into nanosheets to yield ''mille feuille'' structures, they showed efficient removal of parvoviruses (gustafsson et al. b) : maybe a new filtration media for viral clearance? a number of air filtration devices designed for individual protection against airborne viruses also include cellulosic materials. while published data on the filtration performance of cellulosic media for removal of viruses from air are scarce, industrial research is more apparent, in particular in asian countries (china, japan) where airborne pollution is a matter of great concern-the main objective being to add virucidal properties to the filtration media so as to inactivate accumulated viral particles. therefore, some 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anti-microbial air filter the use of masks and respirators to prevent transmission of influenza: a systematic review of the scientific evidence methods for air cleaning and protection of building occupants from airborne pathogens incidence of enteric viruses in groundwater from household wells in wisconsin vulnerability of drinking-water wells in la crosse, wisconsin, to entericvirus contamination from surface water contributions human enteric viruses in groundwater from a confined bedrock aquifer ranking filter methods for concentrating pathogens in lake water new tools for the study and direct surveillance of viral pathogens in water advances in viral clearance pathogen inactivation: the definitive safeguard for the blood supply respiratory and facial protection: a critical review of recent literature nanofiltration of plasmaderived biopharmaceutical products place of nanofiltration for assuring viral safety of biologicals robustness of nanofiltration for increasing the viral safety margin of biological products cellulose nanomaterials in water treatment technologies an overview of viral filtration in biopharmaceutical manufacturing virus transfer from personal protective equipment to healthcare employees' skin and clothing methods for primary concentration of viruses from water samples: a review and metaanalysis of recent studies antiviral effects of polyphenols: development of bio-based cleaning wipes and filters positively charged filters for virus recovery from wastewater treatment plant effluents detection of astroviruses, enteroviruses, and adenovirus types and in surface waters collected and evaluated by the information collection rule and an integrated cell culture-nested pcr procedure membrane processes in biotechnology: an overview photocatalytic oxidation for antimicrobial control in built environment: a brief literature overview enteric viruses in raw vegetables and groundwater used for irrigation in south korea hepatitis a in puglia (south italy) after years of universal vaccination: need for strict monitoring and catch-up vaccination a solvent/detergent-treated and -nm filtered factor viii: a new safety standard for plasma-derived coagulation factor concentrates virus interactions with membrane filters respiratory protection the healthcare infection society working group on respiratory and facial protection, guidance on the use of respiratory and facial protection equipment persistence of the pandemic influenza a (h n ) virus on n respirators face masks to prevent transmission of influenza virus: a systematic review recovery of viruses from water by a modified flocculation procedure for second-step concentration simple respiratory mask testing the efficacy of homemade masks: would they protect in an influenza pandemic pathogen inactivation and filtration efficacy of a new anti-microbial and anti-viral surgical facemask and n against dentistry-associated microorganisms evaluation of viral concentration methods from irrigation and processing water emerging viral infections-a potential threat for blood supply in the st century hepatitis a virus in environmental water samples from the amazon basin development and validation of a concentration method for the detection of influenza a viruses from large volumes of surface water primary biological aerosol particles in the atmosphere: a review effective virus inactivation and removal by steps of biotest pharmaceuticals igiv production process a new series of graded collodion membranes suitable for general bacteriological use, especially in filterable virus studies filtration of vaccinia virus through gradocol membranes the filtration of herpes virus through graded collodion membranes the size of the virus of poliomyelitis as determined by ultrafiltration analysis the sizes of the viruses of human and swine influenza, as determined by ultra-filtration respiratory protective devices-particle filters-requirements, testing, marking effect of chaotropic and antichaotropic agents on elution of poliovirus adsorbed on membrane filters ultrafilter membranes and ultrafiltration reaerosolization of ms bacteriophage from an n filtering facepiece respirator by simulated coughing enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools quantitative detection of human adenoviruses in wastewater and combined sewer overflows influencing a michigan river concentration and processing of waterborne viruses by positive charge mds cartridge filters and organic flocculation detection of rotavirus a in sewage samples using multiplex qpcr and an evaluation of the ultracentrifugation and adsorption-elution methods for virus concentration implementation of a -nm pore-size filter in the plasmaderived factor viii manufacturing process filtration of the virus of footand-mouth disease through a new series of graded collodion membranes composite nonwovens in medical applications evaluation of positively charged alumina nanofiber cartridge filters for the primary concentration of noroviruses, adenoviruses and male-specific coliphages from seawater viral pathogens in water: occurrence, public health impact, and available control strategies a pilot study on the use of geometrically accurate face models to replicate ex vivo n mask fit bacteriophages in health and disease, advances in molecular and cellular microbiology no. . cab international bioseparation in antibody manufacturing: the good, the bad and the ugly concentration of coliphages from large volumes of water and wastewater protecting healthcare workers from pandemic influenza: n or surgical masks? comparison of zetaplus s and nitrocellulose membrane filters for the simultaneous concentration of f-rna coliphages, porcine teschovirus and porcine adenovirus from river water challenges in environmental detection of human viral pathogens new electropositive filter for concentrating enteroviruses and noroviruses from large volumes of water development of a virus concentration method and its application to detection of enterovirus and norwalk virus from coastal seawater one-year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in japan organic flocculation: an efficient second-step concentration method for the detection of viruses in tap water electrophoretic mobilities of virus adsorbing filter materials detection and genetic analysis of human sapoviruses in river water in japan relative abundance and treatment reduction of viruses during wastewater treatment processes-identification of potential viral indicators pathogen inactivation and removal methods for plasma-derived clotting factor concentrates pathogen-reduction methods: advantages and limits virus safety of plasma products using nm instead of nm filtration as virus removing step antimicrobial properties of chitosan and mode of action: a state of the art review evaluation of viral clearance in purification processes assessment of human adenovirus removal in a fullscale membrane bioreactor treating municipal wastewater concentration of enteroviruses, adenoviruses, and noroviruses from drinking water by use of glass wool filters evaluation of electropositive filtration for recovering norovirus in water incidence and molecular characterization of hepatitis a viruses in korean surface water between headform and n filtering facepiece respirator interaction: contact pressure simulation and validation simulation and evaluation of respirator faceseal leaks using computational fluid dynamics and infrared imaging fluid purification media and systems and methods using same polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis a virus and human rotavirus, from oyster, water, and sediment samples concentration of viruses from environmental waters using nanoalumina fiber filters recovery and purification process development for monoclonal antibody production hollow-fiber ultrafiltration for simultaneous recovery of viruses, bacteria and parasites from reclaimed water rapid concentration of bacteriophages from large volumes of freshwater: evaluation of positively charged, microporous filters influence of salts on virus adsorption to microporous filters ultra-fine polysaccharide nanofibrous membranes for water purification concentration of live retrovirus with a regenerated cellulose hollow fiber mechanism of human immunodeficiency virus (hiv) removal by regenerated cellulose hollow fiber (bmm) analytical methods for food and environmental viruses optimization of adenovirus and recovery from tap water using small disk filters a size-exclusion nanocellulose filter paper for virus removal molecular detection and characterization of gastroenteritis viruses occurring naturally in the stream waters of manaus, central amazonia, brazil isoelectric points of viruses antibacterial mask, antibacterial filter for the mask, and antibacterial method using the mask or the filter. japanese patent no. jp a national institute for occupational safety and health (niosh) ( ) cfr part : respiratory protective devices; final rules and notice surgical mask filter and fit performance recent advances in bioprocessing application of membrane chromatography cationic charge modified filter media pre-analytical and analytical procedures for the detection of enteric viruses and enterovirus in water samples pathogen inactivation techniques virus filtration process design and implementation survival of bioaerosols in hvac system photocatalytic filters the rise and fall of polyanionic inhibitors of the human immunodeficiency virus type protection of the respiratory system against nanoparticles inhalation evaluation of mds electropositive microfilters for simultaneous recovery of multiple microbe classes from tap water ultrafiltration-based techniques for rapid and simultaneous concentration of multiple microbe classes from -l tap water samples component pathogen inactivation: a critical review chitosan as antimicrobial agent: applications and mode of action intravenous immunoglobulin g: trends in production methods, quality control and quality assurance second-step concentration of viruses in drinking and surface waters using polyethylene glycol hydroextraction rotavirus concentration from raw water using positively charged filters mechanistic modeling of viral filtration respiratory protection against bioaerosols: literature review and research needs comparison of nanoparticle filtration performance of niosh-approved and ce-marked particulate filtering facepiece respirators simple respiratory protection-evaluation of the filtration performance of cloth masks and common fabric materials against - nm size particles evaluation of hollow-fiber ultrafiltration primary concentration of pathogens and secondary concentration of viruses from water assessment of adenovirus, hepatitis a virus and rotavirus presence in environmental samples in florianopolis, south brazil virus removal from factor ix by filtration: validation of the integrity test and effect of manufacturing process conditions purification of retroviral vectors for clinical application: biological implications and technological challenges the impact of combined sewage overflows on the viral contamination of receiving waters comparison of microporous filters for concentration of viruses from wastewater maintenance of influenza virus infectivity on the surfaces of personal protective equipment and clothing used in healthcare settings nine-year study of the occurrence of culturable viruses in source water for two drinking water treatment plants and the influent and effluent of a wastewater treatment plant in downstream processing of oncoretroviral and lentiviral gene therapy vectors overview of current scalable methods for purification of viral vectors possibility of hepatitis b virus (hbv) removal from human plasma using regenerated cellulose hollow fiber (bmm) an attempt to prepare hepatitis b virus (hbv)-free plasma by ultrafiltration using microporous regenerated cellulose hollow fiber influence of salts on electrostatic interactions between poliovirus and membrane filters downstream processing of monoclonal antibodies-application of platform approaches removal of human enteric viruses by a full-scale membrane bioreactor during municipal wastewater processing poliovirus concentration from tap water with electropositive adsorbent filters concentration of enteroviruses from large volumes of water pathogen reduction of blood components composition for use in decreasing the transmission of human pathogens devices and methods for decreasing human pathogen transmission factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises review of aerosol transmission of influenza a virus nanosize electropositive fibrous adsorbent a high-performance electropositive filter viral safety of nanogam Ò , a new nm-filtered liquid immunoglobulin product viral safety of c -inhibitor nf electret air filters a new material for airborne virus filtration effective detection of human noroviruses in hawaiian waters using enhanced rt-pcr methods different electrostatic methods for making electret filters mechanism of removing monodisperse gold particles from a suspension using cuprammonium regenerated cellulose hollow fiber (bmm hollow fiber) structure of cuprammonium regenerated cellulose hollow fiber (bmm hollow fiber) for virus removal structure and filtration performances of improved cuprammonium regenerated cellulose hollow fiber (improved bmm hollow fiber) for virus removal mask and graft polymer extract. japanese patent no detection of adenoviruses and rotaviruses in drinking water sources used in rural areas of methods for sampling of airborne viruses large-scale production and purification of vlpbased vaccines rational design and optimization of downstream processes of virus particles for biopharmaceutical applications: current advances evaluation of an adsorptionelution method for detection of astrovirus and norovirus in environmental waters glass wool for virus concentration at ambient water ph level virus concentration on aluminum and calcium salts enterovirus concentration on cellulose membranes ayliffe's principles and practice of disinfection, preservation and sterilization, th edn electrostatic forces in fibrous filters-a review the antiviral activities and mechanisms of marine polysaccharides: an overview nanofibrous microfiltration membranes capable of removing bacteria, viruses and heavy metal ions detection and monitoring of virus infections by real-time pcr disrupting the transmission of influenza a: face masks and ultraviolet light as control measures application of enteric viruses for fecal pollution source tracking in environmental waters evaluation of the performance of dialdehyde cellulose filters against airborne and waterborne bacteria and viruses use of dialdehyde starch treated filters for protection against airborne viruses a simple and novel method for recovering adenovirus in small volumes of source water surveillance of adenoviruses and noroviruses in european recreational waters occurrence of human adenoviruses at two recreational beaches of the great lakes evidence of airborne transmission of the severe acute respiratory syndrome virus the particle size of hepatitis c virus estimated by filtration through microporous regenerated cellulose fibre antivirus bacterial cellulose protective material and its preparation method. chinese patent no air filter type bacterial cellulose mask and production method thereof. chinese patent no viral clearance using disposable systems in monoclonal antibody commercial downstream processing key: cord- -ycc csyh authors: rollinger, judith m.; schmidtke, michaela title: the human rhinovirus: human‐pathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery date: - - journal: med res rev doi: . /med. sha: doc_id: cord_uid: ycc csyh as the major etiological agent of the common cold, human rhinoviruses (hrv) cause millions of lost working and school days annually. moreover, clinical studies proved an association between harmless upper respiratory tract infections and more severe diseases e.g. sinusitis, asthma, and chronic obstructive pulmonary disease. both the medicinal and socio‐economic impact of hrv infections and the lack of antiviral drugs substantiate the need for intensive antiviral research. a common structural feature of the approximately hrv serotypes is the icosahedrally shaped capsid formed by identical copies of viral capsid proteins vp ‐ . the capsid protects the single‐stranded, positive sense rna genome of about , bases in length. both structural as well as nonstructural proteins produced during the viral life cycle have been identified as potential targets for blocking viral replication at the step of attachment, entry, uncoating, rna and protein synthesis by synthetic or natural compounds. moreover, interferon and phytoceuticals were shown to protect host cells. most of the known inhibitors of hrv replication were discovered as a result of empirical or semi‐empirical screening in cell culture. structure–activity relationship studies are used for hit optimization and lead structure discovery. the increasing structural insight and molecular understanding of viral proteins on the one hand and the advent of innovative computer‐assisted technologies on the other hand have facilitated a rationalized access for the discovery of small chemical entities with antirhinoviral (anti‐hrv) activity. this review will (i) summarize existing structural knowledge about hrv, (ii) focus on mechanisms of anti‐hrv agents from synthetic and natural origin, and (iii) demonstrate strategies for efficient lead structure discovery. © wiley periodicals, inc. med res rev, , no. , – , human rhinoviruses (hrv) are the major cause of upper respiratory tract symptoms, the socalled common colds in humans. their name reflects the primary site of infection. because hrv are nonenveloped, icosahedral viruses of small size with a diameter of about nm ( pico small in latin) that consist of an rna genome, they were assigned to the family picornaviridae. currently, this virus family of the order picornavirales comprises the eight genera enterovirus, hepatovirus, cardiovirus, kobuvirus, teschovirus, erbovirus, aphthovirus, and parechovirus with species and a multitude of serotypes. because of high similarity in genome sequence and genome organization (fig. ) , the former genera rhinovirus and enterovirus have been combined recently, keeping the existing name enterovirus (www.picornastudygroup. com/taxa/species/species.htm). an overview on the current taxonomy of picornaviruses pathogenic for humans as well as on newly proposed species of hrv is given in table i . at present the genus enterovirus includes four approved human enterovirus (hev) species (hev-a, -b, -c, and -d) and two approved hrv species (hrv-a and -b) (www. picornastudygroup.com/taxa/species/-species.htm). since , the global distribution of highly divergent hrv strains was reported. [ ] [ ] [ ] [ ] based on the results of sequence, genomic, and phylogenetic analyses, it was proposed that these strains represent a new hrv species, hrv-c. [ ] [ ] [ ] in , a further proposal concerning a new potential hrv-d species was published after sequencing and analysis of all known hrv genomes. the approved and newly proposed species of the genus enterovirus share z % homology (average amino acid identity) in the precapsid protein p as well as in c and cd. , [ ] [ ] [ ] [ ] different antigenic properties provide the basis for a further division of species into serotypes (table i) . about rhinovirus serotypes are currently known. according to the currently approved taxonomy, most of them ( and hrv hanks) belong to hrv-a and of them to hrv-b. , the genome of all known hrv-a and -b serotypes as well as of several field isolates of hrv-a, -b, and -c has been sequenced completely. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] viruses classified as hrv-c could not be grown in cell culture until now. phylogenetic analyses have been performed with partial sequences, , as well as with the whole genome. , the most recent and comprehensive analysis of all known hrv genomes revealed that (i) hrv-a and hrv-c share a common ancestor, which is a sister group to hrv-b, (ii) hrv-c represents a third species, and (iii) a basal divergence within hrv-a of three distinct strains that led to the proposal of a fourth species hrv-d. hrv-a and -b most often induce a mild, usually self-limited upper respiratory illness in humans characterized by nasal stuffiness and discharge, sneezing, sore throat, and cough. the conventional term is common cold. the common cold is a heterogeneous group of diseases caused by numerous viruses that belong to several different families e.g. rhinoviruses, coronaviruses, enteroviruses, and adenoviruses. but, hrv represent the most common etiological agent worldwide. a large number of distinct strains circulate each year. moreover, in a family or even in a single specimen, multiple hrv serotypes were detected simultaneously. [ ] [ ] [ ] by using rt-pcr and culture, it was shown that hrv induce - % of upper respiratory tract infections in adults as well as children. [ ] [ ] [ ] [ ] [ ] higher incidence has been described from september to november, [ ] [ ] [ ] and from april to may. , in some years and perhaps some geographical areas, spring was a more important time for rhinovirus transmission. , although overall rates of respiratory illness are lower in summer, rhinoviruses are the most frequently isolated at this time of year. the incidence is inversely proportional to age. , by age years, % of the children have antibodies against rhinoviruses. in addition to common cold, hrv are also involved in acute otitis media in children. , moreover, data supporting a causative association with more severe lower respiratory tract infections of infants, elderly persons, and immunocompromised patients have been accumulated. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] studies of childhood and adult asthma have shown that hrv infections can also trigger exacerbations in patients with asthma, [ ] [ ] [ ] [ ] chronic obstructive pulmonary disease, [ ] [ ] [ ] [ ] [ ] [ ] and cystic fibrosis. [ ] [ ] [ ] the recently discovered novel rhinovirus genotype hrv-c was associated with community outbreaks of influenza-like, acute upper respiratory infections and severe low respiratory tract infections of infants e.g. febrile wheeze, bronchiolytis, and asthma exacerbations, which peaked in fall and winter. [ ] [ ] [ ] [ ] [ ] , , in addition, the presence of hrv-c in the middle ear in patients with acute otitis media was demonstrated. hrv spread occurs by means of virus-contaminated respiratory secretions that contain a high virus concentration. [ ] [ ] [ ] besides direct hand-to-hand transmission, small-and largeparticle aerosol transmission of rhinoviruses has been shown. , , children are important ''vectors'' for hrv transmission to family members. moreover, studies with natural hrv-infected adults provided evidence that daily activities of infected people can lead to contamination of environmental surfaces with hrv e.g. light switches, telephone dial buttons and handsets, and virus transfer to fingers of healthy individuals for infection. , because viral contamination of the hands plays an important role in transmission of hrv from person-to-person, interruption of this step of virus transmission presents a potential target for intervention. this was experimentally proved by treatment of hands by iodine , or salicylic and pyroglutamic acid. observations from experimentally induced infections in normal adult volunteers helped to understand the pathogenesis of hrv infections. [ ] [ ] [ ] [ ] [ ] [ ] the % human infectious dose of rhinovirus is low and the infection rate between and %. after the deposition of hrv on nasal or conjunctival mucosa, viruses are transported to the posterior nasopharynx by mucociliary action of epithelial cells. specific receptors on epithelial cells in the adenoid area are used for binding and entry. already - hr after intranasal inoculation, infectious virus can be detected. virus shedding peaks on the second day after infection and decreases rapidly thereafter. but, small amounts of viruses were discovered in nasal secretions for up to weeks after infection. virus and/or viral rna were demonstrated in the upper as well as lower respiratory tract. , , , using in situ hybridization, arruda et al. ( ) detected viral rna in a low number of ciliated cells in nasal biopsies. in the nasopharynx, a small portion of virus-positive ciliated as well as nonciliated cells was positive for viral rna. in papadopoulos et al. provided evidence that hrv may also lytically infect human bronchial epithelial cells in cell culture as well as in experimentally infected volunteers and induce the production of interleukin- , - , and - . in agreement with these results, hrv rna was detected in - % of children and - % of adults with pneumonia. [ ] [ ] [ ] [ ] taken together, the results of natural cold studies as well as of experimental infection in human volunteers clearly demonstrate that hrv are able to replicate in the upper as well as in the lower airways. hrv infection triggers vasodilation and increased vascular permeability in the nasal mucosa, leading to nasal obstruction and rhinorrhoea. the mechanism is still incompletely understood because no histopathological changes were observed in nasal biopsy specimens from infected persons. this led to the suggestion that clinical symptoms are primarily caused by the inflammatory response of the host to the virus infection and not by the cytopathic effect (cpe) of hrv. results of immunological investigations suggest a modest correlation between the concentrations of il- and il- in nasal secretions and the severity of symptoms in upper and lower hrv-induced respiratory tract disease. , on day - after virus challenge, il- and il- concentrations were significantly greater in nasal secretions from experimentally infected symptomatic subjects than in those from infected asymptomatic or sham-challenged subjects. il- has been proposed as a mediator of neutrophile infiltrations that are observed during symptomatic infections. in experimental rhinovirus infection the onset of symptoms e.g. nasal stuffiness and discharge, sneezing, and cough was observed - hr after intranasal inoculation of the virus. in contrast to rhinovirus infections in adults, fever is found in % of children with upper respiratory tract infections. other symptoms in children and adults may be hoarseness, headache, malaise, and lethargy. sometimes viral infection is accompanied by bacterial complication, leading for instance to acute otitis media in about % of infected children, sinusitis, and pneumonia. [ ] [ ] [ ] , experimental infection was also used to study the causation between rhinovirus infection and asthma as well as copd exacerbations. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] it was shown that hrv infection enhances airway reactivity and predisposes allergic patients to develop late asthmatic reactions. , rhinoviral colds were associated with an increase in histamine responsiveness that was accompanied by a bronchial mucosal lymphocytic and eosinophilic infiltrate. in a recent study, an increased hrv-induced clinical illness severity in asthmatic compared with normal subjects was demonstrated. strong relationships were shown between virus load, lower airway virus-induced inflammation, and asthma exacerbation severity. the results of this study also indicated that augmented th or impaired th or il- immunity are likely important mechanisms. mallia et al. provided evidence that low dose experimental rhinovirus infection in patients with copd induces symptoms and lung function changes typical of an acute exacerbation of copd. viral replication and increased pro-inflammatory cytokine response were associated with symptomatic colds, increases in lower respiratory tract symptoms and reductions in forced expiratory volume in s or peak expiratory flow rate. the epidemiological data and pathology of hrv infections explain their high medical and socio-economic impact. millions of children and adults are taken ill with common cold every year, need medical consultations, are unable to attend school and go to work. , direct costs include hospitalization, medical fees, and symptomatic treatment. moreover, exacerbations are the major cause of asthma and copd morbidity, mortality, and health care costs associated with these diseases. , to date, specific drugs that prevent or reduce rhinovirus infection are not available. common cold can be treated only symptomatically with analgesics, decongestants, antihistamines, or antitussives and antibiotics are often wrongly prescribed. , because of the large number of circulating hrv serotypes, treatment with specific antiviral drugs is considered to be more striking than vaccination. therefore, the search for new highly active synthetic and/or natural anti-hrv compounds is absolutely essential and represents an important area of antiviral research. such an anti-hrv drug would have to be (i) with broad spectrum activity because of the high number of hrv serotypes, (ii) administered very early in infection to demonstrate a good antiviral effect because of the fast infection kinetics, (iii) very safe because of the broad application by millions of people, and (iv) directed against a highly conserved target with low risk of resistance development. due to the very high error rates and the lack of proofreading ability in rna polymerases of picornaviruses, naturally drug-resistant variants may exist in virus populations or resistant viruses can emerge under treatment. as with hiv, another highly variable rna virus, the risk of resistance development and/or selection of resistant virus variants could be minimized by applying combination of drugs directed against different targets. because clinical symptoms are suggested to be primarily caused by the inflammatory response of the host to the virus infection mediated by specific cytokines, a further advantage of drug combinations could be an additional immune-suppressive activity. the knowledge of structural components, nonstructural proteins that are necessary for viral multiplication, and stages of the viral life cycle is an essential precondition for the development of measures to prevent and treat hrv infection. the structure of hrv particles is well known. infectious virions consist of an icosahedral protein shell (capsid) that surrounds and protects the genome, a single positive-stranded rna molecule of approximately , nucleotides. the organization of the enterovirus genome is shown in figure . the viral genomic rna is infectious and encodes a single, long, open reading frame flanked by untranslated regions (utr) at the and end. a small viral protein (vpg) is covalently linked to the end. the end is polyadenylated like cellular messenger rnas. structural components within these utrs e.g. the cloverleaf and the internal ribosome entry site (ires) of the utr play an important role in rna replication as well as protein synthesis. the nucleotide sequence of some regions within these structures is highly conserved among enteroviruses. their blockade could significantly inhibit viral replication. the molecular structure of hrv- a, hrv- , hrv- , hrv- , and hrv- was determined by x-ray crystallography. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the results show that the viral capsid is composed of protomers of each of the three outer structural proteins vp , vp , and vp and of vp in the interior. a star-shaped plateau at the fivefold axis of symmetry, surrounded by a deep depression (canyon) and another smaller depression at the threefold axis were detected. moreover, a hydrophobic pocket was found beneath the canyon floor. with exception of hrv- and hrv- , this pocket is occupied by a fatty acid, the so-called pocket factor. these host cell molecules have been suggested to play an important role in the viral life cycle by providing transient stability to the capsid during its movement from one host cell to another. the outer surface of virions contains neutralization antigenic as well as host cell binding sites. the latter allow the virus to attach to molecules of the host cell membrane (adsorption), the receptors, and to start their life cycle. [ ] [ ] [ ] based on their receptor use, two groups of hrv can be distinguished. the majority of hrv serotypes, the major group uses intercellular adhesion molecule- (icam- ) as their receptor. the viruses belonging to the minor group attach to low density lipoprotein (ldl) receptor, very-ldl (vldl) receptor, and ldlr-related protein on the cells whereat multiple receptors are involved. [ ] [ ] [ ] hrv of the major group apply the canyon as attachment site for binding to icam- . in contrast, ldl receptors of minor group viruses bind near the tip of the five-fold vertex. hrv- has been shown to utilize a sialylated glycoprotein as a cellular receptor. furthermore, a hrv- variant as well as wild-type hrv- can use heparan sulphate proteoglycans for cell attachment in addition to icam- . , the interaction of rhinoviruses with their receptors leads to virus concentration on the cell surface. it induces the release of the pocket factor and conformational changes in the capsid and mediates viral entry via endocytosis. [ ] [ ] [ ] [ ] whereas icam- binding directly causes uncoating, , release of the rna genome from the capsid of ldl-bound minor group rhinoviruses is triggered by acidification of the endosomal, ph-dependent pathway. [ ] [ ] [ ] this detailed knowledge of the capsid structure and function as well as of the virus-receptor interaction offers a good possibility to develop antiviral drugs that interfere with the first steps of the viral life cycle, adsorption as well as uncoating. after uncoating, rhinovirus proteins are synthesized by the translation of a single, open reading frame using cellular ribosomes. the resulting polyprotein of approximately kd is cleaved by viral proteases a pro and c pro into final products ( structural and nonstrutural proteins) immediately after translation. , at first, both proteinases release themselves from the polyprotein by selfcleavage. the primary cleavage of the viral polyprotein between p and p is mediated by a pro . thereafter, cd pro is released from the p precursor by autocatalytic cleavage. next, c pro and its precursor cd pro process proteins of the p (capsid proteins), p , and p (nonstructural proteins) region. interestingly, a pro cleaves also the eif gi/ii component of the translation initiation factor eif f necessary for host cell protein synthesis, [ ] [ ] [ ] and c pro and/or cd pro the rna polymerase transcription factors tfiid, tfiic, sl- , and ubf. therefore, effective inhibition of a pro and c pro would not only inhibit virus replication but could also prevent the shutoff of cellular protein and rna synthesis. moreover, the active site of proteinases is highly conserved among enterovirus serotypes. this high conservation in conjunction with their important role in virus multiplication predestines these enzymes as targets for antiviral therapy. the viral rna polymerase d ( d pol ) represents another very important nonstructural protein of hrv. it forms a complex with both cellular and viral proteins, the rna replication complex. this enzyme synthesizes viral minus-strand rna and uses it as template strand for the synthesis of genomic viral rna. vpg ( b) is the primer for negative-as well positive-strand rna synthesis. negative-strand, but not positive-strand rna synthesis, is stimulated by a pro . further viral accessory proteins include b, c, bc, and ab. besides d pol these proteins can play an important role in inhibition of viral rna synthesis by antiviral compounds. in summary, the knowledge of the structure of the viral capsid, proteases, and polymerase and their important function in the viral life cycle predestine these proteins as potential anti-hrv targets. hrv grow in several human and some primate cells expressing the minor group ldl receptors and/or the major group receptor icam- . human cells susceptible to hrv infections include embryonic kidney, amnion, diploid fibroblasts from embryonic lung, tonsil, liver, intestine, and skin, adult fibroblast lines from aorta and gingival and the kb, hep- , and hela continuous cell lines. but, the susceptibility of hela cells and human fibroblasts to virus infection may vary. hrv multiplication also occurs in primary human airway fibroblasts and differentiated bronchial epithelium. , , the proportion of infectible epithelial cells was shown to be between and %. , but, enhanced levels of viral production were detected in poorly differentiated in comparison to differentiated epithelial cells. the degree of viral infection correlated with il- and il- induction in these cells virus growth causes a typical cpe characterized by ballooning, refractiveness, granularity, and shrinkage of infected cells. the hrv-induced cpe, infectious virus titers, viral protein expression, and rna synthesis can be chosen as parameters to evaluate the anti-hrv activity of compounds in cell-culture based assays. there are several methods for antiviral screening against hrv. the plaque reduction assay has been traditionally performed and accepted as the ''gold standard'' in antiviral testing. [ ] [ ] [ ] however, this test is laborious, time consuming, and the evaluation is subjective. therefore, it is not suited for the routine antiviral testing. it was more and more replaced by methods based on quantification of protection from virus-induced cpe after drug treatment. so, the cpe in sample-treated and untreated cells has been compared by light microscopy. , , but this evaluation is also subjective. another more objective approach is the spectrophotometric quantification of cpe results in neutral red or crystal violet uptake assays, , - and the tetrazolium dye reduction method. , it allows an excellent and rapid antiviral screening of large numbers of compounds using small amounts of extracts, natural, or synthetic compounds. active samples can be scheduled for additional testing using other assays e.g. virus yield or plaque reduction assays, and for studies on the mechanism of action. the activity of potential antiviral drugs has to be approved in vivo. because of the high degree of species-specific variations in icam- preventing infection by major group hrv, practical animal models have been absent for a long time. chimpanzees were infected with several hrv serotypes but without developing clinical signs. hrv do not induce infection in rabbits, guinea pigs, and weanling mice injected with hrv by different routes. one minor group hrv, serotype , was adapted to grow in mouse fibroblasts and used in a mouse model of rhinovirus infection in which growth could be demonstrated. based on the fact that the ldl receptor family is highly conserved between human and mouse, newcomb et al. examined whether hrv- b, another minor group virus, may infect mouse airways in -to -week-old female c bl/ mice. the authors demonstrated that this hrv serotype replicates and induces airway inflammation in vivo. these results strongly correspond to those of bartlett et al. who established three novel mouse models of rhinovirus infection in balb c mice. in the first model, -week-old balb/c mice were infected with hrv- b. in the second model, transgenic balb/c mice, expressing a mouse-human icam- chimera, were inoculated with the major group hrv- . rhinovirus-induced exacerbation of allergic airway inflammation is mimicked in the third model. due to the lack of a small-animal model for hrv infection until , the experimental human challenge model has to be used to approve effects of potential antiviral drugs under controlled conditions in preclinical studies. volunteers were experimentally inoculated with various serotypes e.g. hrv- , hrv- , hrv , hrv- , and hrv- to examine the efficacy of potential antiviral drugs under standardized conditions. [ ] [ ] [ ] [ ] [ ] [ ] examples and results of these studies with capsid-binders, protease, and rna synthesis inhibitors, as well as interferons are described in the following sections. antiviral agents that inhibit virus attachment, capsid uncoating, protein and rna synthesis of picornaviruses are the best studied, , [ ] [ ] [ ] and will be in the focus of this section. inhibition of virus attachment and/or uncoating interrupts the viral life cycle at its beginning and prevents hrv infection. options to prevent these early steps of the viral life cycle include (i) virus neutralization by hrv-specific antibodies, (ii) receptor blockade by antibodies directed against the cellular receptors icam- or ldl, (iii) by soluble receptor molecules, or (iv) by compounds interacting with the viral capsid. because of the high number of serotypes circulating often in parallel, application of hrv-specific antibodies is thought to be no promising approach for prevention or therapy of rhinovirus infection. in contrast, antibodies directed against the cellular receptor or soluble receptor molecules of major or minor group hrv could inhibit and % of hrv serotypes, respectively. therefore, the strategy to prevent virus-receptor interaction by receptor antibodies or soluble receptor molecules has been extensively evaluated in vitro as well as in vivo. the antiviral activity of icam-and ldl-specific antibodies was confirmed in cell culture. , furthermore, the prophylactic effectiveness and safety of intranasally administered rhinovirus murine icam- antibody was assessed in two double-blind, placebo-controlled, randomized studies of volunteers experimentally inoculated with hrv- . in the result, no toxicity related to antibody application was recognized. the higher dosage of mg/subject of rhinovirus murine receptor antibody did not reduce overall infection or illness rates, but was associated with a - day delay in the onset of virus shedding and cold symptoms. viral titers and nasal symptoms were significantly reduced on the second day after challenge. in summary, the monoclonal antibody to the cellular icam- was demonstrated to be not effective enough. a new strategy was the creation of multivalent fab fusion proteins against icam- . a new molecule, named cfy demonstrated a better avidity and in vitro potency against hrv over conventional mabs. cfy is under development as nasal spray with the name of coldsol. antagonism of virus-receptor interaction was considered as another promising way to prevent hrv attachment to host cells. soluble forms of fully or truncated icam- , [ ] [ ] [ ] and ldl or vldl-receptor concatemers [ ] [ ] [ ] exhibited antiviral activity against major and minor group hrv, respectively, in cell culture. soluble forms of icam- compete with receptor binding sites on the virus capsid, hinder an early infection event such as entry or uncoating, or directly inactivate hrv due to the formation of empty capsids. , [ ] [ ] [ ] a soluble ldl receptor fragment neutralized viral infectivity by aggregation. concatemers of the third ligand binding module of the vldl-receptor did not lead to viral aggregation but blocked the receptor binding sites and possibly inhibited viral uncoating by cross-linking the viral capsid subunits via multi-module binding. the antiviral activity of a truncated, soluble form of icam- was proved in hrv- infected chimpanzee. in randomized, double-blind, placebo-controlled trials, the safety and efficacy of intranasal administration of tremacamra, a soluble icam- in experimental hrv- -induced colds in humans, was shown. no further development was reported for these agents. a further option to prevent virus attachment was described for low-molecular-weight compounds, the so-called capsid-binding agents, which enter the small hydrophobic pocket within viral capsid protein beneath the icam-binding canyon of hrv. , zhang et al. showed that drug may integrate into mature viruses by diffusion as well as into progeny viruses during assembly. when hrv- and hrv- were grown in the presence of pleconaril, a higher occupancy occurred than when the drug was introduced into the alreadyassembled viruses. in doing so, capsid-binders induce conformational changes of the canyon of hrv- and hrv- , hinder virus-receptor interactions, and prevent attachment to host cells. , , [ ] [ ] [ ] in addition, uncoating of both hrv serotypes was shown to be inhibited as a result of a potential loss of flexibility of the viral capsid after drug binding. in contrast to hrv- and hrv- , capsid-binding compounds did not prevent attachment of hrv- a. results from x-ray studies showed that drug binding into the hydrophobic pocket of hrv- a replaces the pocket factor but induces only very small conformational changes. therefore, kim et al. suggested that the observed conformational changes are too small to affect receptor binding. but, capsid-binding compounds prevented attachment of hrv- possessing a pocket factor like hrv- a without distinct deformation of the pocket. , , further results from comparative antiviral studies with different capsid-binding compounds and hrv, representative for the major and minor group, did not reveal a correlation between inhibition of adsorption and receptor grouping or antiviral grouping. the reasons for the difference in the mode of action of capsid-binding compounds related to attachment inhibition are not fully understood until now. taken together, inhibition of rna uncoating was found for all investigated serotypes after drug binding independent of receptor grouping whereas prevention of virus attachment was found to be an additional mode of action for individual viruses and/or drugs. till now, various potent compounds belonging to diverse chemical classes have been described as uncoating inhibitors. just to give an impression of diversity, the structures of disoxaril and pleconaril, , - pirodavir and the oxime ether, , , , , the isoxazole derivate compound, , , the imidazole derivative sch , [ ] [ ] [ ] the chalcone ro - , , , dichloroflavan and isoflavan, , the pyridine derivative mdl , , , and the phenoxybenzene mdl- , that exhibit a potent anti-hrv activity (table ii) are shown as examples in figure . they inhibit most of hrv serotypes and a couple of them also affect enteroviruses, however, with varying susceptibility. based on variability of susceptibility to capsid-binders of different length, hrv serotypes were classified into two different groups, a and b. several of the given examples of compounds were also clinically tested. studying the development of clinically effective capsid-binders, the long road to the discovery of a clinically effective anti-hrv drug becomes apparent. one well-described example represents the discovery and optimization of capsid-binders from sterling winthrop pharmaceutical group, the so-called win compounds. first inhibitors originated from juvenile hormone mimetics that demonstrated some activity against hrv- a. determination of the x-ray structure of hrv- helped to understand the compounds' binding sites at the virus capsid. results from subsequent x-ray studies of hrv-win compound complexes revealed the location and nature of binding sites and provided information concerning interactions within these sites. , , this knowledge was used for optimization and design of new compounds. optimized win compounds, for example disoxaril and pleconaril ( fig. ) , consist of a methylisoxazol ring, a substituted phenoxy group, and a five-membered heteroatom ring and inhibit a broad spectrum of rhinoviruses and enteroviruses (table ii) . , [ ] [ ] [ ] in , the first broad-spectrum win compound disoxaril (win ) was tested in clinical trials. the development of crystallurea in human volunteers treated with high doses as well as its low bioavailability ( %) prohibited subsequent development. thereafter, results from sar and qsar analysis were used to further enhance the potency and spectrum of activity. in , another compound, win , was clinically tested. it was not effective in humans infected with hrv- and hrv- . moreover, it was rapidly metabolized and induced a reversible hepatitis. consequently, the further clinical development was stopped. the better understanding of pharmacokinetic properties of capsid binders and synthetic chemistry efforts led to the discovery of pleconaril, an orally bioavailable, welltolerated capsid-binder that inhibits most rhinovirus as well as various enterovirus serotypes. , [ ] [ ] [ ] in , schiff et al. published the efficacy of pleconaril in an experimentally induced coxsackievirus a infection in humans. in phase ii placebo-controlled, natural cold trials, the drug produced a moderate reduction of - . day in the medium time to elevation of illness compared with placebo. these results were confirmed in two subsequent pivotal studies. besides the moderate clinical efficacy, these studies revealed that % of baseline isolates were not susceptible to pleconaril and % developed reduced susceptibility (defined as -fold increase in baseline value). in a subsequent study the relationship of pleconaril susceptibility and clinical outcomes in the treatment of common cold caused by rhinoviruses was demonstrated. based on drug interaction, marginal treatment effect, and possibility of transmission of resistant viruses, the fda did not approve the applied oral administration of pleconaril for the treatment of common cold. the molecular mechanism of drug interaction of orally given pleconaril was shown to be based on hepatic cytochrome p a activation. to reduce adverse effects, shering-plough under license of viropharma completed a phase ii clinical trial with an intranasal formulation of pleconaril for the potential treatment of common cold in high-risk populations in . the results were not published until now. pyridazine analogues developed by janssen research foundation represent another example for the long road to discovery of a clinically effective capsid-binder. in , the broad-spectrum activity of pirodavir ( fig. ; table ii ) against rhinoviruses was published. in the same year the results of a randomized, double-blind, placebo-controlled trail to assess the therapeutic efficacy of intranasal pirodavir in natural common colds were described. possibly as a result of poor water solubility and rapid hydrolysis of pirodavir, no clinical benefit was found. the problem of ester hydrolysis was resolved by the development of oxime ether analogues of pirodavir by biota. an example is shown in figure . like pirodavir these new analogues are potent inhibitors of rhinoviruses. an advantage over pirodavir is their improved bioavailability. bta- , an antiviral analogue with long half-life and good oral bioavailability, was scheduled to a phase ii clinical trial in . the results have not yet been published. in summary, despite extensive research leading to the discovery of potent anti-hrv capsid-binders, no agent has been approved for prevention and/or therapy of rhinovirus-induced diseases so far. nearly, the same conclusion has to be drawn for protease inhibitors. because of their pivotal role for viral polyprotein processing and the high conservation of critical amino acids, a pro as well as c pro represent potential anti-hrv targets. results from cell culture-based assays provided evidence that inhibition of hrv replication is in principle possible. for example, processing of the hrv- polyprotein was prevented by pyrrolidine dithiocarbamate treatment in virus-infected hela cells. in contrast to other enteroviruses, [ ] [ ] [ ] [ ] pretreatment of cell monolayers with different nitric oxide donors leading to s-nitrosylation of a pro and c pro had neither an effect on virus replication nor on hrv-induced il- elaboration. the proteolytic activity of a pro of hrv- was specifically inhibited by two elastase-specific inhibitors, and an antiviral peptide representing a derivative of the caspase inhibitor zvad.fmk. , homophthalimides, e.g. ly ( fig. ; table ii) , were described as inhibitors of a pro as well as c pro . in contrast to protease c, no structure-activity relationship studies have been reported for hrv a protease. moreover, protease a accomplishes only one cleavage in hrv polyprotein, while protease c performs all other cleavages. after elucidation of the crystal structure of c pro , computer modeling of structural features of protease inhibitors became possible. furthermore, structure-based design was used to develop mechanism-based inhibitors of the c protease with potent antiviral activity against multiple hrv serotypes. , highly active compounds incorporate various michael acceptor moieties, irreversibly bind to c pro , and exhibit anti-hrv- activity in hela cells. , structure-activity studies were performed to optimize protease inhibitors. , [ ] [ ] [ ] [ ] [ ] these efforts resulted in the identification of a highly active anti-hrv compound, ag (rupintrivir; fig. ; table ii ) that entered clinical trials. in cell culture, ag inhibited a broad spectrum of laboratory hrv as well as clinical isolates. , , in a single-cycle, time-of-addition assay it demonstrated antiviral activity when added up to hr after infection. inhibition of hrv replication strongly correlated with reduction in the level of il- and il- release into cell supernatant, leading to the suggestion that this agent may not only block virus replication but also diminish symptoms. the pharmacokinetics and safety of rupintrivir were proved in two double-blind, randomized, placebo-controlled studies. intranasal rupintrivir, administered as single doses of and mg or every hr, six times per day, for days, was safe and well tolerated. three double blind, placebo-controlled clinical trials were conducted to assess rupintrivir nasal spray ( % solution) for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers. rupintrivir prophylaxis reduced the proportion of subjects with positive viral culture by % and viral titers but did not decrease the frequency of colds. drug treatment led to the reduction of the mean total daily symptom score by %. subjects receiving rupintrivir also demonstrated significantly lower viral titers and rna levels than placebo-treated subjects on days , , and and on days and , respectively. there was no influence on the proportion of subjects with positive viral culture and the frequency of colds. clinical development was terminated because rupintrivir did not act in a subsequent natural infection study in patients. in parallel research efforts, an orally bioavailable inhibitor of table ii) , was discovered. , like rupintrivir, this compound is an irreversible inhibitor incorporating a michael acceptor moiety that forms a covalent bond with the c protease active site cysteine. it demonstrated an antiviral activity against all hrv and related picornaviruses tested. in a phase clinical study, compound was shown to be safe and well tolerated. according to a publication of patick, no further clinical development was planned for this compound. the blocking of viral rna synthesis during replication represents another site for chemotherapeutic interdiction. it was shown that, rhinoviral rna can be targeted in a sequence-specific manner by deoxyribozymes, morpholino oligomers, and small interfering ribonucleic acids. the efficacy of the latter two approaches was confirmed in cell culture. in addition, -furylmercury chloride ( fig. ; table ii ), table iii) , and pyrrolidine dithiocarbamate (fig. ) interfered with rhinoviral rna synthesis and inhibited hrv replication in cell culture-based assays. , the nucleoside analog ribavirin ( fig. ; table ii ) that inhibits a broad spectrum of rna as well as dna viruses acts also against hrv- in hela cells. , the cellular inosine monophosphate dehydrogenase that controls de novo synthesis of purine nucleosides represents the principal target in the mode of action of ribavirin. moreover, when ribavirin is incorporated into picornavirus rna, it pairs equally well with either uracil or cytosine inducing mutations that can be lethal to rna viruses. further identified mechanisms of action for ribavirin include inhibition of genomic rna capping, enhancement of host t-cellmediated immunity against viral infections through helping to switch the host t-cell phenotype from type to type . another compound with potent anti-hrv activity in vitro is enviroxime ( fig. ; table ii ), a benzimidazole derivative. , it inhibits viral plus strand rna synthesis. in particular the a protein, which is involved in the initiation of plus strand rna synthesis, was implicated as likely target of drug activity. , however, results from another study suggest that enviroxime targets a complex of proteins and/or cellular factors and that the exact mechanism remains to be studied. although there was a statistically significant reduction in clinical score in a prophylactic study with hrv- -infected volunteers, enviroxime failed in experimentally induced hrv- and hrv- infection, , and in clinical studies, - because of poor bioavailability and side effects. in an attempt to overcome the marked hydrophobicity, water insolubility, and toxicity, wyde et al. incorporated enviroxime into liposomes and then tested the anti-hrv activity and toxicity of the liposome-incorporated enviroxime in cell culture. the liposome preparation of enviroxime inhibited hrv- a and hrv as effective as the parent compound and was -to z -fold less toxic. in contrast to free enviroxime, the liposome preparation was readily and successfully delivered by small-particle aerosol to the upper and lower respiratory tract of mice. in another attempt to overcome the disadvantages of enviroxime, several benzimidazole as well as nonbenzimidazole analogs were synthesized and studied. [ ] [ ] [ ] [ ] even though some compounds were better bioavailable and could be administered orally, none of these compounds was tested in clinical studies. besides virus-specific targets, cellular inhibitors like interferons may represent a therapeutical approach. among other activities, interferons exhibit antiviral activity. the advantages of interferon application include the broad spectrum of activity and low risk of resistance development. human leukocyte and fibroblast as well as recombinant human a interferons prevent the hrv-induced cpe in cell culture whereas a variation in sensitivity was observed. [ ] [ ] [ ] intranasally applied recombinant interferon a and interferon b have been shown to be effective in humans when provided prophylactically both in experimental and natural rhinovirus colds. [ ] [ ] [ ] [ ] [ ] [ ] significant reductions in illness frequency, mean symptom score, nasal secretion weights, and frequency of virus isolation were observed. in contrast, recombinant interferon g did not prevent hrv infection or illness and may enhance the symptoms. little to no therapeutic effect was found in patients with common cold after interferon treatment. , moreover, blood-tinged mucus and nasal bleeding were described as side effects. , combining interferons with dichloroflavan, enviroxime, chalcone ro- - produced synergistic increases in antiviral activity in vitro against hrv- and hrv- . an attempt to demonstrate synergy between the anti-hrv effect of recombinant human rhuifn a and enviroxime in hrv- and hrv- -infected volunteers failed. according to the authors, the main reason for this failure may be the rapid removal of enviroxime from the nose when given intranasally. a. impact of natural products nature provides an astonishing pool of secondary metabolites biosynthesized from living organisms such as plants, fungi, protozoan, insects, and other animal sources. in contrast to synthetic compounds, natural products are characterized by an overwhelming chemical diversity. previously, the chemical diversity space between these two groups was evaluated with respect to drug substances by feher and schmidt. it is shown that combinatorial compounds densely populate a small area, whereas natural products cover a wider range quite similar to the chemical space occupied by drug substances. the authors accordingly suggest that combinatorial libraries that mimic the distribution properties of natural products might be more biologically relevant. one may assume that secondary metabolites evolved as reaction to their target receptors related to defence, protection, attraction, and signalling. these adaptation processes have enriched not only the metabolites' structural diversity but have also optimized drug-like metabolic traits likely to have favorable pharmacokinetic properties. , it is this evolutionary concept that gives the pool of natural products the greatest source of scaffold diversity with molecules of biological relevance. newman and cragg analyzed the number of drugs approved between and and circumstantiated that especially the anti-infective area is strongly dependent on natural products and structures derived from natural scaffolds. the anti-infectives including the antiviral vaccines are with . % or launched drugs by far the major category with only about % being synthetic in origin. from to , vaccines and antiviral drugs have been approved. excluding the high number of vaccines ( ) and biologicals ( ) , most of the small antiviral molecules are based on nucleoside structures or on peptidomimetics; only . % are classified as totally synthetic drugs. however, till now, neither a synthetic nor a naturally derived anti-hrv drug substance has been approved for the treatment or prevention of hrv infections. intensive research and development efforts in the field of natural products revealed several inhibitors of viral attachment and entry, and inhibitors of viral protease from natural sources. the efficacy of natural products is not only reflected by statistics of launched drugs but also by empirical knowledge gained over centuries by successful application of naturalbased ethnomedicinal products such as plants, culinary herbs, and spices. phytochemical and pharmacological work performed with ethnomedicinal anti-hrvs mainly from plants revealed a high number of active metabolites from different chemical classes, e.g. coumarins, flavonoids, alkaloids, quinones, terpenoids, polyphenols, and polysaccharides. natural products include complex extracts and their chemical entities, which are biosynthesized by nature. for an unambiguous presentation of anti-hrv natural products it is of prior importance to first distinguish between a single chemical entity from nature, i.e. an isolated, purified natural compound, on one hand, and a natural preparation comprising hundreds to thousands of constituents, mainly secondary metabolites, on the other hand. if natural preparations are derived from plants, they might also be labelled as botanicals, phytoceuticals, or phytotherapeutic agents. these multicomponent preparations might show a varying profile of their constituents depending on the used species, origin, collection time, plant parts, extraction procedures, preparation methods, and manufacturing processes, just to mention a few important elements. these parameters affect the final product in terms of the qualitative and quantitative composition of chemical constituents, which may have an impact in biological activity. accordingly, studies performed with phytochemically not specified extracts or nonstandardized preparations often suffer from irreproducible and incomparable results a wide variety of natural preparations showed to be acting therapeutically in hrv and other viral infections with often complementary and overlapping antiviral mechanisms of action. [ ] [ ] [ ] most of these remedies are described in ethnopharmacological sources or handed down for generations. they usually consist of simply prepared natural items whose chemical composition is complex. many of the contained secondary metabolites, possibly active principles, have never been examined chemically or biochemically using modern medical knowledge. they are however components of plant medicines, which have stood the test of time and as such may offer clues of great interest to medicinal chemists. a clear advantage of the application of these products is their absent or relatively low toxicity due to a usually long-term empirical trial. although the knowledge of the immuno-pathogenesis of rv-induced diseases remains limited, the host defense function of the airway epithelium plays an important role in the innate-immune response to hrv-infection. host cells respond by the production of mediators with antiviral activity such as type i interferons and nitric oxide, and produce cytokines and chemokines that influence the subsequent induced innate-and specific-immune response. these processes are beneficial in facilitating clearance of virus from the respiratory tract, but also cause immuno-pathology. following hrv-infection, disease severity is dependent on direct, harmful effects of the virus as well as tissue damage as a result of the host antiviral immune response. accordingly, a number of agents with phenomenological effects against common cold have shown to exert their activity more in the field of regenerating tissue damage than on a direct anti-hrv effect. several herbal remedies consisting of a multitude of secondary metabolites from different chemical classes may attribute in a beneficial way for the treatment of common cold by reducing symptom severity and duration due to their immune-modulating, anti-oxidative, and anti-inflammatory properties. beside these commonly observed bioactivities of natural products, multicomponent mixtures like botanicals often show overlapping symptomatic effects as well as synergistic and/or additive properties. thus, it is a challenging endeavor to track down an observed phenomenological effect of a complex mixture on a molecular level. the following section explores the significance and current knowledge of selected botanicals for the prevention and therapy of common cold. questions about ( ) clinical evidence of efficacy, ( ) the constituents or at least the chemical classes that are involved in the observed anti-hrv effect, and ( ) the involved pharmacological targets were covered as far as possible. echinacea preparations include expressed juice from aerial parts as well as extracts of roots or aerial parts, or both, from one or more species of the genus echinacea (e. angustifolia, e. purpurea, and e. pallida). they are the most recognized botanicals for prevention and treatment of common cold and flu, and account for the second top-selling herbal products in the us-market. accordingly, echinacea has come under much scientific scrutiny. the high number of studies dealing with the effectiveness of echinacea for preventing and treating the common cold from clinical trials was recently reviewed by woelkart et al. the authors summarized the findings of the meta-analyses regarding the randomized controlled trials evaluated in the cochrane database, the randomized clinical trials analyzed by shah et al., and the experimental hrv-infection studies pooled by schoop et al. to sum up, the clinical data on echinacea so far are not fully consistent, mainly based on problems inherent in assessing the efficacy of echinacea preparations, such as lack of comparability of available preparations, study design, and outcome. nevertheless, the meta-analyses showed some evidence that preparations based on the aerial parts of echinacea purpurea might be effective for the early treatment of colds in adults. echinacea showed to decrease the odds of developing the common cold by % and the duration of a cold by . days. similarly, the evaluation of three induced rhinovirus prevention studies revealed the odds of experiencing a clinical cold were % higher with placebo than with echinacea. stepping into a molecular level, several constituents found in echinacea species could potentially affect the symptoms of common cold. chemically identified substances include polysaccharides and glycoproteins, caffeic acid derivatives (especially cichoric acid and echinacoside), and lipophilic polyacetylenes and alkamides. pharmacological studies have shown that cichoric acid, alkamides, glycoproteins, and polysaccharides possess immunomodulatory activity. additionally, alkamides have been reported to exert not only antiinflammatory effects but also cannabinomimetic properties, which are suggested as molecular mode of action of echinacea alkamides as immunomodulatory agents. raduner et al. showed that some echinacea alkamides exert cannabinoid type receptor-dependent and independent immunomodulatory effects on cytokine expression. different echinacea constituents were evaluated for their anti-oxidative effects measuring the inhibition of in vitro cu(ii)-catalyzed oxidation of human low-density lipoprotein. thereby, the major caffeic acid derivatives, cichoric acid and echinacoside, showed the highest anti-oxidative effects, which was even higher when combined with a natural mixture of alkamides. sharma et al. used cytokine antibody arrays to investigate the changes in the pro-inflammatory cytokines and chemokines released from human bronchial epithelial cells exposed to hrv . application of two chemically characterized echinacea extracts showed a reversion of the stimulated release of numerous pro-inflammatory cytokine-related molecules, e.g. for the cytokine il- , and the chemokines il- and eotaxin. in a similar study, an echinacea extract rich in polysaccharides and another rich in alkamides and caffeic acid derivatives were as well able to neutralize the effects of hrv-infected epithelial cells. using gene expression analysis both studies revealed the anti-hrv benefit of echinacea preparations being involved in multiple immune response signaling pathways. taken together, the numerous pharmacological findings from literature, the potential of echinacea preparations, and their constituents to combat or prevent common cold can be deduced to immune modulating, anti-inflammatory, and anti-oxidative properties that may also act in some combination of these event, rather than acting directly on hrv. garlic cloves have been used traditionally to treat a number of infectious diseases. however, only few confirmatory studies have been published regarding the traditional antiviral uses. the clinical effectiveness of garlic on the prevention of common cold was investigated by josling in , who published a double-blind, placebo controlled study assessing patients more than a -week treatment period with an allicin-containing garlic supplement. common cold infections and symptoms were recorded in a daily diary. patients in the treatment group had significantly fewer colds than patients in the placebo group ( vs. , po . ) who also had a longer duration of symptoms ( . vs. . days, po . ). as soon as the garlic is chewed, cut, or pressed, its main ingredient, the sulphur containing alliin, is broken down by the enzyme alliinase to the thiosulfinate allicin. by steam distillation allicin is transformed to diallyl disulfide and diallyl trisulfide that are responsible for the distinctive smell of garlic. further, allicin transformation compounds, such as e-and z-ajoene, are not found in fresh garlic, but in lipophilic extracts. by investigation of different garlic extracts and isolates against a number of different human pathological viruses, weber et al. could show that allicin was the most active virucidal component from fresh garlic and fresh extracts. results of the direct pre-hrv- -infection incubation assay let suggest allicin to bind to the viral protein capsid, leading to a subsequent inhibition of viral adsorption and penetration. although the garlic thiosulfinates are endowed with significant cytotoxicity, the antiviral effects were obtained in nontoxic concentrations. beside the direct anti-hrv effect of fresh garlic extract and allicin, a number of human immune functions were found to be enhanced in vitro by aqueous garlic extract, its polar, and thiosulfinate fractions. in north america, panax quinquefolium, the ginseng species indigenous to both canada and the united states, has been a popular herbal remedy to combat stress, and to modulate both natural and acquired immune responses. american ginseng root extracts, rich in poly-furanosyl-pyranosyl-saccharides, have been found efficacious in the prevention of upper respiratory infections in immunocompetent healthy adults. , in a randomized, double-blind, placebo controlled trial, mg of a proprietary american ginseng root extract was given to community-dwelling elderly adults (age year) twice a day more than a ''cold and flu'' season period of months. one month into the study, all participants received an influenza vaccination. during the first two months, no significant differences in duration and incidence were observed when compared to placebo. however, during the last two months significantly fewer subjects of the ginseng group reported acute respiratory syndromes than the placebo group ( vs. %). additionally, the duration of respiratory symptoms was reduced by % in the ginseng group. in a similarly arranged trial, healthy adults (ages - years) with a history of at least two colds the previous year commenced a month study at the beginning of a cold and flu season. they received two mg capsules daily of standardized american ginseng root extract or a placebo. outcomes measured were number of colds including symptom severity and total number of symptomatic days. a therapeutic effect was reported regarding symptom severity and fewer symptom days that were and . % lower in the ginseng group than in the placebo group. a phase ii randomized, controlled trial of dosing schedules of american ginseng root extracts, rich in poly-furanosyl-pyranosyl-saccharides, evaluated the safety, tolerability, and efficacy in a pediatric population already suffering from an upper respiratory tract infection. the results showed no serious adverse events and a good tolerability of both ginseng doses; however, frequency and severity of symptoms were not significantly different among each of the three treatment groups, i.e. standard dose, low dose, and placebo. the most prominent constituents of the genus panax are the triterpene saponins ginsenosides. they are known to have numerous pharmacological activities such as anti-cancer, anti-diabetes, antiviral, and anti-atherosclerosis effects. some compounds of this chemical class showed to be responsible for the immunostimulant activity of ginseng. on the other hand, the efficacy of a polysaccharide-rich extract of american ginseng was compared with an extract rich in ginsenosides on systemic and gut-associated immune function. the authors of this study investigated the lymphocytes isolated from spleen, mesenteric lymph nodes and peyer's patches, and immune cell proportions and cytokine production from sprague-dawley rats. they could show that the polysaccharide-rich ginseng extract modifies the rats' systemic immune responses and affects the gut-associated immunity in a manner distinct from that of the ginsenoside-containing extract of american ginseng. a direct antiviral activity of ginseng constituents could be attested for the polysaccharides on rotavirus infection in ma cells. the triterpene saponins, however, did not exhibit any rotavirus infection-inhibitory activity in this study. a moderate in vitro virucidal effect (id mm) of the ginseng saponin chikusetsusaponin iii against herpes simplex virus type i was detected by fukushima et al. this compound exhibited an intracellular inhibitory activity, but could only marginally affect the viral proteins postinfection. the ancient chinese formula bu-zhong-yi-qi-tang (japanese name hochu-ekki-to) is a traditional herbal medicine in china and japan that is composed of ten species of medicinal plants, namely astragali radix, atractylodis lanceae rhizoma, ginseng radix, angelicae radix, bupleuri radix, zizyphi fructus, aurantii nobilis pericarpium, glycyrrhizae radix, cimicifugae rhizoma, and zingiberis rhizoma. this formula is reported to have various immunomodulatory, [ ] [ ] [ ] and anti-inflammatory activities. yamaya et al. recently investigated the effects of hochu-ekki-to in cultures of human airway epithelial cells infected with hrv- . the output of virus, associated levels of viral rna, and the production of icam- , cytokines and acidic endosomes in cells were measured. in airway epithelial cells hochu-ekki-to was able to decrease virus output and susceptibility to hrv infection by decreasing icam- and by blocking the entry of viral rna into the cytoplasm from the endosomes. glycyrrhizin, a major component of one herbal ingredient of hochu-ekki-to, i.e. glycyrrhiza glabra, was able to reduce supernatant virus titers dose-dependently, with a maximum effect between . and . mm. however, no clinical trials with representative numbers of subjects are published so far. the human rhinovirus k . umckaloabo (pelargonium sidoides) p. sidoides and p. reniforme form the origin of the popular drug umckaloabo. this herbal remedy from south africa has found entrance in western medicine mainly as aqueous ethanolic root extract from p. sidoides for the treatment of infections of the respiratory tract. the efficacy of umckaloabo compared with placebo has been evaluated in adults suffering from common cold by lizogub et al. the applied herbal preparation was well tolerated by the patients. the study demonstrated only a weak efficacy of umckaloabo compared to placebo after days. after days, however, the p. sidoides extract significantly reduced the severity of symptoms and shortened the duration of the common cold compared with placebo. just recently, timmer et al. selected randomized controlled trials examining the efficacy of p. sidoides preparations for the treatment of various acute respiratory infections and analyzed their efficacy and safety. the authors concluded that umckaloabo may be effective in alleviating symptoms of acute rhino-sinusitis and the common cold in adults. it may be effective in relieving symptoms in acute bronchitis in adults and children, and sinusitis in adults. reliable data on the treatment for other acute respiratory infections however were not obtained. identification of the metabolites from umckaloabo revealed a high number of different chemical classes, such as phenolic and cinnamic acids, tannins, flavonoids, and coumarins. antibacterial activities of umckaloabo against different pathogens have been reported. phenols, coumarins, and tannins have been identified to contribute with moderate antibacterial activities, however, cannot explain the effect of the whole extract (reviewed by kolodziej , ). additionally, p. sidoides extracts have been reported to significantly activate the nonspecific immune system by induction of tnf and no-release, and ifn-like activities. these effects are assumed to contribute to the controversially discussed potential of p. sidoides extract for the treatment of upper respiratory tract infections. only one study reports a direct antiviral effect, i.e. a clear dose-dependent anti-herpes simplex virus acitivity for the aqueous root extract of p. sidoides. further pharmacological studies are needed to elucidate potential direct anti-hrv properties of umckaloaba and its constituents. carrageenan, a mixture of different polysaccharides, which is mainly extracted from red seaweeds, has been extensively used in food, cosmetic and pharmaceutical industry as a thickener and gelling agent. it has previously shown an antiviral efficacy against several viruses. , in a recently published study, lambda-, kappa, and iota-carrageenan were investigated for their anti-hrv inhibiting potential. at a concentration of mg/ml iotacarragenan, a sulphated polysaccharide, was able to fully inhibit virus-induced cell death in hrv- infected hela cells. based on their studies, grassauer et al. concluded that iotacarrageenan is effective against different hrv-serotypes on primary human epithelial cells. it is hypothesized by the authors that iota-carrageenan might create a hostile environment for hrv and thereby block viral entry and replication. because of its safe application and proved in vitro efficacy, iota-carrageenan deserves consideration as a candidate for clinical trials for prevention and therapy of hrv-induced common cold. the level of knowledge on the impact of the six botanicals on hrv-infection discussed above is different and heterogeneous. the best studied herbal remedy associated with common cold, i.e. echinacea, showcases the innate problem connected with multi-component mixtures: starting from the late nineties till june some original articles have been published to this topic and tried to elucidate questions concerning efficacy, molecular mechanism, and bioactive ingredients of echinacea. although some evidence is provided for the effects of extracts, chemical classes as well as well-defined constituents on specific targets and pathways, the findings cannot be deduced to a common denominator. further, results from clinical trials often suffer from lack of comparability, because of using different study designs, outcome measures, and overall the application of different preparations. a proper quality analysis and characterization of the preparation under investigation is mandatory and should follow the recommendations and guidelines for reporting clinical trials for herbal medicine. as underlined before (sections . . and . .), the chemical complexity of a natural preparation might be beneficial in terms of synergistic, additive, and overlapping effects caused by the multitude of evolutionary trimmed metabolites, which may attribute with modulating multi-target effects. on the other side, exactly this fact is hardly compatible with the proper assignment of an activity to a defined chemical entity according to western medical practise. in contrast to a single compound (synthetic or naturally based), the chemistry of a botanical is not only complex but also varying. the analytical profile and in term the pharmacological profile of the investigated samples can differ substantially. accordingly, the quantitative and qualitative comparison of different studies resulting from botanicals is by far more complex than those performed with pure single compounds, and may also explain why so little emphasis from pharmaceutical industry has been put into the further development of even promising natural preparations. in general, the search for potent, selective, nontoxic compounds that might be developed further to a drug substance is a multidisciplinary, time-and cost-consuming process. therefore, strategies for a target-oriented discovery of lead-structures either from nature or synthesis are in high demand. some of them will be discussed in the following section, providing examples from anti-hrv research. as already mentioned, nature provides an extremely rich pool of bioactive natural products. it is however a challenging endeavor to find exactly those compounds that show an activity on the focused target. in natural product research hints from folk medicine are a valuable starting point to dig for lead structures of certain interest. the majority of active principles from higher plants has been discovered as a result of ethnopharmacologically directed pharmacognostic research. , oral or written indications for a beneficial application of a natural material first need a critical evaluation as to the selection of the correct material, its medicinal preparation, the kind of application, and overall the pharmacological profile. a rational criticism of often anecdotal efficacy from traditional medicine is a mandatory attitude to avoid overinterpretation of handed-down information. in case of pretended anti-hrv remedies, the reported biological efficacy obviously suffers from being restricted to respiratory diseases, which might be caused by a panel of bacterial or viral infections. thus, an approved remedy may affect the microbes, or show an immune modulating or even antiinflammatory effect, or a combination of these. the holistic access is an innate character of ethnopharmacology and needs to be tracked down to the specifically involved target/s. in a recently published study focusing on the discovery of hrv-capsid binders from nature using a pharmacophore-based virtual screening of an ethnopharmacologically biased d-multiconformational database, some secondary metabolites were predicted to act as capsid-binding inhibitors of hrv. for an in depth phytochemical and pharmacological investigation, it was mandatory to focus on one promising natural material. thus, we consulted the ethnobotanical source materia medica, which was written by pedanius dioscorides in the st century ad. the treatise consisting of five books comprises some , or so drugs derived from minerals, animals, and the majority of them from plants ( ). it represents a great repository of botanical, medical, and pharmacological lore. scrutinizing the natural materials underlying the obtained virtual hits we came across asafetida, which is a gum resin gained from the roots of a variety of foul-smelling ferula species from the apiaceae family. based on the descriptions given in the materia medica there is strong evidence that the juice (i.e. resin) of the popular ancient silphion originating from media and syria corresponds to asafetida (book iii, cap. ). yielded by incision of the root and stalk and frequently mixed with sagapenon, i.e. the resin of f. persica willd (book iii, cap. ), it is reported to be effective in the context of upper respiratory diseases, e.g. ''for chronic harshness of the throat,'' ''it clears the voice,'' ''shrinks the uvulas,'' ''suitable for a cough,'' ''for pleurisy,'' ''for chest pain;'' sagapenon is described as follows: ''it clears thick matters from the lungs,'' ''given to those who are chilled.'' these descriptions finally helped to prioritize those virtual hits, which have been reported to be constituents of asafetida. the pharmacological investigation of asafetida and its constituents farnesiferol b and c ( fig. ; table iii ) revealed a distinct anti-hrv- effect in the low micromolar range using a cpe inhibitory assay. the results of this study provided a rationale for the ancient usage of asafetida for upper respiratory tract infections. on the other hand, the traditionally manifested evidence for asafetida for the treatment of common cold symptoms substantially helped in the selection of this plant material in the search for anti-hrv capsid binders. typically, an ethnopharmacologically based discovery of an active (anti-hrv) extract is followed by a bioassay guided fractionation, and the isolation of those constituents that are responsible for the extracts' bioactivity. the concept of a bioassay-guided approach is the fractionation accompanied by simultaneous detection of the activity during the separation steps, which results in a continuous enrichment, and finally in the isolation of the active ingredient/s. in this way a large number of anti-hrv agents from natural sources have already been discovered. semple et al. investigated the active principle of the asteraceae plant pterocaulon sphacelatum, which has been used in traditional medicine of aboriginal people of australia as a favored treatment for respiratory infections, especially colds. by means of an antiviral activity-guided fractionation measuring the poliovirus-induced cpe assay, the authors identified the flavonoid , , -trimethoxy- , , -trihydroxyflavone, i.e. chrysosplenol c ( fig. ; table iii ) as potent and specific inhibitor of the picornaviral replication. , , -trimethoxycoumarin ( fig. ; table iii) was also isolated as a major constituent from the ethanolic extract of p. sphacelatum, but showed no activity against poliovirus. interestingly, this coumarin exhibited a significant effect in the hrv- -induced cpe assay in a recently performed virtual parallel screening study performed in our laboratory. in contrast to the isolated coumarin, chrysosplenol c was already known as a member of the -hydroxy- -methoxyflavones, which represent potent and specific inhibitors of picornaviral, especially rhinoviral replication. [ ] [ ] [ ] in , vanden berghe, haemers, and vlietinck provided a profound survey of antiviral agents from higher plants, and demonstrated the impact of ethnobotanical knowledge in their search for antiviral compounds from african medicinal plants. the selection of investigated plant species was mainly based upon their use in the treatment of viral diseases by african traditional healers. the antiviral activity of different plant species belonging to families was investigated; thereof species exhibited prominent antiviral properties against one or more of the tested viruses. the most pronounced activity against picornaviruses was recorded within the genus euphorbia. all compounds detected as antiviral constituents from the respective extracts were identified as -methoxyflavone derivatives, especially -methylethers of quercetin and kaempferol ( fig. ; table iii ). they showed no significant cytotoxicity and were highly active in tissue culture against all human picornaviruses. in tissue culture, cells infected with different picornaviruses (among them also hrv) no cpe was observed, when cells had been treated with mg/ml of these -methoxyflavones. the selection of natural materials based on ethnopharmacology is a profound rationale for lead structure discovery and highly superior to random selection. this however rarely applies to marine organisms, fungi, and microbes. although these organisms are esteemed as highly valuable source for bioactive metabolites, hardly any records from folk medicine are given for them. a medium-sized activity screening using a robust cell-based or in vitro-assay with reasonable effort as to time and costs is a strategy to get a first insight into the antiviral activities of extracts, fractions, and compounds from synthesis or nature. for the discovery of novel naturally based hrv c-protease inhibitors, singh et al. used a small peptide containing q-g scissile bond as substrate for the in vitro screening of extracts. the authors isolated the novel benzoisochromanquinone ( )-thysanone ( fig. ; table iii ) from an extract of the fungus thysanophora penicilloides with potent hrv c-protease inhibitory activity. a continued screening revealed a pronounced hrv c-protease inhibitory activity for the extract of the chinese herb polygonum cuspidatum. by bioassay-guided fractionation -methoxystypandrone ( fig. ; table iii ), a naphthoquinone, with an ic value of . mm, was isolated from the plant material. the total syntheses of this natural compound and further analogues allowed for a structure-activity relationship, and particularly the comparison between activities of ortho-vs. para-quinones. to measure the selectivity of the compound series against cystein proteases other than hrv c-protease, the compounds were evaluated against papain. the simple , -phenanthraquinone ( fig. ; table iii ) was the most active compound of the series and showed an ic value of . mm with a distinctly higher degree of selectivity than -methoxystypandrone. a cpe reduction assay was applied for the identification of raoulic acid, a bicyclic c terpene acid ( fig. ; table iii ) isolated from raoulia australis (asteraceae). raoulic acid exerted an antiviral activity against coxsackie virus b , b , enterovirus , and hrv- and - with ic values in the submicromolar range. no activity was recorded against influenza a and b viruses. in the course of the systematic screening of microbial and natural products for anti-hrv activity, ishitsuka et al. identified , -dihydroxy- , , -trimethoxyflavone ( fig. ; table iii) from the leaves of the chinese medicinal plant agastache rugosa (lamiaceae) as natural compound with high activity against all picornaviruses except mengovirus. the authors synthesized the orally active , -diacetyloxy- , , -trimethoxyflavone and performed investigations in tissue cultures and in mice to determine the compound's mode of action. this was assumed to be the process of viral replication, thus located between viral uncoating and initiation of viral rna synthesis. the activity of flavan ( fig. ; table iii ) was discovered serendipitously during a screening program by using a plaque inhibition assay. based on these results, bauer et al. synthesized , -dichloroflavan (bw c, fig. ; table ii) , which revealed an activity against a number of hrv serotypes in the range between . and mm. as soon as an active lead compound is identified it is of utmost importance to scrutinize the derivatives' activity to (i) obtain insight into the chemical requirements mandatory for the focused biological activity, (ii) improve the compound's pharmacological profile in terms of potency, selectivity, cytotoxicity, etc., and to (iii) improve its bioavailability. the nonphenolic aporphine alkaloid glaucine is a prominent constituent of the aerial parts of gaucium flavum (papaveraveae). in a recently published study, spasova et al. investigated the antiviral potential of glaucine ( fig. ; table iii) , glaucine derivatives, and its semi-synthesized -aminoethylglaucine cinnamoyl-and hydroxycinnamoyl amides. beside the anti-oxidative potential of the newly synthesized compounds, they all exerted an antiviral activity against the replication of hrv- . the best anti-hrv activity was observed for oxoglaucine ( fig. ; table iii; ic table iii; ic mm, si . and ic mm, si , respectively). the early findings of the anti-hrv active naturally derived -methoxyflavone inspired chemists and pharmacologists in the synthesis and the pharmacological evaluation of derivatives thereof decorated with various substitution patterns. several reports published during the last two decades reviewed the findings of antiviral flavonoid research. , , by investigating the antiviral activity of a wide variety of naturally occurring flavonoids, tsuchiya et al. found chrysosplenol b and c ( fig. ; table iii ) contained in chrysosplenium plants, and axillarin ( fig. ; table iii ) as potent anti-hrv agents. based on their findings, the flavone skeleton decorated with a methoxy group in position and a -hydroxyl group revealed as mandatory for an anti-hrv activity. a series of antipicornaviral -hydroxy- -methoxyflavone derivatives was synthesized by de meyer et al. in order to establish a structure-activity relationship. thereby, different substitution patterns of the a-ring system with methyl, hydroxy, methoxy, halo, nitro, and amino was performed. their activity against polio and hrv was compared with those of naturally occurring flavonoids. further, the importance of the hydroxyl-group and of the -methoxyl-group was confirmed by investigation of different derivatives lacking these features. the results showed that -hydroxy- -methoxyflavones with a monosubstituted a-ring are less active than the corresponding compounds having a polysubstituted a-ring. within the tested series of compounds, , -dihydroxy- -methoxy- , -dimethylflavone emerged not only as noncytotoxic but also as most potent substance in both antiviral test systems. the lowest concentrations for this compound that protect % of the cells from cpe of hrv serotypes were in the range from . to . mg/ml. in contrast to quercetin, this flavone was also reported to have no mutagenic properties (measured up to . mg/plate) in a short-term microbial assay. the mechanism studies performed with -methoxyflavones have shown an interference with an early stage in the viral rna synthesis; no induction of resistance was observed. in contrast to this mode of action, the anti-hrv chalcones and flavans are reported to interact directly with specific sites on the viral capsid proteins, thereby preventing uncoating and the consequent liberation of viral rna. , , due to its anti-hrv potency, low toxicity, and promising bioavailability, dichloroflavan was evaluated for its protective efficacy against experimental hrv-infection in two clinical, double-blind, placebo-controlled trials. however, the drug candidate failed either when administered orally or intranasally. , unfortunately, till now no clinical trials evaluating the efficacy of -methoxyflavones in common cold have been performed. the common idea of all computational approaches is to extract knowledge from a more or less large set of data in order to make predictions of new events. within the lead discovery process, computational approaches, such as virtual screening, docking, quantitative structure-activity relationship, have largely enhanced the impact of computational chemistry and nowadays chemoinformatics plays a predominant role in drug research. the key goal of the use of such methods is to reduce the overall cost associated to the discovery and development of a new drug by identifying the most promising candidates to focus the experimental efforts on. a number of books and reviews on the impact of computational chemistry for lead structure determination highlight these efforts. [ ] [ ] [ ] [ ] in general, in silico methods can be divided into (i) ligand-based approaches, which rely on known active compounds. based on their physicochemical properties crucial for biological affinity, activities are predicted by extrapolation on not-yet tested substances, e.g. machine learning techniques and classical quantitative structure-activity relationship (qsar). ligand-based approaches are invaluable tools in cases where no structural information about the pharmacological targets is available. (ii) on the other hand, structurebased approaches use experimentally determined d structures of the targets, such as molecular docking or structure-based pharmacophore modeling for virtual screening. these methods allow for gaining insight into protein-ligand interactions at an experimentally determined (static) level (however not considering flexibility). a unique platform containing d coordinates of experimentally solved protein structures (by x-ray crystallography or nmr) is the protein data bank (pdb) currently comprising more than , structures of biomolecules and protein-ligand complexes. additionally, there are several pdb-related web services and tools, which enable to use the pdb-portal in a rich diversity of information services for students and scientists. particularly in the early stage of drug development, such as lead discovery and lead optimization, computational approaches allow for a target-oriented and rationalized proceeding, and thus may substantially help to maximize the success rate. a recently published review on the impact of computer-assisted approaches in antiviral research thoroughly describes underlying in silico techniques, and highlights the benefits of computational approaches for the discovery of antiviral lead structures. in anti-hrv research the capsid protein and the protease c revealed to be promising targets (as described before). inhibitors are assumed to have a major impact for the treatment of hrv-infections. additionally, these targets are structurally elucidated, and some potent ligands are known as well. these facts enable the performance of sensible computer-assisted approaches, both ligand-and structure-based. some studies using an in silico approach for the discovery of potential anti-hrv agents focusing on the mentioned targets will be reported in the following paragraphs. for compounds acting as potential hrv-capsid binders, some classical qsar and d qsar studies have been performed. while in classical qsar, the relationship between d calculated properties derived from chemical structures and measured biological activities are explored statistically, d qsar techniques are aimed at deriving a correlation and in turn activity prediction based on spatial arrangements of chemical properties and atoms. applying the d qsar technique comfa (comparative molecular field analysis) statistical models are derived, which are visualized in color-coded contours around the molecule. therein spots indicate where electrostatic properties and spatial arrangements are favorable for biological activity. in the studies of diana et al., artico et al., and verma et al., qsar techniques helped on one hand to analyze and rationalize the structural features of active compounds essential for the interaction to the hrv canyon's binding pocket, and on the other hand to search for new classes of capsid binders, thus to narrow the synthetic challenges for specific anti-hrv agents, respectively. [ ] [ ] [ ] [ ] [ ] in all these investigations hydrophobicity was found to be one of the most important determinants of substance activity. qsar combined with simplex representation of molecular structure was applied by kuz'min et al. based on the selectivity index (cc /ic ) and the hrv- inhibitory concentration of a set of [(biphenyloxy)propyl]isoxazole derivatives. on the basis of qsar analysis and computational design, three new isoxazoles with high activity prediction were selected and synthesized. they all revealed a strong coincidence between experimental and predicted anti-hrv activity and selectivity index. terminal benzene substituents with negative electrostatic potential and a molecule length of approximately . - . Å have been suggested as mandatory features within this chemical class for a hrv- inhibitory activity. hrv-serotypes show a high level of conservation at the protease c binding site; sequence alignment and secondary structure predictions suggested an overall architecture and mechanism of hrv- c proteases that correlates with cellular cystein-and serine proteases, such as chymotrypsin and trypsin. , the identity among c proteases from different families is however modest and provides space for the development of specific inhibitors for hrv- c protease. in a recently published review on selective inhibitors of picornavirus replication, de palma et al. summarized all currently known chemical structures acting as peptidic or nonpeptidic inhibitors of this viral target. in , reich et al., used the hrv c protease co-crystal structure information to rationalize the target-oriented synthesis of hrv c protease inhibitors from the class of substituted benzamides. activity data and subsequent crystallographic studies pointed out important requirements for the inhibition of the c protease. similarly, maugeri et al. used a structure-based approach, and performed docking studies based on the c protease crystal structure with a virtual library consisting of benzamide derivatives. quantum-mechanic calculation proposed substituents with most promising biological activities. this workflow guided the design and synthesis of substances virtually assumed to act as substrate analogues. synthesis of some of these compounds and biological testing confirmed the underlying hypothesis. quantitative molecular modeling studies were performed to better define and predict interactions between bicyclic -pyridone derivatives that showed to be irreversible inhibitors of the c protease. in these studies molecular mechanics simulations to evaluate chemical rate of covalent bond formation and free energy calculation combined with crystallographic studies were applied to explain the differences in activity of some irreversible peptidomimetic inhibitors. these data were used as a basis for further optimization of these compounds. , in a recent study performed by kuo et al. some , compounds were subjected to a high troughput screening in the search for novel inhibitors for both c and cl proteases from picornavirus and coronavirus, respectively. five nonpeptides were identified with ic values r mm against severe acute respiratory syndrome-coronavirus cl-protease; one molecule was found to additionally inhibit the c proteases of coxsackievirus, enterovirus, and rhinovirus. this compound (id ) contains a dihydropyrazole ring decorated with two phenyl groups and a lengthy n-butyl-benzimidazolylamino-toluene. it was used as starting point for the selection of further four analogs showing ic values in the range of . - mm against the tested viral proteases. by means of docking-based computer modeling, the authors tried to rationalize the binding discrepancies responsible for individual and common protease inhibitors, thus to provide a rational base for the development of nonpeptide multiple-function inhibitors against coronaviruses and piciornaviruses. in anti-hrv research, first application scenarios have been conducted using pharmacophore models. according to the official iupac definition by wermuth et al., a pharmacophore describes the d arrangement of steric and electronic features necessary to trigger or block a biological response. pharmacophores can be represented by three-dimensional chemical features, which include hydrogen bond donors and acceptors, aromatic rings, hydrophobic groups, as well as positive and negative ionisable moieties. additionally, the shape of ligands can be represented by shape features, which essentially describe the van der waals radii of the ligand atoms. the pharmacophore concept has proven to be successful, not only in rationalizing structure-activity relationships but also by its large impact in developing appropriate d-tools for efficient virtual screening. , steindl et al. elaborated ligand-and structure-based pharmacophore models implementing the essential feature of the covalent binding to the cysteine in the active site of the hrv- c protease. thus, the in silico approach focused on defining a new pharmacophore feature representing a target structure for nucleophilic addition in the ligands, which is a crucial step for protease inactivation. the generated hypotheses retrieved known c protease inhibitors in the virtual screening cycle, and proposed potential (unconfirmed) ligands of the d protease binding site from available databases. the viral capsid of several hrvs has been elucidated by crystallization and resolution of the d-structure. the hrv coat protein complexed with its highly active inhibitor win was used as starting point for the generation of structure-based pharmacophore models by . the models were used for virtual screening of a large commercially available d database. for final selection of virtual hits worth to be subjected to biological testing, docking studies and principal component analysis were performed. six candidates were tested for their ability to inhibit hrv serotype by multiple-cycle cpe inhibition assay. although all of them showed a certain antiviral potential, one longitudinal piperazine derivative inhibited the virus at a concentration below mm. some of the test candidates showed difficulties in the interpretation of experimental results due to their relatively high cytotoxicity and bad solubility. this circumstance asks for more cautious estimation of molecular properties for compound selection as stated by the authors. in a subsequently performed study, the best validated pharmacophore model was used for the identification of naturally derived hrv coat protein inhibitors. for virtual screening experiments the in-house generated d-database dios was used (as described before). based on the virtually predicted ligands and considering knowledge from traditional use, sesquiterpene umbelliferons from the gum resin of ferula sp., i.e. asafetida, were finally selected as most promising candidates. for biological evaluation, the antiviral activities of asafetida and its isolated constituents were assessed by an exploratory determination of the inhibition of the cpe induced by hrv serotypes a, , , and . the results revealed a dose-dependent and selective anti-hrv activity against serotype for asafetida and its virtually predicted constituents, farnesiferols b and c ( fig. ; table iii ; ic : . and . mm, respectively). to scrutinize the selectivity of these two compounds against hrv- in comparison to the other tested serotypes, the amino acid sequences of hrv- and hrv- vp were aligned. since all amino acid residues involved in ligand binding showed % match in both serotypes, the experimentally determined selectivity profile could not be explained by different binding pockets. the serotype alignment does however not reflect potential protein flexibility during binding, which might differ between the hrv serotypes. additionally, off-target effects could be a reason for the observed selectivity. in a recently performed virtual parallel screening approach, we tried to identify potential targets of human pathological relevance for constituents isolated and identified from the medicinal plant ruta graveolens. using the screening platform pipline pilot included in discovery studio, low-energy conformers of the identified molecules from r. graveolens were subjected to parallel screening. for this purpose, the inte:ligand pharmacophore model collection was used. it currently comprises . models covering unique pharmacological targets. based on the predicted ligand-target interactions, the authors focused on three biological targets, namely acetylcholinesterase, the hrv coat protein, and the cannabinoid receptor type . virtual hits and nonhits were assayed on their respective targets for a critical evaluation of the performed target-fishing approach. beside other predicted bioactivities, determination of their cpes on hrv- revealed the virtual hit arborinine ( fig. ; table iii; ic : . mm) and the nonpredicted hit , , -trimethoxycoumarin ( fig. ; table iii ; ic : . mm) as the most active anti-hrv constituents. it could be shown that the applied in silico strategy has the capacity of catalyzing drug discovery profoundly for all those diseases where molecular targets or molecular ligands are well defined to create reliable pharmacophore models. hrv, a prominent member of the picornaviridae family, infects humans more frequently than any other virus. infections with hrv mainly lead to upper respiratory diseases, such as the common cold, but may also cause more severe lower respiratory tract disorders. although the symptomology and severity of the common cold is relatively mild and the course of disease self-limiting, the socio-economic impact is tremendous in terms of recouping lost productivity due to sick leave. in the last decades, a number of anti-hrv agents from different origin-synthetics, natural compounds, biologicals, botanicals, and nutritionals-have been discovered. different concepts have been used as strategy for their discovery either starting from a phenomenological effect, e.g. empirical knowledge from folk medicine, or from a targetbased molecular level, e.g. icams, capsid binders, and hrv protease inhibitors. some of the outcomes revealed potent and promising activities that partly have been evaluated for the management of hrv-induced common colds in clinical trials mainly with sobering benefit. since the hrv infection is not life-threatening in most cases, a potential therapy has to be safe and effective with an almost unrecognizable level of side effects. these preconditions render the search for anti-hrv-agents into a high challenging endeavor and explains why no antiviral agent is approved for the prevention or treatment of hrv-infection until today, despite the significant efforts. moreover, the high variability of rhinoviruses suggests the need of more than one active principle covering different modes of action for an effective treatment. therefore, there is a high need for an ongoing search for new synthetic as well as natural compounds on a molecular level. the increasing knowledge about the hrv life cycle, gene, and protein sequence combined with the improved technologies in the field of experimental and computational methods have continuously enabled further insights into the spectacular world of hrv. only with this fundamental research, virtual screening approaches, such as qsar, pharmacophore-and docking-based screening cycles, or computational compound design, are applicable on a rational base. with the gained expertise from different disciplines and an adequate infrastructure for further research, it is encouraging to hope that discovery and clinical development efforts will continue in the search for agents that may treat or 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chemistry human rhinovirus c protease: generation of pharmacophore models for peptidic and nonpeptidic inhibitors and their application in virtual screening docking versus pharmacophore model generation: a comparison of highthroughput virtual screening strategies for the search of human rhinovirus coat protein inhibitors ca: accelrys. . discovery studio rollinger extended her studies to the fields of phytochemistry, ethnopharmacology, molecular modelling and its application in natural product science completing the habilitation thesis entitled ''the search for bioactive natural products rollinger is a project leader and an associate in various national projects, as well as executive guest editor for current pharmaceutical design. she has been awarded the phoenix science award ( ) and received several additional awards until she was a postdoctoral research fellow at the institute of immunology, university marburg, where she studied picornavirusimmune cell interactions and the virus-induced cytokine induction. thereafter, she was a postdoctoral fellow at the institute of virology and antiviral therapy key: cord- - jh y authors: li, fufang; li, binbin; huang, yongfeng; feng, yuanyong; peng, lingxi; zhou, naqin title: research on covert communication channel based on modulation of common compressed speech codec date: - - journal: neural comput appl doi: . /s - - -y sha: doc_id: cord_uid: jh y as is well known, multimedia has been widely used in voip and mobile communications. research on how to establish covert communication channel over the above popular public applications has been flourishing in recent years. this paper tries to present a novel and effective method to construct a covert channel over common compressed speech stream by embedding sense information into it. in our method, after analysing the characteristic features of the excitation pulse positions of the itu-t g. . and g. a speech codec, we design a novel and effective covert communication channel by finely modulating the codes of excitation pulse positions of the above two codecs in line with the secret information to be hidden. to improve the embedding capacity of the proposed method, we also use all the odd/even characteristics of pulse code positions to conduct information hiding. to test and verify the proposed approach, experiments are conducted on several different scenarios. experimental results show that our methods and algorithms perform a higher degree of secrecy and sound information embedding efficacy compared with exiting similar methods. covert communication uses human perceptual defects and fuzziness to embed sensitive information into digital carrier media such as text, sound, images, and video signals in a manner that is not easy to be perceived. voice communication is one of the most popular ways for people's daily information exchange. it has a very wide application base and a large amount of communication capacity. therefore, information hiding based on voice carrier has a very wide range of application requirements in the military, security, business, and other fields. the research on information hiding of carriers has very important research significance and broad application prospects. steganography entails the embedding of secret information in digitalized carriers to conceal both its transmission behaviour and contents, thus ensuring its secrecy and security during transmission, which has become an important technical tool for secure secret information transferring [ ] [ ] [ ] . voice-over-internet protocol (voip)based low-bit rate voice services have developed rapidly with the widespread and popularization of internet technologies. therefore, studies on steganographic techniques based on low-bit rate voip are highly significant in theory and in practice [ ] . voip-based steganography had been developed most rapidly among multitudes of steganographic methods that use streaming media as carriers. this is because the secret information embedded within voip communications is highly dynamic, which renders these steganographic methods robust against tampering of attackers. furthermore, voip speech codec can provide excellent levels of imperceptibility and hiding capacity [ , ] . in the research of information hiding based on speech coding, many researchers have carried out fruitful work. reference [ ] proposed a novel qim (quantization index modulation) steganography based on the replacement of quantization index set in linear predictive coding (lpc), by treating each quantization index set as a point in quantization index space. their algorithm had significantly improved the embedding efficiency. tian [ ] tried to present a novel steganalysis method based on statistic characteristics of fundamental frequency, so as to prevent the illegitimate use of voice-over-ip-based information hiding. they introduced the statistics for zero-crossing count (zcc) and the average values of mel-frequency cepstral coefficients (mfccs) to characterize inactive frames, and then, they propose a support vector machinebased steganalysis for inactive speech frames. experiment results showed that their method significantly outperforms the previous ones. wang [ ] put forward a new steganography algorithm by embedding data while pitch period prediction is conducted during low-bit rate speech encoding. their approach has great compatibility and can achieve high quality of speech and prevent detection of steganalysis without causing further delay by data embedding and extraction. article [ ] proposed a method of text emotional modulation steganography based on machine learning. by intelligently expanding the text emotional lexicon and using cosine similarity algorithm to conduct the steganography algorithm, their method obtained sound efficiency, security, concealment, and robustness. by revealing the inactive frames of voip streams which are more suitable for data embedding than the active frames of the streams, paper [ ] presented a novel high capacity steganography algorithm for embedding data in the inactive frames of low-bit rate audio streams. their algorithm had achieved perfect imperceptibility and high data embedding rate up to bits/frame, in which the data embedding capacity is very much larger than most of the existing algorithms. yang [ ] proposed a highly imperceptible, high capacity steganographic algorithm based on the noise coding frames of the g. . codec. literature [ ] studied a novel method to embed sensitive data into g. . speech codec by using the characteristic of the codec. their algorithm can obtain good efficiency and performance. guo [ ] put forward an imperceptible information hiding approach on . kbit/s codec of g. . . to increase the data embedding capacity, they also propose a voice activity detection method that uses the residual signal energy of the speech signal. in recent years, research on information hiding based on a speech coding has also attracted the attention of many researchers. literature [ ] demonstrates the respective performance advantages of the two standards in the error concealment process through the frame loss experiment and subjective test on g. . and amr_wb (adaptive multi-rate wideband). el-khamy et al. [ ] give out a new robust audio steganography technique based on optimum two-dimensional complete complementary codes (ccc). when adopted their method to embed colour images data, and with various attacks applied to the carrier audio signals, simulation experimental results showed that the extracted image had good quality, which proves high robustness and capacity of their proposed algorithm. literature [ ] counts the unimportant bits of the compressed low-bit speech stream, proposes embedding hidden information in the speech stream, and designs a voice concealed communication system of ip network using this algorithm to improve the security of secret information transmission. literature [ ] proposes an algorithm based on code word characteristics, which enables g. a to carry out qim steganography to detect effectively in a short time. paper [ ] discussed the application of speech-based information hiding in the area of medical scenario. literature [ ] carries out a comprehensive test for the frame-by-frame bitwise negation of g. a speech and finds that the speech has hidden bits, and a method of information hiding combined with matrix coding is proposed. as is discussed above, steganography based on compressed speech codec has been deeply studied by many researchers. however, the robustness, concealment, and hiding capacity of existing algorithms are still difficult to meet the needs of practical applications. therefore, it is urgent to study new information hiding methods that have both high privacy and high hiding capacity. to explore a more effective way to establish a novel convert communication channel over compressed speech stream, this paper presents a steganography method based on modulation of the excitation pulse positions of the g. . low-bit rate codec and g. a speech codec as the research scenario. in the proposed method, the secret sensitive information is hidden in the above speech stream by finely modulating the codes of excitation pulse positions. experimental results show that the proposed steganography algorithm of g. . speech codec with an embedding rate of . % and secret information transfer rate of bits/s results in \ . % degradations in perceptual evaluation of speech quality (pesq) scores, which indicates a high degree of imperceptibility. as to the proposed steganography algorithm of g. a speech codec, our method can achieve bite/s secret information communication with % embedding rate, while the capacity and the degradation rate of the pesq score for the carrier speech is less than %, indicating that the hidden method studied has good privacy and efficacy. this paper consists of six sections: sect. (that is, this chapter) briefly introduces the original purpose and related work of the research; sect. introduces the related g. . and g. a speech codec; sect. discusses the model of information hiding and extraction construction method of emotional lexicon based on deep learning; sect. gives the information hiding algorithm based on matrix encoding and emotional word replacement; sect. shows the experiment results and analysis; sect. shows the summary and prospect part. kbit/s high bit rates) algorithms to minimize the perceptually weighted error signal [ ] . according to g. . speech recommendation, the analogue input signal is firstly analysed and processed by using the rules of g. . standard, and then, the parameters, such as lsp parameters, codebook indices, and gains, are calculated. secondly, the parameters are digitalized so as to facilitate transmission in the next step. at last, the digital parameters are packed and wrapped according to g. . standard and then send to the transmission channel. by this way, the encode process of g. . speech codec has been finished. at the receiving end, firstly, the wrapped parameters are unpacked, and thus, the parameters can be obtained by the decoder. secondly, the decoder uses these regained parameters to reconstruct the analogue speech signal [ ] . the encode and decode process of g. . speech codec is shown in fig. . the g. a protocol, which proposed by international telecommunication union, telecommunication standardization sector (itu-t), is a -kbps speech coding standard and is a simplified version of the speech coding standard g. . the g. a protocol uses conjugate structured arithmetic codebook excited linear prediction (cs-acelp) as its coding principle. the cs-acelp encoder is based on a codebook excited linear predictive coding (celp) model. the encoding procedure of g. a speech codec is: ( ) the analogue speech signal is sampled at khz after being band-pass filtered by the speech channel, quantized into a -bit linear pcm digital signal, and then, they are inputted to the g. a encoder. ( ) the g. a encoder first processes the -ms-long speech frame as follows: linear prediction analysis and quantization of lpc coefficients; open-loop pitch period estimation; adaptive codebook search; fixed codebook search. ( ) then, calculate the celp model parameters (lp filter coefficients, adaptive codebook, and fixed codebook index and gain) frame by frame. ( ) finally, encode and transmit these parameters, and thus, the encoding process is finished. the detailed bit allocation of the bits of g. a coding parameters is shown in table . the g. a decoder decodes the g. a code stream and extracts the corresponding line spectrum pair (lsp) coefficients and two fractional pitch delays, two fixed codebook vectors, and two sets of adaptive codebook and fixed codebook gains. these parameters are used to recover the excitation signal and synthesis filter parameters. according to the mechanism of speech generation, linear prediction (lp) filter filtering is used to implement speech synthesis (i.e. speech reconstruction). after the synthesized speech is obtained, post-filtering is performed to improve the speech quality. the method of embedding and extraction secret information based on modulation of the excitation pulse positions of the common compressed speech codec is illustrated in fig. . the low-bit rate acelp of g. . codec uses adaptive codebook searches. a g. . acelp frame is composed of four subframes, and each subframe has four excitation pulse positions. acelp codec uses bits to identify every excitation pulse position in each subframe. to embedding sensitive secret information into the acelp codec bitstream, we finely modulate the excitation pulse positions' code according to the bit of secret data to be embedded. let's suppose the secret information to be embedded is the information embedding subalgorithm of algorithm _ (named as algorithm _ _ ) and the extraction subalgorithm (named as algorithm _ _ ) are as follows: let's suppose the bitstream of the secret information to be embedded is since the pulse positions of every subframe in the g. . high bit rate mp-mlq codec must be integers in the [ - ] interval, and the pulse sequence numbers of each subframe are either all even or all odd, there are only (fewer than - ) possible variations in position for any pulse in a subframe. as the code of pulse positions has less than bits, each pulse may then hide a maximum of bits of secret information. however, not every pulse position code has bits, so it is more appropriate to hide only bits of secret data in each position. this is consistent with the findings of our preliminary experiments. suppose again the bitstream of secret information to be embedded is the fixed code book encoded by g. a is an algebraic code book structure. each code book vector contains four nonzero pulse signals, and the amplitude of each pulse is positive or negative. the positions of each pulse are shown in table . from table , it can be seen that the g. a code has -pulse excitation for each frame of speech signal ( samples), the first, second and third pulses have eight possible positions, and each pulse position is represented by bits. the fourth pulse has two groups of odd and even numbers, with possible positions. the position of the pulse is represented by bits. therefore, the positions of the four pulses in a frame in g. a coding are presented by bits. the detailed distribution of the position of the pulses in g. a fixed code book is shown in table . the ideas of secret information embedding method in information hiding algorithm of g. a coding based on excitation pulse position modulation are shown in fig. . the proposed information embedding subalgorithm of algorithm _ _ is as follows: • step read the information that needs to be hidden and transforms the information into a binary data form of . • step the hidden information of bit (either or ) is read and compared with the parity of the current pulse position value (either or ). if they are also even or odd, the information can be hidden directly without numerical processing of pulse position; otherwise, the position of pulse will be added or subtracted by , so that the bit information to be hidden can be the same as the parity of pulse position value. at this time, the parity of the lowest bit of the pulse position value (binary or ) indicates that bit of information has been hidden. the bit of pulse excitation is found in all parameter bit streams decoded by the g. a decoder. comparing the parity of the hidden information in each pulse coding, i.e. the lowest bit's value of the binary form of the pulse position, the hidden bit of binary information is extracted from the corresponding pulse sequence by (ip) & ( hex). the secret information extraction method in information hiding algorithm of g. a coding based on excitation pulse position modulation is shown in fig. . to evaluate the proposed algorithms, we examine the changes in objective speech quality of pesq scores in different types of voices, and under condition of before and after the embedding of secret information, at different levels of hiding capacity. in the experiments, we used selfedited clips of chinese and foreign male and female voices that were sampled at khz to perform the pesq tests. by using the experimental scheme described in sect. . , objective speech quality tests of pesq values are carried out. the average pesq scores of these self-edited clips of voices (that do not contain any hidden data) are slightly lower than that of standard g. . acelp speech codec. to facilitate an intuitive observation of steganographic efficacy, the results of this experiment are represented as reduction percentage of pesq scores. the average pesq scores and their reduction percentages of before and after steganography by conducting algorithm _ are shown in table , while table illustrates how pesq scores are changed when hiding capacity and embedding rate increase. in table , the first row of data field shows the original average pesq scores of the edited clips of each kind of voice in which no sensitive data have been embedded in. the other rows in the data field of table indicate the average pesq values and their percentages compared to the original pesq values under different embedding rates of sensitive information by using algorithm _ . the corresponding rows in the data field of table , respectively, indicate the average pesq value of the carrier voice and its decrease percentage compared to the original pesq value under different embedding rates and data transmission rates. from tables and , we can also see that, even when the embedding rate goes up to . % and the hiding capacity was bits/s, the reduction percentage in pesq score was less than . % and thus indicates an excellent level of imperceptibility and efficiency can be achieved by algorithm _ . and table tells us the average pesq scores of the speech carrier's decrease with increasing hiding capacity and embedding rate when steganography is performed by using algorithm _ . experiment of algorithm _ is also based on the experimental scheme described in sect. . . the average pesq scores of these voices without information hidden are also slightly lower than the standard pesq score of g. . mp-mlq speech codec. again, to facilitate an intuitive observation of steganographic efficacy, the results of this experiment are represented as percentage reductions in tables and . the first row of the data field of table is as the same as the first row of table . the other rows in the data field of neural computing and applications rates. tables and show that the degradation of pesq score is less than . % even when the embedding rate is . % and the hidden information transmission rate is bits/s, which indicates an excellent level of imperceptibility. and in table , we can see that the average pesq score of the speech carrier decreases with increasing hiding capacity and embedding rate when steganography algorithm _ is performed. to further increase data hiding capacity, we perform an experiment with algorithms _ and _ being used in combination. these experiments are also implemented using the scheme described in sect. . . to investigate different combinations of these algorithms, we perform experiments based on the four scenarios that are described below: ( ) bits of information is hidden in the first pulse of each subframe, except bit of information that is hidden in the reserved bit; hence, each frame (which contains subframes) can hide bits of information in total. ( ) bits of information is hidden in the first pulse of each subframe, and bit of information is hidden in the last bit of the last subframe, while more bit of information is hidden in the reserved bit; hence, each frame hides bits of information in total. ( ) in each subframe, bits of information is hidden in the first pulse and bit of information is hidden in each of the last two pulses, and bit of information is hidden in the reserved bit. hence, each frame is hiding bits of information in total. ( ) in each subframe, bits of information is hidden in the first pulse while the bit of information is hidden in the last bit in each of the remaining pulses, and more bit of information is hidden in the reserved bit. since the first and third subframes have pulse positions while the second and fourth subframes have pulse positions; each frame is therefore hiding bits of information in total. for the pesq reduction and its reduction percentage of the above fourth scenario is too sharp, the fourth scenario is not suitable or practicable for information hiding. the experimental results of the three practicable aforementioned scenarios are shown in tables and . table shows the average pesq scores and their reduction percentage according to schema described in sect. . by using combination of algorithms _ and _ . table describes how the reduction percentage of the average pesq scores is affected to hiding capacity and embedding rate increases when algorithms _ and _ are used in combination. in table , the first data row is as the same as the first row of table . the other data rows of table indicate the average pesq scores and their percentages compared to the original pesq scores under different embedding rates of sensitive data by using combination of algorithms _ and _ . the data rows in table , respectively, show the average pesq values of the carrier voice and their reduction percentage compared to the original pesq values under different embedding rates and data transmission rates. in tables and , it is shown that a combination of algorithms _ and _ greatly increases the hiding capacity while the pesq reduction percentages are acceptable. the pesq scores of the speech carrier decreased with increasing hiding capacity and embedding rate. nonetheless, when the hiding capacity reaches bits/s and embedding rate goes up to . %, the reduction percentage of the pesq score was still \ . %, which shows that the proposed scheme is rational, sound, and effective. algorithm for scenario of g. a codec (algorithm _ ) the experiment schema of this scenario is the same as sect. . . at first, the fourth pulse was used as the basis of information hiding, and the amount of hidden information was gradually increased on other pulses in succession. the average pesq scores and its' reduction percentages before and after steganography is performed by using the presented algorithm are shown in table . the first row of table is similar to the first row of table , which shows, under the g. a codec being conducted, the original average pesq scores of the edited clips of each kind of voice in which no sensitive data have been embedded in. the other data rows in table , respectively, indicate the average pesq value of the carrier voice and its' percentages (or reduction percentages) compared to the original average pesq values under different embedding rates and data transmission rates. as is shown in table , taking the fourth pulse position as the basis of hiding information, when each subframe only hides bit of information, the hidden speech quality is about . % of the speech without hidden information, showing that the hiding effect is very good. when each fourth pulse of every subframe embeds bits of sensitive information, the percentage of pesq scores is still [ %, showing the speech quality is also very sound. while more than one pulse of each subframe or all the four pulses are used for embedding information (as is shown in the last row of the table), the quality of pesq scores of the carrier speech decreases sharply, showing that these occasions are unsuitable for information hiding. to examine the steganographic efficacy of our algorithms in further depth, we choose our algorithm _ and _ to [ , ] and algorithm _ with algorithm in ref. [ ] , which are more similar with our methods. the experiments are conducted using the scheme described in sect. . . the results of the comparative experiments in terms of pesq scores and their reduction percentages when steganography was performed using algorithm _ and the algorithms described in refs. [ , ] are shown in table . and the comparative experiment results of our algorithm _ and the algorithms described in ref. [ ] are shown in fig. . in table , the first column of data field shows the original average pesq scores of the edited clips of each kind of voice in which no sensitive data have been embedded in (note: the g. . codec being conducted). the other columns in the data field of table , respectively, indicate the average pesq values and their decreased percentages are compared to the original pesq values under corresponding embedding algorithms of our proposed algorithm _ and similar algorithm of refs. [ , ] being conducted. as is shown in table , as compared with the algorithms described in refs. [ , ] , the pesq scores and their reduction percentages are lower, when our steganographic algorithm is used. it is thus shown that the proposed algorithm has a greater degree of imperceptibility or in other words higher levels of steganographic efficacy and secrecy and robustness, comparing with other two contrast algorithms. in fig. , the horizontal axis represents the four kinds of edited clips of speech voices, while vertical axis represents the reduction percentage of the pesq scores when different steganography algorithms are used. from fig. , we can see that the pesq scores' reduction percentage of algorithm _ is lower than that of the similar algorithm described in paper [ ] , which shows that our algorithm has a greater degree of secrecy, imperceptibility, and robustness. with the widespread popularity of information and communication applications, the secure and confidential distribution and transmission of sensitive information have a wide range of practical value. obviously, covert communication based on information hiding can be widely used for the secret transmission of confidential information of institutions, organizations, enterprises, and individuals. especially in the recent application scenario of the key data sharing of the novel coronavirus (i.e. covid- ) outbreak information, covert communication based on information hiding can not only realize the safe sharing of related information, but also effectively protect the privacy of related personnel and even personal safety. next, we discuss the design of a stealthy communication experiment system for the safe and secret sharing of key sensitive data. the structure diagram of the hidden communication experimental application system based on information hiding is shown in fig. . it can be seen from fig. that the system is composed of a sending terminal, a sending agent, a virtual covert channel, a receiving agent, and a receiving terminal (similar to the receiving terminal). the sending terminal and the receiving terminal are served by various types of devices participating in the sharing of confidential information, such as personal/portable computers, mobile phones, and handheld digital devices. these devices need to have the conditions or capabilities to deploy sending/receiving agents. the sending and receiving agents implement the processing procedures of the information hiding algorithm proposed in this paper. they are responsible for embedding the confidential information that needs to be sent or shared into the voice carrier, or extracting the confidential information embedded in the voice carrier. the virtual hidden channel is realized by transmitting the encrypted voice data on the public shared channel. the process of sharing or transmitting secret sensitive information is as follows: first, the sender terminal calls the sending agent to embed the confidential and sensitive information in ordinary speech coded data (such as g . or a); then, the sending agent hands over the encrypted speech coded data the public channel is then sent to the receiver; finally, the receiver terminal calls the receiving agent to present the confidential information hidden in the voice-encoded data, thereby completing the secure sharing of confidential and sensitive information. in this paper, we have developed a covert communication channel by steganography on common compressed speech codec such as g. . and g. a. we present four steganography algorithms based on different strategies. it is experimentally demonstrated that the proposed steganographic method can obtain high levels of imperceptibility, security, and robustness with sound hiding capacities. as for g. . speech codec, we can achieve a data hiding capacity of . %, . %, or . % (secret information transmission rate of bits/s, bits/s or bits/s) results in . %, . %, or . % reductions in pesq scores, respectively, while for g. a, we can also obtain a data hiding capacity of . %, . %, or % (secret information transmission rate is bits/s, bits/s or bits/s) results in \ . %, \ . %, or \ . % reductions in pesq scores. the above experimental results show our algorithms can obtain high degree of imperceptibility, security, and robustness. the comparative experiment also demonstrated that our algorithm has a higher level of overall efficacy than similar algorithms in the related literature. the information hiding algorithms studied in this paper can be widely used in various application scenarios for the confidential transmission of confidential information, such as protecting the important sensitive information of various institutions or the privacy of personal financial and health information. although the algorithms proposed in this paper have good comprehensive performance and efficiency, the capacity of information hiding still does not meet the needs of high-speed covert communication, and the contradiction of increasing hidden capacity and increasing privacy has not yet been fully resolved. in the future, we will focus on applications of the methods developed in this work. in addition, we will further investigate the possibility of performing steganography based on other speech codec parameters so as to discover even more effective steganography approach in the area. an information hiding algorithm for speech perceptual hashing authentication system in g. bitstream voip steganography and its detection-a survey an overview of digital audio steganography steganography in vector quantization process of linear predictive coding for low-bit-rate speech codec a novel steganography approach for voice over ip steganography in inactive frames of voip streams encoded by source codec detecting steganography in inactive voice-over-ip frames based on statistic characteristics of fundamental frequency a large-capability self-adaptive steganography algorithm based on g. . research on information security in text emotional steganography based on machine learning a synchronization scheme for hiding information in encoded bitstream of inactive speech signal clustering optimization strategy for g. . speech coder and its application a steganographic method based on high bit rates speech codec of g. . an information hiding method based on g. a speech study on convert communication system based on g. compressed voice stream highly secured image hiding technique in stereo audio signal based on complete complementary codes research on effective and intelligent resource management in internet computing steganography integration into a low-bit rate speech codec an immune-inspired semi-supervised algorithm for breast cancer diagnosis implementation of itu-t g. speech codec in ip telephony gateway research of error concealment and control based on g. . and amr_wb a steganalysis method for g. a compressed speech stream based on codeword distribution characteristics key: cord- -qhlo l authors: axell-house, dierdre b.; lavingia, richa; rafferty, megan; clark, eva; amirian, e. susan; chiao, elizabeth y. title: the estimation of diagnostic accuracy of tests for covid- : a scoping review date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: qhlo l objectives: to assess the methodologies used in the estimation of diagnostic accuracy of sars-cov- real-time reverse transcription polymerase chain reaction (rrt-pcr) and other nucleic acid amplification tests (naats) and to evaluate the quality and reliability of the studies employing those methods. methods: we conducted a systematic search of english-language articles published december , -june , . studies of any design that performed tests on ≥ patients and reported or inferred correlative statistics were included. studies were evaluated using elements of the quality assessment of diagnostic accuracy studies (quadas- ) guidelines. results: we conducted a narrative and tabular synthesis of studies organized by their reference standard strategy or comparative agreement method, resulting in six categorizations. critical study details were frequently unreported, including the mechanism for patient/sample selection and researcher blinding to results, which lead to concern for bias. conclusions: current studies estimating test performance characteristics have imperfect study design and statistical methods for the estimation of test performance characteristics of sars-cov- tests. the included studies employ heterogeneous methods and overall have an increased risk of bias. employing standardized guidelines for study designs and statistical methods will improve the process for developing and validating rrt-pcr and naat for the diagnosis of covid- . after its emergence in december , the virus now known as sars-cov- was identified and sequenced in early january , allowing for the rapid development of diagnostic testing based on the detection of viral nucleic acid (i.e., real-time reverse transcription polymerase chain reaction [rrt-pcr]). because infected patients can present with non-specific symptoms or be asymptomatic, the development of accurate diagnostic tests for both clinical and epidemiological purposes was a crucial step in the response to the covid- pandemic. in the united states, the spread of sars-cov- rapidly outpaced the capacity to test for it, resulting in the food and drug administration (fda) relaxing regulatory requirements to increase testing availability. the fda granted the first emergency use authorization (eua) for a sars-cov- rrt-pcr diagnostic test on february , . consequently, hundreds of tests for sars-cov- , among them rrt-pcrs, other types of nucleic acid amplification tests (naats), and automated and/or multiplex methods based on proprietary platforms, obtained fda emergency use authorization (eua). as of august th , , the fda has granted euas to diagnostic tests, including molecular tests, antibody assays, and antigen tests. although the fda began requiring the submission of validation methods and results as part of eua application for sars-cov- diagnostic tests, these tests were not initially required to undergo the rigorous assessment that would normally be part of the fda approval process. researchers also began developing alternative nucleic-acid based methodologies to detect sars-cov- , including reverse-transcription loop-mediated isothermal amplification (rt-lamp), and others. concurrently with rapid test production, publications emerged reporting clinical diagnostic test performance characteristics, such as "sensitivity" and "specificity", though some lacked the rigorous methodologies usually required to formally estimate diagnostic accuracy. here we present a scoping review of the literature with two main objectives: ) to assess the methodologies used in the estimation of diagnostic accuracy of sars-cov- tests and ) to evaluate the quality and reliability of the studies employing those methods. searches were performed through medline (ovid), embase (elsevier), scopus, web of science, cinahl, and pubmed following the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines between december , and june , . the following search string was used: ( -ncov or sars-cov- or sars-cov or covid- or covid or covid) and ("positive agreement" or "negative agreement" or "overall agreement" or "diagnostic accuracy" or "positive rate" or "positivity rate" or "test performance" or "reference standard" or "gold standard" or sensitivity or specificity or "percent agreement" or "concordance" or "test agreement" or "predictive value" or "false negative" or "false positive") and ("polymerase chain reaction" or pcr or "reverse transcriptase" or "nucleic acid amplification test" or naat or isothermal or "rt-lamp" or "rt-pcr" or "molecular test"). the literature hub litcovid's "diagnosis" section was screened in its entirety once and then daily for relevant titles. we liberally screened articles by title and abstract for further evaluation. articles were included if they met the following criteria on screening: ) peer-reviewed publication, ) study evaluated diagnostic test accuracy of naat, ) diagnostic test performed on ≥ patients, ) diagnostic/clinical sensitivity, specificity, other correlative statistics, or test positive rate were either identified by name or were included in the publication as a numerical value and we could reproduce the calculations. exclusion criteria included: ) pre-print status, ) guidelines, consensus, review, opinion, and other summary articles ) entirely pregnant or pediatric populations, ) overlap of study population with another included publication. four authors independently extracted data and two authors reviewed data for accuracy. for study characteristics, we extracted: first author name, country, study design, patient population, total number of patients or samples included in test performance calculations, and number of cases according to rrt-pcr (tables - ) or total number of cases based on positive result of any platform tested (table ) . for patient characteristics, we extracted age and sex. for index test and reference standard characteristics, we extracted: test type (naat) or definition (clinical diagnosis, composite reference standards), specimen (naat), specimen dry/collection liquid status (for studies evaluating abbott id now), proprietary automated and/or multiplex systems -henceforth called "platforms" (naat), and target genes of primers (naat). for outcomes, we extracted the values of test performance characteristics with their designation according to the original authors, without our interpretation. for this reason, we indicate these outcomes as "reported" (r): reported sensitivity (rsn), specificity (rsp), positive predictive value (rppv), negative predictive value (rnpv), accuracy (racc), positive percent agreement (rppa), negative percent agreement (rnpa), overall agreement (roa), and kappa coefficient. additionally, we extracted "positive rate," a non-standard term used by the included studies to refer to the number of positive naats in a population of patients suspected to have covid- (table ) , or to the number of positive samples in a total population of positive samples after repeat testing (table ) . we constructed x contingency tables and reproduced test performance characteristic calculations to demonstrate the methods of how the original authors obtained the values (supplementary table ) . we report additional pertinent study data in supplementary table : enrollment dates, number of sites of enrollment, symptomatic status, and chest radiology status. no articles were excluded on the basis of quality in order to present the most comprehensive summary of the currently available evidence. we presented the extracted data in tabular form mirrored by a descriptive synthesis in two broad categories: diagnostic accuracy studies for rrt-pcr (tables - ) , and diagnostic accuracy or comparative agreement studies of two naats (tables - ). tables are thematically divided based on the reference standard strategy, or approach to obtaining comparative agreement measures. diagnostic accuracy studies for rrt-pcr were arranged alphabetically in tables by first author last name (tables - ) . diagnostic accuracy and comparative agreement studies for two naats were arranged by decreasing order of studies per methodology, then alphabetically by methodology or platform (tables - ) for easy comparison. due to significant diversity in methods and reporting of results, we reported grouped summary data for study characteristics, patient characteristics, and outcomes. we used the framework of the quality assessment of diagnostic accuracy studies (quadas- ) to evaluate our selected articles (supplementary table ). we collected data, or noted their absence, for a narrative description of risk of bias and concerns of applicability based on the quadas domains. for assessment of bias in patient selection, we evaluated author conflicts of interest, study design type, inclusion/exclusion criteria, method of patient enrollment, and reporting of patient demographics and characteristics (i.e. symptomatic status). for assessment of bias in reference standard and index test, we evaluated the accuracy of the reference standard, the description of duration of symptoms at time of testing, whether the threshold to determine a positive test was prespecified, and researcher blinding to reference standard and index test results. for assessment of bias in flow and timing, we evaluated whether the reference standard was the same for all patients, the sequence and timing of the performance of the reference standard and index test, whether test performance characteristics were calculated based on sample numbers or patient numbers, and whether indeterminate or invalid results were included in test performance calculations. our search yielded articles, with unique articles after deduplication. after screening title and abstract, articles underwent full text evaluation. ultimately, articles were included in our review ( figure ). three studies, with to patients, report a "positive rate" as the number of positive rrt-pcr out of the number of suspected cases of covid- , with a range of . % to % (table ) . [ ] [ ] [ ] the studies do not report these values as "sensitivity" directly, however these concern that their low calculated positive rates ( . % and . %, respectively) were indicative of a failure of rrt-pcr to diagnose covid- . , in terms of quality assessment, the studies lack specific details as to how patients were classified as having suspected covid- infection. the accuracy of clinical diagnosis based on case definitions is unclear but is likely not ideal for diagnosis. additionally, duration of symptoms at the time of clinical diagnosis or rrt-pcr testing was not provided (supplementary table ). eight studies, with a range of to , patients per study, attempted to determine the accuracy of rrt-pcr by comparing the initial rrt-pcr result to the result after multiple repeated samples from the patient submitted for rrt-pcr testing, which was called the reference standard (table ) . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] three studies reported this value as a "positive rate," ranging from . % to %, , , and five reported sensitivity, with a range of . % to . %. [ ] [ ] [ ] , of these studies, only he et al included an rsp of %, calculated from patients who remained negative for sars-cov- after repeated sample testing (supplementary table ). green et al. included patients in their study regardless of whether they were tested once or multiple times, using data from these subsets of patients to make assumptions for estimating clinical test characteristics. in addition, this study also conducted multiple different naats and rrt-pcrs on patients, whereas other studies employing this strategy used only one type of naat. additionally, the authors do not clarify whether patients who had repeat sars-cov- test were consistently tested with the same naat/rrt-pcr test or a different one. they also calculated test performance characteristics differently from other studies: two estimates of sensitivity were calculated, one in which the rate of false negatives for single-tested patients was %, and one in which the "false negative" rate was the same as in repeat-tested patients in their study of approximately . %. however, the details of how they calculated test characteristics were not presented. to clarify the two assumptions made in the calculations, we in terms of quality assessment, most of the studies were performed with non-cohort design, and six consisted of only patients who were determined to have covid- by rrt-pcr, i.e. cases only (table ) . , , [ ] [ ] [ ] five of the studies had inclusion criteria which caused pertinent patients to be excluded by necessitating patients to have had a well-performed ct chest or x-ray (supplementary table ). this excluded several patients who would otherwise have been pertinent to the study of test diagnostic accuracy. , , , , the studies involved repeating rrt-pcr several times for a reference standard, but each patient received a different number of repeat tests over a different time period, resulting in each patient receiving a different reference standard. one study tracked negative-to-positive conversion over to days, and another tracked over to days, leading to concern that potentially a patient could have been infected in the time between the initial test and the final test and confounding results. one study counted invalid results as negative results and indeterminate results as positive results when calculating test performance characteristics, otherwise the rationale and ways invalid and indeterminate results were handled were not reported in these studies. three studies determined the accuracy or agreement of rrt-pcr or automated rrt-pcr platforms/instruments compared to a reference standard based on the results of several tests as a "composite reference standard" (table ) . [ ] [ ] [ ] there were between and patients per study. suo et al considered a positive result of either repeated measurements of rrt-pcr or serology to indicate a positive test according to the reference standard; reported sensitivity of initial rrt-pcr result was %, rsp %, rppv %, and rnpv %. zhen et al compared rrt-pcr performed according to the us cdc protocol to a composite reference standard in which the consensus result of or more out of molecular assays was considered the correct result. the rrt-pcr had an rppa of %, an rnpa of %, and cohen's kappa coefficient of . . cradic et al did not study rrt-pcr but studied three automated molecular assays and used a composite reference standard of the consensus result of two or more of the three assays. while abbott id now had a rppa of %, the roche cobas and diasorin simplexa assays had a rppa of %. these studies either did not report how samples were selected for evaluation (supplementary table ), , or reported that only samples which had sufficient residual volume and had been properly stored were selected. days after the initial test, after the patient had been discharged from the hospital, leading to potential exposure for initial infection or reinfection. the performance other nucleic acid amplification test methods compared to standard rrt-pcr fourteen studies compared other nucleic acid amplification test methods to detect sars-cov- to rrt-pcr (table ) suo et al evaluated digital droplet polymerase chain reaction (ddpcr), with rsn %, rsp %, rppv %, rnpv of %, and racc %. bulterys et al study evaluated an isothermal amplification method with rsn . % and cohen's kappa . . wang, cai, and zhang et al evaluated one-step single-tube nested quantitative polymerase chain reaction (osn-qrt-pcr) with cohen's kappa of . . regarding evaluation of quality (supplementary table ) , the majority of studies did not report how patient samples were selected for evaluation. , , , , [ ] [ ] [ ] [ ] in the study conducted by bulterys et al, sample selection was a convenience selection of samples with residual volume that had been stored correctly. most studies did not report symptomatic status of the patient - , - or patient demographics. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] problematically, many of the studies did not report when the reference standard was conducted on the patient samples compared to the index test, or whether actions that could potentially alter test results (such as freeze/thaw cycles) occurred between reference standard or index test. [ ] [ ] [ ] [ ] , , four studies calculated test performance characteristics based on number of samples rather than number of patients. , , , the management of indeterminate and invalid test results went largely unreported. [ ] [ ] [ ] [ ] [ ] , the performance of naat platforms compared to rrt-pcr as the reference standard fifteen studies compared automated naat platforms to various rrt-pcr assays to determine test performance characteristics ( other studies evaluated ausdiagnostics (rsn %, rsp . %), hologic panther fusion (rppa . %, rnpa . %), luminex nxtag (rsn . %, rsp %), mesa biotech accula (rppa . %, rnpa %), or qiastat-dx (rsn %, rsp %) compared to rrt-pcr. with regards to quality evaluation (supplementary table ), most studies did not report method of sample collection/patient recruitment, , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] and four studies conducted a convenience selection of samples, including enrichment for positive samples. [ ] [ ] [ ] eight studies conducted test performance calculations on sample numbers instead of patient numbers. , , , , , [ ] [ ] [ ] four studies conducted calculation of test performance characteristics with indeterminate or inconclusive results as "positive," , , , and the management of indeterminate/inconclusive as well as invalid results went unreported in an additional three studies. , , no study reported the blinding of researchers to the reference standard or index test results. ten studies, containing between and patients per study, evaluated the agreement between two different types of naat platforms ( in the studies, some platforms were identified as the "comparator" or "reference" platforms, including cepheid xpert xpress, abbott realtime, , hologic panther fusion, , and roche cobas , , and these were listed as "platform # " in table . three studies did not identify any studied platform as the "comparator" or "reference standard," and instead only reported general, non-directional measures of agreement such as overall agreement, cohen's kappa, or alternatively, the calculations of ppa and npa were identical no matter their method of calculation (supplementary table ) . , , regarding quality evaluation (supplementary table ), the samples used for calculating test performance characteristics were reported to be selected for enrichment of positive samples, , for diversity of viral load, , otherwise curated, or the method of selecting samples was unreported. , , , symptomatic status of the patients was largely unreported. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] five studies included samples where one test was conducted, then interim freezing, cooling, or other storage, before performance of the second test. , [ ] [ ] [ ] two studies did not report the sequence of testing of the two platforms or interim handling or storage of the samples. , the status of researcher blinding to either platform result was not reported in any study. in our scoping review of articles concerning test performance characteristics of rrt-pcr and other naat used for the diagnosis of covid- , we were able to observe several overarching themes. clinical diagnosis by the case definition for covid- used in the early period of the pandemic does not correlate well with positive rates of covid- rrt-pcr ( table ). the result of the initial rrt-pcr performed on a patient, if negative, may not be reflective of the result after multiple repeated rrt-pcrs for that patient ( table ) . several alternative naat methods, many of which are easier or faster to perform, may be comparable to standard rrt-pcr (table ). proprietary multiplex, automated, and/or point-of-care methods are comparable to in accuracy to rrt-pcr (table ) and to each other (table ), although the abbott id now sars-cov- test appears to have lower comparative agreement to other platforms. , [ ] [ ] [ ] [ ] [ ] these findings should be viewed cautiously as the sars-cov- tests in these studies have not undergone rigorous evaluation necessary for fda approval due to the emergency state generated by the covid- pandemic. in addition, during our scoping review, we found substantial heterogeneity among available studies in terms of test types, reference standards, metrics, and details of study design and methodology. we categorized the included studies by four different reference standard strategies: clinical diagnosis/case definitions (table ) , repeated index testing (table ) , composite reference standard (table ) , and rrt-pcr (table and ) . additionally, we identified a fifth category, where instead of using a reference standard, comparative agreement between two naat platforms was calculated (table and ). the main limitation of the first group of studies (table ) was the use of a "case definition" as the reference standard to report a "positive rate" of rrt-pcr. during novel disease outbreaks, standard case definitions are often developed to assist clinicians in case identification before a diagnostic test is available. unfortunately, the studies included in this group were unable to use a clear case definition; instead they refer to a population of "suspected cases," for which the definition is not reported. [ ] [ ] [ ] because this group enrolled patients prior to february , in china, during the time in which the chinese national guideline for diagnosis and treatment of covid- (ngdtc) published five different versions of the covid- case definition, the case definitions in use at the time of these studies varied. a recent study estimated that if a single guideline (specifically, version of the ngdtc) had been used to identify cases from the beginning of the outbreak to february , , there would have been more than three times as many identified cases in hubei province. this is relevant to our review because the two largest studies that evaluated the rrt-pcr positive rate of patients with a clinical diagnosis of covid- took place in wuhan, hubei province, and included patients evaluated before february , , (supplementary table ). this increased case estimate due to diagnosis of covid- based on case definition complicates the legitimacy and reported accuracy of the "positive rate" of rrt-pcr referred to in these studies. the second group assessed rrt-pcr test performance characteristics via repeated index rrt-pcr testing ( table ). most studies in this group reported "sensitivity" by dividing the number of participants with positive baseline rrt-pcrs by the total number of participants who eventually had a positive rrt-pcr after repeated measurements. while such an approach may have some advantages over the use of a case definition alone as a reference standard, this strategy is, nonetheless, an imperfect solution with its own set of inherent limitations. sars-cov- infection is transient and the associated viral loads are time-varying because of the natural pathophysiology of the infection. therefore, the time interval between each repeated test becomes crucially important, and even relatively small time differences (and/or lack of uniformly used intervals) could complicate the interpretation of re-test results and their quality as reference standards. furthermore, repeated use of the same test as a reference standard for itself does not eliminate the inaccuracies or limitations of the test. such comparisons ultimately reflect the reliability of the test (assuming a short, uniform time interval between tests), rather than providing a true view of test accuracy. the third group of three studies calculated test performance characteristics of rrt-pcr according to a composite reference standard (table ). using arbitrary rules to combine multiple different and imperfect tests inevitably creates a reference standard with some degree of bias. furthermore, all three studies in this group included the test under evaluation as part of the composite reference standard, which leads to additional bias, described below. use of a biased composite standard is likely to lead to reduced sensitivity, among other errors affecting true test performance characteristics. the fourth group of studies evaluated sars-cov- diagnostic tests that are under development as well as proprietary testing platforms (most of which are based on standard rrt-pcr methods). these studies used traditional rrt-pcr as a reference standard; results are summarized in tables and , respectively. importantly, while these studies were not designed to estimate the accuracy of rrt-pcr, their results indicate that the index tests did not identify significantly more positive samples than rrt-pcr. finally, the last group of studies compared sars-cov- naat platforms (table ) . these comparative accuracy studies examined the agreement between two non-reference standard tests. although most of the testing platforms evaluated in these studies were based on standard rrt-pcr, the agreement between two non-reference standard tests is not equivalent to test accuracy, as mentioned previously. this scoping review is limited by the lack of reporting of several key study features in the majority of the articles evaluated, which is an important indicator of quality and potential bias. based on the quadas- criteria, most of the included studies had concern for bias (supplementary table ). the most prominent concerns were unclear inclusion/exclusion criteria, unclear method of enrollment/selection of patients and samples, and unclear handling of indeterminate/inconclusive and invalid results. additionally, many of the studies were conducted in a so-called "two gate" (case-control) design, in which cases and controls were known and selected ahead of time, rather than performing the test on a group of patients or samples with suspected covid- . these factors likely incorporate bias that significantly confounds the results of the studies, thus, the accuracy of the tests in other settings with different prevalences (such as asymptomatic screening, other age groups) may not be truly generalizable. furthermore, few studies were able to evaluate both the index and reference tests simultaneously or within a short period of time, which is key to avoiding biases caused by changes in the patient's true disease status; this bias can also affect the diagnostic accuracy of the index test. the best approach to determining diagnostic test performance characteristics in the absence of a "gold" standard is an open question in diagnostic accuracy methodology. while many methods have been described, there are only a few well-defined statistical approaches that use a reference standard in lieu of a gold standard, reviewed elsewhere. latent class analysis is one commonly used approach in situations in which neither the true error rates of the reference standard nor the true prevalence of the disease are known. this approach uses the results of a set of imperfect tests to estimate parameters related to sensitivity, specificity, and prevalence often using maximum likelihood methods. however, this is not the only method available and every method has its own strengths and limitations. therefore, careful interpretation by studies that attempt to estimate test characteristics is warranted to account for and clarify the inherent limitations of assessing accuracy-related metrics when a gold standard is unavailable. evaluation of the performance characteristics of sars-cov- diagnostic tests is vital to control of the ongoing covid- pandemic. while more than sars-cov- molecular diagnostic tests have received fda euas, we have described in this scoping review that the performance of few of these tests has been assessed appropriately. the lack of robust test performance that we noted in many studies published to date is undoubtably due in part to the critical need for tests, which resulted in accelerated test development. however, our scoping review also uncovered imperfect methods for estimating diagnostic test performance in the absence of a gold standard and demonstrate that the accuracy of these tests should be interpreted with caution. future studies would benefit from employing statistical methods such as latent class analysis and other methods referenced above to accurately analyze their data. indeed, instituting national requirements for test performance analysis and reporting, perhaps based on the existing fda guidelines on diagnostic tests, would advance the goal of standardizing the evaluation sars-cov- diagnostic test performance. such an initiative would lead to statistically robust conclusions regarding the accuracy of the index test, which will in turn support hospitals and clinicians as they determine the optimal test to use for covid- diagnosis. table . abbreviations-aigs: automatic integrated gene detection system, bal: bronchoalveolar lavage, ci: confidence interval, ddpcr: digital droplet polymerase chain reaction, e: envelope, iamp: isothermal amplification, iqr: interquartile range, κ: kappa statistic, n/a: not applicable, n: nucleocapsid, no.: number, nps: nasopharyngeal swab, nr: not reported, ops: oropharyngeal swab, orf ab: open reading frame ab, osn-qrt-pcr: one-step single-tube nested quantitative real-time polymerase chain reaction, racc: reported accuracy, rdrp: rna-dependent rna polymerase, ref stnd: reference standard, rnpa: reported negative percent agreement, rnpv: reported negative predictive value, roa: reported overall agreement, rppa: reported positive percent agreement, rppv: reported positive predictive value, rrt-pcr: real-time reverse transcription polymerase chain reaction, rsn: reported sensitivity, rsp: reported specificity, rt-lamp: reverse transcription loop-mediated isothermal amplification, rt-raa: reverse-transcription recombinase-aided amplification, s: spike, y: years. a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding presymptomatic transmission of sars-cov- -singapore preferred reporting items for systematic reviews and meta-analyses: the prisma statement synthesis without meta-analysis (swim) in systematic reviews: reporting guideline 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fusion and a laboratory-developed test targeting the envelope gene for detection of sars-cov- clinical performance of the luminex nxtag cov extended panel for sars-cov- detection in nasopharyngeal specimens of covid- patients in hong kong comparison of the accula sars-cov- test with a laboratory-developed assay for detection of sars-cov- rna in clinical nasopharyngeal specimens evaluation of the qiastat-dx respiratory sars-cov- panel, the first rapid multiplex pcr commercial assay for sars-cov- detection comparison of abbott id now and abbott m methods for the detection of sars-cov- from nasopharyngeal and nasal swabs from symptomatic patients performance of abbott id now covid- rapid nucleic acid amplification test in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a new york city academic institution five-minute point-of-care testing for sars-cov- : not there yet clinical evaluation of three sample-to-answer platforms for the detection of sars-cov- comparison of cepheid xpert xpress and abbott id now to roche cobas for the rapid detection of sars-cov- the detection of sars-cov- using the cepheid xpert xpress sars-cov- and roche cobas sars-cov- assays comparison of two high-throughput reverse transcription-polymerase chain reaction systems for the detection of severe acute respiratory syndrome coronavirus clinical evaluation of a sars-cov- rt-pcr assay on a fully automated system for rapid ondemand testing in the hospital setting effect of changing case definitions for covid- on the epidemic curve and transmission parameters in mainland china: a modelling study evaluation of diagnostic tests when there is no gold standard. a review of methods using a combination of reference tests to assess the accuracy of a new diagnostic test value of composite reference standards in diagnostic research diagnostic test evaluation methodology: a systematic review of methods employed to evaluate diagnostic tests in the absence of gold standard -an update food and drug administration cfdarh. statistical guidance on reporting results from studies evaluating diagnostic tests key: cord- -rdhuc n authors: anderson, nancy l. title: pet rodents date: - - journal: saunders manual of small animal practice doi: . /b - - - / - sha: doc_id: cord_uid: rdhuc n nan many small rodents are commonly kept for companionship and enjoyment. this chapter provides information needed to diagnose and treat the most frequently encountered problems of mice, rats, gerbils, hamsters, guinea pigs, and chinchillas. • cages should be made of stainless steel, hard plastic, or glass. these materials are cleaned and sanitized easily and are resistant to gnawing or corrosion from urine and fecal matter. minimum floor space and height requirements are listed for each species in • guinea pigs can be housed in open-topped enclosures with walls higher than inches. ensure that dogs, cats, wild animals, and small children do not have unsupervised access to these cages. • clean cages as needed, usually to times per week for most rodents. a scrub brush, dish soap, and water work well. if cages are not kept clean, ammonia, other irritants, moisture, and bacteria concentrations rise to harmful levels, predisposing animals to disease. • disinfect the cage twice a month with part sodium hypochlorite (household bleach) mixed in parts water. let the bleach solution stand for at least minutes. rinse the cage well afterward. • all solid-floored cages need to be covered in bedding. shredded paper, non-resinous wood shavings, wood wool, and corn cobs are all acceptable. provide at least inches of bedding. most rodents enjoy burrowing in deeper bedding when it is provided in one corner of a cage. do not, however, fill the entire cage with deeper bedding. this usually leads to poor sanitation as a result of owners' failure to recognize buildup of hidden wastes such as moisture from leaking water bottles, cached foods, urine, and feces. • wire mesh floors can be used successfully only if the dimension of the mesh is correct. size the openings to be just large enough for an adult to retract a tarsal joint back through the mesh. larger holes make it difficult for the animals to walk and cause pressure sores. smaller openings may cause injuries such as tibial fractures and self-mutilation while struggling to free trapped appendages. bedding above the wire keeps waste from dropping through the wire and therefore is not recommended. wire bottom cages do not work well for breeding animals because neonatal rodents must be surrounded by nesting material to maintain moisture in the nest and prevent dehydration. young rodents often cannot walk correctly on mesh sized for adult feet. • all pet rodents require visual security. tubes, jars, or cans made of nontoxic, nonabrasive substances work well for this purpose. also provide objects for gnawing. rodents possess open-rooted teeth, and constant wear is necessary to maintain normal dentition. mice, rats, gerbils, and hamsters enjoy and benefit from exercise wheels. • a good room temperature range for most pocket pets is °f to °f. keep rodents with disease at °f to °f unless hyperthermia is of concern (some chinchillas). • provide to hours of darkness to to hours of light. this light cycle is essential if breeding is desired. • hamsters, guinea pigs, and chinchillas that are exposed to temperatures below °f may hibernate for a few days or until the ambient temperature rises. heart rates may be less than bpm during hibernation. m key point feed pet rodents laboratory animal chow appropriate for their species (table - ). seed diets are deficient in protein and contain excessive fat. • seeds, as well as vegetables and other foods, may be fed as treats but not to provide more than % of calories. intermittent exposure to vegetables and seeds causes mild, transient diarrhea. • supplementation of vitamin c is recommended for all guinea pigs. • adult chinchillas that are not obese should be fed high quality, fresh grass hay ad libitum. obese animals may need to have the hay rationed. chinchillas require / to / -cup of fresh pellets per animal each day. feeding pellets free choice leads to obesity, and the high protein and calcium levels in these diets may predispose animals to urinary tract disease. most pellets also do not provide sufficient fiber to maintain normal gastrointestinal (gi) motility. • store food in tightly sealed containers at less than °f; keep food refrigerated if possible. • feed all diets within days of milling to ensure the highest nutritional value. encourage owners to check dates on packages and ask pet store managers about providing dates on bulk items. • if possible, feed pet rodents except guinea pigs from overhead racks. these devices reduce wastage and eliminate fecal contamination of food. covered hoppers, heavy crocks, or stainless steel bowls that are attached to the side of the cage to eliminate spillage are acceptable and recommended for guinea pigs. • feed breeding females and their litters from the floor of their cages until the young are large enough to reach overhead feeders or crawl in and out of crocks. • cannibalism of neonates commonly occurs as the result of stress associated with cage cleaning. to minimize cannibalism, clean the cage and provide a to -day food supply to days before parturition. m key point provide fresh water in clean containers daily. • do not provide water in open crocks. these are contaminated or spilled easily and are a common cause of dehydration and poor sanitation. • sipper tubes and water bottles work well. clean with dish soap and water daily and disinfect them weekly. guinea pigs expel food from their mouths into their sipper tubes, so more frequent cleaning is needed. • some water bottles have special valves to minimize backflow. supplement guinea pigs' water daily with mg vitamin c/l. if the water is not dechlorinated, it will inactivate the vitamin c. quarantine all newly acquired animals in a different room from current pets for a minimum of days. feed and handle quarantined animals last. recommend that caretakers wash their hands and change clothes before handling current pets. avoid the introduction of adult animals because this frequently results in fighting. instead, place animals together while young and allow them to mature together. avoid keeping more than one male per cage because this also usually leads to fighting. a systematic history and physical examination are mandatory. many disease syndromes are caused by poor husbandry. pets that have been kept isolated from other rodents or acquired from a private breeder are less likely to harbor infectious disease than animals obtained from a pet store, laboratory, or wholesaler. see table - for normal physiologic data. obtain the following information: • observe the pet in its cage for mentation, activity, locomotion, dyspnea, head posture, haircoat, and any grossly observable abnormalities. • note respiratory and heart rates before restraint when possible. observe the condition of cagemates. m key point if dyspnea or severe depression is detected, warn the owner that the animal is critically ill and could die of stress brought on by an examination. • handle such animals as little as possible. initially, treat severely ill animals symptomatically, then • note the type of diet and bedding as well as the level of sanitation and compare these with what was described in the history. • observe quantity and quality of feces and urine. diarrhea, soft stools, absence of stools, copious urine, and discolored urine all can be signs of illness. • coprophagy is a normal behavior in rodents. • check the diet and water supply for freshness, quantity, source, and accessibility. • evaluate the presence and suitability of cage furniture. adequate visual security and the ability to exercise and gnaw are extremely important. • an accurate weight in grams is extremely important for evaluating an animal's body condition, calculating drug dosages, and monitoring treatment. the easiest method of weighing a pet rodent is to place it in a box and then subtract the weight of the container. • restraint of pet rodents is easy with experience. pets that have been handled frequently and gently by the owners require only minimal restraint. gentle pressure directs the animal as needed. grasp less cooperative patients (except chinchillas and guinea pigs) by the scruff over the back of the neck with thumb and forefinger ( fig. - ) . take care to pinch enough skin to prevent the animal from turning around, yet leave enough slack for respiration. on smaller specimens, hold the base of the tail, if present, between the fourth and fifth fingers to provide additional restraint. • hold docile guinea pigs with the palm of one hand supporting the chest while the other hand supports the hind quarters ( fig. - ). place the thumb and forefinger of the first hand in the axillas for additional control. • take care to minimize damage to the fur when handling chinchillas because they lose hair easily. grasp the animal by the tail and scoop it up into the palm of the same hand ( fig. - ). if necessary, grasp the thorax just behind the axillas. • calm uncooperative rodents by placing an appropriately sized towel over the head. complete the physical examination by wrapping the patient in a towel and exposing only needed areas. even oral, ophthalmic, and aural examinations can be performed with minimal effort if the animal is given the chance to relax in the towel "burrow." • remove particularly aggressive patients from their cages by scooping them up in a can or bucket; then slide them out onto a slick surface and pick them up or transfer them to a holding area or scale. m key point lift the hind quarters of mice and rats by the base of their tails to facilitate scruffing. never use the tip of the tail for restraint, or the skin of the tail may slough. once the animal is restrained properly, examine the head. assess the cranial nerves. check the nose for presence and character of discharge. examine the mouth for ptyalism, swelling, overgrown incisors, or discharges. to inspect the oral cavity, place an avian speculum across the mouth just caudal to the incisors. use a light source and a pair of hemostats as retractors to improve access. alternatively, use an otoscope with a pediatric head to examine the premolars and molars of guinea pigs and chinchillas for overgrowth. examine the cheek pouches of hamsters for swelling, impaction, or discharge. an ophthalmic examination, including a fundic examination, is important. • use a slit lamp to identify superficial pathology, especially corneal ulcers or foreign bodies. • if indicated, perform fluorescein stain and conjunctival scrapings or cultures. • note the presence of conditions such as discharge, asymmetry, and exophthalmos. m key point gerbils, rats, and mice produce red tears (chromodacryorrhea) with stress or disease. do not confuse them with hemorrhage. • guinea pigs suffering from hypovitaminosis c often produce dry, white tears. • check ears for discharge, foreign bodies, and mites. bluish discoloration of the ears is a sign of cyanosis. bright red injected coloration is associated with septicemia. sores behind the ears and on the neck are often a sequela of aural disease. • evaluate submandibular, axillary, inguinal, and popliteal lymph nodes for size and consistency. enlargements usually indicate infectious or neoplastic disease. • reevaluate respirations and heart rate after the stress of handling and compare them with the resting rate noted when the animal was in the cage. note dyspnea or respiratory sounds. auscultate animals weighing more than g. counting every third or fourth beat and multiplying by the appropriate factor allows recording of heart rates up to bpm. • palpate the abdomen. pay special attention to differentiating pregnancy from the bladder, kidneys, abdominal masses, enlarged cecum, and fecal balls in the colon. while palpating the abdomen, examine the mammary chain of all female rodents for signs of mastitis, lactation, or neoplasia. also check male mice and rats for mammary neoplasia. mammary tissue extends from the base of the neck to the base of the tail. gerbils typically have an elliptical sebaceous gland on their ventral midline. do not confuse this with neoplasia or infection. check the rectum and perineal area for signs of diarrhea, prolapse, irritation, parasites, and bite wounds. note that coprophagy is normal in rodents. • evaluate the urogenital tract for signs of inflammation, foreign bodies, urine scalding, and vaginal discharge. locate and palpate the testicles in males. the easiest method of determining sex in pet rodents is to compare the anogenital distance, which is twice as long in males as in females. other characteristics that allow the determination of sex are as follows: • visualization or palpation of testicles or extrusion of the penis from the prepuce indicates a male. • the presence of two external openings (i.e., anus and urethra) indicates a male. • the presence of three openings (i.e., anus, vagina, and urethra) indicates a female. • examine the skin and fur for conditions such as crusts, alopecia, masses, herniations, and wounds. • check tail and feet for swellings, coloration, sores, length of toenails, and condition of footpads. • evaluate the extremities for trauma or other abnormalities. apply cellophane tape to crusted areas of the skin and view under a microscope as an aid in diagnosing ectoparasites such as lice, mites, and fleas. skin scrapings are beneficial in detecting mites and dermatophytes. dermatophytes are diagnosed best through culture of broken hairs or crust on dermatophyte test medium. use small, cotton-tipped swabs to obtain ear swabs from animals weighing more than g. mix debris with mineral oil and view under low magnification to test for ear mites, or roll onto a glass slide and gram stain to look for bacterial or yeast infections. collect urine by placing the rodent in a clean meshbottomed cage with a plastic liner. after enough urine has been produced, collect it off the bottom of the cage with hematocrit tubes or a syringe and a -gauge needle. perform cystocentesis on non-pregnant animals weighing more than g with a -to -gauge needle. collect feces over several hours to provide a volume sufficient for fecal flotation. flotation allows the detection of nematodes and some trematodes and cestodes. cellophane tape applied to the perineal area and then viewed under a microscope often reveals oxyurid eggs. use a fresh saline smear or fecal sedimentation to diagnose protozoal parasites. fecal cultures are useful in diagnosing bacterial diarrhea. radiology is an extremely useful tool. machines capable of exposures as low as kvp and to mas effectively image mice. most radiograph machines are capable of generating diagnostic radiographs of guinea pigs, chinchillas, and mature rats at settings used for kittens. positioning is accomplished with masking tape or velcro straps. sedate unruly animals. techniques used in cats for contrast studies of both urinary and gi systems are modified easily for use in pocket pets. • use lateral or medial saphenous veins to obtain samples in animals heavier than g. liberally clip the area to allow exposure of the vessel before attempting venipuncture. place a -to -gauge needle in the vein and collect blood into microtainers or hematocrit tubes as it drips from the hub of the needle (fig. - ) . take extreme care not to col-lapse and lacerate the vein with overzealous aspiration if a syringe is attached to the needle. • it is also possible to use the cephalic vein in guinea pigs. • jugular veins are good alternatives in thin individuals under sedation. • an alternative technique that is useful in smaller animals is to coat the skin over the vein with a thin layer of petroleum jelly and then to puncture the vessel. blood is collected with a hematocrit tube as it exits the wound. samples up to % of the animal's weight are considered safe, even in stressed animals. m key point attempt tail bleeding only as a last resort in mice, rats, gerbils, and hamsters. these techniques often are not acceptable to owners. to bleed the tail, warm the tail with water or compresses to dilate the tail vessels. in large rats, perform venipuncture with a needle and obtain blood in the usual fashion. in smaller animals, lacerate the tip of the tail. blood from the wound is collected as described previously. see tables - and - for hematology and chemistry values. incorporate oral medications into a treat, or administer them in liquid form. if the medication is palatable, administer it by placing the tip of a dosing syringe into the diastema. m key point take care not to place the tip into the contralateral cheek pouch, or the patient may store the medication and expel it later. administer medication in small amounts. ensure that the animal swallows the medication in its mouth before more is administered. this technique is useful for force-feeding pellet gruels to anorexic pets if the caregiver is patient. medication or food that is administered too quickly will be spit out or aspirated. for rodents that are intractable or for administration of unpalatable substances, pass a stomach tube per os. • metal feeding needles, red rubber urinary catheters, or infant feeding tubes work well. selection is based on the size of the animal and individual preference. metal feeding needles with ball tips frequently are used in patients weighing less than g . the metal provides the necessary stiffness to pass a tube of small diameter. the ball at the end of the needle makes it difficult (but not impossible) to pass the tube into the trachea. these tubes have the potential to create esophageal tears with improper restraint or when excessive force is applied. • measure the length from the tip of the nose to the last rib. ventroflex the head slightly, and place the tip of the tube through the diastema and over the tongue. if the tube does not pass easily down the esophagus to the premeasured distance, check the tube size and/or reposition the tube before attempting further advancement. the needle is easily palpable percutaneously if it is placed correctly. it is usually safe to administer up to ml/ g body weight. • a flexible catheter is ideal for use as a stomach tube in larger rodents ( fig. - ). use a speculum to prevent chewing on the tube. an otoscope head, avian speculum, or piece of wood or plastic with a hole drilled in the center works well. measure and mark the tube for the distance from the tip of the nose to the last rib. place the speculum in the mouth and over the tongue. pass the tube while holding the speculum in place and slightly ventroflexing the head. resistance is encountered if the tube is malpositioned or is an inappropriate size. the tube must pass over the tongue before it can be advanced down the esophagus. this is difficult in some animals. palpate the throat to confirm the presence of the feeding tube in the esophagus. m key point because the placement of a stomach tube is a blind procedure, administer a small volume of sterile saline into the tube before administering the medication to ensure that the tube is not in the trachea. misplaced medications are fatal. • this method is also useful for administration of nutrition to anorexic patients. place a pharyngostomy tube if repeated dosing is necessary, using the technique for cats. flush pharyngostomy tubes with water at least every to hours. nasogastric tubes are not recommended because they are difficult to place and maintain patency because of their small size. securely suture all tubes to the skin. place a tube collar made of radiographic film or use rear leg hobbles to prevent removal of tubes. • nutritional support is critical in rodents because of their high metabolic rate. provide supplements in animals that are anorexic for longer than hours. if the gi tract is capable of digestion, use a slurry of pellets mixed with a high-calorie supplement. if the tube diameter is too small for this mixture, use avian hand-feeding formula or a mixture of vegetable and cereal baby foods in place of the pellets. if the ability of the gi tract to tolerate enteral feeding is questionable, first try isotonic electrolyte or dextrose solutions. parenteral nutrition is used successfully in research animals and may be feasible in select pet cases. administer sc injectable medications or fluids over the shoulder blades or in folds of skin on the flank. • avoid irritating substances in rats and mice because their mammary tissue extends into these areas. the resulting inflammatory response is thought to increase the occurrence of mammary neoplasia. m key point in general, avoid streptomycin and the carrier procaine in all pet rodents because of a high incidence of toxicity and hypersensitivity reaction. give im injections in the semimembranous and semitendinous muscles. inject only small volumes of nonirritating substances, or tissue damage with resulting self-mutilation may occur. use the epaxial or triceps muscles if repeated injections are necessary. m key point use intraperitoneal (ip) injections only as a last resort for large volumes of fluids or for irritating injections that cannot be administered via an iv or io route. express the bladder and aseptically prepare the abdomen. restrain the rodent with its head down to move the abdominal organs cranially. give the injection . to cm lateral to the midline in the caudal abdomen. aspirate before injecting to ensure that the injection is not being given into the bladder or bowel. never use this technique in pregnant animals. give iv injections into any of the veins as previously described. in addition, the penile vein may be used in hamsters and guinea pigs. placement of iv catheters is possible in animals heavier than g. for small rodents, give a bolus of fluids every - hours, followed by a diluted heparin flush. a pediatric iv pump is used for continuous infusion of fluids to mark distance from nose to last rib larger animals. maintenance of catheters in active animals is extremely difficult. for io injections, place a spinal needle into the proximal tibia or femur following the technique used for placing an intramedullary pin. once the needle is seated, remove the stylet. aspirate and check the hub of the needle for bone marrow. the tip of the needle should be in the bone marrow cavity that directly drains into the central venous system in normal bones (i.e., the cortex must be intact). administer drugs, blood, or fluids at a rate similar to that used for iv catheters. • in chinchillas and guinea pigs, withhold food for hours before anesthesia. withhold food from smaller, mature rodents for hours. withhold food from immature animals for up to / hour depending on age and condition. • use heat lamps and heating pads to prevent hypothermia. have a prewarmed incubator available for recovery. preoperative or intra-operative warmed sc or iv fluids are strongly recommended. place iv or io catheters whenever possible. • administer atropine preoperatively to reduce airway secretions. acepromazine, diazepam, or midazolam work well as premedications for other anesthetics. avoid acepromazine in gerbils because it potentiates seizures. see table - for anesthetic drugs and dosages. surgical anesthesia is reached when toe, tail, and ear pinch fail to generate a withdrawal reaction. depth of anesthesia is best monitored by pulse and respiratory rate and character. pulses drop to within normal ranges after induction. further reduction, especially to less than % of the original stabilized value, is an indication to lighten the plane of anesthesia. monitor the electrocardiogram (ecg) of small patients by clamping the alligator clips onto the hubs of all-metal -gauge needles or steel sutures placed through the skin at the usual sites. tape cables to the table to maintain placement. doppler units taped over the chest also provide accurate heart rates. pulse oximeters are easier to use, more sensitive, and more expensive than the instruments mentioned previously. these instruments are easily taped to the patient's ear, foot, or tail and provide heart rates as well as information regarding oxygenation. respirations are often shallow and rapid during induction. they become deep and regular as a surgical plane of anesthesia is reached. the corneal reflex varies markedly between individuals and anesthetic agents. if the animal has a corneal reflex after induction and then loses it, reduce the anesthetic. induce gas anesthesia using small face masks purchased from laboratory supply houses or make them from syringe cases and latex gloves ( fig. - ) . induction in an anesthetic chamber is also possible. all rodents induced and maintained on gas anesthesia require some form of non-rebreathing system. usual induction is achieved between % and % for isoflurane and % and % for halothane. maintenance for isoflurane and halothane varies from . % to %. there is marked individual variation in the amount of anesthetic required for induction and maintenance. use of % nitrous oxide in oxygen reduces anesthetic concentration requirements for other gases. m key point some chinchillas and guinea pigs hold their breath while being induced with gas anesthetics and then take deep rapid breaths. if the concentration of anesthetic gases is high enough, this behavior results in death. the risk of this behavior is reduced by premedication with tranquilizers, initial induction with nitrous oxide with later addition of primary anesthetic gas after relaxation, and low induction settings. changes in respirations, especially erratic or apneustic patterns and decreased respiratory rates, indicate deepening anesthesia. most pet rodents are not intubated for anesthesia because of their small size. when necessary, as in prolonged oral and other procedures, endotracheal intubation is accomplished with the animal in dorsal or ventral recumbency, depending on the clinician's preference. small non-cuffed or cole endotracheal tubes work well. a stylet usually is required to provide enough stiffness for the tube to pass the larynx. extend the animal's head and neck. grasp the tongue with forceps and use gentle traction. the tip of the tube then is advanced above the tongue and just past its base. the hard palate is used to deflect the tip of the tube ventrally into the glottis. this is a blind procedure that is difficult to master. use of a laryngoscope is helpful in larger rodents. another technique is to place an over-the-needle catheter in the trachea and move it up retrograde through the larynx to act as a guide. the catheter is removed after the endotracheal tube is in place. it is extremely important that the tube be checked for patency. rodents produce copious respiratory secretions, which frequently clog endotracheal tubes. the small diameter allows these tubes to collapse or kink, resulting in asphyxiation of the patient. check patency at least every minutes by applying positive pressure ventilation at to cm water and watching for excursion of the chest wall. if extending the head and neck does not result in air flow, suction the tube. if this is either not successful or impossible, remove the tube and continue anesthesia with a mask or reintubate the animal with a new tube. because of the small diameter of the trachea, endotracheal tube-induced tracheitis and subsequent swelling of the trachea may become a life-threatening situation. doses and routes for injectable anesthetics are listed in table - . needed doses for injectable anesthetics are tremendously variable among species and individuals. most injectable anesthetics provide safe sedation for minor procedures, but very few induce a safe surgical plane of anesthesia on a consistent basis. • ketamine in combination with diazepam is easily obtainable, is given intramuscularly, and has a wide margin of safety, but it does not provide good analgesia. • intraperitoneal injections of barbiturates provide surgical anesthesia but have a low margin of safety and a significant mortality rate. barbiturate anesthesia can result in fatal ileus. euthanasia is performed easily by induction of inhalant anesthetic through a mask or chamber followed by an overdose of barbiturates given intraperitoneally, iv, or intracranially. euthanasia by ip injection of barbiturates alone causes pain in some animals. • surgical techniques for pet rodents are similar to those used in cats and birds. • hemostasis is critical because of small blood volumes. • electrosurgery for incisions and cautery is highly recommended. • if necessary, give fresh blood transfusions drawn from a donor of the same species and mixed with sodium heparin ( iu/ml) at a rate of . ml/ ml of blood directly into an iv or io line. • the lack of a filter creates a potential for thrombosis. • transfusion reactions are possible. administer postoperative analgesics to all rodents undergoing surgical or dental procedures. common analgesics include buprenorphine, butorphanol, ketoprofen, carprofen and meloxicam. see table - for dosages. the most common surgeries are laceration repair and removal of dermal or sc masses. • most rodents will not gnaw on skin sutures. • if this occurs, use steel sutures, subcuticular sutures, or tissue glue. • if an animal still chews at its suture line, physical restraint, such as a tube or an elizabethan collar, is required. castration is a common procedure in guinea pigs. this usually is performed when owners want to house more than one male together or do not wish to breed their female any longer. common abdominal surgeries include cystotomy for urolith removal in guinea pigs and rats, and cesarean section (c-section) in guinea pigs and chinchillas because of dystocia. use a technique similar to those described for dogs and cats. preplaced stay sutures are recommended to define incision edges for closure. use - polyglactin or polydioxanone (pds) on a taper needle and suture in a simple continuous pattern to close the body wall. close the skin with a subcuticular suture (absorbable) or interrupted skin monofilament, nonabsorbable suture. fracture fixation is accomplished best with intramedullary pinning or kirschner apparatus. rodents gnaw on bandages until they remove them. if they are unable to remove a splint, self-mutilation often results in self-amputation. if a cast or splint is necessary, physical restraint often is required. healing usually takes to weeks. incisors can be trimmed with nail trimmers, but this technique often fractures the tooth, causing abscesses of the root. instead, use a high-speed dental burr or a flat cutting disk on a dremel hand tool. trim molars with a high-speed drill or pediatric rongeurs. a mouth speculum that deflects the tongue and other soft tissues is essential to prevent lacerations and provide working space ( fig. - ) . intubate the trachea to prevent aspiration pneumonia when working on molars. if a tooth is abscessed, extract both it and the occlusal tooth. • if necessary, approach cheek teeth via an incision through the cheek. • use a fine dental elevator to loosen the teeth. • patience and firm but gentle pressure are needed, or the root or surrounding bone may fracture. • the roots of the maxillary incisors curve dramatically back into the head. take care to follow the curve of the tooth. • packing an infected tooth socket with a calcium hydroxide paste may decrease the occurrence of persistent infection. remove the paste in to days. • administer meloxicam, carprofen or ketoprofen postoperatively to control pain. see table - for dosages. in chinchillas with dental malocclusion, the roots of the molars can become impacted, causing swelling of the mandible or exophthalmos and epiphora. these teeth are extremely difficult to extract without causing extensive bony and soft-tissue damage. discourage breeding of animals with malocclusion, unless it was acquired as a result of trauma or infection, because this trait is hereditary. most pet and laboratory mice are derived from mus musculus, which is the common house mouse. mice sold in the pet trade are randomly bred and less likely to suffer from the genetic problems associated with inbred laboratory rodents. mice possess brown fat tissues between their scapulae that also are known as hibernating glands; these are thought to provide an energy store. the spleen in male mice is % larger than that of females. ectoparasites usually are found in new acquisitions. • alopecia and pruritus, especially on the back of the head and dorsal midline, usually are associated with lice (polyplax serrata), mites, or fleas. • mite infestation (e.g., mycoptes musculinus, myobia musculini, radfordia affinis) often causes a greasy haircoat and folliculitis. transmission of lice and mites occurs via direct contact. fleas are transmitted by other household pets, such as cats and dogs. • diagnosis is based on clinical signs, history, visualization of parasite, skin scrape, and cellophane tape test. • treatment of fleas and lice is with pyrethrin powder. ivermectin is recommended for treatment of mites (see table - ). • change the bedding and thoroughly clean the cage between treatments to prevent reinfestation. occasionally, the surrounding environment needs to be treated with a premises spray used for killing fleas. • alopecia also may be the result of dermatophytes (see chapter ). lesions are often hyperkeratotic. • diagnosis is made by skin scrape or isolation on culture. most dermatophytes found in pet rodents do not fluoresce under a wood's lamp. • treatment is with lime-sulfur dip or griseofulvin (table - ). • ulcerative dermatitis is a common syndrome caused by staphylococcus aureus characterized by pododermatitis, mastitis, and abscesses in other areas. • administer antibiotics based on culture and sensitivity tests. chloramphenicol is recommended pending culture results (see table - ). the application of hot packs, local drainage, and topical medications are also beneficial in selected cases. • mastitis also may be caused by escheria coli or pasteurella, klebsiella, pseudomonas, or streptococcus species. mastitis usually is caused by poor sanitation, abrasive bedding, or overly aggressive young. • preputial gland abscesses are fairly common in males and are usually caused by e. coli or s. aureus. local flushing and topical treatment are usually adequate. • sc abscesses can be the result of the aforementioned bacteria or actinobacillus spp. or corynebacterium kutscheri. corynebacteria is associated with widespread abscesses, septic arthritis, gangrene, and ulcerated draining tracts. diagnosis is based on finding grampositive pleomorphic rods on gram stain and isolation on culture. • the bacteria are usually sensitive to ampicillin, chloramphenicol, and tetracycline (see table - ). • lymphoma and mammary neoplasia are common causes of sc masses. mammary neoplasia is usually malignant in mice, and metastasis to the lungs is common. (mice have five pairs of mammary glandsthree thoracic and two abdominal.) • obtain thoracic radiographs before surgery. • give a guarded prognosis for long-term survival. • other possibilities for sc masses are fungal granulomas, nodules from the psorergates simplex mite, hematoma, hernia, non-neoplastic lymphadenopathy, or emphysema. • otitis externa usually is caused by ear mites, although bacteria or fungi also may cause primary or secondary otitis. • clinical signs include erythema, pruritus, waxy debris, and excoriations behind the ears. • mites may be diagnosed by identification on otoscopic examination or microscopic examination of ear swabs (see "techniques"). • treatment requires cleaning debris from ears with a commercial ear cleanser followed by administration of three doses of ivermectin at -week intervals or topical acaricides used daily for to weeks (see table - ). • bacterial and fungal otitis is diagnosed by identification of organisms or gram-stained specimens and isolation on culture. • treatment is similar to that used in cats. • otitis media/interna usually are caused by hematogenous spread or local invasion of bacteria from a primary abscess. • clinical signs include head tilt, circling, facial nerve paralysis, and otitis externa. • rule out mouse hepatitis virus as the cause of the head tilt (see "gastroenterology"). • if treatment of the primary disease is successful, the otitis media usually resolves, although a residual head tilt may persist. • if a cluster of pseudomonas infections occurs in a population, evaluate the water source and produce for contamination. use sodium hypochlorite in the drinking water at ppm to control an outbreak while water quality is being restored. • damage to the pinnae can be associated with trauma, dermatitis, pox virus, hypersensitivity reactions, and vasculitis. dry gangrene is a common sequela and is usually self-limiting. i have observed a steroid-responsive pruritus in pet mice. the pruritus is severe enough to result in significant self-mutilation. this condition has been nonresponsive to treatment with ivermectin, lime-sulfur dips, griseofulvin, multiple antibiotics, oral prednisolone, and antihistamines. attempts at bacterial and fungal culture have failed to identify a pathogen. an inflammatory response is observed on histologic examination. mice with this condition respond to repository methylprednisolone injections every to weeks ( . mg/kg im). most owners have not elected to continue injections for longer than a few months. once the injections are stopped, the pruritus returns, often requiring euthanasia of the affected mouse. • bilateral alopecia found around the muzzle associated with no other abnormalities usually is caused by friction from overhead feeders. • alopecia occurring in smaller, weaker individuals is often the result of barbering. removal of the mice in best condition from the cage results in normal appearance of barbered mice in to weeks. • tailhead alopecia and scabbing are usually the result of aggression. separate affected animals, or additional trauma may occur. • other rare causes of alopecia are endocrinopathies, leprosy, and hereditary alopecia in nude mice. • epiphora is a common condition of pet mice. the most common causes in pets owned for longer than months are ammonia fumes and overgrown incisors. • ammonia causing contact irritation is diagnosed by examining an uncleaned cage and checking for odor. • treatment is improved sanitation. • overgrown incisors are diagnosed easily by oral examination. treat by trimming the affected teeth and providing opportunities for gnawing. • foreign bodies and the resultant corneal ulcers can cause epiphora. an eye examination, including fluorescein stain, is indicated. treat by removing the foreign body and administering an ophthalmic antibiotic (gentamicin, tetracycline, or chloramphenicol in affected eye, q h-q h). • in newly acquired pets, epiphora is often the first clinical sign of an upper respiratory infection. pasteurella pneumotropica is the most common pathogen, although salmonella spp., mycoplasma, sendai virus, lymphocytic choriomeningitis, and mouse pox also may cause epiphoria. the ocular discharge later appears mucopurulent (see "respiratory"). retinal degeneration can be either hereditary or (in albino mice) may be caused by exposure to highintensity lighting. the resulting blindness often goes undetected because patients adapt well and behave normally in their cages. • clinical signs include dyspnea (often described as chattering), mucopurulent oculonasal discharge, hunched posture, and anorexia. animals with a chronic history of this disease are often cachexic. • radiology aids in determination of the extent and severity of the pneumonia and the absence or presence of distant foci of infection. • treatment with tylosin is successful in controlling the disease if it is not too advanced. tetracycline, enrofloxacin, and chloramphenicol also have been used (see table - ). • many patients need nutritional support. • mice with pyometra or other abscesses require surgical debridement. • recovered animals are carriers and stress elicits clinical signs. strictly quarantine these animals. a common cause of pneumonia in newly acquired mice is sendai virus. acute fatalities are seen in suckling or weanling mice. • transmission is by aerosol or direct contact. • clinical signs in adults are caused by secondary bacterial infections and are similar to those in mrm. • diagnosis is based on clinical signs and serologic testing. • treat with antibiotics to control the secondary bacterial infection and provide supportive care as needed. • prohibit breeding for to weeks. • a killed vaccine is available. • recovered animals are resistant to new infection. common primary or secondary pathogens causing respiratory signs in mice are streptococcus pneumoniae, corynebacterium kutscheri, pasteurella pneumontropica, pseudomonas aeruginosa, and klebsiella pneumoniae. treatment is empiric or based on culture and sensitivity of a tracheal swab sample. dyspnea often is caused by metastasis to the lungs from mammary adenocarcinomas. primary lung tumors, especially pulmonary adenomas, also occur frequently. although not frequently diagnosed, cardiac disease can result in signs of respiratory disease. diagnosis is based on radiographic evidence of cardiomegaly and pulmonary edema. • tapeworms usually do not cause clinical signs. occasionally, heavy burdens may cause diarrhea or weight loss. the chief concern is the zoonotic potential of one species, hymenolepis nana, which is directly transmissible to humans. • diagnosis is made from visualization of eggs in the feces. treat with praziquantel or niclosamide (see table - ). improve sanitation, and remove indirect hosts (e.g., fleas, beetles, roaches). • pinworms (syphacia obvelata, aspiculuris tetraptera) may cause anal pruritus and, in severe cases, rectal prolapse. • diagnosis is based on clinical signs and observation of eggs on cellophane tape after application to the perineal region. treat with piperazine or mebendazole every to days for three treatments or with fenbendazole once daily for days (see table - ) and provide improved sanitation. • the protozoal parasite spironucleus muris causes diarrhea and slow growth associated with a pot-bellied appearance in young mice. • diagnosis is by fecal wet mount, although falsenegative findings are common. • treat with oxytetracycline (see table - ). supportive care to combat dehydration and hypothermia is extremely important. • control is achieved with improved sanitation. • giardia spp. and, rarely, eimeria falciformis show signs similar to spironucleus. giardiasis is zoonotic. treat with metronidazole. treat eimeria with sulfadiazine/trimethoprim (see table - ). most other protozoa are considered nonpathogenic. • cysticercus fasciolarus causes nonpathologic cysts of the liver. these cysts are the infective form of taenia taeniaformis in carnivores. viral diseases • diagnosis is based on clinical presentation, serology, and presence of syncytial giant cells in the epithelium of the small intestine. • treat supportively, and quarantine affected individuals. the prognosis is grave. • although less commonly seen in pet mice, reovirus occurs in older suckling mice. it is characterized by an oily diarrhea, which results in a greasy haircoat. other signs are conjunctivitis, stunted growth, and tremors. transmission is by ingestion. • diagnosis is based on clinical signs, histology, and serology. • treat supportively. the long-term prognosis is grave. initial survivors are weak and jaundiced, suffer from alopecia, and eventually die. • transmissible murine colonic hyperplasia (mch) caused by citrobacter freundii is characterized by diarrhea followed by rectal prolapse and stunted growth. transmission is feco-oral. • diagnosis is made by clinical signs and fecal culture. • treat with neomycin, tetracycline, or sulfamethazine until sensitivity results are available (see table - ). severe thickening of the distal half of the colon is observed at necropsy. • salmonellosis, also known as mouse typhoid, is transmitted by latent carriers or contaminated feed or bedding. incubation lasts for to days. • clinical signs are lethargy, anorexia, purulent conjunctivitis, arthritis, and diarrhea. • diagnosis is based on clinical signs and fecal culture. treat supportively. use of antibiotics is controversial. • quarantine survivors. • sanitation is extremely important because salmonella spp. are zoonotic. on gross postmortem examination, erythema of distal ileum and congestion of the spleen and liver are seen. with more chronic infections, necrotic foci are seen in the liver, spleen, and lymph nodes. prevent infection by feeding a fresh laboratory chow from a reputable source. thoroughly wash all produce and then dip it in a diluted bleach solution. rinse completely before feeding. • tyzzer disease is caused by bacillus piliformis. transmission is feco-oral. • clinical signs are precipitated by stress and consist of anorexia, diarrhea, and high mortality in weanlings. • diagnosis is made by clinical signs or isolation on culture. enteritis and multiple gray-yellow necrotic foci in the liver are seen on gross postmortem examination. • administer tetracycline for to days (see table - ) and reduce stress to control the disease. breeding systems vary; from one to six females may be placed with one male. all animals are housed together, and the young are removed after weaning. females in proestrus have swollen vulvas. vaginal plugs are present post-copulation. female mice that have been bred within days abort if a new male is placed in the cage. inappropriate light cycle, inappropriate age, crowding, and poor nutrition are the most common causes of infertility in pet mice. pyometra due to pasteurella pneumontropica, mycoplasma spp., or other bacteria is also common. desertion of litters is usually a result of stress, lack of nesting materials, agalactia, or mastitis. • urethral obstruction from proteinaceous plugs of inspissated ejaculum may develop in aged male mice. pseudomonas, e. coli, or proteus are the most frequently cultured pathogens. before complete obstruction, chronic hematuria may be noticed by the owner. • antibiotics, which are chosen based on the results of urine culture, are often curative with early presentation. complete obstruction requires surgical removal. • glomerulonephritis is very common in geriatric mice. it frequently is secondary to chronic viral infection. clinical signs are anorexia, lethargy, dehydration, and cachexia. urinalysis demonstrates proteinuria. as the disease progresses, the urine becomes isosthenuric, the blood urea nitrogen (bun) and creatinine levels rise, and other electrolyte abnormalities typical of chronic renal failure occur. treat supportively. prognosis for long-term survival is grave. • coccidia (e.g., klosseilla muris) occasionally is found in the urine. the clinical significance of its presence is unknown. • mice can be asymptomatic carriers of leptospirosis; however, this is rarely seen in pet mice. • diagnosis is based on darkfield microscopy of urine, serology, or histopathology. euthanasia of carriers is recommended. • infectious polyarthritis or mouse rheumatism is caused by streptobacillus moniliformis. in humans, it is known as rat bite or havernill fever. transmission is by direct contact. clinical signs are cachexia, keratitis, edema and ulceration of the appendages, and ankylosing arthritis. • diagnosis is based on the bacterial culture findings or the presence of caseous pericarditis and arthritis on necropsy. • treat with antibiotics chosen through the results of culture and sensitivity tests. use penicillin while awaiting results. supportive care is important. animals that recover remain arthritic. control is achieved through quarantine and sanitation. • the most frequently diagnosed neurologic disease in pet mice is head tilt resulting from bacterial otitis media (see "otitis"). the second most common cause of neurologic signs is trauma. • diagnosis is based on history and clinical signs. consider neoplasia in aged mice with slowly progressive signs. • lymphocytic choriomeningitis is a zoonotic arenavirus. transmission is airborne, transplacental, or by direct contact, fomites, or insect vectors. acute signs usually occur in mice that are to weeks old. approximately % of infected individuals show acute clinical signs, which include lethargy and photophobia followed by convulsions and paralysis. in animals that are latently infected, glomerulonephritis develops later. mice infected after weaning and before year in age lose weight, appear arthritic, and show signs of conjunctivitis and photophobia. the virus runs its course in several weeks. animals that recover show no residual signs. • diagnosis is based on clinical signs and the presence of immunofluorescent antibody (ifa). pleural effusion, splenomegaly, and hepatic lipidosis are found on necropsy. treat supportively. house survivors separately. • prevent the disease by improving sanitation, providing pest control, and cleaning produce. consider euthanasia because of the zoonotic potential of the virus. • mouse poliomyelitis/encephalomyelitis, also known as theiler disease, causes clinical signs in in , infected mice. two-thirds of healthy mice are carriers. transmission is by oral or respiratory routes. mice younger than weeks of age show signs of encephalitis. animals that are to weeks old are weak in the rear legs and progress to paralysis. the tail may remain mobile. affected mice continue to eat and be alert. albino mice are predisposed to show clinical signs. • diagnosis is based on clinical signs, serology, or histopathology that shows necrosis of the ganglionic cells of the anterior horn of the spinal cord. • treat supportively. consider euthanasia because of poor prognosis. • seizures in mice commonly result from otitis media, trauma, liver or kidney failure, toxin, bacterial meningitis, neoplasia, or viral encephalitis. • leukemia in mice is usually viral in origin. transmission is trans-mammary or trans-placental. • clinical signs are anemia, dyspnea (with thymic involvement), and those signs that are compatible with chronic disease. • diagnosis is based on complete blood count (cbc), bone marrow aspirate, or histopathology. prognosis is grave. • eperythrozoon coccoides is a rickettsial red blood cell (rbc) parasite of mice. affected mice are usually asymptomatic. occasionally, fever, anemia, and splenomegaly develop in infected animals. transmission is through the louse polyplax serrata. control is by extermination of the louse. • treat with tetracyclines. pet rats are derived from the norwegian or brown rat (rattus norvegicus), which did not originate from norway, but from asia. breeds of rats are called strains when they are inbred extensively and stocks when strains are hybridized. rats have brown fat, as discussed in the section on mice. they do not possess a gallbladder. their mandibular symphysis is articulated normally. rats are neophobic; therefore, make gradual changes in food or environment when possible. • fleas, mites (e.g., radfordia ensifera, ornithonyssus bacoti), lice (i.e., polyplax spinulosa), ear mites (i.e., notoedres muris), and dermatophytes cause similar signs in both mice and rats. treatment also is similar (see "mouse"). • sc masses in rats are similar to mice. pasteurella pneumotropica is a very common pathogen in mastitis and sc abscesses. • treat with chloramphenicol until culture results are available (see table - ). • mammary cancer develops in % to % of adult female rats and in approximately % of male rats. always submit biopsy specimens for histologic examination. most, but not all, of these tumors are fibroadenomas, which are benign. prognosis for longterm survival after surgical removal is good. other common neoplasms include interstitial cell tumors of the testes, which cause sc swellings in the inguinal region, and squamous cell carcinomas of the zymbals gland of the external ear canal. • ulcerative dermatitis occurs in rats as well as mice. staphylococcus aureus is the causative agent. c. kutscheri follows a similar course in rats and mice (see "mouse"). • ringtail is the formation of constrictive bands of fibrous tissue around the tail in nestling rats. these bands result in gangrene of the distal tail. this disease occurs when environmental humidity is less than %. • treat by making a longitudinal incision of the ring to release the stricture and apply topical dimethyl sulfoxide (dmso), steroid, and antibiotic solution ( ml dmso, ml mg/ml amikacin, ml mg/ml dexamethasone) four times daily. • to prevent ringtail, keep humidity above %, use solid-bottom cages and provide ample nesting material. prognosis for life is excellent. prognosis for retention of the distal tail is guarded. • epiphora and blepharospasm are caused mostly by ammonia fumes, overgrown incisors, or foreign bodies (see "mouse"). • sialodacryoadenitis virus is a coronavirus that is endemic in many rat populations. • clinical signs vary from mild keratoconjunctivitis to blepharospasm, chromodacryorrhea, severe uveitis, hyphema, buphthalmos, periorbital swelling, and pneumonia. the clinical course of the disease lasts to days. rats maintain normal activity levels and appetite. • treatment is not necessary for mild infections. place rats showing marked ocular disease or discomfort on the appropriate ophthalmic ointments (e.g., atropine, antibiotic, steroid) based on presentation. administer parenteral antibiotics to animals that show signs of respiratory problems. recovery is usually complete unless the eye ruptures or selfmutilation occurs. • control is achieved by not introducing new animals for weeks. • in contrast to mice, sendai virus rarely causes clinical signs in rats. • mucopurulent ocular discharge also may be caused by infection with mycoplasmosis, streptococcus pneumoniae, pseudomonas spp., and other less common bacterial or viral agents that cause pneumonia. • cataracts are primary hereditary defects or occur secondary to severe uveitis or diabetes mellitus. retinal dystrophy and colobomas are also inheritable traits in rats. retinal degeneration occurs in rats housed under intense lighting. • mrm is extremely common in pet rats. its presentation is similar to the disease in mice (see "mouse"). • streptococcus pneumoniae is normal flora for rats. however, during stressful situations, bacteremia may occur, resulting in pneumonia. clinical signs are similar to mrm. differentiation is based on culture and the presence of extensive fibrinopurulent pleural effusion on necropsy. • ampicillin controls clinical signs if treated early in the course of disease (see table - ). prevent the condition by minimizing stress. • corynebacterium kutscheri and pasteurella pneumotropica cause signs similar to mrm (see "mouse"). there is a serologic test for c. kutscheri. see the mouse section for a discussion of pseudomonas aeruginosa. • pneumocystosis carinii is an uncommon protozoa that infects the lung. cysts and trophozoites live in the alveoli. clinical signs occur only in immunocompromised or geriatric individuals. signs are cachexia, cyanosis, and dyspnea. • diagnosis is based on clinical signs, tracheal wash, response to therapy, or histologic examination. • treat with sulfadiazine/pyrinrethamine (see table - ). • myocardial degeneration and subsequent congestive heart failure are fairly common in geriatric rats. diagnosis is based on radiographs of the thorax and clinical signs. treat supportively, and use furosemide and digitalis at cat dosages to alleviate pulmonary edema. • polyarteritis nodosa is an idiopathic condition of geriatric rats that results in thickening and tortuosity of arteries, especially in the mesentery, pancreas, and testicles. affected areas are predisposed to clot formation and aneurysms. • nematode (syphacia muris), cestode, and intestinal protozoal parasite infestations are similar to those in mice. • capillaria hepatica has no clinical significance. yellow streaks on the liver are an incidental finding at necropsy. the causes and treatment of malocclusion are similar to those for mice. • epizootic diarrhea of suckling rats is a viral disorder found in rats to days old. the infection causes a mild diarrhea. most animals recover. occasionally, stunting occurs. treat supportively. • salmonellosis in rats is similar to that in mice. • if breeding is desired, take females showing signs of estrus (e.g., lordosis, hyperactivity, quivering ears, and swollen vulva) to a male rat's cage for hours, or keep one male in a cage with up to six females. check females for a post-copulatory plug to confirm breeding. remove females just before parturition, and house females individually while raising the young. a vaginal discharge is seen . to hours before labor. parturition is accompanied by stretching and extension of the rear legs. all neonates usually are delivered within to hours. • two extremely common conditions in geriatric rats are nephrocalcinosis and chronic progressive nephropathy. clinical signs are compatible with those of chronic renal failure. enlarged or small irregular kidneys may be found on physical or radiographic examination. isosthenuria and marked proteinuria are found in urinalysis. • definitive diagnosis is based on renal biopsy. • treat supportively. prognosis for long-term survival is grave. • trichasomoides crassicauda is an uncommon parasite of the urogenital tract. the adult worms usually reside in the kidney, but they occasionally may wander into the genital tract. the ova are passed in the urine. • clinical signs are hematuria and stranguria. proliferative mucosa of the bladder occasionally may be palpated as an abdominal mass. • treatment is somewhat successful with methyridene (see table - ). sanitation is critical in control of this disease. • klossiella muris is an incidental coccidia of the urinary tract. • many geriatric pet rats have chronic progressive radiculoneuropathy. • clinical signs are compatible with cauda equina syndrome, including posterior paresis progressing to paralysis, urine retention, and incontinence. prognosis is grave. • treat supportively or euthanize. • streptobacillus moniliformis, a normal bacteria found in the oral, nasal, and pharyngeal cavities of rodents, is isolated from % of middle ear infections and % of chronic pneumonias in rats. the bacteria is nonpathogenic for gerbils and guinea pigs. • clinical signs vary with the site of infection. head tilt and circling, septic arthritis, and respiratory disease commonly are seen. • diagnosis is based on isolation on culture. the clinical signs mimic many other diseases, especially mrm and pseudomonas infection (see "mouse"). • head tilt in rats also may be the result of trauma or neoplasia, especially pituitary adenomas. • hemobartonella muris is an rbc parasite of rats that is nonpathogenic unless the rat is immunocompromised or splenectomized. transmission is through the louse polyplax spinulosa. • clinical signs result from hemolytic anemia and hemoglobinuria. • treat with tetracyclines (see table - ). mesocricetus auratus, better known as the golden or syrian hamster, is a primarily nocturnal rodent that originated in the middle east. almost all hamsters in the united states are the offspring of three siblings imported in the s. many color variations are available. long-haired hamsters are called "teddy bear" hamsters. the stomach has two compartments, a non-glandular forestomach, which functions like a rumen, and a glandular stomach. hamsters are very territorial. they possess flank glands, which are larger in males, that are rubbed against objects to mark their territory. females are larger than males. except during estrus, they use this size advantage to attack males. do not allow groups to estivate together or recently awakened animals may cannibalize sleeping hamsters. m key point hamsters are extremely sensitive to antibiotics. penicillins, clindamycin, lincomycin, streptomycin, tylosin, erythromycin, and cephalosporins eliminate the normal intestinal flora, allowing overgrowth of pathogenic bacteria, particularly clostridium difficile. diarrhea, which is almost always fatal within to days, subsequently occurs. even antibiotics considered to be safe can have this effect. treat by discontinuing antibiotics, providing a lactobacillus supplement, and giving supportive therapy. • hamsters are susceptible to demodex criceti and d. aurati mites. d. criceti is limited to skin folds. d. aurati causes hyperpigmentation, alopecia, and seborrhea sicca affecting the dorsal midline. demodex is carried by many normal-appearing hamsters. • clinical signs occur in immunosuppressed animals, as would occur with stress, chronic infection, pregnancy, or malnutrition. • diagnosis is based on clinical signs and deep-skin scrapings. • treat with amitraz every weeks for two treatments past two consecutive negative skin scrapings. use the manufacturer's recommended dilution for dogs. • sarcoptes mites infrequently cause facial alopecia. diagnosis is based on skin scraping. treat with ivermectin (see table - ). do not confuse this condition with alopecia caused by contact with feeders or barbering. • notoedres mites affect only the external ear canal in female hamsters but may affect the ears, feet, geni-talia, and tail in males. diagnosis is made by observation of mites on samples from ear swabs, skin scrapings, or both. treat with ivermectin (see table - ). • other less common causes of alopecia in hamsters are dermatophytosis, endocrinopathies, and genetic defects. • dermal sc masses are usually abscesses caused by pasteurella pneumotropica, s. aureus, or streptococcus spp. treatment is based on results of culture and susceptibility testing. use chloramphenicol until culture results are available. other frequent causes of sc swellings are distended cheek pouches and testicles, mastitis, hernias, neoplasia, and lymphadenopathy. • epiphora and conjunctivitis are caused most frequently by increased environmental ammonia concentrations, incisor overgrowth, foreign body, or lymphocytic choriomeningitis (see "rat"; "mouse"). • mucopurulent discharge is caused by secondary infection by pasteurella or streptococcus spp. • hamsters are predisposed to rupture of the eye following trauma or infection. surgical enucleation is advised. electrosurgery is extremely helpful in controlling bleeding but do not apply heat to the stump of the optic nerve or vessels, or thermal injury to the brain may result. place gelfoam in the socket to enhance clot formation. • hamsters are susceptible to viral respiratory infections of humans. • clinical signs include nasal discharge, sneezing, otitis media, fever, and pneumonia. uncomplicated cases last to days. complications are usually the result of secondary bacterial infections. • treat supportively. use of antibiotics is indicated if copious nasal discharge, dyspnea, anorexia, or marked lethargy is observed. overuse of antibiotics may cause diarrhea-related death in hamsters that might have recovered uneventfully if left untreated. • most dyspnea in hamsters is caused by blunt thoracic trauma. hamsters often bite when startled. reflex actions on the part of humans, especially children, cause hamsters to be flung against hard objects. • diagnosis is by history and presence of fresh epistaxis. • treat supportively. emergency shock therapy, consisting of supplemental heat, oxygen administration, parenteral fluids, and glucocorticoids, frequently is required. • sendai virus can cause death in suckling hamsters housed with mice. adults show no clinical signs (see "mouse"). • primary bacterial pneumonia most frequently is caused by yersinia pseudotuberculosis, pasteurella pneumotropica, or streptococcus. clinical signs are compati-ble with those of pneumonia seen in other species, as well as weight loss and conjunctivitis. all three agents have a tendency to form distant abscesses, especially in the uterus. • diagnosis is based on clinical signs and isolation on culture. • treat with chloramphenicol until antibiotic susceptibility results are available. abscesses require surgical debridement; however, anesthesia in affected animals is very risky. recovered hamsters are carriers and must be quarantined from other rodents. prognosis is guarded. • cardiac thrombosis is seen in % of geriatric hamsters. most thromboses occur in the left atrium and are secondary to degenerative cardiomyopathy, cardiac amyloidosis, sepsis, or calcification of the great vessels. congenital myocardial necrosis also occurs. • clinical signs include cyanosis, dyspnea, and acute death. enlargement of the cardiac silhouette and pulmonary edema sometimes can be seen on thoracic radiographs. • furosemide and digitalis (using standard cat doses) may temporarily alleviate clinical signs. • hamsters can carry the zoonotic tapeworm h. nana (see "mouse"). • treat with niclosamide or praziquantel (see table - ) and provide improved sanitation. • pinworms (aspicularis tetraptera, syphacia muris, s. obvelata) occur in hamsters as well as in mice. • treat with fenbendazole (see table - ). • hamsters are predisposed to dental caries. a large percentage of affected teeth become abscessed, causing facial swelling, ptyalism, and anorexia. • diagnosis is based on clinical signs, oral examination, skull radiographs, and isolation on culture. • extract the tooth and administer antibiotic therapy based on results of susceptibility testing. prognosis is variable depending on the condition of the animal, tooth affected, and extent of the abscess (see "mouse"). • overgrown incisors also occur, as in mice. • the cheek pouches are very distensible. impaction of the pouches occurs on occasion. • clinical signs vary from ptyalism to swelling from abscess. in simple cases, removal of the material from the pouch with fine forceps is sufficient. sedation usually is not required. • if a fungal or bacterial infection of the pouch is present, remove the exudate, submit samples for gram staining and bacterial or fungal culture, and flush the pouch with diluted iodine solution. if cellulitis is present, administer systemic antibiotics as well. fistulas often heal spontaneously. • proliferative ileitis (i.e., wet-tail disease) is thought to be caused by a campylobacter-like organism with or without concurrent bacterial or viral infections. more than % of animals with clinical signs die. the highest morbidity and mortality rates occur in hamsters to weeks of age. teddy bear hamsters may be more susceptible to infection than shorter-haired varieties. transmission is feco-oral. • clinical signs include diarrhea, which mats on the ventrum and perineum, and results in anorexia, dehydration, and a hunched posture. the abdomen frequently seems painful on palpation. bowel loops often are distended on palpation because of ileal obstruction or intussusception. rectal prolapse usually occurs. • administer neomycin, gentamicin, metronidazole, or tetracycline (see table - ). supportive care is critical. prognosis is grave, even with treatment. gross postmortem findings include gas and yellow diarrhea in the distal intestinal tract, mucosal thickening in the ileum and distal jejunum, peritonitis, and liver abscesses. • other common causes of bacterial diarrhea include e. coli, tyzzer disease, or salmonella spp. (see "mouse"). in hamsters older than year of age, liver cysts that are derived from the biliary duct often develop. less frequently, similar cysts arise from the pancreas, epididymis, and seminal vesicles. this syndrome is called polycystic disease. no clinical signs are associated with cysts in these structures, which are an incidental finding on abdominal palpation. no treatment is recommended. • timing is critical to prevent injury to the male when breeding hamsters. transfer the female to the male's cage in the early evening days after a creamy, viscous vaginal discharge is noticed. monitor the pair carefully. remove the male immediately if the female is aggressive. remove the male after mating or after to hours even if mating has not occurred. two days after successful copulation, a gray malodorous vaginal discharge is observed. pregnancy is highly likely if there is no translucent vaginal discharge to days post-breeding. pseudopregnancies last to days. normal gestation is to days. before par-turition, a hemorrhagic vaginal discharge appears, and the female may pant. hamsters rarely suffer from pregnancy toxemia (see "guinea pig"). • infertility may be caused by pyometra (see p. pneumotropica and lymphocytic choriomeningitis). • cannibalism is most frequently a result of stress or mastitis. • in almost % of geriatric hamsters, renal amyloidosis develops. the disease tends to develop more rapidly in females. • clinical signs include edema and ascites due to protein loss in the urine, as well as the typical signs of chronic renal failure. • treat supportively. prognosis for long-term survival is grave. • head tilt is usually secondary to otitis media. also consider lymphocytic choriomeningitis or neoplasia as differential diagnosis (see "rat"). • in hamsters fed all-seed diets and deprived of exercise, cage paralysis syndrome often develops. usually pets are presented for acute posterior paresis which, in reality, was slowly progressive. the distinction is important in ruling out trauma. in mild cases, the hamster is able to move its hind limbs but unable to support weight. vitamin d and e supplementation, along with nutritional improvement and providing exercise, is curative in to weeks. in severe cases, recovery is negligible or incomplete. lymphoma and lymphosarcoma may be viral in origin. diagnosis is made by biopsy or fine-needle aspiration of affected lymph nodes. rule out lymphadenopathy caused by lymphadenitis from infection with streptobacillus moniliformis (see "rat"). although many hamsters initially respond well to chemotherapy protocols established for cats and dogs, prognosis for long-term survival is grave. • rarely, demodex spp. mites cause alopecia in gerbils. • diagnosis is based on skin scraping. • treat with rotenone ointment or amitraz dips every weeks for three to six treatments. use manufacturer's recommended dilution for dogs. • acute moist dermatitis usually is caused by s. aureus infection. infection on the face often begins with the harderian glands. the gland secretion is viscous and causes matting, with secondary staphylococcal infection occurring under the mats. attempts at grooming spread the infection to the feet and abdomen. • diagnosis is based on clinical signs and isolation on culture. • administer enrofloxacin, tetracycline, or chloramphenicol and apply warm, moist compresses to remove dried debris. remove possible irritants from cage (e.g., pine or cedar shavings, ammonia). occasionally, surgical removal of a chronically infected or inflamed gland is needed. • alopecia of the facial area, especially when it is symmetric, is usually the result of self-trauma from feeders, cage bars, or overzealous burrowing. • treat by changing cage construction or providing better visual security. • gerbils that catch their tails in crevices or are restrained inappropriately by their tails often are presented for avulsion of the skin from their tails. • treat initially by controlling hemorrhage and hypovolemic shock. • amputate the tail after patient stabilization to prevent ascending infection. in some animals, the infection is localized to the distal tail, which is sloughed in approximately to weeks. • generalized alopecia is normal in some weanling gerbils. the hair grows in as the animals mature. • melanomas are found most frequently on the ears, feet, or base of the tail. • diagnosis is based on biopsy. • treat by surgical removal. • sebaceous gland disease is usually the result of bacterial infections or neoplasia. • diagnosis is based on cytologic examination, culture, histologic examination, and response to antibiotic therapy. • treat bacterial infections with parenteral or topical antibiotics based on the severity of signs. • sebaceous gland adenomas, basal cell tumors, and squamous cell carcinomas are the most frequently encountered neoplasms. • take a radiograph of the thorax to diagnose metastases. prognosis for long-term survival is based on tumor type, stage, and character. • treat by surgical excision. chromodacryorrhea and epiphora occur as in mice. tapeworms (i.e., h. nana and h. diminuta) and pinworms (i.e., syphacia obvelata, dentostomella translucida, and aspicularis tetraptera) occur as in mice. incisor overgrowth occurs as in mice. • salmonella spp. cause transient diarrheas in gerbils. the source of infection is usually unwashed greens, contaminated feed, or carrier rodents of another species. most recover. animals that die have a fibrinosuppurative peritonitis. • treat supportively. use antibiotics in severe cases based on results of culture and susceptibility testing. • tyzzer disease, caused by bacillus piliformis, is seen most often in weanlings at to weeks of age and in post-partum females. • clinical signs include anorexia, lethargy, rough haircoat, and sometimes diarrhea. gross postmortem findings include yellow-gray nodules in the liver and hemorrhage at the ileocecal junction. • diagnosis is based on postmortem examination or response to therapy. • treat with oxytetracycline (see table - ) and supportive care. hepatic lipidosis and gallstones are frequent sequela to lipemia in gerbils fed diets with excessive fat. • breeding is most successful if animals are paired at weaning and kept in these pairs. male gerbils aid in raising the young. pairing older animals causes fighting. an average of % of neonates fail to survive to weaning. this is usually the result of agalactia and crushing. • chronic hemorrhagic discharge from the vulva is usually the result of cystic ovaries or ovarian tumors. most tumors occur in animals older than years of age and consist of granulosa cell tumors or theca cell tumors. leiomyomas of the uterus also cause similar clinical signs. • rule out urinary tract disease by performing a urinalysis via cystocentesis. large masses may be visualized on abdominal ultrasound. definitive diagnosis is based on vaginal cytology followed by exploratory laparotomy. • ovariohysterectomy is curative for cystic ovaries and tumors if they have not metastasized. • chronic renal failure develops in most gerbils older than . years of age. • clinical signs are polyuria, polydipsia, weight loss, and anorexia. urinalysis demonstrates proteinuria, hematuria, casts, and an increase in white and red blood cells. • treat supportively. prognosis for long-term survival is grave. • up to % of gerbils in certain family lines suffer spontaneous epileptiform seizures. the seizures are induced by stress and are self-limiting. seizures usually start as the gerbil reaches months of age. • treatment is unnecessary. chinchilla laniger and c. brevicaudata are nocturnal rodents from the andes mountains in south america. most animals kept in the united states are the descendants of animals. aside from pets, chinchillas are raised commercially for their pelts. the most common coat color is gray; the most valuable coat color is black. m key point chinchillas are sensitive to antibiotics (see "hamster"); therefore, avoid use of penicillins, lincomycin, erythromycin, and cephalosporins. house chinchillas in a cool environment because they are prone to overheating. if heat stroke occurs, treat with tepid water baths and supportive therapy. • chinchillas require dust baths to keep their skin in condition. use commercially available chinchilla dust only. sand substitutions do not condition the coat and occasionally cause conjunctivitis. offer dust at least once a week. • dermatophytosis occurs as in guinea pigs. • fur chewing is a serious problem in chinchillas that are farmed for pelts and often is seen in pet chinchillas that are recent culls from a ranch. the etiology of fur chewing is unknown. some cases seem to be related to chronic disease, malnutrition, poor caging, or stress. theories for undiagnosed cases include genetic abnormality; undiagnosed dermatophytosis; or adrenal, pituitary, or thyroid gland abnormalities. • diagnostics such as skin scraping, fungal culture, fecal, cbc, history profile, and biopsy are recommended. in general, if changes in diet and husbandry do not elicit a response or an underlying treatable disease condition is not discovered, prognosis for cure is grave. • one source advocates plucking all remaining underfur in chewed areas in an attempt to stimulate new hair growth. place collars after this procedure until the fur has grown in completely. • cystic sc masses may be caused by the intermediate stage of multiceps serialis. transmission is by ingestion of feed contaminated with canine feces. • diagnosis is made by histopathologic or cytologic examination of tissue samples. treat by surgical removal of the masses. • otitis caused by pseudomonas spp. occurs as in rats. • conjunctivitis occurs as in mice. • cataracts are congenital or developmental. • asteroid hyalosis occurs as a degenerative change. pneumonia occurs as in guinea pigs. parasites tapeworms (i.e., h. nana) occur as in mice. malocclusion of incisors and cheek teeth occurs as in guinea pigs. • diarrhea is caused most often by coccidia or giardia spp. or a bacterium. • clinical signs range from soft stools and weight loss to fluid diarrhea, dehydration, bloating, septicemia, and sudden death. • the protozoal parasites are best diagnosed on fresh saline smear or necropsy. • bacterial diarrhea is most often caused by contaminated feed and is diagnosed by isolation on culture. clostridium spp., pseudomonas aeruginosa, e. coli, salmonella enteritidis, and pasteurella spp. are the most common isolates. • treat supportively and use appropriate antiprotozoal or antibiotic drugs. • pasteurella pseudotuberculosis causes acute death from septicemia or a chronic weight loss with intermittent diarrhea. enlarged mesenteric lymph nodes are a hallmark of this disease. • diagnosis is based on clinical signs, histopathologic examination of tissue samples, and isolation on culture. • treat with sulfa drugs until sensitivity results are available. prognosis for recovery is poor. gross postmortem examination reveals yellow to white necrotic foci in the liver. • check male chinchillas four times per year for penile hair rings. roll back the prepuce and expose the penis. roll hair rings off the penis after application of a water-soluble lubricant. treat ulcerations topically or systemically as needed. • dystocia is fairly common in chinchillas (see "guinea pig"). • metritis is suspected when post-partum vaginal discharge, failure to return to a normal estrus cycle, anorexia, weight loss, polydipsia, polyuria, and chewing at flank and abdomen are present. • diagnosis is based on history, physical examination, abdominal radiographs, culture, ultrasound, and cbc. it usually is caused by bacteria introduced by the male or spread from an internal abscess. retained placentas, macerated fetuses, and dystocia are predisposing factors toward metritis. • treat with ovariohysterectomy after stabilization. females used only for breeding purposes may be treated with antibiotics alone, but the prognosis is poor. • female chinchillas are aggressive toward male chinchillas when not in estrus. breeding operations usually have separate cages for females and an interconnecting run for the male. females are kept out of the male's run by their larger size or collars. the young are precocious and do not need a nest. chinchillas only produce two litters per year. • clinical signs include hot, swollen mammary glands. suspect mastitis if previously healthy neonates become restless, then lethargic. • perform bacterial cultures on milk samples, and treat with antibiotics based on susceptibility testing. administer sulfa drugs until susceptibility results are available. local hot packing is also beneficial. occasionally, surgical drainage is required. foster neonates to another female if possible, or use puppy or kitten milk replacers to hand-raise babies. • chinchillas seem to be particularly sensitive to listeria monocytogenes. clinical signs can mimic p. pseudotuberculosis and include anorexia, lethargy, abortion, generalized central nervous system (cns) signs, hepatitis, mild enteritis, and mild emphysematous pneumonia. necropsy shows yellow foci in the liver. • diagnosis is based on isolation on culture. • treat with sulfa drugs (see table - ) until sensitivity results are available. the prognosis is poor. • other less common causes of neurologic disease in chinchillas include lymphocytic choriomeningitis, streptococcus spp., balisascaris procyonis (i.e., aberrant migration of raccoon roundworm), lead poisoning, and thiamine deficiency. guinea pigs (caviae porcellus) are nocturnal rodents that originated in the andes mountains. they are known for their dietary need for vitamin c. they are used as a food source in their native lands. there are three basic types: english, which have short hair; abyssinian, which have short, cowlicked hair; and peruvian, which have long hair. male guinea pigs are known as boars and the females as sows. guinea pigs become neophobic as they mature. offer a variety of foods early in life and make changes in diet or environment gradually. guinea pigs stampede when excited. square cages and strategically placed barriers on external walls prevent the trampling of small or weak animals. the smooth muscle of the bronchial tree is quite developed in guinea pigs. this places them at high risk for asthmatic-type anaphylactic reactions. both male and female guinea pigs have one pair of inguinal mammary glands; however, only the female's are well developed. m key point antibiotic toxicity (see "hamster"): guinea pigs also may be sensitive to tetracyclines. • fleas occur as in mice. • lice (i.e., gliricola porcelli, gynopus ovalis) usually cause no clinical signs except occasional alopecia, seborrhea, and trauma secondary to pruritus. • diagnosis is made by observation of lice on skin scraping. • treat with ivermectin, % malathion dust, or pyrethrin shampoo (see table - ). • the mite trixacarus caviae causes severe pruritus and is zoonotic. it mainly affects the dorsal midline and is difficult to find on skin scraping. it occurs most frequently in recently post-partum females, in which alopecia is the predominant clinical sign. treat with excellent sanitation and ivermectin (see table - ). • chirodiscoides caviae lives on the hair shaft of the perineal regions. it does not cause clinical signs. • treat with % carbaryl or lime-sulfur dip ( : ) (see table - ). sanitation is critical in preventing reinfestation. • about % to % of guinea pigs are carriers of trichophyton mentagrophytes. • clinical signs are alopecia and seborrhea sicca, usually starting on the face and spreading along the dorsum. • treat with lime-sulfur dips or griseofulvin (see table - ) combined with topical povidone iodine or chlorhexadine shampoos. • other causes for alopecia are barbering, alopecia of the flanks in late-gestation females, and generalized alopecia of young at weaning. subclinical hypovitaminosis c causes a poor hair coat and seborrhea sicca, as well as anorexia and large, malodorous stools. • "lumps" is the lay terminology for cervical lymphadenitis, which is characterized by lymphadenopathy in the ventral neck region. • pododermatitis and sore hocks are very common in guinea pigs. predisposing factors are untrimmed toe nails, poor sanitation, and wire flooring. s. aureus is the most commonly cultured pathogen. • clinical signs range from small ulcers on the soles of the feet to abscesses and gangrene. radiography is essential in determining whether bony involvement is present. untreated pododermatitis usually develops into osteomyelitis, which is very difficult to cure. • treat mild cases by improving sanitation and grooming. place affected individuals in solidfloored cages with paper bedding. use sulfa drugs (see table - ) until results of susceptibility testing are available. surgically remove or curette abscesses, and apply topical therapy and hot packing. amputation may be necessary when severe osteomyelitis exists. • conjunctivitis and epiphora occur as in mice. • inclusion body conjunctivitis is caused by chlamydia psittaci and is self-limiting in to weeks. • perform a conjunctival scraping to differentiate inflammatory conjunctivitis secondary to infection from allergy. i have observed an idiopathic, topical steroid-responsive lymphoplasmacytic conjunctivitis in guinea pigs. • white, dry ocular discharge is an early sign of hypovitaminosis c. • "pea-eyes" is the lay terminology for subscleral fatty deposits or protrusion of the lacrimal gland through the lower conjunctiva. the condition is thought to be hereditary. treatment is not required. • cataracts occur and are either congenital or developmental. • diabetes mellitus in guinea pigs also may cause cataracts. usually, no other clinical signs are present and urine glucose is greater than mg/dl whereas blood glucose remains within normal limits. • corneal or scleral calcification is usually an incidental finding. a thorough workup, including serum chemistry profile and radiographs, is recommended to ensure that generalized metastatic calcification is not present. • pneumonia in guinea pigs usually is caused by infection with s. pneumoniae, s. zooepidemicus, or bordetella bronchiseptica. s. aureus, p. aeruginosa, klebsiella pneumoniae, and pasteurella multocida also are cultured frequently. transmission is by direct contact, fomites, or aerosol. hypovitaminosis c and stress often predisposes guinea pigs to bacterial respiratory infections. weanlings are particularly susceptible. clinical signs and diagnosis are similar to other small mammals (see "mouse"). • take radiographs to rule out abscesses, pleural effusion, or pericardial effusion in refractory cases. • treat with chloramphenicol, sulfa drugs, or enrofloxacin (see table - ) and vitamin c (table - ) until results of culture and susceptibility testing are available. cats, dogs, rabbits, and rats are reservoirs for bordetella spp. as in other rodents, respiratory infections may lead to otitis interna/media. bordetella spp. also cause pyometra and abortions. • nasal discharge is most frequently a sign of upper respiratory tract infection but also may be associated with allergies or volatile irritants. • the diagnosis of allergic rhinitis is made by exclusion and through response to antihistamines or environmental changes. • bronchogenic papillary adenoma develops in approximately % of guinea pigs older than years of age. • diagnosis is often an incidental finding when thoracic radiography is performed for another problem. • occasionally, clinical signs are seen as a result of pressure on the heart or great vessels. • dyspnea most frequently is caused by heat stress or trauma. other causes are pregnancy toxemia, gastric bloat, volatile irritants, pleural effusion, pneumonia, or pulmonary edema. rhabdomyomatosis is a common necropsy finding. gross lesions appear as pale foci located on the endomyocardium and valves. histologic examination reveals myocardial cells that have stored excessive glycogen. do not confuse these areas with thrombi, abscesses, or neoplasia. their clinical significance is unknown. • paraspidodera ucinata is the cecal pinworm of guinea pigs. they are generally asymptomatic, but heavy infestations can cause diarrhea and weight loss. • diagnosis is based on fecal examination or cellophane tape test. • treat with piperazine or fenbendazole (see table - ). • coccidiosis caused by eimeria caviae is a fairly common cause of diarrhea in guinea pigs recently purchased from pet stores. • clinical signs are tenesmus, diarrhea, dehydration, and death. • diagnosis is based on fecal examination. on gross postmortem examination, petechiation and thickening of the colon are seen. • treat supportively and administer sulfa drugs (see table - ). • cryptosporidium wrairi and giardia spp. are found rarely. they cause a chronic enteritis. balantidium spp. are thought to be nonpathogenic. • malocclusion in guinea pigs is diagnosed on oral examination. • clinical signs are ptyalism and anorexia. the premolars are the most commonly affected teeth. • long-standing hypovitaminosis c predisposes guinea pigs to malocclusion. • treat malocclusion as in other rodents (see "dental procedures"). • hypovitaminosis c (i.e., scurvy) is associated with soft, malodorous feces. degeneration of the epithelium of the intestinal tract adversely affects digestion and absorption and allows secondary bacterial infections. • diagnosis of scurvy is based on clinical signs, the exclusion of other causes of diarrhea, and response to vitamin c therapy (see table - ). • salmonellosis usually is contracted through contaminated feed. • clinical signs range from sudden death to diarrhea and anorexia. the diarrhea is frequently light colored. sepsis is common and may cause conjunctivitis, shock, pneumonia, abortion, and neurologic symptoms. • diagnosis is based on isolation on culture of feces or other appropriate tissue samples. • treatment is controversial because recovered individuals remain carriers. use sulfa antibiotics or enrofloxacin (see table - ) until sensitivity testing results are available. supportive care is essential. • e. coli, arizona, and clostridium are other commonly cultured diarrhea-causing organisms. clostridium are diagnosed most easily by finding large numbers of spores on a gram stain fecal specimen. treat with metronidazole (see table - ). • yersinia pseudotuberculosis either causes an acutely fatal diarrhea or localizes into regional lymph nodes. • diagnosis is based on culture. • treat by surgical removal or drainage of abscessed lymph nodes. mesenteric lymph node involvement necessitates abdominal surgery. treat with sulfa drugs or enrofloxacin until susceptibility testing results are available (see table - ). • one male usually is housed with four to six females for breeding purposes. signs of estrus are vulvar swelling, lordosis, and opening of the vaginal closure membrane. fetuses are palpable at to weeks of gestation. parturition occurs within hours after the pubic symphysis has reached mm. neonates weighing less than g have a grave prognosis for survival even with intensive care. neonates normally do not nurse for the first to hours. litters with five or more fetuses usually result in abortion. • dystocia commonly occurs in females bred after the age of to months. after this age, the symphysis fuses and is unable to open the to cm required to allow passage of a fetus. dystocia in younger guinea pigs may be caused by obesity, large fetal size, fetal malpresentation, subclinical ketosis, or uterine inertia. on presentation, check the pelvic symphysis. if active contractions are present and the symphyseal gap is less than cm, perform a c-section. normal parturition is very rapid, with a rest of only to minutes between fetuses. • perform a c-section if active straining does not produce a fetus within to minutes. radiograph sows with a history of weak contractions to determine the stage of pregnancy and evaluate the size of the fetuses. if well-developed skeletons of appropriate size are seen and the pubis has not yet fused, give oxytocin and calcium (see table - ). if no fetuses are produced within to minutes, perform a c-section. • if poorly developed fetuses are seen radiographically, consider fetal death, ketosis, or a nonreproductive disorder as possible causes of dystocia. m key point pregnancy toxemia usually is seen in obese sows with large litters in late pregnancy. other risk factors include systemic disease or diet change causing anorexia, genetics, stress, and first litter. • clinical signs are tachypnea, depression, malodorous breath, seizures, and icterus. a urine ph of less than with marked proteinuria is compatible with pregnancy toxemia. a marked hyperkalemia and elevation of liver enzymes often occurs. thrombocytopenia may be present. • treat with iv or io saline, dextrose, glucocorticoids, and calcium. surgical abortion of the fetuses may be attempted, but the anesthesia risk is quite high. prognosis for survival is grave. do not rebreed affected females. do not breed sows heavier than g. • large litters can cause a hemorrhagic syndrome. compression of the portal vein and liver causes hepatic dysfunction, which results in vitamin k and clotting factor deficiency. • treat with vitamin k supplementation (see table - ). response is poor in severely compromised patients. affected individuals are at risk of ketosis developing. prognosis is guarded. • vaginitis in guinea pigs frequently is caused by foreign bodies, usually bedding. • diagnosis is made on vaginal examination. • treat by flushing the vagina to remove the foreign material. • vaginal discharge also can be caused by pyometra, uterine torsion, urinary tract infection, or urogenital neoplasia. • diagnosis is based on findings on abdominal palpation, vaginal cytology and culture, urinalysis, abdominal radiographs, ultrasound, and exploratory. • treatment varies with the condition and is similar to that used in cats. • ovarian teratomas and uterine tumors occasionally are diagnosed and usually resolve with ovariohysterectomy. • a symmetric alopecia with concurrent abdominal enlargement may be seen in female guinea pigs with cystic ovaries. • diagnosis is based on abdominal palpation, cytology, and ultrasound. • treat by performing an ovariohysterectomy. if the guinea pig is not a good candidate for surgery, human chorionic gonadotropin (hcg, usp units im, repeat in week) may temporarily resolve clinical signs. • male guinea pigs are prone to preputial foreign bodies. a preputial discharge is the usual presenting complaint. • diagnosis is based on physical examination. • treat by removing foreign bodies and performing local flushing. chronic problems require a change in bedding. • male guinea pigs produce sebaceous secretions in the folds around their perineal area. clean these areas with soap and water semiannually to prevent localized pyoderma. • if pyoderma occurs, treat with topical therapy and oral antibiotics. bacterial cystitis and urolithiasis are relatively common in guinea pigs. diagnosis is based on a history of stranguria, hematuria, painful abdomen, and anorexia, in addition to abdominal palpation, urinalysis, urine culture, abdominal radiographs, and ultrasonography. treatment consists of antibiotics based on results of culture and susceptibility testing and surgical removal of calculi, if present. prevention of recurrence is difficult if the calculi are not caused by a bacterial infection. addition of vitamin c to the drinking water as well as changing the brand of diet are sometimes helpful in preventing recurrence of metabolic stones. klossiella cobayae is a coccidia that lives in the renal tubules. it has no clinical significance. the most common orthopedic problem seen in guinea pigs is overgrown toenails. this leads to pododermatitis and sore hocks as well as to degenerative joint disease and a predisposition to tibial fractures. tibial fractures are the most common fracture seen in guinea pigs. they most frequently occur after foot entanglement. internal fixation with an im pin or application of a kirschner apparatus is the repair of choice. m key point signs of hypovitaminosis c or scurvy start to develop in guinea pigs as early as - days if they are placed on diets % deficient in vitamin c. early signs are soft, malodorous stools, weight loss, poor hair coat, and anorexia. later, petechia, gingivitis, cutaneous and oral sores, swollen costochondral junctions, joint pain and hemorrhage resulting in lameness, and conjunctivitis become apparent. treat supportively and administer parenteral vitamin c ( mg/day). • lymphocytic choriomeningitis occurs as in mice. • guinea pig paralysis syndrome starts with mild pyrexia and urinary incontinence, followed by weight loss and posterior paresis that progresses to paralysis. currently, the etiology is unknown, but it does not appear to be contagious. • treat with supportive care. prognosis for long-term survival is grave. • head tilt is usually the result of otitis or trauma (see "mouse"). cavian leukemia has a viral etiology. the liver, spleen, and lymph nodes are the primary organs involved. there is no current treatment. quarantine exposed individuals. death usually occurs within days after discovery of lymphoblasts in the peripheral blood. neutrophils normally have red granules. kurloff bodies are normally occurring eosinophilic intracytoplasmic inclusion bodies that are found in mononuclear cells. they are seen most frequently in females and appear to correspond positively with estrogen levels. metastatic calcification occurs in most guinea pigs older than year of age. it is more severe in females than in males. the stomach is one of the first organs affected. dysfunction in motility causes obstruction. the tendency appears to be exacerbated by high calcium and low phosphorus diets. exotic animal formulary the biology and medicine of rabbits and rodents veterinary clinics of north america key: cord- -zgzq fe authors: kumar, durgesh; meena, mahendra kumar; kumari, kamlesh; patel, rajan; jayaraj, abhilash; singh, prashant title: in-silico prediction of novel drug-target complex of nsp of chikv through molecular dynamic simulation date: - - journal: heliyon doi: . /j.heliyon. .e sha: doc_id: cord_uid: zgzq fe literature reported that nsp of chikv is an important target for the designing of drug as it involves in the replication, survival etc. herein, about eighteen million molecules available in the zinc database are filtered against nsp using raspd. top five hit drug molecules were then taken from the total screened molecules ( ) from zinc database. then, a one pot-three components reaction is designed to get the pyrazolophthalazine and its formation was studied using dft method. authors created a library of compounds using the product obtained in the reaction and filtered against nsp of chikv based on docking using igemdock, a computational tool. authors have studied the best molecules after applying the the lipinski's rule of five and bioactive score. further, the authors took the best compound i.e. cmpd and performed the md simulations and tdmd simulations with nsp protease using amber . md trajectories were studied to collect the information about the nsp of chikv with and without screened compound and then, mm-gbsa calculations were performed to calculate change in binding free energies for the formation of complex. the aim of the work is to find the potential candidate as promising inhibitor against nsp of chikv. chikungunya virus (chikv) causes chikungunya fever (chikf) and this virus spread through the biting of mosquito [ , ] . it causes severe infection and the symptoms of chikf are high fever, polyarthralgia, myalgia etc. [ , , ] till date, there is no effective vaccine or drug for this disease available in the market, although few candidates as vaccine are under clinical trials [ ] . alphavirus is an enveloped viruses with a single stranded (þss) rna with non-structural proteins (nsp ) and structural proteins, capsid, envelope glycoproteins (e , e and e ) and k peptide [ , , , , ] . nsp of chikv is also known as macro-domain and have been initially obtained from databank [ ] . researchers reported baicalin as one of the potential drug molecule against the chikv based on binding affinity and π-π interaction between baicalin with tyr residue of nsp of chikv [ , ] . heterocyclic compounds have attracted the attention of the researchers due to biological potency in different aspects and they can be synthesized by number of steps as well one pot synthesis. further, one pot synthesis or the multi-component reactions are preferred due to less time consumption in the synthesis as well less or no time is wasted in the purification of the compound [ , , , ] . in silico methods are being explored by the researchers due to the efficiency and strategic approach. computational tools are used to create a library and filtering them to get the biological potent compound against a receptor [ , , , , , , , , ] . in this work, authors have designed a multi-component reaction (mcr) to produce pyrazolophthalazine via the one pot reaction between benzaldehyde, , -dihydrophthalazine- , -dione and oxazolidine- , -dione (ozd) and its feasibility was studied through dft method using gaussian . then, a library of molecules was designed based on pyrazolophthalazine. designed library was used for virtually screening against nsp of chikv, to get potential lead molecules based on minimum total binding energy, drug-likeness, and bioactivity score [ ] . the filtered compounds were subjected to molecular docking using pardock and their interaction profile was analyzed using ds visualizer, pymol, chimera. further, temperature dependent molecular dynamic simulations (tdmd) and mm-gbsa of screened compound-nsp of chikv complex was performed to analyze the structural stability of the complex. herein, cs chemdraw was used to draw the chemical reaction using from benzaldehyde, , -dihydrophthalazine- , -dione and oxazolidine- , -dione (ozd) to get pyrazolophthalazine i.e., the product molecule. it was used to design drug library by changing alkyl group (from r to r ) in aryl of aldehyde and these molecules were considered to be potential drug molecule targeting nsp of chikv [ , ] . literature reported that the oxazolidine- , -diones are based on five member heterocyclic compounds and many biological activity are reported. they have shown promising role as aldose reductase inhibitors, hypoglycaemic and hypolipidemic agents. a novel mcrs for the formation of novel pyrazolophthalazine molecules through a reaction between oxazolidine- , -dione, benzaldehyde and , -dihydrophthalazine- , -dione is designed as shown in scheme and was studied using dft. it is a proposed mechanism for the reaction shown in scheme . initially, there is a reaction between r i.e. ozd, has active methylene group and r i.e. benzaldehyde, carbonyl group. the reaction will give an unsaturated compound (im ) with an elimination of water molecule via knoevangel reaction. further, im reacts with r i.e. , -dihydrophthalazine- , -dione to give im . herein, the lone pair present on nitrogen of , -dihydrophthalazine- , -dione (nucleophilic site) attack on the unsaturated carbon (electrophilic site). it is azamichael addition followed by the rearrangement to im . further, im loose a molecule of water and cyclization occurs to give p, the product of interest. the adduct formation is justified based on energy diagram using b lyp/ - g*method [ , ] . the following parameters of global reacting indices were calculated from reactant to product such as total energy (e), e homo , e lumo & lumo-homo energy gap (Δe) are calculated [ , ] . the proposed mechanism of mcrs was studied using density functional theory (dft) calculation as in scheme . a library of molecules was created via different susbtitutents on pyrazolophthalazine using cs chemdraw as in table [ , ] . in designing, only aromatic aldehydes are varied to a library of the new compounds which may have better potency against the nsp of chikv. in this designed molecules only aromatic aldehydes have been varied by changing alkyl group r to r . library of designed molecules were used to screen against nsp of chikv through igemdock. authors have been taken best five molecules on the basis of minimum total binding energy. further, screening of best five molecules was filtered through admet properties. the crystal structure of nsp of chikv was obtained from the rcsb protein data bank (pdb id: gpo) in complex with adp-ribose at resolutions of . Ǻ respectively. the removal of extra atoms like water, missing atoms and added explicit hydrogen in the both model (ligand & target protein) was done using pymol (biovia ) and ucsf chimera- . . software [ ] . raspd is used for preliminary screening of potential molecules from zinc database based on minimum binding free energy. this is very fast protocol for accurate prediction of hit candidates for any target protein. in this way, authors screened drug molecules from zinc database based on binding affinity range of - . to . kcal/mol, but top five drug molecules were selected from screened molecules on the basis of molecular weight (mw < ) with minimum binding free energy for molecular docking and simulations [ ] . in this protocol, method a (protein-ligand complex) was used to estimation of binding free energy and these molecules are listed in table . screening is a method to design the drug in short span of time. it is used to investigate potential molecules against nsp of chikv. the purpose of this method is used to predict a best pose of molecule and it was selected best ligand conformations based on pose and their binding free energy [ ] . the designed library of molecules and screened top five molecules from raspd were screened against nsp of chikv using igemdock software [ ] . in this, top five best molecules from the designed library based on binding energy were taken and on other side, screened molecules from raspd are ignored due to high binding energy in comparison of designed best molecules. igemdock computes a ligand conformation and orientation relative to the active site of target protein based on ga and summarized results in term of minimum total binding energy of the complex in tables and [ ] . the bioactive properties like tpsa, chemical structure, logp and lipinski's "rule of five" value using molinspiration were calculated [ , ] . several other biological parameters of best five compounds were calculated using swiss adme as in tables and . thus, the absorption (% abs) was calculated by given equation according to the method [ ] . herein, authors used gusar, a webserver to predict ld values for rats with four types of administration like oral, intravenous, intraperitoneal, subcutaneous and inhalation. the acute toxicity of cmpd , , , & has been calculated for screened molecules. these results were obtained through gusar for prediction of rat acute toxicity and acute rodent toxicity with four type of administration are mentioned in scheme . synthesis of pyrazolophthalazine via the one pot three component reaction between benzaldehyde, , -dihydrophthalazine- , -dione and oxazolidine- , -dione (ozd). table . the acute rate toxicity end-points are based on the log representation of ld value (mg/kg) for the rats [ ] . pardock is an automated web server for rigid docking was used to determine the binding modes of compounds in the receptor [ ] . e is the total energy; eel is energy due to electrostatic interactions; evdw is due to van der waals interactions; ehpb is the due to hydrophobic interactions. the docking result was further validated using the molecular dynamics (md) simulations method. md simulations is used to predict the binding of compounds to the target protein and study the changes in binding strength with change in the temperature [ ] . md simulations, an important approach is to study the physical movements of the atoms of the receptor in presence and absence of the compound for a known time [ ] . three dimensional structure of the cmpd was drawn using marvin sketch and its optimization is done with the gaussian on applying b lyp/ - g(d) basis [ , ] . further, other parameters of screened cmpd for the md simulations were produced using antechamber module of amber suite molecular dynamics software utilizing generalized amber force field (gaff) [ ] . the input files were used to run by xleap command using amber ff sb force field for the created parameter and coordinate files. subsequently, solvate box tip p . was added with a Å buffering distance [ ] . during thermalization, initial velocities were produced based on the maxwell-boltzmann distribution with a temperature of k and constant volume (ntb ¼ ) for ps (nstlim ¼ Â dt ¼ . ) simulation time. further, the receptor or its complex was equilibrated at k and bar using the berendsen thermostat for constant pressure (ntp ¼ ) for another ps simulation time. once the equilibrium is reached, md simulations were performed for ns [ ] . different trajectories based on md simulations were analyzed [ ] . authors also checked the rmsd value by the variable temperature (non-isothermally) of drug-target complex at ns and according to david and konard approximation and this approximation is said to be temperature dependent md simulations. in tdmd simulation for ns, the input file (temp k to k) was used to set print energy output files every steps (ntwx&ntwr ¼ ) and save coordinates every (ntwx ¼ ) as in amber input. md simulations trajectories of complex system were used to determined relative change in binding free energy according to the mm-gbsa method [ , , , ] . in order to calculate binding free energies like for cmpd -nsp of chikv, nsp of chikv and cmpd was calculated for high accuracy results [ , ] . the binding free energy (Δg bind ) of the drug-target complex is calculated on the given eq. ( ). where, Δg bind and Δg bind , vacuum are the free energy difference between the bound and unbound forms of a complex in solvated and vacuum respectively. Δg solv,d-t, Δg solv,d and Δg solv,t represented the change in free energy between the solvated and vacuum states of a cmpd , nsp of chikv and cmpd -nsp of chikv complex. the change in solvation free energy from different systems are calculated by given eqs. ( ), ( ), ( ), and ( ). e mm -mm energy; e int -internal energy; e el -electrostatic energy and e vdw -energy due to van der waals interactions. bonding orbital calculations were performed by full nbo program as executed in the gaussian [ ] . different physiochemical descriptors scheme . the proposed mechanism for the formation of pyrazolophthalazine via one pot three component synthesis. (continued on next page) d. kumar et al. heliyon ( ) e ( ), ( ) , and ( ) as follows. authors have designed the chemical reaction for the synthesis of biologically potent pyrazolophthalazine as in scheme and it is considered as a novel compound to target nsp of chikv. initially, there is a reaction between r i.e. ozd, has active methylene group and r i.e. caronyl of aromatic aldehyde. the reaction will give an unsaturated compound (im ) with an elimination of water molecule via knoevangel reaction. further, im reacts with r i.e. , -dihydrophthalazine- , dione to give im . herein, the lone pair present on nitrogen of , -dihydrophthalazine- , -dione (nucleophilic site) attack on the unsaturated carbon (electrophilic site). it is aza-michael addition followed by the rearrangement to im . further, im loose a molecule of water and cyclization occurs to give p, the product of interest as in scheme . energy level (homo & lumo) of reactant, intermediate and the product are determined. the energy differences between the orbital energies are shown in table . the energy values of homo orbital and lumo orbital of product molecules were lying at an energy value of - . ev and - . ev respectively. the lumo-homo energy gap was obtained at - . ev in the isolated gas molecular calculations. if lumo-homo energy gap is higher implies the kinetic energy is higher and high chemical reactivity [ ] (see figure ). figure . the energy profile diagram of product formation using b lyp/ - g* basis set as in scheme through dft method. igemdock is used for the virtual screening of the compounds against the target protein [ , ] . the details of the binding energy of the designed compounds against the nsp of chikv is given in table and screened molecules by raspd from zinc data base against nsp of chikv is in table . then, the top five compounds are taken based on the least binding energy of the complex system for molecular docking. binding energy of the molecules or compounds from the obtained from the zinc database were further studied using igemdock, it found that the molecules (zinc_ ) showed minimum total binding energy but it was less than the designed best five compounds as in table . this molecule showed only one π-π interaction with tyr- and two h. bond interaction with leu and val in figure . best five molecules from the designed library as in table as well as the screened molecules from zinc database as in table are taken for the prediction of physicochemical properties, lipophilicity, water solubility, pharmacokinetics, drug-likeness and bioactivity score as in tables and . all best five compounds follow the criteria of biological parameters. if a compound having gpcr ligand values > . is mostly likely to possess considerable biological activities, while ligand values - . to . are expected to be moderately active and ligand values < - . , presumed to be inactive. for the prediction of acute toxicity, the adverse effects of a compound may result due to one or more than one time exposure. in present work, authors have determined the median lethal dose (ld ) of top five hit from the designed library (cmpd , , , & as in table ) via four types of administration: oral, intravenous, intraperitoneal and subcutaneous. ld is the amount of molecule, can causes the death or kill the % of test animal. therefore, the toxicologists can use different animals but rats and mice are usually considered for the study. it is expressed per g of the body weight of the small animals. the active site of receptor is a binding pocket due to hydrogen bonding, hydrophobic interactions. screened compounds showed the promising antiviral activity against nsp of chikv based on binding energy. molecular docking method was used for predicting the binding energy of newly formed drug-target complex. in this study, best five compounds from designed library were docked with active site of nsp of chikv using pardock to elucidate their molecular interactions as in table and figure . the best docking pose for each ligand was also recorded for better results. this analysis was showed that molecules fit to bind in the cavity of nsp of chikv and by forming a stable d-t complex. as is evident from figure a , b, drug interacts with nsp protease of chikv forming a most stable complex establishing hydrogen bond interactions with their minimum distance. the insight of various other residues are present in d-t complex interaction is depicted in d plot and these residues are play key role in the formation of stable d-t complex. the docking results were further evaluated in terms of rmsd value and binding free energy through mm-gbsa protocol. the amber program was used for md simulation to study the stability and flexibility of the nsp of chikv with and without cmpd receptor and its complex using different trajectories like rmsd, rmsf and hydrogen bond. rmsd plot showed that most of the complex system was relatively stable within - . Å for - ns simulation time as in figure . rmsf plot was used to understand the flexibility of the nsp of chikv with and without cmpd as in figure . less fluctuation are observed in the complex in comparison of the nsp of chikv alone. further, the hydrogen bond plot and analysis for the complex of nsp of chikv- are given in figure and table respectively. it was used to find the existence of hbs between a donor and acceptor, % occupancy and angle during the simulations. residues asp , arg , thr , leu , gly , ala and val are present in the active site of nsp of chikv and showed noteworthy fluctuations compared to other native residues. the total numbers of average hbs are formed during md simulations were predicted. the analysis to find the maximum number of hydrogen bonds is done and suggested maximum of intermolecular hydrogen bonds. average number of hbs for different donor-acceptor average distance cutoffs is . (strong bonding) with larger average angle. it was found that hbs between drug molecules and residues are gly_ @o with donor h & n of drg_ as in table . it was assume that formed hbs have distance between accepter residue o atom in the backbone with donor h & n in the drug molecules showed shorter distance ( . Å) and the angle of n-h-o is . with . % occupancy at k for ns simulations time observed. binding free energies was calculated of drug (cmpd ), target and drug-target complex using mm-gbsa methods are shown in table . figure . screened drug molecule from zinc data base showed only oneπ-π and hydrogen bonding are represented as a stick model. change in enthalpy (Δh) as in figure , differences of (drug-target complex) with target and drug was found to be - . kcal/mol. change in free energy (Δg) was determined by calculating change in entropy (Δs) and change in enthalpy (Δh) for the formation of complex between nsp of chikv and cmpd . for any spontaneous process or reaction, the change in free energy should be negative. tΔs was calculated for the complex system is - . kcal/mol as in table . Δg for the binding of the complex between nsp of chikv and cmpd comes out to be - . kcal/mol by using eq. ( ) . in the literature, authors were explained md simulation of backbone of nsp of chikv with and without cmpd (target protein and drug-target complex) at k (isothermally) and atm pressure. herein, based on david and konrad approximation, authors varied the temperature from to k (non-isothermally) and atm pressure for md simulation of drugtarget complex. the system minimization, heating, and equilibration were carried out in the same manner used for the optimization of drug-target complex described above. in this way tdmd simulations were performed for ns at , , and k and the rmsd trajectories are given in figure . note: where, in ad meaning the compound falls in applicability domain of models while out of ad means the compound is out of applicability domain of models. table . actual molecular docking results of cmpd drug molecule onto active site of apr ligand and also represented their interactions with distance analysis. the simulations and change in relative enthalpy energy results confirmed that actual stability of system at and k in figure and table . a total of snapshots were taken in a ns md simulations time to calculate the binding free energy difference using eq. ( ). further, rmsf curve for target and its complex with drug molecules was studied at ns at k, k, k, k and k as in figure . the hydrogen bond plot for the complex of nsp of chikv- for ns at , , , and k was studied and maximum number of hydrogen bonds for the complex at , , , and k are , , , and respectively and shown in figures , , , , and respectively. it means on increasing the temperature, number of hydrogen bonds decreases. further, hydrogen bond analysis for the complex of nsp of chikv- for ns at , , , and k was given in tables , , , , and respectively. prediction of the structural stability of intermolecular hydrogen bonds and total number of hydrogen bonds formed with nsp of chikv. md simulation of the drugtarget complex is used to study the stability during the trajectory period. hydrogen bond profiles between the selected drugs and nsp of chikv were calculated using the amber . this analysis revealed that average hydrogen bonds are formed during the simulations period sharing four to five hydrogen bonds with gly , arg , ile , lys , leu and these five hydrogen bonds showed poor hydrogen bond interactions with weak fractions of time at k for ns time period in figure and table . the same pattern was also observed in the case of variable temperature but at k, tyr showed maximum fraction of time in figure and table . dft calculations of best five compounds have been performed and frontier molecular orbitals taken as in table . homo-lumo energy gap plays an important role in stabilizing the interactions between compound and nsp of chikv. by using energy values of homo and lumo for top five screened hit drug molecule from designed library to calculated μ, χ, η and ω by using eqs. ( ), ( ), ( ) , and ( ) . table summarizes the homo, lumo and homo-lumo energy gaps (Δe) of top five hit drug molecules calculated at dft level in the b lyp/ - g* basis set. the energy different between homo and lumo is used to understand the chemical reactivity and kinetic of molecules. if a compound has small energy gap indicates more polarizable and therefore have high chemical reactivity and termed as soft molecule. global term is a primary descriptor for the chemical reactivity of compounds. chemical hardness is a measure to study the stability of compound. it explains the resistance towards polarization of the electron cloud under small perturbation. chemical potential is a form of energy and can be absorbed or released on changing the number of the species in a chemical reaction. larger the value of electronegativity indicates more the attractiveness for electrons. electrophilicity is a measure for the energy stabilization of compound. it is used to understand the the reactivity of compounds involved in chemical reactions. in the present work, finding the promising candidate against nsp of chikv was explored via screening, docking, md simulations, mm-gbsa calculation. a library of compounds is created based on the product obtained in one pot three component reaction. then, the compounds were subjected to docking and bioactive score. further, the results of screened compounds were compared with results of the compounds based on the compounds obtained from the raspd. then, nsp of chikv with and without cmpd were studied using md simulations for ns and change in binding energy is determined by mm-gbsa method. Δg for the formation of complex was found to be - . kcal/mol. subsequently, the effect of temperature was studied for the formation of the complex between the nsp of chikv and cmpd using the md simulations. the rmsd values and fluctuation increased on increasing the temperature. therefore, it is understood that the best inhibition is observed at k by the cmpd . author contribution statement durgesh kumar: performed the experiments; analyzed and interpreted the data; wrote the paper. kamlesh kumari, prashant singh: conceived and designed the experiments; wrote the paper. rajan patel: analyzed and interpreted the data. abhilash jayaraj: performed the experiments; analyzed and interpreted the data. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. modeling dynamic introduction of chikungunya virus in the united states mapping of chikungunya virus interactions with host proteins identified nsp as a highly connected 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alpha and coronaviruses mechanism of action of flavonoids as anti-inflammatory agents: a review nano-sized copper as an efficient catalyst for one pot three component synthesis of thiazolidine- , -dione derivatives copper nanoparticles in an ionic liquid: an efficient catalyst for the synthesis of bis-( -hydroxy- -oxothiazolyl)methanes copper nanoparticles in ionic liquid: an easy and efficient catalyst for the coupling of thiazolidine- , -dione, aromatic aldehyde and ammonium acetate phosphotungstic acid: an efficient catalyst for the synthesis of bis-( -hydroxycoumarin- -yl) methanes in water covid- : emergence of infectious diseases, nanotechnology aspects, challenges and future perspectives development of a theoretical model for the inhibition of nsp protease of chikungunya virus using pyranooxazoles understanding the binding affinity of noscapines with protease of sars-cov- for covid- using md simulations at different temperatures selective docking of pyranooxazoles against nsp of 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toxicity on the basis of pass prediction pardock: an all atom energy based monte carlo docking protocol for protein-ligand complexes structural basis for modulation of a g-protein-coupled receptor by allosteric drugs molecular dynamics simulation of proteins: a brief overview a well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the resp model simmerling, ff sb: improving the accuracy of protein side chain and backbone parameters from ff sb binding free energy based structural dynamics analysis of hiv- rt rnase h-inhibitor complexes ptraj and cpptraj: software for processing and analysis of molecular dynamics trajectory data solvation free energy of biomacromolecules: parameters for a modified generalized born model consistent with the amber force field exploring protein native states and large-scale conformational changes with a modified generalized born model ligand affinities predicted with the mm/pbsa method: dependence on the simulation method and the force field absolute binding free energy calculation and design of a subnanomolar inhibitor of phosphodiesterase- theory and applications of the generalized born solvation model in macromolecular simulations results obtained with the correlation energy density functionals of becke and lee, yang and parr computational studies of global and local reactivity descriptors of some trimethyltetrathiafulvalenylthiophene molecules (tmettf-tp) by density functional theory (dft) green synthesis of tetrazines and their role as human cytomegalovirus (hcmv) protease inhibitor theoretical model to investigate the alkyl chain and anion dependent interactions of gemini surfactant with bovine serum albumin the authors declare no conflict of interest. no additional information is available for this paper. key: cord- -wgvvdmxe authors: mahmood, tahir; ali, zeeshan title: aggregation operators and vikor method based on complex q-rung orthopair uncertain linguistic informations and their applications in multi-attribute decision making date: - - journal: comp doi: . /s - - - sha: doc_id: cord_uid: wgvvdmxe complex q-rung orthopair uncertain linguistic set (cqrouls) is a combination of complex q-rung orthopair fuzzy set (cqrofs) and uncertain linguistic variable set (ulvs) as a proficient technique to express uncertain and awkward information in real decision theory. cqrouls contains uncertain linguistic variables, truth and falsity grades, which gives extensive freedom to decision makers for taking a decision as compared to cqrofs and their special cases. in this article, a new concept of fuzzy set, called cqrouls using cqrofs and ulvs is explored, and this can examine the qualitative assessment of decision makers and gives them extensive freedom in reflecting their belief about allowable truth grades. based on the established operational laws and comparison methods for cqroulss, the notions of complex q-rung orthopair uncertain linguistic weighted-averaging aggregation operator and complex q-rung orthopair uncertain linguistic weighted geometric aggregation operator are explored. some special cases and the desirable properties of the explored operators are also established and studied. additionally, the notion of visekriterijumska optimizacija i kompromisno resenje (vikor) method based on cqroulss is explored, with the help of a numerical example, it is verified and also its comparative study is established. moreover, based on the above analysis, we establish a method to solve the multi-attribute group decision making problems, in which the evaluation information is shown as cqroulns. finally, we solve some numerical examples using some decision making steps and explain the verity and proficiency of the explored operators by comparing with other methods, the advantages and graphical interpretation of the explored work are also discussed. multi-attribute decision making (madm) is a proficient technique to solve various problems in our real-decision environment. madm is used for the purpose of examining to rank the family of alternatives and to examine the best one. but due to the increase in day by day complexity and difficulty in the environment of the decision-making process, the decision-maker cannot face much longer these kind of problems, which are in the form of numerical values. to solve these kind of problems, the theory of fuzzy set was explored by zadeh ( ) , as characterized by the grade of truth belonging to the unit interval. atanassove's ( ) modified the theory of fs to explore the idea of intuitionistic fs (ifs), contains the grade of truth and the grade of falsity with the condition that is the sum of both is restricted to the unit interval. various scholars have studied and utilized it in the environment of different areas (krishankumar et al. ; seker ; zhang et al. a; demircioglu and ulukan ) . but there are various problems where the sum of the truth and falsity grades is exceeded form unit interval. for example, when a student is qualified for the ph.d. test, the group of teachers who set in the interview and indicates their grade whose in the form of yes is . , and in the form of no is . . it is clear that the sum of both is exceeded form unit interval. to address such types of problems, yager ( ) presented the pythagorean fs (pfs) with the condition that is the sum of the squares of the both is not exceeded form unit interval. the pfs has utilized in various areas. for example, the divergence measure for pfs was elaborated by zhou et al. ( ) . song et al. ( ) presented the pythagorean fuzzy analytic hierarchy process. the chebyshev distance measures for pfs was explored by chen ( ). riaz et al. ( ) examined the topsis method by using the pythagorean m-polar fuzzy soft sets. sarkar and biswas ( ) established the entropy measure, linear programing and modified technique for ideal solution by using the pfss. but these were still a problem, when a group of teaches indicates their opinion in the form of yes is . and in the form of no is . , then probably the sum of the squares of the both is exceeded form the unit interval. for addressing such types of difficulties, yager ( ) again explored the q-rung orthopair fs (qrofs) with a condition that is the sum of q-powers of the truth and falsity grades is not exceeded form unit interval. qrofs is extensive proficient technique to resolve real-decision activities. qrofs have received extensive attention form a scholars and various researchers have applied it to in various areas tang et al. ; qin et al. ; liu and huang ) . as for the above existing studies, it has been analyzed that they have investigated the madm problems under the fs, ifs, pfs, qrofs or its generalizations, which are only able to deal with the uncertainty and vagueness that exists in preferences given by the decision makers. none of these models are able to represent the partial ignorance of the data and its fluctuations at a given phase of time. however, in complex data sets, uncertainty and vagueness in the data occur concurrently with changes to the phase (periodicity) of the data. to handle these, ramot et al. ( ) presented complex fs (cfs), which is characterized by the grade of truth in the form of complex-valued, whose real and imaginary parts are belonging to unit interval. allah and salleh ( ) modified the theory of cfs is to explore the idea of complex ifs (cifs), contains the grade of truth and the grade of falsity in the form of polar coordinates with a condition that is the sum of the real part (also for the imaginary part) of the both is restricted to the unit interval. various scholars have studied and utilized it in the environment of different areas (garg and rani a, b; ngan et al. ) . but there are various problems where the sum of the real part (also for imaginary part) of the truth and the real part (also for imaginary part) of the falsity grades is exceeded form unit interval. for example, when a decision maker gives the grades for yes is . e i π ( . ) and for no is . e i π ( . ) . it is clear that the sum of the real part (also for imaginary part) of the both is exceeded form unit interval. to address such types of problems, ullah et al. ( a) presented the complex pfs (cpfs) with a condition that is the sum of the squares of the real part (also for imaginary part) of the both is not exceeded form unit interval. the cpfs have utilized in various areas (akram and naz ) . but these was still a problem, when a decision maker indicate their opinion in the form of yes is . e i π ( . ) and in the form of no is . e i π ( . ) , then probably the sum of the squares of the real part (also for imaginary part) of the both is exceeded form the unit interval. for addressing such types of difficulties, liu et al. ( a, b) explored the complex qrofs (cqrofs) with a condition that is the sum of q-powers of the real part (also for imaginary part) of the truth and the real part (also for imaginary part) of the falsity grades is not exceeded form unit interval. cqrofs is extensive proficient technique to resolve real-decision activities. it is actually complicated for a decision maker to give directly the quantitative assessment data, in various real decision problems. for addressing such kinds of complications, the theory of linguistic variable (lv) was explored by zadeh ( ) as an efficient technique to address with complicated and awkward information. further, various scholars have modified the theory of lv is to explore uncertain linguistic variable (xu ) . the theory of intuitionistic fuzzy uncertain aggregation operators was explored by liu and jin ( ) . liu et al. ( ) established the intuitionistic uncertain linguistic bonferroni mean operators. presented the intuitionistic uncertain linguistic partitioned bonferroni mean operators. lu and wei ( ) explored the pythagorean uncertain linguistic aggregation mean operators. examined the pythagorean uncertain linguistic partitioned bonferroni mean operators. the q-rung orthopair fuzzy uncertain linguistic aggregation operators was explored by liu et al. ( b) . from the above analysis it is clear that, various researchers have utilized the aggregation operators in the environment of ifs, pfs, and qrofs (wang et al. , a xing et al. a, b; ullah et al. a ullah et al. , ghorabaee et al. ; shen and wang ; jana et al. a, b; liu et al. b; zhang et al. b, c, d, e; zhan et al. a, b; jiang et al. ) to evaluate the ambiguities which occurred in the problem of madm. but there is still a problem when a decision-maker provides the information in the form of groups and say to find the best one, it is very difficult to find the relation between them especially when it is in the form of two-dimensional information in a single set. for instance, when a decision maker gives . e i π ( . ) for complex-valued truth grade, . e i π ( . ) for complex-valued falsity grade, and Ṡ ,Ṡ for uncertain linguistic term, then the existing notions like ifs, pfs, qrofs, cifs, cpfs, cqrofs, and their extensions. for handling such kinds of problems, the aims of this manuscript are summarized as follows: . to present the new cqrouls and their special properties. . the aggregation operators called averaging and geometric aggregation operators based on cqroulss are explored and also studied with their important properties. . moreover, based on the above analysis, we establish a method to solve the multi-attribute group decision making problems, in which the evaluation information is shown as cqroulns. . to explore vikor method based on novel cqroulns and compare with some other methods and to examine the reliability and effectiveness of the explored methods. . finally, we solve some numerical examples using some decision making steps and explain the verity and proficiency of the explored operators by comparing with other methods; the advantages and graphical interpretation of the explored work are also discussed. the purpose of this article is summarized in the following ways: in sect. , we review the cqrofss, and the notion of uncertain linguistic variable set (ulvs) and their basic properties. in sect. , the novel approach of cqrouls is explored, which is the combination of cqrofs and ulvs is a proficient technique to express uncertain and awkward information in real decision theory. cqrouls contains uncertain linguistic variable, truth and falsity grades, which gives extensive freedom to a decision makers for taking a decision is compared to cqrofs and their special cases. in this article, a new concept of fuzzy set is called cqrouls using cqrofs and ulvs is explored, and this can examine the qualitative assessment of decision makers and gives them extensive freedom in reflecting their belief about allowable truth grades. in sect. , based on the established operational laws and comparison methods for cqroulss, the notions of complex q-rung orthopair uncertain linguistic weighted averaging aggregation operator and complex q-rung orthopair uncertain linguistic weighted geometric aggregation operator are explored. some special cases and the desirable properties of the explored operators are also established and studied. additionally, the vikor method based on cqroulss are also explored and verified it with the help of numerical example. in sect. , based on the above analysis, we establish a method to solve the multi-attribute group decision making problems, in which the evaluation information is shown as cqroulns. finally, we solve some numerical examples using some decision making steps and explain the verity and proficiency of the explored operators by comparing with other methods; the advantages and graphical interpretation of the explored work are also discussed. for better understanding the established work in the next section, we concisely review some useful notions of cqrofs (liu et al. a, b; zadeh ) and their operational laws. the notion of linguistic term set and uncertain linguistic term set are also discussed. further, the symbols x u , μ, and η are represented by the universal grade of truth, and the grade of falsity. where q sc , δ sc ≥ . definition (liu et al. a (liu et al. , a zadeh ) a cqrofs is of the form: definition (liu et al. a (liu et al. , a zadeh ) for any two cqrofns definition (liu et al. a (liu et al. , a zadeh ) for any two cqrofns , the score and accuracy function is given by: based on the above two notions, the compassion between two cqrofns is given by: definition (xu ) for a linguistic term setṠ Ṡ j / j , , , . . . , z − with odd cardinality, where, z is the cardinality ofṠ, andṠ j is a linguistic variable. a possible linguistic term set is given by: {Ṡ ,Ṡ ,Ṡ ,Ṡ ,Ṡ ,Ṡ ,Ṡ } {ver y poor , poor, slightly poor, f air, slightly good, good, ver y good} by using the values of z , then z − . the linguistic terms are expressed by pythagorean fuzzy sets for five and seven terms. further, to improve the quality of the proposed work, in this study, we present the novel approach of cqrouls and their fundamental operational laws. basically, the cqrouls is a mixture of cqrofs and uls to cope with unpredictable and unreliable information in our day to day life. based on the existing notion which is discussed in sect. , the explored approaches are follow as: is called refusal grade, the complex q-rung orthopair uncertain linguistic number (cqrofn) or complex q-rung orthopair uncertain linguistic variable (cqroulv) is represented by . , then . , the expectation and accuracy function is given by: based on the above two notions, the compassion between two cqroulns is given by: . if s · q c qu l− s · q c qu l− , then: proof straightforward. to improve the quality of the proposed work, in this study, we present the aggregation operators using the cqrouls and also study their special cases. basically, we explored the averaging and geometric aggregation and with their weight vector for cqrouls. based on the established notions which are discussed in sect. , the explored operators are follow as: definition for a collection cqroulns , j , , . . . , n, the cqroulwa operator is given by: where ω w ω w− , ω w− , . . . , ω w−n t denotes the weight vectors with a condition n j ω w− j . , j , , . . . , n, the aggregated value of the eq. ( ) is again a cqrouln, we have proof by using the method of induction, we prove eq. ( ); the steps of the induction is as follows: . for choosing the value of parameter n , then eq. ( ) is hold true. . for choosing the value of parameter n and using def. ( ), we have the eq. ( ) is truly holds for n . further we check for n k moreover, we have to check for n k + , then the result has been proved. further, we evaluate some properties for cqroulns like idempotency, monotonicity and boundedness. proof suppose q c qu l− j q c qu l , then by using eq. ( ), we have the result has been proved. the imaginary part of the complex-valued truth grade is same. next, we prove the real part of the complex-valued falsity grade hence the expectation values of the . so by using the above properties, we have get the result, such that the result has been proved. , then by using the theorem and theorem , such that the result has been proved. definition for a collection cqroulns , j , , . . . , n, the cqroulwg operator is given by: where ω w ω w− , ω w− , . . . , ω w−n t denotes the weight vectors with a condition n j ω w− j . , j , , . . . , n, the aggregated value of the eq. ( ) is again a cqrouln, we have proof straightforward. (similar to theorem ). further, we evaluate some properties for cqroulns like idempotency, monotonicity and boundedness. straightforward. (similar to theorem ). proof straightforward. (similar to theorem ). proof straightforward. (similar to theorem ). remark if we choose the values of imaginary part is zero in eq. ( ), then the eq. ( ) is reduced for q-rung orthopair uncertain linguistic sets. similarly, if we choose the values of q sc in eq. ( ), then the eq. ( ) is reduced for complex pythagorean uncertain linguistic sets and if we choose the values of q sc in eq. ( ), then the eq. ( ) is reduced for complex intuitionistic uncertain linguistic sets. the vikor approach, pioneered for multi-attribute optimization problems, concentrate on ranking the alternatives and considered a compromise solution. the decision making problem, which can be solve by vikor, is express as follows. considered the m alternatives and n attributes x , x , . . . , x m and˜ a ,˜ a , . . . ,˜ a n with respect to weight vectors such that w (w , w , . . . , w n ) h , n j w i , the compromise ranking by vikor methods is started with the form of l p -metric (he et al. ) . in the vikor method, the maximum group utility can be gotten by mins i and minimum individual regret can be gotten by mins i , where s i l , i , and s i l ∞, i . the steps of the vikor method is follow as: step : computing the virtual positive ideal x * j and the virtual negative ideal x − j values under the attributes˜ a j , we have step : computing the values of group utility s i and s i , we have step : computing the values of q i , i , , . . . , m, we have where s * min i (s i ), s − max i (s i ), s * min i s i and s − max i s i , the symbol v is the balance parameter which can balance the group of utility and individual regret. there are three possibilities: . if v > . represents the maximum group utility is more than minimum individual regret. . if v < . represents the minimum individual regret is more than maximum group utility. . if v . represents the maximum group utility and minimum individual regret are same importance. step : using the values of s, s and q and ranking the alternatives, then we will obtain the compromise solution. step : when we get the compromise solution x ( ) in steps , then it satisfied the following two conditions. condition : acceptable advantages: q x ( ) − q x ( ) ≥ m− , where q x ( ) is the q value in the second position of all ranking alternatives produced by the value of q and m number of alternatives. condition : acceptable stability: alternative x ( ) must also in the first position of all ranking alternatives produced by the values of s or s . if one of the above condition is not met, then we collected the compromise alternatives and not one compromise solution. . if condition is not hold, then we will examine the alternatives x ( ) and x ( ) should be compromise solution. . if condition is not hold, then the maximum m eximane by the formula q x (m) − q x ( ) < m q m− , we examined the alternatives x ( ) , x ( ) , . . . , x (m) are compromise solution. based on the above analysis, we will construct the vikor method for cqroulss. ⎠ , the aim of vikor method is follow as: step : normalize the decision matrix, there are two types of attribute such as benefits b and cost c types attributes, the normalized can be done by the following formula; step : computing the virtual positive ideal x * k and the virtual negative ideal x − k values under the attributes˜ a j , we have step : computing the values of group utility s i and s i , we have where r i , r j represents the distance between two cqroulns, which is defined as: step : computing the values of q i , i , , . . . , m, we have the symbol v is the balance parameter which can balance the group of utility and individual regret. there are three possibility: . if v > . represents the maximum group utility is more than minimum individual regret. . if v < . represents the minimum individual regret is more than maximum group utility. . if v . represents the maximum group utility and minimum individual regret are same importance. step : using the values of s, s and q and ranking the alternatives, then we will obtain the compromise solution. step : when we get the compromise solution x ( ) in steps , then it satisfied the following two conditions. condition : acceptable advantages: is the q value in the second position of all ranking alternatives produced by the value of q and m number of alternatives. condition : acceptable stability: alternative x ( ) must also in the first position of all ranking alternatives produced by the values of s or s . we take the method from ref. which is a invest selection problem (table ). the investment company want to in with one of the following company is denoted by x i (i , , , ) and measured by four attributes, whose detail is discussed in table . the attributes in table , is the form of cqroulns, with weight vectors w ( . , . , . , . ). based on vikor method, we solve the following matrix (table ) . step : we normalize the table using the eqs. ( ) and ( ), then the new decision matrix is follow as: step : we computing the virtual positive ideal x * j and the virtual negative ideal x − j values under the attributes˜ a j using the eq. ( ), then step : we compute the values of group utility s i and s i using the eqs. ( ), ( ) and ( ), if we ignoring the values of uncertain linguistic terms, then step : we compute the values of q i , i , , . . . , m using the eq. ( ), then (tables , ) step : using the values of s, s and q and ranking the alternatives, then we will obtain the compromise solution see table . step : we obtain the compromise results using the condition and condition , such that q(x ) , and the second position is q(x ) . , then m d m− − . , so q(x ) − q(x ) . > . which holds the conditions q(x ) − q(x ) ≥ − , but the alternative x is the best ranked by s and s , which holds the condition . by calculating, we get so, the condition holds accurately, therefore by condition , x , x , x and x are the compromise solutions. when condition cannot hold, we used condition and solved the problems. the comparison between the proposed methods and existing methods for numerical example ( ), are discussed in table . based on vikor methods for existing and proposed approach, the best alternative is˜ a . the methods introduced in this manuscript express a wider range of fuzzy information, and they can ask for the sum of square of real part (also for imaginary) of membership degree and the sum of square of real part (also for imaginary) of non-membership degree is table decision matrix for complex intuitionistic uncertain linguistic numbers normalized decision matrix table ranking results of the table symbols compromise solution x , x , x , x greater than one. our proposed methods are more general and more effective. because the vikor methods for ciuls and cpuls are all the special case of the vikor methods for cqrouls. when parameter q the vikor methods for cqrouls reduces the vikor methods for ciuls. when parameter q the vikor methods for cqrouls reduces the vikor methods for cpuls. besides, our approach is more flexible, and decision makers can choose different values of parameter q according to the different risk attitudes. according to the comparisons and analysis above, the vikor methods based on cqrouls proposed in this paper are better than the existing other methods for aggregating the complex intuitionistic uncertain linguistic information and complex pythagorean uncertain linguistic information. therefore, they are more suitable to solve the difficult and complicated problems. the purpose of this communication is to explore the madm problem by using the averaging and geometric aggregation operators based on cqroulss, to improve the quality of the explore approach. based on the above analysis, we consider the family of the alternatives and the family of attributes, whose representations are stated as: ( j , , , .., m) and their attributes c at −k (k , , , . . . , n) ⎞ ⎠ ⎞ ⎠ , l , , , .., l, whose related information is given in sect. . the steps of the madm problem based on cqroulns are follow as: step : by using the cqroulns is to construct the decision matrix r l dm r l jk m×n , and then normalized it with the help of two methods which are discussed below: . when the values of attributes c at −k , k , , . . . , m in the form of benefit kinds, then . when the values of attributes c at −k , k , , . . . , m in the form of cost kinds, then step : to integrate the decision matrix, by using the cqroulwa operator or cqroulwg operator, which is explored below: step : with the aggregated values in step , we examine the expectation values by using the eq. ( ). step : the expectation values, which we obtained in the step , rank to all the alternatives and find the best one. step : the end. to examine the people of which city is more effected form coronavirus disease in the duration of lockdown. to resolve the issues of security, which city is more securable form covid- and which is in the dangerous zone and it may be passable the effected people form covid- is increases day by day in the duration of lockdown. for this mission the pakistan telecom authority (pta) gives the responsibility of the following four cities which is possible to effect the people of that areas form covid- , whose representation is follow as: a al− : islamabad areas; a al− : rawalpindi areas; a al− : karachi areas; a al− : gilgit areas. the information of the effected people form covid- of four cities is collected by the four attributes, whose representation is follow as: c at − : food distribution during at lockdown; c at − : water supply during at lockdown; c at − : electric supply during at lockdown; c at − : money distribution during at lockdown; for evaluating these types of problems, we consider the weight vector, whose information is of the form ω w ( . , . , . , . ) t , with a condition n j ω w− j . additionally, the linguistic terms set is stated byṠ Ṡ ,Ṡ ,Ṡ ,Ṡ ,Ṡ ,Ṡ . the examining information is considered in the form of cqroulvs, whose expression is follows: the complex q-rung orthopair uncertain linguistic decision information is available in table . the steps of the madm problem based on cqroulns are follow as: step : by using the cqroulns is to construct the decision matrix r l dm r l jk m×n , whose information are available in table . further, we normalized the decision matrix with the help of two methods which are discussed in eqs. ( ) and ( ), the information of the normalized decision matrix is discussed in table , which is follow as: step : to integrate the decision matrix, by using the eqs. ( ) and ( ) based on the cqroulwa operator or cqroulwg operator, the aggregated values are discussed below: step : the aggregated values in step , we examine the expectation values by using the eq. ( ), which is follow as: step : the expectation values, which we are obtains in the step , rank to all the alternatives and we find the best one, which is follow as: form the above discussion, we obtain the result the city islamabad and rawalpindi are more effected form corona various diseases , which is a al− and a al− , by using weighted averaging and weighted geometric aggregation operators, which is islamabad and rawalpindi areas. it is required for government of pakistan to supply the necessities' of the people in the duration of lockdown and strictly say to the people of the effected city stay at home to save our life (table ) . step : the end. table decision matrix, whose information is in the form of complex intuitionistic uncertain linguistic numbers normalized decision matrix, whose information is also in the form of complex intuitionistic uncertain linguistic numbers fig. graphical representation using the information of table the comparison of the elaborated approach in this manuscript are examined with the help of some existing operators are discussed in table , whose information is discussed in table , which is stated below. form the above discussion, we obtain the result the cities which is more effected form corona various diseases are a al− and a al− , by using weighted averaging and weighted geometric aggregation operators, which is islamabad and rawalpindi areas. it is required for government of pakistan to supply the necessities' of the people in the duration of lockdown and strictly say to the people of the effected city stay at home to save our life. the graphical interpretation of the information, which is discussed in table , are understand with the help of fig. . the cqrouls is a mixture of cqrofs and ulvs is a proficient technique to express uncertain and awkward information in real decision theory is explored. the advantage of the proposed work for q (complex pythagorean uncertain linguistic information) proposed work for q (complex q-rung orthopair uncertain linguistic information) graphical representation using the information of table cqruls is that it contains the uncertain linguistic variable, truth and falsity grades with a conditions that is the sum of q-power of the real parts (also for imaginary parts) of the truth and falsity grades are not exceeded from unit interval. further, to explore the proficiency and validity of the established operators based on the novel cqroulvs, we choose some existing operators based on intuitionistic uncertain linguistic variables (liu and jin ) , pythagorean uncertain linguistic variables (lu and wei ) , q-rung orthopair uncertain linguistic variables (liu et al. b) , complex intuitionistic uncertain linguistic variables (special case of the explored operators), complex pythagorean uncertain linguistic variables (special case of the explored operators), and complex q-rung orthopair uncertain linguistic variables. further, we choose the complex pythagorean uncertain linguistic information, which is discussed in table , and solve it by using some existing methods (liu and jin ; lu and wei ; liu et al. b ). the aggregated values of the normalized decision matrix, whose information is given in table , are discussed in table . the comparison of the elaborated approach in this manuscript are examined with the help of some existing operators are discussed in table , whose information is discussed in table , which is stated below. form the above discussion, we obtain the result the cities which is more effected form corona various diseases are a al− , by using weighted averaging and weighted geometric aggregation operators, which is rawalpindi areas. it is required for government of pakistan to supply the necessities' of the people in the duration of lockdown and strictly say to the people of the effected city stay at home to save our life. the graphical interpretation of the information, which is discussed in table , are understand with the help of fig. . further, we choose the complex q-rung orthopair uncertain linguistic information, which is discussed in table , and solve it by using some existing methods (liu and jin ; lu and wei ; liu et al. b) . the aggregated values of the normalized decision matrix for q sc , whose information is given in table , are discussed in table . the comparison of the elaborated approach in this manuscript are examined with the help of some existing operators are discussed in table , whose information is discussed in table , which is stated below form the above discussion, we obtain the result the cities which is more effected form corona various diseases are a al− , by using weighted averaging and weighted geometric aggregation operators, which is islamabad areas. it is required for government of pakistan to supply the necessities' of the people in the duration of lockdown and strictly say to the people of the effected city stay at home to save our life. the graphical interpretation of the table decision matrix, whose information is in the form of complex pythagorean uncertain linguistic numbers proposed work for q (complex q-rung orthopair uncertain linguistic information) decision matrix, whose information is in the form of complex q-rung orthopair uncertain linguistic numbers averaging fig. graphical representation using the information of table information, which is discussed in table , are understood with the help of fig. . for further improvement of this manuscript, we consider the example from ref. (liu and jin ) , and solve it by using the established operator and existing operators, whose discussion is explained below. example this example is taken form ref. (liu and jin ) , example . the authors have chosen four alternatives and four attributes and their weight vectors is follow as: ω w ( . , . , . , . ) t . for these information, the decision matrix which is taken form ref. (liu and jin ) is discussed below. the authors chose the intuitionistic uncertain linguistic information, which is discussed in table , and solved it by using some existing methods (liu and jin ; lu and wei ; liu et al. b ). form the above analysis, its clear that e i π ( . ) e , then with the information of the table , we converted it to the information of the table , which is in the form of polar co-ordinates. the aggregated values of the normalized decision matrix, whose information is given in table , are discussed in table . the comparison of the elaborated approach in this manuscript is examined with the help of some existing operators as discussed in table , whose information is discussed in table , which is stated below. form the above discussion, we obtained the result in the cities which is more affected by covid- are a al− and a al− , by using weighted-averaging and weighted-geometric aggregation operators, which is islamabad and rawalpindi areas. it is required by the government of pakistan to supply the necessities' of the people in the duration of lockdown and strictly inform the people of the affected city to stay at home to save their lives. the graphical interpretation of the information, which is discussed in table , can be understood with the help of fig. . from the above discussions, we get the result; our established approach is more refillable and extensive consistence then existing methods (liu and jin ; lu and wei ; liu et al. b ), due to its constraints. therefore, the established approaches in this manuscript are more reliable and more efficient then cifs and cpfs to cope with uncertain and awkward information in realistic decision theory. decision matrix, whose information is in the form of intuitionistic uncertain linguistic numbers table decision matrix, whose information is in the form of complex intuitionistic uncertain linguistic numbers fig. graphical representation using the information of table conclusion various theories have developed in the environment of fuzzy sets. but, one of the most important theory is not explored till date, which is effectively dealing with some issues, no notions deal with such kinds of issues. for instance, when a decision maker gives the complex q-rung orthopair uncertain linguistic types of information. for coping such kinds of issues, in this paper, the theory of complex q-rung orthopair uncertain linguistic set (cqrouls) is a combination of complex q-rung orthopair fuzzy set (cqrofs) and uncertain linguistic variable set (ulvs) is a proficient technique to express uncertain and awkward information in real decision theory is explored. cqrouls contains uncertain linguistic variable, truth, and falsity grades, which gives extensive freedom to a decision makers for taking a decision is compared to cqrofs and their special cases. cqrouls can examine the qualitative assessment of decision makers and gives them extensive freedom in reflecting their belief about allowable truth grades. based on the established operational laws and comparison methods for cqroulss, the notions of complex q-rung orthopair uncertain linguistic weighted averaging aggregation operator and complex q-rung orthopair uncertain linguistic weighted geometric aggregation operator are explored. some special cases and the desirable properties of the explored operators are also established and studied. additionally, the vikor method based on cqroulss are also explored and verified it with the help of numerical example. moreover, based on the above analysis, we establish a method to solve the multi-attribute group decision making problems, in which the evaluation information is shown as cqroulns. finally, we solve some numerical examples using some decision making steps and explain the verity and proficiency of the explored operators by comparing with other methods, the advantages and graphical interpretation of the explored work are also discussed. in the future, we will evaluate some more 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pythagorean fuzzy sets based on belief function and its application in medical diagnosis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations , . e i π key: cord- -q sx dm authors: cacabelos, ramón title: pharmacogenomic biomarkers in neuropsychiatry: the path to personalized medicine in mental disorders date: journal: the handbook of neuropsychiatric biomarkers, endophenotypes and genes doi: . / - - - - _ sha: doc_id: cord_uid: q sx dm neuropsychiatric disorders and dementia represent a major cause of disability and high cost in developed societies. most disorders of the central nervous system (cns) share some common features, such as a genomic background in which hundreds of genes might be involved, genome-environment interactions, complex pathogenic pathways, poor therapeutic outcomes, and chronic disability. recent advances in genomic medicine can contribute to accelerate our understanding on the pathogenesis of cns disorders, improve diagnostic accuracy with the introduction of novel biomarkers, and personalize therapeutics with the incorporation of pharmacogenetic and pharmacogenomic procedures to drug development and clinical practice. the pharmacological treatment of cns disorders, in general, accounts for – % of direct costs, and less than – % of the patients are moderate responders to conventional drugs, some of which may cause important adverse drugs reactions (adrs). pharmacogenetic and pharmacogenomic factors may account for – % of drug variability in drug disposition and pharmacodynamics. approximately – % of cns drugs are metabolized via enzymes of the cyp gene superfamily; % of neuroleptics are major substrates of cyp a enzymes, % of cyp d , and % of cyp a ; % of antidepressants are major substrates of cyp a enzymes, % of cyp b , % of cyp c , % of cyp d , and % of cyp a ; % of benzodiazepines are major substrates of cyp c enzymes, % of cyp d , and % of cyp a . about – % of caucasians are carriers of defective cyp d polymorphic variants that alter the metabolism of many psychotropic agents. other genes participate in the efficacy and safety of psychotropic drugs. the incorporation of pharmacogenetic/ pharmacogenomic protocols to cns research and clinical practice can foster therapeutics optimization by helping to develop cost-effective pharmaceuticals and improving drug efficacy and safety. to achieve this goal several measures have to be taken, including: (a) educate physicians and the public on the use of genetic/ genomic screening in the daily clinical practice; (b) standardize genetic testing for major categories of drugs; (c) validate pharmacogenetic and pharmacogenomic procedures according to drug category and pathology; (d) regulate ethical, social, and economic issues; and (e) incorporate pharmacogenetic and pharmacogenomic procedures to both drugs in development and drugs in the market to optimize therapeutics. central nervous system (cns) disorders are the third problem of health in developed countries, representing - % of deaths, after cardiovascular disorders ( - %) and cancer ( - %) . approximately, million europeans suffer brain disorders. the total annual cost of brain disorders in europe is about € billion, with € billion of direct medical expenditures (€ billion, inpatients; € billion, outpatients; € billion, pharmacological treatment), € billion of indirect costs (lost workdays, productivity loss, permanent disability), and € billion of direct non-medical costs. mental disorders represent € billion ( % of the total cost, excluding dementia), followed by neurological diseases (€ billion, %). senile dementia is becoming a major problem of health in developed countries, and the primary cause of disability in the elderly. alzheimer's disease (ad) is the most frequent form of dementia ( - %), followed by vascular dementia ( - %) , and mixed dementia ( - %) . these prevalent forms of agerelated neurodegeneration affect more than million people at present, and probably more than million people will be at risk in the next - years worldwide. the prevalence of dementia increases exponentially from approximately % at - years of age to more than - % in people older than years. it is very likely that in those patients older than - years of age most cases of dementia are mixed in nature (degenerative + vascular), whereas pure ad cases are very rare after years of age. the average annual cost per person with dementia ranges from € , to , , depending upon disease stage and country, with a lifetime cost per patient of more than € , . in some countries, approximately % of the global costs of dementia (direct + indirect costs) are assumed by the patients and/or their families. about - % of the costs in dementia are attributed to pharmacological treatment, including anti-dementia drugs, psychotropics (antidepressants, neuroleptics, anxiolytics), and other drugs currently prescribed in the elderly (antiparkinsonians, anticonvulsants, vasoactive compounds, antiinfl ammatory drugs, etc). in addition, during the past years more than drugs have been partially or totally developed for ad, with the subsequent costs for the pharmaceutical industry, and only drugs with moderate-to-poor effi cacy and questionable cost-effectiveness have been approved in developed countries. [ ] [ ] [ ] the lack of accurate diagnostic markers for early prediction and an effective therapy of cns disorders are the two most important problems to effi ciently diagnose and halt disease progression. the pharmacological treatment of cns disorders, in general, accounts for - % of direct costs, and less than - % of the patients are moderate responders to conventional drugs, some of which may cause important adverse drugs reactions (adrs). in the case of dementia, less than % of the patients can benefi t from current drugs (donepezil, rivastigmine, galantamine, memantine), with doubtful cost-effectiveness. the pathogenic mechanisms of most cns disorders (e.g., psychosis, depression, anxiety, alzheimer's disease, parkinson's disease, huntington's disease, multiple sclerosis, etc) are poorly understood. this circumstance makes it diffi cult the implantation of a molecular intervention to neutralize causative factors. in fact, more than % of the , genes integrating the human genome are expressed in the cns at different periods of the life span, and only a few neurotransmitters (e.g., noradrenaline, dopamine, acetylcholine, gaba, histamine, and less than ten neuropeptides) are the actual targets of conventional psychopharmacology. common features in cns disorders include the following: (a) polygenic/ complex disorders in which genomic and environmental factors are involved; (b) deterioration of higher activities of the cns; (c) multifactorial dysfunctions in several brain circuits; and (d) accumulation of toxic proteins in the nervous tissue in cases of neurodegeneration. for instance, the neuropathological hallmark of alzheimer's disease (ad) (amyloid deposition in senile plaques, neurofi brillary tangle formation, and neuronal loss) is but the phenotypic expression of a pathogenic process in which more than genes and their products are potentially involved. drug metabolism, and the mechanisms underlying drug effi cacy and safety, are also genetically regulated complex traits in which hundreds of genes cooperatively participate. structural and functional genomics studies demonstrate that genomic factors, probably induced by environmental factors, cerebrovascular dysfunction, and epigenetic phenomena, might be responsible for pathogenic events leading to premature neuronal dysfunction and/or death. pharmacogenetic and pharmacogenomic factors may account for - % of drug variability in drug disposition and pharmacodynamics. about - % of caucasians are carriers of defective cyp d polymorphic variants that alter the metabolism of many psychotropic agents. the incorporation of pharmacogenetic/pharmacogenomic protocols to cns research and clinical practice can foster therapeutics optimization by helping to develop cost-effective pharmaceuticals and improving drug effi cacy and safety. [ ] [ ] [ ] extensive molecular genetics studies carried out in the past decades have demonstrated that most cns disorders are multifactorial, polygenic/complex disorders in which hundreds of genes distributed across the human genome might be involved (tables . - . ). , for example, genes have been associated with dementia (table . ), with schizophrenia (table . ), with depression (table . ), with anxiety, with stroke, with different types of ataxia, with epilepsiy, with meningioma, with glioblastoma, with astrocytoma, with parkinson's disease, and more than genes with cerebrovascular disorders. , many of these genetic associations could not be replicated in different settings and different populations due to many complex (methodological, technological) factors. , , furthermore, the same genomic defect can give rise to apparent diverse phenotypes, and different genomic defects can converge in an apparently common phenotype, this increasing the complexity of genomic studies (e.g., patient recruitment, pure controls, concomitant pathology, epigenetic factors, environmental factors). several candidate genes for schizophrenia may also be associated with bipolar disorder, including g , disc , nrg , rgs , ncam , dao, grm , grm , grin b, mlc , syngr , and slc a . genes associated with bipolar disorder include trpm ( q . ), gpr (xq ), citron ( q ), chp . ( p . ), gchi ( q - ), mlc ( q ), gabra ( q -q ), bcr ( q ), cux , flj ( q -q ), and napg ( p ). another paradigmatic example of heterogeneity and complexity is dementia, one of the most heterogeneous disorders of the cns. the genetic defects identifi ed in ad during the past years can be classifi ed into three main categories: (a) mendelian or mutational defects in genes directly linked to ad, including (i) mutations in the amyloid beta (aβ)(abp) precursor protein (app) gene ( q ); (ii) mutations in the presenilin (ps ) gene ( q . ); and (iii) mutations in the presenilin (ps ) gene ( q -q ) , , (table . ). (b) multiple polymorphic variants of risk characterized in more than different genes distributed across the human genome can increase neuronal vulnerability to premature death (table . ). among these genes of susceptibility, the apolipoprotein e (apoe) gene ( q . ) is the most prevalent as a risk factor for ad, especially in those subjects harbouring the apoe- allele, whereas carriers of the apoe- allele might be protected against dementia. apoe-related pathogenic mechanisms are also associated with brain aging and with the neuropathological hallmarks of ad. (c) diverse mutations located in mitochondrial dna (mtdna) through heteroplasmic transmission can infl uence aging and oxidative stress conditions, conferring phenotypic heterogeneity. , , it is also likely that defective functions of genes associated with longevity may infl uence premature neuronal survival, since neurons are potential pacemakers defi ning life span in mammals. all these genetic factors may interact in still unknown genetic networks leading to a cascade of pathogenic events characterized by abnormal protein processing and misfolding with subsequent accumulation of abnormal proteins (conformational changes), ubiquitin-proteasome system dysfunction, excitotoxic reactions, oxidative and nitrosative stress, mitochondrial injury, synaptic failure, altered metal homeostasis, dysfunction of axonal and dendritic transport, and chaperone misoperation , - ( fig. . ). these pathogenic events may exert an additive effect, converging in fi nal pathways leading to premature neuronal death. some of these mechanisms are common to several neurodegenerative disorders which differ depending upon the gene(s) affected and the involvement of specifi c genetic networks, together with cerebrovascular factors, epigenetic factors (dna methylation) and environmental conditions (nutrition, toxicity, social factors, etc). , [ ] [ ] [ ] [ ] [ ] [ ] [ ] the higher the number of genes involved in ad pathogenesis, the earlier the onset of the disease, the faster its clinical course, and the poorer its therapeutic outcome. , [ ] [ ] [ ] [ ] [ ] high throughput microarray gene expression profi ling is an effective approach for the identifi cation of candidate genes and associated molecular pathways implicated in a wide variety of biological processes or disease states. the cellular complexity of the cns (with different cell types) and synapses (with each of the neurons in the brain having around - synapses with a complex multiprotein structure integrated by different proteins) requires a very powerful technology for gene expression profi ling, which is still in the very early stages and is not devoid of technical obstacles and limitations. transcripts of , genes have been measured in different cns regions. each region possess its own unique transcriptome fi ngerprint that is independent of age, gender and energy intake. less than % of genes are affected by age, diet or gender, with most of these changes occurring between middle and old age. gender and energy restriction have robust infl uences on the hippocampal transcriptome of middle-aged animals. prominent functional groups of age-and energy-sensitive genes are those encoding proteins involved in dna damage responses, mitochondrial and proteasome functions, cell fate determination and synaptic vesicle traffi cking. the systematic transcriptome dataset provides a window into mechanisms of neuropathogenesis and cns vulnerability. with the advent of modern genomic technologies, new loci have been associated with different neuropsychiatric disorders, and novel pathogenic mechanisms have been postulated. cryptic chromosome imbalances are increasingly acknowledged as a cause for mental retardation and learning disability. with subtelomeric screening, nine chromosomal anomalies and submicroscopic deletions of pter, qter, pter, qter and qter have been identifi ed in patients with mental retardation. increased dna fragmentation was observed in non-gabaergic neurons in bipolar disorder, suggesting that non-gabaergic cell may be selectively vulnerable to oxidative stress and apoptosis in patients with bipolar disorder. [ ] [ ] [ ] [ ] with laser microdissection, rna amplifi cation, and array hybridization, expression of more than , genes was detected in ca and ca hippocampal neurons under normoxic conditions. the comparison of each region under normoxic and ischemic conditions revealed more than , ischemia-regulated genes for each individual cell type. microarray technology has helped to elucidate gene expression profi les and potential pathogenic mechanisms in many other cns disorders including schizophrenia and bipolar disorder, [ ] [ ] [ ] speech and language disorders, parkinson's disease, , huntington's disease, prion disease, drug addiction, , alcoholism, brain trauma, epilepsy, [ ] [ ] [ ] cockayne syndrome, rett syndrome, friedreich ataxia, neuronal ceroid lipofuscinosis, multiple sclerosis, amyotrophic lateral esclerosis, acute pneumococcal meningitis, and the role of lipids in brain injury, psychiatric disorders, and neurodegenerative diseases. [ ] [ ] [ ] interactions between genomic factors and environmental factors have been proposed as important contributors for brain neuropathology. in schizophrenia, neurodevelopmental disturbances, neurotoxins and perinatal infections, myelin-and olygodendrocytes abnormalities and synaptic dysfunctions have been suggested as pathophysiological factors. individual genotoxicants can induce distinct gene expression signatures. exposure of the brain to environmental agents during critical periods of neuronal development can alter neuronal viability and differentiation, global gene expression, stress and immune response, and signal transduction. the binomial genome-neurotoxicants effect can be documented in cases of drug abuse or alcohol dependence. functional gene expression differences between inbred alcohol-preferring and nonpreferring rats suggest the presence of powerful genomic infl uences on alcohol dependence. alcohol dependence and associated cognitive impairment may result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. it has been suggested that cycles of alcohol intoxication/withdrawal, which may initially activate nuclear factor-kappa b (nf-κb), when repeated over years downregulate p (rela) mrna expression and nf-κb and p homodimer dna-binding. downregulation of the dominant p homodimer, a potent inhibitor of gene transcription apparently results in depression of κb regulated genes. alterations in expression of p homodimer/nf-κb regulated genes may contribute to neuroplastic adaptation underlying alcoholism. gene expression profi ling of the nucleus accumbens of cocaine abusers suggests a dysregulation of myelin. humans who abused cocaine, cannabis and/or phencyclidine share a decrease in transcription of calmodulin-related genes and increased transcription related to lipid/cholesterol and golgi/er function. another important issue in the pathogenesis and therapeutics of cns disorders is the role of micror-nas (mirnas). mirnas are small ( nucleotide), endogenous noncoding rna molecules that posttranscriptionally regulate expression of protein-coding genes. computational predictions estimate that the vertebrate genomes may contain up to , mirna genes. mirnas are generated from long primary transcripts that are processed in multiple steps to cytoplasmic nucleotide mature mirnas. the mature mirna is incorporated into the mirna-induced silencing complex (mirisc), which guides it to target sequences located in ′ utrs where by incomplete base-pairing induce mrna destabilization or translational repression of the target genes. an inventory of mirna expression profi les from regions of the mouse cns has been reported. this inventory of cns mirna profi les provides an important step toward further elucidation of mirna function and mirna-related gene regulatory networks in the mammalian cns. the introduction of novel procedures into an integral genomic medicine protocol for cns disorders is an imperative requirement in drug development and in the clinical practice to improve diagnostic accuracy and to optimize therapeutics. this kind of protocol should integrate the following components: (i) clinical history, (ii) laboratory tests, (iii) neuropsychological assessment, (iv) cardiovascular evaluation, (v) conventional x-ray technology, (vi) structural neuroimaging, (vii) functional neuroimaging, (viii) computerized brain electrophysiology, (ix) cerebrovascular evaluation, (x) structural genomics, (xi) functional genomics, (xii) pharmacogenetics, (xiii) pharmacogenomics, (ix) nutrigenetics, (x) nutrigenomics, (xi) bioinformatics for data management, and (xii) artifi cial intelligence procedures for diagnostic assignments and probabilistic therapeutic options (table . ). , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , all these procedures, under personalized strategies adapted to the complexity of each case, are essential to depict a clinical profi le based on specifi c biomarkers correlating with individual genomic profi les. functional genomics studies have demonstrated the infl uence of many genes on cns pathogenesis and phenotype expression (tables . - . ). taking ad as an example, it has been demonstrated that mutations in the app, ps , ps , and mapt genes give rise to wellcharacterized differential neuropathological and clinical phenotypes of dementia. the analysis of genotypephenotype correlations has also revealed that the presence of the apoe- allele in ad, in conjunction with other genes, infl uences disease onset, brain atrophy, cerebrovascular perfusion, blood pressure, β-amyloid deposition, apoe secretion, lipid metabolism, brain bioelectrical activity, cognition, apoptosis, and treatment outcome. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] the characterization of phenotypic profi les according to age, cognitive performance (mmse and adas-cog score), serum apoe levels, serum lipid levels including cholesterol (cho), hdl-cho, ldl-cho, vldl-cho, and triglyceride (tg) levels, as well as serum nitric oxide (no), β-amyloid, and histamine levels, reveals sex-related differences in % of the biological parameters and almost no differences ( . %) when patients are classifi ed as apoe- (−) and apoe- (+) carriers, probably indicating that genderrelated factors may infl uence these parametric variables more powerfully than the presence or absence of the apoe- allele; in contrast, when patients are classifi ed according to their apoe genotype, dramatic differences emerge among apoe genotypes (> %), with a clear biological disadvantage in apoe- / carriers who exhibit (i) earlier age of onset, (ii) low apoe levels, (iii) high cho and ldl-cho levels, and (iv) low no, β-amyloid, and histamine levels in blood. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] these phenotypic differences are less pronounced when ad patients are classifi ed according to their ps ( . %) or ace genotypes ( . %), refl ecting a weak impact of ps -and ace-related genotypes on the phenotypic expression of biological markers in ad. ps related genotypes appear to infl uence age of onset, blood histamine levels and cerebrovascular hemodynamics, as refl ected by signifi cant changes in systolic (sv), diastolic (dv), and mean velocities (mv) in the left middle cerebral arteries (mca). ace-related phenotypes seem to be more infl uential than ps genotypes in defi ning biological phenotypes, such as age of onset, cognitive performance, hdl-cho levels, ace and no levels, and brain blood fl ow mv in mca. however, when apoe and ps genotypes are integrated in bigenic clusters and the resulting bigenic genotypes are differentiated according to their corresponding phenotypes, an almost logarithmic increased expression of differential phenotypes is observed ( . % variation), indicating the existence of a synergistic effect of the bigenic (apoe + ps ) cluster on the expression of biological markers, apparently unrelated to app/ps mutations, since none of the patients included in the sample were carriers of either app or ps mutations. , , these examples illustrate the potential additive effects of ad-related genes on the phenotypic expression of biological markers. furthermore, the analysis of genotype-phenotype correlations with a monogenic or bigenic approach documents a modest genotype-related variation in serum amyloid-β (abp) levels, suggesting that peripheral levels of abp are of relative value as predictors of disease-stage or as markers of disease progression and/ or treatment-related disease-modifying effects. , , the peripheral levels of abp in serum exhibit an apoe-dependent pattern according to which both apoe- (+) and apoe- (+) carriers tend to show higher abp levels than apoe- (−) or apoe- carriers , - ( fig. est concentration of serum histamine is systematically present in apoe- (+) and apoe- (+) carriers, and the highest levels of histamine are seen in apoe- (+) carriers ( fig. . ). central and peripheral histaminergic mechanisms may regulate cerebrovascular function in ad, which is signifi cantly altered in apoe- / carriers. , [ ] [ ] [ ] [ ] [ ] [ ] these observations can lead to the conclusion that the simple quantifi cation of biochemical markers in fl uids or tissues of ad patients with the aim of identifying pathogenic mechanisms and/or monitoring therapeutic effects, when they are not accompanied by differential genotyping for sample homogenization, are of very poor value. differential patterns of apoe-, ps -, ps -, and trigenic (apoe + ps + ps ) cluster-related lymphocyte apoptosis have been detected in ad. fas receptor expression is signifi cantly increased in ad, especially in apoe- carriers where lymphocyte apoptosis is more relevant. , it has been demonstrated that brain activity slowing correlates with progressive gds staging in dementia , , [ ] [ ] [ ] (fig. . ). in the general population subjects harbouring the apoe- / genotype exhibit a premature slowing in brain mapping activity represented by increased slow delta and theta activities as compared with other apoe genotypes. in patients with ad, slow activity predominates in apoe- carriers with similar gds stage , , - ( fig. . ) . ad patients harbouring the apoe- / genotype also exhibit a dramatically different brain optical topography map refl ecting a genotype-specifi c differential pattern of neocortical oxygenation as well as a poorer activation of cortical neurons in response to somatosensory stimuli ( fig. . ). all these examples of genotype-phenotype correlations, as a gross approach to functional genomics, illustrate the importance of genotype-related differences in ad and their impact on phenotype expression. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , similar protocols are applied to schizophrenia, depression, anxiety and other neuropsychiatric disorders. most biological parameters, potentially modifi able by monogenic genotypes and/or polygenic cluster profi les, can be used in clinical trials for monitoring effi cacy outcomes. these parametric variables also show a genotypedependent profi le in different types of dementia (e.g., ad vs. vascular dementia). for instance, striking differences have been found between ad and vascular dementia in structural and functional genomics studies. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , our understanding of the pathophysiology of cns disorders has advanced dramatically in the last years, especially in terms of their molecular pathogenesis and genetics. drug treatment of cns disorders has also made remarkable strides, with the introduction of many new drugs for the treatment of schizophrenia, depression, anxiety, epilepsy, parkinson's disease, and alzheimer's disease, among many other quantitatively and qualita-tively important neuropsychiatric disorders. improvement in terms of clinical outcome, however, has fallen short of expectations, with up to one third of the patients continuing to experience clinical relapse or unacceptable medication-related side effects in spite of efforts to identify optimal treatment regimes with one or more drugs. potential reasons to explain this historical setback might be that: (a) the molecular pathology of most cns disorders is still poorly understood; (b) drug targets are inappropriate, not fi tting into the real etiology of the disease; (c) most treatments are symptomatic, but not anti-pathogenic; (d) the genetic component of most cns disorders is poorly defi ned; and (e) the understanding of genome-drug interactions is very limited. with the advent of recent knowledge on the human genome , and the identifi cation and characterization of many genes associated with cns disorders, , as well as novel data regarding cyp family genes and other genes whose enzymatic products are responsible for drug metabolism in the liver (e.g., nats, abcbs/ mdrs, tpmt), it has been convincingly postulated that the incorporation of pharmacogenetic and pharmacogenomic procedures ( fig. . ) in drug development might bring about substantial benefi ts in terms of therapeutics optimization in cns disorders and in many other complex disorders, assuming that genetic factors are determinant for both neuronal dysregulation (and/or neuronal death) , - and drug metabolism. [ ] [ ] [ ] fig. . effi cacy and safety issues associated with pharmacogenetics and pharmacogenomics (adapted from r. cacabelos , ) however, this fi eld is still in its infancy; and the incorporation of pharmacogenomic strategies to drug development and pharmacological screening in cns disorders is not an easy task. the natural course of technical events to achieve effi cient goals in pharmacogenetics and pharmacogenomics include the following steps: (a) genetic testing of mutant genes and/or polymorphic variants of risk; (b) genomic screening, and understanding of transcriptomic, proteomic, and metabolomic networks; (c) functional genomics studies and genotype-phenotype correlation analysis; and (d) pharmacogenetics and pharmacogenomics developments, addressing drug safety and effi cacy, respectively. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] with pharmacogenetics we can understand how genomic factors associated with genes encoding enzymes responsible for drug metabolism regulate pharmacokinetics and pharmacodynamics (mostly safety issues). [ ] [ ] [ ] with pharmacogenomics we can differentiate the specifi c disease-modifying effects of drugs (effi cacy issues) acting on pathogenic mechanisms directly linked to genes whose mutations determine the disease phenotype. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the capacity of drugs to reverse the effects of the activation of pathogenic cascades (phenotype expression) regulated by networking genes basically deals with effi cacy issues. at present, the terms pharmacogenetics and pharmacogenomics are often used interchangeably to refer to studies of the contribution of inheritance to variation in the drug response phenotype ; however, from historical and didactic reasons (until a more suitable and universal defi nition can be established) it would be preferable to maintain the term of pharmacogenetics for the discipline dealing with genetic factors associated with drug metabolism and safety issues, whereas pharmacogenomics would refer to the reciprocal infl uence of drugs and genomic factors on pathogenetic cascades and disease-associated gene expression (effi cacy issues). [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the application of these procedures to cns disorders is a very diffi cult task, since most neuropsychiatric diseases are complex disorders in which hundreds of genes might be involved , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (tables . - . ). in addition, it is very unlikely that a single drug be able to reverse the multifactorial mechanisms associated with neuronal dysfunction in most cns processes with a complex phenotype affecting mood, personality, behaviour, cognition, and functioning. this heterogeneous clinical picture usually requires the utilization of different drugs administered simultaneously. this is particularly important in the elderly population. in fact, the average number of drugs taken by patients with dementia ranges from six to more than ten per day depending upon their physical and mental conditions. nursing home residents receive, on average, seven to eight medications each month, and more than % of residents have monthly drug regimes of nine or more medications, including (in descending order) analgesics, antipyretics, gastrointestinal agents, electrolytic and caloric preparations, central nervous system (cns) agents, anti-infective agents, and cardiovascular agents. in population-based studies more than % of patients older than years are moderate or chronic antidepressant users. polypharmacy, drug-drug interactions, adverse reactions, and non-compliance are substantial therapeutic problems in the pharmacological management of elderly patients, adding further complications and costs to the patients and their caregivers. in - , . - . % of elderly individuals received at least of potentially inappropriate medications in ten health maintenance organizations (hmos) of the usa. although drug effect is a complex phenotype that depends on many factors, it is estimated that genetics accounts for - % of variability in drug disposition and pharmacodynamics. under these circumstances, therapeutics optimization is a major goal in neuropsychiatric disorders and in the elderly population, and novel pharmacogenetic and pharmacogenomic procedures may help in this endeavour. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the pharmacogenomic outcome depends upon many different determinant factors including (i) genomic profi le (family history, ethnic background, disease-related genotype, pharmacogenetic genotype, pharmacogenomic genotype, nutrigenetic genotype, nutrigenomic genotype), (ii) disease phenotype (age at onset, disease severity, clinical symptoms), (iii) concomitant pathology, (iv) genotype-phenotype correlations, (v) nutritional conditions, (vi) age and gender, (vii) pharmacological profi le of the drugs, (viii) drug-drug interactions, (ix) gene expression profi le, (x) transcriptomic cascade, (xi) proteomic profi le, and (xii) metabolomic networking ( fig. . ) . the dissection and further integration of all these factors is of paramount importance for the assessment of the pharmacogenomic outcome in terms of safety and effi cacy (figs. . and . ). more than % of psychotropic drugs (table . ) are metabolized by enzymes known to be genetically variable, including: (a) esterases: butyrylcholinesterase, paraoxonase/arylesterase; (b) transferases: n-acetyltransferase, sulfotransferase, thiol methyltransferase, thiopurine methyltransferase, catechol-o-methyltransferase, glutathione-s-transferases, udp-glucuronosyltransferases, glucosyltransferase, histamine methyltransferase; (c) reductases: nadph: quinine oxidoreductase, glucose- -phosphate dehydrogenase; (d) oxidases: alcohol dehydrogenase, aldehydehydrogenase, monoamine oxidase b, catalase, superoxide dismutase, trimethylamine n-oxidase, dihydropyrimidine dehydrogenase; and (e) cytochrome p enzymes, such as cyp a , cyp a , cyp c , cyp c , cyp c , cyp d , cyp e , cyp a (table . ) and many others. , polymorphic variants in these genes can induce alterations in drug metabolism modifying the effi cacy and safety of the prescribed drugs. drug metabolism includes phase i reactions (i.e., oxidation, reduction, hydrolysis) and phase ii conjugation reactions (i.e., acetylation, glucuronidation, sulfation, methylation). the principal enzymes with polymorphic variants involved in phase i reactions are the following: cyp a / / , cyp e , cyp d , cyp c , cyp c , cyp c , cyp b , cyp a , cyp b , cyp a / , epoxide hydrolase, esterases, nqo (nadph-quinone oxidoreductase), dpd (dihydropyrimidine dehydrogenase), adh (alcohol dehydrogenase), and aldh (aldehyde dehydrogenase). major enzymes involved in phase ii reactions include the following: ugts (uridine ′-triphosphate glucuronosyl transferases), tpmt (thiopurine methyltransferase), comt (catechol-o-methyltransferase), hmt (histamine methyl-transferase), sts (sulfotransferases), gst-a (glutathion s-transferase a), gst-p, gst-t, gst-m, nat (n-acetyl transferase), nat , and others. polymorphisms in genes associated with phase ii metabolism enzymes, such as gstm , gstt , nat and tpmt are well understood, and information is also emerging on other gst polymorphisms and on polymorphisms in the udp-glucuronosyltransferases and sulfotransferases. the typical paradigm for the pharmacogenetics of phase i drug metabolism is represented by the cytochrome p- enzymes, a superfamily of microsomal nonsteroidal anti-infl ammatory drug drug-metabolizing enzymes. p enzymes comprise a superfamily of heme-thiolate proteins widely distributed in bacteria, fungi, plants and animals. the p enzymes are encoded in genes of the cyp superfamily (table . ) and act as terminal oxidases in multicomponent electron transfer chains which are called p containing monooxigenase systems. some of the enzymatic products of the cyp gene superfamily can share substrates, inhibitors and inducers whereas others are quite specifi c for their substrates and interacting drugs. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] there are more than p genes identifi ed in different species. saito et al provided a catalogue of variants among eight cyp genes, nine esterase genes, and two other genes in the japanese population. the microsomal, membrane-associated, p isoforms cyp a , cyp d , cyp c , cyp c , cyp e , and cyp a are responsible for the oxidative metabolism of more than % of marketed drugs. about - % of the psychotropic agents currently used for the treatment of neuropsychiatric disorders are metabolized via enzymes of the cyp family, especially cyp a , cyp b , cyp c / , cyp c , cyp d and cyp a (table . ). cyp a metabolizes more drug molecules than all other isoforms together. most of these polymorphisms exhibit geographic and ethnic differences. [ ] [ ] [ ] [ ] [ ] [ ] [ ] these differences infl uence drug metabolism in different ethnic groups in which drug dosage should be adjusted according to their enzymatic capacity, differentiating normal or extensive metabolizers (ems), poor metabolizers (pms) and ultrarapid metabolizers (ums). most drugs act as substrates, inhibitors or inducers of cyp enzymes. enzyme induction enables some xenobiotics to accelerate their own biotransformation (auto-induction) or the biotransformation and elimination of other drugs. a number of p enzymes in human liver are inducible. induction of the majority of p enzymes occurs by increase in the rate of gene transcription and involves ligand-activated transcription factors, aryl hydrocarbon receptor, constitutive androstane receptor (car), and pregnane x receptor (pxr). , in general, binding of the appropriate ligand to the receptor initiates the induction process that cascades through a dimerization of the receptors, their translocation to the nucleus and binding to specifi c regions in the promoters of cyps. cyps are also expressed in the cns, and a complete characterization of constitutive and induced cyps in brain is essential for understanding the role of these enzymes in neurobiological functions and in age-related and xenobiotic-induced neurotoxicity. assuming that the human genome contains about , - , genes, at the present time only . % of commercial drugs have been assigned to corresponding genes whose gene products might be involved in pharmacokinetic and pharmacodynamic activities of a given drug; and only % of the human genes have been assigned to a particular drug metabolic pathway. supposing a theoretical number of , chemicals in current use worldwide, and assuming that practically all human genes can interact with drugs taken by human beings, each gene in the human genome should be involved in the metabolism and/or biopharmacological effect of - drugs; however, assuming that most xenobiotic substances in contact with our organism can infl uence genomic function, it might be possible that for , , xenobiotics in daily contact with humans, an average of - xenobiotics have to be assigned to each one of the genes potentially involved in drug metabolism and/or xenobiotics processing. to fulfi l this task a single gene has to possess the capacity of metabolizing many different xenobiotic substances and at the same time many different genes have to cooperate in orchestrated networks to metabolize a particular drug or xenobiotic under sequential biotransformation steps (figs. . and . ). numerous chemicals increase the metabolic capability of organisms by their ability to activate genes encoding various xenochemical-metabolizing enzymes, such as cyps, transferases and transporters. many natural and artifi cial substances induce the hepatic cyp subfamilies in humans, and these inductions might lead to clinically important drug-drug interactions. some of the key cellular receptors that mediate such inductions have been recently identifi ed, including nuclear receptors, such as the constitutive androstane receptor (car, nr i ), the retinoid x receptor (rxr, nr b ), the pregnane x receptor (pxr, nr i ), and the vitamin d receptor (vdr, nr i ) and steroid receptors such as the glucocorticoid receptor (gr, nr c ). there is a wide promiscuity of these receptors in the induction of cyps in response to xenobiotics. indeed, this adaptive system acts as an effective network where receptors share partners, ligands, dna response elements and target genes, infl uencing their mutual relative expression. , the most important enzymes of the p cytochrome family in drug metabolism by decreasing order are cyp a , cyp d , cyp c , cyp c , and cyp a . [ ] [ ] [ ] , , the predominant allelic variants in the cyp a gene are cyp a * (leu his) and cyp a del. the cyp a * mutation inactivates the enzyme and is present in - % of caucasians. the cyp a del mutation results in no enzyme activity and is present in % of caucasians and % of asians. [ ] [ ] [ ] the most frequent mutations in the cyp c gene are cyp c * (arg cys), with reduced affi nity for p in - % of caucasians, and cyp c * (ile leu), with alterations in the specifi city for the substrate in - % of caucasians and - % of asians. [ ] [ ] [ ] the most prevalent polymorphic variants in the cyp c gene are cyp c * , with an aberrant splicing site resulting in enzyme inactivation in % of caucasians, - % of asians, % of africans, and - % of ethiopians and saoudians, and cyp c * , a premature stop codon resulting in an inactive enzyme present in - % of asians, and almost absent in caucasians. [ ] [ ] [ ] , the most important mutations in the cyp d gene are the following: cyp d * xn, cyp d * , cyp d * , cyp d * and cyp d * . [ ] [ ] [ ] , the cyp d * xn mutation gives rise to a gene duplication or multiplication resulting in an increased enzyme activity which appears in - % of the caucasian population, - % of asians, % of africans, and - % of ethiopians. the defective splicing caused by the cyp d * mutation inactivates the enzyme and is present in - % of caucasians. the deletion in cyp d * abolishes enzyme activity and shows a frequency of - % in caucasians, % in asians, % in africans, and - % in ethiopians. the polymorphism cyp d * causes pro ser and ser thr mutations with unstable enzyme activity in - % of caucasians, % of asians, % of africans, and - % of ethiopians. the cyp d * variant causes thr ile and arg cys substitutions which produce a reduced affi nity for substrates in % of asians, % of africans, and - % of ethiopians, and is practically absent in caucasians. [ ] [ ] [ ] , , the cyp d enzyme, encoded by a gene that maps on q . - . , catalyses the oxidative metabolism of more than clinically important and commonly prescribed drugs such as cholinesterase inhibitors, antidepressants, neuroleptics, opioids, some β-blockers, class i antiarrhythmics, analgesics and many other drug categories, acting as substrates, inhibitors or inducers with which most psychotropics may potentially interact (table . ), this leading to the outcome of adrs. [ ] [ ] [ ] , , the cyp d locus is highly polymorphic, with more than different cyp d alleles identifi ed in the general population showing defi cient (poor metabolizers, pm), normal (extensive metabolizers, em) or increased enzymatic activity (ultra-rapid metabolizers, um). , most individuals (> %) are ems; however, remarkable interethnic differences exist in the frequency of the pm and um phenotypes among different societies all over the world. [ ] [ ] [ ] , [ ] [ ] [ ] [ ] on the average, approximately . % of the world population belongs to the pm category. europeans ( . %), polynesians ( . %), and africans ( . %) exhibit the highest rate of pms, whereas orientals ( . %) show the lowest rate. the frequency of pms among middle eastern populations, asians, and americans is in the range of - %. [ ] [ ] [ ] [ ] [ ] cyp d gene duplications are relatively infrequent among northern europeans, but in east africa the frequency of alleles with duplication of cyp d is as high as %. the most frequent cyp d alleles in the european population are the following: cyp d * (wild-type) (normal), cyp d * ( c > t)(normal), cyp d * ( a > del)(inactive), cyp d * ( g > a)(inactive), cyp d * (gene deletion)(inactive), cyp d * ( t > del)(inactive), cyp d * ( a > c)(inac-tive), cyp d * ( g > t)(inactive), cyp d * ( - delaga)(partially active), cyp d * ( c > t)(partially active), cyp d * ( g > c) (inactive), cyp d * ( g > a)(inactive), cyp d * ( c > t)(partially active), and cyp d gene duplications (with increased or decreased enzymatic activity depending upon the alleles involved). [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the spanish population, where the mixture of ancestral cultures has occurred for centuries, the distribution of the cyp d genotypes differentiates major categories of cyp d -related metabolizer types: (i) extensive metabolizers (em)( * / * , * / * ); (ii) intermediate metabolizers (im)( * / * , * / * , * / * , * / * , * / * , * / * , * / * , * / * , * / * ); (iii) poor metabolizers (pm)( * / * , * / * ); and (iv) ultra-rapid metabolizers (um)( * xn/ * , * xn/ * , dupl). in this sample we have found . % ems, . % ims, . % pms, and . % ums. , [ ] [ ] [ ] [ ] the distribution of all major genotypes is the following: * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * / * , . %; * xn/ * , . %; * xn/ * , . %; and dupl, . %. , [ ] [ ] [ ] [ ] in some instances, there is association of cyp d variants of risk with genes potentially involved in the pathogenesis of specifi c cns disorders. when comparing ad cases with controls, we observed that ems are more prevalent in ad ( * / * , . %; * / * , . %)(total ad-ems: . %) than in controls ( * / * , . %; * / * , %)(total c-ems: . %). in contrast, ims are more frequent in controls ( . %) than in ad ( . %), especially the * / * (c: . %; ad: . %) and * / * genotypes (c: . %; ad: . %). the frequency of pms was similar in ad ( . %) and controls ( . %), and ums were more frequent among ad cases ( . %) than in controls ( . %). , , , we have also investigated the association of cyp d genotypes with ad-related genes, such as app, mapt, apoe, ps , ps , a m, ace, agt, fos, and prnp variants. , , , no app or mapt mutations have been found in ad cases. homozygous apoe- / ( . %) and apoe- / ( . %) accumulate in ums, and apoe- / cases were also more frequent in pms ( . %) than in ems ( . %) or ims ( %). ps - / genotypes were more frequent in ems ( %), whereas ps- / genotypes were over-represented in ims ( . %) and ums ( %). the presence of the ps - / genotype was especially high in pms ( . %) and ums ( %). a mutation in the ps gene exon (ps e +) was markedly present in ums ( . %). about % of ums were a m-v i-a/a, and the a m-v i-g/g genotype was absent in pms and ums. the a m-i/i genotype was absent in ums, and % of ums were a m-i/d and ace-d/d. homozygous mutations in the fos gene (b/b) were only present in ums, as well. agt-t t cases were absent in pms, and the agt-m m genotype appeared in % of pms. likewise, the prnp-m m variant was present in % of pms and ums. , , , these association studies clearly show that in pms and ums there is an accumulation of ad-related polymorphic variants of risk which might be responsible for the defective therapeutic responses currently seen in these ad clusters. , [ ] [ ] [ ] [ ] it appears that different cyp d variants, expressing ems, ims, pms, and ums, infl uence to some extent several biochemical parameters, liver function, and vascular hemodynamic parameters which might affect drug effi cacy and safety. blood glucose levels are found elevated in ems ( * / * vs. * / * , p < . ) and in some ims ( * / * vs. * xn/ * , p < . ), whereas other ims ( * / * vs. * / * , p < . ) tend to show lower levels of glucose compared with pms ( * / * ) or ums ( * xn/ * ) (table . ). the highest levels of total-cholesterol are detected in the ems with the cyp d * / * genotype (vs. * / * , * / * and * xn/ * , p < . ). the same pattern has been observed with regard to ldlcholesterol levels, which are signifi cantly higher in the em-* / * . in general, both total cholesterol levels and ldl-cholesterol levels are higher in ems (with a signifi cant difference between * / * and * / * ), intermediate levels are seen in ims, and much lower levels in pms and ums; and the opposite occurs with hdlcholesterol levels, which on average appear much lower in ems than in ims, pms, and ums, with the highest levels detected in * / * and * xn/ * (table . ). the levels of triglycerides are very variable among different cyp d polymorphisms, with the highest levels present in ims ( * / * vs. * / * and * xn/ * , p < . ). these data clearly indicate that lipid metabolism can be infl uenced by cyp d variants or that specifi c phenotypes determined by multiple lipid-related genomic clusters are necessary to confer the character of ems and ims. other possibility might be that some lipid metabolism genotypes interact with cyp d -related enzyme products leading to defi ne the pheno-genotype of pms and ums. no signifi cant changes in blood pressure values have been found among cyp d genotypes; however, important differences became apparent in brain cerebrovascular hemodynamics (table . ). in general terms, the best ( ) . ± . ( ) . ± . ( ldl-cholesterol levels, than in ims ( * / * , p < . ); and diastolic velocities (dv) also tend to be much lower in * / * and especially in pms ( * / * ) and ums ( * xn/ * ), whereas the best dv is measured in * / * ims. more striking are the results of both the pulsatility index (pi = (sv-dv)/mv) and resistance index (ri = (sv-dv)/sv), which are worse in ims and pms than in ems and ums (table . ). these data taken together seem to indicate that cyp d -related ad pms exhibit a poorer cerebrovascular function which might affect drug penetration in the brain with the consequent therapeutic implications. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] some conventional anti-dementia drugs (tacrine, donepezil, galantamine) are metabolized via cyp-related enzymes, especially cyp d , cyp a , and cyp a , and polymorphic variants of the cyp d gene can affect the liver metabolism, safety and effi cacy of some cholinesterase inhibitors. , in order to elucidate whether or not cyp d -related variants may infl uence transaminase activity, we have studied the association of got, gpt, and ggt activity with the most prevalent cyp d genotypes in ad (table . ). globally, ums and pms tend to show the highest got activity and ims the lowest. signifi cant differences appear among different im-related genotypes. the * / * genotype exhibited the lowest got activity with marked differences as compared to ums (p < . vs. * xn/ * ; p < . vs. * xn/ * ). gpt activity was signifi cantly higher in pms ( * / * ) than in ems ( * / * , p < . ) or ims ( * / * , * / * , p < . ). the lowest gpt activity was found in ems and ims. striking differences have been found in ggt activity between pms ( * / * ), which showed the highest levels, and ems ( * / * , p < . ; * / * , p < . ), ims ( * / * , p < . ), or ums ( * xn/ * , p < . ) ) ( table . ). interesting enough, the * / * genotype, with the lowest values of got and gpt, exhibited the second highest levels of ggt after * / * , probably indicating that cyp d -related enzymes differentially regulate drug metabolism and transaminase activity in the liver. these results are also clear in demonstrating the direct effect of cyp d variants on transaminase activity , , (table . ). ( ) . ± . ( ) . ± . ( ) intermediate metabolizers * / * . ± . ( , ) . ± . . ± . * / * . ± . ( , ) . ± . . ± . * / * . ± . ( , ) . ± . ( , ) . ± . ( no clinical trials have been performed to date to elucidate the infl uence of cyp d variants on the therapeutic outcome in ad in response to cholinesterase inhibitors or other anti-dementia drugs. to overcome this lack of pharmacogenetic information, we have performed the fi rst prospective study in ad patients who received a combination therapy with (a) an endogenous nucleotide and choline donor, cdp-choline ( mg/day), (b) a nootropic substance, piracetam ( , mg/day), (c) a vasoactive compound, , dimethyl β-( bromonicotinoyl-oxymethyl)- α-methoxyergoline (nicergoline) ( (fig. . ). among ems, ad patients harbouring the * / * genotype responded better than patients with the * / * genotype. the best responders among ims were the * / * , * / * and * / * genotypes, whereas the * / * , * / * , and * / * genotypes were poor responders. among pms and ums, the poorest responders were carriers of the * / * and * xn/ * genotypes, respectively. , , from all these data we can conclude the following: (i) the most frequent cyp d variants in the spanish population are the * / * ( . %), * / * ( . %), * / * ( . %), * / * ( . %) and * xn/ * ( . %), accounting for more than % of the population; (ii) the frequency of ems, ims, pms, and ums is about . %, . %, . %, and . %, respectively; (iii) ems are more prevalent in ad ( . %) than in controls ( . %); ims are more frequent in controls ( . %) fig. . cyp d -related therapeutic response to a multifactorial treatment in alzheimer's disease over a -year period (adapted from r. cacabelos , ).patients received a combina-tion therapy for year, and cognitive function (mmse score) was assessed at baseline (b) and after , , , , and months of treatment. than in ad ( . %), especially the * / * (c: . %; ad: . %) and * / * genotypes (c: . %; ad: . %); the frequency of pms is similar in ad ( . %) and controls ( . %); and ums are more frequent among ad cases ( . %) than in controls ( . %); (iv) there is an accumulation of ad-related genes of risk in pms and ums; (v) pms and ums tend to show higher transaminase activities than ems and ims; (vi) ems and ims are the best responders, and pms and ums are the worst responders to a combination therapy with cholinesterase inhibitors, neuroprotectants, and vasoactive substances; and (vii) the pharmacogenetic response in ad appears to be dependent upon the networking activity of genes involved in drug metabolism and genes involved in ad pathogenesis. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , taking into consideration the available data, it might be inferred that at least % of the ad population may exhibit an abnormal metabolism of cholinesterase inhibitors and/or other drugs which undergo oxidation via cyp d -related enzymes. approximately % of this population cluster would show an ultrarapid metabolism, requiring higher doses of cholinesterase inhibitors to reach a therapeutic threshold, whereas the other % of the cluster would exhibit a poor metabolism, displaying potential adverse events at low doses. if we take into account that approximately - % of therapeutic outcomes depend upon pharmacogenomic criteria (e.g., pathogenic mechanisms associated with ad-related genes), it can be postulated that pharmacogenetic and pharmacogenomic factors are responsible for - % of the therapeutic response (effi cacy) in ad patients treated with conventional drugs. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , of particular interest are the potential interactions of cholinesterase inhibitors with other drugs of current use in patients with ad, such as antidepressants, neuroleptics, antiarrhythmics, analgesics, and antiemetics which are metabolized by the cytochrome p cyp d enzyme. although most studies predict the safety of donepezil and galantamine, as the two principal cholinesterase inhibitors metabolized by cyp d -related enzymes, , no pharmacogenetic studies have been performed so far on an individual basis to personalize the treatment, and most studies reporting safety issues are the result of pooling together pharmacological and clinical information obtained with routine procedures. , [ ] [ ] [ ] in certain cases, genetic polymorphism in the expression of cyp d is not expected to affect the pharmacodynamics of some cholinesterase inhibitors because major meta-bolic pathways are glucuronidation, o-demethylation, n-demethylation, n-oxidation, and epimerization. however, excretion rates are substantially different in ems and pms. for instance, in ems, urinary metabolites resulting from o-demethylation of galantamine represent . % of the dose compared with . % in pms, which show correspondingly higher urinary excretion of unchanged galantamine and its n-oxide. therefore, still there are many unanswered questions regarding the metabolism of cholinesterase inhibitors and their interaction with other drugs (potentially leading to adrs) which require pharmacogenetic elucidation. it is also worth to mention that dose titration (a common practice in ad patients treated with cholinesterase inhibitors; e.g., tacrine, donepezil) is an unwise strategy, since approximately - % of drug failure or lack of therapeutic effi cacy (and/or adr manifestation) is not a matter of drug dosage but a problem of poor metabolizing capacity in pms. additionally, inappropriate drug use is one of the risk factors for adverse drug reactions (adrs) in the elderly. the prevalence of use of potentially inappropriate medications in patients older than years of age admitted to a general medical or geriatric ward ranges from % to %, and these numbers may double in ambulatory patients. overall, the most prevalent inappropriate drugs currently prescribed to the elderly are amiodarone, long-acting benzodiazepines and anticholinergic antispasmodics; however, the list of drugs with potential risk also include antidepressant, antihistaminics, nsaids, amphetamines, laxatives, clonidine, indomethacin, and several neuroleptics, most of which are processed via cyp d and cyp a enzymes. therefore, pre-treatment cyp screening might be of great help to rationalize and optimize therapeutics in the elderly, by avoiding medications of risk in pms and ums. there are substantial differences between individuals in the effects of psychotropic drugs in the treatment of neuropsychiatric disorders. pharmacogenetic studies of psychotropic drug response have focused on determining the relationship between variation in specifi c candidate genes and the positive and adverse effects of drug treatment. more than different genes are potentially involved in the metabolism of psychotropic drugs infl uencing pharmacokinetics and pharmacodynamics. of all genes affecting drug metabolism, effi cacy and safety, the cyp gene family is the most relevant since more than % of cns drugs are metabolized by cytochrome p enzymes. [ ] [ ] [ ] approximately, % of neuroleptics are major substrates of cyp a enzymes, % of cyp d , and % of cyp a ; % of antidepressants are major substrates of cyp a enzymes, % of cyp b , % of cyp c , % of cyp d , and % of cyp a ; % of benzodiazepines are major substrates of cyp c enzymes, % of cyp d , and % of cyp a (table . ). approximately, % of patients with resistant depression, % of patients non-responsive to neuroleptics, and - % of patients with paradoxical responses to benzodiazepines are carriers of mutant variants of the cyp d , cyp c and cyp a genes, falling within the categories of poor or ultra-rapid metabolizers. other genes infl uencing psychotropic drug activity include the following: abcb ( [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table . ) . historically, the vast majority of pharmacogenetic studies of cns disorders have been addressed to evaluate the impact of cytochrome p enzymes on drug metabolism. [ ] [ ] [ ] furthermore, conventional targets for psychotropic drugs were the neurotransmitters dopamine, serotonin, noradrenaline, gaba, ion channels, acetylcholine and their respective biosynthetic and catalyzing enzymes, receptors and transporters ; however, in the past few years many different genes have been associated with both pathogenesis and pharmacogenomics of neuropsychiatric disorders. some of these genes and their products constitute potential targets for future treatments. new developments in genomics, including whole genome genotyping approaches and comprehensive information on genomic variation across populations, coupled with large-scale clinical trials in which dna collection is routine, now provide the impetus for a next generation of pharmacogenetic studies and identifi cation of novel candidate drugs. [ ] [ ] [ ] cyclic nucleotide phosphodiesterases (pdes) are a family of enzymes that degrade camp and cgmp. intracellular cyclic nucleotide levels increase in response to neurotransmitters and are down-regulated through hydrolysis catalyzed by pdes, which are therefore candidate therapeutic targets. camp is a second messenger involved in learning, memory, and mood, and cgmp modulates brain processes that are controlled by the nitric oxide (no)/cgmp pathway. the analysis of snps in genes of this superfamily revealed that polymorphisms in pde a and pde a are associated with major depressive disorder. in addition, remission on antidepressants was associated with polymorphisms in pde a and pde a. according to these results, it has been postulated that pde a (haplotype gaacc) has a role in the pathogenesis of major depression. another example is the purinergic receptor gene p rx( ), located in a major linkage hotspot for schizophrenia and bipolar disorder ( q - ), which has been associated with bipolar disorder, but nine functionally characterized variants of p rx( ) did not show association with schizophrenia. the possible role of a tag snp (the g/a polymorphism) of the gene encoding the cannabinoid receptor type (cnr ) has been investigated in schizophrenics treated with atypical antipsychotics. no difference in g/a polymorphism was observed between patients and control subjects, and no relation-ships were noted between this polymorphism and any clinical parameter considered as potential intermediate factor; however, the g allele was signifi cantly higher among non-responders vs. responsive patients, suggesting that the g allele of the cnr gene could be a pharmacogenetic rather than a vulnerability factor for schizophrenics. synaptic dysfunction is a potential pathogenic factor in schizophrenia. cholesterol is an essential component of myelin and has proved important for synapse formation and lipid raft function. it has been demonstrated that the antipsychotic drugs clozapine and haloperidol stimulate lipogenic gene expression in glioma cells in culture through activation of the sterol regulatory element-binding protein (srebp) transcription factors. recently, the action of chlorpromazine, haloperidol, clozapine, olanzapine, risperidone and ziprasidone on srebp and srebp-controlled gene expression (acetyl-coa acetyltransferase , acetoacetyl-coa thiolase, acat ; -hydroxy- -methylglutaryl-coa reductase, hmgcr; -hydroxy- -methylglutaryl-coa synthase , hmgcs ; fdps; sterol-c -desaturase like, sc dl; -dehydrocholesterol reductase, dhcr ; low density lipoprotein receptor, ldlr; fatty acid synthase; farsenyl diphosphate synthase, fasn; stearoyl-coa desaturase, delta- -desaturase, scd ) has been investigated in different cns human cell lines, demonstrating that antipsychotic-induced activation of lipogenesis is most prominent in glial cells and that this mechanism could be relevant for the therapeutic efficacy of some antipsychotic drugs. rgs (regulator of g-protein signaling ) modulates dopamine receptor signal transduction. functional variants of this gene (rgs -rs c/g) may infl uence susceptibility to extrapyramidal symptoms induced by antipsychotic drugs. this snp is located in the ′-regulatory region of the gene, and is known to infl uence rgs mrna levels and protein expression. furthermore, rgs (regulator of g protein signaling ) genotypes predict both the severity at baseline symptoms and relative responsiveness to antipsychotic medication. tardive dyskinesia is characterized by involuntary movements predominantly in the orofacial region and develops in approximately % of patients during long-term treatment with typical antipsychotics. polymorphic variants of cyp a , cyp d , and drd genes have been associated with tardive dyskinesia in schizophrenics. , in contrast, the haplotype t- b-glu of the endothelial nitric oxide synthase (nos ) gene (- t > c in the promoter region, -bp variable number of tandem repeats in intron , glu asp in exon ) might represent a protective haplotype against tardive dyskinesia after long-term antipsychotic treatment. the t c variant in the serotonin a receptor (htr a) and the ser gly variant in the dopamine d receptor (drd ) were associated with a risperidone response to exacerbated schizophrenia. the patients with t/t in the htr a gene show less clinical improvement than do those with t/c or c/c. the c allele is more frequent in responders. when combinations of both polymorphisms are considered, patients who have t/t in the htr a gene and encode ser/ser or ser/gly from drd gene have a higher propensity to non-responsiveness compared to other subjects, suggesting that the htr a t c variant could be a potential indicator of clinical improvement after risperidone treatment. there is a signifi cant relationship between a promoter region polymorphism in the serotonin transporter gene and antidepressant response, as well as for associations between candidate neurotransmitter receptor genes and second generation antipsychotic drug response. polymorphic variants of several serotonin receptor subtypes seem to be involved in the efficacy and symptomatic response of schizophrenic patients to atypical antipsychotics. for instance, the − c/g polymorphism of the htr a receptor gene is associated with negative symptom response to risperidone in schizophrenics. interaction between comt and notch genotypes may also predict the treatment response to typical neuroleptics in patients with schizophrenia. the effi cacy of iloperidone in patients with schizophrenia has been associated with the homozygous condition for the rs g/g genotype of the ciliary neurotrophic factor (cntf) gene. dopamine receptor interacting proteins (drips) are pivotally involved in regulating dopamine receptor signal transduction. two snps in the dopamine receptor interacting protein gene, nef , which encodes the drip, neurofi lament-medium (nf-m), were associated with early response (rs , rs ). a snp haplotype spanning nef was over-represented in early responders. since nef is primarily associated with dopamine d receptor function, it is likely that both genes cooperate in eliciting genotype-specifi c antipsychotic response. the improvement in the positive and negative syndrome scale (panss) positive subscore was found signifi cantly greater in patients homozygous for the a allele of the slc a (solute carrier family (noradrenaline transporter), member ) gene, and smaller in patients homozygous for the c- allele of the slc a gene, suggesting that these polymorphisms of the noradrenaline transporter gene are specifi cally involved in the variation of positive symptoms in schizophrenia. weight gain is a problem commonly found in patients treated with neuroleptics, tricyclic antidepressants, and some antiepileptics (e.g., valproic acid). the adipocyte-derived hormone, leptin, has been associated with body weight and energy homeostasis, and abnormal regulation of leptin could play a role in weight gain induced by antipsychotics. the leptin gene promoter variant g a was associated with clozapine-induced weight in chinese patients with chronic schizophrenia. likewise, studies in caucasians suggest that genetic vulnerability in the leptin gene (− g/a) and leptin receptor (q r) may predispose some individuals to excessive weight gain from increased exposure to olanzapine. , the development of selective type metabotropic glutamate receptor (mglu ) antagonists, such as -methyl- -(phenylethynyl)-pyridine (mpep) and -[( -methyl- , -thiazol- -yl)ethynyl]-pyridine (mtep), has demonstrated the potential involvement of these receptors in several cns disorders including depression, anxiety, epilepsy, parkinson's disease, drug addiction, and alcoholism. treatment with mpep and mtep can induce gene expression related to atp synthesis, hydrolase activity, and signaling pathways associated with mitogen-activated protein kinase (mapk) in the frontal cortex, this constituting another potential therapeutic target in some neuropsychiatric disorders. a new marker (rs ) in the grik gene, which codes for the kainic acid-type glutamate receptor ka , has been associated with response to the antidepressant citalopram, suggesting that the glutamate system plays a role in modulating response to selective serotonin reuptake inhibitors (ssris). glycogen synthase kinase- β (gsk b) activity is increased in the brain of patients with major depressive disorders. inhibition of gsk b is thought to be a key feature in the therapeutic mechanism of antidepressants. four polymorphisms of the gsk b gene [rs (− t > c); rs (ivs + a > g); rs (ivs + g > t); rs (ivs + t > a)] have been genotyped in chinese patients with major depression. gsk b tagt carriers showed poorer response to antidepressants. lithium has been used for over years as an effective prophylactic agent in bipolar disorder. response to lithium treatment seems to be, at least in part, genetically determined. it has been suggested that lithium exerts an effect on signal transduction pathways, such as the cyclic adenosine monophosphate (camp) pathway. association studies in patients with bipolar disorders revealed that creb - h snp (g/a change at q . -q ) and creb - h (t/c change) may be associated with bipolar disorder and lithium response. dna oligonucleotide microarrays have been used to evaluate gene expression in the substantia nigra of patients with parkinson's disease (pd). sporadic pd is characterized by progressive death of dopaminergic neurons within the substantia nigra, where cell death is not uniform. the lateral tier of the substantia nigra (snl) degenerates earlier and more severely than the more medial nigral component (snm). genes expressed more highly in the pd snl included the cell death gene, p effector related to pmp , the tnfr gene, tnfr superfamily, member , and the mitochondrial complex i gene, nadh dehydrogenase (ubiquinone) -beta subcomplex, , kda (ndufβ ). genes that were more highly expressed in pd snm included the dopamine cell signaling gene, cyclic adenosine monophosphate-regulated phosphoprotein, kda, the activated macrophage gene, stabilin , and two glutathione peroxidase (gpx) genes, gpx and gpx . this gene expression profi le reveals that there is increased expression of genes encoding pro-infl ammatory cytokines and subunits of the mitochondrial electron transport chain in glial cells, and that there is a decreased expression of several glutathione-related genes in the gnl, suggesting a molecular basis for pathoclisis. these fi ndings may contribute to open new therapeutic avenues in pd, where glial cells might represent potential targets to halt disease progression. pharmacological inhibition of cyclic-dependent kinase (cdk ) protects neurons under distinct stressful conditions. in ad and amyotrophic lateral sclerosis deregulation of cdk causes hyperphosphorylation of tau and neurofi lament proteins, respectively, leading to neuronal cell death. by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionisation-time of fl ight (maldi-tof)-mass spectrometry, several phosphoproteins that are modulated by cdk inhibitors have been identifi ed. these phosphoproteins include syndapin i which is involved in vesicle recycling, and dynein light intermediate chain which represents a regulatory subunit of the dynein protein complex, confi rming the role of cdk in synaptic signaling and axonal transport. other phosphoproteins detected are cofi lin and collapsing response mediator protein, involved in neuronal survival and/or neurite outgrowth. selective cdk inhibitors can also block mitochondrial translocation of pro-apoptotic cofi lin. phosphoproteome and transcriptome analysis of neurons indicate that cdk inhibitors promote both neuronal survival and neurite outgrowth. these compounds might represent novel therapeutic alternatives in neurodegenerative disorders. despite the promising results obtained with structural and functional genomic procedures to identify associations with disease pathogenesis and potential drug targets in cns disorders, it must be kept in mind that allelic mrna expression is affected by genetic and epigenetic events, both with the potential to modulate neurotransmitter tone in the cns. epigenetics is the study of how the environment can affect the genome of the individual during its development as well as the development of its descendants, all without changing the dna sequence, but inducing modifi cations in gene expression through dna methylation-demethylation or through modifi cation of histones by processes of methylation, deacetylation, and phosphorylation. cumulative experiences throughout life history interact with genetic predispositions to shape the individual's behaviour. epigenetic phenomena can not be neglected in the pathogenesis and pharmacogenomics of cns disorders. studies in cancer research have demonstrated the antineoplastic effects of the dna methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid, of current use in epilepsy. novel effects of some pleiotropic drugs with activity on the cns have to be explored to understand in full their mechanisms of action and adjust their dosages for new indications. both hyper-and hypo-dna methylation changes of the regulatory regions play critical roles in defi ning the altered functionality of genes (mb-comt, maoa, dat , th, drd , drd , reln, bdnf) in major psychiatric disorders, such as schizophrenia and bipolar disorder. this complexity requires a multifactorial approach to overcome the hurdles that cns drug development faces at the present time. polymorphic variants in the apoe gene ( q . ) are associated with risk (apoe- allele) or protection (apoe- allele) for ad. , [ ] [ ] [ ] for many years, alterations in apoe and defects in the apoe gene have been associated with dysfunctions in lipid metabolism, cardiovascular disease, and atherosclerosis. during the past years an enormous amount of studies clearly documented the role of apoe- as a risk factor for ad, an the accumulation of the apoe- allele has been reported as a risk factor for other forms of dementia and cns disorders. , [ ] [ ] [ ] apoe- may infl uence ad pathology interacting with app metabolism and abp accumulation, enhancing hyperphosphorylation of tau protein and nft formation, reducing choline acetyltransferase activity, increasing oxidative processes, modifying infl ammation-related neuroimmunotrophic activity and glial activation, altering lipid metabolism, lipid transport and membrane biosynthesis in sprouting and synaptic remodelling, and inducing neuronal apoptosis. , [ ] [ ] [ ] different apoe genotypes confer specifi c phenotypic profi les to ad patients. some of these profi les may add risk or benefi t when the patients are treated with conventional drugs, and in many instances the clinical phenotype demands the administration of additional drugs which increase the complexity of therapeutic protocols. from studies designed to defi ne apoe-related ad phenotypes, , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] , several confi rmed conclusions can be drawn: (i) the ageat-onset is - years earlier in approximately % of ad cases harbouring the apoe- / genotype; (ii) the serum levels of apoe are the lowest in apoe- / , intermediate in apoe- / and apoe- / , and highest in apoe- / and apoe- / ; (iii) serum cholesterol levels are higher in apoe- / than in the other genotypes; (iv) hdl-cholesterol levels tend to be lower in apoe- homozygotes than in apoe- allele carriers; (v) ldl-cholesterol levels are systematically higher in apoe- / than in any other genotype; (vi) triglyceride levels are signifi cantly lower in apoe- / ; (vii) nitric oxide levels are slightly lower in apoe- / ; (viii) serum abp levels do not differ between apoe- / and the other most frequent genotypes (apoe- / , apoe- / ); (ix) blood histamine levels are dramatically reduced in apoe- / as compared with the other genotypes; (x) brain atrophy is markedly increased in apoe- / > apoe- / > apoe- / ; (xi) brain mapping activity shows a signifi cant increase in slow wave activity in apoe- / from early stages of the disease (fig. . ) ; (xii) brain hemodynamics, as refl ected by reduced brain blood fl ow velocity and increase pulsatility and resistance indices, is signifi cantly worst in apoe- / (and in apoe- carriers, in general, as compared with apoe- carriers); (xiii) lymphocyte apoptosis is markedly enhanced in apoe- carriers; (xiv) cognitive deterioration is faster in apoe- / patients than in carriers of any other apoe genotype; (xv) occasionally, in approximately - % of the ad cases, the presence of some dementia-related metabolic dysfunctions (e.g., iron, folic acid, vitamin b defi ciencies) accumulate in apoe- carriers more than in apoe- carriers; (xvi) some behavioral disturbances (bizarre behaviors, psychotic symptoms), alterations in circadian rhythm patterns (e.g., sleep disorders), and mood disorders (anxiety, depression) are slightly more frequent in apoe- carriers; (xvii) aortic and systemic atherosclerosis is also more frequent in apoe- carriers; (xviii) liver metabolism and transaminase activity also differ in apoe- / with respect to other genotypes; (xix) blood pressure (hypertension) and other cardiovascular risk factors also accumulate in apoe- ; and (xx) apoe- / are the poorest responders to conventional drugs ( fig. . ). these major phenotypic features clearly illustrate the biological disadvantage of apoe- homozygotes and the potential consequences that these patients may experience when they receive pharmacological treatment. , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] , fig. . apoe-related cognitive performance in patients with alzheimer's disease treated with a combination therapy for year (adapted from r. cacabelos , ). patients received a combination therapy for year, and cognitive function (mmse score) was assessed at baseline (b) and after , , , , and months of treatment. several studies indicate that the presence of the apoe- allele differentially affects the quality and size of drug responsiveness in ad patients treated with cholinergic enhancers, neuroprotective compounds or combination therapies; however, controversial results are frequently found due to methodological problems, study design, and patients recruitment in clinical trials. from these studies we can conclude the following: (i) multifactorial treatments combining neuroprotectants, endogenous nucleotides, nootropic agents, vasoactive substances, cholinesterase inhibitors, and nmda antagonists associated with metabolic supplementation on an individual basis adapted to the phenotype of the patient may be useful to improve cognition and slow-down disease progression in ad. (ii) in our personal experience the best results have been obtained combining (a) cdp-choline with piracetam and metabolic supplementation, (b) cdp-choline with piracetam and anapsos, (c) cdp-choline with piracetam and cholinesterase inhibitors (donepezil, rivastigmine), (d) cdp-choline with memantine, and (e) cdpcholine, piracetam and nicergoline. (iii) some of these combination therapies have proven to be effective, improving cognition during the fi rst months of treatment, and not showing apparent side-effects. (iv) the therapeutic response in ad seems to be genotypespecifi c under different pharmacogenomic conditions. (v) in monogenic-related studies, patients with the apoe- / and apoe- / genotypes are the best responders, and apoe- / carriers are the worst responders ( fig. . ). (vi) ps -and ps -related genotypes do not appear to infl uence the therapeutic response in ad as independent genomic entities; however, app, ps , and ps mutations may drastically modify the therapeutic response to conventional drugs. (vii) in trigenic-related studies the best responders are those patients carrying the -, -, -, and -genomic clusters. (viii) a genetic defect in the exon of the ps gene seems to exert a negative effect on cognition conferring ps + carriers in trigenic clusters the condition of poor responders to combination therapy. (ix) the worst responders in all genomic clusters are patients with the + genotype. (x) the apoe- / genotype seems to accelerate neurodegeneration anticipating the onset of the disease by - years; and, in general, apoe- / carriers show a faster disease progression and a poorer therapeutic response to all available treatments than any other polymorphic variant. (xi) pharmacogenomic studies using trigenic, tetragenic or polygenic clusters as a harmonization procedure to reduce genomic heterogeneity are very useful to widen the therapeutic scope of limited pharmacological resources. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] , apoe infl uences liver function and cyp d -related enzymes probably via regulation of hepatic lipid metabolism. , , [ ] [ ] [ ] [ ] it has been observed that apoe may infl uence liver function and drug metabolism by modifying hepatic steatosis and transaminase activity. there is a clear correlation between apoe-related tg levels and got, gpt, and ggt activities in ad. , [ ] [ ] [ ] [ ] both plasma tg levels and transaminase activity are signifi cantly lower in ad patients harbouring the apoe- / genotype, probably indicating (a) that low tg levels protect against liver steatosis, and (b) that the presence of the apoe- allele infl uences tg levels, liver steatosis, and transaminase activity. consequently, it is very likely that apoe infl uences drug metabolism in the liver through different mechanisms, including interactions with enzymes such as transaminases and/ or cytochrome p -related enzymes encoded in genes of the cyp superfamily. , [ ] [ ] [ ] [ ] , when apoe and cyp d genotypes are integrated in bigenic clusters and the apoe + cyp d -related therapeutic response to a combination therapy is analyzed in ad patients after year of treatment, it becomes clear that the presence of the apoe- / genotype is able to convert pure cyp d * / * ems into full pms (fig. . ) , indicating the existence of a powerful infl uence of the apoe- homozygous genotype on the drug metabolizing capacity of pure cyp d -ems. , , , behavioral disturbances and mood disorders are intrinsic components of dementia associated with memory disorders. , [ ] [ ] [ ] the appearance of anxiety, depression, psychotic symptoms, verbal and physical aggressiveness, agitation, wandering and sleep disorders complicate the clinical picture of dementia and add important problems to the therapeutics of ad and the daily management of patients as well. under these conditions, psychotropic drugs (antidepressants, anxyolitics, hypnotics, and neuroleptics) are required, and most of these substances contribute to deteriorate cognition and psychomotor functions. apoe-related polymorphic variants have been associated with mood disorders , and panic disorder. gender, age, dementia severity, apoe- , and general medical health appear to infl uence the occurrence of individual neuropsychiatric symptoms in dementia, and medical comorbidity increases the risk of agitation, irritability, disinhibition, and aberrant motor behavior. a positive association between apoe- and neuropsychiatric symptoms and depressive symptoms in ad has been reported, especially in women. in other studies, no association of apoe- with behavioral dyscontrol (euphoria, disinhibition, aberrant motor behavior, and sleep and appetite disturbances), psychosis (delusions and hallucinations), mood (depression, anxiety, and apathy), and agitation (aggression and irritability) could be found. some authors did not fi nd association of apoe- with major depression in ad , or in patients with major depression in a community of older adults, but an apparent protective effect of apoe- on depressive symptoms was detected. others, in contrast, found that apoe- was associated with an earlier age-of-onset, but not cognitive functioning, in late-life depression. apoe−/− mice without human apoe or with apoe- , but not apoe- , show increased measures of anxiety. differences in anxiety-related behavior have been observed between apoe-defi cient c bl/ and wild type c bl/ mice, suggesting that apoe variants may affect emotional state. histamine h autoreceptor antagonists increase anxiety measures in wildtype, but not apoe−/−, mice, and apoe defi cient mice show higher sensitivity to the anxiety-reducing effects of the h receptor antagonist mepyramine than wildtype mice, suggesting a role of h -autoreceptormediated signaling in anxiety-like symptoms in this ad-related animal model. in humans, apoe- carriers with deep white matter hyperintensities in mri show association with depressive symptoms and vascular depression. . reduced caudate nucleus volumes and genetic determinants of homocysteine metabolism accumulate in patients with psychomotor slowing and cognitive defi cits, and older depressed subjects have persisting cognitive impairments associated with hippocampal volume reduction. , depressive symptoms are also associated with stroke and atherogenic lipid profi le. some multifactorial treatments addressing neuroprotection have shown to be effective in reducing anxiety progressively from the fi rst month to the month of treatment. the anxiety rate was declining from a baseline hrs-a score of . ± . to . ± . (p < . ) at month, . ± . (p < . ) at months, . ± . (p < . ) at months, . ± . (p < . ) at months, and . ± . (p < . ) at months of treatment (r = − . , a coef.: . , b coef.: − . ). similar striking results were found in depression, suggesting that improvement in mood conditions can contribute to stabilize cognitive function or that neuroprotection (with the consequent stabilization or improvement in mental performance) can enhance emotional equilibrium. , , , at baseline, all apoe variants showed similar anxiety and depression rates, except the apoe- / carriers who differed from the rest in a signifi cantly lower rates of anxiety and depression (figs. . and . ). remarkable changes in anxiety were found among different apoe genotypes (fig. . ) . practically, all apoe variants responded with a signifi cant diminution of anxiogenic symptoms, except patients with the apoe- / genotype who only showed a slight improvement. the best responders were apoe- / > apoe- / > apoe- / > apoe- / carriers (fig. . ) . the modest anxiolytic effect seen in apoe- / patients might be due to the very low anxiety rate observed at baseline. concerning depression, all apoe genotypes improved their depressive symptoms with treatment except those with the apoe- / genotype which worsen along the treatment period, especially after months (fig. . ). the best responders were patients with apoe- / > apoe- / > apoe- / > apoe- / , and the worst responders were patients harbouring the apoe- / genotype , , , (fig. . ) . the optimization of cns therapeutics requires the establishment of new postulates regarding (a) the costs of medicines, (b) the assessment of protocols for multifactorial treatment in chronic disorders, (c) the implementation of novel therapeutics addressing causative factors, and (d) the setting-up of pharmacogenetic/ pharmacogenomic strategies for drug development. , [ ] [ ] [ ] [ ] the cost of medicines is a very important issue in many countries because of (i) the growing of the aging population (> % disability), (ii) neuropsychiatric and demented patients (> % of the population) belong to an unproductive sector with low income, and (iii) the high cost of health care systems and new health technologies in developed countries. despite the effort of the pharmaceutical industry to demonstrate the benefi ts and cost-effectiveness of available drugs, the general impression in the medical community and in some governments is that some psychotropics and most anti-dementia drugs present in the market are not costeffective. , [ ] [ ] [ ] [ ] conventional drugs for neuropsychi-atric disorders are relatively simple compounds with unreasonable prices. some new products are not superior to conventional antidepressants, neuroleptics, and anxiolytics. there is an urgent need to assess the costs of new trials with pharmacogenetics and pharmacogenomics strategies, and to implement pharmacogenetic procedures to predict drug-related adverse events. , , , cost-effectiveness analysis has been the most commonly applied framework for evaluating pharmacogenetics. pharmacogenetic testing is potentially relevant to large populations that incur in high costs. for instance, the most commonly drugs metabolized by cyp d account for million prescriptions and us$ . billion annually in expenditures in the us, which represent - % of total utilization and expenditures for outpatient prescription drugs. pharmacogenomics offer great potential to improve patients' health in a cost-effective manner; however, pharmacogenetics/pharmacogenomics will not be applied to all drugs available in the market, and careful evaluations should be done on a case-by-case basis prior to investing resources in r&d of pharmacogenomic-based therapeutics and making reimbursement decisions. in performing pharmacogenomic studies in cns disorders, it is necessary to rethink the therapeutic expectations of novel drugs, redesign the protocols for drug clinical trials, and incorporate biological markers as assessable parameters of effi cacy and prevention. in addition to the characterization of genomic profi les, phenotypic profi ling of responders and non-responders to conventional drugs is also important (and currently neglected). brain imaging techniques, computerized electrophysiology, and optical topography in combination with genotyping of polygenic clusters can help in the differentiation of responders and non-responders. the early identifi cation of predictive risks requires genomic screening and molecular diagnosis, and individualized preventive programs will only be achieved when pharmacogenomic/pharmacogenetic protocols are incorporated to the clinical armamentarium with powerful bioinformatics support. - , , . an important issue in ad therapeutics is that antidementia drugs should be effective in covering the clinical spectrum of dementia symptoms represented by memory defi cits, behavioural changes, and functional decline. it is diffi cult (or impossible) that a single drug be able to fulfi l this criteria. a potential solution to this problem is the implementation of cost-effective, multifactorial (combination) treatments integrating several drugs, taking into consideration that traditional neuroleptics and novel antipsychotics (and many other psychotropics) deteriorate both cognitive and psychomotor functions in the elderly and may also increase the risk of stroke. few studies with combination treatments have been reported and most of them are poorly designed. we have also to realize that the vast majority of dementia cases in people older than - % are of a mixed type, in which the cerebrovascular component associated with neurodegeneration can not be therapeutically neglected. in most cases of dementia, the multifactorial (combination) therapy appears to be the most effective strategy. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the combination of several drugs (neuroprotectants, vasoactive substances, acheis, metabolic supplementation) increases the direct costs (e.g., medication) by - %, but in turn, annual global costs are reduced by approximately - % and the average survival rate increases about % (from to years post-diagnosis). there are major concerns regarding the validity of clinical trials in patients with severe dementia. despite the questionable experience with memantine, simi-lar strategies have been used to demonstrate the utility of donepezil in severe ad. this kind of studies bears some important pitfalls, including (a) short duration (< year), (b) institutionalized patients, (c) patients receiving many different types of drugs, (d) non-evaluated drug-drug interactions, (e) side-effects (e.g., hallucinations, gastrointestinal disorders) that may require the administration of additional medication, (f) lack of biological parameters demonstrating actual benefi ts, and (e) no cost-effectiveness assessment, among many other possibilities of technical criticism. [ ] [ ] [ ] , some of these methodological (and costly) problems might be overcome with the introduction of pharmacogenetic/ pharmacogenomic strategies to identify good responders who might obtain some benefi t by taking expensive medications. major impact factors associated with drug effi cacy and safety include the following: (i) the mechanisms of action of drugs, (ii) drug-specifi c adverse reactions, (iii) drug-drug interactions, (iv) nutritional factors, (v) vascular factors, (vi) social factors, and (vii) genomic factors (nutrigenetics, nutrigenomics, pharmacogenetics, pharmacogenomics). among genomic factors, nutrigenetics/nutrigenomics and pharmacogenetics/pharmacogenomics account for more than % of effi cacy-safety outcomes in current therapeutics. [ ] [ ] [ ] , , , some authors consider that priority areas for pharmacogenetic research are to predict serious adverse reactions (adrs) and to establish variation in effi cacy. both requirements are necessary in cns disorders to cope with effi cacy and safety issues associated with either current cns drugs and new drugs. , since drug response is a complex trait, genome-wide approaches (oligonucleotide microarrays, proteomic profi ling) may provide new insights into drug metabolism and drug response. genome-wide family-based association studies, using single snps or haplotypes, can identify associations with genome-wide signifi cance. , to achieve a mature discipline of pharmacogenetics and pharmacogenomics in cns disorders and dementia it would be convenient to accelerate the following processes: (a) educate physicians and the public on the use of genetic/genomic screening in the daily clinical practice; (b) standardize genetic testing for major categories of drugs; (c) validate pharmacogenetic and pharmacogenomic procedures according to drug category and pathology; (d) regulate ethical, social, and economic issues; and (e) incorporate pharmacogenetic and pharmacogenomic procedures to both drugs in development and drugs in the market to optimize therapeutics. 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diagnostics date: - - journal: nan doi: nan sha: doc_id: cord_uid: zz rd h the relu activation function (af) has been extensively applied in deep neural networks, in particular convolutional neural networks (cnn), for image classification despite its unresolved dying relu problem, which poses challenges to reliable applications. this issue has obvious important implications for critical applications, such as those in healthcare. recent approaches are just proposing variations of the activation function within the same unresolved dying relu challenge. this contribution reports a different research direction by investigating the development of an innovative quantum approach to the relu af that avoids the dying relu problem by disruptive design. the leaky relu was leveraged as a baseline on which the two quantum principles of entanglement and superposition were applied to derive the proposed quantum relu (qrelu) and the modified-qrelu (m-qrelu) activation functions. both qrelu and m-qrelu are implemented and made freely available in tensorflow and keras. this original approach is effective and validated extensively in case studies that facilitate the detection of covid- and parkinson disease (pd) from medical images. the two novel afs were evaluated in a two-layered cnn against nine relu-based afs on seven benchmark datasets, including images of spiral drawings taken via graphic tablets from patients with parkinson disease and healthy subjects, and point-of-care ultrasound images on the lungs of patients with covid- , those with pneumonia and healthy controls. despite a higher computational cost, results indicated an overall higher classification accuracy, precision, recall and f -score brought about by either quantum afs on five of the seven bench-mark datasets, thus demonstrating its potential to be the new benchmark or gold standard af in cnns and aid image classification tasks involved in critical applications, such as medical diagnoses of covid- and pd. sars-cov- is responsible for covid- , the 'severe acute respiratory syndrome coronavirus ' (cohen & normile, ) and the current global pandemic announced by the world health organization (who, mar ) . this virus leads to respiratory disease in humans (cui et al., ) , but it may take from to days for the initial symptoms, e.g., fever and cough, to become manifest after an infection (centers for disease control and prevention, ) . however, more severe symptoms can progress to viral pneumonia and typically require mechanical ventilation to assist patients with breathing (verity et al., ) . in some more severe cases, covid- can also lead to worsen symptoms and even death (zhou, et al., ) , as well as it may be an aetiology of pd itself (beauchamp et al., ) . thus, it is important to be able to detect neurodegenerative co-morbidities in vulnerable undiagnosed patients, such as pd, promptly and non-invasively too, for example via cnns that can recognise patterns from spiral drawings, and then applying non-ionising medical imaging techniques (bhaskar et al., ) , which are more appropriate for such patients, to facilitate a prompt diagnosis of covid- to improve clinical outcomes. whilst tremors can be detected from patterns in spiral drawings as indicators of early pd, ground-glass opacities, lung consolidation, bilateral patchy shadowing and relevant other lesionslike patterns can be detected as biomarkers to identify covid- -related pneumonia from any other types, including both viral and bacterial pneumonia (shi et al., ) . improvements in the afs of cnns can help to improve generalisation in both these image classification tasks. different layers of a deep neural network represent various degrees of abstraction, thus capturing a varying extent of patterns from input images (zeiler & fergus, ) . afs provide the cnn with the non-linearity required to learn from non-linearly distributed data, even in presence of a reasonable amount of noise. an af defines the gradient of a layer, which depends on its domain and the range. afs are differentiable and can be either saturated or unsaturated. in table the main activation functions commonly used in deep neural networks, including the convolutional neural network (cnn), with their equations and references, are summarised, and introduced below. saturated afs are continuous with their outputs threshold into finite boundaries, typically represented as s-shaped curves, also named 'sigmoidal' or 'squashing' afs, e.g., the logistic sigmoidal function with its output in the range of and (liew et al., ) . saturated afs are typically applied in shallow neural networks, e.g., in mlps. however, saturated afs lead to the vanishing gradient issue whilst training a network with back-propagation (cui, ) , i.e., results in gradients that are less than , which become smaller with multiple differentiations and ultimately become or 'vanish'. thus, changes in the activated neurons do not lead to modifications of any weights during back-propagation. moreover, the exploding gradient problem can occur, which has an opposite effect to vanishing gradients, wherein the error gradient in the weight is so high that it leads to instability whilst updating the weights during back-propagation. hyperbolic tangent or 'tanh' (see table ) is a further saturated af, but it attempts to mitigate this issue by extending the range of the logistic function from - to , centred at . nevertheless, tanh still does not solve the vanishing gradient problem. unsaturated functions are not bounded in any output ranges and are centred at . the rectified linear unit (relu) ( table ) is the most widely applied unsaturated af in deep neural networks, e.g., in cnns, which provides faster convergence than logistic sigmoidal (lecun et al., ) and tanh afs, as well as improved generalisation (litjens et al., ) . in fact, relu generally leads to more efficient updates of weights during the back-propagation training process (gao et al., ) . the relu's gradient (or slope) is either one for positive inputs or zero for negative ones, thus solving the vanishing gradient issue. nevertheless, despite providing appropriate initialisation of the weights to small random values via the he initialisation stage (glorot et al., ) , with large weight updates, the summed input to the relu activation function is always negative ('dying relu' problem) . this negative value yields a zero value at the output and the corresponding nodes do not have any influence on the neural network (abdelhafiz et al., ) , which can lead to misclassification resulting in lack of ability in detecting a pathology involved in an image classification task accurately and reliably, such as for covid- or pd diagnostics. in an attempt to mitigate the 'dying relu' issue, in cnns and deeper alexnet, vgg , resnet, etc.) , multiple variations of the relu af have been introduced, such as the leaky relu (lrelu), the parametric relu (prelu), the randomised relu (rrelu) and the concatenated relu (crelu), as summarised in table . maas et al. ( ) introduced leaky relu (lrelu) to provide a small negative gradient for negative inputs into a relu function, instead of being . a constant variable , with a default value of . , was used to compute the output for negative inputs ( another variant of relu, named 'exponential linear unit' (elu) is aimed at improving convergence (maas et al., ) (table ) , but it still does not solve the 'dying relu' issue either. klambauer et al. ( ) introduced a variant of elu called 'scaled exponential linear unit' (selu) ( table ) , which is a self-normalising function that provides an output as a normal distribution graph, making it suitable for deep neural networks with the output converging to zero mean when passed through multiple layers. although selu attempts to avoid both vanishing and exploding gradient problems, it does not mitigate the 'dying relu' issue. he et al. ( ) proposed the parametric rectified linear unit (prelu) in an attempt to provide a better performance than relu in large-scale image classification tasks, although the only difference from lrelu is that is not a constant and it is learned during training via back-propagation. nevertheless, due to this, the prelu does not solve the 'dying relu' issue either, as it is intrinsically a slight variation of the lrelu af. similarly, the randomised leaky rectified linear unit is a randomised version of lrelu (pedamonti, ) , whereby is a random number sampled from a uniform distribution, thus being still susceptible to the 'dying relu' issue too. shang et al. ( ) proposed a further slight improvement to the relu named 'concatenated relu' (crelu), allowing for both a positive and negative input activation, by applying relu after copying the input activations and concatenating them. thus, crelu is computationally expensive and prone to the 'dying relu' problem, although it generally leads to competitive classification performance with respect to the gold standard relu and lrelu afs (shang et al., ) . table . the main activation functions commonly used in deep neural networks, including the convolutional neural network (cnn), with their equations and reference. the relu and leaky relu are the most common and reliable ones in cnns. logistic sigmoid ( ) = + − han & moraga ( ) tanh gold & rangarajan ( ) arctan ( ) = − ( ) campbell et al. ( ) softplus despite the wide application of dl-based algorithms for image classification in healthcare, such as the cnn (lecun et al., ) described in . , its classical af, although it mitigates the vanishing gradient issue typical of sigmoid afs, can still experience the 'dying relu' problem. as discussed in . , none of the recently proposed afs, such as the lrelu, the prelu, elu and selu, have not solved this issue yet, as they are still algorithmically similar in their relu-like implementations. this issue can lead to lack of generalisation for cnns, thus hindering their application in a clinical setting. it is worth noting that, as an example, the last fully connected layer of the cnn in kollias et al. ( ) , having , neurons led, due to the 'dying relu' problem, to having only neurons yielding non-zero values. even by coupling a recurrent neural network (rnn) with their cnn, thus having a cnn-rnn (kollias et al., ) , and their last layer then being designed with neurons, only about of them led to non-zero values, whilst the remaining ones experienced the 'dying relu' issue, yielding negligible values. these two examples confirm that classical approaches to relu failed to solve its associated 'dying relu' problem, thus warranting a different approach, which the authors suggest being of quantum nature, as illustrated in . and motivated in . . quantum ml is a relatively new field that blends the computational advantages brought by quantum computing and advances in ml beyond classical computation (ciliberto, et al., ) . quantum ml has not only led to more effective algorithmic performance, but it has also enabled to find the global minimum in the solutions sought after in ml with a higher probability (ciliberto, et al., ) . the main principles of quantum computing are those inherited from quantum physics, such as superposition, entanglement, and interferences (barabasi et al., ) . according to the quantum principle of superposition, the fundamental quantum bit or qubit can have multiple states at any point in time, i.e., a qubit can have a value of either or , such as classical bits, but, differently from and beyond classical bits, a qubit can also have both values and concurrently (barabasi et al., ) . a quantum gate is the unification of two quantum states for them to stay 'entangled' into an individual quantum state, wherein a change in one state would affect the other one and vice versa (jozsa & linden, ) . thus, a system of qubits, each of which holds multiple bits of information concurrently, behaves as one via the quantum property named 'entanglement', hence enabling massive parallelism too (cleve et al., ; solenov et al., ) . however, existing quantum approaches to implement afs in deep neural networks have only adopted the repeat-until-success (rus) technique to achieve pseudo non-linearity due to restrictions to linear and unitary operations in quantum mechanics (nielsen & chuang, ; cao et al., ) . this rus approach to afs involves an individual state preparation routine and the generation of various superimposed and entangled linear combinations to propagate the routine of an af to all states at unison. thus, a deep neural network leveraging this quantum rus technique could theoretically approximate most nonlinear afs (macaluso et al., ) . nevertheless, the practical applications of this approach are very limited due to the input range of the neurons in such architectures being bounded between and π/ as a trade-off of their theoretically generic af formulation. hu ( ) led a similar theoretical research effort in proposing a sigmoid-based non-linear af, which is not periodic to enable a more efficient gradient descent whilst leveraging the principle of superposition in training neurons with multiple states concurrently. however, the classical form of the approach of hu ( ) is the traditional relu, thus still not solving the 'dying relu' problem either. konarac et al. ( ) leveraged a similar quantum-based sigmoid af in their quantum-inspired self-supervised network (qis-net) to provide high accuracy ( %) and sensitivity ( . %) in magnetic resonance image segmentation, improving performance by about % with respect to classical approaches. differently from the related studies mentioned above, the two properties of entanglement and superposition could be pivotal in devising a quantum-based approach to relu in having both a positive solution and a negative one simultaneously, being able to avoid a negative solution by preferring the positive one, whereas traditional classical relu at times would fail by leading to negative solutions only, i.e., the 'dying relu' problem. moreover, this principle enables quantum systems to reduce computational cost with respect to classical approaches, since several optimisations in multiple states can be performed concurrently (schuld et al., ) . as described in sections . and . , dl is highly suitable in classifying medical images due to its intrinsic feature extraction mechanisms. as illustrated in both . and . , the importance of the af is evident in both classical and quantum dl, respectively. although numerous variants of relu functions have been proposed in classical dl models (as revised in section . ) they have not been widely adopted as relu and lrelu. these two afs typically ensure accurate and reliable classification and are readily available in python open source libraries, such as tensorflow and keras. nevertheless, both these afs and any recent afs (see section . ) have not solved the 'dying relu' problem yet. moreover, vanishing and exploding gradient issues have not been fully resolved either. elu and selu may at times provide faster convergence than relu and lrelu, but they are not as reliable as those and are computationally more expensive (pedamonti, ) . such unresolved issues lead to lack of generalisation that may hinder the diagnostic accuracy and reliability of an application leveraging dl techniques for the detection of covid- or pd, thus resulting in a potentially high number of false negatives when the model's performance is evaluated on unseen patient data. the authors have hypothesised that this impaired generalisation is due to the classical approach underpinning such relu-based afs that has been just leveraged and moulded in various ways so far, without breaking its inherent functional limitations. the hereby contribution proposes, for the first time, that a quantum-based methodology to relu would improve the learning and generalisation in cnns with relevant impact for critical applications, such as the above-mentioned diagnostic tasks. in particular, by blending the two key quantum principles of entanglement of qubits and the effects of superposition to help reach the global minimum in the solution, thus avoiding negative solutions differently from classical approaches as in . , this study investigates the development of a novel af 'quantum relu' to avoid the problem of the 'dying relu' in a quantistic manner. this builds on recent research efforts by cong et al. ( ) to develop a quantum cnn that, although demonstrating how quantum states can be recognised, have not yet addressed the 'dying relu' problem, as it simply leveraged the traditional relus instead. patterns from lung ultrasound images and spiral drawings are known diagnostic biomarkers for covid- and pd respectively, pd being at times a delicate co-morbidity of covid- patients, and improvements in generalisation are key to an accurate and reliable early diagnosis that can improve outcomes, especially in the event of co-morbidities. thus, the novel quantum relu will be leveraged in a cnn to improve classification performance in such pattern recognition tasks, as quantified via clinically relevant and interpretable metrics, and compared against the same cnn with current gold-standard afs, including relu and lrelu. the proposed added capability of a quantum relu in a cnn is hypothesised to improve its generalisation for pattern recognition in image classification, such as detecting covid- and pd from ultrasound scans and spiral drawings, respectively. the remaining sections of the paper are structured as follows. section deals with the methods, including sub-section . illustrating the two novel quantum afs, along with their mathematical formulation and respective implementations in python codes (in both tensorflow and keras libraries). sub-section . provides a description of the benchmark datasets selected, along with a standardised data pre-processing strategy, whilst section summarises the results obtained comparing the accuracy, reliability and computational time of a cnn with the proposed quantum afs against salient gold standard afs outlined in table . eventually, section provides a thorough discussion of the results and section summarises the current work and outlines its access, impact, and future applications. despite appropriate initialisation of the weights to small random values via the he initialisation, with large weight updates, the summed input to the traditional relu activation function is always negative, although the input values fed to the cnn. current improvements to the relu, such as the leaky relu, allow for a more non-linear output to either account for small negative values or facilitate the transition from positive to small negative values, without eliminating the problem though. consequently, this study investigates the development of a novel activation function to obviate the problem of the 'dying relu' in a quantistic manner, i.e., by achieving a positive solution where previously the solution was negative. such an added novel capability in a cnn was hypothesised to improve its generalisation for pattern recognition in image classification, particularly important in critical applications, such as medical diagnoses of covid- and pd. thus, using the same standard two-layered cnn in tensorflow for mnist data classification, after identifying the main reproducible (with associated codes available in tensorflow and keras) afs following a critical review of the literature (section ), the following nine classical activation functions were considered: relu, leaky relu, crelu, sigmoid, tanh, softmax, vlrelu, elu and selu. a two-step quantum approach was applied to relu first, by selecting its solution for positive values ( ( ) = , ∀ > ), and the leaky relu's solution for negative values ( ( ) = × , ∀ ≤ , ℎ = . ) as a starting point to improve quantistically. by applying the quantum principle of entanglement, the tensor product of the two candidate state spaces from relu and leaky relu was performed and the following quantum-based combination of solutions was obtained: thus, keeping r(z) = z for positive values (z > ) as in the relu, but with the added novelty of the entangled solution for negative values ( ), the quantum relu (qrelu) was attained (fig. ) . the algorithms to describe the methodology and af were implemented in tensorflow and keras, and presented in listings and respectively, thus avoiding the 'dying relu' maintaining the positivity of the solution mathematically via this new quantum state. ()) model.add(layers.maxpooling d(( , ))) by leveraging the quantum principle of superposition on the qrelu's solution for positive and negative values, the following modified qrelu (m-qrelu) was obtained (fig. ) . the algorithms to describe the methodology and af were implemented in tensorflow and keras, and presented in listings and respectively, still avoiding the 'dying relu' issue: listing provides the snippet of code in python to leverage the m-qrelu in tensorflow, using 'py_func' per listing . its usage in tensorflow is the same as the 'qrelu' in listing but using 'tf_m_q_relu' as an activation function of the second convolutional layer ('conv _act' conv d( , ( , ) , input_shape=( , , ))) #model.add(qrelu()) model.add(m_qrelu()) model.add(layers. maxpooling d(( , ) )) the m-qrelu also satisfies the entanglement principle being derived via the tensor outer product of the solutions from the qrelu. thus, a quantum-based blend of both superposition and entanglement principles mathematically leads the qrelu and the m-qrelu to obviate the 'dying relu' problem intrinsically. as shown in ( ) and ( ), although the two proposed afs are quantistic in nature, both qrelu and m-qrelu can be run on classical hardware, such as central processing unit (cpu), graphics processing unit (gpu) and tensor processing unit (tpu), the latter being the type of runtime used in this study via google colab (http://colab.research.google.com/) to perform the required evaluation on the datasets described in . . the novel qrelu and m-qrelu were developed and tested using python . and written to be compatible with both tensorflow ( . and . tested, . supports tensorflow serving to deploy the novel afs on the cloud) and the keras sequential api. thus, both afs were programmed as new keras layers for ease of use. by selecting the positive quantum state of the summed input of the qrelu and m-qrelu, an optimal early diagnosis could be achieved for patients with covid- and pd. thus, this study demonstrates the qrelu and m-qrelu as a potential new benchmark activation function to use in cnns for critical image classification tasks, particularly useful in medical diagnoses, wherein generalisation is key to improving patient outcomes. to assess which af was suitable for each of the pattern recognition tasks involved in classifying the seven benchmark datasets as per . , the performance of the baseline cnn was assessed via the test or out-of-sample classification accuracy, precision, sensitivity/recall and f -score. precision, recall, and f -score are important metrics to measure the reliability of the classification outcomes. % confidence intervals (cis) were also reported. to enable reproducibility and replicability of the results obtained, publicly available benchmark datasets were gathered and used in this study, as mentioned below. moreover, to this purpose, full python codes (.py and .ipynb formats) in both ten-sorflow (https://www.tensorflow.org/) and keras (https://keras.io/) on how these were used for training the model, as well as to evaluate its performance, are also provided. as a general benchmark dataset for any image classifiers, especially cnns, the mnist data (lecun et al., ) , including , images of handwritten digits ( , images for training, , images for testing), was used for the initial model and af validation. this dataset is in tensor format available in tensorflow (https://www.tensorflow.org/datasets/catalog/mnist). to address the specific needs to improve diagnosis of parkinson's disease (pd) and that of covid- dealt with in this study, further benchmark datasets were used. four benchmark datasets were leveraged to identify pd based on patterns on spiral drawings ( subjects in total), as follows: as in the mnist dataset, images in all benchmark datasets were converted to grayscale and resized to be * . the two-layered cnn, designed as an mnist classifier, was initially validated on the mnist benchmark dataset itself, used for recognising handwritten digits. the qrelu and the m-qrelu were the best and second-best performing activation functions respectively, leading to an acc and an f -score of . ( %) and of . ( %) respectively (table ). the relu, the leaky relu and the vlrelu also led to the best classification performance on the mnist data (acc = . / %, f -score = . / %) (table ) . thus, the proposed qrelu achieved gold standard classification performance on this benchmark dataset. noteworthily, the qrelu and the m-qrelu led the same two-layered cnn architecture to achieve the best (acc = . / %, f -score = . / %) and third (acc = . / %, f -score = . / %) classification performance (table ) on the benchmark dataset named 'spiral handpd' on images of spiral drawings taken via graphic tablets from patients with pd and healthy subjects. as illustrated in table , competitive results were achieved by the qrelu and the m-qrelu versions on a further benchmark dataset on spiral drawings, the 'newhandpd dataset', leading to the sixth and eight classification performance respectively (acc = . / %, f -score = . / %; acc = . / %, f -score = . / %). very competitive outcomes were obtained by the two proposed quantum afs on the kaggle spiral drawings dataset, with m-qrelu (acc = . / %, f score = . / %) and qrelu (acc = . / %, f -score = . / %) leading to the second and fourth classification performance respectively (table ) , as well as when evaluated against the uci spiral drawings dataset (qrelu ranked fifth with acc = . / % and f -score = . / %; m-qrelu ranked sixth with acc = . / % and f -score = . / %) ( table ). the overall increased generalisation brought about by the two novel quantum afs is evident in the outstanding and mutually consistent classification outcomes achieved on both benchmark lung us datasets to distinguish covid- from both pneumonia and healthy subjects with the best (table -qrelu and m-qrelu with acc = . / % and f -score = . / %) and the second (table -qrelu and m-qrelu with acc = . / % and f -score = . / %) classification performance respectively for both qrelu and m-qrelu. despite a higher computational cost (four-fold with respect to the other afs except for the crelu's increase being almost three-fold), the results achieved by either or both the proposed qrelu and m-relu afs, assessed on classification accuracy, precision, recall and f -score, indicate an overall higher generalisation achieved on five of the seven benchmark datasets ( table on the mnist data, tables and on pd-related spiral drawings, tables and on covid- lung us images). consequently, the two quantum relu methods are the overall best performing afs that can be applied for aiding diagnosis of both covid- from lung us data and pd from spiral drawings. specifically, when using the novel quantum afs (qrelu and m-qrelu) as compared to the traditional relu and leaky relu afs, the gold standard afs in dnns, the following percentage increases in acc, precision, recall/sensitivity and f score were noted: • an increase of . % in acc and sensitivity/recall via m-qrelu as compared to relu and by . % with respect to leaky relu, thus avoiding the 'dying relu' problem when the cnn was evaluated on the kaggle spiral drawings benchmark dataset (table ); • an increase by . % in f -score via both qrelu and m-qrelu as opposed to leaky relu, hence obviating the 'dying relu' problem again but when tested on the covid- ultrasound benchmark dataset (table ) . • an increase of % in acc and sensitivity/recall via both qrelu and m-qrelu with regards to both relu and leaky relu, hence solving the 'dying relu' problem when evaluated on the pocus ultrasound benchmark dataset (table ). • an increase by , % in acc and sensitivity/recall via qrelu ( %) when compared to tanh ( % acc and sensitivity/recall), thus avoiding the vanishing gradient problem too, as assessed on the uci spiral drawings benchmark dataset (table ) . furthermore, it is worth noting the proposed quantum afs led to improved classification outcomes as compared to recent advances in relu afs, such as crelu and vlrelu: • qrelu led to acc, precision, sensitivity/recall, and f -score all higher by % those obtained via crelu when evaluating the cnn's classification performance on the mnist data (table ). • m-qrelu resulted in an acc and a sensitivity/recall higher by % than crelu, and an f -score greater by % on the spiral handpd dataset (table ) . • m-qrelu led to an acc and a sensitivity/recall greater by % than vlrelu, and an f -score also higher by % on the spiral handpd dataset (table ) . • m-qrelu resulted in an acc and a sensitivity/recall higher by % than vlrelu, and an f -score greater by % on the kaggle spiral drawings dataset (table ) . • qrelu and m-qrelu led to an acc and a sensitivity/recall greater by % and % than crelu and vlrelu respectively, and an f -score higher by % and % on the covid- ultrasound dataset (table ) . • qrelu and m-qrelu resulted in an acc and a sensitivity/recall higher by % than vlrelu, and an f -score greater by % on the pocus ultrasound dataset (table ) . the results obtained via the qrelu and m-qrelu in a two-layered cnn on the mnist dataset (table ) the two-layered cnn's classification performance via the proposed m-qrelu (acc = %, f -score = %, table ) was also higher by over % than the best performing five-layered cnns, whose hyperparameters were also optimised respectively via both the bat algorithm and particle swarm optimisation (pso) (pereira et al., c) , to aid diagnosis of pd from spiral drawings, such as using the 'spiral handpd' benchmark dataset. a comparable precision was achieved by the two-layered cnn model (table ) when the qrelu and m-qrelu were used as afs with respect to the best classifier so far on the covid ultrasound dataset, i.e., the sixteen-layered pocovid-net model, which builds on the vgg model (born et al., ) . table . results on performance evaluation of the first convolutional neural network having two convolutional layers, built in tensor-flow, and tested on the kaggle spiral drawings benchmark dataset. the size of the images was set to * , as per the mnist benchmark dataset. table . results on performance evaluation of the first convolutional neural network having two convolutional layers, built in tensor-flow, and tested on the university california irvine (uci) spiral drawings benchmark dataset. the kaggle spiral drawings benchmark dataset, which includes drawings from both healthy subjects and patients with parkinson's disease, was used for training and the uci spiral drawings benchmark dataset, which only has spiral drawings acquired during both static and dynamic tests from patients with pd, was deployed for testing. the size of the images was set to * , as per the mnist benchmark dataset. table . results on performance evaluation of the first convolutional neural network having two convolutional layers, built in tensor-flow, and tested on the covid- ultrasound benchmark dataset. the size of the images was set to * , as per the mnist benchmark dataset. further to the extensive review of existing relu afs provided in section . , also considering that classical approaches have been unable to solve the 'dying relu' problem as reviewed in section . , and taking into account the advantages of quantum states in afs (listed in section . ), two novel quantum-based afs were mathematically formulated in section . and developed in both tensorflow (listings and , https://www.tensorflow.org/) and keras (listings and , https://keras.io/) to enable reproducibility and replicability. thus, the mnist two-layered cnn-based classifier in tensor-flow was selected as the baseline model to assess the impact of using either quantum afs (qrelu and m-qrelu) on the classification performance on seven benchmark datasets as described in section . and evaluated based on test acc, precision, recall/sensitivity and f -score, as mentioned in section . . the proposed qrelu leads to the best classification performance on the mnist benchmark dataset (acc = %, f -score = %, table ) to recognise handwritten digits serves as a regression test to validate the hypothesis whereby, using the baseline cnn-based mnist classifier, the highest classification performance is achieved with the presumed best af. this hypothesis has been further confirmed by the m-qrelu achieving the second classification performance (acc = %, f score = %, table ) across all eleven afs evaluated as in . . achieving the same classification performance as the gold standard reproducible and replicable afs in cnns (relu, the leaky relu and the vlrelu)readily available in both tensorflow and kerasthe qrelu can be granted the designation of benchmark af for the task of handwritten digits recognition performed on the mnist benchmark dataset. the benefits of avoiding the 'dying relu' problem become evident when assessing the same two-layered cnn architecture with the qrelu especially (acc = . / %, f -score = . / %, table ), which achieved the best classification performance on critical image classification tasks, such as recognising pd-related patterns from spiral drawings in the 'spiral handpd' benchmark dataset. the higher generalisability achieved via the two proposed quantum afs in further support of the advantage of obviating the 'dying relu' issue is evident from the best classification performance in differentiating covid- from both bacterial pneumonia and healthy controls from the lung us data (table -qrelu and m-qrelu with acc = . / % and f -score = . / %). such an overall higher diagnostic performance is corroborated by the second-best classification outcomes attained on the second benchmark lung us dataset (table ) . whilst traditional relu approaches show highly variable classification outcomes, especially when they experience the 'dying relu' problem (tables , and ), both the qrelu and the m-qrelu were able to ensure a consistently higher classification performance and generalisation across the entire variety of image classification tasks involved, from the benchmark handwritten digits recognition task (mnist), to recognising pd-related patterns from spiral drawings taken from graphic tablets, to aiding detection of covid- from bacteria pneumonia and healthy lungs based on us scans. the advantage of using the proposed afs for covid- detection lies in the potential for their translational applications in a clinical setting, i.e., in leveraging cnns with the qrelu or m-qrelu to detect covid- in patients with neurodegenerative co-morbidities, such as pd, via non-ionising medical imaging (e.g., us). this added capability will come handy in future, as portable mri and ml-enhanced mri technologies will also become more affordable and widespread, thus being improvable with deep learning models (e.g., the two-layered cnn with qrelu or m-qrelu afs in this study). solutions either on edge devices or on the cloud for tele-diagnosis and tele-monitoring required in pandemics similar to the current one (covid- ) could be soon suitable for in-home diagnostic and prognostic assessments too, which should improve personalised care for shielded or vulnerable individuals. moreover, competitive outcomes were obtained via the qrelu and the m-qrelu on three further benchmark datasets, e.g., 'newhandpd dataset', the kaggle and the uci spiral drawings benchmark datasets, with acc and f -score mostly above % (tables - ) using the relatively simple deep neural network leveraged in this study (the two-layered mnist cnn classifier). such results also demonstrate the added capability of the proposed qrelu and the m-qrelu to avoid the vanishing gradient problem occurred using tanh ( % acc and sensitivity/recall), as evaluated on the uci spiral drawings benchmark dataset (table ) . despite the overall increase in generalisability brought about by the qrelu and the m-qrelu, the computational cost of the cnn increased by four times as compared to the other nine afs evaluated, except for the crelu, against which a threefold increase was reported (tables - ) . nevertheless, considering the importance of achieving higher classification performance over lower computational cost for diagnostic applications in a clinical setting, especially for the critical image classification tasks involved in this study, such as the detection of pd (tables - ) and covid- (tables and ) , this increase in computational cost is not expected to impair the wide application of the two novel quantum afs to aid such diagnostic tasks and any other medical applications involving image classification. in fact, the qrelu and m-qrelu have been demonstrated as considerably better than the current (undisputedly assumed) gold standard afs in cnns, i.e., the traditional relu and the leaky relu. in particular, an increase by - % in both accuracy and reliability (especially, sensitivity/recall and f -score) metrics was reported across both pattern recognition tasks, i.e., detection of pd-related patterns from spiral drawings (tables and ) and aiding diagnosis of covid- from us scans ( table ) . the two proposed quantum afs also outperformed more cutting-edge relu afs, such as the crelu and the vlrelu, by - % across all classification tasks considered, i.e., mnist data classification (table ) , spiral drawings pd-related pattern recognition (in particular, tables and ) , and covid- detection from us scans (tables and ) . moreover, the qrelu and the m-qrelu led the baseline two-layered cnn mnist classifier to achieve a comparable classification performance on the mnist dataset as deeper cnns, ranging from three to four layers (lecun et al., ; siddique et al., ; ahlawat et al., ) , including deeper architectures, e.g., resnet and densenet (chen et al., ) . it is worth noting that, when leveraging the qrelu and the m-qrelu, the two-layered cnn with hyperparameters based on the mnist data outperformed (acc = %, f -score = %, table ) deeper and ba-and pso-optimised cnns from published studies by over % (pereira et al., c) in aiding the diagnosis of pd from patterns in spiral drawings (e.g., using the 'spiral handpd' benchmark data). the two-layered cnn model with either qrelu or m-qrelu as afs achieved a comparable precision (table ) to the best-performing classifier on the covid ultrasound dataset, i.e., the sixteen-layered pocovid-net model, which is an extension of the vgg benchmark model (born et al., ) . these outcomes show the two main practical advantages brought about by the avoidance of the 'dying relu' problem in qrelu and the m-qrelu that outweigh the initial consideration on these two quantum afs leading to an overall higher computational cost despite the increased generalisation, which are as follows: . using qrelu or m-qrelu can obviate the need for several convolutional layers in cnns and any cnn-derived models, such as alexnet, resnet, densenet, condensenet, ccondensenet and vgg , as demonstrated above and in section (results), . leveraging qrelu or m-qrelu as afs in cnn can minimise the need for optimisation of cnn's hyperparameters. the implications of the two above-mentioned practical benefits are multiple. firstly, the two proposed afs may not only improve generalisation but also computational cost when considering image classification tasks that involve deeper architectures than the two-layered cnn used in this study. thus, the proposed afs may be viable alternatives to the relu af, which is the current gold standard af in cnns. second, by improving both generalisation and computational cost when deeper architectures may be required, the qrelu and m-qrelu may be suitable for tasks that require scalability of deep neural networks. third, the proposed quantum afs may enable more effective transfer learning, such as for covid- detection in multiple geographical areas, as well as extending trained deep nets to further diagnostic tasks, including prognostic applications too, and aiding self-driving vehicles in image classification tasks essential to ensure passenger safety. overall, the avoidance of the 'dying relu' problem achieved via qrelu and m-qrelu is expected to radically shift the paradigm of blindly relying on the traditional relu af in cnn and any cnn-derived models, and embrace innovative approaches, including quantum-based, such as the two novel afs designed, developed and validated in this study. further to a thorough analysis of the classification performance of the two-layered cnn mnist classifier leveraging the two quantum afs developed in this study, qrelu and m-qrelu, and evaluated against nine benchmark afs, including relu and its main recent reproducible and replicable advances, as well as relevant published studies, the proposed qrelu and m-qrelu prove to be the first two afs in the recorded history of deep learning to successfully avoid the 'dying relu' problem, by design. their novel algorithms describing the methodology and af were implemented in tensorflow and keras, as well as presented in listings - . this added capability ensured accurate and reliable classification for recognising pdrelated patterns from spiral drawings and detecting covid- from non-ionising medical imaging (us) data. furthermore, its availability in both google's tensorflow and kerasthe two most popular libraries in python for deep learning -facilitate their wide application beyond clinical diagnostics, including medical prognostics and any other applications involving image classification. thus, the qrelu and m-qrelu can aid detection of covid- during these unprecedented times of this pandemic, as well as deliver continuous value added in aiding the diagnosis of pd based on pattern recognition from spiral drawings. noteworthily, when leveraging the proposed quantum afs, the baseline cnn model achieved comparable classification performance to deeper cnn and cnn-derived architectures across all image recognition tasks involved in this study, from handwritten digits recognition, to detection of pd-related patterns from spiral drawings and covid- from lung us scans. thus, these outcomes corroborate the benefit of using afs that avoid the 'dying relu' problem for critical image classification tasks, such as for medical diagnoses, making them a viable alternative to the current gold standard af in cnns, i.e., the relu. this study is expected to have a radical impact in redefining the benchmark afs in cnn and cnn-derived deep learning architectures for applications across academic research and industry. improved handwritten digit recognition using convolutional neural networks (cnn) quantum computing and deep learning working together to solve optimization problems big data and machine learning in health care parkinsonism as a third wave of the covid- pandemic? chronic neurology in covid- era: clinical considerations and recommendations from the reprogram consortium. front. neurol pocovid-net: automatic detection of covid- from a new lung ultrasound imaging dataset (pocus) stability and bifurcation of a simple neural network with multiple time delays quantum neuron: an elementary building block for machine learning on quantum computers. arxiv 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change the practice of medicine leaky_relu | tensorflow core v . . tensorflow. . tf.keras.layers.leakyrelu | tensorflow core v . . estimates of the severity of coronavirus disease : a model-based analysis empirical evaluation of rectified activations in convolutional network visualizing and understanding convolutional networks distinguishing different stages of parkinson's disease using composite index of speed and pen-pressure of sketching a spiral clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study the authors would like to thank two research assistants from the university of bradford, ms smriti kotiyal and mr rohit trivedi, for their assistance to the background review relevant for this paper.the authors declare that no ethical approval was required for carrying out the study, as the data used in it were taken from publicly available repositories and appropriately referenced in text. moreover, the authors declare not to have any competing interests and an appropriate funding statement has been provided on the title page of this article. key: cord- -py awuav authors: willi, barbara; spiri, andrea m.; meli, marina l.; grimm, felix; beatrice, laura; riond, barbara; bley, tim; jordi, rolf; dennler, matthias; hofmann-lehmann, regina title: clinical and molecular investigation of a canine distemper outbreak and vector-borne infections in a group of rescue dogs imported from hungary to switzerland date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: py awuav background: canine distemper virus (cdv) is a major pathogen of dogs and wild carnivores worldwide. in switzerland, distemper in domestic dogs is rarely reported. in recent years, the import of dogs from eastern europe to switzerland has steadily increased. in the present study, we describe a distemper outbreak in rescue dogs that were imported from hungary to switzerland by an animal welfare organisation. the data on vaccination and medical history were recorded ( dogs), and the samples were collected to investigate cdv and vector-borne infections ( dogs) and canine parvovirus infection ( dogs). the dogs were monitored for six months. results: one dog was euthanised directly after import. thirteen dogs showed clinical signs after arrival, i.e., diarrhoea ( %), coughing ( %) and nasal and/or ocular discharge ( %); radiographic findings that were compatible with bronchopneumonia were present in four dogs. cdv infection was diagnosed in dogs ( %); dogs ( %) tested pcr-positive in conjunctival swabs. vector-borne infections (babesia spp., leishmania infantum, dirofilaria immitis) were found in dogs ( %). three dogs were hospitalized, and six dogs received ambulatory therapy for up to two months until recovery. none of the dogs developed neurological disease. cdv shedding was detected for a period of up to four months. because dogs were put under strict quarantine until cdv shedding ceased, cdv did not spread to any other dogs. the cdv isolates showed % sequence identity in the ha gene among each other and belonged to the arctic-like lineage of cdv. conclusions: the present study highlights the imminent risks of spreading contagious viral and vector-borne infections through the non-selective import of sick dogs and dogs with incomplete vaccination from eastern europe. cdv shedding was detected for several months after the cessation of clinical signs, which emphasised the roles of asymptomatic carriers in cdv epidemiology. a long-term follow-up using sensitive pcr and strict quarantine measures is of upmost importance in preventing the spread of infection. dog owners and animal welfare organisations should be educated regarding the importance of complete vaccinations and the impact of dog imports on the spread of viral and vector-borne pathogens. canine distemper virus (cdv) is one of the most important viral pathogens in domestic dogs and causes high morbidity and mortality worldwide, particularly in unvaccinated dogs or dogs with incomplete vaccination [ ] . cdv is a small, enveloped rna virus that belongs to the family paramyxoviridae and the genus morbillivirus [ ] . cdv has a wide natural host range that includes a variety of terrestrial carnivores [ ] . dogs are thought to be the major reservoir host for cdv [ , ] . infection occurs by direct contact with oronasal secretions of infected animals [ ] ; indirect transmission plays only a minor role in cdv epidemics because the virus is quickly inactivated in the environment [ ] . the course of the cdv infection is strongly dependent on the immune response in infected animals [ ] . in this context, vaccination is critically important. dogs that develop an adequate immune response can clear the virus from most tissues, whereas in dogs that show an intermediate immune response, cdv infects the epithelial tissues and induces clinical signs. in dogs that have a weak immune response, cdv disseminates to various tissues, and the clinical signs are usually severe with the persistence of the virus until death [ ] . invasion of the central nervous system occurs when viraemia is sufficiently high [ , ] . more than % of all cdv infections are perceived to be subclinical [ ] . in clinically affected dogs, the disease usually starts with fever and a serous-to-mucopurulent conjunctivitis, followed by a dry to productive cough, depression, anorexia, vomiting and diarrhoea [ ] . neurological signs usually develop within one to three weeks after recovery from systemic illness, but can occur weeks to months later [ ] . since the introduction of highly protective cdv modified live virus (mlv) vaccines more than years ago [ ] , the incidence of cdv infection in completely vaccinated dogs has decreased [ ] . however, in regions with a low proportion of vaccinated dogs, in stray dogs and in shelter environments, the incidence of cdv epidemics is high. in switzerland, the last cdv epidemic in domestic dogs occurred in - ; this outbreak was suspected to be attributed to an inadequate vaccination rate in the swiss dog population at that time [ ] . furthermore, a cdv epidemic associated with high morbidity and mortality commenced in the spring of in wild carnivores in switzerland [ ] . the latter was perceived to be part of a large transnational outbreak that spread from eastern to western europe. only one domestic dog was affected in switzerland during this outbreak [ ] . remarkably, the -year-old mixed breed dog died of a cdv-associated neurological disease, although it had received the standard anti-cdv vaccination protocol. according to the animal identity service (anis) in switzerland, the import of dogs increased by % within one year ( - ) [ ] . the imported dogs comprised primarily stray dogs that were adopted by animal welfare organizations or pure breed dogs to meet the increasing demand of miniature breeds in western europe. in the present study, we report on a distemper outbreak in rescue dogs that had been imported from hungary to switzerland. the study provides data on vaccination, medical history, clinical examinations and diagnostic imaging of the dogs and cdv testing, testing for canine parvovirus (cpv) and vector-borne infections. additionally, the study gives prospectively collected follow-up data on the treatment, clinical course and outcome of the infections and the period of cdv shedding. finally, a molecular characterization of the cdv isolates was performed. a group of rescue dogs that derived from a shelter in kecskemét, hungary, was imported to switzerland in october . one dog was euthanised within several days of import because of clinical deterioration; no data regarding this dog were available. the other dogs comprised nine female ( spayed) and five male ( castrated) mixed breed dogs, aged months to years old, and weighing kg to kg ( table ). all of the dogs had received rabies vaccination (rabisin®, biokema sa, crissier, switzerland) six to weeks before import and deworming (containing praziquantel, pyrantel and fenbendazol, uniwerm®, provet, beograd, serbia) seven to nine days before their arrival in switzerland. additionally, the dogs had been vaccinated with one shot of a combined mlv vaccine containing cdv, canine adenovirus- , cpv, leptospira spp. and canine parainfluenzavirus (biocan® dhppi & l, table ), either seven to eight days (dogs to and to ) or one month prior to arrival in switzerland (dog ); dog had been revaccinated in switzerland one week prior to sample collection for cdv pcr (table ) . after arrival on october , , the rescue dogs were directly distributed to private households throughout switzerland (table , fig. ). seven dogs (dogs , , , , , and ) were placed in multidog households. after arrival, the new owners observed clinical signs in of the dogs (table ) , i.e., diarrhoea ( %), coughing ( %), nasal and/or ocular discharge ( %), vomiting ( %), gagging ( %), lameness ( %), apathy and sneezing (each %). because of these symptoms, five dogs (dogs , , , and ) were presented to private veterinarians within one week of arrival. three of these dogs (dogs , and ) were subsequently referred to small animal clinics for additional investigations. haematology and blood biochemistry results were available for dogs (tables and ), of which were cdv-pcr positive (dogs to , see below). at initial presentation, anaemia ( / , %), leucocytosis ( / , %), eosinophilia ( / , %), neutrophilia ( / , %) and monocytosis ( / , %) were common ( table ) . dogs and showed severe pancytopenia and moderate bicytopenia, respectively; both were cdv pcr-positive, co-infected with babesia spp. (dog ) or positive for anti-leishmania infantum antibodies (dog , see below), and they exhibited fever, increased inspiratory lung sounds, purulent ocular and nasal discharge and radiographic signs that were compatible with bronchopneumonia (tables to ). dog showed slight anaemia and leucopenia ( table ) ; this animal was co-infected with cdv and babesia spp. (tables and ) . dog showed a pronounced eosinophilia ( table ) ; this animal was cdv-pcr negative but dirofilaria immitis positive (tables and ). blood biochemistry results revealed only unspecific changes in the dogs (table ) . the radiographic examinations of the thorax revealed moderate-to-severe interstitial lung changes with variable bronchial thickening in four dogs (dogs - ). the changes were generalised and most pronounced in the dorsal (dog ), perihilar (dogs and ) and caudal lung areas (dog , fig. ). the radiographic findings were compatible with bronchopneumonia in all four dogs, and all of the dogs tested cdv pcr-positive. the thoracic radiographs of dog revealed mild right-sided cardiomegaly and mild generalised bronchointerstitial lung changes; echocardiography showed mild tricuspid and aortic regurgitation but no signs of pulmonary hypertension or right ventricular pressure overload. dog tested d. immitis-positive but was negative for cdv. eleven of the dogs tested cdv pcr-positive during the initial examination ( table ). the positive pcr results were most commonly obtained from conjunctival swabs ( of the cdv-positive dogs, table ). the vaccine-specific real-time reverse transcription (rt)quantitative (q)pcr was negative for all ten dogs that were tested, which supports the finding of infection with a wild-type cdv strain. all three vaccines that were tested (biocan® dhppi & l, bioveta, ivanovice na hané, czech republic; nobivac® dhhpi, msd animal health, luzern, switzerland; canigen® sha ppi, vibac, glattbrugg, switzerland) exhibited a positive pcr result. in gel electrophoresis of the pcr products, a appropriate-sized band was detected for all ten dogs, as were the three vaccines, as expected [ ] . the sequence of the amplification product of the biocan® dhppi & l vaccine used in the dogs of the present study was clearly distinct from the sequence alb albumin; ap alkaline phosphatase; alat alanine aminotransferase; na sodium; k potassium; p phosphorus of the cdv isolates of the ten rescue dogs (fig. ) and most closely related to the cdv vaccine strain onderstepoort ( % nucleotide identity to ab ). sequencing of the ha gene of cdv isolates of five of the infected dogs revealed that the dogs were infected with a similar cdv strain ( . - % nucleotide identity among the ha gene of cdv isolates of dogs , , and ); the ha gene of the cdv isolate of dog differed in only one nucleotide position from the ha gene sequences of the other four cdv isolates. the phylogenetic analysis revealed that the isolates from the five import dogs belonged to the arctic-like lineage of cdv (fig. ) . the ha gene sequences of the cdv isolates were most similar to a published ha gene sequence of a cdv strain from a domestic dog from italy (kf , % nucleotide identity, fig. ) [ ] . they were only distantly related to cdv strains isolated during a cdv epidemic in wild carnivores in switzerland (jf and jf , . % nucleotide identity, fig. ) overall, two dogs (dogs and ) exhibited cdv pcrnegative results one month after the initial examination (table ). two months after the initial examination, another three dogs were found to be cdv pcr-negative (dogs to ); three months after the initial examination, dog was cdv-negative and the remaining three dogs tested cdv-negative four months (dog ) and five months (dogs and ) after the initial examination (table ) . all twelve dogs that were tested for cpv at the initial presentation were pcr-negative (table ) . vector-borne infections were detected in dogs ( %, table ): infection with babesia spp. was detected in dogs and ; infection with l. infantum was diagnosed in dog and infection with dirofilaria immitis was found in dog . dog , which tested positive in d. immitis antigen and knott tests, had received a certificate from a laboratory in budapest, hungary, that stated a negative result in the knott test in august . none of the rescue dogs tested positive for ehrlichia canis (table ) . the cdv-infected dogs , and were hospitalized for two to three days and received intravenous infusions of crystalloids, intravenous antibiotic therapy and inhalation (table ) . dog , which was co-infected with babesia spp., was treated with two imidocarb injections two weeks apart. dog , which was co-infected with l. infantum was treated using allopurinol. all three dogs showed rapid clinical improvement with treatment and were discharged with oral antibiotic therapy. repeated haematological examination in dog two weeks later revealed that the pancytopenia had resolved and had returned to moderate neutrophilia, eosinophilia and slight monocytosis. mild anaemia was still present in dog at that time (data not shown). repeated haematology in dog two weeks after the initial presentation showed normal platelets counts and nearly normal pcv values (data not shown). all three dogs were clinically asymptomatic in the -month follow-up period. dog was ambulatory and was treated with oral antibiotics and antibiotic eye drops ( table ). the dog showed several relapses with purulent nasal and ocular discharge after antibiotic therapy ceased and was repeatedly treated with antibiotics for two months. thereafter, there was no relapse, and the dog was clinically asymptomatic in the remaining -month follow-up period. five cdv pcr-positive dogs (dogs to and ) received oral antibiotic therapy (amoxicillin clavulanic acid or doxycycline) for seven to ten days after their arrival in switzerland. dog developed watery diarrhoea two weeks after arrival and was additionally treated with metronidazole, deworming and a highly digestible diet. dog , which was co-infected with babesia spp., received two injections of imidocarb diproprionate two weeks apart and antibiotic ear drops because of otitis externa. at the end of the -month follow-up period, all of the cdv pcr-positive dogs had recovered and none had developed neurological signs. all of the owners of the cdv pcr-positive dogs were instructed by the first author (bw) to quarantine the dogs until they tested cdv pcr-negative. the owners of the cdv-positive dogs in multidog households (dogs , , , and ) were instructed to separate the infected dog from the other dogs in the household. however, several dogs (dogs , , and ) had already had contact with adult dogs within the household at the time when the cdv diagnosis was made. all of the contact dogs had been vaccinated against cdv, although several dogs had only received the initial vaccination series as puppies and had received no booster vaccinations (data not shown). two dogs that were in close contact with dogs and were tested for the cdv infection with pcr one month and two months after the initial cdv diagnosis in dogs and . one contact dog exhibited a single, very weak cdv-positive result in the conjunctival swab in the first sampling but was negative in all of the swabs collected one month later (data not shown). the other contact dog tested pcr-negative in all of the collected samples (data not shown). in all of the other multidog households, no samples were collected for cdv pcr, but no clinical signs of the disease were noted in the month follow-up period. the present study describes a distemper outbreak in rescue dogs in switzerland that had been imported by an animal welfare organization. one dog had to be and of ten wild-type cdv isolates of the rescue dogs (dogs to and to ) is shown. grey-shaded letters: identical nucleotides between vaccine strains and wild-type isolates. black letters: nucleotides that differ between vaccine strains and wild-type isolates. grey bars: schematic drawing depicting the positions of the primers and the probe used in the cdv vaccine specific real-time rt-qpcr assay [ ] . consensus: consensus sequence of % identical bases matching all of the sequences (most of the ambiguities) euthanized directly after import for humane reasons, whereas nine dogs required therapy for up to two months; three of these dogs were hospitalized at veterinary clinics. the imported animals shed cdv for up to four months, which necessitated long-term quarantine measures. moreover, four of the dogs were infected with vector-borne pathogens. the present study underscores the risk of introducing contagious or vector-borne pathogens to central european countries by importing rescue dogs with incomplete vaccination. based on the data of the anis in switzerland, . % of the newly registered dogs in were imported [ ] . the imported dogs primarily comprised small and miniature purebred dogs to meet the increasing demand for these types of breeds in switzerland, and mixed breed dogs that are rescued by animal welfare organisations [ ] . the dogs of both groups are at risk for carrying infectious diseases because of the inadequate vaccination policies and quarantine measures in many breeding kennels and animal shelters in eastern europe. in the animal shelter in hungary where the dogs originate, no quarantine measures or reliable vaccination policies had been implemented. the dogs had only received a single cdv vaccination either one week or one month before importation to switzerland. at this time point, several dogs had likely been infected with cdv. after the outbreak, the rescue organisation was instructed to introduce vaccination policies and quarantine measures within the animal shelter. in switzerland, the vaccination rate in dogs is insufficiently high to provide population immunity against the cdv infection. the proportion of vaccinated dogs in a population must be > % to provide protection for the dog population, in contrast to protection of only a single vaccinated dog [ ] . a vaccination rate of - % has been estimated for swiss dogs based on the numbers of sold vaccine doses in [ ] . mandatory courses for new dog owners have been introduced in switzerland in which freshly adopted dogs come in close contact with each other. together with the decreasing vaccination rate and the increasing number of imported dogs, this situation has clearly increased the risk for canine distemper outbreaks. dog owners and animal welfare organisations should be informed concerning the critical importance of complete vaccination schedules in domestic dogs. in the present outbreak, the infection could be prevented from spreading to other dogs by extensive followup examinations of the dogs and a thorough education of the dog owners concerning the critical importance of strict quarantine measures. luckily, no cdv-positive dogs of this study had already had contact with young unvaccinated dogs at the time of diagnosis. the cdv vaccines are known to induce a strong and long-lasting immunity when no interference with maternally derived antibodies occurs [ , ] . cdv was reported to be shed by infected animals for up to three months [ , ] . we demonstrated cdv shedding in some dogs in the present study even for up to four months. shedding was detected for several months after the cessation of clinical disease. our results underscore the role of asymptomatic carriers in cdv epidemiology and the importance of a long-term followup of cdv-positive dogs for preventing the spread of infection. several studies have reported the excretion of vaccine strains after cdv vaccination [ , ] . this was not observed in the present study: the dogs that tested cdv pcr-positive were shown to be infected with wildtype cdv, although the majority of the dogs had received mlv cdv vaccination within one to seven weeks before pcr testing. consistent with the published data [ ] , pcr from conjunctival swabs was found to be most reliable for detecting cdv in acutely infected animals and during follow-up examinations. however, in one animal, only the nasal but not the conjunctival swab tested positive for cdv. nine of the dogs in the present study were hospitalized or required ambulatory therapy for up to two months. another animal was euthanised directly after import because of clinical deterioration. in all of the affected dogs, the respiratory and gastrointestinal symptoms predominated. in four dogs, signs of bronchopneumonia were evident. remarkably, none of the dogs developed a neurological disease. whether the latter was due to the intrinsic properties of the cdv strain or to the age and immune status of the dogs is unknown. the sequence analyses of the cdv strains detected in the rescue dogs indicated that all of the dogs were infected with the same wild-type cdv strain, which belongs to the arctic-like lineage of cdv [ , ] . the initial description of arctic-like lineage of cdv dates back to the late s, when epizootics were observed in seals in northern europe and siberia [ ] [ ] [ ] . (see figure on previous page.) fig. phylogenetic relationship between selected cdv strains based on the complete haemagglutinin (ha) gene sequence. the cdv isolates analysed in this study appear in bold. nine cdv lineages are shown: asia- , europe, america- , europe-wildlife, africa, arctic-like, asia- , asia- and america- . phocine distemper virus (pdv- ) was used as the outgroup. genbank accession numbers, host species and geographical origin are indicated, if known. the numbers at the nodes were generated from bootstrap resamplings; only values > are shown. the bar represents the mean number of differences per sites. strain af (giant panda isolate) is the resultant of a genetic recombination between "asia- " and a "europe-wildlife" strain [ ] the strains of the arctic-like lineage are closely related to the cdv strains in north america, china and greenland and were recently isolated from domestic dogs in hungary [ ] and domestic dogs and wolves in southern italy [ , ] . the present study shows how fast these strains can spread to central european countries by import of infected domestic dogs. the present study indicates that rescue dogs may also play an important role in the spread of vector-borne infections to central european countries. babesia spp., l. infantum or d. immitis infections were detected in four of the rescue dogs. several dogs were reported to be free of these pathogens based on the laboratory certificates provided by the animal welfare organisation. in the case of dog , d. immitis infection was diagnosed in this study using the antigen enzyme immunoassay and the knott test in november . the dog was found to be negative in the knott test in august . the d. immitis antigen and knott tests are known to turn positive not before five to eight months after infection [ ] ; therefore, the infection could have been missed in the first testing. l. infantum serology specimen, which was positive in dog , has similar limitations in that negative serological results cannot exclude infection and seroconversion can occur many months after infection [ ] . future dog owners should be properly informed concerning the limitations of these tests and the costs of treatment for vector-borne infections. the present study highlights the risks of spreading contagious viral and vector-borne infections by a nonselective import of sick and unvaccinated dogs or dogs with incomplete vaccination from eastern european countries. the animal welfare organisations should be thoroughly informed concerning the critical importance of complete vaccination schedules and quarantine measures in animal shelters to combat the outbreaks and the spread of viral pathogens to other countries. dog owners in switzerland should be educated regarding the risk of and the potential costs of adopting sick dogs or dogs with incomplete vaccination and the critical importance of sufficiently high vaccination rates in domestic dogs in switzerland. the present study describes a distemper outbreak in fifteen dogs originating from an animal shelter in kecskemét, hungary, and the prospective follow-up of the infected animals. the dogs were imported to switzerland by an animal welfare organisation on october , . one dog had to be euthanised within a several days of arrival. no data on that dog were available. of the remaining fourteen dogs, the signalment, vaccination and medical history and clinical signs were recorded (table ) and dogs were monitored for six months. sample and data collection and sample processing nasal and/or conjunctival swabs were collected from dogs for cdv-specific real-time rt-qpcr and rectal swabs from dogs for cpv real-time qpcr as indicated in tables and . edta blood and serum samples were collected to obtain haematology and blood biochemistry results, and for cdv real-time rt-qpcr and testing for vector-borne infections (babesia spp., e. canis, l. infantum and d. immitis) as indicated in tables through . all of the samples were processed within h of collection. in of the cdv-infected dogs (dogs to and to ), monthly follow-up examinations for cdv were performed from conjunctival, nasal and oropharyngeal swabs ( table ). the swabs were collected by the owner based on written and image instructions regarding how to collect and ship the samples. all of the swabs were sent by priority mail to the clinical laboratory, vetsuisse faculty, university of zurich, within one day of collection. follow-up was continued in each dog until the dog tested cdv pcrnegative, except for dog , in which the owner declined further testing despite a pcr-positive result at three months of follow-up. haematology and blood biochemistry tests were performed at the clinical laboratory, vetsuisse faculty, university of zurich (dogs and , to and ), at the clinical diagnostic laboratory, vetsuisse faculty, university of bern (dog ), at a private laboratory (dog : alomed, radolfzell-böhringen, germany) or by a private veterinarian (dog , tables and ). the laboratory's own device-specific reference intervals were applied; for dogs aged to months (dogs , to ), published reference intervals for phosphorous, alkaline phosphatase, urea and creatinine were used [ ] . at the time of the initial examinations, conjunctival, nasal or rectal swabs were incubated for min in μl of phosphate buffered saline (pbs) at °c; the swabs were subsequently turned upside down and centrifuged for min at × g and the supernatant was used for tna extraction (see below). for the follow-up examinations, the two conjunctival and the two nasal swabs, respectively, were pooled in a total of μl of pbs before incubation at °c for min and tna extraction. the tna extraction was performed from μl of edta blood, μl of swab supernatant or vaccine material that was resuspended in μl of pbs (see below) using the magna pure lc (roche diagnostics ag, rotkreuz, switzerland) and the magna pure lc tna isolation kit (roche diagnostics) following the manufacturer's instructions. with each batch of extraction, a negative control consisting of μl of pbs was used to monitor for cross-contamination. the tna was stored at − °c until pcr analysis was performed. for cdv testing, a published real-time rt-qpcr assay was used as previously described [ ] . for the detection of cpv, a published real-time qpcr assay developed for the detection of feline parvovirus (fpv) was applied [ ] . the assay amplifies a bp sequence of the highly conserved vp /vp gene region using the following primers and probe: pv f: ′-actgcatcattgat ggttgca- ′; pv r: ′-ggtatggttggtttc catgga- ′ pv p: ′-fam-cccaatgtctcaga tctcatagctgctgg- -tamra- ′. sequence comparison revealed that the primer and probe-binding sites in the vp /vp gene region of fpv and cpv showed > % sequence identity (genbank accession numbers m , m , m , m , m , m , m , km , jq ). during cdv follow-up, a published canine (c)gapdh pcr assay was applied to ensure that the quality of the swabs collected by the dog owners was adequate [ ] . during follow-up, dogs were stated cdv-negative if they tested pcr-negative for cdv in conjunctival, nasal and oropharyngeal swabs and the swabs showed a cgapdh threshold cycle below . all of the real-time pcr reactions were run using an abi fast real-time pcr system (applied biosystems, rotkreuz, switzerland). negative and positive controls were included in each pcr run. to ensure that the cdv real-time rt-qpcr signal was due to infection and was not due to a recent vaccination, a published real-time rt-qpcr system based on the cdv m gene and m-f intergenic region was used [ ] . the primers of the pcr assay amplify the mlv vaccine and wild type cdv strains, whereas the probe of the assay only binds to the mlv vaccine strains (fig. ) . because no sequence data have been published on the m gene and the m-f intergenic region of cdv u strain contained in the vaccine used in the rescue dogs (biocan® dhppi & l, bioveta), the latter vaccine, together with two other cdv vaccines that are commercially available in switzerland (nobivac® dhhpi, msd animal health; canigen® sha ppi, vibac) were tested to confirm that the cdv strains contained in these vaccines are detected by the assay. the tna from the three vaccines and from the conjunctival or nasal swabs from of cdv pcr-positive dogs (dogs to and to ) were subjected to real-time rt-qpcr. the pcr products were separated using . % agarose gel and appropriate-sized bands ( bp) cut out, extracted using the minelute® gel extraction kit (qiagen, hombrechtikon, switzerland) and sequenced (microsynth, balgach, switzerland). the complete haemagglutinin (ha) genes of the cdv isolates from five dogs were sequenced (dogs , , , and ). the tna extracted from the conjunctival swabs was used as a template. for amplification, five previously published primer pairs were used [ ] ; single nucleotides in one primer pair ( f and r) had to be changed because of mismatches within the primer binding sites for the five cdv isolates ( f_new: '-ctgtacat caccaagtcata- ' and r_new: '-tagaatac catcttgtgaat- '). reverse transcription was performed using the high capacity cdna reverse transcription kit (applied biosystems) according to the manufacturer's instructions. the pcr amplification was conducted using μl of x hf pcr buffer (finnzymes, bioconcept, allschwil, switzerland), nm each primer, μm each dntp (sigma-aldrich, buchs, switzerland), u phusion high-fidelity dna polymerase (finnzymes), and . μl template cdna made up to μl with water. the thermal cycling conditions comprised °c for min, cycles at °c for s, °c for s, °c for min, and a final elongation at °c for min. after the pcr run, the amplification products were separated using % agarose gel; appropriately sized products were excised and purified using the minelute gel extraction kit (qiagen). direct sequencing of the purified amplicons was performed using the amplification primers in a commercial laboratory (microsynth, balgach, switzerland) under standard conditions. vector-borne infections were tested in dogs, as shown in table . the analyses were performed at the clinical laboratory (for e. canis) and the institute of parasitology (for babesia spp., l. infantum and d. immitis) of the vetsuisse faculty, university of zurich, and at private laboratories (dog : labor am zugersee, hünenberg, switzerland; dog : alomed; dog : idexx diavet ag, bäch, switzerland) ( table ). the laboratories' own reference values were used for defining the positive, negative and intermediate results. the microscopic blood smear evaluation for the presence of babesia spp. organisms was conducted in dog by the private veterinarian and in dog by a commercial laboratory (alomed) ( table ) . the obtained sequences were edited and aligned with a consensus sequence using geneious version . . [ ] . only the nucleotides available for all of the included sequences ( nucleotides of the ha gene) were used to calculate the percent nucleotide identities and perform the phylogenetic analyses. for the phylogenetic analyses, the sequences were aligned with known distemper sequences from genbank (see fig. ) using geneious version . . . a bootstrap phylogenetic tree demonstrating the relationship between the isolates was created using the maximum-likelihood method [ ] and a distance matrix corrected for nucleotide substitutions based on the kimura -parameter model [ ] . the dataset was resampled times to generate bootstrap values. the phylogenetic and molecular evolutionary analyses were conducted using the mega version [ ] . nucleotide sequences obtained in this study have been submitted to genbank under accession numbers kr to kr . canine distemper spillover in domestic dogs from urban wildlife canine distemper in terrestrial carnivores: a review canine distemper virus-a 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emergence of canine distemper in bavarian wildlife associated with a specific amino acid exchange in the haemagglutinin protein geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data prospects for inferring very large phylogenies by using the neighbor-joining method a simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences molecular evolutionary genetics analysis version . cross-species recombination in the haemagglutinin gene of canine distemper virus evaluation of enzyme-linked immunosorbent assays, an immunofluorescent-antibody test, and two rapid tests (immunochromatographic-dipstick and gel tests) for serological diagnosis of symptomatic and asymptomatic leishmania infections in dogs submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors thank the animal rescue organisation and the owners of the dogs for providing the data and supporting diagnostic work-up. we thank t. meili, e. goenczi, s. childers and the technicians of the clinical laboratory for their excellent laboratory assistance. the laboratory work was performed using the logistics of the center for clinical studies, vetsuisse faculty, university of zurich. as was supported by a research grant (forschungskredit, fk- - - ) from the university of zurich. the authors declare that they have no potential conflicts of interest to disclose.authors' contributions rhl and bw conceived the study. bw and ams were responsible for the study coordination and the data and sample collections. lb, tb and rj were responsible for the clinical work-up and medical care of the dogs. mlm was responsible for the molecular laboratory aspects, fg was responsible for the analyses of vector-borne infections, br was responsible for the haematology and blood biochemistry tests and md was responsible for all aspects of diagnostic imaging. rhl and bw drafted the manuscript. all of the authors read and approved the final manuscript. key: cord- - trqex authors: dana, dibyendu; pathak, sanjai k. title: a review of small molecule inhibitors and functional probes of human cathepsin l date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: trqex human cathepsin l belongs to the cathepsin family of proteolytic enzymes with primarily an endopeptidase activity. although its primary functions were originally thought to be only of a housekeeping enzyme that degraded intracellular and endocytosed proteins in lysosome, numerous recent studies suggest that it plays many critical and specific roles in diverse cellular settings. not surprisingly, the dysregulated function of cathepsin l has manifested itself in several human diseases, making it an attractive target for drug development. unfortunately, several redundant and isoform-specific functions have recently emerged, adding complexities to the drug discovery process. to address this, a series of chemical biology tools have been developed that helped define cathepsin l biology with exquisite precision in specific cellular contexts. this review elaborates on the recently developed small molecule inhibitors and probes of human cathepsin l, outlining their mechanisms of action, and describing their potential utilities in dissecting unknown function. lysosomes play critical roles in human biology receiving, trafficking, processing, and degrading biological molecules from seminal cellular processes, such as endocytosis, phagocytosis, autophagy and secretion. discovered by the ground-breaking work of de duve, these single-membrane enclosed cytosolic organelle maintain an acidic (~ . ph environment, and house close to sixty proteolytic enzymes [ , ] . among these are the eleven members of the cysteine cathepsin enzymes with a versatile expression and functional profile: cathepsin l (l ), b, c, f, h, k, o, v (l ), x, s, and w [ ] . these enzymes closely mimic the ca clan of the papain structure and catalytic cycle and mediate numerous crucial cellular events. for example, they participate in processes involving cell death, protein degradation, post-translational modifications of proteins, extracellular matrix (ecm) remodeling, autophagy, and immune signaling. given that their functions are aberrantly dysregulated in several human diseases, many are considered prime targets for therapeutic development [ ] . several elegant reviews have recently emerged describing the importance of cysteine cathepsins in both normal physiology and human diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the focus of this review is specifically on human cathepsin l, a ubiquitously expressed endopeptidase whose involvement in several human diseases has emerged in recent years. these include liver fibrosis, type i and ii diabetes, cardiac and bone, immune and kidney disorders [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in addition, its role in a wide variety of highly invasive forms of cancer is the propeptides act as an important regulatory on/off switch as well as a folding catalyst in cathepsin activation. not surprisingly, the nature of propeptides among cysteine cathepsins is highly divergent by both chain lengths and primary sequences. it is thought that this uniqueness is functionally relevant given its ubiquitous presence in most tissues and allows for the selective suppression of enzyme activity (hence unintended autoactivation) during the transport to the the propeptides act as an important regulatory on/off switch as well as a folding catalyst in cathepsin activation. not surprisingly, the nature of propeptides among cysteine cathepsins is highly divergent by both chain lengths and primary sequences. it is thought that this uniqueness is functionally relevant given its ubiquitous presence in most tissues and allows for the selective suppression of enzyme activity (hence unintended autoactivation) during the transport to the endolysosomal compartment. molecules , , of in cathepsin l, two inhibitory propeptides, one containing amino acid (thr -glu ) and the other containing amino acid (glu -asp ) exist. a crystal structure of human procathepsin l revealed that the amino acid inhibitory propeptide chain spans in the opposite directions of substrate binding and forms several high-affinity non-covalent interactions with the surrounding residues in active site [ , ] . interestingly, this opposite direction binding of inhibitory propeptide segment is evolutionarily conserved in other members of cysteine cathepsins, including in cathepsin b. molecules , , of endolysosomal compartment. in cathepsin l, two inhibitory propeptides, one containing amino acid (thr -glu ) and the other containing amino acid (glu -asp ) exist. a crystal structure of human procathepsin l revealed that the amino acid inhibitory propeptide chain spans in the opposite directions of substrate binding and forms several high-affinity non-covalent interactions with the surrounding residues in active site [ , ] . interestingly, this opposite direction binding of inhibitory propeptide segment is evolutionarily conserved in other members of cysteine cathepsins, including in cathepsin b. biogenesis of human cathepsin l. after the full length cathepsin l mrna is transcribed, it is translated in ribosomes. following this, the full-length peptide enters the ribosomes-bound endoplasmic reticulum lumen where signal peptide is removed. pro-cathepsin l then enters the golgi network where it undergoes n-linked glycosylation at asn , followed by mannose phosphorylation and formation of appropriate disulfide linkages. in the last step, modified procathepsin l is shuttled to lysosome by endolysosomal pathways, generating the double chain form of active and mature human cathepsin l. the dominant pathway of regulation of activated and mature cathepsin l is by endogenous protein inhibitors, cystatins, that like propeptide compete with the physiological substrates for binding to the enzyme active site (table ) [ , ] . interestingly, protein inhibitory agents of cathepsin l have also been reported in other organisms. for example, kotsyfakis m. et al. reported the existence of two cathepsin l inhibitory proteins in the carrier of the main vector of lyme disease-carrying parasite, ixodus scapularis. named so because of its abilities to specifically inhibit cathepsin l (ic = . nm; ki = pm) activity, sialostatin l abrogates the protective proteolytic activity of host cells at the infestation sites, thereby promoting the tick's survival [ ] . in addition, it also possess a potent anti-inflammatory and immunosuppressive activity by inhibiting cytotoxic killer t cells [ ] . after the full length cathepsin l mrna is transcribed, it is translated in ribosomes. following this, the full-length peptide enters the ribosomes-bound endoplasmic reticulum lumen where signal peptide is removed. pro-cathepsin l then enters the golgi network where it undergoes n-linked glycosylation at asn , followed by mannose phosphorylation and formation of appropriate disulfide linkages. in the last step, modified procathepsin l is shuttled to lysosome by endolysosomal pathways, generating the double chain form of active and mature human cathepsin l. the dominant pathway of regulation of activated and mature cathepsin l is by endogenous protein inhibitors, cystatins, that like propeptide compete with the physiological substrates for binding to the enzyme active site (table ) [ , ] . interestingly, protein inhibitory agents of cathepsin l have also been reported in other organisms. for example, kotsyfakis m. et al. reported the existence of two cathepsin l inhibitory proteins in the carrier of the main vector of lyme disease-carrying parasite, ixodus scapularis. named so because of its abilities to specifically inhibit cathepsin l (ic = . nm; k i = pm) activity, sialostatin l abrogates the protective proteolytic activity of host cells at the infestation sites, thereby promoting the tick's survival [ ] . in addition, it also possess a potent anti-inflammatory and immunosuppressive activity by inhibiting cytotoxic killer t cells [ ] . table . reported physiological inhibitory ligands of human cathepsin l with their inhibition constants. over the years, several classes of physiological and synthetic inhibitors have been discovered targeting cathepsin l. herein, the focus is on non-physiological inhibitors that could broadly be classified as reversible and irreversible inhibitors. reversible and irreversible inhibitor could be distinguished by means of the mechanistic approaches they utilize for enzyme inactivation. reversible inhibitors 'generally' engage with the target protein using non-covalent interaction; however, in certain cases, they form a quasi-covalent bond which eventually disengages from the active site, say, upon dilution. irreversible inhibitors, on the other hand, permanently modify the protein of interest via the formation of a stable covalent bond. while both reversible and irreversible inhibitors have their pros and cons, it is generally believed that reversible inhibitors are preferred candidates in drug discovery and irreversible inhibitors not so due to their adverse immune responses [ ] . there are, however, several examples of successful drugs being used in clinics that work by an irreversible inhibitory mechanism [ ] . irreversible inhibitors are also widely utilized in functional biology (e.g., in the development of activity-based probes). the majority of synthetic cathepsin l inhibitors have recognizable peptide sequences, often derived from its physiological substrates, that bind to the active site of the protein and often contain strategically placed electrophilic warheads that trap the nucleophilic cys residue (figure ) for activity. knowledge of enzyme structure and its associated substrate specificity thus has played an important role in designing selective inhibitors of cathepsins [ , ] . in a seminal study, choe et al. studied substrate specificity using a highly diversified positional scanning synthetic combinatorial library comprised of , fluorogenic tetrapeptides; this allowed to differentiate individual enzymes binding propensity based on their distinct amino acid preferences [ ] . by capitalizing on this strategy, they successfully developed a selective substrate and substrate-based inhibitor of cathepsin k. the other important contribution to our understanding of subsite binding preferences of cathepsin l enzyme stems from several timely crystal structure studies that helped reveal key difference in the structural landscape of enzyme subsites [ ] [ ] [ ] . in an important study, shenoy et al. solved the crystal structure of ligand bound cathepsin l and documented the structural compositions of ligand binding sites ( figure ) [ ] . their analysis of z-phe-tyr (o-tert-butyl)-c(o)c(h)o bound cathepsin l revealed that (a) s subsite is relatively wide and unrestricted and composed of asp , ser , and cys , (b) s subsite is guided by leu , trp , ala and gly where trp associates with trp and phe and forms an aromatic cluster that accommodates the tert-butyl group (c) side chains of leu and met help form the s subsite that engages in non-polar interactions with the phenyl side chain, and finally, (d) the carboxybenzyl group finds interaction with the gly residue of s subsite ( figure ). however, it turns out that different ligands may find slightly altered binding interactions with the enzyme subsites, depending on their structural features. for example, shenoy et al. showed that although z-phe-tyr (o-tert-butyl)-dmk binds to the same subsites of cathepsin l as observed for the z-phe-tyr (o-tert-butyl)-c(o)c(h)o ligand and finds some alternative interactions with residues from the active site pocket. this finding has been corroborated by other studies as well that suggest that structural features of ligands can influence the subsite composition of the cathepsin l enzyme [ , , , ] . such information has been duly capitalized to develop different classes of inhibitors that are discussed next. in the following section, we discuss the types of cathepsin l-targeting chemotypes that have been utilized for development of small molecule inhibitors. available universal cathepsin inhibitor of this class, showed only a marginal selectivity towards cathepsin l compared to cathepsin b and a moderate -fold selectivity over cathepsin h, as reported by barrett et al. [ ] . although they developed several synthetic analogs of e- with improved potency, they lacked desirable selectivity toward cathepsin l. they proposed the probable binding orientation of e- that follows a non-substrate-like orientation, i.e., it occupies only non-prime sites ( figure ) at the enzyme pocket. phe arg cbz amc sh s s ' s ' s s the key subsite-forming amino acid residues with their corresponding numbers are shown in blue whereas the catalytic diad forming residues are depicted in red. (the authors used pdb: of file to construct the figure [ ] ). in another study, gour-salin et al. reported a number of epoxysuccinyl amino acid benzyl esters in which they systematically varied the amino acid attached to the epoxide ring. this was intended to investigate its effect in determining selectivity toward cathepsin l or s [ ] . their results surprisingly indicate that the specificity of these analogs did not follow the trend, generally observed for substrate, possibly due to e- like binding orientation at the enzyme pocket. among all cathepsin l compared to cathepsin b and a moderate -fold selectivity over cathepsin h, as reported by barrett et al. [ ] . although they developed several synthetic analogs of e- with improved potency, they lacked desirable selectivity toward cathepsin l. they proposed the probable binding orientation of e- that follows a non-substrate-like orientation, i.e., it occupies only non-prime sites ( figure ) at the enzyme pocket. phe arg cbz amc sh s s ' s ' s s the key subsite-forming amino acid residues with their corresponding numbers are shown in blue whereas the catalytic diad forming residues are depicted in red. (the authors used pdb: of file to construct the figure [ ] ). in another study, gour-salin et al. reported a number of epoxysuccinyl amino acid benzyl esters in which they systematically varied the amino acid attached to the epoxide ring. this was intended to investigate its effect in determining selectivity toward cathepsin l or s [ ] . their results surprisingly indicate that the specificity of these analogs did not follow the trend, generally observed for substrate, possibly due to e- like binding orientation at the enzyme pocket. among all the key subsite-forming amino acid residues with their corresponding numbers are shown in blue whereas the catalytic diad forming residues are depicted in red. (the authors used pdb: of file to construct the figure [ ] ). epoxysuccinate inhibitors have historically played a crucial role in deciphering the cysteine protease biology. this class of compounds contains an epoxide ring as an electrophilic warhead that traps the active site catalytic cysteine residue of the protein. e- (l-trans-epoxysuccinyl-leucylamido( -guanidino)butane) (entry ; table ), perhaps the most studied commercially available universal cathepsin inhibitor of this class, showed only a marginal selectivity towards cathepsin l compared to cathepsin b and a moderate -fold selectivity over cathepsin h, as reported by barrett et al. [ ] . although they developed several synthetic analogs of e- with improved potency, they lacked desirable selectivity toward cathepsin l. they proposed the probable binding orientation of e- that follows a non-substrate-like orientation, i.e., it occupies only non-prime sites ( figure ) at the enzyme pocket. in another study, gour-salin et al. reported a number of epoxysuccinyl amino acid benzyl esters in which they systematically varied the amino acid attached to the epoxide ring. this was intended to investigate its effect in determining selectivity toward cathepsin l or s [ ] . their results surprisingly indicate that the specificity of these analogs did not follow the trend, generally observed for substrate, possibly due to e- like binding orientation at the enzyme pocket. among all synthesized compounds, katunuma: clik) with the help of computational modeling based on the stereo-structure [ ] . three of the developed clik inhibitors were hydrolytically stable and showed highly selective inhibition for hepatic cathepsin l in vivo. further, they elucidated the inhibition mechanism of this class of compounds based on the crystal structure of papain-clik (entry , table ) complex [ ] . this crystal structure revealed that clik , unlike e- , binds to both prime and non-prime sites of the active site pocket. notably, the specificity toward cathepsin l was attributed to the existence of phenylalanine residue at the s site, and a hydrophobic interaction mediated by n-terminal pyridine ring ( figure ). the other classes of alkylating agents that initially played an important role in deciphering the mechanistic aspects of cathepsin inhibition were peptidyldiazomethane (entry , table ) and peptidylchloromethane (entry , table ). crawford et al. reported highly potent inhibitors from these two chemotypes that spans the active site and shows significant selectivity improvement over other cysteine proteases [ ] . however, these classes of inhibitors suffer from stability issues and have found limited utility in in vivo assays. peptidylhydroxylamines were first introduced as mechanism-based inhibitors of serine and cysteine proteinases [ ] [ ] [ ] [ ] . bromme et al. adapted this scaffold and prepared a library of n-peptidyl-o-acyl hydroxylamines which exhibited rapid and selective inactivation of several lysosomal cysteine proteinases [ ] . this class of inhibitors occupied the active site of the enzyme and irreversibly inactivated cysteine cathepsins as the free enzyme activity was not recovered when the enzyme-inhibitor complex was exposed to exhaustive ultrafiltration (up to an enzyme/free inhibitor ratio of < : . ) or chromatography on sephadex g- . among developed inhibitors, z-phe-phe-nho-ma (entry , table ) inhibited cathepsin l with most potency and showed significant selectivity, -fold and -fold, over cathepsin s and cathepsin b, respectively. interestingly the stability and efficacy of this class of inhibitors were determined by the nature of substitutions on hydroxylamine oxygen as their electron-withdrawing tendencies showed a positive correlation with inactivation kinetics. although this class of inhibitors exhibited desirable traits with respect to both potency and selectivity, they suffered majorly from aqueous stability issue; half-lives (t / ) only in the range of min in aqueous solution. in another study, bromme et al. developed a series of n-peptidyl-o-acyl hydroxamates with lysine in p position with improved inhibitory profile for cysteine proteases over their serine counterparts [ ] . the maximum inhibition was observed by z-phe-lys-nho-nbz (entry , table ), with -fold selectivity over cathepsin s, and > -fold selectivity over cathepsin b. the authors postulated that the active site cys residue attacks the carbonyl of the hydroxamate and forms a tetrahedral intermediate, whereas the nitrogen of the hydroxamate, which primarily remains deprotonated (pka < ), presumably engages in an electrostatic interaction with the active site his residue ( figure ). inhibitors further provided the option of varying the leaving group that targets s ′ site; more hydrophobic substituents were preferred at this position as comparison of ki/ki values of the inactivation exhibited the following trend for aa: gly < ala < val < leu < phe < -no -ph. the nitrophenyl analog (entry , table ) which incur both hydrophobicity and electron-withdrawing property exerted the maximum potency and selectivity for cathepsin l over other tested cysteine proteases. hydrophobic substituents were preferred at this position as comparison of ki/ki values of the inactivation exhibited the following trend for aa: gly < ala < val < leu < phe < -no -ph. the nitrophenyl analog (entry , table ) which incur both hydrophobicity and electron-withdrawing property exerted the maximum potency and selectivity for cathepsin l over other tested cysteine proteases. hydrophobic substituents were preferred at this position as comparison of ki/ki values of the inactivation exhibited the following trend for aa: gly < ala < val < leu < phe < -no -ph. the nitrophenyl analog (entry , table ) which incur both hydrophobicity and electron-withdrawing property exerted the maximum potency and selectivity for cathepsin l over other tested cysteine proteases. in a follow-up study, bromme et al. synthesized and tested a series of new inhibitors, with the general formula of z-phe-gly-nho-co-aa (aa: amino acid), against papain class of enzymes [ ] . this class of inhibitors covalently modified active-site cys residue via sulfenamidation, like in their n-peptidyl-o-acyl hydroxamate counterparts, as shown by the mass spectrometric analysis. these inhibitors further provided the option of varying the leaving group that targets s ′ site; more hydrophobic substituents were preferred at this position as comparison of ki/ki values of the inactivation exhibited the following trend for aa: gly < ala < val < leu < phe < -no -ph. the nitrophenyl analog (entry , table ) which incur both hydrophobicity and electron-withdrawing property exerted the maximum potency and selectivity for cathepsin l over other tested cysteine proteases. in a follow-up study, bromme et al. synthesized and tested a series of new inhibitors, with the general formula of z-phe-gly-nho-co-aa (aa: amino acid), against papain class of enzymes [ ] . this class of inhibitors covalently modified active-site cys residue via sulfenamidation, like in their n-peptidyl-o-acyl hydroxamate counterparts, as shown by the mass spectrometric analysis. these inhibitors further provided the option of varying the leaving group that targets s site; more hydrophobic substituents were preferred at this position as comparison of k i /k i values of the inactivation exhibited the following trend for aa: gly < ala < val < leu < phe < -no -ph. the nitrophenyl analog (entry , table ) which incur both hydrophobicity and electron-withdrawing property exerted the maximum potency and selectivity for cathepsin l over other tested cysteine proteases. another class of peptidyl inhibitors that spans the active site and utilizes the catalytic machinery of cathepsin l for effective attenuation of enzyme activity is peptidyl acyloxymethanes. krantz et al. developed a library of inhibitory compounds with a general sequence, z-phe-x-ch oco-r; here they systematically varied the amino acid residue at p (denoted as x) and p (denoted as r) positions [ ] . among the synthesized compounds, z-phe-cys(sbn)-ch oco- , -(cf ) -ph (entry , table ) exhibited almost a diffusion-controlled inactivation kinetics toward cathepsin l, however it showed only marginal selectivity over cathepsin b and cathepsin s. by analyzing the structure-activity relationships of the library, the author elucidated the importance of s site in cathepsin l that could potentially be used to harness selectivity among other cysteine cathepsins. in a separate study, torkar et al. designed a library of peptides for cathepsin l that spanned the active site and attenuated the enzyme activity via a non-covalent interaction [ ] . they initially evaluated the compounds' activities against cathepsin l and cathepsin b, and compared their hits against cathepsin k and s. the authors discovered five most selective non-covalent, peptidyl inhibitors of cathepsin l, and transformed them into irreversible inhibitors by strategically appending electrophilic warhead ( figure )-acyloxymethyl ketone (aomk) groups (entry , table ). however, the attachment of the aomk group drastically impacted the selectivity profiles of these inhibitors, suggesting the importance of the adjuvant effect of prime site targeting in determining the efficacy and selectivity of this class of compounds. this class of inhibitors found wide-spread utilities in detecting protease activity and were utilized to develop activity-based probe; this will be discussed in the later sections. a very interesting class of inhibitors, the concept of which was derived from e- , is peptidyl aziridines. martichonok et al. developed a series of aziridine derivatives of e- and tested them against papain and cathepsin l and b [ ] . contrarily to e- , in which (l)-diastereomer is more potent than (d)-isomer, aziridine analogs exhibited the opposite trend while still inactivating the enzymes. more importantly, the efficacy of this class of inhibitors was strongly ph-dependent and showed maximal inhibitory potency at ph ; this is attributed to the protonated form of aziridine ring that is more susceptible to nucleophilic attack by catalytic cys residue. among the developed library, ho-(d)-az-leu-nh-iam (entry , table ) analog exhibited maximal inhibitory potency towards cathepsin l with moderate selectivity over cathepsin b; the corresponding l isomer, ho-(l)-az-leu-nh-iam (entry ) was almost -fold less activity while maintaining the similar trend in general. since only protonated form of aziridine ring undergoes nucleophilic attack, presumably it does not involve water molecule-mediated ring-opening like its epoxide counterpart e- . although promising, a full potential of this class of compounds could presumably be achieved only below ph when aziridine ring nitrogen gets completely protonated. this class of inhibitors thus lacks practical utility as the catalytic cysteine of cathepsin l starts to lose its activity below ph and the majority of the cell-based assays are performed mostly at a ph higher than . . in a notable study, schirmeister et al. developed three different classes of inhibitors with aziridine- , -dicarboxylic acid (azi), an electrophilic warhead, installed at different positions of the peptide chain ( figure ) [ ] . they performed a thorough sar analysis of this type of inhibitors, all off which exhibited time-dependent irreversible inactivation of cathepsin l with no-recovery of enzyme activity, even after extensive dialysis of the enzyme-inhibitor complex. among type-i inhibitors, n-acylated aziridines with aziridine as c-terminal amino acid, a mixture of diastereomeric peptides with procathepsin b sequence leu-gly-gly (entry , table ), exhibited enhanced inhibition toward cathepsin l. this was attributed to an overall unique folding of cathepsin l with a shallowness of the s pocket due to the presence of an additional met residue. type ii class of inhibitors ( figure ) resemble classic aziridine scaffold, n-unsubstituted aziridines with aziridine as n-terminal amino acid, analogously to e- where nitrogen of aziridine remained unsubstituted. among the type ii inhibitors tested, eto-(r,r)-azi-leu-obzl (entry , table ) inactivated cathepsin l with higher second-order rate constant than eto-(s,s)-azi-leu-obzl, although the latter showed better selectivity over cathepsin b. a very interesting class of inhibitors, the concept of which was derived from e- , is peptidyl aziridines. martichonok et al. developed a series of aziridine derivatives of e- and tested them against papain and cathepsin l and b [ ] . contrarily to e- , in which (l)-diastereomer is more potent than (d)-isomer, aziridine analogs exhibited the opposite trend while still inactivating the enzymes. more importantly, the efficacy of this class of inhibitors was strongly ph-dependent and showed maximal inhibitory potency at ph ; this is attributed to the protonated form of aziridine ring that is more susceptible to nucleophilic attack by catalytic cys residue. among the developed library, ho-(d)-az-leu-nh-iam (entry , table ) analog exhibited maximal inhibitory potency towards cathepsin l with moderate selectivity over cathepsin b; the corresponding l isomer, ho-(l)-az-leu-nh-iam (entry ) was almost -fold less activity while maintaining the similar trend in general. since only protonated form of aziridine ring undergoes nucleophilic attack, presumably it does not involve water molecule-mediated ring-opening like its epoxide counterpart e- . although promising, a full potential of this class of compounds could presumably be achieved only below ph when aziridine ring nitrogen gets completely protonated. this class of inhibitors thus lacks practical utility as the catalytic cysteine of cathepsin l starts to lose its activity below ph and the majority of the cell-based assays are performed mostly at a ph higher than . . in a notable study, schirmeister et al. developed three different classes of inhibitors with aziridine- , -dicarboxylic acid (azi), an electrophilic warhead, installed at different positions of the peptide chain ( figure ) [ ] . they performed a thorough sar analysis of this type of inhibitors, all off which exhibited time-dependent irreversible inactivation of cathepsin l with no-recovery of enzyme activity, even after extensive dialysis of the enzyme-inhibitor complex. among type-i inhibitors, n-acylated aziridines with aziridine as c-terminal amino acid, a mixture of diastereomeric peptides with procathepsin b sequence leu-gly-gly (entry , table ), exhibited enhanced inhibition toward cathepsin l. this was attributed to an overall unique folding of cathepsin l with a shallowness of the s pocket due to the presence of an additional met residue. type ii class of inhibitors ( figure ) resemble classic aziridine scaffold, n-unsubstituted aziridines with aziridine as n-terminal amino acid, analogously to e- where nitrogen of aziridine remained unsubstituted. among the type ii inhibitors tested, eto-(r,r)-azi-leu-obzl (entry , table ) inactivated cathepsin l with higher second-order rate constant than eto-(s,s)-azi-leu-obzl, although the latter showed better selectivity over cathepsin b. the type iii inhibitor class (figure ) is comprised of n-acylated bispeptidyl derivatives of aziridine, where aziridine ring rests in the middle of the peptide. boc-phe-(r,r)-(eto)-azi-leu-pro-obzl (entry , table ) of this series was -fold more potent than the (s,s) analog; however both exhibited only a marginal selectivity over cathepsin b with diminished eudysmic ratio. the authors then superimposed and analyzed the structures of certain epoxide and aziridines and postulated that these inhibitors can assume different orientations in the active site while still binding within the enzyme pockets. this scaffold was further explored by vicik et al. who extended the previous work and developed a series of compounds in which boc-(s)-leu-(s)-azy-(s,s)-azi(obn) (figure ), type i analog, spanned from s to s pocket and inactivated cathepsin l with more than -fold selectivity over cathepsin b [ ] . this motif was also used for affinity labeling of cathepsin l, which will be discussed in the later sections. these classes of compounds, especially n-unsubstituted aziridinyl peptides and in special cases n-acylated ones, exhibited a high selectivity and potency and provided the premise for the further development of chemical biology tools much needed for functional studies. table ) of this series was -fold more potent than the (s,s) analog; however both exhibited only a marginal selectivity over cathepsin b with diminished eudysmic ratio. the authors then superimposed and analyzed the structures of certain epoxide and aziridines and postulated that these inhibitors can assume different orientations in the active site while still binding within the enzyme pockets. this scaffold was further explored by vicik et al. who extended the previous work and developed a series of compounds in which boc-(s)-leu-(s)-azy-(s,s)-azi(obn) ( figure ), type i analog, spanned from s to s ′ pocket and inactivated cathepsin l with more than -fold selectivity over cathepsin b [ ] . this motif was also used for affinity labeling of cathepsin l, which will be discussed in the later sections. these classes of compounds, especially n-unsubstituted aziridinyl peptides and in special cases n-acylated ones, exhibited a high selectivity and potency and provided the premise for the further development of chemical biology tools much needed for functional studies. another promising scaffold that acts as a michael acceptor and hijacks the catalytic residue of cysteine cathepsins is peptidyl aryl vinylsulfones. this scaffold was first introduced by palmer et al. as an irreversible inhibitor of cysteine cathepsins [ ] . subsequently, they extended their study by performing an sar analysis of this class of inhibitors which showed pan-cathepsin inhibition with table ) of this series was -fold more potent than the (s,s) analog; however both exhibited only a marginal selectivity over cathepsin b with diminished eudysmic ratio. the authors then superimposed and analyzed the structures of certain epoxide and aziridines and postulated that these inhibitors can assume different orientations in the active site while still binding within the enzyme pockets. this scaffold was further explored by vicik et al. who extended the previous work and developed a series of compounds in which boc-(s)-leu-(s)-azy-(s,s)-azi(obn) ( figure ), type i analog, spanned from s to s ′ pocket and inactivated cathepsin l with more than -fold selectivity over cathepsin b [ ] . this motif was also used for affinity labeling of cathepsin l, which will be discussed in the later sections. these classes of compounds, especially n-unsubstituted aziridinyl peptides and in special cases n-acylated ones, exhibited a high selectivity and potency and provided the premise for the further development of chemical biology tools much needed for functional studies. another promising scaffold that acts as a michael acceptor and hijacks the catalytic residue of cysteine cathepsins is peptidyl aryl vinylsulfones. this scaffold was first introduced by palmer et al. as an irreversible inhibitor of cysteine cathepsins [ ] . subsequently, they extended their study by performing an sar analysis of this class of inhibitors which showed pan-cathepsin inhibition with occasional selectivity towards cathepsin s for certain scaffolds [ ] . however, in a separate study by mendieta et al., the authors developed a structurally novel library of twenty peptidyl -aryl vinylsulfones ( figure ) in which they introduced extensive diversity at the r position. subsequently, they also varied the r position while keeping either morpholine or n-methyl piperazine group intact [ ] . docking studies with most active and selective inhibitor (entry , another promising scaffold that acts as a michael acceptor and hijacks the catalytic residue of cysteine cathepsins is peptidyl aryl vinylsulfones. this scaffold was first introduced by palmer et al. as an irreversible inhibitor of cysteine cathepsins [ ] . subsequently, they extended their study by performing an sar analysis of this class of inhibitors which showed pan-cathepsin inhibition with occasional selectivity towards cathepsin s for certain scaffolds [ ] . however, in a separate study by mendieta et al., the authors developed a structurally novel library of twenty peptidyl -aryl vinylsulfones ( figure ) in which they introduced extensive diversity at the r position. subsequently, they also varied the r position while keeping either morpholine or n-methyl piperazine group intact [ ] . docking studies with most active and selective inhibitor (entry , table ) of this class revealed that the inhibitor extends from s to s sites of cathepsin l and the β-vinylsulfone moiety resides in a close proximity of cys- residue thereby favoring the formation of michael adduct. the authors postulated that considering the efficacy of peptidyl aryl vinyl sulfones, strong anti-cancer candidates could be harnessed by cultivating this scaffold. table ) of this class revealed that the inhibitor extends from s to s ′ sites of cathepsin l and the βvinylsulfone moiety resides in a close proximity of cys- residue thereby favoring the formation of michael adduct. the authors postulated that considering the efficacy of peptidyl aryl vinyl sulfones, strong anti-cancer candidates could be harnessed by cultivating this scaffold. one other very potent class of inhibitors that also includes a michael acceptor is peptidyl aryl vinylsulfonate esters, a superior michael acceptor than vinyl sulfone. they served as potent inhibitors of cruzain-a parasitic cysteine protease from t. cruzi that is homologous to cathepsin l [ , ] . this scaffold was explored by dana et al., who determined the superiority of aryl vinylsulfonate ester over aryl vinylsulfone and aryl vinylsulfonamide counterparts towards cathepsin l inhibition [ ] . thus, they synthesized and screened the efficacy of a library of aryl vinylsulfonate ester compounds against cathepsin l; -bromo phenyl vinylsulfonate was found to be the champion ligand presumably due to favorable interactions between the -bromo phenyl moiety with the prime site residues of cathepsin l. they further designed a hybrid inhibitor, kd- , (entry , table ) by strategically appending the -bromophenyl vinylsulfonate moiety as electrophilic warhead to a modestly potent reversible cathepsin l inhibitor [ ] ; this design was based on the hypothesis that the developed compound will target both the prime site and the non-prime site residues for interaction. kd- indeed exhibited almost a diffusion-controlled inactivation kinetics while maintaining an excellent selectivity profile toward cathepsin l ( figure ). furthermore, kd- was cell-permeable and inhibited the intracellular activity of cathepsin l in human mda-mb- breast cancer cell lines. kd- also enhanced the integrity of cell-cell junctions by effectively attenuating the migratory potential of the cells, as demonstrated by the scratch assay. the authors anticipated that this class of inhibitors may find extensive usage in deciphering context-specific cathepsin l biology. one other very potent class of inhibitors that also includes a michael acceptor is peptidyl aryl vinylsulfonate esters, a superior michael acceptor than vinyl sulfone. they served as potent inhibitors of cruzain-a parasitic cysteine protease from t. cruzi that is homologous to cathepsin l [ , ] . this scaffold was explored by dana et al., who determined the superiority of aryl vinylsulfonate ester over aryl vinylsulfone and aryl vinylsulfonamide counterparts towards cathepsin l inhibition [ ] . thus, they synthesized and screened the efficacy of a library of aryl vinylsulfonate ester compounds against cathepsin l; -bromo phenyl vinylsulfonate was found to be the champion ligand presumably due to favorable interactions between the -bromo phenyl moiety with the prime site residues of cathepsin l. they further designed a hybrid inhibitor, kd- , (entry , table ) by strategically appending the -bromophenyl vinylsulfonate moiety as electrophilic warhead to a modestly potent reversible cathepsin l inhibitor [ ] ; this design was based on the hypothesis that the developed compound will target both the prime site and the non-prime site residues for interaction. kd- indeed exhibited almost a diffusion-controlled inactivation kinetics while maintaining an excellent selectivity profile toward cathepsin l ( figure ). furthermore, kd- was cell-permeable and inhibited the intracellular activity of cathepsin l in human mda-mb- breast cancer cell lines. kd- also enhanced the integrity of cell-cell junctions by effectively attenuating the migratory potential of the cells, as demonstrated by the scratch assay. the authors anticipated that this class of inhibitors may find extensive usage in deciphering context-specific cathepsin l biology. molecules , , of table ) of this class revealed that the inhibitor extends from s to s ′ sites of cathepsin l and the βvinylsulfone moiety resides in a close proximity of cys- residue thereby favoring the formation of michael adduct. the authors postulated that considering the efficacy of peptidyl aryl vinyl sulfones, strong anti-cancer candidates could be harnessed by cultivating this scaffold. one other very potent class of inhibitors that also includes a michael acceptor is peptidyl aryl vinylsulfonate esters, a superior michael acceptor than vinyl sulfone. they served as potent inhibitors of cruzain-a parasitic cysteine protease from t. cruzi that is homologous to cathepsin l [ , ] . this scaffold was explored by dana et al., who determined the superiority of aryl vinylsulfonate ester over aryl vinylsulfone and aryl vinylsulfonamide counterparts towards cathepsin l inhibition [ ] . thus, they synthesized and screened the efficacy of a library of aryl vinylsulfonate ester compounds against cathepsin l; -bromo phenyl vinylsulfonate was found to be the champion ligand presumably due to favorable interactions between the -bromo phenyl moiety with the prime site residues of cathepsin l. they further designed a hybrid inhibitor, kd- , (entry , table ) by strategically appending the -bromophenyl vinylsulfonate moiety as electrophilic warhead to a modestly potent reversible cathepsin l inhibitor [ ] ; this design was based on the hypothesis that the developed compound will target both the prime site and the non-prime site residues for interaction. kd- indeed exhibited almost a diffusion-controlled inactivation kinetics while maintaining an excellent selectivity profile toward cathepsin l ( figure ). furthermore, kd- was cell-permeable and inhibited the intracellular activity of cathepsin l in human mda-mb- breast cancer cell lines. kd- also enhanced the integrity of cell-cell junctions by effectively attenuating the migratory potential of the cells, as demonstrated by the scratch assay. the authors anticipated that this class of inhibitors may find extensive usage in deciphering context-specific cathepsin l biology. recently, another interesting discovery that provided a wealth of information on cathepsin l-inhibitor interactions came from marine cyanobacterial extracts. miller et al. first reported gallinamide a as potent irreversible inhibitor of cathepsin l with an ic value of nm and a -to -fold greater selectivity over cathepsin v and b, respectively [ ] . they further performed molecular docking and molecular dynamics simulations and learned that the peptidyl backbone of the inhibitor spans the active site whereas the side chains engage in favorable interactions with different active site pockets, placing the michael acceptor enamide in close proximity to the catalytic cys residue. in a follow-up study, boudreau et al. performed molecular docking studies to predict the potential modifications of a gallinamide a scaffold that would harness favorable enzyme-inhibitor interactions and enable the development of compounds with improved inhibitory efficacy [ ] . they synthesized a panel of compounds by retaining gallinamide a and only varying the amino acids at p , p , and p positions. (figure ). this led to the discovery of the most potent analog of this series (entry , table ) with sub-nanomolar ic value ( pm) and fast time dependent inactivation kinetics, suggesting an improved binding and reactivity of the inhibitor with the enzyme active site. the authors found that this class of compounds effectively inactivated cruzain, a homologous cysteine protease from t. cruzi, using cell-based assay. gallinamide a and its analogs thus provide a remarkable inhibitory scaffold that could potentially be harnessed to build selective enzyme inhibitors for a variety of therapeutic applications. recently, another interesting discovery that provided a wealth of information on cathepsin linhibitor interactions came from marine cyanobacterial extracts. miller et al. first reported gallinamide a as potent irreversible inhibitor of cathepsin l with an ic value of nm and a -to -fold greater selectivity over cathepsin v and b, respectively [ ] . they further performed molecular docking and molecular dynamics simulations and learned that the peptidyl backbone of the inhibitor spans the active site whereas the side chains engage in favorable interactions with different active site pockets, placing the michael acceptor enamide in close proximity to the catalytic cys residue. in a follow-up study, boudreau et al. performed molecular docking studies to predict the potential modifications of a gallinamide a scaffold that would harness favorable enzymeinhibitor interactions and enable the development of compounds with improved inhibitory efficacy [ ] . they synthesized a panel of compounds by retaining gallinamide a and only varying the amino acids at p , p ′, and p ′ positions. (figure ). this led to the discovery of the most potent analog of this series (entry , table ) with sub-nanomolar ic value ( pm) and fast time dependent inactivation kinetics, suggesting an improved binding and reactivity of the inhibitor with the enzyme active site. the authors found that this class of compounds effectively inactivated cruzain, a homologous cysteine protease from t. cruzi, using cell-based assay. gallinamide a and its analogs thus provide a remarkable inhibitory scaffold that could potentially be harnessed to build selective enzyme inhibitors for a variety of therapeutic applications. figure . gallinamide was reconstructed with altered amino acid sequence at p , p ′, and p ′ positions. the acrylic group, the michael acceptor, is shown in red. one classical inhibitor that has been used over a long period of time to dissect cysteine proteinase activity is leupeptin, a microbial product. leupeptin is a peptidyl aldehyde that occupies the active site cleft of cysteine cathepsins and forms a thiohemiacetal intermediate by trapping catalytic cysteine residue; this complex hydrolyzes over time, thus showing the covalent and reversible nature of the inhibitor (figure a ). leupeptin unfortunately suffers from non-specific inhibition of both serine and cysteine proteinases, thus making it unfavorable for clinical usage and chemical biology applications. this issue was, however, addressed by woo et al. who designed and synthesized six peptidyl aldehyde analogs that were more potent than leupeptin (ic = . nm) and exhibited improved selectivity towards cathepsin l over cathepsin b and calpain ii [ ] . the most potent cathepsin l inhibitor of this series was z-phe-phe-h (ic = . nm) (figure b ) that showed more than -fold selectivity over cathepsin b. interestingly; their data demonstrated the importance of aromatic amino acids, such as phenylalanine and tyrosine, at the p position in determining the potency and selectivity towards cathepsin l; o-alkylation of tyrosine group diminishes the inhibitory efficiency as in z-phe-tyr(bu)-h (ic : . nm). in a follow-up publication, they further tested the efficacy of z-phe-tyr-h (ic : . nm, -fold selective over cathepsin b) (figure b ) in vitro and in vivo [ ] . this compound effectively inhibited parathyroid hormone-stimulated osteoclastic bone resorption in pit formation assays, and suppressed bone weight loss of ovariectomized mouse in a dose-dependent manner when administered intraperitoneally. figure . gallinamide was reconstructed with altered amino acid sequence at p , p , and p positions. the acrylic group, the michael acceptor, is shown in red. one classical inhibitor that has been used over a long period of time to dissect cysteine proteinase activity is leupeptin, a microbial product. leupeptin is a peptidyl aldehyde that occupies the active site cleft of cysteine cathepsins and forms a thiohemiacetal intermediate by trapping catalytic cysteine residue; this complex hydrolyzes over time, thus showing the covalent and reversible nature of the inhibitor (figure a ). leupeptin unfortunately suffers from non-specific inhibition of both serine and cysteine proteinases, thus making it unfavorable for clinical usage and chemical biology applications. this issue was, however, addressed by woo et al. who designed and synthesized six peptidyl aldehyde analogs that were more potent than leupeptin (ic = . nm) and exhibited improved selectivity towards cathepsin l over cathepsin b and calpain ii [ ] . the most potent cathepsin l inhibitor of this series was z-phe-phe-h (ic = . nm) (figure b ) that showed more than -fold selectivity over cathepsin b. interestingly; their data demonstrated the importance of aromatic amino acids, such as phenylalanine and tyrosine, at the p position in determining the potency and selectivity towards cathepsin l; o-alkylation of tyrosine group diminishes the inhibitory efficiency as in z-phe-tyr(bu)-h (ic : . nm). in a follow-up publication, they further tested the efficacy of z-phe-tyr-h (ic : . nm, -fold selective over cathepsin b) (figure b ) in vitro and in vivo [ ] . this compound effectively inhibited parathyroid hormone-stimulated osteoclastic bone resorption in pit formation assays, and suppressed bone weight loss of ovariectomized mouse in a dose-dependent manner when administered intraperitoneally. in another interesting study, yasuma et al. developed a library of compounds by varying the amino acid substituents at p , p , p position of the inhibitor and carried out a thorough sar study [ ] . their study revealed that the configuration of the stereogenic center (s-configuration is favored over r-configuration) at the p position, and not the steric factor, was key to the inhibitory efficacy. apparently, the substituent at p position does not interact with s position residues; rather, proper stereogenicity allows the placement of the inhibitor in vicinity of the catalytic cysteine residue for interaction. s subsite of cathepsin l, on the other hand, preferred a hydrophobic and moderate-size group; α-branched alkyl chains but not the bulkier groups like phenylalanine was favorable. further, the s subsite showed a preference for hydrophobic and bulky moieties such as -and naphthalenylsulfonyl substituents. among synthesized compound, n-( -naphthalenylsulfonyl-lisoleucyl-l-tryptophanal (ic = . nm, -fold selective over cathepsin b; figure ) attenuated the release of ca + and hydroxyproline from bone in an in vitro bone culture system and further restricted bone loss in ovariectomized mice dosed orally. a further modification of this scaffold was reported by lynas et al. [ ] here, authors designed and developed di-and tri-peptidyl α-keto-β-aldehydes, based on substrate and inhibitor specificity profiles of cathepsin l. the compound z-phe-tyr(obut)-cocho (entry , table ) turned out as highly potent and selective inhibitor of cathepsin l with ki value of . nm. this molecule was further adapted by shenoy et al. to assess the structural basis for cathepsin l inhibition [ ] . in their study, the authors crystallized the glyoxal inhibitor with cathepsin l; the β-aldehyde forms a tetrahedral thiohemiacetal and α-keto oxygen atom is stabilized by the oxyanion hole. the tyr(obut) group was found to occupy s site while phenyl and carboxybenzyl groups occupied s and s sites, respectively. this class of inhibitors has successfully been deployed in the functional biology of cathepsin l. azepanone-based compounds were first reported as orally bioavailable and extremely potent inhibitors of cathepsin k, as shown by the pharmacokinetic studies in the rat [ ] . marquis et al. subsequently adopted the template and extended their work to acquire a selective inhibitor of cathepsin l with similar potency [ ] . this class of inhibitors are armored with keto functional group in another interesting study, yasuma et al. developed a library of compounds by varying the amino acid substituents at p , p , p position of the inhibitor and carried out a thorough sar study [ ] . their study revealed that the configuration of the stereogenic center (s-configuration is favored over r-configuration) at the p position, and not the steric factor, was key to the inhibitory efficacy. apparently, the substituent at p position does not interact with s position residues; rather, proper stereogenicity allows the placement of the inhibitor in vicinity of the catalytic cysteine residue for interaction. s subsite of cathepsin l, on the other hand, preferred a hydrophobic and moderate-size group; α-branched alkyl chains but not the bulkier groups like phenylalanine was favorable. further, the s subsite showed a preference for hydrophobic and bulky moieties such as -and -naphthalenylsulfonyl substituents. among synthesized compound, n-( -naphthalenylsulfonyl-l-isoleucyl-l-tryptophanal (ic = . nm, -fold selective over cathepsin b; figure ) attenuated the release of ca + and hydroxyproline from bone in an in vitro bone culture system and further restricted bone loss in ovariectomized mice dosed orally. in another interesting study, yasuma et al. developed a library of compounds by varying the amino acid substituents at p , p , p position of the inhibitor and carried out a thorough sar study [ ] . their study revealed that the configuration of the stereogenic center (s-configuration is favored over r-configuration) at the p position, and not the steric factor, was key to the inhibitory efficacy. apparently, the substituent at p position does not interact with s position residues; rather, proper stereogenicity allows the placement of the inhibitor in vicinity of the catalytic cysteine residue for interaction. s subsite of cathepsin l, on the other hand, preferred a hydrophobic and moderate-size group; α-branched alkyl chains but not the bulkier groups like phenylalanine was favorable. further, the s subsite showed a preference for hydrophobic and bulky moieties such as -and naphthalenylsulfonyl substituents. among synthesized compound, n-( -naphthalenylsulfonyl-lisoleucyl-l-tryptophanal (ic = . nm, -fold selective over cathepsin b; figure ) attenuated the release of ca + and hydroxyproline from bone in an in vitro bone culture system and further restricted bone loss in ovariectomized mice dosed orally. a further modification of this scaffold was reported by lynas et al. [ ] here, authors designed and developed di-and tri-peptidyl α-keto-β-aldehydes, based on substrate and inhibitor specificity profiles of cathepsin l. the compound z-phe-tyr(obut)-cocho (entry , table ) turned out as highly potent and selective inhibitor of cathepsin l with ki value of . nm. this molecule was further adapted by shenoy et al. to assess the structural basis for cathepsin l inhibition [ ] . in their study, the authors crystallized the glyoxal inhibitor with cathepsin l; the β-aldehyde forms a tetrahedral thiohemiacetal and α-keto oxygen atom is stabilized by the oxyanion hole. the tyr(obut) group was found to occupy s site while phenyl and carboxybenzyl groups occupied s and s sites, respectively. this class of inhibitors has successfully been deployed in the functional biology of cathepsin l. azepanone-based compounds were first reported as orally bioavailable and extremely potent inhibitors of cathepsin k, as shown by the pharmacokinetic studies in the rat [ ] . marquis et al. subsequently adopted the template and extended their work to acquire a selective inhibitor of cathepsin l with similar potency [ ] . this class of inhibitors are armored with keto functional group figure . n-( -naphthalenylsulfonyl-l-isoleucyl-l-tryptophanal orients itself into the active site of cathepsin l and finds favorable interactions within the s , s , and s pockets of the enzyme. a further modification of this scaffold was reported by lynas et al. [ ] here, authors designed and developed di-and tri-peptidyl α-keto-β-aldehydes, based on substrate and inhibitor specificity profiles of cathepsin l. the compound z-phe-tyr(obut)-cocho (entry , table ) turned out as highly potent and selective inhibitor of cathepsin l with k i value of . nm. this molecule was further adapted by shenoy et al. to assess the structural basis for cathepsin l inhibition [ ] . in their study, the authors crystallized the glyoxal inhibitor with cathepsin l; the β-aldehyde forms a tetrahedral thiohemiacetal and α-keto oxygen atom is stabilized by the oxyanion hole. the tyr(obut) group was found to occupy s site while phenyl and carboxybenzyl groups occupied s and s sites, respectively. this class of inhibitors has successfully been deployed in the functional biology of cathepsin l. azepanone-based compounds were first reported as orally bioavailable and extremely potent inhibitors of cathepsin k, as shown by the pharmacokinetic studies in the rat [ ] . marquis et al. subsequently adopted the template and extended their work to acquire a selective inhibitor of cathepsin l with similar potency [ ] . this class of inhibitors are armored with keto functional group that act as an electrophilic warhead and traps cysteine proteases by forming a transient covalent bond with the active-site cys residue, rendering inactivated enzyme. the authors initiated their work by scrupulously studying the cathepsin k-inhibitor complex that revealed the influence of p and p substituents of the inhibitor in determining the efficacy and selectivity profile of the compound. based on these observations, they designed and synthesized a series of compound and secured a highly potent cathepsin l inhibitor (k i,app : . nm; entry , table ) that exerted remarkable selectivity over cathepsin k and fairly modest selectivity over both cathepsin b and s. interestingly, sar showed that replacement of p leucine and p benzofuran of cathepsin k inhibitor with bulkier hydrophobic aromatic groups yielded an improved potency and the selectivity towards cathepsin l ( figure ). molecular docking studies further supported this observation as cathepsin k was found to have a shallower s pocket than cathepsin l, thus incorporation of bulkier napthyl group at p position favored cathepsin l inhibition but not cathepsin k. on the other hand, inclusion of another napthyl group at p position promoted a steric clash rather than furthering the desired hydrophobic interactions within the s pocket of cathepsin k, thus incurring a better selectivity profile towards cathepsin l over cathepsin k. this template has proven to be an important tool to study cysteine cathepsins as it has been further extended to achieve potent cathepsin s-selective inhibitor with cellular activity [ ] . molecules , , of that act as an electrophilic warhead and traps cysteine proteases by forming a transient covalent bond with the active-site cys residue, rendering inactivated enzyme. the authors initiated their work by scrupulously studying the cathepsin k-inhibitor complex that revealed the influence of p and p substituents of the inhibitor in determining the efficacy and selectivity profile of the compound. based on these observations, they designed and synthesized a series of compound and secured a highly potent cathepsin l inhibitor (ki,app: . nm; entry , table ) that exerted remarkable selectivity over cathepsin k and fairly modest selectivity over both cathepsin b and s. interestingly, sar showed that replacement of p leucine and p benzofuran of cathepsin k inhibitor with bulkier hydrophobic aromatic groups yielded an improved potency and the selectivity towards cathepsin l ( figure ). molecular docking studies further supported this observation as cathepsin k was found to have a shallower s pocket than cathepsin l, thus incorporation of bulkier napthyl group at p position favored cathepsin l inhibition but not cathepsin k. on the other hand, inclusion of another napthyl group at p position promoted a steric clash rather than furthering the desired hydrophobic interactions within the s pocket of cathepsin k, thus incurring a better selectivity profile towards cathepsin l over cathepsin k. this template has proven to be an important tool to study cysteine cathepsins as it has been further extended to achieve potent cathepsin s-selective inhibitor with cellular activity [ ] . nitrile group containing inhibitors have been widely recognized as covalent and reversible inhibitors of a certain class of enzymes that depend on cysteine-mediated nucleophilic attack for catalysis; the nitrile residue traps the sulfur and forms a thioimidate bond ( figure ) that hydrolyzes over the time rendering free enzyme. odanacatib is one of the prime examples of this class of compounds that has been evaluated as a clinical agent, although with limited success [ , ] . because of nitrile's tunable target engagement nature, this scaffold has been adapted to target other relevant enzymes, including cathepsin. hardegger et al. utilized nitrile warhead and examined the effect of halogen bonding in protein-ligand interactions [ ] . they developed a series of compounds and performed a thorough sar analysis in which the nitrile electrophile faced towards s site and trapped the catalytic cysteine. in the developed analogs, the substituents that occupied the s site were systematically varied by strategically altering the substituents at the para-position of the phenyl group ( figure ). the authors observed an improvement in inhibition profile with the placement of halogen at the para-position of phenyl ring which followed a trend cl < br < i (entry , table ), with the f substituent being an outlier. further analysis of the enzyme-inhibitor co-crystal structures revealed that halogen at the para position of the phenyl ring suitably interacted with gly at the s site; fluorine analog pointed away to avoid the electronic repulsion from the oxygen lone pairs of gly . the authors have also performed computational analysis which taken together with the crystal nitrile group containing inhibitors have been widely recognized as covalent and reversible inhibitors of a certain class of enzymes that depend on cysteine-mediated nucleophilic attack for catalysis; the nitrile residue traps the sulfur and forms a thioimidate bond ( figure ) that hydrolyzes over the time rendering free enzyme. odanacatib is one of the prime examples of this class of compounds that has been evaluated as a clinical agent, although with limited success [ , ] . because of nitrile's tunable target engagement nature, this scaffold has been adapted to target other relevant enzymes, including cathepsin. hardegger et al. utilized nitrile warhead and examined the effect of halogen bonding in protein-ligand interactions [ ] . they developed a series of compounds and performed a thorough sar analysis in which the nitrile electrophile faced towards s site and trapped the catalytic cysteine. in the developed analogs, the substituents that occupied the s site were systematically varied by strategically altering the substituents at the para-position of the phenyl group ( figure ). the authors observed an improvement in inhibition profile with the placement of halogen at the para-position of phenyl ring which followed a trend cl < br < i (entry , table ) , with the f substituent being an outlier. further analysis of the enzyme-inhibitor co-crystal structures revealed that halogen at the para position of the phenyl ring suitably interacted with gly at the s site; fluorine analog pointed away to avoid the electronic repulsion from the oxygen lone pairs of gly . the authors have also performed computational analysis which taken together with the crystal data suggests o· x-c angle and the distance between the interacting atoms primarily influenced the protein-ligand interaction. this work provides an important roadmap for developing improved chemical biology tools where a halogen-protein interaction has successfully been utilized [ ] . molecules , , of data suggests o· x-c angle and the distance between the interacting atoms primarily influenced the protein-ligand interaction. this work provides an important roadmap for developing improved chemical biology tools where a halogen-protein interaction has successfully been utilized [ ] . to examine what effect amide···heteroarene π-stacking interactions may have on chalcogen bonding in the s pocket of cathepsin l, giroud et al. utilized triazine-nitrile scaffold [ ] . the authors synthesized a diverse set of triazine-nitrile compounds with a diversified heteroarenes targeting s pocket; the s and s substituents were kept constant. among the developed compound library, -benzothienyl analog (entry , table ) exhibited maximum inhibitory potential; benzofuranyl, -benzothiazolyl, and -imidazopyridinyl, which are of similar geometry, also followed a similar inhibitory pattern ( figure ). molecular modelling based on co-crystal structures showed favorable chalcogen interaction to the backbone carbonyl of asn (d(s···o = casn ) = . Å and the angle α(oasn ···s-c) = °) at the s pocket; this was further supported by a conformational strain analysis, as chalcogen-enzyme interactions compensated for higher torsional strain in the s-containing ligands when compared to the benzofuranyl and imidazopyridinyl ligands. their study demonstrated the importance of both intermolecular interactions and conformational strain in assessing the effect of heterobicyclic ligands at the s pocket that could be potentially be utilized to develop cathepsin l selective inhibitors. data suggests o· x-c angle and the distance between the interacting atoms primarily influenced the protein-ligand interaction. this work provides an important roadmap for developing improved chemical biology tools where a halogen-protein interaction has successfully been utilized [ ] . to examine what effect amide···heteroarene π-stacking interactions may have on chalcogen bonding in the s pocket of cathepsin l, giroud et al. utilized triazine-nitrile scaffold [ ] . the authors synthesized a diverse set of triazine-nitrile compounds with a diversified heteroarenes targeting s pocket; the s and s substituents were kept constant. among the developed compound library, -benzothienyl analog (entry , table ) exhibited maximum inhibitory potential; benzofuranyl, -benzothiazolyl, and -imidazopyridinyl, which are of similar geometry, also followed a similar inhibitory pattern ( figure ). molecular modelling based on co-crystal structures showed favorable chalcogen interaction to the backbone carbonyl of asn (d(s···o = casn ) = . Å and the angle α(oasn ···s-c) = °) at the s pocket; this was further supported by a conformational strain analysis, as chalcogen-enzyme interactions compensated for higher torsional strain in the s-containing ligands when compared to the benzofuranyl and imidazopyridinyl ligands. their study demonstrated the importance of both intermolecular interactions and conformational strain in assessing the effect of heterobicyclic ligands at the s pocket that could be potentially be utilized to develop cathepsin l selective inhibitors. to examine what effect amide···heteroarene π-stacking interactions may have on chalcogen bonding in the s pocket of cathepsin l, giroud et al. utilized triazine-nitrile scaffold [ ] . the authors synthesized a diverse set of triazine-nitrile compounds with a diversified heteroarenes targeting s pocket; the s and s substituents were kept constant. among the developed compound library, -benzothienyl analog (entry , table ) exhibited maximum inhibitory potential; -benzofuranyl, -benzothiazolyl, and -imidazopyridinyl, which are of similar geometry, also followed a similar inhibitory pattern ( figure ). molecular modelling based on co-crystal structures showed favorable chalcogen interaction to the backbone carbonyl of asn (d(s···o = casn ) = . Å and the angle α(oasn ···s-c) = • ) at the s pocket; this was further supported by a conformational strain analysis, as chalcogen-enzyme interactions compensated for higher torsional strain in the s-containing ligands when compared to the benzofuranyl and imidazopyridinyl ligands. their study demonstrated the importance of both intermolecular interactions and conformational strain in assessing the effect of heterobicyclic ligands at the s pocket that could be potentially be utilized to develop cathepsin l selective inhibitors. in a subsequent study, kuhn et al. systematically compared the effectiveness of four different approaches: (a) selection by a medicinal chemist (b) manual modeling (c) docking followed by manual filtering, and (d) free energy calculations (fep). this systematic protocol enabled them to prioritize building blocks for effective targeting of cathepsin l enzyme [ ] . the authors developed a series of analogs by varying only s substituents and keeping s and s fixed ( figure ). after analyzing the affinity by enzyme kinetics, they found that the fep method was superior over other well-established methodologies; this method not only predicted the most relevant ligands but also identified the topological requirements of the substituents for a more effective engagement in the s pocket. among the developed compounds, cyclopentylmethyl substituent in the s pocket (entry , table ) incurred the most favorable interaction as it optimally filled the front part of the pocket. this strategy certainly provided an edge over other conventional methodologies in predicting the optimal ligands for the s pocket targeting. these findings could benefit the ongoing effort of achieving a suitable therapeutic candidate for cathepsin l enzyme. benzofuranyl, -benzothiazolyl, and -imidazopyridinyl, which are of similar geometry, also followed a similar inhibitory pattern (figure ). molecular modelling based on co-crystal structures showed favorable chalcogen interaction to the backbone carbonyl of asn (d(s···o = casn ) = . Å and the angle α(oasn ···s-c) = °) at the s pocket; this was further supported by a conformational strain analysis, as chalcogen-enzyme interactions compensated for higher torsional strain in the s-containing ligands when compared to the benzofuranyl and imidazopyridinyl ligands. their study demonstrated the importance of both intermolecular interactions and conformational strain in assessing the effect of heterobicyclic ligands at the s pocket that could be potentially be utilized to develop cathepsin l selective inhibitors. in a subsequent study, kuhn et al. systematically compared the effectiveness of four different approaches: (a) selection by a medicinal chemist (b) manual modeling (c) docking followed by manual filtering, and (d) free energy calculations (fep). this systematic protocol enabled them to prioritize building blocks for effective targeting of cathepsin l enzyme [ ] . the authors developed a series of analogs by varying only s substituents and keeping s and s fixed (figure ). after analyzing the affinity by enzyme kinetics, they found that the fep method was superior over other well-established methodologies; this method not only predicted the most relevant ligands but also identified the topological requirements of the substituents for a more effective engagement in the s pocket. among the developed compounds, cyclopentylmethyl substituent in the s pocket (entry , table ) incurred the most favorable interaction as it optimally filled the front part of the pocket. this strategy certainly provided an edge over other conventional methodologies in predicting the optimal ligands for the s pocket targeting. these findings could benefit the ongoing effort of achieving a suitable therapeutic candidate for cathepsin l enzyme. the thiosemicarbazone moiety was first recognized as a relevant covalent and reversible warhead of cathepsin l homologous enzyme cruzain, a protease from t. cruzi. the mechanism of inactivaction involves the formation of a transient covalent bond with the catalytic cys residue ( figure ) [ ] . in an interesting study, kishore kumar et al. first utilized this idea and synthesized a small library of compounds in which the most active class of inhibitors were comprised of one metabromo substituted aryl ring along with another one with optimally substituted functionalities [ ] . the inhibitor places itself in the active site cleft of cathepsin l where meta-bromo substituted aryl ring occupies the s site and thiosemicarbazone motif lies near the active site cysteine. however, when the motif was extended to capture s ′ site interaction by placing the aryl/alkyl group at the terminal nitrogen of thiosemicarbazone, the inhibitory potency was completely diminished. overall, this class of inhibitors showed a good selectivity over cathepsin b and exhibited low cytotoxicity when tested on human cancer cell lines. in follow up studies, kishore et al. and parker et al. further expanded the scope of thiosemicarbazone scaffold and developed diversely the thiosemicarbazone moiety was first recognized as a relevant covalent and reversible warhead of cathepsin l homologous enzyme cruzain, a protease from t. cruzi. the mechanism of inactivaction involves the formation of a transient covalent bond with the catalytic cys residue (figure ) [ ] . in an interesting study, kishore kumar et al. first utilized this idea and synthesized a small library of compounds in which the most active class of inhibitors were comprised of one meta-bromo substituted aryl ring along with another one with optimally substituted functionalities [ ] . the inhibitor places itself in the active site cleft of cathepsin l where meta-bromo substituted aryl ring occupies the s site and thiosemicarbazone motif lies near the active site cysteine. molecules , , of in a subsequent study, kuhn et al. systematically compared the effectiveness of four different approaches: (a) selection by a medicinal chemist (b) manual modeling (c) docking followed by manual filtering, and (d) free energy calculations (fep). this systematic protocol enabled them to prioritize building blocks for effective targeting of cathepsin l enzyme [ ] . the authors developed a series of analogs by varying only s substituents and keeping s and s fixed (figure ). after analyzing the affinity by enzyme kinetics, they found that the fep method was superior over other well-established methodologies; this method not only predicted the most relevant ligands but also identified the topological requirements of the substituents for a more effective engagement in the s pocket. among the developed compounds, cyclopentylmethyl substituent in the s pocket (entry , table ) incurred the most favorable interaction as it optimally filled the front part of the pocket. this strategy certainly provided an edge over other conventional methodologies in predicting the optimal ligands for the s pocket targeting. these findings could benefit the ongoing effort of achieving a suitable therapeutic candidate for cathepsin l enzyme. the thiosemicarbazone moiety was first recognized as a relevant covalent and reversible warhead of cathepsin l homologous enzyme cruzain, a protease from t. cruzi. the mechanism of inactivaction involves the formation of a transient covalent bond with the catalytic cys residue ( figure ) [ ] . in an interesting study, kishore kumar et al. first utilized this idea and synthesized a small library of compounds in which the most active class of inhibitors were comprised of one metabromo substituted aryl ring along with another one with optimally substituted functionalities [ ] . the inhibitor places itself in the active site cleft of cathepsin l where meta-bromo substituted aryl ring occupies the s site and thiosemicarbazone motif lies near the active site cysteine. however, when the motif was extended to capture s site interaction by placing the aryl/alkyl group at the terminal nitrogen of thiosemicarbazone, the inhibitory potency was completely diminished. overall, this class of inhibitors showed a good selectivity over cathepsin b and exhibited low cytotoxicity when tested on human cancer cell lines. in follow up studies, kishore et al. and parker et al. further expanded the scope of thiosemicarbazone scaffold and developed diversely functionalized analogs that exhibited an enhanced inhibitory potency and promising cellular activities while still retaining the selectivity over cathepsin b [ , ] . in their latest study, parker et al. strategically transformed an active inhibitor with limited aqueous solubility into a water-soluble prodrug (entry , table ), by phosphorylation of phenolic hydroxy group; this group was readily hydrolyzable by alkaline phosphatases, rendering the active pharmacophore [ ] . the phosphate prodrug exhibited a remarkable -fold increase in solubility over the parent drug and did not disintegrate in aqueous solution, even after prolonged exposure at the physiological temperature. furthermore, this compound did not show any significant cytotoxicity on normal primary huvecs cells in comparison to other fda-approved cytotoxic drugs, doxorubicin and paclitaxel. this prodrug thus far has shown promise to be a desirable clinical candidate and the authors have proposed to evaluate its in vivo efficacy in a preclinical setup. as noted earlier, cathepsin l, like other cysteine cathepsins, contains an inhibitory propeptide domain that spans the active site of the enzyme in the inverse direction to the regular substrate binding mode. chowdhury et al., in their seminal study, exploited this concept by examining the effect of a series of synthesized tripeptidyl compounds that mimicked cathepsin l inhibitory propeptide [ ] . importantly, the developed tripeptidyl motifs also exhibited nanomolar potency; however, a moderate truncation of the full-length propeptide drastically lost all activities [ ] . notably, while the full-length propeptide showed only -fold selectivity over cathepsin k, the most potent analog of this series (entry , table ) demonstrated a far-improved selectivity ( -fold). the authors further investigated the binding mode of this class of inhibitors by means of co-crystal structure and molecular modeling. this revealed that (a) arginine residue of the inhibitor occupied the s pocket, (b) phenyl alanine residue found favorable hydrophobic interactions within the s pocket, (c) -phenylethyl group pointed toward s pocket, (d) the methionine residue showed optimal interaction within s pocket, and (e) the biphenyl acetyl group extended to the s pocket for favorable interactions [ , ] . this class of inhibitors has shown resistance to enzyme-dependent hydrolysis and demonstrates the reversible mode of enzyme inactivation. overall, this inhibitor class provides a wealth of information on inhibitor binding to cathepsin l and provides a general template for the development of therapeutic candidates for other relevant enzymes as well. in an effort to discover small molecule inhibitors of cathepsin l, myers et al. performed high throughput screening (hts) of the nih molecular libraries small molecule repository (mlsmr); they identified , -disubstituted oxadiazoles (figure a ) as potent hit compounds [ ] . surprisingly, upon synthesis and purification of the putative inhibitory lead compounds, a complete loss of activity was observed. the authors then investigated the compounds' integrity from nih mlsmr library by lc-ms; this showed the presence of additional impurities. to trace back the active impurity, the authors hypothesized the presence of impurities resulting from an acid-catalyzed ring-opening reaction of thiocarbazate. the resulting azapeptides was likely the active pharmacophore that inhibited enzyme via acylation of active site cys; this was validated by synthesis of azapeptides and performing the enzyme assay [ ] . the (s)-stereoisomer of newly synthesized compound (entry , table ; figure b ) indeed attenuated the activity of cathepsin l with an ic value of nm. in follow up studies, the authors further developed a series of compounds with structural diversity and performed a computational analysis to recognize the basis of potent enzyme inhibition [ , ] one of the thiocarbazate analogs developed this way (figure c) showed improved potency over the parent compound. molecular modeling studies performed with parent compound (entry , table ) in complex with papain indicated that indole motif preferably bound to s subsite, -nhboc group engaged in favorable hydrophobic interactions within the s subsite and -ethylphenyl anilide extended to s pocket. to further probe the importance of thiocarbazate moiety, the authors synthesized compound containing oxocarbazate (entry , table ) and azapeptide (entry , table ) motifs. the oxocarbazate showed a fairly improved ic value ( nm) towards cathepsin l, whereas the azapeptide was at best only a modest inhibitor (ic = µm). consistently, the binding mode of oxocarbazate exerted similarity to that of thiocarbazate when investigated by molecular modeling studies [ ] . ) motifs. the oxocarbazate showed a fairly improved ic value ( nm) towards cathepsin l, whereas the azapeptide was at best only a modest inhibitor (ic = µm). consistently, the binding mode of oxocarbazate exerted similarity to that of thiocarbazate when investigated by molecular modeling studies [ ] . in a separate study, shah et al. carried out a thorough enzymatic analysis of the champion thiocarbazate compound (entry , table ) that showed a time-dependent improvement in the inhibition profile; the ic value went down to nm when preincubated with cathepsin l for h [ ] . lc-ms and kinetic analysis of enzyme-inhibitor complex (inhibition rate constants: kon = , m − s − and koff = . × − s − , and binding constant: ki = . nm) demonstrated a slow-binding kinetics and reversibility of inhibition. the selectivity over other members of the enzyme family was modest. interestingly, the compound inhibited propagation of malaria parasite plasmodium falciparum [ic = . µm], and leishmania major [ic = . µm], and did not exhibit any significant toxicity against human aortic endothelial cells and zebrafish. although thiocarbazate motif showed promise as an inhibitory scaffold, the lack of reasonable stability (it decomposes even in dmso) and only modest inhibitory activity in cell-based assays probably ceased any further development of the scaffold [ ] . the authors also extended their studies to evaluate the potential of oxocarbazate inhibitor that showed an improved ic value of . nm upon h preincubation with the enzyme. like as in the case of thiocarbazate, they performed an enzyme kinetic analysis of the enzyme-inhibitor complex and obtained the following parameters: inhibition rate constants: kon = , m − s − and koff = . × − s − , and binding constant: ki = . nm [ ] . the inhibitor blocked sars-cov (ic = ± nm) and ebola virus (ic = ± nm) entry into the human embryonic kidney (hek) t cells, a process that utilizes cathepsin l-mediated proteolysis for host cell infection. the oxocarbazate, when treated with hek t lysate in the presence of dcg- , an activity-based cysteine cathepsin probe, showed reduced cathepsin l labeling when assessed by a western-blot analysis; this further in a separate study, shah et al. carried out a thorough enzymatic analysis of the champion thiocarbazate compound (entry , table ) that showed a time-dependent improvement in the inhibition profile; the ic value went down to nm when preincubated with cathepsin l for h [ ] . lc-ms and kinetic analysis of enzyme-inhibitor complex (inhibition rate constants: k on = , m − s − and k off = . × − s − , and binding constant: k i = . nm) demonstrated a slow-binding kinetics and reversibility of inhibition. the selectivity over other members of the enzyme family was modest. interestingly, the compound inhibited propagation of malaria parasite plasmodium falciparum [ic = . µm], and leishmania major [ic = . µm], and did not exhibit any significant toxicity against human aortic endothelial cells and zebrafish. although thiocarbazate motif showed promise as an inhibitory scaffold, the lack of reasonable stability (it decomposes even in dmso) and only modest inhibitory activity in cell-based assays probably ceased any further development of the scaffold [ ] . the authors also extended their studies to evaluate the potential of oxocarbazate inhibitor that showed an improved ic value of . nm upon h preincubation with the enzyme. like as in the case of thiocarbazate, they performed an enzyme kinetic analysis of the enzyme-inhibitor complex and obtained the following parameters: inhibition rate constants: k on = , m − s − and k off = . × − s − , and binding constant: k i = . nm [ ] . the inhibitor blocked sars-cov (ic = ± nm) and ebola virus (ic = ± nm) entry into the human embryonic kidney (hek) t cells, a process that utilizes cathepsin l-mediated proteolysis for host cell infection. the oxocarbazate, when treated with hek t lysate in the presence of dcg- , an activity-based cysteine cathepsin probe, showed reduced cathepsin l labeling when assessed by a western-blot analysis; this further corroborated the results obtained from the virus pseudotype infection assay. overall, oxocarbazate inhibitor not only provided a promising template for further exploitation but also rendered a new direction for intervening sars and ebola virus infections. ubiquitous expression of human cathepsin l in most human tissues possesses a significant challenge in targeting this enzyme for therapeutic development. this problem is further exacerbated with recent findings that alternative spliced isoforms could exist at distinct cellular locations (e.g., nucleus, cytosol, and ecm space) [ , , ] . while several unique functional roles of cathepsin l are known, it has also been reported that some of its function can also be accomplished by other members of the cathepsin family (i.e., functional redundancy); for example, both cathepsin l and b can mediate a mutually compensatory role in the inflammatory response signaling pathways [ ] . in this regard, accurate function of cathepsin l must be first determined in different cell types individually before considering significant investment in drug development. since the function of an enzyme, such as cathepsin l, depends primarily on its activity profile and given that the activity profiles of differentially processed cathepsin l isoforms may be very different, probes capable of reporting accurate activity status in different cell types (and cellular location) are anticipated to advance our understanding if cathepsin l biology. over the years, the concept of activity-based probes (abps) has emerged as a valuable chemical biology tool for monitoring the enzyme activity (not just the expression profile alone) in cells at the proteome levels [ ] [ ] [ ] [ ] . in most cases, existing covalent inhibitors containing a recognition motif are adopted and transformed to abps by conjugating optimal detection modalities; these include fluorescent probes, affinity labels, radiotracer, and many others ( figure ) . indeed, the use of abps has rather established cathepsins as key diagnostic marker for various disease conditions, and have even enabled optical surgical navigation, leading to an improved surgical precision [ , ] . in the following sections, we discussed cathepsin l-selective molecular probes that have been developed and utilized for monitoring its activity. a thorough overview of cathepsin probe development for imaging purpose could be found elsewhere [ , [ ] [ ] [ ] . ubiquitous expression of human cathepsin l in most human tissues possesses a significant challenge in targeting this enzyme for therapeutic development. this problem is further exacerbated with recent findings that alternative spliced isoforms could exist at distinct cellular locations (e.g., nucleus, cytosol, and ecm space) [ , , ] . while several unique functional roles of cathepsin l are known, it has also been reported that some of its function can also be accomplished by other members of the cathepsin family (i.e., functional redundancy); for example, both cathepsin l and b can mediate a mutually compensatory role in the inflammatory response signaling pathways [ ] . in this regard, accurate function of cathepsin l must be first determined in different cell types individually before considering significant investment in drug development. since the function of an enzyme, such as cathepsin l, depends primarily on its activity profile and given that the activity profiles of differentially processed cathepsin l isoforms may be very different, probes capable of reporting accurate activity status in different cell types (and cellular location) are anticipated to advance our understanding if cathepsin l biology. over the years, the concept of activity-based probes (abps) has emerged as a valuable chemical biology tool for monitoring the enzyme activity (not just the expression profile alone) in cells at the proteome levels [ ] [ ] [ ] [ ] . in most cases, existing covalent inhibitors containing a recognition motif are adopted and transformed to abps by conjugating optimal detection modalities; these include fluorescent probes, affinity labels, radiotracer, and many others ( figure ) . indeed, the use of abps has rather established cathepsins as key diagnostic marker for various disease conditions, and have even enabled optical surgical navigation, leading to an improved surgical precision [ , ] . in the following sections, we discussed cathepsin l-selective molecular probes that have been developed and utilized for monitoring its activity. a thorough overview of cathepsin probe development for imaging purpose could be found elsewhere [ , [ ] [ ] [ ] . radio-labeled inhibitors have long been used as a primary mode for detecting active cysteine proteases both in vitro and in vivo. docherty et al. first used a chloromethyl inhibitor containing radioactive iodine, i-tyr-ala-lys-argch cl, and detected cathepsin b in crude granule fraction of islet cells [ ] . in their follow up study, they also presumably identified cathepsin l in insulin secretory granule using the same radio-isotopically labeled inhibitor [ ] . the mechanism of detection involves covalent modification of the target protein that shows up as a distinct band upon performing autoradiography. this chloromethyl containing inhibitor turned out to be non-selective due to its reactivity towards trypsin, a serine protease. this was followed by the discovery of a radioactive-peptidyldiazomethane compound (entry p , table ), a selective cysteine proteinase inhibitor [ ] . mason et al. adopted the peptidyldiazomethane scaffold, which potently inhibited both cathepsin l and b. this scaffold showed improved inhibition profile upon iodination, as demonstrated by crawford et al. [ ] this inhibitory agent was then transformed to a radio-labeled probe via incorporation of i [ ] . the developed probe was utilized to detect both cathepsin l and b in kirsten-virus-transformed knih t cells. the incubation of cellular extracts with p followed by gel electrophoresis showed the presence of two protein bands at and kda, showing two active forms of cathepsin l. active cathepsin b was also detected at around - kda. interestingly, pulse-chase experiments with [ s]methionine-labeled proteins only detected two separate bands at kda and kda, which correspond to the intracellular inactive precursors of cathepsin l and b respectively. this indicated that the inactive precursor proteins did not react with p , demonstrating its unique ability to quantify only active protein. further, p was utilized to probe active cathepsin l and b in different human tissues as well as in lysosomes and whole cells [ , ] . in a follow-up study, xing et al. developed fmoc-[i ]tyr-ala-chn (entry p , table ) that selectively detected cathepsin l and b over cathepsin s, exhibiting a faster rate of inactivation towards cathepsin l [ ] . the developed compound successfully probed the amount of active cathepsin l and b in different cell-lines; two unknown proteins also got labeled in certain cases. overall, these probes enabled the detection of active cathepsin enzymes with their cellular location, thereby advancing the knowledge of cathepsin l biology. radio-labeled inhibitors have long been used as a primary mode for detecting active cysteine proteases both in vitro and in vivo. docherty et al. first used a chloromethyl inhibitor containing radioactive iodine, i-tyr-ala-lys-argch cl, and detected cathepsin b in crude granule fraction of islet cells [ ] . in their follow up study, they also presumably identified cathepsin l in insulin secretory granule using the same radio-isotopically labeled inhibitor [ ] . the mechanism of detection involves covalent modification of the target protein that shows up as a distinct band upon performing autoradiography. this chloromethyl containing inhibitor turned out to be non-selective due to its reactivity towards trypsin, a serine protease. this was followed by the discovery of a radioactive-peptidyldiazomethane compound (entry p , table ), a selective cysteine proteinase inhibitor [ ] . mason et al. adopted the peptidyldiazomethane scaffold, which potently inhibited both cathepsin l and b. this scaffold showed improved inhibition profile upon iodination, as demonstrated by crawford et al. [ ] this inhibitory agent was then transformed to a radio-labeled probe via incorporation of i [ ] . the developed probe was utilized to detect both cathepsin l and b in kirsten-virus-transformed knih t cells. the incubation of cellular extracts with p followed by gel electrophoresis showed the presence of two protein bands at and kda, showing two active forms of cathepsin l. active cathepsin b was also detected at around - kda. interestingly, pulse-chase experiments with [ s]methionine-labeled proteins only detected two separate bands at kda and kda, which correspond to the intracellular inactive precursors of cathepsin l and b respectively. this indicated that the inactive precursor proteins did not react with p , demonstrating its unique ability to quantify only active protein. further, p was utilized to probe active cathepsin l and b in different human tissues as well as in lysosomes and whole cells [ , ] . in a follow-up study, tyr-ala-chn (entry p , table ) that selectively detected cathepsin l and b over cathepsin s, exhibiting a faster rate of inactivation towards cathepsin l [ ] . the developed compound successfully probed the amount of active cathepsin l and b in different cell-lines; two unknown proteins also got labeled in certain cases. overall, these probes enabled the detection of active cathepsin enzymes with their cellular location, thereby advancing the knowledge of cathepsin l biology. radio-labeled inhibitors have long been used as a primary mode for detecting active cysteine proteases both in vitro and in vivo. docherty et al. first used a chloromethyl inhibitor containing radioactive iodine, i-tyr-ala-lys-argch cl, and detected cathepsin b in crude granule fraction of islet cells [ ] . in their follow up study, they also presumably identified cathepsin l in insulin secretory granule using the same radio-isotopically labeled inhibitor [ ] . the mechanism of detection involves covalent modification of the target protein that shows up as a distinct band upon performing autoradiography. this chloromethyl containing inhibitor turned out to be non-selective due to its reactivity towards trypsin, a serine protease. this was followed by the discovery of a radioactive-peptidyldiazomethane compound (entry p , table ), a selective cysteine proteinase inhibitor [ ] . mason et al. adopted the peptidyldiazomethane scaffold, which potently inhibited both cathepsin l and b. this scaffold showed improved inhibition profile upon iodination, as demonstrated by crawford et al. [ ] this inhibitory agent was then transformed to a radio-labeled probe via incorporation of i [ ] . the developed probe was utilized to detect both cathepsin l and b in kirsten-virus-transformed knih t cells. the incubation of cellular extracts with p followed by gel electrophoresis showed the presence of two protein bands at and kda, showing two active forms of cathepsin l. active cathepsin b was also detected at around - kda. interestingly, pulse-chase experiments with [ s]methionine-labeled proteins only detected two separate bands at kda and kda, which correspond to the intracellular inactive precursors of cathepsin l and b respectively. this indicated that the inactive precursor proteins did not react with p , demonstrating its unique ability to quantify only active protein. further, p was utilized to probe active cathepsin l and b in different human tissues as well as in lysosomes and whole cells [ , ] . in a follow-up study, tyr-ala-chn (entry p , table ) that selectively detected cathepsin l and b over cathepsin s, exhibiting a faster rate of inactivation towards cathepsin l [ ] . the developed compound successfully probed the amount of active cathepsin l and b in different cell-lines; two unknown proteins also got labeled in certain cases. overall, these probes enabled the detection of active cathepsin enzymes with their cellular location, thereby advancing the knowledge of cathepsin l biology. radio-labeled inhibitors have long been used as a primary mode for detecting active cysteine proteases both in vitro and in vivo. docherty et al. first used a chloromethyl inhibitor containing radioactive iodine, i-tyr-ala-lys-argch cl, and detected cathepsin b in crude granule fraction of islet cells [ ] . in their follow up study, they also presumably identified cathepsin l in insulin secretory granule using the same radio-isotopically labeled inhibitor [ ] . the mechanism of detection involves covalent modification of the target protein that shows up as a distinct band upon performing autoradiography. this chloromethyl containing inhibitor turned out to be non-selective due to its reactivity towards trypsin, a serine protease. this was followed by the discovery of a radioactive-peptidyldiazomethane compound (entry p , table ), a selective cysteine proteinase inhibitor [ ] . mason et al. adopted the peptidyldiazomethane scaffold, which potently inhibited both cathepsin l and b. this scaffold showed improved inhibition profile upon iodination, as demonstrated by crawford et al. [ ] this inhibitory agent was then transformed to a radio-labeled probe via incorporation of i [ ] . the developed probe was utilized to detect both cathepsin l and b in kirsten-virus-transformed knih t cells. the incubation of cellular extracts with p followed by gel electrophoresis showed the presence of two protein bands at and kda, showing two active forms of cathepsin l. active cathepsin b was also detected at around - kda. interestingly, pulse-chase experiments with [ s]methionine-labeled proteins only detected two separate bands at kda and kda, which correspond to the intracellular inactive precursors of cathepsin l and b respectively. this indicated that the inactive precursor proteins did not react with p , demonstrating its unique ability to quantify only active protein. further, p was utilized to probe active cathepsin l and b in different human tissues as well as in lysosomes and whole cells [ , ] . in a follow-up study, tyr-ala-chn (entry p , table ) that selectively detected cathepsin l and b over cathepsin s, exhibiting a faster rate of inactivation towards cathepsin l [ ] . the developed compound successfully probed the amount of active cathepsin l and b in different cell-lines; two unknown proteins also got labeled in certain cases. overall, these probes enabled the detection of active cathepsin enzymes with their cellular location, thereby advancing the knowledge of cathepsin l biology. the detecting agents-radionuclide (p , p ), biotin (p ), fluorophore (p , p , p ), clickable acetylene (p ), and lanthanide containing dota (p )-is red-color coded and the photoactivatable benzoyl group (p ) and quencher (p ) is coded in blue. # : no inhibition. gelhaus et al. first developed biotinylated aziridine- , -dicarboxylate and demonstrated its antiplasmodial activity using cell-based studies [ ] . since, the biotinylated compound inhibited plasmodial protease falcipain and cathepsin l, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes involved in parasite pathogenicity. later on, vicik et al. adopted this motif (entry p , table ) and developed an affinity label to probe cathepsin l activity [ ] . the aziridine analog irreversibly inactivates the enzyme via covalent modification, as discussed previously. the conjugated biotin moiety is utilized for affinity pull down and target identification. when (s,s) isomer of the biotinylated probe was incubated with cathepsin l and subjected to gel electrophoresis, electrotransferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong labeling of the enzyme-inhibitor complex was observed. however, when the enzyme was treated with e- , an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with p , the labeling was diminished, clearly demonstrating that p competes for the active site of cathepsin l. in line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. although p has a the detecting agents-radionuclide (p , p ), biotin (p ), fluorophore (p , p , p ), clickable acetylene (p ), and lanthanide containing dota (p )-is red-color coded and the photoactivatable benzoyl group (p ) and quencher (p ) is coded in blue. # : no inhibition. gelhaus et al. first developed biotinylated aziridine- , -dicarboxylate and demonstrated its antiplasmodial activity using cell-based studies [ ] . since, the biotinylated compound inhibited plasmodial protease falcipain and cathepsin l, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes involved in parasite pathogenicity. later on, vicik et al. adopted this motif (entry p , table ) and developed an affinity label to probe cathepsin l activity [ ] . the aziridine analog irreversibly inactivates the enzyme via covalent modification, as discussed previously. the conjugated biotin moiety is utilized for affinity pull down and target identification. when (s,s) isomer of the biotinylated probe was incubated with cathepsin l and subjected to gel electrophoresis, electrotransferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong labeling of the enzyme-inhibitor complex was observed. however, when the enzyme was treated with e- , an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with p , the labeling was diminished, clearly demonstrating that p competes for the active site of cathepsin l. in line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. although p has a the detecting agents-radionuclide (p , p ), biotin (p ), fluorophore (p , p , p ), clickable acetylene (p ), and lanthanide containing dota (p )-is red-color coded and the photoactivatable benzoyl group (p ) and quencher (p ) is coded in blue. # : no inhibition. gelhaus et al. first developed biotinylated aziridine- , -dicarboxylate and demonstrated its antiplasmodial activity using cell-based studies [ ] . since, the biotinylated compound inhibited plasmodial protease falcipain and cathepsin l, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes involved in parasite pathogenicity. later on, vicik et al. adopted this motif (entry p , table ) and developed an affinity label to probe cathepsin l activity [ ] . the aziridine analog irreversibly inactivates the enzyme via covalent modification, as discussed previously. the conjugated biotin moiety is utilized for affinity pull down and target identification. when (s,s) isomer of the biotinylated probe was incubated with cathepsin l and subjected to gel electrophoresis, electrotransferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong labeling of the enzyme-inhibitor complex was observed. however, when the enzyme was treated with e- , an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with p , the labeling was diminished, clearly demonstrating that p competes for the active site of cathepsin l. in line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. although p has a fluorescent cathepsin l specific (some degree of labelling was seen for cat v and b) the detecting agents-radionuclide (p , p ), biotin (p ), fluorophore (p , p , p ), clickable acetylene (p ), and lanthanide containing dota (p )-is red-color coded and the photoactivatable benzoyl group (p ) and quencher (p ) is coded in blue. # : no inhibition. gelhaus et al. first developed biotinylated aziridine- , -dicarboxylate and demonstrated its antiplasmodial activity using cell-based studies [ ] . since, the biotinylated compound inhibited plasmodial protease falcipain and cathepsin l, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes involved in parasite pathogenicity. later on, vicik et al. adopted this motif (entry p , table ) and developed an affinity label to probe cathepsin l activity [ ] . the aziridine analog irreversibly inactivates the enzyme via covalent modification, as discussed previously. the conjugated biotin moiety is utilized for affinity pull down and target identification. when (s,s) isomer of the biotinylated probe was incubated with cathepsin l and subjected to gel electrophoresis, electrotransferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong labeling of the enzyme-inhibitor complex was observed. however, when the enzyme was treated with e- , an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with p , the labeling was diminished, clearly demonstrating that p competes for the active site of cathepsin l. in line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. although p has a the detecting agents-radionuclide (p , p ), biotin (p ), fluorophore (p , p , p ), clickable acetylene (p ), and lanthanide containing dota (p )-is red-color coded and the photoactivatable benzoyl group (p ) and quencher (p ) is coded in blue. # : no inhibition. gelhaus et al. first developed biotinylated aziridine- , -dicarboxylate and demonstrated its antiplasmodial activity using cell-based studies [ ] . since, the biotinylated compound inhibited plasmodial protease falcipain and cathepsin l, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes involved in parasite pathogenicity. later on, vicik et al. adopted this motif (entry p , table ) and developed an affinity label to probe cathepsin l activity [ ] . the aziridine analog irreversibly inactivates the enzyme via covalent modification, as discussed previously. the conjugated biotin moiety is utilized for affinity pull down and target identification. when (s,s) isomer of the biotinylated probe was incubated with cathepsin l and subjected to gel electrophoresis, electrotransferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong labeling of the enzyme-inhibitor complex was observed. however, when the enzyme was treated with e- , an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with p , the labeling was diminished, clearly demonstrating that p competes for the active site of cathepsin l. in line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. although p has a the detecting agents-radionuclide (p , p ), biotin (p ), fluorophore (p , p , p ), clickable acetylene (p ), and lanthanide containing dota (p )-is red-color coded and the photoactivatable benzoyl group (p ) and quencher (p ) is coded in blue. # : no inhibition. gelhaus et al. first developed biotinylated aziridine- , -dicarboxylate and demonstrated its anti-plasmodial activity using cell-based studies [ ] . since, the biotinylated compound inhibited plasmodial protease falcipain and cathepsin l, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes involved in parasite pathogenicity. later on, vicik et al. adopted this motif (entry p , table ) and developed an affinity label to probe cathepsin l activity [ ] . the aziridine analog irreversibly inactivates the enzyme via covalent modification, as discussed previously. the conjugated biotin moiety is utilized for affinity pull down and target identification. when (s,s) isomer of the biotinylated probe was incubated with cathepsin l and subjected to gel electrophoresis, electro-transferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong labeling of the enzyme-inhibitor complex was observed. however, when the enzyme was treated with e- , an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with p , the labeling was diminished, clearly demonstrating that p competes for the active site of cathepsin l. in line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. although p has a modest binding affinity (k i = . µm) to cathepsin l, it exerted a -fold selectivity over cathepsin b. certainly, the affinity labeling technique not only served as an abp for cathepsin l but also provides a premise for developing aziridine-based chemical tools for functional proteomics. although covalent modifiers of proteins have been vastly exploited as chemical tools for target identification and functional proteomics, photoaffinity probes offer unique mode of action. they bind proteins non-covalently (affinity based solely on non-covalent interactions) first and form a non-selective covalent bond with the closest amino acid residue only upon irradiation. torkar et al. took advantage of this technique and developed the first photoaffinity-based probe (entry p , table ) to detect active cathepsin l selectively over other members of the family [ ] . the photoaffinity-based probe was designed based on existing peptidyl acetyloxymethyl ketone (aomk), a known covalent modifier of cysteine proteases. during the design, the aomk group at the c-terminus was replaced by a short di(ethylene glycol) moiety that increased the aqueous solubility and altered the character of the inhibitor from irreversible to a reversible one. the probe was comprised of a lysine residue that was strategically placed to append fluorescent cyanine- (cy ) group for detection. a photoactivatable benzoylphenylalanine amino acid was placed to accommodate the s pocket of cathepsin l. the developed probe detected recombinant cathepsin l upon incubation and subsequent irradiation for min at nm as demonstrated by sds-page. the protein band became invisible when enzyme was incubated with known cathepsin l inhibitors, e- and gb -nh , respectively, prior to the probe treatment [ , ] . the probe also showed preferential selectivity towards cathepsin l when compared to other two fluorescent-based probes. the probe p exhibited a remarkable selectivity for cathepsin l over all other cathepsins in light-mediated labeling experiments with recombinant proteins. the relative labeling percentages (cathepsin b and k: %; cathepsin s: %) were insignificant, except for cathepsin v ( %), the closest homolog of cathepsin l, relative to cathepsin l. interestingly, u -mg glioma cell extracts did not present any cathepsin l for detection with the p probe. however, when the same cell extracts were treated with recombinant cathepsin l added externally and subjected to a labeling experiment, the probe selectively detected the desired protein in complex proteome. the mechanism of protein labeling by p was attributed to putative bond formation between benzophenone and non-conserved met at the s site of cathepsin l. although the developed probe lacks cell-penetrability, the authors envisioned that the technique might have diagnostic and prognostic value where cathepsin l overexpression is high, such as in malignant tissues. although several fluorescent-based molecular probes have been reported for cysteine cathepsins, the superiority of two-photon-based over one-photon based imaging technique inspired na et al. to develop probes with a better cellular imaging profile [ ] . notably, the two-photon fluorescence imaging technique provides increased tissue penetration depth with reduced photobleaching, and a lower tissue autofluorescence. the authors first fabricated a microarray with different peptidyl aldehydes and screened against gfp-labeled cathepsin l enzyme; this led to the identification of two inhibitory hits that potently inactivated enzyme with ic values of . and . nm. the lead inhibitors were further structurally modified to include (a) a two-photon dye, dl- (a , -bis( -hydroxystyryl)pyrimidine derivative), at p position, and (b) disperse red dye, a fluorescence quencher, in the place of aldehyde moiety. the resulting imaging probes (entry p in table is one of such examples) showed a time-dependent increase in fluorescence signals when treated with hepg cell lysates, a mammalian liver cancer cell-line known to overexpresses cysteine cathepsins. enhanced fluorescence signal is due to the release of the quencher upon successful proteolytic cleavage. furthermore, to assess the suitability of p as imaging agents for live cells, hepg cells were incubated with the probe and subjected to a live-cell imaging analysis. there were strong fluorescence signals from endolysomal compartments that disappeared completely when cells were pretreated with e- , validating the target specificity of the probe. as the probe was developed using cathepsin l as a model enzyme, it likely will lack specificity and perhaps interact with other members of the family. still, however, this motif could potentially be utilized to develop selective abps targeting respective cathepsins and assess their activities in a tissue environment, as proposed by the authors. activity-based probes with a single photon fluorescent tag have been successfully utilized for the functional analysis of target proteins both in vitro and in vivo. poreba et al., in their pursuit of developing cathepsin l selective imaging agent, took advantage of this technology [ ] . they realized the importance of developing selective cathepsin l substrate that will bind to the active site of the enzyme over other homologous proteins. to do so, they employed hybrid combinatorial substrate library (hycosul) technology that provides information on optimal chemical space inside the enzyme active sites by strategically scanning diverse peptide library containing both natural and unnatural amino acids. this led to the acquisition of a panel of compounds with desired properties. the potent hits discovered by hycosul technology were further optimized to gain selectivity over other cathepsins while identifying the most efficient substrate based on michaelis menten parameters. unfortunately, when the chosen peptide substrate transformed to an activity-based probe by appending an acyloxymethylketone warhead and a biotin tag, it showed cross-reactivity with cathepsin b. despite biotinylated probe's somewhat compromised selectivity, the authors still assessed its activity-based labeling profile in hek t cells; as expected, cathepsin l labeling was primarily observed with minor amounts of cathepsin b. further optimization by swapping the biotin and arg with cyanine- and cys(bzl) (entry p , table ) groups was carried out next. the newly developed fluorescent abp, p , showed an enhanced selectivity (in comparison to pan-cathepsin probes) against a panel of other recombinant cathepsins (cathepsin v, b, s, and k), as well as cellular extracts derived from hek t and mda-mb- cells. the developed probe served as an effective imaging agent for cellular cathepsin l activity in human mda-mb- breast cancer cells when incubated for h. the selectivity of the probe started to recede with longer incubation time. the importance of optimizing the probe concentration and time-course of the reaction was evident from these experiments. interestingly, p only detected active cathepsin l and not procathepsin l; this certainly signifies the effectiveness of the developed probes as an activity-based probe. the authors further examined the colocalization of the probe with both cathepsin l and b in mda-mb- cells by treating the cells with respective cathepsin antibodies and performing a quantitative pixel analysis from a set of fluorescence microscopy images. this supported the previous observation as the highest weighted colocalization coefficient was obtained for cathepsin l and not for cathepsin b. the developed compound certainly harbors the key traits of an effective activity-based probe, as the authors duly envisioned its significance in deciphering cathepsin l biology in the coming years. as noted above, fluorescence-based imaging probes have been successfully developed to gain access to unknown functionalities of cathepsin enzymes. however, the bulkiness and often multiple charges associated with the fluorophore and/or quencher structures on these probes likely render them poorly cell-permeable and reduce their target affinity. to address this issue, dana et al. adopted a previously reported peptidyl vinylsulfonate inhibitor kd- -a highly potent, selective, covalent and irreversible inhibitor of cathepsin l discussed in section . -and tactically appended a small alkynyl group at the para-position of the cbz group [ ] . this led to the development of a clickable and tagless activity-based probe (catabp) of cathepsin l that retained the key desirable traits of kd- ; i.e., cell permeability, charge profile, molecular weight, potency and selectivity profile [ ] . this strategy eliminated the requirements of including bulky and charged fluorophore/quencher moiety to the probe. one of the inherent advantages of this approach is that the labeling can be performed in live cellular environment with high efficiency. after cell lysis, the labeled cathepsin l can be quantified by performing click chemistry protocol with a fluorescent azide containing dye, resolving the protein using gel-electrophoresis, and directly scanning the gel for fluorescence signal. as anticipated, the developed kdp- probe (entry p , table ), exhibited rapid inactivation kinetics, retained selectivity for cathepsin l, and labeled recombinant active cathepsin l in an activity-dependent manner; the heat-denatured and e- treated cathepsin l showed no labeling when subjected to the identical labeling protocol. a mass-spectrometric analysis of the enzyme-probe complex concluded that the probe was active-site directed, and covalently modified the catalytic cys residue for inactivation. since the kdp- probe was developed with the intention of capturing active cathepsin l in vivo and in human live cell culture, the probe was tested for its cytotoxicity in mda-mb- cells; no cytotoxicity was observed in these cells, even at a concentration as high as µm. incubation of mda-mb- cells overexpressing cathepsin l with kdp- attenuated the intracellular cathepsin l activity in a dose-dependent manner; this was demonstrated by live-cell imaging and wound healing assay. finally, kdp- also interfered with the hatching process of post-fertilized zebrafish embryos, further validating probe's in vivo activity; notably, cathepsin l serves as the key regulator of the hatching process [ ] [ ] [ ] . in conclusion, kdp- demonstrated many desirable attributes of a good probe and is anticipated to find extensive applications in probing cathepsin l function in cells from diverse origins. although fluorophores-containing molecules have been appreciated as useful imaging probes, commonly used fluorophores often suffer from spectral overlapping that limits the number of targets that can be analyzed concomitantly [ ] . to address this issue, poreba et al. recently developed protease-selective lanthanide-labeled probes compatible with mass cytometry which allows subsequent analysis by both mass and imaging mass cytometry (imc) [ ] . these metal-tagged, time of flight activity-based probes (tof) allowed them to determine cellular activities and location of three lysosomal proteases. thus, using cathepsin l, b, and legumain as the model systems, they elegantly crafted an activity-based probe by incorporating (a) a protease-selective peptide sequence for specific enzyme recognition, (b) the acyloxymethylketone as electrophilic warhead to trap the target enzyme, and (c) the dodecanetetraacetic acid (dota) for metal chelation that is tethered to the peptide sequence via a linker (entry p , table ). they further incorporated three different lanthanides, tb, lu, and gd (a mixture of naturally occurring six isotopes), to validate their approach and to further evaluate the influence of isotopes on enzyme binding specificity. the newly developed probes exerted promising selectivity toward both recombinant proteases and proteases from cancer cell lines, hct- and mda-mb- .the authors were able to simultaneous detect the activities of proteases in hct- cells. moreover, each of the protease-specific probes exerted a similar labelling efficiency in hct- cells, regardless of their metal counterparts, which further reinforces the compatibility of this class of probes as cytometry-labelling agents. they extended their investigation to thp- cells, a non-adherent monocyte-like cell line, that expresses both cathepsin b and l but contains very low levels of legumain enzyme. probe treated thp- showed a clear labeling of both cathepsin b and l with no detectable activity of legumain. this finding was consistent with their transcriptional data. finally, the developed probes not only allowed to detect the activome of cathepsin l, b, and legumain in peripheral blood mononuclear cells (pbmc) but also enabled to categorize nk cells in two distinct populations based on protease activome levels. this strategy, unlike many existing technologies, thus allowed the simultaneous detection of target proteases, thereby providing a more holistic understanding of the activome. this certainly merits the future development of tof-based probes for multiplexed enzyme activity detection. the inhibition of cathepsin l has continue to emerge at the forefront of drug development for several human diseases. yet, no inhibitory agents targeting cathepsin l have advanced to clinical trials. while inhibitors of cathepsin b and s are currently being evaluated in clinical trials, recent failure of odanacatib, a cathepsin k inhibitor for osteoporosis, in late stage clinical trial has made pharmaceutical industries wary of targeting cathepsins. the key challenge remains gaining inhibitor selectivity with respect to the other members of cathepsins and directing them to the targeted cell types for selective functional perturbation. with its ubiquitous expression profile, the function of cathepsin l in individual cell types must be precisely defined first; this is especially important since several isoforms of cathepsin l have been reported in the distinct cellular locations, and their activity and functional profile in individual cell types still remain poorly documented. while recently developed cathepsin inhibitors and probes have significantly advanced our understanding of cathepsin l function in both normal and disease cells, more efforts are needed for the development of isoform-selective reagents for further advancement; perhaps new allosteric modules on cathepsin l enzyme can be explored and exploited for precision targeting. fortunately, several structural coordinates for cathepsin l enzyme forms, some with a diverse set of complexing ligands and some without, are now available (table ). these could aid in the development of isoform-selective inhibitory probes that will enable researchers to assess the context-specific needs of targeting cathepsin l in different cellular states. in recent years, disease-associated protease activatable prodrugs have gained much recognition and garnered breakthrough therapies in the area of antibody-drug conjugation (adc) [ ] . the majority of the adcs are constructed by tethering a cytotoxic drug with an antibody via a protease-sensitive module for targeted delivery. cathepsin b-specific module, for example, has been successfully implemented for this role, which led to the development of fda approved therapies; for example, brentuximab vedotin (adcetris ® ) for cd -positive relapsed or refractory hodgkin s lymphoma. in addition, protease cleavable prodrug strategy has also inspired the development of cathepsin b selective probe and even enabled real-time monitoring of drug release [ , ] . interestingly, ueki et al. slightly maneuvered this approach to acquire a prodrug which gets serially activated by histone deacetylase (hdac) and cathepsin l, and subsequently delivers the cytotoxic payload, puromycin, to cancer cells [ ] . this strategy has thus enabled selective targeting of cancer cells, specifically with high hdac and cathepsin l activities. taken together, these developments surely lay the foundation for the development of future cathepsin l-based therapies. much excitement remains as new cathepsin l functions continue to emerge from specific cell types in the coming years. axl x-ray . [ ] axm x-ray . [ ] f x-ray . [ ] i h x-ray . [ ] mae x-ray . [ ] maj x-ray . [ ] mqy x-ray . [ ] ezp x-ray . [ ] ezx x-ray . [ ] f x-ray . [ ] funding: s.k.p. gratefully acknowledges the financial support from the national science foundation (nsf); grant no. , , for this work. acknowledgments: authors also wish to thank senthil perumal for providing critical feedback during the preparation of this manuscript. the authors declare no conflict of interest. the lysosome turns fifty the proteome of lysosomes comprehensive search for cysteine cathepsins in the human genome specialized roles for cysteine cathepsins in health and disease cysteine cathepsins: from structure, function and regulation to new frontiers cysteine cathepsin proteases: regulators of cancer progression and therapeutic response cysteine cathepsins in extracellular matrix remodeling: extracellular matrix degradation and beyond. matrix boil the future of cysteine cathepsins in disease management cysteine proteases as therapeutic targets: does selectivity matter? a systematic review of calpain and cathepsin inhibitors cysteine cathepsins: their role in tumor 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cathepsin l structural basis for the recognition and cleavage of histone h by cathepsin l unreduced cathepsin l in complex with stefin a optimization of triazine nitriles as rhodesain inhibitors: structure-activity relationships, bioisosteric imidazopyridine nitriles, and x-ray crystal structure analysis with human cathepsin l. chemmedchem r)- -( -chloro- -methoxy-benzenesulfonyl)- -[ -( -chloro-pyridin- -yl)-azetidine- -carbonyl]-pyrrolidine- -carboxylic acid ( -cyano-cyclopropyl)-amide caught in the act: the crystal structure of cleaved cathepsin l bound to the active site of cathepsin l key: cord- - n cdex authors: corse, tanner; dayan, linda; kersten, sydney; battaglia, fortunato; terlecky, stanley r; han, zhiyong title: clinical outcomes of covid- patients with pre-existing, compromised immune systems: a review of case reports date: - - journal: int j med sci doi: . /ijms. sha: doc_id: cord_uid: n cdex in the ongoing covid- pandemic, all covid- patients are naïve patients as it is the first-time humans have been exposed to the sars-cov- virus. as with exposure to many viruses, individuals with pre-existing, compromised immune systems may be at increased risk of developing severe symptoms and/or dying because of (sars-cov- ) infection. to learn more about such individuals, we conducted a search and review of published reports on the clinical characteristics and outcomes of covid- patients with pre-existing, compromised immune systems. here we present our review of patients who possess pre-existing primary antibody deficiency (pad) and those who are organ transplant recipients on maintenance immunosuppressants. our review indicates different clinical outcomes for the patients with pre-existing pad, depending on the underlying causes. for organ transplant recipients, drug-induced immune suppression alone does not appear to enhance covid- mortality risk - rather, advanced age, comorbidities, and the development of secondary complications appears required. the ongoing covid- pandemic is caused by the zoonotic coronavirus sars-cov- [ ] , which primarily infects cells in the respiratory system, including nasal ciliated epithelial cells and goblet/secretory cells, unciliated epithelial cells in the respiratory tracts, type ii alveolar pneumocytes, and endothelial cells of the microvasculature of lungs [ , ] . these cells express the angiotensin converting enzyme (ace ) as a transmembrane protein in the plasma membrane, and sars-cov- binds to the extracellular domain of ace to gain entrance into the cell to initiate an infection [ ] . some sars-cov- -infected individuals are asymptomatic [ ] , but most infected persons initially develop upper respiratory infection symptoms, such as fever, cough, sore throat, fatigue, and myalgia, with later development of viral pneumonia with accompanying dyspnea [ , ] . most symptomatic covid- patients recover with supportive care in hospitals; however, a small but significant percentage of covid- patients are at risk of developing acute respiratory distress syndrome (ards) or respiratory failure [ , ] . those particularly at risk are elderly covid- patients (> years) and those with underlying comorbidities, such as diabetes, hypertension, and chronic obstructive pulmonary disease [ , ] . such patients often require intensive care unit (icu) care and oxygen therapy including mechanical ventilation [ , ] . these are critically ill covid- patients and the mortality rate among them is high. an analysis of the clinical outcomes of , covid- patients in china suggested an overall ivyspring international publisher mortality rate of . %, but it was . % among those aged - years, and . % among those aged > years [ ] . in italy, the study by onder et al found that the overall mortality rate is . %, with a mortality rate of % for those aged < years, . % for those aged - years, . % for those aged - years, % for those aged - years, . % for those aged - years, . % for those aged - years, and . % for those aged > years [ ] . analysis by the united states center for disease control and prevention (cdc) of the clinical outcomes of covid- patients in the united states between february and march , showed that the mortality rate of covid- patients also varies with age: < % among patients aged - years; % among patients aged - years; % among patients aged - years; and . % among patients ≥ years [ ] these analyses indicate that the risks of developing severe covid- and dying from it are higher in persons of advancing age. postmortem studies of deceased covid- patients suggest that death from covid- results primarily from respiratory failure due to extensive diffuse alveolar damage, and pulmonary microvascular thrombosis [ ] [ ] [ ] [ ] . thus far, most evidence suggests that an overly active immune system in covid- patients causes activation of a disproportionately high number of pathogenic t cells and inflammatory monocytes/ macrophages, resulting in the development of an inflammatory cytokine storm. this causes severe damage of alveolar epithelial cells and endothelial cells of microvasculature in the lungs, culminating in ards and respiratory failure [ , , ] . therefore, treatment of covid- needs to achieve activation of anti-sars-cov- immunity, and at the same time prevention of inflammatory damage and thromboembolism in the lungs. one of the many unanswered questions regarding covid- is how the naïve immune system reacts, adapts, and mounts an effective defense against the sars-cov- virus. in comparison to covid- patients with mild to moderate symptoms, those with severe symptoms had significantly decreased counts of cd + , cd + , and cd + t cells [ , [ ] [ ] [ ] , decreased numbers of b cells and natural killer (nk) cells [ ] , and exhaustion of functional t cells [ , ] . additionally, there is evidence of decreased numbers of cd + t cells and cd + b cells [ ] . these findings collectively suggest that the development of severe covid- is likely due to weakening of both t-cell-mediated and b-cell-mediated immunities. moreover, these findings raise concerns about what would happen to immunocompromised individuals if they are infected by the sars-cov- virus. would they be predisposed to developing severe covid- , and thus manifest a higher mortality rate than others? from this perspective, it is especially challenging for physicians to develop treatment strategies for sars-cov- -infected patients on maintenance immunosuppressants, as it may require making a difficult decision of continuing or discontinuing the drugs. continuation of immunosuppressants may very well impair the development of immunity against sars-cov- , whereas their discontinuation may exacerbate non-covid- symptoms and permit development of a pro-inflammatory cytokine storm. to evaluate differences in the clinical characteristics and outcomes of covid- patients with a pre-existing compromised immune system, we conducted a search and review of relevant published reports. we conducted a search in the pubmed® database for reports that describe covid- patients with pre-existing compromised immune systems. our search key words contained covid- in combination with one of the following: immunocompromised, rheumatoid, arthritis, lupus, crohn's, celiac, hiv, aids, lung transplant, heart transplant, liver transplant, kidney transplant, renal transplant, agammaglobulinemia, immune deficiency, and immune suppressant. figure shows our search flow. in total, we identified relevant case reports. we subsequently excluded reports as they were opinion articles or contained insufficient information to indicate compromised immunity or lacked a history of immunosuppressant use. the remaining articles included patients. we reviewed the clinical characteristics of these patients and their outcomes. clinical outcomes included: i) a patient either was treated as an outpatient and recovered, or was treated in the hospital and ultimately discharged (positive outcomes); ii) the patient remained hospitalized at the time of the case's publication; or iii) death ensued prior to publication of the case. the information concerning individual patients in the reports we reviewed is described in tables , , and supplementary table . during the review, we noticed that detailed laboratory values were not reported for most patients. therefore, the tables do not contain such information. there were reports [ ] [ ] [ ] describing a total of covid- patients with pre-existing pad (table ). of these, patients had common variable immunodeficiency (cvid), had x-linked agammaglobulinemia (xla) due to loss-of-function mutations in the bruton tyrosine kinase (btk), and patient had autosomal recessive agammaglobulinemia (ara), the etiology of which is unknown. the ages of these patients ranged from to years. three patients were female and were male. all patients had been on maintenance immunoglobulin therapy to prevent common infections. for their sars-cov- infection, patients received treatment with hydroxychloroquine, and of these patients received concomitant antivirals (most commonly lopinavir plus ritonavir) with or without antibiotics (table ) . of the patients with pre-existing cvid, ( %) patients achieved a positive outcome, patients had their conditions improved, and patient died ( table ). the patient who died was a -year-old female. she was one of the patients who developed severe respiratory symptoms that required icu care and ventilation, and she received treatment with tocilizumab before death ( table ). the covid- patient with pre-existing ara was treated as an outpatient and achieved a positive outcome. of the patients with pre-existing xla, patients achieved a positive outcome after a short hospital stay, and remained hospitalized with an improved condition at the time of the case publication (table ) . overall, there was no clear link between a specific comorbidity (or a group of comorbidities) and the clinical outcomes for the patients (table ) . ten reports [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] described a total of heart transplant recipients with covid- ( were male), liver transplant recipients with covid- ( were male), and lung transplant recipients with covid- (table ). on admission, most patients presented with common symptoms, such as cough, fever, and dyspnea. patient ages ranged from to years, and the average time since the transplant was . years. sixteen ( %) patients had comorbidities, with hypertension and diabetes being the most prevalent. all patients were on maintenance immunosuppressant therapy before hospitalization, with tacrolimus and mmf most often employed, followed in use by glucocorticoids, and cyclosporine ( table ) . of the patients, ( . %) continued to use immunosuppressants during hospitalization, while ( . %) had their immunosuppressants discontinued. none of these reports indicated any signs of organ rejection in patients during hospitalization. most patients received treatment with antivirals and hydroxychloroquine during hospitalization, and some received additional drugs including antibiotics, interferon, and tocilizumab ( table ). the ages of the heart transplant recipients with covid- ranged from to years, and the average time since the transplant procedure was years. one patient was treated as an outpatient with a positive outcome. nine ( %) patients had at least one comorbidity with hypertension and diabetes being the most prevalent, and of them continued to use immunosuppressants during hospitalization (one patient had immunosuppressants discontinued during the first days and then was back on the drugs for days). during hospitalization, patients developed ards. of these , one patient died, another developed additional renal failure, required both icu care and ventilation, and ultimately died, and the third achieved a positive outcome (table ) . overall, ( %) patients achieved a positive outcome, and thus the overall mortality rate was %. of special note was that one patient who was a recipient of both heart and kidney transplants; although he required icu care, he achieved a positive outcome after a -day hospitalization (table ). however, there was no clear connection between the presence of a specific comorbidity (or a group of comorbidities) and the clinical outcomes ( table ). the high rate of positive outcomes suggests that heart transplant recipients with covid- on immunosuppressants are not at an increased risk of mortality unless the patient develops complications such as ards and/or requires icu care and ventilation. the ages of the liver transplant patients ranged from to years, and the average time since transplantation surgery was . years ( table ) . of the patients, ( %) had at least one comorbidity, with diabetes and hypertension being the most prevalent, and ( %) continued to use immunosuppressants during hospitalization. two patients were treated as outpatients ( patient received hydroxychloroquine), and both achieved a positive outcome (table ) . among those hospitalized, patients developed ards and patient developed pneumonia and cholestasis (table ) . overall, ( %) patients (including the two outpatients) achieved a positive outcome, patient remained hospitalized at the time of the case publication (this patient developed ards and required both icu care and ventilation), and patients died. of the patients that died, aged years developed ards, aged years developed ards and required icu care and ventilation, and aged years developed pneumonia, cholestasis, and required icu care and ventilation. thus, the overall mortality rate was . %. it should be noted that old age by itself did not appear to be a risk of mortality because two patients aged years and years did not develop secondary complication and achieved a positive outcome (table ) , and additionally, there was not a clear connection between the presence of a specific comorbidity (or a set of comorbidities) and the clinical outcomes (table ) . therefore, liver transplant recipients sustained on immunosuppressants do not seem to be at an increased risk of mortality from covid- , but rather the subsequent development of complications such as ards and the requirement for icu care and ventilation place the patient at high risk of mortality. as shown in table , there were female lung transplant recipients with covid- . both patients were in their s and developed relatively mild symptoms. both had been on glucocorticoids plus other immunosuppressants prior to infection. one patient was treated as an outpatient with a mild covid- disease course resulting in a positive outcome. she continued to use immunosuppressants and did not receive other drugs. the other patient was hospitalized with a mild covid- course, during which she continued to use immunosuppressants and received treatment with antibiotics and a bronchodilator. this patient achieved a positive clinical outcome as well (table ) . in total, there were reports table to determine if there is a relationship between clinical outcomes as measured against age, gender, comorbidity, secondary complications, and requirement for icu care and/or ventilation. as shown in table , of the patients, ( %) achieved a positive clinical outcome, ( . %) remained hospitalized at the time of their case publication, and ( . %) died. however, the mortality rate among those without comorbidities was . %, while it was % among those with at least one comorbidity (table ) . thus, it indicates a substantial comorbidity-associated increase in mortality risk. the ratio of male patients to female patients was . , and the mortality rate for female patients was . %, and . % for male patients. when the patients are divided by age groups, the clinical outcomes reveal a higher mortality rate for older age groups compared to younger age groups: in the < -year age group, the rate of a positive outcome was %, the rate of remaining in hospital was %, and the death rate was %; in the - year age group, the rate of a positive outcome was . %, the rate of remaining in hospital was . %, and the death rate was %; in the - -year age group, the rate of a positive outcome was . %, the rate of remaining in hospital was . %, and the death rate was . %; in the - -year age group, the rate of a positive outcome was %, the rate of remaining in hospital was . %, and the death rate was . %; in the > -year age group, the rate of a positive outcome was . %, the rate of remaining in hospital was . %, and the death rate was . %. the ultimate outcome rates of patients aged > years remain unknown at present time, however, as high numbers of this population were hospitalized at the time of their case publication. nevertheless, these values indicate that older patents (> years) were at high risk of mortality, with the death rate especially high for patients aged > years. in table , we summarize the information concerning the types of comorbidities in relationship to patient ages and clinical outcomes. consistent with what we see for the heart or liver transplant recipients with covid- (table ) , there was no strict correlation between a specific comorbidity and clinical outcomes of the kidney transplant recipients with covid- across the age groups. in table , we specifically summarize the information concerning the comorbidities, secondary complications, and requirement for icu care and ventilation of those kidney transplant recipients with covid- who died. the data further illustrates that death was not associated with a specific comorbidity (or a specific group of comorbidities), nor a secondary complication or the requirement for icu care and ventilation. in summary, kidney transplant recipients with covid- aged < years had the highest rate of positive outcomes at %. the mortality rates varied among different age groups: % for the < -year age group; . % for the - -year age group, . % for the - -year age group, and . % for the > -year age group. the presence of comorbidities and secondary complications increased the risk of mortality, but death was not associated with a specific comorbidity and/or a secondary complication. it should be noted that the true rates of positive outcome for those patients aged > years remain unclear because of the high number of hospitalized patents at the time of their case publication. therefore, the ultimate outcomes of these patients will alter the mortality rates of patients in the - -year, - -year, - -year, and > -year age groups. although the reported patient number is very small, it is nevertheless interesting to note that out of the patients with pre-existing cvid developed severe covid- that required both icu care and ventilation, and of them died (table ) ; whereas the patients with pre-existing xla and ara had non-life threatening covid- (table ) . therefore, individuals with cvid may be predisposed to developing a severe covid- course if they are infected with sars-cov- . from this perspective, it is important to note that the cvid is associated with abnormally developed b cells with impaired antibody-producing function [ ] , whereas xla and ara are associated with an extremely low number of, or no, b cells in the circulation due to blockage of the proliferation and development of pre-b cells in the bone marrow [ , ] . as elevation of serum levels of proinflammatory cytokines are believed to drive the development of covid- complications, such as ards and respiratory failure [ ] [ ] [ ] [ ] , it suggests that the abnormally developed b cells in the sars-cov- -infected cvid patients may play a pathogenic inflammatory role in the course of the development of the severe respiratory conditions. however, future investigation is required to determine if and what activities of the abnormally developed b cells contribute to the development of severe covid- respiratory conditions. the recovery of sars-cov- -infected patients with xla and ara as well as the sars-cov- -infected patients with cvid indicates the presence of a cellular immunity against sars-cov- . this agrees with findings by others. for example, wu et al have shown that a small but significant number of patients recovered from covid- lack sars-cov- -neutralizing antibodies [ ] , and that liu et al have found similar mortality risks between those covid- patients who tested positive for igg antibodies against sars-cov- and those who lacked antibodies against sars-cov- [ ] . therefore, those recovered patients with xla, ara, or cvid must have an activated t-cell immunity against sars-cov- virus. in this context, it is relevant to note the presence of sars-cov- -reactive cd + t cells in recovered covid- patients [ , ] , and that the development of sars-cov- reactive cd +/hla-dr+/cd + t cells strongly correlates with a positive clinical outcome [ ] . future studies of the t cell immunity of the patients with xla or ara or cvid who recovered from covid- will be needed to identify sars-cov- -reactive t cells that play a critical role in the recovery of these patients from covid- . recently, myets et al., published the clinical outcomes of sars-cov- -infected individuals with inborn errors of immunity (iei), including individuals with primary antibody deficiency, the causes of which include xla, ara, and cvid, and some patients with t cell deficiency due to loss-of-function mutations in zap , pgm , stat , and arpc b [ ] . in the study, patients ( . %) were asymptomatic, patients ( . %) developed mild symptoms and were treated as outpatients, and patients ( . %) required hospitalization. of the hospitalized patients, ( . %) developed respiratory insufficiency and ( %) required icu care and invasive ventilation. the overall mortality rate is . % ( / ). the authors concluded that "the risk factors predisposing to severe disease and mortality amongst iei patients were comparable to the general population." and they suggested that "certain components of adaptive immunity do not appear to be essential for controlling sars-cov infection. rather, these adaptive immune deficiencies may even contribute to a milder course by reducing the immune-mediated sequelae." strikingly, of the deceased patients had primary antibody deficiency due to cvid [ ] , suggesting that a pathogenic inflammatory role for cvid in the development of the severe respiratory conditions. however, it should be noted that these cvid patients were generally older than the rest of the cohort and had pre-existing health conditions, suggesting that they were likely predisposed to severe covid- [ ] . our analysis indicates an overall high mortality rate among sars-cov- -infected organ transplant recipients who were continuously on immunosuppressants. however, given the small number of covid- patients with a heart, a liver, or a lung transplant, the statistical significance of the mortality rates for these patients cannot be assessed here. for the kidney transplant recipients with covid- who remained on immunosuppressants, the mortality rate among those < years was % (table ) , and this outcome occurred despite some patients having at least one comorbidity, such as hypertension and diabetes, that have a known association with high mortality risk for covid- patients in general. however, death occurred among the sars-cov- -infected older (> years) kidney transplant recipients who were on immunosuppressants (table ) , and it contributed to an overall mortality rate of . % for the sars-cov- -infected kidney transplant recipients on immunosuppressants (table ) . however, the mortality rate among those without comorbidities was . %, while it was % among those with at least one comorbidity (table ) , indicating a substantial comorbidity-associated increase in mortality risk. interestingly, our review does not indicate a specific comorbidity (or a group of comorbidities) that is responsible for the increased mortality risk (tables and ). therefore, the comorbidity-associated mortality risk is most likely dependent on additional factors that vary from patient to patient. nevertheless, a study by richardson et al., [ ] and another study by cummings et al., [ ] of hospitalized covid- patients in the new york city area found an overall mortality rate of % and %, respectively, and moreover these mortality rates are mostly contributed by the death of old patients with comorbidities. since the overall . % mortality rate of the sars-cov- -infected kidney transplant recipient on immunosuppressants is attributed to death of older (> years) patients with comorbidities and/or secondary complications (table ) , the . % mortality rate does not seem to be abnormally high because it is in line with the rates reported by others for different covid- patients populations. nevertheless, the positive outcome of patients aged < years as well as some older patients suggests that having an immunosuppressant-compromised immune system alone is not a significant risk factor of mortality, but rather the presence or comorbidities, development of secondary complications are more consequential mortality risk factors. one unfortunate finding in our review is the exceptionally high mortality rate ( . %) for sars-cov- -infected kidney transplant recipients aged > years (table ) . why this is the case is not clear. however, it should be noted that within this group of patients, the clinical outcomes could not be predicted even after accounting for the presence of comorbidities and secondary complications. for example, a -year-old kidney transplant recipient with covid- , who had the comorbidities of hypertension and iga nephropathy, who continued to use rapamycin and prednisone, and who received treatment with hydroxychloroquine -still achieved a positive clinical outcome after a mere -day hospitalization [ ] . in contrast, a -year-old kidney transplant recipient with covid- , who had a single comorbidity (hypertension), and who continued to use prednisone, and received treatment with hydroxychloroquine, lopinavir, and ritonavir, died after an unspecified period of hospitalization [ ] . therefore, in the absence of additional information, it is possible to hypothesize that the immune system of older patients, as well as some of the younger patients who died, was overly compromised by the immunosuppressants and could thus no longer effectively defend against sars-cov- infection. it would be interesting to know from blood samples of these deceased patients (if the samples are available), whether there is any link between immune system marker indices and likelihood of death. although, the renal function of a significant number of the sars-cov- -infected recipients with kidney transplantation and on immunosuppressants was not reported, some report did include the baseline renal function as measured by egfr (estimated glomerular filtration rate) or serum levels of creatinine at the time of admission. although renal dysfunction as reflected by decreased egfr or increased serum levels of creatinine was not considered in the published reports as a co-morbidity, it is important to recognize that renal dysfunction of kidney transplant recipients with covid- ought to be recognized as such. nevertheless, numerous reports that we reviewed showed that patients with a decreased egfr plus other comorbidities [ , , , , , , , ] or increased serum levels of creatinine plus other comorbidities [ , , , , , , ] still achieved a positive outcome. interestingly, sharma et al recently completed a retrospective study in which they compared the clinical outcomes of covid- between solid organ transplant recipients and matched nontransplant patients in their medical center, and they found the need for renal replacement therapy in the organ recipients did not affect the incidence of severe covid- and the rate of short-term death [ ] . the findings by sharma et al., further suggest that renal dysfunction in sars-cov- -infected organ recipients does not correlate with clinical outcomes. in a report [ ] of the clinical outcomes of consecutive adult kidney-transplant recipients who were infected by sars-cov- , akalin and colleagues found an overall mortality rate of % at weeks, but a mortality rate of % among patients who were intubated, indicating a respiratory complication-associated significant increase in mortality rate [ ] . however, this report neither provide the outcome of individual patient nor specific information on individual patient. in another report [ ] , katz-greenberg et al., described the clinical outcomes of kidney-transplant recipients (ages to years) who were infected by sars-cov- , and showed that only patients ( males aged and and female aged ) died, suggesting a % mortality that is related to advancing age [ ] , which agrees with our review of the published case reports. nevertheless, it seems intuitive for organ transplant recipients to discontinue immunosuppressants after they become infected with sars-cov- virus -presumably to allow the body to increase the immune activities to fight the infection. our review of the positive clinical outcomes of a significant number of organ transplant recipients with covid- suggests that a weaker immune system may not necessarily be a risk factor of mortality. this point is supported by the recent findings by long et al., showing that in comparison to age-and gender-matched symptomatic covid- patients of similar viral loading, the sars-cov- -infected asymptomatic individuals have a significantly weaker immune response as evidenced by lower igg levels, lower levels of sars-cov- neutralization antibodies, and reduced levels of pro-inflammatory cytokines in serum [ ] . the findings by long et al., suggest that a weaker immune response does not necessarily result in development of covid- symptoms in sars-cov- -infected persons, but rather it seems necessary to prevent over activation of immune cells leading to the development of a pro-inflammatory cytokine storm, which is known to cause severe lung damage and promote life-threatening respiratory failure in covid- patients. therefore, it is possible that one reason why sars-cov- -infected young kidney transplant recipients, and some sars-cov- -infected heart and liver transplant recipients, achieved positive outcomes is that their immune systems happened to be compromised by the immunosuppressants to an extent that a proinflammatory storm could not manifest itself, but the residual immunity was sufficient to fight the virus. perhaps again, a retrospective analysis of blood samples of these organ recipients with covid- would shed light on the relationship between an immunosuppressant-compromised immune system and clinical outcomes. alternatively, covid- animal models [ ] [ ] [ ] may be used to assess how the use of immunosuppressants affects the clinical outcomes of covid- . our review of published reports has enhanced our understanding of the outcomes of covid- patients with pre-existing, compromised immune systems. however, it has raised numerous issues that warrant future investigation. specifically, how it is that a compromised immune system, be it a compromised humoral immunity or immunosuppressant-compromised immune system, does not seem to prevent a positive outcome. the findings do suggest that for sars-cov- -infected organ transplant recipients on maintenance immunosuppressants, the decision of whether they should continue to use immunosuppressants should be individualized. as sars-cov- -infected young kidney transplant recipients who continued to take immunosuppressants during hospitalization fared relatively well (table ) -it would be reasonable to suggest that those currently hospitalized may continue to use immunosuppressants at the regular doses or reduced dose. (of course, these are broad generalizations subject to review by physicians overseeing individual patients.) for older patients, it should be determined on a case-by-case basis, perhaps through a gradual dose-reducing process, decreasing the immunosuppressants to a range that does not worsen covid- symptoms and also does not allow high immune reactivity against the transplanted organs. of course, the laboratory values for all patients should be closely monitored for better understanding of the relationship between a compromised immune system and the outcomes of sars-cov- infections. though the information outlined here provides provisional evidence of the effect of immunosuppression on covid- clinical outcomes, more information is needed on this subject moving forward to better understand of the mechanisms of sars-cov- infection and how covid- can best be treated in humans. supplementary table. http://www.medsci.org/v p s .pdf genomic characterisation and epidemiology of novel 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and transmissibility infection and rapid transmission of sars-cov- in ferrets we wish to thank michael h. oppenheim for his excellent editorial assistance. the authors have declared that no competing interest exists. key: cord- - vd mdu authors: nan title: abstracts from the th european society for animal cell technology meeting: cell technologies for innovative therapies: lausanne, switzerland. - may date: - - journal: bmc proc doi: . /s - - -x sha: doc_id: cord_uid: vd mdu nan . a schematic representation of crispr based synthetic transcription factor technology. b mrna expression levels of protein transport related genes (napg, rab a and arpc b). background an increasing number of biologics are entering the development pipelines of pharmaceutical companies [ ] . today, the preferred production host for therapeutic proteins is the cho cell line. however one of the major hurdles, especially for the production of non-antibody glycoproteins, is host cell-related proteolytic degradation which can drastically impact developability and timelines of pipeline projects. material and methods spike-in: cho cells were cultivated in a chemically defined culture medium at . °c/ % co in shake-flasks. when the cells reached their maximum viable density, they were removed by centrifugation and the conditioned medium was collected. a model mab was spiked into the conditioned medium and incubated at °c ± protease inhibitors. the amount of proteolytic degradation was analysed by western blot and lc-ms. transcriptomics: total rna was extracted after days of cell cultivation. rna sequencing libraries were constructed and processed on the hiseq platform from illumina. generation of matriptase knockout: cho-k cells were transfected with mrna encoding "transcription activator-like effector nucleases" or "zinc finger nucleases" targeting matriptase exon . the transfected cells were subsequently sorted into single cells and analysed for frameshift mutations in both alleles via sanger sequencing. cell cultivation: fed batch cultivation was performed in -ml miniaturized bioreactors (ambr ). approximately proteases are known in rodents. to reduce the number of candidate proteases we showed first that a model mab (prone to proteolytic degradation) incubated in conditioned medium of cho-k cells resulted in clipping of the mab, demonstrating the involvement of secreted/shedded proteases (fig. a) . broad spectrum inhibitors of the different protease classes revealed that only serine protease inhibitors prevented clipping. serine protease inhibitors of higher specificity highlighted the group of "s a trypsin-like proteases" (fig. a) . comparison of the proteolytic degradation profile of several therapeutic proteins between cho-k with another cho cell line (cho-a) revealed less degradation in cho-a. therefore expression of the involved protease(s) is likely lower in cho-a. gene expression profile analysis of both cell lines showed five secreted/shedded "s a trypsin-like serine proteases" more than . fold lower expressed in cho-cho-a versus cho-k (fig. b) . surprisingly, sirna knockdown experiments of these five candidates identified "matriptase" as the major protease involved in degradation of recombinant proteins expressed in cho-k cells ( fig. c upper panel) . next, we generated a cho-k matriptase knockout (ko) cell line. no proteolytic degradation product was detected when the model mab was spiked into conditioned medium of the ko cell line (fig. c lower panel) . also, stable expression of the model mab in the ko cell line resulted in no/significantly less clipping (fig. e) . the protein titer and the cell growth behaviour of the matriptase ko cells were similar to the corresponding wildtype (wt) cells (fig. d) as shown by comparative cultivation in ambr system. conclusions one major challenge for the production of recombinant proteins is cho host cell mediated proteolytic degradation which can negatively impact or even result in termination of projects [ ; ] . using a variety of techniques such as applying protease inhibitors, transcriptomics and sirna mediated knock-down we were able to identify "matriptase" as the major protease involved in degradation of recombinant proteins expressed in cho-k cells. subsequently we generated a matriptase deficient cho cell line. protein candidates of diverse formats, severely degraded in wt cho-k cell line, were not or significantly less cleaved in the matriptase ko cell line. furthermore cell growth, viability and productivity levels were comparable between the wt and the matriptase ko cell line. in summary, we have generated a superior platform-compatible cho production host cell line with the same favourable productivity properties as the parental host cell line [ ; ] , allowing expression of complex glycoproteins prone to clipping. background cho cell lines are common hosts for the production of biopharmaceutical proteins. so far, considerable progress has been made increasing productivity of cell culture to meet the rapidly growing demand for antibody biopharmaceuticals through increased cell densities and longer culture times. the downside is the increase of the process related impurities, bringing new challenges for process and harvest development. among the process related impurities such as host cell proteins (hcps) or dna the potential impact of lipids production and release during cell culture is still poorly understood due to the complex nature and diversity of this class of molecules. thanks to recent advances in analytical tools especially mass spectrometry, the advent of lipidomics offers now the feasibility to study several thousands of lipid species thus unraveling the possibility to understand and potentially control the interactions between high performance bioreactor processes, harvest conditions and purification. in order to analyze and quantify lipids, we developed a three steps method. in a first step, lipids were extracted with methyl tert-butyl ether (mtbe) according to matyash method [ ] . lipids were then separated by liquid chromatography using either hilic of reverse phase column prior to detection and quantification by mass spectrometry. all lipid classes were detected by esi-ms/ms excepted cholesterol (apci-ms/ms). finally we applied this method to analyze the lipid content of different cell lines each expressing a different recombinant protein, during a days fed batch process. lipid from cho cells were successfully extracted with a yield between % and % depending on the different lipid classes. stable isotope labeled lipids were used as internal standard in order to have comparable results between batches. the obtained results (fig. ) show that for a given cell line, lipid distribution is changing over the process. moreover, this distribution may vary significantly depending on the cell line: cl- and in a lower extend, cl- , show an accumulation of triglycerides from day to the end of the process, while cl- doesn't seems to follow this trend. conclusion interestingly, in some cell lines/experimental conditions, we highlighted an overproduction of triglycerides and cholesterol leading to the accumulation of lipid droplets known as energy storage sink. at the metabolic level, these findings suggest a relative overflow of the carbon metabolism. from a process development perspective these findings can be considered on the one hand as a resource waste since the stored energy is not used for protein/biomass biosynthesis and on the second hand as the root cause of additional process challenges especially during the harvest and the first capture steps given the hydrophobic nature of these molecules. implementation of lipidomics analysis enables us to highlight a new type of process variability and to anticipate potential problems for the downstream steps. the application of this methodology on our platform has helped us to design tailor made solutions (pretreatment selection, filter selection,…) at the clarification step which are now implemented in our harvest development platform approach. . matriptase knock-out in cho cells prevents clipping of recombinant proteins. a serine protease inhibitors protect model mab from proteolytic degradation in cho-k cell derived conditioned medium. the model mab was incubated in conditioned medium for h or h at °c, subsequently samples were analyzed by western blot. broad spectrum serine protease inhibitors (aprotinin, leupetin) were added during incubation. aprotinin and leupetin are inhibiting proteolytic degradation. the intact mab (upper band) and the clipped mab (lower band) are indicated by arrows. b gene expression profiling of cho-k versus cho-a by ngs. shown is the gene expression profile of "secreted/shedded members of the s a trypsin-like serine protease family" for cho-k and cho-a cell lines using next generation sequencing. the gene expression analysis highlights that five proteases were more than . fold higher expressed in cho-k cells (labelled with a red asterix). the y-axis shows the transcript abundance as rpkm (reads per kilobase of exon model per million mapped reads). c sirna knock-down identifies matriptase as major clipping protease and cho matriptase ko clone shows no detectable clipping activity. upper figure: sirnas directed against the five protease genes and scrambled (scr.) sirna were transfected and conditioned medium was collected three days after transfection. the model mab was incubated in fresh medium as control (first lane) and conditioned medium from the sirna transfected cells. samples were analyzed by western blot. only sirna targeting matriptase (st ) showed reduced proteolytic degradation. the intact mab (upper band) and the clipped mab (lower band) are indicated by arrows. lower figure: the model mab was incubated for h in conditioned medium collected from wt cho-k as well as the matriptase knockout clone. samples were analyzed by western blot. the intact mab (upper band) and the clipped mab (lower band) are indicated by arrows. no proteolytic degradation could be detected in the samples originating from the matriptase ko clone. d cell growth, viability and productivity in ambr (fed batch with temperature shift). cell growth, viability and volumetric productivity profiles of wt cho-k (red circles, n= ) and matriptase ko clone (blue squares, n= ) cultivated in -ml ambr. no significant differences were seen between wt and matriptase ko clone regarding cell growth and viability. comparable or slightly higher productivity was detected for the matriptase ko clone compared to the wt. e significant reduced proteolytic clipping applying matriptase ko clone. the model mab was stable expressed in cho-k (wt) as well as the cho-k matriptase ko clone. samples were analyzed by western blot. the intact mab (upper band) and the clipped mab (lower band) are indicated by arrows. significant reduced proteolytic degradation could be detected in the samples originating from the matriptase ko clone ( samples each is shown for wt and ko cells) the glycosylation of therapeutic proteins is a critical quality attribute (cqa) and needs to be analyzed during cell line and bioprocess development. the current methods for analyzing glycosylation are mainly based on the enzymatic release of glycans. they are tedious and offer only limited throughput, which makes them unsuitable for cell line development work. in this study we evaluated a novel paia assay for measuring intact glycoproteins with capture beads and fluorescence labeled plant lectins to analyze glycans in a high throughput -well plate format. material and methods analytes: erbitux © , mabthera © , arzerra © and avastin © . two glycoengineered variants of one igg were kindly provided by merck (vevey, switzerland). all analytes were spiked into cho-k cell culture supernatant or buffer, diluted : with a denaturation solution and incubated at or °c for minutes to expose the fc glycans. erbitux samples were analyzed under denaturing conditions to detect fab-and fc-glycosylation and in native conditions for fab glycosylation only. μl of pretreated sample was added to each well of the special -well paiaplate, containing labeled lectin and capture beads. the microplate was incubated for minutes at rpm on an orbital shaker at room temperature and spun down at xg. the read-out was done on a fluorescence microscope (synentec, elmshorn, germany) in less than five minutes. results figure a : lectin binding profiles of different iggs. the analysis of different igg results in lectin binding profiles which show the different degrees in glycosylation. high abundance of sugars leads to high binding rates of the lectin for the respective sugar. avastin has a very low degree of galactosylation and high mannose species compared to mabthera and arzerra (fig. a) . only arzerra is carrying glycans with - linked sialic acids. these findings are in line with results from literature [ ] . figure b : distinction between fc and fab glycosylation in erbitux. without denaturation only the fab glycans are detectable in erbitux. denaturation leads to additional exposure of the fc glycans and thus higher lectin binding rates compared to native erbitux. gna and npl only bind to denatured erbitux indicating that the high mannose glycans are only present on the fc part. the equal sna binding rates for both conditions confirm that the - linked sialic acids are almost exclusively found on the fab part. this is in agreement with published data [ ] . figure c : lectin binding rates correlate with the levels of galactosylation and fucosylation. increasing degrees of glycosylation in the mixtures of the glycan variants from merck lead to higher lectin binding rates for all galactose and fucose markers proving that quantitative analysis can be performed with these assays. the cona lectin which binds to the common core mannose glycan motive remains at the same level, suggesting that the fc glycans were similarly exposed in all samples. the results demonstrate that paia assays are capable of quickly detecting differences in glycan patterns of different antibodies. in addition it was shown that glycan variants of the same igg can be analyzed quantitatively. and finally we could confirm the differences in fab and fc glycosylation in erbitux. we believe that bead-based assays with lectins have a great potential for monitoring product quality early in the development process. background gene-and cell therapy-based medicines are experiencing resurgence due to the introduction of "next generation" transfer viral vectors, which have demonstrated improved safety and efficacy. adeno associated viruses (aav) and lentiviruses are very commonly used in therapeutics and often produced by transient gene expression, using pei-mediated transient transfection in hek- or hek- t cells [ ] . the critical raw materials needed for cgmp vector production must be sourced from approved suppliers and should have gone through a rigorous testing program to reduce the risk of introducing adventitious agents into the production process. correspondingly, the pei transfection reagent must also be sourced from a qualified supplier, and have gone through rigorous testing to ensure reliable transfection efficiencies, and hence reproducible virus production yields. here, we present peipro® and peipro®-hq, the unique pei-based transfection reagents suitable for use in process development and in cgmp biomanufacturing, respectively. unlike commercially available peis, peipro® benefits from extensive research and development in polymer chemistry and formulation for mammalian cell transfection. we further demonstrate that peipro® and peipro®-hq are the reagents of choice for virus production runs in most cell culture systems, hence facilitating the transition from initial optimization during process development up to large-scale therapeutic viral vector production in adherent or suspension cells. manufacturing process of peipro® and peipro®-hq reagents. peipro® and peipro®-hq are fully synthetic reagents, free of any animal-origin components. in comparison to peipro®, a more extensive number of quality controls are performed on peipro®-hq to enable its use as a qualified raw material in gmp processes for the manufacturing of clinical batches of therapeutic products. lentivirus and aav production. irrespective of the cell culture vessel type, transfection using peipro® was performed following our recommandations. as an example, hek- t (lentivirus) and hek- (aav) cells were thawed directly into each medium and passaged every to days before going into a liter benchtop bioreactor. cells were resuspended and cultured for days before transfection with peipro®. hek- t cells were transfected with a third-generation system (four plasmids) for lentivirus production. hek- cells were co-transfected with three plasmids for aav production. lentiviral and aav titers were measured and hours post-transfection (data kindly provided by généthon). peipro® is the reagent of choice for virus production runs in most adherent and suspension cell culture systems from process development up to large scale clinical-grade virus production. irrespective of the cell culture-based system and production scale, peipro® and peipro®-hq have led to efficient viral vector yields in standard laboratory cell systems, such as in flasks, cell factories, and roller bottles, as well as in multilayers flasks or fixed-bed culture systems that take into account time and space concerns for the scaling-up process (table ) . for example, high viral vector yields superior to ig/ml and - vg/ml were obtained respectively for lentiviruses and aavs in suspension hek- t and hek- cells cultured in one of the commercially available synthetic cell culture medium balancd® hek (irvine scientific®). conclusion peipro® and its higher quality grade peipro®-hq are the unique pei suitable for efficient and reproducible production of therapeutic viral vectors. efficient viral vector production yields can be achieved in most cell culture systems, irrespective of the production scale. with appropriate and advanced quality controls, the highest quality grade peipro®-hq is commercially available to accompany academics and biopharmaceutical companies in terms of qualified raw material for their gmp-grade viral vector production needs. b distinction between fc and fab glycosylation in erbitux. erbitux was diluted to a concentration of μg/ml in tris buffer and measured in native and denatured conditions to distinguish fab glycosylation (native erbitux) from fab and fc glycosylation (denatured erbitux). it could be confirmed that sialic acids are almost exclusively present on the fab part of erbitux and that the high mannose glycans are only found in the fc part. c lectin binding rates correlate with the levels of galactosylation and fucosylation. two glycan variants samples of the same igg from merck were mixed in different ratios to yield glycosylation rates of to % in terminal β-galactose and to % in core-fucose, based on data from -ab uplc analysis. the mixtures all contained . μg igg per well. the measured lectin binding rates for all galactose and fucose markers correlate very well with the respective degree of glycosylation in the mixtures. all measurements were performed in triplicates (irvine scientific®) . hek- t (lentivirus) and hek- (aav) cells were thawed directly into each medium and passaged every to days before going into a liter benchtop bioreactor. cells were seeded and cultured for days before being transfected by peipro® (polyplus). for transfection, four plasmids were used for lentivirus and three plasmids were used for aav. lentiviral and aav titer were measured and hours post transfection (data kindly provided by généthon) table (abstract p- ). peipro®, the reagent of choice for virus production runs in most cell culture systems in both adherent and suspension cells. irrespective of the cell culture-based system and production scale, peipro® and peipro®-hq have led to efficient viral vector yields superior to ig/ml and vg/ml, respectively for lentiviruses and aavs background continuous perfusion process is making a comeback as a competing upstream manufacturing technology for the production of biopharmaceuticals compared to the standard fed batch processes. this is primarily because of cost advantages such as reduced capital cost and increased product yield. the change in status of perfusion process from older perfusion to the new-age perfusion is due to availability of better cell retention devices leading to more efficient processes, improved cell lines, cell culture medium capable of supporting high cell densities and better bioreactor control strategies. in this work, we present the advantages, limitations and challenges of fed batch and perfusion type of processes through case studies. table results the perfusion run yielded -fold higher titer compared to fed batch run (fig. a) . considering the number of runs that could be executed in a manufacturing facility within the same calendar days, about fold increase in product output can be achieved with the perfusion process (fig. a) . this difference is attributed to higher ivcc, higher pcd and longevity of cells because of decreased level of toxic metabolite concentrations such as lactate and ammonia. case : understanding product retention in perfusion process the new-age perfusion processes utilize hollow fiber filters. this has been observed to cause retention of product within the bioreactor especially towards the end of the production run. two types of experiments were conducted to study the factors contributing to product retention: -spiking studies: -role of product titer: product was spiked into chemically defined media -role of different cell viability: different broths with varying viability spiked with same product titer -evaluation of different hollow fiber membrane (m , m and m ) on product retention. from spiking studies, it was evident that cell debris and poor quality cell broth (lower viability) were the major factors contributing to product retention (fig. c) . from the different membranes experiments, it was identified that at pilot scale, m showed much higher retention from the first perfusion cycle itself and it increased to more than % towards the end of the batch. however, with m membrane, product retention started only late (after % of batch duration) and it remained low (~ - %). on the contrary, this difference was not observed at l scale due to the usage of membranes with larger filter area ( - folds higher compared to pilot scale). when the filter area per unit volume of perfusate was decreased by half (m _batch ) for the pilot scale, even m showed retention profile similar to m (fig. d ). we presented data to show that perfusion process has -fold increase in product yield on a per-batch basis and a -fold increase when facility throughput is considered. product retention is a technical challenge that requires optimization (perfusion rates and filter membrane types). we believe it is imperative that labs that develop processes for biologics can now consider both perfusion and fedbatch based processes as both these technologies can now closely compete with each other. the choice of the process format going forward should now solely be dependent on the requirement for the biologic rather than the earlier perception that fed-batch is the preferred choice because of its simplicity. background chinese hamster ovary (cho) cell culture has been widely used for production of monoclonal antibodies in the pharmaceutical industry. previous studies have shown that the cell specific productivity in cho cells can be increased by glucose limitation [ ] . introducing a productivity enhancing effect it is possible that this also affects the quality of the product such as glycosylation or other posttranslational modifications. in this work, we are focusing on the impact of glucose limitation and increased productivity on the product quality of a monoclonal antibody produced in a fed-batch cultivation of cho cells. materials and methods cho cells were cultivated both under limiting and non-limiting nutrient conditions in fed-batch. for fed-batch cultivation the reduced range for glucose concentration was chosen between . and . g/ l. reference cultivation was performed between . and . g/l. both cultures were fed with similar volumes of a complex nutrient supplement. all cultivations were performed in chemically-defined, animalcomponent free cho growth media (xell ag). viable cell density and viability were determined using the automated cell counting system cedex (roche diagnostics), glucose and lactate concentrations were detected via ysi (ysi life sciences). amino acid were quantified using hplc-fld, vitamins were quantified using reversed phase chromatography coupled to a triple quadrupol mass spectrometer (varian , selected reaction monitoring). amounts of igg were quantified via protein a hplc, mab purified from another cho cell clone was used as a standard. the analysis of product quality was performed by intact mass analysis using reversed phase chromatography coupled to a microotof-q ii mass spectrometer (bruker daltonik). the cho cell culture cultivated under low nutrient conditions reached a % higher viable cell density than the reference culture (fig. a) . the product titer was even increased by % (fig. b) . the spent media analysis shows that some amino acids and vitamins were present at presumably limiting concentrations after day / , mostly in the low nutrient level culture (down to to μm for tyr, gln, arg, and asn, below μm for pyridoxine, data not shown). the product quality showed significant changes for the changed feeding strategy ( fig. c and d) . as expected, the glycation level decreased from % to % compared to the reference culture. the truncation level of c-terminal lysine at the heavy chain of the mab increased from % to %. the glycosylation was also significantly influenced by the low nutrient level (fig. e) : the nonfucosylated variants increased from % to % (fig. f) , the degree of galactosylation increased from % to % (fig. g) . cultivation under low nutrient level led to % higher viable cell density and a product titer increased by % when compared to reference culture grown under non-limiting nutrient conditions. the analysis of product quality reveals % less glycation of light chain for cho cells grown under low nutrient conditions ( . % vs . % in reference culture). the truncation of c-terminal lysine decreased by % (from % to %), the degree of galactosylation increased by % (from % to %, also observed by takuma et al. [ ] ) and non-fucosylated glycans increased by % (from . % to . %) under low nutrient conditions. the product quality analysis by intact mass proved to be highly robust (average cv for four replicates = %). in summary, cultivation with alternative feed led to higher igg product titer and better product quality (glycation unwanted, higher amount of non-fucosylated glycans leads to higher antibody-dependent cellmediated cytotoxicity (adcc), higher amount of galactosylation to higher complement-dependent cytotoxicity (cdc) and adcc [ , ] ). background we developed an automated, multiwell plate (mwp) based screening system for suspension cell cultures (fig. a) which is now routinely used in cell culture process development. it is characterized by a fully automated workflow with integrated analytical instrumentation. it uses shaken - well plates as bioreactors which can be run in batch and fed-batch mode with a capacity of up to reactors in parallel [ ] [ ] [ ] . a wide ranging analytical portfolio to monitor cell culture processes and also a cooperation with internal high throughput (ht) analytic groups to characterize product quality are available. in addition the use and the benefits of spectroscopic methods for cell culture automation were shown in the past [ , ] . automated cell culture systems enable broader screening within a shorter time frame for many applications in upstream process development. the higher degree of parallelization and automation helps to screen for most promising parameters in a shorter time. the use of broad doe screening design allows in addition the identification of parameters that support high titers while keeping high product quality (multiple factors at the same time). the illustration (fig. b) shows an example how this combination can speed up process development steps. main applications of the cell culture automation are for example the identification of product quality levers and media or feed optimization. the application of the cell culture automation is shown for two examples. the goal in the first application was to identify levers to reduce trisulfides. by a screening of conditions in parallel (in -fold replication, wells in sum) the reduction of trisulfides by . % (normalized to start level) was possible. in addition the levers for trisulfide reduction were identified. the best and start conditions were verified in bioreactor scale (fig. c) . the goal in the second application was to increase product concentration without an impact on product quality. by a screening of conditions in parallel (in -fold replication, wells in sum) the increase of titer from . g/l to . g/l (> factor ) was possible by media platform change and media optimization. an impact on product quality could not been shown. the best conditions were also verified in bioreactor scale (fig. d) . the benefits of using cell culture automation in late stage process development were shown based on two examples of current applications. for this purpose the experimental results of the development work of two late state projects using the in-house developed automated cell culture system were shown. the first example shows the capability of the automated cell culture system by reducing trisulfides significantly in just one experiment. for the other project the final product concentration could be increased by factor . by a media screening and changing to the in-house media platform. these two examples show the potential of cell culture automation as a routine tool in process development. the cell line development process has become faster and is simultaneously generating more clone-and product-related analytical data. in order to select the best producer cell line, extremely heterogeneous data types need to be systematically compared. the timely availability of all data needed to decide which cell line to pursue has become a bottleneck in the cell line development workflow. to ensure sound decision making, new integrated workflow support and data analysis methods are needed. we have developed a new end-to-end platform for bioprocess development, which includes a cell line development workflow system supporting seeding, selection, passaging, analyzing, cryo-conservation, and processing in (micro-) bioreactors. this platform, genedata bioprocess™, enables partially or fully automated cell line selection and assessment processes, and it increases process efficiency and quality. the system tracks the full history of all clones -from initial transfection all the way to their evaluation in bioreactor runs -and combines this information with analytics data on molecules, clones, and product quality. it can directly integrate with all instruments, such as pipetting robots, bioreactors, and bioanalyzers. the system is designed for a wide range of . a schematic illustration of the automated cell culture system. only the core system is shown with a robotic plate handler as key device connecting cultivation, processing and analytical parts. b illustration of an example how cell culture automation can speed up process development steps. c application in the identification of product quality levers. d application in titer optimization biologic molecules, including antibodies (iggs, novel formats) and other therapeutic proteins (e.g., fusion proteins). highlighted use cases describe the identification of top producer cell lines, decision making support, bioreactor data management, and full clone history report documentation (fig. ). genedata bioprocess, which was developed in collaboration with top pharmaceutical companies, can flexibly support various (non-linear) workflows and structure the collected information in a way that fosters collaboration across an organization. while increasing throughput is crucial to ensure the timely availability of optimal producer cell lines, high-throughput is only possible when automated processes in the laboratory and the resulting data collection and aggregation can be streamlined. genedata bioprocess helps to establish more productive processes by offering support and integration for automation stations and measurement devices. thanks to the comprehensive workflow support and the possibility to integrate results from cell line stability experiments, product quality assessment, and bioreactor suitability tests, genedata bioprocess provides a unique way to evaluate cell lines. comprehensive analysis of all data collected in the process helps to ensure the highest possible quality and minimize the time and resources needed for data analysis and management. integration of bioreactor data analysis and visualization with other parameters measured in cell line development, streamlines clone evaluation in micro-bioreactors and supports highthroughput operations. genedata bioprocess comprehensively tracks the full clone history from the origin of the host cell line to the generation of the validated monoclonal producer cell line. for promising clones, the clone history report can be generated with one click. besides supporting cell line development, genedata bioprocess is a comprehensive platform capable of tracking the complete bioprocess development process. in , . million people suffered from cancer [ ] making it to a major concern of our society. since common cancer treatment is limited and not effective for late stage carcinoma, alternative methods are needed to reduce the high mortality rate of cancer patients. one alternative approach is the application of the oncolytic measles virus (omv), because omv has a natural affinity against cancer cells. the major drawbacks of omv is to produce the extremely high amount of at least tcid ( % tissue culture infective dose) per dose [ ] which is needed. to solve this problem, a high titer process must be established including an efficient downstream processing (dsp). we developed an appropriate upstream processing and are able to produce - tcid ml - in a bioreactor with . l working volume [ ] . now, we focus on the dsp part. the following study tested the application of charged depth filters for the omv clarification. in contrast to common dsp schemes, a depletion of virus particles or a loss of infectivity is not desired. the aim is a reduction of protein content and dna with minimal loss of infective omv. further, we investigated the influence of the cell culture medium on the depth filtration process. to explore the influence of the surrounding cell culture medium on the depth filtration performance, omv was either produced in serum-free medium (vp-sfm) or serum-containing medium (dmem + % fcs). the production was done in a str with a working volume of . l as described in [ ] . cells and carriers were separated with an opticap xl -module (polygard-cr; μm; merck). for the depth filtration millistak+ ce filters (merck) were used. the filter material was autoclaved and rinsed with ml of mm tris-hcl (ph= . ). the virus suspension was filtered with a load of l m - using a peristaltic pump (ism c; ismatec) applying a flux of l m - h - (fig. ). samples were collected at the beginning and end of a filtration run. the omv titer (tcid ml - ) of the samples was determined according to kärber and reed [ , ] . protein content was measured with the pierce bca protein assay kit (thermofisher scientific) according to the manufacturer's instructions. dna was measured by a microtiter assay using quant-it picogreen dsdna reagent (thermofisher scientific) according to the manufacturer's instructions. we found that positively charged depth filters were suitable to clarify omv suspensions. the cell culture medium, in which the omv was produced, influenced the outcome of the depth filtration. a log reduction value (lrv) of . was determined for omv present in serum-containing medium (scm), whereas the titer of omv in serumfree medium (sfm) was reduced . log levels. this indicates that without serum in the surrounding liquid, omv will adsorb to the filter material. however, we must evaluate if the missing serum or other components present in sfm are responsible for this effect. total protein was not relevantly reduced by the clarification using charged depth filters. for omv present in scm, the residual protein content was slightly less compared to omv present in sfm (table ). in contrast, host cell dna (hcdna) was bound to the filter material. we achieved a % reduction of hcdna for an omv suspension in sfm. after clarifying an omv suspension in sfm, the remaining hcdna content was even lower being only %. conclusions charged depth filters are suitable for the first clarification step of omv downstream processing. residual protein could pass the depth scheme of the complete cell line development workflow support in genedata bioprocess. showcasing integration of data from diverse measurement instruments, data visualization for decision making support as well as, tracking of full clone history filter almost unhindered, whereas the hcdna content was already reduced to % at maximum. however, the omv titer was also reduced by the depth filtration. this undesired effect was stronger for the omv present in sfm. because the agencies require avoiding serum in clinical-grade production processes, this is disadvantageous. nonetheless, because sfm will be soon standard for omv production, further experiments have to be done preventing the omv reduction during clarification. one option can be to reduce the adsorption strength of the virus to the filter material by the addition of salt. moreover, it is important to establish a standardized protocol for the upstream processing. we determined batch-to-batch variations within the clarification indicating a strong impact of upstream processing (usp) on the outcome of the dsp. therefore, further studies must investigate the influence of usp parameter e.g. time of harvest and ph of the harvest solution on the omv. fed-batch culture is commonly employed to maximize cell and product concentrations in upstream mammalian cell culture processes. typical standard platform processes rely on fixed-volume bolus feeding of concentrated feed supplements at regular intervals. however, such static approaches might result in over-or underfeeding. to mimic more closely the dynamics of a fed-batch culture, we developed a dynamic feeding strategy responsive to the actual nutrient needs of a mab-producing recombinant cho cell line. results and discussion improvements made at different steps during fed-batch development are shown in fig. . at step , all eight cell boost supplements were added to cdm ns according to a doe approach, and batch cultures were performed. this evaluation allowed us to select only those cell boost supplements that were beneficial to the overall culture performance. non-performing cell boost supplements were removed and not considered further. at step , the selected cell boost supplements were added daily to the cultures at different ratios according to a doe approach, and fedbatch cultures were conducted. as expected, daily feed additions to replenish consumed nutrients substantially improved mab and peak cell concentrations as well as viable cumulative cell days (vccd) compared to batch cultivation. further, the results enabled us to fine-tune the feed ratio of selected cell boost supplements. at step , we further optimized the best performing feed ratio by investigation of static and dynamic feed protocols. most fed-batch protocols rely on constant feed additions on distinct days. however, these approaches often lead to substantial over-or underfeeding during bioprocessing. to improve such "static" protocols, we investigated three different "dynamic" approaches as shown in table by applying the selected cell boost supplements with the optimized feed ratio. this investigation allowed us to further improve the bioprocess performance. the best performing approaches, constant and retrospective feed, were further investigated in fully automated bioreactors under controlled conditions. in general, constant cultivation parameters in the bioreactor slightly enhanced mab titers compared to shake flask cultivation. the retrospective feed strategy yielded % higher titers than the constant strategy. overall, the established methodology for fed-batch development allowed us to obtain . × higher mab titers (batch mean: . g/l vs. fed-batch . g/l) in a short time and three simple steps. in addition, the product quality was investigated. compared to the legacy fedbatch process, fed-batches that were conducted with the newly selected basal and feed media altered the distribution of charge and glycan variants. the amount of aggregated product was not altered. the established methodology for fed-batch development is a rapid protocol to select well-performing feed supplements and optimize their ratio to the culture requirements. in three steps, mab titers were boosted . x from . g/l to . g/l. product glycosylation and charge variants could be influenced by the newly selected basal and feed media compared to a legacy fed-batch process. the amount of aggregated product was not altered. the present study investigates the beneficial effect of spiking hyclone™ actipro™ basal medium with hyclone cell boost™ a and cell boost b feed supplements on growth and productivity of a recombinant cho cell line. to evaluate the impact of feed-spiking compared with cultivation in basal medium only, the cell line was grown in bioreactors under controlled conditions to determine cellspecific metabolic rates, nutrient consumption, and byproduct accumulation over the process time. transcriptome analysis of the cultivated cells, using microarrays on four consecutive days to investigate differential gene expression, revealed the beneficial effect of feed-spiking compared with cells grown in basal medium. model cell line was a mab-expressing cho dg (licensed from cellca gmbh) cultivated either in actipro basal medium only (ge healthcare) or in actipro basal medium feed-spiked with additional supplementation with % cell boost a and . % cell boost b (ge healthcare). both cultures were grown in batch mode using dasgip™ cellferm-pro™ stirred-tank bioreactors (eppendorf). the beneficial effect of feed-spiking was analysed by transcriptome analysis using microarray technology ( × k design, agilent). both basal and feed-spiked processes lasted for seven days with viabilities above % until day . on day seven, a sharp decline in viability indicated the end of the batch process (fig. a) . in feed-spiked medium, cells initially grew slower but reached almost twice as high peak cell concentrations ( . × c/ml) than in basal medium only ( . × c/ml). remarkably, the integral of the viable cell concentration over the total process time (viable cumulative cell days [vccd] ) was similar between both process strategies (fig. c) . while mab production plateaued after day in basal medium only (final titer . g/l), a continuous increase to three-fold higher final titers ( . g/l) was observed in feed-spiked medium (fig. b) . the higher titers could be attributed to generally higher cell-specific productivities (qp), which remained rather constant (~ pg/cell/day) in feedspiked cultures. in basal medium, the qp continuously dropped by % (day to ), % (day ), and > % (day to ) from to pg/cell/day in basal medium cultures. in average, the qp was % higher in feed-spiked cultures (fig. d ). transcriptome analysis of differentially expressed genes between cells grown in basal medium or feed-spiked medium were used to identify relevant go terms that indicated a more active proliferative state for feed-spiked cultures (data not shown). the top go terms significantly related to cell cycle and primary metabolism, cellular division, as well as nucleobase formation or regulation. furthermore, gsea revealed several significantly enriched set of genes related to gene transcription, dna replication and repair, cell growth and proliferation, as well as inhibition of apoptosis in feed-spiked cultures. thus, feed-spiking increased the proliferative activity of cultivated cells. several of the identified genes appear as promising targets for cell line engineering, but have not yet been described in relation to high-producing recombinant cell lines and will need to be evaluated in future studies. feed-spiking of basal medium is a convenient and easy way to considerably increase product concentrations in a simple batch culture. differential gene expression revealed genes that appear important for high cell-specific production rates, and this knowledge can be leveraged into cell line engineering approaches or the design of high producing cho cell media. in the latter case, a maximized supply of high biosimilarity must be demonstrated by physicochemical and functional characterization for approval requirements of phase i and phase iii studies in terms of efficacy, safety and immunogenicity. in this study, rounds of upstream and downstream processes were run to reach the cqa limits of the originator molecule. after conducting many different development strategies, the mirror plot images of the intact deconvoluted mass were found to be identical corresponding to similar levels of glycoforms. the uv chromatogram of reversed phase ultraperformance liquid chromatography (rp-uplc) of tryptic peptide mapping demonstrated that the primary structure of tur is identical to the originator as shown in fig. a . post-translational modifications (ptms) such as oxidation, deamidation, n-terminal pyroglutamic acid, c-terminal lysine truncation levels were also comparable for two products. the glycosylation site (hc-asn ) was confirmed by peptide mapping analysis and % glycan site occupancy was proven for tur and originator. the glycosylation pattern for two products were highly similar in terms of major glycans (g f, g f, g f-gn and etc.). man level was lower in tur compared to the originator product which may not have any clinical effect on the molecule. the secondary structure was determined by atr-ftir spectroscopy. absorption bands (amide i and amide ii) were overlapped completely and amounts of α-helix and β-sheet structures were comparable. furthermore; size-exclusion chromatography (sec) analysis revealed that both products have the same level of purity (> %) and aggregate (< %) levels. the level of impurities were determined as below % by ce-sds. the capillary isoelectric focusing (cief) experiments showed that the charge variant profiles of two products are indistinguishable and the isoelectric point of main peak is observed at . for both products. the association/dissociation rate constants and binding affinity for both tur and originator were highly similar and similarity score was calculated greater than %, as shown in fig. b . in this study, state-of-art analytical techniques were used to assess the biosimilarity of tur to the originator adalimumab. head-tohead comparison data clearly demonstrated that tur is highly similar to the originator adalimumab in terms of physicochemical and functional characteristics. based on the analytical similarities, we . process performance of basal medium (black) and feed-spiked (red) bioreactor batch cultures: a cell concentrations and viability, b viable cumulative cell days and specific growth rate, and c antibody concentrations and cell-specific productivity. error bars indicate standard deviation from three independent experiments. the black arrows on day indicate the beginning of decreasing cell-specific productivities and lower cell-specific growth rates in basal medium cultures believe that tur will have comparable pk/pd, potency, and efficacy results to the originator adalimumab. the expanded interest in intensified continuous bioprocessing has highlighted the need to develop a small scale model for perfusion cell culture. the direction in the industry has been to increase target cell densities to ≥ x vc/ml and decrease perfusion rates to ≤ vvd. in order to increase the throughput of our perfusion media development capabilities we sought to develop a small scale model of perfusion using the ambr® instrument (sartorius, germany). we used a modified cell settling model from the previously published by kreye et. al. to achieve the cell retention necessary to reach perfusion relevant viable cell concentrations [ ] . in this work, we will show the application of this small scale model for: ( ) identification of specific productivity performance over a steady-state for tested media, ( ) identification of cspr min for a specific cell line and medium combination, and ( ) confirmation of consistent product quality profiles between the small scale model and benchtop perfusion (data not shown). a chozn® cell line producing an igg was evaluated in several proprietary chemically defined media prototypes generated during the development of the catalog excell® advanced hd perfusion medium: "fed batch medium", "early prototype", "mid prototype", "intermediate prototype" and "late prototype" [ ] . small scale simulation of perfusion experiments were run in ambr® . media exchange was performed times per day in equal amounts. agitation, gassing, and liquid handling were stopped for an optimized period of time to allow cells to settle to the bottom. spent media was removed in an amount proportional to / rd daily exchange volume. agitation, gassing, and liquid handling were resumed and fresh media was added back to the vessels. for benchtop perfusion, cells were inoculated in l applikon bioreactors (applikon, netherlands). at a concentration of~ . x vc/ml, perfusion was initiated using the atf (repligen, massachusetts). perfusion rate was limited at . vvd during steady-state. using the cell settling method described above we have been able to achieve ≥ % cell retention efficiency. all media tested in this work were able to reach and maintain the x vc/ml target cell density at vvd (fig. ) . performance of each media formulation was ranked based on specific productivity (table ) . using "intermediate prototype", minimum steady-state cspr was determined to be . pl/c/d for this cell line. n-glycan analysis of ambr® and bioreactor samples via intact mass spectrometry displayed only slight differences in product quality profile (data not shown). our work has shown a clear distinction between various prototype perfusion media and demonstrated a % increase in specific productivity over "fed batch medium" used in perfusion. additionally, we have shown the application to further characterize the process using this model to determine cspr min for a given medium and cell line. the transient process has been successfully operated at l in a sartorius biostat single use bioreactor (sub), yielding . kg of crude product from a two-week expression culture (table ) . successful scale up of the process to l creates the potential to supply transiently expressed products to support toxicology studies or even early gmp clinical supply, enabling accelerated biopharmaceutical development project timelines. the scale up from rocking bioreactors (rbr) to sub scale identified some scalability issues. lower specific productivity due to increased cell growth and decreased titres were observed in the sub ( fig. iii & iv). to improve the predictability of scale up, a new process was developed and evaluated in the sub vessels utilising a modified transfection method, which resulted in comparable expression levels and specific productivity between rbr and sub scales. two sets of expression vectors comprising heavy chain and light chain plasmids expressing a human igg kappa mab, as previously described [ , ] were used in the process optimisation study. the cell line used for transient expression and the pei mediated transfection method has been described previously [ ] . transfected cultures were run under fed batch conditions for days in l ge healthcare wave bioreactors (rbr), hyclone sub using l and l hyclone bioreactor bags (thermo scientific). the transfection process was modified to address the reduced titres and higher viable cell density (vcd) seen in the sub cultures. shake flask cultures were used to assess the standard (a) and modified transfection processes (b and c) (fig. , i & ii). process c was identified as the process to be studied at sub scale, offering the potential to mitigate the high viable cell densities (vcd) observed. scaling up process c to l and l sub resulted in cultures producing titres exceeding g/l with desired cell growth profiles. scale up of process a into sub vessels resulted in decreased productivity compared to the rbr scale. after optimisation, the sub process c yielded increased specific productivities and expression titres comparable to those seen at rbr scale (table ) . medimmune has successfully completed the first known successful cho transient culture at l scale producing > mg/l of mab at harvest. process optimisation has subsequently demonstrated reproducible titres at l to l scale exceeding g/l with comparable glycosylation profiles between sub and rbr cultures across scales. comprehensive analysis of the impact of trace elements in media on clone dependent process performance and product quality background state-of-the-art biopharmaceutical processes are accounting concomitantly for process performance and product quality. even though high yielding, robust processes are the cornerstones of any process development, product quality parameters such as structural integrity, charge variances and post-translational modifications are progressively becoming the focus of the developmental work. in conjunction with host cell line selection and process performance parameters, media components are crucial for the continued progress in rational modulation of product quality attributes affecting biological activity, immunogenicity, half-life or stability. among media components, trace elements (te) are of particular interest as they play a pivotal role in various cell metabolism pathways. based on a comprehensive doe approach, extensive process performance-and product quality evaluation combined with metabolic flux analysis, the impact of several trace elements on the biopharmaceutical process is assessed. in a comprehensive i-optimal doe approach ( fig. ) , the effect of six te in various concentration levels and combinations in serum-free media was studied for four different cho-k cells lines in an ambr® setup. a scrutiny of the process performance parameters such as cellular growth, productivity, amino acids and vitamins consumptions rates for each of the conditions was performed. the process performance evaluation was accompanied by extensive product quality analysis including size and charge variants, glycosylation patterns, oxidation and methylation. furthermore, a metabolic flux analysis was performed based on the nitrogen balance. based on extensive analytical data, the obtained response surface model provides a clear insight into the impact of particular te and their combinations on process performance and product quality. the high model quality enables discriminations between clone dependent and clone independent effects. with an elevation in titer up to % in the best condition of the cell lines clearly show, that even state-of-the-art media can be outperformed by trace element rebalancing. analyzing specific rates in combination with metabolic flux analysis improves the understanding of metabolic restructuring of the cell lines under distinct te levels and combinations. modulation of trace elements levels had a tremendous effect on the charge heterogeneity and glycosylation structure of the different proteins. this provides a toolbox for the fine tuning and control of product quality parameters. taken together, the data further paves the way to the rational fine tuning of process performance and product quality attributes. background due to regulatory concerns and economic impact, ensuring product quality and consistency is now one of the main challenge faced by the biopharmaceutical industry. for monoclonal antibodies (mab), glycosylation is one of the most important quality attributes as it impacts on mab structure integrity, and ultimately on both clinical efficacy and safety. many factors affect mab glycosylation and its inherent heterogeneity, including the host cell, the culture medium, the mode of operation and the operating conditions. in this context, the capacity to monitor and control on-line the antibody glycosylation, from early-to late-stage process development, would be of salient interest to reduce the time and cost to market. in order to address this unmet need, we have designed an improved spr biosensor assay to measure the kinetics of interaction between a mab and the extracellular domain of the fcγriiia receptor bound at the biosensor surface [ , ] . of salient interest, we also demonstrated that various binding kinetic signatures, especially different dissociation kinetics could be correlated with distinct mab glycosylation patterns and with therapeutic efficacies, as deduced from mass spectrometry and a surrogate adcc assay, respectively. in parallel, we have also harnessed a spr biosensor directly to a bioreactor, which permitted the at-line determination of the concentration of antibodies by hybridoma cells during a bioreactor culture. we now plan on combining both approaches to determine on-line the glycosylation profile of the produced mabs. our ultimate goal is to design a unique and highly innovative bioprocess control tool that can be readily applied in an industrial bio-manufacturing setting. reducing timelines and costs are key factors for bio-pharmaceutical industries to accelerate process development and drug delivery to patients. enhancing throughput of bioprocess development has become increasingly important for the screening and optimization of cell culture processes. this challenge requires high throughput tools. in a previous study [ ] , we showed that ambr® , a robotically driven mini-bioreactor system developed by tap-sartorius, could be advantageous to accelerate process development. the use of ambr® system allows us to test a large number of experimental conditions in a single experiment. therefore, the large amount of production samples to be characterized for product quality attributes (pqa) increases as well: the bottleneck has moved from the generation of samples at the production bioreactor step to in-process analysis. for product quality attribute analysis at lab scale, protein purification is generally carried out on > ml columns which is incompatible with the size of ambr® bioreactors. moreover, the applied methods are relatively low throughput. the development of a high binding capacity resin (up to mg/ml) [ ] , combined with high performing new cell lines which are able to produce up to g/l of recombinant monoclonal antibodies, allow require the development of an efficient and high throughput (hts) purification method robot. the use of robotic equipment for small scale purification purposes is a great opportunity for us to tackle this bottleneck, by enabling highthroughput sample purification at smaller scale ( μl). recombinant monoclonal antibodies were produced by a genetically engineered dihydrofolate reductase (dhfr)-/-dg chinese hamster ovary (cho) cell line. clarified cell culture fluid (cccf) was obtained from and k liter bioreactors after three filtration steps. minipurifications were performed on tecan freedom evo® robot with predictor robocol-umns® containing μl mabselect sure® resin. larger scale purification were executed using an aktaxpress using hitrap column prota. to assess monoclonal antibody purification at small scale, we first tested the repeatability of the minipurification, purifying the samples times on the same columns and using different columns, focusing on the yield of the purification and the impact on product quality attributes, especially the hmws. then, we compared those results to those obtained with the aktaxpress at larger scale purification, comparing the yield of the purification and the pqa of the protein-a eluates obtained with both purification systems. finally, we assessed the capability of the robot to perform hts of buffer and purification conditions, evaluating three different buffers at different concentrations and ph values, and also testing different loading column capacities. in this study we established that the tecan can be used as a robust platform for purification at small scale. we observed similar purification yields, intra and inter run. the analysis of the pqa a level showed there is also very high reproducibility. and the ph of the eluate showed as well strong comparability as well. table shows the coefficient of variation (cv) of the yield, hmws and eluate ph are low, demonstrating the good reproducibility of the purification. the strong reproducibility obtained between the different purifications showed that the tecan and the aktaxpress are similar in terms of purification performance and pqa (fig. a, b) . the tecan is a versatile automated liquid handler allowing the screening of huge purification conditions (fig. c) , the possibility to purify large quantities of samples, while the samples amount is limited. the tecan has the potential to purify more than samples/day, reducing timelines and allowing us to deliver faster to the patients. viable cell density monitoring in bioreactor with lensless imaging geoffrey monitoring cell density and viability of mammalian cell culture bioreactors is a necessary task that presents today a number of remaining challenges. the traditional measurement for bioreactor cell count and viability rely on using the trypan blue exclusion method once a day. while automatic cell counters have reduced the statistical manual error, sampling the bioreactor remains a contamination risk and is prohibiting process control as the sampled volume becomes siginficant. lensless imaging technology is a new method for accurately determining cell concentration and viability without staining. this technique directly acquires the light diffraction properties of each individual cells through their holograms images without any objective, lens or focus settings. living and dead cells have significant holographic patterns that can be distinguished and precisely counted. lensless imaging technique directly acquires the light diffraction properties of each individual cells through their holograms images without any objective, lens or focus settings. living and dead cells have significant holographic patterns that can be distinguished and precisely counted. we compare cell counts and viability between the reference method and our lensless imaging device, the cytonote counter. measures are performed once a day on samples from bioreactors, from the inoculation to the end of the culture. we also assessed the repeatability of our method. another lensless imaging prototype is setup as a measurement chamber directly connected to a perfusion bioreactor, for continuously receiving the bioreactor broth, and therefore reproducing an in situ measure. with a concentration range up to x cells/ml ( fig. ) and viability range at - %, we obtained a correlation factor of . between the two compared methods. the large field of view allows the analyze of several thousand cells within a single image, keeping the statistical variability of the measure as low as %. our measurement chamber prototype has demonstarted its capability for continuous viable cell density and viability monitoring. we are now working at designing a steam strerilizable probe, and we envision lensless imaging to become the future method of choice for on-line monitoring of suspension cells cultures. lensless imaging technology is capable of accurately and precisely monitoring viable cell density and viability with a combination of significant advantages starting from low sample volume use, label free detection, quick measure, simple device, to high number of cell analyzed which let us think that it is a good candidate for very small- comparison between both purification systems and the ability of the system to be used as a high throughput tools for buffers screening. a purification yield (%). b pqa .a (normalized). c impact of the ph and buffer concentration on the pqa .a scale bioreactor and high-throughput measures. its high repeatability is also a key parameters in the effort to narrow batch to batch deviations. in addition we demonstrate that this technique is potentially powerful for in-line and continuous monitoring of a lab bioreactor. we envision lensless imaging to become the future method of choice for on-line and in-situ monitoring of suspension cells and a perfect tool for process control in fed-batch or perfusion mode in single-use bioreactors or traditional steam sterilized vessels. it can certainly become the first vcd measurement technique to work from cell line engineering, to process development, pilot scale, and up to manufacturing scale. time-dependent product heterogeneity in mammalian cell fermentation processes background a consistent product quality is a major goal in the production of biotherapeutics, especially recombinant glycoproteins. whereas it is unlikely that the polypeptide chain changes during a production process, posttranslational modifications and protein folding are sensitive to fluctuations in parameters and conditions. here we focus on protein glycosylation as one important indication for product quality [ ] . during a batch process conditions change continuously. at the beginning, the supply situation for the cell is excellent, but the secreted material stays a long time in the culture fluid. later during cultivation substrate provision decreases, whereas the exposition time of the protein to the culture fluid is much shorter. altogether this leads to product heterogeneity of the secreted protein during a batch culture. four different cell lines, two of human origin and two cho clones, producing four different recombinant glycoproteins were investigated in this study. together with their respective parental cell line the clones were cultured in three replicates in shakers. supernatant from the cultures were harvested at four time points. the removed culture volume was replaced by culture supernatant of the identically cultured corresponding parental cell line. the product was isolated from the supernatant and the glycans were released. one part of the released glycans was labeled with -ab and separated by hilic-fld. the other part of the glycans was permethylated and analyzed by maldi-tof mass spectrometry (fig. a) . the investigated proteins were antibody, antithrombin iii from cho clones and α -antitrypsin, c -inhibitor from human clones. the antennarity of the glycans is quite stable in all production phases. the degree of core fucosylation is very high in all products. a low fucosylation degree of antibodies may be favorable for a higher adcc performance [ ] . some of the products showed an antennary fucosylation, which seemed to change not very much in different cultivation phases. nevertheless, this might be an issue due to an antigenic impact in the patient. the antennary galactosylation changes noticeable for the antibody and α -antitrypsin. in both cases the degree is highest in the first phase. an incomplete galactosylation leads to truncated glycans. this leads inevitable to undersialylated antennas to be seen for α -antitrypsin. the sialylation is the highest in the early phases and decreases during cultivation time. sialylation of a therapeutical protein is important for the half-life in the patient. therefore highly sialylated products are desired [ ] . in further studies the consistency of the galactosylation and the sialylation will investigated for fed batch and long term continuous cultures in comparison to batch cultures. due to the feed solution or the fresh media being present during such processes, the supply situation should be excellent for the whole cultivation time. the differences between the maldi-tof and hilic-fld data originate from complex and unresolved chromatograms (fig. b , chromatograms not shown). for that reason coupling of hilic-fld and ms is very much recommended. background novel biologics are often selected from a large library of lead candidates in the initial stage of preclinical and clinical developments. for this selection, there is a demand for high-throughput production of recombinant proteins of high quality and in sufficient quantity. transient gene expression offers a rapid approach to the production of numerous recombinant proteins for the initial-stage developments of biologics. mammalian cells are major host cells for transient gene expression, but they have the disadvantages of complicated operations and high cost of culture. insect cells are easy to handle and can be grown to a high cell density in suspension with a serum-free medium. insect cells can also produce large amount of recombinant proteins through post-translational processing and modifications of higher eukaryotes. hence, insect cells have been recognized as an excellent platform for the production of functional recombinant proteins [ , ] . in the present study, the production of an antibody fab fragment through transient gene expression in lepidopteran insect cells was examined. the dna fragments encoding the heavy chain (hc) and light chain (lc) genes of an fab fragment of mouse anti-bovine rnasea [ ] were respectively cloned into the plasmid vector pihaneo, which contained the bombyx mori actin promoter downstream of the b. mori nucleopolyhedrovirus (bmnpv) ie- transactivator and the bmnpv hr enhancer for high-level expression [ ] . trichoplusia ni bti-tn- b - (high five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine (pei; mw , ). before transfection, the plasmids and pei were prepared in mm nacl, ph . and incubated at room temperature for min. when the transfection efficiency was checked, a plasmid vector encoding the enhanced green fluorescent protein (egfp) gene was also co-transfected. transfected cells were incubated with a serum-free medium in a static or shake-flask culture. culture supernatants were analysed by western blotting and enzyme-linked immunosorbent assay (elisa). the numbers of green fluorescent cells and total cells in culture broth was determined using a flow cytometer. western blot analysis and elisa of culture supernatants showed that transfected high five cells secreted the fab fragment with antigenbinding activity. in static cultures, transfection and culture conditions, such as hc:lc gene ratio, a serum-free medium, dna:pei ratio, and dna amount per cell, were successfully optimized by flow cytometry of egfp expression in transfected cells and the yield of the secreted fab fragment measured by elisa. the effects of culture temperature and initial cell density were also examined by comparing the cell growth and the production of fab fragments in shake-flask cultures. under optimal conditions (medium, psfm-j (wako pure chemical industries, japan); hc:lc gene ratio, : ; dna, μg/( cells); pei, μg/( cells); initial cell density, x cells/cm ; temperature, °c), the yield of more than mg/l of fab fragment was achieved in days in a shake-flask culture ( fig. ) . transfection did not significantly affect the growth of high five cells as compared with untransfected cells. transient gene expression using insect cells may offer a promising approach to high-throughput production of candidate proteins for the development of biologics. the increasing demand for biopharmaceuticals produced in mammalian cells has led industries to increase volumetric productivity of bioprocesses through different strategies [ , , ] . in this context, fedbatch and perfusion cultures have attracted more interest than conventional batch processes. the efficient application of such alternative processes requires the availability of reliable on-line measuring tools for cell density and cell metabolic activity estimation [ ] . the comparison of different culture strategies for hek cell line producing ifn-γ are presented below: batch, fortified batch and fed-batch. in this context, a new robust feeding strategy based on the monitoring of alkali buffer addition was applied for the estimation of nutrient requirements. this method allows to increase cell density and product titer compared with the other strategies assessed. three different culture strategies were carried out in -litre biostat b-dcu ii bioreactor. first, a reference batch and a batch using fortified medium (nutrient enriched medium) were run and assessed in terms of viable cell density (vcd) and product titer, and set as initial references. then, a fed-batch was performed applying a feeding strategy based on the nutrient requirements estimation by monitoring the alkali buffer addition used for the control of ph. results vcd and product titer achieved for the different culture strategies assessed (batch, fortified-batch and fed-batch) are presented in table . in fortified batch an increase in vcd of % and also % in product titer were obtained compared with batch. in the fed-batch culture carried out (fig. ) , we observed that alkali buffer addition profile matched the vcd evolution trend. thus, the monitoring of alkali buffer addition was used for estimating the nutrients requirements (i.e. the volume of feeding medium) at any time during the fed batch phase. the feeding strategy based on alkali buffer addition enabled to maintain glucose concentration set point therein a narrow range during fed-batch phase (around mm). as a result, higher vcd ( . · cells/ml) was obtained when compared with both batch references: vcd was enhanced to % and % and an increase up to % and % in product titer in respect to batch and fortified batch respectively. the results prove that fed-batch strategy based on the alkali buffer addition is a robust on-line monitoring method that enables to optimize the feeding strategy in a fed-batch cultures. three different culture strategies have been tested in bioreactor with a hek cell line producing ifn-γ. results show as the higher vcd is reached, the higher product concentration is achieved. therefore, from bioprocess development point of view, it is very interesting to implement strategies with higher vcd outcome, such as fed-batch operation mode. in this context, a new robust method for vcd estimation in fed-batch was applied. the alkali buffer addition necessary for maintaining the ph set-point is an on-line reliable and easy measuring variable that provides information about by-products formation (mainly lactic acid). the monitoring of this variable can provide information about the cell concentration, activity and metabolism, to detect changes in culture. besides that, a relationship between alkali buffer addition and vcd can be established since the first is strongly correlated with cell growth and metabolites consumption/formation. the application of alkali buffer addition measure to implement an optimal feeding strategy in fed-batch permits to enhance vcd and product titer when comparing with batch strategies. a novel approach to high throughput screening for perfusion background perfusion systems for suspended mammalian cells raise growing interest in the biomanufacturing industry. continuous manufacturing is growing in the field and is encouraged by health authorities [ , , ] . this work addresses scale down limitations inherent to continuous media exchange and cell retention by using a semi-continuous system. data was generated with a set of different clones that were previously studied in fed-batch mode [ ] . materials and methods cho-k cell lines expressing the same monoclonal antibody (mab) and issued from the same transfection were used as models. . l bioreactors (sartorius) were used for fed-batch and perfusion production runs. the perfusion bioreactors were run using an alternating tangential flow filtration device (repligen, xcell™ atf system). the cell biomass was controlled by removing cells through a bleed line and was controlled using a biocapacitance probe (hamilton, incyte). the perfusion rate (d) was fixed to one vessel volume a day (vvd - ). the semi-continuous runs were made in ml shake tube (tpp, tubespin® bioreactor ). once a day, the tubes were centrifuged ( min, g), the supernatant removed (to mimic a perfusion rate of vvd - ), replaced with fresh media and cells were re-suspended. the clone's growth potential were preserved across the systems (fig. ). clone # always reached the highest viable cell density (vcd), followed by clone # . clone # and # showed similar growth characteristics. it is interesting to note that in the perfusion bioreactor different patterns in terms of vcd were observed although the cell biomass signals were similar for all runs. this reflects the fact that the capacitance measures the biomass and not the absolute cell count [ ] . to estimate the minimum cell specific perfusion rate (cspr min ) in the semi-continuous experiment, the perfusion rate was divided by the maximum viable cell density (vcd max ). this value was compared to the cspr obtained during the th set-point (sp ) of the perfusion runs. as expected, the bleed fraction decreased when the capacitance set-point was increased and went down to % or less of the total perfusion rate (data not shown). since the bleed removes the excess biomass, it is an indication of how close to a limitation the system is. therefore, the cspr calculated at sp was considered as the minimum cspr. the cspr min obtained in both systems were very close ( table ). the semi-continuous system can therefore be used to identify the cspr min before running a continuous bioreactor, it therefore facilitates the decision making early in the development (to define the target cell density for a defined perfusion rate). the specific productivity (q p ) of the clones was quantified at the maximum vcd (semi-continuous) or at sp (perfusion). absolute values are not representative since the cell environment is so different in both systems. nevertheless, a relative ranking proved to be indicative of the respective performances ( table ). the maximum cell growth in fed-batch, semi-continuous and perfusion were also compared, their ranking was always preserved. both indications can be used to assess a performance ranking for different clones. the performance of clones was studied in different cultivation systems: fed-batch/perfusion bioreactors and semi-continuous shake tube. the semi-continuous system was able to precisely predict the cspr min , an important process parameter for perfusion. specific productivity and maximum cell density ranking was preserved across the systems, therefore the scale down experiment can be used to assess a performance ranking for perfusion clone screening. modulating antibody galactosylation through cell culture medium for improved function and product quality jenny y. bang, james-kevin y. tan the production of therapeutic antibodies (abs) requires high titers and excellent product quality to ensure efficient manufacturing and potent drug efficacy. glycosylation, or the attachment of sugars to organic molecules, is a critical quality aspect that can significantly alter ab binding, function, and therapeutic effect [ ] . galactose is a key sugar of interest due to its significant impact on ab function and the ability to control galactosylation through cell culture medium. herein, irvine scientific assessed the ability of media components to modulate galactose levels on a model therapeutic ab. various media compositions were able to modulate galactosylation levels without compromising cell growth and ab titers. in addition, an in vitro assay was utilized to evaluate the functional ability of abs to bind and activate complement-dependent cytotoxicity (cdc). differences in galactosylation significantly altered the abs' ability to induce cell cytotoxicity. furthermore, design of experiment analysis determined the optimal ratio of supplements to maximize galactosylation. this "optimized supplement" was verified and evaluated against other suppliers' galactosylation supplements in terms of growth, titer, glycan analysis, and ab function. the optimized supplement outperformed all other suppliers' supplements and resulted in the best overall cell growth, titer, galactosylation, and ab function. ab against cd were grown in balancd® cho growth a and were fed with balancd® cho feed on days - of the cultures. viable cell density and cell viability were assessed by a beckman coulter vi-cell xr, ab titer was assessed by a pall fortébio qk e , and glycan analysis was assessed by a perkinelmer labchip gxii. for the functional cdc assay, abs were incubated with daudi b lymphoblast cells and normal human complement serum. cell cytotoxicity was assessed with a promega cytotox-glo kit. various supplements were evaluated in fed-batch cultures and resulted in - % ab galactosylation without compromising cell growth and ab titers. design of experiment analysis determined an optimal composition, deemed "optimized supplement," which was evaluated against a panel of galactose-modulating supplements from other suppliers. the optimized supplement resulted in a similar viable cell density (vcd) and cell viability compared to the fed-batch culture control which had no supplements (fig. a) . supplements from supplier resulted in similar to half the vcd of the control while supplements from supplier resulted in very low vcd and percent viability. all of the supplements except those from supplier resulted in ab titers similar to the control (fig. b) . due to the poor growth and subsequently low titer from supplier 's supplement, supplier was not further evaluated. the glycan profiles were analyzed and are presented in (fig. c ). all the evaluated supplements were able to raise galactosylation; however, only the optimized supplement and the x supplier supplement resulted in over % galactosylation. the function of the abs was further evaluated in a cdc assay (fig. d) . abs from the optimized supplement were more effective than the control abs and had a significantly lower half-maximal effective concentration (ec , . μg/ml) than the control ( . μg/ml). abs from the x supplier supplement had a similar ec to the control which may be due to the higher man % of the abs. an optimized supplement was produced through fed-batch evaluation and design of experiment analysis. the optimized supplement outperformed all other supplenments from other suppliers and resulted in the best overall cell growth, glycan profile, and functional ab activity (table ) . industry practice for mammalian cell culture media and feed development typically employs a high-throughput screening (hts) platform along with large sets of experiments [ ] . modern hts systems often include robotic liquid handlers to replace labor intensive steps. to align with advancements in the field, a semi-automated hts platform was developed to facilitate in-house media and feed development for early stage biologics projects. selecting appropriate instruments and integrating them into a seamless system are the keys to a hts platform. the developed hts platform uses deep well plates (dwps) for culture vessels, the liquid handler of the advance microscale bioreactor (ambr ) for media/ feed formulation preparation in an aseptic environment, nyone cell imager for viability and cell growth analysis, tecan freedom evo's liquid handler for activity assay sample preparation, and cedex bioht for high-throughput metabolite analysis. dwps offer comparable cell growth to shake flasks and compatible layout to ambr , which makes the dwp an ideal candidate for the platform. in addition, the user friendly design of experiments (does) interface and liquid handler function of the ambr expediates the formulation preparation of varying doe conditions [ ] . a macro program was written and developed in excel to enable the easy import of does design from major statistics software packages, such as jmp and simca, into ambr . performance qualification of each component were performed prior to implementing the hts platform. comparable cell growth profile and productivity were achived between shake flasks and dwps (fig. a ), indicating compatable cell culture environment for the cells. cell counts using nyone gave identical cell growth ranking as the traditional count from vi-cell xr (fig. b) . freedom evo's liquid handler was optimized to produce comparable activity results to manual operation while expediting the sample preparation with improved consistency (fig. c) . finally, implementing the liquid handler function of ambr to support media and feed formulation significantly reduced the labor for each experiment. summary of the capability comparison between the hts platform and the traditional method are listed in table . a case study of a complex feed screening with definitive screening design was completed using the semi-automatic hts platform. this experiment, containing more than feed formulations in duplicates, was handled by one operator and delivered a % improvement in productivity within a week period (data not shown). in addition, implementing the hts platform for this study also resulted into~ % reduction in labor while improving the traceability of formulation preparation. a semi-automated hts platform was developed to support media and feeds screening and development for early stage biologics projects. the platform utilizes dwps, nyone cell imager, ambr , freedom evo liquid handler system, and bioht metabolic analyzer to accelerate the screening process. this screening platform not only improves process throughput, operational precision, and traceability of formulation preparation, but also reduces the labor for the media and feed formulation preparation. background a perfusion medium requires high concentrations of specific nutrients while balancing other components to support intensified perfusion processes. using a combination of design of experiment (doe), multivariate analysis (mva), and spent media analysis, we developed a catalog "de novo" perfusion medium by working with multiple cho cell lines and proteins. the optimization of the medium in bioreactors using alternating tangential flow (atf) cell retention devices reduced the minimum cspr from over pl/cell/d to under pl/cell/d for most cell lines while increasing specific productivity during day steady states with stable growth rates, viability, volumetric productivity and product quality. high throughput screening (hts) was performed with seven cell lines, while four were used in bioreactors: cho-s, dg , and two chozn® gs lines, each producing different monoclonal antibodies and include a fusion protein. for hts experiments, cells were inoculated at . x vc/ml with a ml working volume in ml tpp® tubes and cultured for days in a multitron shaken at rpm, °c, % rh, and % co . for benchtop perfusion, cells were inoculated at . - . x vc/ml in l applikon bioreactors (applikon, netherlands) with a l working volume. bioreactors were operated at rpm, °c, % do, and a ph of . or . ± . depending on the cell line. oxygen was supplied through an l-sparger or microsparger as needed, and excell® antifoam (milliporesigma, germany) was added at a maximum rate of . % v/v to control foam. at a cell concentration of~ . x vc/ml, perfusion was initiated using the atf (repligen, massachusetts), with a bleed set to maintain cell concentrations at or * vc/ml. two "de novo" prototype media were developed using doe and mva in hts with tpps and an ambr® [ ] and one was chosen for further development after comparing to a basal medium enriched with feed in bioreactors. eleven components were identified as significant effectors of critical parameters for perfusion processes across evaluated cell lines. doe central composite experiments were run and component concentrations were optimized in the selected prototype. in parallel, amino acid specific consumption rates were calculated from bioreactor spent media samples and used to adjust the concentration of amino acids to target a reduced cspr. increasing specific amino acids concentrations resulted in a significant reduction of the minimum cspr across all tested cell lines -for example the cspr of cho-s was reduced from to pl/cell/day (table ). however, even at the lower cspr, spent media analysis revealed excess concentration of some amino acids, so specific accumulating amino acids were reduced and components were streamlined for the final medium: excell® advanced hd perfusion medium. using this medium, a cho-s and a chozn® gs cell line producing a fusion protein were cultured at a cspr of less than pl/cell/day with a vcd of * vc/ml. metabolic profile, productivity, and product quality were constant over the day steady state. the chozn® gs cell line was also tested at * vc/ml with a cspr of pl/cell/day (fig. ). we have developed a catalog perfusion medium from first principles, ensuring broadness of application by using seven cell lines in scaleddown systems and four in perfusion bioreactors. the final catalog medium showed significant improvements in productivity across all cell lines, with reduced csprs when compared to enriched fed-batch medium or initial prototypes (table ) . there is a rising demand for accelerated process development, increased efficiency and economics for biopharmaceutical production processes. furthermore, increased process understanding have evolved from the process analytical tool initiative (pat) and the quality by design (qbd) methodology. in contrast to one-factor-at-atime methods, statistical design of experiment (doe) methods are widely used to develop biopharmaceutical processes. even if highthroughput systems can handle these numbers of experiments in parallel, the heuristic restriction of boundaries and the high number of factors results in stepwise iterations with multiple runs. therefore, the combination of model-based simulations with doe methods (mdoe) for the development of sophisticated cell culture processes is a novel tool for process development [ ] . it is used to reduce the number of experiments during doe and the time needed for the development of more knowledge-based cell culture processes. this concept was applied to the optimization of the initial glutamine and glucose concentrations of a cho batch process. a mechanistic model was adapted and modified from [ ] and used to describe the dynamics of cell metabolism and antibody production of an il- antibody producing cho cell line (see abbreviation of fig. for cultivation details). experiments were simulated and compared to a fully experimental doe. as can be seen from table , user defined constraints were chosen to get a stable and reproducible process with the aim of maximizing the cell density but decreased lactate and ammonia production. at first, the experimental space was estimated by simulating the responses for broad concentration ranges and calculating the multiple response desirability function (fig. a) . this results in a small area (turquoise) suggested as experimental space. experiments were planned within these boundaries and responses were either simulated ( fig. b, cultivations for fitting the model) or compared with the purely experimental responses (fig. c, cultivations). optimal concentrations for glutamine and glucose with respect to the constraints are in the lower right corner and similar for both methods (red frame, fig. ). compared with the fully experimental design, mdoe results in a reduction of % in the number of experiments ( experiments for modelling vs. experiments in experimental doe). the method is intended to optimize cultivation strategies for mammalian cell lines and evaluated these before experiments have to be performed in laboratory scale. this results in a significant time and cost reduction during process development and process establishment. the strategy is especially intended for the use in multi-single-use-devices to speed up process development. . at a target cell density of * vc/ml, volumetric productivity was stable for a day steady state with excell® advanced hd perfusion medium. shorter steady states were tested at * vc/ml background for the large-scale production of therapeutic glycoproteins, fedbatch culture has been widely used for its operational simplicity and high titer. however, repeated feeding of medium concentrates and/ or addition of a base to maintain optimal ph during fed-batch culture lead to increase in osmolality. the hyperosmolality affects glycosylation in a protein-specific manner. however, the mechanism behind such osmolality-dependent variations in glycosylation in recombinant chinese hamster ovary (rcho) cells remains unclear. in this study, to better understand the effect of hyperosmolality on the glycosylation of a protein produced from rcho cells, we investigated n-glycosylation-related gene expression and n-linked glycan structure in fc-fusion protein-producing rcho cells exposed to hyperosmotic conditions. furthermore, to validate the effect of hyperosmolality on protein glycosylation, we performed hyperosmotic culture supplemented with betaine, an osmoprotectant, and then analyzed the n-linked glycan structure and mrna levels of n-glycan branching/antennary genes. after three days of hyperosmotic culture, nine genes (ugp, slc a , slc d , gcs , manea, mgat , mgat b, b galt , and b galt ) were differentially expressed over . -fold of the control, and all these genes were down-regulated. n-linked glycan analysis by anion exchange and hydrophilic interaction hplc showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary n-linked glycans was significantly decreased upon hyperosmotic culture. addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary n-linked glycans (p ≤ . ), while it increased the expression of the n-glycan branching/antennary genes (mgat and mgat b). thus, decreased expression of the genes with roles in the n-glycan biosynthesis pathway correlated with reduced sialic acid content of fc-fusion protein caused by hyperosmolar conditions. conclusions taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on n-glycosylation, especially sialylation, in rcho cells. the identified genes, particularly mgat and mgat b, are potential targets for engineering in cho cells to overcome the impact of hyperosmolality on glycoprotein sialylation. disruptive cost-effective antibody manufacturing platform based on cutting-edge purification process v. medvedev, m. duyck, t. albano, j. castillo univercells sa, gosselies, belgium correspondence: v. medvedev (v.medvedev@univercells.com) bmc proceedings , (suppl ):p- background demand for high-quality monoclonal antibodies is growing exponentially, calling for new production capacities. overcoming current limitations of conventional manufacturing strategies, namely the high capital investment and production cost, can only be achieved through innovative process designs based on the latest technologies. this study presents a process design combining batch-fed technology with continuous multi-column capture. an advanced cell culture clarification method was introduced to simplify downstream operations and increase overall cost-effectiveness of the process, for an optimized production of recombinant proteins. this study was performed with cho cells expressing a monoclonal antibody targeted against the coronoavirus responsible for the middle east respiratory syndrome (mers), developed by organic vaccines tm and the nih, kindly provided to univercells. upstream process: -fed-batch, days culture at l scale with cd-cho chemically defined media and feeds. harvest treatment: -precipitation of impurities in the production bioreactor using organic compounds (< % v/v) and flocculation by electropositive organics (< . % w/v). -acidic ph and physiological conductivity. upstream processing and harvest treatment: culture reached . g/ l ( x cells/ml; % viability), harvest treatment was found to be very effective in terms of impurities clearance. capture: capture strategies were evaluated from the point of view of simplification of downstream operations, with hcp impurities content monitored as a key performance indicator. -protein a affinity chromatography: advanced harvest clarification enabled major improvements in affinity capture, in terms of eluate purity and reduction of host cell impurities (< ppm in all conditions tested). (fig. ). -cation exchange chromatography: cex allows higher capacities (> g/l) than protein a, whilst being more affordable (from -to -fold cheaper). low residual hcp (< ppm) was observed with all cex resins tested. without harvest treatment and clarification preceding the capture studies (either affinity or cex), results showed a lower binding capacity of the resin, a higher content of hcp in the eluate (up to ppm), a higher content of hmw species in the elution fraction (up to -fold higher) and a significant turbidity of the neutralized eluate. -continuous multicolumn chromatography: further options to increase cost efficiency include using a continuous multicolumn setup (table ) . two models were assessed based on two different static binding capacities (sbc), demonstrating that to columns of ml were able to process a l production in less than h. this method provides a great opportunity for designing simplified and low footprint mabs dsp processes, while maintaining similar or achieving superior quality profile compared to standard approaches: -harvests treatments followed by depth filtration proved to be a cost-efficient way to obtain pretreated feed and minimize the burden on downstream operations. -protein a resins exhibited advantages of extracting key contaminants during harvest treatment, while caex confirmed to be a competitive capture strategy. -switching from batch to continuous multicolumn mode allowed to process a complete batch in less than hours, requiring lower media and resins volumes. followed by a single polishing step, such process set-up strongly supports the reduction of operations required to deliver a high-quality product. analyses of product quality of complex polymeric igm produced by cho cells background immunoglobulin m (igm) antibodies are secreted by b cells as the first defense against invading pathogens during primary immune response. some igm antibodies already gained the orphan drug status, which shows their unique capability in therapy of rare diseases. potential fields for applications are discovered with increasing knowledge about these molecules. it seems that the most active forms are pentameric and hexameric igms. unfortunately, recombinant production of igms is rather difficult as secretion and correct polymer formation results in low expression yields and mixtures of polymers. we established stable producing chinese hamster ovary (cho) dg cell lines to analyze cellular and extracellular factors that influence quantity and quality of the produced recombinant polymeric igm in future studies [ ] . one quality parameter is polymer distribution, which can be measured directly in cell culture supernatant using densitometric analyses [ ] . additionally, we developed a very efficient single-step-affinity purification strategy using the poros captureselect igm affinity matrix to analyze pure igms. for more precise measurements of the igm isoform distribution we separated the purified polymers by high performance liquid size exclusion chromatography (sec hplc). our cho dg cell lines grow to peak cell concentration of . x cells/ml in erlenmeyer flasks and . x cells/ml in bioreactors. similar productivity of approximately mg/l was observed for cells cultivated in both cultivation vessels in a nonoptimized batch culture using chemically defined media. analysing how cultivation conditions affect the fraction of polymers may offer clues about the assembly of polymers and the challenges of igm production. we quantified polymeric distribution of igm directly in the supernatant using a densitometric method [ ] . cultivated under standard conditions ( °c, ph ) igm is produced as % pentamers, whereas igm _gl only consists of approximately % pentamers. the purified igm _gl was analysed with sec-hplc and contained % pentamer and % dimer, which is comparable to the results achieved with densitometry. the purification of the igm antibodies was quite challenging as the manufacturer recommend acidic elution, which led to aggregation and inefficient elution of our model igms. therefore, we screened for different elution buffers that prevent denaturation and aggregation. by combining high salt concentrations with moderate ph reduction we optimized elution conditions to - % igm recovery, which corresponded to a five to six fold improvement compared to the manufacturers' conditions. sds-page analysis and sec-hplc showed that our elution strategy resulted in a very pure product after a single chromatographic step. the purification strategy was verified with the igm , igm and igm . our model igms were produced in a ratio of approximately : pentameric to dimeric igm, measured concordantly with both analytical methods. process development on igm purification using the poros capture select human igm affinity matrix enabled the recovery of highly pure fractions. through optimization, by combining mild ph and high salt concentrations, the relatively low elution yields were increased by a factor of - . applying densitometry and sec-hplc we will investigate how culture conditions influence polymer formation in future. currently, no small scale (< . l) cell culture system is commercially available for high cell density perfusion cultivations to use in high throughput screening studies. to increase throughput for process characterization activities at janssen vaccines and prevention, a shaker flask-based scale down model was developed. though, the control possibilities of shaker flask cultures are technically very limited and different compared to a bioreactor controlled process. in addition, the sensitivity of the shaker flask model should allow the detection of the effects of process parameters on critical quality attributes (cqas) of the vaccine produced at large scale. iterative experiments were performed in shake flasks to evaluate the influence of cultivation parameters such as shaking speed, working volume, co % in the incubator and daily base additions on cultivation parameters (as cell growth, ph and do). in addition, a medium exchange was tested to mimic the perfusion mode used in the bioreactor process. the presens shake flask reader was implemented to allow for ph and do monitoring. the conditions for which the performance as reflected in specific virus titer showed the best fit were selected. at these conditions, a series of parallel shaker flask infections were conducted to demonstrate statistical equivalence of performance parameter and cqas (as cell specific iu titer and vp/iu ratio) between the production scale and reduced scale processes and thus to qualify the shake flask as a scale-down model. a daily medium exchange by centrifugation was implemented and cultivation parameters for shake flasks were identified. based on performance parameter (cell specific vp titer) and the cqas of the vaccine (cell specific iu titer and vp/iu ratio), equivalence between the production-scale and scale down systems was confirmed. the scale down model data fall into the % prediction intervals calculated on manufacturing data whereas scale down model data from batch mode experiments (using non optimized cultivation conditions) do not. the shaker flask as a scale down model for the l bioreactor perfusion process was qualified. this model is a tool to screen a subset of process parameters at a higher throughput, thereby reducing process characterization timelines. background until today, the market for therapeutic proteins, especially monoclonal antibodies, is gaining more and more importance in the pharmaceutical field. to meet the increasing demand for these products, the industry made tremendous efforts to generate highly efficient production systems. one of the pharmaceutical industry's research focuses is the improvement of the secretion process in eukaryotic cells. in mammalian cells, the efficiency of protein transportation strongly depends on the translocation of a nascent protein into the er, which is mostly conducted by the signal peptide (sp) coupled to the nterminus. through the interchangeability of signal peptides between products and even species, a large variety can be used to enhance protein expression in already existing production systems materials and methods at first the influence of four different natural sps (sp ( ), ( ), ( ) and ( )) was compared on the secreted amount of an igg model antibody (product a) in fed batches using a cho dg host cell line. in the second part, one promising sp-candidate showing improved secretion (sp ( )) was identified and the influence of this sp on four additional antibody products, which varied in their expressability from good to mid/bad, was investigated. in both approaches, the standard sp was implemented for comparative reasons. in the first approach, four signal peptides sp ( ), sp( ), sp( ) and sp( ) were screened for their potential to improve the product secretion of cho dg cells expressing a model antibody (product a). the results revealed a . -fold increase in average final fed-batch antibody titer of sp( ) when compared to the standard sp approach (standard sp = . g/l; sp( ) = . g/l). in the second approach, the enhancing capacity of sp( ) on secretion of four other igg products (named product b to e, table ) was further evaluated. an improved performance was observed for all products when comparing sp( ) and the standard sp in a fed batch process (fig. ) . with an increase in average final fed-batch titers ranging from to % and up to % in cell-specific productivities. taken together, with a positive influence on the final concentrations of all tested products, the results obtained with sp( ) contribute to -signal peptide sp( ) was identified as a promising candidate with an average . -fold titer increase during screening of four signal peptides. -sp( ) was able to improve production titers up to % compared to standard sp. -sp( ) was able to improve cell-specific productivities up to % compared to standard sp. -future usage of sp ( ) contributes to the further optimization of sartorius stedim cellca's standard cell line development process. new platform for the integrated analysis of bioreactor online and offline data lukasz gricman , milan ganguly , amanda fitzgerald , hans peter more and more experiments are used to assess bioreactor suitability and stability of clones, to evaluate media composition and other process parameters, and to start upscaling campaigns. this has resulted in a major bottleneck due to the increase in data capturing, processing, aggregation, visualization, and statistical analysis. in addition, the association of the data with the experimental context (e.g., fermentation protocols, media recipes, bioreactor control parameters) is not easily accomplished in high throughput. the data generated in the process must not only be analyzed, but also managed and stored to enable easy tracking and relating to historical records. furthermore, the processes are often developed by global teams interacting in complex enterprise it ecosystems. therefore, new and high performing systems for data capture, processing, and analysis need to be integrated in order to enable storage and correlation of experimental context information and various types of time course analytics data. we have developed genedata bioprocess™, a new enterprise platform for bioprocess development. the platform enables automatic capture and visualization of all online and offline data (e.g., ph, o , metabolic data), auto-calculations and aggregations (e.g., ivcd, qp, consumption rates) and multi-parametric assessment of any type of time-series bioreactor data in the context of experimental protocol data (e.g., process parameters, feeds). genedata bioprocess comes with dedicated interfaces for integrating with relevant laboratory instruments, control systems, statistical analysis software packages and custom enterprise solutions. it enables the modeling and tracking of complex nonlinear workflows and supports decision making in bioprocess development. the data can be analyzed in the context of upstream process development, and also be correlated to other unit operations. automation support assists the ever increasing throughput of bioprocess development operations, and the analysis of experimental data and process parameters across unit operations or even different projects. this overall integration enhances process development workflows. highlighted use cases describe the selection of the best producer clones (fig. a) , the identification of optimized media feeding strategies (fig. b) , and the comparison of clone performance across different fermentation scales (fig. c) . a special focus is on the analysis of data from micro-and bench-top bioreactors (such as the ambr ™ and dasgip™ systems) operated in parallel. these bioreactors allow for increased throughput of clone selection and process optimization studies, which in turn leads to an increase in data generation. genedata bioprocess supports integration with such systems and enables a comparison of data regardless of the instrument provider or scale. automated bioreactor data analysis allows development groups to take advantage of even richer datasets and, as data management is built-in to the system, the data can be easily tracked and associated to historical records. another focus is on cross-reactor scale comparisons. data coming from different bioreactor scales can be easily imported into the platform and analyzed to establish the best conditions for upscaling. genedata bioprocess enables the correlation of process parameters (e.g., fermentation protocols, media recipes, bioreactor control parameters), with key performance indicators of the processes (e.g., titer, qp) and the product quality attributes (e.g., aggregation, glycosylation profiles). finally, bioreactor time course data can be tracked together with clone analytics and product quality parameters, which makes the platform uniquely able to support end-to-end biopharma development. upstream bioprocesses are at particular risk of contamination from adventitious agents. the typical . μm filters used at this step protect bioreactors from bacteria and mycoplasma but offer no protection from viral contaminations. a new polyethersulfone (pes) upstream virus filter, viresolve® barrier, has demonstrated high levels of microorganism retention -full retention for bacteria and mycoplasma (> . lrv -log reduction value) and~ lrv for small viruses, such as parvoviruses. it also has improved flow and capacity as compared to virus removal filters designed for monoclonal antibody purification. given the small pore size of virus retentive filters, implementing a virus filter upstream of the bioreactor raises the question of whether critical cell culture media components are removed. therefore, it is important to evaluate the cell culture performance and protein quality attributes using virus-filtered media to ensure that filtration does not negatively impact the process. materials and methods ex-cell® cho media and corresponding feeds were processed through either viresolve® barrier filters or . μm filters (control). media composition post-filtration was evaluated by high performance liquid chromatography (hplc), inductively coupled plasma/ optical emission spectrometry (icp-oes), and nuclear magnetic resonance (nmr). recombinant cho cells were cultured in fed batch culture. cell density and viability were measured by vi-cell tm cell viability analyzer while metabolites were analyzed by bioprofile® flex analyzer. shake flasks and bioreactors were utilized to verify that surfactants, such as poloxamer, (which are essential for shear protection in stirred tank bioreactors and can be difficult to filter) have not been removed during filtration. monoclonal antibody titer was quantitated by protein a hplc. characterization of the antibody product quality was assessed via weak cation-exchange chromatography (charge heterogeneity), size exclusion chromatography (aggregate profile), and -ab fluorescent labeling with np-uplc (glycan species). media and feed compositions were unaffected by filtration through the virus barrier filter. no significant differences in concentrations were observed with icp-oes (trace metals) or hplc (amino acids and water soluble vitamins). nmr showed no change in the organic composition of the media including poloxamer. the aromatic region with vitamin and amino acid signals is shown (fig. a) . cell cultures showed no differences in cell growth or titer, in either shake flasks or bioreactors (fig. b) . cell viability was unaffected, metabolite levels were within limits, and titer was consistent. the protein quality of the secreted antibodies showed no differences in the glycosylation pattern (fig. c) , amount of aggregates or charge variants. the risk of virus contamination in the bioreactor remains a concern for biotherapeutic manufacturers as there is no universal technology that provides a reliable, cost effective solution for virus removal that can be applied to all components of cell culture media. this study evaluated the viresolve® barrier filter that provides an efficient and easy way to protect bioprocesses from adventitious virus contamination. study results demonstrated that media and feed compositions, cell culture performance, and product quality were unaffected by filtration through the viresolve® barrier filter. implementation of vire-solve® barrier filters provides efficient filtration performance, high virus retention, and minimal cell culture impact and offers a viable option to improve the overall virus risk mitigation strategy for the manufacture of biotherapeutics. b tracking of process conditions together with online and offline performance analytics. the system allows to flexibly define tracked parameters and select optimal process conditions. c comparison of process performance across different reactor scales. the open architecture makes genedata bioprocess a provider agnostic system which allows to aggregate and compare data regardless of provider background bi-and multi-specific antibodies, antibody-cytokine fusion proteins, nonimmunoglobulin scaffolds, chimeric antigen receptors (cars), engineered t-cell receptors (tcrs) and tcr-based bispecific constructs can provide significant advantages for use in cancer immunotherapy. however, as highly engineered molecules they pose new challenges in design, engineering, cloning, expression, purification, and analytics. we have thus implemented an infrastructure that addresses these challenges and enables the industrialization of these various novel therapeutic platforms. in close collaboration with leading biopharmaceutical companies, we implemented a workflow, data management and analysis support system, genedata biologics™, enabling the automated design, screening, and expression of large panels of therapeutic candidates using these novel technologies. we have also built tools for developability and manufacturability assessments of these complex molecules. we have ensured that there is a seamless integration of all data generated and that functionalities such as bulk protein and vector generation using our in silico cloning engine, configurable library of template vectors and cloning strategies, fully annotated in silico protein molecules and dna constructs, and dna synthesis verification support, can be used for the newest protein formats and molecule topologies. we implemented data structures and data handling systems, which mirror how these complex next-generation biologics molecules and cell lines are being designed, screened, and analyzed. the result successfully addresses workflows for tcr optimization and engineering. we exemplified this with the generation and evaluation of a panel of engineered tcrs with an alpha chain cdr randomization and successfully supported the analysis and selection of beneficial mutations. the system also successfully supported workflows for the design and generation of a panel of tcr-based bispecifics (tcr coupled with anti-cd ) using automated molecule registration and in silico cloning tools and subsequent capture of expression, purification, and functional and analytical characterization data. on the car-t cell front, the system is able to provide traceability of the work from antibody generation, optimization, car engineering (e.g., attachment to the scfv with cd -zeta and co-stimulatory domains to mimic the natural tcr complex) to the engineering of the t-cell. the genedata biologics platform successfully enabled automation, increased data integrity and traceability during research and development work, and will contribute towards the industrialization of these very exciting novel approaches for cancer immunotherapy. optimal selection of therapeutic antibodies and production cell lines by assessment of critical quality attributes and developability background the increasing cost of bringing a new drug to the market has put significant pressure on biopharma organizations. to increase efficiency in r&d processes and reduce costs, organizations need to evaluate potential drug candidates earlier in the r&d process, eliminate those with undesirable characteristics, and focus on the most promising candidates. after designing and thorough testing of successful candidates, efficient production of new biological entities in mammalian cell lines is necessary. the main goal here is to find a suitable cell line and optimal upstream and downstream processing conditions that not only lead to a satisfactory product yield, but also to a product with the desired biochemical properties. the evaluation of production cell lines, processes, and product quality attributes is performed earlier and in higher throughput for an increasing number of drug candidates. in addition, new methods in molecular and cell biology (e.g., novel genome engineering approaches such as crispr/cas ), in analytics [e.g., process analytical techniques (pats)], in process miniaturization, and in automation promise to make process development more efficient. however, the management and analysis of the increasing amount of experimental data during candidate selection and cell line and process development has become a bottleneck. in addition, quality-compromising steps in biopharma organizations can negatively impact the cost of goods and substantially prolong the drug candidate's time to market. therefore, systems for integrated management and analysis of wellstructured and curated data that comprehensively integrate molecule and sample information, manufacturing process parameters, and process and product quality attributes are needed. critical quality attribute (cqa) assessment should be enabled along the whole bioprocess development workflow, including cell line development, upstream and downstream process development, as well as analytical and formulation development. we have developed a comprehensive platform, genedata bioprocess™, which supports drug candidate developability and manufacturability assessment and bioprocess development. the platform captures and structures the cell line and process parameters together with analytical data for cell lines, processes, and protein products. the protein analytical data being tracked include biological data (such as bioactivity, immunogenicity), and physicochemical properties. these properties include glycosylation, chemical liabilities (such as deamidation and oxidation), aggregation, stability under different conditions (low ph, low and high temperature), solubility, and impurities. genedata bioprocess™ simplifies and streamlines laborious, manual process and supports tools for molecule, clone and process selection. furthermore, the platform allows for seamless integration with laboratory instruments, statistical software packages, and custom solutions. here, we present use cases showing how to identify and annotate liability sites prone to chemical modifications (fig. a) and how to monitor cqas of molecules allowing to assess developability more efficiently. we show how the analytical data generated in the course of a developability assessment are compiled to select the best drug candidate (fig. b) . implemented traffic-light systems indicate where molecules harbor issues such as in case of the antibody tpp- , which is compromised by low temperature and repeated freeze-thaw operations. the same assessment views can also be applied on batches and cell lines. the underlying data can be visualized graphically. as an example, we show glycan types of products obtained from different cell line clones generated in a cell line development campaign for the molecule tpp- (fig. c) . even though the selected clone cli- meets the glycosylation criteria (e. g., < % afucosylation, < % galactosylation, < % sialylation), the produced next-generation biologics molecules are composed from a number of specific subdomains. each type of molecule is composed of a specific set of domains, which must be mirrored in the registration and further research and development workflow. molecule registration and hit-selection using data from a number of assays is shown here using the example of car-t cells. the image is a screenshot from the genedata biologics™ software molecule harbors some stability issues as mentioned above. therefore, more attempts would be needed either in formulation or in reengineering of the complimentarity determining regions (cdrs) in order to provide a developable ttp- -like drug candidate. background environmental process variables are often used as tools to optimize the performance of mammalian cell cultures to achieve higher cell densities and high productivities of r-proteins (q p ). the manipulation of culture temperature in the range of mild hypothermia (mh) ( - °c) [ , ] , as well as different glucose availability scenarios [ , ] , has been shown to improve productivity in different cell lines. however, the manipulation of these variables individually or together has a concomitant effect on the rate at which cells grow, masking the net response exhibited by the cells. in order to identify the effects of these variables, we have taken advantage of the use of the chemostat culture. chemostat cultures were performed at two dilution rate (d)( . or . (h- )), two temperatures ( or °c) and three feed glucose concentrations ( , or mm). the response was analysed considering r-protein production, cell growth and key metabolites. r-tpa protein concentration was determined by immunoassay (trinilize tpa kit); cells were counted using a hemocytometer and cell viability was determined by the method of exclusion using trypan blue (t , sigma, usa); glucose, lactate and glutamate were determined by enzymatic assay using a biochemical analyser ysi (yellow spring instruments). statistical analysis of the results was performed by anova (design-expert for windows). a decrease in cell density was observed in response to an increase of glucose feeding concentration, regardless of temperature or specific growth rate (in this case μ=d) evaluated. the maximum cell densities were reached at mm, achieving . and . x cells/ml at / °c and . (h - ); and . and . x cells/ml at / °c and . (h - ) respectively (fig. a) . the increase in glucose concentration from to mm resulted in an q p increase of and . fold at °c/ . (h - ) and °c/ . (h - ) respectively. a lower increase of . and . fold was reached at °c/ . (h - ) and °c/ . (h - ) respectively (fig. b) . the highest q p s were reached at °c and . (h - ). however, a positive effect of mh was not observed, in contrast to that observed in batch culture [ , , ] . this behaviour suggests that low μ is a main factor on increased r-protein production in batch cultures exposed at mh condition. the specific consumption rate of glucose was significantly increased by the glucose increase from to mm and reduced by mh (fig. c) . at . (h - ) the specific production rate of lactate (q lac ) was increased by glucose increase, independent of the culture temperature used. while at °c/ . (h - ) the q lac decreased with increasing glucose concentration and at °c/ . (h - ) a maximum consumption was observed at mm glucose (fig. d) . the lactate-glucose yield ( fig. e ) not showed relevant changes at . (h - ), while at . (h - ) this yield showed a more efficient utilization of glucose, as glucose concentration was increased. however, this last behaviour was not reflected in an increase of r-protein production. the concentration of glucose has the greatest impact on the behaviour of the culture, and its increase affects positively the protein productivity. the mh did not improve proteins productivity of cho cells producing tpa under the different conditions evaluated; low dilution rate and at high glucose concentration impact positively the protein productivity and the metabolism exhibited by the cells. background mammalian cell cultures are the most commonly used bioprocess for the production of therapeutic recombinant proteins such as monoclonal antibodies (mabs). facing to the increasing demand of these biopharmaceuticals, the fda has initiated the process analytical technology (pat) framework in order to encourage pharmaceutical industries to use innovative technologies to monitor in real time the critical process parameters (cpps), and to ensure the final product quality [ ] . one of the most important cpps for cell culture bioprocesses is the specific growth rate (μ), which is a direct indicator of cellular physiological state. indeed, μ is sensible to culture conditions and its value decreases when cells are in the unfavourable environment for growth [ ] , which may greatly influence mab production and quality. however, until this day, the online monitoring of μ remains a great challenge for mammalian cell culture bioprocesses. igg-producing cho cells were cultured in l stirred bioreactors equipped with an in situ dielectric spectroscopy (hamilton). operating conditions were fixed at rpm, % of air saturation, ph . and °c. permittivity of cell culture was measured every min, which allowed to calculate in real time the vcd by using a previously established linear correlation. then, a model of online estimation of μ was developed based on vcd prediction and cell mass balance equations. several signal noise filters and various calculation methods were evaluated to reach better model stability. cell cultures were performed in both batch and feed-harvest modes. feed-harvest cultures consisted of sequential renewals of / volume of the culture medium by following different strategies. this study proposed an innovative methodology based on dielectric spectroscopy to monitor in real time the cellular physiological state, by online estimating the specific growth rate (μ) of cells. model of online estimation of μ was developed from cultures in batch mode, and was validated by comparing online estimated μ with the experimental ones calculated at the end of the culture. with this model, the moment when μ started to decrease significantly, which indicated that cells were no longer in the exponential growth phase, was identified as the critical moment. to demonstrate the interest of online estimation of μ, the developed model was applied to a feedharvest culture, where the medium renewals were performed at the critical moments indicated by the model. this culture was then compared with the traditional feed-harvest culture where medium renewals were performed by following offline measurements of glucose and glutamine. we found that the online strategy allowed to maintain the value of μ by renewing the medium at the right time, while the values of μ varied a lot when using offline strategy. moreover, by using the online estimation of μ, the glycosylation of igg was kept at a high level (about %) throughout the whole culture. however, for the culture using offline strategy, the glycosylation level decreased progressively and was only about % at the end of the culture (fig. ) . model of online estimation of μ was developed by using dielectric spectroscopy, which allowed to monitor the physiological state of cells in cell culture bioprocesses. implementation of this model in feed-harvest cell culture led to better mab glycosylation, which demonstrates clearly the potential of this methodology in mab production bioprocesses. background monoclonal antibodies are normally synthesised from transfected mammalian cells as heterogeneous mixtures of glycoforms [ ] . however, clinical efficacy may depend upon single glycoforms which have been difficult to isolate [ ] . we have now developed an efficient method for generating single glycoforms by solid phase re-modelling which is superior to previous methods because it allows a sequential series of enzymatic changes without the need for intermediate purification of the antibody. solidphase binding exposes the antibody glycans to enable easier access of the transforming glycosylation enzyme. the antibodies subjected to modification were a chimeric human/ camelid monoclonal antibody (eg ), a humanized monoclonal antibody (il ), a full size chimeric antibody (cetuximab) and polyclonal antibodies obtained from pooled human serum. the antibodies were bound to a protein a column using conditions typical of mab purification (fig. ). after washing out non-bound impurities by a neutral ph buffer, each antibody was subjected to enzymatic modification directed to a targeted glycan profile ( table ). the antibodies were then eluted with a low ph buffer and neutralized. the glycan profiles were analysed following glycan removal with pngase f, labelling with -aminobenzamide and separation on a hilic-hplc column [ ] . prior to enzymatic modification glycan analysis of all antibodies showed variable galactosylation and sialylation typical of human abs. this included a distribution of fg , fg , fg , fs and fs with galactosylation indices ranging from . for il to . for eg . there was minimal sialylation in il but up to % in eg . glycan modifications were made as each antibody was held on a protein a column in accordance with procedures shown in table . agalactosylated glycans were enriched by treatment with the single addition of galactosidase and neuraminidase. this resulted in - % of agalactosylated structures in the mabs and % in the polyclonal antibody. galactosylated antibodies (> % yield) were produced by a single stage reaction involving sialidase and by galactosyltransferase with udp-gal. breakdown of the glycans to a trimannosyl core was accomplished by treatment of the agalactosylated structures with hexosaminidase. this produced a yield of - % of the fm structure with a small remainder of fa . sialylated antibodies (> %) were produced by a stage reaction involving sialidase, galactosyltransferase and finally treatment with , sialyltransferase in the presence of cmp-nana. the latter reaction produced equimolar quantities of monosialylated and disialylated cetuximab and polyclonal antibodies. the results suggest that for human antibodies ( kda) there may be a limitation for sialylation given the steric constraints between the two ch domains of the dimeric structure. the ability to sialylate the smaller camelid antibody ( kda) was greater resulting in a high (> %) level of disialylated glycans. this suggests that the steric constraints for glycosylation may be lower. these sialylated antibodies have significant potential clinical importance for their ant-inflammatory activities. we have modified the glycans of antibodies following immobilization on an affinity ligand column. this allows enzymatic transformation in a solid state that has a distinct advantage over the equivalent transformation in solution because the enzymes and buffers can be washed out on completion of the modification leaving the antibody still attached to the affinity ligand. this enables repeated rounds of an enzymatic reaction or sequential reaction steps without the need for intermediate antibody purification. the antibody can be removed eventually from the column by application of an elution buffer once all desired glycan modification have been made. since affinity ligand purification of antibodies is performed routinely as an initial step of purification after cell culture, the glycan modification can easily be incorporated into this process. the enrichment of the resulting antibody for a targeted glycoform can enhance the potential therapeutic efficacy as it is known that specific glycoforms are required for certain biological effects. [ , ] . this is mainly because microvesicles can be enriched/deprived for specific proteins, based on their functional purpose and their cellular origin. recently, microvesicles purified from the supernatant of t bladder cancer cells were reported to be enriched for bcl- and cyclin d (anti-apoptotic proteins), but deprived for bax and caspase- proteins (pro-apoptotic proteins) contributing towards immunity against programmed cell-death [ ] . however, impact of microvesicles on cho-based bioprocess has not been evaluated yet. therefore, in this investigation, we aimed to evaluate their impact on cell growth and recombinant protein production from cho cells. materials and methods cho-k cells were grown in chemically-defined protein-free culture medium (life technologies- ) in shake flask (gx- p). the different fractions of spent-media (microvesicles and microvesicle-free spent media) were collected using ultracentrifugation method [ , ] . quality of different fractions was ensured using western blotting for exosomal marker, cd (sc- ) and coomassie stained gel for loading control (fig. a) . to evaluate impact on cell growth, cells were seeded with microvesicles and microvesicle-free fraction collected from log-phase of culture and cell counts were performed by vicell using trypan-blue dye exclusion method. for impact on productivity, cell-free supernatant, collected from microvesicle-treated human igg secreting cho culture from stationary-phase of culture with respective control, was evaluated using elisa (ab ). microvesicles collected from % of media (by volume) from routine maintenance cultures compared to working volume for microvesiclesupplementation were used in each experiment. the growth of microvesicle-supplemented cultures had shorter lag-phase and achieved . fold higher maximum cell density ( . x viable cells/ml) compared to untreated standard culture ( . x viable cells/ml) and maintained higher for the remaining period of batch culture (fig. b) . however, microvesicle-free fraction did not had significant impact on growth. the viability of microvesicle-supplemented cultures, similar to microvesicle-free media supplemented, was also higher compared to standard culture suggesting potential use of microvesicles for regulating cho growth in production cultures. this could be possibily because microvesicles have already been reported to be enriched with cell growth/death-regulating proteins and hence facilitating cell growth [ , ] . we have also observed abundance of cell cycle regulators including cyclin d in microvesicle-fraction compared to microvesicle-free spent-media in our laboratory (data not shown); however, further investigation are required to prove the hypothesis. the overall productivity of human igg secreting cho cells was also observed to increase bỹ fold following supplementation of microvesicles to the culture without significantly affecting per-cell productivity. since microvesicle-supplementation facilitates cell growth, increased number of viable producer cells in the culture could be expected to be the basis of observed increase in the overall productivity of the culture [ , ] . the further work is ongoing to in-depth explore the potential of microvesicles for improving recombinant protein production from cho cells. the data indicate that microvesicles secreted from cho cells can improve cell growth and hence recombinat protein production in culture. therefore, strategies need to be developed for sterile isolation of cho microvesicles from routine maintenance cultures and their supplementation into the production culture for improving the performance of cho-based production process. the glycosylation profile of a recombinant protein is one of the most important attributes when defining product quality. producing a protein with desired characteristics requires the ability to modify and target specific glycosylation profiles. traditionally the approach to modify the glycosylation profile of a protein involves supplementing a culture with components that can improve galactosylation. experimentation using this supplemental approach resulted in a dramatic increase in terminal galactosylation, but lacked the ability to easily and repeatedly target specific glycosylation profiles. using novel and proprietary technology, we have developed a feed (glycantune™) and a unique feeding process that will maximize growth and titer while being able to modulate glycan profiles. this new feed can be added as a standalone process that can result in a significant shift from g f to g f and g f (maximum galactosylation). using a unique fed-batch process, glycantune can also be used with a standard feed to dial in targeted glycosylation profiles. through process development, we created a method where a transition point is used to switch from a standard feed to a glycan modulating feed. the timing of the transition point will determine the specificity of the glycan profile. ) . n-linked glycans were digested with pngase f and quantified using pmole maltohexose/maltopentose internal standards labeled with -aminopyrene- , , -trisulfonic acid (atps) as described by laroy et al [ ] or the user guide for the glycan labeling and analysis kit (glycanassure™ user guide, thermo fisher scientific). all ce separations were performed using the applied biosystems™ xl. the timing of transition from efc+ to gtc+ made it possible to target specific glycosylation profiles. modulating g f from % down to %, while increasing g f and increasing g f (fig. ) . transitioning to gtc+ early in culture resulted in a greater shift from g f to g f and g f. transitioning midway or late in culture resulted in a greater proportion of g f compared to g f and g f. supplementation based approaches using glycosylation modulating media components to modify and target specific glycosylation profiles proved to be difficult. these approaches were able to increase terminal galactosylation (g f and g f), but lacked the ability to fine tune glycan profiles. this could result in numerous rounds of titration experiments to target specific glycan profiles that would likely remain inconsistent between cell lines, culture media and feeds, and process scale. the development of a unique process made it possible to predictably target specific glycosylation profiles. transition from standard feeding to glycantune allowed for precise targeting of glycan profiles. transition to glycantune early in culture resulted in an increased shift from g f to g f and g f. a transition late in culture resulted in increased g f and decreased g f and g f. growth performance during precultures and batch curves in plain shaking flasks did not show any differences among tested surfactants or lots thereof, and cell densities reached - · cells/ ml ( fig. a and b) . experiments with hek -f cells at elevated power input in baffled shaking flasks revealed distinct differences between pluronic® f- , f- and kolliphor® p , with f- showing the best performance. peak viable cell densities reached with lots a and b of pluronic® f- and f- were comparable to those in plain shaking flasks, while those for kolliphor® p and lots c and d of pluronic® f- were significantly lower. peak viale cell densities were of - · cells/ml (fig. c) . similar transient transfection efficiency and mean fluorescence of transfected cells independent of applied surfactant and lot thereof indicated no major impact of respective poloxamer (fig. d) . interestingly, experiments using fluorescein-labelled pluronic® showed a time-dependent uptake into hek cells. visual tracking revealed an endocytic uptake of poloxamers by the cells (> fold increase in signal after h) and its co-localisation with cell membrane and lysosomes. sec (fig. e) analyses showed differences between the tested poloxamers. especially tested lots of pluronic® f- revealed notable deviations in the low molecular weight fraction (peak , fig. e ), compared to the other poloxamers. cultures subjected to varying levels of shear stress showed distinct growth differences depending on used poloxamer. while experiments in plain shake flasks did not show any differences in growth, cultivations under elevated shear stress in baffled shake flasks resulted in lower peak viable cell densities with kolliphor® p and some pluronic® f- lots. it remaines unclear whether this can be explained by different membrane protective activities alone, or if other mechanisms, occuring during and after cellular uptake, contribute to this effect. especially for the tested lots of pluronic® f- , sec of surfactants showed differences in the low molecular weight fraction. this fraction mainly represents polyethylen oxide (peo) (revealed by nmr), which is likely to be a remnant from synthesis. these observations indicate that the use of different poloxamers and lots thereof should be carefully evaluated, especially under elevated shear stress. further experiments will focus on investigating distinct sec fractions of poloxamers. overcoming (fig. ) . aurintricarboxylic acid (endonuclease inhibitor; enhancer used in e.g. salivary gland transfection) and polyvinylpyrrolidone (polymer; beneficial in electroporation) were both found to negatively impact peimediated transfection of cho cells, while another tested polymer enhanced growth as well as transfection efficiency. the use of a strong chelator led to a high transfection efficiency, but impaired cell growth. based on the results of the independent substance testings, the medium formulation was modified by the addition of a weak chelator and further components including vitamins. different osmolalities between mosmol/kg and mosmol/kg were tested for the final formulation, but no major impact was seen neither on transfection efficiency nor on viability days post-transfection. the final cho tf medium formulation supported high cell growth of finally tested cho cell lines and with peak viable cell densities above ⋅ cells/ml in batch cultivations with an overall cultivation time of - days (fig. ) . further improvements of the process might be achieved by adapting the protocol, as the results shown are based on a simple precomplexing of dna-pei. moreover, product yields could potentially be increased by using feeds, temperature shifts or commonly used enhancers (e.g. valproic acids). scaling of a cell culture process is an essential part in its development. in a typical approach scaling [ ] is performed by keeping a (critical) process parameter constant throughout the complete bioreactor range. this can lead to non-beneficial results either on the high or the low end of the range. for instance, the specific power input [p/v] of w/m might result in a good agitation in production scale whereas it leads to a nonturbulent mixing behavior in process development scale. to overcome this issue a new approach for an easy scaling procedure was developed. this "utility function" approach for agitation scaling is based on individual functions with a value-based mapping independent of bioreactor scale. process insight information (established either from doe process investigation or existing experience with a process platform) is directly formalized into a set of mappings which transform bioprocess values into perceived benefits ( to ). at each bioreactor scale, parameters (e.g. stirring and gassing) are then chosen to maximize the product of resultant utility functions. the model cho fed-batch process in this trial comprised a cho dg cell line that was transfected to produce a humanized antibody igg . a chemically defined media system was used. the process, including cell line, medium and feeding strategy was designed and developed by sartorius stedim cellca. the aim of the gassing scale-up was to achieve similar cell densities when the addition of pure oxygen starts. for all flexsafe str® bags oxygen was sparged via the micro sparger part of the combi sparger. all other systems used a ring sparger with holes face up. the initial air flow rate was set to an oxygen transfer rate (k l a) of /h at the corresponding agitation rate and volume. all process engineering characterization parameters were determined according to dechema guidelines [ ] . with the use of the utility functions the discrete agitation rate was determined (table ) . the utility functions led to discrete agitation rates where not only homogeneous mixing but also a turbulent flow pattern and a suitable specific power input was guaranteed. the initial gassing rate of air supplied enough oxygen for x cells/ml in all bioreactors. due to the used scaling methods the growth patterns in all bioreactor scales were comparable. peak viable cell densities (vcd) of - x cells/ml were achieved and viability at the point of harvest was above % in all scales. the final product concentration was in an acceptable range of . - . g/l. product quality attributes show comparability over the complete bioreactor range (fig. ) . the harvest criteria of days gave a combination of viability and product concentration that made it easy to process the cell broth during cell removal and other downstream steps. the process implementation of the cho production system -expressing mab was successfully performed with the use of utility functions. cell growth, productivity and product quality is comparable over the complete bioreactor range. background endoplasmic reticulum (er), the central part of the secretory pathways in eukaryotic cells, is responsible for controlling the quality of secreted and resident proteins through the regulation of protein translocation, protein folding, and early post-translational modifications [ ] . a number of physiological conditions such as oxidative stress, hypoglycemia, acidosis, and thermal instability can disturb the er functions, which triggers er stress [ ] . prolonged er stress induces apoptotic cell death [ ] . oxidative stress that naturally accumulates in the er as a result of mitochondrial energy metabolism and protein synthesis can disturb the er function [ ] . because er has a responsibility on the protein synthesis and quality control of the secreted proteins, er homeostasis has to be well maintained. when h o , an oxidative stress inducer, was added to recombinant chinese hamster ovary (rcho) cell cultures, it reduced cell growth, monoclonal antibody (mab) production, and galactosylated form of mab in a dose-dependent manner. antioxidants can reduce the oxidative stress level and suppress the apoptotic cell death by scavenging oxygen free radicals, inhibiting chain reaction of oxidation, and detoxifying peroxide [ ] . however, despite the importance of mass production of mabs, studies on the effect of antioxidants on the production and quality of mabs in rcho cell cultures have not been fully substantiated. to find a more effective antioxidant in rcho cell cultures, six different antioxidants including baicalein, which have used widely in mammalian cell cultures, were evaluated as chemical supplements with two different rcho cell lines producing the same mab in -well plates. then, batch and fed-batch cultures were performed in shake flasks with the supplementation of baicalein, which showed the best effect on culture performance among the antioxidants. the reactive oxygen species (ros) and er stress levels were measured to study the effect of baicalein on mab production and quality. among these antioxidants, baicalein showed the best mab production performance. addition of baicalein significantly reduced the expression level of bip and chop along with reduced ros level, suggesting oxidative stress accumulated in the cells can be relieved using baicalein. as a result, addition of baicalein in batch cultures resulted in . - . -fold increase in the maximum mab concentration (mmc), while maintaining the galactosylation of mab ( fig. and table ). likewise, addition of baicalein in fed-batch culture resulted in . -fold increase in the mmc while maintaining the galactosylation of mab. oxidative stress negatively affected the production and galactosylation of mab in rcho cell cultures. among the various antioxidants tested in this study, baicalein showed the best mab production performance in both batch and fed-batch cultures of rcho cells. baicalein addition significantly enhanced mab production while maintaining galactosylated forms of mab. thus, baicalein is an effective antioxidant for use in rcho cell cultures for improved mab production. background the production of many biopharmaceuticals (e.g. antibodies & proteins for diagnostic and therapeutic purposes) requires the cultivation of mammalian cell lines, which is demanding with respect to various aspects such as complex cell metabolism, variabilities in cell behavior, scale dependencies, influences of changes in cultivation conditions, medium composition etc. although an increasing number of measurement parameters is available, only a part of them is routinely utilized in industrial cell culture processes and their corresponding seed trains. nevertheless, the data base grows, statistical investigation of data gains importance and process data are more easily accessible in the context of industry . . cell cultivation has to consider these complex requirements, e.g. for fed-batch control and seed train design. furthermore, cultivation strategies have to be adapted to new products, cell lines and clones as well as to different production plants when transferring processes. one approach to encounter the variabilities and to include actual information from the process and from data analysis is adaptive model-assisted control [ ] . two software tools enabling adaptive model-assisted control applying unstructured, unsegregated models have been developed and implemented using matlab © , winers and fortran, one tool for fedbatch control and another one for seed train simulation and optimization. one key element of adaptive model-assisted control is the underlying process model. in order to provide an adaptive character, model parameters should be easily identifiable from routine cultivation data, which is available during seed train and fed-batch without additional sophisticated measurements. therefore, the usage of unstructured, unsegregated models is recommended. a) example of an unstructured, unsegregated cell culture model (for adaptive model-assisted control) one example, describing cell growth, cell death, uptake of substrates and production of metabolites via a first order system of ordinary differential equations and monod-type kinetics, is shown in table . this mathematical model includes cell specific model parameters [ ] . ii) b) open-loop control sequence for seed train simulation and optimization [ ] : using model, a priori identified model parameters and starting concentration values, the temporal concentration courses can be predicted for the first scale. subsequently, points in time for passaging and starting values for the next scale can be computed by adding a passaging strategy, seed train conditions and medium concentrations. prediction for the following scales can be obtained iteratively. integrating feedback from the process in terms of cultivation data enables increasing prediction accuracy and responding to possible changes in cell behaviour. process design and optimization, e.g. regarding seed train and fed-batch, is realized by adaptive model-assisted software tools using unstructured, unsegregated models. they enable feedback from the process via routine cultivation data and allow adaptation to diverse circumstances such as different cell lines, products, cultivation conditions, plant configurations etc. ) in polyelectrolyte capsules. significant advantages, such as great mechanical stability, good biocompatibility and good mass transfer properties characterized these capsules based on sodium cellulose sulfate/poly(diallyldimethyl) ammonium chloride (scs/pdadmac) [ , ] . here, we present the possibility to cultivate human t cells, freshly isolated from blood, to high densities in similar semipermeable polyelectrolyte microcapsules within less than days. cells were encapsulated in semipermeable scs/pdadmac polyelectrolyte microcapsules or confined in . % alginate/poly-l-lysine (pll) beads, a standard approach for cell immobilization. the permeability of the microcapsules was estimated using dextran-based molecular weight standards ( and kda) and vitamin b ( . kda). gentle digestion with endocellulase allows an easy release of the cells out of the capsules. cell growth, cytokines production and phenotype were measured in non-encapsulated and encapsulated cells grown under standard culture conditions. moreover, we analyzed the interplay between the secreted cytokines and the scs within the capsules and its putative influence on cell growth. cells mixed in the cellulose sulfate solution under physiological conditions can be safely trapped within a liquid core during capsule formation. encapsulated cells can reached cell densities ≤ x cells ml capsule - , whereas cells confined in alginate/pll beads and non-encapsulated ones reached . x cells ml bead - and . x cells ml, respectively. one major advantage of these polyelectrolyte microcapsules (< mm) is the low mwco (< kda) (fig. a-b) . this restricted permeability allows for a conditioning of the capsule core by autocrine factors, which in turn permits the use of basal cell culture medium instead of expensive t cell specialized media, hence does not necessitate high amounts of rhil- and reduces the cultivation costs. moreover, co-encapsulation of rhil- had a beneficial effect on the growth kinetics in most cases (fig. c) . some evidence is presented that the scs used to form the polyelectrolyte microcapsules, specifically adsorbs il- (table ) -a cytokine which provides an essential signal for t-cell proliferation and differentiation [ ] . therefore, we postulate that the scs used for encapsulation has biomimetic properties, creating an artificial extracellular matrix mimicking heparin sulfate which in turn positively affect t cell proliferation via trans-presentation of il- (fig. d) [ ] . primary t lymphocytes can be expanded under appropriate conditions outside the body. in the latter, t cells grow/expand in specific environments where the cells are tightly packed, leading to multiple cell-cell contacts and manifold interactions with the extracellular matrix. ex vivo suspension cultures of diluted cells cannot provide such a microenvironment. in the microcapsulesbased cultivation system presented, the cells are suspended in a viscous scs-solution. the low molecular weight cut off of the surrounding polyelectrolyte membrane assures that typical signaling molecules produced by the cells are retained thus facilitates the "conditioning" of the cellular microenvironment, while nutrients and metabolites can pass. expensive additives, such as interleukin- (il- ), can be co-encapsulated. expansion then no longer requires specialized t-cell media. moreover, the scs seems to have biomimetic properties, representing an artificial extracellular matrix mimicking heparin sulfate. we consider that the described method may be an appropriate alternative to expand t cells while creating a local microenvironment mimicking in vivo conditions. - ) . equations of balances and kinetics of an employed process model including x v viable cell density, x t total cell density, μ cell-specific growth rate, μ d cell-specific death rate, t time, k s and k monod kinetic constant and monod constant for uptake, k lys cell lysis constant, q cell-specific uptake rate or production rate, respectively, y kinetic production constant, c concentration, glc glucose, gln glutamine, lac lactate, amm ammonia, f feed rate, v volume balances with fed-batch terms kinetics ;uptake if c glc ≥ . mm : q lac,uptake = if c glc < . mm : q lac,uptake = q lac,uptake,max q amm = y amm/gln • q gln background digital manufacturing (dm) is heightening the productivity and robustness of existing processes and facilities. it also enables the efficient development of previously unmanageable products or processes and provided the basis for a wave of innovations. dm is a resident and on-line source of continuous optimization of process performance. it relies upon the comprehensive, real-time interfacing of both human and machine sourced information through one centralized system. more than legacy distributed control system (dcs) and supervisory control and data acquisition (scada), it is an integral interconnection of real-time access to divergent sources of information. as such, it can promise deep analysis and predictions leading to shortened product cycle and advanced process control. this comprehensive analysis is extending beyond operations performance data from the production floor to data driving such activities as raw materials security of supply (sos) and business continuity management systems (bcms). digital biomanufacturing (db) can be viewed as yet another, larger, embodiment of digital biotechnology. db is similar to digital manufacturing in that it promotes innovations in the manufacturing of biologicals by using such things as computer aided design, manufacture, verification and deep process analysis using software sensors (fig. ) . however, the fact that there are living components (cells) involved in the processes puts a distinctly different flavor to the systems employed. it is desirable to use a distinct term here to distinguish it because, as in the terms bioproduction and biopharmacology, db addresses many unique aspects of biologically-based activities. the reasons why the biotech and biopharma industry lags behind other sectors such as the automotive regarding the transformation to digital manufacturing are (i) the complexity and dissipative nature of biological systems, (ii) distributed heterogeneous data and (iii) limited at-line or on-line data sources. however, the costs of genomic sequencing, omics data generation, and computing resources are decreasing rapidly, and at the same time process analytical technologies, computational power and predictive modeling as well as data management infrastructures are greatly improving (table ) . by removing roadblocks that used to limit approaches, these changes have paved the way to transforming the bioeconomy into an industry that is based on digital knowledge. such new and optimized manufacturing technologies as continuous biomanufacturing and d bioprinting can actually demand the interfacing of many sources of information, deep data prior to elisa, the various proteins were incubated at °c in scs prepared as for encapsulation. as control, the scs was replaced by pbs. shown are mean values ± sd, n = analysis including software sensors for metabolic fluxes, and model-based predictions of digital biomanufacturing. the application of predictive models for bioprocess optimization greatly improves established platforms and finally leads to a massively increased mechanistic process understanding. four essential benefits result from the increased bioprocess understanding, development, and control of db. first, personnel are relieved of many manual and repetitive tasks. second, strategic planning and operational efficiency are improved. third, we see real-time optimization of end-to-end manufacturing based on such high-value criteria as projected product quality and profitability. fourth, it enables previously unmanageable operations and creates innovative solutions. monitoring between-batch behavior of real-time adjusted cellculture parameters xavier lories, jean-françois michiels arlenda, mont-saint-guibert, , belgium correspondence: xavier lories (xavier.lories@arlenda.com) bmc proceedings , (suppl ):p- background cell-culture parameters (ccp), such as ph, may be continuously measured online and subject to real-time automated adjustment (e.g. automated addition of a base to prevent the ph to drop too low). this is an efficient method to maintain the parameter within specified limits. this type of control constraint the variability within the predefined limits and does not provide any information on the between-batch variability of the process. online measurements of ccp provide time-dependent curves presenting one or more transitions. different types of transition can be observed: -the process can shift from a state in which adjustment is needed to keep the ccp in range to a state in which it is not. typically, the ccp drifts away from a limit. -the process shifts from a state in which adjustment is not needed to one in which it is. for instance, a drifting ccp reaches the lower or upper limit of the accepted range. the timepoints at which those transitions take place are here called changepoints. those are aspects of the process and, as such, should be controlled. in the multiple changepoints cases, the approach allows the early termination of runs showing very early or very late first changepoint. the identification of the changepoints position is based on simple rules rather than complex statistical modeling to keep the identification methodology simple. once the changepoint are identified, a multivariate bayesian model is adjusted on the appropriately transformed data. prediction regions are obtained and used as control limits [ ] . results obtained for a -changepoint case are shown on fig. . points on the right-hand graph represent new batches. the red triangle represents a failed batch. it appears that the control strategy fails to identify the failed batch. two reasons can be considered: -the limits of the prediction region have been established based on points, such a small sample size is likely to be insufficient for the definition of such a control chart. -the tested batches were produced out of set point. a control chart should be used on a stable process, ran in the same conditions, in order to be really relevant. this work was based on available historical data, which is never an ideal situation. the suggested strategy offers a simple approach to the monitoring of between-batch behavior for cell-culture. once the limits have been defined, the approach is quite straightforward and usable by nonstatistician. however, such strategy, as any other of this type, must be based on a sufficient number of batches for the definition of the control limits in order to have a good estimation of the batch-to batch variability. fig. (abstract p- ) . intelligent software applications support digital biomanufacturing process development and control. • databases using data collected online, at-line, and offline from bioprocesses operating worldwide. • process data are used to generate metabolic network models that represent a specific host cell line in a bioprocess. • modelbased computational simulations improve process understanding and reduce experimental efforts for media design, clone selection, and metabolic engineering. • automated data import and processing allow for a streamlined and standardized metabolic process analysis. • identification of critical metabolic parameters is used for proactive steering and control of production processes background rabies is a zoonotic viral disease with a mortality close to % [ ] . as there is not an efficacious treatment available, post-exposure vaccination is recommended for individuals in contact with the virus. on the other hand, the most common source of virus transmission is saliva of infected animals, mostly dogs, whereby mass vaccination of pets is the most cost-effective way to reduce human infections. in this context, availability of both human and veterinary vaccines is critical [ , ] . our group had previously developed an effective vlp-based rabies vaccine candidate produced in high density hek cell cultures with serum free medium (sfm) [ , ] . one of the aims in vaccine production process is the achievement of a good productivity with a low cost per dose, mainly in the case of vaccines for animal use in which case the sfm is one of the principal expenses. in this work, we show the adaptation of the producer clone to a non-expensive in-house developed culture medium, in order to reduce the global cost of the process and therefore the price per dose. experimental approach first, we compared a direct and a sequential adaptation protocol of our hek rv-vlps producer clone, from % of the commercial sfm (ex-cell , safc) to a new formulation with only % of the sfm and a minimum essential medium (p g), developed in our laboratory specifically for rv-vlps production. this new formulation was called rvpm (rabies vaccine production medium). the specific productivity of rv-vlps in culture supernatants was measured by sandwich elisa, using the th international standard for rabies vaccine that quantify the glycoprotein content (nibsc, expressed in elisa units per ml). further, we evaluated both media for the production of the rabies vaccine, using stirred tank bioreactors operated in continuous mode (biostat qplus, sartorius). the production of the rv-vlps was daily evaluated by elisa and the obtained harvests analysed by the nih potency test for rabies vaccine. after the adaptation process, suspension cultures without aggregates or clumps were obtained, with the same specific growth rate. a lower maximum cell density with the rvpm was reached, achieving x cells.ml - , compared with the sfm that reach cell densities between and x cells.ml - in batch mode. the specific rv-vlps productivity per cell was maintained, obtaining values of . and . eu. cells - .day - for the clone being cultured in sfm and rvpm, respectively. taking into account that this producer clone can be changed directly from one medium to the other without lag phase or cell damage, and that in rvpm the maximum cell density reached was lower, this medium was proposed to be analysed in high cell density in perfusion mode for a continuous culture in bioreactor. therefore, we performed two cultures in parallel to compare the efficacy of each media formulation in perfusion. as shown in fig. , we obtained very similar culture performances in both bioreactors; . eu.ml - and . eu.ml - of rv-vlps for the commercial sfm and rvpm, respectively. after that, the harvests were evaluated by the nih potency test obtaining a rabies vaccine potency of . iu.ml - for both cultures (being iu.ml - the minimum potency required for animal vaccine). thus, the results obtained represent an interesting advance in the optimization of this vaccine production process since the use of this new medium formulation represents a reduction of % of the total cost which will be reflected in a considerable reduction of the price of the vaccine dose. background vaccines are one of the most powerful and effective health inventions ever developedproviding tremendous economic and societal value; yet several factors hinder comprehensive immunization coverage. traditional methods of biologics production, based on stainless steel bioreactors, allow pharmaceutical companies to achieve economy of scale, but are limited by high capital expenditures. such approaches stifle manufacturing innovation and lack long-term cost-effectiveness and sustainability. current innovations can cut biologics' production costs to revolutionize the mainstream use of biologic treatments, focusing on developing fast, potent and cost-effective vaccine production. univercells' mission to make biologics affordable to all initiated a paradigm shift, targeting an innovative single-use manufacturing platform incorporating bioprocess into continuous operations. univercells employs process intensification, using high volumetric productivity bioreactors; and unit steps integration, coupling usp and dsp into continuous operations. the objective is a down-scaled high-productivity process for a cost-effective manufacturing solution. the resulting micro-facilities are easily-deployable in developing countries, breaking entry barriers to biomanufacturing (fig. ) . manufacturing and distribution advancements, from centralized to distributed, foresee affordable treatments' obtainability via supplying local populations with local production units. -bench-scale fixed-bed bioreactor; -carriers made of % pure non-woven hydrophilized pet fibers; -vero cells grown in serum-free and serum containing media; -attenuated polio strains; -cell nuclei on carriers counted by the crystal violet method; -polio virus production estimated by elisa assay (d-antigen content). cultivation of vero cells in medium with serum and in serum-free medium, was carried out in bench-scale compact fixed-bed bioreactors, to determine which culture conditions result in the highest growth rate, the highest cell biomass by carriers and virus production. cells were inoculated at . x cells/cm and infected during the mid-exponential phase, following a complete media exchange. viral infection took place in serum-free media. in-line clarification and purification is targeted to be performed in only a few steps (maximum one of two) without intermediary diafiltration. in such configuration, we measured that vero cells can reach a cell density of - x cells/cm with pdl/day of . - . in serum-containing media. this new facility is expected to manufacture any type of viral vaccine at a very low cost and could be deployed at the site of the manufacturer in emerging countries, killing the two birds of cost of manufacturing and distribution with one stone. the presentation will feature the description of the engineering development, but also the preliminary results of cell growth, infections, and product quality, as well as a description of the cogs calculation. univercells developed a disruptive polio vaccine manufacturing technology exceeding expectations when compared to traditional methodsachieving a superior result via its all-in-one solution of a simple, scalable, and fully-disposable vaccine production platform resulting in long-term cost-effectiveness, flexibility and sustainability: -all upstream, downstream and inactivation steps take place within a closed system with all the equipment contained in a low footprint isolatorcreating a confined area for polio virus handling that facilitates the deployment of micro-facilities. -this leads to a dramatic reduction in capital investment, time required for development and increases production capacity. -in conclusion, this is a simple and elegant solution for the industrial production of human vaccines at a low cost in micro-facilities, making polio vaccines available to all. comparison of media formulations for the vaccine production in l stirred tank bioreactors operated in continuous mode. both cultures were performed in parallel using the corresponding medium for the perfusion. a feeding was performed with the commercial sfm. b the first two days of perfusion feeding was performed with sfm until the cell density reach cells.ml - and, after that, the bioreactor was fed using the rvpm formulation. (↓) on day number , % of the reactor volume was punctually bled maintaining the working volume background vectored vaccines based on modified vaccinia virus ankara (mva) are reported to stably maintain large transgenes, and to be safe, immunogenic and tolerant to pre-existing immunity. mva is usually produced on primary chicken embryo fibroblasts but continuous cell lines are being investigated as more versatile substrates. we have previously reported development of a continuous suspension cell line (cr.pix) derived from the muscovy duck and efficient production process for mva in chemically defined media [ , ] . this process allowed isolation of an hitherto undescribed genotype (mva-cr ) that induced fewer syncytia in adherent cultures and replicated to higher infectious titers in the extracellular volume of suspension cultures [ ] . replication of mva-cr remained restricted predominantely to avian cells, an important property of mva vectors. homologous recombination in cr.pix cells was used to generate viruses with various expression cassettes in deletion site iii [ ] and combinations of the differentiating point mutations of mva-cr in a backbone of wildtype virus. all recombinant viruses were plaquepurified. successful introduction of the mutations was confirmed by sequencing and specifically designed restriction fragment length polymorphisms (rflps). viruses were analyzed by serial passaging, diagnostic pcrs accross deletion sites [ ] , replication kinetics, plaque phenotype and electron microscopy. the genome was further investigated by anchored pcr and long pcr. efficiency of spread of recombinant viruses (fig. a) could be mapped to a point mutation in one of the genes, a r. however, although mva-cr carries mutations in three structural proteins we detected no obvious differences to wildtype by electron microscopy (fig. b) . the replacement of the left viral telomere by the right counterpart was the most surprising result of our new study (fig. c) . this extensive rearrangement affects % of the viral genome and has also increased the area of complementarity between the two telomeres. the recombination site was precisely located and shown via analysis of earlier and subsequent passages to be a stable property of mva-cr . various viruses, including those with larger dual (dsred and gfp) expression cassettes, were serially passaged at least -fold. although the genotype of mva-cr is advantageous for replication, all genomic and genetic markers of wildtype and mva-cr were stably maintained in all passages of the recombinant viruses, independent of wildtype or mva-cr backbone. we confirmed our previous results that suggested that mva-cr replicates efficiently in single-cell suspensions and were able to connect this property with the d y mutation in a , a structural protein on the surface of the virions. mva-cr was also found to differ from wildtype mva by a recombination between left and right viral telomere. due to this event, several genes encoded at the left terminus have been deleted whereas the gene dosis of those originally encoded only at the right terminus may have increased. we do not currently know how much the various point mutations and changes in genomic structure combine to explain the improved replication of mva-cr . as several of the affected genes have been reported in the literature to impact interaction of mva with the host we would expect that in vivo studies may reveal additional novel properties of mva-cr . an extremely important distinction between our earlier study [ ] and this one concerns the source of the viruses. here, we investigated plaque-purified viruses and confirm the high genetic and genomic stability of mva. different expression cassettes inserted into deletion site iii, all diagnostic rflps and pcrs over various sites of the genome and within the viral telomeres remained unchanged throughout at least serial passages -independent of whether recombinant viruses with wildtype or cr -derived backbones were characterized. fig. (abstract p- ) . a one hallmark of mva-cr is a significantly reduced tendency to induce syncytia and an increased dispersion of plaques in cr.pix cell monolayers. this property appears to be supported by the mutation in a r. b electron microscopy reveals no obvious differences between novel genotype and wildtype. background transient gene expression systems using polyethylenimine (pei) are considered to be fast, flexible and cost-efficient for recombinant protein production [ ] . transfection efficiency depends on different factors; one of them is the type of media. production media support cell growth and protein production but not high transfection efficiency (te) mediated by pei [ ] . therefore, media were selected for transfection followed by feeding of production media [ ] to improve te and protein production. two different transfection strategies are compared: conventional transfection by preparing polyplex of a plasmid (pdna) and pei interaction before transfection and insitu transfection by direct addition both of them to the cell suspension and the polyplex formed spontaneously [ ] . cells were seeded hr in chomacs cd media before transfection. at transfection time point an equal amount of cells were resuspended in each media type. transfection was applied either insitu or conventional (polyplex prepared in μl of mm nacl and incubated for min.), media addition was performed hours post-transfection (hpt). media type and transfection condition were illustrated in table . media screen result exhibits the highest transfection efficiency of around % transfected cells by opti-mem medium coming along with low cell growth and viability. to improve the transfection efficiency, basic parameters including cell density, pdna, and pei concentrations were varied and higher transfection efficiency was reached by reducing media or accordingly increasing cell density, pei and pdna concentration for transfection. further optimization results show that the transfection of cho-k cells in opti-mem (transfection medium) for hours followed by addition of cho-macs cd (production medium) for further enhancing the transfection, cell count, and cell viability. the transfection efficiency (te) increased up to ± . % coincide with increases in viable cell concentration (vcc) in comparsion to transfection and cultivation in opti-mem media alone fig. a . both conventional and insitu methods are successfully transfected cho-k to the same similar high te as shown in fluorescence microscope images of fig. b . insitu transfection shows super-priority for suspension cell transfection concerning the reduction of handling steps (one step) compared to the conventional way (two steps). the insitu transfection avoiding the optimization step required for the incubation period to prepare transfection polyplex but require a higher amount of pdna and pei than conventional way as shown in table . in order to deal with the growing demand of large quantities of therapeutic proteins in a timely fashion, expression systems are being optimized to reduce the time of generation of stable clones as well as to increase the levels of protein secretion. this can be achieved by a combination of expression cassette optimization, cell engineering and selection process. we have previously developed the cumate gene-switch, which is a very efficient expression system for protein production [ ] . we have shown that the cumate-inducible promoter (cr ) was the strongest promoter we had tested so far in chinese hamster ovary (cho) cells. with this promoter, we were able to generate stable cho pools capable of producing high levels of a fc fusion protein ( mg/l), outperforming by to fold those generated with cmv and hybrid ef α-htlv constitutive promoters. besides the strength of the cr promoter, we demonstrated that the ability to control both the time and the level of expression during pool generation and maintenance gave a real advantage to the inducible expression system. indeed, we observed that keeping the expression off during selection enabled the generation of pools with superior productivity compared with the pools whose expression was maintained on. moreover, preliminary results suggest that keeping recombinant protein expression down increases the frequency of high producer clones [ ] . knowing that one of the main bottlenecks of the successful bioprocessing of recombinant proteins using cho cells is the rapid isolation of a high producer, our data suggest that the cumate gene-switch system could be a valuable platform for the generation of stable clones. the main regulatory authorities and organizations demand proof of monoclonality for biotechnological producer cells. with increasing pressure to shorten timelines and to improve drug safety, technologically advanced methods have to be established to ensure that production cell lines are derived from a single progenitor cell. sartorius stedim cellca's single cell cloning approach is based on one round of fluorescence-activated cell sorting (facs) using becton dickinson (bd) facsariatm fusion cell sorter combined with photodocumentation by synentec cellavista microscopic imaging system. for the approach, critical process parameters such as different cell lines, viability and cell aggregation levels were investigated separately to assess their contribution to the probability of monoclonality. immediately after single cell cloning into -well plates ( cell/ well) the plates were centrifuged followed by imaging using the cellavista (day ). further cellavista images are taken on day , day and on one day between day and . outgrowth was defined at day . cell lines expressing different recombinant products were investigated to calculate probability of having ≥ cells/well after facs sorting p(d), the apparent probability p(i) of having ghostcells (cells that are out-of-focus and, thus, are not visible during initial microscopic imaging), and the apparent probability p(k) of having ghostcells that outgrow the -well stage (fig. ). using these results, the probability of obtaining a monoclonal cell by using sartorius stedim cellca's single cell cloning approach was determined (table ) by conservative examination: p(monoclonal, conservative) = -(p(d) x p(i)) realistic examination: p(monoclonal, realistic) = -(p(d) x p(k)) cell pools with low viability can theoretically impact the probability of monoclonality by e.g. diminishing microscopic imaging quality (cell debris). therefore, pool cell line with very low viability (≥ %) was used to demonstrate, that the probability of monoclonality is still . % in case of low viability on day of sorting: p(monoclonal, conservative) = p(d) x p(i) = . % p(monoclonal, realistic) = p(d) x p(k) = . % furthermore, cell pools with high aggregation levels can theoretically impact the probability of monoclonality by sticking together during facs sorting and therefore increase the probability p(d) of having ≥ cells/droplet. therefore, pool cell line with high aggregation levels (≥ . %) was used to demonstrate, that the probability of monoclonality is still ≥ . % in case of highly aggregated cell pools on day of sorting: p(monoclonal, conservative) = p(d) x p(i) = > . % p(monoclonal, realistic) = p(d) x p(k) = > . % conclusions in summary, there is no obvious correlation between protein product type and the determined probabilities for monoclonality. furthermore, pools with a viability as low as % and pools with an aggregation level as high as . % can be used for scc resulting in acceptable probabilities of monoclonality. background ich guidance [ ] requires that any cell line used to produce biopharmaceuticals originates from a single progenitor cell. recently, there has been increased scrutiny of the method(s) used to achieve this requirement. here, we review the suitability of the legacy capillary aided cell cloning (cacc) method in light of this changing landscape of expectations. the cacc method is based on the 'spotting' technique [ ] and relies on independent visual conformation by two scientists of the presence of a single cell in a μl droplet. this method achieves a high probability of monoclonality in one cloning round. although the method has since been replaced by facs single cell deposition for routine use, it remains a viable cloning method. -performed by trained scientists -dilute culture to ± cells/ml with ≤ % doublets -draw cell suspension into pipette tip by capillary action; tap tip against the centre of the base of each well of a well plate. -size of resulting droplet =~ μl (fig. a ) -two scientists independently view all wells using a microscope (initially use x magnification with the entire rim of the droplet visible within the field-of-view. next, examine particles using x or x magnification to confirm they are cells) and individually record the number of cells present in each well's droplet (fig. b to d) . -exclude droplet from further analysis if full visualisation is hindered (fig. e to h) . -add growth medium, and incubate plates. record all wells containing colonies; only progress colonies from wells that both scientists agree contains only one cell. -data analysis: -each scientist's observations categorised as: cells, cell or > cell -observed outcome for each well: growth or no growth -probability of monoclonality estimated from data using a statistical model cloning (ldc) increased accuracy of p(monoclonality) with cacc -ldc weakness: no visualisation after seeding (to check both well seeding and subsequent growth of colonies is well described by the poisson distribution), potentially overestimating p(monoclonality) -addressed by cacc: visual examination with colonies arising from wells seeded with cell distinguished from those seeded with > cell -visualisation step further strengthened by: using controls for exclusion of wells; measuring errors based on the presence or absence of colonies in wells where two scientists independently reported cells; and formally analysing the data using a suitable statistical model decreased time and resource requirements with cacc -high p(monoclonality) possible in single round as each well examined individually with only those containing a single cell progressed, and because the error rate for incorrect scoring is considered to be low two scientists miss a cell one cell sitting on top of another and the two thus appearing as one an experiment was performed to estimate error frequency [ ] . conclusion -scientists miss a cell infrequently (in the range . % to . %, [ ] ) -error frequency does not invalidate use of direct observation methods for cell cloning -single cell seen by both scientists is highly likely to be monoclonal -during method development, strategies established to control potential sources of error ( table ) use of a contemporaneous visualisation approach, a strict control strategy, and a suitable statistical model (which takes into account potential errors) results in: -the cacc method being at least as robust as the ldc method -the cacc method being a reliable, single-step method for cloning to achieve a high p(monoclonality) background vector design is a key step in cell line development for the expression of therapeutic biologics. it is essential that the vector design results in high, stable expression of the encoded protein. other considerations include ease of cloning, stability for propagation in e. coli as well as in the mammalian host cell line, and ease of sequence amplification for verification of vector construction and for detection of insertion site and copy number in stably expressing cells. for these reasons, use of the same promoters and polya tails in dual cassette vectors, as is common for expression of the heavy and light chains of monoclonal antibodies, can be problematic. in order to minimize sequence similarities between the two expression cassettes, we have modified the promoters, introns, and polya tails of the light chain and heavy chain expression cassettes in the dual expression vector commonly used for the expression of therapeutic antibodies in the chozn® gs -/cell line development platform. gene synthesis and vector construction of igg and fluorophore-expressing vectors was done by atum. vectors were transfected into chozn® gs -/cells via electroporation. analysis of gfp and rfp expression was achieved using a macsquant instrument. selection and generation of stable pools and single cell clones from transfections with igg -encoding vectors was performed as described in the chozn® platform technical bulletin. titer analysis was performed in static ( well plate), in a day tpp assay and in a day fed batch assay using a qk fortebio. initial screening experiments identified a lead vector, # , and a vector, # , which produced very low titers and relatively few minipools expressing detectable levels of igg . analysis of gfp and rfp expression from the modified vectors indicated relatively high expression from the rfp/hc expression cassette of vector # . a stronger promoter resulting in overabundance of hc, known to be toxic to cells, provides a possible explanation for the poor results with this vector. interestingly, swapping the positions of the lc and hc in # resulted in a vector, # , that outperformed the initially identified lead vector (fig. ). this same change was made to vector # without any resulting improvement in titers (vector # , fig. ). interestingly, vector # had a smaller difference in relative promoter strengths, based on mean channel fluorescence ratio of gfp to rfp, suggesting that overabundance of hc was not an impediment to igg expression from # . poor titers were also seen with a modified version of vector # (vector # , fig. ) in which the glutamine synthethase selection cassette was in the reverse orientation. this second screen identified vector # as the lead vector design (fig. ) . a full comparative study of vector # and the control vector was performed, cumulating in the generation and comparison of single-cell clones from each. these studies have demonstrated the equivalence of these vectors in terms of igg titer. this work has resulted in the identification and characterization of a dual expression vector with minimized similarity between the two expression cassettes, easing the cloning, propagation and analysis of vector integration in stable cell lines while maintaining the high, stable expression of the encoded protein of the original vector design. background traditional cell line engineering strategies mainly include an antibiotics resistance selection. in this process, cells are transfected with the goi (gene of interest) together with an antibiotics resistance gene and those cells are selected that survive treatment with the respective antibiotic [ ] . although the gene responsible for the survival of the cell is transfected together with the goi, resistance is not necessarily linked to high goi expression. thus, a significant proportion of resistant cells may not express the goi at all, necessitating the search for alternative, more closely linked selection systems. sirnas (silencing inducing rnas) are short, noncoding rnas that can bind to complementary mrna and inhibit their translation. this function has been used in many approaches to silence the expression of certain genes [ ] . with their short length, sirnas can be hidden in introns (non-translating regions) of genes, making it possible to couple the expression of a sirna to a gene. this way a cell produces a correlating amount of sirna when transcribing the gene, without adding any further translational burden on the cell. the co-expression of the sirna can be used as a selective marker by one of the following methods: ( ) knock-down of a suicide gene to enable a cell's survival after suicide gene mrna transfection, ( ) down-regulation of a surface marker which is used in macs (magnetic cell separation) to filter out wanted or unwanted cells, and ( ) inhibition of a fluorophore marker for selection using facs without product specific antibodies. for sirna based cell selection systems, sirnas replace the commonly used antibiotics resistance as a marker. cells that produce goi will also produce the sirna that protects the cell from a suicide gene. the selection protein (suicide genes, fluorophores, surface markers, etc.) is transfected as mrna and is only expressed during selection. the general process is outlined in fig. . (a) the traditional antibiotics resistance marker is replaced by an sirna, which is cotranscribed with the goi. unlike in antibiotic resistance, the marker here is not a protein, reducing the translational burden and providing more resources for goi production [ ] . transfection with the suicide gene proved to be % lethal within days, with no outgrowth over two weeks. protection by expression of the sirna was shown to be efficient. currently a comparison of stable cell line development programs based on sirna selection and neomycin selection is ongoing. conclusions the novel selection system should speed up cell line development, as the system kills rapidly and directly selects for cells transcribing the product gene on a high level. we expect to see more high producers earlier in the process, which will allow for an easier and faster selection in the following steps. sirna based selection offers great opportunities. by directly selecting based on goi transcription and not a proxy marker, we expect more relevant cells on a pool level. in addition, the elimination of an antibiotics resistance allows more cellular resources for goi production. the system offers multiple ways of application, either by enriching wanted, or depleting unwanted cells. background single-cell cloning is an essential step used in the upstream development of transformed cell lines for therapeutic protein production. while single-cell clones are typically used to ensure product consistency, such low cell density cultures present a survival challenge; cells grow more slowly or may even not survive at low densities in protein-free media, costing the industry time and money and limiting the pool of candidate colonies for choice of production clones [ , ] . to address this problem, we aimed to develop a highly efficient serum-free medium suitable for optimising single-cell cloning efficiency by studying a range of conditioned media (cm) samples isolated from different chinese hamster ovary (cho) cell lines. materials and methods cho-s, dg and cho-k were adapted to cho-s sfm-ii (gibco) medium for a minimum of three passages. conditioned media was then collected when the cultures reached a cell density of x cells/ml (typically day -day depending on the growth profiles of each cell line and whether they grew in suspension or attached conditions). samples were then centrifuged twice to remove cell pellet/debris and stored at - °c. the ability of conditioned media to support cho colony formation was then assayed using -well plates, seeding the cells at low cell density ( - cells/well) by diluting down cho cultures in media/conditioned media. after incubation at °c for days, cloning efficiency was assayed using a standard xtt assay. initial screening of the nine cm samples was performed using cho-k cells due to their widespread use in industrial antibody production. successful media candidates were subsequently screened using additional cho cell lines. table ) . the k -sfmii-cm product improved cell cloning efficiency for dg cells (avg. increase> . -fold) and cho-s cells (avg. increase> -fold) ( fig. ) and also the adherent cho-k cell line growing in atcc + %fbs. the ability of conditioned media to support cho growth in limiting-dilution conditions ( , and cells/ml) was investigated. from a range of nine conditioned media samples; four compelling products have been identified which improve low-cell density growth of cho-k cells, compared to sfm-ii control media. we feel that these early-stage conditioned media products may increase cloning efficiencies during upstream cho cell line development, resulting in financial savings for industry and increasing the possibilities of identifying particularly highperforming transformed clones. ( ): - . the main rate-limiting step in the upstream stages of protein biomanufacture is the isolation of stable, high producing cell clones. ubiquitous chromatin opening elements (ucoe®s) consist of at least one promoter region with associated methylation-free cpg island from housekeeping genes; they possess a dominant chromatin opening capability and thus confer stable transgene expression. ucoe®-viral promoter (e.g. cmv) based plasmid vectors markedly reduce the time it takes to isolate high, stably producing cell clones. although some ucoe®-viral promoter combinations have been tested, they have not been thoroughly evaluated in chinese hamster ovary (cho) cells. plasmid vectors containing combinations of either the human hnrpa b -cbx ucoe® (a ucoe®) or murine rps ucoe® linked to different viral promoters (hcmv, gpcmv, sffv) driving expression of an egfp reporter gene were functionally analysed by stable transfection into cho-k cells and expression analysed by flow cytometry and qpcr to determine vector copy number. the results at days post-transfection and selection clearly indicate that the rps ucoe®-gpcmv and -hcmv combinations give the highest transgene expression as shown in fig. . the a ucoe®-hcmv/gpcmv constructs were the next efficacious but -fold lower than the rps ucoe® vectors. the sffv promoter linked with either of the two ucoe®s was the least effective with expression levels -fold lower than the rps -cmv constructs. the rps ucoe®-gpcmv/hcmv constructs are now being further modified to include elements that will provide optimal post-transcriptional pre-mrna processing (splicing, polyadenylation, transcription termination, mrna stability) thereby maximising stable cytoplasmic transgene mrna levels and protein production. in the last years, growing number of innovator biologics and biosimilars have formed a competitive environment, where speed and efficiency of generating robust and highly productive cell lines needs to be improved continuously. through various advances, especially in media development and process optimization, product titers as high as g/l were achieved in the pharmaceutical industry (kim et al., ) for standard products such as monoclonal antibodies. nevertheless, other proteins e.g. bispecific antibodies, fc-fusion proteins or fab-related products are difficult-to-express (dte) in chinese hamster ovary (cho) and may result in delays or even in termination of the cell line development process. we developed a new robust pool generation approach (cld . ) addressing both, easy-and difficult-to-express molecules, while reducing timelines down to months (cld standard = months), improving reliability of cell line development as well as clearly increasing obtained titers. in order to create stable cell lines, we transfected our cho dg host cells by electroporation. cells processed using the standard approach were cultivated in selective medium or medium containing additional . nm methotrexate (mtx) for three weeks. after an amplification step with nm mtx for three weeks, stable individual cell pools were expanded and clones were generated by facs-sorting. clones were analyzed for growth performance and product concentration in fed-batch studies. in our new cld . approach, we increased mtx concentrations ( . nm, nm and nm mtx) during the first selection phase of three weeks. afterwards we omitted the nm mtx amplification step. thereby, pool generation finished four weeks earlier than in the standard approach. to evaluate the stability of cell clones derived from mini pools (mps) generated according to the cld . approach, stability studies were performed for eight weeks, including stability fed batches at t= weeks and t= weeks. altogether three different proteins of interest with six cell clones each were tested. we adapted our cell line development process by increasing the initial selection during the first selection phase, thereby allowing the omission of the nm mtx amplification step. we observed that the capacity of amplifiability varied for different products. cell lines with a protein titer ranging from > g/l to . g/l (dte) in shake flask fed-batch showed to be more susceptible to increased initial mtx levels and were thus not amplifiable with nm mtx. in contrast, cell lines with high protein titer > . g/l were observed to adapt to nm mtx easily and were amplifiable. finale shake flask fed-batch data with cld . clones of highexpressing products showed comparable titers to clones from the standard approach. cld . clone titers for dte proteins revealed in average a . -fold increase compared to clones generated in the standard approach. titers of top producing clones were in a range of . g/l to to . g/l (fig. ) . furthermore, stability data of cld . cell clones from different dte products showed a stable specific productivity in a range of +/- % over eight weeks cultivation. fed-batch titer from t= weeks and t= weeks were in a normal range of +/- % of the standard nm projects. our results demonstrate that cld . is a robust and reliable process for standard products (mab) and dte proteins. with our new process, we were able to increase titer of difficult-to-express proteins up to %. by omitting the amplification step ( nm mtx) % of generated clones were stable over eight weeks cultivation time. additionally using the cld . approach, the time line from dna to rcb was reduced to months. background cho cells have become the most popular platform for production of therapeutic proteins [ ] . however, the generation of high-producer cells is a time-consuming and labor-intensive process that requires the screening of large amount of cells to get a clone of high titer and stability. since the expression titer and stability of clone is highly dependent on the site of integration, we demonstrated a new cell line development strategy by using ngs to identify the integration site and using crispr/cas to generate the target integrated high producing cell lines [ , ] . to identify the high expression sites in the cho cells, we employed ngs to analyze the integration sites of a high producing cell line (titer > g/l). the pair-end reads with one read mapped to the vector and the other read mapped to the cho reference genome are extracted to identify the integration sites. to test the expression activity of the integration sites, we employed crispr/cas to specifically integrate the antibody gene into cho genome for expression. our data showed integration sites are in the high producing cell line. among the integration site, is integration site was tested by crispr/ cas for target integration of antibody gene for expression. the is target integrated cell pool present higher expression titer than cell pool generated by target integration into other integration sites (fig. a) . the single cell clones derived from is -target integrated cell pool had low copy number of goi (fig. b) . after normalization with copy numbers, the single cell clones derived from is -target integrated cell pool showed high titer per copy ( ~ mg/l/copy) (fig. c) . this study demonstrated the generation of high-producing cell lines by crispr/cas mediated target integration. this approach will cost less time and labor than traditional method. the active integration site will serve as a platform like a cassette player for therapeutic antibody production. background cho, hek and sp / are the dominant host cells for biologics drug production. achieving high level of recombinant protein production by these cell lines still remains a challenge. in order to understand the potential roles of lipids in protein production, secretion, vesicular transport and energy metabolism, we coupled high-throughput transcriptomics and lipidomics technologies. quantitative lipidomics is an emerging 'omics technology which can help us understand the physiological limitations of each cell line. the two types of major lipid groups in cells are non-polar and polar lipids. polar lipids such as glycerophospholipids (pls) include phosphatidylethanolamine (pe), phosphatidylcholine (pc), phosphatidylinositol (pi), phosphatidylserine (ps), phosphatidylglycerol (pg), and phosphatidic acid (pa). in this study; we integrated two dimensional high performance thin layer chromatography ( d-hptlc) and mass spectrometry (ms) lipid analysis of sp / , cho, and hek cell lines to understand the major differences in the lipid content of these hosts. bligh-dyer method was used to extract the lipids and extracts were analyzed by hp-tlc and ms. the polar lipids were separated into different categories by -d hp-tlc using a chcl -meoh-h o ( : : . , v/v/v) solvent system in the first dimension and a chcl -meoh-acetic acid-h o ( : : : , v/v/v/v) solvent system in the second dimension. non-polar lipids were separated by -d hptlc using hexane-diethyl ether-acetic acid. , -dichlorofluorescein dye was used to visualize both polar and non-polar lipids. further detailed analysis was performed on a qqq mass spectrometer (thermo tsq vantage, san jose, ca) using negative-ion and positive-ion esi modes as well as negative-ion esi mode in the presence of lithium hydroxide. in this study, quantitative lipidomics was coupled with transcriptomics to further understand the physiological pathways of hek, cho-m and sp / cells. initial hp-tlc analysis indicated that major lipids in these industrial cell lines were pe and pc. other polar lipids such as pi, ps, pg, pa, and sm were lower compared to pc and pe in exponential and stationary phases of each cell line. figure represents d hp-tlc results of hek with the relative quantitation of polar lipids. in order to investigate the lipid subgroups, shotgun ms analysis was conducted for both exponential and stationary growth phases of the three cell lines. ms analysis indicated that lysophosphatidylethanolamine (lpe) and lyso-phosphatidylcholine (lpc) amounts were - fold and - fold higher in hek cells compared to sp / and cho cell lines. sphingomyelin (sm) was another lipid subgroup that was shown to have a major difference between sp / and other mammalian cell lines. sm was - fold lower in sp / cell line compared to cho and hek. to understand these metabolic differences, transcriptomics analysis using illumina highseq and gene expression omnibus was conducted on these mammalian cells. the kyoto encyclopedia of genes and genomes (kegg) database was used to map the transcriptomics data to the lipid synthetic pathways. transcriptomics data mapping to kegg pathways demonstrated that differences in lpe and lpc pathways correlate with the expression profiles of secretory phospholipase a (spla ), lysophospholipid acyltransferase (lpeat), lysophosphatidylcholine acyltransferase (lpcat), and lysophospholipase (lypla) [ ] . the hp-tlc and lc/ms findings demonstrated that high levels of lpe and lpc existed in the hek cell line and low levels of sm were observed in the sp / cell line. coupling lipidomics with transcriptomics provides us with an improved understanding of the physiological differences across sp / , cho, and hek cell lines that could be used to guide cell engineering efforts with the goal of increasing the recombinant protein expression capabilities of these three cell lines. biopharmaceuticals are a class of biological macromolecules that include antibodies and antibody derivatives, generally produced from cultured mammalian cell lines via secretion directly into the media. manufacturing at medimmune requires the generation of chinese hamster ovary (cho) clonal cell lines capable of producing the biopharmaceutical product at commercially relevant quantities with optimal product quality. the isolation of cell clones based on random single cell deposition via fluorescence activated cell sorting (facs) provides a heterogeneous panel of expressers. we hypothesize that the application of facs to provide an additional sorting step based on desirable cell attributes that correlate with productivity, product quality or cell growth attributes could lead to the isolation of higher producing cell lines with enhanced product quality attributes. a panel of cell lines expressing a model recombinant monoclonal antibody were characterised in terms of growth, productivity, intracellular recombinant protein and mrna amounts. assays were also developed to investigate cell attributes using the commercially available imagestream instrument, an imaging flow cytometer, which enables the investigation of cellular characteristics that correlate with cell productivity at the single cell level. characterisation revealed the cell lines exhibited a range of values for productivity, growth, and intracellular (ic) antibody mrna and protein expression, ideal for further imagestream characterisation. western blot and qrt-pcr analysis demonstrated that final titre correlated with both ic heavy chain (hc) protein and mrna amounts (pearson correlation coefficient (pcc) = . and pcc = . , respectively). to assess productivity at the single cell level, assays multiplexing ic hc protein and mrna with cell attributes were therefore developed. initial assay development focusing on hc mrna and protein amounts has revealed interesting results; four cell lines displayed two distinct populations, one producing the antibody and another nonexpressing population. the ratio of these populations varied amongst the cell lines. images obtained from the imagestream have shown the cellular localization and expression of hc and lc message and protein (fig. ) . for both message and protein, hc and lc colocalize in the cell. whether there is any relationship between ic hc protein and cell attributes at the single cell level was then also investigated, as well as correlations with cell culture parameters at the population level. at the population level, correlations were found between titre and ic hc protein and mrna (pcc = . and pcc = . , respectively) confirming the data obtained by western blot and qrt-pcr analysis. a panel of cell lines has been characterised at the population level and show a wide range of antibody expression profiles at both the mrna and protein level. in parallel, assays have been developed for the imagestream to measure hc and lc message and protein amounts at the single cell level. protein and message quantification with the imagestream are consistent with more traditional approaches, such as western blots and qrt-pcr, that operate at the population level. the developed assays are now being used to investigate single cell productivity attributes and for the isolation of more productive clones. background productivity and stability are key factors for the selection of cell line in protein drugs production. large amount of target gene integrated in cell genome could lead to the instability of production. therefore, cells with low copies of target gene integrated in high yield sites could be an ideal production cells for manufacturing. it has been known that the transposon system can control the integrated copy number of target gene and can generate high yield producing cells, it could be a great approach to generate stable high yield producing cell lines carrying low copies of target gene through transposon system. we intended to develop a platform to generate high yield producing cell lines carrying - copy of the integrated target gene using transposon system. two cho cell lines, cho-s cells and dxb cells, have been applied. cells were co-transfected with transposon and target gene expression plasmids. after drug selection, the cell pool with highest productivity per target gene copy was applied to single cell cloning. the productivity and copy number of cell clones were determined, and the stability of cell clones was analysed after culture of about generations. in the stable pools of cho-s and dxb cells, the productivities per integrated target gene copy were about - mg/l/copy and - mg/l/copy in a batch culture, respectively. after single cell cloning, the integrated copy numbers in most cell clones were less than three copies per cell. in cho-s and dxb cell clones, the productivities per integrated target gene copy were - mg/l/copy and - mg/l/copy in a batch culture, respectively. the productivity per integrated target gene in cell clones developed by the transposon system was much higher than that in cell clones developed by random integration (fig. a and b) . to evaluate the productivity stability of cell clones developed by the transposon system, ten cell clones at generation , , , and were applied in the analysis. of interest, about % of cell clones were stable at generation , but lost the productivity at generation (fig. c) , implying the most cell clones could maintain the stability within months. using the optimized conditions of the transposon system to develop the stable gene expression cells, the productivity per integrated target gene was higher than random integration. these results suggested that our platform is capable to develop high yield producing cells with - copy of integrated target antibody gene and can be applied to identify high yield integration sites. background mammalian cells show an inefficient metabolism characterized by high glucose uptake and the production of high amounts of lactate, a widely known growth inhibition by-product [ ] . recently, we have observed a different glucose-lactate metabolism in some cell lines. while some cell lines are unable to metabolize lactate, others can co-metabolize simultaneously glucose and lactate under certain culture conditions, even during the exponential growth phase [ ] . these metabolic differences between different mammalian cell lines (cho, hek and hybridoma) have been studied by means of flux balance analysis (fba). three different cell lines were cultured in a -liter bioreactor: cho-s, hek sf and hybridoma kb- . . for the fba, two adapted genome-scale metabolic models were used: a reconstruction of mus musculus for cho and hybridoma [ ] , and a reconstruction of human metabolic model (recon ) for hek [ ] . in cultures where ph was not controlled, two different metabolic phases were observed for cho and hek cells. during the first phase both cell lines produced large amounts of lactate as a consequence of the high glucose consumption rates. interestingly, when ph dropped below . , due to acid lactic secretion and accumulation, a second metabolic phase was identified, in which concomitant consumption of glucose and lactate was observed even during the exponential growth phase. conversely, hybridoma cells were unable to co-consume lactate and glucose simultaneously even under noncontrolled ph conditions. therefore, the hybridoma physiological data used for the fba corresponded to only phase of phcontrolled cultures. a summary of the main cell growth and metabolic parameters obtained from the different experiments performed is presented in table . fba shows ( fig. for hek cell culture) that lactate is produced in phase because pyruvate has to be converted to lactate to fulfill the nadh regeneration in the cytoplasm and only a small amount of pyruvate can be transported into tca through acetyl-coa. cell metabolism in phase is highly inefficient, as the majority of the carbon source is not used for the generation of energy nor biomass. in phase , in which mitochondrial ldh was considered, tca fluxes could be maintained as in phase at the maximal rate encountered; hence, the energy available for cells to grow was similar in both phases, obtaining similar growth rate. two different glucose and lactate metabolism behaviors have been observed in cho and hek cultures depending on the culture conditions: phase ) glucose consumption and lactate production, and phase ) glucose and lactate simultaneous consumption. in contrast, only phase was observed in hybridoma cultures even when ph was non-controlled. fba showed that tca fluxes in phase and phase were similar, obtaining similar cell growth rate, but glucose uptake rate was much lower in phase due to the lactate co-consumption. some authors hypothesize that cells metabolize extracellular lactate as a strategy for ph detoxification [ ] . glucose and lactate co-metabolization resulted in a better-balanced cell metabolism, as can be seen from the metabolic fluxes calculated, with minor effects on cell growth. the observation of glucose and lactate co-consumption metabolic behavior and its deeper study and characterization could open the door of novel culturing strategies with the aim of increasing bioprocesses productivity. background transient protein expression in mammalian cell lines has gained increasing relevance as it enables fast and flexible production of high-quality eukaryotic protein. considerable efforts have thus been made to overcome existing limiting aspects of transient gene expression systems, in terms of cell lines, cell culture-based systems, and protein production in a cost-effective manner. milligram amounts of protein per liter can be produced within several days, allowing a significant shortening of the bioproduction process in comparison to protein production from stable clones. to ensure the robustness of the process, it is essential to have a reliable and easy-to-use transfection method. to palliate for the need of a reliable transfection reagent, we developed peipro®, the only commercially available pei optimised for mid to large-scale transient protein production during process development. peipro® is a non-polydiperse and fully-characterised polymer that has become the gold pei standard due to its reliability, reproducibility in high dna delivery efficiency and in ensuing high protein production yields. here, we present experimental data showing the benefits of using peipro® for protein production in comparison to other peis. we further demonstrate compatibility of using peipro® for recombinant protein production in most commonly used chemicallydefined media. materiel and methods suspension hek- and cho cells were cultured in shaker flasks in various synthetic media, as listed in table . hek- and cho cells were resuspended at × cells/ml of serum-free medium, on the day before transfection. cells were transfected with . - mg of plasmid dna encoding for the luciferase gene reporter using peipro®, pei "max" and l-pei kda (polysciences, warrington, pa) resuspended at mg/ml according to the manufacturer's recommendation. protein expression of the luciferase reporter gene was assayed hours post-transfection by affinity chromatography using protein g (hplc). comparison of peipro® to other commercially available peis was achieved by transfecting suspension hek- and cho cells with plasmid dna encoding for the luciferase gene reporter. luciferase production yields obtained in hek- and cho cells were at least respectively -fold and -fold higher when using a similar amount of peipro in comparison to the other peis (fig. ) . furthermore, peipro® was the only pei that led to similar luciferase production yields when decreasing the amount of plasmid dna per liter of cell culture. conversely, at least mg of plasmid dna and -fold more of pei "max" and l-pei kda were needed to obtain a similar luciferase expression range in both hek- and cho cells. we further assessed the compatibility and versatility of peipro® by measuring protein production yields obtained in most commonly used animal-free synthetic media. as shown in fig. , peipro® leads to high protein production yieds in several commercially avaialble media formulations for hek- anc cho cell lines. peipro® is the only fully characterised pei transfection reagent that is suitable for reliable and reproducible recombinant protein production, irrespective of the scale of production and of the type of adherent and suspension cell culture system. fig. (abstract p- ) . peipro® requires less reagent and similar to lower dna amount compared to other peis. suspension hek- and cho cells were seeded at × cells/ml in serum free medium and transfected with peipro®, pei "max" and l-pei kda (polysciences, warrington, pa) resuspended at mg/ml. luciferase expression was assayed h after transfection using a conventional luciferase assay fig. (abstract p- ) . peipro® is optimized for transfection of hek- and cho cells in several specific synthetic culture media. suspension hek- and cho cells were seeded following the recommended protocol in serum-free media and transfected with peipro® using the standard conditions. igg -fc production was assayed h after transfection using protein g affinity quantification (hplc) monoclonal antibodies (mabs), which are widely used in anticancer therapies, are mainly produced by mammalian cell lines. mab conjugation to biological molecules for enhancing their antitumor activity offers a new powerful tool for anticancer therapies. we have assessed the production of commercially approved anti-her therapeutic antibody trastuzumab (tzmb) [ ] and also its fusion with interferon-α b (ifnα b). two cloning strategies consisting in transfecting cho-s and hek cell lines with two bicistronic or with a single tricistronic plasmids have been assessed. the in vitro efficacy of both antibodies has been tested and compared side by side. tzmb heavy and light chains were cloned in two bicistronic plasmids (pirespuro and piresneo , clontech) and in a tricistronic plasmid derived from pirespuro . ifnα b was spliced to tzmb heavy chain by overlap extension pcr and the resulting tzmb-ifnα b fusion protein was also cloned in the expression vectors in the same way than non-modified tzmb. selected cell pools were cultured in ml shake flasks containing sfmtransfx supplemented with % v/v of cell boost (hyclone), mm of glutamax (gibco) and μg/ml of puromycin and also with μg/ml neomycin in the case of the cells transfected with pires-neo . cells were cultivated in the same conditions as described elsewhere [ ] . purified products (using protein a chromatography (hitrap mabselect sure, Äkta avant )) were quantified by both elisa and sds-page. antigen binding test was performed in sk-br- breast cancer cell line by means of flow cytometry analysis. the biological activity of the different candidates was tested with mtt assay. both tzmb and the fusion protein tzmb-ifnα b have been successfully expressed in cho-s and hek , which use for heterologous protein expression have previously been optimized in prior works [ ] . the tricistronic strategy resulted in the most efficient, showing a . fold increase in terms of productivity with respect to the bicistronic double-transfection for tzmb in cho-s cells and a -fold increase in hek cells (fig. a) . in the case of tzmb-ifnα b, the tricistronic strategy also allowed to achieve higher productivities than the bicistronic one (fig. b) . regarding the differences of specific productivity between both cell lines tested, hek emerged as the best production host candidate, for the two tested strategies (tricistronic and bicistronic) and for the two produced proteins, showing a . -fold increase in terms of productivity with respect to cho-s cells for tzmb using the tricistronic strategy. tzmb and tzmb-ifnα b were analysed in terms of their antigen binding capacity, and both were find to efficiently bind to her + skbr- cells (fig. c) . thus, the antibody affinity to her antigen has not been affected when fused to inf-α b. finally, antiproliferative activity of tzmb and tzmb-ifnα b were assessed on the same sk-br- cells. at a concentration of nm of tzmb, and after a -hour incubation, sk-br- cells presented a % growth with respect to the untreated control. however, no antiproliferative effect was observed for tzmb-ifnα b (fig. d) . the tricistronic strategy provides higher productivity yields in hek and cho-s cell lines for both recombinant proteins (trastuzumab and tzmb-ifnα b). regarding which cell line is the best production host candidate, hek achieved higher productivity than cho-s cells for the two proteins tested. all constructions performed preserved the binding affinity to its antigen, trastuzumab and tzmb-ifnα b bind efficiently to the her antigen present in skbr- cells. finally, tzmb-ifnα b does not present an improved antiproliferative effect with respect to trastuzumab when compared by means of an in vitro assay. the genetic engineering of patient-specific t cells with lentiviral vectors (lvv) expressing chimeric antigen receptors (car) for late phase clinical trials requires the large-scale manufacture of high-titer vector stocks. the state-of-the-art production of lvv is based on -to layer cell factories transiently transfected in the presence of serum. this manufacturing process is extremely limited by its labor intensity, open-system handling operations, its requirements for significant incubator space plus costs and patience risk due to presence of serum. to circumvent these limitations, this study aims to develop a stable and serum-free process to produce lvv with pei-mediated transfection. in addition, this study also focuses on the development of a a c b d fig. (abstract p- ) . expression of trastuzumab (a) and trastuzumab-ifnα b (b) from bicistronic strategy (bc) and tricistronic strategy (tri) with cho-s and hek cells. relative specific productivity units are used for comparing the different strategies. c antigen binding analysis of trastuzumab and trastuzumab-ifnα b. d antiproliferative activity of trastuzumab and trastuzumab-ifnα b on sk-br- cells production system not only using a gfp marker but also a therapeutically relevant transgene (cd -car) [ ] . therefore, three different cell lines (hek , t, ft) were investigated concerning their productivity of lvv and their growing behavior in the in-house serum-free medium transmacs. as part of this, design of experiment was used to investigate the optimal conditions for pei/ dna-transfection. furthermore, this statistical approach was used focusing an ideal ratio between the rd generation plasmids (transfer plasmid cd -car or gfp, envelope plasmid, packaging plasmids). in addition, different enhancers (sodium butyrate, lithium acetate, caffeine, trichostatin a, cholesterol, hydroxyurea, valproic acid) were investigated concerning their effects on productivity comparing hek cultures producing lvv encoding for gfp-marker or cd -car. concerning productivity and growing behavior, hek t was the favored cell line for our serum-free lv manufacturing process. in addition, an additive screen revealed that sodium butyrate alone had the most promising effect on both gfp-lvv and cd -car-lvv production. after pei/dna titration, we finally could increase lvv productivity by lowering pei/dna amount at higher cell densities referred to our standard transfection protocol. furthermore, the titration for the optimal plasmid ration revealed, that for large transfer constructs higher amounts of transfer plasmid are required than for smaller constructs to achieve a high productivity (fig. ) . the outcome of these experiments enabled the development of a robust hek t based process to produce clinical relevant lvv under serumfree conditions. furthermore, it provides an insight how therapeutic genes and the expression of its transgene can influence cell productivity. led to a vast increase in productivity, cho cells yield less than other expression systems like yeast or bacteria [ ] . to improve yields and find beneficial bioprocess phenotypes, genetic engineering plays an essential role in recent research. the mir- cluster with its genomic paralogues (mir- a and mir- b) was first identified as differentially expressed during temperature shift, suggesting its role in proliferation and productivity [ ] . the common approach to deplete mirnas is the use of a sponge decoy which, requires the introduction of reporter genes. as an alternative this work aims to knockdown mirna expression using the recently developed crispr/cas system which does not require a reporter transcript. this system consists of two main components: the single guide rna (sgrna) and an endonuclease (cas ) which induces double strand breaks (dsbs). these dsbs can result in insertion or deletion (indels) of base pairs which can disrupt mirna function and processing [ ] . a cho-k cell line stably expressing an igg was used for knockdown experiments. sgrnas were designed to target the seed region of each mirna member and stable mixed populations were generated (fig. a) . total rna form each mixed population was reverse transcribed into cdna using mirna specific stemloop primers. the expression was quantified by rt-qpcr. to further analyse the range of indels the mir- a and mir- b clusters were amplified by a standard high-fidelity pcr. amplicons were cloned into pcr tm -topo® vector and positive clones were analysed by sanger sequencing. cell growth was monitored using viacount tm viability stain on a guava tm benchtop flow cytometer. productivity was assessed by elisa. students t-test was used for statistical analysis. it was shown that mirna expression was significantly reduced in mixed populations. a knockdown up to % was achieved for mir- a, mir- b and mir- . the knockdown in mir- a and mir- b expression was considerably less -between - % (n= , * p ≤ . , ** p ≤ . , *** p ≤ . ) (fig. b) . furthermore, it was shown that various sizes of indels were generated by targeting the seed region. smaller indels (+ / + /- /- bps) seemed to be more common but larger deletions were detected as well (fig. c) . mir- a, mir- b and mir- b showed increased viability in late stages of the culture. depletion of mir- a reduced growth significantly whereas knockdown of mir- showed increased proliferation as well as boosting igg titers (table ) . in this work, we have shown that crispr/cas can be successfully applied as a tool to knockdown mirna expression in cho cells. the data was generated using mixed pools and it remains to be established if both alleles can be successfully targeted e.g. using nextgeneration sequencing of individual clones. background chinese hamster ovary (cho) cells are the most widely used host cell line for the production of therapeutic antibodies. pre-and posttranslational modifications and optimization of culture methods contributed to increase the productivity, resulting in a very high titre [ , ] . however, it has been pointed out that the intracellular secretion process is a bottleneck in the production of therapeutic antibodies [ ] . in addition, the details of the process of secretion of humanized recombinant antibodies from cho cells have not been well investigated. in this study, we thus analysed the detailed process of secretion of therapeutic antibodies using cho cell lines, which have already been established as high producers, with the aim of obtaining information for the more rational and efficient establishment of high-producer cells. we performed ) chase assay, ) immunofluorescent microscopy observation, and ) size exclusion chromatography (sec) analysis to investigate the duration of secretion, bottleneck position, and formation of recombinant igg, respectively. high-producer cho cells expressing humanized igg [ ] and igg were used. for the chase assay, cells were cultivated in shake flasks with serum-free medium containing μg/ml cycloheximide (chx) to stop nascent peptide synthesis. the amounts of igg both remaining in the cell and secreted into the medium at each time point were measured by quantitative western blotting. for immunofluorescent microscopy observation, cells were cultivated on coverslips with chx for h. immunofluorescent staining against the recombinant igg, endoplasmic reticulum (er), and golgi apparatus was performed after chemical fixation. for sec, cells cultured with chx were re-suspended in a buffer containing tritonx- and injected into a column. the amount of igg in each fraction was measured by quantitative western blotting. the amount of igg in the supernatant increased until - h after the inhibition of protein synthesis by chx; however, it hardly changed thereafter (fig. , upper panel) . at this point in time, however, around % of igg still remained in the cells (fig. , lower panel) , meaning that all of the synthesized igg could not be secreted into the medium and remained in the cells for several hours. this result was almost the same as that of studies using igg -expressing cells [ , ] . the localization of igg in the cells was checked before and after the addition of chx, with the results showing that igg remained in the er and was hardly seen in the golgi apparatus [ ] [ ] [ ] ; igg did not seem to be efficiently transported to the golgi apparatus. the sec experiment showed that most of the igg remaining in the cell seemed to form full-sized antibodies [ , ] , but it could not be secreted despite this. the high-producer cells could not secrete all of the synthesized igg, and around % of igg remained in the cells for several hours. this incomplete secretion is a common phenomenon among cho cells producing different types of recombinant igg. the igg could not be transported from the er to the golgi despite its formation of fullsized antibodies. solving this bottleneck in the transportation of igg from the er to the golgi and/or achieving more efficient glycosylation of igg after the formation of full-sized antibodies might be the next target to improve productivity. background humanized monoclonal antibodies (mabs) are among the most promising drugs, but defined strategies for their modification are still not available. our work deals with humanization of murine mab / h . the superhumanization approach leads to a loss of binding affinity which was partially restored by a single human-to-mouse backmutation (t hr). [ ] this residue was selected by synergistic combination of sequence analyses of antibody framework regions and structural information using novel in silico simulations. for structural stabilization, a conglomeration of tyrosine residues surrounding t hr was identified, the so called "tyrosine cage". [ ] analysis of the "tyrosine cage" was done by alanine scanning mutations with a double mutation variant t hr + y ha (bm ) and a triple mutation variant t hr + y ha + y ha (bm ). in a recent series of experiments we tried to enhance binding affinity by three new variants with backmutations in the variable light chain (vl). originating from t hr, residues in the vl were selected based on their spatial proximity to the cdr loop of the variable heavy chain. affinity improvement of t hr was evaluated by vl-double backmutation variants t hr + f ll (su ) and t hr + q ls (su ) and a triple backmutation variant t hr + f ll + q ls (su ). all five variants were expressed transiently in hek - e cells and binding affinities were investigated in two individual settings with bio-layer interferometry. in the first approach concentrated cell culture supernatants were directly applied and mabs were captured on protein a tips, blocked with d scfv-fc and the association and dissociation of f igg was measured. for the second approach, the culture supernatants were purified and the affinity was determined with streptavidin biosensors. first, biotinylated f igg was bound and then the association/dissociation of the purified h variants was measured. affinity evaluation of concentrated culture supernatants with protein a sensor tips showed a decrease of binding affinity of bm and a loss of binding of bm . the protein a measurement showed an increased binding strength of su , su and su compared to su h and bm . su and su result in a higher binding affinity compared to su . these results can be confirmed with purified variants by the streptavidin bio-assay (fig. ) . alanine scanning of the tyrosine cage demonstrated a reduction of binding affinity (bm ) and a severe loss of binding (bm ), concluding that the tyrosine cage plays an important role for supporting a correct cdr loop conformation. further affinity improvement of the single mutation variant t hr could have been reached via mutations in the vl. it demonstrates the underestimated role of the vl for the interaction with its binding partner. although cho cells are a major expression system for production of recombinant biopharmaceuticals, the molecular and cellular background characterizing a high producer is largely unknown. it has been observed that in producer cell lines important signaling pathways like the akt-signaling are altered in characteristical ways. thus analyzing according signaling events should lead to identification of key elements characterizing high producer cells. to investigate this, our emphasis lies on the phosphorylation status of involved proteins as reversible switches in all signaling pathways. we aimed to establish a workflow for cho-specific phosphoproteomics and focused on igf signaling, as cell culture media often are supplemented with this growth factor. two producer cell lines and the according parental cells were cultivated in a stable isotope labeling with amino acids in cell culture (silac) experiment, followed by quantitative ms phosphoproteomic analysis including chospecific data evaluation. the chosen cho cell lines were cultivated in triplicates in silac media containing isotopically-labeled lysine/arginine (hlys/harg) and in parallel in identical standard media (llys/larg, tcx d, xell). cell density, viability, metabolism and cell cycle distribution were monitored during ml batch culture for - days. at day . igf was added into hlys/harg cultures. min later a part of the cells was harvested. for ms analysis igf-treated (hlys/harg cultures) and control cultures (llys/larg cultures) were combined. the following ms sample preparation workflow included digestion of whole protein lysate and phosphopeptide enrichment via tio beads. nanolc-esi-orbitrap ms (q exactive plus, thermo fisher scientific) of phosphopeptides was excecuted with subsequent identification and quantification in maxquant [ ] . in addition to silac quantification of h/l ratios for investigation of igf effects, aquired data was also used to perform label-free quantification (lfq) in maxquant [ ] for comparison of cell lines. statistical significance was calculated via t-test (p< . ) or anova (permutation-based fdr< . ) in perseus [ ] . results igf effects on growth and production the igf treatment resulted in a prolonged viability for all cell lines. however, an increased vcd was only observed for producer cell line , yielding in an enhanced integral of vcd (ivcd). for the parental cells growth was inhibited by igf, although s-phase cells were enriched at least temporary (fig. a) . regarding antibody production igf led to a decreased qp and product titer, concomitantly with an increase in s-phase cells (fig. a) . this inverse correlation of proliferation and cell specific productivity is known from different productivity enhancing molecules, like butyrate [ ] . ms investigation of signaling events the phosphoproteomic experiment resulted in the identification of . class i-phosphorylation sites. statistical evaluation of phosphopeptide abundances in perseus showed up significant differences between the cell lines and led to producer vs. parental classifications (fig. b) . the quantitative evaluation via silac yielded in about . quantifiable phosphosites in at least biological replicates. rapid phosphorylation changes after growth factor treatment indicated signaling towards protein synthesis, cell cycle and regulation of actin cytoskeleton amongst others. for phosphosites significantly different h/l ratios were calculated between the two groups parental vs. producer, four of them are listed (table ) . the workflow to study phosphorylation states revealed differences in the related cell lines and gave insights into signal transduction as a response on igf. on the one hand, igf-treatment resulted in a fast and widespread upregulation of phosphorylation sites within aktand mapk-signaling. on the other hand, a different phosphorylation status for producer compared to parental cell lines uncovered distinctions in biological processes like rna-and dna-binding and regulation of cytoskeleton. in sum, our sucessfully established phosphoproteomic approach allows to detect important signaling key players in cho cells that subsequently can be targeted through cell engineering or small molecule treatment. to improve antibody production in the cho cell expression system, it seems to be useful to up-or downregulate gene expression including antibody folding, secretion, and cell metabolism. many cell engineering approaches, including gene introduction, knockout and knockdown, have been employed to enhance recombinant antibody production [ ] . however, identifying production enhancer genes is the rate-limiting step for cho cell engineering, because the conventional method requires a series of experiments including genomic integration of the tested genes, selection of stable cell clones and cell culture experiments of several clones. in this study, we propose an approach for rapid evaluation of production enhancer genes based on an episomal expression system. plasmid vector carrying the epstein-barr virus (ebv) encoded nuclear antigen (ebna ) was transfected into cho cell line producing igg antibody. after g selection and single colony isolation, ebna expression was checked with capillary electrophoresis system wes (proteinsimple). ebv ebna -antibody ( eb ) was used for detection as the primary antibody. the expression vector for the gene of interest was prepared by inserting bp of an orip dna sequence into a plasmid vector carrying cag promoter, resulting in the potc vector. pei max (polysciences, inc.) and balancd transfectory cho (irvine scientific) were used for the transfection. the number of viable cells and gfp-positive cells were counted using countess ii fl automated cell counter (thermo fisher scientific). the transfected cells were cultured in cellstar cellreactor tubes. the tubes were incubated in a climo-shaker isf -x (kuhner). antibody production was measured using biolayer interferometry with an octet qk system (fortebio). we constructed four cho cell lines stably expressing ebna , termed igg -eb to eb . in capillary electrophoresis analysis, we observed a clear peak corresponding to the ebna expression in all four cell lines. we tested the transfection efficiency by potc-gfp plasmids. in the best transfection condition, pei/dna ratio of / , igg -eb cell showed the highest gfp-positive cell number ( . × cell/ml) and transfection efficiency ( %) among the four cell lines. therefore, igg -eb cell lines were selected for further study. after the transfection, the number of gfp-positive cells continued to increase even after the passage (fig. ) , suggesting that the potc-gfp plasmid was stably retained and replicated by ebna /orip system in igg -eb cell lines. in preliminary experiments, we introduced three genes, mdh , gss and gclm, into igg -eb cell lines. cotransfection of these three genes led to an increase in igg production from ± mg/l (control) to ± mg/l at day (p< . , t-test, n= ). this result suggests that these three genes work as production enhancer genes. conventional methods based on stable cells take up to months to determine whether the gene of interest is beneficial for recombinant igg production. in contrast, identification of production enhancer genes is achievable within days by our proposed method based on ebna /orip system. the proposed method makes it possible to evaluate production enhancer genes in a rapid manner. the proposed method is a promising approach to identify genes enhancing recombinant antibody production. background g unic™ ( gun) technology comprises a set of protected genetic elements that improve protein production by acting on transcription as well as on translation. the elements can either be inserted into existing (platform) vectors or be provided as complete ready-to-use vectors. the technology can be used in stable and in transient transfection to boost protein production for product development and is being applied in cld for pharmaceutical proteins. in combination with antibiotic selection or dhfr selection, gun technology routinely results in - fold increase in expression of client antibodies or fusion proteins, both in pools and after clonal selection. previously, we have successfully combined gun technology with glutamine synthetase (gs) selection and the cho gs null cells of horizon discovery, resulting in clonal cell lines producing > g/l of a biosimilar mab in fed-batch assay. here we present data on the successful application of the gun technology for the enhanced expression of a large (> kda) human heterotrimeric glycoprotein, a renowned difficult-to-express (dte) protein. all expression vectors comprised a hcmv promoter and bgh polyadenylation sequence in the expression cassettes for the gene of interest, and a selection marker gene with sv promoter and sv polyadenylation sequence. g unic™ vectors also contained genetic elements ( g unic™ technology, proteonic). cho gs null cells (horizon discovery) were transfected in duplicate with reference or gun expression vectors and selected in media lacking glutamine and containing the appropriate antibiotics. the bulk pools were seeded at equal viable cell density after obtaining maximum viability and cultured for days without feeding (batch). expression of the target protein in cell culture supernatants of stable bulk pools was measured by elisa. the three protein subunit genes were expressed from vectors with different selection markers. in the reference constructs (without gun), the α, β, and γ chains were expressed from vectors with marker genes for zeocin, blasticidin, and gs, respectively. a similar vector combination was also generated with gun elements integrated in each vector. in addition, vectors with subunits (γ-α and α-γ), each with a separate gun element, promoter and polyadenylation signal, were generated with a gs marker gene. cho gs -/cells were transfected with the appropriate vector combinations in equimolar ratios and selected in bulk in medium lacking glutamine and or antibiotics. the -vector transfected cell pools recovered first, due to the presence of only antibiotic in the medium (fig. a) . the pools transfected with three gun vectors recovered to maximum viability just a few days after the -vector gun pools. recovery of the reference pools took up to a week longer than the gun pools. production of each pool was assessed in a batch production run in shaker flasks. all -vector gun pools which recovered first produced titers around . g/l, which is almost -fold higher as compared to the production by reference pools (fig. b) . the highest titers of . g/l were obtained in the -vector gun pools. these data show that the g unic™ genetic elements can be successfully used to obtain a significant increase in the titer of difficult-to-express proteins. similar results have been obtained with other dte proteins, including fc-fusion proteins and bi-specific antibodies (not shown). the expression of a large, glycosylated multimeric difficult to express protein can be increased more than ten-fold in cho gs pools by application of g unic™ genetic elements. the highest expression of is obtained using a separate vector for each subunit. characterization of antibody-producing cho cells with chromosome aneuploidy noriko yamano , , sho tanaka background chinese hamster ovary (cho) cells are commonly used as host cells to produce biopharmaceuticals. however, the number of chromosomes in cho cells varies. previously, dg -sc and dg -sc cell lines with modal chromosome numbers of and were isolated from parental cho-dg cells, from which igg -expressing cell lines named igg -sc and igg -sc were established, respectively. the igg -sc cell pool showed a higher specific igg production rate than the igg -sc cell pool [ ] . even though all of the igg -sc clones and half of the igg -sc clones contained the same number of vector integration sites (single integration site), igg-sc cell clones produced more igg following the culture of single-cell clones than any of the igg -sc clones [ ] . in this study, we performed transcriptome analysis to investigate the characteristics of high-producer cells with chromosome aneuploidy. transcriptome analyses using amplified fragment length polymorphism (aflp)-based high-coverage expression profiling (hicep) and de novo mrna-seq were performed on dg -sc , dg -sc , igg -sc and igg -sc . to compare cell lines with different numbers of chromosomes, transcriptome data from mrna-seq were adjusted for cell number using rna reference materials (nmij crm -a; national institute of advanced industrial science and technology) mixed at equal amounts per cell. pathways related to differentially expressed genes were searched using keymolnet (km data). high-chromosome-number cho cells showed larger cell diameters, as determined by vi-cell (beckman coulter) measurement. the predicted volume ratios, based on these diameters, are . (dg -sc :dg -sc ) and . (igg -sc :igg -sc ). the levels of β-actin and the products of most other genes that were detected by mrna-seq differed by approximately % in the comparison between sc and sc (sc > sc ). based on the analysis of gene expression levels per cell volume, approximately % of detected genes showed lower expression in both dg -sc and igg -sc compared with the levels in dg -sc and igg -sc , respectively. in addition, the number of genes whose expression level was decreased in igg -sc compared with that in dg -sc was larger than those showing the opposite pattern. the results of the comparisons between igg -sc and igg -sc indicate that differentially expressed genes were mainly related to cell growth (e.g. myc, smad), apoptosis (e.g. caspase), lipid metabolism (e.g. srebp, pparγ) and epigenetic histone modification (e.g. brca, hat) pathways. the mrna levels of myc, smad, caspase, brca and hat related genes were lower in igg -sc , while those of srebp and pparγ related genes were higher in igg -sc . the effects of these pathways on antibody production should be examined in future. in this study, we found that high-chromosome-number cho cells have lower amounts of mrna relative to their volume. a reduction per unit volume in the expression of genes that are required for survival might generate additional energy for recombinant protein production in high-chromosome-number cells. from an evolutionary perspective, an increased set of chromosomes underlies rapid evolutionary adaptation. although there are issues to be considered, such as stability, there may also be advantages to using high-chromosome-number aneuploid cho cells as a production host cells of recombinant proteins. background human growth factors have an enormous therapeutic potential. among them, the bone morphogenetic protein- (bmp- ) can induce de novo bone formation endowing the protein a high therapeutic potential. however, finding a suitable recombinant production system for such a protein still remains a challenge. recombinant expression of hbmp was investigated in transiently transfected hek- cells and in stable clones established in cho-k cells cultivated in excell and pro-cho medium, respectively. protein stability and interaction of the hbmp with the producer cells were investigated in vitro using commercially available rhbmp . in addition, we investigated a cell-free protein synthesis system harboring translocationally active microsomal structures, hence having the potential to perform post-translational modifications, as an alternative production method. we showed that growth rates and viabilities of the rhbmp producing cells were similar to those of the parent cell line, while entry into the death phase was delayed in case of the recombinant cells. the maximum rhbmp concentration detected in the culture supernatant was low for stable clones but can be greatly improved combining the hek- cells transient expression system and batch reactor cultivation which reflects a better compatibility of the codon usage in the human cells (table ) . hbmp protein is sensitive to slightly acidic ph and to a lesser extend to proteases (fig. a ) and binds to both producers cell lines (fig. b) -all this could incidentally contribute to the low product titers. cell-free protein synthesis has been proposed as alternative for "difficult-to-express" proteins. since native hbmp is glycosylated, a cell-free system based on eukaryotic cell lysates is required for its production. cho cell lysates were chosen, since they had previously been established as the most productive eukaryotic system in our hands [ ] , while concomitantly enabling a direct comparison to the production of hbmp in stable clones established in cho-k . the ability to perform post-translational modifications is a major advantage of eukaryotic systems. the cho lysates prepared by the protocol used here have previously been shown to contain significant amounts of endogenous microsomes derived from the endoplasmatic reticulum during lysis [ ] . to enforce translocation of the target protein into the microsomal structures, a melittin signal peptide was fused to the hbmp cdna. the glycosylation of the protein was assessed by enzymatic treatment (pngase, endoh) and confirmed using c-mannose for the de novo protein synthesis. upon cell-free protein synthesis, the hbmp yield was -fold higher than the best one in the hek- cells. the difference becomes even more dramatic, when productivities are considered (table ) , i.e. the fact that maximum product titers are reached within h in the cellfree system compared to h in the cell-based ones. this demonstrates that the cell-free expression system is most suitable compared to mammalian cell expression method for the production of glycosylated human bmp (table ) [ ] . human growth factors are complex molecules, which make their production in mammalian cells desirable. however, low product titers caused by a variety of both cell and process related effects may hinder the development of highly productive processes. in such cases, cell-free protein production using cho cell lysates containing endogenous microsomes for posttranslational processing, may eventually present an attractive alternative. in particular since these lysates can be used under tightly controlled conditions assuring a higher degree of reproducibility, than, e.g. transient transfection systems. cell-free systems are known to circumvent typical bottlenecks of cellbased ones, e.g. metabolic regulation and cell maintenance mechanisms. in consequence, the production of a recombinant protein is neither inhibited by its accumulation nor by any interaction with the cells, e.g. through the activation of inhibitory signaling pathways. core. preliminary studies showed that the corresponding polyplexes, but also some of the cells that came into contact with them, became magnetic and were manageable by magnetic fields [ ] [ ] [ ] . here, we present a characterization of the influence of structure and composition on the function of these polymers using a library of highly homogeneous, paramagnetic nano-stars with varied arm lengths and densities [ ] . the paramagnetic nano-stars library was synthesized by coating maghemite nanoparticles (γ-fe o ) with a thin silica-shell functionalized with an atomic transfer radical polymerization (atrp) initiator. pdmaema arms were grown from the core particles via atrp. in one case, the pdmaema arms was end-capped with pdegma blocks produced during a second atrp step. all nanostars were characterized by size exclusion chromatography and thermogravimetry to calculate number and length of the pdmaema arms. the core diameter was determined by transmission electron microscopy and dynamic light scattering (dls). the different variants (table ) were analyzed for their ability to complex pdna (pegfp-n ) using various physicochemical methods (dls, zeta sizer). transfection efficiency/cytotoxicity in cho-k cells were determined by flow cytometry. transfected cells were placed in a magnetic field and the influence of the polymer architecture on the magnetic separation was investigated. nonparametric spearman analysis was used to correlate between arm length/arm densities, magnetic properties of the cells and transfection efficiency. based on the hydrodynamic radii of the polyplexes, the investigated nano-stars could be divided into three subgroups (table ) . middle, but also high arm density nano-stars formed smaller polyplexes with hydrodynamic radii ≤ nm, a size that is considered suitable for endocytosis and transfection. transfection efficiencies and cytotoxicities varied systematically with the nano-stars architecture, with viability showing a more pronounced dependency on the characteristics of the transfection agent than the transfection efficiency itself. the arm density was particularly important, with values of approximately . arms/ nm yielding the best results (fig. a) . the end-capping the polycation arms with pdegma significantly improved the serum compatibility (fig. b) . the gene delivery potential of a given nano-star and its ability to render the cells magnetic did not correlate. although, compared to the non-separated cells, egfp-expressing cells were consistently more frequent in the magnetic cell fraction, while the non-magnetic fraction was slightly depleted. when the egfp-expressing cells were further divided into low, middle and high producers, a statistically significant shift towards the high producers was observed in the magnetic cell fraction (fig. c) . a nonparametric spearman correlation analysis was used to statistically evaluate possible links between the molecular characteristics of the nano-stars, the physicochemical properties of the corresponding polyplexes, the transfection conditions, and the cellular reactions. the resulting correlogram is shown in fig. d . transfection agents with magnetic properties enlarge the toolbox for studying non-viral gene delivery, since cellular magnetism is added as a new parameter. this allows, inter alia, a distinction between mere cellular interaction and actual uptake, which is otherwise difficult. viability showed a much more pronounced dependency on the characteristics of the transfection agent/polyplex than the transfection efficiency itself, which should be taken into account during method optimization. end-capping the polycationic pdmaema-arms with pdegma-blocks improved the compatibility of the polycationic nano-stars with serum components. in future optimized, blood-compatible, nano-stars, which can be retained/directed by magnetic fields, could become options for non-viral gene delivery in vivo. the increasing demand for monoclonal antibodies has necessitated the need to increase the productivity of current industrial cell lines. in our earlier study [ ] , we had shown that treatment with er-stress inducer, tunicamycin significantly increased the titers and productivity in recombinant cho cell lines with a simultaneous upregulation of many genes from the unfolded protein response pathway (upr). however the loss in cell viability prevented a sustained increase in titers. in the current study we explore the effect of varying concentrations of tunicamycin and treatment times, such as to modulate the increase in protein folding capacity while preventing induction of apoptosis. anti-rhesus igg-secreting cho cells [ ] were cultured in sf-cdm in ml shake flasks. the cells were treated with varying concentrations ( - ng/ml) of tunicamycin in a batch culture. further, the effect of treatment with tunicamycin for short periods of time ( hrs) was also evaluated. igg titers and mrna expression levels were quantified using elisa and qrt-pcr (illumina), respectively. results cho cells were treated with different concentrations of tunicamycin and cultured in a batch for days (referred as continuous treatment/cte). figure a presents the maximum vcd and % drop in viability under treatment. a dose-dependent inhibitory effect is observed on growth and viability of cells in cte-cultures, with minimal inhibition as lower concentrations. contrastingly, igg titers (fig. b) were higher in treated cultures w.r.t. control in initial phase of the cultures at all the concentrations of tunicamycin. the per-cell productivity (fig. c) also showed a significant increase w.r.t control at all the concentrations of tunicamycin. however, the increased productivity due to tunicamycin was not sustained and levels become similar to control after day (data not shown). to prevent loss of viability due to tunicamycin, the effect of short-term treatment (ste) with tunicamycin was explored. cells treated with tunicamycin for hours were harvested (corresponding to day of cte cultures) and inoculated in fresh media. the ste-cultures showed improved viability and higher maximum vcd as compared to cte-cultures (fig. a) . the fold increase in igg titers was not sustained beyond day - in stecultures ( fig. d ) but significant increase in productivity was seen in the initial phase (fig. e) . further, the cells were adapted over continuous generations under ng/ml tunicamycin. the adapted cells had overall . -fold higher productivity, as compared to control (fig. f) , in a batch culture. to understand the molecular basis of increase in productivity, mrna expression level of key genes was determined. xbp s is a transcription factor involved in activation of chaperones (like grp , calreticulin) and apoptotic genes (such as chop). significant increase in the levels of calreticulin was seen on treatment with tunicamycin (fig. g) . both xbp s and grp were marginally induced when treated with ng/ml of tunicamycin in both cte-and ste-cultures (fig. h) , and significantly up-regulated when treated with ng/ml of tunicamycin. the chop mrna levels also increase with increasing tunicamycin concentrations, with levels in ste-cultures lower than cte-cultures (fig. h) . the results suggest that upr induction may be important to increase productivity in these cte/ste-cultures. note that, tunicamycin had no effect on the expression levels of igg heavy-chain, thus eliminating the involvement of igghc-mrna in increasing productivity (fig. i) . tunicamycin induced er-stress increased productivity in the initial phase of the culture and enhanced upr-mediated folding capacity can be attributed as one of the reasons for it. at lower concentrations of tunicamycin, a fine balance between optimum upr induction and apoptosis can be achieved, as seen in ng/ml tunicamycin ste-cultures. in summary, this study demonstrates an alternate approach to enhance productivity of current industrial cell lines. background chinese hamster ovary (cho) cells have been widely used for the large-scale production of biopharmaceuticals [ ] . to construct antibody-producing cho cells, exogenous genes encoding antibodies are usually integrated into unspecified regions of chromosomes (random integration). however, the chromatin structure differs depending on the location of the chromosomal region, which affects the expression level of the gene of interest [ ] . recently, gene-targeting methods that enable site-specific integration of expression vectors have been developed. however, the regions that are most efficient for exogenous gene expression have not been clarified. we previously constructed a cho genomic bacterial artificial chromosome (bac) library generated from the recombinant cho-dg cell line. it was expected to cover the entire cho genome five times. the chromosomes in cho-dg cells were aligned in decreasing order of size and assigned letters from a to t [ ] . three hundred and four bac clones were mapped to every chromosome of cho-dg . among the karyotypes of cho-dg , cho-k and primary chinese hamster cells, chromosomes a and b are considered as the sole paired chromosomes corresponding to chromosome in primary chinese hamster cells. hence, chromosomes a and b are considered to be stable [ ] . in this study, we constructed antibody-producing cells by using a gene-targeting method, which focused on the stable chromosomes. a gene map of chromosome was constructed by combining the bac-fluorescence in situ hybridization (fish)-based chromosome physical map and sequence data of mapped bac clones. the sequences of bac clones were searched by blast with ncbi and chogenome.org databases. three different regions on chromosomes a and b were selected based on cho genomic bac library sequences as target sites. cho-k cells were stably transfected by lipofection. the target sequences were broken using the clustered regularly interspaced short palindromic repeats (crispr)/crispr-associated protein (cas ) system and humanized igg genes were integrated by non-homologous end joining recombination. transfection without using the crispr/ cas system was also performed. these cell pools were cultivated for six days with serum-supplemented medium, and their levels of antibody productivity were evaluated by elisa. copy number analysis was also performed using real-time pcr. results and discussion construction of gene map of chromosome : eighty-three bac clones were mapped onto chromosomes a and b (each clone contained - kb of the cho genome sequence). as a result of annotations of bac clone sequences, genes were mapped on chromosome . investigation of the differences of productivity among antibodyproducing cells that were constructed by chromosome targeting and/or random integration: cell growth was not affected by the gene targeting site. the specific production rates of antibodyproducing cell pools constructed by gene targeting of chromosome were higher than those of the cell pool constructed by random integration. all cell pools constructed by gene targeting showed lower copy numbers of heavy chain and light chain in genomic dna than those in the cell pool constructed by random integration, despite showing high productivity. our results indicate that high productivity of the cells constructed by gene targeting of chromosome does not depend on the increase of the antibody copy number, and that the environments around these target regions are suitable for exogenous gene expression. the approach of using gene targeting to chromosome may be promising for constructing antibodyproducing cells. retroviral vectors have been widely used as gene delivery tools in various biotechnology fields. however, the random integration feature of retroviral vectors seems to cause problems such as insertional mutagenesis and gene silencing. we previously demonstrated cre-mediated retroviral transgene insertion into a pre-determined site of the founder cells using integrasedefective retroviral vectors (idrvs), where a cre expression plasmid was transfected into the cells prior to retroviral transduction [ ] . recently, we reported novel hybrid idrvs (cre-idrvs) incorporating bioactive cre recombinase protein, and validated site-specific gene integration of an scfv-fc antibody expression unit into the chinese hamster ovary (cho) cell genome [ ] . we also developed an accumulative site-specific gene integration system, which enables repeated integration of multiple transgenes into a pre-determined locus of the cell genome [ ] . here, we attempted repeated integration of transgenes using cre-idrvs. a viral vector plasmid (pqmscv/hd[scfv-fc]) encoding reporter genes and an scfv-fc expression unit flanked with wild-type and mutant loxps was constructed for the production of idrvs. cre-idrvs were produced described previously [ ] . results and discussion figure a shows a schematic drawing of each round of targeted transgene integration using cre-idrvs harboring an scfv-fc expression unit. (fig. b) . genomic dna extracted from the cells were subjected to pcr using specific primer pairs α and β, and γ and δ to confirm site-specific integration. dna fragments with expected sizes were amplified in each cell clone (fig. c) . these results indicate that site-specific repeated integration was achieved using cre-idrvs. in contrast, scfv-fc productivity in cho/hd[scfv-fc]× cells was slightly decreased compared with that of cho/ne[scfv-fc]× (data not shown). although the reason remains unclear, repeat-induced gene silencing might occur due to tandem repeat structure of expression units. we reported improved recombinant antibody production using a production enhancer element [ ] . such a cis-regulatory element might be a feasible approach to enhance the productivity. we demonstrated site-specific repeated transgene integration into a pre-determined chromosomal locus using cre-idrvs for the production of an scfv-fc antibody. if lipids role in the cell have been reduced for a long time to cell membrane formation, it is now understood that lipids plays also a role into energy metabolism, vesicular transport, membrane structure, dynamics and signaling. however, the exact mechanism of how compositional complexity affects cell homeostasis remains unclear. thanks to recent advances in mass spectrometry, it is now possible to study a wide range of lipids, providing a better understanding of lipid homeostasis in high performance cell culture processes. the purpose of this work was to develop a robust lipidomics method applied to mammalian cell cultures in a three step method: extraction, separation and detection (fig. ). both matyash [ ] and folch [ ] extraction method were performed on our cells to reach the highest yield. two separation techniques were also tested: hydrophilic interaction liquid chromatography (hilic) and reverse phase chromatography. finally lipid classes' identification was achieved by tandem mass spectrometry analysis thanks to structure-specific fragmentation ions. the yield obtained with matyash extraction method was higher than with folch method for each lipid class tested. besides, matyash method presents also the advantage to be less toxic and suitable for high throughput analysis since the organic layer is above the aqueous layer. lipids separation by hilic is based on their polar head. since lipid classes are defined by polar head, the lipids are eluted class by class, making their identification easier. the separation of lipids by reverse phase was correct but the method is longer and we observed a massive carryover of triglycerides on the column. finally each lipid class was screened in ms/ms parent ion mode. target daughter ion was set according to the lipid class structure and fragmentation pattern. this detection technique enabled the identification of different lipids. to ensure the absolute quantification of the detected lipids and to guarantee comparable results between batches labeled internal standard were added prior to extraction. this method was optimized in a stepwise process to ensure a sensitive and selective measurement of the lipids. lipids were extracted by matyash method, separated by hilic and detected by tandem mass spectrometry. this method is suitable for both in process sample lipid analysis providing information on the cell lipid content, and for harvest samples, enabling to follow the lipid release during the different harvest steps. this non-targeted lipidomic quantitation method will enable us to better control lipid synthesis during biopharmaceutical fed batch production through clone selection, metabolomics studies and harvest development. background human mesenchymal stem/stromal cells (hmsc) can easily be isolated from e.g. bone marrow, fat tissue or umbilical cord blood and are therefore a central player in regenerative medicine, gene therapy and cell therapy [ ] [ ] [ ] . the necessary gene shuttle is mainly provided by viruses associated with diseases, like retrovirus or adenovirus [ ] [ ] [ ] [ ] . these possible pathogen viruses demand for high safety standards. also, they are prone to genomic alterations and there is the possibility of virus inactivation, triggered due to pre-existing immunity in the patient [ ] [ ] [ ] . in this context, the autographa californica multicapsid nucleopolyhedrovirus (acmnpv) is a safe alternative. the virus replication is hostspecific for insects [ ] , but it is known since the mid- s, that a temporary transduction of mammalian cells is possible [ ] . some modifications of the virus increased the applicability in stem cells. pseudotyping the virus with the vesicular stomatitis glycoprotein (vsv-g) led to an expansion of the transducable cell [ , ] and the integration of the woodchuck hepatitis virus post-transcriptional regulatory element (wpre) prolonged the recombinant protein expression [ , ] . for achieving a baculovirus-induced differentiation of hmscs, the promotor and the expression strength of the recombinant protein are crucial factors. still, there are still few comparative promotor studies [ , ] . however, a successful virus uptake is the prerequisite for a successful protein expression. we therefore investigated factors significantly influencing the transduction process by applying design of experiments (s. fig. a ). the experimental design comprises a two level factorial screening, set-up using design expert v .for the transduction , c/cm were seeded in -well plates with dmem + % fcs and incubated overnight at °c, % co and humidified atmosphere. the recombinant baculovirus using an integrated ef α promoter to control gfp expression, described elsewhere [ ] , was diluted to the respective concentrations in the different surrounding fluids. after discarding the cultivation medium of the hmsc-tert, ml of virus containing solution was added to the cells. the following incubation was varied in duration before replacing the virus solution with growth medium and an incubation overnight. h post transduction (hpt) the cells were washed with pbs, trypsinized with μl trypsin/edta and incubated for min at °c. trypsination was then stopped applying μl soybean trypsin inhibitor and the cells were analyzed using flow cytometry. as shown in fig. a , the virus concentration and incubation time exert the highest influence on the transduction efficiency. obviously, a higher concentration of viral particles and longer incubation of cells with virus increases the probability for hits between cells and virus particles. additionally, the surrounding fluid can have a negative impact on the transduction. this is due to the interaction of medium components with the baculovirus. therefore, pbs containing ca + & mg + is recommended as surrounding fluid for transduction experiments. in fig. b , the transduction conditions resulting in the highest percentage of gfp+ cells are displayed: virus particles per cell (ppc) and an incubation time of h with hmsc-tert. the experiments show, that especially the virus concentration and the incubation time of cells with virus influence the transduction efficiency. based on the results of the screening, further optimization of the transduction conditions will be done using a face centered central composite design with pbs containing ca + & mg + as surrounding fluid and at an incubation temperature of °c. background breast cancer is the second main cause of cancer related deaths for women worldwide and among them the triple negative subtype (tnbc) represents a clinical challenge by being associated with high mortality and having no effective therapies against it [ ] , [ ] . accordingly, there is an urgent need to design new and more effective drugs to treat breast cancer. notch signaling is an evolutionary conserved cell-to-cell communication pathway crucial during embryonic and breast development and tissue homeostasis. this pathway is often hyper-activated by overexpression of notch receptors and/or its ligands in several types of cancers, such as breast cancer (tnbc included), where it contributes to its development, progression and drug resistance [ ] , [ ] , [ ] . our aim is to generate a function blocking antibody against the notch delta-like- (dll ) ligand with therapeutic efficacy against breast cancer. materials and methods dna of human dll full length extracellular domain (dll -ecd) and a truncated version, containing the minimal binding region to the notch receptor (dll -egf ), were cloned into pfuse-fc -igg , and expressed in hek e cells. recombinant proteins were purified from culture media by protein-a affinity and size exclusion chromatography. the human scfv phage display tomlinson i+j library was used to select specific scfv against peptides targeting dll binding regions to notch. the binding ability and specificity of the selected scfv clones was evaluated by scfv-on-phage elisa. our strategy allowed us to obtain mg of pure (> %) and stable dll -ecd-fc as confirmed by sds page and thermofluor assay. dll -egf -fc yield was very low and buffer screenings are ongoing to optimize protein stability. functional studies performed in human breast cancer mcf cells showed that both ligands are biologically active as they increased the expression of the notch-dependent genes hes- , hey-l and hey- . recombinant dll and peptides were used to select for monoclonal antibodies by phage display. after three rounds of panning with dll peptides we identified scfv positive clones, of which presented high affinity to dll -ecd-fc. currently we are performing more phage display selections to increase the number of positive clones. scfv with higher affinities will be reformatted into iggs and their ability to inhibit the notch pathway will be evaluated. the anti-oncogenic effects of anti-dll iggs will be assessed in breast cancer cells in viability/apoptosis, proliferation, migration, and invasion assays. an anti-dll igg with therapeutic efficacy against breast cancer will demonstrate that targeting dll could be one of the key factors for successfully targeting breast cancer. recombinant adeno-associated virus (raav) approaches have an outstanding reputation in gene therapy and are evaluated for cancer therapy [ ] . advantages include long-term gene expression, targeting of dividing and non-dividing cells, and low immunogenicity. established raav production utilizes triple transfection of adherent hek cells, which hardly meets product yield requirements for clinical applications. we transferred the aav production system to hek -f suspension cells. this process is scalable and uses serum-free media streamlining downstream procedures. after optimization of transfection efficiencies and shaker cultivations, we produced titers of × viral genomes per cell in a l bioreactor. the suspension adapted hek-freestyle -f cell line was used for the experiments in chemically defined animal component free media (hek-tf, hek-gm (xell ag), freestyle f (thermo fisher scientific)). samples for viable cell density and viabilities were taken daily and analyzed using an automated cell counting system (cedex, roche diagnostics). transient transfection of × cells/ml was carried out with polyethylenimine max in a : dna-pei ratio (w/w) with μg dna. three plasmids (pgoi, prepcap, phelper) were applied in a molar : : ratio (fig. a) . pretests were performed in orbital shaking tube spin bioreactors. for scale-up, batch processes were carried out in ml shake flasks as well as in l stirred bioreactors at % air saturation and ph . . transfection efficiencies and raav production were quantified by flow cytometry using a goi coding for a fluorescent protein and qpcr of genomic copies, respectively. by optimizing the dna amount for transfection of -f cells more than % of the cells were reproducibly transfected. batch cultivations in shaker flasks revealed that raav were produced in the first - h after transfection. figure b shows viable cell densities and viabilities in relation to the genomic titer. genomic titers were determined from raw cell extracts and up to copies/ml were repetitively achievable. a decrease in viability marked the decline in genomic copies per ml showing that a prolongation of the process e.g. by addition of a feed would probably not increase yield. in a first scale-up, the raav production was transferred to a l bioreactor (fig. c) . transfection efficiencies in bioreactors of up to % were comparable to that obtained in a simultaneous shaker flask experiment. transfection efficiencies were lower compared to prior experiments due to controlled conditions in the bioreactor. nonetheless the titer with up to × genomic copies per cell was elevated compared to that of shaker flasks. first experiments with -f cells in hek tf medium showed promising results of transferring raav production from the adherent system to suspension. after improvement of transfections by the adjustment of dna amounts in small scale experiments, aav production was analyzed in shaker flasks. the batch process showed an expected increase in cell density with low variability between biological replicates (fig. b) . the genomic titer increased according to the viable cell density until day four where a sudden drop started. this observation was made for aav productions in hek-tf, hek-gm and freestyle f medium. for optimal yields, we assume that a slight decrease in viability marks the point in time for harvest. from optimized protocols, a batch process in a l bioreactor was carried out. interestingly the bioreactor cultivation resulted in lower overall viable cell densities but in higher genomic copies per cell compared to shaker flasks (fig. c) . these results are comparable to already published data for suspension cells [ ] . subsequent optimization of the bioreactor protocol will lead to further increase in raav yield. genethon and pall have collaborated to assess pall's single-use icellis fixed-bed bioreactor for viral vector production. clinical use of gene therapies to treat formerly incurable genetic diseases is advancing rapidly. viral vectors are an important tool for introducing genes into target cells. many gene therapies have been developed using adherent cells in -dimensional flatware or roller bottles but using these technologies to reach commercial-scale production represents a significant challenge. the icellis bioreactor enables large-scale viral vector production by providing a -dimensional matrix for cell growth in a compact configuration (fig. ). up to m of surface area is available in a compact bioreactor measuring mm in diameter in a total volume of l with ph, do and temperature control. a key feature of the icellis bioreactor is that it scales by increasing the diameter of the fixed-bed while keeping the height constant with no change in aspect ratios. the height of the fixed-bed can be varied ( , and cm) as well as density of carrier packing ( gm/l or gm/l). the icellis system comes in two formats, the icellis nano bioreactor ( . - . m ) and the icellis bioreactor ( - m ). processes developed in the bench top icellis nano bioreactor can be directly transferred to the corresponding icellis system. the icellis nano bioreactor enables an efficient platform for process optimization. the genethon raav- process was transferred to an icellis nano bioreactor . m ( cm bed height, gm/l density) bioreactor using freestyle media. the initial icellis nano process was established as ( ) seed on day , ( ) transfect at day , ( ) harvest at day and yielded < x vp/cm (n= ). media exchange, cell density at transfection, pdna/cell ratio, and lysis method were then changed to determine the effect on productivity. the modified process was then scaled from . m to . m ( cm bed height, gm/l density) icellis nano bioreactor. -media: a media exchange at hours post transfection with dmem substituted for freestyle medium resulted in an x increase in specific productivity. (abstract p- ) . a schematic overview of raav production in hek cells with triple-transfection system. b viable cell densities (vcd), viabilities and genomic copies per ml (gc) of a raav production with -f batch cultivations in shaker flasks. genomic copies per ml refer to the titer determined in ml culture volume. error bars represent biological and technical duplicate measurements of samples. c viable cell densities and genomic copies per cell of a raav production with -f batch cultivation in a l bioreactor. for reasons of comparability between shaker and bioreactor data genomic copies are given per cell. error bars represent technical duplicate measurements of samples -cell density at transfection: cells were seeded at , cells/cm and reached , cells/cm at day which was determined to be the optimal cell density for transfection. -pdna/cell ratio: reducing pdna by % had no significant effect on productivity. -lysis: use of trion x- at . % with mm nacl at ph resulted in > % virus recovery compared to sampled carriers. -scaling: specific productivity was maintained as the system was scaled from . m to . m . -overall, an average yield of x vg/m was achieved. the icellis technology is being adopted widely for viral vector production. transferring a process to the icellis nano bioreactor can be easily achieved and once in place can be optimized to provide significant productivity increases and cost savings such as reduced pdna. the icellis nano bioreactor is an efficient bench-top system the results of which can be readily scaled to the icellis system. background tissuse multi-organ-chip (moc) platform contributes to the ongoing advancement in systemic substance testing in vitro. current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, often do not accurately predict drug toxicity. especially, cardiotoxicity is one of the main reasons why new compounds are failing in clinical trials. therefore, we aimed to establish an autologous dynamic multiorgan-device integrating cardiomyocytes for substance testing. generic d monolayer and d suspension ipsc derived cardiomyocytes differentiation protocols were established. beating cardiomyocytes were first seen on day in monolayer as well as in spheroid culture. cardiomyocytes show up to % cardiac troponin t positive cells and % myosin heavy chain positive cells by flow cytometry (fig. g, h) . myosin ii heavy chain, α-actinin, myosin / , myosin and caldesmon expression was shown by immunohistochemistry ( fig. a-d) . due to the exclusion of a lactate enrichment of cardiomyocytes, cardiac fibroblasts are also expressed in the spheroids shown by vimentin staining. those cardiac fibroblasts lead to a physiological heterologous cell population similar to the human heart. beating spheroids were cultivated for days under dynamic culture conditions in the multi-organ-chip. the integrated on-chip micropump provides physiological-like pulsatile circulation at a microliter scale and leads to better nutrition and oxygen supply. the next significant step is to combine multiple autologous d organ equivalents in our multi-organ-chip using ipsc differentiation technology. differentiating all cell types from one ipsc donor is crucial to overcome source and rejection problems. combining our multi-organ-chip platform with ipsc differentiation technology will eventually lead to a personalized system for drug and substance testing. lab as a service -automated cell-based assays lena schober, moriz walter, andrea traube laboratory automation and biomanufacturing engineering, fraunhofer ipa, stuttgart, germany correspondence: lena schober (lena.schober@ipa.fraunhofer.de) bmc proceedings , (suppl ):p- background the use of cell-based assays in pharmaceutical industry and academic research is a growing trend that is a driving force to reduce costs for drug development. academic research is gaining information about intracellular targets or functional mechanisms through the variety of different assays. these benefits can be used in preclinical studies and furthermore costly late-stage drug failures may be reduced by the use of cell-based assays. the use of automated systems is also in great demand and will change the testing of substances and research activities. nevertheless, there are a lot of barriers at the moment limiting the successful application of automated systems in this field. by the lack of flexibility and the demand for skilled computer scientists & engineers just the two main aspects stated by experts shall be mentioned. our strong background on automated cell culture technologies and expertise, gained in several projects, let us rethink the overall process chain and overcome established principles. a new service orientated platform for the execution of cell-based assays that are commonly used will be introduced. the main idea is to give access to automated infrastructure for academic research or spin-offs which cannot afford the special infrastructure. nowadays it is known that the development of inhibitory antibodies by hemophiliac patients is closely related with immunogenic epitopes present in the coagulation factors. these proteins are produced in hamsters cells [ - ] which insert a different posttranslational modification profile when compared with the human profile. patients with high-titer/high-responding inhibitors must be treated with bypassing agents that can achieve hemostasis. activated factor vii (fviia) is an attractive candidate for hemostasis, independent of fviii/fix, making this coagulation factor an alternative for hemophilia patients with inhibitory antibodies. however recombinant factor vii is produced in bhk- cells (baby hamster kidney cells) and as well as the others coagulation factors, it may contain immunogenic epitopes [ - ] . in this context, becomes extremely important to produce recombinant proteins with complex posttranslational modifications in a cell line not yet used [ - ] . we have been using the sk-hep- human cell line for the production of recombinant fvii. to generate the recombinant cell line we have used a bicistronic lentiviral vector, -gfp, containing a fvii gene and the gfp selection marker gene. a master cell bank and a work cell bank were generated in gmp conditions. the rfvii analyses were made by elisa assay, western blot, gene expression quantification and biological activity using the prothrombin time (pt) assay. rfvii purification by affinity chromatography using viiselect (ge) column. after purification the rfvii was formulated and dry froze to be used in in vivo experiments. in static conditions sk-hep- cells showed, for a period of months, a stable fvii production with an average of , iu/ml of fvii, % of cell viability and % of cells expressing the gfp gene. after purification with viiselect column it was possible observe a recover of % of the purified protein with % degree of purity (fig. ) . this recombinant purified fvii is being used in in vivo experiments to determine the pharmacokinetics parameters and to evaluate the posttranslation modifications profile. in conclusion, this study reports the use of sk-hep- cell line for high-level production of recombinant factor vii. these cells have proven to be effective in the production of recombinant protein and can be used as a new platform for the production of recombinant proteins. fig. (abstract p- ) . a determination of protein yield of egfr (epidermal growth factor receptor) synthesized in a cho cecf system. analysis of egfr protein yield obtained in a various batches of cecf formatted reaction. cecf synthesis was performed in the presence of c leucine for radio labeling of target proteins. radio labeled proteins were precipitated using tca followed by scintillation measurement. b detection of radio labeled egfr by autoradiography. a no template control (ntc) was prepared containing no egfr dna template background emergence of stem cell-based regenerative medicine recently leaded to the necessity to reach a sustained production of such cells [ ] . hence, new bioreactors and carriers were designed for cell expansion. however, to meet this increasing demand, improvement of both quality and quantity of stem cells remains necessary. soft biocompatible microcarriers mimicking extracellular matrix in term of structure and stiffness should be of valuable utility as substrate stiffness strongly influence in vitro stem cell fate and differentiation [ , ] . our expertise in the field of microbeads design using jetcutting technology [ ] enabled us to engineer +/- μm alginate beads of various g/m monomer ratio. we used jetcutter (genialab gmbh) with μm nozzle at max speed rpm. alginate solutions with concentrations % to % were gelifyed in % cacl etoh % solution. alginates with estimated viscosity (@ %) from to mpa were tested. a further surface treatment with gelatine ( , %, %) and poly-l-lysine ( , %) was carried out to reach an optimal cell anchoring of human adiposederived mesenchymal stem cells (atcc-psc- - ) in mesempro rs medium (gibco). jetcutter technology allowed us to obtain alginate microcarriers with a good homogeneity in size around μm and sphericity comparable to commercial carriers (table ) . best adhesion of human adipose-derived mesenchymal stem cells was obtained on , % gelatine coated alginate carriers (fig. ) . we observed limited apoptosis and human adipose-derived mesenchymal cells stemness was conserved after days in culture (data not shown). cellular bioassays developed with functionally immortalized cell lines aileen bleisch , aleksandra velkova , tom wahlicht , dagmar wirth , tobias may inscreenex gmbh, braunschweig, germany; msys, helmholtz centre for infection research, braunschweig, germany; greiner bio-one gmbh, frickenhausen, germany bmc proceedings , (suppl ):p- background a major challenge of current research is the limited availability of physiologically relevant cells [ ] . thus the development of relevant cellular bioassays that are robust, reproducible and scalable is hindered. to overcome current limitations we developed an immortalization strategy allowing the efficient and reproducible establishment of novel cell lines showing an in vivo-like phenotype. the main feature of our ci-screen technology is the ability to combine the advantage of cell linesthe unlimited cell supplywith the advantage of primary cellsthe physiological relevance. using this technology we have immortalized, amongst others, a human osteoblast cell line (ci-huob) [ ] . in the present study, the in vivo-like phenotype and functionality of the novel ci-huob was examined. therefore, ci-huob cells were used to develop a d cell culture model by using the magnetic d bioprinting technology (nano d biosciences, houston, tx, usa) [ ] . the ci-huob cell line was recently described and cultivated in huob maintenance medium (inscreenex, germany). for spheroid creation ci-huobs were grown in a monolayer, magnetized by adding a magnetic nanoparticle assembly (nanoshuttle, ns, nano d biosciences, houston, tx, usa) at a concentration of μl ns/cm growth area. after an overnight incubation magnetized ci-huob were detached and seeded into cellstar® cell-repellent -well plates (greiner bio-one, frickenhausen, germany). with the help of mild magnetic forces cells were printed into spheroids within h. these consist of . - . cells and were cultured for a period of up to days. the cell viability was analyzed by a propidium iodide (pi) and calcein am staining. to improve spheroid functionality spheroids were cultivated with huob differentiation medium (inscreenex, germany). "mini bone" tissue functionality and thus mineralization was analyzed by an alkaline phosphatase (alkaline phosphatase activity) and an alizarin red s staining (ca + deposits). the combination of ci-huob cells with the magnetic d bioprinting technology enabled the establishment of reproducible and consistent d spheroids. single spheroids per well were formed independent of the amount of cells ( . - . cells) (fig. a) . formed spheroids were stable for a culture period of up to days (fig. b) . neither cell death nor cell proliferation were observed in the bioprinted spheroids which is indicated by the stable size of the spheroids throughout the cultivation (fig. c) . after treatment with a differentiation stimulus the d bioprinted spheroids became fully functional "mini bones". this was highlighted by the alkaline phosphatase activity and the ca + deposits within the d bioprinted spheroids (fig. d,e) . taken together, these results demonstrated that the functional immortalization technology provides physiologically relevant cells in sufficient numbers and that the magnetic d bioprinting technology enabled a fast, consistent cell aggregation and the formation of stable uniform spheroids. importantly, these immortalized cells are capable to differentiate when a suitable stimulus is provided. for differentiation into mini bones, d spheroid cultivation and additional stimulation by small molecules are required. the combination of physiologically relevant cell systems with three dimensional culturing will help to generate in vitro test systems which closely resemble the in vivo physiology and thereby supporting future drug discovery approaches. fig. (abstract p- ) . characterization of spheroid "mini bones". a different number ( . - . cells) of ci-huob cells were printed into spheroids. b . ci-huobs were printed into spheroids and cultivated for indicated time points. c for analyzing spheroid sizes, pictures were taken and quantified by imagej. (d/e) . ci-huob cells were printed into spheroids and cultivated with (huob differentiation medium) or without a differentiation stimulus for two weeks. afterwards, bioprinted spheroids were sectioned by a cryo microtome and d stained for ca + deposits (alizarin red s) or e stained for alkaline phosphatase activity crispr/cas , a novel genomic tool to knock down microrna in vitro and in vivo degrontagged dcas /cpf effectors for multi-directional drug-inducible control of synthetic gene regulation assessing the variability of an innovator molecule n-glycan profile correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up esi and maldi mass spectrometry techniques d-dige screening of high productive cho cells under glucoselimitation -basic changes in the proteome equipment and hints for epigenetic effects dependence on glucose limitation of the pco influences on cho cell growth, metabolism and igg 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cells and continuous harvest of vector from the culture media for gmp fix and flt clinical vector development of a cost-efficient scalable production process for raav- based gene therapy by transfection of hek- cells simon arias , mustapha hohoud , roel lievrouw , fabien moncaubeig b- brussels, belgium; généthon, rue de l'internationale cell-free systems based on cho cell lysates: optimization strategies, synthesis of "difficult-to-express" proteins and future perspectives cell-free protein expression based on extracts from cho cells comparison of cell-based vs. cell-free mammalian systems for the production of a recombinant human bone morphogenic growth factor ires-mediated translation of membrane proteins and glycoproteins in production of recombinant factor vii in sk-hep- human cell line zip - , brazil; department of clinical, toxicological and food science analysis, faculty of pharmaceutical sciences of ribeirão preto human cell lines for the production of recombinant proteins: on the horizon production of recombinant protein therapeutics in cultivated mammalian cells recombinant protein therapeutics from cho cells - years and counting establishment of a cell line expressing recombinant factor vii and its subsequent conversion to active form fviia through hepsin by genetic engineering method expression and fast preparation of biologically active recombinant human coagulation factor vii in cho-k cells implications of the presence of n-glycolylneuraminic acid in recombinant therapeutic glycoproteins uniquely human evolution of sialic acid genetics and biology production platforms for biotherapeutic glycoproteins. occurrence, impact, and challenges of non-human sialylation human cells: new platform for recombinant therapeutic protein production therapeutic glycoprotein production in mammalian cells masthering industrialization of cell therapy products tissue cells feel and respond to the stiffness of their substrate matrix elasticity directs stem cell lineage specification continuous cider fermentation with co-immobilized yeast and leuconostoc oenos cells eternity and functionality -rational access to physiologically relevant cell lines generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies biocompatibility of nanoshuttletm and the magnetic field in magnetic d bioprinting publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal • we provide round the clock customer support • convenient online submission • thorough peer review • inclusion in pubmed and all major indexing services • maximum visibility for your research submit your manuscript at www submit your next manuscript to biomed central and we will help you at every step authors thankfully acknowledge the biotechnology and biological sciences research council for funding this research work. sns thanks esact for providing her with the opportunity to present her work at the meeting. we would like to thank moritz frei for his support for the generation of the ngs transcriptomics data. many thanks to valentine chevallier for her precious advices, to stefanos grammatikos for his support and to the whole upstream process sciences team. we thank david bruehlmann and thomas vuillemin from merck (vevey, switzerland) for providing the igg glycan variants and the -ab-uplc glycan data. polyplus-transfection would like to thank généthon for their kindly provided data.acknowledgements asmita mukerji, reetesh pm, sasi kumar k, pilot plant team acknowledgment to cedric allier from cea leti, grenoble, france. we would like to thank a. schemel and a. ehrlich for technical assistance. the authors would like to mention that this research was supported by the fi-dgr ( ) from spanish government and the project was led by prof. jordi joan cairó badillo. the authors want to thank the biotech process sciences team at merck in corsier-sur-vevey for their support and also the members of the morbidelli group at eth zürich for their input and collaboration. authors would like to thank dr. benjamin youn in manufacturing science and technology (msat) at biomarin for his help on coding the excel macro program for ambr , and dr. donald l. traul from tap biosystems (now part of the sartorius stedim biotech group) for his assistance on ambr operations. thanks to the bioreactor team of dustin davis, amer al-lozi, and jana mahadevan. we organic vaccines tm and the nih, who kindly provided to univercells. we thank polymun scientific immunbiologische forschung gmbh for providing the antibodies igm , igm and igm as a kind gift. this allison kurz and gian andrea signorell, genedata ag, basel, switzerland allison kurz, gian andrea signorell, genedata, basel, switzerland. allison kurz, gian andrea signorell, genedata ag high glucose concentration and low specific cell growth rate improve specific r-tpa productivity in chemostat culture of cho acknowledgements r. boraston for photographs in fig. . we acknowledge atum for their contributions to vector design and construction. austrian bmwfw, bmvit, sfg, standortagentur tirol, government of lower austria and business agency vienna through the austrian ffg-comet-k . this research is supported by sfi grant /ia/ . financial support of the austrian science fund (fwf; grant number p ) is gratefully acknowledged. we would like to thank the australian institute for bioengineering and nanotechnology, university of queensland-brisbane, australia (aibn) for providing the cho clones. this research is partially supported by the developing key technologies for discovering and manufacturing pharmaceuticals used for next-generation treatments and diagnoses both from meti and amed, japan. this work was supported in part by grants for developing key technologies for discovering and manufacturing pharmaceuticals used for next-generation treatment and diagnoses, both from the ministry of economy, trade and industry (meti), japan and from the japan agency for medical research and developments (amed). many thanks stefanos grammatikos for his support and to the whole upstream process sciences team. the authors thank the hessen state ministry of higher education, research and the arts within the hessen initiative for scientific and economic excellence (loewe program) for the financial support. the authors thank xell ag, bielefeld, for providing hek serum-free media (hek gm and hek tf) and for fruitful discussions. the authors would like to thank dana wenzel for cho lysate preparation (fraunhofer izi, potsdam-golm, germany). this work is supported by the european regional development fund (efre) and the german ministry of education and research (bmbf, no. b a). the authors acknowledge são paulo research foundation -fapesp ( / - ), centro de pesquisa, inovação e difusão (cepid), and national institute of science and technology in stem cell and cell therapy -inctc for financial support. this work was supported by grants from the niedersächsisches ministerium für wissenschaft und kultur ( ) and the german ministry for economic affairs and energy (igf n). the infrastructure which was modularly built up, consists of automated liquid handling robots, plate and tube handling robots as well as incubators, refrigerator and analysis systems as for example an imaging system. the aim is to address the need on reproducibility and reliability of results and to offer access to a maximal controlled and automated environment. with the help of a web-based configurator assay selection as well as parameterization of the assays can be done in an easy way. after the order process, test items can be shipped to the lab. assays will be executed on the fully automated platform. by capturing in process data as well as environmental conditions, a real complete data set is leading to comprehensively results. as soon as results are available during the process, the view and analysing can be done in a secure cloud. the service can be used for single experiments in low throughput applications and is therefore a benefit for labs which cannot afford automated infrastructure or the staff for the maintenance for such platforms. extensive monitoring and data capturing during the run leads to a gapless data trail and the possibility of detailed result analysis. due to automated processing the reproducibility is increased associated with direct reduction of costs and time. the centralized service paired with specific know-how allows up-scaling of processes at any time. the web-based interface provides a flexible guidance for the user and the online order gives / access on the infrastructure, leading to a fast reliable result generation. furthermore the secure interaction with additional services e.g. other specific data analysis tool is possible. this dynamic access to automation offers high flexibility for low throughput experiments and will push high quality research and drug development in early stage. development of alternative animal cell technology platforms: cho based cell-free protein synthesis systems for the production of "difficult-to-express" proteins lena thoring , background nowadays, animal cell technologies are commonly used for a broad range of medical and pharmaceutical applications. one main topic of these technologies is the production of proteins used for therapeutical purposes. these in vivo production processes are often time consuming and limited in production of so called "difficult-to-express" proteins including the pharmaceutical relevant class of membrane proteins. to overcome these issues, novel cell-free protein synthesis platforms were developed based on the industrial working horse cho cells [ ] . cell lysates provide a basis for this technology by including all components of the translational machinery and enabling protein production within a few hours. microsomal structures present in cho cell lysates enable posttranslational modification of target proteins and insertion of membrane proteins into lipid bilayer. in this study a cell-free protein synthesis platform was developed based on a combination of cho cell lysates and a continuous exchange reaction format. the continuous exchange reactor consists of a twochamber system, a reaction and a feeding chamber, separated by a semipermeable membrane. due to concentration gradients, energy components can diffuse to the reaction chamber, while inhibitory byproducts are continuously removed. different classes of proteins were selected to evaluate the quality of the cho cecf system including a transmembrane receptor, a single chain variable fragment and an ion channel. cell-free protein synthesis was performed in the presence of c leucine for radio labeling of synthesized proteins. protein yield was quantified by tca precipitation of radio labeled proteins followed by scintillation measurement and molecular mass was detected by autoradiography. posttranslational modifications and activities of proteins were estimated by kinase assays, elisa, endoglycosidase treatment and electrophysiological measurements. the demonstrated results showed a protein production of up to around g/l while detecting correct molecular weights by autoradiography. analysis of the productivity using different lysate batches by the production of the membrane protein egfr revealed only minimal batch-to-batch variations (fig. a) . posttranslational modifications of proteins, including phosphorylation and glycosylation, were detected using western blot and autoradiography (fig. b) . evaluation of localization of membrane embedded eyfp fusion proteins by confocal laser scanning microscopy resulted in the detection of proteins in the microsomal fraction of cho cell lysate. produced single chain variable fragments showed binding specificity in elisa experiments. the activity of synthesized ion channels was underlined by electrophysiological measurements and detected single channel activities. a cell-free system based on cho cell lysates for high yield production of proteins was developed that provides a platform for efficient production of "difficult-to-express" proteins. the combination of a cho lysate based cell-free system and a continuous exchange cell-free system leads to be a highly efficient production system for various classes of "difficult-to-express" proteins. this approach opens up a fast and cost-effective process pipeline for the production of "difficult-to-express" proteins and shows a high potential for industrial applications including screening technologies, protein structure determination and just-in-time protein production processes. key: cord- -rpx ww authors: doukas, sotirios g.; kavali, leena; menon, rohan s.; izotov, boris n.; bukhari, amar title: e-cigarette or vaping induced lung injury: a case series and literature review date: - - journal: toxicol rep doi: . /j.toxrep. . . sha: doc_id: cord_uid: rpx ww introduction: recently, a rapidly increasing number of e-cigarette or vaping induced lung injury (evali) has been reported across the nation. given the ongoing epidemic, it has been suggested that specific chemical substances used as additives in e-cigarettes could be highly related to evali. a history of vaping with positive radiographic changes and low suspicion for active infection are requirements for diagnosis but it still remains a diagnosis of exclusion. the course of the disease, mechanism of lung injury and the optimal management options need to be better understood. here we aimed to discuss the clinical characteristics recognized in a case series of ten hospitalized evali patients with radiological findings of lung injury and provide an up today summary of the known literature of evali-induced lung injury. methods: a retrospective chart review was conducted on ten patients who presented to saint peter’s university hospital in new brunswick, nj from july to february , with a mean hospital stay of five days. according to the cdc recommended definition of the disease, our cases met the current working definition of confirmed or probable cases of evali. results: ten patients, with mean age . years ( % male) and average years of vaping . with % endorsing a simultaneous history of cannabis-related products use, went under a retrospective review. / ( %) had documented medically-managed pulmonary disease history, / ( %) presented with the respiratory-related chief complaint, / ( %) presented with gastrointestinal symptoms and / ( %) had constitutional symptoms. all patients ( %) were found to have bilateral ground-glass opacities on chest imaging. / were admitted, / ( %) had an oxygen saturation of < % requiring oxygen supplementation with / managed in the intensive care unit. conclusion: evali patients with radiological findings of lung injury, although mainly present respiratory symptoms, may very often appear with constitutional and gastrointestinal symptoms. based on the existing literature and our data it is argued that evali may be misdiagnosed and that closer monitoring is required to determine optimal diagnostic and therapeutic management of this condition. our data and the existing literature suggest that laboratory and epidemiologic findings can be contributory for the diagnosis of the disease. the use of electronic-cigarettes (e-cigarettes) or vaping products has rapidly increased in the last few years, especially among adolescents and young adults [ ] [ ] [ ] [ ] . although early studies have raised awareness about the harmful effect of e-cigarettes or vaping products [ ] , the healthcare industry remained largely neutral on the topic until convincing data started surfacing in . electronic-cigarette or vaping induced lung injury (evali) was first reported in [ ] , and as january of , hospitalized cases of evali have been reported from states of united states of america (usa), with deaths [ ] . the majority of patients were young, under the age of years [ , ] . although the mechanism of evali is still not well understood, it has been suggested that heating can transform the compounds and additives in vaping products into respiratory irritants and carcinogens [ ] [ ] [ ] [ ] . blount et al. emphasized the effect of vitamin e acetate, a substance used as a thickening agent in tetrahydrocannabinol (thc) containing products as a potential cause of respiratory endothelial injury, suggesting that it could be associated with transition of surfactant from gel to liquid crystalline form, leading to loss of surfactant's ability to maintain surface tension [ ] . although there has been a lot of emerging data on evali, it still remains a diagnosis of exclusion with no confirmatory diagnostic test [ , , ] . significant overlap based on symptomatology can lead to misattribution and misdiagnosis [ ] . given the rapidly growing number of cases, a detailed report of clinical characteristics recognized in evali cases with radiological findings of lung injury will not only provide a better understanding of the natural history of the disease, but also create a strong foundation for the development of evidence-based guidelines for the diagnosis, prognosis, and treatment of this condition. here we present the clinical characteristics of a series of hospitalized evali cases at our institution, and discuss the importance of our data based on the existing literature describing the unique characteristics of the disease that would be significant for the its proper diagnostic and therapeutic approach. ten patients, - years old, with symptomatology similar to the previous evali reported cases such as dyspnea, cough, fatigue, nausea, and vomiting were admitted to the saint peter's university hospital in the period july -february . the data were obtained according to our institutional review board (irb) approved protocol. the patients that were included in this study fulfilled the centers for disease control (cdc) and prevention proposed criteria for the definition of confirmed or possible evali cases and were eventually discharged with a main diagnosis of evali [ , ] . the use of e-cigarettes within the last days before the onset of symptoms and radiographic imaging of opacities or ground glass appearance are the main features of the suspected evali. confirmed cases require the absence of signs and associated symptoms and laboratory findings indicating absence or low suspicion of ongoing infection. probable cases are defined as having the symptomatology and imaging findings, however with laboratory studies indicating a possible ongoing infection. in both cases, no evidence of alternative diagnosis is required [ , ] . all of the patients included in this study presented in the emergency department of saint peter's university hospital during the period of july to february and were eventually discharged with a primary diagnosis of evali. the age range was - years with a mean of years. five of the patients were males ( %) and five of them were females ( %) ( table ) . all the patients reported marijuana use, however, only % of the patients verified vaping history of cannabis-related products use [thc or cannabichromene (cbc)] (table ) . urine toxicology was performed in all the patients with six of them ( %) being positive. from the patients reported using cannabis-related vaping products three of them had positive thc urine test. the average years of vaping of the patients were . (range month to years) ( table ) . only one patient reported a recent initiation of vaping (one month before the hospitalization). only two out of ten patients ( %) were actively using tobacco products, while three of them ( %) were former tobacco users and five out of ten ( %) never used tobacco before. eight out of ten ( %) had respiratory complaints such as cough, shortness of breath and pleuritic chest pain, six out of ten ( %) presented with gastrointestinal symptoms such as nausea, vomiting, abdominal pain, seven out of ten ( %) of the patient had constitutional symptoms of fever, fatigue and generalized weakness ( table ) . all of the patients were hospitalized with a mean hospital stay of five days (variation - . days) ( table ). two out of ten patients had a past medical history of asthma (table ) and one patient had a history of chronic obstructive lung disease. five patients were evaluated as an outpatient for their complaints and treated with antibiotics without any significant improvement prompting them to come to the emergency department. four out of ten patients were admitted to the intensive care unit (icu) of the hospital due to acute hypoxic respiratory failure (table ) . specifically, three patients were admitted directly to the icu on the day of admission and one of the patients was initially managed on the medicine ward, and on the fifth day of hospitalization the patient's respiratory status deteriorated and the patient was transferred to the icu. at the time of presentation, out of patients ( %) were febrile with a temperature of > . f ( table ). all patients were hemodynamically stable with a systolic blood pressure (bp) of more than mmhg and half of the patients ( %) were tachypneic with respiratory rate (rr) > breaths per minute (table ). six out of ten of the patients ( %) were tachycardia with a heart rate of > beats per minute. also, six out of ten ( %) had an oxygen saturation of < % requiring oxygen supplementation via nasal cannula to maintain their oxygen saturation > % (table ) . four out of the ten patients demonstrated increase in oxygen demands required more aggressive oxygen therapy and admission to icu for close monitoring. although the oxygen saturation levels in two of these patients maintained good at presentation, an increase in oxygen demand and an increase in work of respiration, necessitated the use of non-invasive positive pressure ventilation (nppv) or bipap (table ) . arterial blood gasses (abg) that performed in these two patients before the initiation of nppv showed significant hypoxia but not hypercapnia (table ). specifically, in patient the abg showed ph . , pco , po , hco and o saturation of % and in patient ph . , pco , po , hco and % (table ) . also, another patient, patient number , who was admitted to icu, had an oxygen saturation % at presentation, requiring immediately placement on bilevel positive airway pressure (bipap) ( table ) . abg was performed after initiation of nppv improvement of oxygen levels with ph . , pco , po , hco and o sat %. another patient, patient number , that was directly admitted to icu presented with oxygen saturation of % on l was placed on venti mask to maintain oxygen saturation levels ( table ). imaging studies in all the patients were significant for radiological findings of lung disease. computed tomography (ct) was performed in nine of ten patients ( %), showing opacities or infiltrates similar to previously reported evali cases (table , fig. ). one of the patients had only a chest x-ray showing characteristic peribronchial thickening and right and left sided opacities. one of the patients demonstrated emphysematous changes possibly related to chronic underlying lung disease. this patient is a former smoker with a reported history of pack-years that quitted years ago. imaging on the presentation is shown below in fig. . all the patients were found to have leukocytosis with a white blood cell (wbc) count of > , . also, all the patients were found to have leukocytosis with lymphocyte count. nine out of the ten patients that were tested for legionella urine antigen and all of them were found to be negative. more than half of the patients ( %) were tested for pneumococcal urine antigen, and all of them found to be negative. the respiratory viral panel screening for influenza a (subtype h and h ), influenza b, respiratory syncytial virus a and b, adenovirus, human metapneumovirus, parainfluenza , , and , and rhinovirus, was negative in seven patients; however, two cases were positive for rhinovirus. although rhinovirus was positive in these patients, the clinical course and symptoms were not suggestive for rhinovirus infections. hiv test was offered and performed in seven out of ten patients ( %) and was found to be non-reactive in all of them. procalcitonin levels were assessed in most of the cases ( %). in seven out of eight patients the procalcitonin was assessed was found to be normal (< . ng/ml) with values in ng/ml: . , . , . , < . , . , . , . , indicating a low probability for ongoing bacterial infection. one of the patient's presented with elevated procalcitonin . ng/ml that decreased to the normal value . ng/ml within days (< . consider normal, ng/ml). additional laboratory investigations were performed in individual cases, such as c-reactive protein (crp), erythrocyte sedimentation rate (esr) and mononucleosis test, and quantiferon test. crp was evaluated in two cases. in one of the cases the crp was slightly elevated ( . mg/dl) while in the other case crp was significantly increased (> mg/dl). normal value for crp is considered < mg/dl. esr was assessed in two patients, and in both cases was found to be elevated ( mg/dl and mg/dl, respectively). mononucleosis rapid test was performed in two cases and in both was negative. also, the quantiferon tb gold test was performed in two cases and found to be negative. none of the reported patients underwent bronchoalveolar lavage. all the patients ( %) were treated empirically with intravenous antibiotics for community-acquired and atypical pneumonia and % were treated with a minimum of days of systemic glucocorticoids, including intravenous steroid therapy followed by a tapering course of oral steroids ( table ) . all the icu patients were treated with hi-flow nasal cannula for oxygen support, while two of them required further support with bipap ( table ) . none of the patients were intubated ( table ). no deaths have been recorded. follow up data are presented in table , demonstrated that all the treated patients were not readmitted to the hospital. laboratory findings for wbc counting were normal and imaging findings were found stable or improved. the increase in the prevalence of e-cigarette use in the last two years and the recent epidemic of evali necessitates a careful study of the characteristics and the pathophysiology of this syndrome [ , , ] . here, we present data from a series of ten cases that recognize clinical characteristics in hospitalized evali patients with radiological findings of lung injury. the symptomatology of evali has been addressed by previous clinical studies, identifying both respiratory and non-respiratory symptoms related to the disease. the most common respiratory symptoms include dyspnea, cough, and chest pain, and in most cases accompanied by non-specific, constitutional symptoms, such as fever, chills, fatigue, and malaise [ , , , ] . in line with previous studies, the majority of our patients ( %) presented with respiratory symptoms, such as cough and shortness of breath (table ) . gastrointestinal symptoms, such as vomiting, diarrhea, and abdominal pain, were also very common in patients included in this study. there is strong evidence that patients with evali can often present with gastrointestinal complaints [ , , , ] , however the etiopathogenesis leading to these symptoms is not well understood. also, fever and oxygen desaturation, may or may not be part of the initial presentation, and most of the auscultatory findings are considered non-specific [ , ] . overall, given that symptoms are often non-specific, the diagnosis is usually challenging and a significant number of cases are misdiagnosed with pneumonia [ , ] . our knowledge on the pathophysiology of evali is still growing. the most well-supported theory is that e-cigarette irritants lead to a native immunity reaction producing a sterile exogenous pneumonitis [ ] . recent histologic evaluation of series of evali patients showed non-specific inflammatory changes suggesting acute lung injury, such as fibrinous changes, diffuse damage of alveolar architecture, and bronchiolitis. a broad spectrum of substances found in e-cigarette products such as oils, additives, or even heavy metals, could potentially be involved in evali [ , , [ ] [ ] [ ] . some of them include substances that can be found in both nicotine and non-nicotine e-cigarettes, including additives such as glycerol [ , ] and propylene glycol or even compounds found in the metallic coil of the apparatus [ ] . although the toxicity of these substances might be minimal under normal conditions, rapid heating may induce their chemical transformation into toxic byproducts and promote their massive release in aerosol [ , , ] . after vitamin e acetate was identified in bronchoalveolar lavage (bal) fluid specimens of patients with evali, theoretical and experimental studies have focused on the exploration of the role of vitamin e in evali [ ] . results of hematologic tests may also vary and could be non-specific for diagnosis of evali. similar to other studies, common findings in our patients included leukocytosis with neutrophilia. previous studies also reported an increase in inflammatory markers such as esr and crp [ , ] . inflammatory markers esr and crp were assessed in three of our patients, demonstrating relative elevations. however, given the absence of sufficient data and standardized guidelines, the diagnostic and prognostic utility of these markers is controversial. radiologic findings are closely associated with the severity and the progression of the disease [ , , [ ] [ ] [ ] [ ] . in most of the cases, chest x-ray demonstrates bilateral infiltrates and ct of the chest, often shows extensive bilateral ground-glass opacities [ , [ ] [ ] [ ] [ ] . pleural effusions, pneumomediastinum, and tree in bud opacities have also been described in previous case reports [ , , ] . the imaging studies of our patients showed ground-glass opacities similar to those previously reported in other evali cases (fig. ). however, we should highlight that variation exists and multiple patterns may be seen. bronchoalveolar lavage (bal) could have some applicability in cases where the suspicion of evali is high but clinical and imaging findings are not sufficient for diagnosis. in particular, bronchoscopy may be considered in cases of atypical radiologic findings suggesting alternative pathology, such as vasculitis, or in immunocompromised patients, but it is not strongly recommended for each patient with suspected evali [ , ] . bal has been performed in previous cases, typically showing eosinophils and neutrophils or lipid-laden macrophages with oil red o stain, indicating lipoid pneumonitis and/or eosinophils, but findings may vary [ , , , ] . although positive findings might have some specificity, and applicability, the necessity and the sensitivity are still questionable. recent studies showed that vitamin e, a known component of many thc and cbc products, could be a marker for evali given the high prevalence in bal fluids derived from patients with evali [ ] . in the presented study, bal was not performed given the sufficient exclusion of infectious causes and high suspicion for evali. we could speculate that the low positivity of urinary thc products in our cases may be due to the low frequency of use of cannabis-related-products or use of undetectable in urine toxicology cdc products. clinicians should be aware of the clinical features of evali and obtain detailed and proper e-cigarette/vaping history in all patients and especially the high-risk groups. although significant elements of ecigarette/vaping history have been suggested [ ] , incorporation of a routine screening for vaping in every inpatient and outpatient encounter should be performed. until now there is no standardized approach to vaping history. given the absence of specific inclusion criteria, evali remains a diagnosis of exclusion. the criteria as were previously described by layden et al., require the use of an e-cigarette or dabbing in days before onset followed by radiographic evidence of the disease such as opacities in the chest x-ray or ground-glass opacities on chest ct [ ] . confirmation of the case requires the exclusion of underlying infective causes after extensive workup. according to the same criteria, even if the infection is identified but the clinical picture and disease progression imply that this is not the sole cause of the respiratory disease, the case should be characterized as probable evali [ , ] . absence also of other plausible diagnoses that could explain the clinical picture, such as cardiac, rheumatologic or neoplastic disease, is necessary for either confirmed or probable cases [ , ] . although the nature of evali is a better understanding due to the knowledge to date, significant variations have been noticed in the course of the disease and several case reports have shown that the severity and the clinical picture may vary from patient to patient [ , , , ] . despite the fact that, variations of the severity exist the average number of days of hospitalization of the current study is . , similar to that reported by other case series and extended epidemiologic studies. specifically, layden et al. reported an average of days, while siegel reported an average of . days [ , ] . layden et al., % of patients were intubated at some point during hospitalization [ ] . siegel et al., in a larger patient sample of patients showed that % of them required intubation and mechanical ventilation. in our study, although a higher percentage of patients were found hypoxemic ( %) compared to other studies (layden et al., % siegel et al., %), only three out of ten ( %) of the presented patients required non-invasive positive pressure ventilation (nppv) and none of them were intubated. although limitations could apply due to the small number of patients in our study, the differences in requirements for aggressive airway management compared to other studies could indicate other compounding factors, such as coexisting comorbidities and age [ ] . specifically, in the present study none of the patients had reported cardiovascular disease and two out of the ten had asthma, one patient had chronic obstructive airway disease. this is in line with other studies in literature supporting that age and number of coexisting chronic cardiopulmonary conditions are highly associated with the severity of evali [ ] . as it was expected, the oxygen saturation levels at the admission were lower in the patients that were finally admitted to icu compared to the patients that treated in the medical ward (table ). further investigation of the association between oxygen saturation or arterial po levels at the admission and need for immediately nppv and icu transfer could be useful in the development of an evidence-based approach for the management of patients with evali. even though rehospitalization of patients previously treated for evali have been reported, in our study none of the patients was readmitted to the hospital in a period of months after hospitalization. according to recent epidemiologic studies coexistence of multiple chronic medical conditions may significantly increase the risk for rehospitalization of patients with evali [ ] . as in the previous report, only a small percentage of patients in our sample group had chronic cardiopulmonary disease ( %). in general, our patients appear to have relatively mild disease. we may assume that because our patients are young (< years old) the disease was mild. investigation of the effect of age on severity of evali requires a larger sample that includes a wide range of ages. there is a rapid increase in the number of deaths related to evali. specifically, layden et al. showed that % of the patients did not completely recover and died due to evali [ ] .however, the existing data show that the mortality rates of evali is relatively low [ , ] . the therapeutic approach and management of evali is not clear yet. since the knowledge is limited regarding the optimal treatment in the early and late stages of the disease, in most of the cases, antibiotic therapy is administered empirically, until underlying pneumonia is excluded. the use of steroids for treatment of evali remains controversial and although previous studies recommend the use of high doses of steroids [ , , ] , there is not enough evidence to clarify if there is a dose-dependent steroid effect. also, based on the recent data the extent of the steroid treatment varies and could be related to the severity of the disease. in our study, half of the patients ( %) received steroid treatment. all of the patients that presented with higher severity requiring transfer to icu and nippv were treated with steroids. generally, no clear guidelines exist for the management of evali, but considering that the pathophysiology of the disease includes immune reaction to e-cigarette irritants and the development of inflammation, steroid use is a reasonable approach. given the ongoing sars-cov- epidemic, a disease with clinical symptomatology that presents similarities with evali, an expected question is what are the unique clinical and laboratory findings that would help us differentiate the two diseases. according to previous reports, leukocytosis, favor the diagnosis of evali given lymphopenia is a common finding in covid- . vaping history remains key in making the diagnosis. [ , ] also, patients with evali were found to improve within corticosteroids compared to covid- [ ] . we acknowledge that a number of limitations apply to our study due to the small sample size. further investigations including a larger number of patients could increase the power of the study and provide stronger evidence for our conclusions and suggestions. given the majority of the patients defined themselves as non-hispanic whites ( table ) , investigation of the race effect on evali presentation, progression and mortality requires a larger and more variable sample. our ten-case series data recognized that clinical characteristics in hospitalized evali patients with radiological findings of lung injury, may include both respiratory and gastrointestinal or constitutional symptoms and based on the current literature may be misdiagnosed. in an era of new diseases affecting the respiratory system such as covid- , the clinician should be aware of the unique imaging and laboratory findings associated with evali. the reinforcement and standardization of the importance of e-cigarette and vaping history in both outpatient and inpatient settings are highly recommended, especially in high-risk groups like young adults. based on our data and the existing literature, the presence of lymphocytosis and the epidemiologic data, such as the age of the patient could be contributory in the diagnosis of the disease. future studies focusing on the comparison of clinical, laboratory and imaging findings of evali patients with patients presenting with similar respiratory conditions, with similar clinical manifestations, such as covid- , will contribute significantly in the development of standardized approach for evali diagnosis and treatment. pulmonary illness related to e-cigarette use in illinois and wisconsin-final report update: interim guidance for health care providers evaluating and caring for patients with suspected e-cigarette, or vaping, product use associated lung 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electronic-cigarette-product use -interim guidance the evali and youth vaping epidemics -implications for public health pulmonary toxicity and the pathophysiology of electronic cigarette, or vaping product, use associated lung injury fourth generation e-cigarette vaping induces transient lung inflammation and gas exchange disturbances: results from two randomized clinical trials metal concentrations in e-cigarette liquid and aerosol samples: the contribution of metallic coils tree-in-bloom": severe acute lung injury induced by vaping cannabis oil lung injury associated with electronic cigarettes inhalation diagnosed by transbronchial lung biopsy imaging of vaping-associated lung disease diffuse alveolar hemorrhage induced by vaping characteristics of patients experiencing rehospitalization or death after hospital discharge in a nationwide outbreak of e-cigarette, or vaping, product use-associated lung injury -united states pulmonary lipid-laden macrophages and vaping diagnosing evali in the time of covid- the lancet respiratory medicine, the evali outbreak and vaping in the covid- era covid- pneumonia versus evali, distinguishing the overlapping ct features in the covid- era the manuscript is original and it has not been published or accepted for publication, either in whole or in part, in any form. this manuscript is not under consideration elsewhere. the manuscript has been read and approved by all authors. the authors whose names are listed in this article certify that they have no affiliations with or involvement in any organization or entity with any financial interest, or non-financial interest in the subject matter or materials discussed in this manuscript. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord- - rzbrx authors: dohaney, jacqueline; de roiste, mairead; salmon, rhian a.; sutherland, kathryn title: benefits, barriers, and incentives for improved resilience to disruption in university teaching date: - - journal: int j disaster risk reduct doi: . /j.ijdrr. . sha: doc_id: cord_uid: rzbrx pandemics, earthquakes, fire, war, and other disasters place universities at risk. disasters can disrupt learning and teaching (l&t) for weeks to months or longer. some institutions have developed business continuity plans to protect key organisational services and structures, allowing l&t to continue. however, little research touches on how academics, learners, and communities of practice might respond before, during, and after disasters and how their resilience to disruption can be fostered to reduce impacts on l&t. in this research, we investigated academics’ perceptions of building resilience to major l&t disruptions in the new zealand context. specifically, we explored how academics characterise a resilient academic and institution, and identified the benefits, barriers, and incentives to building resilience. we used a pragmatic theoretical approach with a mixed methods methodology, to categorise the results within three distinct levels (individual, school/department, and institution), supporting the design and implementation of resilience-building strategies for academics and institutional leaders. we found that support, community, leadership, and planning at universities are critical in building and inhibiting resilience. participants reported several ‘high impact’ incentives, addressing multiple barriers, that could be used to kick-start resilience. online and flexible learning are key opportunities for resilience-building, but universities should not underestimate the importance of face-to-face interactions between staff and learners. our results provide a strong starting point for practitioners and researchers aiming to understand how universities can foster resilience to major disruptions and disasters on university teaching. in this research, we investigated academics' perceptions of building resilience to major l&t disruptions in the new zealand context. specifically, we explored how academics characterise a resilient academic and institution, and identified the benefits, barriers, and incentives to building resilience. we used a pragmatic theoretical approach with a mixed methods methodology, to categorise the results within three distinct levels ( individual, school/department, and institution), supporting the design and implementation of resilience-building strategies for academics and institutional leaders. we found that support, community, leadership, and planning at universities are critical in building and inhibiting resilience. participants reported several 'high impact' incentives, addressing multiple barriers, that could be used to kick-start resilience. online and flexible learning are key opportunities for resilience-building, but universities should not underestimate the importance of face-to-face interactions between staff and learners. our results provide a strong starting point for practitioners and researchers aiming to understand how universities can foster resilience to major disruptions and disasters on university teaching. earthquakes, fire, war, pandemics, and other disasters place universities at risk. disasters can disrupt learning and teaching (l&t) for weeks to months, and even longer. such significant disruptions are likely to multiply with increasing urbanisation, over-population and changing extreme weather and health crises, in particular, the h n flu pandemic in (ekmekci & bergstrand, ) . indeed, the differing conceptual approaches embraced by individuals and institutions can determine the framework implemented which will in turn influence their chosen actions and priorities (linkov and trump ) . for example, academic continuity has often focused, almost exclusively, on the use of digital technology to provide l&t during the event. ensuring a common understanding of resilience is, therefore, important in exploring its conceptualisation and possible manifestations within higher education institutions. as institutional scale resilience, or business continuity, has been a focus in the literature, the effects of l&t disruption on individual teaching practice has received little attention. this focus on the organisation is not unique to the academic sector and resilience is often explored at societal, community, or organisational scales (linkov and trump ) . in universities and other tertiary institutions, academics are responsible for how courses are delivered, materials presented, and what content is included. additionally, current institutional approaches tend to be responsive, rather than preventive, and focus predominantly on reducing the institutional economic risk following an event (e.g., wide scale online provision in the wake of covid- when face-to-face tuition was impossible). understanding the role of resilience in higher education and the difficulties faced by academics in making their practice resilient facilitates ongoing resilience rather than responses to singular events. this article explores academic and organisational resilience in university teaching, specifically academics' perceptions of the benefits, barriers, and incentives to building resilience. our investigation is supported by two key research questions: ) how do university staff describe a resilient academic and a resilient institution?, and ) what do university staff describe as the benefits, barriers, and incentives to building resilience? we explore both institutional and academic continuity but look further into individual perceptions of resilience-building in the university context. we applied a mixed methods methodology grounded in a post-positivist pragmatic approach (e.g., feilzer, ) using interviews, focus groups, and questionnaires. the key aim of the research was to identify key problems when l&t is disrupted and provide solutions leading to improved resilience. approval for this research was granted by victoria university of wellington (vuw) human ethics committee (# ). in this section, we first describe the context in which this research was undertaken, followed by the participants involved, and the methods for data collection and analysis employed. vuw is distributed across three campuses within wellington in new zealand and several smaller locations across the region. it is situated within an active earthquake zone and is at risk from several natural disasters (johnston et al., ) , but, until / , the potential impact to university l&t at vuw had not been well explored. in and , the canterbury earthquake sequence occurred causing fatalities, significant damage to the built and natural environment, and disruption of social and economic activities across the canterbury region in new zealand (potter, becker, johnston, & rossiter, ) . the scale of the disruption on the local university (the university of canterbury) (healey, ) , and other earthquake events in new zealand, increased the awareness of potential impacts to vuw. in response, the pro-vice-chancellor of sciences, engineering, architecture and design (sead) established a resilience steering group (i.e., a community of practice and research project) to explore how we can prepare staff and learners and promote l&t continuity in the event of significant disruption. the group ran from july to august and explored issues, such as post-disruption access to infrastructure and services, communication, staff and student support, and existing resilience and contingency plans. here, we present our investigation into how the steering group, and other university staff, perceived resilience to disruption and its improvement. the most likely scenario envisaged was an earthquake but other possible disasters were also considered, including pandemics. our participants were sampled from two groups of people. first, was the resilience steering group (sg) consisting of academics across the sead faculties (including the second and third authors of this paper), professional central-service unit staff, and a resilience coordinator (including the first author of this paper). the sg were asked to participate in the research via email, and ten took part ( academics, and professional staff). the second group of participants were sead academics, not members of the steering group (nsg) (n= ). the purpose of engaging this second group was to: ) assess perceptions of those who had not been immersed in the topic, ) raise awareness of resilience to disruption across sead, ) share ways to build resilience, and ) gauge their initial level of 'buy-in' while being new to the initiative. we aimed to sample across the disciplines, length of teaching experience, and workloads. nsg were recruited via an institutional e-newsletter and l&t community e- mailing list. to meet our cross-discipline sample, three participants were recruited directly by email. all participants (n= ) were asked to complete a hardcopy questionnaire at the start of the interview. questions included: gender, age, nationality, ethnicity, discipline, teaching and research workload, and teaching experience. participants included equal numbers of women ( ) and men ( ), a wide range of origin nationalities ( from nz and overseas), and a broad age-spread ( - , median of ). the ethnicity of the participants was predominantly european new zealander or european, and one māori participant. all participants were permanent (i.e., ongoing) staff ( academic, professional, and teaching-only roles) across a wide range of academic disciplines: architecture and design ( ), atmospheric science ( ), geography ( ), biology ( ), science history ( ), chemistry ( ), software engineering ( ), statistics ( ), mathematics ( ) , and psychology ( ), educational technology ( ), and fine arts ( ). participants also reported a range of teaching experience ( - years, median of ), mostly teaching-heavy course loads ( ), with a range of - courses per year and all but one of these participants co-teach at least one course. overall, participants reported diverse workload proportions with teaching-dominant ( ), service- dominant ( ), or distributed evenly amongst research/teaching/service ( ). we interviewed participants in one-on-one and focus group formats (lasting - mins). sg participants were interviewed first ( focus groups, and one-on-ones), followed by nsg participants ( focus group, one-on-ones). the resilience coordinator (dohaney) led the interviews with the participants and was known to the sg but not known to the nsg, and had personally experienced long-term l&t disruption during the canterbury earthquakes. the semi-structured interview protocol (see supplementary material) focused on (part ) characterisations of resilience to disruption, and (part ) benefits, barriers, and incentives to building resilience. part was used as a meaning-making experience for the sg, allowing us to form characterisations of resilience to disruption for communication with the wider community. part aimed to characterise the benefits, barriers and incentives of resilience- building. part results were coded, summarised, and shared with nsg participants to introduce the resilience initiative and help them understand the context of the project, before proceeding to part . during the interviews, we asked interviewees to take several moments to themselves to read and think about the questions and then write down (hardcopy) their thoughts before discussing them. these participant notes (including the same question prompts) were considered primary data and collected when the interview was completed. participant notes were collected to triangulate the verbal data and capture individual views (within the focus group format). interviews and participant notes were de-identified and transcribed by an external research assistant prior to data analysis. we initially approached the datasets holistically and then interrogated the data at different levels (micro, meso, and macro). the first author completed the data analysis (using atlas.ti qualitative coding software) using a conventional approach to content analysis (cohen et al., . using a pragmatic approach, we documented a wide range of unique perceptions prioritising comprehensiveness over depth of description. the participant's notes were analysed first as they represent individual views and because participants often wrote responses in bulleted lists, which helped to create quick, first- impressions of the data. next, the interview data was coded, and initial codes assigned. the multilevel theory approach allows researchers to investigate phenomena within and between major levels (or scales) of a system. this approach appealed to the researchers, as we intended to operationalise our findings into resilience-building initiatives within the existing levels of the university socio-political-system. we, therefore, organised and presented the themes by level. this approach does not follow a systems analysis (i.e., identifying vulnerabilities within a system) but retains the focus on academics' perspectives once a coding scheme was developed, the second and third authors checked the codes to support code generation, clustering, and scheme refinement, while the fourth author (who had no prior involvement in the project) checked the final code scheme through independently coding a full transcript (randomly-chosen participant), resulting in minor additions to one code category and rewording/clarification of another. code frequencies were used to guide our aggregation, clustering or parsing of information. several codes had low frequencies but clustering data in these areas reduced comprehensiveness of a resilience planning/building solution thereby reducing its utility (i.e., supported by our pragmatic approach) to building a solution for increased resilience. code categories and sub-categories were checked for statistical relationships between subpopulations (i.e., did participants with more teaching experience report more learner-centred benefits to resilience?) but no significant relationships were found; consequently, the findings are not presented by subpopulation. the results and discussion are presented within the four major themes identified in our data analysis: our community of practice's characterisation of resilience to disruption, followed by the benefits, barriers, and incentives to building resilience. the following sections of the paper are intended to assist the thinking and planning of university academics, staff, and leaders as they consider their own local context. our tables (tables - ), in particular, offer individual academics, schools, and academic institutions many factors which influence resilience to disruption. longer excerpts of text supporting tables - are available in the online supplementary material. here, we present the sg's characterisation of academics and higher education institutions resilient to disruption, within our institutional and cultural context. both characterisations were developed as shared ideals rather than rigid standards, or definitions, and should reduce l&t disruption regardless of the severity of the disruption. resilient academics (in the order of most frequently mentioned codes): • are flexible, adaptable, emotionally-resilient, collaborative, empathetic, open-minded individuals (attributes); • respond quickly during a disruption, are digitally literate, organised, prepared and creative-thinkers (capabilities); • have a sound awareness of their courses, learner-centred approaches, l&t delivery options during disruptions, emergency protocols, and the wider institutional system (knowledge). resilient institutions have the following characteristics (in the order of most frequently mentioned codes): • effective communication channels, • a coherent crisis communication strategy, • an established, coherent, l&t disruption plan across all levels of the institution, • strong resilience-building leadership, • existing emergency response plans and management, • existing flexible, blended, and digital learning strategies, • support for staff to undertake resilience-building initiatives, • support for staff to develop digital literacy, • effective and easy-to-use digital infrastructure, • a strong sense of staff and learner community, and • existing rewards/schemes to promote engagement with academic professional development (aligned to resilience qualities, above). the characterisations contain major themes that emerged throughout the study and include (in no particular order): flexibility, communication, community, support, strategic planning, preparedness, and leadership. both sets of characteristics highlight flexible learning pedagogies (i.e., l&t delivered through a variety of modes, contexts and settings; errington, ); therefore, flexible learning research and initiatives may provide a critical remedy for building resilience to disruption. however, the characterisations also include sustainable and long-term resilience measures (kapucu & khosa, ) that shift the focus away from short- term (threat-dependent) solutions, advocated by researchers in business continuity community. it is also notable that these characterisations do not rely on a risk assessment, often interwoven with more organisational scale approaches (e.g., linkov et al. ) . we consider these characterisations as a starting place for understanding resilience to disruption and useful to individuals and groups seeking to improve resilience at their institution. no institution is alike, and these characterisations come from a specific university within new zealand. each university and individual are likely to experience different resilience dimensions to a greater or lesser extent. however, these sets of characteristics provide a firm starting point which can be provided to other higher education institutions and individuals to explore their understanding of resilience and upon which a customised plan, taking into consideration their unique vulnerabilities and areas for improvement, can be created. building from these characterisations of resilient academics and their institutions, we here identify the benefits our participants perceived in preparing for disruption (i.e., increasing resilience), the barriers faced when building resilience, and incentives to help overcome these barriers. while the responses are specific to our institution, we expect these dimensions to be recognising these stages and acknowledging that each stage brings different challenges, we conceptualise and investigate resilience as how individuals, schools, and universities perceive both preparing for and responding to disasters. increasing resilience by preparing for a major disruption has benefits in the immediate and longer-term aftermath of a disaster, for both institutions and individual academics. table shows the ways in which the participants anticipated that preparing for disruption will benefit them in their everyday practice before, during, and after such events. [insert table here]. participants described unique benefits to improved resilience to disruption. in table , sub-categories are labelled with the number of unique mentions by participants (in italics, n= ) and codes are labelled with proportion from the total unique benefits (not italics, x/ ). the most frequently mentioned benefits to developing resilience to disruption were academic-centric, with academics… • being more organised, simplifying their everyday l&t, and working on other things aside from teaching, • focusing on learning outcomes, rather than course logistics, • feeling in control and emotionally prepared for a disruption, • better supporting their colleagues, learners, and families during a disruption, • knowing what to expect and things to consider during a disruption, • having the ability to pass their course on to others in the event of an illness, and • being encouraged to try new l&t strategies. most benefits focus on 'during the event', followed by 'before the event'. academic developers and leaders might use this 'dual-benefit' frame (i.e., preparing for disruption will benefit you both now, and in the event of a crisis) to motivate and support individuals to develop resilience over the long-term and with shorter-term disruptions (e.g., going on sabbatical, illnesses). we also differentiated benefits by key impacted groups (academics, learners and institution) with the most frequently mentioned concerned with academics' competency and emotional state, followed by benefits to l&t, and to the institution. with the dominant themes pointing to a personal and professional development aspect, building resilience can be directly beneficial to the individual, as well as the institution, and can be communicated this way. participants also report that building resilience could reduce the emotional impact of disruptive events giving academics more control over what is happening, allowing them to support their learners, colleagues, and families. perhaps through resilience-building efforts academics might better prepare themselves, emotionally and cognitively, and subsequently reduce the impact of potential events. this is promising, as kemp et al. ( ) report that people with lower levels of emotional stability were more affected by the earthquakes in canterbury. on the other hand, participants did not focus on the benefits to the university nor to students. this tells us that the academic's perception of resilience-building described here would also benefit from student engagement. beaven et al. ( ) report that students want to be a part of the solutions rather than 'dictated to' in times of crisis. there were no significant differences in how the two groups (sg and nsg) described benefits. participants new to the idea of improving educational resilience noted the same number and types of benefits as those who had spent two years working on the project. also, academics across the range of teaching experience identified the same number and types of benefits. none of the sg or nsg participants were opposed to, uninterested in, or dismissive of resilience-building. promisingly, this similarity indicates that benefits to increasing resilience are apparent to those without previous exposure to such thinking. it also signifies that the characterisations developed with the sg above are relevant to those new to the concept of resilience and will be a helpful framework to inform staff. in conjunction with the characterisation of resilience for academics and institutions, the benefits listed in table can have two obvious impacts: to communicate to academics why they should undertake resilience-building initiatives, and to convince senior leaders of the need to invest resources in such initiatives. the number of benefits identified by each participant underlines the ease with which resilience concepts can be communicated. however, barriers exist to building resilience. understanding these barriers can influence the planning and implementation of effective resilience initiatives. the focus in this section is on barriers facing academics while institutional barriers are explored in section . . . the participants described perceived barriers to resilience-building across the different levels of the institution (table a- table are organised by group and level, with codes listed in order of mention-frequency. again, no significant differences were reported in barriers by the two participant groups (sg and nsg) or participants with varying levels of teaching experience. these findings represent strongly consistent views. [insert table here]. the most frequently mentioned barriers were: • lack of staff time, • lack of institutional mandate, buy-in and acknowledgement, • poor staff and learner digital literacy, • existing digital systems are limited (i.e., poorly performing and lack resilience), • academics' unwillingness to change, adapt, and be flexible, • lack of a school-level plan for improving resilience and responding to disruption, • lack of cohesive and nurturing institutional community, • lack of incentives to encourage resilience initiatives, and • academics lack of resourcefulness in finding solutions during disasters. the most common barrier mentioned is supported by research indicating that academics report feeling overworked with fewer resources (adams, ) and sustaining high workloads. therefore, academics will require ongoing support and incentives to ensure resilience-building initiatives are undertaken or maintained. post-earthquake research (mackey et al., ) indicates that lack of digital literacy among staff, as identified in table # , hinders institutional efforts to respond during and after disruption. notably, the adoption of digital learning relies on the integration of new and existing tools, staff, and protocols (blin & munro, ) so to develop resilience, ongoing efforts are needed. also, some teachers moving to flexible or distance learning strategies may express fears about losing ownership over their educational materials ( table # : 'academic freedom'; # : 'distrustful of digital technology') and a potential lack of 'real' contact with students (errington, ) . at the individual-level, several barriers are likely to be difficult to change (e.g. table # 'unwillingness to adapt, change, or be flexible', # 'inability to work with others' and # 'limited pedagogical perspectives') and could be considered 'innate traits'. academics prioritising family and personal life over work responsibilities ( table # interestingly, discipline-based barriers to resilience were much less reported than initially anticipated by the researchers. this result reflects krause's ( ) contention that when it comes to l&t, there may be less of a "hard line" between disciplines than for research. we also asked participants to identify potential incentives to reduce barriers and enhance resilience across the institution. table provides readers with strategies that an institution can use to promote and engage staff in resilience-building. many have cost or time implications, but often address multiple barriers. the number of barriers addressed by each incentive are also listed in the right-hand column of table . [insert table here]. the top three incentives were: • providing one-on-one staff academic/educational development support, • providing buy-out for academics to explicitly focus on these initiatives, and • university leadership demonstrating support and endorsement for resilience initiatives barriers to resilience-building and the incentives to overcome these barriers are intrinsically linked. the key themes identified, how each barrier can be addressed, and a detailed description and consideration of how key incentives can be enacted are described in this section. lack of community was a prominent barrier at the school-and institutional-level identified by most participants ( table ). communities of practice (wenger, ) are known to support changes in l&t practice (vescio, ross, & adams, ) , enhance innovative and flexible l&t learning approaches (errington, ) , and are critical for supporting learners in a crisis (mackey et al., ) . additionally, research has shown that in times of crisis, community support (or the perception thereof) is strongly related to 'people feeling normal again' (bonanno et al., ; gill, ) . we recommend that universities incorporate community-building efforts into their resilience plan, such as shared activities in l&t innovation (cherrington et al., ) . five teaching-dominant participants in our research explicitly linked the lack of cooperative institutional-level community and culture at our university to the lack of recognition and importance of l&t. similar to other academic institutions worldwide (adams, ) , l&t was not perceived as a key criterion for promotion. also, many academics are overworked with less resources (adams, ) and l&t and research are seen, by some, as adversaries (roberts, ) . to support resilience initiatives, acknowledgement, endorsement, and engagement from senior leaders is needed (table c, d, b) . senior leaders can support resilience efforts in many ways, namely through reward and recognition schemes for l&t community initiatives (supported by results here and also in krause, ) . for example, our university redesigned its promotion criteria to more explicitly acknowledge l&t in . notably, while it may be appealing for leaders to use the results of this work to enact 'top- down' changes, a divide often exists between managerial approaches to higher education and academics' desire for autonomy (ramsden, ; winter, ) within a resilience quantification framework, targeting particular academic disciplines may make the most sense, e.g. those with costly teaching laboratories. if such an approach is followed, the incentives and barriers identified here may be helpful in working with academic staff in target high risk areas to purposefully increase these strategic areas of resilience. resources and support are the backbone of l&t professional academic development. staff and learners need to be digitally capable, and infrastructure (virtual and physical) is needed to support flexible learning. lacking both these elements poses a significant barrier to resilience. as learning management system technology evolves or courses move from emergency remote teaching to a purposefully designed online model, staff and learners will require continual training. not all staff will want to engage in flexible learning with the perceived loss of ownership over their courses a noteworthy fear among academics (errington, ) . resilient higher education institutions should prioritise support services integral to long-term, sustainable changes (toohey, ) . professional development in resilience can be facilitated through workshops or one-on-one support with educational technologists and academic developers (table a ), recognising that individual support is less possible in the immediate aftermath of a disaster event due to high demand. we also note, though, that professional development may not be appropriate for all perceptions and behaviours as those behaviours may be linked to internal values and resistant to change (table ) . implementing l&t in some disciplines requires more physical infrastructure than wholly online, flexible, or distance learning approaches can provide. for example, conventional geoscience education relies on face-to-face hands-on laboratory lessons. this infrastructure could be damaged during major disruptions, or access could become limited. when discussed, participants perceived this possibility as an opportunity to develop alternative settings/modes of l&t rather than an insurmountable challenge. for example, a blended learning approach can provide some physical access to materials and equipment, supported with online learning opportunities. we recommend using the online learning environment to foster unique approaches, to increase learner engagement and maintain a 'minimum online presence' (i.e., contact information, unit outlines, weekly agenda, and communication channels), which learners can use to communicate with peers and staff. we see this meaningful online environment as the first 'goal post' towards resilience, even if the online space is simply an alternative for face-to-face activities (e.g., delivery of lectures, readings and assessments). furthermore, we assessed which incentives might be the most potent in reducing barriers to resilience. the authors matched and summed the number of barriers addressed by each of these incentives (table , right-hand column; supplementary information) . some barriers could be more 'easily' reduced or mitigated, others much less so, and barriers had no incentive provided by the participants (table # , , , , , , and ) . however, most of the incentives addressed more than ten barriers, with three "high impact" incentives of: • sharing l&t practice within schools to encourage capacity-building, • practicing crisis scenarios, and • buy-out: providing time from other duties to focus on resilience-building. also talked extensively about the incentive of buy-out (i.e., using funding to pay for teaching or administrative support) providing academics much needed time to test resilience initiatives. in new zealand, buy-out is commonly used to focus on research. we propose that 'resilient teaching buy-out' could be a powerful experience, with far-reaching impacts. we suggest institutions trial buy-out schemes to give staff with the greatest vulnerabilities to disruption (i.e., highest student numbers, most dependent on physical infrastructure, caring responsibilities, etc.) or early adopters (i.e., engaged staff likely to implement initiatives and inspire others around them to build resilience) the time to creatively identify, plan, and implement unique resilience-building solutions. at a minimum, we recommend that a business continuity plan is co-created by academics, senior leaders, and business continuity experts. to promote longevity and sustainability, planning needs to take a long-term view (kapucu & khosa, ) and leaders need to buy-in and promote resilience initiatives. for those seeking guidance in campus responses to disasters, there is a range of existing research which discusses universities' preparedness and overall response to crisis and organisation-level communication management and planning (beaven et al., ; dabner, ; fillmore et al., ; palen, ; seville et al., ; tanner & doberstein, ) . a prepared and responsive university would help to ensure that the institution continues to function as a business, that learners continue their studies, and therefore staff and student wellbeing can be prioritised (bates, ; schweber, ) . this study explored academics' perceptions of resilience to disruption and documented the perceived benefits, barriers, and incentives of resilience-building. our approach contributes to the literature through its multidisciplinary lens by including participants across a spectrum of disciplines (e.g., including psychology, geography, biology, chemistry, and science history), with different teaching commitments, and by including both academic and professional staff. the collation of responses across different academic disciplines and the similarity of those responses across our participants for l&t demonstrates the wider applicability of our results. our focus on academics enhances the understanding of impacts on and incentives for resilience on individual practice. however, our focus on individual practice and perceptions does not replace the need for an organisation-level assessment. further, higher education is infrequently explored and investigated within the resilience literature. we applied a pragmatic approach and mixed methods methodology within a university in new zealand, though we propose that the results can be used to build resilience in many other higher education contexts. the key findings and discussion are grouped into the three distinct levels of the university (individual, school, and institution), so that resilience-building strategies are readily facilitated by individuals, schools, and institutional senior leaders. tables - are presented in a way that allows direct application to practice, reducing the burden for individuals, and streamlining the call-to-action. the benefits of continued resilience to disruption for new and existing staff outside of an immediate threat need to be understood to ensure resiliency gains are built upon, rather than lost, in a return to business as usual. awareness of the continued benefits to resilient teaching may encourage the continued engagement of academics in rethinking and reprioritising their course delivery and teaching practices to ensure continued resilience. our research highlights the importance of support, community, and leadership at universities. we also present dual-benefits of implementing flexible learning strategies, which can support meaningful l&t even if face-to-face interactions cannot take place after a disruption. this enables the framing of resilience initiatives as meaningful, regardless of whether an event happens or not. however, if a disaster does occur, we should not dismiss face-to-face interactions where possible (e.g., earthquake rather than pandemic scenario), because stronger community connections are important for 'feeling normal again'. resilience to disruption will be experienced differently across educational and socio-political contexts, where benefits, barriers, and incentives may be of greater/lesser importance. every university, therefore, needs to build a customised plan taking into consideration its unique vulnerabilities and areas for improvement. such quantification methods have been discussed elsewhere. universities comprise complex, tiered relationships between the levels (individuals, schools, and leaders) that are negotiated through communities of practice with unique decision-making and power distributions. it may be appealing for senior leaders to use the results of this work to enact 'top-down' changes in response to these, commonly, institution-based frameworks, but we recommend a blended strategy (top-down and bottom- up) that supports academics' autonomy and diversity in l&t. in most contexts, we cannot change that disasters happen but we can change how vulnerable our universities are to these events and we can reduce the impacts to our people and property. by documenting barriers and highlighting benefits and incentives, we hope academics, senior leadership, and professional staff can enact changes across all levels of their institution, ultimately leading to reduced risk from disaster and long-term disruptions. we hope that this research will benefit those currently working to continue l&t despite the overwhelming challenges presented by the covid- pandemic and other disasters. table . benefits to improving resilience before a disruption, ( ) during ( ) be focussed on learning outcomes rather than course logistics. ( ) be flexible to changing delivery modes. ( ) be able to continue, during the disruption. there is no financial interest or benefit that has arisen from the direct applications of this research. there are no competing interests to declare. the researchers declare there are no personal relationships with people or organisations that could inappropriately influence this research. lack of institutional mandate, buy-in, and acknowledgement ( / ) unwillingness to adapt, change, or be flexible ( / ) . not overwhelmed by the resilience-building task ( / ) . distrustful of digital technology ( / ) family/personal life prioritised over work responsibilities ( / ) . limited pedagogical perspectives ( / ) . research prioritised over teaching ( / ) . resilience professional development sounds boring ( / ) . lack of recognition and motivation for l&t changes ( / ) competencies ( / ) . poor staff digital literacy ( / ) . lack of resourcefulness community & culture ( / ) . lack of shared l&t culture / ) . lack of school-level resilience planning ( / ) inter-dependency of staff ( / ) . lack of l&t tools, equipment, lecture materials ( / ) . uneven workload ( / ) . inter-dependency of courses ( / ) . lack of staff cover for teaching ( / ) . high staff turnover ( / ) . over reliance on tutors and teaching assistants ( / ) discipline-specific ( / ) . different disciplinary approaches to resilience ( / ) poor institutional leadership ( / ) . lack of longevity in resilience vision and efforts ( / ) . resilience as a standalone initiative; not embedded ( / ) . bureaucracy ( / ) infrastructure ( / ) . limited digital systems, structures and processes ( / ) . limited physical systems lack of rewards to encourage resilience initiatives ( / ) . lack of staff expertise, resources and training ( / ) . lack of funding and material resources lack of pastoral care model for learners ( / ) . learners are not digitally literate ( / ) . learners have diverse needs ( / ) . learners are expected to be ready-to-learn communication ( / ) . 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reality of new zealand organisations emergency preparedness amongst university students designing courses for higher education the impacts of climate change on the risk of natural disasters teaching resilience: a narrative inquiry into the importance of teacher resilience. pastoral care in education a review of research on the impact of professional learning communities on teaching practice and student learning. teaching and teacher education communities of practice: learning, meaning, and identity academic manager or managed academic? academic identity schisms in higher education quantifying and mapping resilience within large organizations key: cord- -qr bzp r authors: kutuzova, galina d.; deluca, hector f. title: gene expression profiles in rat intestine identify pathways for , -dihydroxyvitamin d( ) stimulated calcium absorption and clarify its immunomodulatory properties date: - - journal: arch biochem biophys doi: . /j.abb. . . sha: doc_id: cord_uid: qr bzp r microarray technology has been used to discover , -dihydroxyvitamin d( ) ( , -(oh)( )d( )) induced gene expression changes in rat small intestine in vivo. here, we report gene expression changes related to intestinal absorption or transport, the immune system and angiogenesis in response to , -(oh)( )d( ). vitamin d deficient rats were intrajugularly given vehicle or vehicle containing ng of , -(oh)( )d( )/kg of body weight. intestinal mrna was harvested from duodenal mucosa at min, , , and h post-injection and studied by affymetrix microarrays. genes significantly affected by , -(oh)( )d( ) were confirmed by quantitative rt-pcr with remarkable agreement. the most strongly affected gene in intestine was cyp with -fold increase at h post- , -(oh)( )d( ) treatment. intestinal calcium absorption genes: trpv , trpv , calbindin d( k), and ca( +) dependent atpase all were up-regulated in response to , -(oh)( )d( ), supporting the currently accepted mechanism of , -(oh)( )d( ) induced transcellular calcium transport. however, a , -(oh)( )d( ) suppression of several intra-/intercellular matrix modeling proteins such as sodium/potassium atpase, claudin , aquaporin , cadherin , and rhoa suggests a vitamin d regulation of tight junction permeability and paracellular calcium transport. several other genes related to the immune system and angiogenesis whose expression was changed in response to , -(oh)( )d( ) provided evidence for an immunomodulatory and anti-angiogenic role of , -(oh)( )d( ). the active form of vitamin d , , -dihydroxyvitamin d or calcitriol ( , -(oh) d ), is a seco-steroid hormone that in association with high affinity vitamin d receptor (vdr), a ligand-activated transcription factor, transactivates or transrepresses a variety of genes [ ] [ ] [ ] [ ] . for decades the major physiologic function of vitamin d was believed to be a stimulation of intestinal calcium and phosphorous absorption [ ] . however, the detailed mechanism of vitamin d-induced intestinal cal- cium absorption is still not fully delineated. the current model of vitamin d-induced transcellular calcium absorption in intestine is the induction of ( ) calcium channel proteins, trpv and trpv (transient receptor potential channels vaniloid subfamily, former names ecac and ecac [ ] [ ] [ ] [ ] ); ( ) cellular calcium transfer protein, calbindin-d k [ , , ] ; and ( ) a calcium extrusion protein, calcium atpase (pmca b ) [ ] . pmca b has been implicated in the regulation of calcium absorption by vitamin d, but its expression is not as tightly regulated by , -(oh) d as that of trpv and trpv and calbindin-d k . evidence for this regulation being transcriptional is only partly substantiated [ ] . the data on regulation of epithelial calcium channels (trpv and trpv ), calbindin-d k , and pmca b expression by , -(oh) d seem clear and these genes have vdre in their promoter region [ ] [ ] [ ] [ ] . however, administration of , -(oh) d did not change the expression of calbindin d k and pmca b mrna levels in the ovariectomized rats [ ] . also, there is no correlation between calbindin d k expression and plasma level of , -(oh) d in dairy cattle [ ] . a report has also appeared on the absence of classical calbindin d k -mediated mechanisms for active ca + transport in sheep rumen [ ] . vitamin d receptor (vdr) knockout mice revealed vdr-independent regulation of intestinal calcium absorption and levels of trpv and calbindin d k mrna, raising doubts about the role of calbindin d k as calcium ferry protein [ , ] . high dietary ca + intake in vdr knockout mice resulted in a decreased expression of both trpv and trpv and in a reduction in calbindin d k and pmca b expression [ ] . it is currently recognized that other genes/proteins could be involved in calcium uptake process [ ] . the present study represent a comprehensive gene expression study in vivo of the effect of , -(oh) d on rat intestine. to specify the role of vitamin d in intestinal calcium absorption and to identify new genes that may be involved in these processes, we have used highdensity oligonucleotide arrays and studied the dynamics of gene expression in rat duodenum within h postintrajugular injection of , -(oh) d . animals were maintained and research was conducted in accordance with guidelines set forth by the animal care and research committee (university of wisconsin, madison, wi). holtzman male weanling rats were obtained from sprague-dawley (madison, wi) and maintained on a highly purified vitamin d-deficient diet, containing . % calcium and . % phosphorus (pi) supplemented three times a week with lg dl dl-a-tocopherol, lg menadione, and lg b-carotene in . ml soybean oil (aek). rats were housed in hanging wire cages and maintained on a h light/dark cycle. rats fed the vitamin d-deficient diet were maintained in a room with incandescent lighting, and all potential sources of ultraviolet light and vitamin d were excluded. at weeks of age, blood was taken from the tail for measurement of serum calcium concentrations to assess vitamin d depletion. blood samples were obtained from the tail artery. whole blood was centrifuged at g for min at °c to yield serum. serum calcium concentration was determined using a atomic absorption spectrometer (perkin-elmer, norwalk, ct) on serum diluted : with g/l lacl [ ] . vitamin d-deficient rats were given intrajugularly one dose of ng of , -(oh) d /kg of body weight in ethanol or vehicle (for control group) and a sample of blood was taken immediately before the injection for serum calcium concentration measurement. groups of three rats per time point were deeply anesthetized with isoflurane and decapitated at min, , , and h after injection. blood was collected at the same time for determination of changes in serum calcium concentration. the first cm of intestine (duodenum) was removed, slit open longitudinally and scraped with the glass slide. the mucosa was homogenized with power-gen (fisher scientific, pittsburgh, pa) in guanidine thiocyanate (gtc) extraction buffer, supplemented with % b-mercaptoethanol (polyattract system , promega, madison, wi), flash frozen in liquid n , and stored at À °c. experiments were done in duplicate. poly(a) + rna was isolated from pooled homogenized mucosa from three rats at each time point. the mrna was isolated using the polyattract system (promega, madison, wi) and purified using an rneasy kit (qiagen, chatsworth, ca). the quality, integrity, and quantity of the poly(a) + rna was determined by agarose gel electrophoresis, uv absorption spectrophotometry, and agilent bioanalyser (agilent technologies, palo alto, ca). double stranded cdna was synthesized from lg of polyadenylated poly(a) + rna using the superscript choice system (invitrogen life technologies, carlsbad, ca), all according to the affymetrix gene expression manual (affymetrix, santa clara, ca). following phenol/chloroform extraction and ethanol precipitation, a biotin-labeled in vitro transcription reaction was performed using the cdna template and bioarray high yield in vitro transcription kit (enzo life sciences, farmingdale, ny). the crna was fragmented at . lg/ll final concentration in · fragmentation buffer ( mm tris-acetate, ph . , mm potassium acetate, and mm magnesium acetate). the size range of crna before ( . kb and longer) and after base fragments) fragmentation was checked by agarose gel electrophoresis. affymetrix high-density rat oligonucleotide arrays (genechips rg-u a) were synthesized photolithographically by the manufacturer using the unigene set of sequence clusters. two sets of - base oligonucleotides each were used to probe each target sequenceperfect match (pm) and mismatch (mm) probe sets. perfect match (pm) probe set and mismatch (mm) probe set were the same except mm contained a mismatched base in the center of the oligonucleotide. the mm probe set was used to control for non-specific hybridization of related sequences. the chip contained around probe sets, with about % of the (longer) sequences represented by more than one probe set. target and probe set sequences were obtained from the netaffx analysis center (http://www.affymetrix.com/ analysis/index.affx; affymetrix). the hybridization reaction and the automated hybridization procedure were performed by the gene expression center at the biotechnology center of the university of wisconsin-madison. each probe sample was tested on an affymetrix test array and the quality of the cdna and crna syntheses was determined by the / ratio of housekeeping genes within the array (ubiquitin, rat glyceraldehyde -phosphate dehydrogenase, b-actin, and hexokinase). if the sample passed the quality control on the affymetrix test array, it was hybridized to an affymetrix high-density rat oligonucleotide array genechip u a per protocol recommendation in the affymetrix genechip expression analysis technical manual [see: http://www.affymetrix. com/support/technical/manual/expressionmanual.affx]. genechips were hybridized with lg of biotin labeled crna in ll, in · hybridization buffer [ mm mes, m nacl (ambion), mm edta (ambion), and . % tween (pierce chemical), pm control oligonucleotide b (affymetrix), . mg/ml herring sperm dna (promega), . mg/ml acetylated bsa (invitrogen life technologies), and · eukaryotic hybridization controls (biob, bioc, biod, and cre at . , , , and pm, respectively) (affymetrix)] for h at °c on a rotisserie at rpm. prior to application to the genechip, samples were heated at °c for min, followed by incubation at °c for min and spun at , g for min. following hybridization, the labeled samples were removed from the genechip, stored in the appropriate vial at À °c, and immediately filled with non-stringent buffer a which contains · sspe [ . m sodium chloride, mm sodium phosphate, and mm edta (ambion)] and . % tween . all genechips were post-processed using the automated affymetrix genechip fluidics station . the post-processing protocol for the rg_u genome genechip is as follows: wash# : to quantify the fluorescent signal from each feature on the genechip, all genechips were scanned at a wavelength of nm using the hewlett-packard gene array scanner. fluorescent signals corresponding to hybridization intensities were analyzed with the affymetrix microarray suite (mas) . software using the following settings: detection algorithm defaults, a , . ; a , . ; sf, . and comparison algorithm defaults, c h, . ; c l, . ; c h, . ; c l, . ; perturbation, . . in all the analyses, all probe sets were scaled to a target signal of using the ''scale'' function in the genechip software. to estimate the range of linearity, four different bacterial mrnas were added to the hybridization cocktail at the following concentrations: biob ( . pm), bioc ( pm), biod ( pm), and cre ( pm) [ ] . expression data were analyzed using the affymetrix microarrray suite software version . (mas . ). comparison tables for each time point for , -(oh) d vs. vehicle-treated rats were generated in ex-cel (microsoft). for each comparison, e.g., , -(oh) d treated relative to control (vehicle treated), and for each cdna represented in the array, a ratio (e.g., , -(oh) d /control) and an absolute difference of intensities for , -(oh) d and vehicle treated were calculated. for real-time quantitative pcr (q-pcr), reverse transcription reactions were carried out in parallel on dnasei digested pooled mrna from , -(oh) d or vehicle treated rats at different time points. prior to reverse transcription, total rna was confirmed to be free of contaminating dna sequences by pcr using rat b-actin specific primer pairs designed to differentiate between cdna, genomic dna, and pseudogene genomic dna. reverse transcription reactions (five replicas) were done for each time point for , -(oh) d -or vehicle-treated rats. reverse transcription reactions were as follows: ng mrna, . ll oligo(dt) ( ng/ll), ll dntpmix ( mm each dntp), pg artificial transcript of human b-actin (idt, coralville, ia), and h o to . ll. samples were incubated at °c for min, put briefly on ice, and then incubated at °c for - min. mix# ( ll · superscript ii first strand buffer, ll of . m dtt, ll of mm mgcl , and . ll superscript ii rnase h $ reverse transcriptase u/ll) was added, mixed, and samples were immediately returned to incubate at °c for h. reactions were stopped by incubation at °c for min. to confirm uniformity of reverse transcription within sample and control groups, q-pcrs were done in the presence of artificial transcript (human b-actin). then, reverse transcription reactions with similar quantities of human b-actin within a sample group were pooled. quantitative pcrs were performed using a geneamp sequence detection system (perkin-elmer, norwalk, ct), using the ''standard-curvequantitation'' method [ ] . each reaction contained target-specific forward and reverse primers ( - nm final concentration, table ), · sybr green master mix (applied biosystems, foster city, ca), ll of a : dilution of pooled reverse transcription product and h o to a total volume of ll. a two-step pcr profile was used: min at °c denaturation and amplitaq gold activation, followed by cycles alternating between °c for s and °c for s. dilution series ( : ; : ; : ; : ; and : ) standard curves were performed in quadruplicates for each primer pair using reverse transcription products described above. pcr was done in five replicas for each sample and relative quantities were determined based table sequences of primers and fold change in expression of selected genes chosen for confirmation study by real-time quantitative pcr genbank accession number description real-time pcr primers and probe sets were selected for each cdna by using primer express software (ver. . ; applied biosystems, foster city, ca) and are presented in table . we studied differential gene expression profiles in rat intestine after a single intrajugular injection of , -(oh) d with the purpose of identifying novel genes involved in intestinal ca + and other nutrient absorption. it was shown previously [ ] that serum concentration of ca + in the plasma begins to increase h after treatment with , -(oh) d , peaks at about h, and declines at h. we, therefore, examined gene expression in rat intestine at: min, , , and h. we used affymetrix rat genechips u- a array that contains known rat transcripts ( %) and ests ( %). in comparison, tables (sample vs. control) of gene expression (mas . ), only genes considered (p) with a statistically valid signal increase (change ''i'') were considered genes upregulated by , -(oh) d . only genes present (p) in control with a statistically valid signal decrease in the sample (change ''d'') were considered as down-regulated. to identify genes that were differentially expressed between , -(oh) d (sample) and vehicle (control) treated animals for each time point, we arbitrarily set up cut-off values to . for the fold change in ratio. in some cases, it was hard to assign the reliable fold change for the genes that were absent (a) in control and become present (p) in the sample or vise versa. we used rt-pcr to confirm the effect of , -(oh) d on regulated genes. the list of genes confirmed, maximum fold change in their expression after the stimulation with , -(oh) d , and primers used are presented in table . the absolute magnitude of fold regulation detected with rt-pcr technique was always similar or higher than the fold change detected by microarray analysis, except for plasma membrane caatpase b (tables and ). microarray technology can reliably detect changes in gene expression as subtle as . -to -fold [ ] . we were anxious not to overlook genes that could be very important in understanding of vitamin d mechanism of action that may only change by a factor of (cut-off value currently accepted by users). an example is the well-established vitamin d responsive calbindin d k gene. our genechip data showed its maximal up-regulation only . -fold at h after , -(oh) d treatment ( table ) . all , -(oh) d regulated genes that passed the selection criteria (see above) were classified in terms of their function by referring to the literature and affymetrix analysis center web site and links (see materials and methods). the data on , -(oh) d stimulated gene expression are presented on separate tables and are given at the time of expression maximum fold change. we did not provide the fold change at other time points, which we observed, to avoid the complexity in presentation and to conserve space. in this paper, we have restricted our presentation to , -(oh) d -stimulated differential expression of genes dealing with digestion, absorption, and the immune system. in our experiment, , -(oh) d stimulated the highest level of expression of -hydroxyvitamin d -hydroxylase (cyp ), the major enzyme of , -(oh) d degradation pathway, compared to all other transcripts which is consistent with the previous findings on strong up-regulation of this enzyme by , -(oh) d both in vivo and in vitro [ ] . as we observed, cyp mrna was undetectable in the intestine of vehicle treated rats but after , -(oh) d injection its level increased -fold at h and -fold at h. we observed the increased expression of genes considered to be directly involved in the intestinal ca + absorption. the maximum fold change of the expression level of calbindin d k -the vitamin d-dependent cytosolic calcium binding protein within h after the treatment, was . -fold at h (at h after injection there was . -fold increase) ( table ). plasma membrane ca + atpase transcript (est ai ) was not detectable at all time points in the control (vehicle treated) rats, or at min and h in , -(oh) d -treated animals and had . -fold expression increase at h ( -fold by q-pcr) followed by a further increase in transcript level at h. the activity of this ca + atpase is regulated by calmodulin, which also showed a maximal . -fold increase at h (table ) . calmodulin, as a major intracellular ca + sensor and modulator, is involved in numerous calcium signaling pathways by interaction with diverse group of cellular proteins [ ] . calmodulin antagonists significantly reduced , -(oh) d stimulated calcium uptake in caco- colon derived cells, implying its involvement in ca + influx [ ] . it is well known that blood calcium is regulated by several calcitropic hormones, e.g., calcitonin, parathy-roid hormone (pth), and , -(oh) d . caldecrin, a serum calcium-decreasing factor, is a chymotrypsin-type serine protease, which belongs to the elastase family and inhibits parathyroid hormone or parathyroid hormonerelated, peptide-induced bone resorption. caldecrin is synthesized as preprocaldecrin and is secreted from the cell. preprocaldecrin does not possess serum calcium-decreasing activity but acquires it as well as protease activity, upon trypsin treatment [ ] . in our experiments, , -(oh) d stimulated preprocaldecrin expression . -fold at h. the affymetrix rat genome u a array used in our study did not have probe sets for the epithelial calcium channels trpv and trpv , which are considered to be the major channels for calcium entry in intestine [ ] [ ] [ ] . we analyzed the regulation of expression of trpv and trpv channels by , -(oh) d in rat intestine within the time frame of our study ( h) using real-time quantitative pcr (table ) . a slight increase in expression of trpv was detected at h, but at h, it increased more than -fold ( fig. ) . expression of trpv channel started to increase at h ( -fold increase) and continued to increase up to . -fold at h ( fig. ). in table , we present the list of genes involved in intestinal transport of different compounds that were differentially expressed in intestine of rats within h after administration of , -(oh) d . prepro-uroguanylin À . a these genes also showed up-or down-regulation with other probe sets derived from different genbank accession numbers of the same protein. at h, expression of very long-chain acyl-coa synthetase (vlacs) mrna was decreased . -fold (table ) . vlacs catalyzes the activation of very long-chain fatty acids (vlcfas) to their coa thioesters in peroxisomes. vlacs is identical with fatty acid transporter protein (fatp ) and is involved in cellular uptake and metabolism of very long-chain fatty acids [ ] . at h, up-regulation by , -(oh) d was observed for genes that are currently known to be involved in vesicle transport (endocytosis and exocytosis) between secretory compartments like cis-golgi p protein ( . -fold increase) and sip , a zymogen granule protein (syncollin, . -fold increase), though their other possible functions remain to be elucidated (table ). p is the protein involved in a docking and fusion stage of protein transport from endoplasmic reticulum to golgi [ ] . sip may function as a calcium-sensitive regulator of exocytosis in exocrine tissues triggered by the release of ca + from intracellular stores. membrane-associated syncollin is a component of lipid rafts, where it binds to lipid bilayers in a cholesterol-dependent manner and may form pores [ ] . at h, , -(oh) d down-regulated expression the number of transporters and channels. they include a- and b- subunits of sodium/potassium atpase (na,k-atpase), voltage dependent anion channel , transmembrane protein rnp . , fatty acid translocase/cd (fat/ ), claudin- , urate transporter/channel, hypothetical atpase, and aquaporin- ( table ) . the na,k-atpase, or the sodium pump, consists of a non-covalently linked a-and b-subunits and is a key enzyme that regulates the intracellular na + and k + homeostasis in animal cells [ ] . we observed a -fold decrease in expression of na,k-atpase a- subunit and a . -fold decrease in expression of b- subunit (table ) . it was shown previously that inhibition of na,k-atpase in tight monolayers of epithelial cells with ouabain results in an increase in tight junction permeability to ions and non-ionic molecules [ ] . , -(oh) d caused a . -fold down-regulation of voltage-dependent anion-selective channels (vdacs). voltage-dependent anion-selective channel (vdac), or mitochondrial porin, forms a channel through the mitochondrial outer membrane and also through the plasma membrane, which allows the diffusion of a small anionic molecules [ ] . at h, intestinal expression of transmembrane protein rnp . , involved in vesicular trafficking and protein transport [ ] , decreased . -fold (table ). galectin- that showed a -fold expression decrease in the response to , -(oh) d is a kda b-galactoside binding lectin and was identified as a specific urate transporter/channel. galectin- is also involved in a variety of biological functions such as cell activation, proliferation, adhesion, and apoptosis [ ] . the expression of hypothetical putative p-type atpase that possi-bly catalyzes the transport of heavy metal ions was suppressed by , -(oh) d (table ) . fatty acid translocase/cd (fat/ ), which mediates the long-chain fatty acid (lcfa) uptake, was down-regulated by , -(oh) d . -fold at h (table ). in rodent models of obesity and type diabetes, increased expression of fat/cd (type diabetes) has increased the lcfa uptake into the heart and muscle [ ] . we also observed a . -fold decrease in the expression of aquaporin- (table ) . aquaporins are a conserved group of small hydrophobic integral membrane proteins that form a barrel-like channels for water transport [ ] . at h after , -(oh) d treatment, we observed a significant increase in expression of two transporters ( table ) . organic cation transporter (oct ) expression was up-regulated more than -fold with different probe sets. oct is the member of the polyspecific potentialsensitive organic cation transporter gene family and belongs to the class of proteins responsible for transport of sugar and organic cations that include endogenous compounds such as monoamine neurotransmitters, choline, and coenzymes, but also numerous drugs and xenobiotics [ ] . , -(oh) d caused more than a -fold increase at h in the expression of the multidrug transporter or atp-binding cassette (abcb) transporter or the multidrug-resistance/transporter associated with the antigen processing (mdr/tap ), which is involved in the transport of peptide antigens from the cytoplasm into a membrane-bound compartment of endoplasmic reticulum (er) for association with mhc class i molecules. mdr/tap is also the part of er chaperone complex and functions in association with calnexin and calreticulin-the two lectins (carbohydrate binding chaperones), which interact with and assist the folding of proteins that carry monoglucosylated n-linked glycans. calnexin expression was also increased . -fold, h in response to , -(oh) d (table ). in some carcinomas and malignant tumors, the transcription of tap is drastically reduced [ ] . natural resistance-associated macrophage protein (nramp ) or solute carrier family member expression was reduced . -fold by , -(oh) d at h ( table ) and this was seen with multiple probe sets. nramp is a broad specificity divalent-metal transporter and is expressed at the duodenum brush border where it is responsible for transferrin-independent uptake of dietary iron from the intestinal lumen [ ] . , -(oh) d stimulated differential expression of genes involved in intra-/intercellular matrix modeling , -(oh) d regulated the expression of several genes involved in intracellular and intercellular structure formation (table ) . (table ). it caused a . -fold increase in expression of the lysosomal-associated membrane protein (lamp- ). lamp- functions as the receptor for the selective uptake and degradation of cytosolic proteins by lysosomes and is involved in chaperonemediated autophagy and lysosomal biogenesis. remarkably, lamp- deficiency in humans leads to danon disease, a lysosomal glycogen storage disease, cardiomyopathy, and myopathy [ ] . at h, , -(oh) d treatment increased the expression of other cytoskeleton proteins. these include a tropomyosin non-muscle isoform nm (tpm-c), a component of the actin microfilament complex; cytoplasmic dynein, involved in numerous intracellular motility events, including the transport and positioning of vesicles and organelles, spindle assembly and morphogenesis, and ankyrin, the adaptor molecule that links ion channels and cell adhesion molecules to the spectrin-based skeleton in specialized membrane domains (table ) . at the same time ( h), , -(oh) d induced significant down-regulation of several genes involved in cellcell interactions and cell motility ( table ). the expression of cadherin- was decreased . -fold by , -(oh) d ( table ). the cadherinÕs superfamily of transmembrane glycoproteins is calcium-dependent cell adhesion molecules that play an active role in tissue morphogenesis, patterning and cell adhesion [ ] . , -(oh) d treatment inhibited the expression of the brush border myosin-i (bbmi) ( . -fold at h) (table ). bbmi is a major component of the actin assembly in the microvilli of intestinal cells and is involved in endocytosis. non-functional truncated bbmi affected the rate of transferrin recycling and the rate of transepi-thelial transport of dipeptidyl-peptidase iv from the basolateral plasma membrane to the apical plasma membrane [ ] . rhoa gtpase, a member of the family gtp-binding proteins (small gtpases), is the key regulator of actin cytoskeletal dynamics in cells. its expression was significantly inhibited ( . -fold, h) by , -(oh) d ( table ). the rho proteins, cdc , rac , and rhoa, regulate the actin cytoskeleton. cdc and rac are primarily involved in the formation of protrusive structures, while rhoa generates myosin-based contractility. rhoa gtpase is involved in the formation of stress fibers and in the regulation of tight junctions structure and function as well as in generation of myosin-based contractility. all mutations in rhoa induced time-dependent disruptions in epithelial gate function [ ] . at h, claudin- expression decreased . -fold after , -(oh) d treatment (table ). claudins are membrane proteins involved in the formation of tight junctions strands. elevated expression of claudin- was detected in prostate adenocarcinoma and in the primary ovarian carcinoma cells [ ] . it has been suggested that claudin- is a central component determining the integrity of blood-brain barrier tight junctions in vivo [ ] . at h, , -(oh) d increased expression of the following proteins (table ): ( ) dynactin subunit p , an accessory factor that mediates targeting of cytoplasmic dynein (dynein expression was increased at h as we showed above) to a wide array of intracellular substrates; ( ) mucin -the protein, that interacts with actin cytoskeleton; and ( ) cell division cycle homolog (cdc ), a member of rho family gtpases, which regulate the actin cytoskeleton and are primarily involved in the formation of protrusive structures. in mammalian highly similar to myosin light chain alkali, smooth-muscle isoform À . a these genes also showed up-or down-regulation with other probe sets derived from different genbank accession numbers of the same protein. cells, cdc has been implicated in the regulation of an actin rearrangement, receptor-mediated signal transduction pathways, cell cycle progression, apoptosis and multiple membrane trafficking events, including phagocytosis, exocytosis, and endocytosis. cdc may also govern pathways required for establishment and maintenance of cellular polarity [ ] . expression of skeletal muscle a-actin (a-sma) and thymosin b- (tb- )-the intracellular matrix related genes-was strongly inhibited at h by , -(oh) d (table ). our data are similar with published data on , -(oh) d suppression of the expression of a-sma, that was accompanied by a significant inhibition of mesangial cell proliferation [ ] . elevated expression of thymosin b- was shown to be an indicator of a malignant phenotype of human tumor cells, including mammary carcinomas. suppression of thymosin b- protein synthesis reduced the growth of human thyroid carcinoma cells [ ] . strong inhibition of expression of thymosin b- seen in our experiment (table ) may in part explain the antiproliferative activity of , -(oh) d . at min, , -(oh) d stimulated a strong up-regulation (more than -fold) of variant regions of multiple immunoglobulin chains (data not shown). at h, , -(oh) d caused a strong increase in the expression of interferon-c inducing factor isoform a precursor (igif) or il- , increased expression of cd glycoprotein precursor and chemokine cx c (table ) . both il- and its precursor were strongly induced by , -(oh) d ( . -fold at h and . -fold at h, respectively) ( table ). il- is a pleiotropic factor that shares structural features with il- and functional activities with il- and it was identified due to ability to induce interferon-gamma (ifn-c) production by t cells. the regulation of il- by vitamin d receptor (vdr) or by its ligand, , -(oh) d , was proposed recently [ ] . il- was shown to inhibit the osteoclastogenesis in vitro via granulocyte-macrophage colony-stimulating factor (gm-csf) [ ] and to possess the antiangiogenic and antitumor effects, particularly in combination with il- or il- [ ] . the expression of cd (protectin) precursor, a phosphatidylinositol-anchored glycoprotein, was increased by , -(oh) d ( . -fold, h) ( table ). cd is a member of cell membrane-bound complement regulatory proteins that inhibit the formation of the terminal membrane attack complex of complement. loss of cd offers a selective advantage for breast cancers, resulting in more aggressive tumors and conferring a poor prognosis for patients [ ] . increased expression of cd precursor after , -(oh) d treatment, shown in our experiment, may contribute to the known anticancer activity of , -(oh) d ( table ) [ ] . at h, , -(oh) d strongly inhibited the expression of interferon-c receptor ( . -fold) and decreased the expression of mhc class ii-associated invariant chain and proteasome subunit rcx (table ) . interferon gamma receptor (ifn-cr) is the prototypical th- cytokine (table ) . ifnc receptor knockout non-obese diabetic (nod) mice showed a marked inhibition of insulitis and no signs of diabetes, in a sharp contrast to ifn-c knockout nod mice, that had no change in insulitis and only a relatively minor delay in diabetes [ ] . ifn-c receptor null mice also displayed a reduced pain-related behavior after nerve injury compared to wild-type mice. the decreased expression of mhc class ii-associated invariant chain we observed (table ) is in agreement with previous findings on sig- pancreatitis-associated protein precursor (pap) À . nificant reductions in class ii mhc expression in monocytes exposed to , -(oh) d [ ] . expression of small inducible cytokine a precursor (ccl ) and cytokine il- was strongly increased by , -(oh) d at h (table ). ccl or macrophage inflammatory protein- a is a cc-type chemokine that highly specifically binds to and activates cc chemokine receptor- (ccr ) and acts as a chemoattractant for memory/differentiated t-cells, b-cells, and immature dendritic cells, and possesses the antibacterial activity of a greater potency than b-defensins - and - [ ] . il- is expressed in multiple tissues and was able to induce the proliferation of activated t cells. it plays an important role in the development of memory cd + t cells and natural killer (nk) cells, for which il- serves as the ''fuel.'' nk cells spontaneously kill tumor cell lines in vitro. il- null mice displayed reduced numbers of t cells and lack of nk and nk t cells proving that this cytokine is crucial for murine nk cell development and suggesting the potential use of il- therapy for expansion of nk cells in patients [ ] . interestingly at h, , -(oh) d strongly inhibited the expression of both pancreatitis-associated protein (pap) and its precursor ( . -and . -fold, respectively) ( table ). paps are the products of the c-type lectin supergene family of sugar-binding proteins with ability to agglutinate cells, to form antigen-antibody like precipitates with glycoconjugates and to induce mitosis in cells, which are normally not dividing. pap is considered to be a stress protein and is constitutively expressed in the epithelial cells of the small intestine. pap has been described as a marker protein for pancreatitis and for cystic fibrosis in neonates. overexpression of pap in human pancreatic ductal adenocarcinoma indicates tumor aggressiveness [ ] . the maximum change in the expression of these enzymes was observed at h (table ). , -(oh) d stimulated the increased expression of all three types of trypsin precursors (i and ii-both anionic forms and iii-cationic form) (table ) . trypsinogens, the precursors to the serine protease trypsin, are found in the pancreas and mediate the digestive proteolysis in the small intestine. anionic and cationic trypsinogens are approximately % identical in their primary structure. expression of both pancreatic trypsin inhibitor type i and ii (psti-i and psti-ii) was increased . -and . -fold, respectively (table ). both psti-i and ii are expressed in pancreas, liver, and small intestine. expression of caspase was strongly inhibited by , -(oh) d ( . -fold) and this was seen with different probe sets (table ) . caspase cleaves a variety of important cellular proteins and is considered to be a primary executioner of apoptosis or programmed cell death that can be initiated by a number of stimuli. studies in caspase- null mice showed that this protease is essential for brain development [ ] . , -(oh) d strongly suppressed the expression of angiotensin-converting enzymes: cd /aminopeptidase n ( . -fold, table ) and kininase ii or angiotensin iconverting enzyme (ace) ( . -fold, table ). cd /aminopeptidase n (cd /apn) is a type ii membrane-bound metalloprotease that is expressed on the endothelial cells of angiogenic, but not normal, vasculature. it is essential for later stages of neovascular formation and is an important angiogenic activator, indicating that cd /apn plays a functional role in tumorigenesis [ ] . the cell surface aminopeptidase n is overexpressed in tumor cells. it is now generally agreed that conversion (degradation) of ang iii that causes high blood pressure to the hexapeptide ang iv is aminopeptidase n dependent [ ] . intestine brush-border cells present a high concentration of aminopeptidase n that plays a role in the final digestion of peptides generated from hydrolysis of proteins by gastric and pancreatic proteases. human cd /apn is the receptor for coronaviruses; thus, its inhibitors might protect again sars [ ] . our data are in concert with previous finding on reduction of cell surface cd / apn expression in the phagocytic cells by , -(oh) d [ ] and suggest , -(oh) d as the potential inhibitor table , -(oh) d stimulated differential expression at h of proteases, their inhibitors, and peptidases genes interleukin- b-converting enzyme-related protease cpp (caspase ) À . u a ice-like cysteine protease (lice) or caspase À . a these genes also showed up-or down-regulation with other probe sets derived from different genbank accession numbers of the same protein. of cd /apn expression. interestingly, in our experiment , -(oh) d simultaneously increased the expression of transcription factor c-maf (table ) , which was shown to suppress the cd /apn expression ( - % reduction) in human immature myeloblastic cells [ ] . this could be the explanation for , -(oh) d stimulated down-regulation of cd /apn expression observed in our case. angiotensin i-converting enzyme (ace) plays a central role in the renin-angiotensin system. ace is a carboxypeptidase that hydrolyzes the amino acid peptide angiotensin i into the potent vasoconstrictor angiotensin ii. it was reported that angiotensin ii stimulates angiogenesis in vivo, and angiotensin-converting enzyme (ace) inhibitors block angiogenesis [ ] . in addition to inducing vasoconstriction, angiotensin ii increases blood pressure by a variety of physiological actions, including renal salt and water retention. ace may affect blood pressure through the production of the vasoconstrictor angiotensin ii and the inactivation of the vasodilator bradykinin. ace inhibitors block the formation of angiotensin ii and have been used to treat hypertension and heart failure [ ] . ace null mice have low blood pressure and the inability to concentrate urine [ ] . further, it has been reported that vitamin d supplementation reduces blood pressure in patients with essential hypertension [ ] , which may be in part due to its ability to down-regulate ace. vitamin d is known not only as the principal regulator of bone development and calcium homeostasis but also for its widespread effects in many other tissues in vivo. it is a potent inhibitor of the variety of cancer cells [ ] . , -(oh) d exerts its effects through binding to a high affinity receptor (vdr) that following heterodimerization with the retinoid x receptor acts as a ligand-activated transcription factor and selectively binds to recognition sequences in the promoter region of target genes (vitamin d response elements-vdres), transactivating or transrepressing a large variety of genes [ ] [ ] [ ] . it is known that among the vast majority of genes that are regulated by vitamin d or its analogs only relatively few contain vdre in their promoter region and thus are under direct transcriptional control of , -(oh) d . for the majority of genes that do not have the vdres in their promoter sequences, their regulation is indirect but important response to the cascade of reactions induced by , -(oh) d . in this study, the microarray technique allowed us to investigate the role of vitamin d in intestinal absorption of calcium and other nutrients and provide the comprehensive knowledge of genes regulated by vitamin d in intestine, thus clarifying its widespread effects in vivo. in vitro micro-array technique was applied to study the , -(oh) d stimulated gene expression in several cell lines: in mouse osteoblasts [ ] , in squamous carcinoma cells [ ] , and human colon carcinoma cells [ ] . although there is some similarity in regulation of expression of some genes by , -(oh) d in our system and the squamous carcinoma and human colon carcinoma cells [ , ] (in strong up-regulation of cyp , in up-regulation of calmodulin, and in some other genes not presented in this paper), our studies were done in vivo in highly differentiated tissue that is responsible for nutrient absorption. we do not expect the same pattern of gene expression in immortal cell lines treated with high and unphysiological concentrations of , -(oh) d as we see in vivo in a functional tissue carrying out intestinal absorption. the most interesting for us was to identify , -(oh) d regulated genes involved in ca + homeostasis and also genes involved in nutrient absorption in general. our microarray and q-pcr data showed the increase in the expression level of calcium homeostasis genes, and the differential expression of transporters and channels starting at h after , -(oh) d treatment with the expression maximum fold increase at and h (tables and ). our data confirm previously published data that , -(oh) d up-regulates expression of transcellular calcium transport genes such as calbindin d k , plasma membrane ca + atpase, epithelial calcium channels, trpv , and trpv (table and fig. ) [ , , , , [ ] [ ] [ ] [ ] . molecules cross the intestinal epithelium into the systemic circulation primarily by three pathways: passive diffusion across the cell membranes (transcellular pathway), passive diffusion between adjacent cells (paracellular pathway), or carrier-mediated transport (carrier-mediated transcellular pathway). lipophilic molecules easily cross the cell membrane via transcellular diffusion. hydrophilic molecules, if not recognized by a carrier, traverse the epithelial barrier via the paracellular pathway, which is severely restricted by the presence of tight junctions. historically, a simplified view of this absorptive process was that transcellular movement of nutrients and water via specific pumps, transporters, and channels would account for absorption, while an impermeable tight junction seal adjoining epithelial cells for the requisite barrier function. it has now become clear that transjunctional solute movement occurs in a regulated fashion, and that its regulation may be coupled to transcellular absorptive events. thus epithelial solute transport and tight junction barrier function have to be viewed as related coordinated events [ ] . tight junctions (tj) are the contact points between the apical and basolateral membranes that limit paracel-lular flux of ions, proteins, and other macromolecules (gate function) and serve as a fence between the apical and basolateral plasma membranes in epithelial cells (fence function). the restricted movement of ions across the tight junctions gives rise to transepithelial electrical resistance (ter), which is often used as an index of the integrity of the tight junctions in an epithelial or endothelial tissue. tight junctions are crucial in maintaining the polarized phenotype and the vectorial transport functions of epithelial cells. they are also a specialized membrane microdomains that might function as a molecular platform involved in cell signaling, vesicle protein docking, actin organization, and cell polarity in epithelial cells. each particle of the tight junction strand is composed of transmembrane proteins and cytoplasmic plaque proteins connected to the actin cytoskeleton. occludins, claudins, and the junctional adhesion molecule (jam) are the three classes of transmembrane proteins localized to the tight junctions. tjs also concentrate tumor suppressor proteins and cell polarity proteins [ ] . tight junctions have a highly dynamic structure so their permeability, assembly or disassembly can be regulated by a variety of cellular and metabolic mediators including cytokines [ ] . the controlled and reversible opening of the tight junctions could be an attractive approach to increase the absorption of hydrophilic drugs across the intestinal epithelium because it could be applied to many different hydrophilic drugs and thus would allow them to escape the degradation by intracellular enzymes. we identified the number of , -(oh) d target genes with the maximum fold change in the expression at h that are (co)localized in the proximal vicinity of intestinal tight junctions and thus could influence their integrity and permeability. these include not only transporters and channels, but also several intra-/intercellular matrix related genes and g-proteins (tables and ). the following genes down-regulated by , -(oh) d are involved in the regulation of tight junctions permeability (tables and ) . sodium/potassium atpase. both a- and b- subunits of sodium/potassium atpase (na,k-atpase) were significantly down-regulated by , -(oh) d (table ) . na,k-atpase catalyzes an atp-dependent transport of three na + ions out and two k + ions into the cell per pump cycle. in epithelial cells, na,k-atpase was also involved in the formation of tight junctions through rhoa gtpase and stress fibers. the inhibition of na,k-atpase in tight monolayers of epithelial cells resulted in an increased permeability of tight junctions to ions and non-ionic molecules [ ] . claudin- . we observed a decreased expression of claudin- ( . -fold, table ). the claudins, tight junction-specific adhesion molecules, create paracellular channels and their first extracellular domain is sufficient to determine both paracellular charge selectivity and transepithelial electrical resistance (ter). the tight junctions charge selectivity towards cations or anions in epithelial cells could be reversed by expression of different claudins [ ] . the selective loss of claudin- was the cause for ''leakiness'' of blood-brain barrierÕs tight junctions at experimental autoimmune encephalomyelitis [ ] . water channel aquaporin . aquaporin is the tight junction channel; was down-regulated by , -(oh) d (table ) . thus, the inhibition of its expression might change the tj selectivity towards cations. cadherin- . it plays a very important role in cell-tocell adhesion and was down-regulated . -fold by , -(oh) d (table ) [ ] . rhoa. it is the small gtp-ase that regulates remodeling of the actin cytoskeleton during cell morphogenesis and motility. it was shown that rhoa gtpase is an essential component downstream of na,k-atpasemediated regulation of tight junctions [ ] . thus, , -(oh) d may increase intestinal epithelial tight junction permeability or modulate their selectivity towards ca + and other cations by regulation of expression of proteins structurally involved in tight junction formation. the increased tight junction permeability and/or selectivity, regulated by , -(oh) d , could route ca + absorption through the tight junction-regulated paracellular pathway in the intestinal epithelia. our proposal is in agreement with published data on the , -(oh) d stimulated increase of tight junction conductance and increased paracellular ca + , na + , rb + , and mannitol transport in enterocyte-like cell line caco- , while no significant contribution of the ca + -atpase-mediated transcellular pathway to overall transepithelial ca + transport was detected [ ] . the evidence has accumulated since late th for in vitro intestinal model for ca + and pi transport [ ] and recently for ca + transport in caco- cells [ ] that , -(oh) d enhanced both cell-mediated active and passive paracellular ion movement. so based on our microarray data we propose that , -(oh) d regulates the intestinal absorption of ca + in vivo through both transcellular and paracellular pathways by the stimulation or suppression of the expression the group of genes and also identified , -(oh) d target genes possibly involved in regulation of tight junctionÕs permeability and/or selectivity. at h, , -(oh) d caused more down-regulation of transporters mrna and channels genes than up-regulation (table ) . it was the time of a maximal decrease in the expression for both a- and b- subunits of sodium/potassium atpase (na,k-atpase), voltage dependent anion channel , transmembrane protein rnp . , claudin- , urate transporter/channel, hypothetical atpase, and aquaporin- after the , -(oh) d treatment (table ) . at the same time, , -(oh) d increased the expression of p and sip (syncollin) involved in vesicle transport of proteins (endocytosis and exocytosis) though some of their functions remain unknown ( table ) . the expression of both organic cation transporter (oct ) responsible for transport of sugar and organic cations like drugs and xenobiotics and expression of multidrug transporter , atp-binding cassette (abcb) transporter, which is involved in the transport of peptide antigens for association with mhc class i molecules was significantly increased at h post- , -(oh) d treatment (table ). at the same time, the expression of nramp -transporter for other than ca + divalent cations, like iron, was significantly decreased (table ) . the actions of , -(oh) d on the immune system are thought to be targeted primarily at t helper (th) cells. our data showed that transcription factor c-maf which is involved in the regulation of the th /th balance was up-regulated by , -(oh) d (see table ). our data are in agreement with published data on , -(oh) d acting directly on th cells, enhancing the development of a th phenotype and augmenting the expression of the transcription factor c-maf [ ] . some of the , -(oh) d target genes that we identified have also antitumor effect. these include il- (and its precursor), il- , chemokines cx c and cc chemokine st precursor and cd precursor ( table ) . murine models have shown that il- has both antiangiogenic and antitumor effects particularly in combination with il- or il- and also induces the high levels of ifn-c secretion [ ] . in a new gene therapy approach for treatment of osteosarcoma, recombinant il- was proposed for use in combination with plasmids encoding the il- [ ] . il- is involved in the development of natural killer (nk) cells that can spontaneously kill tumor cell lines in vitro [ ] . the loss of cd offered a selective advantage for development of breast cancers, resulting in more aggressive tumors [ ] . at the same time overexpression of pap (its expression was suppressed by , -(oh) d in our experiment) in human pancreatic ductal adenocarcinoma indicated tumor aggressiveness [ ] . the agents that interfere with blood vessel formation can be used to block tumor progression. our data showed that , -(oh) d strongly down-regulated enzymes involved in angiogenesis which is mandatory for tumor progression: cd /aminopeptidase n and angiotensin i-converting enzyme (ace) or kininase ii. the cell surface aminopeptidase n (apn/cd ) was overex-pressed in tumor cells. however, potent, selective, and, particularly, non-cytotoxic inhibitors of this protein were lacking. angiotensin i-converting enzyme (ace) hydrolyzed the amino acid peptide angiotensin i into the potent vasoconstrictor angiotensin ii. it was reported that angiotensin ii stimulates angiogenesis in vivo, and ace inhibitors inhibit angiogenesis [ ] . our data suggest , -(oh) d as the potential inhibitor of both cd /apn and ace expression which also can contribute to , -(oh) d anticancer activity. further, this could account for the well-known effect of vitamin d on the reduction of hypertension [ ] . microarray data have provided major insight into gene transcription profiles in rat intestine in response to , -(oh) d thus creating a snapshot of molecular events following secosteroid intervention. we proposed that , -(oh) d regulates not only established transcellular calcium absorption but also paracellular calcium transport as well. we showed that , -(oh) d modulated the expression of different classes of genes in rat intestine, not only those directly involved in the absorption of nutrients in small intestine but also genes involved in immune response and angiogenesis. since many genes may not have a vdre in the promoter region, their regulation by , -(oh) d could be indirect through other proteins/factors expressed early in response to , -(oh) d or through increased intracellular ca + concentration. in addition to its central role in the maintenance of extracellular calcium level and bone mineralization, , -(oh) d also acts as a modulator of cell growth and differentiation in a number of cell types, including breast cancer cells. particularly important to us was to discover possible biochemical grounds for anti-proliferative and anticancer effects of , -(oh) d by induction of expression il- , il- , cd (protectin), cx c chemokine, and inhibition of the expression of thymosin-b- and both angiogenesis promoting enzymes cd /apn and ace. the down-regulation of ace may also account in part for the anti-hypertensive actions of vitamin d. these data may help to extend the potential use of , -(oh) d and its analogs in the treatment or prevention of a number of diseases. smith, wendy hellwig, maggie highland, and margaret clagett-dame from the biochemistry department, uw-madison for their assistance and valuable advice with this project and pat mings from the biochemistry department, uw-madison for her help with manuscript preparation. methods in enzymology we cordially thank wayne davis and sandra splinter bondurant from the gene expression center at the biotechnology center of uw-madison, christina gutierrez and chiara cirelli from the psychiatry institute at uw-madison, stan trask from affymetrix, connie key: cord- -kf m zho authors: cho, won kyong; yu, jisuk; lee, kyung-mi; son, moonil; min, kyunghun; lee, yin-won; kim, kook-hyung title: genome-wide expression profiling shows transcriptional reprogramming in fusarium graminearum by fusarium graminearum virus -dk infection date: - - journal: bmc genomics doi: . / - - - sha: doc_id: cord_uid: kf m zho background: fusarium graminearum virus strain-dk (fgv -dk ) is a mycovirus that confers hypovirulence to f. graminearum, which is the primary phytopathogenic fungus that causes fusarium head blight (fhb) disease in many cereals. understanding the interaction between mycoviruses and plant pathogenic fungi is necessary for preventing damage caused by f. graminearum. therefore, we investigated important cellular regulatory processes in a host containing fgv -dk as compared to an uninfected parent using a transcriptional approach. results: using a ′-tiling microarray covering all known f. graminearum genes, we carried out genome-wide expression analyses of f. graminearum at two different time points. at the early point of growth of an infected strain as compared to an uninfected strain, genes associated with protein synthesis, including ribosome assembly, nucleolus, and ribosomal rna processing, were significantly up-regulated. in addition, genes required for transcription and signal transduction, including fungal-specific transcription factors and camp signaling, respectively, were actively up-regulated. in contrast, genes involved in various metabolic pathways, particularly in producing carboxylic acids, aromatic amino acids, nitrogen compounds, and polyamines, showed dramatic down-regulation at the early time point. moreover, genes associated with transport systems localizing to transmembranes were down-regulated at both time points. conclusion: this is the first report of global change in the prominent cellular pathways in the fusarium host containing fgv -dk . the significant increase in transcripts for transcription and translation machinery in fungal host cells seems to be related to virus replication. in addition, significant down-regulation of genes required for metabolism and transporting systems in a fungal host containing the virus appears to be related to the host defense mechanism and fungal virulence. taken together, our data aid in the understanding of how fgv -dk regulates the transcriptional reprogramming of f. graminearum. fusarium graminearum (teleomorph gibberella zeae) is a well known phytopathogenic fungus associated with fusarium head blight (fhb) disease, which causes blights, root rots, or wilts, especially in economically important cereal crops such as wheat, maize, and barley [ ] . fhb is considered an important fungal disease because it drastically reduces grain yield and quality, and produces mycotoxins such as deoxynivalenol (don) and nivalenol (niv) in cereals, which are very harmful to human and animal health [ , ] . the fungus can also infect several dicotyledonous plants including arabidopsis, tobacco, tomato, and soybean [ ] . viruses that infect plant fungi are referred to as mycoviruses. infection by some mycoviruses confers hypovirulence by attenuating pathogenicity to their fungal hosts, which are mostly plant pathogens. mycoviruses tend to be double-stranded rna (dsrna) viruses [ ] , and several fusarium-infecting mycoviruses have been isolated [ ] [ ] [ ] . in addition, several whole genome sequences of dsrna mycoviruses strains derived from f. graminearum have recently been reported [ ] [ ] [ ] [ ] . in many cases, such as those of f. poae (fusarium poae virus , fpv ) [ ] and f. solani (fusarium solani virus , fsv ), viral infection is not associated with phenotypic changes [ ] . however, fusarium graminearum virus strain-dk (fgv -dk ) exhibits interesting phenotypes including reduced mycelial growth and the induction of dark red pigmentation [ ] . several previous studies have provided strong evidence that hypovirulent mycoviruses could be used as substitutes for fungicides [ , ] . a recent study demonstrated that protoplast fusion is the most efficient approach for transmitting mycoviruses among a wide range of phytopathogenic fungi and that this approach will facilitate the use of mycoviruses as a biocontrol agent [ ] . with the increasing availability of whole genome sequences for representative plant fungal pathogens [ ] , extensive and diverse genome-wide analyses can be performed, including transcriptomics, proteomics, and metabolomics [ ] . proteomics approaches for different fusarium species have enabled examinations of extracellular proteins, proteins involved in fumonisin biosynthesis, and proteome profiles upon antagonistic rhizobacteria inoculation and mycovirus infection [ ] [ ] [ ] [ ] . several gene expression analyses based on microarrays have also been conducted [ ] [ ] [ ] [ ] [ ] . for example, genome-wide expression profiling of f. graminearum was carried out to examine responses to treatment with azole fungicide tebuconazole and during perithecium development [ , ] . microarrays provide a valuable tool for detecting and identifying fusarium species that produce specific metabolites such as trichothecene and moniliformin [ , ] . moreover, the recently completed genome sequencing of three major fusarium species provides an important resource for studying pathogenicity and functions of individual genes [ ] . several microarray-based studies have demonstrated transcriptional changes in fungal genes following mycovirus infection, although most of these studies examined only chv - infecting the chestnut blight fungus cryphonectria parasitica. initially, a polymerase chain reaction (pcr)-based approach demonstrated that elevation of camp levels by chv - resulted in reduced accumulation of the gtp-binding (g) protein subunit cpg- [ ] . in addition, cdna microarrays containing , genes from c. parasitica showed transcriptional change in g-signaling pathways following hypovirus infections showing different virulence or phenotypes [ ] [ ] [ ] . infection by a virus leads to changes in diverse biological processes between fungal host and viral factors. it is of interest to examine such alterations at the molecular level. however, no previous reports have examined expression differences between a fungus containing a mycovirus and an infected parent, aside from two papers that used microarray cdna chips based on expressed sequence tags to examine fungal host gene expression upon mycovirus infection [ , ] . here, we examined genomewide transcriptional differences in f. graminearum expression between a strain harboring fgv -dk and its uninfected parent. this is the first report of a genome-wide fungal gene expression analysis during mycovirus infection using a ′ tiling microarray, and our findings show global differences in host cellular pathways in f. graminearum harboring fgv -dk . genome-wide ′-tiling microarray to identify differentially expressed genes in f. graminearum harboring fgv -dk the virus-infected f. graminearum exhibited strong inhibition of mycelia growth as well as reduced levels of don at days after inoculation ( figure ). to visualize how gene expression patterns were affected at different time points, we generated scatterplots ( figure ). radial growth was measured on pda h after inoculation. b the number of spores was measured in cmc broth at °c for days. the number of spores indicated represents the number per ml. data were described previously [ ] . c production of deoxynivalenol and -acetyldeoxynivalenol. for toxin analysis, all strains were grown in minimal medium supplemented with mm agmatine and incubated for days. d n/d: not detected. * the value is significantly different than that of the virus-free isolate as determined by a student's t-test. interestingly, the scatterplots showed that there were no significant differences in the number of differentially expressed genes between h and h; however, it appeared that the changes in gene expression at h were somewhat more extensive than those at h ( figure ). to identify differentially expressed genes, we first performed hierarchical clustering, which identified gene sets of significantly differentially expressed genes at figure differentially expressed f. graminearum genes during fgv -dk infection identified by microarray. scatterplots of normalized signal intensities h (a) and h (b) after inoculation with fgv -dk as compared to virus-free samples. all signal intensities were converted to a log scale. the diagonal lines indicate a two-fold change. the venn diagram illustrates the total number of genes that were significantly differentially expressed at h and h (c), down-regulated genes (d), and up-regulated genes (e). the heat map shows the expression patterns of genes that were hierarchically clustered and differentially expressed at both and h (f). red and green indicate upregulation and down-regulation, respectively. two different time points (additional file : table s and additional file : table s ). most of the identified genes showed at least two-fold differential expression. a total of genes, representing . % of , genes, were differentially expressed at both time points ( figure c ), with ( . %) and genes ( %) identified as differentially expressed at h and h, respectively ( figure c ). moreover, genes ( %) were differentially expressed at both time points ( figure c ). we further analyzed the lists of differentially expressed genes according to those down-regulated vs. those upregulated ( figure d , e). genes with an adjusted p-value less than . were selected as differentially expressed genes. in both time points, genes ( %) were downregulated whereas genes ( %) were up-regulated ( figure d , e). among the down-regulated genes, there were more differentially expressed genes at h ( genes) than at h ( genes) ( figure d ). in contrast, more genes were induced at h ( genes) than at h ( genes) ( figure e ). moreover, genes and genes were commonly found in the group of downregulated and up-regulated genes, respectively ( figure d , e). we then investigated the expression patterns of genes that were differentially expressed at both time points using hierarchical clustering, which sorted the genes into four groups according to expression patterns ( figure f ). group a contained genes that were highly downregulated at h but were up-regulated at h. in contrast, the genes belonging to group b were highly induced at h, and subsequently down-regulated ( figure f ). group c included genes that were strongly repressed regardless of virus infection time, and group d contained genes that showed consistently elevated gene expression across both time points. representative genes showing significant expression change and real-time validation of the microarray data by quantitative real-time reverse transcription pcr representative fungal genes that showed significant gene expression are listed in table . at h, several genes encoding enzymes including phospholipase/carboxylesterase, polyketide synthase, eukaryotic aspartyl protease and dipeptidyl aminopeptidases were highly induced, whereas transcripts involved in transport, such as amino acid transporter permease and abc transporter, were up-regulated at h (table ). in contrast, several of the repressed genes at h included maltose transporter, linoleate diol synthase, and genes with unknown functions, whereas those encoding ferric reductase and abhydrolase were strongly repressed at h (table ) . to validate the microarray data, we selected genes whose expression was significantly affected in the host containing fgv -dk , as demonstrated by the microarray analysis, and determined their relative expression by quantitative real-time reverse transcription pcr (qrt-pcr) ( figure and additional file : table s and additional file : table s ). genes that showed diverse expression patterns were categorized into different functional classes (figure and additional file : table s ). the results of the qrt-pcr were highly consistent with those of the microarray data. for example, according to the qrt-pcr and microarray results, the transcript levels for three genes, including fgsg_ , fgsg_ , and fgsg_ , were highly reduced at both h and h, whereas fgsg_ , fgsg_ , fgsg_ , fgsg_ , and fgsg_ were strongly induced regardless of the time point ( figure a -c). when the mrna level of a gene is too low to quantify, or the p-values from the microarray data are very high, it is highly likely that qrt-pcr results will not correlate with microarray data, as was observed for fgsg_ and fgsg_ ( figure a ). as compared to the microarray approach, qrt-pcr offers a highly sensitive technique for detecting low amounts of transcripts and provides the transcript level for the gene of interest. for example, the expression intensities for genes were relatively low, ranging from to . ( figure a ), whereas the amount of mrnas for nad-dependent aldehyde dehydrogenases (fgsg_ ) and dipeptidyl aminopeptidases (fgsg_ ) were very high and ranged from . to . , particularly in the host containing fgv -dk at h ( figure c ). funcat classification for an overview of the transcriptional reprogramming of f. graminearum harboring fgv -dk we subjected a total of differentially expressed genes to functional catalogue (funcat) annotation to gain insight into their functional classifications [ ] . specifically, we divided differentially expressed genes into four groups (table ). more than half of the differentially expressed genes were not assigned to any functional category. specifically, genes ( . %) in the group of down-regulated genes at h were unclassified. of the functional categories, the vast majority of differentially expressed genes were associated with metabolism ( table ). note that genes ( . %) were downregulated while genes ( . %) were up-regulated at h, whereas there were more up-regulated genes ( ) than down-regulated genes ( ) at h. based on the number of differentially expressed genes, it is likely that genes involved in various metabolic pathways were severely repressed at h and then were gradually induced at h. along with a gene set for metabolism, genes associated with energy were highly down-regulated ( genes) at h (table ). in contrast, genes involved in transcriptional and translational machinery were dominantly up-regulated early after fgv -dk infection. for example, genes associated with the cell cycle and dna processing ( genes), transcription ( genes), protein synthesis ( genes), protein fate ( genes), and those encoding proteins with binding function ( genes) were highly upregulated at h. the number of down-regulated genes associated with cellular transport at h was almost twice that of up-regulated genes. conversely, the number of upregulated genes that govern cellular transport was similar to that of the down-regulated genes at h. next, we analyzed the enriched gene ontology (go) terms of the differentially expressed genes. the identified enriched go terms are listed in additional file : table s . the directed acyclic graph (dag) illustrates the go terms that were over-represented (figures , , , , , and ) . interestingly, go terms related to ribosome biogenesis, such as the ribosome ribonucleoprotein complex assembly (go: ), ribosome assembly (go: ), ribonucleoprotein complex biogenesis (go: ), and ribosome biogenesis (go: ), were highly over-represented ( figure ). moreover, go terms for rna processing (go: ), ncrna processing (go: ), rrna metabolic processes (go: ), rrna processing (go: ), and maturation of ssu-rrna (go: ) were over-represented with high levels of transcripts ( figure ). similarly, go terms related to the nucleolus, such as the small nucleolar ribonucleoprotein complex (go: ), the nuclear lumen (go: ), and nucleolus (go: ), were over-represented ( figure ). transcripts associated with the nucleolus were highly accumulated at h. it was not surprising that the expressions of genes that involve a range of metabolic pathways were significantly affected by fgv -dk infection. consistent with the funcat annotation, go enrichment analysis showed that genes associated with a variety of metabolic pathways were significantly down-regulated, particularly at h. for example, over-represented go terms included cellular aromatic compound metabolic processes (go: ), in the cellular component ontology, go terms associated with membranes (go terms , , , and ) were over-represented ( figure ). interestingly, genes associated with membranes were down-regulated regardless of the time point ( figure ). furthermore, transporter activity was one of the significantly over-represented go terms with respect to molecular function. of a total of go terms for transporter activity, were present at h, including carbohydrate transmembrane transporter activity (go: ), ion transmembrane transporter activity (go: ), and sugar:hydrogen symporter activity (go: ) (figure ). at h, three go terms, including primary active transmembrane transporter activity (go: ), atpase activity, and coupled to transmembrane movement of substances (go: ), were over-represented ( figure ). transcription factors (tfs) play a key role in signal transduction pathways by regulating gene expression to control biological processes [ ] . thus, it is of interest to understand their involvement in fungi-virus interactions at the transcriptional level. whole genome sequences of f. graminearum show that there are at least tfs divided into families [ ] . we identified more differentially expressed tfs at the early time point ( tfs) than at the late time point ( tfs) (additional file : table s ). zn cys ( tfs) was the most prevalent tf family, followed by the c h zinc finger ( tfs) and bhlh ( tfs) families. in addition, centromere protein b, dna-binding region, homeodomain-like, lambda repressor-like, dna-binding, nucleic acid-binding, ob-fold, tf jumonji, and bzip were also identified as tfs showing significant change at the transcript level (additional file : table s ). zn cys ( tfs) was also the most prominent tf family at the late time point, followed by c h zinc finger ( tfs), bhlh ( tfs), and bzip ( tfs) (additional file : table s ). moreover, the gene expressions of tfs belonging to the myb (a negative transcriptional regulator), tf jumonji, zinc finger (cchctype), and zinc finger (nf-x -type) families were significantly changed. recently, a mutant library of putative tfs was established via homologous recombination in f. graminearum, providing a valuable resource to study gene regulation in fungus [ ] . thus, it might be of interest to compare phenotypes of mutants for the differentially expressed tfs. most tf knock-out mutants show distinct phenotypes. for example, five mutants for which members of the c h zinc finger family were deleted (fgsg_ and fgsg_ ) and zn cys (fgsg_ , fgsg_ , and fgsg_ ) displayed abnormal phenotypes compared to wild type controls. interestingly, the deletion of fgsg_ resulted in a hypervirulence phenotype. the expression of all five of these tfs was strongly up-regulated h after virus infection. two genes (fgsg_ and fgsg_ ) were highly expressed in both early and late time points. the ascospore was not formed in fgsg_ mutants, while that for fgsg_ exhibited increased resistance to oxidative stress. fgv -dk drastically induces the expression of fungal host genes required for their replication perhaps the most striking finding of this study is that the host containing fgv -dk accumulates transcripts associated with translation machinery, such as ribosome biogenesis and the nucleolus. ribosomes function in the production of proteins, while the nucleolus is the site of ribosomal rna synthesis and ribosome assembly [ ] . the nucleolus actively participates in a variety of biological processes, including cell cycle regulation, cell growth, stress sensing, and viral infection [ ] . moreover, genes involved in rna processing were highly induced. these genes convert precursor rnas such as non-coding rna (ncrna) and small subunit (ssu) ribosomal rnas molecules into mature rna molecules. taken together, these findings suggest that the entire complex for protein synthesis and processing in fungal host cells was highly activated by fgv -dk . viruses rely on host cell machinery and have evolved sophisticated mechanisms to achieve replication efficiently during virus infection [ ] . given that nucleolus localizing genes were up-regulated, it appears that the virus stimulates gene expression associated with nucleolus. as a result, the nucleolus produces numerous ribosomes to maximize viral replication. in addition, the virus might control protein synthetic machinery in host ribosomes to replicate viral proteins. recently, proteomics-based studies confirmed the involvement of the nucleolus in viral infection and replication [ , ] . previous studies have shown that rna viruses can interact with several nucleolar proteins such as nucleolin, b , and fibrillarin to facilitate virus replication [ ] . thus, it might be of interest to examine the interactions between nucleolar proteins from f. graminearum and fgv -dk viral proteins in future studies. although our data suggest that expression of genes related to ribosomes was strongly affected by virus infection, this might be a common phenomenon in hosts in response to various kinds of virus. thus, we cannot exclude the possibility that ribosomes are indirectly involved in the replication of dsrna viruses. since little is known about dsrna viruses, we refer to many results from singlestranded (ss) rna viruses to support our data. the system for dsrna viruses might differ from that of ssrna viruses. metabolism is the core of cellular functions, and comprises numerous reactions that function in the degradation of nutrients and biosynthesis of cellular components including proteins, lipids, carbohydrates, dna, and rna. our results, as well as a previous report of c. parasitica, found dramatic differences in gene expression associated with metabolic pathways [ ] . however, there are inherent differences between that study and the present one. specifically, our microarray data demonstrated down-regulation of genes involved in metabolism. in contrast, the previous study found that the majority of metabolites, including lipids and carbohydrates, were significantly accumulated [ ] . this difference may be due to different infection times. we found the most dramatic changes in gene expression at the early time point, whereas metabolic probing showed that a variety of metabolites accumulated with increasing infection time [ ] . differential expression of genes related to metabolism might be associated with the altered host phenotype. for example, in the group of down-regulated genes at h, genes for cell type differentiation were also highly enriched, suggesting that host cell differentiation seems to be induced by viral infection. interestingly, several studies provide evidence that filamentous differentiation in fungi is required for virulence [ ] . regardless of viral infection stage, genes related to cell rescue, defense, and virulence were highly up-regulated, suggesting that the host defense system was consistently activated. compared to the whole genome, genes with significantly enriched functions were mostly found in the group showing up-regulation at h, suggesting that the transcriptional regulation in the host harboring mycovirus is more important at the early time point than the late time point. of the altered metabolites, our study as well as a previous report [ ] found dramatic changes in gene expression levels for polyamine production. polyamines play roles in many biological processes, such as cell growth, development, and responses to various stresses [ ] . thus, it is likely that the down-regulation of genes involved in polyamine biosynthesis during the early stage ( h) could be correlated with reduced levels of don, which confers hypovirulence to host f. graminearum (figure ) . a previous study showed that polyamine biosynthesis inhibitors decreased mycelial growth of sclerotinia sclerotiorum [ ] . this result is highly consistent with observed phenotypes in virus-infected f. graminearum showing strong inhibition of mycelia growth (figure ). however, we do not know whether the inhibition of mycelial growth in f. graminearum is directly related to polyamine biosynthesis. changes to the membrane-associated transporting system of the host harboring fgv -dk along with the reduced amounts of many metabolites such as carbohydrates, gene expression for the transfer of such metabolites or ions from one side of the membrane to the other was greatly suppressed in the host harboring fgv -dk . this indicates that virus infection affects the transport of many micro-and macroelements in host cells at the transcript level. moreover, this transport system mediates cell-to-cell communication within the host via plasma membranes. for example, transcripts required for cellular communication were highly accumulated at h, suggesting that the virus might first stimulate cell-to-cell communication in fungal host cells, which is necessary to trigger host defense mechanisms against viral pathogens. indeed, a recent study reported that infection of chlorovirus, paramecium bursaria chlorella virus , affects the transport activity of solutes via plasma membranes in chlorella [ ] . plasma membranes are the first barriers to block pathogen attack and can transmit information and molecules between neighboring cells. viruses utilize plasma membranes to interact with signaling molecules. a previous study suggested that virus infection causes depolarization of the host cell membranes, thus decreasing the transport of solutes by active transporters via plasma membranes [ ] . the impairment of plasma membranes by viruses suggests that all materials required for virus replication should be recruited within the host cell [ ] . taken together, these data suggest that fgv -dk might inhibit the transport of diverse metabolites via plasma membranes to the maximize energy required for virus replication within the nucleolus. fungal-specific tfs are key players in the gene expression regulation in f. graminearum harboring fgv -dk the expressions of members of the zn cys tf family were strongly altered at both the early and late time points. these are known to be fungal-specific, and their functional roles are diverse, including sugar and amino acid metabolism, gluconeogenesis, respiration, vitamin synthesis, cell cycle, chromatin remodeling, nitrogen utilization, peroxisome proliferation, drug resistance, and stress response [ ] . the zn cys family is the largest tf family in f. graminearum, comprising tfs. of the nine tfs that were differentially expressed at both the early and late time points, six belong to the zn cys family, indicating that expression of zn cys is necessary in the host harboring fgv -dk . furthermore, the enriched go terms for nucleic acid binding that were up-regulated at h provide evidence for transcriptional regulation by tfs such as the zn cys tf family (figure ). to characterize the functional roles of the zn cys tf family associated with fgv -dk , it is necessary to examine the phenotypes of knock-out mutants. therefore, the recently generated f. graminearum deletion mutant lines will be very useful resources for characterizing the functions of host tfs associated with hypovirulence. fungi utilize camp-mediated signal transduction pathways to recognize and respond to diverse environmental stimuli. camp signaling is implicated in the regulation of hyphal growth, mating, and gluconeogenesis in many fungi [ , ] . in addition, camp and the g-protein alpha subunit coordinate their activities to regulate differentiation and virulence in some fungi. we found that transcripts for genes associated with responses to camp were highly accumulated in the late stage of virus infection. this result is consistent with previous data that suggested up-regulation of camp levels in the fungal transcriptome by hypovirus infection [ ] . thus, we hypothesize that fgv -dk attenuates the pathogenicity of f. graminearum via camp-mediated signaling and that this process occurs relatively late after virus infection. recent years have seen extraordinary developments in genome-wide experimental methods. of these, microarray analyses facilitate an understanding of the dynamic gene expression patterns of target organisms during environmental stimuli such as biotic and abiotic stresses. here, given the benefits of the available whole genome sequences of f. graminearum, we generated a ′ tiling microarray system covering whole genes. to decipher global transcriptional reprogramming in f. graminearum harboring fgv -dk in detail, samples were harvested at two different time points, thus providing lists of differentially expressed genes early and late in the host containing fgv -dk as compared to an uninfected strain. numbers of differentially expressed genes at the early and late time points were comparable, but the gene lists differed, suggesting time-dependent transcriptional changes. genes that were up-regulated at the early time point included those involved in protein synthesis, such as ribosome assembly, as well as nucleolus and ribosomal rna-processing genes, suggesting that fgv -dk strongly modulates translational machinery in f. graminearum to maximize viral replication. moreover, the accumulation of transcripts associated with transcription, such as tfs, indicated that the transcriptional machinery, which is largely regulated by fungal-specific tfs, might be one of the main targets for virus infection. in contrast, genes involved in various metabolic pathways, particularly those that produce carboxylic acids, aromatic amino acids, nitrogen compounds, and polyamines, were highly down-regulated at the early time point ( h) . interestingly, such components are closely associated with the host defense mechanism. these results suggest that fgv -dk suppresses the production of such defense-related components until the transcriptional and translational machinery in host cells have adjusted to fgv -dk replication. in addition, transport systems associated with membranes were severely damaged by hindering the recruitment of materials for viral replication within host cells. when faced with viral infection, f. graminearum tries to establish a defense mechanism by consistently up-regulating genes associated with defense and virulence at the late time point ( h). taken together, our data provide strong genome-wide transcriptional evidence of how fgv -dk regulates the transcriptional reprogramming in f. graminearum. virus-free and fgv -dk -infected f. graminearum strain-dk were stored in % (v/v) glycerol at − °c and reactivated on pda at °c with a h light-dark cycle. f. graminearum cultures used for rna extractions were grown as described previously [ ] . freshly grown mycelia from pda media plates were inoculated in or ml complete media (cm) broth [ ] and incubated at °c for h or h in an orbital shaker ( rpm). hyphae were collected by filtering through mm paper, washed with distilled water, dried by blotting with paper towels, and frozen at − °c. conidia of virus-free and fgv -dk -infected f. graminearum strains were harvested in ml of cmc broth at days after inoculation. conidial suspensions ( × conidia per dish) were grown in ml of defined media containing mm of agmatine [ ] in × mm petri dishes for days prior to filtrate harvests. three replicates were used for this treatment. mycotoxin was extracted from the isolates and analyzed with a shimadzu qp- gas chromatograph-mass spectrometer, as described previously [ ] . the trichothecenes were measured based on biomass produced by each strain. preparation of total rna samples and cdna synthesis for rt-pcr frozen mycelia were pulverized with a mortar and pestle using liquid nitrogen for nucleic acid extraction. total rnas were extracted by iso-rna lysis reagent ( prime, germany). extracted total rna was treated with dnase i (takara, japan) to remove genomic dna according to the instructions provided by the manufacturer. these total rna samples were precipitated with ethanol and resuspended in depc-treated water. next, μg total rna of each sample was used to synthesize first-strand cdna with an oligo (dt) primer and m-mlv reverse transcriptase (promega, usa) according to the manufacturer's protocols. all synthesized cdnas were diluted : with nucleasefree water for rt-pcr. expression profiling was conducted with the gibberella zeae k microarray, which was designed based on the f. graminearum sequences released in march (http://www.broad.mit.edu/annotation/genome/fusarium _group/). the genome contains a total of , genes. ten -nucleotide-long probes were designed from each gene starting base pairs (bp) ahead of the end of the stop codon and by shifting bp. thus, these probes covered bp in the ′ region of the target gene. mitochondrial genes ( genes) and selected markers such as gfp, gus, hyg, bar, and kan were included. in total, , probes were designed. the average probe size was nucleotides long, and the tm values were adjusted from to °c. the microarray was manufactured by nimblegen inc. (http://www.nimblegen.com/). random gc probes ( , ) to monitor the hybridization efficiency and four corner fiducial controls ( ) to assist with overlaying the grid on the image were included. to assess the reproducibility of the microarray analysis, we repeated the experiment three times with independently prepared total rnas. thus, a total of samples were subjected to total rna isolation and used for microarray analyses. for the synthesis of double-stranded cdnas, the revertaidtm h minus first strand cdna synthesis kit (fermentas, lithuania) was used. in brief, μl oligo dt primer ( μm) and μl ( μg) total rnas were combined and denatured at °c for min and renatured by cooling the mixture in ice. first-strand dna was synthesized by adding μl x first strand buffer, μl ribolocktm ribonuclease inhibitor, μl mm dntp mix, and μl rever-taidtm h minus m-mulv reverse transcriptase enzyme and incubating at °c for h. the reaction was stopped by heating at °c for min. to synthesize the second strand, . μl nuclease free water, μl x reaction buffer for dna polymerase i (fermentas, lithuania), μl x t dna ligase buffer (takara, japan), μl unit/ul dna polymerase i (fermentas, lithuania), . μl unit/μl ribonuclease h (fermentas, lithuania), and . μl unit/μl t dna ligase (takara, japan) were added to the firststrand reaction mixture and the reaction was performed at °c for h. the double-stranded cdna mixture was purified using the minelute reaction cleanup kit (qiagen, usa). for the synthesis of cy -labeled target dna fragments, μg double-stranded cdna was mixed with μl ( optical density) cy - mer primer (sigma-aldrich, usa) and denatured by heating at °c for min. the reaction was further proceeded by adding μl x dntp mix ( mm each), μl deionized water, and μl klenow fragment ( unit/μl, takara, japan) and incubating at °c for h. dna was precipitated by centrifugation at , x g after adding . μl m nacl and μl isopropanol. precipitated samples were rehydrated with μl water. the concentration of each sample was determined using a spectrophotometer. a μg aliqout of dna was used for microarray hybridization. the sample was mixed with . μl x hybridization buffer (nimblegen, usa) and finalized to μl with deionized water. hybridization was performed with an maui chamber (biomicro, usa) at °c for - h. after hybridization, the microarray was removed from the maui hybridization station and immediately immersed in a shallow ml wash i (nimblegen, usa) at °c for - s with gentle agitation, then transferred to a second dish of wash i and incubated for min with gentle agitation. the microarray chip was then transferred to another dish of wash ii and further washed in wash iii for min with agitation. the slide was dried in a centrifuge for min at g and scanned using a gene-pix scanner b (axon, usa). the hybridized microarray chip was scanned with genepix b (axon instruments) preset with a μm resolution for the cy signal. signals were digitized and analyzed by nimblescan (nimblegen, usa). the grid was aligned to the image with a chip design file called the ndf file. the alignment was assessed by ensuring that the grid's corners were overlaid on the image corners. this was further checked by uniformity scores in the program. the analysis was performed in a two-part process. first, pair-report files were generated in which the sequence, probe, and signal intensity information for the cy channel were collected. databased background subtraction using a local background estimator was performed to improve fold-change estimates on arrays with high background signals. the data were normalized and processed with a cubic spline normalization using quantiles to adjust for signal variation between chips [ ] . a probe-level summarization by robust multi-chip analysis (rma) using a median polish algorithm implemented in nimblescan was used to produce call files in order to improve the sensitivity and reproducibility of microarray results [ ] . the multiple correction analysis was performed using the limma package in an r computing environment [ ] . linear models implemented in lmfit and empirical bayes methods implemented in ebayes were applied to assess the differential expression of genes. genes for which the adjusted p-value or false discovery rate was below . were collected and further selected. hierarchical clustering was performed by acuity . (axon instruments) with similarity metrics based on squared euclidean correlation, and average linkage clustering was used to calculate the distance of genes. the microarray data were deposited in the ncbi gene expression omnibus (geo) database with the accession number gse . to gain insight into the functions of the differentially expressed genes, go enrichment analysis was conducted with gominer [ , ] . the , genes were matched to the m. grisea sequencing assembly sc (http://www. broad.mit.edu/annotation/genome/magnaporthe_grisea/) with scores of and up by blastp analysis and were used as the total gene set for go enrichment analysis. the gominer program first categorizes each gene according to its go terms and the mode of gene expression (either down-or up-regulation). modes of expressions are denoted as under, over, and change. the program then calculates p-values using a one-sided fisher exact test for the number of categorized go terms out of the total number of terms. false 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normalization, and summaries of high density oligonucleotide array probe level data linear models and empirical bayes methods for assessing differential expression in microarray experiments gominer: a resource for biological interpretation of genomic and proteomic data gene ontology: tool for the unification of biology. the gene ontology consortium the funcat, a functional annotation scheme for systematic classification of proteins from whole genomes submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution rate (fdr) values were obtained from randomizations. go terms for which the fdr was less than . were selected.qrt-pcr analysis qrt-pcr was performed on bio-rad's cfx ™ real-time pcr system using gene-specific internal primers. each reaction mix ( μl) consisted of ng total cdna, μl x ssofast ™ evagreen w supermix (bio-rad, usa), and pmoles each primer. the thermal profile was as follows: min at °c and cycles of s at °c, s at °c, and melting curve data obtained by increasing the temperature from to °c. elongation factor α (ef- α; fgsg_ ) and cyclophilin (cyp ; fgsg_ ) were used as internal reference genes to normalize mrna levels between samples (ef- α; genbank accession no. xm , cyp ; genbank accession no. xm ). data were analyzed using the bio-rad cfx manager v . . . software (bio-rad, usa). rna was extracted from three independent replicate experiments, and each pcr product was evaluated in at least three independent experiments, including three technical replicates [ ] . additional file : table s . differentially expressed f. graminearum genes at h.additional file : table s . differentially expressed f. graminearum genes at h.additional file : table s . the representative f. graminearum genes selected for qrt-pcr validation.additional file : table s . oligonucleotide primers used for qrt-pcr.additional file : table s . enriched go terms of differentially expressed genes.additional file : table s . f. graminearum transcription factors which were differentially expressed by fgv -dk infection. the authors declare that they have no competing interests.authors' contributions jy, kml, ms, ywl, and kk designed the experiment. jy, kml, and ms performed cultivation of f. graminearum and infection with fgv -dk . jy and km conducted mycotoxin analysis, isolated total rnas, and conducted rt-pcr. wkc, jy, and kk analyzed the microarray data and interpreted the results. jy and kk coordinated the study. wkc, jy, ywl, and kk wrote the manuscript. all authors read and approved the final manuscript. key: cord- -istz iql authors: nan title: procedures to investigate waterborne illness date: - - journal: procedures to investigate waterborne illness doi: . / - - - - _ sha: doc_id: cord_uid: istz iql humanity could not survive without a reliably clean, safe, and steady flow of drinking water. since the early s when typhoid fever and cholera were frequently causes of waterborne illness in developed countries, drinking water supplies have been protected and treated to ensure water safety, quality, and quantity. having access to safe drinking water has always been one of the cornerstones of good public health. not only safe water is limited to drinking water, but recreational water can also be a source for waterborne illness—both from treated waters such as in swimming pools, whirlpools, or splash pads and from non-treated surface waters such as lakes, rivers, streams and ponds. recreational waters may cause illness not only from ingestion of pathogens, but also when in contact with eyes, ears, or skin. some pathogens in water can be acquired by inhalation of aerosols from water that is agitated or sprayed such as in humidifiers, fountains, or misting of produce. this poses a potential risk to those exposed, particularly if they are immunocompromised. humanity could not survive without a reliably clean, safe, and steady flow of drinking water. since the early s when typhoid fever and cholera were frequently causes of waterborne illness in developed countries, drinking water supplies have been protected and treated to ensure water safety, quality, and quantity. having access to safe drinking water has always been one of the cornerstones of good public health. safe water is not limited to drinking water, since recreational water and aerosolized water can also be sources for waterborne illness, from treated waters such as in swimming pools, whirlpools, or splash pads and from non-treated surface waters such as lakes, rivers, streams and ponds. recreational waters may cause illness not only from ingestion of pathogens, but also when in contact with eyes, ears, or skin. some pathogens in water can be acquired by inhalation of aerosols from water that is agitated or sprayed such as in humidifiers, fountains, or misting of produce. this poses a potential risk to those exposed, particularly if they are immunocompromised. often when an outbreak is first suspected, the source is not clear, i.e., food, water, animal contact. investigation is usually needed to find the common source. in some outbreaks the food may first be identified as the source, such as with produce, but the ultimate source could be contaminated irrigation water. investigators have to keep an open mind until laboratory and/or epidemiologic evidence links cases to the primary source. although we frequently think of waterborne illness originating from a microbiological agent, we should be aware that water may also be contaminated by pesticides, fertilizers, and other chemicals which may enter through industrial discharge, agriculture runoff, or deliberate contamination. waterborne illness acquired from microorganisms may be classified as: • toxin-mediated infections caused by bacteria that produce enterotoxins or emetic toxins that affect water, glucose, and electrolyte transfer during their colonization and growth in the intestinal tract; • infections caused when microorganisms invade and multiply in the intestinal mucosa, eyes, ears, or respiratory tract, or contact the skin; • intoxications caused by ingestion of water containing poisonous chemicals or toxins produced by other microorganisms manifestations range from slight discomfort to acute illness to severe reactions that may terminate in death or chronic sequelae, depending on the nature of the causative agent, number of pathogenic microorganism or concentration of poisonous substances ingested, and host susceptibility and reaction. the public relies on public health regulators to investigate and mitigate waterborne illness. mitigation depends upon rapid detection of outbreaks and a thorough knowledge of the agents and factors responsible for waterborne illness. public health and law enforcement agency officials should always be alert to the rare possibility of an intentional contamination of water supplies by disgruntled employees or terrorists. the purposes of a waterborne illness investigation are to stop the outbreak or prevent further exposure by: • identifying illness associated with an exposure and verifying that the causative agent is waterborne • detecting all cases, the causative agent, and the place of exposure • determining the water source, mode of contamination, processes, or practices by which proliferation and/or survival of the etiological agent occurred • implementing emergency measures to control the spread of the outbreak • gathering information on the epidemiology of waterborne diseases and the etiology of the causative agents that can be used for education, training, and program planning, thereby impacting on the prevention of waterborne illness • determining if the outbreak under investigation is part of a larger outbreak by immediately reporting to state/provincial/national epidemiologists in the instance of a bottled water outbreak, halting of distribution and sale of product and recall of product, some of which may already be in consumers' homes, are necessary to prevent further illness. as epidemiologic data accumulate, information will indicate the source of the problem, whether a municipal water treatment plant, bottled water manufacturing plant, or recreational water exposure, and suggest methods for controlling and preventing waterborne illness. this information will guide administrators in making informed decisions to provide the highest degree of waterborne safety. a flowchart, sequence of events in investigating a typical outbreak of waterborne illness (fig. ) shows the sequential steps, as presented in this manual, in investigating a typical outbreak of waterborne illness and illustrates their relationships. a description of each step is presented in this manual. can be recognized at any point during the outbreak investigation. if intentional contamination is suspected follow your notification scheme in emergency response plans (this could include law enforcement, emergency management and other government agencies) the primary purpose of a waterborne disease surveillance system is to systematically gather accurate information on the occurrence of water-related illnesses in a community, thus allowing development of a rational approach for the detection, control and prevention of waterborne illness. other purposes are to (a) determine trends in the incidence of waterborne diseases, (b) characterize the epidemiology of waterborne diseases, (c) gather and disseminate information on waterborne diseases, and (d) develop a basis for evaluating control efforts. it may be useful to coordinate this system with, or integrate it into a foodborne disease surveillance system. however, while the procedures are quite similar from an epidemiologic viewpoint, they may differ with respect to personnel or agencies involved. an effective disease surveillance system is essential for detection of disease caused by either unintentional or intentional contamination of food. an effective waterborne disease surveillance system consists of: • early reports of enteric and other illnesses that may be related to water exposure or consumption • coordinated effort between local and state public health partners, water utility and water recreation staff • systematic organization and interpretation of data • timely investigation of identified outbreaks or clusters of illness • dissemination of outbreak reports and surveillance summaries to all appropriate stakeholders many types of reporting systems may already exist at the local or state/provincial level, and these should be incorporated into a waterborne disease surveillance program. these include (a) mandatory (or voluntary) laboratory-or physician-based reporting of specific infectious diseases, (b) national-based surveillance systems such as calicinet (cdc ) or nors (cdc in the us, (c) physician office, hospital emergency, and urgent-care clinic medical records, (d) public complaints made to health agencies and/or local water utilities, (e) school illness and absentee records, (f) absentee records of major employers, (g) water treatment records kept by water utilities (e.g., turbidity, disinfection levels, occurrence of coliforms), (h) increased sales of antidiarrheal drugs and anti-nausea medications, and (i) source water quality data kept by environmental agencies (e.g., departments of natural resources and geological survey agencies). another type of surveillance mechanism that may supplement or enhance existing reporting systems is a daily log of illness and water quality complaints. agency contemplates initiation or development of a waterborne illness surveillance program, give top priority to identification of appropriate financial, political, strategic, and administrative support. then, identify a key person to create, implement, and manage the system. this person takes responsibility for: • reviewing the types of reporting systems that already exist in your agency or in other agencies that could be incorporated into a waterborne illness surveillance system • identifying the types of information that cannot be obtained from existing reporting systems but that need to be collected or addressed by the waterborne illness surveillance system • identifying ways to merge or integrate the data collected by existing systems with data gathered in the waterborne illness surveillance system • identifying collaborating agencies and staff • develop a mechanism to communicate and update all stakeholders (may be by blast e-mail or periodic conference calls) • providing training in surveillance methods for agency staff and other partners to enhance cooperation • assembling materials that will be required during an outbreak investigation • evaluating the effectiveness of the system. develop procedures to seek and record complaints about waterborne illnesses, water supplies, and water recreational sites. for example, list the telephone number of the waterborne illness investigation unit prominently on local and state public health and water utility websites. to be most effective, have this number monitored / by staff or an answering service. if possible, the utilization of social media such as facebook or twitter should be considered and monitored as many large municipalities (including drinking water utilities) and recreational facilities have an internet presence. if your agency has social media accounts, consider using this vehicle to further disseminate information regarding waterborne illness clusters or outbreaks. identify medical care facilities and practitioners and seek their participation. direct educational activities, such as newsletters and talks at meetings, to stimulate participation in the program. encourage water treatment utilities and operators of recreational water sites to report suspected complaints of waterborne illness to the appropriate local agencies. also, encourage private and hospital laboratories to report isolations of parasitic agents (e.g., giardia, cryptosporidium), viruses (e.g., norovirus and hepatitis a virus), bacteria (e.g., e. coli (pathogenic), salmonella, shigella, vibrio cholerae), and other agents that may be waterborne. develop a protocol for notification and coordination with agencies that might cooperate in investigational activities, including -h-a-day, -days-a-week contacts. a comprehensive contact list should be constructed and updated at least twice a year as individuals may change. notify and coordinate with state/provincial or district agencies, national agencies that have surveillance and water regulatory responsibilities, and other national and international health agencies, as appropriate. for example, it may be useful to find out the level of participation within a certain jurisdiction in national-level outbreak surveillance programs such as nors (cdc, ) or other national surveillance system. delegate responsibility to a professionally trained person who is familiar with epidemiologic methods and with the principles of water treatment and recreational water protection. this person will (a) direct the surveillance program, (b) take charge if waterborne and enteric outbreaks are suspected, and (c) handle publicity during outbreaks. delegate responsibility to others who will carry out specific epidemiologic, laboratory and on-site investigations. if an intentional contamination event is suspected, local and national law enforcement agencies will likely become the lead agency responsible for the investigation. with this in mind, it is critical to identify appropriate individuals and include them in communication and any practice drills that may occur. if a relationship has been established prior to any event, the investigation may run more smoothly. enlist help from a team of epidemiologists, microbiologists, sanitarians/environmental health officers/public health inspectors, engineers, chemists, nurses, physicians, public information specialists, and other (e.g., toxicologists) as needed. free flow of information and coordination among those participating in waterborne disease surveillance and investigation are essential, particularly when several different agencies are involved. water-related complaints are equally likely to be directed at health departments or water utilities but also perhaps to different jurisdictions. therefore, it is essential that these complaints be registered by an agency and that the information is rapidly shared within and perhaps outside of a particular jurisdiction as part of an integrated surveillance system. whenever possible, share the information with participating parties by rapid means such as e-mail and by calling / contact phone numbers. if an intentional contamination event is suspected, contact emergency response and law enforcement for their early involvement. select staff members who will participate in the waterborne disease surveillance program on the basis of interest and ability. inform them of the objectives and protocol of the program. emphasize not only the value of disease surveillance, but also the value of monitoring water quality and treatment performance. if possible, provide printed learning material that can be referenced later. encourage the use of epidemiologic information and approaches in routine disease surveillance and prevention activities. develop their skills so that they can carry out their role effectively during an investigation, and teach them how to interpret data collected during investigations. conduct seminars routinely and during or after investigations to update staff and keep agency personnel informed. train office workers who will receive calls concerning waterborne illnesses to give appropriate instructions. those who participate in the investigation will learn from the experience and often are in a position to implement improvements after completion of the investigation. assemble and have readily available kits with forms and equipment as specified in table a (equipment useful for investigations). restock and maintain kits on a schedule recommended by, and in cooperation with, laboratory staff to ensure their stability and sterility. verify expiration dates, and use kits before this date or discard and reorder. assemble a reference library on waterborne illnesses, investigation techniques, and control measures from reference books, scientific journal articles, manuals, and reputable internet sources (e.g., www.cdc.gov, www.who.int/en/, www.hc-sc.gc.ca/ index-eng.php, www.gov.uk/topic/health-protection/infectious-diseases); make it available to the staff in an easy-to-access format. (see further reading for suggestions). organize a multiagency team with representatives from public health agencies, regulatory agencies, and water utilities and with local political officials to review and exercise existing emergency response plans in the event of a large scale waterborne disease outbreak or other disaster likely to result in waterborne illnesses. local public health agencies have the primary responsibility for the restoration and protection of health of a community following an outbreak event or other emergency. emergency operational procedures should include the following: • an emergency notification list that includes phone numbers and e-mail addresses of key persons/agencies that need to be informed about possible outbreaks and that should receive emergency press releases. every state/province has an emergency management agency and depending upon the scale of the event, it may be useful to coordinate efforts. • clear guidelines for household water consumption following an event. for example, boil-water advisories or instructions to drink only bottled water. statements should be reviewed to ensure current relevance and updated to reflect the most current knowledge. • a plan for dissemination of information to the public; select a coordination point to which all news media and outside agencies will be directed, and designate one person or one telephone number as the contact. (more than one contact person can create confusion). • alternative drinking water sources to be used in cases of emergency and plans for the distribution of this water, if necessary. these include alternative munici-pal systems, bottled water supplies, portable filtration and/or disinfection devices and home treatment units. (special attention should be given to backup supplies for hospitals, nursing homes and other places where lack of safe water would be immediately life-threatening). • identification of specialty laboratories at the state/provincial and national level that are capable of performing (and willing to perform) procedures not routinely done at local laboratories (e.g., large volume water sampling and processing for pathogenic parasites and viruses, serotyping, electron microscopic examination of stool samples, molecular and immunodiagnostic tests for pathogens in environmental and clinical samples). one or more of these tests may be necessary to identify the causative agents in an outbreak and confirm their transmission route. • a plan to exercise procedure with tabletop exercises involving all pertinent partners on a regular basis and implement any necessary adjustments based upon review of after-action findings. notification of a suspected outbreak is often received by health authorities from a laboratory report or a family physician and can be documented on a log such as form a (foodborne, waterborne, enteric illness complaint report). public health investigators will then interview cases and persons at risk who are well (controls) to make epidemiologic associations to find a common source. from here a hypothesis can be formed. further investigation will involve: • collecting clinical samples and water samples • conducting an on-site investigation at the facility alleged to be responsible to determine the mode of contamination or process failure, e.g., low disinfectant level • characterizing the etiologic agents by laboratory analysis using various typing schemes. dna profiling or pulsed-field gel electrophoresis (pfge), of isolates from clinical and water samples to "fingerprint" and link strains of the etiologic agent among cases and to the source prompt handling and referral of water-related complaints, rapid recognition of the problem, and prevention of further illnesses are the foundations of a successful investigation. complaints of water problems are as likely to be reported to a water utility as to a health department. communication is essential between these agencies. this first contact with the public is a vital aspect of an investigation of a potential outbreak and needs to begin by public health professionals as quickly as possible, usually within hours, sometimes by putting less urgent activities aside. as indicated earlier, any action respecting a potential deliberate contamination of water will generate a specific approach to further action. upon receiving a complaint or an alert about a water supply or water exposure or illness potentially attributed to these sources, record the information on form a. alerts may be initiated by reports from physicians, laboratories, or from hospital emergency rooms. alerts may also include an increase in a particular pfge pattern from clinical isolates. an investigation may be initiated to determine if there is a common water exposure among patients with the pfge pattern. the pattern may be compared with similar pfge patterns in pulsenet databases to determine if there are similar occurrences of the pattern in water and clinical isolates nationwide or internationally, e.g., for food that might have been contaminated with water, bottled water. the form provides information upon which to decide whether an incident should be investigated. form a is not difficult to fill out and can be completed by a public health professional, a trained water utility staff member, or trained office worker. assign a sequential number to each complaint. if additional space is needed to record information, use the reverse side or attach additional sheets. always ask the complainant to provide names of other persons at the event, or using the water supply or recreational water under suspicion, whether or not ill, and names of any other persons who are known to be ill with the same symptoms. follow up by contacting these additional persons. emphasize to the persons making alerts or complaints the need to retain a sample of the suspect water and to save clinical specimens (vomitus and stool) from ill persons using a clean utensil in a clean jar or plastic bag and to seal tightly and label clearly with the name of the person and date, and store in a refrigerator (do not freeze). also consider family members not ill for case-control studies. advise complainants to collect a liter (quart) of water immediately, preferably in sterile containers but otherwise in jars that have been boiled or in plastic bags, or if this is not feasible, in other clean containers. tell the complainant to save any ice cubes or refrigerated water, either in their present containers or in unused plastic bags, in the refrigerator or freezer (if already frozen) where they are normally kept. instruct the ill person to hold all clinical specimens and water samples until the health agency evaluates the epidemiological evidence and arranges, if necessary, to collect them. if it is determined that the specimen or sample is not necessary, notify the complainant and advise on proper disposal of the material. unfortunately, the specific etiologic agent cannot be identified in a large proportion of waterborne outbreaks because water samples and clinical specimens (a) were not collected in an appropriate time-frame (not early enough during illness), investigate outbreaks (b) are too old, (c) are too small in volume, especially for giardia and viruses which require liters, (d) have not been examined for the appropriate agent. contaminants may be in the water system for only a short time, and concentrations of toxic substances and numbers of microorganisms may decrease significantly because of dilution or disinfection. if there is a cluster of cases, monitor reports from physicians, complaints about water, or records of laboratory isolation of enteric pathogens that may suggest outbreaks of disease or contributory situations. collect clinical specimens and water as soon as practicable according to the section obtain clinical specimens in this book. extract key information (see* and † entries) from form a and enter it onto form b (foodborne, waterborne, enteric illness and complaint log). record time of onset of the first symptom or sign of illness, number of persons who became ill, predominant symptoms and signs, whether ice or water was ingested, and the name of the water supply or recreational water alleged to have caused the illness, and whether a physician had been consulted, and/or had taken feces or emesis samples, and/or prescribed antibiotics. also, enter on form b names of the places or common gatherings (other than home) at which the stricken persons ingested water during the weeks before onset of illness (see table for an example). enter a code for the water source (e.g., community, non-community, individual well, bottled, stream/ lake, vended, or untreated) . under "history of exposures" column indicate whether the afflicted person had recent domestic or international travel, attended a child care facility, or had recent contact with ill persons or animals. under "comment" column, enter notations of type of agent isolated, results of specimen tests, places where water was consumed during travel, names and locations of restaurants or other foodservice facilities, and other pertinent information including hospitalization, occupation, or place of employment. at this phase of the investigation it will probably not be known whether the illness is waterborne, foodborne, or person-toperson spread. this log can be kept either in hardcopy or in electronic format. see table (below) as an example of a log. interpretation of table . entry -get further details on the patient's symptoms and seek other cases. the report of foreign travel suggests an infection that may have been acquired outside the country. follow-up of such cases may identify an outbreak of international scope. if so, inform state/provincial and national authorities concerned with surveillance of waterborne disease about the situation. entry -possibly food associated; alert food safety officials. entry -initiate investigation; the two cases of conjunctivitis suggest the possibility of a common-source outbreak associated with the motel pool. entry -initiate investigation; cases indicate an outbreak that has a common time-place association. entry -this could be related to entry , because this person reported swimming in the same pool. exposure history: dt domestic travel (out of town, within country); it international travel; cc child care; ci contact with ill person outside household or visitor to household; a an exposure to ill animal; c contact with ill person within household responsible for the investigation, if this was not done when the surveillance protocol was established. delegate sufficient authority and provide resources to the head investigator so that the investigation tasks can be accomplished effectively and efficiently. inform all outbreak investigative team members that any findings are to be reported to this delegated authority. a list of all team members and additional contacts such as administrative contacts, sanitarians/environmental health officers/ public health inspectors, local and regional contacts, physicians, clinical laboratories, or other persons who may become involved in outbreak investigations should be assembled. before beginning the investigation, check the supply of forms and the availability of equipment suggested in table a (equipment useful for investigations) and obtain any needed materials or additional equipment. general resource materials describing signs and symptoms, incubation times, and specifics regarding specimen collection and appropriate kits to be used should be maintained and readily available to those processing the initial calls, which may help to formulate the initial hypothesis. if the alert or complaint suggests a possible outbreak, inform laboratory personnel of the type of outbreak and estimated quantity and arrival time of clinical specimens and water samples collected. this information will give laboratory managers time to prepare laboratory culture media, prepare reagents, and allocate personnel. at a minimum, the laboratory should have six to eight stool culture kits on hand or readily available, since in many cases, stool specimens must be collected within h of onset of illness to isolate and identify certain pathogens (e.g., campylobacter spp., and salmonella spp.). consult laboratory personnel about proper methods for collecting, preserving, and shipping environmental samples and clinical specimens if such information is needed. obtain appropriate specimen containers and sample submission (chain of custody forms) from them. once the investigation is underway, the proper clinical specimens should be collected as soon as possible before patients recover and become less likely to submit specimens. all suspected waterborne outbreaks should be examined and a determination made regarding the feasibility of conducting a thorough investigation even if the time to collect proper clinical specimens has passed. an ill person or family member, physician, hospital staff member, or operator of a water utility or recreational site may report suspected cases of waterborne illness. whatever the source of the report, verify the diagnosis by taking a thorough case history and, if possible, by reviewing clinical information and laboratory findings. (this analysis can be further substantiated by detecting suspected etiologic agents in water). verification of the diagnosis is done in consultation with medical professionals. when a complaint involves illness, complete forms c - (case history: clinical data and case history: food/water history and common sources) either at the time of initial notification, during a personal visit, or during a telephone call to the person reported to be ill. use this same detailed interview approach with every person who has been identified in the initial complaint or alert, even though some may not have been ill. be aware that potential cultural and language barriers can make interviews difficult. a different interviewer may be needed to accommodate these barriers. continue this until sufficient information is obtained to decide whether there is, indeed, an outbreak of waterborne illness. from persons who are at risk of illness but who remained well, also obtain water and -hour food histories, inquire about recreational water exposure in past weeks, and information about their activities in common with the ill persons. information from these persons is as important to make epidemiologic associations as it is from the cases. when it is apparent that an outbreak has occurred and a specific event has come under suspicion, substitute forms d - (case history summaries: clinical data and case history summaries: water/laboratory data) for form c. form d can be used initially in many routine waterborne illness outbreak investigations where it is obvious that a common-source outbreak has occurred or when all of the ill persons consumed water together (e.g., drank from the same public system, consumed ice at an event) or recreated at the same place (e.g., swam in the same lake or used the same hot tubs). this will simplify recording, because most affected persons will give similar information. at this time, notify the district, state, or provincial epidemiologist about the outbreak. if a specific pathogen (e.g., norovirus, e. coli o :h , cryptosporidium spp.) has been identified as the etiologic agent, consider developing a form for recording relevant information. many state/provincial or national public health agencies have standard forms tailored to specific pathogens. include signs and symptoms of the illness and other clinical information, the etiology of the agent, and usual methods of transmission. computer programs (e.g., epi info™) can aid in the design of such standard forms. upon contact with the affected person, identify yourself and your agency and explain the purpose of the visit or call. a professional attitude, appropriate attire, friendly manner, and confidence in discussing epidemiology and control of waterborne illnesses are essential for developing rapport with affected persons or their families and in projecting a good image of the investigating agency. keep in mind that you are not interviewing someone you inspect or regulate, but that you are providing a service to the affected person. exhibit genuine concern for persons affected and be sincere when requesting personal and confidential information. communicate a sense of the urgency of the investigation, and emphasize that their participation will make a positive contribution for the control and prevention of waterborne illness. parental consent must be obtained before interviewing children under years of age. in some locations, consent from the affected person's physician may also be required. after asking open-ended questions about the person's food exposures and illness history, follow up with more specific questions to fill in the details and better ensure a thorough recall. base your level of communication on a general impression of the person being interviewed, considering information about age, occupation, education, or socioeconomic status. tact is essential. use either form c or form d, as appropriate, as a guide. state questions so that the persons who are being interviewed will describe their illnesses and associated events in their own words. try not to suggest answers by the way you phrase questions. fill in form c - (if appropriate) and take additional notes during the interview. ask specific questions to clarify the patient's comments. think questions through before conducting the interview. realize that people are sometimes sensitive to questions about age, sex, special dietary habits, ethnic group, excreta disposal, and housing conditions. nevertheless, any or all information of this type can be relevant. word questions thoughtfully when discussing these characteristics and habits. such information can often be deduced from observations. if doubt remains, confirm your guesses by asking indirect questions. information on recent travel, gatherings, or visitors may provide a clue to common sources or events that would otherwise be difficult to pinpoint. review known allergies, recent immunizations, recent changes in the patient's medical status, and similar information. remember that the agents associated with waterborne disease can also be spread by other means such as consuming food, person-to-person, visiting child care centers, animal-to-person in petting zoos, through walk-in-spray fountains, and pools for young children. as persons describe their illnesses, check boxes next to appropriate symptoms or signs on form c . do not ask about all symptoms or signs listed; however, ask about those marked with an asterisk if the ill person does not mention them. if there are questions, explain symptoms to the patient in understandable terms. the symptoms and signs in the first two columns of form c are usually associated with poisoning or intoxication, although some occur during infections. those in the third, fourth, and fifth columns are usually associated with enteric infections, generalized infections, and localized infections, respectively. those in the last column are usually associated with disturbance of the central nervous system. diseases in any category will sometimes be characterized by a few symptoms and signs listed in the other columns, and not all signs and symptoms occur for any one ailment or for all persons reporting illness. if an illness seems to fall into one of these categories, mention other symptoms in the category and record the patient's response. whenever possible, use physician and hospital records to verify signs and symptoms reported by patients. clinical data may strengthen or dismiss the possibility of waterborne illness. before contacting a physician or a hospital, become familiar with laws and codes relating to medical records to ensure that you have legal access to these records. legal release forms may be necessary to obtain some records. do not distribute names of patients, their other personal identities (e.g., address, phone number), or their clinical information to unauthorized persons. the entries begin with the day of illness, followed by the previous days. if the illness, however, began early in the day or before any of the listed meals, modify the entries on the form so that the -hour history can be completed in the space pro-vided on the form. if the incubation period is days to a week in duration, use additional copies of form c and modify day or day before subtitles. signs and symptoms will sometimes give clues to the transmission route by indicating the organ systems affected. if the early and predominant symptoms are nausea and vomiting, ask about the most recently ingested water or beverage within the past h. in these situations, suspect high-acid water supplies, carbonated beverages and fruit drinks, because these tend to leach metallic ions from water pipes and containers. if diarrhea, chills, and fever predominate, be suspicious of water and beverages ingested - hours before onset of illness for salmonellosis, shigellosis, and norovirus related gastroenteritis. if the incubation period averages - weeks, consider typhoid fever, cryptosporidiosis or giardiasis. diseases with incubation periods exceeding weeks (e.g., hepatitis a and e, amebic dysentery, or schistosomiasis) can be handled as special cases for which longer histories would be sought. others, such as chronic lead and arsenic poisoning, have incubation periods of variable durations and onsets so gradual as to be indeterminable. see table b (illness acquired by ingestion of contaminated water: a condensed classification by symptoms, incubation periods, and types of agents) for details on specific pathogens, table c (illnesses acquired by contact with water: a condensed classification by, symptoms, incubation period, and types of agents), and table d (illnesses acquired by inhalation of microorganisms aerosolized from water. a classification by symptoms, incubation period, and type of agent). other microorganisms not listed in tables b, c, and d that can be potentially spread by water include the bacteria klebsiella pneumoniae, mycobacterium avium complex, acinetobacter calcoaceticus, elizabethkingia meningoseptica, stenotrophomonas maltophilia, pseudomonas putida, serratia marcescens, protozoa isospora, microsporidium, algae schizothrix calcicola. these microorganisms are less frequently identified with waterborne illness, but they may become opportunistic pathogens, particularly for highly susceptible and immunosuppressed persons. further investigation is needed to confirm their role in the spread of waterborne diseases. gather information about all sources of water to which the patient(s) may have been exposed weeks before onset of illness. the water supply and the event that precipitated the illness might not be obvious. persons often have difficulty recalling exposure to all water sources including; ice or water ingested; aerosols and recreational water contact. therefore, if the person does not remember specific exposures to water, ask about the water consumed in usual or routine daily habits and the amounts ingested; exposure to recreational waters; and unusual exposures or events attended during this interval. this may stimulate recall of away-from-home water consumption or contact that was unusual. ask about other risk factors for enteric illness, such as contact with young children and child care centers, animal contact, ingestion of raw foods of animal origin, and usual food preference habits. for persons who have been traveling, ask them where (both cities and rural areas) they have traveled during the incubation period of suspected agents. determine if they drank water from any taps or pumps in rural areas they visited. ask whether unheated (or untreated) tap water or beverages containing unheated (or untreated) water or ice was ingested at restaurants, in hotels or at events in the places they visited. also, ask whether they ingested bottled water and, if so, the brand name. find out whether they drank water from streams, ponds, springs, or other natural water sources. if they did, ask if they observed any abnormal condition of the water such as algal growth, high turbidity or discoloration. ask if domestic or wild animals had access to the water. if they have skin or eye infections or generalized infections, ask them to name all swimming pools, water slides, beaches, lakes, ponds, or other chlorinated and nonchlorinated water courses where they swam, waded or bathed during their trip. also ask them whether they used any hot tubs, spas, whirlpools, or similar devices. this information sometimes provides clues to common sources or to events that otherwise would be difficult to discover. record the information on form c . in a protracted outbreak, or when investigating an outbreak of a disease with a long incubation period, expect recall to be poor. in this situation, obtain from ill persons and others at risk a listing of their water, ice, and beverage preferences and amounts usually ingested, or their purchases of these items within the range of the incubation period of the suspected disease. as a guide, draw up a list of either water, ice, and beverages that are commonly consumed by the affected group or those waters, ice, and beverages previously identified as vehicles of the suspected disease under investigation. summarize data from all copies of forms c - on form d. form d allows rapid review of all exposed persons (ill or not ill) and serves as a basis for analyzing the data. diagnosis of most diseases can be confirmed only if etiologic agents are isolated and identified from specimens obtained from ill persons. get specimens from the ill persons to confirm an etiologic agent. • in large outbreaks, obtain fecal specimens from at least ten persons who manifest illness typical of the outbreak • in smaller outbreaks, obtain specimens from as many of those ill and those at risk as practicable, but from at least two, and preferably ten, ill persons • try to collect specimens before the patient takes any medication. if medication has already been taken, collect specimens anyway, and find out the kinds and amounts of medicine taken and the time that each dose was taken • also get control specimens from persons with similar exposure histories that did not become ill obtain clinical specimens at the time of the initial interview during acute illness or as soon as practicable thereafter. even though this is not always possible, take specimens even after recovery because etiologic agents may remain in low populations or concentrations. if a disease has already been diagnosed, collect specimens as listed in table b . if a disease has not yet been diagnosed, choose kinds of speci-mens that are appropriate to the clinical features. laboratory information obtained from the first patients may be useful to physicians in the treatment of cases detected later. apart from the fact that people are more likely to cooperate while they are ill, some pathogens or poisonous substances remain in the intestinal tract for only a day or so after onset of illness. if the patient is reluctant to provide a fecal specimen explain that the specimen will be tested to identify the causative agent and compare it to any agent recovered from the water. if a disease has not yet been diagnosed, choose specimens that are appropriate to the clinical features. laboratory information obtained from the first patients may be useful to physicians in treating cases detected later. some pathogens (e.g., salmonella, parasites) may be recovered for weeks after symptoms have abated. if applicable for the disease under investigation, take specimens even after recovery because some etiologic agents may remain in low numbers, and changes in serologic titers can be detected. before collecting specimens, review table e (guidelines for specimen collection) and, if necessary, get additional instructions from laboratory personnel and seek their advice on how to preserve the stool specimens if you cannot deliver them to the laboratory immediately. many public health agencies have special fecal specimen kits. demonstrate to the patient how to use the materials in the kit, how to complete the form in the kit and how to mail it if you are not going to pick it up. if mailing specimens, make sure that you are aware of the regulatory requirements that may apply to the transport of infectious material. stool specimen containers for intestinal parasite examination are not suitable for bacterial or viral examinations because they ordinarily contain a preservative, such as formalin or polyvinyl alcohol. if an inappropriate transport medium is used, a specimen can be rendered unsuitable for laboratory examination. feces. if the patient has diarrhea or is suspected of having had an enteric disease, obtain a stool specimen (preferred specimen) or a rectal swab. instruct patients to provide you with their own specimens by one of the following means. . if practicable, give the patient a stool specimen container with a wooden or plastic spoon or a tongue depressor. a clean container available in the home (e.g., a jar, or disposable container that can be sealed) and a clean plastic spoon or similar utensil can be used if laboratory containers are not available. . label the specimen container with the patient's name age/date of birth and date of collection. . collect the stool specimen by one of the following methods: (a) put sheets of plastic wrap or aluminum foil under the toilet seat and push them down slightly in the center, but not so far as to touch the water in the bowl. sheets of paper can be tacked on the rise of a latrine and pushed down to form a depression in which to catch feces. take care to ensure that toilet cleaning chemicals and other microorganisms in the toilet bowl do not contaminate the fecal specimen. after defecating, use a clean spoon or other utensil to transfer about g of feces into a specimen container or other clean container. (b) defecate directly into a large clean dry container or bedpan. use a clean spoon or other utensil to transfer about g or the size of a walnut of feces into a specimen container or other clean container. (c) scrape feces off a diaper with a clean spoon or other utensil to transfer about g of feces into a specimen container or other clean container. . collect fecal swabs by twisting the cotton-wrapped end of the swab into the stool obtained in one of the ways described above. follow instructions given in table e . if necessary, use fecal-soiled toilet paper or cloth diaper and twist a swab into the top of feces. take care to ensure that there is no carryover of toilet paper as they are impregnated with barium salts which are inhibitory to some fecal pathogens. dispose of excess fecal material into the toilet and carefully wrap all soiled articles (e.g., by placing them inside two plastic bags) and dispose of in domestic waste. check that the specimen container is tightly sealed and properly labeled and place into a clean outer plastic bag (special zip lock bags for clinical specimens, if available). store the specimen in a cool place, preferably at °c to await pick-up or despatch. do not freeze. feces from rectal swabs. collect rectal swabs by carefully inserting the swab approximately . cm ( in) beyond the anal sphincter. gently rotate the swab. fecal matter should be evident on the swab. vomitus. if the person is vomiting or subsequently does so, arrange to collect vomitus. tell the patient to vomit directly into a sterile specimen container or a plastic bag. otherwise, transfer some vomitus from a clean receptacle into the container with a clean spoon. refrigerate, but do not freeze, this specimen until it can be picked up or delivered to the laboratory. blood. take blood if a patient has a febrile infection or when infectious agents are suspected (see tables b, c, and d) . blood specimens are collected for: • bacterial culture • detection of antibodies to specific agents • detection of certain toxins before collecting specimens, get additional instructions from laboratory personnel and seek their advice. blood should be obtained by an appropriately trained and accredited person (check appropriate laws). collect blood during the acute phase of illness, as soon as the febrile patient is seen (within a week after onset of illness) and, if comparing of serologic titers, again within weeks (usually - weeks later) during the convalescent phase. draw ml of blood (from an adult) or ml (from a child) or - ml (from an infant). if possible, collect the blood from the same patients from which stool specimens were obtained if both specimens are to be examined. label tubes and vials at every step of serum transfer. do not freeze whole blood because the resultant hemolysis interferes with serologic reactions. collect into a sterile syringe or evacuated sterile tube that does not contain anticoagulants. if practicable, centrifuge the blood at , rpm for min; pour off the serum into small screw-cap vials and store at approximately − °c. if the serum cannot be separated immediately, rim the clot with a sterile applicator stick and refrigerate approximately °c to get maximum clot retraction if the specimen is to be stored unfrozen overnight. if centrifugation cannot be done, store the blood specimens in a refrigerator until a clot has formed, then remove the serum and transfer it with a pasteur pipette into an empty sterile tube. send only the serum for analysis urine. instruct patients to collect urine in the following manner. clean the area immediately around the urethral orifice with a paper pad that has been pre-moistened with % tincture of iodine or other appropriate antiseptic. then begin to urinate into a toilet and collect ml (about oz) of midstream urine into a sterile bottle. use either a second antiseptic-moistened pad or an alcohol-moistened cotton ball or tissue to clean any drops from the top or side of the bottle. other instructions. follow applicable instructions given in table e . before or immediately after collecting clinical specimens, use waterproof permanent markers to label each container with the patients name, complaint number, case identification number, specimen number, date and time of collection, tests requested, and other appropriate information. tightly seal all containers. clinical specimen collection report for each specimen. complete form e (clinical specimen collection report). the complaint number, case identification (id) number, and specimen number must be entered on each report so that laboratory results can later be correlated with other data. on form c record the type of specimen collected, and submit both the specimen and a copy of form e to the laboratory. send a copy of the laboratory report to the patient's physician or call if urgent. if the patient/case or other household member collected any water, ice, or beverage as instructed during initial contact, label containers with the complaint/outbreak and sample numbers. proceed as instructed in table f (general instructions for collecting water samples for microbiological analysis) and complete form f (water/ice collection report) and/or forms g -g as applicable. record conditions of collection as called for on the forms. if a hypothesis associates the illness with water, caution these persons not to use the water source unless the water is first boiled and to discard all previously prepared ice and water-containing beverages until notified otherwise. develop a working case definition to classify exposed persons as either cases or non-cases. start with the most specific symptoms (such as diarrhea and vomiting) rather than broader symptoms such as nausea or malaise. for example in an outbreak of gastroenteritis, a case might be defined as a person from whose stool a specific pathogen was isolated. it may be a person who was at risk and developed diarrhea within a specified period of time. diarrhea will have to be defined, perhaps as three or more loose, watery stools during a -hour period. in some cases, a particular pathogen responsible for the outbreak might have been identified from clinical specimens. a case definition, which is developed later in the investigation, might include either a person having specific signs and/or symptoms within a period of time or a person from whom a specific pathogen was isolated. the ultimate case definition has a tremendous impact on the investigator's ability to make illness and exposure associations and to calculate probability of these associations. sometimes the first symptom or sign provides a clue to developing a case definition. information in tables b, c, d, g, and h can be useful in making case definitions. compare newly identified cases with the definition to see whether each is part of the outbreak. classify cases into categories: • a confirmed case is a person with signs and symptoms that are clinically compatible with the disease under consideration and for which there is either (a) isolation of an etiologic agent from (or otherwise identified in) an appropriate specimen from the patient, or (b) serologic evidence of a fourfold or greater rise in convalescent antibody titer. a confirmed case must also have possible exposure to the etiologic agent within the incubation period of disease. see table e . criteria for confirmation of etiologic agent responsible for outbreaks of waterborne illnesses for definitions of confirmed cases for specific waterborne diseases: • a presumptive case is a person with signs and symptoms that are clinically compatible with the disease under consideration, and for which there is laboratory evidence of infection (e.g., an elevated antibody titer but less than a fourfold increase), but the etiologic agent was not found in specimens from patients or no specimens were collected. a presumptive case must also have possible exposure to the etiologic agent within the incubation period of disease. • a suspected case is a person with signs and symptoms that are clinically compatible with the disease under consideration and history of possible exposure, but laboratory evidence is absent, inconclusive or incomplete. • a secondary case is a person who became infected from contact with a primary (outbreak-associated) case or from a vehicle contaminated by a primary case. onset of illness for secondary cases typically is one or more incubation periods after the outbreak-associated cases. it is not essential, however, to classify cases into these categories. do so only if it aids in developing a final case definition or in making comparative analyses of data. consider doing analyses using case definitions of both confirmed and combined confirmed, presumptive, and highly suspect cases, and compare the results. make a preliminary evaluation of the data collected as soon as possible. if you decide that there is an outbreak, use the information you have to develop a hypothesis about the causal factors. an outbreak is an incident in which two or more persons have the same disease, have similar clinical features, or have the same pathogen (thus meeting the case definition), and there is a time, place, or person association among these persons. a waterborne outbreak is traceable to ingestion of contaminated water or ice or to contact with contaminated water. a single case of either chemical poisoning or a disease that can be definitely related to ingestion of drinking water or contact with water can be considered an incident of waterborne illness and warrants further investigation. waterborne methemoglobinemia in an infant who resides in a rural area having a high concentration of nitrates in well water is an example of a single case of waterborne illness due to ingestion. a rare diagnosis such as primary amebic meningoencephalitis in a person who swam in a body of freshwater and inadvertently ingested the ameba, naegleria fowleri, through the nose is an example of a single incident related to water contact. sometimes it will be obvious from an initial report that an outbreak of waterborne disease has occurred simply because of the number of persons displaying certain signs and symptoms at or near the same time. many complaints, however, involve illness in only one or a few persons. it is often difficult to decide whether ingestion or contact with a particular water source and onset of illness was associated or coincidental. certain diseases that are highly communicable (e.g., shigellosis and epidemic viral gastroenteritis) may result in secondary infections from person-to-person spread or from subsequently contaminated food or water. however, if complaints are received from several persons who are associated with ingesting water or contact with water at the same place, water is likely to be involved. routine review of the log pertaining to potential waterborne illnesses for similar complaints can often be useful in detecting time, place or person associations. an investigation may also proceed based upon the suspicion of an intentional contamination of a water source. a time association exists if the time of onset of similar illnesses is within a few hours or days of each other. place associations exist when persons have ingested water from a particular single source, have swum in, worked in or otherwise been exposed to the same water, have attended the same event, or reside in an area common to all. person associations indicate a shared personal characteristic, such as being of the same age group, sex, ethnic group, occupation, social group, or religion. waterborne illnesses transmitted by a community water supply usually afflict persons of both sexes and all ages throughout the community. non-community water sources, such as bottled water, ice, water from individual wells, or water from areas of recreation should also be considered when making associations. keep in mind that water can contaminate foods during washing or freshening, and it can contaminate utensils and vessels that are used to handle or store foods. water may therefore be a source of contamination of another vehicle. also, water can be ingested as aerosols generated by shower heads, whirlpools, hot tubs, fountains, cooling towers, and irrigation devices. once some of these associations become obvious, question other persons who could be at risk because of their time, place, or person associations with the ill persons. from time, place, or person associations that have been established or suggested by the investigation, formulate hypotheses to explain (a) the most likely type of illness, (b) the most likely vehicle involved, (c) where and the manner by which the vehicle became contaminated, and (d) other possible causal relationships. the section "collection and analysis of data" describes calculations that can aid in the formation of these hypotheses. test hypotheses by obtaining additional information to support or reject them. if the hypothesis includes food contamination, the instructions given in the manual, procedures to investigate foodborne illness, might be useful. guidelines for confirmation of waterborne outbreaks are presented in table g and guidelines for confirmation that water is responsible for illness are presented in table h . if there is strong evidence to support a hypothesis that the outbreak is waterborne, take precautionary actions. the choice of action is dictated by the (a) suspected causal agent, (b) size of the water source, (c) availability of alternate water sources, and (d) expected use of the water. on the basis of available information, estimate the population at risk and engage any public relations staff with your organization to help inform all persons potentially impacted. when dealing with a microbiological contaminant or agent, consider issuing a boil-water advisory with water treatment guidelines (e.g., heating water in a covered container to a rolling boil for at least min and keeping it covered until use). other options that can be explored include chlorinators that can be installed in individual and non-community systems. for community and non-community supplies in which chlorine is already used, increasing the chlorine dosage and opening hydrants and taps to draw the super-chlorinated water through the whole system might be an option. increasing chlorine is sometimes not effective because the chlorine contact time is too short or super-chlorinated water does not reach some parts of the system. furthermore, chlorination is ineffective against cryptosporidium oocysts and requires a long contact time to kill other human pathogens like hepatitis a virus and giardia. for suspected chemical contamination contact a specialist for further assessment and remedial strategies, such as activated charcoal filters. as a last resort, shut off the contaminated system until the source of contamination is found and controlled. be cautious when you take this drastic measure, because it may do greater harm than good by causing lack of water for hospitals, nursing homes, or for firefighters to extinguish fires. if the water is shut off or the treatment facility or distribution system disrupted (as in the case of floods or other disasters), consider means to distribute water from an alternative source to healthcare facilities and homes. if an illness could have resulted from water contact, close the offending water source, post warning signs around it, and patrol the area. where there is a swimming pool, hot tub, spa, fountain, or whirlpool, evaluate the recirculation system and its operation. it may be that increasing disinfectant concentration by super-disinfection could resolve the problem. where there may be chronic operational problems, evaluate ph, disinfectant concentration, and bacteriological laboratory records. choose your course of action, including consultation with appropriate professional experts, depending on the contributing factors existing at the time of investigation. verify the effectiveness of these actions (e.g., boil-water advisory, superchlorination, provision of alternate water source) to protect public health by monitoring illness levels in the population to determine if the outbreak terminates. if the outbreak continues unabated, consider the possibility of other transmission routes. also, verify the effectiveness of repairs to the water system, super-disinfection, and other actions by closely monitoring the quality of the water supply or recreational water to determine if laboratory reports indicate that the water is now safe for consumption or contact. if there is a public health threat, work with any available public relations staff to announce the outbreak in the mass media so that the public who consumed or was otherwise exposed to the implicated water can be alerted to take appropriate action including seeking medical consultation or treatment. provide only objective factual information about the outbreak. coordinate among the investigating agencies to assure that a consistent and accurate message is delivered. it is easy for agencies to miscommunicate before and during a water crisis (see box , false alarm; box , the walkerton outbreak; box , the flint water crisis). it is often preferable to have one spokesperson for all agencies. do not release preliminary information that has not been confirmed. the person giving information about an outbreak should be well informed about the etiologic agent being investigated and prepared to deal with questions. if the health hazard warrants a public warning at the hypothesis stage, tell the public why emergency measures are being invoked and that subsequent information may be cause to modify the action. as the investigation proceeds and the etiologic agent is confirmed and contributory factors are identified, consider terminating emergency measures, and give advice on specific control and preventive measures. attempt to reach all segments of the population at risk; this may require communication in multiple languages. route all news releases or statements to all persons involved in the investigation. in situations involving large outbreaks or highly virulent or toxigenic etiologic agents, set up an emergency hotline for the public to call to ask questions. this is likely to occur if there is an intentional contamination event where there is high publicity and public concern. train staff to handle these calls in a consistent manner so that the advice is the same who gives it. faulty information derived from poorly tested hypotheses can lead to severe political, legal or economic consequences. an example of this occurred in sydney, australia, in when an apparent water contamination event was publicized for the public to take precautionary actions. the false alarm was costly because of rebates to water customers, additional water testing, and for hiring extra staff, as well as a loss of confidence in the facility (see box , false alarm). they may then be disseminated by the mass media with inappropriate interpretations of the public health significance. furthermore, this information may be used as an unrealistic base for water programs or water regulations because of either misinterpretations or pressure from misinformed consumer-advocate groups. all involved parties should follow a written protocol for cross-agency communication and release of information to the public. unreasonable delays are unacceptable. test hypotheses by obtaining additional information to either confirm or refute their validity. do this by case-control or cohort studies, additional laboratory investigations, and on-site investigations (e.g., laboratory reports of water testing). sydney water (a new south wales state-owned corporation) supplies million liters of water each day to . million properties in sydney and its outlying areas. the city has a large and complex catchment with nine major dams and several storage reservoirs. about , km of water main, almost pumping stations and many tunnels deliver water from four main river systems. the water is filtered through eleven treatment plants. seven are owned by sydney water and four are privately owned. these plants provide % of sydney's drinking water and one plant, prospect, provides up to %. in , the quality of sydney's drinking water came under acute review when giardia and cryptosporidium were found in the city's main water supply at the warragamba dam. initially, low levels of these parasites were first detected in the water supply on july, but these were within the acceptable health limits. in days following, much higher levels were recorded, and on july the first "boil water" alert (in which residents were instructed to boil their tap water before use) was declared for the eastern central business district of sydney. however, by late on july high readings were found in samples at the prospect filtration plant, in a reservoir and at a location further down the system, and a "boil water" alert was issued for the south of sydney harbour, and on july a sydney-wide "boil water" alert was issued affecting most of sydney's residents. on august the warning was discontinued. however, high levels were again found on august (the second event), although it was believed that most organisms would likely be dead. more positive readings were found on august , although at lower levels. further contamination was identified on august and an extended boil water alert was again declared. this was progressively lifted suburb by suburb until further contamination was reported on september (the third event). a -week alert was then instituted, which was finally lifted on september . it was determined that the parasitic contamination was caused by low-quality surface water entering the dam. this contaminated source was attributed to moderate rainfall in july, followed by heavy rainfall in august and september which caused intermittent supplies of the raw water to enter the dam. despite high levels of cryptosporidium (up to > , oocysts) and giardia (up to > cysts) being recorded in july and august, , no increase in human cryptosporidiosis or giardiasis was detected in the exposed population. the incident was highly publicized and caused major a public alarm because the number of people affected, the on and off boil water alerts, and the fact that the filtration plant had been advertised as one of the best in the world. the economic and political repercussions were extensive. the cost of the crisis to sydney water was estimated at a$ million which included $ million paid in rebates to customers, $ million in lost revenue, water testing and staff costs and at least $ . million for damages claims. these costs exclude those relating to improvements to the system and infrastructure. the lack of cases of cryptosporidiosis, giardiasis or other water-related health problems led to suggestions that the parasites were either not an infectious type, or not as extensively distributed. an inquiry after the event revealed the publicity as an exaggeration of fact, with australian water technologies, part of sydney water, severely overestimating levels of cryptosporidium and giardia present in the water, with the recorded levels exposed to consumers as not harmful to human health. the handling of the crisis by state-owned sydney water was heavily criticized, causing the resignation of both the chairman and the managing director, and bringing up issues of private vs. public ownership and scientific uncertainty. the eventual consequence of the state inquiry was the establishment of the sydney catchment authority in to assume control of sydney's catchments and dams, while sydney water maintained responsibility for water treatment and distribution and for sewage collection, treatment and disposal. if an outbreak investigation requires resources beyond your agency's capacity, request assistance from other health professionals. it is desirable to have a team including, if feasible, an epidemiologist, an engineer, a microbiologist, a sanitarian/ environmental health office/public health inspector, a chemist, a physician and others, to undertake a detailed waterborne illness investigation. such personnel can usually be provided by local, state/provincial, or national agencies concerned with health, environment, or agriculture, depending on the expertise needed. for events suspected to arise from intentionally contaminated food, contact emergency response or law enforcement agencies. continue to search for and interview both ill persons who have had time, place, or person associations with the identified cases (see the section on "make time, place, and/or person associations"). review recently received complaints in the water-related complaint log (form b). contact other nearby health agencies, hospital emergency rooms, elderly care centers, and local physicians to discover other epidemiologically related cases. call previously contacted persons to see whether they know anyone else who has become ill or had a common association suggested by data in the log. the illness you are investigating may be part of a larger multijurisdictional outbreak, and therefore communicate with adjoining local and state agencies to learn if they are seeing similar illnesses. state or provincial public health agencies can check reportable disease records and state/provincial public health laboratories can start looking for clusters in isolates that they are characterizing. for outbreaks where intentional contamination of water is suspected or confirmed, public health and law enforcement agency officials may conduct the investigation jointly. if it becomes apparent that an outbreak is associated with a specific water supply (source) or recreational water or event, use form d for recording information. at this stage of the investigation, interviews can be expedited by reviewing the event itself to stimulate each person's recall. ask about specific symptoms and signs that are known to be common to the syndrome, as well as, time of ingestion or contact with water and onset of illness. mention each source of water to which the person may have been exposed, and ask each person (whether a case and well persons at risk) which of the water sources had been ingested or contacted. the number of persons to be interviewed depends on the number exposed and the proportion of them who are probably affected; if fewer than persons were at risk, try to interview all of them; if several hundred are involved, interview a representative sample. be sure to obtain clinical specimens from these cases and well persons at risk (controls). it is more difficult to obtain positive results if symptoms from persons have ceased. there may be situations where self-administrated questionnaires are sent to cases and persons at risk. use either form c or form d or modified versions for this purpose. after questionnaires have been completed, summarize the data on form d. also, identify and interview secondary cases if they become apparent. because no two waterborne disease outbreaks are identical, the order of the expanded investigation may not always follow the outlined sequence of procedures. some investigative steps can usually be done simultaneously by different investigators. additional procedures may also be required. the principles and techniques described will suffice for most investigations. modify forms, if necessary, to accommodate the type and amount of information to be collected. make on-site observations. prove or refute hypotheses developed during the epidemiological portion of the investigation. focus on sources and modes of contamination and ways contaminants could survive and pass through water treatment. as applicable, conduct an on-site investigation of source (lakes, streams, areas around groundwater, etc.), treatment facilities, distribution lines, cross connections, water reservoirs, places of recreational water contact and/or sites at which aerosols were generated. such an epidemiologically focused investigation is quite different from sanitary surveys done during routine evaluations of water source sites, treatment plants or recreational water facilities. not all drinking water (even municipal and bottled water) is disinfected; so, it is important to identify whether the water source is treated and if so, how. some treatments (filtration, reverse osmosis, membrane treatments, riverbank filtration, and others) may not be complemented with a disinfection step. sanitary survey information can provide information about potential sources of contamination in the area of a usually pristine water source. microbiological records of a water supplier, particularly if any total coliform positive samples were found by the system in the last months, may help identify a contamination pathway. if significant matters relating to water quality are observed or otherwise identified during the investigation, note them and communicate them to those responsible for the water system and to the proper authorities. do not lose the focus and objectivity of the investigation by confusing matters of quality and aesthetics with factors related to contamination by, and survival of, infectious and toxic agents. use the haccp-system, also known as systems analysis, way of thinking in your investigation. contact the person with the highest responsibility for the operation and maintenance for the implicated water source, water treatment facility, and/or distribution lines. identify the types of records that ought to be reviewed during the investigation and their likely source. do not forget that the responsible authorities also can have records (about water quality, if there has been a change of municipal water supply, industrial water pollution, wastewater pollution). they can be good sources of information about recent pipe breaks and other water system issues that could be related. in many cases they will be aware of the potential for contamination upstream of source water intakes. if applicable, obtain water distribution maps and recent water quality reports from appropriate departments. if you are not familiar with the community in which the investigation is to be done, obtain maps of the area to locate streams, lakes, water treatment facilities, and other community features that might have a bearing on the investigation. check if there are water protection areas and their rules. get plans and specifications on design of treatment facilities from consulting engineers or state agencies that approve these facilities. contact weather bureaus, airports, radio/television stations, or newspapers for information on heavy rainfall, flooding, extremely low temperatures, droughts, or other unusual weather conditions that preceded the outbreak, if this information is unknown to investigators. contact police or fire departments about traffic accidents, which can be the source of the outbreak. review all background data pertaining to the suspect water. as information is gathered, record it on applicable parts of form g. discuss with laboratory personnel that a field investigation will be made, and get their suggestions regarding samples and specimens that should be collected (see tables e and f) . confer with them about special analyses, media, and sampling procedures; make arrangements for rapid transport of samples to the laboratory. the samples must maybe be transported at the right temperature. pick up appropriate forms and sample collection equipment (preferably preassembled in a kit-see table a ). the laboratory can probably help assemble this kit. during the investigation, identify factors that contributed to contamination and survival of the etiologic agents and perhaps also to their growth or amplification or another cause of the outbreak. identified factors and situations that have contributed to waterborne disease outbreaks include those listed in table . focus the investigation on the potential situations listed in table , as applicable. remember that other possibilities can occur. describe circumstances that contributed to contamination and that permitted the etiologic agent to survive so that it reached drinking, agricultural, industrial, or recreational water. also describe circumstances that allowed pathogenic bacteria or algae to multiply in the water. write your findings down on the back of form g (illustration of contamination flow) or on a separate sheet. continually update the listing in table with newly available data. introduce yourself (who you are, where you come from, who ordered you there) to the owner, resident, or persons in charge and state your purpose, when you arrive at the place of the suspected contaminated water source. emphasize that your visit is to confirm or eliminate suspicion that this water was a source of illness. tell him or her that a complete epidemiologic study is in progress and that other possible sources (such as food) will be investigated as well as operations of this site. explain that your investigation is not to fix blame but to identify the cause of the outbreak. emphasize that the findings can yield benefits related to the ability to identify needed improvements, to educate staff and to provide public support. try to create an atmosphere of cooperation. maintain an open mind and try to answer all questions. if you can not answer a question, tell the person that you will come back with an answer. come back to the person within or weeks even if you do not have any new information. give the person your phone number and e-mail address and tell the person to contact you if the person has more information later. privately interview key persons responsible for operating or repairing water facilities. do not forget to interview persons from other work shifts. identify persons who were working there at the likely time of contamination and have since left and interview them as well. ask questions to determine the flow of water and operations from intake through distribution through plumbing systems. ask about any changes in operation, unusual events in the watershed or repairs to the water facilities. ask if you can check records, both in paper form and on the computer (monitoring system), analyses of results, and/or incident reports. plant personnel may not describe water treatment or installations exactly as they existed at the time that a mishap occurred. they may fear criticism or punitive action as a result of their possible role in the causation of the outbreak. their descriptions should be plausible and should account for possible sources and modes of contamination and indicate possibilities for survival of pathogens. if a description does not contain all the information desired, reword questions and continue the inquiry. confirm accounts by private interviews with others knowledgeable of water treatment or operation of the facility. be alert for inconsistencies among the accounts told by different persons. seek resolution of discrepancies in accounts by watching actual procedures as they are being carried out, by taking appropriate samples, or by conducting experiments. a communicative working relationship between the plant management and the investigator influences plant workers' attitudes toward the investigative team. consider the position, feelings, and concerns of the manager and staff; defensive reactions are normal on their part. diagram each phase of the water system or situation under study on form g (illustration of contamination flow). insert special symbols and notes for all sites that might be involved in introducing contamination to the water or where contaminants might have survived treatment. record other information gathered on the appropriate parts of forms g - . review and collate appropriate information on quality control and operational records from the water utility and from responsible agencies. as applicable, obtain information on quality of untreated surface or groundwater from a local, state/ provincial, or national pollution control or geological survey agency. also, seek water distribution maps, well logs, descriptions of geological conditions and indices of groundwater quality from them. for surface water supplies, obtain information on upstream discharges and unusual events that may have affected raw water quality. get data on finished water quality in the distribution system from a local, state/ provincial water surveillance or regulatory agency. water suppliers also frequently have records of raw and finished water quality. review data on quality control tests (e.g., ph, chlorine residual, chlorine demand, bacteriological and chemical tests, turbidity, jar test data, incident reports) that are available. obtain data on cross connection control programs and sewer repairs from the water supplier or other local agencies (e.g., building inspectors, sewage departments). review files for data concerning potential sources of contamination for individual or semipublic water supplies (e.g., diagrams of septic tank systems, sewer line locations, well logs, small individual wastewater plants, accidental industrial pollution of water, traffic accidents involving chemicals, salting of roads or sawmills). check if they have any haccp-systems or water safety plan and, if so, how they monitor their ccps (critical control points) and if they are implementing control measures. ask them about haccp, to see if they understand the system and if results are documented. check if the haccp-system is validated (should be documented) and that they are conducting internal audits. get information on all aspects of normal operations as well as unusual events or conditions to determine whether such events were coincidental with the time of suspected contamination as determined from the epidemic curve. also, consider the time it takes for a contaminant in the raw water or treatment plant to reach households in the affected community. ask responsible persons for this information. compare data on heterotrophic plate and total coliform counts of raw and finished water leaving the treatment facility and of water in distribution lines. also, compare data on chlorine residuals within the plant with that in distribution and check, if they have, that the uv-light is functioning. review other test data (e.g., turbidity and chemical analyses) that may indicate a problem situation. identify locations and dates of sample collection. take photos, if it is allowed, of things you suspect are not right. go back more than one incubation period of the disease under investigation. record this information on form g (record review of on-site investigations, and test results prior to and during outbreak). photocopy appropriate records for confirmation and subsequent review and attach them to the record review form. be alert for evidence of falsification of records. while reviewing records, watch for evidence of the following: • potential contamination of groundwater systems because of proximity to septic tank systems, latrines, animals manure or landfills, industrial contamination of the water supply, small sewage plants, especially old and nearly forgotten ones, and recent heavy rain • high heterotrophic plate or coliform counts, or counts that exceeded the average (median) or typical count • sudden changes in water quality or operating practices that suggest the possibility of contamination or treatment failure • high turbidity, unusual odor, color, or taste, or high coliform counts in raw water, which can indicate potential overloading of the normal treatment process • high levels of ammonium, nitrate and nitrite, which can indicate organic and inorganic contaminants • low chlorine residuals in treated water or higher-than-normal amounts of chlorine used, which can indicate either a high chlorine demand or a sudden high level of contamination • a sudden drop in amount of disinfectant used, possibly indicating failure or interruption of a disinfection process. no functioning alarm system • a sudden change in the amount of a chemical (e.g., alum or ferric sulfate) used, suggesting equipment disfunction or inadequate coagulation or flocculation and thus poor filtration • lack of treatment chemicals if a more corrosive water supply is used (see box , the flint water crisis) • pump failures, draining of distribution lines or reservoirs, or massive pumping to fight fires, which can produce low pressures that can cause contamination through cross connections or back siphonage • repairs to water mains, wells or pumps where contamination could have been introduced record this information on form g or other appropriate form in the g series. as applicable, investigate the water source, treatment facility, distribution and plumbing systems, sites where water was contacted, and sites at which microorganisms amplified and aerosols disseminated. use forms in the g series as guides while observing facilities, gathering data, making measurements and collecting samples. google or bing maps or other similar resources' views of the watershed can be very helpful in identifying potential sources of contamination that you will need to investigate further. these maps can also facilitate your own map and diagram making on form g . the water source may be surface or ground or in some cases a combination of the two. verify this by observations at the site and by talking to the property owner or persons responsible for operation or maintenance of water supply or recreational facilities, as applicable. examination of "weather events" such as heavy rainfall may indicate a potential for surface water contamination (see box , the walkerton outbreak). when a surface water is either suspected or implicated as the source of a contaminated supply, get information about the watershed concerning possible sites of contamination of the suspected etiologic agent. this includes, but is not limited to (a) land use, (b) sewage effluent from treatment plants and septic tanks, (c) industrial plants that may be discharging toxic waste, (d) mining wastes, (e) landfill leachates, (f) slaughterhouse discharge wastes, (g) animal feed lots, (h) both domestic and wild animals that use the source water for drinking, (i) sludge disposal from sewage treatment plants or septic tanks (e.g., land spreading or lagoons), (j) storm water discharge. if this information is not available from records or persons familiar with the site, visit the site and observe possible sources of contamination and pollution (e.g., while traveling by foot, vehicle, boat, or helicopter, as applicable). record this information on form g (source and mode of contamination of surface water). diagram the surface water and sites of contamination on form g . note type and location of sources of contamination and their distances from the water. visit groundwater sources. using form g (source and mode of contamination of groundwaters) as a guide, question owner or operator and inspect groundwater installations to ascertain character of the land and surface and subsurface soil and water. when a well or improved spring is under consideration as the source of the contaminated supply, observe its location relative to possible sites of contamination and to whether its construction allows contaminants to reach the water. determine locations of all sewage outflows or disposal sites (e.g., septic tanks and absorption lines, cesspools, privies, and other sewage disposal facilities), gradients, and distances from the well or spring. determine the type of soil at the site. if the soil is limestone or fissured rock or if there is a high ground or perched water table, pollution may travel many miles. in this case, the search for sources of contamination may have to be expanded for a considerable distance from the well or spring. ascertain whether there were heavy rains, heavy snow melts, or sudden discharge in may, , many people in walkerton, a small ontario, canada, community of about , people, began to simultaneously experience bloody diarrhea and other gastrointestinal infections. on may - , torrential rain had unknowingly contaminated the town's water system, but operators failed to check residual levels for a period of several days, allowing unchlorinated water to enter the distribution system. however, the privatelyowned walkerton public utilities commission insisted there was no problem with the water despite other laboratory tests showing evidence of e. coli contamination. illnesses began about may , with the first death occurring on may and the seventh and last on may . by may , however, many more cases had been diagnosed, the infectious agent determined to be e. coli o :h , and contaminated well water was confirmed as the source of the e. coli; all this allowed the region's medical officer of health to issue a boil water advisory, warning residents not to drink the tap water. two days later, laboratory results identified the presence of campylobacter and e. coli o :h and dna testing showed that the contaminating source was a cattle farm a short distance from a well used for the water supply. by the time the outbreak was over, > were ill and had died. the people who died directly from drinking the e. coli-contaminated water might have been saved if the walkerton public utilities commission had admitted to contaminated water sooner. those in charge of the water utilities at the commission had no formal training in their positions, retaining their jobs through three decades of on-the-job experience. they were later found to fail to use adequate doses of chlorine, fail to monitor chlorine residuals daily, make false entries about residuals in daily operating records, and misstate the locations at which microbiological samples were taken. regulations state that water suppliers are required to treat groundwater with chlorine to sufficiently neutralize contaminants and sustain a chlorine residual of . mg/l of water after min of contact. had utility operators adhered to the protocol, the disaster most likely would have been averted. the operators knew that these practices were unacceptable and contrary to ministry of environment guidelines and directives; they eventually admitted falsifying reports and were sentenced to short jail terms. the ontario government was also blamed for not regulating water quality and not enforcing the guidelines that had been in place. the water testing had been privatized in october . an enquiry found that the water supply, drawn from groundwater, became contaminated with the e. coli o :h strain from manure from cattle on a farm washing into a shallow water supply well after heavy rainfall. the risk of contamination from farm runoff into the adjacent water well had been known since . key recommendations from the enquiry included source water protection as part of a comprehensive multibarrier approach, the training and certification of operators, a quality management system for water suppliers, and more competent enforcement, which were incorporated into ontario new legislation. the bottom line of the enquiry was that officials and municipal water facilities operators and managers across north america need to recognize public waters are a most valued but vulnerable public resource. investment in keeping them safe and secure needs to be a community top priority. from dams that could have resulted in flooding within the duration of the incubation period of the disease under investigation. obtain information on the depth of the well in reference to the ground water table from the owner or by referring to any available well logs on public file or from local drillers. observe well construction and get information about depth of casing, depth and method of grouting, and whether there is an underground discharge. observe whether there is an impervious well platform and whether the pump or casing seal was subjected to flooding. illustrate the situation by showing location of the well in this event is considered a disaster, still unfolding, initiated from a political decision to save money, and ending up with acute and chronic illnesses and deaths to residents of a michigan city, as well as high system remediation and health-related costs to the taxpayer. on april , , flint, genesee county, michigan, switched its water supply from detroit's system to the flint river as a cost-saving measure for the struggling, majority-black city on the recommendation of the state-appointed emergency manager. flint agreed to separate from the detroit water and sewerage department and go with the karegnondi water authority, including the decision to pump flint river water. this was to be an interim measure until a new pipeline from lake huron was constructed in to serve the region. soon after the switch, residents begin to complain about the water's color, taste and odor, and to report rashes and concerns about bacteria. in august and september city officials issued boil-water advisories after coliform bacteria were detected in tap water. in october , the michigan department of environmental quality (mdeq) blamed the cold weather, aging pipes and a population decline. in the same month a general motors plant in flint (continued) reference to possible sites of contamination on form g . note distances between the well and contamination sites and elevations. determine whether any pumps were out of order or had been repaired during the interval of concern. if priming of the pump was done, find out the source of the water used. record this information on form g . test hypotheses of modes of contamination by conducting a dye test and/or sampling the water. (see appropriate sections of this manual for directions.) collect samples of water from these sites and submit them for analysis of the suspected etiologic agent or for any physical, bacterial, or chemical tests that will provide evidence of contamination or movement of the contaminants. (see procedures for collecting water samples) record these results on forms g or g and i (laboratory results summary). when appropriate, confirm hypotheses by a dye or other tracer test. (see section on this subject). determine the means by which the etiologic agent survived treatment or was otherwise not eliminated or inactivated. consider the treatment process as a series of barriers placed between contaminants and consumption of the treated water. the operation of each barrier should be optimized. review available data for each step in the treatment process. records of well-maintained and properly calibrated continuous monitoring equipment will be especially valuable. look for failures in the barriers, which could include (a) lack of disinfection, (b) inadequate concentration of disinfectant or contact time, (c) interruption of disinfection, (d) inadequate filtration, (e) lack of corrosion inhibitors, which may follow inadequate pre-filtration treatments. in in flint, michigan excessive levels of lead were found in drinking water from corrosion of water distribution pipes (see box , the flint water crisis). corrosion inhibitor had not been added. also, look for possible introduction of contaminants within the treatment process, such as in treatment chemicals. stopped using municipal water, saying it was rusting car parts. on january , , the city announced that flint's water contained a high level of trihalomethanes, a byproduct from increased disinfection by the city. though this is in violation of the safe drinking water act, officials told residents with normal immune systems that they have nothing to worry about. in january , detroit's water system offered to reconnect to flint, waiving a $ million connection fee but the offer was declined by the emergency manager. by february, state officials continued to play down any water problems saying that the water was not an imminent "threat to public health." on february , parts per billion (ppb) of lead were detected in drinking water at a flint home and the federal environmental protection agency (epa) was notified. the epa does not require action until levels reach parts per billion, but science indicates that there is no safe level for lead in potable water. officials from epa and mdeq discussed the lead level in the sample, and epa found that the state was testing the water in a way that could profoundly understate the lead levels. on march , , a second testing detected ppb of lead in flint drinking water. a consultant group hired by flint, reported that the city's water met state and federal standards, and it did not specifically report on any lead levels. in may, tests revealed high lead levels in two more homes in flint. in july, an epa administrator told flint's mayor that "it would be premature to draw any conclusions" based on a leaked internal epa memo regarding lead. however, in september, flint was asked to stop using the flint water supply or consider corrosion control for it, because it was causing lead to leach from the water pipes and children had high levels in their blood. state regulators insisted the water was safe. nevertheless, on october , the governor of michigan ordered the distribution of filters, the testing of water in schools, and the expansion of water and blood testing after a briefing on the lead problems with the mdeq and federal officials. at the same time, flint city officials urged residents to stop drinking water. on october , flint reconnected to detroit's water system, and residents were advised not to use unfiltered tap water for drinking, cooking or bathing. on october , the director of mdeq reported that his staff had used inappropriate federal protocol for corrosion control, and soon after, the governor announced that an independent advisory task force would review water use and testing in flint. on december flint added additional corrosion controls, and soon after an emergency was declared. at the end of december, the task force found that the mdeq was accountable for its lack of appropriate action, and the director resigned. on january , , the governor asked the national guard to distribute bottled water and filters in flint, and president obama declared a state of emergency in the city and surrounding county, allowing the federal emergency management agency to provide up to $ million in aid. three days earlier the crisis expanded to include legionnaires' disease, because of a spike in cases, including ten deaths, after the city started using river water. on january , the michigan department of health and human services stated it did not have enough information to conclude that the increase in cases was related to the ongoing flint water crisis, although the. head of michigan's communicable disease division had said three months earlier that the number of legionella cases at that time "likely represents the tip of the iceberg." as of february , the number of reported cases was close to . a flint hospital official was surprised that michigan and local health agencies did not inform the public about the legionnaires' outbreak in genesee county in - until january ; the hospital earlier had spent more than $ , on a water treatment system and bought bottled water for patients. the source of legionella is not known but it was likely in the flint river, and possibly extensive flushing of flint's colored water, which had undesirable odors and tastes, by residents may have caused chlorine residual in the pipes to be washed away, leaving the pipes susceptible to growth of the legionella; in addition, aerosols from the extensive flushing from turned-on faucets might have led to close contact between the residents and the pathogen. the investigation of the cause of the illnesses continues with criminal charges laid against michigan departmental employees. observe treatment processes from the water inlet to the finished water discharge. diagram on form g the treatment process; insert notations of hazardous situations that were observed. collect samples of water at the inlet, after each phase of treatment that may have functioned suboptimally or failed, and at the outlet. test the samples for pathogens that cause a syndrome characteristic of that being investigated, for indicator organisms and for physical and chemical characteristics of the water, as appropriate to the situation. evaluate effectiveness of the disinfection process and resulting residuals. determine the type of disinfectant (e.g., gaseous chlorine, hypochlorite, chlorine dioxide, chloramine, ozone, ultraviolet irradiation) used and whether the disinfection treatment was adequate for the volume of water treated. determine, by talking to water treatment plant employees and reviewing records of the plant or regulatory agency, whether there were any interruptions of disinfection during the two weeks prior to the first onset date. determine contact time between the point of addition of the disinfectant and the first point of use. measure the chlorine residual, ph and temperature of the water just before it leaves the plant. observe the condition, operation, and maintenance of disinfectant dispensing equipment. review plant records to identify any sudden changes in disinfectant demand that causes temporary depletion of disinfectant residuals and allows survival of pathogens. review maintenance records for disinfectant dispensing equipment and quality assurance records for online analyzers. record this information on form g a (disinfection failures that allowed survival of pathogens or toxic substances). calculate disinfectant rate applied and usage (see formulae in form a). for example, to calculate disinfectant rate, if the flow rate is , , gal/day and the dosage is lb/day: the destruction of pathogens is dependent on (a) type and condition of microorganisms present, (b) type of disinfectant used, (c) concentration of available chlorine or other disinfectant, (d) contact time, (e) water temperature, (f) ph, (g) degree of mixing, (h) presence of interfering substances (which may be related to turbidity). utilize treatment records that provide small scale time resolution, such as online monitoring data, to determine whether the process was stable during the time period in question. daily averages may provide evidence of massive failures, but will not provide information about whether consistent treatment was being provided. in general, the relative effectiveness of microorganisms' resistance to free chlorine, from high resistance to low resistance, is as follows: • protozoan oocysts (i.e., cryptosporidium) • protozoan cysts (i.e., giardia, entamoeba histolytica) • viruses (hepatitis a virus, poliovirus) • vegetative bacterial cells (shigella, escherichia coli) protozoan oocysts are highly resistant to chemical disinfectants, but not to physical means such as uv light or ozone (gas). microorganisms within each group and strains among the same species differ somewhat in resistance. the state of injury induced by environmental impacts and selection of resistant strains influence survival. aggregation of microorganisms and/or close association with debris shield them to various degrees from lethal effects of disinfectants and attachment to surfaces such as pipe walls to form biofilms that protect organisms from inactivation by disinfectants. a measurement of microbiological inactivation by disinfectants is the ct value (ct calc ), which is the product of the free residual disinfectant concentration (c) in mg/l that is determined before or at the first user (customer) and the corresponding disinfectant contact time (t) in minutes (i.e., c × t). refer to table i (ct . values for inactivation of giardia cysts at different concentrations of disinfectants, temperatures, and ph values) and table i (ct values for inactivation of viruses at ph - , at different temperatures with different disinfectants for comparing disinfectant efficiencies). make residual measurements during peak hourly flow. for comparisons of ct values between the indicated ph, temperature, and concentration values, use linear interpolation. (for example, for free chlorine, °c, concentration mg/l, ph . = [ − = ; / = ; + ] = ). if no interpolation is done, use the ct . value at the higher temperature, at the higher ph and higher concentration. a simple ct calculation, for example, using a disinfectant concentration (c) at the basin effluent of . mg/l and a detention time (t) of min, is as follows: use this calculation for comparing to values in table i or j. the calculated ct value should be higher than the value stated in the table for specific conditions of disinfection, temperature, ph, and concentration (residual). in this situation, if the temperature of the water was °c, the ph and the concentration of chlorine mg/l, a ct value of would be needed for a . reduction of giardia cysts. the ct value of . would have been inadequate to meet the criteria and could explain the survival of the pathogen under investigation. microbial inactivation efficiencies vary considerably among different disinfectants and are influenced by the characteristics of the water and water temperature. tables i and j show that, in general, ozone is more effective than chlorine dioxide, which is more effective than free chlorine, which is more effective than chloramines. also, in general, longer contact time increases the degree of inactivation, and higher water temperatures as well as lower ph values increase rates of inactivation. rapid mixing of the disinfectant with water increases disinfection efficiencies, whereas dissolved organic matter reacts with and consumes the disinfectant and forms products that have weak or no disinfection activity. certain inorganic compounds and particulate matter also react with disinfectants. the ct value must be determined sequentially whenever a disinfectant is added to water. contact time (t) is the duration in minutes for water to move from the point of application of the disinfectant or the previous point of residual disinfectant measurement to the point where residual disinfectant concentration (c) is measured. it is measured from the first point of disinfectant application and from all subsequent applications until or before the water reaches the first user. determine contact time in pipelines by dividing internal volume of the pipe by the maximal hourly flow rate through the pipe. determine the flow rate from (a) plant records, (b) continuous monitoring device readings, (c) measurements at hourly intervals, or (d) if this sort of information is unavailable, measurements at expected high flow periods. use tracer studies to determine contact time within mixing basins and storage reservoirs. these values represent only % effectiveness because of short circuiting. chlorine, fluoride, and rhodamine wt (but not b) are commonly used tracer chemicals. contact time is usually measured by a step-dose method, but a slug-dose method is used where chemical feed equipment is not available at the designated point of addition or where such equipment does not have the capacity to provide the necessary concentration. (see appropriate epa literature for procedures, and consider getting engineering expertise if these matters are too complex.) estimate whether pathogens had been inactivated. to do this, divide the ct calc value by a value (ct x% ) resulting in a certain percentage inactivation (e.g., . % [ -log] or ct . for giardia cysts and . % [ -log] or ct . for viruses). this gives an inactivation ratio. see table i for ct . values for giardia and table j for ct . values for viruses. following is a sample calculation for data in table i when water temperature is °c, ph in a clearwell (reservoir for storing filtered water) is . , time (t) (either calculated or measured by dye test) is min, and the disinfectant used is chlorine: the desired ct value for . % inactivation of giardia for ph at °c is between and depending on concentration of disinfectant. in this case, the disinfectant measured at the clearwell outlet is . mg/l. therefore, the result, , is larger than the value, , required in the table; hence, these disinfection concentration (c) and time (t) conditions should result in a . % or greater inactivation of giardia cysts. for free chlorine, a -log inactivation of giardia cysts provides greater than a -log inactivation of viruses. the following example, using the data in table i , demonstrates a means to calculate the increased disinfectant dosage needed for a plant during the transition from summer to winter, when the water temperature fell from to °c, chlorine dioxide was the disinfectant used and the t value (calculated or measured) is min. using table i in this situation, the plant should have increased the chlorine dioxide concentration from . mg/l to . mg/l to maintain the same efficiency of disinfection. if this had not been done, it may explain the survival of pathogens in the water supply. the sum of these ratios gives the total inactivation ratio, which should equal or more to provide effective disinfection. make calculations and record information on form g a. the following example shows the way this is done. chlorine is added to three basins. chlorine concentration, contact time, temperature and ph are measured at these locations and recorded as shown in table . data from table i is combined to do the calculation. the resulting sum exceeds . . this ensures that the plant met the recommended or required ct. regulations may require that community and non-community public water systems that use surface water or water under direct influence of surface water meet a criterion (e.g., minimum of . % [ -log] removal and/or inactivation of giardia cysts and a minimum of . % [ -log] removal and/or inactivation of viruses of fecal origin that are infectious to humans). removal and/or inactivation of microorganisms may be accomplished by either filtration plus disinfection or disinfection alone, depending on the water source. water systems using chlorine with ct values that attain minimal level or inactivation of giardia cysts will result in inactivation of . % ( -log) of viruses. evaluate the prefiltration processes (e.g., coagulation, flocculation and sedimentation). coagulation is a process that uses coagulant chemicals and mixing, by which colloidal and suspended materials are destabilized and aggregated into flocs. flocculation is the process that enhances agglutination of smaller floc particles into larger ones by stirring. sedimentation is the process by which solids are removed by gravity separation before filtration. observe whether these processes reduce turbidity. calculate detention (transit) time within the settling tank and seek information about frequency and method of cleaning the tank. for example, if an -ft-deep sedimentation basin has a volume of million gal, and the plant flow rate is million gal/day, detention time in the basin is: (in your country you may want to calculate rates based on metric measurements) several different types of filtration may be used in water treatment facilities. these are conventional, direct (both conventional and direct are referred to as "rapid" filtration), slow sand filtration, and diatomaceous earth filtration. conventional filtration consists of a series of processes including coagulation, flocculation, sedimentation, and filtration. direct filtration consists of a series of processes including coagulation and filtration but excluding sedimentation. slow sand filtration is a process involving passage of raw water through a bed of sand at low velocity (usually less than . m/h), utilizing both physical and biological means to remove particles and microorganisms. in diatomaceous earth filtration, water is passed through a precoat cake of diatomaceous earth filter medium while additional filter medium is continuously added to the feed water to maintain the permeability of the filter cake. if done properly, each filtration method results in substantial particulate removal. when rapid sand filters have a head loss of about - ft, they require back washing. filters are backwashed by reversing the flow of the filtered water back through the filter at a rate between and gal/min/ft of sand-bed area. sometimes water jets at the surface aid in loosening and removing deposited material on the sand. observe an actual backwash and look for indications of short-circuiting or areas of the filter material that seem agglomerated or resist being cleaned by the flowing water. if backwash water is not discharged to waste, evaluate where it is released. slow sand filters eventually become clogged. when this occurs, a scraper or flat shovel is used to remove the top layer of clogged sand, and new sand (equivalent to the depth removed by scraping) replaces the old. test the effectiveness of filtration for each filter unit by observing capacity and filtering area relative to volume and turbidity of the filtered water. also, review turbidity, headloss, and filter rate record. look for anomalies, especially in the few hours after a filter is returned to service, and before the filter is backwashed. review criteria that cause a backwash to be initiated, and establish if these criteria were followed during the time preceeding the outbreak. determine the source of backwash water and the frequency of back washing of filters from records and head gauge readings. check whether the water used to back wash or clean filters came from an untreated source and determine the fate of the backwash water. in the case of illness due to chemical substances, evaluate types of chemicals used and condition, operation and maintenance of chemical feeding equipment. consider sampling backwash water for pathogens under investigation. review plant records for results of monitoring and be alert for changes that suggest treatment failure. record this information on form g b (source of contamination and treatment failures that allowed survival of pathogens or toxic substances.) data in table k (estimated removal of giardia and viruses by various methods of filtration), give a summary of expected minimal removal of giardia and viruses in well operated filtration systems. values can be subtracted from ct values required for disinfection. although contamination is likely to be associated with raw incoming surface water, look for bypass connections where raw or partly treated water can get into treated water. also look for common walls that separate treated and untreated water. consider the possibility that a contaminant was introduced in any of the treatment chemicals themselves, or as an act of sabotage. determine whether any flooding has occurred during the interval of concern. check absentee records for possible enteric illness of the water treatment plant staff. such illness may reflect either sources of contamination or victims. record this information on form g b. at domestic locations, evaluate treatment devices (such as chlorinators, filtration units, softening equipment) as described above, but modified to fit the situation under investigation. record observations and measurements on forms g a and g b, as applicable. the water distribution system can be complex, with multiple entrance points for treated water and different pressure zones in which water can enter but not leave. water flows in the direction in which it is being "requested," so can flow in different directions in the same pipes from one hour to the next. contaminated water can enter a potable water supply from a non-potable water supply when the two are directly connected. such interconnections are referred to as cross connections. to evaluate such situations, trace lines of the treated supply from the point of treatment or entrance into a building to points of use and associated plumbing. look for any interconnections of other water supplies, such as wells, waste lines or holding tanks for water intended for fire control. if cross connections are found, look to see whether backflow prevention devices are inserted between the lines and, if so, whether they are functioning properly. also, look to see whether there is an air gap between the water inlet and vessel or tank. evaluate the arrangement and operation of check valves on connections between the two water systems. review inspection report for backflow prevention devices. contaminated water can also enter a treated supply by siphonage from a contaminated vessel or sewerage to the potable water line having negative pressure. this is referred to as back siphonage. examine all water vessels to see whether they contain submerged inlets or hoses connected to water faucets, and if so, whether properly functioning vacuum breakers are in place. without proper air gaps or properly functioning vacuum breakers, there is a possibility of siphonage of water from plumbing fixtures in upper stories to lower stories when line pressure is negative. this may occur when faucets on lower floors are opened after the water supply valve has been turned off for repairs or when the supply line has had a sudden loss of pressure, as can happen with nearby heavy use of water (e.g., to fight fires or irrigate) or when pressure lines are broken. measure water pressure on upper stories of buildings to determine whether negative pressure occurs. (pressure losses may be transient and of very short duration.) interview building managers and residents about whether there were (a) any repairs of water service during the past month, (b) fires that occurred nearby, or (c) other situations that could have caused negative pressure in the water line. also, if appropriate, review fire and utility department records for information about these situations. get dates of line repairs to see whether they correlate with the time of incubation periods of early cases. measure chlorine residual (of chlorinated water systems) and take samples for microbiological tests at several strategic locations in the distribution systems. perform calculation on comparison of disinfectant residual. if a toxic chemical poisoning is under investigation, talk to home owner, building manager or maintenance staff about whether pesticides or other toxic compounds were sprayed with equipment connected to a hose or a sprinkler system. furthermore, interview building managers and residents about whether there are persons residing there who either are or recently were ill with diarrhea. they may represent sources of the etiologic agent or may identify victims. interview those identified about the onset of their illness and symptoms and examine their plumbing systems. record information obtained during the investigation of distribution and plumbing systems, and record related calculations on form g (source and mode of contamination during distribution and at point-of-use). evaluate implicated waters used for swimming, water skiing, bathing, clothes washing by hand, or agricultural activities, in a manner similar to that described under the section on investigation of surface water source. if the potential site of contact was natural surface water, determine whether the water is likely to be infested by parasites and look for the presence of snails (swimmer's itch). for swimming pools, measure the water's ph, chlorine residual, water temperature, and turbidity, if applicable. also, review pool records for previous information on these characteristics. high turbidity in pools, hot tubs, and spas is a sign of either poor filtration or inadequate disinfection. evaluate whether the resulting water would adequately protect those who swam or waded in it or had any physical contact with it. evaluate filter and chlorination equipment as described for water treatment. backwash filters and collect a sample to get an indication of microorganisms present on the filter (thus obtaining historical information). this approach has been useful for identification of pseudomonas aeruginosa. look for the presence of slime on tub, whirlpool, slide and pool surfaces, and collect some of this material for analysis for p. aeruginosa. if the answer is not obvious, ask ill persons whether they had puncture injuries or wounds or scrapes while immersed in water. record this information on appropriate parts of form g (contamination source and survival of pathogens or toxic substances for recreational waters). collect samples of the water (see section on "collect water samples"), and test them for pathogens and/or indicator organisms, as applicable. the agents listed in table d can multiply in water and if such water is aerosolized, they can be transmitted to human beings via the respiratory route. highly susceptible persons (e.g., the elderly, smokers, immunosuppressed individuals) are the usual victims. look for possible sites where water may have been or is being disseminated as aerosols. consider (a) air conditioning cooling towers and evaporative towers, (b) hot water systems (heaters and tanks), (c) shower heads, (d) faucets with aerators, (e) mist machines used to freshen fruits and vegetables in markets, (f) humidifiers, (g) nebulizers/respiratory therapy equipment, (h) whirlpools and spas, (i) dental drills and cleaners, (j) cooling water apparatus for grinders, (k) splash from hoses, (l) water pressure line breaks, (m) decorative water features, (n) outside misters, (o) other aerosol-producing devices. sample water from all suspect sites for legionella or other waterborne agents that may cause illness when inhaled. it is not possible to recognize by visual inspection the potential for water to be contaminated with legionellae. warm temperatures, especially those between °c ( °f) and °c ( °f), are conducive to growth of legionellae. additionally, stagnant water allows time for legionellae to multiply, especially in dead-end lines, reservoirs and hot water tanks, and in water trapped in shower heads and faucet aerators. if it is deemed appropriate or necessary to sample for detection of legionella in the environment, collect water samples from suspect sources. it is important to use a lab with proven expertise in isolating and characterizing legionella, such as those labs in the u.s. certified under the environmental legionella isolation techniques evaluation (elite) program. the centers for disease control (cdc) have a convenient form for recording case histories (http:// www.cdc.gov/legionella/downloads/case-report-form.pdf). it is not appropriate to sample air for detection of legionella hazards. it may, however, be appropriate to use micromanometers or smoke to trace direction of air flow to determine route of dissemination. micromanometers measure pressure differences, and flow can be assumed to travel from high to low pressure areas. smoke moves from areas of higher pressure to areas of lower pressure and is extremely sensitive to air currents. observe direction and spread of smoke movement. record this information on form g (contamination source and sites of amplification and aerosolization of pathogens). prior to the collection of samples, investigators should consult with the testing laboratory that will be used, to receive specific laboratory sampling instructions and sampling kits. sampling protocols for potable and non-potable sources are dependent on the specific etiological agent and the related analytical procedures performed by the testing laboratory. collect samples promptly to test for possible etiologic agents and for microorganisms indicative of fecal contamination. contaminants in water are in a dynamic state; their presence and quantity differ with time and place. see table f (general instructions for collecting drinking water samples) for guidance on collecting and shipping samples for viral, bacterial, and parasitic analyses. samples for bacteriological tests can be collected in one of three ways: (a) by letting a stream of water flow into a container or by submersing a container into a volume of water, (b) by passing a large volume of water through a filter, (c) by putting moore swabs (see table a for description) or similar absorbent materials in surface water or drains for a few days (see table f ). use bottles that have been cleaned, rinsed, and sterilized, or use sterile plastic bags to collect and store samples for bacteriological examination. for a chlorinated water supply, or when in doubt about the presence of residual chlorine, use bottles containing mg/l sodium thiosulfate to combine with any free chlorine in the sample and prevent lethal effects of chlorine on microorganisms in the sample. this compound will not interfere if used for non-chlorinated water. when collecting water samples, first try to get "historical" samples that might give an indication of the condition of the water at the time it was ingested by those who became ill. obtain historical samples from water in bottles in refrigerators, toilet tanks, hot water tanks (for chemical analyses only), fire truck reservoirs, storage tanks, and taps at seldom-used and dead-end locations, and from ice in refrigerators and commercial ice plants. direct the laboratory to test historical samples for pathogenic organisms or toxic chemicals, as well as indicator organisms, because these samples have a chance of still containing the etiologic agent, whereas samples collected during the investigation several days or weeks after the event may be of water that has been flushed free of contamination or has been significantly diluted. take samples from to points throughout the distribution system. sample dead-end locations if they are found. do not neglect to obtain raw water samples even though treatment is provided. this is important, as it suggests possible sources of contamination and reflects the effectiveness of treatment. compare these test results with records of results on previous samples of raw or treated water. before drawing a sample from a water tap, make sure the tap is connected to the supply to be tested. do not collect samples (other than for legionella) from hose connections, sprays, or swivel faucets; uncouple these connections or choose different outlets. it is unnecessary to flame outlets, as this does not improve the quality of the sample. first, ensure your hands have been thoroughly washed then take a line sample by allowing the water to run to waste for - min. adjust the flow of water so that the thiosulfate will not wash out of the bottle or bag (do not overfill-most laboratory bottles indicate a maximum fill line). keep sample containers closed until the moment they are to be filled. hold the bottle near the base, fill to the "fill line" or within an inch of the top without rinsing, and immediately replace the stopper or cap and secure the hood, if attached. if a whirl-pak™-type plastic bag is used instead of a bottle, hold the base, rip off the perforated top, open the bag by pulling the side tabs apart, grasp the end wires, and place the bag under the flowing water. remove the bag before it is completely filled and squeeze most of the air out; fold over the top of the bag several times and secure by twisting the end wires. take a source or a distribution line sample by opening the tap fully and letting the water run to waste for sufficient time to empty the service line (or if in doubt, for min) and proceed as above. collect samples from open shallow wells and step wells by dropping a clean wide mouth container on a string or rope into the well. allow the container to sink below the water surface and then pull it out of the well. pour contents into a sample jar or bag. collect samples from rivers, streams, lakes, reservoirs, springs, toilet tanks, and non-pressurized storage tanks by holding a ml sample bottle near the bottom and plunging it neck down to a depth of cm ( in) below the surface; turn it right side up, and allow it to fill. don a plastic disposable glove when small vessels used for drinking are sampled in this manner. when collecting these samples, move the bottle in a sweeping, continuous, arc-shaped motion, counter to stream flow or in a direction away from the hand. collect samples at locations approximately one-quarter, one-half, and three-quarters the width of the stream or water course. special apparatus can be used for sampling at various depths. samples can then be taken by positioning large bottles on a rod or pole at the desired depth and location before pulling their stoppers with a wire, string or thin rod. samples of bottom sediments are sometimes useful for the detection of certain pathogens. collect surface scum or regions containing dense particulate colored material when seeking cyanobacteria (blue-green algae). collect slime, if present, when seeking pseudomonas. if large amounts of water are needed, seek assistance and obtain specialized sampling equipment from agencies responsible for water quality. if possible, avoid wading when sampling bodies of water because wading often stirs up bottom sediments. if this is the only way to get a sample, however, wade against any current (e.g., upstream in creek or river) and keep moving forward until sample taking is completed. piers or similar structures, or the front end of a drifting or slow moving boat, make good sampling stations. concentration of bacteria by the use of swabs, filters, or by absorption, is particularly important when waterborne pathogens are sought. to concentrate bacterial pathogens from flowing water (e.g., streams, lakes, sewer lines, or drains), suspend moore swabs (or non-medicated sanitary napkins or non-medicated tampons if moore swabs are unavailable) for - days. these can be held in place by wire just below the surface or at other depths. if rodents are about, put moore swabs in wire baskets. after the sampling period, either put swabs or pads into a plastic bag and pack in ice, or put the swabs or pads directly into an enrichment broth for the pathogen sought. take or send these to the laboratory promptly. concentration of microorganisms can be increased by filtration with a variety of filters (e.g., membrane filters, cartridge filters, or other filter media). when membrane filters are used for pathogenic bacteria recovery, pass at least l of water (relatively free of turbidity) through a sterile . μm membrane filter. for viral analysis, use virus-absorbing electropositive cartridge filter to concentrate l or more water (see table f ). keep filters cool (but not frozen) and ship to a reference laboratory for further processing. for giardia cysts and cryptosporidium detection, collect samples by passing at least l water through a cartridge filter (see table f ). for inorganic chemical analyses, use l polyethylene containers. these should be new, or acid-washed if previously used. collect the water without flushing the lines, preferably in the early morning before water is used. for trace metal analyses, preserve one sample with ml of high-grade nitric acid to a ph of or less. this is particularly important whenever it is suspected that metals may have leached from water pipes or vessels. for organic chemical analysis, use l glass containers with teflon-lined caps. clean and rinse the containers with a good quality laboratory solvent and heat at °c for min. rinse the cap thoroughly with distilled water. fill the container so that there is a minimum of air space. for physical analyses, collect at least l, or other amounts requested by the laboratory. collect ice aseptically in sterile plastic bags or jars. use sterile tongs to collect cubes; sterile spoons for collecting chipped or crushed ice; and sterile chisel, hammer, or pick to chip block ice. put block ice or large chips into plastic bags. if legionella is sought, sample water at sites of any source that may have been aerosolized and send to a lab with proven expertise in legionella isolation and characterization, such those in the cdc elite program. this includes cooling towers, evaporative condensers, water heaters and holding tanks, humidifiers, nebulizers, decorative fountains and whirlpool baths (see section on investigating sites where aerosols are disseminated for a more complete listing). turn off fans of condensers before sampling; if this is not possible, wear a respirator. use ml to l polyethylene bottles that have had sodium thiosulfate added if the water to be tested has been chlorinated. for each sample, don disposable plastic gloves and collect the sample by inverting the bottle and moving it in a continuous arc away from the hand. measure and record water temperature. handle samples as described in table f . rub swab over faucet aerators and shower heads if these are considered as sources of aerosols. break stick and allow tip to fall in a tube containing - ml sterile water (not saline). investigators are often requested to test air to demonstrate the presence of legionella in aerosols. although legionellosis is an airborne disease, legionellae are susceptible to low humidity and become non-viable on drying. therefore, air sampling is an ineffective and inefficient way of determining whether a legionella hazard exists, and it can thus be misleading. label each container with sample number, date, time of collection, and your name or initials. complete the water/ice sample collection report, form f, for the first sample. list additional samples with sample numbers and other pertinent information on the back of the form. in those situations where the laboratory needs additional information, attach the appropriate g series forms. send the original form f and list with samples to the laboratory; retain a copy for your files. inform the laboratory of the type and number of samples and specimens; also, consult with the laboratory on methods to preserve and transport samples, if necessary, and on time of their arrival. if legal proceedings are anticipated, deliver sample personally to the analyst, or seal the sample container in such a way that it cannot be opened without breaking the seal. note on form f the method by which the bottle was sealed. maintain a chain-of-custody log to document the handling of the sample, and have the log signed and dated each time it changes hands. consult with state/provincial regulatory agency on complying with legal requirements for chain-of-custody procedures. recipient should record on the form whether the sample was sealed when the laboratory received it. if analysis cannot be done on the day of collection, chill water samples rapidly and hold them at temperatures at or below °c ( °f), but do not freeze, because populations of bacteria such as escherichia coli and of parasites decrease during frozen storage. hold ice samples frozen; if this is not possible, keep the temperature below °c h ( °f). investigators should consult with the testing laboratory that will be used to receive specific laboratory sample packaging, labeling, and transportation instructions as protocols are dependent on specific transportation regulations (iafta, tdgr) within each jurisdiction. ensure each sample is uniquely identified and labeled (as per the receiving laboratories requirements). many laboratories include barcode labels along with the sample containers within the sample collection kits. ensure that the correct label is affixed onto the correct sample container and that this information is transferred to the shipping manifest accurately (chain of custody form). specimens should be packed and the packages labeled according to applicable regulations governing transport of hazardous materials. generally, the transport of samples of water and ice intended for laboratory analyses are packed and shipped in a manner to ensure the sample does not change from the time of sampling to the time received by the testing laboratory and shipped using the most expeditious means (e.g., personal delivery or overnight mail). typically samples of water or ice are packed with refrigerant (ice packs, dry ice, etc.) in insulated and sealed containers (see table f ). several measurements are routinely called for during on-site investigations. brief instructions are given for those that are commonly done; nevertheless, follow manufacturer's instructions if these are available. color comparison kits are available for testing for free, combined and total residual chlorine. the diethyl-p-phenylenediamine (dpd) test is an example (see table a ). check instrument calibration regularly. use dry reagents, because the liquid forms are unstable. chlorine comparators can be used to test for bromine by multiplying the result by the factor . and to test for iodine by multiplying the result by the factor . . measure temperature. measure water temperature by immersing the sensing end of either thermocouples, transistors, or thermometers into the water. sometimes measurements need to be made at various depths; use thermocouples with wire leads of sufficient length for this purpose. calibrate temperature measuring devices periodically. measure ph. calibrate the ph meter as recommended by the manufacturer with at least two standard buffers (e.g., ph . or . ) and compensate for temperature, if the meter does not do it automatically, before each series of tests. remove a sample of water to be tested and immerse the ph electrode into the sample; record the reading. ph can also be measured by color comparators that employ color indicator solutions or discs. (ranges of ph color indicator solutions are bromophenol blue, . - . ; bromocresol green, . - . ; methyl red, . - . ; bromocresol purple, . - . ; bromothymol blue, . - . ; phenol red, . - . ; cresol red, . - . ; thymol blue, . - . ; and phenolphthalein, . - . .) in this case, water containing more than mg/l chlorine in any form must be dechlorinated with sodium thiosulfate before the ph indicator solution is added to prevent decolorization of the indicator. always report temperature at which the ph is measured. measure turbidity. nephelometric turbidity unit (ntu) is the usual standard unit, but other turbidity measurements (such as particle counts) are used. the ntu requires a nephelometer, which measures the amount of light scattered predominantly at right angles and absorbed by suspended particles (e.g., clay, silt, finely divided organic matter, inorganic matter, soluble colored organic compounds, and microscopic organisms) in the water sample. calibrate turbidimeters with a standard reference suspension. make turbidity measures on the day samples are taken. vigorously shake samples, wait until all air bubbles have disappeared, and then pour sample into turbidimeter tube. read directly from scale on instrument or from an appropriate calibration scale. pump chemical smoke into the air at the exit of the device suspected of releasing aerosols. observe the direction and spread of the smoke. otherwise, measure pressure differentials with a micromanometer. measure other attributes of water. follow instructions given by manufacturers or in standard reference books (see further reading). use fluorescein dye, lithium or other tracers in appropriate soils to determine the means by which contamination from sewage, industrial wastes, or other sites of pollution reached the water supply. fluorescein dye is particularly helpful in evaluating flow of contamination through fissured rock, limestone, gravel, and certain other soils. this dye is not readily absorbed or discolored by passage through these soils or sand, as are many other dyes, but it is discolored by peaty formations or highly acid (ph < . ) soils. make a concentrated fluorescein dye solution by mixing g of fluorescein powder into a liter of water. usually, / to l of this solution are sufficient for the test for up to , l of water. fluorescein dye is also available in liquid and tablet form. one tablet will dye approximately l (~ us gal). pour the calculated amount of fluorescein solution or put a sufficient number of fluorescein tablets into a receptacle at a point of potential pollution. usually this point will be located within yards and at a higher elevation than the water source under study. cesspools, latrines, distribution boxes, sink holes, borings, septic tanks, drains, manholes, toilets and plumbing fixtures are typical places to introduce the dye. if dye is poured into a plumbing fixture or dry hole or boring, add water to wash it down. the amount of dye to use varies with the distance the dye must travel, the expected time of the journey, the size of the aquifer or water channel, and the nature of the soil. take samples of the water when the dye is introduced into the test hole or fixture and then hourly for up to h to detect arrival and departure of fluorescein. if no dye is observed, repeat the test with twice the amount of dye. whenever possible, use a fluorescent light or fluorometer to analyze water samples for evidence of fluorescein. a fluorometer can be set up and calibrated, and a continuous recording can be made. this meter can detect fluorescein in concentrations of μg/l (ppb). fluorescein dye will temporarily color water, which discourages use of the water until the dye is sufficiently degraded or diluted. alternate tracers can be used if specific ion meters are available. the dye stains all it touches. methanol is a good solvent for the dye, and hypochlorite solutions decolorize it; both can aid in removing stains. abrasive soaps are useful for cleaning stained skin; fluorescein-stained clothing should be washed separately. appearance of dye in a water supply is conclusive evidence of seepage from the site where the dye was introduced. failure to detect dye, however, is not conclusive evidence that seepage did not or would not occur if more dye had been added or if weather conditions or subsurface flow had been different at the time of the test than during the outbreak event. illustrate source and direction of contaminated water flow as indicated by the dye test on form g . take photographs of sources of contamination and evidence of staining of the ground at the site or dye-stained color of the water. in situations where a single source of contamination is obvious or where multiple sources are readily apparent, dye studies serve little purpose. drinking water, however, is not the only source of water that may contribute to outbreaks. other sources of water that can contribute to outbreaks include water not intended for drinking, recreational water and water used in agriculture during harvesting and packaging. legionnaires disease is the pneumonia caused by the inhalation of contaminated water aerosol containing the bacteria legionella, with legionella pneumophila being responsible for % of all infections. it is also a common cause of healthcare associated pneumonia. legionella can replicate within free-living amebae in water, allowing it to resist low levels of chlorine used in water distribution systems. risk of infection is more common in warm and humid weather, when water droplets are able to drift further due to higher absolute humidity. fifty percent of all legionella outbreaks have been traced to cooling towers with l. pneumophila serogroup responsible for all cooling tower outbreaks. all aerosol generating devices, however, can be potential sources of legionella. some other sources of aerosolization that may have contributed to or be associated with outbreaks include: whirlpool displays, building's air conditioning systems, water spray fountains, public bath houses, vegetable misting systems in grocery stores, evaporative condensers, showerheads, humidifiers, air scrubbers, car washes, ornamental and decorative fountains, potting soil, respiratory therapy equipment, dental units, road asphalt paving machines, car windshield washer fluid and car air-conditioning systems. in the investigation of a legionella outbreak, (see box , the flint water crisis, which describes a likely legionella outbreak from a commercial water source) due to the varied sources, there is a need to use a broad investigative questionnaire and the collection of environmental data. environmental factors such as dry bulb temperature, relative humidity and wind rose data can provide information regarding drift evaporation, deposition (settling) and the size of the affected zone. although aerosol drift can carry legionella up to mi ( km), the risk of infection is usually highest within ft ( m) of the source. there are also air dispersion models that can be used to determine drift zone and the use of human activity mapping in the identification of potential sources. in general, e. coli and norovirus are the most common pathogens responsible for recreational waterborne outbreaks associated with non-treated water such as beaches and lakes. cryptosporidium, which is resistant to chlorination, is the most common pathogen resulting in outbreaks in treated water venues such as swimming pools and water spray parks. it should be noted that e. coli, the indicator of choice of recreational water samples, is not indicative for the presence of norovirus and giardia, cryptosporidium. e. coli can also be "naturalized" and have been found to survive and multiply in beach sand. beach water sampling results therefore may provide false positive or false negative results and may not be the best indicator for the presence or absence of pathogens. recreational waterborne outbreaks are not just traced to the ingestion of contaminated water (table c . illnesses acquired by contact with water: a condensed classification by, symptoms, incubation period, and types of agents). hot tub rash, or pseudomonas dermatitis/folliculitis commonly occurs in public hot tubs or spas such as those found in hotels. the rash is often a result of skin infection from the bacteria pseudomonas aeruginosa colonizing in the hair follicles after exposure to contaminated water. pseudomonas aeruginosa is an opportunistic pathogen that can survive within the biofilm on the tub surface or within the piping system. outbreaks can occur when there is a heavy bather load resulting in an increase in chlorine demand, which in turn reduces the effectiveness of the disinfectant to control the population of pseudomonas. blue-green algae or cyanobacteria bloom can occur in warm, slow-moving or still water. when conditions are favorable, mostly during hot summer weather, cyanobacteria populations may increase dramatically, resulting in a "bloom" as they rise to the surfaces of lakes and ponds. they resemble thick pea soup and are often blue-green in color. although blooms can occur naturally, water bodies which have been enriched with plant nutrients from municipal, industrial, and agricultural sources are particularly susceptible. some cyanobacterial species may contain various toxins, some are known to attack the liver (hepatotoxins) or the nervous system (neurotoxins); others simply irritate the skin. health effects from cyanotoxin exposure may include dermatologic, gastrointestinal, respiratory and neurologic signs and symptoms (table b . illness acquired by ingestion of contaminated water: a condensed classification by symptoms, incubation periods, and types of agents). water can also be an indirect cause of foodborne outbreaks by providing a media for the survival, transportation and the introduction of pathogens into food products. water used during production, including irrigation, pesticides and fertilizers application and washing, frost protection, harvesting, has long been recognized by food safety scientists as one of plausible and probable sources of the contamination of fresh fruits and vegetables. there have been many outbreaks from produce traced to pathogens being introduced by contaminated irrigation water. although harvested products are sometimes washed with chlorine solution, pathogens may still survive the process through internalization. e. coli o :h may migrate to internal locations in plant tissue and be protected from the action of sanitizing agents by virtue of its inaccessibility. experiments have also demonstrated that e. coli o :h can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant. however, this claim has been refuted by others. salmonella and e. coli can also adhere to the surface of plants, and enter through stomata, stem and bud scars and breaks in the plant surface caused by harvesting and processing. water containing bacteria can be drawn into the produce if it is immersed in or sprayed with water that is colder than the produce itself. e. coli o :h may also use its flagella to penetrate the plant cell walls and attached to the inside of the plant. once attached, it may be able to grow and colonize the surface of the plant. the concerns are not just with bacteria. the present of norovirus in the hydroponic water can result in internalization via roots and dissemination to the shoots and leaves of the hydroponically grown lettuce. irrigation water may be contaminated from runoff from nearby domesticated animals and their lagoons, feedlots, ranches into rivers; from feral/domestic animals with direct access to creeks, ditches, rivers, ponds; from sewage flows into waterways and contaminated wells. in some parts of the world sewage contaminated water is preferred for irrigation despite a potential risk of transporting enteric pathogens, since it carries nutrients (n and p) for the plants. there is sufficient information to conclude that the application method of irrigation water to fresh produce can have an effect on the microbiological risks associated with the crop. in general, keeping water away from the edible parts of ready-to-eat crops that are consumed without cooking can result in a lowered risk of a foodborne outbreak. the least to more risky methods for irrigations for microbial contamination are: subsurface irrigation (buried soak hoses) < drip irrigation < indoor flood irrigation (hydroponics) < outdoors flood irrigation (water-filled furrows) < overhead irrigation (sprinklers). an outbreak of illness arising from exposure to water demands immediate epidemiological investigation to assess the situation, gather, evaluate, and analyze all relevant information, with the goal of ( ) halting further spread of this illness, and ( ) predicting, preventing, and/or attenuating future outbreaks. this twofold mandate of epidemiology is usually described as "surveillance and containment." at the commencement of an investigation, the unknowns usually outnumber the known facts. there is no substitute for prompt, thorough, and careful collection of interview data from ill and well persons who ingested or contacted the suspect water, attended a common event, or who were part of a group of persons where illness occurred. careful analysis of these data, particularly with reference to common patterns of "time," "place," and the characteristics of the persons involved, can often eliminate many vehicles, agents, and pathways quite early in the investigation, and focus on the remaining possible vehicles, routes, and agents. later, laboratory results may confirm the agent, the specific pathology, the route taken by the infection or toxic agent, and indicate what is needed to stop the spread, but early epidemiology can often be invaluable in predicting the outcome and taking preventive steps to contain the problem before the lab results are available. lessons can be learned from most outbreak investigations and are invaluable for increasing our understanding of these pathologies, and preventing their future occurrence. an outbreak is defined as either an unusually large occurrence of an expected illness at that time of year in that place, or the occurrence of a type of illness that does not usually appear at that season and location. the "time" factor should be studied immediately by plotting the onset time of each case on a time-based grid, to create the epidemic curve. although any number of cases can be involved, the minimum number for an "outbreak" to be declared is two associated cases, with special exceptions such as naegleria fowleri where, because of the severity and the possibility that cases may have been missed, a single case constitutes an "outbreak." although the epidemic curve is usually measured in hours or days, protracted exposure to agents in water may mean apparent sporadic cases linked to a common source over months or years. if an "outbreak" is suspected by a sudden increase of cases, determining who is to be categorized as a "case" is not necessarily a simple process. many people notoriously fail to report enteric illness for many reasons: embarrassment, lack of time, no clear idea which agency should be notified, mild self-treatable symptoms, or simply because they prefer not to make a fuss. they may therefore be incorrectly classified at least initially as "nonill." consider also that - % of the general population will have experienced some form of "upset stomach" in the last hours, regardless of exposure to the suspect item, and they may be incorrectly classified, at least initially, as "ill." to reduce the "false negatives" and "false positives" that are expected with self-reporting, the investigator needs to establish a working case-definition. a careful case definition categorizes people as "case" or "control" with the best accuracy possible within the time constraints and resources available. a case definition could be considered "too sensitive" if it classifies as a "case" a person who experiences: "… at least one episode of stomach cramps, nausea, vomiting, or diarrhea in the last hours." this would confuse subsequent analysis, and produce more false positives. similarly, a case definition could be considered "too specific" if it classifies as "not-ill" a person who had experienced only three episodes of diarrhea or vomiting, because they failed to satisfy a case definition requiring "…at least four episodes of vomiting or diarrhea in the last hours." should this last individual, having been declared as not fitting the case definition, be taken into the "notill" group, the error and subsequent analysis is confounded even further. a reasonable case definition therefore attempts to reduce both types of errors, and will depend upon the early indications of what the etiology may be. in the instance of a suspected salmonellosis, a case could be defined as "a person who was in good health before attending the event on monday may rd, and who experienced two or more of the following symptoms anytime up to midnight, sunday may th.: nausea, vomiting, stomach-cramps, diarrhea, headache, or fever." note that a case definition should include a place of exposure if known, a timeframe during which symptoms may have been experienced (salmonellosis has a range from to h. usually - h), and the additional footnote that the individual was not already symptomatic before the suspected "exposure." calculate the percentage of ill persons who manifest each symptom by dividing the number of persons reporting the given symptom by the number of cases ( for the example, table ) and multiplying the quotient by . the distribution of symptoms can be used to identify the most likely pathogen, and aids in requests to the laboratory for microbiological assays of samples and specimens. other symptoms (e.g., prostration, lethargy, weakness) may be included if deemed appropriate or helpful, but the six symptoms in table should always be included. headache, for instance, is associated with many viral infections (e.g., norovirus, rotavirus), but much less so with bacterial infections. fever is usually associated with an invasive bacterial infection (such as salmonellosis or campylobacteriosis), and is not usually seen in outbreaks of simple enteritis (such as with cholera). this information helps to determine whether the outbreak was caused by an agent that produced intoxication, an enteric infection, or generalized illness. in the example given, a predominantly diarrheal syndrome without much fever or headache tends to eliminate some of the viral infections (norovirus or rotavirus) or the host-adaptive/invasive serotypes of salmonella (e.g., s. dublin or s. choleraesuis). median onset time calculations may further reduce possible candidate etiologies. in historical investigations, or where no laboratory confirmation is possible, the symptom profile and onset times can sometimes predict the etiology of the outbreak within reasonable certainty. an epidemic curve (also called an onset curve or onset distribution) is a graphic illustration called a histogram that shows the distribution of the time of onset of first symptoms for all cases that are associated with the disease outbreak. paper printed with square "grid" lines will allow the investigator while on site to represent each case as a single "block." the horizontal axis is the sequence of intervals of time and date. the unit of time that defines the width of each interval depends on the characteristics of the illness under investigation. for example, intervals of days or weeks are appropriate for diseases with long incubation periods, such as cryptosporidiosis or hepatitis a. intervals of a day or half-day are appropriate for outbreaks of enterohemorrhagic e. coli strains or shigellosis, while single-hour, -h, or -h intervals will be more suitable for illnesses with shorter incubation periods, such as chemical poisonings. the vertical axis is always the actual count, or "frequency" of cases (blocks) stacked at each interval. it is often necessary to redraw the onset curve as more accurate information becomes available. if the illness is known, a rule of thumb is that the time interval used for each "block" on the x-axis should be no more than ¼ the incubation period of the disease under investigation. if the illness is not known, select an interval where the data produces a bell-shaped curve; not too flat and not too tall. construct this graph using time-of-onset data from forms c or d, employing an appropriate time scale. once all the onset times for the cases have been plotted on the histogram, determine the range as the interval between the shortest and longest incubation periods. in fig. a , the range is the day period from the th to th march. the median onset time is preferred to the mean because the latter is vulnerable to a few or even a single very small or very large value. the median on the other hand, is the midvalue of a list of all individual onset times, including duplicate entries, that are ordered in a series, from shortest to longest. if the series comprises an even number of values, the median is the mean of the two middle values. most standard reference texts on communicable diseases give onset times as median values. the mode is simply the interval having the largest number of observations. a distribution with a single "peak" is called a uni-modal distribution, while an outbreak with two peaks is called "bi-modal." subsequent modal peaks following the first may indicate either a "secondary wave" of cases or the exposure of other people at a later time. the shape of the epidemic curve helps to determine whether the initial cases originated from a single point-source exposure (such as water or food available for only part of a day), or from repeated exposures for a longer time, or even more gradual person-to-person spread. a point-source epidemic curve is characterized by a sharp rise to a peak, followed by a fall that is almost as steep ( fig. a ). an "explosive" outbreak of this type is common where a municipal water supply is the vehicle, affecting large numbers of people in a very short period of time, but without secondary cases occurring, or any evidence of onward spread within the community. propagated outbreaks are those in which the initial victims ("primary cases") manage to spread the agent to other people ("secondary cases") such as family members, patients, clients, or other contacts in crowded places through aerosols, personal contact, or contaminated water/food/utensils/surfaces, etc. propagation following a point-source exposure is demonstrated by a second increase in reported cases following the decline of the first cluster. sometimes this takes the form of a second "modal peak" separated by approximately one incubation period, but this distinction is soon lost. figure a shows no evidence of propagation; fig. b suggests that propagation may have taken place, although care must be taken to consider other explanations. in addition to ( ) true propagation, where the secondary wave can be expected to appear one incubation period after the first, secondary waves may be also explained by ( ) exposure to the same point source (e.g., food or water supply) at different, but specific times by other people; this might be a repeated offering of contaminated food or water at two or more mealtimes; ( ) a second pathogen (perhaps from the same unhygienic food or water source) which may have a different symptom profile and a different (incubation) time. slow propagation from the beginning of an outbreak with neither an obvious point-source, nor any distinctive "waves" separated by an incubation period as in fig. c , usually indicates one-at-a-time person-to-person spread through closecontact, poor personal hygiene, aerosol (e.g., influenza, or sars), or sexual transmission (e.g., hiv/aids). it can also be explained by (non-propagated) continuing exposure, for example drinking of contaminated surface water following a conflict, natural disaster or other breakdown of infrastructure. as such it is commonly associated with waterborne cholera, shigellosis, typhoid fever, or e. coli infection, and characterized by scattered cases which continue until the chain of infection is cut. slow, constant and/or intermittent exposure to persons over time to pathogenic microorganisms can also result from sewage run-off after a series of heavy rainfalls. in addition to revealing whether the outbreak was due to a single point-source, or had been spread steadily through the community by propagation in some way, another important objective in constructing the epidemic curve is to estimate the incubation period of the illness if it is not already known. with waterborne illness especially, the time of exposure may be further obscured because people usually drink water several times a day. hence, the incubation period cannot always be determined for each case, but the actual time of onset is usually available. the incubation period is the interval between exposure to food or water that is contaminated (with enough pathogens or with a sufficient concentration of toxic substances to cause illness), and the appearance of the first sign or symptom of the illness. each etiology is characterized by a typical incubation period (tables b, c, d, and g). individual onset times will vary due to immune factors, co-morbidities, the dose ingested, and other ingested materials, but the investigator can often make a rough estimate of the average incubation time by examining the aggregation of all onset times as an epidemic curve. the modal peak of a single "cluster" or distribution is the time interval in which most cases commence symptoms. in fig. a this occurs on march , and in fig. d that occurs at the double interval feb - th. where two separate modal peaks (a "bi-modal distribution") suggests secondary cases ("propagation"), then the distance between the first two modes is a good estimate of the incubation period. figure b shows about days between primary and secondary modal peaks, suggesting that the initial exposure is likely to have been days before the first mode. in fig. b this would be sometime on or near the th of the month. if the exposure point is known but the agent is not, then that estimation of the median incubation period will allow many etiologic agents to be excluded due to date of onset of symptoms: between march and april , count per day onset histogram for cases of shigellosis, march to april , illustrating slow spread through a community through either propagation (person-to-person spread via poor hygiene), or exposure by many people to a small well at different times (a non-propagated route) incubation periods that are clearly outside the range of times observed. the list of possible candidates can be further reduced by examining the symptom profile and other characteristics of the illness and suspect food or water vehicle. as time passes, the onset curve also provides an ongoing measure of the potential for propagation, and the incidence rate. all this information can be useful in deciding whether the illness in question is an infection or intoxication and thereby determining which laboratory tests should be requested (tables b, c, d, and g). note that not all water or foodborne illnesses listed in a standard reference such as the "control of communicable diseases manual" (apha ), are directly communicable person-to-person; many require a suitable substrate (food or drink) and adequate time/temperature combinations to attain sufficient numbers or the production of enough toxin to induce a pathological condition. an exposure time can sometimes be estimated from a clear, point-source, singleexposure onset distribution (fig. d ). it has no solid basis in statistics, but has sometimes been found to be useful in practice. the typical incubation periods for most foodborne and waterborne illnesses are readily available for comparison (e.g., control of communicable diseases manual, apha/cdc, ), and in this manual in tables b, c, d, and g. an incidence rate is the number of new cases of a specified disease reported during a given time period in relation to the size of the population being studied, multiplied by a constant, usually , to give percentages. thus new cases of e. coli o :h infection among the residents of a children's summer camp in july is an incidence rate of ( / ) × or ( . ) × = . % for that month. if several people have left and their state of wellness is not known, their impact should be expressed in the form of the possible range of values around the known incidence rate within the two extremes whereby they may all be well or they may all be ill. thus, where six children who were at the camp had departed around the time of the outbreak and their health is unknown, the range could be from a possible ( / ) × (or . %) if all of the six had been well, up to ( / ) × (or . %) if all had been ill. note that the "missing" six are added to the denominator only when we speculate that none were ill, whereas they are added to the numerator and denominator if we speculate that they might all have been ill. in this example, the overall incidence rate would be reported as " %, with a possible range from . to . %." depending upon the situation, it is often necessary to identify exposures which may be related to the illness, and to calculate an incidence rate for each such exposure. for example, in the summer camp illustration (above) it might be useful to enquire if gender, age, location, or some other attribute or activity increased the risk of becoming ill. this should not be interpreted automatically as implying that a given exposure would be associated with the outcome in any situation. by hypothesizing that gender was linked to the risk of illness, for example, does not imply that males are more vulnerable to the illness (the outcome) than females, but it can indicate that gender may have been related to the exposure, which in turn increased the risk. as an illustration, suppose that boys at the camp had been swimming, while the girls had gone on a nature walk. the boys may subsequently show increased incidence rate for e. coli o infection, not because they are more susceptible, but because of their activity. every proportion or percentage statement should be made with clear reference to the appropriate denominator used. incidence rates of waterborne illnesses are usually similar for both sexes at any given age group in the population, but differences in activities or dietary habits or susceptibility due to age or underlying health status can change the risk. the very young, the elderly and the immunocompromised can be at more susceptible, while in some instances, previously exposed populations may have developed a measure of immunity to an infection that may still cause more serious illness among visitors. a further complication arises where the "at-risk" population (perhaps residents at an institution, summer camp, or on a cruise ship) have generally consumed all the food and water for the extended period. careful interviewing of affected persons often uncovers one or more persons who entered the subject community shortly before becoming ill or who visited the community for a short time and became ill after leaving it. example: the south-west part of the county is served by three semi-private water systems. thirty cases of waterborne illness are being investigated in the area. when the numbers of cases are displayed for each water system, no clear grouping or clustering is evident, although the delta supply appears associated with about % more cases than the other two (table a) . however, when the analysis introduces the total population of persons who depend upon each water system (as denominators), a different scenario emerges. the incidence rates (expressed here as percentages) now allow a meaningful comparison (table b ). we can see that persons using the bravo system have roughly five times the risk of illness compared to people who are served by the other two systems. the use of the denominator is vital for most calculations. caution: numerous other factors may also explain the outbreak and these should be carefully examined. for example, the households using the bravo supply may be closer to an unhygienic corner store, drink from a cross-connected public water fountain, or their children may swim in a more polluted pond than the other communities. potential sources such as these should be eliminated before the water supply is announced as the source of the illness. sometimes a spot map may be useful in showing the location of the residence of each case, while on a larger scale, the rates of illness can be shown using city blocks, census tracts, townships, or other subdivisions. different colors or symbols to indicate cases with different time of onset periods (such as weeks) may help to support a hypothesis as to where contamination was introduced, inasmuch as the earliest cases tend to cluster around the point where contamination first occurred. the weakness of this procedure is that if the exposure had been at a restaurant, workplace, or school, plotting the relationship to the location of the home would not be useful. the investigation of waterborne or foodborne disease outbreaks invariably commences after both exposure and illness have happened. this is the classical "casecontrol" study, where a group of ill people ("cases") and a group of non-ill people ("controls") are compared in terms of their exposures. to measure the association between exposure and illness, the data are typically displayed in a × contingency table. table compares cases and controls in terms of their exposure to a suspected factor "x." the table is ready for analysis using odds ratio, as well as the chi-square or the fisher's exact tests where appropriate. one × table will be used for each possible exposure (e.g., each beverage, food, or other material). as many cases as can be identified and contacted, and as many non-ill people (controls) as can be found, should be interviewed as quickly as possible about their exposures to each suspect item. fading memories, the chance of obtaining stillavailable samples of implicated food or water, and the opportunity to obtain fecal specimens before the patient is started on the ubiquitous broad-spectrum antibiotics are all reasons for rapid response. case and control numbers do not have to be the same; the calculations compare ratios so equal numbers in each group are not needed. generally a : to : ratio of cases to controls is perfectly adequate. as interview data from cases and controls are accumulated, leading to formation of hypotheses about the source of the illness, human resources should be deployed in two additional essential tasks: ( ) tracking down and confirming the hypothesized source of the illness, and ( ) promptly issuing warnings to all affected groups about the possible risks from any source that is still accessible, with assurances that further bulletins will be issued as soon as confirmation is received. this precautionary principle is a vital component of risk management in modern public health. waiting for absolute confirmation before releasing warnings and advisories should not be an option in the twentyfirst century. the principle holds that while false alarms can be quickly forgiven, further illness should be avoided at the highest priority. failure to heed this step has contributed to needless suffering and severe damage to reputation, trust and credibility. a case-control approach is necessary because unlike the data in table b , we rarely have full information about all the attendees, and therefore the true incidence/attack rate is not available. very rarely, when all cases and controls are available for interview, we would have the true incidence rates for ill and for not-ill persons and this would allow a "retrospective cohort study" to be carried out. under such circumstances, and using table as an illustration, we could state that of persons exposed to item x, persons ( . %) had become ill compared to ill of not exposed ( . %). where incubation times are longer than a day, there is increasing likelihood that only a small proportion of the non-ill people will be available for interview, and on many occasions, not even all the ill persons can be contacted. the point here is that the investigator is usually working with sub-sets of the true cases and controls. the controls in table and possibly even the cases may have been drawn from larger groups, and therefore we cannot state the incidence rate, for example, as: "… of exposed were ill," because the " " had been artificially assembled, and may not resemble the true incidence at all. we can, however, use exposure rates, for example: "…of ill persons, ( . %) had been exposed to x," and, "…of who were not-ill, only ( . %) had been exposed to x." the overwhelming majority of waterborne or foodborne illness investigations are run as "case-control" studies (or to be more accurate, "case-comparison" studies, as very little true "controlling" is accomplished during the selection of the comparison group). a broadcasted invitation to all who might have been exposed to come forward, typically results in few non-ill persons volunteering information, because nonaffected individuals believe they have little if anything to contribute. this reduces validity even further, and more active recruitment is often necessary to convince them that their information is just as essential for the investigation as are the contributions from the less-fortunate attendees. let us examine a waterborne illness suspected as being due to the consumption of water bottled from a certain spring. you have found people who meet the case definition of illness, and another non-ill people in same neighborhood who report no symptoms at all, and who will be your controls. in table we display the data and ask the question: "is drinking this water related to the risk of illness?" whenever a × table appears, the first step is to calculate the odds ratio (or). an odds ratio tells us if there is a relationship (where or ≠ ), and the strength of the relationship (the or value itself). it also clearly indicates the direction of the relationship: was drinking or not-drinking the dangerous activity? this is easily determined by finding the dominant pair from (a × d) or (b × c). in the example above, (a × d) is greater, so cell "a" links the row "drank" with the column "ill," while cell "d" links "not drink" with "not-ill." this assumption is not as obvious as it may seem; the cause of the illness may have been whatever "other" thing was drunk by those who avoided spring water! it is important to clarify that the odds ratio yields the strength of the association, not the statistical significance. most or values (where many exposures are being assessed) will be close to . (= "no association"), while an or clearly exceeding . signifies a positive association between this exposure and illness, such that this exposure increased the risk of illness. an or < . is protective, meaning that exposure to this factor reduced the risk of illness compared to the other group. for example, an or of . means that the exposed group had only one-quarter the risk of illness compared to the non-exposed group. the non-exposed group therefore has a greater risk (by a factor of ). while this protective effect can be due to true therapeutic protection (e.g., exposure to antibiotics when you have an infection), it is frequently explained as "statistical" protection. as an example, consider an outbreak where everyone consumed only one of two possible types of bottled water. one source, a, contains a pathogen, and b does not. if the ill people were found to be five times more likely to have consumed type a (odds ratio = . ) then the not-ill would have five times the rate of consuming water b, and only one-fifth of the rate of choosing water a (or = . or %). this can also be read as the risk of illness for the non-exposed group, or as the risk of staying well by the exposed group. an easier way to interpret an or less than is to place over the or to reveal a value greater than , but clearly labeled "protective." the or is a ratio between numbers, and therefore not sensitive to the actual numbers of people in individual cells, an important consideration when the numbers of subjects are relatively small. this is illustrated by the common question: "how large does an odds ratio have to be before it is considered evidence of an association?" a popular response is "at least . ," but this must be considered with extreme caution. for instance, with very large studies, an or of . (barely more than . ) can be shown to be very highly significant statistically (p = . ), whereas in a small-n study, an or of even . may not achieve statistical significance. the odds ratio is certainly a useful measurement, and should always be used when a × table is encountered. it will quickly advise you ( ) that there is an association, ( ) the strength of that association, and ( ) the direction of the association, none of which are specifically measured by a test of statistical significance. unfortunately, it is not reliable with small cell sizes, and is unable to answer the question: "how likely is it that these numbers could happen just due to chance?" for this, we need to test the statistical significance. the best advice is to use the or (or relative risk where appropriate) together with a test of statistical significance. most online statistic calculators or laptop versions of sas, spss, epiinfo, etc. will give a selection of useful statistics (odds ratio, relative risk, several versions of chi-square, and fisher's exact test, both onetailed and two-tailed.) in keeping with all scientific enquiry, we begin by advancing the notion (the "null hypothesis") that there is no association between the exposure and the illness, and attempt to support that notion. if insufficient evidence is found to support the null hypothesis, we reject it and cautiously consider that an association may exist between the two variables. this can be described as a statistically significant association. two methods of testing are presented: the chi-square test (written χ and pronounced "ky"-square) for most × (or larger) tables, and the fisher's exact test (only for × tables) when chi-square is not valid due to the numbers in the cells being too small (the following sections give advice about this decision). the original data value in each cell we call the observed, or "o" value, and these are compared with the numbers that you would expect ("e" values) if there were no relationship at all; that is, if the variables were not related, and the data were arranged purely by chance (as stated by the null hypothesis). the chi-square test measures the difference between the o and e values. if they are close, we have to accept that there may be no real relationship; if far apart, we can reject the null hypothesis and cautiously declare that exposure and illness were probably related. numerous online statistical calculators can be used to yield ors, rrs, and chisquare values. if you prefer to do the calculation by hand, construct a × table as shown, with "observed" data, marginal totals, and the grand total. the expected "e" values are found from: to make sure the chi-square analysis is appropriate for your table, you must be sure that all "e" numbers are more than . the quickest way is to first calculate for the cell with the smallest e value; (this will be the cell with the smaller column total and the smaller row total.) in table , the smallest e value will be cell "d," and this is calculated as ( × )/ = . . as this is > , all other e values will be greater than this, so chi-sq. is valid. (note that the smallest e value did not coincide with the smallest o value). . for all four cells the sum (χ ) is: . + . + . + . = . an online statistical calculator will give you this same chi-square (χ ) value. to verify by hand whether the o vs. e difference is statistically significant, compare your chi-sq. value (for a × table only) with . . if your calculated value exceeds . , then this is unlikely to be due to chance, and thus you can begin to believe that this exposure did influence the risk of illness, and you can reject the null hypothesis. statistical results usually include a probability (p) statement. this is the probability that the null hypothesis ("no association") is correct. the . value is the minimum needed for statistical significance, where the p is less than % (p < . ). recall that the p is the probability that no real association exists between exposure and illness. by convention, if p > . (more than %) then the relationship is declared not statistically significant. where p = . or < . , then the relationship is statistically significant. the smaller the p value, (p < . , p < . , etc.) the more confidence you have that a relationship really exists. other critical values exist for assessing calculated chi-sq. values, from larger tables than × , and at more extreme levels of significance. a further chi-square calculation is shown as an appendix. where a table greater than × is found to have more than % of the cells with an e value less than , chi-square is not valid. the solution is to collapse either columns or rows to allow the e values to increase. for example in table a , two cells out of six ( %) have e values less than , but if "high dose" is merged with "medium dose" the resulting increase in observed (o) cell sizes is also reflected in greater e values, while the table becomes × (table b) . some outcome information has been lost, but the chi-square analysis can proceed. if, after trying to collapse cells and/or rows, a × table is reached still with an e value < , the fisher's test is indicated. this procedure is reserved only for × tables where one or more expected (e) values is less than , making the chi-square test not valid. our example is taken from an investigation into an outbreak of shigellosis presumed to be due to water from a well ( table ). the odds ratio has been calculated as ( × )/( × ) = . , meaning ill persons were six times as likely to have drunk well water compared to non-ill the "!" denotes a factorial, meaning that number multiplied by the next smallest number, and so on down to . (e.g.: ! = ) p persons. an attempt to use chi-square is prevented by at least one e value less than of . (cells c and d both show e values as ( × )/ = . ). the starting null hypothesis is "that no relationship exists." this is not quite the end of the calculation however. the goal is to calculate the probability of the original data occurring plus all more extreme probabilities. the original data have to be adjusted by increasing the "dominant" pair of cells by + and the others by − , while leaving the margin totals the same (table ) . because no zero has yet appeared in the matrix of cells, we continue to increase the "dominant" pair by + and obtain a zero. the next calculation is the last. (by convention, ! and ! = ) (table ) . no reference table is required. the total calculated probability ( . ) is exactly the probability that the null hypothesis ("that there was no relationship"), is correct: . %. by convention, for a result to be significant statistically, that probability (p) must be less than % (< . ), so in this instance we are not able to reject the null hypothesis and must conclude that the relationship could have occurred by chance alone more than % of the time. the odds ratio of . is explained as the number of times more likely it was for a shigellosis victim to have drunk well water than for a non-ill person. this increased risk would normally be impressive, but because of the small number of persons in the study, it has been found not to pass the test of statistical significance. a basic write up of the results might read: "a relationship exists between drinking well water and developing shigellosis. a shigellosis patient is six times more likely to have drunk water from the well compared to a non-ill person. this relationship is not statistically significant, however, and could have occurred by chance alone more than % of the time. the null hypothesis of no-relationship cannot be rejected." [ df, p > . , or: . , not statistically significant.] with the odds ratio (or) calculated for all the suspected exposures, and the chisquare test or fisher's exact test calculated for the strongest of these, all the results can be displayed in a composite table. earlier protocols for the investigation of waterborne and foodborne diseases encouraged the use of the "factor-specific attack rate table" (for example the "foodspecific attack rate table"), but where only a "convenience sample" of controls and cases are available, we are unable to derive valid incidence/attack rates. investigators are discouraged from using it as it may produce misleading results. the exposurerate table for cases and controls is preferred in all case-control studies, and compares the rates of exposure to each factor between both the ill and non-ill people. table displays six exposure factors from a hypothetical outbreak involving water contamination. exposure rates are calculated from both cases and controls. the "spring-water" data that we used for the odds ratio calculation example in table appears as the first exposure in table . the column headed "differences in exposure rates" subtracts the exposure rate among the non-ill from the exposure rate among the ill. [use: exp. rate (cases) minus exp. rate (controls), keeping the signs correct]. you are looking for a large positive difference to indicate the most likely culprit. the spring water shows the largest positive difference at + %. the odds ratio of . supports this, again the largest value, indicating that ill persons were times more likely to have drunk the spring water compared to non-ill persons in this group. hence both the large positive difference in exposure rates and the large odds ratio point to the spring water being the likely source of the illness, and it is certainly the strongest association between illness and any of the exposures shown. the chi-square value has also been added ( . ), as well as the associated p value. taken together, the evidence clearly points to this factor as the culprit. in those less-common circumstances in which all the ill and non-ill persons can be contacted for interview, the table can be rearranged to show attack rates (incidence rates) for each of the suspect factors (table ) . here, the column of "differences" shows the attack rate (exposed) minus the attack rate (non-exposed), (i e − i n ), and again a large positive difference will point to the culprit. this measure is called the attributable risk and for the spring water example we obtain + %, the largest value of all the risk factors. also, because of the availability of valid attack rates (incidence rates), the true relative risk (rr) is available, and can be substituted the p (probability) value is a statement of statistical significance. it indicates the probability that there is no relationship between the factor and the illness. so as the number becomes very small (as shown here) we can be increasingly satisfied that a real relationship does exist. see the statistical significance section for the correct way to calculate this ns = not significant the ("true") relative risk (also called the risk ratio) is only used when we have the true incidence data. it is calculated as the attack rate (exposed) over the attack rate (non-exposed), or i the p (probability) value is a statement of statistical significance. it indicates the probability that there is no relationship between the factor and the illness. so as the number becomes very small (as shown here) we can be increasingly satisfied that a real relationship does exist. see the statistical significance section for the correct way to calculate this ns = not significant for the odds ratio. the data in table shows the same data as table rearranged for easy comparison. values in the column of "differences" are not the same as for table and of course the relative risks are not the same as the odds ratios in table , but both of these results still point clearly to the suspect exposure. in both analyses, spring water is clearly the factor most strongly associated with illness. it is important to note that in both tables a high rate (exposure-or attack-) on the left side taken by itself is meaningless until it is compared with the rate from the right side of the table. this again underscores the importance of gathering complete data from the non-ill as well as the ill. an interesting phenomenon is visible in the second factor listed (soft drink). the or is listed as . , which is "protective," meaning that this factor is strongly associated with not being ill. it is the equivalent of or equal to . ( / . ), and the chi-square is seen as quite large, although not enough for statistical significance. this is sometimes seen where two factors are "in competition" with each other; if everyone had drunk one item, and the spring water was the contaminated source, then those drinking the other item would be strongly "protected" because they did not drink the spring water, and this shows clearly. all other factors have or values very close to . . most attack rate tables record some persons who did not ingest the suspect vehicle but who nevertheless became ill. plausible explanations are that (a) some people forget which beverages or foods they ingested; (b) some might have become ill from other causes; or (c) some may have exhibited symptoms with a psychosomatic rather than a physiological origin. it is also not unusual for the table to include some persons who ingested contaminated water or food but did not become ill. plausible explanations are that (a) organisms or toxins are not always evenly distributed in water or food and consequently some persons ingest small doses or perhaps none at all; (b) some persons eat or drink larger quantities than others; (c) some are more resistant to illness than others, and (d) some will not admit that they became ill, or fail to report it. whichever table is used, the combined totals for cases (ill) and controls (well) are fixed and should not change for each exposure unless there are "missing" responses from interviewees. while some procedure manuals include confidence limits around both rr and or, this may be omitted here as the use of the chi-square test or fishers exact test yield the statistical significance for both tables. illness caused by ingestion of waterborne toxicants and some pathogenic organisms can be dose-related in that the risk of developing symptoms, and their severity varies with the quantity ingested. where the suspect water is no longer available (for example, the well may have been quickly super-chlorinated to break the chain of infection before samples were taken), attack rates can be based on the amount of water usually drunk per day by each person. this is easily extended to other non-treated sources of water such as ice cubes, water-reconstituted fruit juices, and flavored crystals. a comparison of attack rates at various water intake levels may provide valuable evidence that water is, or is not, the vehicle responsible for the outbreak. for an example, see table . here, the entire group was people and we have interviewed them all, so we are justified in calculating the attack/incidence rates: in this example, the attack rate increased as the consumption of water increased, which suggests that the illness was directly related to water and the agent it contained. this is a trend established from the group as a whole, and an individual's experience may vary with factors such as (a) preferences of water ingestion, (b) intermittent contamination, (c) unequal distribution of the contaminant, or (d) varying susceptibility of individuals. these data can be compared with rates from persons who ingested no water, but only hot tea, hot coffee, soups, and/or other safe sources of liquids. if unheated water was indeed the vehicle, and the agent was a living biological agent, these persons should have attack rates showing no increase in risk of illness. (outbreaks from a toxic agent may be unaffected by chlorination, boiling, and some types of filtering.) the data can be displayed in a contingency table as follows for analysis using chi-square procedure (table ) . for this example, chi-square equals . and if calculated by computer or online, p will be shown as p = . . reference to form j confirms that for a × table ( df), the calculated chi-square ( . ) exceeds the critical value for statistical significance at the . level ( . ), allowing us to claim statistical significance at p < . . odds ratios are normally associated only with × tables, but here, the or can usefully be calculated on selective cells or groups of cells as long as you clearly explain the selection process. for instance, persons who were ill were . times more likely to have drunk three or more glasses of water per day compared to those who were well. for this calculation we collapse cells into a × table and crossmultiply: (a + c) × (f + h)/(b + d) × (e + g) = ( ) × ( )/( ) × ( ) = / = . . alternatively, because we have all people involved, we can compare the attack rates (ar) for each intake level, and observe the increasing attack rate as the intake increases: for five or more glasses/day, ar: %, for - /day, ar: %, for - / day, ar: %, and for < /day, ar: %. we might summarize as follows: "there was a relationship observed between the quantity of water consumed each day and the risk of illness. the incidence rate increased with the quantity consumed from % for < glasses/day to % for five or more glasses/day. this relationship is statistically significant. the null hypothesis of no association can be rejected." , df, p < . ] water as a vehicle can deliver pathogenic organisms in many ways beyond simply drinking a glass of water, or using a drinking fountain. investigators should be sure to ask about the preparation of ice-cubes, the mixing of fruit flavored crystal drinks, reconstituting concentrated orange juice, brushing and rinsing teeth, and washing hands, utensils, or containers. swimming or playing in muddy pools or even swimming pools have caused waterborne poliomyelitis, and naegleriasis, while swimming in saltwater inlets have allowed inadvertent infections from vibrio parahaemolyticus and v. vulnificus. unwashed plastic jugs containing poster paint residue have caused rapid illness when drink crystals are reconstituted in them, while refillable plastic containers and bottles have a long history of contamination from biological and chemical agents. in the late s, an increase of viral ear, nose, and throat infections among people who were using parkland next to a river was hypothesized to have been due to people waterskiing on the river and creating an aerosol. the river was the receiving body for effluent from a water treatment plant upstream. record all laboratory results on form i, laboratory results summary. compare epidemiological and statistical results with on-site observations, laboratory results and the information summarized on form i. the agent responsible for the outbreak can be determined by (a) isolating and identifying pathogenic microorganisms from patients, (b) identifying the same strain and/or pfge pattern or genetic sequence of pathogen in specimens from several patients, (c) finding toxic substances or substances indicative of pathological responses in specimens, or (d) demonstrating increased antibody titer in sera from patients whose clinical features are consistent with those known to be produced by the agent. when implicating the water as a likely (or presumptive) vehicle of transmission, ideally identification of a pathogen in samples of suspect water will correspond to the one found in clinical specimens from ill persons or that produces an illness that is compatible with the incubation period and clinical features of the ill who were exposed to the water. for organisms that are common in the gastrointestinal tract or that have multiple strains, compare strains isolated from ill persons with strains isolated from the suspected water. additionally, specific microbial markers (e.g., serotype, phage type, immunoblotting, plasmid analysis, antibiotic resistance patterns, restriction endonuclease analysis, nucleotide sequence analysis) or chemical markers identified by chromatography or spectrophotometry can be used for this purpose. for confirmation of water-related transmission, the same pathogen strains should be found in both the ill persons and the epidemiologically implicated water. however, due to the period of time that may have passed after the outbreak was actually reported, and to methodological issues, such as the need for concentrating pathogens in water samples, it is often unlikely that the outbreak-associated pathogen will be found in the water samples. laboratories frequently test water samples for indicator organisms, such as fecal coliforms, escherichia coli, or enterococci, rather than pathogens. the finding of these bacteria in high densities in the water may indicate contamination (from a fecal source) and implicate the water was a possible vehicle. however, the finding of increased indicators in water samples alone is insufficient evidence to confirm the water as the source of an outbreak. the probable source of contamination or the situation that allowed contamination to reach and survive in a water supply (e.g., water supply not disinfected or inadequately disinfected, inadequately filtered, or upstream to sewage or agricultural discharges; cross connection between sewerage and drinking water pipes; well improperly constructed; nearby septic tank system; or livestock in water supply) can often be identified, but the etiologic agent in the water may never be found. success in finding the etiologic agent is most likely where (a) the incubation period of the illness is short, (b) the agent is stable in water and the system is static, or (c) large amounts of the agent are being continually added to the water supply. try to recover and identify the specific agent whenever a water supply is suspected to be the vehicle of transmission, even if finding the etiologic agent is likely to be difficult and not considered practical for routine monitoring of water supplies. if water samples do not reveal a likely causal agent, clinical data as well as time, place, and person associations can cast strong suspicion on a water supply, particularly if indicator organisms are found in the water. tests other than those for pathogens, however, are frequently used to evaluate water supplies on a routine basis. organoleptic tests attempt to evaluate the total effect of all compounds present in water that can be measured by the senses of taste, smell, or sight. results cannot be expressed in terms of specific compounds present, and the measured qualities are usually a result of a mixture of compounds. these tests are often empirical and arbitrary, but changes in the physical qualities of water (such as ph, turbidity, color, odor, or taste) can indicate abnormalities of the water. outbreaks have occurred, however, when turbidity readings have met present standards and when water appeared and tasted good. chemical examination of water is useful for (a) detecting pollution (especially from industrial wastes and pesticides), (b) determining effectiveness of treatment processes, (c) evaluating the previous history of the water, (d) determining hardness, and (e) detecting the presence of specific toxins. results are usually expressed in milligrams per liter (mg/l = ppm, parts per million), or micrograms per liter (μg/l = ppb, parts per billion). historically, acute water-related outbreaks seldom involve chemical substances, so chemical tests are not requested routinely unless either (a) circumstances indicate possible chemical contamination or (b) clinical symptoms suggest chemical poisoning. flowing water in a distribution system can be monitored to determine chlorine residual. free available residual chlorine refers to that portion of the total residual chlorine remaining in chlorinated water at the end of a specific contact period that will react chemically and biologically as hypochlorous acid or hypochlorite ion. the reaction is influenced by ph and temperature. total or combined residual chlorine refers to chlorine that has reacted with ammonia or other substances and is not available for further reactions, as well as the free available chlorine. a chlorine demand exists in a chlorinated water until a free available residual is produced. a free available chlorine residual, e.g., mg/l ( ppm) or higher, maintained throughout the distribution system of a community supply is an indicator of safety from enteric bacteria but not necessarily from pseudomonads, viruses or parasites. outbreaks have occurred when chlorine residue levels have met present standards. analyses for microbial indicator organisms provide information on the microbiological quality of water and guidance as to its safety for consumption or contact. indicator organisms are easier to test for than pathogenic organisms, and some serve as a surrogate measure of fecal contamination in water. the absence of indicator organisms in the water, however, does not guarantee water safety; numerous outbreaks of water-related disease have occurred from water in which no indicator organisms were detected. evaluation of the safety of water should be based upon a combination of results of (a) an on-site study to identify sources and modes of contamination and means by which contaminants survived treatment and (b) appropriate laboratory analyses. microbiological results should be compatible with observed sources of contamination and/or treatment failures found during the investigation. although all natural waters contain bacteria, the number and kind vary greatly in different places and under different climatic and environmental conditions. the number of bacteria isolated and reported, however, often represents only a fraction of the total number present, for several reasons. colonies seen on agar plates develop from either single organisms or clusters or chains of organisms. heterotrophic bacteria represent only those that can use organic matter and grow at the selected temperature ( - °c) within - hours under aerobic/microaerophilic conditions in/on a defined medium when the standard test (spread plate, membrane filter or pour plate) is used. the hpc may also be done using different media under different incubation times/conditions. (higher counts are usually found when the longer incubation periods are used.) also, certain microorganisms are unable to grow aerobically either in or on the medium used. because of these variables, the terms total plate count (tpc), standard plate count (spc) and aerobic plate counts (apc) should not be used. hpcs serve as an index of changing sanitary conditions. in general, counts of good-quality well water are fewer than - colonies per ml. densities in surface water are higher, but quite variable, depending on water temperature, sources of pollution, amount of organic matter present, and soil that washes into the water. the sources of pathogens, toxic substances, or fecal contamination may not increase the hpc of a surface water sample as much as washings from soil. nevertheless, marked changes in the number or kind of microorganisms should be viewed with concern, at least until the reason for the change is discovered. heterotrophic plate counts greater than /ml and some specific antagonistic species may interfere with the growth or recovery of pathogenic or indicator organisms. some heterotrophic species are opportunistic pathogens that may pose a health threat to immunocompromised persons. the coliform group of bacteria comprises those from non-fecal environmental sources, and those from animal and human intestines, including escherichia coli. the environmental species of non-fecal bacteria are found in soil, on fruits, leaves, and grains, and in run-off water, especially after heavy rains. some of these species are capable of surviving in water longer than e. coli. furthermore, some coliform strains and can multiply on decaying vegetation in water, in biofilms in pipelines, or on pump packings, washers, and similar materials. therefore, finding coliforms may not be indicative of fecal contamination, although most water utilities have standards for coliforms in water. fecal coliforms are present in large densities in all human and animal feces, normally much higher than pathogens which are typically only present in infected persons and normally at lower levels. as such, high populations of fecal coliforms can indicate recent sewage pollution of water, but are not always indicative of pathogens present, particularly viruses and parasites. none of the coliform group, however persists as long as most viral or protozoan pathogens in water, and indicator bacteria described below (fecal streptococci and clostridium perfringens). typical chlorination or ozonation of water inactivates coliform bacteria. presence of the coliform group or even a high population of coliform bacteria is not proof that a treated water supply contains pathogens. however, coliforms can provide a warning that either the water treatment was inadequate or contamination occurred after treatment, and that some pathogens may be present. as mentioned above, under some conditions, pathogens may be present where there are few or no coliforms. furthermore, unlike coliforms, many parasites and viruses are resistant to normal levels of disinfectants. coliforms have little or no correlation with the presence of parasitic protozoa or pathogenic viruses. the standard test for the coliform group may be carried out by a membrane filtration technique, a multiple-tube fermentation technique (presumptive test, confirmed test, or completed test), or a presence-absence test. results of the membrane filtration technique are reported as colony forming units (cfu) per ml of water. results of the multiple-tube fermentation technique are reported as the most probable number (mpn) per ml of water. this is a statistical estimation of the total number present, but the actual number can fall within a considerable range. counts derived from these two methods are not necessarily the same, but they have the same sanitary significance. false-negative or false-positive results can also occur with the membrane filtration technique because of interfering background growth of nonfecal microorganisms. results of the presumptive test of the multiple-tube fermentation technique can be misleading, because other microorganisms frequently found in water also produce gas in laboratory media, and may thereby give false-positive results. also, especially in waters containing a large number of microorganisms, some coliforms present may produce gas slowly, leading to false-negative results. the presence of coliform bacteria is corroborated by means of the second phase of the multiple-tube fermentation technique, known as the confirmed test. positive results are usually considered confirmation of the presence of coliforms. a third phase of this test, known as a completed test, further ensures the correct identification of coliform bacteria. a simple modification of the coliform test is to analyze for the presence or absence of coliforms in a -ml drinking water sample. the "presence-absence (p/a) coliform test" allows for simple examination of a larger number of samples. when a positive sample is detected, it is advisable to measure coliform densities in repeat samples by one of the other methods to determine the magnitude of the contamination. thermotolerant coliform (fecal coliform). coliform bacteria will frequently grow at a relatively high temperature, . °c, unlike species or strains normally encountered in the environment, which usually have an optimal temperature near °c. this thermotolerant characteristic has been used in an attempt to separate coliform bacteria into those of so-called fecal and non-fecal origin. this test may provide better indication of fecal contamination than the coliform test, but it is however, unreliable. positive results are not proof that either organisms of fecal origin or pathogens are present. the number of thermotolerant coliforms is considerably lower than the number of total coliforms in contaminated water; therefore, the test is less sensitive for testing treated drinking water. furthermore, escherichia coli o :h , which has been implicated as causing water-related illness, does not grow well at . °c. escherichia coli. e. coli is common in feces of human beings, other mammals, and birds. it can also be found to grow naturally in the environment, specifically in tropical waters. comprised of the larger coliform group, its detection in water is a more definitive indicator of fecal contamination, compared to total or fecal coliforms. however, a positive test result does not identify if the fecal source is human or nonhuman. rather, the finding of e. coli in water serves as an indicator that fecal matter reached the water and provides a warning, but not proof, that pathogenic organisms may also be present. it should be noted that some strains of e. coli are pathogenic (see table b ). simple commercial p/a and quantitative tests have been developed to detect the presence of total coliforms and e. coli in hours by observing color changes and fluorescence of the media under daylight and uv light. such tests may be useful for field evaluation of microbiological water quality. another group of organisms, collectively known as fecal streptococci, is also used as an indicator of fecal contamination. enterococci (enterococcus faecalis, enterococcus faecium) are particularly used for testing recreational waters. like coliforms, enterococci are normal inhabitants of the intestinal tract of human beings and other animals. in human feces, they occur in considerably lower numbers than e. coli. some members of the group, such as e. faecalis, subsp. liquefaciens, however, have been associated with vegetation, insects, and certain types of soils. enterococci generally survive longer than coliforms in fresh water, and therefore the source of contamination may be distant in either time or place from the site where samples were obtained. their resistance is, however, less than that of clostridium perfringens, enteric viruses, and parasites. like e. coli, simple commercial p/a and quantitative tests have been developed to detect the presence of enterococci in hours by observing color changes under uv light, which may be useful for field evaluation of microbiological water quality. clostridium perfringens (sulfite reducing clostridia). c. perfringens is also of fecal origin, but it occurs in feces in much lower densities than e. coli and can also be found in soils. being a spore-former, it can survive for long durations in soil and water, and persist when all other bacteria of fecal origin have disappeared. therefore, it is a useful indicator of remote or intermittent contamination in wells that are not frequently examined by the coliform test; but, it is not, by itself, evidence of recent contamination. chlorine, in the concentration typically used in water treatment, does not inactivate all spores; and thus c. perfringens is not valuable in assessing the efficiency of chlorination for bacterial vegetative cells. its long persistence and its resistance to chlorine make this organism a potential indicator for viral and parasitic organisms that have similar resistance and disinfectant susceptibility. coliphage. coliphages, which are viruses that infect e. coli, are simpler to detect and enumerate, compared to other viruses, and are generally associated with fecal contamination. they have been considered as possible indicators of treatment effectiveness for human enteric viruses. coliphages are categorized into two groups: the somatic phages, which enter e. coli via the cell wall and the male-specific phages, which enter e. coli through the sex pili. the somatic and male-specific phages are common in sewage and the feces of human beings and other animals, but in lower densities than the common fecal indicator bacteria, fecal coliforms, e. coli, and enterococci. some strains appear to be more resistant to chemical disinfection than water-related pathogens or indicator bacteria. be aware of local standards for water distribution systems, private water systems, and recreational water. although drinking water standards, such as the total number of coliforms allowed in a water sample, vary from jurisdiction-to-jurisdiction, it is generally agreed that any fecal contamination (e.g. fecal coliforms, escherichia coli) render the water unacceptable for human consumption and may close down recreational bathing waters. there are numerous pathogens that can be transmitted by water, many of which are also able to cause respiratory symptoms, in addition to the classical gastrointestinal symptoms. for a comprehensive summary of waterborne pathogens see "american waterworks association manual of water supply practices, m waterborne pathogens, nd edition ( ) ." for several reasons, analyses for pathogens are not usually conducted during routine water testing, or are only conducted by specialized laboratories. first, tests for pathogens are pathogen-specific, expensive, and often difficult to perform because they may require specialized trained personnel. secondly, the etiologic agent of the outbreak is often unknown at the time of analysis; hence, many analyses would have to be done blindly. thirdly, pathogens are not always recovered because they are heterogeneously dispersed and diluted in the environment, and their numbers decline in water over time. as a result, they may be absent or present in low densities by the time samples are collected following an outbreak. fourthly, recovery efficiencies are often poor because microorganisms are stressed by disinfectants or the method is sensitive to interferences from the source waters environment, hence not easily recovered by routine methods. additionally, recovery efficiencies for viruses and protozoa may be poor because of the interferences of substances within the sample matrix with method reagents (concentrating l of water down to μl will also concentrate inhibitory chemicals and substances). finally, the time required for isolation and identification is often long, and the number of samples is usually too small to allow the investigator to have much statistical confidence in the results when pathogens are not found. negative results should be reported as "not detected" because they do not ensure that the water sampled was not the source of the pathogen. procedures used for many bacterial pathogens are qualitative because enrichment procedures are used. quantitative procedures (e.g., mpn) require considerable work and are less reliable than those used for coliforms because small populations may be present, and these may be unevenly distributed. despite these difficulties, pathogens that cause a syndrome similar to the one being investigated should be sought. see tables b, c, and d, for descriptions of the disease syndrome associated with the pathogens described in the following material. finding the same pathogen in specimens from patients and in water samples confirms water as a vehicle. summarize investigative data in a narrative report. describe in this report situations that led to contamination of the water and survival of etiologic agents up to the time of consumption. include all events that contributed to the outbreak to guide control and preventive measures. compare your data with the listings in table g (guidelines for confirmation of waterborne outbreaks) and table h (guidelines for confirmation of water responsible for illness), and criteria for confirmation of vehicle responsible for waterborne illness before assigning the etiologic agent and the vehicle. outbreak confirmation is based on (a) time, place, person associations, (b) recovery of etiologic agents from clinical specimens from cases and samples of water, and (c) identification of sources and modes of contamination and means by which pathogens or toxic substances survived treatment. all three of these, however, might not be found in any one investigation. complete form k (waterborne illness summary report). attach the narrative and the epidemic curve. also attach form d (case history summaries: water/laboratory data), all applicable parts of forms g, forms h, form i, and other data that will provide supplemental information to reviewers. send this report through administrative channels to the appropriate agency responsible for waterborne disease surveillance. make the final report as complete as possible, so that the agency can accurately interpret the results and develop a meaningful waterborne disease data bank. in the interest of continuing cooperation, give all participants in the investigation due credit and send each a copy of the report. also, send copies of the report through administrative channels to agencies (a) that have jurisdiction over the implicated water, (b) that initiated the alert, and (c) that participated in the investigation. those concerned with water sources, treatment and recreation, as well as with public health, should make every effort to ensure the complete investigation and reporting of waterborne diseases. without reliable, complete information, trends in waterborne disease incidence and causal factors of the disease are difficult to determine. good surveillance is essential for detecting and evaluating new waterborne disease hazards. the primary purposes of a waterborne disease investigation are to identify the cause, establish control measures, and take actions to prevent future illness. prudence may require some action before all the hypotheses regarding the water supply involved and the source of contamination are confirmed. frequently the local health authority will issue a boil water advisory if a microorganism is suspected to have contaminated the water. refer to "possible precautionary control actions" section for a discussion of these precautionary control measures. if these measures have not already been considered, consider them now. once control measures have been implemented, continue to monitor for disease to evaluate whether the measures were effective. in a waterborne event in sydney, australia (see box ) sydney water severely overestimated levels of cryptosporidium and giardia present in the water raising public alarm. boil water advisories were announced and rescinded several times. however, it is better to announce boil-water advisories than to have thousands ill, as has happened in the past, such as the cryptosporidium outbreak in milwaukee in . deficiencies in treatment must be corrected and defective parts of distribution systems must be repaired, beginning with those that either contributed to or had a high potential for contributing to the outbreak. the effectiveness of these efforts will be directly related to the thoroughness of the investigation. document the source and the manner of contamination and survival of the etiologic agent through the water treatment process. provide clear documentation of contributory factors, so that preventive measures taken will be specific to the problem. if previous sanitary surveys have revealed, or if subsequent ones reveal, that conditions which contributed to the outbreak are widespread, initiate a training and education program. these programs can be developed for water treatment plant or recreational water operators and employees, engineers, homeowners, or other appropriate groups. impress upon them the importance of proper construction and operation of facilities and proper protection, treatment, storage, and distribution of water. follow up with periodic inspections and surveys and verify by sampling, as appropriate, to determine whether faulty conditions have been corrected or allowed to be reintroduced. legal action may be necessary to ensure compliance with official standards and accepted sanitary practices. formulate solutions to problems found during outbreak investigations, and incorporate these into regulations for drinking, agricultural, industrial, domestic, and recreational waters. inform the public, through mass media and other means available to your agency, of hazardous conditions that can affect their water supply, but do so only after hypotheses are confirmed. the public must be told of any potential or actual harm that may result from ingesting or contacting contaminated water and must also be informed of measures that they can take and that official agencies are taking to correct these conditions. the water supply and recreational water facilities must be verified periodically to determine whether critical processes are being monitored and operated within limits of appropriate public health standards (see box , the walkerton outbreak). most waterborne illnesses are preventable, but prevention requires that those in the water treatment industry and in health and water-protection regulatory agencies be constantly vigilant to ensure that the hazards are understood and that questionable water treatment or delivery system construction or practices are avoided. examples investigation guidelines and investigative forms iafp manual, "procedures to investigate waterborne illness, rd ed"; copies of form c; one dozen copies each of forms e and f; two copies of form d and all parts of form g, epi-info software (cdc, atlanta). water sample bottles (bottles for chlorinated water should contain enough sodium thiosulfate to provide a concentration of mg of this compound per l of sample), plastic bags (whirl-pak ® type), ml, -l and -gal sized jars and jugs. sterile and wrapped sampling implements moore swabs (compact pads of gauze made from strips cm [ ft] by cm [ in.] tied in the center with a long, stout twine or wire-for sewer drain, stream or pipeline samples), fiberglass-epoxy bacterial filter cartridge, . μm; tongs, scoop or similar utensils for collecting ice. specimencollecting equipment (for human specimens from cases and controls) sterile containers (with lids) for stool specimens, bottles containing a bacterial preservative and transport medium, mailer tubes or styrofoam box, sterile swabs, rectal swab units, tubes of bacterial transport medium, stool preservative medium for parasites, phlebotomy supplies for blood specimens. kits for testing chemical disinfectants and ph dpd (n,n-diethyl-p-phenylenediamine) chlorine comparator with color disc for chlorine ( . ppm) and chlorine test papers; field-type ph meter or ph comparator with color disc or ph test papers; applicable ph indicator solutions and dpd reagent solution; dissolved oxygen testing unit. dye tracing study equipment fluorescein (yellow-green fluorescent) dye in powder form ( packages containing g each), in tablet form ( tablets), or in liquid form (prepared by mixing g in l of water); fluorometer; filters (primary and secondary) for use with fluorometer; sample holder for continuous sampling or individual sampling; fluorometer recorder. disinfectant and neutralizer . % w/v solution of calcium hypochlorite or . % household liquid bleach; % w/v sodium thiosulfate. virus filtration equipment for viruses and parasites b large plastic container for storing water sample prior to concentration; portable electric or gasoline powered water pump with quick disconnect brass or stainless steel plumbing adapters or hose couplings; two filter holders for -in. water filter cartridges fitted for adapters or couplings; portable water meter fitted for adapters or couplings; four lengths of fiber-reinforced garden hose fitted with adapters or couplings; one length of a strong-walled supply hose fitted with adapters or couplings; -in. prefilter ( μm nominal porosity wound polypropylene yarn filter with hollow perforated stainless steel core) cartridge filter; -in. virus absorbing filter pleated . μm porosity nylon membrane type (positively charged) for waters of ph values up to . , or pleated . μm porosity glass fiber membrane type (positively charged) for waters of ph value of . or lower (e.g., virosorb, -mds, amf/cuno meriden, pleated, . μm, glass filter); ml sterile, ph , % beef extract solution in gal wide-mouth screw capped autoclavable polypropylene container for each sample to be collected; stands to support filter holders during filtration; for parasites -in. polypropylene yarn-wound cartridge filter, . μm porosity (e.g., micro wynd ii™, amf/cuno; meriden, ct. . μm normal porosity). (continued) laptop or tablet, with software; thermocouples of varying lengths with either recording potentiometer, data logger, or digital indicator; devices to take samples below surface and sediment samples; chemical smoke kit and/ or micromanometer; occupational safety and health administration (osha) or equivalent approved respirator; sterile plastic gloves; plastic container liners for ice; waterproof marking pens; waterproof test tube rack; pencils, note pad; roll of adhesive or masking tape; labels; waterproof cardboard tags with eyelets and wire ties; flashlight; matches; test tube rack to fit tubes used; insulated chest or styrofoam container; packing material; camera with flash; spare batteries for all equipment; % ethyl alcohol; propane torch; refrigerant in plastic bags, liquid in cans, rubber or heavy plastic bags that can be filled with water and frozen; heavy-duty bags for ice, "canned ice," or cold-packs (blue ice). a assemble a kit to be kept in the agency responsible for investigating waterborne illness. it should include at least ten water sample bottles; ten -l, or gal jars or jugs; ten specimen collection containers or devices; and one each of the following supporting equipment and sterilizing equipment. date of sterilization should be marked. periodic resterilization or replacement of sterile supplies, media, or transport media is required to maintain the kit in a ready-to-use condition b similar equipment for sampling for either viruses or parasites may be available from national water, environmental, or health agencies how much to collect two rectal swabs or swabs of fresh stool from ten ill persons; samples from ten controls also can be submitted. whole stool is preferred if nonbacterial stool testing considered a fresh stool sample from ten ill persons; samples from ten controls can also be submitted. to enhanced detection, three stool specimens per patient can be collected > h apart as much stool sample as possible from ten ill persons (a minimum of ml of stool sample from each); samples also can be obtained from ten controls. a fresh urine sample ( ml) from ten ill persons; samples from ten controls also can be submitted. label each specimen in a waterproof manner and put the samples in sealed, waterproof containers (i.e., plastic bags). batch the collection and send in overnight mail to arrive at the testing laboratory on a weekday during business hours unless other arrangements have been made in advance with the testing laboratory. contact the testing laboratory before shipping, and give the testing laboratory as much advance notice as possible so that testing can begin as soon as samples arrive. when etiology is unclear and syndrome is nonspecific, all four types of specimens may be appropriate to collect c for more detailed instructions on how to collect specimens for specific parasites, please go to http://www.cdc.gov and search the website of key words d for more detailed instructions on how to collect specimens for viral testing, please go to http://www.cdc.gov and search the website of key words e the containers have been tested for the presence of the chemical of interest prior to use f unused specimen collection containers that have been brought in to the field and subjected to the same field conditions as the used containers. these containers are then tested for trace amounts of the chemical of interest table e (continued) isolation of agent from ill persons and from water and laboratory criteria for confirming etiologic agent as stated in table g . combination of on-site investigation, statistical evidence and laboratory analysis. (see entries below) presumptive vehicle on-site investigation demonstrating source and mode of contamination of water and survival of etiologic agent in water. also, desirable to have laboratory isolations from water of etiologic agent that causes syndrome similar to that observed during the investigation and other supportive epidemiologic data. if so, this might provide sufficient evidence for confirmation. or p-value for water < . when other epidemiologic data supports water hypothesis. also, desirable to have either laboratory isolations from water or on-site investigation that demonstrates source and mode of contamination and survival of treatment that supports the hypothesis. if so, this might provide sufficient evidence for confirmation. or odds ratio or relative risk for water greater than and the lower limit of the % confidence level greater than when other epidemiologic data supports the water vehicle hypothesis. also, desirable to have either laboratory isolations from water or on-site investigation that demonstrates source and mode of contamination and survival of treatment that supports the hypothesis. if so, this might provide sufficient evidence for confirmation. for calculation of data when all persons who may have been exposed to the suspect vehicle (but not all persons will have ingested every beverage/food) have been identified and interviewed (retrospective cohort) form j : chi-sq. analysis can be easily completed using on-line calculators or statistics programs such as epi-info. however, to confirm the result or to do the whole thing yourself, here are the steps: ill well totals step : create a x table as shown with observed data (o values) and marginal totals step : calculate odds ratio: (axd)/(bxc) = ( x )/( x )= . step . . chi-sq. (all four cells) = . + . + . + . = . step : compare your calculated chi-sq. value with the critical value to determine significance (table ) : begin at column p < . … your calculated chi-square value must meet or exceed the critical value to be considered statistically significant at that level. if you fail to meet or exceed the minimal value for p < . , the result is p > . , and the relationship is declared "not significant". step : summarize: exposure was related to illness. ill persons were eight times more likely to have been exposed to this factor than non-ill persons. this relationship is statistically significant. the probability of these data occurring by chance alone is less than . %. reject the null hypothesis of "no association." [odds ratio: . , % cl: . < or < . , chi-sq.: . , df, p < . ] form j : fisher's exact test can be easily completed using on-line calculators or statistics programs such as epi-info. however, to confirm the result or to do the whole thing yourself, here are the steps. ill well totals step step : calculate the probability directly... here, cell 'a' has smallest e value at . p = ! x ! x ! x ! = . ! x ! x ! x ! x ! step : the final probability (p) is the sum of all probabilities (in this case p + p + p ) or approximately . . step : summarize: exposure was related to illness. ill people were almost times more likely to have been exposed to this factor compared to non-ill people. the relationship is statistically significant. the probability of these data occurring by chance alone is less than . % (< . ). reject null hypothesis of "no association". [odds ratio: . , % cl: . < or < . , p = . ]. . when deciding which cells to increase by + , always multiply (a) × (d) and compare with (b) × (c). increase each cell of the pair with the higher product and decrease each cell of the pair with the smaller product, while keeping all marginal totals unchanged. . the final p is an "exact" p (probability) and may be reported as such (p = . ). in this example, it is also < . of course, and can be reported in this way if preferred. . the fisher's test is used when the chi-square test is invalid due to any "e" values < in a × table. in all other circumstances, chi-sq. is an excellent approximation for the fe test. . if original data include a zero in one of the cells, you will calculate only one p value. (the o.r. will be reported as "undefinable" but the direction of the effect will be very clear). . this p is calculated for a one-tailed fe test. it is adequate for this application. two-tailed fe test will require further calculation. . should a relationship not meet the critical value for significance (that is, p > . ), it is described as "not statistically significant". note that a relationship may be observed, but this result is telling you that it could have occurred by chance alone more than % of time if you were to repeat the analysis. that may still require further investigation, but from a statistical standpoint, it cannot be claimed as a statistically significant relationship. water treatment plant design, fifth edition american waterworks association. manual of water supply practices, m waterborne pathogens, second edition the flint water crisis confirms that u.s. drinking water needs improved risk management produce safety-what's going on here? national environmental health association neha-cert ep global health -division of parasitic diseases and malaria page last reviewed surveillance reports for recreational water-associated disease & outbreaks. page last reviewed on internalization and dissemination of human norovirus and animal caliciviruses in hydroponically grown romaine lettuce environmental protection agency technical guidance manual lt eswtr disinfection profiling and benchmarking environmental protection agency guidance manual for the compliance with filtration and disinfection requirements public water systems using surface water sources environmental protection agency. manual of individual and non-public water supply systems. epa no. epa no. water treatment manual: disinfection. environmental protection agency health canada risk analysis of the walkerton drinking water crisis algal bloom-associated disease outbreaks among users of freshwater lakes-us microbial resistance to disinfectants: mechanisms and significance. environ health perspect safe drinking water: lessons from recent outbreaks in affluent nations risk management for assuring safe drinking water ensuring safe drinking water: learning from frontline experience with contamination. american water works association cyanotoxin management and human health risk mitigation in recreational waters a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply private water supplies: technical manual good practice guide to the operation of drinking water supply systems for the management of microbial risk: research project two cases of keratosis follicularis squamosa (dohi) caused by swimsuit friction part two report of the walkerton inquiry: a strategy for safe drinking water public health inspector's guide to the principles and practices of environmental microbiology acute otitis externa: an update collect representative distribution system samples guidance on water supply and sanitation in extreme weather events. who regional office for europe effect of irrigation method on transmission to and persistence of escherichia coli o :h on lettuce transmission of escherichia coli o :h from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization acknowledgments the committee and association thank and cite the following persons for chloramines refer to all forms of chloramine. the ct values may be assumed to achieve greater than . % inactivation of viruses only if chlorine is added and mixed in the water before addition of ammonia. if this condition is not met, the system must demonstrate by on-site studies or other information that it is achieving at least this much inactivation of viruses key: cord- -blam f c authors: levade, inès; saber, morteza m.; midani, firas; chowdhury, fahima; khan, ashraful i.; begum, yasmin a.; ryan, edward t.; david, lawrence a.; calderwood, stephen b.; harris, jason b.; larocque, regina c.; qadri, firdausi; shapiro, b. jesse; weil, ana a. title: predicting vibrio cholerae infection and disease severity using metagenomics in a prospective cohort study date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: blam f c background susceptibility to vibrio cholerae infection is impacted by blood group, age, and pre-existing immunity, but these factors only partially explain who becomes infected. a recent study used s rrna amplicon sequencing to quantify the composition of the gut microbiome and identify predictive biomarkers of infection with limited taxonomic resolution. methods to achieve increased resolution of gut microbial factors associated with v. cholerae susceptibility and identify predictors of symptomatic disease, we applied deep shotgun metagenomic sequencing to a cohort of household contacts of patients with cholera. results using machine learning, we resolved species, strains, gene families, and cellular pathways in the microbiome at the time of exposure to v. cholerae to identify markers that predict infection and symptoms. use of metagenomic features improved the precision and accuracy of prediction relative to s sequencing. we also predicted disease severity, although with greater uncertainty than our infection prediction. species within the genera prevotella and bifidobacterium predicted protection from infection, and genes involved in iron metabolism also correlated with protection. conclusion our results highlight the power of metagenomics to predict disease outcomes and suggest specific species and genes for experimental testing to investigate mechanisms of microbiome-related protection from cholera. summary cholera infection and disease severity can be predicted using metagenomic sequencing of the gut microbiome pre-infection in a prospective cohort, and suggests potentially protective bacterial species and genes. cholera is an acute diarrheal disease caused by vibrio cholerae. it is a major public health threat worldwide that continues to cause major outbreaks, such as in yemen, where over . million cases have been reported since ( , ). transmission of v. cholerae between household members commonly occurs through shared sources of contaminated food or water or through fecal-oral spread ( , ). the clinical spectrum of disease ranges from asymptomatic infection to severe watery diarrhea that can lead to fatal dehydration ( ). host factors such as age, innate immune factors, blood group, or prior acquired immunity partially explain why some people are more susceptible to v. cholerae infection than others, but a substantial amount of the variation remains unexplained ( ). the gut bacterial community can protect against enteropathogenic infections ( ) here we used shotgun metagenomics to analyze an expanded prospective cohort of persons exposed to v. cholerae in bangladesh. our metagenomic analysis yielded improved outcome predictions compared to s rrna sequencing, and identified bacterial genes associated with remaining uninfected after exposure to v. cholerae. we are also able to predict disease severity among infected contacts, albeit with lower power and precision than susceptibility. finally, we highlight several microbiome-encoded metabolic functions associated with protection against cholera. sample collection, clinical outcomes and metagenomic sequencing as described in ( ), household contacts were enrolled within hours of the presentation of an index cholera case at the icddr,b (international center for diarrheal disease research, bangladesh) dhaka hospital. index patients with severe acute diarrhea, a stool culture positive for v. cholerae, age between and years old, and no major comorbid conditions were recruited ( , ). a clinical assessment of symptoms in household contacts was conducted daily for the -day period after presentation of the index case, and repeated on day . we collected demographic information, rectal swabs, and blood samples for abo typing and vibriocidal antibody titers as described in the supplementary methods. during the observation period, contacts were determined to be infected if any rectal swab culture was positive for v. cholerae and/or if the contact developed diarrhea and a -fold increase in vibriocidal titer during the follow-up period ( , ). contacts with positive rectal swabs developing watery diarrhea were categorized as symptomatic and those without diarrhea were considered asymptomatic ( figure ). v. cholerae positive contacts (by culture or deep s amplicon sequencing ( )) at the time of enrollment were excluded, in addition to contacts who reported antibiotic use or diarrhea during the week prior to enrollment. dna extraction was performed for the selected samples and used for shotgun metagenomics sequencing. details on cohorts, sequencing methods and sample processing are described in supplementary methods. we used metaphlan (version . ) ( ) for taxonomic profiling and humann ( ) to profile cellular pathways (from metacyc) and gene families (identified using the pfam database). for identification of biomarkers of susceptibility and disease severity, we used metaml ( ) to apply a random forests (rf) classifier on species, pathways and gene-family relative abundances, as well as strain-specific markers presence/absence. models constructed using each of these features types were compared to a random dataset with shuffled labels, and to a model constructed with clinical/demographic data, using two-sample, two-sided t-tests over replicate cross-validation ( ). we used a stratified -fold cross validation approach, splitting our dataset into validation and training sets ( / and / of samples, respectively) with the same infected:uninfected ratio. we used an embedded feature selection strategy to identify the most metagenomic sequencing of the gut microbiome in household contacts exposed to v. cohort, upon which we base the majority of our analyses. we also performed exploratory analyses on the expanded cohort to determine the potential for predictive models to be generalized to larger samples. we used the shotgun metagenomic dna sequence reads from these samples to characterize four features of the microbiome: ) relative abundances of microbial species, ) the presence/absence of sub-species-level strains, ) metabolic pathway relative abundances, and ) gene family relative abundances (table ) (table s ) . however, such high auc values should be treated with caution because the models can be overfit when a supervised feature selection step is applied on the same data used to train the model ( ). because we did not have a fully independent validation cohort (e.g. from another continent) to test our model, we decided to use the features selected from the midani cohort to make predictions on the expanded dataset. using the same features selected from the midani training dataset, we made predictions on the expanded cohort and achieved aucs between . and . for prediction of infection using the four types of features (table s ) . again, because the expanded cohort partly overlaps with the midani cohort, and includes some repeated samples from the same individuals over time, these results could also be prone to overfitting, but they demonstrate the potential for generalized predictions. finally, we repeated the rf analysis using all features in the expanded dataset, whichh increased predictive performance relative to the original midani cohort (figure s ). once again, genes and pathways outperformed species and strains according to all metrics, with auc reaching ~ . using cellular pathways ( table ) . this improvement in the expanded cohort also highlights the importance of using larger, more balanced datasets as input to predictive models. to put the metagenomic predictions in context, we compared their predictive power and accuracy to clinical and demographic factors ( table s a) . three of these factors (age, baseline vibriocidal antibodies and blood group) are known to impact susceptibility to v. cholerae infection ( , ) and we used them to train rf models ( table s ) . as expected, contacts who became infected tended to be younger and have lower baseline antibody titers than those who remained uninfected ( table s b) , but these small differences were not sufficient to train a significantly predictive model. an rf model trained on the seven clinical and demographic factors did not perform better than a random model with shuffled labels (auc= . , p= . ; to predict symptomatic disease among infected individuals (figure ) , we divided samples into uninfected, symptomatic and asymptomatic groups and again applied the rf approach. we used the f score as a performance metric since it is well suited for uneven class distributions in our uninfected/symptomatic/asymptomatic comparison. applied to the midani cohort, this model predicted outcomes significantly better than random (shuffled labels) using species, strains or pathway data, but not gene families ( table ; see table s for p- values). however, the f scores for the symptomatic/asymptomatic predictions were systematically lower (mean scores ranging from of . to . ) than for the infected/uninfected prediction (means ranging from . to . ). using the expanded cohort, the scores were improved only slightly ( table ) . these results suggest that disease severity is predictable in principle, but with greater uncertainty than the infection outcome. (figures a, s a and s a) . (figure s ), but the overlap was poorer for the uninfected/symptomatic/asymptomatic prediction ( figure s ). this is consistent with the difficulty of predicting disease severity. in general, the most important species were selected by the model because of differences in relative abundance at baseline among uninfected/symptomatic/asymptomatic outcomes ( figure s , s ) . in rare cases, species presence/absence information was predictive. for example, ruminococcus gnavus, is absent (near or below limit of detection) in most of the individuals who become infected, but present in many (but not all) of those who remain uninfected ( figure s ) . thus, there is no single, strong predictor of infection outcomes, but rather a probabilistic combination of many species, each of relatively modest predictive value. table s we also identified gene families in the gut microbiome of persons who remained uninfected during follow-up (figures s and s ) , with some of the top gene families involved in dna repair, transmembrane transporter activity, iron metabolism (indicated with asterisks in figure ) , and genes of unknown function (table s ) . long-chain fatty acid biosynthesis pathways (e.g. cis-vaccenate, gondoate and stearate) were associated with individuals who remained uninfected, while amino acid biosynthesis and catabolic pathways were associated with individuals who developed infection (figures s and s , table s ). we identified three iron- related genes associated with remaining uninfected: ( ) the ferric uptake regulator fur, a major regulator of iron homeostasis, ( ) thioredoxin, a redox protein involved in adaptation to oxidative and iron-deficiency stress, and ( ) the tonb/exbd/tolqr system, a ferric chelate transporter ( - ). in individuals who became infected but asymptomatic, two genes involved in the conversion of riboflavin into catalytically active cofactors, the riboflavin kinase and the fad synthetase, were found as the first and the third most discriminant features (figure , table s ). we next asked which taxa in the microbiome likely encoded these genes. in some cases, specific taxonomic groups corresponded to discrete gene functions. for example, several iron metabolism-related gene families tend to be encoded by prevotella genomes (figure s ) . in other cases, the major contributors to protective gene families were unclassified (figures and s here; see table s for pathways. the contributions of each genus to encoding these pathways are shown as stacked colors within each bar, linearly scaled within the total. see table s for the complete list of pathways the gut microbiome is a potentially modifiable host risk factor for cholera, and identification of specific genes and strains correlated with susceptibility is needed for experimental testing to understand the mechanisms of observed correlations. compared to a previous study using a single marker gene, shotgun metagenomics provides this degree of resolution, potentially to the species and strain level, and to the level of individual genes and cellular functions. we found that gene families in the gut microbiome at the time of exposure to v. cholerae were more predictive of susceptibility compared to taxonomic or clinical and demographic information. selecting a subset of the most informative features further improved predictions, but using these selected features may lead to overfitting. this suggests an upper limit to predictive power that requires validation in larger, independent cohorts. all three bifidobacterium species associated with contacts that developed infection were also associated with asymptomatic rather than symptomatic disease, and prior work on this genus supports several hypotheses for this relationship. first, bifidobacteria are known to produce the scfa acetate that can protect against enteric infection in mice ( , , ). scfas are also known to inhibit cholera toxin-related chloride secretion in the mouse gut, reducing water and sodium loss, and have been observed to increase cholera toxin-specific antibody responses ( - ). bifidobacteria are also major producers of lactate, a metabolite that has been shown to impair v. the authors declare that there are no conflicts of interest. updated global burden of cholera in endemic countries cholera epidemic in yemen, - : an analysis of surveillance data defining endemic cholera at three levels of spatiotemporal resolution within bangladesh clinical outcomes in household contacts of patients with cholera in bangladesh cholera transmission: the host, pathogen and bacteriophage dynamic susceptibility to vibrio cholerae infection in a cohort of household contacts of patients with cholera in bangladesh roles of the intestinal microbiota in pathogen protection members of the human gut microbiota involved in recovery from vibrio cholerae infection bile salts modulate the mucin-activated type vi secretion system of pandemic vibrio cholerae a single gene of a commensal microbe affects host susceptibility to enteric infection probiotic strains detect and suppress cholera in mice anti-biofilm properties of the fecal probiotic lactobacilli against vibrio spp commensal-derived metabolites govern vibrio cholerae pathogenesis in host intestine gut microbial succession follows acute secretory diarrhea in humans human gut microbiota predicts susceptibility to vibrio cholerae infection metaphlan for enhanced metagenomic taxonomic profiling species-level functional profiling of metagenomes and metatranscriptomes machine learning meta-analysis of large metagenomic datasets: tools and biological insights fillat mf. the fur (ferric uptake regulator) superfamily: diversity and versatility of key transcriptional regulators thioredoxin h (trxh) contributes to adversity adaptation and pathogenicity of edwardsiella piscicida tonb-dependent transporters: regulation, structure, and function the prevotella copri complex comprises four distinct clades underrepresented in westernized populations dietary fiber-induced improvement in glucose metabolism is associated with increased abundance of prevotella utilisation of mucin glycans by the human gut symbiont ruminococcus gnavus is strain-dependent mucin glycan foraging in the human gut microbiome regulation of bacterial pathogenesis by intestinal short-chain fatty acids from dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites formation of propionate and butyrate by the human colonic microbiota. environmental microbiology butyrate protects mice from clostridium difficile-induced colitis through an hif- -dependent mechanism bifidobacteria can protect from enteropathogenic infection through production of acetate potential beneficial effects of butyrate in intestinal and extraintestinal diseases facilitate mucosal adjuvant activity of cholera toxin through gpr . the journal of immunology short-chain fatty acids inhibit fluid and electrolyte loss induced by cholera toxin in proximal colon of rabbit in vivo overview on the bacterial iron- cholera toxin promotes pathogen acquisition of host- derived nutrients transcriptomics reveals a cross-modulatory effect between riboflavin and iron and outlines responses to riboflavin biosynthesis and uptake in vibrio cholerae the human gut microbiome: from association to modulation supplementary tables s -s are available at: https://figshare.com/articles/supplementary_tables_-_levade_et_al_ / key: cord- -zz h authors: barral-arca, ruth; gómez-carballa, alberto; cebey-lópez, miriam; bello, xabier; martinón-torres, federico; salas, antonio title: a meta-analysis of multiple whole blood gene expression data unveils a diagnostic host-response transcript signature for respiratory syncytial virus date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: zz h respiratory syncytial virus (rsv) is one of the major causes of acute lower respiratory tract infection worldwide. the absence of a commercial vaccine and the limited success of current therapeutic strategies against rsv make further research necessary. we used a multi-cohort analysis approach to investigate host transcriptomic biomarkers and shed further light on the molecular mechanism underlying rsv-host interactions. we meta-analyzed seven transcriptome microarray studies from the public gene expression omnibus (geo) repository containing a total of samples, including rsv, healthy controls, coronaviruses, enteroviruses, influenzas, rhinoviruses, and coinfections, from both adult and pediatric patients. we identified > genes differentially expressed when comparing the transcriptomes of rsv-infected patients against healthy controls. functional enrichment analysis showed several pathways significantly altered, including immunologic response mediated by rsv infection, pattern recognition receptors, cell cycle, and olfactory signaling. in addition, we identified a minimal -transcript host signature specific for rsv infection by comparing transcriptomic profiles against other respiratory viruses. these multi-genic signatures might help to investigate future drug targets against rsv infection. respiratory syncytial virus (rsv) is the main cause of lower respiratory tract infections in early life [ ] , and one of the major causes of morbidity and mortality, especially in children younger than six months [ ] . re-infections are also common in patients older than two years and adults, although usually less severe [ , ] . patients with premature birth, chronic lung disease, congenital heart disease, and immunodeficiency are more likely to suffer from severe forms of rsv and may require hospitalization [ ] ; however, most of the children hospitalized with severe rsv disease lack known identifiable risk factors [ , ] . it has been shown that host genetic susceptibility to the disease may play a key role in the different pathogenesis produced by rsv in children [ , ] , but many aspects of the host-pathogen interaction a principal component analysis (pca ) of the combined data (rsv samples and healthy controls) was carried out to detect batch effects in the eight datasets analyzed. this analysis revealed that the transcriptomes of the rsv cases in the gse form a separate cluster when compared to the rest of the samples ( figure s ). this anomalous pattern could be explained by different severities, strand or sampling time points with regard to the other samples, or undesirable batch effects. this dataset was therefore eliminated from further analysis. thus, the meta-analysis was finally conducted on a total of seven datasets containing transcriptomic data from samples: rsv, coronaviruses, enteroviruses, influenzas, rhinoviruses, coinfections, and healthy controls; from both adult and pediatric patients (table s ) . a new pca was conducted with the remaining seven datasets and based on the most deg (figure ). the first principal component (pc ; accounting for~ % of the variation), in combination with pc (accounting for~ % of the variation), separates all the samples in two clusters according to their disease status. it is remarkable that, while the healthy controls appear more scattered in the plot, rsv patients are more tightly clustered, suggesting that the rsv alters the transcriptome in a more specific manner. it is also noteworthy that the cases of co-infection included in the meta-analysis do not generate a pattern of sub-clustering in the pca plot. by comparing the transcriptomes of rsv-infected patients to healthy controls, we identified , genes differentially expressed ( over-expressed and under-expressed; fdr of %; table s ). this differential gene expression pattern explains the clustering pattern observed in the pca ( figure ). our functional enrichment study using reactome showed that the deg were enriched in the host cell-cycle pathway in rsv infected patients when compared to controls (r-hsa- , adjusted p-value = . × − ; r-hsa- , adjusted p-value = . × − ; r-hsa- , adjusted p-value = . × − ) (table s ) . among the deg between rsv-infected patients and healthy controls, a number of them affecting immunologic-related pathways are strongly over-represented: (i) immune system (r-hsa- ; adjusted p-value = . × − ), (ii) cytokine signaling in immune system (r-hsa- : stat , stat , mmp- ; adjusted p-value = . × − ), (iii) innate immune system (r-hsa- ; adjusted p-value = . × − ) and (iv) adaptive immune system pathways (r-hsa- ; adjusted p-value = . × − ). we also observed an over-representation of deg belonging to pathways related to (i) interleukins signaling (r-hsa- ; adjusted p-value = . × − ) such as interleukin- family signaling (r-hsa- ; adjusted p-value = . × − ) and (ii) interleukin- signaling (r-hsa- ; adjusted p-value = . × − ; table s ). rsv infection also causes an overrepresentation deg related to the interferon signaling pathway (r-hsa- ; table s ) and interferon-inducible genes such as eif ak , mx and ifitm . over-representation is also observed by comparing the transcriptomes of rsv-infected patients to healthy controls, we identified , genes differentially expressed ( over-expressed and under-expressed; fdr of %; table s ). this differential gene expression pattern explains the clustering pattern observed in the pca ( figure ). our functional enrichment study using reactome showed that the deg were enriched in the host cell-cycle pathway in rsv infected patients when compared to controls (r-hsa- , adjusted p-value = . × − ; r-hsa- , adjusted p-value = . × − ; r-hsa- , adjusted p-value = . × − ) (table s ) . among the deg between rsv-infected patients and healthy controls, a number of them affecting immunologic-related pathways are strongly over-represented: (i) immune system (r-hsa- ; adjusted p-value = . × − ), (ii) cytokine signaling in immune system (r-hsa- : stat , stat , mmp- ; adjusted p-value = . × − ), (iii) innate immune system (r-hsa- ; adjusted p-value = . × − ) and (iv) adaptive immune system pathways (r-hsa- ; adjusted p-value = . × − ). we also observed an over-representation of deg belonging to pathways related to (i) interleukins signaling (r-hsa- ; adjusted p-value = . × − ) such as interleukin- family signaling (r-hsa- ; adjusted p-value = . × − ) and (ii) interleukin- signaling (r-hsa- ; adjusted p-value = . × − ; table s ). rsv infection also causes an over-representation deg related to the interferon signaling pathway (r-hsa- ; table s ) and interferon-inducible genes such as eif ak , mx and ifitm . over-representation is also observed in essential proteins of the innate immune response such as oligoadenylate synthase (oas) family proteins and rnase l, which are responsible for rna degradation hereby blocking viral replication [ ] . in addition, other pathways related to host response to infectious disease (r-hsa- ; adjusted p-value = . × − ) and others involved in the viral infection process, such as r-hsa- (influenza infection; adjusted p-value = . × − ), r-hsa- (hiv infection; adjusted p-value = . × − ), r-hsa- (influenza viral rna transcription and replication; adjusted p-value = . × − ) and r-hsa- (viral mrna translation; adjusted p-value = . × − ), were found to be clearly enriched in rsv patients (table s ). rsv infection also provokes an over-representation of deg related to the neutrophil degranulation pathway (r-hsa- ; adjusted p-value = . × − ). rsv additionally induced over-representation of differential expressed pattern recognition receptor genes as compared to healthy controls (table s ): (i) toll-like receptors (tlr) cascades (r-hsa- ; adjusted p-value = . × − ), including tlr (r-hsa- ; adjusted p-value = . × − ), which are specialized in the recognition of conserved molecular features of different pathogens such as bacteria, viruses, fungi, and parasites; and (ii) c-type lectin receptors (r-hsa- ; adjusted p-value = . × − ), capable of sensing glycans present in viral pathogens to activate antiviral immune responses such as phagocytosis, cytokine production, antigen processing and presentation, and subsequent t cell activation. related to this, aim -like receptors (interferon inducible protein aim ; adjusted p-value = . × − ; table s ) were found to be over-expressed (table s ) . aim -like receptors can act as enzymatic complexes (inflammasomes) and are known to be activated by the presence of microbial dna within the cytosol [ ] . in response to viral and bacterial infection these inflammasomes trigger caspase- activation and subsequently induce the release of mature pro-inflammatory interleukins such as il- , contributing to the immune response to pathogens [ ] (table s ) . other enriched pathways (table s ) are the tlr cascade (r-hsa- ), which acts as co-receptor with cd (adjusted p-value = . × − ) detecting bacterial lipopolysaccharide, and other tlr -related pathways such as trif(ticam )-mediated tlr signaling (r-hsa- ) and myd -independent tlr cascade (r-hsa- ). finally, we found that the rsv infection causes an under-representation of deg related to the olfactory signaling pathway (r-hsa- ; adjusted p-value = . × − ) (table s ). to investigate a specific rsv transcriptome signature, we compared rsv expression patterns against a viral multi-cohort set following a cross-validation strategy that randomly divides the whole dataset into a training and a test set. representative samples of coronaviruses, influenzas, rhinoviruses, enteroviruses, and rsv transcriptomes were present in both the training and the test sets. the volcano plot of figure a shows the genes differentially expressed when comparing rsv-infected children to children infected by other pathogens. to investigate a specific rsv transcriptome signature, we compared rsv expression patterns against a viral multi-cohort set following a cross-validation strategy that randomly divides the whole dataset into a training and a test set. representative samples of coronaviruses, influenzas, rhinoviruses, enteroviruses, and rsv transcriptomes were present in both the training and the test sets. the volcano plot of figure a shows the genes differentially expressed when comparing rsvinfected children to children infected by other pathogens. among the most significant genes in this analysis (indicated in red in figure a ), we searched for the minimum transcriptome signature allowing to discriminate between rsv from other pathogens using the optimal gene model size according to prems algorithm. figure b shows the optimal model characterized by genes that conform to an optimal signature to distinguish rsv from other viral conditions. this -transcript signature (table ; figure a ) identified in the discovery training set distinguished rsv from other viral conditions with area under de curve (auc) of . % ( %ci: - . ), sensitivity of . % and specificity of . %. in spite of the global burden of rsv, our knowledge of how rsv interacts with the host remains incomplete. moreover, the study of the host gene expression response to rsv infection is usually restricted to specific and very homogeneous cohorts, hampering the interpretation of the results, the impact of the conclusions obtained and their translation into a molecular test that could be used in routine clinical practice. the present meta-analysis shows that rsv infection leads to global changes in the host transcriptome, affecting not only the transcriptomic machinery of the airway infected cells [ ] , but also the expression of hundreds of genes in blood cells (table s ). in particular, according to our results, rsv alters the expression of > , genes in the host compared to healthy controls, involving e.g., cell cycle and immune system genes. this host response can be differentiated to those from other viruses with only -transcripts, which might eventually facilitate its use for clinical diagnosis. when applying this signature to the test set, the auc decreases to . % ( %ci: . - . ), with a sensitivity of . % and specificity of . %. when we evaluated the performance of the model to differentiate rsv from each pathogen individually, we obtained auc values ranging from . % for coronavirus, to . % for enterovirus in the training cohort, and values ranging from . % for influenza to . % for enterovirus in the test cohort (table ; figure b ,c). in spite of the global burden of rsv, our knowledge of how rsv interacts with the host remains incomplete. moreover, the study of the host gene expression response to rsv infection is usually restricted to specific and very homogeneous cohorts, hampering the interpretation of the results, the impact of the conclusions obtained and their translation into a molecular test that could be used in routine clinical practice. the present meta-analysis shows that rsv infection leads to global changes in the host transcriptome, affecting not only the transcriptomic machinery of the airway infected cells [ ] , but also the expression of hundreds of genes in blood cells (table s ). in particular, according to our results, rsv alters the expression of > , genes in the host compared to healthy controls, involving e.g., cell cycle and immune system genes. this host response can be differentiated to those from other viruses with only -transcripts, which might eventually facilitate its use for clinical diagnosis. in mammals, the cell cycle is governed by a complex molecular machinery. the data indicate that patients infected with rsv have an over-representation of differentially expressed cell cycle-related genes, a well-known strategy employed by many other viruses to facilitate their replication [ , ] . as expected, we observed deg and pathways related to the immunological response that are particularly relevant in the context of rsv infection, e.g., cytokines, which are small proteins released by cells that are known to play a major role in interactions and communications between cells. rsv infection causes a significant alteration of cytokine pathways and related pathways (adjusted p-value = . × − ; table s ), a finding that is in agreement with other authors, indicating that host genetic variation in cytokines predisposes to suffering complications during rsv infection [ ] . although more studies are needed to confirm this hypothesis, overall, these results indicate that measuring cytokines might help identify children at high risk of suffering rsv complications; this would allow starting treatments in earlier stages of the disease. also, among the genes that are over-represented during rsv infection are the stat family members, which act as transcription activators in response to cytokines. for instance, the absence of stat resulted in airway dysfunction in knockout mice infected with rsv [ ] , and variation at stat has been associated with rsv susceptibility in preterm children [ ] . matrix metallopeptidases (mmps) are proteins mostly known for degrading extracellular matrix proteins, but these are also known to be involved in other processes such as cytokine inactivation [ ] . experiments in mice carried out by kong et al. [ ] indicated that rsv infection raises mmp- levels, whereas the reduction of mmp- resulted in decreased viral replication, suggesting that mmp- may be a potential therapeutic target for rsv disease. our results are in line with these findings as we found this gene up-regulated, supporting the hypothesis that mmp- could be a promising drug target. like other authors, we found up-regulated interferon-inducible genes such as eif ak [ ] , which according to previous results plays a role in rsv susceptibility and immunological response. it has been described that mrna level of this gene (and expression of tlr ) depends at least partially on the patient ethnicity, suggesting that the transcriptional response of individuals may be affected by the populational background [ ] [ ] [ ] . another interferon-induced gene we found up-regulated was ifitm , which, according to zhang et al. [ ] , inhibits rsv infection interfering with the viral entry and replication processes. remarkably, our infected patients expressed as almost % more interferon-induced gtp-binding protein mx gene (log fc = . ) than the healthy controls. this finding is interesting as it has been argued that polymorphisms in mx predispose to severe form of rsv in infants [ ] . we also found an up-regulation of genes belonging to the innate immune response. for instance, oas and oas are responsible for the activation of ribonuclease l, which is part of the innate immune defense, during viral infection [ ] . ribonuclease l is an interferon ifn-induced ribonuclease that, when activated, destroys all single-stranded rnas within the cell (cellular and viral) including rrnas [ ] . the destruction of all rna within the cell is its last attempt to fight back against a virus before the onset of apoptosis [ ] . rsv infection also induces an over-representation of deg related to the neutrophil degranulation pathway; this pathway was found to be related to the immune response to rsv [ ] and other viral respiratory diseases [ ] . it has been shown that neutrophils degranulate into the airway in response to rsv, pointing to a local innate response to the infection. neutrophils degranulation implies the release of antimicrobial substances that may contribute to the control of commensal bacteria in the upper respiratory tract [ ] . according to our meta-analysis, five pattern recognition receptors (cd , tlr , tlr , tlr and tlr ) are overexpressed during rsv infection. variants at these genes might be associated with the development of rsv bronchiolitis in different human populations, including israel, finland, argentina, japan, and greece [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in , kurt-jones et al. [ ] reported that rsv persisted longer in the respiratory organs of infected tlr -deficient mice in comparison to controls, and suggested that therapies that target the expression of tlrs could be useful to fight rsv infections [ ] . moreover, tlr polymorphisms asp gly and thr ile seem to be associated with an enhanced risk of developing severe rsv bronchiolitis [ ] . furthermore, zhou et al. [ ] indicated that the tlr signaling pathway, in conjunction with myd up-regulation, could be responsible for the activation of immune responses to rsv infection in airway epithelial cells [ ] . our results are in agreement with their observations, as we detected the up-regulation of both genes in the blood of rsv-infected patients when compared to healthy controls (table s ) . the up-regulation of the tlr cascade is particularly interesting since this receptor recognizes viral dsrna, ultimately stimulating the production on type i interferons, a family of cytokines that regulate immune response to viral and other intracellular infections [ ] . the over-representation in patients of c-type lectin receptors (clrs) pathway (r-hsa- ; table s ) is also remarkable as it has been described that the detection of viral glycans by these receptors help to fight viral infections. up-regulation of clrs activates antiviral immune responses such as antigen processing and presentation, t cell activation and phagocytosis, suppressing viral dissemination within the host. nevertheless, clrs can be a double-edged sword as some viruses have evolved to use these receptors for viral entry into host cells, avoiding immune recognition, where kidnapping the host cell machinery they produce hundreds of new copies of themselves spreading their copies further into the host [ ] . whole exome sequencing studies have revealed host biomarkers of susceptibility to rsv belonging to the olfactory and taste receptors. single nucleotide polymorphism (snp) variation at these genes has been observed to be associated with rsv infection [ ] . rsv has also been described to cause post-viral olfactory loss [ ] . according to our meta-analysis, rsv infection also has an impact on the deg related to on the olfactory signaling pathway (r-hsa- ; adjusted p-value = . × − ). last but not least, we identified a -transcript blood signature specific for rsv that differentiates it from other respiratory viruses of similar etiology such as influenza, rhinovirus, coronavirus and enterovirus. this indicates that rsv alters the host transcriptome in a specific manner that can be distinguished from other respiratory viruses. the identified signature could be relevant for rsv diagnosis. even though current automatic pcr-based technologies already show good performance [ ] , these transcripts might further our understanding of host molecular processes specifically altered by the rsv, which could eventually lead to the discovery of new drug targets for the treatment of rsv patients. future laboratory validation will be necessary before the discovered signature can be used in clinical settings. a major advantage of the present meta-analysis is that, by gathering data from different studies, we substantially increase sample size and consequently the statistical power to detect deg when compared to controls and other pathogens and reduce the potential bias derived from selecting patients in particular populations, geographic areas and/or seasons. to reduce batch effects the data were carefully normalized and preprocessed (see methods section); a limitation of this procedure is however that it not only reduces artificial sources of variability between datasets, but it might also reduce biological sources. the normalization procedure also reduces the number of probes analyzed to those that are shared between all microarrays. another limitation of our study is that information on the rsv serotype (a/a or b), neither the disease status of the patients, are not available for all the datasets. the present study is a stepping-stone towards understanding how rsv affects the host transcriptome, but further studies are needed to better understand how the host transcriptomic response change among serotypes, and during the progression of the disease. these studies would allow to shed further light in the mechanism responsible of rsv pathogenicity. we queried the public gene expression microarray repository gene expression omnibus (geo) for human gene expression datasets using the following terms: "rsv" and/or "syncytial". we retained only those studies containing microarray expression data from whole blood samples of rsv infected patients. more information on the datasets used is provided in table s . to merge and integrate the public domain rsv microarray studies, we first normalized and preprocessed each dataset separately using the package lumi [ ] for illumina ® microarrays data and the package oligo [ ] for affymetrix ® datasets ( figure s ). we also conducted a principal component analysis (pca) to check for outliers and evaluate the presence of strong batch effects that could affect the analysis of the data. subsequently, we used the r package coconut (combat co-normalization using controls) to combine all datasets into one and reduce batch effects in the meta-analysis [ ] . finally, to determine which genes are significantly up-or down-regulated during the rsv infection (n = ) when compared to both healthy controls (n = ) and other viral conditions as a single group (n = ; table s ), we used the r package limma [ ] and a moderated t-statistic. a linear model was fitted considering the age as a categorical covariate of the model (children / adult); this allowed us to minimize confounding effects considering that the study gse was carried out on adults whereas the rest of studies were conducted in children. multiple testing correction was performed using the false discovery rate (fdr). a summary of the methodological procedure is shown in figure . more information on the datasets used is provided in table s . to merge and integrate the public domain rsv microarray studies, we first normalized and preprocessed each dataset separately using the package lumi [ ] for illumina ® microarrays data and the package oligo [ ] for affymetrix ® datasets ( figure s ). we also conducted a principal component analysis (pca) to check for outliers and evaluate the presence of strong batch effects that could affect the analysis of the data. subsequently, we used the r package coconut (combat co-normalization using controls) to combine all datasets into one and reduce batch effects in the meta-analysis [ ] . finally, to determine which genes are significantly up-or down-regulated during the rsv infection (n = ) when compared to both healthy controls (n = ) and other viral conditions as a single group (n = ; table s ), we used the r package limma [ ] and a moderated t-statistic. a linear model was fitted considering the age as a categorical covariate of the model (children / adult); this allowed us to minimize confounding effects considering that the study gse was carried out on adults whereas the rest of studies were conducted in children. multiple testing correction was performed using the false discovery rate (fdr). a summary of the methodological procedure is shown in figure . we used the reactome pathway database to examine biological pathways associated with the genes differentially expressed in rsv patients. thus, to categorize differentially expressed genes (deg) for overrepresentation of reactome pathways we used panther classification system with the following parameters: reactome pathways overrepresentation test (released on - - ) with reactome version (released on - - ), and homo sapiens as a reference list. statistical significance was evaluated using fisher's exact test and fdr as the multiple test correction method. our hypothesis is that rsv affects the transcriptome in a particular way distinguishable from other viral conditions. from the list of deg obtained when compared rsv patients against other viral conditions (table s ) , we investigated a minimum specific transcript signature of rsv infection. we used parallel regularised regression model search (prems) [ ] in a randomly split dataset removing the healthy controls: training set (n = ) and validation set (n = ). prems explores different logistic regression models constructed from optimal subsets of the candidate genes while increasing the model size iteratively. prems [ ] was chosen instead of other methods because it tends to select signatures with fewer genes without sacrificing model accuracy, which would facilitate its future translation into a clinical test [ ] . the bio-signature was searched among the top deg between rsv and non-rsv categories. finally, the accuracy of the model estimated by prems was calculated as the area under the receiver operator curve (auc) using the r package proc [ ] in both training and test cohorts. all analyses were carried out using r software version . . [ ] . the present integrated multicohort analysis suggests that rsv alters the expression of > genes in the host. a number of them are related to different pathways, namely cell cycle, immunological response to viral infection (including pattern recognition receptors), and olfactory signaling. in addition, rsv modifies the host transcriptome in a very specific manner, different from other respiratory viruses with similar phenotypes. we found a -transcript signature (validated in several independent cohorts), that allows the discrimination of rsv infection from other respiratory viruses. considering the small number of transcripts involved, this signature might be potentially translated into a point of care test. this study is a step forward to a better understanding of the molecular mechanism underlying rsv infection. the biomarkers of rsv infection detected may help discover new drug targets and improve the development of vaccines. using pathway-based approaches such as go term enrichment or ingenuity pathway analysis (ipa) to prioritize the genes whose expression is altered by rsv infection may help discovering new drug targets and improve the development of vaccines. supplementary materials: supplementary materials can be found at http://www.mdpi.com/ - / / / / s . table s . description of samples and cohorts included in the present study. table s . genes differentially expressed according to r package limma. table s . reactome pathway enrichment analysis results. figure s . pca plot of the eight datasets initially selected for the meta-analysis. the data points clustering on the right side of the plot (pc ) correspond to the outlier transcriptome profiles of the rsv samples from the dataset gse ; the control samples in gse cluster with the other controls. this study received support from the instituto de salud carlos iii: project gepem (instituto de salud carlos iii(isciii)/pi / /cofinanciado feder), diavir (instituto de salud carlos iii(isciii)/dts / /cofinanciado feder; proyecto de desarrollo tecnológico en salud) and resvi-omics (instituto de salud carlos iii(isciii)/pi / ; /cofinanciado feder) given to a.s.; and project resvinext (instituto de salud carlos iii(isciii)/pi / /cofinanciado feder), and enterogen (instituto de salud carlos iii(isciii)/ pi / /cofinanciado feder) given to f.m.-t. biomarkers of respiratory syncytial virus (rsv) infection: specific neutrophil and cytokine levels provide increased accuracy in predicting disease severity global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in : a systematic review and modelling study whole blood gene expression in infants with respiratory syncytial virus bronchiolitis risk of primary infection and reinfection with respiratory syncytial virus review of epidemiology and clinical risk factors for severe respiratory syncytial virus (rsv) infection risk factors in children hospitalized with rsv bronchiolitis versus non-rsv bronchiolitis the burden of respiratory syncytial virus infection in young children whole exome sequencing reveals new candidate genes in host genomic susceptibility to respiratory syncytial virus disease increased concordance of severe respiratory syncytial virus infection in identical twins disease severity in respiratory syncytial virus infection: role of host genetic variation respiratory syncytial virus bronchiolitis in children up to years of age in spain: epidemiology and comorbidities: an observational study diagnosis of childhood tuberculosis and host rna expression in africa diagnostic test accuracy of a -transcript host rna signature for discriminating bacterial vs viral infection in febrile children whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection a -transcript host cell signature distinguishes viral from bacterial diarrhea and it is influenced by the severity of symptoms a qpcr expression assay of ifi l gene differentiates viral from bacterial infections in febrile children diagnosis of kawasaki disease using a minimal whole-blood gene expression signature gene expression and regulation in systemic lupus erythematosus how to read a systematic review and meta-analysis and apply the results to patient care: users' guides to the medical literature power failure: why small sample size undermines the reliability of neuroscience genome-wide expression for diagnosis of pulmonary tuberculosis: a multicohort analysis host-response-based gene signatures for tuberculosis diagnosis: a systematic comparison of signatures a -gene set predictive of progression to severe dengue a community approach to mortality prediction in sepsis via gene expression analysis - 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ld knm authors: belova, natalya v.; pimenov, oleg a.; kotova, vitaliya e.; girichev, georgiy v. title: molecular structure and electron distribution of -nitropyridine n-oxide: experimental and theoretical study of substituent effects date: - - journal: j mol struct doi: . /j.molstruc. . sha: doc_id: cord_uid: ld knm the molecular structure of -nitropyridine n-oxide, -no( )-pyo, has been determined by gas-phase electron diffraction monitored by mass spectrometry (ged/ms) and by quantum chemical calculations (dft and mp ). comparison of these results with those for non-substituted pyridine n-oxide and -methylpyridine n-oxide ch( )-pyo, demonstrate strong substitution effects on structural parameters and electron density distribution. the presence of the electron-withdrawing –no( ) group in para-position of -no( )-pyo results in an increase of the ipso-angle and a decrease of the semipolar bond length r(n→o) in comparison to the non-substituted pyo. the presence of the electron-donating –ch( ) group in -ch( )-pyo leads to opposite structural changes. electron density distribution in pyridine-n-oxide and its two substituted compounds are discussed in terms of natural bond orbitals (nbo) and quantum theory atoms in molecule (qtaim). pyridine-n-oxide derivatives have attracted (a) great interest in chemistry and biotechnology because of their accentuated oxidizing properties, high polarity, and their ability to be both charge donors and acceptors. heterocyclic compounds with n-oxide groups have achieved widespread utility foremost because of their pronounced biological activity [ ] . in the literature we can find the evidence of bactericidal, analgesic, anticonvulsant, apoptotic, and antimicrobial activity of n-oxides [ , ] . these properties allow to use n-oxides as inhibitor of hiv- reverse transcriptase [ , ] , as antiviral agent against various sars corona virus strains [ ] , and as antiadhesive and quorum-sensitive inhibitor [ ] . some complexes with pyridine-n-oxides are used in agriculture to regulate plant growth rates [ ] . according to numerous studies, the biochemical activity of n-oxides is due to the complexations with the metalloporphyrins in living organisms [ ] [ ] [ ] . obviously, the reactivity of n-oxides may vary due to different substituents in the ring [ ] . the variation of the substituents gives wide possibilities to chemical modifications of n-oxides and allows to change their complexing properties, and, as the result, to influence the biological activity. apparently, the substitution of pyridine-n-oxide with a strong electron-withdrawing -no group would lead to an electron density redistribution, and thus would widen the scope of its reactivity. it is often assumed that the presence of the nitro group in substituted pyridine-n-oxides is responsible for a substantial increase in the antifungal activity [ ] . nitro-pyridine-n-oxides are also known to be used in photonics for design and development of new organic nonlinear optical (nlo) materials [ ] [ ] [ ] [ ] [ ] . in order to elucidate the factors underlying these and other properties of pyridine-n-oxides a detailed information about the structural properties as well as about the electron density distribution in the molecules is needed. the available literature data about molecular structure of gaseous n-oxides is not sufficient. only four pyridine-n-oxides have been studied by gas-electron diffraction (ged). chiang j.f. et al. [ , ] carried out ged studies of pyridine-n-oxide and three of its para-substituted compounds. however, the structural data reported in [ ] should be criticized. thus, e.g. n-c bond lengths in -no -pyo, -cl-pyo, -me-pyo obtained by chiang j.f. et al. [ ] are even longer than r(c-c) in the benzene ring, and are strongly overestimated compared to the calculated values [ ] and x-ray crystallographic data [ , ] . furthermore, the cnc bond angle values in the pyridine ring of -no -pyo and -cl-pyo given in ref. [ ] are not consistent with sp hybridization of the nitrogen atom and do not agree with calculated [ ] and x-ray data [ , ] . the discrepancies in molecular structure of substituted n-oxides according to ged by chiang j.f. et al. [ , ] and other methods motivated us to perform a new gas-electron diffraction study of substituted-n-oxide. the first ged reinvestigation was performed for -methylpyridine-n-oxide [ ] because of the largest structural contradictions for this molecule. now more sophisticated methods for the analysis of ged data are available, including the possibility of quantum chemical results to be used in the interpretation of the experimental data. this allows us to determine the structural parameters more precisely compared to the studies performed in . the experimental structural data for -methylpyridine-n-oxide obtained in our study [ ] are in a good agreement with the theoretical parameters as well as the experimental x-ray data. the present work continues our research of substituted-n-oxide structures. we performed new gas-electron diffraction study of -nitropiridine-n-oxide (fig. ). this molecule was chosen because of the substituent nature. the nitro-group is known to be strong electron-withdrawing, whereas the methyl group is a good electron donor. thus, the comparison of -no -pyo structure with that of -me-pyo allows to reveal the substituent effects. this work presents also a theoretical study (dft and mp ) of the molecular structure of -no -pyo along with the related molecules. electron charge distribution was studied in the framework of nbo scheme. as an alternative to conventional lewis model which was realized in nbo the topological analysis of ρ(r) in the framework of bader's quantum theory of atoms in molecule (qtaim) [ ] was performed for -no -pyo in the present work. the electron diffraction patterns and the mass spectra were recorded simultaneously using the techniques described previously [ , ] . two series of ged/ms experiments at two different nozzle-to-plate distances have been performed. the conditions of ged/ms experiments and the relative abundance of the ions in mass spectra of -no -pyo are shown in tables and s , respectively. the temperature of molybdenum effusion cell was measured by a w/re- / thermocouple calibrated by melting points of sn and al. the wavelength of the fast electrons was determined from the diffraction patterns of polycrystalline zno. optical densities were measured by a computer-controlled md- (carls zeiss, jena) microdensitometer [ ] . the molecular intensities were obtained in the ranges . ÷ . Å - (short camera) and . ÷ . Å - (long camera). the molecular intensities and the radial distributing curves are shown in fig. and fig. respectively. figure and table s present the mass spectrum recorded simultaneously with ged data. all quantum chemical calculations were performed using the gaussian program set [ ] . the hybrid dft computational methods, namely becke's three-parameter hybrid functional b lyp [ - ] and perdew-burke-ernzerh of hybrid functional pbe [ ] , as well as second order møller-plesset perturbation theory, mp , were used. the correlation-consistent basis sets augmented with diffuse functions (aug-cc-pvtz) have been taken to describe the electronic shells of o, c, n and h atoms. structure optimizations were followed by calculations of the vibrational frequencies in order to ensure that a minimum on the potential energy hyper-surface had been reached. all calculations have been performed for the closed-shell electronic state. the geometrical parameters of the calculated equilibrium structure derived with different theory methods are given in table together with the experimental results.vibrational amplitudes and corrections, Δr=r h −r a , were derived from theoretical force fields (b lyp/aug-cc-pvtz) by sipachev's method (approximation with taking into account the nonlinear kinematic effects at the level of the first order perturbation theory for the transformation of cartesian coordinates into internal coordinates), using the program shrink [ ] [ ] [ ] . selected values are listed in table (excluding non-bonded distances involving hydrogen). the potential energy function of the internal rotation of the nitro group about the c -n bond was investigated. for this purpose, we carried out relaxed potential energy surface scans by the variation of the dihedral angle τ(c c n o ) in steps of ° between ° and ° with b lyp/aug-cc-pvtz method. analysis of this potential function (fig. ) shows that there is only one stable geometric configuration with τ about ° (see figure for atom numbering). it should be noted that the internal rotation barrier is a sufficiently large, . kcal/mol (b lyp/aug-cc-pvtz), . kcal/mol (pbe /aug-cc-pvtz) or . kcal/mol (mp /aug-cc-pvtz). the nbo g program [ ] , implemented for natural orbital analysis in pc gamess [ ] , was used to obtain the net atomic charges and wiberg bond indexes. b lyp/aug-cc-pvtz wave functions were used in the nbo analyses. the topological analysis of electron density distribution function ρ(r) for -no -pyo and pyo was carried out using aimall professional software [ ] . the visualization of molecular models are realized by chemcraft [ ] program. the heaviest ion in mass spectrum observed during the combined ged/ms experiment was [no -pyo] + (fig. , table s ). no ions were detected, which could arise from impurities.this according to quantum chemical calculations, a planar structure with c v overall symmetry of the molecule was assumed. independent r h parameters were used to describe the molecular structure. starting parameters from b lyp/aug-cc-pvtz calculations were refined by a least-squares procedure of the molecular intensities. the differences between all c−c bond distances, n-o bond distances, and c-n bond distances as well as between all c−h bond distances were constrained to calculated values (b lyp/aug-cc-pvtz). vibrational amplitudes were refined in groups with fixed differences. with the abovementioned assumptions, four bond distances and three bond angles (table ) were refined simultaneously with eight groups of vibrational amplitudes (table ) . only two correlation coefficients had absolute values larger than . : (r(n -c ))/(r(c -c ))=- . , and (∠n c c )/( ∠c n c ) = - . . the best agreement factor is r f = . %. results of the least squares analysis are given in table (geometric parameters) and table (vibrational amplitudes). the model with nitro group plane turned relative to the pyridine ring plane was also tested in the ged analysis. this refinement leads to r f = . % for τ(c c n o ) fixed at ° and r f = . % for τ(c c n o ) fixed at °. the statistical hamilton criterion [ ] at the . significance level distinctly reveals that the nitro group rotation angle relative to the plane of the pyridine ring is not more exceed than ° because the critical value r f = . %. table summarizes the structural parameters of -nitro-pyridine-n-oxide. we can note a perfect agreement between calculated and experimental molecular structure parameters. thus, changing the calculation method has no significant influence on the values of structural parameters. the differences in theoretical values for bond distances do not exceed . Å, whereas the calculated values of the bond angles are almost equal, independent of the theoretical level. both, quantum chemistry and ged analysis result in c v overall symmetry of the molecule with a planar pyridine ring. the nitro-group is coplanar with the heterocycle plane. obviously, this position provides a conjugation between the π-system of the pyridine ring and the no group. furthermore, figure presenting the calculated (b lyp/aug-cc-pvtz) -no -group rotation potential curve clearly shows that changing of the torsional angle τ(c c n o ) by more than for ° leads to a significant increase in energy. m. dakkouri and v. typke performed theoretical investigation of the structure of -nitropyridine-n-oxide [ ] . it is astonishing to note that the endocyclic bond lengths of the pyridine n-oxide ring is almost equal, independent of the position of the nitro group. table compares the structure of pyridine-n-oxide and its two substituted obtained by ged, x-ray and dft. taking into account that the structural parameters obtained by different methods have different physical meaning, we can note a good agreement of the values obtained in our research (therein and [ ] ) with the theoretical results (therein and [ ] ) and, also, with the parameters for the crystals [ , ] in contrast to the values recommended by chiang j. f. et al. [ ] . apparently, some increase of the semipolar n→o bond lengths in a crystal phase compared to the free molecules is due to intermolecular hydrogen bonding. all parameters presented in table confirm the hyperconjugation in the pyridine ring and the sp hybridization concept of the nitrogen and carbon atoms in the ring. the exceptions are only the data by chiang j. f. et al. [ ] , which are rather strange as we mentioned above. unfortunately, large uncertainties in ged parameters for pyo [ ] do not allow a comparison of the experimental structures. thus, we can compare only the values obtained in our ged study (therein and [ ] ) and the calculated values. the structural data for free molecules in table show that the insertion of the methyl or nitro group in para -position to the pyo has no significant effect on the structural parameters of the pyridine ring, excluding only the ipso-angle ∠ c c c , which is changes from . ° in pyo to . ° in -me-pyo and . ° in -no -pyo (b lyp/aug-cc-pvtz). it is interesting to note, that the substituent effects on the geometry of heterocyclic ring are rather similar to the tendencies for the benzene ring [ , ] . thus, the presence of the electron-withdrawing -no group leads to the increase of the ipso-angle, whereas electron-donating -ch substituent produces the opposite effect. furthermore, the r(n →o ) semipolar bond lengths appear to be also sensitive to a substituent nature. thus, the presence of electron withdrawing-no group in -no -pyo results in a shortening of r(n→o) in comparison with pyo, while in -me-pyo with the donor substituent the bond length n→o increases. the calculated net atomic charges along with the wiberg bond indexes for pyo and two its derivatives are summarized in table . in all three cases the c-c and c-n bond orders in the ring, as well as the values of the distances from tables and show that these bonds do not correspond to either single nor double bonds. this confirms the existence of π-conjugation in the pyridine rings. the c -n bond distance as well as q(c -n ) for -no analyzing the average values of bond length in the pyridine ring chiang j.f. et al. [ ] have (table ) do not change with the insertion of the substituent in para-position. to study the substituent effect on the aromaticity the nuclear independent chemical shifts nics( ) have been calculated. nics( ) values correspond to the negative isotropic shielding (in ppm) at Å above the ring plane [ ] [ ] [ ] . it is of important to note that in a vast number of papers it has been elaborately shown that (depending on the size of the ring) the in-plane aromaticity which is indicated by the isotropic nics( ) value contains considerable contribution of in-plane σaromaticity resulting from the diamagnetic shielding of the σ electronic framework. thus the nics( ) value regards to be a better descriptor of the π contribution to aromaticity due to the apparent decrease of the local contributions of the σ framework at larger distances from the ring center. all nics values were computed by employing the standard giao method implemented in table ) are in satisfactory agreement with the wiberg bond indexes ( table ) and confirm that the bonds in the pyridine ring are neither single nor double. as additional criteria of π-bonding contribution the bond ellipticity ε is used in qtaim. among the endocyclic bonds of -no -pyo molecule the highest value of ε = . is for c -c bond. accordingly, this bond possesses highest π-character in the ring (in comparison ε = . for benzene [ ] ). surprisingly, the ellipticity value ε = . for c -n bond is rather large, that could be a sign of the partial π-character of this bond in spite of the delocalization index value (δ = . ) corresponds to single (sigma) bond, as well as a bond r(c -n ) length. the net atomic charges obtained by qtam approach are presented in figure and in table . some differences of the net atomic charges obtained by npa, natural population analysis, (table ) and by qtaim approach (table ) should be noted, although the general trends in the values remain from pyo to -no -pyo. according to qtaim results, the atomic charge on the ipso carbon atom c in -no -pyo significantly changes in comparison with pyo because of a neighbor to acceptor -no group. the electron density on c , c and n atoms in -no -pyo is decreased also due to negative mesomeric effect which takes place inside the pyridine ring. the total charge q(ring) of the heterocyclic ring in pyo has a positive value + . e which is increased up to + . e when -no group has been introduced. indeed, the electron-withdrawing -no group is a part of π-conjugated system of the molecule, and as result electron density is shifted from o atom and pyridine ring towards the substituent. the negative total charge of -no group q(r) has a value - . e, that additionally supports the suggestion mentioned above. we would like to point out the magnitude of electron density into pyridine ring is changed quantitatively but topologically electron density distribution does not changed dramatically between non-and substituted pyridine-n-oxides (see figure and table ). at the same time a decrease of the charge on o atom can lead to a decrease in the complex formation ability of the substance. the redistribution of electron density in -no -pyo affects to geometrical structure of the pyridine ring. the introduction of-no is accompanied by an increase of ρ b from . a.u. in pyo to . a.u. in -no -pyo and of ellipticity value ε from . to . for c -c bond, respectively (see table ). that leads to magnification of electrostatic repulsion between these bonds and as result the ipso-angle c c c is changed from . ° in pyo to . ° in -no -pyo (b lyp/aug-cc-pvtz).it is interesting to note the same tendency is observed for crystal data where the ipso-angle c c c increased from . ° to . ° (see table ). we can suspect the similar changes in electron density for crystal phase as for free in our research we are trying to understand the influence of the nature of substituents on the properties of n-oxides. in this work the molecular structure of -nitropyridine n-oxide has been studied by ged/ms and quantum chemical calculations (dft and mp ). both, quantum chemistry and ged analyses resulted in the c v molecular symmetry with the planar pyridine ring and -no group which is coplanar to the six-membered heterocycle. furthermore, the rotation of -no groupmore than for ° leads to the significant increase in energy. a distances in Å and angles in degrees. for atom numbering see figure . b uncertainties in r h σ=(σ sc +( . σ ls ) ) / (σ sc = , r, σ ls -standard deviation in least-squares refinement), for angles σ= σ ls . c p i -parameter refined independently. (p i ) -parameters calculated from the independent parameter p i by a difference ∆= p i -(p i ) from the quantum chemical calculations (b lyp/aug-cc-pvtz). [ ] x-ray [ ] b lyp/augcc-pvtz r(n -o ) . ( ) * q(ring) -total natural charge of the heterocyclic ring (c -c -c -n -c -c ) q(r) -total natural charge of the substituent table . bond lengths and topological parameters of ρ(r) * in some bond critical points of -nitropyridine-n-oxide and pyridine-n-oxide (b lyp/aug-cc-pvtz) * -ρthe electron density(a.u.). ∇ ρ-the laplacian(a.u.). λ , λ , λ -electron density hessian matrix eigenvalues (a.u.). h b -the total electronic energy density (a.u.). ε-the bond ellipticity. δthe electronic delocalization index. ab initio scf and ci study of the electronic spectrum of pyridine n-oxide -and -nitro- -azabenzo[a]pyrens and their n-oxides: hifly mutagenic nitrated azaarenes pyridine n-oxide derivatives: unusual anti-hiv compounds with multiple mechanisms of antiviral action pyridine oxide derivatives: structure-activity relationship for inhibition of human immunodeficiency virus and cytomegalovirus replication in cell culture pyridine n-oxide derivatives are inhibitory to the human sars and feline infectious peritonitis coronavirus in cell culture aromatic amine oxides purification and properties of trimethylamine n-oxide reductase from aerobic photosynthetic bacterium roseobacter denitrificans non-enzymatic reduction of aliphatic tertiary amine n-oxides mediated by the haem moiety of cytochrome p nonenzymatic reduction of brucine noxide by the heme group of ctocrome p the effect of electronic factors on the reactivity of heteroaromatic n-oxides chem zur deutung des uv.-spektrums von pyridin-noxid nonlinear optics in multipolar media: theory and experiments synthesis and processing of improved organic second-order nonlinear optical materials for applications in photonics anchorage of -nitropyridine n-oxide, an optically non-linear entity, onto cdci, and cdl, host matrices a new aromatic organometallic nonlinear optical crystal second-order nonlinear optical materials: recent advances in chromophore design molecular structure of pyridine n oxide molecular structures of -nitro-, -methyl-and -chloro-pyridine-n-oxides charge density studies below liquid nitrogen temperature: x-ray analysis of p-nitropyridine n-oxide at k the molecular structure of -methylpyridine-n-oxide: gas-phase electron diffraction and quantum chemical calculations upgrading the emr- electron-diffraction camera for use with gases, instruments and experimental techniques apparatus for study of molecular structure of valence-unsaturated compounds, instruments and experimental techniques automation of a physicochemical experiment: photometry and voltammetry pittsburgh pa. . c. w. bauschlicher, h. partridge, cr revisited density-functional thermochemistry. iii. the role of exact exchange density-functional exchange-energy approximation with correct asymptotic behavior development of the colle-salvetti correlation-energy formula into a functional of the electron density accurate spin-dependent electron liquid correlation energies for local spin density calculations: a critical analysis toward reliable density functional methods without adjustable parameters: the pbe model local centrifugal distortions caused by internal motions of molecules calculation of shrikage corrections in harmonic aproximation theochem) advances in molecular structure research granovsky, pc gamess version . (firefly chemcraft version . (build ) sigmificance tests on the crystallographic r factor a theoretical investigation of the structure of -nitropyridine-n-oxide and the dependency of the no torsional motion on the applied wavefunction and basis set crystal structure of -methylpyridine n-oxide isothermal and isochoric crystallization of highly hygroscopic pyridine n-oxide of aqueous solution molecular geometry of substituted benzene derivatives. i. on the nature of the ring deformations indused by sublimations group electronegativities from benzene ring deformations: a quantum chemical study nucleus-independent chemical shifts: a simple and efficient aromaticity probe induced magnetic fields in aromatic [n]-annulenes-interpretation of nics tensor components conformational and electronic (aim/nbo) study of unsubstituted a-type dimeric proanthocyanidin nitropyridine- -oxides as excellent π-hole donors: interplay between σ-hole (halogen, hydrogen, triel, and coordination bonds) and π-hole interactions figure . the molecular graphs for pyo (left ) and -no pyo (right )molecules and net atomic charges (e). the bond critical points are green this work was supported by the ministry of education and science of the russian federation (the project fzzw- - ). we would like to thank prof. a. v. belyakov for his help in quantum chemical calculation. the molecule possesses c v molecular symmetry with the planar pyridine ring and -no group which is coplanar to the six-membered heterocycle.quantum chemical calculations for non-substituted pyridine-n-oxide, pyo, and for methylpiridine-n-oxide, -mepyo, have been performed using dft.obtained molecular parameters for pyo, -no -pyo, and -mepyo confirm the π-conjugation in the pyridine ring and the sp hybridization concept of the nitrogen and carbon atoms in the ring.electron density distribution analysis has discussed in terms of natural bond orbitals (nbo) and quantum theory atoms in molecule (qtaim) scheme.the presence of electron withdrawing -no group in -no -pyo results in a shortening of r(n→o) in comparison with pyo, the corresponding bond order increases, and the negative net charge on the oxygen atom decreases, while in -me-pyo with the donor substituent all changes are opposite.according to qtaim n→o semipolar bond is predominantly covalent and possess the cylindrical symmetry, at the same time the electron density concentration is substantially high that is usually appropriate to the bonds with large π-contribution. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord- - bf w authors: betten, anton; mukthineni, tarun title: classifying simplicial dissections of convex polyhedra with symmetry date: - - journal: mathematical software - icms doi: . / - - - - _ sha: doc_id: cord_uid: bf w a convex polyhedron is the convex hull of a finite set of points in [formula: see text] a triangulation of a convex polyhedron is a decomposition into a finite number of -simplices such that any two intersect in a common face or are disjoint. a simplicial dissection is a decomposition into a finite number of -simplices such that no two share an interior point. we present an algorithm to classify the simplicial dissections of a regular polyhedron under the symmetry group of the prolyhedron. a convex polyhedron is the convex hull of a finite set of points in r . a triangulation of a convex polyhedron is a decomposition into a finite number of -simplices such that any two intersect in a common face or are disjoint. a simplicial dissection is a decomposition into a finite number of -simplices such that no two share an interior point. a simplicial dissection is a triangulation but not conversely. the problem is that the intersection of two simplices in a dissection may not be face. standard implementations for enumerating triangulations include topcom and mptopcom [ ] (neither one can enumerate dissections, though). a parallel algorithm to classify regular triangulations with applications in tropical geometry is described in [ ] . regarding the enumeration of all triangulations, see [ ] . for minimal dissections, see [ ] . the goal of this paper is to present an efficient algorithm to classify the simplicial dissections of a regular polyhedron under the symmetry group (or automorphism group) g of the polyhedron p. two dissections of p are equivalent is there is a symmetry g ∈ g which maps one to the other. the classification algorithm utilizes the concept of a partially ordered set under a group action, using the theory developed by plesken [ ] as a framework. the partially ordered set is the search space, which is to be partitioned into orbits. the ranking of the poset introduces level sets, and the orbits partition these level sets. the efficiency of the orbit algorithm is based on an effective use of isomorph rejection. this is the problem of deciding when two objects belong to the same g-orbit. isomorph rejection is necessary to avoid duplicates, and it helps reduce the number of objects in the search space that have to be examined. the ultimate goal of the classification algorithm is to establish the poset of orbits of g. isomorphism testing is expensive, and the algorithm that we propose avoids backtracking at the cost of memory. such trade-off between time complexity and space complexity is common in algorithm design, and it has proved to be useful for other classification problems before. the first author has previously used this technique to classify objects like cubic surfaces, packings in projective space and other objects. in this note, we will develop an efficient algorithm to classify the simplicial dissections of a polyhedra. as an application, we compute and classify the simplicial dissections of the cube. we use the binary representation of the integers from to to denote the vertices of the cube (cf. figure ) , with two vertices adjacent if their hamming distance is one. the hamming distance is the number of components which differ in the binary expansion. the automorphism group of the cube has order and is generated by the three permutations ( , , , )( , , , ), ( , , , )( , , , ), ( , )( , )( , )( , ). the tetrahedra are encoded using the lexicographic rank of their vertex set among the set of -subsets of { , . . . , }: theorem . the number of equivalence classes of simplicial dissections of the cube under its automorphism group of order is exactly . six of these are triangulations as described in [ ] . a system of representatives is given in table , together with the order of the automorphism group. a more detailed drawing of the representatives is shown in table . a tretrahedron is spatial if it has positive volume. out of the list of tetrahedra, are flat. the remaining are spatial and can be used for triangulating the cube. following takeuchi and imai [ ] , triangulations can be identified using large cliques in a certain graph Γ , which we call the disjointness graph. this terminology is somewhat abusive, since the graph measures if the interior point sets of the tetrahedra are disjoint: boundary points may or may not intersect. the vertices of Γ are the spatial tetrahedra. two vertices are adjacent if the associated tetrahedra are non-overlapping, i.e. they do not share an interior point. the adjacency matrix of Γ is shown in table . as pointed out by de loera et al. [ ] , there are four types of tetrahedra under the action of the group. the four types are listed in table . the table lists the volume of each tetrahedron, based on a cube of side length one. the type vector of a triangulation is the vector (a, b, c, d) where a is the number of cores, b is the number of corners, c is the number of staircases, and d is the number of slanted pieces. the corner, staircase and slanted pieces each have volume , whereas the core piece has volume . from this it follows that a triangulation or dissection either has tetrahedra and includes a core piece, or it has tetrahedra, none of which are core. this means that the type vector satisfies in table , the list of dissections from theorem is listed, with tetrahedra separated out by type. the type vector is listed in the column headed type. for triangulations, the de loera number is in the column headed dl. this will be discussed in sect. . poset classification is a technique to classify combinatorial objects. canonical augmentation due to mckay [ ] is a very popular technique. mckay introduces the idea of a canonical predecessor to achieve the isomorph classification. mckay's work relies on the notion of a canonical form. his computer package nauty [ ] can compute canonical forms for graphs efficiently. this has led many authors to reduce the classification of different types of combinatorial structures to that of graphs. the original combinatorial objects are equivalent if and only if the associated graphs are isomorphic. by using nauty to solve the isomorphism problem for the associated graphs, the combinatorial objects at hand are classified as well. for many combinatorial objects, this reduction is efficient and works very well. however, there are combinatorial objects for which this reduction is inefficient. also, there is an interest in solving the isomorphism problem for the original combinatorial objects at hand directly, and avoiding the reduction to graphs altogether. a second approach to classify combinatorial objects is losely based on ideas of schmalz [ ] for the enumeration of double cosets in groups. this has been adapted to the problem of classifying the orbits of groups on various posets. the critical operation in any poset orbit classification algorithm is the isomorphism testing. using the ideas of schmalz, backtracking can be avoided at the expense of higher memory complexity. the poset is examined breadth-first, using the rank of the combinatorial objects at hand. for most combinatorial objects, such rank functions are implicit. for instance, for orbits on sets, the size of the set is the rank of the set. in order to do isomorphism test in linear time, previously computed data in lower levels of the poset is utilized when constructing the next level in the poset. for a recent description of this technique, including some comparisons with canonical augmentation, see [ ] . let (p, ≺) be a partially ordered set with rank function. assume that g is a group that acts on p (with the action written on the right). this means that for all g ∈ g and all a, b ∈ p we have let p i be the set of objects at level i in p. the poset of orbits for the action of g on p has as elements the orbits of g. two orbits o and o are related with there exists a ∈ o and b ∈ o with a ≺ b. for computing dissections of a polyhedron p with automorphism group g, let p be the set of partial dissections. a partial dissection is a set of spacial tetrahedra (simplices) which do not intersect in an interior point. the poset p is ordered with respect to inclusion. the group g of the polyhedron acts on this poset. the rank of a dissection is the number of simpices in it. the level set p i contains all partial dissection size i. the dissections of the cube can be recognized using the volume function from table . dissections containing a core tetrahedra have rank . all other dissections have rank . as all partial dissections correspond to cliques in the disjointness graph Γ of table , the problem of finding dissections is reduced to that of finding suitable cliques in the graph Γ. let us present some results from the classification, computed using orbiter [ ] . the number of orbits of g on each of the levels p i for i = , . . . , is shown in table . the poset of orbits for the action of the group of the cube on the partial dissections is shown in fig. . the labeling of the representatives of the dissections is as in table . de loera, rambau and santos [ ] list six types of triangulations of the cube. in table , the de loera triangulations are listed and identified with orbits in table . an isomorphism from the representative picked by de loera to the representative in table is given. minimal simplicial dissections and triangulations of convex -polytopes orbiter -a program to classify discrete objects how fast can we compute orbits of groups? the polytope of all triangulations of a point configuration triangulations. structures for algorithms and applications parallel enumeration of triangulations. electron isomorph-free exhaustive generation nauty user's guide (version . ) counting with groups and rings topcom: triangulations of point configurations and oriented matroids verwendung von untergruppenleitern zur bestimmung von doppelnebenklassen enumerating triangulations for products of two simplices and for arbitrary configurations of points the authors thank the three referees for helpful comments which have increased the clarity of the exposition. key: cord- -wjf c a authors: friis-nielsen, jens; kjartansdóttir, kristín rós; mollerup, sarah; asplund, maria; mourier, tobias; jensen, randi holm; hansen, thomas arn; rey-iglesia, alba; richter, stine raith; nielsen, ida broman; alquezar-planas, david e.; olsen, pernille v. s.; vinner, lasse; fridholm, helena; nielsen, lars peter; willerslev, eske; sicheritz-pontén, thomas; lund, ole; hansen, anders johannes; izarzugaza, jose m. g.; brunak, søren title: identification of known and novel recurrent viral sequences in data from multiple patients and multiple cancers date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: wjf c a virus discovery from high throughput sequencing data often follows a bottom-up approach where taxonomic annotation takes place prior to association to disease. albeit effective in some cases, the approach fails to detect novel pathogens and remote variants not present in reference databases. we have developed a species independent pipeline that utilises sequence clustering for the identification of nucleotide sequences that co-occur across multiple sequencing data instances. we applied the workflow to sequencing libraries from cancer samples of different cancer and tissue types, non-template controls, and test samples. recurrent sequences were statistically associated to biological, methodological or technical features with the aim to identify novel pathogens or plausible contaminants that may associate to a particular kit or method. we provide examples of identified inhabitants of the healthy tissue flora as well as experimental contaminants. unmapped sequences that co-occur with high statistical significance potentially represent the unknown sequence space where novel pathogens can be identified. the international agency for research on cancer (iarc) lists several biological species with carcinogenic potential in humans [ ] . this list comprises a bacterium (species helicobacter pylori), three parasitic flukes (clonorchis sinensis, opisthorchis viverrini and schistosoma haematobium), and seven viruses: human papillomaviruses (hpv), human immunodeficiency virus- (hiv- ), epstein-barr virus (ebv), hepatitis b and c virus (hbv and hcv), kaposi's sarcoma-associated herpesvirus (kshv), and human t-cell lymphotropic virus type (htlv- ). with the advent and spread of low-cost sequencing technologies, many viruses were discovered in the last decade [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one interesting discovery that fuelled the search for oncoviruses was merkel cell polyomavirus (mcpyv) found to be clonally integrated in merkel cell carcinomas [ , ] . the computational biology community has promptly responded to the growing need for specialised algorithms and pipelines to analyse the wealth of data [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . table s summarises the main features of some of the common approaches. in spite of particularities in the implementation, these methodologies share key conceptual similarities: first, sequencing reads or assembled contigs that originate from the host are identified and discarded, a process termed computational subtraction [ , ] . when the genomes or the concentrations of foreign species are small compared to host genomes, this step eliminates a substantial fraction of the total sequencing reads. second, the remaining non-host sequences are compared to a library of known reference sequences for taxonomic characterisation. the aforementioned methods identify species present across multiple samples, and the recurrence of a given viral entity may indicate an association to disease [ , ] . albeit conceptually valid, this bottom-up approach is inherently limited to the pre-existence of the organism in the reference databases, whereas novel oncoviruses showing partial or no similarity to known sequences will be missed. current efforts aiming at estimating and characterising metagenomic diversity are far from a complete mapping of the (viral) sequence-space [ ] . in fact, it is common to observe that a small but significant amount of unknown sequences, the so-called dark matter [ ] , goes through the current analysis pipelines without proper characterisation and is discarded from further analyses [ , , ] . here, we propose a method capable of identifying the recurrence of sequences across related samples independently of their existence in reference databases. our top-down approach compares samples and establishes recurrence prior to the taxonomic characterisation of the sequences. thus, enabling the identification of both known and novel biological entities. our method has conceptual similarities to the work of andreatta et al. [ ] where clustering of genes is used to find families that are predominantly found in pathogenic bacteria. attending to koch's postulates as modified by fredericks and relman [ ] , sequences from biological entities with a causative or facilitator role would be present in diseased samples and absent in healthy controls. in addition, recent studies documented the presence of contaminating and/or artefactual sequences that source from the laboratory kits and reagents used for sample processing and library preparation [ , [ ] [ ] [ ] [ ] [ ] . if not properly addressed, these confounding observations may lead to erroneous conclusions [ , ] . our method ascertains the statistical associations between recurrent sequences and a collection of features that describe the samples with respect to tissue, disease type, laboratory method, etc. additionally, the presence of other known technical problems, such as cluster invasion on the sequencing flow cells [ ] , might be detected. the study was conducted in accordance with the declaration of helsinki. two ethical boards reviewed the protocol of this study: the regional committee on health research ethics (case no. h- - -fsp ) and the national committee on health research ethics (case no. ). because the study used only samples that were anonymised at collection both boards waived the need for informed consent in compliance with the national legislation in denmark. two hundred and fifty-two cancer samples of different types were collected from various locations in denmark and hungary. cancer samples of malignant melanoma, acute myeloid leukaemia (aml), b-cell chronic lymphocytic leukaemia (b-cll), chronic myelogenous leukaemia (cml), and t-lineage acute lymphoblastic leukaemia (t-all; n = ) were obtained from aarhus university hospital, denmark. b-cell precursor acute lymphoblastic leukaemia (bcp-all), oropharyngeal head and neck cancer, testicular cancer, and t-all (n = ) were obtained from rigshospitalet, denmark (copenhagen university hospital). basal cell carcinoma, and mycosis fungoides (cutaneous t-cell lymphoma) were obtained from bispebjerg hospital (copenhagen university hospital). samples of bladder cancer, breast cancer, colon cancer, as well as ascites fluid of breast cancer, colon cancer, ovarian cancer, and pancreatic cancer were obtained from the danish cancer biobank, herlev hospital, denmark. b-cell lymphoma cell lines were obtained from aalborg university hospital, denmark. vulva cancer samples were obtained from the national institute of oncology, budapest, hungary. libraries were prepared at the center for geogenetics (cgg), university of copenhagen, denmark based on seven different methods for sample processing comprising five different enrichment methods and shotgun sequencing targeting total dna or rna (table s ). the enrichment methods used in the current work were circular genome amplification, sequence capture with retrovirus probes, virion enrichment (dna and rna), and mrna enrichment. further details on sample processing and library preparation have been published elsewhere [ , , ] , except for mrna enrichment which was performed using dynabeads mrna direct extraction kit (thermo fisher scientific, waltham, ma, usa) followed by scriptseq v rna-seq library preparation kit as for total rna analysis [ ] . ultimately, the data set consisted of dna and rna libraries, for which ˆ bp paired end sequencing was performed using the illumina hiseq platform at bgi-europe, copenhagen, denmark. the sequencing libraries thus originated from different cancer samples, non-template controls, and exogenous controls. the distribution of methods, libraries and controls for each sample type is provided in table s . samples were preferably analysed with multiple methods, thus out of samples were analysed with more than one laboratory method (table s ). the datasets went through a sequential pipeline with modules (in order) of preprocessing, computational subtraction of host sequences, low-complexity sequence removal, sequence assembly, clustering, association to metadata features, and taxonomical annotation. figure provides a schematic representation of the pipeline used to identify recurrent sequences across related samples. demultiplexing was performed using a local python script to partition the reads based on exact matches in the fastq header lines to the multiplexed indices provided. preprocessing of reads was performed for all datasets in parallel using adapterremoval [ ] with the following parameters {-trimns, -trimqualities, -minquality , -minlength , -collapse, -outputcollapsed, -outputcollapsedtruncated, -singleton}. read ends were trimmed for low quality base calls. reads were discarded if the length after trimming fell below bp. in these cases, the other read in a pair was kept as a singleton. overlapping paired reads from short inserts were collapsed into a single read if the overlap was longer than bp, according to the default behaviour of adapterremoval. preprocessed reads were filtered if they showed homology to the human reference genome, which included decoys and alternative sequences from version gca_ . (grch ) of the genome reference consortium (downloaded august , ) . mapping to the human genome was done using bwa [ ] version . . -r with the mem alignment algorithm and default parameters. all mapped reads without sequence alignment/map (sam) [ ] flag were discarded. single-unmapped reads from read pairs were kept. human depleted reads were filtered for low-complexity regions using the ncbi-blast associated module dustmasker [ ] and default parameters. reads containing low-complexity stretches of bp or longer were discarded. assembly of the remaining (non-human, high complexity) reads was performed with idba-ud [ ] and parameters {-precorrection}. contigs shorter than bp were discarded. a total of , , contigs, originating from the data sets, went through the entire pipeline. contigs ranged from bp to , bp with an overall n of bp. contigs from all data sets were pooled and clustered based on pairwise sequence homology using cd-hit [ ] , in fast mode. we chose parameters for clustering that maximised grouping of similar sequences while minimising inclusion of unrelated sequences. we table s from where we chose the final settings {-c . -as . -g } the datasets were described with a panel of different binary metadata features, for example tissue or disease characteristics (table s ) . features logically assessed whether they related to a particular dataset or not. features describing less than five datasets were removed. additionally, features that correlated perfectly in terms of matthew's correlation coefficient (mcc =˘ ) were merged. these filters resulted in unique features (table s ) . biological features (n = ) defined sample type, for instance tissue or disease category. methodological features (n = ) described specifics for sample preparation such as extraction kits, enrichment methods, polymerases, primers, buffer, filters used, or the laboratory where the work was performed, etc. technical features (n = ) defined the flow cell lane identifiers and whether resequencing was performed. the distributions of datasets and samples across the features are provided in table s . associations in the clustered contigs and metadata features were evaluated with fisher's exact test using a one-tailed alternative hypothesis (greater) and calculated in r using the function fisher.test [ ] . annotation of taxonomy was performed in two rounds. first aligning contigs with blastn [ ] with parameters {-evalue . } using default {-task megablast} to a frozen version of the ncbi nucleotide database nt (downloaded february ). secondly, using blastx with parameters {-evalue . } of all unmapped contig stretches to a frozen version of the ncbi non-redundant protein database nr (downloaded february ). the best hit by highest bit-score was kept for each contig. the taxonomy database (downloaded february ) was used to translate all genbank identifiers from hits to taxonomy identifiers. the taxonomy identifiers were then used to obtain the complete taxonomical lineage and extract scientific names of species. the abundances of all species in each cluster were used to calculate the species evenness index as defined by mulder et al. [ ] . clusters were annotated as the most abundant species in each cluster. the software to use after the assembly step has been uploaded at https://github.com/ jensfriisnielsen/sequence_recurrence. sequence clusters that have been described in detail throughout the manuscript have been included as supplementary files. clustering performance depends on the adequate selection of parameters. we experimented with a variety of configurations described by c xay ygz where x,y,y,z varied. the variables denote minimum percentage of sequence identity x (c x), minimum percentage of alignment length y (ay y) based on mode y of shortest (as) or longest (al) contig in alignment, as well as using local (g ) or global (g ) alignment mode z (gz). for example, a configuration encoded c as g would represent a clustering that requires global alignments with a % minimum sequence identity over % of the length of the shortest contig. the full list of investigated parameter combinations can be found in table s . we chose the parameters based on the performance of the clustering of expected contaminant sequences from avian leukosis virus (accession id ay ) [ ] and other related avian retroviruses (ars) such as avian myeloblastosis virus [ ] . ars are used in the manufacture of the reverse transcriptase failsafe pcr enzyme (epicentre, madison, wi, usa) included in the utilized scriptseq v rna-seq library preparation kit (illumina, san diego, ca, usa). this kit is commonly used for preparation of rna libraries [ ] . we identified clusters containing contigs that aligned to species of the alpharetrovirus genus (ncbi taxa-id: ) according to blastn and blastx hereafter referred to as ar clusters. all contigs in ar clusters were resolved with blastn and blastx and two metrics were considered for ar clusters. as the first performance metric, we computed the odds ratios (ors) of the associations between the presence of ar in the clusters and the use of the scriptseq kit. we used a ˆ contingency table defining the sets of libraries: ar positive and scriptseq positive (arpssp); ar positive and scriptseq negative (arpssn); ar negative and scriptseq positive (arnssp); ar negative and scriptseq negative (arnssn). or is then defined as the ratio arpsspˆarnssn / (arpssnˆarnssp) and describes the strength of the association between clusters and features. ors above indicate association between the presence of the ar virus and the use of the scriptseq kit. ors for all ar clusters were inspected in different parameter settings ( figure s ). the ors varied mostly block-wise with the parameters. the largest differences observed were between usages of the shortest or longest sequence in alignments with the alignment length filter. associations from the shortest mode tended to have higher dispersion in the range of ors. furthermore, one block of clustering results using global alignment mode, alignment length based on the shortest contig, and a minimum sequence identity of % (c ˆasyg ), had an overall high range of ors as well as the highest minimum values. this suggested that the clustering was able to reproduce the association between ar clusters and the scriptseq kit. in contrast, the clustering with parameter settings c as g had a very broad range of ors corresponding to a skewed clustering where some clusters had incorporated most sequences and left other clusters with only a few contigs. as a second performance metric we computed the species evenness [ ] indices of the ar clusters represented in figure s . the species evenness index is a score that derives from the shannon's diversity index [ ] and compares the abundance of each species within a cluster. an index of zero is assigned to clusters that are constituted uniquely by contigs mapping to a single species. contrarily, scores closer to would indicate that the cluster points to several species and that these are equally abundant. in our experiment, we favoured lower evenness indices as they indicate that clusters were able to single out species correctly. for example, parameter settings c al g generally had a high level of species evenness (median . ) in clusters, suggesting an incorrect separation of species. in stark contrast, a block of parameters using global alignment mode, alignment length based on shortest sequence, % minimal sequence identity, and a minimum alignment length of / / / / % of the shortest sequence (c asyg ) all had a median species evenness of . this group of parameter settings also showed desirable performance in terms of or, as mentioned before. generally it seemed that global mode (g ) had better ors than local mode (g ) when keeping other parameters fixed. additionally keeping % minimal sequence identity (c ) and varying minimal length of alignment in shortest mode (as) seemed stable in both ors and species evenness indices indicating that these close parameter settings were generally good. we chose to proceed with a clustering based on global alignments with a % minimum sequence identity over % of the length of the shortest contig (c as g ). this configuration resulted in a total of , clusters. of these, , clusters contained contigs from at least five different data sets and represented , different contigs. the full distribution of cluster sizes can be found in table s . the associations between the clusters and the binary metadata features were assessed using a fisher's one tailed exact test. there were , significant associations having p-value < . e- , corresponding to a . significance level when using bonferroni's correction for multiple testing [ ] . the significant associations were arranged in unique clusters and with unique features. the distribution of the significant associations showed that recurrent sequences originated from diverse sources and that individual clusters often associated to more than one feature (figure ). furthermore it is evident that the clusters tend to group in their associations. likely, these groupings represent one or more organisms. we investigated the nature of the clusters accounting only for the associated feature with the smallest p-value; hereafter described as the strongest associations. there were unique features involved in all the strongest associations. the distribution of p-values for each feature is represented in figure . the strongest associations were distributed according to biological, methodological and technical associations. these unique features were arranged in biological, methodological and technical features. most p-values were above e- and associations with lower p-values were to a few methodological features annotated as extraction kits: qiaamp dna mini kit (f ) (qiagen, hilden, germany), dnase/rnase: promega dnase (f ) (promega, madison, wi, usa), and dnase/rnase: promega dnase stop solution (f ), purification kit: rneasy minelute, qiagen (f ), library build: nebnext, new england biolabs (f ) (new england biolabs, ipswitch, ma, usa), and scriptseq v rna-seq, illumina (f ); the latter with a minimum p-value of . e- . the boxes span the first and third quartiles. the dark band inside each box represents the median. the whiskers of the boxes extend to the lowest and highest values within a distance of . times the interquartile range. as can be seen, most p-values were above e- , but a few methodological features have associated clusters with very low p-values, such as f , f , f , f , f , and f . the library preparation kit scriptseq v rna-seq, illumina (f ) displays strongly associated clusters with p-values as low as . e- that mapped as species avian myeloblastosis-associated virus. clusters that were annotated as ncbi species parvovirus nih/cqv were associated to laboratory-kit rneasy minelute, qiagen (f ) with minimal p-value . e- . finally, a cluster annotated as acanthocystis turfacea chlorella virus mn . (atcv) was associated to dnase/rnase: promega dnase stop solution (f ) with p-value = . e- . using blast and the ncbi taxonomy database a taxonomic characterisation was attempted for the , contigs in the clusters. this resulted in a taxonomical annotation of clusters using blastn and an additional clusters when using blastx. for clusters, neither blastn nor blastx found significant species in the database. these clusters remained uncharacterised (table ) . we found that almost all clusters significantly associated to biological features could be annotated ( of ) in contrast to non-biologically associated clusters ( of ). a total of unique species were annotated corresponding to clusters. the human microbiome project (hmp) [ ] defines a collection of reference genomes built from metagenomic samples and associates these to specific sites and tissues across human body sites. we used this data set of associations as a confirmation that our pipeline was able to correctly detect and taxonomically characterise recurrent biologically relevant sequences. hmp provides a list of commensal organisms commonly found in the three sites that relate to our samples: the gastrointestinal tract, oral cavity and urogenital tract. we observed the strongest, significant associations between the expected organisms and biopsies from colon cancer, oral cavity cancer, and vulva cancer. the taxonomical characterisation of these clusters is described in table . seven clusters significantly associated to colon cancer biopsies describing four different organisms that inhabit the gastrointestinal tract according to hmp, and clusters significantly associated to oral cavity cancer describing different organisms present in the oral cavity in hmp. finally, we also discovered a cluster significantly associated to vulva cancer annotated as species campylobacter ureolyticus (p-value = . e- ), an inhabitant of the urogenital tract as described by hmp. table . taxonomical characterisation of certain biologically associated clusters. the clusters are significantly associated with lowest p-values to biological features and the species annotations are described by hmp. in cases where several clusters shared the annotated species, the lowest p-value of the associations is given #sig: number of significant clusters. cluster in the methodological associations, we correctly detected the strong known association (p-value: . e- ) of avian myeloblastosis-associated virus (accession l . ) used in the manufacture of the scriptseq v rna-seq library preparation kit (f ). as the clustering parameters were evaluated with this known contaminant, this is expected. furthermore, we annotated clusters as ncbi taxonomy species parvovirus nih-cqv (accession kc . ; ncbi taxa-id ), an established contaminant [ , ] . the associated feature with lowest p-value to the parvovirus clusters suggested a contamination from the rneasy minelute purification kit (f ) manufactured by qiagen (p-value: . e- ). in addition, a single cluster annotated as ncbi taxonomy species acanthocystis turfacea chlorella virus mn . (accession jx . , taxa-id ) with lowest associated p-value (p-value = . e- ) to laboratory kit dnase/rnase: promega dnase stop solution (f ). atcv- was previously reported as a contaminant [ ] . in addition to the sequences that were characterised in the previous step, we found examples of uncharacterised clusters. the contigs in these clusters varied substantially in length ranging from a minimum of bp to a maximum of . kb (n = bp). our approach provides the capability to discover these recurrent novel sequences, but also permits the investigation of their plausible origin. most associations were methodological (table ), probably sourcing from nucleotide sequences contained in various laboratory kits (figure ) . for instance, out of the methodologically associated clusters, there were associated clusters to the laboratory reagent dnase/rnase: promega dnase stop solution (minimum p-value: . e- ). additionally, recurring sequences were attributed to technical issues of the flow cell lanes (minimum p-value: . e- in feature ). in total, unmapped clusters were associated to a biological feature, namely oral cavity cancer, with the longest contig of each cluster at , , , and bp and with respective p-values of . e- , . e- , . e- , and . e- . to further clarify the unresolved biologically associated sequences, we manually investigated the cluster representatives using the newest databases (december ) at the ncbi web-interfaces for blastn, blastx and ccd v. . (conserved domains) [ ] with default parameters and an e-value < . (table ) . all cluster representatives could be explained as commensal bacteria related to the oral cavity as described by hmp. in order of increasing length, the cluster representatives were identified as: prevotella veroralis, prevotella veroralis, prevotella fusca jcm , and peptostreptococcus anaerobius as the best hits with percent sequence identity: %, %, %, and %, respectively. cluster representatives and contained both bacterial and phage-like conserved domains. the super family duf is of unknown function but related to bacteria and the nd super family is the nicotine adenine dinucleotide (nadh) dehydrogenase subunit involved in electron transport. conversely, phage_base_v is related to the tail of phages and rve is an integrase domain that could also be explained as part of a transposon. likely these sequences derived from less well-described parts of the microbiome. usually, virus discovery in shotgun sequencing studies involves processing millions of reads in a viroinformatics pipeline. existing tools typically offer a comprehensive taxonomical description of a single sample that is compared to the taxonomy of other samples to determine their relevance. a downside of this bottom-up methodology is that novel sequences that cannot be sufficiently well characterised in the first round are often discarded in the process. another disadvantage is that potential contaminants will have to be controlled for in the post-processing of the data, an effort that is often omitted [ ] . in the present study, we have presented a methodology to categorise recurring sequences according to experimental origin and metadata features. additionally, using this methodology we could replicate both biological and methodological sequence associations known from the literature as well as pinpoint new unannotated recurring sequences. in this study, we had no datasets and features of healthy biological controls. we included a comparison to published reference genomes from hmp to validate that biologically co-occurring sequences can be found with the presented methodology. in this case, we are most likely observing normal biological inhabitants of the tissue samples, something our metadata scheme does not account for. the disease association of many of these organisms is obviously not fully known, and some of them could be related to disease features outside the cancer domain, features that we did not include in the present study. optimising clustering parameters for one virus family might not result in the optimal separation of other families. here, we optimised clustering parameters to rediscover the association of sequences to a known laboratory kit. using these clustering parameters may result in a non-optimal separation of clusters that biologically belonged together, or the reverse problem-merged clusters that reflected different biological units. optimal separation is likely problem-specific. different taxonomic units would require the use of different clustering parameters to separate. however, choosing taxonomy-specific parameters requires a working hypothesis of the most likely findings. here, we focused on the general problem of associating sequences to features using a known association to guide the choice of clustering parameters. a combination of several features may be the true foundation of particular sequences but this was not explored in this work. there may also be situations where a combination of clusters is the correct association to a particular feature. for instance, a virus that is present with a low titre may be sequenced sporadically resulting in less than full coverage and several non-coherent contigs from different viral genome regions. each cluster may include an incomplete amount of data sets and thereby artificially show a weaker association. merged and viewed as one, the incomplete clusters will have the correct strength of association. a grouping based on taxonomy, or a more data-driven approach that cluster sequence groups based on the associated datasets as seen in figure , could be included as another iteration to properly strengthen the statistical associations. furthermore, forming clusters only by internal sequence identity may also miss pathogenic scenarios such as an oncovirus and any necessary helper viruses that do not share homology to the oncovirus. in the present study, we used a majority vote to assign taxonomy. there could be other ways to assign taxonomy, for instance, using a lowest common ancestor (lca) strategy. a majority vote will likely introduce some false assignments if there are distant taxa involved in the sequence group present in nearly equal fractions. a lca strategy can handle this but may reduce the taxonomic resolution to a level where there is no real gain of information. after determining what the significantly co-occurring sequence groups are, more effort might resolve interesting unmapped contigs. for instance, use of more sensitive alignment algorithms, profile hidden markov models (hmms), gene predictors, artificial neural networks trained on specific signals such as viral capsid sequences [ ] , or pcr extraction followed by sanger sequencing might provide the relevant clues. however, that was not within the scope of this study. the major advantage of the top-down approach is that it works without prior knowledge of the sequences. it is not dependent on reference sequence databases to single out the promising candidates for further analysis. the top-down method can determine the relevance of unknown sequences upfront while also systematically controlling for contamination by design. most of the annotated sequences found in this study were sequenced from cancer specimens. however, it is apparent from the association analysis that several viral sequences detected are possibly contaminants or technical artefacts. furthermore, the unmapped clusters are retained and easily arranged by relevance according to the nature of their associated features. having this information helps precipitate a prioritised list of sequence candidates the quality of the associations will depend on the experimental design, sampling, available metadata, as well as the rigorousness and standardisation of both working routines and annotations. we stress the point that care must still be taken when formulating hypotheses and in the interpretation of associations. virus discovery using high-throughput sequencing and especially characterising clinical samples is a challenge. many viral discovery pipelines rely on similarity to reference databases as the most compelling argument for identifying putative sequences of medical or biological importance. although a necessary step in the analysis, it has the downside of not considering novel sequences not included in reference sets as well as not considering the origins of the discoveries. there are many examples of contamination and technical artefacts; therefore, potential discoveries should be accompanied by convincing evidence that the sequences are not instead associated with the methodology or technology in use. we suggest a different approach that has complementary advantages inherent in the design. we show that we can differentiate between biological and non-biological associations, replicate known associations and potentially add new associations of cancer-associated viruses. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / , table s : virus discovery pipelines, table s : distribution of library types, table s : methods in cancer samples, table s : clustering parameters, table s : feature descriptions, table s : datasets in clusters, figure s : clustering performance. identification of a new human coronavirus cloning of a human parvovirus by molecular screening of respiratory tract samples new dna viruses identified in patients with acute viral infection syndrome characterization and complete genome 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idba-ud: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences r: a language and environment for statistical computing; r foundation for statistical computing basic local alignment search tool species evenness and productivity in experimental plant communities avian myeoloblastosis virus (amv): only one side of the coin a mathematical theory of communication how does multiple testing correction work? a framework for human microbiome research cdd: ncbi's conserved domain database artificial neural networks trained to detect viral and phage structural proteins the authors declare no conflict of interest. the founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. key: cord- - v xuicg authors: ba abduallah, mohamed m.; hemida, maged gomaa title: comparative analysis of the genome structure and organization of the middle east respiratory syndrome coronavirus (mers‐cov) to revealing evidence for virus strain barcoding, zoonotic transmission, and selection pressure date: - - journal: rev med virol doi: . /rmv. sha: doc_id: cord_uid: v xuicg the middle east respiratory syndrome coronavirus (mers‐cov) emerged in late in saudi arabia. for this study, we conducted a large‐scale comparative genome study of mers‐cov from both human and dromedary camels from to to map any genetic changes that emerged in the past years. we downloaded submissions, including full‐length genome sequences of mers‐cov available in genbank from to . we used bioinformatics tools to describe the genome structure and organization of the virus and to map the most important motifs within various regions/genes throughout the genome over the past years. we also monitored variations/mutations among these sequences since its emergence. our phylogenetic analyses suggest that the cluster within african camels is derived by s gene. we identified some prominent motifs within the orf ab, s gene and orf‐ , which may be used for barcoding the african camel lineages of mers‐cov. furthermore, we mapped some sequence patterns that support the zoonotic origin of the virus from dromedary camels. other sequences identified selection pressures, particularly within the n gene and the ′ utr. further studies are required for careful monitoring of the mers‐cov genome to identify any potential significant mutations in the future. belong to the betacoronaviruses. the sars-cov emerged in ; the mers-cov was discovered in , and the sars-cov- was reported in late . the time gap between sars-cov and mers-cov is around years, and between the emergence of mers-cov and sars-cov- is years. the continuous emergence of new coronaviruses candidates may be attributed to the features of their genomes. this may be due to several factors, including the low fidelity of their rna-dependant rna polymerases (rdrp), the possibility of recombination, and the high level of expression of their receptors in many mammalian and avian species. thus, there is an urgent need for the regular monitoring of the genome sequences of coronaviruses from various species of bats, animals, and birds. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the main goal of the current study was to do a com- includes the majority of viral isolates from human and some from camel origins. clade-c includes viruses of camel origin isolated from various countries in africa, including egypt, morocco, nigeria, burkina faso, and kenya. , interestingly, results from the virus neutralization assay revealed that all three clades are closely related, which supports the notion that one vaccine may be able to protect against all three clades. it is believed that mers-cov originated in bats and spilled over to humans via an intermediate host, dromedary camels. , dromedary camels are the main known reservoir until now. genome analysis of mers-cov isolates from human and dromedary camel origins revealed a close relationship between each other, suggesting the zoonotic origin of the virus. like other coronaviruses, mers-cov continues to show some changes at the genome level. thus, new virus clades and sub-clades are recently reported of both human and dromedary camel origins. regular monitoring of the genetic makeup of the virus is very important to track down any potential mutation or recombination. the main goal of this study was to do a deep bioinformatic analysis of the most available mers-cov genome sequences in genbank during to to understand the evolution of the virus and map any potential changes across the viral genome. therefore, the development of potential diagnostic assays, vaccines, or therapy should cope with any potential changes over the viral genome. this monitoring may also contribute substantially to the control of the virus by knowing the currently circulating clades in a certain community. an earlier study showed the circulation of three different genotypes of the virus in some patients during in ksa. one year later, the same group reported the presence of four mers-cov clades during the early emergence of mers-cov. later, during three clades were no longer contributing to the reported human cases suggesting their extinction. the main reason behind these changes was the dynamic changes among the s gene of the virus. , studies on mers-cov isolates from various countries in northern and central africa revealed that the circulating strains of the virus in dromedary camels from these countries belong to lineage-c. this lineage is different from the other two lineages reported earlier in the arabian peninsula. although the three lineages have some genetic variations, their antigenic properties remain identical, as shown by the virus neutralization test. isolates from dromedary camels collected from nigeria, burkina faso, and morocco clustered together into a new sublineage-c due to shared genetic signatures, including deletions in orf b. thus, there is an urgent need for continued study not only of mers-cov but also other coronaviruses in the context of the human-animal interface and to understand the biological diversity of coronaviruses. mers-cov submissions were considered as a full-length genome only if they meet three parameters. first, the length of the sequence must be greater than or equal to -kilobases. second, the submission must have the full utr sequence. third, the submission must have a poly-a tail even represented by one nucleotide of adenine (a). multiple sequence alignment was conducted using the mafft tool (http://ma.cbrc.jp/alignment/soware/). single nucleotide polymorphism (snp) density (excluding indels) was counted from the multiplesequence alignment of orf ab and s gene sequences by use of an in-house script written in python. for data visualization, geneious (version . . ) was used. to avoid losing open reading frames (orfs) within different sequences, orfs were collected by retrieving regions flanked by conserved sequences, as shown in (table ) . conserved sequences were obtained from multiple sequence alignment of complete whole mers-cov genomes. different lengths of orf b and orf b were calculated at its minimum possible size ( nt), allowing any start codon (atg, ttg, or ctg) to initiate the orf. mapping the cleavage sites of the nsps among gene- was carried out as previously described. phylogenetic trees were constructed using mega x software using multiple sequence alignment of the whole genome, orf ab, s gene, orf b, orf , e gene, m gene, n gene and orf b sequences. the trees were constructed using maximum likelihood methods and the tamura-nei model. bootstrap analysis ( pseudo-replicates) was conducted to evaluate the statistical significance of the inferred trees, and only values greater than were displayed. since most isolated sequences did not meet the parameters used in this study for the whole-genome sequences, we conducted the analysis on individual genes. for the comparative study of phylogenetic trees, we initially considered sequences greater than kb since most of the isolated sequences (especially isolates from african camels) did not meet the parameters used in this study for the whole-genome sequences. then, to make the tree much simpler and easily readable, we narrowed we used full-length genome sequences after applying filteration parameters. all relevant data of these sequences are summarized in table s . (table ) . orf ab consists of two overlapping orfs (orf- a and orf- b). the orf- b is produced by ribosomal frameshifting in which the ribosome steps back one nucleotide and continues reading and producing orf- b ( figure ). we analyzed sequences of orf ab (table s ). orf ab is nt in length, and the position of the ribosomal frameshifting is located at nt of jx . distribution of polymorphisms (snps found in more than one strain in a -bp sliding window) showed that snp density in orf ab varies from to ( figure s a ). from multiple sequence alignment, we identified eight variants seem to be restricted to mers-cov isolated from african camels. all these variants exited in studied african camels only (table s ). table ). details about the nsps, their locations, as well as sizes, are shown in table . we analyzed of the s-gene sequences (table s ). the full-length of the s gene is about nt. the organization of the mers-cov-s gene is described in figure b (table ) . we conducted an snp density analysis to test whether the three observed variants of the s gene in african camel samples were not in a highly variable region. the distribution of polymorphisms (snps found in more than one strain in a -bp sliding window) showed that snp density in s gene varies from to ( figure s ). the variant v a is located in a region with a snp density of five, while both r q and a s/l substitutions are located in a region of low snp density (snp density = ). the total number of different nucleotides between samples in the phylogenetic tree of the s gene sequences is shown in table s . the phylogenetic tree of the mers-cov of s gene sequences is presented in figure c . compared to the phylogenetic tree of mers-cov whole-genome and orf ab sequences (figure a (table s ). there was no sample observed with the two mutations (i t and d g) at the same time. in the case of orf- , we analyzed sequences (table s ). the length of orf gene is -nt. however, in some submissions, we found truncated versions of the encoded protein of this orf due to deletions varying from -nt to -nt (table ) . two variants were the orf- mainly consists of two overlapping orfs (orf- a and b). in the case of orf- a, we analyzed sequences (table s ). the orf a gene is -nt in length, and there is -nt overlapping between orf a and orf b genes. the orf a is highly conserved, but snp density increases in the last nt (data not shown). we analyzed sequences of the orf- b gene ( figure revealed that all models of the defective gene orf b and the wild type gene orf b could produce an identical potential orf of a.a. in length. we analyzed mers-cov sequences of orf (table s ). the fulllength of gene orf is nt, which encodes a protein of aa in length. we found two strains encoding a truncated orf protein due to deletions causing a frameshift and stop codon ( (figure and figure s ). we analyzed sequences of e gene and sequences of m gene (table s ). the lengths for e protein and m protein are -a.a. and -a.a., respectively. most of the e and m gene sequences were quite conserved and had no significant observation either at the nucleotide or amino acid level. however, we considered observed variants not significant as they either existed in only one sample or in sequences generated by the same group. the number of retrieved and analyzed sequences of n gene was (table s ). the full-length of gene n is nt, which encodes a table ). we analyzed sequences of gene orf- b (table s ). the fulllength of the wild type gene orf is nt, which encodes a protein of aa in length. this orf is located within the n gene (from to of the jx genome sequencing; figure ). (table s ). on the other hand, strains have truncated orf protein ( -aa shorter than the wild type) due to a mutation at position of jx , changing tca to a tga (stop codon). one of these sequences was isolated from a human in qatar during while the others were of human origin from ksa in (one of them was sequenced in indiana-kj ; table s ). all studied african camels ( samples mers-cov isolated from african camels is shown to be phylogenetically distinct from those circulating in the arabian peninsula. , , in this study, we showed that the cluster of mers-cov african camels into (table ) . we tested the possibility of a defective version of the orf gene that can produce orf protein with a length close to the wild type by starting from different frames rather than frame- , all possible orfs were tested. there was no defective form of gene orf that produced orf protein with a length close to the wild type ( figure s ). however, the biological consequence behind the variation of orf protein length is still unknown. comparative genome sequence analysis of the mers-cov of both dromedary camels and human origins revealed significant evidence for potential barcoding of the african clades based on the s gene sequences. it also provided evidence for the zoonotic origins of the virus from dromedary camels to humans and highlighted the role of selection pressure and compensatory mechanisms in virus genome evolution. isolation of a novel coronavirus from a man with pneumonia in saudi arabia ninth report of the international committee on taxonomy of viruses origin and evolution of pathogenic coronaviruses middle east respiratory syndrome coronavirus and the one health concept genomic variance of the -ncov coronavirus full genome characterization of two novel alpha-coronavirus species from italian bats full-length genome analysis of canine coronavirus type i molecular investigation of a fulllength genome of a q -like ibv strain isolated in italy in a metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in neoromicia bats within south africa molecular identification and characterization of novel coronaviruses infecting graylag geese (anser anser), feral pigeons (columbia livia) and mallards complete genome analysis of equine coronavirus isolated in japan full-length genome sequences of porcine epidemic diarrhoea virus strain cv ; use of ngs to analyse genomic and sub-genomic rnas mers coronaviruses from camels in africa exhibit region-dependent genetic diversity genomics and zoonotic infections: middle east respiratory syndrome detection of distinct mers-coronavirus strains in dromedary camels from kenya dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus transmission spread, circulation, and evolution of the middle east respiratory syndrome coronavirus transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage mega : molecular evolutionary genetics analysis version . for bigger datasets variations in spike glycoprotein gene of mers-cov the severe acute respiratory syndrome a novel coronavirus from patients with pneumonia in china middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in africa and middle east serological evidence of mers-cov and hku -related cov co-infection in kenyan camels the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination we wish to thank the king abdul-aziz city for science and technology (kacst), saudi arabia, for their generous funding through the mers-cov research grant program (number - ), which is a part of the targeted research program (trp). the authors have no competing interest. https://orcid.org/ - - - additional supporting information may be found online in the supporting information section at the end of this article. key: cord- -tw armh authors: ma, junling; van den driessche, p.; willeboordse, frederick h. title: the importance of contact network topology for the success of vaccination strategies date: - - journal: journal of theoretical biology doi: . /j.jtbi. . . sha: doc_id: cord_uid: tw armh abstract the effects of a number of vaccination strategies on the spread of an sir type disease are numerically investigated for several common network topologies including random, scale-free, small world, and meta-random networks. these strategies, namely, prioritized, random, follow links and contact tracing, are compared across networks using extensive simulations with disease parameters relevant for viruses such as pandemic influenza h n / . two scenarios for a network sir model are considered. first, a model with a given transmission rate is studied. second, a model with a given initial growth rate is considered, because the initial growth rate is commonly used to impute the transmission rate from incidence curves and to predict the course of an epidemic. since a vaccine may not be readily available for a new virus, the case of a delay in the start of vaccination is also considered in addition to the case of no delay. it is found that network topology can have a larger impact on the spread of the disease than the choice of vaccination strategy. simulations also show that the network structure has a large effect on both the course of an epidemic and the determination of the transmission rate from the initial growth rate. the effect of delay in the vaccination start time varies tremendously with network topology. results show that, without the knowledge of network topology, predictions on the peak and the final size of an epidemic cannot be made solely based on the initial exponential growth rate or transmission rate. this demonstrates the importance of understanding the topology of realistic contact networks when evaluating vaccination strategies. the importance of contact network topology for the success of vaccination strategies for many viral diseases, vaccination forms the cornerstone in managing their spread and the question naturally arises as to which vaccination strategy is, given practical constraints, the most effective in stopping the disease spread. for evaluating the effectiveness of a vaccination strategy, it is necessary to have as precise a model as possible for the disease dynamics. the widely studied key reference models for infectious disease epidemics are the homogeneous mixing models where any member of the population can infect or be infected by any other member of the population; see, for example, anderson and may ( ) and brauer ( ) . the advantage of a homogeneous mixing model is that it lends itself relatively well to analysis and therefore is a good starting point. due to the homogeneity assumption, these models predict that the fraction of the population that needs to be vaccinated to curtail an epidemic is equal to À =r , where r is the basic reproduction number (the average number of secondary infections caused by a typical infectious individual in a fully susceptible population). however, the homogeneous mixing assumption poorly reflects the actual interactions within a population, since, for example, school children and office co-workers spend significant amounts of time in close proximity and therefore are much more likely to infect each other than an elderly person who mostly stays at home. consequently, efforts have been made to incorporate the network structure into models, where individuals are represented by nodes and contacts are presented by edges. in the context of the severe acute respiratory syndrome (sars), it was shown by meyers et al. ( ) that the incorporation of contact networks may yield different epidemic outcomes even for the same basic reproduction number r . for pandemic influenza h n / , pourbohloul et al. ( ) and davoudi et al. ( ) used network theory to obtain a real time estimate for r . numerical simulations have shown that different networks can yield distinct disease spread patterns; see, for example, bansal et al. ( ) , miller et al. ( ) , and section . in keeling and rohani ( ) . to illustrate this difference for the networks and parameters we use, the effect of different networks on disease dynamics is shown in fig. . descriptions of these networks are given in section and appendix b. at the current stage, most theoretical network infectious disease models incorporate, from a real world perspective, idealized random network structures such as regular (all nodes have the same degree), erd + os-ré nyi or scale-free random networks where clustering and spatial structures are absent. for example, volz ( ) used a generating function formalism (an alternate derivation with a simpler system of equations was recently found by miller, ) , while we used the degree distribution in the effective degree model presented in lindquist et al. ( ) . in these models, the degree distribution is the key network characteristic for disease dynamics. from recent efforts (ma et al., ; volz et al., ; moreno et al., ; on incorporating degree correlation and clustering (such as households and offices) into epidemic models, it has been found that these may significantly affect the disease dynamics for networks with identical degree distributions. fig. shows disease dynamics on networks with identical degree distribution and disease parameters, but with different network topologies. clearly, reliable predictions of the epidemic process that only use the degree distribution are not possible without knowledge of the network topology. such predictions need to be checked by considering other topological properties of the network. network models allow more precise modeling of control measures that depend on the contact structure of the population, such as priority based vaccination and contact tracing. for example, shaban et al. ( ) consider a random graph with a pre-specified degree distribution to investigate vaccination models using contact tracing. kiss et al. ( ) compared the efficacy of contact tracing on random and scale-free networks and found that for transmission rates greater than a certain threshold, the final epidemic size is smaller on a scale-free network than on a corresponding random network, while they considered the effects of degree correlations in kiss et al. ( ) . cohen et al. ( ) (see also madar et al., ) considered different vaccination strategies on scale-free networks and found that acquaintance immunization is remarkably effective. miller and hyman ( ) considered several vaccination strategies on a simulation of the population of portland oregon, usa, and found it to be most effective to vaccinate nodes with the most unvaccinated susceptible contacts, although they found that this strategy may not be practical because it requires considerable computational resources and information about the network. bansal et al. ( ) took a contact network using data from vancouver, bc, canada, considered two vaccination strategies, namely mortality-and morbidity-based, and investigated the detrimental effect of vaccination delays. and found that, on realistic contact networks, vaccination strategies based on detailed network topology information generally outperform random vaccination. however, in most cases, contact network topologies are not readily available. thus, how different network topologies affect various vaccination strategies remains of considerable interest. to address this question, we explore two scenarios to compare percentage reduction by vaccination on the final size of epidemics across various network topologies. first, various network topologies are considered with the disease parameters constant, assuming that these have been independently estimated. second, different network topologies are used to fit to the observed incidence curve (number of new infections in each day), so that their disease parameters are different yet they all line up to the same initial exponential growth phase of the epidemic. vaccines are likely lacking at the outbreak of an emerging infectious disease (as seen in the h n pandemic, conway et al., ) , and thus can only be given after the disease is already widespread. we investigate numerically whether network topologies affect the effectiveness of vaccination strategies started with a delay after the disease is widespread; for example, a day delay as in the second wave of the influenza pandemic in british columbia, canada (office of the provincial health officer, ). details of our numerical simulations are given in appendix a. this paper is structured as follows. in section , a brief overview of the networks and vaccination strategies (more details are provided in appendices b and c) is given. in section , we investigate the scenario where the transmission rate is fixed, while in section we investigate the scenario where the growth rate of the incidence curve is fixed. to this end, we compute the incidence curves and reductions in final sizes (total number of infections during the course of the epidemic) due to vaccination. for the homogeneous mixing model, these scenarios are identical (ma and earn, ) , but as will be shown, when taking topology into account, they are completely different. we end with conclusions in section . . on all networks, the average degree is , the population size is , , the transmission rate is . , the recovery rate is . , and the initial number of infectious individuals is set to . both graphs represent the same data but the left graph has a semi-log scale (highlighting the growth phase) while the right graph has a linear scale (highlighting the peak). (b)) on networks with identical disease parameters and degree distribution (as shown in (a)). the network topologies are the random, meta-random, and near neighbor networks. see appendix b for details of the constructions of these networks. detailed network topologies for human populations are far from known. however, this detailed knowledge may not be required when the main objective is to assert the impact that topology has on the spread of a disease and on the effects of vaccination. it may be sufficient to consider a number of representative network topologies that, at least to some extent, can be found in the actual population. here, we consider the four topologies listed in table , which we now briefly describe. in the random network, nodes are connected with equal probability yielding a poisson degree distribution. in a scale-free network, small number of nodes have a very large number of links and large number of nodes have a small number of links such that the degree distribution follows a power law. small world (sw) networks are constructed by adding links between randomly chosen nodes on networks in which nodes are connected to the nearest neighbors. the last network considered is what we term a meta-random network where random networks of various sizes are connected with a small number of interlinks. all networks are undirected with no self loops or multiple links. the histograms of the networks are shown in table , and the details of their construction are given in appendix b. the vaccination strategies considered are summarized in table . in the random strategy, an eligible node is randomly chosen and vaccinated. in the prioritized strategy, nodes with the highest degrees are vaccinated first, while in the follow links strategy, inspired by notions from social networks, a randomly chosen susceptible node is vaccinated and then all its neighbors and then its neighbor's neighbors and so on. finally, in contact tracing, the neighbors of infectious nodes are vaccinated. for all the strategies, vaccination is voluntary and quantity limited. that is, only susceptibles who do not refuse vaccination are vaccinated and each day only a certain number of doses is available. in the case of (relatively) new viral diseases, the supply of vaccines will almost certainly be constrained, as was the case for the pandemic influenza h n / virus. also in the case of mass vaccinations, there will be resource limitations with regard to how many doses can be administered per day. the report (office of the provincial health officer, ) states that the vaccination program was prioritized and it took weeks before the general population had access to vaccination. thus we assume that a vaccination program can be completed in - weeks or about days, this means that for a population of , , a maximum of doses a day can be used. for each strategy for each time unit, first a group of eligible nodes is identified and then up to the maximum number of doses is dispensed among the eligible nodes according to the strategy chosen. more details of the vaccination strategies and their motivations are given in appendix c. to study the effect of delayed availability of vaccines during an emerging infectious disease, we compare the effect of vaccination programs starting on the first day of the epidemic with those vaccination programs starting on different days. these range from to days after the start of the epidemic, with an emphasis on a day delay that occurred in british columbia, canada, during the influenza h n / pandemic. when a node is vaccinated, the vaccination is considered to be ineffective in % of the cases (bansal et al., ) . in such cases, the vaccine provides no immunity at all. for the % of the nodes for which the vaccine will be effective, a two week span to reach full immunity is assumed (clark et al., ) . during the two weeks, we assume that the immunity increases linearly starting with at the time of vaccination reaching % after days. the effect of vaccination strategies has been studied (see, for example, conway et al., ) using disease parameter values estimated in the literature. however, network topologies were not the focus of these studies. in section , the effect of vaccination strategies on various network topologies is compared with a fixed per link transmission rate. the per link transmission rate b is difficult to obtain directly and is usually derived as a secondary quantity. to determine b, we pick the basic reproduction number r ¼ : and the recovery rate g ¼ : , which are close to that of the influenza a h n / virus; see, for example, pourbohloul et al. ( ), tuite et al. ( . in the case of the homogeneous mixing sir model, the basic reproduction number is given by r ¼ t=g, where t is the per-node transmission rate. our table illustration of the different types of networks used in this paper. scale-free small world meta-random table degree histograms of the networks in table with , nodes. scale-free small world meta-random parameter values yield t ¼ : . for networks, t ¼ b/ks. with the assumption that the average degree /ks ¼ , the above gives the per-link transmission rate b ¼ : . the key parameters are summarized in table . in section , we use this transmission rate to compare the incidence curves for the networks in table with the vaccination strategies in table . some of the most readily available data in an epidemic are the number of reported new cases per day. these cases generally display exponential growth in the initial phase of an epidemic and a suitable model therefore needs to match this initial growth pattern. the exponential growth rates are commonly used to estimate disease parameters (chowell et al., ; lipsitch et al., ) . in section , we consider the effects of various network topologies on the effectiveness of vaccination strategies for epidemics with a fixed exponential growth rate. the basic reproduction number r ¼ : and the recovery rate g ¼ : yield an exponential growth rate l ¼ tÀg ¼ : for the homogeneous mixing sir model. we tune the transmission rate for each network topology to give this initial growth rate. in this section, the effectiveness of vaccination strategies on various network topologies is investigated for a given set of parameters, which are identical for all the simulations. the values of the disease parameters are chosen based on what is known from influenza h n / . qualitatively, these chosen parameters should provide substantial insight into the effects topology has on the spread of a disease. unless indicated otherwise the parameter values listed in table are used. the effects of the vaccination strategies summarized in table when applied without delay are shown in fig. . for reference, fig. shows the incidence curves with no vaccination. since the disease dies out in the small world network (see fig. ), vaccination is not needed in this network for the parameter values taken. especially in the cases of the random and meta-random networks, the effects of vaccination are drastic while for the scale-free network they are still considerable. what is particularly notable is that when comparing the various outcomes, topology has as great if not a greater impact on the epidemic than the vaccination strategy. besides the incidence curves, the final sizes of epidemics and the effect vaccination has on these are also of great importance. table shows the final sizes and the reductions in the final sizes for the various networks on which the disease can survive (for the chosen parameter values) with vaccination strategies for the cases where there is no delay in the vaccination. fig. and table show the incidence curves and the reductions in final sizes for the same parameters as used in fig. and table but with a delay of days in the vaccination. as can be expected for the given parameters, a delay has the biggest effect for the scale-free network. in that case, the epidemic is already past its peak and vaccinations only have a minor effect. for the random and meta-random networks, the table illustration of vaccination strategies. susceptible nodes are depicted by triangles, infectious nodes by squares, and the vaccinated nodes by circles. the average degree in these illustrations has been reduced to aid clarity. the starting point for contact tracing is labeled as a while the starting point for the follow links strategy is labeled as b. the number of doses dispensed in this illustration is . random follow links contact tracing table for the network topologies in table given a fixed transmission rate b. there is no delay in the vaccination and parameters are equal to those used in fig. . to further investigate the effects of delay in the case of random vaccination, we compute reductions in final sizes for delays of , , ,y, days, in random, scale-free, and meta-random networks. fig. shows that, not surprisingly, these reductions diminish with longer delays. however, the reductions are strongly network dependent. on a scale-free network, the reduction becomes negligible as the delay approaches the epidemic peak time, while on random and meta-random networks, the reduction is about % with the delay at the epidemic peak time. this section clearly shows that given a certain transmission rate b, the effectiveness of a vaccination strategy is impossible to predict without having reliable data on the network topology of the population. next, we consider the case where instead of the transmission rate, the initial growth rate is given. we line up incidence curves on various network topologies to a growth rate l predicted by a homogeneous mixing sir model with the basic reproduction number r ¼ : and recovery rate g ¼ : (in this case with exponential, l ¼ ðr À Þg ¼ : ). table summarizes the transmission rates that yield this exponential growth rate on the corresponding network topologies. the initial number of infectious individuals for models on each network topology needs to be adjusted as well so that the curves line up along the homogeneous mixing sir incidence curve for days. as can be seen from the table, the variations in the parameters are indeed very large, with the transmission rate for the small world network being nearly times the value of the transmission rate for the scale-free network. the incidence curves corresponding to the parameters in table are shown in fig. . as can clearly be seen, for these parameters, the curves overlap very well for the first days, thus showing indeed the desired identical initial growth rates. however, it is also clear that the curves diverge strongly later on, with the epidemic on the small world network being the most severe. these results show that the spread of an epidemic cannot be predicted on the basis of having a good estimate of the growth rate alone. in addition, comparing figs. and , a higher transmission rate yields a much larger final size and a longer epidemic on the meta-random network. the effects of the various vaccination strategies for the case of a given growth rate are shown in fig. . given the large differences in the transmission rates, it may be expected that the final sizes show significant differences as well. this is indeed the case as can be seen in table , which shows the percentage reduction in final sizes for the various vaccination strategies. with no vaccination, the final size of the small world network is more than times that of the scale-free network, but for all except the follow links vaccination strategy the percentage reduction on the small world network is greater. the effects of a -day delay in the start of the vaccination are shown in fig. and table . besides the delay, all the parameters are identical to those in fig. and table . the delay has the largest effect on the final sizes of the small world network, increasing it by a factor of - except in the follow links case. on a scale-free network, the delay renders all vaccination strategies nearly ineffective. these results also confirm the importance of network topology in disease spread even when the incidence curves have identical initial growth. the initial stages of an epidemic are insufficient to estimate the effectiveness of a vaccination strategy on reducing the peak or final size of an epidemic. the relative importance of network topology on the predictability of incidence curves was investigated. this was done by considering whether the effectiveness of several vaccination strategies is impacted by topology, and whether the growth in the daily incidences has a network topology independent relation with the disease transmission rate. it was found that without a fairly detailed knowledge of the network topology, initial data cannot predict epidemic progression. this is so for both a given transmission rate b and a given growth rate l. for a fixed transmission rate and thus a fixed per link transmission probability, given that a disease spreads on a network with a fixed average degree, the disease spreads fastest on scale-free networks because high degree nodes have a very high probability to be infected as soon as the epidemic progresses. in turn, once a high degree node is infected, on average it passes on the infection to a large number of neighbors. the random and meta-random networks show identical initial growth rates because they have the same local network topology. on different table without vaccination for the case where the initial growth rate is given. the transmission rates and initial number of infections for the various network topologies are given in table , while the remaining parameters are the same as in fig. meta-random network fig. . the effects of the vaccination strategies for different topologies when the initial growth rate is given. the transmission rates b are as indicated in table , while the remaining parameters are identical to those in fig. . network topologies, diseases respond differently to parameter changes. for example, on the random network, a higher transmission rate yields a much shorter epidemic, whereas on the metarandom network, it yields a longer one with a more drastic increase in final size. these differences are caused by the spatial structures in the meta-random network. considering that a metarandom network is a random network of random networks, it is likely that the meta-random network represents a general population better than a random network. for a fixed exponential growth rate, the transmission rate needed on the scale-free network to yield the given initial growth rate is the smallest, being about half that of the random and the meta-random networks. hence, the per-link transmission probability is the lowest on the scale-free network, which in turn yields a small epidemic final size. for different network topologies, we quantified the effect of delay in the start of vaccination. we found that the effectiveness of vaccination strategies decreases with delay with a rate strongly dependent on network topology. this emphasizes the importance of the knowledge of the topology, in order to formulate a practical vaccination schedule. with respect to policy, the results presented seem to warrant a significant effort to obtain a better understanding of how the members of a population are actually linked together in a social network. consequently, policy advice based on the rough estimates of the network structure should be viewed with caution. this work is partially supported by nserc discovery grants (jm, pvdd) and mprime (pvdd). we thank the anonymous reviewers for their constructive comments. the nodes in the network are labeled by their infectious status, i.e. susceptible, infectious, vaccinated, immune, refusing vaccination (but susceptible), and vaccinated but susceptible (the vaccine is not working), respectively. the stochastic simulation is initialized by first labeling all the nodes as susceptible and then randomly labeling i nodes as infectious. then, before the simulation starts, % of susceptible nodes are labeled as refusing vaccination but susceptible. during the simulation, when a node is vaccinated, the vaccine has a probability of % to be ineffective. if it is not effective, the node remains fully susceptible, but will not be vaccinated again. if it is effective, then the immunity is built up linearly over a certain period of time, taken as weeks. we assume that infected persons generally recover in about days, giving a recovery rate g ¼ : . the initial number of infectious individuals i is set to unless otherwise stated, to reduce the number of runs where the disease dies out due to statistical fluctuations. all simulation results presented in sections and are averages of runs, each with a new randomly generated network of the chosen topology. the parameters in the simulations are shown in table . the population size n was chosen to be sufficiently large to be representative of a medium size town and set to n ¼ , , while the degree average is taken as /ks ¼ with a maximum degree m¼ (having a maximum degree only affects the scalefree network since the probability of a node having degree m is practically zero for the other network types). when considering a large group of people, a good first approximation is that the links between these people are random. although it is clear that this cannot accurately represent the population since it lacks, for example, clustering and spatial aggregation (found in such common contexts as schools and work places), it may be possible that if the population is big enough, most if not all nonrandom effects average out. furthermore, random networks lend themselves relatively well to analysis so that a number of interesting (and testable) properties can be derived. as is usually the case, the random network employed here originates from the concepts first presented rigorously by erd + os and ré nyi ( ). our random networks are generated as follows: ( ) we begin by creating n unlinked nodes. ( ) in order to avoid orphaned nodes, without loss of generality, first every node is linked to another uniformly randomly chosen node that is not a neighbor. ( ) two nodes that are not neighbors and not already linked are uniformly randomly selected. if the degree d of both the nodes is less than the maximum degree m, a link is established. if one of the nodes has maximum degree m, a new pair of nodes is uniformly randomly selected. ( ) step is repeated n  /ksÀn times. when considering certain activities in a population, such as the publishing of scientific work or sexual contact, it has been found that the links are often well described by a scale-free network structure where the relationship between the degree and the number of nodes that have this degree follows a negative power law; see, for example, the review paper by albert and barabá si ( ) . scale-free networks can easily be constructed with the help of a preferential attachment. that is to say, the network is built up step by step and new nodes attach to existing nodes with a probability that is proportional to the degree of the existing nodes. our network is constructed with the help of preferential attachment, but two modifications are made in order to render the scale-free network more comparable with the other networks investigated here. first, the maximum degree is limited to m not by restricting the degree from the outset but by first creating a scale-free network and then pruning all the nodes with a degree larger than m. second, the number of links attached to each new node is either two or three dependent on a certain probability that is set such that after pruning the average degree is very close to that of the random network (i.e. /ks ¼ ). our scale-free network is generated as follows: ( ) start with three fully connected nodes and set the total number of links l¼ . ( ) create a new node. with a probability of . , add links. otherwise add links. for each of these additional links to be added find a node to link to as outlined in step . ( ) loop through the list of nodes and create a link with probability d=ð lÞ, where d is the degree of the currently considered target node. ( ) increase l by or depending on the choice in step . ( ) repeat nÀ times steps and . ( ) prune nodes with a degree m. small world networks are characterized by the combination of a relatively large number of local links with a small number of non-local links. consequently, there is in principle a very large number of possible small world networks. one of the simplest ways to create a small world network is to first place nodes sequentially on a circle and couple them to their neighbors, similar to the way many coupled map lattices are constructed (willeboordse, ) , and to then create some random short cuts. this is basically also the way the small world network used here is generated. the only modification is that the coupling range (i.e. the number of neighbors linked to) is randomly varied between and in order to obtain an average degree equal to that of the random network (i.e. /ks ¼ ). we also use periodic boundary conditions, which as such is not necessary for a small world network but is commonly done. the motivation for studying small world networks is that small groups of people in a population are often (almost) fully linked (such as family members or co-workers) with some connections to other groups of people. our small world network is generated as follows: ( ) create n new unlinked nodes with index i ¼ . . . n. ( ) with a probability of . , link to neighboring and second neighboring nodes (i.e. create links i iÀ , i iþ , i iÀ , i iþ ). otherwise, also link up to the third neighboring nodes (i.e. create links i iÀ , i i þ , i iÀ , i i þ , i iÀ , i i þ ). periodic boundary conditions are used (i.e. the left nearest neighbor of node is node n while the right nearest neighbor of node n is node ). ( ) create the 'large world' network by repeating step for each node. ( ) with a probability of . add a link to a uniformly randomly chosen node excluding self and nodes already linked to. ( ) create the small world network by carrying out step for each node. in the random network, the probability for an arbitrary node to be linked to any other arbitrary node is constant and there is no clear notion of locality. in the small world network on the other hand, tightly integrated local connections are supplemented by links to other parts of the network. to model a situation in between where randomly linked local populations (such as the populations of villages in a region) are randomly linked to each other (for example, some members of the population of one village are linked to some members of some other villages), we consider a meta-random network. when increasing the number of shortcuts, a meta-random network transitions to a random network. it can be argued that among the networks investigated here, a meta-random network is the most representative of the population in a state, province or country. our meta-random network is generated as follows: ( ) create n new unlinked nodes with index i ¼ . . . n. ( ) group the nodes into randomly sized clusters with a minimum size of nodes (the minimum size was chosen such that it is larger than /ks, which equals five throughout, to exclude fully linked graphs). this is done by randomly choosing values in the range from to n to serve as cluster boundaries with the restriction that a cluster cannot be smaller than the minimum size. ( ) for each cluster, create an erd + os-ré nyi type random network. ( ) for each node, with a probability . , create a link to a uniformly randomly chosen node of a uniformly randomly chosen cluster excluding its own cluster. the network described in this subsection is a near neighbor network and therefore mostly local. nevertheless, there are some shortcuts but shortcuts to very distant parts of the network are not very likely. it could therefore be called a medium world network (situated between small and large world networks). the key feature of this network is that despite being mostly local its degree distribution is identical to that of the random network. our near neighbor network is generated as follows: ( ) create n new unlinked nodes with index i ¼ . . . n. ( ) for each node, set a target degree by randomly choosing a degree with a probability equal to that for the degree distribution of the random network. ( ) if the node has reached its target degree, continue with the next node. if not continue with step . ( ) with a probability of . , create a link to a node with a smaller index, otherwise create a link to a node with a larger index (using periodic boundary conditions). ( ) starting at the nearest neighbor by index and continuing by decreasing (smaller indices) or increasing (larger indices) the index one by one while skipping nodes already linked to, search for the nearest node that has not reached its target degree yet and create a link with this node. ( ) create the network by repeating steps - for each node. for all the strategies, vaccination is voluntary and quantity limited. that is to say only susceptibles who do not refuse vaccination are vaccinated and each day only a certain number of doses is available. for each strategy for each time unit, first a group of eligible nodes is identified and then up to the maximum number of doses is dispensed among the eligible nodes according to the strategy chosen. in this strategy, nodes with the highest degrees are vaccinated first. the motivation for this strategy is that high degree nodes on average can be assumed to transmit a disease more often than low degree nodes. numerically, the prioritized vaccination strategy is implemented as follows: ( ) for each time unit, start at the highest degree (i.e. consider nodes with degree d¼m) and repeat the steps below until either the number of doses per time step or the total number of available doses is reached. ( ) count the number of susceptible nodes for degree d. ( ) if the number of susceptible nodes with degree d is zero, set d ¼ dÀ and return to step . ( ) if the number of susceptible nodes with degree d is smaller than or equal to the number of available doses, vaccinate all the nodes, then set d ¼ dÀ and continue with step . otherwise continue with step . ( ) if the number of susceptible nodes with degree d is greater than the number of currently available doses, randomly choose nodes with degree d to vaccinate until the available number of doses is used up. ( ) when all the doses are used up, end the vaccination for the current time unit and continue when the next time unit arrives. in practice prioritizing on the basis of certain target groups such as health care workers or people at high risk of complications can be difficult. prioritizing on the basis of the number of links is even more difficult. how would such individuals be identified? one of the easiest vaccination strategies to implement is random vaccination. numerically, the random vaccination strategy is implemented as follows: ( ) for each time unit, count the total number of susceptible nodes. ( ) if the total number of susceptible nodes is smaller than or equal to the number of doses per unit time, vaccinate all the susceptible nodes. otherwise do step . ( ) if the total number of susceptible nodes is larger than the number of doses per unit time, randomly vaccinate susceptible nodes until all the available doses are used up. one way to reduce the spread of a disease is by splitting the population into many isolated groups. this could be done by vaccinating nodes with links to different groups. however given the network types studied here, breaking links between groups is not really feasible since besides the random cluster network, there is no clear group structure in the other networks. another approach is the follow links strategy, inspired by notions from social networks, where an attempt is made to split the population by vaccinating the neighbors and the neighbor's neighbors and so on of a randomly chosen susceptible node. numerically, the follow links strategy is implemented as follows: ( ) count the total number of susceptible nodes. ( ) if the total number of susceptible nodes is smaller than or equal to the number of doses per unit time, vaccinate all the susceptible nodes. ( ) if the total number of susceptible nodes is greater than the number of available doses per unit time, first randomly choose a susceptible node, label it as the current node, and vaccinate it. ( ) vaccinate all the susceptible neighbors of the current node. ( ) randomly choose one of the neighbors of the current node. ( ) set the current node to the node chosen in step . ( ) continue with steps - until all the doses are used up or no available susceptible neighbor can be found. ( ) if no available susceptible neighbor can be found in step , randomly choose a susceptible node from the population and continue with step . contact tracing was successfully used in combating the sars virus. in that case, everyone who had been in contact with an infectious individual was isolated to prevent a further spread of the disease. de facto, this kind of isolation boils down to removing links rendering the infectious node degree , a scenario not considered here. here contact tracing tries to isolate an infectious node by vaccinating all its susceptible neighbors. numerically, the contact tracing strategy is implemented as follows: ( ) count the total number of susceptible nodes. ( ) if the total number of susceptible nodes is smaller than or equal to the number of doses per unit time, vaccinate all the susceptible nodes. ( ) count only those susceptible nodes that have an infectious neighbor. ( ) if the number of susceptible nodes neighboring an infectious node is smaller than or equal to the number of doses per unit time, vaccinate all these nodes. ( ) if the number of susceptible nodes neighboring an infectious node is greater than the number of available doses repeat step until all the doses are used up. 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networks, epidemics and vaccination through contact tracing estimated epidemiologic parameters and morbidity associated with pandemic h n influenza sir dynamics in random networks with heterogeneous connectivity effects of heterogeneous and clustered contact patterns on infectious disease dynamics dynamical advantages of scale-free networks key: cord- -yafcy ul authors: sisto, antonella; vicinanza, flavia; tuccinardi, dario; watanabe, mikiko; gallo, ida francesca; d’alessio, rossella; manfrini, silvia; quintiliani, livia title: the psychological impact of covid- pandemic on patients included in a bariatric surgery program date: - - journal: eat weight disord doi: . /s - - - sha: doc_id: cord_uid: yafcy ul purpose: the covid- pandemic has radically impacted the world lifestyle. epidemics are well-known to cause mental distress, and patients with a current or past history of obesity are at increased risk for the common presence of psychological comorbidities. this study investigates the psychological impact of the current pandemic in patients participating in a bariatric surgery program. methods: patients were consecutively enrolled during the italian lockdown among those waiting for bariatric surgery or attending a post-bariatric follow-up, and were asked to complete through an online platform the depression anxiety stress scales- and a self-assessment questionnaire of items evaluating the resilience, change in eating behavior and emotional responses referring to the ongoing pandemic. results: % of the enrolled subjects reported of being worried about the pandemic, and % specifically reported of being worried about their or their relatives’ health. % and % felt lonelier and more bored, respectively. % was hungrier with increased frequency of snacking ( %) and % reported more impulse to eat. noteworthy, % felt unable to follow a recommended diet. no difference in terms of psychological profile was recorded among pre and post-bariatric subjects. logistic regression analysis on post-bariatric patients showed a relationship between snacking, hunger, eating impulsivity, and anxiety, stress, and/or depression symptoms. conclusion: the pandemic led to increased psychological distress in patients with a current or past history of obesity, reducing quality of life and affecting dietary compliance. targeted psychological support is warranted in times of increased stress for fragile subjects such as pre- and post-bariatric patients. level of evidence: level v: cross-sectional descriptive study. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. the world health organization (who) declared covid- a pandemic on march th [ ] . italy was the first country to be severely impacted by the virus spread in europe and was in national quarantine starting on march th until may th, with all movements to be work-related or certified the article is part of the topical collection on obesity surgery and eating and weight disorders. the online version of this article (https ://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. emergencies, and schools and most commercial services forced to shut down. the profound and broad spectrum of psychological implications that epidemics can cause on the individual as a whole is well-established. previous studies showed that mental disorders may vary from depression, anxiety, panic attacks, somatic symptoms, and symptoms that characterize post-traumatic stress disorder, to delirium, psychosis and even suicide [ , ] . regardless of exposure to infection, individuals may experience fear and anxiety of getting sick or dying and may also feel helpless or guilty towards those who have been infected [ ] . furthermore, it has been shown that, a month after the severe acute respiratory syndrome (sars) epidemic in , approximately % of people revealed moderate to severe depressive or anxiety symptoms [ ] and, one year after, up to % of patients showed high levels of suffering, which were indicative of psychiatric morbidity [ ] . recent studies in mainland china during the current covid- pandemic have shown that % of interviewees reported high levels of psychological distress with post-traumatic stress disorder and depression resulting from self-isolation and quarantine [ , ] . in this context, subjects suffering from obesity represent a highly fragile population since this condition is often associated with significant psychological comorbidity [ ] , which can further aggravate following a highly traumatic event, causing discontinuous adherence to long-term treatment on top of other obvious psychological repercussions. among subjects suffering from obesity who request bariatric surgery, in particular, there is a high prevalence of mood disorders, anxiety disorders and eating disorders [ ] [ ] [ ] . in relation to the mechanisms of psychic functioning, it also emerges that patients suffering from obesity present cognitive, emotional and behavioral patterns characterized by impulsive traits and difficulties in self-regulating emotional states [ , ] . it should be highlighted that psychological frailty can persist even after surgery: in fact, even if the severity of problematic eating behavior decreases immediately after the bariatric surgery, it tends to increase significantly between the first and third post-operative year [ ] . noteworthy, the prevalence of obesity in italy is over % and rapidly increasing, making the number of patients possibly deriving significant stress and difficulty in adhering to treatment from the current pandemic not to be neglected [ ] . the study aimed at identifying the emotional and relational impact that the covid- health emergency and lockdown had on patients included in a bariatric surgery program, paying particular attention to the difference between patients waiting for surgery and those who had already undergone it. in particular, the elements of psychological frailty, the resources of resilience and the change in eating habits related to the experience of the covid- pandemic were analyzed. a nationwide survey aimed at subjects included in a bariatric surgery program was carried out in italy during the generalized lockdown. patients were enrolled through social media according to a consecutive eligibility criterion. in the period from the th of april to the rd of may patients who were candidates for a bariatric surgery procedure following a specific consultation or those who had already undergone the intervention were asked to answer a series of questionnaires through an online platform. exclusion criteria were as follows: impaired ability to provide informed consent or understand or respond to questions due to intellectual impairment or limited italian language knowledge; age below years old; lack of consent. an anonymous participation and the confidentiality of the information collected were ensured. the ethics committee of the university hospital campus biomedico of rome approved this study and its development. all the procedures performed in the study complied with the ethical standards of the institutional and/or national research committee and the helsinki declaration of and its subsequent amendments or comparable ethical standards. written informed consent was obtained from all participants prior to enrollment. the current study involved the administration of a structured interview and a validated self-report questionnaire listed below for the evaluation of the psychological impact of the covid- emergency and lockdown on the enrolled subjects. patients were evaluated once through an online platform. − a self-assessment interview consisting of personal data and items evaluating the presence of psychological protective and risk factors with respect to the possibility of developing psychological distress. in particular, ability to resilience, change in eating behavior, and the emotional responses to their experience of the pandemic were recorded. the replies "often" and "very often" were grouped to obtain the percentage of patients giving a positive answer to each question (supplementary table ). − depression anxiety stress scales dass- [ ] , a validated instrument of items that provides a gen-eral assessment of the psychological distress and three specific domains: depression, anxiety and stress perceptions, with high scale scores indicating high distress. the statistical package for social sciences (spss), v. , was used for statistical analysis. results are presented as mean ± standard deviation or number and percentage. normality was assessed with the kolmogorov-smirnov test. the primary endpoint was to explore the dass derived condition of depression, anxiety and stress in the postoperative bariatric population during the covid- emergency in italy. the secondary outcome was to explore the relationship between depression, anxiety, stress and fear of covid- infection and eating behavior and lifestyle changes in the post-operative bariatric population. in addition, a subpopulation of subjects waiting for bariatric surgery was enrolled to explore whether there was a difference in the psychological response pattern in these subjects as opposed to those who had already undergone surgery. for continuous variables, a t student test to compare the distribution of the psychological parameters was conducted. chi-square test/fisher's exact test was used for categorical variables. univariate and multivariate logistic regression model was performed to analyze the effect of the covid- pandemic (measured through a questionnaire whose scores were used as independent variable: emotionality domain, resilience domain and eating related) on dass derived condition of depression, anxiety and stress used as the discrete dependent variable. univariate analysis was performed by converting continuous variables into dummy dichotomic variables based on median values, while continuous variables were used for multivariate analysis. multivariate model: variables with significant univariate association (p value ≤ . ) as regressors. r : the nagelkerke's r . univariate and multivariate ordinal regression model was performed to analyze the effect of depression, anxiety, stress and fear of covid- infection (used as exploratory variables) on eating behavior and lifestyle changes (used as discrete dependent variables). three discrete and separately analyzed outcomes from the interview questionnaire were used as dependent variables: increased hunger; increased snacking, and increased impulsivity in eating. the univariate analysis was performed using dummy dichotomic variables based on the dass threshold, which identify the condition of depression, anxiety and stress, while continuous variables were used for multivariate analysis. multivariate model: variables with significant univariate association (p value ≤ . ) as regressors. r : the nagelkerke's r . the results were considered statistically significant when p < . . four hundred thirty-four patients with obesity were enrolled, and their general characteristics are summarized in table . briefly, males ( . %) and females ( . %) were enrolled, with an age between and years old, average . ± . years old. among these, ( . %) had undergone bariatric surgery. recorded marital status was as follows: ( . %) were unmarried, ( . %) were married, ( . %) were divorced and ( . %) were widowers. thirteen subjects ( . %) had completed primary school, ( . %) had a secondary junior school degree, ( . %) had a high school degree, ( . %) had a university degree; ( . %) had a ph.d. occupation was as follows: unemployed ( . %), employed ( . %), freelance ( . %), housewives ( . %) and students ( . %). subjects who had undergone surgery had one of the following types: sleeve gastrectomy (n = , . %), roux-en-y gastric bypass (rygbp) (n = , . %), gastric banding (n = , . %), biliopancreatic diversion (n = , . %) and single anastomosis duodeno-ileal bypass with sleeve gastrectomy (sadis) (n = , . %), table . a stratification based on pre-and post-operative status showed that both populations presented similar general characteristics and exhibited only a significant difference in the marital status, the ones who had already undergone the results obtained through the dass questionnaire and that exploring emotions and eating behavior during the covid- lockdown are summarized in supplementary table . briefly, the study population reported to feel depressed in . % of cases (n = ), had significant anxiety in . % of cases (n = ), and was in psychological distress in . % of cases (n = ). regarding the emotions domain during the covid- pandemic, more than half of the subjects ( %) reported to be generally worried (often or very often). specifically, most subjects reported of being worried about their or their relatives' health ( %). % of subjects also described feeling lonelier and % reported an increased sense of boredom. in relation to the eating style domain, % of the sample reported feeling hungrier with increased frequency of snacking between meals ( %), while % of subjects reported a strong impulse to eat during the pandemic. of note, % of the subjects declared that they felt unable to follow a recommended diet. the results obtained through the dass questionnaire and the covid- related questionnaires based on the operative status of the patients are summarized in table . according to the dass questionnaire, subjects awaiting for bariatric surgery reported similar rates of depression (n = , . % vs. n = , . %, p = . , table ), anxiety (n = , . % vs. n = , . %, p = . , table ) and stress (n = , % table ) compared to those who had already undergone it. the subjects also showed similar scores regarding depression, anxiety and stress (p = ns, table ), as well as the three domains derived from the covid- related questionnaire: emotionality, resilience and eating behavior (p = ns, table ). univariate and multivariate binary logistic regression analysis of covid- related psychological markers of depression, anxiety and stress on post-bariatric subjects are summarized in table . regarding the dass derived depression status, the univariate analysis showed that the three domains from the covid- questionnaire were all significant markers of depression: emotivity (or . ; % ci . - . ; p = . , univariate and multivariate ordinal logistic regression analysis of depression, anxiety and stress as markers of lifestyle and eating related behavioral characteristic during covid- pandemic in the bariatric operated population are summarized in table . regarding the increased hunger, the univariate analysis showed that the three statuses from the dass questionnaire were all significant markers of changes in the hunger domain: depression (b: . ; % ci . - . ; p = . , table ), remained a significant marker of increased hunger and this model explained . % of the variance of increased hunger (r = . ; p = . , table ). regarding the increased snacking, the univariate analysis showed that the three statuses from the dass questionnaire table dass derived depression, anxiety and stress as markers of lifestyle and eating related behavioral characteristic during covid- pandemic in the post-bariatric population oucomes: three discrete and separately analyzed outcomes from the interview questionnaire were used as dependent variables: increased hunger; increased snacking and increased impulsivity in eating. univariate analysis was performed using the dummy dichotomic variables based on the dass threshold which identifies the condition of depression, anxiety and stress, while continuous variables were used for multivariate analysis. table ). being worried about covid- was not a marker of increased snacking (p = ns, table ). a multivariate built as previously explained showed that only depression (b: . ; % ci . - . ; p = . , table ), remained a significant marker of increased snacking, with the model explaining % of the variance of increased snacking (r = . ; p = . , table ). regarding the increased impulsivity in eating, the univariate analysis showed that the three statuses from the dass questionnaire were all significant markers of changes in the impulsivity in eating domain: depression (b: . ; % ci . - . ; p = . , table ), anxiety (b: . , % ci . - . ; p = . , table ), and stress (b: . , % ci . - . ; p = . , table ). being worried about covid- was not a marker of increased impulsivity in eating (p = ns, table ). a multivariate model built as previously explained showed that only depression (b: . ; % ci . - . ; p = . , table ) and stress b: . ; % ci . - . ; p = . , table ), remained significant markers of increased impulsivity in eating, with the model explaining % of the variance of increased impulsivity in eating (r = . ; p = . , table ). the covid- pandemic has radically changed lifestyles and daily routines around the world. in particular, during the lockdown period, citizens were called to self-distancing measures, travel restrictions, limited access to services and family isolation to prevent the spread of the virus. in addition, many families have experienced significant financial difficulties resulting from precarious work and, in some cases, from job loss [ ] . moreover, early during the pandemic, many studies have reported an increased risk of developing severe complications following covid- infection in patients with obesity [ ] [ ] [ ] [ ] . the finding has been given wide attention by media, possibly contributing to worsen the sense of fear of those with a current or past history of weight excess. in line with previous reports [ ] [ ] [ ] [ ] , the present study showed that more than half of the study population was generally very concerned about the current pandemic. the prevalent concerns focused mainly on the fear for one's own health and for that of family members. about one fourth of the subjects reported of feeling depressed, anxious or stressed. moreover, recent research stresses that high levels of suffering related to covid- can lead to emotional dysregulation resulting in maladaptive eating behaviors, such as binge eating, grazing and emotional eating both before and after bariatric surgery [ ] . as previously pointed out, the state of alarm and reactive concern to the current pandemic situation enter a context of particularly fragile patients [ , ] , possibly impacting in particular on the ability to maintain a healthy lifestyle and proper eating behavior. in fact, the present study showed that during the lockdown period, the majority of the study population felt hungrier and increased the frequency of snacking. many declared an uncontrollable urge to eat and, in general, that they felt unable to follow the prescribed diet. from the data obtained, it may be postulated that the difficulty of regulating a pre-existing eating misbehavior was further aggravated during the pandemic, and the increased sense of loneliness and boredom might have contributed to further increase the eating dysregulation. it is also possible that a feedback circle is created between anxiety, concern related to the experience of the covid- pandemic and maladaptive eating behavior. noteworthy, no significant difference between candidates to the intervention and those who had already undergone it were present regarding mental distress, eating disorder and resilience. we may therefore assume that the experience of anguish, stress and anxiety is a transversal element that impacts all people with a current or past history of obesity. the conducted study highlights a prevalence of clinically significant mental distress that varies between and % of symptoms of clinically significant anxiety, depression and stress. the obtained results show, in particular, a relationship between snacking, increased hunger, increased impulsivity in eating, and the presence of significant anxiety and/ or depression symptoms. this picture can be interpreted in the light of previous reports showing that mental distress tends to significantly reduce the quality of life and inevitably has an impact on the compliance to dietary treatments [ ] . odom et al. [ ] highlight, in particular, that the risk of weight regain in obese patients is closely linked to a worsening in the depressive state. mc guire et al. [ ] confirm this fact, claiming that the severity of depression is an important risk factor for weight regain. in the present research, the variable resilience intended as a protective factor with respect to psychic discomfort was also considered. in literature, resilience expresses, in fact, the ability to maintain one's orientation towards existential purposes, despite lasting adversities and stressful events. it foresees an attitude of perseverance in front of an obstacle and openness to change. this concept can therefore be understood as the ability to persistently face the difficulties encountered in the different areas of one's life, maintaining a good awareness of oneself and one's internal coherence [ ] . as hypothesized and in line with what is expressed in the literature, we showed that the presence of resilience resources decreased the possibility of manifesting clinically significant depressive and stress symptoms in post-bariatric patients. the strong connection between mental status and food behavior warrants first of all an accurately targeted psychological support in order to favor compliance to dietary treatments. however, some dietary manipulations including certain foods [ , ] , food supplements [ , ] , and dietary patterns [ ] [ ] [ ] [ ] [ ] [ ] together with pharmacological treatment [ , ] have been shown to impact appetite and mood and/ or proved particularly beneficial in subjects with severe obesity and in post-bariatric patients. such measures should be therefore taken into consideration in the next months in order to favor dietary adherence and metabolic improvement, leveraging a multidisciplinary approach to treat those severely obese seeking bariatric surgery and those who have lost weight but need lifelong follow-up after bariatric surgery [ ] . our study has several limitations. first, the sample size was relatively small and a priori power was not calculated, given the exploratory nature of the study. the design of the study was cross-sectional, making causal correlation assessment not possible. moreover, anthropometric parameters were not part of the questionnaires, and correction for such possible confounders was therefore not performed. finally, no in person interview was performed, and psychological assessment solely relied on online administration of questionnaires. moreover, the covid- specific questionnaire has not been validated yet. however, to the best of our knowledge, no validated questionnaire is currently available in order to explore the domains of interest during a pandemic. our study also features some strengths. first, the use of questionnaires should not only be viewed as a limitation. in fact, given the nationwide nature of the survey, if in-person psychological assessment were to be chosen, more than one mental health professional would have been involved, possibly accounting for bias. therefore, the absence of personal interpretation obtained through the choice of questionnaire-driven evaluation has allowed for the absence of operator-dependent variability. furthermore, patients all over the country replied to the audit, allowing for a generalized picture rather than that of a single region. this is particularly important as covid- has differentially spread across regions, with the north being severely impacted and the south only partially affected. in conclusion, our study shows that covid- had a significant emotional impact on patients participating in a bariatric surgery program, inevitably affecting eating habits. according to the results, it seems that general concern related to covid- , isolation and social distancing, the fear of contagion for oneself and for others may activate depressive states and anxiety and inevitably require good resilience qualities stimulating a process of functional adaptation to one's well-being. accurately targeted psychological support is warranted in times of increased stress for more fragile subjects such as those with severe obesity or those who have undergone bariatric surgery in order to favor dietary and treatment compliance and prevent both psychological and organic complications. the literature supports the broad psychological impact that pandemics over the years have had on the individual as a whole. obesity identifies a particularly fragile population since the condition is often associated with significant psychiatric comorbidity. therefore, a pandemic event may aggravate a pre-existing condition of mental discomfort, particularly affecting the patient's ability to maintain a healthy lifestyle and dietary behavior. the study shows that patients included in a bariatric surgery program are generally very worried about the ongoing covid- pandemic and the fears mainly focus on their own health and that of family members regardless of whether they have undergone surgery. the difficulty in regulating eating behavior is further aggravated by the presence of symptoms of depression, anxiety and stress. however, resources of resilience represent a protective factor in preventing the development of discomfort. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. data collection for the study benefitted from the annual, faculty-funded eightweek undergraduate summer research internship. conflict of interest the authors declare that they have no conflict of interest. the ethics committee of the university hospital campus biomedico of rome approved this study and its development. all the procedures performed in the study complied with the ethical standards of the institutional and/or national research committee and the helsinki declaration of and its subsequent amendments or comparable ethical standards. consent to participate informed consent was obtained from all participants prior to start the survey. completion of the survey pack implied consent. availability of data and material the datasets generated 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spread of covid- : evidence from canada date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: vodag c we estimate the impact of mask mandates and other non-pharmaceutical interventions (npi) on covid- case growth in canada, including regulations on businesses and gatherings, school closures, travel and self-isolation, and long-term care homes. we partially account for behavioral responses using google mobility data. our identification approach exploits variation in the timing of indoor face mask mandates staggered over two months in the public health regions in ontario, canada's most populous province. we find that, in the first few weeks after implementation, mask mandates are associated with a reduction of percent in the weekly number of new covid- cases. additional analysis with province-level data provides corroborating evidence. counterfactual policy simulations suggest that mandating indoor masks nationwide in early july could have reduced the weekly number of new cases in canada by to percent in mid-august, which translates into to , fewer cases per week. when government policies to stem the spread of were introduced in early , the best available evidence supporting them was provided by studies of previous epidemics, epidemiological modeling, and case studies (oecd, ). even when the efficacy of a given precaution in reducing covid - transmission has been established, significant doubts regarding the usefulness of specific policy measures may persist due to uncertainty regarding adherence to the rules and other behavioral responses. for example, even though several observational studies, mostly in medical setting, have shown that face masks reduce the transmission of and similar respiratory illnesses (see chu et al. ( ) for a comprehensive review), a face mask mandate may not be effective in practice if it fails to increase the prevalence of mask wearing (compliance), or if it leads to increased contacts due to a false sense of security. it is therefore important to directly evaluate and quantify the relationship between various policy measures and the rate of propagation of . the low cost and high feasibility of mask mandates relative to other containment measures for has generated keen interest worldwide for studying their effectiveness. this attention has been compounded by substantial variation, across jurisdictions and over time, in official advice regarding the use of masks. figure b in the appendix plots self-reported mask usage in select countries (canada, united states, germany and australia) in the left panel, and across canadian provinces in the right panel. the figure shows large differences in mask usage, both across countries and within canada. we estimate and quantify the impact of mask mandates and other non-pharmaceutical interventions (npi) on the growth of the number of covid- cases in canada. canadian data has the important advantage of allowing two complementary approaches to address our objective. first, we estimate the effect of mask mandates by exploiting within-province geographic variation in the timing of indoor face mask mandates across public health regions (phus) in ontario, canada's most populous province with a population of nearly million or roughly % of canada's population (statistics canada, ). the advantage of this approach is that it exploits variation over a relatively small geographic scale (phu), holding all other province-level policies or events constant. in addition, the adoption of indoor face mask mandates in these sub-regions was staggered over approximately two months, creating sufficient intertemporal policy variation across the phus. second, we evaluate the impact of npis in canada as a whole, by exploiting variation in the timing of policies across the country's ten provinces. by studying inter-provincial variation, we are able to analyze the impact of not only mask mandates, but also other npis, for which there is little or no variation across ontario's phus (regulations on businesses and gatherings, schooling, travel and long-term care). in addition, our province-level data include both the closing period (march-april) and the gradual re-opening period (may-august), providing variation from both the imposition and the relaxation of policies. our panel-data estimation strategy broadly follows the approach of chernozhukov, kasahara and schrimpf ( ), hereafter cks ( ), adapted to the canadian context. we allow for behavioural responses (using google community mobility reports geo-location data as proxy for behaviour changes and trends), as well as lagged outcome responses to policy and behavioral changes. our empirical approach also allows current epidemiological outcomes to depend on past outcomes, as an information variable affecting past policies or behaviour, or directly, as in the sir model framework. we find that, in the first few weeks after their introduction, mask mandates are associated with an average reduction of to % in the weekly number of newly diagnosed cases in ontario, holding all else equal. we find corroborating evidence in the province-level analysis, with a to % reduction in weekly cases, depending on the empirical specification. furthermore, using survey data, we show that mask mandates increase self-reported mask usage in canada by percentage points, suggesting that the policy has a significant impact on behaviour. jointly, these results suggest that mandating indoor mask wear in public places is a powerful policy measure to slow the spread of , with little associated economic disruption at least in the short run. counterfactual policy simulations using our empirical estimates suggest that mandating indoor masks nationwide in early july could have reduced weekly new cases in canada by to % on average by mid-august relative to the actually observed numbers, which translates into to , fewer cases per week. we also find that the most stringent restrictions on businesses and gatherings observed in our data are associated with a decrease of to % in weekly cases, relative to a lack of restrictions. the business/gathering estimates are, however, noisier than our estimates for mask mandates and do not retain statistical significance in all specifications; they appear driven by the smaller provinces and the re-opening period (may to august). school closures and travel restrictions are associated with a large decrease in weekly case growth in the closing period. our results on business/gathering regulations and school closure suggest that reduced restrictions and the associated increase in business or workplace activity and gatherings or school re-opening can offset, in whole or in part, the estimated effect of mask mandates on case growth, both in our sample and subsequently. an additional contribution of this research project is to assemble, from original official sources only, and make publicly available a complete dataset of cases, deaths, tests and policy measures in all canadian provinces. to this end, we constructed, based on official public health orders and announcements, time series for policy indicators regarding face masks, regulations on businesses and gatherings, school closures, travel and self-isolation, and long-term care homes. our paper relates most closely to two recent empirical papers on the effects of mask mandates using observational data. cks ( ) and mitze et al. ( ) study the effect of mask mandates in the united states and germany, respectively. cks ( ), whose estimation strategy we follow, exploit u.s. state-level variation in the timing of mask mandates for employees in public-facing businesses, and find that these mandates are associated with to percentage points reduction in the weekly growth rate of cases. this is substantially smaller that our estimates, possibly because the mask mandates that we study are much broader: they apply to all persons rather than just employees, and most apply to all indoor public spaces rather than just businesses. mitze et al. ( ) use a synthetic control approach and compare the city of jena and six regions in germany that adopted a face mask policy in early to mid april , before their respective state mandate. they find that mandatory masks reduce the daily growth rate of cases by about %. our paper has several advantages compared to the above two papers. first, we exploit both regional variation within the same province (like mitze et al., ) and provincial variation in the whole country (like cks, ), and find similar results, which strengthens the validity of our findings. second, we show that self-reported mask usage has increased after introducing mask mandates. we view this "first-stage" result on mask usage as informative, as the effectiveness of any npi or public policy critically depends on the compliance rate. moreover, this result mitigates possible concerns that the estimated mask mandate effect on case growth may be caused by factors other than mask policy. third, a key difference between our paper and cks ( ) is that we evaluate the effect of universal (or community) mandatory indoor mask wearing for the public rather than the effect of mandatory mask wearing for employees only. while other factors such as differences in mask wear compliance between canada and the u.s. may contribute to the different estimated magnitude of the policy impact, our results suggest that more comprehensive mask policies can be more effective in reducing the case growth rate. other related literature abaluck et al. ( ) discuss the effectiveness of universal adoption of homemade cloth face masks and conclude that this policy could yield large benefits, in the $ , -$ , per capita range, by slowing the spread of the virus. the analysis compares countries with pre-existing norms that sick people should wear masks (south korea, japan, hong kong and taiwan) and countries without such norms. in the medical literature, prather et al. ( ) argue that masks can play an important role in reducing the spread of covid- . howard et al. ( ) survey the medical evidence on mask efficiency and recommend public use of masks in conjunction with existing hygiene, distancing, and contact tracing strategies. greenhalgh et al. ( ) provide evidence on the use of masks during non-covid epidemics (influenza and sars) and conclude that even limited protection could prevent some transmission of . leung et al. ( ) study exhaled breath and coughs of children and adults with acute respiratory illness and conclude that the use of surgical face masks could prevent the transmission of the human coronavirus and influenza virus from symptomatic individuals. meyerowitz et al. ( ) present a recent comprehensive review of the evidence on transmission of the virus and conclude that there is strong evidence from case and cluster reports indicating that respiratory transmission is dominant, with proximity and ventilation being key determinants of transmission risk, as opposed to direct contact or fomite transmission. our paper also complements recent work on covid- policies in canada. mohammed et al. ( ) use public opinion survey data to study the effect of changes in mask-wear policy recommendations, from discouraged to mandatory, on the rates of mask adoption and public trust in government institutions. they show that canadians exhibit high compliance with mask mandates and trust in public health officials remained consistent across time. yuksel et al. ( ) use an outcome variable constructed from apple mobility data along period january to february , . in ontario, these location data are available for each of the first-level administrative divisions (counties, regional municipalities, single-tier municipalities and districts). we follow the approach of cks ( ), but modify and adapt it to the canadian context. the empirical strategy uses the panel structure of the outcome, policy and behavioral proxy variables, and includes lags of outcomes as information, following the causal paths suggested by the epidemiological sir model (kermack and mckendrick, ) . specifically, we estimate the effect of policy interventions on covid- outcomes while controlling for information and behaviour. in contrast to cks ( ) and hsiang et al. ( ) , who study variation in npis across u.s. states or across countries, our identification strategy exploits policy variation at the sub-provincial level (ontario's phus) in addition to cross-province variation, and our data captures both the closing down and gradual re-opening stages of the epidemic. . controls, w it -province or phu fixed effects, growth rate of weekly new tests, and a time trend. to assess and disentangle the impact of npis and behavioral responses on covid- outcomes, we estimate the following equation: where l denotes a time lag measured in days. equation ( ) models the relationship between covid- outcomes, y it , and lagged behaviour, b it−l , lagged policy measures, p it−l and information (past outcomes), i it = y it−l . for case growth as the outcome, we use l = . for deaths growth as the outcome, we use l = . the choice of these lags is discussed in appendix d. by including lagged outcomes, our approach allows for possible endogeneity of the policy interventions p it , that is, the introduction or relaxation of npis based on information on the level or growth rate of cases or deaths. also, past cases may be correlated with (lagged) government policies or behaviors that may not be fully captured by the policy and behaviour variables. in appendix table a , we also report estimates of the following equation: which models the relationship between policies p it , information, i it (weekly levels or growth of cases or deaths) and behaviour, b it . it is assumed that behaviour reacts to the information without a significant lag. we find strong correlation between policy measures and the google mobility behavioral proxy measure. equation ( ) captures both the direct effect of policies on outcomes, with the appropriate lag, as well as the potential indirect effect on outcomes from changes in behaviour captured by the changes in geo-location proxy b it−l . in appendix tables a and a , we also report estimates of equation ( ) without including the behavioral proxy, that is, capturing the total effect of policies on outcomes. since our estimates of the coefficient α in equation ( ) are not significantly different from zero, the results without controlling for the behavioral proxy are very similar to those from estimating equation ( ). outcomes. our main outcome of interest is the growth rate of weekly new positive cases as defined below. we use weekly outcome data to correct for the strong day-of-the-week effect present in covid- outcome data. weekly case growth is a metric that can be helpful in assessing trends in the spread of , and it is highlighted in the world health organization's weekly epidemiological updates (see, for example, world health organization ( )). specifically, let c it denote the cumulative case count up to day t and define ∆c it as the weekly covid- cases reported for the -day period ending at day t: the weekly case (log) growth rate is then defined as: that is, the week-over-week growth in cases in region i ending on day t. the weekly death growth rate is defined analogously, using cumulative deaths data. policy. in the ontario analysis, we exploit regional variation in the timing of indoor mask mandates staggered over two months in the province's regions ("public health units" or phus). figure displays the gradual introduction of mask mandates across the phus in ontario. the exact implementation dates of the mask mandates are reported in table c . mandatory indoor masks were introduced first in the wellington-dufferin-guelph phu on june and last in the northwestern phu on august . we also report results using the growth rate of deaths as supplemental analysis in section . . figures b and b in the appendix respectively display the weekly and daily cases, deaths and tests in each canadian province over time. there are markedly lower numbers reported on weekends or holidays. to deal with zero weekly values, which mostly occur in the smaller regions, as in cks ( ), we replace log( ) with - . we also check the robustness of our results by adding to all ∆c it observations before taking logs, by replacing log( ) with , and by using population weighted least squares; see tables a and a . there is no phu-wide mask mandate in lambton as of august , but its main city, sarnia, enacted a mask mandate on july . figure : ontario -mask mandates over time in the province-level analysis, we assign numerical values to each of the policy indicators listed in table c in appendix c. the values are on the interval [ , ], with meaning no or lowest level of restrictions and meaning maximal restrictions. a policy value between and indicates partial restrictions, either in terms of intensity (see more detail and the definitions in table c ) or by geographical coverage (in large provinces). the numerical values are assigned at the daily level for each region (phu or province, respectively for the ontario and national results), while maintaining comparability across regions. many npis were implemented at the same time, both relative to each other and/or across regions (especially during the march closing-down period), which causes many of the policy indicators to be highly correlated with each other (see appendix table a ). to avoid multi-collinearity issues, we group the policy indicators into policy aggregates via simple averaging: (i) travel, which includes international and domestic travel restrictions and self-isolation rules; (ii) school, which is an indicator of provincial school closure; (iii) business/gathering, which comprises regulations and restrictions on non-essential businesses and retail, personal businesses, restaurants, bars and nightclubs, places of worship, events, gyms and recreation, and limits on gathering; (iv) long-term care (ltc), which includes npis governing the operation of long-term care homes (visitor rules and whether staff are required to work on a single site) and (v) mask which takes value if an indoor mask mandate has been introduced, if not, or value between and if only part of a province has enacted such policy. the five policy aggregates are constructed at the daily level and capture both the closingdown period (an increase in the numerical value from toward ) and the re-opening period (decrease in the numerical value toward zero). in comparison, the policy indicators compiled by raifman ( ) for the usa used in cks ( ) are binary "on ( )"/"off ( )" variables. for consistency with the weekly outcome and information variables and the empirical model timing, we construct the policy aggregates p j it used in the regressions (where j denotes policy type) by taking a weekly moving average of the raw policy data, from date t − to date t. figure plots the values of the policy aggregates over time for each of the provinces. travel restrictions, school closures (including spring and summer breaks) and business closures were implemented in a relatively short period in the middle of march. there is some variation in the travel policy aggregate since some canadian provinces (the atlantic provinces and manitoba) implemented inter-provincial domestic travel or self-isolation restrictions in addition to the federal regulations regarding international travel. restrictions on long-term care facilities were introduced more gradually. in the re-opening period (may-august), there is also more policy intensity variation across the provinces, especially in the business and gatherings category, as the different provinces implemented their own re-opening plans and strategies. mask mandates were first introduced in ontario starting from june in some smaller phus and early july in the most populous phus such as toronto, ottawa and peel (see appendix table c ). in quebec, indoor masks were mandated province-wide on july . nova scotia and alberta's two main cities implemented mask mandates on july and august , respectively. there are two empirical challenges specific to our canadian context and data. the first challenge is the presence of small provinces and sub-regions with very few covid- cases or deaths. in section . , we perform a number of robustness checks using different ways of handling the observations with very few cases (in particular zero cases). the second data limitation is that there are only provinces in canada and public health units in ontario, unlike the u.s. jurisdictions in cks ( ). to account for the resulting small number of clusters in the estimation, we compute and report wild bootstrap standard errors and p-values, as proposed by cameron et al. ( ) . on the flip side, our data has the advantage of a longer time horizon (march to august) and non-binary, more detailed policy variables compared to raifman et al. ( ). behaviour proxy. we follow cks ( ) and other authors in interpreting the location change indices from the google community mobility reports as proxies for changes in people's behaviour during the pandemic, keeping in mind that location is only one aspect of behaviour relevant to . the general pattern in the data (see figure b ) shows sharply reduced frequency of recorded geo-locations in shops, workplaces and transit early in the pandemic (march), with a subsequent gradual increase back toward the baseline (except for transit), and a flattening out in july and august. several of the six location indicators (retail, grocery and pharmacy, workplaces, transit, parks and residential) are highly correlated with each other (see tables a and a ) and/or contain many missing observations for the smaller provinces. to address these data limitations and the possible impact of collinearity on the estimation results, we use as proxy for behavioral changes the simple average of the following three mobility indicators: "retail", "grocery and pharmacy" and "workplaces". to be consistent with the weekly outcome variables and to mitigate day-of-week behavioural variation, we construct the behaviour proxy b it by taking a weekly moving average of the (retail + grocery and pharmacy + workplaces) data, from date t − to date t. , as a result, our empirical analysis uses weekly totals (for cases, tests and deaths) or weekly moving averages (for policies and the behaviour proxy) of all variables recorded on daily basis. alternative methods for computing the standard errors are explored in section . . we drop the "transit", "parks", and "residential" location indicators because, respectively, . %, . %, and . % of the observations are missing in the provincial data, and . %, . %, and . % are missing in the ontario data. the "transit" and "residential" variables are also highly correlated with the three indicators we include in our aggregate behaviour proxy b it . furthermore, the "parks" indicator does not have clear implication for outcomes. in the ontario analysis, . % of the b it values are imputed via linear interpolation. in estimation equation ( ), we take moving average from date t − to date t − for policies and behaviour when the outcome is weekly case growth, and from date t − to date t − if the outcome is tables a and a display the correlation between our behaviour proxy b it and the five npi policy aggregates p j it . importantly, the behaviour proxy and mask mandate variables are not highly correlated, suggesting that the effect of mask mandates on covid- outcomes should be independent of location behaviour changes. information. we use the weekly cases and case growth variables defined above, ∆c it and y it , to construct the information variables i it in equation ( ) . specifically, we use as information the lagged value of the weekly case growth rate y it−l (= ∆ log(∆c it−l ) and the log of past weekly cases, log(∆c it−l ). we also use the lagged provincial (ontario analysis) or national (canada analysis) case growth rate and log of weekly cases as additional information variables in some specifications. a two-week information lag l = is used in the baseline results. in the supplementary regressions using the death growth rate as the outcome, we use information on past deaths and a four-week lag (see section . ). control variables. in all regressions, we control for region fixed effects (phu or province) and the weekly covid- tests growth rate ∆ log(∆t it ), where t it denotes cumulative tests in region i until date t and ∆t it is defined analogously to ∆c it above. we include a time trend: our baseline uses a cubic polynomial in days, but we also report results with no time trend and with week fixed effects. robustness checks also include news or weather variables as controls (see section . ). time period. we use the period may to august for the analysis with ontario phu level data and the period march to august for the national analysis with provincial data. the end date reflects data availability at the time of empirical analysis and writing. the start date for the ontario sample (may ) is approximately two weeks after the last restrictive measures were implemented and four weeks before the first mask mandate was introduced in ontario. robustness checks with different initial dates (may , june and june ) are reported in section . , with our results remaining stable. the initial date for the national sample (march ) was chosen as the first date on which each province reported at least one covid- test (so that cases could be potentially reported). again, alternative initial dates are explored in section . . we start with a simple graphical illustration of the effect of mask mandates on covid- cases growth. figure displays the average log case growth, y it = ∆ log(∆c it ) in ontario phus with or without mask mandates. it shows that, on average, the phus with a mask mandate two weeks prior have lower case growth than the phus without a mask mandate two weeks prior. no mask mandate at t - mask mandate at t - notes: the figure plots the average log weekly case growth ∆log(∆c) in the phus with mask mandate (blue) vs. without (red) mask mandate days prior. table shows the estimates of equation ( ), in which we control for other policies, behaviour and information, as explained in section . . we report wild bootstrap p-values clustered at the phu level to account for the small number of clusters. the odd-numbered mask mandates and regulations on business and gatherings vary at the phu level. long-term care policy changed only province-wide. the other policies (schooling and travel) do not vary during the sample period and hence are omitted from the regressions with ontario phu data. table a in the appendix reports alternative standard error specifications: regular clustering at the phu level (stata command "cluster"), wild bootstrap standard errors clustered at the phu level, and wild columns in table use lagged cases and lagged cases growth at the phu level as information; the even-numbered columns also include lagged cases and lagged case growth at the province level as additional information variables. in the tables, variable indicates a -day lag of variable. we present estimates of equation ( ) from three specifications that handle possible time effects differently. columns ( ) and ( ) in table are the most basic specifications, without including a time trend. the estimates in columns ( ) and ( ) suggest that, controlling for behavioural changes, mandatory indoor face masks reduce the growth rate of infections by - log points (p < . ), which is equivalent to a - % reduction in weekly cases. in order to control for potential province-wide factors affecting the spread of covid- such as income support policies or adaptation to the pandemic over time (so-called covid fatigue), we also estimate ( ) with a cubic time trend in days from the beginning of the sample, in columns ( ) and ( ) of table , and with week fixed effects, in columns ( ) and ( ) . columns ( )- ( ) show that our estimates of the mask mandate policy remain robust to the inclusion of a cubic time trend or week fixed effects. the results indicate that, depending on the specification, mask mandates are associated with a reduction of up to log points in weekly case growth or, equivalently, a % reduction in weekly cases. the magnitude of the mask policy estimate is not very sensitive to whether lagged province-level data are included as additional information. the results in table suggest that indoor mask mandates can be a powerful preventative measure in the covid - context. our estimates of the mask mandate impact across ontario's phus are equivalent to a - % reduction in weekly cases. this estimate is larger than the - % reduction estimated by cks ( ) for the u.s. one possible explanation is that ontario's mask policy is more comprehensive: we evaluate the effect of universal indoor mask-wearing for the public rather than the effect of mask wearing for employees only in cks ( ). differences in the compliance rate may also contribute to this difference; we discuss this potential channel in section . . the results in table also show a statistically significant negative association between information (log of past cases, log(∆c) ) and current weekly case growth (p < . in all specifications), indicating that a higher level of cases two weeks prior is correlated with lower current case growth. while b it allows for behavioural responses to information, the negative estimate on log(∆c) in table suggests that our location-based proxy does not capture bootstrap standard errors clustered by both phu and date. our results are robust to alternative ways of calculating standard errors. using equation ( ), a coefficient of x translates into a − exp(x) reduction in weekly cases ∆c it /∆c it− . and in section . 's province-level results), unlike in cks ( ). in appendix table a , we find strong contemporaneous correlations between the policy measures, log cases, and the google mobility behavioral proxy from estimating equation ( ) . this suggests that the information (lagged cases) and the lagged policy variables included in equation ( ) may absorb lagged behavioral responses proxied by b it−l or other latent behavioral changes not captured by b it−l . we next evaluate the impact of npis on covid- cases growth in canada as a whole by exploiting variation in the timing of policies across the provinces. here, we examine npis for which there is no variation across ontario's phus (i.e., schooling, travel, and ltc) in addition to mask mandates. also, provincial data contain variation in the timing of policy changes in both the closing and re-opening phases, allowing us to study both the imposition and relaxation of restrictions. figure : canada -mask mandates and weekly case growth no mask mandate at t - mask mandate at t - notes: the figure plots the average weekly case growth ∆ log(∆c) in the provinces with mask mandate (blue) vs. without mask mandate (red) days prior. we also tried including each location change measure separately and the results are similar (not shown). all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . as in the ontario analysis, we begin with a graphical illustration of mask mandates and covid- case growth across canadian provinces, in the period march to august , . figure plots the average log weekly case growth in the provinces with vs. without mask mandates. while mask mandates are implemented relatively late in our sample period, average case growth in the provinces with a mask mandate (ontario and quebec) diverged from the average case growth in the provinces without a mandate begin roughly four weeks after the mandates are imposed. table displays the estimates of equation ( ) for weekly case growth, along with wild bootstrap p-values, clustered at the province level (see table a for other methods of computing the standard errors). the odd-numbered columns use lagged cases and lagged case growth at the provincial level as information while the even-numbered columns include in addition lagged cases and case growth at the national level as additional information variables. as in the ontario analysis, we present in table estimates from three specifications: no time trend (columns ( )-( )), including cubic time trend in days (columns ( )-( )) and including week fixed effects (columns ( )- ( )). the most robust result is the estimated effect of mask mandates: they are associated with a large reduction in weekly case growth of to log points, which is equivalent to a to % reduction in weekly cases across the different specifications. the estimates are statistically significantly different from zero in all cases, with a p-value of less than . in columns ( )- ( ) . it is reassuring that these results regarding mask mandates are consistent with the ontario analysis in the previous section. table further shows that restrictions on businesses and gatherings are associated with a reduction in the weekly case growth of to log points or, vice versa, that relaxing business/gathering restrictions is associated with higher case growth. the estimate is equivalent to a to % decrease in weekly cases in our sample period. the business/gathering estimates are, however, more noisy than our estimates for mask mandates and do not retain statistical significance in the specifications with week fixed effects (p = . and . ). tables a and a further suggest that the results on business and gathering npis are driven by the smaller provinces and the re-opening period (may to august). still, these results suggest that lowered restrictions and the associated increase in business/workplace activity or gatherings can be an important offsetting factor for the estimated effect of mask mandates on covid- case growth, both in our sample and in the future. we also find that school closures (the school variable in table ) can be negatively figure assumes a july mask mandate implementation date for ontario (when its most populous phu, toronto, adopted a mask mandate, along with ottawa), and july for quebec (province-wide mandate). all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . associated with case growth. however, the estimates are statistically significant from zero only in the specifications with cubic time trend (columns ( ) and ( )). as seen in figure all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . , provincial school closures occurred in a very short time interval during march, so we may lack statistical power to separately identify its effect from other npis (especially the travel-related). hence, we interpret this result with caution. as in table , the level of past cases, log(∆c), is negatively and statistically significantly associated with current weekly case growth in columns ( )- ( ) . since the specification with cubic time trend in tables and allows for possible nonmonotonic aggregate time trends in case growth in a parsimonious way, we choose it as our baseline specification with which to perform robustness checks in the next section. robustness checks with the other specifications are available upon request. a possible concern about our data for the national analysis is that some npis (e.g. international travel restrictions or closing of schools) were implemented within a very short time interval. thus, we may lack enough regional variation to distinguish and identify the separate effect of each policy. collinearity could also affect the standard errors and the signs of the estimated coefficients. to check robustness with respect to potential collinearity in the npi policies, tables a and a report estimates from our baseline specification, omitting one policy at a time, for ontario and canada respectively. first, it is reassuring that the mask mandate estimates are hardly affected by omitting any of the other policies. this is expected since mask mandates were imposed during a period where other npis changed little (see figure ) . similarly, the effects of business/gathering regulations and school closures in table a are not sensitive to omitting other policies one at a time, which suggests that there is sufficient statistical power and variation to identify them in the national analysis. another concern for our empirical strategy is that the usual formula for our dependent variable, ∆ log(∆c it ), cannot be applied when the weekly case total ∆c it is zero. we follow cks ( ) and replace ln( ) with - in our baseline specifications in tables and . we now check the robustness of our estimates to alternative treatments of zero weekly cases. for easier comparison, the first two columns in table a repeat columns ( ) and ( ) for example, table a shows a correlation of . between the travel and school policy aggregates. aggregating the basic policy indicators into five groups mitigates this issue. here, we test whether any remaining collinearity poses a problem. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . from table for ontario. our main results on mask mandates across ontario phus are robust to replacing log( ) with and to adding to all ∆c it observations before taking logs, as shown in columns ( )-( ) of table a . another way to mitigate the issue of phus with very few cases is to estimate a weighted least squares regression where phus are weighted by population. columns ( ) and ( ) in table a show that the resulting mask estimate has a slightly smaller magnitude and, due to the reduced effective sample size, weaker statistical significance. similarly, table a shows that our province-level estimates, in particular for mask mandates, are also robust to the same manipulations as above. in columns ( ) and ( ) of table a , we restrict the sample to only the largest provinces (british columbia, ontario, quebec and alberta), which have only . % ( out of ) zero observation cases. again, the estimated mask effects are little changed. figure b shows that our estimates and confidence intervals for the effect of mask mandates in the ontario baseline regressions do not vary much by the initial date of the sample. similarly, figure b shows that, in the national analysis, our results about mask mandates and business/gathering restrictions are also robust to alternative sample start dates. we explore alternative time lags, either shorter or longer in duration, centered around the baseline value of days. figure b (with ontario data) and figure b (with province-level data) plot the estimates and confidence intervals from the baseline regressions and show that our mask effect estimates remain fairly consistent for different lags. our behaviour proxy variable (google geo-location trends) likely misses some aspects of behaviour relevant for covid - transmission. one factor that may meaningfully impact behaviour is weather. for example, good weather could entice more people to spend time outside, lowering the chance of viral transmission. columns ( ) and ( ) in table a report national estimates with lagged weather variables (daily maximum and minimum temperatures and precipitation for the largest city in each province ) as additional regressors. our npi estimates, in particular mask mandates, are little changed from the baseline results in columns ( ) and ( ) . all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . another possible concern is that our information variables, lagged cases and lagged case growth, may not fully capture the information based on which people react or adjust their behaviour, possibly affecting the observed weekly case growth. columns ( ) and ( ) in table a add a national-level "news" variable to the baseline specification. the news variable is defined as the number of daily search results from a news aggregator website (proquest canadian newsstream) for the terms "coronavirus" or "covid- " (see appendix c for more details). in column ( ), the lagged news variable approaches the % significance level (p = . ). our estimates on masks and business/gathering remain very close to those in the baseline. the effectiveness of any npi or public policy crucially depends on whether it affects behaviour. in this section, we use self-reported data on mask usage to examine whether mask mandates indeed increase mask use in canada ("first-stage" analysis). we use data from the yougov covid- public monitor, which includes multiple waves of public opinion surveys fielded regularly since early april in many countries. here, we focus on inter-provincial comparison within canada. our variable of interest is based on responses to the question "thinking about the last days, how often have you worn a face mask outside your home (e.g. when on public transport, going to a supermarket, going to a main road)?" the answer choices are "always", "frequently", "sometimes", "rarely", and "not at all". we create a binary variable taking value if the response is "always" and otherwise, as well as another variable taking value of if the respondent answered either "always" or "frequently" and otherwise. we begin with a simple illustration of self-reported mask usage in canada from april to august . figure b plots the average self-reported mask usage (the response "always") in the provinces with and without mask mandates. the figure clearly shows that selfreported mask usage is higher, by up to percentage points, in the provinces with a mask mandate than in the provinces without mask mandates. since figure b does not account for compositional changes in the data, we formally estimate equation ( ), using self-reported mask usage as the behavioral outcome. notes: the data source is yougov. the outcome is a binary variable taking value if the respondent respectively answered "always" (in the left panel) or "always" or "frequently" (in the right panel) to "thinking about the last days, how often have you worn a face mask outside your home?" the figure plots the estimates from a version of equation ( ) where the mask policy variable is replaced by the interaction of the variables corresponding to being in the treatment group (imposed mask mandate) and a series of dummies for each week, ranging from weeks before the mask mandate to weeks after (t = - to + , where t = is the mandate implementation date). the reference point is week before the implementation (t = - ). wild bootstrap (cgmwildboot) standard errors clustered by province with repetitions are used to construct the confidence intervals. sample weights are used. figure shows a graphical event study analysis on mask mandates and changes in mask usage. the event study approach is appropriate for the mask usage outcome variable, since the policy impact is expected to be immediate, unlike the other outcomes we study, for which any impact is expected to occur with a lag and we use weekly totals or moving averages. we replace the mask policy variable in equation ( ) by the interaction of variables corresponding to being in the treatment group (i.e. under a mask mandate), and a series of dummies for each week, ranging from weeks before the mask mandate to weeks after the mask mandate (t = - to + , where t = is the implementation date of the mask mandate). the reference point is one week before the implementation of the mask mandate (t = - ), and we use the same y-axis scale on both panels. the left and right panels of figure present the results from the event study analysis for the "always" and "always" or "frequently" mask usage answers, respectively. we make several observations. first, neither panel shows a pre-trend -the estimates are close to zero before the mask mandates. this addresses the potential concern that provinces that implemented mask mandates may have had a different trend in mask usage than provinces that did not. second, the effect of mask mandates on mask usage is immediate: an increase all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint of roughly percentage points as soon as the mask policy is implemented at (t = ). third, the effect appears persistent rather than transitory, since mask usage after t = does not revert to its level before t = . notes: the time period is april to august , . p-values from wild bootstrap (cgmwildboot) standard errors clustered by province with repetitions are reported in the square brackets. nc denotes national total cases. the data source is yougov. the outcome is a dummy which takes value if the respondent answered "always" to the survey question "thinking about the last days, how often have you worn a face mask outside your home?" sample weights are used. individual characteristics include a gender dummy, age dummy (in years), dummies for each household size, dummies for each number of children, and dummies for each employment status. ***, ** and * denote %, % and % significance level respectively. table displays the estimates on self-reported mask usage (answer "always") in equation ( ) along with wild bootstrap p-values clustered at the province level. the odd-numbered columns use lagged cases and lagged case growth at the provincial level as information while the even-numbered columns include in addition lagged cases and case growth at the national level as additional information variables. as in table and table , we present estimates all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . without time trend, including cubic time trend (in days), and including week fixed effects. our preferred specification with cubic time trend, column ( ) of table , shows that mask mandates are associated with . percentage point increase in self-reported mask usage (p < . ), from a base of self-reported mask usage without mask mandate of . %. , these "first-stage" results show that mask mandates exhibit significant compliance in canada and establish a basis for the significant impact of mask mandates on the spread of covid- that we find. that said, given that mask mandates do not change everyone's behaviour, our estimates in tables and represent intent-to-treat effects. the full effect of the entire population shifting from not wearing to wearing masks is likely significantly larger. there is a heated debate on whether community use of masks may create a false sense of security that reduces adherence to other preventive measures. we also investigate this question using yougov survey data. as tables a and a indicate, we find no evidence that mask mandates in canada have had an offsetting effect on other preventive measures such as hand washing, using sanitizer, avoiding gatherings, and avoiding touching objects in public during the period we study. on the contrary, mask mandates may slightly increase social distancing in one out of the eight precaution categories (avoiding crowded areas) (p < . ). we evaluate several counterfactuals corresponding to replacing the actual mask policy in a province or canada-wide with a counterfactual policy, including absence of mask mandate. letting t be the implementation date of a counterfactual policy, we set the counterfactual weekly case count, ∆c c it , equal to ∆c it for all t < t . for each date t ≥ t , using the definition of y it from ( ), we then compute the counterfactual weekly cases, ∆c c it and the counterfactual similarly, in table a , column ( ) shows that "always" or "frequent" mask usage increases by . percentage points. the finding that the increase in mask usage among the "always" respondents is larger than among the "always" or "frequent" respondents is consistent with some people switching from wearing masks "frequently" to "always." hatzius et al. ( ) document that state mask mandates in the us increased mask usage roughly by percentage points in days. the compliance with mask mandates may differ across countries or regions based on social norms, peer effects, political reasons or the consequences of noncompliance (e.g., fines). if we take the increase of about percentage points in reported mask usage induced by mask mandates at face value, the full effect of mask wearing (treatment-on-the-treated effect) would be roughly triple our estimates. it could be larger still if there is desirability bias in answering the mask usage survey question, so that the actual increase in mask use may be smaller than our estimate. consistent with this result, seres et al. ( ) find that wearing masks increased physical distancing based on a randomized field experiment in stores in germany. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . case growth rate, y c it , as follows: whereŶ it is the regression-fitted value of weekly case growth; β m ask is the coefficient estimate on the mask mandate variable mask in baseline specification ( ) in table or , depending on the counterfactual; mask c is the counterfactual mask policy (e.g. different implementation date, wider geographic coverage or absence of mask mandate); and β log∆c is the coefficient estimate (- . or - . ) on lagged cases log(∆c) in table or , column . the coefficient β log∆c adjusts the counterfactual case growth rate for the negative statistically significant association between the weekly case total two weeks prior and time-t case growth. this effect may be due to people being more careful when they perceive the risk of infection to be higher or less careful vice versa. notes: the left panel assumes that mask mandates were adopted in all phus on june (date of the first mask mandate in on). the right panel assumes that mask mandates were not adopted in any phu. we use the mask estimate (- . ) from column ( ) of table . all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. notes: the left panel assumes that mask mandates were adopted in all provinces on july (the adoption date in toronto and ottawa). the right panel assumes that mask mandates were not adopted in any province. we use the mask estimate (- . ) from column ( ) of table . notes: the left panel assumes that mask mandates were adopted in all provinces on july (the adoption date in toronto and ottawa). the right panel assumes that mask mandates were not adopted in any province. we use the mask estimate (- . ) from column ( ) of table . all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . figures , and show results from two counterfactual policy evaluations. the first exercise, depicted in the left-hand side panel of the figures, assumes that masks are adopted everywhere at the earliest date observed in the data. specifically, figure considers the counterfactual of all ontario phus adopting mask mandates on june , while figures and assume that all provinces adopt a mask mandate on july . using our mask policy estimate from table , figure shows that an earlier face mask mandate across ontario phus could have lead to an average reduction of about cases per week as of august , holding all else equal. for canada as a whole, a nation-wide adoption of mask mandates in early july is predicted to reduce total cases per week in the country by to , cases on average as of august , depending on whether we use the more conservative mask estimate (- . ) from column ( ) of table (see figure ) or the larger estimate (- . ) from column ( ) of table (see figure ). in all cases, the indirect feedback effect via β log∆c (lagged cases as information) starts moderating the decrease in cases two weeks after the start of the counterfactual mask policy. in the right-hand side panel of figures , and , we perform the opposite exercise, namely assuming instead that mask mandates were not adopted in any ontario phu or any canadian province. our estimates imply that the counterfactual absence of mask mandates would have led to a large increase in new cases, both in ontario and canada-wide, especially when using the larger mask coefficient estimate from table (see figure ). finally, in figure b in the appendix, we also evaluate the counterfactual in which british columbia and alberta, the third and fourth largest canadian provinces by population, adopt province-wide mask mandates on july . the results, using the mask estimate from table , suggest a reduction of about cases per week in each province by mid-august. the counterfactual simulations assume that all other variables, behaviour and policies (except the mask policy and t − cases) remain fixed, as observed in the data. this is a strong assumption, but it may be plausible over the relatively short time period that we analyze. moreover, the counterfactuals assume that regions without a mask mandate would react in the same way, on average, as the regions that imposed a mandate. therefore, these results should be interpreted with caution and only offer a rough illustration and projection of the estimated effect of mask mandates on covid- cases. june is the date of the earliest mask mandate in ontario. for the national analysis, july , the effective date for toronto and ottawa, is considered ontario's first significant date of mask mandate enactment: phus with earlier mandates account for less than % of ontario's population. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint closing and re-opening sub-periods we investigate whether policy impact varied in different phases of the pandemic by splitting the full sample period into two sub-periods: "closing" (march to may ) and "reopening" (may to august ). the dividing date of may (referring to the npis in place around may ) was chosen because very few policies were relaxed before may , and very few non-mask policies were tightened after may in our sample period (see figure ) . in table a , we report estimates and wild bootstrap standard errors using our baseline specification with cubic time trend, separately for the closing and re-opening periods. we find that the imposition of school closures and travel restrictions early in the closing period is associated with a very large subsequent reduction in weekly case growth, as can be also seen on figure b -the average observed log growth rate of cases ∆ log(∆c) falls from . (ten-fold growth in weekly cases) to − . ( % decrease in weekly cases) between march and april . long-term care restrictions are also associated with reduced case growth two weeks later during the march to may closing period. we interpret these results with caution, however, since many of these policy measures and restrictions were enacted in a brief time interval during march and there is not much inter-provincial variation (see figure ). no mask mandates were present in the closing period. in the re-opening period, our results in table a are in line with our full-sample results for mask mandates and business/gathering regulations (table ) , with slightly larger coefficient estimates and less statistically significant p-values, possibly due to the smaller sample. travel and school closures are not statistically significant in the re-opening period. this is unsurprising: relaxation of travel policies was minor and endogenous (only re-open to safe areas within canada), and the schools that re-opened (in parts of quebec and, on a part-time basis, in british columbia) did so on voluntary attendance basis, yielding smaller class sizes. we also examine the weekly death growth as an outcome. we only have access to disaggregated deaths data at the province level (not at phu levels in ontario). we thus estimate regression equation ( ) using y it = ∆ log(∆d it ) for each province i as the dependent variable. in addition, we use a -day lag for the policy, behaviour proxy, and information variables to reflect the fact that deaths occur on average about two weeks after case detection; see appendix d for details and references. in table , variable denotes the variable lagged by days. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . table reports the estimates from the same specifications as those for case growth in table . in all specifications, mask mandates are associated with a large reduction in the observed weekly deaths growth rate four weeks later (more than log points, or equivalently more than % reduction in weekly deaths). these results are larger than our case growth results, but consistent with them given the substantial uncertainty. see also figure b , which plots the average weekly death growth in the provinces without a mask mandate four weeks prior vs. that for ontario, the only province with mask mandate four weeks prior in our sample period. the robustness checks in table a , however, show that, unlike for case growth, the mask mandate estimates in table are not robust to weighing by population or to restricting the sample to the largest provinces. this suggests that the estimated effect is largely driven by observations from the small provinces, which have a disproportionately larger number of zero or small weekly death totals. furthermore, given the -day lag, there are only days with observations (from ontario only) for which the mask mandate variable takes value of . due to these serious data limitations, the relation between mask mandates and covid - deaths in table is suggestive at best, and we urge caution in interpreting or extrapolating from these results. that said, our main findings about the growth in cases may have implications about future growth in deaths, particularly if the affected demographics become less skewed toward the young in later periods. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the wearing of face masks by the general public has been a very contentious policy issue during the covid- pandemic, with health authorities in many countries and the world health organization giving inconsistent or contradictory recommendations over time. "conspiracy theories" and misinformation surrounding mask wear abound in social media, fuelled by some individuals' perception that mask mandates constitute significant restrictions on individual freedoms. given the absence of large-scale randomized controlled trials or other direct evidence on mask effectiveness in preventing the spread of , quantitative observational studies like ours are essential for informing both public policy and the public opinion. we estimate the impact of mask mandates and other public policy measures on the spread of in canada. we use both within-province and cross-province variation in the timing of mask mandates and find a robust and significantly negative association between mask mandates and subsequent covid- case growth - to % average reduction in weekly cases in the first several weeks after adoption, depending on the data sample and empirical specification used. these results are supported by our analysis of survey data on compliance with the mask mandates, which show that the mandates increase the proportion of reporting as always wearing a mask in public by around percentage points. however, our sample period does not allow us to determine whether their effect lasts beyond the first few weeks after implementation. we conclude that mask mandates can be a powerful policy tool for at least temporarily reducing the spread of mask mandates were introduced in canada during a period where other policy measures were relaxed, as part of the economy's re-opening. specifically, we find that relaxed restrictions on businesses or gatherings are positively associated with subsequent covid- case growth -a factor that could offset and obscure the health benefits of mask mandates. past case totals were also found to matter for subsequent outcomes, suggesting that riskier behaviour based on favourable lagged information may limit how low mask mandates and other restrictions -short of a lockdown -can push the number of new cases. we have deliberately abstained from studying the direct economic impacts of , focusing instead on the unique features of the canadian data for identifying the effect of npis, in particular mask mandates, on covid- case growth. future research combining epidemiological finding with the economic benefits and costs of various public policies or restrictions would enrich the ongoing policy debate and provide further guidance. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. ( ) and ( ) repeat columns ( ) and ( ) from table where we replace log( ) with - . columns ( ) and ( ) replace log( ) with , and columns ( ) and ( ) add to all ∆c it observations. columns ( ) and ( ) report estimates from a weighted least squares regression with weights equal to the phu population sizes. ***, ** and * denote %, % and % significance level respectively. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . ( ) and ( ) repeat columns ( ) and ( ) from table . we drop each policy at at time in columns ( )- ( ) . ***, ** and * denote %, % and % significance level respectively. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . the time period is april to august , . p-values from wild bootstrap (cgmwildboot) standard errors clustered by province with repetitions are reported in the square brackets. nc denotes national total cases. the data source is yougov. the outcome is a dummy which takes one for the respondent who answers "always" or "frequently" to the survey question "thinking about the last days, how often have you worn a face mask outside your home?" sample weights are used. individual characteristics include a gender dummy, dummies for each age (in years), dummies for each household size, dummies for each number of children, and dummies for each employment status. ***, ** and * denote %, % and % significance level respectively. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . notes: the time period is april to august , . p-values from wild bootstrap (cgmwildboot) standard errors clustered by province with repetitions are reported in the square brackets. nc denotes national total cases. the data source is yougov. the outcome is a dummy which takes value if the respondent answered "always" or "frequently" to each survey question in table c . sample weights are used. individual characteristics include a gender dummy, age dummy (in years), dummies for each household size, dummies for each number of children, and dummies for each employment status. ***, ** and * denote %, % and % significance level respectively. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the time period is feb to july (two weeks before the march -august sample period). daily province-level data. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . notes: the data source is yougov. the figure plots the average self-reported mask usage by week (the fraction of respondents who answered "always" to the survey question "worn a face mask outside your home") in the provinces with vs. without mask mandates. sample weights used to compute the averages. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . figure b : canada -behaviour notes: the behaviour proxy b it is the average of the "retail", "grocery and pharmacy", and "workplaces" google mobility indicators. province-level -day moving averages are plotted. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. notes: we plot the coefficient estimates on mask policy, with % confidence intervals, from equation ( ), for different initial dates of the sample. the initial sample date in the baseline specifications reported in table notes: we plot the coefficient estimates on mask policy, with % confidence intervals, in the upper panel and the estimates on business/gathering policy in the lower panel, from equation ( ) for different initial dates of the sample. the initial date in our baseline specification (table ) is march . the left panels correspond to column ( ) in table ; the right panels correspond to column ( ) in table . all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. table ; the right panels correspond to column ( ) in table . all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint figure b : canada -weekly cases, deaths and tests (growth rate) week ending preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint figure b : canada -weekly cases, deaths and tests (level) all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint figure b : canada -daily cases, deaths and tests - all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint figure b : counterfactuals -mask no mask mandate at t- mask mandate at t- notes: average log weekly death growth in provinces with vs. without mask mandates days prior. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint used hand sanitiser i health avoided going out in general i health avoided small social gatherings (not more than people) i health avoided medium-sized social gatherings (between and people) i health avoided large-sized social gatherings (more than people) i health avoided crowded areas i health avoided touching objects in public (e.g. elevator buttons or doors) notes: the data source is yougov. possible responses to each survey item are "always", "frequently", "sometimes", "rarely", and "not at all". for table a , we create a binary variable taking value if the response is "always" and otherwise. for table a , we create a binary variable taking value of if the respondent answered either "always" or "frequently" and otherwise. all data used in the paper are available at https://github.com/c -sfu-econ/data. as discussed in section . , we assume a lag of days between a change in policy or behaviour and its hypothesized effect on weekly case growth, and a lag of days between such a change and its effect on weekly death growth. first, we consider the lag between infection and a case being reported. as most identified cases of in canada are symptomatic, we focus on symptomatic individuals. for most provinces cases are listed according to the date of report to public health. in provinces where the dates instead refer to the public announcement, we shifted them back by one day, as announcements typically contain the cases reported to public health on the previous day. the relevant lag therefore has two components: . time between symptoms onset and reporting of the case to public health: the ontario data contain an estimate of the symptom onset date ("episode date") for each case. for our sample period the average difference between the date of report and the episode date is . days (median: days) including only values from to days, and . all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . days (median: days) including only values from to days. we assume that the lags in ontario and in other provinces are similar, and use a value of [ ] [ ] days between symptom onset and report to public health authorities. adding these together implies that the typical lag between infection and a positive case being reported to public health is around days. second, we consider the effect of weekly averaging on the appropriate lag for our analysis. suppose a policy or behavioural change starts on date t, impacting the daily growth in infections between dates t − and t and in each subsequent day. then, assuming a lag of days between infection and case reporting, case counts c are affected from date t+ onward. our outcome variable ∆ log(∆c) thus would react to the original policy or behavioral change on date t + . the change is complete on t + , when the week from t + to t + is compared to the week from t + to t + . the midpoint of the change is t + . choosing a lag of l days implies that the policy/behaviour variable phases in from t + l to t + l + . to match the midpoint of this phase-in to the midpoint of the change in the outcome variable, we set l = . the chosen lag matches the lag used by other authors who study policy interventions, e.g., cks ( ). we explore sensitivity to alternative lags in section . . with respect to deaths, our data are, in most cases, backdated (revised by the authorities ex , that is, two weeks longer than our estimate of the time from symptom onset to reporting of a positive test result. we correspondingly set the lag used in our analysis of the death growth rate (section . ) to days. all rights reserved. no reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint the case for universal cloth mask adoption and policies to increase supply of medical masks for health workers incubation period of novel coronavirus ( -ncov) infections among travellers from wuhan, china bootstrap-based improvements for inference with clustered errors causal impact of masks, policies, behavior on early covid- pandemic in the physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov- and covid- : a systematic review and meta-analysis face masks for the public during the covid- crisis the effect of large-scale anti-contagion policies on the covid- pandemic a contribution to the mathematical theory of epidemics the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application respiratory virus shedding in exhaled breath and efficacy of face masks early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia incubation period and other epidemiological characteristics of novel coronavirus infections with right truncation: a statistical analysis of publicly available case data community use of face masks and covid- : evidence from a natural experiment of state mandates in the us transmission of sars-cov- : a review of viral, host, and environmental factors face masks considerably reduce covid- cases in germany public responses to policy reversals: the case of mask usage in canada during covid- flattening the covid- peak: containment and mitigation policies reducing transmission of sars-cov- covid- us state policy database high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus face mask use and physical distancing before and after mandatory masking: evidence from public waiting lines table - - - population estimates, quarterly estimates of the severity of coronavirus disease : a model-based analysis weekly epidemiological update, coronavirus disease (covid- ) estimating clinical severity of covid- from the transmission dynamics in wuhan, china log(∆c) - . *** - . *** - . *** - . *** - . ** - . * - . *** - . *** we show mask usage for the u.s. and germany because related work by chernozhukov et al. ( ) and ( ) repeat columns ( ) and ( ) from table where we replace log( ) with - . columns ( ) and ( ) replace log( ) with , and columns ( ) and ( ) add to all ∆c it observations. columns ( ) and ( ) report results from a weighted least squares regression with the province populations as weights. finally, columns ( ) and ( ) ( ) and ( ) report estimates with lagged weather variables as additional controls. columns ( ) and ( ) add a "news" variable to the baseline specification (see appendix c for more details). ***, ** and * denote %, % and % significance level respectively. weather -we downloaded historical weather data for the largest city in each province from the weather canada website. the data provide daily information on variables: maximum temperature (c), minimum temperature (c), mean temperature (c), heating degreedays, cooling degree-days, total rain (mm), total snow (cm), total precipitation (mm), snow on the ground (cm), direction of maximum wind gust (tens of degrees), and speed of maximum wind gust (km/h). we only use the temperature and precipitation data in table a as possible factors determining outside vs. inside activity.news -we collected data from proquest canadian newsstream, a subscription service to all major and small-market daily or weekly canadian news sources. we recorded the number of search results for each day from feb , to aug , by searching the database for the keywords "coronavirus" or "covid- ". we only counted the results with source listed as "newspaper" since other sources, such as blogs or podcasts, tend to duplicate the same original content. key: cord- -w ysjf authors: nan title: th international symposium on intensive care & emergency medicine: brussels, belgium. - march date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: w ysjf nan ventriculostomy-related infection (vri) is a serious complication in patients with hemorrhagic stroke. in such patients, diagnosis of vris is complicated by blood contamination of csf following ventricular hemorrhage. we aimed to evaluate the diagnostic potential of white blood cells count (wbc), c-reactive protein (crp), and procalcitonin (pct) to identify vris in patients with hemorrhagic stroke during the time of external ventricular drain (edv) in situ. this retrospective study was conducted at the neurosurgical-icu, university hospital of zurich. a total of patients with hemorrhagic stroke and an external ventricular drain (evd) were admitted over a years period at the icu. of those, patients with vris ("vri"), defined by positive csf bacterial culture and increased wbc in csf (> /ul), and patients without vris and with serial csf sampling ("no-vri") were analyzed. patients with csfcontamination or suspected vri (negative csf cultures but antibiotic treatments) were excluded. wbc, crp, and pct were measured daily. csf was sampled routinely twice a week or by t> °c. for the analysis, mean peak values of wbc, crp, pct during the time of evd in situ were compared between groups (t test). data are expressed as mean with ci %. results: between groups, wbc and crp were similar (wbc: . g/l and . g/ l, p= . and crp: . mg/l and . mg/l, p= . in the group vri and no-vri, respectively) ( figure , panel a and b ). in the group vri, pct was low and significantly lower than in the group no-vri ( . ug/l and . ug/l, p= . in the group vri and no-vri, respectively) (panel c). wbc in csf were similar between groups ( . /ul and . /ul p= . in the group vri and no-vri, respectively). in this study, serum-inflammatory markers were not able to screen patients with vris. their routine measurement should be carefully evaluated. introduction: central nervous system (cns) infections constitute a potentially lifethreatening neurological emergency. patients admitted to the intensive care unit (icu) usually present with a severe disease and organ failure, leading to high mortality and morbidity. we have performed a retrospective analysis during a -year period of patients admitted to a polyvalent icu. clinical, demographic and outcome data were collected to evaluate its clinical impact on the outcome of patients with cns infections. we identified patients with the diagnosis of meningitis, meningoencephalitis and ventriculitis, where the median age was , years (range - ). upon clinical presentation, their most frequent signs were fever ( %), meningeal signs ( %), seizures ( %), and a glasgow coma scale score < ( %). all needed ventilation support and % needed cardiovascular support. a definitive microbiological diagnosis was achieved on patients and antibiotic therapy was adjusted on of them. most common microorganisms were streptococcus pneumoniae (n= ), listeria (n= ) and pseudomonas aeruginosa (n= ) (figure ). other gram negative microorganisms were detected and lead to more adverse outcomes. meningitis was the cause of admission on patients and on a minority (n= ) meningitis was considered to be a secondary diagnosis on patients admitted for other causes (traumatic brain injury, subarachnoid or intraparenchymal hemorrhage, postoperatively of neurosurgical tumor). patients that eventually died had at least one risk factor (age> , immunocompromised due to diabetes, corticotherapy, hiv or heart transplantation). patients admitted to the icu were not so aged, but had some comorbidities and risk factors leading to more uncommon microorganisms, increasing the risk of adverse outcomes. this lead to an increase of mortality: % in the icu and an overall of %. study of selenium levels in unresponsive wakefullness (uws) patients with systemic inflammatory response syndrome (sirs) e kondratyeva , s kondratyev , n dryagina the objective of this study was to evaluate the pharmacokinetics (pk) of levetiracetam (lev) in critically ill patients with normal and augmented renal clearance (arc), and determine if the recommended dosage regimen provides concentrations in the therapeutic range ( - mg/l) [ ] . a prospective observational study was conducted in a tertiary hospital. six blood samples were taken during a dose interval at steady state and lev was quantified by hplc. a population pk study was carried out. statistical analysis was conducted to evaluate the differences in pk between patients with and without arc. the suitability of drug concentrations was also assessed. results: seventeen patients were included, with normal creatinine clearance (crcl) ( - ml/min) and with crcl≥ ml/min (arc). ten patients received mg q h, one mg q h and two mg q h. the data were best fitted to a two-compartment model. figure shows lev concentrations during the dosing interval. mean clearance (cl) was l/h and mean volume of distribution of central compartment (v) was l. interindividual variability was and % for cl and v, respectively. no differences were identified between both groups (p> . ) in pk parameters. no correlation was found between lev cl and crcl. trough levels were below the minimum concentration (c min ) mg/l of the therapeutic range in all patients except . furthermore, between - h % of samples were below the c min . conclusions: administered doses were not able to maintain lev concentrations in the recommended therapeutic range. other dosage strategies, such the extension of infusion time with higher doses, could be evaluated in order to obtain a more favourable profile. no correlation between lev cl and crcl was found. the mechanical properties of muscles such as tone, elasticity, and stiffness are often affected in chronic critical ill (cci) patients. a hand-held device known as the myotonpro demonstrated acceptable relative and absolute reliability in a ward setting for patients with acute stroke [ ] . the technology works on the principle of applying multiple short impulses over the muscle bulk via the testing probe. the aim of our study is to assess the feasibility of objective measurement of muscle tone in cci patients with neurological dynamics and serum biomarkers. the study included cci patients with neurological disorders (stroke, traumatic brain injury, neurosurgical intervention for brain tumors) with more than a -weeks stay in icu. dynamic measurements of the muscle properties were taken on the deltoideus, brachioradialis, quadriceps femoris, gastrocnemius using the myo-tonpro. to identify the leading factor in impaired muscle tone also were measured neurological (s , nse), inflammatory (il- ), bacterial load (pct) biomarkers using elecsys immunoassay and the serum level of microbial metabolites using gc-ms (thermo scientific). results: all patients were divided into groups depending on positive and negative clinical dynamics. significant differences were obtained in parameters characterizing changes in muscle tone of lower limbs -f gastrocnemius (tone) - . vs . hz, r quadriceps femoris (the mechanical stress relaxation time) - . vs . ms (p < . , respectively). some significant correlations between five parameters of muscle tone biomarkers and microbial metabolites were revealed. the results of a quantitative measurement of muscle tone objectively reflect the dynamics of neurological status, which in the future may be promising technique for the personalized approach cci in patients. introduction: changes in hormonal status in patients with unresponsive wakefulness syndrome (uws) remains poorly understood. methods: patients in uws were examined at the period from to . patients ( men) with tbi and patients ( men) after hypoxia. acth, cortisol, tsh, free t and t , sth, prolactin and natriuretic peptide were studied in the period from to months uws. in men, the level of total testosterone, lh and fsh was additionally studied. the obtained data was compared with the uws outcome in - months (crs-r scale assessment). none of the studied hormones of the hypothalamic-pituitary-adrenal axis were a reliable criterion for predicting the outcome of uws. most often and consistently was revealed a tendency of disrupt the rhythm of cortisol secretion, with higher rates in the evening hours. the average value of sth was higher in men with the consequences of head injury who had recovered consciousness than in those who remained in uws. significant decrease in testosterone levels, regardless of age, was found in patients with a consequence of tbi. mean levels of lh were higher in patients with tbi and hypoxia who remained unconscious than in patients who later restored consciousness. the average level of fsh was higher in patients who had recovered consciousness . the increase of natriuretic peptide level was observed both in patients who remained in chronic uws and in those who restored consciousness. no certain endocrine background, characterising this category of patients was found. violations of some hormones secretion rhythms, in particular, cortisol can be considered usual for uws patients, especially in patients with tbi. therapeutic hypothermia has not been used before our research in chronically critically ill (cci) patients. temperature decrease in neuronal cells is a strong signal that triggers endogenic cytoprotection programs using early response genes expression. our goal is to determine influences of craniocerebral hypothermia (cch) on level of consciousness in cci patients. we examined patients with different types of brain injuries. males and females, mean age . ± . . patients were divided into groups: main group - patients (vegetative state (vs) - , minimally conscious state (mcs) - ), comparison group - patient (vs - , mcs - ), groups were equal on main parameters (severity, functional state, comorbidity). patients from main group received courses of cch, duration - minutes, scalp temperature - °С, cerebral cortex cooling up to - o c, session end was without slow reheating period, and session's amount was set -until signs of consciousness recovery. cortex temperature check done noninvasively by using detection of brain tissue emi in shf-range. consciousness recovery in vs and mcs patients controlled using crs-r scale. results: cch sessions significantly increased level of consciousness in vs and mcs patient groups. in vs patients vegetative state increased until minimally conscious state and mcs +, and in mcs group until lucid consciousness (p < . ) (figure ). craniocerebral hypothermia is used in chronically critically ill patients for the first time. our research results demonstrated effectiveness of cch as an additive treatment tool in such patients. this let us optimistically determine the perspective of inclusion of cch method in chronically critically ill patient's rehabilitation to increase level of consciousness. despite the clinical benefit of endovascular treatment (evt) for large vessel occlusion (lvo) in ischemic stroke, space-occupying brain edema (be) represents a common complication during the course of disease. routinely, ct imaging is used for monitoring of these patients, notably in the critical care setting, yet novel and easy bed-side techniques with the potential to reliably predict be without repetitive imaging would be valuable for a time and cost effective patient care. we assessed the significance of automated pupillometry for the identification of be patients after lvo-evt. we enrolled patients admitted to our neurocritical-care unit who received evt after anterior circulation large vessel occlusion. we monitored parameters of pupillary reactivity [light-reflex latency (lat; s), constriction and re-dilation velocities (cv, dv; mm/s), and percentage change of apertures (per-change; %)] using a portable pupilometer (neuroptics®) up to every minutes during the first hours of icu stay. be was defined as midline-shift ≥ mm on followup imaging within - days after evt. we assessed differences in pupillary reactivity between patients with and without be (u-test) and evaluated prognostic performance of pupillometry for development of be (roc analysis). in patients ( women, . ± . years) without be, , assessments were compared to assessments in patients ( women, . ± . years) with be. on day , day , and day after evt, patients with be had significantly lower cvs and dvs, and smaller perchanges than patients without be, whereas lat did not differ between both groups. roc-analyses revealed a significant negative association of cv, dv, and per-change with development of be. conclusions: automated pupillometry seems to identify patients at risk for be after evt. a prospective study should validate whether automated pupillometry harbors the potential to reduce unnecessary follow-up ct imaging. the aim of this preliminary analysis is to detect differences between the qualitative and quantitative evaluation of the pupillary function carried out by doctors and nurses of an intensive care unit (icu) of a tertiary level hospital. secondary purpose is to investigate new indications for the use of pupillometry in a population admitted in icu methods: the study has been conducted (currently in progress) at the intensive care unit and ecmo referral center at careggi teaching hospital (florence; italy). the enrolled patients are adult subjects (> years) with alteration of consciousness defined by a glasgow coma scale (gcs) < , following a primary brain injury and/or the use of sedative drugs. the studied parameters, obtained with neurolight pupillometer ® (id-med, marseille, france) are analyzed, integrated and visual/qualitative evaluation of the pupil function shows a lower reliability if compared to automated pupillometry. the estimated error in the proper determination of photomotor reflex is . % (p< . ). no significant difference is reported between quantitative and qualitative pupillometry in the detection of anisocoria. our preliminary results are compatible with previously reported data [ ] [ ] [ ] , even if there was no difference in anisocoria determination. interestingly, a longer latency period among patients treated with opioids has been observed. other results are still in progress. introduction: due to the dynamic of critical care disease, a rapid bedside, noninvasive and highly sensitive and specific method is required for diagnosis. in this study we set out our experience with trancranial color-coded duplex ultrasound (dxt) [ ] . the dxt study identifies cerebral arteries as well as hemorrhagic phenomenon, hydrocephalus, mass-occupying lesions and midline shift. this is the main difference between dxt and conventional transcranial doppler (dtc) which is a blind study and do not provide any image. descriptive, cross-sectional and observational study from december to june . patients were included. inclusion criteria: neurocritical patients. exclusion criteria: no acoustic window, presence of ultrasound artifacts. data collection was performed. it was used a lowfrequency transducer from . - . mhz with trancranial duplex preset ( figure) . the patterns were defined as normal, vasospasm, high resistance, hypermedia and cerebral circulatory arrest, depending on the cerebral flow velocity, lindegaard ratio (lr) and pulsatility index (ip). results: men ( . %) and women ( . %). average age . ( - ). patients diseases: subarachnoid hemorrhage , traumatic brain injury , av malformation , stroke , hemorrhagic cerebrovascular accident and mass occupying lesions . normal pattern: patients (rel. freq . ). vasospasm: patients (rel. freq . ). high resistance: patients (rel. freq . ). hyperemia: patient (real. freq . ). cerebral circulatory arrest: patient (rel. freq . ) conclusions: dxt should be part of the routine of neuromonitoring, it allows real time images especially useful in unstable conditions. although it will be needed a large amount of patients to be statistical significant, dxt is useful considering a non invasive study, bedside and it allows early identification of different clinic conditions. introduction: embolization of the draining vein during endovascular treatment of arteriovenous malformation (avm) may result in venous outflow obstruction and hemorrhage. anaesthesiologist can use deliberate hypotension to reduce blood flow through avm which may be somehow helpful to prevent this scenario. adenosine-induced cardiac arrest may facilitate the embolization too. the goal of our study was to improve the results of endovascular treatment of avm using adenosine-induced cardiac arrest. methods: after obtaining informed consent patients ( male, female) were selected for adenosine-induced cardiac arrest during endovascular avm embolization. main age was , ± years old. of them were evaluated as iii class asa, as iv. endovascular treatment in all cases was performed under general anaesthesia. propofol, fentanyl, rocuronium were used to induce anaesthesia, then all the patients were intubated and ventilated with parameters to keep etco - mm hg. sevoflurane , - , vol% ( cases) or desflurane vol% ( case) were used to maintain anaesthesia. hemodynamic monitoring consisted of ecg, pulsoximetry, non-invasive blood pressure measurement. onyx or/and squid were used as embolic agents. ct was performed to every patient just after procedure as well as neurological examination. results: adenosine dosage was . - . mg/kg. time of consequent cardiac arrest was - sec. there were cases we administered adenosine for time, in one case we had to administer it twice, in one fig. (abstract p ) . circle of willis and pulsed-wave doppler mode of middle cerebral artery - times and times in one more case as well. hemodynamic parameters recovered without any particular treatment in all the patients. embolization has been performed in all the cases uneventfully. postoperative ct showed no hemorrhage. nobody from investigated group had neurological deterioration in postoperative period. our study shows that adenosine-indused cardiac arrest is not very difficult to perform method and it can be useful during avm embolization. a major risk factor for stroke is atrial fibrillation (af). to treat af anticoagulation is needed. there are now several anticoagulants available. however, a lack of head to head data as well as the absence of accurate techniques makes it difficult to compare them and measure determine there efficacy. stroke is known to produce an abnormal clot microstructure which is a common factor in many thrombotic diseases. this pilot study aims to use a functional biomarker of clot microstructure (d f ) and clotting time (tgp) to investigate the therapeutic effects of different anticoagulants in stroke and af. we recruited patients ( af and stroke & af). two samples of blood were taken: before anticoagulation (baseline) and post anticoagulation ( - weeks) . patients were either given warfarin ( %) or axipaban ( %). d f and tgp were measured and compared before and after anticoagulation. results: warfarin increased t gp ( ± secs to ± secs (p< . )), and decreased d f ( . ± . to . ± . (p< . )). apixaban increased tgp ( ± sec to ± sec (p< . )) but did not change df ( . ± . & . ± . ). interestingly we found that in the apixaban group tgp significantly correlated (p= . ) with blood drug concentration levels. in this study we show that d f and tgp can quantify and differentiate between the therapeutic effects of two different oral anticoagulants. showing that warfarin prolongs clotting and weakens the ability of the blood to form stable clots. conversely apixaban prolongs clotting time but does not affect the bloods ability to form stable clots. this shows the utility of the d f and tgp biomarkers in comparing two different treatment options, something no other current marker has proven able to do. where d f and tgp may prove useful tools in a personalized approach to anticoagulation treatment and monitoring in an acute setting. hospital mortality compared to the model with the original hairscore. patients with poor-grade aneurysm subarachnoid hemorrhage (asah) world federation of neurological surgeons (wfns) grades iv and v, have commonly been considered to have a poor prognosis ( - % mortality). though early intervention and aggressive treatment in neuroicu has improved outcome in the past years, it is controversial because most of the patients left hospital severely disabled. the objective of this study was to investigate the clinical and social outcomes in intracranial aneurysm patients with poor-grade asah underwent different intervention therapies. a single center observational registry of poor-grade asah consecutive patients, defined as wfns grades iv and v, treated at tertiary chilean referral center from december to march were enrolled in this study. the clinical data including patient characteristics on admission and during treatment course, treatment modality, aneurysm size and location, radiologic features, signs of cerebral herniation (dilated pupils), and functional neurologic outcome were collected. clinical outcomes were assessed via gose and and sociooccupational outcome, both at discharge and at months. figure ). % mortality is less than previously reported, and survivors had a favorable recovery, confirmed with neuro psychological test. poor-grade asah patients in our study shows a more positive outcome than previously considered. prognosis of subarachnoid hemorrhage (sah) is scarce, indeed almost half patients die or become severely disable after sah. outcome is related to the severity of the initial bleeding and delayed cerebral infarction (dci). infection and more precisely pneumonia have been associated with poor outcome in sah. however, the interaction between the two pathologic events remains unclear. therefore, we hypothesized that dci may be associated to pneumonia in sah patients. thus the aim of our study was to analyze the association between delayed cerebral infarction and pneumonia in patients with sah. in this retrospective, observational, monocentric cohort study, patients included in the analysis were admitted in neurosurgical intensive care unit or surgical intensive care unit in the university hospital of brest (france) for non-traumatic sah. primary outcome was diagnosis of dci on ct scan or mri months after sah. multivariate analysis was used to identify factors independently associated with dci. a total of patients were included in the analysis (female male ratio / , median age [ - ] years). multivariate analysis was adjusted on sedation, intracranial surgery, fisher classification of sah severity, pneumonia occurrence and non-pneumonia infectious event occurrence ( figure ). pneumonia occurred in patients ( . %) and other causes of infections in patients ( . %). dci was found in patients ( . %). factors independently associated with dci were pneumonia (or . [ . - . ]; p= . ) and non-pneumonia infectious events (or . [ . - . ]; p= . ). interestingly severity table (abstract p ). correlation of safety and efficacy markers of thrombolysis and thrombolysis time with distance from stroke centre results expressed as odds ratio with % confidence interval of initial bleeding evaluated by fisher scale was not independently associated with dci. dci is independently associated with the occurrence of pneumonia or other cause of sepsis. those results may highlight the need for rigorous approach for prevention protocol, early diagnosis and treatment of hospital acquired infectious diseases in sah patients. introduction: traumatic brain injury (tbi) can have devastating neurological, psychological and social sequelae. increased psychiatric morbidity after tbi has been shown in both adult and the pediatric population. also, critical illness as such is a risk factor for psychiatric problems in youth. our aim was to assess risk factors for later being prescribed psychiatric medication in survivors of intensive care unit (icu)-treated pediatric tbi. we used the finnish intensive care consortium (ficc) database to identify patients - years of age, treated for tbi in four icu in finland during the years - . we examined electronic health records and ct scans and collected data on drug prescription after discharge. we used multivariable logistic regression models to find statistically significant risk factors for psychiatric drug reimbursement. we identified patients of which patients received psychiatric drug prescription ( %) during follow up. the median time to prescription was months after tbi (interquartile range [iqr] - months). patients received antidepressants, received stimulants and received antipsychotics. increasing age showed a positive association with all drug prescriptions except for stimulants, where an inverse relationship was observed (table ) . using multivariable analyses, we could not find any admission or treatment related factors that significantly associated with being prescribed psychiatric medications. teenage survivors with moderate disability (glasgow outcome scale [gos] ) showed high numbers of psychotropic drug utilization ( % received any medication, % received antidepressants, % received antipsychotics). our data suggests, that the risk of psychotropic drug prescription after tbi depends on factors other than those related to injury severity or treatment measures. the incidence of drug prescription is especially high in patients with moderate disability. the effects of -adamantylethyloxy- -morpholino- -propanol hydrochloride on the formation of steroid neurotoxicity in rats with brain injury a. semenenko , s. semenenko , a. solomonchuk , n. semenenko depending on the nature of the brain injury and the severity of the victims, mortality in traumatic brain injury (tbi) ranges from to % [ ] . one of the targets for pathogenetic influence on the course of tbi is the use of pharmacological agents that are able to counteract the negative effects of excess concentrations of glucocorticoids on brain. the therapeutic effect of new pharmacological derivative adamantylethyloxy- -morpholino- -propanol hydrochloride (ademol) in rats with tbi was evaluated for days. the pseudoperated animals and control group received . % nacl solution and the comparison group received amantadine sulfate. cortisol levels were used to determine the efficacy of the test drugs in tbi. in rats treated with ademol, the level of cortisol in the blood ranged from to ng/ml (p -p ) and was . -fold lower (p< . ) compared to control pathology group on the day of therapy. instead, the effect of amantadine sulfate on the level of cortisol in the blood was significantly less than that of ademol. the concentration of cortisol in rats with amantadine sulfate in the blood ranged from - ng/ml (p -p ), was . times lower (p< . ), compared with the control pathology group, and by . % (p< . ) exceeded the corresponding value in animals treated with ademol. therapeutic treatment of rats with severe tbi with a solution of ademol, preferably better than rats in the group with . % nacl and amantadine sulfate protect the brain from the formation of steroid neurotoxicity by cortisol (p< . ). although cerebrovascular pressure reactivity (prx) well correlate to patient's outcome [ ] , it requires continuous monitoring and mobile average calculation for its determination. we therefore hypothesized that a simplified model of variation between mean arterial pressure (map) and intracranial pressure icp over the first three days of admission would have been able to predict patient outcome: we call this new parameter cerebrovascular pressure correlation index (cpc). we performed a retrospective observational study of all adult patients with severe tbi admitted to icu from january to april inclusive. all consecutive patients with a clinical need for icp monitoring were included for analysis. both for icp and map data were mean value over -hours registration, for a total of observations/day, cpc was therefore calculated as the pearson correlation coefficient between icp values (x axis) and map values (y axis), obtaining one single value every hours. variables included in the model (i.e. cpc, cpp, icp, systemic glucose, arterial lactate, paco , icp, and internal body temperature) were collected for the first days since trauma. for the main outcome only the minimum value of cpc fit the regression analysis (p = . ). the correspondent roc curve showed an auc of . . the associated youden criterion was ≤ . (sensitivity = . ; specificity = . ). of all the variables considered for the secondary outcome only cpcmin fit the regression model (p = . ). table reports the median and iqr range for sg and nsg of all the variables considered in the model. this observational study suggests that cpc could be a simplified model of variation between map and intracranial pressure icp over the first three days of admission predicting patient outcome. introduction: impaired cerebrovascular reactivity (car) after traumatic brain injury (tbi) is a marker for disease severity and poor outcome. it is unclear how dynamic changes in body temperature and fever impact car and outcome. we calculated the pressure reactivity index (prx) using the center-tbi high-resolution intensive care unit cohort, as a moving correlation coefficient between intracranial pressure (icp) and mean arterial pressure (map). minute and hourly values of prx and temperature were averaged in patients with simultaneous recording of icp and abp. demographic data was based the core registry (v . ). linear mixed models were calculated based on minute-by-minute data using r with lme v . - and ggeffects v . . . generalized estimating equation models were used to analyze changes during effervescence (increase of temperature of > °c within hours). we assessed high frequency physiological data during days of patients admitted to the icu with predominantly a closed injury type (n= / ). median age was years (iqr - ), baseline gcs was (iqr - ), and % had at least one unreactive pupil. the main measurement site for temperature was the urinary bladder / ( %). half of the patients ( / ) developed fever(> h with mean t ≥ . °c) with a total of h fever and a median of h fever(iqr - ) per patient. of effervescence episodes ( %) reached the febrile threshold of . °c which was associated with an increase in prx from . (±sd . ) at baseline ( h before) to . (±sd . ) during the febrile peak (p= . ) (figure -a) . linear mixed models showed a quadratic relationship between prx and temperature (p< . ) with an increase in predicted prx with febrile and hypothermic temperatures ( figure b ). the association of increasing body temperature with worsening of car supports prevention of fever in severe tbi. prospective studies are needed to further differentiate between mechanisms involved (i.e. inflammation) and central autonomic dysregulation. fig. (abstract p ) . the patients with a good -month outcome (gose> ) after severe traumatic brain injury showed an increase in root mean square of successive differences between normal heartbeats (rmssd) (compared to baseline -minutes before tracheal succtioning) acute kidney injury (aki) is relatively common in patients with severe traumatic brain injury (stbi) and it can contribute to morbidity and mortality [ ] . nephrocheck is a point-of-care urine test that flags two biomarkers that indicate if a critically ill patient is at risk for aki. we investigated the incidence of subclinical aki in patients with stbi. we performed a prospective observational study of all adult patients with severe tbi admitted to icu from january to april inclusive. all consecutive patients with a clinical need for icp monitoring were included for analysis. urine samples of severe tbi patients was collected at icu admission from patients to measure nephrocheck (nc) test [igfbp ] x was performed using the nephrocheck® astute ™ meter. serum creatinine was collected at admission, during the first three days, at icu dismission and -days follow up to assess renal recovery. the diagnosis of aki was based on kdigo criteria. hemodynamics, electrolytes, peep, p/f, kind of fluid administered, fluid balance, % fluid overload, length of stay, the sequential organ failure assessment score, injury severity scores and mortality were collected. a total of patients ( %) presented a median nc higher values at icu admission. one patient with positive nc value experienced aki at hrs. the positive nc group had more plasma transfusion (p-value . ) and a lower median hematocrit at hrs (p-value . ), but similar hospital length of stay (p= . ) and mortality rate (p= . ) conclusions: nc at icu admission identifies subclinical aki in tbi patients and it maight be used to predictclinical aki. hemodilution (but not fluid overload) seems to be associated with development of subclinical aki. higher nc at icu admission is not associated with worst longterm outcome in tbi patients. severe traumatic brain injury (tbi) is considered a serious public health problem in europe. partly because of the heterogeneity of tbi, considerable uncertainty may exist in the expected outcome of patients. the international mission for prognosis and analysis of clinical trials in tbi (impact) and the corticosteroid randomization after significant head injury (crash) prediction models are considered the most widely validated prognostic models [ , ] . however, studies using these prediction models for benchmarking of outcomes have been scarce. we aimed to compare actual outcomes in a tbi cohort of critically ill tbi patients with predicted outcomes in a quality of care initiative in an academic hospital. in this retrospective cohort study, we included consecutively admitted tbi patients to the icu adults of erasmus mc, university medical center, rotterdam, the netherlands between january and february . we included patients with tbi. -day mortality was %, sixmonth mortality was % and six-month unfavourable outcome was %. the impact core+ct+lab model predicted % -month mortality (vs % actual, p= . ) and % unfavourable outcome (vs % actual, p= . ). the -day mortality prediction by crash prognosis calculator was % versus actual -day mortality of only % (p= . ), whereas -month unfavourable outcome prediction by crash was % (vs. % actual, p= . ) ( figure ). the impact model, although developed more than a decade ago, seemed appropriate for benchmarking purposes in this single center cohort in the netherlands, while crash predictions were less applicable to our setting. introduction: out of hospital cardiac arrest (ohca) continues to be associated with significant mortality and morbidity. centralisation of care has considerably improved patient survival but has resulted in increased morbidity in the form of neurological deficit. accurate neurological prognostication remains challenging incorporating repeated clinical examination and ancillary investigations [ , ] . data was collected retrospectively and analysed for patients admitted post ohca from october to october . patient arrest demographics were collected in conjunction with extensive inpatient investigation findings including ct, traditional pupil assessment, pupillometry and eeg. results: % of patients survived to hospital discharge. patients presenting in a shockable rhythm continue to have higher survival rates ( table ) . % of patients who received immediate cpr survived to hospital discharge in comparison to % of patients who did not receive immediate cpr. % of patients underwent non-contrast ct head. % of patients had traditional pupillary examination performed on arrival. pupillometry was introduced in december ; out of a possible patients had pupillometry during their inpatient stay. eeg was undertaken in % of cases. our data shows receiving immediate cpr and presenting with a shockable rhythm remain positive prognostic factors. ct head as a stand-alone prognostic modality is unreliable with % of patients who survived to discharge, with intact neurology, had an admission ct head reported as hypoxic brain injury. a new neuroprognostic strategy is required in our unit that adds further certainty to likely clinical outcome. this includes increased use of tests such as eeg and pupillometry and the introduction of biomarkers such as neuron specific enolase, somatosensory evoked potential testing and magnetic resonance imaging. introduction: post-resuscitation care of patients following an out-of-hospital cardiac arrest (oohca) is set out by the uk resuscitation council [ ] . this is in line with the european resuscitation council guideline [ ] . the aim of this audit was to review compliancy to this guideline at the intensive care unit at the bristol royal infirmary . a retrospective audit was performed over a six-month period in adults who were admitted to the intensive care unit at the bri following an oohca whom later died during that admission ( patients). the focus was on whether the neuroprognostication and end-of-life (eol) care received was as per the standards set by the uk resuscitation council. the main neuroloical examinations documented were pupillary reflex ( %), corneal reflex ( %) and motor response to pain ( %). . % of patients received an ssep analysis > hours post-rosc, . % underwent an eeg and . % had > serum neuron-specific enolase measurements recorded. all patients ( %) underwent a ct head during their admission. . % of patients were referred to palliative care during their admission. % of patients were prescribed all eol medications. most common prescriptions included alfentanil ( . %) and midazolam ( . %). finally, % of appropriate patients were referred to be potential organ donors. the audit reflected our local practice and that some parameters were not being maintained as set by uk resuscitation guideline. multiple introduction: the prognostication of neurological outcome in comatose out-ofhospital cardiac arrest (ohca) patients is an integral part of post cardiac arrest care. biochemical biomarkers released from cerebral cells after hypoxic-ischemic injury represent potential tools to increase accuracy in predicting outcome after ohca. currently, only neuronspecific enolase (nse) is recommended in european prognostication guidelines. in this study, we present the release dynamics of gfap and uch-l after ohca and evaluate their prognostic performance for long-term neurological outcome in ohca patients. serum gfap and uch-l were collected at , and h after ohca. the primary outcome was neurological function at -month follow-up assessed by cerebral performance category scale (cpc), dichotomized into good (cpc - ) and poor (cpc [ ] [ ] [ ] . outcome prognostic performance was investigated with receiver operating characteristics (roc) by calculating the area under the receiver operating curve (auroc) and compared to nse. results: of included patients had at least one serum gfap or uch-l value at , or h after ohca. gfap and uch-l levels were significantly elevated in patients with poor outcome. gfap and uch-l discriminated excellently between good and poor neurological outcome at all time-points (auroc gfap . - . ; uch-l . - . ) and overall predictive performance measured by auroc of gfap and uch-l was superior to nse (auroc . - . ) ( figure ). however, the roc at the highest specificities of uch-l and gfap overlap those of nse and comparing the sensitivities for uch-l and gfap with those of nse for the highest specificities (> %) revealed higher sensitivities for nse than for uch-l and gfap at and h. gfap and uch-l predict poor neurological outcome in patients after ohca excellently and with a higher overall accuracy than nse, but both biomarkers perform inferior to nse at specificities over % at and h limiting their clinical use to guide decisions on prognosis. blood pressure after cardiac arrest and severity of hypoxicischemic encephalopathy c endisch , s preuß , c storm introduction: blood pressure management in post cardiac arrest (ca) patients ensures sufficient cerebral perfusion to avoid secondary brain injury. in local chain-of-survival improvements affect p-ohca survival [ ] [ ] [ ] [ ] [ ] . also initial rhythm in p-ohca is an important predictor of survival [ , ] . little is known about the relationship between initial rhythm in p-ohca and long-term outcome [ ] [ ] [ ] . our aim was to establish the relation between shockable rhythm and favorable long-term outcome in pohca. all children aged day- years who experienced non-traumatic ohca between - and were admitted to the sophia children's hospital in rotterdam were included. long-term outcome was determined using a pediatric cerebral performance category score at the longest available follow-up interval. the primary outcome measure was survival with favorable neurologic outcome, defined as pcpc - or no difference between pre-and postarrest pcpc. the association between shockable rhythm and the primary outcome measure was calculated in a multivariable regression model, adjusted for the pre-defined variables. from the patients included in the year study period ( %) patients survived to hospital discharge of which patients ( %) had favorable neurologic outcome (median follow-up duration of months). the rate of favorable neurologic outcome rose from % in to % in (p < . for trend) (fig. ) the odds of favorable neurologic outcome at the longest follow-up duration were significantly higher after a shockable initial and unknown rhythm. secondly, trend analysis showed an increase in aed defibrillation and shorter cpr duration. this was followed, finally, by a rise in rosc, survival to hospital discharge and favorable neurologic outcome rate. low socioeconomic status is associated with worse outcome after cardiac arrest. this study aims to investigate if patients´socioeconomic status impacts the chance to receive early coronary angiography after cardiac arrest. in this nationwide retrospective cohort study, patients admitted alive after out-of-hospital cardiac arrest (ohca) and registered in the swedish registry for cardiopulmonary resuscitation were included. individual data on income and educational level, prehospital parameters, coronary angiography results and comorbidity were linked from other national registers. in the unadjusted model there was a strong correlation between income level and rate of early coronary angiography where % of patients in the highest income quartile received early angiography compared to % in the lowest income quartile. when adjusting for confounders (educational level, sex, age, comorbidity and hospital type) there were still higher chance of receiving early coronary angiography with increasing income, or . (ci . - . ) and . (ci . - . ) for the two highest income quartiles respectively compared to the lowest income quartile. when adding potential mediators to the model (initial rhythm, location, response time, bystander cardiopulmonary resuscitation and if the arrest was witnessed) no difference in early angiography related to income level where found. the main mediator was initial rhythm (figure ). higher income is strongly related to the rate of early coronary angiography after ohca. this finding is consistent when adjusting for known confounders. however, the association between income and early angiography seems to be mediated by initial rhythm. patients with low income more often presents with non-shockable rhythms which lowers the likelihood to undergo early coronary angiography. a. the total amount of mortality as a stacked bar: in light-red the number of patients who deceased at scene, in green the number of patients deceased during admission, in red patients who died after discharge. the grey line is the total number of inclusions. b. the rate of bystander aed use, rate of initial shockable rhythm, rate of less than minutes of cpr and rate of favorable neurologic outcome over time. p for trend significant for bystander aed use, less than minutes of cpr and favorable neurologic outcome. trend analysis performed using binary logistic regression for dichotomous data (and a kruskal-wallis test for non-normally distributed continuous data) effect of simulation teaching of cardiopulmonary resuscitation for nursing v spatenkova introduction: simulation teaching is a modern type of critical care (cc) education. the aim of this study was to assess the effect of simulation teaching of cc on a comparison of final examination in different model levels of cardiopulmonary resuscitation (cpr) after the first (cc ) and third, final cc . the success rate of cpr was tested in prospective study ( ) ( ) on two groups with a total of students in cc and cc at the faculty of health studies. three semester of undergraduate nursing simulation education (lectures and training) used the laerdal simman g. quality of cpr was evaluated according to parameters: compression depth, compression rate, chest release and time of correct frequency. we tested if cpr quality differed between the two groups. for the compression depth and compression rate parameters, first the conformity of variance was verified and then two-sample t-test. as the chest release and time of correct frequency are recorded as percentages, the wilcoxon rank-sum test was conducted for these parameters. to ensure good resuscitation, all recorded parameters must be properly performed during resuscitation. thus, pivot tables were used to generate statistics and test if the number of correctly performed resuscitation parameters for cc and cc differ. the compression depth parameter was statistically significantly higher for the cc than for the cc (p= . ). there were no differences in compression rate (p= . ), chest release (p= . ) and time of correct frequency (p= . ). it was also tested how many of the parameters were performed correctly by students at cpr. the chi-square test shows the relative frequency of cpr success is higher for the cc group than for the cc group. at least out of parameters were correctly performed by % of cc students compared to % of cc students. the study showed a significant improvement of cpr in the final cc and supported the three semester simulation education. changes in blood gases during intraoperative cardiac arrest jj wang, r borgstedt, s rehberg, g jansen protestant hospital of the bethel foundation, anaesthesiology, intensive care and emergency medicine, transfusion medicine and pain therapy, bielefeld, germany critical care , (suppl ):p introduction: blood gas analysis (bga) is a common approach for monitoring the homeostasis during surgery. while it is well known that cardiac arrest (ca) leads to circulatory collapse and disturbances in homeostasis, little is known about changes of blood gas during peri-operative ca. we retrospectively analysed patients ≥ years who suffered from peri-operative ca during non-cardiac surgery from / to / . peri-operative ca was defined as need for cardiac compression during anaesthesia care. collected data included ph, paco , pao , return of spontaneous circulation (rosc) and -day mortality after ca. within the study period, we observed peri-operative ca (m= , f= ; age ± ) during anaesthesia procedures (rosc occurred in patients ( %). days after ca, the mortality was % (n= ), % (n= ) were discharged, and % (n= ) still in hospital. % (n= ) of ca patients had an invasive blood pressure monitoring, % (n= ) had bga before and % (n= ) during peri-operative ca. prior to ca, the average values were: ph . ± . , paco ± and pao ± . during ca, the average values were ph . ± . , paco ± and pao ± . table shows the distributions of blood gas before and during ca. there were no statistical differences between the groups (ph: p= . ; paco : p= . ; pao : p= . ). hypercapnia and respiratory acidosis is common in peri-operative ca. these data suggests inadequate ventilation during peri-operative resuscitation. further studies should focus on its impact on the outcome. ]. comparing cases with and without rosc, there were significant more diagnostics done in the group without rosc but more therapeutic consequences seen in the rosc-group (table ) . icu-ca is frequent. diagnostics to detect reversible causes of ca were used rarely in icu-ca ( %), even in patients without rosc. notably, diagnostics often had therapeutic consequences particularly in rosc. further studies are required to define standardized diagnostic algorithms during icu-ca. continuous monitoring of cardiac patients on general ward were improved short term survival of in-hospital cardiac arrest uj go introduction: the importance of early detection in the in-hospital cardiac arrest (ihca) is emphasized. previous studies have reported that clinical outcomes are improved if ihca is witnessed, or if a patient admitted to a monitored location [ , ] . this study aimed to evaluate the association between continuous monitoring and survival of ihca on general ward. a retrospective cohort study of ihca in patients admitted to ward at an academic tertiary care hospital between january and december was performed. the primary outcome was return of spontaneous circulation (rosc). the secondary outcomes were hour survival and survival to hospital discharge. (table ) . cardiac patients with continuous monitoring on general ward showed improving rosc and -hour survival but not survival to hospital discharge in ihca. in-hospital cardiac arrest is associated with poor outcomes. although steroids are frequently used in patients with septic shock, it is unclear whether they are beneficial during cardiac arrest and after return of spontaneous circulation (rosc). of cardiac arrest patients evaluated, were enrolled. advanced life support was conducted according to the resuscitation guidelines. forty-six patients were randomly assigned to receive methylprednisolone mg during resuscitation, and to receive saline (placebo). after resuscitation, steroid-treated patients received hydrocortisone mg daily for up to days, followed by tapering . there was no significant difference between the two groups in scvo andall the secondary outcomes (p> . for all comparisons). the present study found no significant physiologic benefit of corticosteroid administration during and after resuscitation in hospitalized patients with cardiac arrest. the experiences of ems providers taking part in a large randomized trial of airway management during out of hospital cardiac arrest, and the impact on their views and practice. results of a survey and telephone interviews m thomas introduction: the aim is to explore ems experiences of participating in a large trial of airway management during out-of-hospital cardiac arrest (air-ways- ), specifically to explore: . any changes in views and practice as a result of trial participation. . experiences of trial training. . experiences of enrolling critically unwell patients without consent. . barriers and facilitators for out-of-hospital trial participation. an online questionnaire was distributed to ems providers who participated in the trial. in-depth telephone interviews explored the responses to the online questionnaire. quantitative data were collated and presented using simple descriptive statistics. qualitative data collected during the online survey were analysed using content analysis. an interpretive phenomenological analysis approach was used for analysis of qualitative interview data results: responses to the online questionnaire were received from % of airways- study paramedics and study paramedics were interviewed. paramedics described barriers and facilitators to trial participation and changes in their views and practice. the results are presented in five distinct themes: research process; changes in views and practice regardingairway management; engagement with research; professional identity; professional competence. conclusions: participation in the airways- trial was enjoyable and ems providers valued the training and study support. there was enhanced confidence in airway management as a result of taking part in the trial. study paramedics expressed preference for the method of airway management to which they had been randomized. there was support for the stepwise approach to airway management, but also concern regarding the potential to lose tracheal intubation from 'standard' paramedic practice. causes of medical care-associated cardiac arrest on the intensive care unit s entz introduction: cardiac arrest on intensive care unit (icuca) following therapeutic interventions is of imminent importance, because the interventions are comparatively predictable and precautions can potentially be taken. this study investigates medical care associated complications that led to icuca. intensive care database was screened for patients ≥ years who experienced icuca in a tertiary hospital with five icu (two medical, two surgical, one interdisciplinary, with a sum of icu beds) in germany from - . icuca was defined as receiving chest compression and/or defibrillation after admission on icu and classified as "medical care associated" if it was preceded by a therapeutic intervention (i.e. induced by medication, bedding procedures, iatrogenic injuries, procedure associated). subgroups included patients with recurrence of spontaneous circulation (rosc) vs. no-rosc and patients with vs. without vasopressor therapy before intervention. there were icuca in patients of totally , icu patients. medical care associated complications leading to icuca were detected in cases ( %) [incidence . / , (ci . - . )]. icuca following therapeutic interventions occurred because of circulatory insufficiency [n= ( %)], respiratory failure [n= ( %)] and airway associated problems [n= ( %)]. nine of the patients ( %) with care-associated icuca died. table demonstrates therapeutic interventions followed by icuca. care-associated complications were common reasons for icuca. most of events were induced by circulatory insufficiency due to induction of anaesthesia and bedding procedures. further investigations should focus on preventive strategies, such as vasopressor infusion before therapeutic interventions. in-hospital cardiac arrest (ihca) is a lethal event. however, ihca has received less attention than out-of-hospital cardiac arrest (ohca). there have been some studies on ihca; however, there is a lack of information on the evidence and clinical features of ihca compared with information for ohca. we therefore conducted this study to clarify important aspects of the epidemiology and prognosis of ihca in patients with code blue activation. we carried out a retrospective observational study of patients with code blue events in our hospital during the period from january to october . we obtained information on the characteristics of patients including age and gender, ihca characteristics including the time of cardiac arrest, event being witnessed, presence of bystander cardiopulmonary resuscitation (cpr), initial shockable rhythm, vital signs h or h before cardiac arrest, survival to hospital discharge (shd), and the cardiac arrest survival postresuscitation in-hospital (caspri) score. the primary endpoint was shd. we performed univariate and multivariate logistic regression analyses. a total of code blue events were activated during the study period. finally, patients were included in this study. overall, the shd rate was . %. the median time of cpr was min (interquartile range, - min). the rate of initial shockable rhythm was . %. there were significant differences in cpr duration, shockable rhythm, and caspri score between the shd group and non-shd group by univariate-logistic regression analysis. caspri score was found to be the most effective predictive factor for shd (or= . , p= . ) by multivariate-logistic regression analysis. our results demonstrated that caspri score is associated with shd in cpa patients with in-hospital code blue events. caspri score in ihca patients would be a simple and useful adjunctive tool for management of post-cardiac arrest syndrome (pcas). peri-operative cardiac arrest in prematurityincidence and causes at a tertiary care hospital between - g jansen, j popp, e lang, r borgstedt, b schmidt, s rehberg protestand hospital of the bethel foundation, anaesthesiology, intensive care and emergency medicine, bielefeld, germany critical care , (suppl ):p the peri-operative care of premature pediatric patients requires special expertise and is therefore reserved for specialized centers. although premature birth is described as a risk factor for peri-operative complications and cardiac arrest (poca) there are no data on its incidence and causality in this particular population [ ] . the present study investigates the incidence and causality of pediatric poca at a tertiary care hospital and level i perinatal center in germany. in the anesthesia database of the study center, all anaesthesiological procedures in patients < years of age were examined for poca in preterm infants (gestational age < th week of gestational age) between and . the peri-operative period was defined between the beginning of anesthesiological care up to minutes after anesthesia and/or sedation. we defined cardiac arrest as the necessity of chest compressions. the perioperative phase and the cause of the poca, gestational age and birth weight were recorded. between and , ( . %) of the , pediatric anesthesiological procedures were performed on premature infants. in total, poca occurred in of these patients (f= , m= ; average gestional age ± days; average birth weight ± g (incidence . %, ci . - . %). the time of occurrence and the causes of poca are shown in table . poca in premature babies is rare and has an incidence of . %, which is significantly higher than the non-premature babies. the main causes are problems or complications associated with the respiratory tract and its management, as well as massive hemorrhage. introduction: peri-operative cardiac arrest (poca) in children's anesthesia care is a dreaded event. depending on the country and population, studies describe incidences between . - . per , children's anesthetics. there are no data on the current incidence of pediatric poca in germany. the present study investigates the incidence of poca at a tertiary hospital and level i perinatal center in germany. in the anesthesia database of the study center, all anaesthesiological procedures in patients < years were examined for poca. the peri-operative period was defined between the beginning of anesthesia care up to minutes after anesthesia or sedation. cardiac arrest was defined as the necessity of chest compressions. age, weight, asa status, cause of death and survival after days were recorded. results: poca (median weight was g [q ;q ( )]) were observed in , anaesthesiological procedures (incidence . ± . per , [ci . - . ]). table shows the distribution of the individual age groups, incidences and mortalities of poca. peri-operative -day mortality was per , [ci [ ] [ ] [ ] [ ] [ ] . three children died intraoperatively as a result of hemorrhagic shock, one on the picu as a result of malignant hyperthermia. days after poca, more children had died on the icu due to their underlying disease. poca is a rare event. risk factors are an age < days and an asa status ≥ iii. the main cause of peri-operative death in patients < years of age is massive hemorrhage, the -day mortality is determined by the underlying disease. in-hospital cardiac arrest -predicting adverse outcomes t partington, j borkowski, j gross northwick park hospital, anaesthesia/critical care, london, united kingdom critical care , (suppl ):p introduction: cardiac arrest occurs in . per hospital admissions in the uk. return of spontaneous circulation (rosc) is achieved in approximately half of resuscitation attempts, but rate of survival to hospital discharge is substantially lower [ ] . in our centre, post-arrest care accounts for . % of icu admissions. premorbid social function is purported to affect outcomes, but comorbidity scores are more often used for risk stratification. using a novel social function score alongside an existing comorbidity scale, we aimed to identify trends to inform management of patients at risk of deterioration. a six-month prospective observational study was conducted in a major uk hospital from october to april . for all adult inpatient cardiac arrests, medical notes were reviewed and data collected on the following domains: patient demographics comorbidities and functional status admission details post-arrest events statistical analysis was performed using student's unpaired t-test. results: cardiac arrests occurred. % were in medical patients, with the majority male ( %) and aged over ( %). % were emergency admissions, with mean duration of hospital stay pre-arrest days. in cases ( %) sustained rosc was achieved. however, seven of these ( %) were not subsequently admitted to the icu. only six patients ( %) survived to hospital discharge. pre-admission function and comorbidity were worse in patients who did not survive to discharge ( fig. ), but these were not statistically significant in view of small survivor group size. in an increasingly frail inpatient population, a substantial proportion of patients in whom circulation is restored after cardiac arrest are subsequently considered unsuitable for icu admission. given our understanding of inferior outcomes in patients with poor physiological reserve, we encourage early discussion regarding the appropriateness of cpr in selected patients, guided by social function and comorbidity. references: . national cardiac arrest audit / introduction: there are studies that determine events related to poor outcome in cardiac arrest [ ] . in our study, following parametres were determined ohca patients; age median years, asian/europe/syrian, bystander cpr, bystander aed, ems defibrillation, initial cardiac rhythm, prehospital rosc, corneal and pupillary light reflex and day survival. we determineted poor prognostic sign with post-cardiac arrest patients. in this study, we identified the causes of poor outcome in patients with ohca. this was a single-centre, retrospective study. we determined incidence and epidemiological factors including: demographics, initial cardiac rhythm. our study population were non-traumatic ohca. our icu, all ohca patient were evaluated wtih echo, and fluid, inotrope and vazopressor were added according to cardiac performance. results: during our study, patients who were admitted to intensive care unit between - were screened. of these patients were out-of-hospital arrest and of them were in-hospital arrest. development of cerebral oedema during treatment in hospital remains a poor prognostic sign. the evaluation of initial cardiac ritm is useful to predict neurological outcome in post-cardiac arrest patients. survival after ohca remains low. the evaluation of initial cardiac ritm is useful to predict mortality and neurological outcome in postcardiac arrest patients. basic life support (bls) education and training for school children is active in japan. however, the bls action by schoolchildren may be limited by school rules. this study aimed to analyse the time factors for basic life support performance and outcome in classmatewitnessed out-of-hospital cardiac arrest (ohca) and to investigate how schoolchildren act when they detect ohca. methods: nation-wide database for , school children cases with ohca and local extended database for , ems-unwitnessed ohca, both of which were prospectively collected during the period of - , were retrospectively analysed. proportion of schoolchildren-detected ohca was low in classmate cases ( . %, / ) in nationwide database and extremely low in all ems-unwitnessed ohcas ( . %, / , ) in local database. nationwide database analyses revealed that both emergency call and bystander cpr were delayed when a classmate witnessed the ohca case: median, vs. min and vs. min, respectively. classmate-witnessed cases were associated with higher incidences of shockable initial rhythm, aed use and traumatic causes. the rate of neurologically favourable outcome was . % and . %, respectively in classmate-witnessed and other cases: adjusted or; % ci, . ; . - . . of cases detected by schoolchildren in our prefecture, ( %) cases had presumed cardiac aertiology and ( . %) cases were caused by suicide attempts (hanging and fall). school children placed emergency calls as the first action only in ( . %) cases. emergency calls were largely delayed when school children dialled other numbers or left the scene to seek adult help. school children were rarely involved in bystander cpr ( %) and aed placement ( %). school children are rarely involved in entire bls. emergency calls and bystander cpr are delayed when schoolchildren act to seek help. because schoolchildren detect suicide-related ohcas, psychological care to schoolchildren involved in bls may be necessary. prognostic value of neutrophil/lymphocyte and platelet/ lymphocyte predicting cardiopulmonary resuscitation with spontaneous circulation recovery c li the affiliated suzhou hospital of nanjing medical university, suzhou, china critical care , (suppl ):p to investigate the predictive value of peripheral blood neutrophil-tolymphocyte ratio (nlr) and platelet-to-lymphocyte ratio (plr) on inhospital mortality in patients with spontaneous circulation recovery after cardiac arrest. a retrospective analysis was made of patients who recovered from cardiac arrest in our hospital from april to november and were admitted to the intensive care unit for more than hours. they were divided into survival group and death group according to the outcome of discharge.the dynamic changes and differences of nlr and plr in hours and - hours after admission to icu between the two groups were analyzed and compared. multivariate analysis and roc curve were used to explore the predictive value of nlr and plr for in-patient mortality. compared with the survival group, plr in the dead group was significantly lower within hours of admission to the intensive care department (p < . ), while nlr in - hours was significantly higher (p < . ). the nlr of surviving group was significantly lower than that of hours (p < . ), while the nlr and plr of death group were not significantly different (p < . ) from that of hours (p < . ). multivariate logistic regression analysis and roc curve showed that nlr of - h in icu was an independent risk factor for predicting in-patient mortality, and had high sensitivity and specificity in predicting death outcomes. neutrophil to lymphocyte ratio, platelet to lymphocyte ratio can help to judge the outcome of patients with cardiac arrest and recovery of autonomic circulation after cardiopulmonary resuscitation. [ , ] patients with sofa score > (vs sofa score ≤ ) had a higher free iron level ( . μmol/l vs μmol/l, p = . ) ( figure ). we found a positive correlation between free iron level at h and changes of sofa score between h and h (r= . ic [ . ; . ]). out-of-hospital cardiac arrest is associated with a significant change of plasma free iron level. free iron level at admission is associated with short term outcome. further research is warranted to better determine the significance of such changes. the optimal level of arterial oxygen in the post-resuscitation period is unknown. recent studies show conflicting results in regard to hyperoxia and its association with survival after out-of-hospital cardiac arrest (ohca) [ ] . the aim of this trial is to study the association between early hyperoxia after ohca with return of spontaneous circulation (rosc) and -day survival. observational study using data from three swedish national registers (i.e. intensive care, cardiac arrest and national patient registries after a successful resuscitation, a systemic inflammatory response occurs, and the c-reactive protein (crp) level represents the degree of inflammation [ ] [ ] [ ] . this study examined the association between increased inflammation and early-onset pneumonia (eop) in patients treated with extracorporeal cardiopulmonary resuscitation (ecpr) after out-of-hospital cardiac arrest (ohca). this retrospective study included data of patients with ohca treated with ecpr admitted to st. luke's international hospital between april and april . the exclusion criteria were as follows: age < years, therapeutic hypothermia withdrawal due to death or circulatory failure, or sepsis as a suspected cause of cardiac arrest. patients were diagnosed with eop according to clinical signs and symptoms acquired after a hospitalization period of > h and within days of admission. the crp levels were measured daily from admission to day . we studied patients with a median age of years (interquartile range: - years). furthermore, ( %) patients were males, and the median time interval from collapse to adequate flow was ( - ) min. all patients received prophylactic antibiotics, and ( %) of them had favorable neurological outcomes (cpc, - ). eop occurred in ( %) patients, with a significantly higher crp level on day than that in those without eop ( . categorizing reasons for death after ecpr is important for comparing outcomes to other studies, assessing benefits of interventions, and better define this heterogeneous patient collective. a categorizing for death after cardiac arrest in both in-hospital (ihca) and outof-hospital (ohca) arrests has been proposed in non-ecpr patients by witten et al. here, we adopt this categorization to ecpr patients. single-center, retrospective, cohort study of patients without rosc after ihca or ohca and ecpr between and . patients with survival below hours were excluded. patients were allocated to one of five predefined reasons for death. results: va-ecmo patients were included (age . ± . , . % female, % ecpr, day survival . %). reasons for death for patients with va-ecmo for shock (survival %) and ecpr ( %) were: neurological withdrawal of care ( % vs %), comorbid withdrawal of care ( % vs %), refractory hemodynamic shock ( % vs %), respiratory failure ( % vs %), and withdrawal due to presumed patient will ( % vs %) ( figure ). the differences in reasons for death among the two groups were significant (p < . ), driven by withdrawal due to neuroprognostication, comorbidity and hemodynamic instability. categorizing death after va-ecmo into five categories is feasible. there are significant difference between patients with va-ecmo for shock and ecpr. interestingly, only a quarter of patients after ecpr died due to brain damage. introduction: scarcity of potential dead brain donors and the persistent mismatch between supply and demand of organs for transplantation has led the transplant community to reconsider donation after circulatory death (dcd) as a strategy to increase the donor pool. normothermic regional perfusion (nrp) by extracorporeal membrane oxygenation (ecmo) may be the most effective method for preserving abdominal organs in dcd, especially in liver transplantation [ , ] . a pitfall of this method is its complexity and the unavailability of this resource in some hospitals, especially in regional hospitals, where potential dcd donors may exist. aim of this study is to report the use of mobile ecmo team in controlled dcd. from june to november our group has worked as a mobile ecmo team for cdcd outside our center. portable equipment included cannulation material and the ecmo device. the transplant team consisted of transplant coordinator (anesthesiologist-intensivist, ecmo operator and organ extraction supervisor), cardiac surgeon (cannulation), interventional radiologist (cannulation) and one cardiovascular perfusionist (ecmo operator). twenty-five cdcd donations were performed. characteristics of donors and organs retrieved are summarized in figure . from cdcd, livers, lungs, kidneys were obtained. the evolution of grafts and receptors was favorable at day post-transplant. mobile ecmo teams may enable cdcd in hospitals without these resources, thereby increasing the pool of donors and optimizing graft outcomes. what is the useful coagulation and fibrinolysis marker for predicting extracorporeal membrane oxygenation circuit exchange due to intra-circuit thrombus? y izutani, k hoshino, s morimoto, k muranishi, j maruyama, y irie, y kawano, h ishikura fukuoka university hospital, emergency and critical care center, fukuoka-shi, japan critical care , (suppl ):p a thrombus formation is one of the most frequent and adverse complications during extracorporeal membrane oxygenation (ecmo) support. previous studies have reported that increased d-dimer is a useful predictor of thrombus formation within the ecmo circuit. the purpose of this study was to identify coagulation/fibrinolysis markers for predicting the replacement of ecmo circuit due to intra-circuit thrombus during ecmo support. fourteen patients who underwent veno-venous ecmo for acute respiratory failure between january and december were enrolled. these patients received a total of days of ecmo support. of these, days (times) on which the ecmo circuits were replaced was regarded as the replacement group, while the remaining days were considered as the non-replacement group. the several coagulation/fibrinolysis markers were routinely measured every day during ecmo support. we compared with the levels of these markers between two group to identify the most relevant marker for ecmo circuit replacement due to thrombus. the mean duration of ecmo support was ± days, and the mean number of ecmo circuit replacement was . ± . times per patient. ddimer, thrombin-antithrombin complex (tat), plasmin-α plasmin inhibitor complex (pic), and soluble fibrin (sf) were significantly higher in the replacement group rather than in the non-replacement group (p < . , respectively). according to a multivariate analysis, sf was the only independent predictor of ecmo circuit replacement due to thrombus. the odds ratio ( % confidence intervals) for sf ( μg/ml) was . ( . - . ). the area under the curve and optimal cut-off value were . and ng/ml for sf, respectively (sensitivity, %; specificity, %). from these results, we concluded that sf may be the useful marker rather than d-dimer for predicting the replacement of ecmo circuit due to intra-circuit thrombosis. inhomogeneity of lung elastance in patients who underwent venovenous extra corporeal membrane oxygenation (v-v ecmo)-a computed tomography scan study rd di mussi , ri iannuzziello , fm murgolo , fd de carlo , e caricola , na barrett , lc camporota , sg grasso università degli studi di bari "aldo moro", department of emergencies and organ transplant, bari, italy; università degli studi di bari "aldo moro", bari, italy; department of adult critical care, guy´s and st thomas´nhs foundation trust, king´s health partners, london, uk critical care , (suppl ):p in patients with acute respiratory distress syndrome (ards), nonaerated, poorly aerated, and normally aerated regions coexist to variable degrees in lung parenchyma. the recruitment maneuvers aim to reopen collapsed lung tissue. in a theoretical point view, this strategy may also prevent the normal aerated lung tissue hyperinflation [ ] . the objective of our study was to evaluate lung characteristics in terms of hounsfield units (hu), volume and elastance before and after a recruitment maneuver. in patients with severe ards who underwent v-v ecmo, computed tomography scans (ct-scans) at cmh o of continuous positive airway pressure (cpap) and cmh o were performed. the same ct image was selected at the two different levels of pressure. the distribution of lung opacities, in terms of hu, was classified using the "ucla" colour coding table (osirix image processing software, geneva, switzerland). correspondent lung regions of about voxels were selected. the quantitative analysis, in terms of volume air (vair) was performed with maluna software (version . ; maluna, goettingen, germany). elastance was calculated as the pressure(cmh o)/ vair (ml) ratio. results: see figure . lung inhomogeneity occurs also after recruiting maneuvers. our data confirm that the elastance of recruited lung regions is higher than the elastance of the normal aerated lung regions at low positive end-expiratory pressure (peep) (baby lung). on the contrary the "baby lung" frequently develops hyperinflation. the unpredictable pattern of distribution of volume after recruitment maneuverers may explain the controversial role of peep during the ards treatment. . formal recommendations on target, timing, and rate of at supplementation are lacking. we conceived this study to evaluate the effect of prolonged at supplementation in adult patients requiring veno-venous ecmo for respiratory failure on heparin dose, adequacy of anticoagulation and safety methods: before ecmo start patients were randomized to either receive at supplementation to maintain a functional at level between and % (at supplementation group) or not (control group) for the entire ecmo course. anticoagulation was provided with unfractionated heparin following a standardized protocol [ ] . the primary outcome was the dose of heparin required to maintain the ratio of activated partial thromboplastin time between . and . secondary outcomes were the adequacy of anticoagulation measured with anti-factor xa and the incidence of hemorrhagic and thrombotic complications and amount of blood products fig. b) . conclusions: this retrospective analysis was not able to show a survival benefit for additive pp to ecmo support in general. early initiation of pp could be an important factor for improving survival in this setting and should be considered in a randomized controlled trial for further evaluation. cause-specific mortality during extracorporeal membrane oxygenation, a single center review of medical records m panigada, d tubiolo, p properzi, g grasselli, a pesenti fondazione irccs ca´granda ospedale maggiore policlinico, intensive care unit, milano, italy critical care , (suppl ):p introduction: mortality during extracorporeal membrane oxygenation (ecmo) settles around % and the occurrence of bleeding during ecmo is associated with a high mortality rate. however, cause-specific mortality is rarely reported, probably due to the difficulty of its classification. the purpose of the study was to evaluate the agreement between two expert icu physician in the classification of the cause of death of patients supported with ecmo for either respiratory or cardiac support. methods: two intensive care unit (icu) expert staff physicians independently reviewed the entire medical records of all ecmo patients who died before icu discharge from january to september at fondazione irccs ca' granda, milan. they were asked to choose the cause of patient's death among six categories. in case of disagreement, a third expert adjudicated the case. the two reviewers were also asked whether, in their opinion, bleeding during the last hours contributed to death. elso definition of major bleeding [ ] during the last hours was also recorded for each patient. results: two-hundred and two patients were supported with ecmo of whom ( . %) died. most of these patients (n= , . %) died during ecmo. interrater agreement for cause-specific mortality between the two expert physicians was substantial (k . , se . , p< . ) of the discordant cases were categorized as refractory respiratory failure and as multiorgan failure and septic shock respectively. the distribution of cause-specific mortality is shown in figure . major bleeding (elso) was present in ( . %) patients, only in ( . %) of them bleeding contributed to death according to the reviewers. patients treated with early pp while ecmo showed a superior survival to patients treated with late pp or without pp while ecmo. optimal cut off value for duration of ecmo initiation to first pp was calculated using roc-analysis (auc = . ) and the youden-index. highest sensitivity and specificity for beneficial survival were achieved for a beginning of pp in < . days. (log rank= . ). pp: prone positioning p non-invasive mechanical ventilation in veno-venous extracorporeal membrane oxygenation j rilinger, v zotzmann, x bemtgen, pm biever, d duerschmied, c bode, dl staudacher, t wengenmayer heart center freiburg university, department of cardiology and angiology i, freiburg, germany critical care , (suppl ):p introduction: veno-venous extracorporeal membrane oxygenation (ecmo) support can be combined with a variety of different non-invasive ways to deliver oxygen to the patient's lung. several positive effects might be linked to this so called "awake ecmo". so far there is little evidence about indications and outcome of this approach. we report retrospective registry data on all ards patients treated with ecmo support at a university hospital between / and / . in a systematic review of medical records, we distinguished between patients with invasive mechanical ventilation (imv) from the initiation of ecmo therapy (imv group) and patients that received any kind of non-invasive oxygen supply (non-imv group). a total of patients could be analysed. ( . %) patients received non-imv ecmo support. patients receiving non-imv ecmo therapy showed severe underlying pulmonary disease and immunosuppression (fig. ) . these patients had higher rates of lung fibrosis, long-term oxygen therapy, pulmonary hypertension, renal insufficiency and immunosuppression (p< . ). of patients ( %) required imv during the hospital stay in average . ± . [ . - . ] days after ecmo initiation. reasons were hypoxia despite of ecmo, insufficient ecmo-flow, insufficient protective reflexes or patient agitation. patients with initially non-imv ecmo support showed a numerical but not significant lower icu and hospital survival ( . % vs. . %, p= . ). non-imv ecmo support was applied in patients with severe underlying pulmonary disease and/or immunosuppression. in a high proportion of patients the ventilation regime had to be switched from non-invasive to invasive. survival in this very selected cohort was low. in this retrospective analysis no evident benefit for a noninvasive ventilation strategy could be found. the high proportion of patients who switched from non-imv to imv therapy underlines the need for rigorous patient selection. intra-hospital transportation on extracorporeal membrane oxygenation (ecmo) -a single centre experience in ireland. z siddique, s o´brien, e carton, i conrick-martin mater misericordiae university hospital, department of critical care medicine, dublin, ireland critical care , (suppl ):p the objective of this study is to evaluate intra-hospital transportation of patients on extracorporeal membrane oxygenation (ecmo). it is a retrospective analysis of prospectively collected database, performed as part of ongoing quality improvement initiatives. the setting of this study is an -bed, combined surgical and medical adult intensive care unit (icu) located in a -bed hospital that serves as the national referral centre for cardiothoracic surgery, heart & lung transplantation and ecmo in ireland. we reviewed months of data (from to ) regarding patients admitted to our critical care unit who required intra-hospital transfer for diagnostic and/or therapeutic interventions. we also compared the data to available local guidelines. results: patients were transported on ecmo on a total of occasions; the most common indication being ct brain (table ) . ecmo cannulation sites were peripheral in patients, patients were centrally cannulated. median time from start of the transfer until the patient was returned to icu was minutes (range: - ). the ecmo console was placed on a dedicated ecmo trolley apart from two occasions where it was placed on the patient's bed. number of staff required for transport was between to ; with an icu consultant as team leader. ecmo specialist nurses were always present on the transport team. transfers were during normal working hours with happening on a weekend. a total of complications occurred during the transports, of underlying pulmonary disease or status of immunosuppression in ecmo patients without invasive mechanical ventilation which was significant and were not. the significant complication encountered was ventricular tachycardia in a v-a ecmo patient which required electrical defibrillation. no adverse events related to transport were seen following return to icu. in this single-centre study, we have demonstrated safe intra-hospital transport of ecmo patients. the use of local guidelines, appropriate personnel and performance during normal working hours is recommended. a novel approach for flow simulation in ecmo rotary blood pumps a supady , c benk , j cornelis , c bode , d duerschmied heart center freiburg university, cardiology and angiogiology i, freiburg, germany; heart center freiburg university, department of cardiovascular surgery, freiburg, germany; fifty technology gmbh, freiburg, germany critical care , (suppl ):p introduction: extracorporeal membrane oxygenation (ecmo) is used increasingly in critically ill patients suffering from acute respiratory failure, cardiogenic shock or cardiac arrest. however, this therapy can have deleterious side effects such as bleeding or clotting complications and hemolysis. these complications are particularly caused by physical stress acting upon the blood components while passing through the ecmo system, especially within the rotary pump. we here present a novel approach to simulate blood flows through rotary blood pumps used in current ecmo systems in order to better understand the genesis of these complications. geometries of the xenios dp (xenios ag, heilbronn, germany) rotary pump were reconstructed by ct-scans and manual measurements using computer-aided design (cad). the computational fluid dynamics (cfd) simulation was performed using the software preon-lab (fifty technology gmbh, freiburg, germany), which implements a mesh-free lagrangian method requiring minimal preprocessing of the cad data. the geometries are introduced to the simulation model as tessellated surfaces. five operating points have been specified by the rotation of the centrifugal fan and the corresponding inflow and outflow of blood. the blood is approximatively modelled as a newtonian fluid with a density of kg/m . preonlab allows detailed assessment of the blood flow while passing through the rotary pump including analysis of local flow rates, pressure gradients and shear stress acting upon the blood. dead zones in the fluid flow can be detected which gives reference points for optimizations of the pump design. for the first time, we demonstrate a novel approach for flow simulation in an ecmo rotary pump ( figure ). this approach may help better understand hemodynamics within the extracorporeal system to define optimal operating points or re-design components aiming to limit hemolysis, coagulation disorders and bleeding in seriously ill patients. one-year experience of bedside percutaneous va-ecmo decannulation in a territory ecmo center in hong kong km fong, sy au, pw leung, kc shek, hj yuen, sk yung, hl wu, so so, wy ng, kh leung queen elizabeth hospital, intensive care unit, hong kong critical care , (suppl ):p when veno-arterial extra-corporeal membrane oxygenation (va-ecmo) support can be terminated, arteriotomy wounds of the patients of are traditionally closed by open repair in the operation theaters. lots of manpower are involved and timeslots in operating theaters are scarce. transport of the critically-ill is risky. successful va-ecmo decannulation using percutaneous device called proglide has been reported and our group had adopted and modified this approach [ ] . methods: this is a retrospective study analyzing the one-year experience of bedside va-ecmo decannulation. our institution is a -bed tertiary ecmo referral center in hong kong. our first bedside decannulation was performed in november , and since then, this practice had replaced the traditional open repair, unless contraindicated. data from november to october were analyzed. in the study period, patients received va-ecmo. survived to decannulation and received bedside percutaneous decannulation. their median age was ( - ). the default arterial catheter size was fr, with fr in cases and fr in one. five ( %) failed percutaneous closure and they were subsequently surgically repaired without extra corporeal life support (ecls) continues to be associated with high mortality rates. our ability to predict outcome prior to initiation ecls remains limited. here we take a single cell rnaseq approach in an effort to identify novel immune cell types that are associated with-and may contribute to-survival on ecls. whole genome transcriptomic profiles were generated from~ , peripheral blood monocytes obtained from patients at the time of cannulation for veno-arterial ecls (va-ecls). within each subpopulation, differential gene expression analysis was performed to identify new markers associated with survival. findings were validated in a additional cohorts by flow cytometry. surviving patients had significantly higher proportions of cd + nkt cells (cd + /cd + /cd -/cd + ) that were cd + (p = . , fdr < . ) ( figure ). to validate this observation, we performed fc analysis of a second cohort of patients. for each patient, we quantified the proportion of cd + nkt cells that were cd + . using the median proportion as the cutoff, we again found that a high proportion of cd + cells among cd + nkt cells was predictive of hour survival (p= . ). we noted that while high levels of cd + cells among the cd + nkt cells was protective in this cohort of va-ecls patients, this relationship did not hold for patients with sepsis. as only a few the va-ecls patients were septic, we analyzed a third cohort of septic ecls patients. we observed that high levels of cd + cells among the cd + nkt populations was not protective in this population. the proportion of cd + nkt cells that are positive for cd is predictive of survival among patients undergoing va-ecls for noninfection related indications. introduction: the use of calcium sensitizers has grown enormously in the last decade, probably due to their interesting pharmacodynamic properties. levosimendan (ls) is frequently administered in patients under mechanical circulatory support. we performed a retrospective evaluation of patients treated with ls prior to weaning from mechanical support. this evaluation was combined with a review of the literature. a query of our icu patient data management system revealed patients receiving ls prior to or during vad/ecls support. outcome data were obtained from the patients medical records. of our patients, % was successfully weaned off ecls. fourteen patients ( %) died before being discharged of whom while on ecls support. of the weaned patients, died afterwards. of the converted patients needed subsequent veno-venous ecls support for right ventricular support after the implantation. survival to discharge ratio for the whole group was %. more detailed demographic results can be found in table . a pubmed search using the terms "(ecmo or ecls) and ls and weaning" resulted in publications which dealt specifically with weaning of ecls support. several weaning approaches are available, however poor outcome has remains a problem. some recent studies show a possible beneficial effect of ls infusion prior to weaning from ecls. however most of these studies are retrospective or observational at best. because ls is primarily reserved for the most severe cases, outcome interpretation is difficult. overall weaning success ranges from %- % and variation is very dependant of inclusion criteria. the calcium sensitizer ls can be used when weaning off patients from ecls, certainly given its low incidence of complications. future, large randomized trials are however needed in order to confirm this strategy. cardiogenic shock is well described in newly diagnosed pheochromocytoma, and crisis may be precipitated by hemorrhage into tumour. v-a ecmo represents a rescue therapy in a subset of these patients refractory to medical management, facilitating cardiac recovery and subsequent definitive surgery. consent to publish: written informed consent for publication was obtained from the patients. during a spontaneous breathing trial respiratory mechanics can worsen, and respiratory muscle effort can increase, leading to respiratory muscle fatigue, pump failure, hypercapnia and an unsuccessful weaning from mechanical ventilation. this case report discusses the possibility of applying extracorporeal co removal (ecco r) to reduce respiratory muscle effort in a liver transplant recipient who already failed three weaning attempts from mechanical ventilation. the ecco r membrane lung was integrated into a conventional renal replacement therapy circuit and blood flow was increased from to ml/min. measurements of respiratory mechanics (including esophageal pressure, as shown in fig. ) were used to assess the reduction of respiratory effort before and during the application of ecco r. was delivered through a fr-double-lumen-cannula; ml/min blood-flow with lt oxygen sweep-gas-flow and aptt . - baseline were maintained (iv-heparin). in all cases respiratory and metabolic parameters improved without complications ( figure ). ecco r-crrt facilitated extubation ( out imv pts). in out of pts at risk of niv failure, it avoided imv. treatment mean duration was ± hours, mean lenght of icu stay was ± days. all patients survived to the treatment, nevertheless patients died due to irreversible multiple mof. in our aecopd series prismalung®-prismaflex® facilitated weaning from imv and avoided intubation in patients at risk of niv failure without complications. these positive results may be related to minimal invasiveness of the low-flow device used and may constitute the rationale for a larger randomized controlled trial. consent: written informed consent for data publication has been obtained. extracorporeal the primary outcome findings from the supernova trial [ ] demonstrated that the use of extracorporeal carbon dioxide reamoval (ecco r) allows a reduction in tidal volume (tv) to ultraprotective levels (≈ ml/kg predicted body weight or pbw) during mechanical ventilation in ards patients without significant increases in the arterial partial pressure of carbon dioxide (paco ). unfortunately, it was not feasible to directly measure ecco r rates during the trial. we used a mathematical model of whole-body oxygen (o ) and carbon dioxide (co ) transport and biochemistry [ ] to calculate ecco r rates that permit a fit to the data reported for hemolung (alung technologies) and ila (novalung)/cardiohelp (getinge) devices in the supernova trial [ ] . the mathematical model was calibrated under baseline conditions where patients were mechanically ventilated at a tv of ml/kg pbw in the absence of an ecco r device; the o consumption rate, co production rate and pulmonary shunt fraction were adjusted to match the measured baseline arterial partial pressure of o and paco . assuming all baseline parameters were fixed, tv was then reduced to . ml/kg pbw and the mathematical model predicted the ecco r rate to the change in the paco level. model predictions for the devices are shown in table . these predictions suggest that ecco r rates for ila/cardiohelp devices were approximately twice those for hemolung devices during the supernova trial. these results may be useful to evaluate the expected performance of novel ecco r devices. efficiency and safety of a system crrt plus ecco r to allow ultraprotective ventilation protocol in patients with acute renal failure f maldarelli despite renal function replacement techniques (crrt), a patient who develops acute renal failure(aki) in intensive care unit (icu) has a mortality rate of - %. this risk is partly due to the adverse effect of aki on other organs than the kidney. respiratory complications are frequently associated with the development of aki. new machines combining crrt with a carbon dioxide removal membrane (ecco r) allows the setting up of an ultra-protective ventilation ( ml/kg of predicted boby weight (pbw)) to reduce any lung damage from mechanical ventilation (mv). the reduction in tidal volume (vt) is associated with a decrease in lung damage partly triggered by aki. we evaluated the efficacy of a combined system crrt+ecco r to reduce the vt to ultraprotective values in patients with acute respiratory failure and aki. ards is a syndrome with high morbidity and mortality. an emerging treatment option is ecco r, but the benefit its remains unclear. we assess different degrees of ecco r and varying dead space (ds) on ventilator settings in order to minimize mechanical power. we calculated mechanical power as ( ) power=rr*{Δ〖vt〗^ *[ / *el+rr*( +i:e)/( *i:e)*r]+ Δvt*peep} (el: system elastance, r: airway resistance, peep: positive end expiratory pressure, i:e: inspiratory to expiratory ratio). we calculated the combination of respiratory rate (rr) and tidal volume (vt) ("optimal rr" and *optimal vt*) leading to minimal applied power for a stable carbon dioxide elimination of ml/min (vco ) for two scenarios: ) variation of physiological ds from to % of vt at a fixed rate of eccor . ) variation of ecco r of either , , or ml/min at a fixed physiological ds of %. the alveolar ventilation (va) necessary to eliminate the vco was calculated as ( ) va= (-vco *σ_co *r*t*( +k_c ))/(vco /q-p_vco *σ_co *r*t*(( +k_c ))/ ) σco : co solubility in blood, r: gas constant, t: temperature. pvco : venous partial pressure, kc: function of ph ( . for a ph of . ), q: blood flow [ l/min]). increasing ds from to % increases the minimal mechanical power from . to . j/min, primarily caused by an increase of optimal vt ( - ml). optimal rr was only slightly increased ( . - . /min, figure panel a). for varying ecco r removal, necessary ventilation ranges from . to . l/min. this predicts a minimal power between . and . j/min with an unchanged optimal vt ( - ml) and an increasing optimal rr ( . to . /min ( figure panel b)). in order to minimize mechanical power, increasing shunt or co production should be met with increases in rr while increases in ds should be met with increases in vt. our results indicate that during ecco r, mechanical power and thus risk for lung injury can be minimized with higher vt compared to conservative ventilation strategies. validity of empirical estimates of physiological dead space in acute respiratory distress syndrome jd dianti, eg goligher, as slutsky university of toronto, interdepartmental division of critical care medicine, toronto, canada critical care , (suppl ):p increased physiological dead space fraction (v d /v t ) is a hallmark of the acute respiratory distress syndrome (ards) and has been shown to predict ards mortality. v d /v t is also important in estimating the reduction in tidal volume (v t ) and driving pressure (Δp) with extracorporeal co removal (ecco r). v d /v t can be measured with volumetric capnography but empirical formulae using the patient's age, weight, height, gender and paco have been proposed to estimate v d /v t based on estimates of co production (v co ). the accuracy of this approach in critically ill patients, however, is not clear. secondary analysis of a previously published trial [ ] in which v d /v t and v co were measured in ards patients. estimated dead space fraction (v d,est /v t ) was calculated using standard formulae. agreement between methods was evaluated by bland-altman analysis. the predicted change in Δp with ecco r was evaluated using both measured and estimated alveolar dead space fraction (v dalv /v t ). results: vd,est/vt was higher than measured vd/vt, with a low correlation between the (r = . ). vco was underestimated by the predicted approach (table ) , accounting for % of the error in estimating vd/vt. the expected reduction in Δp with ecco r using vdalv/ vt was in reasonable agreement with the expected reduction using introduction: acute respiratory distress syndrome (ards) is a common condition in critically ill patient. however neuromuscular blockers (nmb) result controvertial in early treatment of ards [ ] . we ought to search systematically and realize a meta-analysis on the matter. an electronic search of randomized clinical trials in adult patient treated with early neuromuscular blockers compared without neuromuscular blockers in ards. the primary objective of the analysis was the mortality at to days. secondary endpoints included mechanical ventilation free days, icu acquired weakness and barotrauma. the search obtained studies for the analysis [ ] [ ] [ ] [ ] [ ] [ ] (figure ). the early use of neuromuscular blockers in ards showed no increase in mortality, but the results should be taken with caution. there was no differences in mechanical ventilation free days. barotrauma is less with the use of nmb. ultrasound is fairly sensitive in the detection of lung infiltrates in patients with hematologic malignancies. in patients with pneumonia requiring intensive care (icu) admission, we hypothesise that abnormal right ventricular (rv) function is associated with an increased -day mortality. rv dysfunction in critically ill patients has a well-known association with adverse outcomes [ ] . however, its impact on mortality in patients with pneumonia has not been directly studied. patients admitted to the queen elizabeth hospital birmingham icu between april and july with a diagnosis of pneumonia who had a formal cardiologist tte were included. abnormal rv function was defined by either depressed function, dilated size or moderate to severe risk of pulmonary hypertension (phtn). abnormal lv function was defined by an lv ejection fraction £ % or grade ii or more diastolic dysfunction. patients with a clinical suspicion of pulmonary embolism were excluded. the primary outcome was -day mortality. continuous data is presented as median (iqr). categorical data is presented as % and analysed using a chi-squared test. results: patients were admitted to icu with pneumonia, of which ( %) had a tte. patients were % male, had a median age of ( - ) and -day mortality of %. abnormal rv function was present in % (n= ), with % depressed, % dilated and % with moderate to severe risk of phtn. rv dysfunction was associated with an increased -day mortality compared to normal rv patients ( % vs. %, p< . ). lv function was abnormal in % (n= ) and was not associated with a higher -day mortality compared to normal lv patients ( % vs %, p = . ). rv dysfunction was associated with a higher -day mortality than lv dysfunction ( % vs %, p = . ). conclusions: this is one of the first studies to demonstrate that abnormal rv function is associated with an increased mortality in icu patients with pneumonia. interestingly, abnormal lv function was not associated with an increased mortality. rakuno gakuen university, anesthesiology, hokkaido, japan critical care , (suppl ):p we previously reported a simple correction method of estimating pleural pressure (ppl) by using central venous pressure (cvp) and that it can be used to estimate ppl and transpulmonary pressure in pediatric patients with respiratory failure. however, it remains unknown that this method can be applied to patients with various levels of chest wall elastance and/or intravascular volume. the objective of this study is to investigate whether our method is accurate in various conditions of chest wall elastance and intravascular volume. the study was approved by the animal care and use committee of rakuno gakuen university. ten anesthetized and paralyzed pigs ( . ± . kg) were mechanically ventilated and subjected to lung injury by saline lung lavage. each pig was subjected to different intravascular volume and different intraabdominal pressures; in each condition, the accuracy of our method was tested. specifically, airway flow, airway pressure (paw), esophageal pressure (pes), and cvp were recorded in each condition, then changes in pes (Δpes) and Δppl calculated using a corrected Δcvp (cΔcvp-derived Δppl) were compared. cΔcvp-derived Δppl was calculated as κ × Δcvp, where κ was the ratio of the Δpaw to Δcvp during the occlusion test. means and standard deviations of the two variables that reflect Δppl (Δpes and cΔcvp-derived Δppl) in all pigs with all conditions were . ± . and . ± . cmh o. the bland-altman analysis for the agreement between Δpes and Δcvp showed a bias of - . the activity and functionality of the diaphragm are difficult to measure in patients ventilated in intensive care. ultrasound can be a useful tool for monitoring diaphragm muscle activity during different ventilation modes. few data currently exist on diaphragm muscle activity in critically ventilated patients [ ] . our goal is to evaluate the respiratory muscular work of the diaphragm with different settings of the respirator by means of an ultrasound scan. the ultrasound assessments of the diaphragm were performed with a mhz linear probe at the apposition zone. we measured the thickening of the diaphragm with the respiratory acts, through the thickening fraction (thickening fraction, tf), defined as:tf = (tdimax -tdimin / tdi min)% tdimax: diaphragm thickness at the end of inspiration (maximum thickness) tdimin: diaphragm thickness at the end of expiration (minimum thickness). ventilatory support was divided into classes: -spontaneous breathing (sb) or continous positive airway pressure (cpap); -pressure support ventilation (psv) with low pressure support ( - cmh o); -psv with high pressure support (> cmh o); -controlled mechanical ventilation (cmv). a total of assessments were performed in patients. the evaluations were all possible at the right hemidiaphragm, while on the left they were not possible in % of the cases. the median tf (iq range) of the ventilation classes was respectively: % ( - %) in sb / cpap; % ( - %) in low-psv; % ( - %) in high psv; and % ( - %) in cmv. the kruskal-wallis test confirms a significant difference between the groups (p < . ). the ultrasound of the diaphragm can be a valid tool for monitoring respiratory muscle activity during mechanical ventilation. introduction: extubation failure is defined as reintubation after hours of extubation in mechanically ventilated critically ill patients. it is associated with morbidity and mortality. the aim of our study was to assess reintubation rates in a busy district general hospital and evaluate the impact of high flow nasal oxygen therapy (hfno) on reintubation rates. we performed a retrospective observational study looking at patients admitted to our bedded level critical care unit ( patients a year) for a period of years between st november and st october . we included patients over years of age who were mechanically ventilated and length of stay was greater than hours. exclusions were age < years, tracheostomy and patients requiring ventilation for < hours. data was collected from ward watcher, a sicsag database and electronic patient records. our study failed to show any impact of hfno on reducing extubation failure. further work is needed to develop a standardized approach to weaning and to consider routine application of noninvasive ventilation to reduce reintubation rates [ ] . fig. (abstract p ) . the bland-altman analysis for the agreement between Δpes and cΔcvp-derived Δppl in various conditions. low: low intravascular volume, normal: normal intravascular volume, high: high intravascular volume, abd-: without an abdominal compression band, abd+: with an abdominal compression band oral endotracheal intubation is common to critically ill patients in intensive care unit. oral care for an intubated patient is important to maintain the moisture of oral mucosa. also, the securement method of oral endotracheal tube developed from cloth tape to commercial tube holder. training powerpoint and video for microteaching was prepared to train up icu nurses to perform the new practice. demonstration and re-demonstration was arranged to assess skills of every nurse. afterwards, each nurse answered a quiz to evaluate the understanding of oetth and its special techniques in application. questionnaire was designed to collect the feedback from all nurses too. the result showed there was nurses ( %) out of nurses achieved full marks in the post-quiz which demonstrated their full understanding of the use of oral ett holder and its nursing care. about the feedback from nurse, % of nurses claimed that they were confident in using the new oetth in clinical setting after training. % of nurses agreed in time-saving of nursing care routine with the use of an oetth. however, only % of nurses agreed that the oetth is effective in prevention of oral mucosa injuries and another % of nursing staff disagreed on its function in improving the patient's oral care. in conclusion, some of the nurses did not agree the prevention of oral mucosa injuries by the new securement method with oetth while some nurses welcomed the new oetth as more easy and effective in oral care to intubated patients. execution of percutaneous dilatational tracheostomy using the standard laryngeal mask airway for ventilation: a prospective survey study g gagliardi , v gagliardi , c chiani , g laccania , f michielan aulss -veneto, anesthesia and intensive care, adria, italy; aulss -veneto, university of padua, adria, italy; aulss -veneto, anaesthesia and intensive care, adria, italy; aulss -veneto, anaesthesia and intensive care, padua, italy critical care , (suppl ):p we fulfilled a survey study dealing with bronchoscope-guided percutaneous dilatational tracheostomies (pdt), using the classic laryngeal mask airway (lma) for the airway management [ ] . the aim was to verify the safety and the effectiveness of the aforementioned procedure methods: we performed an observational prospective survey study enrolling patients hospitalized in the intensive care unit. before performing the tracheostomy, the endotracheal tube has been replaced by the laryngeal mask airway. arterial blood gases, ventilation pressures and tidal volumes have been monitored, registered and compared. the median peak inspiratory pressure has been detected stable in all patients. furthermore, during the ventilation with the laryngeal mask, the tidal inspiratory and expiratory volume difference observed between before and after the bronchoscope positioning, has shown a statistically significant variation. finally, in all cases etco , spo . , pao , and blood ph values persisted within the normal range. the standard lma provides for a reliable airway management and allows an effective ventilation while performing the pdt. once positioned in the supraglottic zone, the lma does not need to be moved throughout all the pdt performance, avoiding risks of displacement, glottic harm and airway device damage, and permitting an easy handling of the bronchoscope, which gives an appropriated visualization of the trachea and a more efficient aspiration. in consequence to the large internal diameter of the lma tube, ppeak has continued to be stable in all patients, providing for minor resistance and inspiratory work. eventually, no late complications, such as tracheal stenosis and infections, have occurred. tracheostomies are the most common surgical procedure performed on critically ill patients. randomized control trials comparing tracheostomy timing in intensive care patients have been equivocal. in order to perform non-urgent tracheostomy in our icu, consent is required from the patient or a formal guardian appointed ad hoc by the courts. since tracheostomies are practically the only elective surgery performed in the critically ill, icu requested guardianship almost always indicates a clinical decision to perform tracheostomy. as appointing a guardian and arranging a tracheostomy takes about a week, the decision to appoint a guardian offers a unique "intention to treat" opportunity to evaluate outcomes in patients for whom tracheostomy is planned. we performed a retrospective analysis over years on patients for whom guardianship was sought excluding those requiring urgent tracheostomy and those with a do-not-resuscitate order. patients were divided according to outcome (tracheostomy, extubation or death prior to tracheostomy) and compared. guardianship was sought for ventilated patients. a decision to withhold tracheostomy was made for patients, who were excluded, leaving patients for analysis. tracheostomy was performed for / ( %) patients, / ( %) were extubated and / ( %) died while waiting for tracheostomy (from nonairway related reasons). tracheostomy was performed on mean ventilation day ± . comparing extubated patients to those who had tracheostomy (table) shows similar demographics, but significantly lower mortality and hospital length of stay. a significant proportion of patients initially planned for tracheostomy were successfully extubated. despite demographic similarities, mortality in this group was significantly lower than for patients undergoing tracheostomy. for a selected subgroup of possibly difficult to characterize patients, delaying tracheostomy may be beneficial. figure ). ptis were analysed by speciality and by outcome. complications occurred in cases (incidence . %). there were cases of subcutaenous emphysema, pneumothorax (occuring d post procedure) and case each of stoma and suture site infection. there was unplanned cannula change within days of insertion. % of cases had cuff inflated on discharge from icu. handover of care was suboptimal; follow up care plans were documented in % of cases. a supervising consultant was present for all ptis. there was a trend of increased insertion by consultant and increased reliance on theatre, with corresponding decrease in the number inserted by trainees. pti in our training icu appears safe with low incidence of complications and good senior support for tracheostomy insertion. emphasis must continue on training junior intensivists in pti. transition of care beyond icu requires further work where currently there is suboptimal handover of care and safety netting for non-icu colleagues. supplemental oxygen administration is ubiquitous in the critical care environment, yet evidence is mounting for the deleterious effects of hyperoxia [ ] . concerns over the adverse effects from hypoxaemia often exceed those of hyperoxaemia in developing world settings, and inconsistent availability of blood gas monitoring may limit judicious oxygen titration. the aim of this project was to audit oxygen delivery practice and introduce qi measures to avoid excess oxygen delivery in a tertiary icu in lusaka, zambia. a prospective snapshot of ventilatory parameters were recorded for critically ill patients over a -week period, including positive end expiratory pressure (peep), fio , and time-course spo . systematic education was provided through group and one to one tutorials to empower nursing and medical staff to titrate oxygen safely and appropriately. repeat data collection was then performed over weeks. initially / patients ( %) were over-oxygenated, as defined by fio > . and spo consistently > %. / patients with an fio of > . had peep ≤ cm ( %). no patient had a pao recorded in the past hours. education was provided as well as implementation of unit protocols above all patient beds documenting a stepwise approach to titration peep and fio . post intervention fewer patients were over-oxygenated: / ( %) had fio > . and spo consistently > %, and / with an fio > . ( %) had a peep ≤ cm. in addition, / ( . %) had a pao recorded within hours. this qi project has shown that nurse engagement and systematic education to titrate fio and peep can be achieved in a resource poor setting and may decrease the incidence of hyperoxia in critically ill patients. availability of blood gas monitoring and knowledge of interpretation was a major barrier to oxygen titration tracheal intubation (ti) in adult burn patients might be unnecessary in to % of cases [ , ] . in pediatric burn patients, there is little data on both the rate of ti and the rate of early extubation [ ] . it has been common practice for a child with a facial burn and/or a suspected airway injury to be intubated early due to the risk of losing airway patency. however this risk should be mitigated against the potential risks of ti and mechanical ventilation in children. therefore the aim of this study was to describe the airway status of child burn victims taken in charge of in our pediatric burn intensive care unit. focused on patients arriving with ti, we investigated the rate of early extubation. in addition we compared non intubated patients with those with prolonged ti. this retrospective study described a cohort of patients hospitalized between and . data was retrospectively recorded from the patient's paper clinical chart. the mean age of our patients was . ± . years [mean±sd] with an average burn area of ± %. % had scald burns and % had facial burns. % of the children were admitted in the burn icu with ti. for % of them, tracheal tube was removed within the first hours after admission. the probability of prolonged ti increased independently with the burned skin area (bsa) (p < . ), the presence of facial burns (p = . ), and in case of flame burns (p = . ) ( figure ). among patients with more than % bsa, % were intubated more than h. among patients with less than % bsa, . % were intubated more than h. according to our retrospective data, it seems appropriate to intubate children with % and more bsa, while for patient with less than % bsa, it might be relevant to seek guidance from physician of the nearest burn center. under % bsa, ti seems rarely required. an analysis of the predictive applicability of initial blood gas parameters for the need for intubation and the presence of inhalation injury in patients with suspected inhalation injury c pirrone , m chotalia , t mangham , r mullhi , k england , t introduction: we hypothesise that initial blood gas parameters have a good predictive applicability in detecting the need for intubation and the presence of inhalation injury in patients with suspected inhalation injury. to the best of our knowledge, this has not been directly studied in the literature. patients with suspected inhalation injury admitted to the icu at queen elizabeth hospital, birmingham between april and may were included. the initial blood gas parameters analysed were pao (kpa), paco (kpa), ph, carbon monoxide level (cohb; %) and pao /fio (pf) ratio. receiver operator characteristics (roc) for these parameters were plotted against the need for intubation for more than hours and the presence of inhalation injury as detected by bronchoscopy and laryngoscopy. area under the curve (auc) for each parameter was calculated. results: patients were admitted with suspected inhalation injury to the icu. % were intubated for more than hours. of patients who were intubated, % had inhalation injury as indicated by bronchoscopy or laryngoscopy. table outlines the auc for initial blood gas parameters in detecting the need for intubation for more than hours and the presence of inhalation injury. ph was the parameter with the most prominent auc, with reverse correlation indicating fair accuracy. no clear inflection point was identified, although all patients with ph < . required intubation and had inhalation injury. paco had a fair predictive applicability in detecting the need for intubation. pf ratio, pao and cohb had poor accuracy. conclusions: initial blood gas parameters had a broadly poor predictive applicability for the need for intubation and the presence of inhalation injury in patients with suspected inhalation injury. severe acidosis (ph < . ) was the most useful blood gas parameter. clinicians should be cautious in using blood gas parameters alone to inform intubation decisions. lung cancer surgery is associated with a high rate of pulmonary complications including ards and mandates lung protective ventilation strategies [ , ] . such strategies include non-intubated video assisted thoracic surgery (nivats) with spontaneous breathing [ ] . currently neither data on respirator settings nor on gas exchange have been reported for applying the latter. this data constitutes a prerequisite for meaningful evaluating the respiratory consequences of non-intubated spontaneous breathing during lung cancer surgery. the aim of this case series was for the first time providing such data from lung cancer surgery including pneumonectomy. during a month period patients without contraindications [ ] scheduled for video assisted thoracic surgery (vats) for non-anatomical and anatomical lung resection including one pneumonectomy (px) were offered non-intubated spontaneous breathing. all patients gave informed written consent to the procedure as well as for analysis and publication of data. anaesthetic management included target controlled infusion of propofol and remifentanil, laryngeal mask airway, and pressure support ventilation. we present early data that early trials of cuff deflation within hours of tracheostomy insertion can be achieved using a standardized protocol. its impact on length of stay, duration of ventilation and patient-centered outcomes needs to be investigated in larger multi-centre trials. preventing underinflation of the endotracheal tube cuff with a portable elastomeric device. a randomized controlled study je dauvergne , al geffray , k asehnoune , b rozec , k lakhal hopital laënnec -chu de nantes, service d´anesthésie-réanimation, nantes, france; hotel-dieu -chu de nantes, service d´anesthésieréanimation, nantes, france critical care , (suppl ):p the management of the endotracheal tube cuff pressure (p cuff ) is routine practice for critical care nursing staff. underinflation could lead to ventilator-associated pneumonia [ ] whereas overinflation exposes to tracheal damage [ ] . multi-daily check and adjustment is recommended to ensure that p cuff lies between and cmh o [ ] . to automate this task some devices exist but may be inconvenient, bulky and/or ineffective. their use is not supported by guidelines. a portable elastomeric device could be appealing for p cuff automated regulation. this prospective randomized controlled study tested whether the tracoe smart cuff manager tm reduced the rate of patients undergoing ≥ episode of underinflation (p cuff < cmh o), as compared with routine manual p cuff adjustment. monocentric, randomized controlled study. patients with acute brain injury and receiving mechanical ventilation were prospectively allocated to one of the two arms: manual reading and adjustment of p cuff at least every h (routine care) or adjunction of the smart cuff manager tm (intervention). this study was approuved by an institutional review board. among randomized patients (routine care in , smart cuff manager tm in ), measurements were performed in h. with routine care, a higher rate of patients experienced at least one episode of underinflation ( . vs. . %;p< . ). episodes of underinflation episodes ( % vs. %;p< . ) and manual adjustments ( % vs. %;p< . ) were more frequent with routine care. for overinflation, there was no between-arms difference (p> . ). the adjunction of continuous p cuff control with the tracoe smart cuff manager tm reduced the incidence of p cuff underinflation as compared with manual intermittent adjustments. overinflation was not promoted by this device. direct laryngoscopy as a technique for tracheal intubation is a potentially lifesaving procedure that healthcare professionals in a variety of fields are taught. however, this skill is challenging to acquire and difficult to maintain. poorly performed intubation technique can lead to potentially serious complications [ ] . the intersurgical iview video laryngoscope is a new intubation tool which may have advantages over direct laryngoscopes, such as the macintosh, in the hands of novice personnel. a prospective randomized counterbalanced trial of medical students, who did not have previous airway management experience, was conducted. each student received brief didactic teaching,following this, participants were directly supervised performing laryngoscopy and intubation using the macintosh and iview devices in an alternating pattern. students were permitted up to three attempts to successfully intubate under four conditions, three laryngoscopy conditions using alaerdal intubation trainer and one using a laerdal simman manikin. there was no significant difference in the success rate of intubation or time to intubation between the two devices. the iview outperformed the macintosh in time to intubation in the normal airway in the final scenario, once students gained experience with both devices. no significant difference was found in the number of optimisation manoeuvres, or intubation attempts between groups. areas where the iview outperformed the macintosh included severity of dental trauma and participants' perception regarding ease of use ofthe device. the iview may prove to be a useful teaching tool for novice personnel who are acquiring the skills of tracheal intubation. patients with a primary pulmonary pathology were more likely to respond to aprv. this association has not been described before and warrants further multi-centre exploration in a larger patient group. introduction: airway suctioning is common during mechanical ventilation, using either an open endotraqueal suctioning or closed endotracheal suctioning (ces). closed circuits were developed to prevent arterial desaturation and atelectasis associated to ventilator disconnection. however, ces may cause substantial loss of lung volume. the purpose of this study was to investigate the effects of a compensation method to prevent the loss in aeration during ces. the suctioning technique was performed for seconds, negative pressures limited at mmhg. closed suction catheters with fr (halyard health, georgia, eua) were used. electrical impedance tomography (eit) monitoring and arterial blood gas were collected. a nihonkoden mechanical ventilator (nkv , california, eua) was applied, having a newly developed algorithm for suctioning which overcomes any pressure loss during suctioning (inlinesuction-app). when activated, the app delivers pcv ventilation, adding cmh o of end-expiratory pressure above peep, and delivering driving pressures of cmh o. results: pigs ( ± . kg) with injured lungs and mechanically ventilated. we tested the aspiration procedures using low peep= cmh o, or high peep=± . cmh o with v t o), whereas maintenance of compliance was observed when the app was on (from . ± . ml/cmh o to . ± . ml/cmh o. blood gas in a representative animal showed a drop in pao when app was off (from , to mmhg after min, and to mmhg after min) ( figure ). with app on the pao changed from (pre-suction), to ( min), to mmhg ( min). the new nksoftware, delivering pcv ventilation during suctioning, could prevent atelectasis and functional loss associated to the procedure. tyrosine kinase inhibitor: an effective tool against lung cancer involvement responsible for acute respiratory failure in icu y tandjaoui-lambiotte patients with advanced-stage non-small-cell lung cancer have high mortality rates in the intensive care unit (icu). in the last two decades, targeted therapies have changed the prognostic of patients with lung cancer outside the icu. the fast efficacy of targeted therapies led some intensivists to use them as rescue therapy for icu patients. we performed a national multicentric retrospective study with the participation of the grrroh (groupe de recherche en réanimation respiratoire en onco-hématologie). all patients with non-small-cell lung cancer admitted to the icu for acute respiratory failure between and were included in the study if a tyrosine kinase inhibitor was initiated during icu stay. cases were identified using hospital-pharmacies records. the primary outcome was overall survival days after icu admission. results: thirty patients (age: +/- years old) admitted to a total of icus throughout france were included. seventeen patients ( %) were nonsmoker. adenocarcinoma was the most frequent histological type (n= , %). most patients had metastatic cancer (n= , %). epithelial growth factor receptor mutation was the most common oncologic driver identified (n= , %). during the icu stay, ( %) patients required invasive mechanical ventilation, ( %) catecholamine infusion, ( %) renal replacement therapy and one ( %) extracorporeal membrane oxygenation. eighteen patients ( %) were discharged alive from icu and ( %) were still alive after days (see figure) . moreover, patients ( %) were alive one year after icu discharge. despite a small sample size this study showed that, in the context of lung cancer involvement responsible for acute respiratory failure, the use of tyrosine kinase inhibitor should not be refrained in patients with severe condition in icu. the burned patient is one of the most complex patients whith a very high mortality. those patients with inhalation injury have a worst prognosis, typically associated with respiratory complications. the aim of our study is to evaluate the mortality of burn patientes with inalation injury in a critical burn unit. a prospective, observational and descriptive study was conducted over a period of years. inhalation injury was defined with these criteria (≥ ): history of injury in an enclosed space, facial burns with singed nasal hair, carbonaceus sputum and stridor. if they were intubated it was diagnosed by bronchoscopy. demographic data, tbsa, absi, baux score, apache ii, sofa, mechanical ventilation (mv), complications, length of stay, hospital course and mortality data were collected. results: burns patients were admitted. % ( patients) had inhalation injury. mortality among patients with inhalation injury was , % ( patients). most patients were men and those who died were older and with higher severity scores (fig. ) . we found no significant differences between groups in the need for mv ( % vs. %) or in the percentage of tracheostomy performed ( . vs. . ). however, patients who died had more respiratory complications like ards, and also shock, renal failure and need of renal replancement therapies although infectious complications were similar in both groups. there was no statistically significant difference in volume used during initial resuscitation in the different groups. patients with inhalation injury who died had higher severity scores at the begining. although there were no differences in the need for mv patients who died had more respiratory complications as well as shock, renal failure and need of rrt, but no infectious complications.the volume used during inicial resuscitation, that was always related to the prognosis, was similar in both groups. further studies are needed to see if this greater initial severity corresponds to the degree of inhalation. aerogen, medical affairs, galway, ireland; aerogen, science, galway, ireland critical care , (suppl ):p patients with acute exacerbations such as asthma are prescribed aerosol therapy from presentation in the emergency department to progression through to the intensive care unit. however, the variability in dose delivery to the lung across the possible patient interventions is not well characterized. here, we assess the predicted lung dose of a bronchodilator in a simulated spontaneously breathing adult patient via both facemask and nasal cannula, and via tracheostomy during mechanical ventilation. a standard dose of . mg in . ml salbutamol was aerosolized using the aerogen solo nebulizer (aerogen, ireland). for facemask testing, the nebulizer was used in combination with the aerogen ultra with lpm supplemental oxygen flow. for nasal cannula testing, the nebulizer was used in combination with the airvo system (fisher and paykel, nz) system at both and lpm gas flow rate. tracheostomy-mediated ventilation was assessed in combination with a hme, with the nebulizer placed between the hme and the tracheostomy tube. international standard iso adult breath settings (vt ml, bpm , i:e : ) were used across all tests, and generated using a breathing simulator (asl , ingmar medical, usa) or mechanical ventilator (servo-u, maquet, sweden). the dose delivered to the lung was assessed using a capture filter at the level of the trachea, with drug mass determined using uv spectrophotometry at nm and interpolation on a standard curve. the results of testing are illustrated in figure . the bronchodilator dose delivered to the simulated patient was seen to be relatively consistent between progressive interventions, except during high flow therapy, with the more clinically relevant lpm gas flow rate having a profound effect on the dose. these results may go some way towards explaining how different patient interventions can affect aerosol dose. the the mechanical ventilation (mv) have been identified as an independent factor indicating a worse prognosis for lung cancer patients [ ] . this study was conducted in order to assess the results of noninvasive mechanical ventilation (niv) and/or invasive mechanical ventilation (imv) modalities in lung cancer patients admitted to the icu with acute respiratory failure (arf). in this study, lung cancer patients with respiratory failure who were admitted to the icu between january and december were evaluated retrospectively. results: patients were included in the study. the mortality rate was . %. patients had niv. imv was applied to patients. in the first hours, of the patients who were initially treated with niv were administered imv. the duration of hospital stay, diagnosis of pneumonia and mortality rate were found to be significantly lower in patients treated with niv alone (p≤ . , p= . , p= . ), but glaskow coma score (gcs) was significantly higher in this group (p≤ . ). the mortality rate was similar between the patients who were initially treated with imv and those who were treated with imv in the first hours. charlson comorbidity index (cci) and mv duration were significantly higher in patients who died (p= . , p= . ), but gcs was significantly lower in this group (p= . ). in the linear regression model for the likelihood of mortality, ccl≥ and unsuccessful niv increased the mortality rate by . ( . - . ) and . times ( - . ) respectively (p= . , p= . ). niv has been an effective modality for respiratory support in most lung cancer patients presenting with arf. however, failed niv seems to be a factor for increased mortality. therefore, the choice of respiratory support modality to be applied in this patient group should be decided by considering the gcs, cci and etiology of arf. the interaction between ventilator settings and the occurrence of acute kidney injury is not fully elucidated. this study aimed at investigating the effect of stepwise increase in peep level on the risk of acute kidney injury as evaluated with the renal resistivity index (rri).the primary outcome is to investigate whether increased levels of peep could lead to increase rri and whether rri could predict the occurrence of aki. methods: patients mechanically ventilated for at least hours and without aki at admission were included in the study. rri was calculated at icu admission. posterolateral approach was used for kidney ultrasound. the peak systolic velocity (v max ) and the minimal diastolic velocity (v min ) were determined by pulse wave doppler, and the rri was calculated as (v max -v min )/v max . the exam was performed modifying the peep levels: , and cm h o in random order for minutes. occurrence of aki was defined within days according to kdigo criteria. sixty-four patients were enrolled in the study and incidence of aki was / ( %). demographical and clinical characteristics are reported in table . increase in peep showed a significant increase in rri from peep to peep (p< . ) and from peep to peep (p= . ) ( figure ). the area under the roc curve of rri to predict aki was . at peep , . at peep and . at peep (all p< . ). the youden index analysis showed an rri> . as the best cut off for aki with a sensibility of % and a specificity of %. patients with rri> . were / ( %), / ( %) and / ( %) at peep ,peep and peep respectively. patients ventilated with a peep value associated with rri> . had higher incidence of aki ( / vs / , p< . ). the application of peep can increase intrarenal vascular resistance,which is associated occurrence of aki; peep level should therefore be balanced taking into account the rri. the rri seems able to predict occurrence of aki in mechanically ventilated patients. alveolar and respiratory mechanics modifications produced by different concentrations of oxygen in healthy rats subjected to mechanical ventilation with protective ventilatory strategy d dominguez garcia , r hernandez bisshopp , jl martin barrasa , d viera camacho , a rodriguez gil , j arias marzan , s garcia hernandez high oxygen can damage tissues [ ] . in this study, we analyze the histological and pulmonary mechanics modifications that can occur when identifying different inspiratory oxygen fractions (fio ) in lungs of healthy rats during protective mechanical ventilation. we use sprague-dawley rat. groups were designed, each with animals, the tidal volume ( ml/kg), peep ( cmh o) and respiratory rate ( rpm) were kept constant, changing the fio between the groups. four groups were established: fio . , . , . and . after hours, the lungs were removed for histological study and obtaining the wet/dry index. the histological modifications studied were: alveolar septa (as), alveolar hemorrhages (ah), intraalvelolar fibrin (if) and inflammatory infiltrates (ii). each parameter was rated from to [ ] . peak pressure (pp) and pulmonary compliance were monitored every minutes. different statistical tests will be used to analyze the data. results: references to the damage produced in the as, ah, if, ii and the global histological pattern were identified in the groups with the highest fio and there was more damage (p < . ) ( figure ). the wet/dry index rose significantly as the oxygen concentration increased (p = . ). in the groups to which a fio of . and was administered, the pp selected specific values with respect to the baseline intake from the first minutes, an aspect that was not appreciated in the other groups (p < . ). regarding pulmonary compliance, it will be seen that, in the fio . and groups, it decreased from the first minutes, finding differences with respect to the other groups (p < . ). conclusions: mechanical ventilation applied for hours in healthy animals produces disorders that are more pronounced as oxygen concentration increase. fio greater than or equal to . should be avoided without clinical justification. introduction: patients requiring prolonged acute mechanical ventilation (pamv, defined as + days on mv) are sicker and incur disproportionate morbidity and costs relative to patients on short-term mv (stmv, < days of mv). we quantified specific clinical outcomes among patients requiring pamv vs. stmv in a contemporary database. we conducted a multicenter retrospective cohort study within~ hospitals in the premier database, - . using icd- -cm and icd- codes we identified pamv and stmv patients, and compared their baseline characteristics and hospital events. because of the large sample size, we omitted hypothesis testing. a total of , patients met the enrollment criteria, of whom , ( . %) received pamv. at baseline, patients on pamv were similar to stmv with regard to age (years: . ± . pamv vs. . ± . stmv), gender (males: . % pamv vs. . % stmv), and race (white: . % pamv vs. . % stmv). pamv group had a higher comorbidity burden than stmv (mean charlson score . + . vs. . + . ). the prevalence of each of the indicators of acute illness severityvasopressors ( . % vs. . %), dialysis ( . % vs. . %), severe sepsis ( . % vs. . %), and septic shock ( . % vs. . %)was higher in pamv than stmv, as were hospital mortality and combined mortality or discharge to hospice (figure ), extubation failure ( . % vs. . %), tracheostomy ( . % vs. . %), development of c. difficile ( . % vs. . %), and incidence density of ventilator-associated pneumonia ( . / , patient-days vs. . / , patient-days). conclusions: over / of all hospitalized patients on mv require it for days or longer. pamv patients exhibit a higher burden of both chronic and acute illness than those on stmv. commensurately, all clinical outcomes examined are substantially worse in association with pamv than stmv. identifying the readiness of patients recovering from critical illness for liberation from invasive mechanical ventilation (imv) is not always straightforward [ ] . the scottish intensive care society (sics) trainee audit conducted a scotland-wide study to understand current practices relating to liberation from imv. data were prospectively collected on patient demographics, indication for intubation, spontaneous breathing trial (sbt) practices, physiological markers, icu outcome and icu los. all patients > years ventilated with imv for > hrs from the st nov. - th nov. were eligible for inclusion. exclusion criteria included extubation for end-of-life, death whilst intubated and presence of tracheostomy. logistic regression was performed to detect factors associated with extubation failure (ef). results were analysed via excel and stata v. . . patient benefit and privacy panel approval was granted. total population of patients were included: ( %) male and median apache score (iqr - ). ef at first attempt occurred on occasions ( . %), median icu los of days (iqr - ), mortality rate . %. the cohort successfully extubated first time had a median icu length of stay of days (iqr - ) and mortality rate of . %. methods of sbt and extubation outcomes detailed in table . no sbt prior to extubation had higher odds of ef (or . , ci . - . , p= . ); patient ventilation for < days had a three times higher odds of ef (or . , ci . - . , p= . ). these were independently associated with ef on multivariate analysis conclusions: we found a reintubation rate of . % in scottish icus. type of sbt most commonly used is divergent from the methods advocated in the literature. the lack of sbt and early extubation attempt was associated with failure, which in turn was associated with longer icu los and higher mortality. in patients undergoing prolonged invasive ventilation we hypothesise that abnormal right ventricular (rv) and left ventricular (lv) function are associated with increased -day mortality. whether changes in lv or rv function could aid in the prognostication of these patients has not been directly studied. patients admitted to the queen elizabeth hospital birmingham icu between april and july who were intubated and ventilated for more than days and had a formal transthoracic echocardiogram (tte) whilst in icu were included. abnormal rv function was defined by the presence of depressed function, dilated size or moderate to severe risk of pulmonary hypertension. abnormal lv function was defined by the presence of lv depression (lv ejection fraction £ % or grade ii or more diastolic dysfunction) or a hyperdynamic lv (formally mentioned in tte report). patients who had a neurological cause for prolonged ventilation were excluded. the primary outcome was -day mortality. categorical data is presented as % and analysed using a chi-squared test. continuous data is presented as median (iqr). results: patients required prolonged ventilation, of which ( %) had a tte. patients were aged ( - ), were % male and had a % -day mortality. the median ventilator days were ( - ) and % required a tracheostomy. abnormal rv function was present in % (n= ) and was associated with an increased -day mortality compared to normal rv function ( % vs. %, rr . [ . - . ], p< . ). lv function was abnormal in % (n= ) and was associated with an increased -day mortality compared to normal lv function ( % vs %, rr . [ . - . ], p < . ). abnormal rv function had a trend towards an increased mortality compared to abnormal lv function ( % vs %, rr . [ . - . ], p = . ). in this study, abnormal rv and lv function were present in a quarter of patients undergoing prolonged ventilation and were associated with an increased mortality. introduction: tidal volume delivered by mechanical ventilation (mv) in sedated patients is distributed preferentially to ventral alveoli, causing overdistention and associated collapse in dorsal alveoli, driving volutrauma, atelectrauma and ventilator-induced lung injury [ ] . temporary transvenous diaphragm neurostimulation (ttdn) stimulates diaphragm contraction [ ] . when used in synchrony with mv, ttdn encourages increased dorsal ventilation due to the change in pressure gradients with diaphragm contraction, mimicking a more normal physiological pattern. this may improve gas exchange and reduce injury. a pilot study was conducted using kg pigs undergoing mv in a mock icu. deeply sedated subjects were provided lung-protective volume-control ventilation at ml/kg. ttdn diaphragm contractions were delivered in synchrony with inspiration on every second breath, reducing the ventilator pressure-time-product by - % during mv+ttdn breaths. tidal volume distribution was recorded in each condition using electrical impedance tomography, and compared to never-ventilated, spontaneously breathing subjects (nv). results: dorsal ventilation changed from % during mv breaths to % during mv+ttdn breaths, compared to % in the nv group (p= . ). ventral ventilation changed from % during mv breaths to % during mv+ttdn breaths, compared to % in the nv group (p= . , figure ). conclusions: ttdn diaphragm contraction used as an adjunct to mv yields a more physiological pattern of volume distribution. this translates into less overdistension in the ventral areas and less atelectrauma in the dorsal areas and reduces ventilator-induced lung injury. this technology introduction: by measuring the pes and its derivatives, we can measure the relationship that exist between the diaphragmatic excursion and the oscillation of the esophageal pressure curve: pswing (ps) so we infer that, just as with the pes, the variations of it might be related to a weaning failure [ , ] . however, no nominal value exists in the bibliography to predict the test result. patients who meet with the inclusion criteria start the weaning process through a test of minutes of spontaneous ventilation, t-tube (tt). and also the respiratory rate (rr) and the tidal volume (tv). from this analysis, an average ps (aps) is determined for each moment of the test (aps , initial and aps , final.).a quotient was obtained in relation to these variables using the value previously obtained (quotient dtv/dps x . a total of patients were included (n= ).regarding the evolution during tt, (n= ) ( %) were successful, while (n= ) ( . %) failed when analyzing a rate that relates the variables tv and ps, a quotient was obtained in relation to these variables using the value previously obtained (quotient dtv/dps) for patients who were successful and who failed, (dtv/dps)/ successful patients presented a value of . while those of the failure group presented a value of . , (or , - p= . ) ( table ) . when presenting the relationship between tv and ps through the quotient (dvt/dps)/ , it is observed a tendency to have a higher quotient among patients who failed versus those who did not fail. the process of weaning from mechanical ventilation imposes an additional workload on the cardiovascular system, which may result in impaired myocardial function, increase in left ventricular filling pressure and respiratory distress. among surgical patients, those undergoing heart surgery are particularly susceptible to cardiac dysfunction induced by weaning because of inadequate cardiovascular reserve. the aim of our study was to depict the pathophysiological changes assessed by echocardiography during the steps of weaning and to identify possible predictors of weaning failure (wf). we enrolled consecutive patients undergoing isolated coronary artery bypass grafting in our institution. data were obtained by intraoperative transesophageal echocardiography before sternotomy (t ) and by transthoracic echocardiography at the beginning of weaning (t ) and at the time of extubation (t ). wf was defined as deferral of planned extubation or respiratory failure needing reintubation or non-invasive mechanical ventilation within hours. results: wf occurred in patients ( . %) and involved manifestations of respiratory distress in ( . %). we found a significant association between left ventricle outflow tract-velocity time integral (lvot-vti) and ventricular-arterial coupling measured at t and wf, with lvot-vti emerging as the best predictor of wf with an area under roc curve of . ( figure ); an optimal cutoff value of cm provided % sensitivity and % specificity. significant increase in e/e' measured at t ( . vs . , p . ) suggested a cardiac etiology of respiratory distress in patients who failed the weaning trial. our study showed that serial assessment of hemodynamic parameters by means of echocardiography is feasible in cardiac surgical patients and can provide insight into pathophysiological changes during weaning. although these preliminary data need to be confirmed in a larger population sample, lvot-vti emerged as a promising predictor of subsequent wf. compliance with guidelines for respiratory therapy in preclinical emergency medicine g jansen, n kappelhoff, s rehberg protestand hospital of the bethel foundation, anaesthesiology, intensive care and emergency medicine, bielefeld, germany critical care , (suppl ):p introduction: current guidelines on pre-hospital emergency ventilation are based on the guidelines for lung protective ventilation in the intensive care unit. the present survey was designed to determine the accordance of actual pre-hospital emergency ventilation by german emergency physicians (gep) with these recommendations. recommendations include a respiratory rate (rr) between - /min, a tidal volume (vt) between - ml/kg, a maximum pressure (pmax) < mbar and a positive end-expiratory pressure (peep) of mbar. an anonymous web-based questionnaire encompassing questions was sent to gep from september to december of . gep were asked to specify their level of education, their preferred ventilation settings and the usually chosen parameters employed to guide mechanical ventilation. statistical analysis was performed using the ch²-test with a significance level ≤ . . % of the questionnaires were completed ( / ). % of the participants were trainees (tr), % consultants (co). as target parameters for guidance of ventilation, % of the tr and % of the co use capnometry. the vt controlled % of the tr and % of the co on the basis of body weight. % of the tr and % of the co reported to control oxygenation using spo . table shows our analysis of the given answers. there were no statistically significant differences between the groups. deviations from the guidelines of pre-hospital emergency ventilation settings are common and mainly concern the use of a guidelinecompliant peep. in addition, recommended target parameters for guidance of ventilation were not applied in a significant proportion of gep. prospective observational study including ltx recipients admitted to our icu from february to january , who underwent a spontaneous breathing trial (sbt) using a t-piece for minutes. clinical variables and arterial blood gas samples were recorded before starting sbt and after minutes on the t-piece. diaphragmatic excursion (de) and thickening fraction (dtf) were also assessed using ultrasound(us) after minutes on the tpiece. us-dd was defined as de< mm or dtf< . of at least one hemidiaphragm. patients who successfully completed a sbt, defined according to clinical criteria,were extubated. extubation failure was defined as the need for reintubation within h. results are expressed as medians (iqr) or frequencies (%). ltx recipients were admitted to the icu, of whom underwent an sbt. were male, and the median age was y. main indications for ltx were interstitial lung disease ( . %), copd and cystic fibrosis. were bilateral ltx, and and were left and right unilateral ltx respectively. patients were extubated after sbt and required reintubation within h. presented us-dd, though there were no differences between patients who succeeded and those needing reintubation. in contrast, patients who succeeded showed higher pao /fio after minutes on the t-piece (table ) . similarly, higher reductions in deltapao /fio after minutes on the t-piece were observed in patients who failed. oxygenation after sbt performed using a t-piece may predict extubation failure in ltx recipients with successful sbt. us-dd was not associated with the need of reintubation. descriptive study about the relationship between self-extubation episodes and patient-ventilator interaction s nogales , introduction: to evaluate the relationship between self-extubation and patientventilator interaction, among other physiological variables, in order to predict and to prevent these events. self-extubation (se) are quality indicators in patients under invasive mechanical ventilations (imv) and are related with mortality [ ] . planned secondary analysis of a prospective data base of clinical and physiologic signals of patients receiving imv. we included se episodes ( - ) with continuous record of ventilator and monitor signals (bclink bettercare®). we analysed demographic data, physiological parameters (peripheral oxygen saturation spo , heart rate hr, respiratory rate rr and media arterial pressure map) and patientventilator interaction (asynchrony index ai, ineffective efforts during expiration iee and double cycling dc). we studied a period of hours prior to the se episode. we used the wilcoxon non-parametric test and for a proper analysis a linear mixed effects model. we included episodes of se, mean age ± years, %men, apache ii at admission ± , , ± , days under imv until the episode, reintubation rate . %, icu stay , ± , days, icu mortality %. at the time of the se, % were under sedation, % with physical restraint. the % were in weaning. we observed a trend to increase in spo , rr, hr, map and asynchronies in the -hour period prior to se episode. we compared these variables from this period with a -hour period before and we observed a statistically the data presented in this study show that our results are in accordance with the literature with favorable mortality and early postoperative complication rates and support that this procedure is an excellent alternative for surgery in the elderly patients. it is reported that patients with pulmonary hypertension (ph; systolic pulmonary arterial pressure (spap)≥ mmhg)) have frequent cardiac complications after transcatheter aortic valve implantation (tavi). ph often gets worse in some patients despite the normal cardiac function after tavi. no studies have ever examined prognosis after tavi in patients with or without worsening of ph. therefore, we retrospectively examined the frequency of mid-to long-term heart failure and cardiac death in patients with and without deterioration of ph after tavi. among patients who underwent tavi at our hospital between february and march , we analysed patients with ph (spap≥ mmhg) before surgery. spap was measured in transthoracic echocardiography before and within week after tavi. patients were divided into two groups according to whether spap worsened/ did not change or improved after tavi. we examined the frequency of admission due to heart failure or cardiac death (death caused by heart failure, angina, or myocardial infarction) during the period of years after tavi. ph worsened or did not change after tavi in patients, while it improved in patients. the left ventricular ejection fraction measured within week after tavi showed no difference between the two groups ( . ± . % vs . ± . %, p= . ). the worsened/ no change group was higher in frequency of admission due to heart failure (logrank; p< . ) and cardiac death (logrank; p< . ). despite successful treatment for as by tavi, the frequency of heart failure and cardiac death was higher in patients who did not show improvement of ph after tavi, even in the absence of cardiac function decrease. vigorous intervention for ph worsening after tavi may be helpful to improve prognosis. the there are several different anti platelet drugs that can be used to treat acute cardiac events. currently there are no effective markers that can assess how these drugs modify coagulation profile and quality. a new functional biomarker that measures fractal dimension (df ) and clot formation time (tgp) has been developed [ ] . df quantifies clot microstructure whereas tgp is a real-time measure of clotting time. we aimed to validate df and tgp in st elevation myocardial infarction (stemi) and assess the effect of two p y inhibitors which have different pharmacological mechanisms: clopidogrel and ticagrelor. we prospectively recruited stemi patients in the emergency setting. venous blood samples were collected hours after admission, following treatment with either ticagrelor or clopidogrel, in accordance with the local guidelines at the time. the blood samples were tested using the df and tgp biomarker, platelet aggregometry, clot contraction and standard markers of coagulation. results: patients received clopidogrel and received ticagrelor. the df for clopidogrel was higher than ticagrelor ( . ± . vs . ± . , p= . which corresponds to a decrease in clot mass of % figure ) and the tgp was reduced ( ± sec vs ± sec, p= . a % reduction in time). the results of the study suggest that clopidogrel is less powerful in its effects on clotting characteristics compared to ticagrelor. blood from patients receiving clopidogrel formed quicker and denser clots. this would suggest the risk of secondary events or stent occlusion is lower in those patients on ticagrelor, highlighting that df and tgp may be important in identifying patients at risk of future thrombotic events, the study is ongoing and will investigate the long term outcome in these patients. introduction: new onset atrial fibrillation (noaf) during critical illness frequently resolves prior to discharge. however long-term risks of noaf (i.e. heart failure, ischemic stroke and death)remains high [ ] . previous studies noted that nearly half of noaf cases did not have diagnosis recorded [ ] . addressing this may reduce post critical illness mortality by increasing af surveillance post intensive care (icu) discharge. retrospective data was collected from an electronic health record for icu admissions over a month period from a biomarker is defined as a measurable indicator of some biological state or condition. combined with a good clinical evaluation, they can enable an early and safe diagnostic, thus a faster management for the patient. cardiac biomarker testing is not indicated in routine in the emergency department (ed) because of low utility and high possibility of false-positive results. however, current rates of testing are unknown. the aim of our study was to evaluate the importance of measuring cardiac biomarkers especially troponins, d-dimer, and btype natriuretic peptide in our daily practice, and to identify the latest recommendations for a better use of these biomarkers in the diagnostic and therapeutic approaches. we conducted a prospective observational study, over a months periods performed in the ed of the university hospital center ibn rochd, casablanca, morocco, including all patients admitted during our study period and having a blood test for at least one biological marker. the dataset was analyzed by spss statistics . . a total of patients was enrolled. troponins were tested in . % patients (high sensitive in . % and troponin i tni in . %), ddimer in . %, bnp % and nt pro bnp in . % of cases. the diagnostic impact was significant in . % of cases for troponins, . % of cases for d-dimer and . % for bnp. the therapeutic impact was considered important in . % cases for troponins, . % for ddimer and . % for bnp. cardiac biomarkers have an important role in the ed, not only do they confirm the diagnosis (including the role of troponins in acs) but also eliminate others (with a strong negative predictive value of d-dimer for thromboembolic disease) and prove the cardiopulmonary origin of acute dyspnea (the significant place of bnp in confirming the diagnosis of acute heart failure). a multicenter study on the comparison of inter-rater reliability of a new and the original heart score among emergency physicians from three italian emergency departments the heart (based on history,ecg,age,risk factors,troponin) score is a valid tool to stratify the acs in chest pain. but some reports suggest that its reliability could be low for heterogeneity in the assignment due to the subjective interpretation of the history. we used the chest pain score for the "history". in this study we compare the reliability of the new heartcps and original heart. this is a multicenter retrospective study conducted in italian ed between july and october using clinical scenarios. ten physicians were included after a course on heart and heartcps score. we used scenarios which included clinical and demographic data. each participant independently assigned scores to the scenarios using the heart and heartcps. we tested the interrater agreement using the kappa-statistic (k), the confidence intervals are bias corrected ; we used stata/se . statistical software . a p-value of < . defines statistical significance. the overall inter-rater reliability was good for heart and heartcps: kappa = . (ci %; . - . )and , (ci %; . - . ); with good agreement among all the class of risk for heartcps but moderate in the medium class for heart . we found significant differences of inter-rater reliability among the senior and junior physicians who used the heartcps:k= . (ci %; . - . )and . (ci %; . - . ). heartcps score increased its history inter-rater reliability specially among the junior physicians from k= . (ci %; . - . ) to k= . (ci %; . - . ).the junior physicians seem to be more reliable than senior with the heartcps:k= . ( . - . ) vs k= . (ci %; . - . ). the heartcps showed inter-rater reliability better than original heart among the medium class of risk and the junior group. it could be proposed to young doctors to stratify the acs risk of chest pain. limit: we used scenarios rather than real patients. a hybrid approach as treatment for coronary artery disease: endo-cabg or pci first, does it matter? introduction: the aim of this study is to discuss the short-term results of a hybrid approach combining minimally invasive endoscopic cabg (endo-cabg) with a percutaneous coronary intervention (pci). to bypass the disadvantages and potential complications of conventional cabg via median sternotomy, we developed the endocabg technique to treat patients with single-and multi-vessel coronary artery disease (cad). this procedure is performed with three -mm thoracic ports and a mini-thoracotomy utility port ( cm) through the intercostal space. this technique can be combined with pci: the hybrid approach. the sequence of the procedures (endocabg followed by pci or vice versa) may result in different outcomes. from / to / data from consecutive patients scheduled for a hybrid technique at jessa, belgium, were prospectively entered into a customized database. this database was retrospectively reviewed. subgroup analysis was performed to compare outcomes of patients who first received endocabg with patients who first received pci. a p-value < . is considered significant, a p-value < . is considered as a trend toward significance. four patients underwent revision surgery and patients died within the first days. in patients the left anterior descendens artery (lad) was grafted with the left internal mammary artery (lima), the right coronary artery (rca) was the most stented vessel using pci. patients first treated with pci received more units of fresh frozen plasma after endocabg compared to those who were first treated with endocabg (p= . ). there was also a trend toward significant more transfusion of packed cells in this small subgroup (p= . ). the hybrid approach is a feasible technique as a treatment option for patients with multi-vessel cad. if cabg follows the pci, patients are more likely to receive transfusion. a possible explanation could be the need for dual antiplatelet therapy prior to surgery in this group, but this needs further investigation. prognostic difference between troponin elevation meeting the mi criteria and troponin elevation due to myocardial injury in septic troponin t (ctnt) elevation in critically ill patients is common and is associated with poor outcome. using common assays, - % of patients in the icu will have elevated troponin level. our aim was to determine whether there is any prognostic difference between troponin elevation meeting the mi criteria (rise and fall more than % together with echo and ecg new abnormalities) and troponin elevation due to myocardial injury in septic patients. we enrolled patients with sepsis and mean sofa score , respectively in which ctnt level was measured more than once and analyzed there ecg and echo findings. patients were classified into three groups:definite mi (rise and fall ctnt ≥ % and contemporaneous changes on ecg and/or echo),possible mi (rise and fall ctnt ≥ % and no other findings),myocardial injury (ctnt rise less than %) results: data from patients were analyzed ( % female; mean age . (sd . )). a total of patients had at least one elevated ctnt more than . mkg/l. in ( %) of patients ctnt level rised more than % from the first elevated measurement. ( %) of patients met mi criteria considering new ecg and echo findings. the overall mortality rate in all patients was . %.the mortality rate didn't differ significantly in three groups: in the definite mi group . %, in the suspected mi group %, in the non mi ctnt elevation group , %, p= , . coronary angiography was performed in ( %) of patients from the definite mi group,pci was performed in ( %) of patients. the mortality rate in the invasive group was not significantly lower comparing to the nonivasive group % vs , %, p= , . bleeding complications were significantly more frequent in the definite mi group % vs % and % respectively conclusions: ctnt level elevation is associated with poor outcome regardless coronary or non coronary injury. myocardial revascularization may be beneficial in patients with sepsis and definite mi, but it is also associated with increased bleeding risk. diagnostic interest of "marburg heart score" in patient consulting the emergencies department for acute chest pain chest pain is a common reason for emergency department visits, although this primarily refers to acute coronary syndrome (acs), this symptom may be frequently related to other non-ischemic etiologies. the aim was to validate the marburg heart score as a tool to exclude coronary artery disease in emergency department patients with nontraumatic acute chest pain. methods: a prospective, observational, descriptive and analytic cohort study conducted in the emergency department, from february st to march st, , collecting patients consulting for nontraumatic acute chest pain, the "marburg heart" score was calculated for all these patients. telephone contact was made after weeks to look for an ischemic cardiovascular event. we included patients. the mean age was +/- years, the sex ratio was . . the majority of the patients ( . %) consulted directly to the emergency department, . % were referred by a primary care physician. the median time to consultation after the onset of chest pain was hours. high blood pressure was the most common risk factor ( . %), followed by smoking ( %), diabetes ( . %) and dyslipidemia ( . %). thirty-five patients ( . %) had already coronary heart disease, ecg was pathological in . % of patients, patients had an acs with st segment elevation. at six weeks, . % of the patients had an acute coronary event. according to the patients' answers on the questions of the marburg heart score. the area under the roc curve of this score was . with a negative predictive value of . %; the "marburg heart score" is a simple, valid and reproducible clinical score with a discriminatory power to rule out the diagnosis of coronary artery disease from the first contact with the patient presenting for chest pain in emergencies. the abdominal aortic aneurysm (aaa) surgery is a complex procedure in elderly patients with high cardiovascular risk. anesthesiological techniques should play special attention to the volume status during cross-clamping as well as to the blood loss. goal directed fluid therapies (gdt) in aaa surgery in elderly patients decrease the perioperative morbidity and mortality [ ] . aim of this study is to investigate administration of fluid-based on either a gdt approach or a control method (fluid administered based on static preload parameters and traditional hemodynamic) in all phases of aaa surgery and especially in the phase of clamping and de-clamping. a total of patients asa iii, randomly scheduled for elective, open aaa surgery were included in this clinical trial. they were randomly assigned to two groups i -gdt with targeting stroke volume variation (svv) and ii -control group where fluids were administered at the discretion of the attending anaesthesiologist. in both these groups hemodynamic parameters, central venous pressure (cvp), temperature, blood loss and diuresis were registered during the operation and hours postoperatively. each group was assessed for postoperative complications. gdt group received less fluids and had a higher cardiac index (ci) ( . ± . vs. . ± . l/minute per m , p < . ) and stroke volume index ( . ± . vs. . ± . ml/m , p < . ) than the control group. there were significantly fewer complications in the intervention than control group ( vs. , p = . ). gdt fluid administration enables less use of fluids, improved hemodynamic and fewer postoperative complications in elderly patients undergoing aaa surgery. ultrasonography is a valid diagnostic tool, used to measure changes of muscle mass. the aim of this study was to investigate the clinical value of ultrasound-assessed muscle mass, in patients undergoing cardiothoracic surgery that present muscle weakness postoperatively. for this study, consecutive patients were enrolled, following their admission in the cardiac surgery intensive care unit (icu) within hours of cardiac surgery. ultrasound scans, for the assessment of quadriceps muscle thickness, were performed every hours for days. muscle strength was also evaluated in parallel, using the medical research council (mrc) scale. of the patients enrolled, ultrasound scans and muscle strength assessment were performed in patients. the muscle thickness of rectus femoris (rf), was slightly decreased by . % ([ %ci: - . ; . ], n= ; p= . ) and the combined muscle thickness of the vastus intermedius (vi) and rf decreased by . % ([ % ci: - . ; . ], n= ; p= . ). patients whose combined vi and rf muscle thickness was below the recorded median values ( . cm) on day (n= ), stayed longer in the icu ( ± vs ± hours, p = . ). patients with mrc score ≤ on day (n= ), required prolonged mechanical ventilation support compared to patients with mrc score ≥ (n= ), ( ± vs ± hours, p = . ). the use of muscle ultrasound seems to be a valuable tool in assessing skeletal muscle mass in critically ill patients after cardiothoracic surgery. moreover, the results of this pilot study showed that muscle wasting of patients after cardiothoracic surgery is of clinical importance, affecting their stay in icu. prediction of cardiac risk after major abdominal surgery s musaeva, i tarovatov, a vorona, i zabolotskikh, n doinov kuban state medical university, anesthesiology and intensive care, krasnodar, russia critical care , (suppl ):p the aim is to assess the incidence of cardiovascular incidents in major abdominal surgery [ ] using the revised lee index. a study was conducted of elderly patients who underwent major abdominal surgery in the krasnodar regional clinical hospital no. under combined anesthesia. in the preoperative period, the risk of cardiovascular incidents was assessed using the revised lee index and the functional status was assessed by met. depending on the lee index, groups were identified: group (n = ) -low risk (index value - ), group (n = ) -intermediate risk (index value - ); group (n = ) -high risk (index value> ). we estimated the incidence of critical incidents in groups: hypo-, hypertension, arrhythmias, and bradycardia. in the general population, cardiac risk was . ± . points; functional status - . ± met. the greatest number of critical incidents was recorded in patients with high risk ( . %), the smallest -in patients with low risk ( . %), in patients with intermediate risk - . % (n < , between groups according to chi-square criterion). in the structure of critical incidents, hypotension was most often encounteredin ( %) patients, while some patients revealed several incidents from the circulatory system (n = ). overall, the lee scale showed good prognostic ability (auroc = . ) in predicting hemodynamic incidents. the revised lee index is a useful tool to help assess the risk of cardiovascular incidents and determine patient management tactics in the perioperative period. postoperative cognitive dysfunction (pocd) remains an unresolved problem due to lack of consensus on its etiology and pathogenesis. some believe that pocd is the result of the direct toxic effect of general anesthetics on the nervous system. others claim that surgical trauma activates proinflammatory factors that induce neuroinflammation. wistar rats were allocated into groups: -minor surgery (n= ), major surgery group (n= ). after days of handling and habituation rats undergone surgery under isoflurane general anesthesia ( vol.%). group rats underwent laparotomy with gentle gut massage followed by wound closure. rats in group undergone left side nephrectomy. starting from the th postoperative day spatial memory in rats was studied in morris water maze which is a cylinder metal pool with a diameter of . and a height of . m filled with water (temp. ± o c) up to half. it has a platform with a diameter of cm and a height of cm below the water level. testing was preceded by a training stage, which included sessions daily for days. thus, rats developed spatial memory to the location of the platform. on the th day of the study test stage was conducted to assess spatial memory: rats were launched from points into maze without platform and data were recorded for seconds at each session. time spent on the target quadrant (ttq) and the number of target area crossings (tac) were registered. a second test was conducted days after the first test to evaluate long-term spatial memory. the duration of surgery and anesthesia did not differ significantly between groups. there was a significant difference between groups in average ttq and tac in test (table ). in test minor surgery group showed better results but they were less significant. major surgery is associated with a more pronounced deterioration of spatial memory in rats in early postoperative period compared to minor surgery. cardiac inflammatory markers in icu patients with myocardiac ischemia after non cardiac surgery (a pilot study) p manthou , g lioliousis , p vasileiou , g fildissis national kapodistrian university of athens, athens, greece; national kapodistrian university of athens, general thoracic hospital´´sotiria´´, athens, greece; national kapodistrian university of athens, university of athens, athens, greece critical care , (suppl ):p patients with known coronary artery disease have higher perioperative risk for myocardial ischemia [ , ] . mortality is frequent following cardiac ischemia in the intensive care unit (icu) after non-cardiac surgery. the first group includes patients admitted to the intensive care unit for post-operative follow-up without myocardiac ischemia in the first hours. the second group includes patients with myocardiac ischemia postoperatively and needs intensive care monitoring. cardiac risk assessment was made with the lee index,hemorrhagic risk assessment with the has-bled bleeding score and thrombotic risk assessment with cha ds -vasc score. postoperatively, pathological test values such as bnp, troponin, crp, calcitonin were estimated. the sequential organ failure assessment (sofa) systeme was used to assess sepsis. the nursing activity score (nas) scale was used to measure the workload of various nursing activities in the icu. according to the pilot study, the sample consists of patients. . % had myocardial ischemia. the lee index was significantly higher in patients with myocardial ischemia. the duration of hospitalization, the high dose of vasoconstrictive drugs, the length of stay in the icu, the duration of mechanical stay and the nursing workload were higher in patients with myocardial ischemia. ck-mb and troponin levels differed significantly between the two groups. creatinine, bilirubin and bnp during the hours were significantly higher. patients with myocardial ischemia had significantly higher mortality. cardiac risk assessment, has-bled score and cha ds -vasc score in combination with cardiac enzymes such as troponin could predict myocardiac ischemia in severely ill icu patients. introduction: according to the literature an airway complication followed thyroid gland surgery are: difficult trachea intubation, tracheomalacia, postextubation stridor and bleeding [ , ] . most common cause of death was problem with respiration and airway obstruction [ ] . subsequent hypoxia could require emergency airway and even tracheostomy [ ] . aim of our study was to determine the most common of airway complications and their association with type of surgery in our region. the retrospective cohort study included pts., ( women, men) was performed in odessa regional hospital, oncology centre odessa. there were three types of patients: with euthyroid goiter - ( %), polynodos goiter - ( %) and thyroid cancer - ( %) ( table ) . airway complications were diagnosed after trachea extubation based on indirect laryngoscope, presence of stridor, desaturation. the pearson's criteria was calculated. the ratio of airway complications after thyroid surgery was . % ( pts). the main reasons of airway complications in thyroid surgery included: laryngeal edema - pts ( . %); recurrent laryngeal nerve injury - pts ( . %) and postoperative bleeding pts ( . %). thyroid gland cancer and polynodosal goiter associated with laryngeal edema and recurrent laryngeal nerve injury (pearsen criteria were . -moderate and . consequentially). it's may require more attention from the anesthetists after extubation and readiness for an urgent airway. serum iron level and development of multiple organ dysfunction syndrome in patients in the perioperative period s tachyla mogilev regional hospital, department of anesthesiology and intensive care, mogilev, belarus critical care , (suppl ):p recently there has been attention of researchers to the problem of perioperative anemia. it was found that it increases the risk of death and postoperative complications. threatening complication is multiple organ dysfunction syndrome (mods). the objective was to determine the level of serum iron in the perioperative period in patients with endoprosthetics of large joints, and with the presence of mods in abdominal surgery. a prospective cohort study was conducted in patients, including men and women, age . ± . years. two groups were identified: st (control) -patients after endoprosthetics of large joints (n = ), nd (main) -patients in abdominal surgery with the presence of mods (n = ). the presence of mods was established based on the criteria for the sccm / accp conference. serum iron was monitored using an au analyzer (usa). the study identified several stages: st -before surgery, nd - st day after surgery, rd - rd day, th - th day, th - th day. when studying the indicators of serum iron, its significant decrease (p < . ) in the postoperative period was established. in the st group: st stage - . ( - . ) mmol / l, nd stage - . ( . - . ) mmol / l, rd stage - . ( - . ) μmol / l, stage - . ( . - . ) μmol / l, stage - . ( . - ) μmol / l. in the nd group: st stage - . ( - ) mmol / l, nd stage - . ( . - . ) mmol / l, rd stage - , ( . - . ) μmol / l, stage - . ( . - . ) μmol / l, stage - . ( . - ) μmol / l. moreover, in both groups, iron increased at the th stage against the nd stage (p < . ). when comparing the level of iron between the groups, significant differences were found (p < . ) at the nd, rd and th stages. in patients in the postoperative period, a decrease in serum iron is observed, the level of which rises by the th day, but does not reach the initial values. this decrease is more pronounced in patients with the presence of mods after abdominal surgery. kidney and pancreatic graft thrombosis happened in . % and . %, respectively, and bleeding in . %. forty-one ( . %) developed at least one infection during hospital stay. infection during icu was found in . % and main pathogens were gram negative bacilli sensible to beta-lactam. after icu, the incidence of multi-drug resistant pathogen was . %, predominantly gram negative bacilli. fungal infection was lower %. all-cause hospital mortality rate was . %. infectious complications are the main cause of morbidity and mortality following spk transplantation. the administration of broadspectrum prophylactic antibiotics are leading to the appearance of multi-drug resistant pathogens. knowing local microbiological flora may be helpful, allowing more adequate antibiotic prophylaxis. introduction: cardiopulmonary bypass (cpb) is associated with thrombotic complications. occurrence of thrombosis after cpb is % which takes the third place between cpb-associated complications. our study determined preoperative predictors of thrombosis in children with congenital heart defects. patients with congenital heart diseases in age up to months days (median age - , months, youngest age - days after birth, oldest - months days), underwent cardiac surgery with cpb, were enrolled in this study. all patients were divided into two groups: st -without thrombosis, nd -with thrombosis. protein c, ddimer, von willebrand factor and plasminogen plasma levels were assessed directly before surgery. thrombotic cases were proven by performing doppler ultrasound or mri. thrombotic complications were diagnosed in children ( %). between all thrombotic complications ischemic strokes were diagnosed in % ( cases), arterial thrombosis in % ( cases), intracardiac thrombus in % ( cases) and mechanical mitral prosthetic valve thrombosis %( ). receiver operating characteristic (roc) curves are created for the listed indicators. area under the curve (auc) for protein c , (sensitivity(sn)- %, specificity(sp) - %), d-dimer is , (sn - %, sp %), for plasminogen activity - , (sn %, sp %) and for von willebrand factor level - , (sn %, sp %). an roc curve was created for all three indicators, the auc was . (sn - %, sp - %). these parameters can be recommended as predictors of thrombosis in children after cardiac surgery. cpb is related with a large number of life-threatening complications. in our work, preoperative predictors of thrombosis were identified. based on this data, it is possible to create thrombosis risk scale change the tactics of the anaesthetic approach, the prevention of thrombosis in the postoperative period. further studies are needed to identify other possible predictors of thrombosis. introduction: abdominal ischemia occurs in % of patients submitted to aortic aneurysm repair. its early diagnosis requires an elevated index of suspiction, particularly in more severe patients. we hypothesized that earlier increase and higher levels of c-reactive protein (crp) may help to predict intra-abdominal ischemia. we performed a retrospective study of patients admitted to the intensive care department (icd) after abdominal aorta aneurism surgery. we included all patients admitted during a two-year period, that survived for more than hours. primary outcome was splanchnic ischemia assessed by abdominal ct-scan. we also evaluated the presence of bacteremia, abdominal compartment syndrome and icd mortality. association between inflammatory parameters and ischemia was evaluated by multivariate logistic regression. introduction: crp (c-reactive protein) has been shown to be a useful biomarker in identifying complications after major abdominal surgery. gastrectomy is a high-risk surgical procedure that requires post-operative critical care support to monitor for complications which are predominantly infective in nature. the aims of this study were to determine whether there is a relationship between post-operative crp levels and patients who developed post-operative infective complications. a retrospective analysis was performed on patients undergoing elective gastrectomy for gastric cancer at a single centre between september and july . post-operative crp levels for each day following resection were analysed for all patients. roc curve analysis was used to determine which post-operative day (pod) gave the optimal cut-off. of patients included, the majority were male ( . %), mean age was . years and . % had node-negative disease. a total of patients ( . %) had an infective complication, which includes those who experienced an anastomotic leak. crp levels on post-operative day gave the greatest auc for the gastrectomy group ( . ). crp cut-off of mg/l was significantly associated with infective complications (or . , % ci . - . , p= < . ) and gave a sensitivity of % and specificity % (ppv %, npv %). more patients with a crp > on post-operative day experienced an infective complication ( % vs %, p = < . ) or a leak in particular ( % vs %, p = . ). a crp level of less than mg/l on pod may be useful to predict the development or exclude the likelihood of such infective complications in this group of patients prior to clinical signs (ppv %, npv %). this may prompt and facilitate decision-making regarding early investigation and intervention or prevent inappropriate early discharge from critical care, whilst providing more assurance in identifying those who could be stepped down to ward level care. vasoplegia is commonly observed after cardiopulmonary bypass surgery (cpb) and associated with high mortality. chronic use of reninangiotensin aldosterone system inhibitors (raasi) is associated with its incidence and ensuing need for vasopressor support after cpb. renin serves as marker of tissue perfusion [ ] . we examined the role of renin in the setting of raasi exposure and vasopressor needs in the peri-cpb period. prospective observational study of adult patients undergoing cpb, aged . ± . years ( men, women). blood was collected ) post induction, pre-cpb; ) min post cardioplegia, and ) immediately post bypass. vital signs and perioperative medications were recorded. as control, blood was collected from men and women aged . ± . , not diagnosed with lung disease and not prescribed any raasi. baseline plasma renin in cpb patients tended to be higher than in control subjects (mean= . pg/ml± . vs. . pg/ml ± . , respectively, p= . ). minutes into cpb, mean renin was increased from baseline ( . pg/ml± . , p= . ), and remained elevated immediately post cpb ( . pg/ml± . ). patients using raasi prior to cpb tended to have a larger increase in renin post cpb (delta= . pg/ ml± . ) vs. those not previously on raasi ( . pg/ml± . , p= . ). renin was elevated in patients requiring vasopressor support in the hours post cpb vs. those not requiring pressors ( . pg/ ml± . vs. . pg/ml± . p= . ). in those prescribed raasi and requiring pressors post cpb, there was a tendency toward greater renin increase than those not requiring pressors postoperatively ( . pg/ml± . vs. . pg/ml± . , p= . ). this study suggests a trend toward higher renin levels, particularly during cpb, in patients prescribed raasi, and a positive association between renin and postoperative vasopressor needs. we speculate that increased renin levels may predict postoperative vasoplegia. cardiac surgery is associated with perioperative blood loss and a high risk of allogenic blood transfusion. it has been recognized that high blood product transfusion requirement is associated with adverse clinical outcomes. guidelines on patient blood management therefor aim at reducing blood loss and blood transfusion requirements in cardiac surgery. as there remains controversy about the advantage of minimal invasive techniques on blood loss an transfusion requirements, we wanted to investigate if the average blood loss and transfusion requirement in minimal invasive endoscopic coronary artery bypass graft surgery (endo-cabg) differ from conventional technique. we assessed the influence of pre-operative anticoagulant medication for blood loss. estimated average blood loss after conventional cabg is ml (+/- ) and transfusion requirement , units packed red blood cells . we performed a retrospective cohort study of our cardiac surgical database. from / / to / / , we collected data from patients undergoing endo-cabg. we analyzed blood loss, transfusion as well as pre-operative use of anti-coagulants as a risk factor for blood loss. we found that mean total blood loss in endo-cabg does not differ from conventional cabg, nonetheless mean transfusion requirement was lower in our cohort. use of direct oral anticoagulant is aossciated with increased blood loss and transfusion requirements (table ) . total blood loss is not influenced by minimal invasive technique for cabg (endo-cabg). an explanation for the lower transfusion requirements is the use of a minimal extracorporeal circulation, which is known to reduce the risk of transfusion. another important factor is the implementation of a standardized transfusion-protocol based on available evidence. reducing transfusion requirements is an important component in improving patient outcome after cardiac surgery and is related to multiple factors in perioperative care of our patients. retinal microvascular damage associated with mean arterial pressure during cardiopulmonary bypass surgery v shipulin retinal perfusion corresponds to cerebral perfusion and it is very sensitive to hemodynamic disturbances [ , ] . we investigated the association between retinal microvascular damage and hemodynamic characteristics in patients undergoing coronary artery bypass grafting surgery (cabg) with cardiopulmonary bypass (cpb). methods: patients with coronary artery disease and systemic hypertension were examined. ophthalmoscopy and optical coherence tomography were performed before and - days after cabg. the hemodynamic parameters during cpb were analyzed. results: ( %) patients had changes in the retinal vessels and in the ganglionic fiber structure on - day after surgery: in % of patients the foci of ischemic retinal oedema appeared, in % the decrease of the thickness of ganglionic fiber were observed. these changes may be associated with intraoperative ischemia of the central retinal artery. in ( %) patients the mean arterial pressure (map) during cpb was increased up to mmhg. in ( %) of them the association between map and foci of ischemic retinal oedema were revealed. the ischemic retinal changes were observed significantly more often if the delta of map during cpb was over then mm hg compared with the patients where the delta of map was less than mm hg (p= . ). this is probably due to an intraoperative disorders of the myogenic mechanism of blood flow autoregulation in the retinal microvasculature in patients with coronary artery disease [ ] . the level of map up to mm hg during cpb is associated with retinal blood flow impairment and the foci of ischemic retinal oedema. delta of map more than mmhg was associated with the foci of ischemic retinal oedema and decreased ganglionic fiber thickness in % of cases. atrial fibrillation after cardiac surgery: implementation of a prevention care bundle on intensive care unit improves adherence to current perioperative guidelines and reduces incidence introduction: atrial fibrillation after cardiac surgery (afacs) is a very frequent complication affecting - % of all patients. it is associated with an increase in morbidity, mortality and hospital and intensive care unit (icu) length of stay. we aimed to implement an afacs prevention care bundle based on a recently published practice advisory [ ] , focusing on early postoperative (re)introduction of β-blockers. baseline afacs incidence and β-blocker administration practices in our centre were audited for all patients undergoing valve surgery or coronary artery bypass graft (cabg) during a weeks period. the afacs prevention care bundlean easy to follow graphical toolwas subsequently introduced to the cardiac icu by a multidisciplinary team and audited following a model of improvement approach. after exclusion of patients with preoperative af, differences between pre-and post-implementation groups were compared with chisquare and fisher's exact tests for categorical, and one-way anova for continuous variables, using spss. a total of patients were analysed. patient and surgery characteristics did not differ between groups. significantly more patients received postoperative β-blockers after bundle implementation ( . % pre-vs . % post-bundle, p= . ) with a higher proportion on day ( . % pre-vs % post-bundle, p< . , figure ). the incidence of afacs was significantly reduced from . % to . % (p= . ), with a particularly marked reduction in the age group - years and for isolated aortic valve and cabg surgery. there was no significant reduction in hospital length of stay for this cohort. introduction of an afacs prevention care bundle using a graphical tool improved adherence to current guidelines with regards to early β-blocker administration and significantly reduced afacs incidence. future care bundles should include preoperative interventions and might reduce hospital length of stay. in neonates with univentricular physiology, there is a delicate balance between pulmonary and systemic circulations, with a tendency towards generous pulmonary blood flow, and a risk of systemic underperfusion. preoperatively, the use of hypoxic gas mixture (hm) has been advocated as a therapy to increase pvr, with the aim of improving systemic oxygen delivery. it is a therapy which has been routinely initiated in our institution in the setting of signs of pulmonary overcirculation. we performed a retrospective analysis of all patients in our institution who underwent a norwood procedure and who received hm preoperatively. we compared peripheral saturations, arterial blood gas analysis, serum lactate, regional cerebral and renal saturations and invasive blood pressure, prior to, and then , and hours after hm was commenced. between and (inclusive), patients underwent the norwood procedure. patients received preoperative hm. average fio was % during administration of hm. average peripheral saturations were . % prior to hm, and dropped to . % at hours, and % at and hours after initiation (p < . ). there was no change in any of the measured markers of systemic oxygen delivery, including regional cerebral and renal saturations, lactate, urine output or blood pressure. there was an association between an extended period of hm (> hours) and the need for pulmonary vasodilator therapy post norwood procedure. hypoxic gas mixture in patients with parallel systemic and pulmonary cicrculations causes desaturation and hypoxia. it does not lead to an increase in systemic perfusion and thus an improvement in systemic oxygen delivery. its ongoing use in this fragile population should be considered. introduction: analgesia in the critical patient, and especially in the neurocritical patient, is a basic goal in all therapeutic practices. patients in the icu are frequently administered prolonged and/or high doses of opioids. multiple serious complications due to the use of infusion of opioids at large doses has been described. to reduce high doses of intravenous opioids, multimodal forms of analgesia can be used. prospective observational study of the use of tapentadol enteral and buprenorphine in transdermal patches, at low doses, for the control of pain and its effect on reducing the use of fentanyl infusion in high doses on patients admitted to neuro icu of indisa clinic during consecutive years ( - ). enteral tapentadol (through ng tube) mg/ hours, was considered in patients who required intravenous fentanyl in continuous administration. buprenorphine was also added at low doses ( ug/hr) in a weekly transdermal patch, in cases of neurosurgical spine patients, fractures and long-term neuropathic pain. pain was controlled on behavioral pain scale (bps) and visual analogical scale (vas) scores, according to the conditions of each patient. their hemodynamic, gastrointestinal complications and the appearance of delirium episodes according to cam-icu scale were recorded. results: patients received tapentadol. of them also received transdermal buprenorphine. all managed to maintain adequate level of analgesia, not requiring fentanyl at doses greater than . ug / kg / hr. distribution by diagnoses: neurotrauma patients, guillain barre , spine surgery , hsa , hice , malignant ischemic acv . complications: gastric retention patients ( %), hypotension ( %), acute hypoactive delirium ( . %), acute hyperactive delirium ( %). no drug interactions were found. the introduction of enteral tapentadol and buprenorphine patches in neurocritical patients was safe and resulted in a decrease in the use of endovenous opioids and its adverse effects. we hypothesized that changing the pain management for our post cardiac surgical patients to an assessment-driven, protocol-based approach using fast acting and easily titratable agents will significantly improve patient satisfaction by reducing pain intensity in the first h after surgery as suggested by society of critical care [ ] guideline. we prospectively assessed and ( . vs . ) consecutive patients before and after introducing our pain management protocol. the nursing and medical team received rigorous training on the guideline as well as the correct assessment using appropriate pain scores measured at least hourly (numeric pain score, ≥ is timing of beta-blocker (re)initiation versus incidence of afacs before and after prevention care bundle implementation, per post-operative day and for postoperative days - (insets) moderate to severe or critical care observation tool, > is moderate to severe). we introduced a multimodal approach with a combination of fast acting iv, long acting oral opiates, regular paracetamol and rescue iv boluses for difficult to control situations and we created a prescription bundle on our electronic prescribing record. among other variables we assessed hours spent in moderate to severe pain in the first h after surgery and compared to the data collected before the guideline was introduced. we analysed patients from and from . baseline characteristics were similar between the two groups. in only . % of the patients spent less than hours and . % spend more than hours in moderate to severe pain. the data showed significant improvement in that . % of patients spent less than hours and only % patients who spent more than hours in moderate or severe pain. (p < . , chi square) ( figure ). only % of the patient needed rescue medications. % of time was the protocol inadequate necessitating other approach. introducing an assessment driven, stepwise, protocolized pain management significantly improved patient satisfaction by reducing pain intensity in the first h on our cardiothoracic intensive care unit. introduction: proximal femur fractures are most common fractures in the elderly and associated with significant mortality and morbidity, with high economic and social impact. perioperative pain management influence outcomes and mortality after surgery with early mobilization being possible [ , ] . the goal of the study was to compare the efficacy and safety of the compartment psoas block for perioperative analgesia in elderly patients with proximal femur fractures. the randomized controlled study was held in medical center "into-sana" (odesa, ukraine) from january till july . patients with proximal femur fractures and older than years were included in the study. they were randomly allocated to groupscompartment psoas block group (bupivacaine analgesia was started as soon as possible before surgery and prolonged during and after surgery with additional ischiadicus block before surgery) and general (inhalational) anesthesia with systemic analgesia perioperatively. results: patients were included in this study. perioperative compartment psoas block was associated better pain control, decreased opioid consumption, better sleep quality, earlier mobilization after surgery, decreased incidence of opioid-associated vomiting/nausea and myocardial injury. there were no difference in the incidence of hospital acquired pneumonia and delirium. perioperative compartment psoas block is effective and safe for perioperative analgesia in elderly patients with proximal femur fractures, and is associated with better pain control and decreased complications incidence. parenteral olanzapine is frequently used in combination with parenteral benzodiazepines for hospitalized patients with severe agitation. the fda issued a warning for increased risk of excessive sedation and cardiorespiratory depression with this combination based on post-marketing case reports with overall limited quality of evidence [ ] . the purpose of this study is to evaluate the safety and efficacy of concomitant parenteral olanzapine and benzodiazepine for agitation. this retrospective chart review evaluated agitated patients who received concomitant parenteral olanzapine and benzodiazepine within minutes from / / to / / . the primary end points were rate of respiratory depression requiring mechanical ventilation and hypotension requiring vasopressors. the secondary end points were percentage of patients requiring additional sedatives for agitation during the same time frame, cumulative dose of olanzapine and benzodiazepine (midazolam equivalent) received, and rate of cardiac arrest and death. a total of patients were included with notable baseline characteristics: median age of years old, % with a history of substance abuse, and % with a history of psychiatric illness. for the primary outcomes, . % of patients required mechanical ventilation and % required vasopressors. additionally, . % patients received additional sedating agents to control agitation. refer to table for more details. no cardiac arrests or deaths were observed. concomitant use of parenteral olanzapine and benzodiazepine within minutes for the treatment of agitation appears to have a small risk of respiratory depression without significant hypotension. hip fracture is very common in the elderly,it causes moderate to severe pain often undertreated. ficb is a simple safe method, easy to learn and use. the aim of our study is to assess the efficacy and safety of preoperative ficb compared with intravenous analgesia for elderly patients with femoral fracture and hip surgery in terms of opioid consumption and perioperative morbidity methods: after informed consent obtained, patients - yo asa i-iii with hip fracture were randomized to receive either an us guided ficb( ml of ropivacaine , %) or a sham injection with normal saline ' before surgery. both groups were operated under general anesthesia. postoperative analgesia was done according to vas: vas - mm, paracetamol g iv at h, vas - mm, ketoprofen mg iv at h, vas> , morphine , mg/ kgbw iv. the primary outcome was the comparison of vas score at rest over the first 'following the procedure, at the end of the surgery and at h intervals for h. the secondary outcome were the incidence of the cardiovascular events, of the ponv and of the confusion episodes, the amount of morphine consumption for h results: at baseline, ficb group (a) had a lower mean pain score than the sham injection group (b). the same difference was observed over h of follow-up (p< . ). there was a significant difference between the two groups in total cumulative iv morphine consumption at h and in the incidence of ponv and confusion episodes ( figure ). ficb provides effective analgesia for elderly patients suffering from hip fractures, with lower morbidity and lower opioid consumption compared with intravenous analgesia. pain assessment in chronic disorders of consciousness patients with ani monitoring e kondratyeva, m aybazova, n dryagina almazov national medical reseach centre, minimally conscious research group, st petersburg, russia critical care , (suppl ):p pain and suffering controversies in doc to be debated by the scientific, legal and medical ethics communities. methods: ani (anti nociception index) monitor was used to assess pain in patients with chronic disordersof consciousness (doc) age range to years - in vegetative state/ unresponsive wakefulness syndrome (vs/uws) and minimal consciousness state (mcs). average age: in mcs group , ± , and , ± , in vs/uws group. neurological status was assessed using crs-r scale. the average score on the crs-r scale was ± . in vs/uws and . ± . in mcs. pressure on the nail phalanx was used as a pain impulse. ani and nociception coma scale was evaluated before the application of pain stimulus, immediately after and past minutes. prolactin level was measured before the pain stimulus application and minutes after. ani less than indicates pain, - hypoalgesia, severe pain. the mean value of the ani in mcs patients: before the pain stimulus . ± . , after the pain stimulus application ± . and minutes later . ± . . prolactin level in mcs patients before pain . ± . ng/ml; after pain . ± . ng/ml (p> . ). prolactin in vs/uws patients before pain . ± . ng /ml, after pain . ± . ng / ml (p> . ). conclusions: ani monitor revealed that vs/uws and mcs patients react equally to the pain impulse. prolactin dynamics showed poor statistical mean and can not be consider as a marker of nociception in this group of patients. it is possible that the level of pain impulse was insufficient neuroendocrine response activation or the increase of prolactin level occurs in the long term (more than minutes). in all patients the total hip arthroplasty tha is one of the most common major surgical procedures associated with significant postoperative pain that can adversely affect patient recovery and could increase morbidity. effective perioperative pain management allows an accelerated rehabilitation and improve the functional status of these patients. multimodal analgesia mma combines analgesics with different mechanism of action which by synergistic and additive effects enhance postoperative pain management and reduce complications. the aim of our study is to assess if perioperative association of very low dose of ketamine, a potent nmda antagonist and dexamethasone, by antiemetic and antiinflammatory properties could decrease opioid consumption and postoperative morbidity of patients with tha. after informed consent, patients scheduled for primary hip joint replacement surgery aged - yo asa i-iii were prospective randomized in two groups. both groups were operated under general anesthesia fentanyl/sevoflurane. supplementary, patients in group a received mg iv dexamethasone and mg at h and ketamine mg iv bolus at induction and mg/h iv during surgery. postoperative analgesia was done according to vas, - mm paracetamol g iv at h, - mm ketoprofen mg iv at h, vas> mm morhine , mg/kgbw iv. we recorded perioperative opioid consumption, the number of intraoperative cardiac events, vas score at the end of surgery and at h, the incidence of ponv and persistance of chronic pain at months. we obtain a significant less pain score at the end of surgery p< . in group a, no significant difference at h, a significant less chronic pain at months, a fewer npvo and cardiovascular events in group a, p< . ( figure ). a multimodal approach with very low doses of ketamine and dexamethasone could be efficent in the treatment of pain for elderly patients with hip arthroplasty, decreasing postoperative side-effects and reducing chronic pain persistance. introduction: treatment in an intensive care unit (icu) often necessitates uncomfortable and painful procedures for patients. chronic pain is becoming increasingly recognized as a long term problem for patients following an icu admission [ ] . throughout their admission patients are often exposed to high levels of opioids, however there is limited information available regarding analgesic prescribing in the post-icu period. this study sought to examine the analgesic usage of icu survivors pre and post icu admission. methods: patients enrolled in a post-intensive care programme between september and june . intensive care syndrome: promoting independence and return to employment (ins:pire), is a -week multicentre, multidisciplinary rehabilitation programme for icu survivors and their caregivers. patients' level of analgesia was recorded pre-admission and upon attending ins:pire, their level of prescribed analgesia was categorized using the word health organisation (who) analgesic ladder [ ] . results: . % of patients (n= ) were prescribed regular analgesia preadmission; this increased to . % (n= ) post-admission, representing a significant absolute increase of . % ( % ci: . % - . %, p< . ) in the proportion of patients who were prescribed regular analgesia pre and post icu. in addition, pre-admission, . % (n= ) of patients were prescribed a regular opioid (step and of the who ladder) compared to . % (n= ) post-admission, representing an absolute increase of . % ( % ci: . % - . %, p< . ). this study found a significant increase in analgesic usage including opioids in icu survivors. follow-up of this patient group is essential to review analgesic prescribing and to ensure a long term plan for pain management is in place. introduction: pain, agitation, and delirium (pad) are commonly encountered b patients in the intensive care unit (icu). delirium is associated with adverse outcomes, including increased mortality and morbidity. clinical guidelines suggest that routine assessment, treatment and prevention of pad is essential to improving patient outcomes. despite the well-established improvements on patient outcomes, adherence to clinical guidelines is poor in community hospitals. the aim of this quality improvement project is to evaluate the impact of a multifaceted and multidisciplinary intervention on pad management in a canadian community icu. a pad advisory committee was formed and involved in the development and implementation of the intervention. the -week intervention targeted nurses (educational modules, visual reminders), family members (interviews, educational pamphlet, educational video), physicians (multidisciplinary round script), and the multidisciplinary team (poster). an uncontrolled, before-and-after study methodology was used. adherence to pad guidelines in the assessment of pad by nurses was measured weeks pre-intervention and weeks post-intervention. data on patient-days (pd) and pd were available for analysis during the pre-and post-intervention, respectively. the intervention significantly improved the proportion of pd with assessment of pain and agitation at least times per -hour shift from . % to . % and from . % to . %, respectively ( figure ). proportion of pd with delirium assessment at least once per -hour shift did not significantly improve. a multifaceted and multidisciplinary pad intervention is feasible and can improve adherence to pad assessment guidelines in community icus. quality improvement methods that involve front-line staff can be an effective way to engage staff with pad. oversedation introduction: sedation is a significant part of medical treatment in icu patients. a too deep sedation is associated with a longer time of mechanical ventilation, lung injury, infections, neuromuscular disease and delirium, which can lead to a longer duration of icu hospitalization, as well as an increase of morbility and mortality. many patients spend a considerable amount of time in a non-optimal sedation level. a continuous monitoring system of the sedation level is therefore necessary to improve clinical evaluation. our goal was to evaluate the incidence of non-optimal sedation (under and over sedation) comparing the parameters expressed from ngsedline with clinical evaluations and to correlate oversedation and the incidence of delirium. we have studied a cohort of patients admitted to the icu of spedali civili of brescia university hospital requiring continuous sedation for more than hours. in addition to standard monitoring, the patients have been studied using next generation sedline (masimo). sedation depth was evaluated through rass scale and the presence of delirium was evaluated with cam-icu scale. we collected data from adult patients. our data showed high incidence of oversedation. of our patients had a sr> and had a psi level< . a logistic regression analysis was performed and it showed statistically significant association between incidence of delirium and the age of the patients (p . ). the association between delirium incidence and suppression rate time was at the limits of statistics significance (p . ) and was statistically significant for non neurocritical patients (p . ). our study didn't show an association between delirium and the total time of sedation. non-optimal sedation is an unsolved problem in icu, affecting lot of patients, with a major incidence of over-sedation compared to under-sedation. our study shows an association between sr levels and the incidence of delirium. predictors of delirium after myocardial infarction, insights from a retrospective registry m jäckel, v zotzmann, t wengenmayer, d dürschmied, c von zur mühlen, p stachon, c bode, dl staudacher heart center freiburg university, department of cardiology and angiology i, freiburg, germany critical care , (suppl ):p delirium is a common complication on intensive care units. data on incidence and especially on predictors of delirium in patients after acute myocardial infarction (mi) are rare. by analyzing all patients after acute mi, we aim to identify incidence and potential risk factors for delirium. in this retrospective study, all patients hospitalized for acute mi treated with coronary angiography in an university hospital in were included and analyzed. incidence of delirium within the first days of care attributed to the mi and was defined by a nudesc score ≥ , which is taken as part of daily care three times a day by especially trained nurses. this research is authorized by ethics committee file number / . results: patients with acute mi (age . ± . years, stemi, mortality . %) were analyzed. delirium occurred in ( . %) patients and was associated with a longer hospital stay ( ± . d vs . ± . d, p< . ). patients with delirium were significantly older than patients without ( . ± . vs. . ± . years, p< . ) and had more often preexisting neurological diseases ( . % vs. . %, p< . ) and dementia ( . % vs. . %, p< , ). multivariate logistic regression analysis suggested that odds ratio for delirium was higher in patients after resuscitation or . ( % ci . - . ), preexisting dementia or . (ci . - ) and in patients with alcohol abuse or (ci . - ). while maximum lactate was also connected to delirium or . (ci . - . ), infarct size or type had no effect on the incidence of delirium. in patients with mi, delirium is frequent. incidence is associated with clinical instability and preexisting neurological diseases rather than infarct size. incidence and risk factors of delirium in surgical intensive care unit ma ali, b saleem aga khan university, anaesthesia, karachi, pakistan critical care , (suppl ):p introduction: delirium in the critically ill patients is common and distressing. the incidence of delirium in the icu ranges from % to %. although delirium is highly common among intensive care patients, it is mostly underreported. to date, there have been limited data available related to prevalence of delirium in surgical patients. in a study published in , the risk was observed % in surgical and trauma patients [ ] . the purpose of this study was to find out the incidence and associated risk factors of delirium in surgical icu (sicu) of a tertiary care hospital. we conducted prospective observational study in patients with age more than years and who were admitted to the surgical icu for more than hours in aga khan university hospital from january to december . patients who had preexisting cognitive dysfunction or admitted to icu for less than hours were excluded. delirium was assessed by intensive care delirium screening checklist icdsc. incidence of delirium was computed and univariate and multivariable analyses were performed to observe the relationship between outcome and associated factors. delirium was observed in of patients with an incidence rate of . %. multivariable analysis showed that copd, pain > and . ] were also the strongest independent predictors of delirium while analgesics exposures was not statistically significant to predict delirium in multivariable analysis. delirium is significant risk factor of poor outcome in surgical intensive care unit. . there was an independent association between pain, sedation, copd, hypernatremia and fever in developing delirium delirium is an acute mental syndrome which may cause negative consequences if it is misdiagnosed [ , ] . the aim of this study was to determine the incidence of delirium in different intensive care units and reveal the risk factors. the study was performed with patients hospitalized in intensive care units of anesthesia, neurology and general surgery departments. written informed consent was obstained from patients or relatives. delirium screening test was performed twice daily with camicu (confusion assessment method for the icu). patients who met the study criterias, were evaluated for the possible risk factors of delirium and the data was recorded daily. patients were reevaluated after the treatment. the incidence of delirium was . %. delirium was found to increase with the length of stay (p < . ). the mean age of the patients with delirium was . . this was higher than the patients without delirium ( . ) (p< . ). visual impairment (p< . ), hearing impairment (p= . ), educational status (p= . ), hypertension (p= . ), mechanical ventilation (p = . ), oxygen demand (p= . ), midazolam infusion (p= . ), propofol infusion (p= . ), infection (p < . ), sofa (p = . ), apache ii (p < . ), nasogastric catheter (p= . ), aspiration (p < . ), number of aspirations (p< . ), enteral nutrition (p< . ), albumin (p= . ), steroid (p= . ), hypercarbia (p= . ) hypoxia (p= . ), sleep disturbance (p< . ) were found risk factors for delirium. oral nutrition (p< . ) and mobilization (p= . ) were found to prevent delirium development. various factors are important in the development of delirium. these risk factors should be considered in reducing the incidence of delirium in intensive care units. ). an unplanned and brutal stop of alcohol consumption, as it can occur during icu admission, may lead to an alcohol withdrawal syndrome (aws). the most severe clinical manifestation of aws is described as delirium tremens (dt). there are no current guidelines available for aws treatment in icu. the study's aim was to describe the clinician's practices for dt treatment and the outcome of dt in icu patients. observational retrospective cohort study in two icus of a universityaffiliated, community hospital in france. patient diagnosed for dt during their icu stay, as defined by dsm-v classification, were enrolled in the study. results: patients with dt were included between and . benzodiazepines was administered to % of the patients in order to prevent an aws. as associated measures, vitamin therapy was administered to % of the patients and % had an increased fluid intake (mean . l+/- . ). concerning the curative approach of aws, the treatment's heterogeneity was notable. there was a high frequency of treatment's association ( % of the patients), every patient had benzodiazepines and the use of second line treatments such as neuroleptic, alpha- agonist, propofol was variable ( figure ). complications of dt were the following: need for mechanical ventilation due to unmanageable agitation or acute respiratory distress ( % of the patients) self inflicted injuries such as pulling out of central lines, tubes, surgical drain ( %) falls ( %). seizures ( %). delirium tremens is a severe complication of an untreated aws, which can lead to serious adverse events in icu. the current lack of evidence concerning the management of aws in icu probably explains the heterogeneity of treatments. given the potential severity of aws in icu, further evidences are required to optimize care of aws in icu patients. the incidence and related risk factor of delirium in surgical stepdown unit s yoon , s yang , g cho , h park , k park , j ok , y jung asan medical center, nursing department, seoul, south korea; asan medical center, seoul, south korea critical care , (suppl ):p step down units (sdus) provide an intermediate level of care between the icu and the general medical-surgical wards. the critically ill patients who are in recovery after long-term intensive care or who require monitoring after acute abdominal surgery are admitted to sdus. delirium in critically ill patient is common and leads to poor clinical outcomes. it is, however, preventable if its risk factors are identified and modified accordingly. to determine risk factors associated with delirium in critically ill patients to admitted surgical sdu at asan medical center. this is retrospective study conducted on critically ill patients who were admitted to the sdu from september to april and able to express themselves verbally. delirium status was determined using the short-cam tool. data were analyzed by spss . software, using t-test, fisher's exact test and logistic regression. the incidence of delirium was . %( of patients) and hypoactive delirium( case, . %) was the most commonly assessed, followed by hyperactive delirium( case, . %), mixed type( case, . %). risk factors associated with developing delirium identified from univariate analysis were age(p= . ), admission via icu (p= . ), tracheostomy (p= . ), chronic heart failure (chf) (p= . ), invasive hemodynamic monitoring (p= . ), heart rate (p= . ). after adjusted in multivariate analysis; factors those remained statistically significant were old age (rr we identified risk factors consistently associated with incidence of delirium following admitted to surgical sdu. these factors help to focus on patients at risk of developing delirium, and to develop preventive interventions that are suitable for those patients. patients with sepsis frequently develop delirium during their intensive care unit (icu) stay, which is associated with increased morbidity and mortality. the prediction model for delirium in icu patients (pre-deliric model) was developed to facilitate the effective preventive strategy of delirium [ ] . however, the pre-deliric model has not yet been validated enough outside europe and australia. the aim of this study is to examine the external validity of the pre-deliric model to predict delirium using japanese cohort. this study is a post hoc subanalysis using the dataset from previous study in nine japanese icus, which have evaluated the sedative strategy with and without dexmedetomidine in adult mechanically ventilated patients with sepsis [ ] . these patients were assessed daily throughout icu stay using confusion assessment method-icu. we excluded patients who were delirious at the first day of icu, were under sustained coma throughout icu stay and stayed icu less than h. we evaluated the predictive ability of the pre-deliric model to measure the area under the operating characteristic curve. calibration was assessed graphically. of the patients enrolled in the original study, we analyzed patients in this study. the mean age was . ± . years and patients ( %) were male. delirium occurred at least once during their icu stay in patients ( %). to predict delirium, the area under the receiver operating characteristics curve of the pre-deliric model was . ( . to . ). graphically, the prediction model was not well-calibrated ( figure ). to predict delirium in japanese icus, we could not show the well discrimination and calibration of the pre-deliric model in mechanically ventilated patients with sepsis. introduction: delirium is a serious and common complication and in some cases it treatment is difficult. aim of the study was an evaluation of the prevalence, structure of delirium and efficacy of dexmedetomidine and haloperidol sedation in geriatric patients after femur fracture. after local ethic committee approval case-records of geriatric patients with femur fracture in the period from to in the institute of traumatology and orthopedics in astana were analyzed. patients was divided for groups: in dpatients with delirium treated by i/v dexmedetomidine ( . - . mkg/kg per hour), in g group patients with delirium treated by i/v galoperidol ( . - . mkg/kg). delirium was assessed by rass at day of permission and every day at a.m. the prevalence, structure of delirium and efficacy of sedation were analysed. results: by anthropometric and gender characteristics of the group did not differ. the average age in the d-group with delirium was . ± . years old, which was comparable to the g-group - . ± . years old (p = . ). all study participants had similar comorbidities. delirium in all patients debuted at . ± . days, with an average duration of . ± . days. the effect of dexmedetomidine was better and expressed in % decrease in the duration of delirium in compare to haloperidol (p < . ). dexmedetomidine provided a more controlled and safe sedation compared with haloperidol. the average consumption of narcotic analgesics in the subgroup with dexmedetomidine was two times less than in the subgroup with haloperidol. thus, the average consumption of trimeperidine hydrochloride in patients of group d was . mg versus . mg in group g (p = . ). in gerontological patients with femur fracture treatment delirium by dexmedetomidine was more effective in compare with haloperidol. when using dexmedetomidine, the consumption of narcotic analgesics in postoperative period was % less than with haloperidol. live music therapy in intensive care unit mc soccorsi , c tiberi , g melegari , j maccieri , f pellegrini , e guerra intensive care units (icu) are not comfortable for patients, relatives or next of kin. in the last years many news approaches were described to implement the humanization of medical treatments. the positive effect of music therapy in icu is well described, especially reducing delirium risk [ ] . the aim of this paper is describing the effect in patients and their family of a music live performance in icu. after ethical committee approval (procedure aou / , italy) for three months (november -january ) patients in icu were treated twice a week with live music therapy performed by coral vecchi-tonelli of modena, italy (fig. ). data were collected all awake and conscious patients. vitals parameters, gcs, raas and cam icu were collected before, during and after the treatment, at every performance. after the treatment a feedback questionnaire were given to patients and to next of kin. results: subjects were enrolled in the research with mean age of . years old, delirium rate before the treatment was . % later . %, raas does not show any difference. over % of patients were satisfied, and relatives felt less anxiety. we recorded also a satisfaction also in relatives not enrolled. the study does not demonstrate a delirium risk reduction for the small sample and the length treatment, anyway it was recorded a low delirium rate. the safety and the potential effect of music therapy are well known, surely the research underlines the feeling of patients and their next of kin: icu is the most stressful setting for admitted patients and its humanization is a current topic for medical literature. live performances could be an entertainment moment and probably create a moment of an interaction among patients, their family and medical and nurse: icu become more human. the high level of satisfaction push us to continue this experience. introduction: patients undergoing medical procedures benefit from distraction techniques to reduce the need for drugs alleviating pain and anxiety. this study investigates if medical hypnosis or virtual reality glasses (vrglasses) as adjuvant method reduces the need for additional drugs. in a prospective, randomized, interventional trial, patients undergoing procedures were stratified in four age groups, and randomly assigned into three arms by means of a closed envelope system. all patients received standard care for pain before the procedure; the control group received further drugs for pain and stress as indicated by the visual analog scale (vas; threshold / ) and comfortscore (threshold / ), two index groups received either medical hypnosis or vr glasses as a plus before and during the procedure. vas and comfort were scored continuously and analysed with the kruskal-wallis test. patients, parents and healthcare providers scored their satisfaction at the end. of included patients to years old, % were female. regardless of age, pain and comfort scores were similar before and at the start of the procedure (vas . - . ; comfort - . ), but as of one minute after starting the procedure, both vas and comfort reduced significantly more in both index groups compared to the control (p< . ), remaining far below the threshold for both pain and stress ( figure ). there was no advantage of one index group over the other (p= . ). there were no adverse effects. patients in the vr group were more satisfied than in the standard group (p= . ) or in the hypnosis group (p= . ). there was no significant difference in satisfaction of parents or healthcare providers. from the very start of the intervention, the application of either medical hypnosis or vr glasses significantly reduces pain and anxiety in patients undergoing medical procedures. more studies are needed but both are promising safe adjuvant tools to standard pharmacological treatment. music to reduce pain and distress due to emergency care: a randomized clinical trial ne nouira, i boussaid, d chtourou, s sfaxi, w bahria, d hamdi, m boussen, m ben cheikh mongi slim academic hospital, emergency department, tunis, tunisia critical care , (suppl ):p recent clinical studies have confirmed the benefits of music therapy in managing pain and improving quality of care in the emergency department. the aim wasto evaluate the impact of receptive music therapy on pain and anxiety induced by emergency care methods: a randomized controlled study in patients consulting the emergency department. two groups: the music therapy group; patients needed venous sampling, peripheral venous catheter or arterial catheter. will bless ten minutes music therapy by headphones and a second control group of patients with the same care without music therapy. consent was requested from all participants. the level of pain caused by the act of care was assessed by visual analogic scale. heart rate, blood pressure and the mood of the patient were assessed before and after emergency care. we assessed patient satisfaction, adverse events. patients admitted to the emergency room, patients with communication difficulties and non-consenting patients were not included results: two hundred and forty patients were included randomized in both groups, with music therapy and without music therapy, the results showed comparable characteristics between the two groups: demographic data, pathological history, and initial clinical presentation. after the session of music therapy a difference was noted in the evaluation of the mean vas who was in the group with music of . ± . versus . ± . in the control group p< . ci % [- . ; - . ], and the mean of diastolic blood pressure which was , mmhg in the first group against . mmhg for the control group p = . ci % [- . ; - . ]. as for the mood, the patients were more smiling after the act of care in the group music therapy. all patients were satisfied with their experience and % recommend this therapy to their relatives . music therapy may reduce pain and anxiety in patients during emergency care. the music therapy is the intervention of music and/or its elements to achieve individual goals within a therapeutic.the music has proved to have positive physiological and psychological effects on patients [ ] . patients admitted to the intensive care unit (icu) experience anxiety and stress even when sedated, negatively influencing recovery [ ] . methods: two groups are established, a music therapy group (mg) and a control group (cg). the first one undergoes music therapy interventions, it consists of -minutes sessions of live music. patients of the gc will receive the usual treatment established by the service protocol for weaning management and the data are collected during the same time interval. data collection includes mean arterial pressure (map), heart rate (hr), respiratory rate (rr), oxygen saturation (sao ) and temperature (t). a total of patients were recruited, of which patients had to be excluded for meeting any of the exclusion criteria (n= ). of which (n= ) were randomized in the gm and the rest to the gc (n= ) ic %. regarding delirium in gm ( . %) presented a positive cam-icu, while in the cg were ( . %) (p= . ). when analyzing the variables in the cg and gm, it was observed that there were no differences with respect to hr, rr and map variable ( figure ). according to the results, we can say that music therapy as a nonpharmacological strategy for management of anxiety and delirium in patients of critical care units, might be an useful tool for the management of patients in weaning of mechanical ventilation introduction: coagulopathy and basopenia are common features of anaphylaxis, but the role of coagulopathy in anaphylaxis remains uncertain. the aim of this study is to evaluate the association between coagulopathy and clinical severity or basopenia in patients with anaphylaxis. we conducted a single-center, retrospective study of patients with anaphylaxis about their coagulopathy. levels of fibrin degradation products (fdp) and d-dimer were analyzed with the cause of anaphylaxis, clinical symptoms, medications and outcomes. we also studied the levels of intracellular histamine as a biomarker of basophil degranulation in the peripheral blood in relation to fdp and ddimer. in total, sixty-nine patients were enrolled to the study, and the levels of intracellular histamine were analyzed in patients. the symptoms included respiratory failure (n= ), shock (n= ), abdominal impairment (n= ), and consciousness disturbance (n= ). thirty-two patients needed continuous intravenous vasopressors for refractory shock. the increase of fdp was significantly associated with consciousness disturbance (p= . ) and refractory shock (p< . ). the increase of d-dimer was also significantly associated with refractory shock (p= . ). there was no correlation between the levels of intracellular histamine and either of fdp or d-dimer (p= . and p= . , respectively). the increase of fdp and d-dimer were associated with severe symptoms of anaphylaxis, while they were not correlated with intracellular histamine. these results suggest that anaphylaxis is closely associated with coagulopathy in a mechanism which is different from basophile degranulation in anaphylaxis. cardiac manifestations of h n infection in a greek icu population e nanou , p vasiliou , e tsigou , v psallida , e boutzouka , v zidianakis , g fildissis agioi anargiroi hospital, attiki, greece; agioi anargiroi hospital, icu, attiki, greece critical care , (suppl ):p introduction: cardiovascular involvement in influenza infection occurs through direct effects on the myocardium or through exacerbation of pre-existing cardiovascular disease [ ] . the aim was to study cardiac manifestations in all pts admitted to the icu with severe influenza's attack. clinical, laboratory, electrocardiographic, echocardiographic and hemodynamic data were retrospectively recorded in all pts admitted to the icu due to influenza infection (winter -spring ). diagnosis was established by pcr on bronchial aspirates the next days after admission. myocardial injury was defined by troponin levels > pg/ml ( fold uln). left ventricular systolic dysfunction was defined as ef < % and was characterized as either global or regional. hemodynamic monitoring by fig. (abstract p ) . comparison between mg and cg transpulmonary thermodilution method (picco) was recorded in pts with shock (norepinephrine > . μg/kg/min). values are expressed as mean±sd or as median (ir). results: nine pts ( males) with a mean age . ± . years, apache ii ± . and sofa score . ± . were assessed. icu admission was due to ards ( ) and copd exacerbation ( ) . icu los was . ± . days and mortality rate was %. no history of vaccination or coronary heart disease was referred. results are shown in table . levosimendan was administered in pts with severe cardiogenic shock. in all survivors, shock and indices of myocardial dysfunction subsided till discharge. coronary angiography was performed in pt showing no abnormalities. mortality was attributed to septic shock and multi-organ failure. myocardial involvement, though common in influenza pts admitted to the icu, didn't contribute to a dismal prognosis. the cardioprotective effects of levosimendan could be related to the modulation of oxidative balance. we aimed to examine the effects of levosimendan in patients with cardiogenic shock or with ejection fraction (ef) lower than % on cardiac systo-diastolic function and plasma oxidants/antioxidants (glutathione, gsh; thiobarbituric acid reactive substances, tbars). in patients undergone coronary artery bypass grafting or angioplasty, cardiovascular parameters were measured at t (before the beginning of levosimendan, . mcg/kg/min), t ( h after the achievement of the therapeutic dosage of levosimendan), t (at the end of levosimendan infusion), t (at h after the end of levosimendan infusion), t (at the end of cardiogenic shock). the same time-course was followed for plasma gsh and tbars measurements. we found an improvement in cardiac output, cardiac index and systolic arterial blood pressure. ef increased from mean % to %. a reduction of central venous pressure and wedge pressure was also observed. moreover, indices of diastolic function were improved by levosimendan administration (e/e' from to ; e/a from > to < ) at early t . it is to note that an improvement of gsh and tbars was observed early after levosimendan administration (t ), as well ( figure ). the results obtained have shown that levosimendan administration can regulate oxidant/antioxidant balance as an early effect in low cardiac output patients. the modulation of oxidative condition could be speculated to play a role in exerting the cardio-protection exerted by levosimendan in those patients. table . early administration of vasopressors and their use in the emergency department was associated with survival in septic shock. this seemed to be independent of median map recorded in the ed. we excluded all the traumatic or post-myocardial infarction forms. out of patients, the tuberculous etiology was identified in cases ( , %), mean age was years, , % were men. patients reported a tb contact in their environment, had a medical history of pulmonary tb. after pericardiocentesis, the liquid was citrine yellow in cases and hematic in patients, no patient underwent surgical drainage in our serie. mycobacterium tuberculosis was found in the expectorations in cases and ada was positive in patients. hiv serology was negative in all our patients. a months anti bacillary therapy with isoniazid, rifampin, pyrazinamide, and ethambutol was initiated in all our patients with a good evolution in cases, deaths, chronic constrictive pericarditis, small pericardial effusion and lost to follow-up. althought cardiac tamponade is rarely caused by tuberculosis, this condition remains common in endemic countries such as morocco and affect younger population, hence the importance of a better knowledge of its prevalence and and multidisciplinary management and more importantly the treatment of the underlying cause using combined antibacillary medication that has shown satisfying results. . the main perceived limiting factor is the absence of a standardized didactic program, followed by mentor's availability in residents' perception and by mentor's experience in consultants' one. pocus teaching is present although not optimal and not homogenous in italian acc residency schools. standardisation of residents' ultrasound curriculum is suggested to improve ultrasound teaching. the study included a convenience sample of critically ill patients with supradiaphragmatic cvcs and a cxr for confirmation. us is used for direct confirmation of the guidewire in the internal jugular (ijv) or subclavian (scv) vein and visualizing the guidewire in the right atrium. to evaluate for pneumothorax, "sliding sign" of the pleura was noted on us of the anterior chest. results: patients have been included, % of the catheters have been placed in the scv and % in the ijv. it was possible to confirm the position of the cvc tip for . % ( correct, incorrect cxr) of (figure ). overall, it was not possible to identify the guide in the right atrium cases ( false negatives, of them due to the presence of defibrillator leads). regarding the case where an incorrect position was seen on cxr it was also detected on ultrasound: us of the inserted vein and a negative tte confirmation. in all cases it was possible to exclude a pneumothorax by us. these results show that bedside ultrasound might be a feasible technique to confirm the cvc positioning. it is important to note that the level of the operator's expertise is significant when assessing the feasibility of this method. we only had a limited sample size and the occurrence of only one misplaced catheter. these preliminary results need to be confirmed on a larger scale. central venous catheter (cvc) misplacement occurs more frequently after cannulation of the right subclavian vein compared to the other sites for central venous access. misplacement can be avoided with ultrasound guidance by using the right supraclavicular fossa view to confirm correct guidewire j-tip position in the lower part of the superior vena cava. however, retraction of the guidewire prior to the cvc insertion may dislocate the j-tip from its desired position, thereby increasing the risk of cvc misplacement. the aim of this study was to determine the minimal guidewire length needed to maintain correct guidewire j-tip position throughout an us-guided infraclavicular cvc placement in the right subclavian vein. methods: adult intensive care patients with a computed tomography scan of the chest were retrospectively and consecutively included in the study. the distance from the most plausible distal puncture site of the right subclavian/axillary vein to the junction of the right and left brachiocephalic veins (= vessel length) was measured using multiplanar reconstructions. in addition, measurements of the equipment provided in commonly used - cm cvc kits were performed. the minimal guidewire length was calculated for each cvc kit. the guidewires were up to mm too short to maintain correct j-tip position throughout the cvc insertion procedure in seven of nine commercial cvc kits. four of these are shown in table . when us guidance is used to confirm a correct guidewire j-tip position, retraction of the guidewire prior to the cvc insertion must be avoided to ensure correct cvc-tip positioning. this study shows that most of the commonly used - cm cvc kits contain guidewires that are too short for cvc placement in the right subclavian vein. the reliability of lung b-lines to assess fluid status in patients with long period of supine introduction: ultrasound-guided cannulation is usually done using either longitudinal or transverse approach. the oblique approach utilizes advantages of both these approaches allowing visualization of the entire course of needle including tip and lateral discrimination of artery from vein [ ] . the reported incidence of the complete overlap of femoral vein by the femoral artery is - percent [ , ] . we describe the use of the oblique approach for successful cannulation of such a femoral vein which is not possible by usual approaches (figure ). endothelial cells play a pivotal role in the atherogenic process. endothelial cell dysfunction (ed) is the main risk factor for cardiovascular diseases such as hypertension, coronary heart disease (chd) and peripheral occlusive disease (pod). these diseases significantly increase the risk for perioperative complications. therefore, identifying patients with ed is important and should influence our prospective perioperative strategy. however, sensitive tools to diagnose ed are still missing and do not belong to our standard of care. aim of this study was the validation of a new non-invasive method to detect ed and a correlation with a set of established an new endothelial biomarkers. the cohort includes preoperative patients without anamnestic relevant cardiovascular disease and patients with known peripheral occlusive disease (pod). we used non-invasive endopat® technology from itamar-medical to measure ed by changes in vascular tone before and after occlusion of the brachial artery and calculate a reactive hyperemia index (rhi). in addition, we measured established markers and alternative biomarkers potentially indicate vascular diseases such as substrates and products from the no-metabolism l-arginin, asymmetric/symmetric dimethylarginine (adma/sdma), von-willebrand factor (vwf) and sphingosine- -phosphate (s p). rhi was able to identify patients with pod. rhi was significant lower in patients with clinical signs and symptoms of pod (p< . ). among other markers adma was significant higher in pod patients compared to controls and correlates with rhi. the pad technology is a helpful non-invasive functional test to measure ed and seems able in identify patients with vascular disease. in future, a combination of anamnesis, new diagnostic tools and biomarkers may further increase our sensitivity in identifying risk-patients. single-lumen fr and triple-lumen fr peripherally inserted central catheters (piccs) for cardiac output assessment by transpulmonary thermodilution s d´arrigo achieving effective critical care in low-and middle-income countries is a global health goal [ ] , which includes the provision of effective point of care ultrasound [ ] . we sought to establish zambia's first focused critical care echocardiography training programme in a bedded icu at university teaching hospital, lusaka. the programme was accredited by the uk intensive care society fice programme, with teaching adapted for local disease patterns such as tuberculous pericardial effusions. parasternal, apical and subcostal windows were used to assess ventricular dysfunction, hypovolaemia, pleural effusion, alveolar interstitial syndrome and pneumothorax. zambian doctors working with critically ill patients received an intensive one-day course, followed by mentored scanning at the bedside. teaching was delivered by visiting fellows from the uk who are accredited in echocardiography and experienced ultrasound educators. patients with abnormal mean ci or hr suffer from increased hospital mortality. abnormality of mean svi was not associated with mortality. these data support accurate measurement of ci as a hemodynamic target and the normal range defined for ci. since ci also carries the hr information, ci seems to be the more important target than svi. our data cannot necessarily be interpolated to less invasive and less precise measurements of ci. an evaluative study of the novelty device with the function of auto-aspirating and pressure indicator for safety central venous catheterization ly lin, wf luo, cy tsao national taiwan university hospital, taipei, taiwan critical care , (suppl ):p previous studies have shown that . % of cvc attempts resulted in arterial punctures that were not recognized by blood color. to overcome the problem, our team has developed a concept of pressure detecting syringe that can indicate the artery puncture [ ] . based on previous research, different springs, the actuator of the design, have been evaluated to optimize the proposed device and reduce the risk of cvc procedure. tested devices -the inner-spring is set between the pressure indicator and plunger (fig. a ). three springs are tested. test condition -blood samples were simulated by glucose solution with absolute viscosities of and mpa-s. different blood pressures were applied to simulate the artery and vein (fig. b) . the response time (rt) is defined as the time required to show the indicating signal (is) which is the movement of the piston from the position in fig. b : a - to a - . the rt is strongly influenced by spring (fig. b) but every design can show the is when pressure is higher than mmhg, the assumed minimum artery pressure. the rt of s , the strongest spring design, is about s in the mmhg-pressure and high viscosity condition. during our tests we found the user can realize the is before the position be fully changed from fig. ib : a - to a - . thus, we believe the s rt, the worst case, is still acceptable. we also found the weak spring force may lead to difficulty to empty the syringe because the spring must to overcome the blood pressure and the friction between the piston and barrel. as a result, it was difficult for s to absolutely empty the syringe even if the blood pressure is only mmhg. the spring will be compressed as fig. b : a - and fail to push the piston when pushing the plunger forwardly, which is not acceptable in clinical use. the results indicate the feasibility of using the device to facilitate cvc and we believe the s or s are more suitable for the future application. introduction: models using standard statistical features of hemodynamic vital sign waveforms (vs) enable rapid detection of covert hemorrhage at a predetermined bleed rate [ ] . by featurizing interactions between vs we can train powerful hemorrhage detectors robust to unknown bleed rates. waveforms (arterial, central venous, pulmonary arterial pressures; peripheral and mixed venous oxygen saturation; photoplethysmograph; ecg) of healthy pigs were monitored min prior and during a controlled hemorrhage at ml/min (n= ) and ml/min (n= ). two sets of vs features were extracted: statistical features [ ] and maximal pairwise cross correlations between pairs of vs within a s lag over various time window sizes ( s, s, s, s); and normalized with pre-bleed data of each given animal. for each feature set, a tree-based (ert) model [ ] was trained and tested in a one-animal-out setting to mitigate overfitting on the ml/min cohort, and another trained on the ml/min and tested on the ml/min cohort. we evaluated models with activity monitoring operating characteristics curves [ ] that measure false alert rate as a function of time to detect bleeding. models using cross-correlations show no significant deterioration of performance when applied to detect bleeding at different rates than trained for, while standard models require s longer on average to detect hemorrhage at % false alert rate in the previously unknown setting ( figure ). correlations between vs data encode physiologic responses to hemorrhage in a way independent of the actual bleed rates. this enables training effective hemorrhage detectors using only limited experimental data, and using them in practice to detect bleeding that occurs at rates other than used in training. we validated a dataset of data lines containing hemodynamic variables and treatment options. we selected nine hemodynamic variables as inputs. furthermore, data were collected regarding underlying conditions: heart failure, septic shock, renal failure or respiratory failure or a combination. we applied datastories regression on the dataset (turnhout, belgium, www.datastories.com). six different interventions were analyzed as kpi: administration or removal of fluids, increasing or decreasing inotropes and increasing or decreasing vasopressors. finally, we elaborated and challenged predictive models to generate a decision algorithm to predict each kpi. we first looked at how each hemodynamic parameter impacts the prediction of each kpi individually and performed a standard correlation analysis as well as a more involved analysis of the mutual information content between each kpi and all other hemodynamic parameters individually. confusion matrix and variable importance was obtained for each kpi. the baseline hemodynamic parameters were: gedvi ± ml/m , evwli . ± . ml/kg pbw, svv . ± %, mbp . ± . mmhg, hr . ± . bpm, ci . ± . l/min.m . the results of the regression analysis identified the different variables of importance for each of the different interventions ( fig a) . based on these results the hemodynamic variables (hr, mbp, gedvi, elwi, ci, svv) were used to develop the final hemoguide prediction model ( fig b) . the hemoguide app can be used to advise physicians with respect to basic therapeutic decisions at the bedside or as an educational tool for students. with the collection of new data, the accuracy of the system may grow over time. the next step of the project is to develop a more-sophisticated suite: the icu cockpit. feedback function contributes to accurate measurement of capillary refill time r kawaguchi , ta nakada , m shinozaki , t nakaguchi , h haneishi , s oda chiba university, department of emergency and critical care medicine, chiba, japan; chiba university, chiba, japan critical care , (suppl ):p capillary refill time (crt) is well known as an indicator of peripheral perfusion. however, it has been reported to have an intra-observer variance, partly because of manual compression and naked-eye measurement of the nailbed color change. we hypothesized that a we developed a novel portable crt measurement device with an oled display that feedbacks weather the strength of the nailbed compression is enough and counts the time. we settled the target strength and time as n and seconds according to the study we reported before [ ] . examiners measured crt with and without the feedback function. the pressing strength and time during the measurement were evaluated. there was a significant difference among the pressing strength and time between the crt measurement using the device with and without the feedback function (strength: p< . ; time: p< . ). furthermore, intra-examiner variance was significantly reduced with the feedback function (strength: p< . ; time: p< . ). in all measurements without the feedback function, % was outside the optimal strength while the measurements with the feedback function % achieved the targeted range. without the feedback function, % could not reach the optimal time, while % with the feedback function did. in total, % of the measurements could not achieve the optimal pressing strength and time. the feedback function for crt measurements, guiding examiners to an optimal pressing strength and time, fulfilled the required measurement conditions and reduced intra-examiner variance. our novel portable device would assist an accurate crt measurement regardless of personal work experience. introduction: the aim of the study was to detect the difference of conjunctival microcirculation between septic patients and healthy subjects and evaluate the course of conjunctival microcirculatory changes in survivors and non-survivors over a hours period of time. this single-centre prospective observational study was performed in mixed icu in a tertiary teaching hospital. we included patients with sepsis or septic shock within the first hours after icu admission. conjunctival imaging using idf videomicroscope as well as systemic hemodynamic measurements were performed at three time points: at baseline, hours and hours later. baseline conjunctival microcirculatory parameters were compared with healthy control. a total of patients were included in the final assessment and analysis. median apache ii and sofa scores were ( - ) and ( - ) respectively. ( %) were in septic shock, ( %) required mechanical ventilation. patients were discharged alive from the intensive care unit. we found significant reductions in all microcirculatory parameters in the conjunctiva when comparing septic and healthy subjects. we found a significant lower proportion of perfused vessels and microvascular flow index (mfi) of small vessels during all three time points in non-survivors compared with survivors. in nonsurvivors we observed no significant changes in conjunctival microcirculatory parameters over time. however, survivors had significantly improved mfi of small vessels at second and third time points compared to first time point. microcirculatory perfusion in conjunctiva was altered in septic patients. over hours evaluation survivors in comparison with nonsurvivors had better microcirculatory flow with incremental improvement of microvascular flow index. healthy pigs were centrally cannulated for veno-arterial ecmo and precision flow probes were placed on the pulmonary artery main trunk for reference. ml boluses of iced . % saline chloride solution were injected into the ecmo circuit and right atrium at different ecmo flow settings ( , , , l/min). rapid response thermistors of standard pa-catheters in the ecmo circuit and pulmonary artery recorded the temperature change. after calibration of the catheter constants for different injection volumes in the ecmo circuit, the distribution of injection volumes passing each circuit was assessed and enabled calculation of pulmonary blood flow. analysis of the exponential decay of the signals allowed assessment of right ventricular function. calculated blood flow correlated well with true blood flow (r = . , p < . , figure panel a, individual measurements organ congestion is susceptible to be a mediator of adverse outcomes in critically ill patients. point-of-care ultrasound (pocus) is widely available and could enable clinicians to detect signs of venous congestion at the bedside. the aim of this study was to develop prototypes of congestion scores and to determine their respective ability to predict acute kidney injury (aki) after cardiac surgery. this is a post-hoc analysis of a prospective study in patients for which repeated daily measurements of hepatic, portal, intra-renal vein doppler and inferior vena cava (ivc) ultrasound were performed before surgery and during the first hours after cardiac surgery [ ] . five prototypes of venous excess ultrasound (vexus) scores combining multiple ultrasound markers were developed (figure ). the association between each score and aki was assessed using timedependant cox models as well as conventional performance measures of diagnostic testing. a total of ultrasound assessments were analyzed. we found that defining severe congestion as the presence of severe flow abnormalities in multiple doppler patterns with a dilated ivc (> cm), corresponding to grade of the vexus c score, showed the strongest association with the development of subsequent aki compared with other combinations of ultrasonographic features (hr: . there is an increasing awareness on the consequences of fluid administration in patients leading to the development of methods that evaluate the effects of fluids loading on the cardiocirculatory system. however, most of methods used in the clinical practice investigate the effects of fluids on the cardiac function, instead of investigating those on the determinants of venous return. besides volume of fluids, the determinants of fluid loading are the blood volume distribution and the availability of vascular bed. in this study we aimed to test non-invasively the effects of fluids administration on the venular compartment in the skeletal muscle. in addition to the mean systemic filling pressure (msfp), we calculated changes in the stressed and unstressed volumes (vs, vu) and the venular bed availability. we enrolled critically ill patients in our intensive care unit. we assessed volumes and pressures by the near infra-red spectroscopy on the forearm using graded venous occlusions in steps of mmhg from to mmhg. the msfp, vu and vs were measured as previously reported (microcirculation ; : - ). the vascular bed availability was measured by changes in the volume recruited from the occlusion maneuvers. all the measures were done at baseline and after a fluid load ranging from to ml. values were expressed as median and interquartile range. wilcoxon test was used to compare data and a p< . was considered as significant. introduction: hypotension is a common side effect of general anesthesia (ga) and is associated with organ hypoperfusion and poor perioperative outcome [ ] . post-induction hypotension (pih) is caused by the depressant cardiovascular effect of anesthetic drugs and could be amplified by hypovolemia. the aim of this study was to assess the ability of two echocardiographic fluid responsiveness markers to predict pih: the inferior vena cava collapsibility index (ivc-ci) and the velocity time integral change (Δvti) after passive leg raising. sixty patients > years of age and scheduled for elective surgery were included. ivc-ci and Δvti were measured before ga induction. anesthesia protocol, fluid infusion and vasopressor administration were standardized in all patients. pih was defined as a mean arterial pressure (map) < mmhg or a relative decline from pre-induction value of at least % within minutes of ga induction. receiver operating characteristic (roc) curve analysis was used. the optimal cutoff was selected to maximize the youden index (sensitivity + specificity − ). the measurement of ivc-ci and/or Δvti were unsuccessful in seven patients ( . %). pih occurred in patients (incidence %). the areas under the roc curves ( figure ) preload responsiveness might be detected by the changes of cardiac index (Δcimini) induced by a "mini-fluid challenge" (mini-fc) of ml or even by the changes (Δcimicro) in response to a "micro-fluid challenge" (micro-fc) of ml. however, the smaller the fluid challenge, the larger the "grey zone" of diagnostic uncertainty. we tested whether ( ) micro-and mini-fc monitored by calibrated pulse contour analysis detect preload responsiveness and ( ) adding ml when the result of a micro-fc is within the grey zone improves diagnostic accuracy. in patients with circulatory failure, we infused ml saline over s followed by ml over s. we measured Δcimicro and Δcimini by the pulse contour analysis (picco ). preload responsiveness was defined by an increase in ci (Δciplr) during a passive leg raising test ≥ %. diagnostic uncertainty was described by calculating the grey zone after bootstrapping. Δcimicro were larger in responders than in non-responders ( . for the micro-fc, the area under the receiver operating characteristic curve was . ± . (threshold %), while it was . ± . for the mini-fc (threshold %). for the micro-fc, the grey zone ranged from . % to . % and included ( %) patients. for the mini-fc, it ranged from . % to . % and included ( )% patients, among which were already in the grey zone of the micro-fc. when evaluated by pulse contour analysis, micro-and mini-fc reliably detect preload responsiveness but with a large diagnostic uncertainty. it seems that adding ml more fluid to a micro-fc when its result is within the grey zone does not improve the diagnostic accuracy. the study is ongoing. the starling-sv bioreactance device (cheetah medical) reliably detects passive leg raising (plr)-induced changes in cardiac index (Δci). we tested whether it can also track the small and short-time Δci induced by the end-expiratory occlusion (eexpo) test, and whether shortening the time over which it averages cardiac output ( s in the commercial version) improves the detection. in mechanically ventilated patients, during a -sec eexpo, we measured Δci (in absolute value and in percentage) through calibrated pulse contour analysis (ci pulse , picco device) and starling-sv. for the latter, we considered both ci starling- provided by the commercial version and ci starling- obtained by averaging the raw data over s. we calculated the correlation between Δci pulse and both Δci starling- and Δci starling- , and the area under the receiver operating characteristic curve (auroc) to detect preload responsiveness, defined by a plr test. when considering absolute values, the correlation coefficient r between Δci pulse and Δci starling- was . (p= . ), which was lower than the one between Δci pulse and Δci starling- (rr comparison). when considering percentage changes, no correlation was observed between Δci pulse and Δci starling- . conversely, the correlation coefficient between Δci pulse and Δci starling- was . (p= . ), but it was lower than the one obtained for absolute values (p= . for r comparison). eexpo-induced Δci starling- , both in absolute values and in percentage, detected preload responsiveness with aurocs of . (sensitivity %, specificity %) and . (sensitivity %, specificity %), respectively. shortening the averaging time of the bioreactance signal increases the reliability of the starling-sv device to detect eexpo-induced Δci. moreover, the accuracy of the method is increased when absolute rather than percentage changes of ci are considered. fluids are among the most prescribed drug in intensive care, particularly among patient with circulatory failure. yet, very little is known about their pharmacodynamic properties and this topic has been left largely unexplored. there is a lack of strong scientific evidence in current guidelines for fluid administration in shock. several factors may impact the hemodynamic efficacy of fluids among which the infusion rate. the aim of this study was to study the influence of fluids administration rate on their pharmacodynamics in particular by studying mean systemic pressure (p ms ). we conducted a prospective observational study in patients with circulatory failure to compare two volume expansion strategies. when a patient required a fluid bolus, ml of normal saline were administered and several hemodynamic parameters were recorded continuously: cardiac output (co), arterial pressure (ap), mean systemic pressure (p ms ). infusion rate was let to the discretion of the attending physician and a "slow" and a "fast" group were determined based on the median of the infusion time. fluids effect was measured by the area under the curve (auc), maximal effect (e max ) and time to maximal effect (t max ) for each hemodynamic variable. results: p ms auc was higher in the "fast" group compared to the "slow" group (p= . ). we observed a shorter t max and a higher e max for p ms in the "fast" group compared to the "slow" group (p= . and . respectively). regarding co, t max was also shorter in the "fast" group (p= . ). auc and e max were similar between the two groups. fluid effect dissipated within minutes following the end of fluid infusion for every patient in both groups. the decreasing slope from maximal effect was comparable in the groups, for p ms and co alike. the effect of a ml fluid bolus in septic shock patients vanished within one hour. a faster infusion rate increased maximal effect and shortened the delay to reach it. study is ongoing. fluid management in the control arm of sepsis trials aa anparasan, ac gordon, mk komorowski imperial college london, department of surgery and cancer, london, united kingdom critical care , (suppl ):p in the past, high-volume intravenous fluid resuscitation in severe sepsis and septic shock was common. more recently, concerns over the harmful effects of this practice have led some clinicians to adopt less liberal fluid strategies. we sought to analyse temporal trends in fluid administration in the control arms of recent adult sepsis trials and assess any correlation with patient severity and mortality. a literature search was conducted to identify relevant randomized controlled trials that reported fluid administration published post . we recorded outcomes: total amount of iv fluid administered in the control arms of these trials between hospital admission and hour and hour following trial enrolment, mortality rates at the latest reported time point and apache-ii score at admission. we computed the pearson correlation coefficient and linear regression between study dates and the outcomes. we identified relevant trials [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , which recruited a total of , patients in their control arms, from to . the temporal analysis revealed no obvious trend in the in the total volume of iv fluid given by hour following trial enrolment (correlation p= . ) ( figure ). however, the total volume of fluid given by hour decreased significantly over the period of interest (r=- . , p= . ). in parallel, we observed a decrease in mortality (r=- . , p= . ) but there was no evidence of decrease in illness severity over time (p= . ). we found that in published rcts over the last two decades, the amount of intravenous fluid given to patients with sepsis in the initial hours did not appear to change, however less intravenous fluid was given over the first three days. upcoming large rcts will test the safety and efficacy of restrictive fluid administration approaches in sepsis. clinical practice guidelines recommend prompt intravenous (iv) fluid resuscitation for pediatric sepsis, including an initial fluid bolus of ml/kg [ ] . however, recent evidence is conflicting as to the effectiveness, volume, and consequences of aggressive fluid resuscitation in septic children. therefore, we sought to determine the epidemiology of early iv fluid resuscitation in an integrated health system, specifically at community hospital emergency departments (ed). we studied a retrospective cohort of pediatric patients (ages > month to < years) with sepsis identified in electronic health record data at community eds in southwestern pennsylvania from to . sepsis was defined as ) suspected infection (combination of fluid culture collection and administration of antibiotics and ) organ dysfunction (pediatric sofa score ≥ ) within hours of suspected infection. fluid bolus therapy was defined as electronic documentation of administration of . % normal saline iv bolus within hour of the time of sepsis onset. results: among , patients with pediatric sepsis, ( %) received iv fluid bolus therapy within hour of time of sepsis onset. the volume of fluid administered ranged from ml/kg to ml/kg (figure , panel a), corresponding to a median volume of ml/kg (iqr - ml/kg). patients who received ≥ ml/kg of fluids (n = , %) were younger (mean age years, sd vs. years, sd ; p< . ), more often had blood cultures collected during evaluation ( % vs. %, p= . ), and were more often transferred to another facility ( % vs. %, p< . ) when compared to patients who received < ml/kg of fluids (n = , %). mean fluid bolus volume within hour of time of sepsis onset by hospital ranged from ml/kg to ml/kg (figure , panel b) . in a cohort of community emergency departments, % of septic children received intravenous fluid boluses within one hour, and of those, only one half received volumes concordant with guidelines. (figure ). a wide range of fluid balance exists in septic shock patients cared for in icu. trends of serum albumin in septic and non-septic critically ill introduction: the link between hypoalbuminaemia and poor outcomes in critical care is well established [ ] . limited data are available on serum albumin trends during critical illness [ ] . in this study we assessed trends in serum albumin for up to days in both septic and non-septic critically ill patients. we retrospectively examined the records of adult patients admitted to critical care at the royal liverpool university hospital between and . we then excluded patients who did not have albumin data available for the first days, leaving us with patients. patients ( . %) had sepsis, and of these patients had died by day . of the non-septic patients ( . %), patients had died by day . albumin levels were collected for days from admission to critical care, in addition to other demographic and biochemical data. statistical analysis was performed using repeated measures analysis. septic patients had lower serum albumin than non-septic patients throughout the day period (p< . ). we observed a decrease in albumin by day in all groups, with levels increasing over the subsequent days. there was no difference in daily serum albumin between non-septic patients who survived or died. this is the first study, to our knowledge, to compare albumin trends in septic and non-septic critically ill patients over days. further research is needed to elucidate the optimal recipients and timing of albumin therapy. introduction: burn injury is characterized by marked inflammation, capillary leakage, and profound hemodynamic alterations. early albumin resuscitation is avoided fearing a paradoxical fluid escape into the interstitium. on the other hand, administration of crystalloids in massive amounts causes tissue edema and fluid extravasation, which deteriorates tissue perfusion by increasing oxygen diffusion distance. albumin administration could reduce the amount required to maintain hemodynamic stability in this population. we investigated whether albumin improves tissue perfusion and microcirculation by reducing tissue edema. this is an observational study conducted in the burn unit of maasstad hospital, rotterdam. patients with burns higher than % of total body surface area (tbsa) were included in the study. sublingual microcirculation was measured at admission (t ), (t ), and (t ) hours after burn injury. total vessel density (tvd) and functional capillary density (fcd) were analyzed. fluid management was calculated according to the modified parkland formula. albumin ( %) infusion was started hours after the burn insult. a total of nine patients were recruited between january and december . patients were included in the study after . ± . hours of the insult with a mean tbsa of ± %. the amount of crystalloid infusion was ± ml and ± ml at t and t ,respectively. within the first h (t ) ± ml albumin was given. tvd decreased from . ± . at t to ± . at t (p< . ) (figure ) introduction: spontaneous bacterial peritonitis (sbp) accounts for ≥ % of the bacterial infections that occur in patients with cirrhosis, and sbp has a high mortality rate ( % to %). albumin infusion has been shown to improve the outcome of sbp. the aim of this study is to examine the impact of albumin infusion on hospital length of stay (los) for cirrhotic patients with sbp. we utilized a nationwide electronic health record data set (cerner health facts®) to extract real-world data on adult patients (≥ years old) with cirrhosis and sbp who received antibiotics and admitted between january , , and april , . international classification of diseases (icd- / ) codes were used to identify cirrhosis and sbp. we used laboratory data for calculation of the model for endstage liver disease sodium (meld-na) score and vital signs data for calculation of the quick sepsis related organ failure assessment (qsofa) score at baseline for each encounter. a generalized linear model was used to assess the relationship between albumin infusion and hospital los. results: there were , encounters that identified patients with sbp and cirrhosis, of which , survived hospitalization. albumin was infused within hours of admission ('early albumin') in % (n= ), after hours in % ('late albumin', n= ), and not administered in % ('no albumin', n= ). meld-na was higher at presentation in early albumin cases versus late-or no-albumin cases (mean . and . ). unadjusted los was lower in patients receiving early albumin ( . days versus . days). risk-adjusted analysis demonstrated that early albumin led to a . % reduction in los ( % ci . %- . %, p = < . ). in these real-world data, albumin infusion within hours of admission in patients with cirrhosis and sbp was associated with a shorter hospital stay despite more severe illness. early albumin may not only improve clinical outcomes but may also reduce the costs of hospitalization in cirrhotic patients with sbp. early albumin use in patients with septic shock is associated with a shorter hospital stay: real-world evidence in the united states introduction: septic shock is among the most common critical care illnesses and incidence is rising, with mortality in excess of %. septic shock predisposes patients to multiple organ failure. while albumin is effective in management of circulatory dysfunction in septic shock, its utilization in this population is understudied in the us. we evaluated the impact of albumin utilization on hospital length of stay (los) among septic shock patients. we used a nationwide electronic health record data set (cerner health facts®) to extract real-world data on adult patients (≥ years old) with severe sepsis or septic shock, admitted between january , , and april , , identified by international classification of disease (icd- / ) codes, and receipt of antibiotics and vasopressors. we calculated the charlson comorbidity index (cci) and the acute physiology score (aps) at baseline. a generalized linear model was used to examine the association between albumin and hospital los, especially accounting for the timing of albumin infusion. we identified , unique visits for septic shock patients that survived to discharge. albumin was infused within hours of admission ('early albumin') in %, after hours ('late albumin') in %, and not administered in %. both cci and aps were higher, at presentation, in early albumin cases than late-or no-albumin cases (mean: . and . , and . and . , respectively). unadjusted los was slightly lower in patients receiving early albumin ( . days versus . days). a risk-adjusted analysis demonstrated that early albumin was associated with . % shorter los ( % ci . %- . %, p = . ). albumin infusion within hours of admission was associated with a shorter length of hospital stay. early albumin infusion may lead to better outcomes and reduced costs in patients with septic shock. further research is being conducted to assess other potential benefits of early albumin administration in this patient population. every new septic event follows by hemodynamic instability may lead sequentially to decreased organ perfusion, multiple organ failure. acute renal failure is recognized clinical feature during sepsis (up to - % in all cases). furthermore, urine output close monitoring is a cornerstone diagnostic clinical tool in each septic critically ill patient. in present study, we analyzed the dynamic minute-to-minute changes in the urine flow rate (ufr) and also the changes in its minute-to-minute variability (ufrv) during new septic event in critically ill patients. demographic and clinical data were extracted from the of critically ill patients who were admitted to the icu and developed new septic event (followed by fever and leukocytosis) and analyzed. a foley catheter was inserted into the urinary bladder of each study patient. the catheter was then connected to electronic urinometer, a collecting and measurement system which employs an optical drop detector to measure urine flow. the urine flow rate variability (ufrv) is defined and calculated as the change in ufr from minute to minute. results: ufr and ufrv both decreased significantly immediate after new septic episode until beginning fluid resuscitation (ppvalues < . ) (figure ) . statistical analysis by the pearson method demonstrated a strong direct correlation between the decrease in ufr, ufrv and the decrease in the map (r= . , p= . ; r= . , p= . ) ( figure ), and heart rate (r= . ,p=< . ) since systemic pressure starts to drop. ufrv and ufr demonstrated good clinical response to fluid administration despite the fact that systemic blood pressure did not improve (figure ) . we consider that dynamic changes in ufrv and ufr could potentially serve as a more sensitive signals ofclinicaldeterioration during the new septic event in critically ill patients.we also suggest that those parameters mightbeable to identify the optimal end-point of fluid resuscitative measures in septic critically ill patients. diminished urinary output (uo) is largely used as marker of acute kidney injury (aki) in critically ill patients. we aimed to explore the role of urinary output on incidence and mortality of aki developed during icu admission. the study population consists of all patients admitted between and to one of the dutch icus included in the nice database with an icu length of stay of at least hours, having daily measurement of creatinine and uo. only patients without renal replacement therapy that have a serum creatinine lower than . mg/dl ( . μmol/l) or a uo above . ml/kg/h on the day of the index icu admission were considered at risk for aki. patients were followed during their icu stay and classified according to the highest kdigo criteria reached based on creatinine alone (model ) and creatinine plus uo (model ) using icu admission serum creatinine as baseline. in both models, patients were classified as: no aki, renal impairment at the first day of icu admission, aki stage , aki stage , and aki stage . we identified , patients ( % male, mean age years, median icu-los days). of those, . % of patients had renal impairment at the first day of icu admission. among the remaining patients, . % in model and . % in model were classified as having no aki, . % and . % as aki stage , . % and . % as aki stage , and . % and . % as aki stage , respectively. survival at -day markedly differed according to the aki classification model used (figure) . similarly, adjusted hrs for -day mortality differed among patients with and without aki compared to patients with renal impairment at the first day of icu admission ( figure ) . among patients admitted to the icu % had renal impairment at the first day of icu admission. our findings suggested that uo plays an important role both on aki incidence and mortality and should be carefully interpret in the clinical setting especially in aki stage classification. introduction: acute kidney injury (aki) mostly attributed to renal tubular damage, has a high morbidity and mortality outcome [ ] , so a sensitive tool to assess the degree of tubular affection is needed for early detection and management of this condition. we investigated the ability of furosemide stress test (fst) (one-time bolus dose of mg/kg or . mg/kg if on prior furosemide-intake) to predict progression to akin stage-iii in critically ill subjects with early aki. we studied subjects; consecutive patients in group i receiving fst and consecutive patients in group ii receiving standard medical management for aki; patients ( . %) and patients ( %) met the primary endpoint of progression to akin-iii in groups i and ii respectively. patients with progressive aki had significantly lower urine output following fst in the first hours (p< . ). the area under the roc curves for the total urine output over the first hours following fst to predict progression to akin-iii was . (p = . ). the ideal-cutoff for predicting aki progression during the first fig. (abstract p ) . thirty-day survival according to aki classification model and model . hazard ratios (hrs) for -day mortality adjusted by sex, age, type of admission, apache iv score, sofa score at day of admission (excluded renal sofa score) for patients with aki classified with model and model fig. (abstract p ) . clinical correlation between urine flow rate variability (ufrv) and ufr and mean arterial blood pressure over new septic event (black arrows) and and after initial fluid resuscitation (red arrows). note: the ufrv and ufr decreased progressively in parallel with the falling mean arterial blood pressure and, than, rose again after the administration of fluids hours was a urine volume of less than milliliters with a sensitivity of . % and specificity . % group receiving fst. on the other hand, statistically significant hypotension, hypo-(kalemia, phosphatemia and magnesemia) occurred in group i. the fst in patients with early aki could predict liability for progression of aki, however it should be performed under adequate monitoring. introduction: ischemia-reperfusion (ir) causes renal dysfunction and damage. ir induces renal tubular injury triggered by hypoxia and hyperoxia, mediated by oxidative stress and inflammation. furosemide inhibits na + -k + - clcotransporter in the thick ascending limb of the renal medulla to decrease na + reabsorption, reducing oxygen consumption. we investigated if furosemide could improve renal oxygenation, function and damage by reducing o consumption and oxidative stress after ir. methods: wistar albino rats were divided into groups, with in each group; sham-operated control (c), control + furosemide (c+f), ir and ir+f. after anaesthesia (bl), min supra-aortic occlusion was applied to ir and ir+f groups followed by min (t ) and hours of reperfusion (t ). furosemide μg/kg/h infusion was simultaneously administered to c+f and ir+f after ischemia. systemic hemodynamic, renal blood flow (rbf), renal vascular resistance (rvr), renal oxygen delivery (do ren ), renal oxygen consumption (vo ren ), creatinine clearance (ccr), sodium handling, urine output (uo), cortical (cμo ) and medullar (mμo ) microvascular oxygenation were measured. results: rbf was reduced in ir ( . ± ) and ir+f ( . ± ) at t (p< . ) but it was further reduced in ir+f ( . ± ) (p< . ) at t compared to c and c+f. rvr was increased in ir ( ± ) and ir+f ( ± ) at t compared to c. rvr was normalized in ir ( ± ) but not in ir+f ( ± ) at t compared to c (p< . ). cμo and mμo did not differ between groups after ir insults (figure ). tissue o was reduced at the medulla, but not at the cortex in ir+f group compared to ir. do ren and vo ren were reduced in ir ( ± and ± ml/ min) and ir+f ( ± and ± ) at t (p< . ). pc was higher in ir+f ( . ± . ) compared to ir . ± . (p< . ). vo / tna + was increased in ir+f compared to ir. no change in ccr and uo was observed. furosemide after ir causes further impairment of renal perfusion, energy utilization and renal oxygenation resulting in renal damage. acute renal failure induced by hypoxemia: incidence and correlation study a trifi , h fazzeni , a mehdi , c abdennebi , f daly , y touil , s abdellatif , s ben lakhal la rabta hopital, medical intensive care unit., tunis, tunisia; la rabta hopital, tunis, tunisia critical care , (suppl ):p introduction: acute renal failure (arr) is a common complication in icus and usually caused by hypoperfusion. arf induced by hypoxemia is a concept rarely reported in icu. its incidence and pathogenesis are not well understood. we aimed to study the relationship between hypoxemia and the occurrence of arf. retrospective cohort study including patients with hypoxemia whatever its etiology between january and august . patients with chronic renal failure were excluded. arf was defined and ranked according to the kdigo criteria . arterial blood gas, urea, creatinine and clearance were reordered on the first, third and seventh days of evolution. results: patients were included and groups were obtained: group of hypoxemic patients with arf (arf+, n= ): versus group of hypoxemic patients without arf (arf-, n= ). the incidence of hypoxemie-induced arf was therefore %. clinical characteristics were comparable in both groups with a mean age of ± and a sex ratio of . . the comparative study showed in arf+ group: a lower ph ( . . ], p = . ). the most significant correlation was showed with mdrd clearance at day and p/f ratio at day (rho = . , p = . ). multivariate analysis found that septic shock and non invasive ventilation in hypoxemic patients were the factors related to arf with respectively or= . , % ci= . - . , p= . and or= . , % ci= . - . , p= . . overall mortality was % (n= ) and arf was an independent factor of mortality: or= , and % ci= . - . , p = . . hypoxemia-induced arf is a common complication associated with excess mortality. our study suggests that renal function is correlated with the degree of hypoxemia and that this correlation is rather distinct hours from hypoxemia. in preclinical models of sepsis, we have previously demonstrated that activation of amp activated protein kinase (ampk) using metformin, improves survival and organ function. thus, ampk activation is a potential therapeutic target in sepsis, and we hypothesize that exposure to metformin during sepsis is associated with decreased aki and mortality methods: retrospective analysis of a -hospital cohort of adult icu patients with type diabetes mellitus (t dm) who presented sepsis. we investigated if exposure to metformin during the hospitalization was associated with reduced -day mortality and aki. we used : propensity score matching (psm), propensity score stratification (pss) and propensity score weighting (psw) based on the probability to be exposed to metformin using covariates. for psm an exact match for insulin, amputation, cardiovascular diseases, retinopathy, charlson index, egfr, hba c, and apache iii, were used. sepsis was defined using sepsis criteria, and aki as kdigo stage or . from , patients, we found diabetic adults exposed to metformin during hospitalization and , who were not. metformin exposure during hospitalization is associated with decreased -day mortality and aki in septic adult patients with t dm. these findings suggest that metformin may constitute a potential therapeutic strategy in sepsis, and the potential role of ampk activation as a protective mechanism. however, studies are needed to confirm this association and the specific mechanisms of action. introduction: acute kidney injury (aki) may occur up to % in the intensive care unit (icu). predicting aki recovery may allow for risk stratification of patients, patient and family counseling, and early post-discharge renal care planning. however, predicting aki recovery at an early stage remains a challenge. methods: this is a retrospective study of the epanic multicenter randomized controlled trial database [ ] , which was split into development (n= ) and validation (n= ) cohorts, and patients experiencing aki stage and/or renal replacement therapy (rrt) in the icu were included [ ] . aki recovery was defined as being alive, without any stage of aki, and without need of rrt at hospital discharge. a logistic regression model with backward feature elimination was developed. the model performance was assessed by discrimination, calibration, and net benefit analysis, and internally validated with ten-fold cross validation. only the results in the development cohort are reported. of the patients who developed aki , patients ( . %) recovered from aki. the multivariable model selected age, bilirubin, heart rate, mean arterial blood pressure, surgical diagnostic group on icu admission, mechanical hemodynamic support on icu admission, suspected sepsis on icu admission as aki recovery predictors. the model had a mean area under the receiver operating characteristic curve (auroc) of . (standard deviation (sd) . ), mean calibration slope of . (sd . ), and mean calibration-inthe-large of < . (sd . ) (figure ). at the classification threshold that maximized sensitivity and specificity, mean net benefit with respect to treat-none was . (sd . ) and mean net benefit with respect to treat-all was . (sd . ). by using the routinely collected clinical data, the developed prediction model can fairly identify patients with a higher chance of aki recovery at hospital discharge. introduction: acute kidney injury (aki) is a frequent complication in critically ill patients and is associated with increased morbidity and mortality. sepsis is one of the most common cause of aki. a prospective study was conducted over months (january -june , ).we included patients with septic shock at admission or at any time during hospitalization.the aki staging was based on kdigo criteria.patients were divided into two groups, a group with aki (aki+) and a group without aki (aki-).then we compared the baseline characteristics, laboratory and physiologic data. patients with aki (aki+) were subdivided according to their prognosis. were enrolled patients. the mean (sd) age was . (± ) years.sex ratio was . . fifty-two ( %) patients developed aki.sapsii and sofa score in admission were higher in patients with kidney injury [ vs points (p= . ), . vs points ;(p= . )] respectively.the serum lactate level was significantly higher in (aki +) group patients during the first day of septic shock [ . ± . mmol/l (aki+)vs . ± . mmol/l(aki-);(p= . ) ] and its clearance was lower [( ± . % (aki +)vs ± %(aki-);(p= . )]. a significant difference was observed in c reactive protein level [ ± mg/l (aki +) vs ± mg/l (aki-) ; (p= . )].among (aki+) patients, kadigo iii was observed in . % of cases.nineteen ( . %) patients received hemodialysis.a normal kidney function was recovered in . % of cases.aki+ patients had a higher occurrence in disseminated intravascular coagulation ( vs patients, p= . ),acute respiratory distress syndrome ( vs patients; p= . ) and cardiac dysfunction ( vs patient, p= . ).mortality was higher in aki group ( % vs %; p= . ). the development of septic aki was associated with poor outcomes and prognosis.a better understanding of sepsis induced aki pathway will enable us to develop targeted therapeutic protocols.newer tools,permitting aki early detection, may make these therapies more fruitful. this study aims to show that contrast procedures do not significantly increase the risk of renal injury and should not be deferred. traditionally ciaki is the most important cause of in-hospital renal failure after nephrotoxic drugs and shock. problem is also the non-uniform definition of ciaki proposed by three different initiatives (akin, esur and kdigo). akin, being the most rigorous, defines ciaki as an increase in serum creatinine > . mg/dl or > % of baseline within hours. a retrospective observational single-centre cohort study analyzed patients who underwent a contrast procedure with iomeron . the first group underwent a ct pulmonary angiography (ctpa), and the fig. (abstract p ). internally validated model performance: (top row) roc curve; (middle row) calibration curve; (bottom row) decision curve second a coronary angiography with pci. no patient was previously prepared (raas blockade removal, crystalloid administration etc). we studied demographics, history of ckd and comorbidities and their impact on the ciaki by the akin criteria. a total of patients were divided into two groups (ctpa and pci). ctpa group ( m, f) all had acute pe and the pci group ( m, f) were treated for acs. the mean age was and years respectively. ckd was more prevalent in the pci group ( pt vs. pt) possibly explained by the more advanced atherosclerotic disease. advanced chd (nyha iii/iv) was found in pt (pci) vs. pt (ctpa) while diabetes and shock were equally distributed ( pt and pt) in both groups. the mean amount of contrast was significantly higher in the pci group ( . ml vs. ml). the mean creatinine/egfr measured before and after contrast in the ctpa group was . the goal of this study was to determine whether changing the body mass (bm) with fat-free mass (ffm) in cockcroft-gault (cg) formula could provide a more accurate prediction of aki in obese patients undergoing cardiac surgery. in this retrospective study, we reviewed institutional data of patients who underwent elective cardiac surgery in a tertiary referral university hospital. baseline patient creatinine value was collected and gfr was estimated using the mdrd, ckd-epi and cg formulas. cg formula was further modified by replacing the bm with ffm derived from the bioelectrical impedance analysis. postoperative aki was defined by kdigo creatinine change definitions. accuracy of the egfr values to predict the aki was calculated with roc-auc analysis. all the calculations were performed in different categories of bmi. figure ). the egfr is a poor predictor of aki in obese patients undergoing cardiac surgery. the ffm modified cauckraft-gault formula yield more accuracy in this specific group. retroaki: a ten-year retrospective study of acute kidney injury in intensive and progressive care units introduction: acute kidney injury (aki) is a frequent condition in intensive care units (icu) and progressive care units (pcu), affecting % to % of the patients, depending on the studied population and aki definition. aki has been identified as an independent risk factor of icu mortality and development of chronic kidney desease. the objective of this study was to describe the incidence of each aki stages as defined by kdigo definition (with evaluation of urine output, serum creatinine and initiation of renal replacement therapy (rrt)), in a mixed medical and surgical population of patients hospitalized in icu and pcu over a -year period ( - ). we included all patients who stayed more than hours in icu or pcu of edouard herriot hospital from may to january . data used to classify the patients were the urine output over a sixhour period, serum creatinine and the need for rrt, according to kdigo classification results: , hospital stays were analyzed. median icu/pcu length of stay was days [iqr: . - . ]. among icu patients, % had at least one aki episode graded , or and % had at least one severe episode (stage or ). among pcu patients, % had at least one episode of aki and % a severe episode of aki. patients had an average of . episodes of aki per stay. table represents the incidence of maximal aki stage during one stay. we found that urine output was the more frequent criteria to make diagnosis of aki stage or whereas rrt was more frequent for aki stage . this retrospective study reports a more important aki incidence in our icu/pcu than in previous studies. the difference could be fig. (abstract p ) . when comparing auc in different categories of bmi, the mcg appeared to be the only statistically accurate formula in patients with bmi - . explained by the difficulty to collect urine output from conventional database. serum creatinine and the use of rrt are often the only two criteria used to define and classify aki. these results confirm the high incidence of aki in icu and pcu and the importance to make an early aki screening of patients for whom preventive nephroprotective actions are needed. introduction: icu-patients with acute kidney injury (aki) requiring renal replacement therapy (rrt) are at risk for infections [ , ] . in this study we evaluated the incidence of infection in icu patients with and without less severe aki. finally, impact on outcomes was explored. this is a retrospective study on the pdms (protection data management system) of the adult icus of a university hospital. aki was assessed on kdigo criteria (creatinine (scr) and urine output), during the first -d of icu stay. infection was validated in the pdms by a team of icu specialists. results: during a -year period, a total of subjects were enrolled. aki was diagnosed in . % of patients during icu stay. aki patients were older ( vs. y, p= . ), had higher saps ( vs. , p< . ), and had more urgent icu admission ( % vs. %, p< . ). more aki patients had mechanical ventilation ( % vs. %, p< . ) and vasopressors on d- ( % vs. %, p< . ). aki stage , , and was present in . %, . % and . % of patients. more aki patients had infection ( % vs. %, p< . ) and increasing aki stages were associated with higher infection rates (aki- : %; aki- : %, aki- : %, aki- : %, p< . ) (figure ). we observed - times higher mortality in aki patients with infection, and a stepwise increase of mortality with increasing aki stages. after correction for infection and other confounders we found that all aki stages were associated with in-hospital mortality (ors aki- : . , aki- : . , aki- : . , all p< . ). over half of aki patients experienced an episode of infection and increasing aki severity was associated with higher infection rate. aki patients with infection had marked higher mortality, suggesting that infection was an important driver of outcome. however, after adjustment, aki stages had strong association with hospital mortality. several new biomarkers have been introduced to improve early diagnosis of acute kidney injury (aki). "nephrocheck" (nc; astute medical, usa) is a bedside test calculating "akirisk" (product of urinary concentration of the cell cycle arrest-markers timp- and igfbp ). several studies suggest the usefulness of nc in selected populations. however, the value of early routine measurement of nc is unclear. methods: therefore, we compared the prediction of a combined endpoint (cep: death < days and/or requirement of renal replacement therapy rrt) by nc within h of icu admission (nc ) and h later (nc ) with admission values of serum-creatinine, bun, cystatin c, urinary ngal, apache ii and sofa (roc-analysis). as a secondary endpoint we investigated the additional value of pathological measurements of nc ≥ . critically ill patients showed increased relative uce in the first days of icu admission, which may be attributed to higher protein catabolism. increased relative uce was associated with arc and both had no effect on -day mortality. introduction: this study compared epidemiology, short-and long-term outcomes for patients with community-acquired (ca) and hospital-acquired (ha) acute kidney injury (aki). we retrospectively analyzed all episodes of aki over a period of . years ( - ) on the basis of routinely obtained serum creatinine measurements in , patients whose creatinine had been measured at least twice and who had been in the hospital for at least two days. we used the "kidney disease: improving global outcomes" (kdigo) criteria for aki and analyzed the first hospital admission. a total of were admitted in hospital and fulfilled the inclusion criteria. average observation period per patient was days. the incidence of ca-aki among included hospital admissions was . % compared with an incidence of . % of ha-aki, giving an overall aki incidence of . %. patients with ca-aki were younger than patients with ha-aki ( vs . y) and had significantly less comorbidities, including preexisting cardiac failure, ischemic heart disease, hypertension, diabetes. patients with ca-aki were more likely to have stage aki ( , vs , %, p< . ) and had significantly shorter lengths of hospital stay than patients with ha-aki ( vs d, p< . ). those with ca-aki had better survival than patients with ha-aki (figure ; p< the evidence base for management of fluid removal during renal replacement therapy (rrt) is limited. a recent international survey revealed the extent of practice variation worldwide [ ] . our aim was to summarise the responses from europe-based healthcare professionals who participated in the survey. the international self-administered, cross-sectional, internet-assisted, open survey was disseminated between january and january via website links and emails to members of different critical care societies. results: participants from european countries completed the survey of whom ( %) were intensivists and ( %) worked in university-based hospitals. persistent oliguria / anuria was the most common indication for fluid removal ( % responders). the parameters which guided fluid removal included hemodynamic status ( % responders), cumulative fluid balance since admission ( % responders), and -hour fluid balance ( % responders). % of participants reported using crrt with a median net ultrafiltration rate ml/hr (iqr - ml/hr) for hemodynamically unstable and a rate of ml/hr (iqr, - ml/hr) for hemodynamically stable patients. only % of practitioners checked net fluid balance hourly ( % nurses, % physicians). new hemodynamic instability, defined as new onset or worsening tachycardia, hypotension, or need to start or increase the dose of vasopressors was reported to occur in % fig. (abstract p ). long-term survival patients (iqr . - . ). different strategies to re-gain hemodynamic stability were used. (figure ) main barriers to fluid removal were patient intolerance ( % physicians, % nurses) and interruptions in fluid removal ( % physicians, % nurses). the majority of participants agreed that guidelines and protocols would be beneficial. the practice of fluid removal during rrt is very variable across european countries. nurses and doctors identified a need for evidencebased protocols and clear guidelines. introduction: kidney disease improving global outcomes (kdigo) guidelines suggest the use of anticoagulation in continuous renal replacement therapy (crrt) [ ] . the effectiveness of the anticoagulation is important because replacing the hemofilter and tube interrupts crrt and increases total therapy time. regional citrate anticoagulation (rca) and unfractionated heparin (ufh) are most commonly using methods for crrt anticoagulation [ ] . the aim of this study was to investigate the efficacy, safety and metabolic differences of the patients in icu who underwent crrt and anticoagulation method changed from ufh to rca for different reasons. after ethics committee approval ( - / ) patients who underwent crrt between - at bursa uludag university hospital icu have been investigated and patients who underwent crrt by both rca and ufh included in the study. we divided patients in two groups (rca, ufh), demographic data (sex, age), sofa score, creatinine, urea, mean filter life time (flt) and ultrafiltration flow (uf), platelets, electrolytes (na, k, ca, mg), lactate, nahco and ph of groups at beginning and ending of first rca and ufh hemodialysis collected. we used t-test and bootstraps statistic tests. in agreement with other studies [ , ] , flt and uf was statistically significant lower in ufh group (table ) . there was no statistically significant difference in efficiency (urea and creatinine decrease), ph, lactate, nahco level, platelets count and electrolytes between two groups. to our knowledge, there are no studies comparing these two anticoagulation methods in the same patients. small number of patients and retrospective evaluation are limitations of the study. our results suggest that the implementation of rca method is safe and effective as ufh method with longer flt and uf. regional citrate anticoagulation during crrt in liver failure mj jain, pk kumar g, dg govil, jk kn, sp patel, ms shafi, rh harne, dp pal, sm monanga medanta the medicity, critical care, gurugram, india critical care , (suppl ):p continuous renal replacement therapy (crrt) with regional citrate anti-coagulation (rca) is increasingly being used as a treatment modality in critically ill patients. there is limited experience of use of citrate anticoagulation patients with acute liver failure and acute on chronic liver failure who pose a tough challenge of being at a higher risk for bleeding. an institutional protocol was formulated for use of commercially available citrate solutions and the same was studied to assess filter life and safety of citrate in liver disease. the primary objective was to assess safety of citrate anticoagulation in liver disease. this study was a single centre, prospective, non-randomized, single arm, observational study. all adult patients, with acute liver failure and acute on chronic liver failure requiring crrt were included. blood ionized calcium levels of . to . mmol/l was targeted throughout the therapy and total to ionized calcium ratio of less than . was maintained. rca was stopped if the ratio was more than . for consecutive assessments. incidence of citrate accumulation and toxicity were assessed. average filter life was also assessed. metabolic parameters, electrolytes and strong ion gap were followed till hours after completion on crrt. a total of patients were included in the study. nineteen patients of acute on chronic liver failure and patients of acute liver failure underwent crrt with rca. baseline average serum bilirubin, lactate and inr were . mg/dl, . mmol/l and . respectively. the average filter life was hours minutes. citrate accumulation took place in (n= ) patients and rca had to be stopped for ( n= ) patients due to the same. none of the patients had evidence of citrate toxicity. citrate anticoagulation was well tolerated in patients with acute liver failure in patients with or without pre-existing chronic liver disease on crrt. introduction: the intention of this study is to highlight the levels of citrate load for the general population that increases the risk of citrate complications (insufficient trisodium citrate delivery; net citrate overload and citrate accumulation) [ ] . this was a prospective data collection between february and march in a fourteen bedded critical care unit. eleven consecutive episodes of crrt were collected (a new episode characterized if crrt was discontinued for hours and above). one episode was excluded due to short duration (less than hours). patients undergoing rca-crrt received either a fixed or ml/kg/h effluent dose protocol. median patient age was , male %. average time on crrt was . days ( - ). % of the patients had complications, although % were minor ( figure ). all of the patients with net citrate overload had citrate loads of . mmol/h or above. the main risk factors were found to be shock and liver impairment which occurred in % of cases of which % developed complications. a fixed dose effluent protocol to standardise practice can potentially lead to a higher risk of minor complications. in our experience this is likely due to a lack of appropriate monitoring for rca-crrt complications. despite this, our complication rate of citrate accumulation is in line with that reported in literature. citrate loads in our ml/kg/ hr protocol were . % higher than our ml/kg/hr protocol and strongly related to higher complication rate that worsened in patients with risk factors for poor citrate metabolism. introduction: there is no optimal timing of continuous renal replacement therapy (crrt) in acute kidney injury (aki); however, it is based on volume overload, azotemia, hyperkalemia and severe metabolic acidosis [ ] . an important reason for metabolic acidosis in aki is increased unmeasured anions (ua) [ ] . delta-ph-ua (Δph ua ) detects the degree of metabolic acidosis caused by ua and is calculated by using 'the partitioned ph model' [ ] . in this study, we investigated whether Δph ua was a predictor to start crrt in patients with aki. the study was designed as a multicentric, prospective, observational study in . patients who were ≥ years old and diagnosed with aki [ ] were included. the moment aki was diagnosed, arterial blood gas, albumin, magnesium, inorganic phosphorus, urea, creatinine and Δph ua values were recorded. all patients were divided into two groups as crrt(-) and crrt(+) which consists of patients performed crrt due to traditional criteria. fig. (abstract p ) . incidence of complications introduction: continuous renal replacement therapy (crrt) is labor intensive and requires advanced nursing knowledge and skills. however, % of registered nurses (rn) are less than -year post-registration experiences in our unit. also there is an increasing demand of crrt from crrt days in to crrt days in . the obstacles for crrt in our department, includes variation of regimen, complicated workflow and insufficient training of nurses. a continuous quality improvement project is carried out to standardize the regimen, enhance workflow and provide structured training to nurses in the intensive care unit, to enhance nursing competence. methods: introduction: sepsis and septic shock is a leading cause of mortality in the intensive care unit. we tried to evaluate a novel hemoperfusion cartridge through a retrospective evaluation of patient's data in our centre. we used it as an adjuvant therapy in our patients with sepsis and septic shock due to varied causes. the aim of this study was to evaluate the efficacy of therapeutic hemoperfusion cartridge (hc-foshan biosun medical ® ) in the management of patients with sepsis. we retrospectively analysed data of group (n= sepsis) and group (n= sepsis+hemoperfusison; sepsis treated with hemoperfusion cartridge) admitted between to . group had received hemoperfusion cartridge as adjuvant therapy along with standard of care. demographic data, procalcitonin [ ] and leukocyte levels before and after therapeutic cytokine removal and duration of hc were recorded. while the mean duration of cvvhdf was . hours, the duration of hemoperfusion cartridge (application was . ± . hours). among patients who survived patients were administered hemoperfusion cartridge within hours of icu admission. there was a significant reduction in scores like apache and sofa score post hemoperfusion cartridge therapy procalcitonin and leucocyte levels after therapeutic hemoperfusion cartridge were found significantly lower than the pretreatment values (respectively p= . , p= . ). retrospective analysis showed significant reduction of vasopressors, and improvement in map in group . therapeutic hemoperfusion cartridge with cytokine removal applied with cvvhdf in septic patients have positive contributions to provide survival advantage. removal of activated leukocytes and endotoxin from the blood is a complex therapeutic effect of the device for removing endotoxin. in the main group ( patients with abdominal septic shock) after surgery, the traditional treatment was supplemented with two sessions of endotoxin removal ( hours each with an interval of hours) using "alteco lps adsorber" (sweden). the control group consisted of patients with a similar diagnosis and only traditional treatment. results: % of white blood cells were adsorbed in lps adsorber. among them, granulocytes ( %) were maximally extracted, then cd + monocytes (cd + mo) ( %), hla-dr + mononuclear cells ( %), monocytes ( %). il- , il- , procalcitonin (pct) were not adsorbed. the -day mortality rate in the main group was % and was lower compared to the control group - %. during monitoring, in the main group hours after the first removal of endotoxin, a decrease in the initially increased amount of activated cd + mo by . times, as well as functionally mature defensin + granulocytes (def + gran) by . times was observed. il- , il- , and pct decreased by . ; . ; and . times, respectively. during this period, the control group showed an increase in cd + mo and def + gran, while il- , il- did not change, and pct increased . times. a day after the second removal of endotoxin and then days later, the main group of il- , il- , and pct continued to decline. in the control group, only il- decreased after days, the rest continued to grow. the cellular adsorption of endotoxin-bound cd + mo and mature def + gran is an important part of the mechanism of action of the endotoxin removal device. does the endotoxin adsorption of pmx column saturate in hours? preliminary study c yamashita in the euphrates trial, the polymyxin b-immobilized fiber column (pmx) hemoperfusion (hp) had no significant effect on -day mortality. endotoxin (lps) burden by endotoxin activity assay > . may exceed μg [ ] , so the dose and duration of pmx-hp could be insufficient to lower the lps burden. to confirm this issue, we experimented in a closed-circuit with h continuous lps addition, and pmx can adsorb > μg [ ] . further, lps concentration became constant within h in the single lps spike test for determining pmx-hp duration [ ] . to prove our hypothesis that the single lps spike test reflects the adsorption equilibrium, and not saturation, we added lps intermittently to reaction. methods: lps ( ng/ml) was mixed with ml deactivated fetal calf serum as a reflux solution, as previously described [ ] ; this concentration is much higher than that observed in septic patients. we created a closed circuit that incorporates pmx- r at / th the amount of an adult pmx and performed pmx-hp at ml/min for h. lps was added in two shots (post h: ng, ng/ml; post h: ng, ng/ml). lps was measured using the limulus amebocyte lysate test at , . , , , , and hr. after an initial decrease between and h, lps concentration did not decrease between and h after pmx-hp initiation. post lps pulse addition at h, it increased and then decreased till h. futher, it did not decrease between and h, but it increased and then decreased again after lps pulse addition post h (figure ). lps adsorption rates were . , . , and . % at , , and h, respectively. conclusions: lps adsorption capacity of pmx- r was maintained even after two additional shots of lps, suggesting that the constant lps concentration in the previously reported lps spike test might be indicative of adsorption equilibrium rather than saturation. a coohort study included patients admitted to three intensive care with sepsis / septic shock ( sepsis criteria ) and aki ( akin score). all patients were submitted to cvvhdf with the oxiris filter (baxter, usa) . the main clinical data, il , procalcitonin, endotoxin ( eaa ) and sofa score were evaluated at basal time ( t ) and at the end of the treatment ( t ). all data are expressed as mean ± sd or median and iqr . anova test was used to compare the changes in the time. results: patients were submitted to rrt with the oxiris filter for ± hours . patients had aki stage , patients aki stage and patients had aki stage. at t all groups had an high vasopressor fig. (abstract ) . lps concentration in lps pulse addition test support to maintain map ≥ mmhg. il , procalcitonin eaa and sofa total were also elevated with no difference between the groups. at t creatinine improved better in aki ( p< . vs. t ) and in aki ( p< . vs t ) then in aki group. map increased in aki ( p< . vs t ) and aki ( p < . vs t ) , but not in aki group. il , procalcitonin decreased more in aki ( p < . vs t ) then aki . at t sofa total was higher in aki then aki ( p< . ) and aki ( p< . ). conclusions: aki and aki stage patients submitted to bp with the filter oxiris respond better then aki stage patients . -this transalte in a better clinical course. -crrt with oxiris filter is useful in septic patients with aki, but aki stage septic patients represent an high risk group. a non-interventional, multicenter, non-randomized patient registry for multiple organ dialysis with the advos system multiple organ failure is a challenging problem in the icu. as an advanced dialysis system, the advos procedure can eliminate watersoluble and protein-bound substances, regulate the acid-base balance as well as fluid and temperature. in , a national registry was established to collect data under "real-life" conditions of patients treated with advos without any trial-specific interventions (drks id: drks ). methods: data from / to / from german hospitals (university hospitals in hamburg-eppendorf, mainz, essen, and klinikum weiden) were analyzed. clinical parameters, treatment settings and adverse events were documented. the -and -day mortality rates were compared with extrapolated rates based on the sofa score. results: patients with a median age of years (iqr - ), of whom ( %) were male, were evaluated. patients had a median sofa score of (iqr: - ) before the st advos treatment, which is associated with an expected mortality of %. the number of failing organs was (iqr - ): cardiovascular ( %), lungs ( %), liver ( %), kidneys ( %), coagulation ( %) and cns ( %). treatments with a median duration of (iqr: - ) hours were evaluated. were discontinued, of which ( %) were due to a device error. adverse events were documented, were related to the device (all due to clotting and recovered without sequelae). significant removal of protein-bound (bilirubin: . vs . mg/dl) and water-soluble toxins (bun vs and creatinine . vs . mg/dl). in addition, improvement in acid-base balance was observed: ph ( . vs. . ), bicarbonate ( . vs. . mmol/l) and base excess (- . vs. . mmol/l) ( table ) . -and -day mortality rates were % and %, respectively. in a cohort of patients with multiple organ failure, we observed an improvement in the expected mortality rate, especially if the advos procedure was applied early. adverse events are comparable to other dialysis therapies in intensive care patients. introduction: acute kidney injury (aki) due to ischemia-reperfusion affects onethird of the patients in cardiac surgery. we investigated the potential role of cyclosporine (csa) to prevent postoperative aki and mitigate inflammatory response to extracorporeal circulation (ecc). methods: double-blind, randomized, placebo-controlled single-center study. patients (n= ) scheduled for elective cardiac surgery were randomized to , mg/kg csa or placebo before the surgery. the primary objective was to assess the role of csa to reduce the incidence of postoperative aki. the secondary objective was to study csa induced changes in the inflammatory response to ecc. results: all enrolled patients were analyzed. postoperative aki was more pronounced in the cyclosporine group compared to placebo. or= . ( . - . ), % ci. the cytokine production in response to ecc was not affected by cyclosporine (figure ) . in patients undergoing cardiac surgery, a single preoperative dose of csa does not prevent the postoperative decrease in renal function. csa does not alter cytokine release in response to extracorporeal circulation. elevated post-ecc levels of pro-inflammatory cytokine il- are associated with kidney dysfunction and may be predictive. new generation adsorbent such as oxiris r was introduced as novel technique in renal support for critically ill patients [ ] . septic shock patients require decatecholaminization strategies emphasizing blood purification to remove catecholamine-producing mediators and evacuate overload fluid in interstitials. our -year-old female patient, admitted to icu after surgery with history of ovarium cancer. her septic shock was worsened with ards, hypercoagulable state and aki. vasopressors were set. patient was controlled with mode simv ,ps ,tv ml,peep ,fio %. renal support was implemented by diuretic and cvvh started on the second day. at first,regular adsorbent was used, post-filter mode was set, and periodic fluid removal target was ml/h. but after hours, no significant changes observed. oxiris r added and after hours passed, requirements of vasopressors reduced, tidal volume increased, hemodynamic parameters stabilized, urine production increased. it was continued for days and patient was recovered. our patient had fallen into inadequate cars stage in which not able to counter septic effects on vital organs (figure ). renal would be primary target for filtration and monitoring tool. adsorbent consisted of an and polyethyleneimine was useful to purify blood from endotoxins conjoined with slower filtration. continuous yet cautious process in cvvh evacuate fluid and mediators while maintain steady hemodynamics. biomarkers could not be evaluated due to limited resources, but improving parameters could be signs that showed recovery process had already took place. advanced hemofiltration is a privilege. implementing and enhancing it with new generation adsorbent would increase survivors by extracting unnecessary fluids and eliminating catastrophic endotoxins and mediators. consent to publish: written informed consent for publication was obtained from the patient. analysis of retrospective cohort study data of patients (pt) treated for dka at icu of kaunas clinics during - has been carried out. serum kalemia, glycemia; hypokalemia, hypoglycemia episodes; rate of insulin interruption for hypo-and normoglycemia during ketoacidosis; use of nah co for ketoacidosis, and los in icu were analysed. spss . was used for statistic calculations. traits evaluated as significant at p < . . at the beginning of dka treatment in totally hypokalemia ( . ± . mmol/l) was recorded in / pt ( . %). due to ignoring of blood ph ( . - . ( . ± . ) kalemia was falsely misinterpreted as "normo-" or "hyper-" . - . ( . ± . mmol/l) in / pt ( . %), thus disregarded so complicated by obvious hypokalemia additionally in / pt ( . %). in hypokalemia los in icu was . ± . vs . ± . h, p < . . insulin use has caused hypoglycemia ( . - . ( . ± . mmol/l)) in / pt ( . %), los in icu . ± . vs . ± . h, p < . .insulin use was interrupted in case of normoand hypoglycemia with still persisting ketoacidosis in / pt ( . %), los in icu was found to be . ± . vs . ± . hr, p < . . nah co was given for symptomatic treatment of ketoacidosis during first h of dka in / pt ( . %) with stable hemodynamic: hco - buffer has increased ( . ± . - . ± . mmol/l), p < . , but it didn't control ketoacidosis, and los in icu was . ± . . vs . ± . h, p < . . hypokalemia, hypoglycemia, precocious interruption of insulin use were recorded as complications of dka treatment. all of them have prolonged los in icu. symptomatic treatment of ketoacidosis with nah co had no effect on it, and prolonged los in icu as well. a growing interest exists about co derived parameters in shock management. central venous-arterial pco difference (p cv-a co ) is strictly related to cardiac output; central venous-arterial pco difference to arterial-central venous o content difference ratio, p cv-a co / c a-cv o , has been proposed as anaerobic metabolism when it's > . mmhg/ml [ ] . to evaluate p cv-a co /c a-cv o reliability in detecting anaerobic metabolism, we analyzed it in consecutive patients affected by mala admitted to our icu, considering these patients as a prevalent anaerobic metabolism model. we calculated, by douglas formula, central venous-arterial co content difference to arterial-central venous o content difference ratio, c cv-ca co /c a-ccv o , as a respiratory quotient surrogate. we performed arterial and central venous blood gas analysis simultaneously at admission, we calculated p cv-a co , p cv-a co /c a-cv o and c cv-a co /c a-cv o and we recorded scvo . we verified relationship between p cv-a co /c a-cv o and scvo and arterial ph, arterial lactates, sofa score at admission and c cv-a co /c a-cv o by linear regression analysis. pcv-aco /ca-cvo greatly increases in mala ( . ± . ). pcv-aco / ca-cvo (fig. ) shows significant co-variation with ph (r = . ; p= . ) and sofa score at admission (r = . ; p= . ). pcv-aco / ca-cvo has poor agreement with ccv-aco /ca-cvo (r = . ) and disagrees with it in identifying anaerobic metabolism, in our series, in fact, ccv-aco /ca-cvo is, in patients, < like an aerobic rq value. pcv-aco /ca-cvo shows better agreement with ph, sofa score and lactate level than scvo . in our series, p cv-a co /c a-cv o is good illness and acidosis severity marker, but it seems to be affected by ph value in accord with haldane effect [ ] . p cv-a co /c a-cv o , in our study, doesn't seem to be a reliable anaerobic metabolism marker nor a rq surrogate. it is thought that early administration of basal insulin to patients with diabetic ketoacidosis (dka) may improve outcomes. small studies have shown trends towards decreases in time to closure of anion gap (tcag), rates of rebound hyperglycemia following discontinuation of intravenous (iv) insulin, rates of hypoglycemia, intensive care unit (icu) length of stay (los), and hospital los [ ] [ ] [ ] [ ] . this was a single-center, retrospective chart review of our institution's dka protocol between january and august . patients that received early basal insulin within hours of initiation of iv insulin and before closure of the anion gap (ag) were compared to those that did not receive early basal insulin. the primary outcome was median tcag. secondary efficacy outcomes include: time on iv insulin infusion, time to de-escalation of level of care, hospital los, and re-elevation of ag. secondary safety outcomes included incidences of hyperglycemia, hypoglycemia, and hypokalemia. a total of patients were identified meeting inclusion and exclusion criteria. median tcag was longer in the experimental group ( vs. hours, p < . ). incidence of re-elevation of ag and incidence of hyperglycemia were lower in the experimental group. other outcomes were similar (figure ). early administration of basal insulin to patients with dka resulted in a longer tcag with a lower incidence of re-elevation of ag and hyperglycemia. early administration of basal insulin appears to be safe with respect to hypoglycemia and hypokalemia. glycaemic control continues to be a challenge in critically ill patients. stress induced hyperglycaemia has been associated with increased morbidity and mortality [ ] . conversely, patients receiving intensive glucose control have a higher risk of death [ ] . a quality improvement project was designed to develop a comprehensive insulin protocol that recognized pre-existing diabetes and reduced hypoglycaemia. data was collected prospectively in all adult patients admitted to the rah intensive care unit (icu) between october and august from the national icu audit database and electronic patient records. daily figures were collected for numbers of hypoglycaemic episodes (< mmol/l), "in range" ( - mmol/l) blood sugar measurements and patients with a pre-existing diagnosis of diabetes. data was collected and analysed using microsoft excel. results: patients were identified; patients ( . %) had pre-existing diabetes. a total of blood sugar measurements were reviewed; ( . %) were "in range" and hypoglycaemic episodes ( . %) occurred. there was no significant correlation between number of diabetic patients and measurements within range. of note, there was an increase in number of measurements per patient in the second half of the time period ( vs ). the development of this protocol has improved glycaemic control in our icu. there are considerably fewer episodes of hypoglycaemia and a large proportion of blood sugar measurements are in range. we hope to continue data collection and interrogate the prevalence of pre-existing diabetes further to reduce glycaemic variability. the optimal management of blood glucose levels for critically ill patients remains unclear. hypoglycemia, hyperglycemia and glycemic variability are associated with mortality. the time in targeted blood glucose range (tir) has been suggested to correlate with mortality depending on the status of antecedent glycemic control, but it has not been verified optimal tir and whether there is an optimal disease-specific tir. a retrospective observational study was performed at a single center. in the present study, we enrolled all critically ill patients admitted in intensive care unit from january to october. patients with diabetic ketoacidosis or hyperosmolar hyperglycemic syndrome and patients who had < blood glucose readings were excluded. gathered information included, in part, demographics, comorbidities, severity of illness scores, diagnosis at admission, length of icu stay and hospital discharge status. the primary outcome was -day mortality. we analyzed to find the optimal tir for critically ill patients. several tirs were each tested for correlation with mortality. a total of , patients, . % of whom had diabetes, were studied. tir to mg/dl (or, . ; %ci, . - . ), tir to mg/ dl (or, . ; %ci, . - . ) and tir to mg/dl (or, . ; %ci, . - . ) > % was independently associated with mortality in critically ill patients respectively. the optimal tir did not differ depending on diagnosis at admission. in this retrospective evaluation, tir to mg/dl > % was independently associated with mortality in critically ill patients, especially those with good antecedent glucose control. these findings have implications for the design of future trials of intensive insulin therapy. the prevalence of chronic dysglycemia (diabetes and prediabetes) in patients admitted to swedish intensive care units (icus) is unknown. we aimed to determine the prevalence of such chronic dysglycemia and asses its impact on blood glucose control and patient-centred outcomes in critically ill patients. in this retrospective, observational study, we obtained routine glycated hemoglobin a c (hba c) measured in patients admitted to four tertiary icus in sweden between march and august . based on previous diabetes history and hba c we determined the prevalence of chronic dysglycemia (prediabetes, undiagnosed diabetes and known diabetes). we compared indices of acute glycemic control in the icu and explored the association between chronic dysglycemia and icu-associated infections, mechanical ventilation, renal replacement therapy, vasopressor therapy, and mortality within days. of patients, ( %) had chronic dysglycemia. of these patients, ( %) had prediabetes or undiagnosed diabetes and fig. (abstract p ) . results ( %) had a known diabetes diagnosis. during icu stay, patients with chronic dysglycemia had higher average blood glucose, spent less time in target glucose range, had greater glucose variability, and were more likely to develop hypoglycemia than patients without chronic dysglycemia. chronic dysglycemia was associated with greater need for renal replacement therapy (odds ratio . , % ci . - . ) and increased -day mortality (hazard ratio . , % ci . - . ) after adjustment for simplified acute physiology score . in contrast, chronic dysglycemia was not associated with mechanical ventilation, vasopressor therapy, or icu-associated infections. in four tertiary swedish icus, measurement of hba c showed that / of patients had chronic dysglycemia (prediabetes or diabetes). chronic dysglycemia was associated with marked derangements in glycemic control during icu stay, greater need for renal replacement therapy and with increased mortality at days. case report: modern antidiabetic therapie causes ketoacidosis am heiden, m emmerich krankenhaus bad oeynhausen, institut für anästhesie, bad oeynhausen, germany critical care , (suppl ):p the modern antidiabetic class of sglt -inhibitors, that are known to reduce the risk for cardiac events [ ] , are increasingly used in the last few years. a -year old male patient with diabetes mellitus suffered days after colectomy surgery from abdominal pain and nausea. the patient had an antidiabetic therapy with empaglifozin that was paused until day after surgery (nutrition start on day , weaning on day ). methods: this is a case report of one male patient seen in the icu setting. daily blood values including arterial blood gases, vital parameters and clinical status of the patient were observed and evaluated. the blood gases showed this metabolic acidosis: ph . ; pco . mmhg, bicarbonate mmol/l, be - . mmol/l, lactate . mmol/l, glucose mmol/l. a ketonuria despite normal blood glucose values was noticed, so that the diagnosis of ketoacidosis was clear. after analyzing the possible causes we found out, that empaglifozin in times of catabolism and fasting can cause this severe symptomatic. we terminated the therapie with empaglifozin and under the treatment with insulin the symptoms disappeared within days and the patient could be discharged from the icu on day after surgery. after one episode of ketoacidosis the therapy with sglt -inhibitors should lifelong never be started again. we recommend that intensivists should be aware of the modern sglt -inhibitors because of the shown severe complications and the increased use of this medication. consent to publish: written informed consent for publication was obtained from the patient. while obesity confers an increased risk of death in the general population, numerous studies have reported an association between obesity and improved survival among critically ill patients. this contrary finding has been referred to as the obesity paradox. this retrospective study uses two causal inference approaches to address whether the survival of non-obese critically ill patients would have been improved if they had been obese. the study cohort comprises , adult critically ill patients hospitalized at the intensive care unit of the ghent university hospital between and . obesity is defined as a body mass index of ≥ kg/m . two causal inference approaches are used to estimate the average treatment effect in the untreated (atu): a naive approach that uses traditional regression adjustment for confounding and that assumes missingness completely at random, and a robust approach that uses super learning within the targeted maximum likelihood estimation framework and that uses multivariate imputation of missing values under the assumption of missingness at random. obesity is present in . % of patients. the in-hospital mortality is . % in non-obese patients and . % in obese patients. the marginal associational risk difference for in-hospital mortality between obese and non-obese patients is - . % ( % confidence interval (ci) - . % to . %, p= . ). the naive approach results in an atu of - . % ( % ci - . % to - . %, p= . ), whereas the robust approach yields an atu of - . % ( % ci - . % to . %, p= . ). a robust causal inference approach that may handle confounding bias due to model misspecification and selection bias due to missing data mitigates the obesity paradox, whereas a naive approach results in even more paradoxical findings. the robust approach does not provide evidence that the survival of non-obese critically ill patients would have been improved if they had been obese. bowel management within an icu environment is often difficult. recent data collection from an intensive care unit at the rvi identified either loose stool or constipation on > % of patient days. it was postulated this could be improved with a more tightly controlled bowel management regimen. to test this hypothesis a step-wise bowel protocol was created and introduced. data was collected in the month period following its implementation with the following aims: ) assess effectiveness of the protocol ) further observe the reasons for loose or constipated stool on an diarrhea is an important problem in each critically ill pateints [ ] . we aimed to investigate the frequency and management of diarrhea in our icu. in this study patient retrospectively reviewed, in our icu between . . - . . . patients were divided into two group as diarrhea "positive" and "negative". patients with diarrhea had fluid or loose stools or more times a day. each diarrhea period of the patients with diarrhea was examined separately and compared with the group without diarrhea. nutritional status, enteral product formulation, leukocyte, neutrophil, albumin values, gastric sparing, antibacterial and antimycotic use, los in hospital and in icu were compared. in diarrhea positive group, on the day of hospitalization, laxative and/or enema administration, toxin a in stool, nitrogen balance before and after diarrhea, enteral product change in diarrhea, probiotic, metronidazole or oral vancomycin use were examined. the incidence of diarrhea was . %. the most common diagnosis of icu admision was respiratory failure ( - %) in both groups. diarrhea occurred in two days after laxative and/or enema treatment. enteral nutrition was higher in both groups (≥ %). nasogastric tube feeding was significantly higher in the diarrhea group (p= . ). there was no difference between nutritional product formulation and diarrhea development (p> , ). antibacterial use was high in both groups ( %); however, teicoplanin use was significantly higher in the group diarrhea negative group (p= . ). the los in icu, and hospital was higher in diarrhea group (p< . ). no difference in mortality rates (p> . ). many factors may cause diarrhea in icu, and diarrhea may adversely affect patient treatment and increase morbidity. we think that preventive methods are as important as the treatment of diarrhea. the use of parenteral glutamine is studied in number of rcts and systemic reviews (heyland d , wischmeyer p ), while there is a lack of data about the use of enteral glutamine. the aim of our study was to determine the effect of enteral glutamine supplementation on the incidence of hospital infections and death. design: retrospective cohort study. inclusion criteria: males and females > years of age, tbsa burned %- %, nasogastric intubation.patients were divided in two groups: glutamine group (n= ) and control group (n= ). in the study group enteral glutamine was administered to the patients for days after admission to the icu. baseline characteristics were well balanced between groups. no significant difference was found between groups on patients' age, sex, tbsa, need for mechanical ventilation and rate of inhalation injury. primary outcome was all-cause mortality. secondary outcome was rate of nosocomial infections (skin and skin structure infections (sssi), lower respiratory tract infections, urinary tract infections, bacteremia, sepsis). mortality rate was ( %) and ( %) in the glutamine group and the control group, respectively, p= . . rate of nosocomial infections was ( %) in the glutamine group and ( %) in the control group, respectively, р= . . rates of sssi, lower respiratory tract infections, urinary tract infections and sepsis did not differ significantly between the groups: ( %) and ( %), p= . ; ( %) and ( %), р= . ; ( %) and ( %), р= . ; ( %) and ( %), р= . , respectively. rate of bacteremia was significantly different between the groups: ( %) in the glutamine group and ( %) in the control group, p= . . retrospective design is a significant limitation of our study. enteral glutamine supplementation may reduce the incidence of bacteremia in burn patients, but has no influence on the incidence of other nosocomial infections and mortality. further large clinical trials are needed. with outcomes were assessed with multivariable logistic regression and cox proportional hazard analyses, adjusted for baseline risk factors and randomization. in sensitivity analyses, models were further adjusted for key regulators of ketogenesis to assess whether any effect was direct or indirect. late pn increased plasma hb as compared with early pn, with maximal effect on day (p< . for day to and for the "maximal effect" day in the patients). adjusted for baseline risk and randomization, plasma hb associated with a higher likelihood of earlier live weaning from mechanical ventilation (p= . ) and of earlier live picu discharge (p= . ). as plasma hb replaced the effect of the randomization, the hb effect statistically explained these benefits of the randomization. further adjustment for key regulators of ketogenesis did not alter these findings. plasma hb did not independently associate with the risk of infections and mortality. withholding early pn increased ketogenesis in critically ill children, an effect that statistically mediated part of its clinical benefits. critical care patients are prone to frequent feeding interruptions for various reasons including feeding intolerance. these interruptions can lead to adverse outcomes. the aim of the study was to determine the reasons for and the duration of interruptions of enteral nutrition (en). single-center observational, cross-sectional study in a -bed mixed icu of a tertiary hospital. duration: months. patients, aged . years old (± . ), that stayed in the icu > hrs and were fed with en were included. anthropometric data, bmi, time of initiation of prescribed en, type of en formula, daily calories delivered were recorded. energy intake was calculated according to espen guidelines ( kcal/ kg bw/day). the causes for and duration of interruption were reviewed from the patient's chart. apache ii and mnutric score was calculated for all patients. mnutric score ≤ was used to diagnose malnutrition. all patients included in the study were endotracheally intubated. apache ii was . ± . . % of patients had increased risk of malnutrition. icu stay was . ( . ± . ) days, and the in-hospital mortality was %. there were episodes of en interruptions over a median icu stay of . days. median . interruptions/patient. the most common reason for en interruption was gastric residual volume monitoring followed by diagnostic and therapeutic procedures (figure ). other reasons include surgery, intolerance and/or delayed feeding and extubation. the median lost feeding time was . hours/ day ( . - . ) for all causes, while the mean loss of total energy intake was kcal/day (± )/day. average body weight of the patients was kg (± ). caloric deficit was calculated at kcal/day or % of the prescribed caloric goal. the results of this study showed that interruptions can lead to substantial caloric deficit, malnutrition and adverse events. an interruptionminimizing protocol could be useful in order to reduce the missing hours and to improve the clinical outcomes. relationship of goal-directed nutritional adequacy with clinical outcomes in critically ill patients pc tah there are controversies surrounding the effects of optimal nutritional intake on clinical outcomes in critically ill patients. this study aimed at investigating the relationship of goal-directed energy and protein adequacy on clinical outcomes which includes mortality, intensive care unit(icu) and hospital length of stay (los), and length of mechanical ventilation (lomv). this was a single centre prospective observational study. nutritional requirements were guided by indirect calorimetry and -h urinary urea.nutritional intake was recorded daily until death, discharge, or until day of icu stay. clinical outcomes were collected from patient's hospital record. the relationship between the two groups (< % and ≥ % of overall nutritional requirement) with mortality outcomes was examined by using logistic regression with adjustment for potential confounders. terlipressin, despite being one of the main treatments for acute variceal bleeding, may lead to severe hyponatremia due to its antidiuretic activity.we aimed to identify risk factors for development of hyponatremia during terlipressin treatment. retrospective study of patients admitted to acute intermediate care unit for hypertensive upper gastrointestinal bleeding due to chronic liver disease who received terlipressin(december -decem-ber ).hyponatremia was defined as a decrease in na serum levels ≥ meq and severe hyponatremia as > meq within days of treatment. we studied patients, . % male, mean age of . years (sd . ). alcohol-related liver disease was the most frequent etiology. hyponatremia occurred in patients ( . %). serum na Δbetween - and - meq and serum na Δ>- meq occurred in . and . %, respectively (table ) . severe hyponatremia occurred in patients ( . %) and symptoms were reported in two cases (status epilepticus and altered mental status). patients with higher baseline levels of na were more susceptible to terlipressin-induced hyponatremia and a longer length of stay was observed in patients with serum naΔ>- meq ( . vs . days, p< . ). the prevalence of hyponatremia in our study was lower than previously reported.higher serum na at admission and aih as etiology of cirrhosis were predictors of terlipressin-induced hyponatremia. neither the cumulative dose of terlipressin nor the duration of treatment appear to be related to the development of hyponatremia a Δ h-[na] > mmol/l was associated with larger hazards of mortality ( figure ). an increase in serum sodium in the first hours of icu admission is independently associated with a higher mortality in patients admitted with mild hyponatremia, normonatremia, and hypernatremia. based on our findings, it is possible that mild hyponatremia may be a protective mechanism in critical illness, which questions common practice of routinely correcting serum sodium when it is too low. introduction: acute liver failure (alf) represents a life-threatening organ dysfunction associated with increased mortality and liver transplantation represents the only definitive treatment. the aim of this study was to assess the effects of renal replacement therapy in combination with hemoadsorption in alf patients. twenty-nine patients with alf admitted to the intensive care unit (icu) of fundeni clinical institute were included in the study. after icu admission, consecutive session of hemoadsorption in combination with continuous veno-venous hemodiafiltration were applied. number of organ dysfunctions and sirs criteria were recorded at icu admission. the following data were recorded before and after the hemoadsorption therapies: glasgow coma scale, pao /fio , creatinine, -hours urine output, bilirubin, leucocyte and platelet count, heart rate, mean arterial pressure and vasopressor support, c-reactive protein and procalcitonine. clif-sofa score was calculated before and after the therapy. icu length of stay and -days outcome were noted. the mean age in the study group was ± years. the median number of sirs criteria was [ , ] and the median number of organ dysfunctions was [ , ] . the use of hemoadsorption was associated with a decrease in creatinine (from . ± . to . ± . mg/dl, p= . ), bilirubin (from . ± . to . ± . mg/dl, p= . ) and platelet count ( ± / ul to ± /ul, p= . ). we also observed a decrease in clif-sofa score from . ± . to . ± . (p= . ). overall mortality was . % (n= ). six patients ( . %) underwent liver transplantation with % -days survival. the use of hemoadsorption in patients with alf is associated with improvement in liver and kidney functional tests and may represent a new therapy in bridging these patients to liver transplantation. introduction: impairment of intestinal mucosal barrier function is the initiating factor of sepsis. in order to explore the effect of lactic acid bacteria on intestinal barrier function impaired by sepsis, it is necessary to establish sepsis and lactic acid bacteria ecological models. however, how to construct these models is still unclear. co-cultures with a gradient of lactic acid bacteria and caco- cells were constructed. the symbiotic state was observed under an inverted microscope and lactate dehydrogenase (ldh) toxicity tests, transepithelial electrical resistance(teer) tests and western blots were used to determine effective concentrations of lactic acid bacteria in monolayer cell models. lipopolysaccharide (lps) was used to treat cells, and cell counting kit- , quantitative reverse transcription pcr(rt-qpcr) and enzyme linked immunosorbent assays (elisa) were used to determine the appropriate concentration for sepsis models. the number of living cells decreased significantly when the moi(number of lactic acid bacteria/cell number) reached ( figure , panels a, b). the release of ldh indicated that damage to cells began to increase when the moi exceeded (panels a, b). at an moi of . , resistance values began to increase over time, whereas resistance values began to decrease when the moi reached (panel ). as the number of lactobacilli increased, the expression of tight junction protein increased and then decreased (panel a, b, c). in sepsis model experiments, the cell survival rate began to decrease once the concentration of lps exceeded ^ ng/ml (panel ). rt-qpcr results showed that ng/ml lps significantly increased inflammatory cytokines (panel ), and elisa results consistently showed that tnf-α and il- increased significantly when lps concentrations reached ng/ml (panel a, b). it is feasible to construct a cell monolayer model of lactic acid bacteria and lps. the appropriate moi of lactic acid bacteria is . and the optimal concentration of lps is ng/ml. introduction: sepsis is associated with high mortality and morbidity. as the severity increases, physiological parameters such as ph changes are one of the most notable features in metabolic acidosis secondary to high lactate. currently there is no point of care test other than blood gas measurement that could detect these ph changes. this is challenging especially in prehospital environment. the aim of this study is to develop a novel rapid point of care testing using a sensor to detect ph change in blood. sensors were produced by screen printing graphene and silver electrodes and functionalizing the graphene working electrode with an active layer of melanin. a preclinical sensor model was produced by adding lactic acid to a citrated plasma sample thus altering its ph over a clinically relevant range. the ph sensors were exposed to modified plasma, recording any changes in the voltage. the relationship between the voltage potential and plasma ph was established using weighted least squares regression. a ph dependent change in the measured voltage, with respect to the ph of the solution, was observed with a sensitivity of - . mv/ph +/- . over a physiologically relevant ph range between ph . and ph . . in this first phase proof of concept study a low cost, ph sensor was fabricated and demonstrated to be effective in measuring the ph of the plasma. this is the first time that such a sensor has been demonstrated and validated to work in this preclinical model of acidosis. the technology demonstrated here is a promising candidate for a point of care test whereby abnormal blood ph levels can be detected and monitored outside of a laboratory environment in a rapid manner. further studies are now underway to detect this change in whole blood. (figure ) . over one year only a small proportion of patients (n= , %) were classified as 'intermediate high' risk and potential candidates for reperfusion therapies. the revised national early warning score (news) with modified glasgow prognostic score (mgps) is superior to the news for predicting in-hospital mortality in elderly emergency patients t mitsunaga jikei university school of medicine, emergency medicine, tokyo, japan critical care , (suppl ):p the national early warning score (news) was developed in the ukto identify the risk of death. the previous study showed that the modified glasgow prognostic score (mgps) correlate with frailty in elderly patients [ ] . the aim of this study is to evaluate the predict value of the revised news with mgps for in-hospital mortality (in days) in elderly emergency patients. this study is secondary analysis and was carried out in jikei university kashiwa hospital, in japan, from april to march . the acute medical patients aged and older were included. the news was derived from seven physiological vital signs. the mgps was derived from c-reactive protein (crp) and albumin. discrimination was assessed by plotting the receiver operating characteristics (roc) curve and calculating the area under the roc curve (auc). the aucs for predicting in days in-hospital mortality were . for revised news with mgps and . for the original news. the auc of the revised news with mgps was significantly higher than that of the original news for predicting in-hospital mortality (p < . ) (figure ) . our single-centred study has demonstrated the utility of the revised news with mgps as a high predictor of acute phase in-hospital mortality in elderly emergency patients. the diagnostic performance of the five main emergency department (ed) triage systems has been shown to be poor in distinguishing acute coronary syndromes (acs) from mild severity diseases in chest pain patients. these ed triage systems are either clinically-based, being more sensitive or ecg-based, more specific [ ] . the goal of the study was to evaluate if incorporation of cardiovascular risk factors (cvrf) into ecgbased triage could increase his diagnostic performance. cecidoc is a prospective, observational, single-center study in an academic hospital. all consecutive adult patients admitted for acute chest pain were included. we compared the ecg-based french triage system [ ] to a modified system upgrading patients with a normal ecg but significant cardiovascular risk from a low acuity triage score (waiting period before medical assessment of max. min.) to a high acuity triage score (waiting period before medical assessment of max. min.). the final diagnosis was determined after a -day follow-up. we predefined as being adequate a high-acuity triage score (level or ) for acs and a low-acuity score (level , or ) for mild severity diseases. a total of patients was enrolled over a -month period (age . ± . ; m/f ratio . ). triage scores of patients ( . %) with acs were compared to patients ( . %) with mild severity diseases. taking into account cvrf, the sensitivity of the triage system increased from to % whereas the specificity decreased from to %. area under the roc curve (auc) went from . to . (fig. ) . for chest pain triage at ed, addition of cardiovascular risk factors into ecg-based triage increases his diagnostic performance. approximately % of patients presenting to hospital with an intentional overdose require admission to an intensive care unit (icu) [ ] . there are currently no uk guidelines regarding the optimal use of ct head scans (cth) in this patient cohort [ , ] . this study aims to determine whether we should be performing ct head scans in obtunded patients with suspected overdose requiring admission to intensive care. we performed a retrospective search of the icnarc database for plymouth university hospital trust, looking for patients admitted to the icu with overdose or self-poisoning as a primary diagnosis. patients were identified and of these patients required intubation due to obtundation(gcs< ). there were males and females with an average age of years old. the median length of stay on the unit was day. of the patients has a past medical history of mental illness, and overdosed on prescribed medications. the average gcs recorded on admission was . of the ( %) patients had a cth on admission, of which were part of a trauma scan. were known overdoses and were suspected overdose as per the cth request form. the main rationale behind those requests were to exclude additional intracranial injury. none of those cth showed any signs of acute pathology (figure ) . in this retrospective study, obtunded patients with suspected or known overdose with no history of apparent trauma or injury do not benefit from cth. in the absence of a history of trauma or focal neurological signs our conclusions are that cth provides limited value in the management of these patients. the audit was carried out to objectively investigate the problems associated with technique of folley catheterization in emergency department and indoor units of internal medicine wards [ ] . introduction: cellular and molecular mechanisms, epigenetic aspects of acute clozapine poisoning are studied insufficiently. the aim of this study was to identify morphological and epigenetic alteratons in brain neurons during acute exposure to clozapine combined wit ethanol. the experiments were carried out on male wistar rats weighting - g (n= ). group i (control) received . % nacl solution enterally; group iiclozapine mg/kg in . % nacl solution; group iiiclozapine mg/kg in % ethyl alcohol. after hours euthanasia was performed. autopsy included withdrawal of brain samples for histological examination (n = ) and for determination of global dna methylation level (n = ). the global dna methylation level ( -mc%) was determinated by fluorimetric method. inter-group comparisons were made by kruskal-wallis test. histological examination of paraffin sections of brains stained with hematoxylin and eosin was performed by light microscopy. in acute сlozapine poisoning and its combination with ethanol morphological changes in neurons of the cerebral cortex were detected. in acute сlozapine with alcohol poisoning an increase of global dna methylation level was observed. probably the identified changes have a common pathogenesis which will be clarified in our further studies. there is limited information available regarding the prevalence of adder bites and the complications of envenomation. nhs data suggests there are adder bites annually in the uk with the last fatality in [ ] . we performed an audit into adder bites in south west wales to identify the number attending our emergency departments, their management and clinical course as well as any environmental factors that predict increased likelihood of being bitten or the severity of the bite. a retrospective study of adder bites attending emergency departments in south west wales was undertaken (jan to aug ). measurements included were patient demographics, clinical presentation, type of treatment (conservative vs anti-venom) and outcome. results: patients were included, age range - years ( figure ). the majority of bites occurred in sand dunes ( . %) and all bites were on extremities. anti-venom was administered to . % ( / ) of patients. there was a significant positive association between the use of anti-venom and the length of hospital stay (r = . ; p= . ) and a significant negative correlation between the anti-venom use and both diastolic and systolic blood pressure (p= . and . respectively p= . ). all patients fully recovered. in this study, we demonstrated that with a full clinical assessment on presentation it is safe to decide whether anti-venom is required. the current guidelines are safe and effective in the treatment of adder bites. μmol/l, for pao < . kpa and > . kpa, platelets < * ^ /l and > * ^ /l, and bilirubin > μmol/l. in our population of adult ed patients, the thresholds of vital values associated with increased -day mortality were very close to routinely used values, and most of the thresholds were included in the lowest urgency level in triage and risk-stratification scoring systems. the workload in the emergency room: direct assessment by the therapeutic intervention scoring system- and indirect assessment by the nasa task introduction: the number of emergency room admissions continues to increase each year, which increases the care workload of the emergency department staff, who should to use its theoretical and practical knowledge in order to provide quality care in difficult working conditions. the aim of our study was to assess the emergency room staff workload its impact on health workers and patients and to suggest an improvement strategy to decrease this workload. a prospective, monocentric cohort study with descriptive and analytic approach over one month (december ) conducted at the emergency department of an academic hospital. the workload endured by the emergency room staff was evaluated by the nasa task load index and on patients by the therapeutic intervention scoring system- . there were cumulative days of hospitalization in consecutive patients admitted to the emergency room. the average age was ± years. the average length of stay at the emergency room was about ± h. the average tiss- score was . ± . . factors associated with important care workload were: age ≥ years, diabetes, more than comorbidities, the use of intravenous antibiotics; the use of vasoactive drugs and the use of mechanical ventilation; a high tiss score was predictive of emergency room mortality. in the indirect assessment of the care workload, medical and paramedical staff were interviewed, % of them were under years old with a sex ratio of . . a high level of mental and physical workload was expressed by ed staff with considerable level of frustration; the ed staff suggested mainly to improve the working conditions, communication and to redefine tasks "who does what". our study had shown a significant workload in the emergency room, a process to reduce this workload is being implemented medical simulation is a modern teaching tool increasingly used in specialties such as anesthesia, emergency medicine and obstetrics. however, it's not widely used in specialties like cardiology, althought cardiovascular emergencies are very frequent. the purpose of our study was to assess the effectiveness of simulation-based medical education in the management of cardiovascular emergencies among moroccan graduate students. we conducted a prospective, observational, multi-centrer study including the students of three moroccan universities from the th to the th year of medicine who underwent phases: first a pre-test, then a theoretical and practical training on cardiovascular emergencies after which the students were separated in two groups, one undergoing the medical simulation training (group ) and one who didn't (group ), followed by a theoretical then a practical post-test on resusci anne and simman®. at last, the students were asked to answer a satisfaction survey. the reform procedure in the tunisian army consists in repairing the physical damage and deciding on the applicant's ability to continue working. terrorism increases the impact of the co-morbidity generated and the socio-economic consequences that result from it. the purpose of this work was to study the epidemiological, clinical and evolutionary profile of terrorist injuries, to specify the rates of consequent partial permanent disability (ppi) and the possibilities of returning to work. descriptive retrospective cross-sectional study of reform files on military personnel injured during anti-terrorist operations from fig. (abstract ) . changes in total bcpr rate in family-and friends-witnessed ohca cases with dispatcher-assisted instruction during -week period after the day of disaster during three years january to september . the data collection was carried out on the basis of a collection form. our wounded were male, % of whom belonged to the army. the average age was years and months ± . . half of our wounded were troopers. infantry and special forces were the most exposed military units. half of the accidents were recorded in the kasserine region ( cases). chronic post-traumatic stress disorder (cptss) was found in injured, followed by amputations in injured. the after-effects were psychological in %, physical in % and mixed in % of our injured. the ppi rate ranged from % to % in . % of injuries.. more than half of the injured had returned to their professional activity, % were put on reform for health reasons. our results showed that the esptc was the most recorded sequel, and that the ppi rate was significant in a quarter of our injuries. in our series, a third of our wounded were put on reform for health reasons. to state the importance of initial care and adequate and rigorous follow-up to recover a greater number of war wounded. introduction: the rapid response system (rrs) has been shown to decrease hospital mortality [ ] . the japanese coalition for patient safety has set a major goal for hospitals to more widely implement the rrs. however, prevalence and actual circumstances of use in acute care hospitals (including small scale hospitals) in japan are as yet not well-known. web-based questionnaires were sent to acute care hospitals (of scale beds-or-larger) of prefectures in western japan. each participant hospital selected a certain department which answered the questionnaire. the rrs included the medical emergency team (met), the rapid response team (rrt), and the critical care outreach team (ccot). we investigated the presence and circumstances of in-hospital emergency calls, rrs and other systems, and then illuminated issues to be solved. our study suggests that delays in patient transfer to the icu after rrt activation in the wards were associated with slower physiological improvement.these findings support further and larger studies. blood and blood products use in intensive care unit m akcivan, s bozbay, o demirkiran istanbul university cerrahpasa, anesthesiology and intensive care, istanbul, turkey critical care , (suppl ):p blood and blood product (bp) transfusions are frequently used in intensive care units (icu) [ ] . it is important to know transfusion epidemiology and the effect of adverse transfusion reactions and their effect on mortality and morbidity.we aimed to investigate the blood and bp transfusions in the icu. blood and bp transfusions in icu, between - were reviewed retrospectively. we evaluated each transfusion as a data and examined the pre-and post-transfusion laboratory values, demographic data, cause of icu admission and comorbidities. results: patients who underwent transfusion in the icu, and transfusion data from these patients were included. the most frequent cause of hospitalizations were respiratory failure and sepsis. the rate of patients transfused in the five-year period decreased from . % to . %. the hemoglobin threshold before transfusion decreased from . g / dl to . g / dl. a total of transfusion reactions were observed and the most common transfusion reaction was febrile non-hemolytic reaction. the most commonly transfused product was red blood cell suspension. transfusion reactions were found to be slightly higher in men than women in young age group(< y) (p = . and p= . , respectively). transfusion reactions were found to be more frequent in emergency transfusions (p < . ). the number of transfusions was significantly lower in patients with apache ii score < (p < . ). the need for transfusion was found to be higher in patients with hematological malignancy (p < . ). it was observed that as the mean number of transfusions increased the mortality is also increased (p < . ). transfusion therapies are the treatments that are vital but have a serious mortality and morbidity risk. in particular, intensive care patients should be considered in detail because of their specific features. restrictive transfusion practices have positive results. association between anemia or red blood cell transfusion and outcome in oncologic surgical patients. figure a) . the association between rbc transfusion and adverse events also remained after adjustment (or . [ . - . ] ; p < . ) ( figure b) . in oncologic surgical critically ill patients, there was an independent association between anemia (even moderate anemia) or rbc transfusion and patient outcomes. our findings highlight the need for further research to determine the optimal transfusion strategy in surgical oncologic patients. transfusion impaired skin blood flow when initially high e cavalcante dos santos, w mongkolpun, p bakos, al alves da cunha, c woitexen campos, jl vincent, j creteur, fs taccone erasme hospital, intensive care department, brussels, belgium critical care , (suppl ):p red blood cell transfusion (rbct) increases global oxygen delivery (do ) and may improve microcirculation. however, the effects on blood flow have been found to be conflicting. we studied icu patients with stable hemodynamic status (mean arterial pressure (map) ≥ mmhg for at least hours) and without active bleeding, who received a rbct. skin blood flow (sbf) was determined (periflux system , perimed, index finger; perfusion unit, pu) together with map, heart rate (hr), hemoglobin (hb), lactate levels and scvo before and after rbct. sbf was measured before rbct (t ) and after (t ) for each min. according to previous data indicating the lowest sbf value found in noninfected icu patients was pu, all patients were analyzed according to the baseline sbf (i.e. < pu -low sbf vs. ≥ puhigh sbf). the relative change of sbf (Δsbf) was calculated after rbct and the responders were defined by the function of > %. results: icu patients were studied. rbct was associated with increases in map and scvo but no change in sbf. at baseline, scvo was lower in the responders than in the non-responders (p= . ) and lower in patients with low sbf than in the high sbf (p= . ). there was no difference in hb, map, and lactate, between the patients with low and high sbf. after rbct, map rose in the responders (p< . ) and in the non-responders (p= . ), sbf (p< . ) rose in patients with low sbf, and sbf (p= . ) decreased in patients with high sbf. there was a negative correlation between baseline scvo (r= - . , p< . ) or baseline sbf (r= - . , p< . ) and the relative increase in sbf after rbct. rbct increases skin blood flow only when it is impaired at baseline. severe immune dysregulation is associated with adverse outcomes and is common in intensive care unit (icu) patients [ ] . erythropoietin-stimulating agents (esas) have both anti-apoptotic and immune-modulating properties [ ] . despite potential benefit, both the safety and efficacy of these agents remains unclear [ ] . here we evaluate the impact of esas on morality at hospital discharge in critically unwell adult patients admitted to the icu. we conducted our search strategy in accordance with a predetermined protocol. the use of ffp is associated with an increased incidence of complications such as acute respiratory distress and infections, and the rate of complications increased with the quantities of ffp transfused [ ] . pcc contain several important coagulation factors and it has been suggested that they could replace ffp. this has been shown mainly in case reports or series in which coagulation factor deficit was detected by using poc viscoelastic tests in trauma [ ] or traditional hemostatic tests in obstetric patients [ ] . multicenter observational study of the safety and efficacy of the prothrombin complex concentrate. a survey of anesthetists was conducted in maternity hospitals at various levels of care in the russian federation. data has been collected and processed. as a result, patients were analyzed. pph was determined as a volume of blood loss more than ml during vaginal delivery or cs. the most significant risk factors for pph were: preeclampsia or arterial hypertension and a history of postpartum hemorrhage. . % had no risk factors for pph. it was determined that the use of prothromplex iu decreased the number of patients with transfusion ffp - ml/kg by . % and increased the number of patients without transfusion by . %, compared with patients without use of prothromplex iu (figure ). no complications were detected. the use of pcc safety and efficacy reduce use of ffp during pph. the full analysis included patients on either hfc (n= ) or cryoprecipitate (n= ). the intraoperative and postoperative changes in etp and fibrinogen concentration are shown in table . for fibtem a (intraoperatively) and fibrinogen concentration (intraoperatively and postoperatively), the mean numerical values appeared higher with hfc than cryoprecipitate. fxiii (hfc: . %, . %; cryoprecipitate: . %, . %, at baseline and hr after surgery start), fviii and vwf were maintained throughout surgery in both treatment groups. this was also the case for laboratory tests activated partial thromboplastin time, prothrombin time and platelet count. the forma- coagulation parameters analyses showed broad overlaps between hfc and cryoprecipitate, with satisfactory maintenance of the clot quality parameters, fxiii concentrations and thrombin generation parameters. the study group includes men and women with a mean age of , vs. . years (p= . ) admitted with the diagnosis of multiple trauma. we found a directly proportional and highly significant statistical correlation between base excess and fibrinogen level diagnosed using the mcf/fibtem parameter(r= . , p< . )and an inverse proportional correlation between lactate level and fibrinogen level (r= - . , p= . ). in the roc analysis that uses as a variable the level of base excess and as a criterion of classification the fibrinogen deficit (mcf/fibtem< mm) it can be observed that at a value of be<- mmol/l, we can diagnose a fibrinogen deficit with a sensitivity of . % and a specificity of . % (auc= . ,p< . ). lactate appears to be inferior to the excess base (figure ) , but still has a good diagnostic power, a value of . mmol/l has a sensitivity of . % and a specificity of % (auc= . ,p< . ). the difference between the two roc curves ( . ) is statistically significant (p = . ). both base excess and serum lactate can be used to diagnose fibrinogen deficiency with the mention that base excess appears to have a higher sensibility and specificity ability. based goal-directed algorithm. this approach requires further clinical validation. we conducted a retrospective study comparing transfusion strategies in patients with major trauma between and . we retrieved demographic data and blood products administered from patients with at least one red-blood cell (rbc) transfusion. primary outcome was a reduction of rbc administration. secondary outcomes were mortality, icu length of stay and acute kidney injury. we included patients admitted in the icu due to severe trauma (sapsii: . ± . ), and mainly after emergent surgery ( . %). they featured a mean age of . ± . y, were predominantly male ( . %) and % were in shock. in the first hours of hospital admission a mean of . ± . rbc units were administered. most patients received a fibrinogen-based protocol (fbp) ( %), with an average of ± g of fibrinogen and ± fresh-frozen plasma (ffp) units, versus ± g of fibrinogen and ± ffp units in the ffp group. the fbp was associated with a decrease administration of rbcs in the first hours (r = - . ; p < . ), even after adjustment for severity (p= . ) and for tranexamic acid use (p = . ). it was associated also with a decrease of platelet transfusion (p= . ). fibrinogen-based protocol was not associated with a decrease in mortality, acute kidney injury or noradrenaline dose. treatment of tic in past years has progressively changed to a goaldirected fibrinogen-based approach. in our population, the use of fbp lead to a reduction of rbc administration in severe trauma patients. prospective, multicenter, randomized study comparing administration of clotting factor concentrates with a standard massive hemorrhage protocol in severely bleeding trauma patients the objective of this study was to assess the ability of the quantra® qstat® system (hemosonics) to detect coagulopathies in trauma patients. many level trauma centers have adopted whole blood viscoelastic testing, such as rotational thromboelastometry (rotem®, fig. (abstract ) . study treatment plan instrumentation lab) for directing transfusion therapy in bleeding patients. the quantra qstat system is a cartridge-based point-of-care (poc) device that uses ultrasound to measure viscoelastic properties of whole blood. and provides measures of clot time, clot stiffness and a test of fibrinolytic function. methods: adult subjects were enrolled at two level trauma centers which use a rotem based protocol to guide transfusion decisions. study protocols were approved by the site's ethics committee. for each subject, whole blood samples were drawn upon arrival to the emergency department and again, in some cases, after administration of blood products or antifibrinolytics. samples were analyzed on the quantra (at poc) in parallel to rotem delta (in lab). a total of patients were analyzed. approximately % of samples had a low clot stiffness (cs) values suggestive of an hypocoagulable state. the low stiffness values could be attributed to either low platelet contribution (pcs), low fibrinogen contribution (fcs), or a combination ( figure ) . additionally, % of samples showed evidence of hyperfibrinolysis based on the quantra clot stability to lysis parameter. samples analyzed on standard rotem assays showed a lower prevalence of low clot stiffness and fibrinolysis based on extem, fib-tem results. the correlation of cs and fcs vs equivalent rotem parameters was strong with r-values of . and . , respectively. this first clinical experience with the quantra in trauma patients showed that the qstat cartridge detected coagulopathies associated with critical bleeding and may be useful for directing blood product transfusions in these patients. ability to perform testing at poc may provide additional clinical advantage. the objective of the study was to describe the conditions of use of fibryga® g, a new, highly purified, human fibrinogen (hf) recently granted a temporary import authorization for use in congenital and acquired fibrinogen deficiencies in france. observational, non-interventional, non-comparative, retrospective study conducted in french hospital centres using fibryga®. data from patients with fibrinogen deficiency having received fibryga® from december to july were retrieved from their medical files. indications, modalities, efficacy and safety outcomes were recorded. indications encompassed non-surgical bleeding (nsb) either spontaneous or traumatic, including post-partum hemorrhage (pph), bleeding during surgery (sb) or administration to prevent bleeding during planned surgery. treatment success was defined as control of the bleeding or hemoglobin loss < % for bleeding treatment and as absence of major perioperative hemorrhage for pre-surgical prevention. this analysis included patients aged , ± . years and % were male. all presented an acquired fibrinogen deficiency requiring administration of hf. indications were nsb (n= , . %) including ( . %) pph, sb (n= , . %), and prevention of sb (n= ; , %). cardiac surgeries were the main procedures associated with treatment and prevention of sb. mean total doses of fc were . ± . g, . ± . g and . ± . g for nsb, sb and prevention of sb. success rates were . % ( %ci . - . %), . % ( %ci . - %) and . % ( %ci . - %) respectively. for pph, mean dose of hf was . ± . g with a success rate of . % ( %ci . - %). overall, tolerance was good. fibrinogen concentrate fibryga® is mostly used for bleeding control. in one third of patients, hf was administered preventively to avoid bleeding during surgery. use of fibryga® was associated with favourable efficacy outcomes. functional testing for tranexamic acid effect duration using modified viscoelastometry t kammerer , p groene , s sappel , p scheiermann , st schaefer ruhr-university bochum, institute of anaesthesiology, heart and diabetes center nrw, bad oeynhausen, germany; ludwig-maximilans university, department of anaesthesiology, munich, germany critical care , (suppl ):p tranexamic acid (txa) is the gold standard to prevent or treat hyperfibrinolysis [ ] . effective plasma concentrations are still under discussion [ ] . in this prospective, observational trial using modified viscoelastometry we evaluated the time-course of the antifibrinolytic activity of txa in patients undergoing cardiac surgery. methods: patients were included. modified viscoelastometry (tpa-test) was performed and txa-plasma-concentration, plasminogen-activatorinhibitor- (pai- ) and pai-antigen-plasma-concentrations were measured over h. additionally, in vitro dose-effect-curves from blood of healthy volunteers were performed. data presented as median with interquartile range (q /q ). results: txa plasma-concentration was increased compared to baseline (t : μg ml - ) at every time-point with a peak concentration min (t ) after application (p< . ; see fig. a ). lysis was inhibited from min (lysistime tpa-test : p< . ; lysisonsettime tpa-test :p< . ). maximumlysis tpa-test was decreased at t (t : % ( / ) vs. t : % ( / ); p< . ). of note, after h some patients (n= ) had normalized lysis whereas others (n= ) had strong lysis inhibition (ml< %;p< . ) up to h. high and low lysis groups differed regarding kidney function (cystatin c: . mg l - ( . / . ) vs. . mg l - ( . / . );p= . ) and active pai- ( . ng ml - ( . / . ) vs. . ng ml - ( . / . );p= . ). in-vitro, txa concentrations > μg ml - were effective to inhibit fibrinolysis. in our trial, after h there was still completely blocked lysis in patients with moderate renal impairment. this could be critical with respect to postoperative thromboembolic events [ ] . here modified viscoelastometry could be helpful to detect the individual fibrinolytic capacity. introduction: peri-operative coagulopathy correction based on viscoelastic hemostatic assays (vhas) and single-factor coagulation products has changed the paradigm of bleeding management in cardiac surgery [ ] . in a retrospective study, we analysed patients with emergency surgery for thoracic acute aortic dissection (taad), before and after the introduction of fibrinogen concentrate in clinical practice. data were collected from paper and electronic records. the study was approved by the institutional ethical committee. patients were included in the analysis, operated in , before fibrinogen concentrate was approved for human use, and in - . therapy was guided by a rotational thrombo-elastometry (rotem) algorithm. exclusion criteria were non-compliance with the institutional protocol and intra-operative death. we investigated allogeneic blood transfusion (abt), fibrinogen use, peri-operative bleeding (pob), surgical reexploration and post-operative complications (poc). the groups were similar in gender, age, body weight, additive euro-score and aortic cross-clamp time. fresh frozen plasma, cryoprecipitate and red blood cell transfusion were lower in the fibrinogen group, but not platelet transfusion (table). , % of patients in the study group received fibrinogen concentrate and median dose was g (iqr - ). day postoperative chest tube drainage and surgical reexploration were significantly lower. there were no differences in stroke, renal replacement therapy, mechanical ventilation time and icu stay. in patients with taad surgery, rotem-guided algorithms which include fibrinogen concentrate are associated with less (pob), surgical re-exploration and abt. further research is needed to document the role of vhas and concentrated factors in reducing (poc). andexanet alfa (aa, portola pharmaceuticals, san francisco, ca) represents a modified factor xa agent which is approved antidote for apixaban and rivaroxaban. andexanet alfa may also neutralize the anti-xa effects of betrixaban and edoxaban. this study aims to compare the relative neutralization of these four anti-xa agents by andexanet alfa in different matrices. andexanet alfa was diluted at mg/ml. apixaban (a), betrixaban (b), edoxaban (e) and rivaroxaban (r) were diluted in ph . , . m tris buffer (tb), blood bank plasma (bbp) and in % albuminated buffer (ab) at . - . ug/ml. anti-xa activities of all four agents were measured in three systems and the reversibility indices of aa were profiled. the reversibility index (ri ) of anti-xa effects by aa was determined at - ug/ml. each of the four agents produced varying degrees of inhibition of anti-xa at . - . ug/ml, the ic ranged . - . ug/ml in bbp, . - . ug/ml in ab and . - . ug/ml in tb. andexanet alfa produced a concentration dependent reversal of all four anti-xa agents. in the bbp, the ri values for a ( ug/ml), b ( ug/ml), e ( ug/ml) and r ( ug/ml). in the ab, the ri values for a ( ug/ml), b ( ug/ml), e ( ug/ml) and r ( ug/ml). in the tb, the ri values for a ( ug/ml), b ( ug/ml), e (> ug/ml) and r ( ug/ml). each of the four anti-xa agents exhibit varying degrees of matrix independent anti-xa potencies in different systems, the collective order follows edoxaban > apixaban > betrixaban > rivaroxaban. andexanet alfa produced matrix dependent differential neutralization of the anti-xa effects of these agents. individualized dosing of andexanet alfa may be required to obtain desirable clinical results. the diagnostic and prognostic value of thromboelastogram (teg) in sepsis has not been determined. this study aimed to assess whether teg is an early predictor of coagulopathy [ , ] and is associated with mortality in patients with sepsis. in total, patients with sepsis on intensive care unit admission were prospectively evaluated. we measured teg and conventional coagulation tests(ccts)on preadmission and observed for development of , days and , , days respectively. multivariable logistic regression was utilized to determine odds of icu/hospital mortality. the parameter of teg (maximum amplitude, reaction time; ma/r ratio) was calculated to evaluate sepsis-induced coagulopathy. the admission patients were divided into three groupsma/r group(ma/r= - mm/min); ma/r group(ma/r> mm/min)and ma/r group(ma/r< mm/min). in our cohort of patients with severe sepsis, coagulopathy defined by ma/r ratio was associated with increased risk of icu/hospital mortality. introduction: blood sampling for coagulation assessment is often carried out in either arterial or venous samples in the intensive care unit (icu). there is controversy as to the accuracy of this method due to the inherent differences in physicochemical properties as well as the underlying effects of individual diseases in arterial and venous blood. clot microstructure has shown to be a new biomarker (fractal dimension-d f ) which encompasses the effects of diseases in all aspects of the coagulation system [ , ] . in this study, we compared the effect of all these factors in venous and arterial blood to see if there is a difference in the clot microstructure and quality. patients admitted to a tertiary intensive care unit and busy teaching hospital were recruited. arterial and venous blood was sampled from an arterial line and central venous catheter in situ from the same patient. standard markers of coagulation (pt, aptt, fibrinogen, full blood count), rotational thromboelastometry (rotem), whole blood impedance aggregometry and measured clot microstructure (d f ) were measured on both arterial and venous samples. no significant difference was observed in standard laboratory markers, rotem and platelet aggregation between arterial and venous blood. there were no differences in the fractal dimension (d f ) between the arterial and venous blood samples (d f . ± . vs . ± . respectively, p= . ). samples from patients with critical illness give comparable results from either arterial or venous blood despite their underlying pathophysiological process or treatment. this confirms blood for coagulation testing can be taken from arterial or venous blood. clinicians in the emergency setting use a wide range of hemostatic markers to diagnose and monitor disease and treatment. current methods rely on the anticoagulant effect of citrate on whole blood prior to laboratory analysis. despite the well-recognized modulatory effects of citrate on hemostasis, the use of anticoagulated blood has clear analytical advantages, including repeat sampling and storage. however by altering the physiological state of the blood reproducibility and accuracy of the test is affected. recent studies have shown the potential of a novel functional biomarker of clot formation: fractal dimension (d f ), that may give an improved diagnostic accuracy. in this study we assessed the potential of this new biomarker in scientifically measuring the effects of recalcification of citrated samples. methods: healthy volunteers were included. unadulterated and sodium citrate samples of blood were taken from each volunteer. citrated samples were recalcified using ( m cacl ). in the study we compared unadulterated whole blood d f results to citrated d f results and repeated the citrated d f experiments times for each sample over a hour period to ascertain reproducibility. the d f of citrated blood was significantly lower than that of unadulterated blood ( . ± . vs . ± . , p< . ). the results of the citrate samples when tested times over hrs gave a coefficient of variation of . %. for the first time we show that a functional biomarker of clot microstructure, d f , can precisely quantify and measure accurately the direct effect that the addition of the anticoagulant sodium citrate has on whole blood clot microstructure. the study also shows that the test is reproducible and has potential utility as a biomarker of acute disease in the emergency setting in citrated blood. this procedure now needs to be evaluated in a group of acute disease states. in this study, we analyzed the hematological abnormalities of dengue patients by thromboelastography (teg) at initial and -hour of fluid resuscitation. methods: this is a cross-sectional study evaluating teg readings of dengue patients with different severities presenting to the emergency department. laboratory confirmed dengue patient (positive ns antigen or igg/igm) was consecutively sampled. teg readings were taken at presentation and after -hour of fluid resuscitation. twenty dengue patients with varying severity had a median reaction time (r), α -angle, k time, maximum amplitude (ma) and lysis % (ly ) of . min, . ο , . min, . mm and . % respectively. mean fibrinogen was normal before and after fluid infusion. there is a non-significant reduction in ma with prolongation of other teg parameters between different dengue severities. there is a statistically significant reduction of α-angle and ma between pre and post -hour fluid resuscitation (p= . and p= . ). normal fibrinogen with low ma, which signifies a weak clot strength, may indicate either a platelet reduction, platelet dysfunction or both. reduction in ma and α-angle post fluid resuscitation is an alarming finding. this is in contrast with previous teg studies although none of it used normal saline exclusively, studied initial fluid resuscitation in emergency department settings or studied a subject with dengue. a bigger study, especially in severe dengue is needed to validate our findings. agreement between the thromboelastography reaction time parameter using fresh and citrated whole blood during extracorporeal membrane oxygenation with teg® and teg® s m panigada, s de falco, n bottino, p properzi, g grasselli, a pesenti fondazione irccs ca´granda ospedale maggiore policlinico, intensive care unit, milano, italy critical care , (suppl ):p the r (reaction time) parameter of kaolin-activated thromboelastography (teg) may be used to assess the degree of heparinization of blood during ecmo. a teg analysis is usually performed on two types of samples: fresh (f) or citrated-recalcified (c) whole blood. teg® can perform the analysis on c and f whole blood, the new teg® s (haemonetics corp., ma, usa) only on c whole blood. aim of the study was to compare the response of r to heparin using the two types of samples and two teg devices methods: during a three months period at fondazione irccs ca' granda -policlinico of milan, teg was performed (using teg ® and teg s® with and without heparinase, an enzyme that degrades heparin) on consecutive ecmo patients (as part of the gatra study, nct ) and in consecutive non-ecmo patients in whom a teg was requested for clinical purposes. bland altman analysis and lin's concordance correlation coefficient were used to assess agreement results: a total of paired samples were taken ( in-ecmo and off-ecmo). ecmo patients received . ( . - . ) iu/kg/h of heparin. among non-ecmo patients, of them did not receive any dose of heparin, two of them a very low prophylactic dose ( . and . iu/ kg/h, respectively), and one of them . iu/kg/h of heparin. using teg® , r was - . (- . ; . ) min shorter on c compared to f blood in patients receiving heparin (this difference disappeared using heparinase) and only - . (- . ; . ) min shorter in patients notreceiving heparin. r was - . (- . ; . ) min shorter using teg® s (which performs the analysis only on c blood) than teg® on f blood (figure ) . when evaluating the effect of heparin using teg, clinicians should be aware that results obtained using citrated-recalcified or fresh whole blood are not interchangeable. using citrated-recalcified blood to perform teg might lead to underestimation of the effect of heparin trauma patients are at high risk for venous thromboembolism (vte). the east guidelines recommend low molecular weight heparin (lmwh) for vte prevention and antixa monitoring after initiation of the medication or after adjusting doses in certain populations [ ] . studies have shown standard enoxaparin dosing of mg every hours may result in low antixa levels [ ] . this study aims to evaluate the efficacy of a pharmacist-lead protocol for adjusting enoxaparin dosing based on antixa levels in trauma patients. this single center retrospective chart review included adult trauma patients admitted from / / to / / . per protocol, patients with body mass index (bmi) ≤ kg/m were initiated on enoxaparin mg twice daily, and patients with bmi > kg/m were initiated on enoxaparin mg twice daily. peak antixa levels were drawn to hours after at least the third dose of enoxaparin with a goal therapeutic range of . - . iu/ml. the primary objective was time in days to goal peak antixa level. secondary objectives include vte occurrence, bleeding attributed to lmwh, and dosing regimens utilized. subgroups were analyzed based on body mass index (bmi). of patients identified, patients met inclusion criteria. median time to therapeutic antixa level was days (iqr - ). of patients fig. (abstract ) . agreement between teg® s and r teg® on citrated recalcified and fresh whole blood with bmi ≤ kg/m , patients ( . %) were dosed initially per protocol and / patients ( . %) met goal antixa level at first check (table ) . of patients with bmi > kg/m , patients ( . %) were dosed initially per protocol and / patients ( . %) met goal antixa level at first check. our results indicate the protocol is safe due to lack of bleeding attributed to enoxaparin, but less than % of patients achieved goal antixa level at first check. however, despite low rates of achieving goal antixa level, vte rates also remained low. introduction: most patients in the icu are given prophylactic anticoagulation with a fixed dose of mg once daily of enoxaparin (clexane) if cct is normal and mg if cct is low. studies on non icu patients have shown that afxa is below desired range for venous thromboembolism (vte) prevention. in the icu, many factors might influence afxa levels including weight, creatinine clearance (cct), shock and other medication. atxa activity was not yet reported in a big mixed icu population with variable morbidity. our study hypothesis is that enoxaparin is underdosed in most cases and routine afxa activity should be monitored in all icu patients. preventive enoxaparin ( mg qd) was given to all patients unless therapeutic dose was needed or contraindication existed. levels of afxa activity were taken hours after the rd dose. therapeutic vte preventive effect was defined as afxa activity of . - . . patient data was collected from medical files. the study is still ongoing, preliminary results were analyzed for patients. of patients ( %) had afxa activity below normal (subtherapeutic). weight and cct were negatively correlated with afxa activity (figure ). mean weight in the subtherapeutic afxa was significantly higher than the therapeutic group ( . vs. . respectively, p= . ). cct in the subtherapeutic afxa was significantly higher than the therapeutic group ( . vs. . respectively, p= . ). the normal cct group (> ) had significantly more patients with subtherapeutic afxa ( vs , p= . ). in our icu, % of the patients receive insufficient vte prophylaxis. overweight patients and patients with normal cct should probably receive higher enoxaprin dose. afxa activity should be routinely monitored in icu patients. in this study we use a new bedside biomarker to test its ability to measure anticoagulation effects on patients who present with acute first time deep vein thrombosis (dvt). dvt requires oral anticoagulants to prevent progression to potentially fatal pulmonary embolism and recurrence. therapeutic efficacy monitoring of direct oral anticoagulants (doac) including rivaroxaban is problematic as no reliable test is currently available. advances in hemorheological techniques have created a functional coagulation biomarker at the gel point (gp) which allows quantitative assessment of: time to the gel point (t gp ), fractal dimension (d f ) and elasticity (g') [ , ] . the prospective observational cohort study measured t gp , d f , g', standard coagulation and cellular markers in first time dvt patients at three sample points: pre-treatment and approximately and days following mg bd and mg od rivaroxaban respectively. strict inclusion and exclusion criteria applied. results: dvt patients (mean age years [sd± . ]; male, female) and non-dvt patients were well matched for age, gender and co-morbidities. mean t gp on admission was s (sd± . s) and . s (sd± . s) for dvt and non-dvt respectively. doac therapy significantly increased t gp to . s (sd± . s) after days, and subsequently increased to . s (sd± . s) at days as shown in table . d f , g' and standard hemostatic markers all remain within the normal range. conclusions: t gp demonstrates its utility in determining the anticoagulant effect of rivaroxaban. the significant difference in t gp between males and females needs further exploration. localized stasis as a result of transient provoking factors appears not to generate a systemic strength fig. (abstract p ) . correlation of anti factor xa activity with patient cct and weight. anti fxa activity value below . (red line), was considered "non-effective prevention" introduction: trauma remains the leading cause of death all over the world. to better exploit the trauma care system, precise diagnosis of the injury site and prompt control of bleeding are essential. here, we created a nursing protocol for initial medical care for trauma. the aim of this study was to evaluate the impact of protocoled nursing care for trauma on measures of quality performance. this was a retrospective historical control study, consisted of consecutive severe trauma patients (injury severity score > ). people were divided into two groups: protocoled group (from april to march ) and control group (from april to march ). we set the primary endpoint as mortality for bleeding. the secondary endpoints included time allotted from arrival to start of ct scan and surgery, administration rate of several drugs (sedations, painkillers, preoperative antibiotics, and tranexamic acid). for the statistical analysis, continuous variables were expressed as median (interquartile range) and were compared by wilcoxon rank sum tests given a nonnormal distribution of the data. we included patients in the study: in the control group before the introduction of the protocol, in the protocoled group. as a primary endpoint, the mortality for bleeding was similar between two groups ( % in the control group and % in the protocoled group). as a secondary endpoint, the time to ct initiation [group a ( - ) min vs group b ( - ) min; p < . ], and emergency procedure [group a ( - ) min vs group b ( - ); p < . ] were shortened by the protocol introduction. furthermore, the administration rates of sedations, painkillers, preoperative antibiotics, and tranexamic acid were increased in the protocoled group compared with the control group. although the mortality as a patient-oriented outcome was not affected, improved quality of medical care by nursing protocol introduction may be suggested in this analysis. this single-institutional prospective study included patients with uprf who were admitted to the trauma surgical intensive care unit (tsicu) and survived until discharge to home between and . we evaluated the activities of daily living after the discharge using physical and mental component scores of sf- ® and defined physical dysfunction (pd) as physical function (pf-n) score of or less. we divided the patients in the pd (n= ) and control (without pd, n= ) groups and compared the groups. the patients had experienced blunt injuries, including falls ( %) and pedestrian injuries ( %). the mean age was . years (men: . %); the median injury severity score was (interquartile range: - ); and the mean length of tsicu stay was . days. the average period from the injury until the survey was . months. there was no difference between the pd group and the control group in the patient characteristics, fracture type, pelvic fixation, and complications. at the time of the survey, the pd group had significantly more painful complaints than the control group (pd: . %, c: . %, p < . ), and had more physical and mental problems. the sf- ®subscale score showed a significant positive correlation between physical function and body pain, mental health respectively. the percentage of those who were able to return to work was not different in both groups (pd: . %, c: . %). in the multivariate analysis of pd, only age (odds ratio: . , % ci: . - . , p = . ) was relevant. long-term pd was observed in % of patients with uprf. the elderly were particularly prominent, and there was an association between pain and mental health. cells (rbc) this can lead to inhibition of oxygen transport function and development of hypoxia. currently used methods for analyzing the state of rbc either do not have sufficient accuracy or require lengthy analysis and expensive equipment. the use of a simpler and more informative electrochemical approach to assessing the state of rbc is very promising. electrochemical measurements in rbc suspensions (~ • cells / l) were carried out in a special electrochemical cell [ ] in the potentiodynamic mode in the potential range from - . to + . v using the ipc pro mf potentiostat (kronas, russia); optical measurements were performed using an eclipse ts inverted microscope (nikon, japan), a cfi s plan fluor elwd x / . lens (nikon, japan); rbc morphology was recorded in real time using a ds-fi digital camera (nikon, japan). when examining rbc of patients with severe multiple trauma a decrease in the ability of rbc to change their shape during electrochemical exposure was observed, indicating a decrease in deformability, which can lead to a disruption in the oxygen supply to tissues. at the same time, with the stabilization of the patient's condition a restoration of the ability of rbc to change morphology was detected which in turn could have a positive effect on the rheological characteristics of the blood (fig. ) . the results of the analysis of red blood cells using electrochemical changes in their morphology can be used as an additional method for the diagnosis of critical conditions. severe trauma should be treated immediately. whole-body ct (wbct) is widely accepted to improve the accuracy of detecting injuries. however, it remains the problem of time-consuming. therefore, we focused on the scout image taken in advance of wbct. detecting major traumatic injuries from a single scout image would reduce the time to start treatment. a previous study suggested that even specialists could not easily find chest and pelvic injuries using wbct scout image alone. in this study, we aimed to develop and validate deep neural network (dnn) models detecting pneumo/hemothorax and pelvic fracture from wbct scouts. we retrospectively collected anonymous wbct scouts together with their clinical reports at the osaka general medical center between january , , and december , . we excluded incomplete, younger than years old, postoperative, and poorly depicted images. the part of this dataset from january , , until december , , was used for validation and the rest for training dnn models. pneumo/hemothorax detection model and pelvic fracture detection model were trained respectively. accuracy, and areas under the receiver operating characteristic curves (aucs) were used to assess the models. the training dataset for pneumo/hemothorax contained images (mean age years; % female patients), and for pelvic fracture consisted of images ( years; %). the validation dataset for the former contained images ( years; %), and for the latter consisted of images ( years; %). the models achieved % accuracy and an auc of . for detecting pneumo/hemothorax, % and . for pelvic fracture. our results show that dnn models can potentially identify pneumo/ hemothorax and pelvic fracture from wbct scouts. increasing the number of samples, dnn model could accurately detect severe trauma injuries using wbct scout image. clinical information system (cis) is a computer system used in collecting, processing, and presenting data for patient care. it can reduce staff workload and errors; help in monitoring quality of care; track staff's compliance to care bundles; and provide data for research purpose. however, the transition from paper record format to electronic record involves changes in all kind of workflow in icu. therefore, an effective, efficient and evaluative rollout plan was required to minimize the risk that might arise from the new practice. methods: . small groups training were provided. a working station with different case scenarios were set up for practices. . individual tutorials were conducted to clarify questions. emphasis on patient care was always top priority. . contingency plans were available in case of server breakdown and power failure. downtime drills were conducted to prepare the staff in emergency situations. . step-by-step transition from paper record to electronic format was gradually carried out. a plan was discussed among cis team with clear dates and goals. . new items in cis were first reviewed and amended in team meeting until consensus was made; then were promulgated to all staffs during handover before implementation. fig. (abstract p ) . the effect of therapy on the electrochemically induced change in the morphology of red blood cells in patients with combined trauma . staff compliance and outcomes were then monitored; further review and amendment would be possible if necessary. cis roll-out plan was smooth. all staffs were able to integrate cis into the daily routine. the contingency plans were well acknowledged. new items were followed as planned. ongoing enhancement in cis was put forward on nursing orders, handover summary, and integration with inpatient medication order entry (ipmoe) system. with emerging benefits cis brings along, our staff has more time to devote to direct patient care. human input in data interpretation and clinical judgment on top of cis play an irreplaceable role in patient care. the daily request for laboratory tests in intensive care units is a common practice. although common, this strategy is not supported, since more than % of the exams requested with this rationale may be within the normal range [ ] . misconduct based on misleading results, anemia, delirium and unnecessary increase in costs may happen [ ] . we have developed a strategy to reduce laboratory tests without clinical rationale. observational retrospective study, from july to june . the number and type of laboratory orders requested, the epidemiological profile of hospitalized patients, the use of advanced supports, the average length of icu stay and the impact in outcomes such as mortality and hospital discharge at a private tertiary general hospital in the city of rio de janeiro / rj -brazil were analyzed. a strategy was implemented to reduce the request for exams considered unnecessary. approximately , patients underwent icu during this period. the epidemiological profile and severity of patients admitted to the unit were similar to those observed historically. there was a significant reduction (> %) in the request for laboratory tests and there was no negative impact on outcomes such as mortality, mean length of stay and no greater use of invasive resources. over the period evaluated, the estimated savings from reducing the need for unnecessary exams were approximately $ , per year. the rational use of resources in the icu should be increasingly prioritized and the request for routine laboratory tests reviewed. a strategy that avoids such waste, when properly implemented, enables proper care, reducing costs and ensuring quality without compromising safety. evaluating the medication reconciliation errors in icus after implementing a hospital-wide integrated electronic health record system a rosillette, r shulman, y jani university college hospital, centre for medicines optimisation research and education, london, united kingdom critical care , (suppl ):p introduction: medication errors in intensive care unit (icu) are frequent [ ] and can arise from a number of causes including transition of care. our aim was to investigate the impact of an integrated electronic health record system (ehrs) on medication reconciliation (mr) errors occurring at critical steps: during the transition from an icu to the hospital ward and from the ward to hospital discharge. the objective was to examine the influence of icu admission on long-term medication. we performed a monocentric study in icus of a university-affiliated hospital using drug chart and medical notes review to identify mr errors before, during and after icu admission. data were collected retrospectively from ehrs for consecutive patients discharged from the icu between june- july , and who were newly initiated on specific drugs of interest. results: drugs of interest were initiated in icu. many of these were continued after hospital discharge as shown in table . there was appropriate discontinuation of all the antipsychotics newly initiated in icu. other than anticoagulants, there was no reason documented for continuation of the initiated drugs. the planned durations were documented more often after hospital discharge than icu discharge for the following drug classes (% of patients with a plan after icu discharge to the ward; % after home discharge): antibiotics ( . %; . %), and steroids ( . %; . %), but less so for analgesics ( . %; . %), insomnia ( . %; . %), and gastroprotective drugs ( . %; . %). our study has shown that medications initiated in the icu can be inadvertently continued at icu and hospital discharge due to failure in documenting indication or duration. systems are required to deprescribe icu only drugs at discharge or communicate a plan for ongoing treatment. introduction: the surviving sepsis campaign advocates the use of care bundles to guide the management of sepsis and septic shock [ ] . our study aim was to assess compliance with a locally introduced sepsis pathway and to review intensive care unit admission outcomes. we carried out a prospective audit of patients admitted to the icu at royal surrey county hospital with a diagnosis of sepsis between / / and / / , assessing compliance with local sepsis bundle delivery, outcome of icu admission and degree of associated organ dysfunction. results: patients were identified, male ( . %), with a mean age of . ( - ). mean st hour sofa score on icu was . ( - ). % of patients required vasopressors, with % requiring noradrenaline > . mcg/kg/min, and % requiring an additional vasopressor/ inotrope. % required niv, % invasive ventilation and % rrt. icu mortality was %, in-hospital mortality %, mean icu stay days ( - ), and mean length of hospital stay days . in the presence of septic shock mortality was % with post-resuscitation lactate > , versus % in patients with no vasopressor requirement or lactate < (p< . ). the sepsis bundle was delivered in one hour to patients ( %). where the bundle wasn't completed, antibiotics were delayed in % of cases and blood cultures weren't taken in %. where the bundle was fully delivered, unit mortality was % vs. % where it was not (p< . ), but there was no significant difference in hospital mortality ( % vs. %, p> . ) or rates of vasopressor requirement, niv, ippv or rrt. there is room for improvement in timely delivery of the sepsis bundle in our hospital and various measures are being instituted. though there was no significant difference in hospital mortality, icu mortality was significantly lower in patients when the bundle was fully delivered. surviving sepsis campaign recommends h and h sepsis resuscitation bundle for sepsis. the study was done to assess the feasibility of the guideline and the compliance to sepsis- recommendations at an emergency department. prospective interventional study was conducted during one year. were involved in the study all sepsis cases with a qsofa ≥ . were assessed a composite of six components (measurement of serum lactate, obtaining blood culture before antibiotic administration and provision of broad-spectrum antibiotic before the end of h and provision of fluid bolus in hypotension, attainment of target central venous pressure assessed by cardiac ultrasonography, target lactate to normal level before the end of h ). time base line was the first medical contact at triage zone. secondary outcomes of study were the mortality rate and length of stay at intensive care unit (icu). were involved in the study, patients (mean age ± years, sex ration , ). pulmonary infections were the main cause of sepsis ( %) and urinary tracts infections ( %). at h components were achieved in % of cases [lactates ( %), blood culture ( %) and provision of antibiotics ( %)]. at h components were executed in % of cases (fluid provision achievement in %, ultrasonography assessment in % and normal lactate target achieved in %) (figure ). the reliability-adjusted rate for completion of the hours and hours bundle was at %. patients compliant to composite bundle got the mortality benefit (odds ratios = . , % [confidence interval, . - . ]). the study, however, did not show any benefits of mean intensive care unit (icu) length of stay. faisability of - h bundle ratio was at %. it has shown a significant improvement in adaptation and mortality benefit without reducing mean hospital/icu length of stay. more adapted procedures are needed to improve results targeting full compliance of patients to the - h bundle sepsis management. patterns and outcome of critical care admissions with sepsis in a resource limited setting m edirisooriya maddumage , y gunasekara , d priyankara national hospital of sri lanka, medical intensive care unit, colombo , sri lanka; sri jayawardenepura general hospital, department of critical care, nugegoda, sri lanka critical care , (suppl ):p introduction: paucity of epidemiological data is a major barrier in expansion of critical care services, especially in resource limited settings. we evaluated the patterns and the outcome of critically ill patients with sepsis admitted to a level medical intensive care unit in sri lanka. a retrospective cohort study was performed to describe the characteristics and outcome of patients with sepsis, admitted to a medical intensive care unit. sepsis is defined according to sepsis definition. we examined critically ill patients admitted over a period of months. sepsis was the commonest presentation, accounted for . % of all admissions. mean age was . ± . years. septic shock was present in . % on admission. pneumonia ( . %) was the commonest cause, while leptospirosis ( . %) and meningoencephalitis ( . %) accounted for fig. (abstract p ) . sepsis - h bundle components (% of goals achievment) second and third commonest causes of sepsis respectively. the sofa score on admission ( . ± . vs . ± . , p< . ), occurrence of aki ( % vs . %, p< . ) and the length of icu stay ( . days vs . days, p < . ), were significantly higher in sepsis than in patients without sepsis. icu mortality in sepsis (n= ) did not show a significant difference to nortality (n= ) in those without sepsis ( % vs %, p= . ). patients with leptospirosis had a mean sofa score of . , however the mortality ( . % vs %, p = . ) was similar to others with sepsis. in contrast, mortality related to sepsis was significantly high ( %, p< . ) in the packground of immunosuppression (n= ). respiratory failure secondary to pneumonia was the commonest cause of critical care admission with sepsis. sepsis related icu mortality was high in the background of immunosuppression. introduction: training in placement, and the subsequent safe confirmation of position, of a nasogastric (ng) tube, relies on clinicians completing an e-learning module at our trust. feeding through an incorrectly placed ng tube is a 'never event,' associated with significant morbidity and mortality [ ] . analysis of these incidents reveal that the misinterpretation of chest radiographs, by medical staff, who had not received competency-based training, is the most frequent cause [ ] . e-learning has revolutionized the delivery of medical education [ ] , however, there are barriers to its use [ ] . we hypothesized that, by taking e-learning content, and delivering it face-to-face, we would improve training rates, and thus patient safety. a questionnaire was completed by critical care doctors, concerning their knowledge of the existence of the e-learning module, whether they had completed formal training in ng tube placement, and how confident they were, on confirming correct positioning, using a point likert scale. all clinicians underwent training in the interpretation of ng placement, using chest radiographs. after the session they were asked to re-appraise how confident they felt. results were compared using paired t tests. confidence improved in all, rising from a pre-test average score of . (sd= . ), to post-session . (sd= . ), p=< . . prior to the intervention, % of the doctors were aware of the trust guidelines, but only % had completed the training. after the session, % were aware of the guidelines, and % had completed the training (figure ) . conclusions: e-learning is a useful tool, but has its limitations. by using course content, delivered with more traditional learning methods, we im-proved the number of appropriately trained clinicians, and thus the safe use of ng tubes in our unit. a systematic review of anticoagulation strategies for patients with atrial fibrillation in critical care a nelson, b johnston, a waite, i welters, g lemma university of liverpool, liverpool, united kingdom critical care , (suppl ):p there is a paucity of data assessing the impact on clinical outcomes of anticoagulation strategies for atrial fibrillation (af) in the critical care population. this review aims to assess the existing literature to evaluate the effectiveness of anticoagulation strategies used in critical care for atrial fibrillation. only studies contained analysable data. anticoagulated patients had a lower mortality at days and days post admission to critical care, however there was an increased incidence of major bleeding events compared to the non-anticoagulated population. thromboembolic events were comparable in both cohorts. data from current literature is scarce and inferences regarding the effectiveness of anticoagulation in patients in critical care with af requires further investigation and research. every new admission to the icu prompts a handover from the referring department to the icu staff. this step in the patient pathway provides an opportunity for information to be lost and for patient care to be compromised. mortality rates in intensive care have fallen over the last twenty years, however, % of patients admitted to an icu will die during their admission [ ] . communication errors contribute to approximately two-thirds of notable clinical incidents; over half of these are related to a handover [ ] . nice have concluded that structured handovers can result in reduced mortality, reduced length of hospital stay and improvements in senior clinical staff and nurse satisfaction [ ] . a checklist was created to review the information shared and to score the handover. this checklist was created with doctors and nurses and is relevant for handovers between all staff members. information was gathered prospectively by directly observing handovers on the icu. there is a notable discrepancy in the quality of handovers of new patients ( figure ). this is true of handovers between doctors, nurses and a combination of the two. it is also true of all staff grades. whilst a doctor may have reviewed the patient prior to their arrival, % (n= ) of patients weren't handed over to a doctor. the most commonly missed pieces of information were details of the patient's weight ( %, n= ), their height ( %, n= ), whether the patient has previously been admitted to an icu ( %, n= ) and whether the patient has any allergies ( %, n= ). the handover of new patients to the icu is often unstructured and important information is missed. this can be said for all staff members and grades, and for handovers from all hospital departments. post intensive care syndrome-family (pics-f) describes new or worsening psychological distress in family and caregivers after critical illness but remains poorly studied within specialist groups [ ] . we aim to define the degree of pics-f within our tertiary referral cardiothoracic centre and map change over the course of months. caregivers attended a -week multi-professional clinic alongside patients. peer support was facilitated through a café area and a caregiver group psychology session was offered with individual appointments if required. caregiver surveys were completed including: caregiver strain index; hospital anxiety and depression scale (hads); and insomnia severity index. patients also completed hads questionnaires. repeat surveys were completed at and months. results: over cohorts, caregivers attended, of which were spouses ( %), children ( %), and others ( %), with caregivers completing surveys at months. patients' median apache score was (iqr - . ) and median icu length of stay was days (iqr - . ). most admissions were from scheduled operations ( %). severe caregiver strain was present in / ( %) with changes to personal plans ( %) the most common sub category. hads demonstrated caregivers ( %) with anxiety and ( %) with depression. caregiver anxiety exceded that of patients', only reaching fig. (abstract p ) . each handover was scored according to the information accurately given to icu staff similar levels at months, while depression remained static ( figure ). median number of nights with 'bothered' sleep was (iqr - . ) and % of caregivers expressed problems with sleep. conclusions: significant psychological morbidity in caregivers from our tertiary cardiothoracic centre is in keeping with the general icu population [ ] . caregiver strain was reduced suggesting higher levels of resilience. future work should address mental wellbeing, particularly anxiety, to minimise the effects of pics-f. burnout syndrome is an illness that has increasingly affected health professionals. it is characterized by great emotional stress, physical and mental exhaustion and depersonalization of the individual. more serious cases can lead to job loss or even suicide. the described work identifies the burnout level of the multidisciplinary team through a specific questionnaireburnout syndrome is an illness that has increasingly affected health professionals. it is characterized by great emotional stress, physical and mental exhaustion and depersonalization of the individual. more serious cases can lead to job loss or even suicide. the described work identifies the burnout level of the multidisciplinary team through a specific questionnaire methods: application of a questionnaire suitable for the multidisciplinary group in november . the same was answered by professionals among physicians and nursing team. there was no identification of employees. after analysis of the results it is observed that % of the group presents initial burnout, % with the syndrome installed and about % with characteristics of greater severity. main factors found were: mental and physical exhaustion during the work day, the level of responsibility existing in the activity and the perception of disproportionate remuneration by work performed. all interviewees presented some degree of burnout or high risk to develop it. the most severe cases should be traced through occupational medicine and anti-stress measures with reorganization of work performance should be discussed in order to reduce the prevalence of this syndrome. introduction: burnout affecting the psychological and physical state of healthcare workers is recognized in the last years. burnout has been shown to affect the quality of care. whilst some risk factors have been identified, there are gaps within the literature related to mental health and burnout. the aim of this study is to measure levels of burnout across icu units in the metropolitan setting. to determine the level of burnout we used surveys, the maslach burnout inventory human services survey (mbi-hss) and the centre for epidemiologic studies depression scale (ces-d). with the mbi-hss we analysed different variables of burnout; exhaustion, cynicism and emotional exhaustion. basic demographic data and information regarding workout schedules were collected. we studied prevalence and contributing risk factors using and analysing the outcomes of the self-scoring questionnaires. analysis was performed using descriptive statistical analysis. there were respondents, % scored the threshold for depressive symptoms on the ces-d depression scale. interestingly, % (ci . - . %) of those meeting the score for depressive symptoms identified as having frequent restless sleep compared with % ( . - . %) from those not meeting. gender did not affect depressive symptoms % of females and % of males met the threshold. with the mbi-hss for exhaustion the mean was . (sd . ) which is a high level of exhaustion, the second variable cynicism the mean score was . (sd . ), which was considered high. the final variable was emotional exhaustion the mean was . (sd . ), this is considered moderate levels of emotional exhaustion. fig. (abstract p ) . hospital anxiety and depression scale (hads) scores for patients and caregivers at baseline, months, and months there was high prevalence of burnout in icu in all different categories as well as depressive symptoms. age and gender had no affect on burnout. interestingly, we identified that sleep and shift variables were linked to increased burnout. following the implementation of a fully integrated ehrs on march at our university-affiliated hospital we conducted a prospective study in icus by analysing pharmacists' contributions during data collection periods of days at , , and weeks post implementation. a pharmacists' contribution was defined as contacting the physician to make a recommendation in a change of therapy/ monitoring [ ] . the types of contribution were: a medication errorrectification of an error in the medication process; an optimizationproactive contribution that sought to enhance patient care, and a consult -reactive intervention in response to a request. a panel of experts composed of a senior pharmacist, a consultant, a nurse, and a pharmacy student assessed the impact of each contribution, scoring low impact, moderate impact or high impact. there were pharmacist contributions recorded in the periods. of these, ( . %) were medication errors, ( . %) were optimizations, and ( . %) was a consult ( table ) . % of the contributions were assessed as having medium impact, % as high impact and % as low impact. in general, the consultant assessed fewer contributions as having high impact compared to other members of the panel, with contributions assessed as high impact by the consultant versus by the senior pharmacist. implementing an ehrs in combination with contributions of clinical pharmacists can prevent medication related issues. interestingly the types of incident did not change over time. introduction: most icu's are noisy and may adversely affect patients outcomes and staff performance [ ] . who reports that the noise level in hospitals should not exceed db at daylight and db at night. the aim of this study is to evaluate the noise levels in intensive care unit, to apply awareness training to intensive care staff in terms of noise and to compare the noise levels before and after education. noise measurement areas are separated into points including patient bedsides, nurse desk, staff desk, wareroom, corridor and entrance of intensive care unite. measurements were performed times per day. after day, awareness training were given to staff in terms of harmful effects of noise. after the training, noise measurements were repeated during days. after total days the measurements were terminated. noise was measured with incubator analyzer (fluke model: bio-tek serial no: ). the mean noise values before and after the training were not statistically different from the mean average noise values (p> . ). when the time of measurement were compared, the noise levels were higher between - hours to other measurements before and after the training statistically (p= . ). seventeen different noise measurement areas were compared in terms of noise level, there was no statistically significant difference (p> . ). the differences were examined at the same hours between before and after training. contrary to expectations, noise levels were found to be higher after training statistically (p< . ). all of noise measurements were higher than the threshold values that who recommended. increased noise levels in critical care units may lead to harmful health effects for both patients and staff. our results suggest that much noise in the icu is largely attributable to environmental factors and behavior modifications due to education have not a meaningful effect. critical care medicine has focused on continuous, multidisciplinary care for patients with organ insufficiency in the face of lifethreatening illness. despite significant resource limitation low income countries carry a huge burden of critical illness. available data is insufficient to clearly show the burden and outcomes of intensive care units in these developing countries [ ] . the objective of our study is to evaluate the morbidity and outcomes of patients admitted to the intensive care unit of a tertiary university hospital in hawassa, ethiopia. this was a prospective observational study. data was registered and analysed starting from patient admission to discharge during a month period beginning september . data regarding demographics, sources of admission, diagnosis, length-of-stay and outcomes were analysed. the total number of patients admitted to the icu was , with patients dying over a one year period. the highest admission was from emergency medical unit, % and the lowest source was from pediatrics department, %. out of these, . % were males. the mean age was years ( - ). the most frequent aetiologies of morbidity in the admitted patients were traumatic brain injury ( . %), acute respiratory distress syndrome ( . %) and seizure disorder ( %). average median length of stay was . days (interquartile range: . - . ). the overall mortality rate was . %. the top four causes of death in the icu were respiratory illness at % followed by sepsis with multiorgan failure at %, trauma ( %) and central nervous system infection ( %). infection morbidity and mortality remains very high and needs institution of aggressive preventive strategies. the increase in frequency of trauma patients need to receive due attention. sepsis causes a high number of deaths, though overtaken by respiratory illnesses. improving the overall system of icu may achieve better outcomes in resource limited countries. introduction: icu mortality has been widely studied in the literature in relation to outcome index that primarily value organic failure [ ] . however, early mortality, in the first hours of admission has been little documented in the literature. the aim of this study is to analyze factors related to early mortality in icu. retrospective study at a second-level hospital. time of study was months. patients who died in icu were included, patients were classified according timing of dead, including those who died within the first hours of icu admission. the variables analyzed were age, sex, comorbidity, charlson index, apache ii, need for supportive treatments, more frequent admission diagnosis, origin and support treatment limitation decisions. the statistical study was carried out using the spss statistical program. patients were included during the study period, ( . %) died within the first hours of admission. no differences in the needs of support treatments were observed, more than % of patients received mechanical ventilation and vasoactive therapies. table shows characteristics of patients. half of icu deaths occur within the first hours of admission. severity at icu admisison was the main factor related with early mortality. severe stroke and coronary disease were the most frequent causes of early deaths in icu. in august the royal college of anaesthetists published guidelines on care of the critically ill woman in childbirth and enhanced maternal care [ ] . approximately babies are born across the area covered by leicester university hospitals that includes two large maternity units and is part of the uk ecmo network. this audit sets out to assess current practice and form a basis for future planning, which will likely be representative to most major obstetric centres. a retrospective audit of all patients admitted to 'intensive care units' in leicester over a month period following publication of the guidelines. the focus was on patients admitted to general adult intensive care and excludes all patients cared for in 'enhanced obstetric care' units. simple standards were proposed relating to accessibility, resuscitation, follow up and multi-disciplinary learning. in total women were identified with a broad range of diagnosis. the intensive care services are split across hospitals and we found this led to a number of problems. the presence of trained staff to resuscitate a newborn were easily accessible, no steps to provide necessary equipment pre-emptively were present in any centre. none of our critical care units had a plan for perimortem section. on-going reviews by the obstetric and midwifery teams were very variable. contact with the infant and breastfeeding support was also poor. despite the large number of deliveries significant work needs to be done in order to come in line with the new national guidelines for critically ill woman in childbirth. clearly defined pathways around escalation of care, resuscitation of both the mother and baby, integrating care of the mother and the infant in the first few days of life, and multidisciplinary learning events are being produced de novo in response to these guidelines, some of which will be illustrated in the associated poster. interprofessional collaboration scale [ ] . data were analyzed with ibm spss . results: it was found that cooperative attitudes with an average score of to are considered to be of average significance. interprofessional cooperation at an average score of , states that the level of cooperation is high and the quality of working life averages to , suggesting that it is very good. as far as professional satisfaction is concerned, nurses are happy, content and satisfied with their work, despite workload and burnout conclusions: interprofessional cooperation at the icu of the general hospital of larissa is high, but satisfaction from wages, resources, working environment and conditions is low. in addition, the results showed that improvements in hospital communication between staff, has a positive impact on the quality of professional life (table ) . contrasting with previous reports, decreased admissions per unit population in older and oldest age groups, and those with high comorbidity, suggest resource constraints may have influenced admission discussion and decision-making over the -year study period in wales. further investigation is warranted. icu discharge into weekends and public holidays: an observational study of mortality n mawhood, t campbell, s hollis-smith, k rooney bristol royal infirmary, general intensive care unit, bristol, united kingdom critical care , (suppl ):p introduction: up to a third of in-hospital deaths in icu patients occurs following ward stepdown [ ] . discharge time seems to be associated with in-hospital prognosis, but meta-analyses have not shown a difference in weekday compared to weekend discharge [ , ] . however, papers that examined discharge 'into' out-of-hours days, particularly on fridays, have found differences [ ] . our aim was to assess whether discharge from icu 'into' out-of-hours (ooh -weekends and public holidays) is associated with in-hospital mortality or re-admission to icu, and whether these patients were seen on the wards ooh by medical staff. all adults discharged from the general icu to a ward at the bristol royal infirmary in december - were included. in-hospital mortality rates were assessed for each day, with 'into weekdays' defined as sunday to thursday and 'into ooh' friday, saturday and the day before a public holiday. a subset of patients with data on readmission rate to icu was also examined. all available notes from patients discharged into ooh in were reviewed. the study included patients with a subset of with readmission data. sets of notes were reviewed from patients discharged into ooh (figure ). the in-hospital mortality was significantly higher in patients discharged into ooh ( . % vs . %, p= . ). within the subset, ooh was associated with in-hospital mortality or readmission to icu ( . % vs . %, p= . ), though readmission rate alone was not ( . % vs %, p= . ). of patients discharged into ooh, once on a ward % were reviewed by a specialty doctor but . % were not seen. this is the first study to examine icu discharge 'into' ooh days including public holidays. we found increased hospital mortality in ooh, similar to other studies [ ] . up to a fifth of high-risk icu stepdown patients were not reviewed by a doctor on ooh days. exploring the experiences of potential donors' family members (fm) in a follow up clinic is crucial to analyze the effects of organ procurement (op) on the bereavement process, to gain insight on the reasons of family refusals (fr), and to improve family care during op. a mixed-method study involving fm at and months after patients' death was developed and approved by local ethics committee. fm of potential donors after brain (dbd) and cardiac death (dcd) treated in careggi teaching hospital, florence (italy) were eligible if adult and consenting. invitation letters were sent to the entitled months after death and those who actively responded were involved in an encounter with a multidisciplinary group including a clinical psychologist, two nurses and two cultural anthropologists with expertise in op. organ replacement procedures such as ecmo (extracorporeal membrane oxygenation), lvad (left ventricular assist device) and dialysis are routinely used to treat multi-organ failure (mov). globally transplantation programs struggle with increasing organ shortage. patients (pts) with mov are a potential source for procurement. however, outcome data after kidney transplantation (ktx) from such donors are sparse. we retrospectively studied the cadaveric ktx at the charité berlin in and identified donors with ongoing organ replacement procedures. donor and recipient risk factors were assessed. overall patient and graft outcomes were analyzed at months post-transplant. a total of kidneys were transplanted. we identified ktx from donors with mov ( following cardio-pulmonary resuscitation, with acute renal failure - on dialysis) (figure ). in donors, a venoarterial ecmo was implanted during ecls-resuscitation. one donor needed a veno-venous ecmo due to ards, and donor had a lvad implanted due to cardiac failure. the donor age was ± . years (yrs). in addition, donors had at least one cardiac risk factor. the kidney donor risk index averaged . (sd ± . ) and s-creatinine prior to ktx was . (sd ± . one way to expand the potential donor pool is donation after circulatory death (dcd), and a strategy to reduce the complications related to the ischemic time is the use of normothermic regional perfusion (nrp) with extracorporeal membranous oxygenation (ecmo) [ , ] . we compare the use of standard nrp with an effective adsorption system inflammatory mediators (cytosorb®) in the regional normothermic reperfusion phase via regional ecmo, that involves a reduction in cellular oxidative damage, assessed as a reduction in levels of proinflammatory substances. we report a case series of dcd-maastricht iiia category donors, treated in ecmo with nrp, to maintain circulation before organ retrieval, in association with cytosorb® in patients. during perfusion, from starting nrp (t ), blood samples are collected times, every minutes (t , t , t ). during treatment with cytosorb®, lactate levels progressively decrease, ast and alt increase less than without cytosorb®, as sign of improvement in organs perfusion ( figure ). nrp with cytosorb® might help to successfully limit irreversible organ damages and improve transplantation outcome [ ] . development and implementation of uniform guidelines will be necessary to guarantee the clinical use of these donor pools. introduction: shock is a common complication of critical illness in patients in intensive care units (icus), who are undergoing major surgery. this condition is the most common cause of death in postsurgical icus. nowadays, there are different icu scoring systems for predicting the likelihood of mortality, such as apache or sofa. nevertheless, they are used rarely because they also depend on the reliability and predictions of physicians. in these sense, gene expression signatures can be used to evaluate the survival of patients with postsurgical shock. methods: mrna levels in the discovery cohort were evaluated by microarray to select the most differentially expressed genes (degs) between groups of those that survived and did not survive days after their operation. selected degs were evaluated by quantitative real time polymerase chain reactions (qpcr) for the validation cohort to determine the reliability of the expression data and compare their predictive capacity to that of established risk scales. introduction: this study evaluates the prognostic ability of frailty and comorbidity scores in patients with septic shock. the -day mortality rate of individual medical conditions are also compared. the burden of comorbid illness and frailty is increasing in the critical care patient population [ ] . outcomes from septic shock in patients with chronic ill-health is poorly understood. interstitial lung disease is a group of diseases associated with poor prognosis in the intensive care unit despite major improvement in respiratory care in the last decade. the aim of our study is to assess factors associated with hospital mortality in interstitial lung disease patients admitted in the intensive care unit and to investigate the long-term outcome of these patients. we performed a retrospective study in an intensive care unit of teaching hospital highly specialized in interstitial lung disease management between and . a total of interstitial lung disease patients were admitted in the intensive care unit during the study period. overall hospital mortality was %. two years after intensive care unit admission, / patients were still alive ( %). one hundred eight patients ( %) required invasive mechanical ventilation of whom % died in the hospital (figure ). acute exacerbation of interstitial lung disease was associated with hospital mortality (or= . [ . - . ] ), especially in case of acute exacerbation of idiopathic pulmonary fibrosis. multiorgan failure (invasive mechanical ventilation with vasopressor infusion and/or renal replacement therapy) was associated with very high hospital mortality ( / ; %). survival after intensive care unit stay of patients with interstitial lung disease is good enough for not denying them from invasive mechanical ventilation, except in case of acute exacerbation for idiopathic pulmonary fibrosis patients. if urgent lung transplantation or extracorporeal membrane oxygenation are ruled out, multiorgan failure should lead to consider withholding or withdrawal life support therapies. Αgi is a malfunctioning of the gi tract in icu patients associated with prolonged mechanical ventilation, enteral feeding failure and high mortality risk. the wgap of esicm proposed a grading system for agi. four grades of severity were identified: agi grade i, a selflimiting condition; agi grade ii (gi dysfunction), interventions are required to restore gi function; agi grade iii (gi failure); agi grade iv, gi failure that is immediately life threatening. the aim was to evaluate the feasibility of using agi grades i and ii as predictors of malnutrition and -year mortality in critically ill patients methods: single-center retrospective cohort study in a tertiary university hospital ( - ). agi grade iii and iv patients were excluded. Αnthropometric data, gi symptoms (vomiting,diarrhea), feeding intolerance, gastric residual volumes and abdominal hypertension were recorded. daily prescribed caloric intake was calculated using a standard protocol and daily achievement of caloric intake was recorded. mnutric score was calculated for all patients. a score ≤ was used to diagnose malnutrition. patients ( % men, mean age years) that stayed in the icu for > hours were included in the study. % were at high nutritional risk. -year mortality was %. the prevalence of agi ii was %. age, gender, bmi, mortality and energy intake did not differ significantly between patients with agi ii and those with agi i (table ) . logistic the study aimed to assess the effects of icu admission on frailty and activities of daily living in the ≥ 's population at -months. a prospective observational study with data used as a subset of the vip- trial [ ] . research ethics committee approval from the mater misercordiae university hospital (mmuh). inclusion criteria -≥ years of age and acute admission to icu from may to july . data collected on consecutive patients. frailty and activities of daily living (adl) were assessed using the clinical frailty score (cfs) and the katz index of independence in activities of daily living (katz). results: csf pre-admission frailty was present in % of patients, increasing to % at months ( figure ). % of survivors at -months had a cfs score increase by ≥ point. pre-frail and frail cfs patients suffered an average -point deterioration in their instrumental activities of daily living (iadl). % of katz patients were fully functional preadmission, deteriorating to % at months. % of patients declined by adl at months. % of the deceased were deemed fully functional initially. we demonstrate an association between an icu admission event and enduring functional decline at months. icu admission resulted in patients acquiring on average . new iadl limitations despite their initial cfs. this is echoed in a study by iwasyna et al. who also showed similar deteriorations in iadl and cognitive impairment [ ] . katz benefits may be best used in describing functional decline. % of patients developed at least one new limitation. however, the cfs takes into account iadl's and thus may be more sensitive in predicting the functional outcomes of an icu event at months. frailty: an independent factor in predicting length of stay for critically ill t chandler, r sarkar, a bowman, p hayden medway maritime hospital, critical care, gillingham, united kingdom critical care , (suppl ):p frailty has attracted attention in the healthcare community in recent years, as it is associated with worse outcomes and increased healthcare costs [ ] . our objective was to study the impact of frailty as recorded by clinical frailty scale(cfs) to prospectively evaluate the effect of frailty on hospital length of stay (los). a retrospective analysis of consecutively admitted critical care (cc) patients' data (jan' -oct' ) was performed. electronic health records were used to collect demographics, cfs and clinical outcomes. statistical analysis was performed using stata. students t-test, simple and multiple (adjusted for age, disease severity/icnarc score) linear regression were used for comparison between groups and to see group effect. we excluded extreme outliers (los> days; n= ). frailty was defined as cfs> . out of the patients (male %), ( %) were emergency admissions, the rest elective (table ) . ( %) were non-frail. the mean los were days (d) ± and d± (p< . ) in the frail and non-frail patients respectively. for emergency patients, los were d(± ) and d(± ) for the groups, (p< . ). for elective patients; los were d(± ) and los d(± ), (p= . ) for frail and nonfrail respectively. after adjusting, los was significantly higher in frail patients by days ( %ci , ; p< . ), by days ( %ci , ; p= . ) and by days ( %ci , ; p< . ) for total cohort, elective and emergency admissions respectively. the los was days higher in frail than non-frail (p< . ) for cc survivors. frailty was associated with significantly increased los in this cohort, independent of age and illness severity. hospital capacity planning should take this into consideration when modelling bed allocation fig. (abstract p ) . clinical frailty score -month trend robust clinical governance requires analysis of patient outcomes during an icu admission [ ] . on one adult icu weekly mortality meetings are used for this purpose and aid multidisciplinary reflections on individual patient deaths. however, such reviews run the risk of being subjective and fail to acknowledge themes which may relate to preceding or subsequent deaths. this paper describes a new mortality review process in which: a) reviews are structured using the structured judgement review (sjr) framework [ ] ; and b) themes are generated over an extended period of time to create longitudinal learning from death. the sjr framework has been developed by nhs improvement for the new medical examiner role, looking at inpatient deaths. we adapted this to better suit the icu creating a novel review structure. this involves explicit judgement comments being recorded, and the use of a scoring system to analyse the quality of care during the patient's stay with a focus on elements of care delivered on the icu. tabulation of this information allows analysis over time, identifying trends across all patients, and in specific subgroups. this framework has been rolled out at the st george's cardiothoracic icu weekly mortality meetings. themes that have emerged include parent team ownership, delayed palliative care referrals and inadequate documentation of mental capacity. this will continue as part of a three-month trial and following review of this trial may be extended to other critical care units in the trust. this system allows greater insight into patient deaths in a longitudinal fashion and facilitates local identification of problems at an early stage in a way that is not possible within the traditional mortality review format. the nature of the process means that key areas for change can be identified as a routine part of the clinical week. [ ] . in this study, we evaluated three distinct machine-learning methods for predicting possible patient deterioration after surgery. the data was collected retrospectively from the catharina hospital in eindhoven. this dataset contained all the surgeries conducted in the hospital from up to . the variables in this dataset were tested on their ability to differentiate between patients with a normal recovery versus patients with an unplanned icu admission after being admitted to the ward. the dataset contained variables related to either the preoperative screening, surgery or recovery room. all variables were tested for statistical significance using a univariate logistic regression (lr), from which a subset of statistically significant (p< . ) variables was created. these variables were used to train three different types of models, namely, the lr, support vector machine (svm) and bayesian network (bn). the network structure of the bn was designed using expert knowledge and the probabilities were inferred using the data. the three models were validated using five-fold cross-validation, resulting in the following areas under the receiver operating characteristic curve: . ( . - . ) for lr, . ( . - . ) for svm and . ( . - . ) for bn (fig. ) . the results indicate that machine learning is a promising tool for early prediction of patient deterioration. the bn was included because it permits incorporating clinical domain knowledge into the learning process. however, its performance resulted inferior to the lr and svm. in future work, we will investigate alternative domainaware methods, and compare the performance with that of the clinical experts. intensive care unit (icu) admission decisions of patients with a malignancy can be difficult as clinicians have concerns about unfavourable outcomes, such as mortality [ ] . a diagnosis of a malignancy is associated with an almost -fold increased likelihood of refusal of icu admission [ ] . recent large long-term mortality studies of patients with a malignancy admitted to the icu are scarce. therefore, our aim was to compare mortality of patients with either a hematological or a solid malignancy to the general icu population, all with an unplanned icu admission. all adult patients registered in a national intensive care evaluation registry with an unplanned icu admission from to were included. subsequently, we divided these patients into cohorts: cohort (all patients with a hematological malignancy), cohort (all patients with a solid malignancy), and cohort (a general icu population without malignancy). as primary outcome, we used -year mortality, and as secondary outcome, icu and hospital mortality. we included , ( . %) patients in cohort , , ( . %) patients in cohort and , ( . %) in cohort ( table ). the year mortality of patients of cohort , , and was . %, . % and . %, respectively (p< . ). age, comorbidities, organ failure, and type of admission (i.e. surgical or medical) were positively associated with -year mortality in all cohorts (p < . ). one-year mortality is higher in both patients with a hematological malignancy and patients with a solid malignancy compared to the general icu population. in addition, several factors were positively associated with -year mortality, i.e., age, comorbidities, medical icu admission, and organ failure. future research should focus on predictive modelling in order to identify patients with a malignancy that may benefit from icu admission. introduction: drug abuse is associated with immunosuppression in multiple mechanisms. despite that, the only study retrospectively reviewing drug abusers in the icu demonstrated less infections and better outcomes. we compared matched patient populations in order to fully understand whether drug abuse is a risk factor for infection and a predictor of poorer prognosis as is perceived by most physicians. we hypothesized that the drug abusers admitted to the icu will fare as good as or better than non-abuser icu patient populations. methods: this is a prospective study done between the years - on the entire patient population of the detroit medical center. after the drug abuse population was identified, controls were matched according to age and admission icu units. patients charts were reviewed and data regarding baseline demographics, infectious complication and outcome was extracted. data was retrospectively collected for drug abusers and matched controls. comorbidities and hospital admission diagnosis were significantly different between the two groups. disease severity scores were significantly higher in the drug abuser's patient group (dapg) on admission and during the icu stay. dapg had significantly more organ failure: more need for ventilation ( . % vs . % in the dapg (p< . )), more ards ( % vs . %, p= . ), more renal failure ( % vs . %, p= . ) and more need for renal replacement therapy ( . % vs . %, p< . ) .they had longer hospital length of stay (los). there was no difference in icu or hospital mortality. multivariable modeling did not find drug abuse to be an independent risk factor for hospital mortality, icu mortality (hosp: or = . , p = . ; icu: or= . , pp = . ), but was a risk factor for a longer hospital los (me= . , p < . ). drug abuse is not an independent risk factor for mortality or icu los. drug abusers should be evaluated like other patients based on baseline comorbidities and disease severity. this is a small audit which although it did not include general icu still reflects the need for encouraging clinicians and patients to speak freely regarding escalation plans. medical decsions is clinician led however this audit was carried by nursing staff as we have a duty to be advocate for our patients involvement in medical care [ ] . a retrospective analysis of independent risk factors of late death in septic shock survivors c sivakorn , c permpikul , s tongyoo (fig. ) . the pap and katz scales seem to be adequate for predicting mortality of critically ill patients admitted to a medical icu. this finding may help in the elaboration of future icu mortality scoring systems, as well as in more rational use of resources. however, further multicenter studies are needed to better elucidate these results. adherence this last group was chosen because of its experience and specific training in the field of bioethics as a control group or reference. a total of respondents participated in the study. . % were emergency physicians, . % intensivists, . % emergency nursing, . % icu nursing, . % resident doctors, . % medical students and . % other professions. we observed variability in the responses observed not only between different groups of professionals but even within the same group reflecting the difficulty in decision making. variability was observed regarding decisions in end of life ethics conflicts. a high degree of similarity with the group of master in bioethics was observed in the responses issued by medicine students. the barriers and facilitators to framing goals of patient care (gopc) and factors motivating decision making is relatively unexplored [ , , ] . a three part survey of physicians at an australian hospital in a culturally and linguistically diverse suburb ( table ) . identification of levels of confidence and barriers and facilitators to gopc discussion and decision making was the main outcome measure. factors influencing decision-making was analysed through scenarios. results: out of eligible participants responded; female, male, clinical experience - years. level of confidence was ranked between "somewhat confident and very confident." all but one respondent had six months of icu experience. no differences in the level of confidence among physician groups. barriers and facilitators were identified; poor prognosis and patient or family request were most common facilitators; conflict between treating teams and the patient/surrogate and language barriers were most common barriers. factors driving gopc decision-making included clinical, value judgement, communication, prognostication, justice and avoidance. numerous barriers and facilitators were identified. factors driving decision making did not just consider clinical factors; conflict and we aimed to investigate physician-related factors contributing to individual variability in end-of-life (eol) decision-making in the intensive care unit (icu). qualitative study with semi-structured interviews with specialists in critical care, (experience - years) from swedish icus. data was analyzed in accordance to principles of thematic analyses. most of the respondents felt that the intensivist's personality played a major role in eol decisions (table ) . individual variability was considered inevitable. views on acceptable outcome: respondents experienced that the possible outcome for patients was interpreted very differently and subjectively among colleagues, and what seemed an acceptable patient-outcome for one doctor, was not acceptable for another. values: most of the respondents were well aware that they might be affected by their own values and attitudes in the decision-making process. interestingly, several respondents mentioned that they thought that patients that were marginalized by society, especially drug-abusers could be at risk for receiving decisions to limit life sustaining treatments (lst) more often than others. none of the respondents thought that their own religious beliefs played any part in decision making. fear of criticism: among the less experienced respondents there was a clear sense of fear of making a questionable assessment of the patient's medical prognosis. there was a fear for criticism from colleagues that were not directly involved in the decision-making, and may have made another decision. this created a wish among younger respondents to defer or avoid participating in decision-making. physician-related, individual variability in eol decisions primarily consisted of differing views on acceptable outcome, values and fear of criticism. can (figure ). within each quartile of sofa score, mortality was highest in patients with pneumonia and peritonitis and lowest in patients with cellulitis (see figure ). the sepsis- consensus definition identified organ dysfunction as the hallmark feature of sepsis [ ] . in developing sepsis- , the sequential organ failure assessment (sofa) score was chosen for its prognostic value and relative ease of implementation clinically [ ] . we propose an update based on epidemiologic data from two intensive care databases that more effectively captures organ dysfunction in the context of sepsis- . using the mimic-iii (exploration) and e-icu (validation) databases, we extracted patients with suspicion of infection to form the study cohort. the predictive power of each sofa component was assessed using the area under the curve (auc) for in-hospital mortality. a logistic model with the lasso penalty was used to find an alternative statistically optimal score. results: by utilising alternate markers of organ dysfunction (e.g. lactate, ph, urea nitrogen) we demonstrated a significant improvement in auc for several versions of the new score, sofa . ( figure ). the sofa score can be updated to reflect current advances in clinical practice. using epidemiologic data, we have shown that substitution of existing components with more powerful measures of organ dysfunction may provide an improved score with greater predictive power. moreover, sofa . exhibits equivalent ease of implementation, but better reflects organ dysfunction in the context of sepsis- . introduction: risk of acute organ failure (aof) in cancer patients(pts) on systemic cancer treatment isunknown. however, % of non-hematologic and % of hematologic cancer pts will need admission to intensive care unit (icu). ipop-sci- / is a prospective cohort study designed to ascertain the cumulative incidence of aof in adult cancer pts. single centre prospective cohort study with consecutive sampling of adult cancer pts admitted for unscheduled inpatient care while on, or up to weeks after, systemic cancer treatment. primary endpoint was aof as defined by quick sofa. six months accrual expected an accrual of pts to infera population risk aof with a standard error of %. between / and / pts were on systemic anticancer treatment, had unscheduled inpatient care and were eligible for inclusion and were included. median age was years, % were male, % had adjusted charlson comorbidity index (cci) &gt; and hematologic cancers accounted for % of pts. the cumulative risk of aof on hospital admission was % ( %ci: - ); and of aof during hospital stay was % ( %ci: - ). aof was associated with older age, cci &gt; ,hematologic malignancy, shorter median time from diagnosis and &gt; prior line of therapy. on admission, % of pts were considered not eligible for artificial organ replacement therapy (noaort) and % of pts who developed aof while inhospital were judged noaort. overall, ( %) of aof pts wereadmitted to icu, . % for aort. median follow up . months (min ; max ). inpatient mortalitywas %, with icu mortality rate of %, with median cohort survival . months ( %ci: . - . ). on multivariate analysis, aof was an independent poor prognostic factor (hr . ; %ci . - . ). risk of aof in cancer pts admitted for unscheduled inpatient care while on systemictreatment is %, and risk of icu is %. aof in cancer pts was an independent poor prognostic factor. a severity-of-illness score in patients with tuberculosis requiring intensive care u lalla, e irusen, b allwood, j taljaard, c koegelenberg tygerberg academic hospital, internal medicine, division of pulmonology and icu, cape town, south africa critical care , (suppl ):p we previously retrospectively validated a -point severity-of-illness score aimed at identifying patients at risk of dying of tuberculosis (tb) in the intensive care unit (icu). parameters included septic shock, human immunodeficiency virus with cd < /mm , renal dysfunction, ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (pao :fio ) < mmhg, diffuse parenchymal infiltrates and no tb treatment on admission. the aim of this study was to validate and refine the severity-of-illness score in patients with tuberculosis requiring intensive care. we performed a prospective observational study with a planned post-hoc retrospective analysis, enrolling all adult patients with confirmed tb admitted to the medical intensive care unit from february to july . descriptive statistics and chi-square or fisher's exact tests were performed on dichotomous categorical variables, and t-tests on continuous data. patients were categorized as hospital survivors or non-survivors. the -point score and the refined -point score were calculated from data obtained on icu admission. results: forty-one of patients ( . %) died. the -point scores of nonsurvivors were higher ( . +/- . vs . +/- . ; p= . ). a score ≥ vs. < was associated with increased mortality ( . % vs. . %; or . ; %ci, . - . ; p= . )( table ) . post-hoc, a pao :fio < mmhg and no tb treatment on admission failed to predict mortality whereas any immunosuppression did. a revised -point score (septic shock, any immunosuppression, acute kidney injury and lack of lobar consolidation) demonstrated higher scores in non-survivors ( . +/- . vs. . +/- . ; p< . ). a score ≥ vs. ≤ was associated with a higher mortality ( . % vs. . %; or . ; %ci, . - . ; p< . ) ( table ) . the -point severity-of-illness score identified patients at higher risk of death. we were able to derive and retrospectively validate a simplified -point score with a superior predictive power. chronic critical illness remains a scientific challenge, from its conceptualization to its impact on patient prognosis [ ] . we evaluated the long-term evolution of icu survivors by identifying the real burden of prolonged critical illness on survival, quality of life and hospital readmissions. we conducted a prospective cohort in brazilian hospitals including icu survivors with an icu stay > h. we compared the patients diagnosed with chronic critical illness with the other patients. telephone follow-up at and months. quality of life was measured by the sf- questionnaire. it was observed that % of patients had some definition of chronic critical illness. chronic critically ill patients had higher mortality at months (p= . ). this difference is mainly due to higher intrahospital mortality (p= . ). mortality after hospital discharge was similar between groups. there was no difference in hospital readmission rate at months. various scores are developed to predict pulmonary complications such as ariscat for patients at-risk of postoperative pulmonary complication [ ] and lips for patients at-risk of lung injury [ ] . the aim of this study was to compare these scores with ours for predicting pulmonary complications in mechanically ventilated patients in sicu. this prospective observational study was conducted in sicu at a university hospital. adult patients admitted to sicu and required mechanical ventilation > hours were included. primary endpoint was the composite of pulmonary complications including pneumonia, ards, atelectasis, reintubation, and tracheostomy. multivariate analysis was performed to identify risk factors of pulmonary complications and the predictive score was developed. the roc analysis was performed to compare power of ariscat, lips and our newly developed score for predicting pulmonary complications. outcomes in intensive care units have been reported to be better in higher-volume units [ , ] . we compared outcomes for high-risk patients between low and higher volume units. audit data from irish icus is analysed and reported by the intensive care national audit & research centre (icnarc) in london. icnarc report risk-adjusted mortality rates in all patients and in low-risk patients(predicted mortality rate < %) for each unit, using the icnarch- model to predict the risk of death. we used this data to calculate the proportion of high-risk patients(predicted mortality > %) in each unit, the mortality rate for high-risk patients, the riskadjusted mortality rate and we compared the overall risk-adjusted mortality between low and high volume units. the median number of annual new-patient admissions among participating units was ; units below this were defined as lowvolume and those above as high-volume units. the proportion of all admissions to each unit who were high-risk ranged from % to %(mean %). unit mortality rates for high-risk patients ranged from % to %. the ratio of observed to expected mortality(standardized mortality ratio -smr) for high risk admissions in each unit ranged from . to . (mean . ). in fig. introduction: adl weakening is often seen after intensive care and called postintensive-care syndrome (pics). this is also seen in even outside icu and proposed to be called post-acute-care syndrome (pacs), especially in elderly patients. in patients with infection, sofa score is famous for predicting in-hospital mortality, but there are no tools for predicting adl weakening during admission. to search for risk factors for adl weakening during admission other than the age, we conducted a retrospective observational study. the subjects were surviving patients with infection, aged from to who were admitted to our department from april , to may , . information of basic characteristics, laboratory data on admission and adjunctive therapies were extracted from our database. we use barthel index (bi) as adl evaluation, and the bi at discharge were evaluated by nurses. we stratified patients by bi at discharge of over or not, and investigated factors that predicted it. we compared each factor between groups, and perform a logistic regression analysis with those that had a significant effect clinically or statistically. despite improved outcomes of intensive care unit (icu) patients, sleep deprivation remains a major concern after icu discharge. multifaceted causes make it difficult to treat and understand [ ] . not many studies have explored sleep deprivation beyond icu. this is evidenced by findings from a recent systematic review [ ] which included studies with only one study [ ] reporting sleep deprivation beyond icu. the aim of this paper is to present findings of sleep deprivation beyond icu from a larger study that examined the experience of critical illness in icu and beyond in the context of daily sedation interruption. hermeneutic phenomenology was used to conduct the study. participants aged years and above who fulfilled the enrolment criteria were enrolled into the study. the cohort comprised male and female participants. in-depth face to face interviews at two weeks after discharge were conducted and repeated at six to eleven months. interviews were audio taped, transcribed and thematically analysed. significant statements were highlighted and categorized for emergent themes. six participants continued to experience sleep deprivation up to eleven months after icu. two cited dreams about icu, three could not explain why they continued to fail to sleep and one stated that he continued hearing icu alarms in the silence of the night. sleep deprivation continues beyond icu due to nightmares, delusional memories and unexplained reasons. further research is needed to establish causes of sleep deprivation and explore ways to promote sleep in critical illness survivors after icu discharge. frailty is being increasingly seen as an independent syndrome. frail patients now account for an increasing proportion of hospital and critical care admissions [ ] . we aimed to compare frailty and mortality in our intensive care unit. clinical frailty score (cfs) was incorporated within the electronic health record (ehr) . we performed this retrospective analysis on the data collected between jan' and oct' . the predictor and outcome for this study were frailty and hospital mortality respectively. all demographic data, acute physiology score, critical care and hospital outcome data were automatically collected in the ehr and recorded. we used a cut off of cfs> and above to define non-frail and frail respectively. chi-squared test, simple and multiple logistic regression were used. adjustment was done for icnarc score and age. total number of patients was , of which ( . %) died in hospital. within the patients< years (n= ), ( %) were recorded as frail or vulnerable. the number of elective and emergency admission were ( %) and ( %) respectively. in the frail and nonfrail, mortality rates were % and . % (p< . ) respectively, with odds ratio of . ( % ci . , ; p< . ) ( age is a well-known risk factor for critical care (cc) outcome and is incorporated into many prognostic tools; however, this has been criticized for assumption of normal physiology for young at baseline. in recent years, frailty in cc prognostication has been of interest, with meta-analysis correlating worsening outcomes with increasing frailty [ ] . in this study, we compared the effect of frailty versus age for determining hospital survival for critically ill patients. we conducted a prospective cohort in brazilian hospitals including survivors of an icu stay > h. we compared chronic critically ill patients (icu stay> days) and the other patients. we performed psychological and functional presential assessment in patients within hours of icu discharge and by telephone at and months. the prevalence of chronic critically ill patients was %. regarding outcomes, chronic critically ill patients had a higher incidence of depressive symptoms than other patients in the immediate post-icu discharge (p = . ), as well as a higher incidence of muscle weakness (p < . ). however, in subsequent evaluations, we found no difference between groups regarding psychological symptoms -depression, anxiety and post-traumatic stress. higher functional dependence was observed in critically ill patients, but without difference in the quality of life score, both in the physical (p = . ) and mental (p = . ) domains. chronic critically ill patients, when compared to patients with stay> h, have a higher incidence of depressive symptoms at icu discharge. this difference disappears in the follow up. chronic critically ill patients present higher levels of functional dependence but without repercussions on quality of life scores. introduction: activation of the inflammatory response after cardiac arrest (ca) is a welldocumented phenomenon that may lead to multi-organ failure and death. we hypothesized that white blood cell count (wbc), one marker of inflammation, is associated with one-year mortality in icu treated ca patients. we used a nationwide registry with data from five academic icus to identify adult ca patients treated between january st and december st . we evaluated the association between the most abnormal wbc within hours of hospital admission and one-year mortality. we accounted for baseline risk of death using multivariable logistic regression (adjusted for age, gender and h sequential organ failure assessment [sofa] score). a total of , patients were included in the analysis. of those patients , ( %) were alive one year after ca. we plotted wbc against baseline risk of death and through graphic examination of a locally weighted scatterplot smoothing (lowess) curve found the lowest risk of death to be associated with a wbc of (e /l) ( figure mrps were identified by a specialist icu pharmacist during this programme and classified by their significance on a scale of one to four. logistic regression was used to determine if demographic factors were associated with the occurrence of a clinically significant mrp -a significance score of two or above (figure ) . the adjusted model included age, icu los, hospital los, apache ii, number of days of renal replacement therapy, number of days of ventilation, the number of medications prescribed at icu discharge, and the who analgesia classification at ins:pire. there were increased odds of having a clinically significant mrp for hospital los (or results: · % (n= ) of patients required at least one pharmacy intervention. the median number of interventions required per patient was one (iqr - ); the maximum number was six. mrps were recorded in this cohort. the most common intervention was clarifying duration of treatment (n= ), followed by education (n= ), and correcting drug omissions (n= ). the bnf drug class most frequently associated with mrps was neurological (n= ), which comprises analgesics (n= ) and psychiatric medications (n= ) ( figure ). this was followed by cardiovascular medications (n= ), gastrointestinal medications (n= ), nutritional medications (n= ), and others (n= ). many icu survivors experience mrps. the most common class of mrp was neurological, reflecting the high incidence of chronic pain and psychiatric illness in this population following discussion with icu staff, ward staff and fy doctors, a formal standardized handover system was introduced. this involved a verbal handover to the appropriate fy by an icu doctor and the patient drug chart to be rewritten in icu at the time of handover. the next change was to display posters on the wards to alert staff that the medical team are to be contacted when a patient comes to the ward from icu and to ensure the drug chart is completed. the baseline data showed a median time delay of hours, with one patient waiting hours for a drug chart. following the interventions the median time delay has decreased to hours within months as demonstrated in figure . the changes have received positive feedback from icu staff, ward staff and fy doctors. the aim of reducing the time delay by % has been achieved with the median time delay now hours. this has improved patient safety by significantly reduced delays in medications and through the introduction of a standardized handover. this has also provided an opportunity for junior doctors on the wards to seek clarification regarding medications and the clinical management plan for the patient. this has established a communication channel between icu and the wards making patient care safer and more effective. telemonitoring outside the icu is scarce. but with innovative wearables measuring respiratory and heart rate wirelessly, culture on intrahospital telemonitoring should definitely change. however, culture has been known to be one of the most crucial success factors in innovation, especially in health care. human design thinking is a promising tool in health care innovation but rarely used in a multidisciplinary team to initiate an innovation culture and stimulate sustainable collaboration. the aim of this study was to initiate a pilot project with a multidisciplinary team to start using wearables for early warning score (ews) on a clinical ward. human design thinking was used to write a value proposition on wearables in clinically admitted neutropenic hematologic patients in an academic center. a multidisciplinary team was performed to cover all disciplines involved in the technical, clinical and administrative parts of the project. a vendor was chosen based on its product specifications in relation to the present hospital monitoring infrastructure. in design thinking sessions, critical appraisal of multiple telemonitoring factors was performed by sub teams and a canvas projectplan was constructed. the project team was formed of registered nurses, physicians, itspecialists, electronic health record consultants; a critical care physician was appointed as project leader. the main critical factors were: unseamlessly transmitting of both heart and respiratory rates including appropriate movements filtering to the nurse's smartphones direct uploading into electronic health record with automated ews calculation nurse driven protocol on ews follow up. philips healthcare with their intellivue guardian wearable biosensor was the chosen vendor ( figure ). design thinking in a multidisciplinary health care team could positively influence the innovation culture. scientific evaluation of this wearable will focus on both nurse's acceptance and data storage and is expected in the summer of . severity, readmission and lengh of stay were lower in patients receiving discharges directly to home. it seems like a safe way to discharge low-risk short stay patients. it seems to save resources and reduce costs, as well as the need for hospital beds. however, futher estudies are needed to actualy evaluate this safety. forty-four cultures were analyzed with eplex ( figure ). complete agreement with conventional diagnostics was observed in / cases. no false-positive results were observed, yielding a sensitivity and specificity of % and % respectively for target pathogens. time to result was, on average, . h faster with eplex compared to conventional diagnostics. antimicrobial therapy could have been optimized in patients based on the eplex result, but treatment was only changed in one case (e.coli ctx-m+) receiving meropenem . h before the antibiogram was available. the eplex blood culture panels provide high accuracy and significantly faster results. the current implementation offers substantial potential value at a minimal cost, and is a feasible approach to -h/ days blood culture diagnostics in many hospital settings. however, efforts to increase adherence are needed. the rapid increase of extended spectrum β-lactamases (esbl)-producing pathogens worldwide makes it difficult to choose appropriate antibiotics in patients with gram-negative bacterial infection. cica-beta reagent (kanto chemical, tokyo, japan) is a chromogenic test to detect beta-lactamases such as esbl from bacterial colonies. the purpose of the study was to reveal whether cica-beta reagent could detect esbl-producing pathogens directly from urine rather than bacterial colonies to make a rapid bedside diagnosis of the antibiotic susceptibility of gramnegative pathogens. we conducted a prospective observational study from july to october . patients were eligible if they were performed urinary culture tests and gram negative pathogens were detected at least + from their urine samples. the urine sample was centrifugated at x g for min. the supernatant of sample was re-centrifugated at x g for min and the pellet was mixed with cica-beta reagent. the test was considered positive when the enzymatic reaction turned from yellow to red or orange. (fig. ) . the bundle approach could be an effective strategy to prevent hospital-acquisition of drug-resistant pathogens in icus. fig. in the aspect-np trial, c/t was noninferior to mem for the treatment of habp/vabp. we evaluated outcomes from that study in the subgroup of pts failing current antibacterial therapy for habp/vabp at enrollment. methods: aspect-np was a randomized, controlled, double-blind, phase trial in which mechanically ventilated pts with habp/vabp received g c/t or g mem every h for - days. pts with > h of active gram-negative antibacterial therapy within h prior to first dose of study therapy were excluded, except those pts failing current treatment (i.e. signs/symptoms of the current habp/vabp were persisting/worsening despite ≥ h of antibiotic treatment). primary and key secondary endpoints, respectively, were -day all-cause mortality (acm) and clinical response at test of cure (toc; - days after end of therapy) in the intent to treat (itt) population. pts failing current antibacterial therapy for habp/vabp were prospectively categorized as a clinically relevant subgroup. at baseline, failing current therapy for habp/vabp was reported in / ( %) c/t and / ( %) mem itt pts, mostly piperacillin/ tazobactam ( %), rd/ th-generation cephalosporins ( %), fluoroquinolones ( %), and aminoglycosides ( %). baseline demographic and clinical characteristics in this subgroup, including prior therapy regimen, were generally similar between treatment arms. there were greater proportions of patients with esbl+ enterobacterales ( %) and pseudomonas aeruginosa ( %) in the c/t arm than the mem arm ( % and %, respectively). lower -day acm was seen with c/t than mem, as evidenced by % confidence intervals for treatment differences that excluded zero ( figure ); statistical significance cannot be assumed because subgroup analyses in this study were not corrected for multiplicity. conclusions: c/t was an effective treatment for habp/vabp pts who had failed initial therapy. catheter-related blood stream infection (crbsi) is common serious infections and associated with increased mortality in intensive care units (icu). one of the most important strategy to prevent crbsi is to minimize the duration of central venous catheterization. we built a medical team consisting of doctors, nurses and pharmacists in icu to discuss whether patients needed central venous catheter (cvc) in terms of monitoring hemodynamics and administering drugs, and recommend catheter removal to attending physicians every day in april . the purpose of this study is to evaluate whether our team-based approach could shorten the total duration of catheterization and reduce crbsi. this was a retrospective historical control study conducted from april to october in the icu of a tertiary care hospital in japan. every patient admitted to the icu during the study period was eligible if they were inserted cvc. patients were divided into groups: conventional (from april to march ) or intervention (from april to october ). we set the primary endpoint as onset of crbsi. the secondary endpoints included the duration of central venous catheterization, the length of icu stay and hospital mortality. crbsi was defined as bloodstream infection in patients with cvc, not related to another site. we included patients: in the conventional group and in the intervention group. the reduced, though nonsignificant, tendency of crbsi was observed in the intervention group [hazard ratio, . ( % confidence interval, . - . ; p = . )]. the intervention group was significantly associated with reduced duration of central venous catheterization ( days vs days; p < . ). no difference was observed in the length of icu stay and in-hospital mortality between groups. the team-based approach to assess cvc necessity could shorten the duration of central venous catheterization and might reduce crbsi. introduction: empiric antibiotic therapy decisions are based upon a combined prediction of infecting pathogen and local antibiotic susceptibility, adapted to patients' characteristics. the objective of this study was to describe the pathogen predominance and to evaluate the probability of covering the most common gram-negative pathogens in icu patients with respiratory infections. methods: data were collected from multiple us and european hospitals as part of the smart surveillance program ( ). mic (mg/l) testing was performed by broth microdilution, with susceptibility defined as follows for p. aeruginosa & enterobacterales: ceftolozane/tazobactam results: hospitals from countries provided gram-negative respiratory isolates from patients located in an icu in the us ( %), eastern europe ( %) and western europe ( %) in . the most common pathogens isolated were p. aeruginosa ( %), k. pneumoniae ( %), e. coli ( %), and a. baumannii ( %). among enterobacterales, % ( / ) were esbl positive. figure provides the probability of covering the most common respiratory gram-negative pathogens from icu patients. co-resistance between commonly prescribed first line β-lactam antibiotics is common: when nonsusceptibility (ns) of one agent was present, susceptibility to other βlactams was generally < %. ceftolozane/tazobactam provided the most reliable in vitro activity in both empiric and adjustment prescribing scenarios compared to other β-lactam antibiotics. ceftolozane/tazobactam ensured a wide coverage of the most common gram-negative respiratory pathogens demonstrating high susceptibility levels and provided the most reliable in vitro activity in both empiric and adjustment antibiotic prescribing scenarios. further studies are needed to define the clinical benefits that may translate from these findings. evaluation of compliance of icu staff for vap prevention strategies on the outcome of patients a kaur fortis hospital, critical care, mohali, india critical care , (suppl ):p ventilator-associated pneumonia is the most common nosocomial infection diagnosed in adult critical care units. it is associated with prolonged duration of mechanical ventilation, increased icu stay and increased mortality. it continues to be a major challenge to the critical care physicians despite advances in diagnostic and treatment modalities. the primary objective of the study was to determine the compliance of icu staff towards vap prevention bundle and secondary objective was to determine the incidence, risk factors and outcome of vap patients. single center, prospective, observational study carried out from february to july . patients mechanically ventilated for more than hours and satisfying the inclusion and exclusion criteria were enrolled in the study. vap was diagnosed using the cdc criteria and clinical pulmonary infection score. vap preventive strategies were employed and compliance of icu staff was assessed. a total of patients were admitted to icu over the set time period and out of them patients were ventilated for more than hours. among them only patients fulfilled the inclusion and exclusion criteria and were enrolled in the present study. excellent compliance was observed in head end elevation, sedation vacation, stress ulcer prophylaxis, and heat moist exchanger filter use, good compliance in oral care and hand hygiene and moderate to poor compliance in subglottic suctioning. the incidence of vap was . % with a vap rate of . / ventilator days. there was a significant correlation between primary diagnosis, hemodialysis, massive blood transfusion and development of vap (p< . )). mean duration of ventilation (p< . ) and mortality (p< . ) were highly significant in vap patients. conclusions: improvement in compliance towards vap bundle and reduction of risk factors can help decrease incidence of vap and related morbidity and mortality. preventive strategies are effective in reducing ventilation-associated pneumonia (vap) in adults [ , ] . in paediatric population there are no data about vap prevention, so we introduced a new bundle (vap-p) based on the available evidence for adults. this was designed as a before-after study. we enrolled all patients admitted to -bed medical-surgical paediatric icu at gemelli hospital in rome, requiring mechanical ventilation for at least hours. patients with pre-existing tracheostomy were excluded. vap-p has been introduced since in order to improve quality of assistance. our bundle consisted in twice a day oral hygiene with chlorhexidine swab, daily check of oral bacterial colonization and aspiration prevention. comparison was made with an historical group including patients admitted before vap-p introduction (since to ). all data about demographics, antimicrobial therapy, icu stay and treatments, were collected. results: patients were included ( after and before vap-p introduction). ( %) events of vap were recorded in vap-p group compared to ( %, p= . ) vap-p group had less vap per days of mechanical ventilation ( / compared to . / p= . ). multivariate analysis yielded an or of . ( %ci . - . ) for vap incidence after bundle introduction. mortality rate was slightly reduced in vap-p group ( . %vs . % p=ns). patients who developed vap required more days on mechanical ventilation and had higher mortality rate ( vs days p< . and %vs % p= . , respectively). our vap-p seems effective in reducing vap incidence in critically ill paediatric population. introduction: ceftolozane/tazobactam (c/t) is a new antibiotic against mdr gramnegative bacteria infections, whose target population are the critically ill patients. even though / g dose safety administered as a hour-infusion has been already assessed, these patients can be under renal replacement therapy (rrt) and suffer changes in their volume of distribution (vd) that may affect antibiotic concentrations. the objective was to determine concentration reached by g c/t ( hour infusion) in septic patients on rrt (cvvhdf) and interdose behavior. we have used rrt machine prismaflex with oxyris filter and m . hplc-uv method was used for simultaneous quantification of c/t. study population consisted of three obese critically ill patients with sepsis, on cvvhdf while receiving g c/t every hours. samples were taken of prefilter, post filter blood and effluent, min before infusion and , and hours after the end of it. we found great interpatient variability with the lowest cconcentration values in the patient with more hemodynamic instability using oxyris filter. even though cmax was less than reported in healthy subjects, we found similar values of auc and t ½ in comparison with healthy population studies. cmax of t was also compromised in comparison with values reported in healthy subjects, but with higher auc and t ½. cvvhdf contributes to c/t clearance. m filter showed the least clearance and higher values of auc and t ½. extraction rate was similar in all patients and filters (figure ) . cmax achieved may be impaired because of the varying vd caused by obesity and rrt, but not affecting the antibiotic characteristics and behaviour. we conclude that because of the variety of clinical conditions, c-concentration is compromised particularly in hemodynamically unstable patients. however, the small sample doesn´t let us extrapolate these results. the extended infusion seems to be adequate to achieve the interdose antibiotic concentration. the use of biomarkers in sepsis is useful for early diagnosis and prognosis. the desired marker should be sensitive, specific, fast and accurate. procalcitonin (pct) measurement is approved by the fda even its efficacy is still under question. the determination of alfatorquetenovirus (ttv) could be a useful marker [ ] . we analyzed samples from patients admitted to icu with clinical suspicion of sepsis. analytical data of c-reactive protein (crp), neutrophils and procalcitonin were collected. the sofa and apache ii scales were calculated and patients stratified according to these values in good and poor prognosis. ttv quantitative determination was carried by using a quantitative crp . we calculated area under the curve (auc) of ttv plasma levels as a function of time. the statistical analysis involved u-mann-whitney and spearman test, using chi for qualitative variables. results showed a not significant (ns) inverse relationship between the ttv auc and the patient proinflammatory level. a tendency (ns) was found between poor prognosis and the pct median values and crp being higher in the poor prognosis.group. a trend showed lower ttv dna count related to worse prognosis. an inverse relationship was found between pct and crp values and the ttv copies /ml plasma, ns correlation in the case of pct. there was a clear trend between the neutrophils´expansion and the regression line slope, obtained between ttv loads in the first two study steps. fig. (abstract p ) . patient pk/pd measurements value> . ), suggesting that the adsorptive mechanism wasn't primarily mediated by plasma protein. ha was saturated after adsorption of a total of . ± . mg of van. the adsorptive kinetics showed an exponential reduction of van mass that reached a plateau after minutes of circulation. in our study, simulating in vivo conditions of hp using ha during sepsis, a rapid and clinically relevant removal of van has been shown. after hours of hp, we suggest to assess van plasma concentration and a loading dose of van should be considered. however, not knowing the potential interactions with other drugs, further in vivo studies are warranted to confirm these findings. assessing the volume of blood taken for blood culture and culture positivitydo we need to take less blood? it is commonly accepted that larger blood culture (bc) volumes (bcv) increase the yield of true positive cultures, and optimally cc of blood should be obtained per set ( bottles). only scarce data exists on the matter of optimal bcv. it is unknown what is the minimal volume that is acceptable for bc. the objective of this study was to determine the association between bcv and the rate of positive bc. blood taken for cultures in bd bactec plus aerobic/f negative bottles was collected from icus and acute care floors at hospitals at the dmc over months. blood volume was estimated automatically from blood background signal data in the bd bactec fx instrument. cultures were analyzed for each bottle. data was summarized for every month as the average volume and number of cultures taken and rate of positive bc for every unit. units were classified according to unit type (icu, medicine, surgery, mixed, emergency department (ed), organ/bmt or "other" which did not fit the previous categories) and analyzed as a group. a total of cultures were taken in units. there is a positive association between bv and positive bc rate for ed and "other" units (irr= . , p= . for the ed, irr= . , p< . for "other" unit). all other units had no association between bv and positive bc rate (figure ). secondary analysis, excluding pediatric units, gave very similar results. when comparing bv between unit types, the ed and "other" unit had significantly lower bv ( . ml in the ed and . ml in "other" unit compared to . ml in the icu, . ml in surgery, . ml in mixed and . ml in bmt). the correlation between bv and positive bc rate is probably limited to units taking very low bv for cultures. units taking volumes above ml show no improvement in positive bc rate when higher volumes are taken. better prospective studies should be done to further establish the minimal bcv needed and spare unnecessary blood loss to hospitalized patients without compromising bc yield. de-escalating antibiotics in sepsis with the use of t mr in a bed greek university icu c vrettou, e douka, i papachatzakis, k sarri, e gavrielatou, e mizi, s zakynthinos st icu department, university of athens, evangelismos general hospital, icu, athens, greece critical care , (suppl ):p in septic patients, the early use of appropriate empiric antibiotic therapy reduces morbidity and mortality. de-escalation refers to narrowing the broad-spectrum antibiotics once the pathogen and sensitivities are known. t magnetic resonance (t mr) is a novel method of detecting eskape pathogens. we aim at investigating if using t mr technology can expedite de-escalation of broad spectrum antibiotics. this is a prospective observational study conducted in our -bed university icu. inclusion criteria were critically ill patients age> y.o., with newly diagnosed sepsis and clinical suspicion of eskape bloodstream infection. a sample for t mr and a blood culture (bc) sample were collected simultaneously from the patients enrolled. the t mr bacteria panel test was run according to the manufacturer's guidelines and the bcs were processed according to the hospital standard procedures. we recorded clinical data and administered antibiotics. results: patients were included in the study. mean time to culture positivity was hours while mean time to t mr result was . hours. in patients the results of t mr were in concordance with the bcs. in the remaining cases, the bcs were negative while the t mr detected one or more eskape pathogens. there were no false negative results. de-escalation in at least one drug was applied to patients ( . %). no escalation was applied to patients ( . %) and antibiotic escalation in ( . %). conclusions: t mr provides a quicker detection time that could shorten the time to targeted therapy. in our population this corresponded to early (within - h) antibiotic de-escalation in approximately / of the included patients. antibiotic stewardship in icu. a single experience l forcelledo , e garcía-prieto , l lópez-amor , e salgado , j fernández dominguez , m alaguero , e garcía-carús the increasing antibiotic resistance in microorganisms urged interventions such as the antibiotic stewardship programs in icu focused on reducing the inappropriate use of antibiotics by improving the antibiotic selection, the dosage, administration route and length as well as improving clinical outcomes and reducing antibiotic resistance. retrospective study where antibiotic consumption was analysed and measured in days of therapy (dots) between and in a medical-surgical icu of a university hospital where a multimodal educational program was established. specific training in infectious diseases in critically ill patients, periodic clinical and formative sessions fig. (abstract p ) . correlation of blood culture positivity rate with blood culture volume by unit type were performed for icu staff and specific leaders within the icu staff designated. results: patients were admitted to icu. there was a reduction of , % in dots (figure ), reduction in antimicrobial resistance rates ( , in , , in [days of resistant microorganism/ patientdays]) without an impact in icu global mortality ( , % in , , % in ). the resistant bacteria registered were acinetobacter baumannii, s. aureus mr, blee and carbapenemase-producing enterobacteriaceae, pseudomonas aeruginosa mr and clostridium difficile. the safe in antimicrobial consumption was € ( % reduction). the icu stay decreased from , days ( ) to , ( ) , with no variation in mean apache ii ( , ) . the bigger decrease in antibiotic consumption was in colistin related to the reduction in resistance bacteria, in special acinetobacter baumannii, in linezolid and in piperacilin/tazobactam, even more remarkable in due to shortage of supplies which meant an increase in meropenem. the application of an antibiotic stewardship program in icu succeeded in reducing antibiotic consumption, antibiotic resistance and costs without an impact in clinical outcomes like mortality or icu stay. clinical outcomes of isavuconazole versus voriconazole for the primary treatment of invasive aspergillosis: subset analysis of indian data from secure trial p kundu, s kamat, a mane pfizer limited, medical affairs, mumbai, india critical care , (suppl ):p the secure trial was designed to compare the safety and efficacy of isavuconazole (a) versus voriconazole (v) for primary treatment of invasive mould disease caused by aspergillus and other filamentous fungi. the present analysis is aimed at comparing the indian subset of patients with that of the overall trial population and to ascertain any similarity or difference in the primary efficacy endpoint and safety/tolerability in these two groups. in secure trial, patients in one group received (i) & another patients received (v). the indian subset had patients. we have done a qualitative analysis as the sample size of the indian subset was small. non-inferiority of (i) to (v) in terms of all cause mortality from first dose to day was assessed in overall patients. the treatment difference between (i) and (v) group in the indian subset of patients was analyzed. proportion of patients who had to discontinue treatment due to teaes was analyzed. the all-cause mortality in the overall trial population met noninferiority margin (table ). in the indian subset, it was higher for (i) than (v). there was a lower incidence of ocular, hepatobiliary, skin & subcutaneous tissue disorders in the (i) treated patients (see table ). in indian subset, the above adverse events were less in the (i) group, but statistical inference could not be done due to small sample size. however, similar trend of less number of patients discontinuing therapy due to teaes in the (i) treated patients was seen in the overall patients & the indian subset. the all-cause mortality in the indian subset was higher in the (i) patients. a trend similar to the overall population regarding safety parameters favoring (i) was seen in the indian patients. considering the significantly higher prevalence of ia in india, suitably powered study design is necessary to draw definitive conclusions on the non-inferior efficacy & better safety & tolerability of (i) over (v) in patients of ia. introduction: ventilator-associated pneumonia (vap) is one of the most frequent healthcare-associated infections, correlated with increased mortality,extended hospital stay and prolonged mechanical ventilation. considering the latest outbreak of multiresistant a. baumannii infections in the critically ill patients with vap, there is a growing concern regarding challenges of the antibiotherapy in these patients. although ceftazidim-avibactam is considered to have limited effects on a. baumannii, it is reported to have a synergic activity in combination with other antibiotics. we performed a retrospective, observational study which included icu patients diagnosed with vap(cpis > ). oxa a. baumannii was isolated from the tracheal secretions using a rapid molecular diagnostic platform(unyvero a system). patients were divided in two groups according to the antibiotherapy:group a meropenem + colistin and group b meropenem + colistin + ceftazidim-avibactam.statistical analysis was performed using graphpad applying t-test and kaplan-meier curves, having the in-hospital mortality as primary outcome and days of mechanical ventilation and hospital stay as secondary outcomes. mean age(y.o) in group a was and in group b and in both groups mean charlson comorbidity index was points. survival percent was higher in the group treated with ceftazidim-avibactam ( % vs %, p = . )- (fig. ) . length of stay was significantly decreased in group b ( . days vs days in group a, p = . ). number of days under mechanical ventilation was also decreased in the ceftazidim-avibactam group ( vs ) but the data was not statistically significant. in light of the important thread of multiresistant a. baumannii and the lack of therapeutic measures, the synergistic activity of ceftazidim-avibactam use in combination with other antibiotics may be a promising approach to lower the mortality and hospitalization in critically ill patients diagnosed with vap. impact of patient colonization on admission to intensive care on and days mortality g dabar , c harmouch , e nasser ayoub , y habli , g sleilaty , j infections caused by multi resistant bacteria are a major health problem, especially in icus, and it may be associated with high mortality rates. colonization precedes infection in most instances; therefore it may be a marker of a poor outcome. we tried to determine the impact of colonization on mortality at and days in a population of patients admitted to one medical and one surgical icu in the same institution. medical records review over three years - of all patients admitted to one surgical et one medical icu at hotel dieu de france hospital staying more than h. colonization to resistant bacteria was defined as mrsa, esbl, mdr, and vre. all patient received a nasal and rectal screen on icu admission, in intubated patients tracheal aspirate was considered as colonization in the absence of clinical respiratory tract infection. demographics, apache, sofa, immunosupression, charleston comorbidity index, length of stay, mechanical ventilation, hospitalization and antibiotic use in the previous month were collected. mortality at and days was assessed through medical records or phone call. pearson chi-square was calculated for the association of colonization and mortality at and days, and subsequently odd ratio was estimated. introduction: critically unwell patients have been observed to respond unpredictably to traditional intermittent dosing (id) schedules of vancomycin, likely due to the complex physiological derangements caused by critical illness. continuous infusion (ci) of vancomycin has been suggested to overcome such problems by allowing more regular therapeutic drug monitoring and subsequent effective dose titration [ ] . this study conducted at a tertiary intensive care unit, reports our experience following implementation of a continuous vancomycin infusion protocol. prospective data was collected over two consecuative periods of three months, initially capturing plasma levels for id (target level of - mg/l) followed by reviewing plasma concentration levels in a ci protocol (target level of - mg/l). patients recieving renal replacement therapy were excluded. a total of intermittent vancomycin prescriptions were administered and dosing levels observed. in the three month ci period, patients received ci vancomycin and levels subsequently checked. the ci protocol resulted in increased blood sampling ( samples in ci group vs. samples in id cohort). two non serious incidents were reported in the ci cohort relating to preparation of vancomycin. both groups had a comparable median time to therapeutic range ( hours). however, ci vancomycin group had a greater proportion of first samples outside the desired therapeutic range ( %vs %) (figure ). as the therapy continued, ci vancomycin demonstrated a greater propensity towards consistent therapeutic levels than that observed with id. % of patients on a ci regime achieve the desired target levels compared to % in the id cohort (fig. ) . it was positive for single or multiple microbes in ( . %) and ( . %) samples respectively. single or multiple resistance genes were detected in ( %) and ( %) samples respectively. bfpcr was positive only for bacteria in ( . %), virus in ( . %) and for both in ( . %) cases. influenza a was found in ( . %) cases. the most common organisms in community and hospital acquired pneumonia were streptococcus pneumoniae ( / ) and a. baumannii ( / ) respectively. bacterial cultures were concordant with bfpcr in / ( %) of positive cases. decisions to change antibiotics could be taken earlier based on bfpcr (p< . ) than if were based solely on culturesboth in culture positive ( . ± . vs . ± . hrs) and negative cases ( . ± . vs . + . hrs) where antibiotics would have remained unchanged. based on bfpcr antibiotics were escalated in ( %) patients and teicoplanin ( / ) was most often stopped. bal bfpcr were obtained significantly earlier, identified more organisms and bacterial resistance than culture reports and lead to more frequent and earlier antibiotic changes. severe community-acquired pneumonia (scap) is a frequent cause of hospitalization and mortality. ceftaroline is efficacious for treatment of cap (port risk class iii or iv). most severe patients were excluded from the clinical trials, so the efficacy of ceftaroline in these kind of patients is unknown methods: this is a health record-based retrospective before-after study in a tertiary care hospital. all scap patients admitted in icu between november and february receiving ceftaroline were included. control group included patients with same inclusion criteria but receiving ceftriaxone. propensity scores to adjust for potential baseline differences between groups were performed. levofloxacin or azythromicin were administered in both groups. primary outcome was the change in sofa score over the first h and secondary were days of mechanical ventilation, respiratory failure at h, need of rescue antibiotics, length of stay and mortality results: there were patients in ceftaroline group and in ceftriaxone group. baseline characteristics were similar except from more intubated patients in ceftaroline group (figure ). there were less respiratory failure at h in patients with ceftaroline treatment (- . % vs. - . %; p , ), but no differences in other organ failures, mortality, days of mechanical ventilation or los. there were more need of rescue antibiotics in ceftriaxone group ( . % vs . . %; p , ). we found more streptococcus pneumoniae isolation in ceftaroline group ( ( . %) vs ( . %); p = . ); more empiric use of oseltamir ( ( . %) vs ( . %); p = . ), but no more influenzae infections ( ( . %) vs ( . %); p = . ). s. aureus was detected in patient in ceftaroline group and in in ceftriaxone group. introduction: acute respiratory failure (arf) due to pulmonary infections is a usual cause of intensive care unit (icu) admission. immigration patterns and iatrogenic immune-suppression have made tuberculosis (tb) a common disease in western europe. severe tb requiring icu care is rare. nevertheless, mortality associated with active tb and arf is poor [ ] . adult patients with tb admitted to icu from - were identified retrospectively. diagnosis was based on: positive cultures of sputum, bronchial aspirates or bronchioalveolar lavage fluid. demographic characteristics, reasons for admission, hiv status, anti-tb treatment and mortality were recorded. total of patients with tb were admitted to icu. mean apache ii score was , ± , . sixteen were male. mean age , ± , years. eight ( %) were hiv-positive, ( %) diabetes mellitus type , ( %) chronic liver disease. six ( %) had other causes of immunesuppression. main causes for icu admission were arf due to non- mycobacterium tuberculosis pathogens in %, acute liver failure in %, septic shock due to non-respiratory cause in %. overall, % were on anti-tb treatment at time of admission. tb involved the lung parenchyma in all patients. pleural involvement was present in % and lymph node in %. extrapulmonary sites were present in %: urogenital, gastrointestinal, bone marrow. pathogens identified in over-infections: % gram positive coccus, % gram negative bacilli, % fungal, % mdr-pathogen. one patient hiv-positive suffered arf due to pneumocystis jiroveci. overall, % died during icu stay. besides its latent evolution, mortality of tb patients admitted to icu is extremely high. arf due to over-infection seems to be the main cause for icu admission and mortality. better preventive approach of these patients may improve their outcome. introduction: human african trypanosomiasis (hat) is rarely encountered by critical care clinicians, but is an important differential for fever in the returning tropical traveler. late disease is characterized by seizures, fever and multi-organ failure [ , ] . we present an anonymized case presenting from an endemic area in zambia referred for tertiary critical care management. the patient was too obtunded to give informed consent and his relatives could not be contacted despite extensive efforts. a middle-aged man with no past medical history from rural zambia presented to a local clinical officer post with fever and arthralgia. he was treated twice with anti-malarial medication without resolution of symptoms. two months later he was admitted febrile and obtunded to a local hospital with worsening confusion. he was transferred hours by ambulance to our facility in lusaka, which is the only public tertiary critical care unit in zambia results: gcs on arrival was e m v without localizing neurology. microbiology investigations were negative, including for toxoplasma, cryptococcus, hiv or malaria. the patient suffered a generalized seizure followed by a sustained gcs of and was admitted to the icu for invasive ventilation and seizure control. peripheral blood smears demonstrated trypanosomes consistent with hat secondary to trypanosoma brucei rhodesiense. he was commenced on melarsoprol but rapidly deteriorated, with signs of melarsoprol-induced arsenic encephalopathy and subsequent tonsillar herniation. his death was confirmed by neurological criteria. conclusions: icu management of fulminant hat involves supportive neurocritical care plus melarsoprol, a toxic arsenic compound with common side effects of hepatotoxicity and dysrhythmia. arsenic encephalopathy occurs in % of late hat, with a fatality rate of % [ ] . early diagnosis is associated with a % survival rate in developed world travelers repatriated from endemic areas [ ] . lithium chloride to prevent endothelial damage by serum from septic shock patients (in vitro study) a kuzovlev the aim of the study was to investigate into effectiveness of lithium chloride (licl) as agent that prevents damage to the monolayer of endothelial cells under the action of serum from multiple trauma patients with septic shock. methods: serum from pts with septic shock (sepsis- ) and healthy donors was withdrawn. monolayer of ea.hy endothelial cells were incubated for hrs at °c with healthy person's serum and with septic patient's serum without licl and with it at concentrations of . mmol, . mmol, mmol, mmol. licl was added hour before the change of serum. after incubation cells were washed and fixed with % paraform solution and permeabilized with % triton x- solution. fixed cells were stained with primary antibodies to vecadherin and then incubated with secondary antibodies conjugated with oregon green fluorescent dye as well as with phalloid red and hoechst dye . images were processed by fluorescence microscope and imagej . p and metavue . programs. western blotting was used to detect antibodies to ve-cadherin, claudin and gsk- beta. statistics included mann-whitney test and chi-square test. incubation of a monolayer of endothelial cells with % serum of septic shock patients led to loss of ve-cadherin contacts and decrease of claudine. preincubation with licl . mmol did not prevent dismantling of claudine, actin, ve-cadherins; . mmol licl prevented it (p> . ), but at higher concentrations ( mmol, mmol) almost completely protected endothelial monolayer from destruction of intercellular contacts (p< . ). serum had almost no effect on the phospho-gsk- β level after min, min, min and hr, but caused a significant ( %) decrease in its level after and hrs. licl ( mmol) caused a significant increase in phospho-gsk- β already mins and up to hrs after exposure. licl prevents septic damage to the monolayer of endothelial cells in vitro in a gsk- beta mediated way. introduction: the autonomic nervous system (ans) controls both heart rate and vascular tone, which are known to be impaired during septic shock (ss) . acute inflammation is presumed to increase arterial stiffness of large arteries in experimental studies [ ] . the objectives of this work are to verify if standard ss resuscitation modulate mechanical vascular properties and to verify if alterations in these vascular properties and ans activity are correlated. a protocol of fecal peritonitis septic shock and standard resuscitation (fluids and noradrenaline) was applied on pigs. the arterial blood pressure waveform was recorded in the central aorta and in the femoral and radial arteries. the characteristic arterial time constant tau was computed at the three arterial sites, based on the twoelement windkessel model [ ] . the total arterial compliance (ac) and the total peripheral resistance (tpr) were also estimated. baroreflex sensitivity (brs), low frequency (lf, . - . hz) spectral power of diastolic blood pressure, and indices of heart rate variability (hrv) were computed to assess ans functionality. results: septic shock induced a severe vascular disarray, decoupling the usual pressure wave propagation from central to peripheral sites, as shown by the inversion of pulse pressure (pp) amplification, with a higher pp in the central aorta than in the peripheral arteries during shock. the time constant tau together with ac and tpr were independently decreased. a decrease in brs, lf power, and hrv describe an ans dysfunction. after the administration of fluids and noradrenaline, both vascular and autonomic dysfunction persisted and these were found to be significantly correlated. measures of mechanical vascular function and ans activity could represent an useful end-point to guide further clinical investigations and refine our understanding of ss mechanisms, especially under medical treatment. introduction: lipopolysaccharide (lps), is a component of gram-negative bacteria known for its activation of the host immune system. the phospholipid transfer protein (pltp) has previously been shown to promote the binding of lps to lipoproteins, to limit inflammation and to lower mortality following injections of lps or bacterial infection. the aim of the present study was to investigate the role of pltp and lipoproteins in the detoxification of lps from the peritoneal cavity. injection of lps intra-peritoneally (ip) ( mg/kg) to wild type (wt) and pltp knocked-out mice (pltp-ko) (n = per group). mass concentration and activity of lps were quantitated by lcmsms analysis of -hydroxymyristate and lal bioassay, respectively. lipoprotein fractions in plasma were separated by ultracentrifugation (n= vs n = ). following intra-peritoneal injection, clearance of intra-abdominal lps was faster and plasma neutralization was more efficient in wt than in pltp-ko mice ( figure ) . indeed, lps found in plasma of wt mice was proportionally less active, sustaining a higher capacity for wt mice to neutralize lps (figure b) . quantitative dosage of lps in portal blood, minutes after ip injection, revealed that plasma lps associates rapidly with the lipoprotein fraction (hdl plus ldl), and in higher proportions as compared to pltp-ko mice ( [ - ] % vs [ - ] %, respectively; p < . ). in line with previous studies, these observations now indicate that, lps readily associates with lipoproteins in a neutralizing process pltp mediated. finally, even with a heavy lps load ( mg/kg), the bulk of lps was still found in the lipoprotein fraction ( [ - ] %), suggesting that lipoproteins plus pltp in wt mice have a high capacity to detoxify intraperitoneal lps. in a model of peritonitis, lipoproteins and pltp were found to constitute key playors for peritoneal clearance and neutralization of lps. it emerges as a key pathway for the resolution of the inflammatory response in peritonitis. introduction: autotaxin (atx, enpp ) is a secreted enzyme present in biological fluids that catalyses the production of lysophosphatidic acid (lpa). lpa is a bioactive phospholipid evoking various cellular responses in most cell types. upregulated atx levels have been reported in various chronic inflammatory diseases. given the established role of lpa in the inflammatory response, we investigated a possible role for the atx/lpa axis in lps-induced endotoxemia. methods: lps was injected intraperitoneally ( mg/kg) in mice producing % atx levels (atx df/+ , heterozygous null mutant mice), in mice producing - % reduced atx levels upon inducible inactivation (r creer t /enpp n/n mice) and in mice expressing - % increased atx levels (enpp -tg mice). kaplan-meier survival analysis was performed. atx activity was measured using the toos activity assay. results: atx df/+ mice that produce almost % reduced serum atx levels show increased survival compared to their littermate controls. for the inducible inactivation of atx, enpp n/n targeted mice were crossed with the r cre-er t mice and tamoxifen induction enabled temporal control of floxed gene expression. r creer t /enpp n/n mice were more protected against lps-induced endotoxemia compared to control mice. enpp -tg mice overexpressing autotaxin and showing a -fold increase in plasma levels do not display improved survival rates compared to control group. conclusions: atx participates in systemic inflammation, as reduced atx levels in circulation decrease lethality of mice from caused by lps. the excess amount of circulating atx does not exacerbate the systemic inflammatory response to lps. introduction: pneumonia (pn) is a prevalent and severe infectious lung disease. host genetics plays an essential role in the pathogenesis of infectious diseases including pn [ ] . the aim of the study was to analyze the variability of genes associated with neutrophil activation in pneumonia. to identify differential expressed genes (degs) in communityacquired (cap) and hospital-acquired pneumonia (hap) dataset «genome-wide blood transcriptional profiling in critically ill patients -mars consortium» (gse ) from gene expression omnibus was analyzed (logfc≥ . , fdr-corrected p-value< . ). degs associated with neutrophil activation were selected according to gene ontology go: («neutrophil activation»). with the use of gtex portal and blood eqtl browser, we searched for esnps (expression single nucleotide polymorphisms) in whole blood for neutrophil activation genes differentially expressed in cap/hap. these esnps were further analyzed for their association with pn via the global biobank engine (gbe). a total of degs from gse correspond to go: genes ( up-and down-regulated) of which genes were common to cap and hap. functional enrichment of degs based on disgenet detected top- diseases associated with these genes (fdr-corrected p-value< . ): myeloid leukemia, chronic; sepsis; asthma; lung diseases; allergic asthma. for these genes esnps common to gtex portal and blood eqtl browser were identified. more than half of all variants were located on the second chromosome and influenced the expression of tnfaip and il rap genes. among all esnps we identified variants associated with pn in the gbe (table ) . we identified genes related to neutrophil activation, genetic variability of which was associated with pneumonia. sepsis was induced in wild-type c bl mice (n= ) and cse knockout mice (n= ) by i.p. injection of cfu/mice mdr p. aeruginosa. similar experiments were repeated after cyclophosphamide induced neutropenia. survival was recorded for days. mice were sacrificed for determination of bacterial load and myeloperoxidase (mpo) activity as a surrogate marker of myeloid cell recruitment. cytokines were measured in serum by legendplex inflammatory panel. total leukocytes from mice spleens, with or without pretreatment with the h s donor gyy , were incubated with x cfu/ml mdr p. aeruginosa. bacterial clearance was recorded. we observed a significant decrease in survival of cse -/mice as compared to cse +/+ mice ( % vs. %; p: . ). this survival advantage was eliminated in neutropenic mice ( % for both groups, p: . ). cse -/mice had increased pathogen load in the liver ( . ± . vs . ± . , p: . ) and lung ( . ± . vs . ± . , p: . ). mpo activity was lower in cse -/mice in the liver ( ± vs ± , p: . ) and lung ( ± vs ± , p: . ). cse +/+ mice had increased serum levels of il- ( . ± . vs . ± . of cse -/-, p: . ); mcp- ( . ± . vs . ± . , p: . ) and gm-csf ( . ± . vs . ± . , p: . ). phagocytic activity of leukocytes from cse -/mice was reduced compared to cse +/+ mice. this deficit was eliminated after gyy pretreatment (fig. ) . deficiency of host-derived h s leads to increased susceptibility to mdr p. aeruginosa infection due to an inefficient neutrophil chemotaxis and neutrophil mediated phagocytosis. acknowledgement funded by the itn horizon marie-curie european sepsis academy introduction: neuroinflammation often develops in sepsis along with increasing permeability of the blood-brain barrier (bbb), which leads to septic encephalopathy [ ] . the barrier is formed by tight junction structures between the cerebral endothelial cells [ ] . we investigated the expression of tight junction proteins related to endothelial permeability in brain autopsy specimens in critically ill patients deceased with sepsis, and analyzed the relationship of bbb damage and measures systemic inflammation and systemic organ dysfunction. case series included all adult patients deceased with sepsis in the years - with brain specimens taken at autopsy available. specimens were categorized according to anatomical location (cerebrum, hippocampus, cerebellum). the immunohistochemical stainings were performed for occludin, zo- and claudin. patients were categorized as having bbb damage if there was no expression of occludin in the endothelium of cerebral microvessels. results: % ( / ) developed multiple organ failure before death. . % ( / ) had septic shock. the deceased with bbb damage had higher sofa maximum scores ( vs. , p= . ), and had more often procalcitonin levels above ( % vs. %, p= . ). bbb damage in cerebellum was more common in cases with c reactive protein above mg/l as compared with crp less than ( % vs. %, p= . ). absence of zo- expression in cerebral meningeal samples associated with bbb damage ( % vs. %, p= . ). positive blood cultures (n = ) were associated to absence of zo- expression in cerebellar glial cells ( % vs. %, p= . ). in fatal sepsis, damaged bbb defined as loss of cerebral endothelial expression of occludin ( figure ) is related with severe organ dysfunction and systemic inflammation. loss of zo- in endothelial cells associates with bbb damage, and sepsis contributes to zo- loss in cerebellar glial cells. oxylipins are oxidative breakdown products of cell membrane fatty acids. animal models have demonstrated that various vasoactive oxylipin pathways may be implicated in septic shock pathophysiology but these have been poorly studied in humans. oxylipin profiling was performed on serum samples collected on enrolment to the vanish (vasopressin vs. norepinephrine as initial therapy in septic shock) trial. samples were analysed with liquid chromatography-mass spectrometry. patients were followed up until days. results: samples were collected from of ( . %) patients on inclusion to the trial and ( . %) had died by days. non-survivors were found to have higher levels of a number of oxylipins including: , -dihydroxyeicosatrienoic acid (dhet) (p< . ), , -dhet (p= . ), (s)-hydroxyeicosatetraenoic acid (p= . ), -hydroxyoctadeca-pentaenoic acid (p= . ) but lower levels of the precursor eicosapentaenoic acid (p= . ). when corrected for multiple comparisons with the benjamini-hochberg test, only , -dhet remained significant (p= . ). although there was a difference in median , -dhet levels between survivors and non-survivors, many values were below the level of detection (n= / ( . %)). as such, we also analysed - -dhet as a binary variable (figure ). patients with detectable , -dhet were more likely to die (hr . [ % ci . - . ], p< . ) and have a higher median lactate (p = . ) and total sofa score (p< . ) than those patients where baseline , -dhet was undetectable. our study suggests the oxylipin , -dhet may be associated with septic shock severity and -day mortality. these results are consistent with the known vasodilatory actions of this class of oxylipin. more work is needed to confirm its exact role in septic shock and whether this pathway is amenable to therapeutic intervention. introduction: activation of neutrophils is a mandatory stage and a sensitive marker of systemic inflammatory conditions that can lead to the development of multiorgan failure. the aim of the study was to investigate into the antiinflammatory effects of lithium chloride on human neutrophils in vitro. study was carried out on neutrophils isolated from the blood of healthy donors. % of neutrophils were activated by mkm fmlp, % -by ng/ml lipopolysaccharide (lps); then their activity was evaluated by fluorescent antibodies to cd b and cd b degranulation markers. intact and activated neutrophils were treated with a solution of lithium chloride ( mmol). immunoblotting was used to assess gsk b activity in neutrophils. mann-whitney criterion and p< . were used for statistics. results: lithium chloride mmol decreased the level of expression of cd b on intact neutrophils by % (p= . ), cd b by % (p= . ). fmlp increased cd b expression on neutrophils by . times (p= . ), cd b by . times (p= , ). addition of lithium chloride solution to fmlp activated neutrophils reduced the expression of cd b (p= . ) and cd b (p= . ). lps increased cd b and cd b expression by . times (p= . , p= . , respectively); addition of lithium chloride reduced the expression of cd b (p= , ) and cd b (p= . ) on neutrophils. fmlp led to a dephosphorylation of gsk- b by % (p< . ), lithium chloride increased its phosphorylation by % (p < . ). adding lithium chloride to activated fmlp neutrophils restored the level of gsk- b phosphorylation by % compared to controls (p< . ). lithium chloride modulates the inflammatory activation of neutrophils by bacterial components through the phosphorylation of gsk b in neutrophils. human host immune responses to lipopolysaccharide: a comparison study between in vivo endotoxemia model and ex vivo lipopolysaccharide stimulations using an immune profiling panel dm tawfik introduction: sepsis, a leading cause of mortality among critically-ill patients in the icu, recently recognized by the who as a global health burden. patients that suffer from sepsis exhibit an early hyper-inflammatory immune response which can lead to organ failure and death. in our study, we assessed the immune modulations in the human in vivo endotoxemia model and compared it to ex vivo lipopolysaccharides (lps) stimulation using transcriptomic markers. methods: eight healthy volunteers were challenged with intravenous lps in vivo. in parallel, blood from another volunteers was challenged with lps ex vivo. blood was collected before and after hours of lps challenge and tested with the immune profiling panel (ipp) prototype using the filmarray® system. the use of ipp showed that markers from the innate immunity dominated the response to lps in vivo, mainly markers related to monocytes and neutrophils. comparing the two models, in vivo and ex vivo, revealed that most of the markers were modulated in a similar pattern ( %). some cytokine markers such as tnf, ifn-γ and il- β were under-expressed ex vivo compared to in vivo. t-cell markers were either unchanged or up-modulated ex vivo, compared to a down-modulation in vivo. interestingly, markers related to neutrophils were expressed in opposite directions, which might be due to the presence of cell recruitment and feedback loops in vivo. the majority of ipp markers showed similar patterns of expression post-lps challenge in both models, except for several markers related to neutrophils and t-cells. the ipp tool was able to capture the early immune response in the human in vivo endotoxemia model, which is a translational model mimicking immune host response in septic patients. introduction: serum levels of tyrosine kinase receptor mer and its ligand gas predict mortality in septic patients in the intensive care unit. however, whether their early measurement at emergency department (ed) presentation also predicts mortality and organ failure still needs to be clarified. in this multicentre observational study, septic patients admitted to italian eds were included [ ] . at ed presentation blood samples were taken for routine biochemical analyses and serum mer and gas measurement. urinalyses, blood gas analyses and chest x-ray were routinely performed. mortality at and days, as well as the presence of organ damage such as acute kidney injury (aki), thrombocytopenia, pt-inr derangement and sepsis-induced coagulopathy (sic) were evaluated according to baseline levels of mer and gas . in conclusion, neither mer nor gas are early predictors of mortality in septic patients at ed presentation. however, mer independently predicted the development of sic, thrombocytopenia and pt-inr derangement in this population. glycocalyx shedding correlates with positive fluid balance and respiratory failure in patients with septic shock n takeyama, y kajita, t terajima, h mori, t irahara, m tsuda, h kano aichi medical university, department of emergency and critical care medicine, aichi, japan critical care , (suppl ):p endothelial hyperpermeability would play a major role in septic shock related organ failure. the aim of this study is to clarify the relationship between glycocalyx shedding and respiratory failure, sofa score, plasma angiopoietin (ang)- level and patient survival. methods: plasma samples were collected from septic shock patients from admission to icu discharge and healthy volunteers. plasma syndecan (syn)- and ang- were measured and clinical data was also collected. septic shock patients were classified into groups according to the time-course change of syn- levels. excess syn- (> ng/ml) during to days and remaining high following to days were assigned to group i. excess ang- during to days and decreased following to days were assigned to group ii. moderate increase (< ng/ml) during to days were assigned to group iii. results: plasma syn- levels are positively associated with increased ang- levels (r = . , p= . ), suggesting that ang- is involved in endothelial hyperpermeability. fluid balance and ventilator-free days (vfd) are significantly increased in group i as compared with group iii. sofa score, apache ii and patient outcome does not show any differences between groups i, ii, and iii. the positive correlation between glycocalyx shedding and fluid balance indicates plasma syn- may be a valuable marker for endothelial hyperpermeability. the negative correlation between glycocalyx shedding and vfd indicates plasma syn- may be a valuable marker for respiratory failure. the plasma level of syn- for prognosis and organ failure excluding ards in patients with septic shock requires further investigation. serial procalcitonin measurements in the intensive care unit at hiroshima university hospital k hosokawa, s yamaga, m fujino, k ota, n shime hiroshima university hospital, department of emergency and critical care medicine, hiroshima, japan critical care , (suppl ):p introduction: serum procalcitonin (pct) is a promising biomarker for differentiating bacterial infections from other inflammatory states. moreover, including serial pct measurements in the management of acute respiratory infection reduces the duration of antibiotic therapy without increasing the mortality. however, limited real-world information is available regarding the use of pct in intensive care units (icus). we extracted and analysed data from january to december , from all the orders and results of pct measurements in the icu ( beds) at hiroshima university hospital. a total of , pct measurements from icu patients were included. in patients, pct was tested ≥ times during a single icu stay. serial pct measurements showed a fade-out pattern ( [ %] patients), a second day-peaked decrease pattern ( [ %] patients), and a series of negative patterns ( [ %] patients). compared to patients who demonstrated the fade-out pattern, those who demonstrated the second day-peaked decrease pattern had higher mortality rates ( % vs. %, p < . ). approximately one-third patients in the icu who had decreasing serial pct values demonstrated the second day-peaked decrease pattern. since this group of patients had poorer survival, further studies are needed to clarify the association between a late rise in pct levels and delayed therapeutic intervention. the research was performed on full-term newborns; no clinical signs of bacterial infection were diagnosed. on the , , days the plasmà concentration of il- ß, il- , il- , tnf-α, g-csf, sfas, fgf, no was determined by capture elisa; cd cd , cd cd , cd cd , cd , cd , cd , hla-dr, cd , cd , cd cd , lymphocytes in apoptosis -immunophenotype analysis. by applying the statistical cluster population analysis of the immunological criteria under study we have evaluated the feasibility of sepsis diagnostics at the admission to the intensive therapy unit. the diagnostic rule for sepsis has been formulated by applying the "decision tree" approach to the "r" statistic medium. the cluster analysis confirms the presence of two clusters (presence of absence of sepsis: these two components explain the . % of the point variability). the diagnostic rule for the early diagnostics of sepsis is as follows: disease develops providing during the first hours cd ≥ . %, no≤ . mkmol/l or cd ≤ . %, cd ≤ . %, cd ≥ . % or cd ≤ . %, cd ≤ . %, cd ≤ . % and lymphocytes annexinv-fitc+pi-≥ . %. newborns featured the confirmed sepsis development. the accuracy of this diagnostics amounts to . %; sensitivity to . %; specificity to . %; diagnostic false positive share to . %; diagnostic false positive share to . %; positive result accuracy to . %; negative result accuracy to . %. the aggregate determination of cd , cd , annexinv-fitc+ pi-, cd and the plasma concentration of no enables the pre-clinical diagnostics of sepsis development. efficacy of pancreatic stone protein in diagnosis of infection in adults: a systemic review and metaanalysis of raw patient data j prazak , p egimann , i irincheva , mj llewelyn , d stolz , lg de guadiana-romualdo , r graf , t reding , hj klein , ya que fig. (abstract p ) . impact of h lactate and bio-adm values in patients with elevated lactate level at admission. the green curve in the left km-plot illustrates data from patients with events; the red curve patients with events. the green curve in the right km-plot illustrates data from patients with events; the red curve patients with events. of note, differences in numbers between admission (n= ) and h (n= ) is related to initial mortality introduction: adrenomedullin (am) is a peptide synthesized in vascular endothelial cells and cleared by the lungs. the use of am as an inflammatory biomarker and his predictive value has been studied in critically ill patients, but not yet in veno-venous extracorporeal membrane oxygenation (ecmo). the purpose of this study was to describe the plasmatic levels of am in patients supported with ecmo for acute respiratory failure methods: am (normal values < . nmol/l) was measured at time points: immediately before (t ), -h (t ) and -h after (t ) ecmo initiation and immediately before (t ) and -h (t ) after ecmo removal, in consecutive patients with severe respiratory failure supported with ecmo enrolled in the gatra study (nct ) at fondazione irccs ca' granda -policlinico of milan. data are reported as median ( th - th percentile). statistical analysis was performed using logistic and random effects regression models (to account for repeated measurements within individuals) results: a total of measurements were taken in consecutive patients. am (nmol/l) decreased along the course of ecmo: t = . ( . - . ), t = . ( . - . ), t = . ( . - . ), t = . ( . - . ), t = . ( . - . ) (mean diff.= - . , %: ci - . , - . ). am was lower in patients with viral compared to bacterial ards (mean diff.= - . , %ci - . , - . ) (figure ). am was higher in more severe patients (sofa>= , n= ) compared to less severe patients (sofa< , n= ): . ± . vs . ± . nmol/l, respectively p< . . basal values of am could not predict mortality at days (or= . , %ci: . - . ) after conditioning for sofa score and respiratory failure etiology conclusions: am plasmatic values seem to be higher in more severe patients and in patients with bacterial ards. am decreased along the ecmo course but could not predict mortality in our group of patients fig. (abstract p ) . plasmatic adrenomedullin during ecmo heparin binding protein (hbp) is released from activated neutrophils upon stimulation of b integrins. this pro-inflammatory effect generates the hypothesis that it can be a sepsis biomarker for patients admitted at the emergency department (ed) methods: the prompt study (clinicaltrials.gov nct ) took place at the ed of six greek hospitals. participants were admitted with suspected acute infection and at least one vital sign change. hbp was measured by an enzyme immunosorbent assay in plasma. sepsis was diagnosed by the sepsis- criteria. the primary study endpoint was the sensitivity for the diagnosis of sepsis. outcome prediction was the secondary endpoint. a total of patients were enrolled; had sepsis. the most common infections among patients without and with sepsis were upper respiratory tract infections in . % and . %; community-acquired pneumonia in . % and . %; and acute pyelonephritis in . % and . %. median hbp was . and . ng/ml respectively (p: . ). following analysis of the area under the curve (auc) it was found that the best discriminatory cut-off for sepsis was . ng/ml. the comparative diagnostic performance of hbp versus qsofa score is shown in figure . the odds ratio for sepsis with hbp above . ng/ml was . (p: . ). at the same cut-off point the sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) for the prediction of early death after hours was %, . %, . % and % respectively. hbp is more sensitive but less specific than qsofa for the diagnosis of sepsis in the ed. the rule-out prediction of early death seems the great merit. chronobiological and recurrence quantification analysis of temperature rhythmicity in critically ill patients introduction: rhythmicity and complexity of several circadian biomarkers, such as melatonin, cortisol and temperature have been found to be modified by critical illness. we examined the potential alterations of core body temperature (cbt) fluctuations and complexity in three groups (n= ): patients with septic shock upon icu admission (group a, n= ), patients who developed septic shock at icu hospitalization (group b, n= ) and controls (group c, n= ). the hourly, average cbt was computed for h upon icu admission and discharge in groups a and c, as well as during septic shock onset in group b. cosinor analysis of cbt curves was performed leading to the estimation of mesor (mean value), amplitude (the difference between peak and mean values) and acrophase (phase shift of maximum values in hours). complexity of cbt signals was evaluated with recurrence quantification analysis (rqa). no significant alterations in any circadian feature within groups were found, except for amplitude. controls exhibited increased entry cbt amplitude ( . ± . ) compared to groups a ( . ± . , p < . ) and b ( . ± . , p < . ). higher entry cbt amplitude in groups b and c was related with lower saps ii (r = - . and - . , p < . ) and apache ii scores (r = - . and - . , p < . ) respectively, reduced icu and hospital stay in group b (r = - . and - . , p < . ) and entry sofa score in group c (r = - . , p < . ). recovery cbt time series appeared more periodic in relation with icu entry, for all groups. a more random cbt signals pattern upon results: among . . individuals, . received inpatient treatment for sepsis. % had severe sepsis. % of sepsis and % of severe sepsis patients had an explicitly coded hai. the proportion of hai was higher in patients that received icu-treatment than in patients without icu-treatment ( % in icu/ % in non-icu sepsis, % in icu/ % in non-icu severe sepsis patients). tab. shows the foci of explicitly coded hai. nosocomial pneumonia was the most common hai in all patient groups. clabsi occurred more frequently in icutreated patients; % were affected. cauti and c. diff infections were more common among non-icu-treated sepsis patients. more than one quarter of non-icu-treated sepsis patients had a c. diff infection. hai are common causes of sepsis and pose a significant healthcare burden. the proportion of patients affected and the distribution of foci differ between non-icu-and icu-treated sepsis patients with important implications for sepsis management within hospitals. impact of sepsis protocol triggered by ramathibodi early warning score (rews) in ipd sepsis on clinical outcomes s matupumanon , y sutherasan , d junhasawasdikul , p theerawit sepsis is now early identified and managed during triage in the emergency department. however, there is less focus on the effect of patients' management at the ward level. we aim to evaluate the impact of the implementation of the sepsis protocol on clinical outcomes in in-patients with new-onset sepsis. we conducted a prospective observational cohort study among adult medical patients admitted to the general wards in a university hospital. a -month pre-protocol period (august to august ) was assigned to a control group, and a -month protocol period (september to october ) was allocated to a protocol group. an in-patient sepsis protocol comprised nurse-initiated sepsis protocol by ramathibodi early warning score (rews)≥ plus suspected infection, prompt antibiotic, lactate measurement, and fluid resuscitation was implemented. (table ) . the implementation of in-hospital sepsis protocol was associated with significant improvement in patients' outcomes, namely lactate measurement, starting antibiotic within hr, fluid management, and the shorter length of icu stay. icu routine nursing procedures interfere with cerebral hemodynamics in a prolonged porcine fecal peritonitis model sl liu , dc casoni , w z'graggen , d bervini , d berger , sj jakob routine nursing procedures (np) can interfere with blood pressure and cardiac output and may therefore alter cerebral hemodynamics in critical illness. this may be risk factor of sepsis-associated encephalopathy. methods: sedated and mechanically ventilated pigs were randomized to fecal peritonitis or controls (n= , each). after hours of untreated peritonitis, the animals were resuscitated for hours (resuscitation period). np [assessment of sedation (as), tracheal suctioning (ts), change in body position (cp), lung recruitment maneuver (rm)] were performed at baseline and h, h, h and h after start of rp. systemic and cerebral hemodynamics and o saturations were recorded continuously. shock is the most common cause of death in the postsurgical icu, including septic shock and hypovolemic shock, reaching the - % mortality in septic shock. the inadequate response of the immune system to the infection triggers a potent inflammatory cascade, where the c-reactive protein (crp) is an essential key in the amplification and maintenance of this cascade. the gene encoding to crp is located on the proximal long arm of human chromosome ( q ). the gt polymorphism in the promoter sequence of crp gene (rs ) has been associated with invasive pneumococcal disease. thus, we analyze the relationship between rs polymorphism and the risk of developing septic shock in postsurgical patients. an observational, retrospective and single-center study was conducted on a sample of caucasian patients undergoing major abdominal surgery, of which one part developed septic shock and another part developed systemic inflammatory response syndrome, who were used as control. the rs polymorphism was analyzed by vasoactive medications are commonly used in sepsis treatment but may correlate with peripheral ischemia and the well-publicized complication of limb and digit loss. yet, the association between limb and digit threat and the intensity, duration, and pattern of vasopressor exposure are unknown. we studied adults ( - ) at hospitals in an integrated health system who met criteria for sepsis- . we identified the time to clinically apparent limb or digit threat using clinical adjudication among those with vasopressor-dependent sepsis (i.e. > hour of vasopressors at sepsis onset) who had a surgical evaluation within -days of sepsis onset. we defined daily vasopressor intensity as to vasopressors administered. then, we created a time-dependent model for threat with mortality as a competing risk with a weight function to estimates the varying contribution of vasopressors over time. we determined the subdistribution hazard (sh) ratio of threat for various patterns of vasopressor exposure and intensity, adjusted for age, baseline risk factors, and sequential organ failure assessment (sofa) score at sepsis onset. of , adults with sepsis, , ( %) were vasopressordependent (age, [iqr, - ]; , [ %] males; max sofa score, [sd ] ). of these, , ( %) died and ( . %) had evaluations for limb or digit threat [iqr, - ] days after sepsis onset. the model-based weight function showed the contribution of vasopressors to threat was stable over time ( fig a) . overall, a unit increase in cumulative vasopressor exposure was associated with risk of threat (sh ratio, . [ %ci, . - . ], p<. ). for various patterns of vasopressor exposure, greater intensity associated with increased risk of threat ( fig b) . compared to constant exposure, an increasing and peak pattern associated with the greatest sh (fig c) . cumulative vasopressor exposure was associated with an increased risk-adjusted hazard of limb or digit threat following sepsis. fig. (abstract p ) . relationship between vasopressor exposure and limb or digit threat following vasopressor-dependent sepsis. panel a demonstrates the estimated contribution of daily vasopressor intensity prior to surgical evaluation for limb or digit threat, with mortality as a competing risk. panel b and c explore the relationship between threat and both cumulative vasopressor exposure and the pattern of exposure following sepsis onset. (b) the maximum cumulative vasopressor exposure was associated with the highest risk of limb or digit threat (shr . ) when compared to reference exposure pattern (shr . , reference). (c) increasing (shr . ) and peak (shr . ) patterns of cumulative exposure were associate with an increased sh of limb threat, while a decreasing pattern was associated with a lower risk (shr . ) when compared to constant intensity (shr . , reference). abbreviations: shr: subdistribution hazard ratio proportion of encounters transitioning from phenotype at presentation within hrs, by arrival phenotype assignment and probability of membership. (c) tsne plots for α-type, ß-type, y-type, and ∂-type, with core (dark), marginal (light), and non-members (grey) in plots on the left and core, marginal, non members, and transitioning members (black) on the right fig. (abstract p ). isolated microorganisms critical care references: . wertz et al. critical care explorations : e the process investigators choosing wisely guidelines for the provision of intensive care services, version . ics structured patient handovers references: . care of the critically ill woman in childbirth the proqol manual: the professional quality of life scale:compassion satisfaction, burnout & compassion fatigue/secondary trauma scales references: . shimabukuro-vornhagen a et al. ca the code: professional standards of practice and behaviour for nurses, midwives and nursing associates p introduction: the aim of this study was to compare factors associated with the icu mortality for vap due to multidrug-resistant (mdr) klebsiella spp. in case of monobacterial (mo) vs polibacterial (po) origin. methods: retrospective data analysis of patients treated in icu with mdr klebsiella spp. strains as pathogens of vap during three year period was carried out. results: data of patients were evaluated. mo vs po of mdr klebsiella spp. vap cases was found to be ( . %) vs ( . %), p = . . the icu mortality was / ( . %) in mo, and / ( . %) in po one, p = . . statistical significant differences of survivors vs non-survivors in mo and po vap due to mdr klebsiella spp. were found in medians of neutrophilosis p introduction: we study the population structure and resistome of mdr enterobacterales and pseudomonas aeruginosa isolates, c/t-susceptible or -resistant, recovered from low respiratory, intraabdominal and urinary tract infections of icu patients of portuguese hospitals (step study results: in e. coli, two vim- producers were found (st -b -h -o :h -ctx-m- and st -c-h -o :h ) (c/t-mic= . / - / mg/l). a kpc- -st -cladev-h -o :h ( / mg/l) was also detected. the most frequent esbl-e. coli clone was st cpr klebsiella pneumoniae ( patients), candida spp. ( patients). the comparison subgroup consisted of patients with bacteremia caused by non-escape pathogens. we evaluated the days of mechanical ventilation, duration of antibiotic therapy (amt), icu length of stay (los), hospital los and mortality (table ). results: mortality in patients with bacteremia caused by non-eskape pathogens was . %, candida spp vancomycin mass removal over minutes of hemoperfusion using ha . bars refer to vancomycin mass (mg): blue (experiment ) and red (experiment ) bars using blood while green (experiment ) bar using balanced solution. yellow dashes are mean mass values of the three experiments (with standard deviations) and yellow line represents the reduction curve over time table (abstract p ). results. * p-value versus non-eskape subgroup mechanical ventilation p translational value of the microbial profile in experimental sepsis studies sp tallósy , a rutai , l juhász , mz poles , k burián , d Érces , a szabó , m boros invasive hemodynamic monitoring and blood gas analyses were performed on anesthetized animals between - h of sepsis. the respiratory, cardiovascular, renal, hepatic and metabolic dysfunctions were evaluated with the species-specific sequential organ failure assessment (sssofa) score, the microbial profile was determined with selective media and maldi-tof ms in the initial inoculum and in the abdominal fluid taken h after sepsis induction. results: strong correlation was found between the initial dose of the inoculum (cfu) and the sssofa scores for organ dysfunction (rats: r = . , p= . ; pigs: r= . , p = . ) p introduction: pancreatic stone protein (psp) has shown promise as a biomarker of infection however, its diagnostic potential has not been systematically evaluated. we performed a systematic review and meta-analysis of available data on psp to evaluate its value for detecting infection in adults and determining a plasma or serum threshold value. methods: the pubmed and cochrane library database were searched for studies on psp in adult patients and their raw data were analyzed to estimate the best psp cut-off value that could detect infected patients using the youden's index. the cut-off sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) were computed and compared to those for procalcitonin (pct) and c-reactive protein (crp). finally, we explored the potential value of a model combining all three biomarkers to detect infection. results: from a total of potentially eligible published studies, containing patients were included in quantitative analysis. among them, patients suffered from a clinically confirmed infection. the median appropriate statistical tests were used using spss . cd was expressed as % age of neutrophils expressing positivity. results: sixty patients were analyzed. all parameters were compared between survivors and non survivors. demographics were comparable. most common source of sepsis was lungs and majority were admitted due to medical reason. non-survivors had significantly increased number of days with septic shock. at day median values of all the biomarkers and the sofa score were significantly higher in the nonsurvivor group (p< . ). there was a decreasing trend of all biomarkers and sofa score amongst survivors. on multivariate logistic regression analysis, increased cd and crp levels between baseline and day , increased days with septic shock and increased sofa references: introduction: we characterized the association of c-reactive protein (crp) with extracellular vesicles (evs) in plasma from sepsis patients and assessed a commercial crp adsorbent (pentrasorb, pentracor, hennigsdorf, germany) to deplete free and ev-associated crp. in addition, we characterized the potential pro-inflammatory effects of ev-bound crp on monocytes and endothelial cells monocytes and human umbilical vein endothelial cells (huvecs) were stimulated with isolated evs ( , g, min) monocyte il- secretion was quantified by elisa; the activation of huvecs was assessed by their expression of icam- and e-selectin using confocal microscopy. results: septic plasma (n= ) contained . ± . mg/l crp vs. . ± . mg/ l for healthy controls (n= ). both, total evs and crp + evs were significantly elevated in septic plasma as incubation of septic plasma with pentrasorb resulted in depletion of free crp ( . ± . mg/l before vs. . ± . mg/l after adsorption) as well as in a significant reduction in crp evs from crp-depleted septic plasma induced significantly lower il- levels. huvec icam- or e-selectin expression, however, did not increase upon stimulation with septic evs. conclusions: treatment of septic plasma with pentrasorb efficiently removes free crp and detaches crp from the ev surface, resulting in reduced proinflammatory effects flow cytometry confirmed the association of monocytes with platelets and platelet-derived evs as well as the uptake of evs by monocytes. conclusions: storage of isolated monocytes induces a shift towards cd expressing proinflammatory monocytes, which seems to be mediated by residual platelets and platelet-derived evs. it remains to be clarified whether evs released from activated platelets can also trigger a shift towards proinflammatory, intermediate monocytes in vivo ethical approval was provided by ucl research ethics committee ( / ). paired parametric analyses were performed and data displayed as mean +/- % ci. results: plasma calprotectin concentration began to increase . hours after endotoxin administration, was significantly higher than baseline by hours ( . ng/ml vs. ng/ml, p < . ), peaked at hours (mean ng/ml, figure ) and normalized by hrs. calprotectin peaked earlier than comparator soluble mediators (procalcitonin hrs, crp, hrs) and exhibited % sensitivity; all participants demonstrating a minimum -fold increase from baseline (mean . x). calprotectin displayed greater baseline variability (sd . ng/ml) than either crp or procalcitonin. conclusions: our results indicate the potential of plasma calprotectin as a biomarker for bacterial infection. it increases earlier and peaks more rapidly than standard biomarkers. whilst higher baseline variability was observed p a multicenter randomized controlled study on landiolol for the treatment of sepsis-related tachyarrhythmia: subanalysis of the j-land s study o nishida kagoshima university graduate school of medical and dental sciences, department of emergency and intensive care medicine methods: we analyzed a retrospective cohort of electronic health records from adult sepsis patients at upmc hospitals from to . we defined sepsis- by i.) suspected infection (e.g., administration of antibiotics or body fluid culture) & ii.) organ dysfunction (e.g., or more sofa points) in the first hours of care. data were organized by hour and included vital signs, lab values, and treatments (e.g., total hourly iv fluids (ml) and norepinephrine equivalent dose). for each hour we describe, i.) available data elements, ii.) presence of sepsis- , and iii by hour , most patients had vital signs ( %; n= , ), basic labs ( %; n= , ), fluid cultures ( %, n= , ), while serum lactate was completed in % (n= , ) conclusions: early sepsis care patterns are variable. iv fluids were given during early hours, when uncertainty about sepsis was greatest, while vasopressors were administered after sepsis- elements were present. p effects of abdominal negative pressure treatment on splanchnic hemodynamics and liver and kidney function in a porcine fecal peritonitis model sl liu department of intensive care medicine splanchnic hemodynamics and laboratory parameters were measured at baseline (bl, start of rp), and h, h and h after start of rp. two/three-way rm-anova or mixed-effects analysis, and student t tests were performed. results: npt in controls had no effect. after sepsis induction, mean arterial pressure (map) decreased by ( - ) mmhg, cardiac output (co) by . ( . - . ) l/min, and arterial lactate increased by . ( . - . ) mmol/l. sepsis and resuscitation was associated with increasing hepatic and renal arterial flows (p≤ . , both), and increasing prothrombin time npt in sepsis resulted in numerically less noradrenaline administration ( . ± . ug/ min/kg in sepsis with npt vs. . ± . ug/min/kg without npt, p= . ) and positive fluid balance ( . ± . ml/h/kg with npt vs. . ± . ml/h/kg without, p= . ). conclusions: in our experimental fecal peritonitis model, npt did neither impair splanchnic hemodynamics nor abdominal organ function. whether npt helps to reduce noradrenaline and volume administration in abdominal sepsis should be evaluated in further studies. p association between a c-reactive protein gene polymorphism (rs ) with the risk of develop septic shock in postsurgical patients of major abdominal surgery p martínez-paz valladolid, spain; hospital of medina del campo notably, the three groups received a comparable pro kg dose of acetaminophen. no difference was found between groups in term of toxic effects. patients carrying the cyp a p showed a more pronounced effect on body temperature in respect of wt and ugt a p °c respectively, but it does not reach statistical significance (fig. b). only % of the patients reach a temperature < °c at t and only % < . °c. conclusions: polymorphisms in enzymes involved in the metabolism of acetaminophen are relatively common. cyp a p seems to lead to higher peak plasmatic concentration and a slightly increased efficacy in fever control panel a: variations of acetaminophen plasmatic levels after minutes (t ) and hours (t ) after administration of an iv dose of g of paracetamol in wt patients and patients carrying mutation; panel b: body temperature variations in wt patients and patients carrying mutations clinical research, investigation, and systems modeling of acute illness (crisma) center, department of biostatistics we determined phenotype cohesiveness using probability of assignment at presentation, defining core members as ≥ % and marginal as < % probability. we determined how members transitioned to other phenotypes over hrs using t-distributed stochastic neighbor embedding (tsne) plots and determined the odds ( %ci) of transition. results: we studied , adult sepsis encounters (median age c) the odds of ever transitioning from presenting phenotype increased significantly for marginal members vs publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the department of education of the basque government (piba - ) and the university of the basque country upv/ehu (ppg / , giu / ) for their financial support. a great disaster affects the family-and friend-performance of bcpr by diminishing the willingness of family and friend bystanders to follow the instruction provided by dispatchers. the experimental method ifitem could be an alternative of fibtem in cases when internal coagulation pathways assessment is prioritized (i.e. heparinized patients on extracorporeal supports). patients undergoing limitation of life-sustaining therapy had lower karnofsky scale scores. therefore, this scale may be useful to guide end-of-life decisions in the future, but further studies with larger number of patients are needed. readmission after discharge home from critical care: a qualitative study c robinson , f nicolson , p mactavish , t quasim , jm mcpeake nhs greater glasgow and clyde, nhs greater glasgow and clyde, glasgow, united kingdom; university of glasgow, nhs greater glasgow and clyde, glasgow, united kingdom critical care , (suppl ):p readmissions to acute care occur in a high number of critically ill patients within days of hospital discharge [ ] . biomedical drivers such as frailty and pre-existing co-morbidities have been identified as drivers for readmission. however at present there is limited data on the influence of social problems on readmission. this study, using a grounded theory approach, sought to understand from a patient/caregiver perspective what the drivers for readmission to acute care were. ethical approval was granted from the west of scotland research ethics service ( /ws/ ). a grounded theory approach was used to explore from a patient and caregiver perspective what the drivers for readmission are [ ] . using a clinical database, we identified those patients who had an icu admission ≥ days who were readmitted to acute care within days of hospital discharge. the researcher attended the ward and after discussion with the direct care team conducted a semi-structured interview with patient and/or caregiver. the interview was recorded and transcribed verbatim. the transcripts were analysed to generate initial codes, followed by the development categories and sub-categories. theoretical sampling was undertaken. results: participants were interviewed. ( . %) were patients and ( . %) were caregivers. the themes that have emerged from the data were: pain and polypharmacy; lack of social support and/or isolation; strained relationships with primary care providers and information provision across the patient journey. subsequent theory development is underway to understand how this learning could help reduce readmissions in future. in conclusion, both social and biomedical drivers are likely to contribute to acute care readmission in this group. future interventional work is required in order to identify modifiable factors to reduce this burden for patients and the healthcare service. frailty has shown to have prognostic relevance for patients with critical illness. since a wide range of tools has been described to screen for frailty, we aimed to describe the association of two frailty screening tools, the clinical frailty scale (cfs) score and the modified frailty index (mfi) in critically ill patients. we performed a post-hoc analysis of a multicenter cohort of patients admitted to six canadian intensive care units (icu) between february and july . frailty was identified using the clinical frailty scale (cfs) and the modified frailty index (mfi). concordance of the frailty screening tools was evaluated with partial spearman rank correlation and intraclass correlation (icc). discrimination and predictive ability of the tools for hospital mortality, -year mortality, hospital readmission and adverse events were compared using concordance statistic (c-statistic) and calibration plot adjusting for age, sex, sequential organ failure assessment (sofa) score and icu admission source, respectively. the cohort included patients. prevalence of frailty was . % ( % confidence interval [ci] . %- . %) with the cfs and . % ( % ci . %- . %) with the mfi. concordance between the two tools was low [(icc of . ; % ci . - . ) and partial correlation coefficient of . ( % ci . - . )], even after adjustment. hospital and -year mortality were greater for frail compared to non-frail patients using of both tools. similarly, both tools found frail patients were less likely to be living independently after hospital discharge, and more likely to be rehospitalized when compared to non-frail patients. while the cfs and mfi show low concordance, both showed good discrimination and predictive validity for hospital mortality. both tools identify a subgroup of patients more likely to have worse clinical outcomes. the post-intensive care syndrome (pics) is a myriad of physical, psychiatric and cognitive disorders secondary to critical illness, leading to a decreased quality of life and an important socioeconomic burden. this study aimed to identify if the conformity to a pics prevention bundle was able to reduce the incidence of the syndrome at icu discharge. all patients admitted to the icu from january st to december st were included. the conformity to each of the ten components of the pics prevention bundle was assessed daily, and the patients were evaluated for anxiety, depression, cognitive dysfunction, muscular weakness, mobility impairment and nutritional risk at icu discharge and at a -to- -months follow-up consultation. the patient cohort was divided in terciles according to bundle conformity for the analysis. results: from the enrolled patients, ( %) were evaluated at icu discharge, and ( %) attended to the follow-up consultation. there was no difference in baseline characteristics between the cohorts. there was no correlation between the prevalence of pics at discharge and bundle conformity during icu stay ( % vs. % vs %, p . ), though there was a decrease in nutritional risk and days in mechanical ventilation (table ) . after to months there was a reduction on the prevalence of any kind of pics, mobility impairment, muscular weakness and nutritional risk. the patients that developed pics were older and had a higher simplified acute physiology score iii at icu admission. a higher adhesion to a pics prevention bundle was not able to prevent the occurrence of the syndrome. post intensive care syndrome (pics) is well recognized following general icu care [ ] . intensive care syndrome:promoting independence and return to employment (ins:pire) is a multidisciplinary complex intervention designed to address pics [ ] . with a paucity of evidence on pics after cardiothoracic intensive care, we aim to evaluate pics and the feasibility of the ins:pire intervention in this population. those attending the clinic received weeks of intervention including individual appointments with icm nurse, physician, pharmacist, and physiotherapist. a café area facilitated peer support alongside psychology group sessions. primary outcome was quality of life measured by eq- d- l. further surveys included: pain, mental health, and selfefficacy. questionnaires were taken at baseline, and months. results: over cohorts, patients attended, % male, median age years (iqr - ), median apache score of (iqr - . ), and median icu length of stay was days (iqr - ). a total of ( %) patients completed surveys at one year. scheduled admissions represented % of those attending. mean euroqol eq-vas score was / (sd +/- ) at baseline increasing to / (sd +/- ) by year (table ) . those with problems in at least one domain of eq- d- l fell from % at baseline to % at -year with the breakdown shown in table . severe problems were seen in % falling to % at year. hads demonstrated an anxiety or depression rate of %. brief pain inventory identified patients ( %) with ongoing chronic pain. mean self-efficacy was / (sd +/- ) at baseline and / (sd +/- ) at year. cardiothoracic intensive care patients have ongoing and persistent features of pics with significant effects on health-related quality of life. further, the ins:pire multi-professional complex intervention is feasible within this specialist group. screening approach might be implemented whenever screening of the total icu population is not deemed feasible. influenza is an acute viral illness with a significant financial burden. point of care testing for influenza is available and has demonstrated accuracy [ , ] , the current gap in knowledge is the question around the opportunity cost of influenza testing. if poct is financially a less costly test this could free up scarce resource. the study adopts a cost minimisation approach. the point of care test is the roche cobas® liat® machine which can detect flu a/b and is compared with the west of scotland specialist virology centre's established in house multiplex real time pcr assay.the model was developed using microsoft excel and has arms comparing analysis of the above mentioned tests. the model estimates that the total cost of poct per patient tested is £ . compared with £ . for lab testing ( figure ). this is a saving of £ . per patient when poct is used. the result swings in favour of the lab test when poct specificity falls to . %. if the lab could provide the result of influenza testing within hours the result would swing in favour of lab testing. zanamivir which will potentially be used increasingly in the intensive care setting can more than double the difference between the tests in favour of poct. this research suggests that poct offers potential cost savings in the icu setting. this is the case as long as poct specificity is higher than a threshold of . % and the lab take longer that hours to return the result. the sensitivity analysis should allow for external validity given the usual variations in icu practice. the aim of the present study is to describe the demographic, clinical, microbiological aspects and the outcome of patients with intensive care unit-related (icu-related) bacteremia. moreover, we aimed to study the patient outcome in association with colistin susceptibility. retrospective, single-center study in a -bed icu for months, from / / to / / . icu-related bacteremia was defined as bacteremia in patients with icu stay > hours or icu readmission (first admission ≥ month before). only the first episode of bacteremia was considered. the primary outcome was -day mortality. data regarding clinical, demographic and outcome characteristics were retrieved from the patient files. the hospital's ethics committee approved the present protocol. moreover, the patients with bacteremia due to colistin-resistant pathogens were compared with the patients affected by colistin sensitive microbes. forty episodes of gram-negative icu bacteremia were collected during the aforementioned period in patients ( . % male) with a mean age and apache ii of . ± . years and ± . , respectively. the event had taken place at an average of . days. the responsible isolates were resistant to carbapenems in . % of the episodes. the majority of the events were due to a single isolate ( %). acinetobacter baumannii and klebsiella pneumoniae presented the majority of the implicated microbes ( % and . %, respectively). the crude -day mortality was %. finally, we could not detect any difference in mortality between the colistin sensitive and the colistin-resistant pathogens ( figure ). the present study denotes that, in a setting of extremely drugresistant pathogens with limited treatment options, gram-negative bacteremia in the icu is associated with increased mortality. image : characterization of resistance mechanisms affecting ceftolozane/ tazobactam in enterobacterales and pseudomonas aeruginosa icu isolates using whole genome sequencing (step study) m hernández-garcia , cc chaves , jm melo-cristino , ds silva , ar vieira , mp f. pinto , jd diogo , eg gonçalves , jr romano , rc cantón hospital ramón y cajal-irycis, microbiology department, madrid, spain; introduction: clostridium difficile infection (cdi) is the main cause of hospital acquired diarrhoea [ ] . the aim of this study was to compare characteristics of cdi during yr and . a retrospective observational study was carried out in lithuanian university of health sciences hospital -the largest teaching facility of tertiary care in country. according to department of infection control records, patients (pt) with (w.) diarrhoea and the first positive stool test for c.difficile toxin a/b were included. age, charlson comorbidity index (cci) score, profile of hospital department (medical (md), surgical or icu) where cdi was diagnosed, type of cdi (healthcare-associated (ha), hospital or community-acquired) and rate of risk factors (rf) have been estimated in both and . ibm spss . ; pearson's chi-square, fisher's exact tests were used for statistics. p < . was statistically significant. results: in total pt from , from were enrolled. in n= ( %) pt were ≥ yr old, in -n= ( %), (p= . ). in cci> was estimated in n= ( %) pt in comparison of n= ( %) in , (p= . ). in n= ( %) of cdi cases were ha, in -n= ( %), (p= . ). in n= ( %) of cdi were diagnosed in md in comparison of n= ( %) in , (p= . ). in weeks prior to cdi n= ( %) pt have been admitted to hospitals, n= ( %) have been treated w. antibiotics, n= ( %) -w. ppis, n= ( %) -w. h antagonists, n= ( %) -w. immunosupressants in comparison of n= ( %), n= ( %), n= ( %), n= ( %) and n= ( %) in , respectively, (p> . ). overall rate of cdi cases among in-hospital patients increased tenfold by yr and . in , more elderly patients had cdi and severe comorbidities were less frequent in comparison with . in , more cases of cdi were hospital-acquired and have occured in medical departments. rate of risk factors of cdi remained unchanged.these results indicate a possible relationship between ttv dna count and immunological alteration. the ttv quantitative determination could be useful as a proinflammatory marker in sepsis, with some benefits: low cost, easy determination and good correlation with immune system functionalit. it will be necessary to perform a larger study to check our hypothesis and to establish a ttv level threshold that may allow to anticípate the disease prognosis. introduction: acute kidney injury (aki) is a serious complication in sepsis and associated with high morbidity and mortality. the combination antimicrobial regimens with vancomycin (vcm) and broad-spectrum betalactams (bsbl), such as piperacillin tazobactam and cefepime, have been identified as potentially nephrotoxic combinations, but existing studies have not provided sufficient evidence. the aim of this study was to evaluate detailed association between the combination antimicrobial therapy and the risk of aki in septic patients. this investigation was a post hoc analysis of prospective nationwide cohorts enrolling consecutive adult patients with sepsis in intensive care units in japan. in this study, progression of aki was defined as one or more elevation of renal sub-score in sequential organ failure assessment score from day to day . we regarded anti-pseudomonal penicillins, fourth generation cephalosporines, and carbapenems as bsbl. multivariable logistic regression analysis including a two-way interaction term (vcm x bsbl) was performed to assess the add-on effects of each antimicrobial agent on the progression of aki. the final study cohort comprised patients with sepsis. among them, received vcm without bsbl, received bsbl without vcm, received both vcm and bsbl, and received other type of antimicrobials. the administration of vcm was associated with an increased risk of aki in patients with bsbl [odds ratio (or), . ( . - . ); p= . ]. however, the tendency was not evident in patients without bsbl [or, . ( . - . ); p= . ]. the interaction effect on the progression of aki between vcm and bsbl were statistically significant (p for interaction= . ). the regression model including two-way interaction term suggested that the combination of vcm and bsbl might synergistically increase the risk of aki in patients with sepsis. increasing resistance to carbapenems due to carbapenemase productionone of main actual problems of antibacterial resistance in burn icu. production of several types of carbapenemases (kpc, ndm and oxa- ) is common in k. pneumoniae strains. carbapemenase production is a marker of extreme antibacterial resistance. the aim of our study was to investigate the epidemiology of nosocomial infections caused by producing kpc, ndm and oxa- k. pneumonia strains in burn icu. total of patients with nosocomial infections caused by carbapenem resistance strains of k. pneumoniae were included in the study, from whom had lower respiratory tract infection, had skin and skin structure infection. initial identification of isolates was performed in laboratory by automatic microbiological analyzer. for all of k. pneumoniae isolates presence of bla ndm , bla oxa- and bla kpcgenes were examined by pcr method. baseline characteristics of patients: me (iqr) of age - ( ; ) years, me (iqr) of tbsa - ( ; ) percent, me (iqr) of icu los - ( ; ) days. inhalation injury was diagnosed in ( . %) patients. total of patients died, mortality rate was . %. all patients were diagnosed with nosocomial infection caused by k. pneumoniae. from k. pneumonia strains ( . %) were found to be producing kpc, ( . %)producing ndm and ( . %) -producing oxa . only ( . %) carbapenem resistance k. pneumoniae isolates were not producing carbapenemases. from patients infected by oxa producing k. pneumoniae patients died, mortality rate was %. from patients infected by oxa or ndm producing k. pneumoniae patients died, mortality rate was . %. from patients infected by non-carbapenemase producing k. pneumonia no one died. carbapenemase producing strains are widely spread among carbapenem resistance strains of k. pneumoniae in burn icu. mortality of patients infected by producing oxa or ndm k. pneumoniae strains reaches . %. the rationale for blood purification as adjunctive therapy during sepsis involved the capacity in removing endogenous and exogenous toxins, but currently no recommendations exists [ ] . a critical point may be the potential interaction with antimicrobial therapy, which remains the mainstay of sepsis treatment. the aim of our study was to investigate the vancomycin (van) removal during blood purification using an in vitro model of hemoperfusion (hp) with ha cartridge (jafron, zhuhai city, china), most widely used in china and actually available in europe. this is an experimental study. three independent experiments were performed: we injected mg of van in ml of whole blood from healthy donors (experiment and ) or in ml of balanced solution (experiment ) in order to assess membrane saturation. a closed-circuit (blood flow of ml/min) simulating hp ran using ha . samples were collected from arterial line at , , , , , , , , minutes; van plasma concentrations were measured and removal was evaluated using mass balance analysis. differences in mass removal was assessed using kruskal-wallis test. results: figure shows van mass at each timepoints. we observed no difference between in blood and in balanced solution experiments (p- the aim of this study is to determine if routine bbv testing in the icu contributes to the discovery of undiagnosed bbv infections. icu patients may require renal replacement therapy (rrt). sharing rrt equipment carries a risk of bbv transmission, which mainly relates to hepatitis b (hbv), hepatitis c (hcv) and hiv. since , all glasgow royal infirmary icu patients undergo routine bbv screening, with rrt machines allocated for patients with specific bbv statuses. routine bbv testing is beneficial to both the individual and society. hcv is a pertinent health issue in scotland. the scottish government aims to eliminate hcv by and is researching innovative and costeffective methods to identify undiagnosed infections. this single-centre retrospective observational study examined prospectively collected clinical data from icu admissions. proportions were compared using a two-proportion z-test and a logistic regression model was carried out to determine if deprivation quintile was independently associated with the seroprevalence of bbvs. the bbv seroprevalence in the cohort studied: . % (hbv), . % (hcv), . % (hiv). the seroprevalence of hbv in the cohort studied was similar to that of scotland (p= . ), but the seroprevalence of hcv (p< . ) and hiv (p= . ) were statistically significantly higher than that of scotland. due to the small number of reactive test results for hbv and hiv, the relationship between deprivation and bbv seroprevalence was explored for hcv only. the only independent variable associated with a reactive anti-hcv test result was "current or previous illicit drug use" (adjusted odds ratio of . ; % confidence interval of . - . ; p< . ). this study shows that routine bbv testing in the icu is useful in discovering new bbv infections. this is the first observational study focusing on the value of routine bbv testing in an icu setting to our knowledge. continuous infusion vancomycin protocol is a safe, acceptable and effective alternative to intermittent dosing of vancomycin in critical care. ceftaroline is an efficacious treatment in patients with severe cap, admitted in icu. it relates to earlier resolution of respiratory failure and less rescue antibiotics. we need an adequately pragmatic trial to confirm our findings organ dysfunction in scrub typhus, incidence and risk factor a sarkar , a guha , r dey [ , , , , ] . its preads by bite of larval stageof thromboculid mites or chigger [ ] . clinical features may include fever, headache, myalgia, lymphadenopathy, eschar, skinrash. it may also cause pneumonia, renal failure, shock, meningoencephalitis, multiple organ failure [ , ] . our study aims to discuss the incidence of organ dysfunction in a comprehensive way taking the overall population of patients with identified scrub typhus infection. there is lack of data in eastern india regarding the incidence and risk factors of developing multiorgan dysfunction syndrome (mods) in scrub typhus. in this retrospective study we studied the incidence of various organ involvement and the risk factors associated with the development of mods in scrub typhus. we collected data from december to november in tertiary care hospital at kolkata. we have included all patients who are having fever, scrub typhus igm antibody positive, age more than years. sofa score was used in evaluating patients with mods. exclusion criteria involves patient who are having coinfectional ong with scrub typhus. in a cohort (n= ), patients with multiorgan dysfunction syndrome was seen in patients ( . %), the mean age in group of patients with mods was . +/- . years (mean+/-sd). in group of patients with mods, fever duration in days was of +/- . days (mean+/-sd), interval from treatment to defervescenc in days was . +/- . days (mean +/-sd). among patients with mods, hematologic involvement was seen in patients ( . %), hepatic involvement was seen in patients ( . %), renal involvement was seen in patients ( . %), neurologic involvement was seen in patients ( %), respiratory involvement was seen in patients ( . %), cardiovascular was seen in patients ( . %), icu shifting was necessary in patients ( . %), mechanical intubation was needed in patients ( . %) in multiorgan dysfunction syndrome patients. hospital mortality in patients with mods was patients ( . %). no mortality was seen in patients without mods. other parameters were evaluated among patients with mods. they include eschar in patient ( . %), seizure in patients ( . %), hepatoslenomegaly in patients ( . %), leucopenia in patients ( . %), leucocytosis in patients ( . %), thromnbocytopenia in patients ( . %),decreased hemoglobin in patients ( . %), transaminitis in patients ( . %). the risk factors associated with the development of mods are platelet counts, bilirubin, transaminitis, glasgow coma scale, time interval from treatment to defervescence, hemoglobin, total leucocyte count and fever duration. scrub typhus is an important cause of acute febrile illness in this part of the country and is frequently associated with organ dysfunction. however, the overall mortality is low which is similar to other studies done before [ ] . score at baseline were significant (p< . ) predictors of mortality.highest area under the roc curve was obtained for number of days with septic shock ( . ) followed by increased cd between baseline and day ( . ). though serial pct levels significantly increased amongst non-survivors, it did not predict mortality. serial level of biomarkers in icu patients may predict mortality. larger trials are needed to confirm the results. plasma strem- levels were retrospectively measured at day - , - and - in septic shock patients from the immunosepsis cohort (nct ), included between / and / , using a validated elisa method. the associations between strem- , mhla-dr, -day survival status, and occurrence of icu-acquired nosocomial infection (ni) were assessed. neither strem- nor mhla-dr levels at d / were associated with the occurrence of icu-acquired ni. however, -day mortality was significantly higher in patients with d - strem- value superior to the median ( . % vs . %, p= . ; median= pg/ml). a significant inverse correlation was found between mhla-dr at d - and strem- at d - (sp - . , p< . ) and at d - (sp - . , p< . ). at d - , when stratifying patients based on strem- ( pg/ml) and mhla-dr ( ab/c), patients combining elevated strem- and low mhla-dr presented with significantly higher day mortality ( . % vs . %, p = . , chi-squared test) and ni incidence ( . vs %, p= . ) compared with patients with low strem- / high mhla-dr. this study shows for the first time that trem- pathway activation is associated with septic shock-induced immunosuppression, as shown by an inverse correlation between strem- at baseline and mhla-dr expression at d - . persisting high strem- values and low mhla-dr expression in septic shock patients are significantly associated with higher rate of icu-acquired infection and mortality. introduction: sepsis mortality remains high [ ] . the surviving sepsis campaign (ssc) recommends to guide resuscitation on normalization of lactate levels [ ] , however this is debated [ ] . we have shown that plasma levels of bio-adrenomedullin (bio-adm) were associated with patient outcome during sepsis [ ] . we therefore aimed to evaluate the added value of bio-adm to lactate measurement in the adrenoss cohort. this is a post-hoc analysis of the adrenomedullin and outcome in severe sepsis and septic shock (adrenoss) cohort study. the adre-noss study is a prospective observational study conducted in twenty-four centers and included septic patients [ ] . we studied the relationship between the association of initial evolution of lactate plasma levels and bio-adm level at h and outcome in patients for whom both markers were available at admission and one day later (" h"). bio-adm levels below pg/ml were considered as low, and high if greater than pg/ml [ ] . in patients with high lactate levels (> mmol/l) at admission (n= ), lactate normalization (< mmol/l) at h was associated with better outcome than in patients with persistently high lactate at h ( day mortality . % vs . % respectively, hr . [ . - . ], p< . ) ( figure ). among patients with decreasing lactate, high and low bio-adm levels at h identified patients with different outcomes ( day mortality % vs % for low vs high bio-adm respectively, hr . [ . - . ], p< . ). high and low bio-adm levels at h also differentiated outcome of patients with persistently elevated lactate (hr . [ . - . ], p< . ). in patients with low initial lactate, neither lactate or bio-adm had no added prognostic. our data suggest that measurement of bio-adm in addition to lactate may help physicians to refine risk stratification and therefore to guide resuscitation during sepsis. the effect of fluid replacement in sepsis, severe sepsis and septic shock in first hrs in clot quality and microstructure s pillai , g davies the inflammatory response in sepsis can lead to a spectrum of coagulation system defects [ ] . sepsis and severe sepsis is associated with a hypercoagulable state where the clot microstructure is known to be a tight and highly elastic clot, which is potentially resistant to fibrinolysis ( figure ). conversely, septic shock is associated with a hypocoagulable state where the clot microstructure is loose and structurally weak. the study aim to investigate the effect of fluid resuscitation and replacement in clot microstructure over hours. methods: patients ( sepsis, severe sepsis and septic shock) were included in the study. all these patients received standard fluid replacement therapy with crystalloids. blood samples were collected at hours, hours and hours. clot microstructure, standard markers of coagulation and inflammatory markers were measured. in sepsis group following fluid administration, the d f reduced initially and then remained stable ( . - hours, . - hours, . - hours, normal d f range . ± . ). in severe sepsis group, the d f reduced initially, then increased ( . - hours, . - hours, . - hours) and in septic shock, the df was very low to start with and there were only slight increase with fluid administration ( . - hours, . - hours, . - hours). the hypercoagulable state and clot quality in both sepsis and severe sepsis group improved with fluid resuscitation, however despite an early improvement in clot quality, ongoing fluid resuscitation resulted in markedly reduced functional clot with very low clot strength and functionality. this study demonstrates that d f as a marker of clot quality and function may have potential in fluid and component replacement in critical illness and injury. this study analyses the prognostic ability of white blood cell count (wbc), neutrophil:lymphocyte ratio (nlr) and c-reactive protein (crp). hypo-and hyperimmune responses have been associated with increased mortality from septic shock [ ] . patients with septic shock (sepsis . ) admitted to queen elizabeth hospital birmingham, between december and july were included. the primary outcome was -day mortality. data was tested for normality and presented as median (iqr) and analysed using a mann whitney u test. categorical data was presented as % and analysed using a chi-squared test. a p value of < . was used to determine significance. a multivariate binary logistic regression analysis was conducted using age, apache ii, charlson comorbidity index, performance status, and initial lactate as covariates. a hosmer lemeshow test of > . indicated good fit. results: patients were admitted with septic shock. the majority ( %) were male, with a median age of ( - ) and a -day mortality of %. on day , wbc was lower in patients who died compared to patients who survived ( [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] patients who died of septic shock had a lower wbc, nlr and crp response early on compared to survivors. this may represent early immunoparesis that allows infection to propagate unchecked. however, this was not independently associated with mortality when confounding factors were accounted for. a specific metabolite of mitochondriaitaconic acid is formed upon proinflammatory activation. the attempts of various researches to find the itaconic acid in peripherical blood of patients with sepsis were unsuccessful [ ] . some phenylcarboxylic acids (phcas) are known to be microbial metabolites and sepsis biomarkers; they also affect the mitochondrial functions [ ] . concentrations of phcas (phenyllactic, p-hydroxyphenylacetic, phydroxyphenyllactic acids) and mitochondrial metabolites (succinic, itaconic acids) in serum samples from patients on the st day of diagnosis of sepsis and serum samples from patients with late stages of sepsis (sepsis- ) were measured by gas chromatographymass spectrometry; control group - donors. results: itaconic acid was found in low concentrations ( . - . μm) only at early stage of sepsis. the multiple increase in levels of phcas and mitochondrial metabolites were detected in patients with late stage of sepsis in comparison with early stage and donors, p< . . increased succinic acid (up to - μm) concentration is the result of succinate dehydrogenase inhibition by microbial metabolism intermediates (phcas), which was confirmed by in vitro experiments in isolated mitochondria (fig. ) . itaconic acid may be a promising marker in early stage of sepsis, which needs to be proved. prediction of severe events in clinical sepsis is challenging. for such prediction we aimed to compare the novel biomarker calprotectin in plasma, with routine biomarkers. in a prospective study, blood samples were collected from consecutive patients who triggered the sepsis alert in the emergency department in our hospital. c-reactive protein (crp), procalcitonin, neutrophils, and lymphocytes were analysed according to routine practice. p-calprotectin was analysed using a specific particle enhanced turbidimetric assay (gentian diagnostics as). the composite endpoint, which was termed severe event, was defined as death or admission to the intensive care unit (icu)/high dependency unit (hdu) within hours from arrival. the study included patients with written informed consent, of whom were considered to have infection (defined as obtained blood culture and subsequent antibiotic therapy for at least days or until discharge or death), and had no infection. seventy-four patients ( %) with infection developed a severe event. mean pcalprotectin was . mg/l (standard deviation (sd) . ) among patients with infection and . mg/l (sd . ) among patients without infection (p= . ). in patients with infection mean p-calprotectin was . mg/l (sd . ) among those with and . mg/l (sd . ) among those without a severe event (p= . ). analysis of area under the receiver-operating characteristic (roc) curve for prediction of severe events showed superiority for p-calprotectin compared with procalcitonin and neutrophil-lymphocyte-ratio, both regarding all sepsis alert cases and regarding the patients with infection (p< . for all comparisons), fig . in addition, there was a trend toward superior performance compared to crp (p= . and . ). in sepsis alert patients, p-calprotectin was elevated in those who subsequently developed severe events. p-calprotectin was superior to traditional biomarkers for prediction of severe events. introduction: rapid diagnosis of acute infections and sepsis is critical in emergency departments (eds). current tests have slow turnaround times, low sensitivities, and/or signals from contaminant or commensal organisms. empirical antimicrobial treatment may result in severe adverse events and contributes to antimicrobial resistance. diagnostics to distinguish bacterial from viral infections and noninfectious etiologies support clinicians in efforts toward antimicrobial stewardship. in a prospective, non-interventional study in the eds of sites in greece (prompt study nct ), we evaluated hostdx sepsis, a host response test for suspected acute infections and suspected sepsis. hostdx sepsis measures human mrna targets and employs advanced machine learning to differentiate patients with bacterial and viral infections, and noninfectious etiologies. adult patients presenting with suspected acute infection and at least one vital sign change were enrolled. whole blood rna was quantified using nano-string ncounter. predicted probabilities of bacterial and viral infection were calculated (bvn- algorithm). patients were adjudicated in a retrospective chart review by independent infectious disease specialists blinded to hostdx sepsis results. among patients adjudicated as bacterial ( ), viral ( ), noninfected ( ), or indeterminate ( ) the area under the receiver operating characteristics (auroc) of hostdx sepsis for predicting bacterial vs. viral/non-infected patients was . , and auroc for viral vs. bacterial/non-infected patients was . (fig. ) . our results indicate that hostdx sepsis distinguishes bacterial from viral infections and other etiologies with high accuracy. hostdx sepsis is currently developed as a rapid point-of-care device with a turnaround-time of less than minutes. hostdx sepsis may therefore assist ed doctors in making appropriate treatment decisions earlier, towards the ultimate goal of antimicrobial stewardship. we studied the diagnostic value of a leukocyte deformability assay that rapidly quantifies the immune activation signatures of sepsis in an undifferentiated population of adults presenting to the ed. ed clinicians must balance the benefits of early intervention against the risks of indiscriminate use of resource-intensive interventions. there are no currently available rapid diagnostics with acceptable performance to achieve this balance. we prospectively enrolled adult patients within hours of presentation with signs of suspicion of infection in two eds in the usa. edta-anticoagulated blood was drawn and analyzed using deformability cytometry [ ] . procalcitonin (pct) levels were also measured. patients were retrospectively adjudicated for sepsis- by physician committee using the entire medical record. diagnostic performance characteristics and receiver operating curves were used to examine the diagnostic performance of the assay as well as pct. of the patients enrolled, . % were adjudicated as septic. the leukocyte deformability assay demonstrated % sensitivity, % specificity, and % negative predictive value for a single cutoff. the auc was . ( figure ). pct with a cutoff of . ng/ml had % sensitivity, % specificity, and % negative predictive value. the auc for pct (as continuous variable) was . . the leukocyte deformability assay of immune activation signatures demonstrated superior diagnostic performance for sepsis when compared to pct. the assay's diagnostic performance and rapid turnaround time of minutes may positively impact patient outcomes while minimizing indiscriminate use of valuable resources in the ed. it is already known in literature that high levels of midregional proadrenomedullin (mrproadm) are related with organ disfunction in infections despite of source and pathogens [ ] . similarly, microcirculatory impairment has been reported in sepsis. we examine the correlation between microcirculatory disfunction and mrproadm as a sign of early organ failure. we included consecutive adult patients with suspected infection, sepsis or septic shock admitted to our intensive care unit (icu) as first hospital admission with an expected icu stay of > hours. mrproadm was measured daily during the first five consecutive days and sublingual microcirculation was assessed with incident dark field (idf) technology at t , t , and t . we collected information on saps ii, apache scores, and sofa score for each timepoint. results: ten patients had septic shock, sepsis and infection. three patients died during icu stay. a mrproadm clearance of % or more between t and t was found associated with the improvement of mfi (mann-whitney u test, median increase . % versus . %, p= . ) (figure ) . a mrproadm > . nmol/l at the icu admission was associated with a worse sofa score at all the timepoint. moreover, mrproadm levels at admission was found significantly related with icu mortality (auc . [ . - ]; p= . ). mrproadm shown no relation with absolute value of mfi. the study shows a good correlation between the clearance of the biomarker and the improvement in mfi. moreover, our results support previous findings on the prognostic value of mrproadm in terms of sofa and icu-mortality. clinical performance of a rapid sepsis test on a near-patient molecular testing platform r brandon , j kirk , t yager , s cermelli , r davis , d sampson , p sillekens , i keuleers , t vanhoey immunexpress, seattle, united states; immunexpress, immunexpress, seattle, united states; biocartis nv, biocartis, mechelen, belgium critical care , (suppl ):p the purpose of this study was to clinically validate a new, rapid version of the septicyte™ assay on a near-patient testing platform (biocartis idylla™). septicyte™ lab is the first-in-class sepsis diagnostic to gain fda-clearance but has a complex workflow and a turnaround time (tat) of~ hours. the assay in idylla™ cartridge format is called septicyte™ rapid. septicyte™ lab was translated to the biocartis idylla™ near-patient testing platform and analytically validated. for this study, . ml of peripheral blood paxgene tm solution from previously collected patient samples was pipetted directly into the cartridge and inserted into the idylla™ reader. patients were part of an independent cohort (n= ) from intensive care units located in the usa and europe. septicyte™ rapid results were reported as a septiscore™ between and with higher scores representing higher probability of sepsis. assay performance determined included technician hands-on-time (hot), assay tat, failure rates, and area under roc curve based on comparison to retrospective physician diagnosis. average hot was minutes, and average tat was minutes. clinical samples could be processed immediately with septicyte™ rapid and did not require hour pre-incubation of paxgene blood, greatly improving tat. correlation of septiscore™ values between lab and rapid, based upon a subset of samples run on both platforms, was very high (r > . ). estimated roc auc performance for discriminating sepsis from non-infectious systemic inflammation (nisi/sirs) was similar to that previously reported for septicyte™ lab. this is the first demonstration of a validated, fully-integrated, rapid, reproducible, near-patient, immune-response sepsis diagnostic, providing actionable results~ hr, to differentiate sepsis from non-infectious systemic inflammation / sirs. accuracy of septicyte™ for diagnosis of sepsis across a broad range of patients r brandon , k navalkar , d sampson , r davis , t yager immunexpress, seattle, united states; immunexpress, immunexpress, seattle, united states critical care , (suppl ):p the purpose of the study was to demonstrate sepsis diagnostic performance of the biomarkers of septicyte™ in subjects other than critically ill adults, and in hospital locations other than icu. septicyte™ lab was the first immune-response sepsis diagnostic assay to gain fda-clearance (k ) and, as part of gaining this clearance, clinical validation was performed on adult patients admitted to intensive care (icu) only [ ] . we therefore performed an in silico analysis across a broad range of patients using the septicyte™ host immune response biomarkers and algorithm. peripheral blood gene expression data, including public and private datasets, were chosen based on quality, annotation, and clinical context for the intended use of septicyte™. multiple comparisons were performed within datasets to better understand the diagnostic performance in certain cohorts including healthy subjects. diagnostic performance was determined using area under curve (auc). results: table shows some characteristics of the selected datasets and patients, including number of datasets (n= ) and comparisons (n= ), number of cases (n= ) and controls (n= ) used in comparisons, patient category and hospital location. septicyte™ aucs for the three groups of adults, adult / pediatric and pediatric / neonates were . , . , and . respectively, which is similar to that previously reported ( . - . ) [ ] . these results suggest that the septicyte™ signature has diagnostic utility beyond adults suspected of sepsis and admitted to icu. this signature has now been translated to the near-patient testing platform biocartis idylla™ (as septicyte™ rapid) which promises rapid (~ hour) diagnosis of sepsis in a broad patient population following further validation. introduction: especially extracorporeal cardio pulmonary bypass (cpb) is known to induce severe inflammation. postoperative inflammation is associated with a sepsis like syndrome including endothelial barrier disruption, volume depletion and hypotension. sphingosine- -phosphate (s p) is a signaling lipid regulating permeability and vascular tone. in septic humans decreased serum-s p levels could be identified as marker for sepsis severity. we addressed three main issues: ( ) are serum-s p levels affected by cardiac surgery? ( ) are potential alterations of serum-s p levels related to changes of acute-phase proteins, s p sources or carrier? ( ) is the invasiveness of the surgery a factor that may influence serum-s p levels? methods: elective major cardiac surgery patients were prospectively enrolled in this study. serum samples were drawn pre-, post-procedure and on day and day after surgery. we analyzed s pand its potential sources: red blood cells (rbc) and platelets. we further quantified levels of other inflammatory markers and documented other clinical parameters. median serum-s p levels in all patients before the procedure were . (iqr . - . ) nmol/ml. serum-s p levels decrease after surgery, whereas all other inflammatory markers increase. serum-s p levels dropped by % in the on-pump and % in the off-pump group. changes of serum-s p levels are associated with s p sources and carriers: albumin, hdl and vwf:ag activity. patients with a full recovery of their serum-s p levels after surgery compared to their individual baseline presented with a lower sofa score (p> . ) and shorter icu stay (p< . ). serum-s p levels are disrupted by open heart surgery and levels might be negatively affected by endothelial injury or loss of s p sources. low serum-s p levels may contribute to prolonged icu stay and worse clinical status. future studies may investigate the beneficial effects of s p administration during cardiac surgery. the aim of study is to measure and correlate the expression of ncd , mhla-dr, pct (procalcitonin) and qcrp (quantitative creactive protein) to predict development of sepsis and its outcome. in this tertiary centre based longitudinal cohort study, a total patients were enrolled in whom sepsis was suspected on the basis of clinical diagnosis and supported by lab investigations. they were divided into two groups sepsis/case and non-sepsis/control. disease severity in icu was assessed by sequential organ failure score (sofa). blood samples for routine lab investigations and biomarkers were taken at the time of admission in icu before administration of first dose of antibiotics at time d /d . assessment of biomarkers was done simultaneously with tlc at d /d , d and during follow up of patients till their final outcome. there was no significant (p> . ) mean change in pct, qcrp, sofa, ncd , mhla-dr from day to day , however, mean change was higher among cases than controls.on comparison of mhla-dr between the groups across time periods, mhla-dr was significantly (p= . ) lower among septic patients than controls at both day and day . all biomarker correctly predicted cases among different percentage of patients with different sensitivity and specificity. there was no significant (p> . ) association of mortality with the study biomarkers except for pct. in our study, diagnostic value of pct in differentiating sepsis from non-sepsis was similar to ncd among all biomarkers studied. no advantage of ncd or mhla-dr was found over pct in diagnosis and correlation with disease progression and mortality. introduction: aqp is a water channel protein contributing to astrocyte and immune cells migration, blood-brain barrier maintenance and cell survival [ ] [ ] . aqp genetic variants represent biomarkers associating with outcome after traumatic brain injury and intracerebral hemorrhage [ ] [ ] . linking aqp genetic polymorphism to the course of sepsis has not been studied. methods: study cohort included icu patients diagnosed according to sepsis- consensus. aqp rs polymorphism was studied by analyzing pcr products in a % agarose gel using an aqp specific polynucleotide tetraprimer set. data were analyzed by log rank test (medcalc . . ), and odds ratios/hazard ratios were computed. statistical significance was determined by fisher test (ft) or mann-whitney test. results: of sepsis patients had the minor mutation a for snp rs located within the regulatory ' region of the aqp gene. septic shock occurred more frequently in homozygotic carriers of aqp c allele vs. patients with aa or ca genotype: or= . ( %ci: . - . ), p= . (ft). lethality in septic shock patients, n= , significantly increased compared to sepsis patients with no shock, n= ( % vs. %, p= . , ft). maximum sofa values were significantly lower in patients with minor allele a compared to cc carriers of ( . vs. . , respectively, p= . ). in post-surgery group of patients, carriers of ac or aa genotypes had significantly increased survival compared to patients with cc genotypes: chi-square= . ; hr= . ( %ci: . - . ) for lethality; p= . (figure ) . association of minor allele a of aqp snp rs with survival in sepsis patients seems secondary to linking the snp to decreased development of multiorgan failure and septic shock that contribute to mortality. validation of presepsin as a biomarker of sepsis in comparison to procalcitonin, il- and il- v chantziara , f kaminari , c sklavou , s fortis , p kogionou , s perez , a efthymiou saint savvas hospital, icu, athens, greece; saint savvas hospital, cancer immunology and immunotherapy center, athens, greece critical care , (suppl ):p sepsis is an everyday challenge for the intensivist and biomarkers are useful tools for identification and treatment of this syndrome. we sought to validate presepsin as a biomarker of sepsis in comparison to pct(procalcitonin) and interleukins (il- ,il- ). we enrolled patients, men and women average age ( . - ) years old, apache ii ( . - . ), saps ii ( . - . ), sofa ( . - ). patients were septic on admission (according to surviving sepsis campaign: international guidelines for management of sepsis and septic shock: ), had a septic episode during their hospitalization in the icu while patients never endured sepsis. we measured presepsin, procalcitonin, il- , il- during sepsis and on remission. results: all septic patients had increased values of presepsin, pct, il- and il- during sepsis with a cutoff value for presepsin pg/ml, while the values of these biomarkers were significantly decreased during remission or in comparison to non-septic patients(presepsin p = . , pct p≤ . , il- p≤ . , il- p= . . all patients who were not septic survived while among septic patients died ( % mortality). presepsin correlated significantly with pct, il- and il- (p< . ). presepsin is a valid biomarker of sepsis and correlates significantly with all the other values of pct, il- and il- . clinical sepsis phenotypes are proposed at hospital presentation. these phenotypes, biomarker profiles, and outcomes are not yet reproduced in prospective data. even less is known about the biologic mechanism the drives these distinct groups. thus, we sought to validate clinical phenotypes and to determine markers of innate immunity, coagulation, tolerance and tissue damage in a prospective cohort. we prospectively studied patients with sepsis- criteria within hours of presentation at hospitals in pennsylvania ( - ) using automated electronic alerts. using clinical variables, we predicted phenotypes (α, β, γ, δ) for each patient using euclidean distance anchored to published seneca phenotype centroids. discarded blood was analyzed in a subset (n= ) for markers of innate immunity (e.g. il- , il- ), coagulation (e.g antithrombin iii, eselectin), tolerance (e.g. ho- , igfbp ), and tissue damage (e.g. serum lactate, bicarbonate) results: among patients, α-type was present in ( %), β-type in ( %), γ-type in ( %) and δ-type in ( %, figure a ). on average, β-type was older and more comorbid (mean , sd yrs; mean elixhauser . , sd . ) with renal dysfunction (median creatinine . [iqr . - . ] mg/dl, p< . all). the δ-type had more acidosis (mean hco - . , sd . meq/l), higher serum lactate (median . [iqr . - . ] mmol/l, p < . both) and inpatient mortality ( %, figure b) . the γand δ-type had greater markers of innate immunity and abnormal coagulation (e.g il- , icam p< . both), while markers of increased tissue damage (lactate) and poor tolerance (ho- ) were present in δ-type, compared to α-type (figure c) . the distribution and characteristics of clinical sepsis phenotypes were reproduced in a prospective validation cohort. similar to the seneca study, distinct biomarker profiles of tissue damage, innate immunity and poor tolerance were present for the δ-type. the effect that neoadjuvant chemotherapy and hyperthermic intraperitoneal chemotherapy (hipec) may have in the postoperative kinetics of biomarkers remains unknow. some studies demonstrate that neoadjuvant chemotherapy and hipec do not invalidate the use of inflammatory markers in postoperative patient monitoring, but none have compared biomarkers kinetics between patients who underwent hipec or only cytoreduction surgery. our main purpose was to identify a difference pattern in c-reactive protein (crp). we conducted a single-center observational study from january to november , including all patients who underwent cytoreductive surgery with or without hipec. crp was measured daily until seven post-operative day. we compared patients with and without hipec. a total of patients were included, were female. mean age was yrs ( - ). no clinical and demographical differences were observed between groups. no documented infection was found. after surgery crp increased markedly in both groups. crp time-course from the day of surgery onwards was significantly different in hipec patients ( . ± . mg/dl vs . ± . mg/dl; p= . ). multiple comparisons between hipec and non hipec patients were performed and crp concentration was significantly different on the th and th pod (figure ). no differences were found in other biomarkers (leucocytes and platelets) neither in body temperature. after a major elective surgical insult crp levels markedly increase independently of hipec. serum crp time-course showed a higher pattern in hipec patients despite no infection detected. decreased thrombin generation potential is associated with increased thrombin generation markers in sepsis associated coagulopathy d hoppensteadt , f siddiqui , e bontekoe , r laddu , r matthew , e brailovsky , j fareed. introduction: sepsis associated coagulopathy (sac) is commonly seen in patients which leads to dysfunctional hemostasis in which uncontrolled protease generation results in the consumption of clotting factors. the purpose of this study is to determine the thrombin generation potential of baseline blood samples obtained from sac patients and demonstrate their relevance to thrombin generation markers. baseline citrated blood samples were prospectively collected from patients with sac at the university of utah clinic. citrated normal controls (n= ) were obtained from george king biomedical (overland park, ks). thrombin generation studies were carried out using a flourogenic substrate method. tat and f . were measured using elisa methods (seimens, indianapolis, in) . functional antithrombin levels were measured using a chromogenic substrate method. the peak thrombin levels and auc levels were lower in the sac patients in comparison to higher levels observed in the normal plasma ( table ). the sac group showed much longer lag time in comparison to the normal group. wide variations in the results were observed in these parameters in the sac group. the f . and tat levels in the sac group were much higher in comparison to the normal. the functional antithrombin levels were decreased in the sac group. these results validate that thrombin generation markers such as f . and tat are elevated in patients with sac. however, thrombin generation parameters are significantly decreased in this group in comparison to normal. this may be due to the consumption of prothrombin due to the activation of the coagulation system. thus, persistent thrombin generation with simultaneous consumption of clotting factors such as prothrombin contributes to the consumption coagulopathy observed in sepsis patients. introduction: procalcitonin (pct) is used in the icu as an inflammatory marker to monitor bacterial infections and guide antibiotic therapy. whether pct can predict bacteremia and therefore could prevent expenses attached to bloodcultures is unknown . we investigated whether pct can predict the outcome of blood cultures in the icu and reduce expences. a single centre observational cohort study was performed in a dutch community teaching hospital . adult patients who were staying in the icu and were suspected of bacteremia were included. simultaneously with drawing of blood cultures, samples for pct measurement were obtained. expenses for pct measurement and bloodcultures were calculated. in the study period of one year, a total of patients were included. three patients were excluded because of incomplete data. out of the included patients, ten patients had positive blood cultures. there was a significant difference in pct levels between patients who had positive bloodcultures versus patients with negative bloodcultures ( . ng/ml vs . ng/ml) ( figure ). the negative predictive value for negative blood cultures is % when pct is below ng/ml, there was no difference in crp levels between the two groups ( mg/l vs mg/l, p= . ).a set of negative blood cultures in our centre costs euros. positive blood cultures however costs significantly more depending on the micro-organisms found. pct only costs . euros per measurement. so when blood cultures are omitted when the pct level is below ng/ml, a cost reduction of % can be achieved. a pct value below ng/ml is a good predictor of a negative blood cultures in icu patients suspected of bacteremia. pct guided bloodculture management in these patients could lead to a significant cost reduction introduction: level of cfdna in plasma is a promising prognostic candidate biomarker in critical illness [ ] . oxidized cfdna (ocfdna) have not been studied as a biomarker although its functional role in cellular stress have attracted attention of researches [ ] . the goal of our study was to assess the early prognostic value of plasma cfdna/ocfdna for sepsis in a nicu setting. the cohort included nicu patients diagnosed with stroke, intracerebral hemorrhage (ich), anoxia, encephalopathy. cfdna was isolated from day plasma and stained with picogreen. oxidized dna was determined using dna immunoblotting with anti- -oxo-desoxiguanosine antibodies. genotyping of allelic variants of the tlr rs gene was performed using a pcr and designed allele-specific tetraprimers followed by electrophoretic separation of the products statistics was performed by the fisher test and mann-whitney test. results: sepsis was diagnosed by sepsis- criteria in patients ( . %). average nisu staying was , ± , days. circulating dna plasma levels on day predicted the future sepsis development (figure ): or for cfdna was . ( %ci: . - . ), p< . ; or for ocfdna was . ( %ci: . - . ), p= . . power of both performed tests with alpha= . : . . log rank test demonstrated better predictive value of cfdna vs. ocfdna (figure) . concentrations of cfdna, but not ocfdna, on day significantly positively correlated with maximum sofa values during hospitalization, day and pre-outcome leukocyte count and neutrophil-to-lymphocyte ratios in a limited cohort of nisu patients with tlr rs cc genotype and not in other patients with genotype tlr ct+tt. increased level of plasma cfdna better then ocfdna predicts sepsis development in nisu. further studies are warranted to clarify the fig. (abstract p ) . pct values in patients with positive blood cultures and patients with negative blood cultures possible utility of tlr rs polymorphism determining for sepsis risk stratification early on nisu admittance. admission was related with higher severity of illness and extension of icu stay for all groups. reduced cbt fluctuations upon icu admission was found to more severely ill patients with worse clinical outcomes, while the more periodic cbt patterns were correlated with high cbt rhythmicity and better outcome. the impact of sex on sepsis incidence and mortality have been elucidated in previous studies, and sex is increasingly recognized as one key factor in sepsis [ ] . some studies indicate that women have better immunologic responses to infections [ ] . later investigations assume this advantage is linked to immune modulating genes located on the x-chromosome [ ] . the purpose of this study is to reveal sex differences in incidence of and mortality of sepsis in a large population-based cohort. methods: adult participants in the hunt study ( - ) were followed from inclusion through end of . incident bloodstream infections (bsi) from all local and regional hospitals in nord-trøndelag county were identified through linkage with the mid-norway sepsis register, which includes prospectively registered information on bsi used as a specific indicator of sepsis. we estimated age-adjusted cumulative incidence of first-time bsi and compared the risk of a first-time bsi and bsi mortality in men and women using age-adjusted cox proportional hazard regression. during a median follow-up of . years individuals experienced at least one episode of bsi, and died within days after a bsi. cumulative incidence and cumulative mortality curves are shown in fig. a introduction:the proportion of hospital-acquired infections (hai) among sepsis patients is unknown in germany. systematic differences in hai foci between sepsis patients with and without icu treatment are insufficiently described. retrospective cohort study based on nationwide health claims data of the german statutory health insurance aok. incident inpatient sepsis cases were identified in / among insured persons > y without preceding sepsis in months prior to index hospitalization. sepsis was defined according to explicit sepsis icd- -codes (incl. severe sepsis/septic shock). hai were defined based on specific icd- -codes for surgical site infection, catheter- introduction: elevated renin is associated with an increased risk of death in patients with vasodilatory shock (vs). recent data show that patients with vs and elevated renin levels have improved survival when treated with angiotensin ii (ang ii) + standard care (sc) vs placebo + sc. patients with acute respiratory distress syndrome (ards) can develop angiotensin-converting enzyme (ace) defects that can lead to elevated renin levels and insufficient endogenous ang ii production. we hypothesized that patients with severe ards and elevated renin shock would have improved survival when treated with ang ii + sc vs placebo + sc. in the randomized, placebo-controlled, double-blind athos- study, patients with severe vs receiving > . μg/kg/min of norepinephrine or the equivalent were randomized to intravenous ang ii (n= ) or placebo (n= ). in a post hoc analysis, we assessed the subset of patients with elevated renin (defined as a renin level greater than the median value of the overall athos- population) and ards (defined by a pao /fio ratio < ) at the time of randomization. survival to days was compared between the ang ii group (n= ) and the placebo group (n= ). in patients with elevated renin and ards, baseline age, acute physiology and chronic health evaluation ii score, and blood pressure were similar in the ang ii and placebo groups. the median serum renin level was . pg/ml (iqr: . - . ) compared to the normal range for serum renin: - pg/ml. a significantly higher proportion of patients receiving ang ii survived to day compared to those in the placebo group ( % vs %; p= . ). elevated renin identified patients with vs and ards who were most likely to gain a survival benefit from ang ii. elevated renin is likely caused by an ace defect and may describe an important subset of patients with a biotype that responds well to ang ii therapy. introduction: elevated renin levels have been shown to be associated with an increased risk of death and more severe acute kidney injury (aki) in patients with vasodilatory shock (vs). recent data show that patients with vs and elevated renin levels have improved survival when treated with angiotensin ii (ang ii) + standard care (sc) vs placebo (pbo) + sc. we hypothesized that vs patients with severe aki and elevated renin levels would have improved survival and enhanced renal recovery with ang ii treatment. in the randomized, pbo-controlled, double-blind athos- study, patients with severe vs received > . μg/kg/min of norepinephrine or the equivalent and were randomized to intravenous ang ii + sc (n= ) or pbo + sc (n= ). in a post hoc analysis, we assessed the subset of patients with elevated renin (defined as a renin level greater than the median value of the overall athos- population) and severe aki (defined as those with aki requiring renal replacement therapy [rrt] at baseline). survival and renal recovery were assessed in patients treated with ang ii + sc (n= ) and pbo + sc (n= ). in patients with elevated renin and severe aki, baseline age, acute physiology and chronic health evaluation ii score, and blood pressure were similar between ang ii + sc vs pbo + sc. the median baseline serum renin level in the whole group was . pg/ml (iqr: . - . ; normal range for serum renin: - pg/ml). a significantly higher proportion of patients receiving ang ii + sc vs pbo + sc survived to day ( % vs %, respectively; p= . ). ang ii recipients also had a higher rate of discontinuation from rrt by day ( % vs %; p= . ). in this study, elevated-renin shock patients with aki treated with ang ii + sc gained a survival benefit and earlier discontinuation from rrt compared to those receiving pbo + sc. elevated renin is likely caused by an angiotensin-converting enzyme defect and may identify those patients with a biotype that responds well to ang ii therapy. most clinical trials conclude the ineffective use of anticoagulation for sepsis-induced coagulopathy [ ] . however, post hoc analyses of randomized control trials report positive results [ ] , suggesting anticoagulation is effective in specific populations exhibiting coagulopathy. further, anticoagulants should be administered in the early phase [ ] ; however, methods for precisely predicting the progression of sepsis-induced coagulopathy are not established. this study aimed to create and evaluate a prediction model of coagulopathy progression using machine-learning techniques. we performed a subgroup analysis of data from a retrospective cohort study involving adult septic patients in japanese institutions from january to december and used the japanese association for acute medicine disseminated intravascular coagulation (dic) score as a dic severity index test. the predictive ability of Δdic ([dic score on day ] -[dic score on day ]) was evaluated using various statistical methods. using variables available at the outset, we compared the predictive ability of random forest (rf) and support vector machine (svm) with that of multiple linear regression analysis. a total of adults with sepsis were included in the analysis. the root mean square error in Δdic score for the multiple linear regression analysis model was . compared with values of . and . for rf and svm, respectively. thus, the rf method predicted the progression of sepsis-induced coagulopathy more accurately than multiple linear regression analysis. conclusions: rf, a machine-learning technique, was superior to multiple linear regression analysis in predicting the progression of sepsis-induced coagulopathy. this prediction model might enable us to use anticoagulation in an early phase. this study examined the efficacy and safety of landiolol, an ultrashort-acting β -blocker, for treating sepsis-related tachyarrhythmia, according to patient background characteristics. the j-land s study (japiccti- ) was conducted in patients with sepsis, diagnosed according to the sepsis- criteria, and tachyarrhythmia (atrial fibrillation, atrial flutter, or sinus tachyarrhythmia). the patients had a mean heart rate of ≥ beats/min and required catecholamine administration to maintain a mean blood pressure of ≥ mmhg. the efficacy endpoint was the percentage of patients whose heart rate could be controlled within - beats/min at h of registration. the safety endpoint was the incidence of adverse events within h of registration. subgroup analyses of efficacy and safety were performed after stratifying the patients according to various patient background characteristics. a total of patients were randomized, to landiolol and to the control group. the efficacy endpoint, percentage of patients with a heart rate of - beats/min at h of registration, was significantly higher in the landiolol group ( . % vs . %; mantel-haenszel test: p = . ). the incidence of adverse events was . % and . % in the landiolol and control groups, respectively, and there was no difference between the two groups. most adverse events were related to sepsis or septic shock. the subgroup analyses showed that no patient background characteristic clearly affected the efficacy and safety of landiolol. landiolol is a well tolerated and effective therapeutic agent for controlling heart rate in patients with sepsis-related tachyarrhythmias; its safety and efficacy were not affected by the patient background characteristics investigated. tissue oxygenation monitoring in sepsis r marinova, at temelkov umhat alexandrovska, anesthesiology and intensive care, sofia, bulgaria critical care , (suppl ):p near-infrared spectroscopy (nirs) was proposed as a concept in the end of th century. this method offers noninvasive monitoring of oxy-and deoxyhemoglobin in tissues.nirs could be measured on the thenar or forehead within few santimeters of the skin. it was first applied as a monitoring in cardiovascular surgery. patients with sepsis have changes in the microcirculation which are important target for therapy. invasive monitoring of oxygen delivery and consumption has been used in patients with sepsis but as every invasive technique such a monitoring hides risks. nirs offers a noninvasive method for tissue oxygenation monitoring (sto ) and could be useful in patients with sepsis and septic shock. the aim of the study is to compare noninvasive tissue oxygenation monitoring with hemodinamic monitoring and lactate values in patients with sepsis methods:the study includes critically ill patients in icu of umhat alexandrovska, sofia. of the patients fullfil the criteria for septic state. the other patients do not have sepsis. in both group of patients are measured tissue oxygenation with invios monitor, mean arterial pressure, oxygen saturation in mixed venous blood and lactate values during h after icu admission. patients with sepsis are reported with significantly lower values of tissue oxygenation, compared to patients without sepsis. the values of tissue oxygenation correlate well with the mixed venous blood oxygenation, mean arterial pressure and lactate values but not significantly with apache scores. conclusions: nirs when used for tissue oxygenation monitoring correlates well with the hemodinamic monitoring and lacate values in patients with sepsis and could be used as an noninvasive monitoring for guiding teurapeutic strategies. tissue oxygenation monitoring has no linear correlation with the severity of illness in patients with sepsis and could not be reccomended as a guidance in the early ressuscitating stage of sepsis. further investiganions in these field are needed.the sequenom´s massarray platform and a recessive inheritance model was selected (cc vs tt/ct). the possible association between the cc recessive form of the rs polymorphism and the septic shock risk was analyzed, demonstrating a statistically significant relationship (p= . ) between both conditions. among patients who developed septic shock, . % presented a recessive inheritance pattern while . % showed the ct/tt genotype. on the other hand, those patients with the recessive form of the rs polymorphism were selected and a statistical analysis was performed comparing those patients who developed septic shock from those who did not develop it, obtaining a statistically significant relationship (p= . ) between the presence of the recessive form of polymorphism and the likelihood of developing septic shock. the recessive form of rs polymorphism is a risk factor for septic shock in post-operative patients of major abdominal surgery. introduction: sepsis remains one of the major causes of morbidity with mortality rates as high as % worldwide, representing significant clinical challenge to confront highly intangible therapeutic needs. rnabased structures are emerging as versatile tools encompassing a variety of functions capable to bypass the current protein-and cellbased therapies. rna aptamers act as disease-associated protein antagonists. here, the effects of an aptamer, apta- , were evaluated in animal models that mimic systemic inflammation in humans. high dose of lps endotoxin was used to induce systemic inflammation in mice and in non-human primate animal models. apta- was administered intravenously in two doses post lps infection. animals were monitored and blood samples collected up to hours after apta- administration. healthy-and lps-only treated animals served as control groups. complex analyses of clinical parameters, hematology, serum biochemistry, inflammation and tissue damage markers were performed. results: apta- increased survival of endotoxin challenged animals up to % in a dose-dependent manner and exerted profound effects on wellbeing and recovery of healthy eating habits. administration of apta- led to delayed coagulation and enhanced fibrinolysis; maintained the complement cascade activated while preventing it from further amplification. expression of pro-inflammatory cytokines was reduced while anti-inflammatory increased. endogenous pro-inflammatory molecules (damps), secreted from injured cells, were preserved at healthy level in animals treated with apta- . systemic inflammation and sepsis lead to severe dysregulation of several arms/axis of innate immune response. our studies showed that apta- affects various components of this system and restores the organism's control over its dysregulated immune response. thus, apta- might be a promising potential therapeutic candidate to treat life-threatening conditions such sepsis. several preclinical studies demonstrated beneficial effects for methane (ch ) administration in various inflammatory conditions. our aim was to investigate the consequences of post-treatment with inhaled ch in a clinically relevant intra-abdominal sepsis model. anesthetized minipigs were subjected to fecal peritonitis ( . g/kg, - x cfu i.p.; n= ) or sham-operation (sterile saline i.p; n= ). invasive hemodynamic monitoring with blood gas analyses was started between - hours, organ dysfunction parameters (pao /fio ratio; mean arterial pressure; lactate, bilirubin, creatinine; urine output and platelet counts) were determined according to a modified porcinespecific sequential organ failure assessment (ps-sofa) score system, the perfusion rate (pr) of sublingual microcirculation was measured by incident dark field illumination imaging. the animals were divided into non-treated septic or septic shock groups (n= - ) and ch treated septic or septic shock (n= - ) subgroups, ch inhalation started from the th hr ( . % ch in normoxic air; ml/min). despite the standardized induction, heterogeneous severity of organ damage was evolved. in septic and septic shock groups the median values of ps-sofa score reached ( . - . ) and ( . - ), respectively. septic shock was characterized by significant elevations of creatinine and bilirubin levels, while the platelet count decreased (from to * /l). inhalation of ch increased the sublingual pr by % in the septic group, the creatinine and bilirubin levels were decreased by % and %, respectively. ch post-treatment significantly decreased the ps-sofa score (to ; . - . ) and resulted in lower values in septic shock group (to ; . - . ). methane post-treatment effectively influences sepsis-related end organ dysfunction. up to a severity threshold it may be a promising additional organ protective tool. evaluation of sepsis awareness among various groups in turkey: a survey study s erel, o ermis, Ö nadastepe, l karabıyık gazi university school of medicine, anesthesiology and intensive care, ankara, turkey critical care , (suppl ):p introduction: sepsis is a common life-threatening condition in critically ill patients [ ] . public awareness is important for early recognition of sepsis and improvement of outcomes [ ] . we aimed to evaluate sepsis awareness among different groups of people. methods: prospective paper-based surveys were issued between st july and st august to patients, the relatives of the patiens, hospital staff and general public who gave consent to participate in the study. the questionnaire included ten questions about demographic informations, occupational informations of hospital stuff and sepsis awareness. a total of participated in the survey. of these participants, ( . %) were patients, ( . %) were relatives of patients, ( . %) were physicians, ( . %) were medical students, ( . %) were nurses, ( . %) were other hospital stuff and (% . ) were other people. of these participants, ( . %) had heard of the word "sepsis". ( . %) responded correctly regarding the definition of sepsis. ( . %) of the participants heard the word "sepsis" during their education, but only ( %) heard it through the media. in the groups of high school graduates, university graduates and postgraduates, the rate of hearing the word sepsis and correctly identifying sepsis is significantly higher than the primary school graduates or illiterate groups. (p< . ). physicians, nurses and medical students were heard of the word "sepsis" significantly more than other groups (p< . ). physicians and medical students responded more accurately to the definition of sepsis than other groups (p< . ). public awareness of sepsis is limited compared to healthcare workers. increasing public knowledge of sepsis through education and through media may contribute to raising public awareness and improving outcomes. the association between clinical phenotype cohesiveness and sepsis transitions after presentation jn kennedy , eb brant , km demerle , ch chang , s wang , dc angus , cw seymour key: cord- -q sug uu authors: szűcs, zsolt; naesens, lieve; stevaert, annelies; ostorházi, eszter; batta, gyula; herczegh, pál; borbás, anikó title: reprogramming of the antibacterial drug vancomycin results in potent antiviral agents devoid of antibacterial activity date: - - journal: pharmaceuticals (basel) doi: . /ph sha: doc_id: cord_uid: q sug uu influenza a and b viruses are a global threat to human health and increasing resistance to the existing antiviral drugs necessitates new concepts to expand the therapeutic options. glycopeptide derivatives have emerged as a promising new class of antiviral agents. to avoid potential antibiotic resistance, these antiviral glycopeptides are preferably devoid of antibiotic activity. we prepared six vancomycin aglycone hexapeptide derivatives with the aim of obtaining compounds having anti-influenza virus but no antibacterial activity. two of them exerted strong and selective inhibition of influenza a and b virus replication, while antibacterial activity was successfully eliminated by removing the critical n-terminal moiety. in addition, these two molecules offered protection against several other viruses, such as herpes simplex virus, yellow fever virus, zika virus, and human coronavirus, classifying these glycopeptides as broad antiviral molecules with a favorable therapeutic index. seasonal infections by influenza a and b viruses are each year responsible for significant morbidity and mortality [ ] . besides, zoonotic influenza a viruses occasionally enter the human population to cause serious pandemics with a high number of fatalities [ ] . antiviral drugs are essential for influenza treatment and prevention, including in the context of pandemic preparedness. at the moment, four drug classes are available: the m ion channel blockers and neuraminidase inhibitors, approved in all countries [ ] , and two polymerase inhibitors, recently approved in a few countries [ , ] . for each of these drugs, emergence of resistant mutants is possible; this is particularly problematic when the mutant viruses are fit and human-to-human transmissible [ ] [ ] [ ] [ ] . hence, additional drug classes with a distinct mechanism of action remain essential [ ] [ ] [ ] . several recent investigations demonstrated that some antibiotics from bacterial origin display interesting antiviral properties [ ] representing a unique example of drug repurposing [ ] . to synthesize the first variant, we used literature procedures to prepare vancomycin aglycone ( ) and its hexapeptide derivative ( ) by the edman degradation (scheme ) [ , ] . then, by the copper-catalyzed diazotransfer reaction, the n-terminal azido derivative ( ) was successfully prepared in analogy to our previous work [ , ] . pharmaceuticals , , of to synthesize the first variant, we used literature procedures to prepare vancomycin aglycone ( ) and its hexapeptide derivative ( ) by the edman degradation (scheme ) [ , ] . then, by the copper-catalyzed diazotransfer reaction, the n-terminal azido derivative ( ) was successfully prepared in analogy to our previous work [ , ] . the subsequent copper-catalyzed azide-alkyne click reaction using alkyne compound (scheme ) yielded triazole derivative . the second compound modified on the n-terminus ( ) was prepared by the same method using the already described maleimide derivative [ ] as the alkyne compound in the final step. the third derivative ( ) in this group was synthesized using derivative and hexanesulfonyl chloride by sulfonamide formation, similar to what we previously published for teicoplanin congeners [ ] . the subsequent copper-catalyzed azide-alkyne click reaction using alkyne compound (scheme ) yielded triazole derivative . the second compound modified on the n-terminus ( ) was prepared by the same method using the already described maleimide derivative [ ] as the alkyne compound in the final step. the third derivative ( ) in this group was synthesized using derivative and hexanesulfonyl chloride by sulfonamide formation, similar to what we previously published for teicoplanin congeners [ ] . structures of previously prepared alkynes and used for the synthesis of derivatives and . as for the c-terminal modifications, we decided to prepare two derivatives carrying a , , -triazole ring, since this moiety often generates bioactive analogues [ ] . in order to minimize the structural difference between the side chain in the n-versus c-terminal position, we used -azidoethylamine as a small linker for the synthesis of the appropriate c-terminal analogues (scheme ). after preparation of the amines, the reaction of compound with amine using the pybop reagent gave amide derivative (scheme ). using the same conditions, the reaction between compound and maleimide as the alkyne compound yielded compound . in the case of the cterminal sulfonamide derivative we also used the similar, small linker moiety. in this approach, an amine functionalized sulfonamide was first synthesized from n-hexanesulfonyl chloride and ethylenediamine, then the peptide coupling reaction between this compound and compound using pybop yielded compound . structures of previously prepared alkynes and used for the synthesis of derivatives and . as for the c-terminal modifications, we decided to prepare two derivatives carrying a , , -triazole ring, since this moiety often generates bioactive analogues [ ] . in order to minimize the structural difference between the side chain in the n-versus c-terminal position, we used -azidoethylamine as a small linker for the synthesis of the appropriate c-terminal analogues (scheme ). as for the c-terminal modifications, we decided to prepare two derivatives carrying a , , -triazole ring, since this moiety often generates bioactive analogues [ ] . in order to minimize the structural difference between the side chain in the n-versus c-terminal position, we used -azidoethylamine as a small linker for the synthesis of the appropriate c-terminal analogues (scheme ). after preparation of the amines, the reaction of compound with amine using the pybop reagent gave amide derivative (scheme ). using the same conditions, the reaction between compound and maleimide as the alkyne compound yielded compound . in the case of the cterminal sulfonamide derivative we also used the similar, small linker moiety. in this approach, an amine functionalized sulfonamide was first synthesized from n-hexanesulfonyl chloride and ethylenediamine, then the peptide coupling reaction between this compound and compound using pybop yielded compound . scheme . synthesis of amines - for the c-terminal modifications. after preparation of the amines, the reaction of compound with amine using the pybop reagent gave amide derivative (scheme ). using the same conditions, the reaction between compound and maleimide as the alkyne compound yielded compound . in the case of the c-terminal sulfonamide derivative we also used the similar, small linker moiety. in this approach, an amine functionalized sulfonamide was first synthesized from n-hexanesulfonyl chloride and ethylenediamine, then the peptide coupling reaction between this compound and compound using pybop yielded compound . modified on the c-terminus. antibacterial tests were carried out by the broth microdilution method on a panel of eight grampositive bacterial strains, using vancomycin and teicoplanin as reference compounds. neither of the new compounds exhibited significant activity against any bacterium, proving successful elimination of antibacterial activity, as anticipated (table ) . antibacterial tests were carried out by the broth microdilution method on a panel of eight gram-positive bacterial strains, using vancomycin and teicoplanin as reference compounds. neither of the new compounds exhibited significant activity against any bacterium, proving successful elimination of antibacterial activity, as anticipated (table ) . with regard to antiviral activity, the two n-terminal triazole derivatives and displayed robust activity against the three influenza a or b viruses tested. upon microscopic inspection, no virus-induced cytopathic effect (cpe) was observed in virus-infected cells treated with . µm of compound or compound ( figure a ). the quantitative antiviral efficacy (ec ) and cytotoxicity (cc ) values, both determined by the mts cell viability assay, are summarized in table . with ec values of~ µm and a cc value of µm (compound ) and µm (compound ), the molecules had a selectivity index (ratio of cc to ec ) of and , respectively. both molecules exhibited clear inhibition of influenza virus replication, since they strongly reduced the virus yield in the supernatant ( figure b ), giving ec values of . µm (compound ) and . µm (compound ), which is -to -fold lower than the ec for ribavirin ( µm) . at these concentrations, the compounds were devoid of cytotoxicity, as assessed by mts cell viability assay in mock-infected cells ( figure c ). the n-terminal n-hexanesulfonyl derivative and c-terminally modified compound proved inactive. for compound , this was somewhat surprising since the analogous teicoplanin pseudoaglycone derivative showed high activity [ ] . on the other hand, this result is in line with our previous findings on ristocetin and teicoplanin aglycone derivatives, indicating that even minor structural differences in the peptide core can lead to significantly different anti-influenza virus activity [ ] . the c-terminally modified compounds and were only slightly active against one or both influenza a virus strains. compared to compounds and , compound displayed an -fold higher antiviral ec value by mts assay ( table ); its lower potency was also evident in the virus yield reduction assay ( figure b ). this points to the importance of the modification site, since the c-terminally modified compounds were clearly inferior to the n-terminal analogues. pharmaceuticals , , of with regard to antiviral activity, the two n-terminal triazole derivatives and displayed robust activity against the three influenza a or b viruses tested. upon microscopic inspection, no virusinduced cytopathic effect (cpe) was observed in virus-infected cells treated with . µm of compound or compound ( figure a ). the quantitative antiviral efficacy (ec ) and cytotoxicity (cc ) values, both determined by the mts cell viability assay, are summarized in table . with ec values of ~ µm and a cc value of µm (compound ) and µm (compound ), the molecules had a selectivity index (ratio of cc to ec ) of and , respectively. both molecules exhibited clear inhibition of influenza virus replication, since they strongly reduced the virus yield in the supernatant ( figure b) , giving ec values of . µm (compound ) and . µm (compound ), which is -to fold lower than the ec for ribavirin ( µm) . at these concentrations, the compounds were devoid of cytotoxicity, as assessed by mts cell viability assay in mock-infected cells ( figure c ). the nterminal n-hexanesulfonyl derivative and c-terminally modified compound proved inactive. for compound , this was somewhat surprising since the analogous teicoplanin pseudoaglycone derivative showed high activity [ ] . on the other hand, this result is in line with our previous findings on ristocetin and teicoplanin aglycone derivatives, indicating that even minor structural differences in the peptide core can lead to significantly different anti-influenza virus activity [ ] . the c-terminally modified compounds and were only slightly active against one or both influenza a virus strains. compared to compounds and , compound displayed an -fold higher antiviral ec value by mts assay ( table ); its lower potency was also evident in the virus yield reduction assay ( figure b ). this points to the importance of the modification site, since the c-terminally modified compounds were clearly inferior to the n-terminal analogues. encouraged by the promising anti-influenza virus activity of compounds and , we tested the two compounds against a range of dna-and rna-viruses evaluated in human embryonic lung (hel) fibroblast, hela or vero cells. for each virus, appropriate reference compounds were included. protection against virus-induced cytopathicity as well as compound cytotoxicity were determined by the mts cell viability assay. as shown in table , the two compounds exhibited broad protection against a large variety of viruses, including herpesvirus types and and vaccinia virus. they retained full effectivity against a thymidine kinase deficient form of hsv- , which was -fold (acyclovir) and -fold (ganciclovir) resistant to antiherpetic drugs. moreover, the compounds proved effective against two emerging pathogens for which no therapy is currently approved, i.e., coronavirus (inhibited by compounds and ) and zika virus (inhibited by compound ) . at a non-toxic concentration of µm of compound , no coronavirus e-induced cytopathicity could be observed microscopically (figure a ), which agrees with an ec value of µm as determined by mts cell viability assay (table ). in addition, treatment of infected cells with µm of compound resulted in a -fold reduction of the viral rna copy number in the supernatant, yielding an ec value of µm for reduction of virus yield ( figure b ). hence, we established, by virus yield assays, that compound suppresses the replication of influenza virus and coronavirus, and for the other viruses, activity was indicated by the protection against viral cpe. this broad activity against distinct viruses fits with our hypothesis that these molecules may act by disrupting the viral endocytosis process, similarly to what we reported for a glycopeptide active against influenza virus [ ] and what was described for ebola virus and mers and sars coronaviruses [ , ] . this should become clear from mechanistic work ongoing in our laboratory. vancomycin hydrochloride was a gift from teva pharmaceutical industries ltd. (debrecen, hungary). vancomycin aglycone hexapeptide, trifluoromethanesulfonyl azide, compounds and were prepared as described elsewhere [ , ] . tlc was performed on kieselgel f (merck) with detection either by immersing into ammonium molybdate-sulfuric acid solution followed by heating vancomycin hydrochloride was a gift from teva pharmaceutical industries ltd. (debrecen, hungary). vancomycin aglycone hexapeptide, trifluoromethanesulfonyl azide, compounds and were prepared as described elsewhere [ , ] . tlc was performed on kieselgel f (merck) with detection either by immersing into ammonium molybdate-sulfuric acid solution followed by heating or by using pauly's reagent for detection. flash column chromatography was performed using silica gel (merck . - . mm) and silica gel silanized ( . - . mm). the h nmr ( mhz, mhz) c nmr ( mhz, mhz) and d nmr spectra were recorded with a bruker drx- and bruker avance ii spectrometers at k. chemical shifts are referenced to me si and to the solvent residual signals. maldi-tof ms analysis of the compounds was carried out in the positive reflectron mode using a biflex iii mass spectrometer (bruker, bremen, germany) equipped with delayed-ion extraction. , -dihydroxybenzoic acid (dhb) was used as matrix and cf coona as cationizing agent in dmf. elemental analysis (c, h, n, s) was performed on an elementar vario microcube instrument. synthesis of azido vancomycin aglycone hexapeptide ( ): mg ( . mmol) vancomycin aglycone hexapeptide ( ) was obtained from mg ( . mmol) vancomycin hydrochloride ( ) by edman degradation as described in the literature [ , ] . sodium azide ( mg, . mmol) was added to dry pyridine ( . ml) cooled to - • c. tf o ( . mmol, µl) was added dropwise over the course of about min. the reaction mixture was stirred for another h at - • c. then, mg ( . mmol) of was dissolved in ml pyridine, then µl ( . equiv., . mmol) et n was added followed by the solution ( . ml) of the freshly prepared trifluoromethanesulfonyl azide, and finally µl of a mg/ml cuso · h o solution. the solution was stirred at room temperature overnight, then the solvents were evaporated. the crude product was dissolved in dilute nh oh, then the ph was set to - with n hcl, the resulting cloudy mixture was extracted with n-buoh three times, the butanolic phase was washed with water, then evaporated and purified by flash column chromatography using step gradient elution (mecn:h o = : → : → : → : ). the title compound was obtained in mg yield ( %) as an off-white powder. nmr: see synthesis of compound : mg ( . mmol) of was dissolved in . ml of dmf:h o : mixture. next, mg ( . equiv., . mmol) of alkyne was added, followed by mg cuso · h o. the reaction mixture was stirred at room temperature for h. by this time tlc (cellulose, n-proh: cc. nh oh = : ) indicated good conversion. the solvents were evaporated until a syrupy residue was obtained. ether was added, and the product was filtered off after precipitation and washed with additional ether to remove the excess alkyne. purification was carried out by c reverse phase column chromatography (h o:mecn = : → : → : ) followed by gel chromatography using sephadex lh- in meoh. the title compound was obtained as a white powder in mg yield ( %). nmr: see table s in supporting information. elemental analysis: see table s after h stirring at room temperature, tlc indicated good conversion. the reaction mixture was worked up and purified by c reverse phase column chromatography as described above. the title compound was obtained as a yellow powder in mg yield ( %). nmr: see table s in supporting information. elemental analysis: see table s in supporting information. maldi-ms m/z calcd. for c h cl n o s + na + [m + na] + : . . found: . . synthesis of compound : mg ( . mmol) of was dissolved in ml dry pyridine and . ml dry dmf, then µl ( . equiv., . mmol) of n-hexanesulfonyl chloride was added. after stirring h at room temperature, ethyl acetate and ether was added, the resulting precipitate was filtered and washed with ether. the crude product was purified by flash column chromatography using toluene: meoh = : → : as eluent, followed by gel chromatography using sephadex lh- with meoh: h o = : as eluent. the yield was mg ( %). nmr: see table s in supporting information. elemental analysis: see table s synthesis of -( -( -( -((decyloxy)methyl)- h- , , -triazol- -yl)- , , , -tetraoxatridecyl)- h- , , -triazol- -yl)ethanamine ( ): mg of [ , ] ( . mmol) and mg ( . mmol) of -azidoethylamine were dissolved in ml dry dmf under argon. µl ( . equiv., . mmol) et n was added, then mg ( mol%, . mmol) cu(i)i. the reaction mixture was stirred at room temperature for an hour. after evaporation of the solvents, the crude product was purified by flash column chromatography using dcm: meoh = : (+ . % v/v nh oh) as eluent. the title compound was obtained as an off white solid in % yield ( mg). addition of ethyl acetate, filtered out and washed with diethyl ether. the product was purified by flash column chromatography using step gradient elution (toluene: meoh = : → : ) then by gel column chromatography (sephadex lh- , acetone:h o = : ). the title compound was obtained as an off-white powder in mg yield ( %). nmr: see table s in supporting information. elemental analysis: see table s in supporting information. maldi-ms m/z calcd. for c h cl n o s + na + [m + na] + : . . found: . . for antiviral testing, mm compound stocks were prepared in % dmso and stored at • c. the compounds were fully soluble under these conditions. in the antiviral tests, the highest concentration tested was µm, corresponding to a non-toxic dmso content of . %. the virus strains (a/h n : a/ned/ / and a/pr/ / ; a/h n : a/victoria/ / ; and b/ned/ / ) were propagated in embryonated hen eggs. the antiviral procedure was published elsewhere [ , ] . madin-darby canine kidney (mdck) cells were seeded at cells per well into -well plates, using infection medium (ultramdck medium (lonza) with mg/l sodium bicarbonate, mm l-glutamine, and µg/ml n-tosyl-l-phenylalanine chloromethyl ketone (tpck)-treated trypsin). one day later, virus (moi: . ) was added together with : serial compound dilutions, to reach a total volume of µl per well. besides the test compounds, two references were included, i.e., zanamivir and ribavirin (positive controls) plus a condition receiving medium instead of compound (negative control). in parallel, the compound dilutions were also added to a mock-infected plate (in which medium was added instead of virus), to determine compound cytotoxicity. each plate contained two wells in which all reagents yet no cells were added, to serve as blanks in the mts calculations. after three days incubation at • c, viral cpe was first monitored by microscopy. then, the supernatants were replaced by mts reagent (celltiter ® aq ueous mts reagent from promega) diluted : in pbs, and h later, absorbance at nm was measured in a plate reader. to monitor the inhibitory effect of the compounds on virus replication, mdck cells were seeded, infected (with a/pr/ / virus; moi: . ) and treated with : serial compound dilutions. the plate contained three virus controls (receiving no compound) and two cell controls (receiving no virus and no compound). at day p.i., supernatants were collected and frozen at - • c, to quantify the virus yield by one-step qrt-pcr analysis of viral copy number [ ] . two µl supernatant was mixed with µl resuspension buffer and µl lysis reagent (cellsdirect one-step rt-qpcr kit; invitrogen) and heated during min at • c. next, µl lysate was transferred to a qpcr plate containing the qrt-pcr enzymes and buffer (cellsdirect one-step rt-qpcr kit; invitrogen), and influenza virus m -specific primers and probe [ ] . the program consisted of: min at • c; min at • c; and cycles of s at • c followed by s at • c. absolute quantification of vrna copies was performed by including an m -plasmid standard. the ec values were calculated by interpolation and defined as the compound concentration causing -fold reduction in vrna copy number, as compared to the virus control receiving no compound. it was ascertained that the cell controls showed no detectable qpcr signal. the viruses were propagated and evaluated in the following cell lines: human embryonic lung (hel) fibroblast cells, used for human coronavirus e [ ] , herpes simplex virus type (hsv- strain kos, including a thymidine kinase deficient hsv- /tkmutant), herpes simplex virus type (hsv- , strain g) and vaccinia virus (strain lederle); human cervixcarcinoma hela cells, used for respiratory syncytial virus (rsv, strain long), and african green monkey kidney vero cells, used for yellow fever virus (vaccine strain d) and zika virus (strain mr ). the medium used for virus infection was dulbecco's modified eagle's medium supplemented with % fetal calf serum. to prepare virus stocks, confluent cell cultures in -cm flasks were infected with the virus and frozen after to days incubation at • c (or • c in case of human coronavirus e), when full-blown cpe was visible. after freeze-thawing and centrifugation, the clarified lysates were stored at − • c. for the antiviral experiments, the cells were grown in -well plates until confluent. virus was added (moi: . ) together with : serial dilutions of the compounds. for each virus, appropriate reference compounds were included. the compound dilutions were also added to a mock-infected plate, to determine compound cytotoxicity. when manifest cpe was reached, i.e., after to days incubation at • c (or • c in case of human coronavirus e), cpe and compound cytotoxicity were quantified by the mts assay, and ec and cc values were calculated as explained above for influenza virus. to assess inhibition of human coronavirus e replication, hel cells were infected and treated with : serial compound dilutions. the plate contained three virus controls (receiving no compound) and two cell controls (receiving no virus and no compound). at day p.i., supernatants were frozen at − • c to determine the virus yield by one-step rt-qpcr assay. two microliters supernatant was mixed with µl lysis mix containing lysis enhancer and resuspension buffer at a : ratio (cellsdirect one-step rt-qpcr kit; invitrogen), and heated for min at • c. five microliters of lysate was transferred to a pcr plate containing . µl rt-qpcr mix (cellsdirect one-step rt-qpcr) and . µl superscript iii rt/platinum taq enzyme, and coronavirus- e n-gene specific primers and probe (forward primer -ttagagagcgtgttgaaggtg- ; reverse primer -gttctgaattcttgcgcctaac- ; probe -fam-tctgggttg-zen-ctgttgatggtgcta-ibfq- ). the rt-qpcr program consisted of min at • c, min at • c, and cycles of s at • c and s at • c. an n-gene plasmid standard was included for absolute quantification. compound activity was expressed as the ec value, i.e., the concentration causing -fold reduction in vrna copy number, as compared to the virus control receiving no compound. it was ascertained that the cell controls showed no detectable qpcr signal. starting from vancomycin, we have successfully prepared two derivatives with strong activity against influenza virus. the modifications that we introduced were based on our previous work on the glycopeptide antibiotic teicoplanin. interestingly, some of these modifications yielded compounds and having the same antiviral potency as the analogous teicoplanin pseudoaglycon derivatives [ , ] but lacking antibacterial activity. the short work described here validates that the glycopeptide scaffold is an underexplored entity to conceive new antivirals with a broad activity spectrum, that besides influenza virus includes emerging pathogens like coronavirus and zika virus. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : nmr data for compound , table s : nmr data for compound , table s : nmr data for compound , table s : nmr data for compound , table s : nmr data for compound , table s : nmr data for compound , table s : nmr data for compound , table s mg ( . mmol) of compound [ ] and mg ( . equiv., . mmol) of azidoethylamine were dissolved in ml dry dmf under argon. µl ( . equiv., . mmol) et n was added followed by mg ( mol%) cu(i)i. the reaction mixture was stirred for h at room temperature x ch ), . , . , . ( c, × ch ), . ( c, × ch ). maldi-ms m/z calcd. for c h n o s + na + [m + na] + : . . found: . . synthesis of compound : vancomycin aglycone hexapeptide ( mg, . mmol) was dissolved in . ml dry dmf. mg ( . equiv., . mmol) of compound was added followed by µl ( . equiv., . mmol) et n and mg ( . equiv., . mmol) of pybop. the reaction mixture was stirred for h at room temperature, after which tlc (n-proh:nh oh = : , cellulose) indicated complete conversion. the product was precipitated by the addition of ml of cold etoac:et o = : mixture, filtered off and washed thoroughly with diethyl ether. the crude product was purified by gel column chromatography using sephadex lh- in mecn:h o = : mixture, followed by flash column chromatography in mecn:h o = : mixture. the product was obtained as a white powder in mg yield ( %) mmol) was added followed by mg n-( -aminoethyl)hexane- -sulfonamide (~ equiv., . mmol) obtained in step risk factors for serious outcomes associated with influenza illness in high-versus low-and middle-income countries: systematic literature review and meta-analysis influenza a virus transmission via respiratory aerosols or droplets as it relates to pandemic potential the war against influenza: discovery and development of sialidase inhibitors in vitro characterization of baloxavir acid, a first-in-class cap-dependent endonuclease inhibitor of the influenza virus polymerase pa subunit t- (favipiravir) and related compounds: novel broad-spectrum inhibitors of rna viral infections adamantane-resistant influenza a viruses in the world ( - ): frequency and distribution of m gene mutations influenza virus resistance to neuraminidase inhibitors global transmission of oseltamivir-resistant influenza influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance. influenza other respir. viruses emerging antiviral strategies to interfere with influenza virus entry antiviral therapies on the horizon for influenza advancements in host-based interventions for influenza treatment fighting viruses with antibiotics: an overlooked path drug repurposing approaches for the treatment of influenza viral infection: reviving old drugs to fight against a long-lived enemy teicoplanin inhibits ebola pseudovirus infection in cell culture glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) antiretroviral activity of semisynthetic derivatives of glycopeptide antibiotics polycyclic peptide and glycopeptide antibiotics and their derivatives as inhibitors of hiv entry inhibition of feline (fipv) and human (sars) coronavirus by semisynthetic derivatives of glycopeptide antibiotics inhibition of hepatitis c virus replication by semi-synthetic derivatives of glycopeptide antibiotics an analogue of the antibiotic teicoplanin prevents flavivirus entry in vitro anti-influenza virus activity and structure-activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains intracytoplasmic trapping of influenza virus by a lipophilic derivative of aglycoristocetin diazo transfer-click reaction route to new, lipophilic teicoplanin and ristocetin aglycon derivatives with high antibacterial and anti-influenza virus activity: an aggregation and receptor binding study synthesis of fluorescent ristocetin aglycone derivatives with remarkable antibacterial and antiviral activities synthesis of isoindole and benzoisoindole derivatives of teicoplanin pseudoaglycone with remarkable antibacterial and antiviral activities a few atoms make the difference: synthetic, cd, nmr and computational studies on antiviral and antibacterial activities of glycopeptide antibiotic aglycone derivatives semisynthetic teicoplanin derivatives as new influenza virus binding inhibitors: synthesis and antiviral studies synthesis and biological evaluation of lipophilic teicoplanin pseudoaglycone derivatives containing a substituted triazole function structure-activity relationship studies of lipophilic teicoplanin pseudoaglycon derivatives as new anti-influenza virus agents structure-activity relationship studies of a series of antiviral and antibacterial aglycon derivatives of the glycopeptide antibiotics vancomycin, eremomycin, and dechloroeremomycin structural modifications of the active site in teicoplanin and related glycopeptides. . reductive hydrolysis of the , -and , -peptide bonds teicoplanin, antibiotics from actinoplanes teichomyceticus nov. sp. viii. opening of the polypeptide chain of teicoplanin aglycone under hydrolytic conditions structural modifications of the active site in teicoplanin and related glycopeptides. . deglucoteicoplanin-derived tetrapeptide the edman degradation of vancomycin -preparation of vancomycin hexapeptide synthesis and evaluation of vancomycin aglycon analogues that bear modifications in the n-terminal d-leucyl amino acid click chemistry: , , -triazoles as pharmacophores superior inhibition of influenza virus hemagglutinin-mediated fusion by indole-substituted spirothiazolidinones influenza virus entry via the gm ganglioside-mediated platelet-derived growth factor receptor beta signalling pathway mutational analysis of the binding pockets of the diketo acid inhibitor l- , in the influenza virus pa endonuclease synthesis and anti-coronavirus activity of a series of -thia- -azaspiro we acknowledge kind assistance of l. persoons and her team, and of ria van berwaer and julie vandeput. the authors declare no conflict of interest. key: cord- -z l tsir authors: johnson, sonia; dalton-locke, christian; vera san juan, norha; foye, una; oram, sian; papamichail, alexandra; landau, sabine; rowan olive, rachel; jeynes, tamar; shah, prisha; sheridan rains, luke; lloyd-evans, brynmor; carr, sarah; killaspy, helen; gillard, steve; simpson, alan title: impact on mental health care and on mental health service users of the covid- pandemic: a mixed methods survey of uk mental health care staff date: - - journal: soc psychiatry psychiatr epidemiol doi: . /s - - - sha: doc_id: cord_uid: z l tsir purpose: the covid- pandemic has potential to disrupt and burden the mental health care system, and to magnify inequalities experienced by mental health service users. methods: we investigated staff reports regarding the impact of the covid- pandemic in its early weeks on mental health care and mental health service users in the uk using a mixed methods online survey. recruitment channels included professional associations and networks, charities, and social media. quantitative findings were reported with descriptive statistics, and content analysis conducted for qualitative data. results: , staff from a range of sectors, professions, and specialties participated. immediate infection control concerns were highly salient for inpatient staff, new ways of working for community staff. multiple rapid adaptations and innovations in response to the crisis were described, especially remote working. this was cautiously welcomed but found successful in only some clinical situations. staff had specific concerns about many groups of service users, including people whose conditions are exacerbated by pandemic anxieties and social disruptions; people experiencing loneliness, domestic abuse and family conflict; those unable to understand and follow social distancing requirements; and those who cannot engage with remote care. conclusion: this overview of staff concerns and experiences in the early covid- pandemic suggests directions for further research and service development: we suggest that how to combine infection control and a therapeutic environment in hospital, and how to achieve effective and targeted tele-health implementation in the community, should be priorities. the limitations of our convenience sample must be noted. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. been launched, there has been less focus on the needs of people already living with mental health conditions, and on how mental health services are supporting them at a time of potential staff shortages and service reconfigurations [ ] . potential risks to provision of mental health care worldwide include staff absences due to sickness and the need to self-isolate, and workforce redeployment, for example from community to inpatient settings. in the community, staff in many countries have been required to limit face-toface contacts to essential tasks such as the administration of injectable medication [ ] . beyond the immediate changes to services seen in the early stages of the pandemic, there are many potential challenges that are specific to mental health care. these include difficulties in implementing infection control and social distancing guidance in settings where people may be very distressed or cognitively impaired [ ] , especially in mental health wards and the supported accommodation settings where many people with complex mental health problems live [ ] . face-to-face meetings are usually central to mental health care: severe restrictions to this seem likely to greatly alter staff and service user experiences. there is also a considerable risk that, even after restrictions are lifted, there will be a lasting exacerbation of health and social inequalities that affect people with longer term mental health problems, for example, through increased economic disadvantage, inequalities in health care, or sequelae of increased trauma and abuse [ , ] . since the start of the pandemic, experts from around the world have published views about potential negative impacts of the pandemic on mental health services [ , , ] and the suggestion has also been recurrently made that it could provide an opportunity for positive service developments [ ] [ ] [ ] . however, there is a lack of research directly assessing and reporting the experiences and perspectives of those currently working in the mental health system. our aim was to inform further research and service responses by conducting, in the early stages of the covid- pandemic, a survey of the perspectives and experiences of staff working in inpatient and community settings across the uk health and social care sectors. the king's college london research ethics committee approved this study (mra- / - ) , which involved mental health staff in the uk completing an online questionnaire. in the absence of a measure of pandemic impact on mental health care and mental health service users, we rapidly developed an online questionnaire to collect cross-sectional quantitative and qualitative data from mental health care staff. all staff working in face-to-face mental health care in the uk, or managing those who provide such care, were eligible to participate. all specialties were included, as were nhs, private healthcare, social care, and voluntary sector services. the lead developer of the questionnaire, sj, an academic and practising inner london psychiatrist, read key sources identified in an accompanying rapid review of relevant literature [ ] , including academic and professional journals, news media, and organisational websites, and followed relevant social media topics. the drafting of the questionnaire was further informed by the nihr mental health policy research unit (pru) working group for this study (about people, including clinicians, researchers, and people with relevant lived experience), and the pru lived experience working group. both groups discussed the study at online meetings and identified important topics for inclusion. nine further clinicians provided email summaries of the challenges which they were currently facing and how they were being addressed. feedback was obtained from the pru working group on a first draft of the questionnaire, together with additional input from experts in fields including mental health care for older people, children and adolescents, people with drug and alcohol problems, offenders, and people with intellectual disabilities. the questionnaire was revised and converted into an online format using the ucl opinio platform. pilot testing was then conducted with clinicians, who provided feedback on length, acceptability, and relevance, and on problems with specific items. following this, a final version of the questionnaire was agreed. a mixture of structured and open-ended questions was included. participants were asked which sector and region they worked in but not which organisation, maximising anonymity. participants could skip questions if they wished, and internet cookies were used to prevent participants completing multiple questionnaires. a branching structure was adopted, with initial questions asking all participants to rate the relevance of each item on lists of: -challenges at work during the covid- pandemic. -problems currently faced by mental health service users and family carers (from a staff perspective). -sources of help at work in managing the impact of the pandemic. this was followed by sections for staff in specific settings and specialties. questions also elicited details of adaptations and innovations introduced to manage the impact of the pandemic, and their perceived success, and enquired about concerns for the future and any aspects of current practise that they would like to keep after the pandemic. participants were asked between and questions depending on their eligibility for branching questions for specific settings or specialties. depending on the detail provided to open-ended questions, the survey typically took - min to complete. a copy of the survey is available at this web address: https ://opini o.ucl.ac.uk/s?s= . our aim was to achieve rapid recruitment of a large and varied sample by dissemination through multiple channels including: in the final week of recruitment, we targeted under-represented sectors, including relevant voluntary sector organisations and supported housing providers. we also sought to increase representation of staff from black, asian, and minority ethnic groups by focused social media recruitment via the mental elf, including a video in which a prominent black psychiatrist encouraged participation, and contact with the networks of pru researchers who work on issues of diversity. quantitative data: we aimed to give an overview of the impact of the pandemic. we produced descriptive statistics using stata to summarise relevant aspects of the quantitative data. missing data are reported in the footnotes of the relevant tables in the supplementary report. qualitative data: qualitative analysis was conducted to expand on quantitative findings [ ] . a preliminary analytical coding framework was developed by sj guided by the study research questions, quantitative analysis results, and themes emerging from the initial survey responses. the responses to open-ended questions were left unedited and compiled under topics relevant to the research questions. coding matrices were developed in microsoft excel, with the emerging codes in the columns and cases in rows. directed descriptive content analysis was then conducted [ , ] . for this, all survey responses were indexed in the coding matrices by a group of researchers, mostly phd students or researchers with relevant lived experience. topics that came up repeatedly in the data and could not be categorised with the initial coding framework were given a new code. coding work was coordinated by so (associate professor) and nvsj, uf, and ap (post-doctoral researchers) to increase consistency and accuracy when applying the predetermined codes, and to discuss adding codes to the initial framework when necessary. sj and as (clinical professors) helped to understand clinical contexts and resolve coding difficulties. finally, the coding team developed summaries of each code and presented these in tables ranked in order of frequency, shown in the supplementary report. involvement of this large team allowed us to complete analysis within weeks. we summarise key findings here: our accompanying supplementary report gives much more detail. data were collected from april to may . in total, , people started the survey (including many who clicked 'start' but provided no or minimal data) and , got to the end. we report results for participants who completed at least one question from each of the three main sections open to all respondents. this produced a sample of , . there were , responses to open-ended items, yielding , words for rapid qualitative content analysis. a large majority of participants worked in the nhs ( , , . %). approximately a third described themselves as nurses ( , . %), as psychologists ( . %), as psychiatrists ( . %), as social workers ( . %), and as peer support workers ( . %). over a third identified as a manager or lead clinician in their service ( , . %). over two-thirds worked with working age adults ( , , . %), . % worked with older adults ( ), just under a third worked with people with learning disabilities ( , . %), around a fifth worked with people with drug and alcohol problems ( , . %), and another fifth worked with people with eating disorders ( , . %). participants could report working with multiple service user populations and/or in multiple settings. the majority worked in england ( , , . %) with around a third of these based in london ( , . %) and a fifth in the north west ( , . %); three-quarters worked in cities or towns with populations greater than , ( , , . %). four-fifths were female ( , , . %) and almost nine-tenths were from white ethnic groups ( , , . %). full demographic details, including age, caring responsibilities, and covid- status, can be found in table x of the supplementary report (references to tables in the supplementary report are herein indicated with an 'x' after the table number to distinguish them from tables in the main text). participants rated a list of current challenges at work, some general and others setting-specific, on a five-point scale from 'not relevant' to 'extremely relevant'. table shows the five work challenges rated highest in each type of setting; tables x- x report this in further detail. in inpatient wards and crisis houses, infection control challenges, related to table top five rated work challenges* for each setting (see tables x- x and x- x in the supplementary report for further details) * includes 'current work challenges' (c) asked of staff from all settings and 'additional work challenges' (a) that are specific to each service type ** a respondent may work in more than one setting (e.g., an inpatient service and a crisis assessment service), but will provide only one answer per challenge *** the 'additional work challenges' (a) sections, which are specific to specific settings and specialties, appear in the survey after the 'current work challenges' (c) section, which is open to staff from any setting. therefore, the reduced n for a challenges compared to c challenges represents respondents who completed the first sections of the survey, but then did not go on to complete the later branched sections of the survey both service users and staff becoming infected, were rated highest, alongside increased boredom and agitation amongst service users due to lack of activity and contact on the ward. crisis service staff rated as most relevant lack of services to which they could refer on or signpost. community team staff rated items related to changes in ways of working and adoption of remote technologies highest, along with reduced availability of other services. the small group of residential service participants gave a high relevance rating to their environment being more challenging, because residents cannot go out and/or engage in usual activities. table x shows ratings by profession and table x shows ratings by managerial roles. there were fewer obvious differences by profession than by setting, but managers and lead clinicians more often reported challenges relating to supporting colleagues with stressors due to the pandemic, and increased workload during the pandemic as very or extremely relevant ( . % and . %, respectively) compared to those not in these roles ( . % and . %, respectively). half of staff in inpatient and residential settings reported that they could not consistently follow the rules set on infection control ( , . %), and just over a third reported that they could not do this in community and other settings ( , . %). table shows the impediments to this most often identified from qualitative content analysis of responses, with more detail in tables x- x. tensions between meeting clinical needs and infection control were reported across settings, for example in responding to emergencies on wards or when service users in the community needed home visits, on which infection control measures were very difficult to implement. the built environment was the most frequently cited challenge in the community, and ward layouts impeded infection control in hospital. in each setting, there were also reports of conflicting or unclear guidance. reports of not having the facilities and processes to adhere to guidance, for example in putting on and disposing of personal protective equipment (ppe), were especially prominent in the community. unclear or conflicting guidance and procedures, and service users who are unable to understand and adhere to infection control rules, were reported across settings. substantial numbers were also concerned about perceived conflicts between protective equipment and therapeutic relationships, for example when trying to engage service users with paranoid ideas while wearing a mask. we also asked participants to report, if data were available to them, the extent of activity change in the service in which they worked (table x ). responses varied, but reports of reduced activity considerably exceeded those of increased activity, especially regarding inpatient admissions (though less so for compulsory admissions) and new referrals to crisis services and community services. however, in community services, including psychological treatment services, similar numbers of staff said that they were having more weekly contacts as said they were having fewer. table summarises staff perceptions of the current relevance of various types of difficulty for the service users and carers with whom they were in contact (table x reports this in greater detail and by service user group). across all groups, staff tended to rate social difficulties as most relevant, for example, loneliness and lack of usual support from table top five reasons infection control rules could not be followed for inpatient and community settings* (with frequencies), responses to an open-ended question (see tables x- x in the supplementary report for further details) * a respondent may work in more than one setting (e.g., an inpatient service and a crisis assessment service) ** includes staff working in inpatient services, crisis houses, and residential services *** includes staff working in crisis assessment services, community teams and psychological treatment services, community groups, and other settings inpatient and residential settings** community settings*** family and friends. several other types of problem were also rated by many staff as very or extremely relevant, including lack of normal support from mental health and other services, deterioration in mental health in the pandemic period, worries about infection, and being at high risk if infected. responding to open-ended questions, staff identified a range of groups of service users about whom they were particularly concerned, some because of impacts on their clinical condition, others because of their social characteristics or circumstances, or because of specific difficulties providing an adequate service for them. table summarises groups frequently identified as of particular concern, and table x gives more detail. we also asked staff whether they were seeing people with mental health difficulties that appeared to arise from the pandemic (table x) . some described symptoms directly related to covid- , such as delusional beliefs regarding covid- infection or quarantine, and health anxiety or obsessive-compulsive symptoms related to infection. others described relapses in people who had long been stable that they felt were linked to the stresses of the crisis. some also reported apparently first presentations of mental health problems such as psychosis or mania among healthcare workers. table summarises responses to a question about which sources of help were currently most important to staff in managing the impact of covid- at work. across all professions, the most important sources of help were support and advice from employers, colleagues, and managers, closely followed by new digital ways of working and the resilience and coping skills of service users and carers, the latter presumably seen as making the crisis less burdensome for staff, at least at its onset. patterns of response were not markedly different across professional groups (tables x- x). table summary of staff perspectives on which of their service users' and carers' problems are most relevant, in order of % rated very or extremely relevant (n = , ) (see table x participants in crisis and community services were asked whether services they worked in had changed opening hours or locations, and how their practices had changed (table x ). services that had increased their hours during the crisis, for example with weekend opening, were described, as well as reductions in other services. most staff working in crisis services reported that home visits were continuing when strictly necessary. a mixture of responses was obtained from community services (including both community mental health teams and psychological treatment services), with some reporting continuing face-to-face contacts and home visits as needed, others having stopped them. responses regarding psychological treatment were split between aiming to provide a full table frequently cited examples of the groups of service users about whom staff participants have been especially concerned during the pandemic: qualitative content analysis of open-ended responses (see table x in the supplementary report for further details) people who are cognitively impaired (e.g., due to dementia or learning disability), who may find situation hard to understand and struggle to follow guidance people with psychotic symptoms that may be exacerbated by current events and interfere with their ability to follow guidance people with complex emotional needs (who may have a "personality disorder" diagnosis), who may be destabilised by abrupt loss of support and routines; people with anxiety or ocd, especially those for whom covid- interacts with contamination-related symptoms women with perinatal mental health problems, lacking usual support and assessment around the time of birth people with drug and alcohol problems, for whom treatment and support are often severely disrupted and following guidance may be difficult people with eating disorders, at risk from disruption to usual eating, exercise, and social routines and to food access people of concern due to impacts related to social circumstances or characteristics people who live alone/are currently socially isolated and lonely older people with mental health problems, due to loss of usual support (e.g., family visits) and additional physical health vulnerability people who are in households where there is domestic violence or conflict children in homes that may not be safe or where there is family conflict people living in poverty/poor housing, or who are homeless, for whom the lockdown is especially difficulty people of particular concern due to service disruptions inpatients who have experienced service disruptions, including precipitate discharge, delayed discharge because of infection concerns, lack of leave or visits, and increased isolation and lack of activity or therapies on the wards people who are difficult to reach in the community without usual visiting/outreach/face-to-face appointments and may not be seeking help that is needed people at risk because of disrupted availability of medical responses, e.g., for people who harm themselves and are discouraged from visiting/ reluctant to visit emergency departments open-ended questions elicited adaptations and innovations made to manage the impact of the pandemic (table x ). the most widely reported shift was greatly increased adoption of remote technologies, as discussed below. some participants also reported adopting new digital tools for assessment and therapy, such as apps and websites. other innovations included new crisis services, such as crisis assessment centres rapidly established as alternatives to hospital emergency departments and new crisis phone lines, and re-organised services, resulting in extended hours, increased access for specific groups, or shorter waiting lists (e.g., for psychological treatment). reported changes in the types of help offered included community services arranging practical help, such as food deliveries for service users, and providing resource packs to help service users to be active at home. also frequently described were new or expanded forms of support for staff, including 'wobble' rooms (quiet rooms for staff who feel overwhelmed), staff helplines, increased supervision, wellness check-ins, and more use of informal support mechanisms. also reported was a general shift towards a more flexible approach, reducing bureaucracy and removing barriers to change, leading to a more agile way of working and a more responsive service. many staff also valued the many benefits to their well-being, productivity and efficiency in being able to conduct some of their client contact or administrative tasks away from the office. further quantitative and open-ended questions explored views and experiences of the shift to remote working (tables x- x) . almost all staff in community services ( , , . %) , and a large majority in crisis services ( , . %), were replacing some face-to-face contacts with phone or video calls. the shift to video calls did not appear to have been very extensive, however, with the majority ( , . %) reporting use of this technology as their main means of contact with % or fewer of the service users with whom they have contact. views about this were mixed. video calls for communication between staff attracted the greatest enthusiasm, with more than two-thirds ( , . %) from both community and crisis services agreeing or strongly agreeing that they are a good way to hold staff meetings; this was echoed in open-ended questions. a majority ( , . % of respondents to this question) agreed or strongly agreed that video calls were a good way to assess progress of some people already known to the service, but only . % ( ) agreed or strongly agreed that they can be a good way of making the initial assessments. responses to open-ended questions ( table , tables x- x) likewise identified concerns about being able to make a good assessment remotely, as well as about forming rapport: they tended to suggest digital technologies were useful for clients with less complex needs, for "light-touch" interventions or for low-intensity therapeutic approaches and follow-up appointments. a majority ( , . %) agreed or strongly agreed that use of remote rather tables x- x in the supplementary report for further details) what's working well in tele-health what can prevent tele-health from working allows prompt responses saves travelling time is better for the environment may be more convenient for both staff and service users allows staff to connect easily with each other, even if based in different places and different teams allows home working best alternative for now: remote working is allowing services to keep going despite infection control restrictions innovative use of it and digital tools can allow group programmes or individual therapies to continue successfully benefits for some clients: some clients are happy with video-call technology and even prefer it access is improved for some people, especially if travel and public places are challenging may be an efficient way of helping people with less complex needs inadequate resources: equipment and internet connections of low quality processes and preferred platforms not clearly established staff may lack training and confidence impacts on communication and therapeutic relationships may be harder to establish and maintain a good therapeutic relationship may be harder to make an assessment, especially at first contact may be challenging for longer, more in-depth sessions digital exclusion: people who lack equipment and resources to connect people who don't have skills or confidence to connect (including people with cognitive impairments) people lacking a suitably private environment for remote appointments service user preferences: some service users strongly prefer confidential conversations to be faceto-face, or may feel suspicious or anxious about remote means if they do accept remote contacts, some prefer simpler phone or messaging modalities some service users do not engage with remote contacts than face-to-face consultations had resulted in not having contact with some service users who had not engaged with remote appointments. two-thirds ( . %) answered yes when asked whether they wished to retain longer term any changes made during the pandemic. table x summarises responses. a large majority involved keeping some aspects of remote working, with many feeling that selective use of technology platforms to connect staff with each other and with service users has potential long-term benefits for efficiency and the environment, particularly if technical difficulties are resolved and appropriate protocols developed. others wished to retain some new service initiatives, such as crisis centres in the community, or the increased flexibility and ease of making changes experienced at this time. responses to a question about concerns for the future were numerous and detailed (table x) . while many participants reported that referrals to their service had decreased in the early phase of the pandemic, many feared that need would increase significantly in future and that lack of capacity and staff burnout may impede response to this. anticipated drivers of increased future need included traumas, bereavement, and complex grief experienced by frontline staff, service users, and the wider public; mental health problems not managed effectively among people who have disengaged or not sought help during the pandemic; increased levels of domestic abuse and family conflict; and the effects of wider societal disruption and increased inequalities due, for example, to unemployment and homelessness. fears were also expressed that reduced levels of service might persist inappropriately after the current emergency period, that changes made in response to the crisis might be used to justify reduced funding in future, or that staff would be expected to continue with working patterns that they had agreed to only because of the crisis. extension of remote working beyond the circumstances in which it had proved helpful was a further concern. several respondents were concerned about the disproportionate impact of the pandemic on black, asian, and minority ethnic staff and service users, and about potentially increased racism and xenophobia. a wide range of challenges are reported by practitioners across the mental health sector, some specific to service settings or groups of service users and carers. while many commentators have predicted a significant and widespread impact of covid- , we are able to provide a more detailed report that is rooted in direct experience of the effects of the pandemic on mental health care, albeit only in one country and only from the perspective of practitioners. in the context of the pandemic, infection control is an immediate need whose complexity in mental health settings is a significant finding from our study. lack of ppe was sometimes identified as a problem. more prominent, however, were challenges relating to processes, to the physical environment in which mental health care is delivered, and to tensions between infection control requirements and providing safe care and maintaining therapeutic relationships with people who may be distressed, suspicious, or struggling to comprehend the situation. inpatient and residential services, and crisis services, where continuing face-to-face contacts appear more frequent than in routine care, are not surprisingly the settings in which staff are most immediately concerned with the spread of infection: the price of failure is potentially very high, as indicated by a recent care quality commission report on excess deaths related to covid- among people subject to the mental health act [ ] . the shift to remote working, strikingly rapid given that tele-health has been discussed over many years but with limited implementation, has been widely discussed; we examine staff perspectives on this in detail in the current study. both our quantitative and qualitative data suggest clear support for its partial adoption in the longer term: remote contacts are seen as valuable for staff meetings, and for convenient and environmentally friendly follow-up of well-engaged clients with access to and a positive view of technology. however, staff give a very clear warning that there are still important technological, social, and procedural barriers to be addressed, and that its use should remain selective, complementing rather than replacing face-to-face contact. this and other innovations that we document above suggest that, as in other domains of healthcare, there has been considerable agility and flexibility in at least some service contexts during the current crisis, with urgent needs overcoming well-documented barriers to implementing new ways of working. however, while responses to our question about innovations that staff would like to retain were numerous, serious concerns regarding both the short and long-term future were also widely expressed: these data were collected at a very early stage in the covid- pandemic. mental health services in the uk were already under pressure prior to the pandemic [ ] and swift attention, strategic planning, and resources will be required to meet widely anticipated additional demands from people affected directly or indirectly by the impact of the pandemic. this is only one perspective on the impact of the pandemic on mental health care, albeit one rooted in direct experience: it will be essential to investigate service user and carer perspectives, and to measure impacts on the mental health system more systematically as further data become available. given the unprecedented pace of change in the world and in mental health services, we prioritised gaining a broad overview of impacts and responses, but much detail will have been missed. our questionnaire was by necessity an ad hoc and not an established and validated tool. omissions were noted as the study progressed: it was assumed that impacts of the "lockdown" for service users were negative, but positive experiences are noted too, for example of reduced pressure or easier access for people who struggle to travel [ ] . more importantly, we designed the questionnaire early in the pandemic when the evidence of differential effects on some ethnic groups was less striking [ ] : closed questions do not focus on this, although these effects and issues of racism are included in open-ended responses on concerns for the future. our sample, gathered by disseminating our questionnaire through a range of channels, is not representative of those who work in mental health care settings, and may either over-represent people who have strong concerns about the situation or those who wish to report successful new practices. we managed to include a range of professions and work settings, but did not recruit as successfully as we had hoped outside the nhs-more targeted efforts and time are likely to be needed to reach relevant staff from other sectors. many people with mental health difficulties also come into contact with gps, pharmacists, paramedics, and a&e doctors and nurses, especially if they are not under secondary services; we have not included these perspectives. we are especially concerned that, while we do not have any definitive overall figure for the uk mental health care workforce, it is clear that the number of non-white participants in our survey is relatively small, despite targeted efforts to increase their number and a strong emphasis on anonymity and confidentiality, as advised in the previous discussions of this frequently experienced difficulty [ ] . further efforts to engage and form partnerships are likely to be needed here too. london also appears over-represented and rural areas, which may have distinctive challenges, under-represented, and we have not at this stage disaggregated data by country, region, or area type. we present here a series of snapshots capturing, from a staff perspective, the situation in mental health care services in the rapidly evolving early stages of the covid- pandemic. this work cannot yield definitive answers and should be interpreted alongside other perspectives, but offers researchers, service commissioners, managers, and policy makers directions for service development and further rapid research. regarding immediate priorities, our findings point to specific challenges to be addressed to achieve more successful infection control. remote working is a further immediate focus for research and service developments. participants' accounts suggest that it has been helpful in keeping services going and maintaining some level of contact in the community, and aids communication between staff. there is now a need to develop clearer processes in collaboration with service users for its targeted use, to implement guidance and evidence that already exists [ ] , and to explore ways of overcoming barriers to its effective use. mental health providers in the uk and elsewhere have demonstrated unprecedented capacity for rapid adaptation and innovation during the early pandemic period. recovery from the pandemic is a potential opportunity to establish new ways of working, for example with greater co-production with service users, and more widespread implementation of effective interventions and technologies [ ] . this will require sufficient resources, rapid production and translation of evidence, effective planning that engages all stakeholders, and great attention to workforce support and prevention of burnout. it is reassuring to see that staff share many of our concerns about the covid- pandemic: premature discharges, isolation, difficulties with infection control, and accessing care. many of these are reflected in the madcovid project's materials (https ://madco vid.com/). telemedicine drew mixed views from staff; we would like to highlight some difficulties. not everyone has a safe space to speak, may only have privacy in their bedroom or none at all. telemedicine works better for those in better, not-overcrowded housing, so risks widening inequalities in access to care. for many of us, our home is our safety, and it is important to have distressing conversations elsewhere. leaving the therapy room, we can leave some of our trauma behind. video calls may feel invasive-as though the clinician is in your bedroom-bringing up traumatic issues inside the home, where we cannot escape them. any continuation of remote working will need to consider the safety implications of this, assessing its suitability for each individual. it is vital that difficulty adhering to infection control guidance does not lead to blaming inpatients for viral spread. this is particularly important with restraint, where staff mentioned struggling to put on appropriate ppe in time to deal with an unfolding emergency. wide area variations in restraint rates (https ://www.mind.org.uk/media -a/ /physi cal_restr aint_final _web_versi on.pdf [ ] ; https ://weare agend a.org/ wp-conte nt/uploa ds/ / /restr aint-foi-resea rch-brief ing-final .pdf [ ] ), alongside personal experience, make us question whether restraint is ever truly unavoidable. if it places both staff and service users at risk of covid- infection, it is doubly dangerous. however challenging the situation, efforts must be renewed to reduce the iatrogenic distress, fear, and anger which can lead to its use. historically slow-moving services have implemented change at breakneck speeds in response to this crisis despite significant difficulties. service users have campaigned for changes for decades. it is time to implement these changes with the same urgency. the survey dataset is currently being used for additional research by the author research group and is, therefore, not currently available in a data repository. a copy of the survey is available at this web address: https ://opini o.ucl.ac.uk/s?s= . conflicts of interest sj, as, ble, so, and sc are grant holders for the nihr mental health policy research unit. ethics approval the king's college london research ethics committee approved this study (mra- / - ). consent to participate information on participation was provided on the front page of the survey. by starting the survey, participants agreed that they had read and understood all this information. it was explained on the front page of the survey that responses may be used in articles published in scientific journals and that these articles will not include any information which could be used to identify any participant. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. 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consequence of the covid- pandemic physical restraint in crisis: a report on physical restraint in hospital settings in england briefing on the use of restraint against women and girls key: cord- - cml shh authors: dopfer, christian; wetzke, martin; zychlinsky scharff, anna; mueller, frank; dressler, frank; baumann, ulrich; sasse, michael; hansen, gesine; jablonka, alexandra; happle, christine title: covid- related reduction in pediatric emergency healthcare utilization – a concerning trend date: - - journal: bmc pediatr doi: . /s - - - sha: doc_id: cord_uid: cml shh background: the covid- pandemic has disrupted healthcare systems worldwide. in addition to the direct impact of the virus on patient morbidity and mortality, the effect of lockdown strategies on health and healthcare utilization have become apparent. little is known on the effect of the pandemic on pediatric and adolescent medicine. we examined the impact of the pandemic on pediatric emergency healthcare utilization. methods: we conducted a monocentric, retrospective analysis of n = , pediatric emergency department visits between january st and april th of and , and compared healthcare utilization during the pandemic in to the same period in . results: in the four weeks after lockdown in germany began, we observed a massive drop of . % in pediatric emergency healthcare utilization (mean daily visits . ± sem . in vs. . ± sem in , p < . ). this drop in cases occurred for both communicable and non-communicable diseases. a larger proportion of patients under one year old (daily mean of . % ±sem . in vs. . % ±sem . in , p < . ) and of cases requiring hospitalisation (mean of . % ±sem . in vs. . % ±sem . in , p < . ) occurred during the pandemic. during the analysed time periods, few intensive care admissions and no fatalities occurred. conclusions: our data illustrate a significant decrease in pediatric emergency department visits during the covid- pandemic. public outreach is needed to encourage parents and guardians to seek medical attention for pediatric emergencies in spite of the pandemic. the novel severe acute respiratory syndrome coronavirus (sars-cov- ) causing coronavirus disease first emerged in wuhan, china at the end of [ ] . despite efforts to contain the virus, covid- cases emerged all over the world, and the who declared a public health emergency of international concern on january th. on march th , the sars-cov- outbreak was officially designated a pandemic [ ] . globally, nations responded to this pandemic with lockdown strategies including social distancing, school closures and shelter-in-place orders for non-essential workers. at the same time, healthcare systems in europe and elsewhere prepared for an unprecedented health emergency by reallocating resources to care for covid- patients [ ] [ ] [ ] [ ] . public awareness of the pandemic was associated with a vast reduction in healthcare utilization in italy and other countries [ ] [ ] [ ] [ ] . as a result, increased adult and pediatric morbidity and mortality due to delayed healthcare utilization were reported [ , ] . initially, germany took a moderate approach, appealing to the responsibility and solidarity of its citizens. however, school closures were implemented beginning on march th, and an official lockdown of public life, including mandatory closure of all non-essential retail businesses, was implemented starting on march rd . severe cases of covid- remained comparatively low in germany (n = , confirmed sars-cov- infections with . fatalities as of may th [ ] ), and the healthcare system has thus far been spared an untenable influx of critically ill patients. in this setting, we investigated pediatric emergency healthcare utilization in our tertiary care center in hanover, germany. we compare the rate and type of emergency department (ed) visits during lockdown in march and april of to the equivalent period in the spring of . we analysed data from n = , healthcare visits occurring between january st and april th and january st and april th in the pediatric ed of hanover medical school in hanover, lower saxony. we also compared healthcare utilization between calendar weeks - (march th to april th ) in and calendar weeks - (march th to april th ) in . the ed is open to patients h a day, seven days a week, with full-time coverage by a designated nurse and pediatric physician. during daytime hours, from am to pm monday through thursday and am to : pm on fridays, a designated pediatric ed physician provides care. patients presenting to the ed outside these times are seen by pediatric house staff and/or an oncall pediatric hospitalist. all medical and demographic data were extracted from routine electronic clinical records. every onsite healthcare visit was documented in an electronical case reporting system, including the patient's age and sex, date and time of visit, complaints, and suspected or confirmed diagnoses, as well as whether hospitalization was required. in our hospital, standard practice dictates that the diagnosis or primary complaint of each ed patient is recorded during the visit. diagnoses are coded according to the international statistical classification of diseases and related health problems system (icd)- -gm- by trained coding personnel. coding personnel receive intensive training in icd coding, and is specialized and experienced in this field. coding is continuously supervised, monitored, and reviewed for interrater agreement. interdisciplinary meetings between medical and coding personnel to discuss unclear cases are held regularly. as analyses for this paper were retrospective, coding was blinded to the study objectives. all consultations between march th ( : am) and april th ( : pm) and from march th ( : am) to april th ( : pm) were included; no cases were excluded. all data was extracted in depersonalized form. icd diagnoses were grouped into categories that were defined as follows: the category "communicable diseases" was defined as either diagnoses classified in group "a/b" of the (icd)- -gm- coding system (certain infectious and parasitic diseases) or as organ system-specific reporting of an infectious disease (e.g. for airway infections, diagnoses such as cold (j ) or unspecified type of upper airway infection (j . ) were included). for analyses of suspected infectious diseases, the coding of the following signs and symptoms were included: fever, cough, stridor, dyspnoea, vomiting, and diarrhoea. all other diagnoses or complaints were defined as "noncommunicable diseases". for the illustrations in figs. and , patients with signs, symptoms, and findings from icd- groups with low prevalence or from the category "r" (symptoms, signs and abnormal clinical and laboratory findings, not elsewhere classified), or those with unknown complaints were grouped into the category "others". spss version . and graphpad prism version . were used. normal distribution of all variables was assessed, and depending on data distribution, group differences were evaluated by student´s t or mann whitney u testing. p values below . were considered significant. the analyses were approved by the data security management and institutional review board of hannover medical school. in total, n = , visits occurred during the analysed period between january st and april th and january st and april th of in the pediatric ed of our hospital. the mean age of all patients included in this analysis was . years (± sem . yrs.), and . % of cases were female. given that primary pediatric care in germany is usually provided by pediatric practices during weekdays and lockdown did not include closure of pediatric practices, in both observational periods more visits to the ed occurred during weekends. overall, a mean of . ± sem . visits per day on saturday/ sunday as compared to . ± sem . per weekday occurred. when we compared daily visit frequencies between and , we observed a striking reduction in the number of patients presenting to the ed after march th , the day that school closures and a nationwide lockdown began (fig. a) . in calendar weeks to of , case numbers decreased by . % compared to the same time period in (table ; fig. b ). this reduction in emergency healthcare utilization was observed during daytime as well as overnight (table ; fig. c , d). while the mean age of patients did not differ significantly between calendar weeks to in each year, a (table ; fig. e ). although most shifts in daily healthcare utilization were small in absolute numbers (e.g. a mean of one less intoxication case per day in vs. ), most differences were highly significant when examining the entire observational period (table ) . no significant differences in the daily proportion of school children below the age of six years between the two analysed periods was observed, nor did we find a difference in sex distribution (table ) . next, we analysed the complaints or diagnoses recorded in the ed in both years. diagnoses of primary complaints were reported in n = , visits ( %). a clear drop in the frequency of daily visits for noncommunicable diseases and for confirmed infections or signs thereof ( fig. a/ this was confirmed by a separate analysis comparing the frequency of daily visits for noncommunicable diseases in calendar weeks to of to the same period in . here, a significant decrease ( %) ( table ; fig. c ) was observed. daily visits for suspected or confirmed communicable diseases displayed a significant decrease ( . %) in the four weeks after lockdown began (table ; fig. c ). the proportion of daily patients with infectious diseases or signs and symptoms of infection, however, did not differ significantly between calendar weeks to from one year to another (table ; fig. e ). we also investigated the specific complaints leading to pediatric ed visits in the calendar weeks to in and . in both years, a proportion of diagnoses was either unknown or unspecific (in total . % of diagnoses in and . % of diagnoses in ). for reported diagnoses, the pattern of distribution was similar in both years. this held true even for critically ill patients. for example, during the four week period we analysed, we observed three seizures in , and two seizures in . as shown in fig. a , however, some variation was observed, such as a larger proportion of patients presenting with malignant or neoplastic diseases in ( . % vs. . % in ). this change was confirmed by comparing absolute patients numbers (fig. b ). the only disease category with increased daily er visits after the lockdown began was that of malignant/neoplastic disease (table ; fig. ). for the vast majority of noncommunicable diseases, daily visits were reduced (fig. b) . for example, complaints or diagnoses affecting the gastrointestinal system showed a significant fig. ) . similarly, the proportion of organ-specific infectious diseases or signs thereof was analysed for the two time periods. as shown in fig. a , the majority of daily diagnosed infections or typical complaints thereof concerned the respiratory or gastrointestinal tracts. in absolute numbers, presentations due to organ-specific infectious disease decreased significantly during calendar weeks to of as compared to the same period in (table ; fig. b ). respiratory infections or signs thereof were reduced significantly by %, ear and throat infections or signs showed a significant reduction of . %, and gastrointestinal infections or signs thereof were also reduced significantly by % (table ; fig. b ). finally, we analysed the rates of hospitalisation in the first month after pandemic-related school closures in as compared to the same period of time the previous year. although the absolute numbers of daily hospitalisations of pediatric ed patients dropped significantly ( . % decrease; table ; fig. a ), the proportion of hospitalisations among all patients presenting to the ed almost doubled in compared to (table ; fig. b ). the duration of hospitalisation, however, was not different, neither overall (table ; fig. c ) nor for particular subgroups such as patients below the age of one year, those with infectious diseases, or those requiring intensive care. fig. ed cases of patients presenting with noncommunicable disease as well as those with infectious disease or signs thereof were significantly reduced during the covid- pandemic. a: daily number of visits between january st and april th (blue) vs. (red) by patients with noncommunicable diseases (a) and infectious diseases or signs thereof (b). c/d: reduced visits per day due to noncommunicable (c) or infectious diseases or typical signs thereof in calendar weeks to . e: no change in the proportion of daily visits due to infectious diseases or typical signs thereof (bars display mean + sem with overlaying dots representing single daily values (c-e), *** p ≤ . ) we observed a similar pattern of diagnoses leading to hospitalisation from the ed in both years. infectious disease, perinatal pathologies, injuries, gastrointestinal issues and neoplastic disease belonged to the most common indications for admission. the largest changes in absolute patient numbers were observed in patients with neoplastic diseases ( . % in vs. . % in ), those with pathologies of the perinatal period ( . % in vs. . % in ) and those with diseases of the heart and circulatory system ( . % in vs. % in ). during calendar weeks to in , two patients presenting to the ed were admitted to our hospital´s pediatric intensive care unit (picu). no fatalities occurred amongst these patients. in the same four-week period in , two patients were admitted from the ed directly to the picu, and no deaths were observed. only one case of pediatric covid- was diagnosed during the analysed period. our data demonstrate a significant decrease in pediatric ed visits during the covid- lockdown in germany. this finding is consistent with other reports on the substantial reduction in healthcare utilization in europe and elsewhere during the current crisis [ , ] . although a pandemic-related reduction in primary healthcare utilization for critically ill patients has been reported [ , , ] , little is known on the changes in pediatric emergency medicine thus far. lazzerini et al. recently reported twelve pediatric cases of delayed treatment due to parental concern of nosocomial covid- infection from a healthcare facility [ ] . tragically, half the patients in this study were admitted to a picu and four of them died. we do not know whether the reduction in healthcare utilization in our ed in occurred due to lower morbidity or avoidance of hospital visits associated with the current pandemic. although we can only speculate on the causes behind this dramatic reduction in case numbers, the following reasons appear to be most relevant. firstly, a massive flow of alarming medical information has resulted in high levels of uncertainty and fear across europe and elsewhere [ ] . although children are less prone to severe courses of covid- [ ] , the fear of contracting sars-cov- while visiting a doctor´s office or hospital may have affected parental decision-making during the early phase of this unprecedented health emergency [ ] . in this context, effective public health communication is a key factor, not only in fighting the spread of covid- , but in preventing healthcare avoidance for those in need [ , ] . currently, limited scientific understanding of the virus and its spread impedes evidence-based guidance for the public, which poses a significant challenge to health communicators worldwide [ ] . however, pediatricians should emphasize to parents and young patients that the risks of avoiding hospital care in emergencies may exceed that of contracting sars-cov- . the fear of infection may not be entirely irrational in times of reported patient overflow, overcrowded waiting areas, and insufficient personal protective equipment for medical staff [ , ] . but in germany, where the healthcare system thus far has not been overwhelmed by covid- , the risk of nosocomial infection within a hospital setting appears, at least for now, to be low [ ] . during the period of time analysed in this study, in spite of extensive testing, only one child tested positive for sars-cov- in our pediatric emergency department. the second likely reason for the reduction in ed visits, in particular for infectious diseases, lies in the lockdown strategies themselves. even before governmental enforcement, many families practised rigorous social distancing. this measure, together with school closures, cancelation of public gatherings and shelter-in-place orders, led to a dramatic decrease in the spread of communicable disease. for example, the influenza season in germany came to a sudden and almost complete halt by calendar week of [ ] . this may also affect overall morbidity and mortality in children. while the publication from italy reporting on pediatric deaths associated with parental fear of seeking emergency healthcare is alarming [ ] , overall pediatric mortality in europe appears have dropped during the covid- pandemic [ ] . another possibility is that, out of respect for the limited resources of the medical workforce during the pandemic, parents may have been inclined to "watch and wait" when their children fell ill, as opposed to immediately presenting to the ed. the increased proportion of infants and hospital admissions from the ed in calendar weeks to of suggests that children at particular risk for severe disease were brought to the ed despite the pandemic, but that those with non-severe illnesses were presented less frequently. fortunately, no fatalities were observed in our analysis. although an increased rate of daily hospital admissions occurred, we found comparable durations of hospital stay and numbers of icu admission between and . this supports the idea that, despite the pandemic, critically ill patients presented to our ed in time. the overall low case numbers of severely ill children must to be taken into account when interpreting our data. although significant, many shifts in daily emergency care utilization such as the reduction in cases of intoxication or diagnoses affecting the digestive system were small in absolute numbers and should not be overinterpreted. the incidence of picu admissions, as well as of specific severe disease types such as seizures or neonatal sepsis, were low in both time periods studied, prohibiting far-reaching conclusions. the northern german pediatric intensive care network "pin", which is headquartered in hanover and serves as a reference centre for more than pediatric hospitals in northern germany, also reported no change in the number or severity of picu admissions in calendar week - in as compared to . another limitation of our work lies in the fact that, due to the nature of our ed documentation system, in many instances only the primary complaint was recorded, rather than a concrete diagnosis. emergency medicine chart reviews, by nature, are flawed, because the underlying data was not collected for scientific purposes [ ] . furthermore, only the acute reason for presentation was analysed, which does not account for the many patients at our university hospital who suffer from complex chronic conditions. we do not know why more children with neoplastic diseases presented to the ed after lockdown as compared to the same period in time in ; and further analyses are needed to address healthcare utilization and complaints in specific subgroups such as chronically ill children during the current crisis. thus far, germany has taken a moderate approach to lockdown strategies with a combination of legal restrictions to shut down public life and an appeal to the responsibility and solidarity of its citizens. the healthcare system, particularly in northern germany, was in the fortunate situation of having weeks to adapt and prepare for this unprecedented situation and has been able to provide adequate care to covid- patients [ , ] . in spite of the low prevalence of covid- in our region, our data shows the profound impact of the pandemic on pediatric emergency health care utilization. the current pandemic puts pressure on healthcare systems around the globe. our data clearly indicate that the covid- outbreak is associated with a significant reduction in pediatric emergency healthcare utilization. although we did not observe any critical cases due to delayed hospital admission in our analysis, the risk of avoidance or delay of healthcare provision to critically ill children and adolescents may only become apparent in the months and years to come. germany is still in the fortunate position of having sufficient resources for patient care, but our data suggest that the current pandemic results in a dramatic reduction in the use of emergency medical care for children in our system, presumably based on fear of hospital acquired infections or out of respect for the limited resources of the healthcare system. this may affect resource-limited healthcare systems that are struck by covid- to greater extent -as the fear of infection or the ensuing chaos could be more pronounced in these regions. it is important to note that the phenomenon of reduced pediatric healthcare utilization may extend beyond emergency medicine to insufficient healthcare provision and preventive medicine for children in general. as such, public health communication should aim at minimizing possible pediatric "collateral damage" caused by delayed emergency healthcare utilization during the covid- pandemic. we hope our analysis helps to adapt healthcare and communication strategies accordingly. a pneumonia outbreak associated with a new coronavirus of probable bat origin risk communication during covid- fast reshaping of intensive care unit facilities in a large metropolitan hospital in milan, italy: facing the covid- pandemic emergency hospital surge 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endangering health workers worldwide successful containment of covid- outbreak in a large maternity and perinatal center while continuing clinical service european mortality monitoring activity -euromomo how emergency departments prepare for virus disease outbreaks like covid- publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank all doctors and medical personnel involved in the care of covid- patients for their exceptional work. the dataset necessary to interpret, replicate and build upon the findings reported in the article will be made available on reasonable request and can be obtained by contacting the corresponding author.ethics approval and consent to participate the analyses were approved by the data security management and institutional review board of hannover medical school (approval # _bo_k_ ). this manuscript does not contain any individual person's data in any form (not applicable). the authors have no competing interests to report. key: cord- -imc woht authors: schulte-schrepping, jonas; reusch, nico; paclik, daniela; baßler, kevin; schlickeiser, stephan; zhang, bowen; krämer, benjamin; krammer, tobias; brumhard, sophia; bonaguro, lorenzo; de domenico, elena; wendisch, daniel; grasshoff, martin; kapellos, theodore s.; beckstette, michael; pecht, tal; saglam, adem; dietrich, oliver; mei, henrik e.; schulz, axel r.; conrad, claudia; kunkel, désirée; vafadarnejad, ehsan; xu, cheng-jian; horne, arik; herbert, miriam; drews, anna; thibeault, charlotte; pfeiffer, moritz; hippenstiel, stefan; hocke, andreas; müller-redetzky, holger; heim, katrin-moira; machleidt, felix; uhrig, alexander; bosquillon de jarcy, laure; jürgens, linda; stegemann, miriam; glösenkamp, christoph r.; volk, hans-dieter; goffinet, christine; landthaler, markus; wyler, emanuel; georg, philipp; schneider, maria; dang-heine, chantip; neuwinger, nick; kappert, kai; tauber, rudolf; corman, victor; raabe, jan; kaiser, kim melanie; vinh, michael to; rieke, gereon; meisel, christian; ulas, thomas; becker, matthias; geffers, robert; witzenrath, martin; drosten, christian; suttorp, norbert; von kalle, christof; kurth, florian; händler, kristian; schultze, joachim l.; aschenbrenner, anna c.; li, yang; nattermann, jacob; sawitzki, birgit; saliba, antoine-emmanuel; sander, leif erik title: severe covid- is marked by a dysregulated myeloid cell compartment date: - - journal: cell doi: . /j.cell. . . sha: doc_id: cord_uid: imc woht summary coronavirus disease (covid- ) is a mild to moderate respiratory tract infection, however, a subset of patients progresses to severe disease and respiratory failure. the mechanism of protective immunity in mild forms and the pathogenesis of severe covid- , associated with increased neutrophil counts and dysregulated immune responses, remains unclear. in a dual-center, two-cohort study, we combined single-cell rna-sequencing and single-cell proteomics of whole blood and peripheral blood mononuclear cells to determine changes in immune cell composition and activation in mild vs. severe covid- ( samples from individuals) over time. hla-drhicd chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild covid- . severe covid- was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and hla-drlo monocytes. our study provides detailed insights into the systemic immune response to sars-cov- infection and it reveals profound alterations in the myeloid cell compartment associated with severe covid- . immune responses in blood samples in two independent cohorts of covid- patients. activated hla-dr hi cd c hi cd + monocytes were increased in patients with mild covid- , similar to patients with sars-cov- negative flu-like illness ('fli'). in contrast, monocytes characterized by low expression of hla-dr, and marker genes indicative of anti- inflammatory functions (e.g. cd , plac ) appeared in patients with severe covid- . the granulocyte compartment was profoundly altered in severe covid- , marked by the appearance of neutrophil precursors due to emergency myelopoiesis, dysfunctional neutrophils expressing pd-l , and exhibiting an impaired oxidative burst response. collectively, our study links highly dysregulated myeloid cell responses to severe j o u r n a l p r e -p r o o f results dual center cohort study to assess immunological alterations in covid- patients in order to probe the divergent immune responses in mild vs. severe covid- , we analyzed blood samples collected from independent patient cohorts at two university medical centers in germany. samples from the berlin cohort (cohort ) (kurth et al., ) , were analyzed by mass cytometry (cytof) and single-cell rna-sequencing (scrna-seq) using a droplet-based single-cell platform ( x chromium), while samples from the bonn cohort (cohort ) were analyzed by multi-color flow cytometry (mcfc) and on a microwell-based scrna-seq system (bd rhapsody). we analyzed a total of million cells by their protein markers and > , cells by scrna-seq in samples from covid- patients and controls, including patients with fli ( fig. a+b , s a, table s ). we first characterized alterations of the major leukocyte lineages by mass cytometry on whole blood samples from covid- patients collected between day and day after symptom onset, and compared them to age-and gender-matched controls and fli patients. we designed two antibody panels to specifically capture alterations in mononuclear leukocytes (lymphocytes, monocytes and dcs, panel ), and in granulocytes (panel , table s ). high-resolution spade analysis was performed with target nodes and individual nodes were aggregated into cell subsets based on lineage-specific markers, such as cd for monocytes and cd for neutrophils (fig. s b) . uniform manifold approximation and projection (umap) analysis revealed distinct clustering of samples from covid- patients, fli, and healthy controls, with marked changes of the monocyte and granulocyte compartment (fig. c) . leukocyte lineages were compared in the earliest available samples in covid- patients (day to ), fli, and controls (table s , fig. d ). since leukocyte counts were not available for all control samples, we compared the control samples for cytof ('ctrl cytof') to data from our recently published healthy control cohorts ('ctrl flow') (kverneland et al., ; sawitzki et al., ) . the proportions of all major lineages were highly similar, irrespective of the methodology (fig. d) . cell counts of the published cohort could therefore be used as a reference to report absolute cell counts for leukocyte lineages in covid- samples. in line with recent reports xintian et al., ) , we observed elevated leukocytes and increased proportions of neutrophils in patients with severe covid- ( fig. d) , whereas only proportional increases in neutrophils were evident in fli and mild covid- patients (fig. d) . total lymphocytes and t cells were strongly reduced in all covid- and fli patients, whereas non-classical monocytes were specifically depleted in covid- (fig. d) . increased neutrophils in severe covid- and loss of non-classical monocytes in both mild and severe disease, were validated in cohort by mcfc (fig. s c, table s + ). given the dramatic changes in various immune cell populations (fig. c+d) , we next assessed their composition and activation state by droplet-based scrna-seq in samples from covid- patients ( mild & severe, cohort , table s ) collected between day and day after symptom onset. a total of , single-cell transcriptomes of pbmc were analyzed together with , pbmc from publicly available control datasets ( control donors, table s ). umap and high-resolution cell type classification identified all cell types expected in the mononuclear compartment of blood with a high granularity in the monocytes, identifying five distinct clusters (cluster - ) (fig. a+s a, table s ). monocytes in clusters - expressed cd , cluster comprised the non-classical monocytes marked by fcgr a (encoding cd a) and low expression of cd . separate visualization of cells in mild and severe cases revealed highly disease severity-specific clusters (fig. b) . a distinct subset of cd + monocytes (cluster )( fig. a ) marked by high expression of hla-dra, hla-drb and co-stimulatory molecule cd (fig. s d) , engagement of which has been linked to prolonged expansion of antigen-specific t cells (hirano et al., ) , was selectively detected in mild covid- (fig. c ). in addition, we identified another closely related cd + hla-dr hi monocyte population (cluster ), which was characterized by high expression of ifn-stimulated genes (isgs). however, upon closer analysis, this cluster was found to originate from a single donor with mild covid- (fig . a-c, fig. s d ). both cluster and cluster expressed high levels of isgs ifi and isg (fig. s d) . in patients with severe covid- , monocytes showed low expression of dr and high expression of alarmins s a / / (cluster , fig. a-c, fig. s d ). the most prominent change in severe covid- was the appearance of two distinct cell populations (cluster + ), absent in pbmc of patients with mild covid- and control donors ( fig. a) . published markers (kwok et al., ; ng et al., ) identified cluster and as neutrophils and immature neutrophils, respectively (fig. a+b) . immature neutrophils (cluster ) expressed cd , pglyrp , defa and defa , whereas neutrophil cluster expressed fcgr b (cd b), cxcl , and lcn (lipocalin ) (fig. c, fig. s a ). their migration within the pbmc fraction on a density gradient marked these cells as low-density neutrophils (ldn). in the second cohort, pbmc from covid- patients ( mild, severe, table s ), sampled between and days after symptom onset, and controls, were collected for scrna-seq on a microwell-based platform (bd rhapsody). high-quality single-cell transcriptomes for , pbmc were assessed and their population structure was visualized using umap (fig. d, table s ). data-driven cell type classification (aran et al., ) and cluster-specific marker gene expression identified all cell types expected in the pbmc compartment and revealed additional clusters and substructures (fig. d+s b) . similar to cohort , monocytes exhibited significant plasticity and were subclassified into clusters (fig. d , clusters - ). disease severity-associated changes seen in cohort were validated in cohort (fig. e) . immature and mature neutrophil clusters were detected in both cohorts (clusters - ) and showed near identical marker gene expression (fig. c) . similar to cohort , a prominent shift in subpopulation occupancy was observed in the monocyte clusters (fig. d+e) . based on the union of the top genes for monocyte and neutrophil clusters, we found a high correlation between the independently defined functional states within the monocyte j o u r n a l p r e -p r o o f compartment, and mature and immature neutrophils in cohort and cohort (fig. s c) . violin plot representation of important marker genes illustrated distinct phenotypic states and underscored the high similarity of the two cohorts (fig. s d) . disease-severity dependent alterations of the monocyte compartment and the appearance of two ldn populations were detected in two cohorts of covid- patients. predominance of hla-dr hi cd c hi inflammatory monocytes in mild and hla- dr lo cd c lo cd + cd + monocytes in severe covid- the monocyte compartment is particularly affected by covid- , indicated by a loss of cd lo cd hi non-classical monocytes (fig. c+d) . disease severity-dependent shifts in monocyte activation were identified by scrna-seq (fig. ) . we further explored the phenotypic alterations of the monocyte compartment using mass cytometry ( fig. a,c,d) . increased levels of activated hla-dr hi cd c hi monocytes in mild covid- patients was confirmed by mcfc in cohort (fig. e) . in severe covid- , we detected increased expression of cd and cd (cluster ) and/or decreased expression of hla- dr, and total cd + cd + monocytes were elevated compared to controls. cluster expressed high levels of cd , which is induced during macrophage differentiation (huang et al., b). thus, an alternative activation pattern of classical monocytes appeared to be covid- specific and was associated with severe disease. besides activated lymphocytes, also monocytes upregulate cd expression (davison et al., ) , which promotes tissue infiltration and retention (cibrián and sánchez-madrid, ) . similarly, cd expression on alternatively activated monocytes might also promote diapedesis and tissue infiltration j o u r n a l p r e -p r o o f (reymond et al., ) . together, this activation pattern may contribute to the reduction of circulating monocytes in covid- . hla-dr lo monocytes persist in severe covid- next, we dissected covid- associated phenotypic alterations of monocytes by scrna- seq. marker genes of the monocyte clusters derived from fig. a showed that mild covid- associated clusters and were characterized by an isg-driven transcriptional program (fig. s a) , and gene ontology enrichment analysis (goea) assigned these clusters to 'type i interferon signaling pathway' (fig. s b) . a monocyte cluster marked by low expression of hla-dr and high expression of s a and cxcl (cluster , hla-dr lo s a hi ) was strongly associated with severe covid- (fig. s a, b, s d) . for further in-depth analysis, we subclustered the monocyte compartment of the pbmc dataset of cohort ( fig. d, s c, table s ) resulting in subclusters (fig. a) . cluster was marked by high expression of hla-dra and hla-drb and co-stimulatory molecule cd and was therefore designated hla-dr hi cd hi activated inflammatory monocytes (fig. a+b , s d+e). we identified two major clusters ( , ) and a smaller cluster with low hla-dr expression, which were associated with severe covid- (fig. b, s d+e) . low hla-dr expression is an established surrogate marker of monocyte dysfunction (venet et al., ) which results in reduced responsiveness to microbial stimuli (veglia et al., ) , suggesting that cluster and are composed of dysfunctional monocytes. genes of the s a family were expressed in both hla-dr lo clusters (fig. b ), albeit to a higher degree in cluster (hla-dr lo s a hi , e.g. s a , fig. s d , s e, well as pre-maturation markers like mpo and plac (fig. b) , recently linked to immature monocyte states in sepsis patients (reyes et al., ) . in line with these findings, clusters , and were significantly enriched in a gene signature derived from sepsis-associated monocytes ( fig. c ) (reyes et al., ) . moreover, blood monocytes isolated from covid- patients showed a blunted cytokine response to lps stimulation, particularly monocytes from patients with severe covid- (fig. d) . accordingly, hla-dr lo monocyte clusters ( , , ) were detected almost exclusively in severe covid- (fig. e) . we next analyzed time-dependent cluster occupancies per patient in cohort (fig. e+f) . activated hla- dr hi cd hi monocytes (cluster ) were found in all cases of mild covid- , even at late time points (fig. e+f) . in contrast, hla-dr lo cd hi monocytes (cluster ) were present mainly early in severe disease, while hla-dr lo s a hi monocytes (cluster ) dominated the late phase of disease (fig. e+f) . violin plots of isg (fig. s d ) and visualization of marker genes ( fig. s e) indicated differential expression patterns of ifn signature genes in individual monocyte clusters. to reveal the kinetics of isg expression, we plotted the expression of isg and ifi in the complete monocyte population for all patients that had been sampled at least twice (fig. g) . expression levels were highest at early time points and consistently decreased over time, clearly indicating that the ifn response in covid- is inversely linked to disease severity and time (fig. s f+g) . in contrast, decreased expression of hla-dra and hla-drb in severe covid- is evident early on and sustained over time. j o u r n a l p r e -p r o o f transcription factor prediction indicated a stat signaling-driven gene expression program in monocytes in covid- (fig. h) neutrophils and the remaining clusters as mature neutrophils (fig. s a) . accordingly, pro- and pre-neutrophils were enriched for transcriptional signatures of neutrophil progenitors derived from published single-cell data ( and pro-neutrophils in cluster and showed the highest proportion of cells with a proliferative signature (fig. s b) . clusters , , (originally in cluster in fig. a ) expressed mature neutrophil markers fcgr b (cd ) and mme (cd ) (fig. s a) . including cd , olfm , lcn , and bpi, previously associated with poor outcome in sepsis (fig. b, s a ) (kangelaris et al., ) . all ldns also expressed high levels of alarmins s a and s a (fig. d) , whereas other s genes (e.g. s a , s a ) were strongly induced in selected neutrophil alterations of the neutrophil compartment were further interrogated by mass cytometry of whole blood samples of covid- patients (n= mild + severe, cohort ), fli patients (n= ), and age-and gender-matched controls (n= ) (table s ), using a panel designed to detect myeloid cell maturation and activation states as well as markers of immunosuppression or dysfunction (table s ) . unsupervised clustering analysis of all neutrophils in all samples revealed major clusters (fig. a ) of immature (cluster , , , ), mature (cluster , , ) and remaining clusters of low abundancy (cluster , , ). based on their differential expression of cd b, cd , cd , cd and cd , clusters and were identified as pro-neutrophils and cluster as pre-neutrophils (kwok et al., ; ng et al., ). the fourth immature cell cluster ( ) showed very low expression of cd b and cd , reminiscent of pro-neutrophils, but lacking cd , cd and cd (fig. a) , suggesting a hitherto unappreciated pro-neutrophil-like population. the mature neutrophils segregated into non-activated (cluster ), partially activated (cluster ) and highly activated cells (cluster ), based on the loss of cd l and upregulation of cd , as well as signs of proliferative activity (ki + ) (fig. a) . neutrophils from covid- patients clearly separated from those of controls and also fli patients in umap analysis (fig. b) , and neutrophils in patients with severe covid- were distinct from those of patients with mild disease (fig. b) . cells from control donors accumulated in areas enriched for mature non-activated cells (cluster ) and immature pre- neutrophil-like cells (cluster ). in contrast, neutrophils from fli patients were mainly mature non-activated (cluster ) and mature highly activated (cluster ) cells. neutrophils from covid- , particularly from patients with severe disease primarily occupied immature pre- and pro-neutrophil-like clusters. plotting cell cluster-specific surface marker expression onto the umaps (fig. c ) as well as statistical analyses of cell cluster distribution and surface marker expression among different patient groups supported these observations (fig. d+e) . samples from fli patients contain a high proportion of highly activated mature neutrophils, but barely any immature neutrophils. in contrast, severe covid- is associated with the appearance of immature pre-and pro-neutrophils (fig. d+ e) . interestingly, immature cell clusters in severe covid- showed signs of recent activation like upregulation of cd (mortaz et al., ) , rank and rankl (riegel et al., ) , as well as reduced cd l expression (mortaz et al., ) . in addition to loss of cd l, immature and mature neutrophils from severe covid- showed elevated pd-l expression compared to control samples (fig. e) we next assessed the dynamics of the changes within the myeloid cell compartment over time. we grouped samples according to collection time as 'early' (within the first days) or late (during the following days) after onset of symptoms. in both cohorts, we observed a tendency towards (cohort ) or significantly higher (cohort ) proportions of granulocytes in severe vs. mild covid- patients, both at early and late time points (fig. s a) . we observed a persistent release of immature neutrophils (e.g. cluster ) in severe covid- (fig. s b) showing high expression of cd and pd-l , but downregulation of cd l as a sign of activation, dysfunction and immunosuppression (fig. s c ). in addition, severe covid- patients show further increased frequencies of mature, partially activated neutrophils (cluster ) at later time periods (fig. s b) . thus, the neutrophil compartment of severe covid- patients is characterized by a combination of persistent signs of inflammation and immunosuppression, which is reminiscent of long-term post-traumatic complications (hesselink et al., ) . we also analyzed time-dependent phenotypic changes in the monocyte compartment by mass cytometry. non-classical monocytes started to recover in covid- patients during the later stages of the disease (fig. s a) . hla-dr hi cd c hi monocyte cell clusters also declined at later time points in mild covid- ( fig. s d,e,f) , which correlates well with the longitudinal changes of ifi and isg as well as hla-dra, and hla-drb expression profiles (fig. g+s f) . in contrast, overall proportions of hla-dr hi cd c hi monocytes in severe covid- remained low throughout the course of the disease. proportions of cd hi macrophage-like cluster and cd + cd + monocytes were generally higher at later stages in severe covid- , which resembled the kinetics of hla-dr lo s a hi monocytes identified by scrna-seq (fig. f ). this indicates a prolonged alternative activation of monocytes in severe covid- (fig. s e) . table s ). integrated visualization of all samples of cohort (fresh/frozen pbmc, fresh whole blood, , cells, fig. s a ) revealed the expected blood leukocyte distribution, including granulocytes ( fig. a, s a , table s ). cell type distribution identified by scrna-seq profiles (fig. s b ) strongly correlated with mcfc characterization of the same samples (fig. s c) . for further analysis of the granulocyte compartment, we first combined the whole blood samples with the fresh pbmc to guide the clustering of all major immune cells resulting in a total of , cells (fig a) . from these samples, we identified all neutrophil clusters and extracted the cells derived from whole blood for subsampling, which revealed a structure of clusters (n= , cells, fig. b+c ). using marker-and data-driven approaches as applied to ldn (fig. d, s a) , we identified fut (cd ) + cd + cd b + pro-neutrophils, itgam(cd b) + cd + pre-neutrophils, along with mature neutrophil clusters ( fig. b -d, s d, table s ). heterogeneous expression of various markers involved in mature neutrophil function including cxcr , fcgr a (cd ), fcgr a (cd ) or mme (cd ), pointed towards distinct functionalities within the neutrophil compartment (fig. e, s d+e) . seven of the nine neutrophil clusters identified in whole blood in cohort , could also be mapped to the fresh pbmc transcriptomes in cohort (fig. s f) , indicating that scrna-seq of fresh pbmc in covid- patients reveals relevant parts of the neutrophil space. the transcriptional phenotype of pro-and pre- neutrophils (cluster + ) was corroborated in cohort ( fig. b-d, s d) . heatmap and umap visualization of the cell type distribution identified pro-and pre- neutrophils mainly at late time points in severe covid- ( fig. f-g) . furthermore, mature neutrophils with a high ifn-signature (cluster ) were associated with severe covid- (fig. e, s g ). this cluster was also enriched for markers identified by cytof as differentially expressed in patients with severe covid- ( fig. ) , such as elevated expression of cd (pd-l ) and fcgr a (cd ) (fig. h ). in addition to cd , cells in cluster expressed genes indicative of a potentially suppressive or anti-inflammatory state, including zc h a (fig. e) , which is known to suppress hepatitis c virus replication and virus-induced pro-inflammatory cytokine production (lin et al., ) . cluster was also enriched for cells from covid- patients, mainly from severe but also mild cases (fig. f is mainly driven by e f family members and pre-neutrophils mainly depend on ets tfs (fig. s h) . pseudotime analysis strongly supported the differentiation trajectory from pro-neutrophils (cluster ) via pre-neutrophils (cluster ) to mature neutrophils in cluster and ( fig. s i-j) . particularly cd (pd- l) was enriched in cluster compared to cluster , supporting the potential of neutrophils to progress towards a suppressive phenotype in severe covid- (fig. s j) . interestingly, cd is expressed in pre-neutrophils and persisting in cluster further highlighting the newly emerging character of this cluster (volkmann et al., ) . finally, we studied whether the persistent emergence of immature, potentially dysfunctional neutrophils in severe covid- patients can be captured under routine diagnostic conditions. therefore, samples of covid- patients ( table s , cohort ) were characterized by routine hematology analyses using a clinical flow cytometry system (sysmex analyzer). indeed, the assumption of rescue myelopoiesis in severe covid- was supported by significantly higher counts in the population of immature granulocytes (ig, representing promyelocytes, myelocytes, and metamyelocytes) in this patient group ( fig. k). we also found significant differences in the neutrophil compartment, when analyzing the width of dispersion with respect to granularity, activity, and cell volume defined as ne- wx, ne-wy and ne-wz, respectively. as compared to patients with mild course, severely ill patients displayed increases in width of dispersion of activity and cell volume as surrogates for increased cellular heterogeneity, immaturity and dysregulation in severe covid- ( fig. k), resembling previously described alterations in sepsis patients (stiel et al., ) . furthermore, neutrophils of severe covid- patients were partially dysfunctional as their oxidative burst upon stimulation with standardized stimuli (e.coli or pma) was strongly impaired in comparison to control and mild covid- neutrophils, whereas phagocytic activity was preserved (fig. l , table s ). collectively, the neutrophil compartment in peripheral blood of severe covid- patients is characterized by the appearance of ldn, fut (cd ) + cd + cd b + pro-neutrophils, and itgam(cd b) + cd + pre-neutrophils, reminiscent of emergency myelopoiesis, as well as cd (pd-l ) + zc h a + mature neutrophils reminiscent of gmdsc-like cells, which might exert suppressive or anti-inflammatory functions. dysfunctional phenotype, plac was recently shown to suppress production of il- β and il- (segawa et al., ) . in fact, we observed that inflammatory cytokine production, including il- β release, was impaired in monocytes from patients with severe covid- (fig. ) . pbmc fractions in severe covid- contained immature neutrophils, including pro-and pre- neutrophils, which was not observed in mild cases (fig. ) . these immature ldn showed a surface marker and gene expression profile reminiscent of granulocytic mdscs including genes such as s a , s a , mmp , arg (uhel et al., ) , and olfm , which has been recently associated with immunopathogenesis in sepsis (alder et al., ) . emergence of pro-neutrophils in severe covid- was also detected by single-cell proteomics on whole blood samples. strikingly, both immature and the mature neutrophils showed increased expression of cd and pd-l (fig. +s ), similar to recently described alterations in sepsis (meghraoui-kheddar et al., ). in addition to the altered phenotype, we also observed an altered functionality. neutrophils from patients with severe covid- showed an impaired oxidative burst response, while their phagocytic capacity was preserved (fig. ) . single-cell transcriptomics of whole blood samples revealed mature activated neutrophils in both mild and severe covid- (fig. b, cluster ) , however, expression of cd (pd-l ) was only found in severe covid- (cluster ), and it increased in later stages of the disease. expression of pd-l on neutrophils has been associated with t cell suppression (bowers et methodology: js-s, dp, tk, sb, lb, edd, mg, dw, mb, tsk, as, od, hm, ars, cc, dk, ev, cjx, ad, ct, sh, clg, ml, ew, tu, mb, rg, (table s ) . (table s ) . within the monocyte space of cohort (related to fig. , table s ). cluster ranked by adjusted p-values. c, back-mapping of monocyte clusters of cohort (fig. c) onto the pbmc umap of cohort (fig. d) . the legend shows the association of the colors to the clusters together with the labeling of the clusters based on expressed marker genes (according to fig. and fig. s d-f). d, violin plots of marker gene expression in the monocyte clusters identified in the complete pbmc space of cohort (fig. c,d ) e, dot plot of the top marker genes sorted by average log fold change calculated for the monocyte clusters (fig. c) . severe covid- patients ( figure a+b, table s ). information on age, sex, medication, and co-morbidities is listed in covid- patients ( figure a+b , table s ) were included in the study. in patients who were not able to consent at the time of study enrollment, consent was obtained after recovery. information on age, sex, medication, and co-morbidities are listed in table s . covid after one wash in dpbs cells were directly processed for scrna-seq (bd rhapsody) or multi-color flow cytometry (mcfc). frozen pbmc were recovered by rapidly thawing frozen cell suspensions in a °c water bath followed by immediate dilution in pre-warmed rpmi- + % fbs (gibco) and centrifugation at g for min. after centrifugation, the cells were resuspended in rpmi- + % fbs and processed for scrna-seq. antibody cocktails were cryopreserved as described before (schulz et al., ) . all anti-human antibodies pre-conjugated to metal isotopes were obtained from fluidigm corporation (san francisco, us). all remaining antibodies were obtained from the indicated companies as purified antibodies and in-house conjugation was done using the maxpar x labeling kit (fluidigm). tlrpure; innaxon, uk). after stimulation, cell-free supernatants were collected and tested for il- β, ifnγ, and tnfα, respectively, using the cytokine bead assay legend-plex mix&match test was used to report differences in ig count, whereas mixed-effect-analysis and sidak's multiple comparison test was applied to report statistical differences of ne-wx, ne-wy and ne-wz between mild and severe covid- patients. x genomics chromium single-cell rna-seq pbmc were isolated and prepared as described above. afterwards, patient samples were hashtagged with totalseq-a antibodies (biolegend) according to the manufacturer's protocol for totalseq tm -a antibodies and cell hashing with x single cell ' reagent kit v . . µl cell suspension with x cells were resuspended in staining buffer ( % bsa, jackson immuno research; . % tween- , sigma-aldrich; x dpbs, gibco) and µl human trustain fcx tm fcblocking (biolegend) reagent were added. the blocking was performed for min at °c. in the next step µg unique totalseq-a antibody was added to each sample and incubated for min at °c. after the incubation time . ml staining buffer were added and centrifuged for min at g and °c. washing was repeated for a total of washes. subsequently, the cells were resuspended in an appropriate volume of x dpbs (gibco), passed through a µm mesh (flowmi tm cell strainer, merck) and counted, using a neubauer hemocytometer (marienfeld). cell counts were adjusted and hashtagged cells were pooled equally. the cell suspension was super-loaded, with , cells, in the chromium tm controller for partitioning single cells into nanoliter-scale gel bead-in- emulsions (gems). single cell ' reagent kit v . was used for reverse transcription, cdna amplification and library construction of the gene expression libraries ( x genomics) following the detailed protocol provided by x genomics. hashtag libraries were prepared according to the cell hashing protocol for x single cell ' reagent kit v . provided by biolegend, including primer sequences and reagent specifications. biometra trio thermal cycler was used for amplification and incubation steps (analytik jena). libraries were quantified by qubit tm . fluorometer (thermofisher) and quality was checked using bioanalyzer with high sensitivity dna kit (agilent). sequencing was performed in paired-end mode with a s and s flow cell ( × cycles) using novaseq sequencer (illumina). the qubit dsdna hs kit (thermofisher) and the size-distribution was measured using the agilent high sensitivity d assay on a tapestation system (agilent technologies). sequencing was performed in paired-end mode ( * cycles) on a novaseq and nextseq system (illumina) with novaseq s reagent kit ( cycles) and nextseq / high output kit v . ( cycles) chemistry, respectively. data pre-processing of x genomics chromium scrna-seq data cellranger v . . ( x genomics) was used to process scrna-seq. to generate a digital gene expression (dge) matrix for each sample, we mapped their reads to a combined reference of grch genome and sars-cov- genome and recorded the number of umis for each gene in each cell. data pre-processing of bd rhapsody scrna-seq data after demultiplexing of bcl files using bcl fastq v . from illumina and quality control, paired-end scrna-seq reads were filtered for valid cell barcodes using the barcode whitelist provided by bd. cutadapt . was then used to trim nexterape-pe adapter sequences where needed and to filter reads for a phred score of or above (martin, ) . then, star . . b was used for alignment against the gencode v reference genome (dobin et al implemented in seurat. we excluded cells based on the following quality criteria: more than % mitochondrial reads, more than % hba/hbb gene reads, less than expressed genes or more than , expressed genes and less than detected transcripts. we further excluded genes that were expressed in less than five cells. in addition, mitochondrial genes have been excluded from further analysis. lognormalization (seurat function) was applied before downstream analysis. the original gene counts for each cell were normalized by total umi counts, multiplied by , (tp k) and then log transformed by log (tp k+ ). after normalization, the count data was scaled regressing for total umi counts and principal component analysis (pca) was performed based on the , most variable features identified using the vst method implemented in seurat. subsequently, the scrna-seq data from cohort was integrated with publicly available x scrnaseq data of healthy controls using the 'harmony' algorithm (korsunsky et benchmarking data from healthy controls and x v . scrna-seq data from cohort ). we downloaded the count matrices for the publicly available scrna-seq data and filtered the cells using the above-mentioned quality criteria prior to data integration. for two-dimensional data visualization we performed umap based on the first dimensions of the 'harmony' data reduction. the cells were clustered using the louvain algorithm based on the first 'harmony" dimensions with a resolution of . . differential expression (de) tests were performed using findmarkers/findallmarkers functions in seurat with wilcoxon rank sum test. genes with > . log-fold changes, at least % expressed in tested groups, and bonferroni-corrected p-values< . were regarded as significantly differentially expressed genes (degs). cluster marker genes were identified by applying the de tests for upregulated genes between cells in one cluster to all other clusters in the dataset. top ranked genes (by log-fold changes) from each cluster of interest were extracted for further illustration. the exact number and definition of samples used in the analysis are specified in the legend of fig. a and summarized in table s . clusters were annotated based on a double-checking strategy: ) by comparing cluster marker genes with public sources, and ) by directly visualizing the expression pattern of cytof marker genes. significant degs between each monocyte cluster and the rest of monocyte subpopulations were identified by findmarkers function from the seurat package using wilcoxon rank sum test statistics for genes expressed in at least % of all monocyte clusters. p-values were corrected for multiple testing using bonferroni correction and genes with corrected p-values lower or equal . have been taken as significant degs for go enrichment test by r package/clusterprofiler v. . . (yu et al., ) . to systematically compare the similarity of marker gene expression in the identified monocyte/neutrophils subpopulations between the two cohorts, the spearman correlation coefficients were calculated based on the union of the top marker genes of each cluster sorted by fold change in the two cohorts, based on their average expression of all cells in the specific subpopulation. the pairwise comparisons were performed, and the correlation coefficients were displayed using a heatmap. the neutrophil space was investigated by subsetting the pbmc dataset to those clusters identified as neutrophils and immature neutrophils (cluster and ). within those subsets, we selected top , variable genes and repeated the clustering using the snn-graph based louvain algorithm mentioned above with a resolution of . . the dimensionality of the data was then reduced to pcs, which served as input for the umap calculation. to categorize the observed neutrophil clusters into the respective cell cycle states, we applied the cellcyclescoring function of seurat and visualized the results as pie charts. a gene signature enrichment analysis using the 'aucell' method (aibar et al., ) was applied to link observed neutrophil clusters to existing studies and neutrophils of cohort . we set the threshold for the calculation of the area under the curve (auc) to marker genes from collected publications and top of the ranked maker genes from each of neutrophil clusters from cohort . the resulting auc values were normalized the maximum possible auc to and subsequently visualized in violin plots or umap plots. scrna-seq umi count matrices were imported to r . . and gene expression data analysis was performed using the r/seurat package . . (butler et al., ) . demultiplexing of cells was performed using the htodemux function implemented in seurat. after identification of singlets, cells with more than % mitochondrial reads, less than expressed genes or more than , expressed genes and less than detected transcripts were excluded from the analysis and only those genes present in more than cells were considered for downstream analysis. the following normalization, scaling and dimensionality reduction steps were performed independently for each of the data subsets used for the different analyses as indicated respectively. in general, gene expression values were normalized by total umi counts per cell, multiplied by , (tp k) and then log transformed by log (tp k+ ). subsequently, the data was scaled, centered and regressed against the number of detected transcripts per cell to correct for heterogeneity associated with differences in sequencing depth. for dimensionality reduction, pca was performed on the top , variable genes identified using the vst method implemented in seurat. subsequently, umap was used for two-dimensional representation of the data structure. cell type annotation was based on the respective clustering results combined with data-driven cell type classification algorithms based on reference transcriptome data (aran et al., ) and expression of known marker genes. scrna-seq count data of , cells derived from fresh and frozen pbmc samples purified by density gradient centrifugation and whole blood after erythrocyte lysis of cohort (bonn, bd rhapsody) were combined, normalized and scaled as described above (see fig. s a). after variable gene selection and pca, umap was performed based on the first principal components (pcs). no batch correction or data integration strategies were applied to the data. visualization of the cells (fig. s a) showed overlay of cells of the same type (e.g. t cells clustered within the same cluster, irrespective of cell isolation procedure). in other words, cell type distribution was unaffected by the technical differences in sample handling. data quality and information content was visualized as violin plots showing the number of detected genes, transcripts (umis) and genic reads per sample handling strategy split by pbmc and granulocyte fraction. scrna-seq count data of , cells derived from fresh and frozen pbmc samples of cohort (bonn, bd rhapsody) purified by density gradient centrifugation were normalized and scaled as described above. after variable gene selection and pca, umap was performed and the cells were clustered using the louvain algorithm based on the first pcs and a resolution of . . cluster identities were determined by reference-based cell classification and inference of cluster-specific marker genes using the wilcoxon rank sum test using the following cutoffs: genes have to be expressed in more than % of the cells of the respective cluster, exceed a logarithmic fold change cutoff to at least . , and exhibited a difference of > % in the detection between two clusters. the exact number and definition of samples used in the analysis are specified in the legend of fig. d and summarized in table s . to compare shifts in the monocyte and neutrophil populations in the pbmc compartment of covid- patients, the percentages of the cellular subsets -as identified by clustering and cluster annotation explained above for the two independent scrna-seq data sets (cohort and cohort ) -of the total number of pbmc in each data set were quantified per sample and visualized together in box plots. to determine the statistical significance of differences in cell proportions between the different conditions, a dirichlet regression model was used, due to the fact that the proportions are not independent of one another. the r/rdirichletreg (maier, ) package was used. the p-values were corrected for multiple testing using the benjamini-hochberg procedure. the monocyte space was investigated by subsetting the pbmc dataset to those clusters identified as monocytes (cluster - ), removing cells with strong multi-lineage marker expressions, and repeating the variable gene selection (top , variable genes), regression for the number of umis and scaling as described above. the dimensionality of the data was then reduced to pcs, which served as input for the umap calculation. the snn-graph based louvain clustering of the monocytes was performed using a resolution of . . marker genes per cluster were calculated using the wilcoxon rank sum test using the following cutoffs: genes have to be expressed in > % of the cells, exceed a logarithmic fold change cutoff to at least . , and exhibited a difference of > % in the detection between two clusters. the exact number and definition of samples used in the analysis are specified in the legend of fig. a and summarized in table s . j o u r n a l p r e -p r o o f for each patient and time point of sample collection, the proportional occupancy of the monocyte clusters was calculated, and the relative proportions were subsequently visualized as a function of time. scrna-seq count data derived from fresh pbmc samples purified by density gradient centrifugation and whole blood after erythrocyte lysis of cohort (bd rhapsody) were normalized, scaled, and regressed for the number of umi per cell as described above. after pca based on the top , variable genes, umap was performed using the first pcs. cell clusters were determined using louvain clustering implemented in seurat based on the first principle components and a resolution of . . cluster identities were assigned as detailed above using reference-based classification and marker gene expression. subsequently, the dataset was subsetted for whole blood samples after erythrocyte lysis and clusters identified as neutrophils and immature neutrophils, and re-scaled and regressed. after pca on the top , variable genes, the neutrophil subset data was further processed using the data integration approach implemented in seurat (stuart et al., ) based on the first pcs removing potential technical biases of separate experimental runs. umap and clustering were performed as described above on the top pcs using a resolution of . . differentially expressed genes between clusters were defined using a wilcoxon rank sum test for differential gene expression implemented in seurat. genes had to be expressed in > % of the cells of a cluster, exceed a logarithmic threshold > . . the exact number and definition of samples used in the analysis are specified in the legend of fig. a and summarized in table s . after cell type classification of the combined scrna-seq data set of fresh pbmc and whole blood samples of cohort described above, , cells derived from whole blood samples after erythrocyte lysis were subsetted. percentages of cell subsets in those whole blood samples of the total number of cells were quantified per sample and visualized in box plots separated by disease stage and group. to categorize the cells within the neutrophil clusters into the respective cell cycle states, we applied the cellcyclescoring function of seurat and visualized the results as pie charts. trajectory analysis was performed using the destiny algorithm v . . (angerer et al., ) . generate umap representations all events of a given population of interest were down- sampled to , cells and then embedded using the tumap function (r uwot package, https://cran.r-project.org/package=uwot) parameterized by local neighborhood , learning rate . , and using the indicated markers ( stat fkbp lgals ifitm ifit isg ifi mx ifi ifit herc oasl mx ifih ifi ifi l oas serpinb il r serping cd rnase ifi oas adar lgals bp spi defa defa hsp aa mpo elane prtn cd bpi cd clec a fut hexa pde d c qbp ceacam anxa gsn clec a nlrc olfm cybb lcn lgals ltf mmp hp cd camp s a s a cd tspo rab a s p itgam s a s a ifi isg ly e ifi gbp ccr c ar ifih ddx fcgr a aim zc h a tlr abca icam inpp b fbxl slc a clec d itga sell s a cxcr tlr clec e fcgr a adam slc a nlrp tlr c ar coro a cmtm tnfrsf a tnfrsf b rac nlrp ptprc ptgs sirpa ncoa mme s a % exp. transcriptome meta-analysis deciphers a dysregulation in immune response-associated gene signatures during sepsis scenic: single-cell 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of type i interferons in development of severe covid- least-squares means: the r package lsmeans pad mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures arginine deficiency is involved in thrombocytopenia and immunosuppression in severe fever with thrombocytopenia syndrome single-cell landscape of bronchoalveolar immune cells in patients with covid- the molecular signatures database hallmark gene set collection mcpip suppresses hepatitis c virus replication and negatively regulates virus-induced proinflammatory cytokine responses dysregulated myelopoiesis and hematopoietic function following acute physiologic insult antibody responses to sars-cov- in patients with covid- longitudinal analyses reveal immunological misfiring in severe covid- ebola virus disease is characterized by poor activation and reduced levels of circulating cd + monocytes single cell rna sequencing of human liver reveals distinct intrahepatic macrophage populations dirichletreg: dirichlet regression for compositional data in r cutadapt removes adapter sequences from high-throughput sequencing reads validation of diagnostic gene sets to identify critically ill patients with sepsis deep immune profiling of covid- patients reveals patient heterogeneity and distinct immunotypes with implications for therapeutic interventions the innate immune system: fighting on the front lines or fanning the flames of covid- ? two new immature and dysfunctional neutrophil cell subsets define a predictive signature of sepsis useable in clinical practice barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry platinum-conjugated antibodies for application in mass cytometry the cd +hla-drlo/neg monocyte: an immunosuppressive phenotype that restrains responses to cancer immunotherapy pathological inflammation in patients with covid- : a key role for monocytes and macrophages ultra-high-throughput clinical proteomics reveals classifiers of covid- infection frequencies of circulating mdsc correlate with clinical outcome of melanoma patients treated with ipilimumab neutrophil extracellular traps (nets) contribute to immunothrombosis in covid- acute respiratory distress syndrome persisting low monocyte human leukocyte antigen-dr expression predicts mortality in septic shock update on neutrophil function in severe inflammation different phenotypes of non-classical monocytes associated with systemic inflammation, endothelial alteration and hepatic compromise in patients with dengue heterogeneity of neutrophils detection of sars-cov- -specific humoral and cellular immunity in covid- convalescent individuals cytof workflow: differential discovery in high- throughput high-dimensional cytometry datasets a comprehensive single cell transcriptional landscape of human hematopoietic progenitors immunopathogenesis of coronavirus infections: implications for sars biological basis and pathological relevance of microvascular thrombosis a subset of neutrophils in human systemic inflammation inhibits t cell responses through mac- decoding human fetal liver haematopoiesis extracting a cellular hierarchy from high- dimensional cytometry data with spade mortality rates of patients with covid- in the intensive care unit: a systematic review of the emerging literature immunotherapies for covid- : lessons learned from sepsis an immune-cell signature of bacterial sepsis dnam- and pvr regulate monocyte migration through endothelial junctions human polymorphonuclear neutrophils express rank and are activated by its ligand, rankl immunosuppression for hyperinflammation in covid- : a double-edged sword? convergent antibody responses to sars-cov- in convalescent individuals differential redistribution of activated monocyte and dendritic cell subsets to the lung associates with severity of covid- hepatic acute-phase proteins control innate immune responses during infection by promoting myeloid-derived suppressor cell function invariant nkt cells reduce the immunosuppressive activity of influenza a virus-induced myeloid-derived suppressor cells in mice and humans regulatory cell therapy in kidney transplantation (the one study): a harmonised design and analysis of seven non- randomised, single-arm, phase / a trials human neutrophils in the saga of cellular heterogeneity: insights and open questions myeloperoxidase can differentiate between sepsis and non-infectious sirs and predicts mortality in intensive care patients with sirs emerging principles in myelopoiesis at homeostasis and during infection and inflammation surface barcoding of live pbmc for multiplexed mass cytometry minimizing batch effects in mass production through regulation of autophagy and is associated with adult still disease neutrophil diversity in health and disease neutrophil fluorescence: a new indicator of cell activation during septic shock-induced disseminated 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the outcome of critical covid- patients a single-cell atlas of the peripheral immune response in patients with severe covid- pathological findings of covid- associated with acute respiratory distress syndrome increased formation of neutrophil extracellular traps is associated with gut leakage in patients with type but not type diabetes myeloid-derived suppressor cells: their role in the pathophysiology of hematologic malignancies and potential as therapeutic targets clusterprofiler: an r package for comparing biological themes among gene clusters clinical course and risk factors for mortality of adult inpatients with covid china: a retrospective cohort study overly exuberant innate immune response to sars-cov- infection. ssrn electron a dynamic immune response shapes covid- progression neutrophil extracellular traps in covid- cov- infection induces profound alterations of the myeloid compartment • mild covid- is marked by inflammatory hla-dr hi cd c hi cd + monocytes • dysfunctional hla-dr lo cd hi and hla-dr lo s a hi cd + monocytes in severe • emergency myelopoiesis with immature and dysfunctional neutrophils in severe covid- analysis of patients with with mild and severe covid- reveals the presence of dysfunctional neutrophils in the latter that is linked to emergency myelopoiesis in brief, the neutrophil space was subsetted to only severe patients (early and late) and only the most prominent clusters of the latter (clusters , , , ). the normalized data were scaled and regressed for umis and a diffusion map was calculated based on the top , variable genes with a sum of at least counts over all cells. based on the diffusion map, a diffusion pseudo time was calculated to infer a transition probability between the different cell states of the neutrophils. subsequently, the density of the clusters along the pseudotime and marker gene expression for each cluster were visualized. enrichment of gene sets was performed using the 'aucell' method (aibar et al., (aibar et al., ) implemented in the package (version . . ) in r. we set the threshold for the calculation of the auc to the top % of the ranked genes and normalized the maximum possible auc to . the resulting auc values were subsequently visualized in violin plots or umap plots. key: cord- -l q authors: shupler, m.; mwitari, j.; gohole, a.; anderson de cuevas, r.; puzzolo, e.; cukic, i.; nix, e.; pope, d. title: covid- lockdown in a kenyan informal settlement: impacts on household energy and food security date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: l q a covid- lockdown may impact household fuel use and food security for ~ million sub-saharan africans who rely on polluting fuels (e.g. wood, kerosene) for household energy and typically work in the informal economy. in an informal settlement in nairobi, surveys administered before (n= ) and after (n= ) a mandatory covid- -related community lockdown documented socioeconomic/household energy impacts. during lockdown, % of participants indicated income decline or cessation and % reported being food insecure. three quarters of participants cooked less frequently and half altered their diet. one quarter ( %) of households primarily using liquefied petroleum gas (lpg) for cooking before lockdown switched to kerosene ( %) or wood ( %). these results indicate the livelihoods of urban kenyan families were deleteriously affected by covid- lockdown, with a likely rise in household air pollution from community-level increases in polluting fuel use. to safeguard public health, policies should prioritize enhancing clean fuel and food access among the urban poor. approximately . billion people worldwide use polluting fuels, including solid fuels (e.g. wood, charcoal) and kerosene, for household cooking, heating and lighting. in sub-saharan africa (ssa), hap was attributed to % of mortality and % of underlying morbidity in . amid the coronavirus disease (covid- ) pandemic, there is concern about increased disease severity in ssa due to higher baseline prevalence of infectious diseases (e.g. tuberculosis, pneumonia) and non-communicable respiratory/cardiovascular diseases, [ ] [ ] [ ] [ ] which are all causally linked to hap exposure. [ ] [ ] [ ] to combat the adverse health, environmental and social effects of reliance on polluting fuels and associated hap exposures, , several african countries (e.g. kenya, ghana, cameroon), have set aspirational targets for rapid market expansion of liquefied petroleum gas (lpg) for cooking. , despite being a fossil fuel, lpg is considered 'clean' because it burns very efficiently, emitting no black carbon and low levels of fine particulate matter (pm . ) , and contributes to forest protection. , lpg is a clean energy alternative actively promoted in ssa, with several consumer-tailored solutions, including consumer-finance mechanisms (e.g. pay-asyou-go (payg), mobile payments) being evaluated to increase its affordability. however, national scale-up efforts may become increasingly difficult because of the financial repercussions of the covid- pandemic, government-mandated community lockdowns to mitigate its spread and logistical/trade constraints affecting supply/distribution. with affordability of lpg being a critical barrier to uptake in ssa before the pandemic, , income loss during lockdown can further diminish its scalability, and increase levels of food insecurity. a world food programme report predicts a doubling of hunger risk from the covid- pandemic. in africa, up to % of urban development consists of informal settlements. these settlements suffer from overcrowding, with limited water/sanitation facilities and cooking typically done in a single, multipurpose room. household energy sources can be variable in these environments outside the context of a pandemic; cooking fuels may be routinely 'stacked' (combination of clean and/or polluting fuels) , or primary fuels switched from clean to polluting fuels ('reverse switching') due to unexpected/seasonal changes in income. , thus, financial hardship brought about by a mandatory lockdown can potentially spur community-level reverse switching, leading to household pm . quality guidelines, , which can be intensified by tightly-packed housing, as smoke can infiltrate neighboring homes. a lockdown can therefore have severe negative socioeconomic and health consequences, , with limited success in mitigating covid- spread in these densely populated areas, where cramped conditions and shared facilities make physical distancing infeasible. in kenya, a mandatory lockdown was instated on march , , two weeks after the first recorded case of covid- , followed by a dusk-to-dawn curfew ( pm- am) instituted on april , . with % of the urban kenyan population living in informal settlements (> million living in nairobi), the effects of an unprecedented community lockdown on household energy decisions, food security and air pollution exposures of the urban poor are uncertain. this pre-post study provides valuable information on impacts of the covid- lockdown among a vulnerable population living in mukuru kwa reuben informal settlement in nairobi. the study setting of mukuru kwa rueben is an informal settlement with over , residents along the nairobi ngong river, situated on polluted lands in the industrial area of nairobi. a population-based survey on socioeconomic factors and fuel use patterns was administered to the main cook (or another member of the household, if unavailable) via door-to-door sampling from december -march (before the lockdown went into effect). field workers that administered the surveys also serve as community health volunteers in kwa rueben and had an earned level of trust among study participants. questions about familiarity with pay-as-you-go lpg (payg) consumer finance mechanisms were asked to a subset of participants; a sensitivity analysis was conducted to compare socioeconomic characteristics between the payg sample and full sample via pearson's chi-squared tests (categorical data) and two-sample t-tests (continuous data) for independence. all survey data was collected via smartphones and securely transferred to an online storage system using mobenzi research, a secure digital platform that has been used successfully in previous health monitoring studies. , after nairobi went into lockdown on march , , a telephone-based survey was conducted from april - among consenting participants from the baseline survey, to document post-lockdown impacts on food security and cooking behaviours. a sensitivity analysis was conducted to compare demographics among the baseline and follow-up sample in the pre-post study using the same statistical tests as above. additionally, real-time ambient pm . levels before and during the lockdown period were a total of randomly selected participants living in mukuru kwa reuben completed a baseline survey (before lockdown), of whom % (n= ) were the main cook of the household. the mean age was years old, and % of respondents were female. two-thirds of respondents had a monthly household income less than or equal to , kenyan shilling (ksh) (~$ usd). almost half ( %) of households experienced seasonal fluctuations in their income. nine out of ten households comprised one or two rooms (table s ). three-quarters ( %) of respondents reported having access to drinking water in their home and nearly all ( %) used a communal standpipe as their main water source. among baseline survey respondents, half ( %; n= ) used lpg as their primary fuel, % (n= ) used kerosene and the few remaining households used charcoal/charcoal briquettes ( %; n= ), electricity ( %; n= ) or wood ( %; n= ) ( figure -left) . a quarter ( %) of households . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . stacked two or more fuels, with the most common fuel combination being lpg (primary) and kerosene (secondary) ( % of households; n= ) ( figure -right). four out of five of households ( %) cooking with lpg at baseline started using it less than two years earlier (see supplemental information; table s ). over half ( %; n= ) of households not cooking with lpg had used it in the past, and > % of these households expressed an interest in cooking with lpg (table s ). nearly all ( %) families cooked inside the home in a single room. households cooking with lpg self-reported a weekday mean cooking time ( . hours) . hours less than that of woodusing households ( . hours) ( table ). only % of respondents obtained their primary cooking fuel for free. the frequency of fuel purchases among households paying for their cooking fuel varied dramatically by primary fuel type; almost all lpg primary users ( %) buying refills ( % of households used kilogram cylinders (table s )) once a month or every - months. conversely, two-thirds of participants primarily cooking with kerosene ( %) and over half using charcoal ( %) purchased their fuels daily (table ) . despite self-reported monthly mean lpg fuel costs being the least expensive ($ ksh/month (~$ usd/month or $ . usd/day)) when compared to all polluting fuel types, the mean cost of lpg per single purchase was highest because it was acquired in monthly increments (table ) . almost half ( %) of participants using kerosene as a primary fuel perceived the cost of lpg to be expensive or very . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . expensive, compared with % of lpg users. one quarter ( %) of kerosene primary fuel users perceived lpg to be dangerous or very dangerous, compared with only % of lpg users (table ). approximately % of households used electricity for lighting, but only % used it exclusively (table ) . households without electricity mostly used kerosene lamps as the main lighting source ( %). nearly half of electrified households used candles in times of power cuts, while other households also resorted to kerosene lamps ( %), oil/gasoline/lpg lamps ( %) or flashlights/lanterns/torches ( %). only % of sampled households ever heat their homes, with most ( %) only for months or less annually. . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . note: some variables may not add up to %; categories were condensed for brevity due to increasing interest in kenya about the potential for pay-as-you-go (payg) lpg smartmeter technology to transition individuals to lpg for cooking, a subset of participants (similar in demographic profile to the full baseline sample (table s ) ) were questioned before the lockdown about their perceptions of payg lpg technology. the most attractive reported feature was ability to pay for gas in small amounts ( %), followed by increased safety ( %) and customer service ( %) ( table ) . elimination of the need to travel for gas refills was not a highly motivating feature ( %). half ( %) of respondents had not heard of payg lpg; the remaining half were equally split between those intending and not intending to register ( % each) for the equipment, and % of . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint respondents had already registered or were existing customers ( table ) . interest in registering for payg technology among households already using lpg as a primary fuel ( %) was half that of households currently using kerosene ( %). the average monthly household income among the participants not interested in registering for payg lpg was higher than the average of the overall study population, with approximately % earning a monthly income greater than , ksh ($ usd), compared to % among the full sample (table s ). the (table s ) . nearly all participants ( %) reported decreases in income during lockdown, with ( %) indicating that . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint their decreased income was insufficient to purchase food needed by the household (table ) ; a third of households ( %) indicated total cessation of income amid the lockdown. over half of households ( %) cooked less frequently while in lockdown, while % cooked more frequently. the change in cooking frequency varied by primary fuel type with % of households using wood cooking more frequently compared with only % of households using lpg and % using kerosene (table s ) . half ( %) of participants indicated a change in the type of food they cook. common dietary changes were reductions in meat/fish, milk/milk tea and bread/chapati, with higher consumption of vegetables (table s ). in addition to insufficient income, lower food availability ( %) and increases ( %) or decreases ( %) in the number of household inhabitants during lockdown were main reasons alterations in dietary behaviour. a quarter ( %) of households switched their main food vendor/retailer during the lockdown, with % of households resorting to farming/livestock as their new, main food source. one quarter of participants ( %) paid more for their primary cooking fuel during lockdown than before the pandemic ( table ). the percentage of households using lpg that paid a higher rate after lockdown ( %) was three times higher than the proportion of households using kerosene that paid a higher rate after the lockdown mandate ( %) ( table s ). more specifically, the percent of households using lpg paying a total amount of > , ksh for refills increased dramatically ( %) from % at baseline to % during lockdown, compared with only an % rise ( % to %) in the proportion among kerosene users (table s ) . despite fuel cost increases, availability was not an issue experienced by participants during lockdown; % of participants obtained their fuel in under minutes (one way) travel time. . ( . ) more residents ( %) . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . primary fuel switching occurred among % (n= ) of households in response to the lockdown, with lpg users ( %) being three times more likely to switch than kerosene users ( %). previous lpg users switched to kerosene (n= ) or wood (n= ), and households previously using kerosene switched to wood (n= ) (figure -left) . the prevalence of wood as a primary fuel increased by a factor of five ( % to %) during lockdown, in conjunction with a % decline in primary lpg use for cooking ( % to %) (figure -right) . further, approximately a third ( %) of participants that continued to use lpg during the lockdown reported less use (table ) . . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . kwa rueben after the lockdown in kenya took effect (early april ) was observed ( figure s ). . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . this timely pre-post study documented an extremely high proportion ( %; n= ) of households experiencing food insecurity due to income decline/cessation during the covid- lockdown in a kenyan urban informal settlement ( table ) . as a result, % of families resorted to farming/livestock as their main/only source of food, and half ( %; n= ) switched their diets to commonly include higher intake of vegetables, as they were no longer able to afford meat/fish, milk and bread (table s ) , increasing the likelihood of protein deficiency. a decrease or loss of income during lockdown resulted in % of households switching their primary cooking fuel from clean lpg to a polluting fuel (kerosene, wood) (table ) , as these fuels could be purchased daily for small sums of money (kerosene/wood) or gathered for free (wood). while lpg was reported as the least expensive fuel on a monthly basis (table ) , the need to purchase a cylinder refill, and documented increase in the price of lpg compared with kerosene during lockdown (table s ) , likely played critical roles in families opting to purchase kerosene instead. with % of households that primarily cooked with kerosene before the lockdown also switching to firewood while in lockdown, a % rise in prevalence of firewood cooking fuel among the study population ( % to %) ( figure ) likely contributed to a rise in community-level hap concentrations. increased hap exposure can be experienced by both the households where the switching to polluting cooking fuels occurred and nearby families, due to infiltration of biomass . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . smoke to the surrounding area, especially with tightly-packed housing in an informal settlement. although % of families cooked less frequently during home lockdown, the length of hap exposures may still increase, as meals prepared with fuelwood required an average daily cooking time that is . hours longer than with lpg (table ) , and because families were confined inside the home in close proximity to the cooking area ( % of households had less than three rooms) ( table ). elevated hap exposures among the study population may offset potential health benefits from the decrease in ambient air pollution levels during covid- lockdown ( figure ), which were documented in studies conducted in high-income countries. , increased pm . exposure due to hap is a risk factor for several chronic respiratory/cardiovascular health conditions, and may also increase the collective population's vulnerability to suffering from more severe symptoms of covid- ; recent studies have linked covid- severity to increased chronic exposure to ambient air pollution. the limited number of households solely relying on electricity for lighting demonstrates its unreliability in the study setting; renewable energy cooking solutions (e.g. solar/grid electricity) are far from having the scalability needed to reach large sectors of the population. with over % of non-lpg users that had previous experience cooking with lpg expressing interest in future use (table s ) , lpg represents an encouraging clean energy source in informal settlements in nairobi. however, to overcome the affordability barrier to lpg uptake expressed by % of baseline non-lpg users (table s ) , which may increase in the aftermath of the covid- pandemic, there is an urgent need for consumer-centric approaches, including consumer-finance mechanisms (e.g. pay-as-you-go (payg), mobile payments) to offer families the opportunity to pay on an as-needed basis. the high cost of lpg refills ( % of lpg households used kg cylinders) explains the appeal of small incremental payments via payg smart-meter technology among participants (table ). with the launch of two commercial companies in nairobi that offer payg lpg, this innovation represents a promising solution for increasing lpg penetration in the study setting. two-thirds of current kerosene users reported an intention to register with a payg lpg company ( %) or being interested in learning more about the technology ( %) ( table ) . a recent intervention study assessing the acceptability of payg payments for pellets (with gasifier stoves) in peri-urban kenya found that payg . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . displaced traditional fuels in many households, and participants had higher willingness to pay for payg technology, compared with non-payg stoves. participants not interested in registering for payg lpg in the study community had higher average monthly incomes than the study population average, suggesting that the barrier of lpg affordability addressed by payg lpg was of less relevance to these households. nevertheless, % of lpg primary users were interested in registering for payg lpg technology because of other advantages, including perceived increased safety ( %), customer service ( %) and the ability to monitor their lpg usage ( %). expanding the lpg market in kenya and across ssa presents a viable, medium-term pathway to achieving universal clean household energy access (united nation's sustainable development goal (sdg) ). however, there are new challenges brought on by the covid- pandemic: in addition to a lower purchasing power among low-income populations, a recent clean cooking alliance survey of companies involved in fuel supply chains (stove/fuel manufacturers, distributors), ngos and investors showed that % of enterprises had moderate/severe disruptions in their work activities, with % ceasing operations. nearly two-thirds of clean cooking enterprises echoed significant concerns about reduced customer ability to pay for clean cooking fuels during the pandemic. while this pre-post study had a lower-than-expected follow up rate ( %), primarily as a result of participants not willing to be re-contacted or discontinuing mobile phone plans due to financial hardship, a follow up sample of participants, largely representative of the baseline population (table s ) , was powered to detect significant changes in food access and fuel use patterns during lockdown. the publicly available data from laser scattering pm . monitors used in this study, while not gravimetrically corrected to improve accuracy, provide crude evidence of ambient air pollution fluctuations, particularly with concentrations aggregated across multiple sensors to minimize measurement error. while the socioeconomic and fuel pricing data is selfreported, the dramatic results provide strong evidence of the negative side-effects of confinement measures on the livelihoods of africans largely working in the informal economy. while the covid- pandemic presents a precarious situation for countries in ssa with weak healthcare systems, , lockdown measures aiming to mitigate the spread of covid- can intensify existing challenges to combat global hunger and gain momentum in the clean energy sector. there is a need for covid-related interventions to be specifically tailored to the . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . socioeconomic context to minimize unnecessary financial fallout. while the long term effects of the pandemic on consumers' decisions regarding household energy are unclear, the cobenefits to health, climate and livelihoods should make universal access to clean household energy a priority for governments and global health leaders moving forward. the authors have no conflicts of interest to declare. . r core team. r: a language and environment for statistical computing. [internet] . vienna, austria: r foundation for statistical computing; . available from: http://www.rproject.org/ . cc-by-nc . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. 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the lancet the dynamics of covid- : weather, demographics and infection timeline health and health-related indicators in slum, rural, and urban communities: a comparative analysis. glob health action dietary protein quality and malnutrition in africa global estimation of exposure to fine particulate matter (pm . ) from household air pollution transient climate and ambient health impacts due to national solid fuel cookstove emissions exposure to air pollution and covid- mortality in the united states: a nationwide cross-sectional study covid- lockdowns cause global air pollution declines with implications for public health risk. medrxiv can atmospheric pollution be considered a co-factor in extremely high level of sars-cov- lethality in northern italy? environmental pollution supply considerations for scaling up clean cooking fuels for household energy in low-and middle-income countries enhancing clean cooking options in peri-urban kenya: a pilot study of advanced gasifier pellet stove adoption pandemic threatens access to clean cooking energy, potentially worsening air pollution and covid- 's impact [internet]. clean cooking alliance weak health systems and ebola. the lancet global health covid- : africa records over cases as lockdowns take hold national surveillance using mobile systems for health monitoring: complexity, functionality and feasibility. bmc infectious diseases collecting maternal health information from hiv-positive pregnant women using mobile phone-assisted faceto-face interviews in southern africa the authors would like to sincerely thank the field team in mukuru kwa reuben for their flexibility in transitioning from in person to telephone-based surveys. without their extensive efforts and dedication to research during the covid- pandemic, this study would not be possible. key: cord- -ps jyjkl authors: nan title: full issue pdf date: - - journal: jacc: cardiooncology doi: . /s - ( ) - sha: doc_id: cord_uid: ps jyjkl nan disease, coronary artery disease, arrhythmias, and pericardial disease, depending on the exact cardiotoxic agent ( ) . in this state-of-the-art review, we focus on long-term cardiac diseases after treatment for childhood cancer. we discuss the prevalence, risk factors, prevention, prediction, and surveillance of cardiac disease in this population (central illustration). we systematically searched pubmed for studies that described cardiac adverse events in children treated with cardiotoxic cancer treatments. we limited the search to full-text articles written in english and articles published within the last years. we selected articles with a study cohort of which > % were treated for childhood cancer before the age of years. for studies describing the prevalence or cumulative incidence of heart failure, we reviewed articles with a minimum of ccs; a minimum of ccs was required for the other outcomes. studies on primary prevention strategies were identified from previous cochrane searches ( ) ( ) ( ) . based on these criteria, studies were considered to be described in this review ( figure ). the full search strategy is provided in supplemental table . ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . anthracyclines are clearly associated with cardiomyocyte damage. although the exact mechanism of anthracyclineinduced cardiotoxicity has not been fully elucidated, early studies indicate that cardiotoxicity through reduction-oxidation reaction cycling and the generation of reactive oxygen species may be the cause. more recently, topoisomerase b has been proposed to be a mediator of doxorubicin-induced cardiac injury ( ) . systolic dysfunction can eventually progress to heart failure. heart failure is one of the most frequent cardiac late effects in ccs ( , ) , and contributes to significant morbidity and non-cancer-related mortality later in life ( , ) . a large cohort from the childhood cancer survivor study investigated the occurrence of heart failure, defined by the common terminology for criteria adverse events as grade to . based on questionnaires concerning long-term ccs, the reported cumulative incidence is . % by years of age ( ) . these results confirmed earlier reports that anthracyclines and chest rt are strongly associated with heart failure ( ) . recently, it has been shown that even low-to-moderate chest rt doses increase the risk of heart failure substantially ( , ) . in the dutch later (late effects after childhood cancer) cohort, feijen et al. ( ) reported a cumulative heart failure incidence of . % years after childhood cancer diagnosis in ccs who received cardiotoxic cancer treatment. higher exposure to mitoxantrone and cyclophosphamide were suggested as novel treatment-related risk factors ( ) . although mitoxantrone has traditionally been classified as an anthracycline, it has been suggested that mitoxantrone results in cardiotoxicity through mechanisms different from anthracyclines ( , ) . mitoxantrone has a nonlinear dose-response relationship with heart failure risk ( , ( ) ( ) ( ) , and compared to doxorubicin, mitoxantrone is times more cardiotoxic. in addition, a younger age at diagnosis and presence of traditional cardiovascular risk factors may play a role in the development of heart failure ( ) . the influence of sex on the development of myocardial dysfunction is still incompletely conclusive ( , , , , ) . coronary artery disease. the risk of coronary artery disease (cad) is substantially increased in ccs. in the childhood cancer survivor study, the cumulative incidence of cad by age years was . % in survivors with and without exposure to cardiotoxic cancer treatments ( ) . this risk is dependent on chest rt dose with no established safe dose; this risk is also higher in males. the cumulative incidence of symptomatic cad at age years the main risk factors for cardiac disease in childhood cancer survivors are anthracyclines, mitoxantrone, and chestdirected radiotherapy dose. primary prevention strategies may reduce the risk of anthracycline-induced cardiomyopathy. there is an increased prevalence of traditional cardiovascular risk factors in childhood cancer survivors; screening and early management are important to modify risk. multivariable risk prediction models may help to individualize prevention and surveillance strategies. increases to % in males exposed to > gy of radiation ( , ) . the st. jude lifetime cohort study detected cad based on either history, electrocardiogram (ecg), or echocardiography in . % of asymptomatic ccs . years after cardiotoxic therapy ( ) . however, evidence from (non)invasive coronary angiography is scarce. a study evaluating computed tomography in asymptomatic hodgkin lymphoma ccs years old or younger (n ¼ ) exposed to chest rt showed coronary artery lesions to be very proximal, placing large portions of the myocardium at risk ( ) . valvular ( , , ) . chest rt has been identified as an important risk factor that increases at higher doses ( ) . other risk factors are treatment with anthracyclines, hypertension, congenital heart disease, and younger age at diagnosis, although these have not been uniformly shown in all studies ( , , ) . mild tricuspid regurgitation was most prevalent in studies describing valvular disease, but this is also very common in the general population ( , , ) . in lymphoma ccs who were exposed to chest rt, valvular heart disease, defined as mild or higher for left sided valves and moderate or higher for right sided valves, was most frequently detected in the aortic and mitral valves ( ) . valvular abnormalities after chest rt are most likely caused by direct irradiation injury to the valve cusps or leaflets, causing thickening, fibrosis, and calcification ( , ) . these processes progress with age and increase in prevalence over time ( , ) . hence, ccs without echocardiographic abnormalities after a short followup period are still at risk of severe valvular heart disease. pericardial disease. besides paraneoplastic and infectious causes, pericardial disease can arise from chest rt. late constrictive pericarditis, in particular, can lead to disabling symptoms and a poor prognosis ( ) . however, data on pericardial disease in ccs are limited. the childhood cancer survivor study showed a -fold higher risk of pericardial disease in all ccs versus siblings ( - year cumulative incidence, . %) and a dose-response relation with chest rt ( ) . a single-center study in ccs older than years after diagnosis (n ¼ , ; % no cardiotoxic therapy), reported symptomatic pericarditis in only ccs ( ) . although the diagnosis of constrictive pericarditis is difficult by echocardiography, thickening of the pericardium as well as hemodynamic consequences (e.g., "septal bounce," abnormal respiratory variations in doppler findings) can be suggestive. upon high clinical suspicion, cardiac computed tomography, magnetic resonance imaging (mri), and/or invasive hemodynamic evaluation may be needed to confirm the diagnosis ( ) . arrhythmias. the prevalence of symptomatic cardiac arrhythmias in long-term ccs is reportedly low ( , , , ) . in , ccs, the cumulative incidence of grade to arrhythmia by years of age was . % ( ) . a subsequent study (n ¼ , ) showed that chest rt > gy, anthracycline dose $ mg/m , dyslipidemia, and hypertension are risk factors for symptomatic arrhythmia ( ) . myocardial fibrosis caused by chest rt may contribute to the occurrence of arrhythmias. other frequently used cancer agents for pediatric cancers such as cisplatin, cyclophosphamide, and tyrosine kinase inhibitors may also be associated with supraventricular and ventricular arrhythmias ( , ) . prolonged qtc interval, which has arrhythmogenic potential, has been shown in ccs who received anthracyclines with and without chest rt ( , ) . also, rhythm disturbances such as premature ectopic beats and atrioventricular blocks have been reported in ccs ( ) ( ) ( ) . the literature on ecg abnormalities in large cohorts of long-term ccs is sparse ( , ) , data on the use of ambulatory ecg monitoring to define the prevalence of brady-and tachyarrhythmias induced by cardiotoxic cancer treatments are needed, but must be carefully weighed against the potential patient burden and clinical significance. childhood cancer survivors preventive measures for cancer treatment-induced cardiotoxicity. as the risk of cardiac disease is high in chest rt and anthracycline-treated survivors, and as omitting or diminishing the use of cardiotoxic treatments is not always possible, prevention is critical ( ) . advanced radiotherapy techniques to minimize exposure to the heart have been developed; the impact of those improvements is reflected by the decrease in cad in more recent treatment eras ( ) . extensive research has been devoted to the identification of possible cardioprotective interventions during anthracycline treatment that do not have negative effects on antitumor efficacy or other noncardiac adverse effects. below we discuss preventive measures that have been studied during anthracycline treatment. we focus primarily on randomized controlled trials (rcts) as they provide the highest level of evidence to answer this type of question. because of developmental changes and the differences in the body composition of children, data from adults cannot be reliably extrapolated to children ( ) . d e x r a z o x a n e . dexrazoxane is one of the most widely investigated cardioprotective pharmacologic interventions. it has been shown in adult cancer patients to prevent clinical and subclinical cardiac damage ( ) . the few published pediatric rcts have included participants diagnosed with leukemia, lymphoma, and sarcoma ( ) ( ) ( ) . these studies suggest that there are no significant differences in clinical heart failure between dexrazoxane and control patients ( , ) , although dexrazoxane might have a protective effect on asymptomatic cardiotoxicity ( , ) . all studies included relatively short-term follow-up, and the impact on outcomes after longer follow-up is yet unknown. currently, dexrazoxane is not routinely used in clinical practice for all children treated with anthracyclines. this might be explained by a concern over interference with antitumor efficacy and the occurrence of secondary malignancies ( ). however, high-quality evidence to support an increased risk of secondary malignancy is lacking. a cochrane systematic review identified no significant differences between treatment groups ( ) , which is in line with more recently published randomized trials ( , ) . a recently published nonrandomized study in pediatric patients with acute myeloid leukemia mortality. an overview of validated prediction models in ccs is provided in supplemental table . heart failure prediction models. practical models to predict heart failure onset before the age of the discriminatory abilities of the model were further shown by a cumulative incidence of heart failure at age years of . % in the low-risk group, whereas this was . % in the high-risk group. importantly, . % of the ccs were at low risk according to the model and thus unlikely to develop heart failure. ( ) . thus, early, at years after diagnosis, cardiovascular risk factors have been shown to provide little incremental information to prediction models for heart failure and ischemic heart disease ( , ) . in a more recent study, diabetes, hypertension, and dyslipidemia were used in the prediction of heart failure and ischemic heart disease in ccs who were , , , or years of age at time of prediction, with relative risks comparable to moderate doses of anthracyclines ( ). cardiovascular risk factors were present in approximately % of the ccs at the age of years and were strong predictors of heart failure and ischemic heart disease. although the discrimination of the prediction models improved with the addition of cardiovascular risk factors, the c statistics were modest for both events ranging from . to . in the derivation cohort with successful replication in the other one-half of the cohort. both the heart failure and the ischemic heart disease predictions models showed good calibration. a small, very-highrisk group was identified with cumulative incidences of heart failure or ischemic heart disease of w % at age years; survivors in this very-high-risk group may benefit from more frequent surveillance and/or early interventions to modify their risk. however, low-risk survivors who may be excluded from further surveillance could not be identified with these models as cumulative incidences of heart failure (w . % to . %) and ischemic heart disease (w % to . %) were still significantly higher compared to siblings at the age of years. in the context of prediction model-guided surveillance, this can be seen as the benefit of early detection of asymptomatic cardiac dysfunction among those who will develop heart failure (true positives) weighted against the potential harm of an unnecessary diagnostic workup and/or treatment in those who will not develop heart failure (false positives). through decision modeling using simulations it has been shown that routine echocardiographic surveillance for asymptomatic cardiomyopathy every years may be more cost-effective, especially in those treated with an anthracycline dose < mg/m ( ). decision modeling provides weaker evidence on the clinical impact compared to an rct, but it requires no follow-up and is less expensive to perform. such analyses could be performed to assess clinical impact and cost-effectiveness before conducting an rct. there are different methods and techniques available to detect anthracycline treatment induced cardiomyopathy. much of the research in detection of cardiac diseases is focused on improving early detection of myocardial dysfunction. we will describe diagnostic methods that have been studied over the past decade in ccs. an abnormal gls in % of the cohort who were exposed to anthracyclines and/or chest rt and had normal lvefs. both cumulative anthracycline dose > mg/m and any cardiac rt dose were associated with an increased risk for abnormal gls ( ) . it is currently unknown whether an abnormal gls is associated with development of an lvef < % or clinical heart failure in ccs. diastolic dysfunction after cardiotoxic cancer treatment has also been described in ccs ( , ) . in the st. jude lifetime cohort, diastolic dysfunction grades to (based on peak mitral flow velocity, mitral septal and lateral early diastolic velocity, and left atrial volume) was detected in % of all ccs who were exposed to cardiotoxic treatment and in . % with normal lvef ( ) . one must be aware of the dif- table ). the current lack of mechanistic insights for ( , ) tyrosine kinase inhibitor- ( ) , and trastuzumab-related cardiotoxicity ( ) . inflammation studied primarily to reduce major adverse cardiovascular events was associated with a significant reduction of lung malignancy ( ). meta-analyses of therapies lowering low-density lipoprotein cholesterol (ldl-c) demonstrated that prevention of cardiovascular events was proportional to the absolute reduction in ldl-c levels with no major safety concerns, specifically no increase in malignancies ( , ) . however, available data from previous ldl-c-lowering clinical trials have not systematically adjudicated data for malignancy, because these were not considered disease-related events ( ) . an increased rate of malignancy associated with the use of the combination of ezetimibe and simvastatin was unexpectedly reported in the seas (simvastatin and ezetimibe in aortic stenosis) trial ( ) , which compared simvastatin-ezetimibe to placebo in , adults with mild-to-moderate aortic stenosis over a median follow-up of . years ( ). ezetimibe is a nonstatin drug that inhibits the intestinal absorption of cholesterol by targeting the transmembrane protein, nieman-pick c -like ( ) . it is recommended for further ldl-c reduction in combination with a statin ( values are median ( th, th percentiles) or n (%). wilcoxon rank-sum test of differences between with and without primary malignancies for continuous variables. chi-square test of frequencies between with and without malignancies for categorical variables. *the no-malignancy group summary statistics are based on patients without malignancy diagnosis prior to death, loss to follow-up or end of the study. acei ¼ angiotensin-converting enzyme inhibitor; acs ¼ acute coronary syndromes; arb ¼ angiotensin receptor blocker; hdl-c ¼ high-density lipoprotein cholesterol; hs-crp ¼ high-sensitive c-reactive protein; iqr ¼ interquartile range; ldl-c ¼ low-density lipoprotein cholesterol; mi ¼ myocardial infarction; trs p ¼ timi risk score for secondary prevention ( ) . of the skin, as well as limiting malignancy events to only those with available pathology reports. we also performed a competing risk analysis integrating allcause death as a competing outcome in the model using fine and gray's method. finally, we also evaluated the hrs in both arms with increasing duration of follow-up as done in previous publications ( , ) . all analyses were performed using sas software, simvastatin/ezetimibe (red) and placebo/ezetimibe (blue). the primary malignancy endpoint was defined as new, relapsing, or progressive malignancy (excluding nonmelanotic skin malignancy). the cumulative incidence plots were presented graphically using kaplan-meier product-limit method. ci ¼ confidence interval; hr ¼ hazard ratio. simvastatin/ezetimibe (red) and placebo/ezetimibe (blue). the cumulative incidence plots were presented graphically using kaplan-meier product-limit method. ci ¼ confidence interval; hr ¼ hazard ratio. table ) . results were similar between treatment groups for deaths due to malignancy by location table . values are n (%). (p > . for each comparison) (supplemental table ). further analyses by gender showed no significant differences between treatment groups for both the primary malignancy endpoint (p > . for each comparison) (supplemental tables and ) and deaths due to malignancy (p > . for each comparison) (supplemental tables and ). finally, we did not observe any differences in the extent of malignancy by treatment arm ( table ) . in subgroup analyses, the rates of the primary malignancy endpoint were similar between treatment groups for each of the pre-specified high-risk subgroups (each p for interaction > . ) ( figure (figure ). during the years of follow-up, there was no divergence in the km curves over time ( figure ) ; the year-by-year hrs for the primary malignancy endpoint comparing the two treatment arms did not demonstrate a progressive trend over time ( table ) . the number of events and the -year kaplan-meier rates are shown. total cholesterol: th percentile ¼ . mg/dl, th percentile ¼ . mg/dl and th percentile ¼ . mg/dl. abbreviations as in figure . some concern has been expressed that statin and other ldl-c-lowering drugs might be carcinogenic and consequently part of the malignancy data was collected retrospectively. fifth, although the analyses of outcomes stratified by subgroups were pre-specified, the power was low in these subgroups. finally, we did not measure phytosterols or other potential protective or tumor-promoting factors in blood to evaluate the effect of ezetimibe on the potential mechanistic pathways related to malignancy. we found that ezetimibe did not increase the rates of malignancy nor deaths due to malignancy in , patients with recent acs treated with simvastatin and followed up for a median of years totaling , patient-years of follow-up. acknowledgment therapies to reduce ldl-c such as statins or ezetimibe have significantly improved cardiovascular outcomes; this benefit has been ascribed to pleiotropic effects beyond lowering ldl-c. however, such pleiotropic mechanisms have raised safety concerns about long-term use. a potential carcinogenic effect has been of particular concern as earlier studies sug- -year risks in cantos were % (range % to %) and % (range % to %) for total cancer; % (range % to %) and % (range % to %) for colorectal cancer; and % (range % to %) and % (range % to %) for lung cancer. conclusions lifetime and -year risk of total, colorectal, and lung cancer can be estimated reasonably well in patients with established cvd with readily available clinical predictors. with additional study, these tools could be used in clinical practice to further aid in the emphasis of healthy lifestyle changes and to guide thresholds for targeted diag- ( ) . for the current study , patients were included, after exclusion of patients younger than years or older than years. detailed descriptions of the ucc-smart cohort and the cantos trial have been published elsewhere ( ) ( ) ( ) . the studies were approved by institutional review boards and all participants provided written informed consent. an overview of eligibility criteria is provided in supplemental table . missing data (per variable # . % for ucc-smart and # . % for cantos) were singly imputed by weighted probability matching using multivariable regression for the baseline and outcome data. complete case analysis yielded similar model coefficients. continuous variables were truncated at the st and th percentile to limit the effect of outliers on the model coefficients (i.e., leverage) ( ) . to prevent overfitting, predictors were preselected based on presence in previously published risk prediction models of multiple cancer types. antiplatelet use (aspirin, p y -adp receptor antagonist, or other, such as dipyridamole) was added as a predictor, due to its inclusion in multiple previously published prediction models for colorectal cancer and due to the common use of antiplatelet therapy in patients with cvd. furthermore, it was required that the variables were readily clinically available, as well as present in the derivation dataset. this led to the following predictors: age, sex, smoking status, weight, height, alcohol use, use of antiplatelet medication, and diabetes mellitus (supplemental table details an overview of predictor selection). in addition, crp was added as a predictor after a literature search for predictors of cancer was performed ( , , , ) . definitions of the predictors in the ucc-smart cohort and cantos trial are provided in supplemental table . was calculated for each following life-year. next, for each subsequent age year the probability of being healthy and alive at the start of that time interval (age year) (e t þ ) was calculated by multiplying the survival probability (e t ) by the event-free survival probability during that year ( À a t À b t ). these steps were repeated from the age at baseline of an individual patient to the maximum age of years, and together these predictions form an individual lifetable ( , ) . the cancer-free life expectancy was determined as the age at which the median estimated cancer-free survival curve is %. for -year and lifetime risk of cancer, the cumulative cause-specific risks were truncated at years after the age at baseline, and at the age of years, respectively. cantos study populations are shown in table ( ), leading to restricted variation in cancer types. our cancer prediction models performed reasonably well, and calibration plots before and after ( ). second, complex models based on machine learning would not be expected to provide improved performance or better prognostication in the current study, consistent with other large-scale external validation studies ( , ) . from a clinical perspective, a key concern should be whether the predictions from the risk calculator are well calibrated ( ) . for example, the -year-old male in the example calculation sheet has a predicted . % risk of lung cancer, while the risk may in fact be . %, or %. this type of miscalibration has been common in earlier evaluations of lung cancer prediction models ( ) . indeed, the investigators state that "calibration is a more clinically relevant for risk ( ) . it was recently rediscovered and presented through a "decision curve" ( , ) , which is unfortunately missing from the current report. future work should consider the clinical decisionmaking perspective more fully, with net benefit in a decision curve as a step towards a more comprehensive cost-effectiveness analysis. in sum, the presented risk calculator is very promising given its high-quality data sources, large values are mean ae sd or %. *p < . across diuretic categories. †defined as nt-probnp > , pg/ml or bnp > pg/ml. ‡defined as troponin-t > . ng/ml or troponin-i > . ng/ml. ace ¼ angiotensin converting enzyme; arb ¼ angiotensin receptor blocker; attr ¼ transthyretin amyloidosis; bmi ¼ body mass index; bnp ¼ b-type natriuretic peptide; dbp ¼ diastolic blood pressure; egfr ¼ glomerular filtration rate; gdmt ¼ guideline directed medical therapy; hf ¼ heart failure; lvef ¼ left ventricular ejection fraction; mra ¼ mineralocorticoid receptor antagonist; nt-probnp ¼ n-terminal pro-b-type natriuretic peptide; nyha ¼ new york heart association; sbp ¼ systolic blood pressure; shfm ¼ seattle heart failure model. table ). figure a) . similarly, for the uk þ diuretic dose þ nyha functional class model, survival was . years, . years, and . years for the low-, intermediate-, and high-risk groups, respectively (log-rank p < . ) ( figure b ). we also tested the ability of the risk scores to predict a combined outcome of mortality or cardiac transplantation ( table ). optimism-adjusted aucs at years ( adding diuretic dose to the uk score increased the auc from . to . and additionally adding nyha functional class, increased it to . . for diuretic dosing, points were assigned for mg/kg, point for > to . mg/kg daily dose, points for > . to mg/kg daily dose, and points for > mg/kg daily dose. *difference in discrimination slopes between models, where the discrimination slope is the difference between the mean predicted probabilities for events and nonevents. †sum of net proportions of persons with and without the event correctly assigned to a different risk category; ranges from À to . ‡similar to the idi except compares difference between median predicted probabilities. idi ¼ integrated discrimination improvement; nri ¼ net reclassification index; other abbreviations as in tables and . for diuretic dosing, points were assigned for mg/kg, point for > to . mg/kg daily dose, points for > . to mg/kg daily dose, and points for > mg/kg daily dose. abbreviations as in tables to . cheng et al. study limitations. our cohort included both wtattr and hattr. it is possible that risk markers may be differentially predictive in these distinct cohorts because natural disease progression is more aggressive with hattr, and there may also be differences between mutation types. of note, the mayo risk score ( ) only the latter remained significant in multivariable analysis. cardiovascular diagnosis usually includes imaging; thus cardiologists and patients have a tendency to focus on those results. although cardiovascular imaging has revolutionized the diagnosis of cardiac amyloidosis, the role in prognosis is less clear. when the addition of imaging variables to the staging systems is considered, the availability, reproducibility, and incremental value must also be considered. in contrast to the current study, nyha functional rodney falk appropriately coined the term "toxicinfiltrative cardiomyopathy" in reference to the toxicity of circulating light chains (al) to cardiomyocytes in al ( ). similar mechanisms of cardiomyocyte toxicity due to ttr oligomeric intermediates have been reported in attr ( , ) . cardiac amyloidosis is not a simple infiltrative con- doacs in cancer such as those related to trial designs or the risk for contamination or crossover between the groups. data synthesis and analysis. we extracted data from the original primary publications ( ) ( ) ( ) ( ) the flowchart describes study search, screening, and selection processes. sabatino et al. were included in the systematic review and metaanalysis ( ) ( ) ( ) ( ) . the study flowchart is described in figure . table . supplemental figure . overall, the risk for selection table . figure d) . overt bleeding that was associated with a decrease in the hemoglobin level of $ g/dl, led to a transfusion of $ u of blood, occurred in a critical site, or contributed to death overt bleeding that did not meet the criteria for major bleeding but was associated with the use of medical intervention, contact with a physician, interruption of the assigned treatment, discomfort, or impairment of activities of daily living. mcbane et al. ( ) ; overt bleeding plus a hemoglobin decrease of $ g/dl or transfusion of $ u of packed red blood cells, or intracranial, intraspinal/epidural, intraocular, retroperitoneal, pericardial, intra-articular, or intramuscular with compartment syndrome, or fatal bleeding overt bleeding not meeting the criteria for major bleeding but associated with medical intervention, an unscheduled contact with the health care team, or temporary anticoagulant cessation gi ¼ gastrointestinal; other abbreviations as in table . sabatino et al. over the past few years, the use of doacs has revo- values are n (%). *basal cell or squamous cell carcinoma of the skin, primary brain tumor or known intracerebral metastases, and acute leukemia were not included in the caravaggio trial. †basal cell or squamous cell carcinoma of the skin were not included in the select-d trial. ‡basal cell or squamous cell carcinoma of the skin were not included in the hokusai vte cancer trial. §other than brain tumors. ǁ data not available, because brain tumors were included under "other tumors". abbreviations as in tables and . the data from the caravaggio trial concerning the risk of major bleeding were particularly noteworthy. it appears that this finding was not related to any differences in the definition of major bleeding, because this was relatively consistent across trials ( table ). in contrast, the differences in reported major bleeding risk might have resulted from the heterogeneity in enrolled populations. patients with primary brain tumors, brain metastases, and acute leukemia were excluded from the caravaggio trial. patients with a recent diagnosis of testicular/retroperitoneal germ cell cancer and orchidectomy # weeks prior scheduled for cisplatin-based chemotherapy or active surveillance were recruited. participants were stratified into groups by management: ) active surveillance for stage low-risk disease; ) to cycles of adjuvant bleomycin, etoposide, and cisplatin (bep) chemotherapy for stage high-risk disease; or ) to cycles bep chemotherapy for metastatic disease. red circles illustrate study assessments. blood samples were simultaneously drawn from each table ) . this remained numerically greater than baseline thereafter, but was not statistically significant. there were trends toward abbreviations as in table . table ) . there was no differences between groups (p ¼ . ). endothelial dysfunction is a key feature in de novo thrombosis and rupture of pre-existing atherosclerotic plaque ( ) . as such, it is an important component to the risk of thrombotic cardiovascular events in the early period following cisplatin-based chemotherapy. in our cell-based study, exposure of haecs to cisplatin was associated with decreased t-pa mrna expression and activation of akt and erk / . in previous work, cisplatin has been shown to reduce endothelial cell survival and induce apoptosis in human dermal microvascular endothelial cells (hmec- ) ( ) and in the vasa nervorum in rats ( ) . these data support the hypothesis that cisplatin induces direct endothelial toxic effects resulting in increased stress kinase signaling and a propensity for thrombosis via reduction in the capacity for endogenous fibrinolysis. although adverse thrombotic effects are most frequently observed in the early period following chemotherapy, cisplatin is detectable in serum for several years after treatment ( ) . this chronic exposure may provoke low-grade endothelial stimulation and a consequent pro-atherogenic environment ( , ) . our subsequent assessment was with forearm venous occlusion plethysmography, the gold standard for assessing endothelial function ( ) . in appears to be the time period of increased cardiovascular risk. while these effects predominantly occur in the early period after treatment, cisplatin is detectable in serum several years after treatment ( ) . it remains possible that chronic exposure to low levels of cisplatin may cause low-grade endothelial stimulation that contributes to the pathophysiology underlying cardiovascular events occurring more than a decade after initial exposure ( , ) . nephrotoxicity is a major, dose-limiting side effect of cisplatin that affects % to % of patients ( , ) . we found evidence of early nephrotoxicity following cancer an acute decrease in fmd was seen as early as with the first cycle of carboplatin and paclitaxel chemotherapy ( ) . in conjunction with the current findings, one would conclude that the common denominator is platinum drugs and that there is no gender-related difference. furthermore, one would conclude that platinum drugs seemingly induce relatively acute changes in endothelial cells that translate into a reduction in flow-mediated vasodilation and alterations in nitric oxide bioavailability. interestingly, a prior study in testicular cancer survivors showed that those patients who were exposed to cisplatin-based chemotherapy nearly to more than years ago had a more severe reduction in fmd and higher levels of circulating endothelial cells than those not exposed ( ) . the fact that cisplatin levels are detectable even nearly years from therapy supports the theory of long-term exposure and endothelial injury ( ) . a difference in vascular reactivity between cisplatin-exposed and cisplatin-nonexposed was not seen in the current study by cameron et al. ( ) . it is important, however, to realize that the vascular response was blunted in both groups, even in comparison with patients with metabolic syndrome (considered to account for the vascular outcomes in testicular cancer survivors as mentioned) ( ) . such results can also not be unequivocally attributed to hypogonadism ( , ) . ( ) ( ) ( ) ( ) . in patients that survive beyond years, mortality rates are to times higher than the general population, corresponding with a % shorter life expectancy regardless of age at transplantation ( ) . the incidence of heart failure (hf) has been reported to be between . % to . % in those who have survived at least years after hsct ( , , , ) , and the risk of cardiovascular (cv) related mortality is to times higher in hsct survivors than in the general population ( were measured on separate occasions to reduce bias. studies followed current guidelines for evaluation of lv function and cardiotoxicity ( , ) . measurements were averaged from consecutive heart cycles. d-lvef was manually traced using the modified simpson's biplane method ( ) . d-lvef was calculated with semiautomated software for endocardial detection, and was subsequently manually adjusted. a d pyramid volume acquisition was obtained from to cardiac cycles, adjusting for depth and sector width ( to ), resulting in an average volume rate of frames/s (range to frames/s). sex-specific cutoffs for d-lvef were used as recommended ( ) . gls ( , , , ) . however, this study cannot discern the precise timing of lvsd onset, and one could potentially speculate that cardiac injury occurred at time of therapy, with a further worsening in lvsd over time. another possible explanation is that young patients are at higher risk of heart disease due to organ immaturity and growth disturbances caused by cardiotoxic therapies ( ) . this may explain the smaller lv size in survivors compared with control subjects, even after consideration for cv disease and confounders. sex has previously been implicated as a risk factor for cardiotoxicity ( , ( ) ( ) ( ) , but this association was not found in our study. cyclophosphamide has historically been noted to have cardiotoxic effects ( ) . however, published data linking alkylating agents to long-term heart disease is scarce. in contrast, anthracycline exposure is known to cause myocyte depletion, and is shown to increase the risk of hf by -fold in long-term ( , , , , ) . in addition to cardiotoxic therapies, we found that traditional cv risk factors have a potential role in modifying the risk for lvsd in long-term survivors of allo-hsct. cv risk factors are commonly reported in hsct survivors, occurring more frequently in allo-hsct compared with autologous-hsct survivors, resulting in a higher prevalence of heart conditions in allo-hsct survivors ( , , ( ) ( ) ( ) . survivors in this study had a high prevalence of cv risk factors, but at a level comparable to other studies ( , , , , ( ) ( ) ( ) . abbreviations as in table . treated with hsct at various ages ( , , , , , ) . the significance of hypertension in contributing to prevailing cardiac disease in hsct survivors has previously been shown ( , ) . however, in our study, given that the onset and duration of hypertension is unknown, it is unclear if the cumulative consequences of hypertension were fully manifested. indeed, the echocardiograms showed little evidence of concentric remodeling beyond a small but significant difference in indexed lv end-diastolic volume. however, hypertension is known to cause myocardial fibrosis that leads to reduced longitudinal shortening of the heart ( ) . this may explain why hypertension was found to be an independent predictor of gls. hypothyroidism has been reported in % of longterm survivors of hsct with busulfan conditioning ( ) . hypothyroidism was found in . % in our cohort, although no association with lvsd was found in our data. dyslipidemia has been reported in % to % of survivors of hsct ( , , , , , ) . in our study, gvhd was found to be a highly prevalent complication of allo-hsct survivors. there is limited evidence that gvhd directly mediates myocardial damage. however, active chronic gvhd values are median ( th, th percentiles), n (%), or mean ae sd. *significant difference between anthracycline $ mg/m and < mg/m in bonferroni post hoc analysis (< . ). †significant difference with both treatment groups (anthracycline $ mg/m and < mg/m ) with no anthracycline group in bonferroni post hoc analysis (< . ). ‡no significant difference between treatment groups in bonferroni post hoc analysis. §echocardiographical parameters are adjusted for covariates of age, heart rate, body mass index, and diastolic blood pressure. ksignificant difference between anthracyclines $ mg/m and no anthracycline in bonferroni post hoc analysis (< . ). ¶ase cube formula ( ) . table . has been shown to be associated with a higher risk of cv death ( ) . it is thought that the chronic inflammation processes instigated by gvhd results in endothelial damage leading to accelerated atherosclerosis ( ) . we did not find gvhd (acute or chronic) to be consistently associated with lv systolic function, and found a very modest association with remodeling ( , ) . a remaining challenge with gls is the lack of consensus in defining absolute cutoffs for dysfunction, variability, and intervendor differences ( ) . the gls cutoff used in our analysis was based on our normal data, using the same equipment and operator. this value (À %) is conservative when compared with a meta-analysis performed by yingchoncharoen et al. ( ) , which found the % ci for normal gls to be À . % to À . % ( ) .  phosphate-buffered saline) and sacrificed weeks later. as observed in previous studies ( , ) , body mass at the time of sacrifice was lower (À %) in the group administered with doxo ( table ) . lower body mass with doxo was associated with lower heart, quadriceps, gastrocnemius, and epididymal white adipose tissue mass, food consumption (À %), and water intake (À %) ( table ). there were no significant differences in the mass of other tissues and organs (supplemental table ). we first sought to establish that total ros bioactivity was increased in huvecs exposed to plasma from doxo-treated mice compared with sham control mice. to do so, we assessed total cellular ros using the cellrox fluorescent probe (thermo fisher [waltham, massachusetts]; catalog# c ) and found that ros bioactivity was greater in huvecs incubated with plasma from doxo-treated mice compared with sham control mice (p < . ) ( figures a and e ). next, we aimed to determine if enhanced mitochondrial ros contributed to the greater total huvec ros induced by doxo administration by assessing mitochondrial ros bioactivity using the mitosox fluorescent probe (thermo fisher, catalog# m ). we observed greater mitochondrial ros in huvecs exposed to plasma obtained from doxo compared with sham-treated animals (p < . ) (figures b and e ). to determine if this greater ros bioactivity was linked to changes in mitochondrial volume, we figure ) . to determine associations of these plasma metabolites with mitochondrialspecific aortic ros, we performed linear regression analyses. we found that plasma diphosphate (p ¼ . ) and lactate (p ¼ . ) tended to be positively associated with aortic mitochondrial ros (supplemental figure ) . a full report of metabolite abundance is provided in supplemental table . dot blot array. to expand on this initial analysis of potential circulating signals influenced by doxo treatment, we next targeted inflammatory pathways, given that doxo administration is associated with higher levels of pro-inflammatory proteins in the circulation ( ) . to address this aim, we assessed plasma concentrations of different cytokines and chemokines via a dot-blot array. we found that all but of these markers did not differ significantly in plasma from doxo-treated mice compared with sham control mice ( table ). the exceptions were the antiinflammatory cytokine interleukin- , which was only slightly (< %), but significantly (p ¼ . ) lower in the doxo mice, and vascular endothelial growth factor-a (vegf-a), which stood out as being % lower in the doxo treated group (p < . ). moreover, plasma vegf-a was inversely associated to accomplish this, we supplemented plasma from table ). doxo chemotherapy has been shown to impair endothelial function (i.e., reduce edd) in conduit arteries of cancer survivors ( ) . however, the underlying mechanisms and associated therapeutic targets have not been established, because mechanism-focused investigations to date have been largely limited to endothelial cell culture models ( , ) . in the present study, we first determined that these findings also are in agreement with previous work reporting excess production of mitochondrial ros and impaired vascular endothelial function in mice with genetic sod insufficiency ( ) . it should be noted that other ros generating pathways may also contribute, including oxido-reductases such as endothelial no synthase or nadph cytochrome p reductase ( ) . another key finding from our experiments was that changes in the composition of the circulating milieu may contribute to doxo treatment-associated anthracycline treatment in these same children ( ) . the decrease in nrg after anthracyclines has also been observed in adults undergoing anthracyclinebased chemotherapy, which is interpretable as a sign of vascular endothelial cell injury ( ). endothelial cell-derived nrg acts through the erbb receptor tyrosine kinase family to regulate the growth and survival of cardiac myocytes, and protects from anthracycline cardiac cytotoxicity ( , ) . mitoq and other coenzyme q preparations are available over the counter, and coenzyme q supplementation has been studied not only as a way to limit injury to the cardiovascular system, but also kidney and reproductive organs ( , ) . these prom- table ). fulfilled criteria for myocarditis was based on european were receiving dexamethasone as palliative treatment for metastatic disease (supplemental table ). and with other noncardiac iraes. crp in patients with no iraes was < mg/l at baseline and between c and c also was not significantly different (p ¼ . ). in this retrospective study of lung cancer patients receiving ici, there was an % incidence of mace to the rural population of eastern nc ( , ) . indeed, higher inflammatory signals and incidence of pneumonitis were also observed in this lung cancer population in comparison to those reported previously ( , ) . furthermore, in the lung cancer patients who received radiation, it has been suggested that the synergistic effect of radiotherapy and immunotherapy for priming of an endogenous antigenspecific immune response may contribute to a higher incidence of mace by t-cell recognition of shared antigens ( ) . values are mean ae sd or n (%). *analysis was not performed secondary to limited sample size. bb ¼ beta-blocker; bnp ¼ brain natriuretic peptide; cad ¼ coronary artery disease; ccb ¼ calcium channel blocker; ckd ¼ chronic kidney disease; copd ¼ chronic obstructive pulmonary disease; crp ¼ c-reactive protein; cva ¼ cerebrovascular disease; dm ¼ diabetes mellitus; egfr ¼ estimated glomerular filtration rate; hld ¼ hyperlipidemia; htn ¼ hypertension; nlr ¼ neutrophil-lymphocyte ratio; raas ¼ renin-angiotensinaldosterone system; wbc ¼ white blood cell count; other abbreviations as in table . timing of the development of mace with a median delay of days and a median of doses from the first ici administration in this study was similar to that previously reported ( , , ) . in view of this timing of onset from earlier studies, there is a general recommendation that baseline and surveillance cardiac testing (echocardiogram, ecg, tni and bnp) be considered during this potential mace window period, noted as after the second and after the fourth ici cycle administration ( ) . most of the present mace cases were not associated with a decrease in ef from baseline, which is consistent with previous studies ( , ) , suggesting that relying solely on ef in ici-treated patients with mace may be limited for the detection of ircs. tni was observed to be mildly elevated at the time of mace. however, elevations in tni have also been observed in cancer patients receiving cancer therapy, including icis without any cardiotoxicities ( , ) , thus suggesting that its utility may also be limited for the detection of irc. as demonstrated by this study, symptoms of irc may be variable, including nonspecific symptoms of our institution that observed a significant increase in crp with nivolumab-related pneumonitis that was mitigated with use of tocilizumab ( ) . similar inflammatory-mediated mechanisms such as interleukin (il)- may be observed in car t cell-related cardiotoxicities ( ) further highlighting and extending the utility of crp in the detection of ircs. another inflammatory marker that has been previously studied in noncardiac iraes and observed to be elevated at the time of irae is nlr ( , ) . nlr is easily obtained, inexpensive, and a routine test, and is calculated from the total white blood cell count that reflects the ratio of the innate (neutrophils) and the adaptive (lymphocyte) immune pathways, whereby an elevation results in an imbalanced toxic inflammatory response with release of cytokines ( ) . we observed an increase in nlr in patients who experi- however, ici treatment can result in immune-related adverse events (iraes) targeting any organ. cardiovascular iraes, particularly myocarditis, have received considerable attention due to their potentially fatal outcome ( , ) . ici were initially tested and approved as single therapy in patients with metastatic melanoma, a cancer type that historically had few treatment options and poor survival ( ) . later, these therapies were tested in patients with lung cancer and renal cell carcinoma ( , ) . in the latter studies, ici were tested either in combination with or following exposure to classic chemotherapies or targeted therapies. as a result, there was a growing need to define the cardiovascular sequelae in lung cancer patients treated with ici, where the cardiovascular risk was complicated by exposure to cardiotoxic nonimmune-based cancer therapies and a high prevalence of conventional cardiovascular risk factors ( ) . there was also a need to identify biomarkers or imaging approaches that screened for early cardiotoxic effects from immunotherapy and to identify patients at risk who would benefit from closer monitoring ( ) . histopathological analysis of the emb specimen showed intense myocardial inflammation comprised of numerous giant cells associated with many mononuclear cells, and prominent myocyte necrosis ( figure ). immunohistochemistry showed that the inflammatory cells were mainly cd positive giant cells and macrophages and cd -positive t-lymphocytes, many of them exhibiting granzyme b, perforin, and tia cytotoxicity markers. pd-l was expressed only by inflammatory cells and not by cardiomyocytes. cd and cd lymphocytes as well as nkp -positive nk cells were poorly represented (not shown). immunohistochemistry for viral protein detection was performed on heart biopsies and showed foci of positive cardiomyocytes surrounded by inflammatory infiltrates, indicating endomyocardial viral protein synthesis activities. steroids were gradually tapered and discontinued at months in light of the viral analysis. cardiac magnetic resonance imaging showed at weeks: ) resolution of myocardial edema (average myocardial t ¼ ms (figure ) , t values of ms, and extravascular volume decreased to %); ) recovery of lvef to %; and ) improvement of right ventricular function with an ejection fraction of % at weeks to % at months follow-up. the cancer had not progressed at months follow-up, and the patient reported normal functional status. ici therapy was not restarted at the time of last follow-up. this case highlights the heterogeneity of pathogenesis of acute myocarditis on ici therapy. we describe here acute myocarditis with long-term ici therapy, secondary to giant cell myocarditis due to enterovirus. giant cell myocarditis is uncommon; its pathogenesis is poorly understood, and prognosis is poor. recent data from an international registry including patients with histologically proven severe myocarditis demonstrated that giant cell myocarditis bears the worst prognosis ( ). the favorable outcomes in our patient, although the number of patients eligible to ici treatment is increasing dramatically, the pathogenesis of myocarditis needs to be further investigated. based on only extensively studied cases ( ) emerging clinical syndromes ( ) . the american heart association (aha) has recently proposed diagnostic criteria for ici myocarditis diagnosis based mainly on clinical characteristics, abnormal biomarkers, and/or cardiac imaging abnormalities ( ) . the recent publication of ici myocarditis definitions by the aha has the value of standardizing criteria to improve reporting. steroids are the first-line front treatment of ici myocarditis, despite poor evidence to support this, followed by other immune modulators, plasma exchange, or even ctla- agonist (abatacept) infusions. in the present study case, steroids were introduced and then discontinued after the confirmation of active viral markers on emb. indeed, the prognosis of t-cell ici myocarditis is partly driven by early initiation of steroid treatment ( ), which should not be held when awaiting histological or viral results. endomyocardial biopsy is recommended whenever possible in fulminant myocarditis ( ) . it is, however, likely to be overlooked in cancer patients. this case report emphasizes the risk for opportunistic viral infection during long-term ici therapy and utility of biopsy. the first mechanism to be considered in the setting of ici myocarditis is t-cell mediated, prompting steroids as a first-line therapy early after hospital admission. endomyocardial biopsy should be performed whenever possible to rule out infectious causes of myocarditis to guide therapy and further our understanding of the potential varying presentations of ici myocarditis. were paramount in preventing discordant care, especially when her clinical status had many rapid changes while she was in the intensive care unit. she completed car t cell therapy and was discharged to acute rehabilitation after a one-month hospitalization. she was unable to be restarted on gdmt due to hypotension. her pa d was mm hg at discharge. the plan was to obtain a positron emission tomography scan after acute rehabilitation to reevaluate her cancer. her initial course at the facility was uncomplicated, and she was able to participate in physical therapy. she had weekly appointments in the oncology clinic and an appointment with the advanced hf team. the pro- patients ( ) . hypotension is typically treated with intravenous fluids to maintain systolic blood pressure > mm hg. third-spacing of fluids with capillary leak is common, and this can lead to noncardiogenic pulmonary edema. a single-center retrospective study demonstrated that % of patients developed the composite outcome of arrhythmias, decompensated hf, or cardiovascular mortality ( ). the cardiac toxicities associated with car t cell therapy have led many centers to view significant cardiovascular disease as a relative contraindication ( ). in of the main clinical trials for car t cell therapy in patients with refractory dlbcl, patients were excluded if their lvef was # % to % ( ). our team decided to proceed with car t cell therapy in the patient described in this report because of her lack of other comorbidities, preserved performance status, and normal invasive hemodynamics. it was also her only treatment option for refractory dlbcl. the cardiomems device-which is typically used to manage outpatients with hf-was an essential component of our patient's medical treatment ( ) . for example, at periods in the patient's hospitalization she was found to have diffuse pulmonary infiltrates, hypoxemia, and hypotension. her pa d was and mm hg the incidence of secondary metastatic tumors to the heart is to times more common than primary malignant tumors, and estimated to be in the range of . % to . % at autopsy series in the general population, and up to . % in patients with known malignancies ( , ) . the incidence of cardiac metastases has increased over the past decades, likely due to improved life expectancy of oncological patients and advances in diagnosis ( , ) . the most common primary malignancies metastasizing to the heart include lung cancer, breast cancer, malignant melanoma, germ cell tumors, and hematological malignancies ( - ). the most frequent mode of metastatic spread of scc of the cervix is via the lymphatics to the para-iliac and para-aortic lymph nodes ( ). hematogenous spread is unusual and most commonly involves the lungs, bone, liver, and brain ( ). cardiac metastases from cervical cancer are exceedingly rare ( ) ( ) ( ) . the vast majority of cases have involved the right heart, presumably due to hematogenous spread of cervical cancer to the inferior vena cava via the uterine veins, coupled with filtration of tumor cells by the lungs. patients with cardiac metastases from cervical cancer present with a myriad of symptoms that depend on tumor location and size, leading to heart failure, arrhythmias, or tamponade ( - ). many cases were diagnosed postmortem. antemortem diagnosis was usually achieved by multimodality imaging including transthoracic and transesophageal echocardiography, ct angiography of the heart, cmr, and f-fdg pet/ct ( - ). tissue diagnosis should be pursued for prognostic implications and optimizing treatment, but may not be feasible. our patient had no symptoms related to the cardiac metastasis. the tumor was discovered by f-fdg pet/ct during the process of initial clinical staging of the disease. f-fdg pet/ct is a particularly useful imaging modality in this setting. together with cmr for detailed tumor characterization, these imaging modalities provide a comprehensive assessment of disease status that is essential for optimal shared decision making ( , ) . the median survival of patients diagnosed with cardiac metastasis from cervical cancer is months ( to months) ( ) ( ) ( ) . the management of these patients should be individualized and discussed within a dedicated multidisciplinary tumor heart team ( - ). a multimodality treatment strategy including chemoradiation, biological therapy, and surgery is necessary to prolong survival and maintain quality of life. in this regard, in a prospective randomized trial, bevacizumab has been shown to improve survival when added to the commonly used platinum-paclitaxel and topotecan-paclitaxel chemotherapy regimens ( ) . of note, treatment with bevacizumab has been shown to be associated with increased incidence of adverse cardiovascular side-effects such as uncontrolled hypertension, thromboembolism, cardiac ischemia, and heart failure ( ) . the decrease in lvef in our patient occurred immediately after resection and remained stable, and our patient did not have severe cardiac limitations during follow-up. her low lvef was readily managed with cardiac medications. near-complete resection of the interventricular septum via a right ventriculotomy parallel to the interventricular septum. reconstruction of the septum using a bovine pericardial patch (asterisk) sewn into the left ventricular side of the septum. lad ¼ left anterior descending coronary artery; other abbreviations as in figure . surgical resection should be considered for isolated cardiac metastasis in patients with favorable response to initial therapy and in whom complete resection is deemed feasible ( - ). our patient had excellent response of the disease in the cervix and of the single pelvic bone metastasis with no evidence of new disease, with the exception of the cardiac metastasis, which remained metabolically active. this is not an infrequent clinical phenomenon in patients with primary or secondary malignant tumors of the heart ( ), presumably due to differential response of the tumor to cancer therapy in different tissue environments ( ) . given the patient's young age, good functional status, imaging suggesting feasibility of complete resection, as well as lack of an alternative effective treatment, we proceeded with surgery followed by intensive chemoradiation and bevacizumab with curative intent. although the pathological examination in our case did not reveal residual tumor in the tissue specimen, surgical resection had an important impact on the design of her concurrent therapy and prognosis. the local recurrence supports the presence of tumor and findings of the pre-operative f-fdg pet/ct, highlighting the potential for false-negative tumor histological examinations following neoadjuvant chemotherapy ( ) . the radiological evidence of local recurrence in the area of slim resection margins-the basal-posterior septum-underscores the technical challenge of achieving complete r resection with clean margins when treating primary or secondary malignant tumors of the heart ( , ). in summary, cardiac metastases from cervical cancer should be included in the differential diagnosis of secondary malignant tumors of the heart. we believe that the patient-specific, multimodality management with curative intent strategized and delivered by an experienced and dedicated multidisciplinary team was key to achieving exceptional survival in our patient. twitter: @ayeletdaniels. distribution in the basal inferolateral and anterolateral free wall of the left ventricle. the etiology of this scar was unclear because the patient had no prior cardiac history, and to our knowledge, ibrutinib had not been known to be associated with myocardial scar. no abnormality was noted in the right ventricular apical region, which was the likely origin of his va. initially, the patient was managed with intravenous amiodarone for recurrent vt. after extensive multidisciplinary discussions among cardiology, electrophysiology and oncology teams, ibrutinib was discontinued due to its potential association with vt. the patient was started on metoprolol, and an implantable cardioverter-defibrillator (icd) was placed for secondary prevention. in terms of af, his cha ds -vasc score was , and anticoagulation was deferred, particularly given the potential bleeding risks with ibrutinib ( ). although it remained uncertain if ibrutinib played an integral role in the pathophysiology of vt, the medication was discontinued with close outpatient cardiology and oncology follow-up. because he had been in remission for several years, he was not started on alternative therapy for wm. on follow-up visits with close surveillance every months with cbc, comprehensive metabolic panel, and igm levels, he continued to be without evidence of symptomatic cytopenias, hyperviscosity, hepatosplenomegaly, lymphadenopathy, neuropathy, amyloidosis, cryoglobulinemia, or cold agglutinemia and did not meet criteria for reinitiation of therapy. on icd interrogation at months after cessation of ibrutinib, there were no further arrhythmias. ibrutinib is an oral, irreversible bruton's tyrosine kinase inhibitor used to treat a broad spectrum of b-cell proliferative disorders, including chronic lymphocytic leukemia (cll) as first-line therapy, mantle cell lymphoma, marginal zone lymphoma, and wm ( ) . cardiovascular toxicities associated with ibrutinib are the association of ibrutinib with vas is becoming increasingly recognized ( , , ) . in randomized controlled trials, the incidence of all-grade vas in patients treated with ibrutinib (n ¼ , ) compared with patients in the control arm (n ¼ ) was . % versus . %, and for grade or greater vas was . % versus % ( ). in analyses from a u.s.-based comprehensive cancer registry cohort, male sex, previous af, hf, coronary artery disease, diabetes, widened qrs, and valvular disease were associated with the development of any arrhythmias (va and supraventricular tachyarrhythmias) ( ). among those without baseline hf or coronary artery disease, the estimated , person-year incidence rate for vas was compared with . among similar nonibrutinibtreated subjects, which suggested an observed versus expected relative risk of . (p < . ). regarding drug dosage, in study in which approximately % of the patients experienced vas, % were taking at least mg of ibrutinib per day, and only % were taking mg or lower per day ( ). among those with ibrutinibassociated vas, the median time-to-event was months (range . to . months) ( ). ibrutinib has been reported to cause polymorphic vt without qtc prolongation as well as in the absence of structural heart disease ( , ) . tomcsányi et al. ( ) reported the case of a -year-old woman on ibrutinib for cll with underlying left bundle branch block and af who experienced ibrutinib-induced polymorphic vt in the absence of other causes ( ) . the initiation of vt was not characterized by short-long-short cycles as is seen in torsade de pointes. in polymorphic vt not related to prolonged qt, an alteration in the cardiac calcium homeostasis associated with ryanodine receptor-calmodulin-dependent protein kinase pathways is suspected. as such, it has been hypothesized that an interaction between these and pi k-akt pathways could potentially lead to polymorphic vt with ibrutinib ( ). although the awareness of ibrutinib-associated va is increasing, there are no published management guidelines. our patient was initially treated with amiodarone, then started on a beta-blocker. in the case described by tomcsányi et al. ( ) , the patient was treated with amiodarone and remained arrhythmia-free for months. amiodarone was thereafter discontinued due to pulmonary toxicity, and discontinuation led to the recurrence of vt. sotalol was found to be ineffective, as were class i antiarrhythmic agents ( ) . as ibrutinib is primarily metabolized by hepatic cytochrome p a (cyp a ), caution must be exercised when used in combination with amiodarone and the calcium-channel blockers diltiazem and verapamil, given that they inhibit cyp a and potentially result in increased serum levels of ibrutinib ( ) . amiodarone may increase the plasma concentration of ibrutinib by -to -fold ( ). ibrutinib interruption or dose adjustment may be appropriate prior to the initiation of amiodarone. furthermore, the class ia antiarrhythmic quinidine and the beta-blockers carvedilol and nadolol also interact with ibrutinib, increasing the serum concentrations of the antiarrhythmic drugs ( ) . according to the ibrutinib package insert, ibrutinib interruption is recommended for any nonhematological toxicity that is grade or greater. once the toxicity has resolved to grade or baseline, ibrutinib may be reintroduced at a low dose ( ) . for severe or refractory arrhythmias, such as seen in our patient, drug discontinuation may be necessary. in general, icd implantation is recommended for the secondary prevention of sudden cardiac death due to life-threatening vt/ventricular fibrillation in patients in whom a completely reversible cause cannot be identified ( ) . our patient had hemodynamically unstable vt episodes requiring resuscitation, and although ibrutinib was felt to be the culprit, the presence of delayed enhancement on cardiac magnetic resonance imaging made it difficult to rule out underlying structural heart disease, such as an infiltrative cardiomyopathy. additionally, an icd is indicated if it is expected to improve overall mortality. because our patient's long-term prognosis from wm was favorable, it was felt that he would benefit from an icd, particularly because ibrutinib or another therapy could be indicated in the future. in patients with cancer whose overall prognosis is poor (i.e., low expectation of survival with an acceptable functional status beyond year), icd therapy is not recommended. as ibrutinib is often used in patients with cll whose prognosis is generally favorable, icds for secondary prevention have a potential for long-term benefit. an additional advantage of an icd is providing prognostic parameters, such as the burden of atrial arrhythmia, nonsustained vt, and treated episodes of vt/ ventricular fibrillation, which could help to risk-stratify patients in the future prior to reintroduction of potentially cardiotoxic drugs. ibrutinib is a bruton's tyrosine kinase inhibitor associated with a well-known side effect of af. reported cases of vas associated with ibrutinib are rare. we present a case of vt storm in a patient receiving ibrutinib for wm. as the recognition of ibrutinib-associated vas is increasing, more data are needed to guide best management strategies for vas triggered by ibrutinib. the following clinical scenarios focused on cancer prevention highlight the value of systematic cardiovascular risk assessment in individuals at high risk of cancer who are treated with cancer risk-reducing therapies. recommendations regarding the evaluation of lipid abnormalities and the role of coronary artery calcium (cac) scan are provided, balancing existing evidence with the need for rigorous data that specifically applies to cancer populations. in terms of additional potential cardiovascular risk factors, brca has been implicated in the repair of dna double-stranded breaks, and its loss-of-function is associated with reduced cardiac performance and accelerated cardiomyocyte death in murine models. human studies investigating the association between brca / mutations and incident cardiovascular disease (cvd) have shown conflicting results, potentially due to variable sample sizes and ethnic-specific differences in the pathogenesis of cvd ( ). bilateral prophylactic oophorectomy is associated with a % reduction in the risk of epithelial ovarian cancer in brca / mutation carriers. in women younger than years of age, the risk of cardiovascular death after surgical oophorectomy is % higher than that of healthy control subjects ( ) . premature menopause was shown to predict future coronary heart disease and stroke in the mesa (multi-ethnic study of atherosclerosis) cohort and represents an important risk-enhancing factor ( ). the patient has a borderline-range ( % to . %) estimated ascvd risk in the presence of a risk-enhancing factor, namely premature menopause. given this, we would advocate for a discussion regarding initiation of moderate-intensity statin therapy. however, the patient has expressed a preference to avoid taking additional medications. in this case, a cac scan could help refine ascvd risk assessment ( ) . an agatston score of $ and/or a cac score $ th percentile for the patient's age, sex, and race would suggest a benefit to statin initiation, although there are no randomized clinical trial data supporting the use of cac in treatment decisions. a cac score of would increase this patient's -year coronary heart disease risk to % using the mesa risk score calculator, and would further support the use of lipid-lowering therapy. nicotine cessation should be a primary strategy to reduce cvd risk in this patient, while the importance of healthy lifestyle habits, normal-range blood pressure and lipid profile, weight loss, and glycemic control should also be emphasized. aspirin and sulindac are commonly used, contemporary pharmacoprophylactic therapies in patients who are at high risk of colorectal cancer, with the former being the preferred drug for individuals with sporadic adenomas and the latter for those with fap. in the late s, celecoxib was approved for use in patients with fap. the risk-reduction benefit appeared to be significant with a % lower colorectal polyp burden rate in fap patients treated with celecoxib and a % lower incidence of duodenal polyps. however, in a large study of colorectal adenoma prevention, long-term use of celecoxib ( or mg twice daily) was associated with a . -fold increased risk of death from cvd, myocardial infarction, stroke, or heart failure ( ). in addition, a meta-analysis of randomized controlled trials showed that celecoxib use was associated with a -fold increased risk of myocardial infarction. a subsequent prospective investigation, assessing the safety of a lower dose of celecoxib ( mg daily), did not demonstrate a statistically significant hazard; as such, the cardiovascular risk mediated by celecoxib remains incompletely understood ( ) . in , due to high cardiovascular event rates, rofecoxib was removed from the market, and a black-box warning was issued for celecoxib the following year. this patient is in an intermediate-risk ascvd category, but has additional factors that increase his likelihood of future cardiovascular events: chronic nonsteroidal anti-inflammatory drug (nsaid) use and a strong family history of premature ascvd. importantly, the presence of diabetes mellitus in an individual to years of age is an indication for the use of moderate-intensity statins regardless of ascvd score, and in an individual it is important to understand that patients with fap who are treated with preventive total colectomy have favorable long-term survival outcomes. the notion that cvd represents an important competing risk to cancer mortality should be conveyed and serve as a motivating factor to introduce lifestyle changes and consideration of pharmacological therapies. consideration of chronic nsaid initiation in this patient, especially in the context of conventional cardiovascular risk factors, should prompt referral to an internist or cardiologist. numerous risk-reducing interventions are utilized by cardiologists and oncologists in patients at risk of developing cardiovascular disease or cancer. some therapies used to lower the incidence of cancer in individuals at increased risk for malignancy (e.g., prophylactic bilateral oophorectomy in carriers of brca / mutations, sulindac in patients with familial adenomatous polyposis) may potentially increase the risk of cardiovascular disease. consideration of such interventions, particularly in individuals with coexisting cardiovascular risk factors or comorbidities, should prompt referral to cardiology for careful risk assessment and modification that integrates traditional risk factors and the effects of cancer risk-reducing therapies. ascvd ¼ atherosclerotic cardiovascular disease; cv ¼ cardiovascular; nsaid ¼ nonsteroidal antiinflammatory drug. importantly, decisions regarding long-term nsaid therapy should balance the reduced polyp and potential colorectal cancer rate with the increased cvd risk. attainment of optimal blood pressure and glycemic control should be targeted. in addition, a cac scan could be considered. if highly abnormal, it could be used to counsel and motivate this patient to further modify his lifestyle ( ) . it would also be reasonable to check a lipoprotein(a) level given the strong family history of premature atherosclerosis. due to the expected long-term use of sulindac, statin therapy and improved lifestyle habits would be appropriate risk-reduction interventions in this case. cardiovascular risk assessment in individuals who are deemed to be at high risk for cancer is distinct from that in patients with an established cancer. specific therapies aimed at reducing the risk of cancer may augment cardiovascular risk and deserve careful consideration. two important tenets of cardiovascular risk assessment include evaluation of lipid abnormalities and understanding the role of cac imaging in refining ascvd risk stratification in this unique population. although more research is needed regarding the value of cac burden in patients with cancer, available evidence suggests that elevated cac scores are predictive of both future cardiovascular events and cancer. in a recent study, cac > , when compared with a score of , was associated with a . -fold increase in the risk of cv death and a % increase in the risk of cancer death ( ). the most common cause of death among individuals with cac was cancer ( %), whereas patients with cac > experienced most of the mortality due to cvd. in a study of patients with locally-advanced nonsmall-cell lung cancer treated with thoracic radiation therapy, an increased cac score, measured from planning radiation therapy computed tomography, was associated with an elevated risk of all-cause mortality (hazard ratio: . ; confidence interval: . to . ; p ¼ . ) ( ) . in a separate analysis of breast cancer patients who had received radiation therapy, higher pre-rt cac scores were associated with a higher likelihood of acute coronary syndrome at years of follow-up (hazard ratio: . ; confidence interval: . to . ; p ¼ . ) ( ) . coronary calcification is therefore a predictor of cvd in the cancer population and further research is needed to better characterize the contribution of cause-specific mortality and cardiovascular events in cancer patients according to cac levels ( ) . last, breast arterial calcifications detected on screening mammogram are an independent marker for the presence of coronary artery disease and may help identify women at higher risk of ascvd. lipid-lowering therapies should be considered in patients with cardiovascular risk factors or disease, as per the standard ascvd risk score. particular medical interventions to decrease the risk of cancer, such as prophylactic oophorectomy or long-term nonsteroidal anti-inflammatory drugs, may increase the risk of ascvd and should be regarded as deleterious factors. in the coming decade, cardio-oncologists may be asked to assess and manage cardiovascular toxicities in individuals who receive cancer risk-reducing interventions but who are free of cancer. given the increasing data supporting an overlap between the molecular and clinical underpinnings of cancer and heart disease, oncologists should consider involving their cardiovascular medicine colleagues for risk assessment and preemptive management. in addition, longitudinal cardiovascular safety evaluations of specific patient subgroups in whom prophylactic anticancer therapies are considered should be performed to provide an evidence-based understanding of the risks and benefits of such therapies. cardiotoxic risk ( ) ( ) ( ) ( ) ; and the intersection between cardiovascular disease and cancer in our patients ( ) ( ) ( ) . in the second quarter of alone, we had nearly , article usage sessions, and each quarter, this number has grown. the work of our authors is being read for utilization in clinical practice and research, which is always the most important metric for a jacc journal. as a physician scientist, i recognize that the choice of "which journal" is not an easy one and requires careful consideration. we remain committed to serving our authors and working tirelessly to ensure that the peer review process is respectful, fair, constructive, and as seamless as possible. our average time to first decision in second quarter was days, and we will strive to maintain this standard. we seek to partner and work collaboratively with our authors to ensure the highest quality contribution to our community. i am grateful to the reviewers, who provide timely, incisive insight and lend their valued expertise to help ensure the quality of our journal. we have had nearly peer reviewers contribute their evalua- over the next year, we will improve access to jacc: cardiooncology through additional platform changes to improve searchability and integration across the jacc journals. clinical, translational, and basic science original research manuscripts will continue to serve as our foundation. as we evaluate each manuscript, we will continue to ask ourselves the following key questions: are the findings valid? is the methodology rigorous? is the topic of clinical importance? are the findings incremental to our current understanding of the topic and do they fulfill an evidence gap? what is the potential clinical impact and the potential for advancing the field? we will continue to publish state-of-the-art reviews and primers, and similarly ask ourselves: is this an authoritative, critical appraisal of the literature? is it comprehensive, yet focused? is this data-driven and accurately reflective of the current evidence? we will look to clinical case challenges to provide evidence-based descriptions of unique cases that thoughtfully illustrate the diagnostic and therapeutic dilemmas that we as clinicians face as we care for our patients ( ) . our viewpoints will continue to express opinion pieces on important and timely topics, and present thought-provoking, community-building, evidence-based perspectives. we will continue to grow our international engagement events, podcasts, live journal clubs, and dynamic case presentations, each occurring at least once per quarter. we also will launch a new "how to" series that will offer practical, evidence- oncologists primarily consulted general cardiology ( %), as compared with cardio-oncology ( %) for evaluation of treatment-related cardiotoxicity. overall, a lack of awareness ( %) and lack of and education in the growing field of cardiooncology. considering these respondents likely represented a more engaged group of practitioners, there was a lack of general cardio-oncology knowledge, uncertainty of local available resources, and low reported rate of cooperation between cardiologists and oncologists. although we were not able to assess the reasoning for these observed practice patterns, as it was beyond the scope of this study, enhanced collaboration between disciplines will be important and necessary to deliver optimal cardiovascular and oncologic care to this patient group. previous studies indicate that approaches vary between cardiologists and oncologists when using cardio-oncology services for the treatment of patients with cancer. peng et al. ( ) reported that most cardiologists ( %) felt that they should monitor for cardiotoxicity even in the absence of symptoms. however, the same study indicated that only . % of oncologists shared this view. furthermore, % of oncologists felt that cardiologists should be involved only when patients developed cardiotoxicities, but only . % of cardiologists agreed with that opinion. most cardiologists believed that access to cardiooncology services would improve prognosis ( . %), whereas only . % of oncologists shared this view. our study did not directly address this same question, but found that only % of oncologists and % of cardiologists indicated that they felt "very comfortable" interacting with their colleagues for comanagement of cancer and heart disease. sadlerd@ccf.org. twitter: @dsadlermd, @floridaacc, @flasco_org, @accintouch, @dr_mike_fradley, @dr_roohikhan. transthyretin amyloid cardiomyopathy: jacc state-of-the-art review natural his a new staging system for cardiac transthyretin amyloidosis cardiovascular disease mortality after chemotherapy or surgery for testicular nonseminoma: a populationbased study testicular cancer: a mechanisms of cisplatin nephrotoxicity white-paper- .pdf a new equation to estimate glomerular filtration rate crosstalk between vascular redox and calcium signaling in hypertension involves trpm (transient receptor potential melastatin ) cation channel marked impairment of protease-activated receptor type -mediated vasodilation and fibrinolysis in cigarette smokers: smoking, thrombin, and vascular responses in vivo intra-arterial substance p mediated vasodilatation in the human forearm: pharmacology, reproducibility and tolerability role of the endothelium in the vascular effects of the thrombin receptor (protease-activated receptor type ) in humans vascular effects of apelin in vivo in man the vasodilator action of nebivolol in forearm vasculature of subjects with essential hypertension fire simulation and cardiovascular health in firefighters vascular damage in testicular cancer patients: a study on endothelial activation by bleomycin and cisplatin in vitro antiangiointravascular ultrasound imaging for coronary thrombosis after cisplatin-based chemotherapy caspases and calpain are independent mediators of cisplatin-induced endothelial cell necrosis arterial events in cancer patients-the case of acute coronary thrombosis vascular toxic effects of cancer therapies treatmentrelated cardiovascular toxicity in long-term survivors of testicular cancer comprehensive characterisation of the vascular effects of cisplatin-based chemotherapy in patients with testicular cancer impact of platinum-based chemotherapy on the progression of atherosclerosis vascular fingerprint and vascular damage markers associated with vascular events in testicular cancer patients during and after chemotherapy cardiovascular risk in long-term survivors of testicular cancer long-term platinum retention after platinumbased chemotherapy in testicular cancer survivors: a -year follow-up study hematopoietic stem cell transplantation in europe : more than transplants annually reduced mortality after allogeneic hematopoietic-cell transplantation late mortality after allogeneic hematopoietic cell transplantation and functional status of long-term survivors: report from the bone marrow transplant survivor study vascular endothelium as 'novel' target of graft-versus-host disease pericarditis in patients with chronic graft-vs-host disease national institutes of health hematopoietic cell transplantation late effects initiative: the cardiovascular disease and associated risk factors working group report prevalence of outpatient cancer treatment in the united states: estimates from the medical panel expenditures survey (mpes) chemotherapy-induced cardiotoxicity: detection, prevention, and management long-term chemotherapy-related cardiovascular morbidity cardiovascular disease in adult survivors of childhood cancer epirubicin versus doxorubicin: which is the anthracycline of choice for the treatment of breast cancer? gp phox-containing nad(p)h oxidase increases superoxide formation by doxorubicin and nadph enzymatic defenses of the mouse heart against reactive oxygen metabolites: alterations produced by doxorubicin redox cycling of anthracyclines by cardiac mitochondria. i. anthracycline radical formation by nadh dehydrogenase adriamycin-induced interference with cardiac mitochondrial calcium homeostasis cumulative and irreversible cardiac mitochondrial dysfunction induced by doxorubicin role of endothelium in doxorubicin-induced cardiomyopathy a critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin chemotherapeutic drugs and mitochondrial dysfunction: focus on doxorubicin, trastuzumab, and sunitinib glabridin prevents doxorubicin-induced cardiotoxicity through gut microbiota modulation and colonic macrophage polarization in mice vegf-b gene therapy inhibits doxorubicin-induced cardiotoxicity by endothelial protection endothelial function in young adult survivors of childhood acute lymphoblastic leukemia cellular senescence promotes adverse effects of chemotherapy and cancer relapse mitochondria-targeted antioxidant (mitoq) ameliorates age-related arterial endothelial dysfunction in mice sex differences in anthracycline-induced cardiotoxicity: the benefits of estrogens shortterm interleukin- treatment improves vascular endothelial function, endurance exercise capacity, and whole-body glucose metabolism in old mice doxorubicin acts through tumor necrosis factor receptor subtype to cause dysfunction of murine skeletal muscle oxidant stress and endothelial cell dysfunction redox regulation of mitochondrial function increased accumulation of doxorubicin and doxorubicinol in cardiac tissue of mice lacking mdr a p-glycoprotein redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses innovative medicine: basic research and development antioxidants and cancer prevention antioxidant supplementation and breast cancer prognosis in postmenopausal women undergoing chemotherapy and radiation therapy endothelial nitric oxide synthase-dependent superoxide generation from adriamycin doxorubicin accumulation in individually electrophoresed organelles doxorubicin induces endotheliotoxicity and mitochondrial dysfunction manganese superoxide dismutase and aldehyde dehydrogenase deficiency increase mitochondrial oxidative stress and aggravate age-dependent vascular dysfunction rapid-onset endothelial dysfunction with adriamycin: evidence for a dysfunctional nitric oxide synthase mechanism of guanine-specific dna damage by oxidative stress and its role in carcinogenesis and aging nuclear factor-{kappa}b activation contributes to vascular endothelial dysfunction via oxidative stress in overweight/obese middle-aged and older humans chronic supplementation with a mitochondrial antioxidant (mitoq) improves vascular function in healthy older adults the mitochondria-targeted anti-oxidant mitoquinone decreases liver damage in a phase ii study of hepatitis c patients a double-blind, placebo-controlled study to assess the mitochondria-targeted antioxidant mitoq as a disease-modifying therapy in parkinson's disease vitamin c mitigates oxidative/nitrosative stress and inflammation in doxorubicin-induced cardiomyopathy protective effects of mito-tempo against doxorubicin cardiotoxicity in mice doxorubicin inactivates myocardial cytochrome c oxidase in rats: cardioprotection by mito-q. biophys the role of endothelins and their receptors in heart failure plasma endothelin- and doxorubicin cardiotoxicity plasma endothelin- as a marker for doxorubicin cardiotoxicity doxorubicin-induced oxidative stress and endothelial dysfunction in conduit arteries is prevented by mitochondrial-specific antioxidant treatment coenzyme q for prevention of anthracycline-induced cardiotoxicity anthracycline causes impaired ventricular-arterial coupling in breast cancer patients after treatment with anthracycline-containing adjuvant chemotherapy serum cardiovascular growth factors during doxorubicin chemotherapy in children with leukemia circulating neuregulin during the transition from stage a to stage b/c heart failure in a breast cancer cohort cardiac endothelial cells regulate ros-induced cardiomyocyte apoptosis through neuregulin- beta/ erbb signaling neuregulin- protects ventricular myocytes from anthracycline-induced apoptosis via erbb -dependent activation of pi -kinase/akt endothelial cells regulate physiological cardiomyocyte growth via vegfr -mediated paracrine signaling protective mechanisms of coenzyme-q may involve up-regulation of testicular p-glycoprotein in doxorubicin-induced toxicity reno-protective efficiency of coenzyme q on adriamycin-induced nephrotoxicity fundamental mechanisms of immune checkpoint blockade therapy immune checkpoint blockade therapy for cancer: an overview of fda-approved immune checkpoint inhibitors immunerelated adverse events associated with immune checkpoint blockade fulminant myocarditis with combination immune checkpoint blockade cardiovascular toxicities associated with immune checkpoint inhibitors: an observational, retrospective, pharmacovigilance study immune checkpoint inhibitors-related cardiotoxicity cardiotoxicity associated with ctla and pd blocking immunotherapy cardiotoxicity of immune checkpoint inhibitors association between immune-related adverse events and clinical efficacy in patients with melanoma treated with nivolumab: a multicenter retrospective study risk factors for immune-related adverse events associated with anti-pd- pembrolizumab healthy north carolina : social determinants of health indicators inflammatory signature difference in rural urban and regional occupational exposure in lung cancer current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the european society of cardiology working group on myocardial and pericardial diseases myocarditis in the setting of cancer therapeutics: proposed case definitions for emerging clinical syndromes in cardio-oncology what is the normal value of the neutrophil-to-lymphocyte ratio? immune checkpoint inhibitor-related adverse cardiovascular events in patients with lung cancer tocilizumab for the management of immune mediated adverse events secondary to pd- blockade myocarditis in patients treated with immune checkpoint inhibitors influenza vaccination and myocarditis among patients receiving immune checkpoint inhibitors serial troponin for early detection of nivolumab cardiotoxicity in advanced non-small cell lung cancer patients prognostic value of troponin t in cardiac risk stratification of cancer patients undergoing high-dose chemotherapy cardiovascular events among adults treated with chimeric antigen receptor t-cells (car-t) car t cell-induced cytokine release syndrome is mediated by macrophages and abated by il- blockade vinten-johansen j. involvement of neutrophils in the pathogenesis of lethal myocardial reperfusion injury impairment of the pd- pathway increases atherosclerotic lesion development and inflammation proatherogenic immune responses are regulated by the pd- /pd-l pathway in mice ctla -igg ameliorates homocysteine-accelerated atherosclerosis by inhibiting t-cell overactivation in apoe-/-mice impact of baseline steroids on efficacy of programmed cell death- and programmed death-ligand blockade in patients with non-small-cell lung cancer trends in stage distribution for patients with nonsmall cell lung cancer: a national cancer database survey pembrolizumab versus chemotherapy for pd-l -positive non-small-cell lung cancer cardiovascular toxicities associated with immune checkpoint inhibitors fulminant myocarditis with combination immune checkpoint blockade improved survival with ipilimumab in patients with metastatic melanoma nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer pembrolizumab plus axitinib versus sunitinib for advanced renal-cell carcinoma cardiovascular toxic effects of targeted cancer therapies management of cardiac disease in cancer patients throughout oncological treatment: esmo consensus recommendations characterization of immune checkpoint inhibitor-related cardiotoxicity in lung cancer patients from a rural setting grounding cardio-oncology in basic and clinical science acc/aha key data elements and definitions for cardiovascular endpoint events in clinical trials: a report of the american college of cardiology/american heart association task force on clinical data standards (writing committee to develop cardiovascular endpoints data standards) myocarditis in the setting of cancer therapeutics: proposed case definitions for emerging clinical syndromes in cardio-oncology pd- modulates radiation-induced cardiac toxicity through cytotoxic t lymphocytes cardiovascular toxicities associated with immune checkpoint inhibitors: an observational, retrospective, pharmacovigilance study key words immune checkpoint inhibitors, inflammatory markers, myocarditis, neutrophil-tolymphocyte ratio management and outcomes of severe aortic stenosis in cancer patients healthcare cost and utilization project (hcup) aha/acc focused update of the aha/acc guideline for the management of patients with valvular heart disease: a report of the american college of cardiology/american heart association task force on clinical practice guidelines transcatheter aortic valve replacement in oncology patients with severe aortic stenosis cardiac complications of thoracic irradiation roadmap for biomarkers of cancer therapy cardiotoxicity early changes in cardiovascular biomarkers with contemporary thoracic radiation therapy for breast cancer, lung cancer, and lymphoma normalization of ejection fraction in subjects with systolic heart failure. is it really normal? a myocardial deformation study assessment of early radiation-induced changes in left ventricular function by myocardial strain imaging after breast radiation therapy late-onset giant cell myocarditis due to enterovirus during treatment with immune checkpoint inhibitors i mmune checkpoint inhibitor (ici) therapy has significantly improved the prognosis of many advanced cancers cardiovascular adverse effects such as myocarditis are uncommon, but bear high mortality rates of nearly % ( ). ici myocarditis typically occurs early, and potential factors associated with ici myocarditis are combination therapy however, the clinical presentation of ici myocarditis is heterogeneous, with preserved left ventricular ejection fraction in one-half of the cases, and high rates of conduction abnormalities and ventricular arrhythmias ( ). ici myocarditis can also occur late, although notably, there is limited information on late-onset myocarditis with long-term ici therapy a -year-old male patient presented with myalgias and dyspnea after the th course of nd-line ici monotherapy (nivolumab [anti-pd- ] mg/kg every weeks) for metastatic renal cell carcinoma. the electrocardiogram showed sinus tachycardia, low qrs voltage, and t-wave inversion in the anterior leads with no st-segment changes. cardiac biomarkers were elevated (troponin i , ng/l, b-type natriuretic peptide ng/l). left heart catheterization revealed normal coronary arteries. cardiac magnetic resonance issn cardiovascular imaging the authors attest they are in compliance with human studies committees and animal welfare regulations of the authors fatal toxic effects associated with immune checkpoint inhibitors: a systematic review and meta-analysis cardiovascular toxicities associated with immune checkpoint inhibitors: an observational, retrospective, pharmacovigilance study clinical features, management, and outcomes of immune checkpoint inhibitorrelated cardiotoxicity myocarditis in patients treated with immune checkpoint inhibitors fulminant myocarditis with combination immune checkpoint blockade fulminant versus acute nonfulminant myocarditis in patients with left ventricular systolic dysfunction comparative immunologic characterization of autoimmune giant cell myocarditis with ipilimumab aberrant pd- ligand expression contributes to the myocardial inflammatory injury caused by coxsackievirus b infection myocarditis in the setting of cancer therapeutics: proposed case definitions for emerging clinical syndromes in cardio-oncology management of immune-related adverse events in patients treated with immune checkpoint inhibitor therapy report a -year-old woman with advanced diffuse large b-cell lymphoma (dlbcl) was referred for chimeric antigen receptor (car) t cell therapy. she was diagnosed with dlbcl years prior to presentation and had no significant past medical history. she received a total of cycles of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (r-chop), with a lifetime exposure of doxorubicin > mg/m . she underwent an autologous hematopoietic stem cell four years later, her dlbcl recurred, requiring more cycles of r-chop. one month after she completed the last cycle, her transthoracic echocardiogram (tte) demonstrated a left ventricular ejection fraction (lvef) of % to % and global longitudinal strain of À % (normal: À % to À %). two months following therapy, she was admitted to the hospital with acute decompensated heart failure (hf) coronary computed tomography demonstrated mild coronary artery disease, and cardiac magnetic resonance imaging showed no evidence of infiltrative or inflammatory disease. she was presumed to have anthracycline-induced cardiomyopathy and was initiated on furosemide and guideline-directed medical therapy (gdmt), including lisinopril . mg and metoprolol succinate . mg daily. up-titration of gdmt was limited, and spironolactone or eplerenone could not be added, secondary to hypotension. she was referred to a cardio-oncologist for risk stratification prior to car t cell therapy. she had good exercise tolerance, riding a stationary bike for to min at a time. a right heart catheterization (rhc) demonstrated mildly elevated biventricular filling pressures with normal cardiac output (right atrium mm hg, pulmonary artery / / mm hg it was felt that car t cell therapy was her only therapeutic option. multidisciplinary discussions between her hematologist/oncologist, cardio-oncologist, and advanced hf team were held to determine feasibility. derangements in blood pressure and oxygenation, ranging from mild hypotension and hypoxemia to issn dr. bishop has received nonfinancial support from articulatescience llc; and has received personal fees from united healthcare, seattle genetics, celgene, juno therapeutics, novartis, crispr therapeutics, kite pharma, and pharmacyclics. dr. uriel has received grant support from abbott the authors attest they are in compliance with human studies committees and animal welfare regulations of the authors' institutions and food and drug administration guidelines, including patient consent where appropriate. for more information chimeric antigen receptor t-cell therapies for lymphoma chimeric antigen receptor t-cell therapy for cancer and heart: jacc council perspectives cardiovascular events among adults treated with chimeric antigen receptor t-cells (car-t) yakoub-agha i. an international survey on the management of patients receiving car t cell therapy for haematological malignancies on behalf of the chronic malignancies working party of ebmt design and rationale of haemodynamic guidance with cardiomems in patients with a left ventricular assist device: the hemo-vad pilot study advances in diagnosis and treatment of metastatic cervical cancer cardiac metastasis in cervical cancer a rare case of cardiac metastatic mass case report of cardiac metastasis from cervical squamous carcinoma and its literature review tumors metastatic to the heart cardiac tumors: jacc cardiooncology state-ofthe-art review surgery for tumors of the heart improved survival with bevacizumab in advanced cervical cancer differential growth and responsiveness to cancer therapy of tumor cells in different environments akim@uchc.edu. twitter: @agnesskim , @hmadgula ibrutinibassociated atrial fibrillation ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia cardiovascular toxicities associated with ibrutinib the risk of atrial fibrillation with ibrutinib use: a systematic review and meta-analysis ventricular arrhythmias and sudden death in patients taking ibrutinib ventricular arrhythmias following ibrutinib initiation for lymphoid malignancies highlights of prescribing information ibrutinib, an approved tyrosine kinase inhibitor as a potential cause of recurrent polymorphic ventricular tachycardia ventricular tachycardia caused by ibrutinib dr. kopecky has served as a consultant for prime therapeutics; has received research support from true health; has served as data safety and monitoring board chair for applied clinical intelligence; has served as a board member for mayo clinic support services; and has served as a task force member for mayo clinic cv p&t. dr. gersh has served as cro for trials involving edwards percutaneous valve devices through baim institute; has served on the dsmb for the reprise study ); has served as a general consultant for myokardia; and has served on the steering committee of the garfield study (thrombosis research institute) risk of cardiovascular disease in women with brca and brca mutations increased cardiovascular mortality after early bilateral oophorectomy early menopause predicts future coronary heart disease and stroke: the multi ethnic study of atherosclerosis association of coronary artery calcium in adults aged to years with incident coronary heart disease and death cardiovascular risk associated with cardiovascular safety of celecoxib, naproxen, or ibuprofen for arthritis coronary artery calcium and the competing long-term risk of cardiovascular vs. cancer mortality: the cac consortium elevated coronary artery calcium quantified by a deep learning model from radiotherapy planning scans predicts mortality in lung cancer is the coronary artery calcium score associated with acute coronary events in breast cancer patients treated with radiotherapy? breast arterial calcium: a game changer in women's cardiovascular health? jacc: cardiooncology: poised to serve a maturing, collaborative field proceedings from the global cardio-oncology summit jacc: cardiooncology videos resiliency and our cardio-oncology community the novel coronavirus disease (covid- ) threat for patients with cardiovascular disease and cancer covid- clinical trials: a primer for the cardiovascular and cardiooncology communities al amyloidosis for the cardiologist and oncologist efficacy of neurohormonal therapies in preventing cardiotoxicity in patients with cancer undergoing chemotherapy safety of continuing trastuzumab despite mild cardiotoxicity: a phase i trial efficacy of dexrazoxane in preventing anthracycline cardiotoxicity in breast cancer repeated remote ischemic conditioning reduces doxorubicin-induced cardiotoxicity osimertinib-induced cardiotoxicity: a retrospective review of the fda adverse events reporting system (faers) increase in blood pressure associated with tyrosine kinase inhibitors targeting vascular endothelial growth factor car t-cell therapy-related cardiovascular outcomes and management: systemic disease or direct cardiotoxicity? cardiovascular safety of degarelix versus leuprolide for advanced prostate cancer: the pronounce trial study design hypertension in cancer patients and survivors: epidemiology, diagnosis, and management increased cancer prevalence in peripartum cardiomyopathy ischemia and bleeding in cancer patients undergoing percutaneous coronary intervention infiltrative lymphoma-associated bradycardia and cardiac conduction abnormalities deaths: leading causes for the economic burden of chronic cardiovascular disease for major insurers health of patients with cancer and cancer survivors an international survey of healthcare providers' knowledge of cardiac complications of cancer treatments practices in management of cancer treatmentrelated cardiovascular toxicity: a cardio-oncology survey acc multistate chapter cardio oncology network to improve physician participation, awareness, and education in cardio oncology myocardial ischemia: leading the differential diagnosis of polymorphic vt in a -year-old man was acute coronary syndrome.heart failure (hf): he did not have symptoms or signs of hyperviscosity syndrome or hf. systemic amyloidosis associated with wm leading to amyloid cardiomyopathy was also considered.drug toxicity: a high index of suspicion was maintained about the potential association between ibrutinib and ventricular arrhythmia (va). electrocardiogram at the time of evaluation by emergency medical services showed af with a ventricular rate of beats/min and nonspecific st/t-wave abnormalities. complete blood count, serum electrolytes, and renal function were within normal limits at the time of admission, and serial serum troponin levels were undetectable. a transthoracic echocardiogram showed normal biventricular size and function with no regional wall motion abnormalities. furthermore, coronary angiography demonstrated very mild luminal irregularities without obstructive disease. in the cardiac intensive care unit, he was noted to have multiple premature ventricular complexes and nonsustained vt with a left bundle branch block morphology and right superior axis, indicating an origin at the right ventricular apex (figure ). he underwent cardiac magnetic resonance imaging, which demonstrated a small area of mid-myocardial delayed enhancement in a nonvascular key: cord- -w wrwj authors: rothstein, rachel; olympia, robert p. title: school nurses on the front lines of healthcare: the approach to maintaining student health and wellness during covid- school closures date: - - journal: nasn sch nurse doi: . / x sha: doc_id: cord_uid: w wrwj in response to the novel coronavirus disease (covid- ) pandemic, most states in the united states enacted statewide school closures, ranging in duration from month to the remainder of the academic year. the extended durations of these closures present unique challenges, as many families rely on the school as a source of physical activity, mental health services, psychosocial support, child care, and food security. while the school doors may be closed, the school nurse can still play a vital role in emergency management. this article discusses challenges and proposes solutions to maintaining student health and wellness during extended school closures due to the covid- pandemic. furthermore, it is inevitable that until a vaccine for coronavirus is developed and readily available, many schools will continue to see future closures, though likely for shorter periods of time, as they respond to local outbreaks. what is the purpose of the "school nurses on the front lines of healthcare" series? the "school nurses on the front lines of healthcare" series will present cases reflecting emergencies commonly encountered in the school setting, focusing on an evidence-based approach to the initial management, stabilization, and disposition of the ill or injured child. special features unique to each article are extra credit points and report cards. extra credit points are trivia questions or clinical pearls scattered throughout the article related to the topic at hand. report cards are concise tables summarizing key points in each article that you can photocopy and laminate or photograph and keep on your smart device for easy access. during an otherwise normal afternoon in early march, your superintendent urgently assembles the school nurses, n asxxx . school nurses on the front lines of healthcare the approach to maintaining student health and wellness during covid- school closures principals, and other key faculty members, as well as local public health officials, to discuss the growing threat of covid- . your team ultimately decides to close the schools for the next weeks, effective immediately. shortly thereafter, your state governor declares a state of emergency and announces statewide closure of schools for a minimum of weeks. parents in the community begin contacting your team with concerns. many parents are essential workers and cannot obtain child care. others are concerned about food security, in the absence of school meal programs. the list of student health and wellness concerns continues. your school district is fortunate to have an emergency procedure in place for infectious disease outbreaks, and the school closure provides you and the school staff time to review and update your emergency and disaster plan, addressing many aspects of wellness for your students and their families. while the term social distancing is likely new to many, the concept of school closures as a tool for reducing spread of infectious diseases originated long before the covid- pandemic. during the - influenza h n pandemic, over schools, housing nearly , students, closed across the united states (klaiman et al., ) . while the current durations of school closures exceed those of other infectious disease outbreaks in recent history, consistent are the challenges to maintaining student health and wellness in light of closures. the national association of school nurses (nasn) recognizes the school nurse as a "vital member of the school team, who collaborates with community agencies to develop comprehensive emergency response procedures" (nasn, , para. ). likewise, the school nurse provides expertise in school health as a "leader and integral partner with school staff and outside agencies in developing comprehensive school plans/procedures" (nasn, , para. ) . nasn is continually providing up-to-date resources on covid- for school nurses, which incorporate emerging information from the centers for disease control and prevention (cdc) and other groups (cdc, b; nasn, ) . furthermore, the uncertainty surrounding future "peaks" of covid- or other infectious disease outbreaks emphasizes the importance of emergency and disaster planning. as school districts enter this unfamiliar phase of emergency management, the school nurse plays a critical role in addressing student health and wellness during school closures. the document, "considerations for school nurses when providing virtual care," highlights the role of virtual care in addressing student needs, continuing case management, providing resources to families, and mitigating health disparities (nasn, ) . nasn also provides, "guidance for school nurses to safely send and receive resources between school and home during covid- ," to ensure safe transfer of both student and school property, including backpacks, student medication, school supplies, electronic devices, and ongoing food services to socially and medically disadvantaged families (nasn, ). finally, "ideas for school nurse activities during the covid- pandemic" provides a list of activities that school nurses can complete in light of closures (nasn, ) . regarding school reopenings, nasn provides template letters, addressing state leaders, local leaders, and school superintendents, advocating for the inclusion of school nurses in planning processes (nasn, currently, sectors of the workforce qualify as "critical infrastructures" and are essential to the country's continued functioning, potentially leaving . million children, who otherwise would be in school, without adequate supervision (bayham et al., ) . while there is no definitive age at which it is appropriate to leave a child home alone, many resources are available to guide parents in this decision. leaving children home alone may present risks to both physical health and mental well-being. on the national level, the families first coronavirus response act includes a provision that expands protection for employees that during a public health emergency are unable to work due to a need for leave to care for their child because the school or day care has been closed or the child care provider is unavailable (moss et al., ) . similarly, the coronavirus aid, relief and economic security act includes a $ . billion expansion in funding of the child care and development block grant, to help states establish child care assistance for essential workers ( johnson-staub, ). examples of such interventions include temporary pandemic child care centers in ohio and regional enrichment centers in new york city. to assist your community's essential workers in obtaining child care coverage, view your state's emergency child care provisions and actions in response to covid- on the hunt institute website in table . also, the khan academy websites listed in table offer free online video classes, including music, games, crafts, and educational materials, for children to follow along while parents work or accomplish tasks in the home. the cdc recommend that all children aged to years participate in hour of moderate to vigorous physical activity each day, with an emphasis on aerobic activities such as walking, running, or biking, and that all children to years old participate in hours of physical activity daily, through activities spread over the course of the day (cdc, c). in addition to maintaining a healthy body weight and fostering musculoskeletal, cardiovascular, and mental health, physical activity in children is crucial to "self-expression, building self-confidence, social interaction and integration" (world health organization, , para. ). schools help children incorporate physical activity into their daily routines through participation in physical education (pe), recess, sports, and other extracurricular activities. as a result of school closures, children lose access to these structured and supervised sources of physical activity. the extended duration of school closures resembles summer recess, a period during which physical activity levels decline and sedentary time and body mass index rise (brazendale et al., ) ; unlike summer recess, the pandemic limits children's access to alternative sources of physical activity in the community, due to closures of public spaces such as parks, sports fields, tracks, and gyms. many families lack resources and equipment in the home for children to continue their usual activities. finally, social distancing limits the social interaction and bonding that physical activity often affords children. prior to the covid- pandemic, nearly % of children in the united states were overweight or obese, and children spent an average of . hours in front of a screen per day (brazendale et al., ; cdc, ) . both statistics are likely to rise in light of the pandemic. families now have a critical opportunity to adopt new attitudes and strategies to make physical activity a priority. to provide families in your community with outdoor physical activities that follow social distancing guidelines, visit the healthy children website in table . the online pe network website in table provides a list of grade-specific physical activities for the home, as well as monthly and weekly calendars to make these activities a part of children's daily routines. finally, in place of traditional screen time, the gonoodle website in table provides free online physical activity and mindfulness videos for young children to follow. approximately . million children in the united states participate in the national school lunch program, which provides discounted or free school lunches to children of low-income households (u.s. department of agriculture [usda], ). similarly, the school breakfast program provides school breakfast to nearly . million children in the united states. (usda, ). food insecurity disproportionately affects children residing in rural and large urban areas, as well as singleparent, black and hispanic households (no kid hungry, ). childhood hunger can contribute to malnutrition, difficulty concentrating, impaired academic performance, stress, poor social skills, and other adverse physical and mental health outcomes (no kid hungry, ). as a result of school closures, children lose access to these routine daily sources of meals. as of may , , children in these programs have missed a collective one billion school meals (no kid hungry, ) . to locate resources to help families in your community maintain food security, visit the no kid hungry and usda websites in table . even children residing in food secure households may encounter difficulty in maintaining healthy eating during school closures. for many children, school lunches provide the most nutritious meals of the day, by ensuring inclusion of fruits, vegetables, and milk. during school closures for summer recess, children typically consume fewer fruits, vegetables, and milk and more added sugars, saturated fats, and sodium (brazendale et al., ) . unlike summer recess, the pandemic presents unique barriers to obtaining healthy foods, due to grocery store closures, delivery delays, and item shortages, while access to "fast foods," which typically contain more saturated fat, sugar, salt, and calories, remain readily available. in efforts to expand reach during the pandemic, food banks are creating drive-through food distribution centers, preparing and distributing meals to children, prematurely opening summer meal programs, and keeping existing afterschool meal programs accessible. to locate a food bank in your community and to learn more about your local food bank's response to covid- , visit the feeding america website in table . to provide your community with strategies to make nutrition a priority during the pandemic, visit the nutrition website in table . to provide families with healthy eating tips, visit the cdc's website in table . for a list of healthy recipes that children enjoy, visit the academy of nutrition and dietetics' eat right website in table . the eat right campaign also helps parents find safe opportunities to engage their children in meal planning and preparation, which may help children become more curious about trying new foods. schools represent the largest providers of mental health services for children. of the % of children in the united states receiving mental health services, over % do so in school, specifically through school counselors, nurses, psychologists, and social workers (centers for health and health care in schools, ). in addition to counselling, schools provide crucial behavior management and crisis intervention services. as a result of school closures, children lose access to these critical resources. this is particularly concerning for children of racial and ethnic minority groups, who historically face greater difficulty in accessing mental health services. school closures also disrupt the structured daily routines that are critical for all children, especially those with underlying mental health and behavioral disorders. finally, in social distancing from peers, children lose access to a critical social support network. social isolation can further negatively affect mental health outcomes. school nurses searching for new strategies to address student mental health during the pandemic may schedule a zoom meeting with the crisis management institute ( ). the pandemic also presents unique psychosocial stressors, which may negatively affect the well-being of children and exacerbate underlying mental health disorders. children may experience additional home stressors, as parents face unemployment, economic hardship, and difficulty balancing work with their caregiver responsibilities. essential workers may experience greater stress in the workplace and spend less time with their children, which can negatively affect both the child's well-being and the parent-child relationship. physical activity, healthy eating, mindfulness, and sleep hygiene may all reduce psychosocial distress and improve well-being in children. to assist families in improving sleep hygiene, visit the child mind institute website in table . in addition to physical activity videos, the gonoodle website in table also provides online meditation and mindfulness videos for young children to follow. over seven million cases of child abuse and neglect are reported in the united states each year, four children of which die each day (safe horizon, ). professionals expect these numbers to increase in light of the pandemic, as household stress is a significant predictor of child abuse and neglect. in addition, victims have limited ability to seek refuge, due to closure of shelters, religious venues, and other community resources, as well as fear of contracting covid- in the emergency department. victims may connect with an advocate by visiting the national hotline website in table , by calling - - - or by texting loveis to . children may experience the illness, hospitalization, and death of a loved one from covid- . these hardships may be difficult to understand based on developmental age and can have long-term psychiatric consequences. to provide families in your community with guidance on how to help children cope with covid- , visit the nasn website in table . to engage young children in these discussions, age-appropriate covid- activity workbooks and coloring books are available on the cdc websites in table . children may also discuss covid- concerns with a crisis counsellor by visiting the crisis text line website in table or by texting home to . school closures significantly disrupt children's daily routines. literature on the structured day hypothesis demonstrates that healthy behaviors are "beneficially regulated when children are exposed to a structured day (i.e., school weekday)" versus weekends and summer recess, through, "compulsory physical activity opportunities, restricting caloric intake, reducing screen time occasions, and regulating sleep schedules" (brazendale et al., , p. ) . several nationally recognized organizations emphasize the importance of adhering to a structured schedule despite school closures. as an example, table displays a potential schedule for a late elementary school student, which incorporates resources mentioned in table . the khan academy website listed in table provides more comprehensive daily schedules for students in preschool to morning : to : wake up, get dressed, wash up, eat breakfast : to : start assigned schoolwork : to : play outside or complete an online pe network physical activity (table ) second grade, third to fifth grades, sixth to ninth grades, and th to th grades, as well as academic lessons/assignments by subject for students who are not receiving ongoing remote education during school closures (khan academy, ). finally, gasol foundation suggests creating themes for each weekday, to reinforce healthy behaviors and foster family time. examples on this website include "workout wednesdays," when family members participate in a family workout together, or "feelings friday," when family members gather to discuss their emotions surrounding covid- (gasol foundation, ) . families can utilize these resources to create a schedule that optimizes health and wellness during the pandemic. the return to school introduces new challenges to maintaining student health and wellness. several resources are available on the cdc's, "child care, schools and youth programs-plan, prepare, and respond," to assist schools, youth programs, camps sports and child care programs with the process of safely reopening or with maintaining safety for those that have already reopened (cdc, a). you visit the hunt institute website (table ) to learn more about your state's emergency child care provisions in response to covid- . likewise, you visit the no kid hungry and feeding america websites (table ) to locate resources to assist your community in maintaining food security. you disseminate tables and , as well as these state-and community-specific resources, to parents in your community. you receive feedback from several families that these tools assisted them in adopting a new and healthy routine, despite school closures. families in your community feel better equipped should the governor extend the duration of school closures. moreover, you feel better prepared, should your school district encounter intermittent "peaks" of covid- until a vaccine is developed and readily available, or an entirely new infectious disease outbreak occurs in the near future. if you have a clinical question, send your question to dr. olympia (rolympia@ hmc.psu.edu). questions will be selected and discussed as part of the "school nurses on the front lines of medicine" series. ■ robert p. olympia https://orcid .org/ - - - child care for essential workers during coronavirus outbreak understanding differences between summer vs. school obesogenic behaviors of children: the structured days hypothesis children's mental health needs, disparities and school-based services: a fact sheet screen time vs. lean time childcare, schools, and youth programs coronavirus disease (covid- ) frequently asked questions how much physical activity covid- resources for school counselors coronavirus quarantine: how covid- affects childhood obesity, healthy habits and vulnerable populations covid- and state child care assistance programs: immediate considerations for state ccdf lead agencies daily schedule for school closures variability in school closure decisions in response to h n : a qualitative systems improvement analysis the families first coronavirus response act: summary of key provisions coronavirus disease home page safe horizon national school lunch program global strategy on diet key: cord- -me th authors: cox, e.; cools, v.; houvenaghel, a. title: effect of antisecretory drugs on experimentally induced weanling diarrhoea in piglets date: journal: vet res commun doi: . /bf sha: doc_id: cord_uid: me th in newly-weaned to -week-old piglets, diarrhoea was induced by a combined infection with transmissible gastroenteritis (tge) virus and enterotoxigenic e. coli (etec) strains. in untreated control animals this dual inoculation resulted in profuse diarrhoea, vomiting, hypovolaemic shock and death of % of the animals within five days of tge virus inoculation. antisecretory drugs were administered intramuscularly for three consecutive days after experimental infection. the neurolepticum chlorpromazine, at mg/kg/ h, resulted in a significant inhibition of diarrhoea and vomiting, and in an increase in weight gain and survival. sedation and hypothermia, however, were serious side-effects. the α( ) agonist clonidine, at μg/kg/ h, induced a significant antidiarrhoeal effect and a reduction in mortality. the drug, however, provoked decreased activity of α( )-adrenergic excitation and incoordination. the β-adrenergic antagonist propranolol, at . mg/kg/ h, and the calcium channel blocker verapamil, at mg/kg/ h, had no beneficial effect on the experimentally induced diarrhoea. with improved knowledge of the mechanisms of action of specific diarrhoeaproducing organisms, many therapeutic agents have been examined which might exert inhibitory effects at one or another level of the diarrhoeic pathways. enterotoxigenic e. coli (etec) strains adhere with fimbriae to villous epithelial cells, colonize the small intestinal surface and elaborate heat-labile (lt) and/or heat-stable (st, type a and/or b) enterotoxins, which stimulate cyclic adenosine monophosphate (camp)-and/or cyclic guanosine monophosphate (cgmp)-mediated enteric fluid loss by inducing small intestinal hypersecretion. treatment of secretory diarrhoea could be performed using drugs which inhibit this hypersecretion and/or stimulate intestinal absorption (donowitz, wicks and sharp, ; powell, ) . neuroleptics, e.g. chlorpromazine, are reported to induce inhibition of campmediated fluid secretion (donowitz, wicks and sharp, ; greenough and rabbani, ) . adrenergic receptor agonists, especially the aj-agonists, are another promising group of potentially antidiarrhoeal drugs, acting primarily by increasing sodium and chloride absorption and by inhibiting bicarbonate secretion (donowitz, wicks and sharp, ; powell, ) . the fl-adrenergic antagonist, propranolol, has been shown to be antisecretory for cholera toxin and dibutyryl camp in rabbit and rat intestine (donowitz, wicks and sharp, ) . in animal studies verapamil, a well-known calcium channel blocker, increases basal ileal absorption of water, sodium and chloride. some calcium channel blockers have also been shown to prevent secretion induced by cholera toxin and by st-enterotoxin of e. coli (donowitz, wicks and sharp, ) . in pigs post-weaning diarrhoea often results from a combined infection with e. coli and a viral enteropathogen (mostly rotaviruses, sometimes coronaviruses) (tzipori, chandler, makin and smith, ; lecce, ; wilson and francis, ) . the enteropathogenic viruses damage the small intestinal villi, resulting in villous atrophy and subsequently in small intestinal malabsorption (moon, ) . the dual infection induces a more severe diarrhoea than the infection with etec alone (tzipori, chandler, makin and smith, ) . we therefore established a combined hypersecretion-malabsorption diarrhoea model in newly-weaned piglets by inoculating them with transmissible gastroenteritis (tge) virus, an enteropathogenic coronavirus, and etec. in preliminary experiments it has been shown that this combined viral-bacterial infection induces profuse diarrhoea and hypovolaemic shock, which provokes % mortality within five days (cox, cools, charlier, schrauwen and houvenaghel, ) . in the present study the effects of chlorpromazine, clonidine, propranolol and verapamil were evaluated in this model. the experiments were performed on newly-weaned, to -week-old, female piglets, weighing . to . kg, from primiparous sows purchased from the same commercial farm. the piglets were of mixed breed (pietrain x belgian landrace). they were individually housed at °c and allowed to drink uht sterilized whole cow's milk ad libitum. to enhance etec colonization of the small intestine, the piglets were orally pre-treated on days , and with chloramphenicol (chloromycetin, parke-davis; mg/l milk). this pre-treatment with chloramphenicol renders the dual infection more severe (cox, schrauwen and houvenaghel, ) , probably by making the gut more susceptible to etec (kaufman, ) . after starvation for the first three hours of day , the piglets were orally inoculated with tge virus ( . x lo pig infective dose/animal). twenty-four hours later (day ) the piglets were starved again for three hours, after which ml of a . % nahcos solution was given intragastrically to neutralize the gastric acidity and so prevent etec destruction. fifteen minutes later they were intragastrically inoculated with ml of a bacterial suspension of two etec strains. both strains, , :$ :s,a ; lt, sta and stb enterotoxin positive respectively, were grown on brain heart m usion e agar (oxoid) at °c for h. bacteria were suspended and diluted in sterile physiological saline to an a,,,, of . , approximately . x lo bacteria/ml as determined by viable count. antisecretory drug treatment was started h after the etec inoculation and was continued for three consecutive days (table i) . six piglets were injected with chlorpromazine hydrochloride (rhone-poulenc) at a dose rate of mg/kg/ h and six others were injected at a dose rate of mg/kg/ h. another group of six piglets woln~lnwv w mwwwwww was injected with clonidine hydrochloride (boehringer ingelheim) at . mg/kg/l h, whereas six animals received . mg/kg/l h. a further six piglets were injected with propranolol hydrochloride (ici) at a dose rate of . mg/kg/s h. finally, six piglets were treated with verapamil hydrochloride (knoll ag) at a dose rate of mg/kg/s h. to reduce side-effects the first or last dosage was lower for several of the drugs (table i) . all the drugs were dissolved in distilled water and injected intramuscularly. the other nine piglets were kept as untreated controls. during the experiment clinical signs, severity of diarrhoea, milk intake, weight change, body temperature and survival were recorded daily. the severity of the diarrhoea was evaluated by arbitrarily scoring the consistency of the faeces ( = normal; = pasty; = semi-liquid; = watery). scoring was always performed by the same person. shocked piglets died spontaneously or were killed with an overdose of methomidate (hypnodil, janssen). the other piglets were killed with methomidate days after the start of the experiment. all piglets were examinedpost mortem. the genetic susceptibility of the piglets to kssac-etec adhesion to their small intestinal villi was determined by the in vitro technique described by girardeau ( ) . the daily diarrhoea score was expressed as median and ranges or individual values and statistically analysed using the mann-whitney u or wilcoxon tests. the one-way analysis of variance and simple contrasts were used to assess the statistical significance between the values of the different parameters after experimental infection and those before the infection on day . the mann-whitney u test was used to demonstrate significant differences for time-matched values between control and treatment groups. the differences in survival time between the treatment groups and the control group were evaluated using the mantel modification of the gehen generalized wilcoxon test (knapp and wise, ) . the in vitro adhesion assay performed immediatelypost mortem revealed that the villi of seven piglets were genetically resistant to adhesion of gac-etec to their enterocytes (table i) . data from these piglets are not included in the results. as previously described (cox, charlier and houvenaghel, ) , piglets developed diarrhoeic faeces during the chloramphenicol pre-treatment period (figure , day ) . in the control group diarrhoea seriously worsened after inoculation with tge virus and etec (pco. ). daily milk intake decreased (day , + ml: day , + ml; p > o.os), vomiting occurred in % of the animals (table i) and there was a significant weight loss ( figure ) and decrease in body temperature (table ii) . piglets became dehydrated as shown by bilateral retraction of the eyeballs and a decrease in skin turgor. cyanosis of the extremities and depression occurred, and % of the animals developed hypovolaemic shock (table i ). in the terminal stage some animals lay in lateral decubitus, with sunken flanks and a prominent pelvis. mean survival time of shocked piglets was . + . days after tge virus inoculation. the gross changes at necropsy were cyanosis of the extremities, a filled stomach, severe congestion of the gastric mucosa, watery contents in and congestion of the small intestine and sometimes of the colon, and petechial haemorrhages in the kidneys. figure . weight change after a combined infection of newly-weaned piglets with tge virus and etec (infected control group; n = ) and effect of treatment with chlorpromazine at mg/kg/ h (chlorpromazine group; n = ) or with clonidine at pg/kg/l h (clonidine group; n = ). the values represent means + sem. significant differences from the values prior to infection (day ) are indicated by *(p<o.o ); **(p<o.ol) and ***(pc . ) treatment with the low dose of chlorpromazine at mg/kg/ h was without beneficial effect on diarrhoea score, vomiting or survival (table i) . however, the larger dose of this neurolepticum, mg/kg/ h, resulted in a significant decrease in the diarrhoea score on day (figure l) , a complete inhibition of vomiting (table i) , a less pronounced (p > . ) weight loss ( figure ) and an increase, although not a significant one, in percentage survival (table i) . chlorpromazine administration, especially in the larger doses, also provoked marked sedation and a resulting further reduction in daily milk intake ( + ml on day , as against + ml in the control group; p > . ), and a significantly more pronounced decrease in body temperature on day (table ii) as compared to the control piglets. the decrease in body temperature was observed within one hour of the first administration of the neurolepticum and became more pronounced after the second. on the third day we therefore injected . mg/kg instead of mg/kg. administration of clonidine at the low dosage, . mg/kg/l h, was without influence on the parameters studied, whereas at a dose rate of . mg/kg/l h there was a decrease in diarrhoea score (figure ; day values) and a significant reduction in mortality (table i) . vomiting (table i) , daily milk intake and weight change ( figure ) were not significantly influenced by this a -adrenoceptor agonist, whereas body temperature did not undergo the significant decrease after experimental infection observed in the control pigs (table ii ). in both clonidine groups serious side-effects were observed. administration of the low dose induced decreased activity, whereas with the higher dose, although activity was usually decreased, there was sometimes excitation or even incoordination. no analgesic effects were noted min or h after clonidine injection as evaluated by gently pinching the coronets and the skin of the feet and thorax. propranolol at . mg/kg/ h and verapamil at mg/kg/ h were without significant beneficial effect on diarrhoea score, vomiting, weight change or survival rate ( table i ). the decrease in body temperature observed in control piglets after tge virus and etec inoculation was not influenced by the calcium antagonist, whereas administration of the b-adrenergic antagonist resulted in an increase in temperature (table ii) . etec strains are involved in the multifactorial post-weaning diarrhoea syndrome in piglets, stimulating electrolyte and fluid secretion by the crypt cells (lt and sta enterotoxins) or diminishing absorption by the villous cells (lt enterotoxin). the mechanism of action of stb is still unclear. recently rotavirus has also been shown to exert a role in the decrease of villous absorption (tzipori, chandler, makin and smith, ; lecce, ) . since rotavirus is enzootic in belgian piggeries (debouck, ) and the experiments were performed on conventionally bred piglets, tge virus, another enteropathogenic virus, was selected to induce a combined viral-bacterial enteritis. whereas rotavirus only infects the upper part of the villi, tge virus multiplies in and destroys the major part of the villous epithelium (moon, ) , resulting in more profuse diarrhoea and vomiting. in the very young piglets tge is almost invariably fatal, whereas animals over three weeks old usually survive the infection (aitken, ) . in the present study most dually infected piglets in the control group and also in some of the treatment groups developed hypovolaemic shock as a result of vomiting, induced by the tge virus, and diarrhoea, probably induced by a combination of malabsorption (tge virus) and hypersecretion (etec) (cox, charlier and houvenaghel, ) . the diarrhoea occurring during the chloramphenicol pre-treatment can be explained by the change in diet from sow's to cow's milk, by the loss of maternal immunity, by environmental changes (tzipori, chandler, makin and smith, ; lecce, ) and perhaps also by a direct effect of the antibiotic on the intestine. it has been shown that chloramphenicol suppresses the anaerobic gut flora thus probably increasing the intestinal susceptibility to potentially pathogenic micro-organisms (kaufman, ) , whereas in calves a malabsorption diarrhoea has been reported following oral treatment with chloramphenicol (mero, rollin and phillips, ) . we recently evaluated the effect of the chloramphenicol pre-treatment on our diarrhoea model. four piglets were treated with chloramphenicol for three days and were subsequently not inoculated with tge virus and etec strains. faeces became pasty to semi-liquid in two animals during treatment. after treatment, however, diarrhoea resolved within one day (cox, charlier and houvenaghel, ) . the clinical signs in the markedly shocked piglets in our study agree with earlier observations on e. coli infections in gnotobiotic piglets (kenworthy and de g. mitchell, ) , whereas our necropsy findings correspond to those observed in piglets which have died from post-weanling diarrhoea. in the last decade there has been steady progress in research on potentially useful drugs which can reduce fluid loss in secretory diarrhoea (greenough and rabbani, ; powell, ) . these drugs, however, have never been tested in a combined secretion-malabsorption diarrhoea. in animals chlorpromazine inhibits camp-induced hypersecretion provoked by the lt enterotoxin of e. coli and vibrio cltolerae, prostaglandins and dibutyryl camp, as well as cgmp-mediated hypersecretion due to the st enterotoxin of e. coli (liinnroth and jennische, , donowitz, wicks and sharp, ) . this neurolepticum probably also stimulates intestinal absorption of sodium and chloride, as demonstrated for promethazine in the rabbit ileum (cohen, sharp and donowitz, ) . in human medicine the beneficial effect of chlorpromazine as a therapeutic antisecretory drug was first reported in patients with severe diarrhoea due to cholera enterotoxin (greenough and rabbani, ) . however, sedation was a serious disadvantage in its clinical use. in newborn piglets with experimentally etec-induced diarrhoea, a single intramuscular administration of chlorpromazine at mg/kg one hour after the onset of diarrhoea completely normalized the intestinal fluid content within four hours but seriously sedated the animals, whereas at a dosage of l- mg/kg there were no side-effects but an obvious antisecretory effect still occurred (liinnroth, andrcn, lange, martinsson and holmgren, ) . these authors confirmed their findings in a field trial in which the duration of diarrhoea in a spontaneous outbreak due to etec was significantly reduced by a single intramuscular injection of chlorpromazine at mg/kg in addition to oral electrolyte solution and standard antimicrobial agents. in our present study, in which no additional therapy was performed, we only observed significant antidiarrhoeic and antiemetic effects resulting in increased survival following the administration of mg/kg/ h of the neurolepticum for three consecutive days. however, it must be mentioned that the piglets in the present study were newly weaned and had been inoculated with an enteropathogenic virus prior to etec infection, resulting in a more pronounced small intestinal fluid loss. sedation and suppression of vomiting are well-known pharmacological properties of neuroleptic agents (booth, ; baldessarini, ) . these drugs also induce hypothermia (booth, ; baldessarini, ) , which explains the significantly more pronounced decrease in body temperature in the chlorpromazine ( mg/kg) group as compared to the control group. despite important side-effects such as sedation, decreased daily milk intake and hypothermia, chlorpromazine at mg/kg/ h would probably be useful in the treatment of severe diarrhoea of mixed viral-etec origin in weanling piglets. clonidine has been reported to inhibit castor oil-induced secretion diarrhoea in rats (lal, shearman and ursillo, ; spraggs and btmce, ) . in ligated jejunal loops in -week-old piglets this drug reduces the net secretion of water and electrolytes induced by the st enterotoxin of e. coli (ahrens and zhu, ; zhu and ahrens, ) . the a -adrenergic agonist, however, was without effect on the st-induced increase in chloride efflux from isolated porcine enterocytes (ahrens and panichkriangkrai, ) . in a study on its antidiarrhoeal action in man, schiller, santa ana, morawski and fordtran ( ) postulated that cloniclme has two actions which might contribute to this effect. firstly, the drug directly stimulates the rate of intestinal water and electrolyte absorption both in vitro and in vivo by means of an a -adrenergic receptor. secondly, clonidine has been found to inhibit intestinal motility. in the present porcine study, administration of clonidine at pg/kg/l h for three consecutive days resulted in beneficial effects on diarrhoea and survival rate. however, the drug also induced important side-effects such as decreased activity or excitation and incoordination, thus compromising its use as an antidiarrhoeal drug in practice. the sedative effects are similar to those of chlorpromazine (blaschke and melmon, ) . our observations do not confirm the analgesic effects of clonidine previously reported in pigs, horses and man especially after epidural administration (wintzer, krause, siedentopf and frey, ; gordh, feuk and norlen, ) . the @-adrenergic antagonist, propranolol, has a species-specific action. it has been reported to be antisecretory for cholera toxin and dibutyryl camp in rat and rabbit but not in mice small intestine (larsen, ; donowitz, wicks and sharp, ) . in human medicine, an increase in body temperature in response to propranolol is an infrequent side-effect, reflecting an allergic reaction (weiner, ) . the calcium antagonist, verapamil, has been observed to increase basal ileal absorption of sodium, chloride and water and to block jejunal secretory response to the st enterotoxin of e. coli (forsyth, wong and maenz, ; donowitz, wicks and sharp, ) . the results from our porcine study, however, failed to reveal any beneficial effects of verapamil or propranolol, at least in the dosage studied, on the experimentally induced diarrhoea. our results suggest that, of the drugs studied, only chlorpromazine and clonidine are likely to be of value in the treatment of severe diarrhoea of mixed viral-etec nature in newly-weaned piglets. their observed side-effects are, however, a serious disadvantage for their practical use. mechanisms of antisecretory agents and prospects for novel drugs studies of the antidiarrheal action of clonidine. effects on motility and intestinal absorption adrenoceptors and the delay of castor oil-induced diarrhoea by clonidine in rats escherkhia coli and rotavirus infections in four-week-old gnotobiotic piglets fed milk or dry food drugs that inhibit adrenergic nerves and block adrenergic receptors fimbriae and enterotoxins associated with escherichia coli serogroups isolated from pigs with colibacillosis clonidin als sedativum beim pferd. berliner und miinchner tieriirzliche wochenschriji antisecretory effects of berberine with morphine, clonidine, i-phenylephrine, yohimbine or neostigmine in pig jejunum this work was supported by grant oa from the belgian institute for the encouragement of research in industry and agriculture (iwonl).the authors thank professor dr m. pensaert (university of ghent, belgium) for supplying the tge virus, and dr p. pohl and dr p. lintermans (national institute for veterinary research, brussels) and smith kline-rit (genval) for the supply of the etec strains. chlorpromazine, clonidine, propranolol and verapamil were generous gifts from rhone-poulenc, boehringer ingelheim, ici-pharma, and knoll ag respectively. the technical assistance of mr j. bollt, mrs c. de schepper and mrs r. hens is gratefully acknowledged. key: cord- -l a tqn authors: bielińska-wąż, dorota title: graphical and numerical representations of dna sequences: statistical aspects of similarity date: - - journal: j math chem doi: . /s - - - sha: doc_id: cord_uid: l a tqn new approaches aiming at a detailed similarity/dissimilarity analysis of dna sequences are formulated. several corrections that enrich the information which may be derived from the alignment methods are proposed. the corrections take into account the distributions along the sequences of the aligned bases (neglected in the standard alignment methods). as a consequence, different aspects of similarity, as for example asymmetry of the gene structure, may be studied either using new similarity measures associated with four-component spectral representation of the dna sequences or using alignment methods with corrections introduced in this paper. the corrections to the alignment methods and the statistical distribution moment-based descriptors derived from the four-component spectral representation of the dna sequences are applied to similarity/dissimilarity studies of β-globin gene across species. the studies are supplemented by detailed similarity studies for histones h and h coding sequences. the data are described according to the latest version of the embl database. the work is supplemented by a concise review of the state-of-art graphical representations of dna sequences. in an article published by fuchs in nature in we read "future generations may be able to determine whether the sequencing of the human genome in indeed led to a paradigm shift in biology and biomedicine as some predicted, or whether the impact of this event was more gradual instead" [ ] . the author observes that "so far, the history of biology has been characterized by a continous shift from the whole organism down to the molecular level, from the descriptive characterization of species over macroscopic observations and morphological and physiological studies to today's molecular dissection of individual genes". novel experimental techniques require new computational methods. in order to create good models describing the experimental results, researchers from different areas of science joined computational biology and medical sciences. as a consequence, a new interdisciplinary field adapting methods from many different branches of mathematics, physics, chemistry, and computer science emerged. a fundamental task coming from sequencing is to understand the code written in the sequence of four letters. a lot has been done to reveal some global characteristics of long dna sequences. for example herzel et al. [ ] created a model that describes thousands of nearly identical dispersed repetitive sequences present in dna sequences of higher organisms. the hypothetical model sequences consist of independent equidistributed symbols with randomly interspersed repeats. the model that can be analyzed analytically predicts that the entropy of dna sequences measuring the information content is much lower than suggested by earlier empirical studies. a systematic analysis of statistical properties of coding and noncoding dna sequences has been performed by mantegna et al. [ ] . the authors compared the statistical behavior of coding and noncoding regions in eukaryotic and viral dna sequences by adapting two tests developed for the analysis of natural languages and symbolic sequences. the authors analyzed some similarities and dissimilarities of statistical properties of coding and noncoding regions. in particular they found that for the three chromosomes they studied, the statistical properties of noncoding regions appear to be closer to those observed in natural languages than those of the coding regions. statistical studies aiming at characterization of correlation structures of dna sequences has been a subject of many studies (for review see [ , ] ). in particular foss [ ] using spectral density of individual base positions demonstrated long-range fractal correlations as well as short-range periodicities. arneodo et al. [ ] used the wavelet transform to demonstrate the existence of long-correlations in genes containing introns and noncoding regions. buldyrev et al. [ ] in order to answer the question in computational molecular biology whether long-range correlations are present in both coding and noncoding dna sequences have used standard fourier transform analysis and detrended fluctuation analysis. for that purpose, the authors performed analysis of the sequences available in genbank in . for noncoding sequences, they obtained the presence of long-range correlations. azbel in his work [ ] demonstrated a universality in a dna statistical structure using an autocorrelation function. however, no long-range correlations have been found in any of the studied dna sequences. peng et.al. [ ] studied long-range correlations by constructing a map of the nucleotide sequence onto a walk which they referred to as a dna walk. using such an approach they found long-range correlations in intron-containing genes and in nontranscribed regulatory dna sequences, but not in complementary dna sequence or intron-less genes. visualization technique proposed by peng et al. is based on a one dimensional dna walk showing the relative occurrence of purines and pyrimidynes along the sequence. silverman and linsker introduced vectorial representation of the bases in three dimensions [ ] . they used the unit vectors of d space to construct a fourier transform. such fourier transform graphs representing the sequences were used as measures of dna periodicity. another visualization technique based on dna walk plotted in three-dimensional cartesian coordinate system has been introduced by berger [ ] . in his work berger gave also a good review of visualization techniques based on dna walk and their applications for an analysis of dna sequences i.e. a study of correlation information, sequence periodicities, and other sequence characteristics. more examples of studies focused on statistical properties of dna sequences and also on their biological interpretation may be found in [ , ] . another class of studies is developing methods aiming at detailed sequence comparisons. most commonly used in computational biology and medical sciences are global and local alignment methods, for example clustal w [ ] , blast [ ] , needleman-wunsch algorithm [ ] , and t-coffee [ ] (for review see [ , ] ). an alternative to the alignment methods are alignment-free methods that can be divided into two groups: numerical similarity/dissimilarity analysis of dna sequences and similarity/dissimilarity analysis based on graphical representations of dna sequences. there is a variety of numerical alignment-free methods (for a review up to see [ ] ). recently new numerical alternative methods have been developed, as for example [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . another group within numerical alignment-free methods are multidimensional graphical representations. conceptually, they are analogous to the graphical representations but their visualization is difficult (if possible at all). in particular d numerical representations [ ] [ ] [ ] , d representation [ ] , d representation [ ] have been introduced. due to interdisciplinary character of research on dna, many groups of methods have been developed independently and very often without any knowledge about analogous results obtained in different groups of scientists. in particular, dna walk has been independently discovered by the scientists working on statistical properties of dna sequences [ ] and by scientists working on graphical representations. even among researchers working on graphical representations one can find analogous visualization tools discovered independently (see subsequent chapters). this work is focused on graphical representations of dna sequences. biological sequences are often very long, and it is not obvious how to represent them graphically in an easy way that shows the main features of these objects. the size of the plots is restricted by the human abilities of perception. how to restrict the graphs representing the sequences to two-dimensional plots and how to avoid degeneracies has been the subject of numerous studies which resulted in many graphical representations (see subsequent chapters). graphical representations offer both numerical and visualization tool for similarity/dissimilarity analysis. these methods are still restricted to small groups of users. computing codes calculating optimal sequence alignment are implemented using dynamic programming and are freely accessible in the internet and that makes them attractive for potential users. however, they are computationally expensive, and methodologically offer too simplistic similarity/dissimilarity analysis. they restrict the multidimensional similarity space of complex objects and show only one aspect of similarity. it becomes more and more popular to replace the alignment methods by alternative ones. in particular, hönl and ragan consider numerical alignment-free methods that can replace multiple-sequence alignment to infer a phylogenetic tree that represents the history of a set of molecular sequences [ ] . graphical representations have been also used for the construction of phylogenetic trees. since multiple alignment strategy does not work for all types of data, liao et al. [ ] proposed to use the similarity matrix based on their d graphical representation of dna sequences [ ] to construct phylogenetic tree. the authors consider mitochondrial sequences belonging to different species. the same graphical representation has been also used by the authors to obtain the phylogenetic relationships of h n avian influenza virus [ ] and the phylogenetic relationships of coronaviruses [ ] . another d graphical representation [ ] has been used by yu et al. to construct the phylogenetic tree of coronaviruses and lentiviruses [ ] . d graphical representation has been also used to construct a phylogenetic tree [ ] . wang and zhang studied molecular phylogeny of h n avian influenza viruses in asia using d and d graphical representations of dna sequences [ ] . graphical representations of dna sequences have been also generalized for the analysis of similarity/dissimilarity between rna secondary structures, as for example [ , ] . d graphical representation has been used for the characterization of the neuraminidase rna sequences of h n [ , ] and of h n [ ] strains. also graphical representations of the proteins have been created [ ] [ ] [ ] [ ] . graphical representations of the biological sequences (dna, rna, proteins) can be applied to all problems that require similarity/dissimilarity analysis. similarity analysis is not unique to sequences in biology. for instance, the problem of similarity has been developed and applied in computational pharmacology and has resulted in methods such as qsar, qspr [ ] [ ] [ ] [ ] [ ] [ ] [ ] which aim at the prediction of molecular properties. the basic paradigm of quantitative structure-property relationship (qsar) is that compounds with similar structure have similar properties. this implies a smooth transient behavior in the relation between structure and property/activity, i.e., for any small change in the structure, the magnitude of the physico-chemical property or biological activity changes smoothly rather than in an abrupt, in all-or-none type, way. the molecular similarity measures are based on a large number of descriptors, i.e. of the numerical indices characterizing molecules. the basis for these studies is the development of various kinds of mathematical descriptors [ , ] . in the theory of molecular similarity it is commonly accepted that different descriptors and different similarity measures reveal different aspects of similarity. a pair of complex objects may be similar in one aspect and not similar in another aspect. using different similarity measures, usually one obtains contradictory results which may be relevant in different contexts. the first qsar studies on biological sequences using graphical representations of sequences have been already performed [ ] . the present work describes the development of fundamental studies related to graphical representations of dna sequences. first, the corrections to alignment methods are proposed in order to enrich the information related to different aspects of similarity. new similarity measures are created for the alignment distributions. second, a critical review of graphical representations and their numerical characterization is given. in the last chapter, new aspects of four-component spectral representation, graphical representation of dna sequences, recently introduced by the author of this work [ ] , are described. it is shown in the last chapter of this work that by using the four-component spectral representation one can recognize the difference in one base between a pair of sequences so it can be used for single nucleotide polymorfism (snp) analyses which is subject of many investigation, as for example, in a recent work by bhasi et al. [ ] . another important problem is to identify protein coding regions of genomic sequences [ , ] . first attempts of identifying protein coding genes using graphical representations of dna sequences based on z curve [ ] or based on trinucleotides [ ] have been already performed. it has been shown that the similarity relations are different for exons, and sequences with introns using the four-component spectral representation (see subsequent chapters). such an observation suggests that the four-component spectral representation that reveals detailed aspects of similarity, as for example the comparisons of asymmetry of the gene structure, can be used to study this problem [ ] . in this section i introduce corrections that reveal some aspects of similarity which cannot be identified in the standard alignment methods. the similarity space of complex objects is multidimensional. only simple d objects can be classified in a unique way using a single similarity measure. complex objects may be similar in one aspect and very different in another one. for example, in the case of atoms, if a similarity measure based on their atomic numbers is considered then the periodic table of elements is obtained. however, considering ionization energies as descriptors, the similarity relations between atoms change. a final, single similarity measure is a result of averaging over different aspects of similarity or, a consequence of neglecting most of the aspects of similarity. the new similarity measures representing different aspects of similarity can be considered separately, or they can be combined in any way to search for their correlations with different biological functions. the information about similarity of the sequences derived from the alignment methods is rather limited. for example, according to the standard alignment methods, in the following two different cases: the similarity value is the same ( %). the non-zero contributions to the final result come from different positions in the sequences. in the first case, the a bases are spread over the whole sequence (positions , give non-zero contributions). in the second case, the a bases are cumulated. in this sense, the alignment methods are degenerate: different structures give the same result. then, in the alignment methods these structures are undistinguishable. this is certainly one of the weakest points of the alignment methods. the degree of degeneracy may be very large and increases with the lengths of the sequences. obviously, the degree of degeneracy in the model example is larger than . one can add more than two cases that give the same score as the two model cases. for example, the bases that give non-zero contribution can be also c, t, or g and the positions of the aligned bases can be different. such details usually have biological consequences but they are not taken into account in the alignment methods. in order to describe different aspects of similarity in more detail, let us define a discrete alignment distribution n p for a pair of dna sequences: , if the p-th positions in the two sequences are occupied by two identical bases, , otherwise. ( ) let us introduce a variable x p running along the sequence where p = , , . . . k is the position in the sequence and r is the resolution that can be selected, depending on the length of the distribution, in a way convenient for the calculations changing the units of lengths. k is the length of the sequences or subsequences for which the alignment is calculated. two bases belonging to different sequences, both located on the p-th positions are represented by a pair of numbers, {x p , n p }. let us consider multiple alignments. analogously, as for a pair of sequences according to the standard alignment methods, in the following two cases . the similarity value is the same ( %). thus, the alignment method is highly degenerate (different bases on different positions give non-zero contributions and these situations are undistinguishable). as a consequence, additional similarity information should be added for a proper description of the objects. this information is necessary to remove the degeneracy, i.e. to distinguish between different cases. analogously, as for a pair of sequences, we can define a discrete alignment distribution n p of several (m) dna sequences: , if the p-th positions in allmsequences are occupied by the identical bases, , otherwise. m bases belonging to different sequences, located on the p-th positions are represented by {x p , n p }, where x p is defined in eq. . as it is known from the statistics, distributions can be characterized in a convenient way by their moments. distribution moments are the basic quantities in statistical spectroscopy. the aim of statistical spectroscopy [ ] [ ] [ ] , is to construct global characteristics of a spectrum. the individual eigenvalues, the experimental energy levels or the intensities of spectral lines are considered as statistical ensembles. such an approach may be used in many areas of physics to study different kinds of problems. let me just mention several applications of statistical spectroscopy. originally, methods of statistical spectroscopy were used in nuclear physics [ ] where the character of the interparticle interactions is not exactly known. assuming different forms of the hamiltonian matrix and comparing distributions of the densities of the energy levels derived from this matrix and from the experiment some information about the hamiltonian may be derived. statistical spectroscopy may also be used to study the locations of the individual eigenvalues of the hamiltonian. in a way this is an inverse problem to the one from which the statistical spectroscopy originated: from global characteristics of the eigenvalues one tries to obtain some information about details of the spectrum. examples of generating individual energy levels using methods of statistical spectroscopy may be found in the theory of nuclear, atomic, molecular, and solid-state spectra [ ] [ ] [ ] . approximating the eigenvalues by statistical quantities (spectral density distribution moments) is not limited by dimensions of the matrices and that is an advantage comparing to the standard methods based on diagonalization of the hamiltonian matrix. in particular, we have studied statistical properties of spectra of the heisenberg hamiltonian [ ] . the distribution of the eigenvalues have been found to be gaussian-like, well approximated by several-term gram-charlier expansions [ ] . the exact spectra (obtained by the diagonalizations of the hamiltonian matrices) have been compared with the ones derived from the moment-generated spectral density distributions. this approximation gives a very good description of the spectrum in its central part however, as one should expect, deteriorates at the extremes. relations between the exact and the moment-generated spectra are analyzed for several kinds of the lattices as a function of the number of moments. it has been observed that the quality of the statistical description improves with an increase of the dimension of the problem and with a lowering of the symmetry of the lattice. another attractive application of the statistical spectroscopy is a description of the shapes of molecular electronic bands [ ] [ ] [ ] [ ] . initially, the method of generating of envelopes of the intensities has been introduced for the transitions in crystals [ ] and in atoms [ ] . replacing the calculations line by line by the statistical approach with much shorter computing time became also attractive in molecular physics. the shape of a molecular band may be defined as an envelope of the rovibrational lines which constitute the band. the method of determining the shapes of molecular electronic bands consists of several steps. first the expressions for the intensity distribution moments for the considered system are derived. then these expressions are used to calculate the moments corresponding to the solution of the pertinent quantum chemical model. finally, a smooth function for which several lowest moments are equal to the exact ones, is derived. this function is an approximation to the envelope of the electronic band in a molecular spectrum. in particular, i have used this algorithm to derive the intensity spectrum corresponding to the transitions in h molecule using -moment trial function [ ] . in that paper i have also shown that the quality of the approximation depends on the choice of the trial function rather than on the number of moments taken into account. adding moments of the order higher than does not improve the results when the gram-charlier expansion is taken as the trial function. this process may even be divergent. in some cases a -moment gram-charlier expansion may give worse results than the -moment one. for example, this happens in a spectrum derived from a model based on the harmonic oscillator potential. in the case of h molecule a non-standard -moment trial function has to be applied in order to get a high quality approximation of the spectrum (treated as a statistical distribution). distributions are commonly, and very conveniently, characterized by their moments. in the present work i describe dna sequences as distributions and apply the distribution moments to study similarity between these sequences. a similarity measure˜ q based on the q-th moment of the discrete alignment distribution n p is defined as where q = , , , . . . . in this work r = . therefore, the values of x p are equal to p (eq. ). the normalization constant c is defined so that the zeroth moment of the distribution is equal to one (˜ = ): comparing sequences, usually one is interested in the quantities that are independent of the lengths of the sequences. for that purpose, moments for which the mean value is equal to ( = ) and the variance is equal to ( = ) can be used as similarity measures: table shows a model example of the alignment distribution n p for a pair of sequences. the choice of the query sequence has no influence on the results. the length of sequence is and the length of sequence is . if k = is chosen then for p > the distribution is defined as zeros: n = n = n = . therefore, the table model example of alignment distributions (r = ) table shows a model example of the multiple alignment distribution for m = (eq. ). analogously, the value of k may be set equal to the length of any of the three sequences. thus, independently of the choice of k the moments remain the same. in this work,˜ q and q are proposed as new similarity measures that can be treated as corrections to the alignment methods. they describe such features of similarity that cannot be identified in the alignment methods. in particular, the two model cases defined at the beginning of this chapter can be distinguished using the new measures. the new numerical characterization of dna sequences is exemplified using the βglobin gene of different species. the species and the locations of the sequences in genes as well as the lengths of the sequences, n and n , are listed in table . tables , , , , , , , show similarity matrices based on the new measures for the sequences listed in table . tables , , , correspond to the coding sequences of the first exon, exon c ds , and tables , , , correspond to the second exon, exon c ds . the similarity matrices are based on different measures:˜ (tables , ), (tables , ), described by x = , x = , and x = . in this case˜ = / = and x = is the middle of the sequence. one can normalize the similarity matrix based on˜ dividing all its elements by k + . then one can easily see whether the mean value is larger or smaller than / . if˜ is equal to / then the location of the mean value of the distribution is in the middle. if it is greater than / it is shifted towards the end of the distribution. since q are independent of the lengths of the sequences it is convenient to keep at least one similarity measure (˜ ) that carries the information both about the lengths of the sequences and about the distributions of the aligned bases. therefore,˜ is not normalized in this work. an example of the similarity measure independent of the lengths of the sequences is that describes the asymmetry of the aligned distributions. for the symmet- ric distributions, in particular if identical sequences are compared, is equal to zero. it is negative for the left-skewed distributions and positive for right-skewed distributions. one can observe, that the asymmetry of the aligned distributions for exon c ds (table ) is different from the one for exon c ds ( table ). the number of the negative values of is for exon c ds and for exon c ds . for example, in the case of human-mouse sequences, is negative for exon c ds and it is positive for exon c ds . another similarity measure independent of the lengths of the sequences is . this is the kurtosis parameter, that is the measure of the peakedness of the distribution. analogously as for the lower order moments, is different for different parts of a gene (tables , ). for example, the similarity measure based on for galluslemur sequences is . for exon c ds and . for exon c ds . the similarity relations based on between all the sequences are shown in fig. . the horizontal axis represents the values listed in table table versus values listed in table ( fig. , panel b) and the values listed in table versus values listed in table means that the information coming from is similar to the one coming from . therefore, the corrections can be neglected. the information coming from is different than the one coming from which is seen in figs. and , panels a, where table model example of four-component multiple alignment distribution (k = , m = , r = ) in order to further enrich the information that can be derived from the alignment methods, one can introduce the four-component alignment distributions, separately for a, c, t, and g bases. a specific γ -component of this distribution, referred to as γ -distribution, is defined as where γ = a, c, t, g denotes one of the bases. now, for each of the γ -distributions one can calculate the corrections (the appropriate moments). such kind of distributions can be created for a pair of sequences (m = ) and also for the multiple alignment studies (m ≥ ). a model example of the four-component distribution with m = is shown in table . the same definitions of the distributions (eqs. , , ) can be also used after the maximally scoring alignment of the sequences has been found. a model example of the optimized multiple alignment distribution based on eq. for m = is shown in table . the maximally scoring alignment is obtained if two gaps are introduced in sequence . obviously, the information is more detailed if four-component optimized distribution is created. table shows such a distribution (eq. ) for the same sequences as shown in table . the application of the new similarity measures to all kinds of the distributions is simple and straightforward. in this way, different aspects of similarity can be revealed. an attractive, alternative to the time consuming alignment methods, are graphical representations. they reveal different aspects of similarity, offer both numerical characterization of similarity and the visualization. also the computing effort is in this case very small. in this section graphical methods are discussed. in the original approaches, dna sequences were plotted as either three-dimensional [ ] or two-dimensional [ ] [ ] [ ] curves. the shapes of the curves were determined by a walk in a space spanned by four vectors that represent the four bases. in the first article on this subject, hamori proposed a graphical representation method in which the information about the dna sequence has been mapped into a three-dimensionalspace curve. a unit vector of a characteristic direction has been assigned to each of the four nucleotides: adenine a, cytosine c, thymine t, and guanine g. in this approach the shape of the curve (called h-curve) representing the sequence of nucleotides is obtained by joining the vectors in the order of the nucleotides in the sequence. changing the resolution one can see short-range details or global trends of the distribution of nucleotides. for example, h-curve is shifted in characteristic direction if the sequence is rich in certain nucleotides. it is also easy to recognize the locations of the repeating elements in the sequence. the first mathematical representation hamori also published in nature in under the title "novel dna sequence representation" [ ] . the same year another article about a new graphical representation titled "simpler dna sequence representations" has been also published in nature by gates [ ] . in this approach, guanine is represented by a unit vector in the positive x-axis direction, complementary cytosine is represented by a negative x-axis unit vector, and adenine and thymine are represented by unit vectors in the positive and negative y-axis directions, respectively. using such an approach all sequences can be represented in two dimensions in a unique manner, while using the hamori approach, dna structure may be viewed from any chosen perspective in two-dimensional plots. obviously, a chosen perspective of a d curve in d space gives only a part of the total information about the sequence. however, also in the graphical representation proposed by gates some information may be lost, as it is shown in a subsequent part of this work. about years later, nandy (independently of gates) published an article "a new graphical representation and analysis of dna sequence structure: i. methodology and application to globin genes" [ ] . the idea is very similar to the one presented by gates. in the scientific correspondence [ ] , the author explains that he has just brought to his attention that a similar technique was presented by gates and indicates some advantages of his method: the nontrivial choice of the coordinate system a-g, c-t (purine-pirymidine) instead of the axis system proposed by gates (c-g, a-t) may give more significant biological information. one year later (independently of nandy), leong and morgenthaler proposed two new graphical representations of dna sequences [ ] . the first one is a slight modification of the gates method: they change the unit vectors corresponding to the particular bases. the x-axis represents c and a and y-axis g and t. according to the authors such a change allows to exhibit the distribution of purines (a and g) and pyrimidines (c and t). the authors noticed that some information may be lost if a walk moves several times over the same ground. however, the authors found a good solution to identify in the plot the regions in which the parts of the sequences are hidden: the scale is visible even for long sequences and the numbers that label the bases in the plot are pointed every one hundred. the authors have not proposed any numerical characteristics and a way of indication in the plot of the hidden parts (by labeling or coloring) seems to be a good solution. leong and morgenthaler also proposed another, interesting graphical representation: gap plots that give the information about the distances between particular bases. independently of the graphical representations introduced by gates [ ], nandy [ ] , leong and morgenthaler [ ] , similar graphs, also based on vectorial representations of the four bases and constructing d dna walks, have been constructed by mizraji and ninio [ ] and by lobry [ ] . lobry also used orthogonal directions but his choice of the unit vectors representing the four bases was different than the ones used in refs. [ ] [ ] [ ] . surprisingly, these two important contributions remained rather unnoticed. the specific choice of the basis vectors done by mizraji and ninio seems to solve many problems. the vectors have been chosen so that it is easy to distinguish between coding and noncoding parts of the sequence and the graphs are nondegenerate. mizraji and ninio also proposed a graphical representation which shows purine/pirymidyne distributions along the sequence. however, the authors did not propose any numerical representation associated with these graphs. as a consequence, four similar d graphical representations have been created. they differ from each other in the choice of the coordinate systems: x-axis: g-c (gates), a-g (nandy), c-a (leong and morgenthaler), a-t (lobry). the most popular became the graphical representation proposed by nandy, called nandy plots. however, such a two-dimensional representation may lead to some parts of the sequence being hidden if the walk is performed back and forth along the same trace (so called repetitive walks). labeling and coloring only approximately localizes the regions in the sequences where the hidden parts are located. a d walk does not retain the history of the graph. this is not a linear method: a particular part of the graph may come from different parts of the sequences. the advantage is a small size of the graph representing long sequences and very often the information coming from such a plot may be sufficient. in order to eliminate, or to minimize, the degeneracy caused by the repetitive walks, many different methods have been introduced. for example, guo et al. [ ] introduced a new graphical representation, also based on a walk in d space changing the angles between the basis vectors: the four nucleic acid bases are represented by the vectors: a by − , d ; t by d , − ; g by , d ; and c by d , , where d is a positive integer. the authors have shown that the degree of degeneracy of the new graphs is lower than for nandy plots, but it is still present and depends on the value of d. a further modification of the graphical representation based on a walk in d space has been introduced by changing the vectors in such a way that the basis vectors corresponding to pyrimidines (t, c) are located in the first quadrant of the cartesian coordinate system and to purines (a, g) in the fourth one [ , ] . the unit vectors representing four nucleotides are as follows: [ ] and also h-l curve representation proposed recently by huang et al. [ , ] . an interesting d ladder-like graphical representation has been also proposed by li and wang [ ] . their graphs are based on the division of bases according to their chemical properties. the four bases can be classified into groups: . purine r = a, g, pirymidyne y = c, t; . strong h bond s = c, g, weak h bond w = a, t; and . amino m = a, c, keto k = g, t. the method is also based on a walk in d space with basis vectors ( , ), ( , ) for characteristic sequences (m, k), (r, y) and (w, s). recently, we have proposed another method aimed at some improvement of the original d walk method (nandy plots) [ ] . we have called this representation d-dynamic graph because its numerical representation, i.e. the set of descriptors, is analogous to the one used in the dynamics (see subsequent chapter). this method is based on nandy plots but it removes the degeneracy coming from the repetitive walks. the dna sequence is represented as a set of material points in d space. figure , panel a, shows the method of plotting the d-dynamic graph for a model sequence ctc and fig. , panel a, for ctct one. the first base in the sequence is c and we make a shift along the vertical axis in the positive direction. at the end of this vector (position ( , )) we locate the point with the mass equal to . the second base in the sequence is t and we make the second shift along the vertical axis in the negative direction starting from the end of the last vector. at the end of the second vector again we locate the point with the mass also (position ( , )) and so on. if the ends of vectors meet several times at the same point then the mass of this point increases (it is equal to the sum of all masses located in this point). the total mass in the graph is equal to the total number of bases in the sequence ( in fig. and in fig. ). different masses are represented by different symbols in the plots. please note that both sequences ctc and ctct are represented by the identical nandy plots (figs. , , panels b) since the last shift in fig. is made along the same trace as the previous one. d-dynamic graph removes this degeneracy (the masses of the points ( , ) are different: for ctc and for ctct). the difference between the two sequences is also revealed in the mass-density distributions which we create for x and y directions [ ] . the masses are projected onto two orthogonal directions and then summed for each x and y. in the model examples the results of the projection and of the summation of the masses are shown in figs. and (panels a). for example, in the x direction, they are and in figs. and , respectively. the mass-density distributions are composed of single lines located at the coordinates corresponding to the projected masses (x = for ρ x and y = , y = for ρ y ). the intensities of the lines correspond to the projected masses. the center panels in figs. and correspond to mass-density distributions for x direction (ρ x ) and the right ones for y directions (ρ y ). these distributions create another way of visualization of the d-dynamic graphs. however, the main reason for the creation of the mass-density distributions is deriving new descriptors related to d-dynamic graphs (see the subsequent chapter). the modifications of the original d walk methods resulted also in graphs which became linear-like representations ( d), extending along one direction in d space. in such kind of methods only the horizontal axis is associated with the positions of the bases. therefore these methods are free of the effects of self-overlapping of the graphs. the cost we have to pay for the reduction of the degeneracy, is worse visualization of long sequences. a combination of a linear-like method with a dna walk has recently been proposed by zhang [ ] . the author has chosen basis vectors in such a way that the walk is performed along a horizontal axis. one nucleotide is represented by a pair of basis vectors instead of a single vector: ( , ),( , ) corresponds to a, ( , ), ( , − ) corresponds to t, ( , − ), ( , ) corresponds to c, and ( , − ), ( , − ) corresponds to g. since one base is represented by a double vector, the author calls his graphical representation of dna sequences a dv-curve. a recently introduced graphical representation of dna sequences based on the neighboring dual niclueotides (dinucleotides) [ , ] is another example of a linear representation. the authors plot a dinucleotide (dn) curve representing the distributions of pairs of nucleotides along the sequence. several years ago a four-horizontal-line graphical representation has been proposed by randić et al. [ , ] . instead of considering the four directions along the cartesian coordinate axes, they draw four horizontal lines separated by unit distances. each line is associated with one base: a, t, g, and c, from the top. the sequence is written at the bottom of the lowest line, with unit distances between the neighboring bases. the dots (or rectangles) are put on the lines if a particular base appears in the sequence. this graphical representation resembles medieval musical scripts having staff of four lines [ ] . for a better visualization the adjacent points are connected by a line, and zigzag-like curve is obtained. the idea proposed by randić of the visualization of dna sequence by zigzag curves has been extended by different combinations of labeling the lines and by different number of graphs representing one sequence (characteristic graphs). usually, the horizontal lines are not plotted. another linear graphical representation has been proposed by li and wang [ ] . the graphical representation is composed of three characteristic graphs, each of them consisting of two horizontal lines. each line in each graph is assigned to more than one base. then, the sequence is represented by more than one characteristic graph. the lines in particular graphs are labeled by the following bases: a similar graphical representation has been proposed by song and tang [ ] . in this approach, the three classifications are applied to construct six characteristic graphs representing one sequence. two graphs correspond to one classification. for example in two graphs corresponding to classification purine (r) -pirymidyne (y), the middle lines correspond to purines and pirymidynes in the first and in the second graph respectively. the other two lines correspond to these bases that are not purines or not pirymidynes, respectively, i.e.: a, y, g label the lines in the first graph (top, middle, and bottom respectively) and c, r, t label the lines in the second graph. another example of an analogous graphical representation has been proposed by liao and wang [ ] . the sequence is represented by three graphs, and each of them is consisting of two horizontal lines. the lines are labeled as follows: in another analogous graphical representation, proposed by wang and zhang [ ] , also consisting of three characteristic graphs, the lines are labeled by the following bases: graph : non-a = g, c, t and a, graph : non-g = a, c, t and g, graph : non-c = a, g, t and c. a slight modification of this method has been proposed by yao and wang [ ] . the authors proposed to use cells instead of horizontal lines. they considered different shapes of cells, for example a rectangle. each corner of the rectangle is assigned to a particular base. the cells are placed next to each other. particular bases in the sequence are put in a proper corner (each base is located in its own cell). the adjacent dots are connected by a line and a zigzag curve representing the sequence is obtained. the methods described above, based on several horizontal lines, can be also considered as spectral-like representations (lines with some intensities appear in the positions corresponding to the bases in the sequences). this point of view has been expressed in a recent article by randić [ ] . the author presents four-horizontal-line graphs and chaos-game d maps [ , ] in the form of spectrum-like graphical representations. recently, i have introduced another spectral-like graphical representation called four-component spectral representation [ ] . the method is very sensitive. within this model, differences in only one base can be detected. by using linear graphical representations of dna sequences the problem of degeneracy can be overcome. however, in technical terms, the visualization of long sequences is rather inconvenient. a good solution for this drawback is introducing a resolution parameter for linear representations as it was done for the four-component spectral representations (for details see sect. ). another solution is to combine the compact form of the plots characteristic for d walks and zigzag curve method, as proposed by randić et al. [ , ] . in the last approach, the sequence is represented by a zigzag spiral, known in the literature as the worm curve. the worm curve represents a path of a robot [ ] . it does not intersect itself and uses a little space for the graphical representations of long sequences. another compact graphical representation, four-color map, has also been proposed by randić et al. [ ] . the map is constructed as a spiral of square cells. the first base is located at the central square of the spiral, and the last base finishes the spiral. then four different colors are assigned to particular squares representing different bases: red for g, blue for t, green for c, and yellow for a. the original d method proposed by hamori has been also extended by various authors. in particular, a modified hamori curve representation of dna sequences has been recently introduced by pesek and zerovnik [ ] . moreover, methods based on a walk in d space with different vectors corresponding to particular bases were introduced: vectors located along tetrahedral directions a( ,− ,− ), g(− , ,− ), c(− ,− , ), t( , , ) [ ] or agc-t curve, where the vectors are chosen as a( , , ), g( , , ), c( , , ), t( , , ) [ , ] . examples of other d graphs are representations of one sequence by a set of characteristic d curves [ ] [ ] [ ] [ ] [ ] [ ] . another d graphical representation, called z curve, combines the properties of several characteristic curves [ ] . a single z curve contains the information about the distributions of purine/pirymidyne, amino/keto and strong h bond/weak h bond. recently, new d graphical representations based on the frequencies of occurring of pairs of nucleotides (dual nucleotides or dinucleotides) or trinucleotides in dna sequences have been created. four nucleotides form dinucleotides and trinucleotides. by assigning different vectors to each pair or to each trinucleotide in d space, d-curves are obtained. the curves contain the information about neighboring bases and their distributions along the sequence. dual nucleotides can be also divided into groups according to their chemical properties, as for example purine dinucleotides (ag, ga), pirymidyne dinucleotides (ct, tc), amino ones (ac, ca), keto ones (tg, gt), weak h-bond (at, ta) and strong h-bond (cg, gc). d graphical representation of one sequence by four characteristic curves based on dinucletides has been proposed by cao et al. [ ] . other d graphical representations based on dinucleotides (pn-curves) [ ] , (dn-curves) [ ] , (d-curves) [ ] , or based on trinucleotides (tn-curves) [ ] have been also proposed. graphical representations constitute a tool allowing visual inspection of the sequences. moreover, each graph can be characterized by the quantities called in the theory of molecular similarity, descriptors. the descriptors representing numerically some properties of the sequences can be used for similarity/dissimilarity analysis of the sequences. the computing time of the calculations of the descriptors is low and the numerical comparison of long sequences becomes attractive. the algorithm of the computation of the descriptors is independent of the visualization tool. therefore, the graphical representations can be recognized as both numerical and graphical tools separately. however, each descriptor represents some specific properties of the graphs and it is not obvious how to characterize graphical objects by numerical values (for review of methods related to the creation of mathematical descriptors of dna sequences up to see [ ] ). one of the methods, most commonly used to describe graphs numerically, is transforming the plots to matrices. the method has been initially introduced by randić et al. for d graphical representations [ ] . the authors introduced distance matrices, d/d. the numerator in the matrix element (i, j) stands for the euclidean distance between vertices i and j, and the denominator stands for the graph theoretical distance (the number of arcs separating the two vertices). the authors proposed the leading eigenvalues of the matrices as the descriptors. the normalized leading eigenvalue of a d/d matrix offers a measure of the degree of folding of a chain-like structure or a curve. the authors introduced also higher-order matrix k d/ k d that is constructed by taking matrix elements of d/d matrix to power k. in the limit k → ∞, the resulting matrix reduces to a binary matrix ∞ d/ ∞ d. as the descriptors the authors also proposed the leading eigenvalues of these matrices. such kind of descriptors can be viewed as an index of flexibility (or stiffness) of the structure. the methods of transforming graphs to matrices stimulated introducing new kinds of matrices. different kinds of matrices associated with the graphs have been introduced by song and tang [ ] . the authors introduced the euclidean matrix e, whose (i, j) element is defined as the euclidean distance between vertices (dots) i and j of the curve. they also introduced m/m matrix whose elements are defined as a quotient of the euclidean distance between two vertices of the curve and the number of arcs between the two vertices. the third kind of matrix introduced by these authors is l/l matrix whose elements are defined as a quotient of the euclidean distance between two vertices of the curve and the sum of geometrical lengths of arcs between the two vertices. as the descriptors the authors chose the leading eigenvalues of m/m and l/l matrices. the authors considered characteristic linear curves and their descriptors characterize the distribution of bases with different chemical structures. the authors also considered higher-order l/l matrices. new kind of matrices has been also proposed by liao et al. [ ] . the authors introduced covariance matrices associated with the graphs. usually, the leading eigenvalues of the matrices are taken as descriptors. a discussion of the properties of such kind of descriptors may be found in a recent article by yuan et al. [ ] . some authors propose to consider more eigenvalues or matrix elements as descriptors of the sequences. wang and zhang proposed to take as a descriptor the sum of the maximal and minimal eigenvalues for the matrices associated with their graphical representation, called three non-base representation [ ] . the authors suggested that the information reflected only by the leading eigenvalue might not be comprehensive enough. liao et al. [ ] took all (two) eigenvalues of the × covariance matrices. li and wang proposed as descriptors normalized matrix norms instead of the eigenvalues [ ] . randić et al. considered as the descriptors average matrix elements of the matrices associated with the four-color map representation of dna sequences [ ] . liao and wang proposed as descriptors the average bandwidths [ ] . they can be obtained by summing the distance matrix elements along each of the lines parallel to the main diagonal if the matrix is in the canonical form. qi and fan took all elements of the matrix as descriptors of the sequences of equal lengths [ ] . pesek and zerovnik proposed to take as the numerical characterization of the modified hamori curve a product of first ten and last ten eigenvalues of the descending ordered eigenvalue list of the matrix l/l [ ] . numerical representation of d or d graphical representations of dna sequences based on transforming the graphs into matrices and deriving the descriptors from these matrices has been widely used by many authors. these descriptors characterizing a sequence can be used as components of similarity measures between a pair of sequences. examples of similarity analysis of dna sequences using this method may be found in [ , , , , , , , , , , , , , , ] . numerical representation of a graphical representation can be also performed directly from the coordinates or from the properties of the graphs without transforming the graphs to matrices. gates plotted each sequence as a graph of the cumulative manhattan distance (from the origin) against the sequence position [ ] . manhattan or city-block distance considered by gates is calculated as the arc length between points. for sequences of equal lengths it is convenient to plot the differences of the graphs. as descriptors of the sequences he proposed the means of the manhattan and euclidean "fractal" dimensions. raychaudhury and nandy proposed mean x and y coordinate values, and the radius of the graph as descriptors of dna sequences [ ] . guo and nandy introduced also improved mean x and y coordinate values, and the radius of the graph, reducing the degeneracy of the previously defined descriptors of dna sequences [ ] . yao et al. extended these descriptors to three dimensions defining d radius and adding mean z-coordinate as a descriptor [ ] . we have extended the set of these d descriptors to higher-order moments of the mass-density distributions. the mean x and y coordinate values are equal to the firstorder moments (m x, , m y, ) of the mass-density distribution, ρ x and ρ y respectively. in particular, if in a d-dynamic graph we put all masses equal to , then the ddynamic graph becomes the nandy plot and all the moments of the two graphs are identical. introducing the masses different than , the mean x and y coordinate values become the coordinates of the center of mass of the graph and are different than for the nandy plot. as the new descriptors we proposed moments of the mass-density distributions ρ x and ρ y up to the sixth order [ ] and up to the eighth order [ ] . higher-order moments give more specific information about the distribution of masses. for example, second-order moments (m x, , m y, ) give the information about the width of ρ x and ρ y . we have shown that the third-(m x, ), fourth-(m x, ), fifth-(m x, ), and sixth-order (m x, ) x-moments of the mass-density distributions representing histone h coding sequences have different values for plants than for vertebrates [ ] . in the present work, d-plots m x,q − m x,q are proposed instead of d-plots (descriptors versus labels of the sequences) that were shown in [ ] . d-plots are shown in fig. . in all the plots we observe clusterization of evolutionary similar organisms: plants are located in different parts of the plots than the vertebrates. the differences between histone h coding sequences across the species are not big and it is rather difficult to find the descriptors that reveal the clusterization. please note that y-moments and also x-moments for the order smaller than do not lead to clusterization in this case. in particular, this means that using the nandy plots for which the descriptors are taken as the mean values (first-order moments) of x and y we cannot get the clusterization. i have also found another set of descriptors (related to the four-component spectral representation) that reveal clusterization for histone h and h coding sequences (for details see the subsequent chapter). analogous ( d visualization) is introduced in the present work for the recently proposed molecular descriptors. figure shows moment-based classification of the molecules: m − m (top), m − m (middle), and m − m (bottom). we have shown that the new molecular descriptors (moments of the intensity distributions) have different values for two kinds of molecules: nitriles and amides. in our recent paper, d plots have been presented (descriptors versus labels of the molecules) [ ] . figure shows d plots. we observe that the descriptors representing nitriles are located in different parts of the plots than those representing amides. figures and represent different objects: dna sequences and molecules, respectively. however, the idea is the same. the clusterization of the descriptors indicates that these descriptors can be a good tool for similarity/dissimilarity analysis. the descriptors cluster (have similar values) for similar objects so they exhibit some properties of the considered objects. moreover, some of the plots reveal similar shapes, as for example, middle and bottom parts of fig. . this may suggest correlations between some of the descriptors. however, the shape is similar but not identical. the problem of correlation and extracting the minimal set of moments we studied in ref. [ ] . we concluded that a universal set of independent moments does not exist. usually lowest moments are sufficient to describe the object but also the information coming from higher-order moments cannot be neglected in some cases. as the new descriptors of dna sequences we also proposed the angles between the x axis and the principal axis of inertia of the d-dynamic graph (axes for which the tensor of moment of inertia is diagonal) [ ] . we also introduced the principal moments of inertia as the descriptors of dna sequences associated with the d-dynamic graph [ ] . they are associated with the rotations about the principal axes. the moment of inertia of an object about a given axis describes how difficult is to induce an angular rotation of the object about this axis. if the mass is concentrated close to the axis of rotation, it is easier to accelerate into spinning fast and the moment of inertia is smaller. as a consequence, these descriptors give the information about the concentrations of masses around the axes. another kind of new descriptors has been recently proposed by huang et al. [ ] . the authors proposed to take as the descriptors the set of characteristic vectors rep-resenting all bases in the sequence. guo and wang obtained smooth curves from the zigzag curves and took curvatures of the smooth curves as descriptors of the sequences [ ] . yu et al. proposed two kinds of descriptors: a set of coordinates of tn curves, and the probabilities of occurring of particular trinucleotides among all trinucleotides in the sequence [ ] . yu et al. composed d vector associated with the d-curve as a descriptor of dna sequences [ ] . another kind of non-standard descriptors has also been introduced for four-component spectral representation (for the details see the next chapter). the descriptors are the numerical characteristics of the sequences. the next step would be the creation of similarity measures between sequences. in most of the similarity studies the set of descriptors characterizing a sequence is treated as components of a vector. usually, as the similarity measure the euclidean distance between the components of the vectors corresponding to a pair of sequences is taken. in particular, for identical sequences, this similarity measure is equal to zero. recently, non-standard measures have been introduced. for example huang et al. defined a measure that changes from to and is equal to for identical sequences [ ] . chen et al. constructed cosine value that is a similarity measure of the mean x, y, z coordinates of their graphs [ ] . we have used the manhattan distance normalized by the mean value of the descriptors for the similarity studies of the sequences represented by the d-dynamic graphs [ , ] . for identical sequences this measure is equal to zero, as it is assumed for most of similarity studies. another non-standard similarity measure, also normalized to zero for identical sequences, is introduced in this work for four-component spectral representation (for details see subsequent chapter). however, in the alignment studies the similarity measure changes from to for identical sequences. such a measure is also defined for four-component spectral representation ( [ ] , see next chapter). another similarity measure, also normalized to for identical sequences, we have used for comparisons of d-graphs. this similarity measure introduces nonconventional treatment of graphs and their similarity analysis. we have not calculated the descriptors but the similarity measure has been directly obtained from the graphs. in our studies we treated the graphs as rigid bodies, as in the classical dynamics. as a similarity measure for a pair of sequences represented by the graphs we took mass overlaps [ ] . using the genetic methods, very efficient in problems of optimization, we found the locations of a pair of graphs for which their mass overlap reaches maximum. in this position the similarity measure is defined as a mass overlap of a pair of graphs. in the process of maximization of the mass overlap we considered shifts and rotations of the graphs. recently, i have introduced another graphical representation [ ] . in this section, the details and new aspects of this representation are described. graphically, this representation resembles the molecular spectrum so i call it spectral representation. the dna sequence is represented by a four-component function (or, graphically, by a four-component spectrum). a single dna sequence is represented by four abstract spectra: one for bases a, one for c, one for t and one for g. this means that i decompose each sequence to four components. each γ -component i call-γ spectrum where γ = a, c, t, g denotes one of the bases. each γ -component is given by a function that is a superposition of the gaussian functions: where n is the length of the sequence, and the parameter r is the resolution of i γ (x). for the visualization of long sequences it is convenient to take small r . the resolution parameter r determines the differences between the maxima of the gaussians. the details of spectra are better visible when r is large, i.e. when the neighboring maxima are well separated. with an increasing r the resolution becomes larger. if r = then the maximum corresponding to the first base ( p = ) is located at x = = and the maximum corresponding to the last base is located at x = n = n − . generally, the locations of the consecutive bases in one of the fourth γ -spectra correspond to x = , r, r, . . . , (n − )r , i.e. each single gaussian function makes the contribution to one of the fourth γ -spectra. if the neighboring γ bases are closely packed then the intensities (i γ ) increase. if the sequence does not contain one of γ bases then the contribution to γ -component may be zero and all the contributions are located in one of the three other γ -spectra. generally, the distributions of particular bases along the sequences are asymmetric and this information is reflected in the form of i γ (x). in principle, x may change from −∞ to +∞. however, in practical terms, i γ (x) = if x < −r or x > nr. therefore one can assume that the graphs extend for x ∈ −r, nr . in this way the first and the last bases are considered in the same way as the other ones. however, for the numerical characterization related to this graphical representation the range from −∞ to +∞ is considered. as the numerical characterization of the four-component spectral representation i propose the properly scaled distribution moments. analogously to the definitions of the moments of a discrete distribution (eqs. - ), the q-th moment of the continues distribution i γ (x) reads where is the normalization constant and r(x) is the range of x for which the integrand does not vanish. the normalization has been introduced for the numerical characteristics of the sequences. visualization is independent of the numerical calculations and it is more clear to consider unnormalized plots defined as γ -spectra in eq. . good descriptors of the distributions are also the centered moments m for which the first moment is equal to , and also m ( ) for which the first moment is equal to and the second one is equal to . considering several lowest moments it is convenient to perform integrations over the whole range of x (from −∞ to +∞). the integration can be performed analytically and where in the graphical representation defined in eq. , the summations are performed from p = to p = n for each γ . however the contributions of many terms are zero. only the terms for which the occupation number is different than zero give non-zero contribution to the γ -spectrum and their number is n γ which is the number of γ bases in the sequence and let us take an example of a model sequence atat. the nonvanishing terms that make the contribution to a-spectrum are for p = , . in case of t-spectrum p = , and for g and c-spectra all the contributions are zeros. as a consequence, the four-component spectrum is the descriptors associated with the four-component spectral representation (d γ q ) have been defined as properly scaled distribution moments [ ] . in particular and for q ≥ . as it has been shown in the article [ ] , due to the division by r, d [ ] . in particular, these diagrams can be used for an identification of genes. in this kind of visualization, different types of classified objects are clustered in different areas of the plots. as a similarity measure between a pair of sequences labeled by i and j is proposed, where q = , , , [ ] . though q may be easily increased up to higher-orders, as we shall see, the information about similarity sequences is specific enough up to the fourth order. let us note that d γ q is consistent with standard measures used in biology: for the identical sequences the similarity value equals % and it decreases (approaching ) if the difference between the two d γ q increases. the average information about the similarity of a pair of sequences is contained in the measure where are referred to as the weights, n γ (i) is the number of γ bases in the i-th sequence, and is the length of the i-th sequence. in order to study the problem of convergence of the method with respect to the higher-order moments i consider, for a pair of sequences labeled by i and j, the similarity measure where n is the maximum order of moments taken into account. all definitions may be easily generalized for multiple similarity studies. if j sequences labeled by i ≡ {i , i , . . . i j } are matched then the measures are defined as and the weights are equal to the relative numbers of γ bases in all the considered sequences and the measures defined in eqs. , and may change from % to %, analogously to the ones defined, respectively, in eqs. , and . an alternative similarity measure is defined in this work as s γ q is equal to if the descriptors of the i-th and the j-th sequences are the same (d γ q (i) = d γ q ( j)) and approaches if the difference between the two descriptors increases. this similarity measure is analogous to the one that we have introduced in the molecular similarity studies [ ] . i also introduce a similarity measure between the sequences labeled by i and j that carries the information about several (n) properties where i < i < · · · < i n and w i . . . w i n denote the weights. s i ,i ,...i n γ (i, j) is also normalized to the values belonging to the range from (identical properties) to . for example, if we consider similarity of three properties: the width, the asymmetry and the curtosis of the γ -spectrum that are described by s γ , s γ and s γ respectively, then n = , i = , i = , i = and the similarity measure is in this work all the weights in eqs. and are equal to . the units of descriptors d i k (eq. ) are normalized for i k ≥ . as a consequence, for example s , γ is a convenient measure for comparison of sequences of different lengths, if we are interested in the similarity information that is not related to the lengths of the sequences. if the information about the mean value d or about the width d of γ -spectra needs to be compared then s i ,i ,...i n γ , where i k are or may be considered. all the panels (a-d) in the figure represent the same model sequence. the difference is the resolution: r = , r = , r = , r = in panels a, b, c, d respectively. the particular bases are represented by gaussians centered at p = ( p − )r , where p = , , . . . . the first base is represented by a gaussian with the maximum located at = for all the cases and the last one at = , = , = , = for r = , r = , r = , r = respectively. for smaller r the bases are located close to each other and as a consequence the neighboring gaussian functions overlap and we observe the envelope of the spectrum. in particular, if all the bases are the same, the spectrum becomes rectangular (fig. , panel a) . increasing the resolution, the range for which the spectrum is different than zero becomes larger and we have a chance to look into details of the spectra. the details are the locations of particular bases along the sequence. for long sequences, the balance between the details of spectra and the range of the plot determined by the location of the last gaussian n = (n − )r has to be found. theoretically, the resolution may change from a small positive value to infinity. however changing the resolution not always results in a change of the information coming from the spectrum. for example, if in the model example the resolution is taken as smaller than then also rectangular representation is obtained. figure shows i a spectrum for this model example where r = . . the difference between r = (fig. , panel a) is the range ( = . for r = . ) and the maximum values of i a . for smaller resolution the range of the spectrum is smaller and the neighboring maxima are located close to each other. as a consequence of closely located gaussian functions exp[−(x − p ) ], the resulting maxima of spectrum i a are larger (around in fig. and around in fig. , panel a) . however the qualitative information is the same in fig. , panel a, and in fig. . in case of real sequences, there is a natural separation between the neighboring bases. usually the resolution r = and even smaller is sufficient for a good visualization. in fig. , spectral representation of histone h coding sequence of arabidopsis thaliana is shown (i = , table ). the length of the sequence is n = . the resolution has been taken as r = . the numbers of particular bases are n a = , n c = , n t = , and n g = . the largest number of a bases can be easily seen (large number of lines with large intensities as an effect of overlapping closely located gaussians representing a bases). the same sequence but with the resolution ten times smaller is shown in fig. . the resolution r = . seems to be sufficient to distinguish between those ranges of x for which the density of bases is larger comparing to ranges that are poor in the considered bases. a very convenient way of a direct comparison of the difference between a pair of sequences labeled by i and j is plotting the difference i γ i j . clearly, for both sequences i γ (x) must be represented with the same resolution in order to compare the distribution of γ bases along the sequence. figures and show the differences between a pair of sequences. in fig. the differences with resolution r = between the spectra representing histone h coding sequence of human (i = , table ) and histone h coding sequence of maize ( j = , table table ). the distributions of particular bases along the sequences. in particular, the number of lines in i a i j , i t i j , i g i j is smaller than for i c i j . this means that the difference of the distributions of c bases along the sequences is the largest comparing to the differences of the distributions of other bases. moreover comparing the number of lines that are positive to the ones that are negative, for a particular plot, one can easily estimate the differences between the numbers of the particular bases. for example, n c = for the sequence of human and n c = for the sequence of maize so the number of negative lines for i c i j is larger then the number of the positive ones. analogously, the table ) number of negative lines for i g i j can be seen: n g = for the sequence of human and n g = for the sequence of maize. since the number of a and t bases are larger for the sequence of human then for the sequence of maize, one can observe in i a i j and i t i j plots more positive lines than the negative ones. in fig. the differences with the resolution r = between the spectra representing histone h coding sequence of human (i = , table ) and histone h coding sequence of mouse ( j = , table ) are shown. as a result of the difference between the numbers of a bases one can observe in i a i j more positive lines than the negative ones: n a = for the sequence of human and n a = for the sequence of mouse. the difference in c bases is also clearly seen. there are more negative than positive x fig. differences between the spectra for histone h coding sequence of human m and histone h coding sequence of maize m (i = , j = , table ) lines in i c i j plot: n c = for the sequence of human and n c = for the sequence of mouse. generally, comparing fig. and fig. one can see that the differences human-maize spectra are larger then the differences human-mouse spectra (the number of lines in fig. is larger then the number of lines in fig. ). x fig. differences between the spectra for histone h coding sequence of human m and histone h coding sequence of mouse v (i = , j = , table ) as the descriptors of the four-component spectral representation, i have proposed d γ q . figure shows d g − d g diagram for ten sequences listed in table and for one additional sequence (one point in the figure represents descriptors of one sequence). the additional sequence is histone h coding sequence of human (m ). in many articles the ten sequences were treated as a model set to introduce new graphical and numerical representations. however, there was a mistake in the old version of the embl database. obviously, the length of this coding sequence should be and not as it was in the old version of the embl database. the additional base is g, located at the last position of the sequence. the descriptors d γ q of spectral representation are very sensitive. the difference by only one base can be detected using these descriptors. moreover, the approximate location of this base can be indicated. the descriptors characterizing the same sequence calculated using the old and new version of the embl database have been denoted using different symbols in fig. . their locations are different in the diagram. it is remarkable that the difference by this very base may be recognized in the plots. table figures , , , show the diagrams also for the sequences listed in table . in particular, fig. shows diagrams for g-descriptors, fig. for adescriptors, fig. for c-descriptors, and fig. for t-descriptors. panels a in the figures correspond to d table indicates the location in the sequence of the base that is different for a pair of sequences. the additional g base in the new sequence causes the shift to larger values of the mean of the distribution (d g becomes larger, fig. , fig. , panel a). the width of the distribution also increases (d g for the new sequence is larger than for the old one, fig. table lows: considering the properties of g and a-spectra (g and a-descriptors shown in figs. , , respectively) one can observe clusterization of evolutionary similar organisms: plants and vertebrates that are represented by different symbols in the plots (plants-circles, vertebrates-triangles). considering the properties of cspectra (fig. ) one can find the properties that are specific for plants and different than for vertebrates and also one can find the properties that are com- table mon for plants and vertebrates. for example in panels a and c where d it is interesting to note that most of the similarity measures (both the standard ones and many alternative ones) indicate larger or equal similarity values between histone h coding sequences of chicken (labeled by i = , in table ) and plants (labeled in this table by j = , , , ) than between these of chicken and of vertebrates (labeled table similarity measures between a pair of sequences labeled by i and j sim(i, j), where i and j are defined in the first column of table sim sim ( , ) sim ( by j = , , ) . however, using new similarity approach it is possible to extract such components of the similarity measures that cluster the sequence of chicken with the ones of vertebrates rather than with the ones of plants [ ] . table shows similarity values obtained using different similarity measures "sim". using alignment method (sim=cl) the similarity value "chicken-plant" cl( , ) is the same as the similarity value "chicken-vertebrate" cl ( , ) . considering different aspects of similarity, using d γ , one can see that the clusterization of the sequence of chicken with vertebrates is obtained for γ = g, a, c. however the asymmetry of the gene structure for t bases is identical for the sequence of chicken and of plants (d t ( , ) = ) and the similarity value is small in case "chicken-vertebrate" (d t ( , ) = ). figures and show the diagrams for the sequences listed in table (histone h coding sequences of different species). in particular, fig. shows d and d γ is linear. the most regular linear dependence is for g-descriptors (fig. , panel d) . however, using the diagrams for the descriptors independent of the lengths of sequences (fig. ) , for a and g-descriptors (panels a, d respectively) the clusterization of plants and vertebrates is observed. for c-descriptors, the effect of clusterization is smaller. the effect of clusterization is not observed for t-descriptors. t-descriptors representing sequences of plants and vertebrates even overlap. these observations are the same as in the case of histone h coding sequences. figures , , , , show the relations between the standard calculations clustal w (c l) and the new measures (eqs. , , ) for the sequences listed in table (histone h coding sequences). table figures , , , , , , , , are plotted in the same way as it has been done in chapter (figs. , , ) . each point in the plot corresponds to one case: comparison of sequence of species no. i with sequence of species no. j using different methods. for example, the horizontal axis in fig. corresponds to the similarity matrix between sequences of different species using clustal w method (c l) and the vertical axes correspond to the similarity matrix between the same sequences using different components of alternative similarity measures d γ q . as a consequence each plot represents two similarity matrices, which gives a better visualization of the relations between two different similarity measures. in the figures, the functions x = y, where x and y represent, respectively, the horizontal and vertical axes, are plotted table (dashed lines). comparing the distributions of the points around the dashed lines it is easy to recognize these aspects of similarity for which the relations are the same. if the points are concentrated close to the lines then the similarity relations represented by x and y axes are also close to each other. the similarity matrix c l based on clustal w approach for the considered sequences is given in [ ] . small range of similarity measures indicates small differences table between the sequences of different species. the range of values of c l is from % to %. the ranges of values of d γ q for q = , q = , and q = are smaller than for q = for all γ . d γ changes from about % to % for all γ . the differences between sequences across species using d γ are smaller than using d since each γ -component is related in a different way to the standard measure, one may expect that it carries independent similarity information. averaging the measures over γ , and then averaging over q, d m e an q (eq. ) and d n (i, j) (eq. ) are obtained. figure shows the relations of d n with the standard measure. the convergence of d n measures to the standard measure cl we have discussed in [ ] . in the present paper this effect is shown in detail adding d table term. d is very different from cl (the points are located far away from the dashed line, panel a, fig. ). adding higher-order terms, the points are pushed towards the dashed lines (panels b, c, d fig. ). figures , , , , show similarity relations for β-globin gene across species using similarity measures defined in eqs. and . these data are the standard ones for alternative methods. since the sequences in the database are not complete for some species, they are unified in this work and the appropriate locations in the gene are listed in the tables. in particular, the sequences of mouse and of chicken belong to the standard set of data used by many authors. however, several bases are ambiguous for the third exons for the sequences of the two species. as it was already mentioned, the method used in this work is so sensitive that even a difference in a single base can influence the results. therefore the sequences of mouse and of chicken are omitted from this consideration. moreover, in gorilla and chimpanzee sequences the stop codons are not available in the database. therefore for all the species the stop codons are excluded from the calculations. this means that the length of the coding sequence n cds is three times larger than the corresponding length of the protein sequence for all the species. in this way (excluding the stop codons) all the data used in the calculations are consistent. table the locations in the gene, the numbers of γ bases, n γ k , for each k-th exon according to the latest version of the embl database are specified in tables , table . . the whole first exons which are given in the embl database only for three species with the length n w , denoted exon , . the coding sequences with the lengths n cds = k= n k , denoted cds. γ are also shown in fig. . in this figure the measures are compared for different parts of the β-globin gene. the horizontal axes correspond to the sequences with introns, plusi. the vertical axes correspond to the coding sequences of particular exons: column to exon c ds , column to exon c ds and column to exon c ds . the first row of subfigures correspond to a bases, the second row to c bases, the third row to t bases and the fourth row to g bases. we observe that the points are concentrated around the dashed lines in the middle column (exon c ds ) comparing to the first and to the third columns. small deviations from the dashed lines mean that the second exon is most representative in the whole sequence, plusi (the similarity relations across species fulfilled by plusi and and by exon c ds are closer to each other than the relations fulfilled by plusi and by the other exons). we have also shown that the similarity relations across species fulfilled by cds and and by exon c ds are closer to each other than the relations fulfilled by cds and by the other exons [ ] . if we compare the distributions of the points between different bases (rows) one can extract some properties common for particular bases and for some parts of the genes. by a common property we understand close to zero s , γ (small values correspond to large similarities). in particular small differences between sequences across species are revealed for g bases for the first and for the second exons (panels j, k) and also for c and for t bases for the second exon (panels e, h). generally, larger differences are seen for longer sequences. however also for plusi one can extract properties more common for the species (small ranges of s , a [plusi] and s , t [plusi]-first and third rows). figure shows similarity relations for different exons using standard alignment method clustal w version . [ ] . as it was mentioned before, the alignment methods do not take into account which bases are aligned. the alignment of all the bases gives the contribution to the final result and, as a consequence, the similarity is large for all the parts. it is not possible to extract detailed properties of similarities. the information coming from these calculations is averaged. finally, the similarity table values for different exons are the same for all the species since most of the points are concentrated close to the dashed lines. complete sequences for the first exons are given only for three species (table ). the whole sequences of the first exons for human and gorilla differ by only one base. as we see in fig. this is g base. the descriptors d a , d c , d t are exactly the same for human and gorilla sequences. the difference caused by this single base is recognized by d g (panel d). summarizing, four-component spectral representation has been used for similarity/dissimilarity analysis of histone h coding sequences across species (figs. , , , , , , , , , , , ) , of histone h coding sequences across species (figs. , ) , and of different parts of β-globin gene across species (figs. , , , , ) . since many authors use slightly different data for β-globin gene, the locations of different subsequences in this gene and their full description listed in the tables may be helpful for some alternative similarity studies. the numbers of particular bases in all the sequences are also given. it has been shown that the four-component spectral representation can be used for the classification studies (clusterization of the descriptors representing histones h and h coding sequences of plants and of vertebrates). analogous clusterization is also obtained using some descriptors related to d-dynamic graphs (sect. ). the sensitivity of the four-component spectral representation has also been shown. in particular, a difference between a pair of sequences by only one base can be recognized. also the approximate location of the difference and the base which is different in the compared sequences can be also determined. it has been shown that if higher-order terms of similarity measure based on the descriptors of the four-component spectral representation are added and normalized in the same way as in the alignment methods then a convergence to clustal w results may be obtained. this means that the results obtained with the alignment method may be interpreted as an average of the considered components of the alternative similarity measures. calculating an average is always related to some loss of information, i.e. large degree of degeneracy may appear. as we know, this is an inconvenient feature of similarity/dissimilarity analysis. for example, using the alignment methods the two situations . aaaa aaaa . tttt tttt cannot be distinguished. therefore, using the four-component spectral representations one has a chance to decompose the similarity information and remove the degeneracy. reducing the degeneracy can also be obtained by adding the corrections to the alignment methods related to different aspects of similarity, as it is proposed in sect. of this work. it has been shown that each part of β-globin gene demonstrates different similarity relations across species. the relations also change when different aspects of similarity are compared (asymmetry of the gene structure or kurtosis of the distributions). therefore using different descriptors or different graphical representations the results may be or very often should be contradictory. different alternative methods describe different aspects of similarity. in particular, most of alternative studies that have been performed for exon cds of β-globin gene often give contradictory results. for example the similarity value of exon cds human-goat is larger than human-mouse if the methods described in the works [ , , , , ] are used. the reverse situation i.e. similarity value between the sequences of exon cds human-goat is smaller than human-mouse if methods taken from [ , , , , , , [ ] [ ] [ ] are applied. many authors introducing new graphical representations for beta-globin gene try to avoid considering chimpanzee and gorilla sequences not only because the data are not complete but also because the results are often different than our expectations. we expect the largest similarity for human-chimpanzee sequences. however detailed similarity/dissimilarity analysis of beta-globin gene using four-component spectral representation indicates that this is not true for all parts of the beta-globin gene and for all γ -components of similarity measures. according to the definition of the new measures, s , γ becomes smaller if the sequences are more similar. considering the second exon, i obtain the largest similarity in the case of human-chimpanzee sequences. this means that s , γ is the smallest for the two sequences for all γ , and in particular s , γ = for γ = a, c, t . the difference between the two sequences is only in the distribution of g bases. it is interesting to note that s , γ = for the second exon, both for c and for t bases, in three cases: human-chimpanzee, human-gorilla and gorilla-chimpanzee sequences. however for other exons, s , γ is not always the smallest in the case of human-chimpanzee sequences comparing to human-other species sequences. if the sequence with introns, plusi, is considered then s , c is the smallest for humanchimpanzee sequences and for γ = a, t, g, s , γ are the smallest for human-gorilla sequences. each descriptor may be related to different biological function. since we are at the beginning of the way of understanding in which contexts particular descriptors may play the key role, the creation of new alternative methods aiming at similarity/dissimilarity analysis of biological sequences is of particular importance. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. parts of the first exons starting with the start codon (coding sequences of the first exons) with the length n , denoted exon c ds , . the second exons with the length n biological sequence analysis introduction to computational biology: maps, sequences, and genomes: interdisciplinary statistics advances in molecular similarity topological indices and related descriptors in qsar and qspr symmetry, spectroscopy and schur a new view on similarity of dna sequences annual review of nuclear and particle science the advanced theory of statistics symmetry and structural properties of condensed matter the oxford companion to music key: cord- -vlj ay d authors: zhao, hui; meng, qiang; wang, yadong title: probability estimation model for the cancellation of container slot booking in long-haul transports of intercontinental liner shipping services date: - - journal: transp res part c emerg technol doi: . /j.trc. . sha: doc_id: cord_uid: vlj ay d the intercontinental liner shipping services transport containers between two continents and they are crucial for the profitability of a global liner shipping company. in the daily operations of an intercontinental liner shipping service, however, container slot bookings from customers can be freely cancelled during a booking period, which causes loss of revenue and low utilization of ship capacity. though a pain-point of the liner shipping industry, the container slot cancellation problem has not yet been well investigated in the literature. to fill this research gap, this study aims to estimate the probability for the cancellation of container slot booking in the long haul transports of the intercontinental liner shipping service by considering the primary influential factors of cancellation behavior. to achieve the objective, a container slot booking data-driven model is developed by means of a time-to-event modeling technique. to incorporate the effect of booking region on the cancellation probability, we introduce the frailty term in the model to capture the regionality of the container shipping market. our case study with real slot booking data shows that the developed model performs well in forecasting the loaded containers of the slot booking requests. in addition, we shed light on how the internal factors of slot booking and external factors of shipping market influence the probability of cancellation. the liner container shipping companies transport containers on the fixed shipping routes with regular schedules (wang et al., ; wang et al., a) . nowadays, there are about international liner container shipping services in operation carrying about % of the cargoes in international trade by high-capacity container ships (unctad, ) . the intercontinental liner shipping services operated by the global liner container shipping companies transport containers from one continent to another. this kind of shipping service plays a vital role in the global supply chain and global trade (yang et al., ; wang et al., b) . fig. depicts an intercontinental shipping service between asia and north america operated by oocl. a string of container ships is deployed in an intercontinental shipping service to periodically visit the corresponding ports for container loading and discharging operations. for example, six container ships are needed to provide the weekly service frequency for the service shown in fig. . a loop of all the ports in the service is called a shipping "voyage" hereafter. a ship could complete about - voyages a year, and each voyage of these ships is numbered by a voyage code in practice. a directed link between two adjacent ports of a voyage is called a shipping "leg", and a shipping leg connecting two ports on different continents is referred to as a long-haul leg. asian shipping companies usually name a long-haul leg from asia to other continents as a head haul (hh), and the opposite direction as a back haul (bh). the long-haul legs are the longest legs in terms of distance in the shipping service and generate more profit than other legs. for the sake of presentation, we call this kind of transport the "long-haul transport". specifically, container transport via a hh leg is called a hh transport, and that via a bh leg is called a bh transport. it should be noted that although a hh leg connects two countries, e.g., china and the usa shown in fig. , it can transport containers originating from the other countries/ regions owning to the container transshipment operations (schinas and psaraftis, ) . the container slot booking process is the first phase to make an intercontinental liner shipping service provided by a shipping company, as illustrated below (see fig. ): given a planned voyage of a container ship serving an intercontinental liner shipping service, container slots allocated to a port are released for customers booking about - days before the container ship departs from the port in practice. about - days before ship departure time, the shipping company closes the slot booking, loads containers, and issues necessary shipping documents to customers. a container slot booking requested by a customer (shippers/freight forwarders) includes the number of slots, origin port /destination port as well as the other necessary cargo information. the shipping company should reserve the container slots of ships for the customer according to their booking information. the customers then prepare cargoes and transport their containers to ports. however, during the slot booking period, some of the bookings can be cancelled by a customer before ships depart from an original port, resulting in the low ship capacity utilization rate and low profit. especially for the long-haul legs, the cancellation of slot bookings can incur more loss of profit due to their higher freight rates. this study focuses on the slot booking period for a long-haul leg in an intercontinental container liner shipping service since the long-haul transport is the most profitable in the intercontinental shipping service. it is imperative for a liner container shipping company to have a solid model that can estimate the cancellation probability of container slot booking for the long-haul legs based on historical container booking data. this is because such a model enables the shipping company to predict the number of cancelled bookings and the number of containers that could be loaded on a ship. if the number of expected loaded containers is less than the allocated slots of a ship prior to ship departure, the shipping company will continue to accept slot bookings or prompt the shipping services to fill the ship capacity. nevertheless, several factors affect the cancellation probability of container slot bookings, such as freight rate, shipping market condition, booking time, and customer information . therefore, this study aims to develop a container slot booking data-driven model for estimating the probability that a slot booking is cancelled before the loading time by considering the influence of the attributes of the slot booking (e. g., booking time, booked volume), the container shipping market (e.g., shipping index, market volatility) and customer information the developed model can assist the container shipping company to improve the loading rate of the ship and to understand how the internal and external influential factors affecting the slot cancellation. the developed model also lays a foundation for the emerging studies on liner shipping revenue management . for example, it is essential to consider the cancellation of slot bookings in pricing policy which is an important method to raise revenue in container shipping revenue management (von westarp and schinas, ) . container shipping companies implement differential pricing to customers, thus rebates can be given to customers who have lower cancellation probabilities to encourage the fulfillment of slot booking. this model can also benefit the studies of overbooking in container shipping which is a good policy to reduce the waste of ship space incurred by cancellation. there are scant studies on the cancellation of container slot booking. one reason is that container shipping companies are generally conservative and less likely to share their slot booking data for academia. there are only two articles directly related to container slot booking cancellation analyses. wang and meng ( ) used the statistical methods to forecast the net container slot booking, defined as the difference between the booked and cancelled slots, depending on historical container slot booking information. however, their methods could not consider any influential factors to estimate the individual-level cancellation probability. zhao et al. ( ) extracted the possible attributes/factors that influence the customer behavior of slot booking cancellation from the real container slot booking data. their empirical study shows that the relationship between influential factors and cancellation rate is highly nonlinear and calls on a further study to analytically model the relationship. to the best of our knowledge, there are no studies dealing with the estimation of the slot booking cancellation probability although it has been highlighted and requested by several scholars (e.g, lee et al., ) . there are some similarities between container slot cancellation modeling and air ticket/cargo cancellation analyses. the air ticket or air cargo cancellation behaviors have been extensively examined in literature by using different methodologies (e.g., garrow and koppelman, ; hueglin and vannotti, ; iliescu et al., ; graham et al., ; chiew et al., ; morales and wang, ) . although the airline ticket cancellation studies could provide some enlightenment for container slot cancellation modeling, estimating the probability of cancellation in container slot booking is a new and worthy research topic because of the following exclusive features. first, the container transport demand is generated from international trades that exhibit regional characteristics. the intercontinental liner shipping services connect multiple trade zones covering various countries with different shipping policies, shipping market conditions, import/export volume, and cargo type. in other words, the container transportation demand from different countries exhibits diverse characteristics in container slot booking and cancellation. in this regard, the container slot cancellation behavior of customers in the same market region can be correlated and affected by the regional property of trade. thus, it is more practical to assume that some container slot bookings are correlated. however, the studies on air ticket/cargo cancellation postulate that all the bookings are independent. second, the container slot bookings can be freely cancelled without any penalty in practice. for the seat booking in airline, some bookings are not allowed to be cancelled or could be cancelled with a high cancellation fee. however, the container slot cancellation fee is not widely adopted in the container liner shipping industry. the competition among container shipping companies is fierce, thus charging a penalty for cancellation could lead to loss of customers. for example, few container shipping companies (e.g., maersk line) had attempted to charging cancellation fee for some services, but they finally withdrew this fee because other shipping companies would not follow. in addition to cancellation fees, a few third-party platforms also provide shipping companies with some methods to avoid losses caused by the cancellations. one example is new york shipping exchange (nyshex), a digital freight marketplace, acting as a neutral contract arbiter for shippers and carriers in an attempt to prevent cancelled bookings and cargo rolling. another thirdparty platform, cubits, also launched a business that used blockchain technology to develop shipping bitcoins to resolve cancellation and rolling issues. unfortunately, this business was eventually discontinued due to insufficient participation by shipping companies. in other words, the third-party platform solutions are still at the infant development stage. at the current practices, as cancellations by customers are quite common, the shipping companies take the overbooking strategy to hedging the cancellation risk. once the number of show-up containers exceeds the capacity of the ship, some containers can only be postponed to the next voyage. as a consequence, it is a common practice for a customer to book with several container shipping companies to guarantee the slots. this phenomenon results in a high cancellation rate in the container liner shipping industry. third, there are several key factors affecting the cancellation rate of container slot booking, including the attributes of slot bookings (e.g., volume), shipping market conditions (e.g., level of shipping index), and customer information. when estimating the probability of cancellation, both the internal and external factors should be considered simultaneously. however, the studies on the cancellation in airline rarely consider the external factors of the market. h. zhao et al. transportation research part c ( ) the objective of this study is to develop a container booking data-driven model for estimating the probability that a slot booking is cancelled prior to the loading time in a long-haul leg of an intercontinental liner container shipping service. the model focuses on the slot bookings of one direction (hh or bh) in an intercontinental container shipping service. the models of two shipping directions should be separately built because container transportation demand in the two directions has different characteristics . the input data are the historical data of the slot bookings (e.g., booking time, loading time, origin/destination), the data of internal influential factors (e.g., freight rates), and the external data of shipping market (e.g., freight index and user information). these data enable us to build a tangible model that can estimate the cancellation probability of container slot bookings with consideration of the aforementioned characteristics. to achieve our objective, we develop a time-to-event model with a hazard function. time-to-event models are a family of models to analyze the time before an interested event happens (hosmer et al., ) . the event in this study is the cancellation of the slot booking prior to ship departure. we define the time to cancellation and transform the estimation of cancellation probability to the estimation of the distribution of time to cancellation. to consider both internal and external factors affecting the cancellation, we use the hazard regression approach to formulate their influences on the cancellation. additionally, to reflect the specific feature of market segmentation in container liner shipping, we introduce an interesting frailty factor to reflect the correlation of customer's slot booking behavior from the same market. in other words, the customers within a market region (i.e. country) are assumed to have correlated behaviors. the contributions of this study are presented. first, to the best of our knowledge, this study is the first attempt to estimate the cancellation probability of container slot booking. as pointed out by a few researchers (e.g, zurheide and fischer, ) , the lack of research into the cancellation probability estimation hiders the studies on container slot allocation and revenue management in the shipping industry. to some extent, this study fills up this gap. second, this study develops a time-to-cancellation model with origin country frailty to consider the property of the market regions. we introduce the frailty term in survival analysis to capture the correlated behavior of cancellation caused by the regionality of international shipping market, which is a novel application of the frailty theory in the container liner shipping industry. this model can assist container shipping service operators to forecast the loading container volume using the data in the slot booking period. third, real data from a container liner company are used to calibrate and evaluate the model. thus, the results of this study can lead us to have more practical insights. fourth, some behavioral insights into the container shippers' cancellation behavior are analyzed, which is helpful in deciding the customer segmentation and identifying the peak time of cancellation during the booking period. the remainder of this study is organized as follows. section elaborates container slot booking data and the research problem. section develops the container slot booking data-driven model. section presents a case study. section provides valuable insights on the cancellation behavior of customers. section concludes this study and points out some future research directions. let us consider an intercontinental liner shipping service with hh and back bh directions, which is operated by a global liner shipping company. a number of container ships are deployed in the shipping service to maintain a regular service frequency (e.g., weekly service). before a deployed ship departs from a particular port, customers will book the container slots of the ship to transport their containers from a loading port to a discharging port by providing the necessary container slot booking information. customers can request to cancel these bookings before the ship leaves the loading port. after receiving a container slot booking or cancellation request, the information technology (it) department of the shipping company updates its slot booking database. the container slot booking and cancellation data format, illustrated by the first three columns in table , can be exacted from the database. the data example shown in the last column of table is created with respect to the shipping service in fig. . cancellations could be incurred at any time after the shipping company accepting the slot booking and before the containers are loaded to the ship. the cancellation time, shown by the last row of table , is the time when the slot booking is cancelled by the customer. these time data are recorded only if a slot booking is cancelled. thre are two situations of slot booking cancellations. in the first situation, customers inform the shipping company to cancel their bookings, and the cancellation time is the time when the shipping company receives the slot booking cancellation request. in the second situation, customers just do not transport their container to the port before the port cut-off time, and this kind of slot bookings is "no-show" slot bookings. for the no-shows, the cancellation times are assumed to be one day before the ship departure time, which is usually the port cut-off time in practice. for the convenience of presentation, we refer to both cancelled and no-show bookings as "cancelled bookings" in this study. let us examine the influential factors of the cancellation behavior of customers. based on the empirical study by zhao et al. ( ) , the following factors have the impact on the slot booking cancellation: the time attributes of slot booking, the origin/destination of slot booking, the volume of the booked slots (in teus), the attributes of the booked voyage, the customer information, and the freight rate of booking. these factors are the necessary inputs for the model development. additionally, we include the market region of slot booking to reflect the regionality of container liner shipping, and add the average rates and the fluctuations of shipping market index as external factors in order to reflect market conditions. the influential factors considered by this study are presented in table . next, we further analyze the influence factors presented in table as follows: we choose the booking time, booked slots in teu as well as the freight rate for the slot booking as the first type of influence factors because they have an impact on the cancellation behavior of customers . the booking date in table is separated into two attributes presented in table , with bk_m as the month of booking and bktdp as the time of booking. bktdp is measured by days prior to departure. in these two time attributes, the bk_m reflects the season of booking and the bktdp describes how early the booking request is proposed. the second group of covariates is the booked teu which is the number of booked teu proposed by customers. in addition, to investigate the influence of the attribute of voyage, we use the month of the ship departure time (dp_m in table ) to imply the seasonality of voyage time. the customer information is also an important influential factor of the cancellation behavior. such information includes the types of the customer (e.g., freight forwarders, manufactures), customer size (e.g., large retailers), customer location, and relationship with the shipping company. unfortunately, these data are confidential for shipping companies and not available for this study. to incorporate the customer influence, we use the size of the customer's booking to describe the customer information, which is measured by the customer's booking volume in a year (in teu). this booking volume of customer can measure the closeness of cooperation between customers and companies, and can also be used to judge large and small customers. (ii) regional characteristics of container shipping. as aforementioned in section . , the international trades of different origins have various characteristics. the regional characteristics of cargo, namely cargo origins, are the factors for the cancellation probability estimation model development. fig. gives the examples of three bookings in the hh transport of the shipping service shown in fig. to clarify the definition of regional characteristics. the origin countries of bookings , , and are china, philippines, and vietnam respectively. the containers of these three bookings are all transported to the usa through the long-haul voyage. bookings and are first transported to the ports in china by either land transportation or feeder shipping service and then transported to the usa through the hh transport. although they are all transported via the ports in china, these three bookings belong to the different international trades. to reflect this regionality, the original country of a container, rather than the loading country, is also incorporated in the model development. (iii) shipping market. in addition to the voyage information and booking information, we propose the influential factors of shipping market in order to reflect the influence of a specific market environment. two aspects of the shipping market, namely, the average and the fluctuation of the freight rates in the shipping market are included as the inputs of our model development. as it is difficult to obtain the real freight rates of all the shipping companies, in this study, we use the china (export) containerized freight index (ccfi) which is a conventional index of the freight rates of the shipping market. the latest ccfi data of slot booking is used to reflect the average freight rate of shipping market. moreover, we calculate the variance of the last three weeks' ccfi data (ccfi_var in table ) to imply the fluctuation of the shipping market. a high ccfi_var implies a fluctuating market while a low ccfi_var indicates a stable market. with both ccfi and ccfi_var, the influence from the shipping market on the cancellation probability can be analyzed. let us detailedly introduce the research problem. for a given intercontinental container shipping service (e.g., the shipping service in fig. ) and a direction of long-haul transport (e.g., hh transport in fig. ), the historical slot booking and cancellation data are available. these data cover both the non-cancelled and cancelled bookings of the long-haul transport of the service. the factors influencing the behavior of cancellation are listed in table and they can be extracted from the historical data and ccfi index. the aim of this study is to develop a slot booking data-driven model to estimate the probability of slot booking cancellation by considering the key influential factors. we denote a particular container slot booking by b with the given booking information. the influential factors, shown in the third column of table , are grouped into vector z b , namely: . this study is to develop a model f( ⋅ ) which can estimate the cancellation probability of booking b considering the influential factors z b as follows: where prob clc(b) is the probability that booking b is cancelled during the booking period while prob noclc(b) is the probability that the booking b is not cancelled. this model depends on the slot booking data. with the model, the shipping company can estimate the cancellation probability of the accepted slot bookings. the empirical study conducted by zhao et al. ( ) has concluded that the logistic regression model cannot be used to estimate the slot booking cancellation probabilities. therefore, we adopt the time-to-event modeling approach here because it has been shown to be effective in forecasting cancellations in literature (e.g., iliescu et al., ) . to do so, a frailty term should be introduced to reflect the correlations of the cancellation behavior within an origin country. the time-to-event models are a class of analytical methods on modeling the feature of the time before an interested event happens (hosmer et al., ) . this model has been widely applied in the demography, biology, and medicine industries, but rarely applied in shipping industry. in this study, the event is the cancellation of a container slot booking. accordingly, the time to cancellation (ttc, or lifetime) of a container slot booking is defined as the time period from its booking time to the cancellation time. based on ttc, we can transform the problem of estimating the cancellation probability to the problem of modeling the distribution of the ttc (yu et al., ) . therefore, in this study, the time-to-cancellation model (ttcm) aims at formulating the distribution of the ttc. we have two assumptions for the ttcm: (i) every booking has the possibility of being cancelled or not showing. (ii) the cancellation probability of a booking is the probability that the ttc of the booking is less than the days from booking time to ship departure time. in other words, if the slot booking can "survive" to the ship departure time, it will not be cancelled. we formulate the ttc as a random variable t with the probability density function (pdf) f(t). according to the assumption (ii), the cancellation and non-cancellation probabilities of a booking b with booking time t b can be calculated by there are two key functions in time-to-event analysis: survival function denoted by s(t) and hazard function denoted by λ(t). these two functions are elaborated in appendix a. there is a natural relationship between pdf, hazard function, and survival function (hosmer et al., ) : therefore, modeling the distribution of t can be converted to the modeling of λ(t) which implies the instantaneous rate of occurrence of the cancellation. the deducing of this relationship can be found in appendix a. we formulate the hazard function λ(t) as the cox proportional hazard function according to the cox hazard regression method (kay, ) . the cox regression method provides an effective and tractable form of hazard function to incorporate the covariates. this regression method assumes that there is a baseline hazard function and the effect of the covariates is proportional to this baseline hazard. assume that the characteristics of an individual booking can be described as a vector of covariates z. according to the cox regression method, the hazard function is assumed to have the form: where β is the vector of coefficients; λ (t) is the baseline hazard function which is the hazard function when z = . as expressed by eq. ( ), the hazard function λ(t) is proportional to the baseline hazard function λ (t), and the proportion is determined by the covariates z and coefficients β. λ (t) can be specified in various analytical forms. in order to consider the regionality of shipping market, we introduce the term "frailty" based on this classical hazard regression model. the frailty approach is a statistical modeling concept which aims to account for heterogeneity caused by unmeasured covariates from the clustered data (li and fan, ) . in statistical terms, a frailty model is a random effect model for time-to-event data, where the random effect (i.e. the frailty) has a multiplicative effect on the baseline hazard function. individuals in a cluster (origin country in this study) are assumed to share the same frailty, which reflects the behavioral correlations of the individuals. in this study, the frailty term from the origin country i is assumed to be w i , and it is further assumed that the individuals in the same origin country share an identical frailty term. thus, the hazard function of the individual slot booking j from an origin country i can be formulated by where z ij is the covariate vector containing the values of the influential factors. the corresponding survival function of a slot booking j from origin country i can be written as follows: where Λ ij (t) = ∫ t λ ij (x)dx is the cumulative hazard function. in eq. ( ), the baseline hazard function λ (t) reflects the shared feature of the bookings among all origin countries, while frailty term w i is a weight to reflect the influence from different origin countries (i.e. regionality). the w i is usually considered as the unobserved effect from the origin countries and assumed to be independent identically distributed random variables that follow a certain distribution. conventionally, a gamma distribution with e(w i ) = and var(w i ) = θ is assumed for computational convenience (hougaard, ) . the variation of frailty term θ indicates the variability between different origin countries. it is also a useful statistic to rank the cancellation risk of various container shipping markets and assist shipping companies to make variant revenue management strategies for different market sections. the baseline hazard function λ (t), the parameter θ, and the coefficients β are all to be estimated in the ttc analysis model in this study. the shape of λ (t) depends on the characteristics of the container slot booking and cancellation data. the form of baseline hazard function λ (t) is flexible, and can be specified as a non-parametric form or parametric form. this study uses both the two forms of baseline hazard functions to compare their performances. in the parametric form of baseline hazard function, it is conventional to assume that the ttc follows a weibull distribution with two parameters, and the shape of the baseline hazard function is determined by the weibull distribution. however, the baseline hazard function λ (t) that fits the slot booking data may have a complicated shape that cannot be captured by a given form of function. to formulate a more flexible shape of λ (t), we use the spline hazard functions as the non-parametric forms of λ (t). the spline function is capable of approximating complex shapes with a piecewise polynomial function. the spline function is formulated as a linear combination of basis functions which have several forms such as b-spline and mspline basis functions. in this form of baseline hazard function, the parameters to be estimated are the coefficients of the linear combination. the formulations of the three baseline hazard functions are shown in appendix b. the maximum log-likelihood method is used to estimate parameter of the baseline hazard function λ (t), denoted by μ, the coefficients β, and parameter θ in the ttcm. under the above two assumptions, the cancelled bookings and non-cancelled bookings contribute differently to the likelihood function since they provide different information about t. fig. depicts four container slot bookings (i.e. b , b , b , and b ) that belonged to the same voyage and to be loaded on the same port. the horizontal axis is the time axis with time means the time when the ship departures from the loading port. all the booking/ cancellation time is measured as the number of days before the ship departure time . the lines (b to b ) in fig. show the observable "lifetime" (i.e. ttc) of the slot bookings. the start point of each line is the booking time, and the end point is either the cancellation time for cancelled booking or the loading time for the loaded booking. in the four slot bookings, b and b were cancelled before ship departure, and b was a no-show booking. b was a non-cancelled booking. the booking time and/or cancellation time of each booking are shown in the table of fig. . as illustrated by fig. , the observed lifetimes of b , b , and b are days, days, and days respectively, which is exactly the ttc of these bookings. however, not all the bookings can provide the full information of the lifetime. for the bookings that are finally loaded onboard (i.e. b ), what we know is that its lifetime is no < days, the time to loading (ttl), and it survives until the ship departure time . therefore, the likelihood contributed by cancelled and non-cancelled bookings should be formulated separately. we will first discuss the likelihood function of a general cox hazard regression model formulated in the form of eq. ( ), and then extend it to the likelihood function for the model with frailty term w i . we denote the observed ttc/ttl of a slot booking j by t j . t j is the time to cancellation for cancelled bookings (e.g., b , b , and b in fig. ) , and time to loading for non-cancelled bookings (e.g., b in fig. ) . for the cancelled bookings, the contribution of likelihood is the probability that the cancellation happens at the t j days after booking, which can be formulated as follows: fig. . four examples for container slot booking and/or cancellation times. the non-cancelled bookings provide the information that no cancellation happens before the ship departure time. thus, the likelihood contributed by a non-cancelled booking is one minus the cancellation probability denoted by p [ , namely: thus, the likelihood function of the whole sample set (i.e. both cancelled and non-cancelled) can be formulated by where α j is the cancellation indicator which equals to if the booking is cancelled and otherwise, and j is the full sample set of the historical slot booking data. in the case of the model formulated in section . , it is assumed that there are n origin countries denoted by i = , ,…,n, and an individual booking in origin country i is denoted by j with j = , , …, j i where j i denotes the number of bookings in origin country i. as there are n origin countries, we start from the partial likelihood function of an origin country i. according to eq. ( ), the partial likelihood of the slot bookings from origin country i can be expressed by the formulation of partial likelihood function and details for obtaining this function are shown in appendix c. based on eq. ( ), the full likelihood of the sample data, including slot bookings from all the origin countries, is the product of all the partial likelihoods. the logarithm of the full likelihood function is given by where μ is the vector of parameters in the baseline hazard function λ (t). the detailed formulation of eq. ( ) is shown in appendix c. the coefficients β, the parameter for frailty term θ, and parameters μ are all to be estimated. thus, the maximum likelihood estimation problem in this study can be defined as follows: where μ * , β * and θ * are the optimal solutions. eq. ( ) is an unconstrained non-linear optimization problem. in the literature on timeto-event models, a conventional algorithm to solve this maximum log-likelihood problem is the levenberg-marquardt algorithm (hosmer et al., ) . however, this algorithm finds only a local minimum, which is not necessarily the global minimum. therefore, it is interesting to use a global optimization technique to find a better solution for the maximum log-likelihood problem. under this consideration, we choose the basin-hopping algorithm to solve the maximum log-likelihood problem. the basin-hopping algorithm is a stochastic global optimization method that tries to find the global minimum of an objective function. the idea of this algorithm is to search the global minimum around a local minimum. in each iteration, this algorithm first chooses a starting point, completes a local minimum, and then applies a random perturbation to the coordinates of the local minimum to get the next starting point. a detailed description of the algorithm can be found in the study of wales and doye ( ) . with the parameters estimated by the maximum log-likelihood method, the hazard function of ttcm can be expressed by where μ * , β * and θ * are the parameters obtained by solving the problem, and w ∧ i is the estimated frailty term. using the estimated θ * , the frailty term w i can be estimated using a bayes method (rueten-budde et al., ). with this estimated hazard function, the probability that a booking is cancelled before the ship departure is equivalent to the probability that the cancellation event incurred before the departure date, namely: where t b ij is the booking time of a slot booking j from origin country i, measured as the days before ship departure, and s ij (⋅) is the survival function. t ij is the random variable to describe the ttc of the booking. it should be noted that although this study only considers the covariates listed in table , the methodology is a general one and can be used for various data sets. more factors can be incorporated in the model development by extending the covariate vector z ij and reestimating coefficients β * , e.g., such customer information as customer types, relationships, and sizes can be included in z ij of ttcm. we use the real slot booking data of an international container shipping company to evaluate the performance of the developed model. the data are from the head-haul (westbound) transport of the intercontinental container shipping service from asia to europe as shown in fig. . table gives an overview of the data collected for the case study. it can be seen that the data cover the information of slot bookings from may , to april , , and contain , slot booking requests with about % of them cancelled or failed to show up. the notation of the covariates and data used in the model of this case study are summarized in table : table is the lifetime of the slot booking in the historical data which is calculated as the difference between the slot booking time and cancellation time. α ij is a binary parameter to indicate whether the booking is cancelled (α ij = ) or not (α ij = ). for this case study, there are origin countries listed in table . accordingly, there are frailty terms w i , which are assumed to be i.i.d. random variables following the gamma distribution as discussed in section . in addition, to assess the effect of frailty terms, we also consider a model without frailty term but treat the origin country as indicator covariates in z ij . the ccfi data used in this case study from may , to april , are shown in fig. . to choose the most significant covariates and the best form of the baseline hazard function, we test the ttcm with all the covariates shown in table , and various combinations of baseline hazard functions and frailty term. we randomly select % of the , slot booking data to calibrate models and others for evaluating their performances. table lists the covariates and their significance testing results, baseline hazard functions, log-likelihood, and lcv/aic results of the six models under different combinations of baseline hazard function forms and frailty term. all the possible models are calibrated using the same set of training data and compared by the criteria of lcv/aic. the smaller lcv/aic indicates a better model performance (commenges et al., ) . as shown by table , the lcv/aic for the models with frailty are all smaller than the corresponding models without frailty. models , , and , which have origin countries as frailty terms, perform better than models , , and , considering origins as covariates when evaluated by either log-likelihood or lcv/aic criteria. therefore, the ttcm with frailty factors developed in this study performs better than the conventional proportional hazard regression models used by the existing studies on the forecasting of cancellations. this implies that the frailty model could effectively reflect the correlations of the bookings within an origin country, which is the special market feature in container shipping. for the form of the baseline hazard function, model with m-spline baseline hazard function outperforms model with weibull form and model with bspline form. hence, we select model , ttcm with frailty factor and m-spline baseline hazard function, to estimate the probability of cancellation in this case study. for the selected model, the significance test results are shown in the second column of table . for the ttcm with m-spline function and frailty factor, as shown by the first row of table , the voyage month (i.e. dp_m) and booking month (i.e. bk_m) are insignificant at the significant level of . . considering the correlations between dp_m and bk_m, we eliminate the insignificant covariate, dp_m, from the model. for covariate bk_m, which is a set of indicator variables from january to december, we test the significance of the indicators and find that january is the only significant indicator variable in all the months. thus, the indicator variable "booked in january" is selected as a covariate in the model with notation bk_m_jan. finally, we select the most significant variables (i.e. bk_m_jan, bktdp, bk_teu, ccfi_var, ccfi, frt, and cst) of the model in table , and recalibrate the model with these significant covariates. before the recalibration, we validate that the proportional hazard regression model can be used to model the data in this study. we first test the assumption of proportional hazard (ph). readers interested in the statistical test of ph assumption are referred to the study by grambsch and therneau ( ) . for all the covariates, only the freight rate cannot pass the test of ph assumption. hence, the coefficient of freight rate is interpreted as "average effects" of the covariates (hinkle et al., ) . this average effect is sufficient to estimate the probability of cancellation, as we are only concerned with whether the ttc exceeds the ship's departure time and not with its change over time. the freight rate is kept as a covariate in the model since its coefficient is significant. the second test is the correlationship of the covariates because high correlation can cause multicollinearity in survival model. table shows the correlation coefficients of the covariates. all the correlation is negligible except for that between bk_m_jan and ccfi with a moderate positive correlation at . . this is not a high correlation according to the rule of thumb for interpreting the size of a correlation coefficient (hinkle et al., ) . as the correlation is moderate and both of the two factors are statistically significant, we decide to keep these two frailty: yes, if origin countries as frailty term; no, if origin countries as indicator variables in covariates. *significant at level . . the correlation coefficients of covariates. fig. . expected loaded teu, real loaded teu, and total booked teu from different origin countries. factors. it should be noted that the estimated cancellation probability could be sensitive to the input data. the tests of significance and correlationship should be conducted whenever the input data are updated, and the most effective covariates are selected. we use the basin-hopping algorithm to solve the maximum log-likelihood problem, and the estimated parameters/coefficients are presented in table . the implications of these parameters will be discussed in section . herein, we will first use the calibrated parameters shown in table to validate the developed model in estimating the loaded containers for the container shipping service. we use the predicted expected number of loaded teus, which is the product of cancellation probability and booked teus, to evaluate the performance of ttcm (cox ) . the probability that the booking is cancelled before departure date is estimated by the model with parameters in table . according to eq. ( ), the expected loading teu of slot booking i of origin country j can be calculated by where b ij is the booked teu of slot booking j of origin country i; − p is the probability that the slot booking is not cancelled, and el ij is the expected loaded teu of this booking; z ij is the vector of covariates of the booking; β ∧ , θ ∧ , and μ ∧ are the estimated parameters in the ttcm. we use the testing data, which takes % of the slot booking data, to forecast the expected loaded teu. to illustrate the estimated loaded containers, the slot bookings are aggregated by country as shown in fig. and by voyage as shown in fig. . we compare the estimated loaded teu with the real loaded and booked teu in these two figures, and the error rate is shown by the lines in figs. and . this error rate is calculated by where k are the indexes of groups (i.e. origin countries or voyages), and the g k is the set of bookings that belong to a certain group k; el ij is the expected loaded teu estimated by the ttcm; rl ij is the real number of loaded teu of the slot booking. as shown in fig. , in the predicted loaded teu of the countries, countries have error rates < %, and only one country (i.e. au) has a high error rate. the average error rate is about . %. similarly, the average error rate of the estimated loaded teu of the voyages in fig. is about . %, which shows the satisfactory prediction power of the ttcm. in other words, the test data show that the developed model is effective in estimating the loaded teu for shipping companies in practice. fig. . expected loaded teu, real loaded teu, and total booked teu from different voyages. in this subsection, we will discuss the implications of the estimated parameters shown in table , and analyze the effects of the influential factors. the frailty terms are the effective indicators of the influence of origin countries on the cancellation probability (rueten-budde et al., ) . an origin country with a higher w ∧ i indicates a high risk of cancellation of the slot bookings. table shows the bayesian estimations of frailty terms of the origin countries/regions. with the estimated frailty values, the rank of the origins in terms of the slot booking cancellation risk can be obtained: the estimated frailty term of au is the smallest in table , indicating that the slot bookings from au are less likely to be cancelled than other origins; hk shows the highest estimated frailty at . , and the slot bookings from hk are at the highest risk of being cancelled than that from other origins. the estimated frailty of au, jp, cn, th, id are all below implying that if a slot booking is from these origins, the risk of being cancelled will be reduced when other influential factors are the same. in comparison, the frailty values of origins ph, sg, bd, vn, and hk are all larger than , indicating an increase in the risk of slot booking cancellation. the rank of cancellation risk can assist the container shipping companies to adjust their strategies on customer management and to pay more attention to the origins with a high risk of slot booking cancellation. the effects of the covariates (i.e. the influential factors) can be evaluated by the coefficient and hazard ratio (hr) (cox ) . the hazard ratio hr = e β is the increasing ratio of hazard when raising the covariate which has the coefficient β. this ratio can reflect the proportional change of the hazard when increase the corresponding covariate by one unit. the hazard ratio of the covariates is shown in the second column of table . for the external factors, the coefficient of ccfi is negative, which means that the bookings are less likely to be cancelled when the market shows a high freight rate. according to the hazard ratio . , an increase of on the index will decrease the hazard rate by about % when other factors stay the same. on the other hand, high variance of ccfi (i.e. ccfi_var in table ) indicates a higher risk of cancellation with hr at . . for the internal covariates in table , the freight rate shows a coefficient at . and hr at . . this means that the shipping company should consider the increase in cancellation if they decide to raise freight rates. customer's booking volume shows a negative effect indicating large customers are less possibly to cancel their bookings, which is reasonable in practice. the booked teu also shows a negative coefficient at − . , indicating a decrease of cancellation hazard for bookings with larger booked teu. however, for the slot booking data in this case study, this impact is insignificant in practice. fig. shows the histogram of the bk_teu data, and more than % of the bookings booked less than teus. therefore, the range of the booked teu is limited, and the change of cancellation risk caused by different number of booked teu is not as significant as the other attributes. for the internal covariates on time and season, the indicator variable of january (i.e. bk_m_jan in table ) has a positive coefficient and hr at . , which means the hazard of cancellation in january is relatively increased by % compared with other months. the other internal covariate on time is the booking time (i.e. bktdp in table ), but its effect cannot be directly observed from hr since the booking time is in both the covariates and baseline hazard function when calculating the cancellation probability. we examine the impact of booking time using the cancellation probability defined by eq. ( ). suppose there is a slot booking b from country i with booking time t b . according to the relationship between the ttc and hazard function, the distribution of ttc depends on t b which is a covariate in the hazard function. therefore, we denote the ttc of booking b when bktdp = t b as random variable t t b . the probability that a booking b will be cancelled before ship departure can be calculated by where Λ (t) = ∫ t λ (x)dx is the cumulative baseline hazard function, β ∧ bk is the estimated coefficient of booking time, z b is the covariate vector of booking b, and Δ z is the product of the covariates and their coefficients other than β bk ⋅ t b . accordingly, the probability of non-cancellation is − p b (t b ). the influence of booking time depends on the increasing/decreasing intervals of p b (t b ). that is, the earlier the booking is proposed in the decreasing interval the less possible it will be cancelled. however, in the increasing interval, earlier bookings are more likely to be cancelled than late bookings. thus, we can analyze the relationship between booking time and cancellation probability by taking the derivative of p b (t b ), namely: where the first and third parts of the derivative function are positive, but whether g(t b ) is positive or not depends on β ∧ bk . if β ∧ bk > , the probability of cancellation before ship departure is strictly increasing with booking time t b ; otherwise the change of probability depends on the baseline hazard function λ (t b ) and cumulative baseline hazard function Λ (t b ). in this case study, the results in table shows β ∧ bk = − . , thus the increasing/decreasing intervals of p b (t b ) should be obtained by λ (t b ) and Λ (t b ). using the baseline hazard function calibrated in this case study, we find g is negative when < t b < . therefore, the decreasing interval of function p b (t b ) estimated by this study is t b ∈ ( , ), and p b (t b ) is increasing in other intervals. to illustrate the relationship between booking time and cancellation probability, we give the values of the other covariates and show the slot booking cancellation probability at different booking time in fig. . the other covariates are set as the average value in the slot booking data in this case study. the origin country is vietnam and the values of other covariates are shown in fig. . we use the coefficients in table to calculate the cancellation probability. the decreasing interval of p b (t b ) is small enough (i.e. days) to ignore in the practice of slot booking management, and generally the bookings proposed early have a higher probability of cancellation which can be calculated by p b (t b ). as shown in fig. , the cancellation probability will sharply drop if the slots are booked in about days before ship departure. in the time interval from to days before ship departure, the change of cancellation probability is not obvious in comparison with other intervals. in the industrial practice, the time intervals are useful for the shipping companies to find the peak cancellation period in the slot booking stage, and pay more attention to manage the slot bookings which are booked at the time intervals with higher cancellation probability. it should be noted that the influential factors discussed in this section are based on the database of a container shipping company and the index data of the shipping market. to ensure the comprehensiveness of our analysis, we empirically discuss the factors that are not included in our model due to the unavailability of data. according to our interview with some managers in a container liner shipping company, the cause of trade itself, the competent authority, and some emergencies are three notable influential factors. the influence of trade itself is mainly reflected in the behavior of the shipper or the consignee. if the cargoes are not completed on schedule, the packaging of the cargoes are defective, the parties to the trade temporarily reduce the cargo order, or the trade is suspended due to the unqualified goods, the container slot bookings can be cancelled. such cancellations due to trade and cargoes are more common in container shipping practice. the second influential factor is the behavior of some competent authorities, such as customs and commodity inspection authorities. low customs clearance efficiency or high inspection and quarantine standards may cause the cargo to fail to clear customs in time and thus fail to be on board the ship. the third influential factor is emergencies in some areas. for example, due to the outbreak of covid- , some goods intended to be exported and the slots booked for them had to be cancelled. we only discuss them empirically here because these factors are difficult to quantify, and the data are difficult to obtain. quantitative research on these influencing factors is a valuable future research direction. to deal with the pain-point of slot booking cancellation for intercontinental container shipping, this study developed a slot booking data-driven model to estimate the cancellation probability of container slot bookings in the slot booking time period. a ttcm based on ttc analysis method was developed to quantitatively consider the internal factors of a slot booking and external factors that influence the probability of cancellation. to improve the performance of the model and reflect the effects of market regionality, we used the frailty term to formulate the correlation of the cancellation behaviors of the bookings that belong to the same origin country. the case study shows that the developed model is useful in assisting the shipping companies to estimate the loaded containers by their slot booking data in the slot booking time period. we recommend further studies to enahnce the prediction power of the model by considering more customer information discussed in section . it is another research direction to use the cancellation probability to optimize the pricing policy for container liner shipping company. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. according to eq. (a ), there is a natural relationship between λ(t) and s(t) as follows: where Λ(t) = ∫ t λ(x)dx is called cumulative hazard function. thus, the survival function can be obtained if we know the formulation of the hazard function λ(t). in this study, according to the above definition, the cancellation hazard function implies the instantaneous rate of occurrence of the cancellation conditioning that the booking has not been cancelled before time t. in time-to-event analysis, hazard function λ(t) can be formulated to several forms, and the characteristics of the slot bookings, as well as the internal and external influential factors, can be considered when modeling the hazard function. the two forms of baseline hazard funcitons are elaborated as follows: (i) weibull baseline hazard function. for the parametric form, it is conventional to assume that the ttc t follows a weibull distribution of two parameters with the pdf f (t|μ , μ ) = μ μ t μ − exp( − μ t μ ) (b ) where μ = {μ , μ } is the parameters of the weibull distribution. according to the relationship between hazard function and pdf presented by eq. (a ), the weibull baseline hazard function is where μ = {μ , μ } is to be estimated using the slot booking data. the weibull distribution assumption is widely assumed in the studies on time-to-event analysis and shows effective performance for some data (e.g., haque and washington, ; li et al., ) . (ii) spline baseline hazard function. we use both b-spline and m-spline functions in this study to estimate the baseline hazard functions. given the degree and knots, a set of basis functions can be determined. the detailed formulation of the two splines and the concepts of degree and knots can be found in the study by ramsay ( ) . assume there are q basis functions denoted by sp q (t), q = , , ..., q, the spline baseline hazard function can be formulated as λ (t|μ) = ∑ q q μ q sp q (t) (b ) where μ = {μ , μ , ..., μ q } are the parameters for the linear combination of the basis functions, and μ is to be estimated using the slot booking data. suppose that the data set that characterizes an individual booking is {( : i = , , ..., n; j = , , ..., j i } , where z ij is the vector of attributes, t ij is the ttc (or ttl), and α ij is the cancellation indicator of individual j in the origin country i. the cumulative hazard function of slot booking j from an origin country i can be formulated as where Λ (t) = ∫ t λ (x)dx is the baseline cumulative hazards. accordingly, the survival function, defined by eq. (a ), with frailty term can be obtained as follows substitute the hazard function λ ij (t|w i ), to eq. ( ), we get the partial likelihood function note that the w i is a random variable with gamma distribution. therefore, we obtain the expectation over w i . according to eq. ( ), for both cancelled and non-cancelled bookings in origin country i, the likelihood function can be presented as follows bayesian estimation of hazard models of airline passengers' cancellation behavior choice between semi-parametric estimators of markov and non-markov multi-state models from coarsened observations. scand predicting air travelers' no-show and standby behavior using passenger and directional itinerary information business travelers' ticketing, refund, and exchange behavior the impact of mobile phone distraction on the braking behaviour of young drivers: a hazard-based duration model applied statistics for the behavioral sciences applied survival analysis: regression modeling of time to event data: second edition, applied survival analysis: regression modeling of time to event data: second edition frailty models for survival data data mining techniques to improve forecast accuracy in airline business a hazard model of us airline passengers' refund and exchange behavior proportional hazard regression models and the analysis of censored survival data fractional price matching policies arising from the ocean freight service industry variable selection for cox's proportional hazards model and frailty model competing risk mixture model and text analysis for sequential incident duration prediction revenue management for container liner shipping services: critical review and future research directions forecasting cancellation rates for services booking revenue management using data mining monotone regression splines in action investigating hospital heterogeneity with a competing risks frailty model new frontiers through short sea shipping a fuzzy approach for container positioning considering sustainable profit optimization global optimization by basin-hopping and the lowest energy structures of lennard-jones clusters containing up to atoms liner ship route schedule design with port time windows integrated method for forecasting container slot booking in intercontinental liner shipping service optimal port call adjustment for liner container shipping routes intercontinental liner shipping service design on service network improvement for shipping lines under the one belt one road initiative of china using survival models to estimate bus travel times and associated uncertainties exploratory data analysis for the cancellation of slot booking in intercontinental container liner shipping: a case study of asia to a revenue management slot allocation model for liner shipping networks this study is supported by the research projects "liner shipping container slot booking patterns and their applications to the shipping revenue management" (r- - - - ) and "container haulage problems: model development, effective algorithm design and applications" (r- - - - ) funded by the nol fellowship programme of singapore. is defined as the survival function indicating the probability that the ttc of a booking is no less than t. the relationship between survival function and the pdf of t can be formulated bybased on the assumptions in section and the survival function, the probability that a slot booking is cancelled during the booking time period can be expressed by prob clc(b) = p[t < t b ] = − s(t b ) where t b is the booking time of slot booking b measured by days before departure. accordingly, the probability that the slot booking b will not be cancelled can be formulated as prob noclc(b) = − prob clc(b) = s(t b ).hazard function. hazard function indicates the rate of an interested event happening at a particular time (hosmer et al., ) . hazard function can be presented as follows key: cord- - lfsedz authors: sardar, tridip; nadim, sk shahid; rana, sourav; chattopadhyay, joydev title: assessment of lockdown effect in some states and overall india: a predictive mathematical study on covid- outbreak date: - - journal: chaos solitons fractals doi: . /j.chaos. . sha: doc_id: cord_uid: lfsedz in the absence of neither an effective treatment or vaccine and with an incomplete understanding of the epidemiological cycle, govt. has implemented a nationwide lockdown to reduce covid- transmission in india. to study the effect of social distancing measure, we considered a new mathematical model on covid- that incorporates lockdown effect. by validating our model to the data on notified cases from five different states and overall india, we estimated several epidemiologically important parameters as well as the basic reproduction number (r( )). combining the mechanistic mathematical model with different statistical forecast models, we projected notified cases in the six locations for the period may , , till may , . a global sensitivity analysis is carried out to determine the correlation of two epidemiologically measurable parameters on the lockdown effect and also on r( ). our result suggests that lockdown will be effective in those locations where a higher percentage of symptomatic infection exists in the population. furthermore, a large scale covid- mass testing is required to reduce community infection. ensemble model forecast suggested a high rise in the covid- notified cases in most of the locations in the coming days. furthermore, the trend of the effective reproduction number (r(t)) during the projection period indicates if the lockdown measures are completely removed after may , , a high spike in notified cases may be seen in those locations. finally, combining our results, we provided an effective lockdown policy to reduce future covid- transmission in india. • a new mathematical model on covid- that incorporates lockdown effect • several model parameters as well as the basic reproduction number are estimated • we provide ensemble model forecast under five different lockdown scenarios • correlation between important parameters with the lockdown effect are derived • combining all the results, we proposed an effective lockdown policy cov- ), were recorded worldwide [ ] . coronaviruses are enveloped non-segmented positive- sense rna viruses that belongto the coronaviridae family and the order nidovirales, and are widely distributed among humans and other mammals [ ] . the novel coronavirus, covid- started in mainland china, with a geographical emphasis at wuhan, the capi- tal city of hubei province [ ] and has widely spread all over the world. many of the initial cases were usually introduced to the wholesale huanan seafood market, which also traded live animals. clinical trials of hospitalized patients found that patients exhibit symptoms consistent with viral pneumonia at the onset of covid- , most commonly fever, cough, sore throat and fatigue [ ] . some patients reported changes in their ground-glass lungs; normal or lower than average white lymphocyte blood cell counts and platelet counts; has seriously disrupted the life, economy and health of citizens. this is a great concern for everyone how long this scenario will last and when the disease will be controlled. mathematical modeling based on system of differential equations may provide a com- the model we developed in this paper is based on the interaction of seven mutually ex- population in the exposed compartment (e) increased by new infection coming from susceptible compartment. a fraction κ of the exposed individuals become symptomatic infected and remaining fraction ( − κ) become asymptomatic infected after the disease incubation period σ . exposed population also decreased due to natural death at a rate µ. asymptomatic infected compartment (a) increased due to a fraction ( − κ) of infec- tion coming from exposed compartment. since, asymptomatic covid- cases are hard to detect therefore, we assume that asymptomatic infection are not notified. population in this compartment is decreased due to natural recovery and deaths at a rate γ and µ, respectively. population in the symptomatic infected compartment (i) increased due to a fraction κ of infection coming from exposed compartment after the incubation period σ . this compartment decreased due to natural recovery at a rate γ , natural death at a rate µ and those infected population who are notified & hospitalized at a rate τ . notified & hospitalized infected population (c) increased due to influx of infection coming from symptomatic infected class at a rate τ . this population decreased due to natural death at a rate µ, disease related deaths at a rate δ, and recovery from covid- at a rate γ . we assume that population of this compartment do not mix with the general model without lock-down a diagram of our model is provided in fig . information of our model parameters is provided in table . disease-free state is locally asymptotically stable whenever the corresponding basic re- production number (r ) is less than unity (see supplementary appendix). by using a nonlinear lyapunov function, it is also seen that the disease-free equilibrium is globally asymptotically stable whenever r < (see supplementary appendix). in addition, the several important epidemiological parameters (see table ) of our mathematical where, ∆t i is the time step length andθ is the set of unknown parameters of the mod- rate for our covid- mathematical model (see table and table and table ) to obtained the forecast during the mentioned time period. forecast based on % reduction in current lockdown rate: we followed the same procedure as previous two scenarios with % decrement in the estimate of lockdown rate (see table and table ) to obtained the forecast during the mentioned time period. forecast based on % reduction in current lockdown rate: we followed the same procedure as previous three scenarios with % decrement in the estimate of lockdown rate (see table and table ) to obtain the forecast during the mentioned time period. are same and its expression is provided below: the effective reproductive number (r t ) is defined as the expected number of secondary infection per infectious in a population made up of both susceptible and non-susceptible hosts [ ] . if r t > , the number of new cases will increase, for r t = , the disease become endemic, and when r t < there will be a decline in new cases. following [ ] , the expression of r t is given as follows: where,ŝ is the fraction of the host population that is susceptible. r can easily be estimated by plugin the sample values of the unknown parameters (see table ) of the model without lockdown ( . ) in the expressions of r . following procedure is adapted to estimate r t during may , till may , under two lockdown scenarios: • using current estimate of the lockdown rate and different parameters of our mathe- matical model (see table and • using different parameters (see table and table ) ically measurable parameters of our mathematical model (see fig ) . there are several important parameters of our mathematical model (see table ) and among them there τ , respectively from their respective ranges (see table ). partial rank correlation and its corresponding p-value are examined to determine the relation between two mentioned parameters with the lockdown effect and r , respectively. table s ). that low percentage (about % to %) of symptomatic infected in the population (see table ). however, in tn and pj, relatively higher percentage (about % to %) of symptomatic infection is found (see table ). in overall india, our estimate shows that currently about % of new infection are symptomatic (see table ). except for gj, in other five locations, estimate of the transmission rate (β ) are found to be in same scale (see table ). relatively higher value of β is found in gujrat (see table ). low value the estimates of ρ (below %) are found to be low (see table ). this indicates small table ). thus, lockdown is overall successful in those five states. however, this is not the case for overall india, our estimate suggest that about % of the total susceptible population in india maintained proper social distancing during the lockdown period (see table ). our estimate of the basic reproduction number (r ) (see table ), in the six locations suggest that τ has a negative correlation on r . thus, more testing will isolate more infection from the community and therefore may reduce the covid- community trans- mission. furthermore, high positive correlation of κ with r (see fig ) indicates the table in overall india (see table ). these numbers are much higher than the total cumulative cases between march , till may , , in whole india. a global sensitivity analysis of κ and τ on the lockdown effect suggest that both of these parameters have high positive correlation with the lockdown effect in all the six locations (see fig. ). therefore, lockdown will be effective in those region where higher percentage of symptomatic infection is found in the population and also larger covid- mass testing will be required to isolate the cases. we may see a rise in the daily covid- cases in all of the six locations (see fig. ). table ), % reduction: daily notified case projection using % reduction in the estimated value of the lockdown rate (see table ), % reduction: daily notified case projection using % reduction in the estimated value of the lockdown rate (see table ), % reduction: daily notified case projection using % reduction in the estimated value of the lockdown rate (see table ), and no lockdown: daily notified case projection based on no lockdown scenario, respectively. ) . respective row subscripts are same as fig. . all data are given in the format estimate ( % ci). fig. . different lockdown scenarios are current rate: cumulative case projection using the estimated value of the lockdown rate (see table ), % reduction: cumulative case projection using % reduction in the estimated value of the lockdown rate (see table ), % reduction: cumulative case projection using % reduction in the estimated value of the lockdown rate (see table ), % reduction: cumulative case projection using % reduction in the estimated value of the lockdown rate (see table ), and no lockdown: cumulative case projection based on no lockdown scenario, respectively. all data are provided in the format estimate ( % ci). coronavirus covid- global cases by the center for systems science and en- gineering html#/bda fd b e ecf , clinical virology a novel coronavirus outbreak of global health concern wuhan wet market closes amid pneumonia outbreak india covid- tracker report : impact of non-pharmaceutical interventions (npis) to reduce covid mortality and healthcare demand a realistic two-strain model for mers-cov infection uncovers the high risk for epidemic propagation an updated estimation of the risk of transmission of the novel coronavirus ( -ncov) effectiveness of airport screening at detecting trav- ellers infected with novel coronavirus ( -ncov) modelling the epidemic trend of the novel coronavirus outbreak in china estimation of the transmission risk of the -ncov and its implication for public health interventions nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study novel coronavirus -ncov: early estimation of epidemiological parameters and epidemic predictions age-structured impact of social distancing on the covid- epidemic in india a mathematical model for simulating the transmission of wuhan novel coronavirus early dynamics of transmission and control of covid- : a mathematical modelling study epidemic analysis of covid- in china by dynamical modeling modelling strategies for controlling sars outbreaks prudent public health inter- vention strategies to control the coronavirus disease transmission in india: a mathematical model-based approach statewise population data life expectancy at birth an optimal cost effectiveness study on zimbabwe cholera seasonal data from dram: efficient adaptive mcmc mathematical analysis of a power-law form time depen- dent vector-borne disease transmission model convenient functions for ensem- ble time series forecasts, world's biggest lockdown may have cost rs - lakh crore to indian econ- omy modern epidemiology. lippincott the mathematics of infectious diseases population biology of infectious diseases: part i infectious diseases of humans: dy- namics and control the stability of dynamical systems the theory of the chemostat: dynamics of microbial competition dynamical models of tuberculosis and their ap- plications conceptualization: tridip sardar, joydev chattopadhyay. data curation: tridip sardar, sk shahid nadim. formal analysis: tridip sardar, sk shahid nadim, sourav rana. investigation: tridip sardar, sk shahid nadim, sourav rana. methodology: tridip sardar, sk shahid nadim software: tridip sardar, sourav rana supervision: tridip sardar, joydev chattopadhyay. validation: tridip sardar writing original draft: tridip sardar, sk shahid nadim, sourav rana, joydev chattopadhyay. writing review & editing: tridip sardar, sk shahid nadim worlds-biggest-lockdown-may-have-cost-rs- - -lakh-crore-to-indian-economy/ all the authors declare that they have no conflicts of interest. key: cord- - pfrk uk authors: simusika, paul; tempia, stefano; chentulo, edward; polansky, lauren; mazaba, mazyanga lucy; ndumba, idah; mbewe, quinn k.; monze, mwaka title: an evaluation of the zambia influenza sentinel surveillance system, – date: - - journal: bmc health serv res doi: . /s - - - sha: doc_id: cord_uid: pfrk uk background: over the past decade, influenza surveillance has been established in several african countries including zambia. however, information on the on data quality and reliability of established influenza surveillance systems in africa are limited. such information would enable countries to assess the performance of their surveillance systems, identify shortfalls for improvement and provide evidence of data reliability for policy making and public health interventions. methods: we used the centers for disease control and prevention guidelines to evaluate the performance of the influenza surveillance system (iss) in zambia during – using attributes: (i) data quality and completeness, (ii) timeliness, (iii) representativeness, (iv) flexibility, (v) simplicity, (vi) acceptability, (vii) stability, (viii) utility, and (ix) sustainability. each attribute was evaluated using pre-defined indicators. for each indicator we obtained the proportion (expressed as percentage) of the outcome of interest over the total. a scale from to was used to provide a score for each attribute as follows: < % (as obtained in the calculation above) scored (weak performance); – % scored (moderate performance); ≥ % scored (good performance). an overall score for each attribute and the iss was obtained by averaging the scores of all evaluated attributes. results: the overall mean score for the iss in zambia was . . key strengths of the system were the quality of data generated (score: . ), its flexibility (score: . ) especially to monitor viral pathogens other than influenza viruses, its simplicity (score: . ), acceptability (score: . ) and stability (score: . ) over the review period and its relatively low cost ($ , per annum). identified weaknesses related mainly to geographic representativeness (score: . ), timeliness (score: . ), especially in shipment of samples from remote sites, and sustainability (score: . ) in the absence of external funds. conclusions: the system performed moderately well in our evaluation. key improvements would include improvements in the timeliness of samples shipments and geographical coverage. however, these improvements would result in increased cost and logistical complexity. the isss in zambia is largely reliant on external funds and the acceptability of maintaining the surveillance system through national funds would require evaluation. seasonal influenza virus infections are responsible for an estimated , - , respiratory deaths globally every year [ ] . influenza infections have been described in zambia [ ] since the s, but because of limited laboratory diagnostic capacity prior to [ ] , little was known about the contribution of influenza viruses to the respiratory disease burden, rendering difficult public health planning for the prevention and control of influenza-associated illness. at present, no influenza treatment or immunization guidelines are available in zambia, and no measures for mitigating the transmission and disease burden associated with influenza infection are in place. public health surveillance is the ongoing, systematic collection, analysis, interpretation, and dissemination of data regarding a health-related event for use in public health action to reduce morbidity and mortality and to improve health [ ] . data disseminated by a public health surveillance system can be used for immediate public health action, program planning and evaluation, and formulation of research hypotheses [ ] . the lack of a routine influenza surveillance program in zambia placed the country at a disadvantage as such a system would serve as an ever-ready early warning system with the potential to detect and confirm the etiology of respiratory disease outbreaks, including seasonal and pandemic influenza viruses, and trigger a response. the ministry of health (moh) recognized a need to gather influenza epidemiological and virological data from zambia that would not only help decision makers to formulate policies targeted toward influenza prevention in zambia but also to provide a more comprehensive understanding of the dynamics of influenza viruses worldwide and in african tropical zones in particular. in addition, virological data would help to identify seed viruses for the production of seasonal influenza vaccines that can be effectively used for the southern hemisphere. in recognition of these needs, in the zambia ministry of health established an influenza sentinel surveillance system (isss) targeting outpatients with influenza-like illness (ili) and in-patients with severe acute respiratory illness (sari). the world health organization (who) recommends that established influenza surveillance systems undergo a comprehensive evaluation periodically, beginning - years after implementation in order to ascertain how well the system fulfills its purposes [ , ] . such evaluations are useful to identify shortfalls, improve performance and provide evidence of data reliability for policy making and public health interventions. however, despite recent progress in describing the epidemiology and burden of influenza in sub-saharan africa [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , only a few countries have implemented a comprehensive evaluation of their surveillance systems [ ] [ ] [ ] [ ] [ ] . to ensure that the national isss objectives are met, that they are in line with who requirements and that reliable data are generated for public health interventions, we conducted an evaluation of the performance of the zambia ili and sari isss from january to december . the inception and consolidation of the surveillance system occurred during - . the outputs of the surveillance systems during this period have been previously published [ ] and they are not included in this review. overview of the influenza sentinel surveillance system during [ ] [ ] [ ] [ ] [ ] [ ] [ ] in zambia, an isss was first established in in lusaka province (the most populated province of the country where the capital city is located) [ ] and subsequently expanded to the copperbelt province (the second most populated province). the objectives of the zambia-isss were to: (i) monitor the temporal trends of influenza virus circulation; (ii) monitor the circulating influenza virus types and subtypes annually, including pandemic strains; (iii) assess the proportion of patients meeting the ili and sari case definition attributable to influenza virus infection; (iv) assess risk factors for influenza-associated severe illness; (v) assess the burden of influenza-associated illness; and (vi) obtain and share clinical samples for annual selection of influenza virus strains for influenza vaccine formulation under the who-global influenza surveillance and response network. during - , ili influenza sentinel surveillance was conducted in three urban outpatient clinics targeting outpatients with ili and three large referral hospitals targeting inpatients with sari ( fig. , panel a) . both pediatric and adult cases were investigated. a case of ili was defined as an outpatient of any age presenting with a recorded temperature ≥ °c and cough or sore throat of duration of ≤ days [ ] . a case of sari was defined as a hospitalized person who had illness onset within days of admission and who met age-specific clinical inclusion criteria. a case in children aged days to < years included any hospitalized patient with cough or difficulty breathing and at least one of the following danger signs: unable to drink or breastfeed, lethargic, vomits everything, convulsion, chest indrawing or stridor in a calm child. a case in persons aged ≥ years included any hospitalized patient with fever (≥ °c), cough and shortness of breath or difficulty breathing [ ] . all patients with sari were eligible for enrollment; whereas patients with ili (the first patient every day from monday to friday) were targeted for enrollment per week and per facility. activities at the sentinel sites situated in lusaka were coordinated by a dedicated surveillance officer from the national influenza center (nic). for sites situated in the copperbelt province, supervision was conducted by a focal point at the provincial medical office. due to high staff turnover, training was conducted once a year while supervisory visits to sites were done at least quarterly. clinical staff at sentinel sites tasked with surveillance implementation were remunerated according to time spent on surveillance activities. the zambia isss is co-funded by the zambia-moh, the u.s. centers for disease control and prevention (u.s. cdc) and who. data were collected from eligible cases using who adapted and standardized case investigation forms (cif) by trained nurses in each sentinel site. for patients meeting the sari case definition the number of patients eligible for enrollment was also collected. a combination of nasopharyngeal and oropharyngeal swabs were collected from consenting patients, placed in universal transport medium (copan, murrieta, california, usa) and transported to the university teaching hospital virology laboratory, the who-recognized nic in lusaka, via a cold chain for testing. samples with respective cif forms collected from sites situated in lusaka were sent to the laboratory within h, while those from the copperbelt province were refrigerated and sent within two weeks of collection [ ] . verbal informed consent was obtained from all patients prior to data and specimen collection. for children aged < years, verbal consent was obtained from a parent or legal guardian. at the nic, samples and accompanying cifs were logged by recording key data in the laboratory log book. specimens were tested for the presence of influenza viruses using the u.s. cdc real-time reverse-transcription polymerase chain reaction (rt-pcr) protocol for characterization of influenza viruses [ ] . during - , all specimens from children aged < years with sari were tested for other respiratory viruses including parainfluenza virus (piv) types , and , respiratory syncytial virus (rsv), adenovirus, rhinovirus, human metapneumovirus (hmpv), coronavirus (oc , nl , e, hku ) and bocavirus using an ftd multiplex realtime rt-pcr assay [ ] . since , rt-pcr influenzapositive samples with a cycle threshold value < were cultured at the nic, after which further viral identification of the samples was done using haemagglutination (ha) and haemagglutination inhibition (hai) testing. confirmed virus isolates were shared with who collaborating centers for further characterization; both viral isolates and clinical specimens were shared twice a year. data from the cif including demographics and clinical information, exposure risk and past medical history were captured in a customized influenza sentinel surveillance ms access® database with inbuilt data validation checks. data entry was done daily as forms were received. monthly reports of the total number of samples received and processed (including positive specimens by subtype) were reported to the ministry of health public health units. a separate patient line-list was also sent monthly to the sentinel sites; the results report included total samples processed by sentinel site and total confirmed influenza infections. weekly influenza surveillance data and influenza-positive specimens were reported to who afro using an excel template. virological and epidemiological data were reported to who flunet / fluid. the evaluation of the isss was based on cdc guidelines [ ] and focused on the performance of the system from january to december . the objective of the evaluation was to determine: (i) whether the surveillance system was designed and operated in such a way as to be capable of detecting and monitoring seasonal influenza viruses, including pandemic strains; (ii) the usefulness of the collected data to assess influenza disease burden in the general population; (iii) the impact of the system in informing public health intervention and policies; and (iv) the ability of the system to contribute to the annual selection of influenza strains for vaccine development. the performance of the system was assessed using nine surveillance attributes, namely: (i) data quality and completeness for selected key variables, (ii) timeliness, (iii) representativeness, (iv) flexibility, (v) simplicity (vi) acceptability, (vii) stability; (viii) utility; and (ix) sustainability. for consistency and comparability of findings we used the evaluation method and scoring system utilized for influenza surveillance evaluations conducted in other african countries [ ] [ ] [ ] [ ] [ ] . each of the above mentioned attribute was evaluated using pre-defined quantitative or qualitative indicators. a total of indicators were developed for the evaluation. the number of indicators evaluated for each attribute is provided in table ; whereas the individual indicators and the calculations and data sources used to evaluate each indicator are provided in tables - . data for calculation of the indicators for data quality and completeness, timeliness, stability and utility were obtained from various sources including the main influenza sentinel surveillance database, the laboratory database, annual reports and other documents and records. in order to assess simplicity, acceptability, stability and utility, a selfadministered questionnaire as shown in additional file below was designed targeting staff involved in surveillance at the sentinel sites. the questionnaire was designed to capture data based on staff perceptions of the program. data collected from the surveillance system were also compared with who minimum data collection standards for ili and sari surveillance [ ] . each quantitative indicator was evaluated as the proportion (expressed as percentage) of the outcome of interest over the total. a scale from to was used to provide a score for each quantitative indicator as follows: < % (from the above calculation) scored (weak performance); - % scored (moderate performance); ≥ % scored (good performance). for indicators for which a proportion over a total could not be obtained (qualitative indicators) a score was assigned based on the same scale using expert consensus. the scores assigned to each indicator were averaged for all indicators evaluated for each attribute to provide a mean score for each surveillance attribute. an overall score for the surveillance system was obtained by averaging the scores of all evaluated indicators for each quantitative indicator we first obtained the proportion (expressed as percentage) of the outcome of interest over the total [ ] [ ] [ ] [ ] . for instance, for the indicator on completeness of laboratory testing (one of the indicators used to evaluate the data quality and completeness attribute) we divided the number of samples with available influenza results by the total number of samples collected and received by the laboratory. subsequently, similar to other influenza surveillance evaluations conducted in africa, we used a scale from to to provide a score for each quantitative indicator as follows: < % (as obtained in the example above) scored (weak performance); - % scored (moderate performance); ≥ % scored (good performance) [ ] [ ] [ ] [ ] . for indicators for which a proportion over a total could not be obtained (qualitative indicators) a score was assigned based on the same scale using expert consensus. thereafter, the scores assigned to each indicator were averaged for all indicators evaluated within each attribute to provide an overall score for each surveillance attribute assessed in this study. an overall score for the surveillance system was obtained by averaging the scores of all evaluated indicators as previously described [ ] [ ] [ ] [ ] . all data generated by the surveillance system during the review period were included in the evaluation. the analysis was implemented using stata version . (sta-tacorp, college station, texas, usa). this surveillance evaluation was deemed non-research by the zambia-moh and the us cdc. consent to participate was voluntary and verbally obtained, because this was a non-research and the procedure was nonrisks to client ethical clearance was deemed unnecessary by the university of zambia biomedical research ethics committee. from january to december , , patients were enrolled in the isss. of these, ( . %) and ( . %) had ili and sari, respectively. influenza results were available for , of , ( . %) enrolled patients; / ( . %) and / ( . %) among patients with ili and sari, respectively. influenza viruses were detected in / , ( . %) specimens; in / ( . %) and / ( . %) specimens among patients with ili and sari, respectively. of the influenza-positive specimens, ( . %) were influenza a(h n )pdm , ( . %) were influenza a(h n ), ( . %) were influenza a not-subtyped and ( . %) were influenza b viruses. the weekly number of influenza-positive specimens is provided in fig. , panel b . the questionnaire was completed by / ( . %) surveillance staff at sentinel sites. of these, ( . %) were nurses, ( . %) were clinical officers, ( . %) were paramedics and ( . %) was a laboratory technician. the overall mean score for the isss in zambia was . (moderate to good performance) ( table ) . during the evaluation period, of the seven data indicators, six had good performance and one had moderate performance ( table ). approximately % of identified sari cases were enrolled in the surveillance program, while the enrollment target was %. the enrollment target for ili cases was exceeded ( % of the target of cases per week and per facility). more than % of cif were accurately completed for key variables (including site, age/date of birth, sex, date of consultation/admission, date of symptoms onset, date of sample collection and signs and symptoms included in the case definitions) and more than % of samples collected had available influenza results and a positive ribonucleic protein result (an indicator of presence of human mucosal cells in the sample). the proportion of collected variables included in the who minimum data collection standard was . %. information on the use of antivirals and the presence of some underlying medical conditions were not included in the patients' cifs. the mean score for data quality and completeness was . (moderate to good performance) ( table ) . during the evaluation period, one timeliness indicator had good performance and one had moderate performance (table ). approximately % of samples were not received or tested within the expected time period. . % of the samples collected in lusaka province were received within the expected time period, whereas only . % of the samples collected in the copperbelt province met the expected timeline. no significant difference in the proportion of samples positive for influenza was observed between the sites situated in lusaka ( . % / ) and ndola ( . %; / ) (p = . ). the mean score for data timeliness was . (moderate to good performance) ( table ) . during the evaluation period, one indicator had good performance and one had weak performance (table ) . age representativeness was strong, with patients enrolled across all age groups. geographic representativeness was poor, with sentinel sites situated only in / ( . %) provinces. the mean score for representativeness was . (moderate performance) ( table ) . during the evaluation period, both indicators had good performance ( table ). the isss was used as a platform to monitor the circulation of respiratory viruses other than influenza (even though only for two years). the mean score for flexibility was . (good performance) ( table ) . during the evaluation period, of the seven indicators, six had good performance and one (time of enrollment of sari/ili cases) had moderate performance ( table ). the six indicators used to assess the perception of surveillance personnel at sentinel sites to implement different surveillance activities were rated well by responding personnel. over % of staff, however, reported that surveillance procedures from the identification of cases to the final packaging of samples took > min, showing only moderate performance. the mean score for simplicity was . (moderate to good performance) ( table ) . during the evaluation period, all four evaluated indicators had good performance (table ). more than % of surveillance staff was satisfied or very satisfied with the surveillance reports or the feedback of laboratory results. surveillance activities did not pose a heavy workload on surveillance staff in addition to the required clinical work. the mean score for acceptability was . (good performance) ( table ) . during the evaluation period, of the eight indicators, five had good performance and three had moderate performance (table ). all surveillance sites provided samples weekly during the evaluation period. the main aspects that affected stability were elevated frequencies of electricity cuts and generator failures. approximately % of surveillance staff did not receive annual refresher training during the last year and approximately % of expected weekly reports were not sent to the zambia moh within the expected time frame (one week from the end of the reported period). the mean score for stability was . (moderate to good performance) ( table ) . during the evaluation period, of the four indicators, three had good performance and one had moderate performance (table ). in terms of international utility the zambia isss reported regularly to the who flunet, generated samples that were shared with the who collaborating centers and contributed to regional studies. in terms of domestic utility, / ( . %) key national objectives of the isss were met at the time of this evaluation. the mean score for utility was . (moderate to good performance) ( table ) . during the evaluation period, the two indicators had poor performance (table ). only % of the isss budget was provided by the zambia moh and the remaining by international agencies. the mean annual running cost of the system was approximately $ , ($ per sample collected and tested in ). whereas, a sustainability plan was drafted in , the document was not finalized and implemented at the time of this evaluation. the mean score for sustainability was . (weak performance) ( table ). we evaluated the performance of the zambia isss over a -year period, years from its inception. overall, based on the evaluated indicators, the system performed satisfactorily with a mean system score of . (moderate to good performance). the system demonstrated its utility according to its objectives by (i) monitoring the temporal trends of influenza virus circulation; (ii) monitoring the circulating influenza virus types and subtypes annually and enabling the detection of pandemic influenza a(h n )pdm in [ ] and in subsequent years, (iii) assessing the contribution of seasonal influenza viruses to mild (ili) and severe (sari) respiratory illness; (iv) assessing the burden of influenza-associated illness [ ] ; and (v) generating isolates to contribute to the annual influenza vaccine formulation. nonetheless, the isss was not able to identify groups at increased risk of influenza-associated severe illness (as the necessary variables were not collected in the cif), one of the objectives of the surveillance system. strengths of the system included the quality of data generated, its flexibility to monitor viral pathogens other than influenza viruses [ ] , its potential expandability, its stability over the review period, and its relatively low cost. identified weaknesses were poor geographic representativeness, lack of timeliness in shipping samples from remote sites, and low sustainability in the absence of external funds. while quality and completeness of collected key epidemiological and virological data was good overall, an under-enrollment of sari cases was noted. many factors could have affected site performance including low staff numbers, high turnovers rates of staff as well as participant refusals. it should be noted that nurses employed by the moh and tasked with surveillance activities also provided direct patient care. having dedicated surveillance staff may increase enrollment rates. however, hiring new staff would decrease the sustainability of the surveillance system. each quantitative indicator was evaluated as the proportion (expressed as percentage) of the outcome of interest over the total (indicator value). a scale from to was used to provide a score for each quantitative indicator as follows: < % (from the above calculation) scored (weak performance); - % scored (moderate performance); ≥ % scored (good performance) each quantitative indicator was evaluated as the proportion (expressed as percentage) of the outcome of interest over the total (indicator value). a scale from to was used to provide a score for each quantitative indicator as follows: < % (from the above calculation) scored (weak performance); - % scored (moderate performance); ≥ % scored (good performance) the surveillance system performed moderately in terms of timeliness in transporting samples from the field to the laboratory. timeliness was mainly affected by the delays experienced from sites located outside lusaka. conversely, when samples were received at the central laboratory the processing time was overall satisfactory. while timeliness of shipping of samples from sites situated outside lusaka province could be improved by using couriers on a more regular basis this may increase costs. consideration should be given to timeliness of the system and its geographical representativeness especially in the african setting where more regular shipments of samples from areas distant from central locations are not only affected by cost, but also by logistical constraints. the geographic representativeness of the system was weak. establishing additional surveillance sites in other key areas of the country could provide a better representativeness of the circulating influenza viruses. however, it is unclear whether expanding the number of surveillance sites would improve the virological and epidemiological understanding of influenza in zambia. in a study that included surveillance data from african countries, the detection rate of influenza virus among sari and ili cases was similar across the participating countries, suggesting that similarities of the burden of publications and reports . % b • circulating influenza types/ subtypes, including pandemic strains (yes) [ ] • proportion of ili/sari illness attributable to influenza virus infection (yes) [ ] • risk factors for influenzaassociated severe illness (no) • burden of influenza-associated illness (yes) [ ] sustainability each quantitative indicator was evaluated as the proportion (expressed as percentage) of the outcome of interest over the total. a scale from to was used to provide a score for each quantitative indicator as follows: < % (from the above calculation) scored (weak performance); - % scored (moderate performance); ≥ % scored (good performance). for indicators for which a proportion over a total could not be obtained (qualitative indicators) a score was assigned based on the same scale using expert consensus b indicator value calculated by dividing the number of achieved outcome by the total number of outcome considered (i.e. / = . % or / = . %) influenza-associated illness across different geographical areas of the continent may be expected [ ] . increasing the number of surveillance sites in zambia should be considered in light of increased cost, logistical constraints (e.g. regular supervision and transportation of specimens) and impact on laboratory testing capacity and potentially on data quality. instead of expanding the geographic coverage of the system, efforts could be targeted to improve timeliness of sample shipment and enrollment of patients meeting the sari case definition at the existing sentinel sites as well as collecting more detailed epidemiological information on enrolled cases (e.g., relevant risk factors for influenza-associated severe illness and in-hospital outcomes that are recommended by the who guidelines for global influenza surveillance, but are currently not fully captured by the system). in particular, the collection of additional information on underlying medical conditions may enable the continuous monitoring of high risk groups for influenzaassociated severe illness. this would also align the zambia isss to the minimum data collection requirements suggested by who [ ] . the flexibility of the system was demonstrated by its ability to investigate the viral etiology of pathogens other than influenza [ ] . while the recruitment of dedicated surveillance officers may not be justifiable for a vertical influenza surveillance program it may be considered for an integrated multi-pathogen surveillance platform. this could be potentially cost-beneficial for a sentinel surveillance system where a limited number of surveillance officers may be needed in selected sentinel sites as compared to national passive surveillance programs. integrated and well-run multi-pathogen surveillance systems, if adequately staffed may provide high quality and timely epidemiological and virological data with a "modest" financial investment. the national burden of illness attributable to specific pathogens can be estimated from sentinel surveillance data through special studies as demonstrated in zambia and other african countries for influenza [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this would be particularly relevant for policy makers if multiple pathogens are included so as to provide the relative burden associated with different etiological agents. risk factors for influenzaassociated severe illness (e.g. hospitalization or death) have also been assessed using sentinel surveillance systems [ , [ ] [ ] [ ] and this could be implemented also in zambia for influenza and other pathogens. the stability of the system was demonstrated by its ability to enroll and process samples every week of the review period. this may be related to the simplicity and acceptability of the system and the availability and use of standard operating procedures at sites. nonetheless, tasking of surveillance staff with clinical and surveillance work may have affected the enrollment rate as previously discussed. in conclusion, the system performed satisfactorily over recent years. it contributed to the body of knowledge on the burden of influenza [ , ] and other respiratory viruses [ ] among common respiratory syndromes in zambia and globally through sharing of influenza data [ , ] and virus isolates. nonetheless, despite its moderate cost, the isss in zambia is largely reliant on external funds and the acceptability of maintaining the surveillance system through national funds would require evaluation. improvement of the system would entail the improvement of the timeliness of sample shipments, enrollment of patients meeting the sari case definition and an increased geographical coverage; however, the latter would increase costs and impose logistical constraints. furthermore, while the surveillance system is stable and able to identify circulating influenza strains, the data being generated is not fully utilized as zambia lacks guidelines on antivirals use and vaccination policy for influenza. a better use of the available data, in association with special studies, could be made to inform and promote 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hiv-prevalence setting national burden of hospitalized and non-hospitalized influenza-assocaited severe acute respiratory illness in kenya burden and epidemiology of influenza-and respiratory syncytial virus-associated severe acute respiratory illness hospitalization in madagascar the national and provincial burden of medically attended influenza-associated influenza-like illness and severe acute respiratory illness in the democratic republic of congo evaluation of two influenza surveillance systems in south africa evaluation of the influenza sentinel surveillance system in madagascar evaluation of the sentinel surveillance system for influenza-like illnesses in the greater accra region evaluation of the influenza-like illness surveillance system in tunisia evaluation of the influenza sentinel surveillance system in the democratic republic of congo does the length of specimen storage affect influenza testing results by real-time reverse transcriptionpolymerase chain reaction? an analysis of influenza surveillance specimens mortality amongst patients with influenza-associated severe acute respiratory illness, south africa identification of viral and bacterial pathogens from hospitalized children with severe acute respiratory illness in lusaka, zambia risk factors associated with hospitalisation for influenza-associated severe acute respiratory illness in south africa: a case-population study risk factors for influenza-associated severe acute respiratory illness hospitalization in south africa impact of human immunodeficiency virus on the burden and severity of influenza illness in malawian adults: a prospective cohort and parallel case-control study global role and burden of influenza in pediatric respiratory hospitalizations, - : a systematic analysis springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank all members involved in ili and sari surveillance for data collection and their participation to the evaluation. all authors take responsibility for the integrity of the data and the accuracy of the data analysis. in addition, all authors have read and approved the manuscript in its current state. this work was supported by the zambia ministry of health and the us centers for disease control and prevention (co-operative agreement number: nu p - - ). the ministry of health, provided surveillance personnel, laboratory space, reagents and equipment to perform tests and sites for sample data collection and personnel in drafting and analyzing of the manuscript. us cdc provided funds and support for reagents and surveillance sites including technical support to analyze the data, training of key personnel on evaluation of surveillance systems and writing of the manuscript. all data generated or analyzed during this study are included in this published article. all authors declare that they have no commercial or other associations that may pose a conflict of interest. ethical approval and consent to participate this surveillance evaluation was deemed non-research by the zambia-moh and the us cdc.consent to participate was voluntary and verbally obtained, because this was a non-research and the procedure was non-risks to client ethical clearance was deemed unnecessary by the university of zambia biomedical research ethics committee. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the us centers for disease control and prevention, usa or the zambia ministry of health. not applicable. key: cord- -jmcuq c authors: chen, hui; cui, yang; han, xuling; hu, wei; sun, min; zhang, yong; wang, pei-hui; song, guangtao; chen, wei; lou, jizhong title: liquid–liquid phase separation by sars-cov- nucleocapsid protein and rna date: - - journal: cell res doi: . /s - - - sha: doc_id: cord_uid: jmcuq c nan well refolded proteins were concentrated and further purified with anion exchange chromatography column and size exclusion chromatography. all proteins were quantified by a /a (below . ) measurement to guarantee the exclusion of nucleic acid. the n protein rna n ( nt; supplementary information, table s ) and a ( nt; supplementary information, table s ) were in vitro transcribed and purified as previously described . h ( nt), a ( nt) and poly-u rna oligos of different lengths (u , u , u , u ; supplementary information, table s ) with hex labeling were purchased from sangon biotech (shanghai china). all rna sequences were listed in supplementary information, table s . all the proteins were constructed with c-terminal cystine and conjugated with maleimide-alexa fluorescence according to the manufacturer's instruction. briefly, μm proteins were buffer change into labeling buffer ( × pbs, ph . , mm tcep), mm c -maleimide alexa or c -maleimide alexa were added and incubated h at room temperature. excess dye was removed by using a zeba spin desalting column and finally stored at - °c. phase separation assays were performed in × pbs (ph . ) supplemented with . % peg unless otherwise noted. a microwell -well optical-bottom plates with polymer base was coated with % bsa at °c overnight and then washed with pbs three times. phase separation was induced with rna addition and then transferred in -well plate at room temperature for h. imaging was performed with olympus fv equipped with a × oil immersion objective, and - regions were randomly selected for image acquisition. all images were processed with fv -asw, the percentage area of the image occupied by droplets was calculated based on the method from the published research . for negative stain em, approximately μl of sample were applied to glow-discharged copper em grids covered with a thin layer of continuous carbon film, and then the grids were stained with μl of % uranyl acetate. the images were recorded at a magnification of , , yielding a pixel size of . Å on talos f c -kv electron microscope equipped with a k × k ceta camera (fei). samples were prepared by mixing indicated concentration of protein and rna. approximately μl of sample were applied to glow-discharged holey carbon grids the particle size of n protein was detected by a dynapro nanostar (wyatt technology) which was equipped with a nm laser, detector angle was fixed at °. every single experiment was consisted of ten independent repeats of -s acquirement with a -s interval. particle size was characterized from signal intensity with isotropic spheres model based on cumulant fit. assembly of severe acute respiratory syndrome coronavirus rna packaging signal into virus-like particles is nucleocapsid dependent the initiation, propagation and dynamics of crispr-spycas r-loop complex key: cord- -l gvpat authors: singh, kamaljit; kaur, hardeep; chibale, kelly; balzarini, jan; little, susan; bharatam, prasad v. title: -aminopyrimidine based -aminoquinoline anti-plasmodial agents. synthesis, biological activity, structure–activity relationship and mode of action studies date: - - journal: eur j med chem doi: . /j.ejmech. . . sha: doc_id: cord_uid: l gvpat -aminopyrimidine based -aminoquinolines were synthesized using an efficacious protocol. some of the compounds showed in vitro anti-plasmodial activity against drug-sensitive cq(s) ( d ) and drug-resistant cq(r) (k ) strains of plasmodium falciparum in the nm range. in particular, -isopropyloxycarbonyl- -methyl- -( -nitrophenyl)- -[( -chloroquinolin- -ylamino)butylamino] pyrimidine depicted the lowest ic( ) ( . nm) value ( -fold less than cq) against cq(r) strain. structure–activity profile and binding with heme, μ-oxo-heme have been studied. binding assays with dna revealed better binding with target parasite type at rich puc dna. most compounds were somewhat cytotoxic, but especially cytostatic. molecular docking analysis with pf dhfr allowed identification of stabilizing interactions. faced with the challenges of drug resistance, poor health systems, lack of affordable, safe and convenient treatment options, efficient treatment of malaria, one of the most devastating parasitic diseases, represents an unmet medical need. malaria is a major public health concern in more than countries inhabited by more than . billion people e % of the world's population and is responsible for almost million deaths every year [ ] . the majority of malaria victims in developing countries are pregnant women or children under the age of five, possessing little or no immunological protection. the disease is estimated to result in w million new annual infections worldwide. though the majority of the cases and approximately % of the malaria deaths are found in sub-saharan africa, the disease is now increasing in asia and latin america. malaria is caused by protozoan parasites of the genus plasmodium that infects and destroys red blood cells eventually leading to death, if untreated. the persistent threat of emergence of multidrug resistant plasmodium falciparum, universal chloroquine resistance [ , ] , suspected resistance to artemisinins [ , ] , and lack of effective, appropriate and affordable treatment options have given a new impetus to the research leading to broadening of the range of therapeutic targets. thus, creating a new armamentarium of drugs with promising antimalarial activity coupled with understanding of their mode of action may lead to the development of a new generation of treatments both for malaria control and eradication. the common feature of the drugs based on quinine is the presence of a quinoline unit, usually a -chloroquinoline (chloroquine and amodiaquine , chart ), and are known to cause parasite death by blocking the polymerization of the toxic heme, into an insoluble and non-toxic pigment, hemozoin, resulting in cell lysis and parasite cell autodigestion [ e ] . the mode of action of , -diaminopyrimidine based drugs, typified by pyrimethamine [ ] and the lead compound wr [ ] is through the inhibition of p. falciparum dihydrofolate reductase (pf dhfr) enzyme, required for the biosynthesis of tetrahydrofolate involved in the biotransformation of thymidylate synthase-catalysed deoxyuridylate to deoxythymidylate (dump / dtmp), through a methyl group transfer reaction during dna biosynthesis [ e ] . in addition, a number of polyamines inhibit ornithine decarboxylase activity in p. falciparum through binding with plasmodial dna [ , ] . recently, investigations in hybrid antimalarial agents, combining -aminoquinoline with other pharmacophore having antimalarial activity have been found to be less prone to resistance to parasite and has thus offered an effective means to overcome the problem of drug resistance. we envisaged that linking -chloro- -aminoquinoline unit, critical for antimalarial activity through a diversely functionalized lateral side chain with other antimalarial moiety such as aminopyrimidine, might furnish conjugate hybrids [ ] capable of showing useful antimalarial activity. in this communication, we report synthesis of a set of compounds that possess basic, hydrophobic as well as hydrogen bonding substituents, required for targeting either or both heme as well as dna, thus providing new antimalarial agents active against chloroquine resistant strains of p. falciparum. we have evaluated their anti-plasmodial activity, cytotoxicity and cytostatic activity, binding studies with dna and heme (monomeric as well as m-oxo dimeric) using uvevisible, fluorescence spectrophotometry as well as nmr analysis. compounds aes were synthesized as outlined in scheme , via a common intermediate . , -dihydropyrimidin- ( h)-ones were prepared through hcl-catalyzed biginelli condensation of appropriate aldehyde (r cho), alkylacetoacetate (r ch coor ) and urea [ ] . dehydrogenation of using pyridinium chlorochromate in dcm furnished pyrimidinones [ ] . refluxing with pocl yielded which upon nucleophilic substitution reaction with appropriate -amino- -chloroquinoline gave aes in a synthetically useful manner [ ] . structures of e were established on the basis of spectral ( h nmr, c nmr, ms, ft ir) as well as microanalytical analysis. the yields of the -aminopyrimidines are reported in table . antiplasmodial activity of pyrimidines linked to cq as in has not been described in literature albeit the related dihydropyrimidin- ( h)-ones (dhpms) have previously been reported [ ] . using the synthetic protocol shown in scheme allowed considerable diversification around the pyrimidine core for conducting sar analysis. the in vitro anti-plasmodial activities of aes were determined in primary and secondary screening against cq s and cq r strains of p. falciparum. the half maximal inhibitory concentration (ic ) of aes are summarised in table (fig. ) . evidently, the compounds have anti-plasmodial activity in the nm range and against the cq r strain of p. falciparum, in some cases activity was found to be even superior to cq. systematic variation of the length as well as nature of the spacer connecting the pharmacophores discerned useful trends in the anti-plasmodial activity of these analogs. comparing aeg, bearing linear alkyl spacers, revealed an increase in the anti-plasmodial activity with increase in length of the spacer up to methylene groups ( ae c, table ). further lengthening of the spacer chain length resulted in significant reduction in activity against both the cq r as well as the cq s strains. replacing the c- phenyl group in c by a methyl group to create m resulted in nearly times increase in anti-plasmodial activity (ic . nm) against the cq s strain. in fact, compound m was found (table ) to be the most active compound of the series, against the cq s strain. however, m exhibited a high resistance factor (w ) within the series but is . times less active than cq, against the cq r strain. replacement of the ethyl ester with isopropyl ester ( nes), in general showed a decrease in in vitro antiplasmodial activity against the cq s strain, although a reverse trend was observed for the cq r strain, especially in case of s, p and r, where replacing c- phenyl by p-nitrophenyl ( s) or onitrophenyl ( p and r) groups, in addition to incorporating an isopropyl ester, resulted in an increase of activity in that order, rendering r as the most potent (ic . nm, times more potent than cq, table ) of all these compounds. these results are in accordance with the trend observed in case of n, n-bis( chloroquinolin- -yl)alkane diamines, wherein the alkyl spacer consisting of four carbon atoms showed optimum potency [ ] . the better anti-plasmodial activity of phenyl-substituted pyrimidine compounds against the cq r strain may be attributed to optimal fitting of these compounds in the active site of pfdhfr leading to a favorable conformation for p-p interaction with the heme functionality. moreover, the introduction of a nitro substituent on the phenyl ring at the c- position of the pyrimidine core results in a significant increase in anti-plasmodial activity as well as resistance against the cq r strain, although it has little effect on activities against the cq s strain (table ) . also, the corresponding o-, m-or p-nitro derivatives showed considerable variation in antiplasmodial activity ( table ). comparison of compounds ( n, q, s) having an identical spacer reveals that the p-no substituted s (ic . nm) is more active than the o-/m-no substituted compounds q and n, respectively, but are less active than the unsubstituted ethyl ester analog b. further, comparison with the butyl spacer analog suggests that o-no phenyl derivative r is more potent than the m-no counterpart o, as well as its ethyl ester analog c, against the cq r strain. compound r was found to be the most active compound in this series against the cq r strain with anti-plasmodial activity (ic . nm), times more than cq (ic . nm) and comparable to artesunate (ic . nm) ( table ) . comparing j (ic . nm) possessing the branched chain spacers with identical carbon linear spacer analog b (ic . nm, table ) led to significant decrease in activity. thus, compound j displayed a -fold decrease in activity compared to b, against the cq r strain. likewise, k possessing a branched c- spacer depicted a decrease (ic . nm, table ) in activity against linear alkyl (c- ) spacer analog d (table ), but higher than j. comparing j and k, with branched chain spacers, not only was the anti-plasmodial activity of the latter against both cq s and cq r strains increased, but it also showed an increased resistance factor ( table ). the replacement of linear chain spacers with o-and p-linked aryl groups ( h and i, respectively), led to much higher ic (table ) values compared to former against cq r strain, while only moderate activity was observed in case of i against cq s strain. these findings could be attributed to the increased steric bulk ( h more than i) affecting the interaction of the iron center of heme with the compounds. this has been corroborated by performing the titration of monomeric heme with both h and i. : molar ratio of heme and i. hence, the flexibility, chain length and steric constraints of the spacer linking quinoline moiety and pyrimidine unit seem to play a role in anti-plasmodial activity of these derivatives. although the in vitro activity of aec, p and especially r (ic . nm) was superior to cq (ic . nm) against the cq r strain, these compounds suffer from high clogp values (table ) , which are suggestive of the fact that these possess limited aqueous solubility, which in fact is not a serious limitation in view of recent advancements in formulation methods. antiviral activity of the compounds aec, len, p and r, which were active against the cq r strain was also evaluated against (i) parainfluenza- virus, reovirus- , sindbis virus, coxsackie virus b , punta toro virus in vero cell cultures, (ii) herpes simplex virus À (hsv- ; kos), herpes simplex virus- (hsv- ; g), vaccinia virus, vesicular stomatitis virus, herpes simplex virus- (tk À kos acv r ), cytomegalovirus, varicella-zoster virus in hel cell cultures, (iii) vesicular stomatitis virus, coxsackie virus b , respiratory syncytial virus in hela cell cultures, (iv) influenza a virus (h n and h n ) and influenza b virus in mdck cell cultures and (v) feline corona virus (fipv) and feline herpes virus activity in crfk cell cultures (si tables s es ). unfortunately, no significant antiviral activity was noted at subtoxic concentrations. most compounds were somewhat cytotoxic to the different cell lines (confluent nonproliferating cultures), but especially cytostatic against proliferating (vero/mdck/crfk) cell cultures. chloroquine has been shown to inhibit hiv through blockade of viral entry via inhibition of endosomal acidification [ , ] . compounds aec, len, p, r were also tested against hiv- as well as hiv- (si table s ) in human t-lymphocyte (cem) cell cultures. however, none of the compounds were active at subtoxic concentrations. generally, the compounds exhibit a relatively high cytostatic activity but displayed a fairly safe selectivity index (except m and n) ( table ) in the range of . e against mdck cell cultures. the most active compound r with an ic value of . nm against cq r strain exhibited a highest selectivity index (si ¼ ), l with an ic value of . nm against cq r strain exhibited a high selectivity index ( . ). however, m having an ic value of . nm was most toxic with a selectivity index of . . the plausible mechanism of in vitro anti-plasmodial action against cq r strain has been investigated for c and r, found to be most potent of the series of the compounds reported herein (ic . nm, c and ic . nm, r). these compounds are also expected to bind to heme [fe(iii)ppix] (hydroxo or aqua complex of ferriprotoporphyrin ix) in solution and inhibit aggregation to bhematin, in much the same way as cq itself. the higher clogp values ( table ) of these compounds being higher than cq necessitated the use of aqueous dmso as solvent. also in % aqueous dmso solution, heme is expected to be monomeric, while in purely aqueous solutions, it exists in aggregated form. further, it is known that cq binds to heme dimer (m-oxo heme) in vitro and can also inhibit the formation of b-hematin, the in vitro analog of hemozoin [ e ] . therefore the binding assay was also extended to heme dimer also. the interaction of c and r with monomeric heme was followed by spectrophotometric titration at ph . and . (the approximate ph of food vacuole), as described previously [ ] . the spectral changes (fig. a , si figure s ), upon addition of increasing amounts ( e mm) of r to a solution of heme ( . mm, hepes buffer ph . ) showed substantial decrease of intensity of the fe(iii) ppix soret band at nm with no shift in the absorption maximum. the longer wavelength q-bands ( and nm) of the metalloporphyrin also decrease in intensity (si figure s ) [ ] . a sharp isosbestic point was observed at nm (ph . ) and at nm (ph . ). the association constants for the complexes formed between monomeric fe (iii) ppix and r at ph . and . were calculated from titration data and are presented in table . the association constant of c ( fig. a ) is found to be greater (log k . ) than r (log k . ). decrease in the apparent ph from . to . caused a fairly modest decrease in log k values of both c and r, indicating a strong binding to heme even at acidic ph. the binding of c and r was also assessed from their h nmr titration with heme. thus, while h nmr spectrum of r depicted considerable broadening of signals under the conditions ( % dmso-d in d o) of recording and was inconclusive, the variation in the chemical shift in signals corresponding to quinoline unit of c were clear (fig. ), upon addition of heme. a : stoichiometry of the most stable complexes of both r and c with monomeric heme at ph . and . was deduced from the job's plot (si figure s ) [ , ] . the absorbance at nm got to maximum when mole fraction of r approached . . this is in good agreement with the association constant (log k) values corresponding to the most stable : :heme: r species present in solution, obtained by fitting the titration data in hyp spec -a nonlinear least square fitting programme. the measured log k values for both r and c (table ) are also in good agreement with the values reported in literature for cq [ , , ] . thus, the formation of synthetic fe(iii)ppix- r complex, as demonstrated above is suggestive of inhibition of formation of b-hematin and presumably results in anti-plasmodial activity of these compounds in a fashion similar to that of cq. titration of constant concentrations of heme in aqueous naoh solution at the physiological ph . of plasmodial food vacuole [ ] allowed understanding of the interaction of r and c with m-oxo heme. with addition of increasing amount of r to the solution of m-oxo heme, the absorbance of soret band ( nm) decreased appreciably with significant red shift ( e nm, fig. a ). the association constants and stoichiometry for both r and c were determined from titration data and are presented in table .the calculated association constant of r (log k . ) is greater than c (log k . ) and corresponds to : stoichiometry of the r:moxo heme complex. the comparison of association constants of r and c for monomeric and m-oxo heme reveals that both compounds bind strongly with m-oxo heme than monomeric heme and inhibit hemozoin formation by blocking the growing face of heme resulting in the observed anti-plasmodial activity. further, all the compounds were also screened for in vitro inhibition of bhematin formation using b-hematin inhibitory assay in order to further confirm their mechanism of action based on interference with the heme detoxification process [ , ] . as shown in table , there is a general correlation between antiplasmodial activity and inhibition of b-hematin formation, but the same generalisation does not hold for compound h, as, in spite of having % b-hematin inhibition (table ) , it is one of the least active (ic . nm, cq r and ic . nm, cq s ) compounds of the series. however, it must be noted that anti-plasmodial activity not only depends upon the b-hematin inhibition but also on other factors such as degree of accumulation of drug in food vacuole. further, r showed dose dependent inhibition of bhematin formation (si figure s ) with an ic value lower than those reported for cq and quinine, thus demonstrating a better ability to interact with fe(iii)ppix. overall, these compounds showed strong b-hematin inhibition which seems to be their preferred mode of action. dna binding has been considered previously as one of the possible mechanisms for anti-plasmodial activity and has been studied using spectrophotometry [ ] , spectrofluorimetry [ ] , dna melting [ ] , viscometry [ ] etc. recently, some aminoquinoline derivatives have been shown to interact with dna [ ] presumably through ionic interactions between phosphate groups of dna and protonated amine sites resulting in stabilization of the helical configuration of dna against thermal denaturation [ , ] . additionally, interactions between aromatic nucleuses of the drug with nucleotide bases might also contribute. binding of the plasmid dna with cq resulted in the elevation of the thermal melting temperature (t m ) of dna in addition to other effects [ e ]. we therefore investigated the binding of the most active of the series, c and r toward gc rich calf thymus dna (ct dna) as well as at rich puc dna through stepwise addition of small increments of dna to a solution the compounds at constant concentration as well as physiological ph (fig. a and b) . a progressive decrease in the characteristic quinoline ring absorptions at and nm attributable to the intercalation of the quinoline into dna was indicated. the isosbestic point at nm indicated that the spectra of limiting systems (i.e. the spectra of free and completely bound drug) intersect and permitted the selection of a single wavelength for study of complex formation. the binding constant (log k: c . and r . ) was calculated from benesiehildebrand equation [ ] and suggested strong interaction with ct dna. comparison of binding constants reveals that c binds times more strongly with ct dna than r. altering the dna base composition to see its effect on the drug binding, we studied the interaction of r with gc rich ct dna by fluorometric titration. the emission band at nm of r (fig. ) , decreases in intensity with increasing concentration and eventually gets completely quenched. the binding constant (log k) calculated from the fluorescence data is . . similar titration with at rich puc dna (log k ¼ . ) revealed higher affinity of r toward at rich puc dna unlike cq which is known to interact strongly with gc rich dna [ ] . the strong interaction of the pyrimidine analog with at rich puc dna further suggests that these compounds might target parasite dna which has unusually a high at content [ ] in addition to b-hematin inhibition. the binding of ligand to nucleic acid of dna (especially at rich dna) may possibly induce conformational changes depending upon the strength and mode of its interaction with nucleic acids and thus result in increase in the thermal denaturation temperature, as observed for cq. groove binding or electrostatic binding of the phosphate backbone of dna gives rise to small changes in thermal denaturation temperature compared to the intercalation pathway due to stabilization of watson crick base paired duplex [ , , , ] . in the direction of evaluating strength of interaction of c and r with dna, the thermal behavior (si figure s ) of ct dna in the presence of drug was evaluated. the thermal denaturation temperature of ct dna ( c), in the presence of c and r recorded an increase in dt m of c and . c, respectively (si table s ), suggesting primary groove binding and/or partial intercalative nature of the interaction. many of the anti-plasmodial agents are known to interact with pf dhfr for their anti-plasmodial activity. especially table in vitro anti-plasmodial activity, cytotoxicity, resistance factor and selectivity index of compounds a-s. ic pyrimethamine (chart ) is known to produce anti-plasmodial effect by binding to pf dhfr. the best known lead compound for this inhibitory activity is wr ( ) . it is worth comparing the performance of the compounds designed in this work with that of , using molecular docking methods. the crystal structure (pdb id: j i, a structure of wild-type pf dhfr-ts complexed with wr , nadph, dump) was considered for molecular docking analysis. after appropriately preparing the protein structure of pf dhfr, docking of and r were carried out. fig. shows the top scoring binding pose of r in the active site of pf dhfr. the docking score for r is À . , which is much better than that of the lead compound (À . ). the origin of this improvement in docking score can be traced to the increased number of stabilizing interactions between r and pf dhfr, in comparison to the interactions between and pf dhfr. r shows hydrogen bonding interaction with tyr and asn residues; these two interactions are common to r as well as to . in addition, r shows hydrogen bonding interaction with ser and leu residues. these additional stabilizing interactions ensure that r adopts a slightly different (and improved) pose in comparison to that of . these improved interactions can be considered as deterministic factors for the improved anti-plasmodial activity of r. an efficacious transformation for converting readily available , -dihydropyrimidinones (dhpms) to -aminopyrimidine based aminoquinolines is presented. the compounds showed anti-plasmodial activity identical to or superior to cq. structureeactivity relationship has been drawn leading to the identification of at least one compound r as lead compound in this series. the tested compounds did not depict any antiviral activity and were cytotoxic. binding interaction of representative potent compounds with heme and m-oxo heme using uvevisible and nmr experiments furnished log k values identical with cq binding and pointed to : stoichiometry of the most stable complexes in solution. dna (both gc and at rich) binding affinity using absorption and spectrofluorometric data indicated stronger interaction of c ( fold) than r with ct dna, however, r showed higher affinity toward at rich puc dna suggesting targeting of parasite at rich dna by r in addition to b-hematin inhibition as possible mode of action of these compounds. dna melting experiments suggested primary groove binding and/or partial intercalative nature of interaction of c and r with ct dna. thus, the strong electrostatic interaction of newly designed pyrimidine analogs with at rich dna and blockage of heme polymerisation by complexation of drug with heme contribute to the observed antimalarial activity in nano molar range. further, molecular docking analysis of r in the active site of pf dhfr indicated superior binding compared to wr . thus, drug r acts on multiple targets (heme, enzyme involved in biosynthesis of dna (dhfr), parasite dna) which accounts for its high anti-plasmodial activity. all liquid reagents were dried/purified following recommended drying agents and/or distilled over Å molecular sieves. thf was dried (na-benzophenone ketyl) under nitrogen. h nmr ( mhz) and c ( mhz) nmr spectra were recorded in cdcl on a multinuclear jeol ft-al- spectrometer with chemical shifts being reported in parts per million (d) relative to internal tetramethylsilane (tms, d . , h nmr) or chloroform (cdcl , d . , c nmr). mass spectra were recorded from indian institute of integrative medicine (csir), jammu, under electron impact at ev on a bruker daltonics esquire spectrometer. elemental analysis was performed on flash ea (thermo electron corporation) analyzer at department of chemistry, guru nanak dev university, amritsar and the results are quoted in %. ir recorded on ftir shimadzu fourier-transform spectrophotometer in the range e cm À using chloroform as medium. melting points were determined in open capillaries and are uncorrected. for monitoring the progress of a reaction and for comparison purpose, thin layer chromatography (tlc) was performed on precoated aluminum sheets merck ( f , . mm) using an appropriate solvent system. the chromatograms were visualized under uv light. for column chromatography silica gel ( e mesh) was employed and eluents were ethyl acetate/hexane or ethyl acetate/ methanol mixtures. to the stirred solution of ( mmol) and potassium carbonate ( mmol) in dry thf ( ml), a solution of appropriate aminoquinoline ( . mmol) in dry thf ( ml) was added. the reaction mixture was stirred for h at room temperature. the reaction mixture was filtered and thf was removed under vacuum. the residue was purified by column chromatography using meoh/ etoac as eluent to obtain corresponding , which was recrystallized from dcm/hexane. using this procedure the following compounds were isolated. to the stirred solution of appropriate -aminoquinoline in dry thf ( ml) mixture of (in a : molar ratio) and potassium carbonate in dry acetonitrile was added. the reaction mixture was refluxed for h and then filtered. acetonitrile was removed under vacuum and the residue was purified by column chromatography using etoac/hexane as eluent to give h and i which were recrystallized from dcm/hexane. yellow solid. rf: . ( % hexane/etoac two clones of p. falciparum are used: (a) d clone of nf which is known to be sensitive to all anti-plasmodials, (b) k strain originating from thailand that is resistant to chloroquine and pyrimethamine, but sensitive to mefloquine. the cultures were naturally asynchronous ( e % ring stage) and were maintained in continuous log phase growth in rpmi medium supplemented with % washed human aþ erythrocytes, mm hepes, nm nahco , and albumaxii (lipid-rich bovine serum albumin) (gibco, grand island, ny) (cm). all cultures and assays are conducted at c under an atmosphere of % co and % o , with a balance of n . stock drug solutions were prepared in % dmso (dimethylsulfoxide) at mg/ml. the compound was further diluted to the appropriate concentration using complete medium rpmi supplemented with nm cold hypoxanthine and albumaxii. assays were performed in sterile -well microtitre plates; each plate contained ml of parasite culture ( . % parasitemia, . % hematocrit). each compound was tested in triplicate and parasite growth compared to control and blank (uninfected erythtocytes) wells. after h of incubation at c, . bq of [ h] hypoxanthine is added to each well [ ] . cultures were incubated for a further h before they are harvested onto glass-fiber filter mats. the radioactivity was counted using a wallac microbeta scintillation counter. the results were recorded as counts per minute (cpm) per well at each drug concentration, control and blank wells. percentage inhibition was calculated from comparison to blank and control wells, and ic values calculated using graph pad prism . . the preliminary screen uses the d strain. the compounds were tested at concentrations ( , , , , . , and . mg/ml). if the compound did not affect parasite growth at mg/ ml it was classified as inactive, between and mg/ml, the compound was designated as partially active, and if < mg/ml, the compound was classified as active and was further evaluated by three-fold serial dilutions in a repeat test. for secondary screening both the d clone and the k line were used. the compound was diluted three-fold over at different concentrations with an appropriate starting concentration based on the preliminary screen. the ic is determined by a sigmoidal dose response analysis using graph pad prism . . for each assay, the ic and ic values for each parasite line were determined against the known anti-plasmodials chloroquine and artesunate. the antiviral assays [except anti-human immunodeficiency virus (hiv) assays] were based on inhibition of virus-induced cytopathicity in hel [herpes simplex virus type (hsv- ), hsv- (g), vaccinia virus, and vesicular stomatitis virus], vero (parainfluenza- , reovirus- , coxsackie b , and punta toro virus), hela (vesicular stomatitis virus, coxsackie virus b , and respiratory syncytial virus) and mdck (influenza a (h n ; h n ) and b virus) cell cultures. confluent cell cultures in microtiter -well plates were inoculated with cell culture inhibitory dose- (ccid ) of virus ( ccid being the virus dose to infect % of the cell cultures) in the presence of varying concentrations of the test compounds. viral cytopathicity was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. the anti-hiv activity and antiproliferative activity were evaluated against hiv- strain iiib and hiv- strain rod in human t-lymphocyte cem cell cultures. briefly, virus stocks were titrated in cem cells and expressed as the % cell culture infective dose (ccid ). cem cells were suspended in culture medium at w  cells/ml and infected with hiv at w ccid . immediately after viral exposure, ml of the cell suspension was placed in each well of a flat-bottomed microtiter plate containing various concentrations of the test compounds. after a -day incubation period at c, the giant cell formation was microscopically determined. compounds were tested in parallel for cytostatic activity in uninfected cem cells. binding stoichiometries of drug with monomeric & m-oxo dimeric heme were monitored by uvevisible spectrophotometry using job's method of continuous variation [ , ] . the concentration of drug & heme in solution was kept constant and changes in absorbance at nm (monomeric)/ nm (dimeric) were monitored as a function of the mole fraction. well plate containing mixture of ml of . mg\ml of hemin chloride dissolved in dmso, ml of . m sodium acetate buffer (ph . ) and ml of different concentrations of drug solution or ml of solvent (control), was incubated at c for h. the plate was centrifuged at rpm for min, and supernatant was discarded. the remaining pellet was re-suspended with ml of dmso to eliminate unreacted hemin [ , ] . the plate was centrifuged again and supernatant similarly discarded. the precipitate was dissolved in ml of . n naoh & absorbance was read at nm. the percentage of inhibition of ferriprotoporphyrin ix biomineralisation was calculated using formula: inhibitionð%Þ ¼ xf½ðabs of controlÞ À ðabs of drugÞ= ðabs of controlÞg. ic values were determined by using graph pad prism . . . . dna binding studies with c and r stock solution of c and r ( mm) were prepared in ar grade methanol. the dna binding experiments were carried out by making dilution of the stock with : buffered methanol. stock solution of dna was prepared by dissolving dna pellet in te buffer ( mm trisehcl, . mm edta, ph . ). the dna concentration was estimated from its absorbance intensity at nm with a known molar absorption coefficient value of dm mol À cm À . the purity of dna was established from ratio of absorbance intensity at nm and at nm. the observed ratio of . ensured that dna was free from protein. nm at various temperature in the presence and absence of drug in a : ratio of the dna and drug with a ramp rate of . c/min in a % dmso/te buffer (ph . ) with . mm nacl on a shimadzu pc spectrophotometer equipped with a peltier thermo regulator. the thermal melting temperature was calculated by plotting da/dt vs temperature using microsoft excel. the molecular docking analysis was carried out using flexx molecular docking module [ , ] (flexx . . ) available in the sybyl . software package from tripose [ ].this docking approach adopts an incremental construction algorithm for identifying appropriate pose of the substrate in the active site of the enzyme. it generates about possible poses of the substrate in the active site. most of the poses obtained in this analysis adopted similar binding mode in the enzyme cavity and hence the top scoring pose was considered in this analysis. to perform the above analysis, the crystal structure j i (wild-type plasmodium falciparum dihydrofolate reductase-thymidylate synthase (pfdhfr-ts) complexed with wr , nadph, and dump) was downloaded from the protein data bank [ ] (pdb) and subjected to protein preparation and receptor description [ ] . the structures of the substrates were prepared by first building the molecules in d and optimizing the structures using tripos force field. the ionic, tautomeric states of substrates were considered while performing molecular docking analysis. the pose shown in fig. includes the neutral state of the substrate in the active site of the enzyme. yield: %. ir (kbr): n max , , , , cm À h ( mhz ( s, h, ch ), . (d, j ¼ . hz, h, ch ), . (d, j ¼ . hz, h, ch ), . (s, h for c h n o cl: c, . ; n, . . ms: m/z (m þ ) yield: %. ir (kbr): n max , , , , cm À h ( mhz -ethoxycarbonyl- -methyl- -phenyl- -[ -( -chloroquinolin- -ylamino)- -methylpentylamino] pyrimidine ( k) viscous liquid. rf: . ( % meoh/etoac) white solid (s, h, c & c ech ), . (q, j ¼ . hz, h, ch ), . (q, j ¼ . hz, h, ch ), . (q, j ¼ . hz, h h, . ; n, . . ms: m/z . (m þ ) white solid (s, h, c & c ech ), . (q, j ¼ . hz, h, ch ), . (q, j ¼ . hz, h . ; n, . . ms: m/z . (m þ ) -ylamino)propyl amino]pyrimidine ( n) yellow solid. rf: . ( % meoh/etoac) c nmr ( mhz ylamino)butylamino] pyrimidine ( o) yellow solid. rf: . ( % meoh/etoac) (s, h ylamino)ethylamino] pyrimidine ( p) yellow solid (s, h -ylamino)propyl amino] pyrimidine ( q) yellow solid. rf: . ( % meoh/etoac) (s, h, c ech ), . (br, h, ch ), . (q, j ¼ . hz, h ylamino)butylamino] pyrimidine ( r) yellow solid. rf: . ( % meoh/etoac) (s, h, c ech ), . (br, h, nh), . (m, h n, . . ms: m/z . 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incremental construction without manual intervention a fast flexible docking method using an incremental construction algorithm insights into antifolate resistance from malarial dhfr-ts structures a common feature-based d-pharmacophore model generation and virtual screening: identification of potential pfdhfr inhibitors key: cord- - v weno authors: sauter, marian; draschkow, dejan; mack, wolfgang title: building, hosting and recruiting: a brief introduction to running behavioral experiments online date: - - journal: brain sci doi: . /brainsci sha: doc_id: cord_uid: v weno researchers have ample reasons to take their experimental studies out of the lab and into the online wilderness. for some, it is out of necessity, due to an unforeseen laboratory closure or difficulties in recruiting on-site participants. others want to benefit from the large and diverse online population. however, the transition from in-lab to online data acquisition is not trivial and might seem overwhelming at first. to facilitate this transition, we present an overview of actively maintained solutions for the critical components of successful online data acquisition: creating, hosting and recruiting. our aim is to provide a brief introductory resource and discuss important considerations for researchers who are taking their first steps towards online experimentation. in midst of the covid- pandemic [ ], many researchers are bound to rethink lab-based behavioral experiments [ ] . there is an emerging need for online testing solutions (for a flowchart, see figure ) for day-to-day research activities, thesis work and experimental practical courses alike. even without a forced shutdown of physical labs, online experiments have gained popularity [ ] in the last decade [ ] [ ] [ ] [ ] [ ] . they offer great advantages in terms of participant diversity (in terms of age, gender, origin, culture and social status) [ , ] , time and resource efficiency [ ] . a big strength of online studies is that they scale very well, as recruiting larger samples does not require a higher workload and particularly hard-to-reach populations become more readily accessible (e.g., [ ] [ ] [ ] [ ] [ ] ; see also figure ). this can be especially useful for reaching clinical samples or for conducting experimental cross-cultural studies. this article is mainly aimed at cognitive psychology and behavioral neuroscience researchers who have none or limited prior experience in conducting behavioral experiments within an online ecosystem. our focus is on providing a conceptual overview of the critical components of online experimentation. we further summarize the most well-established tools for implementing these components and provide information about good starting points on the road to online studies. finally, we offer some considerations and rules of thumb for succeeding with online acquisition, mainly focusing on feasibility and data quality. the critical procedural pillars of any behavioral study are: ( ) programming an experiment in the preferred software (e.g., e-prime, psychopy, psychtoolbox, etc.); ( ) setting-up the testing machine (e.g., lab-computer, multi-unit testing facility, etc.) and ( ) recruiting participants to conduct the study. the process of bringing experiments online requires the same pipeline but can be more demanding in terms of harmonizing these steps to ensure that each part of the pipeline is compatible with the other parts ( figure ). for comprehensibility, we will outline each of these three steps in the next section. this will include a conceptual overview, but also specific examples of solutions (providers, software) which enable the corresponding step in the pipeline. the features and pricing are subject to change. for this reason, in this overview we discuss the main integrative possibilities, which we believe, will not change as quickly (for an up-to-date description of the detailed offerings, one should consult the respective websites). some of the described solutions are quite modular and specialized (table : b, c, d) in solving only individual steps of the process, whereas other providers offer a more holistic integrated-service ecosystem (table : a). in section . . we will discuss the considerations one should make when picking an ecosystem, but we will abstain for making strong recommendations and claims at this point. notably, we limited this overview to software that appears to be under active development to ensure steady security updates (with updates in ). brain sci. , , x for peer review of machine (e.g., lab-computer, multi-unit testing facility, etc.) and ( ) recruiting participants to conduct the study. the process of bringing experiments online requires the same pipeline but can be more demanding in terms of harmonizing these steps to ensure that each part of the pipeline is compatible with the other parts ( figure ). for comprehensibility, we will outline each of these three steps in the next section. this will include a conceptual overview, but also specific examples of solutions (providers, software) which enable the corresponding step in the pipeline. the features and pricing are subject to change. for this reason, in this overview we discuss the main integrative possibilities, which we believe, will not change as quickly (for an up-to-date description of the detailed offerings, one should consult the respective websites). some of the described solutions are quite modular and specialized (table : b, c, d) in solving only individual steps of the process, whereas other providers offer a more holistic integrated-service ecosystem (table : a). in section . . we will discuss the considerations one should make when picking an ecosystem, but we will abstain for making strong recommendations and claims at this point. notably, we limited this overview to software that appears to be under active development to ensure steady security updates (with updates in ). the compiled experimental files are then uploaded to an online host, which generates a link, making the study accessible online (potentially with the aid of a study management system). participants are recruited through recruitment platforms and access the online experiments on the host. the data is stored on the hosting server. equivalently to studies designed for in-lab testing, the first step in online experimentation is the programming of the experiment (table : b). in comparison to the hegemony of java, python, c++ and matlab libraries for experimental programming of lab-based studies, javascript (js) is the language of choice for online experiments. even though it is usually ranked as the most popular programming language in the world, js has not been a hallmark in behavioral testing. current solutions for online-experimental generation often provide a graphical user interface (gui), enabling users to drag-and-drop modular components into an experimental sequence. as this rather simplistic, general solution is sometimes insufficiently flexible for more complex experimental designs, a good experimental environment should provide the possibility to extend these modular components with scripts and code-based solutions. arguably, the easiest transition from in-lab to online testing is granted by psychopy builder [ ] [ ] [ ] [ ] and opensesame [ , ] . both environments are very popular for traditional testing and allow for a rather straight-forward restructuring towards their online counterparts (psychojs and osweb), if only their drag-and-drop modules were used to create experiments. all sections in which scripting was used (e.g., python inserts) will need to be rewritten into javascript by the experimenter. fortunately, python (especially its 'object-based' subset) and javascript generally only differ in terms of syntax and not programming logic [ ] , so the rewriting is comparably easy. additionally, psychopy auto-translates base-python to js (but not functions from specific libraries). there are the compiled experimental files are then uploaded to an online host, which generates a link, making the study accessible online (potentially with the aid of a study management system). participants are recruited through recruitment platforms and access the online experiments on the host. the data is stored on the hosting server. equivalently to studies designed for in-lab testing, the first step in online experimentation is the programming of the experiment (table : b). in comparison to the hegemony of java, python, c++ and matlab libraries for experimental programming of lab-based studies, javascript (js) is the language of choice for online experiments. even though it is usually ranked as the most popular programming language in the world, js has not been a hallmark in behavioral testing. current solutions for online-experimental generation often provide a graphical user interface (gui), enabling users to drag-and-drop modular components into an experimental sequence. as this rather simplistic, general solution is sometimes insufficiently flexible for more complex experimental designs, a good experimental environment should provide the possibility to extend these modular components with scripts and code-based solutions. arguably, the easiest transition from in-lab to online testing is granted by psychopy builder [ ] [ ] [ ] [ ] and opensesame [ , ] . both environments are very popular for traditional testing and allow for a rather straight-forward restructuring towards their online counterparts (psychojs and osweb), if only their drag-and-drop modules were used to create experiments. all sections in which scripting was used (e.g., python inserts) will need to be rewritten into javascript by the experimenter. fortunately, python (especially its 'object-based' subset) and javascript generally only differ in terms of syntax and not programming logic [ ] , so the rewriting is comparably easy. additionally, psychopy auto-translates base-python to js (but not functions from specific libraries). there are plenty other experiment builders available: gorilla [ ] , inquisit web [ ] , labvanced [ ] and testable [ ] , from the integrated-service providers (see table : a) and lab.js [ , ] , jspsych [ , ] , psytoolkit [ ] [ ] [ ] , tatool web [ ] from the function-specific solutions (see table : b). their advantages and shortcomings should be evaluated on a lab's basis depending on individual needs. generally, as all experiment builders (except for inquisit) operate on a javascript backend, they offer similar flexibility. they differ in available features (example tasks or modules), but as all builders have online documentations, often with demonstration tasks available, researchers can quickly see whether they fit their specific needs. we see the most difficulties in transferring experiments online for psychtoolbox [ ] users, as matlab ® 's compile-to-javascript approach offers no trivial translation of experiments to browser-based software [ ] . [ ] links can be shared to any platform or social media but extensive documentation is not available; [ ] no active participant pool; [ ] testable offers a mixed payment model; testable is also free for all departments in [ ] ; [ ] up to participants and one study; [ ] only available upon request. in lab-based studies, the final resting place of the finished experiment is the testing machine. for online studies, the experiment needs to be made available for online distribution by hosting it on a server (table : c). this is potentially the most confusing step in the pipeline of creating an online study. some labs with a lot of experience in online experimentation host their studies on their own servers. this comes with the advantages of low maintenance costs, full control and flexibility. on the downside, it requires some expertise for setup and continued maintenance. the more feasible alternative is centralized hosting providers. here, hosting and study management is a service, and as such, all providers require a fee. the general idea behind study management systems is to simplify the hosting and participant handling process, like user management, automated data storage or creation of unique participation links. the whole range of features offered by different providers can be evaluated by visiting their websites. for example, one of the easier but not especially flexible hosting services is offered by open lab [ ] . it takes all studies created with lab.js and tests some participants for free. their unique selling point is arguably its integration with open science framework (osf) [ ] . participant data are directly uploaded to osf, which could make it potentially interesting for multi-lab open science initiatives (it should be noted that there is neither a documentation, nor a privacy policy nor information about the responsible person or company publicly available.) another interesting example is pavlovia [ ]. you can upload html /javascript studies and there is documentation for importing studies created with lab.js, jspsych and the psychopy builder (psychojs). it offers easy integration with recruitment tools and a gitlab platform [ ] where experimenters can share their complete code. an example for more easily setting up one's own hosting platform is just another tool for online studies (jatos) [ , ] . jatos similarly takes html /javascript studies and documents how to import studies created via lab.js, jspsych and opensesame web. it offers a wide range of options and is a very comprehensive study management tool. finally, we want to highlight how important experiment-server compatibility is. in the examples above, we pointed out that a specific hosting service supports studies programmed by specific experimental builders. no host supports all experimental builds and no experimental build is compatible with all hosts. thus, a decision should always be made on the level of the overall ecosystem and not on the individual components of the pipeline (building vs. hosting vs. recruiting). the dominant advantage of running experimental studies online lies in its efficiency. it is feasible to collect responses from hundreds of participants within hours. thanks to the possibility of world-wide sampling, data collection can literally be completed over night. once the experiment is created and accessible online (usually with a link), participants can be recruited. due to higher participant numbers compared to most lab-based studies, handling this process manually is not advisable (for tools see table : d). orsee [ , ] and sona [ ] are participant pool management systems, which offer comprehensive automation tools. however, both require researchers to maintain their own (usually limited in size) participant pool. additionally, only a limited number of participants can be recruited from the local university, via social media and (institutional) mailing lists. maintaining an active pool of potential participants is the main advantage of amazon mechanical turk (mturk) [ ] [ ] [ ] , prolific academic [ , ] and qualtrics panel [ ] . all three providers offer participant recruitment and payment handling services. note that there is also cloudresearch [ ] , which is a recruitment service that uses the mturk platform, but unlike mturk itself, is specifically directed at researchers and offers better participant handling and targeting tools. as one essentially only needs a link to the study, they integrate well with the study management systems and experiment builders mentioned above (see table for details). while differences in their features are too narrow for the scope of this article, we will discuss some important points on data quality in section . generally speaking, what researchers need for online experimentation is the same as what they need for lab-based studies (figure ) : ( ) a programmed experiment, ( ) a server to host the study and ( ) a recruiting platform which advertises to participants. as outlined in the previous sections, there are many solutions for each of these steps. some solutions provide a single and holistic framework for all three aspects (table : a), whereas other solutions are specifically tailored to one of the aspects and need to be integrated into an ecosystem by the experimenter. here, the benefits and drawbacks mirror what we already know from software solutions in other domains. integrated-service providers enable time savings by reducing compatibility issues, providing customer support, and reducing administrative load. on the flip side, they sometimes lack transparency, lack flexibility (minimal compatibility with other solutions), and are generally expensive. non-profit and open-source solutions usually require more integration considerations and some of them lack direct customer support. instead, they provide forums and community feedback, low or no costs, and more peer-reviewed benchmarks. ideally, the decision on which online ecosystem to use, should be made in accordance to the lab's capabilities and needs as well as criteria of quality (see section ). as all platforms are javascript-based, they offer similar functionality and most experimental paradigms should theoretically be realizable on all platforms. in principal, a wide range of in-lab research questions can be targeted with online task implementations. the individual journey of a task from in-lab to online, however, can be quite different, as some tasks might need little adjustment, while others would require a major overhaul in order to provide informative results. general recommendations about which tasks are suitable for online testing and which platforms are best for the respective tasks are hard to make, as labs' use cases are too diverse. it will ultimately be a question of money, the labs' know-how and specific institutional infrastructure. of note, switching from other software packages to an integrated-service provider has often the drawback that previously programmed experiments cannot be run anymore and even slight adaptations to the experiments (for example control studies that reviewer asked for) are impossible without completely reprogramming the experiment. therefore, when deciding how to transfer experiments to the online world, researchers should not only consider what the provider offers, but also how they can adapt their research to the new environment. from an open science perspective, it should also be considered, that not all platforms allow experimental scripts to be exported. the authors personally had good experiences with osweb (for building) combined with jatos (for hosting) as well as psychopy (for building) with pavlovia (for hosting) [ ] and prolific (for recruitment). similarly, the authors would not recommend setting up experimental studies on self-maintained webservers without the aid of a study management system (e.g., jatos) because of the need to account for everything that can go wrong, such as handling data storage, assigning participant codes, assuring participants do not participate more than once, handling payment and so on. the dominant concern with running experiments online is data quality. while the most obvious concerns (e.g., motivation, distractions, stimulus timing) can be dealt with an appropriate design and incentive strategy, we would like to stress the importance of recording and analyzing dropouts [ ] . unlike laboratory studies, participants may drop out at rates of up to %. in a dropout analysis of local studies, zhou and fishbach [ ] found that % had a dropout rate of over %. alarmingly, the authors of the analyzed studies were unaware of these dropouts. they also found that out of published mturk studies, only six disclosed dropout rates. crucially, dropout rates can interact with the experimental condition [ , ] . to arrive at sound conclusions, it is therefore obligatory to report and analyze dropout rates. further, it is imaginable that stimulus presentation times or response times are unreliable because of variations in internet speed or display settings throughout the experiment. however, almost all online solutions operate by downloading (pre-buffering) the entire experiment onto the participant's machine. additionally, modern screen refresh rates are almost exclusively set to hz (de facto standard), making certain specifications of online studies a bit more predictable. among others [ ] [ ] [ ] , two recent large studies [ , ] investigated timing precision (unintended variability in stimulus presentation) of several online and offline solutions. the online-based comparison found good overall precision for gorilla ( ms), jspsych ( ms), psychojs (− ms) and lab.js ( ms). notably, these means are inflated by particularly bad performance using the safari browser and mac os x. the offline-based comparison, psychopy and opensesame achieved precisions of ms to ms, with only minor exceptions [ , ] , most notably with audio playback. a study investigating response timing, for example, found an additive timing offset of ms (similar across conditions) in online recordings compared to lab studies, while reproducing all expected task-based effects in various tasks (stroop, flanker, visual search, attentional blink) [ ] . in addition to timing, there could be concerns that participants might be less committed when they sit at home and are not directly observed by the experimenter. several studies have shown that decreased attention to the task is not necessarily found [ , ] and data quality is comparable to lab-based studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, in a recent study, participants completed several attention checks (in between outcome measures) and there was no difference between lab participants and online participants in any of the measures. however, the study showed that online participants had higher self-reported distraction (use of cell phone, talking to another person, etc). in any case, experimenters should adjust their experiment to account for the sample diversity (see the following section) and participants' motivations [ ] . crump et al. [ ] recommend that the latter can be accomplished by giving accuracy as feedback following each trial; giving prompts to encourage speeded responding when participants do not meet deadlines; and by giving summary assessments of performance after blocks of trials. there are some aspects researchers should consider when starting out with running online studies or transferring lab-based experiments to online systems [ , , ] (see figure ). to a certain extent, creating successful online experiments is similar to app development: one needs to think of a coherent framework and constantly worry about what the users are doing with the 'product' and whether they are using it as intended-without many opportunities for direct feedback. experimental instructions should be easy enough to be understood by a more diverse sample that is not necessarily used to behavioral testing. further, measures need to be taken to detect and discourage poor performance, that is 'fake' participation. finally, online studies need to be shorter than classical lab-based studies. lab-based studies typically attract young psychology students who are weird (western, educated, and from industrialized, rich, and democratic countries) [ ] . the samples drawn from online recruitment platforms are more representative of the general (online) population [ , ] . study participants have potentially never participated in a behavioral response time experiment. for this reason, experimenters need to be more thorough when creating experimental instructions and ascertain that they can stand on their own without verbal explanations (note: this is also a good recommendation for lab-based studies). it is crucial that the instructions are comprehensible by people of a wider age range representing many cultures and socio-economic backgrounds [ ] . in the authors' experience, a pictorial step-by-step instruction leads to less misunderstandings or even dropouts compared to a single page of text. it is advisable that instructions are forced to stay on the screen for some time before continuation is allowed or an instruction check is added ( [ ] , experiment ). in order to check whether participants have truly understood the instructions, a test run and online evaluation before beginning the main experiment is advised. additionally, study management systems also incorporate some monitoring functions to check that participants stayed on track. for example, it is possible to monitor how often the browser tab running the experiment was minimized during the experiment and viewing distance can be controlled [ ] . notably, on some platforms, explicit measures need to be taken to prevent participants from completing a study twice [ ] . the interaction between experimenter and participant is comparably indirect in online experiments. therefore, participants might be less inclined to be attentive simply for the sake of helping the experimenter with their research. it should therefore be considered to state the relevance of the research explicitly. it was shown that mturk participants perform better, when the task is presented as meaningful [ ] . for many participants drawn from recruitment services, the dominant motivation for participation is monetary compensation. while the amount of payment should be similar to lab-based studies for ethical reasons, the data quality is not necessarily affected by higher monetary incentives. in a category learning experiment by crump et al. [ ] (see experiment ), participants were paid either $ . (low incentive group) or a base amount of $ and a bonus of up to $ . depending on their performance (high incentive group). they found that the incentive structure had no effect on learning or error rates. however, they found that they could collect data more quickly and had fewer dropouts when payment was higher. typically, participants are paid a fixed amount after successful completion of the study-regardless of how long it takes them to complete it. this is why some participants try to complete the experiment as fast as possible without sticking to the instructions ('fake' participation). in order to ensure good data quality, the experimenters might need to adapt the experimental design to discourage such behavior. this implies that the best experiments to run online include a validation mechanism. generally, forced-choice paradigms (both rt and accuracy types), in which one of the alternatives is the correct choice, are especially suitable because the experimenter can evaluate the participants' performance during runtime, while judgment studies (e.g., moral dilemma tasks) are harder to evaluate and objective performance or attention checks might need to be included into the design. in the authors experience, an easy option for alternative-forced-choice tasks is to repeat the trial each time participants answered incorrectly. the authors also experienced less dropouts when a progress bar (comparable to surveys) was added. gamification of the study in general promises to yield better results [ ] . the interaction between experimenter and participant is comparably indirect in online experiments. therefore, participants might be less inclined to be attentive simply for the sake of helping the experimenter with their research. it should therefore be considered to state the relevance of the research explicitly. it was shown that mturk participants perform better, when the task is presented as meaningful [ ] . for many participants drawn from recruitment services, the dominant motivation for participation is monetary compensation. while the amount of payment should be similar to lab-based studies for ethical reasons, the data quality is not necessarily affected by higher monetary incentives. in a category learning experiment by crump et al. [ ] (see experiment ), participants were paid either $ . (low incentive group) or a base amount of $ and a bonus of up to $ . depending on their performance (high incentive group). they found that the incentive structure had no effect on learning or error rates. however, they found that they could collect data finally, online experimental studies should be short. participants would possibly not sit min in front of their screen and produce quality data. since structured investigations are still missing, we asked germans through appinio [ ] at which time they would abort an online experiment that offered minimum wage. most respondents said 'after minutes' ( %), followed by 'after minutes' ( %), 'after minutes' ( %) and 'after min or never' ( %). keeping these considerations in mind, for a certain subset of investigations (certainly not all), carefully developed online studies have a huge potential. many of the noise factors can be combated with a large sample size and intelligent preparatory work. taking behavioral experiments online is facilitated by numerous steadily maintained tools ranging from simple libraries to complex ecosystems. researchers need to wisely choose the software based on their own prior experience, the lab's resources and the requirements of the general area of study. colleges and universities across the us are canceling in-person classes due to coronavirus-cnn introduction to behavioral research on the internet economic games on the internet: the effect of stakes quantifying the web browser ecosystem psychological experiments on the internet. section iii: computer techniques for internet experimentation the online laboratory: conducting experiments in a real labor market running behavioral operations experiments using amazon's mechanical turk evaluating online labor markets for experimental research: amazon.com's mechanical turk. political anal amazon's mechanical turk: a new source of inexpensive, yet high-quality, data? conducting behavioral research on amazon's mechanical turk a league of their own: demographics, motivations and patterns of use of , male adult non-medical anabolic steroid users in the united states should we trust web-based studies? a comparative analysis of six preconceptions about internet questionnaires the bbc internet study: general methodology does the shape of a cup influence coffee taste expectations? a cross-cultural, online study. food qual standards for internet-based experimenting builder-psychopy v experiments in behavior made easy psychopy-psychophysics software in python building experiments in psychopy opensesame: an open-source, graphical experiment builder for the social sciences comparing python to other languages gorilla in our midst: an online behavioral experiment builder labvanced-online experiments made easy lab.js: a free, open, online study builder a javascript library for creating behavioral experiments in a web browser psytoolkit: a software package for programming psychological experiments using linux a novel web-based method for running online questionnaires and reaction-time experiments what's new in psychtoolbox- . perception switching to online experiments for their projects. therefore, we decided to make testable available to all, waiving all our fees until the end of this academic year open science framework (osf) the first single application for the entire devops lifecycle-gitlab | gitlab just another tool for online studies" (jatos): an easy solution for setup and management of web servers supporting online studies another tool for online studies subject pool recruitment procedures: organizing experiments with orsee sona systems: cloud-based subject pool software for universities running experiments on amazon mechanical turk evaluating amazon's mechanical turk as a tool for experimental behavioral research prolific | online participant recruitment for surveys and market research prolific.ac-a subject pool for online experiments panel management software-recruit, target, & save money | qualtrics survey solutions for researchers introduction to online experiments the pitfall of experimenting on the web: how unattended selective attrition leads to surprising (yet false) research conclusions lópez-de-ipiña, d. presentation accuracy of the web revisited: animation methods in the html era presentation and response timing accuracy in adobe flash and html /javascript web experiments best practices: two web-browser-based methods for stimulus presentation in behavioral experiments with high-resolution timing requirements the timing mega-study: comparing a range of experiment generators, both lab-based and online online timing accuracy and precision: a comparison of platforms, browsers, and participant's devices online psychophysics: reaction time effects in cognitive experiments is there a cost to convenience? an experimental comparison of data quality in laboratory and online studies attentive turkers: mturk participants perform better on online attention checks than do subject pool participants separate but equal? a comparison of participants and data gathered via amazon's mturk, social media, and face-to-face behavioral testing comparing online and lab methods in a problem-solving experiment home is where the lab is: a comparison of online and lab data from a time-sensitive study of interruption psychophysics in a web browser? comparing response times collected with javascript and psychophysics toolbox in a visual search task reaction time data in music cognition: comparison of pilot data from lab, crowdsourced, and convenience web samples comparing the similarity of responses received from studies in amazon's mechanical turk to studies conducted online and with direct recruitment reaction time effects in lab-versus web-based research: experimental evidence crowdsourcing a normative natural language dataset: a comparison of amazon mechanical turk and in-lab data collection testing the effectiveness of the internet-based instrument psytoolkit: a comparison between web-based (psytoolkit) and lab-based (e-prime . ) measurements of response choice and response time in a complex psycholinguistic task types of motivation affect study selection, attention, and dropouts in online experiments lessons learned in adapting a lab-based measure of working memory capacity for the web tapped out or barely tapped? recommendations for how to harness the vast and largely unused potential of the mechanical turk participant pool most people are not weird controlling for participants' viewing distance in large-scale, psychophysical online experiments using a virtual chinrest nonnaïveté among amazon mechanical turk workers: consequences and solutions for behavioral researchers breaking monotony with meaning: motivation in crowdsourcing markets does gamification work?-a literature review of empirical studies on gamification appinio-marktforschung in minuten this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this research received no external funding. the authors declare no conflict of interest. key: cord- -ht hb rc authors: van den brand, judith m. a.; stittelaar, koert j.; van amerongen, geert; reperant, leslie; de waal, leon; osterhaus, albert d. m. e.; kuiken, thijs title: comparison of temporal and spatial dynamics of seasonal h n , pandemic h n and highly pathogenic avian influenza h n virus infections in ferrets date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ht hb rc humans may be infected by different influenza a viruses—seasonal, pandemic, and zoonotic—which differ in presentation from mild upper respiratory tract disease to severe and sometimes fatal pneumonia with extra-respiratory spread. differences in spatial and temporal dynamics of these infections are poorly understood. therefore, we inoculated ferrets with seasonal h n , pandemic h n (ph n ), and highly pathogenic avian h n influenza virus and performed detailed virological and pathological analyses at time points from . to days post inoculation (dpi), as well as describing clinical signs and hematological parameters. h n infection was restricted to the nose and peaked at dpi. ph n infection also peaked at dpi, but occurred at similar levels throughout the respiratory tract. h n infection occurred predominantly in the alveoli, where it peaked for a longer period, from to dpi. the associated lesions followed the same spatial distribution as virus infection, but their severity peaked between and days later. neutrophil and monocyte counts in peripheral blood correlated with inflammatory cell influx in the alveoli. of the different parameters used to measure lower respiratory tract disease, relative lung weight and affected lung tissue allowed the best quantitative distinction between the virus groups. there was extra-respiratory spread to more tissues—including the central nervous system—for h n infection than for ph n infection, and to none for h n infection. this study shows that seasonal, pandemic, and zoonotic influenza viruses differ strongly in the spatial and temporal dynamics of infection in the respiratory tract and extra-respiratory tissues of ferrets. humans may be infected with different categories of influenza a virus-seasonal, pandemic, and zoonotic-each with their own epidemiology and pathogenesis. seasonal influenza viruses cause annual epidemics during autumn and winter in temperate regions. they predominantly cause upper respiratory tract disease with rare extension to the lower respiratory tract, resulting in severe and even fatal pneumonia [ ] . pandemic influenza viruses, like the pandemic h n (ph n ) virus in , cause sporadic pandemics with variable mortality. in fatal cases of ph n infection, virus antigen expression occurred throughout the respiratory tract and was associated with both upper and lower respiratory tract disease [ ] . zoonotic influenza viruses, such as highly pathogenic avian influenza (hpai) h n virus, are sporadically transmitted from poultry and other animals to humans, but do not transmit efficiently from human to human [ ] . human infection with hpai h n virus involves primarily the lower respiratory tract, resulting in diffuse alveolar damage (dad) and a fatality rate of almost % in confirmed cases [ ] . the pathogenesis of both human and avian influenza virus infections in ferrets resembles that in humans [ ] . in part, this is because the distribution of receptors for human and avian influenza viruses in the respiratory tract of ferrets is similar to that in humans [ ] . therefore, the ferret is often used in animal models to study the pathogenesis of different influenza virus strains and to evaluate the efficacy of vaccines and antiviral agents against influenza [ , , , ] . in these studies, it is critical to collect respiratory tract samples for virological, pathological, and molecular analyses at both the appropriate time point after infection and the appropriate location along the respiratory tract. this is because influenza virus infection is a highly dynamic process, both temporally and spatially. we recently compared the pathogenesis of infections with seasonal human h n , ph n , and hpai h n virus in ferrets [ ] . our results showed that, at days post inoculation (dpi), ph n caused pneumonia intermediate in severity between that caused by seasonal h n and hpai h n . this was associated with virus replication throughout the lower respiratory tract for ph n , while seasonal h n replicated mainly in the bronchi, and hpai h n replicated mainly in the alveoli. however, the location of virus replication and extent and severity of associated pathological changes were recorded at a single time point. other experiments have used multiple time points (usually , , , and dpi) to study the dynamics of influenza virus infection in the ferret respiratory tract [ , , , , , , ] ; however, these experiments often lacked detailed pathological or virological analyses of samples collected along the full length of the respiratory tract on all time points. the goal of our study was to describe and compare the temporal and spatial dynamics of different influenza virus infections and associated pathology in the respiratory tract of the ferret. to this end, we inoculated ferrets with either seasonal human h n , ph n , or hpai h n virus, and performed detailed virological and pathological analyses at time points from . to dpi, as well as measuring virus excretion, clinical signs, and hematological parameters. additionally, we compared the results of histopathological analyses with digital microscopical scoring of tissue sections. clinical data and gross pathology. the survival rate was % in all groups. clinical signs (table ) were mild with sneezing from to dpi and nasal discharge from to dpi, as shown by increased licking on the nose. nasal and pharyngeal swabs revealed excretion of virus from . to dpi with a peak on dpi in the nasal swabs (figure a and b). the mean body weight loss was around % (figure d ). on gross pathology, a few dark red and raised areas were seen in the lungs of some animals and were consistent with mild pulmonary consolidation. estimated areas of lung affected ranged from to % (figure a ) between . and dpi. the relative lung weight was comparable to that of non-infected ferrets (figure b ). there was a mild splenomegaly from . to dpi (data not shown). the trachea-bronchial lymph nodes were slightly enlarged between . and dpi, with a peak on dpi (table ) . histopathology and virus antigen expression. by histopathology, there was a mild to severe multifocal rhinitis with necrosis of the epithelium and mild multifocal tracheitis. in the lungs, there was a mild multifocal bronchitis and bronchiolitis with intra-epithelial neutrophils, mild peribronchiolar and perivascular cuffing, mild broncho-adenitis, and mild multifocal alveolitis with mild intra-epithelial infiltration of neutrophils with mild epithelial necrosis and mild edema in the alveolar lumina. by immunohistochemistry, influenza virus antigen expression was visible as diffuse to granular red staining, which usually was stronger in the nucleus than in the cytoplasm ( figures , , , , , ) . antigen expression in the nose was present from . to dpi, while no antigen expression was seen in the tracheal, bronchial, bronchiolar, and tracheo-bronchial glandular epithelium at any time point, and little antigen expression in few type ii pneumocytes in the alveoli. changes in the histological lesions and antigen lowest titers for h n . (b) virus titers in pharyngeal swabs demonstrate initially comparable virus titers for ph n and h n and lower titers for h n , and on dpi comparable titers for ph n and h n and lower titers for h n . (c) body temperatures in ferrets inoculated with ph n and h n are higher than for h n and the sham-inoculated group. the large sem for h n is due to the low body temperatures of moribund animals. (d) body weight loss in animals inoculated with ph n and h n was comparable and higher when compared to h n . doi: . /journal.pone. .g expression over time are described in the supporting information (supporting information s ). semiquantitative histological scoring (table ) showed that the extent and severity of the alveolar lesions were comparable for all days of sampling with no obvious changes. no expression of virus antigen was seen in extra-respiratory tissues on any days. by digital scoring, there was on average to % of air in the pulmonary tissue (table ) . additionally, the percentage of antigen-expressing tissue and counts of virus antigen expression were absent and negligible, respectively ( table ) . virology of tissues. comparable with the pattern of antigen expression in the respiratory tissues, high virus titers were seen in the nasal concha from . to dpi with a peak on dpi (figure ) . low virus titers were present in the trachea, bronchi, lungs, tracheo-bronchial lymph nodes and tonsil ( figure and table ). all other tissues did not contain replication competent virus. hematology and comparison of leucocytes in blood and alveolar lumina. total leucocyte counts after infection were slightly increased on all days except for dpi compared to those in negative control animals (table ). in blood, there was a slight increase in the number of mononuclear cells on dpi and of neutrophils on dpi, followed by a mild decrease up to dpi after which again a small increase of both was observed ( figure ). in the alveolar lumina, the number of mononuclear cells was only slightly increased on dpi ( figure ). generalized linear model of viral excretion. we used generalized linear models (glm) to define possible predictors of viral excretion, based on viral production and damage scores in upper (nose and trachea) and deeper regions of the respiratory tract (bronchi, bronchioles and alveoli). these models were used to determine significant linear relationships between these scores and viral excretion (as measured by viral titers in nose and throat swabs; see methods). the final glm predicting excretion of h n included only viral production in the upper regions of the respiratory tract as a significant predictor (lr x = . , df = , p, . ). excretion of h n was a positive linear function of viral production in the upper regions ( table ). as such, viral excretion of h n could be directly estimated based on viral production scores in the nose and trachea, strongly suggesting that these regions were the main sources of excreted virus. clinical data and gross pathology. the survival rate was %. clinical signs per group were observed from to dpi ( table ) . the activity status varied over the different time points with the highest score (status ) seen in one animal between and dpi. dyspnea was seen only in this animal. inappetence, sneezing and nasal discharge was seen in all animals. virus excretion was shown in nasal and pharyngeal swabs with a peak on dpi and higher values in the nasal swabs. the virus titers for the pharyngeal swabs were comparable to those of the animals inoculated with h n ( figure b ). an increase in body temperature was seen with a peak on dpi (figure c ). the mean body weight loss was up to % on dpi (figure d ). by gross pathology, multifocal pulmonary consolidation was seen on . dpi with grey-red raised, and slightly firmer than normal areas. on dpi, the percentage of affected lung tissue was increased (figure a ). on dpi the lesions were dark red and firmer with increased relative lung weight. on dpi the percentage of affected lung tissue decreased again (figure a ). the relative lung weight was increased from to dpi and decreased on dpi (figure b ). the trachea-bronchial lymph nodes were enlarged from . to dpi, with a peak on dpi (table ). there was a mild splenomegaly from . to dpi (data not shown). histopathology and virus antigen expression. by histopathology, there was a mild to severe multifocal rhinitis with necrosis of the epithelium and mild to moderate multifocal tracheitis. in the lungs, there was a mild to severe multifocal bronchitis and bronchiolitis with intra-epithelial and intraluminal neutrophils, mild to severe necrosis of epithelium, mild to severe peribronchiolar and perivascular cuffing, mild to severe bronchoadenitis, and mild to severe multifocal alveolitis with infiltration of neutrophils in alveolar walls and lumina, mild to moderate epithelial necrosis and mild to moderate edema in the alveolar lumina with hypertrophy and hyperplasia of epithelium ( figures and ). the tracheo-bronchial lymph nodes and tonsils demonstrated lymphadenopathy, and in the palatine roof of two animals there was severe inflammation and necrosis of the sero-mucous glands. antigen expression in the nose was present from . to dpi starting on . dpi in the respiratory epithelium and on dpi in the olfactory epithelium. in the trachea there was antigen expression from . to dpi and in the bronchi and bronchioles from . to dpi with peak values on dpi. bronchial glandular (table ) . when digital scoring for antigen expression was performed, antigen expression as well as the number of positive counts showed that virus antigen was clearly present on . dpi with peak scores on dpi, albeit with high variation between the different animals (table ) . virology. in the respiratory tissues, the changes in virus titers over time showed a comparable pattern to that of viral antigen expression, with high virus titers from . to dpi ( figure ). in the extra-respiratory tissues, virus was isolated from the olfactory bulb, cerebellum, cerebrum, and heart (table ). hematology and comparison of leucocytes in blood and alveolar lumina. total leucocyte counts showed higher values on all time points when compared to those from negative control animals (table ). on dpi, there was an increase of neutrophils in the blood and in the alveoli. on dpi, there was a severe decrease in the number of neutrophils in the alveoli, but the number of neutrophils in the blood remained high. the number of mononuclear cells in the alveoli increased during the first days while the number of mononuclear cells in the blood decreased. on dpi, the numbers of neutrophils and mononuclear cells were decreased in both the blood and the alveoli. generalized linear model of viral excretion. similarly to h n , the final glm predicting excretion of ph n included only viral production in the upper regions of the respiratory tract (nose and trachea) as a significant predictor (lr x = . , df = , p, . ). excretion of ph n was a positive linear function of viral production in the upper regions (table ). clinical data and gross pathology. on . dpi, one animal died and one animal was euthanized because of its moribund state. on dpi, another animal died. from to dpi all animals developed severe clinical signs characterized by decreased activity, dyspnea, and inappetance (table ) . two animals showed nervous signs, characterized by ataxia, drifting to the right, walking into a corner and aggression. virus was excreted in nasal and pharyngeal swabs, with lower virus titers than the ph n and h n groups during the first days for the nose and the first days for the pharynx (figures a and b) . in contrast to what was seen in the other virus groups, the nasal swabs showed lower titers than the pharyngeal swabs. this indicated lower replication in the nose, as was also suggested by the low levels of virus antigen expression and low virus titers in the nose ( figure ). the increase in body temperature showed a peak on dpi. at later time points, the average temperature decreased below baseline values due to the progressively moribund state of most of the ferrets (figure c ). the body weight loss increased during time and was comparable to that of the ph n group ( figure d ). by gross pathology, there was multifocal pulmonary consolidation on . dpi with dark red, raised, and firmer than normal areas affecting an average of % of the lung tissue ( figure a ). on dpi, there was fluid in the bronchi and lung parenchyma, and the percentage of affected lung tissue was increased. on dpi, the percentage of affected lung tissue had increased dramatically to almost % ( figure a ). the relative lung weights were increased on all time points and were much higher than in the other virus groups (figure b ). tracheobronchial lymph nodes were enlarged starting on . dpi, and up to two times the normal size on dpi. there was a mild splenomegaly on all days (data not shown). histopathology and virus antigen expression. by histopathology, there was a mild to moderate multifocal rhinitis with necrosis of the epithelium and mild multifocal tracheitis. in the lungs, there was a moderate multifocal bronchitis and bronchiolitis, with intra-epithelial and intraluminal neutrophils, mild to moderate epithelial necrosis, and mild peribronchiolar and perivascular cuffing; a mild to severe broncho-adenitis; and a severe multifocal to diffuse alveolitis, with severe epithelial necrosis, intra-epithelial and intraluminal infiltration of neutrophils, severe intraluminal edema, and hypertrophy and hyperplasia of epithelial cells (figures , , , , ) . the tracheo-bronchial lymph nodes and tonsils demonstrated lymphadenopathy. in the extra-respiratory tissues there was lymphadenopathy in a sternal lymph node and there was hyperplasia and necrosis in the gut associated lymphoid tissue (galt) of the jejunum and in the spleen of few animals. in the nose there was antigen expression from to dpi with highest values on and dpi. antigen expression in the trachea was present from . to dpi, predominantly on dpi. the bronchi demonstrated antigen from to dpi, and in the bronchioles from . to dpi. antigen expression in the alveoli was present in type ii pneumocytes (figure ), and less in type i pneumocytes and alveolar macrophages. it was seen from . to dpi with large percentages from to dpi. in the tracheobronchial lymph nodes there was antigen expression from to dpi and in the tonsils on and dpi in mononuclear cells. one animal had virus antigen expression in squamous stratified epithelium in the tip of the nose and one animal showed expression in endothelial cells in the pharynx. in the extrarespiratory tissues there was virus antigen expression in mononuclear cells in the galt of the jejunum on and dpi and in the spleen on dpi. no antigen expression was seen in other extrarespiratory tissues. changes in the histological lesions and antigen expression over time are described in the supporting information (supporting information s ). digital scoring demonstrated that the amount of air-containing tissue was lower than in the other virus groups, and decreased over time (table ) . digital scoring for antigen expression showed positive values on all time points with highest values on dpi, but with high variation ( table ) . virology of tissues. in the nose, the virus titers were higher than expected based on virus antigen expression in the nose, indicating a different origin of the virus ( figure ). in the lungs, virus titers on dpi were higher than on the other days. this could be due to slower replication on . dpi and necrosis of virusreplicating cells on dpi. in the extra-respiratory tissues, virus was isolated from the olfactory bulb, cerebrum, cerebellum, heart, spleen, liver, kidney, adrenal gland, pancreas, and jejunum in one or more animals from . to dpi. the highest virus titers were seen on dpi in olfactory bulb and jejunum, and on and dpi in the spleen (table ) . hematology and comparison of leucocytes in blood and alveolar lumina. the total leucocyte counts were decreased from to dpi ( table ). the number of neutrophils in the alveolar lumina was low on . dpi but increased strongly with a peak value on dpi. however, in the blood, after a small peak on dpi, there was a decrease of the number of neutrophils ( figure ). the number of mononuclear cells in the alveolar lumina showed an increase on dpi. this increase was later than in the ph n group, while the number of mononuclear cells in the blood did not increase. the number of lymphocytes in the blood was decreased on all days (table ). generalized linear model of viral excretion. the final glm predicting excretion of the h n included significant predictors (lr x = . , df = , p = . ): viral production and damage in the deeper regions of the respiratory tract (bronchi, bronchioles and alveoli; p = . and p = . , respectively); their product (p = . ); damage in the upper regions of the respiratory tract (nose and trachea; p = . ); its product with viral production in these regions (p = . ); and its product with viral production in the deeper regions (p = . ). excretion of h n was a negative linear function of each of the three single predictors and a positive linear function of each of the three products. coefficients are given in table . clinical signs and gross pathology. no clinical signs were seen and there was a survival rate of %. the body weight loss in the control group increased slightly in time ( figure d ). by gross pathology, the lungs had few dark red and slightly raised areas consistent with mild pulmonary consolidation. the area of affected lung tissue varied between and % of the lung area (figure a) . the relative lung weight remained constant at around . % on all time points (figure b ). tracheo-bronchial lymph nodes were slightly enlarged (table ) and mild splenomegaly was present on all time points (data not shown). histopathology and virus antigen expression. by histopathology, there was mild multifocal rhinitis, tracheitis, bronchitis and bronchiolitis with few neutrophils, mild perivascular and peribronchiolar cuffing, mild broncho-adenitis, and mild focal alveolitis with infiltration of few neutrophils in the alveolar septa. no virus antigen expression was seen in any of the tissues. changes in the histological lesions over time are described in the supporting information (supporting information s ). digital scoring demonstrated that the amount of air-containing tissue was slightly lower on and dpi ( table ) . virology. all swabs and tissues were negative in virus titration for influenza virus. hematology and comparison of leucocytes in blood and alveolar lumina. no trends were observed in total leucocyte, segmented neutrophil, mononuclear cell and lymphocyte counts in the blood, or in comparison of leucocyte counts between blood and alveolar lumina (figure and table ). our time course experiments show how strongly the spatial and temporal dynamics of infection and associated lesions in the ferret respiratory tract differ between a seasonal human influenza virus (h n ), a pandemic human influenza virus (ph n ), and a zoonotic avian influenza virus (h n ): h n infection was restricted to the nose and peaked at dpi; ph n infection also peaked at dpi, but occurred at similar levels throughout the respiratory tract; and h n infection occurred predominantly in the alveoli, where it peaked for a longer period, from to dpi ( figure ). the associated lesions followed the same spatial distribution as virus infection, but their severity peaked between and days later ( table ) : at to dpi for h n and ph n , after which lesions decreased in severity by dpi; and at dpi for h n , when all h n -infected ferrets had either died or been euthanized on humane grounds. an important implication of these results is that location and timing of sample collection need to be chosen carefully in any comparative study of infection and pathology by different influenza viruses, otherwise the comparison will not be valid. the main source of excreted virus likely differs between h n and h n infections, based on comparison of virus titers in nasal and pharyngeal swabs ( figure ) with virus antigen expression in respiratory tract tissues ( figure ). for h n , the temporal dynamics of nasal swab virus titers, pharyngeal swab virus titers, and virus antigen expression in the nose were comparable. together with the lack of virus antigen expression in the lower respiratory tract, these results suggest that the nose is the main source of excreted h n . this is supported by the final glm for h n viral excretion, which included only viral production in the upper regions of the respiratory tract as significant predictor. in contrast, for h n , virus titers in both nasal swabs and pharyngeal swabs were already near peak levels at dpi, while virus antigen expression in the nose remained relatively low until dpi. together with the higher levels of virus antigen expression in the trachea, bronchioles, and especially alveoli at to dpi, these results suggest that the lower respiratory tract is the main source of excreted h n , at least until dpi. in fact, the final glm for there is high antigen expression with associated lesions characterized by bronchiolitis in bronchiolar epithelium of ferrets inoculated with ph n on dpi that was low on dpi, consistent with severe loss of bronchiolar epithelium, and for h n there was no expression on dpi and moderate antigen expression with associated lesions on dpi. there were no antigen expression and no lesions for h n . he and immunoperoxidase counterstained with hematoxylin, . doi: . /journal.pone. .g h n viral excretion suggests that viral production and damage in the deeper regions of the respiratory tract (bronchi, bronchioles and alveoli) may have a strong negative impact on viral excretion. this may be due to physical obstruction of the deeper airways due to damage caused by h n virus infection and immune responses. for ph n , it is more difficult to determine the main source of excreted virus, because virus antigen expression was high at all levels of the respiratory tract. however, the final glm for ph n viral excretion revealed that only the viral production in the nose and trachea was a significant predictor of viral excretion, suggesting that as for the h n virus, the upper regions of the respiratory tract may be the main sources of excreted virus. the pattern of h n and h n antigen expression in the ferret respiratory tract corresponded only in part with the pattern of attachment for these viruses (for the ph n virus, the pattern of attachment in ferrets has not been determined). attachment of an influenza virus to a host cell is the first step in the virus replication cycle and is considered to be necessary, but not sufficient, for subsequent infection of that cell. previously, we found that h n attached to many tracheal epithelial cells (predominantly ciliated cells), rare or few bronchial and bronchiolar epithelial cells, and a moderate number of alveolar epithelial cells (predominantly type i pneumocytes, which have a low metabolism) in the lower respiratory tract of the ferret [ ] . however, in the current study, we found very little h n infection (based on virus antigen expression) in any cell types of the ferret lower respiratory tract ( figure ). this suggests that other factors were necessary for h n to infect these cell types. these factors may be the virus load that may need to be higher for infection with h n and the influence of surfactant that protect against influenza in humans and pigs [ ] . we previously found that h n did not attach to tracheal or bronchial epithelial cells, and attached to rare or few bronchiolar epithelial cells and a moderate number of alveolar epithelial cells (predominantly type ii pneumocytes, which have a high metabolism) in the lower respiratory tract of the ferret [ ] . in the current study, we found abundant h n infection in alveolar and bronchiolar epithelial cells, which fits with the pattern of virus attachment ( figure ). however, we also found h n infection in tracheal and bronchial epithelial cells, which does not. next to the fact that this could be related to a different h n strain (a/ vietnam/ / [ ] , while in the present experiment we used a/indonesia/ / ), the replication of h n in alveolar and bronchiolar epithelial cells was so abundant that tracheal and bronchial epithelial cells became infected despite the relatively weak attachment of h n to the latter cell types. different parameters were used to measure the severity of lower respiratory tract disease in our ferrets. of these parameters, relative lung weight and affected lung tissue allowed the best quantitative distinction between the virus groups: they showed clear differences between virus groups from dpi onwards (table ) , with relatively little within-group variation (figure ). these parameters have been used with success before for studies on severe acute respiratory syndrome [ ] and on influenza [ ] . immunohistochemistry score for virus antigen expression in the table . histopathology scores in ferrets inoculated with different influenza viruses. (table ) , alveolar edema score, and alveolar hemorrhage score (table ) also showed clear differences between the virus groups. however, between-group differences differed according to the dpi, and within-group variation was large. comparing the histopathology scoring for extent and severity of alveolitis and alveolar damage with the digital scoring for aircontaining space in pulmonary tissue showed that the latter demonstrated less statistically significant results, and therefore is less useful for discriminating between viruses (table ) . comparing the antigen expression scoring in the alveoli by hand with the digital scoring and the virus titers, the p-values are similar and allow distinction between the virus groups ( table ) . the other parameters did not allow a clear distinction between the three virus groups. besides quantitative differences in severity of lower respiratory tract disease, the three virus infections also showed clear differences in the qualitative character of lower respiratory tract disease, related to the tropism of the virus and the ability of the host to counter infection. this is particularly well illustrated in the alveoli. for the mildest disease, caused by h n infection, there was rare evidence of virus infection in the alveolar epithelium ( figure ). there was a mild acute inflammatory response with small numbers of neutrophils and macrophages, peaking at dpi. damage to the alveolar epithelium was so mild that there was no visible evidence of epithelial cell hypertrophy or hyperplasia to reepithelialize the alveolar walls (table ) . for the intermediate disease, caused by ph n infection, virus infection already was high at . dpi and peaked soon afterwards at dpi (figure ). there was a moderate acute inflammatory response characterized by influx of neutrophils, peaking at dpi (figure ). at the same time, there was evidence of damage to the alveolar wall, characterized by necrosis of the epithelium and edema and hemorrhage in the alveolar lumina, which was maximal at to dpi (table ) . after dpi, the alveolar lesion progressed to a more chronic inflammatory response, with neutrophils being replaced by mononuclear cells, which peaked at dpi (figure ). at the same time, repair of the alveolar epithelial damage was visible as hypertrophy and hyperplasia of type ii pneumocytes, which was maximal at dpi. during this period, the level of virus infection decreased to a low level. by dpi the damage to and inflammation of the alveoli was completely resolved, except for a remnant of mononuclear cells in table . digital histopathology scores in ferrets inoculated with different influenza viruses. table . virus isolation from tissues in ferrets inoculated with different influenza viruses. the alveolar lumina, and virus infection was no longer detectable. the resolution of edema and hemorrhage could be explained on the one hand by the action of alveolar macrophages [ ] , and on the other hand by re-epithelialization of the alveolar walls [ ] . for the most severe disease, caused by h n infection, the virus infection reached about the same level as that by ph n infection at dpi but remained high until death or euthanasia at dpi, indicating that the host was not able to control virus infection. there was concurrent alveolar damage, which was so severe and extensive that all ferrets either died or had to be euthanized on humane grounds by dpi. this was despite clear attempts at reepithelialization, based on prominent type ii pneumocyte hypertrophy and hyperplasia from dpi onwards (table ). there was a marked acute inflammatory response characterized by neutrophil influx peaking at dpi but much greater than in ph n infection, with a neutrophil count that was nearly twice as high (figure ). this could be explained by a higher induction of pro-inflammatory cytokine production (e.g., il- and il- ) and no production due to h n infection [ , ] . maines et al. [ ] demonstrated a relatively high production of il- and il- in the lower respiratory tract of ferrets infected with h n , but not with ph n . similarly, humans infected with h n had high levels of il- and il- in the blood [ ] . another major difference with ph n infection was the low influx of mononuclear cells, with a mononuclear cell count of less than half that in ph n infection by dpi, suggesting that the inflammatory response never progressed beyond the acute stage. comparison of the dynamics of neutrophil and mononuclear cell influx in the alveolar lumina with neutrophil and monocyte counts in peripheral blood helps to explain the differences in hematological dynamics among the three virus infections (figure ). for h n infection, the slight and transient increase in the numbers of neutrophils and monocytes in the blood at dpi correlates with the mild influx of these cell types into the alveolar lumina at that time. this corresponds with a low demand for neutrophils and monocytes during h n infection. for ph n infection, the marked increase in first the number of neutrophils and then of monocytes in the blood correlates with the moderate influx of first neutrophils and then mononuclear cells in the alveolar lumina. this corresponds with increased demand for both cell types, both of which can be met adequately by the myelopoietic compartment. neutrophils and monocytes are back to normal levels in the blood by dpi, indicating resolution of the respiratory tract inflammation. for h n infection, the initial increase of neutrophils in the blood at dpi correlates with a massive influx of neutrophils into the alveolar lumina. the steep decline in neutrophils in the blood on subsequent days, despite continued high influx in alveolar lumina, corresponds to a demand that has outstripped the available supply by the myelopoietic compartment. this is corroborated by the presence of immature neutrophils with rod-shaped nuclei in the blood from dpi onwards ( table ). the lack of increase in monocytes in the blood corresponds with the failure of the acute inflammatory response in the lungs to transform into a more chronic inflammatory response. in addition to changes in neutrophil and monocyte counts, there was an overall leucopenia from dpi (table ) . such a leucopenia has been described previously for h n infection in both humans and ferrets [ , , , ] , whereas ferrets infected with ph n showed higher leucocyte counts [ ] . together, this comparison between inflammatory cell dynamics in alveoli and blood provides insight into the interpretation of hematological analyses of influenza pneumonia. besides the respiratory tract, there was evidence, by virus isolation, of extra-respiratory spread for h n and ph n , but not for h n ( table ). the failure to detect virus antigen in any of these virus-isolation-positive tissues except jejunum may be because virus isolation is more sensitive than immunohistochemistry, or because virus was present but not replicating in these tissues. previously, h n virus antigen expression in extrarespiratory tissue has been found in the central nervous system table . leucocyte concentrations in the peripheral blood of ferrets inoculated with different influenza viruses. and liver of experimentally inoculated ferrets [ , , ] . in our experiment, we additionally found virus antigen in other extrarespiratory tissues: for ph n in sero-mucous glandular epithelial cells in the palatine roof, and for h n in mononuclear cells in tracheo-bronchial lymph nodes, tonsils, sternal lymph node, spleen, and galt of the jejunum, and in squamous stratified epithelium on the tip of the nose. the implication of these sites of replication for the pathogenesis and transmission of these viruses is yet to be elucidated. the extensive h n infection in extra-respiratory tissues known from previous studies [ , , , ] likely occurs by spread from the respiratory tract via the blood [ , , ] . however, the expression of h n antigen in olfactory mucosa ( figure ) together with high virus titres in the olfactory bulb (table ) suggest that h n may reach the central nervous system from the nasal cavity via the olfactory route, as demonstrated recently by schrauwen et al. [ ] . this h n infection of the central nervous system resulted in clinical disease, judging by the presentation of neurological signs (table ). in addition to extra-respiratory spread of h n , there also was evidence of ph n in extrarespiratory tissues, especially the central nervous system (table ) , and ph n expression in olfactory mucosa ( figure ). this suggests that ph n also may be neurotropic and enter the central nervous system via the olfactory route. animals were housed and experiments were conducted in strict compliance with european guidelines (eu directive on animal testing / /eec) and dutch legislation (experiments on animals act, ). the protocol was approved by the independent animal experimentation ethical review committee of the netherlands vaccine institute (permit number ) and was performed under animal biosafety level conditions. animal welfare was observed on a daily basis, and all animal handling was performed under light anesthesia using a mixture of ketamine and medetomidine to minimize animal suffering. after handling atipamezole was administered to antagonize the effect of medetomidine. three viruses were used: seasonal h n virus (a/netherlands/ / ), isolated from a patient during the influenza season [ ] ; pandemic (h n ) virus (ph n ) (a/netherlands/ / ), isolated from a specimen of a human patient who had recently visited mexico during the pandemic in [ ] ; and hpai h n virus (a/indonesia/ / ) as described earlier [ ] . the h n virus was chosen as a representative from a recent seasonal h n influenza epidemic. like virtually all recent h n viruses the present virus exhibits the oseltamivirsensitive neuraminidase-associated agglutination of turkey erythrocytes [ ] . the ph n virus which had been initially isolated in embryonated chicken eggs was chosen as a representative virus from the pandemic because it has been used in previous experiments by others [ ] and ourselves [ , , ] . the h n figure . histopathologic changes in the alveoli during time in ferrets inoculated with ph n . on . dpi there was mild dad that was characterized by neutrophils in the alveolar walls. on dpi the alveolar walls were thickened with mild necrosis of the lining epithelium and increased number of neutrophils, in the lumina there were more mononuclear cells, little amount of edema fluid mixed with fibrin, hemorrhage and little cellular debris. on dpi there was increased presence of necrosis, edema, hemorrhage and hyperplasia. on dpi there were both new lesions with acute necrosis and old lesions with type ii hyperplasia present, with increased amounts of edema and hemorrhage. on dpi the type ii hyperplasia and hypertrophy was more pronounced with increased amount of edema and high numbers of mononuclear cells. on dpi there was severe epithelial hyperplasia and hypertrophy as well as intraluminal edema and hemorrhage. on dpi the walls were mildly thickened. he, (first column), (second column). doi: . /journal.pone. .g virus was chosen as a representative of clade (subclade . . ) of hpai h n virus, and has been used in several previous experiments [ , ] . the different isolates were passaged three times in madin-darby canine kidney (mdck) cells and titrated according to standard methods. subsequently, the viruses were clarified and reached an infectious virus titer of . median tissue culture infectious dose (tcid ) per ml for h n virus, and . tcid for both ph n and h n virus [ , ] .the inoculum for the control group was prepared as follows: mdck cells were grown up to a monolayer of to % in two cm flasks (a and b). virus medium (emem supplemented with hepes, sodium bicarbonate, bsa fraction v, l-glutamin, penicillin, streptomycin, trypsin and amphothericin-b) was added and the cells were incubated for days at uc. to mimic cellular damage in mdck cells during virus infection when preparing the virus stock we aimed to reach a cytopathic effect (cpe) of %. therefore, the cells in bottle b were collected by disrupting the monolayer with mm glass beads (vwr, amsterdam, the netherlands), sonication ( times seconds in melting ice) and freezing at uc, leading to lysis of all cells. the cells in bottle a were not damaged and the final inoculum consisted for % of the supernatant of bottle a and for % of the supernatant of bottle b. for every virus and the sham inoculated group, groups ( groups for h n ) of four ferrets were inoculated under anesthesia with ketamine ( - mg/kg; nimatek, eurovet animal health b.v., bladel, the netherlands) and medetomidine hydrochloride ( . mg/kg; domitor, orion pharma, espoo, finland) with each of these three viruses with tcid in a -ml volume intratracheally and in a . -ml volume intranasally evenly divided over the two nostrils. intratracheal and intranasal inoculation was performed to ensure that the inocula would reach both the lower and the upper respiratory tract [ , ] . after inoculation, the ferrets received atipamezole hydrochloride ( . mg/kg antisedan; orion pharma, espoo finland) and were monitored daily for clinical signs until maximally dpi [ ] . the animals were predestined to be sacrificed at hours ( . day), , , , , and dpi (for h n not at and dpi) in order to avoid any bias that could follow from clinical observation, or earlier when they were moribund before the selected time point of euthanasia (h n only). the animals were euthanized by exsanguination after anesthesia with ketamine. at euthanasia, body weight was measured, nose, throat and rectal swabs were collected, blood was taken, and samples were taken from both respiratory and extrarespiratory tissues for virological, pathological, and immunohistochemical analyses. the inoculum used for the sham control group induced comparable body weight loss, affected lung tissue, histological scores, digital scoring, and leucocyte counts as the h n group. as an extra comparison, we therefore used non-inoculated healthy ferrets with the same age and background as the other ferrets as negative control animals to score and compare the above parameters. ferrets were used in this experiment since they resemble disease in humans when infected with influenza a viruses [ , ] . hundredand-four eleven-month-old purpose-bred ferrets, seronegative for antibodies against circulating influenza viruses h n (a/bris- figure . histopathologic changes in the bronchioles during time in ferrets inoculated with ph n . on . dpi there was infiltration of neutrophils in the bronchiolar epithelium and lumen with mild peribronchiolar cuffing. on dpi the number of neutrophils increased with necrosis of the epithelium, intraluminal cellular debris and peribronchiolar cuffing. on dpi the necrosis was more severe than on dpi. on dpi there was denudation of the basement membrane and little regeneration of the bronchiolar epithelium with hyperplasia, peribronchiolar cuffing and occasionally cellular debris in the lumen. on dpi there was increased epithelial hyperplasia with moderate cuffing. on dpi the epithelium covered the basement membrane of the bronchiole with hypertrophic cells and moderate clinical scores in all groups were assessed every day. activity status was scored as follows: , alert and playful; , alert and playful only when stimulated; , alert but not playful when stimulated; , neither alert nor playful when stimulated. for diarrhea, sneezing, nasal and conjunctival discharge, inappetence and dyspnea we scored: , not present; , present [ ] . inappetence was measured by the amount of food that was still present in the cages at the time of feeding. as a control we also assessed the amount of food that was present in the stomach and intestine of the animals on the day of necropsy. dyspnea was characterized by open-mouth breathing with exaggerated abdominal movement. additionally, we used four eleven-month-old purpose-bred ferrets, seronegative for antibodies against circulat- aleutian disease virus. the four ferrets were euthanized immediately by the same method as described above and necropsied to provide control data of non-inoculated ferrets, and blood was taken blood for hematologic analyses and respiratory tract tissues for histopathological scoring. for every virus four animals per time point were euthanized by exsanguination under ketamine/medetomidine anesthesia at hours ( . day), , , , , or dpi and were necropsied according to a standard protocol. the trachea was clamped off to prevent the lungs from deflating upon opening the thoracic cavity, allowing visual estimation of the area of affected lung parenchyma. the lungs were weighed and the relative lung weight was calculated by the following formula: (lung weight on day of death/body weight on day of death)* , and presented as percentage. the following tissues were collected for histological examination: left lung, left nasal concha, nasal septum, larynx, trachea, bronchus, tracheo-bronchial lymph node, left tonsil, heart, liver, spleen, kidney, pancreas, duodenum, jejunum, colon, adrenal gland, left olfactory bulb and left brain (cerebrum and cerebellum). lung samples were taken in a standardized way, not guided by changes as seen in the gross pathology. tissues were stored in % neutral-buffered formalin (lungs after careful inflation with formalin), embedded in paraffin, sectioned at mm, and stained with hematoxylin and eosin (he) for examination by light microscopy. semiquantitative assessment of influenza virus-associated inflammation in the lung (four slides with longitudinal section or cross-section of cranial or caudal lobes per animal) was performed on every slide as reported earlier [ ]: for the extent of alveolitis and alveolar damage we used: , %; , - %; , - %; , . %. for the severity of alveolitis, bronchiolitis, bronchitis, and tracheitis we scored: , no inflammatory cells; , few inflammatory cells; , moderate numbers of inflammatory cells; , many inflammatory cells. for the presence of alveolar edema, alveolar hemorrhage, and type ii pneumocyte hyperplasia we scored: , no; , yes. finally, for the extent of peribronchial, peribronchiolar, and perivascular infiltrates we scored: , none; , one to two cells thick; , three to ten cells thick; , more than ten cells thick. slides were examined without knowledge of the treatment allocation of the animals. the cumulative scores for size and severity of inflammation of all slides provided the total score per animal. to assess the number of neutrophils and alveolar macrophages in the alveolar lumina and the number of neutrophils in the alveolar walls, we counted them in arbitrarily chosen objective fields per he-stained slide, with a total of fields per animal. neutrophils were identified on the basis of their size (approximately to mm in diameter) and the morphology of their nucleus (heterochromatic and segmented, with to lobes joined by thin strands). neutrophils in the alveolar walls were counted when present in small capillaries, but were excluded if present in larger blood vessels. pulmonary alveolar macrophages were identified on the basis of their morphology and location: large, oval to round cells with a distinct cell border, foamy cytoplasm and large, oval to bean-shaped nucleus, located in the alveolar lumen separate from the alveolar wall [ ] . for detection of influenza a virus antigen, tissues were stained with a primary antibody against the influenza a nucleoprotein as described previously [ ] . in each staining procedure, an isotype control was included as a negative control and a lung section from a cat infected experimentally with h n was used as positive control [ ] . semiquantitative assessment of influenza virus antigen expression in the lungs was performed as reported earlier [ ] : for the alveoli, twenty-five arbitrarily chosen, objective, fields of lung parenchyma of four lung sections were examined by light microscopy for the presence of influenza virus nucleoprotein, without the knowledge of the identity of the animals. the cumulative scores for each animal were presented as number of positive fields per fields. for the nose, trachea, bronchi and bronchioles, the percentage of positively staining epithelium was estimated on every slide and the average of the four slides was taken to provide the score per animal: , %; , - %; , - %; , . %. for computerized scoring of slides we used the cross-section of the left caudal lung of all animals to make a digital scan using the nanozoomer with accompanying software (nanozoomer digital pathology and ndp.scan and ndp.view, hamamatsu, higashiku, hamamatsu city, japan). of every scan, pictures of the alveoli were made in a randomized order with the objective. from every picture, we calculated: the quantity of tissue (visualized in blue staining) and the quantity of red staining consistent with virus antigen expression using zeiss ks version . image analysing system (carl zeiss vision gmbh, eching, germany). the images were pixels with . mm per pixel. the percentage of air-containing space present in the pulmonary tissue was calculated as % minus the total percentage of pulmonary tissue (blue staining) that was present in the pictures. the quantity of virus replication was calculated either as the percentage of antigen expression (red staining) relative to quantity of tissue (blue staining), or as the number of times red staining was detected in a picture. after collection on day and the day of euthanasia, nose, pharyngeal and rectal swabs were collected in virus transport medium (emem containing bovine serum albumin (fraction v), penicillin, streptomycin, amphothericin-b, l-glutamine, sodium bicarbonate and hepes), aliquotted and stored at uc. upon necropsy, samples were collected from the following tissues: nasal concha, trachea, bronchus, tracheo-bronchial lymph node, tonsil, heart, liver, spleen, kidney, pancreas, duodenum, jejunum, colon, adrenal gland, olfactory bulb and brain. specifically from the lungs, sections of the cranial, median and caudal lobe of the right lung and of the accessory lobe from each animal were collected and pooled (total average weight of about . - . g/animal); lung samples were taken in a standardized way, not guided by changes as seen in the gross pathology. tissue samples were homogenized with a fastprep- (mp biomedicals, eindhoven, the netherlands) in influenza infection medium (emem containing bovine serum albumin (fraction v), penicillin, streptomycin, amphothericin-b, l-glutamine, sodiumbicarbonate, hepes and trypsin) and centrifuged briefly before titration. quadruplicate -fold serial dilutions of tissue and swab supernatants were used to determine the virus titers in confluent layers of mdck cells as described previously [ ] . on day and the day of euthanasia ( . , , , , , or dpi or when an animal was sacrificed due to a moribund state) blood was collected from the euthanized animals. total leucocyte counts were determined in blood collected in edta vacutainer tubes (vacuette, greiner bio-one gmbh, kremsmünster, austria) using an automated hematology analyser sysmex poch- i (sysmex europe gmbh, norderstedt, germany). thin blood films were prepared from edta blood and stained with may-grünwald-giemsa (merck, darmstadt, germany). differential cell counts were obtained by counting cells per slide, and the numbers of lymphocytes, mononuclear cells, blastocytes, rodshaped neutrophils, segmented neutrophils, eosinophils, basophils and normoblasts were calculated by multiplying these percentages by the leucocyte counts obtained for the same sample. by using the total leucocyte counts we calculated the absolute number of different leucocytes in /l. the non-parametric mann-whitney test was used to compare several parameters of the different virus infection per day for; percentage of affected lung tissue, relative lung weight, extent and severity of alveolitis and alveolar damage, percentage of air containing space in pulmonary tissue, virus titers in lung tissue, antigen expression in the alveoli by immunohistochemistry, percentage of antigen expression in the alveoli by digital scoring, and number of antigen expression counts in the alveoli by digital scoring. the outcomes were considered significant when p, . . generalized linear models (glm) were used to determine the most appropriate predictors of viral excretion for each virus, based on viral production and damage in the respiratory tract. these models were used to determine the linear equations of the form y = g(a i x i +b i ) that best fit the data, where y is viral excretion, x i is a predictor, and a i and b i are the predictor and intercept coefficients, respectively. viral excretion was measured as the sum of nasal swab and pharyngeal swab viral titers. predictors initially included in the models were measures of viral production and measures of damage in upper (nose and trachea) and deeper regions of the respiratory tract (bronchi, bronchioles and alveoli), as well as all two-ways interactions. measures of viral production for each region were calculated as the product of the viral titers and immunohistochemistry scores in the respective regions. this was done to take into account the fact that virus isolated in a particular region of the respiratory tract may not be produced locally. measures of damage for each region were calculated as the sum of the severity scores in the respective regions. the severity scores were used because they were similarly assessed in all regions of the respiratory tract. only predictors or interactions of predictors with p, . were retained in the final glm. supporting information s detailed description per virus of histopathologic changes and antigen expression by immunohistochemistry. (doc) pathology of human influenza revisited comparison of the pathology caused by h n , h n , and h n influenza viruses the pathology of influenza virus infections number of human h n cases comparative pathology of select agent influenza a virus infections human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals the ferret: an animal model to study influenza virus efficacy of vaccination with different combinations of mf -adjuvanted and non-adjuvanted seasonal and pandemic influenza vaccines against pandemic h n ( ) influenza in ferrets local innate immune responses and influenza virus transmission and virulence in ferrets pathogenesis of avian influenza a (h n ) viruses in ferrets pathogenesis and transmission of triple-reassortant swine h n influenza viruses isolated before the h n pandemic modeling host responses in ferrets during a/california/ / influenza infection course of seasonal influenza a/brisbane/ / h n infection 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infection fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia avian influenza a (h n ) infection in eastern turkey in update on avian influenza a (h n ) virus infection in humans avian influenza (h n ) viruses isolated from humans in asia in exhibit increased virulence in mammals inefficient transmission of h n influenza viruses in a ferret contact model pathogenesis of influenza a/h n virus infection in ferrets differs between intranasal and intratracheal routes of inoculation lethality to ferrets of h n influenza viruses isolated from humans and poultry in depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h n ) virus isolated from humans influenza a virus (h n ) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts the multibasic cleavage site in h n virus is critical for systemic spread along the olfactory and hematogenous routes in ferrets modification of the ferret model for pneumonia from seasonal human influenza a virus infection pathogenesis and transmission of swine-origin a(h n ) influenza virus in ferrets neuraminidase receptor binding variants of human influenza a(h n ) viruses resulting from substitution of aspartic acid in the catalytic site: a role in virus attachment? in vitro and in vivo characterization of new swine-origin h n influenza viruses pandemic h n vaccine requires the use of an adjuvant to protect against challenge in naive ferrets comparison of rna hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro textbook of veterinary histology. philadelphia: lea & febiger avian h n influenza in cats we thank c. van hagen, r. boom, w. van aert, r. van lavieren, l. leijten, p. van run, e. veldhuis kroeze, d. van riel, d. van de vijver, and t. bestebroer for technical assistance, a. gomersbach and w. vos for biotechnical assistance, f. van der panne for figure preparation, a. nigg for assistance with the digital scoring and m. van leeuwen (uvdl, utrecht) for reading the thin blood slides. key: cord- -g onwhg authors: tahir ul qamar, muhammad; shahid, farah; aslam, sadia; ashfaq, usman ali; aslam, sidra; fatima, israr; fareed, muhammad mazhar; zohaib, ali; chen, ling-ling title: reverse vaccinology assisted designing of multiepitope-based subunit vaccine against sars-cov- date: - - journal: infect dis poverty doi: . /s - - -w sha: doc_id: cord_uid: g onwhg background: coronavirus disease (covid- ) linked with severe acute respiratory syndrome coronavirus (sars-cov- ) cause severe illness and life-threatening pneumonia in humans. the current covid- pandemic demands an effective vaccine to acquire protection against the infection. therefore, the present study was aimed to design a multiepitope-based subunit vaccine (mesv) against covid- . methods: structural proteins (surface glycoprotein, envelope protein, and membrane glycoprotein) of sars-cov- are responsible for its prime functions. sequences of proteins were downloaded from genbank and several immunoinformatics coupled with computational approaches were employed to forecast b- and t- cell epitopes from the sars-cov- highly antigenic structural proteins to design an effective mesv. results: predicted epitopes suggested high antigenicity, conserveness, substantial interactions with the human leukocyte antigen (hla) binding alleles, and collective global population coverage of . %. taken together, amino acids long mesv was designed by connecting cytotoxic t lymphocytes (ctl), helper t lymphocyte (htl) and b-cell epitopes with suitable adjuvant and linkers. the mesv construct was non-allergenic, stable, and highly antigenic. molecular docking showed a stable and high binding affinity of mesv with human pathogenic toll-like receptors- (tlr ). furthermore, in silico immune simulation revealed significant immunogenic response of mesv. finally, mev codons were optimized for its in silico cloning into the escherichia coli k- system, to ensure its increased expression. conclusion: the mesv developed in this study is capable of generating immune response against covid- . therefore, if designed mesv further investigated experimentally, it would be an effective vaccine candidate against sars-cov- to control and prevent covid- . viruses have the potential to become dangerous life threat and cause irreparable loss to human beings. hardly the world learns to cope with one strain of virus when another emerges and poses a threat to the future of humanity. a similar situation has emerged when a new strain of novel coronavirus (cov) that has not been previously identified in humans reported in december, [ , ] . coronaviruses are the largest among rna viruses belonging to coronaviridae, roniviridae and arteriviridae families. coronaviridae are unsegmented, ′ polyadenylated and ′ capped positive sense singlestranded rna viruses cause various respiratory diseases in humans [ , ] . covs are classified into four classes: alpha, beta, delta, and gamma. amongst them, beta and alpha covs have been reported for infecting humans [ ] . recent cov strain has received tremendous attention from researchers, as it causes a global pandemic of coronavirus disease (covid- ) [ ] . severe acute respiratory syndrome coronavirus (sars-cov- ) was identified as the causative agent of this pandemic [ ] . the study of genome sequences has cast a shadow that sars-cov- is closely related to the sars-cov which is the causative agent of the sars disease in / [ ] . initial diagnostic procedures indicated that the sars-cov- is primarily spread through respiratory droplets from sneezing/coughing, body contact and to some extent through fecal contact [ ] . the sars-cov- may show symptoms within days after exposure, or in some cases it takes more than days. symptoms of patients infected with covid- include fever, runny nose, cough, and dyspnea [ ] . although the entire genome sequence of the virus has been published, the origin and proliferation mechanism of the new coronavirus is still ambiguous as stated by the world health organization [ ] . initial reports claimed that bats, snakes, pangolins or civet could be a possible animal source, but the claims are under debate and needs substantial research to prove it [ , [ ] [ ] [ ] . researchers are currently working to sort out the sars-cov- source, including possible intermediate animal vectors. the samples taken from a respiratory system-throat swab or lung fluid are helpful in diagnosing its infection in patients [ ] . a special clinical diagnostic reverse transcription-pcr based test was developed [ ] . over clinical trials are currently underway to test new and repurposed compounds against sars-cov- [ , ] . several medications such as hydroxychloroquine, remedesivir, and dexamethasone are being tested in clinical trials [ ] [ ] [ ] [ ] . several vaccines including subunit vaccines [ , ] , nanoparticle based vaccines, viral vector vaccines (adenovirus vector, ankara vector), inactivated vaccines, fusion-protein based vaccines, recombinant protein, dna vaccines, and live-attenuated vaccines are also being developed and in pre-clinical trials, but these vaccines are long months away from the market [ ] [ ] [ ] [ ] [ ] . immunoinformatics approaches can be applied to examine viral antigens, prediction of its epitopes and assessment of its immunogenicity [ , ] . moreover, this approach could be both time and cost-effective [ , , ] . excessive respiratory infection can also resolve with t-cell reactions and antibodies [ ] . furthermore, rapid identification, isolation, disease prevention, and control measures are required to hinder its spread of sars-cov- at homes, communities and healthcare units [ , ] . in various studies, therapeutic approaches against the ebola virus, zika virus and middle east respiratory syndrome corona virus (mers-cov) were developed using immunoinformatics approaches [ , , ] . the purpose of this study was to pinpoint the potential t-cell and b-cell epitopes from sars-cov- structural proteins which can be further joined through adjuvant and linkers to design a multiepitope-based subunit vaccine (mesv). many in silico approaches were used to validate the structural and physiochemical properties of the mesv. to examine the binding interaction and stability of mesv with human pathogenic receptors, molecular docking analysis has also been carried out. in addition, in silico immune simulation was also performed to validate the immunogenic potential of designed mesv. at the end, the mesv codons were optimized for escherichia coli system and in silico cloning was performed to ensure its expression profiling. flow chart of methodology used in present study is graphically presented in fig. . main structural proteins, surface glycoprotein (s [genbank: qhd . ]), envelope protein (e [genbank: qhd . ]) and membrane glycoprotein (m [qhd . ]) of sars-cov- were taken as targets for epitopes screening and vaccine designing against sars-cov- . their amino acid sequences were collected in fasta format from genbank (https://www.ncbi.nlm.nih.gov/genbank/) [ ] . allergenicity and antigenicity (at a threshold of . ) of selected proteins were evaluated through aller-top v . (https://www.ddg-pharmfac.net/allertop/) and vaxijen v . (http://www.ddg-pharmfac.net/vaxijen/vaxi-jen/vaxijen.html) respectively [ , ] . three dimensional ( d) structure of s protein was retrieved from rcsb protein data bank (pdb; https://www.rcsb.org/) [ ] . however, d structures of other two proteins (e and m) were predicted using homology modeling approach, as their resolved structures are not available yet. raptorx (http:// raptorx.uchicago.edu/) and modeller v. . (https:// salilab.org/modeller/) were employed for homology modeling [ ] . predicted models were then visualized by chimera (https://www.cgl.ucsf.edu/chimera/) [ ] . galaxy refines server (http://galaxy.seoklab.org/) and modrefiner (https://zhanglab.ccmb.med.umich.edu/modrefiner/) was used to refine the predicted models [ , ] . besides, the refined structure needs to be validated based on experimentally validated d structure of proteins. refined structures were therefore applied in the prosa web (https:// prosa.services.came.sbg.ac.at/prosa.php) providing a quality score for a given structure [ ] . the quality score beyond the usual range of native proteins indicates a possible error in protein structure. ramachandran plot was created by rampage server (http://mordred.bioc.cam. ac.uk/~rapper/rampage.php), where the principle of pro-check is applied to validate the protein structure [ ] . structural analysis was performed to later investigate the positions of b-cell epitopes on target proteins. the epitopes of b-cells help to detect viral infections in the immune system. abcpred (http://crdd.osdd.net/ raghava/abcpred/) was used to forecast -mer b cell epitopes for target proteins at . threshold [ ] . epitopes evident on the outer surface were picked, and other intracellular epitopes were removed. the vaxijen server tested the antigenicity of the selected epitopes at a threshold of . . b-cell epitope identification was based upon antigenicity, flexibility, linear epitope predictions, hydrophilicity, and surface accessibility [ ] . parker hydrophilicity prediction algorithms, emini surface accessibility prediction method, kolaskar and tongaonkar antigenicity scale, and karplus and schulz flexibility prediction tool were used to perform hydrophilicity, accessibility of surface, antigenicity and flexibility analysis respectively [ ] . as discontinuous epitopes become more evident and have higher dominant properties than linear epitopes, discotop . server (http://www.cbs.dtu. dk/services/discotope/) was used to forecast discontinuous epitopes from d structures of surface glycoprotein, membrane protein and envelope protein [ ] . the position of epitopes on d structures of proteins was visualized by pymol (https://pymol.org/ /) [ ] . in vaccine designing, t-cell epitopes play a crucial role. more specifically, it reduces the cost and time compared with laboratory experiments [ ] . iedb consensus method (http://tools.iedb.org/mhcii/) was used to predict - mer mhc class-i and - mer mhc class-ii epitopes. the results of this method are very important due to a large number of human leukocyte antigen (hla) alleles used in the calculation. the sequence was given in a fasta format and all the alleles were selected for prediction. epitopes with less than consensus score believed to be good binders and chosen for further research. antigenicity and allergenicity of the selected epitopes were checked by vaxijen v . and allergen fp v . respectively [ ] . protein digest server (http://db.systemsbiology.net: /proteomicstoolkit/proteindigest.html) was used to predict epitopes digesting enzymes. toxinpred (http:// crdd.osdd.net/raghava/toxinpred/) was used for nontoxic/toxic properties prediction of epitopes. non-toxic epitopes were selected for further analysis [ ] . the degree of conservation of predicted t-cell and b-cell epitopes within the protein sequence was analyzed by iedb conservancy analysis tool (http://tools.iedb.org/conservancy/). epitopes having conservancy among all selected proteins were shortlisted for further analyses [ ] . the expression and distribution of hla alleles vary depending on the world's ethnicities and regions, thereby impacting the effective production of mesv [ ] . the population coverage was calculated using the iedb population coverage tool (http://tools.iedb.org/population/), and for this purpose mhc class-i and mhc class-ii epitopes and corresponding hla-binding alleles were considered. this tool estimates population coverage for each epitope for various regions of the world based on the distribution of hla binding alleles [ ] . epitopes with the following characteristics are generally preferred to design a subunit vaccine: (a) highly antigenic, (b) immunogenic, (c) non-allergenic, (d) non-toxic, and (e) with significant population coverage. therefore, only those epitopes were selected further to construct mesv following the above parameters. an adjuvant was attached with the eaaak linker to the first cytotoxic t lymphocytes (ctl) epitope to improve the immune response. other epitopes were linked using aay, gpgpg, and kk linkers after validation of their interaction compatibility to preserve their independent immunogenic activity. βdefensin has been used as an adjuvant in the present research since it is a simple amino acids long peptide that acts as an immunomodulator and as an antimicrobial agent both [ ] . first, blastp analysis was carried out using default parameters to confirm that the designed mesv sequence is non-homologous against the homo sapiens proteome [ ] . protein with less than % is commonly known to be a non-homologous. physiochemical properties of the designed mesv were accessed by the protparam tool [ ] . protparam predicts various physiochemical properties like (half-life, theoretical isoelectric point [pi], instability index, grand average hydropathy, and aliphatic index) based on the amino acid approximations involved in the pk [ ] . allertop v. . server was used to analyze the allergenicity of the mesv construct [ ] . the secondary structure of the mesv construct was evaluated using a psipred workbench [ ] . this test also evaluated various vaccine properties such as alpha helices, extended chain, degree of beta turns, and random coil. the d structure of mesv was predicted using the de novo modeling approach of cabs fold server (http://biocomp.chem.uw.edu.pl/cabsfold/), since the designed mesv was a series of epitopes and no appropriate template was available [ ] . this server is based on a cabs modeling approach that combines a multi-scale modeling pipeline with an exchange replica monte carlo scheme. predicted mesv d structure was modified using a galaxy refine server [ ] . the ramachandran plot analysis was carried out using the rampage server (http://mordred. bioc.cam.ac.uk/~rapper/rampage.php) [ ] , to confirm the quality of the refined mesv structure, followed by the structural validation analysis using the prosa web server [ ] . the errat server (https://servicesn.mbi.ucla.edu/ errat/) was also used to evaluate the calculation of unbounded interactions in the mesv structure [ ] . besides, linear b-cell epitopes were predicted from the mesv using the abcpred server [ ] . ellipro tool (http:// tools.iedb.org/ellipro/) was used to predict the conformational b-cell epitopes of the designed mesv using default settings (maximum distance: a°; minimum score: . ), provided by iedb-ar v. . . it predicts epitopes by estimating residual protrusion index (pi), protein shape, and neighbor residue clustering [ ] . all together for the appropriate evocation of immune response, the interaction amongst the antigenic molecule and immune receptor molecule is essential. molecular docking was performed to analyze the interaction between mesv construct and human immune receptors. toll-like receptors- (tlr ) has been thoroughly studied, and studies found its key role in antiviral immune response generation. gramm-x (http://vakser.compbio. ku.edu/resources/gramm/grammx/) was used for the mesv docking with tlr (pdb id: ziw) [ ] . pymol was utilized for visualization of the docked complexes [ ] . moreover, for the achievement of the conventional sketch of interactions among docked proteins, an online server pdbsum (http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=index.html) was utilized. it analyzes the protein-protein interactions among docked molecules [ ] . an in silico immune simulation was performed using c-immsim . server (http:// . . . /c-immsim/ index.php?page= ) to validate the immunological responses of the designed mesv. c-immsim simulates the three main components of the functional mammal system (thymus, lymph node, and bone marrow) [ ] . the input parameters for the immune simulations are as follows: volume ( ), hla (a , a , b , b , drb _ , drb _ ), random seed ( ), number of steps ( ), number of injection set to . the rest of the parameters were considered to be the default. codon optimization is a method to improve the translation effectiveness of foreign genes in the host if the use of codon is different in both organisms. codon optimization was carried out followed by in silico cloning, after the careful evaluation of mesv properties and immune response. to make this method consistent with the commonly used prokaryotic expression system; e. coli k [ ] , the java codon adaptation tool (http://www.jcat.de/) [ ] was used for mesv codon optimization. the other available choices were selected to evade: (i) termination of rho-independent transcription, (ii) binding-site of prokaryote ribosome, and (iii) cleavage-sites of restriction enzymes. codon adaptation index (cai) [ ] along with the gc (guanine and cytosine) contents were assessed. sticky ends of the restriction sites of hindiii and bamhi restriction enzymes were added to allow restriction and cloning, in the start/n terminal and end/c terminal of the modified mesv sequence, respectively. the modified nucleotide sequence of mesv was additionally cloned into the e. coli pet a (+) vector by using snapgene tool (https://www.snapgene.com/ ), to assure its in vitro expression. sequence and structural analysis of the target proteins all target structural proteins were found to be nonallergenic and highly antigenic. e protein was the most antigenic followed by m and s protein with . , . and . antigenic values, respectively. the d structure of s protein was retrieved from protein-data-bank using id: vyb [ ] . the d structure of e protein was determined using homology modeling. chain-a of envelope small membrane protein of sars-cov (pdb id: x ) was found to be the best template (percent identity . %) for e protein of sars-cov- . however, no suitable template was found for m protein, so its structure was predicted by raptor x [ ] . visualization of the models was done by chimera (additional file : fig. s ). the quality factor (z-score) and ramachandran plot values of refined predicted models are mentioned in additional file : table s (additional file : fig. s -s ) . total linear epitopes (s- , e- , and m- ) were selected. among the chosen linear epitopes, 'ilpvsmtkts vdct' of s protein showed the highest antigenicity ( . ) and predicted score (additional file : table s ). the positions of epitopes on their respective protein structures were visualized by pymol (additional file : fig. s ) . identification of b cell epitope was based on antigenicity, flexibility, linear epitope predictions, hydrophilicity, and surface accessibility. parker hydrophilicity prediction algorithms, emini surface accessibility prediction method, kolaskar and tongaonkar antigenicity scale, and karplus and schulz flexibility prediction tool were used to perform hydrophilicity, accessibility of surface, antigenicity and flexibility analysis respectively (additional file : fig. s -s ) . to further improve the specificity and variety of b-cell epitopes, discotop . server was used to calculate surface abundance concerning residual contact number and use the novel amino acid score to forecast discontinuous epitopes. d structures of the target proteins were used to predict discontinuous epitopes; % specificity, − . thresholds and . angstroms propensity score radius. fifty-five discontinuous epitopes of s protein, epitope of the e protein and epitopes of m protein were calculated (additional file : table s ) . epitopes that are bound to multiple alleles, highly antigenic, non-allergenic and % conserved were screened out, and their antigenicity and allergenicity were checked. based on these criteria, mhc class-i (s- , e- , and m- ) and mhc class-ii (s- , e- and m- ) were shortlisted (additional file : table s ). protein digest server was used to estimate epitopes/peptides digesting enzymes. epitopes digestible with many enzymes are not stable. less enzyme digested epitopes, on the other hand, are very stable and favored vaccine candidates (additional file : table s ). total three ctl epitopes (s- and m- ), six htl epitopes (e- and m- ), and four b-cell epitopes (s- and m- ) were selected to construct mesv ( table ) . the selected epitopes showed . % of the world population coverage (fig. ) . results revealed that predicted epitopes are showing promising population coverage of the countries strongly affected by covid- including, germany, france, spain, saudi arabia, england, italy, iran, the philippines, the united states, and sweden. a mesv construct was further developed using all selected epitopes. using the eaaak linker, an adjuvant ( amino acid long b-defensin) was bound at the beginning (to the mev n-terminal). eaaak linker reduces connections to other protein areas with efficient detachment and improves stability [ , ] . epitopes were merged in a sequential manner with aay, gpgpg, and kk linkers, respectively, based on the compatibility of their interaction. two hundred seventy-six amino acids represented the final mesv construct (fig. ) . first, blastp analysis was carried out against the homo sapiens proteome, and the results revealed that mesv does not resemble any human protein (higher or equal to %). the vaccine structure was then tested for toxicity, allergenicity, and antigenicity. mesv was found to be non-allergenic, highly antigenic ( . ), and non-toxic. the mean half-life of the construct was calculated as h in vitro, > h in yeast and > h in vivo. molecular weight and theoretical pi of the vaccine were . kda and . respectively. grand average hydropathicity was calculated as . . a positive score of the grand average of hydropathy suggests its hydrophobic nature. the secondary structure analysis show cabs fold server was used to predict the tertiary structure of the mesv (fig. ) . the structure was refined by the galaxy refine server. ramachandran plot analysis of improved model showed that . % amino acids are in favored region, . % amino acids in the allowed region and . % amino acids in the outlier region. further analysis showed that the qrmsd is . , molprobity is . , poor rotamers are . %, clash score is . and z-score is − . . in quality check analysis by errat, the refined model score was . . . aay linkers (blue) used to join the ctl epitopes, gpgpg linkers (green) used to join the htl epitopes and kk linkers (gray) used to join the b-cell epitopes fig. a mesv construct sequence. epitopes sequence is in black. the adjuvant sequence is highlighted in brown color, eaaak linker sequence is highlighted in blue, aay linkers are highlighted with orange, gpgpg linkers are highlighted with green and kk linkers are highlighted with maroon color; b mesv construct refined d structure pipes representation (alpha helix: green; beta strands: blue; loops: gray); c ramachandran plot analysis of predicted structure shows . % residues are present in the favored region b-cell epitopes screening from mesv b-lymphocytes also produce antibodies that provide humoral immunity, in addition to the secretion of cytokines. eighteen linear/continuous (additional file : table s ) and six conformational/ discontinuous epitopes (additional file : table s ) from the mesv sequence were predicted without altering abcpred . and ellipro prediction parameters. to start the immune response, an appropriate interaction among the antigenic molecule and immune receptor molecule is needed. to decode the binding potential of mesv to the innate immune receptors, bioinformatic modeling driven molecular docking of the designed mesv to a representative innate immune receptor tlr was performed. the docking evaluation forecast that the best complex with a net global energy of − . kj/mol. visual analysis of the complex leads to the observation of the mesv's deep binding in the center of tlr and favors rigorously hydrogen and weak van dar waals interactions with specific tlr residues. pdbsum was used to gain insights and pin down possible residues of mesv making stable bonds with tlr (fig. ) . within Å, the mesv was observed to form hydrogen bonds with tlr potential residues. all secondary and primary immune responses tend to contribute significantly to the pathogen and may be consistent with the actual immune response. the in silico host immune system response to the antigen is shown in fig. . the primary response was characterized by high igg + igg and igm concentration, followed by igm, igg + igg and igg at both the secondary and primary stages with concomitant antigen reduction. additionally, robust interleukin and cytokine response was observed. all of this indicates the mesv's successful immune response and clearance after subsequent encounters. in silico cloning within e. coli system in silico cloning was done to assure the expression of mesv derived from sars-cov- in widely used e. coli hosts. first, the codons of mesv were modified according to the use of codons of e. coli expression system (strain k ). the optimized mesv construct contains nucleotides, cai value of . ( . - . ), and an optimal range of gc content of . % ( - %) demonstrating the strong potential for reliability and positive protein expression. in the following step, both ends of mesv optimized nucleotide sequence were attached to buffer compatible restriction enzymes bamhi and hindiii restriction sites to assist the purification/cloning process. finally, the refined mesv sequence was cloned to the several cloning sites of the pet a (+) vector between the restriction sites. the clone was . kb long (fig. ). covs have long been considered as insignificant pathogens causing "colds" in humans. in the twenty-first century, two extremely pathogenic covs named sars-cov and mers-cov emerged from the livestock reservoirs and cause deadly outbreaks. a new strain of cov officially named as sars-cov- was identified recently, which started a deadly global pandemic of covid- . the final dimension and impact of this pandemic are currently uncertain due to the rapidly changing situation [ ] . after the recombination of various virus genomes particles, the novel virus infects the host cells rapidly. no reliable medication is currently available for the said infection. covid- infection is a severe problem of morbidity and mortality worldwide. unfortunately, the unavailability of the vaccinations against covid- has impacted several precious lives, in different regions of the world. the emergence of covid- results in a significant global disease burden, for which preventative measures are urgently needed. to successfully eradicate the disease, researchers have been trying to collect data associated with covs to understand its transmission, pathophysiology, and biology [ ] . the rapid development of structural and genomic databases combined with computational tools helps in the design and discovery of new vaccine candidates. recent advancements in the immunological bioinformatics area have resulted in a variety of tools and servers that can lessen the time and cost of traditional vaccine advancement. due to the problems in the selection of suitable antigen candidates and immunodominant epitopes, the development of effective multiepitope vaccines remains toilsome. thus, the prediction of appropriate antigenic epitopes of a targeted protein by the immunoinformatics approaches is very essential for designing a mesv [ ] . here, we explored the development of epitope-based vaccines targeting the structural proteins (s, m, and e) of the sars-cov- . these proteins play a crucial in the replication cycle and the virus particle structure. the sprotein plays an important part in binding the virus to the host cell surface receptors and consecutive fusion to promote the viral entrance in the host cell [ ] [ ] [ ] . m and e proteins are important for replication, particle assembly within human cells, and viral entry [ , ] . tand b-cell epitopes of the target proteins were predicted to support the host's immune response. the research was performed at primary, secondary and tertiary structural levels of proteins. iedb analysis resource and abcpred predicted b-cell conserved epitopes. the position of epitopes on d structures of proteins was visualized by pymol. discotop server was used to predict discontinuous epitopes. to further improve specificity and selectivity, allergenicity, toxicity, and physiochemical properties of predicted epitopes were checked. digestion analysis verified that the peptides predicted during the analysis were stable and safe to use. an appropriate mesv should be designed with b-cell, htl, and ctl epitopes and cause effective reactions to a specific virus [ ] . few groups developed sars-cov- subunit vaccines but only used a single protein for the vaccine design [ , , ] and the use of ctl epitopes only without taking into account the importance of htl or b cell epitopes [ ] . however, we have incorporated b-cell epitopes in addition to t-cell epitopes from multiple structural proteins, because of the functions they play in inducing antibody production and mediating its effective features [ ] . besides, the humoral response of memory b-cells can be easily overcome by the onset of antigens, while the cell-mediated immunity (t-cell immunity) in many cases leads to long-life immunity [ ] . ctl limits pathogen spread through the secretion of unique antiviral cytokines and the identification and destruction of infected cells [ ] . therefore, the present vaccine construct has an advantage over already reported constructs. the hla alleles retain their response to t-cell epitopes which are highly polymorphic in different ethnic groups. to gain more population coverage, the t-cell epitopes are paired with more alleles. so we chose the htl and ctl epitopes with their respective hla alleles to predict the worldwide distribution of the alleles. the results showed that the chosen epitopes and their corresponding alleles preferably cover various geographical vaccine candidates were chosen form ctl, htl, and b cell epitopes depending on their antigenicity, toxicity, immunogenicity, population coverage, and allergenicity. the mesv was designed by joining the htl, ctl, and b cell epitopes with gpgpg, aay, and kk linkers respectively. linkers are introduced as an indispensable element in the mesv development to enhance folding, stabilization, and expression. multi-epitope based vaccines are poorly immunogenic when used alone, and need adjuvant coupling [ ] . adjuvants are ingredients in a vaccine formulation that protects against infection and affect certain immune responses, growth, stability, and durability of antigens [ ] . therefore, amino acids long, an adjuvant β-defensin, was integrated with the eaaak linker whose length is , at n-terminal. the eaaak linker is used to integrate the first epitope and adjuvant to facilitate efficient separation of the bifunctional fusion protein domains [ ] . the final vaccine stretch with the addition of adjuvant and linkers was discovered to be amino acid long. the analysis of physiochemical characteristics of the mesv construct has shown that it is stable, basic, and hydrophobic. mesv was basic, according to the theoretical pi value, which can ensure stable physiological ph interaction. the calculated aliphatic index and instability index scores showed that the vaccine protein may be stable and thermostable. a positive score of the grand average of hydropathy suggests its hydrophobic nature. mesv has been found to be immunogenic, strongly antigenic, and non-allergenic. this suggests the ability of the epitopic vaccine to elicit a strong immune response without allergic reactions. the d structure prediction provides extensive knowledge of the spatial arrangement of essential protein components and provides excellent support for the study of ligand interactions, protein functions, dynamics, and other proteins [ , ] . after refinement, the desirable characteristics of the mesv construct improved considerably. the ramachandran plot analysis shows that most residues are present in favored and allowed regions with very few residues in the disallowed region, which shows a satisfactory overall quality of the model. the good quality of designed mesv construct is further indicated by rmsd value, poor rotamers, clash score, and mol-probity. various structure validation methods have been used to detect errors in the modeled mesv construct. the errat quality factor ( . %) and z-score (− . ) proved that the overall structure of the refined mesv is of good quality. an adequate interaction between the antigen molecule and the immune receptor molecules is important for triggering an immune response. the refined mesv construct was then docked against tlr to examine adequate binding to immediate immune response. stable interactions were observed among the mesv and tlr in molecular docking analysis, and less energy was needed for proficient binding. b-and t-cell epitopes consisting multi-epitope vaccine should hypothetically activate both humoral and cellular immune reactions. with substantial il- and il- activities, our vaccine demonstrated the highest production of ifn-γ. antibodies also provide extracellular sars-cov- protection. we have also noticed excess immunoglobulins that are active, i.e., igm, igg, and their isotypes that may be involved in switching isotype. besides, the irrelevant simpson index (d) recommends a diverse immune reaction that is conceivable as a subunit vaccine contains various b-cell and t-cell epitopes. the translation efficiency of foreign genes inside the host system varies because of the incompatibility of mrna codons, which require codon optimization for higher expression [ ] . cai value obtained was . and gc content ( . %) was also within the optimum limit suggesting possible higher expression in the e. coli k- system. the main aim of mesv in silico cloning was to direct genetic engineers and molecular biologists on the expected expression level and the potential cloning sites in a particular expression system i.e., e. coli k system. we applied the next-generation vaccine designing approach in this research to create a mesv construct, capable of generating immunological responses against the sars-cov- . we believe that our vaccine will successfully produce humoral and cell-mediated immune responses. interaction and binding patterns between receptor and vaccine protein were stable and higher. moreover, in immune simulation, effective immune responses were observed in real life. thus, mesv designed carefully using such a methodology could become an important asset in combating viral infections. computational/immunoinformatics approaches rely on experimental methodologies to generate initial raw data for further analyses. the data quality and efficiency of computational algorithms being applied, can limit the accuracy of immunoinformatics predictions. therefore, further in vivo and in vitro investigations are however required to ensure the real potential of designed mesv to combat covid- . taken together, we characterized sars-cov- structural proteins (s, e, and m) for antigenic epitopes and proposed a potential mesv utilizing various immunoinformatics and computational approaches. the findings of this research could save time and related costs for the study of experimental epitope targets. the mesv can activate all host immune system components and has adequate physicochemical and structural properties. it also appears to interact very stably with an innate immune receptor tlr , making it more likely to be introduced into the host immune system. to reveal its effectiveness in the fight against covid- , however, additional in vitro and in vivo experiments are warranted. structural basis of sars-cov- cl pro and anti-covid- drug discovery from medicinal plants pharmacoinformatics and molecular dynamics simulation studies reveal potential covalent and fda-approved inhibitors of sars-cov- main protease clpro epitope-based peptide vaccine design and 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evolution influences the infectious dose of mers-cov necessary to achieve severe respiratory disease structural basis and designing of peptide vaccine using pe-pgrs family protein of mycobacterium ulcerans-an integrated vaccinomics approach synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein pre-fusion structure of a human coronavirus spike protein the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles membrane binding proteins of coronaviruses coronavirus envelope protein: current knowledge multi-epitope vaccines: a promising strategy against tumors and viral infections design of a multiepitope-based peptide vaccine against the e protein of human covid- : an immunoinformatics approach development of epitope-based peptide vaccine against novel coronavirus (sars-cov- ): immunoinformatics approach t cell epitope-based vaccine design for pandemic novel coronavirus -ncov. chemrxiv the role of antibody and complement in the control of viral infections cd + regulatory t cells: mechanisms of induction and effector function muonic hydrogen and the proton radius puzzle a novel design of a multi-antigenic, multistage and multi-epitope vaccine against helicobacter pylori: an in silico approach recent advances of vaccine adjuvants for infectious diseases design of the linkers which effectively separate domains of a bifunctional fusion protein computational screening of medicinal plant phytochemicals to discover potent pan-serotype inhibitors against dengue virus investigating the molecular mechanism of staphylococcal dna gyrase inhibitors: a combined ligand-based and structure-based resources pipeline novel immunoinformatics approaches to design multi-epitope subunit vaccine for malaria by investigating anopheles salivary protein authors would like to acknowledge guangxi university and government college university faisalabad for providing facilities for this study. supplementary information accompanies this paper at https://doi.org/ . /s - - -w.additional file : figure s . d structural representation of sars-cov- structural proteins: (a) s protein, (b) e protein and (c) m protein. figure s . (a) the e protein contains α-helix ( . %, ) and random coil ( . %, ) ; (b) the z-score ( . ) of the e protein; (c) the ramachandran plot of refined structure shows . , . and . % residues in favored, allowed and disallowed region, respectively. figure s . (a) the m protein contains α-helix ( . %, ), β-strand ( . %, ) and random coil ( . %, ) ; (b) the z-score (− . ) of the m protein; (c) the ramachandran plot of refined structure shows . , . and . % residues in favored, allowed and disallowed region, respectively. figure s table s . structural details of the sars-cov- structural protein predicted models.additional file : table s . linear b cell epitopes predicted through abcpred . server (nt: nontoxic).additional file : table s . emini surface accessibility of sars-cov- structural proteins.additional file : table s . discontinuous epitopes predicted through discotop . server. additional file : table s . mhc class-i allele and mhc class-ii binding peptides with their antigenicity scores. additional file : table s . digestion, allergenicity, toxicity and physiochemical profiling of selected peptides (na: not allergic; nt: nontoxic).additional file : table s . linear b cell epitopes predicted in vaccine construct.additional file : table s . conformational epitopes in d structure of vaccine. availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. all authors have no competing interests. key: cord- -lcgeingz authors: nan title: th international symposium on intensive care and emergency medicine: brussels, belgium, - march date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: lcgeingz nan introduction: increasing evidence supports a central role for "immunosuppression" in sepsis. it is necessary to develop biomarkers of immune dysfunction that could help to identify patients at risk of poor outcomes [ ] . the decreased expression of human leucocyte antigen (hla)-dra is proposed as a major feature of immunodepression and its persistent decrease is associated with mortality in sepsis [ ] . in a previous study, we evidenced that fcer a (fc fragment of ige receptor ia) is the gene showing the lowest expression levels of the entire transcriptome in sepsis [ ] . here we studied the association between fcer a expression and mortality in infected surgical patients. methods: fcer a and hla-dra expression levels were quantified by droplet digital pcr in blood of infected surgical patients. patients died within days ( . %). spearman test was used to evaluate the association between gene expression and the sequential organ failure assessment (sofa) score. areas under receiver operating curves (auroc) were used to determine the gene expression cut-off values predicting mortality. kaplan-meier survival curves were obtained and differences in survival between groups were evaluated using the log rank test. cox regression was employed to assess mortality risk at days. results: gene expression levels of fcer a and hla-dra correlated inversely with patients' severity (r: - . p< . ; r: - . , p< . respectively). both genes showed significant aurocs to predict survival, but fcer a showed the best accuracy (fig. ) . patients with introduction: severe pulmonary and renal conditions such as acute respiratory distress syndrome (ards), respiratory failure, and deterioration in kidney function often occur in patients with nosocomial pneumonia (np). the emergence and course of infection is genetically determined, hence host genetic landscape may influence an ability to resist infection. methods: variants for genotyping were selected using the phewas catalog which presents genotypic data for caucasian patients, phenotypes and single nucleotide polymorphisms (snps) with p < . [ ] . snps with the lowest p-values for phenotypes with both, respiratory and renal manifestations were selected: intergenic variants rs and rs , rs (edil ) and rs (cyp a ). cyp a gene was associated with pneumonia and ards in our previous investigations, so we included in our analysis three sites of cyp a gene (rs , rs and rs ) studied on a smaller sample. genotyping was performed on sites for a sample results: allele rs -g of the cyp a gene was protective against ards and an increase in creatinine level (fig. ) . the rs -g allele was associated with lung complications and with the development of severe respiratory insufficiency (fig. ) . conclusions: the snps rs and rs can influence the aggravation of pulmonary and renal symptoms through genetically mediated response to infection. introduction: an uncontrolled inflammatory response plays a major role in the sepsis related organ dysfunction. mesenchymal stem cells(mscs) can improve survival of sepsis experimental models by modulating the inflammatory response. macrophages have been considered as important immune effector cells and their polarization imbalance aggravates the disordered inflammation reaction. the project aims to identify the effects of mscs on macrophages polarization against dysregulated inflammatory response. methods: raw . cells were plated in the lower chambers of transwell system in the presence or absence of lipopolysaccharide (lps). then, mscs were seeded in the upper chambers and incubation for different time. finally, transforming growth factor beta (tgfβ) receptor (tgf-βr) inhibitor was added in transwell system. the phenotype of raw . cells were analyzed by flow cytometry, the levels of inflammatory cytokines were detected by enzyme-linked immunosorbent assay (elisa). results: our data showed that lps increased the level of interleukin (il)- in raw . cells (p< . ) (fig. ). in line with il- expression, lps induced the expression of m macrophage (p< . ). moreover, lps stimulated raw . cells co-culture with mscs in transwell system, mscs inhibited the expression of il- and m macrophages, while increased m macrophages (p< . ). compared with lps group, the concentration of tgf-Β was obviously increased in mscs treatment groups (p< . ), furthermore, there were no significantly difference between mscs directed and indicted groups. more significantly, tgf-βr inhibitor abolished the impact of mscs on lps stimulated raw . cells (p< . ) (fig. ) . conclusions: mscs polarized m macrophages into m macrophages and decreased pro-inflammatory cytokine levels by paracrining tgf-β. introduction: sepsis is dysregulated response to an infection, which can lead to progressive microcirculatory dysfunction, release of reactive oxygen intermediates (roi) and life-threatening organ dysfunction. our aim was to investigate the relationship between organ damage -characterized by the sequential organ failure assessment (sofa) scores, microcirculatory failure and roi production, in a large animal model of experimental sepsis. methods: fecal peritonitis was induced in anesthetized minipigs (n= ; . g/kg autfeces containing - x cfu bacteria i.p.), control animals (n= ) received sterile saline i.p. invasive hemodynamic monitoring and blood gas analyses were performed between - hrs, the signs for failure of circulatory, respiratory and urinary systems were evaluated in accordance with the sofa score. the microcirculatory perfusion rate in the sublingual region was measured by orthogonal polarization spectral imaging technique (cytoscan a/r). the leukocyte-origin roi production was determined by lucigenine (mostly o -. ) and luminol-based (h o ) chemiluminescence methods. results: between - hrs after induction the sofa score indicated moderate organ failure in animals (m: . ; p: . , p: . ) and the change was statistically significantly higher in pigs, suggesting severe organ dysfunction (m: . ; p: . , p: . ). the microcirculation was significantly deteriorated in all cases, independently of sofa score data. the h o production was significantly lower in septic animals as compared to controls, while the lucigenine enhanced roi production correlated with the sofa score-indicated moderate and severe organ dysfunction. conclusions: sublingual microcirculatory parameters are not correlating with the severity of sofa score-indicated organ dysfunction in abdominal sepsis. the measurement of roi production of the whole blood seems to be better biomarker for the detection of the progression of events from moderate to severe organ damages. introduction: the purpose of this study was to characterize differences in sepsis management in patients with and without left ventricular (lv) dysfunction. septic patients with lv dysfunction have higher mortality, and limited guidance exists for sepsis management of patients with lv dysfunction. the possibility exists that the cornerstones of sepsis management may contribute to these poor outcomes. methods: a retrospective chart review was conducted from may -january at two centers. adult patients who had a diagnosis of sepsis, were treated with vasopressors for > hours, and had an echocardiogram within months were included. patients were divided into two groups: reduced ejection fraction (ef) of < % and preserved ef defined as ef ≥ %. information about patient outcomes and sepsis management were collected. the primary outcome was the need for mechanical ventilation (mv). categorical and continuous data were analyzed using the chi-squared and mann-whitney u tests, respectively. the irb has approved this project. results: a total of patients with ef < % and patients with ef ≥ % were included. no significant differences in fluid management, vasoactive agent maximum rate or duration, or steroid use were observed. net fluid balance between low and preserved ef was positive . liters vs. . liters (p = . ), respectively. the number of patients that needed mv was higher in the low ef cohort ( % vs. %, p = . ), and this cohort had fewer mv-free days ( , iqr - vs. (iqr - ), p= . . conclusions: no significant differences were observed with regard to sepsis management, reflecting current guidelines. the significantly increased need for mv is a provocative result. a potential mechanism is the inability of a patient with reduced lv dysfunction to maintain appropriate cardiac and respiratory function in the face of fluid overload. prospective analysis of the role of fluid balance in septic patients with lv dysfunction is warranted. introduction: the relationship between myocardial injury and systemic inflammation in sepsis response is not well understood [ ] . it´s proposed to evaluate the association between myocardial injury biomarkers, high-sensitive troponin t (hs-ctnt) and n-terminal pro-brain natriuretic peptide (nt-probnp), with inflammatory mediators (il- , il- Β , il- , il- , il- / il- p , il a, il- and tnf-α ) and biomarkers, c protein reactive (cpr) and procalcitonin (pct), in septic patients methods: this was a prospective cohort study performed in three intensive care units, from september to september enrolling patients with sepsis (infection associated with organ dysfunction), and septic shock (hypotension refractory by fluids infusion requiring vasopressor). blood samples were collected up to h after the development of first organ dysfunction (d ) and on the th day after inclusion in the study (d ) results: ninety-five patients were enrolled, with median age years (interquatile? - ), apache ii: median ( - ), sofa: median ( - ); . % were admitted in icu with sepsis and . % with septic shock. hospital mortality was . %. in d , nt-probnp correlated with il- (r = . , p < . ) and il- (r = . , p < . ). in d , hs-ctnt and nt-probnp correlated with pct (r = . , p < . and r = . , p < . ; respectively). nt-probnp d was higher in nonsurvivors than in survivors on mortality in seventh day (p = . ) and in-hospital mortality (p = . ). hs-ctnt d (p = . ) and nt-probnp d (p < . ) were significantly higher in non-survivors on in-hospital mortality. nt-probnp d (or . ; ic % . - . , p= , ) and hs-ctnt d (or , ; ic % . - . , p= , ) were independently associated with in-hospital mortality conclusions: nt-probnp plasma levels at d correlated with il- and il- , and both nt-probnp and hs-ctnt at d correlated with pct. in addition, nt-probnp has been shown to be an important predictor of mortality introduction: heparin-binding protein (hbp) acts proinflammatory on immune cells and induces vascular leakage through cytoskeletal rearrangement and cell contraction in the endothelium and is a promising novel prognostic biomarker in sepsis and septic shock. however, studies on repeated measures of hbp are lacking. our objective was to describe the kinetics of plasma hbp during septic shock and correlate it to hemodynamic parameters. methods: we included patients with septic shock (sepsis- ) on admission to helsingborg hospital's intensive care unit (icu) during september to february . patients were sampled from icu admission and every hours for hours or until death or icu discharge. the plasma samples were analyzed for hbp and converted using the natural log (lnhbp) for normality. lnhbp was then evaluated against mean arterial pressure (map) as primary analysis and against systemic vascular resistance index (svri) as a secondary analysis, using mixed-effects linear regression models, treating patient id as a random intercept and adjusting for hemodynamic parameters. results: a total of patients were included with median age years, females ( %), surgical admissions ( %), median sofa-score points on day one and deaths from all causes within days ( %). plasma hbp ranged from to ng/ml with a median of ng/ml (lnhbp range . to . , median: . ). an increase lnhbp was significantly associated with a decrease in map (coef. - . mmhg, % ci: - . to - . , p= . , n= ), when adjusting for heart rate (hr), noradrenaline (na), vasopressin (vp), dobutamine (dbt) and levosimendan (ls). in a secondary subgroup analysis, an increase in lnhbp was also significantly associated with a decrease in svri (coef. - . dyne*s*cm- *m- , % ci: - . to - . , p= . , n= ), when adjusting for map, hr, na, vp, dbt, ls and cardiac index. conclusions: repeated measures of plasma hbp during septic shock were correlated with important hemodynamic parameters in this small pilot study. introduction: mid-regional pro-adrenomedullin (mr-proadm) comes from the synthesis of the hormone adrenomedullin (adm), which is overexpressed during inflammation and progression from sepsis to septic shock. thus, mr-proadm can be a useful biomarker for the clinical management of septic patients [ ] . the aim of our study was to understand the ability of mr-proadm to predict -day ( -d) mortality and to find a correlation between mr-proadm and sequential organ failure assessment (sofa) score in the first hours from intensive care unit (icu) admission. methods: we evaluated consecutive septic shock patients according to sepsis iii definitions. clinical data from the medical records included demographics, comorbidities, laboratories, microbiology and biomarker levels. whole blood samples for biomarker profiling were collected at , and hours from icu admission. mr-proadm measurement was detected in edta plasma using a sandwich immunoassay by trace® (time resolved amplified cryptate emission) technology (kryptor thermo fischer scientific brahms). results: overall -d mortality rate was . %. mr-proadm [odds ratio (or) = . ], sofa score (or = . ) and lactate (lac) levels (or = . ) in the first hours were associated with -d mortality in univariate logistic analysis (p value < . , table ). -d mortality rate was not associated with procalcitonin (pct) levels (or = . ). further linear regression analysis showed significant correlation between mr-proadm and sofa score at hours from icu admission (p value< . , fig. , table ). conclusions: mr-proadm demonstrated superior accuracy to predict -d mortality compared to pct levels and is directly linked to sofa score at hours from admission. mr-proadm may aid early identification of poor prognosis septic patients who could benefit a more intensive management. introduction: study of the expression of cell free dna (cfdna) in the search for new biomarkers for infection, sepsis and septic shock. methods: the population studied was all patients included in the sepsis protocol from march to january , hospitalized patients of a federal public hospital. plasma samples were collected for quantification of cfdna, which after centrifugation were stored at - °c and then thawed and analyzed by fluorescence using a varioskan flash fluorometer). cfdna values were expressed as ng/ml. the patients were divided into groups: infection and sepsis/septic shock. we analyzed mortality, sequential organ failure assessment score (sofa score), qsofa (quick sofa), comorbidities, cfdna and laboratory parameters of patients. results: among the patients, % were classified as infection and % sepsis/septic shock. overall lethality was %, infection . %, and sepsis/septic shock . % (p< . ). the mean of cfdna, sofa and lactate was higher according to the classification of infection and sepsis/septic shock: cfdna ( . ± . and . ± . , p= . ), sofa ( . ± . and . ± . , p< . ), qsofa (positive in % and %, lactate ( . ± . and . ± . , p< . ). we analyzed leukocytes, creatinine, crp (c reactive protein), inr (international normalized ratio), as predictors of severity and only crp showed no association with disease severity (p= . ). levels of cfdna and qsofa showed worse prognostic utility as a predictor of sepsis / septic shock when compared to lactate and sofa: or . ( % ci . - . ), p= . for cfdna, or . ( % ci . - . ), p= . for sofa and or . ( % ci . - . ), p= . for lactate. negelkerke r square was , for cfdna. in addition, area under the curve for cfdna mortality was . ( % ci . - . ) and sofa . ci % . - . ). conclusions: our study suggests that cfdna and qsofa have worse prognostic accuracy when compared to lactate and sofa, variables already used in clinical practice and easily measured. introduction: the aim of this study is to develop a "molecular equivalent" to sequential organ failure assessment (sofa) score, which could identify organ failure in an easier, faster and more objective manner, based on the evaluation of lipocalin- (lcn /ngal) expression levels by using droplet digital pcr (ddpcr). sepsis has been classically defined as the exuberant, harmful, pro-inflammatory response to infection. this concept is changing [ ] and the presence of a life-threatening organ dysfunction caused by a dysregulated host response to infection is now considered a central event in the pathogenesis of sepsis [ ] . methods: lcn expression levels were quantified by ddpcr in blood of a total of surgical patients with a diagnosis of infection. spearman analysis was used to evaluate if lcn correlated in a significant manner with sofa score. area under the receiver operating curve (auroc) analysis and multivariate regression analysis were employed to test the ability of lcn to identify organ failure and mortality risk. results: spearman analysis showed that there was a positive, significant correlation between lcn expression levels and sofa score (fig. ) . aurocs analysis showed that lcn presents a good diagnostic accuracy to detect organ failure and mortality risk (fig ) . in the multivariate regression analysis, patients showing lcn expression levels over the optimal operating points (oops) identified in the aurocs showed a higher risk of developing organ failure (table ) and a higher mortality risk (table ) . conclusions: quantifying lcn expression levels by ddpcr is a promising approach to improve organ failure detection and mortality risk in surgical patients with infection. introduction: sepsis is an inflammatory state due to an exacerbated immune response against infection. in cancer patients, sepsis presents a -fold higher mortality than in general population and leads to longer intensive care unit (icu) and hospital lengths of stay. it has been shown that reduced levels of circulating immunoglobulins (ig) might be a surrogate marker of unfavorable outcome in sepsis [ ] . the aim of this study was to evaluate the association between ig levels in plasma and -day mortality rate in cancer patients with septic shock. methods: from december to november , we conducted a prospective study in the intensive care unit (icu) of cancer institute of state of sao paulo, an -bed icu linked to university of sao paulo. patients ≥ years old with cancer and septic shock were enrolled. descriptive statistics were computed for demographic and outcome variables. laboratory data and ig levels were collected at icu admission and at days , and . a multivariate analysis was performed to evaluate predictors of -day mortality. results: a total of patients were included in the study. the -day and -day mortality were . % and . %, respectively. no significant differences in igm and igg levels were observed between survivors and non-survivors. in both groups, the median igm levels were low and the median igg levels were normal. in the multivariate analysis for -day mortality, a favorable status performance measured by the eastern cooperative oncology group (ecog) was associated with better survival; metastatic disease, higher sequential organ failure assessment (sofa) score at admission and higher levels of initial lactate were associated with increased mortality. conclusions: low levels of serum endogenous immunoglobulins are not predictors of -day mortality in cancer patients with septic shock. introduction: cytovale has developed a rapid biophysical assay of the host immune response which can serve as a rapid and reliable indicator of sepsis. neutrophils and monocytes undergo characteristic structural and morphologic changes in response to infection. one type of response is the generation of neutrophil extracellular traps (nets), these have been proposed as potential mediators for widespread tissue damage. during netosis there is a fundamental reorganization of a cell's chromatin structurea signal that we have shown is sensitively measured by the cytovale cytometer. we hypothesized that quantification of plasticity (deformability) of leukocytes in the peripheral blood provides an early indicator of sepsis. the cytovale assay uses microfluidic cytometry to measure the plasticity of up to , white blood cells from edta-anticoagulated, peripherally-collected whole blood and provides a result in minutes. methods: in two prospective studies conducted in two academic medical centers in baton rouge, la, the cytovale test was performed on peripheral blood samples obtained from patients who presented to the emergency department with signs or symptoms suggestive of infection. the two studies included high acuity patients ( patient study) and low acuity patients ( patient study). an adjudicated reference diagnosis of sepsis or no sepsis was established for each subject, using consensus definitions, by review of the complete medical records. results: the receiver operator curve (roc) performance of the cytovale assay for both studies demonstrated an area under the curve (auc) greater than . (fig. ) . conclusions: measurement of neutrophil and monocyte plasticity by a novel assay provides an accurate and rapid indication of sepsis in patients who present to an emergency room with signs or symptoms of infection. plasma hepatocyte growth factor in sepsis and its association with mortality: a prospective observational study introduction: sepsis and septic shock are commonly associated with endothelial cell injury. hepatocyte growth factor (hgf) is a multifunctional protein involved in endothelial cell injury and plays a pivotal role in sepsis. this study assesses its correlation with relevant endothelial cell injury parameters and prognostic value in patients with sepsis. methods: a prospective, observational cohort study was conducted in patients with sepsis admitted to the department of critical care medicine at the zhongda hospital from november to march . the plasma hgf level was collected on the first h after admission (day ) and day , then was measured by enzyme-linked immunosorbent assay. the primary endpoint was defined as all-cause -day mortality. furthermore, we analyzed the correlation of hgf with relevant endothelial cell injury markers. results: eighty-six patients admitted with sepsis were included. hgf levels of non-survivors were elevated upon day ( . ± . pg/ml vs. . ± . pg/ml; p = . ) and day ( . ± . pg/ml vs. . ± . pg/ml; p = . ) compared with that in survivors, and showed a strong correlation with von willebrand factor (r = . , p < . ), lactate (r = . , p = . ), pulmonary vascular permeability index (r = . , p = . ), first h fluid administration (r = . , p < . ) and sequential organ failure assessment score (r = . , p = . ) (fig. ) . plasma levels were able to discriminate prognostic significantly on day (auc: . , %ci: . - . ) and day (auc: . , %ci: . - . ) (fig. ) . conclusions: hgf levels are associated with sepsis and are correlated with established markers of endothelial cell injury. elevated hgf level in sepsis patients is a predictor of mortality. methods: adult patients with septic shock by the sepsis- classification due to lung infection or primary bacteremia or acute cholangitis are screened using two consecutive measurements of ferritin and of hla-dr/cd co-expression for mals (ferritin above , ng/ml) or immunosuppression (hla-dr/cd less than %) and randomized into immunotherapy with either anakinra (targeting mals) or recombinant ifnγ (targeting immunosuppression) and into placebo treatment. main exclusion criteria are primary and secondary immunodeficiencies and solid and hematologic malignancies. results: patients have been screened so far. most common infections are community-acquired pneumonia ( . %), hospitalacquired pneumonia ( . %) and primary bacteremia ( . %). mean +/-sd sofa score is . +/- . and charlson's comorbidity index . +/- . ; patients have mals ( . %); two immunosuppression ( %); the majority remain unclassified for immune state. conclusions: current screening suggests greater frequency of mals than recognized so far in a setting of septic shock due to lung infection or primary bacteremia or acute cholangitis. development of an algorithm to predict mortality in patients with sepsis and coagulopathy d hoppensteadt , a walborn , m rondina , j fareed study was to develop an equation incorporating biomarker levels at icu admission to predict mortality in patients with sepsis, to test the hypothesis that using a combination of biomarkers of multiple systems would improve predictive value. methods: plasma samples were collected from patients with sepsis at the time of icu admission. biomarker levels were measured using commercially available, elisa methods. clinical data, including the isth dic score, sofa score, and apache ii score were also collected. -day mortality was used as the primary endpoint. stepwise linear regression modeling was performed to generate a predictive equation for mortality. results: differences in biomarker levels between survivors were quantified and using the mann-whitney test and the area under the receiver operating curve (auc) was used to describe predictive ability. significant differences (p< . ) were observed between survivors and non-survivors for pai- (auc= . ), procalcitonin (auc= . ), hmgb- (auc= . ), il- (auc= . ), il- (auc= . ), protein c (auc= . ), angiopoietin- (auc= . ), endocan (auc= . ), and platelet factor (auc= . ). a predictive equation for mortality was generated using stepwise linear regression modeling. this model incorporated procalcitonin, vegf, the il- :il- ratio, endocan, and pf , and demonstrated a better predictive value for patient outcome than any individual biomarker (auc= . ). conclusions: the use of a mathematical modeling approach resulted in the development of a predictive equation for sepsis-associated mortality with performance than any individual biomarker or clinical scoring system. furthermore, this equation incorporated biomarkers representative of multiple physiological systems that are involved in the pathogenesis of sepsis. the effects of biomarker clearances as markers of improvement of severity in abdominal septic shock during blood purification t taniguchi , k sato , m okajima introduction: sepsis associated coagulopathy (sac) is commonly seen in patients which leads to dysfunctional hemostasis. the purpose of this study is to determine the thrombin generation potential of baseline blood samples obtained from sac patients and demonstrate their relevance to thrombin generation markers. methods: baseline citrated blood samples were prospectively collected from patients with sac at the university of utah clinic. citrated normal controls (n= ) were obtained from george king biomedical (overland park, ks). thrombin generation studies were carried out using a flourogenic substrate method. tat and f . were measured using elisa methods (seimens, indianapolis, in). functional antithrombin levels were measured using a chromogenic substrate method. results: the peak thrombin levels were lower ( ± nm) in the dic patients in comparison to higher levels observed in the normal plasma ( ± nm). the auc was lower ( ± ) in the dic group in comparison to the normals ( ± ). the dic group showed much longer lag time ( . ± . ) in comparison to the normal group ( . ± . ). wide variations in the results were observed in these parameters in the dic group. the f . levels in the dic group were much higher ( ± pmol) in comparison to the normal ( ± pmol). the tat levels also increased in the dic group ( . ± . ng/ml) in comparison to the normal ( . ± . ng/ml). the functional antithrombin levels were decreased in the dic group ( ± %). conclusions: these results validate that thrombin generation such as f . and tat are elevated in patients with dic. however thrombin generation parameters are significantly decreased in this group in comparison to normals. this may be due to the consumption of prothrombin due to the activation of the coagulation system. the decreased functional at levels observed in the dic group are due to the formation of the complex between generated thrombin and antithrombin. introduction: sepsis-associated disseminated intravascular coagulation (dic) is a complex clinical scenario involving derangement of many processes, including hemostasis. assessment of markers including inflammation, endothelial function, and endogenous anticoagulants may provide insight into dic pathophysiology and lead to improved methods for assessment of patient condition and response to treatment. methods: citrated plasma samples were collected from patients with sepsis and suspected dic at icu admission and on days and . dic score was determined using the isth scoring algorithm (e.g. platelet count, pt/inr, fibrinogen and d-dimer). cd ligand (cd l), plasminogen inhibitor (pai- ), nucleosomes, procalcitonin (pct), microparticle tissue factor (mp-tf) and prothrombin . (f . ) were measured using commercially available elisa kits. protein c activity was measured using a clot-based assay. interleukin (il- ), interleukin (il- ), interleukin (il- ), tumor necrosis factor alpha (tnfα), and monocyte chemoattractant protein (mcp- ) were measured using biochip technology. results: significant differences in levels of protein c (p= . ), pct (p= . ), il- (p= . ), il- (p= . ), pai- (p= . ), were observed between survivors and non-survivors. significant variation of protein c (p= . ), nucleosomes (p= . ), pct (p< . ), il- (p= . ), il- (p= . ), il- (p= . ), tnfα (p= . ) and mcp- (p= . ) were observed based on severity of dic score. conclusions: markers from multiple systems perturbed in dic were associated with mortality, suggesting that while these systems may not be routinely evaluated in the normal course of patient care, dysfunction of these systems contributes significantly to mortality. in addition, numerous inflammatory cytokines showed an association with dic score. this suggests that the measurement of additional markers in sepsis-associated dic may be of value in the prediction of mortality and may be helpful in guiding treatment for these patients. introduction: the endotoxin activity assay (eaa) is a rapid immunodiagnostic test based on chemiluminescence. it was approved by the fda in as a diagnostic reagent for risk assessment of severe sepsis in the icu. ascertaining endotoxin levels in the bloodstream is important in targeting patients and determining the appropriate timing for initiation of treatment. it has high sensitivity and specificity for endotoxin, and is considered to be useful in predicting clinical symptoms and determining prognosis. the usefulness of the eaa has yet to be fully clarified. methods: a total of patients admitted to the icu between january and june with suspected sepsis or sepsis were enrolled. the eaa was conducted within hr after admission. patient characteristics were determined, together with levels of il- , procalcitonin, presepsin, and pao /fio . thereafter, the patients were classified into groups depending on their eaa value: ) < . ; ) from ≤ . to < . ; ) from ≤ . to < . ; ) from ≤ . to < . ; and ) ≤ . ). the transition of various markers was also examined. the spearman rank correlation, wilcoxon rank sum test, and a nonrepeated anova were used for the statistical analysis. a p-value of < . was considered statistically significant. the eaa values showed a positive correlation with both the apache ii (r= . ) and sofa scores (r= . )(p< . ), although that with the latter was stronger. a significant correlation was also observed with levels of procalcitonin (r= . ) and presepsin (r= . early diagnosis is important to allow early intervention. the current clinical methods are insufficient for early detection. we hypothesized that intraperitoneal microdialysis allows detection of peritonitis prior to changes in standard clinical parameters in a pig model. methods: bacterial peritonitis was induced in pigs by bowel perforation and intraperitoneal fecal instillation, one pig underwent sham surgery. intraperitoneal microdialysis catheters were placed in each abdominal quadrant. the observation time was hours. results: in peritonitis pigs the intraperitoneal lactate increased during the first two hours and remained elevated throughout the observation time (table ) , whereas the arterial lactate remained within reference range (< . mm). intraperitoneal glucose decreased significantly. hemodynamics were hardly influenced during the first two hours, and decreased thereafter. sham surgery did not influence in any of the parameters. conclusions: a rapid and pronounced increase in intraperitoneal lactate and decrease in intraperitoneal glucose was observed after instillation of intraabdominal feces. systemic lactate increase was absent, and the hemodynamic response was delayed. postoperative intraperitoneal microdialysis is applicable in detecting peritonitis earlier than standard clinical monitoring and should be evaluated in a clinical study in order to explore if early intervention based on md data will reduce icu length of stay, morbidity and mortality. introduction: procalcitonin (pct) is a serum biomarker suggested by the surviving sepsis campaign to aid in determination of the appropriate duration of therapy in septic patients. trauma patients have a high prevalence of septic complications, often difficult to distinguish from inflammatory response. pct values typically declined after h from trauma and increased only during secondary systemic bacterial infections. the aims of the study are to evaluate reliability and usefulness of pct serum concentration in trauma. methods: we retrospectively analyzed data from trauma patients admitted to icu at bufalini hospital -cesena, from july to august . we collected data about antimicrobial therapy, injury severity score (iss), first arterial lactate in emergency room, sofa score and sepsis severity. plasma pct concentration was measured using an automate analyzer (modular e-brahms) on st day of antimicrobial therapy and every h hours. antimicrobial therapy was stopped according to a local protocol; however medical judgment was considered the overriding point for therapeutic decision. results: median iss of patients was . , inter quartile range (iqr) . . pct mean concentration at the starting of antimicrobial treatment was . μg/l (d.s . ), median . (iqr . ). no significative correlation (spearman´s rho test) was found between pct at day of antimicrobial therapy and iss (rho - . ), between first arterial lactate in er and pct (rho . ). daily course of pct was not related to distance from trauma (rho - . ). in of patients ( . %) pct measurement led physician to save days of antimicrobial therapy compared with standard clinical practice. we couldn´t find any cut off value. conclusions: our experience suggests that pct could help physician to optimize duration of antimicrobial therapy in trauma patients. no standard approach can be recommended at present. introduction: long duration of antimicrobial treatment may predispose to colonization and subsequent infections by multidrugresistant organisms (mdro) and clostridium difficile. progress (clinicaltrials.gov registration nct ) is an on-going trial aiming to use pct for the restraining of this calamity. methods: adult patients with sepsis by the sepsis- classification and any of five infections (pneumonia community-acquired; hospital-acquired or ventilator-associated; acute pyelonephritis; primary bacteremia) are randomized to pct-guided treatment or standard of care (soc) treatment. in the pct arm antibiotics are discontinued when pct on or after day is decreased by more than % of the baseline or remains below . ng/ml; in the soc arm antibiotics are discontinued at the discretion of the attending physician. patients are followed for six months. primary endpoint is the rate of infections by mdro and/or c.difficile or death. serial stool samples are cultured for mdro and screened for glutamate dehydrogenase antigen and toxins of c.difficile. results: patients have been enrolled so far. mean ± sd sofa score is . ± . . most common diagnoses are community-acquired the progress trial is the first trial assessing the probable benefit from pct guidance to reduce ecological sequelae from long-term antibiotic exposure. analysis of baseline patient characteristics indicates that progress is a real-world trial so that results can have major clinical impact. prospective multi-site validation of -gene host response signature for influenza diagnosis s thair , s schaffert , m shojaei , t sweeney there are no blood-based diagnostics able to identify influenza infection and distinguish it from other infections. we have previously described a blood-based -gene influenza meta-signature (ims) score to differentiate influenza from bacterial and other viral respiratory infections. methods: we prospectively validated the ims in a multi-site validation study by recruiting individuals ( patients with suspected influenza, healthy controls) in community or hospital clinics across australia. we assayed the ims and genes from viral genome of influenza strains to generate the blood flu score (bfs) as a measure of viremia using nanostring from whole blood rna. results: using clinically determined phenotypes, the ims score distinguished patients with influenza from healthy (auc= . ), non-infected (auc= . ), bacterial (auc= . ), other viruses (auc= . ) ( figure a) . interestingly, probes of bfs were found in all phenotypic groups (non-infected, bacterial, and other viral infections) to varying degrees, and positively correlate with the ims score (r= . ). ims aurocs improve when the bfs is used to inform the phenotypic groups: healthy (auc= . ), non-infected (auc= . ), bacterial (auc= . ), other viruses (auc= . ) ( figure b ). patients who were clinically influenza negative but had a high ims and bfs were admitted less often, yet had~ -fold higher mortality than those who were clinically influenza negative with low ims and no bfs (table ) . conclusions: collectively, our prospective multi-center validation of the ims demonstrates its potential in diagnosis of influenza infections. introduction: previous findings of our group suggest that patients with gram-negative hospital-acquired severe sepsis have better prognosis when sepsis is developing after recent multiple trauma through stimulation of favorable interleukin (il)- responses [ ] . under a similar rationale, we investigated if preceding osteomyelitis may affect experimental osteomyelitis. methods: sham or experimental osteomyelitis was induced in male new zealand white rabbits after drilling a hole at the upper metaphysis of the left tibia and implementing diluent or log of staphylococcus aureus using foreign body. after three weeks, the foreign body was removed and experimental pyelonephritis or sham surgery was induced after ligation of the right pelvo-ureteral junction and instillation of log of escherichia coli in the renal pelvis. survival was recorded and circulating mononuclear cells were isolated and stimulated for the production of tumour necrosis factor-alpha (tnfa) and il- . at death or sacrifice, tissue outgrowth and myeloperoxidase (mpo) were measured. results: four sham-operated rabbits (s), rabbits subject to sham surgery and then pyelonephritis (sp) and rabbits subject to osteomyelitis and then pyelonephritis (op) were studied. survival after days of group sp was . % and of group op % (log-rank . ; p: . ). lab findings are shown in figure . il- production was blunted. negative correlation between e. coli outgrowth and tissue mpo was found at the right kidney of the op group (rs: - . , p: . ) but not of the sp group (rs: - . , p: . ). conclusions: preceding staphylococcal osteomyelitis provides survival benefit to subsequent experimental osteomyelitis through downregulation of innate immune responses leading to efficient phagocytosis. introduction: activation of neutrophils is a mandatory step and a sensitive marker of a systemic inflammatory response syndrome (sirs) which is closely related to development of multiple organ failure. the search for drugs that can prevent sirs and reduce mortality in critically ill patients remains significant. the aim of this study was to study the anti-inflammatory effect of the synthetic analogue of leu-enkephalin (dalargin) on human neutrophils. methods: the study was conducted on isolated from the blood of healthy donors neutrophils. their activation was assessed by fluorescent antibodies to markers of degranulation cd b and cd b (sd b-fitc and cd b-alexafluor (bd biosciences, usa). as inductors of inflammation lipopolysaccharide (lps) and the peptide formyl met-leu-pro (fmlp) were used. mkm fmlp and dalargin in concentrations of and μ g / ml were added to neutrophils at a concentration of ppm / ml and incubated for min at °c; then antibodies were added and incubated for min on ice; then fluorescence was assessed by flow cyto flow meter beckman-coulter fc . non-parametric criteria were used; data were presented as a median and %- % interquartile intervals. the statistical significance was estimated using mann-whitney test. the difference was considered statistically significant at p< . results: synthetic analogue of leu-enkephalin in various concentrations has an anti-inflammatory effect on both intact and preactivated with bacterial components neutrophils, reducing their activation and degranulation in a dose-dependent manner (figs. , ) . conclusions: synthetic analogue of leu-enkephalin prevents neutrophil activation by bacterial compounds. this has a potential of translation into clinical practice for sepsis treatment. introduction: the endothelin system plays important roles in circulatory regulation through vasoconstrictor et-a and et-b receptors and vasodilator et-b receptors (etar; etbr, respectively). tissue hypoxia during the progression of sepsis is associated with microcirculatory and mitochondrial disturbances. our aim was to investigate the possible influence of etar antagonist, etbr agonist or combined treatments on oxygen dynamics, microcirculatory and mitochondrial respiration parameters in experimental sepsis. methods: male sprague-dawley rats (n= /group) were subjected to faecal peritonitis ( . g/kg faeces ip) or sham-operation. septic animals were treated with sterile saline solution, or received the etar antagonist etr-p /fl peptide ( nmol/kg iv), etbr agonist irl- ( . nmol/kg iv) or same doses as combination therapy, hr after sepsis induction. invasive hemodynamic monitoring and blood gas analyses were performed during a -min observational window. introduction: sepsis often induces immunosuppression, which is associated with high mortality rates. nivolumab is a human igg- antibody directed against the programmed cell death (pd- ) immunecheckpoint inhibitor, which disrupts pd- -mediated signaling and restores antitumor immunity. nivolumab is an approved anti-cancer drug that may have the potential to improve sepsis-induced immunosuppression. methods: this multicenter, open-label study investigated the safety, pharmacokinetics and pharmacodynamics of a single intravenous infusion of or mg nivolumab in japanese patients with immunosuppressive sepsis (lymphocytes ≤ /μl). the dosing of nivolumab was set using the predicted steady state concentration of nivolumab at mg/kg every weeks (q w), which was the approved dosage for cancer patients at the time of planning. results: five and eight patients were assigned to the and mg groups, respectively. the mean (standard deviation) peak serum drug concentration in the mg group was comparable to the predicted median concentration ( % pi [prediction (figures and ). adverse events (aes) were observed in four patients in each group. drug related-aes were observed in only one patient in the mg group (table ) . no deaths related to nivolumab occurred. conclusions: a single dose of mg nivolumab appeared to be well tolerated and sufficient to maintain nivolumab blood concentration in patients with sepsis. results suggest both and mg nivolumab therapy could improve relevant immune indices. introduction: the systemic inflammatory response syndrome (sirs) accompanies tissue trauma and infection and, when severe or dysregulated, contributes to multiple organ failure and critical illness. observational studies in man and animal have shown that low-dose acetyl-salicylic acid promotes resolution of inflammation and might attenuate excessive inflammation by increasing the synthesis of specialised pro-resolving lipid mediators (spms). methods: we randomly assigned patients with sirs who were expected to stay in icu for more than hours to receive enteral aspirin ( mg per day) or placebo for days or until death or discharge from the icu, whichever came first. the primary outcome was il- serum concentration at h after randomisation. the secondary outcomes included safety and feasibility outcomes. in one center, additional blood samples were taken during the first three days for exploratory analysis of spms using reversed-phase highperformance liquid chromatography -tandem mass spectrometry (rp-hplc-ms/ms). results: from march through december a total of patients across four general icus in australia underwent randomization (table ) . compared to placebo patients, il- serum concentration after h in aspirin-treated patients was not significantly lower ( [ - ] pg/ml vs [ . - ] pg/ml; p= . ). there were no significant differences for control vs. aspirin-treated patients in the change of pro-resolving/anti-inflammatory lipids between the time points (figure , ). there were no between-group differences with respect to icu or hospital mortality, number of bleeding episodes or requirements for red cell transfusions (table ) . conclusions: in patients admitted to the icu with sirs, low-dose aspirin did not result in a decreased concentration of inflammatory biomarkers compared with placebo. introduction: sepsis is associated with excessive ros production, nf-kb, inos and inflammatory mediators overexpression. vitamin c is a cellular antioxidant, it increases enos and decreases nf-kb; it has several immune-enhancing effects and is crucial for endogenous vasopressors synthesis. vitamin c reserves in sepsis are often as poor as in scurvy [ ] . in recent studies, intravenous high vitamin c dose seems to reduce organ failure and improve outcome in septic shock. methods: we treated all septic shock patients admitted to our icu in months (from / to / ) with intravenous vitamin c . g/ h and thiamine mg/ h (for its synergistic effects) [ ] as adjunctive therapy for consecutive days and we compared data to septic shock patients admitted in the previous months period. we enrolled patients: received vitamins supplementation, standard of care. we analysed -days mortality, sofa at and hours, pct variation from baseline in first days, vasoactive therapy length and daf (days alive and free from vasopressors, mechanical ventilation and rrt in days follow up). patients with end stage kidney disease were ruled out. we analysed data with mann-whitney and wilcoxon tests. results: vit c group showed lower -days mortality ( % vs . %: ns); sofa improvement at (- . ± . vs - . ± . : p= . ) and hours (- . ± vs - . ± : p< . ) was higher in vit c group; vit c patients had faster pct reduction without statistical significance. mean vasoactive therapy length was quite similar. daf was . (± . ) days in vit c group and . (± . ) in controls (p= . ). control patients needed rrt, none in vit c group. conclusions: despite small study size, we found that vit c has positive effects on survival and improves sofa score (fig. ) and daf (fig. ) in septic shock. no vit c patient developed oxalate nephropathy nor worsened renal function. introduction: toxin-producing gram-positive organisms cause some of the most severe forms of septic shock [ , ] . adjunctive therapies such as intravenous immunoglobulins (ivig) have been proposed for these patients [ , ] . however, at patient presentation, the presence of a toxin-producing organism is most often unknown. methods: we reviewed the use of ivig in our patients requiring extracorporeal membrane oxygenation (ecmo) in a -year period between february and march . results: in % ( / ) of the patients that received ivig for presumed toxin-mediated shock, group a streptococcus or panton-valentine leukocidin producing s. aureus was isolated, but the clinical characteristics of these patients were not significantly different from the ones with other final diagnoses, except for a predisposing influenza infection and the presence of an often very high procalcitonin level. these patients were extremely unwell at presentation with a sofa score of ± , high lactate levels ( . ± . mmol/l) and need for vasopressors (equivalent norepinephrine dose of . ± . μ g/kg/min). they had very high inflammatory parameters with a procalcitonin ≥ ng/ml in more than half of patients ( / ). ivig use in these patients was generally safe, with only possible transfusion reaction. the mortality of % ( / ) was lower than predicted based on the sofa scores. conclusions: ivig administration can be considered in a selected group of patients presenting with acute and very severe septic shock, as part of a multimodal approach [ ] . introduction: extra corporeal treatments are used in septic patients to decrease the inflammatory mediators, but definitive conclusions are lacking . more over in many studies the effect of aki isn't evaluated and this may be an important bias. . the aim of this study is to evaluate in septic patients with aki: the effect of the adsorbing membrane oxiris on the immunological response -the different response in survivors and non survivors methods: from our local data base we analyzed retrospectively septic shock patients with aki (kdigo classification) submitted to crrt with the adsorbing membrane oxiris (baxter, usa ) . at basal time ( t ) and at the end of the treatment ( t ) we evaluated the following variables: il il procalcitonin endotoxin (eaa). all data are expressed as mean ±sd or median and iqr. student t test or mann-whitney was used to compare values changes. p < . was considered statistically significant. results: thirty patients with sepsis /septic shock and aki were enrolled in this study. patients had aki , patients aki , patients aki . the duration of treatment was ± hours. patients had citrate as anticoagulation and heparine continous ev. at table are shown the main results of this study in all the patients. survivors vs non survivors had a significant decrease of il , procalcitonin and eaa. conclusions: data of this study confirm on clinical ground previous study "in vitro" [ ] that the adsorbing membrane oxiris has important immunological effect during septic shock with aki. this must be confirmed in a rct. introduction: sepsis is common and often fatal, representing a major public health problem. hemoadsorption (cytosorb) therapy aims to reduce cytokines and stabilise the overall immune response in septic shock patients. methods: a prospective, multi-centre, investigator initiated study to evaluate hemoadsorption (cytosorb) therapy in septic shock patients admitted to a tertiary icu's in india during to . all centres followed a common protocol and received ethics committee approval. results: a total of patients were administered cytosorb in addition to standard of care. a total of patients ( %) survived out of patients. among survival group, patients ( %) were administered cytosorb within hours of icu admission resulting in significant reduction in sepsis scores, apache ii ( . vs . ) and sofa ( . vs . ) post cytosorb therapy. also there was reduction in inflammatory markers like cytokines il in most of the patients. all patients in survivor group showed a significant improvement in map ( . vs . ) and reduction in vasopressors (epinephrine . to . mcg/kg/min, nor-epinephrine . to . mcg/kg/min) after cytosorb therapy. no device related adverse effect was observed in any of the patients. among the non-survivor group, ( patients, %) we observed that cytosorb was administered after hours of icu admission. although a few patients showed improvement in sofa score, majority did not show a significant improvement with map ( . vs . mm of hg) and required increased demand in vasopressors. conclusions: in this multi-centered prospective iis study, we could observe clinical benefits of hemoadsorption (cytosorb) therapy in septic shock patients if the therapy was initiated early. larger randomised study are required to establish the above clinical benefits in larger patient population. a single centre experience with hemoadsorption (cytosorb) in varied causes of sepsis and mods y mehta , c mehta , a kumar , j george , a gupta , s nanda , g kochar , a raizada introduction: sepsis and the multiorgan failure is a leading cause of mortality in the intensive care unit. promising new therapies continue to be investigated for the management of septic shock. we tried to evaluate a novel hemoadsorption therapy (cytosorb) through a retrospective evaluation of patient's data in our centre. we used it as an adjuvant therapy in our patients with sepsis due to varied causes. methods: we retrospectively analysed data of introduction: septic shock is a life-threatening multiple organ dysfunction that has high morbidity and mortality in critically ill patients, due to a dysregulated host response to infection. the aim of this study was to evaluate the efficacy of therapeutic cytokine removal (cytosorb®) in the management of patients with septic shock. methods: we retrospectively analyzed patients admitted to icu with septic shock between june and november . patients included in the study were diagnosed according to the third international consensus definitions for sepsis and septic shock (sepsis- ), received maximal supportive care including continuous veno-venous hemodiafiltration (cvvhdf) for acute kidney injury and cytosorb® haemoadsorption column was added to return limb of the cvvhdf circuit. demographic data, procalcitonin and leukocyte levels before and after therapeutic cytokine removal and duration of cytosorb® haemoadsorption column application and apache ii scores were recorded. results: the mean age of patients included in the study was ± . years ( % male) and the mean body mass index was . ± . . the mean apache ii score was . with an expected and actual mortality rates of % and %, respectively. % of the patients were admitted with sepsis and % of them with septic shock. . % (n= ) of the cases were solid organ transplant recipients. cvvhdf was applied in all patients during therapeutic cytokine removal. treatment was combined with ecmo in patients. while the mean duration of cvvhdf was . hours, the duration of cytosorb® haemoadsorption column application was . ± . hours. procalcitonin ( . ± ng/ml vs ± ng/ml) and leucocyte levels ( ± / mm vs ± mm ) after therapeutic cytokine removal were found significantly lower than the pretreatment values (respectively p= . , p= . ). conclusions: therapeutic cytokine removal applied with cvvhdf in septic shock patients have positive contributions to biochemical parameters and provide survival advantage. introduction: recent studies have focused on demonstrating the potential benefits of immunomodulation in the management of septic patients. the aim of our study was to assess the effects of a hemoadsorption column (cytosorb®) in critical ill septic patients. methods: after ethical approval was obtained, we prospectively included patients admitted to the general icu of fundeni clinical institute. three consecutive sessions of renal replacement therapy (continuous venovenous hemodiafiltration) in combination with cytosorb® were applied after icu admission. clinical (heart rate, arterial pressure, temperature, glasgow coma scale) and paraclinical data (pao , serum bilirubin and creatinine, platelet count, white blood cell count, ph, c-reactive protein and procalcitonine), vasopressor support and need for mechanical ventilation were recorded before and after the three sessions. results: the mean age in the study group was ± years. median number of organ dysfunction at the time of icu admission was [ ] [ ] [ ] [ ] [ ] and the mean sofa score was . ± . . the use of cytosorb® was associated with a non-significant increase in pao /fio ratio from ± to ± (p= , ) and creatinine levels from . ± . to . ± . mg/dl (p= . ). although we observed a non-significant increase in c-reactive protein levels from ± mg/l to ± mg/ l (p= . ), we noted a significant decrease in procalcitonine levels from a median of . [ . , . ] ng/dl to a median of . [ . , . ] ng/dl (p= . ). a significant decrease in platelet count was also noted from ± /mm to ± /mm (p= . ). mean sofa score decreased non-significantly from . ± . to . ± . (p= . ). conclusions: the use of cytosorb was associated with a slight nonsignificant improvement in organ function and a decrease of procalcitonine levels. thrombocytopenia remains one of the most important complications of renal replacement therapy. introduction: circulating cell-free neutrophil extracellular traps (nets) would induce a microcirculatory disturbance of sepsis. the removal of nets remnants from the circulation could reduce nets-dependent tissue injury. to address this issue, we evaluated the effect of hemoperfusion with a polymyxin b cartridge (pmx-dhp; toray, japan), which was originally developed for the treatment in patients with gram-negative bacterial infection, on circulating cell-free nets in patients with septic shock and in phorbol myristate acetate (pma)-stimulated neutrophils obtained from healthy volunteer. methods: ex vivo closed loop hemoperfusion was performed through a circuit formed by connecting the small pmx module to a tube and a peristalsis pump. whole blood from healthy volunteers incubated with or without pma or from septic shock patients were applied to circuit and perfused. blood was collected at , and hr after perfusion. circulating cell-free nets were assessed by myeloperoxidase (mpo)-, neutrophil elastase (ne)-, and cell free (cf)-dna. results: plasma mpo-dna, ne-dna and cf-dna levels were significantly increased at hr after pma stimulation when compared with plasma levels without pma. when either blood from septic shock patients or pma-stimulated neutrophils obtained from volunteers were applied to circuit, circulating mpo-dna, ne-dna and cf-dna were significantly reduced in perfusion with pmx filter than in perfusion without pmx filter at times and hr. conclusions: in the ex vivo experiments, mpo-dna, ne-dna and cf-dna were found to decrease after ex vivo perfusion through pmx filters. selective removal of circulating components of nets may improve the remote organ damage in patients with septic shock. a retrospective study of septic shock patients who were treated with direct hemoperfusion with polymyxin b-immobilized fibers based on the levels of endotoxin activity assay s sekine, h imaizumi, i saiki, a okita, h uchino tokyo medical university, anesthesiology/icu, tokyo, japan critical care , (suppl ):p introduction: the purpose of this study was to evaluate the outcomes for septic shock patients with direct hemoperfusion with polymyxin b-immobilized fibers (pmx-dhp) and endotoxin activity assay (eaa). methods: according to the levels of eaa, patients were classified for three groups (low group (gl); eaa < . , intermediate group (gm); eaa > . or eaa < . , high group (gh); eaa > . ). in order to evaluate the severity of illness, acute physiology and chronic health eva-luationii (apache ii) score, the sequential organ failure assessment (sofa) score, catecholamine index (cai) were recorded. and the presence of pmx-dhp treatments were also recorded. blood samples were obtained to measure eaa levels, inflammatory markers (procalcitonin (pct), c-reactive protein (crp), and white blood cell count (wbc)), serum lactate level as an indicator of tissue hypoxia, and for blood culture. apache ii score, sofa score, cai, inflammatory markers, serum lactate levels (lac) and blood culture results were examined for diagnosis of septic shock and prognosis of -days mortality. each values were also compared to eaa levels. results: septic shock patients were included (gl/ gm/ gh: / / ). in gh, apache ii and sofa score was significantly higher than that in gl (p< . ). eaa levels were significantly increased in gramnegative bacteremia patients compared to the patients with grampositive bacteremia or fungemia. there was no relationship between eaa levels and other inflammation markers, cai, and lac. in gm, days mortality in patient with pmx-dhp treatments was lower than that of without pmx-dhp treatments ( . ( / ) vs . ( / ), p= . ). in gh, -days mortality in patient with pmx-dhp treatments was same as that of without pmx-dhp treatments ( . ( / ) vs . ( / ), p= . ). conclusions: these results of this study suggest pmx-dhp treatment may improve the outcome of septic shock patients with intermediate eaa levels. introduction: numerous inconclusive randomized clinical trials (rcts) in sepsis in the past years suggest a need to re-think trial design to improve resource allocation and facilitate policy adoption decisions. the inclass study (clinicaltrials.gov nct: ) is an ongoing rct evaluating clarithromycin as an immune modulator in high-risk septic patients with clinical and cost-effectiveness outcomes. we aim to compare the original one-shot trial with an alternative sequential design that balances trial costs and value of information. methods: adult patients with sepsis, respiratory failure and total sofa score of at least , are randomized to receive intravenous clarithromycin or placebo adjunctive to standard-of-care therapy. for the cost-effectiveness study, efficacy is measured in quality-adjusted life years (qalys) by eq- d- l questionnaire at days. the endpoint is the incremental net monetary benefit (inmb) of clarithromycin compared to placebo, defined as wtp x (increment in qaly) -(increment in costs), where wtp is willingness to pay per qaly gained. fixed and variable costs of trial execution (including administrative, insurance, supplies, tests) are calculated; hospitalization cost is extracted from patient records; medical care beyond day is recorded; cost of adoption in the general population is estimated. previous data from rcts using clarithromycin are used to form a prior belief about the inmb. known incidence of sepsis with respiratory failure allows estimation of the population to benefit from trial decision. a bayesian model is used to determine the sequential design that maximizes trial value. results: we will compare the performance of the sequential trial design with the one-shot design of inclass trial in terms of sample size, cost, social-welfare, and probability of correctly identifying the best treatment. conclusions: in this protocol we validate a bayesian model for sequential clinical trials and assess the benefits for the patient population and health care system. the effect on the outcome of critically ill patients with catecholamine resistant septic shock and acute renal failure through implementation of adsorption therapy g schittek introduction: cytosorb-adsorption has been described as an effective way for hemodynamic stabilisation in septic shock [ ] . aim of this study was to examine whether the adsorption-therapy could influence patient-outcome with catecholamine resistant septic shock (crss) and acute renal failure(arv). furhtermore we tried to identify clinical constellations that would predict an effective use of adsorbers [ , ] . initial il- in patients with catecholamine-reduction through adsorption was non-significantly different to those with no reduction ( ng/l [ , ] vs. ng/l [ , ]). mortality did not differ significantly between the groups ( % vs %). length of intensive care unit stay (los) did differ significantly ( days [ , ] vs days [ , ] ). conclusions: il- can be reduced with adsorption. patients with catecholamine-reduction did not differ in regard to their initial il- . los was shorter for patients treated with adsorption. according to our experience adsorption can be taken into consideration when crss is beginning. introduction: in our intensive care unit (icu), we have already started expanded application to the contact precautions. applied patients are; ) emergency admission, ) patients who had already had bacteria* that are required to contact precautions, ) scheduled surgical patients with prolonged icu stay, although we have not yet decided the started period of expanded application exactly. *detected bacteria(db);mrsa, cd, mdrp, esbl, pseudomonas a, pisp, prsp, vrsa. the aim of this study was to determine the adequate starting period of expanded application to the contact precautions in the scheduled surgical patients in the mixed icu. methods: we performed retrospective observational study on patients who were admitted to our icu after planed surgery from may to dec. . we detected the patients who acquired bd newly and investigated the relation to the length of icu stay. the relationship between detection rate and categorized date was also analyzed using logistic regression adjusted for age, gender, apache , and sofa score. using youden´s index and roc curve, we also calculated cutoff point of the duration of icu stay related to detection rate. finally, we made the logistic regression model of each cutoff day(day to ) and compared odds ratio(or) and auc of each models using stata. results: category day or more, especially day or more had significantly higher detection rate of db compared to day ( results: pao /fio was lower than mmhg in ( %) patients. compared to patients in group , patients in group were less severely ill at admission but presented a higher sofa and cpis score and a greater incidence of ards and shock at pneumonia onset (fig ) . ( %) patients in group had a microbiological diagnosis of pneumonia, compared to patients ( %) in group (p= . ). pao /fio ≤ mmhg was associated with less probability of having microbiological diagnosis of pneumonia (or . , % ci . to . , p= . ). when adjusted for other variables significantly associated with positive microbiology, pao /fio ≤ mmhg remained significantly associated with less probability of a microbiological diagnosis (adjusted or . , % ci . to . , p= . ). hospital mortality was significantly higher in patients in group compared to group ( % vs %, p= . ). however, no difference was found in non-response to treatment, icu and hospital stay, icu mortality (table ) and -days survival (fig ) . conclusions: a significant higher number of patients with vap didn't have a definitive etiological diagnosis when using the proposed threshold criteria of pao /fio ≤ mmhg. pao /fio ratio does not seem a good predictor of etiology in patients with vap. introduction: immunological dysfunction is common in critically ill patients but the optimal method to measure it and its clinical significance are unknown. levels of tumor necrosis factor alpha (tnf-α) after ex-vivo whole blood stimulation with lipopolysaccharide has been proposed as a possible method to quantitate immunological function. we hypothesized that patients with a lower post-stimulation tnf-α level would have increased rates of nosocomial infections (nis) and worse clinical outcomes. methods: a secondary analysis of a phase randomized, multicentre, double-blinded placebo controlled trial [ ] . there were no differences in allocation groups; all the patients were analyzed as one cohort. on enrolment, whole blood was incubated with lps ex-vivo and tnf-α level was measured. patients were grouped in tertiles according to delta and peak tnf-α level. the primary outcome was the development of nis; secondary outcomes included -day mortality. results: data was available for patients. baseline characteristics and outcomes are reported in tables and . patients in the highest tertile for post lps stimulation delta tnf-α compared to the lowest tertile were younger, had a lower acuity of illness and had lower baseline tnf-α. when grouped according to peak post-stimulation tnf-α levels, patients in the highest tertile had higher serum tnf-α at baseline. both comparisons showed no difference between nis and clinical outcomes between tertiles. in multi-variate analysis peak or delta tnf-α were not associated with the occurrence of nis. conclusions: admission ex-vivo stimulated tnf-a level is not associated with the occurrence of nis or clinical outcomes. further study is required to evaluate the ability of this assay to quantify immune function over the course of critical illness. results: sanitary and epidemiological examination revealed the connection between infection and intravenous infusion of dexamethasone performed concurrently with chemotherapy. in patients fever with chills and hypertension developed within hours after infusion of the infected drug; empirical intravenous antibiotic therapy started immediately after collecting blood culture. in patients fever appeared after - days outpatiently, so they received antibiotics per os. all these patients had permanent vascular access, and bsi was detected either the next chemotherapy course when fever reappeared ( pts) while using vascular access, or as a result of a specific examination ( pts). in all cases empirical antibiotic therapy started on the first day of fever, drug correction was performed in patients according to results of bacteriological research. septic shock developed in patient, pneumonia in patients. permanent vascular access was preserved only in case. all patients were cured and continued to receive antitumor treatment. conclusions: detection of more than case of b. cenocepacia bsi should be the reason for sanitary and epidemiological examination. a favorable outcome of bsi treatment is associated with the early start of antibiotic therapy and its correction after microbiological examination. emerging conclusions: implementation of asp in hospital allows to decrease incidence of eskape-bacteremia and candidemia, which may lead to improved clinical outcomes in icu's patients (fig ) . association of multi-drug resistant (mdr), extended-drug resistant (xdr) and pan-drug resistant (pdr) gram negative bacteria and mortality in an intensive care unit(icu) s chatterjee , s sinha , a bhakta , t bera , t chatterjee , s introduction: colistin-resistant klebsiella pneumoniae (cr-kp) is increasingly reported around the world. it is worrying to note emergence of resistance to last line of defence against mdr gram negative infections in regions endemic to carbapenem resistance. we report the first outbreak of cr-kp co-producing carbapenemases in an adult intensive care unit (icu) from south india. methods: retrospective analysis of all patients with carbapenem resistant klebsiella pneumoniae blood stream infection (bsi) was done between january and december . microbiological and clinical variables along with outcomes were analysed. results: seven patients had cr-kp with no prior exposure to colistin. all seven were modified hodge test (mht) negative making probability of blakpc unlikely. in resource limited setting, analysis beyond mht could only be performed for cr-kp samples. / samples belonging to cr-kp isolates produced the blandm- whilst / cr-kp isolates did not produce either blakpc or blandm carbapenemases prompting hypothesis of blaoxa- or blavim as the causative factor. compared to carbapenem resistance only group, cr-kp group had higher apache ii, icu length of stay and mechanical ventilation duration. day mortality was noted to be . % for carbapenem resistant and % for cr-kp groups. aggressive infection control measures were undertaken with successful containment of cr-kp strains along with reduction in overall bsi. conclusions: infection control measures form the backbone of patient care in centres showing endemicity for carbapenem resistant klebsiella to prevent colistin resistance and also to reduce occurrence of overall blood stream infections. rapid diagnosis of carbapenem resistance: experience of a tertiary care cancer center with multiplex pcr s mukherjee tata medical center, critical care medicine, kolkata, india critical care , (suppl ):p introduction: sepsis due to carbapenem resistant organisms has high mortality; inappropriate empirical antibiotic is one of the main causes of this poor outcome. on the contrary, "too much" broad spectrum empiric antibiotics will increase drug resistance, even in community, because of selection pressure. so, early diagnosis of resistance pattern (carbapenemase genes) is crucial. aim of this study is to compare rapid diagnostic test like polymerase chain reaction (pcr) with conventional culture sensitivity (c/s) to identify carbapenem resistance. methods: this is a prospective observational study done in tata medical center, kolkata, india. real time multiplex pcr technique has been developed "in house" in our microbiology lab and can identify ndm, ndm , kpc, oxa - , oxa - , oxa - & vim carbapenemase genes. blood cultures were sent as per clinical & laboratory diagnosis of sepsis in icu patients. culture positive samples had been used for conventional c/s by vitek system along with pcr study to identify carbapenemase genes. result of pcr technique was been compared with conventional c/s method. results: multiplex pcr results were available within - hours of positive blood culture compared to conventional c/s method that takes - days. among positive blood cultures, samples were positive for carbapenemase genes. most common gene identified was oxa - ( %), followed by ndm ( %). our pcr technique has very high sensitivity, specificity, positive & negative predictive value ( . %, . %, . % & . % respectively) while comparing with final c/s report by vitek system (table ) . there was only one false negative diagnosis for carbapenem resistance. conclusions: real time multiplex pcr for carbapenemase gene can be helpful for early diagnosis of carbapenem resistance and can help us to choose / modify antibiotics or to use 'targeted therapy'. it is more practical to "rule -in" infection rather than "rule -out" by this technique. carbapenemase producing enterobacteriaceae colonization in an icu: risk factors and clinical outcomes m miranda, jp baptista, j janeiro, p martins centro hospitalar e universitário de coimbra, intensive care unit, coimbra, portugal critical care , (suppl ):p introduction: carbapenemase-producing enterobacteriaceae (cpe) colonization has been increasingly reported in intensive care units (icus) since their first identification more than years ago. colonization with cpe seems to constitute a risk factor for mortality. the aim of our study was to identify associated risk factors and clinical outcomes among patients with fecal colonization by cpe admitted to a portuguese tertiary hospital icu. methods: a -year retrospective study was performed in patients with previous unknown cpe status (colonization or infection), admitted to our icu. rectal swabs were performed and analyzed using real-time polymerase chain reaction testing. clinical records were reviewed to obtain demographic and clinical data. results: of patients admitted, ( . %) harbored cpe, ( . %) were colonized at admission and ( . %) acquired cpe colonization during icu stay. the most frequent carbapenemase genes detected were kpc ( . %) and vim ( . %). cpe carriers had high rates of hospitalization (previous or ongoing), invasive procedures (mainly intraabdominal surgery), malignancy (hematopoietic or solid tumor), introduction: gram-negative pathogens-particularly pseudomonas aeruginosa and enterobacteriaceae-predominate in nosocomial pneumonia (np) and ciai both. these infections are becoming difficult to treat with available treatment options due to growing antimicrobial resistance in india. ceftazidimeavibactam has in-vitro activity against gram-negative organisms producing class a, class c and some class d beta-lactamases. we carried out a qualitative analysis to assess the safety and efficacy outcomes of the indian population cohorts involved in the re-prove and reclaim trials. methods: in line with the global reprove protocol, indian patients enrolled in the study with np, were randomly assigned ( : ) to mg ceftazidime and mg avibactam or mg meropenem. in the reclaim study, indian patients with a diagnosis of ciai were enrolled in the study and were randomly assigned ( : ) to receive either ceftazidime-avibactam ( mg of ceftazidime and mg of avibactam) followed by metronidazole ( mg); or meropenem ( mg). the primary efficacy outcome measure in the reprove and reclaim studies was clinical cure rate of caz-avi compared with that of meropenem at toc (test-of-cure) visit in pre-defined analysis sets. in both studies, non-inferiority was concluded if the lower limit of the twosided % ci for the treatment difference was greater than - · % in the primary analysis sets. as the indian subset study was not statistically powered to detect a difference in the subgroup, we descriptively analysed the efficacy results in the indian population and compared them with the overall results in the global trial. in addition, the study also analysed the safety of caz-avi in the indian patients by monitoring the number and severity of adverse events. introduction: early administration of effective intravenous antimicrobials is recommended for the management of the patients with sepsis. although meropenem (mepm) is one of the first-line drugs in patients with sepsis because of its broad spectrum, the optimal dose in the critical care settings especially during continuous renal replacement therapy (crrt) has not been established since therapeutic drug monitoring of mepm has not been popular. methods: eighteen critically ill patients who received crrt were enrolled in this study. one gram of mepm was administered over hour, every hours, and blood samples at , , , and hours after administration were collected on day , and . all samples were stored at - °c until analysis. the measurement of the blood concentration of mepm was performed using high performance liquid chromatography with ultraviolet detection (hplc-uv introduction: meningitis is one of the complications of severe traumatic brain injury, and it is often associated with encephalitis (incidence from . - . % to - %). the aim of the investigation was to study the dynamics of the concentration of meropenem in serum and cerebrospinal fluid (csf) with intravenous and intrathecal administration of meropenem. methods: in eight patients with bacterial meningoencephalitis blood serum and csf were studied prior to the administration of meropenem and - min, , . and hrs after it. antibiotic regimen: mg of vancomycin ( mg bid) and meropenem ( mg tid diluted in ml of saline iv + mg bid diluted in ml of saline bolus slowly intrathecally). meropenem infusion was carried out for minutes, mins after it ml of blood and ml of csf were sampled. prior to antibiotics administration blood and csf were taken for microbiological examination. to determine the concentration of antibiotics iquid chromatography/mass spectrometry was used. the samples were analyzed on an agilent infinity liquid chromatograph coupled to a sciex qtrap mass detector (sciex, us introduction: the prophylactic use of probiotics has emerged as a promising alternative to current strategies viewing to control nosocomial infections in a critically-ill setting. however, their beneficial role in vap prevention remains inconclusive. our aim was to delineate the efficacy of probiotics for both vap prophylaxis and restriction of icu-acquired infections in multi-trauma patients. methods: randomized, placebo-controlled study enrolling multitrauma patients, requiring mechanical ventilation for > days. participants were randomly assigned to receive either probiotic (n= ) or placebo (n= ) treatment. a four-probiotic formula was applied and each patient received two capsules per day from day to day post icu admission. the content of one capsule was given as an aqueous suspension by nasogastric tube, while the other one was spread to the oropharynx after being mixed up with water-based lubricant. the follow-up period was days, while icu stay and mortality were also assessed. ], while no difference in -day mortality rate was identified between groups ( . % probiotics vs . % placebo). conclusions: the prophylactic administration of probiotics exerted a positive effect on the incidence of vap or other icu-acquired infections and icu stay in a critically-ill subpopulation being notorious for its high susceptibility to infections, namely multi-trauma patients. use of a c-reactive protein-based protocol to guide the duration of antibiotic therapy in critically ill patients: a randomized controlled trial i borges introduction: the rational use of antibiotics is one of the main strategies to limit the development of bacterial resistance. in this study we aimed to evaluate the effectiveness of a c reactive protein (crp) based protocol in reducing antibiotic treatment time in critically ill patients. methods: an open randomized clinical trial was conducted in two adult intensive care units of a university hospital in brazil (clini-caltrials.gov: nct ). patients were randomly allocated to: i) intervention -duration of antibiotic therapy guided by crp levels, and ii) control -duration of therapy based on best in the intention to treat analysis, the median (q -q ) duration of antibiotic therapy for the index infection episode was . ( . - . ) days in the crp group and . ( . - . ) days in the control group (p= . ). in the cumulative suspension curve of antibiotics, a significant difference in the exposure time between the two groups was identified, with less exposure in the crp group (p= . ). in the pre-specified per protocol analysis, with patients allocated in each group, the median duration of antibiotics was . ( . - . ) days in the crp group and . ( . - . ) days in the control group (p= . ). mortality and relapse rates were similar between groups. conclusions: daily levels of crp may aid in reducing the time of antibiotic therapy in critically ill patients, even in a scenario of judicious use of these drugs. introduction: the macrophage activation syndrome (mas) or hemophagocytic lymphohistiocytosis(hlh) is a life threatening complication characterized by pancytopenia, liver failure, coagulopathy and neurologic symptoms and is thought to be caused by the activation and uncontrolled proliferation of t lymphocytes and well differentiated macrophages, leading to widespread hemophagocytosis and cytokine overproduction [ , ] .the etiology is unknown, but is considered to have an infectious trigger.the aim of our study is to evaluate the impact of hlh in our beds infectious diseases icu, during months period ( - ). methods: a retrospective study based on electronic databases, including all patients admitted in our icu, that have matched at least out of criteria for hlh diagnosis ( ):fever; hepatosplenomegaly; > cytopenia (hb < g/dl, plt</dl, pmn -n< /mmc); hypertriglyceridemia> mg/dl, fibrinogen< mg/dl; hemophagocytosis-bone marrow, spleen, and/or lymphnodes; nk activity reduced/ absent; ferritin level> ui/l; cd > . we have evaluated the etiology established with cultures, serology, and molecular methods, treatment with corticosteroids, iv immunoglobuline, cyclosporine, etoposide and outcome ( ) . results: patients were admitted to icu, patients( . %) met the criteria for hlh. the average length of stay in icu was days; patients died ( %) without relation with the followed treatment. conclusions: hlh is not a rare condition in infectious diseases icu. the etiology is more frequent established compared with literature data. treatment (corticosteroids, immunoglobuline, cyclosporine, etoposide) is not associated with increased survival forecasting hemorrhagic shock using patterns of physiologic response to routine pre-operative blood draws introduction: irreversible hemorrhagic shock (ihs), a critical condition associated with significant blood loss and poor response to fluid resuscitation, can induce multiple organ failures and rapid death [ ] . determining the patients who are likely to develop ihs in surgeries could greatly help preoperative assessment of patient outcomes and allocation of clinical resources. methods: machine learning model of ihs is developed and validated via porcine induced bleed experiment. healthy sedated yorkshire pigs first had one ml rapid blood draw during a stable period, and then were bled at ml/min to mean arterial pressure (map) of mmhg. subjects had ihs defined as map< mmhg. arterial, central venous and airway pressures collected at hz during the blood draw [ fig ] were used to extract characteristic sequential patterns using graphs of temporal constraints (gtc) methodology [ ] , and a decision forest (df) model was trained on these patterns to determine subjects at high risk of impending ihs. results: in a leave-one-subject-out cross-validation, our method confidently identifies % ( % ci [ . %, . %]) of the subjects who are likely to experience ihs when subject to substantial bleeding, while only giving on average false alarm in , such predictions. this method outperforms logistic regression and random forest models trained on statistically featurized data [tab , fig ] . conclusions: our results suggest that by leveraging sequential patterns in hemodynamic waveform data observed in preoperative blood draws, it is possible to predict who are prone to develop ihs resulting from blood loss in the course of surgery. future work includes validating the proposed method on data collected from human subjects, and developing a clinically useful screening tool with our investigations. work partially funded by nih gm . introduction: the h s and oxytocin(oxy) systems are reported to interact with one another [ ] . h s plays a major role in the hypothalamic control of oxy release during hemorrhage [ ] . there is scarce information about oxy receptor(oxyr) expression in the brain in general and what is there is ambivalent. oxyr has been immunohistochemically(ihc) detected in the human hypothalamus but not in the hippocampus, in contrast to rodents [ ] , which underscores the need for additional characterization in relevant animal models. thus the aim of this study is to map the expression of the oxy and h s systems in the porcine brain in a clinically relevant model of hemorrhagic shock (hs). methods: anesthesized atherosclerotic pigs (n= ) underwent h of hs (map +/- mmhg) [ ] , followed by h resuscitation. ihc detection of oxy, oxyr, the h s producing enzymes cystathionine-γ -lyase (cse) and cystathionine-β -synthase(cbs) was performed on formalin fixed brain paraffin sections. results: oxy, oxyr, cse and cbs were localized in the porcine brain. proteins were differentially expressed in the hypothalamus (fig ) , parietal cortex and cerebellum (fig ) . cell types positively identified were: magnocellular neurons of the hypothalamus, cerebellar purkinje cells and granular neurons, and hippocampal pyramidal and granular neurons of the dentate fascia. arteries and microvasculature were also positive for oxyr and cse. conclusions: our results confirm the presence of oxy and oxyr in the hypothalamus similarly to the human brain. novel findings were: oxyr in the cerebellum and cse expression in the hypothalamus and cerebellum. the coexpression of oxyr and cse may link and help better understand neurochemical systems and physiological coping in hemorrhagic shock. funding: crc introduction: septic shock is one of the main causes of intensive care unit (icu) admission, leading to mortality up to % of patients. acute kidney injury (aki) frequently occurs and is associated to great morbidity and mortality. hemodynamic optimization may reduce the incidence of aki, but the use of vasopressors to increase mean arterial pressure (map) could have deleterious effect on renal perfusion. we aimed at investigating the effect of map and norepinephrine (ne) on the incidence of aki in septic shock patients methods: retrospective study based on prospectively collected data on digital medical records (digistat) at our icu. introduction: in patients with distributive shock, increasing mean arterial pressure (map) to a target of > mmhg can improve tissue perfusion. patients unable to achieve the target map of > mmhg despite adequate fluid resuscitation as well as catecholamines and vasopressin standard care (sc), may benefit from the noncatecholamine vasopressor angiotensin ii to increase map. this posthoc analysis examined whether patients from the athos- study with a baseline (bl) map < mmhg and treated with sc plus either angiotensin ii (ang ii) or placebo achieved a map of > mmhg for consecutive hours, without increasing the dose of sc therapy. methods: patients were assigned in a : ratio to receive ang ii or placebo, plus sc. randomization was stratified according to map (< or > mmhg) at screening. in patients with bl map < mmhg, we evaluated whether patients achieved a map of > mmhg for the first hours after initiation (map measurements taken at hours , , and ), without an increase in the dose of sc. results: among treated patients, had bl map < mmhg (ang ii, ; placebo, ). median bl map (iqr) was ( - ) and ( - ) mmhg for placebo and ang ii groups, respectively. patients with bl map < mmhg who were treated with ang ii were more likely to achieve map ≥ mmhg for consecutive hours after initiation without an increase in sc dose ( %, %ci - ), compared with placebo-treated patients ( %, %ci - , or= . , p< . ). conclusions: in this post-hoc analysis of patients with bl map < mmhg, patients receiving ang ii plus sc were significantly more likely to achieve a map > mmhg for the first consecutive hours after initiation than patients receiving sc only. this suggests that administering ang ii may help patients with catecholamine-resistant distributive shock to achieve the consensus standard target map. norepinephrine synergistically increases the efficacy of volume expansion on venous return in septic shock i adda, c lai, jl teboul, l guerin, f gavelli, c richard, x monnet hôpitaux universitaires paris-sud, hôpital de bicêtre, aphp, service de médecine intensive-réanimation, le kremlin-bicêtre, france critical care , (suppl ):p introduction: through reduction in venous capacitance, norepinephrine (ne) increases the mean systemic pressure (psm) and increases cardiac preload. this effect may be added to the ones of fluids when both are administered in septic shock. nevertheless, it could be imagined that ne potentiates in a synergetic way the efficacy of volume expansion on venous return by reducing venous capacitance, reducing the distribution volume of fluids and enhancing the induced increase in stressed blood volume. the purpose of this study was to test if the increase in psm induced by a preload challenge were enhanced by ne. methods: this prospective study had included septic shock adults. to reversibly reproduce a volume expansion and preload increase at different doses of ne, we mimicked fluid infusion through a passive leg raising (plr). in patients in which the decrease of ne was planned, we estimated psm (using respiratory occlusions) at baseline and during a plr test (plr high ). the dose of ne was then decreased and psm was estimated again before and during a second plr (plr low ). . the increase in cardiac index induced by plr low was significantly greater than that induced by plr high (p< . ). Δ psmhigh -Δ psmlow was moderately correlated with the diastolic arterial pressure at baseline-high (p= . , r= . ) and with the ne-induced change in mean arterial pressure (p= . , r= . ). conclusions: ne enhances the increase in psm induced by a plr, which mimics a fluid infusion. this suggests that it may potentiate the effects of fluid in a synergetic way in septic shock patients. this may decrease the amount of administered fluids and contribute to decrease the cumulative fluid balance. introduction: arginine vasopressin (avp) can be used in addition to norepinephrine (ne) for ne-resistant septic shock. however, a subgroup who will response to avp is unknown. the purpose of this study was to determine factors which could predict the response to avp in patients with ne-resistant hypotension. methods: this was a single-center, retrospective analysis of patients who administered avp for ne-resistant hypotension in our intensive care units (icus). eligible patients were adult patients who administered avp in addition to ne due to hypotension (mean arterial pressure (map) < ) in our icus between august and december . we divided all patients into two groups by response to avp; responders and non-responders. the responders were defined as an increase of map ≥ mmhg at h after avp initiation. we conducted univariate and multivariate logistic regression analysis to evaluate the effect of variables on avp response. results: a total of patients were included; responders ( %), non-responders ( %). there was no significant difference for map at the time of avp initiation ( vs mmhg; p = . ), initiation dose of avp ( . vs . u/min; p = . ), and dose of ne at the time of avp initiation ( . vs . μ g/kg/min; p = . ). map at h after avp initiation was significantly higher in responders than non-responders ( vs mmhg; p < . ). responders were older ( vs ; p = . ) and had lower heart rate (hr) ( vs. ; p = . ) and lactate ( . vs. . mmol/l; p = . ) at the time of avp initiation. the multivariate logistic analysis revealed that hr ≤ (or . , % ci . - . , p < . ), lactate ≤ (or . , % ci . - . , p < . ) and age ≥ (or . , % ci . - . , p = . ) were significantly associated with the response to avp. conclusions: hr, lactate levels and age before avp initiation can predict the response to avp in icu patients with ne-resistant hypotension. the maximum norepinephrine dosage of initial hours predicts early death in septic shock d kasugai , a hirakawa , n jinguji , k uenishi nagoya university gtaduate school of medicine, department of emergency and critical care, nagoya, aichi, japan; fujita health university, department of disaster and traumatology, fujita health university, toyoake, japan; fujita health university hospital, department of emergency and general internal medicine, fujita health university hospital, toyoake, japan critical care , (suppl ):p introduction: the mortality of septic shock refractory to norepinephrine remains high. to improve the management of this subgroup, the knowledge of early indicator is needed. we hypothesize that maximum norepinephrine dosage on the initial day of treatment is useful to predict early death in septic shock. methods: in this retrospective single-center observational study, septic shock patients admitted to the emergency intensive care unit (icu) of an academic medical center between april and march were included. cardiac arrest before icu admission and those with do-not-resuscitate orders before admission were excluded. the maximum dosage of norepinephrine initial hours of icu admission (md ) was used to assess -day mortality. results: one-hundred-fifty-two patients were included in this study. median sofa score was ( - ), and median md was . ( . - . ) mcg/kg/min. vasopressin and steroid were administered in ( %) and ( %) cases. nineteen patients ( %) died within a week. non-survivors had higher md , higher sofa score, and higher rate of vasopressin use. the higher md predicted -day mortality (area under curve . , threshold . mcg/kg/min, sensitivity %, specificity %). after adjustment of inverse probability of treatment weighing method using propensity scoring, md higher than . mcg/kg/min was independently associated with -day mortality (or: . , %ci: . - . , p < . ). conclusions: the maximum dosage of norepinephrine higher than . mcg/kg/min initial hours was significantly associated with day mortality in septic shock, and may be useful in the selection of higher severity subgroup. the impact of norepinephrine on right ventricular function and pulmonary haemodynamics in patients with septic shock -a strain echocardiography study k dalla sahlgrenska university hospital mölndal, göteborg, sweden critical care , (suppl ):p introduction: septic shock is characterized by myocardial depression and severe vasoplegia. right ventricle performance could be impaired in sepsis. the effects of norepinephrine on rv performance and afterload in septic shock are not immediately evident. the aim of the present study was to investigate the effects of norepinephrine on rv systolic function, rv afterload and pulmonary haemodynamics. methods: eleven, volume-resuscitated and mechanically ventilated patients with norepinephrine-dependent septic shock were included. infusion of norepinephrine was randomly and sequentially titrated to target mean arterial pressures (map) of , and mmhg. at each target map, strain-and conventional echocardiographic were performed. the pulmonary haemodynamic variables were measured by using a pulmonary artery thermodilution catheter. the rv afterload was assessed by calculating the effective pulmonary arterial elastance (epa) and pulmonary vascular resistance index (pvri). results: the norepinephrine-induced elevation of map increased central venous pressure ( %, p< . ), stroke volume index ( %, p< ), mean pulmonary artery pressure ( %, p< . ) and rv stroke work ( %, p= . ), while neither pulmonary vascular resistance index nor epa was affected. increasing doses of norepinephrine improved rv free wall strain from - % to - % ( %, p= . ), tricuspid annular plane systolic excursion ( %, p= . ) and tricuspid annular systolic velocity ( %, p= . ). there was a trend for an increase in cardiac index assessed by both thermodilution (p= . ) and echocardiography (p= . ). conclusions: the rv function was improved by increasing doses of norepinephrine, as assessed both by strain-and conventional echocardiography. this is explained by an increase of rv preload. pulmonary vascular resistance is not affected by increased doses of norepinephrine. peripheral perfusion versus lactate-targeted fluid resuscitation in septic shock: the andromeda shock physiology study. preliminary report g hernandez , r castro , l alegría , s bravo , d soto , e valenzuela , m vera , v oviedo , c santis , g ferri , m cid , b astudillo , p riquelme , r pairumani , g ospina- tascón table . conclusions: this preliminary results suggest that using crt as a target for fr in septic shock appears to be feasible, and not associated with impairment of tissue perfusion-related parameters as compared to lactate-targeted fr. grant fondecyt chile introduction: shock patients often become resistant to catecholamines which often require the addition of a non-catecholamine vasopressor. preclinical studies suggest that in the presence of aadrenoceptor antagonism, the renin-angiotensin aldosterone system exerts the major vasopressor influence. we sought to determine the effects of angii or lypressin (lyp [porcine vasopressin]) on blood pressure in a norepinephrine (ne)-resistant hypotension pig model. methods: phentolamine (phn), a reversible α-blocker that antagonizes the vasoconstriction by ne, was continuously infused to induce hypotension. after ne-resistant hypotension was established, lyp or angii was then co-infused with phn. mean arterial pressure (map) and heart rate were continuously recorded (fig. ) . results: as shown in fig. conclusions: in a background of α-adrenoceptor blockade, at clinically comparable doses, the vasopressor effect of ang ii was maintained while those of ne and lyp were attenuated. these data suggest that the blood pressure effect of vasopressin-like peptides may require a functioning α-adrenoceptor. patients with shock who are resistant to increasing doses of catecholamines may also have vasopressin resistance potentially making angiotensin ii a preferred vasopressor for these patients. introduction: resuscitative endovascular balloon occlusion of the aorta (reboa) has been increasingly used for the management of both traumatic and non-traumatic hemorrhagic shock. however, there is limited evidence for its use in gastrointestinal bleeding (gib), especially in the icu setting. we successfully treated a patient with massive gib using reboa in the icu. we will discuss the difficulty performing the procedure and its countermeasure. methods: a case report. results: an -year-old woman was transferred to our hospital with shock. coffee grounds material was found in a nasogastric aspirate after intubation and upper gastrointestinal endoscopy identified a pulsating large duodenum ulcer without active bleeding, for which an elective procedure was planned. she was admitted to our icu, responded to initial resuscitation, and thereafter extubated. her systolic blood pressure (sbp) suddenly dropped to mmhg with massive hematochezia at that night, and did not increase despite resuscitation with blood products, crystalloid and norepinephrine. to buy time until measures for stop bleeding, we planned to place reboa in the icu. following the placement of a sheath in the left femoral artery, we tried to place a fr intra-aortic balloon occlusion catheter, which unintentionally and repeatedly went into the right common iliac artery because her left femoral artery was tortuous. after compressing the right lower abdomen, we managed to introduce reboa in zone . it took approximately minutes to successfully place the catheter. the patient's sbp increased immediately after the balloon inflation and bleeding was endoscopically controlled. introduction: the natural components of the pomegranate fruit may provide additional benefits for endothelial function and microcirculation. we hypothesized that chronic supplementation with pomegranate extract might improve glycocalyx properties and microcirculation during anaerobic condition. methods: eighteen healthy and physically active male volunteers aged - years were recruited randomly to the pomegranate and control groups ( in each group). the pomegranate group was supplemented with pomegranate extract for two weeks. at the beginning and end of the experiment, the participants completed a high intensity sprint interval cycling-exercise (anaerobic exercise) protocol. the systemic hemodynamics, microcirculation flow and density parameters, glycocalyx markers, and lactate and glucose levels were evaluated before and after the two exercise bouts. results: no significant differences in the microcirculation or glycocalyx were found over the course of the study. the lactate levels were significantly higher in both groups after the first and repeated exercise bouts, and were significantly higher in the pomegranate group relative to the control group after the repeated bout: . ( . - . ) vs. . ( . - . ) mmol/l, p = . . conclusions: chronic supplementation with pomegranate extract has no impact on changes to the microcirculation and glycocalyx during anaerobic exercise, although an unexplained increase in blood lactate concentration was observed. introduction: extracorporeal membrane oxygenation in adults in accompanied by high mortality. our ability to predict who will benefit from ecmo based on currently available clinical and laboratory measures is limited. the advent of single cell sequencing approaches has created the opportunity to identify cell populations and pathophysiological pathways that are associated with mortality without bias from a priori cell type classifications. identification of such cell populations would provide both an important prognostic markers and key insight into immune response mechanisms and therefore a possibility for advanced drug matching that may impact clinical response to ecmo in these patients. methods: whole genome transcriptomic profiles were generated from a total of , peripheral blood monocytes obtained from patients at the time of cannulation for ecmo (fig ) . differential gene expression analysis was performed with the monocle package for the r statistical analysis framework. time-to-event data were analyzed in a survival analysis with a log-rank test for differences. results: genes encoding several members of the heat shock family of proteins were up-regulated in cells from non-survivors. notably, these genes were expressed by a small fraction of cells ( . % on average). nevertheless, the proportion of cells expressing these genes was a significant predictor of survival to days (p = . by log rank test), with a particularly pronounced effect in the first days after initiation of ecmo support (fig ) . conclusions: the proportion of cells expressing genes encoding members of the heat shock proteins is predictive of survival on ecmo. majority of pt ( %) had no known predisposing conditions, followed by immobility ( %) and cancer ( %). in ecg analysis tachycardia and v -v t wave inversion were the most common findings whereas hypoxemia± hypocapnia were the most prominent features in abg analysis. pt ( %) had bleeding complications (none intracranial), ( . %) during rtpa, ( . %) in the first h and only pt required transfusion. mortality rate was %: % directly due to pe (all during cpr) and % due to late complications (newly diagnosed cancer and infections). conclusions: in our experience, fibrinolytic therapy is safe and effective but in submassive pe should be applied after thorough assessment of risks and benefits on individual basis aiming to patient tailored precision medicine. [ ] trials evaluated the role of levosimendan in preventing low cardiac output syndrome in patients undergoing cardiac surgery. the studies were similar in their design and recruited patients with preoperatively low lvef undergoing either isolated cabg or valve surgery combined with cabg (table ). in both, a -hour levosimendan infusion was started at induction of anesthesia. neither study met the primary efficacy composite enpoints, but both showed a clear tendency for better outcome in patients undergoing a cabg compared to a valve procedure. we are currently evaluating the solidity of a co-analysis based on shared end-points. we are planning a shared analysed of the data related to the cabg settings and analyze the aggregated mortality data for both studies at and months by cochran-mantel-haenszel odds ratio. data from individual studies would be analysed as fixed effect and breslow-day test was used to evaluate homogeneity of the odds ratios results: in the placebo groups of the two studies, the mortality is similar; . % ( / ) in levo-cts and . % ( / ) in licorn, corroborating the working hypothesis that the two studies can be coanalysed. in a preliminary combined analysis (fig ) , -day mortality was . % ( / ) in the placebo group and . % ( / ) in the levosimendan group. odds ratio was significantly in favor of levosimendan ( . ; % confidence interval . - . ; p= . , fig. ) conclusions: the levo-cts and licorn trials can be co-analysed in their sub-setting of patients requiring isolated cabg surgery for mortality at and months. a preliminary analysis on mortality reinforce the hypothesis that, in isolated cabg surgery, levosimendan lowers post-operative mortality significantly both at and months, when started at the induction of anesthesia introduction: emergency medical system (ems) -based st elevation myocardial infarction (stemi) networks allows not only stemi diagnosis in the pre-hospital phase but also reduces treatment delays; treat your fatal complications and the immediate activation of the catheterization laboratory. the aim of study was to investigate the effect of out-of-hospital by mobile intensive care (micu) versus hospital beginning treatment in hospitalization length and survival of patients with stemi diagnosis introduction: contrast induced nephropathy (cin) is a complex acute renal failure syndrome, which can occur after primary percutaneous coronary intervention (pci) and is an important cause of morbidity and mortality in this subgroup of patients. the aim of our study was to establish the incidence and predictors of cin after primary pci. we performed a retrospective analysis of stemi patients treated with primary pci in the period from january until september of . cin was defined as an absolute increase in baseline serum creatinine of ≥ . mg/dl ( μmol/l) or > % relative rise within hours after primary pci. we analyzed demographic characteristics, risk factors, clinical status at hospital admission, laboratory parameters, left ventricle ejection fraction and data regarding pci procedure. results: the study included patients, with an average age of . ± . years, . % of the patients were males. an average of . ± . ml of contrast medium per patient was utilized. cin developed in ( . %) patients and overall intra-hospital mortality was . %. in multivariate analysis, the independent predictors of cin were age> years ( introduction: left main coronary artery (lmca) disease is a disease of the main coronary branch that gives more than % of blood supply to the left ventricle, it carries high mortality without surgical intervention; [ ] however the influence of lmca surgery on morbidity icu measures needs to be explored. we aim to determine whether lmca is definitive risk factor for prolonged icu stay as a primary outcome and whether lmca is definitive risk factor for early morbidity methods: retrospective descriptive study with purposive sampling analyzing patients underwent isolated coronary artery bypass surgeries (cabg). patients were divided into groups those with lmca disease as group ( patients) and those with coronary arty disease requiring surgery but without lmca disease as group ( patients) then we will correlate with icu outcome parameters including icu stay length, postoperative atrial fibrillation, acute kidney injury, re-exploration, perioperative myocardial infarction, post operative bleeding and early mortality. results: patients with lms had significantly higher diabetes prevalence ( . % vs %, p= . ). however, we did not find a statistical significant difference regarding icu stay, or other morbidity and mortality outcome measures conclusions: diabetes was more prevalent in patients with lms. the latter group showed similar outcome as those without lms in this study these findings may help in guiding decision making for future practice and stratifying the patients care. introduction: multimorbidity in patients admitted for acute myocardial infarction [ami] is associated with higher risk for in-hospital mortality and adverse clinical outcomes. we investigated to what extent an increasing number of comorbidities affects the age-stratified excess risk of death and other clinical outcomes among patients with myocardial infarction. methods: we analyzed nationwide administrative data of ` admissions for an acute myocardial infarction between and . we calculated multivariate regression models to study the association of four comorbidities (chronic kidney disease [ckd], diabetes mellitus, heart failure [hf], and atrial fibrillation) and excess risk of in-hospital mortality, length of hospital stay [los] , and -day readmission and stratified the analysis for different age categories. results: the incidence of admissions for ami increased continuously during the observed decade without an increase in in-hospital mortality, los, and -day readmission. among admitted patients with ami, there was a stepwise increase in risk for adverse outcomes for each comorbidity. compared to patients with no comorbidity, patients with comorbidities had -fold increased risk for mortality (adjusted odds ratio [or] . , % confidence interval [ci] . to . ) and a similar risk for readmission (or . , ci . to . ). the los was . days (ci . to . ) in patients with no comorbidity and increased by . days (ci . to . ) with each additional comorbidity. these associations were stronger in younger compared to older patients. ckd was the strongest predictor of in-hospital mortality and los, while hf was the strongest predictor of -day readmission. conclusions: this study of nationwide admitted patients with ami found a stepwise increase in the risk for adverse outcome with increasing number of comorbidities, particularly in the younger patient population. younger, multimorbid patients may thus have the largest benefits from multidisciplinary treatments. introduction: certified cardiac arrest centers, sophisticated post cardiac arrest care and prehospital ecls teams aim to increase survivor rates with a preferable neurological outcome after cardiac arrest. centers also provide emergency ecls and ecls pick ups for cardiogenic shock patients before arresting. few data answer the question of the long-term quality of life after ecls therapy. methods: in a retrospective single center register we included patients after emergency ecls (ecpr and cardiogenic shock) between / and / discharged alive and performed a follow-up after years on average at / . in our center criteria to initiate ecls therapy in cardiogenic shock or under cardiac arrest are an observed collaps, shockable rhythm, absence of frailty and severe comorbidities. all patients were requested to take part in a telephone interview. thus, we analyzed survival, cpc scores and sf scores. results: patients with hospital survival after ecls were screened. % (n= ) had survived until / ; patients were not accessible; had ceased. survivors (mean±sd; min-max; ± ; - years, women) answered sf questionaires ± ; - months after ecls ( % cardiogenic shock, % ecpr with shockable rhythm in %). the participantsĆ pc scores were in median . the results of the sf were physical functioning ± , physical role functioning ± , bodily pain ± , general health ± , vitality ± , social role functioning ± , emotional role functioning ± and mental health ± . survivors who did not take part at the sf had a cpc score of in median (n= , personally signed refusals, language barriers, vegetative states). conclusions: after emergency ecls therapy and hospital survival % of our patients survived the following years up to over years with a preferable neurological outcome and a general mentally and physically satisfactory quality of life. a vague outcome in % limits the results of our study. introduction: successful weaning from va-ecmo requires the restoration of a sufficient cardiac function to ensure an adequate tissue perfusion. skin blood flow (sbf) is among the first to deteriorate during circulatory shock and the last to be restored after resuscitation. sbf would be a good predictor of successful weaning from va-ecmo. methods: patients with va-ecmo, who required a first weaning attempt, were included. weaning procedure (wp) was performed by a reduction of va-ecmo blood flow to l/min for minutes. the weaning criterion was an aortic velocity-time integral (vti) > cm. successful weaning from va-ecmo was defined as hemodynamic stabilization and without the need to increase the vasopressor dose during the next hours. sbf, assessed by skin laser doppler (peri-flux , perimed, right index finger); perfusion unit: pu), together with global hemodynamic parameters were obtained before and after min of weaning. receiver operating characteristic curves (roc) were generated to assess the ability and reliability of baseline parameters to predict a successful weaning. results: we studied wps in patients with va-ecmo for pulmonary embolism (n = ), post cardiotomy (n = ), acute coronary syndrome (n = ), myocarditis (n = ). these were successful (sw) in and unsuccessful (nsw) in . at baseline, hemodynamic variables, lactate, ecmo blood flow were similar in both groups (table ). sbf was greater in sw than nsw patients (table ). during wp, ci rose from baseline and was similar in sw and nsw (p= . ) ( table ). vtis were higher in sw than nsw ( ( - ) vs ( - ), respectively, p= . ). sbf decreased in sw and remained low in nsw (table ) . from the roc curves analyses, baseline sbf had the highest area under the roc curve with a cut off ≥ pu (sensitivity %, specificity %) (figure ). conclusions: sbf is a good predictor of successful weaning from va-ecmo introduction: postoperative cognitive dysfunction (pocd) is defined as a temporarily decline in cognition associated with surgery. long-term pocd ( months after surgery) occurs in - % of cardiac patients and is associated with a higher morbidity and mortality. endo-cabg is a new minimally invasive endoscopic coronary artery bypass grafting (cabg) technique that requires retrograde arterial perfusion which may be associated with a higher incidence of neurological complications. the aim of this study is to assess the incidence of pocd after endo-cabg. methods: sixty consecutive patients undergoing an endo-cabg were enrolled. pocd was assessed following the recommendations of the " statement of consensus on assessment of neurobehavioral outcomes after cardiac surgery". a comparative group of patients undergoing percutaneous coronary intervention (pci) and a control group of healthy volunteers were also enrolled. additional tests included the digit span test and digit symbol-coding test. patients were tested at baseline and at month follow-up. pocd is defined as a reliable change index (rci) ≤ - . (significance level %), or z-score ≤ - . in at least two different tests. results: after enrolling patients in each group, respectively in the endo-cabg-group, in the pci-group and healthy controls were analysed. patients suffering from a cva within three months after their procedure were automatically classified as having pocd (pci: n= ; endo-cabg: n= ). the total incidence of pocd was not different between groups (pci: n= ; endo-cabg: n= , p= . ). conclusions: our results suggest that the risk of pocd after endo-cabg is low and comparable with the risk of pocd after pci. introduction: rhabdomyolysis ( rml) post aortic surgery probably affects the renal outcome adversely [ , ] . there is no robust data regarding the same in literature. methods: retrospective single center data review; prior approval from institutional review board. patients were divided to two groups group -with rml ( ck above cut off levels u/litre) and group without rml. the determinants of rml and the impact of the same on outcome; predominantly renal function was evaluated. chi-square tests are performed for categorical variables whereas, student t tests (un-paired ) are performed with continuous variables. correlation is performed between creatine kinase and creatinine rise. p value . (two tailed) is considered for statistical significant level. results: out of patients, patients ( . %) developed rhabdomyolysis ( group rml) and did not( group non rml). demographic and intraoperative factors had no significant impact on the incidence of rml. there was a significantly higher incidence of renal complications including new postoperative dialysis in the rml group. other morbidity parameters were also higher in the rml group. conclusions: there is high prevalence of rml after aortic dissection surgery -identification of risk factor and early intervention might help to mitigate the severity of renal failure introduction: we investigate whether central venous pressure (cvp) pressure waveform signal can be informative in detection of slow bleeding in post-surgical patients. we apply a novel machine learning method to analyze cvp datasets to characterize bleeding in a porcine model of fixed rate blood loss. methods: thirty-eight pigs were anesthetized, instrumented with catheters, kept stable for minutes, and bled at a constant rate of ml/min to mean arterial pressure of mmhg. cvp waveforms were extracted from inspiration and expiration phases of respiration and statistically featurized. the proposed machine learning method, canonical least squares (cls) clustering, identifies correlation structures that differ between subsets of observations. we extend it to supervised classification. both clustering and classification methods yield human-interpretable models that reflect distinctive patterns of correlations within cvp waveforms. results: we conducted three experiments to discover structure in the physiological response to bleeding. first, we clustered respiration cycles with full knowledge of blood loss. the color-coded cluster assignments are shown in the figure . they are consistent with escalation of bleeding. second, we deployed clustering on only cvp features without blood loss. temporal structure was complemented with some subject-specific clusters (fig ) . third, we ran cls classification to decide whether an observation came from before or after the onset of bleeding (performance shown in the results: over the last decade, the number of patients with hlhs who underwent norwood has increased. interstage mortality has decreased, and is currently - %. significant morbidity was not seen at a rate higher than in the international literature. discharge planning, and community access to allied health professional services remained a concern. conclusions: the paediatric congenital cardiac surgical service in the united arab emirates is relatively new (compared to some services around the world). interstage mortality in hlhs is improving as a result of programme development, surgical progress and postoperative care. in the interstage period, there is currently no home monitoring programme in place. some patients were found to have had very extended hospital admissions. improved community support may reduce interstage mortality further, as well as improve the social situation of many of these patients. postoperative complications were observed in ( . %) patients. we lined out the prevalence of cardiac complications, such as heart failure and rhythm disturbances, observed in ( . %) and ( . %) patients respectively. hospital mortality rate was . % ( / ). the cause of mortality in all cases was acute heart failure, due to the initial severity of the disease, and in ( . %) cases an acute myocardial infarction was diagnosed. duration of postoperative period was . ± . days. conclusions: off-pump coronary artery bypass grafting can be safely performed with relatively low incidence of mortality and postoperative morbidity. prognostic value of mid-regional pro-adrenomedullin and midregional pro-atrial natriuretic peptide as predictors of multiple organ dysfunction development and icu length of stay after cardiac surgery with cardiopulmonary bypass in adults introduction: one of the most harmful complications after cardiac surgery with cardiopulmonary bypass is a syndrome of multiple organ dysfunction (mods). we consider that mid-regional proadrenomedullin (mr-proadm) and mid-regional pro-atrial natriuretic peptide (mr-proanp) plasma concentrations can be used as predictors of mods development and los in icu. methods: thirty six adult patients (mean age years, male) with cardiovascular diseases undervent cardiac surgery with cardiopulmonary bypass (heart valve(s) replacement - ( . %) patients, aorta and it`s branch surgery - ( . %) patients, valvular surgery and coronary artery grafting - ( . %) patients). nyha heart failure class ii was in ( . %) patients, iiiin ( %) patients, ivin ( . %) patients. in the dynamics levels of mr-proadm and mr-proanp were measured in the venous blood with the kryptor compact plus analyzer (thermo fisher scientific, germany) before day and on the st and th days after surgery. all patients were divided into subgroups according to the lengths of stay in the icu and the development of mod in the postoperative period. the data are shown as median and th and th percentiles. the data were compared by mann-whitney u-test, pvalue of < . was considered statistically significant. results: levels of mr-proanp did not significantly change at the study stages and did not have a significant difference between subgroups. the levels of mr-proadm increased in the first postoperative day and remained elevated for days. this increase was significantly higher in subgroups of increased los in icu and with mods. the data are shown in the table . conclusions: mr-proadm can be used as predictor of mods and los in the icu for adult patients underwent cardiac surgery with cardiopulmonary bypass. introduction: prolonged intensive care unit (icu) stay after cardiac surgery is associated with increased mortality and cost .the aim of this study was to investigate factors influencing prolonged icu stay. methods: consecutive patients who underwent cardiac surgery from june to october in our cardiothoracic department, were retrospectively investigated. group a consisted of pts with prolonged stay defined as more than days and group b the rest of the cohort. the following characteristics and perioperative factors were compared between the groups: smoking, diabetes, copd, redo(re-operation), ejection fraction (ef)< %, emergent procedure, cardiopulmonary bypass time (cpb)> min, low cardiac output syndrome (lcos), acute kidney injury(kdigo) and mortalitychi square test was used for the statistical analysis. introduction: hemorrhagic complications of extracorporeal membrane oxygenation (ecmo) pose a major morbidity and mortality. optimal anticoagulation strategies balancing risks of bleeding and thrombosis in children are poorly understood. we aimed to identify factors associated with non-surgical bleeding in the first ecmo hours. methods: we evaluated all pediatric (< yrs) post-cardiotomy patients requiring ecmo between dec -july stratifying them by presence/absence of surgical bleeding. non-surgical bleeding was defined as chest tube output > cc/kg/hr during the first -hours not requiring reoperation. patient characteristics and coagulation parameters at various time points after ecmo initiation were compared between groups, and receiver operator characteristic (roc) curves were constructed to identify models and thresholds with optimal predictive performance. figure . conclusions: deranged coagulation parameters, particularly kaolin rtime may predict non-operative bleeding in pediatric ecmo patients. these findings may guide therapeutic anticoagulation while avoiding hemorrhagic sequelae in at risk patients. introduction: elevated cardiac troponin (ctn) level in patients (pts) admitted in the intensive care unit (icu) is multifactorial and has been associated with a worse prognosis. the aim of the study was to review the frequency and the main cause of ctn elevation and to calculate a discriminating index. methods: we retrospectively assessed all pts admitted in our eightbed general icu during a -month period with at least one measurement of ctn during their icu stay. we recorded clinical characteristics, the level of ctn on admission, the maximum ctn during icu stay and the possible causes of elevation. variables are expressed as mean ± sd or as median and interquartile ratio (ir), according to the normality of their distribution. student´s Ô test or the mann whitney u tests were used to compare the group of elevated ctn with the group of normal ctn. the prognostic performance of elevated ctn was evaluated by the receiver operating characteristics (roc) curve. statistical analysis was performed using spss version . (spss, inc., chicago, illinois). results: in out of pts that ctn was measured at least once, abnormal levels (> . pg/ml) were found in ( %) of them, and the maximum ctn value was ( . ) pg/ml. the clinical characteristics of the pts are depicted in table . sepsis was the main cause of troponin elevation, which complicated by acute kidney injury (aki) in pts ( %). maximum ctn, aki and the difference of maximum -admission ctn (Äctn) differed significantly between pts who survived and pts who died (p= . and . , respectively). the area under the curve (auc) was . and the optimal prognostic cut-off value of Äctn was pg/ml with a sensitivity of . and a specificity of . conclusions: raised cardiac troponin values is a frequent finding in icu pts and sepsis is the driving cause. aki and the difference between maximum and admission ctn measurements differ significantly between pts who survive and pts who die. an elevation of ctn during icu hospitalization > pg/ml seems to be a threshold indicating poor prognosis regarding both mortality and aki. the prognostic role of nt-pro-bnp in septic patients with elevated troponin t level introduction: sepsis is frequently accompanied with release of cardiac troponin t (tnt) and nt-pro-bnp, but the clinical significance of this myocardial injury and cardiac dysfunction remains unclear [ ] . tnt is known to be an independent predictor of mortality, whereas the prognostic role of nt-pro-bnp is uncertain. methods: here, we report data of va-ecmo-patients, treated with dobutamine, levosimendan, suprarenin or no inotropic agens, in respect of -day survival. all data were collected retrospectively ( / to / ) at a single center, all patients with a survival below hours were excluded. while treatment of va-ecmo patients is strongly guided by standard operation procedures at our institution, no recommendation on positive inotropic therapy could be made. results: a total of va-ecmo patients were evaluated, of which patients were treated with levosimendan within hours after cannulation. day survival in the whole cohort was . %. a total of patients did not receive any positive inotropic therapy at hours after implantation (survival . %). survival was best in the levosimendan plus dobutamine group %, followed by dobutamine mono-therapy . % and levosimendan mono . %. survival with suprarenin mono was . %, suprarenin plus levosimendan . % and suprarenin plus dobutamine , %. pooling data, we found no evidence that levosimendan and/or dobutamine (survival . %, n= , p= . ) improves survival over no inotropic therapy (fig ) . therapy with any combination including suprarenin however resulted in poor survival ( . %, n= , p= . ). adjustment for lactate levels or ecpr did not change the results. conclusions: this retrospective analysis of va-ecmo patients shows no evidence that early inotropic therapy improves outcomes in va-ecmo patients. this conclusion is obviously biased by retrospective design. until randomized data are available, suprarenin however should be avoided. survey of non-resuscitation fluids in septic shock a linden-sonderso introduction: positive fluid balance is associated with poor outcome in septic shock. the objective of the present study was to characterize non-resuscitation fluids in early septic shock. methods: consecutive patients > years of age were screened for inclusion criteria during a -month period in icus in sweden and in canada. inclusion criteria were septic shock per sepsis- definition within hrs of icu admission. a maximum of patients per center were included. type, indication and volume of non-resuscitation fluids were recorded during the first days of admission. fluids other than colloids, blood products and crystalloids given at rate > ml/kg/h were considered to be non-resuscitation fluids. the study was registered on clini-caltrials.gov (nct ). data are presented as median (interquartile range). results: a total of patients were included between march st and june th (see table for demographics). patients received ( - ) milliliters (ml) of non-resuscitation fluids introduction: we aimed to ascertain the extent and make-up of fluid overload in critically ill patients and to identify whether delivery of more concentrated medications could reduce this. positive fluid balance is associated with increased mortality [ ] . a recent study has shown that the predominant component of fluid overload was from iv medications and maintenance fluid [ ] . methods: we reviewed sequential patients admitted to our icu with an apache ii score of greater than and a length of stay (los) greater than hours. the patients' electronic admission summary was interrogated to establish: length of stay (los) fluid balance at hours, total volume administered as iv medications, total volume administered as maintenance fluid and total fluid administered introduction: in children less than kilograms, maintenance fluids are routinely added to the resuscitation requirements calculated using parkland's or other formulae. the contribution of this component for fluid resuscitation in children can add a significant quantity to total estimated fluid requirements. for example, in a child who is kilograms with a % burn, the maintenance fluid requirement is mls per hours and the resuscitation component per parkland's will be x x %= mls. hence, the maintenance requirement can exceed the resuscitation requirement in this child if the burn surface area is less than a % burn. the contribution of maintenance fluids to the total fluid requirements in small children with thermal injuries is under-recognised and not frequently studied. methods: to understand the contribution of maintenance fluids to the total fluid requirements in children less than kilograms who need resuscitation for thermal injuries of different sizes, we numerically simulated . children who had similar weights but different burn sizes and . children with similar burn size but different weights. the results are as shown in fig introduction: accurate quantification of fluid in resuscitation of thermal injuries is important for benchmarking, comparing and improving outcomes. in adults, it is usually expressed as mls/kg/%tbsa. in children, maintenance fluids are added to the resuscitation requirements. this is kept constant and the resuscitation component is titrated to meet pre-defined end points-usually urine output. maintenance fluids are not uniformly stratified across the weight ranges. we propose that quantification of fluids in mls/ kg/%tbsa in children does not accurately capture fluid needs for resuscitation due to the maintenance component of the fluid requirement. methods: we conducted this retrospective study in children admitted to a single-center burns intensive care unit (bicu) between january and december . children ≤ kilograms with tbsa ≥ % admitted within hours of their injury were included. oe (observed to expected ratio) and fluid in mls/kg/% tbsa were calculated as shown in figure . results: there were children in the cohort with half requiring invasive mechanical ventilation in the bitu and nearly a quarter requiring inotropic support. the demographic details are as shown in table . the oe ratio at the end of hours in the cohort was . ( . - . ). the total fluid given was . ( . , ) mls/kg/ % tbsa. the titrated resuscitation component was . ( . , . ) mls/kg/tbsa. total fluid (which included the maintenance fluid) had a poor correlation with oe ratio r = . (fig ) . exclusion of the maintenance fluid had a better correlation with the oe ratio r = . conclusions: to capture differences in the titratable resuscitation component rather than differences in the maintenance requirements, fluid should be quantified in children by excluding the maintenance component when expressed as mls/kg/%tbsa. dynamic arterial elastance for predicting mean arterial pressure responsiveness after fluid challenges in acute respiratory distress syndrome patients p luetrakool , s morakul , v tangsujaritvijit introduction: dynamic arterial elastance (eadyn; pulse pressure variation/stroke volume variation; ppv/svv) is a dynamic parameter of arterial load that can be continuously monitored. previous study proposed that eadyn was able to predict mean arterial pressure (map) responsiveness after fluid challenge [ ] [ ] [ ] [ ] [ ] . the objective of this study was to assess whether the eadyn was able to predict map responsiveness in acute respiratory distress syndrome (ards) patients ventilated with low tidal volume. methods: we performed a prospective study of diagnostic test accuracy in adult ards patients with acute circulatory failure and fluid responsiveness. all patients are continuously monitored blood pressure via arterial line connected with flotrac® transducer and vigileo® monitor. once the attending physicians decided to load intravenous fluid, we recorded ppv/svv and also other hemodynamic parameters before and after fluid bolus. map responsiveness was defined as an increase in map ≥ % from baseline after fluid challenge. results: twenty-three events were included. nine events ( . %) were map-responsive. cardiac output, heart rate and stroke volume were similar in both map-responder and map-nonresponder group. baseline map, diastolic blood pressure (dbp) and pulse pressure (pp) were significantly different after fluid challenge in map-responder group. eadyn of preinfusion phase was failed to predict map conclusions: one of the arterial load parameters such as eadyn derived from non-calibrated pulse contour analysis method was unable to predict map responsiveness in ards patients with low tidal volume ventilation. the our aim is to test the hypothesis that in fr septic shock patients, fluid load will determine a significant increase in pmsf but not in cvp. we prospectively included all mechanically ventilated patients with diagnosis of septic shock with invasive hemodynamic monitoring (transpulmonary thermodilution volumeview-ev ed-wards©). we collected hemodynamic and metabolic data and pmsf with the inspiratory holds technique, before and after a fluid challenge (fc) of ml of ringer lactate in minutes). fr was defined as an increase in cardiac output (co)> %. results: measures were obtained in patients. in case we observed fr. we found a significant increase in pmsf after a fc (mean difference(md) . ± . mmhg, p=. ). cvp increased significantly (md . ± . mmhg, p=. ). pmsf increased significantly in non-fr (md ± mmhg, p=. ) but not in fr while cvp was higher after fc only in fr (md . ± . mmhg, p=. ). venous return gradient (pmsf-cvp) globally increased after fc (md ± mmhg, p=. ), but only in non-fr such increase was significant (md ± mmhg, p=. ). no correlation was found between the variation co and venous return gradient. we did not find any improvement in metabolic parameters after the fluid challenge. conclusions: pmsf and combined cvp variations do not correlate with fr in our cohort of septic shock patients. inspiratory holds may not be adequate to infer pmsf in such context. further studies are warranted to investigate the effect of fc on pmsf in this field. evaluation of pre-load dependence over time in patients with septic shock i douglas , p alapat , k corl , m exline , l forni , a holder , d kaufman , a khan , m levy , g martin , j sahatjian , w self , e seeley , j weingarten , m williams , c winterbottom , d hansell is an effective method to predict fluid responsiveness (fr) or cardiac response to preload expansion. we have previously shown that fluid responsiveness is a dynamic state, changing frequently over a hour monitoring period. methods: fresh is a currently enrolling prospective randomized controlled study, evaluating the incidence of fr and patient centered outcomes in critically ill patients with sepsis or septic shock (nct ). patients randomized to plr guided resuscitation were evaluated every - hours over the first hours of care and classified as fr if the sv increased > % when measured with non-invasive bioreactance (starling sv, cheetah medical). the time of first fr was noted. results: a total of plr assessments were performed in patients over a hour monitoring period. % were female, and the average age was years. plrs were evaluated over time, with time representing initial fluid resuscitation ( figure ). when individual subjects were evaluated over time, % of subjects who became fr only after hours showed evidence of lv/rv dysfunction ( figure ). conclusions: fluid responsiveness or preload dependence frequently changes for septic shock patients over the first hours of care. evidence suggests it is beneficial to periodically perform an assessment of preload responsiveness to guide fluid administration, as preload dependence is a dynamic and changing state. preload dependence provides additional information beyond fluid responsiveness. those patients who remain primarily fluid non-responsive (preload independent) are more likely to demonstrate echo confirmed lv/rv dysfunction, as the delay in return to cardiac function may be related to underlying cardiac deficits. further evaluation may be indicated in preload independent patients. introduction: hydroxyethyl starch (hes), a synthetic colloid, has been used as a volume expander, and is associated with renal impairment in patients with sepsis. however, a small dose of hes ( %, / . ) has sometimes been used in acute ischemic stroke. therefore, we investigated whether a small dose of hes was linked with renal deterioration in patients with acute ischemic stroke. methods: a consecutive patients with acute ischemic stroke within days from onset were included between january and may (fig ) . we collected admission serum creatinine (scr), estimated glomerular filtration rate (egfr), and renal function was assessed using kdigo definition of acute kidney injury on hospital days to as to patient's hospitalization period. is crucial for venous return and volaemic status, and as such it is a useful parameter in physiology and clinical settings alike. we tested whether: near infra-red spectroscopy (nirs) could be effective at measuring msfp both in healthy individuals and in conditions with a rise in interstitial pressures; after an occlusion pressure is relieved, the decrease in venular blood volume could allow calculation of τ (time constant) and thus venous resistances (rv). in order to verify these hypotheses we used a forearm nirs probe on healthy individuals at rest and during different degrees of maximal voluntary contraction (mvc). methods: healthy subjects volunteered in the study that took place at sant'andrea hospital in rome (italy). all subjects had venular pressures and volumes assessed via a nirs probe positioned on the forearm using a pressure-cuff in steps of mmhg from to mmhg, at rest and at % and % mvc. for each patient msfp, unstressed volume (vu) and stressed volume (vs) were measured. a temporary mmhg occlusion was obtained and volume time course was calculated upon release, to derive τ . results: p-v relationship was found to have a -slopes shape reflecting venular network changes. we measured vu, vs, and obtained msfp values of . ± . mmhg, p< . ; during exercise no changes in vu and vs were noted but msfp values rose; value was found to be . ± . sec at rest and . ± . sec after exercise, reflecting a reduction in rv. conclusions: nirs measurements on healthy subject may have implications in the clinical assessment of critical care patients where changes in interstitial pressure are possible. introduction: in the pathogenesis of multiple organ dysfunction syndrome (mods) important role plays the development of hepatic dysfunction. a known method for assessing hepatic blood flow is reohepatography (rhg). however, it requires the analysis of a large number of parameters of the rheogram curve. the aim of this study was to develop a method for assessing arterial hepatic blood flow based on the rhg in patients with mods after abdominal surgery. methods: patients in the department of anesthesiology and intensive care unit were included in a prospective study ( men and women, age . ± . years, weight . ± . kg.). all patients were divided into two groups: group -patients after orthopedic and trauma surgery (n = ), group -patients after abdominal surgery with mods (n = ). patients in the groups did not have statistical differences by sex, age, body weight, height. rhg was carried out using the "reo-spectr" (russian federation). we have compared the rhg indicators between the groups ( table ) . we have developed a method for assessing hepatic arterial blood flow, which consists in determining the area under the arterial part of rhg curve using the simpson's rule. its normal values range from . mΩ *s to . mΩ *s. the method is non-invasive, can be applied at the patient´s bed. its advantage is simplicity, it can be used for rapid diagnosis and monitoring the effectiveness of treatment. area under the rhg curve in the group were . ± . mΩ *s and . ± . mΩ *s in the group (p < . ). conclusions: patients after abdominal surgery with mods have impaired hepatic blood flow, which may be associated with liver pathology caused by main surgical disease (obstructive jaundice) and hemodynamic disorders caused by acute cardiovascular failure. the method we developed allows us to determine disorders of hepatic arterial blood flow in the early stages before signs of liver dysfunction appear. comparison of pulse oximetry hemoglobin with laboratory measurement of arterial and central- results: patients: % male, median years ( - ); p:f ratio ( - ); peep ( - ); apache iii . ( ); median ventilation time days ( - ). fair agreement was seen in subjective assessment vs objective measures with binary assessment of rv size and function. ordinal data analysis showed poor agreement with rvfws ( figure ) and rv dimensions. if onestep disagreement was allowed the agreement was good ( table , ). significant overestimation of severity of abnormalities was seen comparing subjective assessment with rv eda and tapse, s' and fac. there was no difference in agreement values when accounting for clinician echo experience, perceived expertise (at level of cardiologist) or type of qualifications. conclusions: relatively low levels of agreement were seen with subjective assessment vs objective measures of rv size and function assessed by echo. it seems prudent to avoid subjective rv assessment in isolation and a combination of objective and subjective measures should be used. introduction: even short periods of hypotension are associated with increased morbidity and mortality. using high-density numerical physiologic data, we developed a machine learning (ml) model to predict hypotension episodes, and further characterized risk trajectories leading to hypotension. methods: a subset of subjects with / hz physiological data was extracted from mimic , a richly annotated multigranular database. hypotension was defined as > measurements of systolic blood pressure ≤ mmhg and mean arterial pressure ≤ mmhg, within a -minute window. derived features using raw measurements of heart rate, respiratory rate, oxygen saturation, and blood pressure were computed. random forest (rf), k-nearest neighbors (knn), and logistic regression models were trained with -fold cross validation to predict instantaneous risk of hypotension using features extracted from the data leading to the first episode of hypotension (cases) or icu discharge in subjects never experiencing hypotension (controls). for a given subject, risk trajectory was computed from the collation of instantaneous risks. results: from a source population of subjects, subjects met our definition of hypotension, and subjects without hypotension comprised the control group. features were generated from the four vital signs. the area under the curve (auc) for random forest classifier was . , out-performing logistic regression (auc . ) or k-nearest neighbors (auc . ) (fig ) . risk trajectories analysis showed average controls risk scores < . (< % risk of future hypotension), while the hypotension group had a rising risk score ( . to . ) in the hours leading to the first hypotension episode, and significantly higher scores leading into subsequent episodes (fig ) . conclusions: hypotension episodes can be predicted from vital sign time series using supervised ml. subjects developed hypotension have an increased risk compared to controls at least hours prior to the episode. introduction: in critically ill patients or in patients undergoing major surgery, monitoring of co is recommended [ ] [ ] [ ] . less-invasive advanced hemodynamic monitoring with pwa is increasingly used in perioperative and critical care medicine. in this study, we evaluate the measurement performance of an uncalibrated pulse wave analysis (pwa) device (mostcareup, vygon, ecouen, france) compared with cardiac output (co) assessment by pulmonary artery thermodilution (patd) in patients after cardiac surgery. methods: in patients after cardiac surgery, we performed seven sets of patd measurements to assess patd-co. simultaneously, we recorded the pwa-co and compared it to the corresponding patd-co. to describe the agreement between pwa-co and patd-co we used bland-altman analysis showing the mean of the differences and %-limits of agreement and calculated the percentage error. results: we included patients in the analysis. the bias between pwa-co and patd-co was . l*min- . upper and lower % limits of agreement were + . l*min- and - . l*min- . the percentage error was . %. conclusions: pwa-co estimated with using the mostcareup device shows good agreement with pulmonary artery thermodilutionderived co in patients after cardiac surgery. introduction: non-invasive continuous blood pressure monitoring devices have been investigated, however, these devices did not have sufficient accuracy and precision. we developed a continuous monitor using the photoplethysmographic technique and tested the accuracy and precision of this system to ensure it was comparable to conventional continuous monitoring methods used for critically ill patients. methods: the study device was developed to measure blood pressure, pulse rate, respiratory rate, and oxygen saturation, continuously with a single sensor using the photoplethysmographic technique. patients who were monitored with arterial pressure lines in the icu were enrolled. the physiological parameters were measured continuously for minutes at -minute intervals using the study device and the conventional methods. the primary outcome variable was blood pressure. results: pearson fs correlation coefficient between the conventional method and photoplethysmography device were . for systolic blood pressure, . for diastolic blood pressure, . for mean blood pressure, . for pulse rate, . for respiratory rate, and . for oxygen saturation. percent errors for systolic, diastolic and mean blood pressures were . % and . % and . %, respectively. percent errors for pulse rate, respiratory rate and oxygen saturation were . %, . % and . %, respectively. conclusions: the non-invasive, continuous, multi-parameter monitoring device presented high level of agreement with the invasive arterial blood pressure monitoring, along with sufficient accuracy and precision in the measurements of pulse rate, respiratory rate, and oxygen saturation. conclusions: stroke volume measurement using bioreactance technique had strong correlation with odm while pwtt had moderate correlation. both devices had small bias with wide limits of agreement and percentage error compared with odm. therefore, these devices are not interchangeable with odm. however, using trends in stroke volume to guide treatment might still be acceptable. introduction: hemorrhage is the most common cause of trauma deaths and the most frequent complication of major surgery. it is difficult to identify until profound blood loss has already occurred. we aim at detecting hemorrhage early and reliably using waveform vital sign data routinely collected before, during, and after surgery. methods: we use waveform vital sign data collected at hz during a controlled transition from a stable (non-bleeding) to a fixed bleeding state of pigs. these vital signs include airway, arterial, central venous and pulmonary arterial pressures, venous oxygen saturation (svo ), pulse oximetry pleth and ecg heartrate, continuous co, and stroke volume variation (lidco). we used gated recurrent units (gru), long short-term memory (lstm) and dilated, causal, one-dimensional convolutional neural (table ) . however, outside of the very low fpr range (cf. rocs in fig. and ), our models appear inferior to a referenced random forest (rf) classifier. conclusions: our work demonstrates the applicability of deep learning models to diagnose hemorrhage based on raw, waveform vital signs. future work will address why the rf classifier can address the greater homogeneity of subjects when they bleed compared to an apparently wide dispersion of their statuses when being stable. this work is partially supported by nih gm . can myocardial perfusion imaging with echo contrast help recognise type acute myocardial infarction in the critically ill? introduction: many instances of significant bleeding may not occur in highly monitored environment, contribution in the delay in recognition and intervention. we therefore proposed a noninvasive monitoring for early bleeding detection using photoplethysmography (ppg). methods: fifty-two yorkshire pigs were anesthetized, stabilized and bled to hemorrhagic shock, and their invasive arterial blood pressure (abp), and ppg data were collected [ ] . time series of vital signs were divided into data frames of minute updated every seconds and beat to beat features were computed. the final feature matrix contained abp features and ppg features. a supervised machine-learning framework using least absolute shrinkage and selection operator regularized logistic regression model was constructed to score the probabilities for hemorrhage of each data frame. data in stabilization was set as negative and data in bleeding was set as positive. model performance was evaluated by receiver operating characteristic (roc) area under the curve (auc) with leave-one-out cross validation, and its precision was assessed with activity monitoring operative characteristic (amoc). results: two different models were proposed using abp and ppg features separately. figure showed the ppg model could classify the hemorrhage with auc = . , where the auc of abp model was . . figure showed the ppg model could detect the hemorrhage on average . minutes (equals to ml blood loss) if the false alarm rate of / was tolerated, whereas the average detection time of abp model were . minutes at same threshold of false alarm rate. conclusions: we proposed a novel non-invasive bleeding detection approach using ppg signals only. this method potentially can improve the identification of hemorrhage with in patients and environments where invasive monitoring is unavailable. table , catheter and procedure characteristics are shown in table . the median angle of bed position was °. no patients were positioned in neutral or tp. all procedures were successful with a mean of . punctures per patient, and a maximum of . the median procedure time was . minutes. no major complications occurred in any of our patients. conclusions: central venous catheterisation in moderate upright position is feasible and can be done safely when using realtime ultrasound by well-trained physicians. we recommend performing clinical assessment and pre-procedural ultrasound to choose the optimal puncture site and position in order to attain an optimal ultrasound visualisation of the vessel and patient comfort. methods: a retrospective analysis of patients presenting to tertiary-care emergency department who required cvc for vasopressor administration was carried out. all central venous cannulation into the right brachiocephalic vein was performed with ultrasound guidance using the high frequency linear probe. right brachiocephalic vein was visualised in its long axis. the needle was positioned just beside the centre of ultrasound probe degrees below the coronal plane and degrees angle to the ultrasound probe and advanced just behind the clavicle. results: the mean puncture time taken to perform this procedure, calculated from the needle piercing the skin until to the aspiration of blood from the brachiocephalic vein through the needle, was ± . s. no procedure-related complications were detected. conclusions: the oblique needle trajectory of right brachiocephalic vein cvc in adult is feasible and able to visualised well the anatomical structure, hence avoid complications. introduction: central venous cannulation, a routine procedure on intensive care units, is associated with a low complication rate. as a consequence, the routine use of chest x-ray (cxr) or ultrasound (us) to assess these complications is under discussion. our aim was to identify risk factors for central venous catheter (cvc) placement associated complications that can help decide whether or not follow-up using cxr and/or us is indicated. methods: multicenter prospective, observational study. consecutive critically ill adult patients who underwent cvc placement. either the internal jugular vein or subclavian vein was cannulated. complication rates were determined. predicting factors were obtained through a questionnaire filled in by physicians after placing a cvc. if the questionnaire was incomplete or data was missing, analyses were performed using the available data. patient characteristics were duplicated if a patient recieved more than one cvc. outcomes were iatrogenic pneumothorax and malposition. pneumothorax was detected using us, whereas cxr was used to determine cvc malposition. table . usguidance, insertion site, and setting were predictive for complications. the overall cvc placement associated complication rate is low and multiple risk factors associated with the occurrence complications were identified. a complication rate this low, strongly suggests that routine post-procedural diagnostics is superfluous. therefore, we suggest, provided that uneventful execution of the procedure is assured, post-procedural diagnostics are only necessary in selected cases with (multiple) risk factors. introduction: the use of ultrasound for subclavian vein cannulation (scv) has developed poorly due to the difficulty of visualizing this vein via the classical infraclavicular approach. we explored the feasibility of ultrasound-guided subclavian vein catheterization via a supraclavicular approach methods: prospective study conducted over six-month period in intensive care unit. after approval of the ethics committee, we included patients over years of age and requiring central venous access. exclusion criteria were: hemostasis disorders, puncture area infections and cervico-thoracic vascular malformations the procedure consisted of catheterization of the vsc with a supraclavicular approach under ultrasound guidance using an ultrasound in plane approach (fig and ). data collection included clinical and ultrasound data: scv depth, diameter and length, catheterization time, number of needle redirection, cannulation success and complications. results: thirty four patients were included. age: ± (mean ± sd), % of whom were male. the success rate of scv catheterization was % (one failure). the depth of the scv was ± . mm and its diameter was ± . mm. the puncturable length of the scv was ± mm and the puncture angle was ± °. the time required to obtain an adequate ultrasound image was ± seconds. the interval between the beginning of the puncture and the insertion of the guidewire into the vein was ± sec. the total catheterization time was ± seconds. the number of needle redirection . +/- . redirects. the quality of the ultrasound image was excellent or good in . % of cases. an arterial puncture was observed in two patients conclusions: this preliminary study demonstrated the feasibility of the subclavian vein cannulation via the supraclavicular approach. more study are required to confirm its safety and to compare this approach to the infraclavicular acces using ultrasound. introduction: lung ultrasound b-lines, a comet-like reverberation artefacts arising from water-thickened interlobular septa, indicate extravascular lung water which is a key variable in heart failure management and prognosis. aim of this study is to measure the correlation between lung ultrasound b-lines and nyha functional classification. methods: this is a months prospective study on congestive heart failure patients conducted in urban emergency departments in malaysia. following enrolment, patients had their functional capacity categorised based on nyha classification, followed by point of care ultrasound (pocus) lung scan using a mhz linear probe. the scanning was performed by trained emergency physicians. the longitudinal scan done at the recommended zones of both left and right lungs and the total number of b-lines identified were summed up as the comet score. comet score of , , and were categorised based on amount of blines of less than , - , - and more than b-lines respectively. results: hundred and twenty-two patients were analysed ( males( . %) and females( . %)) ranging from to years old. comet score of , and were found to be statistically significant with presence of paroxysmal nocturnal dyspnoea, elevated jugular venous pressure, lung crackles, bilateral pitting oedema and chest radiographic findings. a moderate correlation between nyha classes with comet score , and (rs= . (p< . )) was documented. conclusions: our study demonstrated a moderate correlation between nyha classes and lung ultrasound b-lines. lung ultrasound may be a potential tool to objectively determine the functional capacity in patients with congestive heart failure and monitor its changes in response to treatment and disease progression. the introduction: point of care ultrasound (pocus) is a tool of increasing utility in the management of the critically ill patient. guidelines exist for training and accreditation in pocus [ , ] however the widespread use of pocus has been hampered by a lack of mentors. online communication with end-to-end security, such as whatsapp ™ are increasingly used in medicine as a communication aid [ ] . some individuals are using such communications to share pocus images for review-the overall sentiment around these tools is unknown. methods: an online survey of pocus users was conducted via twitter ™. the question was "in situations where an expert opinion on an ultrasound is not immediately available, is it acceptable to get an expert review via an online medium such as whatsapp, and would you be happy to be that expert?" results: votes were received. voters were a mix of pocus users from the usa, europe, and australia. % said the medium was acceptable, and that they would be happy to provide expertise. % voted "no", with % voting "other" (fig ) . conclusions: in this international survey of pocus users, % were happy to provide and receive mentorship using remote software such as whatsapp. distance mentorship for pocus training should be explored. [ ] . a description of the development and refinement of insight -a feasibility and clinical effectiveness randomized controlled trial. methods: a modified delphi exercise was used to select the most beneficial ultrasound windows and imaging questions to ask for each window in scheduled inter-professional ultrasound. nurses, doctors and physiotherapists from critical care were given the same information regarding potential utility of each window. the windows and associated questions were individually ranked; each window and question tested against three further criteria; and filtered by ease of training to level standard; clinical usefulness; time of practical delivery and applicability across an inter-professional group. results: the modified delphi exercises and prioritization exercise ranked ease of adoption by training; feasibility within the time frame and clinical usefulness to develop a core insight scan of domains, each with set binary questions (tables and ) conclusions: we have developed a research intervention that will allow us to test the effectiveness of inter-professional scheduled whole body assessment of critically ill patients by ultrasound. we now plan to conduct a clinical effectiveness trial with an internal pilot to confirm feasibility. to search for optimal pressing time, the plots from the color sensor during nail bed compression were analyzed. we found two phases in the color sensor plots. in the initial part of compression, the plots changes rapidly (rapid phase) and then the slope of plots reduces (slow phase). the pressure release during the rapid phase could destabilize the measurement. the longest period of the rapid phase was . s among all the study subjects. thus, a pressing time of s seems to be needed to obtain stable crt measurements. conclusions: on our study for the investigation of standard pressing time and strength for crt measurements, pressing the nail bed with - n and s appears to be optimal. detection of pancreas ischemia with microdialysis and co sensors in a porcine model introduction: pancreas transplantation is associated with a high rate of early graft thrombosis. current postoperative monitoring lack tools for early detection of ischemia, which could precipitate a graft-saving intervention. we are currently exploring the possibility of ischemia detection with microdialysis and co -sensors in the organ tissue or on the surface in a porcine model. methods: in anesthetized pigs, co -sensors and microdialysis catheters are inserted into the parenchyma or attached to the surface of the pancreas. pco is measured continuously and lactate is sampled with microdialysis every min. ischemia is induced by sequential arterial and venous occlusions for minutes, with minutes of reperfusion in between. results: pco increased and decreased in response to ischemia and reperfusion within minutes. lactate increased and decreased with the same pattern, but with a considerable delay as compared to pco . an example is depicted in figure . the values are presented in introduction: reliable automated handheld vital microscopy (hvm) image sequence analysis is a prerequisite for use of sublingual microcirculation measurements at the point-of care according to the current consensus statement. we aim to validate a recently developed advanced computer vision algorithm [ ] versus manual analysis in a wide spectrum of populations and contexts. methods: our collaborators were invited to contribute raw data of published or ongoing institutional review board approved work. inclusion criteria were use of the cytocam hvm device, manual analysis with the ava software, and image quality as independently assessed by massey score of < in > % of recordings in a random subset of each study. subjects from studies were included, covering clinical and experimental populations, major shock forms and interventions to recruit the microcirculation (table ) . results: , , red blood cells were tracked by the algorithm across , frames in measurements in real time. a good to excellent correlation was found between algorithm-determined and manual capillary density (p< . , r . - . , figure ). capillary perfusion was classified using space-time diagram derived red blood cell velocity (rbcv), yielding good correlation with manual analysis for functional capillary density und proportion of perfused vessels. microcirculatory alterations during disease and interventions were equally detected by the algorithm and manual analysis. change in flow short of severe abnormality was reflected in absolute rbcv but not microcirculatory flow index. conclusions: we demonstrate the validity of automated software for hvm image sequence analysis across broad populations, disease conditions and interventions. thus, microcirculatory assessment at the bedside may finally complement point-of-care evaluation of disease severity and treatment response in critically ill patients and during surgery. introduction: in , naumann et al introduced the poem score as a real-time, point-of-care score to assess sublingual microcirculation [ ] . our study aimed to determine the reproducibility of the poem score. methods: two expert operators used a sidestream darkfield (sdf) videomicroscope (cytocam, braedius, netherlands) to separately acquire four high-quality video clips and assign a poem score to each image in adult mechanically ventilated patients. each operator was blinded to the other's images and analysis. video clip scores and acquisition times were recorded. results: of the patients enrolled in this study, % (n= ) required vasopressors. we categorized poem scores - as "normal" and poem scores - as "impaired." (fig ) . with only one instance of interrater disagreement (i.e., a single image scored as versus ), cohen's kappa ( . ) confirmed a strong correlation between interpreters. the mean time to complete a study session was minutes. conclusions: the present inability to quickly characterize the quality of sublingual microcirculation as either normal or impaired at the point of care limits real-world clinical application of this resuscitative endpoint. the rapidly obtained poem score appears to be reproducible between bedside interpreters. future studies should assess the effect of poem score-guided resuscitation. . sublingual microcirculatory images were obtained using a cytocam-idf device (braedius medical, huizen, the netherlands) and analyzed using standardized published recommendations. results: the median age of participants was years. we found no significant difference in proportions of hemodynamic responders before and after marathon ( % vs %, p= . ). also we did not find differences between plr induced changes of total vessel density (tvd) and proportion of perfused vessels (ppv) of small vessels before and after marathon. correlations between changes of sroke volume and changes of tvd or ppv of small vessels during plr were not significant. conclusions: marathon running did not change microcirculatory responsiveness. introduction: clinical measurement of mitochondrial oxygen tension (mitopo ) has become available with the comet system [ ] . a question with any novel technique is whether it is feasible to use in clinical practice and provides additional information. in elective cardiac surgery patients we measured cutaneous mitopo and tissue oxygenation (sto ). methods: institutional research board approved observational study in patients undergoing cardiopulmonary bypass (cpb). mitopo measurements were performed on the left upper arm (comet, photonics healthcare b.v.) by oxygen-dependent delayed fluorescence of aminolevulinic acid (ala)-induced protoporphyrin ix [ ] . priming of the skin was done with ala (alacare, photonamic gmbh) applied the evening before surgery. sto measurements (invos, medtronic) were done in close proximity to the comet sensor. results: at the time of writing of patients were enrolled and mitopo measurements were feasible in this clinical setting. mitopo appeared sensitive with a high dynamic range. for example, highdose vasopressor therapy decreased mitopo and blood transfusion increased a low mitopo but not a high mitopo . in the example in figure , mitopo is clearly dependent on cpb flow and the restored cardiac circulation is able to maintain good cutaneous oxygenation after cpb even before returning of cellsaver blood. sto had the tendency to provide relatively stable values within a small bandwidth and little response to even major hemodynamic changes. conclusions: mitopo shows the effect of interventions on mitochondrial oxygenation and provides additional information compared to standard monitoring and sto . introduction: traumatic asphyxia is a rare condition in which breathing and venous return is impaired due to a strong compression to the upper abdomen or chest region, and induces swelling, purplish red appearance, and petechiae around the face and neck. to our knowledge, there are no reports describing details of traumatic asphyxia including the clinical course and the therapeutic reactivity from cardiac arrest. we focused on cardiac arrest among all traumatic asphyxia patients treated at our hospital, and investigated their clinical features and therapeutic reactivity. methods: sixteen cases of traumatic asphyxia involved with our hospital between april and march were reviewed by using the pre-hospital activity record, medical record, and hyogo prefectural inspection record. these patients were divided into three groups. the first group had already cardiac arrest at the time of rescue from the trapped place (group a; cases). the second group became cardiac arrest after the rescue (group b; cases). the third group did not experience cardiac arrest (group c; cases). results: all cases had abnormal findings in skin or conjunctiva (table ) . total mortality rate reached %, but among cases of group a and b who resulted in cardiac arrest, there were cases with injury severity score or more and abbreviated injury scale in the chest or more. they had pneumothorax, flail chest, pericardial hematoma. seven of them restored spontaneous circulation, and two cases achieved neurologically full recovery. conclusions: there are some cases of traumatic asphyxia whose therapeutic reactivity is very good even after cardiac arrest, so it is important not to spare efforts for life support in such cases. rhythm and % witnessed arrest, five hundred ten ( %) patients had a good functional outcome at -months. physiological derangements were each negatively associated with outcome in bivariate analysis at the p < . level. a summary score of physiological derangements was included with potential confounders in the final regression model, and was independently associated with outcome with the chance of a good outcome decreasing by % for each increase of one physiologic derangement ( % ci . - . ). conclusions: uncorrected physiological derangements are independently and cumulatively associated with worse outcome after cardiac arrest. although causality cannot be established, it is reasonable to consider that the correction of physiological parameters may be an important step in the chain of survival after resuscitation. characteristics introduction: glan clwyd hospital (gch) was recently designated one of three cardiac arrest centres for wales. it has offered a / percutaneous coronary angiography (pci) service to a geographically dispersed north wales population of approximately , since june . prior to this, urgent coronary angiography was available on a more limited basis to patients requiring pci. the aim of this study was to investigate factors associated with hospital mortality after critical care admission following cardiac arrest. methods: retrospective review of the ward watcher critical care database at gch to identify patients who had undergone cpr in the hours prior to critical care admission in - . patients likely to have sustained ooha of cardiac aetiology (ooha-c) were identified from primary and secondary diagnoses and free text entry. data were subsequently analysed using excel and spss. the project was registered as a service evaluation with gch audit department. results: there were cardiac arrest admissions over this period, increasing from in - to in - . of these were ooha, of which were considered ooha-c. although ooha-c hospital mortality appeared to decrease over the time period ( %% to %), this was not statistically significant (p= . ). factors associated with survival to hospital discharge are presented in the tables below. on logistic regression, only pci and low ph within the first hours of critical care remained statistically significant (p= . and p< . respectively). conclusions: although we have been unable to make a distinction between patients presenting following stemi and nstemi, and appreciating a potential influence of selection bias, the significant association between pci and survival to hospital discharge supports the introduction of clinical pathways enabling pci access following ooha-c [ ] . chest radiography. [ ] here, we aimed to derive and validate rules to estimate p_max.lv using anteroposterior chest radiography (ches-t_ap), which is performed for critically-ill patients urgently needing determination of personalised p_max.lv. methods: a retrospective, cross-sectional study was performed with non-cardiac arrest adults who underwent chest_ap and computed tomography (ct) within h (derivation:validation= : ). on chest_ap, we defined cd (cardiac diameter), rb (distance from right cardiac border to midline) and ch (cardiac height, from carina to uppermost point of left hemi-diaphragm) (fig , ) . [ ] setting p_zero ( , ) at the midpoint of xiphisternal joint and designating leftward and upward directions as positive on x and y axes, we located p_max.lv (x_max.lv, y_max.lv). the coefficients of the following mathematically-inferred rules were sought: x_max.lv=a *cd-rb; y_max.lv=ß *ch+γ . (a : mean of (x_max.lv+rb)/cd; ß , γ : representative coefficient and constant of linear regression model, respectively ) . conclusions: evaluable echocardiographic records were reached in most of the patients. etco positively correlated with all parameters under consideration, while the strongest correlation was found between cimax and etco . therefore, cimax is a candidate parameter for real-time monitoring of haemodynamic efficacy of chest compressions during cpr. introduction: the uk resuscitation council has set out guidelines for management of patients post cardiac arrest [ ] . this is in line with european resuscitation council guideline. we set out to find if we are following the guideline. methods: we did a retrospective audit over the course of years looking at the data of patients who had in hospital and/or out of hospital cardiac arrest and after the return of spontaneous circulation were admitted to the intensive care unit (icu). we focused on whether the care they received was as per the standards set by the uk resuscitation council. results: we had in the hospital and out of hospital cardiac arrests; patients had less than minutes of cpr, had more than minutes cpr and patients the data was not recorded; patients needed more than minutes to reach from the site of arrest to the icu. the partial pressure of carbon dioxide was > . kpa in patients at two or more occasions. target map was not documented in patients; blood sugar target was not documented in patients and was not maintained within limits in patients. target temperature was not documented in patients. the withdrawal of treatment was not delayed for hours in patient out of . in patients neurological tests were not documented. multimodal assessment tools were not used in patient. electroencephalography and serum neuron specific enolase were not used to diagnose brain deaths as they were not available at our trust. patients were discharged, died in the icu and died in hospital after discharge from icu. conclusions: the audit reflected our local practice and showed that our mortality was in line with the acceptable limits; poor documentation of plan of care which posed problems in analyzing the care that these patients received; some of the parameters were not being maintained as set by uk resuscitation guideline. introduction: high-quality chest compressions (cc) with minimized interruptions are one of the most essential prerequisites for an optimal outcome of resuscitation. therapy of reversible causes of cardiac arrest often requires intra-hospital transportation (iht) during ongoing cpr. the present study investigated cc quality during transportation depending on the position of the provider. methods: paramedics were enrolled into a manikin study with four groups: a reference group with the provider kneeling beside manikin on the floor (group ), and groups performing cc during a simulated iht of meters: walking next to the bed (group ), kneeling beside the patient in bed (group , fig. ) or squatting above the patient in bed (group , figure ). indicators of cc quality were measured as defined in the erc guidelines (pressure point and depth, compression frequency, complete relief, sufficient pressure depth) [ ] . all paramedics performed cc during each scenario (group - ). results: there were no statistical differences in quality of cc between groups , and . notably, group performed significantly worse in respect to the proportion of cc with correct pressure point (p = . vs group ), correct cc depth (p= . vs. group , p= . vs. group , p= . vs. group ). the results are shown in table . conclusions: carrying out guideline-compliant cc [ ] during iht is feasible with multiple provider positions. based on the present results, kneeling or squatting position next to the patient ( figure and ) is recommended, whereas "walking next to the bed" while performing cc should be avoided. methods: a retrospective review of clinical notes was undertaken for patients admitted to icu following return of spontaneous circulation but whom remained comatose. this audit encompassed three-month periods before and after introduction of the care bundle in october . audit standards were assigned from target parameters documented in the bundle and reflected guidance from the cheshire and merseyside critical care network. results: patients were included in our audit; admitted prior to and admitted following implementation of the care bundle. in patients whom targeted temperature management was indicated, improved adherence to thermoregulation between - °c was observed ( vs %). significant improvements were since in the observance to target values for oxygen saturation ( vs . %, p= . ) and mean arterial pressure ( vs . %, p< . ) following the introduction of the care bundle. improved observance of ventilation targets was also seen; maintenance of p a co > . kpa ( vs %, p= . ) and tidal volumes < ml/kg ideal body weight ( to . %, p= . ). conclusions: the introduction of a post-cardiac arrest care bundle in our icu has improved care by providing discrete physiological targets to guide nursing staff and standardising management between clinicians. variations in care are associated with poorer patient outcomes [ ] and introduction of this bundle has reduced disparities in practice. array of cardiac diseases and reported survival rate is low in spite of advances in resuscitation and ems services. methods: single-centre retrospective study analyzed outcomes of ohca patients admitted to cardiac icu between .- . we studied demographic data, initial rhythm, type of cpr, comorbidities and various post admission diagnostic findings in order to identify their impact on survival. results: ohca comprised , % of all admissions. mean los was . days ( - ). mean age was , y ( - ), m: f ratio : and bystander cpr was performed in only % ohca patients. the most common initial rhythm was vf ( . %), followed by vt ( . %), pea was found in , % and asystole in . % of pt more than half of pt received adrenalin ( %) and defibrillation ( %) and only % required a temporary pacemaker. % of pt had an ecg consistent with mi after rosc, % underwent coronary angiography resulting in pci in % of cases. in pt ( %) therapeutic hypothermia protocol was performed. most ohca pt had hypertension ( %) and hyperlipidaemia ( %) as the most common risk factors followed by cardiomyopathy ( %), diabetes ( %) and cad ( %). only % had a preexisting significant valvular disease and the rest were extracardial comorbidities: chronic renal disease ( %), copd ( %) and cerebrovascular disease ( %). patients survived ( %) and gcs on admission was the only significant impact factor on survival along with comorbidities (mean gsc was in survivors vs. in deceased). interestingly, age, initial rhythm, troponin i level, ph and therapeutic hypothermia had no impact on survival. conclusions: our data demonstrate the importance of early onsite resuscitation as the most important factor of neuroprotection and outcome and puts an emphasis on the importance of cpr education for layman population. prediction of acute coronary ischaemia and angiographic findings in patients with out-of-hospital cardiac arrest j higny , a guédès , c hanet , v dangoisse , l gabriel , j jamart introduction: coronary artery disease (cad) is the leading cause of out-of-hospital cardiac arrest (ohca). however, diagnosis of acute coronary ischaemia (aci) remains challenging, particularly in patients without st-segment elevation on the post-resuscitation ecg. in this regard, a consensus statement recommends the implementation of a work-up strategy in the emergency room (er) to exclude noncoronary causes of collapse within hours. methods: retrospective single-centre study performed on consecutive patients with resuscitated ohca who underwent a diagnostic coronary angiography (ca). we present data on coronary angiograms for patients who underwent cardiac catheterization after resuscitation. afterwards, we sought to identify parameters associated with aci. results: st-segment elevation was noted in patients ( %). stsegment depression or t-wave abnormalities were noted in patients ( %). invasive coronary strategy allowed to identify an acute culprit lesion in cases ( %). patients with st-segment elevation underwent an immediate angioplasty for an acute coronary occlusion. patients without st-segment elevation underwent an ad hoc percutaneous coronary intervention for a critical lesion. stable cad was found in cases ( %) and a normal angiogram was found in only cases ( %) (figure ). conclusions: aci was the leading precipitant of collapse. stsegment elevation was highly predictive of coronary occlusion. in addition, a culprit coronary lesion was identified in nearly % of patients undergoing ca despite the lack of stsegment elevation. finally, our findings suggest that the identification of risk criteria may help to improve the recognition of aci after ohca. the prediction of outcome for in-hospital cardiac arrest (pihca) score e piscator , k göransson , s forsberg , m bottai , m ebell , j herlitz , t djärv figure. predictive value for classification into < % likelihood of favorable neurologic survival was . %. false classification into < % likelihood of favorable neurologic survival was . %. the phica score has potential to be used as an aid for objective prearrest assessment of the chance of favorable neurologic survival after ihca, as part of decision making for a dnar order. introduction: prognosis of survival in patients with cardiac arrest remains poor. during and after cardiopulmonary resuscitation, pathophysiological disturbances in relation with a cytokine storm, are described as "post-resuscitation" disease like a combination of cardiogenic and vasodilatory shocks. veno-arterial extracorporeal membrane oxygenation (va ecmo) allows to restore adequate perfusion but little is known about its effect on left ventricular (lv) function and about the role of cytokines. methods: this study was performed in an experimental model of cardiac arrest performed in groups of anesthetized and mechanically ventilated pigs. cardiac arrest was obtained by application of electrical current to epicardium inducing ventricular fibrillation. after a no-flow period of minutes, medical resuscitation with catecholamines and vasopressors was performed in "control" group while va ecmo was started in "ecmo" group and va ecmo in combination with cytosorb (extracorporeal blood purification therapy designed to reduce excessive levels of inflammatory mediators such as cytokines) was started in "ecmo-cyto" group. lv function was assessed with transthoracic echocardiography and arterial pressure with aortic pressure catheter. results: hemodynamic stability was obtained after ± and ± minutes in ecmo and ecmo-cyto groups, respectively. no return of spontaneous circulation was observed in control group. at minutes following cardiac arrest, lv area fractional change on short axis was normalized in ecmo and ecmo-cyto groups ( ± and ± %, respectively). vasopressor requirements were significantly lower in ecmo-cyto group than in ecmo group. conclusions: after cardiac arrest (no-flow) of minutes duration, va ecmo allowed complete lv recovery and hemodynamic stability within minutes of "post-resuscitation" disease. cytosorb added to va ecmo could contribute to reduce post-resuscitation vasodilatation. impact of rapid response car system on ecmo in out-of-hospital cardiac arrest: a retrospective cohort study m nasu , r sato , k takahashi introduction: extracorporeal life support (ecls) has been reported to be more effective than conventional cardio-pulmonary resuscitation (cpr). in ecls, a shorter time from arrival to implantation of extracorporeal membrane oxygenation (ecmo; door-to-ecmo) time has been reported to be associated with better survival rates. this study aimed to examine the impact of the physician-based emergency medical services (p-ems) using a rapid response car (rrc) on door-to-ecmo time in patients with out-of-hospital cardiac arrest (ohca to study the interest and the educational contribution in the short and medium term of medical simulation compared to a classical training. methods: cohort, prospective, observational, single-center, randomized study with control group including residents ( in anesthesia resuscitation and in emergency medicine). all benefited from a theoretical training with a reminder of the latest recommendations on the management of cardiac arrest and anaphylactic shock. they were randomized into groups and received practical training on a high-fidelity simulator for the management of either cardiac arrest (acc group) or anaphylactic shock (ca group). each group was evaluated at weeks (t ) and at months on two scenarios: refractory ventricular fibrillation (fv) scored on points and grade anaphylactic reaction (ra ) scored on points. each group served as the control group for the pathology in which they did not receive specific simulator training. the results are expressed on average with their standard deviations with "p" < . . introduction: simulation is a tool for improving the quality and safety of care, and its recognized as an essential method of evidence-based education. emergency medicine is a discipline in which there is a constant concern for the safety of patients. the emergency physician is often called upon to take charge of critical situations that use knowledge, know-how and knowledge as skills that must be mastered and whose theoretical learning alone is insufficient. methods: it´s a prospective study including residents in emergency medicine performing their specialty courses in emergency services and emergency medical assistance in the region of sousse from january to june . they were randomized into two groups: the one benefiting from a traditional education and the other from an education based on simulation sessions. the chosen scenario was the management of a cardiac arrest. a pre-test and a post-test were performed in both groups. results: we included emergency residents who did not receive specialized training in the management of cardiac arrest, there was a female predominance with an average age of , there was no significant difference regarding the pretest between the two groups with . there was no significant difference with respect to the pre-test score between the two groups . ± . / for the control group versus . ± . / for the simulation group. there was a significant progression after the course with an average posttest score of . ± . in the simulation group while this score was . ± . in the control group with a statistically significant difference (p < . ). conclusions: simulation learning has led to a better acquisition of cognitive knowledge by learners. the simulation is not intended to replace bed-based teaching, nor theoretical or faculty teaching, but it is an essential complement . in tunisia, the simulation must continue its current integration in the initial and continuous training of doctors. introduction: recent studies have shown that obesity and its related metabolic dysfunction exacerbates outcomes of ischemic brain injuries in some brain areas, such as the hippocampus and cerebral cortex when subjected to transient global cerebral ischemia (tgci). however, the impact of obesity in the striatum after tgci has not yet been addressed. the objective of this study was to investigate the effects of obesity on tgci-induced neuronal damage and inflammation in the striatum and to examine the role of mtor which is involved in the pathogenesis of metabolic and neurological diseases. methods: gerbils were fed with a normal diet (nd) or high-fat diet (hfd) for weeks and then subjected to min of tgci. hfd-fed gerbils showed the significant increase in body weight, blood glucose level, serum triglycerides, total cholesterol, and low-density lipoprotein cholesterol without affecting food intake. results: in hfd-fed gerbils, neuronal loss occurred in the dorsolateral striatum days after tgci and increased neuronal loss were observed cholesterol days after tgci; however, no neuronal loss was the in ndfed gerbils after tgci, as assessed by neuronal nuclear antigen immunohistochemistry and fluoro-jade b histofluorescence staining. the hfd-fed gerbils also showed severe activated microglia and further increased immunoreactivities and protein levels of tumor necrosis factor-alpha, interukin- beta, mammalian target of rapamycin (mtor) and phosphorylated-mtor in the striatum during pre-and postischemic conditions compared with the nd-fed gerbils. in addition, we found that treatment with rapamycin, a mtor inhibitor, in the hfd-fed gerbils significantly attenuated hfd-induced striatal neuronal death without changing physiological parameters. conclusions: these findings reveal that chronic hfd-induced obesity results in severe neuroinflammation and significant increase of mtor activation, which could contribute to neuronal death in the stratum following tgci. abnormal mtor activation might play a key role. associations between partial pressure of oxygen and neurological outcome in out-of-hospital cardiac arrest patients introduction: exposure to hyperoxemia and hypoxemia is common in out-of-hospital cardiac arrest (ohca) patients following return of spontaneous circulation (rosc) but its effects on neurological outcome are uncertain and study results are inconsistent. methods: exploratory post-hoc substudy of the target temperature management (ttm) trial [ ] , including patients after ohca with rosc. the association between serial arterial partial pressures of oxygen (pao ) during hours following rosc and neurological outcome at months, evaluated by cerebral performance category (cpc), dichotomized to good (cpc - ) and poor (cpc - ), was investigated. in our analyses, we tested the association of hyperoxemia pao > kpa and hypoxemia pao < kpa, time weighted mean pao , (twm-pao ) (fig ) , maximum pao difference (Δ pao ) and gradually increasing pao levels ( . - . kpa) with poor neurological outcome. a subsequent analysis investigated the association between pao and a biomarker of brain injury, peak serum tau levels. results: patients were eligible for analysis. patients ( %) were exposed to hyperoxemia or hypoxemia after rosc (table ) . our analyses did not reveal a significant association between hyperoxemia, hypoxemia, twm-pao exposure or Δ pao and poor neurological outcome at -month follow-up after correction for co-variates (all analyses p= . - . ) (fig ) . we were not able to define a pao level associated with the onset of poor neurological outcome. peak serum tau levels at either or hours after rosc were not associated with pao . conclusions: hyperoxemia or hypoxemia exposure occurred in one third of the patients during the first hours of hospitalization and was not significantly associated with poor neurological outcome after months or with the peak s-tau levels at either or hours after rosc. introduction: cerebral hypoperfusion may aggravate the developing neurological damage after cardiac arrest. near-infrared spectroscopy (nirs) provides information on cerebral oxygenation but its clinical relevance during post-resuscitation care is undefined. we wanted to assess the possible association between cerebral oxygenation and clinical outcome after out-of-hospital cardiac arrest (ohca). methods: we performed a post hoc analysis of a randomised clinical trial (comacare) where both moderate hyperoxia and high-normal arterial carbon dioxide tension (paco ) increased regional cerebral oxygen saturation (rso ) as compared with normoxia and low-normal paco , respectively. rso was measured from ohca patients with nirs during the first h of intensive care and neurological outcome was assessed using the cerebral performance category (cpc) scale at months after cardiac arrest. we calculated the median rso for patients with good (cpc - ) and poor (cpc - ) outcome and compared the results using the mann-whitney u test. we compared the rso over time with outcome using a generalised mixed model. finally, we added median rso to a binary logistic regression model to control for the effects of possible confounding factors. results: the median (interquartile range [iqr]) rso during the first h of intensive care was . % ( . - . %) in patients with good outcome compared to . % ( . - . %) in patients with poor outcome, p = . . we did not find significant association between rso over time and neurological outcome ( figure ). in the binary logistic regression model rso was not a statistically significant predictor of good outcome (or . , % ci . - . , p = . ). conclusions: we did not find any association between cerebral oxygenation during the first h of post-resuscitation intensive care and neurological outcome at months after cardiac arrest. fig. introduction: near-infrared spectroscopy (nirs) provides a noninvasive means to assess cerebral oxygenation during postresuscitation care but its clinical value is unclear. we determined the possible association between cerebral oxygenation and the magnitude of brain injury assessed with neuron-specific enolase (nse) serum concentration at h after out-of-hospital cardiac arrest (ohca). methods: we performed a post hoc analysis of a randomised clinical trial (comacare) comparing two different levels of carbon dioxide, oxygen and arterial pressure after ohca and successful resuscitation. we measured rso continuously with nirs from patients during the first h of intensive care. we determined the nse concentrations at h after cardiac arrest from serum samples using an electrochemiluminescent immunoassay kit. the samples were tested for haemolysis and all samples with a haemolysis index > mg of free haemoglobin per litre (n = ) were excluded from the analyses. we calculated the median rso for all patients and used a scatterplot and spearman's rank-order correlation to assess the possible relationship between median rso and nse at h. in addition, we compared the nse concentrations at h after cardiac arrest in patients with good (cerebral performance category scale [cpc] - ) and poor (cpc - ) neurological outcome at months using the mann-whitney u test. results: we did not find significant correlation between median rso and serum nse concentration at h after cardiac arrest, rs = - . , p = . (figure ). the median (iqr) nse concentration at h was . ( . - . ) μg/l and . ( . - . ) μg/l in patients with good and poor outcome, respectively, p < . . conclusions: we did not find any association between cerebral oxygenation during the first h of post-resuscitation intensive care and nse serum concentrations at h after cardiac arrest. the association between lactate, cerebral oxygenation and brain damage in post-cardiac arrest patients introduction: patients admitted to the intensive care unit (icu) after being successfully resuscitated from a cardiac arrest (ca) have a large cerebral penumbra at risk for secondary ischemic damage in case of suboptimal brain oxygenation. therefore, resuscitation during icu stay should be guided by parameters that adequately predict cerebral hypoxia. the value of lactate as resuscitation parameter may be questioned in post-ca patients since the brain critically depends on aerobic metabolism. we aimed to investigate the relationship between arterial lactate, cerebral cortex tissue oxygenation (scto ) by near infrared spectroscopy (foresight) and unfavorable neurological outcome at days (cpc score - ) methods: subanalysis from the neuroprotect post-ca trial. lactate values and scto were recorded hourly in post-ca patients during hours ttm and subsequent rewarming. results: in total paired lactate/ scto measurements were analysed. we found no correlation between paired lactate and scto² (fig. ) . moreover, temporary trends in lactate did not correlate with corresponding trends in scto during the same one-hour time interval (r²= . ) (fig ) . if lactate values above . mmol/l are considered to be abnormal, lactate could not adequately detect clinical important brain ischemia (scto < %): sensitivity % and specificity % (table , ). nevertheless, time weighted lactate at h (or . ; p . ), h (or . , p . ), h (or . ; p . ) and h (or . ; p . ) were inversely correlated with unfavorable neurological outcome at days (fig , ) . conclusions: although lactate was a marker of prognosis in post-ca patients, it should not be used to guide resuscitation since lactate values were not correlated with scto and changes in lactate do not correspond with changes in scto during the same time interval. simplified introduction: the aim of the study was to investigate whether simplified continuous eeg monitoring (ceeg) [ ] post-cardiac arrest can be reliably interpreted by icu physicians after a short structured training, and whether acceptable interrater agreement compared to an eeg-expert can be achieved. methods: five icu physicians received training in interpretation of simplified ceeg (fig ) consisting of lectures, hands-on ceeginterpretation, and a video tutorial -total training duration day. the icu physicians then interpreted simplified ceeg recordings. basic eeg background patterns and presence of epileptiform discharges or seizure activity were assessed on -grade rank-ordered scales based on a standardized eeg terminology [ ] . an experienced eeg-expert was used as reference. results: there was substantial agreement (κ . ) for eeg background patterns and moderate agreement (κ . ) for epileptiform discharges between icu physicians and the eeg-expert. sensitivity for detecting seizure activity by the icu physicians was limited ( %), but with high specificity ( %). among icu physicians interrater agreement was substantial (κ . ) for eeg background pattern and moderate (κ . ) for epileptiform discharges. conclusions: after a one-day educational effort clinically relevant agreement was achieved for basic eeg background patterns after cardiac arrest. assessment of epileptiform patterns was less reliable, but bedside screening by the icu physician may still be clinically useful for early detection of seizures. interpretation of simplified ceeg requires awareness of its limitations and support from an eeg-expert when clinically indicated. introduction: hypoxic-ischemic injury on head computed tomography (ct), which manifests with varying degrees of cerebral edema and loss of gray-white matter differentiation, is a poor prognostic sign after resuscitated out-of-hospital cardiac arrest that may influence early clinical decision-making. agreement among physicians on the presence of hypoxic-ischemic injury on early head ct is unknown. methods: we recruited faculty physician participants ( emergency medicine, critical care, neurocritical care, and general radiology; average . years of practice) across academic medical centers each with > admissions for resuscitated out-of-hospital cardiac arrest each year. participants, blinded to clinical context, reviewed unique head cts obtained within hours of cardiac arrest that were randomly selected from a local registry. a blinded neuroradiologist also reviewed all scans (gold standard). participants determined if hypoxic-ischemic injury was present on each ct, and agreement was determined using multi-and dual-rater kappa statistics with % confidence intervals. results: overall agreement among physicians regarding the presence of hypoxic-ischemic injury on head ct was fair (kappa . ; % ci, . - . ) with agreement consistent across most specialties (table ) . when compared to the neuroradiologist, individual physician agreement ranged widely, from poor (kappa . ) to substantial (kappa . ), with of physicians having fair or worse agreement compared to the gold standard interpretation. conclusions: the finding of hypoxic-ischemic injury on early head ct after cardiac arrest had high interobserver variability as interpreted by acute care physicians and general radiologists. pending the development of objective diagnostic criteria, clinicians should bear in mind the subjectivity and subtlety of cerebral edema or loss of graywhite matter differentiation soon after return of spontaneous circulation in these patients. figure ). baseline characteristics and differences between the wlst and no-wlst groups are shown in table . utilization of neuro-prognostication tests is shown in table . while ct and eeg were commonly employed, ssep and mri were used less frequently. basic multimodal neuroprognostication (arbitrarily defined as at least one ct or mri, plus eeg, plus ssep) was performed only in . % of all patients undergoing wlst but the rate increased significantly over six years (p< . ) and was higher in the time period after , compared to the one prior to ( figure ). this association remained significant after adjustment for confounders such as age, arrest rhythm, downtime, targeted temperature management, apache ii score and organ failure in a logistic regression model (p= . ). in an institution with access to a wide range of imaging and neurophysiology tests, mri and ssep remained underutilized but the rate of basic multimodal neuro-prognostication increased significantly over the study period, especially in the period after . introduction: although multiple reports using animal models have confirmed that melatonin appears to promote neuroprotective effects following ischemia/reperfusion-induced brain injury, the relationship between its protective effects and the activation of autophagy in cerebellar purkinje cells following the asphyxial cardiac arrest and cardiopulmonary resuscitation (ca/cpr) remains unclear. methods: rats used in this study were randomly assigned to groups as follows; vehicle-treated sham-operated group, vehicletreated asphyxial ca/cpr-operated group, melatonin-treated shamoperated group, melatonin-treated asphyxial ca/cpr-operated group, melatonin plus (+) p-pdot (the mt melatonin receptor antagonist)-treated sham-operated group and melatonin+ p-pdot-treated asphyxial ca/cpr-operated group. results: our results demonstrate that melatonin ( mg/kg, ip, time before ca and times after ca) significantly improved the survival rates and neurological deficits compared with the vehicle-treated asphyxial ca/cpr rats (survival rates ≥ % vs %). we also demonstrate that melatonin exhibited the protective effect against asphyxial ca/cpr-induced purkinje cell death. the protective effect of melatonin in the purkinje cell death following asphyxial ca/cpr paralleled a dramatic reduction in superoxide anion radical (o ·-), intense enhancements of cuzn superoxide dismutase (sod ) and mnsod (sod ) expressions, as well as a remarkable attenuation of autophagic activation (lc and beclin- ), which is mt melatonin receptor-associated. furthermore, the protective effect of melatonin was notably reversed by treatment with p-pdot. conclusions: this study shows that melatonin conferred neuroprotection against asphyxial ca/cpr-induced cerebellar purkinje cell death by inhibiting autophagic activation by reducing expressions of ros, while increasing of antioxidative enzymes, and suggests that mt is involved in the neuroprotective effect of melatonin in cerebellar purkinje cell death induced by asphyxial ca/cpr. introduction: fucoidan is a sulfated polysaccharide derived from brown algae and possesses various beneficial activities, such as antiinflammatory and antioxidant properties. previous studies have shown that fucoidan displays protective effect against ischemiareperfusion injury in some organs. however, few studies have been reported regarding the protective effect of fucoidan against cerebral ischemic injury and its related mechanisms. methods: therefore, in this study, we examined the neuroprotective effect of fucoidan against cerebral ischemic injury, as well as underlying mechanisms using a gerbil model of transient global cerebral ischemia (tgci) which shows loss of pyramidal neurons in the hippocampal cornu ammonis (ca ) area. fucoidan ( and mg/kg) was intraperitoneally administered once daily for days before tgci. results: pretreatment with mg/kg of fucoidan, not mg/kg fucoidan, attenuated tgci-induced hyperactivity and protected ca pyramidal neurons from ischemic injury following tgci. in addition, pretreatment with mg/kg of fucoidan inhibited activations of resident astrocytes and microglia in the ischemic ca area. furthermore, pretreatment with mg/kg of fucoidan significantly reduced the increased -hydroxy- -noneal and superoxide anion radical production in the ischemic ca area after tgci and significantly increased expressions of superoxide dismutase (sod ) and sod in the ca pyramidal neurons compared with the vehicle-treated-group. we found that treatment with diethyldithiocarbamate (an inhibitor of sods) to the fucoidan-treated-group notably abolished the fucoidanmediated neuroprotection in the ischemic ca area following tgci. conclusions: these results indicate that fucoidan can effectively protect neurons from tgci-induced ischemic injury through attenuation of activated resident glial cells and reduction of oxidative stress following increasing sods. thus, we strongly suggest that fucoidan can be used as a useful preventive agent in cerebral ischemia. the effects of cold fluids for induction of therapeutic hypothermia on reaching target temperature and complications-a sub-study of the tth study a holm , m skrifvars , fs taccone ). there was no difference in early bleeding incidences (fig ) . during late observation, ttm patients had fewer minor bleeding ( . % vs. %) and more intracranial bleeding ( . % vs. %; fig ) . adjusted calculated risk ratio for major bleeding (including intracranial) for ttm was . ( %ci . - . ) at baseline and . ( %ci . - . ) over time. conclusions: bleeding complications were common. although the risk ratio for major bleeding increased over time in ttm patients, residual and unmeasured confounding in addition to selection and detection bias may limit the clinical relevance of this finding. methods: patients with neurological deficit > by nhiss were included. the t°of the brain was recorded non-invasively using radiothermometer rtm- -res (russia). we measured t°in symmetric regions of left & right hemispheres, calculated the average t°of brain, fig. (abstract p ) . temperature of patients given and not given pre-icu fluids (table ) . conclusions: observed moderate brain t°heterogenecity in hp, marked increase brain t°heterogenecity in is & sharp decline of t°h eterogenecity in cci. supposedly, correcting the impairment of cerebral tb (increase or decrease t°) through physical (selective cerebral hypothermia, magnetic stimulation etc.) or pharmacological (sedation) can contribute to positive therapeutic results in is & cci. nonivasive radiothermometry of the brain can be an objective method of patients' condition evaluation & their rehabilitation potential. introduction: basilar artery stroke has a multitude of different presentations and may not be captured on plain computed tomography (ct). it can progress to severe disability, locked in syndrome and death [ ] . with the advent of thrombolytic and endovascular therapies, prompt diagnosis can change the outcome. we present a case of basilar artery stroke, which was heralded by tongue spasticity and dysarthria, indicative of pseudobulbar palsy. methods: case reviewed with consent. a literature search was conducted using pubmed and medline. results: a -year-old presented with pulmonary oedema and hypertension. he was transferred to our intensive care unit for treatment of a suspected anaphylaxis. his marked lingual swelling was associated with dysarthria. glyceryl-trinitrate and labetalol infusions were started for hypertension. he developed left sided weakness and deteriorated over several days to the point that he could only move his right foot (table ) . magnetic resonance imaging (mri) showed midbrain ischaemia and angiogram showed no flow in the basilar artery (fig , ) . conclusions: common presenting features of basilar artery occlusion include dysarthria, vertigo, vomiting, headache and motor defects; these may evolve gradually or be intermittent [ , ] . presentation with pseudobulbar palsy is described in early literature [ ] . delayed recognition of the stroke led to aggressive treatment of hypertension, potentially compromising perfusion to the penumbral area [ , ] . this case highlights the need for a wide index of suspicion with posterior strokes. consent: informed consent to publish has been obtained from the patient prognosis is related to gcs < or = on admission (p = . ) and to malignant cerebral edema (p = . ). conclusions: our study has shown some predictive factors closely related to mortality and morbidity in patients with acute ischemic stroke. gcs at admittance < or = and onset of malignant cerebral edema lead to a worst prognosis at discharge from nicu. coherence analysis of cerebral oxygenation using multichannel functional near-infrared spectroscopy evaluates cerebral perfusion in hemodynamic stroke tj kim table ). in addition, severe stroke patients were more likely to have higher phase coherence in interval iii (p = . ). conclusions: our results demonstrated that the higher phase coherence of oxyhb in myogenic signal, which was originated locally from smooth muscle cells in brain was related to impaired cerebral perfusion. this suggests that monitoring cerebral oxygenation using fnirs could be a useful noninvasive measuring tool for evaluating impaired cerebral autoregulation in stroke patients. is esmolol associated with worse outcome at the acute phase of ischemic stroke that receives thrombolysis? introduction: ischemic stroke patients experienced frequent early neurological deterioration (end) events. since ischemic stroke has also been shown as inflammatory disease, the neutrophil-tolymphocyte ratio (nlr) may associated with end events. however, the direct study regarding this association has not been addressed. poor grade sah, use of vasopressors, mechanical ventilation, intracranial pressure monitoring, external ventricular drainage, blood transfusions and renal replacement therapy were all more frequent among nonsurvivors (all p< . ). mortality was also higher with initial lactate above mmol/l, in those admitted to public hospitals and when admission to icu was delayed more than hours after ictus. after adjusting for common predictors (age, gender and wfns) saps non-neuro, sofa non-neuro, early vasopressor use and admission to a public hospital were independently associated with hospital mortality. moreover, the area under the curve for prediction of mortality with saps , sofa and wfns was . ( figure ). hospital, austria. the association of intensity and duration of intracranial hypertension episodes with -month glasgow outcome score (gos) was visualized using the methodology introduced by güiza et al. [ ] . results: in both cohorts, it could be demonstrated that the combination of duration and intensity defined the tolerance to intracranial hypertension, and that a semi-exponential curve separated episodes associated with better outcomes from those associated with worse outcomes. the association with worse outcomes occurred at a lower pressure-time burden than what has been previously observed in patients with tbi. nevertheless, the percentage of monitoring time spent by every patient in the zone associated with poor gos was independently associated with worse -month neurological outcome, even after correcting for age and fisher score ( introduction: apnea test is an essential component in the clinical determination of brain death, but it may incur a significant risk of complications such as hypotension, hypoxia and even cardiac arrest [ ] . we analyzed the risk factors associated with failed apnea test during brain death assessment in order to predict and avoid these adverse events. methods: medical records of apnea tests performed for brain-dead donor between january and january in our institution, were reviewed retrospectively. age, gender, etiology of brain death, use of catecholamine and results of arterial bleed gas analysis (abga), systolic/diastolic blood pressure (sbp/dbp), mean arterial pressure (map) and central venous pressure (cvp) prior to apnea test initiation were collected as variables. a-a gradient and pao /fio were calculated for more precise assessment of the respiratory system. in total, cases were divided into a group which was completed apnea test and the other which was failed the test. introduction: tunisia has already suffered recurrent outbreaks since . outbreak started relatively earlier this year. we were interpellated by the frequency of neuroinvasive presentation of the disease. methods: we report a case series of patients presented to icu with niwnd. results: we report cases of niwnd with different severe presentations overlapping neurological manifestation including encephalitis (n= / ), meningitis (n= / ) and flaccid paralysis (n= / ). almost all patients live in the locality of sousse. six patients presented a long course of isolated fever before developing neurological signs. cerebrospinal fluid was consistent with encephalitis within the patients. cerebromedullar mri identified brain lesions (n= / ), myelitis (n= / ) and polyradiculoneuritis (n= / ).three patients had electromyography for flaccid paralysis showed diffuse axonal polyneuropathy with motoneuron involvement. ten cases had a positive wnv igm antibody and nine had a positive wnv igg antibody in serum. urine polymerase chain reaction was positive for wnv in / patients. ten patients were mechanically ventilated. all patients were managed symptomatically. two received high doses of methylprednisolone for days, one patient received polyclonal immunoglobulin intravenous and one patient had plasmapheresis. two patients died consecutive to brainstem lesions. two patients recovered significantly and discharged with no complications. five other patients evolved to persistent flaccid paralysis with a minimal consciousness state and weaning difficulties requiring tracheostomy. the last remaining patient is still evolving. conclusions: modification of the regional climatic conditions accounted probably for the early outbreak of niwnd. this initial case series displays the severity and the poor outcomes of niwnd with higher incidence compared to past epidemics. noninvasive estimation of intracranial pressure with transcranial doppler: a prospective multicenter validation study c robba , c fig. ], mean bias was - . mmhg (limits of agreement are ± sd . mmhg). . % measures were outside the limit of agreement in the overall population. however, when icp was high, % of measures were out of the limit of agreement. the auc [ fig. introduction: surgical treatment of aortic aneurysm needs extracorporeal circulation (ecc), aorta clamp and hypothermia, and it is often related to poor systemic perfusion and blood flow velocity. one of the main concerns of intensive care team is to prevent secondary neurological injury after long time without blood flow pulsatility, such as brain edema and seizure. the most common parameters for neuromonitoring would be intracranial pressure and eeg, however, for non-neurological patients this information is unusual and prevents optimal management. methods: we aimed to assess brain compliance and neurological condition of icu patients on immediate post-operative recovery of bentall-de bono procedure and/or other aortic aneurysm surgical treatment using a novel non-invasive intracranial pressure (icp) device. this device uses mechanical displacement sensor capturing extracranial continuous volumetric variation of the skull and this information proportionally reflects intracranial dynamic [ ] . results: twenty patients were included in this study. ecc mean time was minutes for patients and only one did not need it. eleven presented altered icp curves with poor brain compliance (p /p ratio > . ) assessed by icp curve morphology analysis. volemic optimization and neuroprotective measures were taken based on this icp information for acute case management. among these patients with altered icp curves, eight were discharged from icu with good clinical condition and glasgow coma scale of . overall mortality rate was six out of twenty ( %) and three of these had altered icp curves. conclusions: brain monitoring of cardiovascular post-operative patients is important to prevent secondary neurological complications and can be a helpful tool for neuroprotective acute management on icu. the technique supplies electrical current to muscle, combined with passive cycling. prior to a clinical trial, we first investigated the effects of one session of fes in healthy volunteers. methods: healthy male volunteers (n= ) were recruited. the participants had their postural sway assessed on a pressure sensitive board, and measurement of maximal inspiratory pressure (mip). ultrasounds were taken assessing thickness of the quadriceps and rectus abdominis. they performed minutes of supine passive cycling, with fes supplying the lower limbs and abdomen. after a minute rest, the tests were repeated. a further participants performed just the initial baseline tests, to help assess muscular factors affecting balance and sway. results: the current needed for palpable contraction was significantly correlated to weight in the abdomen (r= . , p< . ) and quadriceps (r= . , p< . ). current required to stimulate the abdominal muscles was also correlated to depth of the subcutaneous fat layer (r= . , p< . ) and echogenicity of the muscle (r= . , p= . ). pre-cycling, left and right vastus lateralis thickness inversely correlated to postural sway in the antero-posterior (r=- . , p< . ) plane. compared to pre-cycling, postural sway in the antero-posterior and lateral planes increased significantly after cycling. there was a significant decrease in mip after cycling and greater reductions in mip were found in participants who had thinner rectus abdomni. conclusions: sway at baseline is related to quadriceps thickness, which atrophies during critical illness, and could worsen balance. mip is reduced during fes and the severity of reduction is related to the thickness of the abdominal wall muscles at baseline, suggesting that fes can fatigue the diaphragm and abdominal muscles. in awake healthy volunteers, fes is a safe, comfortable technique. introduction: in most cases postoperative cognitive dysfunction (pocd) is transient, but still some patients suffer from persistent cognitive impairment which is associated with increased length of hospital stay, early withdrawal from labor market and higher mortality. available data on the prevalence of pocd after cardiac surgery is very diverse from % to % upon discharge and up % months after surgery. we aimed to investigate the prevalence of short-term and long-term pocd after off-pump coronary artery bypass grafting (cabg) surgery. methods: psychometric testing was performed in (mean age . ± . ) patients before, days and months after the surgery. we used following tests to assess cognitive capacity: auditory verbal learning test (avlt), digit span test (dst), digit-letter substitution test (dlst), stroop's test and trail making test (tmt). a decline in comparison to preoperative test results for % or more in two or more tests was declared as pocd. results: the prevalence of pocd after days was . % ( patients) and . % ( patients) after months. when comparing patients who developed pocd with those who did not we found the former were older ( . ± . vs . ± . years; p< . ), had lower education level ( . ± . vs . ± . years; p< . ) and had longer surgery duration ( . ± . vs . ± . minutes; p< . ). the most affected cognitive domains were long term memory (avlt) and executive function (tmt) and least affectedworking memory (dst) and selective attention (stroop's test). conclusions: in our prospective study the prevalence of long-term pocd after cardiac surgery was slightly less ( . %) in comparison to available data (from % to %). it might be due differences in psychometric testing and interpretation of its results among authors. advanced age, low cognitive reserve and long duration surgeries are linked with higher incidences of pocd. introduction: postoperative cognitive dysfunction (pocd) is a common and widely described phenomenon in surgical patients. advanced age, major surgery, certain general anesthetics, genetic factors, sleep deprivation and other factors were described as contributing factors to pocd. the hospital stay itself is a major 'social' trauma for patients; social isolation, sleep deprivation and changes in daily regimen may effect neurocognitive behavior of patients. in this trial we tried to assess the link between pocd and the length of hospital stay in cardiac surgery patients. methods: patients who underwent 'off-pump' coronary artery bypass grafting (cabg) surgery selected for this trial. neuropsychological testing was performed prior to the operation and upon discharge. we used auditory verbal learning test (avlt), digit span test (dst), digit-letter substitution test (dlst), stroop test and trail making test (tmt). a % or more decline in two or more tests in comparison to preoperative test results was declared as pocd. patients were allocated into two groups according to the length of hospital stay: the short-stay group (group ) included patients (n= ) who were discharged on the th day after surgery or earlier and the long-stay (group ) group consisted of patients (n= ) who were discharged on the th day after surgery or later. patients received similar anesthesia, postoperative care and were operated by the same surgical team. reasons for prolonged duration of hospital stay were mainly surgical. results: patients ( . %) in group and patients ( . %) in group had pocd upon discharge (p< . ). mean length of hospital stay were ± . and ± . days in group and group patients respectively (p< . ). conclusions: prolonged length of hospital stay increased the prevalence of pocd in our trial. studies with various types of surgical procedures and larger patient populations needed to further understand the effect of length of hospital stay to pocd. the influence of multiple trauma with head trauma on posttraumatic meningitis: a nation-wide study with hospital-based trauma registry in japan introduction: posttraumatic meningitis is one of severe complications and results in increased mortality and longer hospital stay among head trauma patients. however, it remains unclear whether there is a difference in the incidence of post-traumatic meningitis due to single traumatic brain injury (tbi) and multiple trauma including head injury. methods: this study was a retrospective observational study during years we included trauma patients registered in japanese trauma data bank whose head ais score was > in this study. multivariable logistic regression analysis was used to assess potential factors associated with posttraumatic meningitis such as csf fistula, skull base fracture, type of injury that divided into single tbi and multiple trauma. introduction: the aim of this study was to determine if regional cerebral oxygenation (rsco ) can be used as an indicator of tissue perfusion in icu patients with tbi [ , ] , and to determine the prognostic value of cerebral oxygenation rsco in survival prediction. methods: patients were enrolled retrospectively from january through july in the icu of derince kocaeli training hospital. patients with trauma patients and traumatic braine injury patients who were admitted to the icu from the emergency room were included in the study. the sedation levels of the patients were followed up with bis. the rsco , bis was taken as well as blood lactate level, mean arterial blood pressure and cardiac output at baseline time, , , , and hours. results: no significant difference was also detected between the value of rsco in all patients . it was average sco (right) . ± . and average rsco (left) . ± . . conclusions: cerebral regional oxygen saturation might be helpful as one of the perfusion parameters in patients with tbi but it could have no prognostic value in mortality prediction. however, further studies with larger sample size are still needed to validate these results. introduction: tbi in elderly is an increasingly cause of admission in icu. data regarding management and prognosis of these patients are lacking. validated prognostic models refer to younger patients and do not adequately consider the influence of pre-injury functional status, which often compromises with aging. frailty has been defined as a state age-related of increased vulnerability and decline in autonomy of daily life activity. aim of the study is to evaluate the impact of frailty on outcome in tbi elderly patients. methods: moderate and severe tbi patients > years, admitted in neuroicu from january to may , were prospectively enrolled. data of age, comorbidity, glasgow coma scale (gcs), pupils' reactivity, ct scan characteristics, neurosurgical intervention and gose (extended glasgow outcome scale) at -months were collected. frailty status was measured by clinical frailty scale (cfs) [ ] and patients were divided as frail (cfs> ) and not frail (cfs< ). bad outcome was defined as gose< . results: ( %) of the studied patients were frail. frailty was not related to age. frail patients had more comorbidities and worse pupils' reactivity at admission (table ) . other variables did not differ between groups. in univariate analysis neurological diseases, gcs, tsah (traumatic subarachnoid haemorrhage), compressed/absent basal cisterns, non-reactive pupils and cfs were significantly associated to bad outcome. in multivariate analysis only gcs and cfs remained associated to bad outcome ( table ) . conclusions: pre-injury frailty is strongly associated to outcome in tbi elderly patients. the age of the patients was . ± . years. patients were operated on for intracranial traumatic ( cases) and non-traumatic hematomas ( ), brain tumors ( ) and the need for plastic of postoperative skull defects ( ). general endotracheal total intravenous anesthesia with fentanyl, propofol, rocuronium, or tracrium was used. after tracheal intubation, - nerves were blocked (e.g., supraorbital, supratrochlear, zygomaticotemporal, auriculotemporal, great auricular, greater and lesser occipital nerves), depending on the surgical site. . - . % ropivacaine was used. for blockade of one nerve used . - . ml of local anesthetic. fentanyl was applied on section of a periosteum, dura matter and at inefficiency of blockade of nerves. anesthesiology monitoring included hr, ecg, spo , nib, respiratory parameters, eeg (csi), body temperature, blood glucose and lactate levels. in and - hours post-surgery, the intensity of pain was ranked by alert patients using vas. results: the volume of local anesthetic for blockade in one patient was . ± . ml. in ( . %) from patients, an additional fentanyl injection was required to skin incision due to an increase in blood pressure and heart rate by % of the baseline values, and an increase in csi until un. patients available to productive contact in hours post-surgery ranked the pain by vas at ( ; ) point, and in - hours post-surgery ranked it at ( ; ) p. conclusions: at patients with craniotomies scalpe-block with lowvolumes of a ropivacaine showed high efficiency ( . %). were transferred to hospital ward or ( . %) to the center of intensive nursing care; ( . %) went to the surgical recovery room. acute renal failure, hypernatremia and hyperphosphatemia were independent predictors of mortality as described in table . conclusions: hypernatremia and hyperphosphatemia were independent predictors of mortality in critically ill patients. introduction: the strong ion difference (sid) is essential for the assessment of acid-base equilibrium, thus requiring an accurate measurement of plasma electrolytes. currently there is no gold standard for electrolyte measurements and sid computation. differences in electrolyte values obtained with point-of-care (poc) and central laboratory (lab) analyzers have been reported [ , ] . in previous studies [ , ] we have shown that changes in pco induce electrolyte shifts from red blood cells to plasma (and vice versa), yielding variations in sid. aim of the present in-vitro study was to induce sid changes through acute changes in pco and compare values of electrolytes and sid obtained with poc and lab techniques. methods: blood samples from healthy volunteers were tonometered (equilibrator, rna medical) with gas mixtures at fractions of co (fco ) of , , and %. electrolytes were measured quasisimultaneously with a poc analyzer (abl flex, radiometer) and a routine lab method (cobas ise, roche). for both techniques a simplified sid was computed as sodium + potassiumchloride. results: bland-altman analysis of sid calculated with poc and lab showed a proportional bias (slope = . , r = . , p < . ), indicating a variable agreement between methods according to the average sid value (fig. ) . sid values measured with poc and lab at different fco differed significantly (p< . , fig. ) . a similar discrepancy was observed for chloride (p < . , fig. ), while sodium (p= . ) and potassium (p= . ) were similar. conclusions: sid measured with poc and lab differed significantly, mainly due to a variable discrepancy in chloride. our findings suggest that our poc analyzer is superior to the lab in measuring electrolytes and thus compute sid. introduction: this study evaluated the safety of half dose insulin (hdi) versus standard dose insulin (sdi) for the treatment of hyperkalemia in a medical intensive care unit (micu) population with renal insufficiency. recent emergency medicine data demonstrated a lower incidence of hypoglycemia in patients with renal insufficiency when hdi was used for the treatment of hyperkalemia [ ] . there is limited data describing the safety of hdi in a micu population with renal insufficiency. methods: this was a retrospective, chart review of patients admitted to the micu with a diagnosis of aki and/or ckd stage - with a serum potassium ≥ . meq/l from january to september . sdi is defined as units of regular iv insulin and hdi as units. the primary outcome was the incidence of hypoglycemia within hours of insulin administration. secondary outcomes included severe hypoglycemia and change of serum potassium after insulin administration. results: a total of patients were screened and were included for analysis. the incidence of hypoglycemia occurred in / patients ( . %) and / patients ( . %) who received sdi and hdi, respectively. one patient in the sdi group and two patients in the hdi group developed severe hypoglycemia. the mean decrease in serum potassium after insulin administration was . meq/l in both groups. patients in the hdi group who were re-dosed with units of regular insulin did not have any hypoglycemic events. conclusions: in a micu population with renal insufficiency, sdi and hdi regimens appear safe and effective for the treatment of hyperkalemia. introduction: sepsis and septic shock are common causes of admission in the intensive care unit with a high mortality rate [ , ] . hence, electrolyte disturbances are common in this group of patients. acute hypernatremia is one of the multiple features of homeostasis disturbances and available data in the literature suggest that its incidence can reach % [ , ] . (fig , ) . the main source of sepsis was pneumonia with affected patients ( . %). conclusions: hypernatremia is significantly associated with higher mortality in septic patients. (abstract p ) . the outcome versus the sodium levels higher in the group - % vs . % (p= . ). there were no significant differences between the groups in length of stay in the icu. in group , there was an increase of serum phosphorus level and in the group the tendency to decrease. however, statistically significant differences were obtained only on the nd day after surgery . ± . mmol/l (group ) vs . ± . mmol/l (group ) (p= . ). the roc curve was constructed to assess the predictive significance of serum phosphorus levels (fig. ) . auc was . ; % ci . - . ; p= . ; sensitivity . %, specificity . %. the kaplan-meier survival analysis (fig. ) introduction: the rate of extubation failure might be higher in obese patients than in non-obese patients. effect of obesity on mortality is controversial [ , ] (obesity paradox). several pathophysiological changes contribute to an increase of respiratory complications [ ] . we sought to identify incidence of extubation failure in obese and non-obese patients. methods: the primary endpoint of this post-hoc analysis of a prospective, observational, multicenter study [ ] performed in intensive care units was extubation failure, defined as the need for reintubation within hours following extubation. only patients with body mass index (bmi) recorded were included. results: between december , and may , , among the patients with bmi available undergoing extubation, obese patients ( %) and non-obese patients ( %) were enrolled. extubation-failure rate was . % ( / ) in obese patients, and . % ( / ) in non-obese patients (p= . ). delay of reintubation did not differ between obese and nonobese patients (figure ). length of intubation > days was significantly more frequent in obese patients ( / , %) than in non-obese patients ( / , %, p< . ). precautions to anticipate extubation failure were more often taken in obese patients ( / , %) than in non-obese patients ( / , %, p< . ). spontaneous breathing trial (sbt) characteristics differed between obese and non-obese patients (table ) . physiotherapy was more often used in obese patients ( / , %) than in non-obese patients ( / , %, p= . ). conclusions: incidence of extubation failure did not differ between obese and non-obese patients. in obese patients, clinicians anticipate more a possible extubation failure, delaying the moment of extubation, performing more physiotherapy and providing an optimal sbt. introduction: in the acute phase of critical illness, growth hormone (gh) resistance develops, reflected by increased gh and decreased insulin-like growth factor-i (igf-i), mimicking fasting in health. the epanic rct observed fewer complications such as muscle weakness and faster recovery with accepting a macronutrient deficit in the first icu week, as compared with early full feeding [ , ] . we characterized its impact on the gh axis in relation to the risk of acquiring muscle weakness. methods: in this epanic rct sub-analysis, for matched patients per group, and all patients assessed for muscle weakness (n= ), serum gh, igf-i, igf binding protein (igfbp ) and igfbp were measured upon icu admission and at day or the last icu day for patients with shorter icu stay (d /ld). for matched patients per group, gh was quantified every min between pm and am, and deconvolved to estimate gh secretion. groups were compared with wilcoxon test or repeated-measures anova. associations between changes from baseline to d /ld and muscle weakness were assessed with logistic regression analysis, adjusted for baseline risk factors, baseline hormone concentrations and randomization. results: in the fully fed group gh, igf-i and igfbp increased, whereas igfbp decreased from admission to d /ld (all p< . ). accepting an early macronutrient deficit prevented the rise in gh and igf-i and the decrease in igfbp (all p< . ) but did not affect igfbp , whereas basal, but not pulsatile, gh secretion was lowered (p= . ). a stronger rise in gh and igf-i was independently associated with a lower risk of acquiring muscle weakness (or ( %ci) per ng/ml change . ( . - . ) for gh; . ( . - . ) for igf-i). conclusions: accepting an early macronutrient deficit suppressed basal gh secretion and reduced igf-i bioavailability during critical illness, which may counteract its protection against muscle weakness. introduction: aim of the study was to relate hypokalemia (hypok) and hypoglycemia as diabetic ketoacidosis (dka) treatment complications and precocious insulin interruption also use of sodium bicarbonate with length of stay (los) in intensive care unit (icu). methods: analysis of retrospective cohort study data of patient (pt) treated for dka at icu of hospital kaunas clinics of lithuanian university of health sciences during - has been carried out. serum kalemia, glycaemia; rate of episodes of hypok, hypoglycaemia and precocious insulin interruption; use of sodium bicarbonate, in relation with los in icu were analysed. spss . was used for statistic calculations. traits evaluated as significant at p< . . results: at the beginning of dka treatment hypok ( . ± . mmol/l) was recorded in / ( %) pt. due to disregarding of blood ph ( . - . ( . ± . ) kalemia was falsely misinterpreted as "normo-" or "hyperkalemia" . - . ( . ± . mmol/l) in of ( %) pt, as normo-and hyperkalemia thus not treated and complicated by hypok additionally in / ( %) pt. in hypok los in icu was . ± . vs . ± . h, p< . . insulin use has caused hypoglycaemia ( . - . ( . ± . mmol/l)) in / ( %) pt, los in icu . ± . vs . ± . h, p< . . insulin use was interrupted in case of normo -and hypoglycaemia with still persisting ketoacidosis in / ( %) pt, los in icu was found to be . ± . vs . ± . h, p< . . sodium bicarbonate was given for symptomatic treatment of acidosis during the first h of dka in / ( %) pt with stable hemodynamic: hco buffer has increased ( . ± . - . ± . mmol/l), p< . , but ketoacidosis has still persisted, los in icu was . ± . vs . ± . h, p< . . conclusions: hypok ( %), hypoglycemia ( %), precocious interruption of insulin use ( %) have prolonged los in icu almost twice. symptomatic treatment of ketoacidosis with sodium bicarbonate ( / pt) didn't control it and has prolonged los in icu. introduction: cystathionine-γ -lyase (cse), a regulator of glucocorticoid (gc)-induced gluconeogenesis [ ] , correlates with endogenous glucose production in septic shock [ ] . the hyperglycemic stress response to noradrenaline (noa) is mediated by the kidney [ ] and less pronounced with low cse [ ] . gc receptor (gr)-mediated gene expression is differentially regulated: the gr monomer is considered to repress inflammation, and gc side effects are attributed to the gr dimer; recent reports challenge this view [ ] . gc-induced gluconeogenic gene expression is reduced in gr dimerization deficient (grdim) mice [ ] . the aim of this study is to investigate renal cse expression and systemic metabolism in grdim and grwt mice in a resuscitated model of lps-induced endotoxic shock. methods: anesthetized grdim (n= ) and grwt (n= ) mice were surgically instrumented, monitored, resuscitated and challenged with lps. noa was administered to maintain map and c glucose was continuously infused. h after lps, cse expression was determined via immunohistochemistry of formalin-fixed paraffin sections (n= p.gr.). results: grdim required . -fold more noa than grwt and had . fold higher glucose and . -fold higher lactate h after lps. this was concomitant with elevated endogenous glucose production ( -fold), % lower glucose oxidation and . -fold higher renal cse expression in grdim. conclusions: increased cse expression together with higher glucose production (confirming [ , ] ) and glucose levels in grdim mice suggest an association that may link cse to gc signaling. the higher noa administration in grdim mice could contribute to these effects. introduction: to achieve safe glycemic control in critically ill patients frequent blood glucose (bg) measurements and according titration of insulin infusion rates are required. automated systems can help to reduce increased workload associated with diabetes management. this bi-centric pilot study combined for the first time an intraarterial glucose sensor with a decision support system for insulin dosing (sgcplus system) in critically ill patients with hyperglycemia. methods: twenty-two patients ( females, males, with preexisting diabetes mellitus, age . ± . years, bmi . ± . kg/ m , creatinine level . ± . mg/dl, saps (simplified acute physiology score) . ± . , tiss- (therapeutic intervention scoring system) . ± . who were equipped with an arterial line and required iv insulin therapy were managed by the sgcplus system during their medical treatment at the intensive care unit. results: sgcplus-based bg determinations were performed and . ± . sensor calibrations per day were required. sensor glucose readings correlated well with reference bg (figure ). mean treatment duration was . ± . days. time to target was ± min ( - mg/dl) and ± min ( - mg/dl). mean blood glucose was ± mg/dl with seven blood glucose values < mg/dl. mean daily insulin dose was ± u and mean daily carbohydrate intake ± g /day (enteral nutrition) and ± g/day (parenteral nutrition). acceptance of sgcplus suggestions was high (> %). the novel intraarterial glucose sensor demonstrated to be highly accurate. the sgcplus system can be safely applied in critically ill patients with hyperglycemia and enables good glycemic control. introduction: we aimed to assess the effect of frailty as assessed by clinical frailty scale (cfs) and karnofsky performance score (kps) on critical care (cc) and hospital mortality in this group at a nonspecialist tertiary critical care unit. methods: patients admitted to critical care were identified from our electronic database by screening for liver disease or cirrhosis in the admission diagnoses. those with an aetiology of liver disease other than alcoholic liver disease (ald) were excluded. data was collected on patient demographics, length of stay, status at discharge from critical care and hospital and cfs. kps was also calculated where sufficient in-formation was available in the medical record. data was analysed using logistic regression multivariate analysis with stata software. [ ] . results: tg diagnosis criteria and severity grading criteria for acute cholangitis and acute cholecystitis were judged from numerous validation studies as useful indicators in clinical practice and adopted as tg diagnostic criteria and severity grading without any modification. provide initial treatment, such as sufficient fluid replacement, electrolyte compensation, and intravenous administration of analgesics and full-dose antimicrobial agents, as soon as a diagnosis has been made. in new flowchart for the treatment of acute cholecystitis (ac) in the tg , grade iii ac was indicated for gallbladder drainage, but some grade iii ac can be treated by laparoscopic cholecystectomy (lap-c) at advanced centers with specialized surgeons experienced in this procedure and for patients that satisfy certain strict criteria. we also redefine the management bundles for acute cholangitis and cholecystitis. introduction: c-acetate breath tests provide a non-invasive assessment of gastric emptying [ ] and could, hence, be used to judge tolerance to enteral nutrition. result values like t (time for % absorption) correlate with scintigraphic measurements. the data evaluation is based on model equations like the β -exponential function (bex) [ ] . it considers a mono-phasic breath gas response. this may not be the case during critical illness, which could reduce precision too low for a reliable personalized assessment [ ] . methods: we recently developed an evaluation of irregular gastric emptying patterns, which separates absorption from post-absorptive distribution and retention of tracer and from the terminal respiratory release of the oxidized tracer [ ] . using breath test data of icu patients (mean saps +/- ) the precision of this approach was compared with a bex analysis to explore how often an extended analysis is warranted and whether it improves the reliability of estimates. results: patients had a release profile consisting of series of peaks with a periodicity of - min. a first dominant peak carries about % of the released moiety, as reported [ ] for controls. for these patients the precision in t for the bex approach was +/- % of that observed for the new approach. for the other patients, the secondary peaks had a similar periodicity but were more pronounced, indicating persisting peristaltis, which has been linked to tolerance to enteral nutrition [ ] . the bex approach achieved a precision of +/- % relative to the new one, challenging its applicability for these patients. introduction: clinical scoring systems used to prognosticate the severity of acute pancreatitis (ap), such as apache ii, are cumbersome and usually require hours or more after presentation to become accurate, at which time the window for early therapeutic intervention has likely passed. sirs at presentation is sensitive but poorly specific for severe ap. we postulated that sirs and accompanying hypoxemia would specify at presentation patients with ap who have severe inflammation and are at risk for clinically severe disease. methods: patients with ap who had sirs and hypoxemia at presentation were enrolled in an open-label study evaluating the safety and efficacy of cm -ie, a calcium release-activated calcium (crac) channel inhibitor (nct ). hypoxemia was defined as an estimated pao < mm hg calculated using a log-linear equation and the spo on room air at the time of presentation. a contrastenhanced computed tomography (cect) was performed at presentation and a cbc with differential, d-dimer and crp were analyzed daily. the cect was read by a blinded central reader who assessed the degree of inflammation using the balthazar scoring system (table ) . results: patients, seven men and six women, have been randomized in the study. the mean estimated pao at presentation was mm hg. patients had sirs criteria present and the other patients had sirs criteria present. the median value for age was . (iqr - ), initial neutrophil-lymphocyte ratio (nlr) . ( . introduction: to investigate whether circulating immune profiles were able to serve as early biomarkers in predicting persistent organ failure (pof methods: thirty-nine patients with predicted severe acute pancreatitis (psap) and healthy control subjects were prospectively enrolled in our study. we measured the expression of monocytic human leukocyte antigen-dr (mhla-dr), the proportions of dendritic cells (dc) and its subtypes (including myeloid dendritic cell (mdc) and plasmacytoid dendritic cell (pdc)), the different cytokineproducing cd + t helper (th) cells and regular t (treg) cells. plasma crp and several inflammatory mediators levels were measured by elisa. results: compared with healthy controls, there is a significant decrease in the expression of mhla-dr, the frequencies of total circulating dcs and its subsets, and percentage of th cells in patients with psap. however, we found significantly higher frequencies of th cells, higher proportion of treg cells than healthy subjects. of interest, we observed that there was a significant decrease in the positive percentage and mean fluorescence intensity (mfi) of mhla-dr, the proportions of total dcs and pdc, and th cells in patients with pof compared with transient organ failure (tof). besides, there is a significantly higher frequency of th cells in pof than those in tof. area under the receiver-operating characteristic curve analysis showed that disease severity scores had a moderate discriminative power for predicting pof in patients with psap. more importantly, the expression of mhla-dr and the percentage of dcs and pdc had a significantly higher auroc and thus, better predictive ability than disease severity in patients with psap. conclusions: circulating immune profile show multiple aberrations in patients with psap who have developed pof. both the expression of mhla-dr and the percentage of total dc and pdc may be early good biomarkers for predicting risk of pof in patients with psap. introduction: pancreatic fistula (popf) due to anastomosis insufficiency is a common ( - %) complication after pancreaticoduodenectomy and often discovered with delay, causing severe morbidity, icu stay and deaths. microdialysis (md) catheters have been shown to detect inflammation and ischemia in several postoperative conditions and organs. the aim was to investigate if md catheter monitoring could facilitate earlier detection of popf than current standard of care. methods: in a prospective, observational study patients ( to years) were investigated. a md catheter was fixed to the pancreaticojejunal anastomosis. samples for analysis of glucose, lactate, pyruvate and glycerol were acquired hourly during the first hours, then every - hours to discharge. popf was defined according to the international study group of pancreatic fistula update definition. results: patients who developed popf (n= ) had significantly higher glycerol levels (p< . ) in microdialysate than did patients without popf (n= ) during the first h. thereafter, the difference diminished. a glycerol concentration > μmol/l during the first h detected patients who later developed popf with a sensitivity of % and a specificity of %. lactate and lactate to pyruvate ratio were significantly higher (p< . ) and glucose was significantly lower (p< . ) in patients with popf from about h. fig. shows microdialysis measurements in patients with (red lines) and without (blue lines) popf. conclusions: a high level of glycerol in microdialysate is an early (first hours) indicator of popf. glucose, lactate and lactate to pyruvate ratio are indicators of peritonitis caused by the leakage. thus, md monitoring detects popf several days earlier than current methods and may play an important clinical tool in the future. we are currently conducting a rct to explore if md monitoring will improve prognosis in these patients the phenomenon of total impaired of metabolic activity of gut microbiota in critically ill septic patients introduction: during a critical condition, dramatic disturbances occur not only in the change of species diversity, but in gut microbiota metabolism as well, that might lead to nonreversible breakdowns of host homeostasis and death [ ] . metabolic activity of microbes can be assessed by the measurement of the levels of aromatic microbial metabolite (amm) in blood serum, which are associated with the severity and mortality of icu patients. critically ill patients are characterized by the totally different sfs profile than in healthy people, particularly by the absence of phpa; but dominated by p-hphaa and p-hphla [ ] . the purpose of our study is to assess the gut metabolic activity via amm in sepsis. methods: in this study simultaneously serum and fecal samples (sfs) were taken from icu patients: -with sepsis, -chronic critical ill (cci) patients and control - sfs from healthy people. after liquid-liquid extraction from serum and fecal samples, phenylcarboxylic acids (amm) were measured using gc/ms (thermo scientific). results: the sum of the level of most relevant amm in serum samples were higher in patients with sepsis (median - . μm) than in cci patients ( . μm) and healthy people ( . μm). at the same time the opposite pattern was observed in the fecal samples - . , . and . μm, respectively. the ratios of sums amm gut/serum were higher in healthy people than icu patients (fig. ) introduction: the aim of this study is to describe the characteristic of bioelectric impedance vector analysis (biva) and muscular ultrasound during the first week after admission in the icu, and their correlation with indices of metabolic support. biva is a commonly used approach for body composition measurements [ ] . muscular ultrasound represents a valid tool to provide qualitative and quantitative details about muscle disease [ ] . methods: consecutive patients admitted to icu and expected to require mechanical ventilation for at least hours were enrolled in the study. within the first hours of icu admission (t ), patients were evaluated with muscular ultrasonography comprehensive of diaphragm thickness (dth) and rectus femoris cross-sectional area (csa). at the same time, biva and biochemical analysis. all the same measures were repeated at day (t ) and (t ) (figure (table ) . dividing the patients in two groups based on prealbumine changes (t vs t : increase, anabolic vs decrease, catabolic), those in which prealbumine increased had a higher reduction in muscle mass ( figure ). conclusions: this study showed how the pa tends to be reduced in the first week of icu stay. it is correlated with a concomitant introduction: the modified nutrition risk in critically ill (mnutric) has been developed in order to identify critically ill patients who may receive benefit from nutrition support [ ] . several evidences showed the association between the mnutric score and clinical outcomes [ , ] , however there are no data in thai critically ill patients. the purpose of this study was to find the association between mnu-tric score and -day mortality in medical intensive care unit (icu) patients, ramathibodi hospital. methods: we retrospectively reviewed the medical patient records from june to january . a mnutric score of each patient was calculated to evaluate the risk of malnutrition. statistical analysis of the association between mnutric score and -day mortality, length of stay in icu and hospital were performed. results: a total of critically ill patients were included in the study. the -day mortality was . % in patients with high mnutric score ( - ) and . % in patients with low mnutric score ( - ). modified nutric score was significantly correlated with day mortality (r = . , p< . ), length of stay in icu (r = . , p< . ) and length of stay in hospital(r = . , p< . ). in the receiver operating characteristic (roc) curve analysis, the auc of mnutric score and -day mortality was . ( % confidence interval (ci), . - . ) (fig ) . optimal cut-off value of showed sensitivity of . % and specificity of . % in mortality prediction (youden's index, . ). additionally, patients who received adequate nutrition supplement within days was . % for calorie and . % for protein. there was no association between nutrition support and -day mortality. conclusions: in thai medical intensive care population, the mnutric score was associated with -day mortality in critically ill patients. fig. (abstract p ) . within the first hours of icu admission (t ), patients will be evaluated with muscular ultrasonography comprehensive of diaphragm thickness and rectus femoris (medial vastus) cross-sectional area. at the same time, anthropometric measure will be collected (such as body height, ideal body weight, real body weight declared, right arm circumference) as well as biva measure (xc, r, pa, lean body weight and % of extracellular body weight) and biochemical analysis (inclusive albumin, pre-albumin, blood count, lymphocyte count, magnesium, phosphorus, reticulocytes, renal and hepatic function test). the day after, the fluid balance will be calculated as well as the nitrogen balance. all the same measures will be repeated at day (t ) and days (t ) introduction: ultrasonography is an essential imaging modality in critical care to diagnose and guide for therapeutic management of shock, multiple organ failure, etc. enteral tube feed intolerance occurs frequently in hospitalized patients and more so in critically ill patients. in present study, we consider that nursing staff may be able to use bedside ultrasound as an alternative to standard aspiration protocol or radiographic studies to assess gastric volume and nasogastric (ng) tube in patients with enteral feed intolerance. methods: in present prospective, single-center study, we performed ultrasound residual stomach volume and ng tube placement assessments of adult critically ill patients (figure ) compared to standard protocol of stomach volume assessment (routine daily shift -ml syringe aspirations) and ng (nasogastric) tube placement verified by abdominal x ray. we used an abdominal (linear ultrasound transducer) probe ( - mhz). the residual volume was calculated according to formula: gv (ml) = + . x right-lateral csa- . x age). results: hundred simultaneous double (ten critically ill patients) ultrasound measurements sessions were performed by nursing staff of our intensive care (icu) (fig ) . double simultaneous measurements of the ultrasound assessments were compared to standard nurse icu protocol for assessment of residual volume of stomach. the new ultrasound assessment method demonstrated excellent intra-class reliability (icc- . ( . - . , p< . ) and strong correlation with standard residual volume assessment method (icc- . ( . - . , p< . ). ng tube placement was successfully verified by ultrasound measurements in all ten critically ill patients and, thereafter, confirmed by abdominal x-rays. conclusions: preliminary results of our study demonstrated good correlations between both methods of ng tube placement and residual stomach volume: standard icu nurse protocol and ultrasound assessment. evaluating the documentation of nasogastric tube insertion and adherence to safety checking l roberts introduction: enteral feeding into a misplaced nasogastric (ng) tube is recognised by the national patient safety agency as a never event. ng tubes are commonly indicated in level / patients, thus we set out to evaluate current practice in critical care. the aim was to evaluate: documentation of insertion, adherence to safety guidance pertaining to checking safe use, chest x-ray interpretation. methods: this prospective cohort study was based on inpatients in critical care who had insertion of ng tubes over four weeks; there were insertions. data was analysed from patients' medical notes and the hospital's imaging system. results: % of insertions were documented using proformas. . % of proforma documentations included or more details: type of tube, tube length at the nostril, nex measurement, aspirate adequacy, chest x-ray adequacy, whether it was safe to feed. only . % of hand-written documentations included or more details. % of initial aspirates were obtained on insertion, of these, % had an appropriate ph between and . . this led to % of patients having chest x-rays to confirm initial placement of the ng tube. only % of chest x-rays adequately satisfied the four criteria. written documentation in medical notes stating if it was safe to feed was completed in % of cases. conclusions: we found that proformas ensure a higher level of detail and uniformity in the documentation of ng tube insertions. there was a high incidence of chest x-rays performed to confirm correct placement of tubes due to difficulties in obtaining aspirates and failure to follow guidelines. a need for a uniform, ward-specific proforma on ng tube insertion has been identified, as well as a teaching session on chest x-ray interpretation and on techniques to aid obtaining aspirates. we have established critical care's shortcomings in ng tube insertion documentation and tube safety checking. introduction: pressure ulcers(pu) are considered as important types of public health problems, due to high mortality and cost. we aimed to investigate the efficiency of curcumin and fish oil on prevention and treatment of pu using a feasible mice model. methods: mice were randomly divided into control(group ), curcumin(group ), fish oil(group ), curcumin and fish oil(group ) groups. mm skin bridge between two gauss magnets was formed on the back of mice, followed by ischemia reperfusion cycles as hours of rest after hours of magnet placement [ ] . a single dose of curcumin and fish oil was injected intraperitoneally. tissue samples had taken th day of first compression, rates of pu, inflammation, reepithelisation, neovascularisation and granulation were examined histopathologically. the data analyzed by pearson chi-square test. results: third degree pu were observed in all groups.there was no significant difference between groups in terms of inflammation.the formation of reepithelisation showed a significant difference between groups.partial reepithelisation ratios in group and group was elevated.there was significant difference between groups in terms of neovascularisation, the highest rate as % was observed in group .formation of granulation was observed at maximum rate as . % at group . conclusions: depending on positive results of curcumin, fish oil, cur-cumin+fish oil on wound healing it may be advised to use them in treatment of acute pu.after similar rate of pu with control group we consider that it should be beneficial to evaluate the effect of these therapies with more studies by changing the mode of administration, time of initiation and duration of therapy. introduction: inflammation is a key driver of malnutrition during acute illness and has different metabolic effects including insulin resistance and reduction of appetite. whether inflammation influences the response to nutritional therapy in patients with disease-related malnutrition remains undefined. we examined whether the effect of nutritional support on the risk of mortality differs based on the inflammatory status of patients. methods: this is a secondary analysis of a multicentre trial in eight swiss hospitals, where patients with a nutritional risk score (nrs) of ≥ upon hospital admission were randomly assigned to receive protocol-guided individualized nutritional support according to nutrition guidelines (intervention group) or a control group. the inflammatory status was defined based on admission crp levels as low inflammation (cpr < mg/dl), moderate inflammation (crp - mg/dl) and high inflammation (crp > mg/dl). results: we included a total of , patients of which . %, . % and . % had low, moderate and high inflammation levels on admission. while overall there was a significant reduction in day mortality associated with nutritional support (adjusted or in the overall cohort . , %ci . - . ), the subgroup of patients with high inflammation did not show reduced mortality (adjusted or . , %ci . - . , p for interaction = . ). there was no difference in other secondary endpoints when stratified based on inflammation. nutritional support did not affect crp levels over time (kinetics). conclusions: this secondary analysis of a multicentre randomized trial provides evidence, that the inflammatory status of patients influences their response to nutritional support. these findings may help to better individualize nutritional therapy based on patients initial presentation. introduction: low plasma glutamine levels have been associated with unfavourable outcomes in critically ill patients. this study aimed to measure plasma glutamine levels in critically ill patients and to correlate glutamine levels with biomarkers and severity of illness. methods: we enrolled critically ill patients admitted to three icus in south africa, excluding those receiving glutamine supplementation prior to admission. we collected clinical, biochemical and dietary data. plasma glutamine levels were determined within hours of admission, using liquid chromatography mass spectrometry and categorized as low (< μmol/l), normal ( - μmol/l) and high (> μmol/l). results: of the patients (average age . ± . years, % male), % were mechanically ventilated, with a mean apache ii score of . ± . and a mean sofa score of . ± . . plasma glutamine levels were low in . % (median plasma glutamine of . μmol/l). baseline plasma glutamine correlated inversely with crp (r=- . , p< . ) and serum urea (r=- . , p< . ), and positively with serum bilirubin (r= . , p< . ) and serum alt (r= . , p= . ). significantly more patients with low admission glutamine levels required mechanical ventilation (chi = . , p< . ) and had higher apache scores (p= . ), higher sofa scores (p= . ), higher crp values (p< . ), higher serum urea (p= . ), higher serum creatinine (p= . ), lower serum albumin (p< . ) and lower bilirubin levels (p= . ). using multiple logistic regression analysis, apache score (odds ratio, [or] . , p= . ), sofa score (or . , p= . ) and crp (or . , p< . ) were significant predictors of low plasma glutamine levels. roc curve analysis revealed a crp threshold value of . mg/l to be indicative of low plasma glutamine levels (auc . , p< . ). conclusions: . % of critically ill patients had low plasma glutamine levels on admission to icu. this was associated with increased disease severity and higher crp. introduction: the east of england deanery operational delivery network in the united kingdom came together as a group of intensive care units to comply an evidence-based care bundle. one of the branches of this care bundle is on parenteral nutrition and states: 'parenteral nutrition should not be given to adequately nourished, critically ill patients in the first seven days of an icu stay.' this is based on evidence [ ] [ ] [ ] that showed that 'in patients who are adequately nourished prior to icu admission, parental nutrition initiated within the first seven days has been associated with harm, or at best no benefit, in terms of survival and length of stay in icu.´the objective of this second cycle was to assess whether or not we are adhering to the guidelines, last year we were failing to hit targets and after some action i reassessed how we performed in the year compared to . methods: a retrospective audit of the whole year of for all patients admitted to icu who had parenteral nutrition started at any point during their stay. results: there is a significant improvement in the percentage of patients who are being started incorrectly on tpn before days ( % compared to %) (fig , ) . i also found a total reduction in the number of patients prescribed tpn, a reduction in the number of bags being used and a reduction in length of hospital stays. conclusions: as we have recently switched over to an electronic icu programme for all documentation and prescriptions, as part of our plan and act in the pdsa cycle we are organising for several things to be put in place on the new system on prescription: pharmacy authorisation, links to guidelines and alert/justification boxes. i will do a further cycle in another year. jg and mpc contributed equally. introduction: recent rcts revealed clinical benefit of early macronutrient restriction in critical illness, which may be explained by enhanced autophagy, an evolutionary conserved process for intracellular damage elimination [ ] . however, in the absence of specific and safe autophagy-activating drugs, enhancing autophagy through prolonged starvation may produce harmful side effects. a fasting-mimicking diet (fmd) may activate autophagy while avoiding harm of prolonged starvation, which also improved biomarkers of age-related diseases in an experimental study [ ] . we evaluated if short-term interruption of continuous feeding can induce a metabolic fasting response in prolonged critically ill patients. methods: in a randomized cross-over design, prolonged critically ill patients receiving artificial feeding were randomized to be fasted for hours, followed by hours full enteral and/or parenteral feeding, or vice versa. patients were included at day in icu and blood glucose was maintained in the normal range. at the start and after and hours, we quantified total bilirubin, urea, insulin-like growth factor-i (igf-i) and beta-hydroxybutyrate (boh) in arterial blood. insulin requirements were extracted from patient files. changes over time were analyzed by repeated-measures anova after square root transformation. results: as compared to hours of full feeding, hours of fasting decreased bilirubin (- . ± . mg/dl; p= . ) and igf-i (- . ± . ng/ml; p< . ), and increased boh (+ . ± . mmol/l; p< . ), without affecting urea concentrations (fig ) . fasting reduced insulin requirements (- . ± . iu/hour; p< . ). conclusions: short-term fasting induces a metabolic fasting response in prolonged critically ill patients, which provides perspectives for the design of a fmd, aimed at activating autophagy and ultimately at improving outcome of critically ill patients. introduction: recent evidence has led to changed feeding guidelines for critically ill patients, with a shift towards lower feeding targets during the acute phase [ ] . when micronutrients are not provided separately, prolonged hypocaloric feeding could induce micronutrient deficiencies and increase risk of refeeding syndrome once full feeding is restarted, which are both potentially lethal complications [ ] . since there is limited evidence how to optimize micronutrient provision in order to avoid deficiencies, we hypothesized that there is a great variation in current practice. methods: within the men section of the european society of intensive care medicine (esicm), we designed a questionnaire to gain insight in the current practice of micronutrient administration. in email blasts, invitations were sent to all esicm members, with currently more than respondents. the survey will be closed at december , . results: first, we will describe demographic characteristics of the respondents, including geographical location, icu and hospital type, and function. second, we will describe some aspects of the current practice of micronutrient administration. we will identify the proportion of respondents having a protocol, on which evidence such protocol is based and whether it takes into account the stability and daylight sensitivity of micronutrients. next, bearing refeeding syndrome in mind, we will identify whether there are respondents who never measure and/or separately administer micronutrients and phosphate. finally, we will make a top of the most measured and most supplemented micronutrients. conclusions: this survey will deliver more insight in the current practice of micronutrient provision across different types of icus and may identify areas for future research. furthermore, we will evaluate whether there is need to increase awareness for refeeding syndrome. introduction: large gastric residual volumes (grvs) have been used as surrogate markers of delayed gastric motility to define enteral feeding intolerance (efi). recent studies have challenged the definition of efi. study objectives: ) investigate the potential relationship between grvs and clinically outcomes, ) develop an algorithm for early identification of patients at increased risk of mortality due to efi. methods: a retrospective study of inpatient encounters from electronic health record charts within the dascena clinical database. , patients were included in the study; patients had efi. eight vital signs (diastolic/systolic bp, heart rate, temperature, respiratory rate, grv, glasgow coma scale, and feeding rate) and their trends were input to the classifier. machine learning classifiers were created using the xgboost gradient boosted tree method with -fold cross validation. results: rate of change in grv (Δ grv) was measured over a -day period, beginning at the time of efi onset (figure a) . figure b shows a high likelihood of mortality for patients with none or modest grv reduction. patients with an increase in grv over the five-day period after efi onset had the highest mortality likelihood. a stratification algorithm was developed to identify efi patients who died inhospital despite grv reduction at , , and hours in advance of efi onset. area under the receiver operating characteristic (auroc) curves demonstrated high sensitivity and specificity of algorithm predictions of in-hospital death up to hours in advance of efi onset (table ) . conclusions: the analysis suggests an association between grv and mortality, especially in patients with persistent grv increase over the -day period after efi onset and the potential of algorithmic models to predict efi development. prospective validation of these fig. (abstract p ) . changes in metabolic markers of fasting over time for both randomization groups algorithms may assist in clinical trial design to develop treatments for patients at highest risk of experiencing serious outcomes due to efi. a quality improvement project to improve the daily calorific target delivery via the enteral route in critically ill patients in a mixed surgical and medical intensive care unit (icu) b johnston, d long, r wenstone royal liverpool and broadgreen university hospital trust, critical care, liverpool, united kingdom critical care , (suppl ):p introduction: 'iatrogenic underfeeding' is widespread with the calo-ries study reporting only %- % of prescribed daily kcal was actually delivered to patients [ ] . in the present project, quality improvement methodology was utilised with the aim of delivering greater calories by implementing -hour volume-based feeding and allowing increased feeding rates for, 'catch up' of missed daily feed volume. methods: baseline data assessing the percentage of daily kcal delivered to ventilated patients was collected in september . data was presented and new intervention guidelines agreed based upon the pepup protocol [ ] . nurse champions were identified and were responsible for cascade training of the pepup protocol. educational tools to help determine daily calorific requirement and volume of feed required were provided. repeat data was collected at months (cycle ) after pepup implementation. results: ten patients were included in cycle . during cycle the percentage of kcal achieved via enteral feeding was %. following intervention this increased to % (p< . ) during cycle . this increased further to . % of daily kcal when calories obtained from propofol were included. conclusions: a -hour volume-based feeding regimen is a simple and cost-effective method of improving enteral feeding targets. through the use of quality improvement methodology, we demonstrated that this approach is achievable. the success of this project has led to the adoption of the protocol in other icu units in a regional critical care network. effect of non-nutritional calories on the calory/protein ratio in icu patients s jakob, j takala university hospital bern, dept of intensive care medicine, bern, switzerland critical care , (suppl ):p introduction: nutritional diets are composed to match the needs of critically ill patients. while effective calory needs can be measured or calculated, the needs of proteins are more controversial. we aimed to calculate non-nutritional calories and assess how they influence the ratio of calories to protein delivered to the patients. methods: in this retrospective analysis, nutritional and nonnutritional calories and protein delivery were calculated in consecutive icu patients receiving enteral nutrition in . introduction: marked protein catabolism is common in neurocritical patients. optimal nutritional monitoring and protein nutritional adequacy could be associated with outcome in neurointensive care unit (ncu) patients. we aimed to evaluate the impact of monitoring and optimal support of protein using nitrogen balance on outcome in neurocritical patients. methods: a consecutive patients who were admitted to ncu were included between july and february . nitrogen balance was calculated using excreted urine urea nitrogen during icu admission. follow-up nitrogen balance monitoring was performed in patients. we divided patients into two groups based on the results of nitrogen balance (positive balance and negative balance). moreover, we evaluated improvement of nitrogen balance in patients. we assessed the outcome as length of stay in hospital, length of stay in ncu, and in-hospital mortality. we compared the clinical characteristics and outcome according to nitrogen balance. results: among the included patients (age, . ; and male. . %), ( . %) patients had negative nitrogen balance. the negative balance group was more likely to have lower glasgow coma scale (gcs), longer length of stay in hospital, and longer length of stay in ncu. in patients with follow-up nitrogen balance monitoring, improvement of nitrogen balance group had lower in-hospital mortality ( . % vs. . %, p = . ), and received adequate protein intake ( . g/kg/day vs. . g/kg/day, p = . ) compared to no change group (table ) . there was no significant difference in baseline nitrogen balance, baseline body mass index, and gcs between two groups. conclusions: this study demonstrated that critical illness patients in ncu are underfeeding using nitrogen balance, however, adequate provision of protein was associated improvement of nitrogen balance and outcome. this suggests that adequate nutrition monitoring and support could be an important factor for prognosis in neurocritical patients. increased protein delivery within a hypocaloric protocol may be associated with lower -day mortality in critically ill patients introduction: to test the hypothesis, using real world evidence that increasing protein delivery and decreasing carbohydrates (cho) may improve clinical outcomes. methods: retrospective analysis of existing electronic medical records (emr) of patients admitted to the intensive care units (icu) at the geisinger health system. logistic regression analysis was used to determine correlation between protein delivered (which was proportional to the concentration of protein in the formula utilized) and clinical outcomes. results: medical encounters for a total number of , icu days were collected and analyzed. average age was . years ( . % male) and . % were obese and overweight. primary diagnoses included sepsis or septic shock, acute and/or chronic respiratory failure (or illness), cardiovascular diseases, stroke and cerebrovascular diseases among others. median hospital los was . days, . days in the icu, median days of invasive mechanical ventilation of . -day readmission rate among patients discharged alive was . %. patients in the high protein group received lower amounts of chos (data not shown). unadjusted -day post-discharge mortality was inversely proportional to the amount of protein delivered (table ) . conclusions: a significant improvement in mortality is observed with increased protein delivery while decreasing carbohydrate loads. prospective randomized trials are warranted to establish causality. introduction: acute kidney injury (aki) is associated with high mortality. the risk increases with severity of aki. our aim was to identify risk factors for development and subsequent progression of aki in critically ill patients. methods: we analysed patients without end-stage renal disease who were admitted to the icu in a tertiary care centre between january to december and did not have aki on admission. we identified risk factors for development and non-recovery of aki as defined by the kdigo criteria. results: the incidence of new aki in days was % (aki i %, aki ii %, aki iii %). multivariate analysis revealed bmi, sofa score, chronic kidney disease (ckd) and cumulative fluid balance as independent risk factors for development of aki. among patients who developed aki in icu, % had full renal recovery, % partial recovery and % had no recovery of renal function by day . aki patients without renal recovery in days had significantly higher hospital mortality ( %) compared to the other groups. independent risk factors for non-recovery of renal function were ckd, mechanical ventilation, diuretic use and extreme fluid balance before and after first day of aki. (table ) the association between cumulative fluid balance before aki and hours after aki with risk of aki non-recovery are shown in figure and . conclusions: aki is common and mortality is highest in those who do not recover renal function. cumulative fluid accumulation impacts chances of aki development and progression. (table ). all were in r . / ( %) of those with an admission ck> had aki or . all ( %) patients who required crrt for aki associated with rm were at risk for aki regardless of initial ck: vascular surgery ( / ), multi-organ dysfunction ( / ), and/or pre-existing renal disease ( / ). conclusions: raised ck is common in icu but its cause is multi factorial thus an isolated measure > does not require immediate high output treatment for rm aki. aki is more common in patients who have more than ck> on sequential days or those whose first ck was > as rm may be contributing. a single ck> in patients with a clear reason to develop rm should also start treatment. surgical outcomes of end-stage kidney disease patients who underwent major surgery p petchmak , y wongmahisorn , k trongtrakul introduction: acute kidney injury (aki) occurs in more than % of successfully resuscitated out-of-hospital cardiac arrest patients treated with targeted temperature management (ttm) [ ] . the effect of the duration of cooling on aki has not been well studied. in this post-hoc analysis of the tth randomized controlled trial that compared vs -hours of ttm ( °c) after cardiac arrest [ ] , we studied the impact of ttm length on the development of aki. fig. . duration of ttm had a significant impact on the development of creatinine values during the first days in the icu, p< . . this was primarily driven by an increase in creatinine during rewarming on day for the hour and day for the -hour group (fig ) . conclusions: in a trial of vs hours of ttm after out-of-hospital cardiac arrest, the length of ttm did not affect the incidence of aki. fig. (abstract p ) . creatinine over time patients [ ] , but there are no published data on longer-term renal outcomes in adult patients. the purpose of this study was to assess longer-term trends in serum creatinine in this cohort. methods: a retrospective study was conducted of all patients admitted to an adult regional referral centre for ecmo at a uk university hospital between and . those who survived for > months were included. demographics, baseline serum creatinine, presence of aki during icu admission, and serum creatinine at hospital discharge were determined. serum creatinine and dependence on renal replacement therapy (rrt) were assessed at and months post ecmo. results: patients had a complete (or near-complete) data-set available. the mean age was . years, % of whom were male. / had aki during their critical care admission. none were dependent on rrt at or months post ecmo. most patients had lower serum creatinine results at hospital discharge compared to their pre-hospitalisation baseline, but creatinine concentrations at and months post ecmo tended to be higher than at hospital discharge ( figure ) . conclusions: in this cohort of ecmo patients who were discharged from hospital alive, serum creatinine tended to be lower at hospital discharge compared to baseline and rose again in the following months. decreased creatinine production due to deconditioning and muscle wasting may offer a biological rationale for the lower creatinine results at hospital discharge [ ] . therefore, caution should be exercised in the use of serum creatinine at hospital discharge to assess renal dysfunction -further research is warranted. introduction: aki complicates more than half of icu admissions [ , ] and is associated with development of chronic kidney disease (ckd), need for renal replacement therapy (rrt) and increased mortality [ ] . we prospectively evaluated all icu admissions during a one-year period in order to determine incidence, etiology and timing of aki as well relevant clinical outcomes. methods: prospective observational study of all patients admitted from jan to dec to a multidisciplinary icu in greece. patients with end-stage renal disease and anticipated icu stay less than hrs were excluded. aki diagnosis and classification was based on kdigo criteria [ ] . lowest creatinine level within months before admission or first creatinine after icu admission served as reference. (fig ) . conclusions: although aki alert does not include urine output criterion or aki risk factors, it remains a helpful tool to point out patients with aki. education and diagnostic algorithms are still needed to early diagnose and treat aki patients. influence of severity of illness on urinary neutrophil gelatinaseassociated lipocalin in critically ill patients: a prospective observational study c mitaka, c ishibashi, i kawagoe, d satoh, e inada untendo university, anesthesiology and pain medicine, tokyo, japan critical care , (suppl ):p introduction: neutrophil gelatinase-associated lipocalin (ngal) is a diagnostic marker for acute kidney injury (aki). ngal expression is highly induced not only in kidney injury, but also in epithelial inflammation of intestine, bacterial infection, and cancer. however, the relationship between ungal and severity of critically ill patients has not been well understood. the purpose of this study was to elucidate whether ungal is associated with severity of illness and organ failure in critically ill patients. methods: we prospectively enrolled patients with sepsis (n= ) and patients who underwent esophagectomy with gastric reconstruction for esophageal cancer (n= ). sepsis was defined according to sepsis- . ungal levels were measured on icu day , , , and . ungal levels and aki rate in patients with sepsis were compared with those in patients who underwent esophagectomy. aki was defined according to kdigo. acute physiology and chronic health evaluation (apache) ii score and sequential organ failure assessment (sofa) score were calculated. results: median ungal level ( ng/mg creatinine) was significantly higher in patients with sepsis than that ( ng/mg creatinine) in patients who underwent esophagectomy on day . median apache ii score and median sofa score in patients with sepsis were significantly higher than those in patients who underwent esophagectomy. four patients with sepsis developed aki, and out of them underwent continuous renal replacement therapy, whereas no patients who underwent esophagectomy developed aki. ungal levels were positively correlated with apache ii score and sofa score in patients with sepsis. ungal levels were remarkably elevated (> ng/mg creatinine) in urinary tract infection (n= ), loops enteritis (n= ), and obstructive jaundice due to cholangiocarcinoma (n= ). conclusions: these findings suggest that ungal level is associated with severity of illness and organ failure in patients with sepsis. ungal levels might be influenced by severity of illness and inflammation. to assess the quality of the course us renal images had to be evaluated in "post-renal obstruction" (p-ro) or "no p-ro". the rate of correctness (roc farius ) was determined. in we, once again, contacted the students to attend a web-based online "follow-up". this online survey was created with "google formular". new and unknown us images were presented and rated in "p-ro" or "no p-ro" (roc fup introduction: septic-induced kidney injury worsen the patient's prognosis [ ] . renal resistance index (rri) is correlated with an increased mortality in septic patients [ ] . the aim of this study was to describe the evolution of rri in a rat sepsis model. methods: the local ethics committee approved the study (apa-fis# - ). sepsis was induced in -month-old male rats by caecal ligation and puncture (clp) [ ] . the rri was assessed before and h after clp by pulse doppler on the left renal artery (rri=(peak systolic velocityend diastolic peak)/ peak systolic values expressed as % per column. abbreviations in alphabetical order: aki acute kidney injury; akin acute kidney injury network definition; ckd chronic kidney disease. there were statistical differences between subgroups with and without aki for the subgroups of patients with previous ckd (p = . *), sepsis at admission (p = . **), hypotension (p= . ***) fig. (abstract p b) . target comparing accuracy and precision of aki alert and actual aki diagnoses velocity) (fig ) . rri were compared by a paired wilcoxon test (r software v. . . ). a p value < . was considered significant. results: rats were included. hours after sepsis induction, all rats were in septic shock with cardiac dysfunction. the rri increased after sepsis induction compared to baseline ( . ± . vs . ± . , p< . ) and mean renal artery velocity decreased ( . ± . vs . ± . , p< . ) (fig ) . systolic and diastolic peaks velocity of the renal artery were unchanged. conclusions: sepsis induced changes in rri and mean velocity on the left renal artery whereas no changes in systolic or diastolic velocities were seen. these results are consistent with available clinical datas. the rri could be an additional tool to assess renal failure in septic rats. further studies are needed to confirm the validity of this marker during sepsis. kidney failure is one of the most common organ dysfunction during sepsis. the rri could be an additional tool in small animals to assess the effects of potential therapeutic targets on renal function induced by sepsis. (fig ) . the egfr improved more with the heparin group ( % vs %; p= . ) (fig ) . interruptions of the filter circuit were as expected less with the citrate group ( mins vs mins; p= . ). finally, inotropic requirements increased following therapy interruptions, more so with patients receiving citrate ( . % vs . %; p= . ). conclusions: our analysis suggests that using citrate anticoagulation for rrt results in a monitoring cost saving of approximately £ per hours, alongside the other conferred savings previously reported. furthermore, results demonstrate the efficacies of both systems are similar in the initial hours, although there is a suggestion that heparin systems improves renal parameters more quickly. finally, interruptions and 'filter downtime' caused an increase in the patient's inotropic requirements, however results suggestive that this is greater in the citrate group. mmol/l respectively. demographic characteristics of the study group and the main parameters of the procedure were presented in fig . conclusions: regional citrate is a safe and effective anticoagulation method for crrt in children, when it is applied following a protocol. it significantly prolongs circuit survival time and thereby should increase crrt efficiency. we did not find any serious adverse effects of regional citrate anticoagulation. - ) , deceased at year n= ( %). the mdrd trend is more indicative than creatinine of decline of renal function in the post operative period (fig ) . crrt was used in . % ( pts) and was associated to a greater los and mortality (fig ) . preoperative bilirubin, bun and creatinine are among the greatest risk factors for its use ( table . at year follow up n= pts ( . %) were on hemodialysis. conclusions: aki requiring crrt in after lt is associated with higher mortality and los. identify patients at risk and adopt preventive strategies in the perioperative period is mandatory. introduction: we developed a new co removal system, which has a high efficiency of co removal at a low blood flow. to evaluate this system, we conducted in vivo studies using experimental swine model. methods: six anesthetized and mechanically ventilated healthy swine were connected to the new system which is comprised of acid infusion, membrane lung, continuous hemodiafiltration and alkaline infusion. in vivo experiments consist of four protocols of one hour; baseline= hemodiafiltration only (no o gas flow of membrane lung); membrane lung = "baseline" plus o gas flow of membrane lung; "acid infusion" = "membrane lung" plus continuous acid infusion; "final protocol" = "acid infusion" plus continuous alkaline infusion. we provided an interval period of one hour between each protocol. we changed the respiratory rate of the mechanical ventilation to maintain pco at - mmhg during the experiment. results: the amount of co eliminated by the membrane lung (vco ml) significantly increased by . times in the acid infusion protocol and our final protocol compared to the conventional membrane lung protocol, while there was statistically no significant difference observed in the levels of ph, hco -, and base excess between each study protocol. minute ventilation in the "final protocol" significantly decreased by . times compared with the hemodiafiltration only protocol (p < . ), the membrane lung (p= . ) and acid infusion protocol (p= . ). we developed a novel ecco r system which efficiently removed co and is easy-to-setup to permit clinical application. this new system significantly reduced minute ventilation, while maintaining acid-base balance within the normal range. further studies are needed for the clinical application of this easy setup system comprising of the materials typically used in a clinical setting. , and psychomotor agitation ( %) while the most common symptoms of hypertensive emergency were chest pain ( . %), dyspnea ( . %) and neurological deficit ( %). clinical manifestations of hypertensive emergency were cerebral infarction ( . %), acute pulmonary edema ( . %), hypertensive encephalopathy ( . %), acute coronary syndromes ( . %), cerebral hemorrhage ( ,. %), congestive heart failure ( %), aortic dissection ( . %), preeclampsia and eclampsia ( . %). conclusions: hypertensive urgencies were significantly more common than emergencies ( . % vs. . %, p< . ). there was no statistically significant difference in the number of patients with hypertensive urgency and emergency in relation to age, gender, duration of hypertension, except for the - age group, where urgency was statistically significantly higher (p= . ). introduction: emergency department (ed) crowding is a major public health concern. it delays treatment and possible icu admission, which can negatively affect patient outcomes. the aim of this study was to investigate whether ed to icu time (ed-icu time) is associated with icu and hospital mortality. methods: we conducted an observational cohort study using data from the dutch nice registry. adult patients admitted to the icu directly from the ed in academic centers, between and , were eligible for inclusion. for these patients nice data were retrospectively extended with ed admission date and time. ed-icu time was divided in quintiles. the data were analyzed using a logistic regression model. we estimated crude and adjusted (for disease severity; apache iv probability) odds ratios of mortality for ed-icu time. in addition, we assessed whether the apache iv probability (divided into quartiles) modified the effect of ed-icu time on mortality. results: a total of , patients were included. baseline characteristics are shown in table . the median ed-icu time was . [iqr . - . ] hours. icu and hospital mortality were . and . %, respectively. the crude data showed that an increased ed-icu time was associated with a decreased icu and hospital mortality (both p< . , figure a ). however, after adjustment for disease severity, an increased ed-icu time was independently associated with increased hospital mortality (p< . , figure b ). figure shows that only in the sickest patients (apache iv probability > . %), the association between increased ed-icu time and hospital mortality was significant (p= . , figure d ). we found similar results with respect to icu mortality. conclusions: this study shows that a prolonged ed-icu time is associated with increased icu and hospital mortality in patients with higher apache iv probabilities. strategies aiming at rapid identification and transfer of the sickest patients to the icu might reduce inhospital mortality. reliability and validity of the salomon algorithm: -year experience of nurse telephone triage for out-of-hours primary care calls e brasseur, a gilbert, a ghuysen, v d´orio chu liege, emergency departement, liège, belgium critical care , (suppl ):p introduction: due to the persistent primary care physicians (pcp) shortage and their substantial increased workload, the organization of pcp calls during out-of-hours periods has been under debate. the salomon (système algorithmique liégeois d'orientation pour la médecine omnipraticienne nocturne) algorithm is an original nursing telephone triage tool allowing to dispatch patients to the best level of care according to their conditions [ ] . we aimed to test its reliability and validity under real life conditions. methods: this was a -year retrospective study. out-of-hours pc calls were triaged into categories according to the level of care needed: emergency medical services (amu), emergency department visit (maph), urgent pcp visit (upcp), delayed pcp visit (dpcp). data recorded included patients' triage category, resources and potential redirections. more precisely, patients included into the upcp + dpcp cohort were classified under-triaged if they had to be redirected to an emergency department. patients from the amu+maph cohort were considered over-triaged if they did not spend at least resources, emergency specific treatment or any hospitalization. results: calls were actually triaged using the salomon tool, of which . % were classified as amu, . % as maph, . % as upcp and . % as dpcp (fig ) . as concerns the amu+maph cohort, the triage was appropriate in . % of the calls, with an over-triage rate of . %. as concerns the upcp + dpcp cohort, . % of the calls were accurately triaged and only . % were under-triaged. sal-omon sensitivity reached . % and its specificity . %. these results indicate that salomon algorithm is a reliable and valid nurse telephone triage tool that has the potential to improve the organization of pcp out-of-hours work. introduction: inappropriate visits to the emergency department (ed), such as patients manageable by a primary care physician (pcp), have been reported to play some role in the ed crowding [ ] . indeed, non-urgent patients directly managed by pcps could reduce ed workload [ ] . triage and diversion to alternative care facilities, eventually co-located within the ed, could offer a solution [ ] provided fig. (abstract p ) . distribution of different calls, their triage using the salomon algorithm and the inappropriate triages (over and undertriages) based on the preselected criteria the availability of a reliable triage tool for their early identification. we created a new triage algorithm, persee (protocoles d'evaluation pour la réorientation vers un service efficient extrahospitalier) and tested its feasibility, performance and safety. methods: after initial evaluation with a -level ed triage scale [ ] , ambulatory self-referred patients classified as level or below benefited from a simulated triage with persee identifying categories of patients: ed ambulatory patients and primary care (pc) treatable patients. we collected patients data and resources. patients requiring less than resources, no specific emergency treatment and no hospitalization were considered as manageable in a pc facility. results: patients were included in the study of whom . % were self-referred (fig ) . among those self-referrals, . % were triaged as level or below. . % patients were triaged as ambulatory patients of whom % were as pc treatable. we noted a redirection rate of % of the global visits or % of the self-referrals, an error rate of %, a sensitivity of . % and specificity of . %. conclusions: using advanced ed triage algorithm in addition to classical ed triage might offer interesting perspectives to safely divert self-referrals to pc facilities and, potentially, reduce ed workload. introduction: generally, prehospital medical provider should minimize staying prehospital scene to reach the patient to definitive care as soon as possible in prehospital medical activity. in addition, some textbook and report saids that medical provider minimize the number of procedure or limit minimum requirement procedure because unnecessary procedure may extend the staying time in prehospital scene. however, there are few studies evaluating this hypothesis and that this "extension is significant or not. therefore, we perform this study. methods: we evaluated the operated air ambulance(doctor-heli) case from st april to st march , in gifu university hospital using our mission record. we evaluated about time from landing to ready for taking off(activity time), operation doctor, mission category (i.e. trauma), number of procedure in the each activity and work load. we only focused on prehospital care and exclude transportation from hospital to hospital . in addition, we exclude the case which are not suitable for analysis. results: cases were operated in these period. cases were suitable for analysis. average activity time in prehospital scene was . ± . . there was weak correlation between the number of procedure and activity time. (r= . ) the length of the activity time did not depend on mission category. if the doctor perform and over procedures, staying time was minutes longer, this was significantly longer than that of under and under procedures. conclusions: we confirmed that we have to minimize the number of procedure or limit minimum requirement procedure in prehospital scene. and our result suggest we may have to limit appropriate number of procedures. introduction: organ failure is a critical condition, but the prevalence is largely unknown among unselected emergency department (ed) patients. knowledge of demographics and risk factors could improve identification, quality of treatment, and thereby improve the prognosis. the aim was to describe prevalence and all-cause mortality of organ failure upon arrival to the ed. methods: this was a cohort-study at the ed at odense university hospital, denmark, from april , to march , . we included all adult patients, except minor trauma. organ failure was defined as a modified sofa-score > within six possible organ systems: cerebral, circulatory, renal, respiratory, hepatic, and coagulation. the first recorded vital, and laboratory values were extracted from the electronic patient files. primary outcome was prevalence of organ failure; secondary outcomes were - -day and - -day mortality. results: of , contacts . % were female and median age (iqr - ) years. the prevalence of new organ failure was . %, individual organ failures; respiratory . %, circulatory . %, cerebral . %, renal . %, hepatic . %, and coagulation . %. the - -day and - -day all-cause mortality was . % ( % ci: . - . ) and . % ( % ci: . - . ), respectively, if the patient had new organ failures at first contact in the observation period, compared to . % ( % ci: . - . ) and . % ( % ci: . - . ) for patients without. seven-day mortality ranged from hepatic failure, . % ( % ci: . - . ) to cerebral failure, . % ( % ci: . - . ), and the - -day mortality from cerebral failure, . % ( % ci: . - . to renal failure, . % ( % ci: . - . ). conclusions: new organ failure is frequent and serious, with a prevalence of . % and a one-year mortality of % with wide variation according to type of organ failure. results: we proceeded to a descriptive study that showed that % of patients were male and % of them were female with a sex ratio of . .the average age of patients was years old and ranged between and years old.we found that patients of our population had medical background, dominated by diabetes in cases, high blood pressure in cases and asthma in cases.the results also showed that . % of patients had a history of abdominal surgery while % of them had history of other types of surgery.the patients were oriented according to their severity level as following: % care unit of emergency department, . % close monitoring room .the vaspi score was ranged between and with an average of ± . it was higher than in . % of cases.the results of physical examination found an isolated pain in , % of cases, a reactionnal pain syndrom in % of cases, a peritoneal syndrome in % of cases and an occlusive syndrome in % of cases.the final diagnosis was mostly represented by the following causes: . % of gastroenteritis . % of constipation and % of ulcer disease.the final orientation of patients according to the diagnosis led to hospitalization in % of cases and to outpatient clinic in % of cases while % of them did not need any more care. conclusions: appropriate diagnostic evaluation and decision for or against hospitalization is a challenge in the patient who comes to the emergency department with acute abdominal pain it need an adequate evaluation and management. introduction: we assessed patients' impressions of a selfadministrated automated history-taking device (tablet) to gather information concerning emergency department (ed) patients prior to physicians' contact. the quality of communication was compared with the traditional history-taking. methods: the algorithm content was developed by two emergency physicians and two emergency nurses through an iterative process. item-content validity index (i-cvi) was measured by five experts rating the relevance of each item (from : not relevant to : highly relevant) [ ] . next, quality control was realized by research team. to assess the feasibility, we used a computerized randomization. low acuity, ambulatory adult patients presenting to the ed were assigned either to a control group (cg, n= ) beneficiating form a traditional history-taking process or to the experimental group (eg, n= ) assigned to use the tablet with further history-taking by the ed physician. communication was analyzed by the health communication assessment tool [ ] and satisfaction assessed by questionnaires. results: after two rounds, validity was excellent for each item (i-cvi > . ). the universal agreement method was of . . refusals (n= ) to participate were analyzed: they fear using an electronic device or the experimentation. content satisfaction revealed that % of patients understood the questions. % of patients indicated that the device was easy to hold and use. medical communication was not affected by the device (p= . ). we noticed that, among the subsections, physicians significantly introduced themselves better in the eg (p= . ). conclusions: in this feasibility study, patients were highly satisfied. the use of a self-administrated automated history-taking device does not generate miscommunications and allow physicians better introduce themselves. . a positive point we have established is the possibility for the detorsion of a twisted retention ovarian cyst after its transvaginal aspiration. we used this method only in cases when the onset of torsion did not exceed hours. . % of all emergency conditions associated with retention cysts were recurred by conservative therapy, and . % of patients with the retention cysts rupture were successfully treated in this way. conservative management is possible in the case of a small loss of blood (up to . - . ml), hemodynamic stability and the absence of signs of continuing bleeding. the detorsion and resection of the cyst when torsion is not more than °and even longer than hours, in most cases did not reveal necrosis in the appendages. conclusions: improvement of organs of preservation and reproduction in women. criteria for admission to an intensive care unit of a tertiary hospital: analysis of the decisions of the outreach intensivist and day in-hospital mortality introduction: the aim of this study was the analysis of icu admission criteria and evaluation of in-hospital mortality of patients assessed by our critical care outreach team. criteria for admission to the icu should be defined to identify the patients most likely to benefit from icu admission. this triage process is complex, associated with several factors, including clinical characteristics of the patients, but also subjective factors because it depends on the judgment of the intensivist who decides whether to admit or not the patient and is obviously conditioned to the structure and size of the icu. methods: the outreach intensivist records the patient observation in a form with questions (reversibility of acute illness, objective of admission in icu, comorbidities, functional reserve and intuitive prognosis of the doctor). analysis of months (january through june , ) of admission decisions in icu, mean delay, icu mortality, and day in-hospital mortality ( hm). results: the intervention of the intensivist in "outreach" was requested on occasions. the main places of observation were the emergency room ( . %) and the wards ( . %). the hm increased with the degree of comorbidity decompensation. functional reserve also influenced hm, reaching . % in partially dependent patients and . % in totally dependent patients. there was agreement between the mortality and the physician´s intuitive prognosis in % of the cases. conclusions: a larger sample is needed to draw sustainable conclusions, however, the evaluation algorithm correlated well with hospital mortality. decompensated comorbidities and low functional reserve have a negative impact on prognosis, regardless of acute disease. there was agreement between mortality and the physician´s intuitive prognosis. electrochemical methods for diagnosing the severity of patients with multiple trauma introduction: multiple trauma is one of the leading causes of death worldwide [ ] . timely diagnosis and treatment is crucial in this state. one of the promising areas is the use of new electrochemical methods they are simple, flexible, efficient and of low cost. among these methods, attention is paid to the measurement of open circuit potential (ocp) of the platinum electrode and cyclic voltammetry (cva). the ocp is a reflection of the balance of pro-and antioxidants in the body, and the amount of electricity (q) determined by cva is proportional to the antioxidant activity of the biological environment. methods: a total of patients with severe multiple trauma ( . ± . y.o., men and women) were enrolled; apacheii . ± . ; iss . ± . ; blood loss ± ml. blood plasma was collected from patients. measurement of the ocp was carried out according to [ ] , cva analysis -according to the original method on a platinum working electrode. results: a shift in the ocp towards more positive potential values (fig. ) , while the antioxidant activity of blood plasma decreased (fig. ) . a more significant change of ocp, as compared to the q values, may indicate not only a deficiency in the components of the antioxidant defense system of the body, but also an increase in the concentration of prooxidants (e.g., reactive oxygen species), which are involved in oxidative stress. who underwent surgical fixation). information was collected from tarn, icnarc and surgical team databases. our primary outcome was itu resource utilisation (itu los and mechanical ventilation days). our secondary outcomes were morbidity and mortality (hospital los, infection burden, inotrope use and death before discharge). data was collected and analysed in microsoft excel and r. results: patients were included (group = , group = , group = ). mortality was significantly higher when comparing the post groups undergoing conservative ( %, / ) vs. surgical fixation ( %, / ), p-value = . . regarding potential temporal changes, there was no significant difference in mortality between the non surgical groups; pre- (group : / ) and post (group ), p-value . . group patients did spend more time mechanically ventilated (p-value . ) and used more antimicrobials (p-value . ) ( table ) . conclusions: patients undergoing surgical rib fixation at the rlh had significantly improved mortality with more days spent mechanically ventilated. pilot study on ultrasound evaluation of epiglottis thickness in normal adult a osman introduction: as the prevalence of epiglottitis is decreasing due to immunization, the difficulty in early detection remained. the aim of this study is to determine the thickness of epiglottis in normal adult with the utilization of bedside ultrasound. methods: this was a prospective observational study of convenience selection among healthy staff in emergency department, university malaya medical centre. the identification and measurement of epiglottis were performed using a mhz linear transducer by trained emergency physicians and registrars in em. subjects were scanned in either standing or upright seated position with the neck neutral or mildly extended. the epiglottis, thyroid cartilage and vocal cord were visualized and the epiglottis anteroposterior(ap) diameter was measured. difference in categorical parameters were analyzed by independent-sample t-test. the relationship between height, weight and epiglottic size was analyzed using pearson's correlation. results: fifty-six subjects were analyzed with males and females age ranging from to years old. the epiglottis ap diameter ranged from . cm to . cm, with average of . cm. there was significant difference in epiglottic ap diameter between male (m= . cm, sd= . ) and female (m= . cm, sd= . ; t( )= . , p=< . , twotailed). moderate positive correlation between height and epiglottic ap diameter (r= . ) and weight (r= . ) was documented. conclusions: our study demonstrated the identification and visualization of epiglottis was feasible and easy with the use of bedside upper airway ultrasonography. there was a little variation in the ap diameter of epiglottis in adults. indoor vs. outdoor occurrence in mortality of accidental hypothermia in japan y fujimoto , t matsuyama , k takashina introduction: the impact of location of accidental hypothermia (ah) occurrence has not been sufficiently investigated so far. thus we aimed to evaluate the differences between indoor and outdoor occurrence about baselines, occurrence place, mortality, and length of icu stay and hospital stay. methods: this was a multicenter retrospective study of patients with a body temperature ≤ °c taken to the emergency department of hospitals in japan between april and march . we divided the included patients into the following two group according to the location of occurrence of ah (indoor versus outdoor). the primary outcome of this study was in-hospital death. secondary outcomes were the length of icu stay, and hospital stay. results: a total of patients were enrolled in our hypothermia database. there were and patients with the outdoor and indoor occurrence. the indoor group was older ( versus . years-old, p< . ) and worse in adl than the outdoor group. the proportion of in-hospital death was higher in the indoor group than the outdoor group ( . % [ / ] versus . % [ / ], p< . ). the multivariable logistic regression analysis demonstrated that adjusted odds ratio of the indoor group over the outdoor group was . ( %ci; . to . ) ( table ) . as for secondary outcomes, both of the length of icu stay and hospital stay in survivors were longer in the indoor group than the outdoor group. conclusions: our multicenter study indicated that indoor occurrence hypothermia accounts for about % of the total in this study, and the proportion of in-hospital death was higher in the indoor group. we have to raise an alert over the indoor onset accidental hypothermia and need to take countermeasures for prevention and early recognition of ah in indoor location. conclusions: during acute asthmatic attack, arterial hyperlactatemia is frequently present at ed arrival. nevertheless, the plasma lactate level was no significant difference between ed admission and hr after treatment. the introduction: this is a case series of traumatic aortic injury (tai) which was diagnosed by transesophageal echocardiography (tee) in the emergency department. the number of patients with blunt thoracic aorta injury arriving at emergency department is on the rise and survival rate is time-dependent on early diagnosis. tee offers several advantages over transthorasic echocardiography (tte) including reliability, continuous image acquisition and superior image quality. methods: all trauma patients who presented to emergency department from st january until th november at hospital raja permaisuri bainun, perak, malaysia with suspected tai were evaluated with transesophageal echocardiography. over the years period, tee was performed in patients. patients had positive findings suggestive of tai. results: the first case was an old lady who presented after a deceleration injury in a car accident. tee was performed due to hemodynamic instability and found an intimal flap along the ascending aorta. the second case, a stanford type a (figure ) , was complicated with pericardial tamponade. the intimal flap was visualised from the aortic arch extending to the descending aorta by tee. the third case was a case of intramural haematoma involving distal aortic arch extending to the descending aorta which survived until corrective surgery. in the fourth case, tee revealed a motion artefact which mimicked an intimal flap in the ascending aorta. in the fifth case, tee showed intimal flap at aortic isthmus which was not detected by tte. in the last case, a traumatic aortic dissection was complicated by aortic regurgitation (figure ) . conclusions: tee can be a useful point of care tool use by emergency and critical care physicians for early diagnosis of blunt traumatic aorta injury. introduction: reboa is an endovascular intervention intended to preserve central perfusion in the context of shock due to noncompressible torso haemorrhage. more so, it is less invasive than the traditional approach of resuscitative thoracotomy (rt) and aortic crossclamping. though its use dates back to the korean war, it has not been widely adopted in trauma management, as evidence demonstrating clear benefit compared with conventional rt is lacking [ ] . we aimed to evaluate feasibility, outcomes and complications after reboa for haemorrhagic shock and traumatic cardiac arrest. methods: we performed a systematic literature review, searching scopus and pubmed databases using relevant terms (july ). we included studies enrolling patients with haemorrhagic shock or cardiac arrest after civilian trauma who had undergone reboa and reported hospital mortality (our primary outcome). abstract-only studies and single-patient case reports were excluded. we collated and analysed data using review manager v . . the newcastle-ottawa scale was used to assess risk of bias. results: sixteen in-hospital studies met inclusion criteria (n= ). ten were case series and six were cohort studies comparing reboa outcomes with those of rt. there were wide differences between studies' inclusion criteria, case-mix (including cardiac arrest), injury severity, insertion details, and reported outcomes. overall hospital mortality post-reboa was . %. meta-analysis of cohort studies indicated notably lower mortality in patients undergoing reboa (or . , . - . ) than rt with low statistical heterogeneity between studies (i = %), shown in fig . conclusions: whilst our findings are limited by methodological differences and biases in the included studies, almost % of patients undergoing reboa for haemorrhagic shock and/or cardiac arrest survived to discharge. furthermore, reboa appeared to offer a consistent mortality benefit compared with rt. introduction: trauma related coagulopathy remains a primary contributor to mortality on battlefields and in civilian trauma centres. fibrinogen is considered to be the first to drop below critical level and correspondingly compromised coagulation process. however, it is unclear if fibrinogen concentrate at a very early stage is feasible and effective to prevent from coagulopathy. methods: a total of acutely injured patients in austria, germany and czech republic were screened and enrolled in this controlled, prospective randomized placebo controlled double blinded multicentre and multinational trial. upon the completion of randomization, fibrinogen concentrate ( mg/kg, fgtw©, lfb france) or placebo was reconstituted and given to the patients at the scene or during helicopter transportation from the scene to nearby hospitals. blood samples were taken at baseline (scene of accident before study drug administration), at the emergency room, three hours, nine hours and twentyfour hours after admission to the hospital as well as after three and seven days after admission, for measurements of blood gases and coagulation, together with clinical data and outcome records. results: the demographic and injury characteristics and the estimated blood loss, iss, and gcs at the scene were similar in both groups. in the placebo group, fibrinogen concentration dropped from mg/dl at injury site to mg/dl () at er admission and clot stability reduced from . mm ( , mm) to mm (p= . ) (fig ) . fibrinogen concentrate administration prevented the drop of fibrinogen level (baseline of mg/dl to mg/dl and improved clot stability from mm at baseline to mm at er. conclusions: pre-hospital administration of fibrinogen concentrate in traumatic bleeding patients is feasible and effective in preventing the development of coagulopathy. data from this study support the use of fibrinogen to prevent trauma related coagulopathy. fibrinogen concentrate vs cryoprecipitate in pseudomyxoma peritonei surgery: results from a prospective, randomised, controlled phase study results: the per-protocol set included pts (hfc, n= ; cryo, n= ). the mean total intraoperative dose of hfc was . g vs . pools of cryo (containing approx . g of fibrinogen). median duration of surgery was . h. overall haemostatic efficacy of hfc was non-inferior to cryo and was rated excellent or good for % of pts receiving hfc and cryo, with similar blood loss. intraoperatively, only red blood cells were transfused (median: unit). intraoperative efficacy is shown in table . infusions were initiated . h earlier with hfc than cryo due to faster product availability. preemptive hfc led to a greater mean increase vs cryo in fibtem a ( figure ) and plasma fibrinogen (figure ). there were serious adverse events (saes) in the hfc group and in the cryo group, including thromboembolic events (tees; deep vein thromboses, pulmonary embolisms). no aes or saes were deemed related to the study drug. conclusions: hfc was efficacious for treatment of bleeding in pts undergoing surgery for pmp. no related aes and no tees occurred in pts treated with hfc. fig. (abstract p ) . fib mcf t to t with % ci fig. (abstract p ) . fibtem a prior to and following the preemptive dose of hfc/cryoprecipitate introduction: patients in the intensive care unit often suffer from thrombocytopenia. in dealing with this problem, we need to figure out not only the cause of thrombocytopenia but also the risk of bleeding. however, there is no reliable method for evaluating bleeding risk. methods: in this preliminary study, four thrombocytopenic patients who required platelet transfusion before undergoing invasive procedure were enrolled. written informed consent was obtained from all patients for participation in the study. bleeding was graded using the who bleeding scale. thrombogenic activity was evaluated using total thrombus-formation analysis system (t-tas), rotational thromboelastometry (rotem), and multiplate impedance aggregometry. for t-tas analysis, we prepared a novel microchip, named hd chip, which is suited for analyzing low platelet samples rather than those with normal platelet counts. , key patient groups in which it was wasted and the use of standard laboratory tests (slts) to guide its use. the purpose was to assess the potential benefit a point of care viscoelastic haemostatic assay (vha) could have on ffp transfusion and waste. the national blood transfusion committee and nhs blood and transplant committee have published data showing that up to % of ffp is transfused inappropriately [ ] . methods: blood bank data was obtained evaluating haemorrhaging patients in whom ffp was requested across a nine-month period in . patient bleeds were categorised by speciality. the mean time ffp dispensed and wasted was recorded, as were timings of slt requests. where available, the inr result was recorded. results: patients were identified. transfusions were requested. table shows that the highest transfusion requirements are for acute medical emergencies and major trauma. % of transfusion were surgical specialities, it would be expected that these patients would have anaesthetic or critical care input. units were wasted. acute medical emergencies wasted the highest amount of ffp ( units). table demonstrates that . % of transfusions had an inr available one hour prior to ffp being dispensed. conclusions: we conclude that use of slts to guide ffp transfusion is low. this suggests transfusion decisions are being made clinically. a point of care vha could give treating physicians better access to timely haemostatic data. introduction: we developed the process for the out-of-hospital packed red blood cells (prbc) transfusion in the hems of castilla-la mancha clm according to criteria of medical indications, security, monitoring and tracking. haemorrhage is a preventable cause of death among population suffering accidents or bleeding injuries in regions with low population density where health services should reach people in remote areas. hems of clm is the first out-ofhospital emergency service in spain that provides prbc transfusion there where the accident takes place. this program has been developed jointly between hematologists of the center for transfusions ct and the hems team. methods: observational retrospective study with data collected from june to august . the medical helicopter was provided with two prbc o rh(d) negative (fig ) . shock index was selected as indication for transfusion. to achieve feasibility and preservation of the prbc it was established a prospective monitoring and microbiological culture for both groups: case group for the prbc kept in the hems and control group in the hospital (fig ) . controls and comparison of hematologic analysis were performed immediately and days after collection. statistics used spss . (signification p< . ). results: prbc were evaluated, case - control. analyses were tested days and after collection. hemolysis was not observed. all cultures were negative. results obtained of the prbc after days transported in the hems related to monitoring parameters were not different than those observed on prbc conserved in the ct. prbc were transfused to patients in out-of-hospital assistance. neither post-transfusional reactions or undesirable events have been registered. prbc units are changed every days. conclusions: the process designed (collection, conservation, tracking and tests) to make prbc available in the medical helicopter has demonstrated to keep the standard conditions and properties to be transfused in critically ill patients out-of-hospital. outcomes in patients with a haematological malignancy admitted to a general intensive care unit a corner east sussex healthcare nhs trust, intensive care, eastbourne, united kingdom critical care , (suppl ):p introduction: recent published data have challenged the view that critically ill patients with a haematological malignancy have a poor prognosis [ ] . reports have largely originated from tertiary centres. the aim of this audit was to evaluate the intensive care unit (icu), in hospital and one year mortality for a cohort of patients admitted to a mixed medical and surgical icu in a district general hospital. methods: details were obtained for all patients with a haematological malignancy admitted to eastbourne and hastings icu between march and august . patient characteristics, type of malignancy, reason for admission, degree of organ support and survival rates at icu discharge, hospital discharge and year postadmission were collected. results: patients, % male, were identified. median (interquartile range, iqr) age was ( - ) years. % had neutropenia. the commonest malignancies were acute leukaemia %, lymphoma % and myeloma %. reasons for admission were respiratory %, cardiac % and renal %. organ supports used were noradrenaline %, intubation and mechanical ventilation %, renal replacement therapy (rrt) % and dobutamine %. overall survival rates are shown in figure . patients were discharged from hospital following a period of mechanical ventilation. for these patients, median (range) age was ( - ) years. all were male. median (iqr) time in hospital prior to admission was ( - ) days, / patients required vasoactive support, / required rrt, median icu length of stay was ( - ) days. / were admitted following surgery for an unrelated condition. to date, only / patient has survived years post icu admission. conclusions: although survival rates were disappointing, particularly in those patients requiring mechanical ventilation, selected patients have the potential for a good outcome. these results outcomes have been presented to our haematology department to aid patient counselling. analyses. cox regression was used for the survival analysis. organ failure was defined as the occurrence of renal failure based on acute kidney injury network (akin)-creatinine or need for; vasopressors, invasive ventilation or continuous renal replacement therapy (crrt) the first days after admission. length of stay was only analysed in survivors. results: the study included unique patients. prolonged aptt was associated with mortality with a % confidence interval (ci) of hazard ratio . - . . prolonged aptt correlated also with the occurrence of renal failure and the need for vasopressor and crrt with % ci of odds ratio (or) . - . , . - . and . - . (fig ) . increased pt-inr was associated with the need for vasopressors and invasive ventilation with % ci of or . - . and . - . . both aptt and pt-inr correlated with length of stay with % ci of or . - . and . - . . conclusions: activated partial thromboplastin time on admission to the icu is independently associated with mortality. both aptt and pt-inr are independently associated with length of stay and the need of organ support. all regression models were adjusted for saps score which means that aptt prolongation and pt-inr increase on admission represent morbidity that is not accounted for in saps . introduction: the goal was to assess if daily venous thromboembolism (vte) assessment was being done in our critical care (cc) unit, and if not, what changes could be made. a mortality review showed the need for a dynamic vte assessment in cc patients, who are subject to daily changes influencing vte risk. a daily risk assessment was introduced, and a 'tab' on our clinical information system, metavision(r)(mv) was created. recently published national institute for health and care excellence guidelines on vte risk assessment in cc provided us cause to assess our compliance [ ] . methods: data was collected from mv. review of daily vte assessment was made and a percentage completion of daily vteassessments was calculated per patient.interventions were done using standard improvement methods through pdsa cycles. results: baseline data, of patients, was collected in july, .compliance with daily vte assessment was %. the results were presented at the clinical governance forum(cgf), and posters were displayed in cc. the second cycle, of patients, was collected in october. compliance had increased to %.following discussion from presenting results at the cgf, the vte tool was appropriately modified.the responsibility of vte assessment was also shifted to becoming more shared, including all clinical staff, rather than mainly consultants. the third cycle, of patients, was collected in november. compliance had increased to %.introducing a nursing care bundle with vte is in progress. conclusions: despite the identification of a risk in our clinical practice and the development of an appropriate it tool to facilitate improved practice, the advent of new national guidance revealed poor compliance with agreed standards. this shows the difficulties with achieving practice change in complex multiprofessional clinical environments. a sustained effort is required focusing on dissemination and engagement across the whole team. introduction: we describe the changes in anti factor xa (afxa) activity, thrombin generation and thromboelastography (teg) in critically ill patients with and without acute kidney injury (aki) following routine administration of tinzaparin as part of venous thromboembolism (vte) prophylaxis. methods: pilot prospective observational study. patients divided into those with and without aki were administered tinzaparin by subcutaneous injection as per established local guidelines. patients who did not receive tinzaparin were recruited as a 'control'. plasma afxa activity and thrombin generation were measured at intervals over a hour period. teg parameters were collected at t and t . results: afxa activity: results are shown in figure . / patients failed to achieve a prophylactic afxa level of > . at any point. / patients achieved a level of > . however in all cases this was at the lower end of the prophylactic range and was achieved for only a short time (median . hours). / achieved a level of > . for the whole h period. there was no difference between the aki and no aki groups. endogenous thrombin generation: there is no significant difference in thrombin generation between the aki and no aki groups. there is a significant decrease in thrombin generation between h and h (p< . ) and a significant increase between h and h (p< . ) (figure ). there is no significant difference between h and h (p= . ). teg: all teg parameters for all patients were within normal range conclusions: standard vte prophylactic dose tinzaparin rarely achieves an afxa range that has been suggested for vte prophylaxis. however, as assessed by thrombin generation, a hypo-coagulable state is generated in response to lmwh. there is no difference between critically ill patients with or without aki that would suggest the need for dose reduction in this context. (abstract p ) . thrombin generation at h, h and h. t = time of tinzaparin administration, with the sample taken just prior to administration. patients from aki group shown with dotted line and from no aki shown with solid line % which takes the third place between cpb-associated complications . current data demonstrates the importance of researching of changes in haemostatic system in paediatric patiens after cpb. provided below data is an intermediate result of our research. methods: patients in age up to mohth days (median age - , months, youngest age - days after birth, oldest - months days), who underwent cardiac surgery with cpb to treat congenital heart diseases, were enrolled in this study. all patients were divided into two groups: stwithout tc, ndwith tc. protein c (pc) and fibrin-monomer (fm) plasma levels were assessed in there points: before surgery, -hours and hours after surgery. thrombotic cases were provided by doppler ultrasound or mri. results: thrombotic complications were diagnosed in chidren ( %). between all tc ischemic strokes were diagnosed in % ( cases), arterial thrombosis in % ( cases), intracardiac thrombus in % ( cases). in group with tc fm-mean values in points , and respectively were . ; and mcg/ml, meamwhile in group without thrombosis - . ; . and . mcg/ml .pc-mean value in st groupwere ; and %, in the nd group - ; and % respectively in the points , and . statistically significant differences between groups in rd point (p< . ) and correlation between pc and fm (r=- . ; p< . ) were detected. conclusions: cpb causes hypercoagulation with increasing of pc consumtion and fm level. moreover, cp associated with a high risk of tc on the rd day after cardiac surgery. further studies to investigate prognostic values of fm and pc in thrombosis are required. these studies would help to asses fm and pc as markers of tc and possibility of pc-prescribing for prevention and treatment of these complications. introduction: thrombocytopenia is a common condition in critically ill patients and an independent predictor of mortality. the relevance of a supranormal platelet count remains unclear. septic patients with disseminated intravascular coagulation (dic) are also known to have a high mortality, but the influence of sepsis on mortality rates in coagulopathic patients is less well characterised. our objectives were to: ) evaluate mortality amongst patients with sepsis and nonsepsis associated dic. ) assess incidence of dic during the first days of admission. ) assess the relationship between platelet count and mortality. methods: records of adult critical care patients admitted to the royal liverpool university hospital between - were retrospectively reviewed. the presence of sepsis (using the definition of sirs with infection), coagulopathy, degree of thrombocytopenia and day mortality were noted. modified isth dic score was used to define dic. results: the overall mortality rate was %. patients were identified as having sepsis ( %) and non septic patients ( %). mortality rates of patients with sepsis were significantly higher than without sepsis ( % vs % respectively, p< . ). in patients with dic, their dic scores tended to be 'positive' for the first days of admission. fibrin-related markers were often not available for dic scoring. mortality rates amongst patients with sepsis-associated dic were greater than patients with non-sepsis related dic. thrombocytopenia severity was associated with mortality, and patients with platelets above the upper limit of normal had lower mortality rates ( % when platelets > x ^ /l, % when platelets < x ^ /l). conclusions: sepsis-associated coagulopathy is associated with a higher mortality rate than non-sepsis associated coagulopathy. supranormal platelet counts may be associated with a mortality benefit. introduction: deep vein thrombosis (dvt) is a major problem in icu and affects overall lethality. dvt is widespread complication in icu, especially in elderly patients, when early activisation may not be achieved. aim of this study is comparison of haemostatic potential and analgesia methods of elderly patients who underwent major urological surgery during their stay in icu. methods: a cross-sectional study was employed. participants were ≥ y.o., underwent major urological surgery, have had normal initial hemocoagulation data (thromboelastography was performed to all of them), had received analgesia with epidural catheter or iv by opioids use and were treated in icu > days due to non-coagulopathy states, were included. data were collected from october till october . the patients were examined with thromboelastograph "mednord" for thromboelastogramm (teg) and with esaote usg for thrombi occurrence in lower limb deep veins. the anticoagulants were prescribed under the esa guidelines . results: participants (n= ) were divided in two groups -non-opioid analgesia with epidural catheter (n= ) and opioid analgesia (n= ). we received moderate decrease in anticoagulants dosage to the patients with epidural analgesia with the same teg goals compared to the patients with opioid analgesia. other factors as comorbidities may provoke dvt events, but was not evaluated in this study. the dvt events were monitored by expert with the use of usg to locate thrombi in the vein. conclusions: use of epidural catheter analgesia provides moderate decrease of anticoagulants dosage compared to opioid analgesia patients; however strict control of teg data must be presented. comorbidity need to be monitored for early detection and prevention of dvt events. introduction: patients with morbid obesity (mo) have a high risk of thromboembolic events. in patients with a bmi > , the hypercoagulable state is due to impairment of all parts of the blood coagulation as well as anticoagulation mechanisms by obesity. methods: the hemostasis system was studied in patients with a bmi> kg/m with various pathologies that were admitted to icu. all patients were divided into groups depending on the type of therapy: group (n= ) received monotherapy with enoxaparin sodium . % . ml sc times a day every h; group (n= ) received combination therapy with enoxaparin sodium . % . ml sc times a day every h and pentoxifylline mg times a day every h. to study the hemostasis system, we used lpteg immediately after hospitalization, on , , days. results: in both groups, prior to treatment: contact coagulation intensity (icc) was increased by . %, intensity of coagulation drive (icd) -by more than . %, clot maximum density (ma) -by . %, index of retraction and clot lysis (ircl) - . % above normal. patients of the st group: icc increased by . %, icd was close to normal values, ma increased by . %, ircl was increased by . %. patients of the nd group on the th day: icc decreased by . % compared with the norm; the coagulation and fibrinolysis parameters were close to normal values and the decrease in fibrinolysis activity reaches to normal. conclusions: combined therapy of thromboembolic complications in patients with obesity sodium enoxaparin sodium and pentoxifylline is more effective than enoxaparin sodium monotherapy because it affects all parts of the hemostatic system. introduction: a laryngeal injury secondary to blunt neck trauma can lead to life-threatening upper airway obstruction [ , ] . ultrasound enables us to identify important sonoanatomy of the upper airway [ ] . the purpose of this report is to discuss role of pocus airway in blunt neck trauma and to determine airway management based on standard schaefer subgroups classification. methods: three cases of blunt neck trauma presented to our centre with either subtle or significant clinical signs and symptoms. standard airway management was performed prior to pocus airway using mhz linear transducer and it findings were later compared to flexible fibreoptic laryngoscopy and computed tomography (ct). results: pocus airway had identified one out of cases to have schaefer and the remaining as schaefer . all pocus airway findings were confirmed with flexible fibreoptic laryngoscopy and ct scan (figs , ) . based on schaefer, supportive care and early steroid administration are advisable for group and . for groups to , immediate open surgical repair is deemed necessary due to extension of injuries.all cases were intubated using glidescope.all including those presented with schaefer were managed conservatively and discharge well with proper follow-up. conclusions: upper airway ultrasound is a valuable, non-invasive and portable for evaluation of airway management even in anatomy distorted by pathology or trauma. an organised approach using pocus airway as an adjunct can expedite care and prevent early and long term complications in facilities without flexible laryngoscope and ct. introduction: high-flow nasal oxygen (hfno) and helmet noninvasive ventilation (hniv) are increasingly used for the early management of acute hypoxemic respiratory failure (ahrf). we compared the physiological effects of hfno and hniv during ahrf. methods: in this randomized cross-over study, we enrolled patients with acute-onset (< days), non-cardiogenic respiratory distress (respiratory rate> /min), pulmonary infiltrates at the chest-x-ray and hypoxemia (spo < % while breathing on room air). all patients received hniv (peep cmh o, pressure support adjusted to achieve a peak inspiratory flow of l/min) and hfno (flow l/min) for one hour each, in a randomized cross-over manner. at the end of each period, arterial blood gases, inspiratory effort (esophageal pressure) and respiratory rate were recorded. self-assessment of dyspnea and device-related discomfort ( [ ] [ ] [ ] [ ] [ ] ). conclusions: as compared to hfno among critically ill patients with ahrf, hniv ameliorates oxygenation, limits inspiratory effort and relieves dyspnea, without affecting paco , respiratory rate and comfort. introduction: pre-intubation hypoxemia is a predictor of negative patient outcomes including in-hospital mortality. while successful first intubation attempt is also an important factor of patient outcomes, little is known about whether physicians achieve successful first intubation attempt for the hypoxemic patients in the emergency department (ed). the aim of this study is to investigate the first-pass success for patients with pre-intubation hypoxemia in the ed. methods: this is an analysis of the data from the second japanese emergency airway network study (jean- study)a multicenter, prospective, observational study of eds in japan. we included all patients who underwent intubation in the ed from through . we excluded patients ) aged < years and ) patients who underwent intubation for cardiac arrest. we grouped pre-intubation hypoxemia as follows: non-hypoxemia (oxygen saturation [spo ], ≥ %), moderate-hypoxemia (spo , %- %), and severehypoxemia (spo , < %). primary outcome was the first-pass success rate. to demonstrate the association between pre-intubation hypoxemia and the first-pass success in the real-world setting, we fit two unadjusted logistic regression models ) using grouped preintubation hypoxemia as a categorical variable and ) using the preintubation spo as a continuous variable. results: among , patients who underwent intubation in the ed (capture rate, %), , patients were eligible for the analysis. compared to the non-hypoxemia, the first-pass success rate was low in moderate-hypoxemia ( % vs %; or= . [ %ci, . - . ]) and severe-hypoxemia ( % vs %, or= . [ %ci, . - . ]). additionally, there was a linear association between pre-spo and lower first-pass success rate (or for the success, per one pre-spo decrease, . [ %ci, . - . ]). conclusions: based on the large, multicenter data, the first-pass success rate was low in hypoxemic patients compared to nonhypoxemic patients in the ed. introduction of rapid-sequence induction guideline to reduce drug-associated hypotension in critically unwell patients introduction: the aim of this project was to assess whether the introduction of a rapid sequence induction (rsi) agent guideline changed drug choice and the incidence of peri-intubation vasopressor use at st john's hospital, livingston. it is well documented that emergency airway management in the critically ill can be a source of significant morbidity and mortality [ , ] and the choice of induction agent matters [ ] . methods: an rsi agent guideline was instituted for all critically ill patients being intubated in icu and the ed [ figure ]. following this, we set up an intubation registry to collect data from all intubation events. this data was then compared to a previous audit of intubations completed in . results: the choice of agent used pre-and post-intervention are summarized in figure . forty-five intubation events were included in the initial audit in , of which, ( %) required vasopressor support immediately following intubation. of the intubation events following the guideline's introduction, ( %) required vasopressors. ketamine use changed from % to %, propofol use from % to % and midazolam from % to %. thirty-eight of these intubation events ( %) were compliant with the guideline. conclusions: the introduction of the rsi guideline dramatically affected the choice of induction agent and reduced the incidence of significant hypotension requiring vasopressors ( % versus %). overall compliance with the guideline was excellent ( %). introduction: the purpose is to test the feasibility of using the i-gel® device for airway maintenance during bronchoscopic-guided percutaneous dilatational tracheostomy (pdt). usually pdt is accomplished via the tracheal tube. failure to position the endotracheal tube correctly can result in further complications during the procedure. the alternative implies extubation and reinsertion of an i-gel® airway device. methods: the pdt was performed using the blue dolphin method in patients in intensive care unit. before undertaking bronchoscopicguided percutaneous dilatational tracheostomy (pdt), the patient's tracheal tube (et) was exchanged for i-gel®, as a ventilatory device for airway maintenance. the insertion of the i-gel®, the quality of ventilation, the blood gas values, the view of the tracheal puncture site, and the view of the balloon dilatation were rated as follows: very good ( ), good ( ), barely acceptable ( ), poor ( ), and very poor ( ) [ ] . results: the i-gel® successfully maintained the airway and allowed adequate ventilation during percutaneous tracheostomy in all patients. the ratings were or in % of cases with regards to ventilation and to blood gas analysis, for identification of relevant structures and tracheal puncture site, and for the view inside the trachea during pdt. conclusions: the i-gel® successfully maintained the airway and allowed adequate ventilation during percutaneous tracheostomy in all patients. the ratings were or in % of cases with regards to ventilation and to blood gas analysis, for identification of relevant structures and tracheal puncture site, and for the view inside the trachea during pdt. no damages to the bronchoscope, reports of gastric aspiration or technical problems were detected. the bronchoscopic view obtained via an i-gel® seems to be better than that obtained through an endotracheal tube (et) or through traditional laryngeal mask [ ] . introduction: the purpose of this study was to investigate the efficiency of nasal airway inserted in the oral airway (on airway) in securing the airway patency during mask ventilation [ ] (fig ) . methods: fifty eight patients undergoing general anesthesia were randomly assigned to either oral airway group (group o) or on airway group (group n). in both group, mg/kg of propofol was infused intravenously and mask ventilation was performed in the sniffing position without head extension or jaw thrust. the patients were ventilated with a volume-controlled ventilator with o flow of l/min, tidal volume of ml/kg (ibw), and respiratory rate of /min. before the start of mask ventilation, airway was placed in the oral cavity. oral airway was used in group o and on airway was used in group n. peak inspiratory pressure (pip), tidal volume and etco were compared between the two groups. the location of airway tip was graded by fiberoptic bronchoscope as; : airway obstructed by tongue, : epiglottis visible, : airway touches epiglottis tip, : airway passes beyond epiglottis tip [ ] . methods: a prospective uncontrolled observational study in - in ukrainian hospitals. sma-pts from - mo were involved. all pts. ready for extubation: afebrile, no infiltrations on chest x-ray, normal wbc. however, each sma-pts. failed sbt (t-tube or psv). we evaluated: extubation success (no reintubation in hours), icu los, one year survival. three pts. were excluded: two pts. by staff decision, family have choosen tracheostomy. sma-pts. included. a cuff leakage test performed -with a negative, dexamethazone mg iv was administered. after extubation niv was started by ventilogik ls in st mode via nasal mask giraffe. the epap and ipap settings were titrated to reach the chest excursion and target levels of spo ( - %) and etco ( - mmhg). a sputum was draining by mechanical insufflation-excuflation (mie) and aspirator results: all pts, were extubated successful. the mean icu los was . days ( - days), one year survival rate was %, respiratory failure fully compensated by niv, there was no icu admission. every sma-pts. are in good condition, gaining weight introduction: aerosol delivery has previously been assessed during simulated adult hfnt, delivered by various stand-alone humidification systems [ ] . the objective of this study was to evaluate aerosol delivery during simulated hfnt delivered by a mechanical ventilator, across three clinically relevant gas flow rates. methods: ml of mg/ml salbutamol was nebulised using an aerogen solo nebuliser (aerogen, ireland). an adult head model was connected to a breathing simulator (asl , ingmar, us), vt ml, bpm and i: e, : (fig ) . hfnt was supplied via the servo-u ventilator (maquet, getinge, sweden), using the integrated nebulisation option. tracheal dose was recorded at two nebuliser positions; a (after the humidification chamber) or b (before of the cannula), at three gas flow rates ( lpm, lpm and lpm) (n= ). the mass of drug captured on a filter placed distal to the trachea (tracheal dose) was quantified using uv spectroscopy at nm. results: presented in table . conclusions: to our knowledge, this is the first study to successfully demonstrate aerosol delivery during simulated hfnt, delivered by a mechanical ventilator. increasing gas flow rate was associated with a reduced tracheal dose (p= < . ). at lpm, a significantly greater tracheal dose was observed when the nebuliser was positioned before the nasal cannula (p= < . ). at lpm, a greater tracheal dose was yielded when the nebuliser was positioned after the humidifier (p= < . ). introduction: tracheotomies are often performed in critically ill patients who are in need of prolonged mechanical ventilation and respiratory care. our aim was to evaluate the possible effect of percutaneous and surgical tracheotomies on thyroid hormone levels. methods: eighty seven adult patients were included in our study from january to september . patients were in need of prolonged mechanical ventilation and tracheotomies were performed after consent was taken. we have excluded patients with preexisting thyroid diseases. forty five patients were undergone percutaneous tracheotomies and forty two patients were undergone for surgical. thirty eight female patients and forty nine male, age range - . we studied tsh, t and ft serum levels using chemiluminescence immunoassay method before either procedure and hours post each procedure.: statistical analysis was performed using spss . significance was estimated at the level of p< . results: tsh levels were increased in surgical group compared to percutaneous group at hours post procedure but the difference was not found statistically significant (p> . ). the rise in post operative levels of t compared to preoperative was found statistically significant for surgical tracheotomy group (p< . ).elevated ft levels for both groups have shown statistically significant difference between preoperative and postoperative period for the surgical tracheotomy group (p< . ) conclusions: we analyzed the effect of surgical versus percutaneous tracheotomy on thyroid hormones and it was found that both introduction: insertion of a tracheostomy for weaning purposes is associated with prolonged critical length of stay (los) and several adverse patient outcomes [ ] . previous work has suggested that protocolised weaning may reduce weaning times [ ] . we aimed to assess the impact of protocolised weaning on los following introduction of a standardised weaning protocol in . conclusions: introduction of a standardised weaning protocol for patients with a tracheostomy in our unit has had a beneficial effect on several patient outcomes, notably duration of weaning and length of critical care admission. introduction: delirium is a relatively frequent neurologic complication in liver transplantation (lt) recipients, which is an important cause of increased morbidity, mortality, extended icu stay, and increased cost of medical care. extubation of the endotracheal tube at an appropriate timing is an essential part of intensive care after lt, suggested to improve graft perfusion and systemic oxygenation, and thus decrease intensive care unit (icu) stay and positively affect prognosis. the aim of this study was to compare the incidence of delirium between early and late extubation groups after lt. methods: medical records from patients who received lt from january to july in a single university hospital were retrospectively reviewed. patients were divided into groups: those who underwent early extubation after lt (group e, n = ) and those who underwent extubation within few hours of icu admission after surgery (group c, n = ). the data of patients´demographics, perioperative management, and postoperative complications were collected. early extubation was defined as performing extubation in the operating room after lt. a propensity score matching analysis was performed to minimize the effects of selection bias. results: postoperative delirium occurred in / ( . %) in group e and / ( . %) in group c, respectively (p = . ). after propensity score matching, there was no difference in icu stay (p = . ), time to discharge after surgery (p = . ), and incidence of delirium between groups (p = . ). conclusions: although this study is retrospective in nature, limited by small sample size, early extubation did not affect the incidence of delirium after lt. further prospective studies on this area are required. weight estimation and its impact on mechanical ventilation settings in queen elizabeth hospital intensive care unit a nasr, a iasniuk, a roshdy queen elizabeth hospital, icu, london, united kingdom critical care , (suppl ):p introduction: documented weight in the intensive care unit (icu) can be the total, ideal, adjusted or predicted body weight (pbw). lung protective ventilation depends on tidal volume (vt) delivery which is based on accurate calculation of patients´weight [ ] . the weight is most probably documented on admission to the icu using estimation or one of many available equations. the aim of this study is to assess the documented versus the pbw and its impact on tidal volume delivery for mechanically ventilated patients in queen elizabeth hospital icu. methods: data was collected prospectively from all ventilated patients over a period of weeks in june . vt delivered in the first hour was calculated for each patient. documented body weight and height of each patient was obtained from the nursing chart. pbw was calculated and compared with the documented weight. the difference in vt attributable to the difference in weight has been subsequently calculated. results: ventilated patients were included ( males). the mean tidal volume delivered according to the documented body weight was . ml/kg versus . ml/kg based on pbw. vt more than ml/ kg was delivered in % of patients based on documented weight versus % when correcting the weight according to the pbw equation. conclusions: inaccuracy in documenting weight on patients´admission to the icu is a potential cause of delivering unsafe tidal volume [ ] . the harm can extend to drug dosage, nutrition provision and renal replacement therapy. introduction: ventilator-associated pneumonia (vap) is the leading cause of death among mechanically ventilated critically ill patients [ ] . chest radiography (cxr) is essential in the diagnosis of vap. in the past decade lung ultrasonography has proven to be a valuable tool in the diagnosis and monitoring of lung diseases. the aim of the study is to assess sensitivity and correlation between cxr, lung ultrasound and clinical pulmonary infection score (cpis). methods: in this retrospective, non-randomized study seven patients with proved vap were enrolled. in all patients cpis and lung ultrasound score (lus) [ ] were assessed. comparison of patients that had lus≥ and cpis≥ points was performed. the correlation between lus and cxr was done using the pearson model. results: we found significant difference between positive cxr patients with lus≥ and cpis≥ ( % vs %, p< . ). there is a very high correlation between cxr and lus. these results render lung ultrasound as a highly sensitive tool in the diagnosis of vap. conclusions: our study shows that lung ultrasonography could be used as a reliable supplementary method in the diagnosis of vap. the benefits of lung ultrasound include the ability to perform it at the patient´s bed without need for transportation, no radiation exposure and repeatability. the high correlation between cxr and lung ultrasound makes echography a valuable adjunct in the diagnosis of vap. color introduction: it is difficult to differentiate between pneumonia and atelectasis as cause of lung consolidation in intensive care unit patients. tools like the clinical pulmonary infection score are of little help (sensitivity % and specificity % for detecting pneumonia) [ ] . the objective of this study was to determine the accuracy of ultrasound assessed vascular flow within the consolidation to distinguish these causes. methods: adult patients with pulmonary symptoms and lung consolidation on lung ultrasound that were scheduled for chest-ct were included. vascular flow was analyzed with color doppler imaging (flow velocity scale was chosen at . m/sec.). the final diagnosis made by the treating physician was regarded as the gold standard. results: patients were included of which nine ( %) were diagnosed with pneumonia. vascular flow in the consolidation was present in seven ( %) out of nine patients with pneumonia, compared to three out of ( %) patients with atelectasis (p = . ). the diagnostic accuracy in differentiating between pneumonia and atelectasis was %. the sensitivity and specificity were % and % respectively. the positive predictive value was % while the negative predictive value was %. conclusions: vascular flow in lung consolidations assessed by lung ultrasound in icu patients aids in differentiating between pneumonia and atelectasis. it outperforms the frequently used clinical pulmonary infection score. methods: three intubated patients for various causes of respiratory distress undergoing mechanical ventilation were subjected to tee. at the level of mid-esophagus, the descending aorta short-axis view ( °) the imaging plane is directed through the transverse axis of the descending aorta. sector depth was increased to image the left pleural space beneath the aorta. for the right lung, the tee is rotated to the right at the level of atria until lung is seen or until the image of the liver is seen and the probe was withdrawn until the right lung is seen. recruitment manoeuvres were performed after identifying pbl atelectasis. atelectatic lungs were visually observed to open up during and after the recruitment manoeuvres. results: the time to acquire the image of pbl atelectasis from the time of insertion by tee is short. the images of posterior lung and the effect of lung recruitments is successfully viewed (fig ) . no immediate complication seen. conclusions: tee provides an excellent view of pbl atelectasis and able to directly monitor the success and failures of recruitment manoeuvres. introduction: high respiratory driving pressure (Δ prs) is strongly associated with increased risk of lung injury and increased mortality during mechanical ventilation. Δ prs consists of the pressure required to distend the lung the transpulmonary driving pressure (Δ pl) and the pressure required to distend the chest wall. Δ pl is the pressure that increases the risk of lung injury. data on Δ pl is limited because its measurement requires an esophageal catheter. we aimed to assess changes in Δ prs and Δ pl during proportional assist ventilation (pav+) at different experimental conditions. methods: we retrospectively analyzed patients ventilated with pav+ who had esophageal pressure measurements before and after dead space or chest load addition. we calculated end-inspiratory plateau pressure (pplateau), Δ prs, respiratory system compliance (crs) and Δ pl during occluded breaths in pav+ (figure ). data were compared with wilcoxon signed rank test and p value< . was considered significant. results: patients were analyzed. dead space increase ( patients) did not affect the studied parameters. chest load ( patients) significantly increased pplateau (p= . ) and Δ prs (p= . ) and decreased crs (p= . ) but Δ pl remained the same (p= . ). median (iqr) changes were . ml/cmh o ( . - . ) for crs, . cmh o ( . - . ) introduction: particle flow in exhaled air from mechanically ventilated patient's mirrors the opening and closing of small airways and can be detect by optical particle counter [ ] . we hypothesized that this particle flow is affected by cardiac function. methods: exhaled air from mechanically ventilated patients was analyzed using a customized optical particle counter pexa, figure . introduction: we assessed the diagnostic accuracy of mechanical power (mp) and driving pressure (dp) alone and combined with stress index (si) to identify ventilator settings likely to produce ventilator induced lung injury caused by tidal hyperinflation [ ] [ ] [ ] . methods: secondary analysis of a previous database of ards patients [ ] . computerized tomography markers of tidal hyperinflation (were used as a "reference standard". analysis of the area under the receiver-operating characteristics curve (auc) was used using a two-fold cross-validation. results: in a cluster of patients, a "training set" of not hyperinflated patients was compared with a "validation set" of hyperinflated patients. (figure - ) . conclusions: si seems to be more accurate than mp and dp in identifying tidal hyperinflation in patients with ards. specificity and sensibility were not improved combining si with mp or dp. the introduction: the pao /fio (p/f) ratio is widely used to assess the severity of lung injury. conceptually, the p/f ratio should be independent of the fio and solely depend on the pulmonary condition. however, effect of fio modulation on the p/f ratio has not been well characterized in ventilated intensive care (icu) patients. the purpose of the present study was to investigate the relationship between fio and the p/f ratio in icu patients on mechanical ventilation. methods: in a prospective, interventional study patients with a swan ganz catheter in situ were included. the p/f ratio was calculated at fio levels ranging from . to . with minute intervals. during the study other ventilator settings were not modulated. to understand the physiological effects of fio modulation on gas exchange and hemodynamics, mixed venous oxygen saturation and cardiac output were assessed. shunt fraction was calculated as described by west [ ] . results: patient characteristics and ventilator settings are reported in table . all patients were admitted to the icu after elective cardiac surgery. modulation of fio did have a significant effect on the p/f ratio, following a u-shaped pattern (p < . ) (figure ). the shunt fraction varied with altering fio levels, also exhibiting a u-shaped pattern (p < . ) (figure ). cardiac output was not affected by fio . conclusions: in contrast to current thinking, the p/f ratio varied substantially with altering fio levels in mechanically ventilated icu patients. this is an important novel physiological observation. in addition, it demonstrates that the assessment of the severity of respiratory failure by using the p/f ratio should be standardized to a fixed fio level. conclusions: in patients undergoing prolonged mechanical ventilation, we must take into account all the factors that may affect our patients. the assessment of diaphragmatic dysfunction is key to preventing weaning failure. an optimal level of consciousness as well as a good management of secretions are key to a successful weaning. prognostic value of the minute ventilation to co production ratio as a marker of ventilatory inefficiency in the icu r lopez , r pérez , Á salazar , i caviedes , j graf introduction: ventilatory inefficiency for co clearance may provide better severity stratification in acute respiratory failure than oxygenation [ ] . ventilatory inefficiency (vi) is best assessed by the bohr-enghoff physiological dead space [ ] . we recently reported that the minute ventilation to co production ratio (ve/vco ), a simplified vi index from exercise testing that obviates the paco measurement, correlates better than other vi indices to physiological dead space in mechanically ventilated patients [ ] . here we report the prognostic performance of this index using a survival analysis. mean±sem ve/vco was higher in patients who died than those who survived ( ± vs ± , p< . , figure ). we found a ve/ vco cutoff value of . mortality was higher in patients with high-ve/vco (≥ ) as compared to those with low-ve/vco ( % vs %, p= . ) with an odds ratio of . [ %-ci . - . ]. cumulative mortality was higher in the high-ve/vco than in the low-ve/vco group (log-rank p= . , figure ). conclusions: in this unselected cohort of mechanically ventilated patients an early high ve/vco ratio was associated to -days mortality. the ve/vco ratio may be a simple and non-invasive vi index with prognostic value in this population. introduction: sodium thiosulfate (sts) is a clinically relevant and safe hydrogen sulfide donor that improved acute lung injury (ali) and brain ischemia/reperfusion injury in previous studies [ , ] . methods: in a prospective, controlled, randomized, and doubleblinded trial, twenty adult, anesthetized, mechanically ventilated and surgically instrumented swine with preexisting coronary artery disease [ ] underwent h of hemorrhagic shock (hs; removal of % of the calculated blood volume and subsequent titration of mean arterial pressure to mmhg). post-shock resuscitation ( h) comprised re-transfusion of shed blood, crystalloids, and norepinephrine. animals were randomly assigned to "placebo" or "sts" ( . g·kg - ·h - for h). before, at the end of and every h after shock, hemodynamics, blood gases, and lung function were recorded. results: survival rates did not differ between groups. sts-infusion attenuated the hs-induced impairment of lung mechanics and pulmonary gas exchange (table , ), resulting in a significantly higher horovitz/peep-ratio ( figure ). conclusions: sts during acute resuscitation from hs may protect comorbid swine against hs-induced ali. introduction: alveolar epithelial cell (aec) death is a main mechanism of severe respiratory failure in acute respiratory distress syndrome (ards). classically, cell death is classified into necrosis or apoptosis. recent studies have reported that not only apoptosis but also certain types of necrosis are molecularly regulated and that these regulated necrosis can be therapeutic targets for various diseases. however, the relative contribution of necrosis and apoptosis to aec death in ards has not been elucidated. our study aimed to elucidate which type of cell death is dominant in aec death and to evaluate whether the regulated necrosis is involved in lps-induced experimental ards. methods: we established ards model by instilling μ g of lps intratracheally to mice. to estimate the relative proportion of apoptosis and necrosis in aec death, we measured cytokeratin m level (total cell death marker) and m level (apoptosis maker) in bronchoalveolar lavage fluid (balf) by elisa, and quantified propidium iodide-positive necrotic cells and tunel-positive apoptotic cells in the lung sections. moreover, we performed pathway enrichment analysis of gene expression data from pcr array to evaluate whether regulated necrosis pathway is associated with the ards model. results: both m and m levels were increased in the ards mice. the m /m ratio (an indicator of the proportion of apoptosis to total cell death) in the ards mice was significantly lower than that of healthy controls. moreover, the number of propidium iodidepositive necrotic cells was significantly higher than that of tunelpositive apoptotic cells in ards mice. in the pathway enrichment analysis, the necroptosis pathway, a regulated necrosis pathway, was associated with lps-induced experimental ards. conclusions: aec necrosis is more dominant than apoptosis in lpsinduced ards model. moreover, necroptosis may contribute to ards pathogenesis. aec necrosis including necroptosis is a potential therapeutic target for ards. clinical ards diagnosis is not associated with a unique circulating neutrophil cell surface phenotype t craven , s duncan , s johnston , c haslett , k dhaliwal , t introduction: acute respiratory distress syndrome (ards) is a form of non-cardiogenic oedema due to alveolar injury secondary to an inflammatory process. the clinical diagnosis is defined by the berlin criteria but this may not reflect the underlying biological process. the activated neutrophil is central to the pathogenesis of ards, characterised by altered cell surface markers. methods: three cohorts of seven participants were recruited. the first cohort suffered from mild, moderate or severe ards as defined by the berlin criteria [ ] . the second cohort was composed of ventilated patients on the intensive care unit with acute inflammatory lung disease (diagnosis of clinical suspicion) but did not meet the berlin criteria for ards. a third cohort was composed of age and sex matched healthy volunteers. procurement of human tissue was approved by a regional ethics committee ( /ss/ or /s / or amrec: -hv- ) and with the informed consent of the participant or their personal legal representative. patients were excluded if aged under or over years of age, were expected to survive for less than hours, if the attending physician refused, due to the absence of suitable indwelling vascular catheter, if the haemoglobin concentration was below . g/dl, or if the patient was enrolled in a trial of novel anti-inflammatory agent. whole blood (lysed erytocytes) underwent flow cytometry to determine cd b, , b, , l and . results: a description of the enrolled cohorts can be found in table . there were no significant differences between the mechanically ventilated, critically ill cohorts for any cell surface molecule in the multiplicity adjusted p values (fig ) . the results support the conjecture that clinical diagnostic criteria should not be used as a surrogate to stratify patients according to biological changes, with implications for the testing of biological therapies. introduction: aim of the present study was to compare the global and regional diagnostic accuracy of lung ultrasound (lus) compared to lung computed tomography (ct) scan in patients with the acute respiratory distress syndrome (ards). ards is characterized by a diffuse, inhomogeneous, inflammatory pulmonary edema. lung ct scan is the reference imaging technique, but requires transportation outside the intensive care and exposes patients to x-rays. lung ultrasound (lus) is a promising, inexpensive, radiation-free, tool for bedside imaging. methods: lung ct scan and lus were performed at peep cmh o. lus was performed using a standardized assessment of regions per hemithorax: superior and inferior; anterior, lateral and posterior. each region was classified for the presence of normally aerated, alveolar-interstitial syndrome, consolidation regions and pleural effusion. agreement between the two techniques was calculated, and diagnostic parameters were assessed for lus using lung ct as a reference. both a global and a regional analysis were performed. results: thirty-two sedated and paralyzed ards patients (age ± years, bmi . ± . kg/m and pao /fio ± ) were enrolled. global agreement between lus and ct was . ± . . the overall sensitivity and specificity of lus are shown in table . similar results were found with regional analysis (anterior/lateral/posterior lung regions is a common practice in our icu. during the interruption eit belt was positioned. when the presence of spontaneous breathing activity was evident by clinical assessment and ventilator traces analysis, nmba were administered to reach full paralysis, in accordance with the treating physician. eit tracing were analyzed offline and the change in eeli after nmba bolus, as compared to before nmba administration, was measured. respiratory mechanics and arterial blood gas (abg) data were collected results: we enrolled ards patients, undergoing controlled mechanical ventilation with muscle paralysis. baseline respiratory mechanics and abg data are shown in table . in out of patient the bolus of nmba led to an increase of eeli. in case, the nmb administration led to no changes in eeli. the mean change in eeli was ± ml conclusions: in our small population of ards patients, the administration of a bolus of nmba after the regain of spontaneous breathing activity led to an increase in eeli in out of patients. further study are needed to ) correlate this increase to global and regional respiratory system compliance and ) correlate this increase to the time needed to wean the patient from nmba introduction: to analyze the use of the orthostatic board as an auxiliary device for the treatment of severe ards by assessing its risks and benefits. methods: we selected patients, females and males, hospitalized in a neurological icu, between june and july , in a physiotherapeutic follow-up with diagnosis of severe ards. the patients were submitted to orthotics assisted for to minutes and monitored hr, pam, fr, sato at °and °of inclination and the pao / fio ratio after the procedure. the mean number of sessions per patient was . . all patients were undergoing anticoagulation in rass - , in the treatment of the cause of ards. the mean time of mechanical ventilation was . days. results: among the patients selected, . % presented tachycardia above bpm, requiring intervention in . % and interruption of the procedure in . %. pam arterial hypotension < mmhg was observed in . %, requiring intervention (increase of vasopressor dose and / or change of plank angulation) in % and interruption of the procedure in . %. hypoxemia sato < % was observed in . %, without interruption, but an improvement in pao / fio was observed in only . % of the patients. conclusions: assisted orthostatism as an auxiliary device for the treatment of severe ards was shown to be an alternative, with improvement of pao / fio in . % of the patients, safe and without significant hemodynamic repercussions that could lead to interruption of the procedure. introduction: the eolia trial found that vvecmo compared to conventional mechanical ventilation (cmv) did not improve mortality in patients with severe ards [ ] . the cmv strategy consisted of airway pressures below cmh o. in patients with severe ards higher airway pressures are required to maintain lung aeration. grasso et al. measured the transpulmonary pressure (p l ) in patients with severe ards and increased peep until p l was cmh o, accepting airway pressures above cmh o. fifty percent of patients responded to an increase in airway pressure and did not require vvecmo [ ] . we hypothesized that a p l guided open lung concept (olc) improves oxygenation and prevents conversion to vvecmo in patients with severe ards. methods: a retrospective study was conducted in a tertiary referral icu. the records of patients referred to our icu for advanced medical care were reviewed. inclusion criteria were severe ards according to the berlin definition and the eolia trial inclusion criteria for vvecmo. results: mechanical ventilation was limited to a p l of < cmh o instead of plateau pressures below cmh o. the p l guided olc resulted in an increase in p/f ratio and none of the patients required vvecmo. during the first hours peak airway pressure was increased, but was reduced within hours while peep was maintained ( fig. ). at hours both peak airway pressures and peep were reduced to baseline values while p/f ratio remained stable. only one patient ( . %) died of disseminated invasive aspergillosis. conclusions: the p l guided olc improved oxygenation and none of the patients required vvecmo. these findings support a ventilation strategy guided by transpulmonary pressures instead of plateau pressures in patients with severe ards. introduction: the mortality benefit conferred by early prone positioning in the treatment of acute respiratory distress syndrome (ards) has been well established. we also know that aprv improves oxygenation, and more recently has been shown to reduce ventilator dependent days and icu length of stay [ , ] . however, controlled ventilation remains the mainstay mode of ventilation used during prone position. literature looking at combined aprv and prone positioning is scarce. we aim to explore and report our institutional experience with respect to feasibility and outcomes in combining aprv and prone positioning, and perform a literature review in this area. methods: we undertook a single-centre retrospective cohort study within a surgical icu of a tertiary hospital in singapore between jan -oct . patients with ards who received combined prone positioning and aprv were reviewed retrospectively. a literature review of patients with ards who received combined intervention was also performed. results: adult patients aged - years old diagnosed with ards received a combination of aprv and prone positioning for a duration of - h ( table ). all the patients tolerated aprv with prone positioning well. our patients saw an improvement of p:f ratio ranging from - upon completion of combination therapy. out of patients were extubated within hours of turning supine, was weaned to tracheostomy mask after days and died while on the ventilator. only case report and randomized clinical trial were found on this topic upon literature review, which corroborated our findings. conclusions: in our experience, aprv is a practical and feasible alternative mode of ventilation that can be employed in the prone position, yielding significant p:f ratio improvements. the synergistic effects on improving oxygenation herald potential, especially in the subset of severe ards patients with refractory hypoxemia, where extracorporeal membrane oxygenation is unsuitable or unavailable. introduction: the recirculation during veno-venous extracorporeal membrane oxygenation (vv ecmo) had been a drawback, which could limit sufficient oxygenation. purpose of this study is to compare the short-term oxygenation in acute respiratory distress syndrome (ards) patients under vv ecmo according to their cannula configurations, especially in the national environment of the absence of newly developed double-lumen, single cannula. introduction: vv-ecmo is most commonly used in severe potentially reversible respiratory failure. this report looks at two patients in whom vv-ecmo was used to facilitate surgical airway stenting. methods: case -a -year-old with recurrent respiratory arrests, on a background of neurofibromatosis type and kyphoscoliosis. he had complex airway pathology, including, airway neurofibromas and granulation tissue, tracheobronchomalacia, severe kyphoscoliosis and a permanent tracheostomy tube. rigid bronchoscopy was performed and following debridement of granulation tissue, a trouser-leg stent was deployed. case -a -year-old with progressive stridor due to recurrence of a malignant melanoma, which was causing mid-lower tracheal compression. three tracheal stents were deployed via a rigid bronchoscope. in both cases, percutaneous bi-femoral vv-ecmo was established prior to general anaesthesia and decannulation took place the following day. results: in these cases, vv-ecmo provided stable extracorporeal gas exchange without conventional tracheal intubation. cardiopulmonary bypass and veno-arterial ecmo have been described in patients at risk of compression of the heart and distal airway [ ] . however, if the major threat is airway collapse, vv-ecmo can provide cardio-respiratory support without the problems associated with arterial cannulation and with lower anticoagulation requirements. introduction: ecco r facilitates the use of low tidal volumes during protective or ultraprotective mechanical ventilation when managing patients with acute respiratory distress syndrome (ards); however, the rate of ecco r required to avoid hypercapnia remains unclear. methods: we determined ecco r requirements to maintain arterial partial pressure of carbon dioxide or co (paco ) at clinically desirable levels in ventilated ards patients using a six-compartment mathematical model of co and oxygen (o ) biochemistry [ ] and whole-body transport [ ] with the addition of an ecco r device for extracorporeal veno-venous removal of co . the model assumes steady state conditions and is comprehensive from both biochemical and physiological perspectives. o consumption and co production rates were assumed proportional to predicted body weight (pbw) and adjusted to achieve pao and paco levels at a tidal volume of . ml/(kg of pbw) as reported in lung safe [ ] . clinically desirable paco levels during mechanical ventilation were targeted at mm hg for a ventilation frequency of . /min as previously reported [ ] . results: model simulated paco levels without and with an ecco r device at various tidal volumes are tabulated in tables and , respectively. table shows a substantial increase in paco at a tidal volume of ml/(kg of pbw) that is more pronounced when further reducing the tidal volume. additional simulations showed that predicted ecco r rates were significantly influenced by ventilation frequency. conclusions: the current mathematical model predicts that ecco r rates that achieve clinically acceptable paco levels at tidal volumes of - ml/(kg of pbw) can likely be achieved with current technologies; achieving such paco levels with ultraprotective tidal volumes of - ml/(kg of pbw) may be challenging. figure a ). pulmonary infections for each subtype of immunosuppression are shown in figure b . conclusions: ards vv-ecmo patients with underlying immunosuppression have higher mortality rates and higher rates of ecmo weaning failure. immunosuppressed patients suffer from a different spectrum of pulmonary infections in comparison to not immunosuppressed patients. introduction: acute asthma attack in children is a life-threatening emergency that requires urgent medical intervention. in the present study, we aim to clarify the effect of non-invasive ventilation (niv) on the heart rate (hr), respiratory rate (rr), and fraction of inspired oxygen (fio ) in children with acute severe asthma (asa) who failed to respond to standard medical treatment; and to evaluate the associated complications and length of stay (los) at the pediatric intensive care unit (picu). methods: this is a retrospective descriptive study of prospectively collected data. it was carried at the picu of a tertiary university hospital, saudi arabia. the study included children ≤ years old with asa admitted to the picu from november to november and required niv. outcome measures include the effect of niv on the hr, rr, fio , and los. the study included children with asa and ( %) of them required niv. of those patients, ( %) were excluded due to incomplete data, and ( %) patients were included in the final analysis. they were ( %) male and ( %) female with a mean age of months and a median pediatric index of mortality (pim ) score of . %. of them, ( %) had moderate asthma scores (≥ - ) and ( %) had severe asthma scores (≥ ). the median duration of niv was hours and the median los in the picu was three days. at hours, only rr showed a significant decrease compared to initiation of niv (p-value < . ) (fig ) ; while hr, rr, and fio were significantly improved at hours from initiation of niv (p-value < . ) (fig ) . conclusions: non-invasive ventilation, in association with standard medical treatment, was associated with clinical improvement in children with asa not responding to standard medical treatment alone. niv was not associated with significant complications or side effects. neurally adjusted ventilatory assist (nava) is a partial support ventilatory mode which triggers and tailors the level of assistance delivered by the ventilator to the electrical activity of the diaphragm. the objective of this study was to compare nava and pressure support ventilation (psv) in patients who were difficult to wean. methods: a total of difficult-to-wean patients who were able to sustained psv in the critical care medicine unit (icu) of the zhongda hospital, southeast university were enrolled in the study (fig ) . patients were classified according to the reason for weaning failure and were randomly assigned to receive nava or psv during weaning ( table ). the primary outcome was the duration of weaning. secondary outcomes included the proportion of successful weaning and patient-ventilator asynchrony. results: there were % ( / ) and % ( / ) patients in the psv and in the nava group never weaned from mechanical ventilation (p = . ). the duration of weaning was significantly shorter in the nava group [ . ( . - . ) days], than in that in the psv group [ . ( . - . ) days] (p = . ). the proportion of patients with successful weaning was % (n= / ) in nava group which was much higher than that in psv group ( %, n= / ) ( table ) . compared with psv, nava improved the rate of successful weaning in patients with single reason ( % vs. %, p = . ) but not in patients with multiple reasons for difficult weaning ( % vs. %, p = . ). nava decreased ineffective efforts and improved the trigger and cycling-off delays when compared with psv. mortality was similar in the two groups (fig ) . in patients who were difficult to wean, nava decreased duration of weaning and increased the probability of successful weaning. nava which improved patient-ventilator asynchrony, is safe, feasible and effective over a prolonged period of time during weaning. conclusions: only mrc score is independently associated with sbt failure and difficult or prolonged weaning. hgs is also associated with these two outcomes related to mv weaning and may serve as a simple tool to identify icuamw. introduction: there is evidence to support that in patients with hypoxemic respiratory failure (ahrf) under non invasive ventilation (niv), high tidal volume (tv) and high respiratory rate (rr) are associated with niv failure and possibly poor prognosis. we postulated that high minute ventilation (mv); or tv x rr; is associated with mortality in ahrf, when niv is initiated. methods: single-center, prospective and observational study. we included consecutives ahrf adults requiring niv. ahrf was defined as acute dyspnea with new pulmonary infiltrates on chest radiography and paco below or equal to mmhg. we registered demographic and clinical parameters (including rr, mv, arterial blood gases, heart rate and blood pressure) at baseline and after hours of first session of niv, apache ii score, diagnosis, need for intubation and icu mortality. we performed a multivariate analysis to assess independent factors associated with mortality and roc . ) and (auc = . ; p = . ), respectively for mortality, future exacerbations and readmissions. the optimal cut-off point for the mwt ratio to predict mortality was . and to predict future exacerbations and readmissions was . . the mwt ratio performed at icu discharge reveals interesting discriminative properties to predict early mortality, future exacerbations and readmissions in ae/copd patients. diffuse alveolar haemorrhage in an intensive care unit -search and you will find m matias , e ribeiro , j baptista , p martins introduction: the incidence of diaphragmatic ruptures after thoracoabdominal traumas is . - % [ ] and up to % diaphragmatic hernias present late [ ] when there is a complication. we report two cases of delayed traumatic diaphragm rupture to highlight the diagnostic difficulties. methods: case (image ) presented left diaphragmatic hernia containing the stomach, spleen, bowel and pancreas. the patient reported a motor vehicle accident dating months. he had thoracoabdominal trauma with several broken ribs on the left side. he then reported occasional pain in his left shoulder and occasional dyspnoea. case (image ) showed right diaphragmatic hernia containing right hemicolon, right hepatic lobe and gallbladder, he reported occasional dyspnoea and recent right chest pain. he had a years car accident in which three ribs broke on the right side. results: almost % of the patients with delayed diaphragmatic rupture presented with complications between and months after trauma, singh [ ] reported a diaphragmatic rupture presenting years after the traumatic event. the physical examination is often not helpful. conclusions: those cases emphasizes on the delayed presentation, patients may be asymptomatic or produce only mild, nonspecific symptoms, such as vague abdominal pain, chest pain or recurrent dyspnoea for months or years. the best tool to guide the clinician toward the appropriate diagnosis is a high index of suspicion whenever there is a history of high velocity trauma, regardless of how remote. factors associated with asynchronies in pressure support ventilation (psv), a bench study introduction: critically ill patients frequently have increased risk of ocular surface disorders (osds) due to poor eyelid closure and reduced tear production due to sedation during mechanical ventilation. we conducted a study to look at the incidence of osds in our icu with the current eye care practices and the impact of a protocolised eye care on the incidence and outcome and to determine the correlation of risk factors with the incidence of osds methods: this study was done in our mixed medical surgical icu. it had a prospective cohort design and was done as before and after study in two phases (phase i and phase ii). in phase i existing eye care practices were continued. in phase ii protocolised eye care was implemented and incidence of osds was noted in both phases. introduction: both fentanyl and morphine are known as opioid analgesics, which blocks the brain from receiving pain signals, the route of administration and the adverse effects affect their use. we compare the efficacy of intranasal fentanyl versus intravenous morphine adults population presenting to an emergency department (ed) with acute post traumatic severe pain. methods: we conducted a prospective, randomized, double-blind, placebo-controlled, clinical trial in a tertiary emergency department between october and june . adults with severe post traumatic was included to receive either active intravenous morphine ( mg immediately and then mg every min if persistence of severe pain maximum mg) and intranasal placebo or active intranasal concentrated fentanyl ( μ g /kg maximum μ g) and intravenous placebo. exclusion criteria: significant head injury, allergy to opiates, nasal blockage, or inability to perform pain scoring, pain scores were rated by using a digital scale at , , , and minutes. routine clinical observations and adverse events were recorded. conclusions: iscs were related to k over-use in our bicu. burnt patients are at risk of hepatic injury [ ] , but k related hepatic injury likely occurred. its not clearly understood mechanisms may involve a cumulative dose effect. although involvement of concomitant medications is being investigated, k restriction policy seemed to contain hepatic disorders. introduction: in november , our institution switched from using alfentanil to fentanyl for analgesia and sedation in adult patients receiving ecmo. there is no published evidence comparing the clinical use of alfentanil vs fentanyl for sedation in ecmo patients, although some reported increased fentanyl sequestration into the circuit [ ] . for these reasons, we conducted a retrospective observational study to explore whether there were any significant differences in patient outcome or adjunctive sedation before and after the switch. methods: outcome data and total daily doses of alfentanil or fentanyl as well as adjunctive sedation/analgesia for each patient where obtained from our clinical information system (philips icca®). data was included from ecmo patients who were sedated with alfentanil or fentanyl from / / to / / until ecmo decannulation. patients not requiring either opiate or who were switched between the two during ecmo therapy were excluded. all medicines prescribed for the management of sedation or agitation were included. for each patient an average total daily dose of each drug, was calculated. data was analysed using stata®. results: both groups were found to be statistically equivalent for mode of ecmo, age, apache score and charlson score (p= . ) except for bmi (p= . ). no difference in patient outcomes were found between groups (table ) . patients in the alfentanil group were found to have received significantly higher median average total daily dose of quetiapine and midazolam (table ) . conclusions: no differences in patient outcomes were found between patients sedated with alfentanil compared to fentanyl. we introduction: the european society of intensive care medicine consensus statement recommends that for comatose survivors of cardiac arrest hours without sedation is the minimum acceptable before neurological assessment. they highlighted the need to investigate the pharmacokinetics of opioid drugs in post-cardiac arrest patients, especially those treated with controlled temperature [ ] . methods: following approval by research ethics committee, we measured the blood concentration of fentanyl in post-cardiac arrest patients treated with ttm following cessation of continuous infusion. the fentanyl was discontinued when the patients were rewarmed to a temperature of . degrees celsius and a blood sample taken hours later. the blood was analysed using a commercial elisa kit (neogen corporation). using the total dose of fentanyl administered, the half-life of fentanyl was calculated for each patient. patient physiological data, cyp a and abcb polymorphism and drug history were compared with half-life. results: the median fentanyl concentration at hours was . mcg/l with a very wide range ( . - . mcg/l). the results for calculated half lives are shown in figure . there was no correlation between fentanyl level and bmi, illness severity (saps ll), creatinine clearance, transaminase or lactate level. there was no correlation between co-administration of drugs of metabolised by the cyp a and abcb enzyme systems or genotype. conclusions: there is marked variation in the concentration of fentanyl at hours in patients managed with ttm following cessation of fentanyl infusion. the calculated clearance of fentanyl in some patients is greater than hours and a hour cut off is not safe. introduction: objective of this study was to compare the effects of three analgesic regimens, one opioid and two multimodal ones, on cardiovascular stability and pain intensity in patients undergoing elective surgery under general endotracheal anesthesia during the h postoperative period. methods: sixty elderly patients, asa ii, undergoing elective knee sugary were assigned to receive ) morphine or mg iv q h, depending on body weight, and paracetamol g iv q h (mp group), or multimodal nerve block: ) femoral nerve block, single shot (fnb group) or ) fascia iliaca compartment nerve block single shot (ficnb group). measurement of pain intensity was performed with numerical introduction: opioids are frequently used in the intensive care unit (icu) to relieve pain and facilitate tolerance of life-support technologies. when discontinued abruptly, patients may develop a cluster of symptoms known as opioid-associated iatrogenic withdrawal syndrome (oiws). this phenomenon is poorly described in critically ill adults although it is associated with unfavourable outcomes, such as prolonged icu stay. the objective of this study was to describe the signs and symptoms of oiws in adult icu patients. methods: a prospective observational study was conducted in two tertiary care centres in patients requiring mechanical ventilation and regular opioids for more than hours. after an opioid dose reduction of at least %, patients were assessed daily for signs and symptoms of withdrawal using a standardized form. concomitantly, the presence of oiws was assessed daily by a physician using modified dsm- criteria. all physician evaluations were blinded and performed independently. inter-rater reliability for dsm- evaluations was assessed with the kappa coefficient. results: a total of patients were screened and twenty-nine enrolled. the majority were male ( . %) with a median age of . the median apache ii score was . withdrawal occurred in . % of patient within a median of three days (iqr to days) from opioid weaning. according to investigator assessment, restlessness, agitation, anxiety, hallucinations, insomnia/sleep disturbance, mydriasis and elevated blood pressure were more prevalent in oiws-positive patients. dsm- evaluations identified dysphoric mood, muscle aches, lacrimation/rhinorrhea, pupillary dilation/piloerection/sweating, diarrhea and yawning more frequently in oiws-positive patients. the kappa coefficient showed good agreement ( . ). conclusions: oiws in critically ill adults presents with a large spectrum of signs and symptoms that occur within a median of three days from onset of opioid weaning. further studies are needed to confirm these preliminary findings. withdrawal reactions after discontinuation or rate reduction of fentanyl infusion in ventilated critically ill adults s taesotikul introduction: propofol is a well-known sedative, commonly used in intensive care units (icu s), that on rare occasions has been reported to cause green urine and has also been associated with pink or transient white urine discoloration. it can cause several adverse effects, such as low blood pressure, pain on injection, apnea, hypertriglyceridemia and when administered in high doses it may lead to the "propofol infusion syndrome". methods: we present two examples of interesting urine discolorations observed unexpectedly in our icu in patients under propofol sedation requiring mechanical ventilation. results: dark green urine discoloration as presented in fig. is the result of a phenolic metabolite of propofol that is produced in the liver and is subsequently excreted in the urine, thus changing its color. it is considered a reversible phenomenon that resolves after propofol discontinuation.respectively, pink urine discoloration as presented in fig. can also be the result of propofol infusion. the increase in urine excretion of uric acid caused by propofol, in combination with a low urinary ph can lead to the formation of uric acid crystals and turn the urine pink. discontinuation of propofol and urine alkalization can reverse the phenomenon. conclusions: green or pink urine discoloration due to propofol is generally a benign, reversible condition. its presence should not compel the physician in charge to perform unnecessary testing, although other causes of discoloration should be considered. as far as green urine discoloration is concerned, other factors such as drugs, dyes, certain nutritional supplements or even a pseudomonas urinary tract infection may be at fault. on the other hand, pink urine syndrome due to propofol infusion seems to be even rarer. although its presentation is not alarming, it may well increase the risk of uric acid lithiasis, a fact that the physician in charge should always keep in mind. conclusions: hepatic changes related to propofol are frequently observed and should be systematically monitored to ensure patient safety. fig. (abstract p ) . dark green urine discoloration introduction: clevidipine (clev) and propofol (prop) are lipid-based medications used in the intensive care unit (icu) for hypertension and sedation, respectively. no data exists regarding potential adverse effects of concurrent therapy with this combination. this study aims to evaluate the incidence of hypertriglyceridemia (htg) and pancreatitis in icu patients using concurrent clev and prop. methods: this was a single-center, retrospective chart review in patients utilizing clev and prop concurrently from february to november . patients were included if they were years and older, on clev and prop concurrently for at least hours with no more than hours of interruption at a time, had at least one triglyceride (tg) level during concurrent therapy, and admitted to the medical or surgical icu. the incidence of htg (defined as tg equal to or greater than mg/dl) and pancreatitis (provider assessment based on american college of gastroenterology guidelines) was evaluated. patients with and without htg were compared to identify risk factors for the development of htg. results: of patients screened, patients were included which comprised observations. the incidence of htg was . % with no patients developing pancreatitis. patients with htg had a higher median age compared to without htg ( . vs. ), p= . . in patients with htg the median dose of clev and prop were mg/h and . mcg/kg/min, respectively, which was higher but not statistically significant when compared to patients without htg. cumulative lipid load (g/kg/d) was non-significantly higher in patients with htg ( . vs. . ), p= . . conclusions: the incidence of htg was comparable to what is cited in literature for prop alone. patients with htg were older, had higher median clev and prop doses, and a larger cumulative lipid load compared to patients without htg. introduction: the society of critical care medicine guidelines for pain, agitation and delirium suggested use of nonbenzodiazepine sedatives like dexmedetomidine which is associated with a reduced duration of mechanical ventilation, shorter length of hospital stay and a lower incidence of delirium [ ] . enteral clonidine represents a potentially less costly alternative for agitated patients with prolonged dexmedetomidine infusion. limited literature exists examining this transition for management of agitation [ ] . methods: the critical care management initiated an action plan on the transition of patients with prolonged dexmedetomidine infusion to oral clonidine. a protocol was prepared with clinical pharmacist's assistance. risk factors were assessed and inclusion criteria were applied as per protocol. dexmedetomidine infusion rate was reduced gradually with oral clonidine administration in selected patients. other rescue managements were implemented as per protocol. oral clonidine was then tapered down by reducing frequency of administration over few days. results: post intervention data in showed significant decrease of dispensed doses and cost of the injections compared to . the annual cost saving was % equating to , usd (table , figure ). conclusions: transitioning to clonidine may be safe and less costly method of managing agitated critically ill patients on prolonged dexmedetomidine infusion. more studies are needed to evaluate the efficacy and safety of this practice. incidence of dexmedetomidine associated fever at a level trauma center na beaupre, jt jancik hennepin county medical center, pharmacy department, minneapolis, united states critical care , (suppl ):p introduction: we evaluated the incidence of dexmedetomidine associated fever (daf) in a level trauma center's medical intensive care unit (micu). hypotension and bradycardia are the most commonly reported adverse effects associated with dexmedetomidine (dex) infusion. case reports suggest dex can cause fevers and the clinical trials that led to the approval of dex demonstrated fever rate to be - % [ ] . methods: this was a single-center, retrospective chart review of patients admitted to the micu at hennepin county medical center between march and july of that were started on a dex infusion. patients were included if they were years and older, on a dex infusion for at least hours, and had temperature data available. fever was defined as > . c and other causes of fever including infections, medications, withdrawal, recent surgery, thromboembolic disease, thyroid disorders and seizures were excluded from analysis. results: of the patients screened, were included. the mean age was years and . % were males. of all the patients included, the mean change in temperature after initiation of dex infusion was + . c from baseline. the mean initial dose was . mcg/kg/hr. four of patients ( . %) had a daf. of those that had a daf, the median initial dose was . mcg/kg/hr; the median time of infusion was . hours; and the median cumulative dose was . mcg/kg/hr. the median time to fever after initiation of dex was hours, with a range of to hours. the median time to fever cessation after discontinuation of dex was hours. conclusions: in our population, the incidence of dexmedetomidine associated fever was relatively rare at . % and similar to current literature rates. the results obtained showed a statistically significant fact that fewer points on the test, from to points, received older patients who underwent an urgent surgical procedure, over years of age, of which % . also statistically significant data were obtained that patients who used a higher amount of sedatives during emergency surgery, % had a worse test result than under points due to increased preoperative anxiety. the older population is more susceptible to postoperative delirium, especially in emergency surgery situations, which they carry, unpreparedness for surgery, increased use of medication for fig. (abstract p ) . flowchart of enrolled patients calm, unpredictability of the duration of surgery, and therefore anesthesia as well the use of anticholinergics, which is sometimes impossible to avoid in operative procedures such as gall bladder surgery. the results of the study suggest that in cases of emergency surgery, the use of protocols for postoperative delirium should be planned regularly to prevent or at least mitigate the clinical picture of delirium that can lead to complications postoperatively. introduction: delirium is a serious and often underestimated condition with implications for morbidity, mortality and healthcare costs. as it presents in a wide range of settings from admission to discharge, early prediction and risk assessment are essential. e-pre-deliric is a delirium prediction score which has been validated in itu patients but not in other populations, and we conducted a quality improvement project using this score to assess its utility in other settings. methods: data was gathered from three patient categories: those undergoing elective surgery (es), admissions to the emergency observation unit (eou) in the a&e, and patients with fractured neck of femur (nof). clinical notes were reviewed to collect data to calculate e-pre-deliric score at admission, along with a number of other clinical variables including incidence of delirium, and statistical analysis performed. results: a total of patients were included, with in the es group, in the eou group, and in the nof group respectively, with an overall average e-pre-deliric score of . %. es had a . % average e-pre-deliric score, a mean age of and no cases of delirium. the eou group had an average age of , a . % average e-pre-deliric score and no incidence of delirium. the nof group had a mean age of and an average e-pre-deliric score calculated on admission of . %. this was the only group in which patients developed delirium. a % cut off was demonstrated to be the most accurate to predict delirium in this population with a sensitivity of . and a specificity of . . conclusions: despite the limitation of a small sample size, this project has shown that e-pre-deliric score could be a useful tool to predict patients at high risk of delirium in a non-itu setting, with a % cut off in hip fracture patients. further investigation should be conducted into the potential use of e-pre-deliric in non-itu patients. comparison of long-term mortality between patients with and without delirium during admission in medical intensive care units in a university hospital n kongpolprom king chulalongkorn memorial hospital, pulmonary unit, bangkok, thailand critical care , (suppl ):p that delirium is linked with preoperatory comorbidities. the complexity of surgery has a big influence on the development of delirium, especially in the cases of aortic dissection. delirium was associated with intraoperatory blood transfusions. finally, our data point to a bridge between postoperatory electrolytic disturbances, as well as inflammation as factors potentially triggering delirium onset. introduction: we did a retrospective case note study of mortality due to sepsis of our unit over three months as observational study in which we noted the causes of deaths, origin of sepsis, organism, patient characteristics and icnarc physiology scores and icnarc h model predicted risk of acute hospital mortality percentage. methods: icnarc data base was used to gather the data and coding was used to identify the patients with sepsis for three months. patients mortality attributed to sepsis were identified from mortality list.causes of death were noted from patients notes and death certificates.cyber lab was used to access the data and case note were ordered for review.patients characteristics were noted including dnacpr orders and treatment withdrawal orders. scores (apache scores, icnarc physiology scores, icnarc h predicted risk models of acute hospital mortality percentage) were noted. results: mortality percentage was found to be % as per codig which was reduced to % as % deaths were attributed to other causes. % patient had dnacpr in first hrs. average length of stay was . days with median of . days.median age was yrs in surviving age group and years in other. icnarc physiology score with predicted risk of . %. commonest cause was found pneumonia % followed by urine tract infection. % patients were with no source identification. conclusions: conclusion was made that we do need to improve the coding as significant percentage was mentioned as sepsis as cause of death where clinicians differed. pneumonia was found to be the commonest killer in critical care followed by urine tract infection. it was pointed to be useful to carry out further audit targeting pneumonia .review of icnarc case mix program, development of icnarc physiology score, which provides excellent local use with downside of lacking international comparison was done also. introduction: hospitals vary widely in the quality of care they provide for septic patients. since many septic patients present to their nearest hospital, local variations in care quality may lead to geographic disparities in access to optimal sepsis care. we sought to better understand geographic access to high quality sepsis care, taking advantage of publicly reported data on sepsis management and outcomes in a large us state. methods: we performed a cross-sectional analysis of geographic access to high quality sepsis care, taking advantage of a new york state initiative that mandates public reporting of sepsis quality data to the state government. we linked these data to the locations of hospitals in new york state from the us centers for medicare and medicaid services and population data from the us census bureau for . we defined hospital sepsis performance using self-reported risk-adjusted mortality rates (ramr) and defined high-performing hospitals as those with a ramr < %, which represents the lower end of short-term mortality typically observed in sepsis. we used arcgis to generate drive-time estimates and assess population access to high performing acute care hospitals for sepsis care. results: hospitals publicly reported treating , cases of sepsis from a population of , , persons. overall access to an acute care hospital was excellent at the -minute drive threshold ( . %), good at the -minute threshold ( . %), and marginal at the -minute threshold ( . %). we classified hospitals ( . %) as high-performing based on a ramr < %. high-performing hospitals reported , ( . %) of the total sepsis cases. high-performing hospitals were geographically dispersed across the state, although population access diminished substantially with increasing drive times ( . % at -minutes, . % at -minutes, and . % at minutes; figure ). conclusions: one in six people do not have timely access to a high performing hospital for sepsis care using a -minute threshold. [ ] . this poses a significant safety risk. a previous study found that the implementation of a multidisciplinary medication safety group in intensive care increased reporting of errors and near misses [ ] . the purpose of our work was to set up a multidisciplinary group to provide a forum to review and improve medication safety at all stages of the process. here we discuss some of the initiatives and outcomes implemented in the last months. methods: ccmsg was formed in , under the leadership of the critical care pharmacy team, with representation from medical and nursing disciplines. the group meet fortnightly to analyse trends in medication errors, implement changes to local practice and review outcomes to improve patient safety. the cohesive, multidisciplinary nature of the group allows medication safety initiatives to be delivered in the most effective way. results: on average, ccmsg reviewed medication errors per month. the most common high risk drug classes involved are seen in table . medication safety initiatives implemented were based on these trends and included writing guidelines and policies, bedside education, teaching and training, informatics optimisation and operational changes. examples are seen in table . conclusions: initiation of a ccmsg provides a cohesive approach to facilitate the implementation of targeted safety initiatives, which are proven to reduce some of the most common medication errors in critical care. in addition, these often result in optimisation of operational and financial inefficiencies. introduction: cis/hospital electronic medical records downtime can cause major disruptions to workflow, patient care, key communication and information continuity [ ] . here we describe the consequences of deploying a business continuity plan (bcp) designed to support a critical care clinical informatics system (cis) failure, during an -hour unplanned downtime in a large central london icu. the institutional bcp was developed through an iterative process based on cis provider recommendations and internal workflow knowledge. it consisted of a web offline chart (woc) that is accessible at every computer connected to the network (in the event of a cis server fault), and via hard copy from designated back up computers connected to a printer (in the event of whole network loss). operational and clinical consequences were recorded during informal and formal debrief of the informatics team. the decision making around´drop-to-paper´was reviewed. -the bcp permitted´drop-to-paper´, service continuity and controlled uptime -patchy network loss and lack of a general institutional bcp delayed initial system failure diagnosis (network vs primary server); reduced reliability of´read-only´data and delayedd rop-to-paper-day-to-night handover during downtime led to loss ofḿ emory´of key patient data/events, and should have accelerated decision to´drop-to-paper-transfer of prescriptions was time consuming, distracting (occupied cis team) and prone to error conclusions: previous end-to-end testing of the bcp had not identified many of the observations and recommendations that came from the analysis of an actual period of unplanned downtime. we recommend sharing of similar experiences and scheduled high-fidelity simulated downtime in other institutions to replicate real world conditions, particularly in a critical care setting. . ) were predictors of icu transfer. we developed a simple score to predicting icu transfer from previous variables and performed analysis of auc of roc, which was compared to that of apa-che ii. the result showed the auc of roc of a new score was slightly higher than the apache ii, namely . vs. . respectively. conclusions: the immunocompromised patients take two times higher risk than the immunocompetent ones regarding icu transfer. the other risk factors are lower gcs, lower sbp, and higher rr. a newly developed score may be a promising tool for predicting and triaging site of care in patients who require imcu admission. introduction: this research aims to explore the role of situation awareness in the decision-making of patient discharge from the intensive care unit (icu). the discharge of these patients is a complex and, moreover, a challenging transition of care. readmissions are undesirable given the association with a more extended hospital stay and a possible chance of higher mortality. little is known on how the decision-making process takes place and accordingly, the role of situation awareness of patient discharge from the icu. in order to improve the quality of care of patient discharge from the icu, further research is necessary. methods: this research concerns a qualitative study in which various health care providers, working in an icu adults of a large teaching hospital, were interviewed. through purposive sampling, six nurses, two physician assistants, two intensivists and a physiotherapist were included. on the obtained data a thematic analysis was applied, based on the principles of the grounded theory. results: the discharge decision of icu patients seems mainly based on the team´s situation awareness, with the initiating role of the intensivist and the guiding role of the nurse. furthermore, there is an additional role for the physician-assistant and a consultative role for physiotherapy in the process of the decisionmaking. worries of patients and family seem not to affect the decision-making directly. in the decision-making process, the well-being of the patients and the possibility to provide the most suitable and best possible care were central. organizational factors, such as an urgent demand for icu beds do count but seem not to push the decision to transfer patients from the icu to the regular hospital ward. conclusions: the decision to dismiss icu patients is a complex process with different disciplines and a variety of factors involved. obtained knowledge and insights into the role of situation awareness provide starting points for improving the quality of the discharge process of icu patients. conclusions: despite the fact that older people was more severe illnes, and similar frequency of respiratory failure, the use of mechanical ventilation, the use of central venous catheter and arterial catheter was less frequent. the addition of a simulation fellow within the intensive care team and introduction of in situ simulation n bhalla, d hepburn, g phillips royal gwent hospital, intensive care unit, newport, united kingdom critical care , (suppl ):p introduction: traditionally, simulation based medical education has been carried out in off site simulation centres, however, we trialled the addition of a simulation fellow, within our intensive care team, to run an in situ simulation (iss) program on our intensive care unit over a month period. methods: our multi-disciplinary iss program, led by a simulation fellow, incorporated participants, observers and facilitators including doctors (junior trainees up to consultants of varying medical specialties), nursing staff, healthcare support workers, operating department practitioners, physiotherapists and medical students. we ran simulated emergency scenarios and technical skills sessions. with every scenario, we collected data on participant and observer feedback using the world health organisation participant feedback form and conducted a satisfaction survey at the end of our trial period. results: our results, highlighted in table , show participants found iss led by a simulation fellow realistic, well structured and organised. it was useful for testing and understanding our response systems, fig. (abstract p ) . patient journey of group : those patients discharged home days after step down from critical care identifying strengths and gaps and establishing individual roles/functions within emergencies; overall leaving us feeling better prepared for critical care emergencies. from our satisfaction survey, % of participants found the simulation fellow a useful addition to the intensive care team and expressed the need for more in situ simulation. conclusions: the addition of a simulation fellow allowed for numerous disciplines within the critical care team to be involved in challenging emergency scenarios (fig , ) , with the additional realism of being on the intensive care unit playing the role they would in real life; as well as having opportunity for spontaneous discussion and learning. from this they reported great benefit and satisfaction. following our initial success with this program, we plan to have a simulation fellow as an ongoing role within our critical care team. impact of multidisciplinary team in readmission in a brazilian cardiac intensive care unit c bosso , p introduction: the aim of this study is to determine the importance of the multidisciplinary team at readmission rates in a cardiac intensive care unit (cicu). methods: retrospective study with analysis of patients in a cicu of a medium size brazilian hospital. the years of and represent the reduced team (physician, nurse and physiotherapist) and and the complete multidisciplinary team (additional presence of phonoaudiologist, psychologist, pharmacist, dentist and nutritional professional). the risk of mortality was determined by saps score. in order to compare the teams, it was utilized odd ratio of a logistical sample to the discrete data, and t-student test to the continuous data. the data analysis was executed from the software rstudio ( . . ), and the significance level adopted was %. results: the number of patients was of n= ( from the reduced team and from the multidisciplinary team). the age, sex and bmi didn`t present significant difference between groups. the average age of the sample was ± years old (p= . ). the male sex represented % (p= . ), and the bmi was around . ± . (p= . ). the main diagnoses were similar in both groups -coronary angiography with stent ( %), unstable angina and non st elevation myocardial infarction ( %). table shows the average, standard deviation, p-value to t-student test to saps score and lengh of stay (days), according to both reduced and multidisciplinary teams. table exposes the mortality rate and readmission for both teams. the figure shows the odds ratio and its ic % to the comparison of the mortality, readmission, hours readmission and hours readmission rates between the teams. conclusions: the multidisciplinary team performance reduced the number of hospital readmissions in and hours in a cicu. methods: during the initial audit hours' worth of waste from one itu bed was manually divided into the categories above. results: based on these figures it was estimated that a saving of £ per year would be made (£ . per bed space) over the course of a year should domestic waste bins be placed across the bed icu/hdu. a business case was made, and every bay had a domestic waste bin installed with poster signs for explanation.the reaudit in which all domestic waste across the unit was weighed produced an even greater figure of a saving of £ per bed space (£ ) per year. conclusions: introducing a domestic waste bin may save approximately £ per year per bed. in a typical itu such as lewisham ( itu beds/ hdu beds) that may mean a saving of £ per year (with % capacity). there are also environmental benefits, burning of plastics releases harmful dioxins. the authors wish to make intensive care units and indeed all areas of the hospital aware of the cost and environmental impact associated with disposing of waste in incorrect categories. we hope that our quality improvement project demonstrates how easily money may be saved and environmental footprint reduced. association between resilience and level of experience in intensive care doctors in india j gopaldas, a siyal manipal hospital, bangalore, critical care medicine, bangalore, india critical care , (suppl ):p introduction: attrition of doctors in intensive care unit (icu) is one of the highest amongst all medical specialities globally, and is strongly associated with stress and burn out syndrome (bos). factors that contribute to bos are low pre-morbid resilience and low level of icu experience. studies from india have shown high levels of stress in intensive care doctors (> %), but there are no published studies measuring pre-morbid resilience and risk of burnout in relation to years of experience amongst icu doctors. our main aim was to measure cross sectional resilience levels in icu doctors compared between those with less than years of experience to those with years or more. a secondary aim was to assess the impact of other factors that may contribute to low scores. methods: an anonymised survey was conducted involving doctors in icus across different states in india, using the connor-davidson resilience scale (cd-risc ), which is validated in indian population. results: a statistically significant correlation was found between low levels of resilience in icu doctors with under years of experience . ) , and the significance level adopted was %. a logistic regression model was used to test the difference between the mortality and readmission rates in < and ≥ groups, which enabled the calculation of odds ratios. chi-square test was used to evaluate categorical variables and t-student test to some quantitative variables. the roc curve was constructed to verify the sensitivity of prediction of mortality through different saps scores. results: among the < and ≥ groups, respectively % and % was male (p = . ). mean weight of the> years was ± kg and < years was ± (p < . ). odds values indicated a significant difference only for the mortality rate, which was more than double among ≥ . readmissions in any time, h and h as well the mortality is shown in table and odds in figure . there was a significant difference in saps points between groups ( table ). the ≥ group presented an average of points higher on the severity scale when compared with those in the < group. there was no significant difference in lengh of stay. the highest amount provided by saps scores was % and a specificity of % for hospital mortality not group < years. in ≥ group the highest sensitivity was % and the specificity was %. roc curve for saps is shown in figure . conclusions: the extremely elderly patients of a cicu is more severe, with higher mortality and have the same lengh of stay and readmission rates. introduction: the purpose was to assess the prevalence and impact of non-urgent interruptions (nui) within critical care (cc).a root cause analysis of a never event in our cc discussed nui as a contributory factor, paralleled by learning from serious incidents.the negative impact of nui is well evidenced, resulting in delayed task completion, increased stress, and affecting patient safety. methods: any nui during a consultant ward round (cwr) or invasive procedure (ip), not relating directly to the current clinical episode, was included. qualitative data was collected by a survey, assessing the cc multidisciplinary teams(mdt) perception of nui. results: one third of reviews during the cwr, and %of ips, had a nui. adverse effects included prescription omissions, delayed cwr, near-miss with a cvc, and failed picc insertion. overall, % of staff considered nui a problem; % had experienced nui that led to distraction in train of thought. % felt that nui had led to an error: % of doctors, versus % of nurses. % overall felt nui contributed to stress at work. reasons for interruptions included: feeling overloaded, needing to resolve concerns before forgetting/being distracted, unable to prioritise, and to shift responsibility.lack of leadership or clinical supervision providing a point of contact for problems during shifts was mentioned as contributory. senior staff raised that whilst attempts have been made to level hierarchy, allowing a voice for all to express concerns contributes to interruptions. potential solutions included awareness on impact of nui, jobs book,´sterile cockpitd uring ips, and increased clinical supervision during shifts. conclusions: we have demonstrated the prevalence and consequences of nui within cc is significant.the impact on staff is significant, both for contribution to errors and also the negative impact on stress in the workplace. identified potential solution will be implemented. the impact of an education package on the knowledge, skills and self-rated confidence of medical and nursing staff managing airway & tracheostomy/laryngectomy emergencies in critical care l o´connor , k rimmer , c welsh methods: the factors affecting the delivery of intensive care was elucidated by a comprehensive review of the intensive care literature. a further understanding of intensive care delivery in south africa was obtained by "making sense of the mess" with eight workshops and interviews using a systems approach. systemic intervention served as the meta-methodology and methods and techniques from interactive planning, critical systems heuristics, soft systems methodology and the viable system model were employed. results: making sense of the mess emphasised the complexity of intensive care delivery, on both a situational and a cognitive level. it became clear that a single methodology would not suffice, but that a pluralist methodology was required to guide improvement in intensive care delivery. based on this understanding, nine principles were formulated to guide the development of a framework. systemic intervention was again used as the meta-methodology. interactive planning was identified as the key methodology, incorporating methods and techniques used in the making sense of the mess phase to build a systemic framework for the improvement of intensive care delivery. embedded in the proposed framework are matters relating to systemicity, complexity, flexibility, empowerment, and transformation of intensive care delivery. the proposed framework allows for multiple-perspectives, including that of marginalised stakeholders, the mitigation of multivested interests and power relationships (fig ) . it is both flexible and adaptable to promote learning about the complex problems of intensive care delivery and it accommodates the strengths of various relevant approaches to complex problem solving. conclusions: the proposed framework aims to facilitate sustainable improvement of intensive care delivery and to ensure the "just-use" of resources to foster distributive justice. the perioperative management of adult renal transplantation across the united kingdom: a survey of practice c morkane , j fabes , n banga , p berry , c kirwan introduction: there is a limited evidence base to guide perioperative management of patients undergoing renal transplantation and no national consensus in the uk. we developed an electronic survey to provide an overview of uk-wide renal transplant perioperative practice and determine the need for future guidelines on patient management. methods: a -question survey was developed to encompass the entire renal transplant perioperative pathway with input from clinicians with expertise from renal transplant surgery, anaesthesia, nephrology and intensive care. the survey was sent to lead renal anaesthetists at each of the transplant centres across the uk. results: twenty-two centres ( %) returned complete responses. there was limited evidence of guideline-based approaches to preoperative work-up, with marked variety in modality of preoperative cardiorespiratory function testing performed. questions regarding intraoperative fluid management (fig ) , blood pressure targets and vasopressor administration (fig ) identified a broad range of practice. of note, the routine use of goal-directed fluid therapy based on cardiac-output estimation was reported in six ( %) centres whilst nine centres ( %) continue to target a specific central venous pressure (cvp) intra-operatively. a dedicated renal ward was the most common postoperative destination for renal transplant recipients ( % of centres), whilst a renal or transplant-specific hdu provided postoperative care in ( %) centres. the need for care in an icu setting was decided on a case-by-case basis. conclusions: this questionnaire highlighted a high degree of heterogeneity in current uk practice as regards the perioperative management of renal transplant recipients. development of evidence-based national consensus guidelines to standardise the perioperative care of these patients is recommended. fig. (abstract p ) . framework for the improvement of intensive care delivery introduction: postoperative care of high risk patients in the icu used to be considered the gold standard of care in terms of reducing perioperative mortality [ ] . new evidence comes to question this practice [ ] . the primary objective of our study was to detect any benefit of postoperative icu care after elective surgery in terms of patient's outcome, length of hospital stay, complications and cost. methods: a -month retrospective analysis of high perioperative risk patients who were about to be subjected into an elective operation were included into the study. subsequently they were allocated into two groups. group i patients were those admitted into the icu for postoperative care while those admitted into the standard ward consisted group ii. demographic data, length of hospital stay, outcome, need of mechanical ventilation, complications and total cost were recorded. results: a total of patients were recorded, in each group. there was no statistical difference regarding the demographic data between the two study groups. seven patients died before hospital discharge ( in group i and in group ii, p> . ). there was no impact of icu admission on length of hospital stay (p= . ) which is primarily affected by the need of mechanical ventilation (p= . ) and reoperation (p< . ). the total cost and the postoperative cost of hospital care did not statistically differ among study groups. conclusions: according to our study the need of postoperative care of high risk patients in the icu is rather questionable in terms of perioperative mortality, length of hospital stay and cost of care. introduction: tivap is a preferred vascular access device for patients with solid tumors and radiological-guided insertion is a standard of care. however, many hospitals have no access to interventional radiology service. our study aimed to determine whether it is safe to place tivaps in icu for immediate administration of chemotherapy. methods: we analysed prospectively maintained database of our department and collected data for adult pts with tivaps implanted between / and / . the median age was (range - ) years, % were women. all procedures were performed by trained physicians with experience in ultrasound (us). puncture technique was used and tip location was controlled with electrocardiographic (ecg) and us with subsequent chest x-ray confirmation. pts were followed up for at least days after the procedure for complications, functioning of tivap and surgical wound healing. results: all tivaps were successfully implanted in pts. infraclavicular route was used in cases ( . %). difficulties with indwelling guide wire were observed in ( . %) pts but did not precluded implantation. placement complications included pneumothorax (n = ), catheter malposition (n = ) and artery bleeding (n = ). these complications required additional therapy but were managed successfully and resolved without consequences. in the rest cases internal jugular vein (jv) was used. complications were not observed. ecg and us navigation provided optimal tip location control in these situations. surgical wound healed after - days and chemotherapy initiation did not affect healing. all tivaps had adequate functioning days after placement. conclusions: it is feasible to implant tivaps in icu. these devices can be used on the implantation day without jeopardizing patient safety. jv catheterization seems to be optimal approach and us navigation and ecg are sufficient methods for placement control. introduction: there is increasing use of clinical information systems to improve patient safety and quality of care in critical care. with all these systems, a rigorous business continuity access (bca) plan needs to be in place so patient safety is not compromised [ ] and ensure continuity of care. here we evaluate the types of medication errors that occurred during a period of unscheduled downtime; potential contributory factors [ ] and the number of errors involving critical medicines [ ] were analysed. methods: during the unscheduled downtime, all prescribing and administration of medicines were transferred to a paper based system using the patients' web offline chart (woc -philips healthcare). pharmacists at the time double checked the paper charts that were transcribed, to mitigate errors but this was not consistent due to the timing of the event. we retrospectively compared the paper drug charts against the electronic prescriptions and noted all errors for patients. results: in total medication errors were identified & allergy omission ( table ) . pharmacists double checked % of the paper charts. conclusions: our data highlights the risks associated with unscheduled electronic patient management system downtime and the heterogeneity of the types of errors & potential contributory factors. it underscores the need for robust local bca plan implementation, critical review of the woc document and regular staff training around potential unscheduled system downtime. introduction: the transfer of patient care (toc) between the intensive care unit (icu) and hospital ward is associated with a high risk of medical errors [ ] .according to uk national data between - % of patients have an error or unintentional medication change made when moving between care settings [ ] . currently different prescribing systems without interoperability are used between icu areas & ward settings in our institution, resulting in medications needing to be re-prescribed on transfer. we aimed to evaluate the time delay in medication re-prescribing, number of unintentional omissions of drug doses and reasons, as well as percentage of critical medicines [ ] omitted in the first h following discharge. methods: over a month period, discharged patients ( % of all discharges) from two icu units were included. the icu discharge letter which contained the medication list on transfer was compared against the ward based electronic drug chart to identify all unintentional omitted medication doses during the first hours. the starting time point was when the patient physically left icu. results: / ( %) of patients had their medication prescribed more than hours post discharge. there were a total of / , ( %) unintentional omitted doses (table ) . of these / ( %) were considered critical medicines ( table ) . conclusions: this data confirms the risk associated with toc especially around medicines. the need of interoperable electronic prescribing systems is one solution and could improve patient safety by streamlining the process. introduction: staff perceptions of safety may contribute to workforce stress and be organisationally important [ ] . this study explored the feasibility of capturing perceptions of safety with a bedside professional reported (bpr) shift safety score, and explored relationships between bpr and measures of staffing and workload. methods: uk health research authority approval was obtained (id ). data were collected for consecutive days at imperial college healthcare trust ( general critical care beds on sites).the bpr asked all icu staff to rate each shift as "safe, unsafe, or very unsafe". responses were described and correlated with data on organisational staffing (care hours per patient day chppd) and nursing intensity (total number of organs in failure/ total number of nurses). results: a total of bpr scores were recorded (response rate %). we noted heterogenous responses between sites and days, and within shifts, only % of shifts were unanimously rated. whilst % of shifts were rated by staff as "unsafe" or "very unsafe", organisational metrics recorded only % as 'unsafe'. we did not find a correlation between measures of staffing (chppd) and perceptions of safety ( figure ). preliminary analyses suggest that staff perceptions of safety are not well correlated with nursing intensity (figure ), although these numbers commonly inform staffing metrics. conclusions: completing the bpr tool was feasible and acceptable to staff. responses showed variations in perceptions of safety and a gap between organisational metrics and individual perceptions. introduction: delivery of intensive care (icu) is complex because of multiple stakeholders with varied perspectives and conflicting goals that interact and are interdependent. to inform the development of a framework for the improvement of icu delivery in south africa, it was essential to first understand icu delivery or "make sense of the mess". a systemic approach such as systems thinking is required to holistically explore and understand the complexity of icu. no methodology is perfect and methodological pluralism as proposed by systemic intervention, a systems thinking approach, was used for a more flexible and responsive intervention. the methods used was the making sense of the mess phase of interactive planning, stakeholder analysis as describe by critical systems heuristics, rich pictures from soft systems thinking and viable systems model diagnosis. making sense of the mess was done in phases: first the mess was formulated with rich pictures generated in workshops and interviews. the discussions of the rich pictures by the respective stakeholders were transcribed and analysed using braun and clark's thematic analysis. secondly, based on the data generated from phase a diagnosis of the viability of the icu system was made. results: the data from the phases were very rich and complex and themes emerged (figure ). these themes were interdependent and resulted in disorganised icu delivery with limited opportunities for learning to improve icu delivery with dichotomies that existed at various levels of icu. it was a problem to present the complex data in the traditional linear manner due to the interdependence of the themes. the analysis is presented as stories, a known approach in the complexity discipline, where the themes of the analyses are portrayed. the making-sense-of-the-mess phase confirmed the complexity of icu delivery, at both a situational and a cognitive level and with this understanding a framework for the improvement of icu delivery could be developed. introduction: improving prescribing practice involves changing prescriber behaviour. education is assumed to change behaviour but other approaches may be more effective (figure ) [ ] . changes to the presentation of information and the configuration of choices have potential to rectify common prescribing errors through subtle 'nudges' [ ] . the implementation of clinical information systems (cis), including electronic prescribing, provides an opportunity to deploy strategies such as standard orders, dose limits, and product level prescribing. with an infinite number of configuration options available, clinical leaders need to know which interventions are most effective. we evaluated several of these strategies in a before and after observation study methods: interventions, utilising cis nudges, were chosen to improve four areas of prescribing practice in a tertiary critical care unit using methods matched to the top levels of the hierarchy. data were collected for months before and after interventions to map changes in compliance with a pre-defined standard except for the standardisation intervention where months' data were collected due to low prescription numbers. no education on changes was given during the baseline data collection so any change in performance after the go-live date is entirely attributable to the intervention. results: the change in performance for each level ranks the intervention levels in the order (highest first) forced function, automation and standardisation ( table ). the use of point of prescribing reminders was not associated with a significant difference in performance. conclusions: the effectiveness of intervention levels seen in practice is consistent with that of the model. further studies could be undertaken to strengthen these conclusions but in the meantime the approach to changing practice using cis nudges should focus on standardisation or above. introduction: intensive care unit (icu) sound pressure levels (spl) are persistently above world health organisation recommendations for clinical areas [ ] . this may impact patient recovery. standard spl monitoring records single values for each h period (laeq ). we hypothesise this reporting rate is unsuitable for icu. methods: we measured spl october -may , logging frequency (hz), spl (db), and loudness (perception of sound) every second [ ] . the resulting dataset was of a size that conventional statistics programs would require computational resources not easily obtainable on standard university commodity hardware. we processed the full dataset without sampling by using distributed task dispatching, parallelism and scheduling of a cluster computing framework (apache spark). we created a system consisting of a single workstation ( cores; gb ram) running ubuntu . lts, oracle java . , apache spark . , scala . , r core . , r studio . and sparklyr . . . we utilised the sparklyr library in r studio to run arbitrary r code using the dplyr library. we analysed aggregate data in r core & used ggplot (v ) to create visuals. results: we achieved more complex analysis than standard spl reporting with relatively modest computing resources. specifically we identified lower spl peaks in the early hours & loudness levels considerably higher than parallel spl. conclusions: simple laeq do not facilitate reflection on practice thus impetus for change is limited. loudness data highlight the patient experience of spl in the icu is more intrusive than laeq indicates due to high sensitivity to sounds~ - khz, a common frequency range for alarms. higher fidelity increases understanding of spl which can lead to targeted interventions to reduce patient disturbance. introduction: survivors of critical illness face significant long term impairments in mental and physical function. early mobilisation (em) in the intensive care unit has been suggested to improve functional outcomes and reduce delirium in the icu. we hypothesized that implementing a protocol for em in the icu would improve mobilisation rates while remaining safe. methods: design: prospective non-blinded observational cohort study, based on a quality improvement project. data was collected conclusions: only of variables in boyd criteria were significant associated with morbidity or mortality. the physiologic score and operative score were significant higher in the patient on mortality and morbidity after sicu admission. effects of structural hospital characteristics on risk-adjusted hospital mortality in patients with severe sepsisanalysis of german national administrative data d schwarzkopf introduction: the quick sequential organ failure assessment (qsofa) score is a simple tool used to identify severe patients with infection. as this score is calculated from three variables that can be measured at the scene of trauma-systolic blood pressure, respiratory rate and consciousness-the prehospital qsofa score may also be a good predictor of mortality in trauma patients. so we evaluated the discriminative ability of the prehospital qsofa score in patients with trauma for in-hospital mortality. methods: this is a retrospective multicenter study using the data from nationwide trauma registry in japan. we included patients with trauma aged ≥ years old transferred to hospitals from scene. primary outcome is in-hospital mortality. results: the mean age was . ± . years old and patients ( %) were male. in-hospital mortality occurred in patients ( %). in-hospital mortality in each qsofa score was / ( . %), / ( %), / ( %) and / ( %) in qsofa score , , and , respectively (p< . for trend). area under receiver operating characteristics curve (auroc) of the aqsofa score for inhospital mortality was . ( % confidence interval . - . ). if we use the cutoff ≥ , sensitivity and specificity of the qsofa score were . and . . conclusions: in patients with trauma, the prehospital qsofa score was strongly associated with in-hospital mortality. we can identify patients with very low risk of death by using the cutoff ≥ of the prehospital qsofa score. introduction: only one prospective study is available of the validation of the diagnostic and prognostic role of qsofa (quick sofa score) in the emergency department (ed). a prospective study was conducted in greek eds. methods: the prompt study (clinicaltrials.gov nct ) run in the ed of six hospitals in greece among patients with suspected infection and presence of at least one of fever, hypothermia, tachycardia, tachypnea and chills. clinical data were collected and the -day outcome was recorded. sepsis was defined by the sepsis- criteria. results: the sensitivity and the specificity of at least signs of qsofa for the diagnosis of sepsis was . % and . % respectively and for the prognosis of -day mortality . % and . % respectively. the odds ratio for -day mortality when qsofa was equal to or more than was . among patients with charlson's comorbidity index (cci) equal to or less than ; this was . among patients with cci more than (p: . between the two ors by the breslow-day's test; p: . by the tarone's test). conclusions: data validated the sensitivity of qsofa for the diagnosis of sepsis. cci was an independent predictor of severity. qsofa could better predict unfavorable outcome among patients with low cci. comparative accuracy between two sepsis severity scores in predicting hospital mortality among sepsis patients admitted to intensive care unit n sathaporn, b khwannimit prince of songkla university, internal medicine, hat yai, thailand critical care , (suppl ):p introduction: recently, the new york sepsis severity score (nysss) was developed to predict hospital mortality in sepsis patients. the aim of this study was to compare the accuracy of nysss with the sepsis severity score (sss) and other standard severity scores for predicting hospital mortality in sepsis patients. methods: a retrospective analysis was conducted in a medical intensive care unit of a tertiary university hospital. the performance of severity scores was evaluated by discrimination, calibration, and overall performance. the primary outcome was in-hospital mortality. results: overall , sepsis patients were enrolled, patients ( . %) were classified to septic shock by sepsis- definition. hospital mortality rate was . %. the nysss predicted hospital mortality . +/- . %, which underestimated prediction with smr . ( %ci . - . ) . however, the sss predicted hospital mortality +/- . %, which slightly overestimated mortality prediction with smr . ( %ci . - . ). the nysss had the moderate discrimination with an auc of . ( % ci . - . ), in contrast to the sss presented good discrimination with an auc of . ( %ci . - . ). the auc of sss was statistically higher than that of nysss (p< . ). nevertheless the apache iv and saps ii showed the best discrimination with auc of . . the auc of the nysss and sss was significant lower than that of apache ii, iii, iv, saps ii and saps ( figure ). the calibration of all severity scores was poor with the hosmer-lemeshow goodness-of-fit h test < . . the nysss was the lowest overall performance with brier score . . the apache iv present the best overall performance with brier scores . . conclusions: the sss indicated better discrimination and overall performance than the nysss. however the calibration of both sepsis severity scores and another severity score were poor. furthermore, specific severity score for sepsis mortality prediction needs to be modified or customized to improve the performance. introduction: metabolic markers, especially lactate, have been shown to predict mortality in acutely unwell patients. we hypothesised that early changes in metabolic markers over time would better predict mortality and length of stay, with patients who correct their metabolic derangement having lower risk of death and reduced length of stay (los). methods: single centre, retrospective cohort study in a bed icu. we included all patients who had an arterial measurement of lactate, paco , base excess (be) and ph on admission and at hours after admission to icu between / / and / / . the 'clearance' of these markers was calculated using the equation ((value at admissionvalue at hours)/value at admission). clearance calculations only included those patients with deranged results on admission (lactate> mmol/l, be<- mmol/l, ph< . , paco > . kpa). roc analysis was used to predict in-hospital mortality and length of stay, using both the initial admission values, and using the clearance value, as well as icnarc and apache ii scores for comparison. if a patient was admitted twice in the time period, only the first admission was included. results: patients were included (sex ratio . , mean age . ). table ). none of the values tested had a auc greater than . for predicting length of stay. conclusions: the clearances of metabolic markers over the initial hours after icu admission does not provide better prognostic information than the value at admission. initial lactate level was the best predictor of mortality, but compared poorly to icnarc score. metabolic markers do not accurately predict length of stay. . - . ) vs . (iiq . - . ), p= . ]. the other hemogram parameters did not differ between groups (table ) . when adjusted for severity score, in patients submitted to emergent surgery, the mpv value was still independently associated with mortality (or . ci . - . , p= . ), and its roc curve (auc) was . to mortality (figure ). conclusions: mpv is a cheap and easily accessible marker which can add prognostic value in this specific population. in the future, we will validate it in a larger cohort of cancer pts admitted to intensive care. haematological malignancy in critical care: outcomes and risk factors c denny introduction: about % of patients admitted to hospital with a haematological malignancy will become critically ill [ ] . life expectancy in these patients is poor with a month mortality of % or more in specialist units [ ] . in contrast, patients without critical illness can expect a year survival rate exceeding % for many cancers. this disparity results in differences of opinion on the best strategy for such patients among haematologists and critical care physicians. we conducted a local quality improvement project to quantify mortality and risk factors in critically ill patients with a haematological malignancy in our hospital. methods: patients admitted to the critical care unit of broomfield hospital, a district general hospital with tertiary specialist services, from january to december with haematological malignancy were included in the analysis. patients in remission for more than years and patients admitted following elective surgery were excluded from analysis. death in critical care or in hospital after critical care discharge were the primary outcomes. mortality was correlated with demographic data using simple statistical measures and regression analysis. results: patients were included in the analysis. overall mortality was %(n= ). survivors tended to be younger ( vs years) but had similar clinical frailty scores. early critical care admission (within hours) was associated with better survival ( . vs . %). nonsurvivors had a greater incidence of sepsis and respiratory failure, and required more ventilatory and vasopressor support. mortality was higher in patients requiring more than one organ support. conclusions: the overall mortality in our data is lesser than previously published data but supports the conclusion that mortality is determined primarily by the number of organs supported with the effects of malignancy playing a secondary role. (figure ). increasing levels of frailty were associated with increasing risks of death at year (p< . ) (figure ). frailty significantly increased -year mortality hazards in unadjusted analyses (hr . ; %ci; . - . ; p< . ) and covariate-adjusted analyses (hr . ; %ci . - . ; p= . ) ( table ) . conclusions: frailty was common and associated with greater age, more severe illness and female gender. frailty was significantly associated with heightened mortality risks in both unadjusted and covariateadjusted analyses. frailty scoring may encapsulate variables affecting mortality which are omitted in current predictive systems, making it a promising risk stratification and decision-making tool in icu. fig. (abstract p ) . unadjusted survival curves stratified by frailty status. frail patients were statistically significantly less likely to survive to year plateau at day = , delta peak= and hpr= . . were assigned respectively a point value of , , and to these predictors based on their beta coefficient in the predictive model. the score yielded a roc-auc: (auc= . ; %ci, [ . - . ]; p= . ). using the validation data set (n= ), the score had an roc-auc= . and similar estimated probabilities for mortality. conclusions: the paw-mps seems to demonstrate interesting discriminative properties to predict mortality. what is the role of the pulmonary embolism severity index (pesi) and rv/lv ratio as clinical risk assessment tools for patients undergoing ultrasound-assisted catheter-directed thrombolysis (uacdt)? introduction: to evaluate if the pulmonary embolism severity index (pesi) score correlates with rv/lv ratio, biomarkers of cardiac injury, fibrinogen and length of stay(los). also to evaluate the correlation between rv/lv ratio with biomarkers of cardiac injury, fibrinogen and los for patients who underwent uacdt. methods: a retrospective review of patients with sub-massive pulmonary embolism (pe) who underwent ultrasound-assisted catheterdirected thrombolysis (uacdt) was performed. pesi score, rv/lv ratio, length of stay(los), fibrinogen levels, troponin levesl, and brain natriuretic peptide(bnp) levels, were calculated and collected prior to uacdt. spearman's rank correlation coefficient was calculated for all non-parametric variables. results: patients, males and females, were included in the study. the mean (±sd) age was ± years. the mean pesi score was ± . mean rv/lv ratio was . ± . . a significant correlation between the rv/lv ratio and both fibrinogen and troponin level (p= . , p= . ) was noted. no significant correlation existed between pesi score and rv/lv (p= . ). no significant correlation existed between both rv/lv ratio and pesi score with length of stay (p= . ) after uacdt. there were no noted mortality or complications. conclusions: pesi score is used as a prognostic factor for the patients with pe, however, our study shows that pesi score does not correlate with rv/lv ratio or length of stay after the uacdt. there was inverse correlation between rv/lv ratio and fibrinogen. there was also positive correlation between rv/lv ratio and troponin for patients with and without heart failure. according to our data, there may be limited use of pesi score and rv/lv ratio for risk stratification of pe patients undergoing uacdt. introduction: conventional scores for prediction of risk and outcome, such as sapsii and sofa, have not been validated for patients admitted to level ii critical care units (intermediate level or imcus). we compared the performance of sapsii and sofa scores with the intermediate care unit severity score (imcuss) in a general population admitted to imcu. methods: we conducted a prospective observational cohort study in a -bed level ii-iii icu from a university-affiliated hospital, during a three-month period. we applied sapsii, sofa day one and imcuss to all patients admitted during that period. primary outcome was a composite of hospital mortality and need to increase level of care. additionally, we tested the relevance of each variable within each score to predict the outcome. results: we included patients with a mean age of . ± . years. patients were considered "step-down" (transferred from our level iii beds), and the remaining originated from the emergency conclusions: months after completion, the primary care management intervention had no effect on mental health-related quality of life and physical function among survivors of sepsis. increase in ptsd symptoms in the control group may suggest a possible protective effect of the intervention. introduction: critically ill patients and their families are often confronted with an overwhelming amount of clinical information shortly after hospital admission. their reliance on internet resources for additional information is increasing, particularly for unfamiliar medical terminology. yet, little is known about whether these online resources meet the recommended reading level and complexity appropriate for the average reader. methods: an online search of websites containing four common critical care diagnoses in the icu (respiratory failure, renal failure, sepsis and delirium) was performed. a total of readability formulas were used. the flesch-kincaid grade reading level (grl) and flesch reading ease (fre) were used in the final analysis. document complexity was evaluated using the pmose/ikirsch formula. results: websites on respiratory failure were written at the th grl with fre of . . renal failure resources had a th grl with fre of . . sepsis websites had an th grl with fre of . . delirium websites had a th grl with fre of . . when comparing website types (government, non-profit and private), anova showed a difference in fre across all groups and government websites had a conclusions: online resources used by intensive care unit patients and families tend to be written at higher than the recommended th grl, with government sites better meeting this target than nonprofit and private organizations. online resources should be improved to lower this unfortunate barrier to patient education. introduction: the recent enactment of the data protection act , the general data protection regulations, and a series of data breaches in the healthcare sector, have renewed interest in how our patients' information is collected, used and shared. the complex framework of laws and regulations governing the use and disclosure of personal data may lead to professional and financial consequences if information is disclosed inappropriately. disclosures to the police when they concern incapacitous patients are particularly challenging, as the disclosure may have no direct benefit to the patient and may cause the patient considerable harm. methods: we have reviewed the relevant laws and regulations to identify the circumstances in which doctors must release information regarding incapacitous patients to the police. the laws and regulations are examined to identify the extent of the disclosure required, and any requirements for the disclosure to be lawful. we have also identified laws which confer a power to disclose information about incapacitous patients, and the circumstances in which these powers can be used. results: in conjunction with a local police constabulary we have developed an information request form which makes it easier for those requesting and disclosing information to understand the legal basis of the disclosure. we have also developed guidelines to allow practitioners to understand where a disclosure is obligatory or discretionary. conclusions: the next stage of the project is to audit disclosures of information in the intensive care unit, and identify whether information is being released lawfully and following the correct procedure. introduction: family members are affected both physically and psychologically when their relative is admitted to icu. there is limited knowledge describing their experiences and structured interventions that might support them during their relative's critical illness. the aim of this review is to describe published literature on the needs and experiences of relatives of adult critically ill patients and interventions to improve family satisfaction and psychological well-being. methods: design: scoping review. standardised processes of study identification, data extraction on study design, sample size, sample characteristics and outcomes measured (figure ) . results: from references, studies were identified for inclusion four key themes were identified: ) different perspectives on meeting family needs ) family satisfaction with icu care ) factors impacting on family health and well-being and capacity to cope ) psychosocial interventions conclusions: family members of patients in icu experience unmet information and assurance needs which impacts on their physical and mental health. structured written as well as oral information show some effect in improving satisfaction and reducing psychological burden. icu's who are able to support interventions based on meeting family information needs, in addition to reducing psychological burden and increasing satisfaction will enable each family to provide more support to their relative within the icu. introduction: unmet informational needs lead to dissatisfaction with care and psychological distress. identifying interventions to help meet specific needs is a crucial and necessary step in providing family centred care in icu. we aimed to implement and evaluate the impact of delivering a structured communication strategy on levels of anxiety, uncertainty and satisfaction with care and decision making in families of critically ill adults. methods: a quasi experimental study with pre and post test design. a convenience sample of family members were recruited from july to february . the intervention group (n= ) received both oral and printed information to guide them in preparing for a structured family meeting. the control group (n= ) received usual fig. (abstract p ) . article selection process for scoping review routine care and existing family informational support. anxiety, uncertainty and family satisfaction were measured in the two groups on icu admission and icu discharge. results: mean anxiety, uncertainty and satisfaction with care and decision making scores pre and post intervention were compared. there were no significant differences in mean anxiety, uncertainty or satisfaction scores between the two groups before the intervention (p> . ). mean scores on anxiety ( . vs . ), and uncertainty ( . vs . ) were lower post intervention, but not significantly so ( figure & ). total satisfaction, satisfaction with care and satisfaction with decision making mean scores were similar in both groups before and after the intervention (p. . ). conclusions: providing relatives with a combination of targeted written and oral information delivered by nursing and medical staff reduced anxiety and uncertainty with this reduction being evident through to discharge from icu. although not statistically significant, there was what may be seen as a suggestion of a clinically significant drop in anxiety and uncertainty following the intervention introduction: clinical studies in intensive care unit (icu) patients are warranted in order to improve healthcare. the aim of this study was to analyse barriers and challenges in the process of achieving informed consent from icu patients. methods: we analysed patients considered for inclusion in a prospective observational study of venous thromboembolism in the icu, i.e. the norwegian intensive care unit dalteparin effect (norides) study. data were collected from the screening log, consent forms and associated research notes of the norides study. results: we observed that of ( %) eligible patients according to inclusion and exclusion criteria were omitted from the nor-ides study due to barriers and challenges in the process of receiving informed consent. were categorized as psychiatric diseases consisting of known psychosis or recent suicide attempt, likely or actual treatment withdrawals and due to language barriers among non-norwegians. among the patients included in the norides study, ( %) consents were from patients and ( %) obtained from their next of kind. from the patient consents, ( %) consents were oral and ( %) were written. patients were physically unable to sign, and patients did not recognize their own signature. the study further pointed at some specific challenges in the process of consent, herein questionable competence to give consent, failure to remember being asked/included, inability to separate research from treatment etc. there were also difficulties in evaluating who was next of kin and how to reach them. conclusions: barriers and challenges in obtaining informed consent from icu patients led to exclusion of one fifth of the eligible patients in our study. informed consent directly from patients was obtained from less than half of the included patients. obstacles in the process of achieving informed consent were practical, medical, ethical and/or legal. determinants of end-of-life decision-making in the intensive care unit p eiben, c brathwaite-shirley, s canestrini king´s college hospital nhs foundation trust, london, united kingdom critical care , (suppl ):p introduction: although the majority of intensive care unit (icu) deaths follow the decision to forgo life sustaining treatment (lst), variability in patterns is commonly observed [ , ] . we reviewed end of life (eol) practice at our institution in order to explore: (i) patient characteristics affecting eol decision-making, (ii) communication among surrogate decision-makers, and (iii) eol management. methods: we retrospectively analyzed data from consecutive patients who died in our ten-bed icu over months (study period). patient demographics, apache ii, functional status, diagnosis on admission, icu length of stay (los) were collected; family/next-of-kin (nok) involvement and rationale for lst limitation were recorded ( conclusions: our analysis shows that in our institution eol deliberations follow a shared decision-making process. lack of family/nok involvement and incomplete documentation was exceptional. the significant difference in los between w-group and nw-group, in the face of similar apache ii, warrants further investigation. vae calculator rheumatology review . van der jagt m. crit care consensus on circulatory shock and hemodynamic monitoring. task force of the european society of intensive care medicine cardiac output monitoring: how to choose the optimal method for the individual patient perioperative cardiovascular monitoring of highrisk patients: a consensus of guidelines for nutrition support therapy in the adult critically ill patient references . nice guideline for aki: prevention, detection and management serial creatinine results pre-and post ecmo references . polit et al. research in nursing & health reference . sherliker et al national blood transfusion committee, nhs blood and transplant arch otolaryngol head neck surg fig. (abstract p ). rsi agent guideline references . nuckton tj nejm icm baseline characteristics reference elso guidelines for cardiopulmonary extracorporeal life support s -leitlinie invasive beatmung und einsatz extrakorporaler verfahren bei akuter respiratorischer insuffizienz .auflage p handgrip strength does not predict spontaneous breathing trial failure or difficult or prolonged weaning of critically ill patients g friedman total burn care introduction: we aimed to evaluate safety and efficacy of light sedation with dexmedetomidine (dex-ls) in acute brain injury (abi) patients. methods: retrospective analysis on icu patients with traumatic/medical abi, out of the neuroprotection window and undergoing dex-ls. data of pre-infusion and infusion periods were compared. results: patients (age ± , males . %) were included. traurespectively. conclusions: dex-ls among icu patients affected by abi turned out to be feasible and safe. it enabled discontinuation from mv and maintenance of spontaneous breathing in the majority of cases %) delirious patients and of ( . %) non-delirious patients could be discharged from the hospital. we evaluated the -year mortality in the hospital survivors. results: totally, patients participated in our study. the majority of them ( . %) were male with the median age of [ , . ] years and the median apache ii score on the first day of icu admission of risk of delirium was associated with preoperatory euroscore ii (p= . ) and history of previous cardiac surgery (p= . ). moreover, in the intraoperatory period the risk of delirium was associated with red blood cell transfusion, intervention for aortic dissection (p= . ), hypothermic circulatory arrest (hca) with anterograde cerebral perfusion (acp) (p= . ) (table ). in the postoperatory period risk of delirium was associated with levels of creatinine clearance (p= . ) and c-reactive protein (crp) (p= . ). conclusions: delirium is relatively frequent in the cardiac surgical icu patient journey of group : those patients discharged directly home from critical care unit poor compliance with co-signing in icca ( %, n= ) compared to paper ( %, n= ) (figure ) and the reported difficulty in co-signing ( %, n= ) reveals significant usability concerns and potential safety issues. % (n= ) found icca intuitive, though % (n= ) found navigating the interface difficult and reported concerns with losing saved work ( %, n= ). conclusions: this study highlights important usability issues that may impact staff satisfaction th national audit project of the royal college of anaesthetists and the difficult airway society. major complications of airway management references . guidelines for provision for intensive care services (gpics), version medicines optimisation: the safe and effective use of medicines reducing harm from omitted and delayed medicines. a tool to support local implementation p understanding the delivery of intensive care in south p mobilising ventilated patients early with interdisciplinary teams (move it) singapore general hospital, department of respiratory and critical care p validation of boyd criteria and possum-score on mortality and morbidity in general surgical intensive care unit k chittawatanarat, y chatsrisuwan faculty of medicine pts with central nervous system neoplasms or submitted to elective surgeries were excluded. descriptive analysis and χ test, pearson´s, wilcoxon rank-sum, uni and multivariate logistic regressions were used when appropriate. results: from a total of pts identified, . % (n= ) were admitted after emergent surgery and . % (n= ) for medical reasons. global icu mortality was . % (n= ). in comparison to survivors, the patients that died had a similar age were recorded data regarding demographics, clinical variables, paw (at admission and at day ), high pressure ratio (hpr = number of days with high pressures: peak ≥ and/or plateau ≥ ; and/or driving pressure ≥ ; and/or auto-peep ≥ ; divided by los), trends of paw (paw at day -paw at admission) and outcomes. the patients were divided into two groups: a construction group (n= ) and a validation group(n= ). the paw-mps was developed and validated by analyzing in a multivariate regression model the different paw ± . ; pco , ± mmhg paw were respectively for peak, plateau, driving, and auto-peep at admission: ± , . ± , . ± and three independent mortality risk factors were identified centro hospitalar do porto p five-year mortality and morbidity impact of prolonged icu stay n van aerde , g hermans laboratory of cellular and molecular medicine we investigated differences in mortality and morbidity after short (< days) and prolonged (≥ days) icu-stay. methods: prospective, -year follow-up study of former epanicpatients (clinicaltrials.gov:nct , n= ). mortality was assessed in all. for morbidity analyses, all long-stay and a random sample ( %) of short-stay survivors were contacted. primary outcomes were total and post- -day -year mortality in multivariable cox regression analysis, icu-risk factors comprised hypoglycaemia, corticosteroids, nmba, benzodiazepines, mechanical ventilation, new dialysis, new infection, liver dysfunction, whereas clonidine may be protective. among long-and short-stay -year survivors hgf, mwd and pf sf- were lower in long-stayers mwd: % ( %ci: %- %) vs % ( %ci: %- %) multivariable regression identified associations with benzodiazepines (hgf and pf-sf ), vasopressors (pf-sf ) and opioids ( mwd) ptsd related symptoms were accessed with the post traumatic stress syndrome questions inventory (ptss- ) at the post icu follow up clinic, six months after the acute stress event. the post icu consultation was carry out by an icu doctor and an icu nurse. exclusion criteria: previous severe psiquiatric disorders, not able to respond the questionnaire medical %, surgical % and trauma %. patients ( %) were on imv and the median ventilation days was . ptsd scores ranged from to . delusional memories were conclusions: in this study the rate of ptsd was lower . % and related with a lower saps ii and the presence of memories of the icu stay. no relation was found with delusional memories, imv or superior icu length of stay. patients with lower illness severity and without imv, should be elective to the follow up-clinics. p long-term effects of a sepsis aftercare intervention k schmidt united states; jena university hospital patras general university hospital, intensive care unit, patras, greece; patras general university hospital, division of infectious diseases results: ( . %) patients were readmitted within hours and ( . %) in to days. the two groups didn't differ in age, gender, charlson comorbidity index and length of stay on both admissions. elective surgery was the most common type of admission ( . %) followed by medical ( . %), emergency surgery ( %) and trauma ( . %). the mean time to readmission in the late group was . (± . ) days. patients in the late group had higher apache ii score on their first and second admission, ( . ± . vs . ± . ; p= . ) and ( . ± . vs . ± . ; p= . ) respectively. respiratory insufficiency was the most common cause of readmission in both groups followed by sepsis and cardiac arrest. finally in the early group p introduction: in intensive care units, perceived inappropriate treatments (pit) have been associated with negative impact on caregivers univariate analysis revealed that burn-out, pit and intention to leave were greater in units where nurses´teams included no activity in the icu, compared to "shared" work in icu and idtcu. in multivariate analysis, perception of non beneficial treatment of patients with life support witholding was associated with: bad collaboration with other units p profile of intensive care unit (icu) patients on whom life-sustaining medical treatment were withdrawn or withheld s chatterjee variables collected-age, sex, apa-che iv score, diagnostic-category and co-morbidities. primary outcomes were icu and hospital mortality. secondary outcomes included icu and hospital length of stay(los) female sex, n (%) ( . %) diagnosis on admission: medical, n (%) rrt at time of wlst, n (%) ( . %) dnr order, n (%) ( . %) organ donation services involved, n (%) ( . %) introduction: high flow nasal cannula(hfnc) is a new modality in respiratory failure management [ ] . this study objectively held to compare the physiological outcomes in the non-invasive ventilation(niv) treatment of cardiogenic acute pulmonary oedema(apo) patient in the emergency department(ed) delivered by helmet cpap(hcpap) and hfnc. methods: single-centre randomized controlled trial on patients presenting with cardiogenic apo. primary endpoint was a heart rate reduction.secondary endpoints included: improvement in subjective dyspnoea scales, respiratory rate, blood oxygenation, intubation rate and days mortality rate. results: patients were enrolled and randomized ( patients to hcpap; to hfnc) ( to . ± . ). intubation rate was lower in hcpap ( . % for hcpap versus . % for hfnc) and days mortality rate is lower in hcpap ( . % for hcpap versus . % for hfnc). conclusions: both hcpap and hfnc significantly improved patient condition in patient presenting to the ed with cardiogenic apo. however, hcpap was better than hfnc in improving physiology outcomes, lower intubation rate and mortality rate in patient introduction: the aim of the study was to compare the confusing assessment method of the intensive care unit (cam-icu) and the nursing delirium scoring scale (nu-desc) for assessment of delirium in the icu. furthermore we wanted to test the interpersonal variation of the nu-desc. delirium is proved to be associated with increased mortality [ ] . nu-desc is an observational five-item scale that does not require patient participation and is adapted to the fluctuating nature of delirium. each item can be scored from to . delirium is defined with a score > . the nu-desc has recently been translated into danish (nu-desc dk) but has not been validated.methods: icu patients, who met the inclusion-criteria for the cam-icu were scored with both cam-icu and nu-desc dk. patients were scored of two independent nurses at approximately the same time every day.results: a total of patients were enrolled, and comparisons between cam-icu and nu-desc dk were registered ( figure ).there was agreement between nu-desc and cam-icu in of registrations (hereof registrations were delirium negative). in interpersonal variation, registrations were made. the conclusion was identical in % of registrations, but only % agreed in all scoring-scale items (all negative).conclusions: a high agreement between nu-desc and cam-icu was found however the comparison was based on predominately patients with negative delirium score. the interpersonal variation of nu-desc scoring was substantial. a future validation of the nu-desc dk as a screening tool in the icu requires thorough training and instructions to minimize interpersonal variation. introduction: an increasing number of patients are being discharged directly home from critical care units and this is currently viewed as a negative quality indicator [ ] . the purpose of this audit was to characterise a cohort of patients who can be safely discharged directly home from adult critical care at st thomas´hospital (sth). methods: retrospective observational study of two groups of patients; ) those discharged directly home from critical care, ) those discharged within two days of step down to a ward from critical care (admissions st june- st october ). the clinical notes of these patients were reviewed via online systems. results: baseline demographics of the patients in group and patients in group were similar (mean age of years, versus years, p= . ); average length of stay in critical care was also similar ( . days versus . days respectively p= . ). in group , of icu days were after considered fit for step down versus of days in group , p= . (fig , ) . in group , drug related presentations were more common ( % versus % p= . ), fewer patients had specialist follow up post discharge ( % versus %, p< . ). in group , patients ( %) were readmitted within days, to critical care. in group , patients ( %) were readmitted, to critical care (p= . and . respectively); none of these readmissions were felt to have been preventable.conclusions: there is a cohort of patients suitable for discharge directly home from critical care who did not spend significantly longer in icu awaiting discharge than those who were stepped down to the ward. identifying these patients early, potentially by their diagnosis, and creating a pathway including access to specialist follow up clinic could allow prompt discharge directly from critical care, thus improving patient satisfaction and reducing hospital-acquired morbidity healthcare costs [ ] . the evaluation of the usability of a critical care information system ( introduction: critical care information systems (ccis) support clinical processes by storing and managing data, but poor usability can lead to staff dissatisfaction and increased workload, promoting workarounds that may compromise patient safety [ ] . the purpose of the study was to evaluate the usability of a philips intellispace critical care and anaesthesia (icca) ccis, recently implemented in beds across three critical care units of a large uk teaching hospital. methods: a prospective, mixed method observational study conducted in may , comprising of ( ) an audit assessing the ease of linking bedside devices to icca, ( ) an audit assessing the usability of co-signing medications in icca compared with a non-icca paper factors that commonly drive workforce metrics may not correlate with staff perceptions of safety. the bpr is a pragmatic, staff driven, tool to augment other measures of safety and is applicable to various icu settings. further research is needed to explore staff perceptions in order to understand the importance of this organisationally, and for staff stress. ventilator-free duration in icu, central venous catheter duration, urinary catheter duration, rates of deep vein thrombosis (dvt) and stress ulcer prophylaxis, rates of de-escalation antibiotic therapy, dvt prophylaxis duration, stress ulcer prophylaxis duration, icu and hospital mortality, -day mortality, rate of central venous catheter infection, length of stay in icu and hospital between two groups were analyzed. results: rate and duration of dvt prophylaxis in the intervention group were . % and ( , ) days respectively, in the control group were . % and ( , ) days, the differences between two groups were statistically significant(p< . ) ( table ). there were no differences in ventilator-free duration in icu, central venous catheter duration, urinary catheter duration, rate of stress ulcer prophylaxis, rates of de-escalation antibiotic therapy, stress ulcer prophylaxis duration, icu and hospital mortality, -day mortality, rate of central venous catheter(cvc) infection, length of stay in icu and hospital between two groups ( table ) . conclusions: electronic checklist in ward rounds can increase the rate of dvt prophylaxis and reduce the duration, but it cannot improve the prognosis of critically ill patients. introduction: the goal of the project "i see you" is family-centeredcare based on family meetings that improve the experience of the patient´s family members during hospitalization in the icu. the meetings focus on relaying information, raising knowledge and addressing the social and emotional needs of families. providing support along with information was found to be the strongest predictor of family satisfaction and could lead to improve cooperation between family and staff [ ] .methods: meetings and questionnaire: family meetings consist of a multidisciplinary team, a group facilitator and combined with a multimedia presentation about the unit and equipment. in addition, they focus on social and emotional needs: managing daily routine, sharing problems, fears and anxieties and more. at the end of the session a questionnaire was given to assess the impact of the intervention. sharing data: at the end of the first quarter, the data from meeting was summarized and sent to the staff alongside tools for effective communication.results: the project began in february . to date, family members of patients have attended the sessions. the topics discussed by the participants include: contact with the patient, prevention of infections, procedures, visits, conversations with doctors, medical confidentiality; guardianship; tracheotomy and social issues (fig ) . a sample of questionnaires was transferred to participants report satisfaction at a very high level.conclusions: the meeting received a very positive feedback from the participants. the project has achieved its goals and therefore it has been decided to be continued.introduction: possum score and boyd criteria are used to predict the outcome for high risk surgical patients. the aim of this study was to validation of these two measurement tools on mortality and morbidity in a university-based surgical intensive care unit (sicu) in thailand.methods: nine hundred and fifty two patients were enrolled onto this prospective review. all patients who had been admitted to sicu in a university-based hospital were included. all patients were collected for boyd criteria and possum score and outcomes and morbidity during sicu admission and discharge. introduction: aromatic microbial metabolites (amm), such as phenyllactic (phla), p-hydroxyphenylacetic (p-hphaa), and phydroxyphenyllactic (p-hphla) are involved in the pathogenesis of septic shock and are associated with mortality [ ] . according to previous studies, amm have a high prognostic value in patients with abdominal infection [ , ] . we hypothesize that amm have the prognostic value in patients with pneumonia in icu. methods: data of patients with community-acquired pneumonia was obtained on admission to icu. the levels of amm (phla, p-hphla and p-hphaa) were measured in blood serum using gas chromatography with flame ionization detector and compared in groups of patients: with favorable and with lethal outcome (mann-whitney utest). spearman's correlations between amm and clinical and laboratory data were calculated. using method of logistic regression and roc analysis, we measured the prognostic value of amm. (table ) . it was revealed, that some amm have similar prognostic characteristics in comparison with sofa and curb- scales; high level of amm is associated with high risk of death (roc-analysis - fig. ) .conclusions: serum concentrations of amm can be used as independent and practical criteria for the assessing of prognosis in patients with infection in icu. introduction: frailty in the critically ill is associated with increased morbidity and mortality but the optimal timing of frailty assessment, how to best measure frailty, reasons for adverse outcomes and how critical illness impacts frailty are unknown [ ] . in preparation for a multi-center study designed to address these knowledge gaps, we conducted a pilot study whose aim was to assess feasibility as determined by recruitment rates, ability to assess frailty at icu admission and hospital discharge, ability to measure icu and hospital processes of care and ability to conduct -month assessments. conclusions: a multi-center study is feasible but follow-up losses due to mortality and inability to return for assessment will require sample size adjustment. frailty characterization is method dependent, can be done on hospital discharge but varies with time of assessment. these findings will need to be confirmed in our larger study currently in progress. introduction: given the ageing of the world´s population, the demands of critical care resources for elderly patients has increased during the past decade. however, little is known about quality of life and outcomes of elderly icu survivors. the aim of the study is to assess outcomes of elderly icu survivors at least months after discharge: quality of life and mortality. methods: it is a retrospective study performed in a medical adult icu between january to december . the study included all elderly survivors ( ≥ years) after icu admission. outcomes were assessed by telephone interviews at least months after icu discharge. the primary outcome was assessing the quality of life after icu stay, measured by euro qol d questionnaire. the eq- d descriptive system contains five dimensions (mobility, self-care, usual activities, pain and discomfort, and anxiety and depression). for each dimension, there are five levels (no problems, slight problems, moderate problems, severe problems and unable to/extreme problems figure . conclusions: most elderly survivors patients showed a good health related quality of life using the euroqol d- l after icu discharge. fig. (abstract p ) . quality of life (euroqol d) scores after icu discharge introduction: sepsis survivors face mental and physical sequelae even years after discharge from the intensive care unit (icu). effects of a primary care management intervention in sepsis aftercare were tested. exploratory analyses suggest better functional outcomes within the intervention group compared to the control group at six and months after icu discharge. longer term effects of the intervention have not been reported. methods: a randomized controlled trial was conducted, enrolling patients who survived sepsis (including septic shock), recruited from nine german icus. participants were randomized to usual care (n= ) or to a -months intervention (n= ). the intervention included training of patients and their primary care physicians (pcp) in evidence-based post-sepsis care, case management provided by trained nurses and clinical decision support for pcps by consulting physicians. usual care was provided by pcps in the control group. the primary outcome of the trial was the change in mental healthrelated quality at -months after icu discharge. secondary outcomes included measures of mental and physical health. data were collected by telephone interviews using validated questionnaires at the -months follow-up ( months after the -year intervention).results: [ . %, intervention, control] of patients completed the -months follow-up. unlike the intervention group, the control group showed a significant increase of posttraumatic symptoms (diff. ptss- to baseline, mean (sd) . ( . ) control vs.- . ( . ) intervention; p= . ). there were no significant differences in the mcs and all other secondary outcomes between intervention and control group.introduction: survivors of sepsis often show symptoms of posttraumatic stress disorder (ptsd). only few studies report on courses of more than month after discharge from the icu. the aim of this study was to identify predictors for changes in ptsd symptoms over time up to month. methods: follow-up data of the smooth triala rct to evaluate a primary care management intervention on sepsis survivorswere analyzed. included patients were surveyed by phone for ptsdsymptoms at one, , and months after discharge from icu using the post-traumatic-stress-scale (ptss- ). scores changes between follow-up periods were analyzed using latent-change scores in structural equation models. predictors were clinical and sociodemographic baseline characteristics as well as physical, cognitive and functional sepsis sequelae assessed by validated questionnaires.results: patients were included of which participated in the month follow-up. a decrease of ptsd symptoms between and months was predicted by higher education (b=- . , p= . ), while higher pain intensity at one month predicted an increase (b= . , p= . ). increasing ptsd symptoms between and months were predicted by reporting more than two traumatic memories at one month (b= . , p= . ), more sleep problems (b= . , p= . ) and worse cognitive performance at months (b=- . , p= . ) as well as more neuropathic symptoms at months (b= . , p= . ).conclusions: sepsis patients that suffer from physical, cognitive and functional impairments after icu discharge may be at increased risk for developing late-onset ptsd. these predictors need to be replicated by future studies. early versus late readmission to the intensive care unit: a ten-year retrospective study v karamouzos , n ntoulias , d aretha , a solomou , c sklavou , d logothetis , t vrettos , m papadimitriou-olivgieris , d velissaris , f fligou conclusions: icu patients whose life-sustaining treatment was withdrawn or withheld had higher illness-severity scores, were older, had longer icu los and higher mortality than those in active-treatment group. healthcare introduction: caring for the critically ill patient is a complex task and becomes tougher when a death process takes place. a number of needs and coping strategies emerge from the healthcare providers before these issues but are mostly displayed out of individual skills and intuition. if those approaches are unappropriate and the needs are not met, patients' death process may be burdensome for caregivers. this could affect the quality of care for patients and families during the whole end-of-life care process. the aim of our study was to explore the different needs and coping strategies used by icu healthcare providers when facing patients in the dying process. methods: qualitative and collective case study. ten semi-structured interviews were conducted in icu personnel ( physicians and nursing professionals). a thematic analysis was done using nvivo software. local ethics committee approved the study. results: respondents were % women, had . ± . years-old and . ± . years of icu experience. main needs identified in icu healthcare providers refer to a lack of tools for doing emotional containment when delivering bad news to families, handling personal mourning, the need to perceive consistency regarding end-of-life care management across the icu team, and a wish of having regular training from a psychologist. main identified coping strategies included closing rituals, finding quiet spaces to spend time, and asking for counselling with more expert colleagues. a need for systematic, although basic training on these issues from qualified professionals is demanded. conclusions: usually, basic needs from patients and families in the process of dying are well addressed, but healthcare providers' needs are underrecognized and coping strategies mostly unknown. visibilization of those needs and basic but formal training in emotional containment, self-care and coping strategies are greatly desired. introduction: in the intensive care unit (icu), patients often exhibit cognitive impairments that prevent them from participating in decisions related to therapeutic options at the end of life. consequently, their families are often asked to speak for them when difficult decisions must be made. the main of this study was to determine the frequence in wich family want to share in end of life decisions and factors associated with this desire.methods: a prospective study was conducted in one mixed icu in montevideo. relatives of patients were invited to participate in this study after hours in the icu and completed a survey that included the hospital anxiety and depression scale. results: we analized relatives from patients hospitalized in the intensive care unit. the relationship with the patient was as follows: % spouses, % siblings, % grown children, % parents, and % other family members and friends. of them, . % reported a desire to share in end of life decisions. anxiety and depression symtoms were present in % and % respectively. factors asociated with the desire of involvment in end of life decisions by bivariate analysis were: female sex ( % vs %, p= . ), presence of anxiety ( % vs %, p= . ) and patient ecog - ( % vs %, p= . ). multivariate analysis shows that the presence of anxiety is the only independent factor associated with the desire to participate in end of life decisions (or . , ic % . - . ; p= . ). conclusions: have a loved one in icu is often associated with anxiety and depression after hours of admission. only % of the relatives want to participate in end of life decisions. the presence of anxiety is independently associated with the want to share in decisions making process. introduction: intensive care aims to treat failure of vital organ systems. sometimes, a patient's condition is of such a degree that intensive care is no longer beneficial, and decisions to withdraw or withhold intensive care are made. this means that life-sustaining treatments are terminated or not initiated. we aimed to identify variables that are independent factors for the decision to withdraw or withhold intensive care. methods: registry study using extracted data from a national quality registry the swedish intensive care registry (sir) - . data are delivered to the registry by nurses and doctors daily, during each patients' stay in the intensive care unit (icu). a total of , intensive care cases reported to the sir from - . results: data regarding each patient´s age, sex, diagnoses, condition at admission (expressed as simplified acute physiology score version , saps ), comorbidities and registered decisions to withdraw or withhold intensive care were analyzed. of the , cases reported, . % were women and . % men, and . % were - years old. a total of . % received a decision to withdraw or withhold intensive care, accounting for . % of all women and . % of all men, p< . . independent variables associated with increased odds of receiving a decision to withdraw or withhold intensive care were older age, worse condition at admission, and female sex. female sex was associated with an increased odds of receiving a decision to withdraw or withhold intensive care by % (ci . - . %) after adjustments for condition at admission and age. conclusions: older age, worse condition at admission and female sex was found to be independent variables associated with an increased odds to receive a decision to withdraw or withhold intensive care.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - wmrjlhy authors: wicker, l. v.; canfield, p. j.; higgins, d. p. title: potential pathogens reported in species of the family viverridae and their implications for human and animal health date: - - journal: zoonoses public health doi: . /zph. sha: doc_id: cord_uid: wmrjlhy the viverridae is a family of nocturnal carnivores including civets, genets and african linsangs. while a list of known organisms isolated from a species is an essential tool for population management, this review represents the first attempt to collate published reports of organisms isolated from viverrids. a wide range of organisms, including viruses, eight bacterial species, one internal arthropod species, representatives from eight genera of protozoan, genera of nematode, seven genera of cestode, eight genera of trematode and six genera of external arthropod (mites, ticks and louse), have been reported in literature spanning over a century of research. many of these are capable of infecting multiple hosts, including humans. this is of concern given the anthropogenic factors that bring humans and domestic species into close contact with viverrids, facilitating transmission and spillover of organisms between groups. these factors include trade in viverrids for human consumption, captive management in zoos, rescue centres or on commercial breeding farms, and the increasing overlap of free‐ranging viverrid distribution and human settlement. the family viverridae is a diverse group of small, nocturnal carnivores, including species of civet, genet and african linsang distributed across southern europe, africa and asia (wilson and reeder, ) . viverrids are commonly exploited for human consumption, being farmed and hunted for their fur, meat, 'civet' scent, to produce civet coffee, or to be kept as zoo animals or pets (schreiber et al., ; balakrishnan and sreedevi, ; shepherd and shepherd, ; d'cruze et al., ) . while habitat loss has contributed to historical population declines, hunting for human consumption is considered the most significant threat to their global conservation (schreiber et al., ; shepherd and shepherd, ). an inventory of organisms known to be associated with a particular species is considered essential baseline information for species management (munson, ) . this information guides rational development of husbandry protocols for captive populations (hope and deem, ) , facilitates design of surveillance programmes to investigate change in species disease profiles over time (lonsdorf et al., ) and highlights the threatening role infectious diseases might play in endangered species management (gilbert et al., ) . given the potential for transmission of diseases between wildlife, humans and livestock (jones et al., ) , a complete list of pathogens and indigenous microflora also enables evidence-based risk assessment of human and animal contact with the species in question . no attempt has yet been made to collate the current knowledge of potential pathogens detected from species within the family viverridae (shepherd, ; bongiovanni et al., ) despite the family's long history of exploitation for human consumption (abebe, ; shepherd and shepherd, ) , the management of threatened viverrids for conservation (roberton et al., ) , the peri-urban habitation of many species within the family (king et al., ; ninomiya et al., ; sato et al., ) and their susceptibility to a number of important zoonotic pathogens including the novel coronavirus responsible for the outbreak of severe acute respiratory syndrome (sars) (guan et al., ; tu, ) , rabies virus (matsumoto et al., ) and highly pathogenic avian influenza h n (hpai h n ) (roberton et al., ) . while many organisms have been identified in a very wide range of viverrid species globally, this information is scattered throughout the scientific and grey literature. an accessible list of organisms isolated from viverrids does not currently exist. this review represents the first collation of currently recognized organisms isolated from the viverridae, derived from an extensive and intensive assessment of the literature. in doing so, it expands on our understanding of viverrid health and the key epidemiological factors of infectious disease involving free living and captive wildlife. more importantly, this review assesses the significance of isolation of organisms from viverrids in terms of the implications for public health. anthropogenic factors including wildlife trade, the keeping of viverrids in captivity and land use change all encourage increased contact between viverrids and other species, thereby facilitating the transmission and spillover of these organisms between humans, viverrids and other animals. a systematic review of the scientific literature resulted in this first collation of all organisms previously reported for species from the carnivorous family viverridae. all available fields of the databases google scholar, web of science, pubmed and scopus were searched using all possible combinations of the following key terms: 'viverrid*' or 'civet' or 'linsang' or 'binturong' or 'genetta' (the scientific name for the genus genetta was used to reduce the number of papers on 'genetics' returned by the search), and 'disease' or 'pathogen' or 'vir*' (to locate both virus and viral) or 'bacteria*' (to locate both bacteria and bacterial) or 'parasit*' (to locate parasite or parasitic) or 'fung*' (to locate both fungus and fungal). no limitations were placed on date of publication. additional papers were identified by scanning the reference lists and 'cited by' lists of selected papers. for all organisms, complete primary references describing original research were sought. while this review resulted in important baseline information, a number of limitations are acknowledged. it is undoubtedly incomplete as many more reports are likely to exist in the medical records of zoos and rescue centres that hold viverrids, unavailable to a database literature review. inaccuracies are also likely included due to the reliance on traditional approaches to identification, such as descriptions of morphology (nadler and de leon, ) . increased availability of molecular methods has significantly improved accuracy in the detection and identification of organisms (bhadury et al., ; thompson et al., ) , resulting in a revision of numerous taxonomic trees (brown-elliott et al., ; balajee et al., ) . future research may find some organisms listed here no longer considered valid species, and advancing molecular techniques mean that future revision of this list of recognized organisms of viverridae are inevitable. a total of peer-reviewed publications reported the identification of organisms from species within the family viverridae. these included viruses, eight bacterial species, a single internal arthropod species and representatives from genera of nematode, seven genera of cestode, eight genera of trematode, six genera of external arthropod, and eight genera of protozoan, many of which were not identified to species level. these organisms are presented in detail in tables - . of the ten viruses reported for viverridae (table ) , three were zoonoses of considerable public health significance. these were the sars coronavirus (sars cov) (guan et al., ; poon et al., ) ; hpai h n (roberton et al., ; wu et al., ) and a number of variants of the rabies virus (enurah et al., a; wilde et al., ; tremlett et al., ; nel et al., ; susetya et al., ; pfukenyi et al., ; matsumoto et al., ) . three important viruses of domestic carnivores were also reported, including canine distemper virus (cdv) (machida et al., ; hur et al., ; chandra et al., ; l opez-peña et al., ; hirama et al., ; chen et al., ; takayama et al., ) , feline parvovirus (caused by the feline panleukopenia virus, fpv) (ikeda et al., ; demeter et al., ) and canine parvovirus (cp) (santos et al., ; xiao-ying et al., ; duarte et al., ) . in more recent years, the use of advanced molecular techniques has enabled the identification of five novel viruses of uncertain significance. these were an orthoreovirus (mammalian reovirus mpc/ ) isolated (guan et al., ; tu, ; kan et al., ; wu et al., ; wang and eaton, ) family (enurah et al., a; wilde et al., ; tremlett et al., ; nel et al., ; susetya et al., b; pfukenyi et al., ; matsumoto et al., ) ikoma lyssavirus (machida et al., ; hur et al., ; chandra et al., ; l opez-peña et al., ; hirama et al., ; chen et al., ; takayama et al., ; techangamsuwan et al., ) family from masked palm civet (paguma larvata) in china (shao et al., (shao et al., , ; the ikoma lyssavirus, a novel divergent lyssavirus (marston et al., ) ; two picornaviruses (genet faecal theilovirus (gftv) and a kobuvirus genotype); and a picobirnavirus (genet faecal picobirnavirus) (bodewes et al., ; ) . reports of eight bacterial species, all of which are multihost organisms with zoonotic potential, were found (table ). nine serovars of leptospira interrogans, including some known to cause leptospirosis in humans (mill an et al., ) , were cultured (smith et al., ; tsai et al., ) or identified on serology (mill an et al., ; moinet et al., ) , and the gram-negative bacteria bartonella henselae, responsible for cat scratch fever in humans, was isolated from a wild, peri-urban-dwelling masked palm civet (sato et al., ) . a number of reports also describe isolation of enteric bacteria from viverrids. these include salmonella enterica serovar glostrup (falade and durojaiye, ) ; klebsiella pneumoniae (enurah et al., b) ; and plesiomonas shigelloides (bardon, ) , an escherichia coli with a broad antibiotic resistance panel from a captive masked palm civet (ahmed et al., ) , and salmonella enterica enterica (serovars enteritidis, nagoya and , :i), yersinia pseudotuberculosis and campylobacter spp. from peri-urban, free-ranging masked palm civets (lee et al., ) . of the vectorborne protozoans recognized for viverrids (table ) , those carried by ticks include hepatozoon canis (laird, ) and six babesia spp. (l eger and l eger, ; heisch, ; peenen et al., ; tolosa and regassa, ) . the trypanosomes, trypanosoma brucei and t. congolense (njiokou et al., ; tolosa and regassa, ) , and leishmania donovani, the protozoan responsible for visceral leishmaniasis in humans, were reported (hoogstraal and heyneman, ) . species from four genera of non-vector-borne protozoan were reported (table ). these included the zoonotic intracellular protozoan toxoplasma gondii (janitschke and werner, ; lopes et al., ) ; the coccidian isospora spp. (colon and patton, ; su et al., ) ; eimeria spp. (colon and patton, ) ; and three species of giardia including giardia dasi (abraham, ) , g. hegneri (chu, ) and the zoonotic g. duodenalis, one of the few organisms confirmed using molecular (polymerase chain reaction (pcr) on faeces of a captive common palm civet, paradoxurus hermaphroditus) rather than morphological techniques (beck et al., ) . some of the earliest papers found during this review reported on nematode (roundworm) parasites. over two centuries of research has reported on nematodes isolated from a very wide range of viverrid species (table ) . while many were not identified to species level, nematodes representing genera from families have been reported in published papers. these included toxocara spp. (warren, ; maung, ; alvarez et al., ; colon and patton, ; su et al., ) ; ascaris spp. (xavier et al., ; ajibade et al., ; pradhan et al., ) ; the hookworms ancylostoma spp. and uncinaria longespiculum (baylis, ; chowdhury and schad, ; coumaranem and mohan, ; colon and patton, ; su et al., ) ; strongyloides sp. threadworms (su et al., ) ; crenosoma sp. lungworms and viverrostrongylus brauni (myers and kuntz, ; asakawa et al., ; colon and patton, ) ; mammomonogamus sp. (colon and patton, ) ; trichuris spp. whipworms (colon and patton, ; su et al., ) ; capillaria sp. (colon and patton, ; su et al., ) ; rictularia spp. (baylis, ; chen, ; schmidt and kuntz, ; kumar et al., ; mahali et al., ) , spirura sp. (casanova et al., ) ; spirocera sp. and cyathospirura seurati (su et al., ) ; physaloptera sp. (casanova et al., ) ; gnathostoma sp. (probably g. spinigerum) (colon and patton, ) ; brugia spp. and dirofilaria sp. (edeson and wilson, ; masbar et al., ) ; and trichinella spp. (wang et al., ) . nematodes were commonly identified by the examination of morphological features of adult worms, eggs, larvae or oocysts under light microscopy. only a single reference described the use of molecular diagnostic techniques, in this case pcr and genetic sequencing to accurately identify zoonotic trichinella spiralis in infected muscle tissue of a masked palm civet in china (wang et al., ) . platyhelminthes (flatworms) from two classes (cestoda and trematoda), many of which have zoonotic potential, were also reported ( table ). cestode tapeworms from four families included echinococcus sp. and taenia spp. (mahannop et al., ; ndiaye et al., ; millan and casanova, ; lahmar et al., ); diplopylidium spp. and joyeuxiella pasqualei (casanova et al., ; millan and casanova, senegal genet (g. senegalensis) not discussed sudan (wild) (hoogstraal and heyneman, ) z, zoonotic potential acknowledged; m, protozoan affects multiple hosts other than viverridae. (chu, ; abraham, ; beck et al., ) z, zoonotic potential acknowledged; m, protozoan affects multiple hosts other than viverridae. (schrank et al., ; warren, ; maung, ; alvarez et al., ; casanova et al., ; colon and patton, ; su et al., ) family (baylis, ; chen, ; schmidt and kuntz, ; casanova et al., ; kumar et al., ; mahali et al., ) family (casanova et al., ) ); mesocestoides spp. (casanova et al., ; su et al., ) ; and spirometra erinaceieuropaei and diplobothrium sp. (uchida et al., ; xavier et al., ) . flukes from the class trematoda reported for viverrids included species from eight genera, in six families. these were as follows: echinochasmus hangzhouensis and artyfechinostomum sp. (xida, ; varadharajan and kandasamy, ) ; metagonimus yokagawai (uchida et al., ) ; opisthorchis viverrine and metorchis albidus (nicoll, ; casanova et al., ) ; schistosoma japonicum (carney et al., ; he et al., ) ; brachylaema spp. (baugh, ; casanova et al., ) ; and paragonimus spp. in a wide range of civet species (chen, ; dissanaike and paramananthan, ; sachs et al., ; gang et al., ; aka et al., ; colon and patton, ; su et al., ) . the vast majority of platyhelminthes reported for viverrids were identified by examining morphology of different stages within the parasite's life cycle. only one paper cited the use of a molecular biochemical test, an enzyme-linked immunosorbent assay (elisa), to identify echinococcus sp. antigen in common genet (genetta genetta) faecal samples from tunisia (lahmar et al., ) . both external and internal arthropods from seven genera, in four families -sarcoptidae (mites), ixodidae (ticks), trichodectidae (chewing lice) and porocephalidae (pentostomes)were reported for viverrids (table ) . the sarcoptid mite, notoedres cati, a common parasite of domestic and wild felids, was reported for urban-dwelling masked palm civet in japan (ninomiya et al., ) . many species of ixodid tick, most of which are themselves carriers of zoonotic pathogens, were also reported, including amblyomma testudinarium (grassman et al., ) ; ixodes ovatus (tanskul et al., ; robbins et al., ) ; a number of haemaphysalis spp. (hoogstraal and trapido, ; hoogstraal, ; tanskul et al., ; tolosa and regassa, ; mediannikov et al., ) ; rhipicephalus spp. (grassman et al., ; tolosa and regassa, ) ; dermacentor tawianensis (hoogstraal et al., ) and d. auratus (hoogstraal and wassef, ) . the chewing louse, felicola bengalensis was collected from a wild civet (changbunjong et al., ) , and one internal arthropod parasite, the pentostome armillifer moniliformis, was reported for a range of civet species (stabler and self, ; krishnasamy et al., ) . identification of arthropods was based on morphology of varying life cycle stages of the organisms collected from viverrids. while previous authors have suggested that very little is known about the pathogens of viverrids (williams and thorne, ; shepherd, ; bongiovanni et al., ) , this review identified a wide range of viral, bacterial, knowledge of the significance of isolation of an organism for animal health builds on the baseline information provided by a simple inventory of organisms for the species. observation of clinical signs in infected individuals contributes to our understanding of pathogenicity. detailed descriptions of clinical signs seen in viverrids were included in a small number of reports, including an investigation into morbidity or mortality in viverridae (roberton et al., ) , and the documentation of experimental infection of viverrids with sars cov (wu et al., ) . however, most (chen, ; dissanaike and paramananthan, ; sachs et al., ; gang et al., ; aka et al., ; colon and patton, ; su et al., ) z, zoonotic potential acknowledged; m, platyhelminth affects multiple hosts other than viverridae. (hoogstraal and trapido, ; hoogstraal, ; tanskul et al., ; tolosa and regassa, ; mediannikov et al., ) rhipicephalus (hoogstraal and wassef, ; hoogstraal et al., ) family (stabler and self, ; krishnasamy et al., ) z, zoonotic potential acknowledged; m, arthropod affects multiple hosts other than viverridae. reports listed here provide no information on the presence or absence of clinical signs associated with infection, as many document researches carried out on stored biological samples (moinet et al., ) , on samples collected at necropsy (santos et al., ; duarte et al., ) , or on samples, such as faeces, collected opportunistically without seeing the individual animal (colon and patton, ; su et al., ) . in other reports, including all reported nematodes in viverrids (table ) , clinical signs were simply not discussed. in these cases, it is not possible to elucidate whether isolation of these organisms from clinically unwell viverridae explains the presence of disease, or whether it is simply an incidental finding. knowledge of the role a host plays in the life cycle of an organism provides further understanding of the significance of its isolation, and assists in the prevention or management of outbreaks of disease caused by the organism. viverrids are understood to be infective hosts, capable of transmitting disease, for a small number of organisms. these include the sars cov, for which masked palm civets were important amplification and transmission hosts in the epidemic of respiratory disease caused by the organism in southern china in (guan et al., ) . viverrids also transmit the infective life cycle stage of a number of macroparasites including the sarcoptid mite, notoedres cati (ninomiya et al., ) and helminthes toxocara genettae (sanmartin et al., ) , ancylostoma sp. (coumaranem and mohan, ) , rictularia sp. (schmidt and kuntz, ; kumar et al., ) , taenia sp. (mahannop et al., ; millan and casanova, ) and diplopylidium monoophorum (millan and casanova, ) . however, for many more, the significance of viverrids as hosts is probably insignificant. for example, wild common genets are dead end hosts for leptospira interrogans serovars (icterohemorragiae and ballum) (mill an et al., ) ; and binturong (arctictus binturong), small-toothed palm civet (arctogalidia trivirgata), masked palm civet, common palm civet and otter civet (cynogale bennettii) are believed to be accidental hosts for the pentostome armillifer moniliformis (stabler and self, ; krishnasamy et al., ; ) . in many other cases, the opportunistic identification of organisms from a small sample size, a recognized impediment to disease surveillance in wild species (stitt et al., ) , hampers our ability to understand the role of viverridae in the organism's life cycle. the amount of useful epidemiological information obtained is particularly limited where a multihost organism, such as the ixodid tick amblyomma testudinarium, is identified from just one individual viverrid (grassman et al., ) . positive results obtained through opportunistic sampling of small numbers of individuals remain useful in that they provide information on the presence of potential new carriers for an organism, but further research is required to understand the role that viverrids play in the organism's life cycle, or their significance in the epidemiology of the diseases which they cause. some of the organisms listed in this review may be of significance for the conservation of threatened populations of viverrid or other wild carnivores. canine distemper virus (cdv), feline panleukopenia virus (fpv) and canine parvovirus (cp) ( table ) are all highly contagious and cause significant morbidity and mortality in susceptible carnivore species. infected carnivores shed a high viral load in faeces and other bodily secretions (deem et al., ; steinel et al., ) , facilitating environmental contamination and rapid spread of disease. infection of susceptible animals does not require direct contact as transmission may be via inhalation of aerosolized respiratory secretions or ingestion of contaminated material from the environment (deem et al., ; steinel et al., ) . spillover of canine distemper virus from the domestic dog has already impacted several wild carnivore populations, including african wild dogs (lycaon pictus) (fanshawe et al., ) , santa catalina island foxes (urocyon littoralis catalinae) (timm et al., ) , black-footed ferrets (mustela nigripes) (williams et al., ) and lions (panthera leo) (roelke-parker et al., ) , and has been implicated as a significant concern for the conservation of amur tiger (panthera tigris altaica) in russia (gilbert et al., ) . multihost viruses such as those listed for viverridae may pose a threat to the conservation of threatened wild viverrids where their distribution overlaps areas inhabited by unvaccinated domestic carnivores, and must be considered in population management, preventative health measures or in case of disease outbreak. conversely, viverrids themselves may contribute to the maintenance and cycling of pathogens in an environment which, in turn, could threaten the conservation of other endangered species. in addition to the potential significance for viverrid and other animal health, many of the organisms listed for viverridae raise concerns for human health. wild species are a common source or carrier of zoonotic pathogens (wolfe et al., ; jones et al., ) , and there is a growing interest in understanding the host, pathogen and external (environmental and anthropogenic) factors that facilitate their transmission from wild animals to people and other animals (bengis et al., ; woolhouse and gowtage-sequeria, ; greger, ; chomel, ; rhyan and spraker, ) . the vast majority of organisms reported here for viverrids have the capacity to infect a wide range of species a characteristic which, while common amongst pathogens in general - % of all domestic carnivore pathogens infect more than one host (cleaveland et al., )is of concern as it is a significant risk factor for outbreak of disease (cleaveland et al., ; greger, ) . given the prevalence of multihost organisms reported for viverrids, particularly those with zoonotic potential, an examination of the factors bringing these forest dwelling mammals into unnaturally close proximity with humans is warranted. for the viverridae, these include the trades (legal and illegal) in wildlife for human consumption, keeping of viverrids in captivity (wildlife farms, zoos or rescue centres), and human contact with free-ranging viverrids in areas where human and viverrid habitation overlap, or from human incursion into wild spaces. the global wildlife trade is widely regarded as an interface where close contact between humans and wild animals creates an ecosystem favouring disease emergence and expression (bengis et al., ; kuiken et al., ; karesh et al., ; pavlin et al., ; cutler et al., ; bausch and schwarz, ) . stressed, sick and injured animals are transported in mixed-species shipments (bell et al., ) to wet markets or other trade nodes where animals are kept in crowded, unhygienic conditions, exposed to both a wide range of other wild and domestic animals for sale (kan et al., ; woo et al., ) , and to potentially immunologically na€ ıve, urban-dwelling wildlife consumers (swift et al., ) . this combination of conditions facilitates emergence, transmission and amplification of pathogens (greger, ) . viverrids are frequently encountered in the wildlife trade in africa and asia (shepherd, ; nijman, ; shepherd and shepherd, ) where they are consumed for their meat (bell et al., ; yang et al., ; van song, ) or utilized in traditional medicines (kumara and singh, ; ashwell and walston, ; tsegaye et al., ) . while the number of organisms reported for this animal source (refer to 'country (source)' column, tables - ) is low compared to other animal sources, this is likely due to the paucity of research in this area rather than a reliable indication of the health of viverrids in the trade, or the potential significance of this source as a concern for public health. in fact, although few in number, all organisms reported for viverrids sourced from the wildlife tradethe sars coronavirus (table ) (tu, ) , an ixodid tick (table ) (robbins et al., ) , a giardia sp. protozoan (table ) (abraham, ) , the vectorborne parasite responsible for african sleeping sickness trypanosoma brucei (table ) (njiokou et al., ) and a trematode fluke of genus paragonimus (table ) (chen, ) are zoonotic, multihost organisms with the potential to cause considerable socio-economic impact chan et al., ) . in fact, a viverrid played a central role in the pandemic of human respiratory disease caused by one of the most significant diseases to emerge from the wildlife trade system to datethe sars cov (table ) . masked palm civets in this crowded, multispecies wet market in southern china were implicated as the main source of infection in humans via exposure to viral particles in bodily fluids aerosolized during slaughter and preparation for human consumption (guan et al., ; who, ; li, ; chan et al., ) . the socioeconomic impact of this outbreak, which resulted in over human cases and a fatality rate of %, was considerable (dong et al., ; chan et al., ) . this process of preparation of carcasses for human consumption presents a considerable risk for transmission of disease to and from viverrids (wang, ; woo et al., ) , a risk which is amplified by the illegal nature of much of the wildlife trade, meaning that slaughter and preparation for human consumption frequently occur outside government-regulated slaughterhouses, unbound by official hygiene standards (wcs, ) . any one of the zoonotic pathogens reported for viverrids in this review may be transmitted to humans during this process, but the rabies virus, another significant viral zoonosis commonly reported for viverrids, is one for which this process is a documented transmission risk (table ). the broad tissue tropism of rabies (carey and mclean, ) facilitates aerosolization and environmental contamination during the butchering of rabid animals, resulting in confirmed cases of transmission to people following slaughter of domestic dog and cat in china and vietnam (kureishi et al., ; wertheim et al., ; nguyen et al., ) . given the conditions in which the wildlife trade currently operates, the considerable scale of trade in viverrids and their susceptibility to such a wide range of multihost organisms, it is likely that species from this family could again play a central role in the emergence, amplification and transmission of disease to people within this system (dong et al., ) . maintenance of wild species in captivity provides another opportunity for unnatural human-wildlife proximity, facilitating interspecies sharing of organisms (daszak et al., ) . viverrids are kept in captivity in zoos (cosson et al., ) , conservation breeding programmes for threatened species (roberton et al., ) and, in far greater numbers, on commercial wildlife farms. civets have traditionally been farmed for their fur (shi and hu, ) and to supply the demand for 'civet' scent (used in the production of perfume), a practice which drives farming of civets in ethiopia (tolosa and regassa, ) and india (balakrishnan and sreedevi, ) . more recently, farming of civet in asia has expanded to supply a growing demand for their meat in wildlife restaurants and markets (patou et al., ) . while published data on wildlife farming are scant, the number of individual civets on farms is likely to be large by , , individual civets were registered on farms in china (patou et al., ) . another growing industry is the production of civet coffee, where coffee beans are fed to civets and subsequently harvested, undigested, from their faeces. traditionally collected from the faeces of free-ranging animals in the vicinity of coffee farms, coffee producers are increasingly turning to caged production to satisfy demand for this product, which has the dubious distinction of being the 'world's most expensive coffee' (d'cruze et al., ; techangamsuwan et al., ) . a wide variety of zoonoses have been reported for captive viverrids (refer to 'country (source)' columns, tables - ), including the very high profile zoonotic virus hpai h n (table ) . this was confirmed on pcr and serology from a number of owston's civets (chrotogale owstoni) which died following an outbreak of respiratory and neurological diseases in a conservation breeding programme in vietnam (roberton et al., ) . while no evidence of viverrid-to-human transmission of hpai h n exists, isolation of pathogens with pandemic potential from any mammalian host is significant as it may provide conditions suitable for the virus to adapt to mammalian respiratory epithelial receptors, enabling efficient mammalto-human, and possibly also human-to-human, transmission, paving the way for a potentially devastating pandemic (rimmelzwaan et al., ; peiris et al., ) . isolation of this virus from captive civets is also significant in that some countries with the highest number of human cases and fatalities due to hpai h n , such as indonesia, china and vietnam (who, ) , overlap with those where civets are also farmed in the greatest numbers (patou et al., ; d'cruze et al., ) , a risk compounded by the poor standards of husbandry and lack of veterinary care reported for wildlife farms (wspa, ; wcs, ) . knowledge of susceptibility to zoonotic pathogens guides the development of appropriate quarantine and biosecurity protocols in captive institutions required to safeguard both human and animal health. understanding transmission routes of reported organisms also assists in identifying the practices associated with keeping viverrids in captivity which pose the greatest risk to public health. for many of the organisms reported here for captive civets, including the enterobacteriaceae klebsiella pneumoniae and salmonella enterica serovar glostrup in nigerian zoos (falade and durojaiye, ; enurah et al., b) and escherichia coli in a japanese zoo (ahmed et al., ) , the faecal-oral transmission route is common. appropriate hygiene and husbandry standards can reduce the spread of these organisms amongst animals, and between animals and humans, in captive environments. however, for those workers tasked with the collection of undigested coffee beans from civet faeces in the production of civet coffee (d'cruze et al., ) , they present a significant, ongoing occupational hazard which must be addressed. captive viverrids have also been reported as hosts for a number of diseases listed by the world health organization (who) as 'neglected tropical diseases' (ntd), including rabies in an african civet which displayed neurological signs prior to death (table ) (enurah et al., a) , soiltransmitted helminthiasis (ascarid roundworms in large indian civet and masked palm civets and ancylostome hookworms in common palm civet and small indian civet, table ) (baylis, ; ajibade et al., ; pradhan et al., ) and human african trypanosomiasis in a farmed african civet (table ) (njiokou et al., ) . these pathogens are considered 'neglected' because they are poorly studied and commonly ignored by donors, policy makers and public health officials (maudlin et al., ) ; however, they continue to cause widespread morbidity and mortality in the poorer regions of the world (hotez et al., ; who, ) . identification of the pathogens responsible for ntds in captive viverrids contributes to our understanding of potential animal reservoirs for these diseases, knowledge which is vital to the development of interventions aimed at their control or elimination (zinsstag et al., ) . ecotourism is a further anthropogenic factor which brings people into contact with free-ranging viverrids, facilitating the cycling of organisms between these groups. as ecotourism, and hence visitation to national parks, expands globally, an increase in human-wildlife contact has led to a corresponding increase in the risk of transmission of disease (muehlenbein et al., ) , a risk which was illustrated in the serengeti national park in when an african civet, showing clinical signs consistent with rabies, bit a child in an unprovoked attack. a novel lyssavirus, the ikoma lyssavirus, was subsequently isolated from the civet (marston et al., ) . although the pathogenicity of ikoma lyssavirus to humans is unknown, and further research is required to understand its prevalence and distribution (marston et al., ) , this case highlighted the risk of disease emergence when immunologically na€ ıve people come into close and unnatural proximity with wild animals. as human populations expand, and wild spaces are lost, there is an increasing overlap between human habitation and the distribution of wild species, particularly for species which have adapted well to human settlement (campbell, ) . tolerance of human presence by some viverrids has led to high density urban and peri-urban populations, where they are frequently seen as pests, scavenging food from garbage bins, gardens and orchards, and living in the roofs and wall spaces of houses (ninomiya et al., ; campbell, ) . direct contact between viverrids and people in an urban environment leading to disease transmission was reported in japan, where cat scratch fever (caused by the gram-negative bacteria bartonella henselae) was transmitted to a man via a scratch from a free-ranging masked palm civet (table ) (sato et al., ) . peri-urban wildlife may also serve as sylvatic reservoirs for zoonotic disease (weiss and mcmichael, ) , as reported for african civet, believed to contribute to the maintenance and cycling of the mongoose biotype of the rabies virus (sabeta et al., ) , and masked palm civet, considered a maintenance host of the enteric pathogens salmonella, campylobacter spp. and yersinia (table ) in japan (lee et al., ) . while the list of known organisms for viverrids will grow as future work incorporates improved methodology and advanced molecular techniques, this review provides an important baseline inventory of organisms reported for species from the family viverridae. a number of these have clear and serious implications for viverrid health, and as such, this information facilitates sensible health management of both wild and captive viverrids, and provides a knowledge base from which future research, surveillance programmes and disease outbreak investigations can be developed. as the vast majority of organisms listed here are also capable of infecting a wide range of hosts, including humans, many may also pose a risk to public health. this is of concern given the anthropogenic factors, including trade of viverrids for human consumption, keeping of viverrids in captivity, and land use changes which 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wildlife meats in hunan province, central china, before and after the severe acute respiratory syndrome outbreak human benefits of animal interventions for zoonosis control key: cord- - lngdjow authors: solnier, julia; fladerer, johannes-paul title: flavonoids: a complementary approach to conventional therapy of covid- ? date: - - journal: phytochem rev doi: . /s - - - sha: doc_id: cord_uid: lngdjow covid- , the highly contagious novel disease caused by sars-cov- , has become a major international concern as it has spread quickly all over the globe. however, scientific knowledge and therapeutic treatment options for this new coronavirus remain limited. although previous outbreaks of human coronaviruses (covs) such as sars and mers stimulated research, there are, to date, no antiviral therapeutics available that specifically target these kinds of viruses. natural compounds with a great diversity of chemical structures may provide an alternative approach for the discovery of new antivirals. in fact, numerous flavonoids were found to have antiviral effects against sars-and mers-cov by mainly inhibiting the enzymes -chymotrypsin-like protease ( clpro) and papain-like protease (plpro). in this review, we specifically focused on the search for flavonoids, polyphenolic compounds, which are proven to be effective against human covs. we therefore summarized and analyzed the latest progress in research to identify flavonoids for antiviral therapy and proposed strategies for future work on medicinal plants against coronaviruses such as sars-cov- . we discovered quercetin, herbacetin, and isobavachalcone as the most promising flavonoids with anti-cov potential. historically, viral diseases have always emerged and posed major issues to public health. several viral outbreaks-such as the severe acute respiratory syndrome coronavirus (sars-cov) in - , the h n influenza virus in , and the middle east respiratory syndrome coronavirus (mers-cov) in -have caused serious global health concerns in recent years (cascella et al. ) . over the past years, there has been a noticeable increase in the emergence of different novel coronaviruses responsible for a wide range of human and veterinary diseases (fehr and perlman ) . most recently, a new viral epidemic with numerous cases of unexplained low respiratory tract infections occured in wuhan, hubei province, china, as it was first reported to the world health organization (who) on december (world health organization b). the novel virus strain was identified as the severe acute respiratory syndrome coronavirus (sars-cov- ) triggering coronavirus disease (he et al. a ). on march , the who declared covid- a pandemic (world health organization a). coronaviruses (covs) are highly diverse, enveloped, positive-sense, single-stranded rna viruses (? ssrna) (he et al. a) , which constitute the biggest group of viruses within the nidovirales order, containing the largest genomes for rna viruses (fehr and perlman ) . in total, about covs have so far been recognized to be able to infect different species, including humans, mammals, fowl, and other animals ). among them, seven human covs, belonging to the alpha-and beta-covs groups , have been identified as being capable of infecting humans, including e, nl , oc hku , mers-cov, sars-cov and the novel sars-cov- (centers for disease control and prevention ; fehr and perlman ; zhu et al. ). the name 'coronavirus' is inspired by its most defining feature: the club-shaped spikes projecting from the surface of the virion. the spikes sticking out of the envelope's surface give the virus the appearance of a crown (fehr and perlman ) . the nucleocapsids of covs, enclosing the genomic rna, are helically symmetrical. this is in fact unusual for positive-sense rna viruses, and far more common for negative-sense rna viruses (fehr and perlman ) . the two overlapping open-reading-frames (orf a and orf b) of sars, translated into the viral enzymes c-like protease ( clpro) and papainlike protease (plpro), which are vital for virus multiplication, constitute approximately two-thirds of the genome (adedeji et al. ). the other onethird of the genome encodes structural proteins of the virus, such as the spike (s), envelope (e), membrane (m) and nucleocapsid (n) proteins (adedeji et al. ) . the interaction between the s-protein and the receptor is the primary determinant for a coronavirus to infect a host species (lim et al. ) . to date, it is known that sars-cov attaches to its receptor angiotensin-converting enzyme (ace ), while mers-cov was found to bind to dipeptidyl-peptidase (dpp ) in order to penetrate human cells (fehr and perlman ) . so far, it has been observed that the new coronavirus sars-cov- behaves much like sars by using the same entry mechanism to human cells (rabi et al. ) and sharing a . % genome sequence identity to sars-cov zhou et al. ) . several studies have demonstrated that novel sars-cov- likely binds to the human ace receptor, but with a higher affinity than the original sars virus strain (gurwitz ; letko et al. ; rabi et al. ; wrapp et al. ; xu et al. ) . genetic data demonstrated that sars-cov- possesses overlapping open-reading-frames (orf a and orf b) similar to those of sars-and mers-cov ( fig. ) , translated into the viral enzymes clpro and plpro. sars-and sars-cov- share a clpro sequence similarity of %, and a plpro sequence identity of % (mckee et al. ) . therefore, clpro and plpro present two key targets for the development of anti-sars-cov- therapeutics as both are crucial for viral replication; and they share significant homology with proteases of several other related coronaviruses (goetz et al. ). even though the sars and mers outbreaks stimulated research on human covs, there are, to date, no antiviral therapeutics available that specifically target these viruses (rabaan et al. ) . several potential vaccines, including recombinant attenuated viruses, live virus vectors, or individual viral proteins expressed from dna plasmids, have been developed for sars-cov; however, none of them are yet approved for clinical use (fehr and perlman ) . there are several reports which propose potential drugs, although their clinical efficacy has not yet been confirmed for sars-cov- infection and covid- disease. these drugs include: chloroquine, lopinavir/ ritonavir, remdesivir, umifenovir, nucleoside analogs, neuraminidase inhibitors, dna synthesis inhibitors (e.g. tenofovir disoproxil, and lamivudine), ace based peptides, novel vinylsulfone protease inhibitors, teicoplanin, -chymotrypsin-like protease ( clpro)and papain-like protease (plpro) inhibitors mckee et al. ) . to date, the application of remdesivir appears to be the most promising strategy for covid- . in preclinical studies, it has been shown that remdesivir (gs )-an inhibitor of rna polymerase with in-vitro activity which was used against various rna viruses, including ebola-could be effective for both prophylaxis and therapy against human cov infections (gordon et al. ) . alpha-interferon and lopinavir/ritonavir have also been suggested for the treatment of covs (cascella et al. ). since therapy options for coronaviruses, such as for covid- , comprise only preventive and supportive measures, natural products may have a fundamental role in supportive and prophylaxis treatments, and present an alternative approach for cov-management. flavonoids form the largest group of polyphenolic compounds in higher plants (nileeka balasuriya and vasantha rupasinghe ) , with more than structures identified . they represent an important class of plant secondary metabolites, widely distributed throughout the plant kingdom . flavonoids are categorised into several subgroups, which include chalcones, flavanes, flavanols, flavanones, flavanonols, flavones, flavonols, isoflavones or catechins, and procyanidins, all of them consisting of a common flavan ( -phenylchroman) basic structure (fig. ) . these polyphenolic substrates perform a series of protective functions in the human body. many of them are bioactive compounds capable of interfering with nucleic acid or proteins, meaning that they have diverse pharmacological properties (panche et al. ) . it has been reported that flavones and catechins appear to be the most powerful antioxidants, preventing the effects of reactive oxygen species in the body (nijveldt et al. ; panche et al. ) . there are numerous studies highlighting the broad range of biological activities of flavonoids, including antioxidant (d'amelia et al. ), anti-cancer (lejeune et al. , antimicrobial (abreu et al. ; solnier et al. ) , antiviral (wang et al. ) , and antiinflammatory (catarino et al. ) activities (panche et al. ) . more importantly, various flavonoids have been found to inhibit different targets of coronaviruses sars and mers , such as blocking the enzymatic activites of viral proteases like -chymotrypsin-like protease ( clpro), papain-like protease (plpro) and helicase or interfering with spike (s) proteins. a few flavonoids were shown to suppress the activity of angiotensin-converting enzyme (ace) (guerrero et al. ; nileeka balasuriya and vasantha rupasinghe ), which not only plays an important role in cardiovascular diseases such as in hypertension, but may also represent a key determinant in viral infections, and pneumonia (actis-goretta et al. ) . another important issue presents the anti-inflammatory potential of flavonoids in viral diseases, such as activating and stimulating the host immune response to viral infections (dong et al. ), but also suppressing overwhelming inflammatory reactions, which are often associated with a higher mortality rate of sars-cov- infections (mckee et al. ) . for instance, some flavonoids have been reported to interfere with the activation of nlrp inflammasome (lim et al. ) which upregulates the production of inflammatory cytokines, and thus can cause respiratory distress syndrome that frequently occurs within sars coronavirus diseases (chen et al. ) , and sars-cov- infections (shah ) . however, most studies on flavonoids-showing their numerous health-beneficial properties-are conducted in vitro based on the fact that these polyphenolic compounds often deal with low bioavailability, little stability and poor distribution when tested in vivo using animal and/or human cell models (nileeka balasuriya and vasantha rupasinghe ) . there are different strategies reported to enhance these functions, such as the introduction of structural modifications (srinivas ) , absorption enhancers or nanotechnology (ajazuddin and saraf ; zhao et al. ) . the aim of this review is to evaluate the information on flavonoids as possible leads for developing therapeutics to treat sars-cov- . therefore, we searched pubmed for studies reporting on the effects of flavonoids on human coronaviruses. most of the studies targeted the enzymatic activities of the viral proteases cl and pl in vitro using fret (fluorescence resonance energy transfer)-based methods. cl and pl proteases represent valuable targets for the development of anti-coronaviral drugs, as these proteins are essential for the viral transcription and replication complex of all coronaviruses (anand et al. ; chen et al. b; park et al. ) , translated from two open reading frames orf a and orf b which are found in sars-, sars- -, and mers-cov (chuck et al. ; lin et al. ). according to the literature, we were able to identify flavonoids which might present potential agents to treat sars-cov- (table ) . detailed analysis of their structureactivity relationships, has ultimately led to the identification of the three most promising compounds with broad-spectrum antiviral activity. the various studies have focused primarily on the interference of flavonoids with some viral proteases such as cl and pl of sars-and mers-cov by using common enzymatic, fluorogenic (fret)-based methods and molecular docking studies, as summarized in table . clpro and plpro are both key targets as they process several viral polyproteins which are involved in the replication and transcription of the genomic rna within host cells (mckee et al. ) . further, they share significant homology with viral proteases of several other coronaviruses-especially with those of sars-, sars- -, and mers-cov . besides cl protease, papain-like protease has been the focus of numerous studies on the development of chemotherapeutic drugs against cov-induced diseases. plpro is not only responsible for processing viral polyproteins, but is also involved in deubiquitination (cleaving ubiquitin chains) and deisgylation, which represent relevant factors in the host immune response to viruses (cho et al. ) . by removing ubiquitin and isg proteins from host cell proteins, the innate host immune response is likely to be compromised (ratia et al. ; xian et al. ). plpro of both sars-and mers-cov has been shown to possess deubiquitinating activities (park (park et al. ) and may, therefore, have an important part in the virus life cycle. hence, antiviral therapeutics targeting plpro could also prevent the antagonist activities of plpro on host innate immune response (ratia et al. ). in fret-based assays, the proteolytic activity is detected through cleavage of a fluorogenic peptide and measuring the increase in fluorescence intensity by continuously monitoring the reaction . fret is a non-destructive method, widely exploited to study protein interactions (margineanu et al. ) , and is also applied to detect signals in living cells ). while the cell-free fret assays can provide easy and fast access to proteins including some difficult targets (sierecki et al. ) , the cell-based fret methods enable in situ analysis of a variety of biological targets and proteinprotein interactions in a more natural environment (silverman et al. ). the studies of park et al. ( ) , lin et al. ( ) and others could prove that flavonoids tested in cell-based assays generated higher activity against cl and pl, than excerting unspecific activity against other enzymes. furthermore, the unspecific aggregation of proteins often caused by flavonoids could be reduced significantly by the addition of triton x without affecting anti-coronaviral activity in these studies. in a number of studies molecular docking technology was used for screening purposes. despite the valuable use of this method to rapidly discover novel inhibitors of sars-cov- , there are some disadvantages which include low accuracy and high rate of false-positive results ). however, compounds which are identified through such virtual screening methods can be further examined in highthroughput enzymatic in-vitro assays, followed by more in-depth in vivo investigations, such as studies on efficacy and toxicity. in general, the combination of docking studies and bioactivity assays can improve screening accuracy by providing supportive data. chalcones isolated from angelica keiskei were shown to inhibit both sars-cov proteases plpro and clpro in enzymatic, fret-based (table ) and molecular docking studies (park et al. ). inhibition was achieved through a competitive manner against clpro, and a non-competitive mode against plpro (park et al. ) . xanthoangelol e, an alkylated chalcone substituted with a -ooh group (table ) , exerted the most relevant inhibition against clpro (ic = . and . lm) and plpro (ic -= . lm) using fluorogenic methods (park et al. ) ( table ). analyses of the relationships of the alkylated chalcones (tables and ) against clpro showed that xanthoangelol e substituted with -perhydroxyl- -methyl- -butenyl (pmb) group was more effective than xanthoangelol d with a -hydroxy- methyl- -butenyl group (ic = . and . lm). similarly, the hydroxyl group of the a-ring caused apparently higher activity than when substituted with a methyl group. comparing xanthoangelol d and -hydroxyderricin, it seemed that the substitution of the a-ring with a -hydroxy- -methyl- -butenyl group (xanthoangelol d, table ) generated higher inhibition than when substituted with the dimethylallyl group ( hydroxyderricin: ic = . and . lm, table ) (park et al. ). based on the results it can be assumed that a perhydroxyl group on the substituted hemiterpene might be crucial for enzyme binding and may affect conformational stabilization of the polyhydroxylated chain through intramolecular hydrogen bonding (park et al. ) . in general, the bioactivity of highly potent compounds against sars plpro apparently depended on modifications in their molecular structure, such as prenylation in the a and b ring and methylation. for instance, the prenyl moiety of the -prenylated chalcones isobavachalcone and xanthoangelol e led to considerable inhibition against sars plpro (ic -= . and . lm), highlighting the importance of the hydrophobic substituent. further, it is generally assumed that prenylation of flavonoids may enhance their bioactivity and bioavailability (grienke et al. ; mukai ) , presumably by altering lipophilicity and thereby affinity for membrane targets (shen et al. ) . thus, chemical modifications of flavonoids, such as the introduction of hydrophobic substituents, may also demonstrate a reasonable strategy to enhance their antiviral activity against sars-cov- . some flavonoids isolated from broussonetia papyrifera have been proposed as useful lead-compounds for the development of anti-cov agents against sars and mers ) and therefore, might be also considered to treat sars-cov- . among them, broussochalcone a, broussochalcone b, and -hydroxyisolonchocarpin derived from b. papyrifera showed proteolytic activity against both proteases cl and pl of sars-and mers-cov (table ) through non-competitive inhibition. as table demonstrates, the compounds showed generally higher inhibitory potential against sars-cov plpro than when tested against the other viral proteases using fluorogenic methods, which is likely related to genomic variations in the single amino acid sequences. broussochalcone b was found to be the most effective substrate for inhibiting clpro of mers-cov (ic = . lm) in the study of park et al. ( ) . in addition to inhibitory effects on cysteine proteases, the compounds isolated from b. papyrifera caused inhibition against alpha-glucosidase . inhibition of glycosidases, especially of alpha-glucosidases, affects maturation, transport, secretion, and the functioning of glycoproteins which can enhance cell-cell and/or cell-virus recognition processes (ryu et al. b ). therefore, it plays a relevant role in the treatment of viral infections, but also in other diseases such as diabetes mellitus type , and cancer (ryu et al. b ). in accordance with a previous study (ryu et al. b) , the alpha-glucosidase inhibitory activity of the substrates from b. papyrifera depends on the number and positions of prenyl groups present in the molecule . kim et al. ( ) could demonstrate that isobavachalcone and -o-methylbavachalcone extracted from the seeds of cullen corylifolia have great inhibitory potential against sars-cov plpro. both compounds inhibited plpro in a dose-dependent manner, with ic values of . and . lm (table ) (kim et al. ) . isobavachalcone ( , , -trihydroxy- -( -methyl- -butenyl) chalcone) also showed inhibitory activity against mers-cov clpro (ic -= . lm, table ), when tested within the screening of a flavonoid library using a fluorogenic (fret-based) method (jo et al. ) . based on the results summarized in table , the chalcones isobavachalcone, -o-methylbavachalcone, broussochalcone a and b, including xanthoangelols, can be considered as prominent inhibitory compounds of sars-cov plpro (ic b . lm, table regarding the broad-spectrum activity of chalcones, isobavachalcone presents the major compound in this group -with a good scaffold to bind with proteases of both sars-and mers-cov. hence, further in-vitro investigations with isobavachalcone on viral proteins of sars-cov- , as well as some pharmacological studies in-vivo regarding toxicity and bioavailability of the compound, might be a valuable approach in the field of research for covid- . there are a few plant sources of isobavachalcone other than those mentioned in table , as it is abundantly found in species belonging to plant families fabaceae (e.g., anthyllis hermanniae, glycyrrhiza glabra, glycyrrhiza uralensis, sophora prostrata) and moraceae (e.g., dorstenia poinsettifolia, dorstenia turbinata, maclura tinctoria, treculia acuminata) (kuete and sandjo ). the flavanes -( -methylbut- -enyl)- ', , -trihydroxyflavane, broussoflavan a, kazinol a and kazinol b isolated from broussonetia papyrifera interfered with viral proteases cl and pl of both sars-and mers-cov (table ) through non-competitive inhibition ). among all flavanes tested, -( -methylbut- -enyl)- , , -trihydroxyflavane, a c alkyl group (prenyl)-substituted flavan (table ) , exhibited the most potent inhibition against sarsand mers-cov clpro (ic = . lm and . lm). considering the structure-activity relationships of flavonoids from b. papyrifera (tables and ) , it was observed that molecules having a c -oh group and saturated chromenone with a dihydroxy group at ring c have stronger inhibitory potential than those with a closed prenyl group such as kazinol b (ic = . lm, table ) . given the results, the flavane -( -methylbut- enyl)- , , -trihydroxyflavane may serve as good template for continuing in-vitro experiments on viral proteases of sars-cov- due to similar efficacy against proteases of both sars-and mers-cov, and the homology of proteases from the sars-cov- strain (fig. ) . epigallocatechin gallate (egcg) and gallocatechin gallate (gcg), two flavanol derivatives, demonstrated inhibitory activity against sars-cov clpro, when tested in proteolytic (fret-based) assays and molecular docking studies of nguyen et al. ( ) . the catechin gcg (ic = lm) inhibited clpro activity (table ) by interacting with amino acid residues in the active site pocket of clpro (nguyen et al. ). analyses of structure-activity relationships of these compounds (tables and ) suggest that the galloyl moiety at position -oh of egcg and gcg (table ) , which is absent in the other catechins, seemed to be relevant for causing clpro inhibition. gcg ( s, r type), which is a c- epimeric isomer of egcg ( r, r type), generated higher clpro inhibition than that of egcg (ic = lm). in a following docking study, it was shown that the galloyl group of gcg, was essential for hydrogen bond interactions with the amino residues leu , gly , ser , and his of the clpro active pocket site (nguyen et al. ) . for further studies with these compounds on proteases of sars-cov- it should be considered that different conformation and structural modifications in the molecules likely increase antiviral activity. in a study investigating the chinese medicinal plant isatis tinctoria root-which was used for the prevention of sars-cov during the sars-outbreaks, and is known for its antiviral properties against influenza, hepatitis a and japanese encephalitis infections (lin et al. ; wu et al. )-hesperetin was discovered as highly potent inhibitor of sars-cov clpro (ic = . lm) when tested in a cell-based cleavage assay (lin et al. ) . in comparison with other reported inhibitors of sars-cov clpro, such as cinanserin and cinanserin hydrochloride (ic -= lm and lm, respectively) (chen et al. a) , hesperetin may present a promising lead in the development of sars-cov- cl protease inhibitors. however, further screenings on proteolytic activity of hesperetin against plpro might be also important. several citrus fruits are the major plant sources of flavanones, including hesperetin-the aglycone of hesperidin. bavachinin, a flavanone extracted from the seeds of cullen corylifolia, was found to have inhibitory potential against sars-cov plpro (ic = . lm, table ) (kim et al. ) . amelopsin, also known as dihydromyricetin, had only weak inhibitory activity against sars-cov clpro (ic = lm, table ) when tested in a proteolytic (fret-based) assay and molecular docking studies (nguyen et al. ). in the same study, it was observed that the additional oh group at position in the b ring as well as the absence of , double bonds in the c-ring of amelopsin (table ) considerably reduced clpro inhibitory activity when compared to other compounds tested, such as quercetin (ic = lm, table ) (nguyen et al. ). amentoflavone, a biflavone isolated from torreya nucifera, demonstrated a prominent inhibitor of sars-cov clpro (ic = . lm) in a fretbased assay (ryu et al. a) . in that study, amentoflavone proved to be far more potent than the parent compound apigenin (ic = . lm), and the other flavone luteolin (ic = lm), as well as the flavonol quercetin (ic = . lm). by comparing their structures (table ) , those compounds with a c- -substituted hydroxyl group, such as luteolin and quercetin, excerted stronger inhibition than apigenin. furthermore, biflavone derivatives of amentoflavone with methylation of -, -, and -hydroxyl groups, decreased inhibitory activity (ryu et al. a) . based on the prominent in vitro inhibition activity of amentoflavone against clpro of sars-cov, the compound may serve as a good starting point for further investigations on clpro of sars-cov- due to . % genome sequence identity zhou et al. ) , including more than % similarity of clpro amino acid sequences between sars-cov and sars-cov- (mckee et al. ). however, the biflavonoid should be also tested against other proteases of sars-and mers-cov in order to determine a broad spectrum antiviral efficacy against sars-cov- . there are numerous plant sources from which amentoflavone can be isolated, these include for instance gingko biloba, hypericum perforatum, lobelia chinensis, polygala sibirica, ranunculus ternatus, and several plants from selaginella species, such as selagenella tamariscina, selaginella nipponica, and selaginella pulvinata (yu et al. ) . two other flavones, pectolinarin and rhoifolin, and the flavonol herbacetin were discovered as inhibitors of sars-cov cl enzyme when tested in proteolytic assays (ic b . lm, table ) and induced-fit docking experiments (jo et al. ) . in docking studies, jo et al. showed that the additional -hydroxyl group of herbacetin seems to be crucial for its high binding affinity to the polar s site and the hydrophobic s site of sars-cov clpro (jo et al. ) . furthermore, carbohydrate groups of rhoifolin and pectolinarin were responsible for the high affinity to sars-cov clpro (jo et al. ). in another screening using a fret-based method, herbacetin ( , , , , -pentahydroxyflavone, table ) also caused inhibition against clpro of mers-cov at an ic value of . lm (table ) (jo et al. ) . since herbacetin shows similar inhibitory effects against both cl proteases of sars-and mers-cov (ic = . and . lm), it may also demonstrate proteolytic activity when tested against sars-cov- cl protease. several clpro inhibitors that have been suggested as candidates for sars-cov- clpro, were found to be effective against clpro of both sars-and mers-cov (he et al. b ). herbacetin can be found, for instance, in ephedrae herba (hyuga et al. ) , linum usitatissimum (qiu et al. ) and rhodiola rosea (péter zomborszki et al. ) . papyriflavonol a, a double prenylated flavone derivative isolated from broussonetia papyrifera, presents one of the most significant inhibitors of sars plpro at an ic value of . lm (table ) , causing higher activity than other flavonols such as kaempferol (ic = . lm), quercetin (ic -= . lm), and quercetin-beta-galactoside (ic -= . lm). similarly, geranylated flavonols from the fruits of paulownia tomentosa were found to be the main group of active constituents that successfully interferes with sars-cov plpro (cho et al. ). among twelve plpro-inhibitory flavonoids isolated, five new geranylated flavonols named tomentin a-e bearing an unusual , -dihydro- h-pyran motif with a cyclized geranyl chain (table ) caused high inhibition (ic values - . lm) within a fret-based assay (table ) . prenylated and geranylated flavonoids like papyriflavonol a, and tomentins (ic b . lm, table ) can be proposed as interesting leads for sars-cov- plpro inhibitors due to % homology of plpro sequences between sars-cov and sars- cov- (mckee et al. ) . it can be assumed that the hydrophobic substituents of prenylated flavonoids e.g., papyriflavonol a, show higher affinity to sars-cov plpro than to other proteases, which might be due to certain structural differences in the protein sequences. further, the polarity of the compounds such as an increased number of hydroxyl groups, including the addition of sugar residues in the flavone backbone (table ) , seems to affect inhibitory potential, as demonstrated by kaempferol (ic = . lm), quercetin (ic -= . lm), and quercetin-b-galactoside (ic = . lm). comparing quercetin with quercetin b-glycosides-the sugar moieties such as that of quercetin- -b-galactoside, and quercetin -b-d-glucoside did not enhance inhibitory activity against sars-and mers-cov proteases. in molecular docking studies, quercetin- -b-galactoside created hydrophobic interactions with residues asn , glu , leu and met of sars clpro (chen et al. ) . in particular, the residue q represented a principle factor in the binding between quercetin- -b-galactoside and sars clpro (chen et al. ) . analyses of structure-activity relationships of quercetin- -b-galactoside derivatives suggested that hydroxyl groups on the quercetin moiety are crucial for inhibitory activity, which enable compound-target interaction through hydrogen bonding (chen et al. ) . quercetin and quercetin -b-glucoside ( , , , , pentahydroxyflavone -b-glucoside) exerted inhibition when tested against mers clpro with respective ic values of . and . lm (table ) (jo et al. ) . in a following docking study, jo et al. showed that a rhamnose substitution instead of glucose (in quercetin -b-glucoside), results in a stronger binding with mers-cov clpro (jo et al. ) . in general, quercetin causes a broad range of activities against proteases of sars-and mers-cov (table ) . based on that, and the number of sources cited, such as the ic values reported in different studies, quercetin is among the most promising agents to treat sars-cov- . quercetin represents one of the most abundant dietary flavonoids that naturally occurs in a great variety of plant products (medicinal herbs, food, beverages, etc.) (anand david et al. ; pandey and rizvi ) . a few of these rich sources from which quercetin can be isolated include: allium cepa, allium fistulosum, asparagus officinalis, camellia sinensis, capparis spinosa, coriandrum sativum, ginkgo biloba, hypericum perforatum, moringa oleifeira, punica granatum (howell and d'souza ) , sambucus canadensis (anand david et al. ; li et al. ) . the isoflavones, daidzein (ic = lm) and puerarin (ic = lm) lacking the b-ring in their -phenyl- h- -benzopyran- -one backbone (table ) , showed only weak inhibition of sars-cov clpro, as demonstrated in table . neobavaisoflavonestructurally related to daidzein-prenylated at c , and corylifol a extracted from the seeds of cullen corylifolia, were found to have good inhibitory potential against sars-cov plpro (ic = . and . lm) (kim et al. ) . as mentioned before, flavonoids with lipophilic substituents like prenyl or geranyl side chains in the a or b ring (table ) appear to be very good scaffolds to bind with pl proteases, and may therefore be considered as templates for the development of sars-cov- plpro inhibitors. isoflavonoids are predominantly found in legumes; prenylated derivatives can be mainly isolated from the plant familiy fabaceae (Š mejkal ). flavones and flavonols a screening of natural compounds against sars-cov helicase led to the identification of scutellarein and myricetin-two strong inhibitors of sars nsp (table ) using an atp hydrolysis assay yu et al. ) . the non-structural proteins (nsps), which are processed by the viral proteases pl (residing in nsp ) and cl (residing in nsp ), comprise the rna-dependent rna polymerase and the ntpase/helicase-two essential determinants for virus replication (adedeji et al. ) . the sars helicase nsp protein has been shown to have doublestrand (ds) rna and (ds) dna unwinding activity and can translocate along the nucleic acids by hydrolyzing atp . both compounds, the flavone scutellarein and the flavonol myricetin, successfully impaired atpase activity of nsp with respective ic values of . lm and . lm (table ). this effect was mediated through inhibition of atpase activity, but did not affect dsdna-unwinding activity of sars helicase, as it was demonstrated in a fluorometric, fret-based, dsdna unwinding assay of yu et al. ( ) . myricetin naturally occurs in high concentrations in different fruits (e.g., cranberry) (singh et al. ), and vegetables such as spinach, cauliflower etc. (sultana and anwar ) . scutellarein can be found in members of the genus scutellaria such as scutellaria baicalensis-an important traditional chinese plant used against diverse infections including inflammatory or respiratory diseases (zhao et al. ) . another study evaluating derivatives of -oaryl-methylquercetin for their antiviral activity against sars-cov, three derivatives with '-cl, '-cn, and '-cl aromatic substituents showed selective inhibition of sars-cov ntpase/helicase activity by performing a fret-based dsdna unwinding assay (park et al. ) . the introduction of an arylmethyl substituent, such as -clphch , -cnphch and -clphch , at the position -oh of quercetin (table ) , led to stronger inhibition against sars-cov helicase (ic values of . , . , . lm, respectively) than that of quercetin (ic = . lm) (park et al. ). the spike (s) protein of covs is responsible for viral entry into target cells through binding to the angiotensin-converting enzyme (ace ) receptor on host cell membranes , and thus presents another important target in the anti-sars-cov- drug development. besides the inhibitory potential of luteolin against sars cl protease (ic = lm, table ), the flavone was found to successfully impede the entry process of sars-cov into host cells by interfering with the s protein of sars-cov (ec value = . lm) in a two-step screening method combining frontal affinity chromatography-mass spectrometry (fac/ms) and pseudotyped virus-infection assay (yi et al. ). likewise, quercetin was found to block host cell entry (ec = . lm) when tested in hiv-luciferase/ sars pseudotyped virus assay (yi et al. ). the use of pseudotyped viruses is reported to be a powerful, rapid, and highly sensitive method for studying the entry process of viruses to host cells (yi et al. ) . since sars-cov and sars-cov- share high sequence similarity of the s proteins, these compounds may be also expected to impede the entry of sars-cov- into host cells. furthermore, it is proven that the s protein of sars-cov- binds with - -fold higher affinity to ace than that of sars-cov (mckee et al. ). thus, inhibition of ace via a competing substance, seems to be a reasonable approach for preventing sars-cov- infections. quercetin had potent inhibitory effects on ace in vitro, and in vivo when tested in rat models (actis-goretta et al. ; al shukor et al. ; guerrero et al. ; nileeka balasuriya and vasantha rupasinghe ) . however, ace inhibitors-which are standard therapeutics for the treatment of high blood pressure (actis- goretta et al. ) , and may have a vital role in viral infections and pneumonia (henry et al. )-have not yet been shown to inhibit ace . baicalin, a flavone glycoside and major constituent of scutellaria baicalensis, was found to have antiviral in vitro activity against sars-cov ( . to lg/ml after h; and to lg/ml after h) when screened against clinical isolates of sars-cov with foetal rhesus kidney- cells tested in a plaque reduction assay . for comparison, lopinavir-a protease inhibitor used against hiv infections, and which is currently in clinical trials for anti-sars-cov- infections-showed antiviral efficacy against sars-cov infected fetal rhesus kidney- -cells at a concentration of lg/ml, using the same assay (chu et al. ) . baicalin is also a known inhibitor of hiv- reverse transcriptase (li et al. ) , and was shown to possess inhibitory potential against influenza in vitro and in vivo by acting as immune modulator promoting ifn-c production in mice and human cd ? and cd ? t cells (chu et al. ; ding et al. ) . in general, some hiv protease inhibitors e.g., lopinavir, ritonavir or nelfinavir could interfere with sars-cov replication in-vitro, and had binding affinity to sars-cov clpro ). based on available evidence, it is controversial whether antiretroviral drugs can be suggested for the treatment of coronavirus diseases such as sars-cov- . however, flavonoids which were found to have anti-hiv activity may be still worth investigating for their anti-sars-cov- potential. for example, baicalein, another major component from scutellaria baicalensis, has been described for its anti-hiv properties such as its binding to hiv- integrase (hu et al. ; zhao et al. ) , and thus might be proposed as interesting lead compound for sars-cov- drug discovery. in addition to the inhibitory potency of kaempferol against sars-cov proteases (table ) , the flavonol was able to block the a ion channel of sars-cov, a protein encoded by the open-reading-frame (orf) a, which is involved in the virus release mechanism (schwarz et al. ). schwarz et al. demonstrated that glycosides of kaempferol are stronger inhibitors, highlighting the importance of sugar residues (schwarz et al. ) . the kaempferol glycoside juglanin having an arabinose residue, was shown to be the most prominent substrate (ic value = . lm) (schwarz et al. ) . procyanidins such as procyanidin a , and procyanidin b isolated from cinnamomi cortex had inhibitory potential against wild-type sars-cov using a plaque reduction assay (ic = . and . lm, table ). however, it was reported that the interplay of all procyanidins in the extracts from cinnamomi cortex appeared to be crucial for causing strong activity against wild-type sars-cov (zhuang et al. ). plaque reduction assays do not indicate any specific inhibition mechanism of the compounds, but provide a fast and high throughput pre-screening method. analyses of studies based on flavonoids as anti-cov agents revealed compounds (table ) as possible agents against sars-cov- . among them, compounds were investigated in at least two independent studies. of these compounds, quercetin, herbacetin, isobavachalcone, and kaempferol, show activity against at least two viral targets. according to the high ic values of kaempferol ( . lm against sars-cov clpro, and . lm against mers-cov plpro), we do not consider this compound a promising candidate to treat sars-cov- . therefore, the flavonols, herbacetin and quercetin, along with isobavachalcone ( fig. ) were identified to be the most attractive antiviral leads against sars-cov- .-due to their ic values and broad-spectrum activity against proteases of sars-and mers-cov described in different studies (table ) , as well as their availability from different plant sources. given the similarity of the genomic sequences encoding the clpro and plpro catalytic sites of sars-cov- , sars-cov, and mers-cov (fig. ) , clpro and plpro inhibitors of sars-and mers-cov are expected to be valuable leads for treating sars-cov- infections and covid- . for instance, remdesivir-one of the most important antiviral drugs which has been suggested for therapeutic use against sars-cov- -showed broad-efficacy against both sars-and mers-cov (sheahan et al. (sheahan et al. , . based on the high cc values of the active flavonoids, such as that of herbacetin cc = . lm ) and quercetin cc = . lm (chiow et al. ) , they can be used in higher concentrations in therapeutic treatments without causing any substantial cytotoxic effects. prenylated flavonoids like isobavachalcone, are reported be relatively non-toxic to non-cancer cells (Š mejkal ) , and can be found in significant amounts in the plant families moraceae, and fabaceae (kuete and sandjo ) . as quercetin, herbacetin and isobavachalcone are readily available in high amounts from several plant sources, this might be also beneficial for future investigations on sars-cov- , and the development of anti-cov therapeutics. although the daily intake of plant food may appear to be a readily accessible source of active flavonoids, there is no scientific evidence that a high consumption of flavonoid-rich food and/or supplements, like that of quercetin, provides significant protection against viral diseases in humans e.g., against sars-cov- . due to low bioavailability, fast metabolism and elimination of flavonoids, the biological functions of these dietary polyphenols in-vivo are likely to be compromised. on the other hand, there are some external, non-host related, factors limiting the bioavailability of dietary flavonoids which include: environmental factors (i.e., storage, sun exposure), food processing factors (i.e., cooking), the interaction with other polyphenols present in plant-based food (i.e., antagonistic actions), the chemical structure (i.e., polymer or in glycosylated structure) and the concentration of the dietary compounds (d'archivio et al. ) . besides strong inhibitory effects of flavonols and chalcones on sars-and mers-cov, these flavonoids also have some anti-inflammatory (choy et al. ; ur rashid et al. ) and immune-modulating properties (hosseinzade et al. ) , which can be highly beneficial in the host immune response to viral infections. as an example, quercetin which is welldocumented for its wide spectrum of antiviral functions (zakaryan et al. ) , had antiviral effects against rhinovirus (rv) infections in-vitro and in-vivo using rv-infected mice, by reducing the expression of pro-inflammatory cytokines and chemokines (ganesan et al. ). hence, due to different important functions they can be valuable for either preventing and treating viral infections, as well as for alleviating symptoms (e.g., fever, cough, etc.) that may occur during the course of viral infections like that of sars-cov- . in general, quercetin can be considered a highly potent candidate for treating sars-cov- , as it has affinity to a variety of anti-cov drug targets. the flavonol interfered with the viral replication of sars-cov by blocking the enzymatic activities of clpro, plpro (table ) and helicase (ic = . lm) ), but also impaired sars plpro cleavage activity of ubiquitin and interferon-stimulated gene (isg) (ic = . and . lm, respectively) , which can be important for both the viral replication and the host immune response to viruses (cho et al. ) . moreover, quercetin interacted with sars-cov at host cell entry level by binding to the s glycoprotein (ec = . lm) that docks to the host receptor ace ). further, quercetin demonstrated proteolytic activity against mers-cov clpro (ic = . lm, table ). for that purpose, there are a few issues that need to be addressed. since polyphenolic compounds are known for their aggregating tendency, doubts have been raised about the reliability of in vitro bioassay data of flavonoids, as false-positive results may occur through non-specific binding of phenolic compounds to proteins (pohjala and tammela ) . the formation of aggregates can cause a non-specific inhibition by sequestering enzyme molecules, absorbing or adsorbing them within their structure and thus causing denaturation (coan et al. ; pohjala and tammela ) . quercetin, which is one of the best studied flavonoid, showing virucidal activity against various enveloped viruses (e.g., influenza (wu et al. ) , parainfluenza type , herpes simplex (chen et al. ) ), was among the flavonoids that form large aggregates and thus may operate as a promiscuous inhibitor affecting various unrelated targets (pohjala and tammela ) . however, it has been reported that the addition of triton x- , a solubilizing agent, to proteolytic assays, can help to reduce aggregate formation and complexation, such as that of flavonoids (jo et al. ; pohjala and tammela ) . besides the limitations of the bioassays, there is another aspect-the in-vitro molecular assays and cell-based assays do not provide any information about the bioavailability of the compounds. however, this aspect is not within the scope of the review, but for developing flavonoids further, this aspect should be considered. therefore, it is important to address the bioavailability of flavonoids as one of the critical limiting factors in their therapeutic use. different strategies to enhance the stability, solubility and systemic distribution of flavonoids have been reported, which can be employed. these include nanotechnology, co-crystallization, absorption enhancers and structural transformations (e.g. prodrugs, glycosylation) (ajazuddin and saraf ; zhao et al. ) . the use of the flavonoids quercetin, herbacetin, and isobavachalcone in adjuvant therapy with other proposed antiviral drugs may present another interesting approach to combat sars-cov- infections. combination therapy often leads to better outcomes in antiviral treatments. for instance, it was found that synergistic effects between quercetin and the antiviral agent aciclovir resulted in enhanced antiviral activity against pseudorabies herpesvirus infection in-vitro (ahmad et al. ) . similarly, a-glucoside inhibitors applied together with ribavirin, a standard antiviral drug, could improve antiviral efficacy against dengue infection in vitro and in vivo (chang et al. ). furthermore, it should be also evaluated whether the pure flavonoid itself like quercetin, herbacetin, and isobavachalcone, or adminstered in combination with other flavonoids and/or natural compounds can achieve optimal health benefits. the increasing development of drug-resistance in antiviral treatment might be another critical matter for the therapeutic use of flavonoids. literature search in pubmed, has led to the identification of numerous flavonoids exerting antiviral in vitro activity against sars and/or mers coronavirus, primarily by inhibiting the enzymatic activity of cl and pl. flavonols and chalcones were found to be the main groups of flavonoids containing the highest number of effective compounds against cl and pl proteases. in particular, herbacetin, quercetin and isobavachalcone (fig. ) were identified as promising antiviral leads against sars-and mers-cov based on their broad-spectrum activity against the viral proteases cl and pl of both covs, the number of relevant literature data, and the availability of the compounds from different plant sources. considering the fact that quercetin could interfere with sars-cov at different levels, such as at the viral entry and replication process, as well as with cl protease of mers-cov, we specifically propose this compound to be a highly valuable anti-sars-cov- candidate. quercetin may prevent host cell entry of sars-cov through binding to the s protein and inhibiting ace. further, the flavonol can impair the viral replication of sars-and mers-cov by blocking the enzymatic activites of cl and pl including sars-helicase, and can inhibit the deubiquitination and deisgylation process of sars plpro, which might be also an issue in the host immune response. other potent substrates able to interact with the enzymatic activity of sars-and mers-cov proteases, were found to be prenylated flavonoids (e.g., isobavachalcone). however, despite some promising inhibitory activities of flavonoids against sars-and mers-cov in vitro, none of these compounds have been tested in vivo using animal and/or human cell models. therefore, more detailed investigations on pharmacological mechanisms, long-term toxicology, bioavailability, as well as some additional studies on possible herb-drug interactions are required. in fact, natural compounds like flavonoids, continue to be a wealthy source for the discovery of novel antiviral agents. it was reported that natural substrates from traditional chinese medicine (tcm) seemed to have a positive impact on the recovery process of patients suffering from sars-cov diseases, by mitigating possible side effects of conventional therapeutics . since flavonoids are well-documented for their broad spectrum of health-beneficial properties, especially anti-inflammatory and antioxidative effects, they could be of great value for strengthening the host immune response to viral diseases, and alleviating infection-related symptoms, such as down-regulating overwhelming inflammatory responses (e.g. cytokine production). for instance, besides the antiviral effects of quercetin, the flavonol was found to prevent tissue damage by scavenging free radicals, and could reduce the release of inflammatory cytokines such as interleukine il- (kinker ) . due to high similarities of sars-cov, sars-cov- , and mers-cov-sharing several homologous viral proteases, the proposed flavonols herbacetin and quercetin, as well as isobavachalcone may demonstrate attractive antiviral substrates against sars-cov- and/or other coronaviruses. however, further research and more detailed pharmacological investigations in-vivo, particularly on the bioavailability of these compounds, appear to be a promising approach for the discovery of novel herbal substrates used as adjunctive therapeutics in the treatment of coronavirus diseases, such as covid- . furthermore, greater attention should be paid to combinatory effects of flavonoids, especially when used together with other standard antiviral drugs. looking to nature for 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jurisdictional claims in published maps and institutional affiliations acknowledgements open access funding provided by university of graz is greatly acknowledged. author contributions julia solnier and johannes-paul fladerer contributed equally to this work.funding open access funding provided by university of graz. conflict of interest the authors declare that they have no conflict of interest.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. key: cord- -ipaoge authors: sadana, ajit; sadana, neeti title: chapter detection of biomarkers for different diseases on biosensor surfaces part ii date: - - journal: biomarkers and biosensors doi: . /b - - - - . - sha: doc_id: cord_uid: ipaoge abstract in this chapter the authors analyze the binding and dissociation kinetics (if applicable) of ( ) interferon-gamma as a function of aptamer variants and inclusion of spacer, ( ) gst-n protein in pbs and gst-n protein in -fold diluted serum to a localized surface plasmon resonance coupled fluorescence biosensor, ( ) cytochrome c mutant to a superoxide biosensor, ( ) carbonic anhydrase-ii to an -( -aminoethyl)-benzene sulfonamide ligand on an surface plasmon resonance biosensor surface, ( ) glycerol secretion from differentiated (murine t -l ) adipocytes to a microfluidic platform for fluorescence-based assay, and ( ) different concentrations of c-reactive protein in solution to a sandwich-type assay using reflectometric interference spectroscopy (label-free detection method). the previous chapters have described the detection of different biomarkers for different diseases. each chapter analyzed the different biomarker for a particular disease. in this chapter we analyze the binding and dissociation (if applicable) kinetics of other biomarkers on biosensor surfaces. some of the examples analyzed include the following: . binding to and dissociation of different aptamer beacon modifications of interferon (ifn)-gamma in solution using fluorescence resonance energy transfer (fret) and immobilized on an avidin-coated surface (tuleuova et al., ) . . binding and dissociation of glutathione s-transferase fused to the nterminus of a protein (gst-n) protein in phosphate buffered saline (pbs) to a localized surface plasmon resonance coupled fluorescence (lspcf) biosensor (huang et al., ). . binding of a cytochrome c mutant to an amperometric superoxide biosensor (wegerich et al., ). . binding of different concentrations (in micromoles) of carbonic anhydrase-ii (ca-ii) in solution to immobilized -( -aminoethyl)-benzene sulfonamide (abs) using signal-locking surface plasmon resonance (spr) (williams et al., ) . . binding to and dissociation from a microfluidic platform of mm glycerol secreted from differentiated (murine t ) adipocytes (clark et al., ) . . binding of different concentrations of c-reactive protein (crp) to a new sandwich-type assay design using a label-free detection method. some of the other biomarker studies that have appeared in the recent literature or have been presented at conferences include the following: . multichannel mass organic analyzer and microfluidic networks for the automated in situ microchip electrophoretic analysis of organic biomarkers (benhabib et a ., ) . . hybrid magnetic-plasmonic nanoparticles for biomarkers (hirt et al., ) . . engineered knottin peptides: a new class of agents for noninvasive molecular imaging of tumor biomarkers (apte and graves, ) . . identifying secreted biomarkers for murine evasion in cellular models of cancer (kinke, ) . . spr biosensor for parallelized detection of protein biomarkers in diluted blood plasma (pilarik et al., ) . . biomarkers in drug discovery and development: from target identification through drug marketing (colburn and keefe, ) . . validation of analytic methods for biomarkers used in drug development (anonymous, ) . . electrochemical biosensors: toward point-of-care diagnostics (wang, ) . . biosensors for biomarkers in medical diagnostics (mancini and tombelll, ) . . point-of-care biosensor systems for cancer diagnostics/prognostics (sofer et al., ) . . the demonstration of the immunochemical biomarkers in methyl methacrylate-embedded plucked human hair follicles (anonymous, ) . . surface plasmon resonance biosensor based on vroman effect: toward cancer biomarker detection (choi and chase, ). . biogenic nanoporous silica-based sensor for enhanced electrochemical detection of cardiovascular biomarker proteins (lin et al., ) . . nanomonitor: a miniature electronic biosensor for glycan biomarker detection (nagaraj et al., ) . . multifunction dendrimer-template antibody presentation on biosensor surfaces for improved biomarker detection (han et al., ) . . rapid and sensitive detection of protein biomarker using a portable fluorescence biosensor based on quantum dots and a lateral flow strip (li et al., ) . . a biomarker concept for assessment of insulin resistance, beta-cell function, and chronic system inflammation in type diabetes mellitus (pfutzner et al., ) . . multifunctional au nanoparticle dendrimer-based surface plasmon resonance biosensor and its application for improved insulin detection (frasconi et al., ) . recently published reports are also available that describe in detail the different aspects of biomarkers and their applications in a clinical setting and the collaborative efforts that are required for their successful development (laria, ) . for example, they include the highlights of key technologies that are required for the development of imaging biomarkers. more importantly, case studies are presented of individual imaging biomarkers. finally, the future of imaging biomarkers is also presented. we now use fractal analysis to analyze the binding and dissociation kinetics of some of the different biomarkers available in the open literature. the examples were selected at random, with no particular bias toward analyzing a particular biomarker, or a class of biomarkers. havlin ( ) has reviewed and analyzed the diffusion of reactants toward fractal surfaces. the details of the theory and the equations involved for the binding and the dissociation phases for analyteereceptor binding are available (sadana, ) . the details are not repeated here, except that the equations are given to permit an easier reading. these equations have been applied to other biosensor systems (sadana, ; ramakrishnan and sadana, ; sadana, ) . for most applications, a single-or a dual-fractal analysis is often adequate to describe the binding and the dissociation kinetics. peculiarities in the values of the binding and the dissociation rate coefficients, as well as in the values of the fractal dimensions with regard to the dilute analyte systems being analyzed will be carefully noted, if applicable. in this chapter we analyze the binding and dissociation kinetics (if applicable) of ( ) ifn-gamma as a function of aptamer variants and inclusion of spacer in addition to spacer (tuleuova et al., ) , ( ) gst-n protein in pbs and gst-n protein in -fold diluted serum to an lpscf fiber-optic biosensor (huang et al., ) , ( ) cytochrome c mutant to a superoxide biosensor (wegerich et al., ) , ( ) ca-ii to an abs ligand on an spr biosensor surface (williams et al., ), ( ) glycerol secretion from differentiated (murine t -l ) adipocytes to a microfluidic platform for fluorescence-based assay (clark et al., ) , and ( ) different concentrations of crp in solution to a sandwich-type assay using a label-free detection method, reflectometric interference spectroscopy (albrecht et al., ) . here d f,bind or d f (used later on in the chapter) is the fractal dimension of the surface during the binding step. t c is the crossover value. havlin ( ) indicates that the crossover value may be determined by r c w t c . above the characteristic length, r c , the self-similarity of the surface is lost and the surface may be considered homogeneous. above time t c the surface may be considered homogeneous, since the self-similarity property disappears, and "regular" diffusion is now present. for a homogeneous surface where d f ¼ , and when only diffusional limitations are present, p ¼ ½ as it should be. another way of looking at the p ¼ ½ case (where d f,bind ¼ ) is that the analyte in solution views the fractal object, in our case, the receptor-coated biosensor surface, from a "large distance." in essence, in the association process, the diffusion of the analyte from the solution to the receptor surface creates a depletion layer of width (Ðt) ½ where Ð is the diffusion constant. this gives rise to the fractal power law (analyte.receptor) w t ð Àd f;bind Þ= . for the present analysis, t c is arbitrarily chosen and we assume that the value of the t c is not reached. one may consider the approach as an intermediate "heuristic" approach that may be used in the future to develop an autonomous (and not time-dependent) model for diffusion-controlled kinetics. the diffusion of the dissociated particle (receptor [ab] or analyte [ag]) from the solid surface (e.g., analyte [ag]ereceptor [ab] complex-coated surface) into solution may be given, as a first approximation by ðab$agÞz Àt ð Àdf;dissÞ= ¼ Àt p ðt > t diss Þ ( . ) here d f,diss is the fractal dimension of the surface for the dissociation step. this corresponds to the highest concentration of the analyteereceptor complex on the surface. henceforth, its concentration only decreases. the dissociation kinetics may be analyzed in a manner "similar" to the binding kinetics. sometimes, the binding curve exhibits complexities and two parameters (k, d f ) are not sufficient to adequately describe the binding kinetics. this is further corroborated by low values of r factor (goodness of fit). in that case, one resorts to a dual-fractal analysis (four parameters; k , k , d f , and d f ) to adequately describe the binding kinetics. the single-fractal analysis presented above is thus extended to include two fractal dimensions. at present, the time (t ¼ t ) at which the "first" fractal dimension "changes" to the "second" fractal dimension is arbitrary and empirical. for the most part, it is dictated by the data analyzed and experience gained by handling a single-fractal analysis. a smoother curve is obtained in the "transition" region, if care is taken to select the correct number of points for the two regions. in this case, the product (antibodyeantigen; or analyteereceptor complex, ab.ag or analyte.receptor) is given by in some cases, as mentioned above, a triple-fractal analysis with six parameters (k , k , k , d f , d f , and d f ) may be required to adequately model the binding kinetics. this is when the binding curve exhibits convolutions and complexities in its shape due perhaps to the very dilute nature of the analyte (in some of the cases to be presented) or for some other reasons. also, in some cases, a dual-fractal analysis may be required to describe the dissociation kinetics. tuleuova et al. ( ) have developed an aptamer beacon for the detection of ifn-gamma, which is an important inflammatory cytokine. boehm et al. ( ) indicate that it is secreted by immune cells in response to various pathogens. tuleuova et al. ( ) indicate that the levels of this protein provide important information with regard to infectious diseases and the ability of the body to regulate an immune response. panteleo and koup ( ) indicate that there is vigorous production of ifn-gamma in human immunodeficiency virus-infected patients. tuleuova et al. ( ) indicate that previous antibodybased detection techniques for ifn-gamma were very time consuming. jayasena ( ) indicates that aptamer-based affinity strategies are coming into prominence. ellington and szostak ( ) indicate that aptamers are single-stranded dna or rna oligonucleotides that have been selected to bind to target analytes with high specificity and affinity. aptamers have an advantage over antibodies since they are more robust; thus, aptamer-based biosensors can be regenerated and used over and over again. furthermore, balamurugan et al. ( ) , kirby et al. ( ), nutiu ( , and luzi et al. ( ) indicate that aptamers are amenable to modification due to their simplicity and robustness. tuleuova et al. ( ) indicate that fret may be used to convert aptamers into real-time biosensors (urata et al., ; babendure et al., ) . romangani et al. ( ) and karlsson et al. ( ) indicate that ifngamma is an important immune response marker. thus, tuleuova et al. ( ) have designed a novel immune response marker to detect ifn-gamma. the dna aptamer was biotinylated and immobilized on an spr sensing surface by avidinebiotin interactions. the spr biosensor was used to analyze the influence of biotinylation, fluorophore attachment, and spacer incorporation on the ability of the aptamer to bind to ifn-gamma. ifn-gamma is a type ii cytokine, and is critical for innate and adaptive immunity against viral and intracellular bacterial infections and for tumor control. incorrect ifn-gamma expression is associated with a number of autoinflammatory and autoimmune diseases. it plays an important role in immunostimulatory and immunoregulatory effects. the ifn-gamma monomer consists of six alpha-helices and an extended unfolded chain in the c-terminal region (ealick et al., ; thiel et al., ) . tuleuova et al. ( ) analyzed the influence of aptamer modification on the binding and dissociation of ifn-gamma in solution. figure . (a) shows the binding and dissociation of nm ifn-gamma in solution to the modified aptamer b ( -ggg gtt ggt tgt gtt ggg tgt tgt gt-biotin- ; sequence with modification) beacon. a single-fractal analysis is adequate to describe the binding and the dissociation kinetics. the values of ( ) the binding rate coefficient, k, and the fractal dimension for dissociation, d f , for binding and ( ) the dissociation rate coefficient, k d , and the fractal dimension for dissociation, d fd for a single-fractal analysis are given in tables . (a) and (b). tuleuova et al. ( ) indicate that the highest level of the cytokine (ifn-gamma) binding was observed for the modified aptamer b. tuleuova et al. ( ) also included a polyethylene glycol spacer between the aptamer and the biotin so that the nucleotides could be more accessible to the target analyte. modified aptamer beacon. in this case, in addition to the biotin (b) a spacer was included (bs). once again, a single-fractal analysis is adequate to describe the binding and the dissociation kinetics. the values of ( ) the binding rate coefficient, k, and the fractal dimension, d f , and ( ) the dissociation rate coefficient, k d , and the fractal dimension for dissociation, d fd , for a single-fractal analysis are given in tables . (a) and (b). it is of interest to note that as one goes from the modified aptamer beacon b to the modified aptamer beacon bs the binding rate coefficient, k, decreases by % from a value of k ¼ . to k ¼ . , and the fractal dimension, d f , increases by a factor of . from a value of d f ¼ . to d f ¼ . . in this case, changes in the fractal dimension (degree of heterogeneity on the biosensor surface) and in the binding rate coefficient are in opposite directions (tables . (a) and (b)). figure . (c) shows the binding of nm ifn-gamma in solution to the bs( -biotin-c -ggg gtt ggt tgt gtt ggg tgt tgt gt- ) modified aptamer beacon. once again, a single-fractal analysis is adequate to describe the binding and the dissociation kinetics. the values of (a) the binding rate coefficient, k, and the fractal dimension, d f , and (b) the dissociation rate coefficient, k d , and the fractal dimension for dissociation, d fd , are given in tables . (a) and (b). note that in this case there is an increase in the fractal dimension, d f (the highest when compared with the b and b's aptamer modifications), and a decrease in the binding rate coefficient, k (the lowest when compared with b and b's aptamer modifications). in this case, there is a substantial decrease in the binding rate coefficient, k. the decrease is higher than a factor of four. figure . (a) and tables . (a) and (b) show the increase in the binding rate coefficient, k, for a single-fractal analysis with an increase in the fractal dimension, d f . for the data shown in figure . (a), the binding rate coefficient, k, is given by k the fit is reasonable. only three data points are available. the availability of more data points would lead to a more reliable fit. the binding rate coefficient, k, exhibits close to a third (equal to . ) order of dependence on the fractal dimension, d f , that exists on the biosensor surface. this indicates that the binding rate coefficient, k, is sensitive to the fractal dimension, d f , or the degree of heterogeneity that exists on the biosensor surface (tables . (a) and (b)). figure . (b) and table . (a) and (b) show the increase in the affinity, k (¼k/k d ), for a single-fractal analysis with an increase in the fractal dimension ratio, d f /d fd . for the data shown in figure . (b), the affinity, k, is given by the fit is very good. only three data points are available. the availability of more data points would lead to a more reliable fit. the affinity, k (k/k d ), is very sensitive to the fractal dimension ratio (d f /d fd ) as it exhibits close to a fifth (equal to . ) order of dependence on the fractal dimension ratio (d f /d fd ). this is a very convenient way of manipulating the affinity, k, by changing the heterogeneity of the biosensor surface. some ingenuity may be required here, since a change in the degree of heterogeneity on the biosensor surface would change both the binding as well as the dissociation rate coefficients. figure . (a) shows the binding of the ifn-gamma to the biotin þ aptamer b variant (tuleuova et al., ) . once again, a dual-fractal analysis is required to describe the binding kinetics. a single-fractal analysis is adequate to describe the dissociation kinetics. the values of ( ) the binding rate coefficient, k, and the fractal dimension, d f , for a single-fractal analysis, ( ) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f , for a dual-fractal analysis, and ( ) the dissociation rate coefficient, k d , and the fractal dimension for dissociation, d fd , for a single-fractal analysis are given in tables . (a) and (b). in this case, the affinity values k (¼k /k d ) and k (¼k /k d ) are . and . , respectively (tables . (a) and (b)). note that for dual-fractal analysis for the binding phase, an increase in the fractal dimension by . % from a value of d f ¼ . to d f ¼ . leads to an increase in the binding rate coefficient by a factor of . from a value of k ¼ . to k ¼ . . tuleuova et al. ( ) also investigated the influence of a fluorophore (f) in addition to the biotin for the modified aptamer b during the binding of ifngamma. this was one of the aptamer variants. figure . (b) shows that a dual-fractal analysis is required to describe the binding kinetics. a singlefractal analysis is adequate to describe the dissociation kinetics. the values of ( ) the binding rate coefficient, k, and the fractal dimension, d f , for a singlefractal analysis, ( ) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f , for a dual-fractal analysis, and ( ) the dissociation rate coefficient, k d , and the fractal dimension for dissociation, d fd , for a singlefractal analysis are given in tables . (a) and (b). the affinity values k (¼k /k d ) and k (¼k /k d ) are . and . , respectively. note that for dual-fractal analysis for the binding phase, an increase in the fractal dimension by . % from a value of d f ¼ . to d f ¼ . leads to an increase in the binding rate coefficient by a factor of . from a value of k ¼ . to k ¼ . (tables . (a) and (b)). also, note that on comparing the affinity values k and k when the fluorophore is used and not used, the k value is slightly lower and the k value is significantly higher (by about %), respectively. huang et al. ( ) have developed a localized lspcf fiber-optic biosensor for the detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in human serum. these authors indicate that sars is a highly infectious disease. drosten et al. ( ) indicate that sars results in death in a large portion of patients. the sars coronavirus (sars-cov) causes sars, and is detectable in the respiratory secretions of patients after infection (foucher et al., ) . wang et al. ( ) emphasize that sars is highly contagious and exhibits the potential of becoming a large-scale future epidemic if effective therapeutic drugs are not discovered. huang et al. ( ) and che et al. ( ) emphasize the need for a rapid, sensitive, specific, and an accurate diagnostic method so that specific patients may be correctly assessed. huang et al. ( ) indicate that there are methods available to detect sars. however, present methods such as reverse transcriptase polymerase chain reaction are not sensitive enough, and also require a specific laboratory with expertise in molecular diagnostics to confirm sars in the acute phase (fujimoto et al., ; drosten et al., ) . huang et al. ( ) indicate that gold nanoparticles (gnps) have been introduced into biosensing (manso et al., ; cui et al., ) . these gnps possess special properties such as localized surface plasmons. huang et al. ( ) have developed a novel fiber-optic biosensor where the property of lspcf has been combined with the sandwich immunoassay. huang et al. ( ) have used their lspcf fiber-optic biosensor to detect sars-cov protein in diluted serum to a limit of pg/ml. this according to these authors exhibits the potential for the early detection of clinical sars-cov infection. figure . shows the binding of l pg/ml gst-n protein in solution to the lspcf biosensor (huang et al., ) . a single-fractal analysis is required to describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single-fractal analysis are given in tables . (a) and (b). huang et al. ( ) prepared gst-n protein samples in -fold diluted human serum. this allowed them to test their lspcf biosensor in clinical samples. they measured the temporal fluorescence intensity of the biomolecular interaction between the lspcf probes and the gst-n protein. they did this for the e pg/ml rages gst-n protein in solution. figure . (a) shows the binding of pg/ml gst-n protein in -fold diluted human serum in solution. a dual-fractal analysis is required to adequately describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single fractal analysis, and (b) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f for a dual-fractal analysis are given in tables . (a) and (b). for a dualfractal analysis, an increase in the fractal dimension by a factor of . from a value of d f equal to . to d f equal to . leads to an increase in the binding rate coefficient by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the fractal dimension or the degree of heterogeneity on the lspcf biosensor surface leads to an increase in the binding rate coefficient. figure . (b) shows the binding of pg/ml gst-n protein in -fold diluted human serum in solution. once again, a dual-fractal analysis is required to adequately describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single fractal analysis, and (b) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f for a dual-fractal analysis are given in tables . (a) and (b). for a dual-fractal analysis, an increase in the fractal dimension by a factor of . from a value of d f equal to . to d f equal to . leads to an increase in the binding rate coefficient by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the fractal dimension or the degree of heterogeneity on the lspcf biosensor surface leads to an increase in the binding rate coefficient. figure . (c) shows the binding of pg/ml gst-n protein in -fold diluted human serum in solution. a dual-fractal analysis is required to adequately describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single fractal analysis, and (b) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f for a dual-fractal analysis are given in tables . (a) and (b) once again, for a dual-fractal analysis, an increase in the fractal dimension by a factor of . from a value of d f equal to . to d f equal to . leads to an increase in the binding rate coefficient by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the fractal dimension or the degree of heterogeneity on the lspcf biosensor surface leads to an increase in the binding rate coefficient. tables . (a) and (b) show for a dual-fractal analysis the increase in the fractal dimension, d f with an increase in the gst-n protein concentration in solution in the e pg/ml range. figure not shown. for the data shown in table . the fractal dimension, d f is given by: d f ¼ ð : ae : Þ ½gst À n : ae : ( . ) the fit is reasonable. only three data points are available. the availability of more data points would lead to a more reliable fit. the fractal dimension, d f exhibits less than one-half (equal to . ) order of dependence on the gst-n protein concentration in solution in the e pg/ml range. this indicates that the fractal dimension, d f exhibits a mild dependence on the gst-n protein concentration in solution. the superoxide anion radical is present in several pathophysiological situations, such as sepsis (valko et al., ; vaklko et al., ) . electrochemical biosensors can detect this short-lived species (lisdat, ; prieto-simon et al., ) . wegerich et al. ( ) indicate that the redox protein cytochrome c is used as a recognition element. these authors indicate that superoxide dismutase (sod) biosensors used for the detection of the superoxide anion often lack the reproducibility due to immobilization problems. however, cyt c-based superoxide biosensors are more stable and may be used in in vivo applications (buttemeyer et al., ; scheller et al., ) . in this case, the heme protein is reduced by the superoxide, followed by reduction by an electrode. wegerich et al. ( ) indicate that short-chain modified gold electrodes exhibit a highly efficient communication between cyt c and the electrode (frew and hill, ; hinnen et al., ; nahir et al., ; taniguchi et al., ) . they have been used for cyt c based superoxide sensors. wegerich et al. ( ) analyzed the effect of introducing positive charges (lysines) in human cytochrome c on the redox properties and reaction rates of cyt c with superoxide radicals. these authors claim that the eleven mutants analyzed were modified for structural integrity as well as axial coordination of the heme ion. their results indicate that four mutants exhibited a higher reaction rate with the radical as compared with the wild type. these mutants were then used for the construction of the superoxide biosensors. figure . shows the binding of cyt c in solution to the superoxide biosensor (wegerich et al., ) . a single-fractal analysis is adequate to describe the binding kinetics. the binding rate coefficient, k and the fractal dimension, d f , for a single-fractal analysis are . ae . , and . ae . , respectively. these results are also shown in tables . (a) and (b). a single-fractal analysis is also adequate to describe the dissociation kinetics. the values of the dissociation rate coefficient, k d and the fractal dimension, d fd are . ae . and . and . , respectively. in this case the affinity, k (¼k/k d ) is equal to . . williams et al. ( ) have recently analyzed low noise detection of biomolecular interactions with signal-locking surface plasmon resonance. surface plasmon resonance is a popular technique to analyze biomolecular interactions at a surface, especially since it is label-free. these authors indicate that the spr technique is subject to the influence of noise and drift disturbances since that limits the minimum detectable mass change. the spr technique uses the step response of the biomolecular interactions occurring on the biosensor surface. the technique proposed by williams et al. ( ) measures the biomolecular interactions over a very narrow frequency range. this locks the measured response to a very specific narrow band signal. the authors used their technique to analyze the binding kinetics of carbonic anhydrase-ii (ca-ii) and immobilized -( -aminoethyl)-benzenesulfonamide (abs) to a spr surface. carbonic anhydrases are a family of enzymes that catalyze the rapid conversion of carbon dioxide and water to bicarbonate and protons. these anhydrases are classified as metalloenzymes since the active site of most carbonic anhydrases contains a zinc ion. the primary function of this enzyme in animals is to maintain the acid-base balance in blood and other tissues, and to help transport carbon dioxide out of tissues. carbon anhydrase ii is a novel biomarker for gasterointestinal stomal tumors (parkkila et al., ) . these authors indicate that various carbonic anhydrase (ca) isoenzymes have been identified as potential targets against different cancers. they further indicate that high ca-ii expression is associated with a better disease specific survival rate than low or no expression. figure . (a) shows the binding of . mm ca-ii anhydrase in solution to a -( -amino ethyl)-benzene sulfonamide (abs) ligand on a spr biosensor surface (williams et al., ) . a dual-fractal analysis is required to adequately describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single fractal analysis, and (b) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f for a dualfractal analysis are given in tables . (a) and (b). it is of interest to note that as the fractal dimension increase by a factor of . from a value of d f equal to . to d f equal to . , the binding rate coefficient increases by a factor of . from a value of k equal to . to k equal to . . figure . (b) shows the binding of . mm ca-ii anhydrase in solution to a -( -amino ethyl)-benzene sulfonamide (abs) ligand on a spr biosensor surface (williams et al., ) . once again, a dual-fractal analysis is required to adequately describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single fractal analysis, and (b) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f for a dual-fractal analysts are given in tables . (a) and (b). it is of interest to note that as the fractal dimension increase by a factor of . from a value of d f equal to . to d f equal to . , the binding rate coefficient increases by a factor of . from a value of k equal to . to k equal to . . figure . (c) shows the binding of . mm ca-ii anhydrase in solution to a -( -amino ethyl)-benzene sulfonamide (abs) ligand on a spr biosensor surface (williams et al., ) . once again, a dual-fractal analysis is required to adequately describe the binding kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single fractal analysis, and (b) the binding rate coefficients, k and k , and the fractal dimensions, d f and d f for a dual-fractal analysis are given in tables . (a) and (b) it is of interest to note that as the fractal dimension increase by a factor of . from a value of d f equal to . to d f equal to . , the binding rate coefficient increases by a factor of . from a value of k equal to . to k equal to . . figure . (a) and table . (a) show the increase in the binding rate coefficient, k with an increase in the ca-ii concentation in solution in the . e mm range. for the data shown in figure . (a), the binding rate coefficient, k , is given by: k ¼ ð : ae : Þ ½ca À ii : ae : ( . a) the fit is good. only three data points are available. the availability of more data points would lead to a more reliable fit. the binding rate coefficient, k exhibits less than one-half (equal to . ) order of dependence on the ca-ii concentration in solution in the . e . mm range. this indicates that the binding rate coefficient, k is only mildly sensitive to the ca-ii concentration in solution. figure . (b) and table . (a) show the increase in the fractal dimension d f , p with an increase in the ca-ii concentration in solution in the . e mm range. for the data shown in figure . (b), the fractal dimension, d f is given by: d f ¼ ð : ae : Þ½ca À ii : ae : ( . b) the fit is good. only three data points are available. the availability of more data points would lead to a more reliable fit. the fractal dimension, d f exhibits a very mild dependence (equal to . ; close to zero order) on the ca-ii concentration in solution in the . e . mm range. this indicates that the fractal dimension, d f is only mildly sensitive to the ca-ii concentration in solution. the fractal dimension is based on a log scale, and even small changes in the fractal dimension indicate a reasonable change in the degree of heterogeneity on the biosensor surface. figure . (c) and tables . (a) and (b) show the increase in the binding rate coefficient, k with an increase in the fractal dimension, d f . for the data shown in figure . (b), the binding rate coefficient, k is given by: k ¼ ð : ae : Þd : ae : f ( . c) the fit is good. only three data points are available. the availability of mora data points would lead to a more reliable fit. the binding rate coefficient, k exhibits close to a second (equal to . ) order of dependence on the fractal dimension, d f this indicates that the binding rate coefficient, k is sensitive to the fractal dimension, d f or the degree of heterogeneity on the biosensor surface. (b) show the increase in the binding rate coefficient ratio, k /k with an increase in the fractal dimension ratio d f /d f . for the data in figure . (d) the binding rate coefficient ration k /k is given by: the fit is good. only three data points are available. the availability of more data points would lead to a more reliable fit. the binding rate coefficient ratio, k /k exhibits an order of dependence between three and three and one half (equal to . ) on the ratio of the fractal dimensions, d f /d f . this indicates that the binding rate coefficient ratio is very sensitive to the fractal dimension ratio figure . (e) shows the increase in the ratio of the binding rate coefficient, d f /d f with an increase in the ca-ii concentration in solution. the ratio, d f /d f is only mildly dependent on the ca-ii concentration in solution. clark et al. ( ) have recently developed a continuous-flow enzyme assay on a microfluidic chip for monitoring glycerol secretion from cultivated adipocytes. these authors indicate that different studies on using chips to monitor cellular secretion have appeared in the literature (cheng et al., ; lau et al., ; el-all et al., ; kim et al., ; meyvantsso et al., ; urbanskl et al., ) . clark et al. ( ) indicate that physiological studies need to maintain cells or tissues in a controlled environment as one detects their physical, electrical and mechanical properties. these authors indicate micriofluidics facilitates such situations, since they permit creation of highly controlled cell-compatible environments along with measurement and cell maniplation methods. clark et al. ( ) emphasize that the prevalence of obesity-related disorders underscores the need to adipocyte physiology. adipocytes store and release energy. adipocytes store energy as triacylglycerol by lipolysls. ths supplies energy for tissues and organs. getty et al. ( ) and getty-kaushik et al., ( a,b) indicate that the measurement of glycerol is used to determine the function and physiological state of adipocytes. clark et al. ( ) have developed a dual-chip microfluidic system for culturing adipocytes and then monitoring the glycerol using a continuous fluorescent enzyme assay after a perfusion step. the authors used their system to demonstrate transient increases in glycerol secretion during exposure of the cells to isoproterenol, a b-adrenergic agonist. these adrenergic agonists act on receptors. beta receptors are specific molecules found in the body which receive and process signals for the nervous system and various hormones. these beta receptors are located at many places in the body, but are found in high numbers in the heart and blood vessels. here they increase blood pressure when stimulated. thus, they are attractive targets for high blood pressure treatment. clark et al. ( ) analyzed the glycerol secretion data from differentiated adipocytes and response to isoproterenol treatment. figure . (a) shows the binding of glycerol secretion from differentiated (murine t -l ) adipocytes by a continuous-flow enzyme assay on a microfluidic chip. a dual-fractal analysis is required to model the binding and the dissociation kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single-fractal analysis, (b) the binding rate coefficients, k and k and the fractal dimensions, d f and d f for a dual-fractal analysis, (c) the dissociation rate coefficient, k d and the fractal dimension, d fd for a single-fractal analysis, and (d) the dissociation rate coefficients, k d and k d and the fractal dimensions for the dissociation phase, d fd and d fd for a dual-fractal analysis are given in tables . (a) and (b). figure . (b) shows the binding of glycerol secretion from differentiated (murine t -l ) adipocytesin the presence of iso proterenol by a continuous-flow enzyme assay on a microfluidic chip. a dual-fractal analysis is required to model the binding and the dissociation kinetics. the values of (a) the binding rate coefficient, k and the fractal dimension, d f for a single-fractal analysis, (b) the binding rate coefficients, k and k and the fractal dimensions, d f and d f for a dual-fractal analysis, and (c) the dissociation rate coefficient, k d and the fractal dimension, d fd for a single-fractal analysis are given in tables . (a) and (b). albrecht et al. ( ) have recently presented a new assay design for clinical diagnostics based on alternative recognition. these indicate that the assay format has an important impact in the practical handling as well in the sensitivity of the testing results. jaras et al. ( ) indicate that for clinical diagnostics the sandwich assay format is frequently used due to (a) its lower limits of detection compared to other formats, (b) and reliable analysis of the different parameters. albrecht et al. ( ) further indicate that a drawback of the sandwich assay format is the need for immobilization of the capture antibody on the surface. this often results in a significant loss in binding activity. also, there is no guarantee that the binding sites on the antibodies immobilized on the surface are oriented in the 'correct' direction. this hinders the biosensor performance parameters such as sensitivity, loss in function, and stability of the sensor surface. in essence, these authors indicate that the recognition element on the sensor surface needs to exhibit a high affinity and specificity towards the antigen (analyte) in solution on being immobilized on the biosensor surface. albrecht et al. ( ) have presented an immunoassay set-up that uses a small and stable peptide sequence as the immobilized recognition element (receptor) (baltzer, ) . albrecht et al. ( ) indicate that their recognition elements are small helix loop-helix motifs. these recognition elements contain natural binders of the target analyte. furthermore, these motifs are easily accessible. also, these authors indicate concerted modifications made for immobilization at the artificial helices do not affect binding properties. albrecht et al. ( ) have presented a new sandwich-type assay for the detection of c-reactive protein (crp). they used a tailored binder as the capture element on the sensor surface, and an antibody as a detection element. c-reactive protein is a protein found in blood. its levels rise in response to inflammation. thompson et al. ( ) indicate that its physiological role is to bind to phosphocholine expressed on the surface of dead or dying cells in order to activate the complement system via the c q complex. crp is a general marker for inflammation and infection. it can be used as a very rough proxy for heart disease risk. lloyd-jones et al. ( ) emphasize that since many factors are responsible for crp level elevations, thus it is not a very specific prognostic indicator. also the patients with elevated basal levels of crp are at an increased risk of diabetes (pradhan et al., ) , hypertension and cardiovascular disease. figure . (a) shows the binding and dissociation of .  À m crp in solution to the new sandwich assay design that contains a high affinity polypeptide scaffold as the immobilized capture element and an antibody for detection (albrecht et al., ) . as mentioned above a biosensor based on reflectometric interference spectroscopy (rlfs) was used. a dual-fractal analysis is required to describe the binding kinetics. a single-fractal analysis is adequate to describe the dissociation kinetics. the values of (a) the binding rate coefficient k and the fractal dimension, d f for a single-fractal analysis, (b) the binding rate coefficients, k and k and the fractal dimensions, d f and d f for a dualfractal analysis, and (c) the dissociation rate coefficient, k d and the fractal dimension, d fd for a single-fractal analysis are given in tables . (a) and (b). it is of interest to note that for a dual-fractal analysis, an increase in the fractal dimension by a factor of . from a value of d f equal to . to d f equal to . leads to an increase in the binding rate coefficient by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the degree of heterogeneity on the biosensor surface leads to an increase in the binding rate coefficient. in this case, the affinity, k (¼k /k d ) and k (¼k /k d ) are . and . , respectively. figure . (b) shows the binding and dissociation of .  À m crp in solution to the new sandwich assay design (albrecht et al., ) . a dualfractal analysis is once again required to describe the binding kinetics. a single-fractal analysis is adequate to describe the dissociation kinetics. the values of (a) the binding rats coefficient k and the fractal dimension, d f for a single-fractal analysis, (b) the binding rate coefficients, k and k and the fractal dimensions, d f and d f for a dual-fractal analysis, and (c) the dissociation rate coefficient, k d and the fractal dimension, d fd for a single-fractal analysis are given in tables . (a) and (b). it is of interest to note that for a dual-fractal analysis, an increase in the fractal dimension by a factor of . from a value of d f equal to . to d f equal to . leads to an increase in the binding rate coefficient by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the degree of heterogeneity on the biosensor surface leads to an increase in the binding rate coefficient. in this case, the affinity, k (¼k /k d ) and k (¼k /k d ) are . and . , respectively. figure . (c) shows the binding and dissociation of .  À m crp in solution to the new sandwich assay design (albrecht et al., ) . a dualfractal analysis is once again required to describe the binding kinetics. a single-fractal analysis is adequate to describe the binding kinetics. the values of (a) the binding rate coefficient k and the fractal dimension, d f for a single-fractal analysis, (b) the binding rate coefficients, k and k and the fractal dimensions, d f and d f for a dual-fractal analysis, and (c) the dissociation rate coefficient, k d and the fractal dimension, d fd for a single-fractal analysis are given in tables . (a) and (b) it is of interest to note that for a dual-fractal analysis, an increase in the fractal dimension by a factor of . from a value of d f equal to . to d f equal to . leads to an increase in the binding rate coefficient by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the degree of heterogeneity on the biosensor surface leads to an increase in the binding rate coefficient. in this case, the affinity, k (¼k /k d ) and k (¼k /k d ) are . and . , respectively. figure . (a) and table . (a) show for a dual-fractal analysis the decrease in the binding rate coefficient, k with an increase in the crp concentration in solution in the . À .  À m range. for the data shown in figure . (a) the binding rate coefficient, k is given by: k ¼ ð : ae : Þ½crp À : ae : ( . a) the fit is good. only three data points are available. the availability of more data points would lead to a more reliable fit. the binding rate coefficient, k decreases with an increase in the crp concentration in solution in the . À .  À m concentration range, and exhibits less than a negative one half (equal to - . ) order of dependence on the crp concentration in solution. figure . (b) and table . (a) show for a dual-fractal analysis the increase in the binding rate coefficient, k with an increase in the fractal dimension, d f . for the data shown in figure . (b) the binding rate coefficient, k is given by: the fit is very good. only three data points are available. the availability of more data points would lead to a more reliable fit. the binding rate coefficient, k exhibits close to a seven and one-half (equal to . ) order of dependence on the fractal dimension, d f on the biosensor surface. this indicates that the binding fate coefficient, k is very sensitive to the fractal dimension or the degree of heterogeneity on the biosensor surface. figue . (c) show for a dual-fractal analysis the decrease in the fractal dimension, d f with an increase in the crp concentration in solution in the . À .  À m range. for the data shown in figure . (c) the fractal dimension, d f is given by: the fit is good. only three data points are available. the availability of more data points would lead to a more reliable fit. the fractal dimension, exhibits a very mild dependence on the crp concentration in the . À .  À m concentration range. figure . (d) shows the increase in the affinity k (¼k /k d ) with an increase in the crp concentration in solution in the . À .  À m range. for the data shown in figure . (d), the affinity, k is given by: the fit is reasonable. only three data points are available. the availability of more data points would lead to a more reliable fit. the availability of more data points would lead to a more reliable fit. the affinity, k exhibits only a mild, less than one-half (equal to . ) order of dependence on the crp concentration in solution in the . À .  À m range. figure . (e) shows the increase in the affinity k (¼k /k d ) with an increase in the crp concentration in solution in the . À .  À m range. for the data shown in figure . (e), the affinity, k is given by: the fit is very good. only three data points are available. the availability of more data points would lead to a more reliable fit. the availability of more data points would lead to a more reliable fit. the affinity, k exhibits only a mild, less than one-half (equal to . ) order of dependence on the crp concentration in solution in the . À .  À m range. figure . (f) shows the decrease in the dissociation rate coefficient, k d with an increase in the crp concentration in solution in the . À . m  À m range. for the data shown in figure : (f), the dissociation rate coefficient k d is given by: k d ¼ ð : ae : Þ ½crp À : À : ( . f) the fit is poor. there is scatter in the data. this is reflected in the error in the estimated value of the order of dependence of k d on the crp concentration in solution. only the negative sign is applicable since the dissociation rate coefficient, k d decreases with an increase in the crp concentration in solution. figure . (g) and table . (a) and (b) show the increase in the dissociation rate coefficient, k d with an increase in the fractal dimension for dissociation, d fd . for the data shown in figure . (g), the dissociation rate coefficient, k d is given by: the fit is good. only three data points are available. the availability of more data points would lead to a more reliable fit. the dissociation rate coefficient, k d exhibits close to a three and one-half (equal to . ) order of dependence on the fractal dimension in the dissociation phase, d fd . this indicates that the dissociation rate coefficient, k d is very sensitive to the degree of heterogeneity that exists on the biosensor surface in the dissociation phase. figure . (h) shows the decrease in the affinity, k (¼k /k d ) with an increase in the ratio of the fractal dimensions, (d f /d fd ). for the data shown in figure . (h) the affinity, k is given by: k ¼ ðk =k d Þ ¼ ð : ae : Þ ðd f =d fd Þ À : À : : ( . h) the fit is poor. only three data points are available. the availability of more data points would lead to a more reliable fit. the poor fit is expressed as the error in the power to which the ration of the fractal dimensions is raised. only the negative power is applicable since the affinity, k decreases with an increase in the fractal dimension, ratio, d f /d fd . tang et al. ( ) have recently developed an integrated automatic electrochemical immunosensor array for the detection of five hepatitis virus antigens: hepatitis a virus (hav), hepatitis b virus (hbv), hepatitis c virus (hcv), hepatitis d virus (hdv), and hepatitis e virus (hev) (alavian and ballantian, ) . tang et al. ( ) further indicate that hepatitis viruses are one of the leading causes of mortality (bilora et al., ) . thus, an early diagnosis for hepatitis b viruses is critical. tang et al. ( ) further emphasize that the simultaneous determination of multiple virus antigens is helpful in clinical diagnosis since the patient usually suffers from multiple virus antigens (cornberg et al., ; gitlin, ) . tang et al. ( ) emphasize that potentiometric assays are highthroughput systems, are label-free, exhibit low assay cost, and their simplicity permits miniaturization as well as signal quantification (wu et al., ; tang et al., ) . thus, tang et al., ( ) have developed their electrochemical immunosensor array for the simultaneous determination of five-type hepatitis virus antigens in five minutes. the binding of ng/ml of hav to the immunosensor array (tang et al., ) may be modeled by a dual-fractal analysis. figure not shown. the values of the binding rate coefficient, k and the fractal dimension, d f for a single-fractal analysis ar . ae . and . ae . , respectively. for a dual-fractal analysis, (a) the binding rate coefficients, k and k are . ae . , and . ae . , respectively, and (b) the fractal dimensions, d f and d f are . ae . , and . ae . , respectively. note that as the fractal dimension increases by a factor of . from a value of d f equal to . to d f equal to . , the binding rate coefficient increases by a factor of . from a value of k equal to . to k equal to . . once again, an increase in the degree of heterogeneity or the fractal dimension on the immunosensor array surface leads to an increase in the binding rate coefficient. a fractal analysis is used to analyze the binding and dissociation (if applicable) kinetics of biomarkers to different biosensor surfaces. both single-and a dual-fractal analyses are used to analyze the binding and the dissociation kinetics. the dual-fractal analysis is used only if the single-fractal analysis does not provide an adequate fit. for the binding and dissociation of ifn-gamma in solution to the aptamer modification (tuleuova et al., ) , and for a single-fractal analysis, the ( ) binding rate coefficient, k, exhibits close to a third (equal to . ) order of dependence on the fractal dimension or the degree of heterogeneity that exists on the biosensor surface and ( ) the affinity, k (=k/kd), exhibits close to a fifth (equal to . ) order of dependence on the ratio of fractal dimensions, d f /d fd . this indicates that both the binding rate coefficient, k, and the affinity, k, are very sensitive to the nature or the degree of heterogeneity that exists on the biosensor surface. for the binding of different concentrations of ca-ii anhydrase in solution ( ) the binding rate coefficient, k, exhibits a mild (equal to . ) order of dependence on the ca-ii anhydrase concentration in solution, ( ) the binding rate coefficient, k, exhibits close to a second (equal to . ) order of dependence on the fractal dimension, d f , that exists on the biosensor surface, and ( ) the ratio of the binding rate coefficients, k /k , exhibits higher than a third (equal to . ) order of dependence on the ratio of fractal dimensions, d f /d f . the relationships presented above are typical of the ones presented for the biomarkers for the other diseases analyzed and presented in this chapter. they provide a means by which these rate coefficients or affinities may be manipulated in desired directions in order to improve the different biosensor performance parameters. the more sensitive a biosensor is for a specific biomarker for a particular disease the earlier it may be detected. needless to say the early detection of biomarkers for different diseases should lead to a better prognosis. surely, as expected, there is considerable effort and resources being spent in this direction, and correctly so. alzheimer's: a new theory a new assay design for clinical diagnostics based on alternate recognition elements validation of analytic methods for biomarkers used in drug development transcriptional analysis of an e f gene signature as a biomarker of activity of the cyclin-dependent kinase inhibitor pha- in tumor and skin biopsies from a phase i clinical study engineered knottin peptides: a new class of agents for non-invasive molecular imaging of tumor biomarkers polypeptide conjugate binders for protein recognition multichannel mass organic analyzer (momoa) microfluidic networks for the automated in situ microchip electrophoretic analyzers of organic biomarkers multichannel capillary electrophoresis microdevice and instrumentation for in situ planetary analysis of organic molecules and biomarkers surface plasmon resonance biosensor based on vroman effect; towards cancer biomarker detection continuous-flow enzyme assay on a microfluidic chip for monitoring glycerol secretion from cultured adipocytes continuous flow enzyme assay on a microfluidic chip for monitoring glycerol secretion from cultured adipocytes biomarkers in drug discovery and development: from target identification through drug marketing bioavailability and bioequivalence average population and/or individual on chip electrochemical detection of biomarkers for detection of water borne toxins genetic variation, c-reactive protein levels, and incidence of diabetes development of a high sensitivity rapid sandwich elisa procedure and its comparison with 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comparison from a clinical perspective identifying secreted biomarkers for immune evasion in cellular models of cancer advances in imaging biomarkers, innovative technologies, applications in r & d and clinical practice, informatics, and regulatory requirements encyclopedia of sensors lab chip , e biogenic nanoporous silica-based sensor for enhanced electrochemical detection of cardiovascular biomarker proteins narrative reviews: assessment of c-reactive protein in risk prediction for cardiovascular disease biosensors for biomarkers in medical diagnostics carboni anhydrase ii, a novel biomarker for gastrointestinal stomal tumors a biomarker concenpt for assessment of insulin resistance, beta-cell function and chronic systemic inflammation in type diabetes mellitus surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma c-reactive protein, interleukin , and risk of developing type diabetes mellitus a single-fractal analysis of cellular analyte-receptor binding kinetics using biosensors analysis of the role of interferon-gamma, interleukin and a third factor distinct from interferon-gamma and interleukin in human b cell proliferation. evidence that they act at different times after b cell activation a fractal analysis for the evaluation of hybridization kinetics in biosensors fractal binding and disseriation kinetics for different biosensors applications a dual enzyme electrochemical assay for the detection of organophosphorus compounds using organophosphorus hydrolase and horseradish peroxidase point-of-eare biosensor system for cancer diagnostics/prognostics simultaneous determination of five-type hepatitis virus antigens in min using an integrated automatic electrochemuical immunosensor array observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex the physiological structure of human c-reactive protein and its complex with phosphocholine development of an aptamer beacon for detection of interferon-gamma chemical-biological interactions sars associated coronavirus transmitted from human to pig electrochemical biosensors; towards point-of care cancer diagnostics cytochrome c mutants for superoxide biosensors low noise detection of biomolecular interactions with signal locking surface plasmon resonance low noise detection of biomolecular interactions with signal-locking surface plasmon resonance key: cord- -o rlqsq authors: ghosh, arun k.; brindisi, margherita title: organic carbamates in drug design and medicinal chemistry date: - - journal: j med chem doi: . /jm s sha: doc_id: cord_uid: o rlqsq [image: see text] the carbamate group is a key structural motif in many approved drugs and prodrugs. there is an increasing use of carbamates in medicinal chemistry and many derivatives are specifically designed to make drug–target interactions through their carbamate moiety. in this perspective, we present properties and stabilities of carbamates, reagents and chemical methodologies for the synthesis of carbamates, and recent applications of carbamates in drug design and medicinal chemistry. carbamate-bearing molecules play an important role in modern drug discovery and medicinal chemistry. organic carbamates (or urethanes) are structural elements of many approved therapeutic agents. structurally, the carbamate functionality is related to amide-ester hybrid features and, in general, displays very good chemical and proteolytic stabilities. carbamates are widely utilized as a peptide bond surrogate in medicinal chemistry. this is mainly due to their chemical stability and capability to permeate cell membranes. another unique feature of carbamates is their ability to modulate inter-and intramolecular interactions with the target enzymes or receptors. the carbamate functionality imposes a degree of conformational restriction due to the delocalization of nonbonded electrons on nitrogen into the carboxyl moiety. in addition, the carbamate functionality participates in hydrogen bonding through the carboxyl group and the backbone nh. therefore, substitution on the o-and n-termini of a carbamate offers opportunities for modulation of biological properties and improvement in stability and pharmacokinetic properties. carbamates have been manipulated for use in the design of prodrugs as a means of achieving first-pass and systemic hydrolytic stability. carbamate derivatives are widely represented in agricultural chemicals, such as pesticides, fungicides, and herbicides. they play a major role in the chemical and paint industry as starting materials, intermediates, and solvents. furthermore, organic carbamates serve a very important role as optimum protecting groups for amines and amino acids in organic synthesis and peptide chemistry. in recent years, carbamate derivatives have received much attention due to their application in drug design and discovery. however, there are hardly any reviews on this subject in the literature. in the present perspective, we plan to provide an overview of the leading role of organic carbamates in medicinal chemistry, with particular focus on therapeutic carbamates and carbamate-based prodrugs. in this context, we will highlight the chemical methodologies adopted for the synthesis of these carbamate derivatives. also, we will outline successful designs of organic carbamates, including a variety of cyclic ether-derived carbamates, as suitable amide bond surrogates leading to a wide range of novel organic carbamates as potent hiv- protease, βsecretase, serine protease, and cysteine protease inhibitors. this information may be useful in further design of carbamate-based molecules as drugs or prodrugs. peptide-based molecules are an important starting point for drug discovery, especially in the design of enzyme inhibitors. because of their high affinity and specificity toward biological functions, peptide-based molecules also serve as valuable research tools. however, the poor in vivo stability, inadequate pharmacokinetic properties, and low bioavailability have generally limited their broader utility. hence, a variety of peptide mimics are being developed to improve drug-like character along with increased potency, target specificity, and longer duration of action. − to this end, several classes of peptidomimetics are tailored by replacing the native amide bond with unnatural linkages − such as retro-amide, urea, − carbamate, and heterocycles , as peptide bond surrogates. these functionalities confer metabolic stability toward aminopeptidases, the enzymes involved in the metabolism of peptidelike drugs. the carbamate's emerging role in medicinal chemistry is also due to its chemical stability and to its capability to increase permeability across cellular membranes. these attributes of organic carbamates have been exploited in drug design. as a result, the carbamate motif is becoming the choice for peptide bond surrogates. other uses of carbamates are well-known. particularly, the employment of carbamates in various industries as agrochemicals, in the polymer industry, and also in peptide syntheses. − in addition, among the various amineprotecting groups, carbamates are commonly used to enhance their chemical stability toward acids, bases, and hydrogenation. one important feature of organic carbamates is represented by the amide resonance. the amide resonance in carbamates has been studied in detail employing both experimental and theoretical methods by estimating the c−n bond rotational barriers. − the amide resonance in carbamates has been shown to be about − kcal mol − lower than those of amides, owing to the steric and electronic perturbations due to the additional oxygen. three possible resonance structures (a, b, and c, figure ) contribute to the stabilization of the carbamate moiety. carbamate motifs are characterized by a pseudo double bond. this implies the potential deconjugation of the heteroatom-(σ-bond)-carbon-(π-bond)-heteroatom system that restricts the free rotation about the formal single σ-bond. therefore, two isomers, syn and anti, may coexist in carbamates ( figure ) . , although carbamates display close similarity to amides, they show preference for the anti-isomer conformation. the anti rotamer is usually favored by . − . kcal mol − for steric and electrostatic reasons with respect to the syn counterpart. in many cases, the energy difference may be close to zero. as a result, those carbamates are found as an approximately : mixture of syn and anti isomers, as in the case of a number of boc-protected amino acid derivatives. this issue is of key importance since this balanced rotamer equilibria and the low activation energies render carbamates as optimal conformational switches in molecular devices. the influence of the r and r substituents on the free-energy difference between the two conformations has been investigated. beyond steric effects, electronegativity of r must be considered since it may affect the conformation in many ways, including changes in the dipole moment and bond angles. only the anti conformation would be expected in five-, six-, and seven-membered cyclic carbamates. calculations of the dipole moment for the carbamate group support this expectation. solvent, concentration, salts, and ph strongly influence the free energy difference of the syn and anti isomers of carbamates as well. intra-and intermolecular hydrogen bonding may also perturb the syn−anti isomer equilibrium of carbamates. , , a representative example of hydrogen bonding and concentration dependence was provided by gottlieb, nudelman, and collaborators. the authors took into consideration n-boc-amino acids and their corresponding methyl esters. an unusual abundance of syn-rotamer for n-boc-amino acids was detected. n-boc-amino acid esters give the expected spectra, consistent with previous reports of only a single species being observed at room temperature. concentration-dependent h nmr spectra indicate that the proportion of the syn-rotamers increases with concentration, supporting the existence of an aggregation process. since decreasing temperature is another method for stabilizing oligomerization, nmr experiments were also performed at different temperatures. as expected, when the temperature increases, the favored rotamer switches from syn to anti. overall, the collected data strongly supports the concept that the syn rotamers of n-carbamoylated amino acids form intermolecularly h-bonded species and the oh of the carboxylic acid must be involved in this process, as the corresponding esters do not behave similarly. to explain this phenomenon, the formation of a dimer was suggested ( figure ). support of this hypothesis was provided by adding increasing amounts of acetic acid to a solution of a carbamoylated amino acid ester. as expected, the syn rotamer appeared, and its concentration increased as a function of the amount of acid added. in contrast, addition of acetic acid to a solution of the corresponding carbamoylated amino acid did not affect the anti/syn ratio. in this context, moraczewski and co-workers designed a more effective hydrogen-bonding system that selectively perturbs the syn/anti rotamer equilibrium of a target carbamate group. the authors examined the abilities of acetic acid and , -bis(octylamido)pyridine ( ) to perturb the syn/ anti ratio of carbamates and ( figure ). in a cdcl solution, acetic acid moderately stabilizes double hydrogen bonding of the syn rotamer of phenyl carbamate ( figure a) , with no relevant effect on the syn/anti ratio for pyridyl carbamate ( figure b ). in the second case, the carboxylic acid favors donation of a hydrogen bond to the more basic pyridyl nitrogen and forms the complex shown in figure b . on the contrary, in the case of the donor−acceptor−donor triad , it strongly stabilizes the syn rotamer of ( figure d ) over the anti rotamer ( figure c ). there is no effect on the syn/anti ratio for , presumably because of a steric deterrent to the formation of a hydrogen-bonded complex ( figure e ). the carbamate moiety plays a noteworthy role in medicinal chemistry, not only because it is found in drugs but also for its presence in a number of prodrugs. the rate and level of their hydrolysis is a key issue for the duration and intensity of their pharmacological activity. fast hydrolysis of carbamate-bearing drugs may result in weak or shortened activity. on the contrary, carbamate-based prodrugs must undergo extensive hydrolysis at a suitable rate for releasing an active drug and obtaining the expected activity profile. vacondio et al. recently proposed an interesting study in which they compiled a large number of reliable literature data on the metabolic hydrolysis of therapeutic carbamates. the authors were able to exploit the collected data to gain a qualitative relationship between molecular structure and lability to metabolic hydrolysis. a trend was extrapolated, according to which the metabolic lability of carbamates decreased in the following series: aryl-oco-nhalkyl ≫ alkyl-oco-nhalkyl ∼ alkyl-oco-n(alkyl) ≥ alkyl-oco-n(endocyclic) ≥ aryl-oco-n(alkyl) ∼ aryl-oco-n(endocyclic) ≥ alkyl-oco-nharyl ∼ alkyl-oco-nhacyl ≫ alkyl-oco-nh > cyclic carbamates. therefore, carbamates derived from ammonia or aliphatic amines are sufficiently long-lived. an example is represented by cefoxitin ( ), a second-generation cephalosporin antibiotic ( figure ). cyclic fiveor six-membered carbamates are quite stable and do not usually undergo metabolic ring opening. the antibacterial agent linezolid ( ) is a representative example of this class ( figure ). for these drugs, carbamate hydrolysis is not necessarily the half-lifedetermining metabolic reaction. on the contrary, fatty acid amide hydrolase (faah) inhibitor (urb ) showed significant hydrolysis in buffer at physiological ph after h ( figure ). other representative therapeutic carbamate drugs and prodrugs will be discussed in sections and , respectively. organic carbamates play an important role in organic synthesis, especially as subunits of biologically active compounds. accordingly, simple and efficient methods for the synthesis of carbamates are of great interest. a number of methods have been developed for the synthesis of carbamates. . . carbamate synthesis via traditional methods. over the years, a variety of carabamates have been prepared by utilizing the hofmann rearrangement of amides, − the curtius rearrangement of acyl azides, , the reductive carbonylation of nitroaromatics, the carbonylation of amines, the reaction of alcohols with isocyanates, and carbon dioxide alkylation. − the hofmann rearrangement (method i, scheme ) is wellrecognized as a useful method to convert primary carboxamides to amines or carbamates, characterized by the reduction of one carbon in the structure. much effort has been devoted to the development of modified reagents to optimize the hofmann rearrangement since the classical method for this transformation, involving the use of an alkaline solution of bromine, is unsatisfactory and unreliable. a variety of oxidants and bases have been proposed as modified agents, e.g., iodine(iii) reagents such as phi(oac) , meobr, nbs-ch ona, nbs-koh, lead tetraacetate, and benzyltrimethylammonium tribromide. these modified methods, however, require more than equiv or an excess amount of the oxidizing reagent, which is not very convenient. the curtius rearrangement (method ii, scheme ) is the thermal decomposition of acyl azides into the isocyanate intermediate. this method is widely employed in the transformation of carboxylic acids into carbamates and ureas. acyl azides are usually prepared from carboxylic acid derivatives such as acyl chlorides, , mixed anhydrides, , and hydrazides. , subsequent isocyanate intermediates can be trapped by a variety of nucleophiles to provide the carbamate derivatives. the acid chloride method is not suitable for acidsensitive functionalities. one-pot transformations of carboxylic acids into carbamates avoids the isolation of unstable acyl azides. however, protocols involving the use of diphenylphosphoryl azide (dppa) for the one-pot curtius reaction are also characterized by issues related to toxicity and the high boiling point of dppa, which creates difficulties during workup and purification. − other general methods for carbamate preparation involve the use of the highly toxic phosgene, phosgene derivatives, , or isocyanates. significant efforts have been made to find an alternative to the phosgene process. a very attractive substitute for phosgene is carbon dioxide because it is a classic renewable resource (method iii, scheme ). in addition, its use is also very attractive due to its environmentally benign nature (nontoxic, noncorrosive, and nonflammable). carbon dioxide is wellknown to react rapidly with amines to form carbamic acid ammonium salts. the majority of the approaches in this context rely on the creation of the carbamate anion via the reaction of carbon dioxide and amines, followed by the reaction with electrophiles. nevertheless, since the nucleophilicity of the carbamate anion is lower than that of the amine formed in the equilibrium of the salt formation, the subsequent reaction of the carbamate salts with alkyl halides does not selectively provide urethanes. , the formation of carbamates from isocyanates (method iv, scheme ) is fundamentally important to polyurethane industries. synthetic limitations and toxicity issues, however, are associated with the use of phosgene, the most common route to obtain isocyanates. the readily available alkyl chloroformates are the most frequently used reagents for the preparation of carbamates (method v, scheme ). however, these reagents display major drawbacks, as a large excess of base and a long reaction time are required in order to gain acceptable reaction efficiency. moreover, excess reagents are not suitable for the synthesis of molecules bearing multiple functionalities in which the chemoselectivity is critical. . . carbamate synthesis via activated mixed carbonates. a number of organic carbonates have been developed as low-cost and benign alternatives to the phosgenebased routes for the synthesis of organic carbamates. in this context, several new alkoxycarbonylating agents ( − ) based on mixed carbonates have been developed ( figure ). these methods are often used for the synthesis of carbamates in drug design. − mixed carbonates with a p-nitrophenyl moiety are frequently used for the preparation of a large range of carbamates. − for this, p-nitrophenyl chloroformate ( , pnpcocl), when treated with the suitable alcohol in the presence of base, furnishes the corresponding activated carbonates, which have been shown to be useful and effective alkoxycarbonylating reagents for suitable amines (scheme ). examples of carbamate derivatives are shown in table . several alkoxycarbonylating reagents for amino groups having heterocyclic groups, such as n-hydroxyimide, have scheme . traditional synthetic methodologies adopted for the synthesis of carbamates been reported. moreover, the utility and versatility of carbonates and oxalates containing an electron-withdrawing group, such as n-hydroxyimide and benzotriazole derivatives as reagents for various tranformations, have been described. , , takeda et al. reported that -alkoxy[ -(trifluoromethyl)benzotriazolyl]carbonates easily derived from , -bis[ -(trifluoromethyl)benzotriazolyl]carbonate ( , btbc) showed high acylating reactivity toward alcohols as well as amino groups. btbc was prepared from -trifluoromethyl- hydroxybenzotriazole and trichloromethyl chloroformate and purified by washing with dry ether. moreover, it can be stored for several months in a freezer. btbc was allowed to react with primary alcohols in acetonitrile at room temperature to give stable activated carbonates. the carbonates were treated with amines in the presence of -dimethylaminopyridine (dmap), providing the corresponding carbamates (scheme and table ). in connection with our research work aimed at synthesizing biologically active polyfunctional molecules for probing enzyme active sites, we required a more general and synthetically reliable method for the synthesis of various carbamate derivatives. in , we described the utility of di( -pyridyl) carbonate ( , dpc) as an efficient, high-yielding, and convenient alkoxycarbonylation reagent for amines overcoming many of the limitations of existing methodologies. dpc was readily prepared from commercially available -hydroxypyridine and triphosgene in the presence of triethylamine and subsequently reacted with the suitable primary or secondary alcohol (e.g., (+)-menthol) to provide a mixed carbonate. alkoxycarbonylation of primary and secondary amines with the mixed carbonates was carried out in the presence of triethylamine and furnished the corresponding carbamates in good yields (scheme , method a, and table ). potassium hydride was used in the place of triethylamine in the preparation of the mixed carbonates containing tertiary alcohols (scheme and table ). subsequently, we investigated the scope of n,n′-disuccinimidyl carbonate ( , dsc) promoted alkoxycarbonylation of amines with a host of alcohols under mild conditions. rich and co-workers highlighted the convenience of succinimidylbased mixed carbonates for the high-yielding introduction of a -(trimethylsilyl)ethoxycarbonyl (teoc) protecting group to amino acids, without oligopeptide byproduct formation. dsc was found to be a highly effective alkoxycarbonylating reagent for a variety of primary and sterically hindered secondary alcohols. dsc is commercially available, or it can be conveniently prepared from n-hydroxysuccinimide following a procedure tracing out the synthesis of dpc. the ready availability of dsc, the stability of the mixed carbonates, and the mildness of the reaction procedure render this method a reliable route to organic carbamates (scheme and table ). since azides were extensively employed as incipient amines in the context of amino sugar and amino acid syntheses, their conversion into the corresponding carbamate derivatives could provide a novel, effective route for medicinal chemistry applications. in this context, a facile synthetic protocol to transform various azides into the corresponding functionalized journal of medicinal chemistry urethanes in high yields has been developed. in general, mixed carbonates of variously protected alcohols were prepared by reaction of excess dsc or dpc, as described previously. exposure of mixed carbonates to catalytic hydrogenation conditions with azides in the presence of % palladium on charcoal in tetrahydrofuran furnished the corresponding carbamates. interestingly, the use of triethylamine as a promoter has a notable effect on the yield and the rate of the alkoxycarbonylation process (scheme and table ). more recently, yoon and co-workers exploited -substitutedpyridazin- ( h)-ones as electrophilic transfer reagents. , in particular, the authors investigated the carbonylation potency of phenyl , -dichloro- -oxopyridazine- ( h)-carboxylate ( ) to amines for the preparation of phenylcarbamates (scheme and table ). compound is stable in air and in organic solvents at high temperature and is prepared easily from cheap and commercially available , -dichloropyridazin- ( h)-one ( ) in the presence of phenylchloroformate and triethylamine (scheme ). . . recent methodologies for carbamate synthesis. the application of carbon dioxide in organic synthesis has recently attracted much interest. most of the approaches rely on the generation of the carbamate anion via the reaction of carbon dioxide and amines, followed by the reaction with electrophiles, usually alkyl halides. − in this context, a mild and efficient preparation of alkyl carbamates on solid supports was described by jung et al. amines and anilines were coupled with merrifield's resin through a co linker in the presence of cesium carbonate and tetrabutylammonium iodide (tbai). carbon dioxide was supplied by bubbling it into the reaction suspension, where n,n-dimethylformamide (dmf) was the solvent of choice (scheme ). the reaction conditions are convenient for purification, and the reactions undergo complete conversions. the method is convenient for the generation of large combinatorial libraries for rapid screening of bioactive molecules. chiral substrates susceptible to racemization have survived the conditions (table ) . later, these authors reported a one-pot synthesis of n-alkyl carbamates starting from primary amines (scheme ). carbamates were generated via a three-component coupling of primary amines, co , and an alkyl halide in the presence of cesium carbonate and tbai in anhydrous dmf (scheme and table ). direct n-alkylation of the intermediate carbamate a in the presence of additional cesium carbonate by using a different alkyl halide gave rise to the desired n-alkyl carbamate b (scheme ). isolation of the intermediate a proved to be unnecessary, offering shortened synthetic sequences. it is interesting to note that tbai helps to minimize the overalkylation of the produced carbamate, presumably by enhancing the rate of co incorporation and/or stabilizing the incipient carbamate anion through conjugation with the tetrabutylammonium cation. sakakura and co-workers reported urethane synthesis by the reaction of dense carbon dioxide with amines and alcohols by a procedure that is not only phosgene-free but also completely halogen-free (scheme ). dialkyl carbonate synthesis from an alcohol and co is catalyzed by metal complexes such as dialkyl(oxo)tin and dialkyl(dichloro)tin. however, the alcohol conversion is very poor. similarly, the direct reaction of an amine, an alcohol, and carbon dioxide in the presence of dialkyltin compounds produced urethane only in a poor yield. the low conversion observed was attributed by the authors to thermodynamic limitations and catalyst deactivation by coproduced water. in order to overcome this issue, a new reaction system utilizing acetals as a chemical dehydrating agent, with subsequent alcohol regeneration (scheme ), was developed. in order to obtain urethane in good yields, dense-phase co under high pressure was necessary to lower the major side reactions, namely imine formation from acetone and alkylation of amines by alcohols. however, developing less toxic and more active catalysts based on metals other than tin was required. later, these authors reported novel nickel-based catalytic systems for dehydrative urethane formation from carbon dioxide, amines, and alcohols (scheme ). interestingly, adding nitrogenbased bidentate ligands efficiently improved the catalytic activity of ni(oac) -based catalysts (scheme and table ). bipyridines and phenanthrolines with strong coordinating abilities (low steric hindrance and high electron densities) were the better choice for obtaining urethanes in high yields. it is important to note that the ni-phenanthroline system is more active and less toxic than dialkyl(oxo)tin under the same reaction conditions. it is also noteworthy that the catalytic activity of the ni(oac) -( , ′-dimethylbipyridine) system is highly dependent on the ligand/metal ratio (table ). peterson and co-workers proposed a method for rapid sar development of compounds bearing urea or carbamate functionalities (scheme ). for carbamate formation, an journal of medicinal chemistry amine, in principle, could proceed through the carbamic acid− isocyanate reaction, and subsequent reaction with an alcohol may provide a carbamate product. while this is precedented by an intramolecular reaction variant to produce cyclic carbamates, the desired intermolecular coupling was not fruitful under the proposed reaction conditions. carbamic acids produced from secondary amines, however, did react with alcohols under mitsunobu conditions (dibenzyl azodicarboxylate, dbad, and tributylphosphine) in a dbu-catalyzed reaction with gaseous carbon dioxide, providing the corresponding carbamates (scheme and table ). this reaction did not proceed through the isocyanate intermediate but rather through an s n displacement of the activated alcohol. this hypothesis is supported by the observed inversion of stereochemistry upon conversion of a chiral secondary alcohol to the corresponding carbamate (table ) . very recently, jiao and co-workers reported a practical, pdcl -catalyzed efficient assembly of organic azides, carbon monoxide, and alcohols for the direct synthesis of carbamates via isocyanate formation and application in situ (scheme ). mild and neutral reaction conditions and generation of harmless n as the byproduct render this protocol very useful, particularly for the synthesis of bioactive compounds. moreover, the employment of co at atmospheric pressure and the use of a small amount of pdcl catalyst ( mol %) in the absence of any ligand represent a real alternative to customary carbamate synthetic methods (table ) . the synthesis of carbamates through the generation of carbamoyl chlorides is not convenient because of the requirement of the toxic phosgene. also, such carbamoyl chlorides are highly reactive, prone to hydrolysis, unstable, and not suitable for long-term storage. for these problems, batey and co-workers identified the use of carbamoylimidazolium salts as convenient n,n′-disubstituted carbamoyl transfer reagents, showing increased reactivity over carbamoylimidazoles as a result of the imidazolium effect (scheme ). − these salts are readily prepared by the sequential treatment of secondary amines with n,n′-carbonyldiimidazole (cdi) and iodomethane (scheme ). authors envisaged that the carbamoylimidazolium salts, while relatively unreactive with alcohols, would react with nucleophlic alkoxides to produce the corresponding carbamates (table ). in the case of phenols, tertiary amines are appropriate bases for the in situ generation of the reactive phenoxides. the lower acidity of aliphatic alcohols presumably prevents the formation of the alkoxide anion, which would serve as the reactive nucleophile. less acidic alcohols react with carbamoylimidazolium after their conversion into more nucleophilic sodium alkoxides (scheme ). the use of solid-supported reagents has become ubiquitous due to enhanced reactivity and selectivity, milder reaction conditions, convenient work-ups, and decreased solvent waste. the modified hofmann rearrangement, proposed by gogoi et al., is operationally simple, inexpensive and applicable to a variety of aliphatic and aromatic amides for the synthesis of methyl carbamates (scheme ). kf/al o represents a useful and interesting solid-supported strong base, which replaces organic bases in a variety of reactions. sodium hypochlorite is an inexpensive, convenient, and safe alternative to the currently employed oxidants. this prompted the authors to investigate kf/al o along with naocl as an efficient reagent system for hofmann rearrangement. kf/al o basicity stems from the formation of koh in the initial preparation of the solid-supported material by the reaction of kf with alumina supports. under these highly basic reaction conditions, hypochlorite ion is the predominant form of chlorine, reacting with the amide to form an n-chloroamide, which later undergoes rearrangement to the isocyanate. in the presence of methanol, the isocyanate is rapidly converted into the corresponding methyl carbamate (table ) . modifications of the curtius rearrangement have also been explored. lebel and co-workers have reported a useful protocol for the preparation of tert-butyl carbamates from the corresponding carboxylic acids. their reaction with di-tert- (scheme a and table ) . these authors extended the same methodology to the direct synthesis of carbamates of aromatic amines using aromatic carboxylic acids (scheme b and table ) . in particular, the reaction of a chloroformate or di-tert-butyl dicarbonate and sodium azide with an aromatic carboxylic acid produced the corresponding acyl azide, presumably through the formation of an azidoformate. in contrast to what was observed with aliphatic carboxylic acids, using similar reaction conditions, aromatic carboxylic acids led mainly to the formation of the corresponding tert-butyl ester, likely via the displacement of an azide leaving group with tert-butoxide. this may be ascribed to the higher stability of aromatic acyl azides with respect to their aliphatic counterparts. therefore, for these substrates, the curtius rearrangement can be promoted only at higher temperatures ( vs °c). , as mentioned, alkyl chloroformates are the most frequently used reagents for the preparation of carbamates, although the need of an excess amount limits their usefulness. a promising method for preparing carbamates involves the use of a catalytic promoter. − lately, indium-mediated reactions have gained significant consideration due to the high reactivity and unique properties of indium reagents, among them nontoxicity and inertness toward air and water. − moreover, pretreatment is not required for activating indium metal. in this context, jang and co-workers developed a simple, efficient, and selective method for synthesizing carbamates from amines, employing a catalytic amount of indium and only an equimolar amount of alkyl chloroformate (scheme ). the method shows the generality for a wide variety of sterically diverse amines and alcohols and can also be applicable for the selective protection of amino groups under mild conditions (table ) . arndtsen et al. proposed another application of indium-based reagents for the generation of n-protected amines in a single step (scheme and table ). since organoindium reagents readily transfer their organic groups to an imine carbon, only one-third of an equivalent is required, and the only byproduct is represented by indium trichloride. tetraorganoindium reagents can also be employed in a similar fashion for transferring all four organic groups. therefore, one-fourth of an equivalent of indium is necessary for their reaction with imines. copper(i) chloride ( %) was found to be the most efficient catalyst. sodeoka and colleagues reported the use of -alkoxycarbonyl- -nitro- , , -triazole reagents as useful intermediates for the preparation of carbamates (scheme ). to achieve a rapid and clean reaction, the features of the leaving group have a key role. an ideal leaving group should have a highly electronwithdrawing element in order to increase the electrophilicity of the carbonyl carbon, and the nucleophilicity should be low to avoid side reactions. it should also be easily separated from the reaction product. -nitro- , , -triazole (nt), although showing nucleophilicity, could be easily removed from the table . nickel-catalyzed urethane synthesis from co a conversion of amine. b urethane/consumed amide × . c ni/l = : . d ni/l = : . reaction due to its insolubility in dichloromethane or chloroform. nt-based reagents have a series of benefits such as high stability, since they can be stored for long periods without decomposition. reactions of these nt reagents with primary and secondary amines proceeded quickly to give the corresponding carbamates in > % yield (scheme a and table ). in contrast to aliphatic amines, aromatic amines were less reactive. however, the addition of triethylamine was found to be effective in promoting the reactions (scheme b and table ). the reductive carbonylation of aromatic nitro compounds to the corresponding carbamates has remained a subject of great interest both from mechanistic and application standpoints (scheme ). in this section, we will briefly mention the methodologies involving the use of an alcohol, although other procedures employing chloroformates have also been recently reported. , cheng and collaborators report the use of ru(co) − and ru (co) complexes for the catalysis of this reaction and highlighted the key effect of alcohol on the selectivity of carbamates (table ) . the results clearly indicate that low selectivity of carbamate is closely related to the ability of the alcohol to reduce nitroarenes to amino derivatives. therefore, the employment of an alcohol that cannot reduce nitroarene greatly increases the selectivity of carbamate. later, the binuclear rhodium complex [(ph p) rh (μ-oh) ]· c h was employed as an effective catalyst for the reductive carbonylation of nitrobenzenes to carbamate esters (table ) . palladiumbased catalysts have also been explored (table ) . − carbamate synthesis via transfunctionalization of substituted ureas and carbonates in the presence of di-n-butyltin oxide (dbto) as the catalyst was reported by chaudhari and colleagues (scheme a and table ). the carbonate reactivity pattern seems to be driven by the leaving group ability of the alkoxides and phenoxide to form the carbamate observed in aminolysis of carbonates. it has been shown that basicity of reacting urea plays a vital role in the catalytic activity of this reaction. indeed, aliphatic ureas show higher reactivity compared to aromatic ureas due to their higher basicity. the basic dbto is supposed to work as a nucleophile by attacking the carbonyl carbon of the carbonate, thus generating the catalytically active species dibutyl alkoxy carbonato tin [a]. use of dialkyl carbonates as environmentally friendly and nontoxic phosgene substitutes in alkoxycarbonylation reactions has also been exploited by porco et al. (scheme ). particularly, the authors examined the scope of zr(iv)catalyzed carbonate−carbamate exchange processes to prepare carbamates from dialkyl carbonates employing -hydroxypyridine (hyp) as a catalytic additive (table ) . recently, padiya and co-workers reported a useful method for preparing carbamates in an aqueous media (scheme ). interestingly, they found that , ′-carbonyldiimidazole (cdi), although unstable in water, rapidly reacts in aqueous media with amine to give good yields of the corresponding nsubstituted carbonylimidazolide. carbonylimidazolide derived from the primary amine reacts in situ with a nucleophile such as phenol, providing the corresponding carbamate. the product precipitates out from the reaction mixture and can be obtained in high purity by filtration, making the method simple and scalable (table ) . cdi was also found to mediate the lossen rearrangement, which occurs in the transformation of an activated hydroxamic acid into the corresponding isocyanate (scheme ). the proposed methodology is experimentally efficient and mild, being characterized by imidazole and co as the only stoichiometric byproducts. this method is a green and unconventional alternative to the curtius and hofmann rearrangements (table ) . another method based on the lossen rearrangement was recently proposed. the methodology envisaged the reaction of a hydroxamic acid with an alcohol, promoted by , , -trichloro- , , -triazine (cyanuric chloride; tct) in the presence of an excess of n-methyl morpholine (nmm) (scheme and table ). carbamates are inherent to many fda approved drugs. this structural motif is also a key functionality in numerous medicinal agents with clinical potential. in this section, a series of therapeutic carbamates with a variety of applications is outlined. . . miscellaneous carbamates with clinical relevance. . . . rivastigmine. rivastigmine ( , figure ) tartrate (exelon, novartis pharma) is a carbamate derivative that reversibly inhibits the metabolism of acetylcholinesterase (ache) and butyrylcholinesterase (buche) preferentially in the central nervous system (cns). it is used for the treatment of mild-to-moderate alzheimer's disease (ad) dementia and dementia due to parkinson's disease. , the drug can be administered orally or via a transdermal patch. the transdermal patch reduces side effects such as nausea and vomiting. rivastigmine undergoes extensive metabolism by che-mediated hydrolysis to the decarbamylated metabolite, without involvement of the major cytochrome p (cyp ) isozymes. the metabolite may undergo n-demethylation as well as conjugation. the pharmacokinetic half-life of rivastigmine in ad patients is around . h. when given orally, rivastigmine is well-absorbed, with a bioavailability of about % administered as a mg dose. , . . . muraglitazar. muraglitazar ( ) contains a carbamate functionality. it is a potent, novel nonthiazolidindione peroxisome proliferator-activated receptor dual agonist (pparα/γ) that demonstrated highly efficacious glucose and lipid lowering activities in vivo, along with an excellent adme profile. in a double-blind randomized clinical trial, muraglitazar resulted in a statistically significant improvement in plasma triglyceride, hdl cholesterol, apob, and non-hdl cholesterol concentrations at week . muraglitazar reduced triglyceride concentrations to a larger extent than did pioglitazone, regardless of baseline triglyceride levels. muraglitazar and pioglitazone treatment was associated with slight ( − %) increases in ldl cholesterol. however, muraglitazar development was discontinued due to major adverse cardiovascular side effects. . . . roxifiban. roxifiban ( ) is a carbamate derivative with a methyl ester prodrug. it is a potent, nonpeptide antagonist of the glycoprotein iib/iiia receptor. , the free acid resulting from roxifiban hydrolysis blocks the binding of fibrinogen to the receptor, thereby inhibiting platelet aggregation and providing a mechanism for antithrombotic mebendazole ( ) is a methyl carbamate derivative showing broad-spectrum anthelmintic properties. it demonstrated efficacy in the oral treatment of ascariasis, uncinariasis, oxyuriasis, and trichuriasis. like other benzimidazole anthelmintics, mebendazole's primary mechanism of action is consistent with tubulin binding. mebendazole was discontinued in . . . . flupirtine and retigabine. flupirtine ( ) and retigabine ( ) are ethyl carbamate derivatives. flupirtine is a centrally acting nonopioid analgesic that was identified within an antiepileptic drug discovery program by the u.s. national institutes of health. the doses used in a small clinical trial exceeded those established for analgesic activity. on the basis of this data, subsequent structural optimization resulted in retigabine. retigabine has anticonvulsant properties that appear to be mediated by opening or activating neuronal voltage-gated potassium channels. flupirtine showed n-methyl-d-aspartate (nmda) receptor antagonist properties. . . . felbamate. felbamate ( , felbatol, meda pharmaceuticals) is an alkyl carbamate derivative. it is an antiepileptic drug. the mechanism of action of felbamate involves a dual mechanism involving inhibition of n-methyl-d-aspartate (nmda) receptor response and positive modulation of γamino butyric acid subtype a (gaba a ) receptor, thus decreasing neuronal excitation. felbamate is rapidly absorbed (t max = − h) with an oral bioavailability > %. felbamate undergoes moderate metabolism via cyp a and cyp e isoenzymes, which are amenable to inhibition and induction effects. , the clinical use of scheme . synthesis of carbamates by modified curtius rearrangement it is a nonnucleoside reverse transcriptase inhibitor (nnrti). the drug is used as part of highly active antiretroviral therapy (haart). , however, its use is associated with variable treatment response and adverse effects, in most part because of the large differences in pharmacokinetics. cyp b is the main enzyme catalyzing the major clearance mechanism of efavirenz ( -hydroxylation to -hydroxyefavirenz) in vivo. , . . . zafirlukast. zafirlukast ( , accolate, astrazeneca) is a cyclopentyl n-aryl carbamate derivative. it is a selective and competitive receptor antagonist of the cysteinyl leukotrienes d- and e- , which is indicated for the prophylaxis and treatment of mild-to-moderate persistent and chronic asthma. both o → ch and o → nh bioisosteric analogues of zafirlukast were found to be potent. the carbamate moiety present in zafirlukast provided an excellent in vitro and in vivo profile and high oral bioavailability. zafirlukast undergoes hepatic metabolism, where hydroxylation by cytochrome cyp c is the major biotransformation pathway. the metabolites of zafirlukast do not significantly contribute to its overall activity. . . . mitomycin c. mitomycin c ( , mmc, mutamycin) is a complex carbamate derivative. it is an antitumor antibiotic that was identified in the s in fermentation cultures of the gram-negative bacteria streptomyces caespitosus. mmc is a site-specific, nondistorting dna cross-linking agent. , however, recent reports suggest that dna may not be the primary target of the drug. in particular, interaction of mmc with rrna and subsequent inhibition of protein translation has been proposed. mmc is customarily used as a chemotherapeutic agent in the treatment of several types of cancer, such as bladder, colon, and breast cancers. . . therapeutic carbamates as hiv protease inhibitors. hiv protease is an aspartic acid protease responsible for the cleavage of the gag−pol polyprotein into functional proteins essential for the production of infections progeny virus. inactivation of hiv- protease either by site-directed mutagenesis or by chemical inhibition results in the formation of immature, noninfections virus particles. as a consequence, hiv- protease is an attractive target in antiviral therapy. hiv protease is a c -symmetric, -amino acid homodimeric aspartyl protease in which each protein subunit contributes one asp-thr-gly motif to the single active site. the x-ray crystallographic analysis of the native protein and subsequent protein−ligand complexes and extensive research programs on other aspartyl proteases, including human renin, provided a path toward accelerated drug discovery programs targeting hiv protease. − a number of fda-approved hiv protease inhibitor drugs contain an important carbamate functionality. in this section, currently approved protease inhibitor drugs are discussed (figure ) . . . . ritonavir. ritonavir ( , norvir, abt- , a- , abbvie, inc.) structure possesses a thiazolyl methyl carbamate functionality. it is a peptidomimetic inhibitor of both the hiv- and hiv- proteases and was approved by the fda in march . this first-generation protease inhibitor was developed at abbott laboratories. the discovery of ritonavir was based on of . μm, bioavailability of %, and a plasma half-life of . h. ritonavir has a high molecular weight; however, it showed excellent pharmacokinetic properties. this is possibly due to the increased stability of the thiazole groups to oxidative metabolism and also due to its effect on cytochrome p oxidative enzymes. ritonavir is a type ii heme ligand that fits into the cyp a active site cavity and irreversibly binds to the heme iron via the thiazole nitrogen. amprenavir was identified as a potent, orally bioavailable hiv- protease inhibitor with a low molecular weight and a mean ic of nm. it is marketed with a twice-a-day dosing format. amprenavir structure bears a stereochemically defined tetrahydrofuranylcarbamate engaging in a weak backbone interaction with the protease. , in vitro and in vivo studies have shown that amprenavir is primarily metabolized by cyp a , and the two major metabolites result from oxidation of the tetrahydrofuran and aniline moieties. properties. drv also maintains high potency against multidrugresistant hiv- strains. the design of drv originated from the backbone binding concept envisaging that an effective protease inhibitor maximizes rich networks of hydrogen-bonding interactions with the backbone atoms throughout the active site of the protease. the bis-thf moiety present in drv was designed based on the x-ray structure of inhibitor-hiv- protease complexes. the bis-thf carbamate moiety of drv was found to be essential for enzyme affinity (see figure for details). drv demonstrated exceptional potency against both wild-type hiv isolates and a wide range of resistant variants. hiv- variants. in , drv received full approval for the treatment of therapy-naive adults and children. drv is metabolized by the isoenzyme cyp a . , however, in the presence of a low dose of ritonavir, drv exhibits very good pharmacokinetic properties in patients. metabolism prodrugs are chemically modified forms of the actual pharmacologically active drug that undergo in vivo transformation to release the active drug molecule. this is a wellestablished strategy to improve drug disposition properties (physicochemical, biopharmaceutical, or pharmacokinetic properties) of pharmacologically relevant compounds and thereby increase their drug-like profile. , a prodrug strategy helps to overcome a variety of hurdles in drug formulation and delivery such as (i) poor oral absorption and aqueous solubility, (ii) poor lipid solubility, (iii) chemical instability, (iv) rapid presystemic metabolism, (v) toxicity and local irritation, and (vi) lack of site-selective delivery. a functional group on the parent drug may be used to form a chemical bond with the promoiety. generally, the linker should be self-removing or cleavable so that the parent drug can be released spontaneously or under a certain triggering condition, such as the presence of an enzyme or a change in ph. the promoiety coupled to the parent drug provides the ability to improve the drug-like properties or overcome the barriers in delivering the drug to its target cells. carbamates are the esters of carbamic acid, preferentially used in the design of prodrugs as a means of achieving first-pass and systemic hydrolytic stability. carbamates are typically enzymatically more stable than the corresponding esters. they are, in general, more susceptible to hydrolysis than amides. thus, bioconversion of carbamate prodrugs requires esterases for the release of the parent drug. upon hydrolysis, carbamate esters release the parent phenol or alcohol drug and carbamic acid, which, due to its chemical instability, breaks down to the corresponding amine and carbon dioxide. carbamates of primary amines can also fragment into isocyanates and alcohols on treatment with bases, a further potential pathway for metabolic degradation. , the oh-catalyzed hydrolysis of these carbamate esters (r′-nhco-or) is strongly dependent on both the pk a of the proton on the leaving group (roh) and the degree of substitution on the nitrogen of the carbamate ester. since phenols have a lower pk a with respect to alcohols, carbamate esters of phenols are generally more chemically labile than those of alcohols. in the case of alcohols, both the n-monosubstituted and n,n-disubstituted carbamates are chemically stable toward hydrolysis. in phenols, n,ndisubstituted carbamates are chemically stable, whereas nmonosubstituted carbamates are the most labile toward chemical hydrolysis. short-lived carbamates have also been used as prodrugs of heteroaromatic amines (e.g., capecitabine, ) and amidines (lefradafiban ( ), dabigatran). . . alcohol and phenol carbamate prodrugs. most of the therapeutically relevant carbamate prodrugs have been designed as substrates of specific enzymes. antibody-directed enzyme prodrug therapy (adept) , and gene-directed enzyme prodrug therapy (gdept) are new strategies for targeting tumors. carboxypeptidase g (cpg ), an enzyme of bacterial origin, has been shown to catalyze the cleavage of an amide, carbamate, or urea linkage between glutamic acid and an on the basis of this specificity, a large number of prodrugs have been designed and synthesized for cpg . as shown in figure , the prodrug (zd p) is activated by hydrolysis at the carbamate bond by cpg to the corresponding potent di-iodophenol mustard ( ) . was found to possess the best profile in terms of enzymatic kinetics, cytotoxicity, and in vivo efficacy. it was selected for clinical development. the half-life (t / ) of the drug is approximately min, which is enough for diffusion into the tumor cell from the local release site and to minimize peripheral toxicity. , irinotecan was designed to deliver camptothecin as a predominant topoisomerase i inhibitor for anticancer therapy. irinotecan hydrochloride salt (cpt- , camptosar; pfizer) is a parenteral aqueous soluble carbamate prodrug of antineoplastic topoisomerase i inhibitor (sn- , -ethyl- -hydroxy-camptothecin). the potent antitumor activity of irinotecan is due to rapid formation of active metabolite in vivo (figure ). in this molecule, a dipiperidino ionizable promoiety is linked to the phenol functionality by a carbamate bond, thus improving the overall aqueous solubility. − the bioconversion back to occurs primarily by human liver microsomal carboxylesterases, ces a and ces , which release the ionizable piperidinopiperidine promoiety and , the active form of the drug. beyond minimizing the rate of enzymatic hydrolysis of its prodrug, sustained drug action can also be provided by decreasing the rate of drug metabolism. this is the case of bambuterol ( , bambec, astrazeneca), a bis-dimethyl carbamate prodrug of the β -agonist terbutaline ( ), which is used as a bronchodilator in the treatment of asthma. the phenolic moiety of terbutaline is subjected to rapid presystemic metabolism. in bambuterol, protection of this functionality also avoids first-pass intestinal and hepatic metabolism. this prodrug is inactive, however, after oral administration; it is slowly converted to terbutaline, mainly outside the lungs, by a series of hydrolysis and oxidation reactions (maily catalyzed by plasma cholinesterase, pche, and by cyp , figure ). , this allows a once-daily bambuterol treatment with respect to the three daily terbutaline administrations. an n,n′-dimethyl ethylenediamine spacer, used for the evaluation of cyclization-elimination-based prodrugs of phenols and alcohols, has been used for the development of prodrugs as a part of the adept activation strategy. when activated by a specific enzyme, the terminal amino group on the spacer activates and initiates an intramolecular cyclization reaction to eliminate a phenol or alcohol parent drug with parallel release of the cyclized spacer. in one such application, scherren et al. explored paclitaxel- ′-carbamates. this is particularly interesting because a free ′-hydroxyl group is important for biological activity. in general, carbamate linkages are more stable in vivo than esters and carbonates. since the proteolytic active form of plasmin is located in the tumor, linking a cytotoxic drug to a plasmin substrate may result in tumor-selective delivery. on the basis of this rationale, following plasmin hydrolysis, the spacer is expected to undergo spontaneous cyclization to yield a cyclic urea derivative (imidazolidinone), thereby releasing paclitaxel ( ) , as illustrated in scheme . , . . amine and amidine carbamate prodrugs. the amine group is one of the most common functional groups in many approved drugs. amines in drugs can cause physicochemical hurdles that have the potential to limit their safety and effective delivery to desired sites of action. therefore, a variety of prodrugs of amines have been designed to overcome formulation and delivery barriers. the carbamate functionality has been utilized in many prodrug strategies designed for amines. short-lived carbamates are also used as prodrugs of heteroaromatic amines and amidines. gabapentin ( , neurontin; pfizer, figure ) is a structural analogue of γ-aminobutyric acid (gaba). it is marketed as an anticonvulsant and an analgesic agent. gabapentin shows a number of limitations, including saturable absorption, high interpatient variability, lack of dose proportionality, and a short half-life. gabapentin enacarbil ( , horizant, previously known as xp ) is a carbamate prodrug of gabapentin. the prodrug is benefited by a monocarboxylate transporter type (mct ). mct is expressed in all segments of the colon and upper gastrointestinal tract. the prodrug also helps the sodium-dependent multivitamin transporter (smv t), responsible for absorption of multiple essential nutrients. , following absorption via these pathways, the prodrug is rapidly converted to gabapentin by nonspecific esterases, mainly in enterocytes and to a lesser extent in the liver. during conversion to gabapentin, each molecule of also generates carbon dioxide, acetaldehyde, and isobutyrate (figure ). the oral bioavailability of was improved from to % in monkeys. it showed doseproportional gabapentin exposure in humans. in , xenoport received fda approval (horizant) for the treatment of moderate-to-severe restless legs syndrome. in , horizant was also approved for the management of postherpetic neuralgia (phn) in adults. capecitabine ( , xeloda, roche) was designed to achieve greater selectivity than its active form, -fluorouracil ( , -fu). it is an orally administered carbamate prodrug of -fu, belonging to the fluoropyrimidine carbamate class. it requires a cascade of three enzymes for the bioconversion to the active drug. as shown in figure , the enzymatic bioconversion starts in the liver, where human carboxylesterases and (ces and ces ) cleave the carbamate ester bond. intact capecitabine is absorbed in the intestine, and its bioconversion in the liver releases the parent drug. to some extent, its bioconversion proceeds in tumors, thus avoiding any systemic toxicity. in particular, the remaining transformations to -fu are catalyzed by cytidine deaminase and thymidine phosphorylase. the latter enzyme is highly enriched in tumors, thus providing selective release of -fu in cancer cells. , the absorption of capecitabine is evident since % of an orally administered dose is recovered in urine and the t max of -fu is reached in approximately . − h. capecitabine is currently approved as a first line of therapy for colorectal and breast cancers and is also approved for use in combination with other anticancer drugs. , alkoxycarbonyl derivatives can serve as useful prodrugs for benzamidines. for example, the methoxycarbonyl methyl ester lefradafiban ( , bibu , boehringer ingelheim, germany) is effectively converted to the active platelet aggregation inhibitor fradafiban ( , bibu ) after oral administration. this was revealed by monitoring the plasma concentrations of and by ex vivo platelet aggregation studies. lefradafiban is the orally active prodrug of fradafiban, a glycoprotein iib/iiia receptor antagonist. esterases, but not cyp -dependent enzymes, are involved in the conversion of lefradafiban to fradafiban in vivo (figure ). over the years, we have developed a series of novel hiv- protease inhibitors incorporating cyclic ether-derived carbamates designed based on the x-ray structures of inhibitor-hiv- protease complexes. , in this endeavor, we have specifically developed stereochemically defined cyclic ether templates, where the cyclic ether oxygen could effectively replace a peptide carbonyl oxygen. the advantage of such replacement is to reduce peptidic features and improve metabolic stability of compounds. these cyclic ligands have been incorporated as carbamate derivatives. the evolution of the carbamate structural template is shown in figure . on the basis of the x-ray crystal structure of saquinavir ( )-bound hiv- protease, we first investigated -(r)-tetrahydrofuranylglycine so that the -(r)-thf ring oxygen would interact with the asp nh, similar to the asparagine side chain carbonyl oxygen of saquinavir (compound ). − in an effort to reduce molecular weight, the p quinoline was removed, and the amide bond was replaced with a carbamate to provide inhibitor with significant reduction of molecular weight ( da from da). the x-ray crystal structure of bound hiv- protease revealed that the ring oxygen of the -(s)-tetrahydrofuran ( -(s)-thf) is within proximity to form a hydrogen bond with the asp nh bond in the s subsite. the importance of the carbamate moiety is evident. the carbamate nh forms a hydrogen bond with the backbone carbonyl of gly , and the carbamate carbonyl functionality makes a tightly bound water-mediated hydrogen bond with the backbone nh's of the flap ile and ile ′ in the active site. our further investigation of the -(s)-thf in inhibitors containing a hydroxyethylene isostere led to a series of exceptionally potent inhibitors. as shown in table , -(s)-thf-containing carbamate drivatives (compounds − ) provided very potent inhibitors in antiviral assays. the potency enhancing effect of -(s)-thf carbamate was subsequently demonstrated in inhibitors containing the (r)-(hydroxyethyl)sulfonamide isostere. clinical development of inhibitor (vx ) led to fda approval of amprenavir for the treatment of hiv/aids patients. , further development of carbamate-derived novel hiv- protease inhibitors is shown in figure . we have designed a variety of inhibitors incorporating cyclic sulfones and bicyclic ligands (figure , compounds − ) . , these ligands were conceived in order to maximize hydrogen-bonding interactions with the protease backbone as well as to fill in the hydrophobic pocket in the s subsite. on the basis of the x-ray structure of saquinavir-bound hiv- protease, we then designed a fused bicyclic tetrahydrofuran (bis-thf) ligand to form hydrogen bonds with backbone aspartates in the s subsite as well as to fill in the hydrophobic site adjacent to the p -quinoline ring of saquinavir ( figure ) . , an x-ray structural analysis of -bound hiv- protease revealed that the bis-thf carbamate mimics the majority of p −p -amide bonds of saquinavir. a detailed structure−activity study also established that the stereochemistry of the bis-thf ring, and the position of the ring oxygens is critical to potency. with the development of a bis-thf carbamate that could form a network of hydrogen bonds in the s subsite of hiv- protease, we investigated transition state isosteres that can be functionalized to form hydrogen bonds in the s ′ subsite. our basic hypothesis was to design inhibitors that form a network of hydrogen bonds with the protease backbone atoms throughout the active site of hiv- protease, from s to s ′ subsites. this backbone binding strategy to combat drug resistance led to the development of a series of very potent carbamate-derived protease inhibitors. , , as shown in figure , we incorporated the bis-thf ligand in the (r)-hydroxyethylsulfonamide isostere bearing p-methoxysulfonamide as the p ′ ligand so that the methoxy oxygen can interact with aspartate backbone atoms in the s ′ subsite. the resulting inhibitors exhibited notable potency. , inhibitor with a ( r, as, ar)-bis-thf as the p ligand is significantly more potent in an antiviral assay than corresponding inhibitor with an enantiomeric bis-thf ligand. an x-ray structure of -bound hiv- protease revealed that the carbamate nh formed a hydrogen bond with the backbone gly carbonyl group and that carbamate carbonyl of is involved in an interesting tetra-coordinated hydrogen-bonding interaction with the structural water molecule, inhibitor sulfonamide oxygen, and the flap ile nh residues. also, the structure revealed interactions with the backbone atoms in both the s and s ′ subsites. , further replacement of the p-methoxy group at the s ′ to a p-amino group led to inhibitor ( figure ) . this inhibitor showed marked enzyme inhibitory activity as well as antiviral activity. an in-depth antiviral study revealed that maintained excellent antiviral activity against multidrug-resistant hiv- variants. − the x-ray structural studies of darunavir-bound hiv- protease showed extensive active site interactions ( figure ) . particularly, it formed a network of hydrogen bonds with the protein backbone throughout the active site. darunavir also exhibited favorable pharmacokinetic properties. subsequently, clinical development led to its fda approval as darunavir for the treatment of hiv/aids patients. , the carbamate functionality of darunavir ( ) was assembled as shown in scheme . ( r , a s , a r ) - -hydroxyhexahydrofuro[ , -b]furan (bis-thf) was treated with disuccinimidyl carbonate to provide activated mixed carbonate . reaction of this activated carbonate with hydroxyethylsulfonamide isostere provided darunavir. , the backbone binding inhibitor design strategies to combat drug resistance have been further utilized by us and others to advance a number of other preclinical and clinical inhibitors with carbamates. , figure shows selected bis-thfderived carbamates ( − ) with marked enzyme and antiviral activities. − like darunavir, inhibitor-bound x-ray structures of these inhibitors showed a network of hydrogen bonds in both s and s ′ subsites of hiv- protease. the inhibitor side chains as well as the bis-thf bicyclic framework also effectively filled the hydrophobic pockets in the active site. we have outlined a selected number of cyclic ether-derived carbamates that have been developed based on the backbone table . carbamates from cdi-and tct-mediated lossen rearrangement , scheme . tct-mediated lossen rearrangement for carbamate synthesis binding concept in figure . , particularly, incorporation of these stereochemically defined oxacyclic ligands such as cp-thf, tp-thf, tris-thf, and fluoro-bis-thf provided exceptionally potent inhibitors ( and − ) with clinical potential. − the importance of the carbamate functionality in these inhibitors is particularly worthy of note. x-ray crystal structures of these inhibitors in complex with hiv- protease provided the ligand-binding site interactions responsible for their respective antiviral potency against wild-type and multidrug-resistant viruses. in general, inhibitors are involved in hydrogen-bonding interactions with asp , asp , gly , asp , asp ′, and asp ′ in the hiv- protease active site. furthermore, the ring cycles adequately fill the hydrophobic pockets in the active site. inhibitors the search for an effective treatment for alzheimer's disease (ad) remains a major challenge in medicine. one of the pathological hallmarks of ad is the formation of β-amyloid (aβ) peptides in the cortex of ad patients. aβ-peptides are generated from β-amyloid precursor protein (app) by sequential cleavage by β-secretase (also known as bace or memapsin ) and γ-secretase. due to this central role of aβproduction, both β-secretase and γ-secretase have been implicated as important therapeutic targets for ad intervention. , as a result, design and synthesis of selective βsecretase and γ-secretase inhibitors have become an intense area of research over the years. . . development of β-secretase inhibitors. following the discovery of β-secretase, the first-generation β-secretase inhibitors were designed and synthesized by ghosh, tang, and co-workers. as shown in figure , utilizing a carbamate derivative of the leu−ala isostere , potent pseudopeptide inhibitors and were identified. the x-ray crystal structure of -bound β-secretase was determined to provide molecular insight into the ligand binding site interactions. the in-depth structural analysis thus provided critical drug design templates and led to the beginning of structure-based design approaches to peptidomimetic/nonpeptide β-secretase inhibitors. , the x-ray structure of -bound β-secretase revealed that the p asparagine side chain carboxamide nitrogen formed an intermolecular hydrogen bond with the p glutamic acid carbonyl group. on the basis of this molecular insight, a number of − membered cycloamide-carbamate-based macrocyclic inhibitors were designed and synthesized. as shown in figure , acyclic carbamate derivatives ( and ) were less potent than their corresponding cyclic inhibitors. inhibitor , with a -membered macrocycle containing a trans-olefin, amide and carbamate functionalities within the macrocycle, showed good β-secretase inhibitory activity. saturated inhibitor is less potent against bace , but it showed enhanced potency for bace . x-ray structural studies of inhibitor -bound secretase revealed that the carbamate carbonyl forms a hydrogen bond with the gln side chain carboxamide residue. interestingly, unsaturated inhibitor showed slight selectivity against memapsin (k i = nm). the design of a selective inhibitor is important for reducing toxicity through off-target effects. particularly, selectivity over other aspartic proteases, such as bace , pepsin, renin, cathepsin d (cat-d), and cathepsin e, may be important for the reduction of side effects and drug efficiency. on the basis of our detailed structure−activity studies and xray structural analysis, we have designed a variety of highly selective and potent bace inhibitors. in this perspective, we will highlight only the development of bace inhibitors bearing carbamate functionalities. as shown in figure , inhibitor is a potent bace inhibitor. however, it did not show selectivity against bace or cat-d. subsequent structure-based design led to the development of selective inhibitors and , which contain a pyrazolylmethyl and oxazolymethyl carbamate at the p position, respectively. inhibitor showed excellent bace potency and selectivity over bace and cathepsin d. the x-ray crystal structure of -bound β-secretase revealed that the carbamate carbonyl formed a hydrogen bond with the thr- backbone nh. also, figure . examples of phenol carbamate prodrugs and their metabolic activation. ethylenediamine spacer and release of paclitaxel the pyrazole nitrogen formed a strong hydrogen bond with the thr- side chain hydroxyl group. the p -sulfonyl functionality formed a number of hydrogen bonds in the s subsite as well. on the basis of this molecular insight, oxazole-derived was designed to provide a more stable and selective inhibitor. the synthesis of inhibitors and is outlined in scheme . urethanes and were prepared by treatment of , -dimethylpyrazolylmethanol ( ) or , dimethyl- -oxazolemethanol ( ) with triphosgene in the presence of triethylamine, followed by l-methionine methyl ester hydrochloride ( ) . saponification of the resulting methyl esters provided the corresponding acids. coupling of amine with acids and , as described previously, and subsequent oxidation of the sulfides with m-chloroperbenzoic acid furnished inhibitors and . freskos and co-workers have reported a series of β-secretase inhibitors that incorporated polar carbamate derivatives as the p ligand. , this strategy led to improve the cat-d selectivity. it was hypothesized that the s subsite of cat-d is more lipophilic and less tolerant of polar groups. as can be seen in figure , benzyl carbamate derivative displayed -fold selectivity over cat-d. however, polar -pyridylmethyl derivative improved selectivity nearly -fold. the corresponding -pyridyl methyl compound provided a reduction in selectivity (∼ -fold). -(s)-tetrahydrofuranyl carbamate showed a nearly -fold selectivity over cat-d. these inhibitors have also shown good to excellent ic values in hek cells. . . development of γ-secretase inhibitors. over the years, many structural classes of potent and selective γ-secretase inhibitors have been reported. a number of inhibitors displayed drug-like properties and also inhibited aβ production in animal models. in this section, we will review inhibitors with carbamate functionality. on the basis of γ-secretase inhibitor (ly- ), peters and co-workers designed a series of carbamate derivatives of dibenzazepinone as potent and metabolically stable γ-secretase inhibitors. as shown in figure nm. absolute stereochemistry of the active enantiomer was not determined. piperidine carbamate also showed good potency. carbamate derivative displayed a good membrane aβ ic value; however, it showed poor cyp properties. further modification led to compound with good inhibitory activity and improved cyp properties. bergstrom and co-workers reported a series of carbamateappended n-alkyl sulfonamides as γ-secretase inhibitors. , figure depicts selected examples that show potent aβ inhibitory activity. sulfonamide derivative was identified as a potent γ-secretase inhibitor. exploration of carbamateappended n-alkylsulfonamides resulted in potent inhibitors such as − . tertiary carbamate showed significant reduction of brain aβ in transgenic mice compared to that of its benzyl derivative. this compound also showed improved brainto-plasma ratio and good absolute brain concentration. hepatitis c virus (hcv) is a bloodborne virus that is found worldwide. there are multiple strains or genotypes of the hcv virus. hcv infections lead to progressive liver damage, cirrhosis, and liver cancer. in recent years, there have been a number of new and effective antiviral drugs developed for the treatment of hepatitis c. these include the development of hcv ns / a protease inhibitors and inhibitors hcv ns a. in this section, carbamate-derived therapeutics will be discussed. . . carbamate-derived serine protease inhibitors. serine proteases are a large family of proteolytic enzymes that play a variety of critical roles in many physiological processes. , deregulation of serine proteases has been related to the pathogenesis of diseases such as stroke, inflammation, alzheimer's disease, cancer, and arthritis. therefore, significant research efforts have been focused in the discovery of serine protease inhibitors. the active site of all serine proteases consists of a catalytic triad of ser, his, and asp. the nucleophilic attack by the hydroxyl group of serine at the carbonyl carbon of the scissile bond of the substrate, via general base catalysis by histidine, leads to the tetrahedral transition state. the tetrahedral intermediate ultimately collapses, leading to cleavage products. − these key active residues are conserved in all serine proteases. x-ray structural studies revealed that these residues are superimposable in the majority of serine proteases. , therefore, selectivity over other serine proteases represents a key issue to be taken into consideration during inhibitor design. most early inhibitors acted via a covalent mechanism in which an electrophilic group formed a covalent bond with the serine hydroxyl of the catalytic triad. the electrophilic groups are commonly referred to as serine traps or warheads. however, covalent inhibitors lack selectivity and specificity against other proteases in the same class or clan. the rational design of covalent serine protease inhibitors usually involves the selection of a good substrate to be linked to a serine trap/warhead. chloromethyl ketones, diphenyl phosphonate esters, trifluoromethyl ketones, peptidyl boronic acids, α-ketoheterocycles, and β-lactam derivatives are usually employed as warheads. on the basis of these warheads, a variety of irreversible and reversible covalent serine protease inhibitors were designed. in this section, representative serine protease inhibitors containing a carbamate functionality will be outlined. carbamate derivative (figure ), a diphenyl phosphonate ester containing a cbz group and bearing a single amino acid side chain, showed very good inhibitory activity against human plasma kallikrein, useful for the treatment of hereditary angioedema. , thrombin is an attractive therapeutic target for drug development against pulmonary embolism, thrombosis, and related diseases. , compound showed good potency and selectivity against human thrombin. it is stable and displayed no activity against acetylcholinesterase and no selectivity over cysteine proteases. peptidyl boronic acid-based thrombin inhibitors were developed by dupont-merck. in particular, n-boc derived inhibitor is a potent inhibitor (k i = . nm). , imperiali and co-workers introduced trifluoromethyl ketones as specific serine protease inhibitors, particularly for chymotrypsin and elastase. researchers at astrazeneca designed numerous peptidyl trifluoromethyl ketone derivatives as potent human elastase inhibitors. − further optimization of features resulted in the development of a number of orally active inhibitors. in particular, methyl carbamate derivative inhibitor was shown to be a very potent inhibitor (k i = nm) with excellent oral bioavailability in laboratory animals. , optically pure compound with an (s)-configuration at the p isopropyl side chain became a candidate for clinical development for potential treatment of elastase-implicated respiratory diseases. peptidomimetic boronic acid-based hepatitis c virus (hcv) ns / a protease inhibitors were designed and synthesized for the treatment of chronic hcv infections. hcv infections can lead to progressive liver damage, cirrhosis, and liver cancer. the ns / a serine protease plays a critical role in virus replication and has become an antiviral drug development target. , the first specific and potent hcv protease inhibitor with good oral bioavailability was ciluprevir, which contains a carbamate functionality ( , biln , figure ). this noncovalent macrocyclic peptidic inhibitor was the result of a substrate-based approach for the design of active site inhibitors. this inhibitor is very active in enzymatic (ic = nm) and cell-based replicon assays (ic = . nm) of hcv genotype . ciluprevir was later discontinued due to cardiac toxicity in animal models, but its development paved the way to boceprevir (victrelis, schering-plough, approved by fda in may ) and telaprevir (vx- , vertex pharmaceuticals and johnson & johnson). , in particular, for the development of boceprevir, the introduction of a ketoamide moiety, together with p and p optimization, led to inhibitor showing a k i of nm. its x-ray crystal structure in complex with the enzyme also provided insight for further optimization. indeed, a cyclopropylalanine residue was found to be optimal at p , and the resulting carbamate derivative showed a k i of nm. although inhibitors and displayed good enzyme inhibitory potency, they did not display cellular activity in a subgenomic hcv replicon assay, possibly because of their strong peptidic character. the discovery that an nmethylated leucine at p was critical for both enzymatic potency and cellular activity led to the potential of cyclopropylfused proline being envisaged as an optimum, conformationally constrained surrogate for this part of the inhibitor. combination of the p -optimized ligand with previously optimized p and p residues provided carbamate derivative with a k i = . nm and ic = nm. finally, truncation and p optimization, by the employment of a cyclobutyl moiety, led to compound (k i = nm), the direct boceprevir ancestor. subsequently, compounds and ( figure ) with a carbamate containing p proline core showed very potent inhibitory activity (ic = nm for and nm for ). similarly, macrocyclic inhibitor with an α-amino cyclic boronate showed good potency (ic = nm). the electron-withdrawing effect of the ester and amide functionalities was also utilized in the design of α-ketoesteror α-ketoamide-derived transition state inhibitors. a range of hcv ns / a protease inhibitors were designed and synthesized, incorporating α-ketoamide templates at the scissile site. structure-based design led to a variety of potent acyclic and cyclic inhibitors with ketoamide templates, as exemplified in compounds (ic tripeptidyl α-ketobenzoxazole inhibited human neutrophil elastase (hne) with an ic of nm. the ketooxazolinederived inhibitor displayed very potent activity against hne (ic = . nm). . . hcv ns a inhibitors. carbamate derivatives also play a key role as inhibitors of hcv ns a, which represents a new and promising target for hcv therapy. hcv ns a is a zincbinding phosphoprotein, and its role in the hcv virus life cycle is still not clear. however, it plays a critical role in hcv rna replication. also, it is involved in virion morphogenesis. due to the lack of enzymatic function, inhibitors of this viral-encoded protein have been pursued. researchers at bristol-myers squibb screened a library of compounds for their ability to inhibit hcv rna replication. this led to the identification of a lead compound specifically interfering with rna replication and later proving to inhibit the activity of ns a protein. subsequent optimization was focused on broadening the genotype specificity and improving pharmacokinetic properties of compounds. symmetry of the molecule played an important role in inhibitory potency. this finally led to the discovery of daclatasvir ( , bms- , figure ) a first-in-class inhibitor of the hcv ns a replication complex. daclatasvir was approved in europe in august, . ledipasvir ( , gs- , gilead sciences, figure ) is another carbamate-containing hcv ns a inhibitor with potent antiviral activity against hcv genotypes a and b. harvoni, a combination of ledipasvir and sofosbuvir (a nucleotide polymerase inhibitor), was approved by the fda in october for the treatment of chronic hcv genotype infection. this also represents the first approved regimen that does not require administration with interferon or ribavirin. , . carbamates as cysteine protease inhibitors cysteine proteases, also known as thiol proteases, are proteolytic enzymes responsible for the degradation of proteins. these enzymes are divided into three classes based on their sequence homology: the papain, caspase, and picornaviridae families. the papain family of proteases is the most known and studied. cysteine proteases have been identified in a variety of diverse organisms, such as bacteria, eukaryotic micro-organisms, plants, and animals and are divided into the clans ca, cd, ce, cf, and ch in the merops peptidase database. the largest subfamily among the class of cysteine proteases is the papain-like cysteine proteases, originating from papain as the archetype of the cysteine proteases. clan ca proteases utilize catalytic cys, his, and asn residues that are invariably in this order in the primary sequence of the protease. clan ca, family c (papain-family) cysteine proteases are wellcharacterized for many eukaryotic organisms. also, the best characterized plasmodium cysteine proteases, namely, the falcipains, belong to papain-family (clan ca) enzymes. clan cd presents two catalytic residues, his and cys, in sequence; clan ce has a triad formed by his, glu, or asp and cys at the c-terminus; in clan cf, the asparagine residue of the catalytic triad is replaced by a glutamate residue and the catalytic triad is ordered as glu, cys, and his; clan cg has a dyad of two cysteine residues, and clan ch presents a cys, thr, and his triad with the catalytic cysteine at the n-terminus. the proteolytic mechanism involves the formation of a thiolate−imidazolium ion pair, which provides a highly nucleophilic cysteine thiol. over the years, many cysteine protease inhibitors have been designed by appropriately linking electrophilic warheads to the specific recognition sequence of peptide substrates. reversible inhibitor warheads include aldehydes, α-ketoamides, α-ketoesters, and α-ketoacids. these inhibitors interact with the protease active site, forming the tetrahedral intermediate, but are eventually hydrolyzed, regenerating both the enzyme and the inhibitor in an equilibrium reaction. irreversible inhibitors of cysteine proteases include epoxides, aziridines, haloketones, vinyl sulfones, and acyloxymethylketones. these inhibitors inactivate the target through alkylation of the active site cysteine thiol, permanently disabling enzyme function. , , − the occurrence of severe acute respiratory syndrome (sars) in and the subsequent identification of a novel coronavirus as the etiological agent recognized cysteine proteases sars-cov clpro and sars-cov plpro (papainlike protease) as possible targets for drug design. , subsequent structure-based design based on a previous inhibitor's x-ray co-crystal structure with the enzyme provided carbamate derivative ( figure ) as a potent sars-cov clpro inhibitor (ic = μm). a wide variety of human rhinovirus c (hrv c) protease inhibitors were developed by the incorporation of α,βunsaturated carbonyl moieties as warheads. hanzlik et al. reported the first hrv c protease inhibitors containing a peptide portion and incorporating α,β-unsaturated esters. the peptide parts were selected based on the substrate cleavage site. the representative carbamate-containing inhibitor ( figure ) showed an ic value of nm. human cathepsin k plays a critical role in bone resorption. in an effort to block bone resorption, noncovalent cathepsin k inhibitors were developed. kim et al. provided carbamate derivative ( figure ) as a noncovalent and reversible cathepsin k (ic = . μm) and l inhibitor (ic = . μm). glaxowellcome scientists developed carbamate-containing ketoamide-based cathepsin k inhibitors such as ( figure ) (ic = . nm). starting from a potent ketone-based inhibitor with unsatisfactory drug-like properties, , incorporation of p −p elements from the ketoamide-based inhibitor led to a hybrid series of ketone-based cathepsin k inhibitors with improved bioavailability, as exemplified in inhibitor ( figure ) (ic = nm). cathepsin s has been suggested for the development of agents against a range of immune disorders. a new class of nonpeptidic and noncovalent cathepsin s inhibitors was reported in . subsequent structural optimization resulted in a very potent and competitive noncovalent carbamate-containing inhibitor ( figure ) (ic = nm). metabolizing enzyme inhibitors carbamates have been employed in the design of serine hydrolase inhibitiors. in this section, we will focus on inhibitors of endocannabinoid metabolizing enzymes, in which the carbamate functionality plays an important role. the endocannabinoid system is known to be a ubiquitous neuromodulatory system with a wide range of action that can be found in every primitive organism. it is composed of cannabinoid receptors (cbrs), endogenous cannabinoids (endocannabinoids, ecs), and the enzymes responsible for their production, transport, and degradation. , ecs are a class of signaling lipids, such as n-arachidonoyl ethanolamine (anandamide, aea), oleamide, and -arachidonoyl glycerol ( -ag), that exert their biological actions through the interaction with two g-protein coupled receptors, cb and cb . they modulate a range of responses and processes including pain, inflammation, appetite, motility, sleep, thermoregulation, and cognitive and emotional states. the actions of these signaling lipids are rapidly terminated by cellular reuptake and subsequent hydrolysis operated by a number of enzymes. an attractive approach involved the modulation of the ec system and aimed at eliciting the desirable effects of cbrs activation through the pharmacological inactivation of the main endocannabinoid metabolizing enzymes, namely, monoacylglycerol lipase (magl) and α/β-hydrolase domain containing and (abhd and abhd ). these three serine hydrolases account for approximately % of -ag hydrolysis in the cns, whereas fatty acid amide hydrolase (faah) is responsible for aea inactivation. inactivation of these enzymes would elevate the endogenous concentrations of all of its substrate and consequently prolong and potentiate their beneficial effects on pain and anxiety without evoking the classical cb r agonists side effects (hypomotility, hypothermia, and catalepsy). monoacylglycerol lipase (magl) is the primary enzyme responsible for the hydrolysis of -ag in the cns. about % of the total -ag hydrolysis in the brain is ascribed to magl. magl is a kda membrane enzyme belonging to the superfamily of the serine hydrolases with a catalytic triad represented by ser , his , and asp . it is ubiquitously present in the brain (cortex, hippocampus, cerebellum, thalamu, and striatum), where it localizes to presynaptic terminals, even if lower levels are found in the brainstem and hypothalamus. a concomitant distribution in membranes as well as in the cytosol has been reported. magl shares a common folding motif called the α/β-hydrolase fold. studies in recent years have shown that magl inhibitors elicit antinociceptive, anxiolytic, and antiemetic responses. magl inhibitors have also been shown to exert anti-inflammatory action in the brain and protect against neurodegeneration through lowering eicosanoid production. recently, the potential of magl inhibitors for the therapy of fragile x syndrome has been reported. the early discovered magl inhibitors were molecules able to target the cysteine residues present in the active site of the enzyme. later, the research has been focused on the synthesis of compounds covalently binding to ser of the catalytic triad. among them, carbamate (urb , figure ) was the first selective inhibitor of -ag degradation, although its potency remained limited (ic = μm on rat brain). selective magl inhibitors bearing a carbamate scaffold were developed by cravatt and coworkers. inhibitor (jzl , figure ) exhibited selectivity toward faah in vitro (ic = . nm and μm for human recombinant magl and faah, respectively). more recently, cravatt and co-workers reported a distinct class of o-hexafluoroisopropyl (hfip) carbamates bearing a reactive group that is bioisosteric with endocannabinoid substrates. the representative compound, (kml , figure , ic = . nm, human magl), displays excellent potency and in vivo. in comparison to previously described o-aryl carbamates, inhibitor showed enhanced selectivity over faah and other serine hydrolases. abhd gene encodes a ∼ kda protein containing an nterminal transmembrane region followed by a catalytic domain that includes the canonical gxsxg active-site motif of serine hydrolases. abhd is a unique and highly conserved enzyme in mammals and is mainly expressed in the brain, liver, kidney, and brown adipose tissue. as a member of the serine hydrolase class, abhd is predicted to hydrolyze esters, amides, or thioester bonds in substrates that could include small molecules, lipids, or peptides. although, the full range of substrates regulated by this enzyme in vivo is currently unknown. recent studies have also shown that abhd carbamate inhibitors produce anti-inflammatory and neuroprotective effects in a mouse model of traumatic brain injury. among them, optimized inhibitor ( figure ) displayed an ic value of nm and notable selectivity. faah is a membrane-bound enzyme belonging to the amidase family. the analysis of its crystal structure revealed a core composed of a characteristic ser-ser-lys catalytic triad. the catalytic residues of faah are buried deep within the enzyme and are accessible by two narrow channels. the importance of faah was demonstrated by the generation of faah knockout mice. faah −/− mice showed an elevated resting brain concentration of aea and manifested (i) an analgesic phenotype in both the carrageenan model of inflammatory pain and in the formalin model of spontaneous pain, (ii) a reduction in inflammatory responses, and (iii) improvements in slow wave sleep and memory acquisition. , the urb class of compounds was the first class of inhibitors identified for faah, and it is well-represented by (urb , figure , ic = . nm). the n-( phenyl)hexylcarbamate analogue (jp , figure , ic = nm) is another very potent compound representative of the biphenyl series of inhibitors. gattinoni and co-workers developed a series of oxime carbamate inhibitors. compound ( figure , ic = nm) displayed good affinity and selectivity toward faah. more recently, butini et al. developed a new class of potent and selective faah reversible carbamate inhibitors. among them, compound (nf , figure , k i = . nm on mouse brain faah) showed excellent activity. the compound showed impressive selectivity toward all the enzymes and receptors of the endocannabinoid system. in this perspective, the role of carbamates in drug design and medicinal chemistry has been highlighted. in particular, the perspective covers physical properties of carbamates and the development of novel chemical methodologies overcoming the historical safety and toxicity issues related to their preparation. furthermore, the importance of carbamate-derived compounds in medicinal chemistry and their widespread employment as drugs and prodrugs have been discussed. also showcased is the exploitation of organic carbamates in the development of numerous aspartic acid, serine, and cysteine protease inhibitors. we hope that this perspective will stimulate further use of organic carbamate as a structural motif in drug design and medicinal chemistry. the authors declare no competing financial interest. financial support of this research by the national institutes of health (gm ) and purdue university is gratefully acknowledged. we would like to thank our colleagues anthony tomaine, heather osswald, and anindya sarkar (purdue university) for helpful discussions. ad, alzheimer's disease; cat-d, cathespsin d; faah, fatty acid amide hydrolase; ppar, peroxisome proliferator-activated receptor; gaba, γ-amino butyric acid; haart, highly active antiretroviral therapy; bis-thf, bis-tetrahydrofuran; bace , beta-site amyloid precursor protein cleaving enzyme ; mdr, multidrug resistant; nnrti, non-nucleoside reverse transcriptase inhibitors; adept, antibody-directed enzyme prodrug therapy; gdept, gene-directed enzyme prodrug therapy; mct , monocarboxylate transporter type ; smvt, sodiumdependent multivitamin transporter; ces , carboxylesterases ; magl, monoacylglycerol lipase ■ references unusually low barrier to carbamate c−n rotation amide resonance in thioand seleno-carbamates: a theoretical study role of conjugation in the stabilities and rotational barriers of formamide and thioformamide. an ab initio valence-bond study on the stabilization of the syn-rotamer of amino acid carbamate derivatives by hydrogen bonding the dipole moment and structure of the carbamate group chemical aspects of the restricted rotation of esters, amides, and related compounds hydrolysis in drug and prodrug metabolismchemistry qualitative structuremetabolism relationships in the 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hydrolases structural adaptations in a membrane enzyme that terminates endocannabinoid signaling mice lacking fatty acid amide hydrolase exhibit a cannabinoid receptormediated phenotypic hypoalgesia the endogenous cannabinoid system protects against colonic inflammation characterization of the sleep-wake patterns in mice lacking fatty acid amide hydrolase inhibition of fatty-acid amide hydrolase accelerates acquisition and extinction rates in a spatial memory task. neuropsychopharmacology design, synthesis, and structure-activity relationships of alkylcarbamic acid aryl esters, a new class of fatty acid amide hydrolase inhibitors mechanism of carbamate inactivation of faah: implications for the design of covalent inhibitors and in vivo functional probes for enzymes a new group of oxime carbamates as reversible inhibitors of fatty acid amide hydrolase discovery of potent inhibitors of human and mouse fatty acid amide hydrolases key: cord- - nta b w authors: slomka, marek j.; densham, anstice l. e.; coward, vivien j.; essen, steve; brookes, sharon m.; irvine, richard m.; spackman, erica; ridgeon, jonathan; gardner, rebecca; hanna, amanda; suarez, david l.; brown, ian h. title: original article: real time reverse transcription (rrt)‐polymerase chain reaction (pcr) methods for detection of pandemic (h n ) influenza virus and european swine influenza a virus infections in pigs date: - - journal: influenza other respir viruses doi: . /j. - . . .x sha: doc_id: cord_uid: nta b w please cite this paper as: slomka et al. ( ) real time reverse transcription (rrt)‐polymerase chain reaction (pcr) methods for detection of pandemic (h n ) influenza virus and european swine influenza a virus infections in pigs. influenza and other respiratory viruses ( ), – . background there is a requirement to detect and differentiate pandemic (h n ) (h n v) and established swine influenza a viruses (sivs) by real time reverse transcription (rrt) pcr methods. objectives first, modify an existing matrix (m) gene rrt pcr for sensitive generic detection of h n v and other european sivs. second, design an h rrt pcr to specifically detect h n v infections. methods rrt pcr assays were used to test laboratory isolates of siv (n = ; european and north american), h n v (n = ) and avian influenza virus (aiv; n = ). diagnostic sensitivity and specificity were calculated for swabs (n = ) and tissues (n = ) collected from field cases and pigs infected experimentally with sivs and h n v. results the “perfect match” m gene rrt pcr was the most sensitive variant of this test for detection of established european sivs and h n v. h rrt pcr specifically detected h n v but not european sivs. validation with clinical specimens included comparison with virus isolation (vi) as a “gold standard”, while field infection with h n v in swine was independently confirmed by sequencing h n v amplified by conventional rt pcr. “perfect match” m gene rrt pcr had % sensitivity and · % specificity for swabs, · % and · % for tissues. h rrt pcr demonstrated sensitivity and specificity of % and · %, respectively, for the swabs, and % and % for the tissues. conclusions two rrt pcrs for the purposes of (i) generic detection of siv and h n v infection in european pigs, and for (ii) specific detection of h n v (pandemic influenza) infection were validated. the pandemic (h n ) influenza a virus (h n v) emerged in april and was first detected in a cluster of human respiratory cases in mexico and the usa; initially, the virus could not be subtyped using available molecular tests. [ ] [ ] [ ] genetic and antigenic characterisation of the h n v virus showed it to be distinct from current seasonal h n influenza a viruses circulating in humans. , rapid spread of h n v to other continents and frequent escalating human transmission resulted in an official declaration of a new human influenza a pandemic by the world health organization (who) on june . , regardless of their host of origin, influenza a viruses usually have the potential to infect and, during dual infections, re-assort in other host species. pigs are particularly susceptible to infections from other hosts because receptors for influenza a viruses of both human and avian origin are present in their upper respiratory tract. , swine influenza viruses (sivs) circulating in pig herds include h n , h n and h n subtypes and these have been observed globally. [ ] [ ] [ ] [ ] h n sivs were first isolated in the s, and phylogenetic studies have shown that these ''classical swine'' isolates were originally closely related to the human h n ''spanish 'flu''' pandemic influenza virus of , but subsequently these classical h n sivs evolved into a unique genetic lineage. h n sivs have also evolved distinctly in pigs following the human ''asian 'flu''' pandemic of . during the early s, ''avian-like'' h n sivs became common in european farmed pigs and have replaced classical swine h n . , however, classical swine h n remained present in the usa and many parts of asia. [ ] [ ] [ ] [ ] evolution of sivs in north america has included emergence of a triple re-assortment h n in that contained genes of human, avian, and classical h n swine influenza origin genes. , these triple reassortant viruses have become endemic in north america, and, through additional reassortment events, triple reassortant variants of the h n and h n subtypes have been detected. , , in the case of h n sivs isolated in europe, the h gene is of human h seasonal influenza origin. , the first instance of infection of pigs with h n v was reported in may in canada where it was suspected that pigs were infected through contact with infected humans. , sivs are known to infect humans , and poultry, [ ] [ ] [ ] [ ] while human influenza a viruses are also known to transmit to pigs. continuing human cases of h n v during sustained veterinary concerns that this virus may become established in pigs, with a degree of concern that this host may then serve as a source for further influenza a reassortment and future zoonotic transmission. although swine influenza is not listed as a notifiable disease by the world organisation for animal health (oie), , siv surveillance programmes have been carried out in europe and north america and have provided valuable epidemiological information. [ ] [ ] [ ] [ ] these surveillance programmes have been based on conventional testing as recommended by the oie, using attempted virus isolation (vi) in cell culture and embryonated fowls' eggs (efes), followed by typing with defined antisera to identify the haemagglutinin (h) and neuraminidase (n) subtypes using well-established haemagglutination and neuraminidase inhibition tests (hi and ni), respectively. in recent years, highly sensitive and specific real time reverse transcription polymerase chain reaction (rrt pcr) technology has been exploited by veterinary institutes and reference laboratories to develop and validate appropriate tests for avian influenza viruses (aivs). these include validated rrt pcr tests for notifiable ai caused by h and h subtype viruses, following concerns resulting from the spread of h n highly pathogenic (hp) ai in poultry in the eastern hemisphere, together with a number of accompanying zoonotic cases. many veterinary laboratories have already embraced generic ai rrt pcrs that detect all six-teen h subtypes of influenza a, typically through amplifying within the highly conserved m gene, and also as specific assays for aivs of h and h subtypes. while public health institutions have recently described rrt pcrs for the detection of h n v in the context of the current human pandemic, [ ] [ ] [ ] [ ] in this study, we describe and validate similar approaches for the detection of h n v in pigs. full genome sequence analysis of the current h n v reveals the virus to be putatively of swine origin. , segment , which includes the matrix (m) gene that encodes the matrix m and m proteins, appears to be of eurasian siv origin, while segment , which encodes the h haemagglutinin (ha), is related to american classical siv h genes. , this study outlines the adaptation of an existing m gene rrt pcr assay for the generic detection of sivs and h n v, plus a novel rrt pcr that amplifies within the h gene for the specific detection of h n v in european pigs. viruses ninety-nine laboratory isolates of influenza a viruses were obtained from the influenza virus repository at the veterinary laboratories agency (vla-weybridge), grown in -to -day-old specific-pathogen-free embryonated fowls' eggs (efes) and typed using standard protocols. , these included swine influenza virus (siv) isolates, avian influenza virus (aiv) isolates plus five h n v isolates, which included two from humans and three from pigs (table ) . these efe-grown influenza a viruses were diluted at least -to -fold prior to rna extraction to give levels of virus that approximate to those present in clinical specimens. biological infectivity of efe-grown influenza a viruses was determined as the median egg infectious dose (eid ) per ml. ninety-seven frozen () °c) archived respiratory tissues were originally obtained during - from the routine swine influenza surveillance programme in united kingdom, in which acute respiratory disease of a suspected viral aetiology forms the primary submission selection criterion. all were tissue pools that included varying proportions of trachea and lung specimens. virus isolation (vi) in efes at the time of submission divided these into and tissue specimens from pigs that were infected with siv (table ) and uninfected, respectively. in addition, nasal swabs from pigs in united kingdom were collected by the vla regional laboratories at thirsk and bury st edmunds during the summer of . these were obtained from swine that had been submitted for endemic disease investigations. thirty-nine clinical specimens were received between september and november from nine pig herds at nine different locations in six countries and included respiratory swabs and respiratory tissues ( table ) . fifteen of these specimens (six swabs and nine tissues) were shown to be avian influenza viruses, with highly pathogenic (hp) h and h isolates indicated positive for h n v by non-rrt pcr approaches (table ) , i.e. amplification of rna extracted from the clinical specimen by conventional rt pcr using primers that had been designed specifically for the ha gene of current h n v isolates, available at: http://www.who.int/csr/resources/publications/swineflu/genomeprimers_ .pdf amplicons were electrophoresed in % agarose and stained with redsafeÔ (intron biotechnology, kyungki-do, korea) for visualisation, excised and purified from agarose using the qiaquick Ò gel extraction kit (qiagen, crawley, uk). nucleotide sequencing was performed using the big dye Ò terminator v ae cycle sequencing kit (applied biosystems, warrington, uk), and checked by blast analysis at: http://blast.ncbi.nlm.nih.gov/blast.cgi?program= blastn&blast_programs=megablast&page_type= blastsearch&show_defaults=on&link_loc=blast home=blasthome, which confirmed these specimens as h n v positive. in the case of four of these h n v specimens, virus was grown in efes (table ) and rna extracted from infective allantoic fluid, then similarly pcr amplified and sequenced to provide confirmation of h n v infection. uk pigs positive for avian-like swine h n archived tissues were pools that contained varying proportions of respiratory tissues, including trachea and ⁄ or lung, except for a ⁄ swine ⁄ england ⁄ ⁄ where two distinct tissues were tested from the same pig. these samples were used to assess (i) m gene rrt pcrs (original avian protocol and two variants) and (ii) h - rrt pcr. ct values for the most sensitive m gene rrt pcr for a given tissue specimen is shown in normal type, while ct values for the two less sensitive m gene rrt pcr variants are shown in italic type. clinical specimens from pigs infected experimentally six landrace hybrid pigs (age - weeks) were each infected experimentally by inoculation with ml h n v (a ⁄ california ⁄ ⁄ ), which contained ae eid per animal via the intranasal route. twenty-three nasal swabs were obtained by swabbing the infected pigs daily from to days postinfection (dpi), and pig tissues (lung n = , thoracic trachea n = ) were obtained from animals killed humanely for post-mortem examination on , and dpi (table ) . swabs one hundred and ten swabs from the field ( from vla regional laboratories during summer ; six from proven field cases of h n v infection, table ) and swabs from pigs infected experimentally (table ) were stored at ) °c in ml brain heart infusion broth containing antibiotics (bhib). swabs from the vla regional laboratories and h n v experimentally infected pigs (n = ) were tested by vi in -to -day-old efes by means of double inoculations via the allantoic and amniotic cavities, followed by a second efe passage in the event of the first passage being vi negative. influenza a virus growth was detected by a positive haemagglutination assay (ha) result obtained by testing harvested amnio ⁄ allantoic fluid with chicken red blood cells. in the case of six swabs from h n v-field-infected pigs, infection with this pandemic virus was independently proven by conventional pcr amplification, sequencing and blast searching as described earlier (table ) . organ homogenates (approximately % w ⁄ v in bhib) were prepared from archived uk tissue specimens ( from siv-positive pigs ( table ) and siv-negative pigs by vi) and tissues from h n v-field-infected pigs (september-november ; table ) by grinding with sterile sharp sand. for nine of tissues from h n v-fieldinfected pigs, infection with this pandemic virus was independently proven by conventional pcr amplification, sequencing and blast searching as described earlier (table ). ten tissue specimens from the experimentally infected pigs (table ) were disrupted in bhib using a general laboratory homogenizer (omni international, marietta, ga, usa) to provide a similar % w ⁄ v suspension. all tissue homogenates were clarified by centrifugation for minute prior to vi and rna extraction. vi was carried out by inoculating clarified tissue homogenates into -to -day-old efes as described earlier for pig swabs. the mini viral rna kit (qiagen) was used to extract rna from allantoic fluids, bhib swabs fluids and clarified tissue homogenates. this was performed either manually by the ''spun column'' method in accordance with the manufacturer's protocol, or by robotic rna extraction by the same kit chemistry adapted to a universal biorobot (qiagen). comparison of the m gene primer and probe sequence were performed on the influenza research database website http://www.fludb.org. several programmes available on the site were used for the comparison of available swine influenza sequence information including the snp analysis, blast and alignment viewer. the primers and hydrolysis probe used initially were those from the m gene rrt pcr originally described by spackman et al. (table ) for global and generic detection of aivs at final concentrations of ae and ae lm, respectively. the reverse primer was modified in two variations of the m gene rrt pcr assay to investigate the detection of sivs and h n v isolates. the first variant (''perfect match'' m gene rrt pcr) included modification of the aiv reverse primer used by spackman et al. to provide a perfect match primer (reverse modified, i.e. ''rev-mod'') with the corresponding region in the m gene of h n v isolates ( the second variant (''combo'' m gene rrt pcr) included an equimolar mix of the original aiv reverse primer plus the above rev-mod primer, with each included at ae lm final concentration. otherwise cycling conditions, temperatures and chemistry details for both variants were as outlined for the m gene rrt pcr, in which the original m gene rrt pcr and the two variants produced the same -bp product. two microlitres of extracted rna were tested in each m gene rrt pcr in a final ll volume using mx realtime pcr instruments (stratagene, amsterdam, the netherlands). fluorescence thresholds for all rrt pcr experiments were determined using default settings in the supplied stratagene mxpro software, and these were inspected visually after every experiment to ensure consistency. the antisense hydrolysis probe ( nucleotides long) was pro-rev: fam- ¢-ctg gct tct tac ctt tt*c ata taa gtt ctt c- ¢ [ - ], which was labelled with the fam fluorophore at the ¢ end and with black hole quencher (bhq ) at an internal t nucleotide (indicated as t*), while the ¢ terminal c nucleotide was modified to a dideoxy c to prevent probe extension by polymerase activity (eurogentec, liège (luik), belgium). primers and probe were included at final concentrations of ae and ae lm, respectively, and ll of extracted rna were tested in each h - rrt pcr in a final ll volume. other chemistry details and cycling conditions were as outlined for the h ha rrt pcr where stratagene mx realtime pcr instruments were used. occasionally, ''h - '' rrt pcr products were visually checked by electrophoresis in % (w ⁄ v) agarose gels according to standard procedures. table . these were designed to investigate any uk clinical specimens that may be positive by m gene rrt pcr but negative by ''h - '' rrt pcr and negative by vi. three ha gene alignments that included european siv isolates of the h n (avian-like swine), h n and human-like swine h n subtypes were constructed using the megalign software from the lasergene package. the primerselect programme was then used to design appropriate primers to generate small (i.e. < bp) amplicons that were of sufficient size for sequencing and identification by blast searching. five clinical specimens (one uk archived swine tissue submitted in january and four nasal swabs from analysis of the m gene primers and probe to available swine influenza sequences shows several different patterns based on geographical and temporal origins of the viruses (table ). m gene sequences are available from over siv isolates from north america, europe and asia, with different sequence patterns in each region (data not shown). the sequence for the forward m gene rrt pcr primer and the probe remains highly conserved for most isolates (table ). however, the reverse primer has extensive variability at primarily five different positions for h n v and siv isolates compared to the ''avian'' m gene rrt pcr primers and probes originally designed by spackman et al. current sivs and h n v isolates have from zero to four nucleotide mismatches in the reverse primer ( table ) that could potentially contribute to a decrease in sensitivity for some viruses. this influenced the design of the ''perfect match'' m gene rrt pcr ''rev-mod'' primer for h n v detection, which is located at the same corresponding position as the ''m) r'' primer for the ''avian'' m gene rrt pcr (table ) . h n v isolate a ⁄ california ⁄ ⁄ and siv isolate a ⁄ swine ⁄ england ⁄ ⁄ (h n ) were grown in efes and eid titres determined. rna was extracted from these influenza a preparations and used to construct fold dilution series, and both were tested by the three m gene rrt pcr assays, namely the ''avian'', ''combo'' and ''perfect match''. the -fold dilution series from a ⁄ california ⁄ ⁄ (h n v) was also tested by the ''h - '' rrt pcr. the detection limit corresponded to a viral titre (pre-rna extraction) of · eid ⁄ ml for the ''perfect match'' m gene and ''h - '' rrt pcrs, which typically registered at ct values - . when a dilution series of quantified t in vitro rna transcripts were tested, the detection limit of the ''perfect match'' m gene rrt pcr at these ct values was rna copies and rna copies for the h n v and h n targets, respectively. this discrepancy in sensitivity may relate to differences in the m gene reverse primer binding sequence for the two influenza a viruses ( fifty-one laboratory isolates of established sivs were divided into five categories based on combinations of subtype and temporal and geographical origin (table ) . for the two classical h n sivs isolated in united kingdom in and , the ''avian'' and ''combo'' m gene variant rrt pcr assays appeared to be the most sensitive. however, when contemporary uk and european sivs were tested, the ''perfect match'' m gene rrt pcr was the most sensitive for detecting of the isolates in the avian-like swine h n , h n and human-like swine h n categories (table ) . these three categories are representative of the sivs that have been circulating in europe for up to years. it should be noted that the ''combo'' m gene rrt pcr assay was slightly less sensitive, detecting the contemporary european sivs at ct values that were higher, but by no more than for the majority of these isolates ( table ). the one exception was a ⁄ swine ⁄ eire ⁄ ⁄ (h n avian-like), which was most sensitively detected by the original ''avian'' m gene rrt pcr (table ) . in contrast, the north american sivs were most sensitively detected by the ''combo'' m gene rrt pcr assay in which the lowest ct values were obtained, followed by the ''avian'' m gene rrt pcr. the ''perfect match'' m gene rrt pcr assay was the least sensitive for detection of the north american sivs as the ct values obtained were consistently higher than in the other two assays (table ) . it can be inferred from these findings that the ''perfect match'' m gene rrt pcr assay would be the most sensitive to use for generic testing of contemporary european sivs as well as any pig infections caused by h n v. however, for north american pigs, the ''combo'' m gene rrt pcr provided the most sensitive approach to generic siv testing for of north american isolates, the one exception being a ⁄ swine ⁄ nebraska ⁄ ⁄ (h n ) where the ''avian'' m gene rrt pcr registered a slightly lower ct value compared to the ''combo'' m gene rrt pcr (table ) . representative isolates of all sixteen h-types were included in the aivs tested (table ) . forty-two of were detected most sensitively by the ''avian'' m gene rrt pcr as this test consistently gave the lowest ct values, the one exception being a ⁄ duck ⁄ belgium ⁄ ⁄ (h n ) ( table ). this was followed by the ''combo'' m gene rrt pcr, while the ''perfect match'' m gene rrt pcr assay proved to be the least sensitive for the detection of the aiv isolates. the low sensitivity of the ''perfect match'' m gene rrt pcr assay resulted in ''no ct'' being recorded for six aiv isolates, possibly because their rna had been extracted from highly diluted virus preparations. one aiv rna (a ⁄ mallard ⁄ sweden ⁄ , h n ) was so highly diluted that a high ct value ( ae ) was obtained with the ''avian'' m gene rrt pcr, but ''no ct'' registered by both the ''perfect match'' and ''combo'' m gene rrt pcr assays (table ). the ''h - '' rrt pcr assay was used to test siv and aiv isolates plus five h n v isolates (table ). in the tests, comparable ct values were obtained for the five h n v isolates to those observed in the ''perfect match'' m gene rrt pcr assay ( table ) . none of the european siv laboratory isolates, including the h n ( avian-like swine and two classical) and eight h n sivs, were detected by the ''h - '' rrt pcr assay. the ''h - '' rrt pcr assay was designed specifically for differential detection of h n v influenza a viruses. non-detection of contemporary, established european h sivs by the ''h - '' rrt pcr as well as h aivs indicated that it fulfils this condition (table ) . however, positive fluorescence signals were obtained in ''h - '' rrt pcr tests with two of the north american h n sivs, namely a ⁄ swine ⁄ wisconsin ⁄ h ys ⁄ and a ⁄ swine ⁄ nebraska ⁄ ⁄ (table ) . tests with the five other north american h n sivs produced negative fluorescence signals, but gel electrophoresis in % w ⁄ v agarose showed that all seven of the north american h n isolates tested produced a band of the predicted -bp size ( table ) . h gene sequences are available in the public databases for two of these north american h n isolates, namely a ⁄ swine ⁄ ontario ⁄ ⁄ (accession no. dq ) and a ⁄ swine ⁄ indiana ⁄ ⁄ (accession no. m or cy ). a degree of conservation was observed in the primer binding sequences for the ''h - '' rrt pcr, while mismatches in the probe-binding region appeared to account for the failure to generate fluorescence signals (data not shown). ''h - '' rrt pcr testing of both north american h n isolates also produced the -bp band of predicted size. a ⁄ swine ⁄ indiana ⁄ k ⁄ (h n ) (af ) produced fluorescence in this test, but a ⁄ swine ⁄ ontario ⁄ ⁄ (h n ) (dq ) was fluorescence negative ( table ). the a ⁄ swine ⁄ indiana ⁄ k ⁄ h gene sequence has a perfect match with the primer and probe-binding regions for the ''h - '' rrt pcr, while the corresponding h gene sequence in a ⁄ swine ⁄ ontario ⁄ ⁄ included significant mismatches in its probe-binding region (data not shown). the non-influenza a viruses were tested by the three m gene rrt pcr assays and by the ''h - '' rrt pcr assay and only negative results were obtained. ninety-seven frozen archived respiratory tissues that had been obtained from pigs in the united kingdom since were tested. these were divided into two groups, those that had been obtained from pigs that were (i) siv positive (n = ) and (ii) siv negative (n = ) based on vi at the time of sample submission. all archived tissue specimens from positive pigs were retested by vi in the course of this study, as well as by the three m gene rrt pcr approaches and ''h - '' rrt pcr ( table ). the tissues from siv-positive pigs included tissues from pigs infected with avian-like swine h n viruses between and , six from pigs with h n virus infections from to and seven from pigs infected with human-like swine h n virus during - ( table ). three of these archived tissue samples from vi-positive pigs gave no ct value with any of the m gene rrt pcr assays. no ct value was obtained by the ''avian'' m gene rrt pcr with three further samples, although these were positive by the other two m gene assays ( table ). one tissue specimen was negative by the ''combo'' m gene rrt pcr but gave high ct's by the other two m gene rrt pcrs ( table ) . as in the case of the more contemporary siv laboratory isolates from united kingdom and european origin (table ) , the ''perfect match'' m gene rrt pcr was shown to be the most sensitive of the m gene rrt pcr variants for the majority of the archived siv-positive tissue specimens, as for of tissues the m gene ''perfect match'' rrt pcr registered the lowest ct compared to the two other m gene rrt pcrs ( table ). the one exception was the oldest tissue specimen, from which a ⁄ swine ⁄ england ⁄ ⁄ (h n ) had been isolated; in this case, the original avian m gene rrt pcr was most sensitive ( table ) . these test results with archived tissue specimens obtained from the field reinforced the observation that the ''perfect match'' m gene rrt pcr is the most sensitive for detecting contemporary european and uk sivs. all archived uk tissue samples from siv-positive pigs were negative by the ''h - '' rrt pcr assay (table ) . only four of these archived tissues were vi positive during the current validation study, this may reflect the age and ⁄ or storage history of the tissues, plus the use of only two egg passages for vi. sixty-six archived tissue specimens from siv-negative pigs from united kingdom (organ pools of lung, trachea and tonsil, or lung only) were tested by the ''perfect match'' m gene rrt pcr assay to assess its specificity. all gave ''no ct'' results except one, which was submitted in january and produced a ct value of ae by the ''perfect match'' m gene rrt pcr. repeat vi testing during the current validation study failed to recover viable influenza a virus from this specimen. however, investigation of this specimen by conventional rt pcr and amplicon sequencing identified an h gene that was highly suggestive of an h n siv. all of these archived tissues from siv-negative pigs were ''no ct'' by the ''h - '' rrt pcr. nasal swabs collected in the field by vla regional laboratories one hundred and four nasal swabs obtained from pigs in united kingdom during the summer of were tested by vi in efes, the ''perfect match'' m gene rrt pcr assay and the ''h - '' rrt pcr assay. one hundred of these swabs were negative by all three tests. however, four swabs that originated from two uk pig herds gave negative results by vi and ''h - rrt'' pcr but resulted in positive amplifications by ''perfect match'' m gene rrt pcr, with ct values of ae and ae for one herd and ae and ae for the second herd. investigation by conventional rt pcr and sequencing revealed an h gene that was suggestive of h n siv. of the specimens ( respiratory swabs and respiratory tissues) received between september and november from nine geographically diverse pig herds (table ) , fifteen (six swabs and nine tissues) originating from eight pig herds had been shown to be h n v positive by non-rrt pcr approaches (table ) . these and a further specimens ( swabs and six tissues) that included two lung specimens from an epidemiologically related ninth herd in northern ireland (ni ''c''; table ) were tested by both the ''perfect match'' m gene and ''h - '' rrt pcrs. twenty-two of the swabs were positive by both rrt pcrs, with ct value ranges of ae - ae and ae - ae for the ''perfect match'' m gene and ''h - '' rrt pcr assays, respectively ( table ). the submission derived from herd ''c'' in ni that included two lung tissues gave positive results by both rrt pcrs for one specimen, but the second gave high ct values ( ae and ae by ''perfect match'' m gene and ''h - '' rrt pcrs, respectively) that were just above the positive cutoff for the two tests (table ) . however, the clear positive result for the first lung specimen had identified h n v infection in this pig herd. one swab from the norwegian herd gave ''no ct'' by both rrt pcr assays, but the other nasal swabs from the same herd gave similar positive ct values by both methods. the tissues were all positive by both tests, with ct value ranges of ae - ae and ae - ae for the ''perfect match'' m gene and ''h - rrt'' pcr assays, respectively (table ) . thirty-three clinical specimens collected from pigs infected experimentally with h n v were tested by vi, the ''perfect match'' m gene rrt pcr assay and the ''h - '' rrt pcr assay (table ). these samples included nasal swabs taken between and dpi, and post-mortem tissues (lung, n = ; thoracic trachea, n = ). twentytwo of the swabs gave positive results by vi and both rrt pcr assays, while one swab was weakly positive by the two rrt pcrs and vi negative ( dpi, pig ; table ). the ct ranges for these swabs were ae - ae and ae - ae for ''perfect match'' m gene and ''h - '' rrt pcr assays, respectively. the post-mortem tissues from the experimentally infected pigs gave concordant results by all three tests, i.e. seven were positive and three were negative ( table ). the ct ranges for these positive tissues were ae - ae and ae - ae for the ''perfect match'' m gene and ''h - '' rrt pcr assays, respectively. the sensitivity and specificity were calculated for the ''perfect match'' m gene and ''h - '' rrt pcr assays from the clinical specimens (respiratory swabs and tissues) used in this validation study. for clinical specimens, confirmatory results were available either as vi, or in the case of h n v field specimens, through independent confirmation by amplicon sequencing (table ) . for swabs, a diagnostic sensitivity of % and a specificity ae % were calculated for the ''perfect match'' m gene rrt pcr. with this assay, there were five results that were vi negative, but ''perfect match'' m gene rrt pcr positive (table a ). evidence presented elsewhere in the results strongly suggests that these five were genuine positives. when the swabs were tested by ''h - rrt'' pcr, a diagnostic sensitivity of % and specificity of ae % was determined (table b) . there was only one swab, obtained from an experimentally infected pig at dpi, that was vi negative but positive by ''h - rrt'' pcr (table b) , this swab from pig was also positive by ''perfect match'' m gene rrt pcr (table ). in the case of the archived pig tissues collected in united kingdom between and , the specimens were divided into those from siv-positive (n = ; table ) and siv-negative (n = ) pigs that had been identified by vi at the time of the original submission. by inclusion of additional tissues from h n v-fieldinfected (table ) and experimentally infected (table ) pigs, a diagnostic sensitivity of ae % and a specificity of ae % were determined for the ''perfect match'' m gene rrt pcr (table a) . three archived tissues from pigs that were vi positive gave negative results by ''perfect match'' m gene rrt pcr (table ) , and one archived tissue was positive by ''perfect match'' m gene rrt pcr but vi negative (table a ). when the same tissues were tested by ''h - '' rrt pcr assay, the diagnostic sensitivity and specificity were both %, respectively (table b ). in recent years, the m gene rrt pcr assay has gained widespread acceptance as a validated method for the global detection of all aiv subtypes, and this method is referred to as a suitable screening assay in the oie and eu diagnostic manuals for ai. , although no international recommendations have been made concerning rrt pcr testing for sivs, the highly conserved nature of the m gene in all influenza a viruses led to the m gene rrt pcr being considered as a candidate for an initial screening assay for detecting pigs infected with all sivs, including any infections because of h n v. in this study, we have investigated the application of the ''avian'' m gene rrt pcr, but sequence differences observed in the m gene of h n v isolates also prompted investigation of two new variations of the original m gene rrt pcr. the original ''avian'' m gene rrt pcr reverse primer (m) r), with four nucleotide differences to h n v viruses (table ) , clearly resulted in a significantly lower sensitivity when testing h n v laboratory isolates (table ) . consequently, the original avian m gene rrt pcr should not be used for detection of h n v, and this deficiency of the diagnostic test needed to be addressed. among the three variants of the m gene rrt pcr that were evaluated, the ''perfect match'' with the revised reverse primer (''revmod'') provided the most sensitive results for many of the european sivs (tables and ). m gene sequence analysis showed that the ''rev-mod'' primer compensated for two or three nucleotide mismatches in the corresponding region of the m gene from contemporary european sivs (table ) . however, experiments with t in vitro rna transcripts corresponding to the amplified m gene region of a ⁄ swine ⁄ england ⁄ ⁄ (h n ) suggested that the ''perfect match'' m gene rrt pcr may not be maximally sensitive for detection of all contemporary european sivs, which again reflected sequence mismatches with the ''revmod'' primer. nevertheless, the ''perfect match'' m gene rrt pcr demonstrated successful detection of contemporary european sivs in archived tissues from infected pigs ( table ) , plus five specimens (four nasal swabs from uk herds and one archived tissue, collected in , highly likely to be h n ), that were negative by vi (tables and ) . importantly, the ''perfect match'' rrt pcr successfully detected both h n v laboratory isolates (table ) and clinical specimens from pigs that had been infected with h n v in the field (table ) and experimentally (table ) . the ''combo'' m gene rrt pcr assay was slightly less sensitive for detection of contemporary european sivs and h n v (tables and ) . however, for the testing of most north american sivs that were included in this study, the ''perfect match'' m gene rrt pcr suffered from reduced or even poor sensitivity (table ) . for these sivs, improved sensitivity was restored by employing the ''combo'' m gene rrt pcr, where inclusion of the original ''avian'' m) r reverse primer reduced the number of nucleotide mismatches within the corresponding sequence of the north american sivs' m gene (tables and ). it must also be emphasised that the ''avian'' m gene rrt pcr remains the most sensitive of the three m gene rrt pcrs for the detection of aivs (table ) . the diagnostic sensitivity and specificity of the ''perfect match'' m gene rrt pcr assay were % and ae %, respectively, from the testing of swabs (table a ). the five discrepant results included a vi-negative nasal swab obtained from a h n v-experimentally infected pig at dpi when viral shedding appeared to be waning (ct ae ), and this low titre was mirrored by the corresponding h - rrt pcr (ct ae ; table ). the other four discrepant results were vi-negative swabs collected by uk regional laboratories during summer that were shown to be likely h n siv positives, so affirming these ''perfect match'' m gene rrt pcr results. this inferred a greater sensitivity for the rrt pcrs compared to vi. as regards ''perfect match'' m gene rrt pcr testing of the swine tissues, however, there were four discrepant results, where a diagnostic sensitivity and specificity of ae % and ae %, respectively, were observed (table a ). the uk archived swine tissues were classified as siv positive or negative at the time of original submission based on vi. three of the tissues from siv-positive pigs were negative (no ct) by the ''perfect match'' m gene rrt pcr (table ). it was speculated that at the time of the original submission these pigs may have been classified as siv positive based on a vi result from another localised tissue portion or clinical specimen that was no longer available for the current validation. it was also observed that only of tissues obtained from siv-positive pigs were positive when vi was repeated through two egg passages during the current validation (table ). this may be a consequence of the long-term storage history of these archived tissues, where infectivity may have been compromised by repeated freeze-thawing, or even because of limiting vi to two egg passages. one archived tissue from an siv-negative pig collected in was positive by ''perfect match'' m gene rrt pcr, negative by ''h - '' rrt pcr and vi (table ) , but additional investigations suggested that this was a non-viable h n specimen. the h - rrt pcr assay was designed for the differential detection of h n v infections of pigs. this method successfully detected five h n v laboratory isolates (table ) and the presence of h n v in clinical specimens ( swabs and post-mortem tissues) in fieldinfected and experimentally infected pigs (tables and ) . it was also shown that the h genes from nine h aivs, european h sivs (i.e. classical h n , contemporary avian-like swine h n and h n ) were not detected ( table ) by this assay. in addition, no positive signal was obtained when archived tissue samples obtained from pigs infected with european h n and h n sivs were tested with the h - rrt pcr assay ( table ). this affirmed the ability of the h - rrt pcr assay to differentiate h n v infections from those caused by the sivs endemic in europe. however, this test did show some cross-reactivity with north american h sivs (table ) . this was not unexpected in view of the evolutionary relationship between the h gene of north american triple reassortment sivs and h n v. consequently, it would not be an appropriate test for differentiating h n v in north american pig infections. the diagnostic sensitivity and specificity of the ''h - '' rrt pcr assay for swabs were % and ae %, respectively (table b) ; the one discrepant result was the swab obtained at dpi from pig infected experimentally with h n v when the titre of viral shedding was declining (table ) . for the ''h - '' rrt pcr using tissue samples, both the diagnostic sensitivity and specificity of this assay were % (table b) . fifteen clinical specimens from h n v-field-infected pigs ( table ) were independently proven as h n v positive by conventional rt pcr and amplicon sequencing because vi was not consistently successful for the identification of h n v. this is because h n v does not consistently haemagglutinate chicken red blood cells, which may not be observed until the third passage in efes (data not shown). a further clinical specimens ( swabs and six tissues) from h n v-field-infected herds were not included in the diagnostic sensitivity and specificity calculations, but testing by both rrt pcrs gave highly concordant results as judged by a comparison of ct values (table ) . public health laboratories have already described rrt pcr protocols for the detection of h n v in humans, [ ] [ ] [ ] [ ] and in this study, we have validated rrt pcr testing for detection of h n v for veterinary purposes, specifically for detecting h n v in pigs. this included evaluation of a generic approach to detect known sivs as well as h n v. this was achieved for european sivs by the ''perfect mach'' m gene rrt pcr assay, which sensitively detected all known types of european sivs in addition to h n v. however, it was noted that contemporary irish sivs may be the exception to this observation in europe (table ) . testing of recent irish sivs by the various m gene rrt pcr assays suggests that the original ''avian'' m gene rrt pcr may be the more sensitive approach (raleigh and flynn, personal communication). in the case of north american sivs, the ''combo'' m gene rrt pcr assay was most sensitive for generic detection, and this method was slightly less sensitive for the detection of h n v than the ''perfect match'' m gene rrt pcr assay. the ''h - '' rrt pcr assay was shown to be able to differentiate h n v from siv infections in clinical samples derived from field-infected and experimentally infected pigs relevant to a european setting. validation of the generic and differential rrt pcr assays involved not only testing of a range of laboratory-grown influenza a virus isolates, but also crucially included testing of the same set of clinical specimens from pigs infected with sivs or h n v. primers and probe for the differential ''h - '' rrt pcr were deliberately designed within the relatively conserved ha region of the ha gene of h n v. however, continuing molecular evolution the ha gene may yet result in nucleotide changes within these primer and probe-binding sequences. vigilance for relevant nucleotide changes by sequencing the ha genes of new h n v swine isolates may identify potential future modifications to the ''h - '' rrt pcr. validation of the rrt pcr assays was conducted from the perspective of veterinary contingency planning for the eventuality of h n v outbreaks in the european pig population. to date, h n v infections have been already documented in pigs in the americas, eurasia and australasia , , and examples of field cases have been included in this study ( table ) . as the human pandemic caused by h n v continues to spread globally, it is anticipated that further pig infections with h n v will be reported, and precedent suggests that these will be followed by sustained transmission within pig populations. analogous contingency planning was conducted in , when the westward spread of h n highly pathogenic aiv prompted the validation of a h rrt pcr for the detection of both this h virus and other eurasian h aivs. in conclusion, it is possible to present these approaches as sensitive and specific methods for use in surveillance for siv and current h n v isolates in european pig populations. pneumonia and respiratory failure from swine-origin influenza a (h n ) in mexico swine influenza a (h n ) infection in two children -southern california novel swine-origin influenza a (h n ) virus investigation team. emergence of a novel swine-origin influenza a (h n ) virus in humans antigenic and genetic characteristics of swine-origin a (h n ) influenza viruses circulating in humans origins and evolutionary genomics of the swine-origin h n influenza a epidemic pandemic potential of a strain of influenza a (h n ): early findings world now at the start of influenza pandemic. who statements evolving complexities of influenza virus and its receptors the role of swine in the generation of novel influenza viruses the epidemiology and evolution of influenza viruses in pigs the role of pigs in interspecies transmission the emergence of novel swine influenza viruses in north america swine influenza viruses: a north american perspective multiple genetic reassortment of avian and human influenza a viruses in european pigs, resulting in the emergence of an h n virus of novel genotype oie disease notification in pigs canada. (early may). available at pigs destroyed following flu outbreak laboratory characterization of a swine influenza virus isolated from a fatal case of human influenza human infection by a swine influenza a (h n ) virus in switzerland isolation of viruses antigenically related to the swine influenza virus from an outbreak of respiratory disease in turkey farms in israel interspecies transmission and reassortment of influenza a viruses in pigs and turkeys in the united states the nucleotide sequence of the ha of the haemagglutinin of an h avian influenza virus isolate from turkeys in germany provides additional evidence suggesting recent transmission from pigs isolation from turkey breeder hens of a reassortant h n influenza virus with swine, human, and avian lineage genes further evidence for infection of pigs with human-like h n influenza viruses in china avian and swine influenza viruses: our current understanding of the zoonotic risk diseases of swine oie (world organisation for animal health) swine influenza: manual of diagnostic tests and vaccines for terrestrial animals, chapter . . . paris: oie a review of rt-pcr technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the world organisation for animal health available at evaluation of four real-time pcr assays for detection of influenza a(h n )v viruses molecular detection of a novel human influenza (h n ) of pandemic potential by conventional and real-time quantitative rt-pcr assays detection of novel (swine origin) h n influenza a virus by quantitative real-time rt-pcr avian influenza: manual of diagnostic tests and vaccines for terrestrial animals validated realtime reverse transcriptase pcr methods for the diagnosis and pathotyping of eurasian h avian influenza viruses a simple method of estimating fifty percent endpoints replication, pathogenesis and transmission of pandemic (h n ) virus in non-immune pigs biohealthbase: informatics support in the elucidation of influenza virus host pathogen interactions and virulence development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h and h haemagglutinin subtypes the influenza virus resource at the national center for biotechnology information the condensed protocols from ''molecular cloning: a laboratory manual a method for the rapid construction of crna standard curves in quantitative real-time reverse transcription polymerase chain reaction real-time rt-pcr, chapter report reference: nswpigs _ , ref oie: validated h eurasian real-time reverse transcriptase-polymerase chain reaction and its application in h n outbreaks in the authors thank all laboratories that supplied specimens of swine influenza and pandemic (h n ) influenza used in this study, in particular the vla regional laboratories at bury st edmunds and thirsk for their long-standing contribution to swine influenza surveillance in united kingdom. the contribution of dr dennis alexander to the checking of this manuscript is also gratefully acknowledged, as is the funding that was provided from the uk department of the environment, food and rural affairs (defra). key: cord- -x bxnrtn authors: wu, zhiqiang; lu, liang; du, jiang; yang, li; ren, xianwen; liu, bo; jiang, jinyong; yang, jian; dong, jie; sun, lilian; zhu, yafang; li, yuhui; zheng, dandan; zhang, chi; su, haoxiang; zheng, yuting; zhou, hongning; zhu, guangjian; li, hongying; chmura, aleksei; yang, fan; daszak, peter; wang, jianwei; liu, qiyong; jin, qi title: comparative analysis of rodent and small mammal viromes to better understand the wildlife origin of emerging infectious diseases date: - - journal: microbiome doi: . /s - - - sha: doc_id: cord_uid: x bxnrtn background: rodents represent around % of all mammalian species, are widely distributed, and are the natural reservoirs of a diverse group of zoonotic viruses, including hantaviruses, lassa viruses, and tick-borne encephalitis viruses. thus, analyzing the viral diversity harbored by rodents could assist efforts to predict and reduce the risk of future emergence of zoonotic viral diseases. results: we used next-generation sequencing metagenomic analysis to survey for a range of mammalian viral families in rodents and other small animals of the orders rodentia, lagomorpha, and soricomorpha in china. we sampled , small animals from provinces and then outlined the spectra of mammalian viruses within these individuals and the basic ecological and genetic characteristics of novel rodent and shrew viruses among the viral spectra. further analysis revealed that host taxonomy plays a primary role and geographical location plays a secondary role in determining viral diversity. many viruses were reported for the first time with distinct evolutionary lineages, and viruses related to known human or animal pathogens were identified. phylogram comparison between viruses and hosts indicated that host shifts commonly happened in many different species during viral evolutionary history. conclusions: these results expand our understanding of the viromes of rodents and insectivores in china and suggest that there is high diversity of viruses awaiting discovery in these species in asia. these findings, combined with our previous bat virome data, greatly increase our knowledge of the viral community in wildlife in a densely populated country in an emerging disease hotspot. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. approximately two thirds of emerging infectious diseases (eids) that affect humans originate from bats, rodents, birds, and other wildlife [ ] [ ] [ ] . in many of these reservoir host species, emerging viruses appear to be well adapted, with little or no evidence of clinical disease. however, when these viruses spill over into humans, the effects can sometimes be devastating [ ] [ ] [ ] . previously, our limited knowledge of the viral population and ecological diversity harbored by wildlife have complicated the study of eids. thus, comprehensive understanding of the viral community present in wildlife, as well as the prevalence, genetic diversity, and geographical distribution of these viruses, could be valuable for prevention and control of wildlife-origin eids [ ] . the order rodentia is the largest mammalian order, with families and , species (~ % of all mammal species). they live in close contact with humans and their domestic animals and act as a bond between humans, domestic animals, arthropod vectors (ticks, mites, fleas), and other wildlife [ ] [ ] [ ] . this interface with humans has led to the rodent origin of important zoonotic viruses including members of the family arenaviridae, hantaviridae, reoviridae, togaviridae, picornaviridae, and flaviviridae [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . many of these viruses cause severe disease in humans (e.g., lassa virus; tick-borne encephalitis virus, tbev; lymphocytic choriomeningitis virus, lcmv; sin nombre virus; hantaan virus, htnv; seoul virus, seov; and puumala virus); have only recently been discovered (e.g., whitewater arroyo virus and lujo virus); or appear to have a wider geographical range than originally thought (e.g., junin virus, guanarito virus, machupo virus, and sabia virus), suggesting that further viral discovery studies in wild rodent populations may be valuable for public health [ , - , , - ] . recent reports of rodent viruses have enabled new hypotheses regarding the evolution of hepaciviruses and the origin of coronaviruses (covs) and picornaviruses (picovs) such as hepatitis a virus [ ] [ ] [ ] [ ] . china is a megadiversity country and harbors~ rodent species from families [ ] . to develop baseline data on the origin of existing viral eids and identify other potential zoonotic viral reservoir hosts, we have conducted a series of viral surveys from rodents, bats, and other small animals and have simultaneously constructed online viral databases of these animals (dbatvir and drodvir, http://www.mgc.ac.cn/) since [ ] [ ] [ ] [ ] . in the current study, , small mammal individuals of species from the orders rodentia, lagomorpha, and soricomorpha across china were sampled by pharyngeal and anal swabbing. virome analysis was then conducted to outline the viral spectrum within these samples. on the basis of virome data, we describe the community, genetics, evolution, and ecological distribution characteristics of viruses and determined whether these features change with their host species and locations. the identification of novel mammal viruses provides new clues in the search for the origin or evolution pattern of human or animal pathogens such as hantaviruses (hvs), arenavirus (arevs), covs, and arteriviruses (artevs). pharyngeal and anal swabs were collected from , individual small mammals captured from july to july in provinces across china ( fig. a and additional file : table s ). these comprised rodent species of the families muridae, cricetidae, sciuridae, dipodidae, chinchillidae, and gliridae; two lagomorphs of the family ochotonidae; and three soricomorphs of the family soricidae that reside in urban, rural, and wild areas throughout china. the most common species sampled were apodemus agrarius, niviventer confucianus, rattus norvegicus, rattus tanezumi, rattus losea, and sorex araneus. due to repeated sampling of some species in the same location, swabs were combined into pools for analysis. a total of . gb of nucleotide data ( , , valid reads, bp in length) was obtained. reads classified as eukaryotes or prokaryotes and those with no significant similarity to any amino acid (aa) sequence in the nr database were discarded, leading to , , reads best matched with viral protein sequences in the nr database (~ . % of the total sequence reads). the number of virus-associated reads in each lane varied between , and , . a wide range of dna and rna virus groups were covered by these reads. virus-associated reads were assigned into families of double-stranded (ds)dna viruses, dsrna viruses, retro-transcribing viruses, single-stranded (ss)dna viruses, and ssrna viruses in the virus root (additional file : table s ). dietary habits and other host traits were used to exclude insect viruses, fungal viruses, plant viruses, and phages as described previously [ ] . the remaining , , sequence reads (~ . % of the total viral hits) were assigned into families of mammalian viruses (additional file : tables s and additional file : table s ). the prevalence diagram of each viral family related to province, animal species, and reads number was shown in fig. b . the relative abundances of the viral families in pooled samples of different provinces and animals were calculated by normalizing sequence reads and were shown in fig. c . viral reads from the families herpesviridae, picobirnaviridae, anelloviridae, circoviridae, retroviridae, astroviridae, coronaviridae, and picornaviridae, and the subfamily parvovirinae, were widely distributed in different animal species from different regions of china. viral reads from the families adenoviridae, poxviridae, papillomaviridae, reoviridae, arenaviridae, arteriviridae, hantaviridae, caliciviridae, flaviviridae, hepeviridae, rhabdoviridae, and paramyxoviridae were found in fewer species. many of the sequence reads related to mammalian viruses showed low nucleotide (nt) and aa sequence identity with known viruses. the abundance of virus strains in these mammalian viral families was further confirmed on a sample-by-sample basis by pcr screening. in total, positive results were obtained, and viruses from representative positive samples (representative virus strains represent viruses with identical or almost identical sequences (≥ % nt identity) from the same host species at the same location) were selected for genomic or partial genomic sequencing as quasi-species of these viruses (additional file : table s ). according to the genus and species demarcation criteria in each viral family established by the international committee on taxonomy of viruses (ictv; https://talk.ictvonline.org/), these viruses may represent new species and at least seven new genera. although sequence reads apparently from the families hepadnaviridae and poxviridae were occasionally present in some of the samples, it was not possible to amplify viral sequences from these samples, likely due to low viral loads. no virus was detected from samples of lagomorphs, this finding may reveal that the virome of lagomorphs is far less abundant than that of rodents and soricomorphs. by classifying all positive results into each viral family and host genus and then normalizing the virus number according to the sampling number of each host genus, our analysis revealed significant differences among hosts in terms of virus composition and abundance (fig. a) . animals of the families muridae and cricetidae acted as major reservoirs for diverse mammalian viruses in china. although hvs were detected in animals of the families muridae, cricetidae, dipodidae, and soricidae, most of them were of low abundance when compared with other rna viruses. arevs were only detected in animals of the families muridae and dipodidae with low abundance, and artevs were only detected in animals of the families cricetidae and dipodidae with low abundance. animals of the family muridae and the subfamily arvicolinae were the main hosts of covs and astroviruses (astvs) with high viral richness. notably, although limited samples from animals of the family dipodidae were collected ( animals from four species) for virome analysis, allactaga and dipus still harbored diverse viruses with high abundance. to further describe the viral diversity and abundance differences among geographical locations, the virus number of each family was assigned and normalized by the sample size in each province (fig. b) . since many host rodent species such as rattus and apodemus table s . b the prevalence diagram of each viral family related to province, animal species, and reads number. the x axis represents how many provinces certain viral family presents; the y axis represents how many animal species certain viral family presents; and the sizes of these circles represent the sizes of reads numbers of viral families. c heatmap based on the normalized sequence reads of families of mammalian viruses in each pooled sample. the species are listed in the right text column. location information is provided in the life text column. the names of the mammalian viral families are presented in the top text row. the boxes colored from green to red represent the viral reads, which were normalized by average viral genome size and total sequencing reads in each pool species are broadly distributed throughout china, most rna and dna viruses with high (e.g., covs, astvs, and picovs) or low (e.g., hvs and parvoviruses (parvs)) richness were detected in diverse locations throughout china and showed no obvious geographical preference (except some regions that harbored unusual host species showed geographical restriction of certain viruses). however, artevs and flaviviruses tended to be frequently detected in provinces in the north, west, and middle of china with low or medium abundance, and such viruses were not detected in southern and coastal areas. in contrast, except dipus-related arevs detected in inner mongolia and showed distinct genome sequence, all other old world arevs were only present in three southern areas, yunnan, hunan, and zhejiang. an l-segment-based, pan-hv pcr was used to sequence hvs. a total of samples from provinces were positive for hv (additional file : table s ). in rodents, pcr products of the expected size were amplified from niviventer confucianus, apodemus peninsulae, apodemus agrarius, and rattus norvegicus of the family muridae, myodes rutilus, microtus gregalis, and caryomys eva; eothenomys melanogaster of the family cricetidae; and dipus sagitta of the family dipodidae. in soricomorpha insectivores, expected pcr products were obtained from sorex araneus of the family soricidae. thirteen representative viral strains of the positive results were selected for phylogenetic analysis (fig. a) . eight viruses from three rodent families were assigned into phylogroup iii. four viruses clustered in the htnv clade, and two viruses clustered in the seov clade, with high sequence similarities ( - % aa identities). rtds-hv/im identified from dipus sagitta was located between htnvs and seovs ( - % aa identities). shrew-hv/sx was located outside the rodent hv clade within this phylogroup. four fig. a phylogenetic tree based on the partial l protein sequences of hvs. b phylogenetic tree based on the complete l proteins of arevs. the viruses found in this study are labeled in red font. the evolutionary lineages of involved hosts on the right were drawn based on mt-cyt b from genus to family according to previous reports [ , , [ ] [ ] [ ] . the relationships between viruses and their hosts were linked by red lines viruses of cricetids were assigned to phylogroup iv and comprised puumala virus and tula virus ( % aa identity with known viruses), and two viruses, rtce-hv/nx and rtcl-hv/gz , which formed a separate clade with < % aa identity with any other viruses from phylogroup iv. another shrew hv, shrew-hv/tibet , was located in phylogroup i. nineteen rodent samples from zhejiang, hunan, yunnan, and inner mongolia were positive for arevs. six strains were selected for genome sequencing and further analyses (additional file : table s ), and all were assigned to the old world complex (fig. b , additional file : figures s and s ). four viruses identified from rattus tanezumi, rattus norvegicus, and rattus losea captured in zhejiang, hunan, and yunnan clustered in the wenzhou virus (wenv) clade with high sequence similarity ( - % aa identities for l, % for g, and % for n, additional file : table s ). rtmc-arev/ yn from mus caroli was distant from other arevs and clustered with danv and lcmvs with short branch lengths. rtds-arev/im of dipus sagitta appeared to represent a separate evolution being distant from all other arevs in the old world complex. twelve rodent samples of six species from six provinces were positive for artevs. seven strains were determined for genome sequences (additional file : table s ). five virus strains identified from microtus clarkei, eothenomys inez, eothenomys melanogaster, myodes rufocanus, and cricetulus longicaudatus in five provinces appeared to be closely related to porcine reproductive and respiratory syndrome virus (prrsv) with higher sequence similarity than those with other members of the family arteriviridae ( . - . % versus . - . % aa identity for orf b, compared with equine arteritis virus, lactate dehydrogenase-elevating virus (ldv) of mice, simian hemorrhagic fever virus, and wobbly possum disease virus; additional file : table s ). the other two viruses detected in dipus sagitta showed low sequence similarity with known artevs ( . - . % aa identity for orf b, additional file : table s ). genomic structure and phylogenetic analysis (fig. a , additional file : figures s and s ) indicated that four of the five prrsv-related viruses were assigned into the prrsv species as an intermediate between genotype and . furthermore, these four viruses showed a closer relationship to genotype than genotype in the prrsv species. rtclan-arterivirus/gz clustered with the clade of prrsv species but appeared to represent a separate evolutionary lineage. the other two artevs identified from dipus sagitta in inner mongolia formed separate branches that were evolutionarily distant from all known members of the arteriviridae. a total of rodent samples were identified as containing members of the family flaviviridae. eleven strains (five hepaciviruses, five pestvs, and one tbev) were selected for genome sequencing (additional file : table s ). the five novel rodent hepaciviruses were assigned into different clades under the genus hepacivirus with varied sequence similarity to other rodent hepaciviruses ( . - . % aa identities; fig b and additional file : table s ) [ ] . the five pestvs showed low sequence similarity with known pestvs from artiodactylid hosts (< % aa identity) and formed a distinct novel rodent virus lineage distant from all other members of the genus pestivirus. a tbev, rtmg-tbev/xj in microtus gregalis from xinjiang, showed high (> %) nt and aa identities with known tbev found in ticks (ixodes scapularis) in xinjiang (additional file : figure s ). thirty-one rodent samples were hev positive, and the genome sequences of nine viruses were confirmed (additional file : table s ). all had < . % aa homology in orf compared with hevs from other hosts ( fig. c and additional file : table s ). these nine viruses comprised five lineages; all of which represented novel clades of rodent hevs, except lineage which contained two novel rodent hevs and known rat hevs. one hundred and eighteen rodent samples and five sorex araneus samples were identified as cov positive; strains were selected for sequencing of partial rna-dependent rna polymerase (rdrp), and strains were characterized for genome sequences (additional file : table s ). pairwise similarity and phylogenetic analysis ( fig. a and additional file : figure s ) revealed that viruses formed diverse evolutionary clades in lineage a under the genus betacoronavirus, with sequence identities between . and . % (rdrp aa identity). the other six viruses were all assigned to the genus alphacoronavirus; five of these clustering as a rodent-borne clade within the alphacoronavirus genus, with sequence identities between . and . % (rdrp aa identity). one virus, shrew-cov/tibet , identified in sorex araneus from tibet appeared to have undergone separate evolution, phylogenetically distant from all other α-covs with < . % rdrp aa identity (additional file : tables s and s ). sixty rodent samples were identified as picov positive, and strains underwent genome sequencing (additional file : table s ) [ ] . rodent table s ). viruses closely related to known picovs of other hosts were found (e.g., rodent viruses related to human aichivirus, human rosavirus, and bovine hunnivirus) [ ] . a larger number of astvs were detected in both rodent and shrew samples (additional file : table s ). fifty-five astvs were selected for sequencing. most of the rodent astvs sequenced belonged to four main genetic lineages to within the genus mamastrovirus and had less sequence similarity with astvs in other hosts (fig. c) . one rodent astv, rtrn-astv- /gd , was closely related to astvs of cattle, deer, and pigs with > % nt identity. two shrew astvs, shrew-astv/ sax and shrew-astv/gx , were related to mouse astv with~ % nt identity in the genus mamastrovirus. lineage contained one shrew astv and one mouse astv, with % nt identity with each other. lineage branched out of the genus mamastrovirus and showed a closer relationship with the genus avastrovius. all reads of paravs from different rodent species were closely related to previously reported beilong or tailam viruses [ ] [ ] [ ] . we obtained full-length sequence of rtap-parav/ nx from apodemus peninsulae, which we assigned to jeilongvirus close to the beilong and tailam virus clade ( . - . % aa identities for l) (additional file : figure s and additional file : table s ). we characterized shrew-calv/tibet in sorex araneus as a novel species of the genus norovirus with . - . % aa identities with known noroviruses (additional file : figure s ). the most closely related murine norovirus that was classified as genogroup v showed . % aa identity with this shrew calv. c phylogenetic tree based on the complete orf sequences of hevs. the viruses found in this study are labeled in red font. the evolutionary lineages of involved hosts on the right were drawn based on mt-cyt b from genus to family according to previous reports [ , , [ ] [ ] [ ] . the relationships between viruses and their hosts were linked by red lines thirty-three cv-positive samples were confirmed, and cv strains were identified for genome sequencing (additional file : table s ). pairwise alignment and phylogenetic analysis suggested that of these belonged to the genera circovirus and cyclovirus. the other eight novel rodent or shrew cvs branched out of the root of the genera circovirus and cyclovirus (fig. a) . twenty-eight parv-positive samples were identified, and virus strains were selected for genome sequencing (additional file : table s ). these viruses clustered phylogenetically with bocaparvovirus, dependoparvovirus, amdoparvovirus, and protoparvovirus (fig. b) . eight rodent parvs clustered together and formed a novel lineage of rodent bocavirus under the genus bocaparvovirus, with < . % aa identity with bocaviruses of other hosts. three parvs were classified in the genus protoparvovirus but formed novel evolutionary clades. one parv was clustered with mink parvovirus ( . % aa identity) under the genus amdoparvovirus. the other four parvs formed two lineages under the genus dependoparvovirus with < . % aa identity with known adeno-associated virus (additional file : table s ). thirty-one samples were adv positive (additional file : table s ), and seven strains were selected for sequencing of partial dna polymerase gene. two rodent advs clustered with previously reported murine advs (madv- , madv- , and madv- ; . - . % aa identities, additional file : figure s ) [ , ] . three rodent advs formed a separate genetic lineage within mastadenovirus. two shrew advs were also identified; one closely related to previously reported tree shrew adv- and another located in a novel lineage related to bat adv-fbv . rtac-pv/gz in apodemus chevrieri and rtrn-pv/ gd in rattus norvegicus were full-length sequenced (additional file : table s ). these two rodent viruses showed high sequence similarities with known rodent pvs from apodemus sylvaticus and rattus norvegicus in germany ( % and % aa identities) [ , ] . when we mapped the viral phylogram to the evolutionary lineages of their hosts (figs. and , additional file : figures s -s ), co-evolution or co-divergence between viruses and their rodent and insectivore hosts were observed in most cases such as hvs, arevs, artevs, and hevs. most viruses in each family tended to form different lineages that were phylogenetically consistent with the phylogeny of their hosts from species to family. for example, the division of hv phylogroups was congruent with the phylogenies of their arvicolinae, sigmodontinae, murinae, and soricidae hosts; the separation of arevs in the old world complex was congruent with the phylogenies of their murinae and dipodidae hosts. however, virus phylogeny was not always consistent with host phylogeny, and multiple incongruous relationships between the phylogenies of hosts and viruses were also found. for example, dipus sagitta and rtds-hv/im , rattus tanezumi and rtrf-astv- /yn , cricetulus species and their hevs, and rodent species and many of their hepaciviruses and picovs were phylogenetically incongruent. these suggested that host shifts seem to be common for several virus phylogenetic lineages. novel eids from animal reservoirs pose an increasing threat to global health and security. comprehensive knowledge of the viral population and ecological community resident in animal reservoirs, especially in wildlife, could minimize the impact of potential animaloriginated eids on public health by providing meaningful basic data [ , ] . a predictive analysis based on host and viral traits conducted by olival et al. revealed that the present observed viral richness of wildlife is limited, and there is still an alarming number of "missing viruses" and "missing zoonotic viruses" that merit further systematic surveillance globally, especially in rodents, primates, and bats [ ] . by firstly characterizing the pharyngeal and anal viromes of representative rodents and other small mammals throughout china, the present study identified many novel viruses from rodents and insectivores. when added to our recent work on bats [ , ] , the present results suggest that there is rich as-yet-undiscovered viral diversity in rodents, bats, insectivores, and other mammalian species in china. viral diversity among different mammalian hosts is strikingly different. the presence of a large number of diverse rna and dna viruses with high prevalence and abundance highlights that both rodents and soricomorphs can tolerate diverse viruses, as we described previously in bats [ ] . animals in northern and western regions show similar or even higher viral diversity when compared with those of animals in central and southern areas of china. a recent report showed that central and southern china with higher human population density may have a high estimated risk of zoonotic eid events based on previous data [ ] . here, the identification of diverse viruses and even pathogen-related viruses in northern and western china indicates that the risk of eids originating from wildlife in these regions should not be underestimated. in addition to the viral family-specific findings discussed below, our study has some potentially broad implications. first, we have characterized diverse hvs, arevs, aeteriviruses, picobirnaviruses, covs, hepaciviruses, pestvs, picovs, hevs, astvs, herpesviruses, anelloviruses, advs, cvs, and parvs, extending our knowledge for members of each viral family, and identifying new viral genera and species, albeit that the role of some viruses in human illness is unclear [ , ] . second, we showed that rodent species carry more viruses than previously thought (e.g., identification of viruses of the genera bocaparvovirus and pestivirus), extending the known host range of the viral family and suggesting that rodents should be considered potential carriers and disseminators of viruses in these genera or species. third, we also found that many viruses (e.g., arevs, covs, and picovs) identified from the same or different animal species from different locations shared high sequence identities and close genetic relationships; the phylogenetic relationships we uncovered suggest these rodent-and shrew-borne viruses have the potential for intra-or cross-species transmission concomitant with intra-or inter-species contact and then co-evolve with their hosts in a wide geographical area. finally, the phylogenetic incongruence between hosts and their viruses in many cases suggests that host shifts have happened frequently during the viral evolutionary history, which may create opportunities for the emergence of new viruses that are able to adapt to new hosts. rodent origin hvs and arevs are important causative agents of human hemorrhagic fever and related diseases [ ] . although prevention and control measures have been performed in recent years, hemorrhagic fever with renal syndrome (hfrs) caused by hvs remains a serious public health problem in china, and the number of hfrs cases still accounts for~ % of the total cases worldwide [ , , ] . although many members of the genus mammarenavirus in the family arenaviridae are associated with human diseases worldwide, only lcmv and wenv have been reported in china [ , ] . although some cases of lymphocytic choriomeningitis were clinically diagnosed in china based on serology and pathology, lcmv has not been laboratory confirmed nor full-length sequenced in china. we identified diverse hvs and arevs located in different phylogenetic positions that indicate the presence of novel viruses in new hosts such as caryomys eva (rtce-hv/nx ), eothenomys melanogaster (rtcl-hv/ gz ), and dipus sagitta (rtds-hv/im and rtds-arev/im ). these findings are important for the prevention of hvs and arevs transmitted from rodents. we also identified novel arevs in different rattus species with high sequence identities from yunnan, hunan, and zhejiang provinces. these rattus species hosts have high potential for contact with people in rural china, and the clustering of these viruses from different provinces with wenv variants identified in humans suggests there is a broader geographical distribution of these potentially zoonotic viruses [ , ] . we identified diverse rodent artevs phylogenetically scattered throughout the arteriviridae, and with higher genetic diversity than artevs of other host groups. this suggests that rodents are important wildlife hosts for a variety of artevs species with different evolutionary stages and that rodents may act as the main wildlife reservoirs of artevs. infection by the artev prrsv causes severe disease in swine and global economic losses to the swine industry [ , ] . all previously reported prrsv strains are only detected in swine and can be phylogenetically classified into genotype (or european type, prototype: lelystad virus) and genotype (or north american type, prototype: vr- ) without any intermediate genotype [ , ] . our characterization of four rodent artevs in the prrsv species phylogenetically clustered between the two known genotypes suggests that the virus evolved independently in pigs and rats and finally formed at least three different genotypes under the prrsv species. it also indicates the presence of a closer ancestor of prrsv in rodents than the previously hypothesized mouse ldv. unlike bat covs which are grouped into various evolutionary clades of the genera alphacoronavirus and betacoronavirus [ , [ ] [ ] [ ] [ ] , covs identified in various rodent species from various regions can only be classified into two lineages: lineage a of betacoronavirus and a separate lineage of alphacoronavirus. human cov oc , human cov hku , and bovine cov under lineage a of betacoronavirus are human or animal pathogens that cause mild-to-severe diseases [ ] . the identification of diverse lineage-a rodent β-covs confirms the hypothesis that rodents may be an important reservoir for ancestors of lineage-a β-covs [ , ] . the identification of α-covs under separate lineage from diverse provinces suggests a broader geographical distribution of these rodent-specific viruses. although samples from individuals of the order lagomorpha and soricomorpha were limited, many novel viruses were identified from soricomorpha insectivores, a group of mammals that is poorly sampled, but abundant in natural and human-dominated habitats. the characterization of a shrew-borne cov (shrew-cov/tibet ), calv (shrew-calv/tibet ), astvs, and cvs (shrew-cv/tibet ) and a distinctive lineage of the genera alphacoronavirus and norovirus indicates that these insectivores may harbor a diversity of these and other viruses; some of which may have zoonotic potential. these findings, combined with our previous bat virome data, greatly increase our knowledge of the viral community in wildlife in a densely populated country in an emerging disease hotspot. continued efforts in viral discovery in these and other mammalian hosts in china may reveal greater diversity of viral lineages, as shown recently for arthropods [ , ] , and as hypothesized globally [ ] . collection of animal samples was conducted within the national hfrs surveillance network and national medical vectors surveillance network. capture was conducted using live traps supplied by the state key laboratory for infectious diseases prevention and control. most animals captured were euthanized by carbon dioxide although some individuals of rare species (labeled in red with iucn red list level in additional file : table s ) were released after sampling. pharyngeal and anal swabs were placed in virus sampling tubes (yocon, beijing, china) containing maintenance medium and temporarily stored at − °c. after the sampling was finished, samples were transported to the laboratory and stored at − °c. samples from the same species and from the same site were pooled. the voucher collection of specimens was deposited at − °c by the department of vector biology and control, national institute for communicable disease control and prevention, chinese center for disease control and prevention. viral nucleic acid library construction, next-generation sequencing, and taxonomic assignments swab samples in maintenance medium were re-suspended, processed with a viral particle-protected nucleic acid purification method, and amplified by sequence-independent rt-pcr as described previously [ ] . briefly, the samples were filtered through a . -μm polyvinylidene difluoride filter (millipore, darmstadt, germany) to remove eukaryotic and bacterium-sized particles. the filtered samples were then centrifuged at , ×g for h at °c. the pellets were re-suspended in hank's balanced salt solution. to remove naked dna and rna, the re-suspended pellet was digested in a cocktail of dnase and rnase enzymes, including turbo dnase (ambion, austin, tx, usa), benzonase (novagen, darmstadt, germany), and rnase one (promega, madison, wi, usa) at °c for h. the viral dna and rna were simultaneously isolated using a qiamp minelute virus spin kit (qiagen, valencia, ca, usa). viral first-strand cdna was synthesized using the primer k- n and a superscript iii system (invitrogen, carlsbad, ca, usa). to convert first-strand cdna into dsdna, the cdna was incubated at °c for h in the presence of klenow fragment (neb, ipswich, ma, usa). sequence-independent pcr amplification was conducted using primer k and phusion dna polymerase (neb). the pcr products were analyzed by agarose gel electrophoresis. all dna smears larger than bp were excised and extracted with a minelute gel extraction kit (qiagen). amplified viral nucleic acid libraries were analyzed using an illumina hiseq sequencer (illumina, san diego, ca, usa) for a single read of bp in length. raw sequence reads were filtered using previously described criteria to obtain valid sequences [ , , ] , reads with no call sites, reads with similarity to the sequencing adaptor, and the primer k sequence, and duplicate reads and low-complexity reads were culled. each read was evaluated for viral origin by conducting alignments with the ncbi non-redundant nucleotide database (nt) and protein database (nr) using blastn and blastx (with parameters -e e- -f t). reads with no hits in nt or nr were further assembled by metagenomics assemblers (e.g., metavelvet, idba-ud, and soapdenovo), and the contigs were again aligned with nt and nr to identify any viruses present. taxonomy of the aligned reads with the best blast scores (e value < − ) from all lanes was parsed and exported with megan -metagenome analyzer [ ] . we also tested an assembly-first strategy to analyze sequence data. reads were first assembled by metagenomics assemblers, and output contigs were then aligned to nt and nr. sequence reads classified as the same virus family or genus by megan were extracted and assembled with seqman program (lasergene; dnastar, madison, wi, usa). the accurate locations of the assembled reads and the relative distances between them were determined. a draft genome with several snps of each virus was obtained. based on partial genomic sequences of viruses obtained by assembly, we designed specific nested primers for pcr and rt-pcr to screen for each virus in individual samples from each mammalian species. different degenerate primers targeting conserved regions were also used to identify the presence and prevalence of viruses [ , [ ] [ ] [ ] [ ] [ ] [ ] . locations of reads and the distances between reads of the same virus were determined using alignment results exported by megan . representative positive samples for each virus were selected for genome sequencing. reads with accurate genomic locations were then used for reads-based pcr to identify partial genomes. based on the partial genomic sequences obtained by specific nested pcr, the remaining genomic sequences were determined using inverse pcr, genome walking, and ′and ′-rapid amplification of cdna ends (race). nucleotide sequences of genomes and amino acid sequences of open reading frames (orfs) were deduced by comparing them with other viral sequences. the conserved protein families and domains were predicted using pfam and interproscan (available at: http://www.ebi.ac.uk/services/proteins). routine sequence alignments were performed using clustal omega, needle (available at: http://www.ebi.ac.uk/tools/), megalign (lasergene), and t-coffee with manual curation. mega . (phoenix, az, usa) was used to align the nt and the deduced aa sequences using the muscle package and default parameters. the best substitution model was evaluated using the model selection package. we used maximum-likelihood to process the phylogenetic analyses with , bootstrap replicates [ ] . the aa identities and genetic distances were calculated using the ml method with a pairwise evolutionary distance calculation as the distance metric. the evolutionary lineages of involved hosts were drawn based on mitochondrial cytochrome b (mt-cyt b) according to previous reports [ , , [ ] [ ] [ ] . the congruence between the phylogenies of viruses and their hosts were determined by tanglegram which is generated by matching each virus to their associated host [ , ] . all genome sequences were submitted to genbank (accession numbers are given in additional file : table s ). the illumina hiseq sequence data were deposited into the ncbi sequence reads archive (sra) under accession number prjna . supplementary tables and figures are available with the online version of this paper. additional file : table s . samples of the animal species used in this study and the provinces and dates of collection. table s . the reads of virus under each family. table s . the reads of mammailan virus under each family. table s . origin and accession number of viruses identified in this study. table s . amino acid identity of rodents arenavirus and representatives of other species. table s . the aa identities (%) of the predicted orf a and orf b between rodent arteriviruses and other known members of the family arteriviridae (including a tentative member, wpdv). table s . the aa identities (%) between these hepaciviruses and other known rodent hepacivirus. table s . amino acid identity (%)of rodents hepatitis e and representatives of other genotypes. table s . the aa identities (%) between these btcovs and other known members of the lineage-a beta-covs. table s . the aa identities (%) between these btcovs and other known members of alpha-covs. table s . amino acid identity of rodents picornaviruses and representatives of other genera in p , p , and p regions. table s . pairwise amino acid identities (%) of predicted gene products of rtap-parav/nx compared to other jeilongvirus members. table s . amino acid identity of rodents parvovirus and representatives of other genera. (xlsx kb) additional file : table s . the reads of each viral family related to province and animal species. (xlsx kb) additional file : figure s . arenavirus g. phylogenetic tree showing the relationships (amino acid) between arenaviruses in the g polymerase. the viruses found in this study are labeled in red font. figure s . arenavirus n. phylogenetic tree showing the relationships (amino acid) between arenaviruses in the n polymerase. the viruses found in this study are labeled in red font. figure s . phylogenetic tree showing the relationships (amino acid) between arteriviruses in the pp a proteins. the viruses found in this study are labeled in red font. figure s . genomic organization of the rat-arterivirus- /ningxia and rat-arterivirus/jilin . figure s . phylogenetic tree based on the polyproteins of tbev. the viruses found in this study are labeled in red font. figure s . phylogenetic tree based on the complete spike (s) proteins of covs. the viruses found in this study are labeled in red font. figure s . phylogenetic treebased on the l proteins of paravs. the viruses found in this study are labeled in red font. figure s . phylogenetic tree based on the polyproteins of noroviruses. the viruses found in this study are labeled in red font. figure s . phylogenetic tree based on diverse sequences of partial amino acid of the polymerases of advs. the viruses found in this study are labeled in red font. figure s . the phylogenetic relationships between covs and their hosts. figure s . the phylogenetic relationships between picovs and their hosts. figure s . the phylogenetic relationships between astvs and their hosts. figure s . the phylogenetic relationships between cvs and their hosts. figure s global trends in emerging infectious diseases risk factors for human disease emergence economic growth, urbanization, globalization, and the risks of emerging infectious diseases in china: a review origins of major human infectious diseases epidemic dynamics at the human-animal interface bats and their virome: an important source of emerging viruses capable of infecting humans 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revolution and kpg extinction on mammal diversification evolutionary relationships between bat coronaviruses and their hosts the datasets generated and analyzed during the current study are available in this published article (and its supplementary information files) and the ncbi sequence reads archive (sra) under accession number prjna . not applicable. the authors declare that they have no competing interests. key: cord- -ugxou z authors: bherwani, hemant; nair, moorthy; musugu, kavya; gautam, sneha; gupta, ankit; kapley, atya; kumar, rakesh title: valuation of air pollution externalities: comparative assessment of economic damage and emission reduction under covid- lockdown date: - - journal: air qual atmos health doi: . /s - - - sha: doc_id: cord_uid: ugxou z air pollution (ap) is one of the major causes of health risks as it leads to widespread morbidity and mortality each year. its environmental impacts include acid rains, reduced visibility, but more importantly and significantly, it affects human health. the price tag of not managing ap is seen in the rise of chronic obstructive pulmonary disease (copd), cardiovascular disease, and respiratory ailments like asthma and chronic bronchitis. but as the world battles the corona pandemic, covid- lockdown has abruptly halted human activity, leading to a significant reduction in ap levels. the effect of this reduction is captured by reduced cases of morbidity and mortality associated with air pollution. the current study aims to monetarily quantify the decline in health impacts due to reduced ap levels under lockdown scenario, as against business as usual, for four cities—delhi, london, paris, and wuhan. the exposure assessment with respect to pollutants like particulate matter (pm( . ) and pm( )), no( ), and so( ) are evaluated. value of statistical life (vsl), cost of illness (coi), and per capita income (pci) for disability-adjusted life years (daly) are used to monetize the health impacts for the year and , considering the respective period of covid- lockdown of four cities. the preventive benefits related to reduced ap due to lockdown is evaluated in comparison to economic damage sustained by these four cities. this helps in understanding the magnitude of actual damage and brings out a more holistic picture of the damages related to lockdown. the cost of development has had a major impact on the ecosystem balance. one major fallout is the jeopardized air environment (mage et al. ) . this is mainly due to a plethora of emission of pollutants such as pm , pm . , no , so , etc. from various anthropogenic activities such as transportation, industrial processes, mining activities, waste burning, biomass burning, residential cooking, etc. gupta et al. ; humbal et al. ; bherwani et al. ; gautam et al. ) . deteriorated air causes long-and short-term health effects such as chronic obstructive pulmonary disease (copd), asthma, respiratory mortality, cancer, cardiovascular mortality, etc. (kim et al. ; ghorani-azam et al. ; maji et al. ; cohen et al. ; landrigan et al. ) among humans. this global threat is considered as one the leading cause for mortality and accounts for million lives every year, estimating a total cost of more than % of the gdp (who ). extensive studies have been carried out by various researchers (gulia et al. ; maji et al. & maji et al. in heavily polluted cities to quantify the healthrelated monetary burden upon the exposure of human in the deteriorated air environment. severe acute respiratory syndrome coronavirus (sars-cov- ) outbreak firstly identified in wuhan city, china (who ; gautam and hens ; gautam and trivedi ; gautam a ) has now become a pandemic across the globe, resulting in about . million cases in just about months who ) . the pandemic has declared global health emergency enforcing lockdown measures in activities (i.e., social contact, non-essential business, mobility, etc.) to flatten the epidemic curve. lockdown measures have shut down industries, halted vehicular traffic, and have had a huge impact on the daily routine of the people. due to this, considerable improvement in air quality levels of countries such as spain (tobias et al. ) , india (gautam b) , brazil (nakada and urban ) , and china (sharma et al. ) ,and in cities such as bangkok, tokyo, paris, and new york (he et al. ) has been reported. the plummet in pollutant concentration is obvious due to the restriction in anthropogenic activities. the air quality during the lockdown period for majorly polluted cities (i.e., delhi, wuhan, paris, and london) is of high significance in determining the baseline pollution level and attributable health risk, to evaluate the potential fluctuation in monetary damages. assessment in terms of the number of lives affected is important, but a common baseline matrix such as "money" carries a much better message for the sake of understanding and decision making (gautam a ). an unprecedented attempt has been made to quantify the economic benefit due to reduction in an excess number of health risk subject to mortality/morbidity attributable to lower air pollutant (pm . , pm , no , and so ) concentration in wuhan, paris, london, and delhi. figure indicates the geographical location of the cities. the monitored ground-based pollutant concentration are compared with who ambient air quality guidelines (who ) to evaluate the relative risk associated with each disease and thereby, the subsequent damage cost. the economic benefit/loss is calculated for each city based on the difference in excess number of health cases attributable to pollutant concentration during the lockdown period for and . the study has also considered the overall benefit due to mass reduction in air pollutant level over the potential economic loss to the local and national government as an outcome of lockdown. population for the years of valuation is estimated to evaluate the extent of health damages due to ap in a particular city. population data is collected for and for calculating the number of people affected due to ap. the population of delhi, london, paris, and wuhan has increased from , . , . , and . lakhs in to , . , . , and . lakhs in , respectively (economic survey of delhi ). lockdown is imposed in many countries due to the outbreak of covid- (wang et al. a; dutheil et al. ; gautam b) . in delhi, the lockdown was imposed from march (economic times ), in paris from march , (jon et al. ) , in london form march (the hindu ), and in wuhan, it started from january . to analyze the ap levels, days of lockdown is taken for the four cities and the respective data for ap as shown in table along with dates of lockdown. average of -h mean data of pm . , pm , so , and no values are taken in the period of lockdown that is days for the four cities in , and the same duration is taken in and values are shown in table . number of people are affected by mortality and morbidity due to the exposure of harmful pollutant concentrations by taking the -h mean values for days for these cities is evaluated. the values of people getting affected are calculated on the basis of daily data of pollutants in comparison to daily values of who guidelines. the daily values are averaged for month and shown here. so has been found within limits throughout the study period for both the years. short-term health effects such as chronic obstructive pulmonary disease (copd), asthma, respiratory mortality, cardiovascular mortality, and respiratory diseases due to excess pollution level during lockdown period (number of days (n) = ) for the current and previous years were estimated using linear exposure-response curve (lerc) method. the assessment was carried considering various factors such as relative risk r r (m) due to mean pollutant concentration, population attributable to risk (par) due to exposure of pollutant, baseline incidence cases (i e ) per population corresponding to mortality/morbidity health impacts, and the total population of the city (n). equations ( )-( ) are used for estimating earlier mentioned variables. where c m is monitored daily ambient air concentration of pollutant "k" (daily values of average pollutant loads given in table ). c p is permissible standard for the pollutant "k" (who guideline values). the standards preferred are as put forth by who in . rr is relative risk for the pollutant "k," and the values for r r and i e are discussed in table . ρ(c) is the proportion of population being exposed to the pollutant "k" ( % exposure is considered in our study). i ne is the estimated number of cases. the monetary burden due to health risk is calculated using the methods of vsl, coi, and daly for mortality and morbidity endpoints. for valuation, morbidity and mortality are evaluated via separate methods. the valuation of mortality is done using vsl which is usually available for each country based on assessments done for either labor wages or insurance settlements. the data on vsl is inflated to the year (which is the base year of valuation for this research) in the native currency of the reference and is compiled in table . the exchange rate/conversion factor is used for converting the native currency to usd while the inflation is carried out to bring the value to the base year of . the formula for evaluation of monetary damages of mortality is given as follows: where er is the exchange rate (imf ) and ir is the inflation rate (the world bank ). on similar lines to mortality, morbidity is valued using cost of illness (coi) and daly and is added to get total morbidity-related damage assessment. the data related to daly is taken from the who database and is given for the select cities in table . daly indicates the years of life lost and thus can be valued using the annual income of an individual. the per capita income (pci) of the selected cities with their referenced and converted values to usd ( ) is shown in table . the formula related to daly-related morbidity damage valuation is given in eq. ( ). another important related to morbidity is evaluated cost of treatment of the disease, which is done using coi. coi includes total cost incurred such as medicinal cost, travel cost, hospital admission, and lost day. cost of illness of morbidity of each city is shown in table as compiled from the literature and converted to usd ( ). for the respiratory disease of paris, the literature was not available, and hence, the corresponding values in london are used. similarly, literature indicated very high costs for copd for wuhan, and hence, indian costs for copd are normalized with reference to asthma costs and used for valuation. the formula for evaluation of morbidity based on coi is given as follows: based on the above inputs, the valuations of morbidity-and mortality-related damages of ap are carried out and explained in the "results and discussion" section. in order to understand the true magnitude of the damages, it is compared to economic damages sustained during the lockdown. the prevention of ap provided a benefit at the cost of economic damage. for this, economic damage sustained by each city during the lockdown is calculated and compiled, as shown in table . the gross domestic product (gdp) of the city is compared with the gdp of the country to understand the citywide damages as literature only reported country-level damages. ambient air quality for delhi, london, paris, and wuhan cities for the lockdown month with the number of days for the years and as shown in table is considered for calculating the health damage cost due to air pollutants. during the lockdown days due to limited transportation and industrial output, air pollution is significantly reduced (wang et al. b ). some of the air pollutants came within the world health organization (who) guidelines (jordan ) just a few days into the lockdown. no concentration for all the cities during the lockdown is less than the who limit every day, whereas breach is observed for the year . pollutant such as pm . , pm , no x , and so results in various health damages such as respiratory diseases, copd, cardiovascular diseases, and mortality. these pollutants are considered to calculate corresponding health damage cost. it can be observed that so concentration was below the acceptable permissible limit set by international guidelines (who ) . hence, no health damage due to pollutant was considered. the daily mean of pm . , pm and daily averages for no during the month of lockdown days in and the same month for the previous year was found beyond the acceptable limit, thereby health damage assessment is carried out by certain set of formulas (eqs. ( )- ( )) (maji et al. ) . in order to quantify the impacts in monetary terms, it is essential to understand the mechanism by which the impact happens. the impact pathway approach (bherwani et al. ; ghorani-azam et al. ) reveals how emitted pollutants lead to different adverse outcomes on human wellbeing and other natural environments. in the current research, we are primarily focusing on human health impacts of ap, and the same is quantified in monetary terms. basic parameter of the number of people affected under morbidity and mortality are calculated using eqs. ( )-( ) using the available data. the factors for each of the pollutant parameter are taken from table . the reference concentration for the pollutants is taken from the who guidelines. the cases of morbidity and mortality under each heading of disease for each city are summarized in table . while standard deviations have been reported in pollutant concentration, relative risks, and baseline incidences, the resolution of calculation for multiplicative parameters using standard deviation may yield complex variations in the results. hence, the morbidity and mortality assessments have been done on average values. from table , it can be seen that pm . is the most damaging parameter affecting many people in all cities. the highest cases are reported in delhi due to pm; however, no cases due to no in . since it has the highest number of cases to start with, the lockdown indicates the highest reduction as well. while this will hold true for monetary benefit as well or not is discussed later. wuhan indicates the second-highest number of people affected; however, reduction in appears to be low. london and paris show a similar size of cases. figure indicates the morbidity and mortality between the two years and . the valuation of morbidity is carried out for cardiovascular and respiratory diseases, including general respiratory ailments, asthma and copd. coi is used to include the expenditure of treatment, including the outpatient and in-patient costs related to the ailment. the coi for each city, based on national averages, have been used for diseases, as indicated in table . the valuation of the cost of treatment of morbidity is evaluated using eq. ( ). apart from the treatment expenditure, the reduction in functionality of an individual also leads to massive losses to the economy. this is often evaluated as daly and is valued as well. for valuation, per capita income is used as a surrogate measure for the individuals getting effected along with dalys associated with their diseases. the data related to daly and per capita income based on city gdp averages is given in table and table , respectively. the daly-related valuation is carried out using eq. ( ). the morbidity valuation is shown in table while fig. represents a comparative loss of all cities due to ap morbidity in the years and . it can be seen from fig. that the highest benefit in terms of absolute numbers and in terms of percentage reduction of about % is seen by delhi, which also happens to be one of the most polluted cities in the world. the lockdown scenario must have brought down the pollution levels drastically bringing a huge benefit to the city. in terms of change, paris also sees a considerable reduction with morbidity damages reduced by almost %. the city of wuhan and london see a similar kind of drop in terms of percentage reduction of about to %. while the morbidity damages are in millions of dollars, it is evident that they have reduced considerably as compared to the previous year, indicating that the lockdown has improved air quality thereby saving of millions of dollars and more importantly an improved health and associated intangibles. mortality valuation is done using vsl, which is usually evaluated for low-to moderate-income groups in order to avoid overshooting the valuations. the value of statistical life for individuals staying in different countries is given in table and is used for the mortality air qual atmos health cases as evaluated by the relative risk and baseline incidences reported by who. table gives the mortality valuation under each category of disease for the respective pollutants. the equation used for valuation for mortality damages is given in the "methodology" section as eq. ( ). figure gives a year-wise comparison of the benefits produced by lockdown in in terms of mortality reduction. it is to be noted that mortality damages are reported as total mortality, and within that, there is respiratory and cardiovascular mortality. the total mortalities of particulate matter and nitrogen dioxide have been added to get the total value of damage related to mortality, which then further includes mortalities related to other factors of ap. a similar result is obtained as in case of morbidity. delhi has the greatest benefit with almost % reduction in damages related to mortality due to lockdown followed by paris with % reduction, london with %, and wuhan having % reduction. further, in terms of absolute numbers, the reduction of damages is highest in delhi, followed by london, wuhan, and paris, respectively. overall, mortality and morbidity represent the total loss due to ap in the select cities. the damages have been added and compared to the economic damages caused during the considered lockdown of these cities. the economic damages have been reported at the country level; the ratio of the gdps of the corresponding cities is taken to normalize the damages for cities. the raw data with respect to economic damages and gdp ratios are given in table . the overall damages and economic damages are shown in table . valuation of ap damages for and gives an understanding of the magnitude of damages which occurs due to ap. due to lockdown, there is a drastic reduction in ap as indicated by scenario in table and table , leading to the prevention of ap-related damages. it can be seen from table that the highest prevention happens in terms of absolute magnitude and percentage in delhi. london has the second-highest amount of prevention in terms of absolute numbers but falls behind paris in terms of percentage benefit. wuhan has the lowest prevention in terms of magnitude and percentage. when compared with the economic damages, as indicated in fig. above, actually wuhan has the highest normalized benefit due to ap reduction, followed by delhi, london, and paris, respectively. this is evidently shown in terms of damage prevented per unit economic damage, which is highest for wuhan . , followed by delhi . , london . , and paris . . this study highlights that although the world is fighting with covid- and suffering from huge economic losses, due to reduced cohesive situation for work, the environment is benefitted. this further is demonstrated in monetary terms for reduced health damages related to air pollution. the exposure and impact assessment is carried out for delhi, london, paris, and wuhan, using relative risk and baseline indices of various air pollutants including pm . , pm , and no . the study further incorporates monetization of the impacts for and , to gauge the magnitude of impact reduction of air pollution under covid- lockdown scenario. the monetary assessment of damages is done using econometric approaches like vsl, coi, and pci for daly. the key conclusions are summarized as follows: . pm . is one of the major contributors to the burden of disease related to ap. delhi, being one of the most polluted cities globally, is the worst affected. pm and no impacts are also considerable, while so is found within who guidelines limits for both and . monetarily, the impact is curtailed by average % in mortality and about % in morbidity in the year due to substantial reduction in ap amid reduced anthropogenic activities owing to covid- lockdown. in terms of an absolute number of people affected, an average % reduction is for combined mortality and morbidity for all cities. impacts of no are zeroed for the year . for pm . mortality cases, delhi sees the highest fall in the number of cases for from , followed by paris, wuhan, and london, in that order. it can be seen the trend in mortality damages between the cities is not similar to morbidity, indicating that there could be other compounding factors like age, life expectancy, and standard of living in these cities. . the total of morbidity and mortality damages reduction in is compared with the economic damages being reported for the cities. it is reported that london ( . b$) suffered maximum economic damage during its respective lockdown days, while wuhan suffered the lowest ( . b$). paris ( . b$) and delhi ( . b$) suffered the second-highest and lowest damages, respectively. delhi, london, wuhan, and paris prevented ap damages worth . b$, . b$, . b$, and . b$, respectively, in . this indicates that wuhan and delhi could extract better worth from prevention of damages from lockdown as compared to their lockdown losses. this might be due to the fact that both these cities are highly polluted as compared to paris and london and are economically developing as well. wuhan is actually higher as the economic damages incurred by that city are the lowest. the lowest recovery is made by paris, while london stands at second-lowest. it is apparent that while quantifying ap damages in terms of exposure and affected population is fruitful, there are more insights which can be explored to understand the true magnitude of the damage caused by it. the monetary evaluation gives a common denomination for comparison and for making a rational decision for policy formulation. the above research of four major global cities demonstrates that while the world is suffering from enormous economic damage, the lockdown has created some positive for the air environment and hence the human health as well. while the above analysis might hold true for the majority of the cities suffering from a probable of ap, a detailed analysis should be carried out for the respective city before making decisions. accounting of the cost and benefits due to halting of anthropogenic activities gives us a clear indication of the change of damage being incurred on the ecosystem and on human health. it further suggests that stronger policies are needed to safeguard the 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of rest of the uk prevalence and economic burden of cardiovascular diseases in france in according to the national health insurance scheme database direct medical costs of hospitalizations for cardiovascular diseases in a novel coronavirus outbreak of global healthconcern severe air pollution events not avoided by reduced anthropogenic activities during covid- outbreak air quality guidelines: global update : particulate matter, ozone, nitrogen dioxide, and sulfur dioxide metrics: disability-adjusted life year (daly): quantifying the burden of disease from mortality and morbidity coronavirus disease (covid- ) situation reports. geneva: world health organization publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments authors acknowledge council of scientific and industrial research (csir), india, and its constituent laboratory national environmental engineering research institute (neeri) for providing the support for the research. the manuscript is checked for plagiarism using licensed ithenticate software wide krc no.: csir-neeri/ krc/ /may/csum-drc-dir/ . key: cord- -kc gnj z authors: klempt, petr; brož, petr; kašný, martin; novotný, adam; kvapilová, kateřina; kvapil, petr title: performance of targeted library preparation solutions for sars-cov- whole genome analysis date: - - journal: diagnostics (basel) doi: . /diagnostics sha: doc_id: cord_uid: kc gnj z single next-generation sequencing (ngs) proved to be an important tool for monitoring the sars-cov- outbreak at the global level until today, thousands of sars-cov- genome sequences have been published at gisaid (global initiative on sharing all influenza data) but only a portion are suitable for reliable variant analysis. here we report on the comparison of three commercially available ngs library preparation kits. we discuss advantages and limitations from the perspective of required input sample quality and data quality for advanced sars-cov- genome analysis. the global spread of a new type of coronavirus, sars-cov- , causing the respiratory disease covid- [ ] [ ] [ ] [ ] mobilized both the public and private sector and resulted in a rapid development of solutions focused on sars-cov- detection and analysis. next to a number of solutions utilizing the advantages of rt-qpcr techniques for sars-cov- detection [ , ] , the next-generation sequencing (ngs)-based protocols allows the analysis of sars-cov- genome evolution and variability and the monitoring of its spread within the global population (nextstrain; https://nextstrain.org) [ , ] . these knowledges address the need to elucidate its genomic characteristics (gisaid; https://www.gisaid.org) in order to ensure the efficiency of rt-qpcr testing, assess its transmission through clonal events, and develop a reliable vaccination protocols future therapies, especially considering the fact that rna viruses are prone to accumulate variants in its genome in a relatively short timeline, which in the case of sars-cov- is also related to its capacity to proofread and remove mismatched nucleotides during genome replication and transcription [ ] [ ] [ ] [ ] . in general, the various types of kits stem from two dominant approaches-amplicon and hybridization capture. in general, both approaches show significant advantages and disadvantages in the term of workflow, sample input and ngs library output requirements [ , ] . amplicon-based methods are in general based on a simpler workflow and require smaller amounts of input dna, but they are typically limited to small panels (a few hundred amplicons), and tend to have a high pcr background and lower uniformity of coverage [ ] . the capture-based approaches are more useful for targeting large regions, such as small (viral) genomes and whole exomes, and sequencing data shows better uniformity of coverage. concerning the workflow, the capture-based based approaches are more laborious and require relatively large inputs of nucleic acids [ ] . recently, several companies have developed sars-cov- targeted ngs library preparation kits intended for use on illumina ngs platforms. in this study we report performance analysis of all samples originate from patients positively diagnosed with covid- . sampling took place in five czech hospitals-two in prague, two in brno, one in plzeň. in total, we have received samples of nasopharyngeal swabs from positive cases. isolation of total nucleic acid content from nasopharyngeal swabs was performed by qiasymphony virus/pathogen mini kit and/or qiaamp viral rna mini kit k (qiagen, germantown, md, usa) respectively (depending on their availability) according to manufacturer's instructions to obtain isolates. viral content of the isolates was subsequently quantified by rt-qpcr (direct sars-cov- rt pcr kit, institute of applied biotechnologies, prague, czech republic) in order to evaluate the copy number of sars-cov- , based on who-recommended protocol (www.who.org). in direct sars-cov- rt pcr assay we used the novel coronavirus sequences reported by the united states centers for disease control targeting two genetic sequences of the viral nucleocapsid (n , n genes) and human rnase p (rp gene) as an internal control [ ] . negative rna controls prepared from ng (nc) human breast tumor rna (hbt) (takara bio; saint-germain-en-laye, france). positive controls (pcs) were prepared by spiking ng of hbt with synthetic sars-cov- rna controls (sars-cov- rna control , sars-cov- rna control ; twist bioscience; usa) in order to reach (pc ), (pc ), (pc ), (pc ) copies of both synthetic genomes within one control. the prepared controls were qualified by rt-qpcr (ct values). the number of virus copies in samples was calculated according to ct values measured by adoption of direct sars-cov- rt pcr assay for synthetic rna positive controls diluted to defined viral copy concentration. the direct sars-cov- rt pcr results were validated with ivd ce certified kit ultragene combo screen sars-cov- assay (abl sa group, luxembourg) on rna isolated from nasopharyngeal swab samples using a viral rna isolation kit from cell-free fluids (nucleospin rna virus, macherey-nagel, duren, germany). overall, ngs libraries were prepared from isolates in sets specific for each library preparation solution. thirteen samples were prepared using all three kits. the second library preparation followed the same procedure as the first experiment, with exception of utilizing the knowledge of ct values of samples (ct , - ). ngs libraries were combined into plexes ( -plex and -plex) based on ct values in order to minimize the range span (see supplementary table s ). enriched plexed were evaluated by qubit . and bioanalyzer , pooled equally (with regard to the number of samples in individual plexes) and sequenced using miseq platform using reagent kit v ( cycle) (illumina, san diego, ca, usa). a set of isolates (ct values . - , supplementary table s )- positive (pc , pc , pc , pc ) and negative (nc ) controls-was transcribed into ds cdna using nebnext ® rna first strand synthesis module and nebnext ® ultra™ ii directional rna second strand synthesis module (new england biolabs; ipswich, ma, usa) following manufacturer's protocol. libraries were prepared using nextera flex for enrichment (pre-enrichment part of manufacturer's guide; https://emea.support.illumina.com/content/dam/illumina-support/documents/ documentation/chemistry_documentation/illumina_prep/illumina-dna-prep-with-enrichmentreference- - .pdf). next, libraries were combined within plexes by samples each based on ct values (see supplementary table s ) and enriched using the respiratory virus oligo panel (illumina, san diego, ca, usa, following the manufacturer's protocol (https://www.illumina.com/content/dam/illumina-marketing/documents/products/appnotes/ coronavirus-enrichment-product-list- - - .pdf). enriched plexes were equally pooled based on evaluation by qubit . and bioanalyzer and sequenced on the miseq platform using miseq reagent kit v ( cycle) (illumina, san diego, ca, usa). for the ngs library construction with the amplicon approach, the identical set of samples as the one used in the illumina approach was used. briefly, isolates, four positive and one negative controls were prepared using the cleanplex ® sars-cov- research and surveillance panel (paragon genomics, san francisco, ca, usa), following the manufacturer's protocol (https://www.paragongenomics.com/ wp-content/uploads/ / /ug - -cleanplex-ngs-panel-user-guide.pdf) using a variant of two-pooled multiplex pcr reactions (see manufacturer's protocol). from a total of preparations, only libraries were finally successfully prepared (evaluated from bioanalyzer traces as recommended by the guide) in sufficient quality for sequencing. purified libraries were pooled (due to low concentration of some libraries we were not able to pool each of the libraries equally, see supplementary table s ) and sequenced on the miseq platform using reagent kit v ( cycle) (illumina). libraries from each preparation were pooled based on the quality control evaluation. further, libraries were diluted and denatured according to the miseq denature and dilute guide (february , v version). final loading concentration, as well as sequencing configuration specified in supplementary table s . fastqc v. . . [ ] was used to control sequence quality prior to trimming. low quality, short reads and adapters were trimmed by fastp . . [ ] . the remaining reads were aligned to reference genome nm . (which is identical to nc_ ) using bowtie [ ] with default sensitive (-sensitive d -r -n -l -i s, , . ) parameters. flagging duplicate reads (in enrichment libraries) and calculation of read depth were done by picard [ ] . sequencing and bioinformatics workflow were validated by identification of variants present in the synthetic control samples (pc ) based on mt . and nm . reference genome (table ) . variant calling was performed using the freebayes algorithm with default parameters. the median coverage in all coding sequences (cds) of sars-cov- gene regions (orf a, orf ab, s, orf a, e, m, orf , orf a, orf b, orf , n, orf ) was calculated. during the first attempt, thirty libraries (including positive and negative controls) were prepared in three plexes ( samples each, see supplementary table s ) employing nebnext ® ultra™ ii directional rna library prep kit for illumina (new england biolabs) followed by capture-based workflow utilizing the twist sars-cov- research panel (twist bioscience). bioinformatic analysis of sequenced data showed high variability of data quality among samples within the experiment (e.g., mean coverage and total number of reads) as well as within each of plexes (see figure and supplementary table s ). contrary, even distribution of the total reads over three plexes ( , . and . m pe reads) confirmed the pooling efficiency. varying sample performance within plex (supplementary table s , std . m, . and . m pe reads per plex , and ) requested re-evaluation of provided ct measurement metrics, and led us to standardize ct value of all samples by in-house rt-qpcr sample rating assay (iab; see materials and methods), instead of using of data provided by external laboratories. this solution was subsequently proved by the second neb+twist experiment, which resulted in more even coverage distribution among libraries (one positive control) prepared in two plexes (std within plexes . and . m). these data indicate that plexes composed of samples with similar ct value show higher uniformity of coverage within the plex compared to plexes prepared without respect to re-evaluated ct value ( figure ). diagnostics , , x of re-evaluation of provided ct measurement metrics, and led us to standardize ct value of all samples by in-house rt-qpcr sample rating assay (iab; see materials and methods), instead of using of data provided by external laboratories. this solution was subsequently proved by the second neb+twist experiment, which resulted in more even coverage distribution among libraries (one positive control) prepared in two plexes (std within plexes . and . m). these data indicate that plexes composed of samples with similar ct value show higher uniformity of coverage within the plex compared to plexes prepared without respect to re-evaluated ct value ( figure ). subsequently, knowing the importance of ct evaluation for sample qc, ngs libraries were prepared by the illumina protocol, utilizing the respiratory virus oligo panel in combination with nextera flex for enrichment (neb solutions was used for first and second strand cdna generation, see materials and methods). forty libraries (including positive and negative controls) were enriched in plexes with the goal to minimize the ct sample range per plex to a difference of maximum where possible (exception in plex , see supplementary table s ). generally, lowering the number of samples within each plex of capture-based workflows (can go down to single-plex) raises the chance of equal distribution of sequencing capacity amongst samples (see figure and supplementary table s ), however also dramatically raises the price per sample as well as hands-on time. compare to neb+twist the capture-based illumina approach data showed in average a lower percentage of mapped reads (supplementary table s ), this consequence could be related to the lower specificity of respiratory oligo viral panel designed next to the sars-cov- also for other pathogens (see https://www.illumina.com/content/dam/illumina-marketing/documents/products/appnotes/ngsenrichment-coronavirus-app-note- - - .pdf). unlike capture-based protocols, paragon's amplicon-based workflow keeps samples separated during the whole workflow (supplementary table s ). therefore, it is potentially possible to directly evaluate the success of prepared targeted sars-cov- ngs library (see materials and methods). in our hands, paragon protocol resulted in (plus control) successfully generated libraries out of samples in total. since the same set of samples was also used for the illumina workflow where it resulted in sequenced genomes (plus controls), the further optimization of the paragon protocol would be needed. paragon ngs library preparation workflow was proved to be the fastest protocol out of three mentioned solutions (see comparison below). the main quality control parameter is the fraction of all target bases which achieved at least × coverage or greater. following this arbitrary limit, sample analysis was considered as successful in case it reached a minimal % proportion of targeted bases with coverage ≥ ×. table s ). the impact of ct on this parameter is especially apparent from the comparison of the first and second approach using the neb+twist workflow (see figure and supplementary table s ). also, the viral load in a sample (corresponding to sample ct value ≤ . or positive control value ≤ . ) showed to be the limiting factor in case of each workflow, samples with higher ct value resulted in either poor genome coverage (neb+twist workflow and illumina workflow) or in absence of expected library preparation product (paragon workflow). considering the time-consumption of each library preparation approach, the fastest was amplicon-based paragon workflow with hands on time (hot) min and h min instrument time (int), followed by illumina ( h min hot and h min int) and neb+twist workflow ( h min hot and h min int) ( figure s ). this is in concordance with the expectations based on the manufacturer's manual as well as the general principle of each workflow. it should be noted that illumina workflow allows faster hybridization time (down to min; we used h long incubation) which reduces the time difference between enrichment and amplicon-based approaches. from the perspective of quality of generated sequencing data, the ct value showed to be the major predictor of sample outcome in regards to the goal of reaching × coverage obtained on a particular sample, as well as success of library preparation, regardless of type of workflow. since ct reflects the actual viral load, it has negative correlation with almost all monitored parameters (figures and ). diagnostics , , x of that illumina workflow allows faster hybridization time (down to min; we used h long incubation) which reduces the time difference between enrichment and amplicon-based approaches. from the perspective of quality of generated sequencing data, the ct value showed to be the major predictor of sample outcome in regards to the goal of reaching × coverage obtained on a particular sample, as well as success of library preparation, regardless of type of workflow. since ct reflects the actual viral load, it has negative correlation with almost all monitored parameters ( figures and ) . we can observe a significant negative correlation between ct value and % of mapped reads to the reference genome. lower ct number caused higher number of % mapped reads and therefore results in better coverage. prc align ×, the percentage of bases that were aligned to the reference sequence at least ×; prc align ×, the percentage of bases that were aligned to the reference sequence at least ×; prc align ×, the percentage of bases that were aligned to the reference sequence at least ×; prc dupl, percentage of duplicate sequences (not generated in the case of paragon approach); ct value, reflecting the viral load; total reads, total number of reads which were yielded by sequencing on miseq (illumina); pcr mapped reads, total number of reads which were mapped to target regions; perc_mapped, percentage of mapped reads which were mapped to target regions; median insert, median value of insert length; median coverage, median coverage of reads which were mapped to target regions; mean coverage, mean coverage of reads which were mapped to target regions. we can observe a significant negative correlation between ct value and % of mapped reads to the reference genome. lower ct number caused higher number of % mapped reads and therefore results in better coverage. prc align ×, the percentage of bases that were aligned to the reference sequence at least ×; prc align ×, the percentage of bases that were aligned to the reference sequence at least ×; prc align ×, the percentage of bases that were aligned to the reference sequence at least ×; prc dupl, percentage of duplicate sequences (not generated in the case of paragon approach); ct value, reflecting the viral load; total reads, total number of reads which were yielded by sequencing on miseq (illumina); pcr mapped reads, total number of reads which were mapped to target regions; perc_mapped, percentage of mapped reads which were mapped to target regions; median insert, median value of insert length; median coverage, median coverage of reads which were mapped to target regions; mean coverage, mean coverage of reads which were mapped to target regions. in respect to the aim of obtaining > × coverage, based on presented data, the recommended samples are those with ct value among - . samples within this range required a lower number of total reads and higher ratio of mapped reads (supplementary table s ) compared to those with ct above . in the case of data recorded on the set of samples which were prepared by all three approaches the negative correlation of median coverage to ct values did not show such significant trend because all the samples were in the optimal ct values range . - . (supplementary table s , figure ). nevertheless our data demonstrate that even samples with ct values ≥ can be successfully processed, however will require substantially higher sequencing capacity and tend to be more prone to dropouts. enrichment based approaches required a higher number of reads (min. pe reads, iab _ _ sample, see supplementary table s ) to reach the optimal coverage limit than amplicon approach ( pe reads, iab _ _ ), which is in concordance to expectation that amplicon-based approach have generally better on-target rate compared to capture-based approach [ ] . on the other hand, the evenness of coverage was better in both capture-based protocols in respect to the aim of obtaining > × coverage, based on presented data, the recommended samples are those with ct value among - . samples within this range required a lower number of total reads and higher ratio of mapped reads (supplementary table s ) compared to those with ct above . in the case of data recorded on the set of samples which were prepared by all three approaches the negative correlation of median coverage to ct values did not show such significant trend because all the samples were in the optimal ct values range . - . (supplementary table s , figure ). nevertheless our data demonstrate that even samples with ct values ≥ can be successfully processed, however will require substantially higher sequencing capacity and tend to be more prone to dropouts. in respect to the aim of obtaining > × coverage, based on presented data, the recommended samples are those with ct value among - . samples within this range required a lower number of total reads and higher ratio of mapped reads (supplementary table s ) compared to those with ct above . in the case of data recorded on the set of samples which were prepared by all three approaches the negative correlation of median coverage to ct values did not show such significant trend because all the samples were in the optimal ct values range . - . (supplementary table s , figure ). nevertheless our data demonstrate that even samples with ct values ≥ can be successfully processed, however will require substantially higher sequencing capacity and tend to be more prone to dropouts. enrichment based approaches required a higher number of reads (min. pe reads, iab _ _ sample, see supplementary table s ) to reach the optimal coverage limit than amplicon approach ( pe reads, iab _ _ ), which is in concordance to expectation that amplicon-based approach have generally better on-target rate compared to capture-based approach [ ] . on the other hand, the evenness of coverage was better in both capture-based protocols enrichment based approaches required a higher number of reads (min. pe reads, iab _ _ sample, see supplementary table s ) to reach the optimal coverage limit than amplicon approach ( pe reads, iab _ _ ), which is in concordance to expectation that amplicon-based approach have generally better on-target rate compared to capture-based approach [ ] . on the other hand, the evenness of coverage was better in both capture-based protocols compared to amplicon-based, as shown by supplementary table s and figure , which is also in concordance with previously published results [ ] . it should be noted that illumina´s panel also targets several other viral genome sequences ( viruses in total; performance not evaluated) as well as human targets serving as positive control, which is reflected by increased panel size as well as sequencing requirements (see the link in materials and methods). in all cds regions the read depth is higher compare to median coverage across whole genome (see reads of depth for cds in supplementary table s ). we can observe read depth coverage in all cds region of negative control samples (nc). there are also dropouts in neb+twist method in samples iab _ _ and iab _ _ . this gap corresponds to low mapping rate (% mapped reads) to the reference genome (supplementary table s ). except for the mentioned dropouts, we can observe relatively good read depth across whole cds regions. whole experiment was done during the acute phase of the coronavirus crisis, using routinely-generated clinical isolates. these samples represent certain constraints for successful ngs library preparation and/or data processing, due to limited concentration (often below detection threshold of qubit hs assay), variable quality and viral load and limited volume. therefore, it was not possible to apply all three methods on the whole set of samples, and samples were prepared using all three methods. we present relevant data acquired in this set of samples in figure and supplementary table s (highlighted in red), however due to the observation that performance of given sample is also affected by sample composition (number and quality) of enrichment plexes (the effect of ct), we suppose that the performance in regards to solution used for library preparation is rather relative. in regards to data analysis of obtained viral genome sequences, we decided to validate reference-mapping-based pipeline using the knowledge of presence of variants within synthetic twist control ( - copies per reaction volume, see materials and methods). positive control was prepared from two synthetic controls representing two genome variants: mn . and mt . (twist bioscience). thus, genome variants combined in positive control differed by sites: snps ( t > c, t > g, g > t) and one deletion ( acgatcgagtg > a). we filtered out the mapped reads of low quality (mq < ), bases with lower quality (bq < ), supplementary and secondary alignment, pcr duplicates (except paragon which is an amplicon-based approach), read depth (dp > ×). for high quality variant calling, freebayes algorithm (default parameters) was used [ ] . for the paragon data the variant calling was performed after trimming out the primer sequences to minimize false positive results. surprisingly, only sequencing data from libraries prepared by paragon protocol did not show the presence of variants caused by low coverage at those sites (see table ), despite showing the highest mean coverage in comparison to competitors (see supplementary table s ). moreover, the absence of anticipated snps is in sharp contrast to significantly higher overall number of called variants in data from paragon libraries in comparison to both capture-based protocols (neb+twist and illumina), as indicated by the venn diagram ( figure ). both of these findings (low coverage uniformity and high rate of false positives) are in alignment with previously published results [ , ] . almost % of variants in paragon are common in all tested samples which is caused by pcr biased and low mapping quality (mq < ). all acquired sars-cov- genomes were uploaded to ncbi sequence read archive (sra) under accession numbers sub , sub , sub and sub . in our study we compared the performance of three commercially available ngs library preparation kits. we pointed out factors which are positively/negatively correlated to the data quality of advanced sars-cov- genome (variant) analysis. from this point of view the ct value (reflecting the viral load per sample) was determined to be the key predictor of library preparation success and sample data outcome reflected by suitable coverage (at least × on a particular sample), regardless of type of workflow. samples with the ct value ≥ showed a lower number of total reads and lower ratio of mapped reads, although also these samples (ct ≤ ) can be successfully processed, but require higher sequencing capacity. the samples with higher ct value resulted in low genome coverage-neb+twist workflow and illumina workflow or in absence of expected library preparation product-paragon workflow. this empirically evaluated dependence also applies to the capture-based approaches where the plexes composed of samples with similar ct value show higher uniformity of coverage within the plex compared to plexes prepared without respect to ct value. according to our results the capture-based approaches required a higher sequencing capacity (higher number of reads) to reach the suitable ≥ × coverage and showed lower on-target rate than the amplicon-based approach, but the evenness of coverage was better in both capture-based protocols. supplementary materials: the following are available online at www.mdpi.com/xxx/s , supplementary table s : metrics of samples processed in neb+twist , (neb+twist plex - , neb+twist plex - ), illumina (plex - ) and paragon (no plex) approach.; supplementary table s : miseq sequencing. reagent kits used for sequencing, number of cycles, loading concentration and phix spike-in per each sequencing run. cluster density should be within - k/mm in case of miseq v chemistry; figure s : process scheme of timeconsumption for library preparation by neb+twist, illumina and paragon approaches (extrapolation to samples). epidemiology, virology, and clinical features of severe acute respiratory syndrome -coronavirus- (sars-cov- ; coronavirus disease- ) cov- : an emerging coronavirus that causes a global threat a new coronavirus associated with human respiratory disease in china geographical and temporal distribution of sars-cov- clades in the who european region sensitivity of different rt-qpcr solutions for sars-cov- detection direct-rt-qpcr detection of sars-cov- without rna extraction as part of a covid- testing strategy: from sample to result in one hour genetic diversity and evolution of sars-cov- genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding emergence of genomic diversity and recurrent mutations in sars-cov- a recurrent mutation at position , of sars-cov- is associated with failure of the e-gene qrt-pcr utilized in a commercial dual-target diagnostic assay genetic diversity among sars-cov strains in south america may impact performance of molecular detection coronavirus rna proofreading: molecular basis and therapeutic targeting evaluation of hybridization capture versus amplicon-based methods for whole-exome sequencing assessment of capture and amplicon-based approaches for the development of a targeted next-generation sequencing pipeline to personalize lymphoma management division of viral diseases fastqc: a quality control tool for high throughput sequence data-scienceopen an ultra-fast all-in-one fastq preprocessor fast gapped-read alignment with bowtie the authors declare no conflict of interest.diagnostics , , the authors declare no conflict of interest. in our study we compared the performance of three commercially available ngs library preparation kits. we pointed out factors which are positively/negatively correlated to the data quality of advanced sars-cov- genome (variant) analysis. from this point of view the ct value (reflecting the viral load per sample) was determined to be the key predictor of library preparation success and sample data outcome reflected by suitable coverage (at least × on a particular sample), regardless of type of workflow. samples with the ct value ≥ showed a lower number of total reads and lower ratio of mapped reads, although also these samples (ct ≤ ) can be successfully processed, but require higher sequencing capacity. the samples with higher ct value resulted in low genome coverage-neb+twist workflow and illumina workflow or in absence of expected library preparation product-paragon workflow. this empirically evaluated dependence also applies to the capture-based approaches where the plexes composed of samples with similar ct value show higher uniformity of coverage within the plex compared to plexes prepared without respect to ct value. according to our results the capture-based approaches required a higher sequencing capacity (higher number of reads) to reach the suitable ≥ × coverage and showed lower on-target rate than the amplicon-based approach, but the evenness of coverage was better in both capture-based protocols. key: cord- -j eqmxt authors: robertson, matthew j.; kent, katarzyna; tharp, nathan; nozawa, kaori; dean, laura; mathew, michelle; grimm, sandra l.; yu, zhifeng; légaré, christine; fujihara, yoshitaka; ikawa, masahito; sullivan, robert; coarfa, cristian; matzuk, martin m.; garcia, thomas x. title: large-scale discovery of male reproductive tract-specific genes through analysis of rna-seq datasets date: - - journal: bmc biol doi: . /s - - -z sha: doc_id: cord_uid: j eqmxt background: the development of a safe, effective, reversible, non-hormonal contraceptive method for men has been an ongoing effort for the past few decades. however, despite significant progress on elucidating the function of key proteins involved in reproduction, understanding male reproductive physiology is limited by incomplete information on the genes expressed in reproductive tissues, and no contraceptive targets have so far reached clinical trials. to advance product development, further identification of novel reproductive tract-specific genes leading to potentially druggable protein targets is imperative. results: in this study, we expand on previous single tissue, single species studies by integrating analysis of publicly available human and mouse rna-seq datasets whose initial published purpose was not focused on identifying male reproductive tract-specific targets. we also incorporate analysis of additional newly acquired human and mouse testis and epididymis samples to increase the number of targets identified. we detected a combined total of genes for which no previous evidence of male reproductive tract-specific expression was annotated, many of which are potentially druggable targets. through rt-pcr, we confirmed the reproductive tract-specific expression of novel orthologous human and mouse genes without a reported mouse model. of these, we ablated four epididymis-specific genes (spint , spint , spint , and ces a) and two testis-specific genes (pp d and saxo ) in individual or double knockout mice generated through the crispr/cas system. our results validate a functional requirement for spint / and ces a in male mouse fertility, while demonstrating that spint , pp d , and saxo are each individually dispensable for male mouse fertility. conclusions: our work provides a plethora of novel testis- and epididymis-specific genes and elucidates the functional requirement of several of these genes, which is essential towards understanding the etiology of male infertility and the development of male contraceptives. the world human population reached nearly eight billion people in august . this number continues to rise and is predicted to reach nearly ten billion by the year [ ] . the increasing need to promote family planning through the development of reliable contraceptive options available to both men and women is widely recognized. currently there are numerous contraceptive options available to women; however, identification of a safe, non-hormonal contraceptive option for men is still an ongoing challenge. although several different fertility control alternatives for men have been investigated, none are currently clinically approved for use. our understanding of the mechanisms underlying male reproductive physiology is still at an early stage as the identification and elucidation of the function of key reproductive proteins is still an ongoing effort. identifying druggable protein targets expressed in the male reproductive tract has been the focus of numerous studies dedicated to the development of male contraception. the mammalian epididymis is a segmented organ comprised of a single, highly coiled tubule with functionally and morphologically distinct regions that can be subdivided most simplistically into a proximal, central, and distal region, conventionally named the caput, corpus, and cauda regions, respectively [ ] . as mammalian spermatozoa transit through the epididymis, they acquire the ability to recognize and fertilize an egg, properties that they did not possess upon exiting the testis [ ] . considering its essential role, the epididymis-in addition to maturing germ cells of the testis and spermatozoa-is a prime target for the development of a male contraceptive. to advance progress towards the development of a non-hormonal male contraceptive, several previous high-throughput studies have been published that identified a number of human, mouse, and rat genes as testis-specific or epididymis-specific [ , [ ] [ ] [ ] [ ] [ ] [ ] . in , schultz et al. conducted the first study to identify male reproductive tract-specific genes using microarrays. through affymetrix-based genome-wide geneexpression analysis of meiotic-and post-meiotic spermatogenic cells, together with parallel analysis of available data from the ncbi unigene database, the authors identified mouse genes as testis-specific, which included genes with both known and unknown function at the time [ ] . in the following years, through two additional microarray-based studies of rat testes and purified rat testicular cells, johnston et al. identified [ ] and [ ] additional or overlapping genes as testisspecific. in , as part of the continued effort to identify novel contraceptive targets, the newer rna-seqbased transcriptomics methodology was utilized identifying human genes as testis-specific [ ] . together with antibody-based protein profiling, many of these genes were characterized in terms of the spermatogenic cell populations showing expression [ ] . the first high-throughput transcriptomics study to identify epididymis-specific genes was a mouse epididymal transcriptome study, in which rna isolated from each of the epididymal segments was analyzed by microarray analysis, identifying epididymis-specific genes with distinct patterns of segmental gene regulation [ ] . later in , additional transcriptome profiling utilizing whole genome microarrays resulted in identification of previously unreported epididymis-specific transcripts in the mouse [ ] and epididymis-specific transcripts in the rat [ ] . a significant number of the identified mouse and rat genes in these studies were not known at the time, and only the probe identification numbers were presented. when evaluating potential druggability in a targetbased drug discovery process, one must consider the protein properties that are required for safe and effective inhibition. among the most significant is tissue expression specificity to minimize potential adverse effects, protein function and whether protein activity or interaction with other proteins is potentially druggable, sequence similarity to closely related paralogs that may be ubiquitously expressed, and whether genetically manipulated animal models demonstrate a functional requirement for the target of interest [ ] . several noteworthy review publications have mentioned numerous genes whose critical functions, high expression, and specificity to the testes or epididymides make them viable nonhormonal male contraceptive targets [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, among the identified genes, a significant number either ( ) are required for fertility, but are expressed in nonreproductive tissues, or ( ) are reproductive tractspecific, but, when disrupted, lead to subfertility [ ] . in either case, both are ineffective and highly undesirable outcomes for a potential male contraceptive target. therefore, the identification of additional novel male reproductive tract-specific genes would allow for further advances to be made in the quest to develop an effective and safe non-hormonal male contraceptive. in this study, newly acquired and previously published human and mouse rna-seq datasets [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] were processed in parallel through a custom bioinformatics pipeline designed to identify novel reproductive tractspecific and reproductive tract-enriched transcripts. additional databases obtained from illuminating the druggable genome [ ] , mouse genome informatics [ ] , and ensembl biomart [ ] were utilized to stratify the results into subgroups based on protein druggability and on the availability of a mouse model. numerous reproductive tract-specific and reproductive tract-enriched, potentially druggable targets for which no published mouse model exists, congruent in expression across both mouse and human datasets were identified through our analysis and verified through conventional polymerase chain reaction (pcr). we present the data in a manner that should be most relevant and of substantial interest to the male contraceptive development field since identification of new targets worthy of consideration for further functional analysis in a knockout animal model and potential drug targeting continues to be of vast importance. through our results, we identified four novel epididymis-specific genes (spint , spint , spint , and ces a) and two novel testis-specific genes (pp d and saxo ) worthy of functional validation in an animal model. through the crispr/cas system, we generated four individual gene knockouts (spint , ces a, pp d , and saxo ) and one double knockout mouse model (spint / ) revealing an essential requirement for spint and spint in male mouse fertility, and the potential utility of pursuing spint in humans as a non-hormonal contraceptive target. despite significant advances in our understanding of the human and rodent testis and epididymis transcriptome, mostly through microarray-based studies, no prior studies have utilized purified human testis cells for the identification of human testis-specific transcripts, no prior studies have utilized the more state-of-the-art rna-seqbased transcriptomics methodology for analysis of human epididymis-specific transcripts, and no prior studies have utilized rna-seq analysis of rodent reproductive tissues or cells to identify rodent reproductive tractspecific transcripts. to address these gaps in knowledge, and to increase the number of identified reproductive tract-specific genes in both species using the most relevant high-throughput transcriptomics methodology, we analyzed in parallel on a custom bioinformatics pipeline a large number of published and newly acquired human and mouse rna-seq datasets. one hundred and sixtytwo previously published human and previously published mouse rna-seq datasets were retrieved from the sequence read archive (sra). the sra value for each sample is listed in additional file : table s and additional file : table s . we also generated new human and new mouse reproductive tissue rna-seq samples (geo accession gse ). the final dataset is comprised of new and previously published human testis datasets [ ] , previously published purified human germ cell datasets [ , ] , previously published purified human sertoli cell datasets [ , ] , new and previously published human epididymis segment datasets [ ] , previously published mouse testis datasets [ ] , new mouse epididymis datasets, previously published purified mouse germ cell datasets [ , ] , and previously published purified mouse sertoli cell datasets [ ] . an additional previously published datasets contributed to the non-reproductive human tissues [ ] and previously published datasets contributed to the non-reproductive mouse tissues [ ] . figure a , b summarizes all the samples acquired for the study. we performed a principal component analysis to visualize the variation in the samples after correcting for batch effects. human reproductive and non-reproductive tissues grouped according to sample type. the reproductive tissue samples clustered by tissue type whether or not they were newly generated or acquired from the sra (fig. c) . mouse data showed a similar variation in the samples based on the tissue type (fig. d) . for both human and mouse reproductive tissues, samples separated by whether or not the rna-seq was performed on isolated cells or the whole tissue. epididymal tissue was distinct from testis tissue in both human and mouse (fig. c, d) . to identify potential male reproductive tract-specific drug candidates, we analyzed the aggregated rna-seq data to find genes that were statistically significant in expression when compared to the non-reproductive tissue that had the maximum expression for that gene. this gene list was then further refined by filtering for genes that were lowly expressed in the non-reproductive tissue that had the maximum expression for that gene (tpm less than or equal to . for human; tpm less than or equal to . for mouse). finally, this tpm filtered list was then filtered for the genes that had a reproductive tissue or cell expression value greater than or equal to . tpm for human, or . tpm for mouse (fig. a) . across all the reproductive tissues, candidate genes were identified in the human and candidate genes were identified in the mouse samples (fig. b , additional file : fig. s ). additional file : table s and additional file : table s summarize the differential fold change, identity of the non-reproductive tissue with maximal gene expression based on the differential gene analysis, fdr, average and standard deviation tpm expression values, and log cpm gene expression value for the human and mouse samples, respectively. the results from the fdr and tpm expression value filtering for the human and mouse samples are summarized in additional file : table s and additional file : table s , respectively. additional file : table s and additional file : table s report the log fold change for the reproductive tissue or cell of interest compared to the tissue with maximal gene expression. the genes identified in additional file : table s and additional file : table s pass the filters in at least one of the reproductive tissues or cells of interest. in additional file : table s and additional file : table s , a value of zero for a given gene and fold expression comparison indicates that for that comparison, the gene did not pass the filters. the majority of genes were downregulated in the reproductive tissue of interest compared to the maximal gene expressing nonreproductive tissue (additional file : fig. s ). from the analysis, the majority of the candidate genes that passed the fdr and tpm filters were identified in the testis-or sperm-related cells in both human and mouse samples (additional file : fig. s ). the majority of candidate genes identified in our screen that were testis-specific were already identified by the human protein atlas [ ] and/or our reanalysis of (see figure on previous page.) fig. summary of the human and mouse rna-seq samples used in the identification of novel male reproductive tract-specific drug targets. the rna-seq samples used in the human (a) and mouse (b) analyses are schematically shown. principal component analysis was performed on the human (c) and mouse (d) non-reproductive and reproductive samples separately. the colors of the circles next to the tissues listed in a and b correspond to the colors used in the circles for the pca in c and d. sample size (n) values in red and/or black denote the number of new (red) and previously published (black) samples included in our analysis. fig. identification of candidate drug male reproductive gene targets. a diagrammatic representation of overall methodology used to identify reproductive tract-specific candidate genes in humans ( genes) and in mice ( genes). the maximum gene expression was determined across all the non-reproductive tissue samples for each gene for a reproductive tissue or cell sample of interest. genes were then filtered for significance using a false discovery rate (fdr) of less than or equal to . based on the differential gene expression analysis for the nonreproductive tissue with maximum gene expression and reproductive tissue or cell sample of interest. genes that passed the fdr filter were filtered such that the average tpm expression value of the maximum expressing non-reproductive tissue was less than or equal to . tpm and the average tpm expression value of the reproductive tissue or cell of interest was greater than or equal to . tpm. b diagrammatic representation of the number of human and mouse candidate genes in terms of ( ) the number of orthologs in the opposite species, ( ) the number of genes previously or not previously identified in a prior transcriptomics-based drug target report, ( ) the availability and phenotypic outcome of any reported mouse models, and ( ) the number of novel genes without a reported mouse model congruent across both species. the main value in each bubble represents the total number of candidate genes identified regardless of tissue or cell identified in. the numbers in parentheses comprise the total number of candidate genes that are either epididymis-specific or specific to testis and epididymis, but not testis and/or testis cell-specific only. the hpa testis datasets (additional file : fig. s and additional file : table s ). thirty-six out of the genes that were identified across all the human epididymis tissue were also identified by the human combined (newly acquired and previously published datasets) testis candidate gene list. finally, the majority of the candidate genes, , identified from the combined newly generated and previously published human testis datasets were shared with genes identified from the various testis cell datasets. we identified more candidate genes in the newly generated human epididymis tissues compared to previously published data: out of genes were unique to the newly generated caput samples compared to only out of genes which was unique to the previously published samples, out of genes were unique in the newly generated corpus samples compared to out of genes which were unique to the previously published corpus samples, and genes were unique to the newly generated cauda samples compared to genes in the previously published cauda data with no overlap between the two cauda gene lists (additional file : fig. s and additional file : table s ). there were candidate genes that overlapped between the newly generated human testis samples and mouse testis sample gene lists, while there were candidate genes that overlapped between the previously published human testis sample and mouse testis sample gene lists (additional file : fig. s and additional file : table s ). across all human epididymis tissue samples, including the newly generated and previously published samples, there were genes in common with the combined list of candidate genes across all the mouse epididymis tissue samples. there was a small overlap between the human and mouse samples when the newly generated human caput, corpus, and cauda tissues were individually compared to the mouse caput, corpus, and cauda tissues; there was an overlap of , , and for the caput, corpus, and cauda, respectively (additional file : fig. s and additional file : table s ). this trend was continued for the candidate gene lists derived from the previously published human caput, corpus, and cauda samples when compared to the candidate gene list from the mouse caput, corpus, and cauda, with , , and genes in common for the caput, corpus, and cauda comparisons, respectively (additional file : fig. s and additional file : table s ). additional file : table s details the genes that are unique and in common for each of the comparisons. to assess the potential usefulness of the candidate genes identified in each human reproductive tissue as drug targets, we assigned the genes to a protein family (i.e., gpcr or ion channel). the majority of identified genes were not from a traditional drug target family like kinases or enzymes. the testis and germ cell datasets provided the most potential targets while the epididymis datasets provided the fewest (additional file : fig. s a ). the protein family classification for each candidate gene identified in each reproductive tissue is detailed in additional file : table s . the majority of the candidate genes do not have a reported mouse model (additional file : fig. s b ). additional file : table s summarizes mouse model availability for each candidate gene identified from human reproductive tissues or cells. figure shows the complete list of novel human genes without a reported mouse model as identified in each of the respective cell and/or tissue datasets. digital pcrs (heatmap) and conventional pcrs demonstrating expression of a subset of the novel human reproductive tract-specific genes without a reported mouse model that we identified are shown in figs. and , respectively. additional file : fig. s shows the complete list of previously identified human genes that remain without a reported mouse model as identified in each of the respective cell and/or tissue datasets. additional file : fig. s shows the complete list of male reproductive tract-specific human genes for which a previously generated mouse model shows male infertility phenotype, as identified in each of the respective cell and/or tissue datasets. through our bioinformatics analysis of previously published and newly acquired rna-seq datasets, we identified a total of genes as reproductive tract-specific in humans (fig. ). of these genes, genes do not have a mouse gene ortholog, while genes have a mouse gene ortholog (fig. ) . of those with a mouse gene ortholog, have a single gene ortholog ( have the same symbol in mouse, while have a different symbol in mouse), while have two or more orthologous mouse genes. seventy-six human genes had - orthologous mouse symbols, genes had - orthologous mouse symbols, and genes (fam a, krta p - , magea , or ag , pramef , pramef , ssx , ssx , and ssx b) had greater than orthologous mouse symbols ( - symbols) (additional file : table s ). of the human genes that we identified as male reproductive tract-specific, have not been previously identified in a transcriptomics-based male reproductive tract-specific study [ , [ ] [ ] [ ] [ ] [ ] [ ] . the sum of our human data confirms the findings of out of genes from djureinovic et al. [ ] . after re-identification of gene symbols from reported affymetrix ids and consideration of orthologous genes (mouse to human and rat to human), our human data confirm the findings of out of genes from johnston et al. [ ] , out of genes from schultz et al. [ ] , out of genes from johnston et al. [ ] , out of genes from johnston et al. [ ] , out of genes from johnston et al. [ ] , and out of genes from jelinsky et al. [ ] . of the genes that have a mouse gene ortholog, have not been previously identified as male reproductive tractspecific, and of these, human genes currently lack mouse phenotype information based on data obtained from ensembl biomart, mgi, impc, and ncbi. three hundred and eighty-six genes were identified as testis-specific through either the reanalysis of djureinovic et al. testis datasets ( genes identified), analysis of our de novo testis datasets ( genes identified), or both (additional file : table s ). three hundred and thirteen genes were congruent across both datasets, while genes were uniquely identified through our reanalysis of djureinovic et al.'s datasets and only genes [ac . , ankrd a , ankrd , fam a, ggtlc , iqcm, potec, prnt, and utf ] were uniquely identified through our de novo datasets (additional file : table s ). interestingly, of the genes we identified through djureinovic et al.'s reanalyzed datasets, were not previously identified in their report [ ] or any of the other previous reports [ , [ ] [ ] [ ] [ ] [ ] . of these genes, we randomly verified of these genes as testis-specific in humans through conventional pcr (fig. ) . we also verified through rt-pcr an additional genes-such as allc, cdkl , cox b , or h , and sppl c-that had been identified through previous studies (additional file : fig. s ). of the genes identified through either testis datasets, have not been previously identified; of these, genes have one or more mouse orthologs; and of these, genes are lacking reported phenotype information. of the novel genes lacking a reported mouse model, genes encode enzymes (adam , cpa , dusp , naa , plscr , prss , and triml ), encode transcription factors (bhmg , foxr , prdm , tgif ly, znf , znf ), encode transporters (slc a , slc a ), and encode proteins of unknown drug target type two hundred and thirty-three novel human reproductive tract-specific genes that each have mouse orthologous genes but with no reported knockout mouse models. the listed genes were identified in one or more datasets as indicated in the venn diagram. underlined genes were also identified in our studies as reproductive tract-specific in mouse ( genes). genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation ( genes) . genes written in dark red were identified in both testis (testis and/or testis cell) and epididymis ( genes). (such as etdb, smim , bend , btg , cnbd , dppa , efcab , erich , fthl , iqcm, mroh b, ms a , oosp , pnma e, ppp r c, rbmxl , rtl , spdye , spem ). all of these genes are listed in fig. , and many of these genes are listed in figs. , , and/or . to the best of our knowledge, no prior studies have utilized purified human testis cells for the identification of human testis-specific transcripts. through our analysis, we identified genes as human testis-specific through one or more of the human germ cell datasets, but not through either of the human testis datasets (additional file : table s ). seventy-six genes were identified exclusively through one or more of the five human spermatogonia datasets (genes such as anp d, c orf , dscr , or g , or d , or e , ssx , tle ), while genes were identified exclusively through the human spermatocyte datasets (genes such as h bfm, mageb , mageb , or b , tcp , and znf ) and genes were identified exclusively through the human spermatid datasets (genes such as ac . , clec a, or e , pramef , spat a a , tmem c, znf ). thirty-four genes were identified through all three cell types' datasets (genes such as ccdc , eloa , fam a, heat r , and spata a ). many of these genes are listed in figs. , , , and/or . of the genes identified as human testis-specific through one or more of the human germ cell datasets, genes have not been previously identified, of which have one or more equivalent mouse orthologs with of these genes having not been knocked out in mouse. of these novel genes with no mouse model, encode enzymes (glt d , prss , satl , sult b , tmprss , tpte , triml , and ttll ), encodes an epigenetic protein (taf l), encode gpcrs (gpr , tas r , tas r , tas r , tas r , vn r ), encodes a kinase (cdkl ), encode ogpcrs (such as or d , or a , or e , or g , or j , and ) and obp b met candidate threshold through our analysis of human testes datasets but did not meet candidate threshold from any of the germ cell or sertoli cell datasets, indicating potential expression in peritubular myoid cells, leydig cells, or other cell outside of the seminiferous epithelium. fam a has not been previously identified, and neither mouse orthologs ( m rik, gm ) have been knocked out. obp b was previously identified through djureinovic et al. [ ] and johnston et al. [ ] ; however, of the equivalent mouse orthologs (lcn , obp a, obp b), only obp a has been knocked out revealing abnormal coat/hair pigmentation [ ] . ism and magec were identified through both human sertoli cell datasets, while also identified through testis and/or germ cell datasets. both genes have been previously identified (ism [ ] , magec [ ] ). ism knockout mice display non-reproductive phenotypes [ ] . consistent with this finding, our mouse data do not identify ism as reproductive tract-specific in mice. magec lacks a mouse ortholog for functional analysis in mice. human sertoli cell-specific krtap - , krtap - , lhx , and psg were identified through one or both human sertoli cell datasets but were not identified through any of the testis or germ cell three hundred and two novel mouse genes with human orthologs without a reported mouse model. the listed genes were identified in one or more mouse datasets as indicated in the venn diagram. underlined genes were also identified through our studies as reproductive tractspecific in human ( genes). genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation ( genes). genes written in dark red were identified in both testis (testis and/or testis cell) and epididymis ( genes). datasets indicating sertoli cell-specific expression in the testes (additional file : table s ). none of these genes have been previously identified as reproductive tractspecific in humans although lhx and psg have mouse orthologs that have been knocked out [ ] [ ] [ ] [ ] [ ] [ ] . human krtap - has mouse orthologs krtap - , gm , gm , and gm , and human krta p - has mouse orthologs krtap - and gm ; none of these mouse orthologs have been knocked out ( fig. and additional file : table s ). psg knockout mice display non-reproductive phenotypes [ ] [ ] [ ] [ ] [ ] ; however, lhx knockout mice display absent testes and sterility due to an essential requirement for lhx during mouse gonad formation [ ] . a lhx -gfpcreer knock-in mouse line-generated by knocking-in gfpcreer at the endogenous lhx locuscrossed with the rosa -tdtomato reporter mouse line revealed cre recombinase activity in retinal amacrine cells, developing limbs, testis, hippocampal neurons, thalamic neurons, and cerebellar neurons [ ] . thus, lhx is not reproductive tract-specific in mice. our mouse data confirm this finding. to the best of our knowledge, no prior studies have utilized rna-seq for analysis of human epididymis-specific transcripts. through our studies, we identified genes as human epididymis-specific through one or more of the human epididymis segment datasets that were not identified through any of the other human male reproductive tissue or cell datasets, indicating true epididymis specificity (additional file : table s ). of these genes identified as human epididymis-specific, genes have not been previously identified, of which have equivalent mouse orthologs with of these genes having not been knocked out in mouse. of these novel human epididymis-specific genes with no mouse model, encodes an enzyme-related gene (spint ) and the remaining encode proteins of unknown drug target type (such as actbl , bsph , mslnl, spag a, spag b, wfdc a, and wfdc ) (fig. ) . seven genes were identified through our de novo sequenced human epididymis segment datasets that were not identified through our reanalysis of the human epididymis segment datasets by browne et al.; two of these genes are considered novel without mouse models: defb a and defb b (fig. , additional file : table s , and additional file : table s ). meanwhile, five genes were identified through our reanalysis of the human epididymis segment datasets by browne et al. that were not identified through our de novo-sequenced human epididymis segment datasets; two of these genes are considered novel without mouse models: actbl and mslnl (fig. , additional file : table s , and additional file : table s ). fifty-two genes met the criteria for identification as epididymis-specific through one or more of the human epididymis segment datasets, while also being identified as reproductive tract-specific through one or more of the testes, germ cell, and/or sertoli cell datasets (additional file : table s ). thus, these targets are not epididymis-specific per se, but may be desirable potential male contraceptive targets considering their broader target availability. of these genes identified as human male reproductive tract-specific and epididymisexpressed, genes have not been previously identified, of which have one or more equivalent mouse orthologs with of these genes having not been knocked out in mouse. all of these novel genes with no mouse model encode proteins of unknown drug target type (al . , ccdc , ccnb , defb , defb , eppin-wfdc , krtap - , magea , pnma e, spem , tex ) ( fig. and additional file : table s ). since model organisms other than mice may be of interest for the future functional study of human genes-especially those for which no known mouse ortholog exists-we list novel reproductive tract-specific human genes without a mouse ortholog in additional file : fig. s , which may be of interest for generating null rat or marmoset models [ ] . digital pcr (heatmap) demonstrating expression of a subset of these novel human reproductive tract-specific genes without mouse orthologs is shown in additional file : fig. s . through our bioinformatics analysis of previously published and newly acquired mouse rna-seq datasets, we identified a total of genes as reproductive tractspecific in mice. of these genes, genes do not have a human gene ortholog, while genes do (fig. ) . of those with a human gene ortholog, have a single ortholog ( with the same gene symbol in human; with a different symbol), while mouse genes have two or more ortholog human genes (fig. ). ninety-two mouse genes have - orthologous human symbols, genes have - orthologous human symbols, and genes (such as o rik, ankrd , fam a b, gm , magea , pramel , spdye a, spdye b, and zfy ) have greater than orthologous human symbols ranging anywhere from twelve to twenty-six symbols (additional file : table s ). of the mouse genes that we identified as male reproductive tract-specific in mouse, have not been identified in a previous transcriptomics-based study [ , [ ] [ ] [ ] [ ] [ ] [ ] . the sum of our mouse data confirms the findings of out of mouse genes from schultz et al. [ ] , out of mouse genes from johnston et al. [ ] , and out of mouse genes from johnston et al. [ ] (additional file : table s ). of the mouse genes that have a human ortholog equivalent, have not been previously identified as male reproductive tract-specific, and of these, genes currently lack mouse phenotype information based on data obtained from ensembl biomart, mgi, impc, and ncbi (fig. ). digital pcr (heatmap) demonstrating expression of a subset of the novel mouse reproductive tract-specific genes with human orthologs and no reported mouse model, and with human reproductive tract enrichment, is shown in fig. . seventeen novel genes without mouse models ( k rik, fthl b, fthl c, fthl d, fthl e, fthl f, gm , gm , magea , magea , magea , magea , magea , mageb , xlr a, xlr b, and xlr c) were identified through the mouse id + spermatogonia datasets that were also identified as spermatogonia-specific through the human datasets (bend , bx . , fam a, fthl , magea , magea , magea , mageb , and pnma e) and were not identified through either mouse or human testis datasets, indicating restricted expression in spermatogonia, spermatogonial stem cells, or both (additional file : table s and additional file : table s ). eight genes ( o rik, ccnb , gm , gm , pet , prr a , prr a , and prr a ) were identified through the mouse id + spermatogonia datasets, genes whose human orthologs-ccnb , dcaf l , magea , magea , magea , magea , magea b, prr a, prr b, prr c, and tle -were also identified through the human spermatogonia datasets. since these genes were also identified through mouse, human, or both species' respective testis datasets, this indicates either strong expression in the spermatogonia compartment or expression outside of and in addition to the spermatogonia compartment. twenty-two genes were identified as mouse sertoli cellspecific as they were not otherwise identified as reproductive tract-specific through our analysis of mouse testes or germ cell datasets (encode project consortium testes and helsel et al.'s id + germ cell datasets) (additional file : table s ). of these genes, have human orthologs; of these genes, are novel and not previously identified as male reproductive tract- specific; of these genes, have not been previously knocked out in the mouse (c ql , gm , mageb , mycs, shc , sowahd, and tmsb b ); and of these genes, genes (gm and mageb ) were identified with human orthologs (pnma e and mageb ) that were also identified through our human analysis as human reproductive tract-specific (additional file : table s and additional file : table s ). unlike the limited number of reproductive tract-specific genes we identified through human sertoli cell-specific datasets, a considerable number of genes- mouse genes-were identified as reproductive tract-specific through analysis of zimmermann et al.'s mouse postnatal day sertoli cell datasets that were also identified through either the mouse testis datasets, mouse id + germ cell datasets, or both (additional file : table s ). of these genes, have one or more human orthologs; of these genes, are novel and not previously identified as male reproductive tract-specific; of these genes, have not been previously knocked out in the mouse; and of these genes, ( l rik, m rik, b rik, g rik, ankrd , ankrd , etd, gm , gm , gm , magea , spata d b, tcp c, tex , tex , and wfdc ) were identified with human orthologs that were also identified through our analyses as human reproductive tractspecific (fig. , additional file : table s , and additional file : table s ). to the best of our knowledge, published rna-seq data of mouse whole epididymis or epididymis segments does not exist, for the identification of epididymis-specific transcripts or otherwise. therefore, we isolated caput, corpus, and cauda segments from adult (postnatal day ) b / mice and subjected the rna to sequencing. sixty-six genes were identified as mouse epididymisspecific as they were not identified as mouse male reproductive tract-specific through our reanalysis of the en- table s ). of these genes, have human orthologs; of these genes, are novel and not previously identified as male reproductive tract-specific; of these genes, have not been previously knocked out in the mouse (ascl , bsph , c s , clec a, cyp j , cyp a b, defb , gm , lce a, lcn , muc , odam, spag a, spag b, spink , svs , tchhl ); and of these genes, genes (bsph , defb , gm , spag a, and spag b) were identified with human orthologs (bsph , defb , spag a, and spag b) that are also human epididymis-specific (fig. , additional file : table s , and additional file : table s ). sixty-five genes were identified as reproductive tractspecific in mouse with expression in both epididymis and testis and/or testis cell. of these genes, have human orthologs; of these genes, are novel and not previously identified as male reproductive tract-specific; of these genes, have not been previously knocked out in the mouse ( n rik, d rik, d rik, d a rik, defb , dgkk, fam a b, gm , hrasls , nxf , shc , spint , trpc os, wfdc ); and of these genes, genes (spint , trpc os, and wfdc ) were identified with human orthologs (spint , trpc os, and wfdc ) that are also human epididymis-specific (fig. , additional file : table s , and additional file : table s ). through the aforementioned studies, we identified spint , spint , ces a, pp d , and saxo as congruent in expression across both mouse and human datasets with expression restricted to either the epididymis (spint , spint , ces a) or the testis (pp d and saxo ) (figs. , , , and ; additional file : table s ; additional file : table s ). spint was also identified as epididymisspecific in mouse (fig. , additional file : table s ); however, in humans, spint p is a pseudogene that is not processed into protein. conventional rt-pcr of a panel of mouse and human tissue cdnas confirmed epididymis-or testis-restricted expression of spint , spint , ces a, pp d , and saxo in both species and spint in mouse (fig. ) . to glean insight into the onset of expression for the epididymis-specific genes, spint , spint , spint , and ces a; whole epididymides from postnatal days (p) , p , p , and p ; and epididymis segments (caput, corpus, and cauda) from p , p , p , and p aged mice were collected and analyzed through rt-pcr (additional file : fig. s ). spint expression begins as early as p (low) and gradually increases through p and p reaching steady levels throughout p to p in all three segments of the epididymis (additional file : fig. s ). in contrast, spint and spint display near identical expression levels with no expression at p , p , or p , and expression apparent at p and later time points, with expression restricted to corpus only at p and p , and caput and corpus, but not cauda at p and p (additional file : fig. s ). rnascope-based fluorescence in situ hybridization revealed a distinct segment-specific pattern of expression for spint that was identical to spint , with both showing expression in most of the epithelial cells restricted to a brief region of distal caput/proximal corpus (additional file : fig. s ) . spint , on the other hand, displayed a pattern of expression in a majority of epithelial cells that begins just a bit further downstream along the corpus, but persisting for much further along the corpus, throughout the corpus and into the cauda (additional file : fig. s ). these results indicate that spint shares a role that is distinct from spint and spint and indicates a potential redundancy between spint and spint and how humans may have lost the evolutionary pressure to keep spint p as a protein-coding gene. to glean insight into the potential spermatogenic cell population(s) expressing pp d and saxo , we performed rt-pcr of mouse testes isolated at postnatal day (p) , a time point enriched for gonocytes; p (onset of expression of type a spermatogonia); p (early spermatocytes); p (late spermatocytes); p (spermatids); and p and p , which display complete spermatogenesis [ ] (additional file : fig. s ). expression of pp d and saxo is detected at similar levels at p and later, but not at p or before indicating expression during spermiogenesis and spermiation (additional file : fig. s ). to determine the male reproductive requirement and potential functional role of the identified novel male reproductive tract-specific genes, spint , ces a, pp d , and saxo were individually ablated by crispr/cas mediated zygote approach. since in humans spint p is a pseudogene, and in mice, spint protein is most similar in sequence to mouse spint , we simultaneously ablated both mouse spint and spint genes, which on mouse chromosome are only separated by . kilobases. the efficiency of generating each mutant is summarized in additional file : table s . each of the genes contained deletions of differing sizes and genomic targets. the genomic sequences flanking the deletion in each mutant are presented in additional file : table s , and representative sanger sequencing results for each mutant are presented in fig. . using the forward and reverse primer pairs presented in fig. a -e and listed in additional file : table s , offspring carrying the mutant alleles were identified through routine genotyping (fig. k-o) . spint , spint / , ces a, pp d , and saxo knockout mouse lines were examined in parallel with littermate controls of equivalent age to determine the effect of gene ablation on spermatogenesis, sperm maturation, and fertility in male mice. none of the knockout strains generated in this study displayed any overtly abnormal appearance, difference in body weight (fig. ) or composition, or difference in behavior when compared to the controls. to determine the male reproductive requirement of each of the genes of interest, spint , spint / , ces a, pp d , and saxo knockout and control adult male mice were housed continuously with two females for months and the size and number of litters were recorded. although spint , pp d , and saxo knockout males sired a number and size of litters during the test mating period that was not significantly different from controls (fig. a-c) , spint / and ces a knockout males sired significantly fewer litters and pups over the test mating period (fig. a-c) . spint / null males displayed a statistically significant % reduction in the number of litters and pups sired per male and statistically significant % reduction in litter size, over the -month mating period (n = controls, n = kos) (fig. ). seven out of males displayed complete infertility, and the two remaining males, who sired pups, sired pups at a significantly reduced number of litters and pups per month with litters of reduced litter size (fig. a-c) . this fertility defect in spint / double ko males was not associated with any significant changes in epididymis and testis histology (additional file : fig. s ) or sperm numbers, motility, and morphology ( fig. g-i) . ces a null males displayed a variegated phenotype with an overall statistically significant % reduction in the number of litters and pups sired per male, but no significant difference in litter size, over a -month mating period (n = controls, n = kos). the fertility defect in ces a ko males was associated with significant changes in epididymis histology (additional file : fig. s ) and significant reductions in sperm motility and progressive motility (fig. h, i) . ces a null males displayed a % reduction in sperm motility and progressive cells, a % increase in static cells, and a % decrease in average path velocity and progressive velocity after hyperactivation. no changes in testis histology were found (additional file : fig. s ) , and despite the sperm motility defect, scanning electron microscopy failed to identify a morphological defect in ces a null sperm in comparison to controls (additional file : fig. s ) . the epididymides and testes weights of spint , spint / , ces a, pp d , and saxo knockout mice were not significantly different from littermate control mice (fig. e, f) . histological analyses of testes from spint , spint / , ces a, pp d , and saxo knockout mice revealed all had seminiferous tubules with intact epithelia and the presence of all germ cell subtypes and all stages of spermatogenesis (additional file : fig. s and additional file : fig. s ). histological analyses of caput, corpus, and cauda from spint , spint / , pp d , and saxo ko mice revealed spermatozoa in tubule lumens of all knockouts with no significant differences in epididymal histology in comparison to controls (additional file : fig. s and additional file : fig. s ). however, ces a knockout mice displayed significant histological abnormalities including lumen dilation (possibly from occlusion), inflammation, and the appearance of abnormal epithelia (additional file : fig. s ). computer-assisted sperm analysis of spint , spint / , pp d , and saxo knockouts phenotype analysis of crispr/cas generated null mice for determining the contraceptive potential of the selected genes. spint / and ces a null mice show significant fertility defects; meanwhile, spint , pp d , and saxo null mice appear normal. fertility (a-c), body and reproductive organ weights (d-f), and sperm parameters (g-i) were all measured between knockout (−/−) and littermate control [wild-type (+/+) and heterozygous (+/−)] mice as indicated. bars represent mean ± sem. *p < . , **p < . , ***p < . . ns, not significant. showed no statistically significant differences across all measured parameters including sperm concentration, sperm motility, and progressive motility (fig. g-i) . ces a knockouts displayed significant decreases in sperm number and sperm motility (fig. h, i) ; however, cauda epididymal sperm isolated from a variety of ces a null animals looked morphologically indistinguishable to controls (additional file : fig. s ). to date, the etiology of idiopathic male infertility is not fully understood, and hormonal male contraceptives have not been effective. therefore, identification of novel reproductive tract-specific genes, and elucidating the functional requirement or lack thereof of these genes, is essential towards understanding the etiology of male infertility and the development of male contraceptives. despite significant advances in our understanding of the human and rodent testis and epididymis transcriptome, mostly through microarray-based studies, no prior studies have utilized purified human testis cells for the identification of human testis-specific transcripts, no prior studies have utilized the more state-of-the-art rna-seq-based transcriptomics methodology for analysis of human epididymis-specific transcripts, and no prior studies have utilized rna-seq analysis of rodent reproductive tissues or cells to identify rodent reproductive tract-specific transcripts. to address these gaps in knowledge, and to increase the number of identified reproductive tract-specific genes using the most relevant high-throughput transcriptomics methodology, we analyzed in parallel on a custom bioinformatics pipeline a large number of published and newly acquired human and mouse rna-seq datasets. through our studies, we identified and verified many novel male reproductive tract-specific transcripts in both species, and through the crispr/cas system, we interrogated the reproductive requirement of a subset of these genes. we found that spint (together with spint ) in mice is required for normal male mouse fertility, and although not required for male fertility, we identified ces a as playing a major biological role in the epididymis. we report the remaining genes that we knocked out-spint , pp d , and saxo as dispensable for male reproductive function, which is essential information to disseminate to the scientific community. our study also verified the male reproductive tract-specific expression of many previously identified genes (additional file : fig. s , additional file : fig. s ) , and genes for which previously published mouse models display male infertility phenotypes (additional file : fig. s ). this later group of already functionally validated genes serves as potential male contraceptive targets worth underscoring to the research community. prior to massively parallel microarray-based and rnaseq-based transcriptomics analyses for the identification of reproductive tract-specific genes, the ncbi unigene database was a valuable resource for many in the male reproductive biology field for identifying testis-specific transcripts [ , [ ] [ ] [ ] . although in our study we only considered prior microarray-based and rna-seq-based studies when considering the novelty of the genes that we identified, it is worth noting that several genes that we identified-not previously identified in microarray-based and rna-seq-based transcriptomics studies-were previously identified through studies that solely utilized the unigene database [ , ] . nineteen human genes that we identified-c orf , ccdc , cpa , fam a, fam d, fam c, fbxo , fbxw , fkbp , galn tl , kcnu , mageb , mroh b, nutm , prdm , rbmxl , spata e , triml , and trpc os-were previously identified by liu et al. [ ] , and seven mouse genes that we identified- d rik, akap , ankrd , hrasls , spesp , tex , and ubqlnl-were previously identified by choi et al. [ ] and liu et al. [ ] . thus, our results confirm the findings of these previous studies. since more than half of all human protein-coding genes are categorized as unknown in terms of drug target potential (additional file - table s ), and only % encode for classically druggable enzymes, gpcrs, ion channels, nuclear receptors, and transporters (additional file - fig. s ) , the potential to find new undiscovered drug targets that can be drugged using classical approaches is somewhat limited. indeed, in our study, we found one hundred and nine genes to be novel in terms of previously published high-throughput transcriptomics studies, without a current reported mouse model, and reproductive tract-specific in both humans and mice (figs. , , and ). many of these genes ( genes; %) fall into the category of unknown and may otherwise be considered "undruggable" due to various challenges with existing targeting approaches [ ] . however, the contraceptive potential of these genes should not be overseen, but rather investigated for potential identification of a high-affinity small molecule that can either interfere with protein-protein interaction (ppi) or target the protein specifically for degradation using a new technology called proteolysis targeting chimeras (protacs). protein-protein interaction targets are not deemed undruggable, based on the discovery of small molecules capable of deeper and higher affinity binding within the contact surfaces of the target protein [ ] . additionally, once a high-affinity small molecule against a specific target protein is identified, an engineered protac molecule can mark a target protein for proteasomal degradation by linking the target protein to the polypeptide co-factor, ubiquitin [ ] [ ] [ ] . there are currently various combinations of protacs developed to overcome the limitations of cell permeability, stability, solubility, selectivity, and tissue distribution [ ] [ ] [ ] [ ] . therefore, disrupting ppis or utilizing protacs provides the potential to greatly promote the development of contraceptive drugs against the "undruggable" nonenzymatic target protein space. if gene knockouts for closely related and ubiquitously expressed paralogs display no abnormal phenotype, then unintended drug targeting of these proteins may result in no side effects in humans. however, the burden of safety for a male contraceptive is extremely high, and if it can be avoided, targeting non-reproductive tractexpressed proteins in humans should be avoided since the functional requirements for these proteins may not be fully understood. further, although mice are one of the best models for human disease, they are unable to communicate when they are unwell, a phenomenon that may occur independent of any measurable phenotypic traits. thus, potential reported side effects in humans during clinical trials may have been present, but missed, during animal studies, or in fact be present in humans and not in mice because of the vast biological differences across these two species. thus, with drug safety in mind, reproductive tract-specific candidates should be prioritized based on somatic cell-expressed protein sequence similarity, especially in the drug binding pocket. according to ensembl, several novel reproductive tract-specific genes without mouse models that we identified and verified-ac . , al . , bhmg , c orf , c orf , c orf , ccdc , spint , tex , tex , tex , and trpc os (figs. , , and ; additional file : table s )-have no known associated paralogs, indicating reproductive tract-specific drug targeting is highly likely. additionally, spag a and spag b are epididymis-specific paralogs (figs. , , and ; additional file : table s ) with no other known paralogs according to ensembl. efcab (figs. and , additional file : table s ) has a ubiquitously expressed paralog, nsrp , with only % amino acid sequence similarity, indicating specific drug targeting potential for this candidate. likewise, erich and mroh b (figs. and , additional file : table s ) have ubiquitously expressed paralogs erich b and mroh a, respectively, with % and % amino acid sequence similarity, indicating reasonable potential for specific drug targeting. prr a, prr b, and prr c are testis-specific paralogs (figs. and , additional file : table s ) with prr d as the next closest paralog according to ensembl. since prr d has less than % amino acid sequence similarity to prr a, prr b, and prr c, but appears to be epididymis-specific according to the human protein atlas [ ] , all four proteins of unknown function make suitable drug candidates. likewise, spat a d , spata d , and spata d are testis-specific paralogs (figs. and , additional file : table s ) with spata a as the next closest paralog according to ensembl. since spata a has less than % sequence similarity to spata d , spata d , and spat a d , but also appears to be reproductive tractspecific according to hpa [ ] , all four proteins with unknown function also appear to be worthy of consideration for potential drug targeting. tpte and tpte are testis-specific paralogs (figs. and , additional file : table s ) with ubiquitously expressed pten as the next closest paralog according to ensembl. since pten has less than % sequence similarity to tpte and tpte , off-target effects appear to be unlikely. likewise, wfdc a, wfdc b, and wfdc are all epididymis-specific paralogs with the closest nonreproductive tract-expressed paralog, wfdc , having less than % sequence similarity to any of the rts paralogs, also indicating high drug specificity potential. several additional novel reproductive tract-specific enzyme and gpcr genes without mouse models that we identified-gpr , prss , prss , sult b , tmpr ss , triml , and ttll (fig. , , and ; additional file : table s )-have non-reproductive tract-expressed paralogs with less than % sequence similarity indicating good drug specificity potential. a novel testis-specific transporter gene without a reported mouse model that we identified, slc a , would make a poor drug candidate since its closest paralog, slc a , is % similar in amino acid sequence and ubiquitously expressed. cpa , iqca l, and ppp r c have ubiquitously expressed paralogs, cpa , iqca , and ppp r b, respectively, with - % protein sequence similarity indicating careful consideration must be made for potential drug targeting without off-target effects. of the seventy-three genes that our study identifies as reproductive tract-specific in humans and for which a published mouse model shows male infertility phenotype (additional file : fig. s ) [ , , ] , it is worth noting that genes-cnbd , defb , fam a, fbxo , meig , meiob, meioc, odf , odf , rec , rnf , spaca , spata , spem , spo , sycp , terb , tex , tex , tnp , and topaz-do not have any associated paralogs and, thereby, may be considered most suitable for further drug development. however, it is also worth noting that it may be possible that genes required for male fertility in mice may not necessarily be required for male fertility in humans. of the seventy-three human reproductive tract-specific genes our study identified with male mouse infertility phenotypes, twenty-seven genes-actl b [ ] , akap [ ] , boll [ ] , brdt [ ] [ ] [ ] [ ] [ ] [ ] , catsper [ ] , ccdc [ ] , fkbp [ , [ ] [ ] [ ] , meig [ ] , meiob [ ] [ ] [ ] ] , nanos [ , ] , odf [ ] , prdm [ , , ] , prss [ ] , rad l [ ] , rbmxl [ , ] , rnf [ ] , sohlh [ , ] , spaca [ ] , spata [ , ] , spem [ ] , spo [ , , ] , sun [ ] [ ] [ ] , sycp [ ] , tex [ ] , tnp [ ] , tssk b [ ] , and zpbp [ ] -are currently associated with mutations underlying human male infertility, confirming a similar functional requirement for these genes in humans may exist. for the remaining genes, however, either these genes are not required for human male fertility as they are required in mice, or associated mutations in male infertile patients have not yet been reported. although many reproductive tract-specific genes have been studied through functional genetics approaches, many remain to be solved. elucidating the function of these novel genes is necessary to build a better understanding of the factors underlying spermatogenesis and sperm maturation, which has implications in understanding the etiology of male infertility and the development of male contraceptives. in this study, four epididymis-specific genes (spint , spint , spint , and ces a) and two testis-specific genes (pp d and saxo ) were deleted in mice to determine their functional requirement in male fertility and potential utility as male contraceptive target. we chose to study these genes because all but saxo encode enzymes or enzyme-related protein products and are thus considered druggable in the classical sense. saxo , a cilia-related gene, was chosen because prior literature demonstrated expression in sperm [ ] . although not druggable in the classical sense, if targeted through non-canonical approaches, one could obtain a fast-acting drug with greater reversibility potential and a decreased likelihood of affecting testicular function and size. the epididymis-specific genes we chose to target for functional analysis, by the very nature of their tissue's expression, also fit this potential drug profile of modulating only the latest stages of sperm development. analyses of testis and epididymis organ weights and histology, sperm parameters and morphology, and fertility revealed no significant differences in spint , pp d , and saxo knockout mice in comparison to littermate controls demonstrating that, individually, spint , pp d , and saxo are not required for male mouse fertility and are not suitable targets for the development of a male contraceptive. however, we found partial effects on male fertility in ces a knockout mice and profound effects on male fertility in spint / double knockout mice. ces a is a member of a multigene family of mammalian carboxylesterases that can hydrolyze ester, thioester, amide, and carbamate linkages in a wide variety of endogenous and exogenous molecular substrates, including triglycerides, thus playing key roles in both metabolism and detoxification [ ] [ ] [ ] [ ] . ces a shares roughly similar percent homology (~ % homology) to all four of its related paralogs, ces , ces , ces , and ces a. human carboxylesterase (ces ) is predominantly expressed in the liver and has been shown to have triglyceride hydrolase activity as overexpression of human ces in cells leads to an increase in cholesteryl ester hydrolysis and free cholesterol efflux [ ] . further, mouse ces g-a protein expressed by one of a cluster of eight syntenic genes (ces a through ces h) orthologous to the human ces gene-has been shown to have triglyceride hydrolase activity as ces g null mice display hyperlipidemia and abnormal lipid homeostasis including increased liver and circulating cholesterol and triglycerides, and altered saturated and unsaturated fatty acid levels [ , ] . therefore, it is likely that ces a exhibits similar carboxylesterase activity in the epididymis hydrolyzing cholesteryl ester and affecting free cholesterol efflux. indeed, recombinant ces a protein has been previously shown to have carboxylesterase activity hydrolyzing cholesterol ester and choline ester [ ] . since sperm cholesterol content is significantly decreased during epididymal maturation [ , ] and a proper cholesterol/phospholipid (c/pl) ratio of the sperm plasma membrane is required for sperm capacitation [ , ] , ces a may be pivotal in regulating sperm membrane cholesterol and lipid levels to ensure the normal function of male gametes in the last steps of the fertilization process. the most closely related paralog to spint is eppin, which, as reviewed in o'rand et al., has at least three physiological functions [ ] . eppin inhibits sperm motility when it binds the semen coagulation protein semenogelin (semg ) on the sperm surface [ ] ; it modulates the proteolytic activity of prostate-specific antigen (psa), a serine protease, against its seminal plasma substrate, semg [ ] ; and it exhibits strong antibacterial activity [ ] . these functions are postulated to prevent premature hyperactivation and capacitation of sperm in the female reproductive tract [ ] , and to protect spermatozoa from proteolytic and bacterial attack during transit in the female reproductive tract [ , ] . thus, it is possible that the physiological function of spin t is similar. however, unlike spint and spint , which have been shown to act as protease inhibitors against a wide variety of prss and tmprss proteases [ ] [ ] [ ] [ ] , when tested against a panel of eight proteases (including psa, trypsin, chymotrypsin, plasmin, urokinase, thrombin, factor xa, and elastase), spint and spint were shown to lack protease inhibiting capability [ ] . this indicates that either the protease inhibiting properties of spint and spint were lost in favor of yet unknown functions or their protease activity has a narrower spectrum of inhibition against unknown targets. since spint / null male mice are severely subfertile, without an apparent difference in epididymis histology, sperm number, sperm morphology, and sperm motility parameters in comparison to the wild-type (wt) mice, this phenocopies the reproductive phenotype of several null mice of testis-, epididymis-, or prostate-specific genes (sof , tmem , and spaca ; pate , and pate ), which reveal a requirement in regulating sperm migration through the oviduct and sperm-oocyte fusion in mice [ , ] . a severe fertility defect associated with normal sperm number, morphology, and motility is also shared among mice lacking the sperm membrane protein adam , thought to be crucial in sperm-zp binding and sperm migration through the uterotubular junction (utj) [ , ] . more than proteins including proteases (ace, adam a, adam , calr , clgn, cmtm a/b, pdilt, pmis , prss , rnase , tex , and tpst ) have been described that affect the processing and/or localization of adam protein in spermatozoa [ , ] . further studies with spint / null mice are required to determine whether sperm behavior in the female reproductive tract, specifically the sperm migration through the utj, is adversely affected. since a large , -bp genomic region-including the intergenic region between the spint and spint genes-was deleted ( fig. ; additional file : table s ), based on the evidence presented in this manuscript, we cannot exclude the possibility that cis-acting elements and/or trans-acting factors affecting the expression of other genes may have contributed to the phenotype of these mice. lack of protein-coding ability of human spint p does not necessarily indicate that this pseudogene is functionally obsolete. pseudogenes have been shown to play roles in gene expression and gene regulation [ ] . for example, pseudogene transcripts can act as competitive endogenous rnas (cerna) through competitive binding of mirna, which results in regulation of gene expression [ ] . to this end, studying the functional requirement of spint in male mice is necessary to further our knowledge of evolutionarily conserved genes between species. since humans are genetically diverse, a limitation to phenotype characterization of genetically manipulated mice is the reliance of a single mouse background to the examination of complex genetic outcomes, such as fertility, that is under the control of many genes with different levels of contribution to the phenotype [ ] . it is possible that a gene that causes complete infertility in an inbred mouse background may only cause partial infertility or subfertility in a different inbred line or more robust outbred background. since the mice used in our study were a cross between c bl/ (b ) mice and dba/ (d ) mice, and thus, these b d f mice are heterozygous for b and d alleles at all loci in their genome, we can eliminate infertility susceptibility of either the b or the d background as the cause for fertility defects in ces a and spint / mice. it does remain to be determined, however, whether the phenotype of the genes we knocked out would be more or less severe on a different mouse background, and if required for male fertility in humans, the level of contribution to male fertility of these genes across genetically diverse men. a limitation to this study is the reliance on mrna abundance positively correlating with protein abundance. future studies are necessary to elucidate the relationship between mrna and protein expression levels of the candidate genes identified in our study. furthermore, despite batch corrections that were made, technical differences in sample preparation and integrity across the various published rna-seq datasets can influence the results of our findings. one of the major advantages to our study design is the use of rna-seq datasets from purified human and mouse germ cells and sertoli cells to identify reproductive tract-specific targets since the use of whole testes for the identification of cell typespecific transcripts in past studies is subject to dilution effects. however, this advantage could also be considered a disadvantage since purified cells from nonreproductive tissues were not used for comparison but, if analyzed, purified cells from non-reproductive tissues may have revealed significant levels of expression in non-reproductive tissues. ultimately, functional studies in animals and humans will help to confirm whether genes identified in our study are essential for male fertility and not any other physiological process. through the integration of hundreds of published and newly acquired human and mouse reproductive and non-reproductive tissue and cell rna-seq datasets, we have generated a list of novel genes expressed predominantly or exclusively in the male reproductive tract that are worthy of consideration for functional validation in an animal model and potential targeting for a male contraceptive. our results further validate a functional requirement for spint / and ces a in male mouse fertility, while demonstrating that spint , pp d , and saxo are each individually dispensable for male mouse fertility. identifying novel reproductive tract-specific genes congruent across species adds insight into organismal biology and valuable information that can be used to identify potential male contraceptive drug target candidates. furthermore, elucidating the individual functional requirement or lack thereof of these novel genes builds a better understanding of the factors underlying spermatogenesis and sperm maturation, which has implications in understanding the etiology of male infertility and further validation of the utility of a potential male contraceptive target. the de novo isolated human testes and epididymides included in this study were obtained from three donors through a local organ transplant program in quebec, canada, called transplant quebec. all procedures were approved by the local ethics committee, and written consent was obtained from each respective donor's family. the donors were of , , and years of age with no preexisting medical condition that could affect reproductive function. donor testes and epididymides were removed under artificial circulation to preserve other organs that were assigned for transplantation. each testis and epididymis were dissected in the laboratory of robert sullivan at université laval. each epididymis was dissected into three segments corresponding to the caput, corpus, and cauda regions and minced into small tissue pieces. testes and epididymides tissue fragments were immediately snap frozen in liquid nitrogen, stored at − °c, and shipped frozen to baylor college of medicine for further processing. eight non-reproductive tissue types (kidney, liver, lung, skin, spleen, and stomach) and female reproductive tissues (ovary and uterus) were obtained from the baylor college of medicine tissue acquisition and pathology core. thirteen nonreproductive tissue types (adipose, adrenal gland, brain, colon, heart, leukocytes, pancreas, prostate, salivary gland, skeletal muscle, small intestine, smooth muscle, thyroid) were obtained as purified rnas from takara bio (kusatsu, japan). human testes and epididymis segments were used for de novo rna-seq analysis; all human tissues and/or resulting rnas were used for rt-pcr verification. mouse tissues [testis, caput, corpus, cauda, ovary, uterus, and non-reproductive tissue types (adipose, bladder, brain, colon, eye, heart, kidney, liver, lung, prostate, skeletal muscle, skin, small intestine, spleen, stomach)] were obtained from dissection of b / mice; the remaining non-reproductive tissues (smooth muscle and thyroid) were obtained as purified rnas from takara bio. mouse epididymis segments were used for de novo rna-seq analysis; all mouse tissues and/or resulting rnas were used for rt-pcr verification. rna for both rna-seq and/or rt-pcr verification was isolated from human and mouse tissues using trizol/ chloroform extraction method followed by rneasy mini kit from qiagen with on-column dnase (qiagen) treatment using the manufacturer's protocol. rnas used for rna-seq were assessed by bioanalyzer for rna integrity. for rt-pcr, rna was reverse-transcribed to cdna using superscript iii reverse transcriptase from thermo fisher according to the manufacturer's protocol. cdna was then pcr amplified using gene-specific primers designed using ncbi primer design tool. primer sequences are listed in additional file : table s . library generation for rna-seq rna-seq libraries were made using kapa stranded mrna-seq kit (kk ). briefly, poly-a rna was purified from total rna using oligo-dt beads; subsequently, it was fragmented to small size; and first strand cdna was synthesized. second strand cdna was synthesized and marked with dutp. resultant cdna was used for end repair, a-tailing, and adaptor ligation. finally, the library was amplified for sequencing on an illumina novaseq platform. the strand marked with dutp was not amplified, allowing strand-specific sequencing. sequence alignment, quantification, and differential gene expression human testes, human epididymis segments, and mouse epididymis segments were sequenced by the department of molecular and human genetics functional genomics core at baylor college of medicine (additional file : table s and additional file : table s ). previously published reproductive and non-reproductive tissue and cell sequences were downloaded from the sequence read archive (sra) [ ] (additional file : table s and additional file : table s ). all sequences were trimmed using trim galore! and aligned against the human genome (grch ) or mouse genome (grcm ) using hisat [ , ] . gene expression in each tissue was quantified using featurecounts, filtered for only protein-coding genes, and batch corrected by removing unwanted variation using the ruvr method from ruvseq [ , ] . differential gene expression was determined for each reproductive tissue against each nonreproductive tissue using the r package edger [ ] . principal component analysis (pca) was performed in the r statistical environment using the log counts per million (cpm) for each gene in the corresponding tissue after using the ruvr method to correct for batch variation as described above. our procedure for identifying a reproductive-specific gene was repeated for each reproductive tissue or cell sample independently. the following selection criteria were applied to each reproductive tissue or cell sample. first, the non-reproductive tissue with the maximum expression, expressed as the log fold change between the non-reproductive tissue and the reproductive tissue or cell of interest, was identified for each gene using the results from the differential gene expression analysis. second, we identified reproductive-specific gene drug candidates using three filters: a false discovery rate (fdr) filter, a maximum transcript per million (tpm) expression value filter on the non-reproductive sample with the maximum expression identified as described above, and a minimum tpm expression value filter on the reproductive tissue or cell sample of interest. a gene was kept if the fdr from the differential gene expression analysis was less than or equal to . for the comparison of the reproductive tissue or cell sample of interest to the non-reproductive tissue with the maximum expression. a gene was considered to be a male reproductive tissue-specific drug target if the average tpm expression value in the non-reproductive tissue with the maximum expression from the differential analysis was less than or equal to . for human ( . for mice), and if the average tpm expression value for that gene was greater than or equal to . for human ( . for mice) in the reproductive tissue or cell sample of interest. the average and standard deviation of the tpm expression value for each gene was calculated from the ruvr batch corrected counts per million expression value for each tissue or cell sample. we consolidated data from ensembl biomart [ ] and mouse genome informatics (mgi) [ ] to create a comprehensive database of mouse gene symbols orthologous to human genes and vice versa. each respective species' stable ensemble gene id was used for each conversion, with gene symbol as the final output. as mentioned, several notable high-throughput gene expression studies using microarrays or rna-seq, focused on identifying male reproductive tract-specific genes, have been previously published [ , [ ] [ ] [ ] [ ] [ ] [ ] . tables and supplementary tables from these studies were gathered to collect the lists of genes previously identified. for microarray-based studies, affymetrix ids were used to confirm the identity of a listed gene, based on current sequence mappings, or in many cases to identify de novo the identity of a gene only known at the time of the study by its affymetrix id and not gene symbol. for mouse and rat studies, gene symbols were converted to orthologous human symbols, to systematically catalog both the rodent and corresponding human symbols as previously identified. for example, affymetrix probe ids were listed as reproductive tract-specific in johnston et al. [ ] . after re-identification of gene symbols based on current ensembl sequence mappings, identified gene symbols remained the same, received an updated/replacement gene symbol identification, genes that were previously unidentified received a new gene symbol identification, affymetrix ids lost mapping to any gene symbol, and remain unidentified. out of the total of affymetrix ids that mapped to current rat gene symbols, rat gene symbols converted to at least one human ortholog gene symbol that was either the same symbol or different. both rat and human symbols, based on new mappings, were considered previously identified. for the complete list of previously identified genes, see additional file : table s . genes were classified as encoding either enzymes, epigenetic-related proteins, g protein-coupled receptors (gpcrs), ion channels, kinases, nuclear receptors, orphan gpcrs (ogpcrs), transcription factors, transporters, or unknown proteins based on data obtained from illuminating the druggable genome [ ] (additional file : table s ). we used data obtained from ensembl biomart [ ] , mgi [ ] , the international mouse phenotyping consortium (impc) [ ] , and pubmed searches to generate a comprehensive database identifying the existence of a mouse model for all mouse genes (additional file : table s ). we then queried our identified candidate human and mouse genes against this list. for a given human gene, we queried the equivalent mouse ortholog gene symbol(s). spint , spint / , ces a, pp d , and saxo knockout mice were produced at baylor college of medicine. b d f (c bl/ × dba ) mice were used as embryo donors, and cd mice were used as foster mothers. mice were purchased from charles river (wilmington, ma). all mice were housed in a temperature-controlled environment with -h light cycles and free access to food and water. mice were housed in accordance with nih guidelines, and all animal experiments were approved by the institutional animal care and use committee (iacuc) at baylor college of medicine. generation of spint , spint / , ces a, pp d , and saxo knockout mice to generate spint , spint / , ces a, pp d , and saxo knockout mice, grna/cas ribonucleoprotein complex was electroporated into fertilized eggs and transplanted into surrogate mothers as previously described [ ] . briefly, to harvest fertilized eggs, card hyperova ( . ml, cosmo bio) was injected into the abdominal cavity of b d f females (charles river), followed by human chorionic gonadotropin (hcg) ( units, emd chemicals). forty-eight hours after card hyperova, b d f males were allowed to mate naturally. twenty hours after mating, fertilized eggs with pronuclei were collected for electroporation. custom crrnas targeting each gene were purchased from millipore-sigma. the sequences for all guide rnas used for crispr/cas mediated gene editing are listed in additional file : table s . crrna and tracrrna (millipore-sigma) were diluted with nuclease-free water. the mixture was denatured at °c for min and allowed to anneal by cooling gradually to room temperature ( h). each grna was mixed with cas protein solution (thermo fisher scientific) and opti-mem media (thermo fisher scientific), and then incubated at °c for min to prepare the grna/cas rnps [final concentration, ng/μl cas for ng/μl of each grna]. the grna/cas rnp solution was placed between electrodes with a mm gap in the ecm electroporation system (btx). fertilized eggs were arranged between the electrodes, and then, the electroporation was performed with the following conditions: v, -ms pulse duration, and pulses separated by -ms pulse interval. for egg transfer, electroporated embryos were transplanted into the oviduct of pseudo-pregnant icr recipients. after days, offspring were obtained by natural birth or cesarean section. the f mice with sequence-predicted heterozygous mutations were used for the mating with additional b d f mice to generate homozygous mutants. the f or later generations were used for the phenotypic analyses. for sanger sequence analysis of mutant mice, genomic dna was isolated by incubating tail tips in lysis buffer [ mm tris-hcl (ph . ), mm edta, mm nacl, . % sds, and μg/ml actinase e solution] at °c overnight. polymerase chain reactions (pcrs) amplifying the genomic region containing the insertion/deletion events were performed using kod xtreme enzyme (toyobo, osaka, japan); pcr products were purified using the qiaquick pcr purification kit (qiagen, carlsbad, ca, usa) and sent for sanger sequencing on an abi xl genetic analyzer (thermo fisher scientific, waltham, ma, usa) using the forward primer. for routine genotyping of mutant mice, genomic dna was isolated by separately incubating ear snips and tail tips in mm naoh solution at °c overnight and inactivating with m tris ph = . . pcrs amplifying wild-type and mutant-specific amplicons were performed using x amfisure pcr master mix (gendepot, barker, tx). primer sequences are listed in additional file : table s . upon sexual maturation ( - weeks of age), knockout and littermate control male mice (n = - mice per genotype) were continuously housed with two - week-old wild-type b d f /j female mice per male for weeks. during the fertility test, the number of pups was counted shortly after birth. the total number of litters and pups per male over the mating trial was calculated and divided by the number of months to generate averages and statistics per genotype. the average number of pups per litter is based on the average litter size per male where a litter is considered one or more pups. knockout and littermate control male mice (n = - mice per genotype) that were weeks of age were used to examine body and reproductive organ weights, and testicular and epididymal histology. testes and epididymides were fixed in bouin's fixative, embedded in paraffin, sectioned at μm thickness, and stained with % periodic acid-schiff (pas) stain followed by counterstaining with hematoxylin solution. histological images were acquired with an aperio at slide scanner (leica microsystems). knockout and littermate control male mice (n = - mice per genotype) that were weeks of age were used to examine sperm numbers and motility parameters using computer-assisted sperm analysis (casa). cauda of both epididymides was isolated, transferred into human tubule fluid (htf) (irvine scientific, santa ana, ca) containing mg/ml of bsa, minced, and placed in a humidified incubator for min at °c with % co . following incubation, the sperm were diluted : in htf, added to a pre-warmed slide, and analyzed using a hamilton-thorne bioscience's ceros ii instrument. several fields of view were illuminated and captured until at least cells were counted. rnascope . hd reagent kit (red) (cat. , advanced cell diagnostics, newark, ca, usa) was used to detect spint , spint , and spint mrna transcripts on pfa-fixed, paraffin-embedded sections from -monthold wild-type epididymis. the probes against mm-spint , mm-spint , and mm-spint were custom-made, and the standard positive control (mm-ppib, cat. ) and negative control (dapb, cat. ) probes were used. the assay was performed according to the manufacturer's instructions. slides were counterstained using dapi and mounted using prolong glass antifade mountant (thermo fisher scientific inc.). multi-channel fluorescent images were acquired with an aperio versa (leica microsystems). all measurements are expressed as mean ± standard error of the mean. statistical differences were determined using student's t test. differences were considered statistically significant if the p value was less than . . supplementary information accompanies this paper at https://doi.org/ . /s - - -z. additional file : tables s , s , s , s , s , and s . table s . summary of human rna-seq datasets. this table contains the sra value for each previously published human rna-seq dataset that was reanalyzed as part of this study. the geo accession number for each new human rna-seq dataset generated and subsequently analyzed in this study is also included. table s . summary of mouse rna-seq datasets. this table contains the sra value for each previously published mouse rna-seq dataset that was reanalyzed as part of this study. the geo accession number for each new mouse sample generated and subsequently analyzed in this study is also included. table s . single-guide rnas targeting the genes' upstream (u) and downstream (d) regions used for generating knockout mice. efficiency of embryo transplantation was presented using the number of total pups delivered by pseudopregnant mice divided by the number of total embryos used for oviduct transplantation (total pups/embryos transplanted). efficiency of genome editing was determined by the number of pups carrying enzymatic mutations divided by the number of pups subjected to genotyping (gm pups/pups genotyped). table s . sanger sequencing of detailed genotype of mutant dna sequences in all the five mouse lines. table s . primers and pcr conditions used for genotyping the mutant alleles of the knockout mouse lines. table s . human and mouse rt-pcr primer sequences used for verification of reproductive tract-specificity. additional file : fig. s . genes that passed the tpm and fdr filters in at least one of the measured reproductive tissues or cells were visualized using a heatmap of the ruvr batch corrected log cpm gene expression values for the human (a) and mouse (b) samples. additional file : table s . human expression summary. contains differential fold change, identity of the non-reproductive tissue with maximal gene expression based on the differential gene analysis, false detection rate (fdr) value, average and standard deviation tpm expression values, and log cpm gene expression value for the human samples. all protein-coding genes ( , genes) that had expression in at least one reproductive tissue or cell is listed. additional file : table s . mouse expression summary. contains differential fold change, identity of the non-reproductive tissue with maximal gene expression based on the differential gene analysis, false detection rate (fdr) value, average and standard deviation tpm expression values, and log cpm gene expression value for the mouse samples. all protein-coding genes ( , genes) that had expression in at least one reproductive tissue or cell is listed. additional file : table s . all human male reproductive tract-specific genes that met the criteria of identification as reproductive tract-specific in at least one male reproductive tissue or purified cell type, with the level of fold change listed under the tissue or cell if all criteria were met. the criteria of selection are as follows: fdr < . ; tpm repro > ; tpm non-repro , max < . a fold change value of indicates the criteria were not met for that that tissue or cell. additional columns indicating .) the equivalent mouse ortholog gene symbols (single or multiple symbols) that exist, and .) if our studies identified any of these mouse orthologs as reproductive tract-specific in mouse, are included. additional file : table s . all mouse male reproductive tract-specific genes that met the criteria of identification as reproductive tract-specific in at least one male reproductive tissue or purified cell type, with the level of fold change listed under the tissue or cell if all criteria were met. the criteria of selection are as follows: fdr < . ; tpm repro > ; tpm non-repro , max < . a fold change value of indicates the criteria were not met for that that tissue or cell. additional columns indicating .) the equivalent human ortholog gene symbols (single or multiple symbols) that exist, and .) if our studies identified any of these human orthologs as reproductive tract-specific in human, are included. received: april accepted: july population division: un the mouse epididymal transcriptome: transcriptional profiling of segmental gene expression in the epididymis the human epididymis: its function in sperm maturation a multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets identification of testis-specific male contraceptive targets: insights from transcriptional profiling of the cycle of the rat seminiferous epithelium and purified testicular cells identification of epididymis-specific transcripts in the mouse and rat by transcriptional profiling the rat epididymal transcriptome: comparison of segmental gene expression in the rat and mouse epididymides stage-specific gene expression is a fundamental characteristic of rat spermatogenic cells and sertoli cells the human testis-specific proteome defined by transcriptomics and antibody-based profiling toward development of the male pill: a decade of potential non-hormonal contraceptive targets epididymal protein targets: a brief history of the development of epididymal protease inhibitor as a contraceptive disrupting the male germ line to find infertility and contraception targets male contraception: another holy grail male contraception: past, present and future metabolic cooperation in testis as a pharmacological target: from disease to contraception non-hormonal male contraception: a review and development of an eppin based contraceptive the control of male fertility by spermatid-specific factors: searching for contraceptive targets from spermatozoon's head to tail epididymal approaches to male contraception an integrated encyclopedia of dna elements in the human genome research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis expression profiles of human epididymis epithelial cells reveal the functional diversity of caput, corpus and cauda regions transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage dynamics of the transcriptome during human spermatogenesis: predicting the potential key genes regulating male gametes generation chromatin and single-cell rna-seq profiling reveal dynamic signaling and metabolic transitions during human spermatogonial stem cell development id levels dictate the stem cell state in mouse spermatogonia human sertoli cells support high levels of zika virus replication and persistence pharos: collating protein information to shed light on the druggable genome : knowledgebase for the laboratory mouse analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics high-throughput discovery of novel developmental phenotypes carcinoembryonic antigen-related cell adhesion molecule controls energy balance and peripheral insulin action in mice shp phosphatase-dependent t cell inhibition by ceacam adhesion molecule isoforms deletion of the carcinoembryonic antigen-related cell adhesion molecule (ceacam ) gene contributes to colon tumor progression in a murine model of carcinogenesis ceacam a-/-mice are completely resistant to infection by murine coronavirus mouse hepatitis virus a carcinoembryonic antigen-related cell adhesion molecule expressed specifically early in pregnancy in the decidua is dispensable for normal murine development the lim homeobox gene lhx is essential for mouse gonad formation generation and characterization oflhx -gfpcreert knock-in mouse line targeted germline modifications in rats using crispr/cas and spermatogonial stem cells spermatogenic cells of the prepuberal mouse. isolation and morphological characterization integrative characterization of germ cell-specific genes from mouse spermatocyte unigene library comparative and functional analysis of testis-specific genes genome engineering uncovers evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice reaching for high-hanging fruit in drug discovery at protein-protein interfaces pharmacological perturbation of cdk using selective cdk inhibition or degradation catalytic in vivo protein knockdown by small-molecule protacs hijacking the e ubiquitin ligase cereblon to efficiently target brd lessons in protac design from selective degradation with a promiscuous warhead assessing different e ligases for small molecule induced protein ubiquitination and degradation cell-penetrating peptides: years later, where do we stand? tat peptide-mediated cellular delivery: back to basics proteomics. tissue-based map of the human proteome single nucleotide polymorphisms: discovery of the genetic causes of male infertility a comprehensive gene mutation screen in men with asthenozoospermia malacards: an amalgamated human disease compendium with diverse clinical and genetic annotation and structured search genetic evaluation of patients with non-syndromic male infertility a systematic review on the genetics of male infertility in the era of nextgeneration sequencing genetics of male infertility: from research to clinic association study of single-nucleotide polymorphisms in faslg, jmjdia, loc , tex , brdt, or w , insr, and tas r genes with male infertility evaluation of candidate polymorphisms for association with oligozoospermia or azoospermia in a large cohort of men of european descent point-of-care whole-exome sequencing of idiopathic male infertility the biology of infertility: research advances and clinical challenges mutation screening of the fkbp gene and its association study with spermatogenic impairment in idiopathic infertile men efficient typing of copy number variations in a segmental duplication-mediated rearrangement hotspot using multiplex competitive amplification genetic defects in human azoospermia single-nucleotide polymorphisms of the prdm (meisetz) gene in patients with nonobstructive azoospermia two single nucleotide polymorphisms in prdm (meisetz) gene may be a genetic risk factor for japanese patients with azoospermia by meiotic arrest single-nucleotide polymorphisms in the human rad l gene may be a genetic risk factor for japanese patients with azoospermia caused by meiotic arrest and sertoli cell-only syndrome excess of rare variants in genes that are key epigenetic regulators of spermatogenesis in the patients with non-obstructive azoospermia mutations in sohlh gene associate with nonobstructive azoospermia human fam a (saxo ) is a microtubule-stabilizing protein specific to cilia and related structures human carboxylesterases: a comprehensive review structure and catalytic properties of carboxylesterase isozymes involved in metabolic activation of prodrugs human carboxylesterases and their role in xenobiotic and endobiotic metabolism the mammalian carboxylesterases: from molecules to functions heterologous expression, purification, and characterization of human triacylglycerol hydrolase hepatic carboxylesterase is essential for both normal and farnesoid x receptorcontrolled lipid homeostasis deficiency of carboxylesterase /esterase-x results in obesity, hepatic steatosis, and hyperlipidemia baculo-expression and enzymatic characterization of ces esterase lipid remodeling of murine epididymosomes and spermatozoa during epididymal maturation development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane regionalization and redistribution of membrane phospholipids and cholesterol in mouse spermatozoa during in vitro capacitation variation in the cholesterol/phospholipid ratio in human spermatozoa and its relationship with capacitation analysis of recombinant human semenogelin as an inhibitor of human sperm motility characterization of an eppin protein complex from human semen and spermatozoa antimicrobial activity of human eppin, an androgen-regulated, sperm-bound protein with a whey acidic protein motif the role of type ii transmembrane serine proteasemediated signaling in cancer delineation of proteolytic and non-proteolytic functions of the membrane-anchored serine protease prostasin mechanisms of hepatocyte growth factor activation in cancer tissues regulation of cell surface protease matriptase by hai is essential for placental development, neural tube closure and embryonic survival in mice three genes expressing kunitz domains in the epididymis are related to genes of wfdc-type protease inhibitors and semen coagulum proteins in spite of lacking similarity between their protein products sperm proteins sof , tmem , and spaca are required for spermoocyte fusion in mice identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice disruption of adam impairs the migration of sperm into oviduct in mouse male mice deficient for germ-cell cyritestin are infertile co-expression of sperm membrane proteins cmtm a and cmtm b is essential for adam localization and male fertility in mice factors controlling sperm migration through the oviduct revealed by gene-modified mouse models pseudogenes regulate parental gene expression via cerna network interspecific recombinant congenic strains between c bl/ and mice of the mus spretus species: a powerful tool to dissect genetic control of complex traits the sequence read archive cutadapt removes adapter sequences from high-throughput sequencing reads transcript-level expression analysis of rna-seq experiments with hisat, stringtie and ballgown systematic selection of reference genes for the normalization of circulating rna transcripts in pregnant women based on rna-seq data featurecounts: an efficient general purpose program for assigning sequence reads to genomic features edger: a bioconductor package for differential expression analysis of digital gene expression data crispr/cas mediated genome editing in es cells and its application for chimeric analysis in mice publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank drs. yumei li all data generated or analyzed during this study are included in this published article, its supplementary information files, and publicly available repositories. the sra values for each of the previously published reproductive and non-reproductive human rna-seq datasets [ , , , , , ] and previously published reproductive and non-reproductive mouse rna-seq datasets [ , , ] are listed in additional file : table s and additional file : table s . all raw and processed data for the new human and new mouse samples generated in this study is deposited in ncbi geo (accession gse ). all mice generated in this study, and any additional information about this study, are available from the corresponding authors upon request.ethics approval and consent to participate human tissue acquisition was approved by the ethics committee at université laval with written consent obtained from each respective donor's family. all animal experiments were approved by the institutional animal care and use committee (iacuc) at baylor college of medicine. additional file : fig. s . summary of number of statistically significant up and down-regulated genes, and quantification of candidate genes with respect to the individual reproductive tissue or cell of interest. the plots in panels (a) and (b) summarizes the number of statistically significant human or mouse genes respectively, that are up-regulated or downregulated in each reproductive tissue or cell of interest compared to the non-reproductive tissue with maximal gene expression. red columns depict the number genes that are up-regulated and blue columns depict the number genes that are down-regulated. changes in gene expression were considered statistically significant for an fdr of less than or equal to . . the total number of candidate genes are designated by the black columns. candidate genes are genes that passed the fdr and tpm expression value filters.additional file : fig. s . venn diagrams comparing the overlap between the candidate male reproductive genes identified by the indicated reproductive tissues. the human testis combined gene list is the list of genes from both new samples we isolated and from previously published testis samples. the human epididymis combined gene list is the list of genes identified in either previously published samples or the newly generated samples across all sections of the epididymis. lastly, the mouse epididymis combined gene list is the list combined list of genes identified across all three sections of the mouse epididymis.additional file : table s . complete cross-sample comparison identifying human and mouse reproductive tract specific genes common to two or more samples and unique to each as identified through our studies.additional file : fig. s . classification of genes into different protein families and identification of the existence of an experimental mouse model. each candidate human gene was classified as an enzyme (enzyme), chromosome and histone modifiers (epigenetic), g-proteincoupled receptor (gpcr), orphan g-protein-couple receptor (ogpcr), kinase (kinase), transcription factor (tf), nuclear receptor (nr), ion channel (ic), chromosome and histone modifying transcript factor (tf; epigenetic), transporter (transporter) and unknown (a). the total number of candidate genes identified in our search for mouse models were plotted. orange columns designate the number of candidate genes where a model was identified while yellow designates candidate genes where a model was not identified (b).additional file : table s . drug target type classification for human genes. genes are listed according to the tissue and/or cell that they were identified as reproductive tract-specific in.additional file : table s . availability of a mouse model for human genes with a mouse ortholog. genes are listed according to the tissue and/or cell that they were identified as reproductive tract-specific in.additional file : fig. s . one-hundred and forty-two previously identified human male reproductive tract-specific genes that remain without a reported mouse model. the listed genes were identified in one or more datasets as indicated in the venn diagram. underlined genes were also identified in our studies as reproductive tract-specific in mouse. genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation. genes written in dark red were identified in both testis (testis and/or testis cell) and in epididymis.additional file : fig. s . seventy-three human male reproductive tract-specific genes that each have a reported mouse model with male infertility phenotype. the listed genes were identified in one or more datasets as indicated in the venn diagram. underlined genes were also identified in our studies as reproductive tract-specific in mouse. genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation. genes written in dark red were identified in both testis (testis and/or testis cell) and in epididymis.additional file : fig. s . rt-pcr confirmation of reproductive tractspecificity in both humans (a) and mice (b). the genes listed in this figure were identified through our studies and previous studies, but currently remain without a reported mouse model. gapdh and hprt are included as housekeeping genes.additional file : fig. s . eighty-nine novel human genes without a mouse ortholog. the listed genes were identified in one or more datasets as indicated in the venn diagram. genes written in blue encode either enzymes, kinases, gpcrs, ogpcrs, transporters, transcription factors, or proteins involved in epigenetic regulation. genes written in dark red were identified in both testis (testis and/or testis cell) and in epididymis.additional file : fig. s . novel reproductive tract-specific human genes that do not have any equivalent mouse orthologs. these genes may serve as potential contraceptive targets, however functional validation would need to be carried out in another model organism than mouse, such as rat or marmoset, which do have orthologs to these genes. the digital pcr (heatmap) depicts the average transcripts per million (tpm) value per tissue per gene from the indicated human rna-seq datasets as processed in parallel through our bioinformatics pipeline. white = tpm, black ≥ tpm. the expression profile of the human housekeeping gene, gapdh, is included as reference. for data obtained from published datasets, superscript values reference the dataset publication as previously mentioned.additional file : table s . previously identified genes. genes previously identified as male reproductive tract-specific through high throughput gene expression studies using either microarrays or rna-seq [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the human ortholog to genes identified in mouse and rat studies is included.additional file : fig. s . developmental expression pattern of spint , spint , spint , pp d , and saxo in epididymis and testis of postnatal and adult mice. whole epididymides were used at postnatal days , , , and and epididymis segments (caput, corpus, and cauda) were used at postnatal days , , , and . whole testes were used at all time points. the housekeeping gene, hprt, was used as reference.additional file : fig. s . multi-channel fluorescence images of bilateral epididymis serial sections stained with custom rnascope probes targeting either spint , spint , or spint mrna (red) and dapi (blue). the position of caput (cap), corpus (cor), and cauda (cau) is labeled in the overview image (left column). the position of the magnification over the epididymis is the same for all three sections (right column).additional file : fig. s . representative periodic acid-schiff staining of spint and spint / knockout and littermate control (wild-type) testes and epididymis segments (caput, corpus, and cauda) at months of age.additional file : fig. s . representative periodic acid-schiff staining of ces a knockout and littermate control (wild-type) testes and epididymis segments (caput, corpus, and cauda) at months of age.additional file : fig. s . representative scanning electron microscopy images of spint / and ces a ko and littermate control mouse sperm.additional file : fig. s . representative periodic acid-schiff staining of pp d and saxo knockout and littermate control (wild-type) testes and epididymis segments (caput, corpus, and cauda) at months of age. all authors have no competing interests. key: cord- - b fep authors: zhang, xiao-jing; qin, juan-juan; cheng, xu; shen, lijun; zhao, yan-ci; yuan, yufeng; lei, fang; chen, ming-ming; yang, huilin; bai, liangjie; song, xiaohui; lin, lijin; xia, meng; zhou, feng; zhou, jianghua; she, zhi-gang; zhu, lihua; ma, xinliang; xu, qingbo; ye, ping; chen, guohua; liu, liming; mao, weiming; yan, youqin; xiao, bing; lu, zhigang; peng, gang; liu, minyu; yang, jun; yang, luyu; zhang, changjiang; lu, haofeng; xia, xigang; wang, daihong; liao, xiaofeng; wei, xiang; zhang, bing-hong; zhang, xin; yang, juan; zhao, guang-nian; zhang, peng; liu, peter p.; rohit, loomba; ji, yan-xiao; xia, jiahong; wang, yibin; cai, jingjing; guo, jiao; li, hongliang title: in-hospital use of statins is associated with a reduced risk of mortality among individuals with covid- date: - - journal: cell metab doi: . /j.cmet. . . sha: doc_id: cord_uid: b fep summary statins are lipid-lowering therapeutics with favorable anti-inflammatory profiles and have been proposed as an adjunct therapy for covid- . however, statins may increase the risk of sars-cov- viral entry by inducing ace expression. here, we performed a retrospective study on , patients with covid- in hubei province, china, among which , received statins. based on a cox model with time-varying exposure, as well as a mixed-effect cox model after propensity score-matching, we found that the risk for -day all-cause mortality was . % and . % in the matched statin and non-statin groups, respectively, with a hazard ratio . . these results imply the potential benefits of statin therapy in hospitalized subjects with covid- . further, they give support for the completion of on-going prospective studies and randomized controlled trials involving statin treatment for covid- , which are needed to further validate the utility of this class of drugs to combat the mortality of this pandemic. the coronavirus disease pandemic has profoundly affected the health and livelihood of millions of people worldwide at an unprecedented scale and speed. to date, there are no definitive treatments specifically targeted to sars-cov- infection for covid- therapy or prevention. moreover, the development of effective vaccines or new therapies for curing is time-consuming and likely well off in the future. thus, repurposing existing approved drugs to mitigate the severity of covid- has been viewed as a more cost-effective and time-sensitive strategy. statins are first-line lipid-lowering therapies with well-tolerated side effects, are low in cost and are broadly available worldwide, including in developing countries. the potent antiinflammatory and immunomodulatory effects of statins suggest they could be beneficial to counter coronoviral infections, including for sars- cov- (castiglione et al., ; dashti-khavidaki and khalili, ; fedson et al., ) . indeed, observational studies and randomized controlled trials (rcts) have demonstrated a significant protective effect of statins on improving proinflammatory cytokine release and immune cell functions among individuals with viral and bacterial pneumonia (fedson, ; papazian et al., ; pertzov et al., ; sapey et al., ) . a more recent report based on molecular docking analysis showed that statins might inhibit sars-cov- entry into host cells by directly binding the main protease of the coronavirus (reiner et al., ) . these data led to speculation regarding the potential therapeutic benefits of statins for the treatment of covid- (arabi et al., ; bifulco and gazzerro, ) . however, concerns have been raised regarding whether individuals on statins are at a greater risk for sars-cov- infection and covid- exacerbation, as this class of drugs has been shown to increase the expression of angiotensinconverting enzyme (ace ), the receptor for the virus, in lab animals (hoffmann et al., ; shin et al., ; tikoo et al., ; wang et al., b) . thus, direct clinical evidence is urgently needed to answer the question as to whether statin use is detrimental or beneficial in hospitalized individuals with covid- . in the clinical setting, statins are often prescribed along with renin-angiotensin-aldosterone system (raas) blockers, in particular, angiotensin-converting enzyme (ace) inhibitors and angiotensin ii receptor blockers (arbs), for subjects with hypertension or cardiac pathologies (ray et al., ) . remarkably, clinical applications of ace inhibitors and arbs for covid- also share a similar dilemma as statins treatment regarding the perceived contraindications of increasing ace expression versus anti-inflammation and cardio-protection (south et al., ) . our recent research has shown that individuals with covid- on ace inhibitors and arbs are at lower risk of -day all-cause mortality than those not treated with ace inhibitors or arbs (acei/arb) (zhang et al., a) . moreover, combination therapy of statins and arbs showed encouraging results in improving the survival of ebola-infected individuals (fedson et al., ) . however, the effects of such combination treatment in individuals with covid- have not been studied. to address these important clinical questions, we conducted one of the largest retrospective cohort studies to date -one involving , clinically confirmed cases of covid- -to determine the association of in-hospital use of statins with clinical outcomes. in the subgroup analysis, we further investigated the additional effects of combining acei/arb with statins on the clinical outcomes of covid- . the time-varying cox model, marginal structure model (msm), and propensity score-matching analysis consistently showed a lower risk of all-cause mortality of covid- in individuals with statin use versus statin nonuse. a total of , cases of confirmed covid- admitted in hospitals from hubei province, china, were included in this analysis. among them, , had in-hospital use of statins (statin group) and the remaining , had no statin treatment (non-statin group) (figure ) . the participants received statin treatment were older ( . versus . years of age, p < . ) and had higher prevalence of chronic medical conditions, including hypertension ( . % versus . %, p < . ), diabetes mellitus (dm) ( . % versus . %, p < . ), coronary heart disease ( . % versus . %, p < . ), cerebrovascular diseases ( . % versus . %, p < . ), and chronic kidney diseases ( . % versus . %, p < . ) than those without statin treatment ( table ) . chest ct revealed bilateral pulmonary lesions were more common in the statin group compared with that in the non-statin group ( . % versus . %, p < . ) ( table ) . larger proportions of subjects in the statin group showed increased neutrophil counts, procalcitonin levels, and d-dimer compared with the non-statin group ( table ). in addition to these inflammatory markers, abnormal serum biochemistry, including increased alt and ast levels and decreased estimated glomerular filtration rate (egfr), indicated more prevalent organ impairments in the participants with statin therapy compared to non-statin use ( table ) . the frequencies of individuals with increased low-density lipoprotein cholesterol (ldl-c) and total cholesterol (tc) levels were higher among the statin group versus the non-statin group at admission. in terms of the whole hospitalization period, lipid profiles were comparable between the two groups ( figure s ). days from onset of symptom to hospitalization and the median follow-up days were longer in the individuals on statin treatment compared to the non-statin group ( table ) . the absolute values of the median and interquartile range (iqr) of each laboratory examination were shown in table s . the laboratory values, including for c-reactive protein (crp), procalcitonin, d-dimer, ldl-c, tc, and creatine kinase (ck), had different reference ranges for hospitals, which are listed in table s . for subjects enrolled in the propensity score matching model (psm) that was conducted to minimize the differences in baseline characteristics, participants from the statin group were matched at a : ratio to , participants from the non-statin group. the baseline characteristics were comparable between the two groups, except the proportion of individuals with spo < % was lower in statin group than the non-statin group (table s ) . lipid profiles also were comparable during hospitalization between statin and non-statin groups after psm ( figure s ). among the participants on statin therapy, ( . %) of them had hypertension. of these participants with hypertension, were also treated with ace inhibitors or arbs regimen (statin+acei/arb group) for antihypertensive management, while were on nonacei/arb antihypertensive drugs (statin+nonacei/arb group) for antihypertensive therapy (figure ) . age and gender distribution were comparable between the two groups. the systolic blood pressure (sbp) levels and prevalence of dm were higher, while the prevalence of copd was lower, in individuals on statin+acei/arb, compared to those on the combination of statin and other types of antihypertensive drugs ( table s ). the absolute values of the median (iqr) of each laboratory examination were shown in table s . after psm, individuals in the statin+acei/arb group were matched at a : ratio to individuals in the statin+nonacei/arb group. the baseline characteristics were comparable between the two groups ( table s and s ). among the subjects received statin treatment, atorvastatin was the most frequently prescribed (accounting for . % of all the statins users), followed by rosuvastatin ( . % of statin users). statin treatment started at the day of hospital admission. the doses difference among statins were converted to an equivalent dose of atorvastatin ( table s ). the therapeutic duration for individuals in the statin group was . ( . - . ) days, with an atorvastatin equivalent dose at . ( . - . ) mg per day ( table ) . there were . % individuals also treated with acei/arb in the statin group compared to . % for the non-statin group ( table ). in the psm cohort, the percentage of individuals on acei/arb treatment was comparable between the statin and non-statin groups ( table ) . the distributions of different brands of statins, therapeutic duration, and daily equivalent dose of statin were comparable between the individuals on the statin+acei/arb group versus those on statin+nonacei/arb group ( table s ) . the median (iqr) day of starting acei/arb treatment was . ( . - . ) days after admission, and the median (iqr) therapeutic duration was . ( . - . ) days (table s ). in the psm cohort, the median (iqr) acei/arb starting time was . ( . - . ) days after admission, and the median (iqr) therapeutic duration was . ( . - . ) days (table s ). the incidence rate of death during a -day follow-up was . cases per person-days in the statin group versus . per person-days in the non-statin group ( in the psm cohort, the crude incidence of death in the statin group (ir, . cases per person-days; death rate . %) was markedly lower than that in the non-statin group (ir, . per person-days; death rate . %). due to the severe symptoms and comorbidities of subjects in the statin group, the matched non-statin group had more severe baseline symptoms and higher proportions of cardiovascular and metabolic comorbidities than the unmatched non-statin group. this might account for the increased death rate in statin nonusers after propensity matching. using a mixed-effects cox model without accounting for the time-varying exposures in the psm cohorts, statin treatment was also associated with a decreased incidence of death (ahr, . , %ci, . - . ; p = . ) ( table and figure ). to adjust for the potential bias from the competing medical issues-caused by a delayed start or discontinued use of statin therapy, we performed analysis using marginal structural model analysis in the individuals with and without statin treatment. in this model, the use of statins was maintained to be associated with a lower -day mortality (ahr, . ; % ci, . - . ; p = . ) than individuals with no use of statins (table ) . in a sensitivity test, we excluded individuals who were admitted to icu or died within hours after admission and performed the same analyses for the resting individuals. similar results were observed in the cox model with time-varying exposure (ahr, . , %ci, . - . ; p = . ), the msm analysis (ahr, . ; % ci, . - . ; p = . ), and the mixed-effects cox model in psm population (ahr, . , %ci, . - . ; p < . ) ( table s ) . we also conducted e-value analysis and found the point estimate of the primary endpoint was . in the mixed cox model. since the value was larger than the strong confounders, it is unlikely that unmeasured confounders would overcome the conclusion regarding statin use is not associated with increased day all-cause mortality among individuals with covid- . a recent retrospective report including covid- cases indicated that statin intake was significantly associated with the asymptomatic status of covid- with an unadjusted or of . (anton de spiegeleer, ). previous observational studies and meta-analyses have shown that statin treatment may be associated with reduced morbidity and mortality in individuals with sepsisassociated ards (mansur et al., ) . however, large rcts of individuals with ards have shown that neither atorvastatin nor simvastatin provided a significant benefit in overall mortality (mcauley et al., ; national heart, lung, and blood institute ards clinical trials network et al., ; papazian et al., ) . the discrepancy in the results between the observational and rcts studies may be due to differences in the heterogeneity of study populations, plausible selection bias, and measured/unmeasured confounders in the observation studies. further analyses of rcts data have shown the existence of subphenotypes in ards and differential response to statin treatment (calfee et al., ; sinha et al., ) . for instance, simvastatin was associated with improved survival in the hyper-inflammatory rather than the hypo-inflammatory subgroups, while atorvastatin therapy in the acute phase was not associated with improved survival in patients with sepsis but improved -day mortality when pretreatment (kruger et al., ) . these findings support efforts to examine the effect of statins in targeted subphenotypes of individuals and to pursue the approach to stratify patients in clinical trials. to minimize the potential bias in our study, we conducted various models and sensitivity tests to evaluate the reliability of our study results. individual accessibility of medical resources, survivor treatment selection bias (glesby and hoover, ) and competing medical issues (redelmeier et al., ) are three potential causes for bias in observational studies of treatment efficacy. in our study, individuals who received statins did not have earlier initiation of medical therapy or hospital admission than statin nonusers ( . days in the statin group versus . days in the non-statin group), implying unlikely imbalanced access to the health system-related lower risk of covid- mortality in the statin group. in addition, the covid- -associated medical expenses were covered by the chinese government during the pandemic, which largely reduced the impact of the socioeconomic level on in-hospital treatment. regarding the potential bias from survivor treatment selection, we performed a cox-model with time-varying exposure to adjust for potential bias caused by differences in the initial time of treatment. meanwhile, we calculated the average hospitalization period for individuals discharged in the statin and the non-statin groups which were similar ( . days versus . days), indicating unlikely prolonged benefit from hospital treatment in statin group. competing medical issues, such as clinicians being more likely to arrange urgent therapy in critically ill patients versus those less critically ill, could lead to a bias resulting in an association between not using statins with poor outcomes. therefore, we conducted an msm analysis adjusting time-varying confounders that simultaneously influences the time of statin initiation and risk of mortality. cox models with and without time-varying exposure and msm analysis were also performed in the patient populations excluding those who were admitted to the icu or died within hours after admission. despite our extensive efforts to utilize multiple models with rigorous controls, there still could be possible biases that can impact on the magnitude of the statin use-associated benefits on all-cause mortality among individuals with covid- , thus calling for the need of further validations of our conclusions via rct studies. encouragingly, there have been several rct studies started (nct , nct , nct , nct , nct , nct , and nct ). we are looking forward to the release of those results. among the subjects with hypertension, the incidence of -day mortality was . cases per person-days versus . per person-days in the statin+acei/arb group and the statin+nonacei/arb group, respectively, with the irr of . ( % ci, . - . ; p = . ) ( table ) . using a cox model with statin and acei/arb as time-varying exposures, there was no significant association between acei/arb therapy and -day mortality in individuals with hypertension and statin treatment (ahr, . ; % ci, . - . ; p = . ) ( table ) . although a mixed-effect cox model in psm cohorts showed acei/arb therapy treatment was associated with decreased incidence of death (ahr, . , %ci, . - . ; p = . ) ( table ) , the significant association did not appear in a marginal structure model (ahr, . ; % ci, . - . ; p = . ) ( table ) . in a sensitivity test, we excluded participants who were admitted to icu or died within hours after admission and performed the same analyses as described above for the resting subjects. similar results were found using the cox model with time-varying exposure, the msm analysis, and the mixed-effects cox model of the psm population (table s ). these results indicated that using acei/arb conferred neither additive beneficial nor detrimental effects in combination with statin treatment about to -day mortality among subjects with pre-existing hypertension. although the use of an ace inhibitor or arb was once speculated to be potentially harmful in patients with covid- (fang et al., ) , numerous observational studies have shown either a protective or neutral effect on mortality (mancia et al., ; zhang et al., b) . while more definitive results from rcts are forthcoming, several professional societies have recommended the continuation use of ace inhibitors and arbs in patients with covid- and pre-existing hypertension (esc, ; ish, ) . to our knowledge, the results from this study were the first clinical evidence supports the notion that the risk of covid- mortality was not increased by using ace inhibitors or arb in combination with statin treatment in individuals with covid- . in terms of secondary endpoints of covid- , we analyzed the association of statin use with incidences of invasive mechanical ventilation, icu admission, ards, septic shock, acute liver injury, acute kidney injury, and acute cardiac injury. after adjusted baseline differences, cox model analysis showed that statin usage was associated with a lower prevalence of using mechanical ventilation (ahr, . ; % ci, . - . , p < . ), icu admission (ahr, . ; % ci, . - . , p = . ) and ards (ahr, . ; % ci, . - . , p = . ) in individuals with covid- (table s ) . after matching baseline differences in two groups by psm, the statin group still showed a lower incidence of invasive mechanical ventilation compared to the non-statin group (ahr, . ; % ci, . - . , p = . ) ( table ) . statin therapy was not significantly associated with other secondary outcomes (e.g., acute kidney injury, liver injury, and cardiac injury) and increased serum ck or transaminase levels in the psm cohort (table ) . among the individuals with covid- and pre-existing hypertension, there were no significant associations between all observed secondary outcomes and statins with or without ace inhibitors or arbs treatment in unmatched subjects (table s ). after matching baseline characteristics, the associations between statin+acei/arb treatment and lower incidences of ards (ahr, . ; % ci, . - . ; p = . ) and cardiac injury (ahr, . ; % ci, . - . ; p = . ) compared to statin+nonacei/arb group were shown in mixed-effect cox model ( table ). there were no significant associations between the co-treatment of statin with acei/arb and other secondary outcomes or the raised serum ck or transaminase levels ( table ). given the putative anti-inflammatory and immunomodulatory effects of statins (castiglione et al., ; dashti-khavidaki and khalili, ; fedson et al., ) , we explored the changes of inflammatory markers in statin users with covid- . the dynamic changes of inflammatory factors in individuals with covid- with and without statin treatment during the -day of hospitalization were fitted using a locally-weighted regression and smoothing scatterplots (lowess) model. circulating crp, interleukin (il- ) and neutrophil counts were three inflammation biomarkers selected to represent the overall status of inflammation (nathan, ; vasileva and badawi, ; wang et al., a) . in subjects with matched baseline differences, the dynamic trajectories of crp showed a downward trend after admission in both groups, with lower levels among the statin users in the whole in-hospital duration ( figure a) . the il- in the statin group showed a lower level at admission and had a less increase than that of non-statin group in the entire duration of follow-up ( figure b) . meanwhile, the dynamic curve of neutrophil counts level showed a more significant downward trend in the statin groups than the non-statin group during hospitalization ( figure c) . furthermore, to eliminate any artifacts due to censoring or death, the analysis also conducted in participants alive. the tendencies were similar when individuals who died during -day of followup were excluded from each group (figure d-f) . the dynamic trajectories for circulating crp, il- , and neutrophil counts were also determined in statin users and statin nonusers before psm and found to show similar patterns to those after psm analysis ( figure s ). because individuals on statins were older and had a greater incidence of chronic diseases (table ) , the benefits of statins in suppressing circulating proinflammatory markers were less remarkable as in the matched cohort with comparable baseline characteristics. overwhelming inflammation response is a pathological hallmark of covid- -associated phenomena and ards and contributes to extrapulmonary organ damage (tay et al., ) . statins can reduce inflammation and the progression of lung injury in experimental models (fan et al., ) . mechanistic studies have shown that statins can suppress tlr /myd /nf-κb signaling and cause an immune response shift to an anti-inflammatory status (gallelli et al., ; yuan et al., ) . more recent evidence has shown statins having pleiotropic effects on nlrp inflammasome activation and cytokine releases in numerous disease conditions (henriksbo et al., ; satoh et al., ; xu et al., ) . during metabolic dysfunction, factors, such as oxidized ldl and advanced glycation end-product, promote nlrp inflammasome activation to magnify the inflammatory responses during pathogen infection (duewell et al., ; sheedy et al., ) . this response may underlie why patients with the metabolic disorder are prone to more severe complications of covid- . the potential benefit of statin on nlrp inflammasome might be also associated with improved outcomes in the setting of covid- . clinical data to date has been inconclusive as to the impact of statins on inflammatory mediators (mcauley et al., ; national heart, lung, and blood institute ards clinical trials network et al., ) . some earlier studies have reported that in bacterial infections or acute lung injury, inflammation mediators (e.g., tnf-α, il- , and crp) were significantly lower either in circulation or in bronchoalveolar lavage among subjects on simvastatin. however, due to a selfcontrol comparison study design and limited sample size, these results need to be interpreted with caution (craig et al., ; novack et al., ). an rct was conducted among patients from icu and treated with atorvastatin, found that the plasma level of il- was not significantly affected by atorvastatin therapy (kruger et al., ) . another study designed to explore the formation of neutrophil extracellular traps (nets), representing responsiveness of neutrophils to bacterial infection, showed that simvastatin treatment in patients with pneumonia resulted in altered formation. in this study, four days of simvastatin adjuvant therapy was associated with improvements in systemic neutrophil function (i.e., netosis and chemotaxis) (sapey et al., ). the different results from such studies may result from diverse disease conditions among the different trials and the heterogeneity in the target populations. thus, to address these concerns regarding the true effect of statins on inflammatory diseases, rcts with appropriate patient stratification will be required. the use of statins in hospitalized subjects with covid- was associated with a lower risk of allcause mortality and a favorable recovery profile. due to the nature of such retrospective studies, these results should be interpreted with caution, however, these data provide supportive evidence for the safety of statin or combination of a statin with acei/arb for treatment in patients with covid- . further rcts to prospectively explore the efficacy of statins on covid- outcomes are urgently needed. our study has several limitations. firstly, the inherent limitation of a retrospective study makes it impossible to infer causality in the association between the use of statins and acei/arb and the ameliorated severity and mortality in covid- . secondly, even if we used multiple statistical models to adjust for potential bias and performed a sensitivity analysis to show that the overall unmeasured confounders were unlikely to undermine our main conclusion, some unforeseen confounders (e.g., prehospital medication and socioeconomic status) may still potentially alter the magnitude of statin effects on all-cause mortality of covid- . thirdly, marginal structural models require the availability of time-varying data on each day surrounding the initiation of statin exposure. the data for time-varying confounders for each day were not fully available, and imputation for days with missing data would also lead to uncertain bias to the conclusion. however, participants started on statin treatment in a very early phase after admission, thus would minimize the impact of imputation. fourthly, the bmi in the statin and non-statin groups were comparable and thus was not adjusted in the following statistical analysis. moreover, due to the urgent status of covid- pandemic, bmi was not always measured and has a relatively high missing proportion. this might lead to an uncertainty of the impact of bmi on the associations between statins use and lower risk of all-cause mortality. fifthly, the role of different types of statins on covid- outcomes was not fully analyzed since the majority of the cases were taking atorvastatin and rosuvastatin, while the number of individuals taking other types of statins was relatively small. sixthly, the study population included only hospitalized subjects, so extrapolation of these conclusions to the general population with covid- -related complications in the non-hospital setting requires caution. g.s., x.m., q.x., p.p.l., and l.r. edited manuscript and provided valuable suggestions for study design and data analysis. j.x., y.w., j.c., j.g., and h.li. contributed equally, designed the project, edited manuscript, and supervised the study. all authors have approved the final version of this paper. the authors declare no competing interests a schematic overview illustrating participant enrollment and the exclusion and inclusion criteria. * participants without the medical history of hypertension or with hypertension but not taking antihypertensive medication were excluded from subgroup analyses. adjusted hr was calculated based on the mixed-effect cox model with adjustment of age, gender, and spo at admission. the % confidence intervals were represented by shaded regions. the table below the graph indicated accumulated numbers at risk, death, discharge, and loss of follow-up at each indicated time point. the number of "at-risk" was defined as the total number of individuals subtracting the number of "death" and the number of "loss of follow-up". participants in the "loss of follow-up" group were those still in hospital, but not meet the criteria for -days follow-up at the end of our study follow-up day. further information and requests for resources and reagents should be directed to the lead contact, hongliang li (lihl@whu.edu.cn). the study did not generate any new reagents or materials. the data and codes related to the findings of this study will be available from the corresponding author after publication upon reasonable request. the research team will provide an email address for communication once the data are approved to be shared with others. the proposal with detailed aims, statistical plan, and other information/materials may be required to guarantee the rationality of requirement and the security of the data. the patient-level data, but without names and other identifiers, will be shared after review and approval of the submitted proposal and any related requested materials. this retrospective, multi-centered study was conducted in hospitals in hubei province, china. a total of , participants diagnosed with covid- following who interim guidance and the new coronavirus pneumonia prevention and control program ( th edition) published by the national health commission of china were included (national health commission of china, ; world health organization, ). participants were admitted between december th , and april th , . the final date of the follow up was april th , . the study protocols were approved by the central ethics committee and were accepted or approved by each collaborating hospital. patient informed consent was waived by each ethics committee. among the original participants with covid- , participants with incomplete electronic medical records, aged less than or over years, with pregnancy or severe medical conditions, including acute lethal organ injury (i.e. acute coronary syndrome, acute stroke, and severe acute pancreatitis) were excluded. individuals with pre-existing hypothyroidism (fellstrom et al., ; national heart, lung, and blood institute ards clinical trials network et al., ) or contraindications for statins use including presented serum levels of ck or aminotransferase of more than five times of the upper limit of normal (uln) at admission were also excluded (fellstrom et al., ; national heart, lung, and blood institute ards clinical trials network et al., ) . to avoid the confounding effects from non-statin lipid-lowering drugs, participants taking statin combined with other lipid-lowering drugs or those taking non-statin lipid-lowering agents were excluded. the number of participants enrolled in this study from hospitals was listed in table s . to explore whether using acei/arb brings additional benefit for individuals with hypertension and taking statins, subjects without hypertension or not taking any antihypertensive medicine during hospitalization were excluded. the flowchart for patient inclusion was illustrated in figure . demographic and clinical characteristics, vital sign, laboratory tests, radiological reports, therapeutic interventions, and outcome data were extracted from electronic medical records using a standardized data collection, as described in the previous reports (lei et al., ; zhang et al., a; zhu et al., ) . the laboratory data included a routine blood test, serum biochemical markers reflecting liver injury, kidney injury, and cardiac injury, lipid profile, il- , crp, procalcitonin, and d-dimer were collected during hospitalization. in-hospital medication and life support intervention included the classification of the drugs, the dosage, the course of treatment, and using mechanical ventilation were also extracted from medical records. data were carefully reviewed and confirmed by an experienced physician team and were double-checked to guarantee the accuracy. the primary endpoint was defined as -day all-cause death. the secondary endpoints were the occurrence of ards, septic shock, acute liver injury, acute kidney injury, acute cardiac injury, invasive mechanical ventilation, and intensive care unit admission. ards and septic shock were defined according to the who interim guideline "clinical management of severe acute respiratory infection when novel coronavirus ( -ncov) infection is suspected". acute kidney injury was diagnosed by an elevation in serum creatinine level ≥ . ummol/l within hours (khwaja, ). acute cardiac injury was defined with serum level of cardiac troponin i/t (ctni/t) above the uln(huang et al., ; yancy et al., ) . acute liver injury was defined using serum alt or alkaline phosphatase above folds of uln (marrone et al., ) . the adverse effect of statin was determined by ck to increase above uln or alt increase above -folds of uln during follow-up (national heart, lung, and blood institute ards clinical trials network et al., ) . to test the association between in-hospital statin therapy and mortality, three statistical models were applied. one approach was cox proportional hazards regression model after propensity scorematching for baseline characteristics, but without considering immortal time bias or time-varying confounders; a second approach was cox proportional hazards regression model accounting for time-varying exposure that adjusted for baseline differences and accounts for immortal time bias (with statin or statin and acei/arb therapy as a time-varying exposure); a third approach was a marginal structural model that adjusts for baseline differences and accounts for indication bias (by examining the impact of time-varying confounders on the daily risk of prescription of statin or acei/arb) and immortal time bias (using statin or statin and acei/arb therapy as a time-varying exposure). to minimize baseline differences between statin and non-statin groups, we performed propensity score-matched analysis (psm). baseline matching variables included age, gender, pre-existing comorbidities (copd, dm, hypertension, coronary heart disease, cerebrovascular disease, chronic liver disease, and chronic kidney disease), indicators of disease severity and organ injuries (neutrophil counts increase, lymphocyte counts decrease, crp level increase, alt increase, creatine kinase [ck] increase, egfr < ml/min/ . m ) and ldl-c increase, cholesterol increase, and use of acei/arb. residual imbalance in spo between statin and non-statin group were further adjusted in the cox regression model. to match the differences between the individuals with statin combined with acei/arb treatment and statin combined with other types of antihypertensive treatment, we matched variables including age, gender, blood pressure (sbp and dbp), pre-existing comorbidities (copd, dm, coronary heart disease, cerebrovascular disease, chronic liver disease, and chronic kidney disease), indicators of disease severity and organ injuries (neutrophil counts increase, lymphocyte counts decrease, crp level increase, alt increase, creatine kinase [ck] increase, egfr decrease) and ldl-c increase, cholesterol increase, numbers of antihypertensive drugs, and spo . pre-existing coronary heart disease, crp increase, ldl-c increase, and d-dimer increase were remaining imbalanced variables after psm and were further adjusted in the cox regression model. we used nonparametric missing value imputation, based on the missforest procedure in the r, to account for the missing data on the laboratory variables of increased crp, ldl-c, egfr, alt, ck, bun, d-dimer and cholesterol as well as decreased lymphocyte counts (waljee et al., ) . a random forest model using the remaining variables in the data set was performed to predict the missing values for chest ct lesions and decreased spo . the internally cross-validated errors were also estimated. statin users and nonusers were paired according to the propensity scores using exact matching with a caliper size of . . the balance of covariates was evaluated by estimating standardized differences before and after matching, and a small absolute value of less than . was considered qualified balancing between the two groups. for the mixed cox analysis, the statin versus non-statin group ratio was paired at : . in subgroup analysis, ratio was paired at : for statin+acei/arb versus statin+nonacei/arb group. the caliper size in the subgroup cohort was . according to the propensity scores. the risk of primary and secondary endpoints and corresponding hazard ratio (hr) were calculated using the cox proportional hazard model comparing the statin group versus the non-statin group and the statin+nonacei/arb group versus the statin+nonacei/arb group. in the cox analysis, individuals discharged were treated as " -at risk" but not censored data for two major reasons. first, individuals with covid- would not be discharged only if their symptoms significantly relieved with continuous viral pcr negative two times. second, individuals discharged from hospitals had another -week of quarantine. any death occurred would be documented. thus, discharged individuals were unlikely to die due to covid- and their survival information was still available after discharge. regression adjustment was applied to remove post-psm residual confounding bias where it included the covariates with a standardized difference greater than . . multi-variable adjusted residual imbalances including age, gender, and spo were performed when analyzing the association between statin treatment and clinical outcomes. pre-existing coronary heart disease, crp, ldl-c, and d-dimer were further adjusted when analyzing the association between acei/arb treatment and clinical outcomes in subjects with statin treatment. we modeled the site as a random effect in the mixed-effect cox model. the proportional hazard assumptions were verified using correlation testing based on the schoenfeld residuals. to examine endpoints as a time to mortality in the statin and the non-statin group, we performed a cox proportional hazards model adjusting for age, gender, blood pressure (sbp and dbp), preexisting comorbidities (dm, hypertension, coronary heart disease, cerebral arterial disease, and chronic kidney disease), indicators of disease severity and organ injuries (lesions in chest ct, neutrophil counts increase, procalcitonin increase, d-dimer increase, alt increase, ast increase, creatinine increase, and spo ), ldl-c increase, cholesterol increase, medications at admission, using invasive mechanical ventilation support, and days from symptom onset to hospitalization covariates with statin therapy as a time-varying exposure. when analyzing the association between statin combined with or without acei/arb and mortality accounting for time-varying exposure, we adjusted for age, gender, pre-existing comorbidities (copd and dm), sbp, medications at admission, using invasive mechanical ventilation support covariates with statin and acei/arb therapy as time-varying exposures. changes in patient conditions influenced the initiation or termination of statin therapy or combined treatment of statin and acei/arb. we performed a marginal structural model (msm) analysis with inverse probability of treatment weighting (iptw) to account for time-varying confounders. when analyzing the association between statin use and mortality in participants with covid- , timevarying confounders are factors that influence the statin therapy initiation and correlated with the risk of mortality. in this analysis, curb- pneumonia severity score (including confusion, blood urea nitrogen, respiratory rate, sbp, and age) (table s ), serum alt levels and ck levels were considered as time-varying confounders, which reflected the patient conditions that might impact clinical decision on initiating statin therapy. baseline characteristic, including age, gender, blood pressure (sbp and dbp), pre-existing comorbidities (dm, hypertension, coronary heart disease, cerebrovascular disease, and chronic kidney disease), indicators of disease severity and organ injuries (lesions in lung ct, neutrophil counts increase, procalcitonin increase, d-dimer increase, creatine increase, and spo ), ldl-c increase, cholesterol increase, medications at admission, using invasive mechanical ventilation support, and days from symptom onset to hospitalization were adjusted in the model. alt level, ck level, and crub- score in days with missing values were imputed by the last-observation-carried-forward approach through the linear mixed-effects model. before imputation, alt and ck levels were standardized through dividing by the upper limit of the reference value of the corresponding institution. when analyzing the association between acei/arb use and mortality in the statin treated patients, time-varying confounders were factors that could influence the acei/arb therapy initiation and correlated with the risk of mortality. in this analysis, curb- pneumonia severity score, serum creatinine, and alt levels were considered as time-varying confounders, which reflected the patient conditions that could impact clinical decision on initiating aceii/arb therapy. imbalanced baseline characteristics, including gender, pre-existing copd and dm, medication at admission, and use of mechanical ventilation and the number of antihypertensive drugs were adjusted in the model. curb- score was assessed every day during hospitalization. serum creatinine level, alt level, and curb- score in days were imputed by the last-observationcarried-forward approach. the treatment selection weights were calculated to evaluate the probability of a patient to receive statin therapy at a specific time k. the weights were updated until the first day of statin therapy and kept constant afterward. the censoring weights were calculated for early patient dropout. the finally stabilized weights were calculated by multiplying the treatment selection weights and the censoring weights. the time-varying intercept was modeled by a smoothing function of time, using restricted cubic splines. then a generalized additive model was performed to estimate the effect of statin use on results, with age and gender adjusted. the marginal structural cox proportional hazards model was performed incorporating the stabilized weights to estimate the effect of statin therapy on clinical outcomes and side effects. we modeled the probability of receiving statin therapy with the assumption that once the patient was started on statin therapy the patient will remain on that treatment. the e-value analysis was conducted to assess the robustness of the association between statin use and all-cause mortality in the cox models to address potential unmeasured confounding effect, using the methodology of vanderweele and ding (haneuse et al., ; mathur et al., ; vanderweele and ding, ) . the e-value is an alternative approach to sensitivity analyses for unmeasured confounding in our studies that avoids making assumptions that, in turn, require subjective assignment of inputs for some formulas. because critically ill patients at admission were less likely to receive statin treatment, this bias could lead to an association between not using statins with poor outcomes. we performed a sensitivity analysis using cox models with and without time-varying exposure and marginal structural model in patient population not including those who admitted to icu or died within hours after admission. longitudinal covariates and baseline confounders were also adjusted as same as the full cohort. variables were used for matching in propensity-score matched analysis and for adjusting in cox analysis at admission. to account for the missing data on the laboratory variables, we used nonparametric missing value imputation, based on the missforest procedure in the r (waljee et al., ) . a random forest model based on the rest of the variables in the data set was constructed to predict the missing values with an estimation of the internally cross-validated errors. continuous variables with non-normal distributions were expressed as median [iqr] . categorical variables were expressed as number and percentage (%). comparisons between groups were performed with mann-whitney u test for nonparametric variables and fisher's exact test or χ test for categorical variables. person-time data (incidence) of two groups with different exposures may be expressed as a difference between incidence rates or as a ratio of incidence rates (irrs). the irrs of endpoint outcomes were calculated to estimate the incidence difference in absolute change in the incidence of two comparison groups. the cumulative rates of death were compared using the kaplan-meier curves. dynamic changes of inflammatory factors tracking from day to day after admission were depicted using the lowess model. a two-side α less than . was considered to define statistical significance. data were analyzed in r- . . (r foundation for statistical computing, vienna, austria) and spss statistics (version . , ibm, armonk, ny, usa). . sbp, systolic blood pressure; dbp, diastolic blood pressure; bmi, body mass index; copd, chronic obstructive pulmonary disease; alt, alanine transaminase; ast, aspartate transaminase; egfr, estimated glomerular filtration rate; ldl-c, low density lipoprotein cholesterol; tc, total cholesterol; ck, creatine kinase; spo , oxygen saturation; iqr, interquartile range. a upper limit of normal (uln) was defined according to criteria in each hospital. b p values were calculated by mann-whitney u test for non-normally distributed continuous variables and fisher's exact test or χ test for categorical variables. . . ( . - . ) h . ir( person-day), incidence rate; irr, incidence rate ratio; ahr, adjusted hazard ratio; ci, confidence interval; acei, angiotensin-converting enzyme inhibitor; arb, angiotensin ii receptor blocker. a there were and participants in unmatched statin and non-statin groups, respectively. after psm with a : ratio, there were and participants in the matched statin and non-statin groups, respectively. b there were and participants in unmatched statin+acei/arb and statin+nonacei/arb groups, respectively. after psm with a : ratio, there were and participants in the matched statin and non-statin groups, respectively. c adjusted for age, gender, blood pressure (sbp and dbp), pre-existing comorbidities (dm, hypertension, coronary heart disease, cerebrovascular disease, and chronic kidney disease), indicators of disease severity and organ injuries (lesions in chest ct, neutrophil counts increase, procalcitonin increase, d-dimer increase, alt increase, ast increase, creatinine increase, and spo ), ldl-c increase, cholesterol increase, medications at admission, using invasive mechanical ventilation support, and days from symptom onset to hospitalization. d adjusted for age, gender, blood pressure (sbp), pre-existing comorbidities (copd and dm), medications at admission, using invasive mechanical ventilation support covariates with statin and acei/arb therapy as time-varying exposures. e curb- pneumonia severity score, serum alt levels, and ck levels were considered as time-varying confounders. additionally, the adjustment factors included age, gender, blood pressure (sbp and dbp), pre-existing comorbidities (dm, hypertension, coronary heart disease, cerebrovascular disease, and chronic kidney disease), indicators of disease severity and organ injuries (lesions in chest ct, neutrophil counts increase, procalcitonin increase, d-dimer increase, ast increase, creatinine increase, and spo ), ldl-c increase, cholesterol increase, medications at admission, using invasive mechanical ventilation support, and days from symptom onset to hospitalization. f curb- pneumonia severity score, serum alt levels, and creatinine levels were considered as time-varying confounders. additionally, the adjustment factors included age, gender, pre-existing copd, medication at admission, and use of mechanical ventilation and the number of antihypertensive drugs. g ahr was calculated based on mixed-effect cox model with adjustment of age, gender, and spo at admission. h ahr was calculated based on mixed-effect cox model with adjustment of age, gender, coronary heart disease, and incidence of increased crp, d-dimer, and ldl-c at admission. i p values were calculated by r package "fmsb". the significant probability of the result of null-hypothesis testing. ir( person-days), incidence rate; irr, incidence rate ratio; ahr, adjusted hazard ratio; ci, confidence interval; icu, intensive care unit; ards, acute respiratory distress syndrome; ck, creatine kinase; alt, alanine transaminase; uln, the upper limit of normal. a adjusted hr was calculated based on mixed-effect cox model with adjustment of age, gender, and spo on admission b adjusted hr was calculated based on mixed-effect cox model with adjustment of age, gender, coronary heart disease, and incidence of increased crp, d-dimer, and ldl-c on admission. c p values were calculated by r package "fmsb". the significant probability of the result of null-hypothesis testing. figure time(days) adjusted hr here, zhang et al. retrospectively analyzed , covid- cases and found that inhospital statin use is associated with a lower risk of all-cause mortality. by zhang et al. table s . absolute values of laboratory indicators on admission in statin and non-statin groups before and after psm table s . the reference range of laboratory indicators in study hospitals table s . characteristics of patients in statin and non-statin groups after psm table s . characteristics of patients in statin+acei/arb and statin+nonacei/arb groups table s . absolute values of laboratory indicators on admission in statin+acei/arb and statin+nonacei/arb groups before and after psm leukocyte count, median(iqr), ^ /l . ( . - . ) . ( . - . ) < . . ( . - . ) . ( . - . ) - . . neutrophil count, median(iqr), ^ /l . ( . - . ) . ( . - . ) < . . ( . - . ) . ( . - . ) - . . lymphocyte count, median(iqr), ^ /l . ( . - . ) . ( . - . ) . . ( . - . ) . ( . - . ) . . c-reactive protein, median(iqr), mg/l . ( . - . ) . ( . - . ) . leukocyte count, median(iqr), ^ /l . ( . - . ) . ( . - . ) . . ( . - . ) . ( . - . ) - . . neutrophil count, median(iqr), ^ /l . ( . - . ) . ( . - . ) . . ( . - . ) . ( . - . ) - . . lymphocyte count, median(iqr), ^ /l . ( . - . ) . ahr, adjusted hazard ratio; ci, confidence interval; acei, angiotensin-converting enzyme inhibitor; arb, angiotensin ii receptor blocker. a adjusted for age, gender, blood pressure (sbp and dbp), pre-existing comorbidities (dm, hypertension, coronary heart disease, cerebrovascular disease, and chronic renal disease), indicators of disease severity and organ injuries (lesions in chest ct, wbc increase, neutrophil counts increase, procalcitonin increase, d-dimer increase, alt increase, ast increase, creatinine increase, spo , ldl-c increase, cholesterol increase, medications at admission, using invasive mechanical ventilation support, and days from symptom onset to hospitalization support covariates with statin therapy as time-varying exposures. b adjusted for age, gender, blood pressure (sbp), pre-existing comorbidities (copd and dm), medications at admission, using invasive mechanical ventilation, numbers of antihypertensive drugs support covariates with statin and acei/arb therapy as time-varying exposures. c curb- pneumonia severity score, serum alt levels, and ck levels were considered as time-varying confounders. additionally, the adjustment factors included gender, blood pressure (sbp and dbp), pre-existing comorbidities (dm, coronary heart disease, cerebrovascular disease, and chronic renal disease), indicators of disease severity and organ injuries (lesions in chest ct, wbc increase, neutrophil counts increase, procalcitonin increase, d-dimer increase, ast increase, creatinine increase, and spo ), ldl-c increase, cholesterol increase, medications at admission, using invasive mechanical ventilation support, and days from symptom onset to hospitalization. d curb- pneumonia severity score, serum alt levels, creatinine levels were considered and statin therapy as time-varying confounders. additionally, the adjustment factors included gender, pre-existing copd and dm, medication at admission, and use of mechanical ventilation and the number of antihypertensive drugs. e ahr was calculated based on mixed-effect cox model with adjustment of age, gender, blood pressure (sbp and dbp), pre-existing comorbidities (dm, hypertension, coronary heart disease, cerebrovascular disease, and chronic renal disease), indicators of disease severity and organ injuries (lesions in chest ct, the incidence of increased wbc, neutrophil counts, alt, ast, procalcitonin, d-dimer and spo , decreased egfr), ldl-c increase, cholesterol increase. f ahr was calculated based on mixed-effect cox model with adjustment of age, gender, blood pressure (sbp), pre-existing copd and dm. c-reactive protein; ldl-c, low density lipoprotein cholesterol bmi, body mass index; copd, chronic obstructive pulmonary disease ast, aspartate transaminase; egfr, estimated glomerular filtration rate; ldl-c, low density lipoprotein cholesterol uln) was defined according to criteria in each hospital. b the statin+acei/arb and statin+nonacei/arb groups were matched based on variables including age, gender, blood pressure (sbp and dbp), pre-existing comorbidities (copd, dm, coronary heart disease, cerebral arterial disease, chronic liver disease, and chronic renal disease), indicators of disease severity and organ injuries (neutrophil counts increase, lymphocyte counts decrease, crp level increase, egfr decrease) and ldl-c increase ir( person-days), incidence rate irr, incidence rate ratio ards, acute respiratory distress syndrome cox model with adjustment of age, gender, blood pressure (sbp and dbp), pre-existing comorbidities (dm, hypertension, coronary heart disease, cerebrovascular disease, and chronic renal disease), indicators of disease severity and organ injuries (lesions in lung ct, the incidence of increased neutrophil counts, alt, ast, procalcitonin, d-dimer and spo , decreased egfr), ldl-c increase, cholesterol increase. b ahr was calculated based on mixed-effect cox model with adjustment of age, gender, blood pressure (sbp), pre-existing copd, and dm. c p values were calculated by r package"fmsb key: cord- -n ezxnjc authors: bertasio, cristina; giacomini, enrico; lazzaro, massimiliano; perulli, simona; papetti, alice; lavazza, antonio; lelli, davide; alborali, giovanni; boniotti, maria b. title: porcine epidemic diarrhea virus shedding and antibody response in swine farms: a longitudinal study date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: n ezxnjc the porcine epidemic diarrhea virus (pedv) causes an acute and highly contagious enteric disease characterized by severe enteritis, vomiting, watery diarrhea, and a high mortality rate in seronegative neonatal piglets. in the last few years, ped had a large economic impact on the swine industries in asia and the us, and in , the pedv also re-emerged in europe. two main pedv variants circulate worldwide but only the s indel variant, considered a mild strain, is spreading in europe. to gain insights into the pathogenicity of this variant, its viral load and temporal shedding pattern were evaluated in piglets from infected farms. quantitative real-time pcr (qpcr) targeting the spike gene, was validated according to the minimum information for quantitative real-time pcr experiments guidelines. the qpcr was applied to longitudinal studies conducted in four swine farms naturally infected with the pedv s indel variant. clinical data, fecal swabs, and blood samples were collected from piglets at – -day intervals for – months. on all four farms, diarrhea was observed in sows during gestation and in farrowing units, and the mortality rates of piglets were , , , and %. different clinical pictures ( - % of diarrhea positivity), viral titer levels (mean . - . log( ) genome copies/ml), and antibody conditions ( - % of positivity) were registered among sows on the four farms. the percentage of qpcr positive piglets varied greatly from the beginning ( – %) to the end ( %) of the infection course. clinical signs were present in % of the qpcr positive animals. viral loads ranged from . log( ) to log( ) genome copies/ml in suckling pigs at – days of age and were not statistically different among farms, despite the different patterns observed in sows. after – weeks, only a few piglets still showed detectable viral levels and clinical signs, and they developed antibody responses. moreover, co-infections with other pathogens and biosecurity procedures limiting the circulation of the virus could have influenced the severity of ped infection. qpcr and clinical data were useful in understanding the dynamics of pedv infections and, therefore, in implementing appropriate control measures. porcine epidemic diarrhea virus (pedv) causes an acute and highly contagious enteric disease, which is characterized by severe enteritis, vomiting, watery diarrhea and a high mortality rate in neonatal piglets. belonging to the family coronaviridae, genus alphacoronavirus, pedv has a single-stranded, positive-sense rna genome of ∼ kb that encodes four structural proteins, spike (s), envelope, membrane, and nucleocapsid, and three nonstructural proteins (kocherhans et al., ) . in particular, the s protein is important in regulating interactions with specific cell receptor glycoproteins to mediate viral entry, inducing neutralizing antibodies (bosch et al., ) , growth adaptation in vitro and in virulence attenuation in vivo (sato et al., ) . since , ped has caused large economic losses in the swine industries of asia and north america (usa and canada) (stevenson et al., ; kochhar, ) . based on the nucleotide sequence of the s spike gene (lee, ) , two main genetic variants have been detected: a "highly virulent" strain, called "non-s indel (insertions and deletions)" (vlasova et al., ; sun et al., ) , and a "mild" strain, called "s indel", identified in pigs with mild clinical signs and no mortality (vlasova et al., ; wang et al., ) . since , in europe, the non-s indel pedv strain has been detected only in the ukraine (dastjerdi et al., ) , while the s indel strain has spread throughout many countries including germany, france, belgium, portugal and italy (grasland et al., ; hanke et al., ; mesquita et al., ; theuns et al., ; boniotti et al., ) . all of the s indel pedv european strains share % nucleotide identity with s indel pedv oh , but in contrast with field observation in the usa , outbreaks with high mortality rates in suckling piglets have been reported in germany and portugal (mesquita et al., ) . in italy, ped has been documented since , with a few cases appearing per year. however, between and , a severe pedv epidemic occurred in italy (martelli et al., ) , which was characterized by mortality rates in neonatal piglets of up to %, whereas it was very low in adults. during - , only sporadic confirmed clinical cases of ped were reported (boniotti et al., ) . during this period, two swine coronavirus clades were identified. the first resembled the oldest global pedv strain cv (pedv/italy/ / ) and the second resembled a new transmissible gastroenteritis coronavirus/pedv recombinant variant (secov/italy/ / ). during summer , animals on two farms displaying mild clinical signs were detected as positive for pedv by pcr (boniotti et al., ) , and at the beginning of a new severe epidemic wave occurred (efsa ahaw panel, ) . recently, the virulence of the s-indel strain has been evaluated and compared to the non-s indel strain through experimental infections (lin et al., ; yamamoto et al., ; chen et al., ) . in one study, -day-old piglets inoculated with an s indel pedv strain (usa/il/ / ) did not develop clinical signs and had only mild histopathological lesions (chen et al., ) . lin et al. ( ) showed that the virulence of the s indel pedv strain was lower than the non-s indel us pedv based on a longer incubation time, a shorter duration of diarrhea, a lower percentage of infected enterocytes and a lower piglet mortality rate. however, the severity of clinical signs and mortality rates ( - %) varied greatly among litters inoculated with the s indel strain. these variations were associated with piglet birth weights and the sow's health and lactation status (lin et al., ) . to evaluate differences in morbidity and the virulence of the s indel strains, field data are extremely important, as many concurrent factors, not reproducible in an experimental infection, can determine the infection severity. presently, the limited available field observations mesquita et al., ; stadler et al., ) indicated that the virulence level of s indel pedv varied greatly. the different immunological states of infected piglets, due to lactogenic immunity, is a likely explanation of such variations. viral load is an important indicator in evaluating the virulence of a strain and the susceptibility of infected animals, and in understanding the mechanisms of viral transmission and circulation within the different farm units. at the moment, very limited data have been published on the pedv viral load during an outbreak under field conditions (bjustrom-kraft et al., ) . in this study, we describe acute outbreaks of ped in three farrow-to-finish and one farrow-to-wean farms in northern italy. we conducted a longitudinal study by sampling the feces and blood of piglet groups from each farm at fixed intervals during a - months period, and then we determined pedv shedding and the antibody presence. in particular, the quantification of pedv in fecal samples was used to better describe the infection dynamics under field conditions. three farrow-to-finish and one farrow-to-wean farms located in the north of italy were chosen for this study, which occurred from january to may . farms were selected based on the following criteria: location in the high density swine production area of the po valley, a short distance from the diagnostic laboratory to facilitate the conservation of samples and quick delivery, sudden onset of enteric clinical signs, mortality in newborn piglets clearly referable to ped and an absence of an anamnesis of ped on the farm. in table , data on the four farms are summarized. the ped clinical and epidemiological characteristics, as well as the disease course, for the four farms are recorded in supplementary table . at each sampling time, clinical evaluations of the farms were determined by vets from our institute, recording the percentages, in four ranges, low ( - %), medium ( - %), high ( - %), and very high (> %), of diarrheic pigs among the different farm units (supplementary table ). fecal consistency was visually evaluated and scored using the following criteria: = normal, = formed and soft, = semi-solid, = watery, and = presence of mucus and blood. the maximum observed fecal score attributed to each farm unit was recorded at each sampling time and reported in supplementary table . at - days from the first appearance of clinical signs on the four farms (f −f ), sows per farm, and , , , and newborn piglets from f , f , f , and f , respectively, were selected from symptomatic litters, identified by ear tag and sampled every weeks (samplings - ), − weeks (samplings − ) and after weeks (sampling ). the number of sampled animals decreased as the study continued due to mortality and animal sales (supplementary table ). the sows were sampled only once at the beginning of the study. the presence of diarrheic piglets was recorded at each sampling time (supplementary table ). fecal and blood samples were collected from sows, whereas blood and rectal swabs were collected from piglets. feces were diluted : (w/v) in minimum essential media (mem). rectal swabs were suspended in ml of mem, vortexed and incubated at • c for min to allow the release of the feces from the cotton. fecal suspensions were clarified by centrifugation for min at , × g to eliminate fecal debris. viral rna was extracted from µl of sample using a commercial kit (nucleomag r vet kit, macherey-nagel, düren, germany), according to the manufacturer's instructions. an exogenous internal control rna (ic) (qiagen, hilden, germany), was added to specimens prior to rna extraction to verify the success of the procedure and the absence of inhibitors. the extraction was carried out on the biosprint instrument (qiagen) using the nucleomag vet protocol. nucleic acids were eluted into µl of elution buffer and immediately subjected to rt-pcr or stored at − • c until used. extracted viral rna was subjected to a "one-step" rt-pcr assay using the commercial quantifast pathogen rt-pcr kit (qiagen) with primers and probe targeting the s gene of pedv that were previously developed at the university of minnesota veterinary diagnostic laboratory ( ), and are reported in table . the optimum concentrations of primers and probe were deduced by titration experiments and are reported in table . pcr reactions were performed on µl of extracted viral rna in a final volume of µl, which also contained µl of × pcr-master mix, . µl of a × internal control assay, . µl of each forward and reverse primer (final concentration nm), . µl of ped_s probe (final concentration nm), and . µl of enzyme mix. the pedv rna was reverse transcribed at • c for min, followed by cycle of taq polymerase activation at • c for min. amplification consisted of cycles at • c for s and • c for s. amplification were performed on a cfx touch real-time pcr detection system (bio-rad laboratories) and data were analyzed with the software bio-rad cfx manager . , using the single threshold method for the cq determination. an experiment was accepted when the cq of the "no template control" (ntc) was > and the ic of a negative control was < . we cloned a fragment of the s gene (nucleotide positions - ; genbank dq . ) into a pcr r . -topo r vector (topo ta cloning r kit, invitrogen) according to the manufacturer's instructions. plasmid dna was linearized by restriction enzyme digestion and then subjected to transcription using the ribomax large scale rna production system (promega) to produce an rna transcript. the rna concentration was determined using an infinite r nanoquant spectrophotometer (tecan). a -fold serial dilution in nuclease-free water of ssrna transcripts ( × - × copies/µl) was used to generate a standard curve and to quantify pedv rna in the samples. triplicates of each dilution were run in each assay. the following equation: where x represents the genome copies/µl, was used to transform the samples' cq values into estimates of genome copies of pedv rna per ml of fecal homogenate. the qpcr assay used to quantify pedv rna was validated according to the minimum information for quantitative realtime pcr experiments guidelines (bustin et al., ) . validation results are summarized in table . to ascertain the specificity of the qpcr used in this study, we tested eight samples that were negative for pedv and positive for different viral agents, including the limit of detection (lod) and limit of quantification (loq) were determined by testing replicates of a -fold serial dilution of ssrna transcript, from an initial concentration of inhibition assay . cycles r , linear correlation index; lod, limit of detection; loq, limit of quantification. × to a final concentration of × − genome copies/µl (supplementary table ). the lod was calculated as the lowest concentration at which % of the positive samples are detected. the loq was defined as the lowest concentration of viral rna that can be determined with acceptable precision (relative standard deviation ≤ %) under the stated conditions of the test. the linear correlation index (r ) and the slope of the calibration curve were calculated using mean values from three replicates of four different runs. the reaction efficiency (e) under our experimental conditions was determined by the following formula: the dynamic range was determined by testing three replicates of a -fold serial dilution of ssrna transcript, from an initial concentration of × to a final concentration of × genome copies/µl (supplementary figure ) . repeatability was evaluated by analyzing three pedv positive samples in triplicate in the same extraction and qpcr run (supplementary table ). intra-assay variance was expressed as the range of the relative standard deviation (rsd%) associated with copy number/µl. reproducibility was determined by testing three pedv-positive samples having different concentration levels. samples were extracted and quantified on four different days (supplementary table ). inter-assay variance was expressed as mean rsd% with minimum and maximum values of copy numbers at each concentration level, calculated for the different concentration levels. to verify the absence of contaminants that can cause the inhibition of qpcr assays after the rna extraction procedure, the extracted rnas of specimens were diluted : (v/v) in water and subjected to qrt-pcr. inhibition was evaluated by calculating the differences in mean cq values between diluted and undiluted samples. the s gene sequences of pedv positive samples on farm , were obtained from one sow and one piglet at the first sampling and from three piglets at days of age, as described by boniotti et al. ( ) (accession number: ky -ky ). an in-house pedv whole virus elisa was used. in brief, a pedv strain cv -based elisa was developed and validated at izsler based on the previously described double-antibody sandwich elisa protocol (sozzi et al., ) . the elisa microplates were coated with the f capture monoclonal antibody (mab). serum samples diluted : or : were mixed with equal volumes of whole pedv inactivated with ß-propiolactone and pre-incubated in an auxiliary microplate for h at • c. then, µl of the pre-incubated mixtures were transferred into the f mab-coated plate and the conjugated horseradish peroxidase mab c was added. following a further h incubation at • c, the plate was washed. the colorimetric reaction was performed, and optical densities (od) were measured at nm using an elisa plate reader. results were calculated by determining the absorbance value reduction, expressed as percentage of inhibition (pi) using the control wells as the reference. the antibody-blocking reaction was considered positive if the pi was ≥ %. for differential diagnoses and to investigate concomitant infections, fecal and serum samples were further investigated to detect transmissible gastroenteritis coronavirus, porcine deltacoronavirus, swine rotavirus a, b, c, and h, porcine reproductive and respiratory syndrome virus; influenza a virus, escherichia coli, clostridium perfringens, and salmonella typhimurium, as previously described (kim et al., ; marthaler et al., a,b) . statistical analyses were performed on quantitative data from fecal specimens of the first sampling both in sows and piglets, using the kruskal−wallis test and dunn's multiple comparisons test (graphpad instat prism . software). at the beginning of the s indel pedv epidemic wave in northern italy (january ), four farms with symptoms referable to ped were selected for a longitudinal study. farm production type, onset of outbreak, mortality rate in suckling pigs and sampled animals for each farm, were described in table table ). watery diarrhea with presence of mucus and blood was observed in piglets of the four farms, but it was also present in gestation and farrowing sows. growing and fattening animals from f , f , and f also showed severe clinical signs including watery diarrhea and anorexia. vomiting was observed in fattening animals on f . dehydration was observed in litters on f and f and cachexia in litters on f and f . agalactia was present in the sows from f , f , and f . for differential diagnoses and to investigate concomitant infections, which potentially could impact the evolution of clinical signs and the course of the disease, fecal and serum samples were further investigated to detect other viral and bacterial pathogens. rotavirus a was present in both the sows and piglets of f , e. coli, expressing the virulence factor f , in f and f , c. perfringens in f and f , and s. typhimurium in f . however, no clinical signs, referable to a specific enteric disease, were present before the beginning of the longitudinal study. symptoms at the first sampling, watery diarrhea or soft diarrhea was observed in % ( / ), % ( / ), % ( / ), and % ( / ) of the sows from f , f , f , and f , respectively (table ) . at the same time high percentages ( - %) of - -day-old piglets were observed to have diarrhea on all of the farms ( table ) . clinical symptoms in piglets progressively disappeared at the following sampling times. however, a second outbreak of diarrhea was observed in out of animals ( %) at two months of age on f . samples were only taken from sows at the initial sampling time, which occurred at , , , and days after the onset of symptoms on f , f , f , and f , respectively. pedv rna was detected in / sows on f , / sows on f , / on f and / on f ( table ). the highest fecal pedv rna shedding titers were detected on f and f ( . and . log genome copies per ml of fecal homogenate), with a mean titer among shedding animals of . and . log copies/ml, respectively ( figure a) . mean lower titers were observed in f and f animals ( . and . log copies/ml, respectively) where the highest titers were . and . log copies/ml, respectively. the fecal viral shedding in piglets from the four farms is summarized in table and figure b . all of the farms had a similar virus-shedding pattern, with high percentages of pedv pcr positive - -day-old animals (sampling ) that decreased in - -day-old animals (sampling ) and was no longer present in - -day-old animals (sampling ) to the end of the study period (table ) . furthermore, on f , a second peak of viral shedding was detected in weaning piglets at days of age (sampling ). two weaning piglets did not show detectable viral shedding in the previous samplings. interestingly, the presence of different strains was assessed by sequencing the s gene of positive samples collected at the first and forth samplings. two genetic variants with a single amino acid substitution ( e > g) were detected at samplings and . on f , . % of -day-old piglets were pcr positive, four of them ( . %) were positive at days of age (sampling ) and only one animal was still positive at -days old (sampling ). moreover, one pig showed intermittent pedv shedding, being pcr positive at the first sampling ( days after birth), negative at -and -days old (samplings and , respectively) and again positive at -days old (sampling ) (supplementary table ). on f , all of the day-old piglets were pcr positive using rectal swab samples but only three of them remained positive at -days old (sampling ). at the following sampling times, when they were -and days old, they were all pcr negative. on f , . % of -day-old piglets were pcr positive but viral shedding was not detected in -day-old piglets through the end of the study. the highest fecal pedv rna shedding titer was observed in - day-old piglets with mean values (among shedding animals) of . , . , . , and . log copies/ml on f , f , f , and f , respectively ( figure b; supplementary table ). no significant differences were statistically evident among the farms. the titer values observed in - -day-old piglets were . (one animal), . (mean value of two animals), . (mean value of three animals) log copies/ml on f , f , and f , respectively (supplementary table ). pedv-antibodies were detected in / , / , / , and / sows on f , f , f , and f , respectively ( table ). in newborn piglets, pedv-antibodies were detected in , , , and % of animals from f , f , f , and f , respectively ( table ). in total, % of the sows had anti-pedv antibodies at delivery but only a few piglets ( %) showed detectable antibodies and a lack of clinical signs at - days of age. most of the piglets on the four farms developed antibody responses within weeks of age, and they remained stable until the end of the study ( - days of age). in the last few years, ped had a large economic impact on the swine industries in asia and the us, and in , the pedv +, presence/positive; −, absence/negative; na, not available (missing sample). *animals died or were not available (na). the number of sampled animals decreased as the study continued due to mortality, earmarks lost (na a ), and animal sales (na b ). also re-emerged in europe. two main pedv variants circulate worldwide but only the s indel variant, considered a mild strain, is spreading in europe. during summer , animals with mild clinical signs on two farms were detected as pcr positive for pedv in northern italy (boniotti et al., ) . thereafter, a new severe ped epidemic wave occurred. to gain insights into the pathogenicity of this variant, we described the results of a longitudinal study conducted during acute outbreaks of ped in three farrow-to-finish (f −f ) and one farrow-to-wean farm (f ), occurred in the beginning of . on the four farms, the mortality rates of suckling piglets were high ( , , and % on f , f , f and f , respectively) ( table ) . however, they did not reach the percentages observed in the us, caused by the original pedv strain (> %) (alvarez et al., ) . moreover, the four farms showed high percentages ( − , > %) of diarrheic animals, in particular, in all the units of the f , in piglets of f and in sows and piglets of f and f , but differences among farms were also observed. the course of ped was particularly severe on f , where we observed a high percentage of animals with diarrhea in suckling, weaning and fattening animals (supplementary table ). moreover, the virus appeared to circulate longer on this farm since positive animals were detected even and days after the first pedv detection within the farm. in particular, at days of age a second peak of viral shedding was observed in three animals. the s gene sequences of the strain identified in these animals showed a the different patterns and severity of clinical signs observed on the four farms may have resulted from the concurrent effects of co-infections with other pathogens. in particular, rotavirus, with a well-known pathogenic aptitude, which is mainly dosedependent, could have had a synergistic effect with pedv on f , causing a slight change in the course of the disease in sows and litters after the first week, and predisposing animals to secondary infections, such as those caused by e. coli f , c. perfrigens, and s. typhimurium. in this study, we determined the fecal pedv shedding in sows and in their piglets from to days to − days of age. in particular, we selected groups of sows and - piglets on each farm. none of the farms had reported pedv infections prior to this study, and thus, we can surmise that the sows had been recently infected at the time of their enrollment in the study, while piglets were infected after birth, through contact with infected sows. more than % of the sows showed detectable levels of pedv at the first sampling but only the % showed diarrhea. the severity of clinical signs caused by pedv is agedependent (jung et al., ) . in adult animals, clinical signs are usually milder or completely absent. moreover, in our study, pedv exposure dose and the gestational stage of the sows could have influenced their health status at the moment of delivery in terms of clinical signs, viral loads and pedv antibody level. on f and f , where the onset of pedv symptoms occurred in the fattening unit, and out of sows, respectively, had pedv antibodies. on f , where the onset of pedv symptoms occurred during the gestation period, out of sows had diarrhea but only had already developed pedv antibodies. on this farm, all of the sows showed high mean viral loads ( . log copies/ml) as determined by pedv rna. sows on f probably had less time to develop antibodies and transmit them to their litters through the colostrum. in fact, all of the piglets were infected and showed high titers of pedv rna in fecal materials. similarly, on f , where the onset of the outbreak took place in the delivery room, out of sows showed detectable levels of pedv antibodies. at the first sampling, a high percentage ( - %) of the - -day-old piglets from all of the farms were positive by qpcr. despite the different immunity levels among the sows on the four farms, and the different proportions of infected piglets, the quantitative pedv rna results in piglets were not statistically different among farms. as proposed by other authors, we assumed that once the pigs were infected and viral replication began, the initial viral dose appeared to have little impact, at the group level, on the average amount of fecal shedding (thomas et al., ) . at weeks of age, the proportion of pcr positive piglets decreased to - . %, and at month of age only one animal was positive. the pedv rna titer also decreased with time, confirming that ped is characterized by high pedv fecal shedding titers a few days post infection and the titers tend to decrease after week. intermittent shedding can be observed until days post infection, as shown in f and f , and such variations in excretion levels and viral loads could be determined by poor management and a lack of biosecurity measures, but it could also be due to a new introduction of the virus, as likely occurred on f . for immunity responses, % of the animals on f showed detectable levels of pedv antibodies up to the end of the study ( days of age). on f , f , and f , the percentage of seropositive animals decreased slightly starting from days of age. the longer and biphasic circulation of the virus on f might have favored subsequent exposures of the animals to the virus, and thus, a more durable immunity. the likely outcome of a massive outbreak of pedv within one herd is a diffuse and long-lasting immunity. indeed, especially in farrow-to-finish farms, such herd immunity and, particularly, the maintenance of seropositive sows could be an important tool to prevent severe cases of ped in newborn piglets by transferring passive maternal immunity with colostrum. goede et al. ( ) reported that durable lactogenic immunity was present in sows previously exposed ( months) to a s indel strain of pedv and that this immunity induced cross-protection to an original virulent pedv. cumbersomely, in this study we did not plan to investigate the presence of maternal antibodies (igg and iga) in the colostrum or to register the piglets' birth weight, two important factors influencing the protective maternal immunity. however, considering the high percentage of diarrheic and pedv positive piglets, and the presence of clinical signs in animals of different ages, including sows, we can suppose that these farms were originally naïve with regard to pedv infection, and thus the lactogenic antibodies were not present at all, or anyway sufficient to effectively protect piglets from infection. thus, future management of pedv infection in farrow-to-finish or farrow-to-wean farms cannot disregard to periodically check and evaluate the iga and igg titers in sow sera and in the colostrum. determining the viral loads and shedding rates of pedv in real field situations during outbreaks is important in evaluating the virulence of a strain and in predicting the susceptibility of infected animals, at different ages and in the various farm units, within a herd. at the moment, very limited data have been published on the pedv viral load during an outbreak under field conditions (bjustrom-kraft et al., ) . the qpcr assay used in this study was validated according to the minimum information for quantitative realtime pcr experiments guidelines (bustin et al., ; table ; supplementary table ). the method validation is an important requirement to allow the comparison of quantitative results from different studies, which would be otherwise difficult to achieve. moreover, understanding the mechanisms of viral transmission and circulation within the farm can be useful in implementing appropriate control measures on the farm to limit the infections spread. considering the ability of pedv to be dispersed rapidly through fecal contamination, particular attention should be paid to biosecurity and hygiene measures, such as the proper disinfection of equipment and sites, manure disposal, suitable procedures for the movements of animals within the farm, and the use of disposable clothes and shoes for personnel, staff and visitors. longitudinal field studies examining natural infections are comparatively uncommon amongst reports of pedv in comparison to the several experimental studies already performed. in fact, many unforeseen events can adversely affect the success of this kind of study: dead of animals, lost of the earmark, selling or moving of the animals due to unexpected need of the farmer. these practical hitches could negatively influence the sampling procedures (e.g., respect of time points) and data registration. therefore, by planning these "observational" studies, so tight inclusion criteria cannot be established and limited field data could be successfully registered. on the other hand, the longitudinal studies, directly conducted in natural outbreaks, are inclusive of the several "farm factors and field effects" which are very difficult to reproduce in experimental trials, and thus, the obtained results, being more reliable and adherent to real conditions could be effectively used in risk analysis and for defining control strategies. in conclusion, longitudinal studies conducted under field conditions during and after a ped outbreak could be useful in determining the level of immunity acquired at the herd level and may be integrated with the data acquired from experimental infections. they provide an added value, in the possibility to study and evaluate the effects of cofactors, such as other infectious agents, and management and environmental conditions, on the evolution and epidemiology of the disease. the study was exempt of ethical approval procedures because animal samplings were performed during the routinely 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outbreak of porcine epidemic diarrhea virus in portugal mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo comparison of enzyme-linked immunosorbent assay and rt-pcr for the detection of porcine epidemic diarrhea virus emergence of porcine epidemic diarrhea virus in southern germany emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences epidemiology and vaccine of porcine epidemic diarrhea virus in china: a mini-review complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium effect of porcine epidemic diarrhea virus infectious doses on infection outcomes in naive conventional neonatal and weaned pigs new rapid semi-quantitative rt-pcr assay developed to detect porcine epidemic diarrhea virus distinct characteristics and complex evolution of pedv strains development and evaluation of a duplex real-time rt-pcr for detection and differentiation of virulent and variant strains of porcine epidemic diarrhea viruses from the united states isolation and experimental inoculation of an s indel strain of porcine epidemic diarrhea virus in japan we thank anna mangeli for the skilled technical assistance and federico scali for the statistical analysis. thanks also to science docs for the english language editing of this manuscript. the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fmicb. . /full#supplementary-material key: cord- -z v s n authors: adhikari, nilanjan; baidya, sandip k.; saha, achintya; jha, tarun title: structural insight into the viral c-like protease inhibitors: comparative sar/qsar approaches date: - - journal: viral proteases and their inhibitors doi: . /b - - - - . - sha: doc_id: cord_uid: z v s n severe acute respiratory syndrome (sars), caused by sars-coronavirus (sars-cov), is a dreadful infection worldwide having economic and medical importance and a global threat for health. it was turned into an epidemic in south china followed by a chain of infections across three generations. a number of pathogeneses in human may occur due to the virus. this infection has not been taken into account before the sars outbreak, and still it is a neglected one. therefore, there is an urgent need to develop small molecule antivirals to combat the sars-cov. no vaccines are available till date though a number of sars-cov c-like and c protease inhibitors were reported. in this chapter, quantitative structure–activity relationship technique is used for development of anti-sars and anti-hrv drugs and outcome discussed in details. this approach may be a useful strategy to design novel and potential anti-sars drugs to combat these dreadful viral diseases. in early , about people were diagnosed across the world with severe acute respiratory syndrome (sars). among them, almost died due to its first outbreak. the disease was broken out and turned into an epidemic in guangdong of south china. two cases of sars infections were noticed in taiwan and singapore due to improper handling of the samples in the research laboratory. during april , a "mini outbreak" of infections took place in a research laboratory of beijing that, in turn, led to a chain of infections across three generations. fortunately, the total number of sars-infected people was only nine that time. this incidence threatens us about the mini outbreaks of sars globally at any time (anand et al., ) . the sars is caused by the sars-coronavirus (sars-cov) that belongs to the family of coronaviridae. this family also includes viruses, such as feline infectious peritonitis virus, murine hepatitis virus, bovine coronavirus, transmissible gastroenteritis virus (tgev), as well as human coronavirus e . the sars is considered as a global threat to the health (khan, ; perlman and netland, ) . moreover, water and food-borne viral gastroenteritis may be caused by the noroviruses that belong to the family calciviridae (atmar, ; patel et al., ) . therefore, there is an urgent need to develop small molecule antiviral drugs to combat these viruses. the picornavirus belongs to the family of viruses namely picornaviridae, calciviridae, and coronaviridae (mandadapu et al., a) . a number of pathogeneses in human may occur due to these viruses leading to economic and medical burden. for example, human rhinovirus (hrv) is the major reason viral proteases and their inhibitors for upper respiratory tract infection (ren et al., ; turner and couch, ; winther, ) whereas nonpolio enteroviruses are responsible for symptomatic infections with - million cases per year in united states (mcminn, ; solomon et al., ) . depending on the similarity of the polycistronic organization of the genome, common and posttranscriptional strategies along with the conserved region of domain homology in viral proteins, the virus families are related phylogenetically though these families are not related morphologically (anand et al., ; cavanagh, ; cowley et al., ) . coronavirus is found to be responsible for causing a number of diseases not only in human but also in animals, though the human coronavirus has not been taken into account seriously before the sars outbreak (anand et al., ) . human coronavirus (hcov) oc and e may be responsible for illness in the upper portion of the respiratory tract along with common cold-like conditions (myint, ) . the hcov e is the only strain till date that can be cultured in cell culture technique efficiently. the symptoms of sars include rigor, malaise, high degree of fever, cough, headache, and dyspnoea. the symptoms may also lead to produce interstitial infiltrates in lungs that may be treated through ventilation and intubation . not only the lungs but also other organs may be affected by sars infection (such as liver, kidney, and gastrointestinal tract). therefore, the sars infection may be treated as a cause of systemic infection. face-to-face contacts may be supposed to be the reason for transmission of the pathogen though other routes are also possible. a number of inhibitors against sars-cov cl pro and hrv c pro were reported and the process of development of new antivirals against this class has been continued for a decade. in the present report, quantitative structure-activity relationships (qsars) techniques have been explored to understand the relation between the sars-cov cl pro and hrv c pro enzyme inhibitory activity with the physicochemical and structural properties of these inhibitors developed till now. this approach may be a useful strategy to design and develop novel and potential sars-cov cl pro and hrv c pro inhibitors to combat the dreadful viral infections. and the sequence of genomic structure (marra et al., ; rota et al., ) . three noncanonical m pro cleavage sites are observed in sars coronavirus polyproteins having val, met, or phe amino acid residues at p position whereas the same cleavage site is found dissimilar in other coronaviruses. therefore, the structural and functional criteria of m pro helps to identify it as an important target for developing anti-sars drugs or other anticoronaviral drugs (anand et al., ) . the structures of hcov e m pro , tgev m pro , and sars-cov m pro demonstrate that these enzymes have three distinct domains. the first two domains (domain i and ii) together possess similarity with chymotrypsin whereas the third one consists of an α-helical fold which is unique (anand et al., ) . the active site which is situated between the first two domains possesses a cys-his catalytic site. antiparallel β-barrels with six strands are composed of the domain i and ii (residues - of i and - of ii, respectively). the domain ii is connected to domain iii (residues - ) through a long loop (residues - ) (anand et al., ) . the hydrophobic amino acid residues are found to compose the domain i β-barrel. the α-helix (residues - ) helps to close the β-barrel like a lid. the domain i is bigger than domain ii, as well as the homologous domain ii of chymotrypsin and c pro of hav (allaire et al., ; bergmann et al., ; tsukada and blow, ) . moreover, a number of secondary structural elements are found to be missing in coronavirus m pro compared to hav c pro (such as strands b ii and cii along with the linking loop). the gly to ser form a portion of the barrel though domain ii possesses maximum consecutive turns and loops. moreover, the structural alignment of coronavirus m pro domain ii with the picornavirus c pro domain ii is found to be different. superimposition of domain i of tgev m pro , with hav c pro domain i results in a root mean square deviation (rmsd) of . Ǻ whereas superimposition of domain ii of both of these enzymes yields a rmsd of . Ǻ. the overall rmsd for the c α atoms between their structures is > Ǻ for all c α positions and the three m pro structures possess similarity among themselves (anand et al., ) . the helical domain iii is the most variable domain that exhibits a better overlapping between hcov m pro and tgev m pro compared to the sars-cov m pro , with each other. moreover, tgev and hcov e (belongs to group i coronavirus) show % sequence similarity whereas sars-cov (belongs to group ii coronavirus) exhibits % and % sequence similarity with hcov e and tgev, respectively . a high degree of conserved region ( %- %) between the domain i and ii is observed while comparing group i coronavirus m pro , and group ii sars-cov m pro . the domain iii comparatively exhibits a lower degree of sequence similarity ( %- %) between these two groups coronaviral enzymes (anand et al., ) . the x-ray crystallography structures of sars-cov m pro , tgev m pro , and hcov e m pro show that these form dimers (anand et al., yang et al., ) . moreover, it was also confirmed that the dimer form is enzymatically active but the monomeric form is not active (anand et al., ; fan et al., ) . the dimerization process is found to be mandatory for enzyme activity and this process helps to discriminate the coronavirus m pro , and the picornavirus m pro distinctly. a catalytic dyad is formed by cys and his at the sars-cov active site, whereas other cysteine and serine protease are found to form a catalytic triad. a water molecule is found to have hydrogen bonding interaction with his and asp . moreover, if the cysteine residue is replaced with serine at the enzyme active site, the enzymatic activity of sars-cov m pro is decreased. for coronaviral main protease, as well as the picornaviral protease, the cysteine residue is located at the same place of the active site of the his imidazole ring plane (distance . - Ǻ). for hydrogen bonding interaction between the side chains, the sulfur atom of cysteine residue should be along with the same plane of imidazole function (anand et al., ) . the substrate binding sites are found to be conserved in all coronavirus main proteases as suggested by the experimental observations (anand et al., , . the x-ray crystallographic study between the inhibitor-sars cov m pro suggests viral proteases and their inhibitors that the imidazole function of his is located at the bottom of the s site of m pro to donate hydrogen bond to the backbone carbonyl function of glutamine. for interaction with glutamine at s site, the histidine amino acid residue has to be remained unaltered over a broad range of ph. this may be possible through two interactions involved in the imidazole ring. it may either stack to the phenyl ring of phe or may accept a hydrogen bond from the hydroxyl function of tyr . replacement of the his is found to abolish the proteolytic activity (hegyi et al., a; ziebuhr et al., ) . all these residues discussed are found to be conserved not only in sars-cov m pro but also in all other coronavirus main proteases. moreover, residues ile , met , glu , and his of the s pocket take part in the conformation of sars-cov m pro (anand et al., ) . regarding the s specificity site, all the coronaviruses m pro consists of a leucine residue at the s cleavage site. this s site is hydrophobic in nature and is composed of side chain amino acid residues, such as his , thr , met , tyr , and met . the longer methionine residue may restrict the s pocket and requires slight spatial orientation to accommodate the substrate leucine residue. due to the presence of ala residue and differences in amino acid sequences, the s pocket is bigger in sars-cov m pro compared to hcov e m pro and tgev m pro . in sars-cov m pro , a stretch of amino acid sequences is observed in - residues that help to enlarge the size by forming a helix which is not observed in other coronaviruses. this bigger size may be effective in the substrate binding (anand et al., ) . apart from the s and s pockets, some other substrate binding pockets should be taken into consideration. at the p position, small amino acid residues may be preferable (such as val, thr, ser, and pro) whereas no specificity at the p position is observed for coronavirus m pro . at the p position, some amino acid residues are found to be conserved in sars-cov m pro (such as met and thr ). moreover, p amino acid side chains are found to interact with the main chain at pro , thr , and gln in sars-cov m pro , and help like a linker between domain ii and iii. apart from antivirals to fight against these coronaviruses, rna interference (rnai) and vaccine development may be a useful strategy though it is a challenging task. the rnai is an important tool for gene silencing. apart from the use of rnai in cancer and genetic disorder , development of sirna inhibitors in sars infection may be a boon for the treatment of the disease (li et al., ) . the replication of the sars may be inhibited effectively through rnai in vero cells. therefore, sirna therapy may be effective to combat sars infection . short hairpin rna (shrna) may be useful to target the n gene sequence of sars coronavirus and to inhibit shrna of sars-cov antigen expression (tao et al., ; zhai et al., ) . these results suggest that gene silencing through rnai may effectively inhibit the sars-cov antigen expression, and, therefore, rnai approach may be effectively utilized as possible therapy for inhibiting sars-cov infection. moreover, the rnai is used to target the replicase enzyme of human sars virus. it not only targets the hsars gene but also produces inhibitory effects on the sars rna virus expression (zhai et al., ) . as far as the development of sars vaccines is concerned, the inactivated sars-cov along with the full-length s protein and an attenuated weak virus, and recombinant sars protein may be used (jiang et al., ; zhai et al., ) . the s protein and the inactivated virus were reported to be used to neutralize antibodies. the attenuated or the weak form of the virus might be used to induce immunity, as well as to neutralize antibodies (finlay et al., ) . the development of recombinant vaccines may be a useful strategy to prevent sars infections. it mainly depends on the best antigen identification, as well as the choice of expression system. the s glycoprotein of sars-cov along with its truncated form may be targeted for development of recombinant vaccine as the best candidate (babcock et al., ; bisht et al., ; buchholz et al., ; yang et al., ; zhai et al., ) . a number of reports were published regarding recombinant s protein vaccine against different sars-cov through aryl delivery system (pogrebnyak et al., ; tuboly et al., ) . qsar is a useful tool to understand the relation between the structural and physicochemical properties of the drug molecules, and their biological activity which may be useful for predicting the activity or toxicity profile of drugs (gupta, ; verma and hansch, ) . the data required for developing the qsar models are collected from the literature (see individual qsar for corresponding references). the ic (molar concentration required to produce % inhibition of the enzyme), ec (effective concentration), or k i (binding affinity) data are obviously considered as the biological activity term or dependent variable. the independent variables include physicochemical parameters [such as hydrophobicity, molar refractivity, dipole moment along with different axes, molecular weight (mw), polar surface area (psa), and polar volume, as well as surface area (sa) and volume] and many topological parameters, such as kier's molecular connectivity indices, balaban indices, etc. regarding the statistics of qsar models, n is used to indicate the number of compounds in the set, r to indicate the correlation coefficient of the qsar model obtained, r refers to the squared correlation coefficient exhibiting the goodness of fit, q indicates square of the leave-one-out cross-validated correlation coefficient (represents the internal validation of the model), r a refers to the adjusted r , f value represents the fischer statistics (fischer ratio) that actually means the ratio between the explained and unexplained variance for a particular degree of freedom, p stands for the probability factor related to f-ratio, see means the standard error of estimate, q is the quality factor that can be a measure of chance correlation. a high q represents the high predictivity, as well as the lack of over-fitting of the model. compounds that misfit in the correlation are considered as outliers and are usually removed from the regression. we discuss here the qsar models obtained for different categories of sars-cov cl pro and hrv c pro inhibitors. this model suggested that the increasing value of the psa may contribute positively to the binding enzyme. compounds with a higher psa (compounds - , table . ) have higher activity than compounds with a lower psa (compounds , , table . ). compound has a lower psa but higher activity whereas compound having the higher psa has lower activity. these molecules are not explained properly by this model. therefore, these molecules (compounds , , table . ) were considered as outliers. they may have different mechanism(s) of action(s). blanchard et al. ( ) reported some sars-cov cl pro inhibitors (fig. . ; table . ). the qsar model for these compounds was as shown by eq. ( . ). this model also exhibited that the psa of the molecule might be conducive to the enzyme inhibitory activity of the compounds. as obvious from table . , compounds and with a higher psa have higher activity than compounds with the lower psa. the sulfone and amino functions of compound and the disubstituted amino acid function of compound may produce higher psa as compared to the trichloro-substituted compound and the monohydroxy trifluoro substituted ester analog (compound ). it also suggested that the enzyme-drug interaction might be taking place in a nonhydrophobic space at the enzyme active site. compound has the lower psa but possesses comparatively higher activity than other compounds. it may be assumed that compound may behave differently. probably, the ester function and the chloro group may have some electronic interaction with the enzyme responsible for the higher inhibitory activity. therefore, compound may be considered as an outlier. jain et al. ( ) synthesized and evaluated some keto-glutamine analogs as potent sars-cov cl pro inhibitors (table . ). for this series of compounds, the qsar model obtained was as shown by eq. ( . ). in this equation, "i" is an indicator parameter that was used with a value of for the presence of the conme . for the absence of this group, its value was zero. the negative coefficient of i suggests that compounds with conme function (compounds - , table . ) are less active than compounds with -pyrrolidinone function (compounds - , table . ). therefore, compounds with -pyrrolidinone functions (compounds - ) are preferable for the higher inhibitory activity. shie et al. ( a) reported a series of potent anilide inhibitors against sars- cl pro (fig. . ; table . ), for which the qsar model obtained was as shown by eq. ( . ). this model showed the importance of dipole moment along the x-axis (d x ), mw, psa, and volume (vol) for controlling the enzyme inhibition. the positive coefficient of the dipole moment along x-axis suggested that the bulky substitutions along x-axis may favor the activity. moreover, the mw was also shown to have a positive impact on the activity, whereas psa was shown to have the negative effect. therefore, it may be suggested that molecules with the bigger size along with bulky substituents may be conducive to the inhibition. moreover, the volume is found to have a parabolic relation with the enzyme inhibition. the optimum value of the volume is . compounds , , , and were considered as outliers as these molecules may work with a different mechanism(s). shie et al. ( b) reported a series of peptidomimetic α,β unsaturated esters as promising sars- cl pro inhibitors (table . ). the qsar model obtained for them was as shown by eq. ( . ). it was observed from eq. ( . ) that increase in the value of both the mw, as well as the psa may be detrimental to the activity. thus, the model suggested that the smaller molecules with less steric bulk might favor the activity. moreover, the enzyme-drug interaction would be more favored in nonhydrophobic space. it was observed that compounds having unsaturation at the r' position (compounds - , table . ) possess lower mw compared to the other molecules in the dataset and possess higher inhibitory activity. in compound , both the phenyl rings might be accommodated in the s and s pockets. moreover, the (dimethylamino) cinnamyl function adopts a coplanar rigid structure at the end terminal, which may help it in forming hydrogen bonding with amino acids residues glu , glu , and glu at the enzyme active site. compound may behave differently and hence, this was considered as an outlier. wu et al. ( ) reported some benzotriazole esters as promising sars- cl pro mechanism-based inhibitors (table . ). the model obtained for this series is as shown by eq. ( . ). it was observed from eq. ( . ) that increasing value of the dipole moment along y-axis (d y ) may lead to a decrease in the activity, whereas increasing value of the mw may be conducive to the activity. it also suggests that compounds with the higher molecular bulk with lower steric effect may be favorable for the higher inhibitory activity. compounds with ester functions (compounds - , table . ) is better active than compounds with acetyl function (compounds - ) as these molecules (compounds - ) possess higher bulkiness. therefore, it may be assumed that the ester analogs impart less steric effect with the enzyme and hence, produce higher activity. chen et al. ( a) reported some diverse chemical entities through virtual screening, surface plasmon resonance and fluorescence resonance energy transfer based assays as promising against sars-cov cl pro (fig. . ; table . ). the qsar model obtained was as shown by eq. ( . ): it is observed from eq. ( . ) that increasing the value of the psa may be detrimental to the activity. thus it suggested that less polar molecules may have better inhibitory activity. due to the presence of electronegative function (such as carboxyl, chloro, etc.), the molecule may have larger psa. compounds and (table . ), though possess lower psa, have higher activity and this could not be explained by this model. thus these compounds might be supposed to involve the different mechanism of action for producing the higher activity. therefore, these compounds are considered as outliers. zhou et al. ( ) reported some isatin analogs as sars-cov cl pro inhibitors (table . ), for which the correlation obtained was as in eq. ( . ): it was observed from eq. ( . ) that increasing the sa of these molecules may impart higher inhibitory activity. bulky substitution at the r position, such as β-napthylmethyl (compounds and , table . ) may impart higher sa and hence, produce higher activity. thus substitution with -conh function at the r position in place of iodo function may have a better effect (compound vs. , compound vs. , and compound vs. , table . ). similarly, bulky aryl function may be more favorable than the alkyl function. the larger sa may help the molecule to occupy more space in the enzyme active site to have better binding interaction as evidenced by the molecular docking analysis (zhou et al., ) . compound having maximum sa exhibits hydrogen bonding with his and cys through the keto functions of the isatin moiety. moreover, the carboxamide function at the r position makes hydrogen bonding with phe and his . the β-napthyl moiety (compound ) fits well into the hydrophobic s pocket whereas smaller and less bulky substituents, such as methyl (compound ), n-propyl (compound ), n-butyl (compound ), and benzyl (compound ) do not accommodate well into the s pocket. it is not clear why the compound behaves aberrantly though possessing a comparable good sa. therefore, compound may be considered as an outlier. tsai et al. ( ) reported a series of sars-cov cl pro inhibitors through pharmacophore mapping and virtual screening approach (fig. . ; table . ). for this, the qsar model obtained was as shown by eq. ( . ). . , p < . , see = . , q = . , q = . , outlier = compounds , , it was observed from eq. ( . ) that increasing the value of the molar refractivity (cmr) and decreasing the value of the dipole moment along y-axis (d y ), as well as the volume (vol) may contribute positively to the enzyme inhibitory activity. it was, therefore, suggested that increasing the total molecular bulk may increase the activity whereas bulky substituent along y-axis may be detrimental to the activity. bulky substitution along y-axis may produce some unfavorable steric interaction with the enzyme. therefore, the bulky molecule with less steric effect may be favorable for the activity. compounds , , and (table . ) may act through different mechanism(s) of action and hence, they were considered as outliers. table . ) through structure-based drug design approach, for which the qsar model obtained was as shown by eq. ( . ). it was observed from this equation that the increase in the value of the sa and the polar volume (pol vol) may be conducive to the activity, whereas the increasing in the value of volume and dipole moment along x-axis (d x ) might be detrimental to the activity. thus it could be suggested that bulky substitutions along x-axis might produce unfavorable steric hindrance that may lower the activity. moreover, this model also revealed that compounds having higher polar volume may favor the activity compared to compounds with lower polar volume. in compound (table . ), one of the nitro groups is closer to the imidazole function of his and thus there may be some electrostatic interaction between them leading to better activity. moreover, the phenyl ring may form π-π interactions with his at the enzyme active site leading to potent activity. compounds and (table . ) might act in a different manner and hence, they were considered as outliers. chen et al. ( b) reported some quercetin- -β-galactoside and its analogs as promising sars-cov cl pro inhibitors (table . ), for which the qsar model obtained was as in eq. ( . ): it was observed from eq. ( . ) that decreasing the value of the psa would have the positive effect on the biological activity. it meant that less polar molecules would be preferred to the high polar molecules. due to the presence of a number of hydroxyl groups, these molecules may interact with the enzyme as hydrogen bond acceptors. the molecular modeling study revealed that the side chain of gln forms four hydrogen bonds with compound (table . ), whereas two hydrogen bonding interactions are observed with the nitrogen atom of glu . it was, however, observed that compound having the highest psa value due to the presence of two galactose rings was less active. probably, compound might behave in an aberrant fashion and hence, it was considered as an outlier. zhang et al. ( ) synthesized and evaluated some phthalhydrazide ketones (table . ) and heteroatomic ester as potential sars- cl pro inhibitors. the qsar model developed for this set of compounds was as shown by eq. ( . ): eq. ( . ) suggested that high polar volume of the compound would not favor the activity. a molecular modeling study had revealed that the halopyridine moiety of the compounds was well accommodated in the s binding pocket where it could have van der waals interactions. moreover, it was observed that the halogen atom does not interact with the enzyme and is directed toward the solvent exposed area. the furyl group of compound is located near the catalytic cys residue where it can have hydrophobic interaction. compounds and being a misfit in the correlation were excluded. ghosh et al. ( ) reported some peptidomimetic sars-cov cl pro inhibitors (table . ), for which a qsar model obtained was as: . ( . ) . ( . ) z = ± − ± ( . ) it was observed from eq. ( . ) that increasing the value of the dipole moment along z-axis (d z ) will lead to decrease the enzyme inhibitory activity. it thus suggested that the bulky substituent along z-axis will not be conducive to the activity. the long chain linear aminobutoxy derivatives (compounds and , table . ) are better than the isoxazole analogs (compounds , , , and , table . ) as the isoxazole moiety may produce more bulkiness that may impart unfavorable steric effect with the enzyme. zhang et al. ( ) reported some arylmethylene ketones and fluorinated methylene ketones as sars-cov cl pro inhibitors (table . ). the qsar model for them was as shown by eq. ( . ), where the indicator variable "i" stands for a value of unity for the ester group. a positive coefficient of it suggested that the ester group may be favorable for imparting the higher inhibitory activity. compounds bearing ester functions (compounds - , table . ) are highly active compared to the nonester derivatives (compounds - , table . ). compounds - are found to be oriented from s to s pocket and the furan oxygen atom forms hydrogen bonds with the amino function of glu . moreover, it is observed that compound though having ester function, may behave in an aberrant fashion. therefore, it was considered as an outlier. the qsar model obtained for a series of chloropyridine esters reported by niu et al. ( ) as potent sars-cov cl pro inhibitors (table . ) was as shown by eq. ( . ), which suggested that increasing the molar refractivity and decreasing the total dipole moment may favor cl protease inhibitory activity this also suggested that the smaller molecules with less steric effect may be conducive to the inhibitory activity. the α-naphthyl (compound , table . ) and the -oxochromene function (compound , table . ) at ar position yield less dipole moment and better molar refractivity compared to the nitrophenyl (compounds and ), the chlorophenyl (compound ) or the viral proteases and their inhibitors pyridyl analog (compound ) and thus compounds and are more potent than compounds - . furyl derivatives (compounds - ) are better inhibitors as compared to the other aryl ester analogs (compounds - ) as they have higher molar refractivity despite having comparatively moderate bulky p-chlorophenyl or the p-nitrophenyl groups at r position. a slight reduction in the activity is noticed for the disubstituted aryl function (compound ) and the alteration of the nitro function at the nd position of the phenyl ring (compound ) in contrast to the th position (compound ), which increases the bulkiness or total bulk, and reduces the activity slightly. it is observed from the molecular modeling study that increasing the length of the side chain may increase the interaction between s and s pocket and the inhibitor that can be reflected by the qsar model. compound possesses the higher molar refractivity while compound possesses the lowest value of total dipole moment but it is not reflected in their activity. probably, these compounds behave differently from other compounds in the dataset and hence were outliers. yang et al. ( ) reported some cinanserin analogs as sars-cov cl pro inhibitors (table . ), for which the qsar model obtained was as shown by eq. ( . ). this equation clearly exhibited that high molecular volume will not be favorable to the activity. thus compounds having aryl (compound ), the heteroaryl (compounds , ), or the long chain amide function (compounds , ) at y position have the lower activity than compounds having at this position the unsaturation (compounds , ) or the ester function (compound ). compound enters into the deep s pocket and has hydrophobic interactions. however, compound has the lowest volume but it does not show the highest activity. probably, this compound (compound ) may behave in a different manner with the enzyme and hence it is considered as an outlier. the qsar model derived for some trifluoromethyl, benzothiazolyl, and thiazolyl ketone compounds with peptide side chain reported by regnier et al. ( ) eq. ( . ) showed that the enzyme inhibitory activity was correlated with the polar volume of the molecules through a parabolic relation. it, therefore, suggests that the activity would decrease upto an optimum value of polar volume (pol vol opt = ) and beyond that will start increasing. compound with the -oxo-pyrrolidin- -yl function (compound , table . ) possesses the higher polar volume and hence, possess the maximum inhibition. comparing the activity of this compound with those of compounds , - , it may be suggested that the -oxo-pyrrolidin- -yl function in compound is favorable than the diethylamino function in compounds - and morpholino function in compound at the x position. moreover, the benzothiazole- -yl function in compounds , is favorable than the thiazole function in compounds , , . the bulky group, such as the morpholino in compound and the benzylmethylamino function in compound are favorable than the smaller substituents, such as the hydroxyl in compound , the amino group in compound , and the diethylamino group in compounds , , and at x position. comparing compound with , it may be inferred that the bulky amino acid moiety (compound ) is favorable than smaller amino acid functions (compound ), as bulky functions may produce the higher polar volume. the molecular modeling study revealed that the benzyloxycarbonyl moiety of compound did not make any hydrophobic interaction rather had hydrogen bonding interactions with glu through its adjacent amino function. ramajayam et al. ( ) reported some pyrazolone analogs as promising sars-cov cl pro inhibitors (table . ), for which the qsar model obtained was as: eq. ( . ) thus suggested that increasing value of the hydrophobicity of these molecules may be detrimental to the activity. compounds with the smaller halogen substitution, such as fluorine at r position (compound , table . ) are better than compounds with the bigger halogen substituents, such as the chloro (compounds and , table . ). further, a dihalo substituted compound, such as compound was shown to be less active as compared to monohalo-substituted analogs (compounds , , and ) . the cyano (compound ) and the nitro (compound ) substitutions also produced the higher activity as compared to the methoxy substitution (compound ) . the docking study suggested that the n -phenyl group was located near to the s ' pocket. one of the oxygen atoms of the nitro group forms a hydrogen bond with gly . the keto function of the pyrazolone ring was also found to form another hydrogen bond with glu . the c- phenyl ring was found to be well-accommodated in the s pocket. the benzylidene ring without any carboxyl functions lost the activity. therefore, it may be assumed that hydrogen bonding interaction is more important than the hydrophobic interaction. the oxygen atom of the carboxyl group forms a hydrogen bond with gln . therefore, apart from s pocket, none of the aryl functions has exhibited hydrophobic interactions, whereas three hydrogen bonding interactions were observed. compounds and considered as outliers. ( . ) . it was observed from eq. ( . ) that the increasing value of the dipole moment along x-axis may be conducive to the activity. thus, the bulky substitution at x-axis of these molecules may be favorable for activity. compounds - (table . ) possess higher dipole moment due to much bulky aryl groups as compared to the compounds - , - and, therefore, have higher activity. compounds and exhibited the aberrant behavior and thus were considered as outliers. nguyen et al. ( ) reported some promising sars-cov cl pro inhibitors through virtual screening (fig. . ; table . ). the qsar model obtained for these compounds was as shown by eq. ( . ), which exhibited that the activity is well correlated with the hydrophobicity of the molecules. the docking study had revealed that compound (table . ) had good hydrophobic interactions with his , phe , leu , cys , his , glu , gly , and his apart from a number of hydrogen bonding interactions (the nitro group with gly , methacrylamide group with phe , one of the oxygen atoms of the nitro group with cys ). the nitrophenyl group was found to be the most crucial moiety to enter into the s pocket for imparting potent inhibition. compounds and though possessed a higher value of hydrophobicity but less activity than expected, hence, they were considered as outliers. some peptidomimetic sars-cov cl pro inhibitors (table . ) were synthesized and evaluated by akaji et al. ( ) and the qsar model obtained for this [eq. ( . ) ] indicated that the activity is controlled by a single indicator parameter "i" used for an imidazolyl- -yl methyl substituent at the r position. the positive coefficient of this indicated that such a substituent would conducive to the activity. the reason of this may be that this substituent might have better steric fitting in the s pocket of the enzyme formed by phe , leu , and glu . compounds and (table . ) were considered as outliers. nguyen et al. ( ) reported some flavonoids from pichia pastoris (fig. . ; table . ) having sars-cov cl pro inhibitory activity. for these compounds, the inhibition activity was shown to be correlated with the psa of the molecule [eq. ( . )], suggesting that highly polar molecules may have better activity. substituents like hydroxy might give better psa, leading to better figure . general structure of some flavonoids as sars-cov cl pro inhibitors. activity and also such substituents might form the hydrogen bonds. a molecular docking study showed that the galloyl group forms hydrogen bonds with leu , gly , ser , and his at the enzyme active site. compounds without any b ring (compounds and ) are less active. compound with no , double bond in the c ring is less active than the compound though possessing the higher psa. compound was found to act as an outlier. it was also observed that the rigid aryl substitution with the hydroxyl group (compound ) was better than the flexible cycloalkyl substitution with the hydroxyl group (compound ). mandadapu et al. ( b) reported some dipeptidyl aldehydes and α-keto amides as potent norovirus cl pro inhibitors (table . ). the qsar model obtained for these compounds was as shown by eq. ( . ) that again exhibited that the hydrophobicity of the compounds may be beneficial to sars-cov cl pro inhibitory activity of the compounds. compounds with cyclohexylmethyl group appeared to be more potent than other compounds. this might be due to the bulkiness of this group providing the higher c log p value and due to its better fitting in the active site of the enzyme. compounds and , however, showed aberrant behaviors and thus were considered as outliers. thanigaimalai et al. ( a) reported a series of dipeptide-type sars-cov cl pro inhibitors (table . ), for which the qsar model obtained was as shown by eq. ( . ). it was suggested from eq. ( . ) that decreasing value of the sa may be conducive to the enzyme inhibition. however, the volume of the molecules was found to exhibit a parabolic relation with the enzyme inhibitory activity. it, therefore, suggested that increase in volume may be responsible for enhancing the activity only up to an optimum value of . beyond this value, the activity would decrease. thus it indicated that molecules with limited bulk or with substituents with limited bulk might be favorable to the activity. thus indole derivatives with less bulky substitution (compounds - , table . ) resulted in higher activity than those with a greater bulk (compounds , ) . compared to the indole analogs, the oxopyrrolidine (compound ), the pyrrole (compound ), the benzothiazole (compound ), and the benzofuran (compound ) analogs were comparatively less active. however, it could not be explained by the model why benzimidazole analog (compound ) had higher activity as compared to the benzothiazole (compound ) and benzofuran analogs (compound ). probably, this compound may behave differently as compared to the other compounds in the dataset. therefore, this compound is considered as an outlier. in the subsequent study, thanigaimalai et al. ( b) reported a series of dipeptide-type sars-cov cl protease inhibitors (table . ) whose activity was shown to be controlled by the molar refractivity (cmr) and the polar volume (pol vol) of the compounds [eq. ( . )]. since the correlation was quadratic with respect to both cmr and pol vol, it suggested that compounds with limited bulk and polarity may have a better binding affinity. several compounds, however, were treated as outliers. , , , , , , turlington et al. ( ) reported a series of n-(benzo [ , , ]triazol- -yl)-n-(benzyl)acetamido) phenyl) carboxamides as promising sars-cov cl pro inhibitors (table . ). the qsar model obtained for these compounds [eq. ( . )] suggested that highly hydrophobic (c log p > . ) molecule with high molar refractivity but the less mw will be conducive to the activity. with the structural insight into the viral c-like protease inhibitors chapter | n-(benzo[ , , ]triazol- -yl)-n-( (table . ) reported by prior et al. ( ) suggested that the psa of the molecules will control their activity and that a -cho group at their x-position, for which an indicator parameter "i" was used, will have an added advantage. while the psa may affect the activity due to a polar interaction of the molecule, the -cho group might be involved in some hydrogen bond interactions with some residue of the active site. (table . ), for which the qsar model was as shown by eq. ( . ). it was observed from eq. ( . ) that increasing value of hydrophobicity may be favorable for the activity and that the moderate dipole moment of the compound will also be conducive to the inhibition of the enzyme. however, a negative value of the indicator variable "i", which was used with a value of for compounds having a β-napthylmethyl function at r -position, indicated that such a substituent would not be preferred, probably such a substituent might create steric hindrance in the interaction of the compounds with the receptor. mohamed et al. ( ) recently reported some substituted pyrazoles and substituted pyrimidines as promising sars-cov cl pro inhibitors (fig. . ; table . ). the qsar model [eq. ( . ) ] obtained for these compounds simply suggested that highly polar fraction of the molecule with the high value of its x-component of dipole moment (d x ) will not be conducive to the activity. galasiti et al. ( ) recently reported a series of dipeptidyl norovirus cl pro inhibitors having potent inhibitory activity (table . ). the qsar model obtained for these inhibitors was as shown by eq. ( . ). this equation simply suggested that while the z-component of the dipole moment will be favorable to the activity, its moderate psa will have an adverse effect. (table . ), for which the qsar model obtained [eq. ( . )] suggested that molecules should have an optimum lipophilicity value for imparting the higher activity. the activity may be further supported by the x-component of the dipole moment and the psa of the molecule. notwithstanding, the high polar volume of the molecule will be delirious to the activity. where the activity is shown to be correlated with two indicator variables, i and i . i and i , with a value of each, indicate the presence of the ipropyl and the t-butyl moiety at r -position, respectively. the positive coefficients of both these parameters suggested that the i-propyl and the t-butyl functions at r -position will be favorable for inhibitory activity. compound behaved aberrantly and therefore it was considered as an outlier. it may be observed that the t-butyl substitution at r -position in compounds and gave better activity than the i-butyl substitution at the same position in compounds and . dragovich et al. ( a) reported a series of michael acceptor type potent hrv c pro inhibitors (fig. . ; table . ), for which the qsar model [eq. ( . ) ] exhibited that the positive effect on the activity of the compounds will be produced by the z-component of the dipole moment and the sa of the compounds till it attains an optimum value. these two properties indicate the same kind of electronic interactions of the molecule with the receptor. dragovich et al. ( b) reported a series of peptide-derived potent hrv c pro inhibitors (table . ), the qsar model [eq. ( . ) ] suggested that the activity would be primarily controlled by the hydrophobicity of the molecule. the polar volume and the total dipole moment of the compounds would also help to increase the activity of the compounds. "i" is an indicator parameter indicating the presence of i-butyl moiety at r position. its negative coefficient suggested that i-butyl function is not favorable at r -position. this might be creating some steric problem. dragovich et al. ( a) reported some ketomethylene group containing peptide-based hrv c pro inhibitors (table . it is observed from eq. ( . ) that decreasing value of the dipole moment along y-axis may increase the activity. this model also showed the importance of two indicator variables, i and i , each of which was used with a value of unity i-butyl and the i-propyl substituent at r position, respectively. the negative coefficient of i suggested that i-butyl group will not be conducive to the activity, while the positive coefficient of i suggested that i-propyl group would be favorable to the activity. these facts are observed from the i-butyl substituted compounds - and - and the i-propyl substituted compounds , - . dragovich et al. ( b) reported some hrv c pro inhibitors (table . ). the qsar model is shown in eq. ( . ) obtained for them suggested that the high polarity of the compound in the x-direction will be detrimental to the inhibition potency of the compound. the simple structure-activity relationship study of (continued ) dragovich et al. ( c) had also reported some tripeptidyl n-methyl amino acids as hrv c pro inhibitors (table . ), for which the qsar model obtained [eq. ( . ) ] had suggested that the activity will be totally governed by the total dipole moment of the compound. this indicated the strong electronic interaction between the molecule and the receptor. however, for some ketone containing tripeptidyl hrv c pro inhibitors (table . ) reported by dragovich et al. ( ) , the qsar model [eq. ( . ) ] exhibited the importance of only z-component of the dipole moment of the compound. in the z-direction, the polarity of the compound may be more favorable for electronic interaction with the receptor. webber et al. ( ) reported some depsipeptidyl hrv c pro inhibitors ( table . ), for which the qsar model obtained was as is shown by eq. ( . ). this model suggested that in this case, the polar volume of the molecule will not be conducive to the activity. = ± + ± − ± + ± − ± p c p d d i = ± + ± − ± + ± − ± + ± − ± p c p d d for some -pyridone containing peptidomimetics as promising hrv c pro inhibitors (table . ) reported by dragovich et al. ( ) , the qsar model [eq. ( . ) ] had, however, suggested that the sa of the molecule will be favorable to the activity, probably because of dispersion interaction between the active surface of the molecule and that of the receptor. however, a very high sa was shown to be detrimental to the activity, probably because of the steric problem. (table . ). for these inhibitors, the qsar model [eq. ( . ) ] indicated that the activity of compounds will simply depend upon the presence or absence of β-naphthyl group or chromene ring at the r-position of the compound. of the two indicator variables, i and i , the former with a value of indicated the presence of β-naphthyl group and the latter with a value of unity indicated the presence of chromene ring at the r-position. the positive coefficients of both the variables indicated the favorable contribution of either of the substituent. both might have steric interactions with the active site of the enzyme. dragovich et al. ( ) reported a series of -pyridone containing peptidomimetics as hrv c pro inhibitors (table . ). the qsar model obtained for these compounds was as shown by eq. ( . ). this model suggested that have less bulky molecules with small x-component of their dipole moment will be favored. simultaneously, the high value of the psa of these molecules may be conducive to activity. further, the positive coefficient of the indicator variable "i" that was defined with a value of unity for a benzyl substituent at r -position indicated that such a substituent should be desired for the better activity of the compound. this benzyl group might have steric interaction with the receptor. a total of qsar models ( for sars-cov cl pro inhibitors and for hrv c pro inhibitors) have been reported here to get an insight into the relation between the enzyme inhibitory activities of the antiviral compounds and their physicochemical and structural properties. qsar models exhibited that the physicochemical parameters, such as dipole moment, psa, polar volume, hydrophobicity, molar refractivity, sa, and molecular volume of the compounds play a crucial role in controlling both sars-cov cl pro and hrv c pro inhibitory activities. moreover, some structural indicator variables were found to play an important role for inhibition of these enzymes. in many cases, the dipole moment and the psa were found to be dominant factors. the bulk of the inhibitors and their flexibility and polarity also appeared to play crucial roles in the inhibition of the enzyme. most of the qsar models exhibited a direct correlation of dipole moment with the cl pro or c pro inhibitory activity, where a majority of them showed the positive effect of dipole moment on activity but few showed the negative effect, too. these positive and negative effects may be attributed to the orientation of the inhibitor molecules in the active site of the enzyme. the psa and the polarity of the inhibitors were some other important factors that were found in some cases to influence the activity. with their positive coefficients in the correlation, they indicated the attractive electronic interactions of the molecules with the enzyme, and with negative coefficient, they indicated the repulsive interaction. in many cases, the polar volume was found to govern the activity. the polar volume also indicated the attractive or repulsive dispersion interaction between the molecule and the receptor. among all, the hydrophobicity of the molecules had its own role. in any drug-receptor interaction, hydrophobicity is found to play an important role because in most of the enzymes the active site has a hydrophobic pocket which plays an important role in the activity of the site. in most of the cases, the bulky portion of the molecule tries to occupy this hydrophobic pocket where it may have hydrophobic interaction. the molecular volume, mw, or molar refractivity all sometimes become synonymous to the hydrophobic property of the molecules. if they are not found related to hydrophobicity and are crucial for the activity, then it means that the inhibitor-enzyme interaction involves dispersion 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papain-like novel protease inhibitors: design, synthesis, protein-ligand x-ray structure and biological evaluation papain-like protease (plpro) inhibitory effects of cinnamic amides from tribulus terrestris fruits key: cord- - jd gn authors: karra, nour; dolinski, rina; akria, luiza; yampoulski, yevgeni; awad, jamal title: a case of hemophagocytic lymphohistiocytosis associated with mediterranean spotted fever in a healthy -year-old female date: - - journal: open forum infect dis doi: . /ofid/ofaa sha: doc_id: cord_uid: jd gn a -year-old female presented with fever, headache, and epigastric pain. though her initial presentation was benign and nonspecific, she soon developed a full-blown cytokine storm with disseminated intravascular coagulation. she was diagnosed with hemophagocytosis secondary to rickettsia conorii infection. a good outcome was achieved thanks to prompt diagnosis and proper treatment. hemophagocytic lymphohistiocytosis (hlh) is a rare, potentially life-threatening syndrome caused by excessive immune activation and cytokine storm [ , ] . cardinal features include fever, hepatosplenomegaly, cytopenias, and histological hemophagocytosis [ , ] . primary genetic hlh is caused by a mutation in a component of the perforin-mediated cytotoxicity pathway and may be either familial or associated with genetic immunodeficiency syndromes (chediak-higashi, griscelli type- , and x-linked lymphoproliferative disorder) [ ] . most commonly it affects infants, and it may also be triggered by a viral infection, particularly epstein barr virus (ebv) [ ] . secondary or acquired hlh may occur in adults sporadically, in association with many triggers, namely infectious, autoimmune, and malignant diseases [ ] . it is believed that the incidence of secondary hlh has been rising in recent years, perhaps due to increased awareness. in the absence of a proven relevant mutation, the henter criteria have been proposed and are commonly applied to diagnose hlh [ , , ] . we describe a case of acquired hlh secondary to mediterranean spotted fever (msf) caused by rickettsia conorii in a -year-old healthy female in the north of israel. the clinical manifestations, workup, differential diagnosis (dd), and outcome in this patient are described and discussed. a -year-old jewish female presented to the emergency room at the galilee medical center nahariya in northern israel with a -week history of headache and days of fever (> . °c). she had also complained of diarrhea lasting days at a prior presentation that had resolved spontaneously. she had been previously healthy with an uneventful medical history. at presentation, she was tachycardic ( bpm) with fever ( . °c). otherwise she was in a good clinical shape, with no alarming signs. her laboratory results were unremarkable (table ) . her beta hcg was negative. she was symptomatically treated with clinical improvement and subsequently discharged with a presumed diagnosis of a mild viral illness. two days later, she returned with persisting complaints of headache, fever, and chills. in addition, she mentioned epigastric pain, nausea, and vomiting. she had been self-medicating with ibuprofen for days. except for tachycardia ( bpm), her vital signs were normal. blood tests revealed a normal white blood cell count and hemoglobin (hb), but she now had thrombocytopenia (platelets [plt] × /ul), mild hyponatremia ( mmol/l), and hypokalemia ( . mmol/l) with a c-reactive protein of mg/l (table ) . chest and abdominal x-rays were unrevealing. due to persisting symptoms and abnormal labs, she was admitted for further evaluation. upon admission, she was tachycardic ( bpm) with a fever of °c. in repeated anamnesis, she denied any travels, exposure to unhealthy individuals, or contact with animals. on physical examination, there was only mild epigastric tenderness without rebound. blood cultures and viral serologies were drawn. due to increased hepatocellular enzymes and slightly increased bilirubin (table ) , the dd included viral hepatitis, acute cholecystitis/cholangitis, or a liver injury secondary to nonsteroidal anti-inflammatory drugs. an abdominal ultrasound showed only a normal size gallbladder with thickened walls ( cm). acute cholecystitis was the most likely diagnosis. ceftriaxone and metronidazole were initiated. twelve hours later, she began deteriorating: her blood pressure dropped, and lab tests showed metabolic acidosis, acute kidney injury, hepatocellular and cholestatic injury with rising bilirubin, prolonged pt, and low fibrinogen ( mg/dl). she had anemia (hb . g/dl), deteriorating thrombocytopenia ( × /μl), and leukocytosis with a left shift ( figure ). schistiyocytes were observed on a blood smear. a diagnosis of disseminated intravascular coagulation (dic) was made, and she was treated with fresh frozen plasma and cryoprecipitate. following blood product transfusion, the patient stabilized. she later developed skin necrosis on the dorsal side of her feet and was treated with intravenous heparin. an urgent computed tomography scan excluded emphysematous cholecystitis or visceral perforation as a cause of dic. significantly thickened gallbladder and periportal edema were demonstrated. re-evaluating the dd, a wide battery of serological tests was taken (table ) . sepsis was the most likely diagnosis. due to thrombocytopenia, hyponatremia, and headache, an atypical infection such as rickettsiosis was considered, and doxycycline treatment was empirically initiated. interestingly, ferritin levels were dramatically elevated ( ng/ml), and fibrinogen levels were still particularly low ( mg/dl). a bone marrow biopsy performed on day of admission exhibited evident hemophagocytosis ( figure ). natural killer (nk) cell activity was preserved ( %), but soluble il- receptor (sil- r) levels were elevated ( u/ml; normal: - u/ml). a diagnosis of hlh was established. as there was no family history of primary hlh, immune deficiencies, or perforin pathway mutations, a plethora of possible triggers was evaluated. rheumatologic and infectious serologies were all negative (table ) . adamts- antibodies and activity were within the normal range. blood, urine, and stool cultures were also negative. blood polymerase chain reaction (pcr) was positive for rickettsia conorii. the hlh- treatment protocol (dexamethasone and etoposide) was immediately initiated, in addition to doxycycline. subsequently, the patient gradually improved during the next few weeks, and her labs normalized ( figure ). an increasing number of acquired hlh cases have been reported in recent years [ ] . however, it has been suggested that hlh may go undiagnosed due to a varying clinical presentation, poor reproducibility of the henter criteria, and insufficient awareness in medical caregivers of this syndrome [ ] . indeed, several challenges might hinder diagnosis [ ] : first, the hlh- diagnostic criteria were initially developed based on pediatric familial cases, and even though widely applied, they have not been validated in adults or in secondary hlh. second, many diagnostic criteria are nonspecific (fever, cytopenias, hypofibrinogenemia) and may be encountered contrarily, hlh patients frequently exhibit findings other than those included in the diagnostic criteria (hypoalbuminemia, elevated liver enzymes, coagulopathy). the clinical overlap between hlh, sepsis, and multi-organ dysfunction syndrome may lead to hlh misdiagnosis, particularly in an intensive care unit setting. finally, without specific markers such as sil- r levels, nk cell activity, or histological hemophagocytosis, these syndromes could be clinically indistinguishable. needless to say, these markers are neither tested routinely nor readily available at medical centers [ , ] . in the present case, the clinical presentation was indeed nonspecific and dynamic. through the course of illness, the patient developed clinical and radiologic findings suggestive of acute acalculus cholecystitis. retrospectively, these findings could be explained by periportal and hepatic infiltration of macrophages and lymphocytes [ ] . upon deterioration, she was re-evaluated, and a comprehensive dd was considered. fever and multi-organ failure in a previously healthy individual are attributed with a high probability to sepsis. as already mentioned, sepsis is a great mimicker of hlh. padhi et al. previously reported that in out of reviewed cases of secondary hlh, a diagnosis of sepsis was initially presumed [ ] . in the present case, the dramatic rise in ferritin beyond ng/ml ("sky high" hyperferritinemia) was suggestive of hlh [ ] . microangiopathic hemolytic anemia and thrombocytopenia could also be attributed to hemolytic uremic syndrome (hus) or thrombotic thrombocytopenic purpura (ttp). however, the multi-organ failure combined with coagulopathy could not be well explained by hus or ttp. nevertheless, a normal admats activity ruled out ttp. although diagnosis of hlh is quite challenging, our patient eventually fulfilled of the henter criteria: fever, bicytopenia, hyperferritinemia, hypertriglyceridemia with hypofibrinogenemia, histologic hemophagocytosis, and elevated sil- r levels. when hlh diagnosis was established, a trigger was sought. pcr was positive for rickettsia conorii, although serologies were negative. this was attributed to the fact that serologies were examined early during the disease course (day / since initial symptom onset). early manifestations of msf commonly include fever, headaches, myalgias, and sometimes nausea and vomiting. a maculopapular rash usually follows, predominantly on the palms and soles, with rickettsia conorii. occasionally an inoculation eschar may be found [ ] . thrombocytopenia, leukopenia, hyponatremia, and increased hepatocellular enzymes are common findings [ ] [ ] [ ] [ ] . indeed, our patient presented with many of these symptoms. however, rash and eschar were not found. in a study by lecronier et al., in only out of msf cases was a rash not present [ , ] . in israel, the common variant is israeli spotted fever (isf) caused by rickettsia conorii subsp. israelensis. the clinical manifestations are similar to msf; however, tick exposure is reported in %, and an eschar is rarely observed ( %) [ ] . unfortunately, sequencing for the specific subspecies of rickettsia conorii was not performed in the present case. ramos-casals reported a total of identified cases of adult hlh in the literature, half of which were triggered by infections [ ] . most cases of bacteria-associated hlh were due to intracellular organisms, primarily tb. notably, < cases of hlh secondary to msf have been reported [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table ) . most cases were reported in france [ , , ] , with others in spain [ ] , italy [ ] , and sri lanka ( table ) [ ] . in nearly all cases, patients presented with fever and a maculopapular rash. headache, nausea, vomiting, and myalgia were also prevalent. interestingly, of the earliest hlh cases caused by rickettsia conorii was reported in israel in [ ] . israel is an endemic country of rickettsia conorii [ ] . thus, in the present case doxycycline was initiated early and based on clinical suspicion only, even though the classical rash and exposure were absent. this clinical approach turned out to be life-saving. other pathogens from the rickettsia family have also been associated with hlh (table ) [ ] . in fact, hlh has been primarily reported with scrub typhus and ehrlichiosis and to a lesser extent with rickettsia conorii and rickettsia japonica (table ) [ , ] . a recent comprehensive review on scrub typhus-associated hlh identified cases, all in the far east [ ] . in adults, treatment mostly relied on rickettsia-specific antibiotics, while only in % were other treatments added ( table ) . as for ehrlichiosis-induced hlh, there are substantial reports [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , mostly in the united states. ten cases in immunocompetent adults have been reported with a good outcome upon doxycycline treatment (table ) [ ] . the pathogenesis of rickettsia-induced hlh is not completely elucidated. the underlying common mechanism in both genetic and reactive hlh is a defective granule-mediated cytotoxicity. uncontrolled activation of t cells and antigenpresenting cells (macrophages and histyocytes) is thought to be caused by enhanced antigen presentation and continuous stimulation of toll like receptors by interferon-ɣ [ , ] . rickettsia is an intracellular pathogen. as such, following phagocytosis, it may trigger an inappropriate th- response, activating macrophages and significant cytokine release. concurrent deficient cytotoxicity of nk and cd cells may result in persistent lymphocyte and histyocyte activation and excessive levels of tumor necrosis factor-α, interferon-ɣ, macrophage colony-stimulating factor, interleukin (il)- , il- , il- , il- , and il- , all important markers of the cytokine storm seen in hlh [ ] . treatment of hlh is generally based on support measures, trigger elimination, and immunosuppressive therapy. however, currently, there are no randomized controlled table trial-based guidelines regarding the ultimate treatment protocol in adults. therapeutic decisions are practically based on clinical experience, expert opinion, and on a case-by-case approach [ ] . the hlh- protocol has been mainly established as a consensus for primary genetic hlh [ ] . in adults, treatment of secondary hlh should be tailored according to the underlying trigger. a treatment algorithm has been recently suggested by la rosée et al. [ ] : in clinically stable patients, it is acceptable to specifically target treatment to the underlying condition. however, in deteriorating patients, corticosteroids may be initiated with the possible addition of intravenous immunoglobulin (ivig). a clear indication for immediate etoposide administration is severe hlh presenting with imminent organ failure [ ] . upon trigger identification, several additional treatments are possible: in ebv-associated hlh, virostatics and rituximab may be added. in leishmaniasisassociated hlh, liposomal amphotericin b is suggested. cytokine-directed agents (anti-il , anti-il ) have emerged in recent years as add-on treatments in macrophage-activating syndrome (mas; ie, hlh secondary to rheumatologic conditions). conventionally, mas is treated with pulse steroids, with the possible addition of cyclosporin a and anakinra (anti-il ) [ ] . etoposide may be added in nonresponsive cases. tocilizumab (anti-il ) may be used in mas and druginduced hlh [ ] . tocilizumab has also been reported as an adjuvant therapy for hlh associated with visceral leishmaniasis [ ] and has been recently employed to treat the hlhresembling hyperinflammation associated with sepsis and covid- [ ] [ ] [ ] . there is a strong consensus that hlh induced by intracellular infections (tuberculosis, leishmaniasis, rickettsiosis) usually does not need hlh- treatment [ ] . notably, most reported cases of rickettsiosis-associated hlh have been successfully treated with only specific antibiotics (mostly doxycycline). however, in acutely ill deteriorating patients, treating the underlying infection may not always be sufficient. in a few reported cases, corticosteroids, ivig, and occasionally anakinra were added (table ) . although prognosis varies between studies, if left untreated, hlh is invariably fatal. in adults, prognosis is worse in those with an underlying malignancy (particularly lymphoma), alcoholism, g pd deficiency, age > years, neurologic involvement, and high ferritin with a slow decline [ , [ ] [ ] [ ] . specifically, the prognosis of msf-associated hlh may depend on the specific rickettsia subspecies, the delay in antibiotic treatment, and the use of immunosuppressive drugs [ ] . an excellent outcome has been reported in msf-associated hlh, in most cases without additional treatment other than antibiotics [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nevertheless, as our patient presented with rapidly deteriorating multi-organ failure and dic, an aggressive treatment approach was opted for. dic is considered a severe complication and has been previously reported as an independent predictor of high mortality in hlh [ ] . here, dic was diagnosed early. skin gangrene in the lower extremities also indicated the severity of our patient's disease. cohen et al. reported a case of isf complicated by dic and purpura fulminans where the patient eventually died [ ] . similarly, a case of jsf complicated by hlh and dic was fatal despite maximal treatment [ ] . in contrast, a good outcome was achieved here. this may be attributed to the patient's young age, baseline well-being, early recognition and treatment of dic, and most importantly prompt initiation of doxycycline and the hlh- protocol. hlh is an elusive clinical syndrome. this case underscores the importance of having a high index of suspicion both for hlh and for msf as a triggering infection in endemic areas, even when the classical rash and exposure details are lacking. hypercytokinemia in familial hemophagocytic lymphohistiocytosis cytokine production regulating th and th cytokines in hemophagocytic lymphohistiocytosis adult haemophagocytic syndrome review of haemophagocytic lymphohistiocytosis hlh- : diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis how i treat hemophagocytic lymphohistiocytosis hemophagocytic lymphohistiocytosis: critical reappraisal of a potentially under-recognized condition diagnostic challenges of hemophagocytic lymphohistiocytosis 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of literature human monocytic ehrlichiosis complicated by hemophagocytic lymphohistiocytosis and multi-organ dysfunction syndrome macrophage activation syndrome secondary to human monocytic ehrlichiosis ehrlichiosis presenting with toxic shock-like syndrome and secondary hemophagocytic lymphohistiocytosis hemophagocytic lymphohistiocytosis: an unreported complication of ehrlichiosis in adults diagnosis and treatment of hemophagocytic lymphohistiocytosis in an adult patient with ehrlichiosis hemophagocytic lymphohistiocytosis (hlh) secondary to ehrlichia chaffeensis with bone marrow involvement ehrlichia-induced hemophagocytic lymphohistiocytosis in two children use of extracorporeal support in hemophagocytic lymphohistiocytosis secondary to ehrlichiosis recommendations for the management of hemophagocytic lymphohistiocytosis in adults tocilizumab as an adjuvant therapy for hemophagocytic lymphohistiocytosis associated with visceral leishmaniasis cytokine release syndrome in severe covid- : interleukin- receptor antagonist tocilizumab may be the key to reduce mortality tocilizumab for the treatment of severe covid- pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of patients in hlh across speciality collaboration, uk. covid- : consider cytokine storm syndromes and immunosuppression prognostic factors of early death in a cohort of adult haemophagocytic syndrome: impact of triggering disease and early treatment with etoposide prognostic factors and outcomes of adults with hemophagocytic lymphohistiocytosis clinical characteristics, prognostic factors, and outcomes of adult patients with hemophagocytic lymphohistiocytosis potential conflicts of interest. there are no conflicts of interest to declare. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed.patient consent. the patient's consent was obtained. ethical approval. a local ethical committee approval does not apply in this case. key: cord- -ouvn r authors: chow, michael y.t.; qiu, yingshan; lam, jenny k.w. title: inhaled rna therapy: from promise to reality date: - - journal: trends pharmacol sci doi: . /j.tips. . . sha: doc_id: cord_uid: ouvn r rna-based medicine is receiving growing attention for its diverse roles and potential therapeutic capacity. the largest obstacle in its clinical translation remains identifying a safe and effective delivery system. studies investigating rna therapeutics in pulmonary diseases have rapidly expanded and drug administration by inhalation allows the direct delivery of rna therapeutics to the target site of action while minimizing systemic exposure. in this review, we highlight recent developments in pulmonary rna delivery systems with the use of nonviral vectors. we also discuss the major knowledge gaps that require thorough investigation and provide insights that will help advance this exciting field towards the bedside. michael y.t. chow, , , yingshan qiu, , and jenny k.w. lam , * rna-based medicine is receiving growing attention for its diverse roles and potential therapeutic capacity. the largest obstacle in its clinical translation remains identifying a safe and effective delivery system. studies investigating rna therapeutics in pulmonary diseases have rapidly expanded and drug administration by inhalation allows the direct delivery of rna therapeutics to the target site of action while minimizing systemic exposure. in this review, we highlight recent developments in pulmonary rna delivery systems with the use of nonviral vectors. we also discuss the major knowledge gaps that require thorough investigation and provide insights that will help advance this exciting field towards the bedside. the diverse roles of rna in the body have led to the emergence of different approaches to harnessing rna for therapeutic use. rna therapeutics can be broadly divided into three functional classes: (i) inhibition of gene expression [e.g., small interfering rna (sirna; see glossary), microrna (mirna), and antisense oligonucleotide (aso)]; (ii) protein encoding (e.g., mrna); and (iii) protein targeting (e.g., rna aptamers) [ ] . despite their diverse mechanisms of action, it is no secret that the biggest barrier to all types of rna therapeutic is delivery; that is, to bring therapeutic rna molecules into the target cells effectively in a safe and reproducible manner. with the us food and drug administration (fda) approval of the first two sirna therapeutics, patisiran and givosiran, both of which target hepatic disorders, the field of rna therapy is ready to look for applications beyond the liver [ , ] . there is an increasing number of studies that report the potential of rna in treating a range of lung diseases including asthma [ ] , cystic fibrosis (cf) [ ] , lung cancer [ ] , and respiratory infections [ ] . inhalation of aerosol is an efficient way to deliver rna to the lung by maximizing local concentration while minimizing systemic exposure. aln-rsv , designed to treat respiratory syncytial virus (rsv) infection, was the first sirna candidate to be delivered through the pulmonary route in clinical trials in [ , ] . since then, several clinical trials on inhaled rna therapy have been initiated (box ). however, no inhaled rna therapeutic has yet been approved for use in clinics. naked rna for inhalation rna is a negatively charged, hydrophilic macromolecule that is incapable of permeating the cell membrane. it is vulnerable to degradation before reaching the target sites due to the abundance of rnase in the body. therefore, it has to rely on delivery vectors to protect it from premature degradation and facilitate its cell entry. interestingly, it has been known for over a decade that naked rna, including both sirna and mrna, can be transfected in the lung following pulmonary delivery, as shown in many in vivo studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . given that the lung comprises various cell types with distinct functions, it is crucial to understand which cell types are susceptible to naked rna transfection for effective clinical translation. to address this issue, ng et al. carried out a comprehensive investigation on the distribution and activity of naked sirna in the lung of mice following intratracheal administration [ ] . the silencing activity of naked sirna was most prominent in lung epithelial cells, dendritic cells, and alveolar macrophages. similar observations were reported by other groups in which the activity of naked sirna was primarily found in lung epithelial cells but not the endothelial cells [ ] , making it attractive for use in lung conditions affecting these cell types without systemic exposure, for example in rsv infection and cf. ng et al. stressed that chemical modification is crucial for naked sirna to induce effective gene silencing by improving metabolic stability and reducing immunostimulation [ ] . using modified naked mrna, tiwari et al. successfully developed a mrna-based approach to express neutralizing antibodies in the lung via intratracheal delivery to prevent rsv infection in mice [ ] . the authors also compared naked mrna with the use of polyethyleneimine (pei, a synthetic cationic polymer discussed later) derivatives as delivery vectors and noticed that the transfection efficiency of naked mrna was either better than, or comparable to, these polymers. despite the promising effect of pulmonary naked rna delivery, the exact mechanism of how naked rna crosses the cell membrane barrier in the lung remains unclear, although it has been suggested that the pulmonary surfactant has a significant role in facilitating rna uptake [ , ] . some studies also showed that the use of delivery vectors could significantly improve rna transfection compared with naked rna in the airways [ ] [ ] [ ] [ ] [ ] . the development of safe and effective inhaled delivery systems in parallel is paramount currently. with the recent success of sirna in the clinic and the intensive investigation of mrna in clinical trials, including mrna vaccines against coronavirus disease (covid- ) [ , ] , we believe that these two rna candidates are likely to be the first to enter the clinic for treating lung diseases. therefore, in this review, we focus on the pulmonary delivery of sirna and mrna. we glossary aerosol: suspension of solid particles or liquid droplets in gas. in humans, drug delivery via the pulmonary route has to be administered in the form of an aerosol. antisense oligonucleotide (aso): short single-stranded dna or rna (~ nucleotides in length) that binds to a target mrna through complementary base pairing, activating rnase h that leads to degradation of mrna, thereby preventing the translation of mrna into protein. cell-penetrating peptide (cpp): short cationic or amphipathic, natural or synthetic peptide (usually < amino acids) that is developed to deliver large cargoes, such as proteins, peptides, and nucleic acids, into cells by promoting cellular uptake. chemical modification: strategy to improve the stability and/or reduce immunogenicity of rna by modifying the structure of rna while maintaining the biological activity of the molecules (e.g., methylation of the ribose '-oh group; alteration of the bases; modification of phosphodiester backbone, etc.). intratracheal administration: introduction of substances directly into the trachea, either through the oral cavity via intubation or through a surgical procedure that creates an incision in the trachea (tracheotomy). microrna (mirna): a short rna (~ - nucleotides in length) that is partially complementary to multiple messenger rna (mrna), preventing the translation of mrna into protein through the rna interference mechanism. mrna vaccine: mrna that encodes the target antigen to elicit immune responses in the body. naked rna: rna that is not associated with any delivery vectors or transfection agents, such as polymers and lipids. nebulization: conversion of liquid medications into a spray of fine droplets that can be breathed in by the patient. nonviral vector: agent or vehicle that transports nucleic acids into the cells without involving the use of viruses. parenteral: nonoral route of drug administration; usually refers to injection or infusion of drug directly into the body, bypassing the skin and mucous membranes. rna aptamer: singled-stranded rna oligonucleotide that serves as ligand and binds to specific targets (e.g., proteins aln-rsv is a naked sirna targeting the rsv nucleocapsid protein for the treatment of the associated viral respiratory infection. it was the first sirna investigated for pulmonary delivery in clinical trials. rsv causes significant illness in immunocompromised patients following lung transplantation, and bronchiolitis obliterans syndrome (bos) is the major cause of morbidity and mortality in these patients [ ] . the phase i clinical trial (nct ) started in and demonstrated that aln-rsv was well tolerated following intranasal administration. the phase iib clinical trial (nct ) showed that aerosolized aln-rsv was effective in reducing the incidence of new or progressive bos in lung transplant patients with rsv infection following inhalation. although aln-rsv failed to progress to a phase iii trial, it marked an important milestone of inhaled rna therapy [ , [ ] [ ] [ ] . excellair is an sirna targeting spleen tyrosine kinase (syk), which is involved in the inflammatory response in the lung epithelium [ ] . it was investigated for the treatment of asthma by inhalation. the phase i trial began in . there was little information published about the outcome of the study, although it was reported that the drug was well tolerated in patients with asthma. the phase ii trial was discontinued in [ ] . eluforsen is a single-stranded rna aso targeting cf transmembrane conductance regulator (cftr) for inhalation to patients with f del cf. the phase i clinical trial (nct ) initiated in showed that cftr activity was restored after intranasal administration of eluforsen. the phase ib clinical trial (nct ) was completed in and demonstrated that inhaled eluforsen was safe, well tolerated, and improved respiratory symptoms in patients with f del cf [ , ] . however, no further clinical development is planned for this candidate. mrt is the first inhaled mrna candidate for cf and delivers mrna encoding fully functional cftr protein. the phase i/ii clinical trial was initiated in may to test the safety and tolerability of mrt . patients with cf received mrna encoding fully function cftr protein through nebulization. interim results were encouraging, showing that mrt was well tolerated at low and mid-dose levels ( - mg) with no serious adverse events reported at any dose level (up to mg). there was a marked improvement of lung function in patients after single dose of mrt at the middose level ii . in early , the fda granted fast track and rare pediatric disease designations for mrt for the treatment of cf iii,iv . discuss and highlight the recent advances of nonviral vector-based pulmonary rna delivery systems development, gather what we have learnt from these studies, and identify the major gaps of knowledge. with these, we provide insights and direction to move the field forward. many rna delivery vectors have been developed for pulmonary delivery and their major functions are to facilitate the uptake of rna by target cells and to protect rna from premature degradation. selected recent studies with different rna delivery vectors that have demonstrated in vivo transfection in animal models are summarized in table . we highlight some important studies in each category and discuss them in more detail herein. lipid-based delivery systems due to their high transfection efficiency, ease of synthesis, and low batch variability, lipids were popular as transfection agents during the early years of gene therapy studies for various routes of administration [ , ] . the transfection efficiency and toxicity of lipid-based systems are affected by the composition of lipids and the ratio of lipids to rna. typically, cationic lipids, such as n-[ -( , -dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (dotma) and , -dioleoyl- -trimethylammonium-propane chloride salt (dotap), are used to form lipoplexes with, or encapsulate, rna [ ] . the addition of neutral helper lipids, such as dioleoyl phosphatidylethanolamine (dope) and cholesterol, in the lipoplexes provide the ability to facilitate rna complexation, increase stability of the lipoplexes, and reduce toxicity [ , ] . one major problem associated with lipid-based systems for pulmonary delivery is their poor structural stability because they readily fuse with pulmonary surfactants in the airways [ ] , leading to premature release of rna before cellular uptake. lipid nanoparticles (lnps), which comprise cationic lipids, cholesterol, and polyethylene glycol (peg), have been developed to improve the structural stability of lipid-based systems [ , ] (table ) . with proper design and engineering, they can encapsulate rna efficiently. lnps are already in use in the clinic for parenteral injection of sirna [ ] and also are in an ongoing clinical trial to deliver mrna (mrt ) to the lung through nebulization for the treatment of cf, with encouraging early results (box ). there are two main categories of polymers: natural and synthetic polymers. natural polymers have the advantages of excellent biocompatibility, biodegradability, and safety profiles [ ] . derived from the shells of crustaceans, the natural polymer chitosan is commonly investigated for pulmonary delivery due to its mucoadhesive and mucopermeable properties, enabling it to cross the mucus layer in the airways efficiently [ ] . it can be used to form polyplexes with rna or as a coating layer on the surface of nanoparticles [ , ] (table ) . however, chitosan is limited by its poor solubility at physiological ph and relatively low transfection efficiency [ ] . to overcome these problems, water-soluble chitosan derivatives, such as piperazine-substituted chitosan, were developed and found to be efficient for pulmonary sirna delivery in healthy mice [ ] (table ) . furthermore, inhalable chitosan/sirna dry powder formulations were successfully prepared by supercritical drying [ ] and spray-freeze drying [ ] (table ) . both studies demonstrated a gene-silencing effect of the powder formulations in lung tissues following intratracheal administration in mouse models of lung cancer, taking these delivery systems one step closer to clinical application. among the synthetic polymers, pei is extensively studied due to its high cationic charge density, good aqueous solubility, and wide ph-buffering capacity [ ] . its high versatility allows it to be functionalized to achieve specific targeting. for example, transferrin-pei was used to target activated t cells in the lung, particularly t helper cells, as potential therapy for asthma by reducing and nucleotides) with high affinity and specificity. small interfering rna (sirna): short double-stranded rna (~ - nucleotides in length with two 'overhang nucleotides) in which the antisense strand binds to the target mrna through complementary base pairing, preventing the translation of mrna into protein via rnai. transfection: a process of introducing nucleic acids into cells artificially. (table ) . however, the relatively high toxicity of this nonbiodegradable polymer remains a considerable concern, even with a low-molecular-weight pei, rendering it difficult to be translated for clinical application. thus, biodegradable synthetic polymers were developed to address this issue. for example, in a recent study, hyperbranched poly(beta amino esters) (hpbaes) [ ] were used to deliver mrna through nebulization, with promising gene expression observed in the lung epithelium of mice without local or systemic toxicity after repeated dosing (table ) . to enhance rna delivery efficiency for lung cancer therapy, yan et al. used a combinatorial library of functional polyester and high-throughput screening to identify matching cancer cells for specific targeting [ , ] (table ) . after screening a library of > polyester candidates, synthetic amine a modified polyester pe k-a was found to be potent for sirna delivery to mice with lung tumors [ ] (table ) . cell-penetrating peptides (cpps) have attracted increasing attention for rna delivery due to their versatility and cell entry ability [ ] . the design of peptide sequence can be inspired by natural peptides, proteins with known functions, or by computational simulation [ , ] . the sequence of amino acids determines the properties of peptides, such as structure, charges, solubility, and polarity, which further affect the interaction with rna, cellular uptake, toxicity, and transfection efficiency. one of the drawbacks of cpps is the lack of cell specificity, which can be addressed by introducing cell-targeting sequences [ ] . furthermore, natural l-amino acids are susceptible to protease degradation; although strategies such as replacing l-amino acids with d-analogs has been proposed, this approach may lower the efficiency of the peptides [ ] . although many cpps appeared to be promising candidates for delivering rna to the lung, only a few have shown in vivo transfection. welch et al. developed disulfide-constrained cyclic amphipathic peptides that form complexes with sirna with good transfection efficiency because the disulfide bond reduction in the cytosol facilitated the release of the cargo as well as proteolytic clearance. efficient gene knockdown was also observed in lung tissues of healthy mice following pulmonary delivery [ ] ( [ , ] . the pegylated kl peptide was also used to formulate mrna as inhalable dry powder by spray-drying and spray-freeze drying techniques, with efficient gene expression observed in the lung of healthy mice [ ] (table ) . to overcome the limitations of a single class of delivery vector, hybrid delivery systems, which are defined as the combination of two or more delivery vectors into a single entity, have been investigated and developed. this formulation strategy aims to increase the strengths of these delivery vectors while decreasing their disadvantages, with lower toxicity compared with their precursors [ ] . the combination of lipids with polymers intends to address the problems of poor structural stability associated with lipids and the low biocompatibility of polymers. thanki et al. developed lipid-polymer nanoparticles (lpns) comprising a poly(lactic-co-glycolic acid) (plga) matrix core coated with lipidoid, with sirna localized in both the core and the shell [ ] . this design compensated the low sirna-loading capacity of plga by introducing cationic lipidoids while controlling the rate of sirna release through degradation of the polymer. enhanced lung retention upon pulmonary administration of the sirna-loaded lpns was observed in animal studies, suggesting their potential for controlled sirna delivery in the lung (table ) (table ). this hybrid system demonstrated effective gene silencing in animal models of pulmonary fibrosis with good stability and low toxicity. another common hybrid strategy is to combine lipids with peptides, with the latter serve as a hydrophilic group and facilitate cellular uptake and transportation. for example, cationic liposomes comprising dotma/dope were blended with epithelial-targeting peptides to deliver sirna to the lung of healthy mice, leading to successful silencing of the epithelial sodium channel at the airway epithelium [ , ] (table ). the results demonstrated the potential application of this hybrid system for cf therapy. similar to lipid-peptide hybrids, the major aim of combining polymers with peptides is to enhance the cellular transport efficiency. polymers are usually conjugated covalently with the peptides. feldmann et al. developed a polymer system called viper (virus-mediated polymer for endosomal escape), which comprised a cationic methacrylated-based copolymer for sirna binding and a membrane lytic peptide melittin, to facilitate endosomal escape [ ] ( table ). the hybrid system demonstrated more effective gene-silencing effects in the lung of healthy mice compared with the unmodified polymer system. guan et al. developed a multimodular synthetic peptide with anchor, cationic, and targeting moieties that can form complexes with biocompatible poloxamine-based copolymers and mrna via self-assembly [ ] (table ) . these ternary complexes showed excellent mrna expression in the lungs of mice with cf with negligible toxicity, making them an attractive gene delivery system for cf and other lung diseases. functionalized nanoparticles in addition to the aforementioned conventional delivery vectors, inorganic metal-based nanoparticles have also been evaluated for the pulmonary delivery of rna. however, these nanoparticles cannot act as transfection vectors by themselves due to their lack of rna-binding ability. therefore, they are usually functionalized on the surface to enhance the transfection efficiency. for example, gold nanoparticles are attractive delivery vectors because of their ease of synthesis and conjugation as well as their superior stability [ ] . gold nanoparticles were conjugated with sirna through gold-thiol bonds and functionalized with m pep, a peptide that selectively targets tumor associated macrophages [ ] (table ) . specific gene-silencing effects were observed in targeted macrophages following intratracheal administration in a mouse lung tumor model. it has been suggested that pulmonary surfactant facilitates the delivery of polymer-based delivery systems in the lung [ ] . to take advantage of this phenomenon, pulmonary surfactant and surfactant protein b-coated dextran-based nanoparticles were developed for sirna delivery, with successful gene-silencing effects observed in healthy mice and in a model of acute lung injury (ali), respectively, following pulmonary administration [ , ] (table ) . exosomes are extracellular vesicles secreted in many cell types. they are involved in communication through the transfer of substances, such as lipids, proteins, and nucleic acids, between cells [ , ] . they have many desirable features for rna delivery, including high biocompatibility with low inherent toxicity and immunogenicity [ ] . zhang et al. presented a new approach to delivering small rna to the lung with the use of exosomes. sirna was loaded into serum-derived exosomes via calcium-mediated transfection. following intratracheal administration, the sirnaloaded exosomes were successfully taken up by lung macrophages to achieve specific gene silencing in a mouse model of ali [ ] (table ) . we carried out a survey of studies published between and early that reported sirna or mrna transfection following pulmonary delivery in animals ( figure ) . a pubmed search was performed using the search terms 'sirna' or 'mrna', 'pulmonary delivery', 'intratracheal', 'inhalation', 'nebulization', and with the filters 'last years' (publication date) and 'other animals' (species) . in total, articles were included in the survey [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . each article was categorized manually according to the type of rna delivery vector used or naked rna; the animal model (disease or healthy); the method of administration to animal; and the type of rna (sirna or mrna). while polymer was the most commonly used rna delivery vector due to its high versatility and ease of preparation, the hybrid delivery system is gaining popularity, many of which include a targeting peptide to improve specificity. with the successful transfection of naked rna in the lung and the technological advances in chemical modifications that greatly enhance the stability and reduce the immune activation of rna [ , ] , many researchers opt for the use of naked rna to transfect lung tissues due to its simplicity ( figure a ). this approach can also eliminate the risk of toxicity and immunogenicity associated with the delivery vectors. it is particularly popular when researchers are interested in examining the biological function of a particular protein rather than its therapeutic potential, such as the role of yes-associated protein (yap), ribosomal protein s (rps ), and pi k/sgk pathway in ali [ , , ] . nevertheless, the successful use of naked rna should not lead us to discard the use of delivery vectors for clinical applications. the transfection efficiency of naked rna may not be robust enough for therapeutic use. also, as mentioned earlier, the cellular uptake mechanism of naked rna in the airways is still unclear. it has been speculated that lung lining fluid and pulmonary surfactants have a critical role in facilitating the transportation of rna into cells [ ] . the composition of lining fluids could vary substantially among patients and pathological conditions. indeed, it has already been observed that pulmonary surfactant protein and lipid composition change significantly as a result of aging [ ] . lung diseases, such as cf, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease (copd), are also associated with surfactant deficiency and altered lipid compositions [ ] [ ] [ ] . consequently, the transfection efficiency of naked rna could be easily influenced by the age and disease status of the patients, leading to problems of reproducibility. moreover, naked rna lacks active targeting ability. given that the lung tissues contain a variety of cell types, it would be desirable to use a delivery vector with a targeting moiety to enhance specificity while reducing transfection in unintended cell types. the potential of rna therapeutics to treat a range of lung diseases prompts us to examine the possibility of developing a universal delivery platform that can be adopted by different rna therapeutics for different conditions. while such a system would accelerate the clinical translation of inhaled rna therapy, delivery barriers could be unique to a specific physiological condition. in that case, it would be more effective to have a carefully designed system to overcome these specific sets of barriers. for example, patients with cf have thick and excessive mucus in the airway; hence, a delivery vector with excellent mucus penetration ability would be favorable. moreover, in preclinical studies, healthy animals are sometimes used to evaluate the efficiency of a delivery vector, with the use of reporter or housekeeping genes as the rna target ( figure b) . this approach may run into a similar problem in that a delivery vector may perform differently in a healthy animal versus a disease model due to the different delivery barriers in the airways. furthermore, a non-disease rna target (which is generally used to evaluate the efficiency of a delivery vector) does not provide any information regarding the pharmacodynamics properties of the proposed therapy. even if a disease model is used, there is still the challenge of the clinical translation because many lung disease models have limitations that prevent their direct translation to human disease [ ] [ ] [ ] [ ] . for instance, asthma is a complex condition that is observed exclusively in humans. the most common model used in preclinical studies is murine allergic airway inflammation. however, the distribution of lung inflammation in mice is different from human asthma, and the animals develop tolerance after repeated allergen exposure [ ] . therefore, a collaborative approach between formulation scientists, pharmacologists and clinicians will facilitate the development of a clinically relevant delivery system for inhaled rna therapy. many studies have focused on the development of rna delivery vectors, but the translation of these vectors into suitable dosage forms for clinical application is currently lacking. here, we summarize the critical steps for the development of successful pulmonary rna delivery system ( figure , key figure) and discuss the areas/factors that should be focused on. in general, it is desirable to use delivery vectors that exhibit cell-targeting properties with the ability to overcome the specific barriers associated with the disease concerned. delivery vectors that are nonbiodegradable or with a low rna-loading capability would be less attractive. a thorough understanding of the excretion pathway is required to ensure that the delivery vectors would not accumulate in the body, especially when synthetic or nonbiodegradable materials are used. vectors with high loading capacity could avoid the use of excessive excipients, thereby minimizing the risk of toxicity. moreover, to evaluate the in vivo efficacy of the delivery system, rna is often administered intratracheally to the lung of animals either as large droplets instilled by pipette or as a fine spray aerosolized by a microsprayer or similar device ( figure c ). clearly, these administration methods are impractical for human use. for clinical practice, rna drugs can be delivered either as liquid aerosols through nebulization or as dry powder for inhalation ( figure ). while the former can deliver high doses of liquid formulations over a period of time (typically~ - min, depending on the dose), the latter is more portable and convenient to use. however, there are stringent requirements for particles to be suitable for inhalation in clinical settings and animal studies cannot reflect the 'inhalability' of a formulation. surprisingly, only a few studies have evaluated the aerosol performance of rna formulations designed for inhalation [ , , ] (box ). a good understanding of the aerosol properties of the formulation could boost the chance of successful clinical translation ( figure ). moreover, nebulizers or dry powder inhalers are needed to generate aerosol for inhalation. given that both devices could damage the fragile rna molecules (especially single-stranded mrna) due to the high shear stress during aerosolization, it is essential to examine the rna integrity, in terms of physical structure and biological activity, in the aerosolized particles ( figure ). it is unclear whether the rna dose is optimized in in vivo preclinical studies because the doseresponse relationship of rna following pulmonary delivery is often not reported. dose trends optimization is critical for clinical translation not only for maximizing the therapeutic efficacy, but also for the practicality of administration. the amount of excipient (which may be included in the formulation to improve stability or to enhance aerosol performance) needs to be considered carefully because this can also affect the final liquid volume or powder mass to be administered. when rna is delivered through nebulization, a high dose would increase the volume and, hence, the administration time, leading to the increased risk of rna degradation due to prolonged exertion of shear stress on the rna molecules. for dry powder formulations, there is a constraint on the amount of powder that could be inhaled by a patient each time. currently, tobi® podhaler® (tobramycin inhalation powder) approved for treating pseudomonas aeruginosa infection in patients with cf, has the highest inhaled dose of~ mg per actuation [ ] . the rna dose has to be taken into consideration during the development of an effective delivery system that is fit for purpose. another area that requires attention during development of a pulmonary rna delivery system is understanding the biodistribution and pharmacokinetic profile of the inhaled rna formulation ( figure ). many studies focus on the evaluation of gene expression in the lung tissues as a whole. while naked rna is shown to manipulate gene expression primarily in the lung epithelial cells and macrophages [ ] , the sites where nanoparticulate rna delivery vectors exert their biological activity are less clear. for delivery vectors that rely on nonspecific cellular uptake mechanisms, rna could be effectively taken up by various cell types and absorbed into the systemic circulation. therefore, it is critical that biodistribution as well as pharmacokinetic profiles are thoroughly investigated. furthermore, the long-term toxicity of the delivery system also needs to be carefully evaluated. rna therapeutics have great potential in lung diseases. here, we have discussed nonviral delivery systems developed for sirna and mrna therapeutics in the lung and briefly summarized the what is the exact cellular uptake mechanism of naked rna in the airways? what are the factors governing its uptake? what are the delivery barriers to different rna delivery systems in different respiratory diseases? is it possible to have a universal pulmonary delivery platform that can be used to deliver different types of rna therapeutics? how can we protect the integrity of rna molecules against shear and thermal stresses effectively during the aerosolization and drying process? how do we improve the translation of inhaled rna therapy from animal studies to clinical applications? the most important factor that affects the aerosol performance of an inhaled formulation is particle size, which is most suitably expressed in aerodynamic diameter. aerodynamic diameter is defined as the diameter of a sphere of density g/cm that has the same settling velocity in still air as the particle of interest. it is generally accepted that particles with aerodynamic diameter between and μm are optimal for lung deposition [ ] . larger particles tend to deposit at the back of the throat and get swallowed subsequently, whereas smaller particles are likely to be exhaled. this criterion applies to both liquid and powder aerosol [ ] . the aerodynamic particle size distribution of an aerosol is often described in terms of mass median aerodynamic diameter (mmad) and geometric standard deviation (gsd) [ ] . the method of choice for measuring particle size distribution of inhaled products is the cascade impactor (ci), which operates on the principle of inertia impaction [ ] . ci comprises multiple stages and separates particles according to their aerodynamic diameters. large particles with high inertia are unable to follow the airstream and impact on earlier stages, whereas small particles remain in the airstream and flow to the next stage, where the process is repeated. the two parameters commonly reported from ci are emitted dose and fine particle dose. the former refers to the total dose that has exited the dispersion device, while the latter represents the amount of aerosol with an aerodynamic diameter below a certain threshold (typically μm) [ ] . these can also be expressed in fractions relative to the loaded dose or recovered dose. an important feature with the use of ci for measuring particle size is that a dispersion device (a nebulizer for liquid dosage forms or a dry powder inhaler for solid dosage forms) needs to be connected to generate the aerosol [ ] . given that the choice of device can have a dramatic impact on the aerosol properties of formulations, it is crucial to identify a suitable device to maximize the aerosol performance of a given formulation. there are different designs of ci, but only three are currently listed in both european pharmacopoeia and united states pharmacopoeia: the andersen cascade impactor (aci), the next generation impactor (ngi), and the multi-stage liquid impinger (msli) [ ] . current preclinical and clinical state of the field. currently, it appears that the development of mrna therapeutics is lagging behind that of sirna therapeutics ( figure d ), possibly because of the relatively poor stability of the long single-stranded rna molecule. with technological advances in rna modification that improve the stability, specificity, and safety of rna therapeutics [ , ] and the recent success of sirna therapeutics, we believe that both sirna and mrna will enter the clinic for the treatment of respiratory diseases in the near future. however, some crucial questions remain to be addressed before a successful rna inhalation delivery system can be realized (see outstanding questions). in light of the current covid- pandemic, development of an inhaled version of mrna vaccine is an area that deserves more attention. currently, there are several clinical studies (clinical trial no i .-nct ; nct ; nct ; nct ; and nct ) that demonstrate the safety and efficacy of mrna vaccines for the treatment of and protection against lung cancer and influenza, respectively [ , ] . mrna vaccines for covid- are being explored in several different clinical trials (clinical trial no.: nct ; nct ; nct ; nct ; nct ; nct ; and nct ). these mrna vaccine candidates are designed to be administered through parenteral injection. if any of these are successful, we believe that an inhaled version of the successful mrna vaccine will be an area to explore because it will provide a non-invasive route of administration with the possibility of self-administration, especially dry powder formulations, which show superior stability. the challenges of manufacture and scale-up, including the 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obstructive and restrictive respiratory diseases antisense oligonucleotide eluforsen improves cftr function in f del cystic fibrosis antisense oligonucleotide eluforsen is safe and improves respiratory symptoms in f del cystic fibrosis pulmonary delivery of therapeutic sirna pulmonary drug delivery. part i: physiological factors affecting therapeutic effectiveness of aerosolized medications pulmonary drug delivery measurement of aerodynamic particle size distribution of orally inhaled products by cascade impactor: how to let the product specification drive the quality requirements of the cascade impactor key: cord- -g i b authors: niles, meredith t.; bertmann, farryl; belarmino, emily h.; wentworth, thomas; biehl, erin; neff, roni title: the early food insecurity impacts of covid- date: - - journal: nutrients doi: . /nu sha: doc_id: cord_uid: g i b covid- has disrupted food access and impacted food insecurity, which is associated with numerous adverse individual and public health outcomes. to assess these challenges and understand their impact on food security, we conducted a statewide population-level survey using a convenience sample in vermont from march to april , during the beginning of a statewide stay-at-home order. we utilized the united states department of agriculture six-item validated food security module to measure food insecurity before covid- and since covid- . we assessed food insecurity prevalence and reported food access challenges, coping strategies, and perceived helpful interventions among food secure, consistently food insecure (pre-and post-covid- ), and newly food insecure (post covid- ) respondents. among respondents, there was nearly a one-third increase ( . %) in household food insecurity since covid- (p < . ), with . % of food insecure households classified as newly food insecure. respondents experiencing a job loss were at higher odds of experiencing food insecurity (or . ; % ci, . – . ). we report multiple physical and economic barriers, as well as concerns related to food access during covid- . respondents experiencing household food insecurity had higher odds of facing access challenges and utilizing coping strategies, including two-thirds of households eating less since covid- (p < . ). significant differences in coping strategies were documented between respondents in newly food insecure vs. consistently insecure households. these findings have important potential impacts on individual health, including mental health and malnutrition, as well as on future healthcare costs. we suggest proactive strategies to address food insecurity during this crisis. the global covid- pandemic, and social distancing efforts implemented to slow its spread [ ] , have disrupted economies and food systems globally and locally, with extensive food security ramifications. food insecurity-the lack of consistent physical, social, and economic access to adequate and nutritious food that meets dietary needs and food preferences [ ]-can lead to serious public health consequences. in , . % of american households were considered food insecure at some very low food security ( to affirmative answers) can be combined and referred to as having food insecurity [ ] . in addition to measuring food security status, the survey also included additional questions related to food access challenges, use of food assistance programs, food purchasing behaviors, concerns about food access and availability, covid- perceptions, and behaviors and demographics. table a details the specific questions utilized in this analysis, which are primarily focused on understanding the relationship of food security status to food access challenges, use of food assistance programs, and concerns about food access and availability. future analyses will explore other questions in the survey. to examine differences in household food insecurity during the first weeks of the covid- pandemic, we created three categories of respondents: ( ) households with food security (n = , including households food secure before and since covid- and households who were food insecure at some point in the year before covid- , but were no longer food insecure during covid- ); ( ) households with consistent food insecurity (n = , both food insecure before covid- and remaining food insecure since covid- ); ( ) households with new food insecurity (n = , categorized as food secure before covid- , but food insecure since . in some cases, we refer to food insecure households, which encompass both consistently food insecure households and newly food insecure households. to determine statistically significant differences between groups we utilized stata [ ] , to run kruskal-wallis tests, wilcoxon rank sum tests, t-tests, and one-way analysis of variance (anova) tests, depending on the distribution of the dependent variable. we used a logistical regression model to determine the factors correlated with food insecurity during the covid- pandemic, with coefficients reported in odds ratios. in this model, we estimate food insecurity outcomes during covid- , including respondents who were classified as either consistently food insecure or newly food insecure. we used all available data to estimate effect sizes and interactions and assumed any missing data were missing at random. reflecting the demographic composition of vermont [ , , ] , the majority of respondents identified as non-hispanic white, lived in rural areas, and had a household income below $ , ( table , table a ). women encompassed % of our sample, which may be reflective of the fact that women are the dominant food shoppers in households [ ] . we found a nearly one-third increase ( . %) in food insecurity prevalence (p < . ) between the year preceding the covid- outbreak, when . % of households ( % ci . - . %) reported experiencing food insecurity at some point, and since the covid- outbreak when the percentage rose to . % ( % ci . - . %) (table a ). among those experiencing food insecurity since the outbreak, . % also experienced food insecurity at some point in the year prior to covid- , and were also food insecure since covid- ; in comparison, . % were newly food insecure. in consistently food insecure households, . % exhibited very low food security since covid- (marked by disrupted eating patterns and reduced intake), while . % had low food security. in newly food insecure households . % exhibited very low food security, while . % had low food security (table a ) since covid- (p < . ). multivariable logit models predicted the factors contributing to higher odds of food insecurity during covid- (e.g., both consistently food insecure respondents and newly food insecure respondents) ( table ). note that we also ran a multinomial logit model to examine whether there were statistically significant differences in newly versus consistently food insecure respondents, which there were not (table a ) . respondents experiencing a job loss had three times greater odds of living in a household experiencing food insecurity (or . ; % ci, . - . ), and those experiencing a furlough (or . ; %ci, . - . ), or a loss of hours (or . ; % ci, . - . ) also had significantly greater odds of being in a household experiencing food insecurity (p < . ). the odds of experiencing food insecurity since the covid- outbreak were higher among households with children (or . ; % ci, . - . ), while households with higher incomes had reduced odds (or . ; % ci, . -. ) (p < . ). finally, women were % more likely to experience household food insecurity during covid- , compared to men (p < . ) (or . ; % ci . - . ), while a college degree (or . , % ci . - . ) was associated with reduced odds of household food insecurity (p < . ). respondents indicated multiple physical and economic barriers to food access during covid- , with respondents experiencing household food insecurity significantly more likely to express greater access, availability, and utilization challenges than respondents in food secure households (p < . ) ( figure , table a ). these challenges included not finding as much or the kinds of food that someone wanted, going to more places than usual to find food, and not being able to afford the food a household wanted. challenges also included those related to food assistance, including at food pantries and through school food programs. consistently food insecure households had a higher average prevalence of food access challenges, as compared to those in newly food insecure households including trouble affording food (p < . ), getting food through a food pantry (p = . ), and knowing where to find help for getting food (p < . ). households with children (or . ; % ci, . - . ), while households with higher incomes had reduced odds (or . ; % ci, . -. ) (p < . ). finally, women were % more likely to experience household food insecurity during covid- , compared to men (p < . ) (or . ; % ci . - . ), while a college degree (or . , % ci . - . ) was associated with reduced odds of household food insecurity (p < . ). respondents indicated multiple physical and economic barriers to food access during covid- , with respondents experiencing household food insecurity significantly more likely to express greater access, availability, and utilization challenges than respondents in food secure households (p < . ) ( figure , table a ). these challenges included not finding as much or the kinds of food that someone wanted, going to more places than usual to find food, and not being able to afford the food a household wanted. challenges also included those related to food assistance, including at food pantries and through school food programs. consistently food insecure households had a higher average prevalence of food access challenges, as compared to those in newly food insecure households including trouble affording food (p < . ), getting food through a food pantry (p = . ), and knowing where to find help for getting food (p < . ). average frequency of challenging food access situations since covid- among respondents with household food security and food insecurity in a survey of vermont households, march-april, (p < . for comparison among all groups). standard errors shown with brackets. differences between newly and consistently food insecure shown through stars (*** p < . ), ** p < . ) and in table a . respondents experiencing household food insecurity during covid- (both newly and consistently food insecure) were significantly more likely (p < . comparison across all groups) to express higher levels of concern and worry about a variety of potential situations related to food access and covid- ( figure , table a ). these situations included potential for food to become more expensive and for households to have a decrease in income, not enough food, loss of access to food programs, and food availability and safety. as compared to newly food insecure households, consistently food insecure households were also significantly more likely to have higher levels of concern and worry about food access for all situations except for food becoming unsafe (p < . , table a ). nutrients , , x for peer review of march-april, (p < . for comparison among all groups). standard errors shown with brackets. differences between newly and consistently food insecure shown through stars (*** p < . ), ** p < . ) and in table a . respondents experiencing household food insecurity during covid- (both newly and consistently food insecure) were significantly more likely (p < . comparison across all groups) to express higher levels of concern and worry about a variety of potential situations related to food access and covid- ( figure , table a ). these situations included potential for food to become more expensive and for households to have a decrease in income, not enough food, loss of access to food programs, and food availability and safety. as compared to newly food insecure households, consistently food insecure households were also significantly more likely to have higher levels of concern and worry about food access for all situations except for food becoming unsafe (p < . , table a ). average level of concern for potential food access situations during covid- among respondents with household food security and food insecurity in a survey of vermont households, march-april, (p < . for comparison among all groups). standard errors shown with brackets. statistically significant differences were also found between newly and consistently food insecure in all cases except for "food will become unsafe" (shown through stars, *** p < . ), ** p < . , * p < . ) and in table a . households newly and consistently experiencing food insecurity were significantly more likely (p < . ) to be implementing coping strategies related to obtaining food as compared to respondents in food secure households. these strategies included those related to disrupted eating patterns (i.e., eating less), buying different, cheaper foods, accepting food from friends and family, and utilizing government programs, credit or food pantries ( figure , table a ). consistently food insecure households, as compared to those newly experiencing food insecurity, were also significantly more likely to currently accept food (p = . ) or borrow money from friends or family (p = . ), use a food pantry (p < . ) and use government assistance programs (p = . ), especially the supplemental nutrition assistance program (snap) (p < . ) (tables a and a ). average level of concern for potential food access situations during covid- among respondents with household food security and food insecurity in a survey of vermont households, march-april, (p < . for comparison among all groups). standard errors shown with brackets. statistically significant differences were also found between newly and consistently food insecure in all cases except for "food will become unsafe" (shown through stars, *** p < . ), ** p < . , * p < . ) and in table a . households newly and consistently experiencing food insecurity were significantly more likely (p < . ) to be implementing coping strategies related to obtaining food as compared to respondents in food secure households. these strategies included those related to disrupted eating patterns (i.e., eating less), buying different, cheaper foods, accepting food from friends and family, and utilizing government programs, credit or food pantries ( figure , table a ). consistently food insecure households, as compared to those newly experiencing food insecurity, were also significantly more likely to currently accept food (p = . ) or borrow money from friends or family (p = . ), use a food pantry (p < . ) and use government assistance programs (p = . ), especially the supplemental nutrition assistance program (snap) (p < . ) (tables a and a ) . households newly and consistently experiencing food insecurity were also significantly more likely (p < . across all group comparisons) to report an intention to implement these same coping strategies in the future for assistance with obtaining food during covid- . among food insecure households, those with consistent food insecurity were more likely to indicate that in the future they would accept food from friends or family (p = . ), use food pantries (p < . ), government assistance programs (p < . ), and to stretch the food they have by eating less (p = . ), as compared to newly food insecure households (table a ) . compared to food secure households, new and consistently food insecure households were significantly more likely (p < . ) to find strategies to address physical or economic food access challenges helpful during covid- (table a ) . these helpful strategies included extra money to help pay for food or bills, an increase in benefits of existing food assistance programs, greater trust in the safety of going to stores and food delivery, support for food delivery costs, more or different food in stores, and information about and help with food assistance programs, among others. consistently food insecure households were also significantly more likely than those in newly food insecure households to find access to public transit, extra money for food or bills, increased benefits of food assistance programs, information about food assistance programs (all p < . ), help with administrative food assistance problems (p = . ), and support for food delivery costs (p = . ) more helpful (table a ) . nutrients , , x for peer review of figure . prevalence of current coping strategies utilized by households with food security and with food insecurity during covid- in a survey of vermont households, march-april, (statistical differences among all groups p < . ). statistical differences between newly and consistently food insecure shown through stars (*** p < . ); ** p < . , * p < . ) and in table a . households newly and consistently experiencing food insecurity were also significantly more likely (p < . across all group comparisons) to report an intention to implement these same coping strategies in the future for assistance with obtaining food during covid- . among food insecure households, those with consistent food insecurity were more likely to indicate that in the future they would accept food from friends or family (p = . ), use food pantries (p < . ), government assistance programs (p < . ), and to stretch the food they have by eating less (p = . ), as compared to newly food insecure households (table a ) . compared to food secure households, new and consistently food insecure households were significantly more likely (p < . ) to find strategies to address physical or economic food access challenges helpful during covid- (table a ) . these helpful strategies included extra money to help pay for food or bills, an increase in benefits of existing food assistance programs, greater trust in the safety of going to stores and food delivery, support for food delivery costs, more or different food in stores, and information about and help with food assistance programs, among others. consistently food insecure households were also significantly more likely than those in newly food insecure households to find access to public transit, extra money for food or bills, increased benefits of food assistance programs, information about food assistance programs (all p < . ), help with administrative food assistance problems (p = . ), and support for food delivery costs (p = . ) more helpful (table a ). this statewide survey in vermont documented a statistically significant increase in food insecurity since the state's first reported case of covid- and the stay-at-home executive order (which began march , ). we demonstrate a nearly one-third increase in household food figure . prevalence of current coping strategies utilized by households with food security and with food insecurity during covid- in a survey of vermont households, march-april, (statistical differences among all groups p < . ). statistical differences between newly and consistently food insecure shown through stars (*** p < . ); ** p < . , * p < . ) and in table a . this statewide survey in vermont documented a statistically significant increase in food insecurity since the state's first reported case of covid- and the stay-at-home executive order (which began march , ). we demonstrate a nearly one-third increase in household food insecurity among respondents, with individuals experiencing job loss or disruption at significantly greater odds of experiencing household food insecurity since covid- , as compared to other demographic controls. further, we find that the majority of consistently food insecure households and nearly one-third of newly food insecure households were classified as having very low food security, marked by disrupted eating and cutting meals or going hungry. fully, two-thirds of vermont respondent households with food insecurity during covid- are already eating less to stretch their food. the findings indicate challenges to all food security dimensions, including economic and physical access, availability, utilization, and stability, and may have profound potential health impacts. we further demonstrate physical and economic barriers to food access during covid- and the respondents' coping strategies in food insecure households. previous research [ , ] suggests links between job loss and food insecurity, indicating that the profound increase in americans experiencing job loss and disruption [ ] will present acute and large-scale impacts across the population. since vermont unemployment claims reflect the national trend, these results likely reflect a broader u.s. phenomenon of rising food insecurity rates, evidenced by early non-peer reviewed studies [ , ] . in addition to these new economic barriers, the pandemic presents many new physical barriers for food access, reductions in public transportation, and new distribution models, and in a rural state like vermont, a lack of income for transportation costs including fuel. in rural areas where food assistance programs, such as food pantries, are limited, closures due to illness, social distancing, or lack of volunteers may be particularly challenging. this presents opportunities to expand food pantries and support mobile pantry units, as well as encourage the expansion of programs such as fruit and vegetable prescription programs, shown to positively affect food security [ ] and improve health outcomes [ ] . ultimately, this research demonstrates a need to increase food assistance programs and provide resources to remove food access barriers now, and likely in the future, during state and national economic and health emergencies. this rise in food insecurity presents many potential health impacts. food insecurity is negatively associated with health outcomes [ , ] and some evidence indicates it is positively associated with poor diet quality [ , ] . further, higher rates of anxiety and mental health disorders among children and adults have been documented in food insecure households [ , ] . indeed, survey respondents in this study experiencing household food insecurity demonstrated significantly higher rates of concern and worry about food. disrupted eating, found in two-thirds of respondent households with food insecurity, is associated with decreased immune function and can negatively impact mental and emotional health [ ] . further research is needed to understand how food insecurity during the covid- pandemic relates to diet quality, particularly if disrupted eating patterns persist and increase. healthcare providers can address food insecurity through simple interventions. screening for food insecurity and providing resources now may reduce short-and long-term consequences, including the potential long-term impacts on child health outcomes associated with the duration of household food insecurity [ ] and higher health care expenditures associated with food insecurity [ ] . the hunger vital sign, a validated two-question food insecurity screening tool based on the usda household food security survey module [ ] , can quickly determine risk for food insecurity in clinical and community settings. this tool is widely utilized, especially in pediatrics [ , ] , and could be made standard in health care and other service settings during covid- and beyond. providers could refer families in need to locally available resources or to united way, which aggregates these resources locally. however, during this heightened time of unemployment, there is also potential for government agencies, particularly those distributing unemployment benefits, to help connect families in need to available resources as well. importantly, this research demonstrates there are still a significant number of food insecure households which, even if aware of food assistance programs, may not use them. low rates of seeking assistance in our results, especially among newly food insecure households, may be partly related to the stigma associated with assistance programs [ , ] . prior research suggests that populations living outside major metropolitan areas may be more likely to use friends and family for support [ ] and to see government assistance programs as a "last resort" [ ] . however, with social distancing and widespread financial challenges, such personal safety nets may be eroded, and these households may be particularly vulnerable. additional research is needed to understand the barriers to using food assistance programs, especially among those that may be newly food insecure since covid- . this study suggests some of the first population-level impacts of covid- and social distancing policies on food insecurity. the limitations are partly rooted in the need to rapidly administer this survey in the early days of the pandemic, to provide data that can be tracked over time. though our respondent population matches statewide census statistics closely on many metrics, this was a convenience sample; further research is expanding these results using similar questions with representative samples across states and populations. it is worth noting that our observed overall rate of food insecurity prior to covid- ( . %) is above the most recently available state figure ( . %) in . there are potentially multiple reasons for this. first, this is likely to be due, in part, to a higher than average number of female respondents and respondents in households with children; both groups have been documented, in vermont and elsewhere, to have elevated rates of food insecurity [ ] . second, our measurement instrument for documenting food security, the usda -item food security module, includes a subjective experience domain that measures concern about household food supplies. according to the local media [ ] , anxiety about household food supplies nutrients , , of preceded the stay home/stay safe order and may explain the higher than expected level of food insecurity prior to covid- . further, we used an internet-based survey, given the necessity of social distancing during covid- and the need for a rapid response, which may limit the capacity of some people to participate, although % of vermonters do have internet plans [ ] . the study's strengths include its large sample size, early administration, population-based assessment, and survey instrument addressing the multiple dimensions of food security. we implemented this survey in the beginning of a stay at home order and covid- economic impacts. as such, it is likely that many respondents experiencing job loss or disruption had not yet received unemployment benefits and federal stimulus checks were not distributed. future research will examine the evolution of food security impacts, and how various interventions, including the care act and unemployment benefits, as well as food assistance expansion and health care screenings, may affect food insecurity outcomes as covid- unfolds. since the coronavirus outbreak (march th), how often did these happen to your household? = never, = sometimes, = usually, = always, not applicable could not afford the amount or kind of food my household wanted to buy could not find as much food as i wanted to buy (e.g., food not in store) could not find the kinds of food my household prefers to eat delivered food to a friend, neighbor, or family member had challenges getting food through a food pantry had challenges getting food through a school food program had challenges knowing where to find help for getting food had to go to more places than usual in order to find the food my household wanted had to stand "too close for safety" to other people, when getting food (less than six feet) table a . cont. on a scale from (not at all worried) to (extremely worried), what is your level of worry for your household about the following as it relates to coronavirus. = not at all worried, = extremely worried, not applicable there will not be enough food in the store food will become more expensive for my household food will become unsafe my household will lose access to programs that provide free food or money for food my household will have a decrease in income and won't be able to afford enough food my household won't have enough food if we have to stay at home and can't go out at all what, if anything, would make it easier for your household to meet its food needs during the coronavirus pandemic? = not helpful, = somewhat helpful, = helpful, = very helpful, not applicable access to public transit or rides different hours in meal programs or stores extra money to help pay for food or bills help with administrative problems (like applying for food assistance) increase benefits of existing food assistance programs (like snap or wic) information about food assistance programs or food pantries more (or different) food in stores more trust in safety of food delivery more trust in safety of going to stores support for the cost of food delivery the base outcome comparison is food secure households. we find no significantly differences between the factors predicting food insecurity since covid- for newly or consistently food insecure households, so report the combined results of a multivariable logit model in the main results of both newly and consistently food insecure households together. interventions to mitigate early spread of sars-cov- in singapore: a modelling study. lancet infect. dis. . [crossref] . united nations food and agriculture organization united nations food and agriculture organization household food security in the united states incremental health care costs associated with food insecurity and chronic conditions among older adults food insecurity and health care expenditures in the united states food insecurity and health outcomes food insecurity status and mortality among adults in ontario association between household food insecurity and annual health care costs food insecurity, healthcare utilization, and high cost: a longitudinal cohort study prevalence of us food insecurity is related to changes in unemployment, inflation, and the price of food unemployment and household food hardship in the economic recession united states department of labor. the employment situation the impact of the coronavirus on child food insecurity covid- forces recalibration of priorities as world embraces new habits pantries padded with produce as north americans prepare for the covid- long haul food insecurity and covid- : disparities in early effects for us adults food access and security during coronavirus survey-version . . harvard dataverse v a meta-analysis of cronbach's coefficient alpha front porch forum paid campaign posting united states census bureau. cp : comparative demographic estimates. available online: data.census.gov household food security survey module: six-item short form statacorp llc: college station united states census bureau. cp : comparative economic characteristics cp : comparative social characteristics in the united states private label manufacturer's association. today's primary shopper about half of lower-income americans report household job or wage loss due to covid- the covid- crisis has already left too many children hungry in america food insecurity in the united states during covid- pandemic a pediatric fruit and vegetable prescription program increases food security in low-income households participation in a farmers' market fruit and vegetable prescription program at a federally qualified health center improves hemoglobin a c in low income uncontrolled diabetics the role of food insecurity in developmental psychopathology food insecurity and dietary quality in us adults and children: a systematic review household food insecurity during childhood and subsequent health status: the early childhood longitudinal study-kindergarten cohort development and validity of a -item screen to identify families at risk for food insecurity implementation of a food insecurity screening and referral program in student-run free clinics food insecurity screening in pediatric primary care: can offering referrals help identify families in need? acad what factors influence snap participation? literature reflecting enrollment in food assistance programs from a social and behavioral science perspective why do low-income women not use food stamps? findings from the california women's health survey. public health nutr accessing food resources: rural and urban patterns of giving and getting food understanding persistent food insecurity: a paradox of place and circumstance map the meal gap people stocking up on toilet paper; shelves empty we would like to thank those who assisted in the dissemination of the survey including key: cord- -re v qt authors: le, thanh ninh; chiu, chiu-hsia; hsieh, pao-chuan title: bioactive compounds and bioactivities of brassica oleracea l. var. italica sprouts and microgreens: an updated overview from a nutraceutical perspective date: - - journal: plants (basel) doi: . /plants sha: doc_id: cord_uid: re v qt sprouts and microgreens, the edible seedlings of vegetables and herbs, have received increasing attention in recent years and are considered as functional foods or superfoods owing to their valuable health-promoting properties. in particular, the seedlings of broccoli (brassica oleracea l. var. italica) have been highly prized for their substantial amount of bioactive constituents, including glucosinolates, phenolic compounds, vitamins, and essential minerals. these secondary metabolites are positively associated with potential health benefits. numerous in vitro and in vivo studies demonstrated that broccoli seedlings possess various biological properties, including antioxidant, anticancer, anticancer, antimicrobial, anti-inflammatory, anti-obesity and antidiabetic activities. the present review summarizes the updated knowledge about bioactive compounds and bioactivities of these broccoli products and discusses the relevant mechanisms of action. this review will serve as a potential reference for food selections of consumers and applications in functional food and nutraceutical industries. over the past twenty years, the heavy dependence of human nutrition on the sustainability of agricultural production has been highlighted owing to rapid population growth and environmental degradation [ ] . increasing food and agricultural productivity has simultaneously imposed external expenses and consequences upon the financial resource, ecological system, and human health by excessive water use, soil occupation, fertilizers, pesticides, herbicides, and food waste treatment [ ] . thus, to meet the united nations' sustainable development goals, the agriculture and food sectors have been confronted with a major challenge: to provide adequate nutrition for global food demand while minimizing the negative impacts on the environment [ ] . furthermore, in recent years, improving public awareness about the healthy lifestyle framework has prompted the search for novel food sources, which are rich in essential nutrients and have positive effects on human health [ ] . as a result, functional foods and nutraceuticals are gaining significant attention since these foods could provide both health benefits to reduce the risk of chronic diseases and basic nutrition [ , ] . besides, many scientific projects and research groups are focusing on the recovery of food wastes and upgrading them into high-value byproducts to serve as functional ingredients in new product development [ ] [ ] [ ] [ ] . particularly, the issues of the food systems, including food safety, food security, and food sustainability, should be significantly addressed in the era of the coronavirus (covid- ) pandemic crisis. the availability of bioactive constituent of food and functional foods may become bioactive compounds are extra-nutritional constituents found in foods, mainly in fruits, vegetables, and grains, which are capable of modulating metabolic processes and providing healthpromoting benefits [ ] . regular consumption of broccoli seedlings could stimulate the natural defense systems and decrease the risk of chronic diseases, owing to their concentration of bioactive compounds several times higher than those of the mature florets [ ] . thus, they are considered a novel plant-derived functional food. previous studies have extensively indicated that broccoli sprouts and microgreens contain a remarkably high amount of glucosinolates, phenolic compounds, and essential nutrients ( figure and tables - ). there are numerous methods employed for the extraction of bioactive compounds from herbs, vegetables, and fruits, including emerging technologies such as pressurized hot water extraction, microwave-assisted extraction, or pulsed electric extraction [ ] [ ] [ ] . the separation, identification, and characterization of these compounds of broccoli seedlings, in particular, and the genus brassica, in general, were investigated by both conventional and non-conventional technologies [ , ] . among these technologies, the broccoli samples were mainly conducted by high-performance liquid chromatography (hplc) coupled to diode array (dad), ultraviolet-visible (uv-vis), electrospray ionization (esi), or mass spectrometry (ms) detectors with c analytical columns. gas chromatography combined with mass spectrometry (gc-ms) was also applied to characterize isothiocyanates, the hydrolysis products of glucosinolates. besides, the spectrophotometric (uv-vis) detection was employed in most of the research to determine the total phenolic and flavonoid contents as the simplest procedure, if it was not required to determine a specific compound (tables - ). it could be summarized that glucosinolates and related compounds are the major group of phytochemicals investigated in broccoli sprouts and microgreens, although phenolic compounds have also been analyzed in many studies ( figure ). bioactive compounds are extra-nutritional constituents found in foods, mainly in fruits, vegetables, and grains, which are capable of modulating metabolic processes and providing health-promoting benefits [ ] . regular consumption of broccoli seedlings could stimulate the natural defense systems and decrease the risk of chronic diseases, owing to their concentration of bioactive compounds several times higher than those of the mature florets [ ] . thus, they are considered a novel plant-derived functional food. previous studies have extensively indicated that broccoli sprouts and microgreens contain a remarkably high amount of glucosinolates, phenolic compounds, and essential nutrients ( figure and tables - ). there are numerous methods employed for the extraction of bioactive compounds from herbs, vegetables, and fruits, including emerging technologies such as pressurized hot water extraction, microwave-assisted extraction, or pulsed electric extraction [ ] [ ] [ ] . the separation, identification, and characterization of these compounds of broccoli seedlings, in particular, and the genus brassica, in general, were investigated by both conventional and non-conventional technologies [ , ] . among these technologies, the broccoli samples were mainly conducted by high-performance liquid chromatography (hplc) coupled to diode array (dad), ultraviolet-visible (uv-vis), electrospray ionization (esi), or mass spectrometry (ms) detectors with c analytical columns. gas chromatography combined with mass spectrometry (gc-ms) was also applied to characterize isothiocyanates, the hydrolysis products of glucosinolates. besides, the spectrophotometric (uv-vis) detection was employed in most of the research to determine the total phenolic and flavonoid contents as the simplest procedure, if it was not required to determine a specific compound (tables - ) . it could be summarized that glucosinolates and related compounds are the major group of phytochemicals investigated in broccoli sprouts and microgreens, although phenolic compounds have also been analyzed in many studies ( figure ). summary of the bioactive compounds analyzed in the last ten years of broccoli sprouts and microgreens. glucosinolates (glss), nitrogen−sulfur compounds (β-d-thioglucoside-n-hydroxysulfates), are important plant secondary metabolites, almost exclusively found in the genus brassica, specific to kale, cabbage, and broccoli [ ] . glss are classified as aliphatic (derived from methionine, isoleucine, leucine or valine), aromatic (derived from phenylalanine or tyrosine), or indole (derived from tryptophan) groups. aliphatic group is the major group of glss in almost all cruciferous seeds and seedlings of b. oleraceae, b. napus, b. rapa, and r. sativus [ ] . in broccoli sprouts and microgreens, compounds of glss were identified (table ) . among these compounds, the most abundant glss are glucoraphanin, glucoiberin, glucoerucin, glucobrassicin, and neoglucobrassicin [ ] . in particular, glucoraphanin accounted for over % of the total glss content, which was quantified in the range of to mg per g of fresh weight (fw) [ ] [ ] [ ] . glucosinolates (glss), nitrogen−sulfur compounds (β-d -thioglucoside-n-hydroxysulfates), are important plant secondary metabolites, almost exclusively found in the genus brassica, specific to kale, cabbage, and broccoli [ ] . glss are classified as aliphatic (derived from methionine, isoleucine, leucine or valine), aromatic (derived from phenylalanine or tyrosine), or indole (derived from tryptophan) groups. aliphatic group is the major group of glss in almost all cruciferous seeds and seedlings of b. oleraceae, b. napus, b. rapa, and r. sativus [ ] . in broccoli sprouts and microgreens, compounds of glss were identified (table ) . among these compounds, the most abundant glss are glucoraphanin, glucoiberin, glucoerucin, glucobrassicin, and neoglucobrassicin [ ] . in particular, glucoraphanin accounted for over % of the total glss content, which was quantified in the range of to mg per g of fresh weight (fw) [ ] [ ] [ ] . notably, glss are not the functional components in cruciferous vegetables, rather their hydrolysis products are the putative bioactive compounds [ ] . when plant tissues are mechanically damaged, glss could be hydrolyzed by the enzyme myrosinase into a variety of degradation products, including isothiocyanates (itcs), thiocyanates, nitriles, epithionitriles, and oxazolidines. in particular, itcs have been proven to present strong anticarcinogenic activities [ ] . as mentioned above, predominant glss in broccoli seedlings are glucoraphanin, glucoerucin, and glucobrassicin, which are enzymatically converted into sulforaphane (sfn), erucin (ern), and iberin, respectively [ ] . sfn is a naturally occurring inducer of phase ii enzymes in human and animal bodies to detoxify cancer-causing chemicals. thus, it can reduce the risk of different cancers, especially those of the bladder, colon, and lung [ ] . to date, sfn was analyzed as a major compound among the total of itcs identified from broccoli sprouts and microgreens ( table ). the total itc content of broccoli seedlings was reported to be around mg per g of fw. additionally, such seedlings were said to contain % sfn [ , ] . uv/vis, ultraviolet-visible spectrophotometry; gc-ms, gas chromatography combined with mass spectrometry; gc−fid, gc with a flame ionization detector. besides glss and itcs, another important group of bioactive constituents present in cruciferous vegetables is the phenolic compounds. they are secondary metabolites produced in plants through the phenylpropanoid and shikimate pathways [ ] . based on their structure, which comprises one or more aromatic rings with attached hydroxyl substituents, phenolic compounds can be categorized into various subgroups, such as phenolic acids, flavonoids, tannins, coumarins, lignans, quinones, stilbenes, and curcuminoids. these compounds have been mainly reported for antioxidant activity. moreover, they are also associated with other health-promoting effects such as anticarcinogenic, antimicrobial, anti-inflammatory, and anti-aging properties [ ] . hydroxycinnamic acids and flavonoid glycosides are among the main phenolic compounds found in broccoli seedlings [ , ] . using quantitative and qualitative analysis, phenolic compounds were characterized in broccoli sprouts and microgreens, including hydroxycinnamic acids and derivatives, and flavonoids and derivatives. their phenolic profile composed mostly of sinapic acid, gallic acid, flavonoids (quercetin and kaempferol), and other hydroxycinnamic acids (chlorogenic, caffeic acid, and ferulic acids) ( table ). in general, total phenolic and flavonoid contents were determined to be in the ranges of - mg per g of fw and - mg per g of fw, respectively [ , , ] . similar to other sprouted seeds, broccoli sprouts and microgreens are known for their high concentration of essential nutrient composition [ ] . essential nutrients are compounds that must be supplied from foods, such as vitamins, minerals, fatty acids, and amino acids. they are required for normal body functions, including dna synthesis, energy production, and biosynthetic pathways [ ] . nonetheless, the number of studies, which determined the nutritional composition of broccoli seedlings and the variations occurring during germination, is significantly smaller than that of glucosinolates and phenolic compounds. these studies indicated that broccoli sprouts are expressly rich in vitamins (a, c, k, and folic acid), minerals (potassium, calcium, magnesium, and selenium), pigments (carotenoids and chlorophylls) and some other important nutrients (amino acids, fatty acids, and dietary fiber) [ ] [ ] [ ] , , , ] . in general, the biochemical composition of broccoli sprouts and microgreens depends mainly on the germination time, and -day-old sprouts displayed the highest nutrient concentration [ ] . among these compounds, carotenoids and chlorophylls were determined in several studies of broccoli sprouts [ , , , , ] . carotenoids are a group of isoprenoid molecules synthesized as secondary metabolites by all photosynthetic plants, including broccoli [ ] . they have lipophilic antioxidant and immunomodulatory activities owing to the conjugated double bonds of the long polyene chain, which are capable of inhibiting reactive oxygen species and reducing oxidative damage [ ] . thus, carotenoids might prevent degenerative diseases, such as cardiovascular diseases, skin damage, diabetes, and several types of cancer [ ] . the major carotenoids found in broccoli are β-carotene, α-xanthophyll (lutein), and β-xanthophylls (zeaxanthin, violaxanthin, and neoxanthin) [ ] . in particular, β-carotene is the most typically studied carotenoids in broccoli sprouts and microgreens, due to its importance in medical science [ ] . it supplies the human diet a substantial amount of vitamin a that is essential for organogenesis, tissue differentiation, immune function, and vision [ ] . the total carotenoid (β-carotene) concentration of broccoli sprouts was reported in several studies with a range of to mg per g of dry weight (dw) [ , ] . chlorophylls are another group of light-absorbing pigments regularly present in broccoli sprout extract. they are primary metabolites that have a porphyrin structure [ ] . similar to carotenoids, they also have antioxidant, anti-mutagenic, and anti-inflammatory potential [ ] . the content of chlorophyll in broccoli sprouts was commonly determined along with total carotenoid content in previous studies, ranging from to mg per g dw [ , ] . additionally, a study showed individual concentrations of carotenoids (lutein and neoxanthin) and chlorophyll (chlorophyll a and b) in broccoli sprouts [ ] . the human consumption of foods or nutraceuticals is not limited to bioactive compounds. in the human body, bioavailability is determined as the fraction of the dose administered that reaches the circulatory system and then distributes into target tissues so that the bioactive compounds are biologically available for exerting health-promoting benefits [ ] . many factors could affect the bioavailability of the dietary phytochemicals, including plant cell wall compositions, chemical structures, processing conditions, environmental stress, as well as an individual's gastrointestinal system [ ] . the effectiveness of a high consumption of broccoli sprouts in reducing the risk of cancer has been attributed mainly to their high content of glss. the daily intake of glss is difficult to estimate owing to the high variability of the constituent in cruciferous vegetables, particularly broccoli. a report for glucosinolate intake was indicated for european people ranging from . - mg/day [ ] . glss are broken down by both the plant enzyme myrosinase in the small intestine and the microbiota in the gastrointestinal tract into itcs, which are reported to have the prominent anticancer effect [ ] . after intake - h, the itcs are absorbed from the small bowel and colon and metabolized by the mercapturic acid pathway. the metabolites are detectable in human urine and blood [ ] . in broccoli sprouts, two of the most abundant glss are glucoraphanin and glucoerucin, and myrosinase hydrolysis of these glss form sfn and ern, respectively. sfn, distinguished itcs in broccoli sprouts, is one of the most potent naturally occurring inducers of phase detoxification enzymes, boost antioxidant status, and protect animals against chemically induced cancer [ , ] . its modes of action are involved with the repressor protein kelch-like ech associated protein (keap ), nuclear factor erythroid p -related factor (nrf ), and genes, which contain an antioxidant responsive element (are) [ ] . thus, it is important for the inclusion of potential cancer chemopreventive agents in dairy foods. moreover, it has shown an inhibitory effect for urease from helicobacter pylori, a human pathogen [ ] . the formation of other breakdown products from glss by intestinal microbiota is not well documented. for example, bifidobacterium strains belonging to the human intestinal microbiota can in vitro metabolize the glss to nitriles. it is also known that several microorganisms are able to convert nitriles into ammonia and organic acids [ ] . phenolic compounds are substantial micronutrients in the human diet and the health benefits of polyphenols depend on the amount consumed and on their bioavailability [ ] . the dietary intake of phenolic compounds has been associated with health-promoting effects, such as antioxidant, anti-aging, antiproliferative, and anti-inflammatory activities [ ] . it has been reported that the average dietary intake of phenolic compounds for humans is about - mg/day, including % of hydroxycinnamic acids, - % of flavonoids, and % of anthocyanins [ ] . bioavailability differs significantly among phenolic compounds so that the most abundant compounds in the human diet are not necessarily those leading to the highest concentrations of active metabolites in target tissues [ ] . there are two directions for the digestion of dietary phenolics, comprising digestion along the gastrointestinal tract and digestion inside the enterocytes. these digestions are based on hydrolase enzymes, which are available in the intestinal lumen, brush border, and enterocyte [ ] . many studies showed that polyphenols are absorbed in both the small intestine and the large intestine by intestinal enzymes after microbial digestions [ ] . in the human small intestine and stomach, gallic acid and caffeic acid are the most well-absorbed compounds ( %), followed by catechins ( %). the least well-absorbed polyphenols are proanthocyanidins and the anthocyanins. they are ph-sensitive, which could be broken down in the stomach and readily absorbable. flavonoid group is one of the group molecules with molecular weights > da so that it has a low bioavailability level ( %) because molecules are improbable to be transported through passive diffusion pathways [ , , ] . the dietary intake of carotenoids, particularly vitamin a, has been linked to the protection of dna, proteins, and lipids from oxidative damage [ ] . digested carotenoids decrease oxidative dna damage so that they could protect colon cells against the stress of reactive oxygen species [ ] . the dietary chlorophylls from fresh fruits and vegetables, which compose of chlorophyll a and b, showed distinct biological potentials, including wound healing, the control of calcium oxalate crystals, the modulation of xenobiotic metabolism, and the induction of apoptosis [ ] . broccoli seedlings and their bioactive components exhibit many potential health-promoting roles. in the last decade, the beneficial effects, including antioxidant, anticarcinogenic, antimicrobial, anti-inflammatory, and several other properties, have been widely investigated in a number of in vitro and in vivo studies and topically applied in clinical trials [ , , ] . particularly, these studies mostly focused on the antioxidant and anticancer activities of broccoli sprouts and microgreens owing to the functions of glucosinolates and phenolic compounds (tables and ). in this section, the findings of different biological activities of broccoli sprouts and microgreens will be discussed with the possible mechanisms of action ( figure ). anti-inflammatory, and several other properties, have been widely investigated in a number of in vitro and in vivo studies and topically applied in clinical trials [ , , ] . particularly, these studies mostly focused on the antioxidant and anticancer activities of broccoli sprouts and microgreens owing to the functions of glucosinolates and phenolic compounds (tables and ). in this section, the findings of different biological activities of broccoli sprouts and microgreens will be discussed with the possible mechanisms of action ( figure ) . the overproduction or incorporation of free radicals, such as reactive oxygen species (ros), cause oxidative damage to biomolecules and consequently lead to many chronic diseases, including neurodegenerative diseases, cardiovascular diseases, and certain age-related cancers [ ] . ros are previously thought to form in mammalian cells almost exclusively as a consequence of mitochondrial metabolism. recently, it has been demonstrated that cellular enzymes known as nicotinamide adenine dinucleotide phosphate (nadph) oxidases produce a considerable amount of ros in the human body. moreover, other cellular sources of ros involve neutrophils, monocytes, endothelial cells, xanthine oxidases, cytochrome p , lipoxygenases, and nitric oxide syntheses [ ] . hence, ros has distinct effects on normal physiological processes, oxidative stress/regulation, metabolic diseases, and chronic inflammation. targeting ros is involved in antioxidant, anti-inflammatory, antidiabetic, and anti-obesity therapeutics [ ] . antioxidants are divided into natural and synthetic compounds, which could remove free radicals, inhibit ros formation, and scavenge ros [ ] . previous studies the overproduction or incorporation of free radicals, such as reactive oxygen species (ros), cause oxidative damage to biomolecules and consequently lead to many chronic diseases, including neurodegenerative diseases, cardiovascular diseases, and certain age-related cancers [ ] . ros are previously thought to form in mammalian cells almost exclusively as a consequence of mitochondrial metabolism. recently, it has been demonstrated that cellular enzymes known as nicotinamide adenine dinucleotide phosphate (nadph) oxidases produce a considerable amount of ros in the human body. moreover, other cellular sources of ros involve neutrophils, monocytes, endothelial cells, xanthine oxidases, cytochrome p , lipoxygenases, and nitric oxide syntheses [ ] . hence, ros has distinct effects on normal physiological processes, oxidative stress/regulation, metabolic diseases, and chronic inflammation. targeting ros is involved in antioxidant, anti-inflammatory, antidiabetic, and anti-obesity therapeutics [ ] . antioxidants are divided into natural and synthetic compounds, which could remove free radicals, inhibit ros formation, and scavenge ros [ ] . previous studies on broccoli sprouts and microgreens have demonstrated that they are recognized for their variety of naturally occurring antioxidants, comprising both nutritional antioxidants like vitamins (especially, ascorbic acid and α-tocopherol), and non-nutritional antioxidants such as carotenoids and phenolic compounds [ ] . it was reported that these compounds are responsible for − % of the total antioxidant capacity in broccoli sprouts [ ] . the antioxidant activity of broccoli sprouts and microgreens has been determined in numerous studies using various methods. the capacity of broccoli sprouts and microgreens for chelating fe + and scavenging free radicals such as dpph ( , -diphenyl- -picrylhydrazyl) and abts ( , -azino-bis- -ethylbenzothiazoline- -sulphonic acid) was demonstrated in numerous studies (table ) . among the possible methods, dpph radical scavenging assay, abts radical cation decolonization assay, and ferric reducing antioxidant power (frap) assay were commonly employed in previous studies ( table ). the dpph method is speedy, simple, and low cost in comparison to other test models. dpph, a dark crystalline molecule, is a stable chromogen radical formed by the delocalization of the spare electron over the molecule. the dpph scavenging assay is based on the electron donation of antioxidants to neutralize dpph radical. the reaction occurs with the loss of the violet color of dpph that is measured at nm, and the discoloration acts as an indicator of the antioxidant effectiveness [ ] . the abts decolonization assay is appropriate for both hydrophilic and lipophilic antioxidants. it uses a spectrophotometer to measure the ability of antioxidants to scavenge the stable radical cation abts + , a blue-green chromophore molecule with absorption at nm. antioxidants can neutralize and decolorize the radical cation abts + by electron or hydrogen atom donations [ ] . the frap assay was originally employed to measure reducing power in plasma, but it has been expanded for other biological fluids and plant extracts. thus, it is applicable for both in vitro and in vivo experiments. the frap mechanism is based on electron transfer rather than hydrogen atom transfer. the assay demonstrates the ability of antioxidants to reduce ferric iron. it measures the reduction of the ferric ion (fe + )-ligand complex to the blue-colored ferrous (fe + ) form at low ph by antioxidants (absorption at nm) [ ] . moreover, another mechanism of antioxidant activity of broccoli sprouts and microgreens have been confirmed in several in vitro and in vivo examinations by inhibiting the activity of prooxidant enzymes such as lipoxygenase (lox), and xanthine oxidase (xo), and activating antioxidant enzymes such as peroxidase (pod), catalase (cat), superoxide dismutase (sod), glutathione peroxidase (gpx), nad(p)h-quinone acceptor oxidoreductase (nqo ), and heme oxygenase- (ho- ) ( table ) . prooxidant enzymes are considered as the main biological source of superoxide radicals, whereas antioxidant enzymes could eliminate the excess of reactive oxygen species and reduce oxidative damage during senescence [ , ] . for example, enzyme activities of cytosolic glutathione peroxidase (gpx ), thioredoxin reductase(tr) in the thyroid, plasma glutathione peroxidase (gpx ), and ferric reducing ability of plasma (frap) significantly increased in response to broccoli sprouts ingestion in -week-old wistar rats [ ] . in summary, previous studies showed that broccoli sprout extracts rich in vitamins, carotenoids, and phenolic compounds showed very high antioxidant activity in both in vitro and in vivo tests (table ) . thus, they could be applied for antioxidant therapeutics to reduce the risk of chronic diseases caused by ros. interestingly, although the antioxidant capacity of broccoli seedlings has been reported comprehensively, it is still attracting considerable attention from investigators and researchers at present [ , , ] . abts, , -azino-bis- -ethylbenzothiazoline- -sulphonic acid decolonization activity; dpph, , -diphenyl - -picrylhydrazy radical scavenging activity; frap, ferric reducing-antioxidant power; teac, trolox equivalent antioxidant capacity; orac, oxygen radical absorbance capacity; pod, peroxidase activity; cat, catalase activity; sod, superoxide dismutase activity; gpx, glutathione peroxidase activity; chel, metal chelating activity; lpo, inhibition of lipid peroxidation; loxi, inhibition of lipoxygenase; xoi, inhibition of xanthine oxidase; nqo , nad(p)h-quinone acceptor oxidoreductase ; ho- , heme oxygenase- ; ma/gc, malonaldehyde/gas chromatography. in the last decade, many studies and projects have supported the protective effects of natural products in cancer prevention. the protective role against cancer has been mostly attributed to the high content of glss, typically found in cruciferous vegetables [ ] . broccoli sprouts and microgreens have been valued as a rich source of glss and their hydrolysis products (icts, particularly sfn), which are a well-known class of cancer chemotherapeutic agents that work by inducing apoptosis and arresting cell cycle progression (tables and ) . the potential mechanisms of action mainly involve the inhibition of proliferation and the induction of apoptosis in cancer (table ) . hence, to demonstrate the anticancer activity of broccoli sprouts and microgreens, many in vitro tests have been carried out to determine the antiproliferative activity. their results indicated that broccoli seedlings exert strong cytotoxicity against different types of cancer cell lines, including hepatocellular carcinoma cells (hepg ), prostate carcinoma cells (pc- , at- , and sum ), lung carcinoma cells (a ), and colorectal adenocarcinoma cells (caco- , and ht- ) ( table ). in these studies, the highly effective ic values were found to be from to µg/ml. on the another hand, the selectivity of broccoli seedlings was reported on normal skin fibroblasts (bj), normal colon fibroblasts (ccd -co), and normal liver cells (fl b) by displaying no toxic effects on their viability after the treatment of broccoli samples (table ) . cancer is essentially a disease of uncontrolled cell division. it is caused by an imbalance between cell proliferation and apoptosis [ ] . its development and progression are generally associated with the disorder of cell cycle regulators' activity. defects in the programmed cell death mechanism also make a key contribution to tumor pathogenesis [ ] . thus, most chemotherapeutic agents exert their cytotoxic activity against cancer cells, since they cause dna damage and activate a complex signaling network resulting in cell cycle arrest and apoptosis induction [ ] . targeting the cell cycle phase and the checkpoint signaling pathway, which leads to the arrests at g /s or g /m phases, would provide a promising opportunity for cancer treatment. besides, triggering cell apoptosis could be an effective strategy for potential chemotherapeutic agents [ , , , ] . to confirm the antiproliferative activity of broccoli sprouts and microgreens, several in vitro studies revealed the mechanism of cell death based on inducing cell cycle arrest and apoptosis (table ) . their results showed cell cycle arrests (obviously at g /g and s phases) and significant increases in cell percentage with subg dna content, which is considered as a marker of apoptosis. besides, mitochondrial changes might activate the intrinsic apoptotic pathway. the loss of mitochondrial membrane potential (mmp) leads to the activation of several proteins linked to apoptosis, such as caspase- and cytochrome c [ ] . hence, a few studies indicated the notable decrease in mmp levels of cancer cells after treatment by broccoli seedlings to prove the mechanism of programmed cell death [ , ] . furthermore, some in vivo carcinogenesis models were employed to demonstrate the anticancer effects and related molecular mechanisms of broccoli sprouts and microgreens, such as c bl/ mice, balb/c mice, skh- hairless mice, and sprague-dawley rats (table ). sfn extracted from broccoli sprouts showed the inhibition of breast cancer stem cells and downregulate the wnt/β-catenin self-renewal pathway in a nonobese diabetic/severe combined immunodeficient xenograft model [ ] . sfn isolated from broccoli seeds and sprouts also exhibited anticancer effects in lung cancer cell lines and nude balb/c mice with lung cancer xenograft by inhibiting the pi k-akt signaling pathway [ ] . the transgenic adenocarcinoma of the mouse prostate model with broccoli sprout intake demonstrated a significant decline in prostate cancer occurrence and hdac protein expression in the epithelial cells of the prostate. hdac is one of the histone deacetylase enzymes that turn off tumor suppressor genes and promote the expression of oncogenes [ ] . the broccoli sprout diet administered to adult her /neu mice showed both preventive and suppressive effects on mammary cancer. these protective effects were associated with tumor-and epigenetic-related gene expression, and changed histone acetylation, dna methylation, and dna hydroxymethylation levels [ ] . the dietary administration of broccoli sprout extract to the rat model inhibited bladder cancer development by inducing glutathione s-transferase and nad(p)h-quinone oxidoreductase in the bladder, which are important enzymes against oxidants and carcinogens [ ] . in vitro ns a , h , h , hcc , h , h cells inhibiting cell proliferation; inducing apoptosis [ ] in vitro ns caco- , ccd -co cells inducing cell cycle arrest and apoptosis; decreasing mmp level; increasing ros generation [ ] in vivo ns female nod/scid mice eliminating breast cscs in vivo; downregulating wnt/β-catenin pathway [ ] in vivo ns female nude balb/c mice inhibiting the pi k-akt signaling pathway [ ] in vivo days female skh- hairless mice stabilizing p ; inducing phase enzyme; inhibiting inos upregulation [ ] in vivo ns male tramp mice in c bl/ background decreasing hdac protein expression [ ] in vivo ns sv and her /neu mice modulating epigenetic pathways; regulating epigenetic-controlled gene expression [ ] in vivo ns female her /neu mice regulating tumor-and epigenetic-related gene expression; increasing tumor suppressor gene expression [ ] in vivo days female sprague-dawley rats inducing gst and nqo [ ] hepg the antimicrobial capacity of broccoli has been reported in several publications; however, most of these studies focused on broccoli florets. there are a few reports that displayed the detrimental effect of broccoli sprouts on pathogenic bacteria. a study investigated broccoli sprouts with high levels of gallic acid, esculetin, ferulic acid, and myricetin have the antibacterial activity against foodborne pathogens, including both gram-positive bacteria (staphylococcus aureus and bacillus subtilis) and gram-negative bacteria (salmonella typhimurium and escherichia coli), with minimum inhibition concentration (mic) values from to µg/ml [ ] . this study revealed that the gram-positive bacteria were more sensitive to broccoli sprout extract than the gram-negative bacteria, similar to the previously published results of other plant extracts [ , ] . the possible reason might be the structural differences in the cell wall between these two classes of bacteria. gram-negative bacteria are surrounded by an additional outer membrane, which is a hydrophilic layer to prevent the access of many substances including natural compounds [ , ] . thus, broccoli sprout extract was demonstrated to be more active on gram-positive bacteria. another in vitro study reported the powerful bactericidal activity of broccoli sprouts against helicobacter pylori by the presence of sulforaphane as the major anti-helicobacter active compound, with highly active inhibition zones (> cm) [ ] . many reports showed that plant-derived bioactive compounds, such as phenolics, flavonoids, and glucosinolates, can inhibit the growth and activity of various microorganisms [ ] . with the diversity in the molecular structure and chemical composition, these compounds can perform distinct antimicrobial effects, such as destabilization of the plasma membrane or inhibition of extracellular enzymes [ ] . the antimicrobial activity against pathogenic bacteria, yeast, and phytopathogenic fungi was also proven by the presence of many antimicrobial peptides in broccoli floret extract [ ] . moreover, the daily intake of sulforaphane-rich broccoli sprouts for months was demonstrated to reduce gastric bacterial colonization and attenuate gastritis in helicobacter pylori-infected mice and patients [ ] . in another clinical trial, the high-sulforaphane broccoli sprouts powder also showed a considerable effect on h. pylori eradication in type diabetic patients with positive h. pylori [ ] . collectively, these findings indicate that broccoli sprout extracts might serve as a potential source of antimicrobial agents in the food and pharmaceutical industry. several studies proved that broccoli sprouts and their bioactive components possess anti-inflammatory activity. hence, the application of broccoli sprouts and microgreens has potential in the prevention and treatment of inflammatory bowel diseases, such as ulcerative colitis and crohn's disease [ ] . the anti-inflammatory mechanisms of broccoli sprouts are probably associated with the nuclear factor-kappa b (nf-κb) and nuclear factor erythroid -related factor (nrf ) signaling pathways [ ] . both in vitro and in vivo experiments, as well as clinical trials, mainly exhibit anti-inflammatory effects of broccoli sprouts, which showed high concentrations of sulforaphane and a group of phenolic compounds, including anthocyanins, isoquercetin, chlorogenic and cinnamic acids, via inhibiting inflammatory mediators such as a nitric oxide (no), decreasing the levels of proinflammatory cytokines such as tumor necrosis factor α (tnf-α), interleukin- (il- ), and il- β, and increasing the levels of anti-inflammatory cytokines such as il- and il- [ , , , , , ] . for example, a study showed that sulforaphane-enriched broccoli sprouts inhibited activation of the nf-κb signaling pathway and the secretions of inflammatory proteins (inducible nitric oxide synthase (inos), cyclooxygenase (cox- ), tnf-α, il- , il- β, and prostaglandin e (pge )) in microglial cells (bv ) and male icr (institute of cancer research) mice. in this study, broccoli samples also upregulated the expression of nrf and heme oxygenase- (ho- ) in normal bv cells and against scopolamine-induced amnesia in mouse brain tissue samples [ ] . to analyze immunological parameters in the wistar rats with iodine deficiency, the significant decrease in il- level, along with no significant differences in il- concentration, were observed after adding broccoli sprouts to the diet [ ] . high-sulforaphane broccoli sprouts also indicated favorable effects on inflammatory markers by reducing concentrations of serum high-sensitive c reactive protein (hs-crp), il- , and tnf-α in type diabetic patients [ ] . broccoli is one of the few vegetables that is considered as a supplementary treatment for type diabetes and for the prevention of its long-term complications [ ] . a few studies investigated the potential benefits of broccoli sprouts and microgreens for patients with diabetes. a study determined the effects of broccoli sprout powder on insulin resistance in type diabetic patients as a new approach by the use of its bioactive constituents. the results showed that broccoli sprout powder containing a high concentration of sulforaphane may significantly decrease in serum insulin concentration and lessen complications of diabetes [ ] . in another report, the potential efficacy of sulforaphane extracted from young broccoli sprouts has been confirmed as an effective option for supplementary treatment in type diabetes. it could induce some peroxisome proliferators-activated receptors, which contribute to glucose homeostasis in hyperglycemia and oxidative conditions [ ] . obesity has become a worldwide health problem and leads to adverse metabolic disorders, such as cardiovascular disease and type diabetes. a couple of studies documented positive actions of broccoli sprouts on obesity by regulating lipid metabolism. in a double-blind clinical trial, broccoli sprout powder as supplementary treatment in type diabetic patients could have beneficial effects on lipid profiles and oxidized low-density lipoprotein ratio (ox-ldl/ldl), as risk factors for obesity and cardiovascular disease [ ] . another mechanism to regulate lipid metabolism of broccoli sprouts may be associated with sulforaphane capacity to induce the nrf pathway [ ] . a recent study revealed that glucoraphanin extracted from broccoli sprouts can decrease the lipid accumulation and increase the nrf activation. it leads to the downregulation of the expression of multiple genes involved in gluconeogenesis and lipogenesis, thereby alleviating obesity and diabetes [ ] . several other health-promoting effects of broccoli sprouts and microgreens have also been explored by in vitro, in vivo, and clinical research. a couple of studies indicated that broccoli sprouts are a good source of bioactive compounds that act as xanthine oxidase (xo) inhibitors [ , , ] . xo is the enzyme that catalyzes the metabolism of hypoxanthine and xanthine into uric acid, so inhibiting xo activity may be potentially useful in the treatment of gout or other xo-induced diseases [ ] . broccoli sprout supplementation during pregnancy and the early newborn period could reduce brain injury following placental insufficiency in the newborn rats. the findings provide a new approach for the prevention of cerebral palsy and disabilities related to placental insufficiency [ ] . the analgesic and antinociceptive effects of broccoli sprout extract were proven by the opioid mechanism using two in vivo experimental models of nociception. this study suggests the potential activity of broccoli sprouts in pain therapy [ ] . in a double-blind study, the short-term ingestion of broccoli sprout homogenates showed the beneficial effects on nasal responses to the influenza virus in smokers by significantly decreasing virus-induced markers of inflammation and reducing the virus quantity. it may be a promising strategy for preventing influenza risk in smokers and other people exposed to airborne pollutants [ ] . in recent years, broccoli sprouts and microgreens are one of the most consumed vegetable products. they have gained recognition as functional foods or nutraceutical foods by the increasing interest of consumers for diets that support health and longevity. consequently, the health-promoting compounds of broccoli seedlings have been extracted and isolated to integrate into food and pharmaceutical product formulations. over the past ten years, the extraction, isolation, and characterization of the functional properties of these broccoli products have been demonstrated by numerous scientific publications. in this review, we summarized for the first time the research findings of the last decade into bioactive constituents, bioactivities, and molecular mechanisms of broccoli sprouts and microgreens. they have been proven to present an abundant source of important natural compounds, including glucosinolates, phenolics, flavonoids, vitamins, minerals, and pigments. in particular, glucosinolates and their hydrolysis products are the main components analyzed, followed by phenolic compounds with a detailed profile. moreover, the previous studies have focused on several biological activities of broccoli seedlings, such as antioxidant, anticancer, antimicrobial, and anti-inflammatory, as well as the potentially beneficial effects for patients with cancers, diabetes, and obesity. in general, they are non-toxic or have low toxicity. hopefully, this updated review, in addition to being a reference for consumers' food selections, might attract more attention to broccoli sprouts and microgreens and their further applications in the food and nutraceutical industries, or even in clinical studies as cancer chemopreventive agents. in the future, more bioactive compounds in broccoli sprouts and microgreens will be isolated, identified, and evaluated. further investigations are required for exploring unrecognized biological functions and their underlying mode-of-action and molecular mechanisms in both in vitro and in vivo models, such as cardiovascular protective, hepatoprotective, neuroprotective, or enzyme inhibitory potentials. besides, well-designed clinical trials should be carried out to confirm these health-promoting benefits of broccoli seedlings on humans. food security and sustainable intensification broccoli microgreens: a mineral-rich crop that can diversify food systems how circular will you eat? the sustainability challenge in food and consumer reaction to either waste-to-value or yet underused novel ingredients in food small-seeded legumes as a novel food source. variation of nutritional, mineral and phytochemical profiles in the chain: raw seeds-sprouted 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analgesia-preclinical in vivo studies effect of broccoli sprouts on nasal response to live attenuated influenza virus in smokers: a randomized, double-blind study funding: this research received no external funding. the authors declare no conflict of interest. key: cord- - jfw y authors: albiol, nil; awol, rahinatu; martino, rodrigo title: autoimmune thrombotic thrombocytopenic purpura (ttp) associated with covid- date: - - journal: ann hematol doi: . /s - - - sha: doc_id: cord_uid: jfw y nan a -year-old woman with a history of hypertension and breast cancer in complete remission was seen in late-march at the emergency ward in a private clinic with dry cough, anosmia, and dysgeusia. physical examination found a low grade fever ( . °c) but was otherwise normal. a thoracic computerized tomography was normal, and a single nasopharyngeal swab (nps) was reported as negative for severe acute respiratory syndrome coronavirus (sars-cov- ) by a polymerase chain reaction (pcr)-based test. a complete blood work-up was normal except for mild lymphopenia (day on table ). because of the pandemic state of sars-cov- and associated symptoms [ ] , the treating physicians considered the patient to have coronavirus disease (covid- ) but with a false-negative result of the pcr nps test. the patient was treated with lopinavir/ritonavir, hydroxychloroquine, and azithromycin. on the fifth day of treatment (day on table ) routine blood tests showed severe thrombocytopenia, moderate anemia with a normal reticulocyte count, and high serum lactate dehydrogenase (ldh) and bilirubin (table ) . treatment with methylprednisolone mg/kg/ h and intravenous immunoglobulin was added, but after no improvement in laboratory findings (day on table ), the patient was transferred to our institution for further management. after initial clinical and laboratory work-up at our institution (day on table ), a diagnosis of autoimmune thrombotic thrombocytopenic purpura (ttp) was rapidly established, based on the presence of microangiopathic hemolytic anemia, severe thrombocytopenia, and very low activity ( %) of adamts- (a disintegrin and metalloproteinase with a thrombospondin type motif, member ) in combination with the presence of an adamts- inhibitor, which is an autoantibody to adamts- [ , ] . a nps sample was retested in our center on admission and was negative for sars-cov- , but serological tests were positive for sars-cov- igg, thus confirming the past covid- [ ] . treatment with plasma infusion on admission led to a rapid rise in the patient's platelet count (day on table ). after placement of a central venous catheter, therapeutic plasma exchange was begun, with a favorable clinical and laboratory course over the next week (day on table ). we present a case of acquired autoimmune ttp whose onset occurred immediately after covid- , since the patient was admitted for this latter infection with normal laboratory values. of course, this could be a mere coincidence rather than a causal relationship, owing to the very high incidence of sars-cov- infection [ ] . as with other hematological disorders, the covid- pandemic may change the way in which the approach to and management of patients may vary from the standard of care, as highlighted by the american society of hematology covid- resources center (specific information on ttp can be found at https://www.hematology.org/covid- / covid- -and-ttp). conflict of interest the authors declare that they have no conflict of interest. ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. informed consent informed consent was obtained from all individual participants included in the study. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. isolated sudden onset anosmia in covid- infection. a novel syndrome? rhinology thrombotic thrombocytopenic purpura: diagnostic criteria, clinical features, and long-term outcomes from through predictive features of severe acquired adamts deficiency in idiopathic thrombotic microangiopathies: the french tma reference center experience antibody responses to sars-cov- in patients of novel coronavirus disease consent for publication consent for publication was obtained from all of the authors. key: cord- -knhkdlq authors: kanduc, darja; stufano, angela; lucchese, guglielmo; kusalik, anthony title: massive peptide sharing between viral and human proteomes date: - - journal: peptides doi: . /j.peptides. . . sha: doc_id: cord_uid: knhkdlq thirty viral proteomes were examined for amino acid sequence similarity to the human proteome, and, in parallel, a control of sets of human proteins was analyzed for internal human overlapping. we find that all of the analyzed viral proteomes, independently of their structural or pathogenic characteristics, present a high number of pentapeptide overlaps to the human proteome. among the examined viruses, human t-lymphotropic virus , rubella virus, and hepatitis c virus present the highest number of viral overlaps to the human proteome. the widespread and ample distribution of viral amino acid sequences through the human proteome indicates that viral and human proteins are formed of common peptide backbone units and suggests a fluid compositional chimerism in phylogenetic entities canonically classified distantly as viruses and homo sapiens. importantly, the massive viral to human peptide overlapping calls into question the possibility of a direct causal association between virus–host sharing of amino acid sequences and incitement to autoimmune reactions through molecular recognition of common motifs. protein sequences of the human proteome as well as of a number of viral proteomes have become available in databanks, offering an opportunity for a thorough comparative analysis of their reciprocal inter-relationship(s). this area of inquiry is of special interest because of our persistent ignorance of the mechanisms at the basis of viral pathogenesis and virus-host interactions [ , ] . to define the molecular structural determinants possibly involved in viral virulence and human susceptibility, we have undertaken a systematic analysis of a large set of viral proteomes searching for amino acid sequences shared with the human proteome. specifically, the study was designed to answer two main questions: (i) what is the quantitative dimension of shortpeptide sequence sharing between viruses and human proteomes? (ii) how many human proteins harbor viral peptide modules? the two questions are of paramount importance in the context of autoimmune diseases. indeed, it is a common belief that an autoimmune reaction is mostly caused by a host receiving an antigen that has amino acid homology/similarity with amino acid sequences in selfantigens of the host [ ] . this may result in the host immune system attacking the organs and tissues expressing the selfantigens with the shared sequences [ ] . in this context and given the sustained increase in the incidence of autoimmune diseases [ , , , ] , the mathematical quantification of peptide overlap extent between viruses and humans is essential to understand the role of structural viral similarity in the pathogenesis of autoimmunity. here we report a virus versus human comparative screening that reveals a massive, indiscriminate, unexpected pentapeptide overlapping between viral and human proteomes. the viral proteomes analyzed in this study were downloaded from www.ebi.ac.uk/genomes/virus.html and the human proteome obtained from uniprotkb (http://www.ebi.ac.uk/ integr ). the human proteome originally contained , proteins at the time of download, but we filtered out ( ) two viral contaminants (i.e. viral proteomes which were being expressed in human cells), and ( ) duplicated sequences and fragments. after the filtering, we were left with a human proteome consisting of , unique proteins. similarity analyses were conducted on viral proteomes for a total of viral proteins, equal to , amino acids. a similarly sized control set of human protein samples (for a total of human proteins, equal to , amino acids) was analyzed for inter-human overlapping. more precisely, for each viral proteome size, we constructed an artificial human sub-proteome drawn from the human protein set. the proteins were selected at random without replacement, and assigned to an artificial sub-proteome at random. we kept adding human proteins to each artificial human sub-proteome until adding the next protein would have caused the total number of amino acids in the artificial sub-proteome to exceed to the number of amino acids in the corresponding viral proteome. this way we eliminated possible bias in the comparison. the artificial sub-proteomes are random samplings of human proteins and represent cross-sections of all human proteins. the list of the human proteins forming the artificial human sub-proteomes is detailed as swiss-prot entries in table . sequence similarity analyses of each of the viral proteomes (or the artificial human sub-proteomes) to the human proteome were carried out using viral (or human) mers sequentially overlapped by four residues. the scans were performed by custom programs written in c and utilizing suffix trees for efficiency [ ] . the viral or human proteins were manipulated and analyzed as follows. the entire viral (or human) proteome was decomposed in silico to a set of -mers (including all duplicates). a library of unique -mers for each virus or human proteome was then created by removing duplicates. next, for each -mer in the library, the entire human proteome was searched for instances of the same -mer. any such occurrence was termed an overlap. cursory analyses (e.g. identification of unique overlapping -mers, counts of unique overlapping -mers, counts of duplications) were performed using linix/unix shell scripts and standard linux/unix utilities. data were plotted and linear least-squares regression was performed to determine whether any linear relationships exist between the level of overlap and the size of viral (or control) proteomes. the same procedure was applied when longer amino acid sequences (esa-, epta-, octapeptides, etc.) were used as probes in the similarity analyses. the similarity analysis for the artificial human subproteomes was performed as for the viral proteomes, with p e p t i d e s ( ) - one modification. the modification is that all the proteins in the artificial proteome were removed from the comparison human proteome when the overlap determination was done. e.g., the artificial human sub-proteome contains human proteins selected from the original set of , proteins comprehensively forming the human proteome. these proteins were removed from the comparison set prior to determining the overlap of artificial human sub-proteome versus human; i.e. for artificial proteome , the overlap comparison was between proteins in the artificial proteome and the remaining , human proteins. and similar for each of the other artificial proteomes. the viruses were chosen based on the following criteria: ( ) known to be pathogenic to human; ( ) of significant health impact (e.g. hcv, rhinovirus, hiv, mumps, measles, sars, hpv, and polio); ( ) phylogenetically different; ( ) proteomes established to a significant degree of completeness. in addition, the viral proteomes were chosen to span a range of proteome sizes, with the smallest viral proteome being amino acids, the largest being , amino acids, and all the viral proteomes combined amounting to , amino acids. the viral proteomes are described in table . as controls, we used similarly sized protein sets drawn from the human proteome. in brief, we constructed artificial sub-proteomes populated by proteins randomly selected without replacement from the human proteome. the constraints we used were that ( ) the size of each artificial sub-proteome was to approximate the size of one of the viral proteomes, and ( ) that the combined size of all the artificial proteomes was to approximate the combined size (in amino acids) of the viral proteomes. the resultant artificial human sub-proteomes are described in table . table has four columns. the first is the artificial subproteome number. the second is the cumulative amino acid length of all the human proteins in that artificial subproteome. the third column is the cumulative amino acid length of the corresponding viral proteome. the fourth column is the number of human proteins in the artificial sub-proteome. for example, in our virus versus human analysis, information for hepatitis c virus (hcv) was used as follows. the virus has taxonomic id and a proteome consisting of amino acids (see table ). corresponding to this virus is the artificial human sub-proteome , which contains human proteins which sum to a cumulative length of amino acids (i.e. just about the amino acids in the hcv proteome). and similar for each of the other viral proteomes. the list of the human proteins forming the artificial human sub-proteomes is detailed as swiss-prot entries in table . sequence similarity analyses of each of the viral proteomes (or the human artificial sub-proteomes) to the human proteome were conducted using viral (or human) mers sequentially overlapped by four residues. basically, we used -mer sequences as probes to scan viral/human proteins against human proteome since pentapeptides are minimal structural units critically involved in biological/pathological interactions such as peptide-protein interaction and (auto)immune recognition ( [ , , , , ] and further refs. therein). longer amino acid sequences (esa-, epta-, octapeptides, etc.) were used as additional probes to control the similarity pattern. quantitative analysis of the pentapeptide overlapping of viral versus human proteomes the numerical values that define virus-to-human similarity level are reported in table . the table documents that all of the viral proteomes under analysis have high and wide pentapeptide overlapping to the human proteome. only a limited number of viral pentamers are unique to the viruses, with no counterpart in the human proteome. the overlapping extent (in terms of percentage of unique viral -mers which occur in the human proteome, see column in table ) is rarely less than %. that means that almost % of the viral pentamer peptides are widely, intensively and repeatedly scattered throughout the human proteome. numerically, the viral versus human overlap is defined by , , total table . p e p t i d e s ( ) - matches. also the viral overlapping distribution in the human proteome (as number of human proteins hosting viral -mers, column in table ) is highest, being close to % with hhv- (i.e. hhv- pentamers are present in , human proteins out of the , ones which comprehensively form the human proteome). given the ample literature documentation and research data in support of the critical role exerted by pentapeptide modules in cell biology as well in antigen/antibody immunorecognition ( [ ] , and refs. therein), the data of table are impressive. they become even more significant, if possible, when compared to the control data obtained for inter-human p e p t i d e s ( ) - pentapeptide overlapping illustrated in table . when human artificial sub-proteomes were analyzed for pentapeptide overlapping to the entire human proteome, we obtained clear numerical evidence that the inter-human level of perfect -mer matching is of the same order of magnitude as the viral overlapping to human proteome detailed in table , i.e. there are , , inter-human versus , , viral perfect -mer matches to the human proteome (see table ). likewise, the number of human proteins involved in viral and inter-human overlaps is highest and practically identical in the numbers: human proteins are involved in the inter-human peptide overlapping for , times and in the viral versus human ( ) total number of -mers in the artificial sub-proteome (including multiple occurrences); ( ) unique -mers from the artificial subproteome occurring in the human proteome; ( ) occurrences in the human proteome of -mers from artificial sub-proteome (including multiple occurrences); ( ) number of human proteins in the human proteome involved in overlap; ( ) % of unique -mers from the artificial sub-proteome which occur in the human proteome (i.e. Â column /column ). a analogous to viral proteomes in size (see table ), and composed by set of human proteins as detailed in table overlap for , times (see data from tables and , respectively). this indicates a massive, repeated, acritical usage of the same pentapeptide blocks in viral and human proteomes. plotting the viral -mer occurrences in the human proteome as a function of the viral proteome length (see data under table , columns and ) produces the graph illustrated in fig. . the following observations are worth noting: first, the level of viral overlaps to the human proteome is directly related to the length of the viral proteome by a linear relationship. the pearson's correlation coefficient of the line is . , and represents an exceptionally strong linear relationship. biologically, fig. suggests that peptide motif sharing is a constant property of the viral proteomes, exclusively depending on the viral proteome length and with no relationship to other structural and/or pathogenic viral features. as a note of interest, it can be seen that htlv- , rubella virus, and hcv present a number of overlaps to the human proteome above the expected number of overlaps predicted by the linear regression line. this indicates that the massive, repeated, acritical, usage of the same pentapeptide blocks in viral and human proteomes is even more accentuated in the case of htlv- , rubella virus, and hcv. to understand the overlap usage in entities so distant in the evolutionary time and so enormously different in the evolutionary history such as viruses and humans, we investigated the profile of viral motif distribution along the human proteome. representative histograms are reported in fig. . it can be seen that, independently of the virus size, the density of the viral -mer motif matches along the human proteome presents constant behavior, with virus portions endowed with high similarity alternating with portions scarcely represented in the human proteome. the same alternating behavior is shown by the histograms of inter-human overlapping by analyzing sub-proteomic sets of low, medium and large size (fig. ) . figs. and confirm the commonality of pentapeptide block usage in viruses and humans and, more in general, document the existence of a basic structural platform in the protein world. as shown in tables and , the viral versus human overlapping is still remarkable by using viral esa-or eptapeptide sequences as probes. non-stochastic nature of the peptide overlapping between the viral and human proteomes under analysis the results illustrated in tables , and are indicative of a widespread peptide overlapping between viral proteins and the homo sapiens proteome. in order to understand how the above reported data are mathematically governed, we explored the viral proteomes and the human subproteomes under analysis for the degree of internal redundancy and the viral versus human proteome overlapping at nmer level (with n from to amino acids). the quantitative data we found are reported in table listing three orders of data: (a) the number of unique occurrences of n-peptides in the viral (or the human) proteome samples; (b) the total number of occurrences (i.e. including multiple occurrences) of n-peptides in the viral (or the human) proteome samples; and c) the relative viral versus human n-peptide overlaps. the first set of data, column through in table , provides evidence that the numerical values for uniquely expressed npeptides are always lower than the actual total values for npeptide occurrences. this is remarkable in light of the enormously high number of potential n-peptides which theoretically are available. as an example, in face of the possible , , pentamers, both the viral proteomes and the human sub-proteomes present a high degree of repetitiveness in their -mer composition. in fact, the viral proteomes and the human sub-proteomes are respectively formed by a total of , and , -mers and, although there is the possibility of forming , , different pentapeptides, these total -mers present , and , repeated pentapeptides, in the viral and human proteins, respectively. the intra-viral (or intra-human) peptide redundancy strikingly persists at higher n-mer level, despite the exponentially increasing numbers of theoretically possible nmers. the intra-repetitiveness is mathematically quantified in table , columns and , for the viral proteomes and the human sub-proteomes, respectively. the second set of data illustrates the overlapping at npeptide level (with n from to amino acids) from the viral proteomes versus the human sub-proteomes (table , columns and ) . again, we note that, although there is a potential pentapeptide reservoir amounting to , , , still , of unique viral pentapeptides ( . % of the total) appear in the human proteins forming the human sub-proteomes. when considering the total viral pentapeptides including multiple occurrences (table , column ), the viral contribution to the human subproteomes raises to , pentamers. these repeated viral overlaps in the human proteins appear to further support the existence of a defined peptide repertoire which is utilized (even repeatedly) in protein composition. a similar observation holds in considering the viral peptide overlapping to the human sub-proteomes at higher n-mer level (table , columns and ) . finally, the third set of data documents the overlaps at npeptide level (with n from to amino acids) from the viral proteomes versus the entire human proteome (table , columns and ). it can be seen that, independently of the vastness of the human protein sample, the extent of the viral overlap is relevant, especially when considering that the human proteome has the possibility of drawing from an enormous number of potential theoretical n-peptides. for example, notwithstanding the astonishingly great repertoire of theoretical possible undecapeptides (that is: , , millions), nonetheless eight viral overlaps (one of which even repeated) are present in the sub-proteomic human samples. the numbers raise to and (including multiple occurrences of the same -mer) when considering the viral -mer overlaps in the entire human proteome. our conclusion is that the mathematical redundancy present in the protein world is not stochastic (i.e. is not pure random chance), but rather reflects strong peptide usage bias since certain peptides are repeatedly used (and shared) in (and among) viral and human proteins. it seems that peptide usage in the protein world (and, consequently, the degree of similarity, repetition, overlapping) is not dictated by pure mathematical laws of distribution. rather, powerful constraints appear to be at work by forcing the use of restricted platforms of n-mers and privileging the repeated usage of the chosen ones. possibly, as anticipated by preliminary data from our labs (kusalik et al., manuscript in preparation), these powerful constraints able to influence and favor specific n-mer synthesis might be found among physico-chemical factors such as hydrophobicity, residue bulkiness, dg of peptide bond formation, plus biological factors such as codon bias. . the difference between biological entities such as viruses and cellular organisms is quite obvious when the functional roles of the proteins found in viruses and in cellular organisms are compared [ ] . on the other hand, according to the data we present here, this obvious difference disappears at the peptide phenetic level. the mathematical quantification of the widespread and high pentapeptide similarity between viral and human proteomes is surprising, raising fundamental questions. in particular, the ample viral-human peptide sharing is significant to (auto)immune phenomena as well as to evolutionary pathways. indeed, in the context of the physio(patho)logical relationships between viruses and humans, it is mandatory to observe that short-peptide motifs can exert a central role in cell adhesion, signal transduction, hormone activity, regulation of transcript expression, and enzyme activity [ , , , , , ] . likewise, the antigen-antibody recognition process can reductionistically be circumscribed to interacting modules of five amino acid residues which, therefore, appear to be sufficient structural immunological determinants ( [ ] [ ] [ ] ] and refs. therein). consequently, we notice that the data shown here call into question the possibility of a direct causal association between virus-host sharing of amino acid motifs and incitement of autoimmune reactions [ ] . indeed, the molecular mimicry hypothesis suggests that, when bacterial/viral agents share epitopes with a host's proteins, an immune response against the infectious agent may result in formation of crossreacting antibodies that bind the shared epitopes on the normal cell and result in the auto-destruction of the cell. in the present case, the molecular mimicry hypothesis implies that viral infections should be a practically infinite source of autoimmunity diseases since this study demonstrates that viral -mer matches are disseminated throughout practically all the human proteome and each viral match is repeated almost more than times (see table ). consequently, autoimmune diseases should theoretically approach a % real incidence, since the viruses we examined practically are more or less disseminated throughout the entire human species. taking hcv as an example, we note that the virus versus human overlap analysis in table produces the mathematical evidence that hcv (as well as htlv- and rubella virus) are even ''more similar'' to the human proteins than that expected by the linear regression relationship reported in fig. . the data reinforce and add to our previous report on the sharing of numerous perfect exact matches between hcv and human proteomes [ ] . since out of hcv -mers occur in the human proteome, the , occurrences include multiple/repeated matches in the human proteins. more exactly, each hcv -mer that overlaps with the human proteome are repeated an average of . times. moreover, the , occurrences occur in , different human proteins (table ). that means that almost % of the , human proteins forming the h. sapiens proteome contain viral hcv pentamers. this datum is of cogent interest in the present worldwide advance of hcv infection, since it further excludes sequence similarity as a possible mechanism in hcvassociated autoimmunity phenomena and, at the same time, might address research towards new unexplored scenarios. these observations, of course, apply to almost all the viruses listed in table . when analyzed in the evolutionary context, the viral and human protein sets studied here yield a closely congruent overlapping pattern to the human proteome at the -mer level as well as at higher n-mer levels, indicating a common compositional mosaicism in viral and human proteins. the absence of specific phenetic n-mer clusterings in the viral versus human proteome (and vice versa) suggests a synergetic platform of development of non-equilibrium systems which can be traced back to the prigoginian model rather than to the column number: ( ) actual unique n-mers in the viral proteomes; ( ) actual n-mers in the viral proteomes (including multiple occurrences); ( ) number of repeated n-mers in the viral proteomes; ( ) actual unique n-mers in the human sub-proteomes; ( ) actual nmers in the human sub-proteomes (including multiple occurrences); ( ) number of repeated n-mers in the human sub-proteomes; ( ) unique viral n-mers overlaps in the human sub-proteomes; ( ) total viral n-mers overlaps in the human sub-proteomes (including multiple occurrences); ( ) unique viral n-mers overlaps in the human proteome; ( ) total viral n-mers overlaps in the human proteome (including multiple occurrences). a peptide length, with n from to amino acids. b the number of possible amino acid combinations is given by n . p e p t i d e s ( ) - darwinian evolutionary scheme, where harmful protein sequences are ''selectively removed'' whereas neutral or useful peptides are left alone. de facto, it seems that viruses and mammalians have drawn and draw from a common ancient melting pot of short-peptide modules in building up their proteomes, either simple or complex. indeed, it seems that human evolution has paid very little attention to a peptide motif being viral or not. the reconstruction of the informational network relating all living organisms might be helped by an integration with the present data which recall a time when the totality of life on earth was a simple heterogeneous community that shared only the ability to synthesize proteins with a ribosomal machine and that freely exchanged genetic material [ ] . r e f e r e n c e s to c or not to c: these are the questions myasthenia gravis: a higher than expected incidence in the elderly enhanced antigenicity of a four-contactresidue epitope of the measles virus hemagglutinin protein by phage display libraries: evidence of a helical structure in the putative active site evolutionary aspects of 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from the transmission dynamics in wuhan, china date: - - journal: nat med doi: . /s - - - sha: doc_id: cord_uid: w x g as of february there were , confirmed cases and , deaths from covid- in mainland china. of these, , cases and , deaths occurred in the epicenter, wuhan. a key public health priority during the emergence of a novel pathogen is estimating clinical severity, which requires properly adjusting for the case ascertainment rate and the delay between symptoms onset and death. using public and published information, we estimate that the overall symptomatic case fatality risk (the probability of dying after developing symptoms) of covid- in wuhan was . % ( . – . %), which is substantially lower than both the corresponding crude or naïve confirmed case fatality risk ( , / , = . %) and the approximator( ) of deaths/deaths + recoveries ( , / , + , = %) as of february . compared to those aged – years, those aged below and above years were . ( . – . ) and . ( . – . ) times more likely to die after developing symptoms. the risk of symptomatic infection increased with age (for example, at ~ % per year among adults aged – years). of case fatality risk warrants these interventions, which seriously disrupt social and economic stability. for a completely novel pathogen, especially one with a high (say, > ) basic reproductive number (the expected number of secondary cases generated by a primary case in a completely susceptible population) relative to other recently emergent and seasonal directly transmissible respiratory pathogens , assuming homogeneous mixing and mass action dynamics, the majority of the population will be infected eventually unless drastic public health interventions are applied over prolonged periods and/or vaccines become available sufficiently quickly. even under more realistic assumptions about mixing informed by observed clustering of infections within households and the increasingly apparent role of superspreading events (for example, the diamond princess cruise ship, chinese prisons and the church in daegu, south korea) , , at least one-quarter to onehalf of the population will very likely become infected, absent drastic control measures or a vaccine. therefore, the number of severe outcomes or deaths in the population is most strongly dependent on how ill an infected person is likely to become, and this question should be the focus of attention. we therefore extended our previously published transmission dynamics model , updated with real-time input data and enriched with additional new data sources, to infer a preliminary set of clinical severity estimates that could guide clinical and public health decision-making as the epidemic continues to spread globally. estimation of true case numbers-necessary to determine the severity per case-is challenging in the setting of an overwhelmed healthcare system that cannot ascertain cases effectively. therefore, as in our prior work , our approach has been to use a range of publicly available and recently published data sources (numbered to below) to build a picture of the full number of cases and deaths by age group. briefly, because the healthcare structure has been overwhelmed in wuhan and milder cases were unlikely to have been tested, we used the prevalence of infection in travelers (both on commercial flights before january and on charter flights from january to february) to estimate the true prevalence of infection in wuhan; we also used the wuhan case numbers from only the first cases to estimate the growth rate of the epidemic (assuming that the ascertainment proportion was constant between december and january ) (fig. ) . specifically, we inferred the epidemiologic parameters listed in extended data fig. by fitting an age-structured transmission model to the following data: . the epidemic curve of confirmed cases of covid- in wu- han with no epidemiologic links to huanan seafood wholesale market (which was postulated to be the index zoonotic source of the covid- epidemic) between december and january ( fig. table ). . the time between onset and death or the time between admission and death for death cases of covid- in wuhan [ ] [ ] [ ] (supplementary table ). . the time between the onset dates (that is, serial intervals) of infector-infectee pairs (supplementary table ). the clinical severity of infectious diseases is typically measured in terms of infection fatality risk (ifr), symptomatic case fatality risk (scfr) and hospitalization fatality risk (hfr). the case definitions underlying these severity measures are as follows: . ifr defines a case as a person who would, if tested, be counted as infected and rendered (at least temporarily) immune, as usually demonstrated by seroconversion or other immune response . such cases may or may not be symptomatic. . scfr defines a case as someone who is infected and shows certain symptoms. . hfr defines a case as someone who is infected and hospitalized. it is typically assumed in such estimates that the hospitalization is for treatment rather than isolation purposes. figure summarizes our estimates of age-specific scfrs and susceptibility to symptomatic infection. both parameters increase substantially with age. if the probability of developing symptoms after infection, p sym , is . , the scfr values are . % ( . - . %), . % ( . - . %) and . % ( . - . %) for those aged < years, - years and > years, respectively. the overall scfr is . % ( . - . %). compared to those aged - years, those aged < years and > years are . ( . - . ) and . ( . - . ) times more susceptible to symptomatic infection. our estimates of scfrs would be lower if p sym were higher than the baseline value of . ; for example, the overall scfr is . % ( . - . %) and . % ( . - . %) if p sym is . and . , respectively. our estimates of age-specific susceptibility are not sensitive to p sym . figure summarizes our estimates of the key epidemiologic parameters of covid- in wuhan. in the baseline scenario i.e., cases due to human-to-human (h h) transmission) between december and january (blue), the daily number of cases exported from wuhan to cities outside mainland china via air travel between december and january (orange) and the proportion of expatriates on charter flights between january and february who were laboratory-confirmed to be infected (green). the numbers of passengers and confirmed cases who returned to their countries from wuhan on chartered flights are provided in supplementary table . bars indicate the % confidence intervals (cis) of the proportion. b, the daily number of deaths in wuhan reported between december and february . (p sym = . ), the basic reproductive number is . ( . - . ). the mean serial interval is . ( . - . ) days, with a standard deviation of . ( . - . ) days. the mean time from onset to death is ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) days, with a standard deviation of ( - ) days. the epidemic doubling time (the time it takes for daily incidence to double) was . ( . - . ) days before wuhan was quarantined and public health interventions implemented within wuhan reduced transmissibility by % ( - %). we estimate that only . % ( . - . %) of symptomatic cases that occurred between december and january were ascertained. figure suggests that our estimates of the basic reproductive number, mean generation time and intervention effectiveness would be slightly lower if p sym were higher than the baseline value of . , whereas our estimates of the other parameters are largely insensitive to p sym . there is a clear and considerable age dependency in symptomatic infection (susceptibility) and outcome (fatality) risks, by multiple folds in each case. given that we have parameterized the model using death rates inferred from projected case numbers (from traveler data) and observed death numbers in wuhan, the precise fatality risk estimates may not be generalizable to those outside the original epicenter, especially during subsequent phases of the epidemic. the experience gained from managing those initial patients and the increasing availability of newer, and potentially better, treatment modalities to more patients would presumably lead to fewer deaths, all else being equal. public health control measures widely imposed in china since the wuhan alert have also kept case numbers down elsewhere, so that their health systems are not nearly as overwhelmed beyond surge capacity, thus again perhaps leading to better outcomes , . indeed, so far, the death-to-case ratio in wuhan has been consistently much higher than that among all the other mainland chinese cities (extended data fig. ). given the intensive efforts of case finding and the sharp drop in community transmission of covid- in chinese cities outside hubei over the past few weeks, the ascertainment rates in these cities were probably very high. as such, we postulate that confirmed case fatality risk in these cities should be in some ways comparable to our scfr estimates for wuhan, which attempt to account for under-ascertainment of cases in wuhan. nonetheless, crude case fatality risks estimated from cities outside wuhan should be, and are, lower than our scfr estimates for wuhan, because the former do not account for the delay between onset and death (thus being artefactually lower) and because healthcare outside hubei is less overwhelmed (thus allowing a truly lower cfr). indeed, as of february , the crude case fatality risk in areas outside hubei was . %, which is ~ - % lower than our scfr estimates of . - . % for wuhan . considering the risk estimates in context, extended data fig. compares infection, case and hospitalization fatality risks for pandemic influenza in and , sars and mers. sars causes moderate to severe disease requiring hospitalization, so the infection fatality risk and case fatality risk are essentially the same as the hospitalization fatality risk. the hospitalization fatality risk for mers is well documented, although the shape and depth of the clinical iceberg remains less well defined. in contrast, because ( ) the majority of covid- infections do not cause severe disease and ( ) hospitals in wuhan have been overwhelmed, presumably having led to prioritized admission of more serious cases, the scfr will be substantially lower than the hfr. however, despite a lower scfr, covid- is likely to infect many more (given emerging evidence of presymptomatic transmission , and growing evidence of extensive community spread in numerous countries ), thus ultimately causing many more deaths than sars and mers. compared with the and influenza pandemics, our estimates are intermediate but substantially higher than , which was generally regarded as a low-severity pandemic. we find that scfr is highest in the oldest age group. unlike any previously reported pandemic or seasonal influenza, we find that risk of symptomatic infection also increases with age, although this may be in part due to preferential ascertainment of older and thus more severe cases. one largely unknown factor at present is the number of asymptomatic, undiagnosed infections. these do not enter our estimates of scfr, but if such asymptomatic or clinically very mild cases existed and were not detected, the infection fatality risk would be lower than scfr. further clarifying this requires new data sources that are not yet available, specifically including agestratified serologic studies. our inferences were based on a variety of sources, and have a number of caveats that are highlighted below, but considering the totality of the findings they nevertheless indicate that covid- transmission is difficult to control. with a basic reproductive number of around two, we might expect at least half of the population to be infected, even with aggressive use of community mitigation measures. perhaps the most important target of mitigation measures would be to 'flatten out' the epidemic curve, reducing the peak demand on healthcare services and buying time for better treatment pathways to be developed. in due course, but almost certainly after the first global wave of infections, vaccines may also be available to protect against infection or severe disease. although our estimates of scfr are concerning, these could be reduced if effective antivirals were identified and widely adopted for the treatment of severe cases. timely data from clinical trials of remdesivir, lopinavir/ritonavir and other potential chemotherapies, as well as supportive care modalities, would be extremely informative. several important caveats are worth mentioning, as follows. first, and most importantly, our modeled estimates have necessarily relied on numerous strong assumptions, given the paucity of definitive data elements such as serosurveys, serial viral shedding studies, robust ascertainment of sufficient transmission chains and incomplete testing of travelers and returnees from wuhan, all of which need to be underpinned by systematic unbiased sampling of the underlying population and by important age and other sub-groups. our estimates of scfr are inevitably affected by under-ascertainment of cases and deaths of covid- . on the one hand, overstretched and overwhelmed healthcare surge capacity in wuhan could result in scfrs that are higher than they would be in a less stressed healthcare setting, as presumably the sicker patients would have been prioritized for admission while leaving the milder cases untested and thus unconfirmed. our prevalence estimates relying on travelers are based on those well enough to travel, so may slightly underestimate prevalence in wuhan by not including those who are already in a serious condition and perhaps hospitalized. we have accounted for the possibility that travelers may underestimate the prevalence of infection in wuhan by using our best estimate, from a separate analysis, of the probability of detection for international travelers ( % ( - %)) . on the other hand, the numerator of the number of deaths could also have been undercounted, although much less likely compared to enumerating the denominator, for the same surge capacity reason or due to imperfect test sensitivity, especially during the first month of the outbreak . if deaths in wuhan were under-ascertained, this would bias our severity estimates downward. another caveat concerns one of our key inputs-the infection prevalence among returnees airlifted out of wuhan on charter flights. their point prevalence might well be lower than that among local residents, because of a generally more advantaged socioeconomic background, and the sensitivity for detecting infected individuals among them might not be %, as assumed. as such, this would be a lower bound of the cross-sectional disease prevalence. if this were the case, then we would have overestimated the reduction in transmissibility conferred by public health interventions in wuhan and overestimated the severity. based on only publicly available data, there is necessarily substantial uncertainty in our estimates of the effectiveness of intra-wuhan public health interventions in reducing transmissibility. calculating the instantaneous reproductive number from a set of line lists that are updated daily would be the most reliable method for detecting changes in transmissibility associated with interventions. there has been refinement of case definitions at both national and provincial levels, such as excluding rt-pcr-test-positive asymptomatics (perhaps, in fact, very mildly symptomatics) from being labeled an officially 'confirmed' case or including test-naïve clinically diagnosed cases with clear epidemiologic links as 'confirmed' . although these should not affect our estimation given our data sources from the earlier phase of the epidemic, such changes in the reporting criteria may influence the interpretation of future data. finally, given that wuhan is no longer the only (albeit the first) location with sustained local spread, it would be important to assess and take into account the experience from elsewhere, both domestically in mainland china and overseas. these secondary epicenters, having learned from the early phase of the wuhan epidemic, might have had a systematically different epidemiology and response that could impact the parameters estimated here - . any methods, additional references, nature research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and estimates of basic reproductive number, mean serial interval, initial doubling time, intervention effectiveness, ascertainment rate and the mean time from onset to death, assuming p sym is . (red), . (green) and . (blue). the markers show the posterior means and the bars show % cris. we made the following assumptions in the model: . the population of wuhan is stratified into m = age groups: - , - , - , - , - , - , - , - and > . the relative susceptibility to infection of age group i is α i with respect to those aged - years (that is, α = ). the scfr of age group i is scfr i . . the probability density function (pdf) of the incubation period, f incubation , is gamma, with a mean of . days and standard deviation of . days . . the pdf of the time between onset and death, f onset-to-death , is gamma. we inferred the values of the mean and standard deviation of f onset-to-death (extended data fig. ). . the pdf of the generation time, f gt , is gamma and the same as that of the serial interval. we inferred the values of the mean and standard deviation of f gt (extended data fig. ). . the infection-symptomatic probability (p sym ; the proportion of infections that progress to develop symptoms) is the same for all age groups. we assume p sym = . in the baseline scenario and . and . in alternate scenarios. . the sensitivity of detecting symptomatic cases exported from mainland china is p det = % ( %- %) for cities that reported case importation between december and january (supplementary table . as such, we assume that the epidemic in wuhan was seeded by a single zoonotic event that generated z infections on november . we inferred the value of z (extended data fig. ). . public health interventions in wuhan reduced local transmissibility by φ . we inferred the value of φ (extended data fig. ). . given that the epidemic curve in wuhan was weeks ahead of that in other mainland chinese cities, we ignored the effect of case importation at wuhan. these assumptions were reflected in the following susceptible-infectedrecovered (sir) model for simulating the covid- epidemic in wuhan, where s i (t), and r i (t) are the number of susceptible and recovered individuals in age group i at time t, and i(t, τ) is the number of infected individuals in age group i at time t who were infected at time t − τ: the next-generation matrix for this sir model is where t g is the mean generation time. the basic reproductive number r is the largest eigenvalue of this matrix, which is βtg n p m i¼ α i n i i . the incidence rates of infection, onset and death for age group i at time t are calculated as follows: the number of new cases (onset) and the cumulative number of cases in age group i on day d are be the summation of the number of new cases, the cumulative number of cases and the cumulative number of deaths across all age groups up to time t, respectively. similarly, is the total number of infected individuals at time t. we inferred the parameters listed in extended data fig. assuming that the remaining parameters are fixed at the values shown in extended data fig. . we use θ to denote the set of parameters that are subject to inference (extended data fig. ). the likelihood function is a product of several components associated with the data in supplementary tables - : the formulation of each component was as follows: . the number of observed international case exportations on each day is assumed to be an imperfect poisson observation of the number of infected travelers leaving wuhan on that day who had or would develop symptoms. let x d be the observed number of such international case exportations on day d between december (d s , ) and january (d e , ) based on the data in supplementary table . we assume that travel behavior is not affected by disease and hence such case exportation occurs according to a non-homogeneous process with rate λ t ð Þ ¼ p sym lw;i t ð Þ n t ð Þ iðtÞ: i let p det be the probability that an infected traveler who has or will develop symptoms is detected in the destination country. the expected number of detected case exportations on day d is and hence x d ≈ poisson(λ d ). as such, the likelihood function associated with the data in supplementary table is where g is the posterior distribution of p det from a separate study that had a mean of % and a % credible interval of - % . . let y d be the observed number of confirmed cases of covid- in wuhan with no epidemiologic links to huanan seafood wholesale market (which is presumed to be the index zoonotic source of the covid- epidemic) on day d between december (d s , ) and january (d e, ) based on the data in supplementary table . these cases are assumed to be a poisson observation of the true number of newly symptomatic cases on that day, with ascertainment rate ε, which remained fixed over this time period. as such, assuming y d ≈ poisson(εω d ), the likelihood function for the data in supplementary table is table . the prevalence of infection and symptoms among travelers are assumed to reflect a representative binomial sample of the same quantities in the wuhan population on their day of departure. the likelihood function associated with the data in supplementary table is is the proportion of individuals who were infected on day d. . we assume that all deaths from covid- infection in wuhan were confirmed. let g be the cumulative number of death cases in wuhan as of february (time t). we assume g ≈ poisson(d(t)) and hence the likelihood function associated with this data is l θ ð Þ ¼ e �dðtÞ dðtÞ g g! . we assume that the age distribution of confirmed cases is a multinomial sampling process from the age distribution of true cases. let c i be the observed number of confirmed cases in age group i in wuhan based on the data in supplementary table . the likelihood function for the data in supplementary table is . we assume that the age distribution of confirmed deaths is a multinomial sampling process from the age distribution of true deaths. given that most covid- deaths were wuhan-related, we assume that the age distribution of confirmed deaths for wuhan is the same as that for mainland china . let b i be the observed number of death cases in age group i in wuhan based on the data in supplementary table . the likelihood function for the data in supplementary table is with regard to the data in supplementary table , let a be the set of death cases whose onset dates are known, and b the set comprising the remaining cases. let v j be the observed time delay between onset and death for the jth case in a and let v l j i be the observed time between hospital admission and death (which serves as a lower bound for the delay between onset and death) for the jth case in b. the likelihood function for the data in supplementary table is where f onset-death and f onset-death are the pdf and cumulative density function (cdf) of the time between onset and death (assumed to be gamma-distributed with mean μ d and standard deviation σ d ). . with regard to the data in supplementary table , let a be the set of infectorinfectee pairs for whom the serial interval (time elapsed between their onset dates) is known and b the set comprising the remaining pairs for whom only the ranges of their serial intervals are known. let s j be the observed value of the serial interval for the jth pair in a, and s l j ; s u j i be the observed range of the serial interval for the jth pair in b. for some infector-infectee pairs, the travel history and onset dates of the infector impose a lower bound on the serial interval (supplementary table ). let s * j be such a lower bound for the jth pair. the likelihood function for the data in supplementary table is where f si and f si are the pdf and cdf of the serial interval. we assume that the serial interval and the generation time have the same pdf. we estimated the model parameters θ using markov chain monte carlo methods with gibbs sampling and non-informative flat priors. point estimates and statistical uncertainty are presented using posterior means and % cris, respectively. reporting summary. further information on research design is available in the nature research reporting summary 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influenza a h n virus: an assessment of clinical severity nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study secondary attack rate and superspreading events for sars-cov- report of the who-china joint mission on coronavirus disease (covid- ) early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia the novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- )-china chinese center for disease control and prevention. dashboard of reported -ncov cases data platform of shanghai observer. line list of -ncov confirmed fatal cases (from publicly available information wuhan municipal health commission. wuhan municipal health commission's briefing on the current pneumonia epidemic in the city hubei municipal health commission's briefing on the current pneumonia epidemic in the province infection fatality risk of the pandemic a(h n ) virus in hong kong sars-cov- viral load in upper respiratory specimens of infected patients viral load of sars-cov- in clinical samples time to use the p-word? coronavirus enters dangerous new phase quantifying bias of covid- prevalence and severity estimates in wuhan, china that depend on reported cases in international travelers the state council of the people's republic of china national health commission of people's republic of china. notice of the general office of the national health commission on the distribution of the plan of prevention and control of the pneumonia caused by the novel coronavirus situation of the epidemic of pneumonia caused by the novel coronavirus in hubei case fatality of sars in mainland china and associated risk factors epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong the epidemiology of severe acute respiratory syndrome in the hong kong epidemic: an analysis of all patients a comparative epidemiologic analysis of sars in hong kong, beijing and taiwan influenza: the mother of all pandemics age and sex incidence of influenza in the epidemic of - , with comparative data for preceding outbreaks: based on surveys in baltimore and other communities in the eastern states epidemiologic characterization of the influenza pandemic summer wave in copenhagen: implications for pandemic control strategies mortality from pandemic a/h n influenza in england: public health surveillance study middle east respiratory syndrome: what we learned from the outbreak in the republic of korea hospitalization fatality risk of influenza a (h n )pdm : a systematic review and meta-analysis middle east respiratory syndrome coronavirus (mers-cov) neutralising antibodies in a high-risk human population incubation period of novel coronavirus ( -ncov) infections among travellers from wuhan, china extended data fig. | a summary of severity estimates among pandemic influenza strains and coronaviruses with pandemic potential in the past the study is a mathematical modeling study. we have provided the full information of data in figures data exclusions not applicable. the study is a mathematical modeling study. we have provided the full information of data in figures replication all the data have been provided in figures, extended data and supplementary tables randomization not applicable. the study is a mathematical modeling study. we have provided the full information of data in figures blinding not applicable.not applicable. the study is a mathematical modeling study. we have provided the full information of data in figures reporting for specific materials, systems and methods we require information from authors about some types of materials, experimental systems and methods used in many studies. here, indicate whether each material, system or method listed is relevant to your study the authors declare no competing interests. extended data is available for this paper at https://doi.org/ . /s - - - .supplementary information is available for this paper at https://doi.org/ . / s - - - .correspondence and requests for materials should be addressed to j.t.w.peer review information joao monteiro was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. key: cord- -kp jznwb authors: roczniewska, marta; rogala, anna; puchalska-kaminska, malwina; cieślak, roman; retowski, sylwiusz title: i believe i can craft! introducing job crafting self-efficacy scale (jcses) date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: kp jznwb job crafting is beneficial for employees and organizations. to better predict these behaviors, we introduce the concept of job crafting self-efficacy (jcse) and define it as an individual’s beliefs about their capability to modify the demands and resources of their job to better fit their needs. this article describes the development and validation of a scale to measure jcse. we conducted a qualitative study to design and four quantitative studies to test the psychometric properties of this scale among polish and american employees in both paper-and-pencil and online versions. three independent (n( ) = ; n( ) = ; n( ) = ) confirmatory factor analyses demonstrated a good fit to a -factor solution comprising jcse beliefs about increasing (a) structural job resources, (b) social job resources, and (c) challenging job demands. the -item jcse scale had good internal consistency, high time stability, and good validity. it correlated positively with general self-efficacy. jcse explained unique variance in job crafting behaviors over and above general self-efficacy, and was more important in predicting job crafting than contextual factors. we demonstrate the role of social cognitions in shaping job redesign behaviors and provide a useful tool to evaluate the effectiveness of interventions dedicated to empowering jcse. job design and redesign are usually seen as top-down processes in which people who manage an organization create jobs and then decide to introduce changes to them [ ] . recently, a new perspective on this phenomenon has been suggested wherein job redesign is understood as a process depending on an individual's initiative. unbeknownst to their supervisors, employees may transform the characteristics of their jobs to match them to their needs and preferences. these proactive, bottom-up, and self-initiated actions have been labeled as job crafting [ , ] . the current covid- pandemic crisis highlighted the importance of flexibility in how jobs are performed. thus, employee job crafting may form an especially important organizational asset. moreover, job crafting has numerous positive consequences for employees and organizations (for a meta-analysis, see: [ ] ). therefore, scholars and practitioners are interested in promoting these acts in the workplace (e.g., [ ] ). when are these behaviors most likely to occur? following social cognitive theory [ ] , we argue that a key step for an employee to engage in job crafting is to build an expectancy that performance of this behavior is within this person's control. in this project we integrate social cognitive theory and the job demands-resources model [ , ] to introduce the concept of job crafting self-efficacy (jcse). we define jcse as an individual's beliefs about his or her own capability to modify demands and resources present at their job to better fit their needs and preferences. we argue that the development of this construct is warranted because scholars and practitioners are interested in finding valid, precise, and useful predictors of job crafting to strengthen these behaviors in the workplace. while the literature points to a list of contextual and individual antecedents to job crafting [ ] , not all of them are malleable (e.g., personality traits) or easily changed by the organizations (e.g., autonomy levels). this gap does not allow utilizing these factors as enhancers in introducing job crafting to the organizations. thus, a factor susceptible to interventions and change is practical. in the meta-analysis, general self-efficacy has been positively linked with job crafting behaviors [ ] ; however, its predictive power was low to medium, and differed for distinct crafting behaviors. we propose that being a context-specific belief, jcse predicts jobcrafting behaviors more precisely than general measures of self-efficacy. by developing a more accurate predictor, we help to explain when job crafting occurs, which is relevant for both research and practice. we contribute to research and practice in three distinct ways. first, we introduce a malleable, context-specific construct to explain when employees engage in job crafting acts. by integrating social cognitive theory and the job demands-resources model, we explicate how such beliefs are formed and affect behaviors. this knowledge is vital for organizational practice, because it guides development of practices and interventions that may help with introducing job crafting in the workplace. next, we investigate the interplay between contextual (i.e., perceived opportunities to craft) and individual (i.e., jcse) factors predicting job self-efficacy to examine their relative importance. an answer to this question may help better guide organizational actions aimed at supporting proactivity in the workplace. finally, we provide an instrument to measure jcse beliefs, which was validated across different formats and two languages. this scale may be used to evaluate the effectiveness of interventions dedicated to empower jcse. the term job crafting was first coined by wrzesniewski and dutton [ ] who described the bottom-up process of job re-design, wherein employees proactively introduce changes to certain aspects of their jobs. these authors proposed that individuals craft their jobs by changing their understanding of the role they perform (cognitive crafting), by altering the amount or type of tasks they pursue at work (task crafting), or by modifying their interactions with other people at work (relational crafting). the purpose of these behaviors is to increase the meaning of work and to transform the jobs people have into ones they want to perform [ ] . tims and bakker [ ] proposed an alternative to wrzesniewski and dutton's conceptualization by framing job crafting within the job demands-resources model (jd-r; [ , ] ). these authors defined job crafting as a bottom-up process of shaping one's job characteristics, that is, demands and resources [ ] . job demands are those aspects of the job that require physical or psychological effort, and are therefore associated with physiological and psychological costs [ ] . some of the demands, like time pressure, are of a challenging character, that is, although they are appraised as stressful, they provide the potential for growth and may have a positive effect on an individual [ ] . other types of job demands, like role conflict, serve as a hindrance to effective goal pursuit, and therefore negatively influence an individual [ ] . job resources represent those aspects of the job that help buffer the potentially negative impact of job demands on individuals. they support goal pursuit and stimulate personal growth [ ] . examples include work autonomy, feedback, and being provided with learning opportunities. jd-r theory poses that interactions between the levels of job demands and job resources determine employee well-being [ ] . indeed, research demonstrates that high hindrance job demands combined with low amounts of job resources result in exhaustion, whereas high levels of both challenge demands and job resources create employee engagement [ ] . given the assumptions of the jd-r theory, tims and bakker [ ] argue that employees craft their jobs to achieve higher well-being by optimizing the levels of demands and job resources in their jobs. these authors proposed four crafting strategies. the first two relate to employees increasing their job resources. individuals can seek more structural job resources, for example, by creating opportunities for development at work or expanding their levels of job autonomy. employees may also seek more social job resources. for instance, they can look for help or advice from their colleagues to better deal with the demands of their job. individuals can also optimize the levels of their job demands. they do so by increasing challenging job demands (e.g., introducing new projects in the company), and by decreasing hindering job demands (e.g., reducing workload). individuals benefit from job crafting. those who more frequently initiate changes in their jobs experience higher job satisfaction and work engagement [ ] , stronger person-job fit [ ] , increased work meaning [ ] , and better health [ ] . although the primary aims of job crafting are self-serving, organizations benefit from individual job crafting acts as well. employees who engage in job-crafting are more productive and have more sustainable employment [ ] . namely, job crafting predicts better in-role and extra-role performance, and weaker turnover intentions [ ] . in addition, ghitulescu [ ] found that job crafting is linked with reduced absenteeism. given the multiple positive consequences of job crafting, it is important to understand how to encourage it in the workplace. although job crafting concerns employees' self-initiated actions, these bottom-up re-design behaviors can be supported through organizational interventions (e.g., [ ] ). employee intention to engage in these acts combined with managerial encouragement or workplace social norms may not be enough to pursue job crafting. we argue that yet another element has to be added to that equation: an expectancy that performance of the behavior is within a person's control, that is, they have the capability to implement it successfully. this element is called self-efficacy. self-efficacy represents people's beliefs in their capabilities to exert control over the environment and execute actions necessary to deal with future situations [ ] . vough and parker [ ] argue that employees who feel self-efficacious are more likely to act proactively. indeed, social cognitive theory postulates that people with high self-efficacy levels are more eager to perform challenging or risky tasks [ ] . because job crafting is a demanding activity that requires moving beyond the job descriptions, we believe that the postulates of social cognitive theory apply to this phenomenon. when people with high self-efficacy realize that they are not completely satisfied with their jobs, they perceive that they are in control to change the situation. this realization may lead them to actually alter the characteristics of the job to fit them better [ ] . indeed, a meta-analysis performed by rudolph and colleagues [ ] revealed that general self-efficacy positively correlates with job crafting behaviors. although self-efficacy may be conceptualized and measured in a more global way as the general belief in one's competence to cope with a broader range of stressful or challenging demands [ ] , it is usually defined and measured as a domain-specific construct. social cognitive theory argues that self-efficacy measures should be context-specific because self-efficacy itself is a context-specific belief [ , ] . people usually hold distinct efficacy beliefs across disciplines, for example, one can feel very self-efficacious as an academic and, simultaneously, extremely inefficacious as a driver. indeed, previous studies suggest that applying context-specific self-efficacy measures allows outcomes to be predicted more successfully (e.g., [ ] ). to better predict job crafting in the workplace we introduce a new, domain-specific selfefficacy construct: job crafting self-efficacy (jcse). we theoretically frame our understanding of jcse within the jd-r resources approach to job crafting. therefore, we define jcse as an individual's beliefs about his or her own capability to modify demands and resources present at their job to adjust them to their needs and preferences. in that, jcse is different from job crafting itself, because the latter describes employee acts of modifications, whereas the former encompasses employee beliefs about the ability to perform these modifications. further, jcse differs from general self-efficacy, because it is contextualized to focus on one's beliefs regarding the ability to alter their job characteristics rather than optimistic self-beliefs to cope with a variety of demands in life. overall, the proposed construct of jcse combines job crafting and selfefficacy, but is distinct from them. because job crafting consists of four conceptually different dimensions in the jd-r model, we propose four corresponding types of jcse beliefs about one's ability to: (a) increase structural job resources; (b) increase social job resources; (c) increase challenging job demands; and (d) decrease hindering job demands. we argue that these beliefs form separate dimensions because they describe independent behaviors that employees may engage in to align their jobs with their own preferences. therefore, we hypothesize that jcse consists of four subdimensions (hypothesis ). based on the assumptions of social cognitive theory, we also expect that these specific beliefs predict matching job-crafting behaviors, for example, individuals who feel self-efficacious with respect to increasing challenging job demands are more likely to start new projects or learn about new developments at work and try them out. we hypothesize that there is a positive link between a specific jcse belief and a corresponding job crafting behavior (hypothesis ). specific jcse beliefs may derive from more generalized self-efficacy beliefs. due to the fact that certain levels of hierarchy in self-efficacy have been detected, where general self-efficacy beliefs consist of domain-specific self-efficacy beliefs (e.g., [ ] ), scholars posit that when individuals encounter recurrent successes in specific contexts, they develop a more generalized self-efficacy. then, this generalized belief may form the basis for an individual's assessment of prospective efficacy in new situations [ ] . therefore, we expect a positive correlation between general self-efficacy and jcse (hypothesis ). however, because jcse beliefs are context-specific, we argue that they predict job crafting behaviors more accurately than general self-efficacy (hypothesis ). self-efficacy, next to organization-based self-esteem and optimism, has been included as a personal resource in an extension to the jd-r model [ ] . xanthopoulou and colleagues [ ] showed that personal resources mediated the relationship between job resources and engagement. this result indicates that the supply of job resources activates employees' self-efficacy, self-esteem, and optimism, and makes them feel more engaged, thus affecting the motivational process described by the jd-r model [ ] . the study also showed that personal resources mediated the relationship between job resources and exhaustion. moreover, previous studies have supported the moderator role of personal resources in the relationship between job demands and well-being (e.g., [ ] ). thus, personal job resources affect the health impairment process described in the jd-r model. overall, we expect that jcse is positively related to work engagement (hypothesis ) and negatively to job burnout (hypothesis ). finally, we examine the interplay between situational conditions and jcses in shaping job crafting. thus, we compare the predictive power of jcse and a newly-developed construct that measures perceived opportunities to craft (poc) in an organization [ ] . this latter construct addresses an individual's perceptions regarding the possibilities his or her job provides to change certain aspects of it. these opportunities seem dependent on organizational-or jobrelated factors rather than individual self-efficacy beliefs. because self-efficacy has both direct and indirect (via intentions) links with actual behaviors [ ] , we expect jcse to explain job crafting behaviors above and beyond poc (hypothesis ). the aim of our project was to develop a four-dimensional measure for job crafting self-efficacy (jcse) that uses a limited number of items and can be applied regardless of cultural and occupational context. to achieve this goal, we conducted a series of studies with diverse procedures and samples. as the jcse scale (jcses) was constructed on the basis of the jd-r approach to job crafting [ , ] , in line with guidelines [ ] we started with conducting a confirmatory factor analysis to verify its four-dimensional factorial structure (hypothesis ). then, in order to achieve intended parsimony of the scale, we reduced the number of jcses items, analyzing its internal and external consistency and judgmental qualities following the guidelines outlined by stanton, sinar, balzer, and smith [ ] . next, we conducted a series of confirmatory factor analyses among distinct samples and formats, that is, paper-and-pencil (study ) and online (study ), as well as in an additional cultural context (study ). subsequently, we analyzed jcses's convergent and criterion validity (study - ) to test hypotheses - regarding jcse relations with other constructs. moreover, we have assessed jcses's discriminant validity (study - ). the overview of jcses development procedure is presented in fig . three work and organizational psychologists, familiar with job crafting research and social cognitive theory, independently generated an initial pool of items. following the recommendation for self-efficacy scales [ ] , self-efficacy items should be represented by confidencestatements with the following semantic structure: "i am confident that i can (perform something) despite (barrier)". this structure is warranted because human behavior is not only a function of intentions and cognitive control, but also is influenced by the perceived and the actual environment [ ] . thus, introducing barriers provides an opportunity for more realistic assessments [ ] . such structure was used in a variety of scales measuring a wide range of selfefficacy beliefs regarding health behaviors, e.g., physical activity [ ] , regulation of eating [ ] or resisting the urge to use drugs [ ] . hence, following these recommendations, generated items comprised job crafting behaviors along with job crafting barriers. we defined barriers as aspects of the immediate work environment and the self that inhibit the translation of motivation and abilities into job crafting. typical barriers were selected on a basis of a semi-structured interviews, where participants were asked about constraints to their crafting behaviors (see: s appendix). examples included lack of energy, hostile relations among employees or a fear of being overwhelmed with additional tasks. we choose the most typical barriers and connected them with specific job crafting behaviors. specific job-crafting behaviors were chosen on the basis of the jd-r approach to job crafting [ ] , e.g., asking one's supervisor for feedback or seeking opportunities for development. before we collected data on the jcses, all authors had discussions about the proposed definition of jcse and the constructed items. after eliminating clearly redundant items, the initial version of the jcse scale consisted of items. importantly, the items of the jcse scale were worded in a simple way (avoiding complex terminology or professional jargon) making the instrument suitable for participants with different levels of education and professional backgrounds. example items for this initial version of the jcse scale include "how certain are you that you would be able to develop your knowledge and abilities despite a heavy workload?" (jcse in increasing structural resources), "how certain are you that you would be able to ask your supervisor for support, despite concerns about being judged?" (jcse in increasing social resources), "how certain are you that you would be able to set new job challenges, even though it leads to more responsibilities or a larger workload?" (jcse in increasing challenges) and "how certain are you that you would be able to reduce how work affects your emotions despite worries about how others will judge you?" (jcse in decreasing demands). analogical to other se scales [ ] , response categories refer to the certainty of being capable of performing the indicated behaviors in one's job. we used a -point scale, ranging from (definitely incapable) to (definitely capable). this first version of the scale was assessed to test the factorial validity of the scale and choose items for the final version of the instrument. a summary of participants' characteristics across studies - is given in table . all studies described in this manuscript were carried out in accordance with the recommendations of departmental ethics committee, who provided specific approval for this research project after the research was reviewed (wke/s /v/ ). we obtained written informed consent from all subjects in study and , and online consent in studies and , in accordance with the declaration of helsinki. all of our studies were based on convenience sampling. study . the first study had a cross-sectional design. participants were employees recruited by seven research assistants who distributed paper-and-pencil questionnaires among working adults in poland. participants were not remunerated for participation. study . the second study was of a cross-sectional nature as well. participants were recruited by four research assistants who distributed invitations to the on-line study among polish working adults. participants took part in a prize draw to win one out of three vouchers for a bookstore worth about eur each. five hundred fifty-four individuals agreed to participate in this research. missing data were deleted list-wise. due to participants quitting after completing the first scale in the procedure (i.e., jcse scale) and missing data in the next scales, the sample for the subsequent validity analysis was lower (n = ) than for cfa (n = ). study . the third study had a cross-sectional design and was conducted online on the u. s. sample. the scale was translated into english beforehand using a backward translation procedure [ ] . participants were recruited through mechanical turk (mturk)-an online data collection website where individuals register to complete tasks for financial rewards. mturk was proven to be a reliable source of data collection in several studies (e.g., [ , ] ). to be eligible to participate in our study, individuals had to be employed and reside in the united states. participants were instructed about requirements of the study and its purpose (i.e., research on work attitudes and behaviors). participants spent approximately minutes on the study. after the completion of the study, each participant was rewarded with $ . in compensation. within the study survey, three validity check items were inserted to control participants' engagement in the study. there were employees who agreed to participate. individuals who failed to pass the attention test (n = ) were deleted from the database. due to missing data and participants quitting after filling in the first measure, that is, jcses, the final samples for cfa was n = and n = for the validity analysis. study . the fourth study was conducted by adopting a time-lagged design. the data were gathered using a paper-and-pencil procedure at two measurement points with a six-week time break between time and time . at time and participants were asked to mark their questionnaire with a unique code. additionally, participants were informed at time that there would be a second measurement point in weeks, and they were kindly asked to attend the study if possible. there were participants at time and at time . participants were not remunerated for participation. in each study we measured job crafting self-efficacy as well as actual job crafting behaviors. in study we additionally tested general self-efficacy and work well-being indicators, i.e., work engagement and job burnout. in study we also measured general self-efficacy. in study we assessed perceived opportunity to craft in the organization. all used measures are described below. job crafting. this construct was assessed in study , and using four sub-dimensions of the polish version [ , ] of the job crafting scale (jcs) originally developed by tims, bakker and derks [ ] . in study we used the english language version of the jcs. the dimensions are: increasing structural job resources (e.g., "i try to develop my capabilities"), increasing social job resources (e.g., "i ask colleagues for advice"), increasing challenging job demands (e.g., "when there is not much to do at work, i see it as a chance to start new projects") and decreasing hindrance job demands (e.g. "i make sure that my work is mentally less intense"). items were rated on a -point scale, ranging from (never) to (very often). general self-efficacy. the variable was measured in study with the general self-efficacy scale (gses; [ ] ) in adaptation by juczyński [ ] . in study the original english version of gses was used. the instrument consists of items (e.g., "i can always manage to solve difficult problems if i try hard enough"). participants respond to these items using a -point scale ranging from (not at all true) to (exactly true). work engagement. this construct was measured in study with the short version of the utrecht work engagement scale (uwes- ; [ ] ) in adaptation by cieslak and colleagues [ ] . the instrument consists of items (e.g., "at work, i am bursting with energy", α = . ). participants respond to these items using a -point likert scale ranging from (never) to (always). job burnout. this variable was measured in study with oldenburg burnout inventory (olbi) [ ] in adaptation by baka and basińska [ ] . the instrument consists of items (e.g., "after my work, i usually feel worn and weary", α = . ). the scale ranged from (totally disagree) to (totally agree). perceived opportunity to craft in organization. the variable was measured in study with perceived opportunity to craft scale (pocs; [ ] ) translated into polish by the authors of this study using a back translation procedure. the scale consists of items (e.g., "at work i have the opportunity to vary the type of task i carry out"). reliability of the scale was high and stable at time (α = . ) and time (α = . ). factorial structure of the jcses analytical strategy. to verify the factorial structure of jcses we performed a series of confirmatory factor analyses (cfa) using mplus . [ ] . missing data were deleted list-wise. in step , we tested for the multivariate normal distribution. significant coefficients of skewness and kurtosis (p < . ) indicate that this assumption cannot be accepted in each reported sample. consequently, we used the maximum likelihood robust (mlr) estimator instead of maximum likelihood (ml) estimator to test the fit. according to a multifaceted approach to an assessment of the model fit, we considered the following fit indicates: comparative fit index (cfi; [ ] ) and tucker and lewis index (tli; [ ] ); root mean square error of approximation (rmsea; [ ] ) along with % confidence interval limits. additionally, we report traditional chi-square statistics. the model is considered to fit the data when the following values are obtained: rmsea < . [ ] , and tli and cfi > . [ ] . we also tested χ /df ratio across models, assuming that values below three generally indicate good model fit [ ] . confirmatory factor analysis of the initial version of the jcses. the theoretically supported four-factor model was tested in study against an alternative one-factor model. this comparison is derived from theory as self-efficacy may also be measured as a more general one-factor construct. the results of the cfa regarding the goodness-of-fit indices of the tested models are presented in table (see: jcse- ). the results revealed that the one-factor model of -item jcses fits the data inadequately, with high rmsea (. ) and the chi-square/df ratio above ( . ). the goodness-of-fit indices of the four-factor model are not highly satisfactory; however, they are better than a one-factor model with the results of rmsea (. ) and the chi-square/df ratio ( . ). the cfi and tli ratios are not satisfactory in both models: one-factor (cfi = . ; tli = . ) and four-factor (cfi = . ; tli = . ). we compared the models using satorra-bentler scaled chi-square [ , ] . the results demonstrated that the -factor model had a significantly better fit than the -factor model, Δχ = . , Δdf = , p < . . overall, none of the two models had good fit parameters. thus, we decided to modify the initial version of the jcses with the aim to reduce the number of items. scale reduction process. to achieve indented parsimony of the scale we selected three items per subscale. research suggests that scales consisting of three items may achieve adequate internal consistency reliability [ ] . following the recommendations of stanton, sinar, balzer, and smith [ ] regarding the reduction of the self-reported scales, we first analyzed the internal consistency and then the judgmental qualities of the jcses. see s table for jcses- 's reliability estimates and factor loadings. internal consistency refers to the overall degree that items of an instrument are intercorrelated and form a homogenous construct. since all cronbach's alpha reliability estimates were high and range from . (jcse in decreasing hindrance demands) to . (jcse in increasing structural resources), indicating good consistency of the scale [ ] , we decided to study the factor loadings of items. according to stanton et al. [ ] , factor analysis is a popular technique used in the scale reduction as retained items that fail to load strongly on the factor of interest may increase the internal consistency of the scale. since all of jcse- items had significant and satisfactory loadings (i.e., more than . ; see [ ] ), we did not have any premises to exclude items upon this criterion. in the last step, an expert judge panel evaluate the qualities of the subscales such as clarity of expression, the semantic redundancy of an item's content with other items, and face validity [ ] . this criterion of assessment is very important, since respondents might negatively view items that are highly redundant with each other. following this recommendation, three work and organizational psychology experts analyzed items and chose those that were clear and distinctive from each other. on this basis, we chose items per dimension, resulting in a total of items for future studies. confirmatory factor analysis of jcses- . to verify the proposed four-factor model of the shortened version of jcses, we performed a confirmatory factor analysis (cfa), using mplus . [ ] on a sample from study . as table shows (see: jcse- ), in line with the results of study , the goodness-of-fit indices suggest that a one-factor model does not fit the data well, with rmsea higher than . (. ) and the chi-square/df ratio above ( . ). the four-factor model is satisfactory as for rmsea (. ) and has an acceptable chi-square/df ratio ( . ). however, two out of three items from jcse in decreasing demands had low factor loadings (below . ) and one of them ("postpone making tough decisions, despite the pressure from your co-workers") did not load significantly on the latent factor (p = . ) (see table ). this result may suggest that these items do not build a coherent latent factor of jcse in decreasing hindering job demands. therefore, we decided to reject the four-factor model and in the next step tested an alternative model of jcse without the dimension of decreasing hindering job demands. confirmatory factor analysis of jcses- . in the next step we verified the hypothesis of the three-dimensional structure of jcse and contrasted it with the one-factor model. as table demonstrates (jses- [pl]), the goodness-of-fit indices of the three-factor model regarding cfi and tli exceed the required . (cfi = . ; tli = . ). moreover, rmsea is low (. ) and the chi-square/df ratio is lower than ( . ) indicating a good fit. the fit of the three-factor model is significantly better in comparison with the jcse as a one-factor model Δχ = . , Δdf = , p < . . additionally, the one-factor model of jces- seems to fit the data rather poorly (rmsea = . , the χ /df = . ). we aimed to replicate the factor structure of jses- among a us sample (n = ). as table shows (jses- [us]), the fit of the three-factor model is significantly better in comparison with the jcse as a one-factor model Δχ = . , Δdf = , p < . . the goodness-of-fit indices regarding cfi and tli are higher than . (cfi = . ; tli = . ), rmsea is low (. ) and the chi-square/df ratio is lower than , indicating a good fit. to sum up, our series of studies showed that the final version of the jcse scale consists of three dimensions-jcse in: (a) increasing structural resources, (b) increasing social resources, and (c) increasing challenging job demands. to achieve parsimony of the scale, each of jcse dimensions contains three items. this three-factor structure was confirmed among distinct samples and formats (see s appendix). the final version of the instrument can be found in s appendix. in the next step, we set out to test the convergent and criterion validity of the jcses. results regarding convergent and criterion validity are based on correlations and multivariable hierarchical regression analyses. hypotheses - regarding criterion validity were verified on samples from cross-sectional online studies: polish (study ) and us (study ), and hypotheses - about criterion validity with data from study . hypothesis concerning jcse's relative role in comparison with perceived opportunity to craft as predictors of job crafting, and time stability of the scale were verified with data from study with a time-lagged procedure (polish sample). results regarding discriminant validity are based on heterotrait-monotrait (htmt) ratio of correlations, using data from study and study . the results of correlations from studies - are displayed in tables and . in line with hypothesis , there are positive and moderate correlations between each dimension of jcse and its parallel job crafting dimension, that is, increasing structural resources (study : r = . , see table , p < . ; study : r = . , p < . , see table ), social resources (study : r = . , see table p < . ; study : r = . , p < . , see table ), and challenging job demands (study : r = . , p < . , see table ; study : r = . , p < . , see table ). supporting hypothesis , we found significant and positive correlations between general self-efficacy and each dimension of jcse, i.e., increasing structural resources (study : r = . , p < . , see table ; study : r = . , p < . , see table ), social resources (study : r = . , p < . , see table ; study : r = . , p < . , see table ) and challenging job demands (study : r = . , p < . , see table ; study : r = . , p < . , see table ). to verify whether each jcses dimension has a stronger impact on specific job crafting behavior than general self-efficacy (hypothesis ), we conducted multivariable hierarchical regression analyses among polish and us samples. in each model with job crafting behaviors as explained variables, general self-efficacy (gse) was entered in the first step (model ), and in the second step we entered a relevant jcse dimension (model ). the increment in rsquare was considered as a measure of the added value of the variable entered in the second step, and thus of its lack of redundancy with respect to what is explained in the first step [ ] . table presents results of regression analyses for job crafting dimensions in studies (poland) and (us). as table indicates, adding jcse in step contributed to additional unique explained variance in each type of job crafting behavior across both samples. this result means that a relevant jcse belief predicts additional variance in job crafting behaviors over and above gse. the results among polish and us samples are comparable. in both samples, gse predicted increasing structural resources (study : β = . , p < . ; study : β = . , p < . ) in model ; however, after entering jcse in increasing structural job resources (model ), gse's impact decreased (study : β = . , p < . ; study : β = . , p < . ). jcse in increasing structural job resources positively predicted increasing structural resources (study : β = . , p < . ; study : β = . , p < . ), and explained additional variance (study : Δr = . , p < . ; study : Δr = . , p < . ;) over and above gse. general self-efficacy also predicted increasing social resources (study : β = . , p = . ; study : β = . , p = . ); however, its impact stopped being significant with job crafting self-efficacy in increasing social resources (study : β = . , p = . ; study : β = . , p = . ) as a predictor in model . jcse in increasing social resources was a positive predictor of increasing social resources (study : β = . , p < ; study : β = . , p < . ) and explained additional variance in increasing social resources (study : Δr = %; study : Δr = %). finally, general self-efficacy was a stronger predictor of increasing challenging demands in model (study : β = . , p < . ; study : β = . , p < . ), than in model (study : β = . , p < . ; study : β = . , p < . ). when jcse in increasing challenging demands was entered in model , both variables were a positive predictor of job crafting through challenges, but jcse was a stronger one (study : β = . , p < . ; study : β = . , p < . ). jcse in increasing challenging demands contributed significantly to the prediction of how often participants sought challenges (study : Δr = . , p < . study : Δr = . , p < . ). in hypothesis we predicted that all types of jcse would be positively related to work engagement (we). in study we found positive and low relations between we and jcse in table . results of the multivariable hierarchical regression analysis in study (n = ) and study (n = ). increasing both structural (r = . , p < . , see table ) and social (r = . , p < . , see table ) job resources. the relation between we and jcse in increasing challenging job demands was positive and moderate (r = . , p < . , see table ). in line with hypothesis , study demonstrated that relations between jsce dimensions and burnout are significant and negative. these relations were similar in strength and low for each jcse dimension, that is, jcse in increasing structural resources (r = -. , p < . , see table ), jcse in increasing social resources (r = -. , p < . , see table ), and jcse in increasing challenging demands (r = -. , p < . , see table ). to verify whether jcse predicts job crafting better than perceived opportunity to craft in an organization (hypothesis ), we utilized a time-lagged design (study ) with predictors measured at time , and explained variables-at time . we used a multivariable hierarchical regression analysis with pocs measured at time entered in step (model ), and a relevant jcse dimension measured at time and added as a second step variable (model ). relevant job crafting behaviors measured at time acted as an explained variable. table presents study correlations and table presents the results of the regression analysis. the results suggest that poc has a lower predictive power than jcse for job crafting behaviors. poc predicted increasing structural resources (β = . , p < . ) in model ; however, after entering jcse in increasing structural job resources (model ), poc's impact decreased (β = . , p < . ). jcse in increasing structural job resources positively predicted increasing structural resources (β = . p < . ), and explained additional variance (Δr = . , p < . ) over and above poc. surprisingly, poc did not predict job crafting in increasing social resources (β = -. , p = . ). as expected, jcse in increasing social job resources positively predicted increasing structural resources (β = . p < . ), model indicated that when jcse in increasing social resources entered the model, the link between poc and job crafting in increasing social resources became significantly negative (β = -. p = . ). finally, poc is a [ ] for both study and . the htmt test 'requires the calculation of a ratio of the average correlations between constructs to the geometric mean of the average correlations within items of the same constructs' [ , p. ]. the . threshold was used to indicate if the two constructs are distinguishable [ ] . the results of both study and study suggest that all jcse dimensions show discriminant validity with respect to dimensions measuring job crafting behaviors, as well as to gse. all ratios resulting from the htmt analyses were lower than . cut-off point, and ranged from . to . . cronbach's alpha values for all jcse dimensions in studies - ranged from . to . , which is acceptable in preliminary research [ ] . what is more, we examined test-rest reliability of time and time responses on the jcse scale by testing correlations between the two measurement points. as table demonstrates, the results of the correlation analyses between job crafting self-efficacy dimensions measured both times are similar and high, that is, r = . , p < . , for jcse increasing structural resources; r = . , p < . , for jcse increasing social resources; r = . , p < . , for increasing challenging demands. in this paper we introduced the concept of job crafting self-efficacy and evaluated the psychometric characteristics of the jcse scale. job crafting self-efficacy is a person's beliefs about his or her own capability to modify demands and resources present at their job to better fit their needs and preferences. the results of our research suggest that jcse predicts actual job-crafting behaviors. these findings are consistent with social cognitive theory [ , ] according to which self-efficacy beliefs 'play a key role in shaping the courses lives take by influencing the types of activities and environments people choose to get into' [ , p. ] .the key strength of our studies is developing a more accurate predictor of job crafting behaviors, which has both theoretical and practical implications. below we expand on these contributions. the present series of studies showed that the final version of the jcses (jcses- ) consists of three dimensions-jcse in: (a) increasing structural resources, (b) increasing social resources, and (c) increasing challenging job demands. in the first two studies we tested a four-factor model of jcse, including the dimension of jcse in decreasing hindering job demands (jcses- ). in study , the four-factor model did not fit the data well; therefore, based on the results of confirmatory factor analysis, we proposed an alternative, three-factor model where we dropped "jcse in decreasing hindering job demands" as a factor. the three-factor model of jcse is supported not only by statistical means, but is also sound given the results of two recent meta-analyses on job crafting. rudolph and colleagues [ ] found that there is low factor loading and a small amount of variance explained in overall job crafting by the dimension of decreasing hindering job demands. moreover, there is no link between this latter construct and general self-efficacy [ ] . it is therefore possible that decreasing hindering job demands operates differently than other job crafting dimensions. similarly, lichtenthaler and fischbach [ ] proposed a distinction between promotion-focused job crafting (increasing resources and challenging demands) and prevention-focused job crafting (decreasing hindering demands). these authors demonstrated that there is a positive relationship between promotion-focused job crafting and health, motivation and performance, while there is a negative relationship between prevention-focused job crafting and the aforementioned variables. it seems, therefore, that promotion-focused job crafting offers a more constructive way of optimizing the fit between one's job and their needs and preferences than a prevention-focused one. thus, we suggest that it is possible to differentiate between personal beliefs about one's abilities to perform promotion-versus prevention-focused job crafting behaviors, and that the jcses- developed here can be used to measure the first group of beliefs. recently, scholars have introduced the facet of job crafting which relates to optimizing (rather than avoiding) job demands [ ] . initial research shows that optimizing demands is positively related to work engagement, which implies that-in contrast to decreasing hindering job demands-it may be a favorable behavior [ ] . however, because optimizing job demands was not a part of the initial job crafting model [ ] , as well as the corresponding scale to measure job crafting behaviors [ ] , we have not included this in our theorizing. however, we encourage future researchers to develop jcse to capture self-efficacy in optimization of job demands. the results of our research support the validity of the jcse- . across polish (study ) and us samples (study ) all jcse dimensions were positively associated with gse. moreover, jcse dimensions were predictive of their parallel job crafting dimensions (studies - ). we also showed that specific jcse dimensions predicted actual job crafting behaviors more accurately than general self-efficacy beliefs. further, jcse- dimensions were positively related to work engagement, and negatively-to job burnout. this result supports the positive role of personal job resources in motivational and health impairment processes as described by the jd-r model [ ] . what is more, results of discriminant validity analysis showed that jcse dimensions are different from dimensions measuring job crafting behaviors, as well as from a measure of gse. thus, the results suggest that jcses, self-efficacy scale and job crafting scale do not overlap. finally, evidence from of our research showed that the distinguished dimensions can be reliably measured with our instrument as jcses- showed good internal consistency estimates (studies - ) and high stability over time (study ). importantly, the results of our studies showed that the three-factor model had significantly better fit than the one-factor model. in this sense, it follows the results for job crafting instruments, where two recent metaanalyses [ , ] revealed that specific job crafting dimensions may have distinct relationships with individual differences, job characteristics, and work outcomes. for example, only increasing structural resources and increasing challenging demands have negative relationships with turnover intentions. on the other hand, increasing social resources and increasing challenging demands are weakly, but positively associated with prevention focus [ ] . these and our findings, combined with the results from cfas, discourage from using a total score when assessing job crafting behaviors. therefore, we highly recommend using the jcses- subscale scores rather than the total score. in our series of studies, we introduced the concept of jcse and consequently contributed to the theory and practice in three distinct ways. first, our research demonstrated that jcse, as a context-specific belief, is a more accurate predictor of job crafting than general self-efficacy. thus, we introduced a predictor that is not only more precise, but its advantage (as compared to, for example, personality traits) is the potential for malleability. self-efficacy is a modifiable belief [ ] , which may be successfully enhanced using psychological interventions [ , ] . techniques enhancing self-efficacy that are used in these interventions are usually based on sources of self-efficacy listed by bandura [ ] , for example, vicarious or mastery experiences. therefore, we propose that it may be possible to promote job crafting behaviors through jcse interventions. for example, during workshops employees may be encouraged to share successful attempts in job crafting and plan how to overcome barriers that may interrupt the job crafting plans that they develop. moreover, since leaders have significant impact on employees [ ] , we believe that they can enhance jcse among their subordinates through acting as role models (i.e., vicarious experience) by demonstrating how they craft their jobs. moreover, leaders can use verbal persuasion to encourage employees to craft. on a day-to-day basis, they may use feedback and appreciate employees' crafting attempts. further experimental investigations are needed to evaluate the effectiveness of this type of interventions. second, the evidence from study also suggests that jcse is more relevant for job crafting than perceived opportunity to craft in an organization. an implication of this result is the possibility that in case of proactive behaviors, such as job crafting, the predictive power of personal resources may be stronger than factors that relate to environmental context. thus, our research demonstrates that is not enough to provide the opportunities or encouragement to craft for employees to start exhibiting these behaviors. our line of work suggests that organizations may benefit from introducing elements enhancing job crafting self-efficacy during job crafting interventions to increase the probability that employees implement job crafting practices in their jobs. moreover, the results of study suggest that jcse, being a personal resource, is linked with enhanced work engagement and reduced job burnout. therefore, our research suggests that there is an opportunity to affect the level of the aforementioned variables through jcse psychological interventions. the final contribution of our research is that the jcses- may be helpful in monitoring employees' beliefs about their capability to modify demands and resources present at their job. this instrument may be used as one of the outcome evaluation tools of job crafting interventions. our research has some limitations. in all of our studies we applied a cross-sectional (study , study , and study ) or time-lagged cross-sectional design (study ), which precludes causal inferences. further studies using a longitudinal design need to be done to explore relationships between jcse and related constructs. however, the main goal of our series of studies was to validate a scale to measure jcse and a cross-sectional design is sufficient for these strictly psychometric purposes. both the strength and directions of the obtained relationships support the theoretical validity of the jcse scale. another limitation is that in all studies we used selfreport measures, which may lead to common method variance (monomethod bias; [ ] ). to minimize this risk we avoided repeated use of the same anchor points in questionnaires, and in study we spaced the predictors and explained variables in time [ ] . to further reduce this method bias, future studies could also include various types of measures, such as peer-ratings or supervisor assessments of job crafting behaviors [ ] . moreover, the results of our research suggest that jcse in increasing social resources may operate differently than other jcse dimensions. results of the regression analysis showed that the impact of gse on increasing social resources ceases to be significant when jcse in increasing social resources enters the model. with regard to environmental factors-when the dimension of jcse in increasing social resources enters the regression model as a predictor, the influence of perceived opportunity to craft in an organization on this parallel job crafting dimension starts to be negative. future studies need to examine how jcse in increasing social resources may operate in relationship with related constructs. job crafting is said to occur in every profession [ ] , on every organizational level [ ] , and on an everyday basis [ ] . in our research we introduced the concept of jcse, which may constitute one of the key predictors of job crafting behaviors. we also developed and validated a measure to assess jcse, which may be used by both researchers and practitioners to empirically examine this phenomenon. job crafting: towards a new model of individual job redesign crafting a job: revisioning employees as active crafters of their work job crafting: a meta-analysis of relationships with individual differences, job characteristics, and work outcomes the job crafting intervention: effects on job resources, self-efficacy, and affective well-being self-efficacy: the exercise of control job demands-resources theory: taking stock and looking forward the job demands-resources model: state of the art managing yourself: turn the job you have into the job you want the job demands-resources model of burnout the job demands-resources model: challenges for future research a meta-analytic test of the challenge stressor-hindrance stressor framework: an explanation for inconsistent relationships among stressors and performance the impact of job crafting on job demands, job resources, and well-being does work engagement increase person-job fit? the role of job crafting and job insecurity work meaning in self and world perspective: a new outlook on the wami scale individual job redesign: job crafting interventions in healthcare how can i shape my job to suit me better? job crafting for sustainable employees and organizations shaping tasks and relationships at work: examining the antecedents and consequences of employee job crafting. 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concern, and proposed remedies a meta-analysis of cronbach's coefficient alpha social cognitive theory: an agentic perspective a meta-analysis on promotion-and prevention-focused job crafting transmission of reduction-oriented crafting among colleagues: a diary study on the moderating role of working conditions do-it-yourself: an online positive psychology intervention to promote positive emotions, self-efficacy, and engagement at work leaders can make or break an intervention-but are they the villains of the piece? in: leading to occupational health and safety: how leadership behaviours impact organizational safety and well-being method variance in organizational research: truth or urban legend? common method biases in behavioral research: a critical review of the literature and recommended remedies what is job crafting and why does it matter? retrieved form website posit organ scholarsh febr are managers also "crafting leaders"? the link between organizational rank, autonomy, and job crafting crafting a job on a daily basis: contextual correlates and the link to work engagement we wish to thank research assistants who helped gather data for study (sylwia dzikowska, andżelika krauze, patrycja Łuszczyńska), study (jolanta kwiecień, anna orzeszek), and study (katarzyna jabłońska, wiktoria jarosz). key: cord- -h mtpcyc authors: mathé-hubert, hugo; colinet, dominique; deleury, emeline; belghazi, maya; ravallec, marc; poulain, julie; dossat, carole; poirié, marylène; gatti, jean-luc title: comparative venomics of psyttalia lounsburyi and p. concolor, two olive fruit fly parasitoids: a hypothetical role for a gh β-glucosidase date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: h mtpcyc venom composition of parasitoid wasps attracts increasing interest – notably molecules ensuring parasitism success on arthropod pests – but its variation within and among taxa is not yet understood. we have identified here the main venom proteins of two braconid wasps, psyttalia lounsburyi (two strains from south africa and kenya) and p. concolor, olive fruit fly parasitoids that differ in host range. among the shared abundant proteins, we found a gh β-glucosidase and a family of leucine-rich repeat (lrr) proteins. olive is extremely rich in glycoside compounds that are hydrolyzed by β-glucosidases into defensive toxic products in response to phytophagous insect attacks. assuming that psyttalia host larvae sequester ingested glycosides, the injected venom gh β-glucosidase could induce the release of toxic compounds, thus participating in parasitism success by weakening the host. venom lrr proteins are similar to truncated toll-like receptors and may possibly scavenge the host immunity. the abundance of one of these lrr proteins in the venom of only one of the two p. lounsburyi strains evidences intraspecific variation in venom composition. altogether, venom intra- and inter-specific variation in psyttalia spp. were much lower than previously reported in the leptopilina genus (figitidae), suggesting it might depend upon the parasitoid taxa. strikingly, a high venom diversity was observed between the closely related drosophila parasitoids leptopilina boulardi and l. heterotoma (hymenoptera, figitidae), with none of the abundant venom proteins in common . to assess whether this variation between two figitid species that differ in their host range similarly exists in other parasitoid taxa, we compared here the venom composition of two braconid wasps, psyttalia lounsburyi and p. concolor (hymenoptera, braconidae, opiinae) that belong to the same complex of species . both psyttalia species are used as biological control agents of the olive fruit fly bactrocera oleae and they differ in their host range. p. lounsburyi is specialized on b. oleae whereas p. concolor successfully develops in b. oleae and at least other fruit fly species . comparison of p. lounsburyi and p. concolor venom was performed using a combined transcriptomic and proteomic approach, and it was extended at the intraspecific level using two geographically distant african strains of p. lounsburyi (south africa and kenya). we also compared data with large-scale venomics results from other braconids, either associated with pdvs, as chelonus inanitus and microplitis demolitor , or devoid of pdvs, as aphidius ervi , since using various parasitism strategies could possibly impact venom evolution and composition. this study will contribute to a better picture of the diversification of venom components at a short evolutionary scale, opening the way to the characterization of underlying mechanisms. structure of the psyttalia venom apparatus. as typically observed for braconidae, the venom apparatus of both psyttalia species is composed of venom gland filaments (which secrete venom), a venom reservoir, and a venom duct that extends into the ovipositor (fig. a) . as was previously described in opiinae , , p. lounsburyi and p. concolor venom gland is multi-lobed, each lobe displaying an external thick layer of tissue and a central lumen filled by a large volume of venom (fig. b,c) . the gland lobes are joined together at the base where the ovoid shaped reservoir is connected. the reservoir is composed of a large muscular layer surrounding a small internal volume of venom, suggesting it may serve as a "pump" for injecting venom at the time of oviposition rather than as a storage organ. the reservoir also shows internal structures that form intricate spirals possibly involved in maintaining the shape of the reservoir, like spiral springs, by passively counteracting the muscular contraction (fig. b,c) . at the base of the venom apparatus of p. concolor, we also observed a "round gland" filled with large vesicles (fig. c) , already described by quicke and called "basal bulb" by wharton . interestingly, the round gland and the intima spirals of the reservoir showed a strong endogenous green fluorescence (fig. d) , suggesting the presence of universal cellular auto-fluorophores such as nad(p)h and flavins, pigments, or cuticular compounds . no vlp or pdv in p. lounsburyi and p. concolor. among the few studies on psyttalia species, two had reported the occurrence of unidentified virus-like particles (vlps) within the venom secretory cells (in p. concolor, previously opius concolor , and the close species o. caricivorae fisher ). we thus performed electron microscopy on p. concolor venom glands to search for vlps, as well as on p. lounsburyi and p. concolor ovaries to ensure the absence of pdvs (polydnaviruses). we did not observe vlps or vesicular material resembling vlps in venom gland cells or venom (fig. a) , nor viral structures or pdv particles in the ovarian cells and fluid close to the eggs (fig. b-d) . accordingly, pl and pc transcriptomes contained no transcript having similarities with genes specific of nudiviruses (from which braconid pdvs derive) or the sister group of baculoviruses. as coronaviruses and cypo-like viruses were also described in p. concolor venom glands , previous observations likely corresponded to small viruses infecting the reproductive tract of the samples, as reported also in other hymenoptera . in the absence of vlps and pdvs, secreted venom proteins are likely the main maternal actors of parasitism success of psyttalia species. comparison of electrophoretic profiles of psyttalia venom proteins. venom samples collected from individuals of p. concolor (pc) and the p. lounsburyi south african (pl sa ) and kenyan (pl k ) strains were the empty space is due to retraction during dehydration. no vesicle is observed in the size range of the vlps described in parasitoids venom ( to nm). bar = nm. (b) picture of whole mounts p. concolor ovaries. oogenesis occurs in egg tubes (et), late oocytes being located in reservoirs (or) and moving down to the calyx (c) where tubes fuse to form the oviduct. bar = μ m. (c,d) tem micrographs of sections through the calyx region of p. lounsbury (c) and p. concolor (d) ovaries, showing the egg chorion (ec), the ovarian fluid (of) and the calyx cells. no pdv particle is observed inside the cells nor in the fluid surrounding the egg chorion. bar = nm. analyzed by d and d gel electrophoresis (figs and ) . on a - % sds-page, the protein content of venom glands was resolved into numerous bands from kda to more than kda (fig. ) . on the silver-stained d gels, to spots were clearly visible, having a to . - isoelectric point, and ranging from to more than kda (fig. ) . some trains of spots were also observed that likely corresponded to post-translational modifications of the same protein. the pl sa and pl k d electrophoretic profiles were very similar with presence/absence or intensity variation detected for only a few bands (fig. ) . the distribution of d spots evidenced more differences, in particular the absence of the pl sa spot number ( kda) in the pl k strain (fig. a,b) . we observed a greater variation at the interspecific level with several profile differences in the - kda range (figs and ) . comparison of transcriptomic and proteomics data between psyttalia wasps. all the major bands and spots on d and d electrophoretic profiles of pl and pc venom, and a number of minor spots ( bands for pl, bands for pc, from at least two d gels per species; spots for pl, for pc, from at least three d gels per species) were excised, and tryptic peptides were analyzed by lc-ms-ms. in parallel, a transcriptomic analysis of pl and pc venom glands was performed, based on illumina sequencing. de novo assembly of the pl transcriptome was improved using additional (full body) and sanger (venom apparatus) sequence data (supplementary figure s ) and it yielded a total of , and , unisequences for pl and pc, respectively (supplementary table s ). data suggested a similar quality of the transcriptomes, based on general features and similarity searches (supplementary table s ), as well as go terms comparison (supplementary figure s ) . the combined transcriptomic and proteomic data resulted in and unisequences for pl and pc, respectively, among which and had a putative function (supplementary tables s and s ) . some of these, such as actin- c or glyceraldehyde- -phosphate dehydrogenase , were typical cellular proteins with no predicted signal peptide (supplementary tables s and s ). whether their identification in venom is due to cellular damage during collection, or these proteins are actually secreted by non-canonical export mechanisms remains unclear. therefore, we only considered as putative venom proteins the unisequences (i) found in venom proteomics, and (ii) either predicted to be secreted or for which the presence of a signal peptide was not tested due to the incompleteness of the sequence. this resulted in a total of and putative venom proteins for pl and pc respectively (tables and ), whose relative abundance was compared using (i) the rpkm normalized number of illumina reads from pl and pc venom apparatus, mapped to the assembled transcriptomes and (ii) the number of peptides matches in mascot searches. interestingly, most of the proteins identified in the proteomics of the reservoir (detection of the most abundant putative venom proteins only, data not shown), such as actin or paramyosin, had a predicted muscular function, as expected from microscopy observations (see above; fig. ). comparison of venomics data from the two psyttalia species evidenced that % and % of the proteins identified in pl and pc were shared with the other species, respectively (tables and ; fig. a ). in comparison, l. boulardi and l. heterotoma shared less than % of the identified venom proteins . when considering only the most abundant venom proteins (rpkm > and mascot matches > ), and out of the pl and pc proteins, respectively, were shared between p. concolor and p. lounsburyi ( fig. b ; tables and ) whereas the two leptopilina species had no protein in common . finally, pl and pc venom proteins ( % and %) had already been identified in the venom of another braconid species ( fig. c ; tables and ) . identified venom proteins. putative venom proteins described below are classified based on their abundance in venom (rpkm values and mascot matches; tables and ) and their occurrence in the venom of (i) both psyttalia species, (ii) p. lounsburyi only and (iii) p. concolor only. venom proteins with a putative function are described in table (with the proposed biochemical function, previous identification in parasitoid venom, and demonstrated or proposed role in parasitism success). several proteins with low rpkm values and lacking n-terminal sequence were not considered since they were typical cellular proteins and the number of proteomic matches was low (tables and ) . proteins identified in the venom of both psyttalia species. proteins of unknown function. several abundant unisequences with no predicted function were characterized by a high number of matches (tables and ) and rather intense protein spots (fig. ) . among these, a family of five related proteins contains a signal peptide and the duf domain of unknown function (supplementary figure s ). they share similarities with venom proteins of the braconids c. inanitus , m. demolitor and m. hyperodae , and of n. vitripennis . leucine-rich repeat protein. two and four unisequences encoding leucine-rich repeat (lrr) proteins were identified in pl and pc, respectively (tables and ; supplementary tables s and s ). one of these (pl_ ) was highly abundant (rank ) in the p. lounsburyi sa strain only, and it corresponded to one of the most intense spot (spot , fig. a ). the complete unisequences contain a n-terminal signal peptide and to canonical lrr motifs similar to the lrr motif in toll-like receptors (tlrs). yet, the majority of tlrs are multidomain table , locus comp _c ). protein not found in the proteomic analysis but with rpkm > and for which a homolog was found in p. lounsburyi. proteins while psyttalia predicted proteins only contain the lrr domain, as already observed for a. ervi venom lrr proteins . as suggested for a. ervi, psyttalia truncated lrr proteins might interfere with the host immune response by targeting the toll pathway. neprilysin-like metalloproteases. three and two unisequences encoded neprilysin-like (nep) zinc-dependent metalloproteases in pl and pc, respectively (tables and ; supplementary tables s and s ) , one of which was in high abundance (rank ; and peptide matches in pl and pc venom, respectively). nep-like proteins occur in the venom of several parasitoid wasps (table ) . annexins are a family of ca + -dependent lipid binding proteins believed to be engaged in membrane transport processes, although recent work suggests a more complex set of functions. annexins normally lack signal sequences for secretion, but some members of the family have been identified extracellularly where they can act as receptors . annexins had never been described so far in the venom of parasitoids. however, some data suggest that different mammalian parasite clades possess annexins with unique properties that can be secreted and are likely involved in host-parasite interactions and host immune-modulation . moreover, some parasitic nematodes secrete an annexin-like effector into host root cells that may mimic plant annexin function during the parasitic interaction . at last, it has been shown that annexins are also involved in the binding and internalization of toxins in eukaryotic cells . arginine kinase plays a crucial role in the energy metabolism of insects and other invertebrates through the use of atp to catalyze the phosphorylation of arginine in phosphoarginine. this enzyme was detected in the venom of pteromalus puparum and leptomastix dactylopii , but its role in the host-parasitoid interaction is unknown. calreticulin is a calcium (ca + )-binding protein with multifunctional properties including chaperone functions . calreticulin was shown to inhibit host cell encapsulation in cotesia rubecula and p. puparum , although the mechanism is still unclear. calreticulin was found in the venom of several phylogenetically distant species and seems thus largely shared among parasitoids. endoplasmin (alternative names: hsp b , gp , grp- ), which belongs to the heat shock protein family, is a molecular chaperone located in the er and involved in the final processing and export of secreted proteins . among parasitoids, endoplasmin has only been detected so far in the venom gland of aphidius ervi . this venom protein was suggested to play a role in the secretion, stabilization, transport and host cell targeting of the different a. ervi venom proteins. enolase is a key enzyme in cell metabolism which is also associated with virulence of several pathogens . an extracellular enolase was recently described in the oviposition injecta from a. ervi and the venom of toxoneuron nigriceps . enolase is also released by teratocytes surrounding the a. ervi embryo . this enzyme was proposed to play an important role in the regulation of the host physiology and the host nutritional exploitation , . esterase/lipase-like esterases and lipases belong to a superfamily of hydrolytic enzymes that act on carboxylic esters . proteins belonging to this functional class were previously found in the venom of several phylogenetically distant species , and appear to be common in parasitoids. the functions of these hydrolase enzymes in host-parasitoid interactions have not been investigated yet. gh β -glucosidases gh β -glucosidases are a family of enzymes found from bacteria to mammals that hydrolyze glycosidic bonds from glycosides and oligosaccharides, and remove non reducing terminal glucosyl residues . among parasitoids, a β -glucosidase enzymatic activity was detected in the venom of pimpla hypochondriaca . a member of this enzyme family was also recently identified, but not abundant, in the venom of microplitis demolitor and, in a low quantity, in a transcriptomic study of the a. ervi venom apparatus . gh β -glucosidases include myrosinases that play a central role in the glucosinolate-myrosinase system, one of the best-studied activated plant defense system . some insects have co-opted this system to defend themselves against enemies, by sequestering plant-derived glucosinolates and producing their own myrosinase-like enzyme [ ] [ ] [ ] . heat shock protein heat shock proteins (hsp ; alternative name: grp- ) are a family of chaperones with distinct sub-cellular localization and function . an hsp protein was identified in the venom of the parasitoid p. puparum whose function remains to be elucidated. leucine-rich repeats (lrrs) are motifs involved in protein-protein interactions . lrrs are generally composed of - amino acid stretches rich in leucine. to our knowledge, lrr domain-containing proteins were only identified in the venom of a. ervi until now . they were suggested to act as scavengers for the pea aphid toll-like receptors, thus impairing the host immune response via the toll pathway. neprilysin-like (nep) proteins are zinc-dependent metalloproteases belonging to the m peptidase family. they are involved in the degradation of a number of regulatory peptides in the nervous or immune system of mammals and insects . although they are typically membrane-bound, ectopeptidases such as nep may also be shed from the membrane through a proteolytic process and found in the surrounding fluid . nep-like proteins were detected in the venom of the braconidae a. ervi , microctonus hyperodae , m. demolitor and t. nigriceps , as well as of the figitidae leptopilina boulardi . they were also found associated with the vlps produced in the ovary of venturia canescens . neplike proteins have been hypothesized to modulate the host immune system by degrading immune-specific peptides . secreted phospholipases a (pla s) are a family of relatively stable enzymes found in venoms. pla has in vitro and in vivo immunomodulatory effects in bee venom , and neurotoxic and myotoxic effects in snake venoms . this enzyme was recently detected in the venom of m. demolitor and t. nigriceps , but its function in the host-parasitoid interaction is unknown. protein disulfide isomerases (pdis) are enzymes involved in the folding and stabilizing of nascent polypeptides in the endoplasmic reticulum (er) through catalysis of disulfide bond formation and isomerization . although this protein is normally recycled back to the er from the golgi via its c-terminal kdel motif, secreted pdis can escape this turnover mechanism . among parasitoids, pdis have only been detected so far in the venom gland of a. ervi . they have a broad substrate specificity and are involved in the folding of toxin peptides in different venomous organisms , . reprolysin-like (rep) proteins are zinc-dependent metalloproteases belonging to the m peptidase family, commonly found as constituents of snake venom. they were previously detected in the venom of the parasitoids pimpla hypochondriaca classical serine carboxypeptidases are enzymes that hydrolyze a peptide bond at the c-terminal end of peptides and proteins. a related enzyme (scpep ) that do not show proteolytic activity but is involved in other functions was described in mice . to our knowledge, serine carboxypeptidase has only been identified so far in the venom of m. demolitor and t. nigriceps . interestingly, serine carboxypeptidases have also been described as candidate virulence factors in several pathogens . serpins (serine protease inhibitors) are a large family of functionally diverse protease inhibitors. they share a conserved structural architecture with an exposed reactive center loop (rcl) of about amino acids, which acts as bait for target serine proteases . the l. boulardi venom serpin lbspny was previously shown to be involved in host immune suppression : it interferes with melanization in the drosophila host through inhibition of the phenol oxidase activation. more recently, serpins were described in the venom of a. ervi and m. demolitor but their role in parasitism success is unknown. another zinc-dependent metalloprotease was identified in each psyttalia wasp, with low inter-species sequence similarity. both proteins are weakly related to venom reprolysin-like proteins of p. hypochondriaca and eulophus pennicornis . however, the sequences were incomplete and the number of matches in the venom was rather low (tables and ; supplementary tables s and s ). gh β -glucosidases. peptides from major d spots at - kda (spots and in pl k and pl sa , respectively, spots , , in pc) matched with two unisequences (one for each psyttalia species) that encoded proteins containing a glycosyl hydrolase family (gh ) domain (pfam ) found in gh β -glucosidases (fig. ) . the high rpkm values and number of peptides matches confirmed that the pl_ and pc_ unisequences, that share % identity (supplementary figure s ) , were among the most abundant in venom (tables and ; supplementary tables s and s ). the pc sequence seems full-length, with a predicted signal peptide of residues, while the pl sequence probably lacks the n-terminal part (supplementary figure s ) . the . kda predicted mw of the mature protein is close to that of the observed spot on d gels, suggesting none or few glycosylation, although several n-glycosylation sites are predicted. yet, several spots were observed, having different isoelectric points, suggesting post-translational modification(s) and thus several isoforms (fig. ) . two pl and one pc additional unisequences, not found in proteomics, shared similarities with gh β -glucosidases, suggesting occurrence of a multigene family (supplementary figure s ) . gh β -glucosidases were previously identified in venomics data from m. demolitor and a. ervi, but they did not correspond to abundant proteins (tables , and ) . finally, the venom of the ichneumonid pimpla hypochondriaca was reported to display β -glucosidase enzymatic activity (table ) . gh β -glucosidases are found from bacteria to mammals. they play an essential role in the metabolism of glycolipids and exogenous glycosides by hydrolyzing glycosidic bonds and removing non-reducing terminal glucosyl residues from saccharides and glycosides. this enzyme family includes for instance the myrosinases, well-known for their role in the "glucosinolate-myrosinase" plant defense system (table ) , and also identified in the cabbage aphid brevicoryne brassicae that feed on crucifers . the alignment of pl and pc venom unisequences with that of well-described plant myrosinase (sinapsis alba) and insect β -glucosidases (b. brassicae, phyllotreta striolata, spodoptera frugiperda) shows conservation of all critical enzymatic site residues , , suggesting that psyttalia enzymes are indeed functional (fig. ) . insect β -glucosidases differ from plant myrosinases in that they have two glutamates in the catalytic site instead of one glutamate and one glutamine. the possible role of psyttalia venom gh β -glucosidase in host-parasitoid interaction is discussed below. heat shock proteins (hsps). molecular chaperones belonging to the heat shock protein (hsp) class, including hsp , calreticulin and protein disulfide isomerase (pdi), were found in variable amounts in the two species (tables and ; supplementary tables s and s ). endoplasmin, also identified in transcriptomic data from both species (rpkm values of . for pl and . for pc) was detected in pl venom only, suggesting a lower abundance in pc venom (tables and ; supplementary tables s and s ). in the endoplasmic reticulum (er), all these hsps cooperate to form multi-chaperone complexes involved in the folding and export of secreted proteins. hsp , pdi and endoplasmin are normally retained in the er through the binding to specific receptors via a k/r/hdel motif. since these proteins are abundant in cells, we cannot totally exclude that their detection in the venom results from tissue contamination during venom collection from the gland. however, one of the venom pdis, identified in both species, contains a substitution that changes the hdel motif into a sdel motif, suggesting a loss of binding and then of retention in the er. moreover, the binding of hsps to er receptors is labile and the retention is not absolute, allowing hsps to be secreted and to play a role in intercellular transport and signaling , . endoplasmin, for example, has been associated as a chaperone with the secretion of pancreatic lipases and their further internalization by intestinal cells in rat . venom hsps may thus play a role in stabilization of other venom proteins and/or their transport and targeting of host cells. among these, endoplasmin is of particular interest because it is a master chaperone for tlrs, a family of lrr domain-containing proteins of which members were found in psyttalia venom (see above). in accordance with a possible role of endoplasmin as a chaperone of venom tlrs, endoplasmin was only detected in the venom of pl that contains higher amounts of lrr proteins than the pc venom (tables and ; supplementary tables s and s ). interestingly these two proteins are also secreted in the venom of the braconid a. ervi . pdis play a central role in the protection of disulfide bounds of secreted proteins . interestingly, the rapid expansion of a large gene family encoding pdis specifically-expressed in the venom glands of cone snails has been recently suggested to facilitate the folding of the numerous conotoxins produced by these marine predators . here, however, phylogenetic analysis shows that psyttalia venomous pdi sequences group with different pdis from braconid wasps (supplementary figure s ) , suggesting that no specific expansion occurred. proteins of low abundance. proteins with similarities with serpin and enolase (tables and ; supplementary tables s and s ) were identified. the pl and pc serpin sequences shared % identity but only the pc sequence was complete, the pl serpin lacking the n-and c-termini (supplementary figure s ) . the pc serpin contains a signal peptide as well as the consensus hinge sequence essential for the conformational change involving the rcl and necessary to inhibit the target protease (supplementary figure s ). an extracellular enolase was already identified in a. ervi injecta and also produced by specialized extra-embryonic cells (the teratocytes) and suggested to be involved in nutritional host exploitation ( toxin-like peptides were previously identified in a. ervi venom but no toxin-like signature was found in pl venom proteins. proteins of low abundance. all the other proteins found in p. lounsburyi venom only were in low abundance (table ; supplementary table s ) . among these, an arginine kinase-like protein was identified, and similarities were found with members of the esterase/lipase-like superfamily, detected in many parasitic wasp species (table ) . proteins detected in p. concolor venom only. proteins of unknown function. two different unisequences (pc_ and pc_ ) were detected ( table ; supplementary table s ) but the sequences were not complete and the presence of a signal peptide could not be assessed. pc_ (rank based on rpkm values) was one of the most intense d spots at less than kda (fig. ) . pc_ was better ranked based on rpkm values, but the corresponding spot was less intense (fig. ) and the number of peptide matches was lower (table ; supplementary table s ) , suggesting a lower abundance in venom. annexin. one unisequence, corresponding to peptide matches, had similarities with annexins ( table ; supplementary table s ). this protein contains a aa n-terminal signal peptide followed by two annexin domains. based on data from their role in mammalian parasites, p. concolor venom annexin might be involved in binding and cell internalization of other venom proteins ( table ). proteins of low abundance. among these, a secreted phospholipase a (pla s) and a retinoid-inducible serine carboxypeptidase (table ; supplementary table s ) were identified (table ). large scale combined "omics" studies have recently increased knowledge of the nature and diversity of the venom content of parasitoid wasps , . yet, very few studies were designed to evaluate how far closely-related parasitoid species differ in venom composition. venom glands of the braconidae m. hyperodae and m. aethiopoides were shown to express a similar set of genes but analyses relied on less than ten genes, and proteomics data were only available for m. hyperodae. more recently, we evidenced striking differences in venom composition of the closely-related figitidae l. boulardi and l. heterotoma , drosophila parasitoids differing in their host range. we observed here a rather similar protein composition of the venom of p. lounsburyi and p. concolor, albeit they also differ in their host specificity level. the host range is determined by both behavioral and physiological traits that include host choice and venom adequacy to the host (i.e. "virulence"). in some taxa, the diversity of venom composition could then be decoupled from the diversity of parasitized hosts in natura if specialization mainly relies on behavioral traits. for instance, although p. lounsburyi is a specialist of the olive fly in the field, it can develop in laboratory conditions on some non-natural hosts such as ceratitis capitata. similarly, although intraspecific variation in p. lounsburyi venom was notably evidenced for a lrr protein, the difference between psyttalia strains from distant geographic origin was much lower than observed between l. boulardi strains . whether the level of diversity of venom differs among taxa (being lower in braconidae than in figitidae), or the extensive venom variation in leptopilina wasps is specific of these genus/species remains an open and stimulating question. unfortunately, a majority of the most abundant unisequences, either common or specific to each psyttalia species, had no predicted function. it is thus difficult to make assumptions about how these wasps counteract the host immune defense and regulate the host physiology, and whether they use similar mechanisms. yet, the identification of a gh β -glucosidase as one of the most abundant venom protein in both species suggests a possible role of this protein in parasitoid wasp's success. in plants, glycoside compounds and hydrolytic enzymes form a classic two-component defense system, with glycosides inducing biological effects after being activated by the enzymes. the vast array of secondary metabolites produced is used as a protection against phytophagous organisms and pathogens , . hydrolysis-products can indeed be repellent or toxic to insects, nematodes, fungi and bacteria, and they also serve as volatile attractants for specialist herbivores and their parasitoids . plant compounds and endogenous glycosidases are usually stored in separated compartments so that activation only occurs upon tissue damage. to reduce toxic effects, some phytophagous insects were shown to downregulate their gut glycosidases while others have even evolved their own glycosylation system to reglycosylate the produced aglycons. the neo-formed glycosides can then possibly be stored, similarly to the ingested plant glycosides that are indeed sequestered by several insect species. by doing so, these insects prevent the production of toxic compounds [ ] [ ] [ ] , and some even use them for their own defense . the main psyttalia hosts, b. olea and c. capitata, oviposit in developing olives and fruits that contain large quantities of various glycoside compounds. olive is particularly rich in phenol-glucosides such as oleuropein, verbascoside and rutin, which accumulate during its development while b. olea is growing inside . oleuropein was shown to be converted into a toxic compound with a glutaraldehyde-like structure -a potent protein crosslinker -by a defense-related olive β -glucosidase . assuming that fly larvae use a sequestering mechanism to survive within fruits during development, the injection of psyttalia venom β -glucosidase inside their body might result in a burst of toxic compounds that could weaken the host and increase the parasitoid probability of success. the release of sugar moieties from glycosides could also increase the amount of energy available for the developing parasitoid larvae. although this is an attractive hypothesis, the fact remains that the alleged role of psyttalia venom β -glucosidase is based on the sequestration of phenolic glycosides by the olive fly larvae, which has not been tested yet. the importance of a tri-trophic understanding of plant-herbivore and herbivore-predator/parasitoid interactions is increasingly recognized. for instance, the role of glucosides/glucosinolates has started to be evaluated not only in insect-plant interactions but also for their cascading effects on the performance of herbivore enemies through metabolic impacts or emission of volatile products . the report of the large production of a β -glucosidase in a parasitoid venom suggests that this enzyme might participate in parasitoid phenotypes such as counter-defense and parasitism success, in addition to its well-described defensive role in plants and herbivore insects. altogether, this study illustrates that parasitoid venom is a complex mixture of proteins whose relative abundance in a given species or group is still hardly explained. the study of a new species often reveals new types of abundant venom molecules, as exemplified here with the β -glucosidase, and it highlights the role of gene duplication in the rapid evolution of venom and acquisition of new features. deciphering the role of major venom proteins in psyttalia parasitism success, especially those with no predicted function, is a key challenge. this will require further exploration using techniques such as rnai, demonstrated as an efficient approach for impairing the production of parasitoid venom proteins . biological material. the south african (sa) and kenyan (k) strains of p. lounsburyi (pl) were previously described . they were reared on a laboratory strain of the fruit fly c. capitata under a : h light/dark cycle at °c. the p. concolor (pc) population was collected in in sicily (italy) and reared for one generation on c. capitata under the same conditions, prior to analysis. c. capitata is a natural host for p. concolor but it is used as a substitute host for p. lounsburyi due to the difficulties of rearing the main natural host, bactrocera oleae (the olive fly). light, fluorescence, and transmission electron microscopy. light and fluorescence microscopies were performed using epifluorescent microscopes fitted with differential interference contrast (dic) optics (imager z , zeiss) fitted with a black and white camera (axiocam mrm, zeiss). images were pseudo-colorized digitally (adobe photoshop). for transmission electron microscopy (tem), blocks were prepared from ovaries or venom glands per sample. dissected samples were pooled into μ l of ringer's saline (kcl mm; nacl mm; cacl mm; tris-hcl mm) in a centrifuge vial on ice. an equivalent volume of fixative ( % glutaraldehyde (sigma) in . m sodium cacodylate buffer, ph . ) was then added and the sample was kept for h at °c. fixed samples were centrifuged ( × g, min) to pellet tissues and remove the fixative prior to post-fixation in % osmium tetroxide in cacodylate buffer. following dehydration in graded series of ethanol solutions, samples were embedded in epon. sample sections were cut with a diamond knife using a lkb ultramicrotome, mounted on copper grids, stained with uranyl acetate and lead citrate, and observed with a zeiss em cr electron microscope at kv. total rna isolation and cdna library construction. the transcriptomic analysis was performed from samples of pl and pc venom glands using illumina rna-seq. to improve de novo assembly for pl, we also generated sanger sequences from samples of venom glands, and sequences from full insect bodies of males and females obtained from six siblings (supplementary fig. s ). pl and pc venom glands were dissected in ringer's saline and stored at − °c. total rna was extracted using trizol reagent (invitrogen) according to manufacturer's instructions, and quality was checked using an agilent bioanalyzer. cdna library construction for illumina rna-seq and sequencing was performed by beckman coulter genomics (usa). cdna library used for sanger sequencing was constructed from μ g of total rna using the creator smart cdna library construction kit (clontech). ligation products were transformed into electromax dh b escherichia coli competent cells (invitrogen). sequencing and assembly. illumina rna-seq sequencing (hiseq , × pb), sequencing ( gs-flx titanium platform) and trimming were performed by beckman coulter genomics. quality of illumina raw reads was controlled using fastqc software and reads were cleaned by removing low quality sequences and reads containing n or adaptor sequences. for sanger sequencing, a total of , clones were analyzed by the genoscope (cea, evry, france) on an abi sequencer using the standard m forward primer and bigdye terminator cycle sequencing kit (applied biosystems, foster city, ca, usa). sanger ests were then trimmed using tigr seqclean software. for each species, we performed de novo transcriptome assembly using velvet/oases assembler (https://www. ebi.ac.uk/~zerbino/oases/) after the filtering process of illumina raw reads. the first assembly step used a multiple kmer approach with kmer size ranging from to (k = , , and coverage = ). a meta-assembly (kmeta = , coverage = ) was then performed using all previously obtained transcripts (> bases long) ( supplementary fig. s ). at both assembly steps, we used cd-hit-est to remove the shorter redundant transcripts entirely covered by other transcripts with more than % identity. finally, a clustering of transcripts was performed using tigr-tgicl. to improve the quality of the assembly for pl, we included the cleaned and sanger sequences as long sequences (minimum size of bases) or otherwise as short sequences, in addition to the short illumina reads. sequence annotation and analysis. to identify similarities with known proteins, the unisequences were compared to ncbi non-redundant protein sequence database, uniprotkb/swiss-prot database, insect predicted proteome databases (drosophila melanogaster v . and nasonia vitripennis v . ) and all braconid venom proteins found in uniprotkb ( venom proteins from different braconid species), using blastx with a cut-off e-value of e- . comparisons with previously published venom gland transcriptomes of a. ervi and leptopilina spp were performed using tblastx with a cut-off e-value of e- . search for nudivirus/baculovirus-related specific genes was performed using tblastn with a cut-off e-value of e- . orf prediction and translation were performed with framedp (https://iant.toulouse.inra.fr/framedp/), signal peptide prediction with signalp (http://www.cbs.dtu.dk/services/signalp/), and prediction of n-glycosylation sites with the netnglyc . server (http://www.cbs.dtu.dk/services/netnglyc/). search for protein domains was achieved using pfamscan (ftp://ftp.sanger.ac.uk/pub/databases/pfam/tools/) and cd-search against conserved domain database (cdd) at ncbi (http://www.ncbi.nlm.nih.gov/structure/ cdd/cdd.shtml). identification of leucine-rich repeats (lrr) in protein sequences was done using lrrfinder (http://www.lrrfinder.com/). search for duf domain-containing, gh β -glucosidase and protein disulfide isomerase sequences was performed using blastp at ncbi (http://www.ncbi.nlm.nih.gov/blast/) and hmmsearch from the hmmer package (http://hmmer.org/). pairwise and multiple amino acid sequence alignments were respectively obtained using the needleman-wunsch algorithm and mafft implemented in geneious software (biomatters). phylogenetic analyses were performed using maximum likelihood (ml) with phyml (http://phylogeny.lirmm.fr/). prottest was used to select the best fit model of amino acid substitution for ml phylogeny (https://github.com/ddarriba/ prottest ). gene functions and go terms were automatically assigned to the predicted proteins based on the identification of domains with pfamscan. only the root domain of the hierarchical domain organization available from ebi was conserved. comparison of go terms between pc and pl unisequences and homogenization of the annotation level were performed using go slim terms. for each species, we used bowtie (http://bowtie-bio.sourceforge.net/) to map back all input trimmed illumina raw reads (minimum size of bases) to the assembled transcriptome with up to nucleotides mismatches allowed. to compare the unisequence expression levels, the number of mapped raw reads for each transcript was normalized with the rpkm (reads per kilobase per million reads), using the r package edger (https://bioconductor.org/packages/release/bioc/html/edger.html). analysis was performed independently on pc and pl wasp venom ( supplementary fig. s ). venom apparatus were dissected from individuals per sample and glands were collected in - μ l of ringer's saline supplemented with a protease inhibitor cocktail (sigma). glands were opened to release the venom and centrifuged for min at × g to remove residual tissues. for d sds-page, samples were mixed with × laemmli buffer containing β -mercaptoethanol (v/v) and boiled for min. proteins were then separated on a - % linear gradient sds-page and the gel was silver stained as previously described , . for isoelectric focusing (ief), samples were prepared by boiling the protein solution for min with % (v:v) of a denaturing solution ( . m dithioerythritol, % sds). after cooling, the samples were mixed with an equal volume of a solution containing . m urea, . m dithioerythritol, and % chaps. ief was performed using slab gel. slab gels were made on glass tubes cm in length ( . mm internal diameter) that were filled with % acrylamide, . m urea, % ampholytes [ % ph - (pharmacia) and % ph - (servalytes)], and % chaps. isoelectric focusing was run in two steps: a first run at ma, . w/tube, v for a total of , v/h, followed by a second run at ma, . w/tube, , v for a total of , v/h. for the second dimension, - % linear gradient sds-page was used. ief gels were incubated with x laemmli buffer containing ß-mercaptoethanol and loaded on top of the d sds-page. after separation, proteins were silver stained as previously described , . identification of proteins by mass spectrometry was performed on d bands and d spots excised from the gels as previously described , . ms/ms data analysis was performed with the mascot software (http://www. matrixscience.com) licensed in house using the combined pc and pl unisequences and non-redundant nr (ncbi). data validation criteria were (i) one peptide with individual ion score above (the mascot significant identity threshold corresponding to p < . is in our case) or (ii) at least two peptides of individual ion score above (corresponding to % probability that a peptide spectrum match is a random event). the mascot score was calculated as − *log (p). the calculated fdr (based on an automatic decoy database search) ranged from to . % depending of the individual gel analysis. mascot analysis was performed with a fragment ion mass tolerance of . da and a parent ion tolerance of . da. carbamidomethyl of cysteine was specified in mascot as a fixed modification, and oxidation of methionine as a variable modification. the maximum missed cleavage allowed was set to . insect immune resistance to parasitoids insights into function and evolution of parasitoid wasp venoms venom proteins from parasitoid wasps and their biological functions diversity of 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action protein disulfide isomerase secretion, surface localization, turnover, and steady state expression of protein disulfide isomerase in rat hepatocytes parameters affecting in vitro oxidation/folding of maurotoxin, a four-disulphide-bridged scorpion toxin modulation of conotoxin structure and function is achieved through a multienzyme complex in the venom glands of cone snails serine carboxypeptidase scpep and cathepsin a play complementary roles in regulation of vasoconstriction via inactivation of endothelin- the peptidases of trypanosoma cruzi: digestive enzymes, virulence factors, and mediators of autophagy and programmed cell death serpin structure, function and dysfunction a serpin from the parasitoid wasp leptopilina boulardi targets the drosophila phenoloxidase cascade we are grateful to m. thaon for help in insect rearing, n. ris and t. malausa for fruitful discussions, m.c. bon for providing p. lounsburyi samples, and k. varikou and v. caleca for providing p. concolor samples. we also thank a. schmitz, j. villalba, and the microscopy platform of the sophia agrobiotech institute (isa) for technical advices. we are grateful to the anonymous reviewers for their careful reading of our manuscript. this work received support from the genoscope ( accession codes: p. concolor and p. lounsburyi raw illumina sequencing data are available in the genbank sequence read archive (sra) database under the accessions srr , srr , srr and srr , and raw sequencing data for p. lounsburyi under the accession srr . all trimmed ests for p. lounsburyi can be found in the genbank dbest repository under the accessions: jz -jz . the transcriptome shotgun assembly (tsa) projects were deposited in genbank under the accessions gcdx (p. concolor) and gceq (p. lounsburyi). versions in this paper are the first versions gcdx and gceq . key: cord- -j w vsw authors: stockman, lauren j; bellamy, richard; garner, paul title: sars: systematic review of treatment effects date: - - journal: plos med doi: . /journal.pmed. sha: doc_id: cord_uid: j w vsw background: the sars outbreak of – presented clinicians with a new, life-threatening disease for which they had no experience in treating and no research on the effectiveness of treatment options. the world health organization (who) expert panel on sars treatment requested a systematic review and comprehensive summary of treatments used for sars-infected patients in order to guide future treatment and identify priorities for research. methods and findings: in response to the who request we conducted a systematic review of the published literature on ribavirin, corticosteroids, lopinavir and ritonavir (lpv/r), type i interferon (ifn), intravenous immunoglobulin (ivig), and sars convalescent plasma from both in vitro studies and in sars patients. we also searched for clinical trial evidence of treatment for acute respiratory distress syndrome. sources of data were the literature databases medline, embase, biosis, and the cochrane central register of controlled trials (central) up to february . data from publications were extracted and evidence within studies was classified using predefined criteria. in total, sars treatment studies, in vitro studies, and three acute respiratory distress syndrome studies met our inclusion criteria. within in vitro studies, ribavirin, lopinavir, and type i ifn showed inhibition of sars-cov in tissue culture. in sars-infected patient reports on ribavirin, studies were classified as inconclusive, and four showed possible harm. seven studies of convalescent plasma or ivig, three of ifn type i, and two of lpv/r were inconclusive. in studies of steroid use, were inconclusive and four were classified as causing possible harm. conclusions: despite an extensive literature reporting on sars treatments, it was not possible to determine whether treatments benefited patients during the sars outbreak. some may have been harmful. clinical trials should be designed to validate a standard protocol for dosage and timing, and to accrue data in real time during future outbreaks to monitor specific adverse effects and help inform treatment. the sars outbreak of - presented clinicians with a new, life-threatening disease for which they had no experience in treating and no research on the effectiveness of treatment options. the world health organization (who) expert panel on sars treatment requested a systematic review and comprehensive summary of treatments used for sars-infected patients in order to guide future treatment and identify priorities for research. in response to the who request we conducted a systematic review of the published literature on ribavirin, corticosteroids, lopinavir and ritonavir (lpv/r), type i interferon (ifn), intravenous immunoglobulin (ivig), and sars convalescent plasma from both in vitro studies and in sars patients. we also searched for clinical trial evidence of treatment for acute respiratory distress syndrome. sources of data were the literature databases medline, embase, biosis, and the cochrane central register of controlled trials (central) up to february . data from publications were extracted and evidence within studies was classified using predefined criteria. in total, sars treatment studies, in vitro studies, and three acute respiratory distress syndrome studies met our inclusion criteria. within in vitro studies, ribavirin, lopinavir, and type i ifn showed inhibition of sars-cov in tissue culture. in sars-infected patient reports on ribavirin, studies were classified as inconclusive, and four showed possible harm. seven studies of convalescent plasma or ivig, three of ifn type i, and two of lpv/r were inconclusive. in studies of steroid use, were inconclusive and four were classified as causing possible harm. despite an extensive literature reporting on sars treatments, it was not possible to determine whether treatments benefited patients during the sars outbreak. some may have been harmful. clinical trials should be designed to validate a standard protocol for dosage and timing, and to accrue data in real time during future outbreaks to monitor specific adverse effects and help inform treatment. the severe acute respiratory syndrome (sars) is a febrile respiratory illness primarily transmitted by respiratory droplets or close personal contact. a global outbreak of sars between march and july caused over , probable or confirmed cases and deaths [ ] . the causative organism has been identified as a novel coronavirus (sars-cov) [ ] [ ] [ ] . the overall mortality during the outbreak was estimated at . % [ , ] . the overriding clinical feature of sars is the rapidity with which many patients develop symptoms of acute respiratory distress syndrome (ards). this complication occurred in approximately % of all patients with sars, and when it occurred was associated with a mortality rate of % [ , ] . at the time of the sars epidemic it was not known what treatments would reduce sars-related illness and deaths. because the urgency of the international outbreak did not allow time for efficacy studies, physicians in canada and hong kong treated the earliest patients with intravenous ribavirin, based on its broad-spectrum antiviral activity [ , ] . corticosteroids and immune-modulating agents were often prescribed empirically. soon after sars-cov was identified as the causative agent, antiviral screening programs were initiated; these programs reported several antiviral agents that inhibited sars-cov replication in vitro. these results led to the experimental use of protease inhibitors and interferon alpha (ifn-a) in the treatment of patients. the most commonly used treatments for sars are associated with adverse effects when used for other conditions (table s ). in october , the who established an international sars treatment study group, consisting of experts experienced in managing sars. the group recommended a systematic review of potential treatment options to identify the targets for proper evaluation in trials should the disease recur [ ] . this paper reports on this systematic review designed to summarise available evidence on the effects of ribavirin, lopinavir and ritonavir (lpv/r), corticosteroids, type i ifn, intravenous immunoglobulin (ivig), or convalescent plasma in relation to ( ) sars-cov replication inhibition in vitro; ( ) mortality or morbidity in sars patients; and ( ) effects on ards in adult patients. we prepared a protocol that defined our scope, inclusion criteria, and outcomes to be assessed. the interventions we included were defined by the who: ribavirin, lpv/r, corticosteroids, type i ifn, convalescent plasma, or ivig. the types of study we included were: ( ) in vitro studies, in which the authors examined inhibition of sars-cov viral replication, and data from an assay in human or animal cell line; ( ) in vivo studies, which included randomised controlled trial (rct), or prospective uncontrolled study design, or retrospective cohort design, or case-control design, or a case series, and patients treated for sars, and ten or more patients; and ( ) studies of ards that included rct, or systematic review, and treatment for ards or acute lung injury, and or more patients. in february , we systematically searched the literature databases medline, embase, biosis, and the cochrane central register of controlled trials (central) for articles that included the selected treatments (table s ) . the full text of each identified study was retrieved and each was independently reviewed by two authors (ls and rb). publications in chinese were selected after review of the english abstract. unpublished data were not sought, as the task of summarising existing published data was extensive and the international sars treatment group indicated that much of the clinical data had already been published. we used the quorom checklist to help ensure the quality of this review (table s ) . data from the full text of studies in english were extracted independently by two authors (ls and rb). data from the chinese literature were extracted with the assistance of a translator. because the chinese articles were reviewed by only one author, the consistency of the translated information with that from english articles was maintained by subsequent discussion with the translator to verify the extracted data. we established explicit criteria to assess the level of evidence for each human treatment study (box ). since the treatments chosen for evaluation were often given in combination, evidence was classified by the treatment that was given to all patients in the cohort or given to some with the author's intention of studying its effects. if putative effects within a study included several drugs, then we extracted data for each intervention. the level of evidence was independently classified by two authors (ls and rb). chinese studies were appraised and classified in the same way using translated information extracted from each report. discrepancies were resolved by consensus. in vitro evidence was available in studies. clinical evidence of sars treatment in humans was reported in studies ( in english, in chinese). three studies addressed treatment of ards ( figure ). in vitro. we found six studies that described the antiviral effect of ribavirin in vitro (table s ) ; four showed an antiviral effect (table s ) . a synergistic antiviral effect between ribavirin and type i ifn (ifn-b a or leukocytic ifn-a) was described in two studies performed in human cell lines and vero cell lines [ , ] . in sars patients. we found studies that described ribavirin treatment in cohorts larger than ten patients (table s ). our formal assessment classified studies as ''inconclusive,'' due to study design or because the effect of ribavirin could not be distinguished from the effects of other treatments (such as steroids and antiviral drugs). four publications presented evidence of possible harm ( ) ( ) ( ) ( ) . three of these studies, each of which included over patients, documented a fall in haemoglobin levels after ribavirin treatment when compared to levels in patients before treatment [ ] [ ] [ ] . of patients treated with ribavirin, / to / ( %- %) developed haemolytic anaemia, a recognised complication with this drug, although it is not possible to rule out the possibility that sars-cov infection caused the haemolytic anaemia, as there is no control group. one study noted that over % of sars patients had some degree of liver dysfunction indicated by alt levels higher than normal, and the number of patients with this complication increased to over % after ribavirin treatment (table s ) [ ] . in the chinese literature six additional reports described patients with sars treated with ribavirin (often with steroids). these six reports were determined to be inconclusive in the evaluation of treatment for sars (tables s and s ). in vitro. of three studies, two demonstrated that lopinavir inhibits cytopathic effects of sars-cov in fetal rhesus monkey kidney cells (table s ). one study showed detectable but reduced activity in vero-e cells [ ] , and one study concluded that neither lopinavir nor ritonavir had an effect [ ] . a synergistic effect of lopinavir with ribavirin has been reported (table s ) . in sars patients. we found two studies of lpv/r (lopinavir mg with ritonavir mg orally every h) in cohorts larger than ten patients (table s ) . patients also received ribavirin and corticosteroids. lpv/r use was compared among three groups of patients: those who received it as an early sars treatment, those who received it as a late treatment, and those who did not receive it at all. when lpv/r was added as an initial treatment to ribavirin and corticosteroid therapy, the death rate was lower than among those who received ribavirin and corticosteroids ( / [ ] . both studies were determined to be inconclusive due to possible bias in the selection of control group or treatment allocation. no additional studies were identified from the chinese literature. in vitro. no studies were found on the cytopathic effect of corticosteroids alone against sars-cov. corticosteroids act as immunomodulatory agents, and therefore studies to measure direct antiviral effects in vitro were not expected. in sars patients. fifteen articles examined corticosteroid treatment in ten or more patients. of these cohorts were also treated with ribavirin (table s ) . we determined that of the studies were inconclusive. of these, in an uncontrolled and nonrandomised study, / ( %) of patients treated with high-dose methylprednisolone ( . - mg/kg prednisolone on day of illness, followed by hydrocortisolone mg every h, and pulse-doses of methylprednisolone . g iv for d) after the first week of illness recovered from progressive lung disease (table s ) [ ] . two studies contained evidence of possible harm from corticosteroids [ , ] . one measured sars-cov plasma viral load across time after fever onset in a randomized, double-blind, placebo-controlled trial; corticosteroid use within the first week of illness was associated with delayed viral clearance. the other study, which was case-controlled, found that patients with psychosis received higher cumulative doses of steroids than patients without psychosis ( , mg versus , mg; p ¼ . ) [ ] . in the chinese literature, we found reports in which steroids were used (table s and table s ). twelve studies were inconclusive and two showed possible harm. one study reported diabetes onset associated with methylprednisolone treatment [ ] . another study (an uncontrolled, retrospective study of sars patients) reported avascular necrosis and osteoporosis among corticosteroid-treated sars patients [ ] . in ards patients. three clinical trials examined the effect of corticosteroids on mortality in patients with established ards (table s ). in two trials, high-dose methylprednisolone given for approximately d was not effective for early ards [ , ] . one small rct that used a regimen of lower dose methylprednisolone ( mg/kg per day), tapered after wk, showed possible evidence of ards improvement (table s ) [ ] . in vitro. twelve in vitro studies with data on the antiviral effect of ifn type i have been reported, and all demonstrated an antiviral effect against sars-cov (six for ifn-a and ten for ifn-b) (tables s and s ) . antiviral effects have been demonstrated in monkey (vero; vero-e ), fetal rhesus monkey kidney (frhk- ), and human (caco , cl , and hpek) cell lines. three reports presented evidence that ifn-b was superior against sars-cov compared to ifn-a and found rifn-a virtually ineffective against sars-cov compared to other ifns [ ] . synergistic effects were reported for leukocytic ifn-a with ribavirin [ ] , ifn-b with ribavirin [ , ] and ifn-b with ifn-c [ , ] . ''inconclusive'' if a study could not be used to inform a decision about treatment efficacy due to having either outcomes which were not reported consistently, an inconsistent treatment regimen, no control group or a control group which was a likely source of bias. a control group was considered a likely source of bias if there were differences in co-morbidities, sex, age and markers of severe disease compared to the treatment group. ''possible harm'' if a study reported adverse effects of treatment that were consistent with adverse effects reported with the use of the drug in the treatment of other conditions. evidence of direct causality was not required. a study could be classified as suggesting possible harm from the drug even if the study had methodological weaknesses. ''possible benefit'' if a study had evidence of benefit for an important outcome measure which was recorded consistently (e.g., case fatality, need for mechanical ventilation, duration of hospitalization, frequency of ards) in patients treated in a defined way compared to a valid control group. a control group was considered valid if randomized, or if patient characteristics and illness severity were comparable to the treatment group. evidence of direct causality was not required. ''definite harm'' if a study contained statistically significant evidence of harm demonstrated in a double-blind randomized trial, which did not contain serious methodological weaknesses. ''definite benefit'' if a study contained statistically significant evidence of harm demonstrated in a double-blind randomized trial, which did not contain serious methodological weaknesses. in sars patients. two studies of ifn-a given with steroids and/or ribavirin were reported (table s ). no significant difference was seen in outcome between ifn-a treatment group and those treated with other regimens. results of both studies were inconclusive due to a lack of a consistent treatment regimen or suitable control group (table s ). in the chinese literature, one additional study reported the use of ifn-a as part of a regimen that included ribavirin and steroids [ ] . we determined this study to be inconclusive because a variety of treatments given masked the effect of ifn-a alone (table s and table s ). in vitro. no studies were found on the cytopathic effect of this treatment on sars-cov. convalescent plasma and ivig act as immunomodulatory agents and therefore studies to measure direct antiviral effects in vitro were not expected. in sars patients. five studies of either ivig or convalescent plasma treatment given in addition to steroids and ribavirin were reported for treatment of sars (table s ) . these studies were inconclusive, because the effect of convalescent plasma or ivig could not be discerned from effects of patient comorbidities, stage of illness, or effect of other treatments (table s ). in the chinese literature, two additional studies reported evidence on the effect of convalescent plasma as a treatment for sars [ , ] . these studies were inconclusive (table s and table s ). evidence collected on the benefit or harm of drugs used to treat sars is summarized in table . the rapid spread and subsequent control of sars precluded controlled clinical treatment trials during the in this report we summarize the results of a systematic evaluation of the findings from published reports of treatments used for sars during the epidemic. publications from the chinese literature were included to capture as much evidence as possible. we developed specific criteria (box ) to look for large, obvious effects of benefit, adverse or poor outcomes, or evidence of potential benefit that could be used to prioritise future research of sars treatments. a summary of this evidence in sars patients is shown in table . despite thirty reports of sars-infected patients treated with ribavirin, there is no convincing evidence that it led to recovery. haemolytic anaemia, a recognized side effect of this treatment, was observed in three studies. we would infer from these findings that any future use of ribavirin for sars should be within the context of a controlled trial with close attention given to adverse effects. corticosteroids were commonly prescribed to sars patients with worsening pulmonary disease or progressing abnormalities on chest x-rays. treatment regimens varied widely but can be classified into two groups, early treatment and rescue treatment given at a later stage of illness. it is difficult to make a clear recommendation about whether corticosteroids should be used to treat sars-associated lung injury in any stage of illness, particularly as the drug is immunosuppressive and may delay viral clearance if given before viral replication is controlled [ ] . of added concern are infectious complications, avascular necrosis, and steroidinduced psychosis-recognized adverse effects of corticosteroid use. fungal superinfection and aspergillosis have been noted in case reports and autopsy findings of sars patients given corticosteroids at high doses or for prolonged periods [ , ] . this review has found evidence of avascular necrosis and steroid-induced psychosis in sars patients. seven studies of treatment with convalescent plasma or ivig, three with ifn type i, and two with lpv/r were inconclusive by the criteria used in our analyses. authors of four of the ivig studies commented that patients seemed to improve upon treatment, but that more controlled trials of this approach are needed to provide evidence of an effect for sars. important caveats should be considered in this review. most of the studies of sars patients were descriptions of the natural course of the disease and had not been designed to reliably assess the effects of the treatments used. patient characteristics such as age and presence of diabetes mellitus have been associated with severe disease and can confound treatment effects. a diagnostic test for early sars illness was not validated or widely available, and in general, treatment was initiated once patients fulfilled a clinical and epidemiological case definition. it is possible that the inclusion of patients without laboratory confirmation of sars-cov infection in this review could cause an underestimate of any true effect of antiviral treatment on sars. the variation in treatment regimens-particularly the wide range in doses, duration of therapy, and route of administration of ribavirin and corticosteroids-is a major obstacle to a clear interpretation of the data in this review. the nonstandardised collection of clinical information limits the conclusions that can be drawn from a retrospective analysis. we suggest that, in the event of a future outbreak of sars-cov or another novel agent, attempts be made to develop treatment protocols and to collect and contribute information for a standardized minimum dataset that could facilitate analysis of treatment outcomes among different settings. as observational studies pose problems of interpretation, the need is great for good-quality randomised trials, despite the difficulties in organising such trials. the group recommended a systematic review of potential treatments for sars. in particular, it was considered important to summarise the available evidence on the use of certain antiviral drugs (ribavirin, lopinavir, and ritonavir), steroids, and proteins called immunoglobulins, which are found naturally in human blood. the who group wanted to know how these treatments affected the virus outside the body (''in vitro'') and whether it helped the condition of patients and reduced the death rate, particularly in those patients who developed the dangerous complication called acute respiratory distress syndrome (ards). this study is a systematic review conducted in response to the who request. what did the researchers do and find? they did no new work with patients or in the laboratory. instead they conducted a comprehensive search of the scientific and medical literature for published studies that fitted their carefully predefined selection criteria. they found sars treatment studies, in vitro studies, and three ards studies that met these criteria. some of the in vitro studies with the antiviral drugs found that a particular drug reduced the reproduction rate of the viruses, but most of the studies of these drugs in patients were inconclusive. of studies on steroid use, were inconclusive and four found that the treatment caused possible harm. what do these findings mean? from the published studies, it is not possible to say whether any of the treatments used against sars were effective. no cases of sars have been reported since but it is always possible that the same or a similar virus might cause outbreaks in the future. it is disappointing that none of the research on sars is likely to be useful in helping to decide on the best treatments to use in such an outbreak. the authors discuss the weaknesses of the studies they found and urge that more effective methods of research be applied, in a timely fashion, in any similar outbreaks in the future. while the systematic review suggests that we do not know which if any of the potential treatments against sars are effective, its recommendations mean that researchers should at least be better prepared to learn from potential future outbreaks. additional information. please access these web sites via the online version of this summary at http://dx.doi.org/ . /journal.pmed. . wikipedia entry on sars (wikipedia is a free online encyclopedia that anyone can edit) medlineplus pages on sars wikipedia entry on systematic reviews, which includes links to other web sites where more detailed information may be found summary of probable sars cases with onset of illness from a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome the severe acute respiratory syndrome the epidemiology of severe acute respiratory syndrome in the hong kong epidemic: an analysis of all patients critically ill patients with severe acute respiratory syndrome acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada a major outbreak of severe acute respiratory syndrome in hong kong future clinical trials for sars, informal meeting and workshop ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines in vitro susceptibility of clinical isolates of sars coronavirus to selected antiviral compounds common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada clinical features and short-term outcomes of patients with sars in the greater toronto area severe acute respiratory syndrome: report of treatment and outcome after a major outbreak temporal patterns of hepatic dysfunction and disease severity in patients with sars ( ) hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients factors associated with psychosis among patients with severe acute respiratory syndrome: a case-control study glucocorticoidinduced diabetes in severe acute respiratory syndrome: the impact of high dosage and duration of methylprednisolone therapy highdose corticosteroids in patients with the adult respiratory distress syndrome early steroid therapy for respiratory failure effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome: a randomized controlled trial increased sensitivity of sars-coronavirus to a combination of human type i and type ii interferons interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) pulmonary pathology of severe acute respiratory syndrome in toronto fatal aspergillosis in a patient with sars who was treated with corticosteroids we acknowledge dr. larry j. anderson for his review of manuscript drafts and consultation on in vitro data for sars treatment, dr. haoqiang zheng for extracting and translating data from the articles published in chinese, ms. vittoria lutje for helping with the literature search strategy, and the who international sars treatment study group for prioritising this research project and identifying treatments for review.the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention.author contributions. ls, rb, and pg drafted the protocol for this review. ls and rb reviewed all abstracts and extracted data. all authors appraised included studies, interpreted results, commented critically on the manuscript, and contributed text to the final version. key: cord- - g xsys authors: nimgampalle, mallikarjuna; devanathan, vasudharani; saxena, ambrish title: screening of chloroquine, hydroxychloroquine and its derivatives for their binding affinity to multiple sars-cov- protein drug targets date: - - journal: j biomol struct dyn doi: . / . . sha: doc_id: cord_uid: g xsys recently chloroquine and its derivative hydroxychloroquine have garnered enormous interest amongst the clinicians and health authorities’ world over as a potential treatment to contain covid- pandemic. the present research aims at investigating the therapeutic potential of chloroquine and its potent derivative hydroxychloroquine against sars-cov- viral proteins. at the same time screening was performed for some chemically synthesized derivatives of chloroquine and compared their binding efficacy with chemically synthesized chloroquine derivatives through in silico approaches. for the purpose of the study, some essential viral proteins and enzymes were selected that are implicated in sars-cov- replication and multiplication as putative drug targets. chloroquine, hydroxychloroquine, and some of their chemically synthesized derivatives, taken from earlier published studies were selected as drug molecules. we have conducted molecular docking and related studies between chloroquine and its derivatives and sars-cov- viral proteins, and the findings show that both chloroquine and hydroxychloroquine can bind to specific structural and non-structural proteins implicated in the pathogenesis of sars-cov- infection with different efficiencies. our current study also shows that some of the chemically synthesized chloroquine derivatives can also potentially inhibit various sars-cov- viral proteins by binding to them and concomitantly effectively disrupting the active site of these proteins. these findings bring into light another possible mechanism of action of chloroquine and hydroxychloroquine and also pave the way for further drug repurposing and remodeling. communicated by ramaswamy h. sarma coronavirus disease pandemic is caused by severe acute respiratory syndrome coronavirus- (sars-cov- ) (gorbalenya et al., ) , is a rapidly spreading disease globally and has so far claimed thousands of lives around the world and caused enormous damage to society and economy (cebm, ) . world health organization (who) has declared that the covid- is a pandemic, and a public health emergency of international concern (wee et al., ) . the novel coronavirus or sars-cov- has four transmission stages in line with other infectious diseases and is generally categorized into asymptomatic, moderate, extreme, and critical. sars-cov- exhibits different symptoms depending on the severity of the disease including fever, dry cough, dyspnea, pneumonia, hypoxemia, encephalopathy, heart failure, and acute kidney injury. there is currently no defined antiviral drug or therapy available for covid- treatment, and mostly the disease is managed symptomatically (yuki et al., ) . several medications are being tested in clinical trials for covid- , including antiviral, antiinflammatory, anti-malarial and other pharmacologically active drugs (rabby, ) . however, recently chloroquine and its derivative hydroxychloroquine are being positioned as a possible treatment for covid- . presently, multi-centric global clinical trials are underway to evaluate the therapeutic potential of chloroquine and hydroxychloroquine as a treatment for novel coronavirus infection. the food and drug administration (fda), usa, has, however, approved both chloroquine and hydroxychloroquine for covid- control and treatment for emergency purposes (scholz & derwand, ) . in order to address the virus infection and replication it is critical to understand proteins involved in the process. functionally, sars-cov- consists of two different types of proteins, which include structural proteins and non-structural proteins (nsps). the structural proteins are involved in the formation of the spherical shape of the virus, which including spike protein (trimeric), membrane protein, envelope protein, and the nucleocapsid protein. while sixteen non-structural proteins (nsps) are formed from the proteolytic cleavage of two polyproteins (pp a and pp b). these nsps are essential for the metabolic and molecular events include transcription and translation (prajapat et al., ) . in this context, key regulatory proteins and enzymes associated with the pathogenesis of sars-cov- were selected as drug targets for chloroquine and its derivatives. chloroquine and hydroxychloroquine are anti-malarial drugs, which are also used for the treatment of rheumatoid arthritis and lupus erythematosus (touret & de lamballerie, ) . these drugs are believed to be relatively safe when administered within the clinically advised limits with mild side effects. furthermore, chloroquine derivatives have been tested on pneumocystis pneumonia (pcp) for their therapeutic activity to repurposing antimalarial drugs for pneumonia (gomes et al., ; yeo et al., ) . pneumonia is a lifethreatening symptom for advanced stage coronavirus infected patients and clinicians are using chloroquine and its derivative hydroxychloroquine to treat the disease. currently, these two drugs are being reused for the treatment of covid- since the infection involves pneumonia (devaux et al., ) . recently, treatment with the combination of both hydroxychloroquine and azithromycin has shown a significant improvement within the covid- patients (gautret et al., ) . similarly, patients treated with chloroquine and hydroxychloroquine have also shown significant recovery from covid- (singh et al., ) . chloroquine and hydroxychloroquine is a cost effective drug that has long been a therapy of choice for malaria prophylaxis due to excellent results and good safety and tolerability. recently, world over chloroquine and its derivative analog hydroxychloroquine has garnered enormous attention as a possible treatment for sars-cov- infection. although in this context, the exact mechanism of action of chloroquine and hydroxychloroquine is still not known. at the same time some reports have cautioned against the use of chloroquine due to the known dose-related toxicity of chloroquine and its derivative. several adverse events mainly involving retinal and psychiatric symptoms are observed with chloroquine. however, such symptoms are dose-dependent and are observed when dosage levels exceed prescribed pharmacological dosage limits. an understanding of sars-cov- disease biology indicates that it is important to target the viral replication in order to effectively control the infection. also, it is perceived that chloroquine and its derivative can prevent the disease onset in covid negative and healthy subjects and treat sars-cov- infection in healthy but asymptomatic carriers. this can be effectively accomplished by understanding the detailed mechanism of action of chloroquine and hydroxychloroquine in preventing coronavirus infection. the mechanism of action of these two drugs is not well known, but it has been demonstrated in vitro that these drugs inhibit sars-cov- by elevating the endosomal ph, and alter ace- terminal glycosylation there by leading to the interruption of virus receptor binding (vincent et al., ) . however, if chloroquine and its derivative hydroxychloroquine acts exclusively by elevating the endosomal ph, then chloroquine should act as a broad-spectrum anti-viral agent since modulation of endosomal ph is a common strategy utilized by viruses for internalization. this appears to be doubtful since chloroquine is not effective against most of the viral diseases like dengue (tricou et al., ) , chickenguniya (lamballerie et al., ) , and hiv (savarino & shytaj, ) . however, both chloroquine and its derivative hydroxychloroquine were shown to be of some use in countering the sars virus (vincent et al., ) . it is a well-established fact that sars virus (sars-cov) and sars-cov- share almost % sequence similarity. with this, it can be postulated that chloroquine might be actively binding to one or more sars-cov- proteins to inhibit viral replication. to study this, multiple chloroquine derivatives reported earlier, were screened for their binding potential to various sars-c v- virus proteins important for its binding, internalization, replication and budding in the host cell using the molecular docking studies. in this work, we have demonstrated the capability of chloroquine and its derivatives, reported for their anti-pcp potential earlier (gomes et al., ) for selective binding to different viral proteins. this work aims at increasing the information for anti-viral mechanism of chloroquine and hydroxychloroquine against the sars-cov- virus. this research can support new anti-viral drug discovery against sars-cov- virus and at the same time can support drug repurposing efforts around chloroquine. also these results can be used as a basis for modifying clinical dosage of chloroquine and thereby rendering it more effective against coronavirus infection. post the approval of hydroxychloroquine by the food and drug administration (fda), usa, the same has been used as a potential drug for the treatment and management of emerging disease covid- . by using in-silico molecular docking studies, the binding potential of chloroquine and its derivatives with different sars-cov- proteins involved in viral replication was evaluated. the d-structures for chloroquine and its derivatives viz. hydroxychloroquine, chloroquine sulfate, chloroquine mustard, chloroquine pyrolidinyl, were taken from pubchem (https://pubchem.ncbi.nlm.nih.gov/) database. while, the d-structures of chemically synthesized chloroquine derivatives from gomes et al., were drawn using chemsketch software and named as cqn a, cqn b, cqn c, cqn d, cqn e, cqn f, cqn g, cqn h, cqn i, cqn j, cqn a, cqn b, cqn a, and cqn b (table ). all the structures of chloroquine derivatives were organized as a compound library following energy minimization using the open babel module in pyrx software. all the compounds in this study were also assessed for their drug likeliness based on the lipinski's 'rule of five' using swissadme server. the key regulatory proteins and enzymes associated with the pathogenesis of severe acute respiratory syndrome coronavirus (sars-cov- ) were selected as potential targets for chloroquine and its analogues . specifically, the proteins table ) . three-dimensional structures of the above drug targets were retrieved from the rcsb protein data bank (www.rcsb.org). before docking, the pdb structures were observed for sequence break and their ligand association by using pymol molecular visualization system (delano, ) . proteins ( m and svb) with sequence break were subjected to homology modeling using the swissmodel server (daina et al., ) , subsequently used for further studies. liganded form of protein ( lu ) were identified, retrieved, and employed for validation of docking (nimgampalle et al., ) . while non-liganded forms of proteins ( m , vsb, vw , vxs, and w b) were subjected to active site prediction using the castp server (tian et al., ) . in this study, molecular docking was performed at known active site of ligand form of proteins ( lu ), whereas, both blind docking and predicted active site docking (site-specific docking) was performed to the remaining non-liganded form of proteins ( m , vsb, vw .e, vxs, w , and w b) to examine the binding affinities of chloroquine derivatives against each drug target of sars-cov- . docking was performed using pyrx . software consist of autodock . (morris et al., ) . the structures of chloroquine and its derivatives were organized as a compound library after subjecting them to energy minimization using the open babel module in the virtual screening software, pyrx. later, the protein structures were refined for heteroatoms and water molecules to demarcate active sites of proteins. further, the gasteiger charges and hydrogen atoms were added to each drug target to maintain coordination between various interactions by using ucsf chimera- . . software (huang et al., ) . then, each drug target was saved in pdb format, and the chloroquine derivative compounds under examination were also uploaded and saved in ligand.pdbqt format. the grid was set around the active site of the drug target for site-specific docking, whereas the grid was maximized to surround the entire protein surface for blind docking. to generate possible binding conformation of chloroquine derivatives, empirical free energy force fields were applied to the lamarckian genetic algorithm (lga) with default parameters (lga: , -energy evaluations) (bommu et al., ) . the grid spacing was generated around the active site residues of each drug target, and the grid parameters for each drug target are given in table . as supplementary file. after completion of docking, the dock results were saved for the observation of binding affinities and binding interactions between ligand-target were analyzed by ligplot software (wallace et al., ) . prior to molecular docking studies, all the synthetic derivatives of chloroquine were tested for their "drug-likeliness" properties based on lipinski's rule of five. as shown in table , all the compounds used in the present study follow lipinski's rule of five (molecular weight < , hydrogen acceptor < , hydrogen donor < , and logp < ) without any exception. these results demonstrate that all the chloroquine derivatives studied in this study are capable of eliciting pharmacological response through their absorption, distribution, metabolism, and excretion. the details of druglikeness and relevant molecular properties of compounds are listed in table . it is important for a chemical compound to demonstrate drug-likeness properties since this would render in order to completely understand the binding potential of chloroquine and its derivatives to different sars-cov- proteins, it is important to identify their natural active sites. active sites of proteins can be predicted by ascertaining the geometric and topological properties of protein structures. castp server was used to predicted active sites for the nonliganded form of proteins ( w , m , vsb, vw , vxs, and w b). the amino acids that form the active site of each viral protein and their exact position on the polypeptide chain are listed in table . it was presumed that chloroquine and its derivatives can bind to the active site of these viral proteins thereby inhibiting their natural function. the active site of each protein identified by this procedure was used for further molecular docking studies. in this study, nineteen compounds including chloroquine derivatives (chloroquine, hydroxychloroquine, chloroquine sulfate, chloroquine mustard, chloroquine pyrolidinyl) and other chemically synthesized chloroquine derivatives (cqn a, cqn b, cqn c, cqn d, cqn e, cqn f, cqn g, cqn h, cqn i, cqn j, cqn a, cqn b, cqn a, and cqn b) were studied to evaluate their binding potential against the various viral proteins and enzymes ( w , lu , m , vsb, vw .e, vxs, and w b) through molecular docking approaches. the results clearly demonstrate that the ability of hydroxychloroquine and chloroquine to bind with different viral proteins albeit with different efficiencies. on the contrary some of the chemically synthesized chloroquine derivatives exhibit the higher binding affinities with tested viral proteins than the hydroxychloroquine (table ). our results point to the fact that potential inhibitors against viral target proteins can be derived by further modifying chloroquine structures. in the results, blind docked ligandprotein complexes are illustrated as figures and the amino acid residues involved in the site-specific docking are listed in table as predicted active sites of the non-liganded form of viral proteins. our results demonstrate that hydroxychloroquine can bind to nsp with a binding affinity of À . kcal/mol and À . kcal/mol in both blind and site-specific docking, respectively (table ). in parallel, even stronger binding to nsp when compared to hydroxychloroquine was demonstrated by chemically synthesized chloroquine derivative, cqn h showing the maximum binding affinity of À . kcal/ mol and À . kcal/mol in both blind and site-specific docking (tables and . supplementary). hydroxychloroquine shows hydrogen bonding with the residues gly and leu , whereas cqn h forms hydrogen bonds with amino-acid residues asn , gly , and gly ( figure ). overall, chloroquine and its derivatives demonstrated a higher binding affinity for nsp viral protein when compared to other proteins included in this study, thereby signifying their potential as inhibitors of nsp function. the main protease is moderately inhibited by hydroxychloroquine with the binding affinity (À . kcal/mol) (tables and . supplementary). amino acid residues of the main protease involved in the formation of five hydrogen bonds with hydroxychloroquine are leu , thr , thr , thr , and thr ( figure ). however, compared to hydroxychloroquine, both cqn h and cqn b show considerable higher binding affinity (À . kcal/mol) to the main protease (tables and . supplementary). the compound cqn h shows hydrogen interactions with the residues of thr , ser ( figure ). our results indicate that some derivatives of chloroquine and its derivatives are capable of binding to the main viral protease. the results obtained from both blind docking and site-specific docking demonstrate that hydroxychloroquine can effectively bind to rna polymerase with binding affinities À . and À . kcal/mol, respectively (table ). the amino acid residues (asn and asn ) of rna polymerase are involved in formation of hydrogen bond with hydroxychloroquine ( figure ) . interestingly, the compound cqn h, demonstrate strong binding with rna polymerase with the highest binding affinity in both blind docking (À . kcal/mol.) and site-specific docking (À . kcal/mol) (tables and . supplementary). cqn h shows the ability to form hydrogen interactions with the rna polymerase residues thr and thr (figure ). the high binding affinity of cqn h indicates towards its potential as an inhibitor of viral rna polymerase. hydroxychloroquine exhibits considerable binding efficiency against spike glycoprotein during both blind docking and site-specific docking with binding affinities of À . and À . kcal/mole, respectively (table ) . additionally, the compounds cqn b, cqn h demonstrated even stronger binding to spike glycoprotein with the binding affinities of À . kcal/ mol. and À . kcal/mol in both blind docking and predicted active site binding, respectively (table ) . interestingly, only single amino acid (ile ) of the viral spike protein is involved in the formation of hydrogen bond with hydroxychloroquine, whereas cqn b shows no hydrogen bonds formation rather it forms van dar waal interactions with various amino acid residues (figure ) . the obtained results demonstrate that the compounds cqn b and cqn h may act as potential inhibitor of viral spike glycoprotein function. our studies demonstrate that hydroxychloroquine can also bind to the receptor-binding domain with a moderate binding affinity (À . and À . kcal/mol) in both blind docking and site-specific docking ( table ). the amino acid residues of the receptor-binding domain involved in the hydrogen bonding are asp , met , phe , and glu ( figures and . supplementary) . on the contrary other chloroquine derivatives like cqn a and cqn b show a strong binding affinity (À . kcal/mol) to the receptor binding domain of the viral spike protein in both blind docking and site-specific docking studies (table ). the high binding affinities exhibited by these compounds represent their potential as inhibitors of receptor-binding domain. cqn a shows hydrogen interaction with the residues ile and asn ( figure ). molecular docking studies with chloroquine and its derivatives with adp-ribose- monophosphatase demonstrated the ability of hydroxychloroquine to bind with the mentioned viral protein with binding affinity À . kcal/mol in both blind docking and site-specific docking studies (table ) . a detailed analysis of the dock showed that hydroxychloroquine was able to form hydrogen bonds with leu , ala , and ala residues ( figure ). two compounds cqn e and cqn i, also show effective binding with adp-ribose- monophosphatase with the binding affinity of À . kcal/mol in blind docking (table ). the amino acid residues involved in the formation of hydrogen bonding with cqn i are val and ala ( figure ) . the compound cqn c also demonstrated a strong binding to adp-ribose- monophosphatase with the binding affinity À . kcal/mol in site-specific docking studies. hydroxychloroquine shows moderate to minimal binding with replicase protein with binding affinities of À . and À . kcal/mol in blind docking and site-specific docking, respectively ( table ). the residues of replicase protein, val , and pro are involved in the formation of hydrogen bonds with hydroxychloroquine ( figure ) . however, another derivative of chloroquine, compound cqn h potentially inhibits replicase protein in both blind docking and site-specific docking with the binding affinities are À . and À . kcal/mol, respectively (table ) and forms hydrogen bonds with arg , pro , and thr residues of the replicase protein (figure ). in the present work, with the use of molecular docking techniques the molecular interactions between chloroquine derivatives and selective sars-cov- viral proteins were studied. since most of these viral proteins can be potential drug targets, this study aims at understanding the potential role of chloroquine and its potent derivative hydroxychloroquine in inhibition of sars-cov- viral infection and replication by assessing the binding efficiency of chloroquine and its derivatives to various viral proteins. these results obtained in this study can also be extrapolated to evaluate the therapeutic efficiency of chloroquine and hydroxychloroquine in controlling sars-cov- infection. also in this study we have attempted to screen for derivatives of chloroquine that can bind to sars-cov- viral drug targets more efficiently than chloroquine with the hope that this can open a case for chloroquine remodeling for better inhibition of sars-cov- multiplication. based on the recent reports, some of the essential regulatory proteins and enzymes associated with the pathogenesis of sars-cov- were selected as drug targets such as the spike glycoprotein that enables virus internalization, rna dependent rna polymerase that supports replication of viral genetic material, chimeric rbd (receptor binding domain) that interacts with the ace , main protease responsible for cleaving the viral polypeptide, non-structural protein , nonstructural protein , non-structural protein (replicase table . drug-likeness properties of chloroquine and its derivatives predicted in swissadme web tool. protein) and adp-ribose- monophosphatase. in order to have a detailed understanding of interaction of chloroquine and allied compounds with sars-cov- viral proteins the potential active site of these proteins and the amino acid residues involved in formation of these active sites were identified. with the molecular docking studies, the hydrogen bonds and van der waals interactions formed as a result of binding of the drugs to the sars-cov- viral proteins were identified (table . supplementary). the normal function of sars-cov- proteins can be inhibited as a result of the binding of these drugs to the active site of these sars-cov- proteins. our results clearly demonstrate that chloroquine and its derivatives can bind to sars-cov- proteins and thereby can disrupt the normal functioning of these proteins. also it was found that some chemically synthesized derivatives of chloroquine are more efficient in binding to viral proteins when compared with chloroquine or hydroxychloroquine. however, some of these derivatives can elicit toxicity and therefore better modeling of chloroquine can lead to the identification of an effective inhibitor of sars-cov- virus (supplementary table . ). as we write this report, extensive research is ongoing around the globe to discover specific and effective drug(s) or drug combination that can halt the spread of covid- pandemic. with the help of computational methods for drug discovery and repurposing, several natural compounds, anti-viral compounds, anti-malarial drugs, antibiotics, and pharmacologically active compounds have been screened and are being investigated for their ability to inhibit sars-cov- protein function (elfiky, ; fantini et al., ; wu et al., ) . however, to the best of our knowledge not much research has been performed to probe the effect of chloroquine, hydroxychloroquine and their derivatives on various drug targets of sars-cov- virus. post internalization of the virus in the host cell, both nonstructural protein & are involved in the formation of multidomain m protease, which plays a key role in the replication process (stobart et al., ) . in this context, the binding potential of chloroquine and its derivatives was assessed for binding to non-structural protein as this interaction can disrupt sars-cov- multiplication within the host cell. our results indicate that chloroquine and its derivatives can effectively bind to the nsp . moreover, it was found that some of the chloroquine derivatives such as cqn b (À . kcal/mol), cqn a (À . kcal/ mol) and cqn h (À . kcal/mol) can bind even strongly to nsp than the binding affinity of hydroxychloroquine (À . kcal/ mol) to nsp . the main protease is also a critical enzyme in the life cycle of sars-cov- virus involved in the cleavage of polyprotein (pp) at the c-terminal end and leads to the formation of non-structural proteins (lindner et al., ) . targeting the main protease is one of the ways to prevent the replication of sars-cov- . the obtained results clearly demonstrate that the chloroquine and its derivatives cqn h and cqn b can bind to the main protease and thereby can potentially inhibit its activity. non-structural protein acts as rna dependent rna polymerase (rdrp). it is a vital enzyme in the replication of rna viruses. therefore, it has been studied in various viruses table . predicted active sites for the non-liganded form of proteins using the castp server. active site predicted from castp w ala , asp , ile , ala , ala , asn , lys , his , gly , gly , gly , val , ala , ala , gly , ly , pro , leu , leu , ser , ala , gly , ile , phe , ala , val , phe , asp , leu m val , glu , his , phe , asp , tyr , tyr , ile , asn , asn , leu , asp , ser , lys , arg , thr , met , asn , leu , lys , tyr , ala , ile , ser , ala , lys , arg , ala , arg , arg , thr , thr , val , ala , gly , his , leu , lys , ala , val , ile , gly , thr , thr , ser , lys , phe , tyr , trp , gly , trp , asp , tyr , pro , lys , cys , asp , arg , glu , val , lys , ser , ser , gly , ala , thr , thr , ala , asn , leu , ser , asp , asp , phe , ser , trp , trp , glu , phe , cys , ser , gln , pro , arg , ile , ala , val , asp , ile , val , thr , leu , arg , val , leu , ile , tyr , ser , asn , arg , tyr . vsb ala , tyr , thr , asn , phe , thr , tyr , pro , asp , lys , val , phe , arg , ser , ser , val , leu , his , ser , thr , gln , asp , leu , leu , pro , phe , phe , ser , asn , val , thr , trp , phe , pro , val , leu , pro , phe , asn , asp , gly , lys , asn , ile , asp , gly , tyr , lys , ile , tyr , leu , gln , gly , phe , pro , leu , val , asp , ile , asn , ile , thr , arg , arg , ser , tyr , leu , thr , pro , asp , val , val , gly , tyr , tyr , leu , gln , pro , arg , thr , asp including the hepatitis c virus and the zika virus (elfiky, ; ganesan & barakat, ) . recently, the fda approved anti-rdrp drugs (ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir) have shown potential inhibitory activity against the rdrp of sars-cov- (elfiky, ) . in our study, chloroquine derivative cqn h demonstrated strong binding affinity to rdrp (À . kcal/mol) which is even stronger that that of hydroxychloroquine with rdrp protein. these results can be valuable in repurposing and redesigning cqn h as a potential inhibitor of rdrp function. spike glycoprotein plays an essential role in the attachment of coronavirus with the ace on the host cell surface. hence, this protein is considered as an important drug target for drug discovery (prajapat et al., ) . our results demonstrate that hydroxychloroquine can effectively bind to both spike glycoprotein and chimeric receptor binding domain along with compounds cqn b, cqn h that can effectively inhibit spike glycoprotein. besides, the compounds cqn a, cqn b can also potentially inhibit receptor binding domain. these results point to the fact that the chloroquine chloroquine sulfate_chebi_ À . (À . ) À . À . (À . ) À . (À . ) À . (À . ) À . (À . ) À . (À . ) . chloroquine pyrolidylin_zinc À . (À . ) À . À (À . ) À . (À . ) À . ( . ) À . (À . ) À . (À . ) . chloroquine mustard_zinc À . (À . ) À . À . (À . ) À . (À . ) À . (À . ) À . (À . ) À . (À . ) chemically synthesized chloroquine derivatives . cqn a ( derivatives can bind to spike glycoprotein, which can potentially lead to disruption of spike protein interaction with the ace- on host cell surface. however, in another recent study is was demonstrated that both chloroquine and hydroxychloroquine can prevent the interaction of spike protein with sialic acids and gangliosides present on the host cell surface (fantini et al., matrosovich et al., . non-structural protein (nsp ) acts as a replicase protein (rna-binding protein) and its dimerization is essential for viral propagation (prajapat et al., ) . therefore, targeting the dimerization of nsp can be an effective strategy to control virus multiplication (egloff et al., , hu et al., . our results show that chloroquine derivative cqn h can bind to the replicase protein with higher efficiency than the hydroxychloroquine. further, the derivatives of chloroquine such as cqn e, cqn i, and cqn c can also effectively inhibit adp-ribose- monophosphatase than the hydroxychloroquine. from these results, it is apparent that these chloroquine derivatives possess therapeutic activity against sars-cov- virus. our studies clearly demonstrate binding of chloroquine, hydroxychloroquine and their derivatives to various sars-cov- proteins. however, these results need to be substantiated with binding experiments to ascertain other drug-protein interaction parameters. at the same time, the specificity of these studies can be ascertained from the fact that one of the derivatives of chloroquine, cqn f did not show binding to any viral protein included in the study. although chloroquine and hydroxychloroquine are losing their popularity due to possible mutagenic activity our literature survey points to the fact that chloroquine and hydroxychloroquine have well established toxicokinetic properties and might only elicit dosage dependent toxicity. also chloroquine and hydroxychloroquine represent an economical option to prevent the spread of covid- pandemic. we believe that from our study informed approach can be taken to understand the drug-protein interaction and then further modify the drug structure which will have a minimal toxic burden and maximal inhibitory potential against sars-cov- viral proteins. our results also enhance the basic knowledge of the interaction of chloroquine derivatives with the various drug targets of sars-cov- and point to a possible novel mechanism of action of the century old drug. from our results, it can be infered that hydroxychloroquine considerably inhibits all the drug targets of sars-cov- . further, it was found that hydroxychloroquine might not only enhance the endosomal ph, but it could also interact with various proteins of sars-cov- . chemically synthesized chloroquine derivatives reveal more effective inhibitory activity against all drug targets of sars-cov- than hydroxychloroquine. among the various derivatives of chloroquine, cqn h and cqn b show potential inhibition against all the drug targets. our results could also be used for further constructive research on chemically synthesized chloroquine derivatives to identify successful drugs for the treatment of covid- . there is no conflict of interest. the current project being an in-silico work did not require any additional financial support. this is purely a working arising from "work from situation". otherwise, the authors are thankful for the financial support from their respective institutes. http://orcid.org/ - - - x ambrish saxena http://orcid.org/ - - - structural probing, screening and structure-based drug repositioning insights into the identification of potential cox- inhibitors from selective coxibs global covid- case fatality rates swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules pymol: an open-source molecular graphics tool new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? the severe acute respiratory syndrome-coronavirus replicative protein nsp is a single-stranded rna-binding subunit unique in the rna virus world ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study zika viral polymerase inhibition using anti-hcv drugs both in market and under clinical trials structural and molecular modeling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov- infection applications of computer-aided approaches in the development of hepatitis c antiviral agents structure of the rnadependent rna polymerase from covid- virus hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial chloroquine analogues as leads against pneumocystis lung pathogens the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- structural basis for dimerization and rna binding of avian infectious bronchitis virus nsp enhancing ucsf chimera through web services structure of mpro from covid- virus and discovery of its inhibitors crystal structure of adp ribose phosphatase of nsp from sars cov- . biorxiv on chikungunya acute infection and chloroquine treatment. vector-borne and zoonotic diseases the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme crystal structure of the sars-cov- non-structural protein sialic acid receptors of viruses crystal structure of adp ribose phosphatase of nsp from sars cov- in the complex with adp ribose. biorxiv autodock and autodocktools : automated docking with selective receptor flexiblity novel inhibitors of rho-kinase mediated neuroinflammatory pathways and their potential application in recovery of injured spinal cord drug targets for corona virus: a systematic review current drugs with potential for treatment of covid- : a literature review chloroquine and beyond: exploring anti-rheumatic drugs to reduce immune hyperactivation in hiv/aids does zinc supplementation enhance the clinical efficacy of chloroquine/hydroxychloroquine to win todays battle against covid- ? medical hypotheses structural basis of receptor recognition by sars-cov- chloroquine and hydroxychloroquine in the treatment of covid- with or without diabetes: a systematic search and a narrative review with a special reference to india and other developing countries chimeric exchange of coronavirus nsp proteases ( clpro) identifies common and divergent regulatory determinants of protease activity castp . : computed atlas of surface topography of proteins of chloroquine and covid- a randomized controlled trial of chloroquine for the treatment of dengue in vietnamese adults chloroquine is a potent inhibitor of sars coronavirus infection and spread ligplot: a program to generate schematic diagrams of protein-ligand interactions who declares global emergency as wuhan coronavirus spreads cryo-em structure of the -ncov spike in the prefusion conformation analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods hydroxychloroquine may reduce risk of pneumocystis pneumonia in lupus patients: a nationwide, population-based case-control study covid- pathophysiology: a review the authors would like to thank iiser tirupati and iit tirupati for their support. key: cord- -hnf vayd authors: ford, richard b.; mazzaferro, elisa m. title: emergency care date: - - journal: kirk and bistner's handbook of veterinary procedures and emergency treatment doi: . /b - - - / - sha: doc_id: cord_uid: hnf vayd nan in the event that you suspect peritonitis and have a negative tap with abdominal paracentesis, a diagnostic peritoneal lavage can be performed. to perform abdominal paracentesis, follow this procedure: . place the patient in left lateral recumbency and clip a -to -inch square with the umbilicus in the center. . aseptically scrub the clipped area with antimicrobial scrub solution. . wearing gloves, insert a -or -gauge needle or over-the-needle catheter in four quadrants: cranial and to the right, cranial and to the left, caudal and to the right, and caudal and to the left of the umbilicus. as you insert the needle or catheter, gently twist the needle to push any abdominal organs away from the tip of the needle. local anesthesia typically is not required for this procedure, although a light sedative or analgesic may be necessary if severe abdominal pain is present. in some cases, fluid will flow freely from one or more of the needles. if not, gently aspirate with a -to -ml syringe or aspirate with the patient in a standing position. avoid changing positions with needles in place because iatrogenic puncture of intraabdominal organs may occur. . save any fluid collected in sterile red-and lavender-topped tubes for cytologic and biochemical analyses and bacterial culture. monitor hemorrhagic fluid carefully for the presence of clots. normally, hemorrhagic effusions rapidly become defibrinated and do not clot. clot formation can occur in the presence of ongoing active hemorrhage or may be due to the iatrogenic puncture of organs such as the spleen or liver. if abdominal paracentesis is negative, a diagnostic peritoneal lavage can be performed. peritoneal dialysis kits are commercially available but are fairly expensive and often impractical. to perform a diagnostic peritoneal lavage, follow this procedure: . clip and aseptically scrub the ventral abdomen as described previously. . wearing sterile gloves, cut multiple side ports in a -or -gauge over-the needle catheter. use care to not cut more than % of the circumference of the catheter, or else the catheter will become weakened and potentially can break off in the patient's abdomen. . insert the catheter into the peritoneal cavity caudal and to the right of the umbilicus, directing the catheter dorsally and caudally. . infuse to ml of sterile lactated ringer's solution or . % saline solution that has been warmed to the patient's body temperature. during the instillation of fluid into the peritoneal cavity, watch closely for signs of respiratory distress because an increase in intraabdominal pressure can impair diaphragmatic excursions and respiratory function. . remove the catheter. . in ambulatory patients, walk the patient around while massaging the abdomen to distribute the fluid throughout the abdominal cavity. in nonambulatory patients, gently roll the patient from side to side. . next, aseptically scrub the patient's ventral abdomen again, and perform an abdominal paracentesis as described previously. save collected fluid for culture and cytologic analyses; however, biochemical analyses may be artifactually decreased because of dilution. remember that you likely will retrieve only a small portion of the fluid that you instilled. during the early stage of repair, granulation tissue, some exudate, and minor epithelialization is observed. place a nonadherent bandage with some antibacterial properties (petroleum or nitrofurazone-impregnated gauze) or absorbent material (foam sponge, hydrogel, or hydrocolloid dressing) in direct contact with the wound to minimize disruption of the granulation tissue bed. next, place an absorbent intermediate layer, followed by a porous outer layer, as previously described. granulation tissue can grow through gauze mesh or adhere to foam sponges and can be ripped away at the time of bandage removal. hemorrhage and disruption of the granulation tissue bed can occur. later in the repair process, granulation tissue can exude sanguineous drainage and have some epithelialization. a late nonadherent bandage is required. the contact layer should be some form of nonadherent dressing, foam sponge, hydrogel, or hydrocolloid substance. the intermediate layer and outer layers should be absorbent material and porous tape, respectively. with nonadherent dressings, wounds with viscous exudates may not be absorbed well. this may be advantageous and enhance epithelialization, provided that complications do not occur. infection, exuberant granulation tissue, or adherence of absorbent materials to the wound may occur and delay the healing process. moist healing is a newer concept of wound management in which wound exudates are allowed to stay in contact with the wound. in the absence of infection a moist wound heals faster and has enzymatic activity as a result of macrophage and polymorphonuclear cell breakdown. enzymatic degradation or "autolytic debridement" of the wound occurs. moist wounds tend to promote neutrophil and macrophage chemotaxis and bacterial phagocytosis better than use of wet-to-dry bandages. a potential complication and disadvantage of moist healing, however, is the development of bacterial colonization, folliculitis, and trauma to wound edges that can occur because of the continuously moist environment. use surfactant-type solutions (constant clens; kendall, mansfield, massachusetts) for initial wound cleansing and debridement. use occlusive dressings for rapid enzymatic debridement with bactericidal properties to aid in wound healing. bandage wet necrotic wounds with a dressing premoistened with hypertonic saline (curasalt [kendall] , % saline) to clean and debride the wounds. hypertonic saline functions to desiccate necrotic tissue and bacteria to debride the infected wound. remove and replace the hypertonic saline bandage every to hours. next, place gauze impregnated with antibacterial agents (kerlix amd [kendall] ) over the wound in the bandage layer to act as a barrier to bacterial colonization. if the wound is initially dry or has minimal exudate and is not obviously contaminated or infected, place amorphous gels of water, glycerin, and a polymer (curafil [kendall] ) over the wound to promote moisture and proteolytic healing. discontinue moisture gels such as curafil once the dry wound has become moist. finally, the final stage of moist healing helps to promote the development of a healthy granulation tissue bed. use calcium alginate dressings (curasorb or curasorb zn with zinc [kendall] ) in noninfected wounds with a moderate amount of drainage. alginate gels promote rapid development of a granulation tissue bed and epithelialization. foam dressings also can be applied to exudative wounds after a healthy granulation bed has formed. change foam dressings at least once every to days. for closed wounds without any drainage, such as a laceration that has been repaired surgically, a simple bandage with a nonadherent contact layer (telfa pad [kendall] , for example), intermediate layer of absorbent material, and an outer porous layer (elastikon, vetrap) can be placed to prevent wound contamination during healing. the nonadherent pad will not stick to the wound and cause patient discomfort. because there usually is minimal drainage from the wound, the function of the intermediate layer is more protective than absorptive. any small amount will be absorbed into the intermediate layer of the bandage. it is important in any bandage to place the tape strips or "stirrups" on the patient's limb and then overlap in the bandage, to prevent the bandage from slipping. place the intermediate and tertiary layers loosely around the limb, starting distally and working proximally, with some overlap with each consecutive layer. this method prevents excessive pressure and potential to impair venous drainage. leave the toenails of the third and fourth digits exposed, whenever possible, to allow daily examination of the bandage to determine whether the bandage is impairing venous drainage. if the bandage is too tight and constricting or impeding vascular flow, the toes will become swollen and spread apart. when placed and maintained properly (e.g., the bandage does not get wet), there usually are relatively few complications observed with this type of bandage. in some cases, it is necessary to cover a wound in which a penrose drain has been placed to allow drainage. in many cases, there is a considerable amount of drainage from the drain and underlying soft tissues. the function of the bandage is to help obliterate dead space created by the wound itself, absorb the fluid that drains from the wound and that will contaminate the environment, and prevent external wicking of material from the external environment into the wound. when the bandage is removed, the clinician can examine the amount and type of material that has drained from the wound in order to determine when the drain should be removed. when placing a bandage over a draining wound, the contact layer should be a commercially available nonadherent dressing and several layers of absorbent wide-mesh gauze placed directly over the drain at the distal end of the incision. overlay the layers of gauze with a thick layer of absorbent intermediate dressing to absorb fluid that drains from the wound. if the gauze and intermediate layers are not thick or absorbent enough, there is a potential for the drainage fluid to reach the outer layer of the bandage and provide a source of wicking of bacteria from the external environment into the wound, leading to infection. some wounds such as lacerations have minor bleeding or hemorrhage that require an immediate bandage until definitive care can be provided. to create a pressure bandage, place a nonadherent dressing immediately in contact with the wound, followed by a thick layer of absorbent material, topped by a layer of elastic bandage material such as elastikon or vetrap. unlike the bandage for a closed wound, the top tertiary outer layer should be wrapped with some tension and even pressure around the limb, starting from the distal extremity (toes) and working proximally. the pressure bandage serves to control hemorrhage but should not be left on for long periods. pressure bandages that have been left on for too long can impair nerve function and lead to tissue necrosis and slough. therefore, pressure bandages should be used in the hospital only, so that the patient can be observed closely. if hemorrhage through the bandage occurs, place another bandage over the first until the wound can be repaired definitively. removal of the first bandage will only disrupt any clot that has formed and cause additional hemorrhage to occur. fractures require immediate immobilization to prevent additional patient discomfort and further trauma to the soft tissues of the affected limb. as with all bandages, a contact layer, intermediate layer, and outer layer should be used. place the contact layer in accordance with any type of wound present. the intermediate layer should be thick absorbent material, followed by a top layer of elastic bandage material. an example is to place a telfa pad over a wound in an open distal radius-ulna fracture, followed by a thick layer of cotton gauze cast padding, followed by an elastic layer of kling (johnson & johnson medical, arlington, texas) , pulling each layer tightly over the previous layer with some overlap until the resultant bandage can be "thumped" with the clinician's thumb and forefinger and sound like a ripe watermelon. the bandage should be smooth with consecutive layers of even pressure on the limb, starting distally and working proximally. leave the toenails of the third and fourth digits exposed to monitor for impaired venous drainage that would suggest that the bandage is too tight and needs to be replaced. finally, place a top layer of vetrap or elastikon over the intermediary layer to protect it from becoming contaminated. if the bandage is used with a compound or open fracture, drainage may be impaired and actually lead to enhanced risk of wound infection. bandages placed for initial fracture immobilization are temporary until definitive fracture repair can be performed once the patient's cardiovascular and respiratory status are stable. wounds with exuberant granulation tissue must be handled carefully so as to not disrupt the healing process but to keep an overabundance of tissue from forming that will impair epithelialization. to bandage a wound with exuberant granulation tissue, place a corticosteroid-containing ointment on the wound, followed by a nonadherent contact layer. the corticosteroid will help control the exuberant growth of granulation tissue. next, carefully wrap an absorbent material over the contact layer, followed by careful placement of and overlay of elastic bandage material to place some pressure on the wound. leave the toenails of the third and fourth digits exposed so that circulation can be monitored several times daily. bandages that are too tight must be removed immediately to prevent damage to neuronal tissue and impaired vascularization, tissue necrosis, and slough. because wound drainage may be impaired, there is a risk of infection. gaping wounds or those that have undermined in between layers of subcutaneous tissue and fascia should be bandaged with a pressure bandage to help obliterate dead space and prevent seroma formation. an example of a wound that may require this type of bandage is removal of an infiltrative lipoma on the lateral or ventral thorax. use caution when placing pressure bandages around the thorax or cervical region because bandages placed too tightly may impair adequate ventilation. to place a pressure bandage and obliterate dead space, place a nonadherent contact layer over the wound. usually, a drain is placed in the wound, so place a large amount of wide-mesh gauze at the distal end of the drain to absorb any wound exudate or drainage. place several layers of absorbent material over the site to further absorb any drainage. place a layer of elastic cotton such as kling carefully but firmly over the dead space to cause enough pressure to control drainage. place at least two fingers in between the animal's thorax and the bandage to ensure that the bandage is not too tight. in many cases, the bandage should be placed once the animal has recovered from surgery and is able to stand. if the bandage is placed while the animal is still anesthetized and recumbent, there is a tendency for the bandage to be too tight. finally, the tertiary layer should be an elastic material such as elastikon or vetrap. many wounds require a pressure relief bandage to prevent contact with the external environment. wounds that may require pressure relief for healing include decubitus ulcers, pressure bandage or cast ulcers, impending ulcer areas (such as the ileum or ischium of recumbent or cachexic patients), and surgical repair sites of ulcerated areas. pressure relief bandages can be of two basic varieties: modified doughnut bandage and doughnut-shaped bandage. to create a cup or clamshell splint, follow this procedure (figures - to - ): . place a nonadherent contact layer directly over the wound. . place stirrups of tape in contact with the skin of the dog, to be placed over the intermediate layer and prevent the bandage from slipping. . place a fairly thick layer of absorbent intermediate bandage material over the contact layer such that the bandage is well-padded. pull the tape stirrups and secure them to the intermediate layer. . place a length of cast material that has been rolled to the appropriate length, such that the cast material is cupped around the patient's paw, and lies adjacent to the caudal aspect of the limb to the level of the carpus or tarsus. in the case of a clamshell splint, place a layer of cast material on the cranial and caudal aspect of the paw and conform it in place. . take the length of cast padding and soak it in warm water after it has been rolled to the appropriate length. wring out the pad, and secure/conform it to the caudal (or cranial and caudal, in the case of a clamshell splint) aspect of the distal limb and paw. . secure the cast material in place with a layer of elastic cotton gauze (kling). . secure the bandage in place with a snug layer of elastikon or vetrap. short or long splints made of cast material can be incorporated into a soft padded bandage to provide extra support of a limb above and below a fracture site. for a caudal or lateral splint to be effective, it must be incorporated for at least one joint above any fracture site to prevent a fulcrum effect and further disruption or damage to underlying soft tissue structures. a short lateral or caudal splint is used for fractures and luxations of the distal metacarpus, metatarsus, carpus, and tarsus. to place a short lateral or caudal splint, follow this procedure: . secure a contact layer as determined by the presence or absence of any wound in the area. . place tape stirrups on the distal extremity to be secured later to the intermediate bandage layer and to prevent slipping of the bandage distally. . place layers of roll cotton from the toes to the level of the mid tibia/fibula or mid radius/ulna. place the layers with even tension, with some overlap of each consecutive layer, moving distally to proximally on the limb. . secure the short caudal or lateral splint and conform it to the distal extremity to the level of the toes and proximally to the level of the mid tibia/fibula or mid radius/ulna. . secure the lateral or caudal splint to the limb with another outer layer of elastic cotton (kling). . cover the entire bandage and splint with an outer tertiary layer of vetrap or elastikon. make sure that the toenails of the third and fourth digits remain visible to allow daily evaluation of circulation. long lateral or caudal splints are used to immobilize fractures of the tibia/fibula and radius/ulna. the splints are fashioned as directed for short splints but extend proximally to the level of the axilla and inguinal regions to immobilize above the fracture site. • packed cell volume drops rapidly to less than % in the dog and less than % to % in the cat • acute loss of more than % of blood volume ( ml/kg in dog, ml/kg in cat) • clinical signs of lethargy, collapse, hypotension, tachycardia, tachypnea (acute or chronic blood loss) • ongoing hemorrhage is present • poor response to crystalloid and colloid infusion • life-threatening hemorrhage caused by thrombocytopenia or thrombocytopathia • surgical intervention is necessary in a patient with severe thrombocytopenia or thrombocytopathia plasma support • life-threatening hemorrhage with decreased coagulation factor activity • severe inflammation (pancreatitis, systemic inflammatory response syndrome) • replenish antithrombin (disseminated intravascular coagulation, protein-losing enteropathy or nephropathy) • surgery is necessary in a patient with decreased coagulation factor activity • severe hypoproteinemia is present; to partially replenish albumin, globulin, and clotting factors type a cats typically possess weak anti-b antibodies of igg and igm subtypes. transfusion of type b blood into a type a cat will result in milder clinical signs of reaction and a markedly decreased survival half-life of the infused rbcs to just days. because type ab cats possess both moieties on their cell surface, they lack naturally occurring alloantibodies; transfusion of type a blood into a type ab cat can be performed safely if a type ab donor is not available. the life span of an rbc from a type-specific transfusion into a cat is approximately days. . indications for fresh whole blood transfusion include disorders of hemostasis and coagulopathies including disseminated intravascular coagulation, von willebrand's disease, and hemophilia. fresh whole blood and platelet-rich plasma also can be administered in cases of severe thrombocytopenia and thrombocytopathia. stored whole blood and packed rbcs can be administered in patients with anemia. if pcv drops to below % or if rapid hemorrhage causes the pcv to drop below % in the dog or less than % to *indicates that this must be done for each donor being tested. minor crossmatch* . obtain a crossmatch segment from blood bank refrigerator for each donor to be crossmatched, or use an edta tube of donor's blood. make sure tubes are labeled prop-erly. . collect ml of blood from recipient and place in an edta tube. centrifuge blood for minutes. . extract blood from donor tubing. centrifuge blood for minutes. use a separate pipette for each transfer because cross-contamination can occur. . pipette plasma off of donor and recipient cells and place in tubes labeled dp and rp, respectively. . place µl of donor and recipient cells in tubes labeled dr and rr, respectively. . add . ml . % sodium chloride solution from wash bottle to each red blood cell (rbc) tube, using some force to cause cells to mix. . centrifuge rbc suspension for minutes. . discard supernatant and resuspend rbcs with . % sodium chloride from wash bottle. . repeat steps and for a total of three washes. . place drops of donor rbc suspension and drops of recipient plasma in tube labeled ma (this is the major crossmatch). . place drops of donor plasma and drops recipient rbc suspension in tube labeled mi (this is the minor crossmatch). . prepare control tubes by placing drops donor plasma with drops donor rbc suspension (this is the donor control); and place drops recipient plasma with drops recipient rbc suspension (this is the recipient control). . incubate major and minor crossmatches and control tubes at room temperature for minutes. . centrifuge all tubes for minute. . read tubes using an agglutination viewer. . check for agglutination and/or hemolysis. . score agglutination with the following scoring scale: + one solid clump of cells + several large clumps of cells + medium-sized clumps of cells with a clear background + hemolysis, no clumping of cells neg = negative for hemolysis; negative for clumping of red blood cells fresh whole blood coagulopathy with active hemorrhage (disseminated intravascular coagulation, thrombocytopenia; massive acute hemorrhage; no stored blood available) stored whole blood massive acute or ongoing hemorrhage; hypovolemic shock caused by hemorrhage that is unresponsive to conventional crystalloid and colloid fluid therapy; unavailability of equipment required to prepare blood components packed red blood cells nonregenerative anemia, immune-mediated hemolytic anemia, correction of anemia before surgery, acute or chronic blood loss fresh frozen plasma factor depletion associated with active hemorrhage (congenital: von willebrand's factor, hemophilia a, hemophilia b; acquired: vitamin k antagonist, rodenticide intoxication, dic); acute or chronic hypoproteinemia (burns, wound exudates, body cavity effusion; hepatic, renal, or gastrointestinal loss); colostrum replacement in neonates frozen plasma acute plasma or protein loss; chronic hypoproteinemia; (contains stable colostrum replacement in neonates; hemophilia b and clotting factors) selected clotting factor deficiencies platelet-rich plasma* thrombocytopenia with active hemorrhage (immune-mediated thrombocytopenia, dic); platelet function abnormality (congenital: thrombasthenia in bassett hounds; acquired: nsaids, other drugs) cryoprecipitate congenital factor deficiencies (routine or before surgery): (concentration of factor hemophilia a, hemophilia b, von willebrand's disease, viii, von willebrand's hypofibrinogenemia; acquired factor deficiencies factor, and fibrinogen) *must be purchased because logistically one cannot obtain enough blood simultaneously to provide a significant amount of platelets; platelets infused have a very short (< hours) half-life. dic, disseminated intravascular coagulation; nsaids, nonsteroidal antiinflammatory drugs. universal donor (e.g., should be administered whenever possible. because there is no universal donor in the cat and because cats possess naturally occurring alloantibodies, all cat blood should be typed and crossmatched before any transfusion. if fresh whole blood is not available, a hemoglobin-based oxygen carrier (oxyglobin, to ml/kg iv) can be administered until blood products become available. table - indicates blood component dose and administration rates. blood products should be warmed slowly to °c before administering them to the patient. blood warmer units are available for use in veterinary medicine to facilitate rapid transfusion without decreasing patient body temperature (thermal angel; enstill medical technologies, inc., dallas, texas). red blood cell and plasma products should be administered in a blood administration set containing a -µm in-line filter. smaller in-line filters ( µm) also can be used in cases in which extremely small volumes are to be administered. blood products should be administered over a period of hours, whenever possible, according to guidelines set by the american association of blood banks. the volume of blood components required to achieve a specific increment in the patient's pcv depends largely on whether whole blood or packed rbcs are transfused and whole blood ml/kg will increase max rate: ml/kg/ max: ml/kg/ volume by % hours hour packed red ml/kg will increase critically ill blood cells volume by % patients (e.g., cardiac failure or renal failure): - ml/kg/hour fresh frozen ml/kg body mass (repeat - ml/minute or use rates as for plasma in - days or in - days whole blood (infuse within - hours) or until bleeding stops); monitor act, aptt, and pt before and hour after transfusion cryoprecipitate general: unit/ kg/ hours - ml/minute or use rates as for whole or until bleeding stops blood (infuse within - hours) hemophilia a: - units factor viii/kg; unit of cryoprecipitate contains approximately units of factor viii platelet-rich unit/ kg ( unit of ml/minute plasma platelet-rich plasma will check platelet count before and hour increase platelet count after transfusion hour after transfusion by , /µl) whether there is ongoing hemorrhage or rbc destruction. because the pcv of packed rbcs is unusually high ( % for greyhound blood), a smaller total volume is required than whole blood to achieve a comparable increase in the patient's pcv. in general, ml/kg of packed rbcs or ml/kg whole blood will raise the recipient's pcv by %. the "rule of ones" states that ml per lb of whole blood will raise the pcv by %. if the patient's pcv does not raise by the amount anticipated by the foregoing calculation(s), causes of ongoing hemorrhage or destruction should be considered. the goal of red blood component therapy is to raise the pcv to % to % in dogs and % to % in cats. if an animal is hypovolemic and whole blood is administered, the fluid is redistributed into the extravascular compartment within hours of transfusion. this will result in a secondary rise in the pcv hours after the transfusion in addition to the initial rise to hours after the rbc transfusion is complete. the volume of plasma transfused depends largely on the patient's need. in general, plasma transfusion should not exceed more than ml/kg during a -hour period for normovolemic animals. thaw plasma at room temperature, or place it in a ziplock freezer bag and run under cool (not warm) water until thawed. then administer the plasma through a blood administration set that contains an in-line blood filter or through a standard driptype administration set with a detachable in-line blood administration filter. the average rate of plasma infusion in a normovolemic patient should not exceed ml/kg/hour. in acute need situations, plasma can be delivered at rates up to to ml/kg/minute. for patients with cardiac insufficiency or other circulatory problems, plasma infusion rates should not exceed ml/kg/hour. plasma or other blood products should not be mixed with or used in the same infusion line as calcium-containing fluids, including lactated ringer's solution, calcium chloride, or calcium gluconate. the safest fluid to mix with any blood product is . % sodium chloride. administer fresh frozen plasma, frozen plasma, and cryoprecipitate at a volume of ml/kg until bleeding is controlled or source of ongoing albumin loss ceases. the goal of plasma transfusion therapy is to raise the albumin to a minimum of . g/dl or until bleeding stops as in the case of coagulopathies. monitor the patient to ensure that bleeding has stopped, coagulation profiles (act, aptt, and pt) have normalized, hypovolemia has stabilized, and/or total protein is normalizing, which are indications for discontinuing ongoing transfusion therapy. plasma cryoprecipitate can be purchased or manufactured through the partial thawing and then centrifugation of fresh frozen plasma. cryoprecipitate contains concentrated quantities of vwf, factor viii, and fibrinogen and is indicated in severe forms of von willebrand's disease and hemophilia a (factor viii deficiency). platelet-rich plasma must be purchased from a commercial source. one unit of fresh whole blood contains to platelets. the viability of the platelets contained in the fresh whole blood is short-lived, just to hours after transfusion into the recipient. because platelet-rich plasma is difficult to obtain, animals with severe thrombocytopenia or thrombocytopathia should be treated with immunomodulating therapies and the administration of fresh frozen plasma. in dogs, blood and plasma transfusions can be administered intravenously or intraosseously. the cephalic, lateral saphenous, medial saphenous, and jugular veins are used most commonly. fill the recipient set so that the blood in the drip chamber covers the filter (normal -µm filter). with small amounts of blood ( ml) or critically ill patients, use a -µm filter. avoid latex filters for plasma and cryoprecipitate administration. blood can emergency care be administered at variable rates, but the routine figure of to ml/minute often is used. normovolemic animals can receive blood at ml/kg/day. dogs in heart failure should receive infusions at no more than ml/kg/hour. volume is given as needed. to calculate the approximate volume of blood needed to raise hematocrit levels, use the following formula for the dog: anticoagulated blood volume (ml) = body mass (kg) × × pcv desired − pcv of recipient pcv of donor in anticoagulant an alternative formula is the following: . × recipient body mass (kg) × (dog) × pcv desired − pcv of recipient pcv of donor in anticoagulant surgical emergencies and shock may require several times this volume within a short period. if greater than % of the patient's blood volume is lost, supplementation with colloids, crystalloids, and blood products is indicated for fluid replacement. one volume of whole blood achieves the same increase in plasma as two to three volumes of plasma. if the patient's blood type is unknown and type a-negative whole blood is not available, any dog blood can be administered to a dog in acute need if the dog has never had a transfusion before. if mismatched blood is given, the patient will become sensitized, and after days, destruction of the donor rbcs will begin. in addition, any subsequent mismatched transfusions may cause an immediate reaction (usually mild) and rapid destruction of the transfused rbcs. the clinical signs of a transfusion reaction typically only are seen when type a blood is administered to a type a-negative recipient that has been sensitized previously. incompatible blood transfusions to breeding females can result in isoimmunization and in hemolytic disease in the puppies. the a-negative bitch that receives a transfusion with a-positive and that produces a litter from an a-positive stud can have puppies with neonatal isoerythrolysis. cats with severe anemia in need of a blood transfusion are typically extremely depressed, lethargic, and anorexic. the stress of restraint and handling can push these critically ill patients over the edge and cause them to die. extreme gentleness and care are mandatory in restraint and handling. the critically ill cat should be cradled in a towel or blanket. supplemental flow-by or mask oxygen should be administered, whenever possible, although it may not be clinically helpful until oxygen-carrying capacity is replenished with infusion of rbcs or hemoglobin. blood can be administered by way of cephalic, medial saphenous, or the jugular vein. intramedullary infusion is also possible, if vascular access cannot be accomplished. the average -to -kg cat can accept to ml of whole blood injected intravenously over a period of to minutes. administer filtered blood at a rate of to ml/kg/hour. the following formula can be used to estimate the volume of blood required for transfusion in a cat: anticoagulated blood volume (ml) = body mass (kg) × × pcv desired − pcv of recipient pcv of donor in anticoagulant the exact overall incidence and clinical significance of transfusion reactions in veterinary medicine are unknown. several studies have been performed that document the incidence of transfusion reactions in dogs and cats. overall, the incidence of transfusion reactions in dogs and cats is . % and %, respectively. transfusion reactions can be immune-mediated and non-immune-mediated and can happen immediately or can be delayed until after a transfusion. acute reactions usually occur within minutes to hours of the onset of transfusion but may occur up to hours after the transfusion has been stopped. acute immunologic reactions include hemolysis and acute hypersensitivity including rbcs, platelets, and leukocytes. signs of a delayed immunologic reaction include hemolysis, purpura, immunosuppression, and neonatal isoerythrolysis. acute nonimmunologic reactions include donor cell hemolysis before onset of transfusion, circulatory volume overload, bacterial contamination, citrate toxicity with clinical signs of hypocalcemia, coagulopathies, hyperammonemia, hypothermia, air embolism, acidosis, and pulmonary microembolism. delayed nonimmunologic reactions include the transmission and development of infectious diseases and hemosiderosis. clinical signs of a transfusion reaction typically depend on the amount of blood transfused, the type and amount of antibody involved in the reaction, and whether the recipient has had previous sensitization. monitoring the patient carefully during the transfusion period is essential in recognizing early signs of a transfusion reaction, including those that may become life threatening. a general guideline for patient monitoring is first to start the transfusion slowly during the first minutes. monitor temperature, pulse, and respiration every minutes for the first hour, hour after the end of the transfusion, and every hours minimally thereafter. also obtain a pcv immediately before the transfusion, hour after the transfusion has been stopped, and every hours thereafter. monitor coagulation parameters such as an act and platelet count at least daily in patients requiring transfusion therapy. the most common documented clinical signs of a transfusion reaction include pyrexia, urticaria, salivation/ptyalism, nausea, chills, and vomiting. other clinical signs of a transfusion reaction may include tachycardia, tremors, collapse, dyspnea, weakness, hypotension, collapse, and seizures. severe intravascular hemolytic reactions may occur within minutes of the start of the transfusion, causing hemoglobinemia, hemoglobinuria, disseminated intravascular coagulation, and clinical signs of shock. extravascular hemolytic reactions typically occur later and will result in hyperbilirubinemia and bilirubinuria. pretreatment of patients to help decrease the risk of a transfusion reaction remains controversial, and in most cases, pretreatment with glucocorticoids and antihistamines is ineffective at preventing intravascular hemolysis and other reactions should they occur. the most important component of preventing a transfusion reaction is to screen each recipient carefully and process the donor component therapy carefully before the administration of any blood products. treatment of a transfusion reaction depends on its severity. in all cases, stop the transfusion immediately when clinical signs of a reaction occur. in most cases, discontinuation of the transfusion and administration of drugs to stop the hypersensitivity reaction will be sufficient. once the medications have taken effect, restart the transfusion slowly and monitor the patient carefully for further signs of reaction. in more severe cases in which a patient's cardiovascular or respiratory system become compromised and hypotension, tachycardia, or tachypnea occurs, immediately discontinue the transfusion and administer diphenhydramine ( mg/kg im), dexamethasone-sodium phosphate ( . to . mg/kg iv), and epinephrine to the patient. the patient should have a urinary catheter and central venous catheter placed for measurement of urine output and central venous pressures. aggressive fluid therapy may be necessary to avoid renal insufficiency or renal damage associated with severe intravascular hemolysis. overhydration with subsequent pulmonary edema generally can be managed with supplemental oxygen administration and intravenous or intramuscular administration of furosemide ( to mg/kg). plasma products with or without heparin can be administered for disseminated intravascular coagulation. the hbocs can be stored at room temperature and have a relatively long shelf life compared with red blood component products. the hbocs function to carry oxygen through the blood and can diffuse oxygen past areas of poor tissue perfusion. an additional characteristic of hbocs is as a potent colloid, serving to maintain fluid within the vascular space. for this reason, hbocs must be used with caution in euvolemic patients and patients with cardiovascular insufficiency. central venous pressure (cvp) measures the hydrostatic pressure in the anterior vena cava and is influenced by vascular fluid volume, vascular tone, function of the right side of the heart, and changes in intrathoracic pressure during the respiratory cycle. the cvp is not a true measure of blood volume but is used to gauge fluid therapy as a method of determining how effectively the heart can pump the fluid that is being delivered to it. thus the cvp reflects the interaction of the vascular fluid volume, vascular tone, and cardiac function. measure cvp in any patient with acute circulatory failure, large volume fluid diuresis (i.e., toxin or oliguric or anuric renal failure), fluid in-and-out monitoring, and cardiac dysfunction. the placement of central venous catheters and thus cvp measurements is contraindicated in patients with known coagulopathies including hypercoagulable states. to perform cvp monitoring, place a central venous catheter in the right or left jugular vein. in cats and small dogs, however, a long catheter placed in the lateral or medial saphenous vein can be used for trends in cvp monitoring. first, assemble the equipment necessary for jugular catheter (see vascular access techniques for how to place a jugular or saphenous long catheter) and cvp monitoring (box - ). after placing the jugular catheter, take a lateral thoracic radiograph to ensure that the tip of the catheter sits just outside of the right atrium for proper cvp measurements (see to establish an intravenous catheter for cvp, follow this procedure: . assemble the cvp setup such that the male end of a length of sterile intravenous catheter extension tubing is inserted into the t port of the jugular or medial/lateral saphenous catheter. make sure to flush the length of tubing with sterile saline before connecting it to the patient to avoid iatrogenic air embolism. . next, insert the male end of a three-way stopcock into the female end of the extension tubing. . attach a -ml syringe filled with heparinized sterile . % saline to one of the female ports of the three-way stopcock and either a manometer or a second length of intravenous extension tubing attached to a metric ruler. . lay the patient in lateral or sternal recumbancy. . turn the stopcock off to the manometer/ruler and on to the patient. infuse a small amount of heparinized saline through the catheter to flush the catheter. . next, turn the stopcock off to the patient and on to the manometer. gently flush the manometer or length of extension tubing with heparinized saline from the syringe. use care not to agitate the fluid and create air bubbles within the line or manometer that will artifactually change the cvp measured. . next, lower the cm point on the manometer or ruler to the level of the patient's manubrium (if the patient is in lateral recumbancy) or the point of the elbow (if the patient is in sternal recumbancy). . turn the stopcock off to the syringe, and allow the fluid column to equilibrate with the patient's intravascular volume. once the fluid column stops falling and the level rises and falls with the patient's heartbeat, measure the number adjacent to the bottom of the meniscus of the fluid column. this is the cvp in centimeters of water (see figure - ). . repeat the measurement several times with the patient in the same position to make sure that none of the values has been increased or decreased artifactually in error. alternately, attach the central catheter to a pressure transducer and perform electronic monitoring of cvp. there is no absolute value for normal cvp. the normal cvp for small animal patients is to cm h o. values less than zero are associated with absolute or relative hypovolemia. values of to cm h o are borderline hypervolemia, and values greater than cm h o suggest intravascular volume overload. values greater than cm h o may be correlated with congestive heart failure and the development of pulmonary edema. in individual patients, the trend in change in cvp is more important than absolute values. as a rule of thumb, when using cvp measurements to gauge fluid therapy and avoid vascular and pulmonary overload, the cvp should not increase by more than cm h o in any -hour period. if an abrupt increase in cvp is found, repeat the measurement to make sure that the elevated value was not obtained in error. if the value truly has increased dramatically, temporarily discontinue fluid therapy and consider administration of a diuretic. delaforcade am, rozanski ea: central venous pressure and arterial blood pressure measurements, vet clin north am small anim pract ( ) the diagnosis of intracellular fluid deficit is difficult and is based more on the presence of hypernatremia or hyperosmolality than on clinical signs. an intracellular fluid deficit is expected when free water loss by insensible losses and vomiting, diarrhea, or urine is not matched by free water intake. consideration of the location of the patient's fluid deficit, history of vomiting and diarrhea, no visible clinical signs of deficit % dry mucous membranes, mild skin tenting % increased skin tenting, dry mucous membranes, mild tachycardia, normal pulse* % increased skin tenting, dry mucous membranes, tachycardia, weak pulse pressure % increased skin tenting, dry corneas, dry mucous membranes, % elevated or decreased heart rate, poor pulse quality, altered level of consciousness* the respiratory system further contributes to acid-base status by changes in the elimination of carbon dioxide. hyperventilation decreases the blood pco and causes a respiratory alkalosis. hypoventilation increases the blood pco and causes a respiratory acidosis. depending on the altitude, the pco in dogs can range from to mm hg. in cats, normal is to mm hg. venous pco values are to mm hg in dogs and to mm hg in cats. use a systematic approach whenever attempting to interpret a patient's acid-base status. ideally, obtain an arterial blood sample so that you can monitor the patient's oxygenation and ventilation. once an arterial blood sample has been obtained, follow these steps: . determine whether the blood sample is arterial or venous by looking at the oxygen saturation (sao ). the sao should be greater than % if the sample is truly arterial, although it can be as low as % if a patient has severe hypoxemia. . consider the patient's ph. if the ph is outside of the normal range, an acid-base disturbance is present. if the ph is within the normal range, an acid-base disturbance may or may not be present. if the ph is low, the patient is acidotic. if the ph is high, the patient is alkalotic. . next, look at the base excess or deficit. if the base excess is increased, the patient has higher than normal bicarbonate. if there is a base deficit, the patient may have a low bicarbonate or increase in unmeasured anions (e.g., lactic acid or ketoacids). . next, look at the bicarbonate. if the ph is low and the bicarbonate is low, the patient has a metabolic acidosis. if the ph is high and the bicarbonate is elevated, the patient has a metabolic alkalosis. . next, look at the paco . if the patient's ph is low and the paco is elevated, the patient has a respiratory acidosis. if the patient's ph is high and the paco is low, the patient has a respiratory alkalosis. . finally, if you are interested in the patient's oxygenation, look at the pao . normal pao is greater than mm hg. the metabolic acidosis early in renal failure may be hyperchloremic and later may convert to typical increased anion gap acidosis. . next, you must determine whether the disorders present are primary disorders or an expected compensation for disorders in the opposing system. for example, is the patient retaining bicarbonate (metabolic alkalosis) because of carbon dioxide retention (respiratory acidosis)? use the chart in table - to evaluate whether the appropriate degree of compensation is occurring. if the adaptive response falls within the expected range, a simple acid-base disorder is present. if the response falls outside of the expected range, a mixed acid-base disorder is likely present. . finally, you must determine whether the patient's acid-base disturbance is compatible with the history and physical examination findings. if the acid-base disturbance does not fit with the patient's history and physical examination abnormalities, question the results of the blood gas analyses and possibly repeat them. the most desirable method of assessing the acid-base status of an animal is with a blood gas analyzer. arterial samples are preferred over venous samples, with heparin used as an anticoagulant (table - ) . potassium primarily is located in the intracellular fluid compartment. serum potassium is regulated by the actions of the sodium-potassium-adenosinetriphosphatase pump on cellular membranes, including those of the renal tubular epithelium. inorganic metabolic acidosis artifactually can raise serum potassium levels because of redistribution of extracellular potassium in exchange for intracellular hydrogen ion movement in an attempt to correct serum ph. metabolic acidosis potassium is one of the major players in the maintenance of resting membrane potentials of excitable tissue, including neurons and cardiac myocytes. changes in serum potassium can affect cardiac conduction adversely. hyperkalemia lowers the resting membrane potential and makes cardiac cells, particularly those of the atria, more susceptible to depolarization. characteristic signs of severe hyperkalemia that can be observed on an ecg rhythm strip include an absence of p waves, widened qrs complexes, and tall tented or spiked t waves. further increases in serum potassium can be associated with bradycardia, ventricular fibrillation, and cardiac asystole (death). treatment of hyperkalemia consists of administration of insulin ( . to . units/kg, iv regular insulin) and dextrose ( g dextrose per unit of insulin administered, followed by . % dextrose iv cri to prevent hypoglycemia), calcium ( to ml of % calcium gluconate administered iv slowly to effect), or sodium bicarbonate ( meq/kg, iv slowly). insulin plus dextrose and bicarbonate therapy help drive the potassium intracellularly, whereas calcium antagonizes the effect of hyperkalemia on the myocardial cells. all of the treatments work within minutes, although the effects are relatively short-lived ( minutes to hour) unless the cause of the hyperkalemia is identified and treated appropriately (box - ). dilution of serum potassium also results from restoring intravascular fluid volume and correcting metabolic acidosis, in most cases. treatment with a fluid that does not contain potassium (preferably . % sodium chloride) is recommended. hypokalemia elevates the resting membrane potential and results in cellular hyperpolarization. hypokalemia may be associated with ventricular dysrhythmias, but the ecg changes are not as characteristic as those observed with hyperkalemia. causes of hypokalemia include renal losses, anorexia, gastrointestinal loss (vomiting, diarrhea), intravenous fluid diuresis, loop diuretics, and postobstructive diuresis (box - ). if the serum potassium concentration is known, potassium supplementation in the form of potassium chloride or potassium phosphate can be added to the patient's intravenous fluids. correct serum potassium levels less than . meq/l or greater than . meq/l. potassium rates should not exceed . meq/kg/hour (table - ) . metabolic acidosis from bicarbonate depletion often corrects itself with volume restoration in most small animal patients. patients with moderate to severe metabolic acidosis may benefit from bicarbonate supplementation therapy. the metabolic contribution to acid-base balance is identified by measuring the total carbon dioxide concentration or calculating the bicarbonate concentration. if these measurements are not available, the degree of expected metabolic acidosis can be estimated subjectively by the severity of underlying disease that often contributes to metabolic acidosis: hypovolemic or traumatic shock, septic shock, diabetic ketoacidosis, or oliguric/anuric renal failure. if the metabolic acidosis is estimated to be mild, moderate, or severe, add sodium bicarbonate at , , and meq/kg body mass, respectively. patients with diabetic ketoacidosis may not require bicarbonate administration once volume replacement and perfusion is restored, and the ketoacids are metabolized to bicarbonate. if the bicarbonate measurement of base deficit is known, the following formula can be used as a gauge for bicarbonate supplementation: base deficit × . = body mass (kg) = meq bicarbonate to administer osmolality osmolality is measured by freezing point depression or a vapor pressure osmometer, or it may be calculated by the following formula: mosm/kg = [(na + ) + (k + )] + bun/ . + glucose/ where sodium and potassium are measured in milliequivalents, and bun and glucose are measured in milligrams per deciliter. osmolalities less than mosm/kg or greater emergency care than mosm/kg are serious enough to warrant therapy. the difference between the measured osmolality and the calculated osmolality (the osmolal gap) should be less than mosm/kg. if the osmolal gap is greater than mosm/kg, consider the presence of unmeasured anions such as ethylene glycol metabolites. the volume of extracellular fluid is determined by the total body sodium content, whereas the osmolality and sodium concentration are determined by water balance. serum sodium concentration is an indication of the amount of sodium relative to water in the extracellular fluid and provides no direct information about the total body sodium content. unlikely to cause hyperkalemia in presence of normal renal function unless iatrogenic (e.g., continuous infusion of potassium-containing fluids at an excessively rapid rate) acute mineral acidosis (e.g., hydrochloric acid or ammonium chloride) insulin deficiency (e.g., diabetic ketoacidosis) acute tumor lysis syndrome reperfusion of extremities after aortic thromboembolism in cats with cardiomyopathy hyperkalemic periodic paralysis (one case report in a pit bull) mild hyperkalemia after exercise in dogs with induced hypothyroidism infusion of lysine or arginine in total parenteral nutrition solutions nonspecific β-blockers (e.g., propranolol)* cardiac glycosides (e.g., digoxin)* urethral obstruction ruptured bladder anuric or oliguric renal failure hypoadrenocorticism selected gastrointestinal disease (e.g., trichuriasis, salmonellosis, or perforated duodenal ulcer) late pregnancy in greyhound dogs (mechanism unknown but affected dogs had gastrointestinal fluid loss) chylothorax with repeated pleural fluid drainage hyporeninemic hypoaldosteronism † angiotensin-converting enzyme inhibitors (e.g., enalapril)* angiotensin receptor blockers (e.g., losartan)* cyclosporine and tacrolimus* potassium-sparing diuretics (e.g., spironolactone, amiloride, and triamterene)* nonsteroidal antiinflammatory drugs* heparin* trimethoprim* from dibartola sp: fluid, electrolyte and acid-base disorders in small animal practice, st louis, , saunders. *likely to cause hyperkalemia only in conjunction with other contributing factors (e.g., other drugs, decreased renal function, or concurrent administration of potassium supplements). † not well documented in veterinary medicine. if refractory hypokalemia is present, supplement magnesium at . meq/kg/day for hours. alone unlikely to cause hypokalemia unless diet is aberrant administration of potassium-free (e.g., . % sodium chloride or % dextrose in water) or potassium-deficient fluids (e.g., lactated ringer's solution over several days) bentonite clay ingestion (e.g., cat litter) alkalemia insulin/glucose-containing fluids catecholamines hypothermia hypokalemic periodic paralysis (burmese cats) albuterol overdosage patients with hyponatremia or hypernatremia may have decreased, normal, or increased total body sodium content (boxes - and [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). an increased serum sodium concentration implies hyperosmolality, whereas a decrease in serum sodium concentration usually, but not always, implies hypoosmolality. the severity of clinical signs of hypernatremia and hyponatremia is related primarily to the rapidity of the onset of the change rather than to the magnitude of the associated plasma hyperosmolality or hypoosmolality. clinical signs of neurologic disturbances include disorientation, ataxia, and seizures, and coma may occur at serum sodium concentrations less than meq/l or greater than meq/l in dogs. therapy of hypernatremia or hyponatremia with fluid containing low or higher concentrations of sodium should proceed with caution, for rapid changes (decreases or increases) of serum sodium and osmolality can cause rapid changes in the intracellular and extracellular fluid flux, leading to intracellular dehydration or edema, even though the serum sodium has not been returned to normal. a rule of thumb is to not raise or lower the serum sodium by more than meq/l during any one -hour period. restoration of the serum sodium concentration over a period of to hours is better. in almost all circumstances, an animal will correct its sodium balance with simple fluid restoration. if severe hypernatremia exists that suggests a free water deficit, however, the free water deficit should be calculated from the following formula: hypernatremia can be corrected slowly with . % sodium chloride plus . % dextrose, % dextrose in water, or lactated ringer's solution (sodium content: meq/l). correct hyponatremia initially with . % sodium chloride. sodium is balanced predominantly by chloride and bicarbonate. the difference between these concentrations, (na , has been called the anion gap. the normal anion gap is between and meq/l. when the anion gap exceeds , consider the possibility of an accumulation of unmeasured anions (e.g., lactate, ketoacids, phosphate, sulfate, ethylene glycol metabolites, and salicylate). abnormalities in the anion gap may be helpful in determining the cause of metabolic acidosis (boxes - and - ). the colloid oncotic pressure of blood is associated primarily with large-molecular-weight colloidal substances in circulation. the major player in maintaining intravascular and interstitial oncotic pressure, the water-retaining property of each fluid compartment, is albumin. albumin contributes roughly % to the colloidal oncotic pressure of blood. the majority of albumin is located within the interstitial space. hypoalbuminemia can result from increased loss in the form of protein-losing enteropathy or nephropathy and wound exudates, or it may be due to lack of hepatic albumin synthesis. serum albumin pools are in a constant flux with interstitial albumin. once interstitial albumin pools become depleted from replenishing serum albumin, serum albumin levels can continue to decrease, which can lead to a decrease in colloidal oncotic pressure. serum albumin less than . g/dl has been associated with inadequate intravascular fluid retention and the development of peripheral edema and third spacing of fluid. oncotic pressure can be restored with the use of artificial or synthetic colloids or natural colloids (see colloids). maintenance fluid requirements have been extrapolated from the formulas used to calculate a patient's daily metabolic energy requirements because it takes ml of water to metabolize kcal of energy (table - ) . the patient's daily metabolic water (fluid) requirements can be calculated by the following formula: administration of an isotonic crystalloid fluid for maintenance requirements often can produce iatrogenic hypokalemia. in most cases, supplemental potassium must be added to prevent hypokalemia resulting from inappetance, kalliuresis, and supplementation with isotonic crystalloid fluids. the most reliable method of determining the degree of fluid deficit is by weighing the animal and calculating acute weight loss. acute weight loss in a patient with volume loss in the form of vomiting, feces, wound exudates, and urine is due to fluid loss and not loss of muscle or fat. lean body mass normally is not gained or lost rapidly enough to cause major changes in body weight. one milliliter of water weighs approximately g. this fact allows calculation of the patient's fluid deficit, if ongoing losses can be measured. when a patient first presents, however, the body weight before a fluid deficit has occurred rarely is known. instead, one must rely on subjective measures of dehydration to estimate the patient's percent dehydration and to calculate the volume of fluid required to rehydrate the patient over the next hours. to calculate the volume deficit, use the following formula: body mass (kg) × (% dehydration) × = fluid deficit (ml) the patient's fluid deficit must be added to the daily maintenance fluid requirements and administered over a -hour period. ongoing losses can be determined by measuring urine output, weighing the patient at least to times a day, and measuring the volume or weight of vomitus or diarrhea. a crystalloid fluid contains crystals of salts with a composition similar to that of the extracellular fluid space and can be used to maintain daily fluid requirements and replace fluid deficits or ongoing fluid losses (table - ) . metabolic, acid-base, and electrolyte imbalances also can be treated with isotonic fluids with or without supplemental electrolytes and buffers. depending on the patient's clinical condition, choose the specific isotonic crystalloid fluid to replace and maintain the patient's acid-base and electrolyte status ( table - ) . crystalloid fluids are readily available, are relatively inexpensive, and can be administered safely in large volumes to patients with no preexisting cardiac or renal disease or cerebral edema. following infusion, approximately % of the volume of a crystalloid fluid infused will redistribute to the interstitial fluid compartment. as such, crystalloid fluids alone are ineffective for ongoing intravascular volume depletion when given as a bolus. the crystalloid fluid bolus must be followed by a constant rate infusion, taking into consideration the patient's daily maintenance fluid requirements and ongoing fluid losses. administration of a large volume of crystalloid fluids can cause dilutional anemia and coagulopathies. * × bw kg + = kcal/day = ml/day. note: this formula will slightly underestimate the requirements for patients that are less than kg and will slightly overestimate the requirements for patients greater than kg. retain fluid in the vascular space, the volume of crystalloid fluid infused (maintenance + deficit + ongoing losses) should be decreased by % to % to avoid vascular volume overload. two major classes of colloids exist: natural and synthetic. natural colloids (whole blood, packed rbcs, plasma) are discussed elsewhere in this text. concentrated human albumin is a natural purified colloid that recently has become more popular in the treatment of advanced hypoalbuminemia and hypoproteinemia and will be discussed here. synthetic colloids are starch polymers and include dextrans and hetastarch. concentrated human albumin is available as a % or % solution. the % solution has an osmolality similar to that of serum ( mosm/l), whereas the % solution is hyperoncotic ( mosm/l). a % albumin solution draws fluid from the interstitial space into the intravascular space. concentrated albumin solutions often are used to restore circulating volume when synthetic colloids are not available. albumin not only is important at maintaining the colloidal oncotic pressure of blood but also serves as a valuable free-radical scavenger and carrier of drugs and hormones necessary for normal tissue function and healing. albumin levels less than . g/dl have been associated with increased morbidity and mortality. concentrated human albumin solutions can be administered as an effective method of restoring interstitial and serum albumin concentrations in situations of acute and chronic hypoalbuminemia. albumin ( %) is available in -and -ml vials and is more cost-efficient as an albumin replacement than procurement and administration of fresh frozen plasma. recommended albumin infusion rates are to ml/kg over hours, after pretreatment with diphenhydramine. although concentrated human albumin is structurally similar to canine albumin, closely monitor the patient for signs of allergic reaction during and after the infusion. dextran- is a synthetic high-molecular-weight polysaccharide (sucrose polymer) with a molecular weight of , d. particles less than , d, are cleared rapidly by the kidneys, whereas larger particles are cleared more slowly by the hepatic reticuloendothelial system. dextran- can coat platelets and inhibit platelet function and so must be used with caution in patients with known coagulopathies. the total daily dosage should not exceed ml/kg/day. hetastarch (hydroxyethyl starch) is a large-molecular-weight amylopectin polymer, has molecules with a molecular weight that exceeds , d, and has an average half-life of to hours in circulation. hetastarch can bind with vwf and cause prolongation of the act and aptt; however, it does not cause a coagulopathy. recommended rates of hetastarch infusion are -to -ml incremental boluses for the treatment of hypotension and to ml/kg/day as a constant rate infusion for maintenance of colloidal oncotic pressure. many are the acceptable ways to administer the fluids prescribed for each patient based on the degree of dehydration, estimation of ongoing losses, ability to tolerate oral fluid, and metabolic, acid-base, and electrolyte derangements. administer the fluids in a manner that is best for the patient and most appropriate for the practice. to determine the rate of intravenous fluid infusion, take the total volume of fluids that have been prescribed and divide the total volume by the total number of hours in a day that intravenous fluids can be delivered safely and monitored. the safest and most accurate way to deliver intravenous fluids, particularly in extremely small animals or those with congestive heart failure, is through an intravenous fluid pump. fluid should not be administered intravenously if the patient cannot be monitored to make sure that the fluids are being delivered at a safe rate and that the fluid line has not become disconnected. supplement fluids over as many hours as possible to allow the patient as much time as possible to redistribute and fully utilize the fluids administered. fluids administered too quickly can cause a diuresis to occur, such that the majority of the fluids administered will be excreted in the urine. if time is limited or if extra time is needed for safe administration of fluids, consider using a combination of intravenously and subcutaneously emergency care administered fluids. intravenous is the preferred route of administration of fluids in any patient with dehydration and hypovolemia. as intravascular volume depletion occurs, reflex peripheral vasoconstriction occurs to restore core perfusion. the subcutaneous tissue are not perfused well and therefore fluids administered subcutaneously will not be absorbed well into the interstitial and intravascular spaces. subcutaneously administered fluids can be absorbed slowly and delivered effectively in the management of mild interstitial dehydration and in the treatment of renal insufficiency. subcutaneously administered fluids should never take the place of intravenously administered fluids in a hypovolemic patient or one with severe interstitial dehydration. intramedullary (intraosseous) infusion works well in small patients in which vascular access cannot be established. shock doses of fluids and other substances, including blood products, can be administered under pressure through an intraosseous cannula. because of the inherent discomfort and risk of osteomyelitis with intraosseous infusion, establish vascular access as soon as possible. the safest and most efficient method of intravenous fluid infusion is through a fluid pump. in cases in which a fluid pump is unavailable, infusion by gravity feed is the next option. infusion sets from various manufacturers have calibrated drip chambers such that a specific number of drops will equal ml of fluid. fluid rates can be calculated based on the number of drops that fall into the drip chamber per minute: fluid volume to be infused (ml) = ml/hour number of hours available many pediatric drip sets deliver drops/ml, such that milliliters/hour equals drops/ minute. carefully record fluid orders so that the volume to be administered is recorded as milliliters/hour, milliliters/day, and drops/minute. this will allow personnel to detect major discrepancies and calculation errors more readily. the volume actually delivered should be recorded in the record by nursing personnel. all additives should be listed clearly on the bottle on a piece of adhesive tape or a special label manufactured for this purpose. a strip of adhesive tape also can be attached to the bottle and marked appropriately to provide a quick visualization of the estimate of volume delivered. includes a large-bore flexible orogastric lavage tube, permanent marker or white tape, lubricating jelly, warm water, two large buckets, a roll of -inch white tape, and a manual lavage pump. to perform the orogastric lavage, follow this procedure: . place all animals under general anesthesia with a cuffed endotracheal tube in place to protect the airway and prevent aspiration of gastric contents into the lungs. . place a roll of -inch white tape into the animal's mouth, and secure the tape around the muzzle. you will insert the tube through the hole in the center of the roll of tape. . next, place the distal end of the tube at the level of the last rib, directly adjacent to the animal's thorax and abdomen. measure the length of the tube from the most distal end to the point where it comes out of the mouth, and label this location on the tube with a permanent marker or piece of white tape. . lubricate the distal portion of the tube, and gently insert it through the roll of tape in the animal's mouth. . gently push the tube down the esophagus. palpate the tube within the esophagus. two tubes should be palpable, the orogastric tube, and the patient's trachea. push the tube down into the stomach. you can verify location by blowing into the proximal end of the tube and simultaneously auscultating the stomach for borborygmi. . insert the manual pump to the proximal end of the tube, and instill the warm water. alternate instilling water with removal of fluid and gastric debris by gravity. repeat the process until the efflux fluid is clear of any debris. . save fluid from the gastric efflux fluid for toxicologic analyses. hackett tb: emergency approach to intoxications, clin tech small anim pract ( ): - , . hypoxia, or inadequate tissue oxygenation, is the primary reason for supplemental oxygen therapy. major causes of hypoxia include hypoventilation, ventilation-perfusion mismatch, physiologic or right-to-left cardiac shunt, diffusion impairment, and decreased fraction of inspired oxygen (table - ) . inadequate tissue perfusion caused by low cardiac output or vascular obstruction also can result in circulatory hypoxia. finally, histiocytic hypoxia results from inability of cells to use oxygen that is delivered to them. this form of hypoxia can be observed with various toxin ingestions (bromethalin, cyanide) and in septic shock. a patient's oxygenation status can be monitored invasively by drawing of arterial blood gas samples or noninvasively through pulse oximetry, in most cases (see acid-base physiology and pulse oximetry). inspired air at sea level has a po of mm hg. as the air travels through the upper respiratory system to the level of the alveolus, the po drops to mm hg. tissue oxygen saturation in a normal healthy animal is mm hg. after oxygen has been delivered to the tissues, the oxygen left in the venous system (pvo ) is approximately mm hg. normally, oxygen diffuses across the alveolar capillary membrane and binds reversibly with hemoglobin in rbcs. a small amount of oxygen is carried in an unbound diffusible form in the plasma. when an animal has an adequate amount of hemoglobin and hemoglobin becomes fully saturated while breathing room air, supplemental oxygen administration will only increase the sao a small amount. the unbound form of oxygen dissolved in plasma will increase. if, however, inadequate hemoglobin saturation is obtained by breathing room air, as in a case of pneumonia or pulmonary edema, for example, breathing a higher fraction of inspired oxygen (fio ) will improve bound and unbound hemoglobin levels. the formula for calculating oxygen content of arterial blood is as follows: where cao is the arterial oxygen content, . is the amount of oxygen that can be carried by hemoglobin (hb), sao is the hemoglobin saturation, and . × pao is the amount of oxygen dissolved (unbound) in plasma. dissolved oxygen actually contributes little to the total amount of oxygen carried in the arterial blood, and the majority depends on the amount or availability of hemoglobin and the ability of the body (ph and respiratory status) to saturate the hemoglobin at the level of the alveoli. oxygen therapy is indicated whenever hypoxia is present. the underlying cause of the hypoxia also must be identified and treated, for chronic, lifelong oxygen therapy is rarely feasible in veterinary patients. if hemoglobin levels are low due to anemia, oxygen supplementation must occur along with rbc transfusions to increase hemoglobin mass. whenever possible, use arterial blood gas analyses or pulse oximetry to gauge a patient's response to oxygen therapy and to determine when an animal can be weaned from supplemental oxygen. the goal of oxygen therapy is to increase the amount of oxygen bound to hemoglobin in arterial blood. oxygen supplementation can be by hood, oxygen cage or tent, nasal or nasopharyngeal catheter, or tracheal tube. in rare cases, administration of oxygen with mechanical ventilation may be indicated. administration of supplemental oxygen to patients with chronic hypoxia is sometimes necessary but also dangerous. with chronic hypoxia the patient develops a chronic respiratory acidosis (elevated paco ) and depends almost entirely on the hypoxic ventilatory drive to breathe. administration of supplemental oxygen increases pao and may inhibit the central respiratory drive, leading to hypoventilation and possibly respiratory arrest. therefore, closely monitor animals with chronic hypoxia that are treated with supplemental oxygen. oxygen hoods can be purchased from commercial sources or can be manufactured in the hospital using a rigid elizabethan collar, tape, and plastic wrap. to make an oxygen hood, place several lengths of plastic wrap over the front of the elizabethan collar and tape them in place. leave the ventral third of the collar open to allow moisture and heat to dissipate and carbon dioxide to be eliminated. place a length of flexible oxygen tubing under the patient's collar into the front of the hood, and run humidified oxygen at a rate of to ml/kg/minute. animals may become overheated with an oxygen hood in place. carefully monitor the patient's temperature so that iatrogenic hyperthermia does not occur. commercially available plexiglass oxygen cages can be purchased from a variety of manufacturers. the best units include a mechanical thermostatically controlled compressor cooling unit, a circulatory fan, nebulizers or humidifiers to moisten the air, and a carbon dioxide absorber. alternately, a pediatric (infant) incubator can be purchased from hospital supply sources, and humidified oxygen can be run into the cage at to l/minute (depending on the size of the cage). high flow rates may be required to eliminate nitrogen and carbon dioxide from the cage. in most cases, the fio inside the cage reaches % to % using this technique. disadvantages of using an oxygen cage are high consumption/ use of oxygen, rapid decrease in the fio within the cage whenever the cage must be opened for patient treatments, lack of immediate access to the patient, and potential for iatrogenic hyperthermia. one of the most common methods for oxygen supplementation in dogs is nasal or nasopharyngeal oxygen catheters: . to place a nasal or nasopharyngeal catheter, obtain a red rubber catheter ( f to f, depending on the size of the patient). a. for nasal oxygen supplementation, measure the distal tip of the catheter from the medial canthus of the eye to the tip of the nose. b. for nasopharyngeal oxygen supplementation, measure the catheter from the ramus of the mandible to the tip of the nose. . mark the tube length at the tip of the nose with a permanent marker. . instill topical anesthetic such as proparacaine ( . %) or lidocaine ( %) into the nostril before placement. . place a stay suture adjacent to (lateral aspect) the nostril while the topical anesthetic is taking effect. . lubricate the tip of the tube with sterile lubricant. . gently insert the tube into the ventral medial aspect of the nostril to the level made with the permanent marker. if you are inserting the tube into the nasopharynx, push the nasal meatus dorsally while simultaneously pushing the lateral aspect of the nostril medially to direct the tube into the ventral nasal meatus and avoid the cribriform plate. . once the tube has been inserted to the appropriate length, hold the tube in place with your fingers adjacent to the nostril, and suture the tube to the stay suture. if the tube is removed, you can cut the suture around the tube and leave the stay suture in place for later use, if necessary. . suture or staple the rest of the tube dorsally over the nose and in between the eyes to the top of the head, or laterally along the zygomatic arch. . attach the tube to a length of flexible oxygen tubing, and provide humidified oxygen at to ml/kg/minute. . secure an elizabethan collar around the patient's head to prevent the patient from scratching at the tube and removing it. the rule of s states that if a patient's pao is less than mm hg, or if the paco is mm hg, mechanical ventilation should be considered. for mechanical ventilation, anesthetize the patient and intubate the patient with an endotracheal tube. alternately, a temporary tracheostomy can be performed and the patient can be maintained on a plane of light to heavy sedation and ventilated through the tracheostomy site. this method, a noninvasive means of determining oxygenation is through the use of pulse oximetry. a pulse oximeter uses different wavelengths of light to distinguish characteristic differences in the properties of the different molecules in a fluid or gas mixture, in this case, oxygenated (oxyhemoglobin) and deoxygenated hemoglobin (deoxyhemoglobin) in pulsatile blood. the process is termed pulse oximetry. oxyhemoglobin and deoxyhemoglobin are different molecules that absorb and reflect different wavelengths of light. oxyhemoglobin absorbs light in the infrared spectrum, allowing wavelengths of light in the red spectrum to transmit through it. conversely, deoxyhemoglobin absorbs wavelengths of the red spectrum and allows wavelengths in the infrared spectrum to transmit through the molecule. the spectrophotometer in the pulse oximeter transmits light in the red ( nanometers) and infrared ( nanometers) spectra. the different wavelengths of light are transmitted across a pulsatile vascular bed and are detected by a photodetector on the other side. the photodetector processes the amount of light of varying wavelengths that reaches it, then transmits an electrical current to a processor that calculates the difference in the amount of light originally transmitted and the amount of light of similar wavelength that actually reaches the photodetector. the difference in each reflects the amount of light absorbed in the pulsatile blood and can be used to calculate the amount or ratio of oxyhemoglobin to deoxyhemoglobin in circulation, or the functional hemoglobin saturation by the formula: where hbo is oxygenated hemoglobin, and hb is deoxygenated hemoglobin. four molecules of oxygen reversibly bind to hemoglobin for transport to the tissues. carbon monoxide similarly binds to hemoglobin and forms carboxyhemoglobin, a molecule that is detected similarly as oxygenated hemoglobin. thus sao as detected by a pulse oximeter is not reliable if carboxyhemoglobin is present. in most cases, pulse oximetry or sao corresponds reliably to the oxyhemoglobin dissociation curve. oxygen saturation greater than % corresponds to a pao greater than mm hg. above this value, large changes in pao are reflected in relatively small changes in sao , making pulse oximetry a relatively insensitive method of determining oxygenation status when pao is normal. because pulse oximetry measures oxygenated versus nonoxygenated hemoglobin in pulsatile blood flow, it is fairly unreliable when severe vasoconstriction, hypothermia, shivering or trembling, or excessive patient movement are present. additionally, increased ambient lighting and the presence of methemoglobin or carboxyhemoglobin also can cause artifactual changes in the sao , and thus the measurement is not reliable or accurate. most pulse oximeters also display a waveform and the patient's heart rate. if the photodetector does not detect a good quality signal, the waveform will not be normal, and the heart rate displayed on the monitor will not correlate with the patient's actual heart rate. the efficiency of ventilation is evaluated using the paco value on an arterial blood gas sample. alternatively, a noninvasive method to determine end-tidal carbon dioxide is through use of a capnograph. the science of capnometry uses a spectrophotometer to measure carbon dioxide levels in exhaled gas. the capnometer is placed in the expiratory limb of an anesthetic circuit. a sample of exhaled gas is aliquoted from the breath, and an infrared light source is passed across the sample. a photodetector on the other side of the sample flow measures the amount or concentration of carbon dioxide in the sample of expired gas. the calculated value is displayed as end-tidal carbon dioxide. this value also can be displayed as a waveform. when placed in graphic form, a waveform known as a capnograph is displayed throughout the ventilatory cycle. normally, at the onset of exhalation, the gas exhaled into the expiratory limb of the tubing comes from the upper airway or physiologic dead space and contains relatively little carbon dioxide. as exhalation continues, a steep uphill slope occurs as more carbon dioxide is exhaled from the bronchial tree. near the end of exhalation, the capnogram reaches a plateau, which most accurately reflects the carbon dioxide level at the level of the alveolus. because carbon dioxide diffuses across the alveolar basement membrane so rapidly, this reflects arterial carbon dioxide levels. if a plateau is not reached and notching of the waveform occurs, check the system for leaks. if the baseline waveform does not reach zero, the patient may be rebreathing carbon dioxide or may be tachypneic, causing physiologic positive end-expiratory pressure. the soda-sorb in the system should be replaced if it has expired. conversely, low end-tidal carbon dioxide may be associated with a decrease in perfusion or blood flow. decreased perfusion can be associated with low end-tidal carbon dioxide values, particularly during cardiopulmonary cerebral resuscitation. end-tidal carbon dioxide levels are one of the most accurate predictors of the efficacy of cardiopulmonary cerebral resuscitation and patient outcome. additionally, the difference between arterial carbon dioxide levels (paco ) and end-tidal carbon dioxide can be used to calculate dead-space ventilation. increases in the difference also occur with poor lung perfusion and pulmonary diffusion impairment. thoracocentesis refers to the aspiration of fluid or air from within the pleural space. thoracocentesis may be diagnostic to determine whether air or fluid is present and to characterize the nature of the fluid obtained. thoracocentesis also can be therapeutic when removing large volumes of air or fluid to allow pulmonary reexpansion and correction of hypoxemia and orthopnea. to perform thoracocentesis, follow this procedure: . first, assemble the equipment necessary (box - ). . next, clip a -cm square in the center of the patient's thorax on both sides. . aseptically scrub the clipped area. . ideally, thoracocentesis should be performed within the seventh to ninth intercostal space. rather than count rib spaces in an emergent situation, visualize the thoracic cage as a box, and the clipped area as a box within the box. you will insert your needle or catheter in the center of the box and then direct the bevel of the needle dorsally or ventrally to penetrate pockets of fluid or air present. . attach the needle or catheter hub to the length of intravenous extension tubing. attach the female port of the intravenous extension tubing to the male port of the three-way stopcock. attach the male port of the -ml syringe to one of the female ports of the three-way stopcock. the apparatus is now assembled for use. . insert the needle through the intercostal space such that the bevel of the needle initially is directed downward. . next, push down on the hub of the needle such that the needle becomes parallel with the thoracic wall. by moving the hub of the needle in a clockwise or counterclockwise manner, the bevel of the needle will move within the thoracic cavity to penetrate pockets of air or fluid. in general, air is located dorsally and fluid is located more ventrally, although this does not always occur. . aspirate air or fluid. save any fluid obtained for cytologic and biochemical analyses and bacterial culture and susceptibility testing. in cases of pneumothorax, if the thoracocentesis needs to be repeated more than times, consider using a thoracostomy tube. place a thoracostomy tube in cases of pneumothorax whenever negative suction cannot be obtained or repeated accumulation of air requires multiple thoracocentesis procedures. thoracostomy tubes also can be placed to drain rapidly accumulating pleural effusion and for the medical management of pyothorax. before attempting thoracostomy tube placement, make sure that all necessary supplies are assembled (box - ; table - ) . to place a thoracostomy tube, follow this procedure: . lay the patient in lateral recumbency. . clip the patient's entire lateral thorax. . aseptically scrub the lateral thorax. . palpate the tenth intercostal space. . have an assistant pull the patient's skin cranially and ventrally toward the point of the elbow. this will facilitate creating a subcutaneous tunnel around the thoracostomy tube. . draw up mg/kg % lidocaine ( mg/kg for cats) along with a small amount of sodium bicarbonate to take away some of the sting. . insert the needle at the dorsal aspect of the tenth intercostal space and to the seventh intercostal space. inject the lidocaine into the seventh intercostal space at the point where the trocarized thoracic drainage catheter will penetrate into the thoracic cavity. slowly infuse the lidocaine as you withdraw the needle to create an anesthetized tunnel through which to insert the catheter. . while the local anesthetic is taking effect, remove the trocar from the catheter and cut the proximal end of the catheter with a mayo scissors to facilitate adaptation with the christmas tree adapter. . attach the christmas tree adapter to the three-way stopcock and the three-way stopcock to a length of intravenous extension tubing and the -ml syringe so that the apparatus can be attached immediately to the thoracostomy tube after placement. . aseptically scrub the lateral thorax a second time and then drape it with sterile huck towels secured with towel clamps. . wearing sterile gloves, make a small stab incision at the dorsal aspect of the tenth intercostal space. . insert the trocar back into the thoracostomy drainage tube. insert the trocar and tube into the incision. tunnel the tube cranially for approximately intercostal spaces while an assistant simultaneously pulls the skin cranially and ventrally toward the point of the elbow. . at the seventh intercostal space, direct the trocar and catheter perpendicular to the thorax. grasp the catheter apparatus at the base adjacent to the thorax to prevent the trocar from going too far into the thorax. . place the palm of your dominant hand over the end of the trocar, and push the trocar and catheter into the thoracic cavity, throwing your weight into the placement in a swift motion, not by banging the butt of your hand on the end of the stylette. for small individuals, standing on a stool, or kneeling over the patient on the triage table can create leverage and make this process easier. the tube will enter the thorax with a pop. . gently push the catheter off of the stylette, and remove the stylette. . immediately attach the christmas tree adapter and have an assistant start to withdraw air or fluid while you secure the tube in place. . first, place a horizontal mattress suture around the tube to cinch the skin securely to the tube. use care to not penetrate the tube with your needle and suture. . next, place a purse-string suture around the tube at the tube entrance site. leave the ends of the suture long, so that you can create a finger-trap suture to the tube, holding the tube in place. . place a large square of antimicrobial-impregnated adhesive tape over the tube for further security and sterility. . if antimicrobial adhesive is not available, place a gauze pad × inches square over the tube, and then wrap the tube to the thorax with cotton roll gauze and elastikon adhesive tape. . draw the location of the tube on the bandage to prevent cutting it with subsequent bandage changes. an alternate technique to use if a trocar thoracic drainage catheter is not available is the following: . prepare the lateral thorax and infuse local lidocaine anesthetic as listed before. . make a small stab incision with a no. scalpel blade, as listed before. . obtain the appropriately sized red rubber catheter and cut multiple side ports in the distal end of the catheter, taking care to not cut more than % of the circumference of the diameter of the tube. . insert a rigid, long urinary catheter into the red rubber catheter to make the catheter more rigid during insertion into the pleural space. . grasp the distal end of the catheter(s) in the teeth of a large carmalt. tunnel a metzenbaum scissors under the skin to the seventh intercostal space and make a puncture through the intercostal space. . remove the metzenbaum scissors, and then tunnel the carmalt and red rubber tube under the skin to the hole created in the seventh intercostal space with the metzenbaum scissors. . insert the tips of the carmalt and the red rubber catheter through the hole, and then open the teeth of the carmalt. . push the red rubber catheter cranially into the pleural cavity. . remove the carmalt and the rigid urinary catheter, and immediately attach the suction apparatus. secure the red rubber catheter in place as listed before. placement of a temporary tracheostomy can be lifesaving to relieve upper respiratory tract obstruction, to facilitate removal of airway secretions, to decrease dead space ventilation, to provide a route of inhalant anesthesia during maxillofacial surgery, and to facilitate mechanical ventilation. in an emergent situation in which asphyxiation is imminent and endotracheal intubation is not possible, any cutting instrument placed into the trachea distal to the point of obstruction can be used. to perform a slash tracheostomy, quickly clip the fur and scrub the skin over the third tracheal ring. make a small cut in the trachea with a no. scalpel blade, and insert a firm tube, such as a syringe casing. alternately, insertion of a -gauge needle attached to intravenous extension tubing and adapted with a -ml syringe case to attach to a humidified oxygen source also temporarily can relieve obstruction until a temporary tracheostomy can be performed. in less emergent situations, place the patient under general anesthesia and intubate the patient. assemble all the equipment necessary before starting the temporary tracheostomy procedure (box - ). to perform a tracheostomy, follow this procedure: . place the patient in dorsal recumbency. . clip the ventral cervical region from the level of the ramus of the mandible caudally to the thoracic inlet and dorsally to midline. . aseptically scrub the clipped area, and then drape with sterile huck towels secured with towel clamps. . make a -cm ventral midline skin incision over the third to sixth tracheal rings, perpendicular to the trachea. . bluntly dissect through the sternohyoid muscles to the level of the trachea. . carefully pick up the fascia overlying the trachea and cut it away with a metzenbaum scissors. . place two stay sutures through/around adjacent tracheal rings. . incise in between trachea rings with a no. scalpel blade. take care to not cut more than % of the circumference of the trachea. . using the stay sutures, pull the edges of the tracheal incision apart, and insert the tracheostomy tube. the shiley tube contains an internal obturator to facilitate placement into the tracheal lumen. remove the obturator, and then insert the inner cannula, which can be removed for cleaning as needed. . once the tube is in place, secure the tube around the neck with a length of sterile umbilical tape. postoperative care of the tracheostomy tube is as important as the procedure itself. because the tracheostomy tube essentially bypasses the protective effects of the upper respiratory system, one of the most important aspects of tracheostomy tube care and maintenance is to maintain sterility at all times. any oxygen source should be humidified with sterile water or saline to prevent drying of the respiratory mucosa. if supplemental oxygen is not required, instill to ml of sterile saline every to hours to moisten the mucosa. wearing sterile gloves, remove the internal tube and place it in a sterile bowl filled with sterile hydrogen peroxide and to be cleaned every hours (or more frequently as necessary). if a shiley tube is not available, apply suction to the internal lumen of the tracheostomy tube every to hours (or more frequently as needed) with a sterile f red rubber catheter attached to a vacuum pump to remove any mucus or other debris that potentially could plug the tube. unless the patient demonstrates clinical signs of fever or infection, the prophylactic use of antibiotics is discouraged because of the risk of causing a resistant infection. after the temporary tracheostomy is no longer necessary, remove the tube and sutures, and leave the wound to heal by second intention. primary closure of the wounds could predispose the patient to subcutaneous emphysema and infection. baker gd: trans-tracheal oxygen therapy in dogs with severe respiratory compromise due to tick (i. holocyclus) toxicity, aust vet pract ( ) urohydropulsion is a therapeutic procedure for removal of uroliths from the urethra of the male dog. the technique works best if the animal is heavily sedated or is placed under general anesthesia (figure - ) . to perform urohydropulsion, follow this procedure: . place the animal in lateral recumbency. . clip the fur from the distal portion of the prepuce. . aseptically scrub the prepuce and flush the prepuce with to ml of antimicrobial flush solution. . have an assistant who is wearing gloves retract the penis from the prepuce. . while wearing sterile gloves, lubricate the tip of a rigid urinary catheter as for urethral catheterization. . gently insert the tip of the catheter into the urethra until you meet the resistance of the obstruction. . pinch the tip of the penis around the catheter. . have an assistant insert a gloved lubricated finger into the patient's rectum and press ventrally on the floor of the rectum to obstruct the pelvic urethra. . attach a -ml syringe filled with sterile saline into proximal tip of the catheter. . quickly inject fluid into the catheter and alternate compression and relaxation on the pelvic urethra such that the urethra dilates and suddenly releases the pressure, causing dislodgement of the stone. small stones may be ejected from the tip of the urethra, whereas larger stones may be retropulsed back into the urinary bladder to be removed surgically at a later time. the type of catheter that you choose for vascular access depends largely on the size and species of the patient, the fragility of the vessels to be catheterized, the proposed length of time that the catheter will be in place, the type and viscosity of the fluid or drug to be administered, the rate of fluid flow desired, and whether multiple repeated blood samples will be required (table - ) . a variety of over-the-needle, through-the-needle, and over-the-wire catheters are available for placement in a variety of vessels, including the jugular, cephalic, accessory cephalic, medial saphenous, lateral saphenous, dorsal pedal artery, and femoral artery. one of the most important aspects of proper catheter placement and maintenance is to maintain cleanliness at all times. the patient's urine, feces, saliva, and vomit are common sources of contamination of the catheter site. before placing a peripheral or central catheter in any patient, consider the patient's physical status including whether vomiting, diarrhea, excessive urination, or seizures. in a patient with an oral mass that is drooling excessively or a patient that is vomiting, peripheral cephalic catheterization may not be the most appropriate, to prevent contamination. conversely, in a patient with excessive urination or diarrhea, a lateral or medial saphenous catheter is likely to become contaminated quickly. whenever one places or handles a catheter or intravenous infusion line, the person should wash the hands carefully and wear gloves to prevent contamination of the intravenous catheter and fluid lines. one of the most common sources of catheter contamination in veterinary hospitals is through caretakers' hands. in emergent situations, placement of a catheter may be necessary under less than ideal circumstances. remove those catheters as soon as the patient is more stable, and place a second catheter using aseptic techniques. in general, once the location of the catheter has been decided, set up all equipment necessary for catheter placement before starting to handle and restrain the patient. lists the equipment needed for most types of catheter placement. after setting up all of the supplies needed, clip the fur over the site of catheter placement. make sure to clip all excess fur and long feathers away from the catheter site, to prevent contamination. for catheter placement in limbs, clip the fur circumferentially around the site of catheter placement to facilitate adherence of the tape to the limb and to facilitate catheter removal with minimal discomfort at a later date. next, aseptically scrub the catheter site with an antimicrobial scrub solution such as hibiclens. the site is now ready for catheter insertion. consider using a central venous catheter whenever multiple repeated blood samples will need to be collected from a patient during the hospital stay. central venous catheters also can be used for cvp measurement, administration of hyperoncotic solutions such as parenteral nutrition, and administration of crystalloid and colloid fluids, anesthesia, and other injectable drugs (figures - and - ) . to place a jugular central venous catheter, place the patient in lateral recumbancy and extend the head and neck such that the jugular furrow is straight. clip the fur from the ramus of the mandible caudally to the thoracic inlet and dorsally and ventrally to midline. wipe the clipped area with gauze × -inch squares to remove any loose fur and other debris. aseptically scrub the clipped area with an antimicrobial cleanser. venocaths (abbott laboratories) are a through-the-needle catheter that is contained within a sterile sleeve for placement. alternately, other over-the-wire central venous catheters can be placed by the seldinger technique. sterility must be maintained at all times, regardless of the type of catheter placed. wearing sterile gloves, drape the site of catheter placement with sterile drapes, and occlude the jugular vein at the level of the thoracic inlet. pull the clear ring and wings of emergency diagnostic and therapeutic procedures figure - : lateral thoracic radiograph of a central venous catheter. note that the tip of the catheter is inserted in its proper location, just outside of the right atrium. the catheter cover down toward the catheter itself to expose the needle. remove the guard off of the needle. lift the skin over the proposed site of catheter insertion and insert the needle under the skin, with the bevel of the needle facing up. next, reocclude the vessel and pull the skin tight over the vessel to prevent movement of the vessel as you attempt to insert the needle. in some cases, it may be difficult actually to see the vessel in obese patients. if you cannot visualize or palpate the needle, gently bounce the needle over the vessel with the bevel up. the vessel will bounce in place slightly, allowing a brief moment of visualization to facilitate catheter placement. once the vessel has been isolated and visualized, insert the needle into the vessel at a -to -degree angle. watch closely for a flash of blood in the catheter. when blood is observed, insert the needle a small distance farther, and then push the catheter and stylette into the vessel for the entire length, until the catheter and stylette can be secured in the catheter hub. if the catheter cannot be inserted fully into the vessel for its entire length, the tip of the needle may not be within the entire lumen, the catheter may be directed perivascularly, and the catheter may be caught at the thoracic flexure and may be moving into one of the tributaries that feeds the forelimb. extend the patient's head and neck, and lift the forelimb up to help facilitate placement. do not force the catheter in because the catheter potentially can form a knot and will need to be removed surgically. remove the needle from the vessel, and have an assistant place several × -inch gauze squares over the site of catheter placement with some pressure to control hemorrhage. secure the catheter hub into the needle guard, and remove the stylette from the catheter. immediately insert a -to -ml syringe of heparinized saline and flush the catheter and draw back. if you are in the correct place, you will be able to draw blood from the catheter. to secure the catheter in place, tear a length of -inch white tape that will wrap around the patient's neck. pull a small length of the catheter out of the jugular vein to make a semicircle. the semicircle should be approximately / inch in diameter. let the length of catheter lie on the skin, and then place × -inch gauze squares impregnated with antimicrobial ointment over the site of catheter insertion. secure the proximal end of white tape around the white and blue pieces of the catheter, and wrap the tape around the patient's neck so that the tape adheres to the skin and fur. repeat the process by securing the gauze to the skin with two additional lengths of white tape, starting to secure the gauze in place by first wrapping the tape dorsally over the patient's neck, rather than under the patient's neck. in between each piece of tape and bandage layer, make sure that the catheter flushes and draws back freely, or else occlusion can occur. gently wrap layers of cotton roll gauze, kling, and elastikon or vetrap over the catheter. secure a male adapter or t port that has been flushed with heparinized saline, and then label the catheter with the size and length of catheter, date of catheter placement, and initials of the person who placed the catheter. the catheter is ready for use. monitor the catheter site daily for erythema, drainage, vessel thickening, or pain upon infusion. if any of these signs occur, or if the patient develops a fever of unknown origin, remove the catheter, culture the catheter tip aseptically, and replace the catheter in a different location. as long as the catheter is functional without complications, the catheter can remain in place. central catheters also can be placed via the seldinger or over-the-wire technique. a number of companies manufacture kits that contain the supplies necessary for over-the-wire catheter placement. each kit minimally should contain an over-the-needle catheter to place into the vessel, a long wire to insert through the original catheter placed, a vascular dilator to dilate the hole in the vessel created by the first catheter, and a long catheter to place into the vessel over the wire. additional accessories can include a paper drape, sterile gauze, a scalpel blade, local anesthetic, -gauge needles, and -or -ml syringes. restrain the patient and prepare the jugular furrow aseptically as for the percutaneous through-the-needle catheter placement. the person placing the catheter should wear sterile gloves throughout the process to maintain sterility. pick up the skin over the site of catheter placement, and insert a small bleb of local anesthetic through the skin. the local anesthetic should not be injected into the underlying vessel (figure - ) . make a small nick into the skin through the local anesthetic with a no. or no. scalpel blade. use care to avoid lacerating the underlying vessel. next, occlude the jugular vein as previously described, and insert the over-the-needle catheter into the vessel. watch for a flash of blood in the catheter hub. remove the stylette from the catheter. next, insert the long wire into the catheter and into the vessel (figures - and - ) . never let go of the wire. remove the catheter, and place the vascular dilator over the wire and into the vessel (figure - ) . gently twist to place the dilator into the vessel a short distance, creating a larger hole in the vessel. the vessel will bleed more after creating a larger hole. remove the vascular dilator, and leave the wire in place within the vessel. insert the long catheter over the wire into the vessel (figure - ) . push the catheter into the vessel to the catheter hub (figure - ) . slowly thread the wire through a proximal port in the catheter. once the catheter is in place, remove the wire, and suture the catheter in place to the skin with nonabsorbable suture. cover the catheter site with sterile gauze and antimicrobial ointment, cotton roll bandaging material, gauze, and kling or vetrap. flush the catheter with heparinized saline solution, and then use the catheter for infusion of parenteral nutrition, blood products, crystalloid and colloid fluids, medications, and frequent blood sample collection. examine the catheter site daily for evidence of infection or thrombophlebitis. the catheter can remain in place as long as it functions and no complications occur. place the patient in sternal recumbency as for cephalic venipuncture. clip the antebrachium circumferentially, and wipe the area clean of any loose fur and debris (figure - ) . aseptically scrub the clipped area, and have an assistant occlude the cephalic vein at the crook of the elbow. the person placing the catheter should grasp the distal carpus with the nondominant hand and insert the over-the-needle catheter into the vessel at a -to -degree angle ( figure - ) . watch for a flash of blood in the catheter hub, and then gently push the catheter off of the stylette (figure - ) . have the assistant occlude the vessel over the catheter to prevent backflow. flush the catheter with heparinized saline solution. make sure that the skin and catheter hub are clean and dry to ensure that the tape adheres to the catheter hub and skin. secure a length of / -inch white tape tightly around the catheter and then around the limb. make sure that the catheter hub does not "spin" in the tape, or else the catheter will fall out. next, secure a second length of -inch adhesive tape under the catheter and around the limb and catheter hub (figure - ). this piece of tape helps to stabilize the catheter in place. finally, place a flushed t port or male adapter in the catheter hub and secure to the limb with white tape. make sure that the tape is adhered to the skin securely, but not so tightly as to impede venous outflow (figure - ) . the catheter site can be covered with a cotton ball impregnated with antimicrobial ointment and layers of bandage material. label all catheters with the date of placement, the type and gauge of catheter inserted, and the initials of the person who placed the catheter. the femoral artery can be catheterized for placement of an indwelling arterial catheter. indwelling arterial catheters can be used for continuous invasive arterial blood pressure monitoring and for procurement of arterial blood samples. place the patient in lateral recumbancy, and tape the down leg in an extended position. clip the fur over the femoral artery and aseptically scrub the clipped area. palpate the femoral artery as it courses distally on the medial surface of the femur and anterior to the pectineus muscle. make a small nick incision over the proposed site of catheter placement using the bevel of an -gauge needle. place a long over-the-needle catheter through the nick in the skin and direct it toward the palpable pulse. place the tip of the catheter so that the needle tip rests in the subcutaneous tissue between the artery and the palpating index finger. advance the needle steeply at a -degree angle to secure the superficial wall of the vessel and then the deep wall of the vessel. the spontaneous flow of blood in the catheter hub ensures that the catheter is figure - : catheter is taped in place with a t-port. situated in the lumen of the artery. feed the catheter off of the stylette, and cover the hub with a catheter cap. flush the catheter with sterile heparinized saline solution, and then secure it in place. some persons simply tape the catheter in place with pieces of / -and -inch adhesive tape. others use a "butterfly" piece of tape around the catheter hub and suture or glue the tape to the adjacent skin for added security. the dorsal pedal artery commonly is used for catheter placement. to place a dorsal pedal arterial catheter, place the patient in lateral recumbency. clip the fur over the dorsal pedal artery, and then aseptically scrub the clipped area. tape the distal limb so that the leg is twisted slightly medially for better exposure of the vessel, or the person placing the arterial catheter can manipulate the limb into the appropriate position. palpate the dorsal pedal pulse as it courses dorsally over the tarsus. place an over-the-needle catheter percutaneously at a -to -degree angle, threading the tip of the needle carefully toward the pulse. advance the needle in short, blunt movements, and watch the catheter hub closely for a flash of pulsating blood that signifies penetration into the lumen of the artery. then thread the catheter off of the stylette, and cover the catheter hub with a catheter cap. secure the catheter in place with lengths of / -and -inch adhesive tape as with any other intravenous catheter, and then flush it with heparinized saline solution every to hours. any vessel that can be catheterized percutaneously also can be catheterized with surgical cutdown. restrain the patient and clip and aseptically scrub the limb or jugular vein as for a percutaneous catheterization procedure. block the area for catheter placement with a local anesthetic before cutting the skin over the vessel with a no. scalpel blade. while wearing sterile gloves, pick up the skin and incise the skin over the vessel. direct the sharp edge of the blade upward to avoid lacerating the underlying vessel. using blunt dissection, push the underlying subcutaneous fat and perivascular fascia away from the vessel with a mosquito hemostat. make sure that all tissue is removed from the vessel. using the mosquito hemostat, place two stay sutures of absorbable suture under the vessel. elevate the vessel until it is parallel with the incision, and gently insert the catheter and stylette into the vessel. secure the stay sutures loosely around the catheter. suture the skin over the catheter site with nonabsorbable suture, and then tape and bandage the catheter in place as for percutaneous placement. remove catheters placed surgically as soon as possible and exchange them for a percutaneously placed catheter to avoid infection and thrombophlebitis. the most important aspect of catheter maintenance is to maintain cleanliness and sterility at all times. an indwelling catheter can remain in place for as long as it is functional and no complications occur. change the bandage whenever it becomes wet or soiled to prevent wicking of bacteria and debris from the environment into the vessel. check the bandages and catheter sites at least once a day for signs of thrombophlebitis: erythema, vessel hardening or ropiness, pain upon injection or infusion, and discharge. also closely examine the tissue around and proximal and distal to the catheter. swelling of the paw can signify that the catheter tape and bandage are too tight and are occluding venous outflow. swelling above the catheter site is characteristic of perivascular leakage of fluid and may signify that the catheter is no longer within the lumen of the vessel. remove the catheter if it is no longer functional, if there is pain or resistance upon infusion, if there is unexplained fever or leukocytosis, or if there is evidence of cellulitis, thrombophlebitis, or catheter-related bacteremia or septicemia. aseptically culture the tip of the indwelling catheter for bacteria. animals should wear elizabethan collars or other forms of restraint if they lick or chew at the catheter or bandage. catheter patency may be maintained with constant fluid infusion or by intermittent flushing with heparinized saline ( units of unfractionated heparin per to ml of saline) every hours. flush arterial catheters more frequently (every hours). disconnect intravenous connections only when absolutely necessary. wear gloves whenever handling the catheter or connections. label all fluid lines and elevate them off of the floor to prevent contamination. date each fluid line and replace it once every to hours. if an intravenous catheter cannot be placed because of small patient size, hypovolemia, hypothermia, or severe hypotension, needles can be placed into the marrow cavity of the femur, humerus, and tibia for intraosseous infusion of fluids, drugs, and blood products. this technique is particularly useful in small kittens and puppies and in exotic species. contraindications to intraosseous infusion is in avian species (which have air in their bones), fractures, and sepsis, because osteomyelitis can develop. an intraosseous catheter is relatively easy to place and maintain but can cause patient discomfort and so should be changed to an intravenous catheter as soon as vascular access becomes possible. to place an intraosseous catheter, clip and aseptically scrub the fur over the proposed site of catheter placement. the easiest place for intraosseous placement is in the intertrochanteric fossa of the femur. inject a small amount of a local anesthetic through the skin and into the periosteum where the trocar or needle will be inserted. place the patient in lateral recumbency, and grasp the leg in between your fingers, with the stifle braced against the palm of your hand. push the stifle toward the abdomen (medially) to abduct the proximal femur away from the body. this will shift the sciatic nerve out of the way of catheter placement. insert the tip of the needle through the skin and into the intertrochanteric fossa. gently push with a simultaneous twisting motion, pushing the needle parallel with the shaft of the femur, toward your palm. you may feel a pop or decreased resistance as the needle enters the marrow cavity. gently flush the needle with heparinized saline. if the needle is plugged with bone debris, remove the needle and replace it with a fresh needle of the same type and size in the hole that you have created. a spinal needle with an internal stylette also can be placed. the stylette will prevent the needle from becoming clogged with bone debris during insertion. secure the hub of the needle with a butterfly length of white adhesive tape and then suture it to the skin to keep the catheter in place. the catheter is now ready for use. the patient should wear an elizabethan collar to prevent disruption or removal of the catheter. the intraosseous catheter can be maintained as any peripheral catheter, with frequent flushing and daily evaluation of the catheter site. the definition of pain has been debated philosophically over the ages and has changed as knowledge has increased. pain is defined as an unpleasant sensory or emotional experience associated with actual or perceived tissue damage. until recognition of a noxious stimulus occurs in the cerebral cortex, no response or adaptation results. rational management of pain requires an understanding of the underlying mechanisms involved in pain and an appreciation of how analgesic agents interact to disrupt pain mechanisms. multiple factors and causes produce pain in human beings and domestic animal species. the causes of pain, psychological and physical, may derive from many different mechanisms within emergency medicine, among them trauma, infectious disease, neglect, environmental stress, surgery, and acute decompensation of chronic medical conditions. the two major classes of pain are acute and chronic pain. box - gives specific categories and causes of pain. the pain sensing and response system can be divided into the following categories: nociceptors, which detect and filter the intensity of the noxious stimuli; primary afferent nerves, which transmit impulses to the central nervous system (cns); ascending tracts, which are part of the dorsal horn and the spinal cord that conveys stimuli to higher centers in the brain; higher centers, which are involved in pain discrimination, some memory, and motor control; and modulating or descending systems, which are a means of processing, memorizing, and modifying incoming impulses. current analgesic therapies may inhibit afferent nociceptive transmission within the brain and spinal cord; directly interrupt neural impulse conduction through the dorsal horn, primary afferent nerves, or dorsal root ganglion; or prevent the nociceptor sensitization that accompanies initial pain and inflammation. the physiologic aspects of pain are believed to be produced by the transmission, transduction, and integration of initial nerve endings, peripheral neuronal input, and ascending afferent nerves via the thalamus to the cerebral cortex. ascending afferent nerves to the limbic system are believed to be responsible for the emotional aspects of pain. there are several classification schemes for different types of pain. acute pain, such as that which results from trauma, surgery, or infectious agents, is abrupt in onset, relatively short in duration, and may be alleviated easily by analgesics. in contrast, chronic pain is a long-standing physical disorder or emotional distress that is slow in onset and difficult to treat. both types of pain can be classified further based on site of origin. somatic pain arises from superficial skin, subcutaneous tissue, body wall, or appendages. visceral pain arises from abdominal or thoracic viscera and primarily is associated with serosal irritation. analgesia, then, is the loss of pain without the loss of consciousness. this is in contrast to anesthesia, which is the loss of sensation in the whole body or a part of the body with the loss of consciousness or at least depression of the cns. untreated pain causes immediate changes in the neurohormonal axis, which in turn causes restlessness, agitation, increased heart and respiratory rates, fever, and blood pressure fluctuations, all of which are detrimental to the healing of the animal. a catabolic state is created as a result of increased secretion of catabolic hormones and decreased secretion of anabolic hormones. the net effect the majority of neurohormonal changes produce is an increase in the secretion of catabolic hormones. hyperglycemia is produced and may persist because of production of glucagon and relative lack of insulin. lipolytic activity is stimulated by cortisol, catecholamines, and growth hormone. cardiorespiratory effects of pain include increased cardiac output, vasoconstriction, hypoxemia, and hyperventilation. protein catabolism is a common occurrence and major concern regarding healing. pain associated with inflammation causes increase in tissue and blood levels of prostaglandins and cytokines, both of which promote protein catabolism indirectly by increasing the energy expenditure of the body. powerful evidence indicates that local anesthetic, sympathetic agonist, and opioid neural blockade may produce a modification of the responses to these physiologic changes. variable reduction in plasma cortisol, growth hormone, antidiuretic hormone, β-endorphin, aldosterone, epinephrine, norepinephrine, and renin is based on the anesthetic technique and the drugs selected. prophylactic administration of analgesics blunts the response before it occurs; analgesics administered following perception or pain are not as effective, and higher doses are generally necessary to achieve an equivalent level of analgesia. effective pain control can be achieved only when the signs of pain can be assessed effectively, reliably, and regularly. the experience of pain is unique to each individual, which makes pain assessment difficult, especially in traumatized and critical patients. most attempts to assess clinical pain use behavioral observations and interactive variables in addition to assessment of physiologic responses such as heart rate and respiratory rate, blood pressure, and temperature. but many factors can influence the processing and outward projection of pain, including altered environments, species differences, withinspecies variations (age, breed, sex), and the type, severity, and chronicity of pain. within-species differences (age, breed, and sex) further complicate the pain assessment. most notable is that different breeds of dogs act differently when confronted with pain or fear. labrador retrievers tend to be stoic, whereas greyhounds and teacup breeds tend to react with a heightened state of arousal around even the simplest of procedures (e.g., subcutaneous injections and nail trims). the individual character and temperament of the animal further influences its response. pediatric and neonatal animals seem to have a lower threshold for pain and anxiety than older animals. in any species, the duration and type of pain make it more (acute) or less (chronic) likely to be expressed or exhibited outwardly. unfamiliarity with normal behaviors typical of a particular species or breed makes recognition of their painful behaviors and responses impossible. the definition and recognition of pain in an individual animal is challenging. because of all the differences discussed, there is no straight line from insult, albeit actual or perceived, to degree of pain experienced. nor is there a formula for treating "x" type of pain with "y" type of analgesic. a goal of analgesia is to treat all animals with analgesic drugs and modalities as preemptively as possible and using a multimodal approach. use analgesic treatment as a tool for diagnosis of pain in the event that recognition of these phenomena is difficult for the patient. in other words, with countless drugs and treatment modalities available, analgesic administration should never be withheld in an animal, even if pain is questionable. it is important to remember that no behavior or physiologic variable in and of itself is pathognomonic for pain. interactive and unprovoked (noninteractive) behavior assessments and trending of physiologic data are useful to determine the pain in an individual animal. this is known as pain scoring. baseline observations, especially those observations from someone who has known the animal well, can be helpful to serial behavior and pain assessments. pain scoring systems have been developed and are reviewed elsewhere; the purposes of these systems are to evaluate and to help guide diagnostic and analgesic treatments (table - ) . regardless of the scale or method used to assess pain, the caregiver must recognize the limitations of the scale. if in doubt of whether pain is present or not, analgesic therapy should be used as a diagnostic tool. classic behaviors associated with pain in dogs and cats include abnormal postures, gaits, movements, and behaviors (boxes - and . stoicism is the apparent apathy and pain: assessment, prevention, and management indifference in the presence of pain and is perhaps the no. sign of ineffective pain relief or persistent pain in many animals, because so many display apathy and classically normal physiologic parameters even in the face of severe distress, overt suffering, or blatant trauma and illness. the absence of normal behaviors is also a clinical sign of pain, even when abnormal behaviors are not observed. acute pain results in many of the aforementioned behavioral and physiologic signs, but chronic pain in small animals is an entirely different and distinct entity. chronic pain is often present in the absence of obvious tissue pathology and changes in physical demeanor. again, the severity of the pain may not correlate with the severity of any pathologic condition that may or may not be present. chronic pain, especially if insidious in onset (cancer, dental, or degenerative pain), may well go unnoticed in dogs and cats, even by family members or intermittent caregivers. inappetance, lack of activity, panting in a species classically designed to be nose breathers, decreased interest in surroundings, different activity patterns, and abnormal postures are just a few signs of chronic pain in cats and dogs. cats are a species that in particular are exemplary in their abilities to hide chronic pain. they will exhibit marked familial withdrawal, finding secluded areas where they may remain for days to weeks when they experience acute and chronic pain. when deciding on a pain management protocol for a patient, always perform a thorough physical examination and include a pain score assessment before injury and pain has occurred, whenever possible. form a problem list to guide your choice of anesthesia and analgesia. for example, using a nonsteroidal antiinflammatory drug (nsaid) in an animal with renal failure would not be wise. remember to account for current medications that the patient may be taking that may augment or interfere with the analgesic or anesthetic drugs. use multimodal techniques and regional therapy and drugs to target pain at different sites before it occurs. once a strategy is decided upon, frequently reassess the patient and tailor the protocol to meet each patient's response and needs. drug therapy (in particular, opioids with or without α -agonists) is a cornerstone for acute pain treatment and surgical preemptive pain prevention. however, local anesthetics delivered epidurally, via perineural or plexus injection, intraarticular or trigger point injection, are also effective analgesics for acute and chronic forms of pain and inflammation. the nsaids that classically have been reserved for treatment of more chronic or persistent pain states now are being used regularly for treatment of acute and perioperative pain once blood pressure, coagulation, and gastrointestinal parameters have been normalized. an opioid is any natural or synthetic drug that is derived from the poppy, which interacts with opiate receptors identified on cell membranes. the drugs from this class constitute the most effective means of controlling acute, perioperative, and chronic pain in human and veterinary medicine (table - ) . their physiologic effects result from the interaction with one or more of at least five endogenous opioid receptors (µ, σ, δ, ε, and κ). µ-receptor agonists are noted for their ability to produce profound analgesia with mild sedation. these drugs diminish "wind-up," the hyperexcitable state resulting from an afferent volley of nociceptive impulses. they elevate the pain threshold and are used preemptively to prevent acute pain. as a class, opioids cause cns depression with their intense analgesia. dose-related respiratory depression reflects diminished response to carbon dioxide levels. cardiac depression is secondary only to bradycardia and is more likely with certain opioids such as morphine and oxymorphone. narcotics produce few if any clinically significant cardiovascular effects in dogs and cats; they are considered cardiac soothing or sparing. because opioids increase intracranial and intraocular pressure, use them more cautiously in patients with severe cranial trauma and or ocular lesions. opioids directly stimulate the chemoreceptor trigger zone and may cause nausea and vomiting. most opioids depress the cough reflex via a central mechanism; this may be helpful in patients recovering from endotracheal intubation irritation. a key characteristic of opioids that makes them desirable for use in emergency and critical care situations is their reversibility. antagonists block or reverse the effect of agonists by combining with receptors and producing minimal or no effects. administer all reversal agents, such as naloxone and naltrexone, slowly if given intravenously and to effect. α α -agonists as a class of drugs, α -agonists warrant special attention because most members of the group possess potent analgesic power at doses that are capable of causing sedation, cns depression, cardiovascular depression, and even general anesthetic states. originally developed for antihypertensive use, α -agonists quickly have attained sedative analgesic status in veterinary medicine (table - ) . like the opioids, α -agonists produce their effects by aggravating α-adrenergic receptors in the cns and periphery. emergency care among them cyclooxygenase- (cox- ), the major constitutive enzyme primarily involved in normal physiologic functions, and cox- , the enzyme responsible for most of the hyperalgesia and pain responses experienced after tissue injury or trauma. some nsaids inhibit cyclooxygenase and lipoxygenase activity. most of the currently available oral and parenteral nsaids for small animal medicine and surgery target the cyclooxygenase pathways predominantly, although one (tepoxalin) is thought to inhibit both pathways. inhibition of cox- and cox- can inhibit the protective effects and impair platelet aggregation and lead to gastrointestinal ulceration. there are definite contraindications and relative contraindications for the use of nsaids. nonsteroidal antiinflammatory drugs should not be administered to patients with renal or hepatic insufficiency, dehydration, hypotension or conditions that are associated with low circulating volume (congestive heart failure, unregulated anesthesia, shock), or evidence of ulcerative gastrointestinal disease. trauma patients should be stabilized completely regarding vascular volume, tone, and pressure before the use of nsaids. patients receiving concurrent administration of other nsaids or corticosteroids, or those considered to be cushingoid, should be evaluated carefully for an adequate "washout" period (time of clearance of drug from the system) before use of an nsaid or before switching nsaids. patients with coagulopathies, particularly those that are caused by platelet number or function defects or those caused by factor deficiencies, and patients with severe, uncontrolled asthma or other bronchial disease are probably not the patients in which to use nsaids. other advice is that nsaids not be administered to pregnant patients or to females attempting to become pregnant because cox- induction is necessary for ovulation and subsequent implantation of the embryo. the administration of nsaids should be considered only in the well-hydrated, normotensive dog or cat with normal renal or hepatic function, with no hemostatic abnormalities, and no concurrent steroid administration. nonsteroidal antiinflammatory drugs can be used in many settings of acute and chronic pain and inflammation. among these are the use in well-stabilized musculoskeletal trauma and surgical pain, osteoarthritis management, meningitis, mastitis, animal bite and other wound healing, mammary or transitional cell carcinoma, epithelial (dental, oral, urethral) inflammation, ophthalmologic procedures, and dermatologic or otic disease. whereas opioids seem to have an immediate analgesic effect when administered, most nsaids will take up to minutes for their effect to be recognized. as such, most perioperative or acute nsaids use is part of a balanced pain management scheme, one that uses narcotics and local anesthetic techniques. nonsteroidal antiinflammatory drugs are devoid of many of the side effects of narcotic administration; namely, decreased gastrointestinal motility, altered sensorium, nausea/vomition, and sedation. nonsteroidal antiinflammatory drugs are also devoid of many of the side effects of steroid administration; namely, suppression of the pituitary adrenal axis. the toxic effects of salicylates in cats are well documented. cats are susceptible because of slow clearance and dose-dependent elimination because of deficient glucuronidation in this species. because of this, the dose and the dosing interval of most commonly used nsaids need to be altered in order for these drugs to be used. cats that have been given canine doses of nsaids (twice daily or even once daily repetitively) may show hyperthermia, hemorrhagic or ulcerative gastritis, kidney and liver injury, hyperthermia, respiratory alkalosis, and metabolic acidosis. acute and chronic toxicities of nsaids have been reported in cats, especially after repeat once daily dosing. ketoprofen, flunixin, aspirin, carprofen, and meloxicam have been administered safely to cats, although like most antibiotics and other medications, they are not approved and licensed for use in cats. an important note, though, is that dosing intervals ranging from to hours have been used, and antithrombotic effects often can be achieved at much lower doses than those required to treat fevers and inflammation. i recommend the use of no loading doses, minimum -hour dosing intervals, and assurance of adequate circulating blood volume, blood pressure, and renal function. because many of the nsaids are used off-label in cats, it is imperative that the clinician carefully calculate the dose, modify the dosing interval, and communicate this information to the client before dispensing the drug. even drugs that come in liquid form (meloxicam), if administered to cats via box-labeled directions used for dogs, will be given in near toxic doses. to worsen the misunderstanding about dosages for cats, drops from manufacturer's bottles often are calibrated drops; when these same liquids are transferred into pharmacy syringes for drop administration, the calibration of course is lost, and the animal potentially is overdosed. a more accurate method of dispensing and administering oral nsaids in cats is to calculate the dose in milligrams and determine the exact number of milliliters to administer, rather than use the drop method. ketamine classically was considered a dissociative anesthetic, but it also has potent activity as an n-methyl-d-aspartate (nmda) receptor antagonist. this receptor located in the cns mediates windup and central sensitization (a pathway from acute to chronic pain). blockade of this receptor with microdoses of ketamine results in the ability to provide body surface, somatic, and skin analgesia with potentially lower doses of opioids and α-agonists. loading doses of . to mg/kg are used intravenously with continuous rate infusions of to µg/kg/minute. in and of itself, this drug possesses little to no analgesic ability and indeed in high doses alone often can aggravate, sensitize, or excite the animal in subacute or acute pain. amantadine is another nmda blocker that has been used for its antiviral and parkinson's stabilizing effects. amantadine has been used for neuropathic pain in human beings but is only available in an oral form. suggested starting doses for cats and dogs range from to mg/kg po daily. when the drug is given orally and intravenously, patients are unlikely to develop behavioral or cardiorespiratory effects with ketamine or amantadine. tramadol is an analgesic that possesses weak opioid µ-agonist activity and norepinephrine and serotonin reuptake inhibition. tramadol is useful for mild to moderate pain in small animals. although the parent compound has very weak opioid activity, the metabolites have excellent binding affinity for the µ-receptor. tramadol has been used for perisurgical pain control when given orally in cats and dogs at a dose of to mg/kg po sid to bid. cats appear to require only once daily dosing. regardless of its affinity for the opioid receptors, the true mechanism of action of tramadol in companion animals remains largely unknown. gabapentin is a synthetic analog of γ-aminobutyric acid (gaba). originally introduced as an antiepileptic drug, the mechanism of action of gabapentin remains somewhat unclear in veterinary medicine. the drug is among a number of commonly used antiepileptic medications used to treat central pain in human beings. the rationale for use is the ability of the drugs to suppress discharge in pathologically altered neurons. gabapentin does this through calcium channel modulation without binding to glutamate receptors. chronic, burning, neuropathic, and lancinating pain in small animals responds well to to mg/kg po daily. local anesthetic agents are the major class used as a peripheral-acting analgesic ( table - ) . local anesthetics block the transmission of pain impulses at the peripheral nerve nociceptor regions. local anesthetics may be used to block peripheral nerves or inhibit nerve "zones" using regional techniques. although all local anesthetics are capable of providing pain relief, agents with a longer duration of action are preferred for pain management purposes. bupivacaine is an example of a long-acting local anesthetic drug that is used along with lidocaine for long-acting pain relief. a single dose of bupivacaine injected at a local site will provide local anesthesia and analgesia for to hours. when lidocaine is administered as an intravenous constant rate infusion ( to µg/kg/minute in dogs, to µg/kg/minute in cats) is effective in the treatment of chronic neuropathic pain and periosteal and peritoneal pain (e.g., pancreatitis). mexiletine, an oral sodium channel blocker, can be used as an alternative to injectable lidocaine for provision of background analgesia. many drugs (table - ) are used in combination with opioids, α -agonists, and ketamine to provide anxiolysis and sedation. injection of local anesthetic solution into the connective tissue surrounding a particular nerve produces loss of sensation (sensory blockade) and/or paralysis (motor nerve blockade) in the region supplied by the nerve. local anesthetics also may be administered epidurally, intrathoracically, intraperitoneally, and intraarticularly. lidocaine and bupivacaine are the most commonly administered local anesthetics. lidocaine provides for quick, short-acting sensory and motor impairment. bupivacaine provides for later-onset, longerlasting desensitization without motor impairment. combinations of the two agents diluted with saline are used frequently to provide for quick-onset analgesia that lasts between and hours in most patients. adding narcotic and/or α agent often maximizes the analgesia and increases the pain-free interval to to hours. epinephrine and preservative-free solutions are recommended. precision placement of anesthetic close to nerves, roots, or plexuses is improved with the use of a stimulating nerve locator. cats seem to be more sensitive to the effects of local anesthetics; as such the lower ends of most dosing ranges are used for blockades in this species. unlike most instances of general anesthesia, during which the animal is rendered unconscious and nerve transmission is decreased by virtue of cns depression, local and regional techniques block the initiation of noxious signals, thereby effectively preventing pain from entering the cns. this is an effective means of not only preventing initial pain but also reducing the changes that take place in the dorsal horn of the spinal cord, spinothalamic tracts, limbic and reticular activating centers, and cortex. frequently, the neurohormonal response that is stimulated in pain and stress is blunted as well. overall, the patient has fewer local and systemic adverse effects of pain, disease processes are minimized, chronic pain states are unlikely, and outcome is improved. regional techniques are best used as part of an analgesic regimen that consists of their continuous administration, narcotics, α-agonists, anxiolytics, and good nursing. lidocaine can be added to sterile lubricant in a one-to-one concentration to provide decreased sensation for urinary catheterization, nasal catheter insertion, minor road burn analgesia, and pyotraumatic dermatitis analgesia. proparacaine is a topical anesthetic useful for corneal or scleral injuries. local anesthetics can be used to infiltrate areas of damage or surgery by using long-term continuous drainage catheters and small, portable infusion pumps. this is an effective means of providing days of analgesia for massive surgical or traumatic soft tissue injury. even without the catheter, incisional or regional soft tissue blocking using a combination of to mg/kg lidocaine and . to mg/kg bupivacaine diluted with equal volume of saline and : with sodium bicarbonate is effective for infiltrating large areas of injury. administration of local anesthetic drugs around the infraorbital, maxillary, ophthalmic mental, and alveolar nerves can provide excellent analgesia for dental, orofacial, and ophthalmic trauma and surgical procedures. each nerve may be desensitized by injecting . to . ml of a % lidocaine hydrochloride solution and . to . ml of . % bupivacaine solution using a . -to . -cm, -to -gauge needle. precise placement perineurally versus intraneurally (neuroma formation common) is enhanced by using catheters in the foramen versus needle administration. always perform aspiration before administration to rule out intravascular injection of agents. this block is used to provide analgesia for thoracic, lower cervical, cranial abdominal, and diaphragmatic pain. following aseptic preparation, place a small through-the-needle ( -to -gauge) catheter in the thoracic cavity between the seventh and ninth intercostal space on the midlateral aspect of the thorax. aseptically mix a . to mg/kg lidocaine and a . to . mg/kg bupivacaine dose with volume of saline equal to the volume of bupivacaine, and slowly inject it over a period of to minutes following aspiration to ensure that no intravascular injection occurs. depending on where the lesion is, position the patient to allow the intrapleural infusion to "coat" the area. most effective is positioning the patient in dorsal recumbency for several minutes following the block to make sure local anesthetic occupies the paravertebral gutters and hence the spinal nerve roots. the block should be repeated every hours in dogs and every to hours in cats. secure the catheter to the skin surface for repetitive administration. administration of local anesthetic around the brachial plexus provides excellent analgesia for forelimb surgery, particularly that distal to the shoulder, and amputations. nerve locator-guided techniques are much more accurate and successful than blind placement of local anesthetic; however, even the latter is useful. to administer a brachial plexus blockade, follow this procedure: . aseptically prepare a small area of skin over the point of the shoulder. . insert a -gauge, / -to -inch spinal needle medial to the shoulder joint, axial to the lesser tubercle, and advance it caudally, medial to the body of the scapula, and toward the costochondral junction of the first rib. aspirate first before injection to make sure that intravenous injection does not occur. . inject one third of the volume of local anesthetic mix, and then slowly withdraw the needle and fan dorsally and ventrally while infusing the remaining fluid. . local anesthetic doses are similar to those for intrapleural blockade. epidural analgesia refers to the injection of an opioid, a phencyclidine, an α-agonist, or an nsaid into the epidural space. epidural anesthesia refers to the injection of a local anesthetic. in most patients a combination of the two is used. epidural analgesia and anesthesia are used for a variety of acute and chronic surgical pain or traumatically induced pain in the pelvis, tail, perineum, hind limbs, abdomen, and thorax (table - ) . procedures in which epidural analgesia and anesthesia are useful include forelimb and hind limb amputation, tail or perineal procedures, cesarean sections, diaphragmatic hernia repair, pancreatitis, peritonitis, and intervertebral disk disease. epidural blocks performed using opioids or bupivacaine will not result in hind limb paresis or decreased urinary or anal tone (incontinence), unlike lidocaine or mepivicaine epidural blocks. morphine is one of the most useful opioids for administration in the epidural space because of its slow systemic absorption. epidural catheters used for the instillation of drugs through constant rate infusion or intermittent injection can be placed in dogs and cats. routinely placed at the lumbosacral junction, these catheters are used with cocktails including preservative-free morphine, bupivacaine, medetomidine, and ketamine. extremely effective for preventing windup pain in the peritoneal cavity or caudal half of the body, the catheters may be maintained if placed aseptically for to days. to provide epidural analgesia or anesthesia, follow this procedure: . position the animal in lateral or sternal recumbency. . clip and aseptically scrub over the lumbosacral site. . palpate the craniodorsal-most extent of the wings of the ileum bilaterally and draw an imaginary line through them to envision the spine of l located immediately behind the imaginary line. . advance a -to -gauge, / -to -inch spinal or epidural needle through the skin just caudal to the spine of l . . the needle will lose resistance as it is introduced into the epidural space. drop saline into the hub of the needle, and the saline will be pulled into the epidural space as the needle enters. discrete intercostal nerve blocks can provide effective analgesia for traumatic or postsurgical pain. identify the area of the injury, and infiltrate three segments on either side of the injury with analgesic. to perform an intercostal nerve block, follow this procedure: . clip and aseptically scrub the dorsal and ventral third of the chest wall. . palpate the intercostal space as far dorsally as possible. . use a -gauge, . -inch needle at the caudolateral aspect of the affected rib segments and those cranial and caudal. . direct the tip of the needle caudally such that the tip of the needle "drops" off of the caudal rib. (this places the needle tip in proximity to the neuromuscular bundle that contains the intercostal nerve that runs in a groove on the caudomedial surface of the rib.) . aspirate to confirm that the drug will not go intravenously. . inject while slowly withdrawing the needle. inject . to . ml at each site, depending on the size of the animal. gaynor js, an acute condition in the abdomen is defined as the sudden onset of abdominal discomfort or pain caused by a variety of conditions involving intraabdominal organs. many animals have the primary complaint of lethargy, anorexia, ptyalism, vomiting, retching, diarrhea, hematochezia, crying out, moaning, or abnormal postures. abnormal postures can include generalized rigidity, walking tenderly or as if "on eggshells," or a prayer position in which the front limbs are lowered to the ground while the hind end remains standing. in some cases, it may be difficult initially to distinguish between true abdominal pain or referred pain from intervertebral disk disease. rapid progression and decompensation of the patient's cardiovascular status can lead to stupor, coma, and death in the most extreme cases, making rapid assessment, treatment, and definitive care extremely challenging. often the patient's signalment and history can increase the index of suspicion for a particular disease process. a thorough history often is overlooked or postponed in the initial stages of resuscitation of the patient with acute abdominal pain. often, asking the same question in a variety of methods can elicit an answer from the client that may lead to the source of the problem and the reason for acute abdominal pain. important questions to ask the client include the following: • what is your chief complaint or reason that you brought your animal in on emergency? • when did the signs first start, or when was your animal last normal? • do you think that the signs have been the same, better, or getting worse? • does your animal have any ongoing or past medical problems? • have similar signs occurred in the past? • does your animal have access to any known toxins, or does he or she run loose unattended? as with any other emergency, the clinician must follow the abcs of therapy, treating the most life-threatening problems first. first, perform a perfunctory physical examination. examination of the abdomen ideally should be performed last, in case inciting a painful stimulus precludes you from evaluating other organ systems more thoroughly. briefly observe the patient from a distance. are there any abnormal postures? is there respiratory distress? is the animal ambulatory, and if so, do you observe any gait abnormalities? do you observe any ptyalism or attempts to vomit? auscultate the patient's thorax for crackles that may signify aspiration pneumonia resulting from vomiting. examine the patient's mucous membrane color and capillary refill time, heart rate, heart rhythm, and pulse quality. many patients in pain have tachycardia that may or may not be accompanied by dysrhythmias. if a patient's heart rate is inappropriately bradycardic, consider hypoadrenocorticism, whipworm infestation, or urinary obstruction or trauma as a cause of hyperkalemia. assess the patient's hydration status by evaluating skin turgor, mucous membrane dryness, and whether the eyes appear sunken in their orbits. a brief neurologic examination should consist of whether the patient is actively having a seizure, or whether mental dullness, stupor, coma, or nystagmus are present. posture and spinal reflexes can assist in making a diagnosis of intervertebral disk disease versus abdominal pain. perform a rectal examination to evaluate for the presence of hematochezia or melena. finally, examination of the abdomen should proceed first with superficial and then deeper palpation. visually inspect the abdomen for the presence of external masses, bruising, or penetrating injuries. reddish discoloration of the periumbilical area often is associated with the presence of intraabdominal hemorrhage. it may be necessary to shave the fur to inspect the skin and underlying structures visually for bruising and ecchymoses. auscultate the abdomen for the presence or absence of borborygmi to characterize gut sounds. next, perform percussion and ballottement to evaluate for the presence of a gas-distended viscus or peritoneal effusion. finally, perform first superficial and then deep palpation of all quadrants of the abdomen, noting abnormal enlargement, masses, or whether focal pain is elicited in any one area. once the physical examination has been performed, implement initial therapy in the form of analgesia, fluid resuscitation, and antibiotics. treatment for any patient with an acute condition in the abdomen and shock is to treat the underlying cause, maintain tissue oxygen delivery, and prevent end-organ damage and failure. a more complete description of shock and oxygen delivery is given in the section on shock. emergency care the administration of analgesic agents to any patient with acute abdominal pain is one of the most important therapies in the initial stages of case management. many patients with acute abdominal pain are clinically dehydrated or are in hypovolemic shock because of hemorrhage. careful titration of intravenous crystalloid and colloid fluids including blood products is necessary based on the patient's perfusion parameters including heart rate, capillary refill time, blood pressure, urine output, and pcv. fluid therapy also should be based on the most likely differential diagnoses, with specific fluid types administered according to the primary disease process. in dogs, a shock volume of fluids is calculated based on the total blood volume of ml/kg/hour. in cats, shock fluid rate is based on plasma volume of ml/kg/hour. in most cases, any crystalloid fluid can be administered at an initial volume of one fourth of a calculated shock dose and then titrated according to whether the patient's cardiovascular status responds favorably or not. in cases of an acute condition in the abdomen from known or suspected hypoadrenocorticism, severe whipworm infestation, or urinary tract obstruction or rupture, . % sodium chloride fluid without added potassium is the fluid of choice. when hemorrhage is present, the administration of whole blood or packed rbcs may be indicated if the patient has clinical signs of anemia and shows clinical signs of lethargy, tachypnea, and weakness. fresh frozen plasma is indicated in cases of hemorrhage resulting from vitamin k antagonist rodenticide intoxication or hepatic failure or in cases of suspected disseminated intravascular coagulation (dic). a more thorough description of fluid therapy is given under the sections on shock and fluid therapy. the empiric use of broad-spectrum antibiotics is warranted in cases of suspected sepsis or peritonitis as a cause of acute abdominal pain. ampicillin sulbactam ( mg/kg iv q - h) and enrofloxacin ( mg/kg once daily) are the combination treatment of choice to cover gram-negative, gram-positive, aerobic, and anaerobic infections. alternative therapies include a second-generation cephalosporin such as cefotetan ( mg/kg iv tid) or cefoxitin ( mg/kg iv tid) or added anaerobic coverage with metronidazole ( to mg/kg iv tid). tissue oxygen delivery depends on a number of factors, including arterial oxygen content and cardiac output. if an animal has had vomiting and subsequent aspiration pneumonitis, treatment of hypoxemia with supplemental oxygen in the form of nasal, nasopharyngeal, hood, or transtracheal oxygen administration is important (see oxygen supplementation under emergency diagnostic and therapeutic procedures). perform a complete blood count in all cases of acute abdominal pain to determine if lifethreatening infection or coagulopathy including dic is present. in cases of sepsis, infection, or severe nonseptic inflammation, the white blood cell count may be normal, elevated, or low. examine a peripheral blood smear for the presence of toxic neutrophils, eosinophils, atypical lymphocytes, nucleated rbcs, platelet estimate, anisocytosis, and blood parasites. a falling pcv in the face of rbc transfusion suggests ongoing hemorrhage. perform a biochemistry panel to evaluate organ system function. azotemia with elevated bun and creatinine may be associated with prerenal dehydration, impaired renal function, or postrenal obstruction or leakage. the bun also can be elevated when gastrointestinal hemorrhage is present. serum amylase may be elevated with decreased renal function or in cases of pancreatitis. a normal serum amylase, however, does not rule out pancreatitis as a source of abdominal pain. serum lipase may be elevated with gastrointestinal inflammation or pancreatitis. like amylase, a normal serum lipase does not rule out pancreatitis. total bilirubin, alkaline phosphatase, and alanine transaminase may be elevated with primary cholestatic or hepatocellular diseases or may be due to extrahepatic causes including sepsis. obtain a urinalysis via cystocentesis whenever possible, except in cases of suspected pyometra or transitional cell carcinoma. azotemia in the presence of a nonconcentrated (isosthenuric or hyposthenuric) urine suggests primary renal disease. secondary causes of apparent renal azotemia and lack of concentrating ability also occur in cases of hypoadrenocorticism and gram-negative sepsis. renal tubular casts may be present in cases of acute renal ischemia or toxic insult to the kidneys. bacteriuria and pyuria may be present with infection and inflammation. when a urinalysis is obtained via free catch or urethral catheterization, the presence of bacteriuria or pyuria also may be associated with pyometra, vaginitis, or prostatitis/prostatic abscess. serum lactate is a biochemical indicator of decreased organ perfusion, decreased oxygen delivery or extraction, and end-organ anaerobic glycolysis. elevated serum lactate greater than mmol/l has been associated with increased morbidity and need for gastric resection in cases of gdv and increased patient morbidity and mortality in other disease processes. rising serum lactate in the face of adequate fluid resuscitation is a negative prognostic sign. obtain abdominal radiographs as one of the first diagnostic tests when deciding whether to pursue medical or surgical management. the presence of gdv, linear foreign body, pneumoperitoneum, pyometra, or splenic torsion warrants immediate surgical intervention. if a loss of abdominal detail occurs because of peritoneal effusion, perform additional diagnostic tests including abdominal paracentesis (abdominocentesis) and abdominal ultrasound to determine the cause of the peritoneal effusion. abdominal ultrasonography is often useful in place of or in addition to abdominal radiographs. the sensitivity of abdominal ultrasonography is largely operator dependent. indications for immediate surgical intervention include loss of blood flow to an organ, linear bunching or placation of the intestinal tract, intussusception, pancreatic phlegmon or abscess, a fluid-filled uterus suggestive of pyometra, gastrointestinal obstruction, intraluminal gastrointestinal foreign body, dilated bile duct, or gallbladder mucocele, or gas within the wall of the stomach or gallbladder (emphysematous cholecystitis). the presence of peritoneal fluid alone does not warrant immediate surgical intervention without cytologic and biochemical evaluation of the fluid present. see also abdominal paracentesis and diagnostic peritoneal lavage. abdominal paracentesis (abdominocentesis) often is the deciding factor in whether to perform immediate surgery. abdominocentesis is a sensitive technique for detecting peritoneal effusion when more than ml/kg of fluid is present within the abdominal cavity. abdominal effusion collected should be saved for bacterial culture and evaluated biochemically and cytologically based on your index of suspicion of the primary disease process. if creatinine, urea nitrogen (bun) or potassium is elevated compared with that of serum, uroabdomen is present. elevated abdominal fluid lipase or amylase compared with serum supports a diagnosis of pancreatitis. elevated lactate compared with serum lactate or an abdominal fluid glucose less than mg/dl is highly sensitive and specific for bacterial/ septic peritonitis. the presence of bile pigment or bacteria is supportive of bile and septic peritonitis, respectively. free fibers in abdominal fluid along with clinical signs of abdominal pain strongly support gastrointestinal perforation, and immediate surgical exploration is required. text continued on p. the following are clinical conditions, patient signalment, common history, physical examination, and characteristic findings of various diagnostic tests. a blank column next to a condition indicates no specific signalment, history, physical examination, or diagnostic test characteristic for a particular disease process. lack of contiguity of body wall surgical ( medical unless perforation present present c-shaped abnormal gas pattern with plication on radiographs surgical (immediate) dilation of bowel cranial to foreign object, radiopaque object in surgical (immediate) stomach or intestines, hypochloremic metabolic acidosis on bloodwork if pyloric outflow obstruction is present elevated or decreased wbc; foreign material, wbcs and medical unless perforation bacteria on abdominal fluid, elevated lactate and decreased present glucose on abdominal fluid target shaped soft tissue density on abdominal u/s, soft tissue surgical (immediate): density with gas dilation cranially on abdominal radiographs medical management of primary cause colonic distension with hard feces on radiographs medical increased or decreased wbc, septic abdominal effusion surgical (immediate) elevated t bili, alt, alk phos, and wbc hypoechoic hepatic medical after biopsy parenchyma on ultasound hepatomegaly elevated t bili, alt, alk phos, and wbc hyperechoic foci in surgical (immediate) gallbladder or sludge on u/s, free gas in wall of gall bladder abdominal effusion, bile pigment in effusion surgical (immediate) elevated t bili, alk phos, alt surgical (immediate) elevated or decreased wbc, elevated t bili, alk phos and surgical (immediate) alt, free gas in hepatic parenchyma on rads, hypoechoic mass with hyperechoic material in hepatic parenchyma on u/s heteroechoic liver with hyperechoic center on ultrasound surgical (immediate) mixed echogenic mass on ultrasound, soft tissue mass surgical (immediate or density on radiographs, elevated alk phos, alt, delayed) t bili, hypoglycemia pain-cont'd elevated t bili, alk phos, alt, amylase and/or lipase, elevated medical in most cases or decreased wbc, hypocalcemia, focal loss of detail in right unless abscess or cranial quadrant on radiographs hypo-to hyperechoic phlegmon is present pancreas with hyperechoic peri-pancreatic fat on ultrasound, abdominal and/or pleural effusion on radiographs and ultrasound pancreatic soft tissue mass effect on radiographs and surgical if mass identified, ultrasound, elevated amylase and lipase, hypoglycemia, otherwise medical elevated serum insulin management of hypoglycemia splenomegaly on radiographs, hyperechoic spleen with no surgical (immediate) blood flow on ultrasound soft tissue mass effect and loss of abdominal detail on surgical (immediate) radiographs, cavitated mass with abdominal effusion on u/s hyperechoic spleen with no blood flow on abdominal u/s, surgical (immediate) abdominal effusion, thrombocytopenia loss of abdominal detail on radiographs, peritoneal effusion medical unless refractory on u/s, hemoabdomen on abdominocentesis hypotension diagnosis based primarily on clinical signs medical fracture of the os penis on radiographs largely medical unless urethral tear diagnosis based primarily on clinical signs medical, although prepuce may need to be incised to allow replacement of penis into sheath prostatomegaly on radiographs and ultrasound hypoechoic medical prostate on u/s, pyuria and bacteriuria and u/a prostatomegaly on radiographs and ultrasound hypo-to surgical (delayed) hyperechoic prostate on u/s, bacteriuria and pyuria on u/a prostatomegaly on radiographs and ultrasound, prostatic medical/surgical mineralization on radiographs and ultrasound hypoechoic kidneys on u/s, pyuria on u/a, elevated wbc, medical azotemia pyuria, bacteriuria on u/a medical pyelectasia in abdominal u/s, azotemia surgical (immediate) renomegaly on radiographs, azotemia renal mass on u/s, renomegaly on radiographs surgical (immediate) renal mass on u/s, azotemia, lack of renal blood flow surgical (delayed) on u/s calculi in renal pelvis on radiographs and ultrasound, azotemia medical unless both kidneys affected ureteral calculi on radiographs and ultrasound, hydronephrosis, medical unless both azotemia kidneys affected ureteral calculi on radiographs and ultrasound, hydronephrosis, surgical (delayed until fluid or soft tissue density on u/s, azotemia electrolyte stabilization) diagnosis largely based on physical examination medical unless cannot pass findings urethral catheter azotemia, no peritoneal effusion, lack of urine output or surgical (delayed until outflow with ureteral catheterization, double contrast electrolyte stabilization) cystourethrogram indicated transitional cellular casts on u/a, hematuria, mass effect or surgical and medical thickened irregular urethra on ultrasound or management cystourethrogram hypoechoic swollen testicle on testicular ultrasound surgical (immediate) fluid or gas-filled tubular structure on abdominal ultrasound or surgical (immediate) abdominal radiographs soft tissue tubular structure on radiographs, fluid-filled uterus surgical ( in the event of a negative abdominocentesis, but peritoneal effusion or bile or gastrointestinal perforation are suspected, perform a diagnostic peritoneal lavage. peritoneal dialysis kits are commercially available but are often expensive and impractical (see p. ). animals that have acute abdominal pain can be divided into three broad categories, depending on the primary cause of pain and the initial definitive treatment (table - ) . some diseases warrant a nonsurgical, medical approach to case management. other conditions require immediate surgery following rapid stabilization. other conditions initially can be managed medically until the patient is hemodynamically more stable and then may or may not require surgical intervention at a later time. specific management of each disease entity is listed under its own subheading. box - lists specific indications for exploratory laparotomy. the best means to explore the abdominal cavity accurately and thoroughly is to open the abdomen on midline from the level of the xyphoid process caudally to the pubis for full exposure and then to evaluate all organs in every quadrant in a systematic manner. address specific problems such as gastric or splenic torsion, enteroplication, and foreign body removal, and then copiously lavage the abdomen with warmed sterile saline solution. suction the saline solution thoroughly from the peritoneal cavity so as to not impair macrophage function. in cases of septic peritonitis, the abdomen may be left open, or a drain may be placed for further suction and lavage. the routine use of antibiotics in irrigation solutions is contraindicated because the antibiotics can irritate the peritoneum and delay healing. when the abdominal cavity is left open, secure sterile laparotomy towels and water-impermeable dressings over the abdominal wound with umbilical tape, and then change these daily or as strike-through occurs. open abdomen cases are often effusive and require meticulous evaluation and management of electrolyte imbalances and hypoalbuminemia. the abdomen can be closed and/or the abdominal drain removed when the volume of the effusion decreases, when bacteria are no longer present, and when the neutrophils become more healthy in appearance. bischoff mg: radiographic techniques and interpretation of the acute abdomen, clin tech small anim pract ( ) anaphylactic shock occurs as an immediate hypersensitivity reaction to a variety of inciting stimuli (box - ). in animals, the most naturally occurring anaphylactic reaction results from wasp or bee stings. most other reactions occur as a result of an abnormal sensitivity to items used in making medical diagnoses or treatment. during an anaphylactic reaction, activation of c a and the complement system results in vascular smooth muscle dilation and the release of a cascade of inflammatory mediators, including histamine, slow-reacting substance of anaphylaxis, serotonin, heparin, acetylcholine, and bradykinin. clinical signs associated with anaphylaxis differ between dogs and cats. in dogs, clinical signs may include restlessness, vomiting, diarrhea, hematochezia, circulatory collapse, coma, and death. in cats, clinical signs often are associated with respiratory system abnormalities. clinical signs may include ptyalism, pruritus, vomiting, incoordination, bronchoconstriction, pulmonary edema and hemorrhage, laryngeal edema, collapse, and death. the most important steps to remember in any emergency is to follow the abcs of airway, breathing, and circulation. first, establish an airway through endotracheal intubation or emergency tracheostomy, if necessary. concurrently, an assistant should establish vascular or intraosseous access to administer drugs and fluids (box - ). the patient should be hospitalized until complete resolution of clinical signs. after initial stabilization and treatment, it is important to maintain vascular access and continue intravenous fluid therapy until the patient is no longer hypotensive, and vomiting and diarrhea have resolved. in cases of fulminant pulmonary hemorrhage and edema, administer supplemental oxygen until the patient is no longer hypoxemic or orthopneic on room air. normalize and maintain blood pressure using positive inotropes (dobutamine, - µg/kg/ minute cri) or pressors (dopamine, to µg/kg/minute iv cri; see shock). if bloodtinged vomitus or diarrhea has been observed, administer antibiotics to decrease the risk of bacterial translocation and sepsis (cefoxitin, mg/kg iv tid; metronidazole, mg/kg iv tid). also consider using gastroprotectant drugs (famotidine, . to . mg/kg iv; ranitidine, . to . mg/kg po, iv, im bid; sucralfate, . to . g po tid; omeprazole, . to . mg/kg po sid). a second and less serious form of allergic reaction is manifested as angioneurotic edema and urticaria. in most cases, clinical signs develop within minutes of an inciting allergen. although this type of reaction causes patient discomfort, it rarely poses a life-threatening problem. most animals have mild to severe swelling of the maxilla and periorbital regions. the facial edema also may be accompanied by mild to severe generalized urticaria. some animals may paw at their face, rub at their eyes, or have vomiting or diarrhea. the treatment for angioneurotic edema involves suppressing the immune response by administration of short-acting glucocorticoid drugs and blocking the actions of histamine by the synergistic use of histamine and histamine receptor blockers (box - ). in some cases, the inciting cause is a known recent vaccination or insect sting. many times, however, the inciting cause is not known and is likely an exposure to a stinging insect or arachnid. differential diagnoses for acute facial swelling and/or urticaria include acetaminophen toxicity (cats), anterior caval syndrome, lymphadenitis, vasculitis, hypoalbuminemia, and contact dermatitis. observe animals that have presented for angioneurotic edema for a minimum of to minutes after injection of the short-acting glucocorticoids and antihistamines. monitor blood pressure to make sure that the patient does not have concurrent anaphylaxis and hypotension. after partial or complete resolution of clinical signs, the animal can be discharged to its owner for observation. in dogs, mild vomiting or diarrhea may occur within to days after this type of reaction. wherever possible, exposure to the inciting allergen should be avoided. • administer short-acting glucocorticoid: complications observed while a patient is under anesthesia can be divided into two broad categories: ( ) those related to equipment malfunction or human error and ( ) the patient's physiologic response to the cardiorespiratory effects of the anesthetic drugs. careful observation of the patient and familiarity with anesthetic equipment, drug protocols, and monitoring equipment is necessary for the safest anesthesia to occur. despite this, however, anesthetic-related complications are frequent and need to be recognized and treated appropriately. many anesthetic drugs have a dose-dependent depressive effect on the respiratory system and cause a decrease in respiratory rate and tidal volume, leading to hypoventilation. respiratory rate alone is not a reliable indicator of the patient's oxygenation and ventilatory status. the respiratory tidal volume can be measured with a wright's respirometer. perform pulse oximetry and capnography as noninvasive measures of the patient's oxygenation and ventilation. ventilation can be impaired as a result of anesthetic drugs, patient position, pneumothorax, pleural effusion (chylothorax, hemothorax, pyothorax), equipment malfunction, rebreathing of carbon dioxide, thoracic wall injury, or alveolar fluid (pulmonary edema, hemorrhage, or pneumonia). problems such as a diaphragmatic hernia, gdv, or gravid uterus can impede diaphragmatic excursions once the patient is placed on its back and can lead to impaired ventilation. the work of breathing also may be increased because of increased resistance of the anesthesia circuit and increased dead space ventilation. this is particularly important in small toy breeds. clinical signs of inadequate ventilation and respiratory complications include abnormal respiratory pattern, sudden changes in heart rate, cardiac dysrhythmias, cyanosis, and cardiopulmonary arrest. end-tidal carbon dioxide, or capnography, gives a graphic display of adequacy of ventilation. rapid decreases in end-tidal carbon dioxide can be caused by disconnection or obstruction of the patient's endotracheal tube or poor perfusion, namely, cardiopulmonary arrest (see capnometry [end-tidal carbon dioxide monitoring]). postoperatively, hypoventilation can occur because of the residual effects of the anesthetic drugs, hypothermia, overventilation during intraoperative support, surgical techniques that compromise ventilation (thoracotomy, cervical disk surgery, atlantooccipital stabilization), postoperative bandaging of the abdomen or thorax, ventilatory muscle fatigue, or injury to the cns. cardiac output is a function of heart rate and stroke volume. factors that influence stroke volume include vascular and cardiac preload, cardiac afterload, and cardiac contractility. the patient's cardiac output can be affected adversely by the negative inotropic and chronotropic and vasodilatory effects of anesthetic drugs, all leading to hypotension. emergency care bradycardia, tachycardia, cardiac dysrhythmias, and vascular dilation can lead to hypotension and inadequate organ perfusion. table - lists the normal heart rate and blood pressure in dogs and cats. bradycardia is defined as a heart rate below normal values. many anesthetic drugs can cause bradycardia. causes of bradycardia include the use of narcotics or α -agonist drugs, deep plane of anesthesia, increased vagal tone, hypothermia, and hypoxia. table - lists the causes of bradycardia and the necessary immediate action or treatment. tachycardia is defined as a heart rate above normal values. common causes of tachycardia include vasodilation, drugs, inadequate anesthetic depth and perceived pain, hypercapnia, hypoxemia, hypotension, shock, or hyperthermia. table - lists the causes and immediate action or treatment for tachycardia. hypotension is defined as physiologically low blood pressure (mean arterial pressure less than mm hg). a mean arterial blood pressure less than mm hg can result in inadequate tissue perfusion and oxygen delivery. the coronary arteries are perfused during diastole. inadequate diastolic blood pressure, less than mm hg, can cause decreased coronary artery perfusion and myocardial hypoxemia that can predispose the heart to dysrhythmias. causes of perianesthetic hypotension include peripheral vasodilation by anesthetic drugs, bradycardia or tachyarrhythmias, hypothermia, inadequate cardiac preload from vasodilation or hemorrhage, decreased venous return from patient position or surgical manipulation of viscera, and decreased cardiac contractility. electrocardiogram monitoring is useful for the early detection of cardiac dysrhythmias during the perianesthetic period. clinical signs of cardiac dysrhythmias include irregular pulse rate or pressure, abnormal or irregular heart sounds, pallor, cyanosis, hypotension, and an abnormal ecg tracing. remember that the single best method of detecting cardiac emergency care vagolytic drugs atropine allow time for the drug to wear off. glycopyrrolate allow time for the drug to wear off. sympathomimetic drugs epinephrine allow time for the drug to wear off; administer a β-blocker; turn off infusion. isoproterenol administer a β-blocker. turn off infusion; administer a β-blocker. allow time for drug to wear off. inadequate anesthetic depth increase anesthetic depth. hypercapnia increase ventilation (assisted ventilation). hypoxemia increase gas flow and oxygenation. hypotension decrease anesthetic depth; administer an intravenous crystalloid or colloid bolus, positive inotrope drug, positive chronotrope drug, or pressor. hyperthermia apply ambient or active cooling measures; administer dantrolene sodium if malignant hyperthermia is suspected. hypothermia provide ambient rewarming. hypocalcemia * administer calcium chloride ( mg/kg iv) or calcium gluconate ( mg/kg). decrease vaporizer setting/anesthetic depth. reverse with opioids or a -agonists. vasodilation administer an intravenous crystalloid bolus ( ml/kg). administer an intravenous colloid bolus ( ml/kg). administer a pressor (epinephrine, phenylephrine dysrhythmias is with your fingertips (palpate a pulse or apex heartbeat) and ears (auscultate the heart). confirm the dysrhythmia by auscultating the heart rate and rhythm, identify the p waves and the qrs complexes, and evaluate the relationship between the p waves and qrs complexes. is there a p wave for every qrs, and a qrs for every p wave? during anesthesia, fluid, acid-base, and electrolyte imbalances can predispose the patient to dysrhythmias. sympathetic and parasympathetic stimulation, including the time of intubation, can predispose the patient to dysrhythmias. if the patient's plane of anesthesia is too light, perception of pain can cause catecholamine release, sensitizing the myocardium to ectopic beats. atrioventricular blockade can be induced with the administration of α -agonist medications, including xylazine and medetomidine. thiobarbiturates (thiopental) can induce ventricular ectopy and bigeminy. although these dysrhythmias may not be harmful in the awake patient, anesthetized patients are at a particular risk of dysrhythmia-induced hypotension. carefully monitor and treat all dysrhythmias (see cardiac dysrhythmias). box - lists steps to take to prevent perianesthetic dysrhythmias. awakening during anesthesia can occur and can be caused by equipment failure and simply, although no one likes to admit it, human error. table - lists causes of arousal during anesthesia and appropriate immediate actions. awaken patient, and administer dantrolene arousal (e.g., malignant hyperthermia) sodium. • stabilize acid-base and electrolyte balance before anesthetic induction, whenever possible. • rehydrate patient before anesthetic induction. • select anesthetic agents appropriate for the particular patient. • be aware of the effects of the drugs on the myocardium. • ensure adequate anesthetic depth and oxygenation before anesthetic induction. • ensure ventilatory support during anesthesia. • monitor heart rate, rhythm, blood pressure, pulse oximetry, and capnometry during anesthesia. • ensure adequate anesthetic depth before surgical stimulation. • avoid surgical manipulation to the heart or great vessels, whenever possible. • avoid changes in perianesthetic depth. • avoid hypothermia. delayed recovery can be caused by a number of factors, including excessive anesthetic depth, hypothermia, residual action of narcotics or tranquilizers, delayed metabolism of anesthetic drugs, hypoglycemia, hypocalcemia, hemorrhage, and breed or animal predisposition. careful monitoring of the patient's blood pressure, acid-base and electrolyte status, anesthetic depth, pcv, and vascular volume intraoperatively and taking care with supportive measures to prevent abnormalities can hasten anesthetic recovery and avoid postoperative complications. gaynor the presentation of a patient with a bleeding disorder often is a diagnostic challenge for the veterinary practitioner (boxes - and - ). in general, abnormal bleeding can be caused by five major categories: ( ) vascular trauma, ( ) circulating inhibitors of coagulation heparin fibrin degradation products development of spontaneous deep hematomas, unusually prolonged bleeding after traumatic injury, bleeding at multiple sites throughout the body involving multiple organ systems, delayed onset of severe hemorrhage after bleeding, and an inability on the practitioner's part to find an organic cause of bleeding. the signalment, history, clinical signs, and results of coagulation often can aid in making a rapid diagnosis of the primary cause of the disorder and in the selection of appropriate case management. when taking a history, ask the following important questions: • what is the nature of the bleeding? • what sites are affected? • how long has the bleeding been going on? • has your animal had any previous or similar episodes? • is there any possibility of any toxin exposure? • if so, when and how much did your animal consume? • is there any possibility of trauma? • does your animal run loose outdoors unattended? • have you ever traveled, and if so, where? • has your animal been on any medications recently or currently? • has your animal been vaccinated recently? • have any known relatives of your animal had any bleeding disorders? • are there any other abnormal signs that you have seen? abnormalities found on physical examination may aid in determining whether the hemorrhage is localized or generalized (i.e., bleeding from a venipuncture site versus bleeding diathesis). note whether the clinical signs are associated with a platelet problem and superficial hemorrhage or whether deep bleeding can be associated with abnormalities of the coagulation cascade. also, make an attempt to identify any concurrent illness that can predispose the patient to a bleeding disorder (i.e., pancreatitis, snakebite, sepsis, immunemediated hemolytic anemia, or severe trauma and crush or burn injury). abnormalities associated with coagulopathies include petechiae and ecchymoses, epistaxis, gingival bleeding, hematuria, hemarthrosis, melena, and hemorrhagic cavity (pleural and peritoneal or retroperitoneal) effusions. disseminated intravascular coagulation is a complex syndrome that results from the inappropriate activation of the clotting cascade, leading to disruption of the normal balance between thrombosis and fibrinolysis. the formation of diffuse microthrombi with concurrent consumption of platelets and activated clotting factors leads to end-organ thrombosis with various degrees of clinical hemorrhage. in animals, dic always results from some other pathologic process, including various forms of neoplasia, crush and heat-induced injury, sepsis, inflammation, and immune-mediated disorders (box - ). the pathophysiologic mechanisms involved in dic include vascular endothelial damage, activation and consumption of platelets, release of tissue procoagulants, and consumption of endogenous anticoagulants. because dic always results from some other disease process, diagnosis of dic is based on a number of criteria when evaluating various coagulation tests, peripheral blood smears, platelet count, and end products of thrombosis and fibrinolysis. there is no one definitive criterion for the diagnosis of dic (box - ). thrombocytopenia occurs as platelets are consumed during thrombosis. it is important to remember that trends in decline in platelet numbers are just as important as thrombocytopenia when making the diagnosis. in some cases the platelet count still may be within the normal reference range but has significantly decreased in the last hours. early in dic the procoagulant cascade dominates, with hypercoagulability. activated clotting time, aptt, and pt may be rapid and shorter than normal. in most cases, we do not recognize the hypercoagulable state in our critically ill patients. later in dic, as platelets and activated clotting factors become consumed, the act, aptt, and pt become prolonged. antithrombin, a natural anticoagulant, also becomes consumed, and antithrombin levels decline. antithrombin levels can be measured at commercial laboratories and in some large veterinary institutions. the end products of thrombosis and subsequent fibrinolysis also can be measured. fibrinogen levels may decline, although this test is not sensitive or specific for dic. fibrin degradation (split) products also become elevated. fibrin degradation products are normally cleared by the liver, and these also become elevated in cases of hepatic failure because of lack of clearance. more recently, cageside d-dimer tests have become available to measure the breakdown product of cross-linked fibrin as a more sensitive and specific monitor of dic. management of dic first involves treating the primary underlying cause. by the time dic becomes evident, rapid and aggressive treatment is necessary. if you are suspicious of dic in any patient with a disease known to incite dic, then ideally, you should begin treatment before the hemostatic abnormalities start to occur for the best possible prognosis. treatment involves replacement of clotting factors and antithrombin and prevention of further clot formation. to replenish clotting factors and antithrombin, administer fresh whole blood or fresh frozen plasma. heparin requires antithrombin as a cofactor to inactivate thrombin and other activated coagulation factors. administer heparin ( to units/kg sq q - h of unfractionated heparin; or fractionated enoxaparin [lovenox], mg/kg sq bid). aspirin ( mg/kg po bid in dogs; every third day in cats) also can be administered to prevent platelet adhesion. management of dic also involves the rule of twenty monitoring and case management to maintain end-organ perfusion and oxygen delivery (see the rule of ). hemophilia a is a sex-liked recessive trait that is carried by females and manifested in males. female hemophiliacs can occur when a hemophiliac male is bred with a carrier female. hemophilia a has been reported in cats and a number of dog breeds, including miniature schnauzer, saint bernard, miniature poodle, shetland sheepdog, english and irish setters, labrador retriever, german shepherd, collie, weimaraner, greyhound, chihuahua, english bulldog, samoyed, and vizsla. mild to moderate internal or external bleeding can occur. clinical signs of umbilical cord bleeding can become apparent in some animals shortly after weaning. gingival hemorrhage, hemarthrosis, gastrointestinal hemorrhage, and hematomas may occur. clotting profiles in animals with factor viii deficiency include prolonged aptt and act. the pt and buccal mucosa bleeding time are normal. affected animals have low factor viii activity but normal to high levels of factor viii-related antigen. carrier females can be detected by low ( % to % of normal) factor viii activity and normal to elevated levels of factor vii-related antigen. von willebrand's disease is a deficiency or defect in von willebrand's protein. a number of variants of the disease have been described: von willebrand's disease type i is associated with a defect in factor viir/protein concentration, and von willebrand's disease type ii is associated with a defect in viiir:vwf. type i von willebrand's disease is most common in veterinary medicine. von willebrand's disease has been identified in more than breeds of dogs, with an incidence that varies from % to % depending on the breed of origin. affected breeds include doberman pinchers, german shepherd dogs, scottish terriers and standard manchester terriers, golden retrievers, chesapeake bay retrievers, miniature schnauzers, and pembroke welsh corgis. two forms of genetic expression occur: ( ) autosomal recessive disease in which homozygous von willebrand's disease individuals have a bleeding disorder, whereas heterozygous individuals carry the trait but are clinically normal. the second variant of genetic expression involves an autosomal dominant disease with incomplete expression such that heterozygous individuals are affected carriers and homozygous individuals are severely affected. von willebrand's disease has high morbidity, but fortunately a low mortality. dogs with % or less than normal vwf tend to hemorrhage. platelet counts are normal, but bleeding times can be prolonged. the aptt can be slightly prolonged when factor viii is less than % of normal. routine screening tests are nondiagnostic for this disease, although in a predisposed breed with a normal platelet count, a prolonged buccal mucosa bleeding time strongly supports a diagnosis of von willebrand's disease. documentation of clinical bleeding with low or undetectable levels of factor viii antigen or platelet-related activities of vwf support a diagnosis of von willebrand's disease. recessive animals have zero vwf:antigen (a subunit of factor iii); heterozygotes have % to % of normal. in the incompletely dominant form, levels of vwf antigen are reduced (less than % to %). clinical signs in affected animals include epistaxis, hematuria, diarrhea with melena, penile bleeding, lameness, hemarthrosis, hematoma formation, and excessive bleeding with routine procedures such as nail trimming, ear cropping, tail docking, surgical procedures (spay, neuter), and lacerations. estrous and postpartum bleeding may be prolonged. a dna test to detect carriers of the vwf gene is available through vetgen (ann arbor, michigan) and michigan state university. patients with von willebrand's disease should avoid drugs known to affect platelet function adversely (sulfonamide, ampicillin, chloramphenicol, antihistamines, theophylline, phenothiazine tranquilizers, heparin, and estrogen). hemophilia b is an x-linked recessive trait that occurs with less frequency that hemophilia a. the disease has been reported in scottish terriers, shetland and old english sheepdogs, saint bernards, cocker spaniels, alaskan malamutes, labrador retrievers, bichon frises, airdale terriers, and british shorthair cats. carrier females have low ( % to % of normal) factor ix activity. clinical signs are more severe than for hemophilia a. congenital deficiencies of factor vii have been reported as an autosomal, incompletely dominant characteristic in beagles. heterozygotes have % factor vii deficiency. bleeding tends to be mild. the pt is prolonged in affected individuals. factor x deficiency has been documented in cocker spaniels and resembles fading-puppy syndrome in newborn dogs. internal or umbilical bleeding can occur, and affected dogs typically die. bleeding may be mild in adult dogs. in severe cases, factor x levels are reduced to % of normal; in mild cases, factor x levels are % to % of normal. factor xii deficiency has been documented as an inherited autosomal recessive trait in domestic cats. heterozygotes can be detected because they have a partial deficiency ( % of normal) of factor xii. homozygote cats have less than % factor xii activity. deficiency of hageman factor usually does not result in bleeding or other disorders. factor xi deficiency is an autosomal disease that has been documented in kerry blue terriers, great pyrenees, and english springer spaniels. in affected individuals, protracted bleeding may be observed. homozygotes have low factor xi activity (< % of normal), and heterozygotes have % to % of normal. the management of congenital defects of hemostasis typically involves replenishing the clotting factor that is present. usually, this can be accomplished in the form of fresh frozen plasma transfusion ( ml/kg). if anemia is present because of severe hemorrhage, fresh whole blood or packed rbcs also can be administered. recent research has investigated the use of recombinant gene therapy in the treatment of specific factor deficiencies in dogs; however, the therapy is not yet available for use in clinical practice. in cases of von willebrand's disease, administration of fresh frozen plasma ( to ml/kg) or cryoprecipitate ( unit/ kg body mass) provides vwf, factor viii, and fibrinogen. doses can be repeated until hemorrhage ceases. -desamino- -d-arginine vasopressin (ddavp) also can be administered ( µg/kg sc or iv diluted in . % saline given over to minutes) to the donor and patient to increase the release of stored vwf from endothelial cells. a fresh whole blood transfusion can be obtained from the donor and immediately administered to the patient, or spun down and the fresh plasma administered if rbcs are not needed. administer a dose of ddavp to any affected dog before initiating any elective surgical procedures. a supply of fresh frozen plasma and rbcs should be on hand, should uncontrolled hemorrhage occur. platelets are essential to normal blood coagulation. after a vessel is damaged, release of vasoactive amines causes vasoconstriction and sluggish flow of blood in an attempt to squelch hemorrhage. platelets become activated by platelet activating factor, and attach to the damaged vascular endothelium. normal platelet adhesion depends on mediators such as calcium, fibrinogen, vwf:antigen, and a portion of factor viii. after adhesion, the platelets undergo primary aggregation and release a variety of chemical mediators including adenosine diphosphate, prostaglandins, serotonin, epinephrine, thromboplastin, and thromboxane a that promote secondary aggregation and contraction. platelet abnormalities can include decreased platelet production (thrombocytopenia), decreased platelet function (thrombocytopathia), increased platelet destruction, increased platelet consumption, and platelet sequestration. thrombocytopathia refers to platelet function abnormalities. alterations in platelet function can affect platelet adhesion, aggregation, or release of vasoactive substances that help form a stable clot (box - ). in von willebrand's disease there is a deficiency in vwf:antigen that results in altered platelet adhesion. vascular purpuras are reported and have been seen in collagen abnormalities such as ehlers-danlos syndrome, which can be inherited as an autosomal dominant trait with complete penetrance and has been recognized in german shepherd dogs, dachshunds, saint bernards, and labrador retrievers. thrombasthenic thrombopathia is a hereditary autosomal dominant abnormality that has been described in otterhounds, foxhounds and scottish terriers. in this condition, platelets do not aggregate normally in response to adenosine diphosphate and thrombin stimulation. evaluation of platelet function is based on a total platelet count, buccal mucosa bleeding time, and thromboelastography. platelet function defects (thrombocytopenia and thrombocytopathia) can affect both sexes. clinical signs can resemble von willebrand's disease. in most cases, buccal mucosa bleeding time will be prolonged, but platelet count and clotting tests will be normal. platelet count can be decreased because of problems with production, increased consumption, sequestration, or destruction. causes of accelerated platelet destruction are typically immune-mediated autoantibodies, drug antibodies, infection, and isoimmune destruction. consumption and sequestration usually are caused by dic, vasculitis, microangiopathic hemolytic anemia, severe vascular injury, hemolytic uremic syndrome, and gram-negative septicemia. primary thrombocytopenia with no known cause has been called idiopathic thrombocytic purpura. in approximately % of the cases, thrombocytopenia is associated with immune-mediated destruction caused by immune-mediated hemolytic anemia, systemic lupus erythematosus, rheumatoid arthritis, dic, and diseases that affect the bone marrow. in systemic lupus erythematosus, % to % of the affected dogs have concurrent idiopathic thrombocytic purpura. when immune-mediated hemolytic anemia and idiopathic thrombocytic purpura are present in the same patient, the disease is called evans syndrome. pf- is a non-complement-fixing antibody that is produced in the spleen and affects peripheral and bone marrow platelets and megakaryocytes. antibodies directed against platelets are usually of the igg subtype in animals. antiplatelet antibodies can be measured by a pf- release test. platelet counts with immune-mediated destruction typically are less than , platelets/µl. infectious causes of thrombocytopenia include ehrlichia canis, anaplasma phagocytophilum (formerly, ehrlichia equi), and rickettsia rickettsii (rocky mountain spotted fever). primary immune-mediated thrombocytopenia has an unknown cause and most frequently is seen in middle-to older-aged female dogs. breed predispositions include cocker spaniels, german shepherd dogs, poodles (toy, miniature, standard), and old english sheepdogs. thrombocytopenia usually is manifested as petechiae, ecchymoses of skin and mucous membranes, hyphema, gingival and conjunctival bleeding, hematuria, melena, and epistaxis. to make a diagnosis of idiopathic thrombocytic purpura, measure the severity of thrombocytopenia (< , platelets/µl), analyze the peripheral blood smear for evidence of platelet fragmentation or microthrombocytosis, normal to increased numbers of megakaryocytes in the bone marrow, detection of antiplatelet antibody, increased platelet counts after starting glucocorticoid therapy, and elimination of other causes of thrombocytopenia. if tick-borne illnesses are suspected, antibody titers for e. canis, a. phagocytophilum (formerly e. equi), and r. rickettsii should be performed. treatment of immune-mediated thrombocytopenia involves suppression of the immune system to stop the immune-mediated destruction and to stimulate platelet release from the bone marrow. traditionally, the gold standard to suppress the immune system is to use glucocorticoids (prednisone or prednisolone, to mg/kg po bid divided, or dexamethasone, . to . mg/kg iv or po q h). more recently human serum immunoglobulin (igg) also has been used ( . to . g/kg iv in saline over hours; pretreat with mg/kg diphenhydramine minutes before starting infusion). vincristine ( . mg/m iv once) can stimulate the release of platelets from the bone marrow if megakaryocytic precursors are present; however, the platelets released may be immature and potentially nonfunctional. treatment with fresh whole blood or packed rbcs is appropriate if anemia is present; however, unless specific platelet-rich plasma has been purchased from a blood bank, fresh whole blood contains relatively few platelets, which are shortlived ( hours) and will not effectively raise the platelet count at all. finally, long-term therapy is usually in the form of azathioprine ( mg/kg po once daily, tapered to mg/kg daily to every other day after week) and cyclosporine ( to mg/kg po divided). if a tickborne illness is suspected, administer doxycycline ( to mg/kg po bid) for weeks or if titers come back negative. thrombocytopenia also can occur in the cat. causes for thrombocytopenia in cats include infections ( %), neoplasia ( %), cardiac disease ( %), primary immune-mediated disease ( %), and unknown causes ( %). in one study of cats with feline leukemia and myeloproliferative disease, % of cases had thrombocytopenia. warfarin and coumarin derivatives are the major class of rodenticides used in the united states. vitamin k antagonist rodenticides inhibit the epoxidase reaction and deplete active vitamin k, causing a depletion of vitamin k-dependent coagulation factors (ii, vii, ix, x) within hours to week of ingestion, depending on the ingested dose. affected animals can spontaneously hemorrhage anywhere in the body. clinical signs can include hemoptysis, respiratory difficulty, cough, gingival bleeding, epistaxis, hematuria, hyphema, conjunctival bleeding, petechiae and ecchymoses, cavity hemorrhage (pleural, peritoneal, retroperitoneal) with acute weakness, lethargy or collapse, hemarthrosis with lameness, deep muscle bleeds, and intracranial or spinal cord hemorrhage. diagnosis of vitamin k antagonism includes prolonged pt. a pivka (protein induced by vitamin k absence or antagonism) test also can be performed, if possible. treatment of vitamin k antagonist rodenticide intoxication and other causes of vitamin k deficiency involves supplementation with vitamin k (phytonadione, mg/kg sq once with -gauge needle in multiple sites, and then . mg/kg po bid to tid for days). never administer injections of vitamin k intramuscularly, because of the risk of causing deep muscle hematomas, or intravenously, because of the risk of anaphylaxis. the pt should be rechecked days after the last vitamin k capsule is administered, for some of the secondgeneration warfarin derivates are fat-soluble, and treatment may be required for an additional weeks. act, activated clotting time; aptt, activated partial thromboplastin time; bmbt, buccal mucosa bleeding time; fdp, fibrin degradation products; n, normal; pt, prothrombin time. thermal burns are fortunately a relatively infrequent occurrence in veterinary patients. box - lists various causes of malicious and accidental burns. the location of the burn is also important in assessing its severity and potential to lose function. burns on the perineum, feet, face, and ears are considered to be the most severe because of loss of function and severe pain. often the severity of thermal injury is difficult to assess in animals because hair coat potentially can mask clinical signs and because the thermal injury can continue after the animal has been removed from the heat source. the skin cools slowly and warms slowly, considerations that become important when initiating therapy for burns. the severity of thermal injury is associated with the temperature to which the animal is exposed, the duration of contact, and the ability of the tissue to dissipate heat. the tissue closest to the heat source undergoes necrosis and has decreased blood flow. the severity of thermal burn injury is associated directly with the temperature to which the animal is exposed, the percentage of total body surface area affected, the thickness of injured tissue, and whether underlying complications with other body systems occur. prognosis largely depends on the total body surface area affected (table - ) . superficial partial thickness, or first-degree, burns offer the most favorable prognosis. the affected epidermis initially appears erythematous and then quickly desquamates within to days. in most cases, fur grows back without leaving a scar. deep partial thickness, or second-degree, burns involve the epidermis and dermis and are associated with subcutaneous edema, inflammation, and pain. deep partial thickness burns heal from deeper adnexal tissues and from the wound edges and are associated with an increased chance of scarring and depigmentation. the most severe type is known as full thickness, or third-degree, burns, in which thermal injury destroys the entire thickness of the skin and forms an eschar. thrombosis of superficial and deeper skin vasculature and gangrene occurs. treatment involves sequential wound debridement. healing occurs by second intention and reepithelialization or by wound reconstruction. in most cases, scarring is extensive in affected areas. burns greater than % of total body surface area will have systemic effects, including impaired cardiovascular function, pulmonary dysfunction, and impaired immune function. burned tissue, with capillary damage, has increased permeability. the release of inflammatory cytokines, oxygen-derived free radical species, prostaglandins, leukotrienes, emergency care histamine, serotonin, and kinins results in increased vascular permeability and leakage of plasma proteins into the interstitium and extravascular space. at the time of presentation, first examine the patient and ascertain whether airway obstruction, impaired ventilatory function, circulatory shock, or pain are present. if necessary, establish an airway with endotracheal intubation or emergency tracheostomy. next, cool the burned area(s) with topical cool water. use care to avoid overcooling and iatrogenic hypothermia. the best approach is to cool only one portion of the patient's body at a time, then dry, and repeat the process for all affected areas to avoid overcooling and iatrogenic hypothermia. establish vascular access and administer appropriate and judicious analgesic drugs and intravenous fluid therapy. whenever possible, avoid placing a catheter through an area of burned or damaged skin. in the early stages of burn injury, shock doses of intravenous crystalloid fluids usually are not required. later, however, as severe tissue exudation occurs, protein and fluid losses can become extensive, requiring aggressive crystalloid and colloid support to treat hypovolemia and hypoproteinemia. flush the eyes with sterile saline and examine behind the third eyelids for any particulate matter. stain the corneas to make sure that superficial corneal burns are not present. treat superficial corneal burns with triple antibiotic ophthalmic ointment. next, assess the total body surface area affected, as this will gauge prognosis. depending on the extent of the damage, decide whether the burn is superficial and local therapy is indicated or whether more severe injuries exist that may involve systemic therapy or possibly euthanasia. in most cases the diagnoses of thermal burns are based on a clinical history of being in a house fire, clothes dryer, or under a heating lamp. too frequently, however, thermal burns become apparent days after an elective surgical procedure in which the patient was placed on a faulty heating pad rather than a circulating warm water or warm air blanket. superficial burns appear as singed fur with desquamating, easily epilated hair. this condition also can resemble a superficial or deeper dermatophytosis if history is unknown. other differential diagnoses include immune-mediated vasculitis or erythema multiforme. unless the superficial dermis is blistered, it may be difficult to distinguish between a thermal burn, chemical burn, or electrical burn if the trauma went unnoticed. management of burn injury largely depends on the depth of injury and the total body surface area affected. partial thickness burns and those affecting less than % of the total body surface area will require support in the form of antibiotic ointment and systemic analgesic drugs. burns affecting greater than % of total body surface area or deep thickness burns require more aggressive therapy. central venous catheters can be placed to administer crystalloid and colloid fluids, parenteral nutrition if necessary, antibiotics, and analgesic drugs. monitor perfusion parameters closely, including heart rate, blood pressure, capillary refill time, and urine output. respiratory function can be impaired because of concurrent smoke inhalation, thermal damage to the upper airways and alveoli, and carboxyhemoglobin or methemoglobin intoxication. respiratory function also can be impaired because of burn injury to the skin around the thoracic cage. thoracic radiographs may reveal patchy interstitial to alveolar infiltrates associated with pulmonary edema, pneumonia, and atelectasis. bronchoscopy often reveals edema, inflammation, particulate matter, and ulceration of the tracheobronchial tree. in some cases, upper airway inflammation is so severe that an emergency tracheostomy must be performed to treat airway obstruction. administer supplemental humidified oxygen at to ml/kg/minute via endotracheal tube, tracheostomy, nasal or intratracheal tube, or hood oxygen if respiratory function and hypoxemia are present. perform blood work including a hematocrit, albumin, bun, creatinine, and glucose at the time of presentation. monitor serum electrolytes, albumin, and colloid oncotic pressure closely because derangements can be severe as burns become exudative. the goal of fluid therapy in the burn patient is to establish and maintain intravascular and interstitial fluid volume, normalize electrolyte and acid-base status, and maintain serum albumin and oncotic pressure. in the first hours following burn injury, direct fluid therapy to maintaining the patient's metabolic fluid requirements. crystalloid fluids in the form of normosol-r, plasmalyte-m, or lactated ringer's solution can be administered according to the patient's electrolyte and acid-base status (see fluid therapy). monitor urine output, and keep it at to ml/kg/hour. avoid overhydration in the early stages of burn injury. in affected burn patients, calculate the amount of fluid that should be administered over a -hour period from the formula − ml/kg × percent total body surface area. administer half of this calculated dose over the first hours and then the remaining half over the next hours. in cats, administer only % to % of this calculated volume. to administer this volume and also avoid fluid overload is often difficult in critically ill patients with pulmonary involvement associated with smoke inhalation injury. avoid colloids in the first hours after burn injury. monitor the patient closely for serous nasal discharge, chemosis, and rales that may signify pulmonary edema. as burns become exudative, weigh the patient at least twice daily. infused fluid should equal fluid output in the form of urine and wound exudates. acute weight loss signifies acute fluid loss and that crystalloid fluid infusion should be more aggressive. ideally, keep the patient's serum albumin equal to or greater than . g/dl and total protein between . and . g/dl using a combination of fresh frozen plasma or concentrated human albumin. adjunct colloidal support can be provided with synthetic colloids including hetastarch or hbocs. keep serum potassium within . to . meq/l using potassium chloride or potassium phosphate supplementation. if potassium supplementation exceeds to meq/l and the patient continues to have severe refractory hypokalemia, administer magnesium chloride ( . meq/kg/day) to enhance potassium retention. if anemia occurs, administer packed rbcs or whole blood (see blood component therapy). lavage wounds daily with lactated ringer's solution or . % sodium chloride solution. place wet-to-dry bandages or bandages soaked in silver sulfadiazine or nitrofurazone ointment over the wounds. depending on the thickness of the burn, epilation and eschar formation and separation may take to days. at each bandage change, debride devitalized tissue to normal tissue. perform staged partial or total escharectomy, and leave the wound to heal by second intention or by reconstruction using skin advancement flaps or grafts. maintain meticulous sterility at all times, given that burn patients are at high risk for infection. administer broad-spectrum antibiotics including cefazolin and enrofloxacin. perform wound culture if a resistant bacterial infection is suspected. the most common cause of electrical injury is associated with an animal chewing on low-voltage alternating current electrical cords in the household. damage is caused by the current flowing through the path of least resistance, causing heat and thrombosis of vessels and neurons. in some cases, the owner witnesses the event. in other cases, the owner presents the patient because of vague nonspecific signs, and characteristic abnormalities on physical examination support a diagnosis of electrocution. burns on the face, paws, commissures of the mouth, tongue, and soft palate may be present. electrocution causes a massive release of catecholamines and can predispose the patient to noncardiogenic pulmonary edema within hours of the incident. clinical signs may be isolated to the pulmonary system, including orthopnea, pulmonary crackles, and cyanosis. assess the patient's lips, tongue, soft palate, gingivae, and commissures of the mouth. early after electrocution, the wound may appear small and white, black, or yellow. later, the wound may become larger as tissue sloughs because of damaged vascular supply. assess the patient's respiratory status. auscultate the lungs to determine whether pulmonary crackles emergency care are present. if the patient is stable, thoracic radiographs may demonstrate an interstitial to alveolar lung pattern in the dorsocaudal lung fields. measure the patient's heart rate, blood pressure, oxygenation as determined by pulse oximetry or arterial blood gas and urine output. immediate treatment consists of judicious use of analgesics for the burn injury, antibiotics (cefazolin, mg/kg q h; cephalexin, mg/kg q h), and humidified supplemental oxygen ( to ml/kg/minute). direct fluid therapy at providing the patient's metabolic fluid requirements. because of the risk of development of noncardiogenic pulmonary edema, avoid overzealous administration of crystalloid fluids. differential diagnoses for the patient with electrical burn injury and electrocution include chemical or thermal burn, immune-mediated glossitis, cardiogenic pulmonary edema, and pneumonia. management of the patient with electrical burn injury and electrocution primarily involves the administration of analgesic agents, supplemental humidified oxygen, and topical treatment of electrical burns. the noncardiogenic pulmonary edema is typically unresponsive to diuretics (i.e., furosemide), bronchodilators (i.e., aminophylline), and splanchnic vascular dilators (i.e., low-dose morphine). the use of glucocorticoids has no proven benefit and may impair respiratory immune function and is therefore contraindicated. oral burns may require debridement and advancement flaps if large defects or oronasal fistulas develop. if oral injury is severe, place an esophagostomy or percutaneous gastrostomy tube to ensure adequate nutrition during the healing process. if an animal survives the initial electrocution, prognosis is generally favorable with aggressive supportive care. chemical burns are associated with a number of inciting causes, including oxidizing agents, reducing agents, corrosive chemicals, protoplasmic poisons, desiccants, and vesicants. the treatment for chemical burns differs slightly from that for thermal burns, so it remains important to investigate the cause of the burn when providing initial treatment, whenever possible. at the scene, advise the owner to wrap the patient in a clean towel for transport. chilling can be avoided by then wrapping the patient in a second or third blanket. placement of ointments by well-doers should be avoided. encourage immediate transport to the nearest triage facility. the first and foremost consideration when treating a patient with chemical burn is to remove the animal from the inciting cause or offending agent. make no attempt to neutralize alkaline or acid substances because the procedure potentially could cause an exothermic reaction, leading to thermal injury in addition to the chemical injury. remove collars or leashes that may act as tourniquets or constricting devices. flush affected areas with copious amounts of cool water for several minutes, not cooling more than % to % of the body at any one time to prevent iatrogenic hypothermia. support breathing by extending the patient's head and neck. carefully clip the fur over affected areas for further evaluation of the extent of the injury. lavage exposed eyes with sterile saline, and stain the cornea to evaluate for any corneal burns. debride any wounds carefully, knowing that the full extent of the wound may not manifest itself for several days. then cover the wounds with antibiotic burn ointment such as silver sulfadiazine and an occlusive dressing. without a history of exposure, the differential diagnosis for any chemical burn includes thermal burn, necrotizing vasculitis, erythema multiforme, or superficial or deep pyoderma. contact local or national animal poison control regarding whether to attempt neutralization. perform daily bandage changes with staged debridement as the full extent of the wound manifests itself. place antimicrobial ointment and silver sulfadiazine ointment over the wound to prevent infection. the routine use of antibiotics may promote the development of a resistant bacterial infection. first-generation cephalosporin can be administered. if a more serious infection develops, perform culture and susceptibility testing to direct appropriate antibiotic therapy. the wound can heal by second intention or may require reconstructive repair for definitive closure. the primary cause of radiation injury in small animal patients is radiation therapy for neoplastic conditions. the goal of radiation therapy is to kill neoplastic cells. an unfortunate side effect is damage to adjacent normal tissue that results in necrosis, fibrosis, and impaired circulation to the affected area. radiation burns result in dermatitis, mucositis, impaired surgical wound healing, and chronic nonhealing wounds. in many cases, the degree of secondary radiation injury to normal tissue can be prevented or decreased with careful radiation planning and mapping of the radiation field, such that radiation exposure to normal tissue is limited to the smallest extent possible. with the advent of three-dimensional imaging modalities such as computed tomography (ct) and magnetic resonance imaging (mri), this has become more routine in veterinary oncology to date. radiation injury can be early and appear at the later stage of the course of radiation therapy. late effects can be delayed and occur months to years after treatment. the degree of radiation injury is categorized based on the depth of tissue affected. first-degree changes cause cutaneous erythema. second-degree changes cause superficial desquamation. thirddegree changes cause deeper moist desquamation, and fourth-degree changes are associated with complete dermal destruction and ulceration. during the early stages of radiation injury, affected tissues may appear erythematous and edematous. wound exudates may be moist, or the skin may appear dry and scaly with desquamation or ulceration. later, the area may scar and depigment or may have induration, atrophy, telangiectasia, keratosis, and decreased adnexal structures. treatment for radiation dermatitis is to irrigate the area with warmed saline and to protect the area from self-mutilation. no-bite, or elizabethan, collars or loose clothing can be used to protect the area for patient-induced injury. mucositis can be treated with topical green tea baths and the administration of an oral solution of l-glutamine powder ( g/m ). local irrigation of xylocaine or lidocaine viscous jelly can be used in dogs but should be avoided in cats because of the risk of inducing hemolytic anemia and neurotoxicity. topical and systemic antibiotics (cephalexin, mg/kg po tid) also can be administered. avoid antibiotics that can be sensitized by radiation (i.e., metronidazole). because most radiation burns are associated with a known exposure to radiation therapy, the cause of the patient's injury usually is known. if an animal presents to you with a scar, however, differential diagnoses may include nasal planum solar dermatitis, pemphigus foliaceus, discoid lupus, superficial necrolytic dermatitis, superficial or deep pyoderma, chemical burn, or thermal burn. treatment of radiation injury involves making the patient as comfortable as possible with analgesic drugs, prevention of self-mutilation, and staged debridement techniques. wounds can heal by second intention or may require reconstructive surgery. distress syndrome (ards), and anesthetic agents. the acute onset of bradycardia, change in mucous membrane color and capillary refill time, change in respiratory pattern, and change in mentation are signs of possible deterioration and impending cardiopulmonary arrest. the diagnosis of cardiopulmonary arrest is based on the absence of effective ventilation, severe cyanosis, absence of a palpable pulse or apex heartbeat, absence of heart sounds, and ecg evidence of asystole or other nonperfusing rhythm such as electricalmechanical dissociation (aka pulseless electrical activity) or ventricular fibrillation. the goals of cpcr are to obtain airway access, provide artificial ventilation and supplemental oxygen, implement cardiac compressions and cardiovascular support, recognize and treat dysrhythmias and arrhythmias, and provide stabilization and treatment for cardiovascular, pulmonary, and cerebral function in the event of a successful resuscitation. even with aggressive treatment and management, the overall success of cpcr is less than % in critically ill or traumatized patients and % to % in anesthetized patients. basic life support involves rapid intubation to gain airway access, artificial ventilation, and cardiac compressions to promote blood flow and delivery of oxygen to the brain and other important tissues (figure - ). perform the abcs or cabs of cpcr, where a is airway, b is breathing, and c is compression and circulation. recently, the paradigm has shifted to cabs. while a team member is grabbing an endotracheal tube, clearing the airway of foreign debris, and establishing airway access through endotracheal intubation, a second person starts external cardiac compressions to deliver oxygen that is in the bloodstream to the vital organs. the patient should be positioned in dorsal (> kg) or lateral (< kg) recumbency for external cardiac compressions. approximately to external compressions should be performed over the patient's sternum. a team member should palpate for a peripheral pulse to determine whether cardiac compressions are actually effective. if a peripheral pulse cannot be palpated for every chest compression, change the patient's position and have a larger individual perform compressions, or initiate open-chest cardiac resuscitation. once the patient is intubated, tie in the endotracheal tube and attach it to an oxygen source (anesthetic machine or mechanical ventilator or ambu bag) for artificial ventilation. the oxygen flow rate should be ml/kg/minute. give two long breaths, and then to breaths per minute. simultaneous ventilation with thoracic compression increases the pressure difference in the thorax and allows more forward flow of oxygenated blood through the great vessels into the periphery. if possible, a third team member can initiate interposed abdominal compressions, compressing the abdomen when the thoracic cage is relaxed, to improve forward flow. if only one person is available to perform the thoracic compressions and ventilation, give two breaths for every compressions (i.e., thoracic compressions followed by two long breaths, and then start thoracic compressions again). the jen chung maneuver can be performed by placing a -to -gauge hypodermic needle through the skin of the nasal philtrum and twisting the needle into the periosteum to stimulate respirations. this maneuver appears to work better in cats than dogs at return to spontaneous respiration. advanced life support during cpcr involves ecg, pulse oximetry and capnometry monitoring, administration of drugs, and the administration of intravenous fluids (in select cases). most of the drugs used during cpcr can be administered directly into the lungs from the endotracheal tube (intratracheal tube). therefore, only in select instances is it necessary to establish vascular or intraosseous access during cpcr (figure - ) . if an animal experiences cardiopulmonary arrest because of extreme hemorrhage or hypovolemia, inappropriate vasodilation caused by sepsis or systemic inflammation, or vasodilation resulting from anesthesia, the administration of shock volumes ( ml/kg/hour in dogs and ml/kg/hour in cats) is appropriate. if a patient is euvolemic and experiences cardiopulmonary arrest, however, an increase in circulating fluid volume actually can impair coronary artery perfusion by increasing diastolic arterial blood pressure and is asystole is one of the most common rhythm disturbances that causes cardiac arrest in small animal patients. one of the most important things to do when the ecg looks like asystole is to make sure that the ecg monitor is working properly and that all ecg leads are attached properly to the patient. if asystole is truly present, reverse any opiate, α -agonist, or benzodiazepine drugs with their appropriate reversal agents. lowdose epinephrine ( . to . mg/kg diluted with ml sterile saline) can be administered directly into the endotracheal tube via a rigid or red rubber catheter. if vascular access is available, epinephrine ( . to . mg/kg) can be administered intravenously. no drug should ever be administered directly into the heart by intracardiac injection. unless the heart is in the veterinarian's hand during open-chest cpcr, intracardiac injection is risky and potentially could lacerate a coronary artery or cause the myocardium to become more irritable and refractory to other therapies, if a drug is delivered into the myocardium and not into the ventricle. for these reasons, intracardiac injections are contraindicated. administer atropine ( . mg/kg iv, io, or . mg/kg it) immediately after the epinephrine. atropine, a vagolytic drug, serves to decrease tonic vagal inhibition of the sinoatrial and atrioventricular node and increase heart rate. administer atropine and epinephrine every to minutes during asystole while cardiac compressions, interposed abdominal compressions, and artificial ventilation are continued. although discontinuation of thoracic compressions can decrease the chance of success during cpcr, you must intermittently evaluate the ecg monitor for any rhythm change that may require different drug therapies. if the cardiac arrest was not witnessed or more than to minutes have passed without successful return to a perfusing rhythm, perform open-chest cpcr, if the client wishes. administer sodium bicarbonate ( to meq/kg iv) every to minutes during cpcr. sodium bicarbonate is the only drug used in cpcr that should not be administered intratracheally because of inactivation of pulmonary surfactant. electrical-mechanical dissociation also is known as pulseless electrical activity and is an electrical rhythm that may look wide and bizarre and irregular with no associated mechanical contraction of the ventricles. the rhythm can appear different from patient to patient. electrical-mechanical dissociation is one of the more common nonperfusing rhythms observed during cardiopulmonary arrest in small animal patients (figure - ) . when electrical-mechanical dissociation is identified, first confirm the rhythm and proceed with cpcr as previously described. electrical-mechanical dissociation is thought to be associated with high doses of endogenous endorphins and high vagal tone. the treatment of choice for electrical-mechanical dissociation is high-dose atropine ( mg/kg iv, it [ times the normal dose]) and naloxone hydrochloride ( . mg/kg iv, io, it). administer epinephrine ( . to . mg/kg diluted in ml sterile . % saline it). if the rhythm does not change within minutes, consider open-chest cardiac massage. ventricular fibrillation can be coarse (figure - ) . patients with coarse ventricular fibrillation are easier to defibrillate than those with fine defibrillation. if ventricular fibrillation is identified, initiate cpcr as described previously (figure - ) . if an electrical defibrillator is available, administer j/kg of direct current externally. when a patient in cardiopulmonary arrest is attached to ecg leads, it is important to use contact electrode paste, water-soluble gel such as ky jelly, or water, rather than any form of alcohol. electrical defibrillation of a patient who has alcohol on the ecg leads can lead to fire and thermal burns. reverse any opioid, α -agonist, and phenothiazine drugs that have been administered to the patient. if fine ventricular fibrillation is identified, administer epinephrine figure - : electrical-mechanical dissociation (emd), also known as pulseless electrical activity (pea). the complexes often appear wide and bizarre without a palpable apex beat or functional contraction of the heart. this is just one example of emd, as many shapes and complexes may be observed. organized according to whether an electrical defibrillator is available. after each intervention step, the ecg should be reevaluated and the next step initiated if v-fib is still seen. if a new arrhythmia develops, the appropriate therapy for that rhythm should be inititated. if a sinus rhythm is seen with a palpable apex beat, postresuscitation measures should be implemented. perform open-chest cpcr immediately if a pathologic condition exists that prevents enough of a change in intrathoracic pressure that closed-chest cpcr will not be effective in promoting forward blood flow (box . to perform open-chest cpcr, place the patient in right lateral recumbency. clip a wide strip of fur over the left fifth to seventh intercostal space and quickly aseptically scrub over the clipped area. using a no. scalpel blade, incise over the fifth intercostal space through the skin and subcutaneous tissue to the level of the intercostal muscles. with a mayo scissors, make a blunt stab incision through the intercostal muscles in the left sixth intercostal space. make sure that the person who is breathing for the patient deflates the lungs as you make the stab incision to avoid iatrogenic lung puncture. after the stab incision, open the tips of the mayo scissors and quickly open the muscle dorsally and ventrally to the sternum with a sliding motion. avoid the internal thoracic artery at the sternum and the intercostal arteries at the caudal aspect of each rib. cut the rib adjacent to the sternum and push it behind the rib in front of and at the caudal aspect of the incision to allow more room and better visualization if a rib spreading retractor is not available. visualize the heart in the pericardial sac. visualize the phrenic nerve, and incise the pericardium just ventral to the phrenic nerve. make sure to not cut the phrenic nerve. grasp the heart in your hand(s) and gently squeeze it from apex to base, allowing time for the ventricle to fill before the next "contraction." if the heart does not seem to be filling, administer fluids intravenously or directly into the right atrium. the descending aorta can be cross-clamped with a rummel tourniquet or red rubber catheter to improve perfusion to the brain and heart. postresuscitation care and monitoring (prolonged life support) postresuscitation care involves careful monitoring and management of the adverse effects of hypoxia and reperfusion injury on the brain and other vital organs. the first hours after an arrest are most critical, because this is the time period in which an animal is most likely to rearrest unless the underlying cause of the initial arrest has been determine and treated (table - ) . until an animal is adequately ventilating on its own, artificial ventilation by manual bagging or attaching the patient to a mechanical ventilator with supplemental oxygen must continue. the efficacy of oxygenation and ventilation can be monitored using a wright's respirometer, pulse oximetry, capnometry, and arterial blood . once an animal is extubated, administer supplemental oxygen ( to ml/ kg/minute) (see oxygen supplementation). the brain is sensitive to ischemia and reperfusion injury. the effects of cellular hypoxia and reperfusion include the development of oxygen-derived free radical species that contribute to cerebral edema. administer mannitol ( . to g/kg iv over to minutes), followed by furosemide ( mg/kg iv) minutes later, to all patients that have experienced cardiopulmonary arrest and have had successful resuscitation. mannitol and furosemide work synergistically to decrease cerebral edema formation and scavenge oxygen-derived free radical species. the combination of cardiac arrest, myocardial ischemia and acidosis, and external or internal cardiac compressions often make the myocardium irritable and predisposed to dysrhythmias following successful cpcr. start lidocaine ( to mg/kg iv, followed by to µg/kg/minute iv cri) in all patients following successful resuscitative efforts. monitor the ecg continuously for the presence of cardiac dysrhythmias and recurrence of nonperfusing rhythms. perform direct or indirect blood pressure monitoring. if a patient's systolic blood pressure is less than mm hg, diastolic pressure is less than mm hg, or mean arterial blood pressure is less than mm hg, administer positive inotropic drugs (dobutamine, to µg/kg/minute) and pressor agents (epinephrine, . to . mg/kg iv, io, it) to improve cardiac contractility, cardiac output, and core organ perfusion. the kidneys are sensitive to decreased perfusion and cellular hypoxia. place a urinary catheter and monitor urine output. in a euvolemic patient, normal urine output should be no less than to ml/kg/hour. if urine output is low, administer low-dose dopamine ( to µg/kg/minute iv cri) in an attempt to dilate afferent renal vessels and improve renal perfusion. maintain acid-base and electrolyte status within normal reference ranges. monitor serum lactate as a rough indicator of organ perfusion and cellular oxygen extraction. the presence of elevated or rising serum lactate in the face of aggressive cardiorespiratory and cerebral support makes prognosis less favorable. cole sg, otto cm, hughes d: cardiopulmonary cerebral resuscitation: a clinical practice review part i, j vet emerg crit care ( ) immediate action depends largely on recognition of the primary or secondary cause of the dysrhythmia and treating the dysrhythmia and underlying cause. diagnosis of cardiac dysrhythmias is based on physical examination findings of abnormal thoracic/cardiac auscultation, the presence of abnormal pulse rhythm and quality, and recognition of ecg abnormalities. the ecg is critical to the accurate diagnosis of dysrhythmias. ventricular dysrhythmias arise from ectopic foci in the ventricles that cause the wave of depolarization to spread from cell to cell rather than spread through fast-conducting tissue. this causes the qrs complex to appear wide and bizarre, unless the ectopic focus originates close to the atrioventricular node high in the ventricle. other ecg features of ventricular dysrhythmias include a t wave polarity that is opposite to the qrs complex and nonrelated p waves. ventricular dysrhythmias may manifest as isolated ventricular premature complexes, couplets, or triplets; bigeminy; or ventricular tachycardia. relatively slow ventricular tachycardia is known as an idioventricular rhythm and is not as hemodynamically significant as faster ventricular tachycardia. idioventricular rhythm usually is less than beats per minute and may alternate spontaneously with sinus arrhythmias (figures - to . supraventricular dysrhythmias arise from ectopic foci in the atria and are commonly associated with atrial dilatation and structural heart disease such as advanced acquired or congenital heart disease, cardiomyopathies, cardiac neoplasia, or advanced heartworm disease. occasionally, supraventricular dysrhythmias may be associated with respiratory or other systemic illness. sustained supraventricular tachycardia in the absence of underlying structural heart or systemic disease is disturbing and should alert the clinician that an accessory pathway conduction disturbance may be present, particularly in labrador retrievers. supraventricular dysrhythmias can manifest as isolated premature complexes (atrial premature complexes or contractions), sustained or paroxysmal supraventricular tachycardia (atrial tachycardia), or atrial fibrillation or flutter. in the dog, atrial fibrillation most commonly is associated with dilative cardiomyopathy. rarely and primarily in giant breed dogs, lone atrial fibrillation can occur with no underlying heart disease. atrial fibrillation and the resultant sustained elevation in ventricular rate are presumed to progress to dilative cardiomyopathy in such breeds. by comparison, atrial fibrillation is relatively uncommon in cats because of the small size of their atria but is associated most commonly with hypertrophic and restrictive cardiomyopathy. the ecg is critical to the diagnosis of a supraventricular dysrhythmia. the ecg usually demonstrates a normal appearance to the qrs complex unless aberrant conduction occurs in the ventricles, in which case the qrs can be wide but still originate from above the atrioventricular node. in most cases of a supraventricular dysrhythmia, some evidence of atrial activity including p waves, atrial flutter, or atrial fibrillation is apparent. in some cases, it may be difficult to diagnose the exact rhythm without slowing the rate down mechanically or through pharmacologic intervention. once a rhythm diagnosis is made, appropriate treatment strategies can be implemented (figures - and - ). treatment of ventricular dysrhythmias largely depends on the number of ectopic foci discharging, the rate and character of the dysrhythmia, and whether the presence of the abnormal beats is of adverse hemodynamic consequence, including risk of sudden death. many ventricular dysrhythmias, including slow idioventricular rhythms, ventricular bigeminy, or intermittent ventricular premature complexes, do not warrant antiarrhythmic therapy unless the patient is hypotensive and the dysrhythmia is thought to be contributing to the hypotension. in such cases, correction of the underlying disease process including hypoxia, pain, or anxiety often alleviates or decreases the incidence of the dysrhythmia. more serious ventricular dysrhythmias that warrant antiarrhythmic therapy (table - ) include sustained ventricular tachycardia (> beats/minute in dogs; > beats/minute in cats), multifocal ventricular premature complexes originating from more than one place in the ventricles, and the presence of r-on-t phenomena where the t wave of the preceding complex is superimposed on the qrs of the next complex with no return to isoelectric shelf in between complexes. treat these ventricular dysrhythmias immediately and aggressively. in dogs, the mainstay of emergency treatment for ventricular dysrhythmias is lidocaine therapy. administer lidocaine ( to mg/kg iv bolus) over a period of minutes to prevent the adverse side effects of seizures or vomiting. the bolus can be repeated an additional times (total dose mg/kg) over minutes, or the patient can be placed on a constant rate infusion ( to µg/kg/minute) if control of ventricular tachycardia is accomplished. also correct the patient's magnesium and potassium deficiencies to maximize the success of lidocaine therapy in the treatment of ventricular tachycardia. procainamide ( mg/kg iv slowly over to minutes) also can be used to control ventricular tachycardia. if procainamide is successful at controlling ventricular tachycardia, administer it as a constant rate infusion ( to µg/kg/minute). side effects of procainamide include vomiting, diarrhea, and hypotension. chronic oral therapy may or may not be necessary in the treatment of acute ventricular tachycardia. the decision to continue antiarrhythmic therapy depends on the underlying disease process and the expectation of persistent arrhythmogenesis of the underlying disease process. oral antiarrhythmic therapy is warranted in cases in which a serious ventricular dysrhythmia is recognized but the animal does not require hospitalization, such as the syncopal boxer with intermittent ventricular dysrhythmias and no evidence of structural heart disease. it deserves emphasis that asymptomatic, low-grade ventricular dysrhythmias probably do not require treatment. if maintenance therapy for ventricular dysrhythmias is needed, use an oral drug based on the underlying disease process, clinical familiarity, class of drug, dosing frequency, owner compliance, concurrent medications, cost, and potential adverse side effects. in the cat the mainstay of antiarrhythmic therapy is the use of a β-adrenergic antagonist. in the acute management of ventricular dysrhythmias in cases of hypertrophic, restrictive, or unclassified cardiomyopathies, consider using injectable esmolol ( . to . mg/kg iv slowly to effect) or propranolol ( . to . mg/kg iv slowly to effect), particularly if the dysrhythmia results from hyperthyroidism. for chronic oral ventricular antiarrhythmic therapy in cats, propranolol ( . to . mg po per cat q h) or atenolol ( . to . mg po per cat q - h) can be used. the decision to treat supraventricular dysrhythmias depends on the ventricular rate and the hemodynamic consequences of the dysrhythmia. for intermittent isolated atrial emergency care procainamide - mg/kg po q - h tocainide* - mg/kg po q h sotalol - mg per dog q h (start low, then titrate up to effect) mexiletine - mg/kg po q h atenolol . - . mg/kg po q - h (start low, titrate upward to effect) *do not use for longer than weeks because of idiosyncratic blindness. premature contractions, couplets, and triplets, usually no treatment is required. when the ventricular rate exceeds beats/minute, diastolic filling time is shortened, causing the heart to not fill adequately. the consequence is decreased cardiac output and decreased coronary artery perfusion. the goal of therapy is rhythm control or, in most cases, rate control. in cases of atrial fibrillation and congestive heart failure, conversion to a normal sinus rhythm rarely can be achieved, although electrocardioversion or pharmacoconversion can be attempted. in the dog a vagal maneuver can be attempted by pressing on the eyeballs or massaging the carotid body. for sustained supraventricular tachycardia, diltiazem ( . mg/kg iv), esmolol ( . to . , titrated upward to a cumulative dose of . mg/kg iv), or propranolol ( . to . mg/kg iv slowly to effect) can be administered in an attempt to slow the ventricular rate in emergent situations. administer oral diltiazem ( . mg/kg po q h), diltiazem (dilacor-xr) ( . to mg/kg po q - h), propranolol ( . to . mg/kg tid, titrated up to a maximum of . mg/kg po q h), atenolol ( . to mg/kg q - h), or digoxin ( . to . mg/kg bid or . mg/m for dogs greater than kg). in the cat a vagal maneuver can be attempted by ocular or carotid massage. (diltiazem [dilacor] to po q - h), propranolol ( . to mg/kg q - h), or atenolol ( . mg q - h) also can be administered. if structural heart disease is present, treat pulmonary edema and start angiotensin-converting enzyme inhibitor therapy. table - summarizes the drugs used in the management of supraventricular dysrhythmias. severe bradycardia often results from systemic disease, drug therapy, anesthetic agents, or hypothermia and thus rarely requires specific therapy except to treat or reverse the underlying mechanisms promoting bradycardia. hemodynamically significant bradyarrhythmias that must be treated include atrial standstill, atrioventricular block, and sick sinus syndrome. atrial standstill most commonly is associated with hyperkalemia and is seen most often in urinary obstruction, renal failure, urinary trauma with uroabdomen, and hypoadrenocorticism. characteristic ecg abnormalities observed in atrial standstill are an absence of p waves, widened qrs complexes, and tall spiked t waves (figure - ). the treatment for hyperkalemia-induced atrial standstill is to correct the underlying cause and to drive potassium intracellularly and protect the myocardium from the adverse effects of hyperkalemia. regular insulin ( . to . units/kg iv) followed by dextrose ( g/unit insulin iv, followed by . % dextrose cri to prevent hypoglycemia) or sodium bicarbonate ( meq/kg iv) can be administered to drive potassium intracellularly. calcium gluconate ( . ml/kg of % solution iv over minutes) also can be administered as a cardioprotective drug until the cause of hyperkalemia has been identified and resolved. also administer sodium chloride fluids ( . % sodium chloride iv) to promote kaliuresis. less commonly, atrial standstill is associated with atrial cardiomyopathy or silent atrium syndrome. persistent atrial standstill has been recognized without electrolyte abnormalities in the english springer spaniel and the siamese cat. short-term therapy for persistent atrial standstill includes atropine ( . mg/kg sq) until definitive treatment by implantation of a cardiac pacemaker can be performed. complete or third-degree atrioventricular block or high-grade symptomatic seconddegree atrioventricular block can be hemodynamically significant when ventricular rates are less than beats/minute in the dog. classic clinical signs include weakness, exercise intolerance, lethargy, anorexia, syncope, and occasionally seizures. advanced atrioventricular block usually is caused by advanced idiopathic degeneration of the atrioventricular node. less commonly, atrioventricular block has been associated with digoxin toxicity, magnesium oversupplementation, cardiomyopathy, endocarditis, or infectious myocarditis (lyme disease). an accurate diagnosis is made based on the ecg findings of nonconducted p waves with ventricular escape beats. first-and second-degree atrioventricular block may not be hemodynamically significant and therefore may not require therapy. initially treat third-degree (complete) or symptomatic high-grade second-degree atrioventricular block (< beats/minute) with atropine ( . mg/kg sq or im). perform a follow-up ecg in to minutes. atropine is rarely successful in treating complete atrioventricular block. also attempt treatment with isoproterenol ( . to . µg/kg/minute iv cri or . mg in ml % dextrose in water iv slowly), a pure β-agonist. definitive treatment requires permanent pacemaker implantation. consultation with a veterinary cardiologist who implants pacemakers is suggested. never attempt to convert or treat the observed ventricular escape beats with lidocaine ( figure - ) . sick sinus syndrome most commonly is recognized in the miniature schnauzer, although any dog can be affected. sick sinus syndrome usually results from idiopathic degeneration of the sinus node in the dog. in the cat, sinus node degeneration usually is associated with cardiomyopathy. dysfunction of the sinus node may manifest as marked bradycardia with periods of sinus arrest followed by junctional or ventricular escape complexes. a variant of sick sinus syndrome is the presence of severe bradycardia followed by periods of supraventricular tachycardia, often termed bradycardia-tachycardia syndrome. the most common clinical signs are syncope, exercise intolerance, and lethargy. in cats, hypertrophic cardiomyopathy is the most common form of acquired cardiac disease observed. congestive heart failure resulting from hypertrophic cardiomyopathy can occur in animals as young as to months of age. hypertrophic cardiomyopathy is characterized by stiff, noncompliant ventricles that do not relax during diastole, causing an increase in left atrial pressures and left atrial enlargement. other cardiomyopathies, including unclassified, restrictive, and dilated, are less common but also can occur in the cat. cats often develop acute exacerbation of clinical signs because of stress or arterial embolization. the rapid diagnosis of chf often is made on owner history, signalment, and physical examination findings (box - ). typical physical examination findings include a cardiac murmur or gallop dysrhythmia, abnormal breath sounds, respiratory difficulty and orthopnea, tachycardia, weak pulse quality, cool peripheral extremities, and pale or cyanotic mucous membrane. initiate immediate treatment based on physical examination findings and index of suspicion. in some cases, it is difficult to distinguish between chf and feline lower airway disease (asthma) without performing thoracic radiographs. let the animal rest and become stabilized before attempting any stressful procedures, including thoracic radiographs. immediate treatment consists of administering supplemental oxygen, decreasing circulating fluid volume with furosemide, dilating pulmonary and splanchnic capacitance vessels with topical nitroglycerine and morphine, and alleviating patient anxiety and stress (box - ). primary differential diagnoses are made based primarily on the patient's breed, age, clinical signs, history, and physical examination abnormalities. the most common differential diagnoses in a patient with chf are cardiac abnormalities and respiratory disease (chronic bronchitis [asthma], pulmonary hypertension, cor pulmonale, neoplasia). postpone diagnostic tests in any patient with suspected chf until the immediate treatments have taken effect and the patient is cardiovascularly more stable. in most cases, lateral and dorsoventral thoracic radiographs are one of the most important diagnostic tools in helping make a diagnosis of chf. increased perihilar interstitial to alveolar infiltrates are characteristic of pulmonary edema. left atrial enlargement may be observed as a "backpack" sign at the caudal cardiac waist. cardiomegaly of the right or left side also may be present in cases of valvular insufficiency. in cats, increased sternal contact and a classic valentine-shaped heart may be observed in cases of hypertrophic cardiomyopathy. perform a vertebral heart score (sum) to measure cardiac size and determine whether cardiomegaly is present (box - ). also obtain arterial blood pressure and ecg readings to determine whether hypotension and dysrhythmias are present. atrial fibrillation, ventricular premature contractions, and supraventricular tachycardia are common rhythm disturbances that can affect cardiac output adversely and influence treatment choices. the echocardiogram is a useful noninvasive and nonstressful method to determine the degree of cardiac disease present. the echocardiogram is largely user-dependent. the quality of the study is based on the experience of the operator and the quality of the ultrasound machine. echocardiography can be a useful tool in making a diagnosis of pericardial effusion, dilated or hypertrophic cardiomyopathy, cardiac neoplasia, and endocarditis. the medical management of chf is designed to improve cardiac output and relieve clinical signs. the immediate goal of therapy is to reduce abnormal fluid accumulation and provide adequate cardiac output by increasing contractility, decreasing preload and ventricular afterload, and/or normalizing cardiac dysrhythmias. strict cage rest is of utmost importance when managing a patient with chf. after initial administration of furosemide, morphine, oxygen, and nitroglycerine paste, clinical signs of respiratory distress should show improvement within minutes. if no improvement is observed, administer repeated doses of furosemide. reevaluate severe cases that are refractory to this standard treatment protocol. vasodilation should be the next step in the management of refractory cases, provided that a normal blood pressure is present. sodium nitroprusside is a potent balanced vasodilator that should be administered ( to µg/kg/minute iv cri), taking care to monitor blood pressure continuously because severe vasodilation and hypotension can occur. the goal of nitroprusside therapy is to maintain a mean arterial blood pressure of mm hg. sodium nitroprusside should not be considered in cases of refractory chf with severe hypotension. for more long-term management of chf, the use of angiotensin-converting enzyme (ace) inhibitors including enalapril ( . mg/kg po q - h), benazepril ( . mg/kg po q h), and lisinopril ( . mg/kg po q h) have become the mainstay of therapy to reduce sodium and fluid retention and decrease afterload. start angiotensin-converting enzyme inhibition as soon as a patient is able to tolerate oral medications. dobutamine ( . to µg/kg/minute cri diluted in % dextrose in water) can be administered to improve cardiac contractility, particularly in cases of dilated cardiomyopathy. at low doses, dobutamine, primarily a β-adrenergic agonist, will improve cardiac output with minimal effects on heart rate. dobutamine must be given as a constant rate infusion with careful, continuous ecg monitoring. despite minimal effects on heart rate, emergency management of specific conditions the vertebral heart sum can be calculated by performing the following steps: . measure the long axis of the heart from the apex to the carina on the lateral view and mark the distance on a sheet of paper. . measure the length of the long axis of the heart in terms of vertebral bodies, starting by counting caudally from the fourth thoracic vertebra; count the number of vertebrae that are covered by the length of the long axis of the heart. . measure the short axis of the heart at the caudal vena cava, perpendicular to the long axis of the heart. . count the number of thoracic vertebrae covered by the short axis of the heart, starting at t . . add the two numbers together to yield the vertebral heart sum; a vertebral heart sum greater than . is consistent with cardiomegaly. sinus tachycardia or ventricular dysrhythmias may develop during infusion. cats are more sensitive to the effects of dobutamine than dogs. monitor carefully for seizures and facial twitching. digoxin is a cardiac glycoside that acts as a positive inotrope and negative chronotrope in the long-term management of chf. digoxin has a long ( hours in dogs, and hours in cats) half-life and so has minimal use in the emergency management of chf. in chronic management of chf resulting from dilated cardiomyopathy or advanced mitral disease, however, digoxin is extremely useful. oral digitalization protocols have been developed but are risky in that dysrhythmias and severe gastrointestinal side effects can occur. cats with chf often have fulminant pulmonary edema, pleural effusion, arterial thromboembolism, or some combination of all three. if the pleural effusion is significant, perform therapeutic thoracocentesis to relieve pulmonary atelectasis and improve oxygenation. once the diagnosis and initial management of chf has been made, formulate a plan for continued management and monitoring. tailor the therapeutic plan to the patient based on the cause of the chf, the presence of concurrent diseases, and response to therapy. an important and often overlooked part of the successful emergency management of chf is the open communication with the owner regarding the owner's emotional and financial commitment for immediate and long-term management to ensure appropriate quality of life for each patient. pathophysiology and treatment, vet j ( ) caval syndrome resulting from severe heartworm disease is caused by the rapid maturation of a large quantity of adult worms in the right atrium and cranial and caudal venae cavae. most cases of caval syndrome occur in regions of the world where heartworm disease is highly endemic and dogs spend a large portion of time living outdoors. caval syndrome is recognized by the following clinical signs and results of biochemical analyses: acute renal and hepatic failure, enlarged right atrium and posterior vena cava, ascites, hemoglobinuria, anemia, acute collapse, respiratory distress, dic, jugular pulses, circulating microfilariae, and sometimes tricuspid insufficiency. immediate action in cases of caval syndrome in dogs involves immediate stabilization of the cardiovascular and respiratory systems with supplemental oxygen, furosemide ( mg/kg iv), and careful crystalloid fluid infusion. diagnosis of caval syndrome is based on clinical signs of cardiogenic shock with right ventricular heart failure, intravascular hemolysis, and renal and hepatic failure. thoracic radiographs reveal cardiomegaly of the right side and enlarged tortuous pulmonary arteries. a right axis deviation may be seen on ecg tracings. clinicopathologic changes observed include azotemia, inflammatory leukogram, regenerative anemia, eosinophilia, elevated hepatocellular enzyme activities, hemoglobinuria, and proteinuria. circulating microfilariae may be observed on peripheral blood smears or in the buffy coat of microhematocrit tubes. heart worm antigen tests will be strongly positive. echocardiographic changes include visualization of a large number of heartworms in the right atrium, pulmonary arteries, and vena cava, tricuspid insufficiency, and right atrial and ventricular enlargement. treatment involves surgical removal of as many of the adult heartworms as possible from the right jugular vein and right atrium. glucocorticosteroids are recommended to decrease inflammation and microangiopathic disease associated with heartworm infection. for more long-term management, administer adulticide therapy several weeks following surgery, followed by routine microfilaricide therapy and then prophylaxis. calvert pericardial effusion often develops as a consequence of neoplasia in the older dog and cat. the most common types of neoplasia that affect the heart and pericardium include hemangiosarcoma, chemodectoma, mesothelioma, and metastatic neoplasia. more rarely, other causes of pericardial effusion include benign idiopathic pericardial effusion, coagulopathy, left atrial rupture in dogs with chronic mitral valvular insufficiency, infection, or pericardial cysts. regardless of the cause of the effusion, the development of pericardial tamponade adversely affects cardiac output. cardiac output is a function of heart rate and stroke volume. stroke volume depends on cardiac preload. the presence of pericardial effusion can impede venous return to the heart and thus adversely affect preload. in addition, as preload decreases, heart rate reflexively increases in an attempt to maintain normal cardiac output. as heart rate increases more than beats/minute, diastolic filling is impaired further, and cardiac output further declines. animals with pericardial effusion often demonstrate the classic signs of hypovolemic or cardiogenic shock: anorexia, weakness, lethargy, cyanosis, cool peripheral extremities, tachycardia, weak thready pulses, hypotension, and collapse. physical examination abnormalities may include muffled heart sounds, thready femoral pulses, pulsus paradoxus, jugular venous distention, weakness, tachycardia, cyanosis, and tachypnea. electrocardiogram findings may include low amplitude qrs complexes (< . mv), sinus tachycardia, ventricular dysrhythmias, or electrical alternans (figure - ) . thoracic radiographs often demonstrate a globoid cardiac silhouette, although the cardiac silhouette rarely may appear normal with concurrent clinical signs of cardiogenic shock in cases of acute hemorrhage. in such cases the removal of even small amounts of pericardial effusion by pericardiocentesis can increase cardiac output exponentially and alleviate clinical signs (table - ) . unless an animal is dying before your eyes, ideally perform an echocardiogram to attempt to determine whether a right atrial, right auricular, or heart base mass is present before pericardiocentesis. before attempting pericardiocentesis, assemble all of the required supplies (box - ) . to perform pericardiocentesis, follow this procedure: . place the patient in sternal or lateral recumbency. . attach ecg leads to monitor the patient for dysrhythmias during the procedure. . clip a -cm square caudal to the right elbow over the fifth to seventh intercostal space. . aseptically scrub the clipped area, and infuse to mg/kg of % lidocaine mixed with a small amount of sodium bicarbonate just dorsal to the sternum at the sixth intercostal space. bury the needle to the hub, and inject the lidocaine as you withdraw the needle. . while the local anesthetic is taking effect, assemble the intravenous extension tubing, three-way stopcock, and -ml syringe. . wearing sterile gloves, make a small nick incision in the skin to decrease drag on the needle and catheter during insertion. . slowly insert the needle and catheter, watching for a flash of blood in the hub of the needle, and simultaneously watching for cardiac dysrhythmias on the ecg monitor. . once a flash of blood is observed in the hub of the needle, advance the catheter off of the stylette further into the pericardial sac, and remove the stylette. . attach the length of intravenous extension tubing to the catheter, and have an assistant withdraw the fluid slowly. . place a small amount of fluid in a red-topped tube, and watch for clots. clot formation could signify that you have penetrated the right ventricle inadvertently or that active hemorrhage is occurring. withdraw as much of the fluid as possible, and then remove the catheter. monitor the patient closely for fluid reaccumulation and recurrence of clinical signs of cardiogenic shock. less rd, bright jm, orton ec: intrapericardial cyst causing cardiac tamponade in a cat, j am anim hosp assoc ( ) foreign bodies within the ear canal (e.g., foxtails) can present as emergencies because of acute inflammation and pressure necrosis of the tissue of the external auditory meatus causing pain and discomfort. clinical signs may be limited to incessant head shaking or scratching of the ear canal. complete examination of the ear canal and removal of any foreign body often requires administration of a short-acting anesthetic agent. once the animal has been restrained sufficiently and placed under anesthesia, carefully examine the ear canal and remove any foreign material with an alligator forceps. stimulation of the ear canal can cause awakening after removal of all debris and detritus, gently wipe the internal and external ear canal with a sterile gauze. place a topical antimicrobial-antifungal-steroid ointment such as otomax in the ear every to hours. if pain and discomfort is severe, systemically effective opioids or nsaids may be required. otitis externa is a common emergency that causes excessive head shaking, scratching, and purulent malodorous aural discharge. clean the ear canal with an irrigating solution such as epiotic and wipe it clean of debris. perform a complete aural examination to determine whether a foreign body or tumor is present and whether the tympanic membrane is intact. heat-fix any discharge and examine it cytologically for bacteria and fungal organisms. following careful cleansing, instill a topical antibiotic-antifungal-steroid ointment. in severe cases in which the ear canal has scarred and closed down with chronicity, consider administering systemically effective antibiotics (cephalexin, mg/kg po tid) and antifungal agents (ketoconazole, mg/kg po q h) instead of topical therapy. systemically effective steroids (prednisone or prednisolone, . mg/kg po q h) may be indicated in cases of severe inflammation to decrease pruritus and patient discomfort. presentation of a patient with otitis interna often is characterized by torticollis, head tilt, nystagmus, circling to the affected side, or rolling. fever, pain, vomiting, and severe depression may accompany clinical signs. most cases of severe otitis interna are accompanied by severe otitis media. both conditions must be treated simultaneously. the most common causes of otitis interna are staphylococcus aureus, pseudomonas, escherichia coli, or proteus spp. otitis interna can develop by infection spreading across the tympanic membrane, through the eustachian tubes, or by hematogenous spread from the blood supply to the middle ear. in most cases of otitis media, the tympanic membrane is ruptured. perform a culture and susceptibility test of the debris behind the tympanic membrane and within the aural canal. carefully clean the external ear canal. medicate with a topical combination antibiotic, antifungal, and antibiotic ointment. administer high-dose antibiotics (cephalexin, mg/kg po q h, or enrofloxacin, to mg/kg po q h). if the tympanic membrane is not ruptured but appears swollen and erythematous, a myringotomy may need to be performed. if clinical signs of otitis media persist despite topical and systemic therapy, radiographic or ct/mri examination of the tympanic bullae may be required. chronic shaking of the head and ears or aural trauma (bite wounds) causes disruption of the blood vessels and leads to the development of unilateral or bilateral aural hematomas. aural hematomas are clinically significant because they cause patient discomfort and are often due to the presence of some other underlying problem such as otitis externa, atopy, or aural foreign bodies. acute swelling of the external ear pinna with fluid is characteristic of an aural hematoma. in some cases, swelling can be so severe that the hematoma breaks open, bathing the patient and external living environment in blood. when a patient has an aural hematoma, investigate the underlying cause. perform a complete aural examination to determine whether an aural foreign body, otitis externa, or atopy are present. carefully examine and gently clean the inner ear canal. treat underlying causes. management of an aural hematoma involves draining the hemorrhagic fluid from the aural tissue and tacking the skin down in multiple places to prevent reaccumulation of fluid until the secondary cause is resolved. many techniques have been described to surgically tack down the skin overlying the hematoma. after the animal has been placed under general anesthesia, lance the hematoma down the middle with a scalpel blade and remove the fluid and blood clot. tack down the skin with multiple through-and-through interrupted or mattress sutures through the ear. some clinicians prefer to suture through and attach a sponge or length of x-ray film to the front and back of the ear for stabilization and support. more recently, a laser can be used to drill holes in the hematoma and tack the skin down in multiple areas. compress the ear against the head with a compression bandage, whenever possible, for to days after the initial surgery, and then recheck the ear. the patient must wear an elizabethan collar until the surgical wound and hematoma heal to prevent selfmutilation. also systemically treat underlying causative factors such as otitis externa with antibiotics, antifungals, and steroids as indicated. investigate and treat other underlying causes such as hypothyroidism or allergies. bass electrocution usually is observed in young animals after they have chewed on an electric cord. other causes of electrocution include use of defective electrical equipment or being struck by lightning. electric current passing through the body can produce severe dysrhythmias, including supraventricular or ventricular tachycardia and first-and thirddegree atrioventricular block. the electric current also can produce tissue destruction from heat and electrothermal burns. electrocution also commonly results in noncardiogenic pulmonary edema caused by massive catecholamine release and increase in pulmonary vascular pressures during the event. ventricular fibrillation can occur, although that depends on the intensity and path of the electrical current and duration of contact. clinical signs of electrocution include acute onset of respiratory distress with moist rales, and localized necrosis or thermal burns of the lips and tongue. often the skin at the commissures of the mouth appears white or yellow and firm to the touch. muscle fasciculations, loss of consciousness, and ventricular fibrillation may occur. thoracic radiographs often reveal an increased interstitial to alveolar lung pattern in the dorsocaudal lung fields. noncardiogenic pulmonary edema can develop up to to hours after the initial incident. the first hours are most critical for the patient, and then prognosis improves. the most important aspect in the treatment of the patient with noncardiogenic pulmonary edema is to minimize stress and to provide supplemental oxygen, with positive pressure ventilation, when necessary. although treatment with vasodilators (low-dose morphine) and diuretics (furosemide) can be attempted, noncardiogenic pulmonary edema is typically resistant to vasodilator and diuretic therapy. positive inotropes and pressor drugs may be necessary to treat shock and hypotension. opioid drugs (morphine, hydromorphone, oxymorphone) may be useful in controlling anxiety until the pulmonary edema resolves. administer broad-spectrum antibiotics (cefazolin; amoxicillin and clavulanic acid [clavamox]) to treat thermal burns. use analgesic drugs to control patient discomfort. if thermal burns are extensive and prohibit adequate food intake, place a feeding tube as soon as the patient's cardiovascular and respiratory function are stable and the patient can tolerate anesthesia. prolapse of the uterus occurs in the immediate postparturient period in the bitch and queen. excessive straining during or after parturition causes the uterus to prolapse caudally through the vagina and vulva. immediate intervention is necessary. examine the bitch or queen for a retained fetus. treatment consists of general anesthesia to replace the prolapsed tissue. if the uterus is edematous, physical replacement may be difficult or impossible. application of a hypertonic solution such as hypertonic ( %) saline or dextrose ( %) to the exposed endometrium can help shrink the tissue. that, combined with gentle massage to stimulate uterine contraction and involution and lubrication with sterile lubricating jelly, can aid in replacement of the organ into its proper place. to ensure proper placement in the abdominal cavity and to prevent recurrence, perform an exploratory laparotomy and hysteropexy. postoperatively, administer oxytocin ( to units im) to cause uterine contraction. if the uterus contracts, it is usually not necessary to suture the vulva. administer antibiotics postoperatively. recurrence is uncommon, even with subsequent pregnancies. if the tissue is damaged or too edematous to replace or if the tissue is devitalized, traumatized or necrotic, perform an ovariohysterectomy. in some instances, replacement of the damaged tissue is not necessary before removal. pyometra occurs in dogs and cats. the disease process occurs as a result of infection overlying cystic endometrial hyperplasia under the constant influence of progesterone. during the -month luteal phase after estrus or following copulation, artificial insemination, or administration of hormones (particularly estradiol or progesterone), the myometrium becomes relaxed and favors a quiescent environment for bacterial proliferation. clinical signs of pyometra are associated with the presence of bacterial endotoxin and sepsis. early, affected animals become lethargic and anorectic. polyuria with secondary polydipsia is often present because of the influence of bacterial endotoxin on renal tubular concentration. if the cervix is open, purulent or mucoid vaginal discharge may be observed. later in the course of pyometra, vomiting, diarrhea, and progressive debilitation resulting from sepsis occur. diagnosis is based on clinical signs in an intact queen or bitch and radiographic or ultrasonographic evidence of a fluid-filled tubular density in the ventrocaudal abdomen, adjacent to the urinary bladder (figures - and - ) . treatment of open and closed pyometra is correction of fluid and electrolyte abnormalities, administration of broad-spectrum antibiotics, and ovariohysterectomy. close pyometra is a life-threatening septic condition. open pyometra also can become life-threatening and so should be treated aggressively. in closed pyometra, conservative medical therapy is not advised. administration of prostaglandins and oxytocin do not reliably cause the cervix to open and can result in ascending infection from the uterus into the abdomen or uterine rupture, both of which can result in severe peritonitis. for animals with an open pyometra, ovariohysterectomy is the most reliable treatment for chronic cystic endometrial hyperplasia. although less successful than ovariohysterectomy, medical therapy may be attempted in breeding bitches as an alternative to surgery. the most widely used medical therapy in the breeding queen and bitch is administration of prostaglandin f α . this drug has not been approved for use in the queen or bitch in the united states. to proceed with medical management of pyometra, first determine the size of the uterus. start the patient on antibiotic therapy (ampicillin, mg/kg iv q h, or enrofloxacin, mg/kg po q h). administer the prostaglandin f α ( µg/kg sq q h) for to days until the size of the uterus approaches normal. measure serum progesterone concentrations if the bitch is in diestrus. as the corpus luteum degrades under the influence of prostaglandin f α , serum progesterone levels will decline. prostaglandin f α is an abortifacient and thus should not be administered to the pregnant bitch or queen. clinical signs of a reaction to prostaglandin f α can occur within to minutes in the bitch and can last for as long as minutes. clinical signs of a reaction include restlessness, hypersalivation, panting, vomiting, defecation, abdominal pain, fever, and vocalization. in a very ill animal, death can occur. the efficacy of prostaglandin f α is limited and may require more than one treatment. the bitch should be bred on the next heat cycle and then spayed because progressive cystic endometrial hyperplasia will continue to occur. acute metritis is an acute bacterial infection of the uterus that typically occurs within to weeks after parturition. the most common organism observed in metritis is e. coli ascending from the vulva and vaginal vault. sepsis can progress rapidly. clinical signs of acute metritis include inability to nurse puppies, anorexia, lethargy, foul-smelling purulentsanguineous vaginal discharge, vomiting, or acute collapse. physical examination may reveal fever, dehydration, and a turgid distended uterus. septic inflammation will be observed on vaginal cytologic examination. an enlarged uterus can be observed with abdominal radiographs and ultrasonography. treatment of acute metritis is directed at restoring hydration status with intravenous fluids and treating the infection with antibiotics. because the primary cause of metritis is e. coli infection, start enrofloxacin ( mg/kg iv or po once daily) therapy. as soon as the patient's cardiovascular status is stable enough for anesthesia, perform an ovariohysterectomy. if the patient is not critical and is a valuable breeding bitch, medical therapy can be attempted. medical management of acute bacterial metritis includes administration of oxytocin ( to units q h for three treatments) or administration of prostaglandin f α ( µg/kg/day for to days) to evacuate the uterine exudate and increase uterine blood flow. either drug should be used concurrently with antibiotics. rupture of the gravid uterus is rare in cats and dogs but has been reported. uterine rupture may occur as a consequence of parturition or result from blunt abdominal trauma. feti expelled into the abdominal cavity may be resorbed but more commonly cause the development of peritonitis. if fetal circulation is not disrupted, the fetus actually may live to term. uterine rupture is an acute surgical emergency. an ovariohysterectomy with removal of the extrauterine puppies and membranes is recommended. if only one horn of the uterus is affected, a unilateral ovariohysterectomy can be performed to salvage the remaining unaffected puppies and preserve the breeding potential for the valuable bitch. if uterine rupture occurs because of pyometra, peritonitis is likely, and copious peritoneal lavage should be performed at the time of surgery. the patient should be placed on to days of antibiotic therapy (amoxicillin or amoxicillin and clavulanic acid [clavamox] with enrofloxacin). vaginal prolapse occurs from excessive proliferation and hyperplasia of vaginal tissue while under the influence of estrogen during proestrus (figure - ) . the hyperplastic tissue usually recedes during diestrus but reappears with subsequent heat cycles. vaginal prolapse can be confused with vaginal neoplasia. the former condition occurs primarily in younger animals, whereas the latter condition occurs primarily in older animals. treatment for vaginal hyperplasia or prolapse generally is not required if the tissue remains within the vagina. the proliferation can lead to dysuria or anuria, however. in some cases, the tissue becomes emergency care dried out and devitalized or becomes traumatized by the animal. such extreme cases warrant immediate surgical intervention. the treatment for vaginal prolapse consists of ovariohysterectomy to remove the influence of estrogen, placement of an indwelling urinary catheter if the patient is dysuric, and protection of the hyperplastic tissue until it recedes on its own. although surgical resection of the hyperplastic tissue has been recommended, excessive hemorrhage after removal can occur, and so the procedure should not be attempted. the patient should wear an elizabethan collar at all times to prevent selfmutilation. administer broad-spectrum antibiotics for a minimum of to days or until the hyperplastic tissue recedes. keep the tissue clean with saline solution. dystocia, or difficult birth, can occur in the dog and cat but is more common in the dog. a diagnosis of dystocia is made based on the time of onset of visible labor and the time in which the last puppy or no puppy has been born, the intensity and timing of contractions, the timing of when the amniotic membranes first appear, the condition of the bitch, and the timing of gestation. causes of dystocia can be maternal or fetal and include primary or secondary uterine inertia, narrowing of the pelvic canal, hypocalcemia, psychological disturbances, or uterine torsion. maternal-fetal disproportion, or large fetus size in relation to the bitch or queen, also can result in dystocia (box - ). obtain an abdominal radiograph for all cases of suspected dystocia at the time of presentation to determine the size of the fetus, presentation of the fetus (both anterior or posterior presentation can be normal in the bitch or queen, but fetal malpositioning can cause dystocia), and whether there is radiographic evidence of a uterine rupture or torsion. if maternal-fetal disproportion, uterine torsion, or uterine rupture is observed, take the patient immediately to surgery. if the puppies or kittens are in a normal position for birth, medical management can be attempted. clip the perineum and aseptically scrub it. wearing sterile gloves, insert a lubricated finger into the vagina and palpate the cervix. massage (or "feather") the dorsal wall of the vagina to stimulate contractions. place an intravenous catheter, and administer oxytocin ( to units im), repeating up to times at -minute intervals. in some cases, hypoglycemia or hypocalcemia can contribute to uterine inertia. administration of a calciumcontaining solution (lactated ringer's solution) with . % dextrose is advised. alternately, administer % calcium gluconate ( mg/ kg iv slowly). if labor has not progressed after hour, immediately perform a cesarean section. uterine torsion is an uncommon emergency seen in the gravid and nongravid uterus and has been reported in dogs and cats. the onset of clinical signs of abdominal pain and straining as if to whelp/queen or defecate is usually acute and constitutes a surgical emergency. in some cases, there may have been a history of delivery of a live or dead fetus. vaginal discharge may or may not be present. radiographs or ultrasound examination reveal a fluid-filled or air-filled tubular density in the ventral abdomen. treatment consists of placing an intravenous catheter, stabilizing the patient's cardiovascular status with intravenous fluids and sometimes blood products, and performing an immediate ovariohysterectomy. if there are viable feti, the uterus should be delivered en mass and the puppies or kittens delivered. the expulsion of one or more fetus before term is known as spontaneous abortion. in dogs and cats, it is possible to expel or abort one or more fetuses and still carry viable fetuses to term and deliver normally. clinical signs of spontaneous abortion include vaginal discharge and abdominal contractions. in some cases, the fetus is found, or there may be evidence of fetal membranes or remnants. causes of spontaneous abortion in dogs include brucella canis, herpesvirus, coronavirus, and toxoplasmosis. in cats, herpesvirus, coronavirus, and feline leukemia virus can cause spontaneous abortion. in both species, trauma, hormonal factors, environmental pathogens, drugs, and fetal factors also can result in spontaneous abortion. the safest method of pregnancy termination in the bitch or queen is by performing an ovariohysterectomy. oral diethylstilbesterol is not an effective mechanism of pregnancy termination in the bitch. a so-called mismating shot, an injection of estradiol cypionate ( . mg/lb im) is effective at causing termination of an early pregnancy but can be associated with severe side effects, including bone marrow suppression and pyometra. estradiol cypionate is not approved for use in the bitch or queen and is not recommended. prostaglandin f α is a natural abortifacient in the bitch if treatment is started within days of cytologic evidence of diestrus (noncornified epithelium on a vaginal smear). the prostaglandin f α causes lysis of the corpora lutea and a rapid decline in progesterone concentration. the prostaglandin f α is administered for a total of eight injections ( µg/kg q h for days), along with atropine ( to µg/kg sq). side effects can occur within to minutes of injection and include restlessness, panting, salivation, abdominal pain, urination, vomiting, and diarrhea. walking the patient for to minutes after each treatment sometimes decreases the intensity of the reactions. bitches in the first half of the pregnancy often resorb the embryos. if prostaglandin f α is administered in the second half of the pregnancy, the fetuses are aborted within to days of treatment. measure serum progesterone concentrations at the end of treatment to ensure complete lysis of the corpus luteum. prostaglandin f α is not approved for pregnancy termination in the bitch. in cats, prostaglandin f α can terminate pregnancy after day of gestation. prostaglandin f α should be used only in healthy queens ( to µg/kg sq q h for days). side effects in the queen are similar to those observed in the bitch but typically have a shorter duration ( to minutes). prostaglandin f α is not approved for use in cats in the united states. the use of prostaglandin f α does not preclude breeding and pregnancy at a later date. biddle d, macintire dk: obstetrical emergencies, clin tech small anim pract ( ) in the dog and cat the majority of injuries to the scrotum are associated with animal fights or shearing and abrasive injuries sustained in accidents involving automobiles. scrotal injuries should be categorized as superficial or penetrating. treatment of superficial injuries to the scrotum includes cleaning the wound with dilute antimicrobial cleanser and drying it. administer antiinflammatory doses of steroids (prednisolone, . to . mg/kg po q - h) or nsaids (carprofen, . mg/kg po q h in dogs) for the first several days after scrotal injury to prevent or treat edema. administer topical antibiotic ointment until the wound heals. in most cases, place an elizabethan collar to prevent self-mutilation. prognosis is generally favorable; however, semen quality may be affected for months after injury because of scrotal swelling and increased scrotal temperature. penetrating injuries to the scrotum are more serious and are associated with severe swelling and infection. surgically explore and debride penetrating scrotal wounds. administer systemically effective antibiotics and analgesics. in extreme cases, particularly those that involve the testicle, consider castration and scrotal ablation. scrotal dermatitis is common in intact male dogs and can be associated with direct physical injury, self-infliction from licking, chemical irritation, burns, or contact dermatitis. in affected animals, the scrotum can become extremely inflamed, swollen, and painful. if left untreated, pyogranulomatous dermatitis can develop. make an attempt to determine whether an underlying systemic illness is present that could predispose the animal to scrotal dermatitis. widespread vasculitis with scrotal edema, pain, fever, and dermatitis has been associated with rickettsia rickettsii (rocky mountain spotted fever) infection. brucella canis also has been associated with scrotal irritation and dermatitis. if scrotal dermatitis follows from an infectious cause, empiric use of glucocorticosteroids potentially can make the condition worse by suppressing immune function. empiric treatment with antibiotics also potentially can confound making an accurate diagnosis. treatment of scrotal dermatitis is to eliminate predisposing causes, if possible. place an elizabethan collar at all times to prevent self-mutilation. bathe the scrotum with a mild antimicrobial soap and dry it to remove any offending chemical irritants. topical medications including tar shampoo, tetracaine, neomycin, and petroleum can cause further irritation and are contraindicated. use oral or parenteral administration of glucocorticosteroids or nsaids to control discomfort and inflammation. scrotal hernias occur when the contents of the abdomen (intestines, fat, mesentery, omentum) protrude through the inguinal ring into the scrotal sac. like inguinal hernias, scrotal definitive therapy for a scrotal hernia involves exploratory laparotomy and surgical reduction of the contents of the hernia, surgical correction of the rent in the inguinal ring, and castration. trauma to the epididymis or testicle can cause testicular pain and swelling of one or both testes. treat penetrating trauma to the testicle by castration to prevent infection and selfmutilation. administer oral antibiotics (amoxicillin or amoxicillin-clavulanate) for to days after the injury. nonpenetrating injuries to the scrotum and testicle rarely may cause acute testicular hemorrhage or hydrocele formation. palpation of the affected area often reveals a peritesticular, soft, compliant area. treatment consists of cool compresses on the scrotum and testicle and administration of antiinflammatory doses of glucocorticosteroids or nsaids. if the swelling does not resolve spontaneously in to days, consider surgical exploration and drainage. increased scrotal temperature and testicular inflammation can affect semen quality for months after the initial incident. testicular torsion, or torsion of the spermatic cord, causes rotation of the testicle, ultimately causing obstruction to venous drainage. testicular torsion often is associated with a neoplastic mass of a retained testicle within the abdomen but also can be observed with nonneoplastic testes located within the scrotum. the predominant clinical signs are pain, stiff stilted gait, and the presence of an abnormally swollen testicle (if located within the scrotum). if an intraabdominal testicular torsion is present, pain, lethargy, anorexia, and vomiting can occur (see acute condition in the abdomen). an intraabdominal mass may be palpable. perform an abdominal or testicular ultrasound, preferably with color flow doppler to evaluate perfusion to the testicle. treatment involves surgical removal of the involved testes. bacterial infections of the testicle or epididymis most commonly are caused by ascending infections of the normal bacterial flora of the prepuce or urethra. common inhabitants include escherichia coli, staphylococcus aureus, streptococcus spp., and mycobacterium canis. brucella canis and r. rickettsii are also capable of causing orchitis and epididymitis in the dog. clinical signs of orchitis or epididymitis include testicular enlargement, stiff stilted gait, and reluctance to walk. physical examination often reveals a fever and self-induced trauma to the scrotum from licking or chewing at the inflamed area. collect a semen sample by ejaculation, and culture it to identify the causative organism. alternately, collect samples by needle aspiration of the affected organ(s) and test serologically for b. canis. treatment of infectious orchitis involves a minimum of to weeks of specific antimicrobial therapy, based on culture and susceptibility testing, whenever possible. if a bacterial culture cannot be obtained, initiate fluoroquinolone therapy (enrofloxacin, mg/kg po q h). doxycycline ( mg/kg po bid for days) has been shown to suppress but not eradicate b. canis infection. testicular inflammation and increased temperature can affect sperm quality for months after infection. the most common causes of acute prostatitis are associated with acute bacterial infection (e. coli, proteus spp., pseudomonas spp., and mycoplasma spp.). less common causes include fungal infection (blastomyces dermatitidis) or anaerobic bacterial infection. acute prostatitis is characterized by fever, caudal abdominal pain, lethargy, anorexia, blood in the ejaculate, hematuria, dyschezia, and occasionally stranguria or dysuria. the patient often appears painful and depressed and may be dehydrated on physical examination. symmetric or asymmetric prostatomegaly and prostate pain may be evident on rectal palpation. in severely affected dogs, clinical signs of tachycardia, hyperemic or injected mucous membranes, bounding pulses, lethargy, dehydration, and fever may be present because of sepsis. death can occur within days if a prostatic abscess ruptures. diagnosis of acute prostatitis is confirmed based on the presenting clinical signs, neutrophilic leukocytosis (with or without a left shift), and positive urine culture results. prostatic samples may be obtained from the prostatic portion of the ejaculate, prostatic massage, urethral discharge, urine, or (less commonly) prostatic aspirate. although semen samples can yield positive bacterial cultures, dogs with acute prostatitis are often unwilling to ejaculate. radiography may reveal an enlarged prostate, but this alone does not confirm the diagnosis of prostatitis. an abdominal ultrasound often reveals prostatic abscessation and allows for the collection of samples from the affected area(s) via prostatic aspirate. aspiration of the affected tissue potentially can wick infection into periprostatic tracks. cytologic examination of the patient's ejaculate or prostatic wash from a dog with acute prostatitis reveals numerous inflammatory cells and may contain bacterial organisms. the treatment of a patient with acute prostatitis is directed at correcting dysuria and constipation associated with prostatic enlargement. enrofloxaxin ( mg/kg po sid) can penetrate the inflamed prostatic tissue and is effective in treating gram-negative and mycoplasma spp. infections. ciprofloxacin does not appear to penetrate prostatic tissue as readily. alternatives to enrofloxacin therapy are trimethoprim-sulfamethoxazole ( mg/kg po q h) or chloramphenicol ( - mg/kg po q h) for a minimum of to weeks. castration is recommended because benign prostatic hyperplasia may be a predisposing factor in the development of acute prostatitis. do not perform castration until the patient has been on antibiotic therapy for a minimum of days, to prevent the surgical complication of schirrous cords. finasteride (proscar, mg/kg po q h), an antiandrogen α-reductase inhibitor, may help reduce the size of prostatic tissue until the effects of castration are observed. if a prostatic abscess is present, perform marsupialization, surgical drainage, or ultrasonographic drainage. surgical therapy is associated with a large incidence of complications, including incontinence, chronic drainage from fistulas and stomas, septic shock, and death. fracture of the os penis is an uncommon condition encountered in male dogs. os penis fractures can occur with minimal soft tissue damage but cause hematuria and dysuria. on physical examination, urethral obstruction and crepitus in the penis are found. a lateral abdominal radiograph is usually sufficient to document the fracture. treatment consists of conservative therapy, in most cases, and consists primarily of analgesia administration. if the urethra also is damaged, place a urethral catheter for to days to allow the urethral mucosa to heal. fractures of the os penis that are comminuted or severe enough to cause urethral obstruction require open reduction and fixation, partial penile amputation, or antescrotal (prescrotal) urethrostomy. lacerations of the penis cause significant bleeding because of the extensive vascular supply to the penis. dogs and cats tend to lick penile lacerations and prevent adequate clot formation. sedation or general anesthesia often is required to evaluate and treat the laceration. after sedation or general anesthesia, place a urinary catheter and examine the penis under a stream of cold water. small lacerations can be managed with cold compresses and one to several absorbable sutures. extensive suturing usually is not required. prevent erection by isolating the patient from females in estrus or allowing excitement or excessive activity. place an elizabethan collar to prevent self-mutilation. initiate systemic antibiotic therapy to prevent infection. the inability to withdraw the penis into the prepuce in male dogs or cats is known as paraphimosis. paraphimosis usually develops following an erection in young male dogs and in emergency care older dogs after coitus. mucosal edema, hemorrhage, self-mutilation, and necrosis requiring penile amputation can occur if left untreated. treatment consists of applying cold water to the penis and reducing edema with application of an osmotic substance such as sugar. examine the base of the penis for hair rings that can prevent retraction of the penis into the prepuce. rinse the penis carefully with cold water and lubricate it with sterile lubricant and replace it into the prepuce. if the penis cannot be reduced easily into the prepuce, anesthetize the patient and make a small incision at the lateral aspect of the preputial opening. replace the penis and close the incision with absorbable suture. place a purse-string suture and leave it in place for several days to prevent recurrence. instill topical antimicrobial ointment with steroids into the prepuce several times a day. in severe cases, a urinary catheter may need to be placed to prevent urethral obstruction, until penile swelling and edema resolve. place an elizabethan collar to prevent excessive licking during the healing process. prolapse of the distal urethra is a condition usually confined to intact male english bulldogs, although isolated incidences also have been reported in yorkshire and boston terriers. the exact cause of this condition is unknown but usually is associated with a condition that causes increased intraabdominal pressure or urethral straining, including sexual excitement, coughing, vomiting, obstructed airway or brachycephalic airway syndrome, urethral calculi, genitourinary tract infection, and masturbation. the urethral prolapse usually appears as a mushroom-tip congested, irritated mass at the end of the penis that may or may not bleed (figure - ) . in some cases, bleeding occurs or worsens with sexual excitement. clinical signs associated with the prolapsed urethra include excessive licking of the prepuce, stranguria, and preputial bleeding. once the mass is observed, other differential diagnoses include transmissible venereal tumor, urethral polyp, trauma, urethritis, and neoplasia. in most cases, however, the prolapse occurs in intact young dogs, making neoplastic conditions less likely. treatment for urethral prolapse should occur at the time of diagnosis to prevent selfinduced trauma and infection. immediate therapy includes manual reduction of the prolapsed tissue and placement of a purse-string suture around an indwelling urinary catheter. the purse-string suture can remain in place for up to days until definitive repair. until the time of surgery, place an elizabethan collar on the patient to prevent self-mutilation. several forms of surgical correction have been described. in some cases, surgical resection of the prolapsed tissue with apposition of the urethral and penile mucosa can be attempted. more recently, a technique involving placement of several mattress sutures to reduce and secure the prolapsed tissue has been described. recurrence of prolapse can occur with either technique, particularly if the inciting event recurs. because there may be a genetic predisposition in this breed and because the prolapse can recur with sexual excitement, neutering should strongly be recommended. local freezing or frostbite most commonly affects the peripheral tissues of the ears, tail, paws, and genitalia that are sparsely covered with fur, are poorly vascularized, and may have been traumatized previously by cold. clinical signs of frostbite are paleness and appearance of a blanched pink to white discoloration to the skin. the skin also may appear black and necrotic. immediate treatment consists of slowly rewarming the affected area with moist heat at . ° c ( °f) or by immersion in warm water baths. analgesics may be required to alleviate patient discomfort. carefully dry the injured areas and protect them from further trauma. the use of prophylactic antibiotics is controversial because it can promote resistant bacterial infection. use of antibiotics should be based on the presence of infection. treatments that are ineffective and may be harmful include rubbing the affected areas, pressure bandages, and ointments. corticosteroids can decrease cellular immunity and promote infection and are therefore contraindicated. many frostbitten areas that appear nonviable can regain function gradually. use care when removing areas of necrotic tissue. affected areas may take several days to a week before fully manifesting areas of demarcation between healthy viable and necrotic nonviable tissue. chilling of the entire body from exposure or immersion in extremely cold water results in a decrease in core body temperature and physiologic processes that become irreversible when the body temperature falls below °c ( °f). mild hypothermia can be °to °c, moderate hypothermia from °to °c, and severe hypothermia below °c. the duration of exposure and the general condition of the animal influences its ability to survive. clinical signs and consequences associated with hypothermia include shivering, vasoconstriction, mental depression, hypotension, sinus bradycardia, hypoventilation with decreased respiratory rate, increased blood viscosity, muscle stiffness, atrial and ventricular irritability, decreased level of consciousness, decreased oxygen consumption, metabolic (lactic) acidosis, respiratory acidosis, and coagulopathies including dic. if the animal is breathing, administer warm, humidified oxygen at to breaths per minute. if the animal is not breathing or is severely hypoventilating, endotracheal intubation with mechanical ventilation may be necessary. place an intravenous catheter and infuse warmed crystalloid fluids. if the blood glucose is less than mg/dl, add supplemental dextrose ( . %) to the crystalloid fluids. monitor the core body temperature and ecg closely. rewarming should occur in the form of external circulating warm water blankets, radiant heat, and circulating warm air blankets (bair hugger). never use a heating pad, to avoid iatrogenic thermal burn injury. severe hypothermia may require core rewarming in the form of intraperitoneal fluids ( to ml/kg of lactated ringer's solution warmed to . °c [ °f]). place a temporary peritoneal dialysis catheter, and repeat the dialysis every minutes until the patient's body temperature reaches . °to . °c ( °to °f). the body temperature should rise slowly, ideally no more than °f per hour. because the response of the body to drugs is unpredictable, avoid administering drugs whenever possible, until the body temperature returns to normal. complications observed during rewarming include dic, cardiac dysrhythmias including cardiac arrest, pneumonia, pulmonary edema, cns edema, ards, and renal failure. heat stroke and heat-induced illness in dogs can be associated with excessive exertion, exposure to high environmental temperatures, stress, and other factors that cause an inability to dissipate heat. brachycephalic breeds, obesity, laryngeal paralysis, and older animals with cardiovascular disease can be particularly affected. hyperthermia is defined as a rectal temperature of °to °c ( °to °f). clinical signs of hyperthermia include congested hyperemic mucous membranes, tachycardia, and panting. more severe clinical signs include collapse (heat prostration), ataxia, vomiting, diarrhea, hypersalivation, muscle tremors, loss of consciousness, and seizures. heat-induced illness can affect all major organ systems in the body because of denaturation of cellular proteins and enzyme activities, inappropriate shunting of blood, hypotension, decreased oxygen delivery, and lactic acidosis. cardiac dysrhythmias, interstitial and intracellular dehydration, intravascular hypovolemia, central nervous dysfunction, slough of gastrointestinal mucosa, oliguria, and coagulopathies can be seen as organ function declines. excessive panting can result in respiratory alkalosis. poor tissue perfusion results in a metabolic acidosis. loss of water in excess of solutes such as sodium and chloride can lead to a free water deficit and severe hypernatremia. a marked increase in pcv occurs because of the free water loss. severe abnormalities in electrolytes and ph can lead to cerebral edema and death. treatment goals for the patient with heat-induced illness are to lower the core body temperature and support cardiovascular, respiratory, renal, gastrointestinal, neurologic, and hepatic functions. at the scene the veterinarian or caretaker can spray the animal with tepid (not cold) water. immersion in cold water or ice baths is absolutely contraindicated. cold water and ice will cause extreme peripheral vasoconstriction, inhibiting the patient's ability to dissipate heat through conductive and convective cooling mechanisms. as a result, core body temperature will continue to rise despite the good intentions of well-doers at the scene. animals that present to the veterinarian that have been cooled to the point of hypothermia have a worse prognosis. once the animal has presented to the veterinarian, the goal is to cool the animal's body temperature with towels soaked in tepid water, cool intravenous fluids, and fans until the temperature has decreased to °f. organ system monitoring and support is based on the severity and duration of the heat stroke and the ability of the body to compensate and respond to treatment. management of the patient with heat-induced illness involves prompt aggressive cooling without being overzealous and creating iatrogenic hypothermia. administer cool intravenous crystalloid fluids to replenish volume and interstitial hydration and correct the patient's acid-base and electrolyte abnormalities. management consists of rule of twenty monitoring (see rule of ), taking care to evaluate, restore, and maintain a normal cardiac rhythm, blood pressure, urine output, and mentation. administer antibiotics if there are any signs of gastrointestinal bleeding that will predispose the patient to bacterial translocation. monitor baseline chemistry tests including a complete blood count, biochemical panel, platelet count, coagulation tests, and urinalysis. treat coagulopathies including dic aggressively and promptly (see also disseminated intravascular coagulation). severe changes in mentation including stupor or coma worsen a patient's prognosis. following initial therapy, monitor the patient for a minimum of to hours for secondary organ damage, including renal failure, myoglobinuria, cerebral edema, and dic. dogs that are going to die of heat-induced illness usually die within the first hours. animals that survive longer than hours have a more favorable prognosis. immediate treatment consists of cooling the patient with cooling measures as for hyperthermia and heat-induced illness (see the previous discussion), and eliminating the cause (i.e., exertion, anesthesia, or neuromuscular blockers such as succinylcholine). if the patient is under general anesthesia, hyperventilate the patient to help eliminate carbon dioxide and respiratory acidosis. administer dantrolene sodium ( to mg/kg iv) to stabilize the sarcoplasmic reticulum and decrease its permeability to calcium. animals with malignant hyperthermia should avoid any predisposing factors, including exertion, hyperthermia, and anesthesia. after an episode of malignant hyperthermia, administer crystalloid fluids intravenously to aid in the elimination of myoglobin. monitor renal function closely for myoglobinuria and pigment damage to the renal tubular epithelium. monitor and correct acid-base and electrolyte changes. walters jm: hyperthermia. in wingfield we, editor: the veterinary icu book, jackson, wyo, , teton newmedia. sometimes it is difficult to assess whether an animal has been bitten by a poisonous or nonpoisonous snake. in colorado, the bull snake closely resembles the prairie rattlesnake. both snakes make similar noise and can be alarming if noticed on a hike or in the backyard. whenever possible, identify the offending reptile but never risk being bitten. know what types of venomous creatures are in the geographic area of the practice. if an animal has been bitten by a nonpoisonous snake, usually the bite marks are small with multiple small tooth punctures, and the bite is relatively nonpainful. usually local reaction is negligible. however, large boas or pythons also can inflict large crushing injuries that can cause severe trauma, including bony fractures. treatment for a nonpoisonous snakebite involves clipping the bite wound and carefully cleaning the area with antimicrobial scrub solution. broad-spectrum antibiotics (e.g., amoxicillin-clavulanate, . mg/kg po q h) are indicated because of the extensive bacterial flora in the mouths of snakes. monitor all snakebite victims for a minimum of hours after the incident, particularly when the species of the offending reptile is in question. if clinical signs of envenomation occur, modify the patient's treatment appropriately and aggressively. the two major groups of venomous snakes in north america are the pit viper and the coral snake. all venomous snakes are dangerous. the severity of any given bite depends on the toxicity of the venom, the amount of venom injected, the site of envenomation, the size of the animal bitten, and the time from bite/envenomation to seeking appropriate medical intervention. the majority of reptile envenomations in the united states are inflicted by pit vipers, including the water moccasin (cottonmouth), copperhead, and numerous species of rattlesnakes. pit vipers are characterized by a deep pit located between the eye and nostril, elliptic pupils, and retractable front fangs (figure - ) . localized clinical signs of pit viper envenomation may include the presence of bleeding puncture wounds, local edema close to puncture wounds, immediate severe pain or collapse, edema, petechiae, and ecchymosis with subsequent tissue necrosis. systemic signs of pit viper envenomation may include hypotension, shock, coagulopathies, lethargy, weakness, muscle fasciculations, lymphangitis, rhabdomyolysis, and neurologic signs including respiratory depression and seizures. neurologic signs largely are associated with envenomation emergency management of specific conditions by the mojave and canebrake rattlesnakes, although a potent neurotoxin, mojave toxin a, also has been identified in other subspecies of rattlesnake. clinical signs of envenomation may take several hours to appear. hospitalize all suspected victims and monitor them for a minimum of hours. the severity of envenomation cannot be judged solely on the basis of local tissue reaction. first aid measures by animal caretakers do little to prevent further envenomation. the most important aspect of initiating therapy is to transport the animal to the nearest veterinary emergency facility. to determine whether an animal has been envenomated by a pit viper, examine a peripheral blood smear for the presence of echinocytes. echinocytes will appear within minutes of envenomation and may disappear within hours. other treatment should be initiated as rapidly and aggressively as possible, although controversy exists whether some therapies are warranted. the mainstay of therapy is to improve tissue perfusion with intravenous crystalloid fluids, prevent pain with judicious use of analgesic drugs, and when necessary, reverse or negate the effects of the venom with antivenin. because pit viper venom consists of multiple fractions, treat each envenomation as a complex poisoning. obtain vascular access and administer intravenous crystalloid fluids (one fourth of a calculated shock dose) according to the patient's perfusion parameters of heart rate, blood pressure, and capillary refill time (see also shock and fluid therapy). opioid analgesics are potent and should be administered at the time of presentation. (see also pharmacologic means to analgesia: major analgesics). diphenhydramine ( . to mg/kg im or iv) also can be administered to decrease the effects of histamine. famotidine, a histamine receptor antagonist, also can be administered ( . to mg/kg iv) to work synergistically with diphenhydramine. although antihistamines have no effect on the venom per se, they may have an effect on the tissue reaction to the venom and may prevent an adverse reaction to antivenin. the use of glucocorticosteroids is controversial. glucocorticosteroids (dexamethasone sodium phosphate [dex-sp], . to . mg/kg iv) may stabilize cellular membranes and inhibit phospholipase, an active component of some pit viper toxins. polyvalent antivenin is necessary in many cases of pit viper envenomation, except in most cases of prairie rattlesnake (crotalus viridis viridis) envenomation in colorado. a recent study demonstrated no difference in outcome with or without the use of antivenin in cases of prairie rattlesnake envenomation. clinically, however, patients that receive antivenin are more comfortable and leave the hospital sooner than those that do not receive antivenin. the exact dose of antivenin is unknown in small animal patients. administer a dose of at least vial of antivenin to neutralize circulating venom. mix antivenin with a swirling, rather than a shaking motion, to prevent foaming. mix the antivenin with a -ml bag of . % saline, and then administer it slowly over a period of hours. pretreat animals with diphenhydramine ( . to mg/kg im) before the administration of antivenin, and then monitor the animal closely for clinical signs of angioneurotic edema, urticaria, tachyarrhythmias, vomiting, diarrhea, and weakness during the infusion. administration of antivenin into the bite site is relatively contraindicated and ineffective because uptake is delayed, and systemic effects are the more life-threatening. management of pit viper envenomation largely involves maintenance of normal tissue perfusion with intravenous fluids, decreasing patient discomfort with analgesia, and negating circulating venom with antivenin. hydrotherapy to the affected bite site with tepid water is often soothing to the patient. the empiric use of antibiotics is controversial but is recommended because of the favorable environment created by a snakebite (i.e., impregnation of superficial gram-positive bacteria and gram-negative bacteria from the mouth of the snake into a site of edematous necrotic tissue). administer amoxicillin-clavulanate ( . mg/kg po q h, or cephalexin, mg/kg po q h). also consider administration of nsaids (carprofen, . mg/kg po q h). monitor the patient closely for signs of local tissue necrosis and the development of thrombocytopenia and coagulopathies including dic (see management of disseminated intravascular coagulation). treat coagulopathies aggressively to prevent end-organ damage. coral snakes are characterized by brightly colored bands encircling the body, with red and black separated by yellow. "red on black, friend of jack; red on yellow, kill a fellow." types of coral snakes include the eastern coral, texas coral, and sonoran coral snakes. clinical signs of coral snake envenomation may include small puncture wounds, transient initial pain, muscle fasciculations, weakness, difficulty swallowing/dysphagia, ascending lower motor neuron paralysis, miotic pinpoint pupils, bulbar paralysis, respiratory collapse, and severe hemolysis. clinical signs may be delayed for as long as hours after the initial bite. immediate treatment with antivenin is necessary in cases of coral snake envenomation before the clinical signs become apparent, whenever possible. support respiration during paralysis with mechanical ventilation. secure the patient's airway with a cuffed endotracheal tube to prevent aspiration pneumonia. clinical signs will progress rapidly once they develop. rapid administration with antivenin is the mainstay of therapy in suspected coral snake envenomation. respiratory and cardiovascular support should occur with mechanical ventilation and intravenous crystalloid fluids. keep the patient warm and dry in a quiet place. turn the patient every to hours to prevent atelectasis and decubitus ulcer formation. maintain cleanliness using a urinary catheter and closed urinary collection system. perform passive range of motion and deep muscle massage to prevent disuse atrophy of limb muscles and function. treat aspiration pneumonia aggressively with broad-spectrum antibiotics (ampicillin, mg/kg iv q h, with enrofloxacin, mg/kg iv q h, and then change to oral once tolerated and the patient is able to swallow) for weeks past the resolution of radiographic signs of pneumonia, intravenous fluids, and nebulization with sterile saline and coupage chest physiotherapy. several weeks may elapse before a complete recovery. the adult black widow spider (latrodectus spp.) can be recognized by a red to orange hourglass-shaped marking on the underside of a globous, shiny, black abdomen. the immature female can be recognized by a colorful pattern of red, brown, and beige on the dorsal surface of the abdomen. adult and immature females are equally capable of envenomation. the male is unable to penetrate the skin because of its small size. black widow spiders are found throughout the united states and canada. black widow spider venom is neurotoxic and acts presynaptically, releasing large amounts of acetylcholine and norepinephrine. there appears to be a seasonal variation in the potency of the venom, lowest in the spring and highest in the fall. in dogs, envenomation results in hyperesthesia, muscle fasciculations, and hypertension. muscle rigidity without tenderness is characteristic. affected animals may demonstrate clinical signs of acute abdominal pain. tonic-clonic convulsions may occur but are rare. in cats, paralytic signs predominate and appear early as a ascending lower motor neuron paralysis. increased salivation, vomiting, and diarrhea may occur. serum biochemistry profiles often reveal significant elevations in creatine kinase and hypocalcemia. myoglobinemia and myoglobinuria can occur because of extreme muscle damage. management of black widow spider envenomation should be aggressive in the cat and dog, particularly when the exposure is known. in many cases, however, the diagnosis is made based on clinical signs, biochemical abnormalities, and lack of other apparent cause. antivenin (one vial) is available and should be administered after pretreatment with diphenhydramine. if antivenin is unavailable, administer a slow infusion of calcium-containing fluid such as lactated ringer's solution with calcium gluconate while carefully monitoring the patient's ecg. the small brown nonaggressive spider is characterized by a violin-shaped marking on the cephalothorax. the neck of the violin points toward the abdomen. brown spiders are found primarily in the southern half of the united states but have been documented as far north as michigan. the venom of the brown spider has a potent dermatonecrolytic effect and starts with a classic bull's-eye lesion. the lesion then develops into an indolent ulcer into dependent tissues promoted by complement fixation and influx of neutrophils into the affected area. the ulcer can take months to heal and often leaves a disfiguring scar. systemic reactions are rare but can include hemolysis, fever, thrombocytopenia, weakness, and joint pain. fatalities are possible. immediate management of an animal with brown spider envenomation is difficult because there is no specific antidote and because clinical signs may be delayed until necrosis of the skin and underlying tissues becomes apparent through the patient's fur to days after the initial bite. dapsone has been recommended at a dose of mg/kg for days. surgical excision of the ulcer may be helpful if performed in the early stages of wound appearance. glucocorticosteroids may be of some benefit if used within hours of the bite. the ulcer should be left to heal by second intention. deep ulcers should be treated with antibiotics. bufo toad species (b. marinus, aka cane toad, marine toad, giant toad; and the colorado river toad or sonoran desert toad b. alvarius) can be associated with severe cardiac and neurotoxicity if an animal licks its skin. the severity of toxicity depends largely on the size of the dog. toxins in the cane toad, b. marinus, include catecholamines and vasoactive substances (epinephrine, norepinephrine, serotonin, dopamine) and bufo toxins (bufagins, bufotoxin, and bufotenine), the mechanism of which is similar to cardiac glycosides. clinical signs can range from ptyalism, weakness, ataxia, extensor rigidity, opisthotonus, and collapse to seizures. clinical signs associated with b. alvarius toxicity are limited largely to cardiac dysrhythmias, ataxia, and salivation. the animal should have its mouth rinsed out thoroughly with tap water even before presentation to the veterinarian. if the animal is unconscious or actively seizing and cannot protect its airway, flushing the mouth is contraindicated. once an animal presents to the veterinarian, the veterinarian should place an intravenous catheter and monitor the patient's ecg and blood pressure. attempt seizure control with diazepam ( . mg/kg iv) or pentobarbital ( to mg/kg iv to effect). ventricular dysrhythmias can be controlled first with esmolol ( . mg/kg). if esmolol is ineffective, administer a longer-acting parenteral β-antagonist such as propranolol ( . mg/kg iv). ventricular tachycardia also can be treated with lidocaine ( to mg/kg iv, followed by to µg/kg/minute iv cri). case management largely depends on supportive care and treating clinical signs as they occur. monitor baseline acid-base and electrolyte balance because severe metabolic acidosis may occur that should be treated with intravenous fluids and sodium bicarbonate ( . to meq/kg iv). monitor ecg, blood pressure, and mentation changes closely. control seizures and cardiac dysrhythmias. eubig pa: bufo species intoxication: big toad, big problem, vet med ( ) lizards of the family hemodermatidae are the only two poisonous lizards in the world. they are found in the southwestern united states and mexico. the venom glands are located on either side of the lower jaw. because these lizards are typically lethargic and nonaggressive, bite wounds are rare. the lizards have grooved teeth that introduce the venom with a chewing motion as the lizard holds tenaciously to the victim. the majority of affected dogs are bitten on the upper lip, which is very painful. there are no proven first aid measures for bites from gila monsters or mexican bearded lizards. the lizard can be disengaged by inserting a prying instrument in between the jaws and pushing at the back of the mouth. the teeth of the lizard are brittle and break off in the wound. topical irrigation with lidocaine and probing with a needle will aid in finding and removing the teeth from the victim. bite wounds will bleed excessively. irrigate wounds with sterile saline or lactated ringer's solution, and place compression on the affected area until bleeding ceases. monitor the patient for hypotension. establish intravenous access, and administer intravenous fluids according to the patient's perfusion parameters. antibiotic therapy is indicated because of the bacteria in the lizard's mouth. because no antidote is available, treatment is supportive according to patient signs. the majority of musculoskeletal emergencies are the result of external trauma, most commonly from motor vehicle accidents. blunt trauma invokes injury to multiple organ systems as a rule, rather than an exception. because of this, massive musculoskeletal injuries are assigned a relatively low priority during the initial triage and treatment of a traumatized animal. perform a rapid primary survey and institute any lifesaving emergency therapies. adhere to a crash plan or the abcs of resuscitation (see initial emergency examination, management, and triage). although musculoskeletal injuries are assigned a relatively lower priority, the degree of recovery from these injuries and financial obligation for fracture repair sometimes becomes a critical factor in a client's decision whether to pursue further therapy. one of the most important deciding factors is the long-term prognosis for the patient to have a good quality of life following fracture repair. the initial management of musculoskeletal injuries is important in ensuring the best chance for maximal recovery with minimal complications after definitive surgical fracture repair. this is particularly important for open fractures, spinal cord compromise, multiple fractures, open joints, articular fractures, physeal fractures, and concomitant ligamentous or neurologic compromise (box - ). immediately after the initial primary survey of a patient, perform a more thorough examination, including an orthopedic examination. multiple injuries often are observed in the patient that falls from height (e.g., "high-rise syndrome"), motor vehicle accidents, gunshot wounds, and encounters with other animals (e.g., "big-dog-little-dog"). address the most life threatening injuries, and palliate musculoskeletal injuries until more definitive repair can be attempted when the patient is more stable. in animals with the history of potential for multiple injuries, search thoroughly and meticulously for areas of injury to the spinal column, extremities, and for small puncture wounds. helpful signs that can provide a clue as to an underlying injury include swelling, bruising, abnormal motion, and crepitus (caused by subcutaneous emphysema or bony fracture). if the patient is alert, look for areas of tenderness or pain. in unconscious or depressed patients, reexamine the patient after the patient becomes more mentally alert. injuries often are missed during the initial examination in obtunded patients because of the early response and attenuation of pain. unconscious or immobile patients must have radiographic examination of the spinal column following stabilization and support. palpate the skull carefully for obvious depressions or crepitus that may be associated with a skull fracture. localization of the injury can be determined by motion in abnormal locations, swelling caused by hemorrhage or edema, pain during gentle movement or palpation, deformity, angular change, or a significant increase or decrease in normal range of motion of bones and joints. perform a rectal examination in all cases to palpate for pelvic fractures and displacement. once the diagnosis of a fracture or luxation has been confirmed, look for any evidence of skin lacerations or punctures near the fracture site. in long-haired breeds, clipping the fur near the fracture site often is necessary to perform a thorough examination of the area. if any wounds are found, the fracture is classified as an open fracture until proven otherwise. in some cases, the open fracture is obvious, with a large section of bone fragment protruding through the skin. in other cases, the puncture wound may be subtle, with only a small amount of blood or pinpoint hole in the skin surface. characteristics observed with open fractures include bone penetration, fat droplets or marrow elements in blood coming from the wound, subcutaneous emphysema on radiographs, and lacerations in the area of a fracture. protect the patient from further injury or contamination of wounds. excessive palpation to intentionally produce crepitus is inappropriate because it causes severe patient discomfort and has the potential to cause severe soft tissue and neurologic injury at the fracture site. sedation and analgesia aids in making the examination more comfortable for the patient and allows localization of the injury and comparison with the opposite extremity. higher-quality radiographs can be performed to determine the extent of the injury when the animal is sedated adequately and pain is controlled. sedate the patient judiciously with analgesic drugs. opioid drugs work well for orthopedic pain, produce minimal cardiorespiratory depression, and can be reversed with naloxone if necessary. handle the fracture site gently to avoid causing further pain and soft tissue injury at the fracture site. rough or careless handling of a fracture site can cause a closed fracture to penetrate through the skin and become an open fracture. cover open fractures immediately to prevent contamination of the fracture with nosocomial infection from the hospital. administer a first-generation cephalosporin (cephalexin, mg/kg po q h, or cefazolin, mg/kg iv q h). the bandage also serves to control hemorrhage and prevent desiccation of the bones and surrounding soft tissue structures. leave the initial bandages in place until the patient's cardiorespiratory status has been determined to be stable and more definitive wound management can occur in a clean, preferably sterile location. examine the neurologic status and cardiovascular status of the limb before and after treatment. determine the vascular status of the limb by checking the color and temperature of the limb, the state of distal pulses, and the degree of bleeding from a cut nail bed. in patients with severe cardiovascular compromise and hypotension caused by hemorrhagic shock, the viability of the limb may be in question until the cardiovascular status and blood pressure are normalized. reduction of the fracture or straightening of gross deformities may return normal vascularity to the limb. when checking neurologic status, examine for motor and sensory function to the limb. swelling may increase pressure on the nerves as they run through osteofascial compartments, resulting in decreased sensory or motor function, or neurapraxia. diminished function often returns to normal once the swelling subsides. serial physical examinations in the patient and response to initial stabilization therapy can lead to a higher index of suspicion that more occult injuries are present, such as a diaphragmatic hernia, perforated bowel, lacerated liver or spleen, or uroabdomen. to prevent ongoing trauma, reduce any fracture and then stabilize the site above and below the fracture. a modified robert jones splint or bandage often works well for fractures emergency management of specific conditions involving the distal extremities. fractures of the humerus or femur are difficult to immobilize without the use of spica or over-the-hip coaptation splints to prevent mobility. inappropriate bandaging of humerus or femur fractures can result in a fulcrum effect and worsen the soft tissue and neurologic injuries. further displacement of vertebral bodies or luxations can cause cord compression or laceration such that return to function becomes impossible. immediately place any patient with a suspected spinal injury on a flat surface, and tape down the animal to prevent further movement until the spine has been cleared by a minimum or two orthogonal radiographic views (lateral and ventrodorsal views performed as a cross-table x-ray technique). wounds associated with musculoskeletal trauma are common and include injury to the bones, joints, tendons, and surrounding musculature (box - ). major problems associated with these cases are the presence of soft tissue trauma that makes wound closure hazardous or impossible, because of the risk of infection. chronic deep infection of traumatized wounds can cause delayed healing and sequestrum to develop, particularly if there is avascular bone or cartilage within the wound. in the early management of an open fracture, the areas should be splinted without pulling any exposed bone back into the soft tissue. the wound should not be probed or soaked, as nosocomial bacteria and other external contaminants can be introduced into the wound, leading to severe infection. because of the risk of actually causing infection, probing, flushing, or replacing tissues back into the wound should be performed at the time of formal debridement when the patient is physiologically stable. immediate bactericidal antibiotic therapy with a first-generation cephalosporin should be started immediately to obtain adequate concentrations of antibiotics at the fracture site. the duration of antibiotic therapy should ideally be limited to - days to prevent the risk of superinfection. treatment of open musculoskeletal injury involves three considerations: initial inspection and wound debridement, stabilization and repair, and wound bandaging. emergency care when associated with a fracture, wound is created from the inside out by penetration of bone fragments through the skin or from a low-energy gunshot. simple or comminuted fracture pattern good stability of the two main bone segments treatment and prognosis are good and similar to those of a closed injury if wound is debrided and stabilized within to hours. when associated with a fracture, wound is created from the outside in. major deep injury with considerable soft tissue stripping from bone and muscle damage simple or comminuted fracture pattern prognosis is good if wound is debrided within hours of injury and provided rigid stabilization with a bone plate or external fixator. results from major external force severe damage and necrosis of skin, subcutaneous tissue, muscle, nerve, bone, tendon, and arteries soft tissue damage may vary from crush injury to shearing injury associated with bite wounds or low-speed automobile accidents. requires immediate and delayed sequential debridement and rigid external fixation can require prolonged healing times guarded prognosis initial inspection and wound debridement include the following steps: . after the patient's cardiovascular status has been stabilized and it has been determined that it can withstand anesthesia, place the animal under general anesthesia and remove the temporary splint. . keeping the wound covered, shave the surrounding fur. . remove the covering and then place sterile lubricant jelly over the wound. shave the fur to the edges of the wound margin. . wash away any entrapped fur and the lubricant jelly. . complete an antiseptic scrub of the surrounding skin. . if the wound is a small puncture (e.g., gunshot pellets or bites), probe the wound with a sterile hemostat. do a thorough debridement if tissues deep to the hole are cavitated. if not deep, create a hole for drainage. . flush the wound with a physiologic solution (lactated ringer's solution is preferred). . debride the wound from outward to inward. cut away damaged areas of skin and deeper tissues to open up underlying cavitations and tissue injury. . continuously irrigate with warm physiologic solution (lactated ringer's solution is preferred). the stream must be strong enough to flush debris out of the bottom of the wound. to accomplish this, attach a -gauge needle to a -ml syringe (will deliver psi). excise any obviously devitalized tissue. . do not remove any bone fragments that are firmly attached to soft tissue. do not cut into healthy soft tissue to find bullet or bone fragments, unless the bullet can cause injury to joints or nerve tissue. . do a primary repair of tendons and nerves if the wound is type i and recent (within hours of the initial injury). if the wound is too severe or if there is obvious infection, tag the ends of the tendons and nerves for later repair. it is best to stabilize and repair open fractures as soon as the patient's cardiovascular and respiratory status can tolerate general anesthesia, provided that adequate stabilization is possible. if this is not possible because of the level of experience of the surgeon or the lack of necessary equipment, it is best to perform wound management and place a temporary splint until definitive repair can be performed. wound bandaging is discussed in the section on bandaging techniques. structural injuries to the joints are common and can involve both ligaments and articular cartilage injuries. cartilage does not heal well; therefore, injuries involving articular cartilage can lead to a significant loss of function and degenerative joint disease (osteoarthritis). cartilage injuries that are superficial evoke a short-lived enzymatic and metabolic response that does not stimulate enough cellular growth to repair the defect. superficial lesions remain as defects but do not progress to chondromalacia or osteoarthritis. deep cartilage lacerations that extend to subchondral bone produce an exuberant healing response from the cells of the underlying cartilage. in many cases, this material undergoes degeneration and leads to osteoarthritis. impact injuries to surface cartilage can cause chondrocyte and underlying bone injury. these lesions rapidly progress to osteoarthritis; however, they may be totally or partially reversible. treatment of grade i injuries requires short-term coaptation splints and has a good prognosis. grade ii injuries require surgical treatment with a suture stent and consistent postoperative coaptation splints to heal and maintain good function. healing of grade iii injuries often is a problem, and suture stents or surgical reapproximation may be indicated. failure to immobilize joints that are frequently flexed (elbow and stifle) can result in late complications of ligament repair. ligamentous injuries of joints, particularly the collateral ligaments of the stifle, elbow, and hock, and carpal hyperextension injuries are commonly missed and may require surgical fixation, including arthrodesis (box - ). fractures in immature animals differ from those in adults in that young puppies and kittens have a great ability to remodel bone. remodeling is dependent on the age of the patient and the location of the fracture. the younger the puppy or kitten and the closer the fracture to the epiphysis or growth plate, the greater the potential for remodeling and the development of angular limb deformities. remodeling occurs more effectively in longlimbed breeds of dogs than in short-limbed breeds. fractures through the growth plate of immature animals may potentially cause angular limb deformities, joint dislocations or incongruity, and osteoarthritis. this form of injury is commonly observed in the distal ulnar growth plate and the proximal and distal radial growth plates. high-rise syndrome in cats is seen in cats that fall from a height usually greater than feet. it occurs most frequently in high-rise buildings in urban areas where cats lie on window ledges and suddenly fall out the window. the most common lesions observed in cats that fall from heights are thoracic injuries (rib and sternal fractures, pneumothorax, and pulmonary contusions) and facial and oral trauma (lip avulsions, mandibular symphyseal fractures, fractures of the hard palate, and maxillary fractures). limb and spinal cord fractures and luxations, radius and ulna fractures, abdominal trauma, urinary tract trauma, and diaphragmatic hernias are also common. the injuries sustained are often found in combination, rather than as an isolated injury of one area of the body. follow the mnemonic a crash plan when managing a cat suffering from high-rise syndrome, treating the animal immediately for shock. following cardiovascular and respiratory stabilization, evaluate thoracic and abdominal radiographs, including those of the spine. evaluate the bladder closely, making sure that the cat is able to urinate effectively. examine the hard palate, maxilla, and mandibular symphysis for fractures. palpate the pelvis and carefully manipulate all limbs to examine for fractures or ligamentous injuries. finally, perform a complete neurologic examination. patients that fall less than five stories often have a more guarded prognosis than patients that fall from higher levels. sometimes the owner witnesses the ingestion of a foreign body during play, such as throwing a stick or fetching a ball. cats tend to play with string or thread that becomes caught around the base of the tongue. in many cases, however, ingestion of the foreign object is not witnessed, and diagnosis is made based on clinical signs and physical examination. foreign bodies lodged in the oral cavity often cause irritation and discomfort, including difficulty breathing and difficulty swallowing. often, an animal paws at its mouth in an attempt to dislodge a stick or bones wedged across the roof of the mouth. irritation, inability to close the mouth, and blockage of the orpharynx can result in excessive drooling. the saliva may appear blood-tinged due to concurrent soft tissue trauma (figs - and - ) . obstruction of the glottis by a foreign body (e.g., tennis ball or toy) can result in cyanosis secondary to an obstructed airway and hypoxemia. in many cases, the object is small enough to enter the larynx but too large to be expelled. if a foreign object is lodged in the mouth for more than several days, halitosis and purulent discharge may be present. many animals are anxious at the time of presentation and may require sedation or a light plane of anesthesia to remove the foreign object. the animal may bite personnel and may have bitten the owner during his or her attempt to remove the object from the mouth en route to the hospital. propofol ( mg/kg iv) or a combination of propofol with diazepam ( . - mg/kg iv) is an excellent combination for a light plane of anesthesia. exercise caution when anesthetizing a patient with a ball lodged in the airway, as further compromise of respiratory function may occur and cause worsening of the hypoxemia. before inducing anesthesia, assemble all supplies necessary to remove the object. make sure that rigid towel clamps, sponge forceps, and bone forceps are on hand, because the foreign object is often very slippery with saliva. hemostats and carmalts may slip and not be useful in the removal of the foreign object. place a peripheral intravenous catheter to secure vascular access prior to anesthetic induction. have available the supplies necessary for an emergency tracheostomy, if the foreign object cannot be removed by usual methods. induce a light plane of anesthesia and then grasp the object with the sponge forceps or towel clamps, and extract. monitor the cardiorespiratory status of the animal at all times during the extraction process. if you are unable to remove the object, and if severe respiratory distress, including cyanosis, bradycardia, or ventricular dysrhythmias, develop, perform a tracheostomy distal to the site of obstruction. once the foreign body has been removed, administer supplemental flow-by oxygen until the animal awakens. if laryngeal edema or stridor on inspiration is present, administer a dose of dexamethasone sodium phosphate ( . mg/kg iv, im, sq) to decrease inflammation. the patient should be carefully monitored for hours, because noncardiogenic pulmonary edema can develop secondary to airway obstruction. esophageal foreign bodies pose a serious medical emergency. it is helpful if the owner witnessed ingestion of the object and noted rapid onset of clinical signs. in many cases, however, ingestion is not witnessed, and the diagnosis must be made based on clinical signs, thoracic radiographs, and results of a barium swallow. the most common clinical signs are excessive salivation with drooling, gulping, and regurgitation after eating. many animals will make repeated swallowing motions. some animals exhibit a rigid "sawhorse" stance, with reluctance to move immediately after foreign body ingestion and esophageal entrapment. after completing a physical examination, evaluate cervical and thoracic radiographs to determine the location of the esophageal obstruction. esophageal foreign objects are lodged most commonly at the base of the heart, the carina, or just orad to the lower esophageal sphincter. if the object has been lodged for several days, pleural effusion and pneumomediastinum may be present secondary to esophageal perforation. endoscopy is useful for both diagnosis and removal of the foreign object; however, it is invasive and requires general anesthesia ( fig. - ) . remove foreign objects lodged in the esophagus with a rigid or flexible endoscope after the patient has been placed under general anesthesia. evaluate the integrity of the esophagus both before and after removal of the material because focal perforation or pressure necrosis can be present. necrosis of the mucosa and submucosa of the esophagus often leads to stricture formation or perforation. attempt to retrieve the object with a flexible fiberoptic endoscope if available. rigid tube endoscopy can also be performed. in many cases, smooth objects that cannot be easily grasped can be pushed into the stomach and allowed to dissolve or may be removed by gastrotomy. if the foreign body is firmly lodged in the esophagus and cannot be pulled or pushed into the stomach, or if perforation has already occurred, the prognosis for return to function without strictures is not favorable. in such cases, referral to a surgical specialist is recommended for esophagostomy or esophageal resection. after removal of the object, carefully examine the esophagus and then administer gastroprotectant agents (famotidine, . mg/kg po bid; sucralfate slurry, . - . g/dog) for a minimum of to days. to rest the esophagus, the patient should receive nothing per os (npo) for to hours. if esophageal irritation or erosion is moderate to severe, a percutaneous gastrotomy tube should be placed for feeding until the esophagus heals. perform repeat endoscopy every days to evaluate the healing process and to determine whether stricture formation is occurring. persistent vomiting immediately or soon after eating is often associated with a gastric foreign body. in some cases, the owner knows that the patient has ingested a foreign body of some kind. in other cases, continued vomiting despite lack of response to conservative treatment (npo, antiemetics, gastroprotectant drugs) prompts further diagnostic procedures, including abdominal radiographs and bloodwork. obstruction to gastric outflow and vomiting of hydrochloric acid often cause a hypochloremic metabolic acidosis. radiopaque gastric foreign bodies may be observed on plain films. radiolucent cloth material may require a barium series to delineate the shape and location of the foreign body ( fig. - ) . treatment consists of removal with flexible endoscopy or a simple gastrotomy. most animals with uncomplicated gastric foreign bodies are relatively healthy, but any metabolic and electrolyte abnormalities should be corrected prior to anesthesia and surgery. small intestinal obstruction can be caused by foreign bodies, tumors, intussusception, volvulus, or strangulation within hernias. regardless of the cause, clinical signs of small intestinal obstruction depend on the location and degree of obstruction, and whether the bowel has perforated. clinical signs associated with a high small intestinal obstruction are usually more severe and more rapid in onset compared with partial or complete obstruction of the jejunum or ileum. complete obstructions that allow no fluid or chyme to pass are worse than partial obstructions, which can cause intermittent clinical signs interspersed with periods of normality (table - ). the most common clinical signs associated with a complete small intestinal obstruction are anorexia, vomiting, lethargy, depression, dehydration, and sometimes abdominal pain. early clinical signs may be limited to anorexia and depression, making a diagnosis challenging unless the owner has a suspicion that the animal ingested some kind of foreign object. obstructions cranial to the common bile duct and pancreatic papillae lead to vomiting of gastric contents, namely hydrochloric acid, and a hypochloremic metabolic alkalosis. obstructions caudal to the common bile duct and pancreatic papillae result in loss of other electrolytes and sometimes mixed acid-base disorders. eventually, all animals with small intestinal obstruction vomit and have fluid loss into dilated segments of bowel, leading to dehydration and electrolyte abnormalities. increased luminal pressure causes decreased lymphatic drainage and bowel edema. the bowel wall eventually becomes ischemic and may rupture. linear foreign bodies should be suspected in any vomiting patient, particularly cats. string or thread often is looped around the base of the tongue and can be visualized in many cases by a thorough oral examination. to look properly under the tongue, grasp the top of the animal's head with one hand, and pull the lower jaw open with the index finger of the opposite hand while pushing up the thumb simultaneously on the tongue in between the intermandibular space. thread and string can be observed lying along the ventral aspect of the tongue. in some cases, if a linear foreign body is lodged very caudally, it cannot be visualized without heavy sedation or anesthesia. linear foreign bodies eventually cause bowel obstruction and perforation of the intestines along the mesenteric border. the foreign material (e.g., string, thread, cloth, pantyhose) becomes lodged proximally, and the intestines become plicated as the body attempts to push the material caudally through the intestines ( fig. - ) . continued peristalsis eventually causes a sawing motion of the material and perforation of the mesenteric border of the intestines. once peritonitis occurs, the prognosis is less favorable unless prompt and aggressive treatment is initiated. reevaluate any patient that does not respond to conservative symptomatic therapy, performing a complete blood count, serum biochemical panel (including electrolytes), and abdominal radiographs. intestinal masses may be palpable on physical examination and are often associated with signs of discomfort or pain when palpating over the mass. radiography and abdominal ultrasound are the most useful diagnostic aids. plain radiographs may be diagnostic when the foreign object is radiodense or there is characteristic dilation or plication of bowel loops. as a rule of thumb, the width of a loop of small bowel should be no larger than twice the width of a rib. diagnosis of small intestinal obstruction or ileus can be based on the appearance of stacking loops of dilated bowel. comparison of the width of the bowel with the width of a rib is often performed. with mild dilation, the bowel width is three to four times the rib width; with extensive dilation, five to six times the rib width ( fig. - ) . in cases of linear foreign bodies, c-areas (comma-shaped areas) of gas trapped in the plicated bowel will appear stacked on one another. blunt, wedge-shaped areas of gas or square linear areas of gas adjacent to a distended bowel loop are characteristic of a foreign body lodged in the intestine. contrast radiography is indicated when confirmation of the suspected diagnosis is necessary and ultrasonography is not available. contrast material may outline the object or abruptly stop orad to the obstruction. the definitive treatment of any type of small intestinal foreign body is surgical removal. linear foreign bodies sometimes pass, but they should never be left untreated in a patient that is demonstrating clinical signs of inappetence, vomiting, lethargy, and dehydration. the timing of surgery is critical because the risk of intestinal perforation increases with time. prior to surgery, correct any acid-base and electrolyte abnormalities with intravenous fluid therapy. administer broad-spectrum antibiotics. perform an enterotomy or intestinal resection and anastomosis as soon as possible once the patient's acid-base and electrolyte status have been corrected. clinical signs of a foreign body in the large bowel are usually nonexistent. in most cases, if a foreign object has passed successfully through the small bowel, it will pass through the large bowel without incident unless bowel perforation and peritonitis occur. penetrating foreign bodies such as needles often cause localized or generalized peritonitis, abdominal pain, and fever. hematochezia may be present if the foreign object causes abrasion of the rectal mucosa. symptomatic patients should have abdominal radiographs performed. colonoscopy or exploratory laparotomy should be performed if survey radiographs are suggestive of a large intestinal obstruction or perforation. in most cases, large intestinal foreign bodies will pass without incident. surgery is required to treat perforations, peritonitis, or abscesses. emergency care figure - : after minutes, the barium has stopped moving and has reached a blunt, intraluminal intestinal foreign body. note that barium appears wedge-shaped or square at the site of the foreign body. foreign bodies in the rectum and anus often are the result of ingestion of bones, wood material, needles, and thread, or malicious external insertion. often the material can pass through the entire gastrointestinal tract and then get stuck in the anal ring. clinical signs include hematochezia and dyschezia with straining to defecate. diagnosis is made by visual examination of the item in the anus, or by careful digital palpation after heavy sedation or short-acting general anesthesia. radiography is helpful in locating needles that have penetrated the rectum and lodged in the perirectal or perinatal tissues. treatment consists of careful removal of the needle digitally or surgically. intussusception is the acute invagination of one segment of bowel (the intussusceptum) into another (the intussuscipiens). the proximal segment always invaginates into the distal segment of bowel. intussusception most commonly occurs in puppies and kittens less than year of age but can occur in an animal of any age with hypermotility of the small bowel, gastrointestinal parasites, and severe viral or bacterial enteritis. intussusception occurs primarily in the small bowel in the jejunum, ileum, and ileocolic junction. clinical signs include vomiting, abdominal discomfort, and hemorrhagic diarrhea. usually, hemorrhagic diarrhea is the first noticeable sign, and in puppies, may be due to parvoviral enteritis, with secondary intussusception. usually, the obstruction is partial with mild clinical signs. more serious clinical signs develop as the obstruction becomes more complete. differential diagnoses include hemorrhagic gastroenteritis, parvoviral enteritis, gastrointestinal parasites, intestinal foreign body, bacterial enteritis, and other causes of vomiting and diarrhea. the diagnosis of intussusception is often made based on palpation of a sausage-shaped firm, tubular structure in the abdomen accompanied by clinical signs and abdominal pain. plain radiographs may demonstrate segmental or generalized dilated segments of bowel, depending on the duration of the problem. ultrasonographs of the palpable mass resemble the layers of an onion, with hyperechoic intestinal walls separated by less echogenic edema. treatment consists of correction of the patient's acid-base and electrolyte abnormalities with intravenous fluids and surgical reduction or removal of the intussusception with resection and anastomosis. although enteroplication has been suggested, the technique has fallen out of favor because of the increased risk of later obstruction. the primary cause of intestinal inflammation and hypermotility must be identified and corrected. gastric dilatation can occur with or without volvulus in the dog. gastric dilatationvolvulus (gdv) occurs primarily in large-and giant-breed dogs with deep chests, such as the great dane, labrador retriever, saint bernard, german shepherd dog, gordon and irish setters, standard poodle, bernese mountain dog, and bassett hound. the risk of gdv increases with age; however, it can be seen in dogs as young as months. deep, narrow-chested breeds are more likely to develop gdv than dogs with broader chests. the overall mortality for surgically treated gastric dilatation-volvulus ranges from % to %, with most deaths occurring in patients that required splenectomy and partial gastrectomy. clinical signs of gdv include abdominal distention, unproductive vomiting or retching, lethargy, weakness, sometimes straining to defecate, and collapse. the owner may think that the animal is vomiting productively because of the white foamy froth (saliva) that is not able to pass into the twisted stomach. in some cases, there is a history of the dog's being fed a large meal or consuming a large quantity of water prior to the onset of clinical signs. instruct the owner of any patient with a predisposition for and clinical signs of gdv to transport the animal to the nearest veterinary facility immediately. physical examination often reveals a distended abdomen with a tympanic area on auscultation. in dogs with very deep chests, it may be difficult to appreciate abdominal distention if the stomach is tucked up under the rib cage. depending on the stage of shock, the patient may have sinus tachycardia with bounding pulses, cardiac dysrhythmias with pulse deficits, or bradycardia. the mucous membranes may appear red and injected or pale with a prolonged capillary refill time. the patient may appear anxious and attempt to retch unproductively. if the patient is nonambulatory at the time of presentation, the prognosis is more guarded. the definitive diagnosis of gdv is based on clinical signs, physical examination findings, and radiographic appearance of gas distention of the gastric fundus with dorsocranial displacement of the pylorus and duodenum (the so-called "double-bubble" or "popeye arm" sign) ( fig. - ) . in simple gastric dilatation without volvulus, there is gas distention of the stomach with anatomy appearing normal on radiography. with "food bloat," or gastric distention from overconsumption of food, ingesta is visible in the distended stomach ( fig. - ) . as soon as a patient presents with a possible gdv, place a large-bore intravenous catheter in the cephalic vein(s) and assess the patient's ecg, blood pressure, heart rate, capillary refill time, and respiratory function. obtain blood samples for a complete blood count, serum biochemistry profile, immediate lactate measurement, and coagulation tests before taking any radiographs. rapidly infuse a colloid (hetastarch or oxyglobin, ml/kg iv bolus) along with shock volumes of a crystalloid fluid (up to ml/kg/hour) (see section on shock). monitor perfusion parameters (heart rate, blood pressure, capillary refill time, and ecg) and titrate fluid therapy according to the patient's response. the use of short-acting glucocorticosteroids is controversial. glucocorticosteroids may help stabilize cellular membranes and decrease the mechanisms of ischemia-reperfusion injury, but no detailed studies have proved them to be beneficial versus not using glucocorticosteroids in the patient with gdv. attempt gastric decompression, either with placement of an orogastric tube or by trocharization. to place an orogastric tube, position the distal end of the tube at the level of the patient's last rib ( fig. - ) and place it adjacent to the animal's thorax; then put a piece of tape around the tube where it comes out of the mouth, once it is in place. put a roll of -inch tape in the patient's mouth behind the canine teeth and then secure the roll in place by taping the mouth closed around the roll of tape. lubricate the tube with lubricating jelly and slowly insert the tube through the center of the roll of tape into the stomach. the passing of the tube does not rule out volvulus. in some cases, the front legs of the patient need to be elevated, and the caudal aspect of the patient lowered (front legs standing on a table with back legs on the ground) to allow gravity to pull the stomach down to allow the tube to pass. once the tube has been passed, air within the stomach is relieved, and the stomach can be lavaged. the presence of gastric mucosa or blood in the efflux from the tube makes the prognosis more guarded. if an orogastric tube cannot be passed, clip and aseptically scrub the patient's lateral abdomen and then insert -gauge over-the-needle catheter. "pinging" the animal's side with simultaneous auscultation allows determination of the location that is most tympanic-that is, the proper location for catheter insertion. once intravenous fluids have been started in the animal, take a right lateral abdominal radiograph to document gdv. if no volvulus is present, the owner may elect for more conservative care, and the animal should be monitored in the hospital for a minimum of hours. because some cases of gdv intermittently twist and untwist, the owner should be cautioned that although the stomach is not twisted at that moment, a volvulus can occur at any time. if radiographs demonstrate food bloat, induce emesis (apomorphine, . mg/kg iv) or perform orogastric lavage under general anesthesia. documentation of gastric dilatation-volvulus constitutes a surgical emergency. figure - : example of "food bloat" with severe gastric distention caused by overconsump-following diagnosis of gdv, continue administration of intravenous fluids. serum lactate measurements greater than . mmol/l are associated with an increased risk of gastric necrosis, requirement for partial gastrectomy, and increased mortality. administer fresh frozen plasma ( ml/kg) to patients with thrombocytopenia or prolonged pt, activated partial thromboplastin time (aptt), or activated clotting time (act). cardiac dysrhythmias, particularly ventricular dysrhythmias, are common in cases of gdv and are thought to occur secondary to ischemia and proinflammatory cytokines released during volvulus and reperfusion. lidocaine ( - mg/kg followed by mcg/kg/minute iv cri) can be used to treat cardiac dysrhythmias preemptively that are associated with ischemia-reperfusion injury, or administration can be started when ventricular dysrhythmias are present. correct any electrolyte abnormalities, including hypokalemia and hypomagnesemia. the use of nonsteroidal antiinflammatory drugs (flunixin meglumine, carprofen, ketoprofen) that can potentially decrease renal perfusion and predispose to gastric ulcers is absolutely contraindicated. administer analgesic drugs (fentanyl, µ/kg iv bolus, followed by - µ/kg/hour iv cri; or hydromorphone, . mg/kg iv) before anesthetic induction. after carrying out a balanced anesthesia protocol, the patient should be taken immediately to surgery for gastric derotation and gastropexy. postoperatively, assess the patient's ecg, blood pressure, platelet count, coagulation parameters, and gastric function (see section on rule of twenty). if no resection is required, the animal can be given small amounts of water beginning hours after surgery. depending on the severity of the patient's condition, small amounts of a bland diet can be offered to hours postoperatively. continute supportive care with analgesia and crystalloid fluids until the patient is able to tolerate oral analgesic drugs (tramadol, - mg/kg po q - h). once the patient is ambulatory and able to eat and drink on its own, it can be released from the hospital; instruct the owner to feed the animal multiple small meals throughout the day for the first week. when the intestines twist around the root of the mesentery, a small intestinal or mesenteric volvulus occurs. the problem is most common in the young german shepherd dog, although it has been observed in other large and giant breeds. predisposing factors include pancreatic atrophy, gastrointestinal disease, trauma, and splenectomy. clinical signs of mesenteric volvulus include vomiting, hemorrhagic diarrhea, bowel distention, acute onset of clinical signs of shock, abdominal pain, brick-red mucous membranes (septicemia), and sudden death. diagnosis is based on an index of suspicion and the presence of clinical signs in a predisposed breed. plain radiographs often reveal grossly distended loops of bowel in a palisade gas pattern. in some dogs, multiple, tear-drop-shaped, gas-filled loops appear to rise from a focal point in the abdomen. usually, massive distention of the entire small bowel is observed ( fig. - ) . the presence of pneumoperitoneum or lack of abdominal detail secondary to the presence of abdominal fluid is characteristic of bowel perforation and peritonitis. in a patient with mesenteric volvulus, immediate aggressive action is necessary for the animal to have any chance of survival. treatment consists of massive volumes of iv crystalloid and colloid fluids (see section on iv therapy), broad-spectrum antibiotics (ampicillin, mg/kg iv qid, with enrofloxacin, mg/kg iv once daily), and surgical correction of the bowel. because of the massive release of proinflammatory cytokines, bacterial translocation, and ischemia, treatment for shock is of paramount importance (see sections on rule of twenty and shock). prognosis for any patient with mesenteric volvulus is poor. obstipation (obstructive constipation) is most common in the older cat. in cases of simple constipation, rehydrating the animal with intravenous fluids and stool softeners is often volvulus. this consistutes an immediate surgical emergency, and the prognosis is often poor. this condition is most common in young german shepherd dogs, but can be observed in any breed. sufficient for it to regain the ability to have a bowel movement. obstipation, however, is caused by adynamic ileus of the large bowel that eventually leads to megacolon. affected cats usually are anorectic, lethargic, and extremely dehydrated. treatment consists of rehydration with intravenous crystalloid fluids, correction of electrolyte abnormalities, enemas, and promotility agents such as cisapride ( . mg/kg po q - h). the use of phosphate enemas in cats is absolutely contraindicated because of the risk of causing acute, fatal hyperphosphatemia. in many cases, the patient should be placed under general anesthesia and manual deobstipation is performed with warm water soapy enemas and a gloved finger to relieve and disimpact the rectum. stool softeners such as lactulose and docusate stool sofener (dss) may also be used. predisposing causes of obstipation such as narrowing of the pelvic canal, perineal hernia, and tumors should be ruled out. adenocarcinoma is the most common neoplasm of the gastrointestinal tract that causes partial to complete obstruction. adenocarcinomas tend to be annular and constricting, and they may cause progressive obstruction of the lumen of the small or large bowel. siamese cats tend to have adenocarcinomas in the small intestine, whereas in dogs, the tumor tends to occur in the large intestine. clinical signs of adenocarcinoma are both acute and chronic and consist of anorexia, weight loss, and progressive vomiting that occur over weeks to months. effusion may be present if metastasis to peritoneal surfaces has occurred. diagnosis is based on clinical signs and physical examination findings of a palpable abdominal mass, radiographic evidence of an abdominal mass and small or large intestinal obstruction, or ultrasonographic evidence of an intestinal mass. treatment consists of surgical resection of the affected bowel segment. the prognosis for long-term survival ( - months) is good if the mass is completely resected and if other clinical signs of cachexia or metastasis are observed at the time of diagnosis. median survival is to weeks if metastasis to lymph nodes, liver, or the peritoneum are absent at the time of diagnosis. in dogs, the prognosis is more guarded. leiomyoma and leiomyosarcoma are tumors that can cause partial or complete obstruction of the bowel. clinical signs are often referred to progressive anemia, including weakness, lethargy, inappetence, and melena. hypoglycemia can be observed as a paraneoplastic syndrome, or due to sepsis and peritonitis secondary to bowel perforation. leiomyomas are most commonly observed at the ceco-colic junction or in the cecum. surgical resection and anastomosis is usually curative, and has a favorable prognosis. incarceration of a loop of bowel into congenital or acquired defects in the body wall can cause small bowel obstruction. pregnant females and young animals with congenital hernias are most at risk. rarely, older animals with perineal hernias and animals of any age with traumatic hernias can be affected. clinical signs are consistent with a small intestinal obstruction: anorexia, vomiting, lethargy, abdominal pain, and weakness. diagnosis is often made based on physical examination of a reducible or nonreducible mass in the body wall. hernias whose contents are reducible are usually asymptomatic. treatment consists of supportive care and rehydration, administration of broad-spectrum antibiotics, and surgical correction of the body wall hernia. in some cases, intestinal resection and anastomosis of the affected area is necessary when bowel ischemia occurs. the potential for bowel perforation should be suspected whenever there is any penetrating injury (knife, gunshot wound, bite wound, stick impalement) of the abdomen. injuries that result in bowel ischemia and rupture can also occur secondary to nonpenetrating blunt emergency care trauma or shear forces (e.g., big dog-little dog/cat). perforation of the stomach and small and large intestines can occur with use of nonsteroidal antiinflammatory drugs. diagnosis of bowel perforation first depends on the alertness to the possibility that the bowel may have been perforated or penetrated. as a general rule, all penetrating injuries of the abdomen should be investigated by exploratory laparotomy. diagnostic peritoneal lavage (dpl) can be performed; however, early after penetrating injury of the bowel, dpl may be negative or nondiagnostic until peritonitis develops. whenever any patient with blunt or penetrating abdominal trauma does not respond to initial fluid therapy, or responds and then deteriorates, the index of suspicion for bowel injury should be raised. the findings of pneumoperitoneum on abdominal radiographs or of intracellular bacteria, extracellular bacteria, bile pigment, bowel contents, and cloudy appearance of fluid obtained by abdominocentesis or diagnostic peritoneal lavage fluid (see sections on abdominocentesis and diagnostic peritoneal lavage) warrant immediate surgical exploration. treatment largely consists of stabilizing the patient's cardiovascular and electrolyte status with intravenous fluids, administration of broad-spectrum antibiotics, and definitive surgical exploration and repair of injured structures. prolapse of the rectum is observed most frequently secondary to parasitism and gastrointestinal viral infections in young puppies and kittens with chronic diarrhea. older animals with rectal prolapse often have an underlying problem such as a tumor or mucosal lesion that causes straining and dyschezia. the diagnosis of a rectal prolapse is made based on physical examination findings. the diagnosis of rectal prolapse is sometimes difficult to distinguish from small intestinal intussusception. in rare cases, the intussusception can invaginate through the large bowel, rectum, and anus. the two entities are distinguished from one another by inserting a lubricated thermometer or blunt probe into the cul-de-sac formed by the junction of the prolapsed mucosa and mucocutaneous junction at the anal ring. inability to insert the probe or thermometer indicates that the rectal mucosa is prolapsed. passage of the probe signifies that the prolapsed segment is actually the intussusceptum. treatment can be performed easily if the prolapse is acute and the rectal mucosa is not too irritated or edematous. the presence of severely necrotic tissue warrants surgical intervention. to reduce an acute rectal prolapse, after placing the patient under general anesthesia, lubricate the prolapsed tissue and gently push it back into the rectum, using a lubricated syringe or syringe casing. apply a loose purse-string suture, leaving it in place for a minimum of hours. de-worm the patient and administer stool softeners. if a rectal prolapse cannot be reduced, or if the tissue is nonviable, surgical intervention is warranted. in patients in which viable tissue does not stay reduced with a purse-string suture, a colopexy can be performed during a laparotomy. first, place tension on the colon to reduce the prolapse, and then suture the colon to the peritoneum of the lateral abdominal wall with two to three rows of - or - monofilament suture material. if the prolapsed tissue is nonviable, it must be amputated. place four stay sutures at -degree intervals through the wall of the prolapse at the mucocutaneous junction. resect the prolapse distal to the stay sutures and then reestablish the rectal continuity by suturing the seromuscular layers together in one circumferential line and the mucosal layers together in the other. replace the suture incision into the anal canal. following surgery, de-worm the patient and administer a stool softener and analgesic drugs. avoid using thermometers or other probes in the immediate postoperative period because they may disrupt suture lines. acute gastritis may be associated with a variety of clinical conditions, including oral hemorrhage, ingestion of highly fermentable nondigestable foods or garbage, toxins, foreign bodies, renal or hepatic failure, inflammatory bowel disease, and bacterial and viral infections. diarrhea often accompanies or follows acute gastritis. hemorrhagic gastroenteritis often occurs as a shock-like syndrome with a rapidly rising hematocrit level. clinical signs of gastritis include depression, lethargy, anterior abdominal pain, excessive water consumption, vomiting, and dehydration. differential diagnosis of acute gastritis includes pancreatitis, hepatic or renal failure, gastrointestinal obstruction, and toxicities (box - ). the diagnosis is often a diagnosis of exclusion of other causes (see preceding text). a careful and thorough examination of the vomitus may be helpful in arriving at a diagnosis. a complete blood count, serum biochemistry profile including amylase and lipase, parvovirus test (in young puppies), fecal flotation and cytology, abdominal radiographs (plain and/or contrast studies), and abdominal ultrasound may be warranted to rule out other causes of acute vomiting. while diagnostic tests are being performed, treatment consists of withholding all food and water for a minimum of hours. after calculating the patient's degree of dehydration, administer a balanced crystalloid fluid to normalize acid-base and electrolyte status. control vomiting with antiemetics such as metoclopramide, prochlorperazine, chlorpromazine, dolasetron, and ondansetron (table - ). if vomiting is accompanied by diarrhea, administer broad-spectrum antibiotics (cefazolin, mg/kg iv q h, with metronidazole, mg/kg iv q h; or ampicillin, mg/kg iv q h, with enrofloxacin, mg/kg iv q h) to decrease the risk of bacterial translocation and bacteremia/septicemia. although antacids (famotidine, ranitidine, cimetidine) do not have a direct antiemetic effect, their use can decrease gastric acidity and esophageal irritation during vomiting. if gastritis is secondary to uremia or nonsteroidal antiinflammatory drug use, administer gastroprotectant and antiemetic drugs (ranitidine, mg/kg po q h; sucralfate, . - g/dog po q h; or omeprazole ( . - mg/kg po q h) to decrease acid secretion and coat areas of gastric ulceration (table - ) . once food and water can be tolerated, the patient can be placed on an oral diet and medications, and intravenous fluids can be discontinued. do not use until a gastrointestinal obstruction has been ruled out. hemorrhagic gastroenteritis (hge) is an acute onset of severe hemorrhagic vomiting and diarrhea most commonly observed in young small-breed dogs (e.g., poodles, miniature dachshunds, miniature schnauzers) to years of age. clinical signs develop rapidly and include vomiting and fetid diarrhea with hemorrhage, often strawberry jam-like in appearance. the hematocrit can rise from % to %. often, the animal is extremely hypovolemic but has no apparent signs of abdominal pain. there is no known cause of hge, although clostridium perfringens, escherichia coli, campylobacter, and viral infections have been suggested but not consistently confirmed. other differential diagnoses of of hematemesis and hemorrhagic diarrhea include coronavirus, parvovirus, vascular stasis, sepsis, hepatic cirrhosis with portal hypertension, and other causes of severe shock. immediate treatment consists of placement of a large-bore intravenous catheter and replenishment of intravascular fluid volume with crystalloid fluids (up to ml/kg/hour), while carefully monitoring the patient's hematocrit and total protein. administer broad-spectrum antibiotics (ampicillin, mg/kg iv q h, and enrofloxacin mg/kg iv q h) because of the high risk of bacterial translocation and sepsis. control vomiting with antiemetic drugs. monitor the patient's platelet count and coagulation tests for impending disseminated intravascular coagulation (dic), and administer fresh frozen plasma and heparin, as needed (see section on disseminated intravascular coagulation). when vomiting has ceased for hours, offer the animal small amounts of water, and then a bland diet (e.g., boiled chicken and rice or boiled ground beef and rice mixed with low-fat cottage cheese). pancreatitis occurs most frequently in dogs but can occur in cats as well. in dogs, the onset of pancreatitis is sometimes preceded by ingestion of a fatty meal or the administration of drugs (e.g., potassium bromide or glucocorticoids). glucocorticoids can increase the viscosity of pancreatic secretions and induce ductal proliferation, resulting in narrowing and obstruction of the lumen of the pancreatic duct. pancreatitis can also occur following blunt or penetrating abdominal trauma, high duodenal obstruction causing outflow obstruction of the pancreatic papilla, pancreatic ischemia, duodenal reflux, biliary disease, and hyperadrenocorticism. in cats, acute necrotizing pancreatitis is associated with anorexia, lethargy, hyperglycemia, icterus, and sometimes acute death. chronic pancreatitis is more common in cats and results in intermittent vomiting, anorexia, weight loss, and lethargy. predisposing causes of chronic pancreatitis in cats include pancreatic flukes, viral infection, hepatic lipidosis, drugs, organophosphate toxicity, and toxoplasmosis. clinical signs of acute pancreatitis include sudden severe vomiting, abdominal pain, and lethargy. depending on the severity of pancreatic inflammation, depression, hypotension, and systemic inflammatory response syndrome (sirs) may be present. subacute cases may have minimal clinical signs. severe pancreatic edema can result in vascular changes and ischemia that perpetuates severe inflammation. hypovolemic shock and dic can also decrease pancreatic perfusion. severe pancreatic edema, autolysis, and ischemia lead to pancreatic necrosis. duodenal irritation is manifested as both vomiting and diarrhea. pain may be localized to the right upper abdominal quadrant or may be generalized if peripancreatic saponification occurs. differential diagnosis of pancreatitis is the same as for any other cause of vomiting. complications that occur in patients with severe pancreatitis include dehydration, acidbase and electrolyte abnormalities, hyperlipemia, hypotension, and localized peritonitis. hepatic necrosis, lipidosis, congestion, and abnormal architecture can develop. inflammatory mediators (bradykinin, phospholipase a, elastase, myocardial depressant factor, and bacterial endotoxins) stimulate the inflammatory cascade and can lead to sirs, with severe hypotension, clotting system activation, and dic. electrolyte imbalances and hypovolemia secondary to vomiting all can lead to multiple organ dysfunction syndrome (mods), and ultimately, death. if a patient survives an episode of acute pancreatitis, long-term sequelae can include diabetes mellitus. monitor patients with recurrent pancreatitis for clinical signs of polyuria, polydipsia, polyphagia, hyperglycemia, and glucosuria. the diagnosis of pancreatitis is based on the presence of clinical signs (which may be absent in cats), laboratory findings, and ultrasonographic evidence of pancreatic edema and increased peripancreatic echogenicity. serum biochemistry analyses can sometimes support a diagnosis of pancreatitis; however, serum amylase and lipase are often unreliable indicators of pancreatitis, depending on the chronicity of the process in the individual patient. both serum amylase and lipase are excreted in the urine. impaired renal clearance/ function can cause artifactual elevations of serum amylase and lipase in the absence of pancreatic inflammation. furthermore, serum lipase levels can be elevated as a result of gastrointestinal obstruction (e.g., foreign body). early in the course of the disease, levels can be two to six times normal, but they may decrease to within normal ranges at the time of presentation to the veterinarian. the transient nature of amylase elevation makes this test difficult to interpret, and it is not highly sensitive if a normal value is found. lipase levels also increase later in the course of the disease. amylase and lipase should be tested concurrently with the rest of the biochemistry profile. other changes often observed are elevations in bun and creatinine levels secondary to dehydration and prerenal azotemia, hyperglycemia, and hyperlipemia. hypocalcemia can occur secondary to peripancreatic fat saponification, and its presence warrants a more negative prognosis. a more specific measure is pancreatic lipase immunoreactivity, which becomes elevated in dogs and cats with pancreatitis. this test, combined with ultrasonographic or computed tomography evidence of pancreatitis, is the most sensitive and specific test available for making an accurate diagnosis. however, because the results of this test take time to obtain, animals must be treated in the meantime. abdominal effusion or fluid from diagnostic peritoneal lavage can be compared with serum amylase and lipase activity. abdominal lipase and amylase concentrations in the fluid greater than that in the peripheral blood are characteristic of chemical peritonitis associated with pancreatitis. wbc counts greater than cells/mm , the presence of bacteria, toxic neutrophils, glucose levels less than mg/dl, or lactate levels greater than that of serum are characteristic of septic peritonitis, and immediate exploratory laparotomy is warranted. if a biopsy sample obtained during laparotomy does not demonstrate inflammation, but this does not rule out pancreatitis, because disease can be focal in nature and yet cause severe clinical signs. abdominal radiographs may sometimes reveal a loss of abdominal detail or a ground glass appearance in the right upper quadrant. pancreatic edema and duodenal irritation can displace the gastric axis toward the left, toward the left with dorsomedial displacement of the proximal duodenum (the so-called "backwards " or "shepherd's crook" sign). ultrasonography and ct are more sensitive in making a diagnosis of pancreatitis. treatment of pancreatitis is largely supportive in nature and is designed to correct hypovolemia and electrolyte imbalances, prevent or reverse shock, maintain vital organ perfusion, alleviate discomfort and pain, and prevent vomiting (see section on rule of twenty). when treating pancreatitis in dogs, all food and water should be restricted. however, food should not be withheld from cats with chronic pancreatitis. give fresh frozen plasma to replenish alpha- -macroglobulins. administer antiemetics such as chlorpromazine (use with caution in a hypovolemic or hypotensive patient), dolasetron, ondansetron, or metoclopramide to prevent or control vomiting. analgesic drugs can be provided in the form of constant rate infusion (fentanyl, - µ/kg/hour iv cri, and lidocaine, - µ/kg/minute iv cri), intrapleural injection (lidocaine, - mg/kg q h), or intermittent parenteral injections (morphine, . - mg/kg sq, im; hydromorphone, . mg/kg im or sq). because the pancreas must be rested, consider using parenteral nutrition. acute hepatic failure may be associated with toxins, adverse reaction to prescription medication, and bacterial or viral infections. the most frequent clinical signs observed in a patient with acute hepatic failure are anorexia, lethargy, vomiting, icterus, bleeding, and cns depression or seizures (associated with hepatic encephalopathy). differential diagnosis and causes of acute hepatic failure are listed in box - . diagnosis of acute hepatic failure is based on clinical signs and biochemical evidence of hepatocellular (ast, alt) and cholestatic (alk phos, t bili, ggt) enzyme elevations. ultrasonography may be helpful in distinguishing the architecture of the liver, but unless a mass or abscess is present, cannot provide a specific diagnosis of the cause of the hepatic damage. management of the patient with acute hepatic failure includes correction of dehydration and acid-base and electrolyte abnormalities, as shown in the following list: • hypoalbuminemia: plasma or concentrated albumin. plasma also is an excellent source of clotting factors that can become depleted. • clotting abnormalities: vitamin k ( . mg/kg sq or po q - h) to • severe anemia: fresh or stored blood • gastric hemorrhage: gastroprotectant drugs (omeprazole, ranitidine, famotidine, cimetidine, sucralfate) • hypoglycemia: dextrose supplementation ( . %- %) • hepatic failure, particularly when hypoglycemia is present: broad-spectrum antibiotics (ampicillin mg/kg iv q h; with enrofloxacin, mg/kg iv q h) • hepatic encephalopathy: lactulose or betadine enemas • cerebral edema: mannitol ( . - . g/kg iv over to minutes) followed by furosemide ( mg/kg iv minutes later). deterioration of clinical signs may signify the development of cerebral edema. applewhite aa, cornell kk, selcer ba: diagnosis and treatment of intussusception in dogs. comp cont educ pract vet ( ) often, systemic hypertension is diagnosed when the animal is seen by the veterinarian because of some other clinical sign, such as acute blindness, retinal detachment, hyphema, epistaxis, and cns signs following intracranial hemorrhage. diagnosis of systemic hypertension is often difficult in the absence of clinical signs and without performing invasive or noninvasive blood pressure monitoring. normal blood pressure (bp) measurements in dogs and cats are listed in table - . hypertension is defined as a consistent elevation in systolic bp > mm hg, consistent diastolic bp > mm hg, and consistent mean arterial blood pressure > mm hg. the effects of systemic hypertension include left ventricular hypertrophy, cerebrovascular accident, renal vascular injury, optic nerve edema, hyphema, retinal vascular tortuosity, retinal hemorrhage, retinal detachment, vomiting, neurologic defects, coma, and excessive bleeding from cut surfaces. emergency care dog - - - cat - - - patients with systemic hypertension should have a thorough diagnostic work-up to determine the underlying cause. although uncommon, hypertensive emergencies can occur with pheochromocytoma, acute renal failure, and acute glomerulonephritis. sodium nitroprusside ( - µ/kg/minute iv cri) or diltiazem ( . - . mg/kg iv given slowly over minutes, followed by µ/kg/minute) can be used to treat systemic hypertension. with the use of sodium nitroprusside or diltiazem, monitor carefully for hypotension. diagnosis is based on consistent elevations in systolic, diastolic, and/or mean arterial bp. because many of the clinical signs associated with systemic hypertension involve hemorrhage into some closed cavity, other causes of hemorrhage, such as vasculitis, thrombocytopenia, thrombocytopathia, and hepatic or renal failure, should be investigated (see section on coagulation disorders). diagnostic testing is based on clinical signs and index of suspicion for an underlying disease and may include a complete blood count; urinalysis; urine protein:creatinine ratio; acth stimulation test; thoracic and abdominal radiographs; thoracic and abdominal ultrasound; tick serology; brain ct or mri; and assays of serum electrolytes, aldosterone concentration, t , endogenous tsh, plasma catecholamine, and growth hormone. management of systemic hypertension involves treatment of the primary underlying disorder, whenever possible. long-term adjunctive management includes sodium restriction in the form of cooked or prescription diets to decrease fluid retention. obese animals should be placed on dietary restrictions and undergo a weight reduction program. thiazide and loop diuretics may be used to decrease sodium retention and circulating blood volume. alpha-and beta-adrenergic blockers may be used, but they are largely ineffective as monotherapeutic agents for treating hypertension. calcium channel blockers and angiotensin-converting enzyme (ace) inhibitors are the mainstay of therapy in the treatment of hypertension in dogs and cats ( diabetic ketoacidosis (dka) is a potentially fatal and terminal consequence of unregulated insulin deficiency and possible glucagon excess. in the absence of insulin, unregulated lipolysis results in the beta-hydroxylation of fatty acids by abnormal hepatic metabolism. as a result, ketoacids-namely, acetoacetic acid, beta-hydroxybutyric acid, and acetoneare produced. early in the course of the disease, patients exhibit clinical signs associated with diabetes mellitus: weight loss, polyuria, polyphagia, and polydipsia. later, as ketoacids stimulate the chemoreceptor trigger zone, vomiting and dehydration occur, with resulting hypovolemia, hypotension, severe depression, abdominal pain, oliguria, and coma. at the time of presentation, often a strong odor of ketones (acetone) is present on the patient's breath. physical examination often reveals dehydration, severe depression or coma, and hypovolemic shock. in extreme cases, the patient exhibits a slow, deep kussmaul respiratory pattern in an attempt to blow off excess co to compensate for the metabolic acidosis. a serum biochemistry profile and complete blood count often reveal prerenal azotemia, severe hyperglycemia (blood glucose > mg/dl), hyperosmolarity (> mosm/kg), lipemia, hypernatremia (sodium > meq/l), elevated hepatocellular and cholestatic enzyme activities, high anion gap, and metabolic acidosis. although a whole body potassium deficit is usually present, the serum potassium may appear artifactually elevated in response to metabolic acidosis. with severe metabolic acidosis, potassium moves extracellularly in exchange for a hydrogen ion. phosphorus too moves intracellularly in response to acidosis, and serum phosphorus is usually decreased. hypophosphatemia > mg/dl can result in intravascular hemolysis. urinalysis often reveals + glucosuria, ketonuria, and a specific gravity of . or greater. the urine of all diabetic animals should be cultured to rule out a urinary tract infection or pyelonephritis. treatment of a patient with dka presents a therapeutic challenge. treatment is aimed at providing adequate insulin to normalize cellular glucose metabolism, correcting acidbase and electrolyte imbalances, rehydration and restoration of perfusion, correcting acidosis, providing carbohydrate sources for utilization during insulin administration, and identifying any precipitating cause of the dka. obtain blood samples for a complete blood count, and serum biochemistry electrolyte profiles. whenever possible, insert a central venous catheter for fluid infusion and procurement of repeat blood samples. calculate the patient's dehydration deficit and maintenance fluid requirements and give appropriate fluid and electrolytes over a period of hours. it is advisable to rehydrate patients with severe hyperosmolarity for a minimum of hours before starting insulin administration. use a balanced electrolyte solution (e.g., plasmalyte-m, normosol-r, lactated ringer's solution) or . % saline solution for maintenance and rehydration. balanced electrolyte solutions contain small amounts of potassium and bicarbonate precursors that aid in the treatment of metabolic acidosis. treat animals with severe metabolic acidosis with an hco − > meq/l or a ph < . with supplemental bicarbonate ( . - . meq/kg). add supplemental dextrose to the patient's fluids as a carbohydrate source during insulin infusion. both insulin and carbohydrates are necessary for the proper metabolism of ketone bodies in patients with dka. the rate and type of fluid and amount of dextrose supplementation will change according to the patient's blood glucose concentration. serum potassium will drop rapidly as the metabolic acidosis is corrected with fluid and insulin administration. measure serum potassium every hours, if possible, and supplement accordingly (see section on fluid therapy for chart of potassium supplementation). if the patient's potassium requirement exceeds meq/l, or if the rate of potassium infusion approaches . meq/ kg/hour in the face of continued hypokalemia, magnesium should be supplemented. magnesium is required as a cofactor for many enzymatic processes and for normal function of the na,k-atpase pump. hypomagnesemia is a common electrolyte disturbance in many forms of critical illness. replenishing magnesium (mgcl , . meq/kg/day iv cri) often helps to correct the refractory hypokalemia observed in patients with dka. patients with hypophosphatemia that approaches . mmol/l should receive potassium phosphate ( . - . mmol/kg/hour iv cri). when providing potassium phosphate supplementation, be aware of the additional potassium added to the patient's fluids, so as to not exceed recommended rates of potassium infusion. to determine the amount of potassium chloride (kcl) to add along with potassium phosphate (kpo ), use the following formula: meq k + derived from kcl = total meq of k + to be administered over hours − meq in which k + is derived from kpo clinical signs of severe hypophosphatemia include muscle weakness, rhabdomyolysis, intravascular hemolysis, and decreased cerebral function that can lead to depression, stupor, seizures, or coma. regular insulin can be administered either im or as a constant rate infusion in the treatment of patients with dka. subcutaneous insulin should not be administered. because of the severe dehydration present in most patients with dka, subcutaneous insulin is poorly absorbed and is not effective until hydration has been restored. in the low-dose intravenous method, place regular insulin ( . units/kg for a cat, and . units/kg for a dog) in ml of . % saline solution. run ml of this mixture through the intravenous line to allow the insulin to adsorb to the plastic tubing. administer the patient's insulin fluid rate according to blood glucose levels ( table - ) . adjust the patient's total fluid volume according to changes in the insulin fluid rate as necessary. in many cases, multiple bags of fluids are necessary because they must be changed when fluctuations in blood glucose concentrations occur in response to therapy. infusion of the insulin mixture should be in a separate intravenous catheter. to replenish hydration, use a second intravenous line for the more rapid infusion of non-insulin-containing fluids. to administer the regular insulin im, first give . unit/kg im and then re-check the patient's blood glucose every hour. additional injections of regular insulin ( . unit/kg other fluid type (ml/hour) > . % nacl - . % nacl + . % dextrose - . % nacl + . % dextrose - . % nacl + . % dextrose < . % nacl + % dextrose im) should be administered based on the patient's response to subsequent injections. once the patient's blood glucose falls to to mg/dl, add . % to % dextrose to the fluids to maintain the blood glucose concentration at to mg/dl. continue intramuscular injection of regular insulin ( . - . unit/kg q - h) until the patient is rehydrated, no longer vomiting, and able to tolerate oral fluids and food without vomiting. even in patients with intramuscular regular insulin therapy, a central venous catheter should be placed for frequent blood sample collection. as the patient begins to respond to therapy, monitor electrolytes, glucose, and acid-base status carefully. hypokalemia, hypophosphatemia, and hypomagnesemia can occur. when the patient's hydration and acid-base status has normalized and the patient is able to tolerate oral food and water, a longer-acting insulin can be administered as for treatment of a patient with uncomplicated diabetes. extreme hyperosmolarity can result in a coma, if uncorrected. in patients with diabetes mellitus, hyperglycemia and hypernatremia secondary to osmotic diuresis and free water loss can lead to severe hyperosmolarity. in dogs, normal serum osmolality is < mosm/l of serum. hyperosmolarity is expected when serum osmolality is > mosm/l. if equipment for determining serum osmolarity is not available, osmolarity can be calculated by the following formula: osm/l = (na + k) + (glucose/ ) + (bun/ . ) patients with severe dehydration, hyperglycemia, hypernatremia, and azotemia may experience cerebral edema without ketonemia. treatment is directed solely at rehydrating the patient and slowly reducing blood glucose levels using a hypotonic solution such as . % nacl + . % dextrose or % dextrose in water (d w). after the initial rehydration period, administer potassium supplementation conservatively. red blood cells and the brain absolutely depend on the oxidation of glucose for energy. hypoglycemia can be caused by various systemic abnormalities that can be related to intestinal malabsorption of nutrients, impaired hepatic glycogenolysis or gluconeogenesis, and inadequate peripheral utilization of glucose. clinical signs of hypoglycemia are extremely variable and can include weakness, tremors, nervousness, polyphagia, ataxia, tachycardia, muscle twitching, incoordination, visual disturbances, and generalized seizures. clinical signs typically occur when serum glucose levels are < mg/dl. the combination of the clinical signs listed previously, documentation of low serum glucose, and alleviation of clinical signs upon glucose administration is known as whipple's triad. whenever a patient presents with hypoglycemia, consider the following important factors: the age of onset, the nature of the hypoglycemic episode (transient, persisent, or recurrent) , and the pattern based on the patient's history . treatment of hypoglycemia is directed at providing glucose supplementation and determining any underlying cause. administer supplemental dextrose ( %- % dextrose, - ml/kg iv; or % dextrose, ml/kg po) as quickly as possible. do not attempt oral glucose supplementation in any patient having a seizure or if the airway cannot be protected. administer intravenous fluids (e.g., normosol-r, lactated ringer's solution, . % saline solution) with . %- % supplemental dextrose until the patient is eating and able to maintain euglycemia without supplementation. in some cases (e.g., insulinoma), eating or administration of supplemental dextrose can promote insulin secretion and exacerbate clinical signs and hypoglycemia. in cases of refractory hypoglycemia secondary to iatrogenic insulin overdose, glucagon ( mg/kg iv bolus, then - ng/kg/minute iv cri) can also be administered along with supplemental dextrose. to make a glucagon infusion of ng/ml, reconstitute ml ( mg/ml) of glucagon according to the manufacturer's instructions and add this amount to ml of . % saline solution. emergency care the diagnosis of eclampsia (puerperal tetany) is often made on the basis of history and clinical signs. clinical signs can become evident when total calcium decreases to < . mg/dl in dogs and < . mg/dl in cats. the disease is often observed in small, excitable dogs, and stress may play a complicating role in the etiology. in most bitches, the disease manifests itself to weeks after parturition. in some cases, however, clinical signs can develop before parturition occurs. hypophosphatemia may accompany hypocalcemia. clinical signs of hypocalcemia include muscle tremors or fasciculations, panting, restlessness, aggression, hypersensitivity, disorientation, muscle cramping, hyperthermia, stiff gait, seizures, tachycardia, a prolonged qt interval on ecg, polydipsia, polyuria, and respiratory arrest. treatment of eclampsia consists of slow, cautious calcium supplementation ( % calcium gluconate, . mg/kg iv over minutes). severe refractory tetanus can be controlled with intravenous diazepam. supportive care includes intravenous fluid administration and cooling (see section on hyperthermia and heat-induced illness). instruct the owner to give the patient oral calcium supplements (e.g., to tablets of tums bid-tid) after discharge from the hospital. also instruct the owner about how to wean the puppies, allowing the bitch to dry up, in order to prevent recurrence. recurrence with subsequent pregnancies is common, particularly in patients that receive calcium supplementation during gestation (table - ) . hypercalcemia can occur from a variety of causes. the gosh darn it mnemonic can be used to remember the various causes of hypercalcemia in small animal patients (box - ) . the gastrointestinal, renal, and nervous systems are most commonly affected, particularly when serum total calcium rises above . mg/dl. clinical signs of severe hypercalcemia include muscle weakness, vomiting, seizures, and coma. ecg abnormalities include prolonged pr interval, rapid qt interval, and ventricular fibrillation. the most serious clinical signs are often seen when hypercalcemia is observed in combination with hyperphosphatemia or hypokalemia. pay special attention to the "calcium × phosphorus product." if this product exceeds , dystrophic calcification can occur, leading to renal failure. renal complications include polyuria, polydipsia, dehydration, and loss of renal tubular concentrating ability. renal blood flow and the glomerular filtration rate (gfr) are impaired when serum total calcium exceeds mg/dl. the extent, location, and number of renal tubular injuries are the main factors in determining whether renal damage secondary to hypercalcemia is reversible or irreversible. emergency therapy of hypercalcemia is warranted when severe renal compromise, cardiac dysfunction, or neurologic abnormalities are present, or if no clinical signs occur but the calcium × phosphorus product exceeds . the treatment of choice is correction of the underlying cause of hypercalcemia, whenever possible. in some cases, the results of diagnostic tests take time, and emergency therapy should be initiated immediately, before a definitive cause of the hypercalcemia is found. emergency management of hypercalcemia consists of reduction of serum calcium levels. administer intravenous fluids ( . % saline solution) to expand extracellular fluid volume and promote calciuresis. to promote diuresis, initial intravenous fluid rates should approach two to three times maintenance levels ( - ml/kg/day). potassium supplementation may be required to prevent iatrogenic hypokalemia. administration of a loop diuretic such as furosemide ( - mg/kg iv) will promote calcium excretion. calcitonin ( iu/kg im q h for cats and iu/kg im q h for dogs) can be administered to decrease serum calcium levels. in severe refractory hypercalcemia secondary to cholecalciferol toxicity, more aggressive calcitonin therapy ( - iu/kg sq q - h) can be attempted. side effects of calcitonin treatment include vomiting and diarrhea. alternatively, bisphosphonates (pamidronate, . - . mg/kg iv) are useful in rapidly reducing serum calcium concentrations. glucocorticosteroids reduce calcium release from the bone, decrease intestinal absorption of calcium, and promote renal calcium excretion. administer glucocorticosteroids only after the underlying cause of hypercalcemia has been determined and appropriate therapy started. because many forms of neoplasia can result in hypercalcemia as a paraneoplastic syndrome, empiric use of glucocorticosteroids can induce multiple drug resistance, making the tumor refractory to the effects of chemotherapeutic agents. hypoadrenocorticism is most commonly observed in young to middle-aged female dogs, but it can occur in animals of any age, gender, and breed. clinical signs, which are referable to deficiency in glucocorticoid (cortisol) and mineralocorticoid (aldosterone) hormones, may develop slowly over time, leading to a waxing and waning course; acute clinical signs occur when > % of the adrenal functional reserve has been destroyed. in such cases, complete adrenocortical collapse can result in an addisonian crisis. lack of aldosterone causes a lack of renal sodium and water retention, and impaired potassium excretion. the most significant clinical signs associated with hypoadrenocorticism are depression, lethargy, weakness, anorexia, shaking, shivering, vomiting, diarrhea, weight loss, abdominal pain, weakness, hypotension, dehydration, and inappropriate bradycardia (box - ) . the diagnosis of hypoadrenocorticism is made based on the patient's clinical signs in combination with electrolyte abnormalities that include hyperkalemia, hyponatremia, and hypochloremia. serum sodium concentration ( - meq/l) is often greatly reduced, and serum potassium is elevated (> . meq/l). a sodium:potassium ratio of < is characteristic of hypoadrenocorticism, although not exactly pathognomonic. electrocardiographic changes associated with hyperkalemia include inappropriate bradycardia, absence of p waves, elevated spiked t waves, and widened qrs complexes. other more variable bloodwork abnormalities include a lack of a stress leukogram, eosinophilia, hypoglycemia, hyperphosphatemia, hypercalcemia, azotemia, and hypocholesterolemia. a definitive diagnosis of hypoadrenocorticism is based on an adrenocorticotropic hormone (acth) stimulation test. in patients with hypoadrenocorticism, baseline cortisol levels are usually low, with a lack of appropriate cortisol release after administration of acth analogue. rarely, animals with "atypical" hypoadrenocorticism lose glucocorticoid secreting ability from the zona fasciculata, but retain mineralocorticoid secretory ability from the zona glomerulosa. atypical addisonian patients have normal serum electrolytes but still have clinical signs of vomiting, diarrhea, weakness, lethargy, inappetence, muscle wasting, and weight loss. the diagnosis is more difficult in such cases because of the presence of normal electrolytes. an acth stimulation test should be considered, particularly in predisposed breeds. treatment of hypoadrenocorticism includes placement of a large-bore intravenous catheter, infusion of intravenous crystalloid fluids ( . % saline solution), and replenishment of glucocorticoid and mineralocorticoid hormones. administer dexamethasone or dexamethasone-sodium phosphate ( . - . mg/kg iv). dexamethasone will not interfere with the acth stimulation test, unlike other longer-acting steroids (e.g., prednisolone, methylprednisolone sodium succinate, triamcinolone). depending on the severity of the patient's condition, consider monitoring using the rule of twenty. administer antiemetics and gastroprotectant drugs to treat nausea, vomiting, and hematemesis. give the patient broad-spectrum antibiotics (ampicillin, mg/kg iv q h) if hematochezia or hemorrhagic diarrhea is present. if severe gastrointestinal blood loss occurs, whole blood, packed red blood cells, or fresh frozen plasma may be required. control hypoglycemia with . %- . % dextrose. use sodium bicarbonate, regular insulin with dextrose, or calcium gluconate to correct severe hyperkalemia with atrial standstill (see section on atrial standstill). chronic therapy for hypoadrenocorticism consists of mineralocorticoid and glucocorticosteroids supplementation for the rest of the animal's life. mineralocorticoid supplementation can be in the form of desoxycorticosterone pivalate (docp) ( . mg/kg im) or fludrocortisone acetate ( . mg/ . - kg body weight daily). fludrocortisone acetate possesses both mineralocorticoid and glucocorticoid activities and can be used as the sole daily treatment of hypoadrenocorticism. (because fludrocortisone is poorly absorbed in some dogs, it may not completely normalize electrolyte abnormalities in these animals.) docp is primarily a mineralocorticoid. give supplemental glucocorticosteroids in the form of prednis(ol)one ( - . mg/kg/day). in dogs, iatrogenic hypoadrenocorticism can be caused by abrupt discontinuation of glucocorticosteroid treatment. long-term glucocorticosteroid supplementation can downregulate the pituitary gland's excretion of endogenous acth and the zona fasciculata's ability to excrete cortisol. however, the zona glomerulosa's ability to secrete aldosterone does not appear to be affected. clinical signs of iatrogenic hypoadrenocorticism include inability to compensate for stress, weakness, lethargy, vomiting, diarrhea, and collapse. treatment of iatrogenic hypoadrenocorticism is the same as for naturally occurring disease. following immediate emergency treatment, the patient should be weaned slowly from exogenous glucocorticosteroid supplementation. severe hyperthyroidism can manifest as a medical emergency as a result of hypermetabolism. clinical signs in affected cats with severe thyrotoxicosis include fever, severe tachycardia (heart rate > bpm), vomiting, hypertension, congestive heart failure with pulmonary edema, and fulminant collapse. clinical signs typically are manifested as an end-stage of chronic debilitation associated with hyperthyroidism and are often preceded by polyphagia, weight loss, cardiac murmur, polyuria/polydipsia (pu/pd), vomiting, and diarrhea. treatment of thyrotoxicosis includes antagonizing the adrenergic activity by administration of a beta-adrenergic blocker (esmolol, ( - µ/kg/minute, or propranolol, . mg/ kg/hour). administration of glucocorticosteroids (dexamethasone, mg/kg) may inhibit the conversion of thyroxine (t ) to the active form triiodothyronine (t ) and decrease peripheral tissue responsiveness to t , effectively blocking its effects. correct hypoglycemia with supplemental dextrose ( . %). use care to avoid overhydration in a patient with cardiac failure or insufficiency. start the patient on methimazole as quickly as possible and consider the use of radioactive iodine therapy. to maintain cerebral perfusion pressure, blood pressure must be normalized. if other concurrent injuries are suspected (e.g., pulmonary contusions), administer synthetic colloid fluids (dextran- , - ml/kg iv, or hetastarch, - ml/kg iv) to normalize blood pressure. although the use of colloids is controversial because of their potential to leak into the calvarium, the benefits of reestablishing cerebral perfusion far outweigh the risks of their use. hypertonic saline ( . % nacl, - ml/kg iv) can also be administered over to minutes to expand intravascular volume. maintain blood glucose within normal reference ranges whenever possible, because hyperglycemia is a negative prognostic indicator in cases of head trauma. if tremors or seizures cause hyperthermia or increased metabolism, active cooling of the patient is warranted (see sections on hyperthermia and heat-induced injury). all patients with head trauma should receive care and monitoring based on the rule of twenty (see section on rule of twenty). examine the patient's level of consciousness, response to various stimuli, pupil size and reactivity to light, physiologic nystagmus, and cranial nerve deficits. in dogs, damage to the midbrain often produces coma and decerebrate rigidity. initial consciousness followed by a unconsciousness or stupor usually involves an injury to the brainstem. brainstem lesions can be caused by compressive skull fractures, extradural or subdural hematomas, or herniation through the foramen magnum from cerebral edema (box - ) . the patient's pupil size and response to light can be used to localize a diagnosis and give a rough prognosis for severity of disease and possibility for return to function. pupils can be normal in size, mydriatic, or miotic. whenever a pupil appears miotic, direct ocular emergency care unconscious with no response to noxious stimuli injury with uveitis or secondary miosis due to brachial plexus injury should be ruled out. the eyes should always be examined to rule out ocular trauma. in a patient with head trauma, a change from dilated to constricted to normal pupil size is suggestive of improvement in clinical function. bilateral mydriatic pupils that are unresponsive to light in an unconscious animal are a grave prognostic sign and usually indicate an irreversible severe midbrain contusion. bilateral miotic pupils with normal nystagmus and ocular movements are associated with diffuse cerebral or diencephalic lesions. miotic pupils that become mydriatic indicate a progressive midbrain lesion with a poor prognosis. unilateral, slowly progressive pupillary abnormalities in the absence of direct ocular injury are characteristic of brainstem compression or herniation caused by progressive brain swelling. asymmetric pupils are seen in patients with rostral brainstem lesions and can change rapidly. unresponsive pupils that are seen in the midposition occur with brainstem lesions that extend into the medulla and are a grave sign. visual deficits are common with intracranial injury. lesions that are less severe and limited to the cerebrum produce contralateral menace deficits with normal pupillary light response. bilateral cerebral edema can cause blindness with a normal response to light if the midbrain is not disturbed. a patient that is severely depressed and recumbent may not respond to menacing gestures, even when visual pathways are intact. ocular, optic tract, optic nerve, or optic chiasm lesions can interfere with vision and the pupillary light response. brainstem contusion and cerebral edema may produce blindness and dilated unresponsive pupils due to disturbance of the oculomotor area. examine all cranial nerves carefully. cranial nerve abnormalities can indicate direct contusion or laceration of the neurons in the brainstem or where they exit the skull. cranial nerves that are initially normal then later lose function indicate a progressively expanding lesion. when specific cranial nerve deficits are present, the prognosis is considered guarded. clinical signs such as rolling to one side, torticollis, head tilt, and abnormal nystagmus are usually associated with petrosal bone or cerebellomedullary lesions that produce vestibular neuron dysfunction. fractures of the petrosal temporal bone often cause hemorrhage and cerebrospinal fluid (csf) leak from the external ear canal. if the lesion is limited to the membranous labyrinth, the loss of balance will be toward the injured side and the quick phase of the nystagmus will be toward the injured side. normal physiologic nystagmus requires that the pathway is between the peripheral vestibular neurons and the pontomedullary vestibular nuclei to the nuclei of the cranial nerves that innervate the extraocular muscles (iii, iv, vi). severe brainstem lesions disrupt this pathway. disruption of the pathway is manifested as an inability to produce normal physiologic nystagmus by moving the patient's head from side to side. in patients with severe central nervous system depression, this reflex may not be observed. next, assess postural changes and motor function abilities. a loss of the normal oculocephalic ("dolls-eye") reflex is an early sign of brainstem hemorrhage and a late sign of brainstem compression and herniation. any intracranial injury may be accompanied by a concurrent cervical spinal cord injury. handle animals with such injuries with extreme care to avoid causing further damage. whenever there is uncertainty whether a spinal cord lesion exists, strap the patient down to a flat surface and obtain radiographs of the spine. at least two orthogonal views may be required to see fractures; however, do not manipulate the patient until radiography has been completed. crosstable views, in which the bucky is turned perpendicular to the patient's spine, with a radiograph plate secured behind the patient, may be required to minimize patient motion. in patients with cerebral lesions, hemiparesis usually resolves within to days. evaluation of cranial nerve function at frequent intervals may reveal an initial injury or a progressively expanding lesion in the brain. signs of vestibular disorientation, marked head tilt, and abnormal nystagmus occur with contusions of the membranous labyrinth and fracture of the petrous temporal bone. hemorrhage and cerebrospinal fluid otorrhea may be visible from the external ear canal. rolling movements indicate an injury to the cerebellar-medullary vestibular system. respiratory dysfunction and abnormal respiratory patterns are sometimes observed with severe head injury. lesions of the diencephalon produce cheyne-stokes respirations, in which the patient takes progressively larger and larger breaths, pauses, then takes progressively smaller and smaller breaths. mesencephalic lesions cause hyperventilation and can result in respiratory alkalosis. medullary lesions result in a choppy, irregular respiratory pattern. clinical signs of respiratory dysfunction in the absence of primary respiratory damage indicate a guarded prognosis. after injury, seizures may be associated with intracranial hemorrhage, trauma, or an expanding intracranial mass lesion. immediately begin medical therapy to control the seizure. administer diazepam ( . mg/kg iv or . - . mg/kg/hour iv cri) to treat seizures. if diazepam is not effective in combination with other treatments to control intracranial edema, consider giving pentobarbital . loading doses of phenobarbital ( - mg/kg iv divided into or doses, given every to minutes) may be beneficial in preventing further seizures. severe refractory seizures or decreased mentation may be associated with cerebral edema and increased intracranial pressure. mannitol, an osmotic diuretic, is effective at reducing cerebral edema ( . - . g/kg iv over to minutes). mannitol also acts as a free radical scavenger that can inhibit the effects of cerebral ischemia-reperfusion injury. mannitol works synergistically with furosemide ( mg/kg iv given minutes after the mannitol infusion). corticosteroids have not been demonstrated to be beneficial in the treatment of head trauma and may induce hyperglycemia. hyperglycemia has been shown to be a negative prognostic indicator in cases of head trauma. also, glucocorticoids can suppress immune system function and impair wound healing. because of the known risks and lack of known benefits of glucocorticosteroids, their use in treatment of head trauma is contraindicated. the prognosis for any patient with severe head trauma is guarded. management of head trauma patients may include intense nursing care for a period of weeks to months, depending on the presence and extent of concurrent injuries. if progressive loss of consciousness occurs, surgery for decompression of compressive skull injuries should be considered. the most common injury associated with head trauma in small animals is a contusion with hemorrhage in the midbrain and pons. subdural or extradural hemorrhage with space-occupying blood clots is uncommon. diagnostic tests of head trauma may include skull radiographs, ct, and mri of the brain. special studies can help detect edema and hemorrhage in the brain and brainstem, and aid in making an accurate diagnosis and prognosis. a cerebrospinal fluid tap is contraindicated in patients with head trauma because of the risk of causing a rapid decrease in intracranial pressure and brainstem herniation. if a compressive skull fracture is present, the patient should be stabilized for surgery to remove the compression. surgery to alleviate increased intracranial pressure is rarely performed in veterinary medicine because of the poor prognosis and results. in some cases, when a lesion can be localized to one area, -to -cm burr holes can be placed through the skull over the affected area of the cerebrum, exposing the underlying brain tissue. blood clots can be removed through the holes. the bone flap may or may not be replaced, depending on the surgeon's preference and the degree of brain swelling. spinal cord injuries may be associated with trauma, disk rupture, fractures, and dislocation of the spinal column. proceed with caution when moving a patient with suspected spinal cord injury. avoid flexion, extension, and torsion of the vertebral column. all animals that are unconscious following a traumatic event should be considered to have cervical or thoracolumbar spinal injury until proved otherwise by radiography, ct, or mri. the animal should be moved onto a flat surface (e.g., board, door, window, picture frame) and taped down to prevent motion and further displacement of vertebrae. sedation with analgesics or tranquilizers may be necessary to keep the animal immobile and to minimize patient motion. whenever possible, avoid the use of narcotics in patients with head trauma because of the risk of increasing intracranial pressure. as in other emergencies, the abcs emergency care should be evaluated, and the patient treated for shock, hemorrhage, and respiratory compromise. once the cardiovascular and respiratory systems have been evaluated and stabilized, a more thorough neurologic examination can be performed. protrusion of an intervertebral disk indicates that the disk is bulging into the vertebral canal as a result of dorsal shifting of the nuclear pulposus disk material. disk extrusion refers to the rupture of the outer disk membrane and extrusion of the nuclear material into the vertebral column. in dogs and cats, there are intervertebral disks that potentially can cause a problem. chondrodystrophic breeds of dogs are predisposed to endochondral ossification and include the dachshund, shih tzu, french bulldog, bassett hound, welsh corgis, american spaniel, beagle, lhasa apso, and pekingese. initial examination of the patient with suspected intervertebral disk disease includes identifying the neuroanatomic location of the lesion based on clinical signs and neurologic deficits and then establishing a prognosis. the neurologic examination should be carried out without excessive manipulation of the animal. the presence of pain, edema, hemorrhage, or a visible deformity may localize an area of vertebral injury. once an area of suspected lesion is localized based on physical examination findings, take radiographs to establish a diagnosis and to institute therapy. in most cases, the animal must receive a short-acting anesthestic for proper radiographic technique and to prevent further injury. lateral and crosstable ventrodorsal (vd) or dorsoventral (dv) radiographs require less manipulation of the animal compared with traditional vd and dv projections. myelography is often required to delineate the location of the herniated disk material. prognosis in spinal cord injury depends on the extent of the injury and the reversibility of the damage. perception of noxious stimuli, or the presence of "deep pain," by the animal when the stimulus is applied caudal to the level of the lesion is a good sign. to apply a noxious stimulus, apply firm pressure to a toe on one of the rear limbs using a thick hemostat or a pair of pliers. flexion or withdrawl of the limb is simply a local spinal reflex, and should not be perceived as a positive response to or patient perception of the noxious stimulus. turning of the head, vocalization, dilation of the pupils, change in respiratory rate or character, or attempts to bite are behaviors that are more consistent with perception of the noxious stimulus. absence of perception of the noxious stimulus ("loss of deep pain") is a very poor prognosis for return to function. focal lesions are usually associated with vertebral fractures and displacement of the vertebral canal. focal lesions in one or more of the spinal cord segments from t to t can cause complete dysfunction of the injured tissue as a result of concussion, contusion, or laceration. the degree of structural damage cannot be determined from the neurologic signs alone. transverse focal lesions result in paraplegia, with intact pelvic limb spinal reflexes and analgesia of the limbs and body caudal to the lesion. clinical signs in patients with spinal injury are summarized in table - . carefully evaluate the cardiovascular and respiratory status of patients with spinal injuries. immediately address specific injuries such as pneumothorax, pulmonary contusions, hypovolemic shock, and open wounds. if there is palpable or radiographic evidence of a vertebral lesion causing compressive injury, surgery is the treatment of choice unless the displacement has compromised most or all of the vertebral canal. displacements through % to % of the vertebral canal are associated with a poor prognosis, particularly if deep pain is absent caudal to the lesion. in the absence of a radiographic lesion and in the presence of continued neurologic deficits, an mri or ct scan or myelography is warranted to localize a potentially correctable lesion. surgical exploration can be considered: with the objectives of providing spinal cord decompression by hemilaminectomy or laminectomy with removal of disk material or blood clots, realign and stabilize the vertebral column, and perform a meningotomy, if necessary. place the patient on a backboard or other rigid surface, taped down for transport and sedated, to be transported to a surgical specialist. the presence of worsening or ascending clinical signs may signify ascending-descending myelomalacia and is characteristic of a very poor prognosis.in acute spinal trauma, the use of glucocorticoids has been the mainstay of therapy; however, controversy exists about whether they actually offer any benefit. traditional glucocorticosteroid therapy is listed in box - . more recently, the use of propylene glycol has proved to be beneficial in the treatment of acute traumatic herniated disk. high-dose glucocorticoids should only be used for the first hours after initial injury. side effects of glucocorticosteroid therapy include gastric and intestinal ulceration. the prophylactic use of gastroprotectant drugs will not prevent gastrointestinal ulcer formation; however, if signs of gastrointestinal ulcer are present, institute gastroprotectant therapy. management of the patient with spinal cord injury includes aggressive nursing care and physical therapy. many patients with spinal cord injury have little to no control over bladder function, which results in chronic dribbling or retention of urine and overdistention of the urinary bladder with overflow incontinence. urinary bladder retention can lead to urinary tract infection, bladder atony, and overflow incontinence. manual expression of the bladder several times a day may be enough to keep the bladder empty. alternatively, place a urinary catheter to maintain patient cleanliness and to keep the bladder decompressed. (see section on urinary catheterization). paralytic ileus and fecal retention are frequent complications of spinal cord injury. to help prevent constipation, provide highly digestable foods and maintain the patient's hydration with oral and intravenous fluids. mild enemas or stool softeners can also be used to treat fecal retention. to prevent decubital ulcer formation, turn the patient every to hours, and use clean, dry, soft padded bedding. apply deep muscle massage and passive range of motion exercises to prevent disuse atrophy of the muscles and dependent edema. the radial nerve innervates the extensor muscles of the elbow, carpus, and digits. the radial nerve also supplies sensory innervation to the distal craniolateral surface of the forearm and the dorsal surface of the forepaw. injuries to the radial nerve at the level of the elbow emergency care cranial to c spastic tetraplegia or tetraparesis hyperreflexive all four limbs severe injury can result in death from respiratory failure. c -t tetraparesis or tetraplegia depressed thoracic limb spinal reflexes (lower motor neuron) hyperreflexive pelvic limbs (upper motor neuron) t -t horner' syndrome (prolapsed nictitans, enophthalmos, and miosis) t -l schiff-sherrington syndrome (extensor rigidity of thoracic limbs, flaccid paralysis with atonia, areflexia, and analgesia of pelvic limbs) result in an inability to extend the carpus and digits. as a result, the animal walks and bears weight on the dorsal surface of the paw. there is also loss of cutaneous sensation, which leads to paw injury. injuries to the radial nerve above the elbow (in the shoulder area) results in an inability to extend the elbow and bear weight on the affected limb. it can take weeks before the full extent of the injury and any return to function are manifested. the animal may need to be placed in a carpal flexion sling or have eventual amputation if distal limb injury or self-mutilation occurs. the sciatic nerve primarily innervates the caudal thigh muscles that flex the stifle and extend the hip. the tibial branch of the sciatic nerve innervates the caudal leg muscles that extend the tarsus and flex the digits. the tibial nerve provides the sole cutaneous sensory innervation to the plantar aspect of the paw and digits. the peroneal branch of the sciatic nerve provides the sole sensory cutaneous innervation to the dorsal surface of the paw ( table - ) . sciatic nerve injury may occur with pelvic fractures, particularly those that involve the body of the ileum at the greater ischiatic notch, or with sacroiliac luxations that contuse the l and l spinal nerves that pass ventral to the sacrum to contribute to the sciatic nerve. with sciatic nerve injury, there is decreased stifle flexion and overflexion of the hock (tibial nerve), and the animal walks on the dorsal surface of the paw (peroneal nerve). clinical signs of tibial or peroneal damage are seen with femur fractures or with inadvertent injection of drugs into the caudal thigh muscles. the femoral nerve innervates the extensor muscles of the stifle. the saphenous branch of the femoral nerve provides the sole cutaneous innervation to an area on the medial distal thigh, the leg, and the paw. the femoral nerve is protected by muscles and is rarely injured in pelvic fractures. clinical signs of femoral nerve injury are inability to support weight on the pelvic limb, absence of a patellar reflex, and analgesia in the area of cutaneous innervation. coma is complete loss of consciousness, with no response to noxious stimuli. in some animals that present in a coma or stuporous state, the immediate cause will be apparent. in other cases, however, a careful and thorough diagnostic work-up must be performed. a coma scale devised to assist in the clinical evaluation of the comatose patient is shown in table - . whenever an animal presents in a comatose state, immediately secure the emergency management of specific conditions c -t nerve roots radial nerve paralysis musculocutaneous nerve inability to flex the elbow axillary or thoracodorsal dropped elbow nerve median and ulnar nerves loss of cutaneous sensation on the caudal surface of the forearm and palmar and lateral surfaces of the paw; inability to flex the carpus and digits c -t nerve roots radial, median, or ulnar nerve injury c -c nerve roots musculocutaneous, suprascapular, and axillary injury c -t horner's syndrome (miosis, enophthalmos, and prolapsed nictitans) airway by placing an endotracheal tube (see section on endotracheal intubation). if necessary, provide respiratory assistance, or at a minimum, supplemental oxygen. control existing hemorrhage and treat shock, if present. take a careful and thorough history from the owner. make careful note of any seizure, trauma, or toxin exposure, and whether prior episodes of coma have ever occurred. perform a careful physical examination, taking note of the patient's temperature, pulse, and respiration. an elevated temperature may suggest the presence of systemic infection, such as pneumonia or hepatitis, or a brain lesion with loss of hypothalamic thermoregulatory control. very high temperatures associated with shock and coma are often observed in animals with heat stroke (see section on heat stroke and heat-induced illness). circulatory collapse or barbiturate overdose can produce coma and hypothermia. abnormal respiratory patterns also may be observed in a comatose patient. hypoventilation may occur with elevated intracranial pressure or barbiturate overdose. rapid respiratory rate may be associated with pneumonia, metabolic acidosis (dka, uremia), or brainstem injury. examine the skin for any bruises or external trauma. examine the mucous membranes and make note of color and capillary refill time. icterus with petechiae or ecchymotic hemorrhage in a comatose patient may be associated with end-stage hepatic failure and hepatic encephalopathy. smell the patient's breath for the odor of ketones that may signify dka or end-stage hepatic failure. motor activity normal gait, normal spinal reflexes hemiparesis, tetraparesis, or decerebrate activity recumbent, intermittent extensor rigidity recumbent, constant extensor rigidity recumbent, constant extensor rigidity with opisthotonus recumbent, hypotonia of muscles, depressed or absent spinal reflexes normal papillary reflexes and oculocephalic reflexes slow pupillary light reflexes and normal to reduced oculocephalic reflexes bilateral unresponsive miosis with normal to reduced oculocephalic reflexes pinpoint pupils with reduced to absent oculocephalic reflexes unilateral, unresponsive mydriasis with reduced to absent oculocephalic reflexes bilateral, unresponsive mydriasis with reduced to absent oculocephalic reflexes occasional periods of alertness and responsive to environment depression of delirium, capable of responding to environment but response may be inappropriate semicomatose, responsive to visual stimuli semicomatose, responsive to auditory stimuli semicomatose, responsive only to repeated noxious stimuli comatose, unresponsive to repeated noxious stimuli *neurologic function is assessed for each of the three categories and a grade of to is assigned according to the descriptions for each grade. the total score is the sum of the three category scores. this scale is designed to assist the clinician in evaluating the neurologic status of the craniocerebral trauma patient. as a guideline and according to clinical impressions, a consistent total score of to represents a grave prognosis, to a poor to guarded prognosis, and to a good prognosis. (modified from the glasgow coma scale used in humans.) from shores a: craniocerebral trauma. in kirk rw, ed: current veterinary therapy x. small animal practice. philadelphia, wb saunders, , p . finally, conduct a complete neurologic evaluation. the presence of asymmetric neurologic signs may suggest an intracranial mass lesion (e.g., hemorrhage, neoplasia, injury). usually, toxicities or metabolic disturbances (e.g., dka, hepatic encephalopathy) cause symmetric clinical signs of neurologic dysfunction, with cerebral signs predominating. in hepatic encephalopathy, pupils are usually normal in size and responsive to light. in toxicities, the pupils are abnormal in size and may be unresponsive to light. obtain a complete blood count, serum biochemistry profile, urinalysis, and specific tests for glucosuria and ketonuria. findings of a drastically elevated blood glucose with glucosuria, ketonuria, and high specific gravity are characteristic of dka. fever and uremic encephalopathy are characterized by severe azotemia with a low urine specific gravity. if barbiturate intoxication is suspected, save urine for later toxin analysis. evaluate urine sediment for calcium oxalate crystalluria that may indicate ethylene glycol toxicity. calculate plasma osmolality (see following section) to check for nonketotic hyperosmolar diabetes mellitus. elevated blood ammonia levels may be associated with hepatic encephalopathy. in uncontrolled diabetes mellitus, hyperosmolarity can result in clinical signs of disorientation, prostration, and coma. plasma osmolarity can be calculated from the formula: mosm/l = (na + k) + (glucose/ ) + (bun/ . ) clinical signs of hyperosmolarity can occur when the plasma osmolarity exceeds mosm/l. treatment of dka or nonketotic hyperosmolar syndrome is aimed at reducing ketoacid production, stimulating carbohydrate utilization, and impeding peripheral release of fatty acids. the treatment of choice is rehydration and provision of supplemental regular insulin and a carbohydrate source (see section on diabetic ketoacidosis). during ketosis, insulin resistance may be present. slow rehydration with . % saline solution or other balanced crystalloid fluids (e.g., normosol-r, plasmalyte-m, lactated ringer's solution), should occur, with the goal of rehydration over to hours. too rapid rehydration can result in cerebral edema and exacerbation of clinical signs. hepatic encephalopathy (he) is characterized by an abnormal mental state associated with severe hepatic insufficiency. the most common cause of he is congenital or acquired c o m a portosystemic shunts. acute hepatic destruction can also be caused by toxins, drugs, or infectious causes. the treatment of he is considered a medical emergency (table - ) . absorption of ammonia and other nitrogenous substances from the gastrointestinal tract is thought to be one of the complicating factors in he. prevent absorption of ammonia and other nitrogenous substances from the gastrointestinal tract by restricting dietary protein to % to % for dogs, and to % to % (on a dry matter basis) for cats. dietary protein should be from a nonanimal plant source (e.g., soybean) whenever possible. caloric requirements are met with lipids and carbohydrates. also prescribe cleansing enemas to rid the colon of residual material, and antibiotic therapy to reduce gastrointestinal tract bacteria. neomycin ( mg/kg q h) can be administered as a retention enema. metronidazole ( . mg/kg po, q - h) or amoxicillin-clavulanate ( . mg po q h) can also be administered. administer lactulose ( . - . ml q h for cats; . - ml q h for dogs) to trap ammonia in the colon to prevent absorption (table - ) . administer lactulose orally to an alert animal, or as a retention enema to a comatose animal. if lactulose is not available, betadine retention enemas will change colonic ph and prevent ammonia absorption. a side effect of lactulose administration (po) is soft to diarrheic stool. a seizure is a transient disturbance of brain function that is sudden in onset, ceases spontaneously, and has a tendency to recur, depending on the cause. most seizures are generalized and result in a loss of consciousness and severe involuntary contraction of the skeletal muscles, resulting in tonic-clonic limb activity and opisthotonus. mastication, salivation, urination, and defecation are common. partial (petit mal) seizures range from limited limb activity, facial muscle twitching, and episodic behavioral abnormalities to brief loss of consciousness. similar clinical signs also can occur with syncopal episodes. conduct a careful cardiac examination in any patient with a history of petit mal seizures. seizures of any form constitute a medical emergency, particularly when they occur in clusters, or as status epilepticus. most seizures are of short duration and may have subsided by the time the animal is presented for treatment. whenever a seizure occurs, however, it is important that the animal does not inadvertently injure itself or a bystander. it is important to evaluate whether the patient has a coexisting disease that can predispose it to seizures, such as hepatic failure, uremia, diabetes mellitus, hypoglycemia, toxin exposure, insulin-secreting tumors, and thiamine deficiency. many toxins are responsible for clinical signs of tremors or seizures (see section on poisons and toxins). treatment of a primary disease entity can help control seizures, in some cases, provided that the underlying cause is investigated and treated. status epilepticus, a state of continuous uncontrolled seizure activity, is a medical emergency. when an animal is in a state of status epilepticus, immediately place a lateral or medial saphenous intravenous catheter and administer diazepam ( . mg/kg iv) to help control the seizure. in most cases, the seizure must be controlled before a diagnostic workup is attempted. whenever possible, however, blood samples should be collected before administration of any anticonvulsant agent because of the risk of incorrect test results. for example, the propylene glycol carrier in diazepam can cause a false-positive ethylene glycol test using an in-house testing kit. whenever possible, check blood glucose levels, particularly in young puppies or kittens, to evaluate and treat hypoglycemia as a cause of seizures. if hypoglycemia exists, administer % dextrose ( g/kg iv). if diazepam partially controls the status epilepticus, administer a constant rate infusion ( . mg/kg/hour in % dextrose in water). diazepam is sensitive to light, and the bag and infusion line must be covered to prevent degradation of the drug. if diazepam fails to control status epilepticus, give pentobarbital ( - mg/kg iv to effect). the animal's airway should be intubated and protected while the patient is kept in the drug-induced coma. protracted cases of seizures may require mannitol and furosemide therapy to treat cerebral edema. administer intravenous fluids (balanced crystalloid at maintenance doses [see section on intravenous fluid therapy]). the patient should be turned every to hours to emergency care prevent atelectasis. insert a urinary catheter for cleanliness, and place the animal on soft dry padded bedding to prevent decubital ulcer formation. depending on the length of time that the patient is rendered unconscious, apply passive range of motion exercises and deep muscle massage to prevent disuse atrophy of the muscles and dependent or disuse edema. monitor the patient's oxygenation and ventilation status by arterial blood gas measurement or pulse oximetry and capnometry (see section on blood gas, pulse oximetry, and capnometry). administer supplemental oxygen to any patient that is hypoxemic secondary to hypoventilation or other causes. severe refractory seizures can result in the development of neurogenic pulmonary edema. lubricate the animal's eyes every hours to prevent drying out and corneal abrasions. depending on the cause of the seizure, administer phenobarbital at a loading dose of to mg/kg iv given in four to five injections, every to minutes; make sure that the patient is rousable in between injections). seizures in cats often are associated with structural brain disease. the occurrence of partial focal seizures is unequivocally associated with a focal cerebral lesion and acquired structural brain disease. an initial high frequency of seizures is also a strong indication that structural brain disease is present. seizure activity in cats may occur as mild generalized seizures or complex partial seizures and may be associated with systemic disorders such as feline infectious peritonitis virus, toxoplasmosis, cryptococcus infection, lymphosarcoma, meningiomas, ischemic encephalopathy, and thiamine deficiency. thiamine deficiency in the cat can be a medical emergency characterized by dilated pupils, ataxic gait, cerebellar tremor, abnormal oculocephalic reflex, and seizures. treatment consists of administration of thiamine ( mg/day) for three days. steffen f, grasmueck s: propofol for treatment of refractory seizures in dogs and a cat with intracranial disorders. j small anim pract ( ) ( ) ( ) ( ) . j am vet med assoc ( ): [ ] [ ] [ ] [ ] [ ] [ ] . an ocular emergency is any serious condition that causes or threatens to cause severe pain, deformity, or loss of vision. treat ocular emergencies immediately, within to several hours after the emergency, whenever possible (box - , - ). to assess the location and degree of ocular injury, perform a complete ocular examination. in some cases, short-acting sedation or general anesthesia in conjunction with topical local anesthetic may be necessary to perform the examination, because of patient discomfort and blepharospasm. the equipment listed in box - may be necessary and may be invaluable in making an accurate diagnosis. to perform a systematic and thorough ocular examination, first obtain a history from the owner. has there been any prior incident of ocular disease? is there any history of trauma or known chemical irritant or exposure? did the owner attempt any irrigation or medical techniques prior to presentation? when was the problem first noticed? has it changed at all since the owner noticed the problem? after a history has been obtained, examine the patient's eyes for discharge, blepharospasm, or photophobia. if any discharge is present, note its color and consistency. do not attempt to force the eyelids open if the patient is in extreme discomfort. administer a short-acting sedative and topical local anesthetic such as . % proparacaine. note the position of the globe within its orbit. if the eye is exophthalmic, strabismus and protrusion of the third eyelid are often visible. exposure keratitis may be present. in cases of retrobulbar or zygomatic salivary gland inflammation, the patient will resist opening the mouth and exhibit signs of discomfort or pain. note any swelling, contusions, abrasions, or lacerations of the eyelids. note whether the lids are able to close completely and cover the cornea. if a laceration of the lid is present, determine the depth of the laceration. palpate the orbit for fractures, swelling, pain, crepitus, and cellulitis. examine the cornea and sclera for penetrating injury or foreign material. the use of lid retractors or small forceps can be very helpful in these cases. if a wound appears to penetrate completely into the globe, look for loss of uveal tissue, lens, or vitreous. do not put any pressure on the globe, because intraocular herniation may result. examine the conjunctiva for hemorrhage, chemosis, lacerations, and foreign bodies. examine the superior and inferior conjunctival cul-de-sacs for foreign material. in such cases, placement of a topical anesthetic and use of a moistened cotton swab is invaluable to sweep the conjunctival fornix to pick up foreign bodies. use a small, fine-tipped forceps to retract the third eyelid away from the globe and examine behind the third eyelid for foreign bodies. next, examine the cornea for opacities, ulcers, foreign bodies, abrasions, or lacerations. place a small amount of fluroescein stain mixed with sterile water or saline on the dorsal sclera. close the eye to disperse the stain over the surface of the cornea, then flush gently with sterile saline irrigation. examine the cornea again for any defects. a linear defect perpendicular to the long axis of the eye should alert the clinician to investigate the conjunctiva for dystechia. record the pupil size, shape, and response to light (both direct and consensual). examine the anterior chamber and note its depth and whether hyphema or aqueous flare are present. is the lens clear and is it in the normal position? lens luxation can cause the lens tissue to touch the cornea and cause acute corneal edema. measure intraocular pressure with a schiotz tonometer or tonopen. finally, dilate the pupil and examine the posterior chamber using a direct or indirect ophthalmoscope to look for intraocular hemorrhage, retinal hemorrhage, retinal detachment, tortuous retinal vessels, optic neuritis, and inflammation. the basic surgical instruments listed in box - may be useful in the treatment of ocular lacerations and other ophthalmic injuries: bite wounds and automobile trauma commonly cause lacerations and abrasions of the lid margins. the lids can be considered to be two-layer structures, with the anterior composed of the skin and orbicularis muscle and the posterior layer composed of the tarsus and conjunctiva. the openings of the meibomian glands in the lid margin form the approximate line separating the lids into anterior and posterior segments. splitting the lid into these two segments facilitates the use of sliding skin flaps to close wound defects, if necessary. clean and thoroughly but gently irrigate the wound with sterile saline solution before attempting any lid laceration repair. use sterile saline solution to irrigate the wound and conjunctiva. a % povidone-iodine scrub can be used on the skin, taking care to avoid getting any scrub material in the soft tissues of the eye. drape the eye with an adhesive ocular drape, if possible, to prevent further wound contamination. trim the ragged wound edges, but be very conservative with tissue debridement. leave as much tissue as possible to insure proper wound contracture with minimal lid deformity. close a small lid wound with a figure-of-eight or two-layered simple interrupted suture of absorbable suture material or nylon in the skin. the lid margins must be absolutely apposed to prevent postoperative lid notching. direct blunt trauma to the eye can cause severe ecchymosis because of the excellent vascular supply of the eyelids. other associated ocular injuries such as orbital hemorrhage, proptosis, and corneal laceration may also occur. trauma, allergic reactions, inflammation of the sebaceous glands (hordeolum), thrombocytopenia, and vitamin k antagonist rodenticide intoxication can all cause ecchymoses of the lids. treat eyelid ecchymoses initially with cool compresses, followed by warm compresses. resorption of blood can occur from to days after the initial insult. ocular allergies respond well to topical application (dexamethasone ophthalmic ointment q - h) and systemic administration of glucocorticosteroids, along with cool compresses. in order to fully assess the conjunctiva for abnormalities, it may be necessary to carefully dissect it away from the underlying sclera. when performing this dissection, do not place undue pressure on the globe because of the risk of herniation of the intraocular contents through a scleral wound. repair large conjunctival lacerations with - absorbable sutures, using an interrupted or continuous pattern. carefully approximate the margins of the conjunctiva to prevent formation of inclusion cysts. when large areas of the conjunctiva have been damaged, advancement flaps may be required to close the defect. subconjunctival hemorrhage is a common sequela of head trauma, and it may also be observed in various coagulopathies. by itself, it is not a serious problem but may signify severe underlying intraocular damage. a complete ocular examination is indicated. other causes of subconjunctival hemorrhage include thrombocytopenia, autoimmune hemolytic anemia, hemophilia, leptospirosis, vitamin k antagonist rodenticide intoxication, severe systemic infection or inflammation, and prolonged labor (dystocia). uncomplicated subconjunctival hemorrhage usually clears on its own within days. if the conjunctiva is exposed because of swelling and hemorrhage, administer a topical protective triple antibiotic ophthalmic ointment every to hours until the conjunctival hemorrhage resolves. toxic, acid, and alkaline chemical injuries to the eye can sometimes occur. the severity of the injury caused by ocular burns depends on the concentration, type, and ph of the chemical and on the duration of exposure. weak acids do not penetrate biologic tissue very well. the hydrogen ion precipitates the protein upon contact and therefore provides some protection to the corneal stroma and intraocular contents. precipitation of corneal proteins produces a ground-glass appearance in the cornea. alkaline solutions and very strong acids penetrate tissues rapidly, causing saponification of the plasma membrane, denaturation of collagen, and vascular thrombosis within the conjunctiva, episclera, and anterior uvea. severe pain, blepharospasm, and photophobia are produced by exposure of free nerve endings in the corneal epithelium and conjunctiva. severe alkaline burns cause an increase in intraocular pressure. intraocular prostaglandins are released, and the intraocular aqueous ph increases, producing changes in the blood-aqueous barrier and secondary uveitis. uveitis with anterior synechia formation, eventual chronic glaucoma, phthisis, secondary cataract, and corneal perforation can occur. healing of the corneal epithelium is usually accomplished by neovascularization and sliding and increased mitosis of the corneal epithelium. severe stromal burns within the cornea heal by degradation and removal of necrotic debris, followed by replacement of the collagen matrix and corneal epithelial cells. the release of collagenase, endopeptidase, and cathepsins from polymorphonuclear cells serves to cause further corneal breakdown. in severe cases, only pmns may be present, and fibroblasts may never invade the corneal stroma. all chemical burns should be washed copiously with any clean aqueous solution available. if any sticky paste or powder is adherent to the conjunctival sac, remove it with moist cotton swabs and irrigation. begin mydriasis and cycloplegia by topical application of % atropine ophthalmic drops or ointment. start antibiotic therapy with triple antibiotic ophthalmic ointment or gentocin ointment every to hours. treat secondary glaucomas with topical carbonic anhydrase inhibitors. to avoid fibrinous adhesions and symblepharon formation, keep the conjunctival cul-de-sacs free of proteinaceous exudate that can form adhesions. analgesics are required for pain. oral nonsteroidal antiinflammatory agents such as carprofen, ketoprofen, meloxicam, or aspirin are recommended. persistent epithelial erosions may require a conjunctival flap left in place for to weeks or placement of a topical collagen shield (contact lens). topical antibiotics, mydriatics, and lubricants (lacrilube or puralube ointment) should also be used. strong acid or alkali burns can result in severe corneal stromal loss. in the past, topical n-acetylcysteine ( % mucomyst) has been recommended. this treatment is very painful. other treatments are also available, such as ethylenediaminetetraacetic acid (edta) ( . m solution) and patient serum to inhibit mammalian collagenase activity. to prepare patient serum, obtain to ml of whole blood from the patient. spin it down in a serum separator tube after a clot forms and then place the serum in a red-topped tube on the patient's cage. (the contents of the tube are viable for days without refrigeration.) apply the serum topically to the affected eye every to hours. avoid using topical steroids because they inhibit fibroblast formation and corneal healing. in severe cases, if conjunctival swelling and chemosis also are present, antiinflammatory doses of oral steroids can be administered short-term. oral steroids and nonsteroidal antiinflammatory drugs should never be administered to the patient concurrently, because of the risk of gastrointestinal ulcer and perforation. corneal abrasions are associated with severe pain, blepharospasm, lacrimation, and photophobia. animals with such intense pain are often difficult to examine until analgesia has been administered. topical use of proparacaine ( . % proparacaine hydrochloride) is usually sufficient to permit relaxation of the eyelids so that the eye can be examined. using a focal source of illumination and an eye loupe, examine the cornea, inferior and superior conjunctival fornixes, and medial aspect of the nictitans for foreign bodies. place a sterile drop of saline on a fluorescein-impregnated strip and touch the superior conjunctiva once to allow the stain to spread onto the surface of the eye. irrigate the eye to remove excess stain and then examine the corneal surface for any areas of stain uptake. if an area of the cornea persistently remains green, there is damage to the corneal epithelium in that area. initial treatment consists of application of a topical mydriatic ( drop of % atropine in affected eye q h) to prevent anterior synechiae and improve cycloplegia. triple antibiotic ointment is the treatment of choice (a / -inch strip in the affected eye q h) until the ulcer heals. in some cases, nonhealing ulcers (e.g., boxer ulcer, indolent ulcer) form in which the epithelial growth does not adhere to the underlying cornea. gently debride the loose edges of the ulcer/erosion with a cotton swab and topical anesthesia. more severe cases in which only minimal healing has occurred after days of treatment require grid keratectomy, in which a -gauge needle is used to gently scratch the surface of the abrasion or ulcer in the form of a grid to promote neovascularization. apply a topical anesthetic before performing the procedure. a collagen contact lens also may be required to promote wound healing. all corneal abrasions should be reevaluated in hours, and then every to days thereafter until they have healed. acute infectious keratitis secondary to bacterial infection is characterized by mucopurulent ocular discharge, rapidly progressing epithelial and corneal stromal loss, inflammatory cellular infiltrates into the corneal stroma, and secondary uveitis, often with hypopyon formation. confirmation of infectious keratitis is based on corneal scrapings and a positive gram stain. initial treatment for bacterial keratitis consists of systemic antibiotics and topical ciprofloxacin ( . % eyedrops or ointment). penetrating injuries through the cornea may result in prolapse of intraocular contents. frequently, pieces of uveal tissue or fibrin effectively but temporarily seal the defect and permit the anterior chamber to re-form. avoid manipulation of these wounds until the animal has been anesthetized, as struggling or excitement can promote loss or dislodgement of the temporary seal and cause the intraocular contents to be extruded. superficial corneal lacerations need not be sutured and can be treated the same as a superficial corneal ulcer or abrasion. if the laceration penetrates more than % the thickness of the cornea, or extends more than to mm, it should be sutured. when placing sutures in the cornea, it is helpful to use magnification. referral to a veterinary ophthalmologist is advised. if a veterinary ophthalmologist is not available, use - or - silk, collagen, or nylon sutures on a micropoint spatula-type needle. use a simple interrupted suture pattern and leave the sutures in place for a minimum of weeks. because many corneal lacerations are jagged and corneal edema forms, most of the wound edges cannot be tightly juxtaposed. in such cases, pull a conjunctival flap across the wound to prevent leakage of aqueous fluid. never suture through the full thickness of the cornea; rather, the suture should pass through the mid-third of the cornea. following closure of the corneal wound, the anterior chamber must be re-formed to prevent anterior synechia formation with secondary glaucoma. taking care to avoid iris injury, use a -or -gauge needle to insert sterile saline at the limbus. any defect in the suture line will be apparent because of leakage of the fluid from the site and should be repaired. incarceration of uveal tissue in corneal wounds is a difficult surgical problem. persistent incarceration of uveal tissue can result in development of a chronic wick in the cornea, a shallow anterior chamber, chronic irritation, edema, vascularization of the cornea, and intraocular infection that can lead to panophthalmitis. referral to a veterinary ophthalmologist is strongly recommended. the most common foreign bodies associated with ocular injuries in small animals are birdshot, bb pellets, and glass. the site of intraocular penetration of the foreign bodies may be obscured by the eyelids. a foreign body entering the eye may penetrate the cornea and fall into the anterior chamber or become lodged in the iris. foreign bodies may occasionally penetrate the lens capsule, producing cataracts. some metallic high-speed foreign bodies may penetrate the cornea, iris, and lens to lodge in the posterior wall of the eye or vitreous chamber. direct visualization of a foreign body is the best means of localization. examination of the eye with an indirect ophthalmoscope or biomicroscope (if available) is invaluable for locating foreign bodies. indirect visualization of the ocular foreign body can also be achieved through radiographic techniques. three separate views should be obtained to determine the plane of location of the foreign object. ct or mri may prove useful, although scatter from the foreign body may make it difficult to directly visualize with these techniques. ocular ultrasound is perhaps the most useful and refined radiographic technique for locating intraocular foreign bodies. before removing any foreign body from the eye, the risk and surgical danger of removing it must be weighed against the risks of leaving it in place. metallic foreign bodies in the anterior chamber are much easier to remove than nonmagnetic ones. attempted removal of foreign objects from the vitreous chamber of the eye has consistently produced poor results. for the best chance of recovery, ocular foreign bodies should be removed by a veterinary ophthalmologist whenever possible. blunt trauma to the globe can result in luxation or subluxation of the lens. the subluxated lens may move anteriorly and make the anterior chamber more shallow. trembling of the iris (iridodonesis) may be noticed when the lens is subluxated. in complete luxation, the lens may fall totally into the anterior chamber and obstruct aqueous outflow, causing secondary glaucoma. alternatively, the lens may be lost into the vitreous cavity. luxation of the lens is almost always associated with rupture of the hyaloid membrane and herniation of the vitreous through the pupillary space. emergency surgery for lens luxation is required if the lens is entirely within the anterior chamber or incarcerated within the pupil, causing a secondary pupillary block glaucoma. acute elevation in intraocular pressure can cause vision loss within hours; thus, lens removal should be accomplished as quickly as possible. referral to a veterinary ophthalmologist is recommended. severe trauma to the globe or a direct blow to the head can result in retinal or vitreous hemorrhage. there may be large areas of subretinal or intraretinal hemorrhage. subretinal hemorrhage assumes a discrete globular form, and the blood appears reddish-blue in color. the retina is detached at the site of hemorrhage. superficial retinal hemorrhage may assume a flame-shaped appearance, and preretinal or vitreous hemorrhage assumes a bright-red amorphous appearance, obliterating the underlying retinal architecture. retinal and vitreous hemorrhage secondary to trauma usually resorbs spontaneously over a -to -week period. unfortunately, vitreous hemorrhage, as it organizes, can produce vitreous traction bands that eventually produce retinal detachment. expulsive choroid hemorrhage can occur at the time of injury and usually leads to retinal detachment, severe visual impairment, and total loss of vision. treatment of vitreal and retinal hemorrhage includes rest and correction of factors that may predispose to intraocular hemorrhage. more complicated cases may require vitrectomy performed by a veterinary ophthalmologist. hyphema refers to blood in the anterior chamber of the eye. the most common traumatic cause of hyphema is an automobile accident. hyphema may also present because of penetrating ocular wounds and coagulopathies. blood within the eye may come from the anterior or posterior uveal tract. trauma to the eye may result in iridodialysis or a tearing of the iris at its root, permitting excessive bleeding from the iris and ciliary body. usually, simple hyphema resolves spontaneously in to days and does not cause vision loss. loss of vision following bleeding into the anterior chamber is associated with secondary ocular injuries such as glaucoma, traumatic iritis, cataract, retinal detachment, endophthalmitis, and corneal scarring. treatment of hyphema must be individualized, but there are severe general principles of treatment. first, stop ongoing hemorrhage and prevent further bleeding whenever possible. this may involve correction of the underlying cause, if a coagulopathy is present. next, aid in the elimination of blood from the anterior chamber, control secondary glaucoma, and treat associated injuries, including traumatic iritis. finally, detect and treat any late complications of glaucoma. in most cases of traumatic hyphema, little can be done to arrest or prevent ongoing hemorrhage. it is best to restrict the animal's activity and prohibit exertion. rebleeding can occur within days, and intraocular pressure must be monitored closely. after to days, the blood in the anterior chamber will change color from a bright red to bluish-black ("eight-ball hemorrhage"). if total hyphema persists and intraocular pressure rises despite therapy, surgical intervention by a veterinary ophthalmologist may be necessary. the primary route of escape of rbcs from the anterior chamber is via the anterior drainage angle. iris absorption and phagocytosis play a minor role in the removal of blood from the anterior chamber. because of the associated traumatic iritis in hyphema, topical administration of a glucocorticoid ( % dexamethasone drops or % prednisolone drops) is advised to control anterior chamber inflammation. a cycloplegic agent ( % atropine) should also be used. the formation of fibrin in the anterior chamber of the eye secondary to hemorrhage can produce adhesions of the iris and secondary glaucoma (see section on glaucoma secondary to hyphema) by blocking the trabecular network. hyphema secondary to retinal detachment (collie ectasia syndrome) and end-stage glaucoma are extremely difficult to treat medically and have a poor prognosis. proptosis of the globe is common secondary to trauma, particularly in brachycephalic breeds. proptosis of the globe in dolichocephalic breeds requires a greater degree of initiating contusion than the brachycephalic breeds because the orbits are so much deeper. therefore, secondary damage to the eye and cns associated with proptosis of the globe may be greater in the collie or greyhound than in the pug. when proptosis occurs, carefully evaluate the cardiovascular system for evidence of hypovolemic or hemorrhagic shock. examine the respiratory and neurologic systems. be sure to establish an airway and treat shock, if present. control hemorrhage and stabilize the cardiovascular system before attempting to replace the globe within its orbit or perform enucleation. during the initial management of the cardiovascular and respiratory systems, the eye should be covered with an ophthalmic grade ointment or sponges soaked in sterile saline to prevent the globe from drying out. proptosis of the globe can be associated with serious intraocular problems including iritis, chorioretinitis, retinal detachment, lens luxation, and avulsion of the optic nerve. stain the surface of the eye with fluorescein to look for topical abrasions or ulcers. carefully examine the sclera, cornea, and conjunctiva for penetrating injuries that may allow aqueous leakage. evaluate the size, location, and response to light of the pupil. a reactive pupil is better than a mydriatic fixed pupil. topical administration of a mydriatic (atropine %) to prevent persistent miosis and synechia formation is indicated, along with topical and oral antibiotics and oral analgesic therapy. reposition the proptosed globe with the patient under general anesthesia. make a lateral canthotomy incision to widen the palpebral fissure. lavage the globe with sterile saline irrigation to remove any external debris. place a copious amount of triple antibiotic ophthalmic ointment on the surface of the eye and then gently press the globe into the orbit using the flat side of a scalpel handle or a moistened sterile surgical sponge. do not probe the retro-orbital space with a needle or attempt to reduce intraocular pressure by paracentesis. when the globe is replaced in the orbit, close the lateral canthotomy incision with simple interrupted sutures. place three non-penetrating mattress sutures in the lid margins but do not draw them together. tighten the lid sutures through small pieces of a red rubber catheter or length of intravenous extension tubing to prevent the sutures from causing lid necrosis. leave the medial canthus of the eye open in order to allow topical treatment. postoperative treatment is directed at preventing further iritis and preventing infection. administer systemic broad-spectrum antibiotics (clavamox, . mg/kg po bid) and analgesic drugs. apply topical triple antibiotic ophthalmic ointment ( / inch in affected eye q - h) and atropine ( % in affected eye q h) to prevent infection, cycloplegia, and anterior synechiae. antiinflammatory doses of systemic steroids can also be added to the treatment if severe periorbital inflammation is present. systemic steroids should never be used in conjunction with nonsteroidal antiinflammatory drugs, because of the risk of gastrointestinal ulceration and perforation. the sutures should remain in place for a minimum of weeks. after this time, remove the sutures and inspect the globe. if proptosis recurs, repeat the treatment. following proptosis, strabismus is common secondary to periorbital muscle injury. even after extensive treatment, vision in the eye may still be lost. nonvisual eyes can remain in place, but phthisis may develop. carbonic anhydrase inhibitors such as acetazolamide and dichlorphenamide decrease aqueous secretion and may effectively reduce intraocular pressure if the trabecular outflow is still functioning at % of its capacity. an eye with a poorly functional trabecular outflow system will respond poorly to therapy with carbonic anhydrase inhibitors. osmotic agents such as mannitol or glycerol may be helpful in controlling glaucoma secondary to hyphema. reduction in vitreous chamber size can make the anterior chamber deeper and may allow increased aqueous outflow. evacuation of blood or blood clots from the anterior chamber is not advisable unless the glaucoma cannot be controlled medically or there is no indication after a prolonged period of time that blood is being resorbed. tissue plasminogen activator (t-pa) has proved to be useful in may be helpful in lysing blood clots and preventing excessive fibrin formation. the t-pa is reconstituted to make a solution of µ/ml, which is then frozen at − °c in . -ml aliquots. the thawed, warmed reconstituted t-pa is injected into the anterior chamber. blind probing of the anterior chamber of the eye and surgical intervention in an attempt to remove blood clots can cause serious complications such as rebleeding, lens luxation, iris damage, and damage to the corneal epithelium, and therefore is not advised. acute glaucoma is a rise in intraocular pressure that is not compatible with normal vision. glaucoma may present as early acute congestive or noncongestive glaucoma, or as end-stage disease. cardinal signs of glaucoma are a sudden onset of pain, photophobia, lacrimation, deep episcleral vascular engorgement, edematous insensitive cornea, shallow anterior chamber depth, dilated unresponsive pupil, loss of visual acuity, and buphthalmia. intraocular pressure usually exceeds mm hg but may be normal or only slightly increased if glaucoma is secondary to anterior uveitis. most forms of clinical glaucoma in dogs are secondary to some other intraocular problem. primary glaucoma is recognized in some breeds, including the bassett hound, cocker spaniel, samoyed, bouvier des flandres, and some terrier breeds either from goniodysgenesis or a predisposition to lens luxation. other common causes of acute glaucoma are anterior uveitis and intumescent lens secondary to rapid cataract development, particularly in dogs with diabetes mellitus. treatment involves investigation of the underlying cause of the sudden rise in intraocular pressure and rapid reduction in intraocular pressure. permanent visual impairment is often associated with chronically buphthalmic globes or the presence of rippling or striae formation on the cornea. referral to a veterinary ophthalmologist is recommended. if the eye is still visual and not buphthalmic, the prognosis is favorable, depending on the cause of the acute glaucoma. treatment to reduce intraocular pressure consists of improving aqueous outflow, reducing intraocular volume with osmotic agents, and reducing aqueous formation (table - ). the use of topical mydriatic agents in acute glaucoma is contraindicated because of the risk of making lens luxation or anterior uveitis worse. referral to a veterinary ophthalmologist for emergency surgery is indicated in cases of iris bombe, intumescent lens, or lens subluxation. administer osmotic agents to reduce the size of the vitreous body and the amount of aqueous. osmotic agents create an osmotic gradient between the intraocular fluids and the emergency management of specific conditions vascular bed, thus allowing osmotic removal of fluid independent of the aqueous inflow and outflow systems. if no other treatments are available, oral glycerol ( %, . ml/kg or . g/kg) can be used to effectively reduce intraocular pressure. an adverse side effect of oral glycerol treatment is protracted vomiting. do not use glycerol in a diabetic patient. mannitol ( - g/kg iv over hour) also effectively reduces intraocular pressure but does not cause vomiting. carbonic anhydrase inhibitors can be used to reduce intraocular volume by reducing aqueous production. oral administration of dichlorphenamide, methazolamide, and acetazolamide ( - mg/kg) is usually not very effective alone in reducing aqueous volume and intraocular pressure and also can cause metabolic acidosis. topical carbonic anhydrase inhibitors appear to be more effective (dorzolamide, trusopt) when used in conjunction with topical beta-blockers (timolol, . % or . % solution q h). the most effective treatment for acute pressure reduction is use of a topical prostaglandin inhibitor (latanaprost). usually just one or two drops effectively reduces intraocular pressure in the emergency stages, until the patient can be referred to a veterinary ophthalmologist the following day. many clinical conditions that are presented as emergencies may be due in part or wholly to the presence of a neoplasm. paraneoplastic signs are summarized in table - . prompt identification of the neoplasia combined with knowledge of treatment, expected response to therapy, and long-term prognosis can aid owners and practitioners in making appropriate treatment decisions. hemorrhage or effusion can occur in any body cavity as a result of the presence of benign or malignant tumors. tumors secrete anticoagulants to allow angiogenesis to grow unchecked. hemorrhage often occurs as a result of rupture of a neoplasm or invasion of a neoplasm into a major vascular structure. effusion may be the result of direct fluid production by the mass or may be due to obstruction of lymphatic or venous flow. hemorrhagic effusions in the abdominal cavity occur most commonly with neoplastic masses of the spleen or liver. the most common causes are hemangiosarcoma and hepatocellular carcinoma. clinical signs associated with acute abdominal hemorrhage, regardless of the cause, are related to hypovolemic shock and decreased perfusion and include pale mucous membranes, tachycardia, anemia, lethargy, and acute collapse. treatment for abdominal hemorrhage includes placement of a large-bore peripheral cephalic catheter and starting one fourth of a shock dose ( ml/kg/hour for dogs, and ml/kg/hour for cats) of intravenous crystalloid fluids, taking care to carefully monitor perfusion parameters of heart rate, capillary refill time, mucous membrane color, and blood pressure. administer intravenous colloids such as dextran- , hetastarch, and oxyglobin ( - ml/kg iv bolus) to restore intravascular volume and normotension. treat severe anemia with whole blood or packed rbcs to improve oxygen-carrying capacity and oxygen delivery (see sections on transfusion medicine and treatment of shock). confirm the presence of hemoabdomen abdominocentesis (see section on abdominocentesis). the presence of nonclotting hemorrhagic effusion is consistent with free blood. packed cell volume of the fluid is usually the same or higher than that of the peripheral blood. an abdominal compression bandage can be placed while further diagnostics are being performed. in cases of acute hemoabdomen, obtain right lateral, left lateral, and ventrodorsal or dorsoventral thoracic radiographs to help rule out obvious metastasis. monitor the patient's ecg and correct dysrhythmias as necessary (see section on cardiac dysrhythmias). surgery is indicated once the patient is stabilized. in some cases, hemorrhage is so severe that the patient should be taken immediately to surgery. when recommending surgery for a hemorrhaging intraabdominal mass, it is important to discuss likely diagnoses and long-term prognosis with the owner. hemangiosarcoma usually involves the spleen or liver or both. the presence of free abdominal hemorrhage is associated with a malignant tumor in % of cases. even when free abdominal hemorrhage is not present, the tumor is malignant in % of cases. approximately % (two thirds) of masses in the spleen are malignant (hemangiosarcoma, lymphoma, mast cell tumor, malignant fibrous histiocytoma, leiomyosarcoma, fibrosarcoma), and approximately one third are benign (hematoma, hemangioma). hepatocellular carcinoma usually affects one liver lobe (usually the left), and surgery is the treatment of choice. with complete surgical excision, median survival in dogs is longer than days. if diffuse disease is observed at the time of surgery, the prognosis is poor. nonhemorrhagic effusions are associated with mesothelioma, lymphoma, carcinomatosis, or any mass that causes vascular or lymphatic obstruction. clinical signs of respiratory distress and abdominal distention with nonhemorrhagic effusions are usually slowly progressive in onset and not as severe as those observed with hemorrhage. treatment is usually aimed at identification of the underlying cause. obtain a fluid sample via thoracocentesis or abdominocentesis. to obtain further cells for cytologic evaluation, aspirate fluid from the thoracic or abdominal mass with ultrasound guidance. cytologic evaluation of the fluid will often elucidate the causative tumor type. an abdominal ultrasound can determine the degree of metastasis. perform therapeutic abdominocentesis or thoracocentesis if the effusion is causing respiratory difficulty. rapid re-accumulation of the fluid potentially can cause hypoproteinemia and hypovolemic shock. mesothelioma is a rare tumor most commonly observed in urban environments. in humans, mesothelioma has been associated with exposure to asbestos. it is sometimes difficult to differentiate between reactive mesothelial cells and malignant mesothelial cells. treatment is aimed at controlling the neoplastic effusion. intracavitary cisplatin has been demonstrated to slow rates of fluid re-accumulation, but is largely a palliative therapy. lymphoma is another tumor type that can cause thoracic or abdominal effusion. cytologic evaluation of the fluid usually reveals abundant lymphoblasts. treatment with multiagent chemotherapy protocols, with or without adjunctive radiation therapy, can prevent tumor remission and stop fluid accumulation. carcinomatosis occurs as a result of diffuse seeding of the abdominal cavity with malignant carcinomas and has a poor prognosis. carcinomatosis may occur de novo or from metastasis of a primary tumor. treatment consists of fluid removal when respiratory difficulty occurs, with or without intracavitary cisplatin as a palliative measure. cisplatin should never be used in cats due to fatal acute pulmonary edema. clinical signs of hemorrhagic thoracic effusion include acute respiratory distress, anemia, hypovolemic or cardiogenic shock, and collapse. hemorrhagic thoracic effusions are rare in association with neoplastic effusions. a notable exception is intrathoracic hemorrhage in young dogs with osteosarcoma of the rib. hemorrhage can result when a primary lung tumor erodes through a vessel. hemangiosarcoma of the lungs or right auricular area can also result in hemorrhagic thoracic effusion. in many cases, hemorrhage may be confined to the pericardial sac with a right auricular mass, causing a globoid cardiac silhouette on thoracic radiographs. treatment consists of pericardiocentesis (see section on pericardial effusion and pericardiocentesis) and placement of a pericardial window, or the mass may be removed if it is in the right auricular appendage and resectable. although surgery can resolve clinical signs of right-sided heart failure, metastatic disease often develops soon afterward. nonhemorrhagic thoracic effusion is more common than hemorrhagic thoracic effusion, and is caused most commonly by mesothelioma, lymphoma, carcinomatosis, and thymoma. clinical signs develop gradually and include respiratory difficulty, cyanosis, and cough. supplemental oxygen should be administered. in many cases, thoracocentesis can be therapeutic and diagnostic. obtain thoracic radiographs both before and after thoracocentesis to determine whether a mass effect is present. following identification of a cause, definitive therapy can be instituted. mesotheliomas are rare and are associated with diffuse serosal disease. they are more common in dogs than in cats. effusions caused by mesotheliomas can affect the pleural or pericardial cavities. treatment is directed at removing effusion fluid and controlling reaccumulation with use of intracavitary platinum compounds, carboplatin, and cisplatin can be used in dogs. (cisplatin and carboplatin should never be used in cats.) chemical or physical pleurodesis may be helpful in controlling reaccumulation of fluid, but it is very painful in small animal patients. thoracic effusion secondary to lymphoma often is associated with an anterior mediastinal mass. t-cell lymphoma is the most common type of mediastinal mass observed in dogs. b-cell lymphoma is associated with a decreased response to chemotherapy and shorter survival times. treatment consists of combination chemotherapy with or without radiation therapy to decrease mass size. carcinomatosis is a diffuse disease of the pleural cavity that often is a result of metastasis from a primary pulmonary carcinoma or mammary adenocarcinoma. treatment is similar to that for mesothelioma and is aimed at controlling the effusion and delaying its recurrence. thymomas have been documented in both dogs and cats. dogs most commonly present with a cough, while cats present with clinical signs of respiratory distress and a restrictive respiratory pattern associated with the presence of pleural effusion. an anterior mediastinal mass is often observed on thoracic radiographs. in some cases, the pleural effusion must be drained via thoracocentesis before a mass is visible. ultrasound-guided aspiration and cytologic evaluation of the mass reveal a malignant epithelial tumor with small lymphocytes and mast cells. prognosis is good if the tumor can be completely excised. treatment consists of surgical removal with or without presurgical radiation therapy to shrink the mass. paraneoplastic syndromes of myasthenia gravis have been documented in dogs with thymomas. if megaesophagus or aspiration pneumonia is present, the prognosis is more guarded because of the high rate of complications. obstructive lesions affecting the urinary tract can be extramural (intra-abdominal, pelvic, or retroperitoneal) or intramural (urethral, bladder, or urethral wall) . transitional cell carcinoma is the most common type of bladder tumor observed in dogs. prostatic adenocarcinoma, or neoplasia of the sublumbar lymph nodes (lymphoma, adenocarcinoma from apocrine gland adenocarcinoma), also can cause urethral obstruction. treatment is aimed at relieving the obstruction and then attempting to identify the cause of the disease. to alleviate the obstruction, pass a urinary catheter whenever possible. perform cystocentesis only as a last resort because of the risk of seeding the peritoneal cavity with tumor cells if transitional cell carcinoma is the cause of the obstruction. institute supportive therapy including intravenous fluids and correction of electrolyte abnormalities. plain radiographs may reveal a mass lesion or may not be helpful without double contrast cystography. abdominal ultrasound is more sensitive in identifying a mass lesion in the urinary bladder. masses in the pelvic urethra are difficult to visualize with ultrasonography. double contrast cystourethrography is preferred. once the patient is stabilized, biopsy or surgery is indicated to identify the cause of the mass and attempt resection. urine tests for transitional cell carcinoma are available for identification of transitional cell carcinoma in the dog. complete surgical excision of transitional cell carcinoma or removal of benign tumors of the urinary bladder yields a favorable prognosis. poorer prognosis is seen with incomplete excision. many transitional cell carcinomas are located in the trigone region of the bladder and cannot be completely excised. the nonsteroidal antiinflammatory drug piroxicam is helpful in alleviating clinical signs for a reported -month median survival. in some dogs, cisplatin and carboplatin may delay recurrence of transitional cell carcinoma. tumors of the prostate gland are always malignant and occur with equal frequency in castrated and uncastrated male dogs. diagnosis of prostatic tumors is based on ultrasonographic evidence of a mass effect or prostatomegaly and on transrectal or transabdominal aspiration or biopsy. surgery, chemotherapy, and radiation therapy generally are unrewarding over the long term, although palliative radiation therapy may relieve clinical signs for to months. luminal tumors of the gastrointestinal tract typically cause obstruction, with slowly progressive clinical signs including vomiting, inappetence, and weight loss, or with acute severe protracted vomiting. extraluminal obstructive lesions usually arise from adhesions, or strangulation may occur, resulting in obstruction. perforation of the mass through the gastric or intestinal wall can cause peritonitis. treatment consists of initial stabilization and rehydration, evaluation for evidence of metastasis, and surgical resection of the affected area in cases of adenocarcinoma, leiomyoma, leiomyosarcoma, and obstructive or perforated lymphoma. gastric and intestinal adenocarcinoma are the most common gastrointestinal tumors observed in dogs. affected animals typically have a history of anorexia, weight loss, and vomiting. obtain an abdominal ultrasound before performing any surgery. fine needle aspirates of the mass and adjacent lymph nodes are usually diagnostic and can determine whether there is local metastasis. many tumors are not resectable, and metastasis occurs in approximately % of cases. dogs with smaller tumors that can be resected typically have longer survival times. leiomyosarcomas occur in the intestines of dogs, and carry a more favorable prognosis than adenocarcinoma if the mass can be completely resected. with complete resection, the average survival time is longer than year. the paraneoplastic syndrome of hypoglycemia has been observed with this tumor type. gastrointestinal lymphoma is the most common tumor of the gastrointestinal tract observed in cats. in comparison, it is relatively rare in dogs. unless there is complete obstruction or perforation of the gastrointestinal tract, surgical treatment for gastrointestinal lymphoma is not indicated. rather, multiple chemotherapy drugs are used in combination to achieve remission and resolution of the clinical signs of anorexia, weight loss, and vomiting. treatment responses unfortunately are poor. mast cell tumors of the gastrointestinal tract typically are manifested as gastrointestinal ulceration and hemorrhage in up to % of patients. the gastrointestinal hemorrhage that occurs with mast cell tumors results from increased acid secretion as a result of histamine receptor stimulation. treatment consists of histamine or proton pump inhibition (ranitidine, famotidine, cimetidine, or omeprazole). bowel perforation is a rare complication. many chemotherapy agents exert their effects on rapidly dividing normal and neoplastic cells. normal tissues that are commonly affected include the bone marrow, gastrointestinal tract, skin and hair follicles, and reproductive organs. some drugs have unique organspecific toxicities that must be monitored. knowledge and recognition of the expected type and onset of complications can alleviate their severity by rapid treatment, when complications occur (see table - ) . neutropenia is the most common bone marrow toxicity observed secondary to chemotherapy in small animal patients (table - ) . in most cases, the neutropenia is dose-dependent. the nadir, or lowest neutrophil count, is typically observed to days after chemotherapy treatment. once the nadir occurs, bone marrow recovery is observed, with an increase in circulating neutrophils within to hours (table - ) . treatment of myelosuppression is largely supportive to treat or prevent sepsis. prophylactic antibiotics are recommended in the afebrile patient with a neutrophil count < /µl. acceptable antibiotics include trimethoprim-sulfa and amoxicillin-clavulanate. granulocyte-colony stimulating factor (g-csf) (e.g., neupogen) is a recombinant human product that stimulates the release of neutrophils from the bone marrow, and its use shortens the recovery time following myelosuppressive drug therapy. disadvantages of g-csf include antibody production in response to the drug within weeks of use and its high cost. to prevent ongoing neutropenia, subsequent chemotherapy dosages should be decreased by %, and the interval in between treatments increased. whenever possible, overlap of myelosuppressive drugs should be avoided. acute gastrointestinal toxicity can occur within to hours after administration of cisplatin and actinomycin d. in many cases, pretreatment with the antiemetics metoclopramide, butorphanol, chlorpromazine, dolasetron or ondansetron can prevent chemotherapyinduced nausea and vomiting. vomiting can also occur as a delayed side effect to days after treatment with doxorubicin (adriamycin), actinomycin d, methotrexate, and cytoxan. in delayed reactions, vomiting and diarrhea are caused by damage to intestinal crypt cells. treatment consists of administration of antiemetics, intravenous fluids, and a bland highly digestible diet. doxorubicin also can cause hemorrhagic colitis within to days of administration. treatment includes a bland diet, metronidazole, and tylosin tartrate (tylan powder). emergency care mild to none not observed vincristine (low-dose), l-asparaginase, glucocorticosteroids moderate - days melphalan, cisplatin, mitoxantrone, actinomycin d severe - days doxorubicin, cyclophosphamide, vinblastine paralytic ileus can be observed to days after administration of vincristine. this side effect is more common in humans than animals and can be treated with metoclopramide once a gastrointestinal obstruction has been ruled out. cardiotoxicity doxorubicin (adriamycin) causes a dose-dependent dilative cardiomyopathy when the cumulative dose reaches to mg/m . in many cases, however, clinical signs do not occur until the cumulative dose is mg/m . the myocardial lesions are irreversible. treatment of cardiac dysrhythmias is dependent on the type of dysrhythmia (see section on treatment of dysrhythmias). discontinue doxorubicin and administer diuretics and positive inotropic therapy for dilative cardiomyopathy in order to delay the progression of congestive heart failure (see sections on treatment of congestive heart failure). if abnormalities are shown on electrocardiography performed before beginning therapy, substitute liposome-encapsulated doxorubicin or mitoxantrone substituted in the chemotherapy protocol. cardioprotectant drugs such as vitamin e, selenium, and n-acetyl cysteine have shown some promise in the prevention of doxorubicin-induced cardiotoxicity. cyclophosphamide can cause a sterile hemorrhagic cystitis. damage to the urinary bladder mucosa and vessels is caused by the toxic metabolite acrolein. clinical signs of sterile hemorrhagic cystitis include a history of cyclophosphamide administration, stranguria, hematuria, and pollakiuria. treatment for sterile hemorrhagic cystitis is discontinuation of the drug, treatment of any underlying urinary tract infection with antibiotic therapy based on susceptibility testing, and intravesicle drug administration. in extremely refractory cases, surgical debridement and cauterization of the bladder mucosa may be necessary. prevention of sterile hemorrhagic cystitis includes emptying the bladder frequently and administering the drug in the morning. concurrent administration of prednisone can induce polyuria and polydipsia. if sterile hemorrhagic cystitis occurs, chlorambucil can be substituted as a chemotherapeutic agent. anaphylactic reactions have been observed with the administration of l-asparaginase, adriamycin, etoposide, and paclitaxel. the risk of anaphylaxis increases with repeated administration, although in some animals anaphylaxis will occur on the first exposure to the drug. treatment consists of administration of epinephrine, diphenhydramine, famotidine, and glucocorticosteroids, as with any other life-threatening allergic reaction (see section on treatment of allergic reactions). to decrease the risk of an adverse reaction, give diphenhydramine ( . mg/kg im) to minutes before drug administration. slowing the rate of intravenous infusion also can decrease the chance of an anaphylactic reaction. cisplatin can cause a fatal irreversible pulmonary edema in cats, even at low dosages. -fluorouracil ( -fu) can cause a severe neurotoxicity in cats that results in ataxia and seizures. never use cisplatin or -fu in cats. poisoning cases benefit from a rapid, organized approach. key points in this approach are giving appropriate advice over the telephone, being able to access information sources, and providing appropriate treatment. there are only a few classes of poisons that account for the majority of toxicities reported in dogs and cats. every veterinarian should develop a familiarity with the clinical management of rodenticide and insecticide toxicity and be prepared with antidotes on hand. beyond the most common toxins, the spectrum of possibilities is endless, and the veterinarian must rely on appropriate information resources. it is important to have available a comprehensive source of pharmaceutical and plant identification resources. remarkably, considering the myriad of potentially toxic substances to which an animal can be exposed, relatively few specific antidotes are commonly used in veterinary medicine. because of the lack of specific antidotes, the veterinarian must treat each toxicity with general methods of poison management, applying basic critical care in the treatment of specific clinical signs associated with the poison exposure or toxicity. the adage "treat the patient, not the poison" often comes into play when the exact toxic substance is unknown, or has no specific antidote. before an animal arrives, the staff should be prepared to ask specific questions over the phone, and provide initial advice for clients, particularly if the animal lives some distance from the hospital (box - .) it is important to have access to a database of information on toxic substances. thousands of potentially toxic substances are available on the market today. the american society for the prevention of cruelty to animals (aspca) animal poison control center provides direct access to veterinary toxicologists hours a day, days a year. for additional information, call the nearest veterinary school or emergency center (box - ). also, see section for a table of emergency hotlines. check your local telephone book for a poison control center listing under emergency numbers, usually found on the front cover. although these numbers are for human poisonings, they have access to extensive poison and toxin databases and can potentially provide useful information for veterinarians, particularly regarding antidotal substances suitable for out of the ordinary toxins and human medications. information on the toxic ingredients in thousands of medications, insecticides, pesticides, and other registered commercial products has been confidentially placed by the government in these poison control centers. as new products are marketed, information regarding toxin ingredients is forwarded to the centers. various e-mail discussion lists can serve as an informative resource for practitioners, but access generally requires an initial subscription and may have the disadvantage of delayed *do not keep the client on the telephone for too long. lengthy histories can be performed once the animal is at your hospital and you have started to initiate treatment. † hair dressing products sometimes have hydrogen peroxide as a % w/v; this concentration is not suitable for induction of emesis. is your animal breathing or does it have respiratory difficulty? what is the color of the gums or tongue? is your animal able to walk? is there any vomiting, diarrhea, trembling, or seizures? does it appear lethargic or hyperactive? what is the substance that your animal ingested (was exposed to)? did you witness the ingestion or exposure? how much did the animal consume? how long ago was the exposure? was the substance swallowed, or is it on the animal's skin or eyes? how is the patient acting? how long has the animal been acting that way? or when was the last time you saw your animal act normally? . first aid instructions for the client: induce vomiting at home and save the vomitus. never induce vomiting if the patient is depressed, appears comatose, or is actively seizing. if the animal has ingested a caustic substance (strong alkali or acids) or a petroleum-based product (kerosene or turpentine), never recommend induction of emesis. hydrogen peroxide ( % w/v † ) ml = tsp/ lb of body weight can repeat once if no vomiting occurs after minutes . remind the owner to bring a sample of the toxin and the vomitus in with the patient. . advise the owner to transport the patient as rapidly as possible to the nearest veterinary hospital. response times. they are useful for ideas on standard and long-term therapy, but not emergency stabilization. an exception to this is the veterinary interactive network (vin), which posts message board communications. previous communications from veterinarians who treated a case with the same poison/toxin can be accessed with a subscription. many manufacturers operate an information service about their products. if the product label or name is available, check for a telephone number that may route you to a specialist. there are six essential steps in treating toxicities: . performing a physical examination . stabilizing the patient's vital signs . taking a thorough history . preventing continued absorption of the toxin . administering specific antidotes when available . facilitating clearance or metabolism of the absorbed toxin it is most important to provide symptomatic and supportive care both during and following emergency treatment. immediately on presentation, perform a brief but thorough physical examination. obtain a minimum database as well as serum, urine, or orogastric lavage samples for later toxicologic analyses. it is important at this time to systematically evaluate the patient's physical status, focusing particularly on the toxins most common to a particular geographic location and the organ systems most commonly affected by toxins in veterinary medicinenamely, the neurologic and gastrointestinal tracts. a checklist is useful when performing a complete physical examination (box - ). the minimium database includes a urine sample, packed cell volume, total protein, serum urea, and serum glucose. the information obtained from these simple cage-side tests is useful for determining dehydration, hemoconcentration, azotemia (renal or prerenal), and hypo-or hyperglycemia. when appropriate, obtain samples for serum biochemistry profiles, serum electrolytes, blood gases, serum osmolality, a complete hemogram, and coagulation profiles. samples of serum, urine, and any vomitus or orogastric lavage contents should be collected and saved for later toxicologic analyses as required later. stabilization of vital signs includes four major goals of treatment: maintain respiration, maintain cardiovascular function, control cns excitation, and control body temperature. in any patient with clinical signs of respiratory distress or respiratory dysfunction, supplemental oxygen should be administered via flow-by, oxygen hood, oxygen cage, nasal, nasopharyngeal, or transtracheal oxygen sources. ventilatory assistance may be necessary. irritant or corrosive substances can cause damage to the oropharyngeal mucosa to such an extent that airway obstruction occurs. when necessary, a temporary tracheostomy should be performed. arterial blood gases, pulse oximetry, and capnometry may be required to monitor oxygenation and ventilation. at the time of presentation, immediately place an intravenous catheter for administration of intravenous fluids, inotropes, antiarrhythmics, and antidotes, if necessary. the initial fluid of choice is a balanced crystalloid solution such as normosol-r, plasmalyte-m, or lactated ringer's solution. fluid therapy can later be changed based on the patient's acidbase and electrolyte status. some toxins can cause severe dysrhythmias and hyper-or hypotension. monitor blood pressure and perform ecg and correct any abnormalities according to standard therapy (see sections on hypotension and cardiac dysrhythmias). what is the pupil size? what is the pupil reactivity to light? is the ocular examination normal? what is the sensitivity to light or sound? nose: is it moist, dry, bubbling, or frothy, or caked with dirt? throat: are there any characteristic odors on the breath? are there any traces of foreign material on the tongue or in the crevices of the teeth or gums? are there petechiae or ecchymosis on the gums or bleeding from the gumline? what is the mucous membrane color? is it normal and pink, or dark red (injected), pale, or icteric? what is the capillary refill time? is it fast, normal, or slow? what is the patient's heart rate? are there any pulse deficits or dysrhythmias auscultated? what is the patient's blood pressure? what is the quality of the femoral pulse? is it synchronous with the heart rate, or are there dropped pulses? is the pulse bounding, normal, thready, or not palpable? what is the patient's electrocardiogram? what is the patient's respiratory rate? what is the patient's respiratory character? is it normal, fast, shallow, or labored? what do you hear on thoracic auscultation? do you hear harsh airway sounds or pulmonary crackles? what is the patient's rectal temperature? is there excessive salivation? is there evidence of vomiting or diarrhea? is abdominal palpation painful? do the intestinal loops feel normal, or are they fluid-filled or gas-filled? what is the color and consistency of the feces? is there a palpable urinary bladder? is there urine production? what is the color of the urine? peripheral lymph nodes should be normal in poisonings. some toxins cause hemolysis, methemoglobinemia, heinz body anemia, and coagulopathies. whole blood, fresh frozen plasma, packed rbcs, or hemoglobin-based oxygen carriers should be available and used if necessary. treat methemoglobinemia with a combination of ascorbic acid and n-acetylcysteine. many toxins affect the cns, producing clinical signs of excitation and/or seizures. diazepam is the drug of choice for most but not all seizures and tremors. if an animal has cns excitation secondary to the ingestion of selective norepinephrine reuptake inhibitors, avoid using diazepam, as it can potentially exacerbate clinical signs. muscle relaxants such as guaifenesin or methocarbamol may be required to control muscle spasm and tremors associated with some toxicities. consider animals that are in status epilepticus because of toxin exposure at high risk. such patients may not require the full dose of anesthetics or sedatives for seizure control. give phenobarbital ( - mg/kg iv) or pentobarbital ( - mg/kg iv to effect) for longer-term management of seizures. core body temperature can easily increase or decrease secondary to increased muscle activity or coma. animals may present as hypo-or hyperthermic, depending on the toxin ingested and the stage of toxicity. manage hypothermia with circulating hot water or hot air blankets, or place bubble wrap or saran wrap around the animal's peripheral extremities. manage hyperthermia by placing lukewarm wet towels on the patient until the rectal temperature has decreased to . °c ( °f). (see section on of hyperthermia and heat-induced illness). if sedatives or anesthetics have been used, initial hyperthermia may initially resolve due to hypothalamic loss of thermoregulatory control, cool water bathing should not be performed. when the patient is first presented to the veterinarian, have the owner complete a toxicologic history form (figure - ) while the animal is being initially assessed and vital signs are being stabilized. when initial stabilization of vital signs has been accomplished, the veterinarian can discuss the patient's history with the owner. in urgent situations, the veterinarian should obtain a brief history as an initial procedure (box - ). knowing when the animal was last seen as normal provides a time frame in which the toxic substance was most likely accessed, allowing differential diagnoses to be ranked in some order of probability by rate of onset. in eliciting a history from the owner about the animal's access to poisons, it is important not to take anything for granted. many owners do not realize how poisonous some substances can be, such as insecticide products, garbage, cleaning chemicals, and over-the-counter drugs commonly used by humans. many owners will deny that an animal could have ingested anything that might be toxic, not wanting to believe that the source of the toxin is within their household or property, particularly if recreational drug exposure is suspected. it is useful to phrase questions in a neutral fashion-for example, "is such-and-such present on the premises?" rather than "could the dog have eaten such-and-such?" if recreational drug exposure is suspected, another way to question the owners is to ask whether they have had any guests in their house recently that may have had such-and-such (e.g., marijuana, cocaine, methamphetamine). this approach serves to minimize the suggestion of any bias or preconceptions. when questioning an owner about recent events, it is useful to realize and acknowledge that disruption in the household routine is a distinct factor in the occurrence accidents, including poisonings. examples of such disruptive events include moving from the house, family member is ill or in the hospital, and renovations or recent construction. while these events are occurring, the safeguards followed by a normally careful owner may be disrupted. often, doors or gates may be left open, animals may be outside instead of inside (or vice versa), and inexperienced people may be pet-sitters. once owners are made aware of the importance of assessing such risks, they are often able to provide insight into otherwise baffling circumstances. various methods can be used to remove toxins from the gastrointestinal tract, including emesis, orogastric lavage, cathartics, and enemas. adsorbents, ion exchange resins, or precipitating or chelating agents may be used. removal of a toxic substance from the body surface may be necessary, depending on the toxin.the use of both emesis and orogastric lavage is less and less frequent in human medicine because of the risk of aspiration pneumonia and doubts about their efficacy. currently, management of poisonings in human medicine relies heavily on the use of activated charcoal combined with sorbitol as a cathartic, when appropriate, and supportive critical care. it should be emphasized, however, that the majority of poisonings in humans are due to drug overdoses (illicit or otherwise) (which have a relatively small volume and rapid absorption), for which this treatment is appropriate. furthermore, adoption of the approach rests on the availability of a hospital intensive care infrastructure, which is not always available in veterinary practice. induce emesis if the animal's physiology and neurologic status are stable (i.e., does not have respiratory depression or is not actively seizing, obtunded, unable to swallow or protect its airway). do not administer the same emetic more than twice. if the emetic doesn't work after two doses, give a different emetic or perform orogastric lavage under general anesthesia. emetics are strictly contraindicated for toxicity from petroleum-based products and corrosives because of the risk of aspiration pneumonia and further esophageal damage. emetics may also be of little value if poisons with antiemetic properties have been ingested, such as benzodiazepines, tricyclic antidepressants, and marijuana (table - ) . various emetics traditionally have been recommended for use in veterinary medicine. many have fallen out of favor because of the risk of causing adverse consequences and side effects. apomorphine ( . mg/kg iv or in the conjunctival sac) remains the standard but is less useful in certain situations in which the poison causes cns excitation or stimulation. it is ineffective in cats. other emetics include xylazine and hydrogen peroxide. do not use table salt because of the risk of severe oropharyngeal irritation and hypernatremia. do not use mustard powder or dishwashing liquid detergent because of the risk of severe oropharyngeal, esophageal, and gastric irritation. orogastric lavage is described in detail in the section on emergency procedures gastric lavage is contraindicated in treatment of toxicity from petroleum-based compounds and acid/alkali ingestion. the procedure can be messy but is very effective if performed within to hours of ingestion of the poison. to prevent aspiration, the patient should be placed under general anesthesia. keep the animal's head lowered during the procedure to prevent aspiration of stomach contents into the trachea. it is sometimes helpful to put the animal in both right and left lateral recumbency to allow complete emptying of gastric contents. repeat the procedure until the fluid runs clear from the stomach. in some cases in which solid material has been ingested, this process can take a long time, so be prepared with a large volume of warm water. following successful evacuation and lavage, administer a slurry of activated charcoal through the orogastric tube before removing it. keep the endotracheal tube cuffed and in place until the animal is semi-conscious, is starting the fight the tube, and is visibly able to swallow and protect its airway. • when was the animal last seen as normal? • what clinical signs developed? • how fast did the clinical signs develop? • when was the onset of clinical signs? • what is the animal's activity level? • does the animal have access to any poisonous substances? • this includes known toxins or chemicals, over-the-counter or prescription medications (including the owner's), and recreational drugs. enemas are useful to facilitate the action of cathartics and in cases in which the poison is a solid material (e.g., compost, snail bait, garbage) (box - ). it is best to use just lukewarm water. commercially available phosphate enema solutions can cause severe electrolyte disturbances (hyperphosphatemia, hyponatremia, hypocalcemia, and hypomagnesemia) and acid-base abnormalities (metabolic acidosis); therefore, they are absolutely contraindicated in small animal patients. use nonsterile nonspermicidal water-soluble lubricants (k-y jelly) old intravenous fluid bag enema bag -to -ml syringe fluid warm water, with or without hand or liquid dish soap the fluid volume required depends on the size of the animal and the state of its lower gastrointestinal tract. as with orogastric lavage, continue the procedure until the water runs clear. if difficulty is encountered emptying the lower gastrointestinal tract, repeat the enema in or hours, rather than be overzealous on the first attempt. cathartics are useful for hastening gastrointestinal elimination of toxins, and they are particularly useful for elimination of most solid toxicants (e.g., compost, garbage, snail baits). cathartics can be used in conjunction with activated charcoal. do not use magnesium-based cathartics in patients with cns depression, because hypermagnesemia can worsen this disorder and also cause cardiac rhythm disturbances (table - ) . activated charcoal ( - ml/kg) is the safest and to date the most effective adsorbent for the treatment of ingested toxins. activated charcoal can be administered after emesis or orogastric lavage or can be administered as the sole treatment. various preparations are available on the market, including dry powder, compressed tablets, granules, liquid suspensions, and concentrated paste preparations. commercially available products are relatively inexpensive and should be used whenever possible for ease of administration. vegetableorigin activated charcoal is the most efficient adsorbent and binds compounds with weak, nonionic bonds. some preparations are combined with sorbitol to provide simultaneous administration of an adsorbent and a cathartic; this combination has been shown to be most efficacious. repeated administration of activated charcoal every to hours has been shown to be beneficial in the management of a toxin that undergoes enterohepatic recirculation. administration of an oily cathartic or mixing the activated charcoal with food only serves to reduce the absorptive surface of the activated charcoal and therefore is not recommended. in general, substances that are very soluble and are rapidly absorbed are not well adsorbed by activated charcoal, including alkalis, nitrates, mineral acids, ethanol, methanol, ferrous sulfate, ammonia, and cyanide. kaolin and bentonite are clays that have been used as adsorbents. both are usually less effective than activated charcoal. however, they are reported to be better adsorbents than activated charcoal for the herbicide paraquat. ion exchange resins can ionically bind certain drugs or toxins. cholestyramine is one such resin, commonly used in human medicine to bind intestinal bile acids and thereby decrease cholesterol absorption. its application in toxicology extends to the absorption of fat-soluble toxins such as organochlorine and certain acidic compounds such as digitalis. ion exchange resins also have been used to delay or reduce the absorption of phenylbutazone, warfarin, chlorothiazide, tetracycline, phenobarbital, and thyroid preparations. precipitating, chelating, and diluting agents precipitating, chelating, and diluting agents are used primarily in the management of heavy metal intoxications, such as alkaloids or oxalates. they work by binding preferentially to the metal ion and creating a more soluble complex that is amenable to renal excretion. those chelating agents in common usage are calcium edta, deferoxamine, and d-penicillamine. calcium edta and deferoxamine should both be on hand in the veterinary hospital because they are necessary to treat zinc and iron toxicity, respectively, both of which have a short window of opportunity for therapeutic intervention. d-penicillamine has a wide application for a number of metal toxicities but tends to be used for long-term chronic therapy because it can be administered orally. various agents used for nonspecific dilution of toxins, including milk of magnesia and egg whites, although old-fashioned, still have wide application in many cases in which low-grade irritants have been ingested. bathing the animal is an important aspect of treatment for topical exposures to toxins such as insecticidal products, petroleum-based products, and aromatic oils. bathing an animal is not an innocuous procedure. to avoid hypothermia and shock, use warm water at all times. actively dry the animal to further minimize the risk of hypothermia. when bathing the animal, use rubber gloves and a plastic apron to avoid exposure to noxious agents. in most cases, a mild dishwashing soap is appropriate. medicated or antibacterial shampoos are less appropriate in this situation. for petroleum-based products in particular, dawn dishwashing liquid that "cuts the grease" works well to remove the oils. if dawn is not available, mechanics' hand cleaners or coconut oil-based soaps can be used instead. as a general principle, best results are obtained by barely wetting the patient's fur until the detergent is worked well into the fur, keeping the amount of water to a minimum until ready for the rinse. oil-based paint is best removed by clipping rather than by attempting removal with solvents, because solvents are also toxic. to remove powder products, brush and vacuum the animal before bathing it to eliminate further toxic exposure. with caustic alkaline or acidic products, the primary treatment is to dilute and flush the skin with warm water; do not attempt neutralization. neutralization can cause an exothermic reaction that causes further damage to the underlying tissues. eliminating poison from the eyes for ocular exposures, irrigate the eyes for a minimum of to minutes with warm (body temperature) tap water or warmed . % sterile saline solution. the use of neutralizing substances is not recommended because of the risk of causing further ocular damage. following adequate irrigation, treat chemical burns of the eyes with lubricating ointments and possibly a temporary tarsorrhaphy. atropine may be indicated as a cycloplegic agent. systemic nonsteroidal antiinflammatory drugs can be used to control patient discomfort. daily follow-up examinations are required because epithelial damage may be delayed, especially with alkali burns, and it is difficult to predict the final extent of ocular damage. topical glucocorticosteroids are contraindicated if the corneal epithelium is not intact. if severe conjunctival swelling is present with a corneal ulcer, parenteral glucocorticosteroids can be administered to help alleviate inflammation, but nonsteroidal antiinflammatory drugs should not be used simultaneously due to the risk of gastrointestinal ulceration or perforation. whenever possible, administer specific antidotes to negate the effects of the toxin and prevent conversion of the substance to the toxic metabolite. three categories of agents are used in the management of poisonings. the first category is specific antidotes. unfortunately, few specific antidotes are available for use in veterinary medicine. some "classic" toxins and antidotes are now considered to be rare, such as curare and physostigmine, thallium and prussian blue, and fluoride and calcium borogluconate. these and a few others have been omitted from the table. the second, broader category of antidotes includes those drugs used in the symptomatic management of clinical signs, which are part of our routine veterinary stock. drugs such as atropine, sedatives, steroids, antiarrhythmics, and beta-blockers fall into this category. the third category comprises nonspecific decontaminants such as activated charcoal, cathartics, and emetics. these were discussed previously. many patients benefit from efforts to enhance clearance or metabolism of the absorbed toxins. some specific therapies have been developed for this purpose, including -methylpyrazole for ethylene glycol toxicity and specific antibodies such as digibind (digoxin immune fab [ovine]) for digitalis toxicity. other strategies are aimed at promoting renal excretion. renal excretion strategies include diuresis, ion trapping, and peritoneal dialysis or hemodialysis (see section on peritoneal dialysis). diuresis and ion trapping are applicable to a large number of toxins and are discussed here in more detail. other toxins respond to urine acidification and urine alkalinization. enhancing renal excretion of substances is most useful for those organic substances that are present in significant concentrations in the plasma. substances that are non-ionic and lipid-soluble, such as certain herbicides, are likely to be less affected by attempts to promote rapid renal elimination. before starting diuresis or ion trapping, intravenous fluid therapy should be adequate as determined by normal central venous pressure, urine output, and mean arterial blood pressure. if any of these values are less than normal, use other measures to ensure adequate renal perfusion, including but not limited to a constant rate infusion of dopamine. simple fluid diuresis can influence the excretion of certain substances. the use of mannitol as an osmotic diuretic may reduce the passive reabsorption of some toxic substances in the proximal renal convoluted tubule by reducing water reabsorption. dextrose ( %) can be used as an osmotic diuretic. furosemide can be used to promote diuresis, but again, there is no substitute for intravenous fluid therapy. the use of mannitol, dextrose, and furosemide is contraindicated in hypotensive or hypovolemic patients. take care to avoid causing dehydration with any diuretic; central venous pressure monitoring is strongly recommended. ion trapping is based on the principle that ionized substances do not cross renal tubular membranes easily, and are not well reabsorbed. if the urinary ph can be changed so that the toxin's chemical equilibrium shifts to its ionized form, then that toxin can be "trapped" in the urine and excreted. alkaline urine favors the ionization of acidic compounds, and acidic urine favors the ionization of alkaline compounds. those toxins that are amenable to ion trapping are mostly weak acids and weak bases. ammonium chloride can be used to promote urinary acidification. contraindications to the use of ammonium chloride include a preexisting metabolic acidosis, hepatic or renal insufficiency, and hemolysis or rhabdomyolysis leading to hemoglobinuria or myoglobinuria. signs of ammonia intoxication include cns depression and coma. when performing urine acidification, frequently check the serum potassium concentration and urine ph. urine alkalinization can be performed with use of sodium bicarbonate. contraindications to the use of sodium bicarbonate include metabolic alkalosis (particularly with concurrent use of furosemide), hypocalcemia, and hypokalemia. as with urine acidification, monitor the serum potassium concentration and urine ph frequently. the major steps in management of poisonings discussed here must be accompanied by application of the fundamentals of critical care. respiratory and cardiovascular support have been discussed previously. renal and gastrointestinal function and analgesia are particularly important in the management of the poisoning patient. maintenance of renal perfusion is a priority in the poisoning patient. fluid, electrolyte, and acid-base balance must be controlled and be accurate. poisoning patients are at particularly high risk for renal damage and acute renal failure, whether by primary toxic insult to the renal parenchyma or by acute or prolonged renal hypoperfusion. for this reason, a protocol that aims at preventing oliguria and ensuing renal failure is one of the therapeutic strategies that should be routinely employed. this protocol is described in box - . gastrointestinal protectant drugs may be indicated for the management of those poisons that are gastrointestinal irritants or ulcerogenic. commonly used gastroprotectant drugs include cimetidine, ranitidine, famotidine, omeprazole, sucralfate, and misoprostol. antiemetics may be used to suppress intractable vomiting. metoclopramide is commonly used, and it is the drug of choice for centrally mediated nausea. antiemetics that work by different mechanisms can be used in combination as necessary. examples are dopamine -receptor antagonists such as prochlorperazine, -hydroxytryptamine antagonists such as ondansetron and dolasetron, and h- receptor antagonists such as diphenhydramine and meclizine. analgesics are more appropriate to treat poisonings than once thought. common effects of poisons including severe gastroenteritis and topical burns or ulcerations may warrant the use of analgesics. longer-acting analgesics such as morphine, hydromorphone, and buprenorphine are particularly useful. nutritional support may be necessary in the form of enteral or parenteral feeding in patients that have esophageal or gastric damage or that need to be sedated for long periods of time. endoscopy may be useful in assessing the degree of esophageal and gastric damage, particularly after ingestion of caustic substances. introduction: acetaminophen (paracetamol) is the active ingredient in tylenol and many over-thecounter cold products. acetaminophen is converted to n-acetyl-p-benzoquinonimine in the liver, a toxic substance that can cause oxidative injury of red blood cells and hepatocytes. clinical signs of acetaminophen toxicity include respiratory distress from lack of oxygen-carrying capacity, cyanosis, methemoglobinemia (chocolate-brown appearance of the blood and mucous membranes), lethargy, vomiting, and facial and paw swelling (cats). the toxic dose of acetaminophen is > mg/kg for dogs, and mg/kg for cats. treatment of acetaminophen toxicity includes induction of emesis or orogastric lavage if the substance has been ingested within minutes. activated charcoal should also be administered. in cases of severe anemia, give supplemental oxygen along with a packed rbc transfusion. administer intravenous fluids to maintain renal and hepatic perfusion. n-acetylcysteine, vitamin c, and cimetidine are the treatments of choice for methemoglobinemia in patients with acetaminophen toxicity. introduction: hydrochloric, nitric, and phosphoric acids cause chemical burns through contact with the skin and/or eyes. localized superficial coagulative necrosis occurs upon contact. usually, the patient's skin is painful to the touch or the animal may lick or chew at an irritated area that is not visible under the haircoat. if the chemical is swallowed, do not induce emesis or perform orogastric lavage, because of the risk of worsening esophageal irritation. rinse the patient's skin and eyes with warm water or warm saline for a minimum of / hour. use analgesics and treat corneal ulcers (see section on corneal ulcers) as required. do not attempt chemical neutralization, because of the risk of causing an exothermic reaction and worsening tissue injury. aflatoxin (aspergillus flavus) is found in moldy feed grains. clinical signs of toxicity occur after ingestion and include vomiting, diarrhea, and acute hepatitis; abortion may occur in pregnant bitches. treatment of suspected aflatoxin ingestion consists of gastric decontamination, administration of activated charcoal, intravenous fluids, and hepatic supportive care (s-adenosyl methionine [same], milk thistle). drinking (ethanol), rubbing (isopropyl), and methyl (methanol) alcohols can be harmful if ingested ( . to . g/kg po). all cause disruption of neuronal membrane structure, impaired motor coordination, cns excitation followed by depression, and stupor that can lead to cardiac and respiratory arrest, depending on the amount ingested. affected animals may appear excited and then ataxic and lethargic. contact or inhalant injury can occur, causing dermal irritation and cutaneous hyperemia. methanol also can cause hepatotoxicity. and diarrhea result from muscarinic overload. nicotinic overload produces muscle tremors. toxicity can result in seizures, coma, and death. and cause severe irritation and corrosion of the mucous membranes and skin. some compounds also can cause clinical signs similar to those observed with anticholinesterase compounds, including muscle tremors, seizures, paralysis, and coma. methemoglobinemia can occur. signs of ethylene glycol intoxication and renal impairment or failure, a negative test for the presence of calcium oxalate crystalluria means that there is no more ethylene glycol in the patient's serum because it has all been metabolized. cats are very sensitive to the toxic effects of ethylene glycol. in many cases, cat may have ingested a toxic dose, but because the sensitivity of the assay is low, test results will be negative. lack of treatment can result in death. there are three phases of ethylene glycol intoxication. in the first to hours after ingestion (stage i), the patient may appear lethargic, disoriented, and ataxic. in stage ii ( to hours following ingestion), the patient improves and appears clinically normal. in stage iii ( to hours following ingestion), the patient demonstrates clinical signs of renal failure (polyuria and polydipsia) that progress to uremic renal failure (vomiting, lethargy, oral ulceration). finally, seizures, coma, and death occur. crosses, old english sheepdogs, and some terriers. clinical signs of ivermectin toxicity include vomiting, ataxia, hypersalivation, agitation, tremors, hyperactivity, hyperthermia, hypoventilation, coma, seizures, signs of circulatory shock, bradycardia, and death. clinical signs often occur within to hours after ingestion or iatrogenic overdose. blood ivermectin levels can be measured, but diagnosis is often made based on clinical signs and knowledge of exposure in predisposed breeds. there is no known antidote. the clinical course can be prolonged for weeks to months before recovery occurs. to treat known exposure, induce emesis or perform orogastric lavage if the substance was ingested was within hour of presentation and the patient is not symptomatic. administer activated charcoal. control seizures with phenobarbital, pentobarbital, or propofol administered as intermittent boluses or as a constant rate infusion. diazepam, which potentially can worsen central nervous stimulation, is contraindicated. administer intravenous fluids to maintain perfusion and hydration, and treat hyperthermia. supportive care may be necessary, including supplemental oxygen (or mechanical ventilation, if necessary), frequent turning of the patient and passive range-of-motion exercises, placement of a urinary catheter to maintain patient cleanliness and monitor urine output, lubrication of the eyes, and parenteral nutrition (see section on rule of twenty). specific antidotes used to treat ivermectin toxicity include physostigmine and picrotoxin. physostigmine therapy was beneficial in some patients for a short period; picrotoxin caused severe violent seizures and therefore should be avoided. introduction d-limonene and linalool are components of citrus oil extracts used in some flea control products. the toxic dose is unknown, but cats appear to be very sensitive to exposure. clinical signs of toxicity include hypersalivation, muscle tremors, ataxia, and hypothermia. treatment of d-limonene and linalool exposure includes treatment of hypothermia, administration of activated charcoal to prevent further absorption, and careful, thorough bathing to prevent further dermal exposure. lead is ubiquitous, and is found in some paints, car batteries, fishing equipment/ sinkers, and plumbing materials. lead can be toxic at doses of mg/kg. if more than than - mg/kg of lead is ingested, death can occur. lead causes toxicity by inhibiting sulfur-containing enzymes, leading to increased rbc fragility, and cns damage. clinical signs of hyperexcitability, dementia, vocalization, seizures, and lower motor neuron polyneuropathy can occur. affected animals may appear blind, or vomiting, anorexia, and constipation or diarrhea may occur. if lead toxicity is suspected, blood and urine lead levels can be measured. treatment of lead toxicity is supportive and is directed at treatment of clinical signs. control seizures with diazepam or phenobarbital. if cerebral edema is present, administer mannitol ( . - . g/kg iv), followed by furosemide ( mg/kg iv minutes after mannitol). sodium or magnesium sulfate should be administered as a cathartic. initiate chelation therapy with dimercaprol, penicillamine, or calcium edta. if a lead object is identified in the gastrointestinal tract on radiographs, remove the object using endoscopy or exploratory laparotomy. hyperthermia, that occurs within - minutes of ingestion. diarrhea and convulsions can develop. if hyperthermia is severe, renal failure secondary to myoglobinuria and disseminated intravascular coagulation can result. delayed hepatic failure has been described days after initial recovery. if metaldehyde toxicosis is suspected, analysis of urine, serum, and stomach contents is warranted. to treat metaldehyde toxicity, procure and maintain a patent airway and control cns excitation and muscle tremors. if an animal has just ingested the metaldehyde and is not symptomatic, induce emesis. if clinical signs are present, perform orogastric lavage. both emesis and orogastric lavage should be followed by administration of one dose of activated charcoal. administer intravenous fluids to control hyperthermia, prevent dehydration, and correct acid-base and electrolyte abnormalities. methocarbamol is the treatment of choice to control muscle tremors. diazepam can be used to control seizures if they occur. introduction mushroom ingestion most commonly causes activation of the autonomic nervous system, resulting in tremors, agitation, restlessness, hyperexcitability, and seizures. in some cases slud (salivation, lacrimation, urination, and defecation) is seen. some mushrooms (amanita spp.) also can cause hepatocellular toxicity. clinical signs include vomiting, anorexia, lethargy, and progressive icterus. hemoglobinuria and pigment damage of the renal tubular epithelium. heinz bodies may be observed on cytologic evaluation of the peripheral blood smear. paint in a sorbitol or glycerol carrier. when large quantities of these osmotically active sugars are ingested, osmotic shifts of fluid cause a sudden onset of neurologic or gastrointestinal signs, including ataxia, seizures, and osmotic diarrhea caused by massive fluid shifts into the gastrointestinal tract. the loss of water in excess of solute can result in hypernatremia, a free water deficit, and increased serum osmolality. following orogastric lavage, treatment of ingestion includes administering warm water enemas to help speed the movement of the paintballs through the gastrointestinal tract. do not administer activated charcoal (usually in a propylene glycol carrier), because the compound's cathartic action will pull more fluid into the gastrointestinal tract. baseline electrolytes should be obtained and then carefully monitored. if severe hypernatremia develops, administer hypotonic solutions such as . % nacl + . % dextrose or % dextrose in water after calculating the patient's free water deficit. because of the large volume of fluid loss, intravenous fluid rates may seem excessive but are necessary to normalize acid-base, electrolyte, and hydration status. in most cases, these patients can survive if the problem is recognized promptly and corrected with careful electrolyte monitoring, aggressive decontamination strategies, and intravenous fluid support. introduction paraquat, a dipyridyl compound, is the active ingredient in some herbicides. the ld of paraquat is - mg/kg. paraquat initially causes cns excitation. it also causes production of oxygen-derived free radical species in the lungs, that can lead to the development of acute respiratory distress syndrome. initial clinical signs include vomiting, diarrhea, and seizures. within to days, clinical signs associated with severe respiratory distress and acute respiratory distress syndrome (ards) can develop, leading to death. chronic effects include pulmonary fibrosis, if the patient survives the initial toxicity period. the prognosis for paraquat toxicity is generally unfavorable. to treat paraquat ingestion, remove the toxin from the gastrointestinal tract as rapidly as possible after ingestion. there are no known antidotes. if the compound was ingested within the past hour and the animal is able to protect its airway, induce emesis. otherwise, perform orogastric lavage. activated charcoal is not as effective as clay or bentonite adsorbents for removing this particular toxin. early in the course of paraquat toxicity, oxygen therapy is contraindicated because of the risk of producing oxygen-derived free radical species. later, oxygen therapy, including mechanical ventilation, is necessary if ards develops. experimentally, free radical scavengers (n-acetyl cysteine, vitamin c, vitamin e, same) have been shown to be useful in preventing damage caused by oxygen-derived free radical species. hemoperfusion may be useful in eliminating the toxin, if it is performed early in the course of toxicity. pennyroyal oil is an herbal flea control compound that contains menthofuran as its toxic compound. menthofuran is hepatotoxic and may cause gastrointestinal hemorrhage and coagulopathies. to treat toxicity, administer a cathartic and activated charcoal and antiemetic and gastroprotectant drugs, and thoroughly bathe the animal to prevent further dermal exposure. petroleum distillates: see fuels phenobarbital: see barbiturates phenylcyclidine (angel dust) introduction phenylcyclidine (angel dust) is an illicit recreational drug that causes both cns depression and excitation, decreased cardiac output, and hypotension. to treat phenylcyclidine toxicity, place an intravenous catheter, and administer intravenous fluids and antiarrhythmic drugs to maintain organ perfusion. administer supplemental oxygen, and administer diazepam to control seizures. urine alkalinization can help eliminate the compound. phenylephrine is an α-adrenergic agonist in many over-the-counter decongestant preparations. clinical signs of intoxication include mydriasis, tachypnea, agitation, hyperactivity, and abnormal flybiting and staring behavior. tachycardia, bradycardia, hypertension, hyperthermia, and seizures can occur. to treat phenylephrine toxicity, place an intravenous catheter and give intravenous fluids to maintain hydration, promote diuresis, and treat hyperthermia. administer prazosin or sodium nitroprusside to treat hypertension, antiarrhythmic drugs as necessary, and diazepam to control seizures. phenylpropanolamine has both αand β-adrenergic agonist effects, and is used primarily in the treatment of urinary incontinence in dogs. the drug was taken off of the market for use in humans because of the risk of stroke. clinical signs of phenylpropanolamine intoxication include hyperactivity, hyperthermia, mydriasis, tachyarrhythmias or bradycardia, hypertension, agitation, and seizures. to treat toxicity, administer prazosin or nitroprusside to control hypertension, a betablocker (esmolol, propranolol, atenolol) to control tachyarrhythmias, diazepam to control seizures, and intravenous fluids to maintain hydration and promote diuresis. urine acidification may aid in facilitating excretion. if bradycardia occurs, do not use atropine. pseudoephedrine is an αand β-adrenergic agonist that is a component of many over-thecounter decongestants and is used in the manufacture of crystal methamphetamine. clinical signs of toxicity include severe restlessness, tremors, mydriasis, agitation, hyperthermia, tachyarrhythmias or bradycardia, hypertension, and seizures. to treat toxicity, administer activated charcoal, intravenous fluids to promote diuresis and treat hyperthermia, chlorpromazine to combat α-adrenergic effects, a beta-blocker (propranolol, esmolol, atenolol) to treat β-adrenergic effects, and cyproheptadine (per rectum) to combat serotoninergic effects. piperazine is a gaba agonist, and causes cervical and truncal ataxia, tremors, seizures, coma, and death. salt used for thawing ice commonly contains calcium chloride, a compound that has a moderate toxic potential. calcium chloride produces strong local irritation and can cause gastroenteritis and gastrointestinal ulcers if ingested. respiratory emergencies consist of any problem that impairs delivery of oxygen to the level of the alveoli or diffusion of oxygen across the alveolar capillary membrane into the pulmonary capillary network. decreased respiratory rate or tidal volume can result in hypoxia and buildup of carbon dioxide, or hypercarbia, leading to respiratory acidosis. conditions most frequently encountered result in airflow obstruction, prevention of normal lung expansion, interference with pulmonary gas exchange (ventilation-perfusion mismatch), and alterations of pulmonary circulation. evaluation of the patient with respiratory distress is often challenging, because the most minimal stress can cause rapid deterioration, or even death in critical cases. careful observation of the patient from a distance often allows the clinician to determine the severity of respiratory distress and localize the lesion based on the patient's respiratory pattern and effort. animals in respiratory distress often have a rapid respiratory rate (> breaths per minute). as respiratory distress progresses, the patient may appear anxious and start openmouth breathing. the animal often develops an orthopneic posture, characterized by neck extension, open-mouthed breathing, and elbows abducted or pulled away from the body. cyanosis of the mucous membranes often indicates extreme decompensation. clinical signs of respiratory distress can develop acutely, or from decompensation of a more chronic problem that was preceded by a cough, noisy respirations, or exercise intolerance. localization of the cause of respiratory distress is essential to successful case management. in any patient with clinical signs of respiratory distress, the differential diagnosis should include primary pulmonary parenchymal disease, airway disease, thoracic cage disorders, congestive heart failure, dyshemoglobinemias (carbon monoxide, methemoglobin), and anemia. careful observation of the patient's respiratory pattern can aid in making a diagnosis of upper airway disease/obstruction, primary pulmonary parenchymal disease, pleural space disease, and abnormalities of the thoracic cage. it is often helpful to rest a hand on the patient and breathe along with the patient's effort, to confirm the periods of inhalation and exhalation. the pharynx, larynx, and extrathoracic trachea comprise the upper airway. obstructive lesions are associated with a marked inspiratory wheeze or stridor and slow deep inspiratory effort. auscultation of the larynx and trachea may reveal more subtle obstructions of normal air flow. stridor can usually be auscultated without the use of a stethoscope. lung sounds are usually normal. the neck should be carefully palpated for a mass lesion, tracheal collapse, and subcutaneous emphysema. subcutaneous emphysema suggests tracheal damage or collapse secondary to severe trauma. in some cases, there is a history of voice, or bark, change secondary to laryngeal dysfunction. differential diagnosis is usually based on the patient's signalment, history, and index of suspicion of a particular disease process. differential diagnoses of upper airway obstruction are listed in box - . diseases of the pleural space often are associated with a restrictive respiratory pattern. inspiratory efforts are short, rapid, and shallow, and there is often a marked abdominal push. the pattern has been referred to as a choppy "dysynchronous" respiratory pattern. depending on the disease present, lung sounds may be muffled ventrally and enhanced dorsally. percussion of the thorax reveals decreased resonance if fluid is present. increased resonance is present with pneumothorax. decreased compressibility of the anterior thorax may be present with an anterior mediastinal mass lesion, particularly in cats and ferrets. a pneumothorax or diaphragmatic hernia is commonly associated with evidence of trauma, with or without rib fractures. respiratory distress due to hemothorax may be exacerbated by anemia. differential diagnoses for patients with evidence of pleural cavity disease include pneumothorax, diaphragmatic hernia, neoplasia, and various types of pleural effusion. primary pulmonary parenchymal disease can involve the intrathoracic airways, alveoli, interstitial space, and pulmonary vasculature. a rapid, shallow, restrictive respiratory pattern may be observed with a marked push on exhalation, particularly with obstructive airway disease such as chronic bronchitis (asthma) in cats. crackles or wheezes are heard on thoracic auscultation. differential diagnoses for pulmonary parenchymal disease include cardiogenic and noncardiogenic pulmonary edema, pneumonia, feline bronchitis (asthma), pulmonary contusion, aspiration pneumonitis, pulmonary thromboembolism, neoplasia, infection (bacterial, fungal, protozoal, viral) , and/or chronic bronchitis. other abnormal respiratory patterns may be evident, and warrant further consideration. tachypnea present in the absence of other signs of respiratory distress can be a normal response to nonrespiratory problems, including pain, hyperthermia, and stress. a restrictive respiratory pattern with minimal thoracic excursions can be associated with diseases of neuromuscular function, including ascending polyradiculoneuritis, botulism, and tick paralysis. if adequate ventilation cannot be maintained by the patient, mechanical ventilation may be indicated. kussmaul respiration manifests as very slow, very deep respirations when a metabolic acidosis is present. this type of respiratory pattern typically is observed in patients with severe diabetic ketoacidosis and renal failure in a compensatory attempt to blow off carbon dioxide. cheyne-stokes respiration is usually observed with a defect in the central respiratory control center. the classic pattern of cheyne-stokes respiration is normal or hyperventilation followed by a period of apnea or hypoventilation. in cases of lower cervical cord damage or damage to the central respiratory control center in the cns, the diaphragm alone may assume most of the ventilatory movement. with diaphragmatic fatigue, severe hypoventilation and resultant hypoxemia may require mechanical ventilation. immediate management of any patient in respiratory distress is to minimize stress at all costs. relatively benign procedures such as radiography or intravenous catheter placement can be fatal in patients with severe respiratory compromise. stabilization should always precede further diagnostic evaluation. in some cases, sedation may be required before performing any diagnostics, to prevent further stress. all patients should receive some form of supplemental oxygen, either by mask, cage, or flow-by techniques. in cases in which a severe pneumothorax or pleural effusion is suspected, perform therapeutic and diagnostic thoracocentesis bilaterally to allow lung re-expansion and alleviate respiratory distress, whenever possible. if thoracocentesis alone is not effective at maintaining lung re-expansion, place a thoracostomy tube (particularly in cases of tension pneumothorax). if hypovolemic/ hemorrhagic shock is present, initiate treatment while stabilizing the respiratory system (see section on shock). if an animal is suspected of having an upper airway obstruction, reestablish airflow. in cases of laryngeal paralysis, tracheal collapse, and brachycephalic airway syndrome, sedation is often very useful in alleviating the distress of airway obstruction. in cases of laryngeal collapse, however, sedation may make the condition worse. if laryngeal edema is severe, administer a dose of short-acting glucocorticosteroids (dexamethasone sodium phosphate) to decrease laryngeal inflammation and edema. if a foreign body is lodged in the pharynx, perform the heimlich maneuver by thrusting bluntly several times on the patient's sternum. objects such as balls or bones may be small enough to enter the larynx but too large to be expelled, and will require rapid-acting general anesthesia to facilitate dislodgement and removal. if the obstruction cannot be removed, bypassing the obstruction with an endotracheal tube or temporary tracheostomy should be considered. in an emergency, a temporary transtracheal oxygen catheter can quickly be placed in the following manner. connect a -or -gauge needle to a length of intravenous extension tubing and a -ml syringe. place the male connector of the syringe into the female portion of the extension tubing. cut off the syringe plunger and connect the resulting blunt end to a length of flexible tubing attached to a humidified oxygen source. run the oxygen at l/minute to provide adequate oxygenation until a tracheostomy can be performed. (see sections on oxygen supplementation and tracheostomy). once the animal's condition has been stabilized, specific diagnostic tests, including arterial blood gas analyses, thoracic radiographs, and/or transtracheal wash, can be performed, depending on the patient's condition and needs. specific therapies for management of upper airway obstruction, pleural space disease, and pulmonary disease are discussed next. upper airway obstruction can occur as a result of intraluminal or extraluminal mass lesions or foreign bodies in the oropharynx (abscess, neoplasia), laryngeal paralysis, trauma, and anatomic abnormalities. clinical signs of an upper airway obstruction are associated with an animal's extreme efforts to inhale air past the obstruction. marked negative pressure occurs in the extrathoracic airways and can cause worsening of clinical signs. mucosal edema and inflammation further worsen the obstruction. therapy for upper airway obstruction is aimed at breaking the cycle of anxiety and respiratory distress. administer the anxiolytic tranquilizer acepromazine ( . - . mg/kg iv, im, sq) to decrease patient anxiety. many animals develop hyperthermia from increased respiratory effort and extreme anxiety. implement cooling measures in the form of cool intravenous fluids and wet towels soaked in tepid water placed over the animal (see section on hyperthermia). administer supplemental oxygen in a manner that is least stressful for the animal. short-acting glucocorticosteroids can also be administered (dexamethasone sodium phosphate, . mg/kg iv, sq, im) to decrease edema and inflammation. if the airway obstruction is severe and there is no response to initial measures to alleviate anxiety and decrease inflammation, establish control of ventilation by placement of an endotracheal tube (see section on endotracheal intubation), tracheal oxygen catheter, or temporary tracheostomy. to obtain airway control, administer a rapid-acting anesthetic (propofol, - mg/kg iv to effect), and intubate with a temporary tracheostomy. an intratracheal oxygen catheter can be placed with sedation and/or a local anesthetic (see technique for transtracheal wash). laryngeal paralysis is a congenital or acquired condition that occurs primarily in largebreed dogs secondary to denervation of the arytenoid cartilages by the recurrent laryngeal nerve. congenital laryngeal paralysis occurs in the bouvier des flandres, siberian husky, and bull terrier. acquired laryngeal paralysis occurs in labrador retrievers, saint bernards, and irish setters. acquired laryngeal paralysis can be idiopathic, acquired secondary to trauma to the recurrent laryngeal nerve, or can be a component of systemic neuromuscular disease. although rare, this condition also occurs in cats. with dysfunction of the recurrent laryngeal nerve, the intrinsic laryngeal muscles atrophy and degenerate. as a result, the vocal folds and arytenoid cartilage move in a paramedian position within the airway and fail to abduct during inhalation, causing airway obstruction. laryngeal paralysis can be partial or complete, unilateral or bilateral. in many cases, a change in bark is noted prior to the development of clinical signs of respiratory distress or exercise intolerance. when a patient presents with severe inspiratory stridor (with or without hyperthermia) initiate stabilization with anxiolytic tranquilizers, supplemental oxygen, and cooling measures. once the patient's condition has been stabilized, definitive measures to accurately document and assess the patient's airway should be considered. place the patient under very heavy sedation with short-acting barbiturates or propofol ( - mg/kg iv) and observe the arytenoid cartilages closely in all phases of respiration. administer just enough drug to allow careful examination without getting bitten. if the arytenoid cartilages do not abduct during inhalation, administer dopram (doxapram hydrochloride, - mg/kg iv) to stimulate respiration. absent or paradoxical laryngeal motion (closed during inspiration and open during exhalation) is characteristic of laryngeal paralysis. correction of the defect involves documentation and treatment of any underlying disorder and surgical repair of the area to open the airway. partial laryngectomy, arytenoid lateralization ("tie-back" surgery), or removal of the vocal folds has been used with some success. aspiration pneumonitis is common following these procedures. brachycephalic airway syndrome is associated with a series of anatomic abnormalities that collectively increase resistance to airflow. affected animals typically have stenotic nares, an elongated soft palate, and a hypoplastic trachea. components of the syndrome can occur alone or in combination. in severe cases, laryngeal saccular edema and eversion, and eventual pharyngeal collapse, can occur secondary to the severe increase in intrathoracic airway pressure required to overcome the resistance of the upper airways. specific airway anomalies can be identified with general anesthesia and laryngoscopy. severe respiratory distress should be treated as discussed previously. treatment requires surgical correction of the anatomic abnormalities. in animals with laryngeal collapse, surgical correction may not be possible, and a permanent tracheostomy may be required. because an elongated soft palate and stenotic nares can be identified before the onset of clinical signs, surgical correction to improve airflow when the animal is young may decrease the negative intra-thoracic pressure necessary to move air past these obstructions. the chronic consequences of everted laryngeal saccules and laryngeal collapse potentially can be prevented. tracheal collapse is common in middle-aged and older toy and small-breed dogs. the owner typically reports a chronic cough that is readily induced by excitement or palpation of the trachea. the cough often sounds like a "goose honk." diagnostic confirmation is obtained by lateral radiography or fluoroscopy of the cervical and thoracic trachea during all phases of respiration. acute decompensation is uncommon but does occur, particularly with excitement, exercise, and increased environmental temperatures or ambient humidity. therapy of the patient with acute respiratory distress secondary to tracheal collapse includes sedation, administration of supplemental oxygen, and provision of cooling measures to treat hyperthermia. cough suppressants (hydrocodone bitartrate-homatropine methylbromide, . mg/kg po q - h, or butorphanol, . mg/kg po q - h) are useful. tracheal collapse is a dynamic process that usually involves both the upper and lower airways. because of this, bypassing the obstruction is often difficult. tracheal stents have been emergency care used with limited success in combination with treatment of chronic lower airway disease. crush or bite injuries to the neck can result in fractures or avulsion of the laryngeal or tracheal cartilages. bypassing the obstructed area may be necessary until the patient is stable and can undergo surgical correction of the injury. if there is avulsion of the cranial trachea, it may be difficult to intubate the patient. a long, rigid urinary catheter can be inserted past the area of avulsion into the distal segment, and an endotracheal tube passed over the rigid catheter, to establish a secure airway. neck injury can also result in damage to the recurrent laryngeal nerve and laryngeal paralysis. foreign bodies can lodge in the nasal cavity, pharynx, larynx, and distal trachea. signs of foreign bodies in the nares include acute sneezing and pawing at or rubbing the muzzle on the ground. if the object is not removed, sneezing continues and a chronic nasal discharge develops. respiratory distress is uncommon, but the foreign body is severely irritating. pharyngeal and tracheal foreign bodies can cause severe obstruction to airflow and respiratory distress. diagnosis of a foreign body is based on the patient history, physical examination findings, and thoracic or cervical radiographs. smaller foreign bodies lodged in the distal airways may not be apparent radiographically but can cause pulmonary atelectasis. foreign bodies of the nose or pharynx can often be removed with an alligator forceps with the patient under anesthesia. if removal is not possible with a forceps, flushing the nasal cavity from cranial to caudal (pack the back of the mouth with gauze to prevent aspiration) can sometimes dislodge the foreign material into the gauze packing. rhinoscopy may be necessary. if an endoscope is not available, an otoscope can be used. foreign objects lodged in the trachea can be small and function like a ball valve during inhalation and exhalation, causing episodic hypoxia and collapse. when attempting to remove these objects, suspend the patient with its head down. remove the object with an alligator forceps, using a laryngoscope to aid in visualization. foreign bodies lodged in the trachea or bronchi require removal with endoscopic assistance. nasopharyngeal polyps (in cats, tumors, obstructive laryngitis, granulomas, abscesses, and cysts) can cause upper airway obstruction. clinical signs are usually gradual in onset. the lesions can be identified through careful laryngoscopic examination performed with the patient under general anesthesia. the nasopharynx above the soft palpate should always be included in the examination. pedunculated masses and cysts are excised at the time of evaluation. biopsy of diffusely infiltrative masses is indicated for histologic examination and prognosis. it is impossible to distinguish obstructive laryngitis from neoplasia based on gross appearance alone. whenever possible, material should be collected from abscesses and granulomas for cytologic evaluation and bacterial culture. extraluminal masses impinge on and slowly compress the upper airways, resulting in slow progression of clinical signs. masses are usually identified by palpation of the neck. enlarged mandibular lymph nodes, thyroid tumors, and other neoplasms may be present. diagnosis is usually based on a combination of radiography and ultrasonography. ct and/or mri are helpful in identifying the full extent and invasiveness of the lesion. definitive diagnosis is made with a fine-needle aspirate or biopsy. many thyroid tumors bleed excessively. the inside of each side of the hemithorax is covered in parietal pleura. the lung lobes are covered in visceral pleura. the two surfaces are in close contact with each other, and are contiguous at the hilum under normal circumstances. pneumothorax refers to free air within the pleural space, accumulating in between the parietal and visceral pleura. the term pleural effusion refers to fluid accumulation in that area but does not reflect the amount or type of fluid present. the mediastinal reflections of the pleura typically are thin in dogs and cats, and usually, but not always, connect. bilateral involvement of pneumothorax or pleural effusion is common. both pneumothorax and pleural effusion compromise the lungs' ability to expand and result in hypoxia and respiratory distress. pneumothorax can be classified as open versus closed, simple versus complicated, and tension. an open pneumothorax communicates with the external environment through a rent in the thoracic wall. a closed pneumothorax results from tears in the visceral pleura but does not communicate with the outside. a tension pneumothorax occurs as a result of a tear in the lung or chest wall that creates a flap valve, such that air is allowed to leave the lung and accumulate in the pleural space during inhalation, and closes to seal off exit of air from the pleural space during exhalation. tension pneumothorax can cause rapid decline in cardiopulmonary status and death if not recognized and treated immediately. a simple pneumothorax is one that can be controlled with a simple thoracocentesis. complicated pneumothorax involves repeated accumulation of air, requiring placement of a thoracic drainage catheter. in many cases, pneumothorax develops as a result of trauma. spontaneous pneumothorax occurs with rupture of cavitary lesions of the lung that may be congenital or acquired as a result of prior trauma, heartworm disease, airway disease (emphysema), paragonimiasis, neoplasia, or lung abscess. pneumothorax also rarely occurs as a result of esophageal tears or esophageal foreign bodies. rapid circulatory and respiratory compromise following traumatic pneumothorax can develop as a result of open or tension pneumothorax, rib fractures, airway obstruction, pulmonary contusions, hemothorax, cardiac dysrhythmias, cardiac tamponade, and hypovolemic shock. any patient that is rapidly decompensating after a traumatic episode must be quickly assessed, and emergency therapy initiated (see section on immediate management of trauma, a crash plan). diagnosis of pneumothorax is usually made based on a history of trauma, a rapid, shallow, restrictive respiratory pattern, and muffled heart and lung sounds on thoracic auscultation. the clinical signs and history alone should prompt the clinician to perform a bilateral diagnostic and therapeutic thoracocentesis before taking thoracic radiographs (see section on thoracocentesis). the stress of handling the patient for radiography can be deadly in severe cases of pneumothorax. although the mediastinum on both sides of the thorax connects, it is necessary to perform thoracocentesis on both sides to ensure maximal removal of free air in the pleural space and allow maximal lung expansion. if negative pressure cannot be obtained, or if the patient rapidly reaccumulates air, place a thoracostomy tube connected to continuous suction. (see section on thoracostomy tube placement). treat all penetrating wounds to the thorax as open sucking chest wounds unless proved otherwise. to "close" an open sucking chest wound, clip the fur around the wound as quickly as possible, and place sterile lubricant jelly or antimicrobial ointment circumferentially around the wound. cut a sterile glove to provide a covering. place the covering over the wound, making sure to cover all of the sterile lubricant, thus creating a seal to close the wound temporarily from the external environment. evaluate the patient's thorax via thoracocentesis while placing a thoracostomy tube. once the patient is stable, the open chest wound can be surgically explored, lavaged, and definitively corrected. all animals with open chest wounds should receive antibiotics (first-generation cephalosporin) to prevent infection. following stabilization, radiographs can be taken and evaluated. pneumothorax is confirmed by evidence of elevation of the cardiac silhouette above the sternum, increased density of the pulmonary parenchymal tissue, free air in between the parietal and visceral pleura (making the outline of the lungs visible), and absence of pulmonary vascular structures in the periphery. parenchymal lesions within the lungs are best identified after as much air as possible has been removed from the thorax. obtain left and right lateral and ventrodorsal or dorsoventral views. a standing lateral view may reveal air-or fluid-filled cavitary masses. if underlying pulmonary disease is suspected as a cause of spontaneous pneumothorax, a transtracheal wash, fecal flotation, and heartworm test may be indicated. treatment of pneumothorax includes immediate bilateral thoracocentesis, covering of any open chest wounds, administration of supplemental oxygen, and placement of a thoracostomy tube if negative pressure cannot be obtained or if air rapidly reaccumulates. serial radiography, ct, or mri should be performed in dogs with spontaneous pneumothorax, because the condition can be associated with generalized pulmonary parenchymal disease. strict cage rest is required until air stops accumulating and the thoracostomy tube can be removed. the patient's chest tube should be aspirated every hours after discontinuing continuous suction. if no air reaccumulates after hours, the chest tube can be removed. exercise restriction is indicated for a minimum of week. if bullae or mass lesions are present, exploratory thoracotomy should be considered as a diagnostic and potentially therapeutic option for long-term management in prevention of recurrence. pleural fluid cytologic analysis is indicated for all patients with pleural effusion before administration of antibiotics. the general term pleural effusion means a collection of fluid in the space between the parietal and visceral pleura but does not indicate what kind or how much fluid is present. clinical signs associated with pleural effusion depend on how much fluid is present, and how rapidly the fluid has accumulated. clinical signs associated with pleural effusion include respiratory distress, reluctance to lie down, labored breathing with an abdominal component on exhalation, cough, and lethargy. auscultation of the thorax may reveal muffled heart and lung sounds ventrally and increased lung sounds dorsally, although pockets of fluid may be present, depending on the chronicity of the effusion. percussion of the thorax may reveal decreased resonance. in stable patients, the presence of pleural effusion can be confirmed radiographically. radiographic confirmation of the pleural effusion should include right and left lateral and dorsoventral or ventrodorsal views. a handling or standing lateral view should be obtained if an anterior mediastinal mass is suspected. the standing lateral view will allow the fluid to collect in the costophrenic recess. in patients with respiratory distress, muffled heart and lung sounds, and suspicion of pleural effusion, thoracocentesis should be performed immediately. thoracocentesis can be both therapeutic and diagnostic. radiography is contraindicated because the procedure can cause undue stress and exacerbation of clinical signs in an unstable patient. pleural effusion can cause severe respiratory distress, and can be the result of a number of factors that must be considered when implementing an appropriate treatment plan. pathology of the pleura is almost always a secondary process except for primary bacterial pleuritis and pleural mesotheliomas. causes of pleural effusion in the cat and dog include pyothorax, feline infectious peritonitis, congestive heart failure, chylothorax, heartworm disease, hemothorax, hypoalbuminemia, lung lobe torsions, neoplasia, diaphragmatic hernia, and pancreatitis (box - ). in stable animals, diagnosis of pleural effusion can be made based • imbalance of transpleural or hydrostatic or protein osmotic forces • change in membrane permeability • decrease in rate of fluid reabsorption • combination of foregoing mechanisms on thoracic radiography or ultrasound. thoracic radiographs can show whether the pleural effusion is unilateral or bilateral. effusions in dogs and cats are usually bilateral. the lung parenchyma and the cardiac silhouette cannot be fully evaluated until most of the fluid has been evacuated from the pleural cavity. following thoracocentesis, radiography should be performed with left and right lateral and ventrodorsal or dorsoventral views. in cases of suspected heart failure, echocardiography also is necessary. pleural fluid cytologic analysis is indicated for all patients with pleural effusion. collect specimens before administering antibiotics, whenever possible, because treatment with antibiotics can make a septic condition (pyothorax) appear nonseptic. the remainder of the diagnostic workup and treatment is based on the type of fluid present (table - ). the fluid may be a transudate, nonseptic exudate, septic exudate, chylous, hemorrhagic, or neoplastic. ultrasonographic evaluation of the thorax can be helpful in identifying intrathoracic masses, diaphragmatic hernias, lung lobe torsions, and cardiac abnormalities. unlike radiography, ultrasonography is facilitated by the presence of fluid in the pleural space. pyothorax refers to a septic effusion of the pleural cavity. the infection is generally the result of a combination of aerobic and anaerobic bacteria. rarely, fungal organisms are present. the source of the underlying organisms is rarely identified, particularly in cats, but can be caused by penetrating wounds through the chest wall, esophagus, migrating foreign bodies (especially grass awns), or primary lung infections. the most common organisms associated with pyothorax in the cat are pasteurella, bacteroides, and fusobacterium. fever is often present in addition to clinical signs of pleural effusion. septic shock is ununcommon. diagnosis of pyothorax is made based on cytologic analysis and the demonstration of intracellular and extracellular bacteria, toxin neutrophils and macrophages, and sometimes the presence of sulfur granules. gram stains of the fluid can assist in the initial identification of some organisms. bacterial cultures are indicated for bacteria identification and antibiotic susceptibility testing. administration of antibiotics before cytologic evaluation can cause a septic effusion to appear nonseptic. emergency treatment for pyothorax involves placement of an intravenous catheter, intravenous fluids to treat hypovolemic shock, and broad-spectrum antibiotics (ampicillin, mg/kg iv q h, and enrofloxacin, mg/kg iv q h). chloramphenicol also is an appropriate antibiotic to use for penetration into pockets of fluid. administration of a beta-lactam antibiotic (ampicillin or amoxicillin) with a beta-lactamase inhibitor (amoxicillin clavulanate or ampicillin sulbactam) is helpful in achieving better coverage of bacteroides spp. treatment of pyothorax differs in the cat and dog. in the cat, placement of one or two thoracic drainage catheters is recommended to allow continuous drainage of the intrathoracic abscess. inadequate drainage can result in treatment failure. fluid should be evaluated and the pleural cavity lavaged with ml/kg of warmed . % saline or lactated ringer's solution every hours. approximately % of the infused volume should be recovered after each lavage. in dogs, or in cats with refractory pyothorax, perform an exploratory thoracotomy to remove any nidus of infection. rarely a foreign body is visible that can be removed at the time of surgery, but this finding is rare. antibiotics are indicated for a minimum of to weeks after removal of the thoracostomy tube. early diagnosis and aggressive treatment result in a good prognosis in the majority of patients with pyothorax. in cats, clinical signs of ptyalism and hypothermia at the time of presentation worsen the prognosis. chylothorax refers to the abnormal accumulation of chyle (lymphatic fluid) in the pleural cavity. the cisterna chili is the dilated collection pool of lymphatic ducts in the abdomen that accumulate chyle prior to entry into the thoracic duct located within the thoracic cavity. the thoracic duct enters the thorax at the aortic hiatus. numerous tributaries or collateral ducts exist. the functions of the lymphatic vessels collectively serve to deliver triglycerides and fat-soluble vitamins into the peripheral vascular circulation. damage of the thoracic duct or lymphatic system or obstruction to lymphatic flow can result in the development of chylous effusion in the pleural or peritoneal space. it is difficult to identify chylous effusions based on their milky appearance alone. to identify a chylous effusion versus a pseudochylous effusion, the triglyceride and cholesterol levels of the fluid must be compared with those of peripheral blood. chylous effusions have a higher triglyceride and lower cholesterol levels than peripheral blood. pseudochylous effusions have a higher cholesterol and lower triglyceride levels than peripheral blood. disease processes that can result in chylous effusions are listed in the box - . clinical signs associated with chylous effusion are typical of any pleural effusion and of the disease process that caused the effusion. weight loss may be evident, depending on the chronicity of the process. the diagnosis is made based on thoracocentesis, cytology, and biochemical evaluation of the fluid (i.e., triglyceride and cholesterol levels). the fluid often appears milky or bloodtinged but can be clear if the patient has significant anorexia. typical cytologic characteristics are listed in table - . lymphangiography can be used to confirm trauma to the thoracic duct, but this is usually not necessary unless surgical ligation is going to be attempted. the diagnostic evaluation must also attempt to identify an underlying cause. therapy for chylothorax is difficult and primarily involves documentation and treatment of the underlying cause. if an underlying cause is not found, treatment is largely supportive and consists of intermittent thoracocentesis to drain the fluid as it accumulates and causes respiratory dysfunction, nutritional support, and maintenance of fluid balance. a variety of surgical techniques, including ligation of the thoracic duct, pleural-peritoneal shunts, and pleurodesis, have been attempted but have had limited success. most recently, the combination of thoracic duct ligation with subtotal pericardectomy has been shown to improve surgical success rates in the treatment of chylothorax. rutin, a bioflavinoid, has been used with limited success in the treatment of idiopathic chylothorax in cats. prognosis in many cases of chylothorax is guarded. extensive hemorrhage into the pleural cavity can cause fulminant respiratory distress due to sudden hypovolemia and anemia and interference with lung expansion. hemothorax typically is associated with trauma, systemic coagulopathy, lung lobe torsions, and erosive lesions within the thorax (usually neoplasia). diagnosis of hemothorax involves obtaining a fluid sample via thoracocentesis. hemorrhagic effusion must be differentiated from systemic blood inadvertently collected during the thoracocentesis procedure. unless the hemorrhage is peracute, fluid in cases of hemothorax is rapidly defibrinated and will not clot, has a packed cell volume less than that of venous blood, contains rbcs and macrophages. hemorrhagic effusions also usually contain a disproportionately higher number of white blood cells compared with peripheral blood. hemothorax commonly is the sole clinical sign observed in animals with vitamin k antagonist rodenticide intoxication and systemic coagulopathy. whenever an animal presents with signs of a hemorrhagic pleural effusion, perform coagulation testing immediately to determine whether a coagulopathy exists. the prothrombin time test is fast and can be performed as a cage-side test (see section on coagulopathy). therapy for hemorrhagic pleural effusions should address the blood and fluid loss. administer intravenous crystalloid fluids and rbc products (see section on transfusion therapy). when necessary, administer coagulation factors in the form of fresh whole blood or fresh frozen plasma, along with vitamin k ( mg/kg sq in multiple sites with a -gauge needle). if severe respiratory distress is present, evacuate the blood within the pleural space via thoracocentesis until clinical signs of respiratory distress resolve. fluid that remains aids in the recovery of the patient, because rbcs and proteins eventually will be reabsorbed. autotransfusion can be performed to salvage blood and reinfuse it into the anemic patient. in cases of neoplastic or traumatic uncontrollable hemorrhagic effusions, surgical exploration of the thorax is warranted. diaphragmatic hernia, or a rent in the diaphragm, can result in the protrusion of abdominal organs into the thoracic cavity and impair pulmonary expansion. organs that are commonly herniated into the thorax include the liver, stomach, and small intestines. diaphragmatic hernia usually is secondary to trauma but can occur as a congenital anomaly. in cases of trauma, rib fractures, pulmonary contusions, traumatic myocarditis, hemothorax, and shock are also often present concurrently with diaphragmatic hernia. respiratory distress can be caused by any one or a combination of the above lesions. animals with prior or chronic diaphragmatic hernias may have minimal clinical signs despite the presence of abdominal organs within the thorax. clinical signs of acute or severe diaphragmatic hernia include respiratory distress, cyanosis, and shock. a diagnosis of diaphragmatic hernia is made based on the patient's history (traumatic event), clinical signs, and radiographs. in some cases, ultrasonography or contrast peritoneography is necessary to confirm the diagnosis. contrast radiographs may show the presence of the stomach or intestines within the thorax following oral administration of barium. never administer barium directly into the peritoneal cavity or in cases of suspected gastrointestinal rupture. treatment of a patient with a diaphragmatic hernia includes cardiovascular and respiratory system stabilization before attempting surgical repair of the diaphragm. if the stomach is within the thorax, or if the patient's respiratory distress cannot be alleviated with medical management alone, immediate surgery is necessary. if the respiratory distress is minimal and the stomach is not located within the thorax, surgery can be postponed until the patient is a more stable anesthetic candidate. at the time of surgery, the abdominal organs are replaced into the abdominal cavity, and the rent in the diaphragm is closed. air must be evacuated from the thorax following closure of the diaphragm. if chronic diaphragmatic hernia is repaired, the complication of reexpansion pulmonary edema can occur. cardiac injury is a common complication secondary to blunt thoracic trauma. in most cases, cardiac injury is manifested as arrhythmias, including multiple premature ventricular contractions, ventricular tachycardia, st segment depression or elevation secondary to myocardial hypoxemia, and atrial fibrillation (see section on cardiac emergencies). myocardial infarction and cardiac failure can occur. careful and repeated assessments of the patient's blood pressure and ecg tracing should be a part of any diagnostic work-up for a patient that has sustained blunt thoracic trauma. rib fractures are associated with localized pain and painful respiratory movements. radiographs are helpful to confirm the diagnosis. careful palpation may reveal crepitus and instability of the fractured ribs. common problems associated with rib fractures emergency care include pulmonary contusions, pericardial laceration, traumatic myocarditis, diaphragmatic hernia, and splenic laceration or rupture. a flail segment results from rib fractures of more than three adjacent ribs that produce a "floating segment" of the chest wall. the flail segment moves paradoxically with respiration-that is, it moves inward during inhalation and outward during exhalation. respiratory distress is associated with the pain caused by the fractures and the presence of traumatic underlying pulmonary pathology. therapy for rib fractures and flail chest includes administration of supplemental oxygen, treatment of pneumothorax or diaphragmatic hernia, and administration of systemic and local anesthesia to alleviate the discomfort associated with the fractures. although controversial, positioning the patient with the flail segment up may reduce pain and improve ventilation. avoid the use of chest wraps, which do nothing to stabilize the flail segment and can further impair respiratory excursions. following administration of a systemic analgesic, administer a local anesthetic at the dorsocaudal and ventrocaudal segment of each fractured rib, and in one rib in front of and behind the flail segment. often, pulmonary function will improve once the pain associated with rib fractures has been adequately treated. in rare cases in which the flail segment involves five or more ribs, surgical stabilization may be necessary. single rib fractures or smaller flail segments are allowed to heal on their own. feline bronchitis has a variety of names (bronchial asthma, asthma, acute bronchitis, allergic bronchitis, chronic asthmatic bronchitis, feline lower airway disease) and refers to the acute onset of respiratory distress secondary to narrowing of the bronchi. cats may present with an acute onset of severe restrictive respiratory pattern associated with lower airway obstruction. acute bronchitis in cats typically has an inflammatory component in the lower airways, resulting in acute bronchoconstriction, excessive mucus production, and inflammatory exudates. in cats with chronic bronchitis, there may be damage of the bronchial epithelium and fibrosis of the airways. these patients often have a history if intermittent exacerbation of clinical signs, intermittent cough, and periods of normality throughout the year. because there appears to be an allergic or inflammatory component in feline bronchitis, clinical signs can be acutely exacerbated by stress and the presence of aerosolized particles such as perfume, smoke, and carpet powders. causes of feline bronchitis include heartworm disease, parasitic infestation (lungworms), and (rarely) bacterial infection. on presentation, the patient should be placed in an oxygen cage and allowed to rest while being observed from a distance. postpone performing stressful diagnostic procedures until the patient's respiratory status has been stabilized. after careful thoracic auscultation, administer a short-acting bronchodilator (terbutaline, . mg/kg sq or im) along with a glucocorticosteroid (dexamethasone sodium phosphate mg/kg im, sq, iv) to alleviate immediate bronchospasm and airway inflammation. clinical signs of feline bronchitis are characterized by a short, rapid respiratory pattern with prolonged expiration with an abdominal push. wheezes may be heard on thoracic auscultation. in some cases, no abnormalities are found on auscultation, but become acutely worse when the patient is stimulated to cough by tracheal palpation. radiographs may reveal a hyperinflated lung field with bronchial markings and caudal displacement of the diaphragm. in some cases, consolidation of the right middle lung lobe is present. a complete blood count and serum biochemistry profile can be performed, but results usually are unrewarding. in endemic areas, a heartworm test is warranted. fecal examination by flotation and the baermann technique is helpful in ruling out lungworms and other parasites. bronchoalveolar lavage or transtracheal wash is useful for cytologic and bacterial examination. long-term management of feline bronchitis includes isolation from environmental exposure to potential allergens (litter dust, perfumes, smoke, incense, carpet powders) and treatment of bronchoconstriction and inflammation with a combination of oral and inhaled glucocorticosteroids and bronchodilators (table - ). antibiotic therapy is contraindicated unless a pure culture of a pathogen is documented. oral therapy with steroids and bronchodilators should be used for a minimum of weeks after an acute exacerbation and then gradually decreased to the lowest dose possible to alleviate clinical signs. metered dose inhalers are now available (aerokat.com) for administration of inhaled bronchodilators and steroids. fluticasone (flovent, mcg/puff ) can be administered initially every hours for week and then decreased to once daily, in most cases. inhaled glucocorticosteroids are not absorbed systemically, and therefore patients do not develop the adverse side effects sometimes documented with oral glucocorticosteroid administration. because it takes time for glucocorticosteroids to reach peak effects in the lungs, administration of inhaled glucocorticosteroids should overlap with oral prednisolone administration for to days. treatment of pulmonary contusions is supportive. administer supplemental oxygen in a manner that is least stressful for the animal. arterial blood gas analysis or pulse oximetry can determine the degree of hypoxemia and monitor the response to therapy. intravenous fluids should be administered with caution to avoid exacerbating pulmonary hemorrhage or fluid accumulation in the alveoli. treat other conditions associated with the traumatic event. possible complications of pulmonary contusions are rare but include bacterial infection, abscessation, lung lobe consolidation, and the development of cavitary lesions. the routine use of antibiotics or steroids in cases of pulmonary contusions is contraindicated unless external wounds are present. empiric antibiotic use without evidence of external injury or known infection can potentially increase the risk of a resistant bacterial infection. steroids have been shown to decrease pulmonary alveolar macrophage function and impair wound healing and are contraindicated. aspiration pneumonia can occur in animals as a result of abnormal laryngeal or pharyngeal protective mechanisms or can be secondary to vomiting during states of altered mentation, including anesthesia, recovery from anesthesia, and sleep. megaesophagus, systemic polyneuropathy, myasthenia gravis, and localized oropharyngeal defects such as cleft palate can increase the risk of developing aspiration pneumonitis. iatrogenic causes of aspiration pneumonia include improper placement of nasogastric feeding tubes, overly aggressive force-feeding, and oral administration of drugs. aspiration of contents into the airways can cause mechanical airway obstruction, bronchoconstriction, chemical damage to the alveoli, and infection. severe inflammation and airway edema are common. pulmonary hemorrhage and necrosis can occur. diagnosis of aspiration pneumonia is based on clinical signs of pulmonary parenchymal disease, a history consistent with vomiting or other predisposing causes, and thoracic radiographs demonstrating a bronchointerstitial to alveolar pulmonary infiltrate. the most common site is the right middle lung lobe, although the pneumonia can occur anywhere, depending on the position of the patient at the time of aspiration. a transtracheal wash or bronchoalveolar lavage is useful for bacterial culture and susceptibility testing. treatment of aspiration pneumonia includes antibiotic therapy for the infection, administration of supplemental oxygen, and loosening the debris in the airways. administer intravenous fluids to maintain hydration. nebulization with sterile saline and chest physiotherapy (coupage) should be performed at least every hours. antibiotics to consider in the treatment of aspiration pneumonia include ampicillin/enrofloxacin, amoxicillinclavulanate, ampicillin-sulbactam, trimethoprim sulfa, and chloramphenicol. the use of glucocorticosteroids is absolutely contraindicated. continue antibiotic therapy for a minimum of weeks after the resolution of radiographic signs of pneumonia. pulmonary edema arises from the accumulation of fluid in the pulmonary interstitial alveolar spaces, and airways. ventilation-perfusion abnormalities result in hypoxia. pulmonary edema can be caused by increased pulmonary vasculature hydrostatic pressure, decreased pulmonary oncotic pressure, obstruction of lymphatic drainage, or increased capillary permeability. multiple factors can occur simultaneously. the most common cause of edema is increased pulmonary hydrostatic pressure resulting from left-sided congestive heart failure. decreased plasma oncotic pressure with albumin < . g/dl can also result in accumulation of fluid in the pulmonary parenchyma. overzealous intravenous crystalloid fluid administration can result in dilution of serum oncotic pressure and vascular overload. obstruction of lymphatic drainage is usually caused by neoplasia. other causes of pulmonary edema include pulmonary thromboembolic disease, severe upper airway obstruction (noncardiogenic pulmonary edema), seizures, and head trauma. increased capillary permeability is associated with a variety of diseases that cause severe inflammation (systemic inflammatory response syndrome). the resultant pulmonary edema contains a high amount of protein and is known as acute respiratory distress syndrome (ards). ards can be associated with pulmonary or extrapulmonary causes, including direct lung injury from trauma, aspiration pneumonia, sepsis, pancreatitis, smoke inhalation, oxygen toxicity, electrocution, and immune-mediated hemolytic anemia with disseminated intravascular coagulation. diagnosis of pulmonary edema is made based on clinical signs of respiratory distress and the presence of crackles on thoracic auscultation. in severe cases, cyanosis and fulminant blood-tinged frothy edema fluid may be present in the mouth and nostrils. immediate management includes administration of furosemide ( - mg/kg iv, im) and supplemental oxygen. sedation with low-dose morphine sulfate ( . - . mg/kg iv) is helpful in dilating the splanchnic capacitance vasculature and relieving anxiety for the patient. if fluid overload is suspected secondary to intravenous fluid administration, fluids should be discontinued. severely hypoalbuminemic patients should receive concentrated human albumin ( ml/kg of a % solution) or fresh frozen plasma. furosemide as a constant rate infusion ( . - . mg/kg/hour) also can dilate the pulmonary vasculature and decrease fluid accumulation in cases of ards. following initial stabilization of the patient, thoracic radiographs and an echocardiogram should be assessed to determine cardiac side, pulmonary vascular size, and cardiac contractility. further diagnostic testing may be required to determine other underlying causes of pulmonary edema. heart failure is managed with vasodilators, diuretics, oxygen, and sometimes positive inotropes. treatment ultimately consists of administration of supplemental oxygen, minimal stress and patient handling, and judicious use of diuretics. in cases of cardiogenic pulmonary edema, administer furosemide ( - mg/kg iv, im) every to minutes until the patient loses % of its body weight. positive inotropic and antiarrhythmic therapy may be necessary to improve cardiac contractility and control dysrhythmias. the clinician should determine whether the cause of the pulmonary edema is secondary to congestive heart failure with pulmonary vascular overload, volume overload, hypoalbuminemia, or increased permeability (ards). pulmonary edema secondary to ards typically is refractory to supplemental oxygen and diuretic therapy. in many cases, mechanical ventilation should be considered. a diagnosis of pulmonary thromboembolism (pte) is difficult to make and is based on clinical signs of respiratory distress consistent with pte, lack of other causes of hypoxemia, a high index of suspicion in susceptible animals, the presence of a condition associated with pte, and radiographic findings. virchow's triad consists of vascular endothelial injury, sluggish blood flow with increased vascular stasis, and a hypercoagulable state as predisposing factors for thromboembolic disease. clinical conditions that predispose an animal to pte include hyperadrenocorticism, disseminated intravascular coagulation (dic), catheterization of blood vessels, bacterial endocarditis, protein-losing nephropathy or enteropathy, hyperviscosity syndromes, heat-induced illness, pancreatitis, diabetes mellitus, inflammatory bowel disease, and immune-mediated hemolytic anemia. definitive diagnosis requires angiography or a lung perfusion scan. clinical signs associated with pte include an acute onset of tachypnea, tachycardia, orthopnea, and cyanosis. if the embolism is large, the patient may respond poorly to supplemental oxygen administration. pulmonary hypertension can cause a split second heart sound on cardiac auscultation. in some cases, a normal thoracic radiograph is present in the face of severe respiratory distress. this is a classic finding in cases of pte. potential radiographic abnormalities include dilated, tortuous, or blunted pulmonary arteries; wedge-shaped opacities in the lungs distal to an obstructed artery; and interstitial to alveolar infiltrates. the right heart may be enlarged. echocardiography can show right heart enlargement, tricuspid regurgitation, pulmonary hypertension, and evidence of underlying cardiac disease, possibly with clots in the atria. measurement of antithrombin (at) and d-dimer levels can be useful in the identification of hypercoagulable states, including dic. treatment of any patient with at deficiency or dic includes replenishment of at and clotting factors in the form of fresh frozen plasma. treatment of pte includes therapy for cardiovascular shock, oxygen supplementation, and thrombolytic therapy (see section on thromboembolic therapy). for short-term treatment, administer heparin (heparin sodium, - units/kg sq once, followed by units/kg q h of unfractionated heparin; or fractionated heparin). thrombolytic therapy may include tissue plasminogen activator, streptokinase, or urokinase. long-term therapy with low molecular weight heparin or warfarin may be required to prevent further thromboembolic events. ideally, management should include treatment and elimination of the underlying disease. smoke inhalation commonly occurs when an animal is trapped in a burning building. the most severe respiratory complications of smoke inhalation are seen in animals that are close enough to the flames to also sustain burn injuries (see section on burn injury). at the scene, many animals are unconscious from the effects of hypoxia, hypercapnia, carbon monoxide intoxication, and hydrogen cyanide gases that accumulate in a fire. carbon monoxide produces hypoxia by avidly binding to and displacing oxygen binding to hemoglobin, resulting in severe impairment of oxygen-carrying capacity. the percentage of carboxyhemoglobin in peripheral blood depends on the amount or carbon monoxide in inhaled gases and the length of time of exposure. clinical signs of carbon monoxide intoxication include cyanosis, nausea, vomiting, collapse, respiratory failure, loss of consciousness, and death. smoke inhalation of superheated particles also causes damage to the upper airways and respiratory tree. the larynx can become severely edematous and obstruct inspiration. emergency endotracheal intubation, tracheal oxygen, or tracheostomy tube may be required in the initial resuscitation of the patient, depending on the extent of airway edema. inhalation of noxious gases and particles can cause damage to the terminal respiratory bronchioles. specific noxious gases that can cause alveolar damage include combustible particles from plastic, rubber, and other synthetic products. pulmonary edema, bacterial infection, and ards can result. in any case of smoke inhalation, the first and foremost treatment is to get the animal away from the source of the flames and smoke and administer supplemental oxygen at the scene. at the time of presentation, carefully examine the animal's eyes, mouth, and oropharynx suction soot and debris from the mouth and upper airways. evaluate the patient's respiratory rate, rhythm, and pulmonary sounds. arterial blood gases should be analyzed with co-oximetry to evaluate the pao and carboxyhemoglobin concentrations. evaluation of sao by pulse oximetry is not accurate in cases of smoke inhalation, as the pao may appear normal, even when large quantities of carboxyhemoglobin are present. radiographs are helpful in determining the extent of pulmonary involvement, although radiographic signs may lag behind the appearance of clinical respiratory abnormalities by to hours. bronchoscopy and bronchoalveolar lavage provide a more thorough and accurate evaluation of the respiratory tree; however, these procedures should be performed only in patients whose cardiovascular and respiratory status is stable. management of the patient with smoke inhalation includes maintaining a patent airway, administration of supplemental oxygen, correction of hypoxemia and acid-base abnormalities, preventing infection, and treating thermal burns (see section on burn injury). if severe laryngeal edema is present, a temporary tracheostomy may be necessary to allow adequate oxygenation and ventilation. glucocorticosteroids should not be empirically used in the treatment of smoke inhalation, because of the risk of decreasing pulmonary alveolar macrophage function and increasing the potential for infection. in cases of severe laryngeal edema, however, glucocorticosteroids may be necessary to decrease edema and inflammation. the use of empiric antibiotics is contraindicated unless clinical signs of deterioration and bacterial pneumonia develop. epistaxis can be caused by facial trauma, a foreign body, bacterial or fungal rhinitis, neoplasia, coagulopathies, and systemic hypertension. acute, severe bilateral hemorrhage without wounds have been classified in several ways according their degree of tissue integrity, etiologic force, degree of contamination and duration, and degree of contamination and infection (table - ) . there are also unique causes of wounds such as burns, psychogenic dermatoses, frostbite, decubital ulcers, and snake bite. the animal should be transported to the nearest veterinary facility for definitive care. the wound should be covered or packed with dry gauze or clean linen to protect the wound, and to prevent further hemorrhage and contamination. if an open fracture is present, the limb should be splinted without placing the exposed bone back into the wound. replacing the exposed bone fragment back through the skin wound can cause further damage to underlying soft tissue structures and increase the degree of contamination of deeper tissues. if a spinal fracture is suspected, the patient should be transported on a stable flat surface to prevent further spinal mobilization and neurologic injury. at the time of presentation, first refer to the abcs of trauma, taking care to evaluate and stabilize the patient's cardiovascular and respiratory status. after a complete physical examination and history, ancillary diagnostic techniques can be performed if the patient is hemodynamically stable (see section on triage, assessment, and treatment of emergencies). initially, every patient with superficial wound should receive some degree of analgesia and an injection of a first-generation cephalosporin, preferably within hours of the injury. evaluate the wound after the patient's cardiovascular and respiratory status have been stabilized. always cover an open wound before taking an animal to the hospital to prevent a nosocomial infection. evaluate limb wounds for neural, vascular, and orthopedic abnormalities. carefully examine the structures deep to the superficial wounds. when there has been a delay in assessment of the wound, obtain samples for culture and antimicrobial susceptibility testing. if the wound is older and obviously infected, a gram stain can help guide appropriate antimicrobial therapy pending results of culture and susceptibility testing. place a support bandage saturated with a water-soluble antibiotic ointment or nonirritating antimicrobial solution (e.g., . % chlorhexidine, if bone or joint tissue is not exposed) around the wound. in addition to a first-generation cephalosporin, other appropriate antibiotic choices include amoxicillin-clavulanate, trimethoprim-sulfadiazine, amoxicillin, and ampicillin. if gram-negative flora are present, administer enrofloxacin. administer the antibiotics of choice for a minimum of days unless a change of antibiotic therapy is indicated. at the time of wound cleansing or definitive wound repair, the patient should be placed under general anesthesia with endotracheal intubation, unless the procedure will be brief (i.e., less than minutes). in such cases, a short-acting anesthetic combination open lacerations or skin loss closed crushing injuries and contusions etiologic force abrasion loss of epidermis and portions of dermis, usually caused by shearing between two compressive surfaces avulsion tearing of tissue from its attachment because of forces similar to those causing abrasion but of a greater magnitude incision wound created by a sharp object; wound edges are smooth and there is minimal trauma in the surrounding tissues laceration irregular wound caused by tearing of tissue with variable damage to the superficial and underlying tissue puncture penetrating wound caused by a missile or sharp object; superficial damage may be minimal; damage to deeper structures may be considerable; contamination by fur and bacteria with subsequent infection is common class i - hours with minimal contamination class ii - hours with significant contamination class iii > hours with gross contamination (analgesia + propofol, analgesia + ketamine/diazepam) can be administered to effect. heavy sedation with infiltration of a local anesthetic may also be appropriate for very small wounds, depending on the location of the wound and temperament of the patient. protect the wound by packing it with sterile gauze sponges soaked in sterile saline, or with watersoluble lubricating gel such as k-y jelly. clip the fur surrounding the wound, moving from the inner edge of the wound outward, to help prevent wound contamination with fur or other debris. scrub the wound and surrounding skin with an antimicrobial soap and solution such as dilute chlorhexidine until the area is free of all gross debris. gross debris within the wound itself can be flushed using a -ml syringe filled with sterile saline or lactated ringer's solution and an -gauge needle. pressure-lavage systems are also available for use, if desired. grossly contaminated wounds can be rinsed first with warm tap water to eliminate gross contamination, and then prepared as just described. debride the wound, removing skin and other soft tissue that is not obviously viable. obviously viable and questionable tissue should remain, and the wound left open for frequent reassessment on a daily basis. remove any dark or white segments of skin. questionable skin edges may or not regain viability and should be left in place for hours, so the wound can fully reveal itself. excise grossly contaminated areas of fat and underlying fascia. blood vessels that are actively bleeding should be ligated to control hemorrhage, if collateral circulation is present. if nerve bundles are ligated cleanly in a clean wound, the nerve edges should be reapposed and anastomosed. if gross contamination is present, however, definitive neurologic repair should be delayed until healthy tissue is present. excise contaminated muscle until healthy bleeding tissue is present. anastamoe tendon lacerations if the wound is clean and not grossly contaminated. if gross contamination is present, the tendon can be temporarily anastomosed and a splint placed on the limb until definitive repair of healthy tissue is possible. thoroughly lavage open wounds to a joint with sterile saline or lactated ringer's solution. infusion of chlorhexidine or povidone-iodine solution into the joint can cause a decrease in cartilage repair and is contraindicated. smooth sharp edges and remove any obvious fragments. whenever possible, the joint capsule and ligaments should be partially or completely closed. after removing bullets and metal fragments, the subcutaneous tissue and skin should be left open to heal by second intention, or should be partially closed with a drain. the joint should then be immobilized. injuries and exposed bone should be carefully lavaged, taking care to remove any gross debris without pushing the debris further into the bone and wound. the bone should be covered with a moist dressing and stabilized until definitive fracture repair can be made. this type of injury typically is seen with shearing injuries of the distal extremities caused by interaction with slow-moving vehicles. perform wet-to-dry or enzymatic debridement until a healthy granulation bed is present. if large areas of contamination are present (e.g., necrotizing fasciitis), en bloc debridement may be necessary. en bloc debridement consists of complete excision of badly infected wounds without entering the wound cavity, to prevent systemic infection. this technique should be used only if there is sufficient skin and soft tissue to allow later closure and it can be performed without damaging any major nerves, tendons, or blood vessels. open wounds often are managed by second intention healing, delayed primary closure, or secondary closure. see section on wound management and bandaging for a more complete discussion on the use of various bandaging materials in the treatment of open wounds. if an animal is presented very shortly after a wound has occurred and there is minimal contamination and trauma, the wound can be closed after induction of anesthesia and careful preparation of the wound and surrounding tissues. close any dead space under the skin with absorbable suture material in an interrupted suture pattern. avoid incising major blood vessels or nerves. close the subcutaneous tissues with absorbable suture material in an interrupted or continuous suture pattern. take care that there is not too much tension on the wound, or else surgical dehiscence will occur with patient movement. close the skin with nonabsorbable suture or surgical staples ( - to - ) . if there is any doubt at the time of repair about tissue status or inability to close all dead space, place a passive drain (penrose drain) so that the proximal end of the drain is anchored in the proximal aspect of the wound with a suture(s). leave the ends long so that the suture can be accurately identified at the time of drain removal. pass the suture through the skin, through the drain, and out the other side of the skin. place the rest of the drain into the wound and then secure it at the most ventral portion of the wound or exit hole in the most dependent area of the body, to allow drainage and prevent seroma formation. close the subcutaneous tissue over the drain before skin closure. during wound closure, be sure to not incorporate the subcutaneous or skin sutures into the drain, or it will not be possible to remove the drain without reopening the wound. bandage the area to prevent contamination. the drain can be removed once drainage is minimal (usually to days). active drains can be constructed or purchased; their use is indicated in wounds that are free of material that can plug the drain. to construct a small suction drain, remove the female portion or catheter hub at the end of a butterfly catheter. fenestrate the tubing so that there are multiple side holes, taking care to avoid making the holes larger than % of the circumference of the tubing. place the tubing into the wound via a small stab incision distal to the wound. use a purse-string suture around the tubing to facilitate a tight seal and prevent the tubing from exiting the wound. following wound closure, insert the butterfly needle into a -to -ml evacuated blood collection tube to allow fluid to drain into the tube. incorporate the tube into the bandage, and replace it when it becomes full. alternatively, the butterfly portion of the system can be removed and the tube fenestrated as described previously. place the tube into the wound and suture it in place to create a tight seal. secure the catheter hub to a syringe in which the plunger has been drawn back slightly to create suction. insert a metal pin or -to -gauge needle through the plunger at the top of the barrel to hold it at the desired level. incorporate the suction apparatus into the bandage and replace it when it becomes full. delayed primary closure should be considered when there is heavy contamination, purulent exudate, residual necrotic debris, skin tension, edema and erythema, and lymphangitis. delayed primary closure usually is made to days after the initial wound infliction and open wound management has been performed. once healthy tissue is observed, the skin edges should be debrided and the wound closed as with primary closure. secondary wound closure should be considered when infection and tissue trauma necessitate open wound management for more than days. secondary wound closure is performed after the development of a healthy granulation bed. this technique also is useful when a wound has dehisced and has formed granulation tissue. if the wound edges can be manipulated into apposition and if epithelialization has not begun, the wound can be cleansed and the wound edges apposed and sutured. this is known as early secondary closure. late secondary closure should be performed whenever there is a considerable amount of granulation tissue, the edges of the wound cannot be manipulated into position, and epithelialization has already started. in such cases, the wound should be cleaned, and the skin edges debrided to remove the epithelium. the remaining wound edges are then sutured over the granulation tissue ( shock is defined as a state of inadequate circulating volume and inability to meet cellular oxygen demands. there are three types of shock: hypovolemic, cardiogenic, and septic. early recognition of the type of shock present is crucial in the successful clinical management of shock syndrome. tissue oxygen delivery is based on cardiac output and arterial oxygen concentration. knowledge of the components of normal oxygen delivery is essential to the treatment of shock in the critical patient. improper handling of animal during further tissue and neurologic damage may occur transport (e.g., improper limb or spine immobilization). inadequate assessment of animal's animal's condition may worsen or animal may general condition or wounded tissues succumb; tissue injuries may be overlooked. inadequate wound protection during further wound contamination may occur at assessment, resuscitation, or veterinary facility. stabilization procedures inadequate wound protection while further wound contamination with fur and preparing the surrounding area debris may occur. insufficient wound lavage wound infection may occur. hydrogen peroxide wound lavage lavage offers little bactericidal activity and contributes to irritation of tissues and delayed healing. lavage has short residual activity and absorption with large wound. overly aggressive initial layered debridement may result in the removal of viable debridement tissue. en bloc debridement debridement results in removal of large amounts of tissue and a large defect for closure. use of drains potential exists for bacteria to ascend along the drain, for drain removal by the animal or breakage of the drain, and for possible tissue emphysema with air being sucked under the skin with patient movement. tube-type drains drains may cause postoperative discomfort; fenestrations may become occluded to stop intraluminal drainage. deeply placed sutures in the presence drain may be incorporated into the repair and of a drain prevent drain removal. active drains high negative pressure may cause tissue injury; highly productive wounds may necessitate changing the evacuated blood tubes several times a day with constructed drains. oxygen delivery (do ) = cardiac output (q) × arterial oxygen content (cao ) where q = heart rate × stroke volume. stroke volume is affected by preload, afterload, and cardiac contractility. where hb = hemoglobin concentration, sao = oxygen saturation, and pao = arterial partial pressure of oxygen in mm hg. thus, factors that can adversely affect oxygen delivery include inadequate preload or loss of circulating volume, severe peripheral vasoconstriction and increased afterload, depressed cardiac contractility, tachycardia and decreased diastolic filling, cardiac dysrhythmias, inadequate circulating hemoglobin, and inadequate oxygen saturation of hemoglobin. during septic shock, enzymatic dysfunction and decreased cellular uptake and utilization of oxygen also contribute to anaerobic glycolysis. an inadequate circulating volume may develop secondary to maldistribution of available blood volume (traumatic, septic, and cardiogenic origin) or as a result of absolute hypovolemia (whole blood or loss of extracellular fluid). normally, the animal compensates by ( ) splenic and vascular constriction to translocated blood from venous capacitance vessels to central arterial circulation, ( ) arteriolar constriction to help maintain diastolic blood pressure and tissue perfusion, and ( ) an increase in heart rate to help maintain cardiac output. arteriolar vasoconstrictions support perfusion to the brain and heart at the expense of other visceral organs. if vasoconstriction is severe enough to interfere with delivery of adequate tissue oxygen for a sufficient period of time, the animal may die. hypovolemic shock can result from acute hemorrhage or from severe fluid loss from vomiting, diarrhea, or third spacing of fluids. early in shock, baroreceptors in the carotid body and aortic arch sense a decrease in wall stretch from a decrease in circulating fluid volume. tonic inhibition of sympathetic tone via vagal stimulation is diminished, and heart rate and contractility increase and peripheral vessels constrict to compensate for the decrease in cardiac output. the compensatory mechanisms protect and support blood supply to the brain and heart at the expense of peripheral organ perfusion. this is called early compensatory shock. early compensatory shock is characterized by tachycardia, normal to fast capillary refill time, tachypnea, and normothermia. as shock progresses, the body loses its ability to compensate for ongoing fluid losses. early decompensatory shock is characterized by tachycardia, tachypnea, delayed capillary refill time, normotension to hypotension, and a fall in body temperature. end-stage decompensatory shock is characterized by bradycardia, markedly prolonged capillary refill time, hypothermia, and hypotension. aggressive treatment is necessary for any hope of a favorable outcome. septic shock should be considered in any patient with a known infection, recent instrumentation that could potentially introduce infection (indwelling intravenous or urinary catheter, surgery or penetrating injury), disorders or medical therapy that can compromise immune function (diabetes mellitus, immunodeficiency virus, parvovirus or feline panleukopenia virus infection, stress, malnutrition, glucocorticoids, chemotherapy). the presence of bacteria, viruses or rickettsiae, protozoa, or fungal organisms in the blood constitutes septicemia. septic shock is characterized by the presence of sepsis and refractory hypotension that is unresponsive to standard aggressive fluid therapy and inotropic or pressor support. septic shock and other causes of inflammation can lead to systemic inflammatory response syndrome (sirs). in animals, the presence of two or more of the criteria in table - in the presence of suspected inflammation or sepsis constitutes sirs (table - ). clinical signs associated with sepsis may be vague and nonspecific, including weakness, lethargy, vomiting, and diarrhea. cough and pulmonary crackles may be associated with pneumonia. decreased lung sounds may be associated with pyothorax. abdominal pain and fluid may be associated with septic peritonitis. vaginal discharge may or may not be present in patients with pyometra. diagnostic tests should include a white blood cell count, serum biochemical profile, coagulation tests, thoracic and abdominal radiographs, and urinalysis. the white blood cell count in a septic patient that is appropriately responding to the infection will be elevated with a left-shifted neutrophilia and leukocytosis. a degenerative left shift, in which leukopenia with elevated band neutrophils suggests an overwhelming infection. biochemical analyses may demonstrate hypoglycemia and nonspecific hepatocellular and cholestatic enzyme elevations. in the most severe cases, metabolic (lactic) acidosis, coagulopathies, and end-organ failure, including anuria and ards, may be present. cardiogenic shock occurs as a result of cardiac output inadequate to meet cellular oxygen demands. cardiogenic shock is associated with primary cardiomyopathies, cardiac dysrhythmias, pericardial fluid, and pericardial fibrosis. abnormalities seen on physical examination often are similar to those seen in other categories of shock, but they can also include cardiac murmurs, dysrhythmias, pulmonary rales, bloody frothy pulmonary edema fluid from the nares or mouth, orthopnea, and cyanosis. it is important to distinguish the primary cause of shock before implementing treatment (table - ) , whenever possible, because treatment for a suspected ruptured hemangiosarcoma differs markedly from the treatment for end-stage dilatative cardiomyopathy. the patient's clinical signs may be similar and include a peritoneal fluid wave, but the treatment for hypovolemia can dramatically worsen the congestive heart failure secondary to dilatative cardiomyopathy. when a patient presents with some form of shock, immediate vascular access is of paramount importance. place a large-bore peripheral or central venous catheter for the infusion of crystalloid or colloid fluids, blood component therapy, and drugs. monitor the patient's cardiopulmonary status (by ecg), blood pressure, oxygen saturation (as determined by pulse oximetry or arterial blood gas analyses), hematocrit, bun, and glucose. ancillary diagnostics, including thoracic and abdominal radiography, urinalysis, serum biochemistry profile, coagulation tests, complete blood count, abdominal ultrasound, and echocardiography, should be performed as determined by the individual patient's needs and the type of shock. the following list, called the "rule of twenty," is a guideline for case management of the shock patient. consideration of each aspect of the rule of twenty on a daily basis ensures temperature < °f or > . °f < °f or > . °f heart rate > beats/minute in dogs < or > beats/minute in cats respiratory rate > breaths/minute or paco > breaths/minute or paco < mm hg < mm hg white blood cell > , cells/µl , cells/µl count or < cells/µl o r < cells/ml or > % bands or > % bands that major organ systems are not overlooked. the list also provides a means to integrate and relate changes in different organ systems functions with one another.* the treatment of hypovolemic and septic shock requires the placement of large-bore intravenous catheters in peripheral and central veins. if vascular access cannot be obtained percutaneously or by cutdown methods, intraosseous catheterization should be considered. once vascular access is achieved, rapidly administer large volumes of crystalloid or colloid fluids. as a rule of thumb, administer / of a calculated shock dose of fluids-that is, / × ( ml/kg/hour) in dogs and / × ( ml/kg/hour) in cats) of a balanced crystalloid fluid ( normosol-r, plasmalyte-m, lactated ringer's solution, or . % sterile saline). reassess the patient's perfusion parameters (heart rate, capillary refill time, blood pressure, urine output) on a continual basis to direct further fluid therapy. synthetic colloid fluids (hetastarch, dextran , or oxyglobin) can also be administered in the initial resuscitation from shock. a guideline is to administer to ml/kg of hetastarch or dextran as a bolus over to minutes and then reassess perfusion parameters. hypertonic saline ( . % nacl, ml/kg) can be used in cases of hemorrhagic shock to temporarily restore intravascular fluid volume by drawing fluid from the interstitial space. because this type of fluid resuscitation is short-lived, hypertonic saline should always be used with another crystalloid or colloid fluid, and it should not be used in patients with interstitial dehydration. if hemorrhagic shock is present, the goal should be to return a patient's blood pressure to normal (not supraphysiologic) levels (i.e., systolic pressure - mm hg, diastolic pressure > mm hg, and mean arterial pressure ≥ mm hg) to avoid iatrogenically causing clots to fall off and hemorrhage to re-start. in critically ill patients, fluid loss can be measured in the form of urine, vomit, diarrhea, body cavity effusions, and wound exudates. additionally, insensible losses (those that cannot be readily measured from sweat, panting, and cellular metabolism) constitute ml/kg/ day. measurement of fluid "ins and outs" in conjunction with the patient's central venous pressure, hematocrit, albumin, and colloid oncotic pressure can help guide fluid therapy (see also section on fluid therapy). maintenance of normotension is necessary for adequate oxygen delivery to meet cellular energy demands. blood pressure can be measured using direct arterial catheterization, or through indirect means such as doppler plesthymography or oscillometric methods. the systolic pressure should remain at or greater than - mm hg at all times. the diastolic pressure is very important, too, as it constitutes two thirds of the mean arterial pressure; it must be greater than mm hg for coronary artery perfusion. the mean arterial pressure should be greater than mm hg for adequate tissue perfusion. if fluid resuscitation and pain management are not adequate in restoring blood pressure to normal, vasoactive drugs including positive inotropes and pressors should be considered (table - ). in cases of cardiogenic shock, vasodilator drugs (table - ) can be used to decrease vascular resistance and afterload. low-dose morphine ( . mg/kg, iv, im) dilates splanchnic vessels and helps reduce pulmonary edema. furosemide ( mg/kg/hour) also can dilate pulmonary vasculature and potentially reduce edema fluid formation in cases of ards. cardiac output is a function of both heart rate and stroke volume. stroke volume or (the amount of blood that the ventricle pumps in minute) is affected by preload, afterload, and contractility. during hypovolemic shock, there is a fall in cardiac preload due to a decrease in circulating blood volume. during septic and cardiogenic shock, there is a decrease in contractility secondary to inherent defects of the myocardium or due to the negative inotropic effects of inflammatory cytokines such as tnf-alpha, myocardial depressant factor, il- , and il- released during sepsis and systemic inflammation. afterload also may be increased because of the compensatory mechanisms and neurohumoral activation of the renin-angiotensin-aldosterone axis in hypovolemic or cardiogenic shock. as heart rate increases to compensate for a decline in cardiac output, myocardial oxygen demand increases and diastolic filling time becomes shorter. because the coronary arteries are perfused during diastole, coronary perfusion can be impaired, and myocardial lactic acidosis can develop, causing a further decline in contractility. in addition to lactic acidosis, acid-base and electrolyte abnormalities, inflammatory cytokines, direct bruising of the myocardium from trauma, and areas of ischemia can further predispose the patient to ventricular or atrial dysrhythmias. cardiac dysrhythmias should be controlled whenever possible. treatment of bradycardia should be directed at treating the underlying cause. administer anticholinergic drugs such as atropine ( . mg/kg im) or glycopyrrolate ( . mg/kg im) as necessary. in cases of third-degree or complete atrioventricular (av) block, administer a pure betaagonist such as isoproterenol ( . - . µg/kg/minute iv cri, or . mg in ml of % dextrose in water iv slowly). perform passive rewarming if the patient is hypothermic. receptor activity dosage (iv) dopamine da , da , α +++ , - µg/kg/minute (blood pressure support)* β +++ - µg/kg/minute (renal afferent diuresis) dobutamine α + , β +++ - µg/kg/minute* (blood pressure support, positive inotrope) norepinephrine α +++ , β + . - . mg/kg/minute; . - . mg/kg phenylephrine α +++ , β . - . mg/kg epinephrine α +++ , β +++ . - . mg/kg, . - . mg/kg/minute +++, strong receptor activity; , no receptor activity; +, weak receptor activity. *monitor for tachyarrhythmias at higher doses. correct any underlying electrolyte abnormalities such as hyperkalemia and hypo-and hypermagnesemia. treat ventricular dysrhythmias such as multifocal premature ventricular contractions (pvcs), sustained ventricular tachycardia > beats per minute, and r on t phenomenon (the t wave of the preceding beat occurs superimposed on the qrs complex of the next beat, and there is no return to isoelectric shelf), or if runs of ventricular tachycardia cause a drop in blood pressure. intravenous lidocaine and procainamide are the first drugs of choice for ventricular dysrhythmias. supraventricular tachycardia can impair cardiac output by impairing diastolic filling time. control supraventricular dysrhythmias with calcium channel blockers, beta-adrenergic blockers, or quinidine (table - ) . (disorientation); is minute; minutes) light sensitive and must be covered in foil and not kept for longer than hours albumin can decrease as a result of loss from the gastrointestinal tract, urinary system, and wound exudates, or into body cavity effusions. albumin synthesis can decrease during various forms of shock due to a preferential increase in hepatic acute phase protein synthesis. serum albumin contributes % of the colloid oncotic pressure of blood, in addition to its important roles as a free radical scavenger at sites of inflammation and as a drug and hormone carrier. albumin levels < . g/dl have been associated with an increase in morbidity and mortality in human and veterinary patients. administer fresh frozen plasma ( ml/kg) or concentrated human albumin ( ml/kg of % solution) to maintain serum albumin ≥ . g/dl. additional oncotic support can be in the form of synthetic colloids, as indicated. colloid oncotic pressure within the intravascular and interstitial spaces contributes to fluid flux. oncotic pressure can be measured with a colloid osmometer. normal oncotic pressure is mm hg. in cases of sepsis and sirs, increased vascular permeability increases the tendency for leakage of fluids into the interstitial spaces. colloids that can be administered until the source of albumin loss resolves include the synthetic colloids hetastarch and dextran ( - ml/kg/day), synthetic hemoglobin-based oxygen carriers (oxyglobin, - ml/kg/day), concentrated human albumin ( % albumin, ml/kg), and plasma ( ml/kg). oxygenation and ventilation can be evaluated by arterial blood gas analysis or by the noninvasive means of pulse oximetry and capnometry (see sections on pulse oximetry and capnometry). oxygen delivery can be impaired in cases of hypovolemic shock because of hemorrhage and anemia, and thus a decrease in functional capacity to carry oxygen, and is not to be used for more than weeks due to idiosyncratic blindness. in cases of cardiogenic shock as a result of impaired ability to saturate hemoglobin due to pulmonary edema in the lungs, or decrease in cardiac output. in septic shock, decreases in cardiac output due to inflammatory cytokines and a decrease in cellular oxygen extraction can lead to lactic acidosis. increased cellular metabolism and decreases in respiratory function can lead to respiratory acidosis as co increases. administer supplemental oxygen as flow-by, nasal or nasopharyngeal catheter, oxygen hood, or oxygen cage. supplemental oxygen should be humidified, and delivered at - ml/kg/minute. if oxygenation and ventilation are so impaired that the pao remains < mm hg with the patient on supplemental oxygen, a paco > mm hg, or severe respiratory fatigue, develops, and mechanical ventilation should be considered. glucose is a necessary fuel source for red blood cells and neuronal tissues, and serum glucose should be maintained within normal reference ranges. glucose supplementation can be administered as . - % solutions in crystalloid fluids, or in parenteral and enteral nutrition products. arterial and venous ph can be measured by performing blood gas analyses. decrease in tissue perfusion, impaired oxygen delivery, and decreased oxygen extraction in the various forms of shock can lead to anaerobic metabolism and metabolic acidosis. in most cases, improving tissue perfusion and oxygen delivery with crystalloid and colloid fluids, supplemental oxygen, and inotropic drugs will help normalize metabolic acidosis. serial measurements of serum lactate (normal, < . mmol/l) can be used as a guide to evaluate the tissue response to fluid resuscitative efforts. serum electrolytes often become severely deranged in shock states. serum potassium, magnesium, sodium, chloride, and total and ionized calcium should be maintained within normal reference ranges. if metabolic acidosis is severe, sodium bicarbonate can be administered by calculating the formula base deficit × . × body weight in kg = meq bicarbonate to administer because iatrogenic metabolic alkalosis can occur, a conservative approach is to administer / of the calculated dose and then recheck the patient's ph and bicarbonate levels. if the base excess is unknown, sodium bicarbonate can be administered in incremental doses of meq/kg until the ph is above . . complications associated with bicarbonate therapy include iatrogenic hypocalcemia, metabolic alkalosis, paradoxical cerebrospinal fluid acidosis, hypotension, restlessness, and death. massive trauma, neoplasia, sepsis, and systemic inflammation can all lead to coagulation abnormalities, including disseminated intravascular coagulation (dic). cage-side coagulation monitors are available for daily measurement of prothrombin time (pt), activated partial thromboplastin time (aptt), and platelet counts. fibrin degradation products (fibrin split products) become elevated in dic, trauma, hepatic disease, and surgery. coagulation proteins (clotting factors) and antithrombin often are lost with other proteins in hypoproteinemia or are consumed when microclots are formed and then dissolved. antithrombin levels can be measured by commercial laboratories. antithrombin and clotting factors can be replenished in the form of fresh frozen plasma transfusions. a more sensitive and specific test for dic is the detection of d-dimers, which can be measured by commercial laboratories. treatment for dic involves treatment and resolution of the underlying disease and administration of antithrombin and clotting factors in the form of fresh frozen plasma ( ml/kg) and heparin (unfractionated, - units/kg sq tid; fractionated [lovenox], mg/kg sq bid). monitor the patient for changes in mental status, including stupor, coma, decreased ability to swallow and protect the airway, and seizures. elevation of the patient's head can help to protect the airway and decrease the risk of increased intracranial pressure. serum glucose should be maintained within normal levels to prevent hypoglycemia-induced seizures. one of the major components of oxygen delivery is the binding to hemoglobin. packed cell volume must be kept above - % for adequate cellular oxygen delivery. acid-base status can adversely affect oxygen offloading at the tissue level if metabolic or respiratory alkalosis is present. oxygen-carrying capacity and hemoglobin levels can be increased with administration of rbc component therapy or with hemoglobin-based oxygen carriers. monitoring of renal function includes daily measurement of bun, creatinine, and urine output. normal urine output in a hydrated euvolemic patient is - ml/kg/hour. fluid ins and outs should be measured in cases of suspected oliguria or anuria. in patients with oliguria or anuria, furosemide can be administered as a bolus ( - mg/kg) or by constant rate infusion (cri)( . - mg/kg/hour). mannitol should also be administered ( . - g/kg over to minutes). dopamine ( - µg/kg/minute cri) can be administered to dilate renal afferent vessels and improve urine output. the patient's white blood cell count may be elevated, normal, or decreased, depending on the type of shock. the decision to administer antibiotics should be made on a daily basis. superficial or deep staphylococcus or streptococcus infection usually can be treated with a first-generation cephalosporin (cefazolin, mg/kg iv tid). if a known source of infection is present, administer a broad-spectrum antibiotic (cefoxitin, mg/kg iv tid; ampicillin, mg/kg qid, or enrofloxacin, - mg/kg once daily) pending results of culture and susceptibility testing. if broader anaerobic coverage is required, metronidazole ( mg/kg iv tid) should be considered. gentamicin ( - mg/kg iv once daily) is a good choice for gram-negative sepsis, provided that the patient is well hydrated and has normal renal function. ideally, patients receiving any aminoglycoside antibiotic should have a daily urinalysis to check for renal tubular casts that signify renal damage. in dogs, the gut is the shock organ. impaired gastrointestinal motility and vomiting should aggressively be treated with antiemetics and promotility drugs (dolasetron, . mg/kg iv once daily, and metoclopramide, - mg/kg/day iv cri). metoclopramide is contraindicated in cases of suspected gastrointestinal obstruction. histamine-receptor blockers such as famotidine ( . mg/kg bid iv) and ranitidine ( . to mg/kg iv bid, tid) or proton-pump inhibitors (omeprazole, . - mg/kg po once daily) can be administered for esophagitis. administer sucralfate ( . - g po tid) to treat gastric ulceration. if the gastrointestinal barrier function is diminished due to poor perfusion, infection, or inflammation, administer broad-spectrum antibiotics such as ampicillin ( mg/kg iv qid) to prevent gastrointestinal bacterial translocation. the course of drug therapy should be reviewd daily and the patient should be monitored for potential drug interactions. for example, metoclopramide and dopamine, working at the same receptor, can effectively negate the effects of each other. cimetidine, a cytochrome p enzyme inhibitor, can decrease the metabolism of some drugs. drugs that are avidly protein-bound may have an increase in unbound fraction with concurrent hypoalbuminemia or when hypoalbuminemia is present. decreased renal function may impair the renal clearance of some drugs, requiring increased dosing interval or decreased dose. nutrition is of utmost importance in any critically ill patient. patients with septic shock may become hypermetabolic and require supraphysiologic nutrient caloric requirements, while others may actually become hypometabolic. enteral nutrition is preferred, whenever possible, because enterocytes undergo atrophy without luminal nutrient stimulation. a variety of enteral feeding tubes can be placed, depending on what portion of the gut is functional, to provide enteral nutrition in an inappetent patient. loss of gastrointestinal mucosal barrier function may predispose the patients to the development of bacterial translocation and may contribute to sepsis. if enteral nutrition is impossible because of protracted vomiting or gastrointestinal resection, glucose, lipid, and amino acid products are available that can be administered parenterally to meet nutrient needs until the gastrointestinal tract is functioning and the patient can be transitioned to enteral nutrition. assessment of pain in animals in shock can be challenging. pain can result in the release of catecholamines and glucocounterregulatory hormones that can impair nutrient assimilation and lead to negative nitrogen balance, impaired wound healing, and immunocompromise. in any animal determined to be in pain, analgesic drugs should be administered to control pain and discomfort at all times. opioids are cardiovascularly friendly, and their effects can easily be reversed with naloxone if adverse effects such as hypotension and hypoventilation occur. if the patient is nonambulatory, rotate the animal from side to side every to hours to prevent lung atelectasis. passive range-of-motion exercises and deep muscle massage should be performed to increase tissue perfusion, decrease dependent edema, and prevent disuse atrophy. animals should be kept completely dry on soft, padded bedding to prevent the development of decubital ulcers. all bandages, wound sites, and catheter sites should be checked daily for the presence of swelling, erythema, and pain. soiled bandages should be changed to prevent strike-through and contamination of the underlying catheter or wound. hospitalization can be a stressful experience for patient and client alike. allowing brief visits and walks outside in the fresh air can improve a patient's temperament and decrease stress. the preemptive use of analgesic drugs on a regular schedule (not prn) should be used to prevent pain before it occurs. pain decreases the patient's ability to sleep. lack of sleep can promote further stress and impaired wound healing. the use of glucocorticosteroids and antiprostaglandins in shock therapy remains a topic of wide controversy. although the use of these agents potentially may stabilize membranes, decrease the absorption of endotoxin, and decrease prostaglandin release, the routine use of glucocorticosteroids and antiprostaglandins can decrease renal perfusion and gastrointestinal blood flow, promoting gastrointestinal ulceration and impaired renal function. the administration of supraphysiologic levels of glucocorticosteroids in patients in any type of shock can increase sodium and water retention, depress cellular immune function, and impair wound healing. in clinical studies of small animal patients, the routine use of glucocorticosteroids and antiprostaglandins has not demonstrated definite improved survival. the risks of therapy do outweigh the anecdotal reported benefits, and therefore the empiric use of glucocorticosteroids and antiprostaglandins in any shock patient is urinary tract emergencies azotemia azotemia occurs when % or more of the nephrons are nonfunctional. the magnitude of the azotemia alone cannot be used to determine whether the azotemia is prerenal, renal, or postrenal in origin, or whether the disease process is acute or chronic, reversible or irreversible, progressive or nonprogressive. before beginning treatment for azotemia, the location or cause of the azotemia must be identified. take a thorough history and then perform a physical examination. obtain blood and urine samples before initiating fluid therapy, for accurate assessment of the location of the azotemia. for example, an azotemic animal with a history of vomiting and diarrhea that appears clinically dehydrated on physical examination, normally should have a concentrated urine specific gravity (> . ) reflecting the attempt to conserve fluid. if this level is found, the azotemia is much less likely to be renal in origin, and the azotemia will likely resolve after rehydration. if, however, the urine specific gravity is isosthenuric or hyposthenuric ( . - . ) in the presence of azotemia and dehydration, primary intrinsic renal insufficiency is likely present. if the azotemia resolves with fluid therapy, the patient has prerenal and primary renal disease. if the azotemia does not resolve after rehydration, the patient has prerenal and primary renal failure. dogs with hypoadrenocorticism can have both prerenal and primary renal disease secondary to the lack of mineralocorticoid (aldosterone) influence on the renal collecting duct and renal interstitial medullary gradient. medullary washout can occur, causing isosthenuric urine in the presence of dehydration from vomiting and diarrhea. the patient often has azotemia due to fluid loss (dehydration and urinary loss) and gastric or intestinal hemorrhage (elevated bun). the prerenal component will resolve with treatment with glucocorticoids and crystalloid fluids, but the renal component may take several weeks to resolve, until the medullary concentration gradient is reestablished with the treatment and influence of mineralocorticoids. drugs such as corticosteroids and diuretics can influence renal tubular uptake and excretion of fluid, and cause a prerenal azotemia and isosthenuric urine in the absence of primary renal disease. treatment of azotemia includes calculation of the patient's dehydration estimate and maintenance fluid volumes, and administering that volume over the course of hours. identify and treat underlying causes of prerenal azotemia (shock, vomiting, diarrhea). monitor urine output closely. once a patient is euvolemic, oliguria is defined as urine output < - ml/kg/hour. urine output should return to normal in patients with prerenal azotemia as rehydration occurs. if a patient remains oliguric after rehydration, consider the possibility of oliguric acute intrinsic renal failure, and administer additional fluid therapy based on the patient's urine output, body weight, central venous pressure, and response to other medical therapies. prerenal azotemia is caused by conditions that decrease renal perfusion, including hypovolemic shock, severe dehydration, hypoadrenocorticism, congestive heart failure, cardiac tamponade, cardiac dysrhythmias, and hypotension. once renal perfusion is restored, the kidneys can resume normal function. glomerular filtration rate decreases when the mean arterial blood pressure falls to less than mm hg in a patient with normal renal autoregulation. renal autoregulation can be impaired in some diseases. passive reabsorption of urea from the renal tubules can occur during states of low tubular flow (dehydration, hypotension) even if glomerular filtration is not decreased. if renal hypoperfusion is not quickly restored, the condition can progress from prerenal disease to acute intrinsic renal failure. prerenal and renal azotemia can coexist in animals with primary renal disease, as a result of vomiting and ongoing polyuria in the absence of any oral fluid intake. the treatment of prerenal azotemia consists of rehydration, antiemetic therapy, and treatment of the underlying cause of vomiting, diarrhea, or third spacing of fluids. acute intrinsic renal failure is characterized by an abrupt decline in renal function to the extent that azotemia and an inability to regulate solute and fluid balance. patients with acute intrinsic renal failure may be oliguric or polyuric, depending on the cause and state of renal failure. in small animals, the most common causes of acute intrinsic renal failure are renal ischemia and toxins. there are three phases of acute intrinsic renal failure: induction, maintenance, and recovery. during the induction phase, some insult (ischemia or toxin) to the kidneys occurs, leading to a defective concentrating mechanism, decreased renal clearance of nitrogenous waste (azotemia), and polyuria or oliguria. if treatment is initiated during the induction phase, progression to the maintenance phase potentially can be stopped. as the induction phase progresses, there is worsening of the urine-concentrating ability and azotemia. renal tubular epithelial cells and renal tubular casts can be seen on examination of the urine sediment. glucosuria may be present. the maintenance phase of acute intrinsic renal failure occurs after a critical amount of irreversible nephron injury. correction of the azotemia and removal of the cause of the problem do not result in return to normal function. in patients with oliguria, the extent of nephron damage is greater than that observed in patients with polyuria. the maintenance phase may last for several weeks to months. recovery of renal function may or may not occur, depending on the extent of injury. the most serious complications (overhydration and hyperkalemia) are observed in patients with oliguria. the recovery phase occurs with sufficient healing of damaged nephrons. azotemia may resolve, but concentrating defects may remain. if the patient was oliguric in the maintenance phase, a marked diuresis develops during the recovery phase that may be accompanied by fluid and electrolyte losses. this phase may last for weeks to months. treatment of acute intrinsic renal failure consists of determining the cause and ruling out obstruction or uroabdomen whenever possible. a careful history can sometimes determine whether there has been exposure to nephrotoxic drugs, chemicals, or food items. if ingestion or exposure to a toxic drug, chemical, or food occurred recently (within to hours), induce emesis with apomorphine ( . mg/kg iv). next, administer activated charcoal either orally or via stomach tube, to prevent further absorption of the toxin. obtain blood and urine samples for toxicologic analysis (e.g., ethylene glycol) and to determine whether azotemia or abnormalities in the urine sediment exist. (see section on ethylene glycol, grapes and raisins, and nonsteroidal antiinflammatory drugs). obtain a complete blood count, biochemical profile, and urinalysis to determine the presence of signs of chronic renal failure, including polyuria, polydipsia, and nonregenerative anemia. radiographs and abdominal ultrasound can help in determining the chronicity of renal failure. normal renal size is . - . times the length of l in dogs and . - . times the length of l in cats. monitor the patient's body weight at least twice a day to avoid overhydration. also monitor urine output; normal output is - ml/kg/hour. in cases of polyuric renal failure, massive fluid and electrolyte losses can occur. place a urinary catheter for patient cleanliness and to facilitate urine quantitation. measure fluid ins and outs (see section on fluid therapy). after the patient has been rehydrated, the amount of fluids administered should equal maintenance and insensible needs plus the volume of urine produced each day. if a urinary catheter cannot be placed or maintained, serial body weight measurements and central venous pressure should be used to monitor the patient's fluid balance and prevent overhydration. if the patient is oliguric (urine output < - ml/kg/hour), pharmacologic intervention is necessary to increase urine output. first, administer furosemide ( - mg/kg or . mg/kg/hour iv cri). repeat bolus doses of furosemide if there is no response to initial treatment. if necessary, administer low-dose dopamine ( - µg/kg/minute iv cri) to increase renal afferent dilatation and renal perfusion. dopamine and furosemide may be synergistic if administered together. if dopamine and furosemide therapy is ineffective, administer mannitol ( . - . g/kg iv) once only. if polyuria is present, management is simplified because of the decreased risk of overhydration. if oliguria cannot be reversed, monitor the central venous pessure, body weight, and respiratory rate and effort, auscultate for crackles, and examine the patient carefully for signs of chemosis and the presence of serous nasal discharge. correct hyperkalemia with sodium bicarbonate ( . - . meq/kg iv) or with insulin ( . units/kg) plus dextrose ( g/unit of insulin iv, followed by . % dextrose iv cri). treat severe metabolic acidosis (ph < . or hco − < meq/l) with sodium bicarbonate. if anuria develops or oliguria is irreversible despite this therapy, begin peritoneal dialysis. obtain a renal biopsy to establish a diagnosis and prognosis (see section on renal biopsy). administer gastroprotectant drugs and antiemetics to control nausea and vomiting. if possible, avoid the use of nephrotoxic drugs and general anesthesia. initiate nutritional support in the form of an enteral feeding tube or parenteral nutrition as early as possible. once the patient enters the recovery phase, diuresis may occur that can lead to dehydration and electrolyte imbalances (hyponatremia, hypokalemia). dehydration and electrolyte imbalances can be treated with parenteral fluid and electrolyte supplementation. postrenal azotemia is primarily caused by urethral obstruction or leakage from the urinary tract into the abdomen (uroabdomen). complete urinary tract obstruction and uroabdomen are both ultimately fatal within to days if left untreated. in dogs, the most common causes of urethral obstruction are urinary (urethral) calculi or tumors of the urinary bladder or urethra. in male cats, feline urologic syndrome (fus) is the most common cause of urethral obstruction, although there has been an increased incidence of urethral calculi observed in recent years. a ruptured urinary bladder is the most common cause of uroabdomen and is usually secondary to blunt trauma. clinical signs of urinary tract obstruction include dysuria, hematuria, inability to urinate or initiate an adequate stream of urine, and a distended painful urinary bladder. late in the course of obstructive disease, clinical signs referable to uremia and azotemia (vomiting, oral ulcers, hematemesis, dehydration, lethargy, and anorexia) occur. the initial goal of treatment of urinary tract obstruction is to relieve the obstruction. in male dogs, a lubricated catheter can be inserted past the area of obstruction with the animal under heavy sedation or general anesthesia (see section on urohydropulsion). depending on the chronicity of the obstruction, serum electrolytes should be measured;an ecg should be obtained before administering any anesthetic drugs, because of the cardiotoxic effects of hyperkalemia (see section on atrial standstill). correct fluid, electrolyte, and acid-base abnormalities. if a urinary catheter cannot be placed, perform cystocentesis only as a last resort, because of the risk of urinary bladder rupture. definitive treatment includes identification and treatment of the underlying cause (tumor versus urinary calculi). in most cases, surgical intervention is necessary. if an unresectable tumor is present, a low-profile permanent cystostomy tube can be placed, if the owner desires. administration of piroxicam (feldene, . mg/kg po q - h) with or without chemotherapy may shrink the tumor mass and delay the progression of clinical signs. a complete discussion of this disorder is beyond the scope of this text (see additional reading for other sources of information). feline lower urinary tract disease can cause urethral obstruction, particularly in male cats. clinical signs include stranguria, dribbling of small amounts of urine, lethargy, inappetence, and vomiting. often, owners call with the primary complaint of constipation, because the cat is making frequent trips to the litterbox and straining. cases with a duration of obstruction < hours are considered uncomplicated; those with a duration > hours are complicated. treatment of urethral obstruction includes stabilizing and normalizing the patient's electrolyte status, induction of sedation or general anesthesia, and relieving the obstruction. obtain blood samples for analysis of electrolyte abnormalities. treat hyperkalemia (k + > . meq/l) with sodium bicarbonate ( . - . meq/kg iv), regular insulin ( . unit/ kg iv) plus dextrose ( g//unit of insulin iv), followed by . % dextrose iv cri to prevent hypoglycemia; or calcium gluconate ( . ml/kg % iv slowly). administer non-potassiumcontaining intravenous fluids in . % saline solution. obtain an ecg to detect atrial standstill (see section on atrial standstill). in some cases, a urethral plug is visible at the tip of the penis. the urethral plug can sometimes be manually extracted or massaged from the penis, and the obstruction temporarily relieved. in such cases, it is still necessary to pass a urethral catheter to flush sediment from the urethra and urinary bladder. unless a patient is obtunded, administer an anesthetic such as ketamine, atropine, or propofol ( - mg/kg iv) with diazepam iv for patient comfort and muscle relaxation. once the patient is under anesthesia or heavily sedated, urinary catheterization should be performed. in some cases, it will be difficult to advance the catheter. lubricate a closedended tomcat catheter and pass the tip into the distal urethra. fill a -ml syringe with sterile saline and sterile lubricant and connect the syringe to the hub of the catheter. pulse the fluid into the catheter as you gently move the catheter tip back and forth against the urethral obstruction. when the catheter has been passed into the urinary bladder, obtain a urine sample for urinalysis. drain the bladder and flush with sterile saline solution until the urine efflux appears clear. remove the tomcat catheter and insert a - fr red rubber tube or argyle infant feeding catheter into the urethra for urine collection and quantitation. secure the urinary catheter to prepuce with a butterfly strip of -inch adhesive tape secured around the catheter and then sutured to either side of the prepuce. the catheter should be connected to a closed urinary collection system for cleanliness and to reduce the risk of ascending bacterial infection. an elizabethan collar should be placed at all times to prevent the patient from damaging or removing the catheter. when the urethral obstruction has been relieved and the catheter placed, continue intravenous fluid diuresis to alleviate postrenal azotemia. monitor the urine for bacteria and other sediment. in some cases, postobstructive diuresis can be severe. carefully monitor fluid ins and outs, along with body weight, to maintain adequate hydration and perfusion. remove the urinary catheter can be removed after to hours. palpate the bladder frequently to make sure that the patient is voiding normally and to detect the recurrence of obstruction. in patients with severe penile or urethral trauma or edema, administer a short-acting steroid (dexamethasone sodium phosphate, . mg/kg iv, im, sq). at the time of initial diagnosis and again at the time of discharge, the clients need to be instructed about the long-term management of feline lower urinary tract disease at home, and informed of the risks and consequences of recurrence. uroabdomen can occur from trauma or leakage from the kidneys, ureter, or urinary bladder. clinical signs of uroabdomen (azotemia, uremia, hyperkalemia) can also occur secondary to third spacing of urine and leakage into muscular tissue from a ruptured urethra. in most cases, urinary bladder trauma and rupture are secondary to blunt trauma. abdominocentesis should be performed in any animal with suspected blunt abdominal trauma, and any fluid obtained should be analyzed for creatinine or potassium and compared with the patient's serum levels. an abdominal effusion that has a low packed cell volume and a potassium or creatinine level greater than that of the patient's serum is consistent with the diagnosis of uroabdomen. uroabdomen is not a surgical emergency. however, medical management consists of placement of a temporary abdominal drainage catheter into the abdomen, to facilitate removal of urine from the peritoneal cavity. to place the catheter, position the patient in dorsal or lateral recumbency, shave the ventral abdomen, as for any exploratory laparotomy. aseptically scrub the clipped area, and instill a local anesthestic (lidocaine, - mg/kg) caudal and to the right of the umbilicus, through the skin, subcutaneous tissues, and rectus emergency care clinical differentiation of acute necrotizing from chronic nonsuppurative pancreatitis in cats: cases acute pancreatitis in dogs mesenteric volvulus in the dog: a retrospective study of cases incidence and prognostic value of low plasma ionized calcium concentration in cats with pancreatitis: cases ( - ) review of feline pancreatitis. part : clinical signs, diagnosis and treatment gastric dilatation-volvulus syndrome in dogs diagnostic approach to acute pancreatitis pathophysiology of organ failure in severe acute pancreatitis in dogs washabau rj: gastrointestinal motility disorders and gastrointestinal prokinetic therapy watson pt: exocrine pancreatic insufficiency as an end-stage of pancreatitis in dogs clinical signs, underlying cause, and outcome in cats with seizures: cases fibrocartilaginous embolism in dogs: clinical findings and factors influencing the recovery rate kirk's current veterinary therapy xiii intervertebral disc extrusion in six cats medical management of acute spinal cord disease risk factors for recurrence of clinical signs associated with thoracolumbar intervertebral disk herniation in dogs: cases intervertebral disk disease in cats long-term functional outcome of dogs with severe injuries of the thoracolumbar spinal cord: cases canine status epilepticus: a retrospective study of cases risk factors for development of status epilepticus in dogs with idiopathic epilepsy and effects of status epilepticus on outcome and survival time: cases ( - ) skills laboratory part i: performing a neurologic examination skills laboratory part ii: interpreting the results of the neurologic examination accuracy of localization of cervical intervertebral disk extrusion or protrusion using survey radiography in dogs medical and surgical management of the glaucoma patient the feline glaucomas: cases ( - ) the canine glaucomas traumatic ocular protrusion in dogs and cats: cases traumatic glaucoma in a dog ocular and orbital porcupine quills in the dog: a review and case series hyphema: pathophysiologic considerations. comp cont educ pract vet van der woerdt a: the treatment of acute glaucoma in dogs and cats administer crystalloid intravenous fluids at maintenance rates using a balanced electrolyte solution perform urinary catheterization and collection to monitor urine output monitor serum urea nitrogen and creatinine every hours treat oliguria, defined as a drop in urine output to less than ml/kg/hour ml/kg) bolus start dopamine at to µg/kg/minute if no response to crystalloid/colloid bolus occurs within minutes consider mannitol ( . to g/kg iv) administration if no response to dopamine occurs within minutes consider furosemide ( to mg/kg iv, or . to mg/kg/hour iv cri) if no response to dopamine or mannitol occurs in to minutes if no response to furosemide, peritoneal dialysis or hemodialysis is indicated immediately, particularly if anuria is present administered with caution, because of the risk of exacerbating increased capillary permeability and causing pulmonary edema. animal patients. chlorphenoxy derivatives exert their toxic effects by an unknown mechanism, and cause clinical signs of gastroenteritis and muscle rigidity severe anemia should be treated with packed rbcs or hemoglobin-based oxygen carriers handbook of small animal toxicology and poisonings macadamia nut toxicosis in dogs the recognition and treatment of the intermediate syndrome of organophosphate poisoning in a dog acute renal failure in four dogs after raisin or grape ingestion pleural effusion in cats pulmonary function, ventilator management, and outcome of dogs with thoracic trauma and pulmonary contusions: cases ( - ) acute lung injury and acute respiratory distress syndrome smoke exposure in cats: cases ( - ) smoke exposure in dogs: cases ( - ) thoracic duct ligation and pericardectomy for treatment of idiopathic chylothorax use of intraluminal nitinol stents in the treatment of tracheal collapse in a dog clinical approach to epistaxis the veterinary icu book. teton newmedia radiographic diagnosis of diaphragmatic hernia: review of cases in dogs and cats tracheal collapse: diagnosis and medical and surgical management acute respiratory distress syndrome brachycephalic syndrome in dogs outcome and postoperative complications in dogs undergoing surgical treatment of laryngeal paralysis: cases ( - ) full recovery following delayed neurologic signs after smoke inhalation in a dog aspiration pneumonitis the veterinary icu book. teton newmedia allergic airway disease canine pleural and mediastinal effusion, a retrospective study of cases suggested strategies for ventilatory management in veterinary patients with acute respiratory distress syndrome laryngeal and tracheal disorders the veterinary icu book. teton newmedia medical and surgical treatment of pyothorax in dogs: cases traumatic diaphragmatic hernia in cats: cases canine pyothorax: clinical presentation, diagnosis, and treatment canine pyothorax: pleural anatomy and pathophysiology treatment of chronic pleural effusion with pleuroperitoneal shunt in dogs: cases ( - ) effects of doxapram hydrochloride on laryngeal function of normal dogs and dogs with naturally occurring laryngeal paralysis an overview of positive pressure ventilation risk factors, prognostic indicators, and outcome of pyothorax in cats: cases ( - ) use of percutaneous arterial embolization for the treatment of intractable epistaxis in dogs systemic inflammatory response syndrome, sepsis, and multiple organ dysfunction cardiogenic shock and cardiac arrest hemostatic changes in dogs with naturally occurring sepsis multiple organ dysfunction syndrome in humans and dogs increased lactate concentrations in ill and injured dogs the role of albumin in health and disease pathophysiologic characteristics of hypovolemic shock usefulness of systemic inflammatory response syndrome criteria as an index for prognosis judgement current principles and application of d-dimer analysis in small animal practice choosing fluids in traumatic hypovolemic shock: the role of crystalloids, colloids and hypertonic saline colloid and crystalloid resuscitation thromboembolic disease: predispositions and management marks sl: systemic arterial thromboembolism retrospective study of streptokinase administration in cats with arterial thromboembolism feline arterial thromboembolism: an update arterial thromboembolism in cats: acute crises in cases ( - ) and long-term management with low-dose aspirin in cases cut multiple holes in the side of a - fr red rubber tube or thoracic drainage catheter, using care not to make the cut wider than % of the circumference of the tube. insert the catheter into the abdominal cavity in a dorsal caudal direction. make sure that all incisions within the abdomen. secure the tube by placing a pursestring suture around the tube entrance site in the abdominal musculature with absorbable suture material. close the dead space in the subcutaneous tissues with absorbable suture. close the skin around the tube with another purse-string suture secured using a finger-trap technique. connect the tube to a closed urinary collection system and bandage the catheter to the abdomen. the tube can remain in place until the patient retrospective evaluation of acute renal failure in dogs uroabdomen in dogs and cats drug-induced nephrotoxicity: recognition and prevention peritoneal dialysis in emergency and critical care acute renal failure caused by lily ingestion in six cats early diagnosis of renal disease and renal failure acute renal failure in four dogs after raisin or grape ingestion disorders of the feline lower urinary tract the use of a low-profile cystostomy tube to relieve urethral obstruction in a dog renal biopsy: methods and interpretation feline idiopathic cystitis: current understanding of pathophysiology and management today's problem when did you first notice that something was wrong with your pet? when was the last time you noticed your pet act normally? what was the first abnormal sign noticed? what other conditions have developed and what are they? how soon did other signs develop? have the signs become better or worse since you first saw them? what is the name of the product? do you have the container with you today? is it a liquid concentrate, dilute spray, or solid? how long ago do you think that your pet was exposed to the poison? where do you think it happened? do you have any over-the-counter or prescription medications that your animal may have had access to? did you give any medications to your animal? is there any possibility of recreational drug exposure?your pet's recent activity did your pet eat this morning or last night? what is he/she normally fed? is there a chance that your pet may have gotten into the garbage? have you fed table scraps or anything new recently? if so, what? has your pet been off your property in the last - hours? does your pet run loose unattended? has your pet had any antiflea/tick medication within the last week?your pet's environment is your animal kept inside or outside of the house? is your pet kept in a fenced-in yard or allowed to run loose unattended? does your pet have access to neighboring properties (even for a short time)? where has your pet been in the last hours? has your pet traveled outside of your immediate geographic location? if so, when? has your pet been to rural areas in the last week? has there been any gardening work recently? does your pet have access to a compost pile? any fertilizers or weed killer used in the last week? any construction work or renovation recently? any mouse or rat poison in your house, yard, or garage? any cleaning products used inside or outside the house within the last hours? if so, which? have you changed your radiator fluid or does a car leak antifreeze? induce and maintain a patent airway and stabilize the patient's cardiovascular and respiratory status. control cns excitation with diazepam, if necessary, and control the patient's body temperature (both hypo-and hyperthermia) . induce vomiting if the patient is alert and can protect its airway; otherwise, perform orogastric lavage with the patient under general anesthesia with a cuffed endotracheal tube in place. alcohols do not bind well with activated charcoal. treat dermal exposure by bathing the area with warm water. introduction: if ingested, sodium or potassium hydroxide can cause severe contact dermatitis or irritation of the gastrointestinal tract. esophageal burns and full-thickness coagulative necrosis can occur. if an animal ingests a caustic alkali substance, feed the animal four egg whites mixed with quart of warmed water. perform endoscopy within hours to evaluate the extent of injury and to place a feeding tube, in severe cases. do not induce emesis , and do not perform orogastric lavage, because of the risk of worsening esophageal irritation. in cases of contact exposure to the skin or eyes, rinse the exposed area with warm water baths for at least minutes. administer gastroprotectant, antiemetic, and analgesic drugs as necessary. avoid neutralization, which can cause a hyperthermic reaction and worsen injury to the skin and gastrointestinal tract. amitraz is the active ingredient in ascaricides and anti-tick and anti-mite products such as mitaban and taktic. the toxic dose is to mg/kg. amitraz exerts its toxic effects by causing α-adrenergic stimulation, and causes clinical signs similar to those observed with administration of xylazine: bradycardia, cns depression, ataxia, hypotension, hyperglycemia, hypothermia, cyanotic mucous membranes, polyuria, mydriasis, and emesis. a coma can develop. treatment of amitraz intoxication includes cardiovascular support with intravenous crystalloid fluids and induction of emesis in asymptomatic animals. if clinical signs are present, orogastric lavage may be required. many toxic compounds are impregnated in a collar form. if the patient has ingested a collar and does not vomit it, it should be removed using endoscopy or gastrotomy. administer activated charcoal to prevent or delay absorption of the toxic compound. yohimbine or atepamizole, both α-adrenergic antagonists, are the treatment(s) of choice to reverse the clinical signs of toxicity. avoid the use of atropine, because it can potentially increase the viscosity of respiratory secretions and cause gastrointestinal ileus, thus promoting increased absorption of the toxic compound. ammonium hydroxide, or cleaning ammonia, can be caustic at high concentrations (see alkalis/caustics) and cause severe injury to the respiratory system if inhaled. pulmonary edema or pneumonia can occur, resulting in respiratory distress. ingestion of ammonia can cause severe irritation to the gastrointestinal tract and cause vomiting and esophageal injury. if ammonia is ingested, administer a dilute solution of egg white.administer gastroprotectant, antiemetic, and analgesic drugs as necessary. if pneumonia or pulmonary edema occurs secondary to aspiration of ammonia into the airways and alveolar spaces, treatment is largely supportive with supplemental oxygen administration, antibiotics, fluid therapy, and mechanical ventilation as necessary. diuretics may or may not be useful in the treatment of pulmonary edema secondary to ammonia inhalation. amphetamines cause cns excitation due to neurosynaptic stimulation, resulting in hypersensitivity to noise and motion, agitation, tremors, vomiting, diarrhea, and seizures. clinical signs of amphetamine toxicity include muscle tremors, tachyarrhythmias, mydriasis, ptyalism, and hyperthermia. amphetamines are rapidly absorbed from the gastrointestinal tract. treatment includes administration of intravenous fluids to maintain hydration and renal perfusion and correction of hyperthermia. administer sedative drugs such as chlorpromazine to control agitation and tremors, and diazepam to control seizures. urinary acidification can promote excretion and prevent reabsorption from the urinary bladder. in severe cases, treat cerebral edema with a combination of mannitol followed by furosemide to control increased intracranial pressure.antifreeze: see ethylene glycol antihistamines introduction antihistamines (loratadine, diphenhydramine, doxylamine, clemastine, meclizine, dimenhydrinate, chlorpheniramine, cyclizine, terfenadine, hydroxyzine) are available as over-thecounter and prescription allergy and anti-motion sickness products. clinical signs of antihistamine toxicity include restlessness, nausea, vomiting, agitation, seizures, hyperthermia, and tachyarrhythmias. treatment of antihistamine intoxication is largely symptomatic and supportive, as there is no known antidote. if ingestion is recent (within to hours) and the patient is not actively seizing and can protect its airway, induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. monitor the patient's heart rate, rhythm, and blood pressure. treat cardiac arrhythmias, if present, with appropriate therapies (see section on cardiac dysrhythmias). administer cooling measures and intravenous fluids to treat hyperthermia. a constant rate infusion of guaifenasin can be used to control muscle tremors. introduction α-naphthylthiourea (antu) is manufactured as a white or blue-gray powder. the toxic dose in dogs is - mg/kg, and in cats is - mg/kg. younger dogs appear to be more resistant to its toxic effects. antu usually causes profound emesis and increased capillary permeability that eventually leads to pulmonary edema. treatment of antu toxicity includes respiratory support. mechanical ventilation may be required in severe cases of pulmonary edema. if an animal does not vomit, orogastric lavage should be performed. administer gastrointestinal protectant, antiemetic, and analgesic drugs. cardiovascular support in the form of intravenous crystalloids should be arsenic introduction inorganic arsenic (arsenic trioxide, sodium arsenite, sodium arsenate) is the active ingredient in many herbicides, defoliants, and insecticides, including ant killers. the toxic dose of sodium arsenate is - mg/kg; that of sodium arsenite is - mg/kg. sodium arsenite is less toxic, although cats are very susceptible. arsenic compounds interfere with cellular respiration by combining with sulfhydryl enzymes. clinical signs of toxicity include severe gastroenteritis, muscle weakness, capillary damage, hypotension, renal failure, seizures, and death. in many cases, clinical signs are acute in onset. treatment of arsenic toxicity involves procuring and maintaining a patent airway. administer intravenous crystalloid fluids to correct hypotension and hypovolemia, and normalize acidbase and electrolyte balance. if no clinical signs are present and if the compound was ingested within hours, induce emesis. if clinical signs are present, perform orogastric lavage followed by administration of activated charcoal. if dermal exposure has occurred, throughly bathe the animal to prevent further absorption. dimercaprol (bal, - mg/kg im q h) can be administered as a chelating agent. n-acetylcysteine (mucomyst) (for cats, - mg/kg po iv, then mg/kg po iv q h for days; for dogs, mg/kg po or iv, then mg/kg po iv q h for days) has been shown to decrease arsenic toxicity in rats. aspirin causes inhibition of the production of prostaglandins, a high anion gap metabolic acidosis, gastrointestinal ulceration, hypophosphatemia, and decreased platelet aggregation when ingested in high quantities (> mg/kg/ hours in dogs; > mg/kg/ hours in cats). clinical signs of aspirin toxicity include tachypnea, vomiting, anorexia, lethargy, hematemesis, and melena. treatment of aspirin toxicity is largely supportive. if the ingestion was recent (within the last hour), induce emesis or perform orogastric lavage followed by administration of activated charcoal. administer intravenous crystalloid fluids to maintain hydration and correct acid-base abnormalities. administer synthetic prostaglandin analogues (misoprostol), gastroprotectant drugs, and antiemetics. alkalinization of the urine can enhance excretion. introduction baclofen is a gaba agonist centrally acting muscle relaxant. clinical signs of toxicity include vomiting, ataxia, vocalization, disorientation, seizures, hypoventilation, coma, and apnea. clinical signs can occur at doses as low as . mg/kg. treatment of baclofen ingestion includes induction of emesis if the animal is asymptomatic. otherwise, perform orogastric lavage. emesis or orogastric lavage should be followed by administration of activated charcoal. perform intravenous crystalloid fluid diuresis to promote elimination of the toxin, maintain renal perfusion, and normalize body temperature. supplemental oxygen or mechanical ventilation may be required for hypoventilation or apnea. if seizures occur, avoid the use of diazepam, which is a gaba agonist and can potentially worsen clinical signs. control seizures with intravenous introduction β-adrenergic agonists, including terbutaline, albuterol (salbutamol), and metaproterenol, are commonly used in inhaled form for the treatment of asthma. animals commonly are exposed to the compounds after chewing on their owners' inhalers. clinical signs of β-adrenergic stimulation include tachycardia, muscle tremors, and agitation. severe hypokalemia can occur. treatment of β-adrenergic agonist intoxication includes treatment with beta-blockers (propranolol, esmolol, atenolol), intravenous fluids, and intravenous potassium supplementation. diazepam or acepromazine may be administered for sedation and muscle relaxation. introduction barbiturates such as phenobarbital are gaba agonists and induce cns depression. clinical signs of barbiturate overdose or toxicity include weakness, lethargy, hypotension, hypoventilation, stupor, coma, and death. treatment of barbiturate toxicity includes maintenance and support of the cardiovascular and respiratory systems. if clinical signs are absent and the patient can protect its airway, induce emesis followed by repeated doses of activated charcoal. perform orogastric lavage if emesis is contraindicated. administer supplemental oxygen if hypoventilation occurs. some animals may require mechanical ventilation. administer intravenous fluids to control perfusion and blood pressure. positive inotropic drugs may be required if dosedependent decrease in cardiac output and blood pressure occurs. alkalinization of the urine and peritoneal dialysis can be performed to enhance excretion and elimination. hemodialysis should be considered in severe cases, if available. automotive and dry cell batteries contain sulfuric acid that can be irritating on contact with the eyes, skin, and gastrointestinal tract. button batteries, which contain sodium or potassium hydroxide, cause contact irritation if chewed. to treat exposure, rinse the eyes and skin with copious amounts of warm tap water or sterile saline solution for a minimum of minutes. if ingestion occurred, administer gastroprotectant and antiemetic drugs. induction of emesis and orogastric lavage is absolutely contraindicated because of the risk of aspiration pneumonia and worsening esophageal irritation. no attempt should be made at performing neutralization because of the risk of causing an exothermic reaction and worsening tissue damage. administer analgesics to control discomfort. benzoyl peroxide is the active ingredient in many over-the-counter acne preparations. ingestion can result in production of hydrogen peroxide, gastroenteritis, and gastric dilatation. topical exposure can cause dermal irritation and blistering. if an animal has ingested benzoyl peroxide, do not induce emesis, because of the risk of worsening esophageal irritation. instead, perform orogastric lavage. administer gastroprotectant and antiemetic medications and closely observe the patient observed for signs of gastric dilatation.bismuth subsalicylate (pepto-bismol): see aspirin bleach, chlorine (sodium hypochlorite) introduction sodium hypochlorite is available in dilute ( %- %) or concentrated ( % industrial strength or swimming pool) solutions for a variety of purposes. sodium hypochlorite can cause severe contact irritation and tissue destruction, depending on the concentration. affected animals may have a bleached haircoat. treatment of exposure includes dilution with copious amounts of warm water or saline baths and ocular lavage. induction of emesis and orogastric lavage is absolutely contraindicated because of the risk of causing further esophageal irritation. to treat ingestion, give the animal milk or large amounts of water, in combination with gastroprotectant and antiemetic drugs, to dilute the contents in the stomach. administration of sodium bicarbonate or milk of magnesia is no longer recommended. nonchlorine bleaches (sodium peroxide or sodium perborate) have a moderate toxic potential if ingested. sodium peroxide can cause gastric distention. sodium perborate can cause severe gastric irritation, with vomiting and diarrhea; renal damage and cns excitation followed by depression can occur, depending on the amount ingested. to treat dermal or ocular exposure, rinse the skin or eyes with copious amounts of warm tap water or sterile saline for a minimum of minutes; treat ocular injuries as necessary, if corneal burns have occurred. if the bleach has been ingested, do induce emesis and perform orogastric lavage. administer milk of magnesia ( - ml/kg). boric acid is the active ingredient in many ant and roach killers. the toxic ingredient (in amounts of - g/kg) can cause clinical signs in dogs by an unknown mechanism. clinical signs include vomiting (blue-green vomitus), blue-green stools, renal damage, and cns excitation and depression. treatment of boric acid or borate ingestion includes gastric decontamination with induction of emesis or orogastric lavage, followed by administration of a cathartic to hasten elimination. activated charcoal is not useful to treat ingestion of this toxin. administer intravenous fluid therapy to maintain renal perfusion. administer gastroprotectant and antiemetic drugs, as necessary. clostridium botulinum endospores can be found in carrion, food, garbage, and the environment. ingestion of endospores and c. botulinum endotoxin rarely can cause generalized neuromuscular blockade of spinal and cranial nerves, resulting in miosis, anisocoria, lower motor neuron weakness, and paralysis. respiratory paralysis, megaesophagus, and aspiration pneumonia can occur. clinical signs usually develop within days of ingestion. differential diagnosis includes acute polyradiculoneuritis (coonhound paralysis), bromethalin intoxication, and tick paralysis. treatment of botulism is largely supportive; although an antitoxin exists, it often is of no benefit. treatment may include administration of intravenous fluids, frequent turning of the patient and passive range-of-motion exercises to prevent disuse muscle atrophy, and supplemental oxygen administration or mechanical ventilation. administer amoxicillin, ampicillin, or metronidazole. recovery may be prolonged, up to to weeks in some cases. bromethalin is the active ingredient in some brands of mouse and rat poisons. it usually is packaged as . % bromethalin in green or tan pellets, and packaged in - . g place packs. the toxic dose for dogs is . g/kg, and for cats g/kg. bromethalin causes toxicity by uncoupling of oxidative phosphorylation. an acute syndrome of vomiting, tremors, extensor rigidity, and seizures occurs within hours of ingestion of high doses. delayed clinical signs occur within to days of ingestion of a lower dose and include posterior paresis progressing to ascending paralysis, cns depression, and coma. treatment of known bromethalin ingestion includes induction of emesis or orogastric lavage, and repeated doses of activated charcoal every to hours for days, because bromethalin undergoes enterohepatic recirculation. supportive care includes intravenous fluids, anticonvulsants, muscle relaxants (methocarbamol up to mg/kg/day iv to effect), frequent turning of the patient, and passive range-of-motion exercises. supplemental oxygen and /or mechanical ventilation may be required in patients with coma and severe hypoventilation. administer mannitol ( . - g/kg) in conjunction with furosemide ( mg/kg iv) if cerebral edema is suspected. the majority of caffeine toxicities occur in dogs that ingest coffee beans. caffeine causes phosphodiesterase inhibition, and can cause cardiac tachyarrhythmias, cns stimulation (hyperexcitability and seizures), diuresis, gastric ulcers, vomiting, and diarrhea. muscle tremors and seizures can occur, resulting in severe hyperthermia. treatment of caffeine toxicity is largely symptomatic and supportive, as there is no known antidote. if clinical signs are not apparent and the patient is able to protect its airway, induce emesis. alternatively, orogastric lavage can be performed, followed by administration of activated charcoal. administer diazepam to control seizures. administer betaadrenergic blockers (e.g., esmolol, propranolol, atenolol) to control tachyarrhythmias. give intravenous fluids to maintain hydration and correct hyperthermia. the patient should be walked frequently or have a urinary catheter placed to prevent reabsorption of the toxin from the urinary bladder. carbamate compounds are found in agricultural and home insecticide products. examples of carbamates include carbofuran, aldicarb, propoxur, carbaryl, and methiocarb. the toxic dose of each compound varies. carbamate compounds function by causing acetylcholinesterase inhibition. toxic amounts cause cns excitation, muscarinic acetylcholine overload, and slud (salivation, lacrimation, urination, and defecation). miosis, vomiting, treatment of carbamate intoxication includes maintaining an airway and, if necessary, artificial ventilation. administer intravenous crystalloid fluids to control the patient's hydration, blood pressure, and temperature. cooling measures may be warranted. induce emesis if the substance was ingested within minutes and the animal is asymptomatic. give repeated doses of activated charcoal if the animal can swallow and protect its airway. control seizures with diazepam ( . mg/kg iv). bathe the patient thoroughly. atropine ( . mg/kg iv) is useful in controlling some of the muscarinic signs associated with the toxicity. pralidoxime hydrochloride ( -pam) is not useful in cases of carbamate intoxication. control muscle tremors with methocarbamol (up to mg/kg iv) or guaifenesin. in humans, ingestion or inhalation of - ml of carbon tetrachloride can be fatal. clinical signs of carbon tetrachloride toxicity include vomiting and diarrhea, then progressive respiratory and central nervous system depression. ventricular dysrhythmias and hepatorenal damage ensue. the prognosis is grave. treatment of carbon tetrachloride inhalation includes procurement and maintenance of a patent airway with supplemental oxygen, and cardiovascular support. to treat ingestion, administer activated charcoal, and give intravenous fluids to maintain hydration and support renal function. chlorinated hydrocarbons include ddt, methoxychlor, lindane, dieldrin, aldrin, chlordane, chlordecone, perthane, toxaphene, heptachlor, mirex, and endosulfan. the toxic dose of each compound varies. chlorinated hydrocarbons exert their toxic effects by an unknown mechanism, and can be absorbed through the skin and the gastrointestinal tract. clinical signs are similar to those observed in organophosphate toxicity: cns excitation, seizures, slud, (salivation, lacrimation, urination, defecation), excessive bronchial secretions, vomiting, diarrhea, muscle tremors, and respiratory paralysis. secondary toxicity from toxic metabolites can cause renal and hepatic failure. chronic exposure may cause anorexia, vomiting, weight loss, tremors, seizures, and hepatic failure. the clinical course can be prolonged in small animal patients. treatment of chlorinated hydrocarbon toxicity is largely supportive in nature, as there is no known antidote. procure and maintain the patient's airway. normalize the body temperature to prevent hyperthermia. if the substance was just ingested and the patient is not demonstrating any clinical signs, induce emesis. if the patient is symptomatic, perform orogastric lavage followed by activated charcoal administration. bathe the patient thoroughly in cases of topical exposure. administer intravenous crystalloid fluids to maintain hydration. these compounds do not appear to be amenable to fluid diuresis. introduction: chlorphenoxy derivatives are found in , -d, , , -t, mcpa, mcpp, and silvex. the ld of , -d is mg/kg; however, the toxic dose appears to be much lower in small treatment treatment of chlorphenoxy derivative toxicity is largely supportive in nature, as there is no known antidote. secure the patient's airway and administer supplemental oxygen, as necessary. control cns excitation with diazepam ( . mg/kg iv). intravenous crystalloid fluid diuresis and urinary alkalinization can promote elimination. administer gastroprotectant and antiemetic drugs, as needed. the toxic effects of chocolate are related to theobromine. various types of chocolate have different concentrations of theobromine and thus can cause clinical signs of toxicity with ingestion of varying amounts of chocolate, depending on the type. the toxic dose of theobromine is - mg/kg in dogs. milk chocolate contains mg/oz ( mg/ g) of chocolate, and has a low toxic potential. semisweet chocolate contains mg/oz ( mg/ g), and baking chocolate contains mg/oz ( mg/ g). semisweet and baking chocolate, being the most concentrated, have a moderate to severe toxic potential, even in large dogs.clinical signs of theobromine intoxication are associated with phosphodiesterase inhibition and include cns stimulation (tremors, anxiety, seizures), myocardial stimulation (tachycardia and tachyarrhythmias), diuresis, and (at very high doses) gastrointestinal ulceration. with treatment, the condition of most dogs returns to normal within to hours (t / = . hours in dogs). potential side effects include gastroenteritis and pancreatitis due to the fat content of the chocolate. treatment of chocolate toxicity includes obtaining and maintaining a protected airway (if necessary), intravenous fluid diuresis, induction of emesis or orogastric lavage followed by administration of repeated doses of activated charcoal, and placement of a urinary catheter to prevent reabsorption of the toxin from the urinary bladder. cholecalciferol rodenticide ingestion can lead to increased intestinal and renal reabsorption of calcium, causing an increase in serum calcium and dystrophic mineralization of the kidneys and liver at - mg/kg. clinical signs include lethargy, anorexia, vomiting, constipation, and renal pain within to days of ingestion. seizures, muscle twitching, and central nervous system depression may be observed at very high doses. as renal failure progresses, polyuria, polydipsia, vomiting/hematemesis, uremic oral ulcers, and melena may be observed. if the compound was ingested recently (within to hours) induce emesis or perform orogastric lavage, followed by administration of activated charcoal. check the patient's serum calcium once daily for three days following ingestion. if clinical signs of toxicity or hypercalcemia are present, decrease serum calcium with loop diuretics (furosemide, - mg/kg po or iv q h) and glucocorticosteroids (prednisone or prednisolone, - mg/kg po bid) to promote renal calcium excretion. in severe cases, salmon calcitonin ( - iu/kg sc q - h in dogs) or bisphosphonate compounds may be required. correct acid-base abnormalities with intravenous crystalloid fluid diuresis and sodium bicarbonate, if necessary. (see section on hypercalcemia.) denture cleaners contain sodium perborate as the active compound. sodium perborate can cause severe direct irritation of the mucous membranes and may also act as a cns depressant. clinical signs are similar to those seen if bleach or boric acid compound is ingested, namely vomiting, diarrhea, cns excitation then depression, and renal failure. treatment for ingestion of denture cleaner includes gastric decontamination along with induction of emesis or orogastric lavage and administration of a cathartic to hasten elimination. activated charcoal is not useful for treatment of ingestion of this toxin. administer intravenous fluid therapy to maintain renal perfusion. administer gastroprotectant and antiemetic drugs, as necessary. deodorants are usually composed of aluminum chloride and aluminum chlorohydrate. both have a moderate potential for toxicity. ingestion of deodorant compounds can cause oral irritation or necrosis, gastroenteritis, and nephrosis. treatment of deodorant ingestion includes orogastric lavage, and administration of antiemetic and gastroprotectant drugs. introduction anionic detergents include sulfonated or phosphorylated forms of benzene. dishwashing liquid is an example of an anionic detergent that can be toxic at doses of - g/kg. anionic detergents cause significant mucosal damage and edema, gastrointestinal irritation, cns depression, seizures, and possible hemolysis. ocular exposure can cause corneal ulcers and edema. treatment of anionic detergent exposure is largely symptomatic, as there is no known antidote. to treat topical toxicity, flush the patient's eyes and skin with warmed tap water or . % saline solution for a minimum of minutes, taking care to avoid hypothermia. to treat ingestion, feed the patient milk and large amounts of water to dilute the toxin. do not induce emesis, because of the risk of worsening esophageal irritation. to dilute the toxin, perform orogastric lavage, followed by administration of activated charcoal. closely monitor the patient's respiratory status, because oropharyngeal edema can be severe. if necessary, perform endotracheal intubation in cases of airway obstruction. monitor the patient for signs of intravascular hemolysis. administer intravenous crystalloid fluids to maintain hydration until the patient is able to tolerate oral fluids. cationic detergents and disinfectants include quaternary ammonia compounds, isopropyl alcohol, and isopropanol. quaternary ammonia compounds have a serious toxic potential treatment treatment of cationic detergent exposure includes careful bathing and ocular rinsing of the patient for a minimum of minutes, taking care to avoid hypotension. secure the patient's airway and monitor the patient's respiratory status. administer supplemental oxygen, if necessary. place an intravenous catheter and administer intravenous crystalloid fluids to maintain hydration. do not induce emesis, because of the risk of causing further esophageal irritation. give milk or large amounts of water orally, as tolerated by the patient, to dilute the toxin. nonionic detergents include alkyl and aryl polyether sulfates, alcohols, and sulfonates; alkyl phenol; polyethylene glycol; and phenol compounds. phenols are particularly toxic in cats and puppies. clinical signs of exposure include severe gastroenteritis and topical irritation. some compounds can be metabolized to glycolic and oxalic acid, causing renal damage similar to that observed with ethylene glycol toxicity. topical and ocular exposure should be treated with careful bathing or ocular irrigation for at least minutes. administer activated charcoal to prevent absorption of the compound. as tolerated, give dilute milk or straight tap water orally to dilute the compound. administer antiemetic and gastroprotectant drugs to control vomiting and decrease gastrointestinal irritation. administer intravenous crystalloid fluids to maintain hydration and decrease the potential for renal tubular damage. monitor the patient's acid-base and electrolyte status and correct any abnormalities with appropriate intravenous fluid therapy. introduction diclone (phigone) is a dipyridyl compound that is a cns depressant. the ld in rats is - mg/kg. dichlone reacts with thiol enzymes to cause methemoglobinemia and hepatorenal damage. to treat dichlone ingestion, induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. procure and maintain a patent airway. perform intravenous fluid diuresis to maintain renal perfusion. n-acetylcysteine may be useful in the treatment of methemoglobinemia. diethyltoluamide (deet) is the active ingredient in many insect repellants (e.g., off, cutters, hartz blockade). the mechanism of action of deet is not fully understood, but it acts as a lipophilic neurotoxin within to minutes of exposure. cats appear to be particularly sensitive to deet. a lethal dermal dose is . g/kg; if ingested, the lethal dose is much less. the toxic dose of dermal exposure in dogs is g/kg. clinical signs of toxicity include aimless gazing, hypersalivation, chewing motions, and muscle tremors that progress to seizures. recumbency and death can occur within minutes of exposure at high doses. treatment of deet toxicity is largely supportive, as there are no known antidotes. procure and maintain a patent airway and perform mechanical ventilation, if necessary. place an intravenous catheter and administer intravenous crystalloid fluids to control hydration and treat hypotension, as necessary. treat seizures with diazepam ( . mg/kg iv) or phenobarbital. because of the rapid onset of clinical signs, induction of emesis is contraindicated. perform orogastric lavage if the compound was ingested within the last hours. administer multiple repeated doses of activated charcoal. cooling measures should be implemented to control hyperthermia. if dermal exposure has occurred, bathe the patient thoroughly to avoid further exposure and absorption. diquat is a dipyridyl compound that is the active ingredient in some herbicide compounds. the ld of diquat is - mg/kg. like paraquat, diquat induces its toxic effects by causing the production of oxygen-derived free radical species. clinical signs of diquat intoxication include anorexia, vomiting, diarrhea, and acute renal failure. massive dehydration and electrolyte imbalances can occur as a result of fluid loss into the gastrointestinal tract. treatment of diquat intoxication is similar to that for paraquat ingestion. if the animal had ingested diquat within hour of presentation, induce emesis. in clinical cases, orogastric lavage may be required. both emesis and orogastric lavage should be followed by administration of kaolin or bentonite as an adsorbent, rather than activated charcoal. place an intravenous catheter and administer crystalloid fluids to restore volume status and maintain renal perfusion. monitor urine output. if oliguria or anuria occurs, treatment with mannitol, furosemide, and dopamine may be considered. ecstasy ( , -methylenedioxymethylamphetamine; mdma) is a recreational drug used by humans. ecstasy causes release of serotonin. clinical signs of intoxication are related to the serotonin syndrome (excitation, hyperthermia, tremors, and hypertension), and seizures may be observed. a urine drug screening test can be used to detect the presence of mdma. treatment of ecstasy intoxication is largely supportive, as there is no known antidote. administer intravenous fluids to maintain hydration, correct acid-base status, and treat hyperthermia. serotonin antagonist drugs (cyproheptadine) can be dissolved and administered per rectum to alleviate clinical signs. intravenous propranolol has additional antiserotonin effects. administer diazepam ( . - mg/kg iv) to control seizures. if cerebral edema is suspected, administer mannitol, followed by furosemide. ethylene glycol is most commonly found in antifreeze solutions but is also in some paints, photography developer solutions, and windshield wiper fluid. ethylene glycol in itself is only minimally toxic. however, when it is metabolized to glycolate, glyoxal, glyoxylate, and oxalate, the metabolites cause an increased anion gap metabolic acidosis and precipitation of calcium oxalate crystals in the renal tubules, renal failure, and (ultimately) death.the toxic dose in dogs is . ml/kg, and in cats is . ml/kg. the toxin is absorbed quite readily from the gastrointestinal tract and can be detected in the patient's serum within an hour of ingestion. colorimetric tests that can be performed in most veterinary hospitals can detect larger quantities of ethylene glycol in the patient's serum. in a dog with clinical treatment begin treatment of known ethylene glycol ingestion immediately. induce emesis or perform orogastric lavage and adminiser repeated doses of activated charcoal. place an intravenous catheter and perform crystalloid fluid diuresis with a known antidote. the treatment of choice for dogs is administration of -methylpyrrazole ( -mp), which directly inhibits alcohol dehydrogenase, thus preventing the conversion of ethylene glycol to its toxic metabolites. the dose for dogs is mg/kg initially, followed by mg/kg at and hours and mg/kg at hours. -mp has been used experimentally at . times the recommended dose for dogs. in cats, treatment with -mp is effective if it is administered within the first hours of ingestion.cats will demonstrate signs of sedation and hypothermia with this treatment. if -mp is not available, administer ethanol ( mg/kg iv loading dose, followed by mg/ kg/hour), or as a % solution (for dogs, . ml/kg iv q h for five treatments, then q h for five more treatments; for cats, ml/kg q h for four treatments). grain alcohol ( proof) contains approximately mg/ml of ethanol. antiemetics and gastroprotective agents should be considered. urinary alkalinization and peritoneal dialysis may enhance the elimination of ethylene glycol and its metabolites. many fertilizers are on the market, and may be composed of urea or ammonium salts, phosphates, nitrates, potash, and metal salts. fertilizers have a moderate toxic potential, depending on the type and amount ingested. clinical signs of fertilizer ingestion include vomiting, diarrhea, metabolic acidosis, and diuresis. nitrates or nitrites can cause formation of methemoglobin and chocolate-brown blood. electrolyte disturbances include hyperkalemia, hyperphosphatemia, hyperammonemia, and hyperosmolality. treatment of fertilizer ingestion includes cardiovascular support, and administration of milk or a mixture of egg whites and water, followed by induction of emesis or orogastric lavage. correct electrolyte abnormalities as they occur (see section on hyperkalemia). administer antiemetic and gastroprotectant drugs, as necessary. administer intravenous fluids to control hydration and maintain blood pressure. n-acetylcysteine may be useful if methemoglobinemia is present. fipronil is the active ingredient in frontline, a flea control product. fipronil exerts its effects by gaba antagonism and can cause cns excitation. treatment of fiprinol toxicity includes treatment of cns excitation, treatment of hyperthermia by cooling measures, and administration of activated charcoal. fire extinguisher fluid contains chlorobromomethane or methyl bromide, both of which have a serious toxic potential. dermal or ocular irritation can occur. if ingested, the compounds can be converted to methanol, and cause high anion gap metabolic acidosis, cns excitation and depression, aspiration pneumonitis, and hepatorenal damage. to treat ocular or dermal exposure to fire extinguisher fluids, flush the eyes or skin with warmed tap water or . % saline solution for a minimum of minutes. do not induce emesis or perform orogastric lavage to treat ingestion, because of the risk of causing severe aspiration pneumonitis. gastroprotectant and antiemetic drugs may be used, if indicated. administer intravenous fluids to maintain hydration and renal perfusion. supplemental oxygen or mechanical ventilation may be required in severe cases of aspiration pneumonitis. fireplace colors contain salts of heavy metals-namely, copper rubidium, cesium, lead, arsenic, antimony, barium, selenium, and zinc, all of which have moderate toxic potential, depending on the amount ingested and the size of the patient. clinical signs are largely associated with gastrointestinal irritation (vomiting, diarrhea, anorexia). zinc toxicity can cause intravascular hemolysis and hepatorenal damage. to treat ingestion of fireplace colors, administer cathartics and activated charcoal and gastroprotectant and antiemetic drugs. place an intravenous catheter for intravenous crystalloid fluid administration to maintain hydration and renal perfusion. specific chelating agents may be useful in hastening elimination of the heavy metals. fireworks contain oxidizing agents (nitrates and chlorates) and metals (mercury, copper, strontium, barium, and phosphorus). ingestion of fireworks can cause hemorrhagic gastroenteritis and methemoglobinemia. to treat firework ingestion, induce emesis or perform orogastric lavage and administer activated charcoal. administer specific chelating drugs if the amount and type of metal are known, and administer gastroprotectant and antiemetic drugs. if methemoglobinemia occurs, administer n-acetylcysteine; a blood transfusion may be necessary. introduction fuels such as barbecue lighter fluid, gasoline, kerosene, and oils (mineral, fuel, lubricating) are petroleum distillate products that have a low toxic potential if ingested but can cause severe aspiration pneumonitis if as little as ml is inhaled into the tracheobronchial tree. cns depression, mucosal damage, hepatorenal insufficiency, seizures, and corneal irritation can occur. if fuels are ingested, administer gastroprotectant and antiemetics drugs. do not induce emesis or perform orogastric lavage, because of the risk of aspiration pneumonia. to treat topical exposure, rinse the skin and eyes copiously with warm tap water or . % saline solution. administer antiemetic and gastroprotectant drugs, as necessary. administer intravenous fluids to maintain hydration and treat acid-base and electrolyte abnormalities. children's glue contains polyvinyl acetate, which has a very low toxic potential. if inhaled, the compound can cause pneumonitis. treatment of polyvinyl acetate should be performed as clinical signs of pneumonitis (increased respiratory effort, cough, lethargy, respiratory distress) occur. introduction superglue contains methyl- -cyanoacrylate, a compound that can cause severe dermal irritation on contact. do not induce emesis. do not bathe the animal, and do not apply other compounds (acetone, turpentine) in an attempt to remove the glue from the skin. the fur can be shaved, using care to avoid damaging the underlying skin. the affected area should be allowed to exfoliate naturally. glyophosate is a herbicide found in roundup and kleenup. if applied properly, the product has a very low toxic potential. clinical signs of toxicity include dermal and gastric irritation, including dermal erythema, anorexia, and vomiting. cns depression can occur. treatment includes thorough bathing in cases of dermal exposure, and induction of emesis or orogastric lavage followed by administration of activated charcoal. administer antiemetic and gastroprotectant drugs as necessary. administer intravenous crystalloid fluids to prevent dehydration secondary to vomiting. even small amounts of grapes and raisins can be toxic to dogs. the mechanism of toxicity remains unknown. clinical signs occur within hours of ingestion of raisins or grapes, and include vomiting, anorexia, lethargy, and diarrhea (often with visible raisins or grapes in the fecal matter). within hours, dogs demonstrate signs of acute renal failure (polyuria, polydipsia, vomiting) that can progress to anuria. to treat known ingestion of raisins or grapes, induce emesis or perform orogastric lavage, followed by repeated doses of activated charcoal. if clinical signs of vomiting and diarrhea are present, administer intravenous fluids and monitor urine output. aggressive intravenous fluid therapy, in conjunction with maintenance of renal perfusion, is necessary. in cases of anuric renal failure, dopamine, furosemide, and mannitol can be useful in increasing urine output. peritoneal or hemodialysis may be necessary in cases of severe oliguric or anuric renal failure. calcium channel blockers such as amlodipine and diltiazem can be used to treat systemic hypertension. supportive care includes treatment of hyperkalemia, and administration of gastroprotectant and antiemetic drugs and (if the animal is eating) phosphate binders. aromatic hydrocarbons include phenols, cresols, toluene, and naphthalene. all have a moderate toxic potential if ingested. toxicities associated with ingestion of aromatic hydrocarbons include cns depression, hepatorenal damage, muscle tremors, pneumonia, methemoglobinemia, and intravascular hemolysis. if an aromatic hydrocarbon is ingested, do not induce emesis, because of the risk of aspiration pneumonia. a dilute milk solution or water can be administered to dilute the compound. perform orogastric lavage. carefully monitor the patient's respiratory and cardiovascular status. administer supplemental oxygen if aspiration pneumonia is present. to treat topical exposure, thoroughly rinse the eyes and skin with copious amounts of warm tap water or . % saline solution. imidacloprid is the compound used in the flea product advantage. clinical signs of toxicity are related to nicotinic cholinergic stimulation, causing neuromuscular excitation followed by collapse. the compound may induce respiratory paralysis. to treat imidacloprid toxicity, procure and maintain a patent airway with supplemental oxygen administration. control cns excitation with diazepam, phenobarbital, or propofol. administer enemas to hasten gastrointestinal elimination, and administer activated charcoal. bathe the animal thoroughly to prevent further dermal absorption. closely monitor the patient's oxygenation and ventilation status. if severe hypoventilation or respiratory paralysis occurs, initiate mechanical ventilation. iron and iron salts can cause severe gastroenteritis, myocardial toxicity, and hepatic damage if high enough doses are ingested. lawn fertilizers are a common source of iron salts. treatment of ingestion of iron and iron salts includes cardiovascular support in the form of intravenous fluids and antiarrhythmic drugs, as needed. induce emesis or perform orogastric lavage for gastric decontamination. a cathartic can be administered to promote elimination from the gastrointestinal tract. antiemetic and gastroprotectant drugs should be administered to prevent nausea and vomiting. in some cases, radiographs can aid in making a diagnosis of whether the compound was actually ingested. iron toxicity can be treated with the chelating agent deferoxamine. ivermectin is a gaba agonist that is used in commercial heartworm prevention and antihelminthic compounds and can be toxic in predisposed breeds, including collies, collie loperamide is an opioid derivative that is used to treat diarrhea. clinical signs of loperamide intoxication include constipation, ataxia, nausea, and sedation. induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. naloxone may be beneficial in the temporary reversal of ataxia and sedation. ingestion of macadamia nuts can cause clinical signs of vomiting, ataxia, and ascending paralysis in dogs. the toxic principle in macadamia nuts is unknown. there is no known antidote. treatment consists of supportive care, including administration of intravenous fluids and antiemetics and placement of a urinary catheter for patient cleanliness. clinical signs resolve in most cases within hours. marijuana is a hallucinogen that can cause cns depression, ataxia, mydriasis, increased sensitivity to motion or sound, salivation, and tremors. along with these findings, a classic clinical sign is the sudden onset of dribbling urine. urine can be tested with drug test kits for tetrahydrocannabinoid (thc), the toxic compound in marijuana. there is no known antidote for marijuana toxicity; therefore, treatment is largely symptomatic. place an intravenous catheter and administer intravenous fluids to support hydration. administer atropine if severe bradycardia exists. induction of emesis can be attempted but because of the antiemetic effects of thc, is usually unsuccessful. orogastric lavage can be performed, followed by repeated doses of activated charcoal. clinical signs usually resolve within to hours. introduction "strike anywhere" matches, safety matches, and the striking surface of matchbook covers contain iron phosphorus or potassium chlorate. both compounds have a low toxic potential but can cause clinical signs of gastroenteritis and methemoglobinemia if large quantities are ingested. treatment of match and matchbook ingestion includes gastric decontamination with induction of emesis or orogastric lavage and administration of activated charcoal and a cathartic. if methemoglobinemia occurs, administer n-acetylcysteine, intravenous fluids, and supplemental oxygen. metaldehyde is the active ingredient in most brands of snail bait. the exact mechanism of toxicity is unknown but may involve inhibition of gaba channels. clinical signs associated with metaldehyde toxicity include severe muscle tremors, cns excitation, and treatment treatment of mushroom toxicity is largely supportive. if the mushroom was ingested within the last hours, induce emesis or perform orogastric lavage and then administer activated charcoal. symptomatic treatment includes intravenous fluids to promote diuresis and treat hyperthermia and skeletal muscle relaxants to control tremors and seizures (methocarbamol, diazepam). if amanita ingestion is suspected, administer hepatoprotectant agents including milk thistle. mycotoxins from penicillium spp. are found in moldy foods, cream cheese, and nuts. clinical signs of intoxication include tremors, agitation, hyperesthesia, and seizures. if tremorigenic mycotoxin toxicity is suspected, a sample of the patient's serum and gastric contents or vomitus can be submitted to the michigan state university veterinary toxicology laboratory for tremorigen assay. there is no known antidote. perform orogastric lavage, followed by administration of activated charcoal. control tremors and seizures with methocarbamol, diazepam, phenobarbital, or pentobarbital. administer intravenous fluids to control hyperthermia and maintain hydration. in cases in which cerebral edema is suspected secondary to severe refractory seizures, administer intravenous mannitol and furosemide. naphthalene is the active ingredient in mothballs and has a high toxic potential. clinical signs associated with naphthalene toxicity include vomiting, methemoglobinemia, cns stimulation, seizures, and hepatic toxicity. a complete blood count often reveals heinz bodies and anemia. do not induce emesis if naphthalene ingestion is suspected. if the ingestion was within hour of presentation, perform orogastric lavage. control seizures with diazepam or phenobarbital. administer intravenous fluids to control hyperthermia and maintain hydration. n-acetylcysteine can play a role in the treatment of methemoglobinemia. a packed rbc transfusion may be necessary if anemia is severe. observe the patient for clinical signs associated with hepatitis. nicotine toxicity occurs in animals as the result of ingestion of cigarettes, nicotine-containing gum, and some insecticides. nicotine stimulates autonomic ganglia at low doses, and blocks autonomic ganglia and the neuromuscular junction at high doses. absorption after ingestion is rapid. clinical signs include hyperexcitability and slud (salivation, lacrimation, urination, and defecation). muscle tremors, respiratory muscle fatigue or hypoventilation, tachyarrhythmias, seizures, coma, and death can occur. if the patient presents within hour of ingestion and has no clinical signs, induce emesis, followed by administration of repeated doses of activated charcoal. in patients with clinical signs of toxicity, perform orogastric lavage. administer intravenous fluids to maintain hydration and promote diuresis, and treat hyperthermia. administer atropine to treat cholinergic symptoms. urinary acidification can promote nicotine excretion. nonsteroidal antiinflammatory drugs (nsaids) include ibuprofen, ketoprofen, carprofen, diclofenac, naproxen, celecoxib, valdecoxib, rofecoxib, and deracoxib. nsaids cause inhibition of prostaglandin synthesis, leading to gastrointestinal ulceration, renal failure and hepatotoxicity. ibuprofen toxicity has been associated with seizures in dogs, cats, and ferrets. the toxic dose varies with the specific compound ingested. to treat nsaid toxicity, induce emesis or perform orogastric lavage, followed by administration of multiple repeated doses of activated charcoal. place an intravenous catheter for crystalloid fluid diuresis to maintain renal perfusion. administer the synthetic prostaglandin analogue misoprostol to help maintain gastric and renal perfusion. control seizures, if present, with intravenous diazepam. administer gastroprotectant and antiemetic drugs to control vomiting and gastrointestinal hemorrhage. continue intravenous fluid diuresis for a minimum of hours, with frequent monitoring of the patient's bun and creatinine. when the bun and creatinine levels are normal or have plateaued for hours, slowly decrease fluid diuresis % per day until maintenance levels are restored. onions, garlic, and chives contain sulfoxide compounds that can cause oxidative damage of rbcs, leading to heinz body anemia, methemoglobinemia, and intravascular hemolysis. clinical signs of toxicity include weakness, lethargy, tachypnea, tachycardia, and pale mucous membranes. vomiting and diarrhea can occur. intravascular hemolysis can cause treatment treatment of onion, chive, and garlic toxicity includes administration of intravenous fluid diuresis, and induction of emesis or orogastric lavage, followed by administration of activated charcoal and a cathartic. in cases of severe anemia, packed rbc transfusion or administration of a hemoglobin-based oxygen carrier should be considered. opiate drugs include heroin, morphine, oxymorphone, fentanyl, meperidine, and codeine. opiate compounds bind to specific opioid receptors throughout the body and produce clinical signs of miosis or mydriasis (cats), and cns excitation, followed by ataxia and cns depression, leading to stupor and coma. hypoventilation, bradycardia, hypoxia, and cyanosis can occur. to treat known overdose or ingestion of an opiate compound, induce emesis (in asymptomatic animals) or perform orogastric lavage, followed by administration of activated charcoal. administer intravenous fluids and supplemental oxygen to support the cardiovascular and respiratory systems. mechanical ventilation may be necessary until hypoventilation resolves. administer repeated doses of naloxone as a specific antidote to reverse clinical signs of narcosis and hypoventilation. if seizures are present (meperidine toxicity), administer diazepam. organophosphate compounds traditionally are used in flea control products and insecticides. common examples of organophosphates include chlorpyrifos, coumaphos, diazinon, dichlorvos, and malathion. the toxic dose varies, depending on the particular compound and individual animal sensitivity. organophosphate toxicity causes acetylcholinesterase inhibition, resulting in clinical signs of cns stimulation, including tremors and seizures. muscarinic acetylcholine overload causes the classic slud signs of salivation, lacrimation, urination, and defecation. miosis, excessive bronchial secretions, muscle tremors, and respiratory paralysis can occur. an intermediate syndrome of generalized weakness, hypoventilation, and eventual paralysis with ventral cervical ventroflexion that may require mechanical ventilation has been described. if organophosphate toxicity is suspected, whole-blood acetylcholinesterase activity can be measured and will be low. treatment of toxicity includes careful and thorough bathing in cases of dermal exposure and, if the substance was ingested, gastric decontamination with induction of emesis or orogastric lavage, followed by administration of activated charcoal, and administration of the antidote pralidoxime hydrochloride . atropine can help control the muscarinic clinical signs. supportive care in the form of cooling measures, intravenous crystalloid fluids, and supplemental oxygen or mechanical ventilation may be required, depending on the severity of clinical signs. introduction ingestion of large amounts of paintballs can cause neurologic signs, electrolyte abnormalities, and occasionally death. paintballs are gelatin capsules that contain multiple colors of if ingestion was recent and if no clinical signs of toxicity are present, induce emesis or perform orogastric lavage, followed by administration of a cathartic and activated charcoal. there is no known antidote. treatment includes supportive care in the form of intravenous fluids and administration of phenobarbital or methocarbamol to control seizures and tremors. diazepam, a gaba agonist, is contraindicated, because it can potentially worsen clinical signs. urine acidification may hasten elimination. clinical signs can last from to days. pyrethrin and pyrethroid compounds are extracted from chrysanthemums, and include allethrin, decamethrin, tralomethrin, fenpropanthrin, pallethrin, sumethrin, permethrin, tetramethrin, cyfluthrin, and resemethrin. the oral toxicity is fairly low; however, the compounds can be significantly harmful if inhaled or applied to the skin. pyrethrin and pyrethroid compounds cause depolarization and blockade of nerve membrane potentials, causing clinical signs of tremors, seizures, respiratory distress, and paralysis. contact dermatitis can occur. to distinguish between pyrethrin/pyrethroid toxicity and organophosphate toxicity, acetylcholinesterase levels should be obtained; they will be normal if pyrethrins are the cause of the animal's clinical signs. treatment of toxicity is supportive, as there is no known antidote. carefully bathe the animal in lukewarm water to prevent further oral and dermal exposure. both hyperthermia and hypothermia can worsen clinical signs. administer activated charcoal to decrease enterohepatic recirculation. atropine may control clinical signs of excessive salivation. to control muscle tremors, administer methocarbamol to effect. administer diazepam or phenobarbital to control seizures, as necessary. rotenone is used as a common garden and delousing insecticide. fish and birds are very susceptible to rotenone toxicity. rotenone inhibits mitochondrial electron transport. clinical signs of tissue irritation and hypoglycemia can occur after topical or oral exposure. if the compound is inhaled, cns depression and seizures can occur. to treat toxicity, perform orogastric lavage, followed by administration of a cathartic and activated charcoal. bathe the animal carefully to prevent further dermal exposure and further ingestion. administer diazepam or phenobarbital to control seizures. the prognosis generally is guarded. treatment of ingestion includes dilution with milk, water, or egg whites. perform orogastric lavage, followed by administration of activated charcoal. administer intravenous crystalloid fluids to maintain hydration. administer antiemetic and gastroprotectant drugs to treat gastroenteritis and vomiting.shampoos, nonmedicated: see detergents, nonionic shampoos, selenium sulfide introduction selenium sulfide shampoos (e.g., selsun blue) have a low toxic potential, and primarily cause gastroenteritis. treatment of ingestion includes dilution with water, milk, or egg whites and administration of activated charcoal. carefully and thoroughly rinse the skin and eyes to prevent further exposure. administer antiemetic and gastroprotectant drugs in cases of severe gastroenteritis. zinc-based (zinc pyridinethione) anti-dandruff shampoos have a serious toxic potential if ingested or if ocular exposure occurs. gastrointestinal irritation, retinal detachment, progressive blindness, and exudative chorioretinitis can occur. treatment of ingestion includes gastric decontamination. induce emesis or perform orogastric lavage, followed by administration of a cathartic and activated charcoal.to treat ocular exposure, thoroughly rinse the patient's eyes for a minimum of minutes. carefully monitor the animal for clinical signs of blindness. implement intravenous fluid to maintain hydration and renal perfusion in cases of severe gastroenteritis. silver polish contains the alkali substance sodium carbonate and cyanide salts, and has a serious toxic potential. ingestion results in rapid onset of vomiting and possibly cyanide toxicity. to treat ingestion, monitor and maintain the patient's respiration and cardiovascular status and administer intravenous crystalloid fluids. induce emesis, followed by administration of activated charcoal. administer sodium nitrite or sodium thiosulfate iv for cyanide toxicity. bath soap (bar soap) usually has low toxic potential and causes mild gastroenteritis with vomiting if ingested. to treat ingestion, include dilution with water, administration of intravenous fluids to maintain hydration, and administration of antiemetic and gastroprotectant drugs to treat gastroenteritis. sodium fluoroacetate is a colorless, odorless, tasteless compound that causes uncoupling of oxidative phosphorylation. the toxic dose in dogs and cats is . - . mg/kg. clinical signs of toxicity include cns excitation, seizures, and coma secondary to cerebral edema. the prognosis is guarded. to treat toxicity, procure and maintain a patent airway, monitor and stabilize the cardiovascular status, and control hyperthermia. perform orogastric lavage, followed by administration of activated charcoal. if clinical signs are not present at the time of presentation, induce emesis. administer intravenous fluids and supplemental oxygen, as necessary. strattera (atomoxetine hydrochloride) is a selective norepinephrine reuptake inhibitor used in the treatment of attention deficit hyperactivity disorder (adhd) in humans. peak serum concentrations occur in dogs within to hours of ingestion, with a peak half-life at to hours following ingestion. clinical signs of toxicity include cardiac tachyarrhythmias, hypertension, disorientation, agitation, trembling, tremors, and hyperthermia. treatment of intoxication is largely symptomatic and supportive in nature. first, induce emesis if the patient is conscious and has an intact gag reflex. orogastric lavage can also be performed. administer one dose of activated charcoal to prevent further absorption of the compound from the gastrointestinal tract. identify cardiac dysrhythmias and treat accordingly. control hypertension with sodium nitroprusside or diltiazem as a constant rate infusion. administer acepromazine or chlorpromazine to control agitation. do not use diazepam, because it can potentially worsen clinical signs. administer intravenous fluids to maintain hydration and promote diuresis. strychnine is the active ingredient in pesticides used to control rodents and other vermin. the toxic dose in dogs is . mg/kg, and in cats is mg/kg. strychnine antagonizes spinal inhibitory neurotransmitters and causes severe muscle tremors, muscle rigidity, and seizures. clinical signs are stimulated or exacerbated by noise, touch, light, and sound. mydriasis, hyperthermia, and respiratory paralysis can occur. if strychnine toxicity is suspected, gastric contents should be collected and saved for analysis. if the animal is asymptomatic at the time of presentation, induce emesis. if clinical signs are present, perform orogastric lavage. both emesis and orogastric lavage should be followed by the administration of activated charcoal. administer intravenous crystalloid fluids to support the cardiovascular system, aid in cooling measures, and improve renal diuresis. treat cns stimulation with methocarbamol, diazepam, or phenobarbital. the animal should have cotton packed in its ears to prevent noise stimulation, and should be placed in a quiet, dark room. treatment of ingestion includes dilution with milk of magnesia or water, administration of antiemetic and gastroprotectant drugs, and administration of intravenous crystalloid fluids to maintain hydration. do not induce emesis, because of the risk of causing further esophageal irritation.sunscreen: see zinc and zinc oxide suntan lotion: see shampoos, zinc-based, and alcohols tar: see fuels tea tree oil (melaleuca oil) introduction tea tree (melaleuca) oil is an herbal-origin flea-control product. the toxic principles in tea tree oil are monoterpenes, which produce clinical signs of neuromuscular weakness, and ataxia. treatment of tea tree oil toxicity includes administration of cathartics and activated charcoal to prevent further absorption. carefully bathe the animal to prevent further dermal exposure. tetanus spores from clostridium tetani organisms are ubiquitous in the soil and feces, particularly in barnyards. cases have been reported in dogs after tooth eruption and after abdominal surgeries performed with cold sterilization packs. anaerobic wound infections can contain tetanus spores. the neurotoxin from c. tetani inhibits spinal inhibitory neurons, causing motor neuron excitation. extensor muscle rigidity ("sawhorse stance"), erect ears, and risus sardonicus (a sardonic grin) are characteristic features of tetanus. administer tetanus antitoxin if toxin has not already been bound in the cns. to eliminate the source of the toxin (e.g., abscess), open and debride all wounds. intravenous administration of ampicillin or penicillin g is the treatment of choice for tetanus. supportive care in the form of skeletal muscle relaxants, intravenous fluids and parenteral nutrition, and nursing care to prevent decubitus ulcer formation is required. in extreme cases, mechanical ventilation may be necessary. triazene compounds include atrazine, prometone, and monuron (telvar). the toxic mechanism of triazene compounds is unknown. clinical signs of toxicity include salivation, ataxia, hyporeflexia, contact dermatitis, hepatorenal damage, muscle spasms, respiratory difficulty, and death. treatment of triazene exposure includes cardiovascular and renal support in the form of intravenous crystalloid fluids, inotropic drugs, and antiarrhythmic agents, as necessary. if the exposure is recent, induce emesis. perform orogastric lavage in animals that cannot protect the airway. emesis and orogastric lavage should be followed by the administration of activated charcoal and a cathartic. carefully bathe the patient to prevent further dermal absorption. a variety of tricyclic antidepressants are available for use in both humans and animals, including amitriptyline, amoxapine, desipramine, doxepine, fluoxetine (prozac), fluvoxamine (luvox), imipramine, nortriptyline, paroxetine (paxil), protriptyline, sertraline (zoloft), and trimipramine. selective serotonin reuptake inhibitors (ssris) are rapidly absorbed from the digestive tract, with peak serum concentrations occurring to hours after ingestion. the elimination half-life for each drug differs in dogs, but typically last to hours. ssris inhibit the reuptake of serotonin, causing serotonin to accumulate in the brain. this can cause "serotonin syndrome," characterized by trembling, seizures, hyperthermia, ptyalism or hypersalivation, cramping or abdominal pain, vomiting, and diarrhea. other clinical signs of ssri intoxication include depression, tremors, bradycardia, tachyarrhythmias, and anorexia. any animal that has ingested an ssri should be promptly treated and carefully observed for at least hours for side effects. the treatment of suspected ssri intoxication involves gastric decontamination if the patient is not depressed and has an intact gag reflex. perform orogastric lavage and administer activated charcoal to prevent further toxin absorption and hasten elimination from the gastrointestinal tract. treat other clinical signs symptomatically. administer intravenous diazepam to control seizures. treat tachyarrhythmias according to type. administer methocarbamol to control muscle tremors. cyproheptadine ( mg/kg), a serotonin antagonist, can be dissolved in water and administered per rectum. vitamin k antagonist rodenticides, which are commonly found in pelleted or block form, inhibit the activation of the vitamin k-dependent coagulation factors ii, vii, ix, and x. clinical signs of hemorrhage occur within to days of exposure. hemorrhage can occur anywhere in the body, and can be manifested as petechiation of the skin or mucous membranes, hemorrhagic sclera, epistaxis, pulmonary parenchymal or pleural hemorrhage, gastrointestinal hemorrhage, pericardial hemorrhage, hematuria, retroperitoneal hemorrhage, hemarthrosis, and central nervous system hemorrhage. clinical signs include respiratory distress, cough, bleeding from the gums or into the eyes, ataxia, paresis, paralysis, seizures, hematuria, joint swelling, lameness, lethargy, weakness, inappetence, and collapse.diagnosis is made based on clinical signs and a prolonged activated clotting time, or prothrombin time. the pivka (proteins induced by vitamin k antagonism) test may be helpful but usually cannot be performed in-house. slight thrombocytopenia may be present secondary to hemorrhage; however, blood levels usually do not reach the critical level of < , platelets/µl to cause clinical signs of hemorrhage. in some cases, severe stressinduced hyperglycemia and glucosuria may be present but resolves within hours. if the rodenticide was ingested within the last hours, induce emesis. alternatively, orogastric lavage can be performed in an uncooperative patient. both emesis and orogastric lavage should be followed by administration of activated charcoal. the stomach contents can be submitted for analysis. following successful treatment, administer oral vitamin k for days after the exposure; or a check prothrombin time days after gastric decontamination. if the prothrombin time is prolonged, administer fresh frozen plasma and vitamin k.if the prothrombin time is normal, gastric decontamination was successful, and no further treatment is necessary.if an animal presents with clinical signs of intoxication, administer activated clotting factors in the form of fresh frozen plasma ( ml/kg), and vitamin k ( mg/kg sq in multiple sites with a -gauge needle). packed rbcs or fresh whole blood may be required if the patient is also anemic. supportive care in the form of supplemental oxygen may be necessary in cases of pulmonary or pleural hemorrhage. following initial therapy and discharge, the patient should receive vitamin k ( . mg/kg po q - h for days), and prothrombin time should be checked days after the last vitamin k capsule is administered. in some cases, depending on the type of anticoagulant ingested, an additional weeks of vitamin k therapy may be required. xylitol is a sugar alcohol that, when ingested by humans, does not cause a significant increase in blood glucose, and therefore does not stimulate insulin release from the human pancreas. in dogs, however, xylitol causes a massive rapid and dose-dependent release of insulin from pancreatic beta-cells. following insulin release, clinically significant hypoglycemia can develop, followed by signs of vomiting, weakness, ataxia, mental depression, hypokalemia, hypoglycemic seizures, and coma. clinical signs associated with xylitol ingestion can be seen within minutes of ingestion and can last for more than hours, even with aggressive treatment. known xylitol ingestion should be treated as for other toxin ingestion. if no neurologic abnormalities exist at the time the patient is seen, induce emesis, followed by administration of activated charcoal. it remains unknown at this time whether activated charcoal actually delays or prevents the absorption of xylitol from the canine gastrointestinal tract. if clinical signs have already developed, perform orogastric lavage and gastric decontamination. blood glucose concentrations should be analyzed and maintained with supplemental dextrose as a constant rate infusion ( . %- %) until normoglycemia can be maintained with multiple frequent small meals. hypokalemia may develop because it is driven intracellularly by the actions of insulin. treat hypokalemia with supplemental potassium chloride by infusion, not to exceed . meq/kg/hour. pennies minted in the u.s. after contain large amounts of zinc rather than copper. other sources of zinc include zinc oxide ointment and hardware such as that found in metal bird cages. zinc toxicity causes intravascular hemolysis, anemia, gastroenteritis, and renal failure. if zinc toxicity is suspected, take an abdominal radiograph to document the presence of the metal in the stomach or intestines. (if zinc-containing ointment was ingested, this will not be visible on radiographs.) induce emesis or perform orogastric lavage, depending on the size of the object ingested. often, small objects such as pennies can be retrieved using endoscopy or surgical gastrotomy/enterotomy. always take an additional radiograph after the removal procedure to ensure that all objects have been successfully removed. administer intravenous fluids to maintain renal perfusion and promote fluid diuresis. administer gastroprotectant and antiemetic drugs. chelation therapy with succimer, calcium edta, dimercaprol, or penicillamine may be necessary. do not administer pulmonary contusions are a common sequela of blunt traumatic injury. a contusion basically is a bruise characterized by edema, hemorrhage, and vascular injury. contusions may be present at the time of presentation or can develop over the first hours after injury. a diagnosis of pulmonary contusion can be made based on auscultation of pulmonary crackles, presence of respiratory distress, and the presence of patchy interstitial to alveolar infiltrates on thoracic radiographs. radiographic signs can lag behind the development of clinical signs of respiratory distress and hypoxemia by hours. in most cases, cage rest is sufficient to temporarily diminish blood loss. sedation (acepromazine, . - . mg/kg iv, im, sq) may be helpful in alleviating anxiety and decreasing blood pressure. the hypotensive effects of acepromazine are potentially harmful if severe blood loss has occurred. if evidence of hypovolemia is present (see section on hypovolemic shock), intravenous fluid resuscitation should be administered. rapid assessment of clotting ability, with a platelet count estimate and clotting profile (act or aptt and pt), should be performed. if epistaxis secondary to vitamin k antagonist rodenticide intoxication is suspected, administer vitamin k and fresh frozen plasma or fresh whole blood.persistent hemorrhage from a nasal disorder can be treated with dilute epinephrine ( : , ) into the nasal cavity with the nose pointed toward the ceiling to promote vasoconstriction. if this fails, the animal can be anesthetized, and the nasal cavity packed with gauze, and the caudal oropharynx and external nares covered with umbilical tape to control hemorrhage. a rhinoscopy should be performed to determine the cause of ongoing hemorrhage. continued excessive hemorrhage can be controlled with ligation of the carotid artery on the side of the hemorrhage, or with percutaneous arterial embolization. systemic thromboembolism is most commonly recognized in cats with cardiomyopathies (hypertrophic, restrictive, unclassified, and dilatative) but can also occur in dogs with hyperadrenocorticism, disseminated intravascular coagulation (dic), systemic inflammatory response syndrome (sirs), protein-losing enteropathy and nephropathy, and tumors affecting the aorta and vena cava. thrombosis occurs through a complex series of mechanisms when the components of virchow's triad (hypercoaguable state, sluggish blood flow, and vascular endothelial injury or damage) are present. in cats, blood flow through a severely stretched left atrium is a predisposing factor to the development of clots and thromboembolism.the most common site of embolism is the aortic bifurcation, or "saddle thrombus." other, less common locations of thromboembolism include the forelimbs, kidneys, gastrointestinal tract, and cerebrum. diagnosis usually is made based on clinical signs of cool extremities, the presence of a cardiac murmur or gallop rhythm, auscultation of pulmonary crackles resulting from pulmonary edema, acute pain or paralysis of one or more peripheral extremities, respiratory distress, and pain and lack of a palpable pulse in affected limbs. the affected nailbeds and paw pads are cyanotic, and nails do not bleed when cut with a nail clipper.client education is one of the most important aspects of emergency management of the patient with thromboembolic disease. concurrent congestive heart failure (chf) occurs in % to % of cats with arterial thromboembolism. more than % of cats are euthanized during the initial thromboembolic event because of the poor long-term prognosis and the high risk of recurrence within days to months after the initial event, even with aggressive therapy. although the long-term prognosis varies from months to years after initial diagnosis and treatment, in the majority of cats thromboembolic disease recurs within months. rectal temperature hypothermia and bradycardia on presentation are negative prognostic indicators.immediate treatment of a patient with chf and thromboembolic disease involves management of the chf with furosemide, oxygen, and vasodilators (nitroglycerine paste, morphine, nitroprusside). additional management includes analgesia (butorphanol, . - . mg/kg iv, im) and prevention of further clot formation. aspirin ( mg/kg po q h) is beneficial bcause of its antiplatelet effects. heparin works in conjunction with antithrombin to prevent further clot formation ( - units/kg iv, followed by - units/kg sq q h in cats, and - units/kg sq q h in dogs). acepromazine can cause peripheral vasodilation and decreased afterload but also can promote hypotension in a patient with concurrent chf. acepromazine ( . - . mg/kg sq) should be used with extreme caution, if at all.thrombolytic therapy can also be attempted, but in most cases is not without risk, and may be cost-prohibitive for many clients. streptokinase ( , units iv over minutes and then , units/hour iv cri for hours) was administered with some success in cats; however, many died of hyperkalemia or other complications during the infusion. tissue plasminogen activator ( . - mg /kg/hour iv cri, up to mg/kg total dose, to effect) has been used with some success but is cost-prohibitive for most clients. side effects of thrombolytic therapy include hyperkalemia with reperfusion and hemorrhage.in cats, the primary cause of arterial thromboembolism is cardiomyopathy. once an animal is determined to be stable enough for diagnostic procedures, lateral and dv thoracic radiographs and an echocardiogram should be performed. ultrasound of the distal aorta and renal arteries should also be performed to determine the location of the clot and help establish the prognosis.other diagnostic procedures to evaluate the presence and cause of thromboembolism include a complete blood count, serum biochemistry profile, urinalysis (to rule out proteinlosing nephropathy), urine protein:creatinine ratio, antithrombin levels, acth stimulation test (to rule out hyperadrenocorticism), heartworm antigen test (in dogs), thyroid profile (to rule out hyperthyroidism in cats, and hypothyroidism in dogs), thoracic radiographs, arterial blood gas analyses, coagulation tests, and coombs' test. selective and nonselective angiography can also be performed to determine the exact location of the thrombus.long-term management of thromboembolism involves management of the underlying disease process and preventing further clot formation. begin therapy with heparin until the aptt becomes prolonged . times; then administer warfarin ( . - . mg/kg/day). monitoring therapy based on prothrombin time and the international normalized ratio (inr, . - . ) is recommended. low-dose aspirin ( - mg/kg q h) also has been recommended. physical therapy with warm water bathing, deep muscle massage, and passive range-of-motion exercises should be performed until the patient regains motor function. future therapy may involve the use of platelet receptor antagonists to prevent platelet activation and adhesion. key: cord- -uih jf w authors: li, diya; chaudhary, harshita; zhang, zhe title: modeling spatiotemporal pattern of depressive symptoms caused by covid- using social media data mining date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: uih jf w by may , the coronavirus disease (covid- ) caused by sars-cov- had spread to countries, infecting more than . million people, and causing , deaths. governments issued travel restrictions, gatherings of institutions were cancelled, and citizens were ordered to socially distance themselves in an effort to limit the spread of the virus. fear of being infected by the virus and panic over job losses and missed education opportunities have increased people’s stress levels. psychological studies using traditional surveys are time-consuming and contain cognitive and sampling biases, and therefore cannot be used to build large datasets for a real-time depression analysis. in this article, we propose a corexq algorithm that integrates a correlation explanation (corex) learning algorithm and clinical patient health questionnaire (phq) lexicon to detect covid- related stress symptoms at a spatiotemporal scale in the united states. the proposed algorithm overcomes the common limitations of traditional topic detection models and minimizes the ambiguity that is caused by human interventions in social media data mining. the results show a strong correlation between stress symptoms and the number of increased covid- cases for major u.s. cities such as chicago, san francisco, seattle, new york, and miami. the results also show that people’s risk perception is sensitive to the release of covid- related public news and media messages. between january and march, fear of infection and unpredictability of the virus caused widespread panic and people began stockpiling supplies, but later in april, concerns shifted as financial worries in western and eastern coastal areas of the u.s. left people uncertain of the long-term effects of covid- on their lives. in december , an outbreak of pneumonia caused by a novel coronavirus (covid- ) occurred in wuhan and spread rapidly throughout the globe [ ] . the covid- outbreak has forced people to change their regular routine lives and practice social distancing. such a sudden change can drastically increase people's stress level and lead to other mental health issues. the difficulties caused by the covid- outbreak in different geographic regions can determine the cause and degree of stress in people, which corresponds to their risk of developing serious depression [ ] . according to a poll [ ] , nearly half ( %) of adults in the united states reported that their mental health has been negatively impacted due to worry and stress over the virus. as the pandemic continues, it is likely that the mental health burden will increase as people's sense of normalcy continues to be disrupted by social distancing, business and school closures, and shelter-in-place orders. the preexisting stress, constant unpredictability, and lack of resources lead to even greater isolation and financial distress. traditional mental health studies rely on information primarily collected through personal contact with a healthcare professional or through survey-based methods (e.g., via phone or online questionnaire). for instance, the patient health questionnaire (phq) is a self-administered version of the primary care evaluation of mental disorders (prime-md) diagnostic instrument for common mental disorders [ ] . however, these survey-based methods are time-consuming and suffer from cognitive and sampling biases, and therefore cannot be used to build large datasets for a real-time depression analysis [ ] . furthermore, understanding of spatial epidemic trends and geographic distribution patterns of covid- provides timely information on people's risk perception of epidemics. however, these important spatial and environmental leading factors are difficult to include in a survey-based method to model covid- related mental stress. geographic information system (gis) and social media data mining have become essential tools with which to examine the spatial distribution of infectious diseases [ ] [ ] [ ] , and can be used to investigate the spatiotemporal pattern of mental stress caused by the pandemic. for instance, social media data (e.g., twitter data) provide a unique opportunity to learn about the users' moods, feelings, and behaviors that reflect their mental health as they experience daily struggles [ ] [ ] [ ] . many articles focused on using feature-based approaches to perform sentiment and emotional analysis using twitter data [ ] [ ] [ ] [ ] . for instance, go and colleagues [ ] investigated the usage of unigrams, bigrams, and their combination in training the classifiers for sentiment analysis of tweets. various supervised classifiers were trained, including maximum entropy, naïve bayes [ ] , and support vector machine (svm) classifiers and their performance on the n-grams was compared. however, some methods previously used [ ] have become outdated; for instance, they took emoticons into account for their sentiment index, but nowadays lots of twitter users use emojis more frequently [ ] . barbosa and feng [ ] showed that n-grams are not useful in classifying tweets, as unused words in tweets can cause problems during classifier training. pak and paroubek [ ] proposed the usage of microblogging features like hashtags, emoticons, re-tweets, and comments to train an svm classifier and showed that it resulted in higher accuracy than training using n-grams. several articles address the effect of using part-of-search (pos) tag features in text classifiers [ , ] . abadi and colleagues [ ] investigated pos, lexicon, and microblogging features. the results showed that the most relevant features are those that combine prior polarity with the pos tags of the words. however, there have been mixed results reported on the usage of pos tags. go and colleagues [ ] showed that the pos tags caused reduced performance, although pos tags can be strong indicators of emotions in text and serve as a helpful feature in opinion or sentiment analysis [ ] . moreover, bootstrapping approaches, which rely on a seed list of opinion or emotion words to find other such words in a large corpus, are becoming more popular and have proven effective [ ] [ ] [ ] [ ] . mihalcea, banea, and wiebe [ ] described two types of methods for bootstrapping the subjectivity lexicons into dictionary-based and corpus-based. their research began with a small seed set of hand-picked subjective words, and with the help of an online dictionary produced a larger lexicon of potential candidate words. a similar bootstrapping model was effectively used to build a sentiment analysis system for extracting user-generated health review about drugs and medication [ ] . however, all the aforementioned methods only detect the general emotion of tweets and lack the ability to model depression levels in detail. latent dirichlet allocation (lda) is one of the most commonly used unsupervised topical methods, where a topic is a distribution of co-occurring words [ ] . however, the topics learned by lda are not specific enough to correspond to depressive symptoms and human judgments [ ] . the unsupervised method can work with unclassified text, but it often causes topics overlap [ ] . later, the lda method was extended by using terms strongly related to phq- depression symptoms as seeds of the topical clusters and guided the model to aggregate semantically-related terms into the same cluster [ ] . however, this approach only detects the presence, duration, and frequency of stress symptoms, ignoring the spatial context or environmental factors that are important in modeling the covid- related mental stress. to identify phq related text and unrelated text, a sentiment analysis index generated by python textblob was used [ ] , which only calculates the average polarity and subjectivity over each word in a given text using a constant dictionary [ , ] . work based on the lda probabilistic generative model was found to have limitations related to interpreting high dimensional human input factors which makes it difficult to generalize generative models without detailed and realistic assumptions for the data generation process [ ] [ ] [ ] . in this article, we propose a corexq algorithm that integrates correlation explanation (corex) learning algorithm and clinical phq lexicon to detect covid- related stress symptoms at a spatiotemporal scale in the united states. we aim to investigate people's stress symptoms in different geographic regions caused by the development of the covid- spread. since twitter data are high-dimensional human input data with diverse terms used to express emotions, we used the corex algorithm, a method intended to bypass the limitations of lda implementation and minimize human intervention [ ] . after that, we developed a fuzzy accuracy assessment model to visualize the uncertainty of the analytical results on the map. the rest of the article is organized as follows: section introduces the material and methods used in the research work including the introduction of data collection and processing methods, basilisk and machine learning classifier, and the proposed corexq algorithm. the results and discussion are presented in sections and , respectively. section draws conclusions. twitter data used in this article were collected through the twitter api from january to april for the continental united states. the collected data contained million tweets (~ gb), which posed significant computationally intensive challenges for the traditional gis computing environment. to address this challenge, we used a jupyter computing environment deployed on the texas a&m high performance computer. we filtered the collected twitter data using coronavirus related entities (e.g., hashtag, trends, and news). then, we removed irrelevant information (e.g., non-english language tweets, punctuation, missing data, messy code, url, username, hashtags, numbers, and query terms) from the filtered tweets. some adjustments and normalizations (e.g., uniform lower case, nonmaize vectorized tweets, standardize time sliced tweets) were also made in order to fulfill the common requirements of machine learning models. however, the stop words were removed later when applying the proposed algorithm to match the tweet phrase with lexicon. after that, the tweets were tokenized using the natural language toolkit's (nltk) tweettokenizer [ ] . we also replaced repeated character sequences by using the length value of three for any sequences of length three or greater ( +), since most users often extend words or add redundant characters to express strong feelings. tweets with an exact geospatial tag and timestamp were mapped to the corresponding county using reverse geocoding method [ , ] . other tweets (e.g., without geotags but containing user-defined location information in the user's profile) were geocoded to their corresponding county using a fuzzy set search method and city alias dataset [ ] . we excluded tweets that did not have geotags nor user-defined location information. one of the key innovations in our research was to map the covid- caused stress symptoms at a temporal scale. in this case, we set the temporal scale to biweekly starting from january , so the number of tweets collected in each county could be sufficient for accurate and reliable analysis. we used the basilisk bootstrapping algorithm to find semantic lexicons that could be used to divide the tweets into two categories: stressed and non-stressed. the bootstrapping approach to semantic lexicon induction using semantic knowledge, also known as the basilisk algorithm, was developed by thelen and riloff in [ ] . this approach can extend to divide the tweets into multiple categories across different areas [ ] . it employs a bootstrapping method to determine high-quality semantic lexicons of nouns. the algorithm takes a huge unannotated corpus from where it finds new related words and assigns them to the different semantic categories (e.g., stressed and non-stressed in our case). it is a form of categorization that is based on the seed words manually provided to the algorithm. these seed words are bootstrapped to identify new words that fall within the two categories. basilisk must be seeded with carefully selected terms for it to be effective. the two categories of seeds used for this task consisted of words each (table ) [ ]. the first category contained words describing stress and were used to bootstrap other words semantically related to stress or carrying a similar context. the second category contains words that describe non-stressed or a relaxing behavior. these two categories can be thought of as words that fall at the opposite ends of a stress level spectrum. before the bootstrapping process, the patterns were extracted on the unannotated corpus. this is used to extract all the noun phrases that were either the subject, direct object or prepositional phrase. the noun phrases were extracted from the corpus using the stanford dependency parser [ ] . it is a natural language parsing program used to find grammatical structure in sentences and can be used to find relationships or dependencies between nouns and the actions or words that form a group and go together. the dependency parser was run on all the sentences in the corpus and dependency relations were extracted for each word in the text (in the conll-u format [ ]). for each tweet, the following dependency information was extracted. the conll-u format of the extracted dependency pattern consists of the index, text, lemma, xpos, feats, governor, and dependency relations ( table ). these extracted dependency relations were used to extract patterns that were used by the basilisk algorithm to generate seeds. these extraction patterns were created for each dependency relation obtained in the previous step. the extraction patterns consisted of noun phrases and the dependency of them with other related words in the sentence. this acted as the input to the bootstrapping method. after the input was generated, the next step was to generate the seeds using basilisk. the seed words from the initial pattern pool enlarge with every bootstrapping step. the extraction patterns were scored using rlogf metric [ ] , which is commonly used for extraction pattern learning [ ] . the score for each pattern was computed as: rlogf(pattern (i) ) = f i n i * log f i , where f i represents the number of category members extracted by pattern (i) and n i is the total number of nouns extracted by pattern i . this formula was used to score the patterns with a high precision or moderate precision but a high recall. the high scoring patterns were then placed in the pattern pool. after this process, all head nouns co-occurring with patterns in pattern pool were added to the candidate word pool. at the end of each bootstrapping cycle, the best candidates were added to the lexicon thus enlarging the lexicon set. the process used related to basilisk, as proposed by thelen and riloff, can be described using the algorithm shown on table (for notation description see appendix a). this performs the categorization task of assigning nouns in an unannotated corpus to their corresponding semantic categories. using the words generated by the basilisk algorithm, we counted the total number of occurrences of any of the keywords in both categories. after the total count of stress and non-stress words in each tweet was obtained, we determined whether the tweet was in the category of stressed or non-stressed or neutral. this was done by finding the maximum of the stress and non-stress word counts in three conditions: ( ) if there were more stress words than non-stress words, we annotated the tweet as expressing stress. ( ) if the number of non-stress words is greater than the number of stress words, we annotated the tweet to express relaxed behavior. ( ) if the count was zero for both stress and non-stress words, we did not annotate the data. thus, tweets and their corresponding labels generated using this process were the initial training set, which was used to train a classifier to classify the other unannotated tweets. table . illustration of the basilisk algorithm [ ] . procedure: lexicon = {seed words} for i := . score all extraction patterns with rlogf . pattern pool = top ranked + i patterns . candidate word pool = extractions of patterns in pattern pool . score candidate words in candidate word pool . add top five candidate words to lexicon . i := i + . go to step . the universal sentence encoder [ ] was used to generate word embeddings. these text embeddings convert tweets into a numerical vector, encoding tweet texts into high dimensional vectors that are required to find semantic similarity and perform the classification task. it takes a variable length english text as input and outputs a -dimensional vector embedding. the encoder model was trained with a deep averaging network (dan) encoder [ ] . after the word embeddings were obtained for each stressed and non-stressed category tweet, a technique was used to make the two classes equalized. to do this, we selected the category with fewer samples and made the other category a similar size by removing samples. this ensured that the training process was not biased towards a particular class. before training the classifier, the data were split into training and validation sets. the data were randomly shuffled and put into the two datasets, with % used as the training dataset. to obtain the best performance, multiple classifiers were used, and performance was compared using accuracy metrics. the classifiers used in the training process were svm [ ] , logistic regression [ ] , naïve bayes classifier [ ] , and a simple neural network. svm handles nonlinear input spaces and separates data points using a hyperplane using the largest amount of margin. as a discriminative classifier, svm found an optimal hyperplane for our data, which helped with classifying new unannotated data points. we used different kernels to train the svm. the hyperparameters were tuned and the optimal value of regularization and gamma were also recorded. the logistic regression classification algorithm can be used to predict the probability of a categorical dependent variable. the dependent variable is a binary variable that contains data coded as (stressed) or (non-stressed). the logistic regression model predicts p(y = ) as a function of x. prior to training, it shuffles the data. it uses a logistic function to estimate probabilities to calculate the relationship between independent variable(s) and the categorical dependent variable [ ] . naïve bayes is another probabilistic classifier which makes classifications using the bayes rule. this classifier is simple and effective for text classification. a simple neural network consisting of three dense layers were used to train our datasets. the loss function and optimizer used in the training is binary cross entropy and rmsprop, respectively. training was done for epochs with a batch size of . table illustrates the performance evaluation of these classifiers. after the model was trained, the model was run on the unannotated tweets to label them. to label the sentence embeddings for the tweets, the same procedure was used as for the training set. the universal sentence encoder extracts features and created vectors that were used to classify the tweets based on the model. the svm classifier with linear kernel was used to predict the probabilities of the tweets because it had the best trained models (see table ). here, a threshold of . was set to determine if the tweet belonged to a particular category or not. if the probability of the tweet was above . for that category, the tweet was classified with the corresponding label. the tweets and labels generated using the above process were then used to train another classifier to generate the final model for classification of the entire unannotated corpus. here, a logistic regression model was used to train tweets and their corresponding labels generated using the above process to ensure that the model was robust and was not overfitted on the initial set of tweets that were filtered out using the basilisk generated keywords. the trained model had an accuracy of . % on the validation data. in this article, we propose a novel corexq algorithm to detect spatiotemporal patterns of covid- related stress. table illustrates the general structure of the corexq algorithm. the input of the algorithm was the stressed-related tweets derived by using the trained models (see sections . . and . . ) to all the processed covid- related tweets. we assessed the level of stress expressed in covid- related tweets by integrating a lexicon-based method derived from established clinical assessment questionnaire phq- [ ] . table illustrates the phq- lexicon examples and their corresponding mental stress symptoms. procedure: . shallow parsing each tweet into tweet_pharse using spacy . for each word_set in phq_lexicon do . calculate average vector of word_set and tweet_pharse using glove . match word_set with tweet_pharse set using cosine similarity measure . append each matched tweet_pharse to word_set . calculate tf-idf vector for all the tweets and transform the calculated value to a sparse matrix x . iteratively run corex function with initial random variables v random . estimate marginals; calculate total correlation; update v random . for each word_set in phq_lexicon . compare v random and word_set with bottleneck function . until convergence the phq- lexicon contains about clinical words, which is difficult to understand and match with the spoken language that is often used on twitter. therefore, we used the following methods to transform phq- lexicon to human understandable language by appending matched tweets to their best match phq- categories. in the first step, each tweet was placed into a set of phrase sets using natural language processing toolkit spacy [ ] (see table , procedure ). after that, the tweets and phq- lexicon were vectorized using global vectors for word representation (glove), wikipedia, and gigaword model (with dimensional word vectors and four million unique tokens) [ ] . glove provides a quantitative way to distinguish the nuance difference of two words (e.g., happy or unhappy), which is useful to match phrases set with the phq- lexicon. those pre-trained vectors were loaded to gensim [ ] to perform average vector and cosine distance calculation (see equations ( ) and ( )). we appended all phrases that have the similarity rate higher than . to their corresponding phq- lexicon (see table , procedures - ). given any words in a phrase, the average vector was calculated using the sum of the vectors divided by the number of words in a phrase: given any two average vectors v a and v b of two phrases, the cosine similarity, cos θ, is represented by next, a sparse matrix (e.g., a vocabulary dense matrix) for stressed corpus was calculated by transforming those tokenized and vectorized tweets using frequency inverse document frequency (tfidf). the mathematical formula of tfidf is illustrated below: where t denotes the terms; d denotes each document; and d denotes the collection of documents. the first part of the formula t f (t, d) calculates the number of times each word in covid- corpus appeared in each document. the second part of id f (t, d) is made up with a numerator d = d , d , . . . d n and a denominator | {d ∈ d : t ∈ d}|. the numerator infers the document space, which is all documents in our covid- stress corpus. the denominator implies the total number of times in which term t appeas in all of our documents d. the id f (t, d) can be represented by we utilized scikit-learn tfidfvectorizer to transform preprocessed tweets to a sparse matrix [ ] (see table , procedure ). after that, the sparse matrix and lexicon are used by the anchored corex model to perform anchored topic modeling [ ] . the total correlation tc [ ] (for notation description see appendix a) of each topic is calculated by anchoring the corex model with the document sparse matrix. the total correlation in our phq- lexicon detection can be expressed using kullback-leibler divergence as below [ ] . where p(x g ) represents the probability distribution and tc(x g ) is non-negative or zero factorizes of p(x g ) (see appendix a for more detail). in the context of phq- detection, x g represents the group of word types among the covid- corpus. note that each vector in the tfidf matrix is based on the distance between two probability distributions, which is expressed as cross-entropy entropy(x) [ , ] . when introducing a random variable y, the tc can explain the correlation reduction in x, which is a measure of the redundant information that the word types x carry about topic y [ ] . the total correlation can be represented by: where i(x : y) = entropy(x) + entropy(y) − entropy(x, y) (for notation description see appendix a). thus, the algorithm starts with randomly initialized variables α i,j and p(y i |x i ), where α i,j are indicator variables of tc that are assigned to if the topic is detected and p(x i ) represents the approximate empirical distribution (see table , procedure ). then, the correlation explanation updates both variables iteratively until the result achieves convergence. in each iteration, the estimate marginals p(y j |x i ) = x p(y i |x)p(x)δ x i and mutual information tc are calculated (notation description see appendix a). next, the update for a t i,j in each t step is calculated by where λ conduct a smooth optimization of the soft-max function [ , ] . finally, the soft labeling of any x (for notation description see appendix a) can be computed by after the soft-max function α converges to the true solution at a particular step α k in the limit λ → ∞ , the mutual information terms can be ranked by the informative order in each factor. to perform semi-supervised anchoring strategies, gallagher and reing proposed the combination with bottleneck function and total correlation [ ] . the bottleneck function can be represented by: the connection with corex and anchor words can be described by comparing equation ( ) with equation ( ). the same term i(x : y) in two equations represents the latent factor and the variable z corresponds to x i . it is worth noting that z is typically labeled in a supervised learning task [ ] and β is a constant parameter to constrain supervising strength so that α = β can imply a word type x i correlated with topic y j . in this case, z was represented by each variable generated by the enriched phq- lexicon. to seed lexicon to detect topics, we can simply anchor the word type x i to topic y j , by constraining the β (see table , procedures - ). the symptoms of covid- related stress were visualized at the county level biweekly from january. here, we used the fuzzy accuracy assessment method to evaluate the uncertainty of final phq stress level for each county [ , ] . we summarized the implementation of fuzzy accuracy assessment for a thematic map as presented by gopal and woodcock to explain our model evaluation for the phq map [ ] . let x be a finite universe of discourse, which is the set of county polygons in the study area. let ζ denote the finite set of attribute membership function (mf) topics categories to the d in x; and let m be the number of categories |ζ| = m, (e.g., nine phq categories). for each x x, we define χ(x) as the mf classes assigned to x. the set: defines the data. the subset s ⊂ x of n data is used. a fuzzy set is associated with each class c ζ where µ c (x) is the characteristic of mf of c. the fuzzy set can be represented as: to implement a decision-making system for fuzzy accuracy, the model uses a boolean function σ that returns results of or based on whether x belongs to the class c with respect to the matrix a. that is, σ(x, c) = if x "belongs" c, and σ(x, c) = if x does not "belong" to c. then σ(x, c) is if the numeric scale of the mf for x in category c(µ c (x)) is maximum among all map categories µ c (x), and we set the boolean function σ as max follows: according to the fuzzy set accuracy assessment, the final phq value for each county was selected based on the max function, meaning each county was colored based on the majority tweet phq value derived from the proposed corexq algorithm. since the accuracy assessment was based on a comparison of the phq label assigned to each county with the evaluation given by the expert (e.g., in each county, the majority tweet phq label). the rating system can thus be expressed as linguistic variables that describe the uncertainty associated with the evaluation of the class label. here, the linguistic variables are described below: . score : understandable: the answer is understandable but may contain high levels of uncertainty; . score : reasonable: maybe not the best possible answer but acceptable; . score : good: would be happy to find this answer given on the map; . score : absolutely right: no doubt about the match. it is a perfect prediction. figure illustrates the fuzzy mf created for the fuzzy accuracy assessment analysis. the x-axis represents the percentage of the tweets that belong to the assigned final phq category. the y-axis represents the value of the degree of the membership function corresponding to the linguistic score. for instance, if a county was assigned to a phq category , and % (e.g., x = . in figure ) of the tweets within this county polygon were labeled as phq- using the corexq algorithm, the corresponding mf should be absolutely right with membership value equal to . the accuracy assessment score was further visualized on the phq stress map to show the spatial uncertainty of the analysis results. the numeric scale of the mf for in category ( ( )) is maximum among all map categories ′ ( ), and we set the boolean function as follows: according to the fuzzy set accuracy assessment, the final phq value for each county was selected based on the max function, meaning each county was colored based on the majority tweet phq value derived from the proposed corexq algorithm. since the accuracy assessment was based on a comparison of the phq label assigned to each county with the evaluation given by the expert (e.g., in each county, the majority tweet phq label). the rating system can thus be expressed as linguistic variables that describe the uncertainty associated with the evaluation of the class label. here, the linguistic variables are described below: . score : understandable: the answer is understandable but may contain high levels of uncertainty; . score : reasonable: maybe not the best possible answer but acceptable; . score : good: would be happy to find this answer given on the map; . score : absolutely right: no doubt about the match. it is a perfect prediction. figure illustrates the fuzzy mf created for the fuzzy accuracy assessment analysis. the x-axis represents the percentage of the tweets that belong to the assigned final phq category. the y-axis represents the value of the degree of the membership function corresponding to the linguistic score. for instance, if a county was assigned to a phq category , and % (e.g., x = . in figure ) of the tweets within this county polygon were labeled as phq- using the corexq algorithm, the corresponding mf should be absolutely right with membership value equal to . the accuracy assessment score was further visualized on the phq stress map to show the spatial uncertainty of the analysis results. since corexq represents topic and potential symptoms as a lexicon-based topic modeling, traditional measures such as regression correlation and log-likelihood are unnecessary for the semantic topics. therefore, to evaluate the baseline performance of the corexq model, we first involved the semantic topic quality coherence measure methods with other common topic models. we compared corexq with lda and non-negative matrix factorization (nmf) [ , ] . in addition, we used frobenius normalized nmf (nmf-f) and generalized kullback-leibler divergence nmf (nmf-lk) for a closer comparison with traditional topic modeling. all models were trained with a since corexq represents topic and potential symptoms as a lexicon-based topic modeling, traditional measures such as regression correlation and log-likelihood are unnecessary for the semantic topics. therefore, to evaluate the baseline performance of the corexq model, we first involved the semantic topic quality coherence measure methods with other common topic models. we compared corexq with lda and non-negative matrix factorization (nmf) [ , ] . in addition, we used frobenius normalized nmf (nmf-f) and generalized kullback-leibler divergence nmf (nmf-lk) for a closer comparison with traditional topic modeling. all models were trained with a randomly selected covid- twitter dataset. the topics generated by those models were scored by topic coherence measures to identify the degree of semantic similarity between high-scoring words in the topic. a common coherence measure is umass which calculates and scores the word co-occurrence in all documents [ ] : where d(w i , w j ) represents the number of documents containing both w i and w j words and d(w i ) counts the ones containing w i , and c represents a smoothing factor. the intrinsic umass [ ] coherence measure calculates these probabilities over the same training corpus. additionally, the extrinsic uci measure [ ] introduced by david newman uses a pairwise score function, which is based on pointwise mutual information (pmi). it can be represented by: where p(w i ) represents the probability of seeing w i in a random document, and p(w i , w j ) is the probability of seeing both w i and w j co-occurring in a random document. those probabilities are empirically estimated from an external dataset such as wikipedia. the higher the topic coherence measure score, the higher the quality of the topics. in our baseline evaluation, we calculated the coherence scores by setting the range of topic numbers from to . the abnormal and low-quality topics were cleared and the average coherence scores (table ) were calculated by the sum of all coherence scores divided by the number of topics. on average, the corexq algorithm has a better umass score than lda and nmf. even though the uci score was slightly lower than two types of nmf algorithms, we can take the external estimation dataset as an uncertainty factor of this coherence model because the result of the comparison was still meaningfully coherent and it has the competitive functionality of the semi-supervised feature, which exceeded the usable range of nmf. in our research, the methods described above were combined to generate the final thematic map. to summarize processes for each detailed procedure, the workflow for the research is shown in figure . first, starting from data collection, we prepared a twitter dataset, basilisk lexicon, and phq- lexicon. then, we cleaned each tweet and extracted its location information using the method mentioned in section . . to engage time series analysis, the whole twitter dataset was formatted and sorted by unix timestamp before being sliced into two-week intervals. third, two lexicons were separately assigned to corexq and basilisk algorithm (mentioned in section . ) with the prepared twitter dataset. in the end, we decomposed the result generated by anchored corex model into spare matrix in order to group by all tweets in county level for visualization. note that each row of the results from the corex algorithm represents the correlations index within an individual tweet explained by nine phq levels so that we can reverse convert the result to its original tweets. the selected top symptoms and topics are present in table . the fuzzy accuracy assessment results of the study are illustrated in figure . on each map, the individual county is colored according to the assigned phq index using the proposed algorithm and fuzzy assessment accuracy assessment method. the numbers on the map represent the spatial uncertainty indices derived from the fuzzy accuracy assessment. each number represents the assessment score calculated from section . . . for most of the hot spots areas in figure , the values are greater than two, which indicates middle to high accuracy results have been reached for those regions. higher scores for an area indicate a larger percentage of the topics being present in this area at specific time region. the fuzzy accuracy assessment results of the study are illustrated in figure . on each map, the individual county is colored according to the assigned phq index using the proposed algorithm and fuzzy assessment accuracy assessment method. the numbers on the map represent the spatial uncertainty indices derived from the fuzzy accuracy assessment. each number represents the assessment score calculated from section . . . for most of the hot spots areas in figure , the values are greater than two, which indicates middle to high accuracy results have been reached for those regions. higher scores for an area indicate a larger percentage of the topics being present in this area at specific time region. the results also present the spatiotemporal patterns from january to april (shown in figure a -g. table shows the detected stress symptoms and topics generated from corexq . each map represents the spatial distribution of stress symptoms over a biweekly period. it indicates that most of the regions have low to medium phq values (topic - ) during january and february, since information about the u.s. covid- outbreak was not publicly available in the u.s. during that time. most counties that have a low phq level contain general covid- related topics that are tied to the cases in asia and general symptoms of covid- (e.g., "wenliang li" (a chinese doctor) [ ] , "south korea confirms", "coughing", "sneezing"). from the end of january, a few hotspots appear in some major u.s. cities such as san francisco, denver, los angeles, and seattle with topics related to "mistakenly released", "vaccine", "pandemic bus", and "china death" (see table , figure b ,c). for instance, the keyword "mistakenly released" reflects news story in february about the first u.s. evacuee from china known to be infected with the coronavirus being mistakenly released from a san diego hospital and returned to quarantine [ ] . people who living in california reacted strongly to this news (figure d) . later, on march (figure c,d) , the phq level started to increase rapidly due to the covid- test stations available, increased number of covid- death cases, and a shelter-in-place order in many states (see table , march). an interesting pattern was found that the number of counties with a high phq value kept growing until april and started to decrease after the second week of april [ ] . figure illustrates the number of increased cases in the u.s. from january to may . results show that the phq stress level in our results matches well with the number of increased cases illustrated in the johns hopkins coronavirus resource centers' statistical analysis results [ ] . this means the number of new cases reduced due to the social distancing practice, and at the same time, the level of people's major concerns in many geographic regions reduced as well. the results also present the spatiotemporal patterns from january to april (shown in figure a g. table shows the detected stress symptoms and topics generated from corexq . each map represents the spatial distribution of stress symptoms over a biweekly period. it indicates that most of the regions have low to medium phq values (topic - ) during january and february, since information about the u.s. covid- outbreak was not publicly available in the u.s. during that time. most counties that have a low phq level contain general covid- related topics that are tied to the cases in asia and general symptoms of covid- (e.g., "wenliang li" (a chinese doctor) [ ] , "south korea confirms", "coughing", "sneezing"). from the end of january, a few hotspots appear in some major u.s. cities such as san francisco, denver, los angeles, and seattle with topics related to "mistakenly released", "vaccine", "pandemic bus", and "china death" (see table , figure b,c) . for instance, the keyword "mistakenly released" reflects news story in february about the first u.s. evacuee from china known to be infected with the coronavirus being mistakenly released from a san diego hospital and returned to quarantine [ ] . people who living in california reacted strongly to this news (figure d) . later, on march (figure c,d) , the phq level started to increase rapidly due to the covid- test stations available, increased number of covid- death cases, and a shelter-in-place order in many states (see table , march). an interesting pattern was found that the number of counties with a high phq value kept growing until april and started to decrease after the second week of april [ ] . figure illustrates the number of increased cases in the u.s. from january to may . results show that the phq stress level in our results matches well with the number of increased cases illustrated in the johns hopkins coronavirus resource centers' statistical analysis results [ ] . this means the number of new cases reduced due to the social distancing practice, and at the same time, the level of people's major concerns in many geographic regions reduced as well. our results also show a meaningful explanation of the spatial pattern caused by people's risk perception to various media messages and news during the pandemic. in march , people in the united states had mild concerns about the uk prime minister boris johnson's talk of "herd immunity" [ ] and social distancing (see table , phq , march). on the other hand, the major stress came from topics such as cases of deaths (e.g., in washington state), lack of food and covid- protection equipment (e.g., panic buy), and the increasing number of confirmed and death cases in our results also show a meaningful explanation of the spatial pattern caused by people's risk perception to various media messages and news during the pandemic. in march , people in the united states had mild concerns about the uk prime minister boris johnson's talk of "herd immunity" [ ] and social distancing (see table , phq , march). on the other hand, the major stress came from topics such as cases of deaths (e.g., in washington state), lack of food and covid- protection equipment (e.g., panic buy), and the increasing number of confirmed and death cases in the united states. figure d ,e shows that most of the hotspots were located in washington, california, and new york, and florida matched with to the march covid- increased cases map (see [ ] . in april, keywords such as "death camps", "living expenses", "white house", and "economy shrinks" (see table ) appeared most often in the high phq value categories, which indicated that people's major concerns shifted to financial worries due to businesses shutting down and the economic depression [ ] . our study was conducted to perform a spatiotemporal stress analysis of twitter users during covid- pandemic by the corexq algorithm. according to the model evaluation results, the proposed corexq had the best baseline performance among other similar algorithms such as lda, nmf-lk, and nmf-f models. in addition to the corexq algorithm, we applied a fuzzy accuracy assessment method to the corexq analysis results to visualize the spatial uncertainty of the analysis results. this enables expert knowledge (e.g., phq rating of tweets) to be integrated in the social media data mining process. the result of our observed pattern reasonably matched the relevant events and epidemic trends. ideally, the analytic result of our collected twitter dataset is expected to support the research of mental health for the entire u.s. population as a sample case. in our cleaned twitter dataset, those tweets were posted by , , users, which represent over . % of the u.s. population. however, a previous investigation found that the % of american adults who use twitter are not uniformly distributed across age [ , ] . another study found that twitter users are getting younger [ ] , but the actual age, gender, and race of twitter users from those investigations have been controversial [ ] . to generalize the psychology health analysis to the whole u.s. population, further work related to the user demographic is required to reduce the influence of the sample bias. the thematic maps we created for phq topics distribution were assessed based on fuzzy sets. the purpose of this commonly used method for categorical maps is to allow explicit accounts for the possible ambiguity regarding the appropriate map label [ , [ ] [ ] [ ] [ ] . a wide variety of statistical techniques have been proposed for the accuracy assessment of thematic maps [ ] . in the future, we can use the standard deviation approach to estimate the quantity derived from the distribution of the tweets as a count on specific category if the assessment is focused on how the number of labeled phq tweets were distributed in each category. even though our datasets were preprocessed and selected with entities on covid- related topic, some of the tweets might be outside of the topic or are influenced by other objective factors. our future focus of uncertainty assessment of the thematic maps could be to extend to spatial uncertainty [ ] , temporal uncertainty [ ] semantic uncertainty [ ] , etc. our assessment task can be considered a criterion referenced task that can focus on a selected phq level and can represent the majority level in any location. the fuzzy area estimation methods were extended based on previous research [ ] . category assessment based on fuzzy sets can estimate the accuracy of classes as a function of levels of class membership [ ] . here, we used biweekly data as a temporal scale for the analysis. our research group continues collecting twitter data for this project, so analysis could be applied to more fine-grained temporal scales in the future. since coivd- is a global pandemic, this project could be extended to a global scale to compare the results across different countries. in the future, the model could be applied to other cases to detect the related stress symptoms and provide real-time spatial decision support for addressing the problem. an end-to-end spatiotemporal analysis system could be built if all of the modules were integrated; this would increase the efficiency of determining the potential symptoms and causes of public mental health problems. in this article, we proposed the corexq algorithm to analyze the covid- related stress symptoms at a spatiotemporal scale. the corex algorithm combined with clinical stress measure index (phq- ) helped to minimize human interventions and human language ambiguity in social media data mining for stress detection and provided accurate stress symptom measures of twitter users related to the covid- pandemic. there was a strong correlation between stress symptoms and the number of increased new covid- cases for some major u.s. cities such as chicago, san francisco, seattle, new york, and miami. people's risk perceptions were sensitive to the release of covid- related public news and media messages. many frequently appearing keywords in the high phq value categories represent the popular media and news publications at that time. before march, most regions had mild stress symptoms due to the low number of reported cases caused by the unavailability of test stations, creating a false sense of security among the public in the united states. the number of cases increased suddenly in march due to governmental confirmation of the seriousness of the pandemic in the united states and shelter-in-place orders in many states. from january to march, a major concern for people was being infected by the disease and there was panic-buying behavior, but this shifted to financial distress later in april along coastal eastern and western united states. our main contributions are as follows: first, we introduced a specialized stress tweets classifier, which narrows down the theoretical algorithms to practical usage on the public health area and demonstrates more effectiveness than traditional sentiment index classifiers. second, we framed corexpq as a topic detection model in our research. we explored the latent connection between the social media activity and phq- depression symptoms and topics in united states. finally, as a supplement methodology for the existing questionnaire-driven mental health research, our integrated system was used to glean depression topics in an unobtrusive way. the proposed algorithm provides an innovative way to analyze social media data to measure stress symptoms under covid- pandemic at a spatiotemporal scale. by doing this, we were able to observe spatiotemporal patterns of stress symptoms and answer the questions of what the major concerns related to the pandemic in different geographic regions at different time scales were. in the future, this model could be applied to other cases to detect related stress symptoms and provide real-time spatial decision support for addressing arising issues. the authors declare no conflict of interest. table a . notation table. pattern pool a subset of the extraction patterns that tend to extract the seed words candidate word pool the candidate nouns extracted by pattern pool are placed in candidate word pool tc total correlation, also called multi-information, it quantifies the redundancy or dependency among a set of n random variables. kullback-leibler divergence, also called relative entropy, is a measure of how probability distribution is different from a second, reference probability distribution [ ] . p(x g ) probability densities of x g i(x : y) the mutual information between two random variables p(y|x) y's dependence on x can be written in terms of a linear number of parameters which are just the estimate marginals δ the kronecker delta, a function of two variables. the function is if the variables are equal, and otherwise. a constant used to ensure the normalization of p(y|x) for each x. it can be calculated by summing |y| = k, an initial parameter. situation report centers for disease control and prevention mental health and coping during covid- |cdc. available online the impact of coronavirus on life in america the phq- : a new depression diagnostic and severity measure the evolution of cognitive bias covid- : challenges to gis with big data gis-based spatial modeling of covid- incidence rate in the continental united states using twitter and web news 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analysis statistical techniques technical note: statistical methods for accuracy assessment of classified thematic maps geostatistics: modeling spatial uncertainty, second edition spatio-temporal uncertainty in spatial decision support systems: a case study of changing land availability for bioenergy crops in mozambique managing uncertainty and vagueness in description logics for the semantic web using known map category marginal frequencies to improve estimates of thematic map accuracy fuzzy set theory and thematic maps: accuracy assessment and area estimation this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - gyo rya authors: bonvehí, pablo e.; temporiti, elena r. title: transmission and control of respiratory viral infections in the healthcare setting date: - - journal: curr treat options infect dis doi: . /s - - -y sha: doc_id: cord_uid: gyo rya purpose of the review: viral respiratory infections have been recognized as a cause of severe illness in immunocompromised and non-immunocompromised hosts. this acknowledgement is a consequence of improvement in diagnosis and better understanding of transmission. available vaccines and antiviral drugs for prophylaxis and treatment have been developed accordingly. viral respiratory pathogens are increasingly recognized as nosocomial pathogens as well. the purpose of this review is to describe the most frequent and relevant nosocomial viral respiratory infections, their mechanisms of transmission and the infection control measures to prevent their spread in the healthcare setting. recent findings: although most mechanisms of transmission and control measures of nosocomial viral infections are already known, improved diagnostic tools allow better characterization of these infections and also lead to the discovery of new viruses such as the coronavirus, which is the cause of the middle east respiratory syndrome, or the human bocavirus. also, the ability to understand better the impact, dissemination and prevention of these viruses, allows us to improve the measures to prevent these infections. summary: healthcare viral respiratory infections increase patient morbidity. each virus has a different mechanism of transmission; therefore, early detection and prompt implementation of infection control measures are very important in order to avoid their transmission in the hospital setting. healthcare-associated viral infections cause an increase in morbidity and mortality among hospitalized patients and also in healthcare-associated costs. viral respiratory pathogens are increasingly recognized as etiological agents of nosocomial infections. they can be acquired in different settings inside the hospital and they can produce upper (uri) or lower respiratory tract infections (lri) [ •]. transmission can occur by air through aerosolization, by drops of respiratory secretions or by inoculation after touching contaminated surfaces [ ] . among immunocompromised hosts, these types of infections can present themselves with subclinical manifestations and can be associated with high morbidity and mortality. for this reason, a high suspicion vigilance must be kept to confirm viral infections in these hosts. it is also known that viral shedding is more prolonged [ , •] . there are several etiological agents that cause these infections, with different ways of transmission depending on each virus. for this reason, it is mandatory to suspect and correctly identify the etiological agent and promptly implement isolation precautions. molecular methods have improved sensitivity for the diagnosis of viral infections and have also facilitated the identification of other viral pathogens that cause respiratory tract infections [ ] . in the clinical setting, it is sometimes difficult to establish if a respiratory viral infection was acquired in the hospital or in the community. this situation is most commonly observed when the length of hospitalization is less than or equal to the incubation period of the disease. in some cases, virological studies such as next generation sequencing can be used to differentiate between these situations [ ] . as a measure to establish the magnitude of respiratory viral infections in the healthcare setting, it must be noted that % of all nosocomial pneumonias are of viral origin. in this context, the incidence of each virus is related to its circulation in the community [ , ] . the objective of this revision is to describe the means of transmission and the measures to control the dissemination of respiratory viral infections in the healthcare setting. there are several viruses associated with upper and lower respiratory infections in humans. we describe the most relevant ones for the clinician. this virus belongs to the mononegavirales order, paramyxoviridae family and pneumovirinae subfamily. main characteristics of rsv include the number and order of genes and lack of hemagglutinin and neuraminidase activity [ ] . rsv is the main cause of respiratory tract disease in small children. viral propagation is so efficient that humans can acquire this infection in their first years of life. immunity achieved after rsv infection is incomplete and reinfections are frequent. although life threatening infections occur mainly during the first years of life, rsv infections can cause a high morbidity due to acute upper and lower respiratory tract infections. in children, rsv causes bronchiolitis and pneumonia and it is a common pathogen in older adults and immunocompromised hosts, where it can either cause upper respiratory infections or pneumonia [ ] . influenza viruses are single-stranded, negative-sense rna viruses belonging to the orthomyxoviridae family and are classified into three main types, a, b and c, based on their antigenic different characteristics. these three types show significant differences related to their genetic organization, structure, possible hosts, epidemiology and clinical manifestations, with influenza a and b the most common human pathogens. these viruses share certain features such as the presence of an envelope derived from the host cell and surface glycoproteins essential for replication. they have a spheric or filamentous shape and measure to nm with surface projections that are spicules of hemagglutinins (ha) and neuraminidases (na), also important for their antigenic mutations, as they are used as antigens for the influenza vaccine [ ] . this virus is an important cause of infections in immunocompetent and in immunocompromised hosts causing mild upper respiratory infections (pharyngitis, coryza, conjunctivitis) and complicated infections (pneumonia, hepatitis, hemorrhagic cystitis or disseminated diseases), respectively. there are seven species of hadv (a to g) and an increasing number of different serotypes were identified through genomic assays. besides this, the improved understanding of viral persistence sites and reactivation requires continuous adaptations of diagnostic tools in order to facilitate timely detection and control of hadv infections. hadv infections are more frequent in closed communities such as the armed forces, nurseries and hospitals with immunocompromised hosts [ ••] . this is a single-stranded rna virus from the paramyxoviridae family classified into five types: respirovirus genus (piv and ) and rubulavirus (piv , a, and b). [ ] piv serotypes may cause bronchiolitis, laryngotracheobronchitis (croup) and pneumonia in different age groups in immunocompetent hosts, with piv more frequently associated with lower respiratory tract infections. among immunocompromised hosts, piv has been found in upper and lower respiratory tract infections in . - . % of hsct recipients. in solid organ transplant patients, lung recipients have demonstrated to be at risk for piv disease and this infection was also associated to allograft rejection or dysfunction [ ] . these viruses are non-segmented rna viruses, belonging to the mononegavirales order, paramyoviridae family, pneumovirinae subfamily and metapneumovirus genus. initially described as a pathogen in children, where it can cause upper respiratory disease, bronchiolitis and pneumonia, has also been later identified as an adult respiratory viral pathogen [ ] . in immunocompromised hosts, hmpn has also been associated to produce upper and lower respiratory tract infections among hsct patients and solid organ recipients, mainly lung transplant patients [ ] . picornaviridae family includes nine genuses, four of which (rhinovirus, enterovirus, parechovirus and hepatovirus) can infect humans. human rhinoviruses (hrv) were classified based on immune serotypes, type of receptor, antiviral sensitivity and genotype. rhinovirus infectivity can be neutralized by specific antisera and the new serotypes are defined based on the absence of cross reactivity with known specific antisera. hrv are rna viruses and more than serotypes have been identified. clinical importance of hrv infections is well demonstrated in children where they are responsible for more than % of asthma exacerbations [ ] . in immunocompetent adults, these are the most common cause of selflimited upper respiratory tract infections and could be responsible for more than % of common colds in autumn and spring seasons [ ] . immunocompromised hosts are particularly prone to severe infections. in lung transplant patients can be a common infection and lead to graft dysfunction. among hematological malignancies hrv can produce lower respiratory tract infections with potential adverse consequences [ ••, , ] . hcov are positive-stranded rna viruses from the coronaviridae family, causing common colds; in children, they have also been associated with bronchiolitis and croup [ ] . there are five hcov identified as hcov-oc , hcov- e, hcov-hku , hcov-nl and hcov-sars. the latter virus was responsible of an epidemic of severe respiratory disease syndrome in some countries in asia (china, hong kong sar, taiwan, singapore, vietnam) and canada in , with approximately % mortality and high attack rate in healthcare workers and family members; it was detected in humans for the last time in [ , ] . more recently, one coronavirus was able to cause severe respiratory disease in humans in the middle east. this coronavirus was named as mers-cov and was isolated in saudi arabia in from respiratory secretions in a man who died from pneumonia. this finding, combined with a retrospective assessment of stored samples from an outbreak observed in healthcare workers in jordan, allowed the recognition of mers-cov as a cause of this new severe respiratory disease [ • ]. the origin of mers-cov has been widely debated. it was originally believed that the reservoir was bats due to the similarity between mers-cov and bat coronavirus. however, the strain found in humans was not related to the one described in bats. another possible source for this type of coronavirus is the camel because they have antibodies against mers-cov similar to those found in humans. most human cases of mers-cov were described in saudi arabia and, to a lesser degree, in the united arabs emirates. later on, other cases outside the arabian peninsula were notified, but all of them were imported from endemic countries in the middle east, and were related to close contact with sick people. most of the patients were healthy males around years [ ] . this is a single-stranded dna virus that has recently been found as a human pathogen. it belongs to the parvoviridae family, parvovirinae subfamily, bocaparvovirus genus; four genotypes have been described (hbov - ) [ , ] . hbov has been associated with upper and lower respiratory tract infections, sometimes as a single agent or as a co-pathogen with other viruses. it is also found in high proportion in fecal samples of children who had the virus already detected in respiratory samples. it has also been reported in stool and blood of immunosuppressed hosts [ ] . this virus is transmitted through droplets or by direct contact (table ) [ ] . droplet transmission is possible when the source is within m. the size of droplets, produced by coughing, sneezing or any other procedure that moves respiratory secretions, is larger than μm. recent data has demonstrated the transmission of rsv through aerosolized particles based on the fact that they appear close to hospitalized children with bronchiolitis and rsv infections. these particles can remain suspended in the air for prolonged periods of time and can infect human epithelial cells [ ] . the survival of rsv in the environment seems to be partly dependent on drying time and dew point. at room temperature rsv in patients' secretions can be viable on polished surfaces for to h. on porous surfaces such as cloth or tissue papers viability is shorter, usually less than an hour. viral infectivity in the hands varies from person to person, generally less than an hour [ ] . the risk of transmission is not the same in all the hospital areas: in pediatric and neonatal units, risk of transmission occurs between % and %, in transplant and oncohematological units fluctuates between % and %, and in other adult wards between % and % [ ] . it must also be considered that healthcare workers can be a source of transmission for rsv, considering that shedding occurs between % to % of them, even without symptoms [ ] . in high transmission sites, as in oncohematological adult patient wards, it is recommended to manage these infections in ambulatory care to decrease the risk of transmission within the hospital. nevertheless, it has been demonstrated that this group of patients can acquire rsv and piv infections in spite of their short time of hospitalization [ , ] . influenza virus can be transmitted through infectious droplets eliminated by patients when coughing or sneezing, or through direct contact with surfaces contaminated by respiratory secretions from symptomatic infected subjects (table ) [ ] . airborne transmission was observed with influenza a h n p strain; therefore, airborne precautions should also be implemented in case of a pandemic situation where high risk of aerosolization can occur (e.g. intubation) [ ] . in closed areas with high risk patients, such as bone marrow transplant units about % of the infections come from healthcare personnel or infected visitors [ •] . another source of nosocomial transmission of influenza are hospitalized patients with prolonged viral shedding, sometimes for weeks or months. high risk patients for prolonged shedding include those under high doses of steroids, oncohematological patients and children [ , , ] . another potential source of transmission at the hospital are those individuals not yet symptomatic, but within - days prior the infection. these cases are difficult to detect, and hence, preventive control measures are usually not implemented [ ] . different studies show that healthcare workers are one of the main vehicles of transmission of influenza within the hospital. there is data showing that many of them continue to work being ill, which certainly amplifies the rate of transmission [ ] . it is transmitted through direct contact with a contaminated patient's environment and also by fomites containing the virus (table ) . adenovirus can also be transmitted through the fecal-oral route and by organ transplantation from infected donors. it can infect an individual, remain latent for a period of time and later cause disease during immunosuppression [ ] . the ability of adenovirus to survive in the environment on certain surfaces like non-porous ones can be as prolonged as days, facilitating its transmission [ ] . it is transmitted through direct contact with patients' secretions or by direct contact with the contaminated environment (table ) . this virus can survive more than h on porous surfaces and more than h on non-porous surfaces. however, it is difficult to recover it from the hands and only % has been detected after min of contact with contaminated secretions [ ] . piv can also be transmitted through droplets, and nosocomial outbreaks have been described [ , , ] . nosocomial transmission has been reported in pediatric and neonatal emergency wards, as well as in nurseries [ ••] . immunosuppressed hosts, particularly hsct and oncohematological patients, are more prone to present piv infections with an incidence of % and %, respectively [ ] . viral asymptomatic excretion has been described in hsct and oncohematological patients, further increasing transmission in the hospital setting [ ] . hmpn this virus is transmitted by respiratory secretions from coughing, sneezing or through direct contact with contaminated surfaces (table ) . it is transmitted mainly within the community, but a few reports of nosocomial outbreaks have been described [ ] . autoinoculation is the most common way of transmission after contact with contaminated objects. aerosolization also contributes to viral spread (table ) [ ] . some epidemiologic data support the possibility of transmission by contact or by air (table ) . however, means of transmission of mers-cov have not been elucidated yet. the epidemiology of mers-cov infection since isolation of the pathogen in is consistent with multiple introductions of the virus into humans from an animal reservoir, with no long-term sustained human-to human transmission [ ] . hbov is probably transmitted through respiratory droplets; however; nosocomial transmission of hbov has not been reported (table ) [ ] . education of healthcare workers, close monitoring of compliance to preventive measures, and infection surveillance are useful tools to reduce the risk of transmission for most respiratory viruses that cause nosocomial infections [ ] . in the following section, preventive and control measures for each respiratory virus are described. the precautions recommended by the cdc to avoid rsv transmission include standard and contact precautions (handwashing with water and soap or alcohol hand rub; appropriate use of gloves in order to decrease autoinoculation and use of gown) [ ] . use of facemask (droplet precautions) and goggles when direct contact with the patient are also recommended by others (table ) [ , ] . eye protection should always be worn when performing procedures that might generate sprays from respiratory secretions, considering that nose and eyes are the main sites for virus inoculation [ , ] . cdc also recommends that infected patients be hospitalized in single rooms or kept in cohort isolation if no individual isolation rooms are available. when cohorting, there should be at least . m of separation between beds [ ] . another measure to be considered is the use of individual equipment for each patient, including toys. other strategies that help prevent rsv transmission in the healthcare setting include: reducing the quantity of persons in contact with hospitalized patients (especially oncohematological), avoiding the presence of children under years of age in the hospital when rsv circulates either in the community or the hospital, and using single-bed rooms for infected patients [ , , ] . some studies have demonstrated that healthcare workers use of eye protection might more efficient than contact precautions in decreasing the risk of rsv transmission within the hospital setting [ ] . other recommendations, with very little or no evidence at all, suggest that healthcare personnel caring for rsv infected patients be exclusive for these individuals and that hospital visitor restrictions be taken during rsv season. the use of drugs for prophylaxis is limited to palivizumab in the high risk pediatric population although there are some data that demonstrates its effectiveness in the prevention of rsv in hsct adult patients [ , [ ] [ ] [ ] . annual influenza vaccination of healthcare workers and high-risk patients for influenza complications and their households is the most effective measure to prevent the disease and its complications. it also contributes to decrease viral transmission in the healthcare setting [ ] . vaccination of healthcare workers is the second most important strategy, just behind handwashing, to prevent nosocomial dissemination of influenza [ ••] . several studies have demonstrated a reduction in morbidity and mortality among patients in different healthcare settings when healthcare workers have been vaccinated against influenza [ ••] . different strategies were successfully implemented in order to increase vaccine uptake in healthcare personnel. among these were educational campaigns regarding benefits of the vaccine for patients and personnel, actions addressed to remove administrative barriers for vaccination, implementation of accessible locations for vaccine administration and of signed declination statements, and mandatory vaccination policies for healthcare workers [ ] . however, the scientific support for this last strategy has been questioned recently [ ] . antivirals against influenza have proved effective to reduce influenza viral transmission. the ones now available are the neuraminidase inhibitors such as oseltamivir, zanamivir and peramivir. oseltamivir which is administered through the oral route has proved to effectively prevent secondary cases of influenza among household contacts if it is given within h of symptoms initiation of the index case. zanamivir, which is administered by inhalation per mouth, has also proved effective in preventing secondary influenza cases in households and healthcare workers [ , ] , but caution is advised, as its efficacy has only been demonstrated in particular circumstances. during a nosocomial outbreak, in high-risk patients and non-vaccinated healthcare workers, antiviral administration should be implemented. when a mismatch between the circulating influenza strain and the vaccine strain is identified, antiviral prophylaxis should be given to all healthcare workers if an outbreak occurs. duration of antiviral administration during a nosocomial outbreak of influenza should be two weeks or one week after the outbreak control [ ] . besides vaccination and antivirals, infection control measures are of utmost importance in order to prevent influenza virus transmission and dissemination in the hospital environment. these measures include handwashing, limitation of visitors and of health personnel in contact with infected patients, cohort or single bed rooms isolation, and droplet precautions. precautions to avoid aerosolization should also be implemented in case of a new pandemic strain or when performing high risk procedures (e.g. intubation) ( table ) [ ] . preventive measures to avoid adenovirus nosocomial infections include patient cohorting, reduction of visitors and contact and droplet precautions, along with the exclusion of infected healthcare workers from clinical duties (table ) [ , ] . handwashing seems to be non-effective in removing adenovirus from contaminated fingers [ ] . there are no available vaccines or antiviral prophylaxis to help prevent adenovirus infection [ •, , ] . in order to prevent nosocomial transmission of piv several measures are proposed. they include contact precautions, reinforcement of hand hygiene and standard precautions in all patients to avoid an outbreak in the hospital setting (table ) [ ] . there are some vaccines under the initial phases of development that take advantage of mucosal immunity, but there are no efficacy data yet [ ] . hmpv infection control measures to prevent nosocomial transmission of this virus include handwashing and contact precautions ( table ) [ ] . there is no consensus regarding recommendations to prevent mers-cov transmission. while who recommends contact isolation and droplet precautions for any suspected case and respiratory precautions only for aerosol generating procedures, the cdc promotes air and contact precautions for all the activities related to patient care (table ). other centers recommend intensification of handwashing, contact precautions, ocular protection and the use of facemasks. currently, any suspected or confirmed mers-cov case should be treated in a medical facility. transfer and transportation of an infected patient in the hospital environment should include a minimum crew, all wearing surgical facemasks. healthcare workers with direct patient contact should follow the recommendations mentioned before and keep updated on new recommendations. any transference of mers-cov infected patients from one healthcare setting to another should be coordinated with public health authorities [ , , ] . although nosocomial transmission has not been reported, it seems prudent to implement at least contact precautions in patients when this virus is detected (table ) . the epidemiology of viral respiratory infections has been better understood and modified in the last years for multiple reasons, including vaccination, improvement in diagnostic assays such as molecular techniques and better knowledge and understanding of mechanisms of transmission. cdc/nhsn surveillance definitions for specific types of infections surveillance definitions janu healthcare infection control practices advisory committee. guidelines for preventing health-careassociated pneumonia, : recommendations of cdc and the healthcare infection control practices advisory committee viral pneumonia in patients with hematologic malignancy or hematopoietic stem cell transplantation respiratory viral infections in solid organ and hematopoietic stem cell transplantation reference is important because nosocomial respiratory tract infections are frequent and severe in this group of patients. this article allows the reader to get updated on respiratory viral infections in immunosuppressed hosts detection of respiratory viruses and legionella spp. by real-time polymerase chain reaction in patients with community acquired pneumonia the role of next generation sequencing in infection prevention in human parainfluenza virus infections in immunocompromised patients nosocomial viral respiratory infections: perennial weeds on pediatric wards adenovirus - . in: mandell, douglas, and bennett's principles and practice of infectious diseases a case control study assessing the impact of non-ventilated hospital-acquired pneumonia on patient outcome treanor: influenza (including avian influenza and swine influenza) - . in: mandell, douglas, and bennett's principles and practice of infectious diseases this reference reviews all the aspects related to respiratory viral infections including those which are of nosocomial origin. it is a very detailed and exhaustive description of the most common and frequent viral respiratory tract infections. it describes each virus, their mechanisms of transmission ison parainfluenza viruses. - . in: mandell, douglas, and bennett's principles and practice of infectious diseases respiratory viral infections in hematopoietic stem cell and solid organ transplant recipients. semin respir crit care med rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses frequency and natural history of rhinovirus infections in adults during autumn chronic rhinoviral infection in lung transplant recipients clinical and molecular epidemiology of human rhinovirus infections in patients with hematologic malignancy world health organization. summary of probable sars cases with onset of illness from middle east respiratory syndrome is a severe and emerging respiratory viral infection that can also be transmitted in the health care setting. this article describes in detail the latest advances in the research of mers-cov, its epidemiology, mechanisms of transmission, virus characteristics middle east respiratory syndrome coronavirus: review of the current situation in the world cloning of a human parvovirus by molecular screening of respiratory tract samples risk of acute gastroenteritis associated with human bocavirus infection in children: a systematic review and metaanalysis outbreak of respiratory syncytial virus (rsv) infection in immunocompromised adults on a hematology ward evidence of respiratory syncytial virus spread by aerosol. time to revisit infection control strategies? breese hall respiratory syncytial virus (rsv) risk of nosocomial respiratory syncytial virus infection and effectiveness of control measures to prevent transmission events: a systematic review. influenza other respir viruses nosocomial respiratory syncytial virus infections: the "cold war" has not ended prolonged outbreak of human parainfluenza virus infection in a stem cell transplant outpatient department: insights from molecular epidemiologic analysis molecular characterization of strains of respiratory syncytial virus identified in hematopoyetic stem cell transplant outpatient unit over years: community or nosocomial infection guidelines for preventing infectious complications among hematopoietic cell transplant recipients: a global perspective the authors of this article address the importance of rsv, adenovirus, pai, hrv, influenza and hmpn in both sot and hsct patients. they point out the main characteristics of these viruses in immunosuppressed hosts such as prolonged excertion and higher morbidity and mortality in relation to normal hosts the influenza viruses and influenza incidence and recall of influenza in a cohort of glasgow healthcare workers during the - epidemic: results of serum testing and questionnaire adenovirus in solid organ transplant recipients prolonged recovery of desiccated adenoviral serotypes , , and from plastic and metal surfaces in vitro potential role of hands in the spread of respiratory viral infections: studies with human parainfluenza virus and rhinovirus infection control of nosocomial respiratory viral disease in the immunocompetent host an outbreak of human parainfluenza virus infection in an outpatient hematopoietic stem cell transplantation clinic dare and talbot make a very good description of health care acquired viral respiratory infections, their different control measures(education, hand-washing, isolation, ppe, cohorting of patients and personnel) and other measures such as influenza vaccination parainfluenza virus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection viral respiratory tract infections in transplant patients: epidemiology, recognition and management how contagious are common respiratory tract infections? clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission an outbreak of respiratory syncytial virus in a bone marrow transplant center detection and control of a nosocomial respiratory syncytial virus outbreak in a stem cell transplantation unit: the role of palivizumab respiratory syncytial virus infection in adults immunization of health-care personnel: recommendations of the advisory committee on immunization practices (acip) influenza vaccination of healthcare workers: critical analysis of the evidence for patient benefit underpinning policies of enforcement safety and effectiveness of neuraminidase inhibitors for influenza treatment, prophylaxis, and outbreak control: a systematic review of systematic reviews and/or meta-analyses oseltamivir and inhaled zanamivir as influenza prophylaxis in thai heaith workers: a randomized, double-blind, placebo-controlled safety trial over weeks adenovirus type epidemic keratoconjunctivitis in an eye clinic: risk factors and control adenovirus: current epidemiology and emerging approaches to prevention and treatment emerging viral respiratory tract infections-environmental risk factors and transmission safety and immunogenicity of an intranasal sendai virus-based human parainfluenza virus type vaccine in -to -year-old children an opportunistic pathogen afforded ample opportunities: middle east respiratory syndrome coronavirus this is also observed in the healthcare setting where respiratory viruses are recognized as a cause of uri and lri. these infections are not only limited to children and may cause severe infections in immunocompromised patients.knowing the mechanisms of respiratory viral transmission in the healthcare setting allows to implement infection control measures to prevent its dissemination and reduce the risk of a hospital outbreak.early detection and strict compliance to infection control measures limit the spread of nosocomial viral respiratory infections. other measures, such as healthcare workers receiving an annual influenza vaccination, should be encouraged. the authors declare that they have no competing interests. this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord- -flzqm wh authors: buchanan, tom title: why do people spread false information online? the effects of message and viewer characteristics on self-reported likelihood of sharing social media disinformation date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: flzqm wh individuals who encounter false information on social media may actively spread it further, by sharing or otherwise engaging with it. much of the spread of disinformation can thus be attributed to human action. four studies (total n = , ) explored the effect of message attributes (authoritativeness of source, consensus indicators), viewer characteristics (digital literacy, personality, and demographic variables) and their interaction (consistency between message and recipient beliefs) on self-reported likelihood of spreading examples of disinformation. participants also reported whether they had shared real-world disinformation in the past. reported likelihood of sharing was not influenced by authoritativeness of the source of the material, nor indicators of how many other people had previously engaged with it. participants’ level of digital literacy had little effect on their responses. the people reporting the greatest likelihood of sharing disinformation were those who thought it likely to be true, or who had pre-existing attitudes consistent with it. they were likely to have previous familiarity with the materials. across the four studies, personality (lower agreeableness and conscientiousness, higher extraversion and neuroticism) and demographic variables (male gender, lower age and lower education) were weakly and inconsistently associated with self-reported likelihood of sharing. these findings have implications for strategies more or less likely to work in countering disinformation in social media. disinformation is currently a critically important problem in social media and beyond. typically defined as "the deliberate creation and sharing of false and/or manipulated information that is intended to deceive and mislead audiences, either for the purposes of causing harm, or for political, personal or financial gain", political disinformation has been characterized as a significant threat to democracy [ , p. ] . it forms part of a wider landscape of information operations conducted by governments and other entities [ , ] . its intended effects include political influence, increasing group polarisation, reducing trust, and generally undermining civil society [ ] . effects are not limited to online processes. they regularly spill over into other parts of our lives. experimental work has shown that exposure to disinformation can lead to attitude change [ ] and there are many real-world examples of behaviours that have been directly attributed to disinformation, such people as attacking telecommunications masts in response to fake stories about ' g causing coronavirus' [ , ] . social media disinformation is very widely used as a tool of influence: computational propaganda has been described as a pervasive and ubiquitous part of modern everyday life [ ] . once disinformation has initially been seeded online by its creators, one of the ways in which it spreads is through the actions of individual social media users. ordinary people may propagate the material to their own social networks through deliberate sharing-a core function of platforms such as facebook and twitter. other interactions with it, such as 'liking', also trigger the algorithms of social media platforms to display it to other users. this is a phenomenon known as 'organic reach' [ ] . it can lead to false information spreading exponentially. as an example, analysis of the activity of the russian 'internet research agency' (ira) disinformation group in the usa between and concluded that over million users shared and otherwise interacted with the ira's facebook and instagram posts, propagating them to their families and friends [ ] . there is evidence that false material is spread widely and rapidly through social media due to such human behaviour [ ] . when individuals share or interact with disinformation they see online, they have essentially been persuaded to do so by its originators. influential models of social information processing suggest there are different routes to persuasion [e.g . ] . under some circumstances, we may carefully consider the information available. at other times, we make rapid decisions based on heuristics and peripheral cues. when sharing information on social media occurs, it is likely to be spontaneous and rapid, rather than being a considered action that people spend time deliberating over. for example, there are indications of people using the interaction features of facebook in a relatively unthinking and automatic manner [ ] . in such situations, a peripheral route to persuasion is likely be important [ ] . individuals' choices to share, like and so on will thus be guided primarily by heuristics or contextual cues [ ] . three potentially important heuristics in this context are consistency, consensus and authority [ ] . these are not the only heuristics that might possibly influence whether we share false material. however, in each case there is suggestive empirical evidence, and apparent realworld attempts to leverage these phenomena, that make them worth considering. consistency. consistency is the extent to which sharing would be consistent with past behaviours or beliefs of the individual. for example, in the usa people with a history of voting republican might be more likely to endorse and disseminate right-wing messaging [ ] . there is a large body of work based on the idea that people prefer to behave in ways consistent with their attitudes [ ] . research has indicated that social media users consider headlines consistent with their pre-existing beliefs as more credible, even when explicitly flagged as being false [ ] . in the context of disinformation, this could make it desirable to target audiences sympathetic to the message content. consensus. consensus is the extent to which people think their behaviour would be consistent with that of most other people. in the current context, it is possible that seeing a message has already been shared widely might make people more likely to forward it on themselves. in marketing, this influence tactic is known as 'social proof' [ ] . it is widely used in online commerce in attempts to persuade consumers to purchase goods or services (e.g. by displaying reviews or sales rankings). the feedback mechanisms of social networks can be manipulated to create an illusion of such social support, and this tactic seems to have been used in the aftermath of terror attacks in the uk [ ] . bot networks are used to spread low-credibility information on twitter through automated means. bots have been shown to be involved in the rapid spread of information, tweeting and retweeting messages many times [ ] . among humans who see the messages, the high retweet counts achieved through the bot networks might be interpreted as indicating that many other people agree with them. there is evidence which suggests that "each amount of sharing activity by likely bots tends to trigger a disproportionate amount of human engagement" [ , p. ] . such bot activity could be an attempt to exploit the consensus effect. it is relatively easy to manipulate the degree of consensus or social proof associated with an online post. work by the nato strategic communications centre of excellence [ ] indicated that it was very easy to purchase high levels of false engagement for social media posts (e.g. sharing of posts by networks of fake accounts) and that there was a significant black market for social media manipulation. thus, if boosting consensus effectively influences organic reach, then it could be a useful tool for both those seeding disinformation and those seeking to spread counter-messages. authority. authority is the extent to which the communication appears to come from a credible, trustworthy source [ ] . research participants have been found to report a greater likelihood of propagating a social media message if it came from a trustworthy source [ ] . there is evidence of real-world attempts to exploit this effect. in , twitter identified fraudulent accounts that simulated those of us local newspapers [ ] , which may be trusted more than national media [ ] . these may have been sleeper accounts established specifically for the purpose of building trust prior to later active use. factors influencing the spread of disinformation. while there are likely to be a number of other variables that also influence the spread of disinformation, there are grounds for believing that consistency, consensus and authority may be important. constructing or targeting disinformation messages in such a way as to maximise these three characteristics may be a way to increase their organic reach. there is real-world evidence of activity consistent with attempts to exploit them. if these effects do exist, they could also be exploited by initiatives to counter disinformation. not all individuals who encounter untrue material online spread it further. in fact, the great majority do not. research linking behavioural and survey data [ ] found that less than % of participants shared articles from 'fake news' domains during the us presidential election campaign (though of course when extrapolated to the huge user base of social network platforms like facebook, this is still a very large number of people). the fact that only a minority of people actually propagate disinformation makes it important to consider what sets them apart from people who don't spread untrue material further. this will help to inform interventions aimed at countering disinformation. for example, those most likely to be misled by disinformation, or to spread it further, could be targeted with counter-messaging. it is known that the originators of disinformation have already targeted specific demographic groups, in the same way as political campaigns micro-target messaging at those audience segments deemed most likely to be persuadable [ ] . for example, it is believed that the 'internet research agency' sought to segment facebook and instagram users based on race, ethnicity and identity by targeting their messaging to people recorded by the platforms as having certain interests for marketing purposes [ ] . they targeted specific communications tailored to those segments (e.g. trying to undermine african americans' faith in political processes and suppress their voting in the us presidential election). research has found that older adults, especially those aged over , were by far the most likely to spread material originally published by 'fake news' domains [ ] . a key hypothesis advanced to explain this is that older adults have lower levels of digital media literacy, and are thus less likely to be able to distinguish between true and false information online. while definitions may vary, digital media literacy can be thought of as including ". . . the ability to interact with textual, sound, image, video and social medias . . . finding, manipulating and using such information" [ , p. ] and being a "multidimensional concept that comprised technical, cognitive, motoric, sociological, and emotional aspects" [ , p. ] . digital media literacy is widely regarded as an important variable mediating the spread and impact of disinformation [e.g. ]. it is argued that many people lack the sophistication to detect a message as being untruthful, particularly when it appears to come from an authoritative or trusted source. furthermore, people higher in digital media literacy may be more likely to engage in elaborated, rather than heuristic-driven, processing (cf. work on phishing susceptibility [ ] ), and thus be less susceptible to biases such as consistency, consensus and authority. educating people in digital media literacy is the foundation of many anti-disinformation initiatives. examples include the 'news hero' facebook game developed by the nato strategic communications centre of excellence (https://www.stratcomcoe.org/news-hero), government initiatives in croatia and france [ ] or the work of numerous fact-checking organisations. the effectiveness of such initiatives relies on two assumptions being met. the first is that lower digital media literacy really does reduce our capacity to identify disinformation. there is currently limited empirical evidence on this point, complicated by the fact that definitions of 'digital literacy' are varied and contested, and there are currently no widely accepted measurement tools [ ] . the second is that the people sharing disinformation are doing so unwittingly, having been tricked into spreading it. however, it is possible that at least some people know the material is untrue, and they spread it anyway. survey research [ ] has found that believing a story was false was not necessarily a barrier to sharing it. people may act like this because they are sympathetic to a story's intentions or message, or they are explicitly signalling their social identity or allegiance to some political group or movement. if people are deliberately forwarding information that they know is untrue, then raising their digital media literacy would be ineffective as a stratagem to counter disinformation. this makes it important to simultaneously consider users' beliefs about the veracity of disinformation stories, to inform the design of countermeasures. personality. it is also known that personality influences how people use social media [e.g. ]. this makes it possible that personality variables will also influence interactions with disinformation. indeed, previous research [ ] found that people low on agreeableness reported themselves as more likely to propagate a message. this is an important possibility to consider, because it raises the prospect that individuals could be targeted on the basis of their personality traits with either disinformation or counter-messaging. in a social media context, personalitybased targeting of communications is feasible because personality characteristics can be detected from individuals' social media footprints [ , ] . large scale field experiments have shown that personality-targeted advertising on social media can influence user behaviour [ ] . the question of which personality traits might be important is an open one. in the current study, personality was approached on an exploratory basis, with no specific hypotheses about effects or their directions. this is because there are a number of different and potentially rival effects that might operate. for example, higher levels of conscientiousness may be associated with a greater likelihood of posting political material in social media [ ] leading to a higher level of political disinformation being shared. however, people higher in conscientiousness are likely to be more cautious [ ] and pay more attention to details [ ] . they might therefore also be more likely to check the veracity of the material they share, leading to a lower level of political disinformation being shared. the overall aim of this project was to establish whether contextual factors in the presentation of disinformation, or characteristics of the people seeing it, make it more likely that they extend its reach. the methodology adopted was scenario-based, with individuals being asked to rate their likelihood of sharing exemplar disinformation messages. a series of four studies was conducted, all using the same methodology. multiple studies were used to establish whether the same effects were found across different social media platforms (facebook in study , twitter in study , instagram in study ) and countries (facebook with a uk sample in study , facebook with a us sample in study ). data were also collected on whether participants had shared disinformation in the past. a number of distinct hypotheses were advanced: h : individuals will report themselves as more likely to propagate messages from more authoritative compared to less authoritative sources. h : individuals will report themselves as more likely to propagate messages showing a higher degree of consensus compared to those showing a lower degree of consensus. h : individuals will report themselves as more likely to propagate messages consistent with their pre-existing beliefs compared to inconsistent messages. h : individuals lower in digital literacy will report a higher likelihood of sharing false messages than individuals higher in digital literacy. other variables were included in the analysis on an exploratory basis with no specific hypotheses being advanced. in summary, this project asks why ordinary social media users share political disinformation messages they see online. it tests whether specific characteristics of messages or their recipients influence the likelihood of disinformation being further shared online. understanding any such mechanisms will both increase our understanding of the phenomenon and inform the design of interventions seeking to reduce its impact. study tested hypotheses - with a uk sample, using stimuli relevant to the uk. the study was completed online. participants were members of research panels sourced through the research company qualtrics. participants were asked to rate their likelihood of sharing three simulated facebook posts. the study used an experimental design, manipulating levels of authoritativeness and consensus apparent in the stimuli. all manipulations were between, not within, participants. consistency with pre-existing beliefs was not manipulated. instead, the political orientation of the stimuli was held constant, and participants' scores on conservative political orientation were used as an index of consistency between messages and participant beliefs. the effects of these variables on self-rated likelihood of sharing the stimuli, along with those of a number of other predictors, were assessed using multiple regression. the primary goal of the analysis was to identify variables that statistically significantly explained variance in the likelihood of sharing disinformation. the planned analysis was followed by supplementary and exploratory analyses. all analyses were conducted using spss v. for mac. for all studies reported in this paper, ethical approval came from both the university of westminster research ethics committee (eth - ) and the lancaster university security research ethics committee (buchanan ). consent was obtained, via an electronic form, from anonymous participants. a short questionnaire was used to capture demographic information (gender; country of residence; education; age; occupational status; political orientation expressed as right, left or centre; frequency of facebook use). individual differences in personality, political orientation, and digital / new media literacy were measured using established validated questionnaires. ecologically valid stimuli were used, with their presentation being modified across conditions to vary authoritativeness and consensus markers. personality was measured using a -item five-factor personality questionnaire [ ] derived from the international personality item pool [ ] . the measure provides indices of extraversion, neuroticism, openness to experience, agreeableness and conscientiousness that correlate well with the domains of costa and mccrae's [ ] five factor model. conservatism was measured using the -item social and economic conservatism scale (secs) [ ] , which is designed to measure political orientation along a left-right; liberal-conservative continuum. it was developed and validated using a us sample. in pilot work for the current study, mean scores for individuals who reported voting for the labour and conservative parties in the uk general election were found to differ in the expected manner (t ( ) = - . , p = . , d = . ). this provides evidence of its appropriateness for use in uk samples. while the measure provides indices of different aspects of conservatism, it also provides an overall conservatism score which was used in this study. digital media literacy was measured using the -item new media literacy scale (nmls) [ ] . this is a theory-based self-report measure of competences in using, critically interrogating, and creating digital media technologies and messaging. in pilot work with a uk sample, it was found to distinguish between individuals high or low in social media (twitter) use, providing evidence of validity (t ( ) = - . , p < . , d = . ). while the measure provides indices of different aspects of new media literacy, it also provides an overall score which was used in this study. participants were asked to rate their likelihood of sharing three genuine examples of 'fake news' that had been previously published online. an overall score for their likelihood of sharing the stimuli was obtained by summing the three ratings, creating a combined score. this was done, and a set of three stimuli was used, to reduce the likelihood that any effects found were peculiar to a specific story. the stimuli were sourced from the website infowars.com (which in some cases had republished them from other sources). infowars.com has been described [ ] as a high-exposure site strongly associated with the distribution of 'fake news'. rather than full articles, excerpts (screenshots) were used that had the size and general appearance of what respondents might expect to see on social media sites. the excerpts were edited to remove any indicators of the source, metrics such as the numbers of shares, date, and author. all had a right-wing orientation (so that participant conservatism could be used as a proxy for consistency between the messages and existing beliefs). this was established in pilot work rating their political orientation and likelihood of being shared. the three stories were among seven rated by a uk sample (n = ) on an -point scale asking "to what extent do you think this post was designed to appeal to people with right wing (politically conservative) views?" anchored at "very left wing oriented" and "very right wing oriented". all seven were rated statistically significantly above the politically-neutral midpoint of the scale. of the three stimuli selected for use in this study, a one-sample t-test showed that the least right-wing was statistically significantly higher than the midpoint, (t ( ) = . , p < . , d = . ). one of the stimuli was a picture of masked and hooded men titled "censored video: watch muslims attack men, women & children in england". one was a picture of many people walking down a road, titled "revealed: un plan to flood america with million migrants", with accompanying text describing a plan to "flood america and europe with hundreds of millions of migrants to maintain population levels". the third was a picture of the swedish flag titled "'child refugee' with flagship samsung phone and gold watch complains about swedish benefits rules", allegedly describing a year-old refugee's complaints. the authoritativeness manipulation was achieved by pairing the stimuli with sources regarded as relatively high or low in authoritativeness. the source was shown above the stimulus being rated, in the same way as the avatar and username of someone who had posted a message would be on facebook. the lower authoritativeness group were slight variants on real usernames that had previously retweeted either stories from infowars.com or another story known to be untrue. the original avatars were used. the exemplars used in this study were named 'tigre' (with an avatar of an indistinct picture of a female face), 'jelly beans' (a picture of some jelly beans) and 'chucke' (an indistinct picture of a male face). the higher authoritativeness group comprised actual fake accounts set up by the internet research agency (ira) group to resemble local news sources, selected from a list of suspended ira accounts released by twitter. the exemplars used in this study were 'los angeles daily', 'chicago daily news' and 'el paso top news'. pilot work was conducted with a sample of uk participants (n = ) who each rated a selection of usernames, including these , for the extent to which each was "likely to be an authoritative source-that is, likely to be a credible and reliable source of information". a within-subjects t-test indicated that mean authoritativeness ratings for the 'higher' group were statistically significantly higher than the 'lower' group (t ( ) = - . , p < . , d z = . ). the consensus manipulation was achieved by pairing the stimuli with indicators of the number of shares and likes the story had. the indicators were shown below the stimulus being rated, in the same way as they normally would be on facebook. in the low consensus conditions, low numbers of likes ( , , ) and shares ( , , ) were displayed. in the high consensus conditions, higher (but not unrealistic) numbers of likes ( k, k, k) and shares ( k, k, k) were displayed. the information was presented using the same graphical indicators as would be the case on facebook, accompanied by the (inactive) icons for interacting with the post, in order to maximise ecological validity. procedure. the study was conducted completely online, using materials hosted on the qualtrics research platform. participants initially saw an information page about the study, and on indicating their consent proceeded to the demographic items. they then completed the personality, conservatism and new media literacy scales. each of these was presented on a separate page, except the nmls which was split across three pages. participants were then asked to rate the three disinformation items. participants were randomized to different combinations of source and story within their assigned condition. for example, participant a might have seen story attributed to source , story attributed to source , and story attributed to source ; while participant b saw story attributed to source , story attributed to source , and story attributed to source . each participant saw the same three stories paired with one combination of authoritativeness and consensus. there were distinct sets of stimuli. each participant saw an introductory paragraph stating "a friend of yours recently shared this on facebook, commenting that they thought it was important and asking all their friends to share it:". below this was the combination of source, story, and consensus indicators, presented together in the same way as a genuine facebook post would be. they then rated the likelihood of them sharing the post to their own public timeline, on an -point scale anchored at 'very unlikely' and 'very likely'. this was repeated for the second and third stimuli, each on a separate page. having rated each one, participants were then shown all three stimuli again, this time on the same page. they were asked to rate each one for "how likely do you think it is that the message is accurate and truthful" and "how likely do you think it is that you have seen it before today", on -point scales anchored at 'not at all likely' and 'very likely'. after rating the stimuli, participants were asked two further questions: "have you ever shared a political news story online that you later found out was made up?", and "and have you ever shared a political news story online that you thought at the time was made up?", with 'yes' or 'no' response options. this question format directly replicated that used in pew research centre surveys dealing with disinformation [e.g. ] . finally, participants were given the opportunity once again to give or withdraw their consent for participation. they then proceeded to a debriefing page. it was only at the debriefing stage that they were told the stories they had seen were untrue: no information about whether the stimuli were true or false had been presented prior to that point. data screening and processing. prior to delivery of the sample, qualtrics performed a series of quality checks and 'data scrubbing' procedures to remove and replace participants with response patterns suggesting inauthentic or inattentive responding. these included speeding checks and examination of response patterns. on delivery of the initial sample (n = ) further screening procedures were performed. sixteen respondents were identified who had responded with the same scores to substantive sections of the questionnaire ('straightlining'). these were removed, leaving n = . these checks and exclusions were carried out prior to any data analysis. where participants had missing data on any variables, they were omitted only from analyses including those variables. thus, ns vary slightly throughout the analyses. participants. the target sample size was planned to exceed n = , which would give % power to detect r = . (a benchmark for the minimum effect size likely to have realworld importance in social science research [ ] ), in the planned multiple regression analysis with predictors. qualtrics was contracted to provide a sample of facebook users that was broadly representative of the uk census population in terms of gender; the split between those who had post-secondary-school education and those who had not; and age profile ( +). quotas were used to assemble a sample comprising approximately one third each self-describing as left-wing, centre and right-wing in their political orientation. participant demographics are shown in table , column . descriptive statistics for participant characteristics (personality, conservatism, new media literacy and age) and their reactions to the stimuli (likelihood of sharing, belief the stories were likely to be true, and rating of likelihood that they had seen them before) are summarised in table . all scales had acceptable reliability. the main dependent variable, likelihood of sharing, had a very skewed distribution with a strong floor effect: . % of the participants indicated they were 'very unlikely' to share any of the three stories they saw. this is consistent with findings on real-world sharing that indicate only a small proportion of social media users will actually share disinformation [e.g. ] , though it gives a dependent variable with less than ideal distributional properties. to simultaneously test hypotheses - a multiple regression analysis was carried out. this evaluated the extent to which digital media literacy (nmls), authority of the message source, consensus, belief in veracity of the messages, consistency with participant beliefs (operationalised as the total secs conservatism scale score), age and personality (extraversion, conscientiousness, agreeableness, openness to experience and neuroticism), predicted self-rated likelihood of sharing the posts. this analysis is summarised in table . checks were performed on whether the dataset met the assumptions required by the analysis (absence of collinearity, independence of residuals, heteroscedasticity and non-normal distribution of residuals). despite the skewed distribution of the dependent variable, no significant issues were detected. , and with likelihood of having seen the stimuli before (r = . , n = , p = . ). selfreported belief that respondents had seen the stories before also correlated significantly with likelihood of sharing (r = . , n = , p < . ), and a number of other predictor variables. accordingly, a further regression analysis was performed, including these additional predictors (gender, education, level of facebook use, belief they had seen the stories before). given inclusion of gender as a predictor variable, the two respondents who did not report their gender as either male or female were excluded from further analysis. the analysis, summarised in table , indicated that the model explained % of the variance in self-reported likelihood of sharing the three disinformation items. neither the authoritativeness of the story source, nor consensus information associated with the stories, was a significant predictor. consistency of the items with participant attitudes (conservatism) was important, with a positive and statistically significant relationship between conservatism and likelihood of sharing. the only personality variable predicting sharing was agreeableness, with less agreeable people giving higher ratings of likelihood of sharing. in terms of demographic characteristics, gender and education were statistically significant predictors, with men and less-educated people reporting a higher likelihood of sharing. finally, people reported a greater likelihood of sharing the items if they believed they were likely to be true, and if they thought they had seen them before. participants had also been asked about their historical sharing of untrue political stories, both unknowing and deliberate. out of participants ( . %) indicated that they had ever 'shared a political news story online that they later found out was made up', while out of indicated they had shared one that they 'thought at the time was made up' ( . %). predictors of whether or not people had shared untrue material under both sets of circumstances were examined using logistic regressions, with the same sets of participant-level predictors. having unknowingly shared untrue material (table ) was significantly predicted by lower conscientiousness, lower agreeableness, and lower age. having shared material known to be untrue at the time (table ) was significantly predicted by lower agreeableness and lower age. the main analysis in this study (table ) provided limited support for the hypotheses. contrary to hypotheses , , and , neither consensus markers, authoritativeness of source, nor new media literacy were associated with self-rated likelihood of sharing the disinformation stories. however, in line with hypothesis , higher levels of conservatism were associated with higher likelihood of sharing disinformation. this finding supports the proposition that we are more likely to share things that are consistent with our pre-existing beliefs, as all the stimuli were right-wing in orientation. an alternative explanation might be that more conservative people are simply more likely to share disinformation. however, as well as lacking a solid rationale, this explanation is not supported by the fact that conservatism did not seem to be associated with self-reported historical sharing (tables and ). the strongest predictors of likelihood of sharing were belief that the stories were true, and likelihood of having seen them before. belief in the truth of the stories provides further evidence for the role of consistency (hypothesis ), in that we are more likely to share things we believe are true. the association with likely previous exposure to the materials is consistent with other recent research [ , ] that found that prior exposure to 'fake news' headlines led to higher belief in their accuracy and reduced belief that it would be unethical to share them. of the personality variables, only agreeableness was a significant predictor, with less agreeable people rating themselves are more likely to share the stimuli. this is consistent with previous findings [ ] that less agreeable people reported they were more likely to share a critical political message. lower education levels were associated with a higher self-reported likelihood of sharing. it is possible that less educated people may be more susceptible to online influence, given work finding that less educated people were more influenced by micro-targeted political advertising on facebook [ ] . finally, gender was found to be an important variable, with men reporting a higher likelihood of sharing the disinformation messages than women. this was unanticipated: while there are a number of gender-related characteristics (e.g. personality traits) that were thought might be important, there were no a priori grounds to expect that gender itself would be a predictor variable. study also examined predictors of reported historical sharing of false political information. consistent with real-world data [ ] , and past representative surveys [e.g. ], a minority of respondents reported such past sharing. unknowingly sharing false political stories was predicted by low conscientiousness, low agreeableness, and lower age, while knowingly sharing false material was predicted only by lower agreeableness and lower age. the effect of agreeableness is consistent with the findings from the main analysis and from [ ] . the finding that conscientiousness influenced accidental, but not deliberate, sharing is consistent with the idea that less conscientious people are less likely to check the details or veracity of a story before sharing it. clearly this tendency would not apply to deliberate sharing of falsehoods. the age effect is harder to explain, especially given evidence [ ] that older people were more likely to share material from fake news sites. one possible explanation is that younger people are more active on social media, so would be more likely to share any kind of article. another possibility is that they are more likely to engage in sharing humorous political memes, which could often be classed as false political stories. study set out to repeat study , but presented the materials as if they had been posted on twitter rather than facebook. the purpose of this was to test whether the observed effects applied across different platforms. research participants have reported using 'likes' on twitter in a more considered manner than on facebook [ ] , raising the possibility that heuristics might be less important for this platform. the study was completed online, using paid respondents sourced from the prolific research panel (www.prolific.co). the methodology exactly replicated that of study , except in the case of details noted below. the planned analysis was revised to include the expanded set of predictors eventually used in study (see table ). measures and materials were the same as used in study . the key difference from study was in the presentation of the three stimuli, which were portrayed as having been posted to twitter rather than facebook. for the authoritativeness manipulation, the screen names of the sources were accompanied by @usernames, as is conventional on twitter. for the consensus manipulation, 'retweets' were displayed rather than 'shares', and the appropriate icons for twitter were used. participants also indicated their level of twitter, rather than facebook, use. procedure. the procedure replicated study , save that in this case the nmls was presented on a single page. before participants saw each of the three disinformation items, the introductory paragraph stated "a friend of yours recently shared this on twitter, commenting that they thought it was important and asking all their friends to retweet it:", and they were asked to indicate the likelihood of them 'retweeting' rather than 'sharing' the post. data screening and processing. data submissions were initially obtained from participants. a series of checks were performed to ensure data quality, resulting in a number of responses being excluded. one individual declined consent. eleven were judged to have responded inauthentically, with the same responses to all items in substantive sections of the questionnaire ('straightlining'). twenty were not active twitter users: three individuals visited twitter 'not at all' and seventeen 'less often' than every few weeks. three participants responded unrealistically quickly, with response durations shorter than four minutes (the same value used as a speeding check by qualtrics in study ). all of these respondents were removed, leaving n = . these checks and exclusions were carried out prior to any data analysis. participants. the target sample size was planned to exceed n = , as in study . no attempt was made to recruit a demographically representative sample: instead, sampling quotas were used to ensure the sample was not homogenous with respect to education (pre-degree vs. undergraduate degree or above), age (under vs. over ) and political preference (left, centre or right wing orientation). additionally, participants had to be uk nationals resident in the uk; active twitter users; and not participants in prior studies related to this one. each participant received a reward of £ . . participant demographics are shown in table (column ) . for the focal analysis in this study, the sample size conferred . % power to detect r = . in a multiple regression with predictors ( -tailed, alpha = . ). descriptive statistics are summarised in table . all scales had acceptable reliability. the main dependent variable, likelihood of sharing, again had a very skewed distribution with a strong floor effect. to simultaneously test hypotheses - , a multiple regression analysis was carried out using the expanded predictor set from study . given inclusion of gender as a predictor variable, the three respondents who did not report their gender as either male or female were excluded from further analysis. the analysis, summarised in table , indicated that the model explained % of the variance in self-reported likelihood of sharing the three disinformation items. neither the authoritativeness of the story source, nor consensus information associated with the stories, nor new media literacy, was a significant predictor. consistency of the items with participant attitudes (conservatism) was important, with a positive and statistically significant relationship between conservatism and likelihood of sharing. no personality variable predicted ratings of likelihood of sharing. in terms of demographic characteristics, gender and education were statistically significant predictors, with men and less-educated people reporting a higher likelihood of sharing. finally, people reported a greater likelihood of sharing the items if they believed they were likely to be true, and if they thought they had seen them before. participants had also been asked about their historical sharing of untrue political stories, both unknowing and deliberate. out of participants ( . %) indicated that they had out ever 'shared a political news story online that they later found out was made up', while out of indicated they had shared one that they 'thought at the time was made up' ( . %). predictors of whether or not people had shared untrue material under both sets of circumstances were examined using logistic regressions, with the same sets of participant-level predictors. having unknowingly shared untrue material (table ) was significantly predicted by higher extraversion and higher levels of twitter use. having shared material known to be untrue at the time (table ) was significantly predicted by higher neuroticism and being male. for the main analysis, study replicates a number of key findings from study . in particular, hypotheses , and were again unsupported by the results: consensus, authoritativeness, and new media literacy were not associated with self-rated likelihood of retweeting the disinformation stories. evidence consistent with hypothesis was again found, with higher levels of conservatism being associated with higher likelihood of retweeting. again, the strongest predictor of likelihood of sharing was belief that the stories were true, while likelihood of having seen them before was again statistically significant. the only difference was in the role of personality: there was no association between agreeableness (or any other personality variable) and likelihood of retweeting the material. however, for self-reports of historical sharing of false political stories, the pattern of results was different. none of the previous results were replicated, and new predictors were observed for both un-knowing and deliberate sharing. for unintentional sharing, the link with higher levels of twitter use makes sense, as higher usage confers more opportunities to accidentally share untruths. higher extraversion has also been found to correlate with higher levels of social media use [ ] so the same logic may apply for that variable. for intentional sharing, the finding that men were more likely to share false political information is similar to findings from study . the link with higher neuroticism is less easy to explain: one possibility is that more neurotic people are more likely to share falsehoods that will reduce the chances of an event that they worry about (for example, spreading untruths about a political candidate who one is worried about being elected). given that these questions asked about past behaviour in general, and were not tied to the twitter stimuli used in this study, it is not clear why the pattern of results should have differed from those in study . one possibility is that the sample characteristics were different (this sample was younger, better educated, and drawn from a different source). another realistic possibility, especially given the typically low effect sizes and large samples tested, is that these are simply 'crud' correlations [ ] rather than useful findings. going forward, it is likely to be more informative to focus on results that replicate across multiple studies or conceptually similar analyses. study set out to repeat study , but presented the materials as if they had been posted on instagram rather than facebook. instagram presents an interesting contrast, as the mechanisms of engagement with material are different (for example there is no native sharing mechanism). nonetheless, it has been identified as an important theater for disinformation operations [ ] . study therefore sought to establish whether the same factors affecting sharing on facebook also affect engagement with false material on instagram. the study was completed online, using paid respondents sourced from the prolific research panel. the methodology exactly replicated that of study , except in the case of details noted below. the planned analysis was revised to include the expanded set of predictors eventually used in study (see table ). materials. measures and materials were the same as used in study . the only difference from study was in the presentation of the three stimuli, which were portrayed as having been posted to instagram rather than facebook. for the consensus manipulation, 'likes' were used as the sole consensus indicator, and the appropriate icons for instagram were used. procedure. the procedure replicated study , save that in this case the nmls was presented on a single page. before participants saw each of the three disinformation items, the introductory paragraph stated "imagine that you saw this post on your instagram feed:" and they were asked to indicate the probability of them 'liking' the post. data screening and processing. data submissions were initially obtained from participants. a series of checks were performed to ensure data quality, resulting in a number of responses being excluded. four individuals declined consent. twenty-one were judged to have responded inauthentically, with the same scores to substantive sections of the questionnaire ('straightlining'). five did not indicate they were located in the uk. ten were not active instagram users: three individuals visited instagram 'not at all' and seven 'less often' than every few weeks. two participants responded unrealistically quickly, with response durations shorter than four minutes (the same value used as a speeding check by qualtrics in study ). all of these respondents were removed, leaving n = . these checks and exclusions were carried out prior to any data analysis. participants. the target sample size was planned to exceed n = , as in study . no attempt was made to recruit a demographically representative sample: instead, sampling quotas were used to ensure the sample was not homogenous with respect to education (pre-degree vs. undergraduate degree or above) and political preference (left, centre or right-wing orientation). sampling was not stratified by age, given that instagram use is associated with younger ages, and the number of older instagram users in the prolific pool was limited at the time the study was carried out. additionally, participants had to be uk nationals resident in the uk; active instagram users; and not participants in prior studies related to this one. each participant received a reward of £ . . participant demographics are shown in table (column ) . for the focal analysis in this study, the sample size conferred . % power to detect r = . in a multiple regression with predictors ( -tailed, alpha = . ). descriptive statistics are summarised in table . all scales had acceptable reliability. the main dependent variable, probability of liking, again had a very skewed distribution with a strong floor effect. to simultaneously test hypotheses - , a multiple regression analysis was carried out using the expanded predictor set from study . given inclusion of gender as a predictor variable, the three respondents who did not report their gender as either male or female were excluded from further analysis. the analysis, summarised in table , indicated that the model explained % of the variance in self-reported likelihood of sharing the three disinformation items. neither the authoritativeness of the story source, consensus information associated with the stories, nor consistency of the items with participant attitudes (conservatism) was a statistically significant predictor. extraversion positively and conscientiousness negatively predicted ratings of likelihood of sharing. in terms of demographic characteristics, men and younger participants reporting a higher likelihood of sharing. finally, people reported a greater likelihood of sharing the items if they believed they were likely to be true, and if they thought they had seen them before. participants had also been asked about their historical sharing of untrue political stories, both unknowing and deliberate. eighty five out of ( . %) participants who answered the question indicated that they had out ever 'shared a political news story online that they later found out was made up', while out of indicated they had shared one that they 'thought at the time was made up' ( . %). predictors of whether or not people had shared untrue material under both sets of circumstances were examined using logistic regressions, with the same sets of participant-level predictors. having unknowingly shared untrue material (table ) was significantly predicted by higher extraversion, lower conscientiousness and male gender. having shared material known to be untrue at the time (table ) was significantly predicted by higher new media literacy, higher conservatism, and higher neuroticism. as in studies and , results were not consistent with hypotheses , and : consensus, authoritativeness, and new media literacy were not associated with self-rated probability of liking the disinformation stories. in contrast to studies and , however, conservatism did not predict liking the stories. belief that the stories were true was again the strongest predictor, while likelihood of having seen them before was again statistically significant. among the personality variables, lower agreeableness returned as a predictor of likely engagement with the stories, consistent with study but not study . lower age predicted likely engagement, a new finding, while being male predicted likely engagement as found in both in study and study . unlike study and study , education had no effect. with regard to historical accidental sharing, as in study higher extraversion was a predictor, while as in study so was lower conscientiousness. men were more likely to have shared accidentally. deliberate historical sharing was predicted by higher levels of new media literacy. this is counter-intuitive and undermines the argument that people share things because they know no better. in fact, in the context of deliberate deception, motivated individuals higher in digital literacy may actually be better equipped to spread untruths. conservatism was also a predictor here. this could again be a reflection of the consistency hypothesis, given that there are high levels of conservative-oriented disinformation circulating. finally, as in study , higher neuroticism predicted deliberate historical sharing. study set out to repeat study , but with a us sample and using us-centric materials. the purpose of this was to test whether the observed effects applied across different countries. the study was completed online, using as participants members of research panels sourced through the research company qualtrics. the methodology exactly replicated that of study , except in the case of details noted below. the planned analysis was revised to include the expanded set of predictors eventually used in study (see table ). social media disinformation measures and materials were the same as used in study . the only difference from study was in the contents of the three disinformation exemplars, which were designed to be relevant to a us rather than uk audience. two of the stimuli were sourced from the website infowars.com, while a third was a story described as untrue by the fact-checking website politifact.com. in the same way as in study , the right-wing focus of the stories was again established in pilot work where a us sample (n = ) saw seven stories including these and rated their political orientation and likelihood of being shared. all were rated above the mid-point of an -point scale asking "to what extent do you think this post was designed to appeal to people with right wing (politically conservative) views?" anchored at "very left wing oriented" and "very right wing oriented". for the least right-wing of the three stories selected, a one-sample ttest comparing the mean rating with the midpoint of the scale showed it was statistically significantly higher, t ( ) = . , p < . , d = . ). one of the stimuli, also used in study - , was titled "revealed: un plan to flood america with million migrants". one was titled "flashback: obama's attack on internet freedom", subtitled 'globalists, deep state continually targeting america's internet dominance', featuring further anti-obama, china and 'big tech' sentiment, and an image of barack obama apparently drinking wine with a person of east asian appearance. the third was text based and featured material titled "surgeon who exposed clinton foundation corruption in haiti found dead in apartment with stab wound to the chest". the materials used to manipulate authoritativeness (facebook usernames shown as sources of the stories) were the same as used in studies - . these were retained because pilot work indicated that the higher and lower sets differed in authoritativeness for us audiences in the same way as for uk audiences. a sample of us participants again each rated a selection of usernames, including these , for the extent to which each was "likely to be an authoritative source-that is, likely to be a credible and reliable source of information". a within-subjects ttest indicated that mean authoritativeness ratings for the 'higher' group were statistically significantly higher than the 'lower' group (t ( ) = - . p < . , d z = . ). procedure. the procedure replicated study , save that in this case the nmls was presented across two pages. data screening and processing. prior to delivery of the sample, qualtrics performed a series of quality checks and 'data scrubbing' procedures to remove and replace participants with response patterns suggesting inauthentic or inattentive responding. these included speeding checks and examination of response patterns. on delivery of the initial sample (n = ) further screening procedures were performed. nine respondents were identified who had responded with the same scores to substantive sections of the questionnaire ('straightlining'), and one who had not completed any of the personality items. twelve respondents were not active facebook users: six reported using facebook 'not at all' and a further six less often than 'every few weeks'. all of these were removed, leaving n = . these checks and exclusions were carried out prior to any data analysis. participants. the target sample size was planned to exceed n = , as in study . qualtrics was contracted to provide a sample of active facebook users that was broadly representative of the us population in terms of gender; education level; and age profile ( +). sampling quotas were used to assemble a sample comprising approximately one third each self-describing as left-wing, centre and right-wing in their political orientation. sampling errors on the part of qualtrics led to over-recruitment of individuals aged years, who make up of the individuals in the - age group. as a consequence, the - age group is itself over-represented in this sample compared to the broader us population. participant demographics are shown in table , column . for the focal analysis in this study, the sample size conferred . % power to detect r = . in a multiple regression with predictors ( -tailed, alpha = . ). descriptive statistics are summarised in table . all scales had acceptable reliability. the main dependent variable, likelihood of sharing, again had a very skewed distribution with a strong floor effect. to simultaneously test hypotheses - a multiple regression analysis was carried out using the expanded predictor set from study . given inclusion of gender as a predictor variable, the one respondent who did not report their gender as either male or female was excluded from further analysis. the analysis, summarised in table , indicated that the model explained % of the variance in self-reported likelihood of sharing the three disinformation items. neither the authoritativeness of the story source, consensus information associated with the stories, nor consistency of the items with participant attitudes (conservatism) was a statistically significant predictor. extraversion positively predicted ratings of likelihood of sharing. in terms of demographic characteristics, age was a significant predictor, with younger people reporting a higher likelihood of sharing. finally, people reported a greater likelihood of sharing the items if they believed they were likely to be true, and if they thought they had seen them before. participants had also been asked about their historical sharing of untrue political stories, both unknowing and deliberate. of the participants, ( . %) indicated that they had ever 'shared a political news story online that they later found out was made up', while out of indicated they had shared one that they 'thought at the time was made up' ( . %). predictors of whether or not people had shared untrue material under both sets of circumstances were examined using logistic regressions, with the same sets of participant-level predictors. having unknowingly shared untrue material (table ) was significantly predicted by higher new media literacy, lower conscientiousness, higher education, and higher levels of facebook use. having shared material known to be untrue at the time (table ) was significantly predicted by higher extraversion, lower agreeableness, younger age, and higher levels of facebook use. again, the pattern of results emerging from study had some similarities but also some differences from studies - . once again, hypotheses , and were unsupported by the results. similarly to study , but unlike studies and , conservatism (the proxy for consistency) did not predict sharing the stories. belief that the stories were true, and likelihood of having seen them before, were the strongest predictors. higher levels of extraversion (a new finding) and lower ages (as in study ) were associated with higher reported likelihood of sharing the stimuli. for historical sharing, for the first time-and counterintuitively-new media literacy was associated with higher likelihood of having shared false material unknowingly. as in studies and , lower conscientiousness was also important. counterintuitively, higher education levels were associated with higher unintentional sharing, as were higher levels of facebook use. for intentional sharing, higher extraversion was a predictor, as was lower agreeableness, younger age and higher levels of facebook use. when interpreting the overall pattern of results from studies - , given the weakness of most of the associations, it is likely to be most useful to focus on relationships that are replicated across studies and disregard 'one off' findings. tables - provide a summary of the statistically significant predictors in each of the studies. it is clear that two variables consistently predicted self-rated likelihood of sharing disinformation exemplars: belief that the stories were likely to be true, and likely prior familiarity with the stories. it is also clear that three key variables did not: markers of authority, markers of consensus and digital literacy. hypothesis predicted that stories portrayed as coming from more authoritative sources were more likely to be shared. however, this was not observed in any of the four studies. one interpretation of this is that the manipulation failed. however, pilot work (see study , study ) with comparable samples indicated that people did see the sources as differing in authoritativeness. the failure to find the predicted effect could also be due to use of simulated scenarios-though care was taken to ensure they resembled reality-or weaknesses in the methodology, such as the distributional properties of the dependent variables. however, consistent relationships between other predictors and the dependent variable were observed. thus, the current studies provide no evidence that authoritativeness of a source influences sharing behaviour. hypothesis predicted that stories portrayed as having a higher degree of consensus in audience reactions (i.e. high numbers of people had previously shared them) would be more likely to be shared. in fact, consensus markers had no effect on self-reported probability of sharing or liking the stories. therefore, the current studies provide no evidence that indicators of 'social proof' influence participant reactions to the stimuli. hypothesis was that people would be more likely to share materials consistent with their pre-existing beliefs. this was operationalised by measuring participants' political orientation (overall level of conservatism) and using stimuli that were right-wing in their orientation. in studies and , more conservative people were more likely to share the materials. further evidence for hypothesis comes from the finding, across all studies, that level of belief the stories were "accurate and truthful" was the strongest predictor of likelihood of sharing. this is again in line with the consistency hypothesis: people are behaving in ways consistent with their beliefs. the finding from study that more conservative people were more likely to have historically shared material they knew to be untrue could also be in line with this hypothesis, given that a great many of the untrue political stories circulated online are conservativeoriented. hypothesis , that people lower in digital literacy would be more likely to engage with disinformation, was again not supported. as noted earlier, measurement of digital literacy is problematic. however, pilot work showed that the new media literacy scale did differentiate between people with higher and lower levels of social media use in the expected manner, so it is likely to have a degree of validity. in study , higher nmls scores were associated with having unwittingly shared false material in the past, which is counterintuitive. however, this may be due to the fact that more digitally literate people should be more able to see that something was false in hindsight. higher nmls scores were also associated with deliberately sharing falsehoods in study . this could be attributable to greater ease with which digitally literate individuals can do such things, if motivated to do so. a number of other variables were included on an exploratory basis, or for the purpose of controlling for possible confounds. of these, the most important was participants' ratings of the likelihood that they had seen the stimuli before. this variable was originally included in the design so that any familiarity effects could be controlled for when evaluating the effect of other variables. in fact, rated likelihood of having seen the materials before was the second strongest predictor of likelihood of sharing it. it was a predictor in all four studies, and for the facebook studies ( and ) it was the second most important variable. this is consistent with work on prior exposure to false material online, where prior exposure to fake news headlines increased participants' ratings of their accuracy [ ] . furthermore, it has been found that prior exposure to fake-news headlines reduced participants' ratings of how unethical it was to share or publish the material, even when it was clearly marked as false [ ] . thus, repeated exposure to false material may increase our likelihood of sharing it. it is known that repeated exposure to statements increases people's subjective ratings of their truth [ ] . however, there must be more going on here, because the regression analyses indicated that the familiarity effect was independent of the level of belief that it is true. when considering work that found that amplification of content by bot networks led to greater levels of human sharing [ ] , the implication is that repeated actual exposure to the materials is what prompts people to share it, not metrics of consensus such as the number of likes or shares displayed beside an article. of the five dimensions of personality measured, four (agreeableness, extraversion, neuroticism and conscientiousness were predictors of either current or historical sharing in one or more studies. consistent with findings from [ ] , studies and found that lower agreeableness was associated with greater probability of sharing or liking the stories. it was also associated with accidental historical sharing in study , and deliberate historical sharing in studies and . in contrast to this, past research on personality and social media behaviour indicates that more agreeable people are more likely to share information on social media: [ ] reported that its role in this was mediated by trust, while [ ] found that higher agreeableness was associated with higher levels of social media use in general. given those findings, it is likely that the current results are specific to disinformation stimuli rather than social sharing in general. agreeableness could potentially interact with the source of the information: more agreeable people might conceivably be more eager to please those close to them, however, while it is possible that agreeableness interacted in some way with the framing of the material having been shared by 'a friend' in study , study had no such framing. more broadly, the nature of the stories may be important: disinformation items are normally critical or hostile in their nature. this may mean they are more likely to be shared by disagreeable people, who themselves may be critical in their outlook and not concerned about offending others. furthermore, agreeableness is associated with general trusting behaviour. it may be that disagreeable people are therefore more likely to endorse conspiracist material, or other items consistent with a lack of trust in politicians or other public figures. lower conscientiousness was associated with accidental historical sharing of false political stories in studies , and . this is unsurprising, as less conscientious people would be less likely to check the veracity of a story before sharing it. the lack of an association with deliberate historical sharing reinforces this view. higher extraversion was associated with probability of sharing in study , with accidental historical sharing in study and , and with deliberate historical sharing in study . higher neuroticism was associated with historical deliberate sharing in studies and . all these relationships may simply reflect a higher tendency on the part of extraverted and neurotic individuals to use social media more [ ] . there are clearly some links between personality and sharing of disinformation. however, the relationships are weak and inconsistent across studies. it is possible that different traits affect different behaviours: for example low conscientiousness is associated with accidental but not deliberate sharing, while high neuroticism is associated with deliberate but not accidental sharing. thus, links between some personality traits and the spread of disinformation may be context-and motivation-specific, rather than reflecting blanket associations. however, lower agreeableness-and to a lesser extent higher extraversion-may predict an overall tendency to spread this kind of material. demographic variables were also measured and included in the analyses. younger individuals rated themselves as more likely to engage with the disinformation stimuli in studies and , and were more likely to have shared untrue political stories in the past either accidentally (study ) or deliberately (studies and ) . this runs counter to findings that older adults were much more likely to have spread material from 'fake news' domains [ ] . it is possible that the current findings simply reflect a tendency of younger people to be more active on social media. people with lower levels of education reported a greater likelihood of sharing the disinformation stories in studies and . counterintuitively, more educated people were more likely to have accidentally shared false material in the past (study ). one possible explanation is that more educated people are more likely to have realised that they had done this, so the effect in study reflects an influence on reporting of the behaviour rather than on the behaviour itself. in each of studies , and , men reported a greater likelihood of sharing or liking the stimuli. men were also more likely to have shared false material in the past unintentionally (study ) or deliberately (study ). given its replicability, this would seem to be a genuine relationship, but one which is not easy to explain. finally, the level of use of particular platforms (facebook, twitter or instagram) did not predict likelihood of sharing the stimuli in any study. level of use of twitter (study ) predicted accidental sharing of falsehoods, while facebook use predicted both accidental and deliberate sharing (study ). for historical sharing, this may be attributable to a volume effect: the more you use the platforms, the more likely you are to do these things. it should be noted that the level of use metric lacked granularity and had a strong ceiling effect, with most people reporting the highest use level in each case. in all four studies, a minority of respondents indicated that they had previously shared political disinformation they had encountered online, either by mistake or deliberately. the proportion who had done each varied across the four studies, likely as a function of the population sampled ( . %- . % accidentally; . %- . % deliberately), but the figures are a similar magnitude to those reported elsewhere [ , ] . even if the proportion of social media users who deliberately share false information is just . %, the lowest figure found here, then that is still a very large number of people who are actively and knowingly spreading untruths. the current results indicate that a number of variables predict onward sharing of disinformation. however, most of these relationships are very small. it has been argued that the minimum effect size for a predictor that would have real-world importance in social science data is β = . [ ] . considering the effect sizes for the predictors in tables , , and , only belief that the stories are true exceeds this benchmark in every study, while probability of having seen the stories before exceeded it in studies and . none of the other relationships reported exceeded the threshold. this has implications for the practical importance of these findings, in terms of informing interventions to counteract disinformation. some of the key conclusions in this set of studies arise from the failure to find evidence supporting an effect. proceeding from such findings to a firm conclusion is a logically dangerous endeavour: absence of evidence is not, of course, evidence of absence. however, given the evidence from pilot studies that the manipulations were appropriate; the associations of the dependent measures with other variables; and the high levels of power to detect the specified effects, it is possible to say with some confidence that hypotheses , and are not supported by the current data. this means that the current project does not provide any evidence that interventions based on these would be of value. this is particularly important for the findings around digital literacy. raising digital media literacy is a common and appealing policy position for bodies concerned with disinformation (e.g. [ ] ). there is evidence from a number of trials that it can be effective in the populations studied. however, no support was found here for the idea that digital literacy has a role to play in the spread of disinformation. this could potentially be attributed to the methodology in this study. however, some participants- in total across all four studies-reported sharing false political stories that they knew at the time were made up. it is hard to see how raising digital literacy would reduce such deliberate deception. trying to raise digital literacy across the population is therefore unlikely to ever be a complete solution. there is evidence that consistency with pre-existing beliefs can be an important factor, especially in relation to beliefs that disinformation stories are accurate and truthful. this implies that interventions are likely to be most effective when targeted at individuals who already hold an opinion or belief, rather than trying to change people's minds. while this would be more useful to those seeking to spread disinformation, it could also give insights into populations worth targeting with countermessages. targeting on other variables-personality or demographic-is unlikely to be of value given the low effect sizes. while these variables (perhaps gender and agreeableness in particular) most likely do play a role, their relative importance seems so low that the information is unlikely to be useful in practice. alongside other recent work [ , ] , the current findings suggest that repeated exposure to disinformation materials may increase our likelihood of sharing it, even if we don't believe it. the practical implication would be that to get a message repeated online, one should repeat it many times (there is a clear parallel with the 'repeat the lie often enough' maxim regarding propaganda). social proof (markers of consensus) seems unimportant based on current findings, so there is no point in trying to manipulate the numbers next to a post as sometimes done in online marketing. what might be more effective is to have the message posted many times (e.g. by bots) so that people had a greater chance of coming across it repeatedly. this would be true both for disinformation and counter-messages. as a scenario-based study, the current work has a number of limitations. while it is ethically preferable to field experiments, it suffers from reduced ecological validity and reliance on selfreports rather than genuine behaviour. questions could be asked, for example, about whether the authoritativeness and consensus manipulations were sufficiently salient to participants (even though they closely mirrored the presentation of this information in real-life settings). beyond this, questions might be raised about the use of self-reported likelihood of sharing: does sharing intention reflect real sharing behaviour? in fact, there is evidence to suggest that it does, with recent work finding that self-reported willingness to share news headlines on social media paralleled the actual level of sharing of those materials on twitter [ ] . the scenarios presented were all selected to be right-wing in their orientation, whereas participants spanned the full range from left to right in their political attitudes. this means that consistency was only evaluated with respect to one pole of the right-left dimension. there are a number of other dimensions that have been used as wedge issues in real-world information operations: for example, support for the black lives matter movement; climate change; or for or against britain leaving the european union. the current research only evaluated consistency between attitudes and a single issue. a better test of the consistency hypothesis would be to extend that to evaluation of consistency between attitudes and some of those other issues. a key issue is the distributions of the main outcome variables, which were heavily skewed with strong floor effects. while they still had sufficient sensitivity to make the regression analyses meaningful, they also meant that any effects found were likely to be attenuated. it may thus be that the current findings underestimate the strength of some of the associations reported. another measurement issue is around the index of social media use (facebook, twitter, instagram). as table shows, in three of the studies over % of respondents fall into the highest use category. again, this weakens the sensitivity of evaluations of these variables as predictors of sharing disinformation. in order to identify variables associated with sharing disinformation, this research programme took the approach of presenting individuals with examples of disinformation, then testing which of the measured variables was associated with self-reported likelihood of sharing. a shortcoming of this approach is that it does not permit us to evaluate whether the same variables are associated with sharing true information. an alternative design would be to show participants either true or false information, and examine whether the same constructs predict sharing both. this would enable identification of variables differentially impacting the sharing of disinformation but not true information. complexity arises, however, from the fact that whether a story can be considered disinformation, misinformation, or true information, depends on the observer's perspective. false material deliberately placed online would be categorized as disinformation. a social media user sharing it in full knowledge that it was untrue would be sharing disinformation. however, if they shared it believing it was actually true, then from an observer's perspective this would be technically categorised as misinformation (defined as "the inadvertent sharing of false information" [ , p. ] ). in fact, from the user's perspective, it would be true information (because they believe it) even though an omniscient observer would know it was actually false. this points to the importance of further research into user motivations for sharing, which are likely to differ depending on whether or not they believe the material is true. in three of the four studies (studies , , ) , the stimulus material was introduced as having been posted by a friend who wanted them to share it. this is likely to have boosted the rates of self-reported likelihood of sharing in those studies. previous work has shown that people rate themselves as more likely to engage with potential disinformation stories posted by a friend, as opposed to a more distant acquaintance [ ] . to be clear, this does not compromise the testing of hypotheses in those studies (given that the framing was the same for all participants, in all conditions). it is also a realistic representation of how we may encounter material like this in our social media feeds. however, it does introduce an additional difference between studies , and when compared with study . it would be desirable for further work to check whether the same effects were found when messages were framed as having been posted by people other than friends. finally, the time spent reading and reacting to the disinformation stimuli was not measured. it is possible that faster response times would be indicative of more use of heuristics rather than considered thought about the issues. this could profitably be examined, potentially in observational or simulation studies rather than using self-report methodology. a number of priorities for future research arise from the current work. first, it is desirable to confirm these findings using real-world behavioural measures rather than simulations. while it is not ethically acceptable to run experimental studies posting false information on social media, it would be possible to do real-world observational work. for example, one could measure digital literacy in a sample of respondents, then do analyses of their past social media sharing behaviour. another priority revolves around those individuals who knowingly share false information. why do they do this? without understanding the motivations of this group, any interventions aimed at reducing the behaviour are unlikely to be successful. as well as being of academic interest, motivation for sharing false material has been flagged as a gap in our knowledge by key stakeholders [ ] . the current work found that men were more likely to spread disinformation than women. at present, it is not clear why this was the case. are there gender-linked individual differences that influence the behaviour? could it be that the subject matter of disinformation stories is stereotypically more interesting to men, or that men think their social networks are more likely to be interested in or sympathetic to them? while the focus in this paper has been on factors influencing the spread of untruths, it should be remembered that 'fake news' is only one element in online information operations. other tactics and phenomena, such as selective or out-of-context presentation of true information, political memes, and deliberately polarising hyperpartisan communication, are also prevalent. work is required to establish whether the findings of this project related to disinformation, also apply to those other forms of computational propaganda. related to this, it would be of value to establish whether the factors found here to influence sharing of untrue information, also influence the sharing of true information. this would indicate whether there is anything different about disinformation, and also point to factors that might influence sharing of true information that is selectively presented in information operations. the current work allows some conclusions to be drawn about the kind of people who are likely to further spread disinformation material they encounter on social media. typically, these will be people who think the material is likely to be true, or have beliefs consistent with it. they are likely to have previous familiarity with the materials. they are likely to be younger, male, and less educated. with respect to personality, it is possible that they will tend to be lower in agreeableness and conscientiousness, and higher in extraversion and neuroticism. with the exception of consistency and prior exposure, all of these effects are weak and may be inconsistent across different populations, platforms, and behaviours (deliberate v. innocuous sharing). the current findings do not suggest they are likely to be influenced by the source of the material they encounter, or indicators of how many other people have previously engaged with it. no evidence was found that level of literacy regarding new digital media makes much difference to their behaviour. these findings have implications for how governments and other bodies should go about tackling the problem of disinformation in social media. conceptualization: tom buchanan. formal analysis: tom buchanan. investigation: tom buchanan. methodology: tom buchanan. disinformation and 'fake news': final report the global disinformation order: global inventory of organised social media manipulation warring songs: information operations in the digital age the ira, social media and political polarization in the united states the disconcerting potential of online disinformation: persuasive effects of astroturfing comments and three strategies for inoculation against them uk phone masts attacked amid g-coronavirus conspiracy theory. the guardian misinformation in the covid- infodemic government responses to malicious use of social media what's the difference between organic, paid and post reach? 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presidential elections. cage working paper series ( ) why summaries of research on psychological theories are often uninterpretable the tactics & tropes of the internet research agency wä nke m. the truth about the truth: a meta-analytic review of the truth effect how do personality traits shape information-sharing behaviour in social media? exploring the mediating effect of generalized trust. information research: an international electronic journal self-reported willingness to share political news articles in online surveys correlates with actual sharing on twitter writing -review & editing: tom buchanan. key: cord- -bw x h authors: little, mark p.; zhang, wei; van dusen, roy; hamada, nobuyuki title: pneumonia after bacterial or viral infection preceded or followed by radiation exposure - a reanalysis of older radiobiological data and implications for low dose radiotherapy for covid- pneumonia date: - - journal: int j radiat oncol biol phys doi: . /j.ijrobp. . . sha: doc_id: cord_uid: bw x h purpose currently, there are about ongoing clinical studies on low dose radiotherapy (ldrt) for covid- pneumonia. one of the underlying assumptions is that irradiation of . – . gy is effective at ameliorating viral pneumonia. we aimed to reanalyze all available experimental radiobiological data to assess evidence for such amelioration. methods and materials with standard statistical survival models, and based on a systematic literature review, we re-analyzed thirteen radiobiological animal datasets published in - in which animals (guinea pigs/dogs/cats/rats/mice) received radiation before or after bacterial/viral inoculation, and assessing various health endpoints (mortality/pneumonia morbidity). in most datasets absorbed doses did not exceed gy. results for six studies evaluating post-inoculation radiation exposure (more relevant to ldrt for covid- pneumonia) the results are heterogeneous, with one study showing a significant increase (p< . ) and another showing a significant decrease (p< . ) in mortality associated with radiation exposure. among the remaining four studies, mortality risk was non-significantly increased in two studies and non-significantly decreased in two others (p> . ). for pre-inoculation exposure the results are also heterogeneous, with six (of eight) datasets showing a significant increase (p< . ) in mortality risk associated with radiation exposure and the other two showing a significant decrease (p< . ) in mortality or pneumonitis morbidity risk. conclusions these data do not provide support for reductions in morbidity or mortality associated with post-infection radiation exposure. for pre-infection radiation exposure the inconsistency of direction of effect is difficult to interpret. one must be cautious about adducing evidence from such published reports of old animal datasets. low dose radiotherapy (ldrt) for coronavirus disease (covid- ) pneumonia was proposed in early april ( , ) . at least clinical studies are currently ongoing in countries ( ). the rationale for clinical benefit, in other words the effectiveness of irradiation at the level of . - . gy in treating viral pneumonia, largely relies on early human case studies or animal studies mostly obtained in the pre-antibiotic era, when a number of attempts were made to treat various non-cancer diseases with ionizing radiation, including virally-or bacteriallyassociated pneumonia. an influential paper underlying a number of proposals made for use of ldrt to treat covid- pneumonia ( , ) was calabrese and dhawan in ( ) who reviewed papers describing various relatively small case series, describing outcomes from low dose radiotherapy with x-rays (ldrt) for pneumonia. their sampling frameworks are unknown, and therefore they are subject to ascertainment bias and are largely uninterpretable. calabrese and dhawan ( ) also identified four radiobiological animal studies of post-inoculation ldrt, all from experiments done in the s, namely fried ( ) using a guinea pig model, lieberman et al. ( ) using a canine model, baylin et al ( ) using a cat model, and dubin et al. ( ) using a murine model, the first two of these for bacterially-induced pneumonia and the last two for virally-induced pneumonia. however, calabrese and dhawan ( ) did not consider four other radiobiological studies relating to post-inoculation rt, nor results of eight others relating to preinoculation rt, and did not attempt any statistical reanalysis of these old data. the aim of the present paper is to look at the totality of published radiobiological data relating to radiation exposure before or after inoculation with a viral or bacterial agent likely to result in pneumonia. because of the age of the data being considered there are shortcomings in the original statistical analysis that was performed -indeed in all but a few cases ( , ) there j o u r n a l p r e -p r o o f was no formal statistical analysis in the original reports. it is the purpose of this paper to report reanalysis of the data abstracted from the original publications so far as that is achievable, using standard statistical survival models in order to assess modification of pneumonia morbidity or mortality risk by radiation exposure before or after inoculation. we aimed to capture all radiobiological datasets relating to moderate or ldrt whether given before or after viral or bacterial inoculation leading to pneumonia. we searched literature by means of a pubmed search (using terms ((radiation or radiotherapy) and pneumonia and viral and animal) or ((radiation or radiotherapy) and pneumonia and bacterial and animal)) conducted on - - , which returned articles. we also searched for citations of the articles of fried ( ), lieberman et al. ( ) , baylin et al ( ) , dubin et al. ( ) , and an authoritative contemporary review (of ) by taliaferro and taliaferro ( ) on the same date. we did not restrict by date or language of the publication. we selected from these searches all relevant articles with information on radiobiological animal experiments in which there was any type of ionizing radiation exposure with determination of mortality or morbidity from bacterially-or virally-induced pneumonia. the datasets used are listed in table . it should be noted that the datasets we used include three of the four cited by calabrese and dhawan ( ), but did not include the paper of fried ( ) which we judged did not contain any quantitatively useful information. in appendix a we provide details of the process used to abstract data from the publications that we identified as being potentially informative. the data was abstracted independently three times by xxx, yyy and zzz. we convert the given free-in-air dose in j o u r n a l p r e -p r o o f legacy units radiation absorbed dose (rad), roentgen (r) or rep in all studies to absorbed dose in gray (gy) via the scaling r/rep = . gy. rad = . gy ( ) . details of the statistical models fitted are given in table , and some further details on adjustments used are also given in the summary table . mortality and morbidity risks in the radiobiological cohorts of lieberman et al. ( ) and dubin et al. ( ) were assessed using a cox proportional hazards models ( ) , with time after radiation exposure, if that followed the inoculation, or time after bacterial or viral inoculation, if that followed the radiation exposure, as timescale, in which the relative risk (rr) (=hazard ratio) of death for animal i at time a after inoculation was given by a linear model in dose: or alternatively using a log-linear model in dose: where i d is the total dose (in gy), α is the excess relative risk coefficient (err) per unit dose (gy). for most of the other datasets a linear logistic model is fitted to the data (generally on number of animals that died in each group): in some cases the more standard loglinear logistic model is fitted to the data (generally on number of animals that died in each group): for the data of fried ( ) , numbering only animals and using as outcome improvement in pneumonia in relation to unirradiated controls, an exact logistic model was used ( ), as non-j o u r n a l p r e -p r o o f exact methods did not converge. it is well known that the excess odds ratio (eor) approximates to the excess relative risk ( ) . all confidence intervals (cis) and two-sided p-values are profilepartial-likelihood based ( ) . in the murine dataset of dubin et al. ( ) in various subgroups risks were assessed in relation to radiation dose administered after inoculation or dose before inoculation. in the murine dataset of quilligan et al ( ) pre-inoculation dose was given to all animals; there is ambiguity in quilligan et al ( ) as to whether radiation exposure may also have been given post-inoculation. there is some uncertainty associated with the number of mice in the first of the control groups in this dataset, so a range is employed, spanning the plausible range of - mice, with as the central estimate ( table ). the model was stratified by the three experiments reported in the data of dubin et al. ( ) and by the three groups used by lieberman et al. ( ) . tables b and b and figure and show the risks in relation to dose for these two datasets. in various other datasets adjustment was made for various other covariates, as detailed in tables b -b . in the fits to the pneumonia intensity data of baylin et al. ( ) we used either loglinear logistic regression (as described above) comparing each pneumonia intensity group and those with greater intensity vs every group with reduced intensity and because of the small number of animals ( ) we also employed exact logistic methods ( ); we also used ordinal regression with log-linear link ( ) fitting to all the ordered intensity groups. in fits of the days of infection data of baylin et al. ( ) we used a linear regression model, estimating the cis via the bias-corrected advanced method ( ) . all models were fitted via epicure ( ), r ( ) or logxact ( ). two-sided levels of statistical significance are reported in all cases, with a conventional threshold for type i error of -sided p< . used to assign statistical significance. all statistical analyses were independently performed by xxx and yyy to check for j o u r n a l p r e -p r o o f concordance. all datasets and analysis files are available in online supporting information (appendix c). we present results of analyses of risk in relation to whether radiation exposure occurred after inoculation or before inoculation. the results are given in summary form in table , which also provides summary details of the models used and assumptions made in fitting, and in more detail in appendix b tables b -b . table , table b ), as also shown by figure . however, table b shows that this is largely driven by a single group, group , as also shown by figure . the three groups in the study of lieberman et al ( ) , were treated with slightly different x-ray energies, kvp, kvp and kvp, respectively, and mean doses also slightly differed, . gy, . gy and . gy, respectively (table b ). there are few indications of trend of degree of pneumonia infection with dose in the feline data of baylin et al. ( ) , whether using logistic, exact logistic or ordinal models ( table , table b ). however, table b indicates that there is a significant decreasing trend of days of acute figure ); results did not appreciably vary with the type of model used (linear logistic, log-linear logistic) or whether or not adjustment was made for the virus concentration (table b ). there is a highly significant increasing trend (p< . ) of mortality risk with dose after endemic coccobacillus infection in the rat data of bond et al. ( ) , whether adjusting for likelihood of infection or not ( as also shown by figure . there is no significant trend (p> . ) with post-inoculation dose in the data of dubin et al. ( ) , whether using linear or log-linear models, which is confirmed also by figure (table b ) . however, there is a borderline significant decreasing trend (p= . ) of mortality with preinoculation dose in this dataset, with eor/gy = - . ( % ci - . , - . ) ( table , table b ) again confirmed by figure . in the murine data of beutler and gezon ( ) there are highly significant (all p< . ) increasing trends of mortality with post-inoculation dose, whether in relation to mouse-adapted or eggadapted pr influenza a virus and irrespective of the type of statistical model (linear logistic, loglinear logistic) used; for example with a linear logistic model the eor per gy is . ( % ci . , . ) ( table , table b ) , and as also shown in figure . the morbidity trends exhibit more heterogeneity, so that for the mouse-adapted virus the trends are generally negative, so that j o u r n a l p r e -p r o o f for example with a linear logistic model the eor per gy is - . ( % ci - . , - . ) (table b ); however, for the egg-adapted virus the trends are generally positive with dose, so that for example with a linear logistic model the eor per gy is . ( % ci . , . ) (table b ) , as also shown in figure . in the swiss mice data of hale and stoner ( ) there is no overall trend (p> . ) of mortality with radiation dose given before inoculation with type iii pneumococcus. however, if attention is restricted to the animals that received inoculation there is a highly significant increasing trend with dose (p< . ), so that the eor per gy is . ( % ci . , . ) ( table , table b ). the same researchers went on to study a wider range of infective agents in the same strain of mice, and observed a generally highly significant (p< . ) increase in mortality risk associated with radiation before inoculation with influenza virus, pneumococcus type iii bacterial infection or trichinella spiralis larval infection ( ), whether adjusted or not for type of first immunizing infection, so that for example without such adjustment and excluding the trichinella spiralis challenge infections the eor/gy = . ( % ci . , . ) ( table b , table ). there is a highly significant (p< . ) increase in mortality risk in the c bl mouse data of quilligan et al ( ) associated with post-influenza-inoculation radiation dose with eor/gy ranging from . ( % ci . , . ) to . ( % ci . , . ) depending on how many mice are assumed to be in the first control group (table b , table ). pneumonitis morbidity and mortality were significantly decreased (p< . ) after . gy whole body air-dose exposure in adult male albino cf- mice in the data of berlin ( ) , so that for pneumonitis morbidity the eor/gy = - . ( % ci - . , - . ) and for pneumonitis mortality the eor/gy = - . ( % ci - . , - . ) (table b , table ). in contrast table (see also j o u r n a l p r e -p r o o f table b ) and figure b show reanalysis of slightly later data of berlin and cochran ( ), which exhibits slightly heterogeneous results, with one set of experiments (given in table iii of berlin and cochran ( )) indicating a highly significant increase (p≤ . ) in influenza mortality, whether or not adjusted for mode of administration of virus, but a different experimental set (reported in table ii of the paper) showing no significant effect (p> . ) of radiation exposure on influenza morbidity or mortality. these experiments use a similar murine system, also given . gy whole body air-dose exposure, as in the earlier paper of berlin ( ). lundgren et al ( ) used a novel type of radiation exposure, aerosolized ceo , which delivers localized β dose to the lungs of c bl/ j mice. there was a small but highly significant increase in mortality risk associated with radiation exposure, with eor/gy = . ( % ci . , . , p= . ) ( table , table b ). we have re-analyzed thirteen radiobiological animal datasets, dating from the late s to the early s, in which bacterial or viral agents were administered to induce pneumonia in animals that were also exposed to varying fractionated doses of radiation before or after inoculation. the statistical analysis in the original papers was limited, indeed in all but two cases ( , ) there was no formal statistical analysis in the publications. we therefore judged it necessary to statistically reanalyze the data from the original publications with standard statistical models. for the six studies that evaluated post-inoculation radiation exposure (which is more ( ); the groups were treated with slightly different x-ray energies, and mean doses also slightly differed (see results and table b ). it is possible that these variations in mean dose and radiation energy may have some bearing on the differences observed ( table ). it is possible that the range of doses used, and the variable degree of fractionation employed may also be factors, although there does not appear to be an obvious pattern linking these to the direction or strength of effect, as shown by tables and . more recently, hasegawa et al ( ) showed that irradiation before influenza vaccination and a j o u r n a l p r e -p r o o f succeeding lethal challenge influenza inoculation exacerbates mortality from influenza, but that vaccination prior to irradiation confers protection against a subsequent challenge influenza inoculation. dadachova et al ( ) reported that targeted radionuclide immunotherapy induces streptococcus pneumoniae killing in vivo, thereby alleviating bacterial pneumonia. as can be inferred, both these publications ( , ) address somewhat different questions to those of this paper. calabrese and dhawan ( ) reviewed three of the studies we consider here ( ) ( ) ( ) , and a fourth ( ) which we judged did not contain any quantitatively useful information, and stated that "these studies constitute the entire set of animal model studies assessing the capacity of x-rays to affect pneumonia-induced clinical symptoms and mortality. each study demonstrated some measure of support for the hypothesis that x-ray treatment could reduce the effects of the pneumonia induced by bacteria or viruses." manifestly this is not the entirety of the literature relating to post-inoculation radiation exposure (tables , ) , and a review of our results (table ) demonstrates that there is little evidence overall of reduction of morbidity or mortality with increasing radiation dose. calabrese and dhawan ( ) also reviewed papers (all published before ) describing or mostly relatively small case series (the uncertainty reflecting whether or not an otherwise unpublished case series mentioned by a discussant in a paper of powell ( ) was included) and concluded that "x-ray therapy was successful in decreasing the mortality rate in untreated patients from about percent to to percent". however, as noted in the introduction, without a well-defined sampling framework such data are likely subject to ascertainment bias and are effectively uninterpretable. the radiation doses employed in some of the case series are also unknown. similar conclusions have been arrived at by others in discussing this paper ( , ) . the major strength of the present analysis is that we use standard statistical models to assess the totality of published radiobiological data on ldrt given either before or after virallyor bacterially-induced pneumonia. moreover, unlike the review of calabrese and dhawan ( ) in this appendix we outline the steps taken to transcribe data from the papers relating to radiobiological animal experiments produced by our literature search. data was transcribed from data was transcribed from tables - (pp. - ) into a table giving dog number, group number, blood culture (coding "…." = - , "+"= , "++"= , "+++"= , "++++"= , " "= ) pneumonia type ("i"= ,"iii"= ), radiation energy (kev)(from table title), radiation st day (gy converting r = . gy), radiation nd day (gy converting r = . gy), radiation rd day (gy converting r = . gy), radiation total (gy converting r = . gy), radiation total excluding day of death (gy converting r = . gy), death day, dead (coding =dead, =no death). for those animals that are indicated as having recovered (animal in table , animals , , , , in table ) a large value ( ) was used as the end of follow-up time j o u r n a l p r e -p r o o f (death day), with death coded as for these animals, so that the records for these animals only contribute to the denominator in the cox model fit. we constructed a we constructed a table from tables ii and iii table ii and table iii . j o u r n a l p r e -p r o o f we used a combination of the data in table (p. ) and table (p. ) to create a table with fields for experiment number, series, sex, age start weeks (minimum), age start weeks (maximum), infection status [coded as likely unexposed to infection, probably exposed to infection, highly probably exposed to infection -so that experiments and were both coded as probably exposed to infection, and all except the last group of experiment were coded as highly probably exposed to infection, the last group of experiment being coded as likely unexposed to infection], minimum radiation dose (in gy, using r = . gy), maximum radiation dose (in gy, using r = . gy), deaths infected, deaths total, survived infected, survived total. these numbers were used to compute entries for numbers of deaths among infected and uninfected animals, which formed the basis of the analysis. the radiation dose used was the mean of the minimum and maximum doses. dubin et al ( table. we continued in this way for all elements of this table. all information about doses at various times before or after inoculation came from the text description above each row in the table. there was information on individual mice in this data. from for the first group, increasing by for each successive group), the radiation dose (gy, using r = . gy), the number of mice that died and the number exposed. likewise we constructed a similar table for morbidity from mouse-adapted virus, based on table iii (p. ) with records containing for each group of animals the experiment number, the suspension group, the negative log viral dilution, the group number (an arbitrary number, which we took to start at for the first group, increasing by for each successive group), the radiation dose (gy, using r = . gy), the number of mice with morbidity and the number in each treatment group. from j o u r n a l p r e -p r o o f observed odds ratio = is low dose radiation therapy a potential treatment for covid- pneumonia? covid- tragic pandemic: concerns over unintentional "directed accelerated evolution" of novel coronavirus (sars-cov- ) and introducing a modified treatment method for ards gov studies found for covid- radiation how radiotherapy was historically used to treat pneumonia: could it be useful today? the roentgen treatment of experimental pneumonia in the guinea-pig roentgen therapy of experimental lobar pneumonia in dogs the effect of roentgen therapy on experimental virus pneumonia; on feline virus pneumonia the effect of roentgen therapy on experimental virus pneumonia; on pneumonia produced in white mice by swine influenza virus sparing effect of x-rays for mice inoculated intranasally with egg-adapted influenza virus, cam strain delay of fatal pneumonia in x-irradiated mice inoculated with mouseadapted influenza virus, pr strain effect of x-rays on immunity; a review unit)). wikipedia regression models and life-tables die artefizielle pneumonie und ihre bestrahlung. experimentalbeitrag zur frage der wirkung der röntgenstrahlen auf entzündungsgewebe statistical methods in cancer research. volume ii -the design and analysis of cohort studies generalized linear models continuous cobalt- irradiation and immunity to influenza virus regression models for ordinal data better bootstrap confidence intervals risk sciences international project version . . . r: a language and environment for statistical computing effect of roentgen therapy on mouse encephalitis the effects of x irradiation on a naturally occurring endemic infection the effect of total body x irradiation on the susceptibility of mice to influenza a virus infection the effect of cobalt- gamma radiation on passive immunity effects of ionizing radiation on immunity effects of inhaled ceo on influenza virus infection in mice protection against influenza virus infection by nasal vaccination in advance of sublethal irradiation feasibility of radioimmunotherapy of experimental pneumococcal infection low dose radiation therapy for covid- pneumonia: is there any supportive evidence? is there any supportive evidence for low dose radiotherapy for covid- pneumonia? low-dose radiation therapy for covid- pneumopathy: what is the evidence? flying by the seat of our pants: is low dose radiation therapy for covid- an option? lack of supporting data make the risks of a clinical trial of radiation therapy as a treatment for covid- pneumonia unacceptable united nations scientific committee on the effects of atomic radiation (unscear) systematic review and meta-analysis of circulatory disease from exposure to low-level ionizing radiation and estimates of potential population mortality risks tables and figures of results table b . improvement in pneumonia morbidity in guinea pigs associated with radiation exposure post inoculation to staphylococcus aureus haemolyticus in the data of fried ( ) key: cord- -cxrjzva authors: kang, minghui; wu, haolin; yang, qiao; huang, li; hu, quanjun; ma, tao; li, zaiyun; liu, jianquan title: a chromosome-scale genome assembly of isatis indigotica, an important medicinal plant used in traditional chinese medicine: an isatis genome date: - - journal: hortic res doi: . /s - - - sha: doc_id: cord_uid: cxrjzva isatis indigotica ( n = ) is an important medicinal plant in china. its dried leaves and roots (called isatidis folium and isatidis radix, respectively) are broadly used in traditional chinese medicine for curing diseases caused by bacteria and viruses such as influenza and viral pneumonia. various classes of compounds isolated from this species have been identified as effective ingredients. previous studies based on transcriptomes revealed only a few candidate genes for the biosynthesis of these active compounds in this medicinal plant. here, we report a high-quality chromosome-scale genome assembly of i. indigotica with a total size of . mb and scaffold n = . mb using single-molecule real-time long reads and high-throughput chromosome conformation capture techniques. we annotated , high-confidence protein-coding genes. based on homolog searching and functional annotations, we identified many candidate genes involved in the biosynthesis of main active components such as indoles, terpenoids, and phenylpropanoids. in addition, we found that some key enzyme-coding gene families related to the biosynthesis of these components were expanded due to tandem duplications, which likely drove the production of these major active compounds and explained why i. indigotica has excellent antibacterial and antiviral activities. our results highlighted the importance of genome sequencing in identifying candidate genes for metabolite synthesis in medicinal plants. the plant family brassicaceae (cruciferae) comprises over genera and~ species with a worldwide distribution [ ] [ ] [ ] [ ] [ ] . numerous crops are derived from this family, including vegetables (brassica and raphanus), ornamentals (matthiola, hesperis, and lobularia), spices (eutrema and armoracia), and medicines (isatis). based on sequenced genomes, several model species have been developed for diverse studies, including arabidopsis thaliana for molecular function studies, brassica for polyploidization and whole-genome duplication (wgd) studies, and eutrema salsugineum for abiotic tolerancerelated studies. however, genetic biosynthesis of the major active compounds in medicinal plants of this family remains poorly investigated. isatis indigotica ( n = ) belongs to tribe isatideae in lineage ii of the family , - . this species is widely cultivated in china as an important medicinal plant because its dried leaves and roots are used as a traditional chinese medicine for curing diseases and viruses [ ] [ ] [ ] . the major active compounds isolated from this species comprise terpenoids, lignans, and indole alkaloids [ ] [ ] [ ] [ ] . these compounds were confirmed to have antiviral , , antibacterial , anti-inflammatory , , and antileukemia , functions. previous studies based on transcriptomes revealed a few candidate genes involved in the biosynthesis of active compounds in this species [ ] [ ] [ ] [ ] . however, the limitations of transcriptome quality and integrity hinder the identification of all candidate biosynthesisrelated genes. in the present study, we used single-molecule sequencing combined with high-throughput chromosome conformation capture (hi-c) technology to assemble the genome and construct the pseudochromosomes of i. indigotica. based on homolog searching and functional annotations, we aimed to identify candidate gene sets involved in the biosynthesis of putative active components. the candidate genes and genomic resources recovered here will be critically important for further experimental verification and artificial syntheses of the active compounds of this medicinal plant in the future. the genome size, genome repeat size, and heterozygosity rate of i. indigotica were estimated using k-mer analysis. the -mer frequency of illumina short reads with the highest peak occurred at a depth of . the genome was estimated to be . mb in size with . % repeats, and the heterozygosity rate was estimated to be . % (supplementary table s and supplementary fig. s ). in addition, the genome size of i. indigotica was estimated to be~ mb based on flow cytometric analyses using vigna radiata as the internal standard (supplementary fig. s ). we sequenced and assembled the genome of i. indigotica using single-molecule real-time (smrt) sequencing technology from pacific biosciences (pacbio) and anchored the assembled contigs to seven pseudochromosomes using hi-c techniques. the final chromosome-scale genome was . mb in length with contigs (contig n = . mb), a scaffold n = . mb, and a maximum pseudochromosome length of . mb (table , supplementary table s , and supplementary fig. s ). the completeness of the genome assembly was evaluated using benchmarking universal single-copy orthologs (busco) . of the plant-specific orthologs, ( . %) were identified in the assembly, of which ( . %) were considered to be complete (supplementary table s ). the assembly base accuracy was also assessed based on illumina short read mapping. in total, . % of the clean reads were mapped to the genome assembly, and . % of them were properly mapped (supplementary table s ). the base error percentage of the genome assembly was estimated to be . % (supplementary table s ). all these evaluations indicate the high completeness, high continuity, and high base accuracy of the present genome assembly. repetitive sequences were identified using a combination of ab initio and homology-based approaches. in total, we identified . % of the assembled sequences as repetitive sequences, including . % retrotransposons and . % dna transposons. long terminal repeat (ltr) retrotransposons were found to account for . % of the genome (supplementary table s ). we annotated proteincoding genes by combining transcriptome-based, homology-based, and ab initio predictions. finally, we predicted a total of , genes, of which had alternatively spliced transcripts. the average transcript length and coding sequence size were and bp, respectively, with a mean of . exons and . transcripts per gene (table ) . overall, , genes ( . %) were assigned functions, and . % and . % of these genes had homologies and annotated proteins in the swiss-prot and trembl databases. further functional annotations using interproscan estimated that . % of the genes contained conserved protein domains, and . % of the genes were classified by gene ontology (go) terms, with . % mapped to known plant biological pathways based on the kyoto encyclopedia of genes and genomes (kegg) pathway database (supplementary table s ). evolution of chromosome structures in brassicaceae has been traced and established through comparative chromosome painting techniques using bac probes of the a. thaliana genome , . using these techniques, lysak and (fig. a) . six tribes (calepineae, coluteocarpeae, conringieae, eutremeae, isatideae, and sisymbrieae) of expanded lineage ii were found to derive from a common ancestor with the proto-calepineae karyotype (pck; n = ). among these tribes, three (eutremeae, isatideae, and sisymbrieae) displayed an additional whole-arm translocation in the second and seventh chromosomes (translocation pck, tpck; n = ) - (fig. a) . ack and pck shared five similar chromosomes. thus, they might descend from a common ancestor; alternatively, pck may have evolved from ack. to determine whether the i. indigotica genome sequence also supported tpck structure in isatideae, we compared the seven pseudochromosomes of i. indigotica with the a. thaliana genome by last and mcscanx. we table s ). the i. indigotica genome has good collinearity in each gb compared with the a. thaliana genome and is consistent with tpck structure in both order and orientation ( fig. and supplementary figs. s , s ). furthermore, we carried out sequence alignments between the genomes of i. indigotica and the other three species that might also display tpck structure (sisymbrium irio for sisymbrieae, e. salsugineum for eutremeae, and schrenkiella parvula for unassigned genera) using last. our analyses suggested that these four species have similar chromosome structures (supplementary figs. s -s ). however, we found obvious inversions in the s. parvula genome and low continuity of sequences in the e. salsugineum and s. irio genomes. these comparisons suggest that the present i. indigotica genome was better assembled in terms of both accuracy and continuity than others with tpck structure. we clustered the annotated genes into gene families among i. indigotica and eight other brassicaceae species with cleome hassleriana as the outgroup. a total of , i. indigotica genes ( . %) clustered into , gene families, of which , ( . %) gene families were shared with nine other species and ( . %) were i. indigotica specific ( fig. c and supplementary table s ). we selected single-copy gene families among species to construct a phylogenetic tree, which showed that i. indigotica was sister to s. irio. we further estimated the divergence time between them as . ( . - . ) million years ago (mya) (fig. a) . the relationships of all species are consistent with those from previous phylogenetic analyses , - , . then, we used synonymous substitution rates (ks) between collinear paralogous genes to identify potential wgd events, based on the assumption that the number of silent substitutions per site between two homologous sequences increases in a relatively linear manner with time. a density plot of ks values for the collinear gene pairs suggested that i. indigotica experienced a recent wgd event with a peak value of~ . , consistent with at-α-wgd for all brassicaceae species , , . an independent wgd event was identified for b. rapa after its divergence from i. indigotica at ks = . - . , previously reported as a brassiceae-specific triplication (br-α-wgd) , - (fig. b) . whole-genome alignment among the i. indigotica, a. thaliana, and b. rapa genomes carried out by last also confirmed the collinear relationship and these wgd events. for each genomic region of i. indigotica, we typically found one matching region in a. thaliana and three matching regions in b. rapa. these comparisons suggest that i. indigotica did not experience an independent wgd event after at-α-wgd (supplementary figs. s , s ). the expansion and contraction of gene families play critical roles in driving phenotypic diversification and enhancing special traits in plants. we discovered expanded and contracted gene families in i. indigotica relative to s. irio (fig. a) . tandem duplication was the main contributor to the gene family expansions. go enrichment analysis of tandem repeat genes suggested that they were enriched in defense response to virus, indole biosynthetic process, lignin biosynthetic process, flavone synthase activity, and glucosyltransferase activity, some of which might be involved in the biosynthesis of active compounds in i. indigotica (supplementary table s ). we also performed go enrichment analysis of the contracted gene families, and the results showed that they were enriched in proton export across plasma membrane, proton-exporting atpase activity, regulation of stomatal movement, and defense response to other organism (supplementary table s ), which are probably related to the environmental adaptation of the species. based on the kegg database, go classification, and the suggested biosynthesis pathways, we used a combined method of homolog searching and functional annotation thaliana gb boundaries were derived from a previous study to identify candidate genes for the biosynthesis of three types of active compounds, namely, terpenoids, phenylpropanoids, and indoles, in i. indigotica [ ] [ ] [ ] , , . sterols are the major terpenoids in i. indigotica, mainly comprising β-sitosterol and daucosterol . β-sitosterol was reported to play a critical role in curing lung inflammation , while daucosterol can inhibit cancer cell proliferation . a total of genes in the present genome, which encoded enzymes, were identified to be involved in terpenoid and sterol biosynthesis (supplementary table s ). based on the functional annotations of these genes, the biosynthesis pathway of β-sitosterol is nearly complete and daucosterol can be further synthesized from β-sitosterol by glucosyltransferases (fig. a) . in addition, the intermediate product geranyl diphosphate can be used not only to synthesize sterols but also to produce secologanin for monoterpene indole alkaloids in numerous medicinal plants such as catharanthus roseus . however, we annotated genes only with geraniol -hydroxylase activity (go: ). the lack of other related genes may account for the absence of secologanin and other related monoterpene indole alkaloids in i. indigotica. table s ). the identified putative pathway mainly comprises the biosynthesis of isovitexin and lariciresinol, while their glycosides were further synthesized by glucosyltransferases (fig. b) . indole alkaloids comprise another active component of i. indigotica [ ] [ ] [ ] with important anti-influenza, anti-inflammatory, and leukocyte inhibition effects , , [ ] [ ] [ ] . based on the kegg maps and previously suggested pathways , , , we identified genes that encoded enzymes involved in the biosynthesis of indole alkaloids (fig. c and supplementary table s ). because of the lack of downstream pathways, other genes for indole alkaloid biosynthesis in i. indigotica need further identification. it should be noted that numerous genes involved in the biosynthesis of the three major types of active compounds increased in copy number because of tandem duplication, for example, geranylgeranyl diphosphate synthase, cinnamate -hydroxylase, coumarate-coa ligase, and indole- -pyruvate monooxygenase ( fig. and supplementary tables s -s ). continuity and completeness are important indicators of genome assembly. pacbio-based genome assembly plus error corrections based on illumina data could greatly improve continuity and completeness [ ] [ ] [ ] [ ] . our genome assembly of i. indigotica by this strategy showed a highly resolved result with an n = . mb and longest contig length = . mb. in addition, we used hi-c data to cluster the contigs into seven pseudochromosomes with a final scaffold n = . mb and longest chromosome length = . mb. the completeness and high quality of the present i. indigotica genome were further confirmed by busco and comparative chromosome analyses . a total of . % of the genes examined by busco were complete, and the chromosome structure of i. indigotica was consistent with the tpck type. we constructed the phylogenetic relationships of i. indigotica based on genomic data and found that i. indigotica of isatideae is sister to s. irio of sisymbrieae among the sampled species, consistent with the results of previously published phylogenetic analyses , [ ] [ ] [ ] . based on the phylogenetic results, we identified expanded and contracted gene families in i. indigotica. the expanded genes in this species were mainly derived from tandem duplications and were obviously enriched in some secondary metabolite pathways. based on homolog searching and functional annotation in our high-quality genome, we further identified candidate genes for the biosynthesis of three main classes of active compounds in i. indigotica: terpenoids, phenylpropanoids, and indole alkaloids. these candidate genes complete or replenish gene sets for biosynthetic pathways of these compounds concentrated in i. indigotica [ ] [ ] [ ] [ ] (fig. ). in addition, we found that in some synthesis steps, the copy number of enzyme-coding genes increased to two or more because of tandem duplications. the increase in copy number may drive the production of major active compounds in i. indigotica and account for its excellent antibacterial and antiviral activities because gene expansions are responsible for enhancing a special trait or the origin of a new trait [ ] [ ] [ ] . overall, in this study, we present a high-quality genome for i. indigotica. we further identify or replenish candidate genes for biosynthesis pathways of the active compounds in this medicinal plant. these genes and genomic resources will provide a solid basis for future biosynthesisrelated studies. we initially extracted high-quality total dna from fresh young leaves of a -month-old plant artificially cultivated in the greenhouse using the cetyltrimethylammonium bromide method. we used a smrtbell template prep kit . (pacbio, menlo park, ca, usa) to construct the dna libraries for pacbio long-read sequencing and sequenced them on a pacbio sequel system. we obtained a total of four smrt cells with . gb of sequencing data (coverage of . ×) from the pacbio sequel platform and generated a total of . million subreads with an n read length of . kb (supplementary table s and supplementary fig. s ). we also prepared paired-end illumina libraries using an illumina genomic dna sample preparation kit and sequenced them on an illumina hiseq x ten system for error correction and k-mer analysis and generated a total of . gb of data and . gb of clean data (supplementary table s ). we initially estimated the genome size of i. indigotica by flow cytometry with vigna radiata as the reference . we then used clean illumina short reads to calculate k-mers (illumina dna short read size of bp) by jellyfish v. . . to confirm the genome size. the sequencing depth was estimated by determining the highest peak value of the frequency curve of the k-mer occurrence distribution. we used smrt link pipeline v. . . . to process the polymerase reads into subreads with readscore = . and minsubreadlength = and used canu v. . to correct errors of the pacbio subreads and assemble the corrected reads into contigs after trimming low-quality bases using wtdbg (https://github.com/ ruanjue/wtdbg). we corrected the assembled contigs by using bp pe illumina data by pilon v. . and finally obtained a . mb contig-scale assembly with a contig n of . mb. the genome contained contigs, and the longest contig was . mb with a . % gc content. these contigs were further anchored to chromosomes by the hi-c technique. we grounded~ g of fresh young leaf tissue into powder in liquid nitrogen for hi-c experiments and constructed a hi-c library following louwers et al. with chromatin extraction and digestion and dna ligation, purification, and fragmentation. finally, we obtained a total of . gb of clean reads for hi-c analyses by the illumina hiseq x ten platform. we first carried out a preliminary assembly by splitting contigs into segments of kb on average and mapping the hi-c data to the contigs using bwa v. . . -r in order to correct contig errors. we then used lachesis software we identified repetitive elements through both repeatmodeler v. . . and repeatmasker v. . . , . repeatmodeler employed recon and repeatscout to predict interspersed repeats and then obtained the consensus repeat library. repeatmasker recovered the repeats in the i. indigotica genome through a homologybased repeat search using the ab initio repeat database and repbase. the overlapping repeats belonging to the same repeat class were combined according to their coordination in the genome. the overlapping repeats belonging to different repeat classes were then split into different types. to improve gene prediction, we further obtained transcriptomes by sequencing high-quality rna from mixed fresh leaf, flower, and stem tissues and sequenced them by the illumina hiseq x ten platform. we removed adapters and discarded reads with > % n bases or reads having more than % bases of low quality (below ) using ngs qc toolkit v. . . and finally generated . gb of clean data. we assembled the de novo and genomeguided transcriptomes with clean reads by trinity v. . . . we also mapped the rna-sequencing (rnaseq) reads to the assembled genome to obtain the mapping rate through hisat v. . . to evaluate the completeness of the genome. we run pasa pipeline v. . . to align the transcripts to the assembled genome to carry out orf prediction and gene prediction. to train the hmm model for augustus, we extracted complete, multiexon genes, removed redundant high-identity genes (cut-off all-to-all identity of %), and finally generated the best candidate and low-identity gene models for training. we aligned the rna-seq data to the hard-masked genome assembly by hisat and searched with an e value of e − . after filtering lowquality results, gene structure was predicted using genewise v. . . . we combined the results from pasa, augustus and genewise to generate the final protein-coding gene set using evidencemodeler v. . . . to obtain the untranslated regions and alternatively spliced isoforms, we used pasa to update the gff file for two rounds and obtain the final gene models. we annotated the functions of the predicated genes against public databases by ncbi blast+ v. . . with a cut-off e value of e − and maximum number of target sequences of , including the swiss-prot and trembl databases . best-hit blast results were then used to define gene functions. we used interproscan v. . - . to identify motifs and domains by matching against public databases. we identified go annotations by using blast go v. . according to the blast results and combined them with interpro go entries. we mapped the existing go terms to enzyme codes by blast go and submitted the predicted proteins to the kegg (kyoto encyclopedia of genes and genomes) automatic annotation server (kaas) to obtain ko numbers for kegg pathway annotation. we used protein sequences of i. indigotica and eight other brassicaceae species (arabidopsis thaliana, capsella rubella, brassica rapa, brassica napus, raphanus sativus, schrenkiella parvula, sisymbrium irio, and eutrema salsugineum) with the outgroup species cleome hassleriana for same-family gene clustering. for genes with alternative splicing variants, the longest transcript was selected to represent the gene. similarities between sequence pairs were calculated using blastp v. . . with a cut-off e value of e − . additionally, orthomcl v. . . was used with default parameters to assess gene family membership based on overall gene similarity combined with markov chain clustering (mcl) v. - . we extracted single-copy orthologous genes from the ten species by orthomcl and aligned the resulting protein sequences by mafft v. . . then, we used gblocks v. . b to extract the conserved sites of multiple sequence alignments and constructed a phylogenetic tree by raxml v. . . . we used c. hassleriana as an outgroup and performed bootstrap analyses to test the robustness of each branch. we used the bayesian relaxed molecular clock approach in mcmctree of paml v. . e to estimate divergence time. we calibrated this tree based on the estimated divergence times in the timetree database gene families that had undergone expansion or contraction were identified in the eight sequenced species using cafe . the cafe parameters included a p value threshold = . and automatic searching for the λ value. the algorithm in cafe takes a matrix of gene family sizes in extant species as input and uses a probabilistic graphical model to ascertain the rate and direction of changes in gene family size across a given phylogenetic tree. to examine wgd in i. indigotica and b. rapa, we extracted all homologous proteins between these two species and a. thaliana using an all-to-all search in blastp v. . . with an e value cut-off of e − . we used mcscanx with default parameters to identify collinear blocks, each containing at least five collinear gene pairs. to infer wgd events, we used the downstream mcscanx script add_ka_and_ks_to_collinearity.pl to calculate the ks values between collinear genes among these three genomes. we further performed whole-genome alignment of the three species by last v. and constructed a dot plot by the downstream program last-dotplot. identification of tandem repeat genes in the i. indigotica genome was based on three criteria: ( ) two or more genes had more than % identity and % coverage according to blastp; ( ) the pairwise gene distance was < kb; and ( ) there were no more than genes lying between the repeat genes on a single scaffold . the genes identified in this way were subjected to functional analysis using go enrichment. the cruciferae of continental north america: systematics of the mustard family from the arctic to panama transfer of the south african genera brachycarpaea, cycloptychis, schlechteria, silicularia, and thlaspeocarpa to heliophila (brassicaceae) systematics and phylogeny of the brassicaceae (cruciferae): an overview phylogeny, genome, and karyotype evolution of crucifers (brassicaceae) brassicaceae: species checklist and database on cd-rom brassicaceae phylogeny and trichome evolution brassicaceae phylogeny inferred from phytochrome a and ndhf sequence data: tribes and trichomes revisited cabbage family affairs: the evolutionary history of brassicaceae plastome phylogeny and early diversification of brassicaceae resolving the backbone of the brassicaceae phylogeny for investigating trait diversity antiviral activity of isatis indigotica extract and its derived indirubin against japanese encephalitis virus antiviral action of radix isatidis and folium isatidis from different germplasm against influenza a virus antioxidant and anti-inflammatory activities of radix isatidis polysaccharide in murine alveolar macrophages alkaloids from the root of isatis indigotica qualitative and quantitative analysis of flavonoids in the leaves of isatis indigatica fort. by ultra-performance liquid chromatography with pda and electrospray ionization tandem mass spectrometry detection phenylpropanoids isolated from tetraploid roots of isatis indigotica research progress of chinese herbal medicine radix isatidis (banlangen) anti-sars coronavirus c-like protease effects of isatis indigotica root and plant-derived phenolic compounds indole alkaloids from the roots of isatis ingigotica and their antiherpes simplex virus type (hsv- ) activity in vitro function research of resistance to salmonella typhimurium infection using banlangen polysaccharide studies on the antinociceptive, anti-inflammatory and antipyretic effects of isatis indigotica root among plant lignans, pinoresinol has the strongest antiinflammatory properties in human intestinal caco- cells indirubin, the active constituent of a chinese antileukaemia medicine, inhibits cyclin-dependent kinases inhibition of leukocyte functions by the alkaloid isaindigotone from isatis indigotica and some new synthetic derivatives biosynthesis of the active compounds of isatis indigotica based on transcriptome sequencing and metabolites profiling elicitation of isatis tinctoria l. hairy root cultures by salicylic acid and methyl jasmonate for the enhanced production of pharmacologically active alkaloids and flavonoids cloning and characterization of a differentially expressed phenylalanine ammonialyase gene (iipal) after genome duplication from tetraploid isatis indigotica fort isolation and characterization of a gene encoding cinnamoyl-coa reductase from isatis indigotica fort busco: assessing genome assembly and annotation completeness with single-copy orthologs painting of arabidopsis chromosomes with chromosome-specific bac clones the abc's of comparative genomics in the brassicaceae: building blocks of crucifer genomes comparative paleogenomics of crucifers: ancestral genomic blocks revisited chromosomal phylogeny and karyotype evolution in x = crucifer species (brassicaceae) deciphering the diploid ancestral genome of the mesohexaploid brassica rapa unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events paleopolyploidy in the brassicales: analyses of the cleome transcriptome elucidate the history of genome duplications in arabidopsis and other brassicales comparative genome mapping in brassica ancient whole genome duplications, novelty and diversification: the wgd radiation lag-time model nested radiations and the pulse of angiosperm diversification: increased diversification rates often follow whole genome duplications a comprehensive metabolite profiling of isatis tinctoria leaf extracts indole alkaloids from the roots of isatis indigotica and their inhibitory effects on nitric oxide production research progress of antiviral active components of radix isatidis effects of lactose-β-sitosterol and β-sitosterol on ovalbumininduced lung inflammation in actively sensitized mice daucosterol inhibits cancer cell proliferation by inducing autophagy through reactive oxygen species-dependent manner genome-guided investigation of plant natural product biosynthesis isovitexin exerts anti-inflammatory and anti-oxidant activities on lipopolysaccharide-induced acute lung injury by inhibiting mapk and nf-κb and activating ho- /nrf pathways dietary lariciresinol attenuates mammary tumor growth and reduces blood vessel density in human mcf- breast cancer xenografts and carcinogen-induced mammary tumors in rats diglycosidic indole alkaloid derivatives from an aqueous extract of isatis indigotica roots novel indole c-glycosides from isatis indigotica and their potential cytotoxic activity antiviral glycosidic bisindole alkaloids from the roots of isatis indigotica inhibition of rantes expression by indirubin in influenza virusinfected human bronchial epithelial cells indirubin inhibits inflammatory reactions in delayed-type hypersensitivity alpha-tryptophan synthase of isatis tinctoria: gene cloning and expression employing a biochemical protecting group for a sustainable indigo dyeing strategy altering regioselectivity cytochrome p bm- saturation mutagenes is for the biosynthesis of indirubin de novo pacbio long-read and phased avian genome assemblies correct and add to reference genes generated with intermediate and short reads improved brassica rapa reference genome by single-molecule sequencing and chromosome conformation capture technologies liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation gossypium barbadense and gossypium hirsutum genomes provide insights into the origin and evolution of allotetraploid cotton genomic signatures of specialized metabolism in plants a role for gene duplication and natural variation of gene expression in the evolution of metabolism gene duplication in the diversification of secondary metabolism: tandem -oxoglutarate-dependent dioxygenases control glucosinolate biosynthesis in arabidopsis genome sequence of mungbean and insights into evolution within vigna species a fast, lock-free approach for efficient parallel counting of occurrences of k-mers canu: scalable and accurate long-read assembly via adaptive kmer weighting and repeat separation pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement studying physical chromatin interactions in plants using chromosome conformation capture ( c) fast and accurate short read alignment with burrows-wheeler transform chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions using repeatmasker to identify repetitive elements in genomic sequences de novo identification of repeat families in large genomes toolkit: a toolkit for quality control of next generation sequencing data trinity: reconstructing a full-length transcriptome without a genome from rna-seq data hisat: a fast spliced aligner with low memory requirements automated eukaryotic gene structure annotation using evi-dencemodeler and the program to assemble spliced alignments augustus: a web server for gene finding in eukaryotes blast+: architecture and applications the swiss-prot protein sequence database and its supplement trembl in interproscan-an integration platform for the signature-recognition methods in interpro blast go: a comprehensive suite for functional analysis in plant genomics kaas: an automatic genome annotation and pathway reconstruction server orthomcl: identification of ortholog groups for eukaryotic genomes mafft multiple sequence alignment software version : improvements in performance and usability selection of conserved blocks from multiple alignments for their use in phylogenetic analysis raxml version : a tool for phylogenetic analysis and postanalysis of large phylogenies paml : phylogenetic analysis by maximum likelihood timetree: a resource for timelines, timetrees, and divergence times estimating gene gain and loss rates in the presence of error in genome assembly and annotation using cafe mcscanx: a toolkit for detection and evolutionary analysis of gene synteny and collinearity adaptive seeds tame genomic sequence comparison importance of lineage-specific expansion of plant tandem duplicates in the adaptive response to environmental stimuli key: cord- -jcueu xh authors: rauch, sheila a.m.; simon, naomi m.; rothbaum, barbara o. title: rising tide: responding to the mental health impact of the covid‐ pandemic date: - - journal: depress anxiety doi: . /da. sha: doc_id: cord_uid: jcueu xh now freely downloadable on the anxiety and depression association of america website (https://adaa.org/sites/default/files/phasedapproachtocovid‐ .ver . % ( ).pdf) is a framework for covid‐ mental health response (see table ). this framework of phased interventions and resources is intended to assist health systems and programs impacted by the pandemic to plan for how to address current mental health issues arising as well as to prepare and plan for the continued needs of their communities, patients, and staff. this article is protected by copyright. all rights reserved. is larger than previous pandemics and has resulted in more deaths than / (cdc, ) , there is much that the mental health field knows from military, disaster settings, and other types of trauma exposures that can help guide how we support our health professionals working in this extremely stressful environment (va/dod, ) . investments in efforts to support mental health and provide indicated prevention and intervention may decrease long-term risk for negative mental health outcomes, including for healthcare workers and other staff on the frontlines of the pandemic. many are predicting that the mental health impact will be grim. we agree that the mental health impact will be significant, but also feel confident that for most, the pandemic and its aftermath will be a significant stressor that people cope with in real time and even if highly distressed acutely, will eventually naturally recover and move on to a new normal and satisfying life. the mental health response must be measured and not overly emphasize a belief and expectation of an epidemic of unmanageable long-term negative mental health impacts. instead, while planning to address mental health needs that will arise, we should project hope and have confidence that most people will recover with time. data from the field of trauma and bereavement strongly support that resilience is the modal outcome even for severe challenges (bonanno et al., ; barbara olasov rothbaum, foa, riggs, murdock, & walsh, ) . nonetheless, a shortage of access to effective mental healthcare was already a problem prior to the pandemic and will worsen even if only a small percentage require intervention (thomas, ellis, konrad, holzer, & morrissey, ). as such, planning needs to consider efficient use of resources and possibly stepped care models to provide the most effective and least resource intensive course of care to respond adequately (richards et al., ; zatzick et al., ) . helping people cope acutely this article is protected by copyright. all rights reserved. while assuring access to those in need of higher levels of mental health intervention are critically important goals. while the evidence base for prevention intervention is less clear about optimal approaches, there are nonetheless many helpful strategies that may help reduce both short-and long-term distress, and their targets are guided by relevant clinical and research lessons learned over many years. key to any mental health response to the current pandemic or other extended potentially highly distressing and/or traumatic events is a response that simultaneously considers both timing (referred to as phase) and associated distress and/or functional impairment (referred to as level). as defined below, the framework includes three phases (initial, post, and longer-term) and three levels (system level, self-directed level, and mental health supported brief intervention). the phased approach to covid- mental health response (pac) now freely downloadable on the anxiety and depression association of america website (https://adaa.org/sites/default/files/phasedapproachtocovid- .ver . % ( ).pdf) is a framework for covid- mental health response (see table ). this framework of phased interventions and resources is intended to assist health systems and programs impacted by the pandemic to plan for how to address current mental health issues arising as well as to prepare and plan for the continued needs of their communities, patients, and staff. in addition, many of the resources presented may be used by healthcare professionals and others on the front lines of care, as well as anyone being significantly impacted by covid- as they see fit. the framework provides a model for response over time and across the wide range and severity of potential impact of the pandemic, including program design considerations and examples of evidence-guided resources when available. for specific areas where this article is protected by copyright. all rights reserved. previous resources were not freely available, select evidence informed brief interventions were created and are downloadable from the framework posting on the adaa website [mask desensitization (with colleagues from the university of chicago, emory university school of medicine, and new york university), selfdirected difficult experience exposure (created by the authors and dr. jeffrey cigrang) , and assessment protocol with brief intervention for a mh provider adapted from (rothbaum et al., ) . this proposed phased approach is intended to help guide efficient allocation of mental health resources to those most in need of assistance at the time that they need it at the level that they need it (e.g., self-directed versus brief intervention with some support versus traditional treatment sessions with a provider). appropriate allocation of expert mental health resources can assist the system in providing evidence based clinical care to those who need it. the initial phase includes the period of time while we are dealing with the ongoing stressor, such as the current covid- pandemic (see table ). this phase would end when the initial risk and impact has ended or reduced to a "new normal" or lower level of risk. the post phase includes the initial responses in the period after the acute exposure to risk and loss is complete and the following three months when expectations are that people impacted will be having various levels of emotional reactions and response (see table ). the long-term phase covers from three months after the individual's experience of impact and loss is over and into the future (see table ). for this pandemic, the phases will overlap for individuals depending on their roles and specific impacting traumatic exposures (traumatic loss of significant this article is protected by copyright. all rights reserved. others, exposure to death at work, etc.). further, grief responses generally occur on a longer timeline with current iterations of prolonged or complicated grief utilizing a or month minimum for diagnosis to account for broad individual, cultural and religious variability in usual acute grief and its evolution to more integrated forms of grief (e.g., for recent commentary and treatment approaches see (iglewicz et al., ; simon et al., ) . within each phase, there are different levels of response. the system supported level focuses on recommendations for leadership and organizations to put in place for those in the relevant phase (see table ). the self-directed level includes resources that individuals can work with on their own. these self-directed interventions are intended to be used based on either self-assessment of need or as an initial intervention for those with mild to moderate distress and/or functional impairment without imminent risk to self or others (see table ). finally, the mental health supported brief intervention level includes brief interventions provided with entry through primary care or mental health providers for those with a higher level of need or for whom self-directed approaches are not possible and/or effective (see table ). as basic principles across all phases and levels, systems and individuals engaging in mental health response are encouraged to: ) ensure basic needs for food, sleep, and lodging are met. for healthcare providers and staff this means management of risk of personal and family covid- infection such as ppe. ) find creative ways to safely enhance social connection and support. ) provide ongoing support for people as they would like-not a single shot. this article is protected by copyright. all rights reserved. them in the past that they can draw on again such as talking with friends and/or family, exercise, yoga, prayer, etc. and monitor or possibly reduce unhelpful coping such as the use of alcohol or other harmful strategies. ) create opportunities for people as they are interested and able to talk about difficult experiences. this can be helpful to process them, but avoid compelling people to tell their stories, especially in groups. let them share as they are ready and willing-be ready to listen but don't force the story. ) avoid group debriefing where everyone is forced to share and listen to details of death or traumatic events. this has shown iatrogenic effects in some studies of trauma and ptsd. other types of group support efforts, however, can be helpful to provide opportunity to build community, emotional support, psychoeducation and reminders about resources (even if virtual). ) provide information about accessible mental health resources for those who may need them. people undergoing stress most often need support from family and community for basic needs, safety, and emotional support. plans for mental health response in the coming months must focus on providing social support and helping people to feel in control of things they can control while focusing professional mental health resources on those who need it most. the framework offers some ideas about how to integrate a phased approach to helping support those on the front lines or anyone being significantly emotionally challenged by the covid- pandemic to meet our this article is protected by copyright. all rights reserved. community needs over time. empirical examination of the evidence informed new resources that are part of the framework will soon be underway and as we learn about needs and response overtime, this framework will be modified to address the best science available. resilience to loss and chronic grief: a prospective study from preloss to -months postloss moving effective treatment for posttraumatic stress disorder to primary care: a randomized controlled trial with active duty military corona virus disease (covid- ) cases date and surveilence complicated grief therapy for clinicians: an evidence-based protocol for mental health practice delivering stepped care: an analysis of implementation in routine practice a prospective examination of post-traumatic stress disorder in rape victims early intervention may prevent the development of posttraumatic stress disorder: a randomized pilot civilian study with modified prolonged exposure understanding and addressing sources of anxiety among health care professionals during the covid- commentary on evidence in support of a grief-related condition as a dsm diagnosis county-level estimates of mental health professional shortage in the united states this article is protected by copyright. all rights reserved reactions when they think about their experiences.in military service members to reduce ptsd symptoms when provided within the primary care environment with an embedded mental health provider as support (cigrang et al., (rothbaum, et al., ) . early intervention may prevent the development of posttraumatic stress disorder: a randomized pilot civilian study with modified prolonged exposure. settings. focus more closely over time on those with identified functional impairment over time evidence based care such as cbt, or pharmacotherapy when indicated guideline key: cord- -wg sa u authors: quah, stella r. title: public image and governance of epidemics: comparing hiv/aids and sars date: - - journal: health policy doi: . /j.healthpol. . . sha: doc_id: cord_uid: wg sa u abstract a comparative analysis of the – infectious disease outbreak, severe acute respiratory syndrome (sars), and the hiv/aids epidemic that has affected the world over the past two decades reveals the significant role of socio-cultural beliefs and attitudes in the shaping of people's lifestyles and approaches to the control and prevention of epidemics. the main research question is: what can we learn from the sars experience about effective prevention of hiv/aids? the sources of data include population figures on the development of these epidemics and findings from two sociological studies of representative samples of singapore's multi-ethnic population. the comparative study illustrates the impact of cultural beliefs and attitudes in shaping the public image of these two different infectious diseases; the relevance of public image of the disease for effective prevention and control of epidemics. traditionally, the human suffering inflicted by longterm epidemics have tended to find expression in literature and the fine arts thus becoming a visible part of the collective memory and shaping the public image of the disease. for example, the impact of the bubonic and pneumonic plague or "black death" had a major influence on painters of the gothic period [ ] ; tuberculosis is featured in eugene g. o'neill's long day's journey into night; franz kafka's diaries; thomas mann's the magic mountain [ ] ; victor hugo's les misérables; dickens' nicholas nickleby, brontë's wuthering heights; verdi's la traviata [ ] . among e-mail address: socquahs@nus.edu.sg. epidemics in the past three decades (hiv/aids, sars, mad cow disease, and avian flu among others), only hiv/aids has lasted long enough to inspire artistic expressions in literature [ ] [ ] [ ] [ ] , theatre [ ] , dance [ ] , and film [ ] mostly used as vehicles for hiv/aids preventive education programs particularly in africa [ ] [ ] [ ] [ ] [ ] . one of the pioneer studies in prevention was published in by zinsser [ ] . ever since, a community of experts worldwide has been dedicated to prevention [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, despite the struggle to convey a more accurate and humane public image of aids in the past decade, the stigma attached to hiv/aids still persists as a formidable obstacle to prevention efforts [ ] [ ] [ ] . figures on the spread of the disease suggest we are losing the battle against hiv/aids especially in developing countries [ , [ ] [ ] [ ] [ ] . in china alone, the estimated number of deaths due to aids as of december (latest figures available), ranged from , to , and , persons infected with hiv/aids. the figures for thailand, the second most affected east asian country, are , - , deaths, and , persons infected with hiv (appendix a, table a . ). in contrast to the dismal hiv/aids situation, the - outbreak of severe asymptomatic respiratory syndrome or sars, offers a completely different picture for the analysis of preventive efforts. although sars, like hiv/aids, was unknown in the medical world and hit unexpectedly, there are some significant differences, particularly in their etiology, epidemiology, natural history and clinical outcomes of the two diseases. hiv/aids is asymptomatic for - years after infection so that hiv-positive persons may continue to spread the disease unknowingly. the main mode of hiv/aids transmission is through direct contact with infected body fluids or blood (sexual intercourse, use of infected needles by drug users and receiving contaminated blood transfusions). sars is caused by the sars coronavirus and characterized by airborne transmission. sars develops very rapidly, with an average incubation period of days or a range of - days after contact. within week of the illness patients show typical influenza-like symptoms such as fever, malaise, and headache with cough and diarrhoea getting worst in the second week of infection. it has been determined that "transmission occurs mainly during the second week of illness". these external signs facilitate prompt action: exposed patients may be placed under fever surveillance twice a day "in an isolation facility or ward for at least days after the last exposure to the source case(s)" [ ] . in the span of months sars infected persons, caused deaths (appendix a, table a . ) and became a widespread visible threat through the serious disruption of normal daily activities of individuals and major sectors of the economy such as transportation, commerce, industrial production, and tourism [ ] [ ] [ ] . the first probable sars case was reported in china on november and the infection spread to other countries around the world but the largest number of locally transmitted infections and deaths were reported in china, hong kong, taiwan, canada, and singapore (appendix a, table a . ). despite the fact that sars caught the world unprepared, hit at great speed, and it is very difficult to eradicate [ ] , the outbreak was contained within months, a relatively brief period of time (appendix a, table a . ). despite the medical differences (in etiology, epidemiology, natural development and clinical outcomes) between these two epidemics, i argue and attempt to demonstrate in this study that we may advance our knowledge on preventive strategies by conducting a systematic comparison of important social aspects of hiv/aids and sars. what can we learn from the sars experience about effective prevention of hiv/aids? more specifically, why were the efforts to contain and prevent the spread of a new epidemic like sars successful while it has taken years so far to contain the spread of hiv/aids and no effective solution is yet in sight? social science research has identified over the past decades a complex array of factors and conditions associated with disease prevention in individuals (micro-level analysis) as well as collectivities (macro-level analysis) but the factors and conditions vary for different diseases and there may be many other factors yet to be identified. still, contrasting the two epidemics in terms of social attitudes and beliefs at the micro-and macro-levels, will help us to elucidate some of the major obstacles to hiv/aids prevention. therefore, this paper focuses on only three possible factors: the impact of perceived severity and susceptibility to infection and the public image of the epidemic (micro-level factors); and the governance of epidemics (macro-level factor). sociology and social psychology offer some interesting explanations of the sluggishness of preventive health behavior in individuals [ , ] . among ten theories identified as the "most often used" today [ ] , the top two explanatory models are the social cognitive theory (sct) and the health belief model (hbm) [ , ] . both social theories are useful in the analysis of preventive action: they focus on the individual's capacity to make his/her own decisions, and the recognition that there are multiple and varied factors involved in a person's health-related actions. the sct explains people's health-related actions primarily in terms of their expectations of the outcome, their confidence in the success of their actions, and their ability to perform the action; but perhaps the most relevant aspect of the sct is its consideration of the individual's environment as one of the important determinants of his/her behavior [ ] . the hbm proposes that the likelihood of a person taking preventive action increases if he/she believes in his/her personal susceptibility to the illness and in the severity of the illness; perceives the preventive action as beneficial; believes that there are no barriers to action or that barriers can be overcome; believes in the net gain -benefits exceed barriers or costs-of taking preventive action [ , , ] . a comprehensive review of studies applying the hbm [ ] found the variables perceived susceptibility and perceived severity to be closely correlated and to have significant influence upon a variety of preventive behaviors. the combination of both perceived severity and perceived susceptibility labeled "perceived threat" has been found to be a more significant explanatory variable than severity and susceptibility used separately [ ] . the hbm variable influencing the perception of effective prevention of hiv/aids in three ethnic communities is perceived severity or seriousness of the disease, together with the perception of personal responsibility for contracting the disease [ ] . i ascertained perceived severity of hiv/aids in terms of the respondents' subjective perception of the likelihood of death using the close-ended question "when you think about aids, how serious do you feel it is? this approach is basically the same as that of janz et al. [ ] who define perceived severity as "one's belief of how serious a condition and its sequelae are". strecher et al. had offered earlier [ ] a wider definition of perceived severity: "personal evaluations of the probable biomedical, financial and social consequences of contracting hiv and having aids". although there are slight variations in the wording of the question asked in interviews and in the definition of perceived severity, the general consensus in the literature is that this conceptual construct, often together with perceived susceptibility, is essential in the analysis of people's motivation to take preventive action. nevertheless, as preventive health behavior is influenced by a multiplicity of factors, the hbm, sct and all the other top eight theories [ ] are limited as they offer only partial explanations of health-related behavior, but they are complementary. i include social constructs from the hbm and the sct and some relevant contextual social factors, to explore the research question "what can we learn from the sars experience about effective prevention of hiv/aids"? combining the analysis of individuals' responses with their collective implications, i attempt to demonstrate that perceived severity together with perceived susceptibility and the public image of the two epidemics help to explain some of the difference in prevention effectiveness. i test two related assumptions: ( ) a higher perception of disease severity and personal susceptibility to sars as compared to hiv/aids, contributed to the higher effectiveness of sars prevention efforts; ( ) the second assumption is two-fold: (a) in contrast to sars, the overall negative social 'image' of hiv/aids as a disease associated with particular types of individuals tends to weaken people's perception of susceptibility; (b) correspondingly, low perceived susceptibility tends to discourage public support for robust preventive efforts at the community level. these assumptions require elaboration. following the hbm, the first assumption to be tested is that a person's perception of the severity of the disease and his/her perceived susceptibility to that disease are likely to motivate him/her towards taking preventive action. all things being equal, such a commitment to prevention would weaken or be altogether absent when perceived severity and/or susceptibility are low or nil. the perceived severity of hiv/aids was ascertained through the question "when you think about aids, how serious do you feel it is? four alternative response categories were provided (see tables and a. ) . for the logistic analysis, the responses were dichotomized into high perceived severity (( ) "very serious, causes death"), and low perceived severity (( ) "not very serious" or "not serious at all" and "don't know"). perceived susceptibility to hiv/aids was ascertained by the level of agreement to the statement "aids doesn't happen to people like me" (tables and a. ). in the study of sars, perceived susceptibility was ascertained through the question "how likely do you think it is for you to contract sars"? respondents expressed their belief in their personal susceptibility by indicating whether they saw the likelihood of contracting sars as "very likely", "likely", "not very likely", "not likely at all", or did not know ( table ). the perception of severity of sars was measured by the question "if you have contracted sars, what is the likelihood of survival"? the four response categories were "very likely", "likely", "not very likely" and "not likely at all" ( table ) . the second assumption is that in contrast to sars, the overall negative public 'image' of hiv/aids as a disease associated with particular types of individuals tends to weaken people's perception of susceptibility and, correspondingly, tends to discourage public support for robust preventive efforts at the community level. the individual's 'image' of the disease shapes his/her perception of seriousness and susceptibility and thus contributes to his/her motivation to take preventive action. that 'image' of the disease and of persons affected, however, is shaped to a large extent by prevailing values and normative beliefs in the community and it is subject to change over time. this process is suggested by many sociological theories including social networks and social support theory [ ] , rational choice theory [ ] , and the sct. the sct offers the concept "reciprocal determinism" [ ] that proposes a dynamic interplay of "the person, behavior, and the environment". a eight preventive measures were considered as part of the respondents' "activities during the past days": covering the mouth with paper tissue or handkerchief when sneezing or coughing; covering the mouth with bare hand when sneezing or coughing; washing hands after sneezing or coughing; using soap or liquid hand-wash when washing hands; wearing a mask over the mouth; using serving utensils (chopsticks or spoons) for shared food when joining others for meals; when touching objects that may possible carry the sars virus (e.g., door handles, buttons in lifts), taking preventive measures (e.g., pressing lift buttons with tissue paper); washing hands as soon as possible after touching objects that may possibly carry the sars virus (e.g., door handles, buttons in lifts). b the original response categories for perceived susceptibility (that is, the perceived likelihood of contracting sars) were: "very likely", "likely", "not very likely", "not likely at all" and "don't know". for the logistic regression analysis the latter group, . % of respondents who had no idea on their susceptibility to sars, were contrasted with all other respondents who did have an assessment of their likelihood of getting infected. c the original response categories for perceived severity (that is, the likelihood of survival) were "very likely", "likely", "not very likely" and "not likely at all". for the logistic regression analysis, these responses were dichotomized into low perceived severity (survival "very likely/likely") and high perceived severity (survival "not very likely"/"not likely at all"). d the respondents' appraisal of the health authorities' crisis management was ascertained by their assessment of the distribution of information in terms of accuracy, clearness, sufficiency, timeliness, and trustworthiness in a scale from very negative (score ) to very positive (score ). the scale had high reliability (α = . ) and the mean score was . (s.d. = . ). in my view, the dynamic interaction of the individual's health-related behavior and the environment is better explained by the individual's subjective perception of the situation (for example a crisis or stressor) and the social context of the situation, as proposed by symbolic interaction and family stress theory [ ] . thus, my assumption that a person's motivation to take preventive action may also be manifested in his or her cooperation with community-based preventive measures is explained clearly by the application of the conceptual premises on community responses to stressor events formulated by reiss and oliveri [ ] . the public's perception of the scope of the problem was highlighted by these authors as part of their concept "community's punctuation of an event". they defined the community's punctuation of an event or perceived scope as "when the problem begins and when it ends and who is involved". they proposed that the community and its leaders would be more inclined to invest concerted efforts to solve a problem if three conditions are met: accountability, duty, and competence [ ] . that is, the community and its policy-makers would be most inclined to mobilize assistance and preventive efforts when these three conditions are met: the persons affected are perceived as not being accountable for the problem; they are regarded as having the duty to request outside help; they are considered as lacking the competence to solve by themselves the problem affecting them. i suggest that these three conditions shape the public image of sars and hiv/aids and add to our understanding of the disparity in prevention effectiveness of these two epidemics at the community level. the public image of hiv/aids was ascertained through one open-ended question about persons living with hiv/aids: "what kind of people do you think are most likely to get aids"? the analysis of responses revealed three types of stereotypes or 'images': 'risktakers', 'deviants', and 'victims'. only a small group of respondents had not particular image of people living with hiv/aids ( table ). the public image of sars may be ascertained indirectly through the respondents' perception of a sense of social responsibility and willingness to make some personal sacrifices in combating the disease. those perceptions reflect people's collective sense of accountability and duty and their recognition of the need for expertise to handle the crisis. referring the respondents to the measures implemented to prevent the spread of sars, they were asked their level of agreement (strongly agree to strongly disagree) with these statements: "people should be willing to make some personal sacrifices"; "people have mostly been socially responsible"; "if you did not develop symptoms of sars after having close contact with someone diagnosed with sars, would you agree to be quarantined for days?"; "if you did not develop symptoms of sars after having non-close contact with someone diagnosed with sars, would you agree to be quarantined for days"? ( table ). the condition of "competence" identified by reiss and oliveri [ ] as discussed above, has to do with expertise in handling the crisis, and thus brings in a final concept of relevance to this discussion: governance. governance is another socially significant aspect of hiv/aids and sars as a strong political will at the national level is needed to invest state resources, and to utilize knowledge and technology creatively for their detection and prevention. but these epidemics are also global problems that challenge national boundaries and the conventional idea of state sovereignty and governance, and test international cooperation, because of their mode of transmission and the increasing movement of people across countries for leisure, trade, and study among other activities. given the easiness and speed of their international transmission, these epidemics have unwittingly pushed forth a new phenomenon that fidler [ ] calls "global health governance" that is, "the proposition that governance of public health issues must include not only state actors but also non-state actors". examining the international impact of sars, this international law expert concluded that "to govern an increasingly borderless world, requires, in essence, increasingly borderless governance" [ ] . the analysis of the sars crisis management in singapore suggests that effective containment and prevention of infectious disease outbreaks requires dedicated and transparent governance at both levels, national and global. and to be successful, global governance requires timely and effective response and collaboration from sovereign nations. but as i shall discuss later, the governance approach to sars differs substantially from that applied to hiv/aids. first, a word of caution: i reiterate that this study is by necessity limited and exploratory because the two epidemics, hiv/aids and sars, are very different microbiologically and epidemiologically as indicated in the introduction; and there is a -year gap between the two surveys. the sars study was conducted as the outbreak progressed in may , years after the hiv/aids study. nonetheless, despite these methodological difficulties, i believe it is important to scrutinize available information on both epidemics in the hope of increasing our understanding of the dynamics of preventive action against hiv/aids. while the two epidemics are different in many respects, they are both serious public health threats that require a collective response [ , ] as indicated earlier. two general types of data are discussed here: population figures and data from personal interviews. the international population figures on the impact and spread of hiv/aids and sars are taken from who's published reports [ , , , [ ] [ ] [ ] . the analysis of behavior and attitudes of individuals is based on data from two separate studies i conducted in singapore as principal investigator. the data on hiv/aids are from a study of attitudes and preventive behavior regarding hiv/aids based on a survey of personal interviews with a representative stratified random sample of adults aged and older following a structured questionnaire. respondents were from the chinese, malay, and indian communities, the three largest ethnic groups in singapore. the sample characteristics and the details on measurement of the variables included in this analysis are described in tables and a. . the data discussed in this paper are part of a larger study on preventive health behavior regarding cancer, heart disease, and hiv/aids supported by a research grant from the national university of singapore. further methodological details are provided elsewhere [ ] . the new findings discussed in this paper were obtained through logistic regression analysis. the data on behavior and attitudes on sars are from a study based on telephone interviews with a representative stratified random sample of adults aged and older, following a structured questionnaire. the three main ethnic groups in singapore (chinese, malays, and indians) were proportionally represented. the interviews were conducted within the span of days, from to may, while the country was facing the sars epidemic. telephone interviews were the only data collection option because it was imperative at the time to follow the public health advice to restrict personal contact to home and the workplace, whenever possible. the main characteristics of the sample and the attitudinal measurements applied are presented in table . further methodological details of this study are described elsewhere [ ] . the new findings presented in this paper were obtained through logistic regression analysis. while the data refer to three ethnic communities in singapore the findings illustrate the impact of social attitudes upon the governance of epidemics in a high density global city. acknowledging the multiplicity of factors that may play a role in shaping the success or failure of illness prevention and containment of epidemics, this study deals only with a small number of variables. the analysis of data in both studies comprised two stages: an initial scrutiny of the main assumed correlations and attitudinal scales using partial correlation and factor analysis; logistic regression to explore the likelihood of occurrence of stereotypical images of people living with hiv/aids, the dependent variable in the hiv/aids study; and likelihood of public support of the sars crisis management, the dependent variable in the sars study. logistic regression is a very useful tool to explore the probability of occurrence of the dependent variable over the probability of it not occurring and the outcome is provided as odds ratios. the odds ratio is the odds of one variable occurring to the odds of another [ , ] . the logistic regression analysis of the public image of hiv/aids comprised three sets of variables: sociodemographic variables (gender, age, ethnicity, marital status and religion); social class factors (occupation, personal monthly income, and educational level); attitudinal factors proposed by the hbm and the sct (tendency to worry about falling ill; future orientation; sense of personal control of one's life; life satisfaction; perceived severity of hiv/aids; perceived susceptibility to hiv/aids; belief in effective prevention of hiv/aids) and perception of hiv/aids. a description of these variables is provided in table a. . five sets of variables were included in the logistic regression analysis of the public support of the sars crisis management: socio-demographic variables (gen- der; age; ethnicity; place of birth; marital status); social class (educational level and personal monthly income); health behavior variables (smoking, exercising regularly, and preventive measures against sars taken over the days preceding the interview); attitudes suggested by the hbm including perceived susceptibility and perceived severity; and attitudes on sars crisis management. to meet requirements of the logistic regression analysis the response categories of the question on perceived susceptibility were dichotomized contrasting the respondents who expressed an estimation of their likelihood of infection on the one hand, with respon-dents who have no awareness of their susceptibility to sars, on the other hand. the response categories for perceived severity were dichotomized into "high" severity (survival not very likely or not likely at all) versus "low" severity (all other responses including "don't know"). the complete list and explanation of all the variables are presented in table . the discussion follows the two related assumptions presented earlier. the first assumption to be tested is that a higher perception of disease severity and personal susceptibility to sars as compared to hiv/aids, contributed to the higher effectiveness of sars prevention efforts. show that the sars outbreak was contained within months of its onset while the hiv/aids epidemic continues undefeated after nearly three decades. would people's sense of susceptibility to these diseases and their severity contribute to that difference? the survey data from singapore on the two epidemics provide a tentative yet useful indication of the differential perception of severity and susceptibility. only . % of the respondents expressed high susceptibility to hiv/aids (table ) compared to . % of respondents in the case of sars ( table ). the corresponding figures on the expression of high perceived severity are . % of the respondents in the case of hiv/aids (table ) and only . % of the respondents in the case of sars ( table ) . the findings from the analysis of the belief in effective hiv/aids prevention in the total sample (appendix a, table a. ) indicate that people who believe that hiv/aids is very serious and fatal (high perceived severity) are significantly more inclined than those with low perceived severity to believe there are effective ways of preventing the disease. this belief in effective prevention of hiv/aids is also found among people with high future orientation, those who are inclined to worry about falling ill; it is expressed by men more than women. the nagelkerke r coefficient of . suggests that . % of the overall variation in the belief in effective prevention of hiv/aids is predicted by the variables in the model. the model predicted correctly the belief in effective hiv/aids prevention . % of the time. in the case of sars, preventive measures were being implemented as the outbreak progressed. the interviews took place in the midst of the crisis as the country and the region were coping with this completely new threat. no clear indication of effective prevention was in sight but through a steep learning process several effective preventive measures were being identified and the information transmitted from the experts to the public daily through various mass media including radio, newspapers, the internet, regular television, and a dedicated television channel set up specifically for that purpose. this special situation may explain the respondents' very low perceived severity of sars and their very high sense of susceptibility to it as ways of transmission encroached into people's daily life, for example: droplets from the sneezing or coughing of an infected person, and the touching of infected commonly used objects such as eating utensils, buttons in elevators, and door handles [ , ] . the second assumption to be explored here is that in contrast to sars, the overall negative social 'image' of hiv/aids as a disease associated with particular types of individuals tends to weaken people's perception of susceptibility and, correspondingly, tends to discourage public support for robust preventive efforts at the community level. as suggested earlier, this assumption may be examined using reiss and oliveri's [ ] concept "community's punctuation of an event" and the three conditions -accountability, duty, and competence -these authors identified as requirements for the community's positive response. in the case of hiv/aids and sars the focus is the community's endorsement of disease prevention and containment plans and their active collaboration in prevention efforts. when does the problem begin and when does it end and who is involved? the answers to these questions mark the community's punctuation of crises and show that the punctuation of the sars outbreak was rather different from that of hiv/aids. the punctuation of the sars outbreak as a crisis was very clear. sars was imported into singapore at the end of february, , when an infected vacationer returned home from hong kong where she caught the infection while staying at the same hong kong hotel where a doctor from guangzhou, china who had treated sars patients there, was residing [ ] . the unknown cause and nature of the disease deterred the assignment of blame or accountability. sars patients were not held accountable for their illness. nor were they assumed to have the expertise to solve the problem on their own although it soon became a duty for people with the publicized symptoms to seek immediate expert medical help [ , ] . the findings from the sars study in table show that . % of the respondents made a positive appraisal of the health authorities' management and control of the sars crisis; . % had the chance to express their opinions to the authorities; . % were prepared to be quarantined for days after close contact with an infected person and . % would agree to be quarantined even if there was non-close contact. further indications of the public's willingness to collaborate in preventive efforts against sars were the very positive attitudes of the majority of respondents: although about one of every two agreed that preventive measures taken against sars "have affected my personal choice and freedom in life", most respondents ( . %) agreed that "people should be willing to make some personal sacrifices" to contain the epidemic and % felt that "people have mostly been socially responsible". the findings from the logistic regression analysis of the sars study data confirm that this sense of social responsibility was a fundamental manifestation of the community's positive and compassionate 'image' of sars patients and it was significantly associated with their endorsement of the health authorities' management of the crisis in the total sample as well as among people with lower education, and ethnic minorities such as the singaporean malays (table ) . among the respondents, the endorsement of the health authorities' crisis management was particularly supported by people who perceived the community as being socially responsible; those who believed that the crisis justified making some personal sacrifices (especially with regard to movement outside their homes, restricting or changing their travel patterns, and abiding by quarantine regulations); those felt that they had the chance to be part of the effort and express their personal opinions; people who had formed an opinion on their personal susceptibility to the infection (in contrast to those who had no or very little information on sars). the nagelkerke r coefficient of . suggests the factors in the model explain . % of the variation in endorsement of the crisis management in the total population. the analysis of the same factors was repeated among three specific subgroups that have shown less positive appraisal of crisis management: the senior cohort (respondents aged and older), the lower educated (people with only primary or lower education), and malays. as illustrated in table , even among the lower educated and the malay, the sense of social responsibility was significantly associated with their endorsement of preventive efforts (dependent variable). however, perceived susceptibility to sars did not influence significantly the appraisal of crisis management by the seniors and the less educated but it did among malays in the expected direction: persons who have no idea of their susceptibility (no awareness of it) were most likely to give a negative appraisal of crisis management. no significant impact of perceived severity upon people's appraisal of crisis management was detected in the total sample or any of the three subgroups. overall, the nagelkerke r coefficients indicate that, compared to the total sample, variables in the model helped explain a larger proportion of the variance in the dependent variable among subgroups such as seniors ( . %), the less educated ( . %) and the malay community ( . %) about % of the time. these figures point to the importance of variations the perception of and responses to crises among different segments of the population given their differences in life experiences, in knowledge and level of information on the problem, and in cultural values and beliefs, among other factors. as indicated earlier, from the contextual perspective proposed by reiss and oliveri [ ] the public image of a crisis or stressor (e.g., an infectious disease epidemic) refers to the public's perception of its scope and its social acceptability. in terms of the scope of the problem -when it begins and when it ends -the quiet and prolonged way in which the hiv virus enters and destroys the immune system represents a major challenge for the mobilization of public interest and support of testing. visible signs of the disease tend to appear only in the late stages. the opposite occurred with respect to sars. two separate studies of the awareness of health threats in the united kingdom confirm these findings on impact of the public image of the problem and punctuation or scope of the event: british lay respondents and journalists were inclined to see aids as a "far-flung" risk of no immediate relevance to their lives [ , ] . regarding social acceptability, if the stressor is seen by the community as the consequence of socially unacceptable behavior, one would expect collective apathy or reluctance or opposition to the investment of public funds and efforts to contain and solve the problem. the findings in tables , and a. suggest that this appears to be the case with hiv/aids. the public image of hiv/aids tends to be shaped by normative expectations or stereotypes in the community. the large majority of the respondents ( . %) associated a particular lifestyle with the contracting of the disease thus forming negative images of people living with hiv/aids. as shown in table , over half of the respondents ( %) saw them as "risk-takers": people who engage in activities that put them at risk of infection such as having multiple sexual partners or procuring the services of commercial sex workers. another % of the respondents associated them with people who engage in 'deviant' activities such as commercial sex workers and injecting drug users who exchange infected needles. a small group ( . %) considered them as "victims" of "fate" or "bad luck" or accidental infection. only . % of the respondents did not have an opinion or image of people living with hiv/aids. practically all in this group had no information on hiv/aids. table presents some of the factors that contribute to the formation of a particular 'image' of people living with hiv/aids. the odds of seeing them as 'victims' (column b) were significantly higher among older people, those who do not think hiv/aids is a serious and deadly disease; and those who do not believe there is an effective way of preventing the illness. interestingly, the same features are exhibited by the small group who did not put a label on people living with hiv/aids (column a): they tend to be older, unaware of the severity of the disease, and unaware of any effective preventive measures. the odds of perceiving hiv/aids sufferers as 'risk-takers' that is, associating a 'risk-taking' life style with hiv/aids infection (column c), decreased by % among men; increased significantly among people who worry about falling ill and those who believe that there are effective ways of preventing the disease. the most negative 'image' or lifestyle associated with hiv/aids infection is that labeled 'deviants' (column d). the odds of having this image of hiv/aids sufferers increase significantly among women in contrast to men; among younger people in contrast to people who are or older; among those believe the disease is very severe, and among people who do not worry much about falling ill. the nagelkerke r coefficients indicate that the variables in the model explain . % of the overall variation in emphasis on the 'victim' image; . % of the emphasis on the 'risk-takers' image; . % of the emphasis on the 'deviant' image, and . % of the variance on the absence of a stereotypical image of hiv/aids sufferers. this variable is explained correctly by the variables in the model . % of the time (table ). these findings fit the international pattern: the presence of stereotypical images of people who get infected with hiv/aids is not restricted to a particular country [ , ] . state regulations on infectious diseases such as notification and surveillance systems have been in place for more than a century in many countries [ , ] and today they are followed by all state members of the united nations including singapore [ ] . but the official approach to the control and prevention of hiv/aids differs widely from that of sars and the difference has to do with the public image of the disease discussed in the preceding sections. the experience of sars in singapore provides an interesting illustration of the positive synergy between national governance and global health governance. the main features of the state's crisis management approach illustrate the situation well. those features were: (a) transparency; (b) public education; (c) multi-pronged approach; (d) legislation. in contrast to the situation in china and some other affected countries at the onset of the epidemic [ , ] , the sars situation in singapore was characterized throughout by transparency on the part of the health authorities in their reporting and distribution of a continuous flow of information to the public on new infections and deaths, locations, and contact tracing efforts and approaches. it was believed that an informed public can collaborate better and participate more effectively in containing the spread of the disease than a public kept ignorant of the seriousness of the situation. news reports on the progress of the epidemic were transmit- notes: total sample size, . * statistically significant at p = . - . . ** statistically significant at p = . - . . *** statistically significant at p = . - . . **** statistically significant at p = . or lower. ted to the public through all printed media, radio and television in all the four official languages (mandarin, malay, tamil and english). exceptional measures were taken to reach as many people as possible throughout singapore: broadcasting was resumed temporarily in some of the chinese dialects that had not been used in tv for nearly two decades; a new dedicated tv channel was set up, sarstv. singapore was the only country affected by sars to set up this public service [ ] . informing the public on the development of the epidemic went hand-in-hand with public health education whereby public information on the current state of knowledge on the disease was constantly updated at various levels of sophistication, from medical and epidemiological data in specialized publications [ , ] to newspaper articles explaining how the coronavirus attacks a healthy cell, to cartoons illustrating the proper use of masks, of serving utensils, and to take one's temperature correctly with a thermometer, and how to wash hands thoroughly, among other things. it was evident to the authorities and the population that the sars epidemic affected the daily life activities of every citizen and demanded drastic changes in lifestyle. this realization led to the implementation of a multi-pronged approach to deal with the crisis. all relevant ministries, statutory boards and other organs of the state were mobilized and non-governmental organizations and the private sector joined the effort. this approach was in fact a typical response in singapore as "ministries and government agencies had honed emergency preparedness to a fine art" [ ] . part of that preparedness was the use of legislation including quarantine laws and other preventive measures. for example, section of the infectious diseases act was amended with effect from april requiring "medical or dental practitioners to obtain information from their patients and transmit such information to the director [of medical services] to investigate the outbreak or prevent the spread of an infectious disease such as sars"; a new "patient declaration form" was used for this purpose during the outbreak [ ] . while transparency, public education, the implementation of a multi-pronged approach, and the use of legislation were characteristics of the national government's response internally, there was also transparency and close collaboration of singapore with the who and other international organizations. in his global analysis of the epidemic, fidler highlights this feature: "singapore was initially scheduled to be removed from the [who's] list of sars-affected areas on may; but, on that date, singapore reported a new case of sars to who, an indication of singapore's commitment to open reporting and cooperation with who" [ ] . a sovereign state's abiding to international guidelines, even at the expense of its own economic interests, illustrates the importance of "global health governance" to deal with infectious disease epidemics and similar health threats in the st century. the control and prevention of hiv/aids has followed a different approach. fidler [ ] correctly highlights "the conceptual and policy shifts" from standard procedures applied to infectious diseases by the who and public health experts. the international health regulations (ihr) "are the only set of international legal rules binding on who member states concerning the control of infectious diseases" [ ] . yet, in fidler's view, one distinguishing feature of the official international approach to deal with the hiv/aids epidemic was that public health experts and the who did not follow the ihr's classical "westphalian" model -that emphasizes state sovereignty and non-intervention -to deal with infectious diseases "but rather turned to international human rights law to provide governance norms for the fight against this new plague" [ ] . this conceptual shift was unique. the approach to hiv/aids was "the first time in history [that] preventing discrimination towards those affected by an epidemic became an integral part of a global strategy to prevent and control an epidemic of infectious disease" [ , ] . danziger [ ] suggests that this conceptual shift was promoted and supported by the "neoliberal democratic ideology" prevalent in western countries by the end of the th century and enthusiastic enough to lead some countries to abandon the compulsory surveillance methods and "placing protection of individual rights on a par with (or even above) the protection of the public health". apart from the ideological angle, the who's concern with human rights and particularly with the matter of discrimination of people living with hiv/aids has its roots in actual manifestations of social stigma associated with the presumed lifestyle of the first persons affected by the disease. in june and july, , five young homosexual men were diagnosed with pneumocystis carinii pneumonia and young homosexual men with kaposi's sarcoma "a rare form of cancer which had until then been associated with elderly americans"; added to the spectrum of aids features in the early s was the confirmation that one of the main forms of transmission is sexual intercourse [ ] . the spread of the epidemic has been so extensive geographically as well as socially, that people living with hiv/aids today come from all walks of life. but as the figures in appendix a, table a . indicate, the highest prevalence of hiv are still found among some distinct lifestyle groups including commercial sex workers and injecting drug users. the survey data discussed in the preceding sections suggest that the public image of hiv/aids reflects the prevalence figures among some specific groups; a large majority of respondents saw hiv/aids sufferers as following their lifestyle by personal choice. thus, their image remains negative despite public education efforts by the who, non-state organizations and non-governmental organizations. an additional aspect is the slow pace of development of the disease: people may be infected for many years without developing any symptoms and may thus continue unwittingly to infect others [ ] . the global governance approach to protect people with hiv/aids from discrimination involves avoidance of disclosure of one's health condition and of routinely or compulsory name-linked testing and other features of standard infectious disease surveillance. danziger [ ] sees this position as "possessive individualism" that demands testing to be done only if the person has consented freely and has received full information on the consequences through the process of informed consent. moreover, danziger points out that with the current stage of knowledge on hiv/aids, "there is little or no gain for a hiv-infected person to be tested and identified as hiv-positive" [ ] . this reasoning creates a dilemma because experts agree that standard infectious disease surveillance is indispensable for the effective prevention and control of infectious diseases [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , ] that is, the beneficiaries of effective control and prevention are the rest of the community. according to danziger [ ] the dilemma has been sorted out internationally by an apparent consensus of stakeholders to consider hiv/aids as "a crisis of human rights" and not as a "public health crisis". however, with the epidemic proceeding unrelentingly, there are signs of concern. a recent development in singapore is illustrative of the range of opinions on this matter: the ministry of health announced on july that "spouses of patients with hiv will be informed of their partner's illness, regardless of whether the infected person agrees" [ ] . the newspaper report cited the case of four women who discovered they had hiv when they did their blood test during their pregnancies. the husband of one of them had been diagnosed with hiv since . previously, patients were counseled and informed consent was required. now the doctor needs only to keep the patient "appropriately informed". the spouses will be informed "in a sensitive manner" by trained personnel of a new hiv prevention unit. the infectious diseases act will be invoked as is the case with all other infectious diseases [ ] . the singapore shift towards a version closer to standard surveillance of hiv/aids is indicative of its concern for the rights of the infected person as well as the rights of the patient's partner, and of all other persons involved. the global governance of hiv/aids prevention needs to address the public image of the disease and the impact that such an image has upon efforts to mobilize the community in prevention efforts. for as long as hiv/aids is seen as a problem of particular lifestyles (that is, of specific types of people), prevention efforts such as condom use and testing are bound to have limited impact. the analysis was based on data from two separate studies of singapore residents' attitudes and behavior. given the differences in questions asked during the respective interviews, the -year difference between studies and the significant differences in the etiology, nature, and development of the two infectious diseases, the findings must be treated with caution. that said, the availability of data from the two studies offered a good opportunity for this exploratory comparison of attitudes towards and the public image of hiv/aids and sars in search of a better understanding of the social obstacles to effective prevention against hiv/aids. this exploratory comparison was guided by two main assumptions. the first assumption was that a higher perception of disease severity and personal susceptibility to sars as compared to the level of perceived susceptibility to and severity of hiv/aids, contributed to the difference in effectiveness of prevention efforts. the data from the singapore study support that assumption partially and revealed a new aspect of the problem. perceived susceptibility was high for sars but relatively low for hiv/aids. the opposite was found for perceived severity. the findings suggest that among the complex set of factors that motivate people to take preventive measures, perceived susceptibility to the disease (your subjective assessment of the likelihood of becoming infected) is more relevant than perceived severity of the disease. the data show a strong tendency for people to consider hiv/aids as peculiar to certain types of people different from themselves. thus their belief that their chances of being infected with the hiv virus are remote is not surprising. these findings on low perceived susceptibility and high perceived severity are also meaningful in the context of the second assumption tested. the second assumption explored in this study was two-fold: (a) that in contrast to sars, the overall negative social 'image' of hiv/aids as a disease associated with particular types of individuals tends to weaken people's perception of susceptibility; (b) that correspondingly, low perceived susceptibility tends to discourage public support for robust preventive efforts at the community level. the findings verify and clarify these assumptions. only out of every respondents expressed high perceived susceptibility to hiv/aids compared to out of every in the case of sars. but, as mentioned above, it was also found that the perceived severity of hiv/aids was significantly higher than the perceived severity of sars. more importantly, the labeling of people living with hiv/aids as 'risktakers' or 'deviants', was expressed by nine out of every of the respondents who believed hiv/aids was a incurable disease (high perceived severity) and believed that the disease is mostly linked to one's lifestyle choice. the challenge for health authorities is to enhance the population's perceived susceptibility to hiv/aids. the first step in this direction is the widespread distribution of accurate, clear and consis-tent information on the 'silent' nature of the disease in its early stages. the second part of this assumption was on the mobilization of the community's endorsement and active participation in the control and prevention of the epidemics. i have discussed the elements involved including the community's punctuation of the crisis and the aspects of accountability, duty, and competence. in the case of epidemics, competence is typically found at the community level and this brings us to the question of governance: how does a government deal with the health crisis represented by an infectious disease epidemic? much has been learned over the centuries around the world but each new epidemic brings new dangers. the sars outbreak tested the state's level of emergency preparedness and commitment to transparency particularly in asian countries, but it also highlighted the need for global governance of health threats that cut across national boundaries. in contrast, the hiv/aids epidemic still represents a challenge in terms of public health, political ideology, human rights, and social discrimination. the lack of success in the control and prevention of the epidemic highlights the fact that after nearly three decades the global governance of hiv/aids is still a work-in-progress. the urgency of the problem is well recognized by most world leaders and specialists with some experts warning that "new threats to [world] stability and secu-rity may emerge as the pandemic escalates" because, among other reasons, large numbers among police and armed forces in many countries and un peacekeeping forces are getting infected [ ] . the current global governance of hiv/aids requires critical scrutiny, as well as active exploration of solutions -among all types of stakeholders with differing ideological and social perspectives -to the slackness of preventive efforts against the two main behaviors that are sustaining the epidemic: "high-risk sexual activity and drug use" [ ] . one aspect of that critical scrutiny is the systematic and comparative analysis of hiv/aids global governance with the global governance of other infectious disease epidemics that have been successful. in sum, the findings on the sars and hiv/aids experiences suggest that health authorities need to navigate effectively the local and global obstacles to prevention by: (a) enhancing the public's understanding of the etiology of hiv/aids, its modes of transmission and effective preventive measures; and (b) correcting the lay public's inaccurate perception of personal susceptibility and 'the punctuation of the event'. tables a. -a. . very serious because it can cause death and has no cure serious but has some partial cure only mildly serious because does not cause death not serious at all scores range from (very serious) to (not serious at all). for the logistic regression analysis these response categories were dichotomized: ( ) "very serious" vs. ( ) all other responses perceived susceptibility to hiv/aids "aids doesn't happen to people like me" mean, . ; s.d., . ; sample size, this statement is part of a series involving cancer, heart disease and aids. respondents were asked to tell the interviewer if they strongly agree (sa), agree (a), disagree (d) or strongly disagree (sd) with each statement scores range from (sa) to (sd), with higher scores indicating higher perceived susceptibility for the logistic regression analysis these response categories were dichotomized: ( ) "sa/a" vs. ( ) all other responses belief in effective hiv/aids prevention "is there an effective way of protecting yourself from aids"? mean, . ; s.d., . ; sample size, the responses were scored as "yes" ( ) vs. "no" ( ) dependent variable: perception of people living with hiv/aids "what kind of people do you think are most likely to get hiv/aids?" during the personal interviews this open-ended question was preceded by identical questions for cancer and heart disease. factor analysis of the responses revealed three categories or 'images': (a) "victims" of "fate" or "bad luck" or accidental infection; (b) "risk-takers": people who engage in activities that put them at risk of infection such as having multiple sexual partners or procuring the services of commercial sex workers; (c) people who engage in 'deviant' activities such as commercial sex workers and injecting drug users who exchange infected needles. a fourth category comprises a small group of respondents who did not label people living with hiv/aids. each of the four categories is examined using separate logistic regression analyses (see table ) ( ) . **** personal control: high ( ) . life satisfaction: high ( ) . perceived severity: high ( ) . **** perceived susceptibility: high ( ) . nagelkerke r . variance predicted correct (%) . a see table a . for the description of measurement of belief in effective prevention. total sample size: . ** statistically significant at p = . - . . *** statistically significant at p = . - . . **** statistically significant at p = . or lower. dk publishing and national gallery of art illness as a metaphor and aids and its metaphors disease and the modern world. to the present day hiv stories: the archaeology of aids writing in france narrative in the time of aids: postcolonial kenyan women's literature the evil of the th century: poetry and aids on finding in a book of poems by norman dubie, a -year-old letter from the bookbinder to my cousin now dead of aids playing for life: performance in africa in the age of aids choreographer mark sieczkarek's 'living with aids' with the dance-factory in ghana nationalizing the gay body: aids and sentimental pedagogy in 'philadelphia'. american literary history narrative in times of crisis: aids stories in ghana playing for life: performance in africa in the age of rats, lice and history emerging issues in infectious disease epidemiology mapping the world of epidemiology. . the disease detectives disease detectives are turning to molecular techniques to uncover emerging microbes epidemiology: a science of patterns st century detectives-the laboratory professional in the prevention of infectious disease the making of a germ panic, then and now disease detectives celebrate years of successful 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we go from here? geneva: who health behavior. emerging research perspectives handbook of health behavior research health behavior and health education. theory, research and practice promoting health: intervention strategies from social and behavioral research how individuals, environments, and health behavior interact. social cognitive theory the health belief model and health behavior the health belief model hiv/aids prevention and public health education social networks and social support rational choice theory: advocacy and critique family stress management. a contextual approach family stress as community frame global atlas of infections -epidemiological fact sheet - update world health organization. summary of probable sars cases with onset of illness from summary table of areas that experienced local transmission of sars during the outbreak period from ordinary least squares and logistic regression analysis medical statistics a defining moment. how singapore beat sars at the epicentre. hong kong and the sars outbreak infectious diseases law & sars. singapore: times editions the politics of emerging and resurging infectious diseases hiv in singapore-past, present and future the sars epidemic under china's media policy online news media as interactive community bulletin boards. coverage of sars in the greater china regions the sars channel in singapore severe acute respiratory syndrome (sars) human rights and aids: the future of the pandemic new rule overrides patient confidentiality in order to protect the vulnerable aids as a social problem. the creation of social pariahs in the management of an epidemic. in: ritzer g, editor. handbook of social problems. a comparative international perspective representations of far-flung illnesses: the case of ebola in britain representations of sars in the british newspapers key: cord- - a u p t authors: söderman, martina; rhedin, samuel; tolfvenstam, thomas; rotzén-Östlund, maria; albert, jan; broliden, kristina; lindblom, anna title: frequent respiratory viral infections in children with febrile neutropenia - a prospective follow-up study date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: a u p t objective: febrile neutropenia is common in children undergoing chemotherapy for the treatment of malignancies. in the majority of cases, the cause of the fever is unknown. although respiratory viruses are commonly associated with this condition, the etiologic significance of this finding remains unclear and is therefore the subject of this study. study design: nasopharyngeal aspirates were collected during episodes of febrile neutropenia in children age – years, being treated at a children’s oncology unit between january and june . real-time polymerase chain reaction was used to determine the presence of respiratory viruses. follow-up samples were collected from children who tested positive for one or more respiratory viruses. rhinoviruses were genotyped by vp /vp sequencing. fisher’s exact test and mann-whitney u test were used for group comparisons. results: at least one respiratory virus was detected in samples from of episodes of febrile neutropenia ( %), with rhinoviruses the most frequently detected. follow-up samples were collected after a median of days (range, – days) in of the virus-positive episodes. the respiratory viral infection had resolved in episodes ( %). the same virus was detected at follow-up in one coronavirus and six rhinovirus episodes. genotyping revealed a different rhinovirus species in two of the six rhinovirus infections. conclusion: the frequency of respiratory viral infections in this group of patients suggests an etiologic role in febrile neutropenia. however, these findings must be confirmed in larger patient cohorts. febrile neutropenia is a common complication in children undergoing chemotherapy for the treatment of malignancies. because septicemia (which is potentially lethal) is difficult to rule out at the onset of fever, empiric treatment with broad-spectrum antibiotics is promptly initiated based on wide indications [ ] . however, in most cases, no underlying cause of the fever can be identified [ ] . febrile neutropenia is associated with long hospitalization [ ] [ ] [ ] which has negative social effects for the child and its family [ ] . in addition, hospitalization and the use of broad-spectrum antibiotics increase the patient's risk of subsequent infection with antibiotic-resistant bacteria [ , ] and fungal infections [ , ] . a better understanding of the etiology of febrile neutropenia is thus needed in order to decrease unnecessary hospitalization and excessive antibiotic use. as infections with respiratory viruses are a common morbidity in children [ ] respiratory viruses likely play a significant role in childhood febrile neutropenia. advances in molecular methods have increased the sensitivity of viral diagnostics tests, with recent studies reporting the detection of respiratory viruses in the nasopharynx in - % of childhood febrile neutropenia episodes using real-time polymerase chain reaction (pcr) [ ] [ ] [ ] ] . however, the clinical significance of positive pcr findings is unclear, as respiratory viruses have also been detected in asymptomatic immunocompetent children [ ] [ ] [ ] [ ] . in addition, some respiratory viruses can be detected weeks after the first infection, which is suggestive of prolonged viral shedding [ , ] . here, we describe the results of a longitudinal study involving repeated sampling and the assessment of a broad panel of respiratory viruses by pcr to clarify whether respiratory viruses play a causal role in childhood febrile neutropenia. children aged - years, who were treated for a malignancy at the childhood cancer unit at astrid lindgren children's hospital in stockholm, sweden, between january and june , were eligible for enrollment in this study. all the patients who met the criteria for febrile neutropenia were asked to participate. patients could be enrolled multiple times if recurrent episodes of febrile neutropenia occurred during the study period. to be enrolled for a new episode, the patient needed to have been afebrile for more than hours and have completed the antibiotic treatment for the previous episode of febrile neutropenia. febrile neutropenia was defined as a body temperature of . °c on one occasion or . °c on two occasions at least minutes apart, combined with an absolute neutrophil count of either . × /l on one occasion or . × /l with a decline to less than . × /l over a subsequent -hour period. oral and written information regarding the study were provided to each patient prior to enrollment, and signed consents were obtained from their caretakers. the study was approved by the regional ethical review board in stockholm. university laboratory (iso: : ) for microbiological analysis. viral nucleic acids were extracted from the npa with a magattract virus mini m kit (qiagen, sollentuna, sweden) and analyzed with in-house real-time pcrs for the following viruses: adenovirus (hadv); bocavirus (hbov); coronaviruses nl /oc / e/hku (hcov); enterovirus (ev); influenza virus a, including a(h n )pdm and b (flu); metapneumovirus (hmpv); parainfluenza viruses - (piv); respiratory syncytial virus (rsv) and rhinovirus (rv) [ ] . a semiquantitative assay was used and the actual cycle thresholds-values (ct-values) were provided, however, the npa has not been validated for quantitative data. in all study subjects who initially tested positive for one or more respiratory viruses, a follow-up sample was collected at the time of their next visit to the hospital. this follow-up sample was collected regardless of the patient's absolute neutrophil count or symptoms and was analyzed in the same manner as the first sample. however, if the patient had a new episode of febrile neutropenia at the time of the follow-up, this sample was considered both a follow-up sample and a new episode sample, which required an additional follow-up sample if the new episode tested positive for a respiratory virus. rv/ev genotyping was performed by vp /vp sequencing for samples that were pcr-positive for rv and/or ev using a recently published method [ ] based on a method and primers originally published by wisdom et al. [ ] . briefly, viral rna was extracted as described above for most samples, but the rneasy lipid tissue mini kit (qiagen, sollentuna, sweden) was used for samples that were negative in the vp /vp pcr after magattract extraction. the rna was used for the nested reverse transcriptase (rt)-pcr with one-step superscript-platinum taq (life technologies, stockholm, sweden) for the first (outer) pcr, and platinum taq for the second (nested) pcr. sequencing was done on an abi instrument and the rv/ev species and type was determined by maximum likelihood phylogenetic trees constructed using phyml [ ] and the sequences have been deposited in genbank under accession numbers kx -kx for rv-a and kx -kx for rv-c. two of the rv-c samples were not submitted to the genbank because the sequences were of suboptimal quality due to a probable infection with more than one rv genotype. blood cultures were collected from all patients for the detection of bacterial infections and analyzed at the karolinska university laboratory, as per routine clinical procedures. clinical data, including the results of biochemical and microbiological analyses, patient's characteristics, treatment during the febrile episode, fever characteristics, and the duration of hospitalization, were collected from the medical records. data were analyzed using graphpad prism . software (graphpad prism, san diego, ca). fisher's exact test and the mann-whitney u test were used for group comparisons of categorical and continuous data, respectively. a p-value less than . was considered statistically significant. a total of patients representing episodes of febrile neutropenia were enrolled in the study. five episodes were excluded due to incomplete sampling. therefore, the analyses included patients with episodes of febrile neutropenia (ranges, - episodes per patient and - days between episodes). the characteristics of the study participants are listed in table . the median age across all episodes was years (range, . - . years), and % of the episodes occurred in females. the patient was undergoing treatment for a hematologic malignancy in % of the episodes and for a solid tumor in % of the episodes. respiratory symptoms were noted in % of the episodes of febrile neutropenia ( table ) . at least one respiratory virus was identified in ( %) of the episodes. a single respiratory virus was detected in ( %) of the episodes (table ) . multiple viruses (range, - viruses) were detected in two episodes ( %), whereas co-presence with a respiratory virus and septicemia was detected in three episodes ( %) ( table ) . of the episodes, rv was the most frequently detected respiratory virus (n = , %), followed by hcov (n = , %), flu (n = , %), rsv (n = , %), piv (n = , %), hmpv (n = , %), hbov (n = , %), and hadv tumor type . . hematologic d malignancy days with antibiotics (n = , %) ( table ). in of the virus-positive episodes, the patient was < years of age. the most common respiratory virus identified in the group < years of age was rv (n = ), followed by piv (n = ), hcov (n = ), hadv (n = ), hbov (n = ) and rsv (n = ). in addition, one episode with co-presence with a respiratory virus and septicemia was identified in the group < years of age. of the episodes involving only respiratory viral infection (i.e., infection with either a single respiratory virus or multiple respiratory viruses) respiratory symptoms were detected in ( %) ( table ) . no symptoms were apparent in four episodes involving rv and one episode involving hcov (table ) . of the episodes involving respiratory symptoms, reported the appearance of respiratory symptoms at time of fever onset ( table ). in the remaining five episodes, the respiratory symptoms appeared over days before the onset of fever. these five episodes represented one flu b and four rv ( table ). bacterial blood cultures were positive in ( %) of the episodes of febrile neutropenia. five cultures were excluded from further analysis and not defined as septicemia because they were determined to be either contaminants or of no clinical relevance by the treating clinician and/ or the laboratory: micrococcus species (n = ), coagulase-negative staphylococcus (n = ), staphylococcus epidermidis (n = ), and unspecified gram-positive bacteria (n = ). eight episodes were therefore considered true septicemia. of these, five episodes involved only septicemia ( %) ( table ) (i.e. they tested negative for respiratory viruses by pcr). three episodes involved co-presence with a respiratory virus and septicemia; gram-positive bacteria (staphylococcus epidermidis (n = ) and alpha streptococcus (n = )) were detected in all three episodes and one episode was also positive for gram-negative bacteria (escherichia coli). in all three episodes involving co-presence of respiratory virus and septicemia the patients were being treated for a hematologic malignancy, and all had respiratory symptoms ( table ). the viruses found in this group were rv (n = ) and hbov (n = ) ( table ). in episodes ( %), no respiratory virus or septicemia were detected and the febrile episode was therefore defined as a fever of unknown origin (table ) . when comparing episodes involving only respiratory viral infection (i.e., infection with either a single respiratory virus or multiple respiratory viruses) with episodes involving only septicemia or fever of unknown origin, no statistically significant differences were observed with respect to age, gender, days with fever, or maximum temperature (table ) . however, the presence of respiratory symptoms was significantly higher in the episodes involving only respiratory viral infection ( %) compared with both the episodes involving only septicemia ( %) and fever of unknown origin ( %) (p = . and p = . , respectively). the episodes with only respiratory viral infection were also more often treated for a hematological malignancy ( %) compared to the fever of unknown origin episodes ( %) (p = . (table ) . a new respiratory virus was detected in six follow-up samples: flu a (n = ), rv (n = ), hcov (n = ), and rsv (n = ). of these six, the episodes for all except one involving rv exhibited respiratory symptoms, and one (flu) represented a new episode of febrile neutropenia. rv genotyping was performed to determine whether the episodes exhibiting repeated pcr positivity involved a new or persistent infection. of samples from the rv-positive episodes, were successfully sequenced, resulting in the identification of rv-a and rv-c species ( table ). before sequencing, four patients, representing six episodes, remained rv-positive at follow-up (table ). one patient was rv-positive in three follow-up samples; however sequencing revealed that two of these follow-up samples contained a new rv specie (fig ) . sequencing also revealed that the three remaining patients all had the same genotype (fig ) . all of the rv-c had cleared by the time of follow-up (table ) , and the four persistent episodes involving rv were associated with rv-a infections ( table ). to the best of our knowledge, this is the first longitudinal study to assess respiratory viral persistence in children presenting with febrile neutropenia. we found that respiratory viral infections are common in children with febrile neutropenia, in the majority of episodes accompanied by respiratory tract symptoms and that the infection had resolved at follow-up in the majority of the episodes. these results support the theory holding that there is a causal relationship between respiratory viral infections and episodes of febrile neutropenia, but proving this theory will require more longitudinal studies with asymptomatic neutropenic control cohorts. in addition, prolonged viral persistence with regard to these respiratory tract infections seems to be uncommon in children undergoing chemotherapy for the treatment for malignancies. in a previous study we detected respiratory viruses in % of episodes of febrile neutropenia [ ] . subsequent studies have confirmed this finding, with detection rates ranging between - % [ , ] . the detection rate of % in our current study is consistent with earlier studies. it is crucial to correctly diagnose febrile neutropenia in immunosuppressed children, as infections with viruses [ , ] , bacteria [ , ] or fungi can be fatal [ ] in this group of patients. the clinical significance of a single time-point pcr finding of certain pathogens has been debated. studies on immunocompetent children have reported that viruses such as hbov, hcov, and rv are detected in asymptomatic children at rates ranging between - % [ ] [ ] [ ] [ ] , which could be suggestive of prolonged viral shedding from an earlier infection or the incubation period prior to a symptomatic- [ ] , or asymptomatic infection [ , , ] . to clarify this issue, more longitudinal studies with appropriate control groups are needed. rhedin et al. conducted a case-control study to investigate the etiologic role of respiratory viruses in a group of children with respiratory tract symptoms seeking medical care at a pediatric emergency center [ ] . the control group consisted of children on routine visits to child welfare centers for vaccination within the childhood immunization program. the detection rate of respiratory viruses in the control group was high ( . %) with rv and hcov the most commonly detected viruses in asymptomatic children and hmpv, piv and rsv detected only rarely. the authors concluded that hmpv, piv and rsv play an etiologic role and suggested that findings of rv and hcov must be carefully interpreted [ ] .we chose a longitudinal design for the present study, and found that % of the infections had resolved by the median follow-up time of days. in addition, two of the rv infections identified at follow-up represented new species of rv from the previous infection, which increases the proportion of resolved infections to %. one major limitation of the current study was the lack of a control cohort of neutropenic patients without fever to address the question as to whether respiratory viruses are frequently found in asymptomatic immunosuppressed children. however, the use of a longitudinal design allowed us to collect follow-up samples from patients both with and without respiratory symptoms. only five patients at the first sampling and two patients at follow-up sampling were positive for a respiratory virus without the presence of respiratory symptoms, which suggests that the frequency of asymptomatic infections is low in this group of patients. interestingly, rv was detected in five of these seven episodes and hcov was detected in the other two, which are in line with other studies [ ] . the presence of respiratory symptoms was significantly higher in the episodes involving only respiratory viral infection, compared with both episodes involving only septicemia and fever of unknown origin, with symptoms appearing with the onset of fever in the majority of the episodes. peck et al. investigated the incidence of respiratory virus in patients receiving a hematopoietic stem cell transplant using longitudinally collected respiratory tract samples regardless of symptoms [ ] . in line with our results, a majority of the respiratory viruses detected could be correlated with respiratory tract symptoms, with the exception of piv, which was detected in asymptomatic patients. infection with rv or hcov was not investigated in that study. in addition, high viral loads were correlated to more symptoms. our real-time-pcr technique was not validated for use in reporting clinical viral loads; thus the presented ct-values must be interpreted with extreme caution. nevertheless, the ct-values < observed in of the episodes of febrile neutropenia are suggestive of high viral loads ( table ) . considered together, our data suggests that respiratory viruses play an etiologic role in febrile neutropenia, but our results should be interpreted carefully, especially those regarding rv, which is commonly detected in asymptomatic patients [ ] . despite the above-mentioned limitations, we believe that our findings of a respiratory virus together with other clinical parameters such as respiratory symptoms, negative blood cultures after hours, and a history of short duration of myelosuppression, may help to reduce the treatment time with broad-spectrum antibiotics and the need for prolonged hospitalizations. we were particularly interested in rv in our study because it was the most frequently detected virus, was still detected at follow-up, and has been reported in asymptomatic immunocompetent children [ ] [ ] [ ] [ ] . the clinical impact of rv has been correlated with severe respiratory diseases in children under years of age [ ] . in our study, only the a and c species of rv were identified. these species are believed to be more pathogenic and more strongly associated with hospital treatment than rv-b [ ] [ ] [ ] . rv-a and rv-c causing both upper and lower respiratory tract infections were the most frequently detected species in another study that investigated rv infection in immunosuppressed children [ ] . in the only case of rv-b reported in that study, the patient had an upper respiratory tract infection. in our study cohort, all episodes with a detected rv infection were hospitalized as a result of their febrile neutropenia, and therefore the severity of the disease in relation to the rv specie was difficult to evaluate. respiratory viruses and their shedding times have not been thoroughly investigated in immunosuppressed children. earlier studies on rsv have shown prolonged viral shedding in immunosuppressed children compared with healthy children [ ] . martin et al. conducted a longitudinal study of healthy immunocompetent children attending a daycare center [ ] and found prolonged shedding (i.e., persistent virus > days) of all viruses examined except flu a and b. jartti et al. reported shedding times of - and - weeks for ev and rv, respectively, in immunocompetent children [ ] . however, rv was not sequenced in that study; therefore, it is unclear whether the sample at weeks represented the same or a new genotype of rv. another study reported that prolonged persistence (> days) of the same rv strain is uncommon (< %) [ ] . shedding times were of special interest in our cohort of immunosuppressed children because viral shedding necessitates isolation from other immunosuppressed children. the design of our present study did not allow us to determine the exact shedding time, which is a major limitation. however, the clearance of all viruses except rv-a and hcov at a median follow-up time of days, together with respiratory symptoms appearing at the time of fever onset in a majority of the patients, suggests that the shedding time for viruses such as flu, hmpv, rsv and piv is limited. the same rv genotypes were detected from follow-up samples in four episodes, with a follow-up time of - days. furthermore, five patients positive for rv reported symptom appearance days or longer before fever onset. that could indicate longer shedding times for rv, which is in line with results from studies on immunocompetent children [ ] . however, this needs to be addressed in additional studies with repeated followup sampling, preferable on a weekly basis. hbov has been associated with acute wheezing in immunocompetent children [ ] . in immunosuppressed children, hbov has been detected both together with other viruses and also detected repeatedly, suggesting prolonged shedding or reactivation [ , ] . in this study, hbov was detected twice, with hadv, rsv and rv in one episode and together with septicemia in another episode; both episodes involved respiratory symptoms. at follow-up sampling, hbov was no longer detectable. in our previous study [ ] , we detected hbov in three episodes: together with one other virus (rv) with septicemia and as the only agent, making it difficult to ascertain 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viral infections in hematopoietic cell transplant recipients and patients with hematologic malignancies influenza a/h n in pediatric oncology patients pathogenesis of bloodstream infection in children with blood cancer a prospective study of septicaemia on a paediatric oncology unit: a three-year experience at the royal liverpool children's hospital, alder hey, uk respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection human rhinovirus species associated with hospitalizations for acute respiratory illness in young us children human rhinovirus c infections mirror those of human rhinovirus a in children with community-acquired pneumonia high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease human rhinovirus c infections in pediatric hematology and oncology patients respiratory syncytial viral infection in children with compromised immune function epidemiology of multiple respiratory viruses in childcare attendees duration of rhinovirus shedding in the upper respiratory tract in the first year of life clinical assessment and improved diagnosis of bocavirus-induced wheezing in children human bocavirus in children with acute lymphoblastic leukemia persistence of human bocavirus dna in immunocompromised children key: cord- -dsjq ks authors: fragkou, paraskevi c.; belhadi, drifa; peiffer-smadja, nathan; moschopoulos, charalampos d.; lescure, françois-xavier; janocha, hannah; karofylakis, emmanouil; yazdanpanah, yazdan; mentré, france; skevaki, chrysanthi; laouénan, cédric; tsiodras, sotirios title: review of trials currently testing treatment and prevention of covid- date: - - journal: clin microbiol infect doi: . /j.cmi. . . sha: doc_id: cord_uid: dsjq ks background: as covid- cases continue to rise globally, evidence from large randomised controlled trials is still lacking. currently, numerous trials testing potential treatment and preventative options are undertaken all over the world. objectives: we summarised all registered clinical trials examining treatment and prevention options for covid- . additionally, we evaluated the quality of the retrieved studies. data sources: clinicaltrials.gov, the chinese clinical trial registry and the european union clinical trials register were systematically searched. study eligibility criteria: registered clinical trials examining treatment and/or prevention options for covid- were included. no language, country or study design restrictions were applied. we excluded withdrawn or cancelled studies and trials not reporting therapeutic or preventative strategies for covid- . participants: and interventions: no restrictions in terms of participants’ age and medical background or type of intervention were enforced. methods: the registries were searched using the term “coronavirus” or “covid- ” from their inception until (th) march .additional manual search of the registries was also performed. eligible studies were summarised and tabulated. interventional trials were methodologically analysed, excluding expanded access studies and trials testing traditional chinese medicine. results: in total, trials evaluating therapeutic management options, studies assessing preventive strategies and studies examining both were retrieved. finally, studies were methodologically reviewed. interventional treatment studies were mostly randomised (n= , %) and open-label (n= , %) with a median number of planned inclusions of (iqr - ). major categories of interventions that are currently being investigated are discussed. conclusion: numerous clinical trials have been registered since the onset of the covid- pandemic. summarised data on these trials will assist physicians and researchers to promote patient care and guide future research efforts for covid- pandemic containment. given the steep upsurge of covid- cases worldwide within an unprecedented short period azvudine, an azidocytidine analogue that inhibits viral reverse transcriptase, has been effective against hiv, hepatitis b and c viruses [ ] . its efficacy against sars-cov- is being tested in ongoing clinical trials (table , table s ). another nucleoside analogue undergoing investigation for covid- pneumonia is emtricitabine/tenofovir alafenamide. chloroquine and hydroxychloroquine are currently licensed for the treatment of malaria and autoimmune diseases [ ] . however, they have also been studied against several viruses with promising in vitro results, never confirmed in humans [ ] [ ] [ ] . as weak bases, they are concentrated in acidic intra-cellular organelles, leading to alkalization and disruption of the low ph-dependent steps of viral replication, including viral-cell fusion and uncoating [ , ] . moreover, they impair the terminal glycosylation of ace receptor in golgi apparatus, thus inhibiting the viral penetration into the host cells [ ] . as they are accumulated in lymphocytes and macrophages, these drugs reduce secretion of proinflammatory cytokines, and particularly of tumour necrosis factor alpha (tnf-α) [ ] . experimental data demonstrated that chloroquine is highly effective in vitro against sars- cov- in an estimated effective concentration that is easily achievable with standard dosing regimens [ ] . however, the efficacy of anti-malaria drugs in clinical practice is still much debated. some preliminary reports from ongoing trials supporting their effectiveness, alone or in combination with azithromycin [ , ] , have been called into question on the basis of their methodology. moreover, these results were challenged by new trials that did not find any substantial benefit from hydroxychloroquine administration [ ] [ ] [ ] . . therefore, clinical trials with a control group are needed to provide reliable answers for clinicians; antimalaria drugs are being tested in randomised controlled trials (table , table s ). virus-induced immune response leading to cytokine storm syndrome (css) and secondary haemophagocytic lymphohistiocytosis (hlh) is probably the underlying pathogenetic mechanism that leads to critical and often fatal covid- infection [ , ] . (table , table s ); tocilizumab, in particular, improved symptoms and laboratory parameters in a small immunomodulators licensed for haematological and rheumatological conditions (such as leflunomide and thalidomide), as well as colchicine that counteracts the assembly of the nlrp inflammasome, are also being studied for their therapeutic use against sars-cov- (table ,table s ) [ ] . the immunomodulatory effects of macrolide antibiotics, as well as their pharmacodynamic property to achieve at least -fold higher concentrations in epithelial lung fluid than in serum, have led researchers to repurpose them against sars-cov- (table , table s observations the antifibrotic agent pirfenidone is being evaluated in at least three randomised clinical trials for its efficacy in the prevention of post-covid- pneumonia fibrosis (table , table s ). pirfenidone targets collagen synthesis by inhibiting transforming growth factor beta (tgf-b), diminishing extracellular matrix deposition and reducing the activity of lung fibroblasts in vitro [ ] . finally, immunostimulatory molecules that enhance the hosts' immune response against the invading pathogen, like ifn-α, interferon beta (ifn-β), the recombinant protein produced by (table , table s ). (table , table s ). (table , table s ). many studies are currently evaluating the efficacy of tcm in covid- prevention in china. importantly, at least vaccines are under development. among them, an mrna-based vaccine encoding the s-protein is being assessed for its safety, reactogenicity and efficacy against sars-cov- (table , table s ). besides the registered trials, other large companies have also announced the initiation of vaccine development [ , ] . other preventative molecules include hydroxychloroquine and the recombinant human interferon α b spray. in the usa, exposed individuals are randomised to hydroxychloroquine or placebo, evaluating the agent's potential as post-exposure prophylaxis (nct , table s ). furthermore, another randomised clinical trial evaluates the efficacy of a -month course of chloroquine in at-risk healthcare personnel (nct , table s ). finally, the live attenuated strain of mycobacterium bovis is expected to be tested as a preventative strategy against covid- among healthcare professionals, in australia and france. in total, interventional treatment and prevention trials were included in the methodological analysis respectively (table ) . among the eligible treatment studies, children recruitment (i.e.< years old) was reported in clinical trials in total: testing darunavir with cobicistat (nct ); on human stem cells transfusion (chictr , chictr ); testing hydroxycholoroquine (eudract number: - - ); using tocilizumab (nct ); and assessing nutritional supplements (nct ) ( table s ).with respect to relevant prevention studies, children were included in vaccine trials (nct , nct ) as shown in table s . phase iv and phase iii treatment trials were the most commonly reported interventional study types (n= , % and n= , % respectively) as demonstrated in table . nonetheless, the majority of registered trials do not disclose the study phase (n= , %). in terms of blinding, open-label ( %), double-blinded ( %), and single-blinded ( %) studies were retrieved. most trials were randomised (n= , %) with a parallel assignment between arms. the median (iqr) number of planned inclusions is ( - ) with a range of to participants. phase iii and phase i prevention studies were the most commonly reported ones (n= , % and n= , % respectively, table ). as with treatment trials, many prevention trials do not report the study phase (n= , %). regarding prevention studies' blinding, double-blinded ( %), open-label ( %), and single-blinded ( %) were found. most studies were randomised (n= , %) with a parallel figure shows the total number of planned inclusions and the number of clinical trials for the ten most frequently studied treatments, with hydroxychloroquine being the treatment associated with a clinical primary outcome was defined in out of therapeutic trials ( % ; table ) . ( %) studies respectively (table ) . regarding prevention studies, out of ( %)disclosed a clinical primary outcome , such as confirmed symptomatic covid- for studies, severe covid- for studies, confirmed or suspected covid- for one study, and safety for (studies evaluating vaccines. the other prevention studies had a virological outcome (confirmed sars-cov- infection with or without symptoms, n= ) or a biological outcome (n= , routine blood tests) (table ) . analyse clinical trials testing these agents. firstly, study design data and details on the interventions being assessed were often lacking. this hampers the available information to researchers and relevant stakeholders, and potentially influences the discovery of successful treatments. secondly, most trials, and especially those registered at the beginning of the pandemic, disclosed low participant numbers, which may impact the robustness of their future results. however, these numbers should be cautiously interpreted, as they represent the anticipated, the progression of computed tomographic (ct) images in patients with coronavirus disease (covid- ) pneumonia the ct progression of covid- pneumonia integrating mechanisms of pulmonary fibrosis induction of pro- inflammatory cytokines (il- and il- ) and lung inflammation by coronavirus- covi- or sars-cov- ): anti-inflammatory strategies pirfenidone attenuates lung fibrotic fibroblast responses to transforming growth factor-β discovery french steering committee. type interferons as a potential treatment against covid- angiotensin converting enzyme (ace) inhibitors and angiotensin receptor blockers in tmprss and adam cleave ace differentially and only proteolysis by tmprss augments entry driven by the severe acute respiratory syndrome renin-angiotensin system blockers and the covid- pandemic: at present there is no evidence to abandon renin camostat mesilate, pancrelipase, and rabeprazole combination therapy improves epigastric pain in early chronic pancreatitis and functional dyspepsia with arbidol as a broad-spectrum antiviral: an update antiviral activity of arbidol against influenza a virus, respiratory syncytial virus, rhinovirus, coxsackie virus and adenovirus in vitro and in vivo pharmacokinetics, metabolism, and excretion of the antiviral drug arbidol in humans arbidol combined with lpv/r versus lpv/r alone against corona virus disease :a retrospective cohort study evaluation of convalescent whole blood for treating ebola virus disease in freetown treatment of severe acute respiratory syndrome with convalescent plasma use of convalescent plasma therapy in sars patients in hong kong retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone treatment in sars patients the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis feasibility of using convalescent plasma immunotherapy for mers-cov infection disappearance of antibodies to sars-associated coronavirus after recovery therapeutic implications of human umbilical cord mesenchymal stromal cells in attenuating influenza a(h n ) virus-associated acute lung injury mesenchymal stem cells improves the outcome of patients with covid- exosomes derived from mesenchymal stem cells exosomes from mesenchymal stem/stromal cells: a new therapeutic paradigm moderna's covid- vaccine could reach healthcare workers this fall | fiercebiotech n johnson identifies lead covid- vaccine candidate n.d who r&d blueprint novel coronavirus covid- therapeutic trial synopsis drug treatment options for the -new coronavirus ( -ncov) covid- infection: the perspectives on immune responses early antiviral treatment contributes to alleviate the severity and improve the prognosis of patients with novel coronavirus disease (covid- ) temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study clinical features of patients infected with novel coronavirus in wuhan, china covid- : combining antiviral and anti-inflammatory treatments nih clinical trial of investigational vaccine for covid- begins | national institutes of health (nih) n.d chloroquine/ hydroxychloroquine prevention of coronavirus disease (covid- ) in the healthcare setting -full text view -clinicaltrials molecular immune pathogenesis and diagnosis of covid- xpress sars-cov- has received fda emergency use (covid- ) globally key: cord- -vk h qau authors: tsoungui obama, h. c. j.; adil mahmoud yousif, n.; hassan mohamed alawam nemer, l.; ngougoue ngougoue, p. m.; ngwa, g. a.; teboh-ewungkem, m.; schneider, k. title: preventing covid- spread in closed facilities by regular testing of employees - an efficient intervention in long-term care facilities and prisons date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: vk h qau background: draconic control measures were introduced to contain the global covid- pandemic, many of which have been controversial, particularly the comprehensive use of diagnostic tests. regular testing of high-risk individuals (pre-existing conditions, older than years of age) has been suggested by public health authorities. the who suggested the use of routine screening of residents, employees, and visitors of long-term care facilities (ltcf) to protect the resident risk group. similar suggestions have been made by the who for other closed facilities including incarceration facilities (e.g., prisons or jails), where in parts of the us, accelerated release of approved inmates is taken as a measure to mitigate covid- . methods and findings: here, the simulation model underlying the pandemic preparedness tool covidsim . (http://covidsim.eu/) is extended to investigate the effect of regularly testing of employees in order to protect immobile resident risk groups in closed facilities. the reduction in the number of infections and deaths within the risk group are investigated as well as the potential economic gain resulting from savings in covid- related treatment costs in comparison to costs resulting from the testing interventions. our simulations are adjusted to reflect the situation of ltcfs in the federal republic of germany. the probability is nearly one that covid- spreads into closed facilities due to contact with infected employees even under strict confinement of visitors in a pandemic scenario without targeted protective measures. regular screening of all employees by pcr tests provides a significant reduction of covid- cases and related deaths in ltcfs. while the frequency of testing (testing rate) and the quality of tests have noticeable effects, the waiting time for obtaining test results (ranging from up to hours) hardly impacts the outcome. the results suggest that testing every two weeks with low-quality tests and a processing time of up to hours yields a strong reduction in the number of cases. rough estimates suggest a significant economic gain. conclusions: the introduction of covid- in closed facilities is unavoidable without thorough screening of persons that can introduce the disease into the facility. these measures provide an economically meaningful way to protect vulnerable risk groups characterized by an elevated risk of severe infections in closed facilities, in which contact-reducing measures are difficult to implement due to imminent unavoidable close human-to-human contacts. the global covid- pandemic that emerged in wuhan, china in december was declared a public health emergency of international concern by the who director-general in late january and drastically changed the way of living across the globe [ ] . sars-cov- is an extremely contagious virus affecting the respiratory system [ ] . while most infections are asymptomatic and mild, severe infections are life-threatening [ , , , ] . if the virus affects the lung it can result in diffuse pneumonia, requiring oxygen supply, hospital, or even icu treatment [ , , , , ] . with no effective treatment against the virus, severe episodes can result in death by multiple organ failure [ ] . moreover, severe (and even mild) infections can cause substantial long-term effects, potentially imposing long-term burdens on healthcare systems [ , ] . from the beginning of the pandemic, older adults and individuals with underlying medical conditions, particularly lung or heart disease, diabetes, obesity, etc. are associated with an increased risk of developing serious complications from sars-cov- infections [ ] . the centers for disease control and prevention (cdc) identified people aged years and older and people living in a long-term care facility (ltcf) as high-risk groups. indeed, every second covid- related death in the federal republic of germany occurred within ltcfs [ ] . likewise the rapidly growing elderly population in u.s. prisons [ ] is at high risk due to the exceedingly high numbers of infections in such facilities [ ] . draconic control measures were implemented by governments across the globe to prevent the spread of the pandemic, including social distancing (cancellation of mass crowdings and events, enforced physical distance, etc.), curfews, quarantine, and home isolation measures, mandatory use of face masks, accompanied by massive deployment of disinfectants, supply of contact tracking mobile-device applications, and diagnostic tests [ , , ] . most commonly used are pcr tests that detect the virus in nasopharyngeal swabs, diluted gargle samples, or peripheral blood. as pcr tests amplify virus-specific rna, they are characterized by very high specificity. the sensitivity of such tests varies across different products on the market. moreover, pcr tests are easy to perform. alternatives to quantitative pcr tests are crispr-based [ , ] , which are rapidly performed, and have high specificity and sensitivity. other tests are antibody or antigen based. such tests are less specific and do not necessarily detect active infections, since antibodies and antigens are present in the blood serum after the infection is cured. the who established guidelines -including regular testing of employees and residents -to protect individuals in ltcfs [ , ] due to high case fatality rates [ ] . residents of ltcfs constitute a substantial group in high-income countries such as the federal republic of germany. with a population of . million, the number of people depending on nursing either in ltcfs or at home in germany increased from . million in to . million in (over % of them being over years old) [ ] . the capacity of ltcfs in germany was beds (full stationary capacity: ) in , with beds ( full stationary) filled ( beds in , being full stationary) [ ] . these are sustained by employees, % of which are care and support personnel [ ] [ ] . the use of routine screening of residents, employees, and visitors before entering an ltcf by diagnostic tests was mentioned in guidelines by public health authorities [ , , ] and also suggested for incarceration facilities [ ] . the impact of such control measures can be studied through the use of mathematical models. here, a mathematical model, based on the freely available covidsim simulation tool, is adapted to estimate the benefit and economic gain of routine screening for covid- infections of employees in ltcfs and retention facilities by pcr tests. in particular, we study the impact of i. the frequency at which employees are tested, (ii) the processing time to obtain test results, and (iii) the quality of the pcr test in terms of sensitivity. while the model is described verbally in the main text, a concise mathematical description can be found in the s appendix. the model is exemplified by parameters that reflect the situation in the federal republic of germany. we study the impact of testing employees in ltcfs or incarceration facilities to protect immobile risk groups from covid- infections using an extended seir model, i.e., by a deterministic compartmental model of ordinary differential equations. in particular, the model is an extension of that underlying the pandemic preparedness tool covidsim [ ] . the flow chart of the model is presented in fig . the model is described verbally with the concise mathematical description found in s appendix. in the description, we focus on ltcfs, although the model equally applies to prisons. a population of size n is divided into three groups, i. the immobile risk group (ri), i.e., residents of ltcfs, (ii) the employees (staff) working in ltcfs (st), who are in close contact with the risk group, (iii) and the general population (ge), i.e., the rest of the population. each group (ge, st, ri), is further subdivided into susceptible, infected, recovered, or dead individuals. infected individuals pass through i. a latency period (not yet infective), (ii) a prodromal period (already partly infective but not yet exhibiting characteristic symptoms), (iii) a fully contagious period (symptoms ranging from non-existent or mild to severe), and (iv) a late infective period (no longer fully contagious). all individuals either recover from covid- and obtain full permanent ignored, as we assume a pandemic in a large population in a relatively short time period. in classical seir models, individuals in the latent, prodromal, infected, and late infected classes simply proceed from one stage to the next at a rate directly related to the residence time in each stage. this simplistic flow implicitly assumes that the times individuals spend in the various compartments are exponentially-distributed, and hence have a large variance. in particular, a proportion of individuals progresses too fast, whereas others progress much too slow. to resolve this, we subdivide the latent, prodromal, fully contagious, and late october , / all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint infectious periods into several sub-stages, through which individuals pass successively. this ultimately leads to more realistic durations and hence dynamics. the characteristics of the population subgroups (ge, st, ri) are incorporated within the contact behavior. namely, the risk group has mainly contacts with other individuals in the risk group and the ltcf employees, whereas their contacts with the general population are limited. the employees (st) have contacts among themselves, with the risk group and the general population. however, the general population has mainly contacts among themselves. given a contact within or between certain sub-populations, the contact occurs at random. the contact behavior is captured by the contact matrix (see s appendix section "the basic reproduction number and the next generation matrix"). susceptible individuals acquire infections through contacts with individuals in the prodromal, the fully contagious, or the late infectious periods at rates β p , β i , β l , respectively, which are identical for each subgroup. the basic reproduction number r is the average number of infections caused by an average infected individual in a completely susceptible population during the infectious period. in a subdivided population (here ge, st, ri), the definition of r is not straightforward and has to be derived from the next-generation matrix [ ] (see s appendix section "the basic reproduction number and the next generation matrix"). importantly, r fluctuates seasonally with a yearly average basic reproduction number ofr . first, infected individuals are latent carriers, before they enter the prodromal period, in which they become partly infective. from there, they enter the fully contagious period, at the beginning of which, it is determined whether the infection proceeds as symptomatic or asymptomatic. the likelihood to suffer from a symptomatic episode is elevated in the risk group (ri). covid- confirmed individuals are subject to case isolation. specifically, a fraction of symptomatic individuals will be detected and isolated in quarantine wards (perfect isolation preventing all contacts). if the wards are full, infected individuals are sent into home isolation (imperfect isolation, preventing only a fraction of contacts). regarding this, there are differences in the subgroups: each symptomatic individual in the risk group will be detected and isolated in quarantine inside the ltcf (perfect isolation), whereas only a fraction of the general population and the ltcf employees will be isolated. infected individuals further progress to the late infective stage, during which they will stay quarantined as determined during the fully contagious stage. importantly, ltcf employees will be tested for covid- on a regular basis. we assume that the test is % specific, i.e., there are no false-positive test results, reflecting pcr-or crispr-based tests [ , ] . if tested positive, they will be isolated either in quarantine wards or at home, in which case all contacts with the risk group are prevented. staff can be tested positive during any of the infected stages (latent, prodromal, fully contagious, late infective), however with different sensitivity depending on the characteristic of the covid- test being used, irrespective of symptoms. in particular, there is a possibility (depending on the sensitivity of the covid- test) that employees are isolated already during the latent period before they are infectious. test results are not obtained instantaneously, but with a time delay. infected staff can still infect others during this time. the waiting time for the test results depends on the available infrastructure. finally, late infected individuals, either recover or die. only symptomatic infections can cause death. the fraction of lethal infections is higher in the risk group. all case isolation mechanisms are not initially present, but implemented with a time delay after the initial occurrence of the disease. in addition to case isolation, general contact reducing interventions, e.g., social distancing, curfews, etc. will be sustained for a specific time interval during the beginning of the epidemic. during the time interval in which case isolation measures are sustained, contacts between the risk group and the general population are reduced, reflecting preventative measure. furthermore, visitors have to provide a negative test result before entering the ltcf. to obtain conservative estimations the latter intervention is ignored in the simulations. the effect of protecting an immobile risk group (ltcf residents) by regularly pcr-testing ltcf employees, who are the most likely to import the disease into the facility, is studied. model parameters are adjusted to roughly reflect the situation in the federal republic of germany, one of the countries that has successfully intervened in the covid- epidemic. the model itself is applicable to any industrial nation with an aging population. the aim is to investigate the effects of protecting the risk group and to estimate the demand for pcr tests. some testing scenarios are not feasible in terms of logistics and available testing capacities, and just serve as a comparison. the parameters used are listed in tables , and s table-s table. germany has a population of roughly n = million. we assume elderly individuals live in ltcfs in which control interventions by pcr testing can be implemented. all employees of ltcfs amount to approximately . this number however includes employees in the administration and external services, who are not regularly working in these facilities. hence, a number of employees was assumed to work regularly in the ltcfs. the first covid- cases were introduced in the middle of february , corresponding to time t = . a basic reproduction number of r = . was assumed. when studying the effect of seasonal variation, r was assumed to fluctuate % over the year, with an annual averager = . and a peak roughly in late december (t r max = ). the average duration of the latent, prodromal, fully contagious and late infective states were assumed to last on average d e = . , d p = , d i = , d l = days, respectively. in the prodromal and late invective states, individuals were assumed to be half as infective as in the fully contagious state. individuals in the risk group were more likely to develop severe symptoms (f sick = % vs. f in essence, the model without testing interventions is equivalent to the one underlying covidsim . or . [cf. , http://covidsim.eu]. hence, we used a combination of general contact reduction and case isolation as proposed in [ ] . clearly, the available capacities of tests, the infrastructure to perform tests, the tables , and s table -s table. the dashed grey line in panels (e-h) corresponds to the value of r (right y-axis). in the presence of seasonal fluctuations in r , qualitatively the same picture emerges (see fig e-h, s fig) . however, the differences between the testing rates are more pronounced, particularly between testing every vs. days. tables , and s table -s table. the dashed grey line in panels (e-h) corresponds to the value of r (right y-axis). (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . tables , and s table -s table. the dashed grey line in panels (e-h) corresponds to the value of r (right y-axis). testing with a processing time of hours was assumed in the absence of seasonal tables , and s table - while this suggests, that the intervention is cost-efficient, the actual gain is likely to be underestimated. namely, long-term effects of infections and additional costs are not accounted. furthermore, costs for testing can presumably be reduced. in particular, the testing intervention results in a -fold reduction of the number of infected individuals even under the most conservative setup. hence, the follow-up costs of infections reduce -fold. elderly citizens and particularly residents of long-term care facilities (ltcf) were identified early as a vulnerable risk group that deserves particular protection, as reflected by the who guidelines in march [ ] . regular testing of ltcf employees and residents for covid- was explicitly mentioned by the john hopkins university in their guidance on protecting individuals residing in long-term care facilities [ ] . furthermore, such recommendations can also be found in the who policy brief on preventing and managing covid- across long-term care services from july [ ] . to evaluate the effectiveness of testing interventions to protect resident risk groups in ltcfs we extended the model underlying the pandemic preparedness tool covidsim [ , http://covidsim.eu]. in particular, the deterministic model formulated as systems of differential equations was extended to separate the risk group of ltcf residents and in our investigations, the model was adjusted to reflect the situation in the federal republic of germany. however, the model is not restricted to one particular country but will be applicable to any other industrialised nation with a similar age structure. the results clearly indicate that regular covid- -screening of ltcf employees by testing successfully reduces the number of cases and deaths in the resident risk group. even with relative conservative assumptions a -fold reduction is achieved. our results indicate that the frequency at which employees are tested has a strong effect. testing once every to days is sufficient and seems to be a realistic. although more frequent testing further improves the intervention, the gain is insignificant. importantly, the waiting time for the return of test results (ranging from to hours) has no noticeable effect. a waiting time of - hours seems to be realistic when compared with the requirements for international air traveling since summer , requiring passengers to provide proof of a negative covid- test, taken no longer than hours before departure. the quality of the test in terms of sensitivity has a clear impact on the outcome. here, pcr tests were assumed to be relatively conservative, considering the fact that these tests are constantly improved. our simple rough estimates of the economic gain of the proposed intervention, comparing the potential costs of covid- treatments with the costs for the testing intervention, is substantial. these estimates are conservative as they do not account for health care costs for long term effects of the infection and capacity shortages in the ltcfs, e.g., due to isolation measures of infected residents. notably, testing a population of ltcf employees every two weeks requires a total of . million tests per year (assuming tests per person per year), or tests per week, which is a realistic number in germany, having a capacity of approximately . million tests per week in september [ ] . notably, similar results can be obtained for serological tests. however, these tests october , / all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint typically have lower specificity, so that false-positive results can no longer be ignored. per test. hence, the economic gain would be further amplified due to the cost efficiency. the proposed intervention considers regular testing only of ltcfs employees (staff) not of residents or the general population upon entry. the reason is that we wanted to study the impact of minimal-invasive control measure. namely, the risk group is twice as large as the target population being tested. hence, also testing the risk group would result in the requirement of unrealistically many tests. tables , and s table -s table, the parameters used are listed in tables , and s table -s table. seasonal (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . tables , and s table -s table, tables , and s table - tables , and s table -s table, table . the parameters used are listed in tables s table -s table, tables , and s table -s table. s appendix. mathematical description s sick dead g=f f sick dead all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint who) who. coronavirus disease (covid- ) severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges clinical features of patients infected with novel coronavirus in wuhan the possible immunological pathways for the variable immunopathogenesis of covid- infections among healthy adults, elderly and children a systematic review of asymptomatic infections with covid- estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship african journal of primary health care & family medicine hyperbaric oxygen therapy in non-ventilated covid- patients tocilizumab for the treatment of severe covid- pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of patients in covid- critical illness pathophysiology driven by diffuse pulmonary thrombi and pulmonary endothelial dysfunction responsive to thrombolysis radiological findings from patients with covid- pneumonia in wuhan, china: a descriptive study. the lancet infectious diseases causes of death and comorbidities in patients with covid- estimates of the severity of coronavirus disease : a model-based analysis. the lancet infectious diseases covid- : impact of long term symptoms will be profound, warns bma risk factors for covid- severity and fatality: a structured literature review covid- jedes zweite corona-opfer lebte im heim. \"{a}rzezeitung aging of the state prison population how covid- 's disruption of the u.s. correctional system provides an opportunity for decarceration comparison of measures adopted to combat covid pandemic by different countries in who regions differentiating covid- response strategies. the innovation analysing governmental response to the covid- pandemic development and evaluation of a rapid crispr-based diagnostic for covid- clinical validation of a cas -based assay for the detection of sars-cov- rna infection prevention and control guidance for long-term care facilities in the context of covid- : interim guidance recommendations for a metropolitan covid- response special emphasis series -guidance on protecting individuals residing in long-term care facilities epidemiology of covid- in a long-term care facility in king county pflegestatistik -pflege im rahmen der pflegeversicherung deutschlandergebnisse covid- in correctional and detention facilities -united states covid- in prisons and jails in the united states incarceration and its disseminations: covid- pandemic lessons from chicago's cook county jail. health affairs (project hope) correctional populations in the united states correctional populations in the united states preventing and managing covid- across long-term care services: policy brief overview of european prison services' responses to the covid- crisis the covid- pandemic preparedness simulation tool on the definition and the computation of the basic reproduction ratio r in models for infectious diseases in heterogeneous populations aktuelle daten und informationen zu infektionskrankheiten und public health for the western pacific whoro. type . for europe whoro. preparedness, prevention and control of covid- in prisons and other places of detention acknowledgments we want to dedicate this work to all the victims of the sars-cov- virus. our grief is with the friends and families of the dreadful disease. the authors like to express their sympathy to all working to find a cure for the virus. the fruitful discussions with prof. martin eichner and his helpful comments and inputs are gratefully acknowledged. key: cord- - uglwvid authors: nadim, sk shahid; chattopadhyay, joydev title: occurrence of backward bifurcation and prediction of disease transmission with imperfect lockdown: a case study on covid- date: - - journal: chaos solitons fractals doi: . /j.chaos. . sha: doc_id: cord_uid: uglwvid the outbreak of covid- caused by sars-cov- is spreading rapidly around the world, which is causing a major public health concerns. the outbreaks started in india on march , . as of april , , , confirmed cases and , deaths are reported in india and more than , , confirmed cases and , , deaths are reported worldwide. mathematical models may help to explore the transmission dynamics, prediction and control of covid- in the absence of an appropriate medication or vaccine. in this study, we consider a mathematical model on covid- transmission with the imperfect lockdown effect. the basic reproduction number, r( ), is calculated using the next generation matrix method. the system has a disease-free equilibrium (dfe) which is locally asymptotically stable whenever r( ) < . moreover, the model exhibits the backward bifurcation phenomenon, where the stable dfe coexists with a stable endemic equilibrium when r( ) < . the epidemiological implications of this phenomenon is that the classical epidemiological requirement of making r( ) less than unity is only a necessary, but not sufficient for effectively controlling the spread of covid- outbreak. it is observed that the system undergoes backward bifurcation which is a new observation for covid- disease transmission model. we also noticed that under the perfect lockdown scenario, there is no possibility of having backward bifurcation. using lyapunov function theory and lasalle invariance principle, the dfe is shown globally asymptotically stable for perfect lockdown model. we have calibrated our proposed model parameters to fit daily data from india, mexico, south africa and argentina. we have provided a short-term prediction for india, mexico, south africa and argentina of future cases of covid- . we calculate the basic reproduction number from the estimated parameters. we further assess the impact of lockdown during the outbreak. furthermore, we find that effective lockdown is very necessary to reduce the burden of diseases. an outbreak of coronavirus disease (covid- ) has resulted in , , con- firmed cases and , , deaths as of april , according to who [ ] . the out- break was first taken place in wuhan, china, in december , with the majority of early cases reported in the city. coronaviruses are single-stranded, positive-sense rna viruses belonging to the coronaviridae family [ ] . it has been confirmed that people have been infected due to viral pneumonia, including seven critically ill cases [ ] , and this epidemic has drawn tremendous attention worldwide. it causes variety of symptoms, including dry cough, fever, weakness, trouble breathing, and bilateral lung infiltration, be the sixth public health emergency of international concern. since the first discovery and identification of coronavirus in , there are three major outbreaks occurred due to coronaviruses and the outbreak was called 'severe acute respiratory syndrome (sars) outbreak ( ) in china [ ] . saudi arabia suffered from 'middle east respiratory syndrome' (mers) outbreak ( ) [ ] and south korea ( ) [ ] . the indian government reported that on january in the state of kerala, across the world and an unprecedented threat to the community's health care, economy and lifestyle. for all, there is a huge worry as to how long this condition can continue and whether the epidemic can be handled. we also study the cases of mexico, south africa and argentina as the lockdown was carried out partially or in a less severe form in these countries. in february the virus was confirmed to reach mexico. however, two recently lockdown measure has been used successfully to control covid- spread. the aim of this study is to investigate the qualitative effect of the imperfect lockdown on the spread of disease dynamics. to achieve this goal, a mathematical model for covid- with the lockdown is proposed and analyzed. in this model, we implemented the imperfect lockdown, which means that the lockdown susceptible population also gets infected during the lockdown period by unnotified infected individuals. we looked at in- dia's situation during the outbreak period and fitted our model with the newly daily cases reported from march th , to april th , . we also looked at the situation of mexico, south africa and argentina during the outbreak period and fitted our model with the new daily cases reported for a certain outbreak period. we are providing a short-term prediction for india, mexico, south africa and argentina of future cases of covid- using the estimated parameters for the period march , , to may , , march to july , march to july and march to july respectively. for the above-mentioned period we aslo estimate the basic reproduction number. it is common for classical epidemic models that a basic reproduction number is a threshold in the context that if the basic repro- duction number is greater than one, a disease will persist, and dies out if it is less than one. in this case, for imperfect lockdown, the basic reproduction number does not rep- resent the required elimination effort; rather, the effort at the turning point is described the paper is organized as follows: our proposed mathematical model which incorporates the lockdown of susceptible individuals and imperfect lockdown efficacy is described in section . the model is analyzed specifically for the existence of backward bifurcation in section . in section we fitted our model to daily new cases. we provided a all the parameters and their biological interpretation are given in table respectively. proof. the system ( . ) can be written as follows .., f (x)) denotes the right hand side functions. it is very obvious that for every j = , ..., the basic reproduction number r is a threshold value that is epidemiologically significant and determines the potential of an infectious disease to enter a population. to obtain the basic reproduction number r of the system ( . ), we apply the next generation matrix approach. the system has a disease-free equilibrium given by , , , , . the infected compartments of the model ( . ) consist of (e(t), i(t), j(t)) classes. fol- lowing the next generation matrix method, the matrix f of the trransmission terms and the matrix, v of the transition terms calculated at ε are, so, the next generation matrix f v − is, calculating the dominant eigenvalue of the next generation matrix f v − , we obtain the basic reproductive number as follows [ ; ] the basic reproduction number r is defined as the expected number of secondary cases generated by one infected individula during its lifespan as infectious in a fully susceptible population. the basic reproduction number r of ( . ) given in . . using theorem in [ ], the following result is established. lemma . . the disease-free equilibrium ε of system ( . ) is locally asymptotically stable whenever r < , and unstable whenever r > . . . existence of endemic equilibrium we are now exploring the existence of endemic equilibrium. let ε * = (s * , l * , e * , i * , j * , r * ) be any endemic equilibrium of system ( . ). let us denote further, the force of infection be by setting the right equations of system ( . ) equal to zero, we have substituting the expression in . into . shows that the non-zero equilibrium of the model ( . ) satisfy the following quadratic equation, in terms of λ * h : the endemic equilibrium of the model ( . ) can be obtained by solving for λ * h from in order to check the possibility of backward bifurcation in ( . ), the discriminant b − ac of the quadratic . , is set to zero and the result solved for the critical value (denoted by r c ) of r . this gives: from which we have seen that backward bifurcation occurs for values of r such that r c < r < . we explore the details analysis of backward bifurcation in the next a stable endemic equilibrium co-exists with a stable disease-free equilibrium for r < . clearly, this phenomenon has significant public health consequences, as it makes the classical requirement of the associated basic reproduction number being less than unity to be necessary, but not sufficient to eradicate the disease. in this section, we explore the phenomenon of backward bifurcation in system ( . ). first, we execute bifurcation analysis by using the center manifold theorem as follows: the jacobian of the system ( . ) at the dfe ε is given as, choose β as the bifurcation parameter, then setting r = gives the system ( . ) at the dfe ε evaluated for β = β * has a simple eigenvalue with zero real part, and all other eigenvalues have negative real part. we therefore apply the center manifold theorem in order to analyze the dynamics of ( . ) near β = β * . the jacobian of ( . ) at β = β * , denoted by j ε |β = β * has a right eigenvector (corresponding to the zero eigenvalue) given by w = (w , w , w , w , w , w ) t , where similarly, from j ε |β = β * , we obtain a left eigenvector v = (v , v , v , v , v , v ) t (corresponding to the zero eigenvalue), where ( . ) we calculate the following second order partial derivatives of f i at the disease-free equilibrium ε to show the existence of a backward bifurcation and obtain since the coefficient b is always positive, system ( . ) undergoes backward bifurcation we have established the following conclusion. theorem . . system ( . ) undergoes a backward bifurcation at r = whenever the inequality . holds. furthermore, it should be noted that for the case when lockdown susceptible individuals do not acquire infection during lockdown period (i.e., r = ), the bifurcation coefficient a becomes thus, since a < in this case, it follows from theorem . globally-asymptotically stable (gas) under some certain conditions, as shown below. setting r = in the model ( . ) gives the following reduced model: it can be shown that the reproduction number associated with the reduced model ( . ), is given by the model ( . ) has a dfe ε = (s , l , , , , ). theorem . . the dfe (ε ) of the reduced model ( . ), is globally asymptotically proof. consider the following lyapunov function d = γk θk (k + l) e + k θ(k + l) i we take the lyapunov derivative with respect to t, since all the variables and parameters of the model ( . ) are non-negative, it follows that therefore by combining all above equations, it follows that each solution of the model equations ( . ), with initial conditions ∈ Ω , approaches ε as t → ∞ for r * ≤ θ < . the above result shows that, for the case when the lockdown efficacy in preventing tion of this technique for model fitting is given in [ ] . the estimated parameters are given in table the estimated values of unknown initial conditions are given by table . the fitting of the daily new hospitalized covid cases of this four country are displayed in figure . using these estimated parameters from table and the fixed parameters from table , we calculate the basic reproduction numbers given in table . in this section, the impact of lockdown is measured qualitatively on the disease transmission dynamics. a threshold study of the parameters correlated with the lockdown of susceptible individuals l is performed by measuring the partial derivatives of the basic reproduction number r with respect to this parameters. we observe that so that ∂r ∂l < for all < r < . we perform the sensitivity of model parameters with respect to the significant re- sponse variable and analyze different control parameters to limit covid cases for the four countries. in order to get an overview of most influential parameters, we compute the normalized forward sensitivity indices of the model parameters with respect to basic reproduction number r . we have chosen parameters transmission rate between hu- man population β, rate of transition from exposed to infected class γ, the rate at which symptomatic infected become hospitalized or notified η, recovery rate for symptomatic infected τ , lockdown success rate l and lockdown efficacy r for sensitivity analysis. we use the estimated parameters from table for the baseline values. the rest of the pa- rameter values are the same as in table . the bar diagram of the normalized forward sensitivity values of r against these parameters is depicted in figure . however, the mathematical definition of the normalized forward sensitivity index of a variable m with respect to a parameter τ (where m depends explicitly on the parameter τ ) is given as: the normalized forward sensitivity indices of r with respect to the parameters β, η, l and r for india are found to be the fact that x β r = , means that if we increase % in β, keeping other parameters be fixed, will produce % increase in r . similarly, x η r = − . means increasing argentina are given in the table . we have seen that the transmission rate between susceptible humans and lockdown efficacy is positively correlated and the recovery rate of symptomatic infected and lockdown success rate is negatively correlated with respect to basic reproduction number. reproduction number r . we have seen a similar patteren for this four countries. in cases india, the contour plots in figure show the dependence of r on dif- ferent parameters. in figure (a) and figure table and table . from the above finding it follows that lockdown success rate and lockdown efficacy is table and table . time point which is very slow(see the figure ). that means extension of lockdown for these two countries is not too much effective. table and table . backward bifurcation phenomenon, where two stable equilibria, namely the disease-free equilibrium and an endemic equilibrium coexist when the corresponding basic number of reproduction is less than unity. this backward bifurcation phenomena of this study is very important, and this occurs only under imperfect lockdown individuals. this is basically telling us even if the basic reproduction number is less than one, but the disease will persists which is against classical epidemiological theory. in such a situation, the policy makers may stop surveillance, and the results will be disaster. our model exhibits the non-existence of backward bifurcation when the lockdown is perfect (r = ). we have seen that the disease-free equilibrium is globally asymptotically stable whenever the as- sociated basic reproduction number is less than unity for the perfect lockdown model. table and table . table and table . covid- pandemic lockdowns coronavirus disease coronavirus disease applications of centre manifold theory dynamical models of tuberculosis and their applications emerging coronaviruses: genome structure preliminary epidemiologic assessment of mers-cov outbreak in south euro surveillance: bulletin europeen sur les maladies trans- missibles= sars and mers: recent insights into emerging coronaviruses mathematical epidemiology of infectious diseases: model 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overall india: a predictive mathematical study on covid- outbreak mathematical analysis of a power-law form time depen- dent vector-borne disease transmission model im- pact of intervention on the spread of covid- in india: a model based study influence of backward bifurcation on interpretation of r in a model of epidemic tuberculosis with reinfection. mathe- matical the theory of the chemostat: dynamics of microbial competition estimation of the transmission risk of the -ncov and its implication for public health interventions mathematics in population biology pandemic potential of -ncov. the lancet infectious diseases reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission backward bifurcation of an epidemic model with saturated treatment function preliminary estimation of the basic reproduction number of novel coronavirus ( -ncov) in china, from to : a data-driven analysis in the early phase of the outbreak. international journal of infectious diseases estimating the unreported number of novel coronavirus ( -ncov) cases in china in the first half of january : a data-driven modelling analysis of the early outbreak the authors are grateful to the anonymous referees for their careful reading, valuable the authors declare that they have no conflicts of interest. appendix a the center manifold theory [ ; ] is used to determine the existence of the backward bifurcation phenomenon of the model ( . ) theoretically. theorem . . let us consider the following general system of ordinary differential equations with a parameter φwithout loss of generality, it is assumed that x = is an equilibrium for system (a- ) for all values of the parameter φ. ( ) matrix a has a nonnegative right eigenvector w and a left eigenvector v corresponding to the zero eigenvalue. let f k be the k-th component of f andthen, the local dynamics of system (a- ) around are totally determined by the sign of (ii) a < , b < . when φ < , with |φ| , x = is unstable; when < φ , x = is locally asymptotically stable and there exists a negative unstable equilibrium; (iii) a > , b < . when φ < , with |φ| , x = is unstable and there exists a locally asymptotically stable negative equilibrium; when < φ , x = is stable and a positive unstable equilibrium appears; (iv) a < , b > . when φ changes from negative to positive, x = changes its stability from stable to unstable. correspondingly, a negative unstable equilibrium becomes positive and locally asymptotically stable. in particular, if a > , b > then a backward bifurcation occurs at φ = . key: cord- -jx kspen authors: tatapudi, hanisha; das, rachita; das, tapas k. title: impact assessment of full and partial stay-at-home orders, face mask usage, and contact tracing: an agent-based simulation study of covid- for an urban region date: - - journal: glob epidemiol doi: . /j.gloepi. . sha: doc_id: cord_uid: jx kspen purpose: social intervention strategies to mitigate covid- are examined using an agent-based simulation model. outbreak in a large urban region, miami-dade county, florida, usa is used as a case study. results are intended to serve as a planning guide for decision makers. methods: the simulation model mimics daily social mixing behavior of the susceptible and infected generating the spread. data representing demographics of the region, virus epidemiology, and social interventions shapes model behavior. results include daily values of infected, reported, hospitalized, and dead. results: results show that early implementation of complete stay-at-home order is effective in flattening and reversing the infection growth curve in a short period of time. whereas, using florida's phase ii plan alone could result in % infected and end of pandemic via herd immunity. universal use of face masks reduced infected by %. a further reduction of % was achieved by adding contact tracing with a target of identifying % of the asymptomatic and pre-symptomatic. conclusions: in the absence of a vaccine, the strict stay-at-home order, though effective in curbing a pandemic outbreak, leaves a large proportion of the population susceptible. hence, there should be a strong follow up plan of social distancing, use of face mask, contact tracing, testing, and isolation of infected to minimize the chances of large-scale resurgence of the disease. however, as the economic cost of the complete stay-at-home-order is very high, it can perhaps be used only as an emergency first response, and the authorities should be prepared to activate a strong follow up plan as soon as possible. the target level for contact tracing was shown to have a nonlinear impact on the reduction of the percentage of population infected. increase in contact tracing target from % to % appeared to provide the largest incremental benefit. emergence of the severe acute respiratory syndrome coronavirus type (sars-cov- ) was first reported on december , in wuhan, china and subsequently declared a global pandemic on march by the world health organization (who) [ , ] . as of sept. , , the number of reported cases worldwide has reached over . million causing , deaths. the number of infected cases continues to rise quite significantly [ ] . the u.s. has been among the hardest hit by the coronavirus pandemic with . + million reported infections and , reported deaths (> % of the total reported deaths worldwide) so far. however, as the new cases, hospital admissions, and deaths began to decline in mid-may, most states in the u.s. began phased lifting of their social intervention measures. for example, florida adopted a three phased approach: phase i (which began in may , ) allowed most business and workplaces to reopen with up to % of their building capacities and with large events constrained to %; phase ii began in june , and allowed all businesses to reopen for up to - % of their capacities and also permitting events in large venues with no more than % of their capacities; phase iii will be akin to a complete reopening for which neither a date nor the criteria have been declared. a summary of florida's phased intervention plan can be seen in figure a (in the appendix). as the reopening entered phase ii, florida, along with many other states, began to see sharp increases in daily new infections (e.g., florida reported over , new cases on july , along with a test positivity rate reaching over %). in this paper, we investigate a few 'what-if' scenarios for social intervention policies including if the stay-at-home order were not lifted, if the phase ii order continues unaltered, what impact will the universal face mask usage have on the infections and deaths, and finally, how do the benefits of contact tracing vary with various target levels for identifying asymptomatic and pre-symptomatic. we conduct our investigation by first developing a comprehensive agent-based simulation model for covid- , and then using a major urban outbreak region (miami-dade county hospitalization (if infected with acute illness); and ) recovery or death (if infected). the ab model reports daily and cumulative values of actual infected, doctor visits, tested, reported cases, hospitalized, recovered, and deaths, for each age category. a schematic diagram depicting the algorithmic sequence and parameter inputs for the ab simulation model is presented in figure . our ab simulation model works as follows. it begins by generating the individual people according to the u.s. census data that gives population attributes including age (see table a ) and occupational distribution (see table a ). thereafter, it generates the households based on their composition characterized by the number of adults and children (see table a ). the model also generates, per census data, schools (see table a ) and the workplaces and other community locations (see table a ). each individual is assigned a household, while maintaining the average household composition, and, depending on the age, either a school or a workplace (considering employment levels). a daily (hour by hour) schedule is assigned to every individual, chosen from a set of alternative schedules, based on their attributes. the schedules vary between weekdays and weekends and also depend on the prevailing social intervention orders (see table a ). simulation begins on the day when one or more infected people are introduced to the region (referred to as simulation day ). simulation model tracks hourly movements of each individual (susceptible and infected) every day, and records for each susceptible the number of infected contacts and their identification at each location. based on the level of infectiousness of each infected contact (which depends on the day of his/her infectiousness period), the model calculates the daily force of infection received by each susceptible from all infected contacts at all hours of the day [ ] . daily force of infection is considered to accumulate. however, it is assumed that if a susceptible does not gather any additional force of infection (i.e., does not come in contact with any infected) for two consecutive days, the cumulative force of infection for the susceptible reduces to zero. at the end of each day, the table a for average lengths of the periods) epidemiological models and other parameters that guide the ab model are described next. figure presents a schematic of the disease natural history of covid- , parameters of which are given in table a . once infected, an individual simultaneously begins the latency and the incubation periods. the individual becomes infectious after the latent period is complete but displays symptoms (unless asymptomatic) at the end of the incubation period. the period between end of latency and end of incubation is referred to as pre-symptomatic, a time when the infectiousness grows rapidly and almost reaches its peak. symptomatic cases either follow a non-acute progression (majority of cases, not requiring hospitalization) or acute progression (requiring hospitalization). cases for whom disease does not become acute, enter a recovery period after infectiousness ends. those with acute disease progression (generally toward the end of the infectious period) are hospitalized. after the hospital stay period, cases either recover or die. for average lengths of recovery and hospitalized periods that are used in the ab model, see table a . there is some evidence based on animal experimentation that recovered individual may become immune to reinfection [ , ] . but other studies remain inconclusive [ ] . hence, due to lack of established data on this matter, our model considers the recovered cases to be immune to further covid- infections. the duration and intensity of infectiousness is considered to be guided by a lognormal density function (see figure ). the function is truncated at the average length of the infectiousness period (which is considered to be . days). asymptomatic cases are assumed to follow a similar infectiousness intensity profile but scaled by a factor ( in the force of infection calculation ( ), see table a ). ` the ab model estimates the probability of infection for a susceptible using the accumulated value of daily force of infection ( ), which is calculated as follows. the first component in ( ) accounts for the force experienced by susceptible individual at home from another infected household members . the second component captures the force experienced at schools/workplaces/community places for work and also at community places visited for daily errands; this happens when a susceptible is in the same location type where infected is at hour . the definition and values of the parameters of ( ) are given in table a . equation ( ) is a modified version of the force of infection equation given in [ ] , which has three components that separately calculate force of infection received at home, at indoor workplaces, and at the outdoor community. for the sake of simplicity, we have considered only the first two components, home and indoor workplaces, where most of the covid- transmission is assumed to be taking place. we have assumed that the mode of virus transmission at indoor community places that are routinely visited by people as part of their daily errands (like grocery stores, home goods stores, dine-in/take-out restaurants, etc.) is similar to that of indoor workplace transmission. the force of infection is gathered by a susceptible individual each day from all infected contacts in his/her mixing groups (home, school/workplace, and community places). the cumulative value of is used at the end of each day to calculate the probability of infection as . j o u r n a l p r e -p r o o f the ab model incorporates all applicable intervention orders like stay-at-home, school and workplace closure, isolation of symptomatic cases at home, and quarantine of household members of those who are infected. the model also considers: varying levels of compliances for isolation and quarantine, lower on-site staffing levels of essential work and community places during stay-at-home order, restricted daily schedule of people during various social intervention periods, phased lifting of interventions, use of face masks in workplaces, schools and community places with varying compliance levels, and contact tracing with different target levels to identify asymptomatic and presymptomatic cases. the timeline for social interventions implemented in the model are summarized in table a . other salient considerations in the implementation of our ab model are as follows. across all age groups, % of the infected cases were considered asymptomatic [ ] . approximately, twenty percent ( %) of florida residents are reported as uninsured and do not have access to a primary care physician [ ] . uninsured people thus considered not to have the doctor referral required for most of the testing facilities in miami-dade county, and hence not tested. all symptomatic cases with health insurance were assumed to visit/consult with a doctor. depending on their symptoms, travel history, and contact history, some of them were given referrals for testing. we considered that only a small percentage of cases visiting/consulting a doctor were given referrals in the early months of the pandemic (until the middle of april ), due to the shortage of testing and restrictive cdc guidelines for who could be tested [ ] . however, as cdc relaxed its test eligibility guidelines [ ] and the capacity to test increased in florida, we gradually increased the probability of getting a test referral from a doctor closer to % by early june for symptomatic cases (see table a ). we also considered in our model that a small fraction (reaching only up to % over time) of the asymptomatic cases are randomly tested through various community testing protocols, e.g., at elderly care facilities, healthcare facilities, workplaces, etc. note that we did not consider co-infection, and therefore all cases that were tested in our simulation model had covid- . hence, each test yielded a positive outcome with a probability equal to the test sensitivity (see table a ). based on the data reported on florida covid- dashboard, a test result reporting delay of up to days on average was considered at the start of the pandemic, which was progressively reduced (see table a ). all symptomatic cases with or without testing were considered to isolate at home with a given probability of compliance. the probability of compliance was considered to vary during the length of the symptomatic period of infection. for this purpose, we divided the symptomatic period into three parts: i, ii, and iii, and assumed a lower isolation compliance in parts i and iii and higher in part ii, when the illness j o u r n a l p r e -p r o o f is more apparent. see table a for the isolation compliance probabilities. susceptible members of the households with one or more infected cases are considered to quarantine themselves. we also assumed a level of compliance for the quarantine (see table a ). we used hospitalization and death data reported for miami-dade county [ ] for each age group to obtain probabilities of hospitalization of the reported cases, and probabilities of death for those who are hospitalized (see table a ). though we have implemented our ab simulation model for a specific region, it is quite general in its usability for other urban regions with similar demography, societal characteristics, and intervention measures. in our model, tables a -a summarize the demographic inputs (age and household distribution, number of schools for various age groups, and number of workplaces of various types and sizes). these data will need to be curated from both national and local census records. social interventions vary from region to region and hence the data in table a will need to be updated. similarly, testing availability, test sensitivity, and test outcome reporting delay may also vary significantly from region to region, and thus table a will also need to be updated. the rest of the data (in tables a , a , a , a , and a ) are related to epidemiology of covid- . these are unlikely to be significantly different, though some adjustments of these based on population demographics may be needed. the ab model utilizes a large number of parameters, which are demographic parameters, epidemiological parameters, and social intervention parameters. we kept almost all of the above parameters fixed at their respective once the model is calibrated and validated with available reported data on infected and dead, we extended the model into the future to predict outcomes. the only parameters that were altered after the calibration period are to reflect the expected changes in social interventions, e.g., order mandating use of face mask, re-closing some community places, expected increase in contact tracing, and changes in community response via daily schedule restrictions. hence, the parameters that were changed after the calibration period included those for daily schedules, transmission coefficients, testing and contact tracing rates, and compliance to isolation and quarantine. most of the parameter values used in the ab model were obtained from government archives and research literature, for which references are provided (see tables a -a ). for some of the parameters for which we could not find an archived data source, we used expert opinion and current media reports. we first present a summary of the key results of our study (see table ), from which a number of key insights can be derived that may apply to other similar urban regions experiencing respiratory/influenza type virus outbreaks. early imposition of stay-at-home order appears to have been quite effective in first flattening and then reversing the growth curve. per our model, if the stay-at-home order was allowed to remain enforced, the pandemic would have subsided with a relatively low percentage of the population ( . %) infected and approximately . % dead within six months of inception; or below daily new infections was used as the criterion to consider that pandemic has subsided in miami-dade county. if the extent of social mixing akin to phase ii reopening of florida is in place for an urban region (without the use of face mask and contact tracing), the pandemic would likely have raged for - months and subside only after reaching herd immunity with over % of the population infected and . % of the population dead. universal use of face masks of surgical variety was shown by the model to reduce average total infected, hospitalized, and dead by %, %, and %, respectively. aggressive contact tracing with a goal to identify % of the asymptomatic and pre-symptomatic was also projected to have a very significant positive impact with an average reduction of % of total infected. the average reductions in total infected with %, %, and % contact tracing targets were found to be %, %, and %, respectively. in what follows, we expound the results from our study. figures and fig. (a) ) and hospitalizations and deaths ( fig. (b) ) if stay-athome order were not lifted figure shows a strong influence of continuing with the stay-at-home order in curbing the covid- growth j o u r n a l p r e -p r o o f within approximately months from its inception with on average less than . % of the population infected, . % hospitalized, and . % dead; or below daily new infections was used as the criterion to consider that pandemic has subsided in miami-dade county. such a quick suppression of a virus outbreak always leaves the possibility of resurgence, for which an effective plan of contact tracing, testing, isolation, and support for those isolated (when needed) should be in place. . (a) and fig. (b) ) and phase ii reopening without face mask and contact tracing ( fig. (c) and fig. (d) ) figure shows the expected outcomes of continuing with the phase i order and the phase ii order. figure outbreaks, it is shown that adjusted odds ratio (aor) of getting an infection after wearing surgical variety face masks versus without wearing face mask is . on average [ ] . this can be interpreted as the likelihood of getting j o u r n a l p r e -p r o o f infected if wearing a surgical variety face mask is one third of what it would be for not wearing a mask. hence, we considered a % reduction in the transmission coefficient ( ) used in calculating the force of infection (see equation ( )), assuming a % compliance in the use of surgical variety masks at workplaces, schools and community places. we also tested the impact of % and % reductions in the transmission coefficient value ( ), which translate to approximately % and % compliance for face mask usage, respectively. the anticipated impact of face mask usage together with phase ii order on the average cumulative numbers of infected are shown in figure (a). it also depicts the risk difference between the average values of cumulative infected without and with the universal use of face mask. it may be noted that since the infections grow slower with the use of face mask, the cumulative risk difference rises to almost k in the middle of august and then settles down close to k when pandemic is predicted to subside by the end of november . figure (b) depicts the daily values of the average infected for phase ii without and with universal face mask policy. as expected, the peak of daily infection with face mask usage is shifted to a slightly later date and the downward trend begins after a smaller percentage ( %) of the total population are infected compared to % without the use of face mask. our agent-based model has several limitations. first and foremost, the simulation model is an abstraction of how a pandemic impacts a large and complex society. though our model deliberately introduces some variabilities, somewhat pre-defined daily schedules are used to approximate a highly dynamic contact process of an urban region. also, the contact process does not account for significant variabilities in the types and lengths of interactions even within each mixing groups. we did not assign geographic locations (latitude and longitude) for households, businesses, schools, and community places, and assumed them to be uniformly distributed over the region. it is common for urban population centers and associated establishments to grow in clusters, for which the contact patterns are expected to be different from those in uniformly dispersed regions. we did not consider special events like parties, games, and street protests, some of which are known to have caused superspreading of the virus and case increases. finally, and perhaps most importantly, the model uses a large number of parameters (listed in tables each scenario of our case study with replicates (with different seeds) takes approximately - hours to run in a standard desktop computer with intel core i with gb memory. in the interest of presenting our observations quickly to the public health decision makers, while covid- is still rampant in the region, we chose to use a limited number ( ) of replicates. as the main purpose of this paper is to conduct a broad what-if analysis, we do not believe that use of a small number of replicates has negatively influenced our observations. the trends and observations derived from our results are only intended to be used for planning and guidance of public health decision makers. as part of our continuing (future) work, we plan to use our model to examine the impact of reopening of k- schools and colleges/universities for the new academic year, which began at the end of august and early september. we also plan to use our ab model to assess efficacy of various prioritization strategies (based on age, risk, and work groups) for the vaccines that are anticipated to be available in limited quantities by the beginning of . the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. county can be found in [a ] . c) use of face mask: boston implemented a facemask policy in early may in an executive order by the state [a ] . d) implementation of contact tracing: massachusetts state government provides a dashboard on the community health outcomes for covid- . details on the success of contact tracing in the communities along with the outcome measures varying over time can be found in [a ] . e) policy for returning to school: school reopening policies also widely vary from state to state and also among counties within a state. information on boston's public school reopening policy can be found in [a ] . it is important to frequently check sources on school policy as they are transient. for example, boston planned to reopen on october , but shifted to october after seeing an increase in the number of cases. ) time varying testing of symptomatic and asymptomatic: limited testing availability has been a serious concern in many u.s. regions that suffered from a high level of disease spread. time varying data on test availability and test outcome reporting delay are difficult to find in indexed literature during a pandemic. hence, these can be assumed from regional news reports, test reporting data, and/or other grey literature. ) number of reported, hospitalized and deaths: daily data and archived data on the number of people reported positive, hospitalized and dead can be found from dashboard in [a ] . ) probability of hospitalization for reported cases and probability of death for hospitalized: this can be calculated from [a ] based on age specific reporting. the information contained in the following tables for miami dade county are likely to be same for other regions like boston city/suffolk county. ) daily schedules for people can be assumed to be same as in table a . ) disease natural history parameters for covid- can be assumed to be same across regions within a country, see table a . ) some of the parameters in table a for calculating the force of infection need to be calibrated (see step ) . however, the remaining parameters in table a can be assumed to be same. ) self-isolation compliance for symptomatic cases, and quarantine compliance for household members can be assumed to be same for different urban regions within a country, as in table a . step : updating of the simulation model once the input data collection is complete, the next step is to update the model parameters as follows. ) update the simulation model with all gathered input data from step : after gathering data, it needs to be curated and transformed into .txt files to be read by the simulation model. some of the data are directly coded in the model, where applicable. ) decide simulation begin date: simulation begin date depends on the outbreak region and is based on the date of the first reported case. up to days before the first reported case can be used as a potential date for simulation model to begin. ) decide simulation end date: simulation end date is chosen as desired by the modeler. ) number of initial infected cases: most departments of health (doh) provide a count and characterization of the number of initial infected cases with travel histories. one can identify these initial infected cases during the first month or so of the outbreak and use those cases to initiate social mixing and community spread. step : calibrate and validate simulation model once the simulation model is updated with the input data for the region, the model is calibrated using a small applicable subset of input data and the model output is validated with actual surveillance data from the region, as follows. ) generate multiple seeds for the uniform random variables that are used to calculate the probabilities of infection, hospitalization, death, testing, symptomatic, disease severity, test sensitivity, compliance for isolation and quarantine, among others. simulation output from each seed is considered a replicate. using output data from all replicates, an average value and a corresponding confidence interval for each output measure are calculated. ) a set of initial values of the transmission coefficients for home, school, work, and community places are assumed (based on current literature and published models for outbreaks of similar diseases). these transmission coefficients (along with other parameters, see table a ) are used for calculating force of j o u r n a l p r e -p r o o f infection, which is then use to calculate the probability of infection. different sets of transmission coefficient values are selected for different reference points in time in the simulation, depending on changes in social intervention status and significant current events. for example, the transmission coefficients are appropriately calibrated (reduced) on the day universal use of facemask is announced. also, percentage testing of asymptomatic and pre-symptomatic are increased when contact tracing begins. street protests combined with independence day holiday in early july, are examples of current events that may require adjustment of transmission coefficient values. ) other parameters that are considered suitable for the ab model calibration are probability of running errand that guides daily schedule and probability of employees reporting to work for essential and nonessential businesses. these values can also be assumed to change over time during a pandemic depending on the phased intervention policies implemented by the government in the outbreak region. ) the simulation is calibrated for a chosen period. in this study, we chose to calibrate the model up to july th, as we had reported data available until that date for validation purposes at the time of calibrating the model. ) results for reported cases, hospitalized, dead for all age groups are gathered from the simulation model for each seed. ) average values (with confidence interval) are computed for the numbers of reported, hospitalized and dead. ) for model validation, the simulated average values for the reported, hospitalized, and dead and compared with actual surveillance data. ) alter calibration parameters as needed to obtain desired level of validation accuracy. measure validation accuracy is calculated as the difference in the seven-day moving average between simulated and surveillance data. step : implement calibrated model for prediction ) run calibrated simulation model for all seeds for a desired prediction period beyond the calibration/validation time. ) extract age specific data for total infected, reported cases, hospitalized, and dead from simulation for each seed. ) report mean and confidence interval. figure a : florida's phased social intervention plan for covid- pandemic [ ] j o u r n a l p r e -p r o o f table a : self-isolation compliance for symptomatic cases and quarantine compliance for household members modeling the impact of social distancing, testing, contact tracing and household quarantine on second-wave scenarios of the covid- epidemic the proximal origin of sars-cov- estimating the infection horizon of covid- in eight countries with a datadriven approach evolution and epidemic spread of sars-cov- in brazil centers for disease control and prevention'. cdc releases consolidated covid- testing recommendations centers for disease control and prevention. covid- pandemic planning scenarios centers for disease control and prevention. interim clinical guidance for management of patients with confirmed coronavirus disease modeliling tranmission and control of the covid- pandemic in australia flute, a publicly available stochastic influenza epidemic simulation model covid- virus outbreak forecasting of registered and recovered cases after sixty day lockdown in italy: a data driven model 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data for county: miami-dade covid- /parameter_estimates coronavirus screening test developed at johns hopkins will we see protection or reinfection in covid- ? epidemic analysis of covid- in china by dynamical modeling. medrxiv covid-abs: an agent-based model of covid- epidmeic to simulate health and economic effects of social distancing interventions estimating disease burden of a potential a(h n ) pandemic influenza outbreak in the united states the city of miami -florida. covid- updates all sectors: county business patterns by legal form of organization and employment size class for u.s., states, and selected geographies all sectors: county business patterns by legal form of organization and employment size class for u.s., states, and selected geographies annual business survey: statistics for employer firms by industry, sex, ethnicity, race, and veteran status for the a predictive decision-aid methodology for dynamic mitigation of influenza pandemic a new coronavirus associated with human respiratory disease in china early estimation of the case fatality rate of covid- in mainland china: a data-driven analysis modified seir and ai prediction of the epidemics trend of covid- in china under public health interventions the impact of social distancing and epicenter lockdown on the covid- epidemic in mainland china: a data-driven seiqr model study. medrxiv the boston planning and development agency annual business survey: statistics for employer firms by industry, sex, ethnicity, race, and veteran status for the state-by-state guide to face mask requirements covid- response reporting covid- state of emergency j o u r n a l p r e -p r o o f journal pre-proof table a : distribution of different types of workplaces in miami-dade county. all industries and community places are classified into essential or non-essential. essential industries remain functional with a percentage of their workforce reporting during interventions like stay-at-home or phased reopening. non-essential industries are considered to operate remotely. essential industries include wholesale trade, waste management and remediation services, agriculture, forestry, fishing and hunting, mining, quarrying, oil and gas extraction, utilities, construction, manufacturing, transportation and warehousing. non-essential industry includes finance and insurance, real estate and rental and leasing, professional, scientific and technical services, management of companies and enterprises, administrative and support for waste management and remediation services, educational services, other services except public administration. essential community includes grocery stores, convenience stores, pharmacies and drug stores, home centers, health care and social assistance. non-essential community includes retail, arts, entertainment and recreation and accommodation and food services. for details on education institutions, see j o u r n a l p r e -p r o o f key: cord- - rt wga authors: pattnaik, debidutta; hassan, mohammad kabir; kumar, satish; paul, justin title: trade credit research before and after the global financial crisis of – a bibliometric overview date: - - journal: nan doi: . /j.ribaf. . sha: doc_id: cord_uid: rt wga abstract this study presents an overview of the state-of-the-art in trade credit research by examining publications between and . applying bibliometric and econometric tools, this study presents a comparative analysis of the extant research across the three sub-domains of banking and finance, production and operations, and accounting. findings suggest that the financial emergency in the global market had resulted in a watershed moment in trade credit research. about % of the literature was found to have emerged after the global economic crisis of . a network analysis grouped the trade credit articles into four major and four minor clusters. the banking and financing cluster exhibited the highest growth followed by the production and operation cluster while the perspectives of accounting are yet to gain traction. conversely, reputation of the publishing hub, empirical studies, and the production and operational dimensions of the research positively and significantly influence citations. alongside thorough introspection, the study also provides new areas to direct the course of future research. emerging from the field of banking and finance, research in trade credit has evolved as a multi-disciplinary scientific domain with contributions from business management, industrial engineering, production and operations, finance, economics, etc. (paul & boden, ) . prior reviews have provided partial qualitative and (or) quantitative perspectives of their respective disciplines (see table ), while the need for a holistic overview of the extant literal corpus is unaddressed. in addition, the precise factors contributing to the growth in literature in this fastevolving academic domain lack empirical support. as an example, in one of the recent reviews, pattnaik et al. ( ) provided a comprehensive overview of the literal corpus on trade credit covered in top finance and economics journals between and february, . however, the study misses out the basic multi-disciplinary nature of the research domain. therefore, views from other important disciplines e.g., industrial engineering, production and operations etc. excluded in the former are included in this endeavor. alternatively, while the former evaluates only articles covered in web of science (wos); this work analyzes , works of research available in scopus. in addition, both the studies also differ in their analytical outfits. the primary concern of the first review is on network analysis and therefore exhibits limitations in the descriptive front. however, this endeavor is rich in both descriptive, network, and predictive contents of the multidisciplinary research domain. j o u r n a l p r e -p r o o f clustering exposes their semantic (dis)association. further, we also explore the thematic components and introspect on the extant literature to provide some future research directions. in addition, we also identify a number of factors influencing the quantum of citations, thus indicating a growth of trade credit research. therefore, apart from the de novo researchers, such a detailed introspection may also inform the established academics in the field about theories which require future validation and the study methods they could deploy. moreover, this study contributes significantly to the extant research by asking the following research questions (rqs): rq . what is the state-of-the-art of the research front-in terms of publications, authorship and citation structure, influence, impact, activity, and productivity of its contributing authors and publishing sources-and how does it vary in the pre and post crisis era of ? rq . what is the thematic layout of the research domain? rq . how do the extant literature, its prolific contributors, publishing sources, and highly cited references converge intellectually? rq . what factors significantly contribute to the growth of the research domain? rq . what is the direction for actionable research in future? in the remaining sections, we summarise the research methods, discuss the study outcomes, provide key summaries, and conclude the paper. (insert table about here) trade credit-in form of receivables-is a financing provision by non-financing firms (garcía-teruel & martínez-solano, ) with or without bank intermediation. in the context of a complex and competitive global market, credit supply is inevitable to retain b b relations and impart business growth (chowdhury & lang, ) while financing supply chain is must for obtaining competitive advantage (pirttilä et al., ) . enhanced fluctuations in the level of industrial production backed by disruptive technologies significantly distress trade credit policies both for the upstream and downstream supply chain. the nature of the product, fluctuations in market demand and the level of hostility in the business environment further impact its demand and supply. buyers benefit from the time value of money by increasing their demand for credit supplies while suppliers, on the other hand, register a growth in sales (schwartz, ) . simultaneously, empirical evidences assert the influential role of a number of qualitative indicators such as the national culture (el ghoul and zheng, ) , the cultural background of finance managers (bedendo et al. ) , and level of social trust (levine et al. ) impacting the demand and supply of trade credit. demand for trade credit is also influenced by the level of financial market development and financing constraints. more importantly, trade credit substitutes bank credit during a crisis (bastos & pindado, ) and therefore is a viable source of finance both in the developed and developing world (mcmillan & woodruff, ; afrifa & gyapong, ; li et al., ) . suppliers' credit played a crucial role in the survival of smes during the crisis. empirical evidence substantiates the argument that trade credit reduces the likelihood of financial distress among smes (mcguiness et al. ) . simultaneously, it impacts the production cycles, optimal ordering quantity, level of inventory, firm performance, and industry growth (haley & higgins, j o u r n a l p r e -p r o o f ; barth et al., ; huang, huang, , huang & hsu, ; fisman & love, ; chung & huang, ; chung et al., ; liao, ; allen et al. ). in the chronological progression of the research domain, the intertwined financing and accounting perspectives of trade credit shows signs of separation. few notable thinkers such as brick and fung ( ) proposed the tax theory of trade credit, hypothesising that businesses offer trade credit to obtain tax benefit. other themes explored in this area include revenue management and budgetary participation among others. thus, the literature analysed exhibit three broader academic domains: first, the banking and financing aspects, and second, the production and operational dimensions, and third the accounting perspectives of trade credit. in order to delineate the potential factors contributing to the popularity of trade credit literature in academia this study applies both quantitative and qualitative tools. in the field of scientometrics citations are predominantly considered to be the indicators of quantity versus that of quality (aksnes et al. ). on the basis of citations, hirsch ( ) proposed the "h-index" while egghe ( ) proposed the "g-index". both hirsch and egghe provide authorial performance indicators measuring the quantitative performance of authors. drawing from such prior evidence, we examined citation as a growth factor for a research domain as every new citation is the birth of a new publication. alternatively, it also adds to the influence and impact of the academic source cited, the specific theme cited, and contributes to the intellectual influence of its contributor(s). given the debatable qualitative indications of citations (aksnes et al. ), we present qualitative perspectives i.e. journal's academic reputation, an author's academic reputation, etc. alternatively, ball & tunger ( ) argue that growth in publications indicates a growing research area while acedo et al. ( ) and finardi & burati ( ) associate growth with authorial collaborations. other factors include an increase in global submissions and publications in prestigious academic outlets (merigó et al., ; baker et al. ) , etc. alongside the hypothetical constructs, this study also applies some of the tested factors affecting citations (growth in publications) by drawing upon schwert's work ( ) . by combining all the theoretical perspectives, the conceptual model of our study is proposed in figure . during academic emergency or external threats to industry (environment)-established research domains grow faster. growth of the broader academic domain is also due to the upsurge in research activities of the interrelated disciplines. in such circumstances, published articles in reputed journals positively affect growth. the growing inter-disciplinary variable mediates between the primary established academic domain and the discipline's overall growth. in other words, by drawing wisdom from the (reputed) publications and the established academic domain, the growing inter-disciplinary sub-domain(s) expand(s) the broader horizon of knowledge further. j o u r n a l p r e -p r o o f data analysed in this study was furnished from scopus. it is one of the largest databases (bartol et al., ) of peer-reviewed works in social science (norris & oppenheim, ) , extensively accessed for quantitative analyses (durán-sánchez et al., ; baker et al. ; donthu et al. ) . figure summarises the study design. (insert figure about here) to carry out the investigation, a broad search term was finalised. the term, "trade credit" or "account* receivable*" or "account* payable*", searched in the title, abstract or keywords enabled access to the extant literature in scopus. redundant documents were eliminated using subject filters. literature confined to business management, accounting, economics, econometrics, finance, social sciences, arts and (or) humanities were included. since our focus was accessing the core bibliographic records on trade credit; only those documents classified as articles, reviews, or conference proceedings were included and the rest excluded. further, due to the dynamic nature of the research domain literature published in is omitted. finally, a total of , documents published between and were considered for analyses. the descriptive analysis in this study was conducted manually using microsoft (ms) excel under the four broad categories of: . publication trend. . authorship pattern. . citation structure. . the influence, impact, activity, and productivity indicators (donthu et al. ; baker et al. ) . refer appendix i for definitions of the descriptive variables. j o u r n a l p r e -p r o o f the study explores the thematic factors of the research domain using principal component analysis (pca) with varimax rotation under kaiser normalisation. it was conducted with the help of the statistical packages for the social sciences (spss) software. the core of such an analysis is based on the co-occurrence counts of the keywords indicating themes (callon et al. ; marrone, ) . ponzi ( ) applied a similar technique to explore the intellectual structure among frequently co-occurring authors. to carry out the analysis, prolific themes appearing at least five times in the shortlisted articles are identified. using ms-excel, the co-occurrence matrix of those themes was later processed in spss to obtain the pearson's correlations. finally, we used the correlations' matrix to explore the thematic components. when two distinct works cite one or more common documents, they exhibit intellectual similarities (kessler, ) ; whereas, co-citation is the citation of two or more existing researches in a later article (small, ) . the network analyses in the forms of bibliographic couplings (kessler, ) and co-citations (small, ) unveil the semantic clustering among the citing and cited documents, contributing authors, publishing sources, etc. (merigó et al., ; donthu et al. ; baker et al. ) . the networks in this study are visualized using vosviewer and gephi software (van eck & waltman, ; van eck & waltman, ; bastian et al., ) . both the programs apply two standardized weights for visualising the networks e.g. the total number of links and the total strength of the links. the size of the nodes in a network indicates its relevance; whereas, the j o u r n a l p r e -p r o o f number and size of the interlinking lines represent the strength of the association among the nodes (van eck & waltman, ). we apply the ordinary least square (ols) regression in spss to explore the potential growth factors of the research domain (dragos et al. ) . drawing primarily from previous research (schwert, ) , the following model is proposed: total citations = article length + number of contributing authors + authorship type (sole-authored or co-authored) + publication year (before or after ) + ajg ratings + bibliographic cluster of the article (major or minor) + study method (primary or secondary) + thematic components' score + error (eq. ) the dependent variable, total citations, is defined as the total quantitative growth of the research domain. article length-defined as the number of pages of the article. number of contributing authors-defined as the total number of authors who contributed to the article. thematic score-respective thematic component scores of the articles. dummy variables include, authorship type (sole-authored or co-authored), publication year ( figure shows the yearly growth in publications, its influence (h-index), and the average annual citations of the articles published between and . figure depicts the annual trend of the intelligentsia, while figure distributes the trade credit articles by its number of contributors. it also compares the authorial distribution of the articles before and after . (insert table figure depicts an exponential growth in publications since . as indicated in table , about % (tp: of , ) of the study articles are published after . thus, the majority of the research on trade credit followed the global economic crisis of . such a trend is not surprising as-during the collapsing phase of banking-academia largely proclaimed trade credit as the primary source of alternate finance that sustained the global economy (giannetti et al. ; chor and manova, ) . conversely, as the demand was non-decreasing post , the proposition of the economic order quantity model by teng et al. ( ) attracted further research attention (see table ). not only did academia recognise trade credit as a major source of finance over these years, trade credit also managed to establish itself as one of the predominant factors influencing factors like economic order quantity (eoq), economic production quantity (epq), the shifts in the volume of production, etc. from another perspective, an evolving research domain triggers further research attention by attracting and engaging more scholars (ball & tunger, ) . the notion is evidentially affirmed in figure . the figure depicts an increasing trend in the number of thinkers. , unique researchers have contributed to the domain between and (see table in conclusion, the protective mechanism of trade credit as an alternative source of finance during banking emergency has historically triggered academic attention between and . with the pandemic onset of covid- shutting down operations, we foresee a similar trend in the production and operational area of trade credit research in the near future. table presents the top articles published before and after . (insert table interestingly, the top three highly cited works exhibit convergence in their respective philosophical outlooks. petersen and rajan ( ) predominantly present the banking and financing views of trade credit research while both dechow et al. ( ) and barth et al. ( ) navigate the broader finance and accounting dimensions. however, the model proposed in the note by huang ( ) modified the production and operational dimensions concerning the trade j o u r n a l p r e -p r o o f credit policies of retailers. while earlier notions predominantly argued against the extension of credit period by retailers, huang ( ) proposed that like suppliers, retailers stimulate their demand by extending the credit period to their customers. thus, the highly cited articles, depicting the existing knowledge in the established academic domain, presumably influence their respective research sub-domains. however, a deeper investigation of the highly cited articles reveal that the accounting dimension of trade credit is dormant without any significant contribution in the post-crisis era thereby presenting scope for future research. in the subsequent discussion, we recognize some of the most prolific and influential thinkers in the broader domain. table lists some of the most prolific authors who have contributed at least five works of research between and . (insert table (insert table table enlists the prominent themes of the extant literature on trade credit which have been discussed in at least articles. (insert table table presents the communalities of the thematic items. table shows the loading of the thematic items to their respective components and also presents the reliability of the thematic scale. (insert table about here) (insert table (kessler, ) while small ( ) argued that frequent co-citations of published articles in the subsequent research evidentially confirms intellectual association. unfortunately, the argument of small ( ) may suffer a time lag effect as more recent publications require time to influence academic evidence in the form of citations (marrone, ) . since our study incorporates articles till , we applied bibliometric coupling analysis on the trade credit articles, authors, and publishing sources. however, to expose the prior seminal ideas, we applied co-citation analysis on the cited references as explained by small ( ) . table presents the descriptive analyses of the bibliographic clusters, figure reveals the publication trend of the clusters in the pre and post crisis era, while figure shows the kind of studies that prevailed within the respective bibliographic clusters. (insert table further, as indicated in figure , most of the articles are empirical investigations while the cluster contributes a smaller number of qualitative researches such as conceptual papers, reviews etc., thereby suggesting few methodological gaps for the aspiring contributors to fulfil. of note is that some of the influential themes of the cluster include terms such as firm value, corporate social responsibility, earnings and cash flows, prediction of future cash flows, etc. some of its recent and original publications draw upon transactions cost theory; for eg. the third cluster is the most competitive cluster of the research front occupying the topmost rank in the majority of the indicators reported in table (tp: the supplier-retailer's relation mediate the above proposition. we argue that apart from establishing the credit quality of the new buyers, the incremental increase of credit supply with age may naturally eliminate the risk of free-riding. cluster most of the latest articles published in the cluster work in the area of green supply chains, operational management, optimal ordering quantity, optimal wholesale price, optimal carbon j o u r n a l p r e -p r o o f emissions, etc. as an example, the amalgamative supply chain operations model of dash ( ) addressing the concern of optimal carbon emissions (oce), optimal ordering quantity (ooq) of a capital-constraint retailer, and the optimal wholesale price (owp) for a manufacturer operating in a viable trade credit financing and bank financing market environment proposed and validated that lower levels of carbon emissions fosters win-win outcomes. future empirical validations of such models can sensitise controlled carbon emissive behaviour as it positively impacts profitability. it can be concluded that the bibliographic coupling analysis affirms three major subdomains of trade credit (banking and financing, accounting, and production and operations) gradually expanding to eight specialized sub-domains. the following section visualizes the intellectual epicentres of trade credit research, and the network among the most prolific authors and contributing sources. schwartz ( ) . interestingly, excluding goyal ( ) , aggarwal and jaggi ( ) , and teng ( ) all the remaining articles are closely knitted. it suggests that the operational dimension of trade credit stand out from the remaining perspectives. interestingly, figure also presents three broad groups of intellectuals. however, x. chen and d. yazdanfar stand out in their referencing pattern exhibiting similarities with n. h. shah and l. -y. ouyang, respectively. in figure , the majority of the journals present the financial perspectives of trade credit in the subsequent section we report the outcome of the regression analysis revealing some key variables influencing the total citations of the discipline over the years. j o u r n a l p r e -p r o o f table presents the description of the studied variables, table presents the correlation matrix of the studied variables while table presents the regression coefficients of the variants. (insert table about here) (insert table about here) (insert table although the directions for future research are already presented during the analysis of the bibliographic networks, we summarise some of the key aspects here. while analysing the gaps in research methods, we suggest future research to be more qualitative in nature. researchers should consider providing more conceptual and theoretical models, specialized pathways, survey-based studies etc. in the emerging clusters to fortify the domains. simultaneously, we call for more studies with empirical insights from emerging economies that would educate global academia which has largely observed this phenomenon simultaneously , paul et. al. ( ) presented receivables as an alternative investment. however, the social nature of trade credit is unexplored. given its importance, we argue that trade credit is not only commercial, it also has social implications, especially when the relation is between the large-scale lender to the micro, small, and medium enterprises (msmes). further, drawing from the model of dash ( ), future research could also explore the impact of green trade credit on firm's accounting, market, and social performances. conversely the behavioral dimensions in the research front is unexplored. with regard to the application of theories in this area, we suggest that there are opportunities to carry out researches using theories such as the prospect theory and dynamic capability theory. prospect theory was originally developed by economists kahneman and tversky ( ) . it constitutes one of the first economic theories formulated using experimental methods. prospect describes how individuals asymmetrically assess their loss and gain perspectives relative to their specific situation. they found that individuals are risk-averse when facing gains but risk-lovers when facing losses. accordingly, the prospect theory describes the actual behavior of people. in the original formulation of the theory, the term prospect referred to the predictable results of a lottery. however, the prospect theory can also be applied to the prediction of other forms of behaviors and decisions in areas such as trade credit. similarly, tenets of dynamic capability framework (teece, ) can be applied in trade credit research to analyze how the firm capabilities and trade credit influence firm performance. the state-of-the-art in trade credit literature indicates an evolving growth trend. a growing discipline attracts more researchers from multiple disciplines resulting in synergetic ( ), - . https://doi.org/ . /j. - . .tb .x chung, k.-j., goyal, s. k., & huang, y.-f. ( . the optimal inventory policies under permissible delay in payments depending on the ordering quantity. international journal of production economics, ( ) financial and economic perspectives notes: this table presents some of the former reviews on trade credit. it includes the author(s) of the study, type of study (tos), the study method, study period, number of articles analysed (na) and the primary focus of the study. nd stands for not defined period/articles. this table presents the publication here, iiap = influence, impact, activity, and productivity; tp = total publications; b = number of publications before ; a = number of publications after ; nca = number of contributing authors; ci = collaboration index; sa = number of sole-authored articles; ca = number of co-authored articles; pcp = proportion of cited publications; c/ca = citations per contributing author; ct = first citation threshold i.e. between and cites; ct = second citation threshold i.e. between and cites; h = h-index; g = g-index; nay = number of active years; and pay = productivity per active year. here, iiap = influence, impact, activity, and productivity; tp = total publications; nca = number of contributing authors of those publications; ci = collaboration index; sa = sole-authored articles; ca = co-authored articles; pcp = proportion of cited publication; tc = total citations; c/cp = citations per cited publication; c/ca = citations per contributing author; ct = first citation threshold i.e. between and citations; ct = second citation threshold i.e. between and citations; ct = third citation threshold i.e. above citations; h = h-index; g = g-index; nay = number of active years; and pay = publications per active year; and na = not available. here, sd = standard deviation, n = number of cases, and na = not applicable. the dummies include: authorship type of the trade credit articles i.e. sole-authored or co-authored; publication year of the articles i.e. before or after ; ajg ratings of the publishing sources of the trade credit articles such as *, , , , or ; bibliographic clustering of the trade credit articles i.e. major or minor cluster; and research type of the trade credit articles i.e. primary (empirical) or secondary (review, conceptual, model building, etc.). apart from reporting the standardized coefficients (sc) of the independent variables influencing the dependent (total citations), the table also presents the collinearity statistics (cs) such as tolerance (t) and the variance inflation factors (vif) of the regressors. of note, the r of the model is . with an adjusted r value of . . the regression is significant at % confidence interval (p-value ≤ . ). here, t = tolerance; and vif = variance inflation factor. citations, citation indicators, and research quality: an overview of basic concepts and theories understanding informal financing twenty-five years of the journal of corporate finance: a scientometric analysis bibliometric analysis -a new business area for information professionals in libraries? accruals and the prediction of future cash flows assessment of research fields in scopus and web of science in the view of national research evaluation in slovenia gephi: an open source software for exploring and manipulating networks trade credit during a financial crisis: a panel data analysis fuzzy decision making: a bibliometric-based review cultural preferences and firm financing choices taxes and the theory of trade debt from translations to problematic networks: an introduction to co-word analysis turnaround in small firms: an assessment of efficiency strategies off the cliff and back? credit conditions and international trade during the global financial crisis determinants of trade credit: a comparative study of european smes what you sell is what you lend? explaining trade credit contracts stem education: a bibliometric overview a bibliometric study of reference literature in the sciences and social sciences economic order quantity under conditions of permissible delay in payments partial least squares: the better approach to structural equation modeling? long range planning inventory policy and trade credit financing science studies: an advanced introduction an index to quantify an individual's scientific research output optimal retailer's ordering policies in the eoq model under trade credit financing optimal retailer's replenishment decisions in the epq model under two levels of trade credit policy an eoq model under retailer partial trade credit policy in supply chain prospect theory: an analysis of decision under risk analysing entrepreneurship education: a bibliometric survey pattern bibliographic coupling between scientific papers corporate resilience to banking crises: the roles of trust and trade credit legal system and trade credit: evidence from emerging economies. emerging markets finance and trade an eoq model with noninstantaneous receipt and exponentially deteriorating items under two-level trade credit application of entity linking to identify research fronts and trends european trade credit use and sme survival interfirm relationships and informal credit in vietnam research in production and operations management: a university-based bibliometric analysis assessing the usefulness of bibliometric indicators for the humanities and the social and behavioural sciences: a comparative study comparing alternatives to the web of science for coverage of the social sciences' literature qualitative research in financial markets, ahead-of-print(ahead-of-print the secret life of uk trade credit supply: setting a new research agenda trade credit: theories and evidence working capital management in the russian automotive industry supply chain scholarly influence in the field of management: a bibliometric analysis of the determinants of university and author impact in the management literature in the past quarter century the intellectual structure and interdisciplinary breadth of knowledge management: a bibliometric study of its early stage of development statistical bibliography or bibliometrics an economic model of trade credit co-citation in the scientific literature: a new measure of the relationship between two documents explicating dynamic capabilities: the nature and microfoundations of (sustainable) enterprise performance. strategic management journal on the economic order quantity under conditions of permissible delay in payments economic order quantity model with trade credit financing for non-decreasing demand citation analysis of ted nelson's works and his influence on hypertext concept software survey: vosviewer, a computer program for bibliometric mapping vosviewer manual using payment behaviour data for credit risk modelling supply chain finance: a systematic literature review and bibliometric analysis financial intermediary development, and industry growth" fisman r., love i. bank credit: evidence from recent financial crises sarria-allende v. in-kind finance: a theory of trade credit bank discrimination in transition economies: ideology, information, or incentives are accruals during initial public offerings opportunistic an eoq model with noninstantaneous receipt and exponentially deteriorating items under two-level trade credit" liao j.-j. multi-delay-in-payments under single-setup-multiple-delivery policy in a global sustainable supply chain chain finance: a systematic literature review and bibliometric analysis international trade during the - crisis: in search of the smoking gun ranking (r) of the articles are based on their respective average citations per year (cpy). here, tc = total citations, and c/ca = citations per contributing author notes: this table presents the pearson's correlation among the potential variables influencing the citations of trade credit articles = type of the trade credit research (empirical or secondary); = empirical research defined as the sum of total publications b defined as the number of publications before a defined as the number of publications after authorship pattern nca number of authors contributing the research article(s). annual increment of authors added to the research domain. ratio between the number of contributing authors to total publications less the number of total publications ( − ). number of articles contributed by a single author. number of articles contributed by multiple authors. the number of articles cited at least once in scopus. ratio between the number of cited publications to the total number of publications. sum of the citations accredited to an article, an author, a journal, a cluster, etc. c/p average citations per publication. average citations per cited publication. average citations per contributing author. citations between and times. citations between and times. ct citations and above. influence, impact, productivity, and activity h h number of publications cited at least h times. gsum of g number of highly cited publications cited at least g times. number of years an article or a theme on trade credit was published or the number of years an academic source &/or a cluster published on trade credit. number of publications in each of the active years. key: cord- -il gs authors: jayapal, manikandan; tay, hwee kee; reghunathan, renji; zhi, liang; chow, kah kiong; rauff, mary; melendez, alirio j title: genome-wide gene expression profiling of human mast cells stimulated by ige or fcεri-aggregation reveals a complex network of genes involved in inflammatory responses date: - - journal: bmc genomics doi: . / - - - sha: doc_id: cord_uid: il gs background: mast cells are well established effectors of ige-triggered allergic reactions and immune responses to parasitic infections. recent studies indicate that mast cells may play roles in adaptive and innate immunity, suggesting an innovative view of the regulation of immune responses. here, we profiled the transcriptome of human mast cells sensitized with ige alone, or stimulated by fcεri aggregation. results: our data show that among , genes examined, genes are differentially regulated in stimulated mast cells when compared with resting/unstimulated mast cells. the major functional categories of upregulated genes include cytokines, chemokines, and other genes involved in innate and adaptive immune-responses. we observed the increased expression of over gene-transcripts following ige-sensitization alone. our data was validated using real-time-pcr; elisa and western blot. we confirmed that ige alone does not trigger mast cell-immediate responses, such as calcium signals, degranulation or protein-phosphorylation. conclusion: this report represents a substantial advance in our understanding of the genome wide effects triggered by "passive sensitization" or active stimulation of human mast cells, supporting mast cells' potential involvement in a wide range of inflammatory responses. mast cells are best known for their role in immunoglobulin e (ige)-dependent allergic responses as one of the most powerful reactions of the immune system [ ] . recent studies suggest that mast cells may also be involved in innate and adaptive immunity by producing high levels of chemokines and cytokines [ ] [ ] [ ] . mast cells are derived from haematopoietic progenitor cells that enter nearly all vascularized tissues, where they complete their maturation and, in some cases, can migrate into epithelia [ ] [ ] [ ] [ ] . after appropriate activation, such as the aggregation of the high affinity ige receptor (fcεri), mast cells can produce a range of pro-inflammatory mediators, including cytokines and chemokines [ , ] . crosslinking of fcεri-bound ige with multivalent antigen initiates the activation of mast cells by promoting the aggregation of fcεri [ , ] . this fcεri-dependent cell activation process has three major outcomes: degranulation, secretion of preformed mediators stored in cytoplasmic granules -these granules contain vasoactive amines, neutral proteases, proteoglycans and some cytokines and growth factors; the de novo synthesis of pro-inflammatory lipid mediators (such as eicosanoids); and the synthesis and secretion of cytokines and chemokines [ ] [ ] [ ] [ ] [ ] . mast cells are regarded as key effector cells in ige-associated immediate hypersensitivity reactions and allergic conditions, as well as in certain immune responses to parasites [ ] . because the ige dependent release of proinflammatory mediators can begin within minutes of antigen challenge, the crucial role of mast cells in acute allergic reactions, such as anaphylaxis and acute attacks of atopic asthma, is now widely accepted [ , , , ] . on the other hand, the role of mast cells in chronic inflammation and other long-term tissue changes that are observed in some ige associated diseases, including asthma, still debated [ ] . however, in recent studies it is suggested that mast cells can markedly enhance antigen-dependent airway hyper reactivity, airway eosinophil infiltration, and the increased number of proliferating cells in the airway epithelium [ ] [ ] [ ] [ ] . these reports and other lines of evidence, suggest that a key role of mast cells in igeassociated immune responses is to amplify both acute and long-term local tissue responses to relatively weak biological signals [ , , , ] . moreover, through their ability to release immunoregulatory cytokines, and perhaps through other mechanisms, mast cells might also influence the development, strength and persistence of thelper cell-associated immunity. however, such immunoregulatory functions of mast cells have not been fully characterized. mast cells have also been proposed to play a role in mediating bacterial clearance by releasing cytokines, and by ingesting and killing opsonized bacteria, suggesting that mast cells have a physiological role in modulating host defenses against infectious agents [ ] . mast cell products, such as tryptase and histamine, can influence the immune-response by recruiting neutrophils or by activating other immune-effector cells [ ] . these previous findings show that mast cells, apart from being principal players in allergies, appear have effects in the initiation and regulation of innate immunity. there is increasing evidence demonstrating that the innate and adaptive immune systems cooperate through shared signaling mechanisms, and very recently it was shown that mast cells may regulate the recruitment of t-cells to lymph nodes [ ] . several groups have carried out limited gene expression profiles of mast cells, without any stimuli [ ] [ ] [ ] ; stimulated by cytokines [ ] ; triggered by antigens and in response to the inhibition of calcium-atpase [ ] ; or stimulated by tlr [ ] ; or focused on the cytokine/ chemokine profile triggered by fcεri aggregation [ ] [ ] [ ] . a recent study has shown that ige alone can induce the release of the interlukin- (il- ) and of the monocyte chemoattractant protein (mcp- ) [ ] . however, many aspects of the genetic responses triggered by ige-antigen on human mast cells are still unclear, such as the overall genetic responses to ige sensitization alone, or widegenome expression profile triggered by fcεri aggregation. thus, we decided to analyze the differential gene expression profile of , fully-annotated genes, after ige sensitization, and following fcεri aggregation. the data we present here show that, over genes were differentially regulated in the stimulated cells, when compared to unstimulated cells (basal). a substantial number of genes were regulated by ige sensitization alone; and following fcεri aggregation, a wide range of genes were triggered, including genes for cytokines, chemokines, transcription factors, anti-apoptosis, and several genes involved in innate and acquired immunity. some of the most prominent findings are the upregulation of proinflammatory cytokines and chemokines, involved in innate and adaptive immunity. we also observed the upregulation of several receptors involved in innate immune reactions. we confirmed that ige alone does not trigger mast cell-immediate responses, such as calcium signals, degranulation or protein-tyrosine phosphorylation, whereas fcεri aggregation did indeed trigger these immediate responses. thus, these results represent a substantial advance in our understanding of the genomewide effects triggered by the physiological "passive sensitization" or "active stimulation" of human mast cells, and suggest that mast cell activation may not only play a pivotal role in allergic responses, but may influence the regulation of other inflammatory immune responses. perhaps the most important factor for mast cell development, survival and proliferation is stem-cell factor (scf or c-kit ligand) [ ] , although several lines of evidence also indicate il- as a crucial factor for mast cell development, survival and proliferation [ ] . human mast cells are very difficult to isolate in numbers that would allow for wide-genome expression profiling studies: for that reason we isolated cd + progenitor hematopoietic cells from umbilical cord blood and differentiated them to mast cells by culturing with the cytokines scf and il- . after culturing cd + progenitor cells with cytokines for - weeks, cells were characterized as mast cells by flow cytometry as positive for the specific mast cell marker intracellular chymase; positive c-kit/cd + and fcεri + , and by toluidine blue staining ( figure ). purity was estimated at > %. our aim is to study the global gene expression pattern induced by ige sensitization and fcεri aggregation on human mast cells. gene expression analysis using cdna or oligo-dna microarrays has proven to be a sensitive method to develop and refine the molecular determinants of several human disorders, including cancer and autoimmune diseases. we analyzed the expression pattern of , transcripts from the stimulated mast cells, and compared the expression patterns with control/unstimulated samples. the complete gene expression data of our experiments, representing , probeid is available at the ncbis gene expression omnibus [ ] , and is accessible through geo series accession number gse ( ) . the microarray analysis revealed that genes (~ . %) were differentially expressed between resting and stimulated mast cells with statistical significance (p ≤ . ), which were hierarchically clustered ( figure ). because of the relatively large number of genes that were differentially regulated, we focused on genes that were upregulated by at least a -fold in any time point of mast cell stimulation. of the genes, genes were initially upregulated (at least -fold) by ige-sensitization alone (table. ), and a total of genes were overexpressed (by -fold or more in at least one time point), during the time course of mast cell activation by ige-alone or after crosslinking fcεri (table. ). in order to examine the global characteristics of these genes, we used the gene ontology consortium database for biological processes [ ] . using this database we analyzed the genes that were upregulated by at least fold, thus, allowing us to separate the genes, into the following functional families: (a) cytokines and cytokine receptors; (b) chemokines and chemokine receptors; (c) other immunoregulatory genes; (d) cell proliferation and anti-apoptosis; (e) adhesion and cytoskeleton remodeling; (f) transcription factors and regulators of transcription; (g) signal transduction; (h) genes involved in other cellular functions "others" (table & figure ) our study revealed that substantial changes in gene expression in response to monomeric-ige sensitization alone. in order to ensure that the human ige (ige, cat: -ai , lot number a , fitzgerald, concord, ma), used in this study was indeed monomeric ige, prior to each experiment; we run a sample of the ige through nondenaturing polyachrylamide-gel-electrophosys (nonreducing-page). no aggregates were observed at any time (data not shown). we focused the analysis of our data on genes that were upregulated by at least -fold, over basal levels, and identified genes that were increased by ige sensitization alone (table. ). we then separated them into different categories, based on their biological function (determined by public databases). among the most prominent findings was the upregulation of genes coding for the cytokines il- β ( . fold), il- ( . fold), and csf ( . fold); genes coding for the chemokines il- (cxcl ) ( . fold), mip β (ccl ) ( . fold), mcp (ccl ) ( . fold), groα (cxcl ) ( . fold) and groγ (cxcl ) ( . fold), were also upregulated. other than these, several genes coding for other receptors involved in immune-responses; immunoregulatory genes; adhesion and/or cytoskeleton remodeling; regulators of apoptosis; signal transduction; transcription factors; were also upregulated by monomeric ige (table. ). thus, these results suggest that "passive" sensitization of mast cells, with monomeric ige, may not only prime mast cells to be ready for the challenge to come, but that mast cells may also have the potential to purity of mast cells an interesting finding in our study was upregulation of genes coding for the pyrogenic and proinflammatory cytokines il- β, il- ( table. a). one of the key roles for il- β is to trigger the upregulation of key proinflammatory proteins [ ] . we also observed the upregulation of ptx (pentaxin-related gene) ( table. c): ptx is a protein involved in inflammation that is rapidly upregulated by il- β. other cytokine genes that were upregulated are csf (a growth promoting cytokine), and the cytokine receptors il r , il ra, tnfrsf , tnfrsf and il rn (table. a). we also found that the level of expression of genes coding for the chemokines il- (cxcl ), ccl (mcp ), ccl (mip β) and cxcl (groα) was increased (table b) . il- plays a major role in inflammatory responses mainly due to its ability to recruit and activate neutrophils. ccl , ccl and cxcl are known to recruit monocytes, nk, basophils, dendritic cells and th cells. moreover, our data also show that several other chemokine genes were upregulated during mast cell stimulation: these include ccl (rantes), cxcl (groγ), and the chemokine receptor ccrl (table. b). these data suggest a key role for fcεri in triggering an a wide range of inflammatory responses, as the over-expression of cytokines and chemokines is a prerequisite to triggering inflammation, including vascular permeability, and leukocyte and lymphocyte recruitment, differentiation and activation. several genes involved in innate and adaptive immuneresponse were upregulated at least by -fold during mast cell stimulation: these include the toll like receptor (tlr ), as well as several genes of the tnfα signaling pathways, including tnfaip (table. c). these findings support previous reports of the role of mast cells in innate immunity and antibacterial activity [ ] . transcripts of genes lif, cd , and cd were also upregulated, as were the major histocompatibility complex genes (hla-dqb , and hke ) and inhibitory receptor of igg, fcgr b (table. c). the generation of transcripts for such genes suggests that mast cells can acquire characteristics typical of cells involved in innate and adaptive immune responses. several genes involved in cell proliferation and survival, such as pdgfa (platelet-derived growth factor alpha polypeptide), pdgfb (platelet-derived growth factor beta polypeptide), pbef (pre-b-cell colony-enhancing factor), tieg (tgfb inducible early growth response), and insig (insulin induced gene ) were upregulated, as well as several anti-apoptosis genes including tnfaip changes in gene expression in human mast cells following ige sensitization and fcεri aggregation figure changes in gene expression in human mast cells following ige sensitization and fcεri aggregation. changes in expression over control of human cord blood derived mast cells that were activated by ige sensitization and fcεri crosslinking for different time points ( hr, hr and hr). labelled crna from cell of each time point were hybridized to human genome focus array and signals were scanned after fluidics. the data was analyzed as described in material and methods and analysis revealed differential expression of genes between resting and stimulated mast cells with statistical significance (p ≤ . ). agglomerative average-linkage hierarchical clustering of the five different experimental conditions was obtained for selected groups of genes using genespring . . each colored box represents the normalized expression level of a given gene in a particular experimental condition and is colored according to the color bar. the data represent average of four independent experiments. sensitised h h h fold change color bar table (table. d ). this supports the fact that, in the initial stages of mast cell activation, several mediators produced are mainly for cell proliferation and survival [ ] . thus, fcεri aggregation may enhance mast cell proliferation and survival, perhaps owing to the autocrine effects of the cytokines, growth factors, and antiapoptotic proteins, triggered by fcεri aggregation. nm_ . ccl chemokine (c-c motif) ligand . . . - . nm_ . cxcl chemokine (c-x-c motif) ligand . . . - . nm_ . ccl chemokine (c-c motif) ligand . . . . nm_ . cxcl chemokine (c-x-c motif) ligand . . . - . nm_ . ccl chemokine (c-c motif) ligand . . . . af ccrl chemokine (c-c motif) receptor- another functional characteristic of immune-cell activation is the coordinated expression of genes involved in cell adhesion and cytoskeleton remodeling (table. e). of particular importance are the genes coding for proteins involved in cell motility, cytokinesis, endocytosis and exocytosis. we found at least -fold upregulation of various genes coding for proteins involved in cell adhesion, such as flrt (fibronectin leucine rich transmembrane protein ), kal (kallmann syndrome sequence), cd (cd antigen), and alcam (activated leukocyte cell adhesion molecule); as well as for several gene-transcripts involved in cytoskeleton remodeling, including rasal (ras protein activator like ), arhe (ras homolog gene family, member e), arf (adp-ribosylation factor ), and flnb (filamin b, β-actin binding protein ) (table. e). the expression of genes involved in cell adhesion and cytoskeleton remodeling is an essential step in immunecell activation. resting immune cells have cytoskeletal structures that sequester antigen, chemokine, and adhesion receptors in accessible regions of the plasma membrane. upon activation, reorganization of the actin cytoskeleton leads to the formation of supramolecular activation clusters, bringing receptors and costimulatory molecules together, as well as important adaptor proteins that promote the sustained activation of the cell. stimulation of immune-effector cells through their antigen receptors initiates cell cycle entry and changes the gene expression pattern, a response generally referred to as "activation". we found that the genes for several transcription factors were upregulated during ige-sensitization and fcεri aggregation, including the transcription factors most active during an immune response, such as nfκb and nfat (table. f). we observed an increase in the transcripts for the nuclear factor of kappa light polypeptide genes , alpha, and epsilon (nfκb , a, and e), and pie chart showing the percentage distribution of the upregulated genes figure pie chart showing the percentage distribution of the upregulated genes. a. percentage distribution of the total amount of genes upregulated. all the genes, observed to be upregulated at least -fold at any given time point, were distributed, according to their biological function described in b the nuclear factor of activated t cells, nfatc (table. f ). other transcription factors upregulated were the oncogenes myc (v-myc myelo-cytomatosis viral oncogene homolog), and maff (v-maf musculo-aponeurotic fibrosarcoma oncogene homolog f) ( table. f). interestingly, the activities of nfκb and nfat together are responsible for the transcription of many proinflammatory genes, including several genes coding for cytokines and chemokines [ , ] . during mast cell activation, many signaling molecules are engaged in diverse responses, ranging from calcium release from internal stores, degranulation, the generation of lipid-derived proinflammatory mediators and the production of cytokines and chemokines. in our study we observed that a substantial number of genes coding for intracellular signaling proteins were upregulated, by at least -fold ( (table. g ). moreover, we show here the upregulation of genes that code for oxidized low density lipoprotein receptor (olr ), and for the low density lipoprotein receptor (ldlr) ( table. g), indicating a potential role for mast cells in cholesterol homeostasis. of particular interest is the upregulation of the gene coding for the lipid kinase, sphingosine kinase (sphk ) ( table. g). we and others have previously reported that sphk plays a critical role in the intracellular signaling pathways triggered by fcεri in mast cells [ , ] , and coordinates several physiological responses triggered by activated mast cells. we confirmed our microarrays findings by real time pcr on selected genes such as il- β il , il- , mcp , rantes and sphk , utilizing an aliquot of the same rna sample that was used for the microarray experiments ( figure ) . the results showed that the messenger rna for the selected genes follows a similar pattern of expression to that observed with the oligo-dna microarray experiment, thus confirming the results and the quality of the data obtained with the high-density microarrays. mast cell activation also results in the sustained de novo production of pro-inflammatory cytokines and chemokines both of which may contribute to the inflammation and pathology underlying allergic disease as well as in innate and acquired immunity. the amounts of these cytokines were measured by elisa and depicted in figure a . fcεri-triggered generation of il- β, il- , il- , ccl (mcp ) and ccl (rantes), whereas ige sensitization alone triggered smaller amounts of il- β and mcp , high amount of il- , and very less amounts of il- and rantes ( figure a ). we verified the differential expression of sphk by western blot analysis ( figure b ). its levels of expression were found to be consistent with that of microarray as well as real time pcr results. thus, these data together with real time pcr data validate microarray results. mast cell activation via fcεri triggers exocytosis of granules containing pre-formed inflammatory mediators in a tyrosine kinase and calcium dependent manner. here we studied, whether monomeric-ige alone, may activate fcεri intracellular signaling pathways, leading to physiological responses of mast cells, by analyzing the overall tyrosine phosphorylation; fluctuations in cytosolic ca + concentration; and degranulation by measuring β-hexosaminidase release (fig. a,b & c) . we show here that in our experimental setting monomeric-ige alone is not able to trigger any changes on the overall protein-tyrosine phosphorylation patterns compared with resting cells; nor was it able to trigger calcium release from internal stores; neither degranulation. on the other hand fcεri-aggregation did indeed trigger all these responses (fig. a,b & c ). binding of ige to fcεri enhances the cell surface expression of fcεri, as a result, its ability to promote the stabilization/accumulation of fcεri on the mast cell surface in the presence of continued basal levels of protein synthesis [ , ] . it is possible that most of the enhanced ige dependent functions that are observed after antigen or anti-ige-induced fcεri aggregation, in cells that have been sensitized with ige, are a consequence of the higher level of fcεri expression. however, a controversial question remains as to whether monomeric ige can also have more direct effects on mast cell functions. many studies over the years have shown no evidence that the binding of monomeric ige can induce detectable signaling or production of mediators by mast cells. however, some groups have reported that monomeric ige can enhance mast cell survival and trigger cytokine production [ , , ] . in con- con sen h h h trast, a recent study by matsuda et al [ ] , fail to find any ability of ige to enhance mast cell survival on withdrawal of scf. interestingly, the study by matsuda et al, also showed that ige sensitization alone can induce the upregulation of cytokines and chemokines at the protein level, namely il- and mcp [ ] . in agreement to this, we show that il- is induced by ige alone at the mrna as well as at the protein level (table , and figures and ) , in contrast we could not detect any significant increment on mcp- levels, we can speculate that this difference could be due to the different amounts ige used ( µg/ml vs . µg/ml). however, we also show the upregulation of various chemokines, including the mcp- -related protein mcp- , which was also upregulated by ige alone (table , and figures and ) . in our present study, we found that several genes related to proliferation were upregulated by ige alone (table ) ; however, whether these genes, if fully transcribed, may be able to trigger mast cell proliferation in the absence of scf is not known. the observation that ige alone can induce the upregulation of a substantial number of genes encoding for cytokines and chemokines, has profound implications in our understanding of the role of mast cell in inflammation. cytokines and chemokines share many activities, including the ability to induce fever and shock syndrome in animal models [ ] . cytokine and chemokine production are universal components of a wide range of disease states, including immune-complex-mediated conditions such as nephritis [ ] , arthritis [ ] , and acute graft rejec- [ ] . these data suggest a potential role for mast cells in triggering, or at least contributing to, strong inflammatory responses. in is also interesting to mention that, several genes encoding for transcription factors were upregulated by monomeric-ige and fcεri aggregation. perhaps the most prominent of these transcription factors, is nfκb. nfκb represents a family of related proteins which dimerize to form transactivating complexes [ ] . nfκb dimmers are sequestered in the cytoplasm by interaction with inhibitory proteins (the iκbs). various stimuli activate kinase signaling cascades that result in the phosphorylation and degradation of iκb, thereby releasing nfκb to translocate to the nucleus, where it activates transcription of target genes. many studies have emphasized the role of this transcription factor in regulating genes at critical points in immune-cell development and activation [ ] . many nfκb targets are antiapoptotic [ ] , which may explain the importance of the nfκb pathway in oncogenesis and resistance to chemotherapy [ , ] . during an immuneresponse several genes are triggered by the nfκb, these include genes coding for the various proinflammatory molecules, such as mips, il- β, il- , il- , tnfα, groα and other cytokines, chemokines and cell adhesion molecules icam, vcam and selectins [ ] . as immune cells progress through development and respond to antigenic challenge, they trigger signal transduction pathways that alter their cellular functions and the activity of transcription factors, changing their effector functions and their gene expression profiles. during mast cell activation, many signaling molecules are engaged in diverse responses, ranging from calcium release from internal stores, degranulation, the generation of lipidderived proinflammatory mediators and the production of cytokines and chemokines. in our study we observed that a substantial number of genes coding for intracellular signaling proteins were upregulated, by at least -fold, during mast cell stimulation (table. g). interestingly, we observed a substantial upregulation of the mrna for sphingosine kinase (sphk ), even by ige alone, this upregulation was also confirmed at the protein level. sphingosine kinases are novel enzymes that phosphorylate sphingosine (a membrane lipid), to generate the bioactive molecule sphingosine- -phosphate (spp), which is implicated in several inflammatory responses. we and others have previously reported that sphk plays a critical role in the intracellular signaling pathways triggered by fcεri in mast cells [ , ] , and coordinates several physiological responses triggered by activated mast cells. we showed that sphk is involved in the calcium signals triggered by fcεri aggregation in human mast cells, as well as playing a critical role for mast cell degranulation [ ] . previously, we reported a pivotal role for mast cell activation: tyrosine phosphorylation, calcium sig-nals, degranulation figure mast cell activation: tyrosine phosphorylation, calcium signals, degranulation. a. analysis of overall protein phosphorylation on tyrosine residues. upper panel; overall tyrosine-phosphorylation pattern was analysed in cell extracts from: control unstimulated cells (basal); cells treated with ige alone for (ige) for min; and after fcεri crosllinking for min (xlfcεri). lower panel; the blots were probed for α-tubulin (control for equal loading). results shown are representative of four separate experiments. b. levels of intracellular free calcium. intracellular calcium measurements of mast cells following addition of ige alone (ige); and following the addition of the anti-human ige, to igesensitized cells (xlfcεri), the intracellular calcium levels were analyzed in a continuous reading for the timesstated in the graph. results shown are the mean plus the standard deviation of triplicate measurements and are representative of four separate experiments. c. degranulation. b-hexosaminidase release was determined from control-unstimulated mast cells (basal); following monomeric-igesensitization for minutes (ige); and following fcεri aggregation by addition of the anti-human ige to sensitized cells for minutes (xlfcεri). results shown are the mean plus the standard deviation of triplicate measurements and are representative of four separate experiments. [ , ] , showing that sphk is key in triggering calcium release from internal stores, and the activation of the phagocyte nadph oxidase. moreover, very recently we demonstrated the role of sphk in inflammatory responses triggered by the anaphylatoxin c a in human neutrophil and macrophages. these responses include: calcium signals, degranulation, cytokine production and chemotaxis triggered by c a [ , ] . the observation that the gene encoding for sphk is activated during ige-sensitization of mast cells, coupled to the findings above may indicate a key role for sphk in mast cell triggered responses. the significance of this research supports the notion that, activation of mast cells appear to be linked to a wide range of pathologies, not only in allergies (as is widely recognized), but potentially in other inflammatory conditions. the method of global gene expression analysis using cdna or oligo-dna microarrays has proven to be a sensitive method to identify and define/redefine the molecular determinants of several human disorders, including cancer and autoimmune diseases, and has provided us with signatures of the immune response [ ] . using this technology, complemented with powerful analytical methods, we compared the gene expression profiles of human mast cells stimulated by ige sensitization, and from a series of time points of fcεri aggregation, with unstimulated/control human mast cells. whether changes in gene expression, under these conditions, are representative of a pathological state is not currently known. it is also not known whether ige/antigen and fcεri aggregation will trigger the same set of genes in an organism, where a number of events may be activating mast cells at the same time. however, taken together, our data brings us better insights into the molecular basis of mast cell activation, and provides meaningful information, regarding the mechanisms by which mast cell activation may contribute to the overall activation of the immune response, having clinical implication for improving not only allergic conditions but potentially other inflammatory diseases, where mast cells may play a role. this study is an attempt to elucidate the molecular mechanisms which mast cells undergo during "priming" ige sensitization and full activation by fcεri aggregation in a global perspective. in conclusion, our present study provides information that mast cells, by generating a broad range of cytokines and chemokines, may be a potent contributor of the immune response by recruiting and/or activating other immune-effector cells including the activation of lymphocytes that may, in turn, continue the spreading of the inflammatory response. moreover, changes in the gene expression pattern of transcription factors, intracellular signaling molecules, and cytoskeletal remodeling and anti-apoptosis pathways occur, which would also contribute to the amplification of the inflammatory response. mast cells are well established innate immune-effector cells, and there is mounting evidence to, at least, suggest that mast cells may contribute to the development of acquired immunity [ , ] , whether in host defense or in allergic or autoimmune diseases. it will be pivotal to define in more detail whether and under which circumstances mast cells may influence the development and/or magnitude of acquired immune responses. unless specifically stated all materials and reagents were purchased from sigma-aldrich (singapore). human umbilical cord blood (cb) samples were collected from normal full-term deliveries of informed individuals with formal consents, meeting the universality institutional review board guidelines, for research using human samples. cd + haematopoietic progenitor-cells were harvested using macs cell isolation kit (miltenyi biotec), following the manufacturer's instructions. the isolated cd + haematopoietic progenitor-cells were cultured for - -weeks in the presence of ng/ml of stem cell factor (scf cat: - , peprotec, rocky hill, nj), and for the first week this was supplemented with ng/ml of interleukin- (il- cat: - , peprotec, rocky hill, nj). cells were shown to be differentiated by staining them for specific mast cell markers as follows: for mast cells chymase, with an anti-human chymase mab (igg -mab , chemicon, temecula, ca), and fitc-conjugated secondary antibody (anti-mouse igg-fitc, sigma-aldrich, singapore); for c-kit with anti-human c-kit mouse monoclonal pe-conjugated (cat. no. ; clone yb .b , bd biosciences -pharmigen, singapore), isotype control anti-mouse ige-pe (cat. no. ; clone mopc- , bd biosciences -pharmigen, singapore); and for fcεri cell-surface expression with antihuman fcεri polyclonal (rabbit-igg-ab ; abcam, cambridge, uk) and fitc-conjugated secondary antibody (anti-rabbit igg-fitc, sigma-aldrich, singapore) also used as isotype control, and analyzed immediately using a coulter epics-elite esp flow cytometer (beckman, germany). purity was estimated at > %. the differentiated mast cells were plated in well plates and allowed to rest for hr. after differentiation, mast cells were plated in well plates and allowed to rest for hr. cells in all wells, except the control well, were sensitized with human monomeric ige ( µg/ml, ige, cat: -ai , lot number a , fitzgerald, concord, ma) overnight. fcεri aggregation was carried out by incubating the cells with monoclonal mouse-anti-human ige ( µg/ml, anti-human-ige, cat: mca , clone c , serotec, oxford, uk) at °c, for hr, hr and hr. rna was extracted from all the samples using the qiagen rneasy mini kit (qiagen, valencia, ca). integrity of rna was checked by formamide gel electrophoresis; quantification of rna was carried out by measuring the a nm . labeling and hybridization was carried out as previously described [ ] . briefly, µg of total rna from each sample was used to synthesize double stranded cdna using t -(dt ) oligonucleotide primer and superscript reverse transcriptase (invitrogen). the resultant cdna was purified and µg of purified cdna was labeled with biotin by transcription in vitro. the labeled crnas were, fragmented in the presence of metal ions and then hybridized to hg-focus array (affymetrix), following hybridization the gene chips were washed and stained after which the chips were scanned by gene array scanner (agilent technologies). data collection and analysis was carried out using micro-array suite . (mas) (affymetrix). the absolute data (signal intensity, detection call and detection p-value) were exported into genespring v . (silicon genetics, redwood city, ca, usa) software for analysis by parametric test based on cross gene error model (pcgem). the anova approach has been used to find differentially expressed genes (p < . ). the benjamini and hochberg false discovery rate multiple testing correction was applied. agglomerative average-linkage hierarchical clustering of the five different experimental were obtained for selected groups of genes with gene spring . software (silicon genetics, redwood city, ca, usa) using standard correlation as similarity matrix. real-pcr was performed, as previously described [ ] , using µg of total rna from the same samples used for microarray experiments. pcr was performed for transcripts of il- β (primers: for amplicon detection, the light cycler rna master sybr green kit (roche) was used as described by the manufacturer. pcrs were performed in a lightcycler ® instrument (roche) as follows: reverse transcription at °c for min, initial denaturation at °c for min; amplification for - cycles of denaturation ( °c, s, ramp rate °c/s), annealing (optimal temperature, s, ramp rate °c/s) and extension ( °c, product length [bp]/ s, ramp rate °c/s). a single online fluorescence reading for each sample was taken at the end of extension step. quantitative results were expressed by identification of the second derivative maximum points, which marked the cycles where the second derivatives of the fluorescence signal curves are at maximum. these points were expressed as fractional cycle numbers. then, these cycle numbers were plotted against the logarithm of the concentrations of serially -fold diluted standard samples to obtain a standard curve. the concentrations of unknown samples were calculated by extrapolation from this standard curve. positive sample specificity was confirmed by determining the melting curve ( °c, s, ramp rate °c/ s; °c, s, ramp rate °c/s; °c, s, ramp rate . °c/s, continuous measurement). supernatants from control cells, cells sensitized, and cells following fcεri aggregation, were collected and stored at - c until use. il- β, il- , il- , mcp- and rantes levels in the supernatants were evaluated using elisa (r&d systems inc., mn, usa) following the manufacturer's instructions. western blots were carried out as previously done [ ] . briefly, µg of lysate for each sample was resolved on % polyacrylamide gels (sds-page) under denaturing conditions and then transferred to . µm nitrocellulose membranes. for overall tyrosine phosphorytaion, the blots were probed using a specific monoclonal anti-phosphotyrosine primary antibody (p-tyr, sc- , santa cruz, ca, usa), and an anti-mouse hrp-conjugated secondary antibody (anti-mouse igg-hrp, a- , sigma). bands were visualized using the ecl western blotting detection system (amersham, singapore). for sphk expression, the blots were probed using a rabbit polyclonal anti-sphk primary antibody (anti-sphk , x p, exalpha, ma, usa), and hrp-conjugated secondary antibody (anti-rabbit igg-hrp, sc- , santa cruz, ca, usa). for loading control the blots were probed with a monoclonal anti α-tubulin (anti-α-tubulin, sc- , santa cruz, ca, usa), and an anti-mouse hrpconjugated secondary antibody (anti-mouse igg-hrp, a- , sigma). bands were visualized using the ecl western blotting detection system (amersham, singapore), and quantified by densitometry analysis. cytosolic calcium was measured as described previously [ ] . briefly, cells were loaded with µg/ml fura -am (molecular probes, leiden, the netherlands) in pbs, . mm ca + and % bsa. after removal of excess reagents by dilution and centrifugation (in pbs), the cells were resuspended in pbs containing . mm ca + and % bsa, for min; or in pbs containing . mm ca + , % bsa, and human-monomeric ige ( µg/ml) for sensitization, for min. after removal of excess ige by dilution and centrifugation (in pbs), the cells were resuspended in . mm ca + supplemented pbs and warmed to °c in the cuvette; unsensitazed cells were placed in the cuvette and cytosolic calcium was measured before and after the addition of monomeric ige. ige-sensitized cells were placed in the cuvette and fcεri was crosslinked by addition of mouse-anti-human ige ( µg/ml). fluorescence was measured at and nm. degranulation was measured using as previously described [ ] . briefly, an aliquot of cells was resuspended in pbs containing . mm ca + and % bsa, and incubated with monomeric ige for min at °c. another aliquot of cells was resuspended in pbs containing human-monomeric ige ( µg/ml) for sensitization, . mm ca + and % bsa, for min. after removal of excess ige by dilution and centrifugation (in pbs), the cells were resuspended in . mm ca + supplemented pbs, and fcεri was crosslinked by addition of mouseanti-human ige ( µg/ml) to cells for min at °c. following the incubation, µl of supernatant, was incubated with µl of mm p-nitrophenyl n-acetyl-β-dglucosaminide for hr at °c. the total β-hexosaminidase concentration was determined by a : extraction of the remaining buffer and cells with % triton x- ; a µl aliquot was removed and analyzed as described. reactions were quenched by addition of µl of . m sodium carbonate buffer. the enzyme concentration was determined by measuring the od at nm. β-hexosaminidase release was represented as a percent of total enzyme. to analyze the expression of the intracellular mast cell chymase, × cells were washed with ice cold pbs, fixed and permeabilised using the fix and perm reagents from caltag (caltag laboratories, burlingame, ca) as follows: after washing, samples were resuspended in ul of reagent a (fixation medium) and incubated for min at rt. the cells were then washed twice with ice-cold pbs, and resuspended in ul of reagent b (permeabilization medium) and incubated for min at rt. cells were washed twice and resuspended in µl of pbs/ % fbs and µl of the anti-human chymase mab (igg -mab , chemicon, temecula, ca) was added and samples were incubated for min at rt. samples were washed twice in ice-cold pbs, then resuspended in pbs/ % fbs and µl of fitc-conjugated secondary antibody (anti-mouse igg-fitc, sigma-aldrich, singapore) was added, and incubated in dark for an min at rt. samples were washed twice with ice-cold pbs and resuspended in µl of pbs/ % fbs for immediate analysis. to analyze the cell surface expression of ckit and fceri, the samples were initially processed as above except that the permeabilisation step was omitted. for c-kit the primary antibody was an anti-human c-kit mouse monoclonal (mca , clone d , serotec, oxford, uk), and the secondary antibody was a fitc-conjugated (antimouse igg-fitc, sigma-aldrich, singapore). for fcεri cell-surface expression, the cells were labeled with the primary anti-human fcεri polyclonal (rabbit-igg-ab . abcam, cambridge, uk), and the secondary antibody was a fitc-conjugated (anti-rabbit igg-fitc, sigma-aldrich, singapore). all the samples were analyzed by flow cytometry was using a facscalibur machine (bd biosciences), and the data analysed using the cell quest™ pro software. mast cells in innate immunity mast cells to the defense mast cells in autoimmune disease mast cells the diverse potential effector and immunoregulatory roles of mast cells in allergic disease transcriptional response of human mast cells stimulated via the fcεri and identification of mast cells as a source of il- the receptor with high affinity for ige signalling through the high-affinity ige receptor fcεri roles of mast cells and basophils in innate and acquired immunity complexity and redundancy in the pathogenesis of asthma: reassessing the roles of mast cells and t cells mast cells can amplify airway reactivity and features of chronic inflammation in an asthma model in mice an essential role of mast cells in the development of airway hyperresponsiveness in a murine asthma model mast cells phagocytosis of fimh expressing enterobacteria histamine-induced activation of human lung macrophages mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection human mast cell transcriptome project identification of novel mast cell genes by serial analysis of gene expression in cord blood-derived mast cells gene expression screening of human mast cells and eosinophils using high-density oligonucleotide probe arrays: abundant expression of major basic protein in mast cells comparative cytokine profile of human skin mast cells from two compartments strong resemblance with monocytes at baseline but induction of il- by il- priming gene expression profiling of ca + -atapase inhibitor dtbhq and antigen-stimulated rbl- h mast cells identification of specific gene expression profiles in human mast cells mediated by toll-like receptor and fcεri gene expression profiles for fcεri, cytokines and chemokines upon fcεri activation in human cultured mast cells derived from peripheral blood marked increase in cc chemokine gene expression in both human and mouse mast cell transcriptomes following fcε-receptor i cross-linking: an interspecies comparison monomeric ige enhances human mast cell chemokine production: il- augments and dexamethasone suppresses the response interleukin- induces a shock-like state in rabbits: synergism with tumor necrosis factor and the effects of cyclooxygenase inhibition monomeric ige stimulates signaling pathways in mast cells that lead to cytokine production and cell survival nf-kappa b and rel proteins: evolutionary conserved mediators of immune responses generic signals and specific outcomes: signaling through ca + , calcineurin, and nf-at dichotomy of ca + signals triggered by different phospholipid pathways in antigen stimulation of human mast cells minimal requirements for ige mediated regulation of surface fcεri karasuyama h: drastic up-regulation of fcεri on mast cells is induced by ige binding through stabilization and accumulation of fcεri on the cell surface increased il- release by monocytes in nephritic patients the physiological and pathophysiological role of chemokines during inflammatory and immunological responses differential roles of il- and tnf-α on graft versushost disease and graft versus leukemia rel/nf-κb transcription factors: key mediators of b-cell activation constitutive nfκb maintains high expression of a characteristic gene network, including cd , cd , and a set of antiapoptotic genes in hodgkin/reed-stemberg cells control of oncogenesis and cancer therapy resistance by the transcription factor nf-κb calcium mobilization via sphingosine kinase in signalling by the fcεri antigen receptor signatures of the immune response fcγri coupling to phospholipase d initiates sphingosine kinasemediated calcium mobilization and vesicular trafficking a molecular switch changes the signalling pathway used by the fcγri antibody receptor to mobilize calcium anaphylatoxin signaling in human neutrophils: a key role for sphingosine kinase antisense knockdown of sphingosine kinase in human macrophages inhibits c a receptordependent signal transduction, ca + signals, enzyme release, cytokine production and chemotaxis mast cells in the development of adaptive immune responses mast cells in allergy and autoimmunity: implications for adaptive immunity melendez aj: expression profile of immune response genes in patients with severe acute respiratory syndrome this work was supported by a bmrc-young investigator award (r- - - - ). we thank a-k fraser-andrews for proofreading the manuscript. mj carried out data analysis and prepared the microarraydata table and figures. hkt isolated the progenitor cells, differentiated the mast cells and carried out the receptor crosslinking. rr carried out the rna extraction, labeling and microarray hybridization. lz carried out rt-pcr. kkc and mr provided the cord blood. ajm designed the study and drafted the manuscript. all authors read and approved the final manuscript. key: cord- -qz ef y authors: xu, shanhui; li, yanran title: yeast as a promising heterologous host for steroid bioproduction date: - - journal: j ind microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: qz ef y with the rapid development of synthetic biology and metabolic engineering technologies, yeast has been generally considered as promising hosts for the bioproduction of secondary metabolites. sterols are essential components of cell membrane, and are the precursors for the biosynthesis of steroid hormones, signaling molecules, and defense molecules in the higher eukaryotes, which are of pharmaceutical and agricultural significance. in this mini-review, we summarize the recent engineering efforts of using yeast to synthesize various steroids, and discuss the structural diversity that the current steroid-producing yeast can achieve, the challenge and the potential of using yeast as the bioproduction platform of various steroids from higher eukaryotes. sterols are essential membrane components that regulate membrane fluidity and permeability in eukaryotic organisms [ ] . animals generally synthesize cholesterol, fungi produce ergosterol, and plants utilize an array of sterols with β-sitosterol and campesterol as the most common ones [ ] . despite the essential roles in membrane modulation, sterols also exhibit important bioactivities. for example, due to the structural similarity to cholesterol, phytosterols can reduce cholesterol absorption in digestive system and reduce the risk of heart disease [ , ] . in higher eukaryotes, sterols are also precursors to a wide array of specialized metabolites with broad structural diversity and biological activities. in insects, cholesterol is converted into insect steroid hormones, such as ecdysteroids that can be potentially utilized as anabolic steroids [ ] . in human, cholesterol is the precursor to an array of steroid hormones such as progesterone and vitamin d [ , ] . in plants, campesterol is the main precursor of the ubiquitous phytohormone brassinosteroids [ ] ; cholesterol, although exhibits very low abundance in plants, is the precursor to a broad spectrum of phytochemicals, such as the medicinally important steroidal alkaloids and steroidal saponins [ ] . intriguingly, some phytosteroids exhibit promising antiviral activities via different mechanisms, which make them intriguing lead structures for novel antiviral drug discovery and development [ ] [ ] [ ] [ ] . plants also produce ecdysteroid analogs, phytoecdysteroids, as natural defense mechanism against phytophagous insects [ ] . moreover, steroids are an important pharmaceutical medication, with the global steroid market projected to reach $ billion in [ ] . steroid drugs generally fall into two categories: anabolic and corticosteroids. anabolic steroids are involved in the treatment of muscle loss or late puberty [ ] ; while corticosteroids can be used as anti-inflammatory drugs or allergic treatment [ ] . during the recent outbreak of covid- , methylprednisolone (median price $ . / mg [ ] ), a corticosteroid, was reported to have positive effects on a faster improvement of the clinical symptoms caused by covid- [ ] . more recently, another corticosteroid, dexamethasone (median price $ . / mg from www. goodr x.com), has been found to able to reduce the death by roughly one-third of the patients on ventilators [ ] . however, most of these specialized steroids are not natively produced at a high level, due to the nature of their biological functions. similar to other specialized metabolites, the structural complexity also makes the chemical synthesis of these compounds challenging and expensive [ ] . recent development of synthetic biology provides an alternative sourcing strategy of these molecules through biotechnological production in the fast-growing, fermentable microorganisms, such as yeast and escherichia coli. the structural and biosynthetic similarity of ergosterol in yeast to the other eukaryotic sterols highlights the potential of using yeast as the bioproduction host for sterols and derivatives [ ] . the previous metabolic engineering efforts in establishing steroid production in yeast have been very well reviewed in wriessnegger's review [ ] . in this review, we will mainly discuss the efforts in engineered steroid bioproduction in yeast since and focus on the metabolic engineering strategies, and the challenges and potential biosynthetic capacities of yeast-based steroid bioproduction. ergosterol, similar to cholesterol in animals and phytosterols in plants, is the most abundant sterol in fungi and plays an essential role in maintaining physicochemical properties of plasma membrane and involves in protein sorting and protein receptor regulation [ ] . the biosynthesis of ergosterol exists in almost all yeast species and is well characterized, especially in the model organism saccharomyces cerevisiae. the biosynthesis of ergosterol in s. cerevisiae can be divided into three modules [ ] : ( ) biosynthesis of mevalonate (mva) from acetyl-coa, ( ) conversion of mva into farnesyl pyrophosphate (fpp) and ) ergosterol biosynthesis from fpp. the third module can be further divided into lanosterol biosynthesis from fpp, ( - ) zymosterol biosynthesis from lanosterol and ( - ) ergosterol biosynthesis from zymosterol. ergosterol, cholesterol, and the major phytosterols, campesterol and β-sitosterol, are different in three aspects: saturation of c -c bond in b-ring, saturation of c -c bond, and the alkane group at c in the side chain ( fig. ) . sterol biosynthesis is highly conserved in eukaryotes [ ] . the synthesis of ergosterol, cholesterol, and phytosterols diverges from lanosterol, and shares a number of downstream intermediates. for example, zymosterol and episterol in the ergosterol synthesis (submodule - ) can also be found in animals and plants, respectively [ , ] . the convergency of the eukaryotic sterol structures and biosynthesis makes it possible to redirect the biosynthesis of ergosterol to cholesterol and phytosterols in yeast. the latestage biosynthesis of ergosterol from zymosterol (module fig. ergosterol synthesis and sterols from mammals and plants. the endogenous ergosterol biosynthesis in yeast is highlighted in yellow. the three different sheds of yellow represented three modules of ergosterol synthesis. major phytosterols that have been synthesized or possibly synthesized in yeast are highlighted in green, and cholesterol is highlighted in blue. the characteristic structural features of sterols from different eukaryotic organisms are highlighted in pink. the enzymes marked in red represent the ones that require nadph/nadp + , and the ones that require atp are marked in blue - ) involves five erg enzymes, erg - . erg is the ∆ -sterol c-methyltransferase, converting zymosterol into fecosterol [ ] . fecosterol is then converted into episterol by erg through the isomerization of c -c double bond to c -c position [ ] . erg is the c desaturase that converts episterol into ergosta- , , ( )-trienol [ ] . erg is the c- sterol desaturase that catalyzes the conversion from ergosta- , , ( )-trienol to ergosta- , , , ( )tetraenol [ ] . erg is the ∆ -sterol reductase that reduces c -c double bond to synthesize ergosterol [ ] . the corresponding genes (erg - ) are all identified as nonessential genes to yeast viability [ ] . previous investigations on the effects of erg gene deletions suggest that erg - enzymes can accept a broad range of sterol structures, which indicates the broad substrate promiscuities of erg - and that these enzymes may not follow a specific order to afford the synthesis of ergosterol [ ] . for example, the disruption of erg accumulates not only zymosterol, but also cholesta- , -dienol, cholesta- , , -trienol and cholesta- , , , tetraenol, which indicates that erg , erg and erg can keep functioning sequentially on zymosterol in the absence of erg [ ] . different combinations of erg gene deletions result in various sterol compositions. more detailed sterol composition can refer to the review published by johnston et al. very recently [ ] . the versatility of yeast ergosterol biosynthesis implicates the potential of yeast to synthesize diversified sterol structures through ergosterol biosynthesis engineering and heterologous pathway reconstitution. to utilize yeast as the steroid bioproduction platform, ergosterol biosynthesis needs to be disrupted and thus, it is necessary to understand the effects of erg gene deletions on yeast. generally, among single deletion of erg - , Δerg and Δerg exhibit the most evident effects on the response of yeast to antifungal drugs (e.g., hygromycin b, tetramethylammonium), cations and heat [ ] [ ] [ ] . inactivation of erg or erg also results in a crucial deficiency on plasma membrane integrity, leading to strong deviation from the wild type in respect of membrane potential and tolerance to licl [ , ] . on the other hand, inactivation of erg from s. cerevisiae does not change the membrane potential and the tolerance to cations (such as li + , k + and na + ) and hygromycin b as much [ ] . the phenotypes of other erg mutants have been carefully summarized and discussed in the recent reviews by johnston et al. and sokolov et al. [ , ] . biotransformation of steroid drugs usually requires hydroxylation and dehydrogenation at different positions of the sterane skeleton, and is normally catalyzed by cytochrome p s and hydroxysteroid dehydrogenases (hsds) [ ] . steroid hydroxylation usually involves α-, α-, β-, α-hydroxylases, while dehydrogenation is generally catalyzed by β-, α-hsds. biotransformation of androstenedione (ad) to testosterone (ts) by s. cerevisiae was discovered as early as in [ ] , with the same function discovered in schizosaccharomyces pombe later [ ] . the biotransformation of steroid drugs by microbial strains was intensively studied in the s, but limited to strain mining without comprehensive enzyme characterizations [ , ] . however, the wild-type strains are limited to specific types of reactions, which cannot be utilized to catalyze biotransformation beyond native catalytic capacity. for example, although s. cerevisiae was reported to be able to catalyze the reduction at c , c and c of c and c steroids [ ] , it cannot catalyze the desired hydroxylation of steroids for the synthesis of hydrocortisone, which requires the c -hydroxylation. it is important to characterize the enzymes responsible for the bioconversion in the corresponding microbial strains, and thus enable the establishment of programmable bioproduction of steroids using biotransformation strategy. more information about the microbial biotransformation in all types of microbes can be found in an earlier comprehensive review by donova et al. [ ] ; here, we will only discuss the biotransformation efforts established in yeast (table ) . α-hydroxylation (fig. , table ) of progesterone is an important step in corticosteroid synthesis [ ] , and the biotransformation has been studied since s [ ] . many microbial strains, such as rhizopus nigricans and aspergillus ochraceus [ , ] , have been reported to be able to catalyze this conversion. however, the enzyme responsible for α-hydroxylation in rhizopus was not discovered until [ ] . cyp c from the filamentous fungi rhizopus oryzae was characterized as the enzyme responsible for the synthesis of α-hydroxylation from progesterone [ ] . coexpression of cyp c with its nature redox partner nadph-cytochrome p reductase (rocpr) in the fission yeast s. pombe led to . % conversion of progesterone into α-hydroxyprogesterone and small amounts of a byproduct β-hydroxyprogesterone (~ . %) [ ] . cyp c was also found to be able to catalyze the α-hydroxylation of ts, -deoxycorticosterone (doc) and cortodoxone (rss), with doc exhibiting the highest conversion of progesterone but with low specificity ( % α-hydroxylated product and . % β-hydroxylated byproduct) [ ] . in , another α-hydroxylase ( α-sh aoch ) from the industrial steroid bioconversion strain a. ochraceus was characterized in s. cerevisiae [ ] . progesterone fed to the α-sh aoch -expressing yeast strain was converted into α-hydroxyprogesterone as the sole product with a high conversion at . %, without coexpression of the native cpr [ ] . in this study, cyp c was expressed in s. cerevisiae as well as a comparison, resulting in a mixture of α-hydroxyprogesterone and β-hydroxyprogesterone (with the ratio of : ). the amino acid sequences of α-sh aoch and cyp c were then compared, which only share % identity at whole length and % identity in a c-terminus ( amino acid long), indicating that they belong to different cytochrome p families [ ] . the β-hydroxyl (fig. , table ) is essential for the anti-inflammatory activity of relating steroid drugs, such as hydrocortisone [ ] . the filamentous fungi absidia orchidis was found to be able to catalyze β-hydroxylation on rss to form hydrocortisone [ ] . recently, the enzymes responsible for the β-hydroxylation in a. orchidis were identified, including a cytochrome p cyp b and its native associating cpr and cytochrome b (cyb ) from a. orchidis [ ] . the three enzymes were then functionally reconstituted in s. cerevisiae to catalyze the synthesis of a hydroxylation β-dehydrogenation -hydroxylation hydrocortisone from rss with the productivity of hydrocortisone at up to mg/l day [ ] . as a comparison, the mammalian β-hydroxylation system with cyp b from homo sapiens and the redox partners for p s, bovine adrenodoxin reductase (adr) and adrenodoxin (adx) were expressed in yeast, which produced mg/l day of hydrocortisone, around sixfold lower than the a. orchidis system. the a. orchidis system (cyp b , cpr and cypb ) was further engineered for enhanced hydrocortisone production. the site-directed mutagenesis of cyp b was conducted and the r d/y f mutant exhibited times higher productivity of hydrocortisone compared with the wild-type cyp b . to eliminate undesired side reactions in s. cerevisiae, two yeast endogenous enzymes, glycerol dehydrogenase gcy and aldosterone reductase ypr that can catalyze the c -keto reduction of rss, were inactivated. additionally, various cprs (cprs from s. cerevisiae, arabidopsis thaliana, vitis vinifera, and a. orchidiswas) were introduced into s. cerevisiae to identify the optimal cpr for the function of cyp b ; with cpr from a. orchidiswas exhibiting the best performance. the hydrocortisone productivity was further enhanced to ~ mg/l day upon tuning the expression level and the ratio among cyp b , cpr and cyb in the engineered yeast strain, then to ~ mg/l day through overexpression of steroid transporter cdr from cochliobolus lunatus, and ultimately towards ~ mg/l day by increasing the cell density to od ~ . interestingly, the engineered yeast strain exhibits an enhanced regioisomeric ratio ( : ) between the desired β-hydrocortisone to α-hydrocortisone, compared to : in a. orchidis, which also encodes α-hydroxylase [ ] . this study demonstrates the high efficiency and regioselectivity of the engineered steroid biotransformation in s. cerevisiae, indicating yeast as a promising platform for the bioproduction of hydrocortisone through biotransformation [ ] . beside -hydroxyl, α-hydroxyl ( fig. , table ) is also an important structural feature and is responsible for the anti-gonadotropic and carcinolytic biological properties of α-hydroxysteroid drugs [ ] , such as α-hydroxy- androstene- , , -trione ( α-oh-at) [ ] . p lun from c. lunatus was originally characterized as a bifunctional hydroxylase that catalyzes both β-and α-hydroxylations of rss, and has been utilized in the industrial production of hydrocortisone from rss through β-hydroxylation [ ] . recently, the production of α-hydroxysteroids was established in s. cerevisiae using p lun to catalyze the formation of α-hydroxyandrostenedione ( α-oh-ad) from androstenedione (ad) (regiospecificity > %) [ ] . to enhance the efficiency and specificity of p lun towards the synthesis of α-oh-ad, the native redox partner cpr lun was identified and co-expressed with p lun with enhanced copy numbers in s. cerevisiae. the gcy and ypr genes were inactivated in the strain to eliminate the side reaction of the c -keto reduction, same as in the biotransformation of hydrocortisone using cyp b . the final engineered yeast strain synthesized α-oh-ad with % w/w yield and % regioisomeric purity converted from ~ mg/l of fed ad [ , ] . boldenone (bd), the precursor to a number of steroid medicines, is normally synthesized from ad through ∆ dehydrogenation and -keto reduction (fig. , table ) [ ] . a number of yeast strains have been found to be able to catalyze steroid β-reduction natively, including pichia pastoris gs [ ] . one recent example of yeast-based bioproduction of bd was achieved through overexpressing the -ketosteroid-Δ -dehydrogenase ksdd from rhodococcus rhodochrous in gs [ ] . both ∆ dehydrogenated product androst- , -ene- , -dione (add) from ad and -keto reduced product bd from add were detected from the engineered yeast strain, indicating the -step formation of bd from ad in gs [ ] . however, the conversion efficiency of ad into bd is low, at around % due to the insolubility of steroid in aqueous solution (ad was fed at g/l). therefore, the cosolvent -hydroxypropyl-βcyclodextrin (hp-β-cd) was supplemented to improve the solubility of steroids in the medium and enhanced the conversion of ad into bd to . % (with molar ratio : of hp-β-cd to ad) [ ] . the conversion was further improved with the bacterial and yeast co-culture strategy in the followup investigations [ , ] . the bacterial strain arthrobacter simplex previously identified to exhibit high ∆ -dehydrogenation activity was used to convert ad into add; and p. pastoris gs was engineered to express βhsd from s. cerevisiae to enhance the conversion of add into bd [ ] . with the optimized medium condition ( . g/l ad with molar ratio : hp-β-cd and -g/l glucose), the conversion of ad into bd achieved % [ ] . the efficient conversion of ad into bd in the bacteria-yeast coculture later on inspired mycobacterium neoaurum-p. pastoris gs coculture, where m. neoaurum can natively convert phytosterols into add (with a small amount of ad supplemented) [ ] . the two-stage fermentation of ksdd -overexpressing m. neoaurum and βhsd-overexpressing gs produced bd at a conversion of % from fed phytosterols under the optimal medium condition ( g/l glucose supplementation and mm β-cd) [ ] . multi-step biotransformation has also been established in yeast (table ) . recently, the early steps of cardenolide formation, from pregnenolone to β-pregnane- β, diol- -one, were reconstituted in s. cerevisiae [ ] , through expressing ∆ β-hsd from digitalis lanata (dl β-hsd), ∆ isomerase from comamonas testosteronii (ct ksi), β reductase from arabidopsis thaliana (atst βr), and a -hydroxylase cyp a from mus musculus (mmcyp a ). to avoid the esterification of pregnenolone which inhibits its conversion into downstream products, acetyl-coa:pregnenolone acetyltransferase (apat) encoded by atf was disrupted. subsequently, to enhance the performance of the mitochondria cytochrome p mmcyp a , the redox partners, adr and adx were co-expressed. although the conversion (< % from . mm fed pregnenolone) is not as high as the aforementioned biotransformation using single or double enzymes, this strategy implicates the potential of programming yeastbased biotransformation for the production of more diverse steroid structures using enzymes of different functions and from various organisms [ ] . yeast has also been utilized to characterize several steroid hydroxylases that catalyze special hydroxylation activities. recently, thanatephorus cucumeris was found to be able to catalyze β-hydroxylation of rss under neutral or acidic condition (table ) [ ] . transcriptome analysis of t. cucumeris was conducted upon rss exposure at ph . and ph . . the highly upregulated gene cyp ap and downregulated gene cyp an at ph . were reconstituted in p. pastoris and presented special hydroxylation activities at c and c . cy ap catalyzes β-hydroxylation on rss and ts in yeast, with % side reaction of β-hydroxylation. cyp an was identified to form a β-hydroxyl on rss and produced β-oh-rss as the sole product [ ] . in addition to the fungal enzymes, cyp d from ajuga hairy roots was characterized in yeast as a c- hydroxylase that functions on β-hydroxy- β-cholestan- -one ( β-ketone) and β-hydroxy- β-cholest- -en- -one ( β-ketol) [ ] . biotransformation of steroid drugs provides an environmentally more friendly approach for the industrial steroid production, compared to chemically catalyzed conversion [ ] . the fast growing, genetically manipulatable, and fermentable yeast strains have been considered as a favorable host for biotechnological production [ ] . as discussed in this session, different yeast strains have been utilized for the production of various steroids through biotransformation strategy and generally with good conversion efficiency. the medium condition of steroid biotransformation in yeast is relatively simple, usually common yeast rich or minimal medium supplemented with organic solvent, such as ethanol and n,n-dimethylformamide (dmf) to help dissolve the steroid substrates [ , ] . addition of β-cyclodextrin often helps the transport of steroid substrates into the cells [ , ] . enzymes responsible for steroid hydroxylation and dehydrogenation have been largely identified within the past years, with many characterized in yeast. the massive strains with steroid transformation abilities discovered in the past decades are important resources for novel enzyme discovery; and the discovery of these enzymes set the foundation to use yeast as a versatile bioproduction platform for diverse steroid structures using the biotransformation strategy. de novo synthesis of steroids in yeast has gained much attention in the past decades [ ] . the complete synthesis of steroid from simple carbon sources exploits the native ergosterol pathway to produce the desired sterol structure by inactivating late stage erg genes. the erg gene, encoding ∆ -sterol c-methyltransferase, can be inactivated to form sterols without c -alkyl, which is a key characteristic of cholesterol. cholesterol is the crucial component in animal cell membrane and precursor to vitamin d and many other hormones [ , ] . cholesterol can also be found in many plants at a trace amount but is an important precursor to many specialized metabolites such as steroidal glycoalkaloids (sgas) involving in the plant defense mechanism [ , ] . the first stable cholesterol-producing s. cerevisiae strain rh was established by souza et al. in [ ] . compared to ergosterol, cholesterol does not own c -methyl and has saturated c -c bond and c -c bond. erg and erg are responsible for the c methylation and c -c desaturation, respectively. to redirect ergosterol synthesis to cholesterol, erg and erg were disrupted, and ∆ -reductase dhcr and ∆ ( ) -reductase dhcr , both from danio rerio, were introduced to s. cerevisiae to generate strain rh (fig. , table ). s. cerevisiae rh can stably produce cholesterol from simple carbon source, providing a yeast-based platform to synthesize specialized cholesterol and derivatives [ ] . for example, rh was applied to produce c-labeled cholesterol by culturing the strain rh with c-labeled glucose. the c-labeled cholesterol was used to investigate influenza m protein-cholesterol interaction by nmr spectroscope [ ] . another yeast strain p. pastoris has also been engineered to produce cholesterol using the same strategy as rh (table ) [ ] . these cholesterol-producing yeast strains were proposed to be humanized strains and might be suitable hosts for the expression of mammalian membrane proteins. a membrane-binding human protein na,k-atpase α β isoform, which requires the interaction with cholesterol and phospholipids to stabilize and enhance the ion transport activity, was used to examine how cholesterol incorporation affects the function of animal membrane proteins. in comparison to the wild-type p. pastoris, na,k-atpase α β isoform exhibited longer protein half-life, correct co-localization in plasma membrane, and enhanced atpase activity in the cholesterol-producing p. pastoris. the enhanced stability and activity of na,k-atpase α β isoform in the cholesterol-producing p. pastoris imply the potential of using cholesterol-producing yeast strains to study and express animal membrane proteins [ ] . cholesterol is also the precursor to a number of specialized metabolites in plant, such as saponins and steroidal alkaloids [ ] . the cholesterol-producing s. cerevisiae strain rh is, thus, often used for enzyme functional characterization to elucidate the biosynthesis of cholesterolderived phytochemicals. one recent example is the characterization of the r-hydroxylase cyp b involved in the synthesis of polyphyllins in the medicinal herb paris polyphylla [ ] . the first step from cholesterol to saponins and steroidal alkaloids is believed to be c -hydroxylation and catalyzed by a cytochrome p . cyp b was identified from functional transcriptome analysis, and the heterologous expression of cyp b led to a descent conversion towards (r)-hydroxycholesterol, which confirmed cyp b as the α-hydroxylase (fig. , table ) [ ] . more recently in , rh was utilized in the elucidation of the biosynthesis of diosgenin, the aglycone of a class of spirostanol-type saponins that is synthesized from cholesterol [ ] . similarly, ppcyp g -ppcyp d and tfcyp b -tfcyp j were identified to catalyze the conversion of cholesterol into diosgenin in p. polyphylla and trigonella foenum-graecum, respectively, through transcriptome analysis and functional screening using nicotiana benthamiana-based co-infiltration. the functions of ppcyp g , ppcyp d , tfcyp b , and tfcyp j were further confirmed through functional reconstitutions in rh , which also enabled the first reported yeast bioproduction of diosgenin (fig. , table ) [ ] . although yeast is mostly used as the characterization platform here instead of robust producers of the cholesterolderived phytosteroids, these studies demonstrate the potential of using yeast as the platform for the bioproduction of various cholesterol-derived phytosteroids. in addition to cholesterol-derived compounds, the Δerg mutant of s. cerevisiae has also been used to investigate plant cholesterol biosynthesis. plant cholesterol biosynthesis also involves ∆ ( ) -reductase [ ] . to provide a platform for the putative solanum tuberosum ∆ ( ) -reductase screening, erg was disrupted in s. cerevisiae and stdwf (a ∆ -reductase from s. tuberosum) was expressed to accumulate desmosterol (t ) [ ] . the expression of ssr , the putative ∆ reductase, in this desmosterol-accumulating yeast strain resulted in the production of cholesterol, which confirms that ssr functions as a ∆ ( ) -reductase and is involved in cholesterol biosynthesis in s. tuberosum (table ) . moreover, co-expression of ssr with stdwf fig. synthesis of steroids without c -alkyl yeast. the characteristic c of cholesterol is marked in red. single solid arrow represents reactions that have been validated in yeast, multiple solid arrows indicate multiple steps, and dashed arrows implicate conversions catalyzed by unknown enzyme(s). compounds that potentially can be synthesized from yeast but not established yet are marked in blue. the endogenous ergosterol biosynthesis in yeast is highlighted in yellow, cholesterol is highlighted in blue, and the steroids derived from cholesterol are highlighted in pink. the enzymes marked in red represent the ones that require nadph/nadp + , and the ones that require atp are marked in blue x x in the Δerg Δerg double mutation and -methylenecholesterol-accumulating yeast strain (t ) also resulted in the synthesis of cholesterol. the synthesis of cholesterol in both engineered yeast strains implies that the Δ ( ) -reduction catalyzed by ssr could occur before the c- methylation catalyzed by erg (table ) [ ] . the versatility of yeast in sterol biosynthesis and engineering provides a unique platform for the clear elucidation of sterol biosynthesis. the Δerg s. cerevisiae, with the lack of -alkylsterols, also provides a favorable platform for the characterization of c -methyltransferase. since ergosterol cannot be detected in the fungal pathogen pneumocystis carinii, it is resistant to many common antifungal agents targeting ergosterol biosynthesis, and the s-adenosylmethionine:c- sterol methyltransferase (sam:smt, adomet) has been shown to be a promising target for the development of antifungal drug active against p. carinii [ ] . however, p. carinii is difficult to study due to the lack of in vitro culture conditions. instead, p. carinii sam:smt was heterologously expressed in the Δerg mutant s. cerevisiae for functional characterization, which revealed the function that this methyl transferase can transfer one or two methyl to c to form both -carbon and -carbon sterols [ ] . although inactivation of erg is not lethal to yeast, it affects yeast plasma physical properties. beside the enhanced hypersensitivity to licl and hygromycin b as previously discussed [ ] , erg inactivation is lethal to yeast with tryptophan auxotroph because that the localization of tryptophan transporter tat relies on the interaction with ergosterol in the plasma membrane [ ] . the lack of ergosterol results in the mislocation of tat in the vacuole and hinders the uptake of tryptophan. however, the production of cholesterol in Δerg mutant can restore the function of tat misroute and recover the tryptophan transport capability [ ] . additionally, ergosterol is very important for the function of pdr [ ] , which is responsible for yeast resistance to weak organic acids. mutant lacking ergosterol is hypersensitive to weak organic acid. these factors need to be considered during erg inactivation related yeast engineering. on the other hand, the Δerg mutant shows higher resistance to tomatidine, compared to the wild-type s. cerevisiae [ ] . the enhanced resistance is likely due to the accumulation of zymosterol, implying the potential of using Δerg yeast mutants for the biosynthesis of steroidal alkaloid and saponins exhibiting antifungal activities [ ] . erg catalyzes c- desaturation, which is a unique characteristic of ergosterol, compared with cholesterol and phytosterol. the inactivation of erg resulted in the accumulation of ergosta- , -dienol- β-ol, the precursor of vitamin d [ ] . the expression of dhcr from homo sapiens in Δerg mutant s. cerevisiae led to the accumulate -dehydrocholesterol, the precursor of vitamin d [ ] . phytosterols generally have saturated c -c bond in the b ring, which is catalyzed by the ∆ -reductase (dwf ). dwf has been previously utilized for the de novo synthesis of hydrocortisone with the function of eight mammalian enzymes in s. cerevisiae [ ] . hydrocortisone was proposed to be converted from campesterol or brassicasterol of saturated c -c in the b ring. the synthesis of hydrocortisone in the engineered s. cerevisiae with the titer up to . mg/l implies the significance of establishing efficient bioproduction of phytosterols in yeast [ ] . although stable synthesis of campesterol was established in s. cerevisiae as early of [ ] , most recent campesterol bioproduction efforts were focusing on engineering of yarrowia lipolytica. the efficient production of campesterol in y. lipolytica was first reported in [ ] . -dehydrocholesterol reductases (dhcr s) from rallus norvegicus, oryza saliva and xenopus laevis were examined in Δerg mutant y. lipolytica for the best production of campesterol (fig. , table ). out of three reductases, dhcr from x. laevis exhibited the highest campesterol production, up to mg/l under the optimal medium optimization with sunflower seed oil as the carbon source under high-cell density fed-batch fermentation [ ] . the campesterol-producing y. lipolytica strain was further enhanced to mg/l by substituting xldhcr with dhcr from d. rerio and expressing peroxisome acyl-coa oxidase (pox ), an enzyme with high activity on the conversion of c - long-chain fatty acids (the main components in sunflower seed oil) into acetyl-coa [ ] . subsequently, the de novo production of pregnenolone, the early-stage intermediate for the synthesis of hydrocortisone, was established through coexpression of the sterol side-chain cleavage enzyme cyp a with the redox partners bovine adx and adr in y. lipolytica (fig. , table ) [ ] . various cyp a orthologs (from homo sapiens, bos taurus, danio rerio, lithobates catesbeiana, gossypium arboretum, taeniopygia guttata, gallus gallus, mus musculus) and redox partners were compared to locate the pair (cyp a from sus scrofa with adx/ adr from bos taurus) that provides the highest titer of pregnenolone, which was further optimized to mg/l through expression level tuning and fermentation engineering [ ] . the characteristic c -c bond of campesterol is marked in red. single solid arrow represents reactions that have been validated in yeast, multiple solid arrows indicate multiple steps, and dashed arrows implicate conversions catalyzed by unknown enzyme(s). compounds potentially can be synthesized from yeast but not established yet are marked in blue. the endogenous ergosterol biosynthesis in yeast is highlighted in yellow, phytosterols are highlighted in green, and steroids synthesized from phytosterols are highlighted in pink. the enzymes marked in red represent the ones that require nadph/nadp + , and the ones that require atp are marked in blue besides steroid drugs, phytosterols are also precursors to an array of specialized metabolites in plants, such as brassinosteroids and withanolides. as discussed previously, phytosterol-producing yeast strains are useful platforms for the functional characterization of enzymes involved in the biosynthesis of specialized phytosteroids. brassinosteroids are a group of phytohormones that play an essential role in plant growth, development, and resistance to both biotic and abiotic stress, and are mainly synthesized from campesterol [ ] . in our most recent work, s. cerevisiae was engineered to express Δ -sterol-c -desaturase (dwf ), dwf and Δ -sterol reductase (dwf ) from a. thaliana to synthesize campesterol. however, campesterol and the precursor -methylenecholesterol were found to be vigorously esterified and were merely accessible to the downstream tailoring enzymes such as cyp b or smt [ ] . the inactivation of the native sterol acyltransferase genes are and are resulted in the synthesis of free phytosterols without much growth deficiency [ ] . the introduction of smt to the free campesterol-producing yeast strain led to the synthesis of β-sitosterol, the titer of which was then enhanced through inactivating erg , the paralog and competing gene of dwf (table ). however, inactivation of erg resulted in dramatically reduced campesterol synthesis and significant growth deficiency, which were partially overcome by adaptive evolution. the free campesterol-producing yeast strain was also utilized as a platform to reconstitute the early part of the brassinolide biosynthetic pathway, using cyp b and cyp a , towards the synthesis of (s)hydroxycampest- -en- -one (fig. , table ) [ ] . on the other hand, withanolides are a group of bioactive steroids mainly found in the solanoideae family, and are believed to be synthesized from -methylenecholesterol [ , ] . the first step converting -methylenecholesterol towards the synthesis of withanolides has been very recently characterized to be catalyzed by a sterol Δ -isomerase ( iso) (fig. , table ) [ ] . iso candidates were proposed from transcriptome analysis of different solanoideae plants, and functionally identified in both plant tissues and the -methyldesmosterol-producing s. cerevisiae (strain t ) [ ] . most steroids with saturated c -c and unsaturated c -c bonds are phytosterol related. to date, except for campesterol production, metabolic engineering efforts in pursuing yeast-based bioproduction of phytosterol and phytosteroids are limiting. yeast is more often utilized as an enzyme functional characterization tool. although often with low efficiency, the structural diversity of phytosteroids produced in yeast has been broadened through this yeast-based enzyme characterization process, and thus shed lights on the synthetic potential of yeast-based steroid bioproduction. there are several challenges remaining in establishing efficient steroid production in yeast. first, cytochrome p s are often involved in steroid biosynthesis, which has been generally considered as a burden in yeast metabolic engineering of specialized metabolites [ ] . in several examples discussed in this review, the co-expression and examination of different redox partners have been conducted for the sufficient electron transfer, enhanced conversion efficiency, and higher regioselectivity [ , , ] . second, steroids, such as campesterol and pregnanolone [ ] , are normally non-exportable, and the accumulation of heterologous steroids within the cells could be toxic to the yeast cells [ ] . further investigations on steroid transport and storage should be conducted to develop engineered transportation of steroids of cytotoxicity in yeast. third, many steroids that can be potentially synthesized in yeast require highly complex synthetic pathways (e.g., brassinolide [ ] , tomatidine [ ] ), which is a general challenge in engineering de novo synthesis of complex structures in microbial hosts and has been carefully discussed in several recent reviews [ , , ] . last but not least, heterologous steroid production often requires the alternation of endogenous sterol synthesis, which may cause extra burden to the engineered yeast strains and growth deficiency. these challenges inevitably affect the engineered biosynthesis of steroids and may result in low production of target steroids. one promising strategy to overcome these challenges is to use non-conventional yeast strains other than s. cerevisiae. among the studies discussed in this review, multiple yeast strains have been utilized, such as s. cerevisiae, y. lipolytica and p. pastoris, which are the most common selections and with different properties. s. cerevisiae has been extensively studied and utilized for genetic and metabolic engineering, with the well-established genetic modification tools [ ] . therefore, it is widely used in enzyme characterizations in addition to bioproduction purpose, such as the functional identification of cyp b [ ] and iso [ ] . y. lipolytica is an industrial oleaginous strain and is usually used for high-yield lipid production, up to % w/w dry cell weight [ ] . the high efficiency of acetyl-coa synthesis and robust lipid storage make y. lipolytica a favorable host for the hydrophobic steroid production [ ] [ ] [ ] . p. pastoris can be cultured with methanol and grow at a relatively low ph, making the fermentation process less prone to microorganism contamination [ ] . moreover, unlike s. cerevisiae known to have hyperglycosylation with the addition of - mannose residues to heterologous proteins on average, p. pastoris exhibits much less glycosylation with an average of ~ - mannoses, providing a favorable yeast host when certain enzymes are difficult to be functionally reconstituted due to incorrect glycosylation [ ] . p. pastoris also encodes an efficient secretion system [ ] , which may enable extracellular steroid synthesis aided by the efficient export of the steroid synthetic enzymes. in addition, using non-conventional yeast strains for specialized metabolite bioproduction has gained much attention recently [ , ] . with the genetic engineering tools developed in these strains [ ] [ ] [ ] , there will be more choice of yeast strains that are feasible for the bioproduction of specialized steroids. directed evolution has been successfully utilized to engineer yeast strains for enhanced performance [ , ] , such as enhanced thermostability with altered sterol composition [ ] . this strategy has also been successfully used to develop yeast adaptive mutants that overcome the growth deficiency caused by altered sterol metabolism in our efforts to develop β-sitosterol and (s)-hydroxycampest- -en- -one-producing yeast strains. the evolved strain was able to synthesize ~ sixfold more free campesterol and double the cell density at stationary phase, compared to the original free campesterol-producing strain [ ] . although the evolved strain still exhibits certain limitations, adaptive evolution is indispensable for the engineering and reconstitution of downstream metabolic pathways. the system biology and metabolic flux analysis, thus, become important to decipher how yeast was able to adapt its metabolism and growth to the altered sterol composition with excessive free sterols. the system biology-based approaches have also been successfully utilized to increase the production of target metabolites and overcome the metabolic burdens in different microorganism hosts [ , ] . the system-guided strategy will provide a promising route to tune the crosstalk between heterologous sterol pathway and the central sterol metabolism for enhanced performance of the engineered steroidproducing yeast strains, which are often of low efficiency. although the system-level analysis of mevalonate pathway has been thoroughly investigated [ ] , few studies have been done on yeast endogenous sterol metabolism, as well as the crosstalk with the heterologous sterol-derived pathways, which, thus, become an appealing target for future investigation to better understand and engineer yeast for enhanced sterol production, especially in the case of de novo steroid biosynthesis. pathway compartmentalization and metabolite channeling have been successfully applied to enhance the efficiency of many heterologous pathways in yeast [ , ] , mainly through enhancing the local substrate concentrations, separating desired reactions from the competing side reactions, and decreasing the release of the toxic intermediates into the cells [ , , ] . the synthesis of steroids in yeast takes place on endoplasmic reticulum (er) membrane with excessive sterols generally esterified and stored in the lipid droplets [ ] . as described in our recent work, the natural compartmentalization of sterol synthesis and esterification in yeast blocked the access of downstream enzymes (e.g., cyb b , smt ) to the sterol substrates, which leads to the failed synthesis of the target products. although inactivation of the acyltransferases are and are enabled the synthesis of free sterol and downstream target metabolites, it also introduced growth deficiency and decreased overall sterol production, which is possibly due to the cytotoxicity of excessive free sterols [ ] . in such a case, compartmentalization and intermediate channeling may provide a unique tool to circumvent the undesired crosstalk between yeast sterol esterification and the heterologous sterol pathway, and decrease the toxicity caused by the presence of heterologous sterols unnatural to yeast. last but not least, feeding steroid intermediates as the precursors is also a promising approach for the production of high-value specialized steroids. there are two main challenges to use biotransformation for steroid production: yeast exogenous sterol uptake mechanism prefers anaerobic condition, and many value-added steroids of complex structural features require large numbers of enzymes due to the lack of sterol intermediates that are commercially available. although the uptake control mutant (upc - ) has been shown to enable aerobic uptake of sterols [ ] , the production of target steroids can also be limited by yeast intracellular capacity of sterols. thus, the choice of establishing de novo synthesis or biotransformation depends on a number of factors, including but not limited to the availability of substrates, chemical characteristics of the substrates, number of heterologous enzymes required, and type of yeast to be engineered. to date, steroids that have been synthesized from yeast can be generally divided into groups: ( ) c or c steroid drugs, such as boldenone and hydrocortisone [ , ] , ( ) cholesterol and its derivatives [ , , ] and ( ) phytosterols and their derivatives [ , ] . the steroid drug biotransformation in yeast strains has been discovered since early last century, yet it is not until the recent years that enzymes responsible for the steroid biotransformation were characterized and their 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wisdom for metabolic engineering: yin-yang recent applications of metabolomics to advance microbial biofuel production compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols an artificial transport metabolon facilitates improved substrate utilization in yeast compartmentalized metabolic engineering for biochemical and biofuel production a microbial biomanufacturing platform for natural and semisynthetic opioids mechanisms of sterol uptake and transport in yeast we thank tiffany chiu, anqi zhou, and dr. sheng wu for their valuable feedback in the preparation of the manuscript. this work was supported by the national science foundation (grant to yl, ) and cancer research coordinating committee research award (grant to yl, crn- - ). conflict of interest the authors declare that there is no conflict of interest regarding the publication of this article. key: cord- -d dvdzb authors: zhang, xiaoqian; wang, zhiwei title: marketization vs. market chase: insights from implicit government guarantees date: - - journal: nan doi: . /j.iref. . . sha: doc_id: cord_uid: d dvdzb abstract local government financing vehicles (lgfvs) and state-owned enterprises (soes) provide implicit guarantee during the issuing of bonds, thereby reducing their funding cost. the credit spreads are lower when issued by a lgfv with a higher administrative level. this means that implicit guarantee is also strengthened with government centralization. we also explain the anomaly of municipal corporate bonds (mcbs)’ spreads decrease after a marketization regulation of removing implicit guarantees. this paper provides strong evidence that the market will chase implicit guarantee when default wave comes even under tight government regulations. implicit government guarantees that favor state-owned firms are becoming more prominent during recessions. it reduces the financing cost, and distorts corporate investment decisions. current loan-level data. in the post-stimulus period, chinese local governments resorted to non-bank debt with the development of the chinese corporate bond market (chen et al., b) . both papers demonstrate the effect of government guarantee on firm financing. the difference between government guarantees between china and the u.s. is highlighted in municipal bonds. in china, municipal bonds are also issued by local government financing vehicles ( lgfvs), which are state-owned enterprises to support the infrastructure investment. there are two areas in particular that make china's municipal corporate bonds are different from american municipal bonds from the following two perspectives. first, individual investors hold municipal bonds directly or through municipal bond funds in the u.s., while financial institutions buy the municipal corporate bonds (mcbs) in china. second, the credit rates of china's mcbs are highly and have never defaulted. as our data shows, almost all the mcbs were high rated in china while there are lots of speculative-grade municipal bonds in the u.s. as babina et al. ( ) show. borisova et al. ( ) find that government ownership is generally associated with a higher cost of debt in countries over - . in contrast, we find china market chases more implicit government guarantees. this paper provides strong evidence that the implicit government guarantee does exist and - -reduces the funding costs by exploring bonds' credit spreads. we find that the spreads of mcb and other local soes are and basis points lower than that those of poe bonds while their roa is significantly lower. central soes have even lower spreads. the implicit government guarantee does exist in mcbs when we control bonds' credit rates. our finding stems from a deeper analysis from three perspectives. first, mcbs and other local soes have about % lower financing cost when we introduce bond characters and regional economic development. this reveals that the main concern is that of misallocation of capital as gertler and klenow ( ) addressed. government credit may cause crowding out of private companies in the same industry (ru, ) . second, we further exploit the effect of administrative levels of mcbs. our results identify political decentralization (bardhan, ) in different administrative level of lgfvs. administrative level decentralization increases funding costs, which is consistent with huang et al. ( ) . third, our in-depth research on guarantees reveals the heterogeneities of implicit and explicit government guarantees. we find that credit spreads are about bps higher with guarantors or explicit government guarantees, while implicit guarantee significantly decreases the spreads of bonds by %, very significantly. the mechanism is adapted from regional government intervention and macro heterogeneities. we use two indicators to identify government intervention following hao and lu ( ) , based on employees or and gdp, respectively. we find that there are significant effects stemming from government intervention. regional macro heterogeneities are also examined from regional economic development, fiscal statement and bank loan credit. we find that the implicit government guarantee has more power over bank loans and decreasing the mcb's financing cost in more developed regions and more bank loans. economic development dominates this effect. in order to handle the endogeneity, we introduce an instrumental variable to predict local gdp per capita, an intersection of regional - -medical and health expenditure in public expenditure per capita and the pandemic. the latter is a dummy variable, displaying either high or low cumulative confirmed cases of coronavirus in this region. the results are robust. the reason why we introduce this iv based on public health is on the basis of the power of local governments pertaining to public health, as pointed out by journalists (for example, the lead article on economist may , ). prominent studies have a tendency to focus on the black side of government intervention, like conflicts among different parties in production under asymmetric information (cailaud, et al., ) , policy burdens and soft budget constraint (lin & tan, ) , making inefficient investment decisions (chen, et al., ) . however, price is endogenous to government policy, which is a clear sign of a key economic force. when governments make decisions based on information they learn from market prices, this affects the amount of information the government can obtain. bond and goldstein ( ) documents that the government discloses details about a variable that would be beneficial to speculators. this in turn, helps the government due to the reduction of risk faced by speculators because of the disclosed stock prices. but bond and goldstein ( ) maintains the concern that the government would conduct major interventions without having precise information about the costs and benefits of doing so. china's bond market and the recent tightening of interventions provide insights of bond and goldstein's concern. china government has much more information on municipal corporate bonds (mcbs) than the speculators, but it kept marketizing mcbs without learning from the information on mcbs' price. the implicit government guarantee should decrease as these marketing policies being practiced, in contrast, we find that market kept chasing these mcbs because of the implicit guarantees which they regard as much safer. our paper is related to the literature on political connections or state ownership. previous research focused on the political connections, originating from the findings of fisman ( ) . fisman and wang ( ) which provide evidence on how political connections enable firms to avoid compliance measures based on their finding that the death rate of workers for connected companies is ~ times that of unconnected firms. gao et al. ( ) recently find that established local politicians engage less often in selective defaults on bank loans. our paper provides a perspective from the bond market to explain the bargaining between government and market. it also provides evidence on the research of state ownership. shi and zhang ( ) institutions have entered the chinese bond market, with a debt holding amount of more than . trillion rmb. we provide an insight from implicit government guarantee on an aspect of the post-stimulus period in china. while our paper draws evidence from china, the insight is also able to explain the relationship between government and market more broadly. this paper may also provide new evidence on some important theoretic literature, such as hart and zingales ( ) , bond and goldstein ( ) , who call for the state to make use of information contained in market prices, as many researchers and policy makers suggest. the paper is organized as follows. section briefly reviews china bond market and regulations, and the implicit government guarantees implied in mcbs. section describes the sample and data. section presents the empirical results. section analyzes the mechanism and section further exploits the bargaining between state and market. section introduces bonferroni tests, extreme bounds analysis and placebo tests as robustness checks. section summarizes our findings and has bypassed its own stock market capitalization and the level of the u.s. bond market in . therefore, the chinese bond market plays an increasingly important role, whether within its own capital market or the global perspective. panel b of figure compares the proportions of government debt over gdp in g countries. china has a much lower government debt ratio than other developed countries except australia, and even some developing countries like india and brazil. the reason may lie in that some chinese government debts have been converted into corporate debt, municipal corporate bonds (mcb), whose issuer is a special corporation called as local government financial vehicle. it is more convenient for the government to conduct financing and investment through these mcbs. chinese government debt - -is implied within corporate debt, which is a unique perspective of focusing on the bargain between state and market. [insert figure ] jiang et al. ( ) provide an overview of chinese capital markets before . they documented that despite the accelerated growth of china's bond markets are growing fast but bank financing still dominates debt financing in china. as panel a of figure shows, chinese bond market grows rapidly after , the period examined by jiang et al. ( ) based on issuing entities, the market is classified into three broad bond categories; government, financial, and corporate bonds. according to amstad and he ( ) , government bonds is . % in china which is lower than us ( . %). why does china issue less government bonds than the u.s.? the reason amstad and he ( ) did not mention is that chinese local government obtains finance through mcbs which is issued by lgfvs. this means, more and more government debts are regarded as corporate bonds which embedded in national balance sheet. lgfvs. lgfvs played an integral role in implementing the fiscal expansion of and . the local government could not issue bond to raise money and is heavily dependent on the central government before . the local government has to establish lgfvs to raise money indirectly by issuing mcbs. bai et al. ( ) and chen et al. ( a) this paper focuses on an aspect of post-stimulus period in china. in this period, the government tightened the regulations on lgfvs. figure shows the revolution of the tightening regulation on china's lgfvs. as the non-standard municipal corporate crisis happened in , and the first soe bond baoding tianwei defaulted in april , the market began to explore the value of implicit government guarantee (jin et al. process, market is still mcbs as a safe-haven asset due to its implicit guarantee nature. [insert figure ] implicit government guarantee, also known as soft budget constraints, is implied in almost all the developed and developing countries. dewatripont and maskin ( ) documents that centralized economy is more likely to have soft budget constraints. the existence of implicit guarantee requires the government to spend more energy to manage the financial security problems caused by this implicit debt, thus triggering the credit crisis of local governments. enterprises will blindly expand aggressive investment activities with the guarantee, constantly expand credit scale, increase leverage ratio and aggravate risk problems. the market's general expectation of soft budget constraint will disrupt the normal development of financial market order. because of their close relationship with the government, soes are strongly motivated to seek help from the government in the form of soft budget - -constraints (lin and tan, ) . banks will tighten credit to higher-risk borrowers with less collateral and higher regulatory costs, which result in credit rationing (stiglitz & weiss, ) . conversely, if banks prioritize the allocation of credit to politically connected customers, such as soes, it is because they often perpetuate implicit or explicit government guarantees. huang et al. ( ) measures the risk of implicit guarantees on chinese shadow interbank products. they find that banks extend more implicit guarantees to shadow bank products when their solvency deteriorates. ( ) verifies that political connections offset the negative relation between investment and political uncertainty because it reduced information asymmetry for connected firms resulting in a delaying of investment in anticipation of future lucrative tax incentives. nagano ( ) documents that firm information asymmetry, a common feature of emerging markets, moderates the negative relationship between the firm's financial constraints and debt security issuance. the issuing of credit spreads are more dependent on the fluctuations in the macroeconomic cycle (gilchrist and zakraisek, ), as well as the quality of issuer (benzion et al., ) we start with a sample of all chinese enterprise bonds (eb hereafter), exchange-traded corporate bonds (etcb hereafter), and medium-term notes (mtn hereafter) issued between and . amstad and he ( ) we only keep state-owned enterprises (soes) or private-owned enterprises (poes), thus bonds constitute our final sample. [insert table ] we collect bond characteristics and financial indicators of issuers from wind database. bonds issuers are required to submit quarterly financial reports by securities law of the people's republic of china. therefore, although % of bond issuers are not listed, their financial indicators are able to be obtained. we use the data from their financial reports in the last quarter to reduce the concern of endogeneity. all corporate-level financial indicators have been winsorized by year. the definitions of variables are shown in appendix table a . bond daily transaction data and macro-economic data are obtained from csmar database. we also use an iv based on the recent shock to the global economy, covid- , to reduce the concern from endogeneity, because pandemics are more exogenous compared with policy shocks. we use credit spread to measure the financing cost of the bonds. spread is the difference between bond yield and the matching central government bond yield, which has the same cash flow characteristics with the same issuance date and maturity following ang et al. ( ) . we also use the second index, spreadcdb, to measure credit spread, defined as the bond yield minus the matching china development bank (cdb) bond yields following chen et al. ( a) . the results are robust. panel a of table compares mcbs and other soe bonds with poe bonds in order to reveal the existence of implicit government guarantee. poes are regarded as being without government guarantee, therefore we use poes as the basis group. the comparison in panel a of table demonstrates the existence of government implicit guarantee in mcbs, central soes and local soes. [insert table ] table reports the average credit spreads of the bonds according to administrative regions year by year. we divide the sample into ten subsamples by sorting deciles of the regional gdp per capital, with the lowest group denoted as and the highest group denoted as . the result implies that bond spread is relatively lower in the developed regions with higher average gdp in average. among them, the anomaly that only exists in may be due to the relatively small number of bonds issued during - -the year, which was greatly affected by extreme values. we will further discuss the impact of macro variables on bond spreads in section . . we also divide the sample by sorting city-level gdp in panel b of table . the result is consistent with panel a of table . [insert table ] ． ． ． ．empirical results first, we propose the following benchmark model to test hypothesis : includes variables of the firm issuing the bond; return on assets (roa), total debt over total asset (leverage), the difference of current operating income and initial operating income over initial operating income (salegrowth), the logarithm of total asset (firmsize), and the difference between the - -year firm established and year bond issuance (age). α s, α p and α t is industrial, provincial and year fixed effect, respectively. table shows the regression results. column ( ) is the baseline model following ang et al. ( ). following erel et al. ( ) , we introduce two impact factors into the regression in column ( ), privatesector controlling the development of poes, and marketcap controlling the financing from equity market in province p. column ( ) implies that the financing cost of soe is . % lower than that of poe. column ( )-column ( ) show the same results that central soe, local soe and mcb have . %, . % and . % lower than poe ceteris paribus. we can find that the coefficient of central soes is even lower than local soes, which implies that central soes are provided a stronger implicit guarantee from the central government. in column ( ), we define mcb i as treated i , which equals to when the bond issuer is lgfv, while it equals to when the issuer is poe. the result shows that mean spread of mcbs is lower than that of poes by . %. [insert table ] to deal with the concern that mcbs usually have higher credit ratings compared to bonds of poes, we further compare the matched sample in table . column ( ) and column ( ) report the regression results in non-matched sample. column ( ) and column ( ) report the results in propensity-score matched sample with the same region, the same year and the same credit rate where we select the closest propensity score without replacement. the results are consistent. the coefficients of mcbvspoe are all significantly negative. lgfv, mcb's issuer, has a significantly lower financing cost than poes, around basis points. [insert lgfv and the level of lgfv is prefectural level, while it equals to when the issuer is poe. countylevel is a dummy variable. it equals to if the issuer is lgfv and the level is county and county-level cities, while it equals to when the issuer is poe. ( ) and column ( ) show. county-level lgfvs have around . % lower spreads than poes as the last two columns demonstrate. our results imply that the provincial government provides the strongest implicit guarantee, followed by the prefecture-level government and county level. we control the credit rating fixed effect in column ( ), column ( ) and column ( ), which are consistent with column ( ), column ( ) and column ( ). our paper provides strong evidence on political decentralization as mentioned by bardhan ( ) and huang et al. ( ) . china state (the central government) has been decentralizing those soes with poor performance. our paper provides evidence to their findings by the side of mcbs. [insert table ] . . implicit guarantee compared with explicit guarantee some credit bonds have guarantor to increase market confidence in anticipation of successful issuance. however, the market will respond differently to the guarantor's qualifications. then we study the impact of different kind guarantor. where guarantee i is a dummy variable to indicate the guarantee state of bond i. we use three variables, dummyguarantee, exgovguarantee and imgovguarantee, respectively. the first indicator, dummyguarantee, equals if the bond is guaranteed, otherwise is . the coefficient of dummyguarantee in column ( ) in table is positive, which means the spread of guaranteed bonds is . % higher than those unguaranteed. the second indicator, exgovguarantee, is a dummy variable to indicate explicit government guarantee, which equals if the bond is guaranteed by government or soe, otherwise is . the result in column ( ) shows that the credit spread is . % higher. the third indicator, imgovguarantee, is a dummy variable to grasp the effect from implicit government guarantee. we only keep the bonds unguaranteed. it equals to if the bond is unguaranteed but the issuer is state owned, otherwise . this is because unguaranteed soe bonds are largely subject to implicit government guarantees for political purposes to prevent bond defaults. column ( ) shows that the coefficient of imgovguarantee is significantly negative, which proves that the spread of state-owned corporate bonds with implicit government guarantees is averagely . % lower. we further introduce the intersection terms with mcbvspoe to ascertain the heterogeneity between mcbs and poes in column ( )-column ( ). the imgovguarantee in column ( )-column ( ) equals mcbvspoe in column ( ) -column ( ). therefore, these three regressions run performance tests between the two explicit guarantees and implicit guarantee. the regression results show that both the guarantees are significant. implicit guarantee, which is indicated definitely as mcbsvspoe, reduces the spread about %, i.e. basis points. for the two explicit guarantees, dummyguarantee and exgovguarantee, both reduce the spread for the control group, poes. the two intersection terms are significantly positive and more than the coefficients of the two explicit guarantees, implying that explicit guarantee increases mcb's spread. therefore, the conclusion from column ( )-column ( ) is much clearer than the above conclusion in column ( )-column ( ) in which mcb and other bonds of soes are much more than poes. considering the heterogeneity between mcb and poe, we find that for poes, explicit guarantee has a significant effect of reducing the spread, but it increases the spread of mcbs because the of market's concerns. [insert table ] this section aims to explain why there are variances in spread in different regions as table shows. first, we exploit the effect of government intervention by the following model, where govint p,t- is the indicator measuring the strength of government intervention in province p. we use two measures following hao and lu ( ) , the ratio of the number of employees in public administration, social security and social organizations to the total number of employees (govint ) and the ratio of government expenditure to local gdp (govint ). table reports the result of equation ( ). column ( )-column ( ) compares mcb with poes. column ( )-column ( ) compares mcb with local soes. both of the coefficients of government intervention based on gdp, govint , are significantly positive, which is helpful to explain our findings of table . there is a relatively low degree of marketization in the provinces with strong government intervention, that goes against the firm's objective of maximizing profit. the government has been interfering with the companies' normal operations and risk is increasing, so the credit spread increases. the intersection terms exploit the heterogeneity. column ( ) and ( ) compares the difference in mechanism of government intervention's impact on spreads between mcbs and poe bonds. column ( ) and ( ) compares the difference in mechanism between mcbs and local soe bonds. the coefficients of intersection terms are all insignificant, implying the robustness of our conclusion. [insert table ] in this section, we discuss about the effects of different macro environments: spread i =β fiscal r,t- +γ bond i +γ issuer j,t- +α s +α t +ε i where fiscal represents the provincial fiscal statement in the region r where the issuer is located. here we consider both provincial-level and city-level fiscal statements. provincial-level variables include - -the logarithm of the per capita gdp (lnpergdp p,t- ), the logarithm of the local gdp of the real estate sector (realestategdp p,t- ), the logarithm of the local tax revenue of the real estate sector (realestatetax p,t- ) and the growth rate of housing prices in each province (housepricegrowth p,t- ). city-level variables include the logarithm of per capita gdp of the city of the lgfv (lnpercitygdp c,t- ) and which is defined as the value of the outstanding loan balance of a financial institution prefectural-level, financial institution divided by the gdp of that city (privatecredit c,t- ). since macro includes the provincial-level variables, we exclude these two macro control variables to reduce multicollinearity. we use mcbs as the sample to test equation ( ) in table because we only focus on the impact of government fiscal situation on mcbs, which have close relationship with the government. column ( )-column ( ) ascertains the effect from each provincial-level fiscal statement. column ( ) considers their effects together. first, from the effect of local gdp, column ( ), column ( ) and column ( ) all imply that spreads decline in developed province or city. column ( ) shows the impact from gdp is the strongest among all the factors. this is consistent with our prior findings. mcb has about . % decrease in spread with every % increase in local gdp per capita, which is even stronger in the city level. second, we also consider the impact from real estate following ang et al. ( ) . we use three variables to indicate the impact from real estate, the local gdp of the real estate sector, real estate tax and the growth rate of housing prices. the regression results show that when the real estate gdp increases by %, the issued credit spread will decrease by . %, and significantly at the % level. the results in columns ( ) and ( ) show that the greater the degree of government intervention, the lower the spread of the mcbs. by adding all provincial variables in column ( ), lnpergdp p,t- and realestategdp p,t- still exert a significant negative impact. this implies that mcbs have lower financing cost in developed province. third, further considering the impact from bank loans, we keep the strongest impact factor, gdp but at city level, and add privatecredit, the ratio of bank loans over gdp in the city. column ( )-column ( ) show significant impact from these two city-level factors. the coefficients are both negative whenever we put them in the regression separately or together. gdp still has significantly negative impact. for bank loan's effect, the higher privatecredit means it is easier for the firm to obtain bank loans. our results show that if privatecredit increases by %, mcb's spread falls . %. but the effect shrinks rapidly, by . % if we consider the effect of economic development as column ( ) shows. this provides evidence that economic development has stronger effect on reducing mcb's financing cost than bank loans do. [insert table ] in order to reduce the endogeneity, we also introduce an exotic instrumental variable to predict lnpergdp. since we use gdp per capital, the population characteristic and investment should be critical to it. the covid- outbreak is an exogenous shock to the global economy, as recent papers examined the relationship between the pandemic and economic development, ex. jia et al. ( ), fang et al. ( ) and qiu et al. ( ) . we introduce an intersection of regional medical and health expenditure in public expenditure per capita with pandemic, and lag item of regional gdp in the first stage to predict regional economic development . the sls regression is as follows, lnpergdp rt =β healthexpenditure , × pandemic+ lnpergdp ,t- +α r r r r +α t +ε ( ) where healthexpenditure is the logarithm of regional medical and health expenditure in public expenditure per capita. pandemic is a dummy, which equals above the median of the cumulative confirmed cases in this region on april , , otherwise is . april , is the date when wuhan, the city that was the epicenter of the coronavirus outbreak, reopened and the lockdown restrictions were eased, indicating that the number of cases has been steady afterwards. we also use the number of deaths and recoveries as well. the results are robust. table still reports the robust results as table shows. [insert year and same province. [insert figure ] we use did model to exploit the structural change of article as follows, where post t is a dummy, equaling to if the bond was issued after september, . α s means the industry fixed effect, and α p means province fixed effect and α t shows year fixed effect. column ( ) and ( ) show the result of psm-did, which we match the bond credit rating, year and province fixed-effect. column ( ) represents that compare to the local soes' bonds with same credit rating and issuance year and province. ( ) shows. the decrease is even lower when we use the matched sample, even . % lower than local soe. column ( ) and column ( ) show the results compared with poe. the coefficients of mcb and intersection term, mcb×post, are significantly negative, which implies that mcb has lower spread than poes by around . % before the shock, then even lower after that. mcb has even lower financing costs by about % whether we use matched sample or not. [insert table ] table further exploits the impact from administrative level before and after article by considering capital-level and prefectural-level mcbs, respectively. for the capital-level mcbs, we divide the samples into ten groups according to provincial gdp where the issuer of mcb located in. the group of highest gdp is denoted as high gdp, and the lowest is denoted as low gdp. as for city level mcbs, we divided the samples into ten groups according to gdp of the city where the - -issuer of mcbs located. table shows that the mean spread of each region is lower after the shock than before. for the capital-level mcbs, we find that mcb in high gdp provinces has . % lower spread than in low gdp provinces before article in column ( ). this difference is even strengthened after the shock, which is . % as column ( ) shows. for prefectural level mcbs, we still find that this difference is significant, . % in average before the shock. but after the shock, there is no significant difference between high and low cities among the prefectural mcbs. thus, we find that the mean spread of mcbs even declined after the implementation article for mcbs at both capital and prefectural level. [insert table ] finally, we will explain why the spread declines even after the government aims to begin marketization of those mcbs. the reason lies in the market concern on bond defaults. we introduce the variable default to indicate our hypothesis. the regression model is as follows, where we use the total default, local soe default and poe default, respectively. ( ) considers these two factors together, the conclusion still holds and we find poes default has more impact. those poes' defaults restore the market's confidence in the mcbs. that is the reason why lgfv could issue bonds at lower spreads. [insert table ] we calculate the bonferroni's upper bound of joint distribution on the true joint p-value. we have controls and an independent variable in the base model, so we use the one-thirteenth p-value ( . / ) as the significant standard. in panel a of table , ***, **, * means p< . , p< . , p< . respectively, and the results still are robust when we use the strict bonferroni bound. the result is consistent with our benchmark model of table . [insert table ] leamer ( ) we find strong evidence that government guarantee reduces the funding cost by examining the credit spread of corporate bonds in china. mcbs have significantly lower funding costs because of the implicit government guarantee. according to administrative level of lgfvs, states provide the strongest implicit guarantee, then prefectural level, and county level is the last. we also find the heterogeneity of explicit guarantee and implicit guarantee between mcbs and poes. although explicit guarantee reduces poe bond's spread, it is a bad signal for the market on mcbs where the spread increases if it is issued with explicit guarantees. in contrast to explicit guarantee, implicit guarantee is good news because the market has more confidence on mcbs rather than poe bonds. we then analyze the mechanism by two perspectives, government intervention and macro heterogeneities. we find that mcb still has lower spread than poe bonds even introducing two indicators of government intervention. regional economic development has the strongest effect whenever we use province-level or city-level gdp per capita. bank loans reduce the spread too, but this effect shrinks rapidly introducing economic development. these results contribute to the literature on policy decentralization and government intervention. furthermore, the bargaining between market and government is exploited, by introducing the indicators of government intervention. divide government expenditure by local government gdp (hao & lu, ) govint indicators of government intervention. divide the number of employees in public administration, social security and social organizations by the total number of employees (hao & lu, ) privatesector the total employees in private and self-employed enterprises over total employees in each province marketcap the market value of listed companies over gdp (erel et al., ) in each province lnpergdp the logarithm of gdp per capital in each province realestategdp the logarithm of gdp of real estate sector in each province realestatetax the logarithm of local tax revenue of real estate sector in each province housepricegrowth growth rate of housing prices in each province lnpercitygdp the logarithm of per capita gdp in each city privatecredit the total outstanding loan over gdp (erel et al., ) in each city healthexpenditure the logarithm of regional medical and health expenditure in public expenditure per capita. a dummy equals above the median of the cumulative confirmed cases in this region on april , , otherwise is . april , is the date of the end of the lockdown and the reopening of the city of wuhan.. municipal corporate bond dummy variable. lgfv is , and poe is rateaaa dummy variable. it equals to if the bond is aaa-rated when issued, otherwise is . lnbondsize the logarithm of bond size (in million rmb) lnmaturity the logarithm of bonds maturity (in year) eb dummy variables. it equals to if the bond is enterprise bond regulated by ndrc, otherwise is . dummy variables. it equals to if the bond is middle-term note, otherwise is . dummy variable. it equals to if the administrative level is province, provincial capitals and cities specifically designated in the state plan (single-listed-city), otherwise is . prefecturallevel dummy variable. it equals to if the level is prefecture-level city, otherwise is . asset-liability ratio (%) salegrowth current operating income minus initial operating income over initial operating income firmsize the logarithm of total asset of the issuer. the difference between the year firm established and year bond issuance figure (a) reports the growth of the chinese bond market capitalization scaled by gdp (in bars) or stock market capitalization (in lines). as for the proportions of government debt over gdp in g countries, which implies that china has a much lower government debt ratio than developed countries except australia, and even some developing countries like india and brazil. figure shows the revolution of the tightening regulation on china's lgfvs. as the non-standard municipal corporate crisis happened in , and the first soe bond baoding tianwei defaulted in april , the market began to explore the value of government's implicit guarantee (jin et al. ) . under the background of deinventory and deleverage, the secure payout of state-owned enterprises is broken, resulting in the market turmoil. implicit guarantee should have been gradually losing its effectiveness. on the other hand, the rapidly increasing implicit debt of local government has drowned central government's attention, and a growing number of mcbs improve the debt risk. the state council issued article request lgfv to be decoupled from local government and the debt cannot be regarded as implicit debt of local government, which decrease the implicit guarantee of local government. article regulates on local government financing. article regulates the lgfv and clearly defining the boundaries between the government and enterprises. the first wave of bond defaults broke out. table summary statistics of china's bond market data. non-mcbs but soe bonds bonds of poes year etcb eb mtn etcb eb mtn etcb eb mtn table compares mcbs and other soe bonds with poe bonds in order to reveal the existence of implicit government guarantee. poes are regarded as being without government guarantee, therefore we use poes as the basis group. columns ( ) to ( ) report the mean and median credit spreads. medians are in the brackets. columns ( ) to ( ) show the difference compared to poes. the significance of mean or median tests is based on one-side t-tests or rank-sum tests (in parentheses). the comparison in table in table , we summarize the spreads according to the administrative regions year by year since bond spreads are affected by regional characteristics. we divide ten subsamples according to deciles of province gdp, and the lowest group is denoted as , while the highest group is denoted as . the result shows that bond spreads are relatively small in provinces with higher gdp and relatively larger in provinces with lower gdp on the whole. we will further discuss the impact of macro variables on bond spreads in section . . we also divide ten subsamples by city gdp in panel b of table , and the result is consistent with panel a of table . the effect of implicit guarantee ( ) ( ) ( ) ( ) ( ) ( ) table shows the regression results. see appendix table a for definition of the variables. *** p< . , ** p< . , * p< . . to deal with the concern that mcbs usually have higher credit ratings compared to bonds of poes, we further compare the matched sample in table . column ( ) and column ( ) reports the regression results in non-matched sample. column ( ) and column ( ) reports the results in propensity-score matched sample with the same region, the same year and the same credit rate where we select the closest propensity score without replacement. see appendix table a for definition of the variables *** p< . , ** p< . , * p< . . table shows mcbs are lower in different administrative level compared to poes. we use capital-level lgfvs in column ( ) and column ( ), and we use prefectural-level lgfvs to compare against poes in column ( ) and column ( ). county-level lgfvs in the last two columns. we control the credit rating fixed effect in column ( ), column ( ) and column ( ), which are consistent with column ( ), column ( ) and column ( ). see appendix table a for definition of the variables .*** p< . , ** p< . , * p< . . table shows the different results among variety of guarantee. see appendix table a for definition of the variables. *** p< . , ** p< . , * p< . . table reports the result of equation ( ). column ( ) and ( ) compare the difference in mechanism of government intervention's impact on spreads between mcbs and poe bonds. column ( ) and ( ) compare the difference in mechanism between mcbs and local soe bonds. see appendix table a for definition of the variables.*** p< . , ** p< . , * p< . . we use mcbs as the sample to test equation ( ) in table because we only focus on the impact of government fiscal situation on mcbs, which have close relationship with government. column ( )-column ( ) exploit the effect from each provincial-level fiscal statement. column ( ) considers their effects together. column ( )-column ( ) show significant impact from these two city-level factors. for bank loan's effect, the higher privatecredite means it is easier for the firm to obtain bank loans. see appendix table a for definition of the variables.*** p< . , ** p< . , * p< . . ( ) ( ) ( ) step : lnpergdp step : spread step in order to reduce the endogeneity, we also introduce an exotic instrumental variable to predict lnpergdp. the covid- outbreak is an exogenous shock to the global economy, as recent papers examined the relationship between pandemic and economic development, ex. jia et al. ( ) , fang et al. ( ) and qiu et al. ( ) . we introduce an intersection of regional medical and health expenditure in public expenditure per capita with the pandemic, and lag item of regional gdp in the first stage to predict regional economic development. we also use the number of death and recoveries. the results are robust. see appendix table a for definition of the variables.*** p< . , ** p< . , * p< . . the shock of article . ( ) ( ) table shows the heterogeneous effects of mcb with local soe and poe, and the structural change from the shock of article . we use the unmatched sample in column ( ) and matched sample in column ( ). the regression results are consistent. column ( ) and column ( ) show the results compared with poe. see appendix table a for definition of the variables.*** p< . , ** p< . , * p< . . high-low - . *** - . *** - . ** - . table further exploits the impact from administrative level before and after article by considering capital-level and prefectural-level mcbs, respectively. for the capital-level mcbs, we divide the samples into ten groups according to provincial gdp where the issuer of mcb located in. the group of highest gdp is denoted as high gdp, and the lowest is denoted as low gdp. as for city level mcbs, we divided the samples into ten groups according to gdp of the city where the issuer of mcbs located. see appendix table a for definition of the variables.*** p< . , ** p< . , * p< . . impact of bond defaults. ( ) ( ) ( ) ( ) table reports the regression results, where we use the logarithm of number of defaulted bonds, we also use the logarithm of value of the defaulted bonds and the cumulative number of defaulted bonds which are all consistent. we still focus on the mcb subsample in the following regressions. column ( ) shows that the greater the number of default bonds, the lower spread of mcb has. column ( ) and column ( ) further consider the impact from local soe's default and poe's default. column ( ) considers these two factors together. see appendix table a for definition of the variables.*** p< . , ** p< . , * p< . . chinese bond market and interbank market. chapter for the handbook of "china's financial system the great wall of debt: real estate, political risk and chinese local government financing cost heterogeneous taxes and limited risk sharing: evidence from municipal bonds. the review of financial studies the long shadow of china's fiscal expansion state and development: the need for a reappraisal of the current literature debt composition and lax screening in the corporate bond market government intervention and information aggregation by prices government ownership and the cost of debt: evidence from government investments in publicly traded firms government intervention in production and incentives theory: a review of recent contributions pledgeability and asset prices: evidence from the chinese corporate bond markets quantifying liquidity and default risks of corporate bonds over the business cycle government intervention and investment efficiency: evidence from china the financing of local government in china: stimulus loan wanes and shadow banking waxes credit and efficiency in centralized and decentralized economies credit-induced boom and bust do acquisitions relieve target firms' financial constraints? politically connected firms public governance and corporate finance: evidence from corruption cases human mobility restrictions and the spread of the novel coronavirus ( -ncov) in china estimating the value of political connections the mortality cost of political connections subnational debt of china: the politics-finance nexus economic fluctuations and growth credit spreads and business cycle fluctuations do politically connected boards affect firm value? the impact of government intervention on corporate investment allocations and efficiency: evidence from china a new capital regulation for large financial institutions political connections and the cost of bank loans the risk of implicit guarantees: evidence from the shadow interbank market in china local crowding out in china hayek, local information, and commanding heights: decentralizing state-owned enterprises in china population flow drives spatio-temporal distribution of covid- in china capital market, financial institutions and corporate finance in china the value and real effects of implicit government guarantees let's take the con out of econometrics political connections, financing and firm performance: evidence from chinese private firms policy burdens, accountability, and the soft budget constraint implicit government guarantee and the pricing of chinese lgfv debt what promotes/prevents firm bond issuance in emerging economies: bank-firm relationship or information asymmetry? inventory capacity and corporate bond offerings impacts of social and economic factors on the transmission of coronavirus disease (covid- ) in china government credit, a double-edged sword: evidence from the china development bank how to explain corporate investment heterogeneity in china's new normal: structural models with state-owned property rights credit rationing in markets with imperfect information mitigating political uncertainty is trust priced? evidence from the bond market does mixed-ownership reform improve soe's innovation? evidence from china's state ownership ebs are regulated by the national development and reform commission (ndrc), a powerful government agency overseeing soes. etcbs are issued in the exchange market and regulated by china securities regulatory commission (csrc) yes key: cord- - rcqbgl authors: shidham, vinod b.; frisch, nora k.; layfield, lester j. title: severe acute respiratory syndrome coronavirus (the cause of covid ) in different types of clinical specimens and implications for cytopathology specimen: an editorial review with recommendations date: - - journal: cytojournal doi: . /cytojournal_ _ sha: doc_id: cord_uid: rcqbgl nan coronavirus disease (covid- ) caused by "severe acute respiratory syndrome coronavirus " (sars-cov- ) was first reported in china. [ ] is is the third extremely pathogenic human coronavirus which has emerged recently after severe acute respiratory syndrome (sars) coronavirus and middle east respiratory syndrome (mers) coronavirus. sars-cov- is mainly transmitted by person-to-person contact in community and health-care settings. [ ] is pattern of spread demands large-scale and proactive measures to avoid further widespread dissemination. sars-cov- survives on contaminated dry surfaces and fomites, which facilitate hand to mucous membranes (of the mouth, nose, and eyes) spread. [ , ] is emphasizes the significance of in-depth knowledge about the perseverance of coronavirus on inanimate surfaces. [ ] various fixatives and biocidal agents are widely used in health-care settings including cytopathology laboratories which may impact (and help negate) the spread of this virus. [ , ] e current review summarizes the available relevant data on this topic about cytopathology laboratory protocols and suggests precautions based on this data. ese recommendations may change as new information comes to light. each institution and laboratory has to adapt and adjust depending on local regulatory limitations. e recommendations suggested at the end of the review discussion should be considered with these things in mind. at present, the diagnosis of covid is mostly being accomplished by performing real-time reverse transcriptase-polymerase chain reaction (rrt-pcr) for sars-cov- on respiratory specimens such as nasopharyngeal swabs. [ ] e primary mode of spread, which has led to the global pandemic, is the respiratory route. a report studying different types of clinical specimens from patients (most cases - days after hospital admission) detected a positive pcr test in about % (n = ) of blood samples, suggesting the systemic nature of the disease in at least a portion of the cases. [ ] is study also reported positive results in % (n = ) of fecal samples. [ ] although tests based on pcr have been reported positive in non-respiratory specimens, it does equate with infectivity and so the non-respiratory route of viral transmission is possible but is not proven at this stage. as mentioned previously, the molecular test to detect sars-cov- is based on rrt-pcr. is test amplifies viral rna in a patient's specimen for the detection of sars-cov- genetic material. [ ] false negatives are possible due to multiple variables including simple technical errors, inadequate collection, improper handling, and shipping. other possibilities for incorrect results include flawed key reagents in the kit. [ ] one study comparing rrt-pcr with computerized tomography (ct), reports that the sensitivity of the test is only %. [ ] further compounding the issue, the sensitivity may be lower in some symptomatic cases due to a smaller viral load, [ ] but usually the viral load is higher in the early stages of the disease. however, newer tests suggest higher sensitivity with the potential of false positives due to very high sensitivity and possible cross contaminations during the pandemic with the widespread presence of virus (at the level of collection of sample and later during the technical procedure). as a result, even if the specimen in the cytopathology laboratory is not expected to be positive or even if the molecular test is negative for sars-cov- , all of the specimens should be considered potentially positive. it is therefore mandatory to practice all the universal/standard precautions with basic protective measures while handling any biological specimen irrespective of sars-cov- status [tables - ] . [ ] [ ] [ ] all cytology personnel should follow the basic protective measures against sars-cov- recommended by the world health organization (who) [ table ]. [ ] although, appropriate disinfectants and precautions related to cytopathological/histological fixation and processing of samples during the current covid pandemic are not known, information can likely be extrapolated from other recent coronaviruses (e.g., sars and mers). similar table : summary of the interim laboratory biosafety guidelines from the cdc for the specimens suspected for or positive for sars-cov- . [ ] . follow standard precautions when handling clinical specimens, all of which may contain potentially infectious materials mentioned in table . . any technique which may generate aerosols or droplets (e.g., squirting [instead of gently delivering as tiny drops] of the specimen through a needle, vertexing) should be avoided, but is required to be practiced then it should be executed in a certified class ii biological safety cabinet (bsc). similarly, for centrifugation suitable physical containment should be practiced with securely capped specimen tubes. ideally, such procedures should also be performed in a class ii biological safety cabinet. . clean and disinfect the equipment(s) and work surfaces after specimens are processed using appropriate disinfectants which are used for disinfecting other respiratory pathogens, such as other human coronaviruses and seasonal influenza viruses. . practice standard procedures applied for other respiratory pathogens, such as other human coronaviruses and seasonal influenza viruses. . if the diagnostic testing specimens are processed outside of a bsl- laboratory, [ ] such as preparation of cytology direct smears, rinsing of fnab aspirates for cell block, the standard precautions (similar to those mentioned under table ) should be practiced as a barrier between the specimen and personnel. [ ] . preparation and fixing of cytology smears should be performed under certified a class ii [ ] biological safety cabinet. table : standard precautions -summary modified for cytopathology laboratory specimens based on the cdc guidelines. [ , ] . ensure that the technologist use personal protective equipment (ppe) such as medical mask, gloves, eye protection, and a long-sleeved gown. . if the specimen with potential of aerosol-generation, such as squirting of aspirates from fine-needle aspiration biopsy procedure is being processed, the personnel should wear at least the protective mask such as niosh certified n , an eu standard ffp , or the equivalent. . all personnel involved in handling and transporting the specimens should be trained for safely handling the process, including spill decontamination methods. . transport the primary specimen container with the patient's label in a leak-proof secondary containers, such as sealable plastic biohazard specimen bag with properly filled laboratory requisition. . adhere to all biosafety practices including transport precautions ( c) depending on the pathophysiology of the organism being considered. . preferably transport fresh, unfixed specimens by hand, and do not ship the specimen with pneumatic-tube systems. . each specimen must be clearly labeled with at least two patient identifiers including full name and date of birth with other details with specific warnings as applicable (e.g., suspected or confirmed sars-cov- virus) on the form. let the laboratory know immediately that such a specimen is on the way. [ ] a. wash your hands frequently with soap and water counting up to (approx. s). b. maintain social distancing and maintain at least m ( ft) distance between yourself and anyone to avoid droplet/ microparticle infection due to coughing, sneezing, and even talking. [ , ] c. avoid touching face (eyes, nose, and mouth) is the most important component as final personal protection. studies showed that rate of unknowingly touching the face is up to - /h. [ , ] e most probable mode of getting infection is from many inanimate surfaces [ table ]. d. if you are sick with fever, cough, or difficulty breathing, seek medical advice early and stay informed to follow updated advice by your health-care provider and official resources. protocols should be effective to disinfect and inactivate sars-cov- . [ ] us, once the processing is complete as cytopathologic preparation and after formalin fixation and paraffin embedding for cell-blocks, sars-cov- if present in the specimen should be inactive. [ ] a study reported that aerosol and fomite transmission of sars-cov- is possible because the pathogen continues to be infectious for hours in aerosols and for days on surfaces. [ ] e findings are comparable to those observed with sars-cov- [ ] providing useful information for the current mitigation efforts during this pandemic. e study observed that sars-cov- survives for up to h on cardboard and plastic/stainless-steel surfaces up to - days. [ ] [ ] [ ] many ostensibly clean surfaces and devices within a pathology department (including but not limited to doorknobs, tables and chairs, countertops, desks, keyboards, phones, microscopes, glass slides, slide trays/boards, on-site adequacy carts, door handles, light switches, toilets, faucets, and sinks) may be contaminated with the virus and should be considered potential fomites. [ ] e duration for which sars-cov- -like viruses have been reported to persist on various surfaces is listed in table . [ ] virus infectivity is defined as the ability of the virus to enter and colonize the host to replicate and reproduce itself with the potential to establish itself as an infection resulting in disease. [ ] a previously suggested benchmark, a log viral reduction factor of more than , is considered as virucidal effectivity against viruses including coronaviruses on surfaces. [ ] formalin and ethanol used in low concentration (usually used as a preservative) decrease the viral infectivity to more than log . [ ] e effect of time and concentrations of various fixatives and biocidal agents on sars and a few other viruses are summarized in table . e table shows that for ethanol, % ethanol with sars-cov (isolate ffm- ) for s reduces the viral infectivity to more than . log ; % ethanol with sars-cov (isolate ffm- ) for s reduces the viral infectivity to more than . log ; [ ] % ethanol with sars-cov (isolate ffm- ) for s reduces the viral infectivity to more than . log ; [ ] % ethanol with mers-cov (strain emc) for s reduces the viral infectivity to more than > . log ; [ ] % ethanol with sars-cov (isolate ffm- ) for s reduces table : summary of measures recommended for routine cytopathology division (in addition to the basic protective measures summarized in table ) during sars-cov- pandemic. general cytopathology division routine standard precautions [ table ]. [ , ] in the case of sars-cov- suspected or known case-avoid exposure to older personnel over years and personnel with compromised immunity. cytoprep laboratory routine standard precautions [ table ]. [ , ] in cases suspected or positive for sars-cov- virus-perform the processing in certified class ii [ ] biological safety cabinet fnab procedure to minimize the risk of exposure with rapid dissemination of the virus, the "on-site adequacy evaluation services" may be suspended during covid pandemic related suspension of elective procedures.** however, if fnab has to be performed without on-site adequacy evaluation: • do not squirt the specimen on the slide but drop gently on the slide without letting the specimen aerosolized. • spread the specimen between two slides with two patient identifiers as routine to make direct smears* to be processed according to individual laboratory protocol(s). • if cell-block is indicated (discuss with associated pathologist), collect the needle rinses directly in % formalin (avoid any alcohol-based fixative to prevent potential compromisation of immunohistochemistry results [ ] without contaminating the needle with formalin if that needle is to be re-used. • (recommended to be performed under class ii [ ] biological safety cabinet for cases suspected or positive for sars-cov- virus). • send all the material (direct smears in slide container(s) and appropriately labeled % formalin container with needle rinses for cell-block) in a leak-proof sealed (ziploc) specimen bag with properly filled requisition form. eus-fna procedure same guidelines as for "# fnab procedure" with extra precaution, because the endoscopes travel through anatomical sites with the highest proportion of viruses in potentially positive cases, especially asymptomatic ones. respiratory specimens such as bronchoalveolar lavage, bronchial lavage, bronchial brush, tracheal brush, sputum, and others such as rare percutaneous sampling of lung lesions routine standard precautions [ table ]. [ , ] in cases suspected or positive for sars-cov- virus-perform the processing in certified class ii [ ] biological safety cabinet. other body fluids such as peritoneal, pleural, pericardial, and other fluids (with potential positivity considered to be similar to blood positivity for sars-cov- virus) except urine routine standard precautions [ table ]. [ , ] in cases suspected or positive for sars-cov- virus-perform the processing in certified class ii [ ] biological safety cabinet. routine standard precautions [ table ]. [ , ] in cases suspected or positive for sars-cov- virus-perform the processing in certified class ii [ ] biological safety cabinet fix the cell-block for at least h (for - mm thick cell-block material, formalin diffuses at the rate of mm/h and % formalin ( . % formaldehyde) would inactivate the virus in the interior of the cell-block). most of the lab protocols require more than h (up to h) fixation. [ ] all specimens received in % formalin and alcoholic fixatives routine standard precautions [ table ]. [ , ] concerning the surfaces of transport containers and paperwork, etc. in cases suspected or positive for sars-cov- virus-perform the processing in certified class ii [ ] biological safety cabinet. splitting of specimens depending on individual institutional/laboratory protocols, some labs may split the specimen between various subspecialty labs. in such cases, the microbiology lab should split the specimen under microbiology precautions and send most of the specimen to the cytopathology lab for further processing. *air-drying both the direct smears spread between slides allows staining the smears with diff-quik and pap stain (as saline-rehydrated alcohol-fixed airdried smears). [ ] e saline used for rehydration should be discarded as biological waste similar to other biological specimens. e air-dried smears are easy to be transported to cytoprep lab in slide containers, which should be sanitized/disinfected by rinsing in % for - min, if the slide containers are reused. **if on-site adequacy evaluation services are resumed, all personnel associated with performing the procedure and performing the onsite adequacy service should follow routine standard precautions [ table ]. [ , ] ***modify as per local regulatory issues and geopolitical limitations based on general information reviewed in this editorial. the viral infectivity to more than . log ; [ ] % ethanol with mhv (strains mhv- and mhv-n) for min reduces the viral infectivity to more than > . log ; [ ] and % ethanol with ccv (strain i- ) for min reduces the viral infectivity to more than > . log . [ ] similarly for formalin, % formaldehyde with sars-cov (isolate ffm- ) for min reduces the viral infectivity to more than > . log ; [ ] . % formaldehyde with sars-cov (isolate ffm- ) for min reduces the viral infectivity to more than > . log ; [ ] . % formaldehyde with mhv for min reduces the viral infectivity to more than > . log ; [ ] . % formaldehyde with ccv (strain i- ) for min reduces the viral infectivity to more than > . log ; [ ] and . % formaldehyde with ccv for h reduces the viral infectivity to more than > . log [ ] [ table ]. about % ethanol reduces the viral infectivity to more than . in s and formaldehyde even in the concentration of % decreased the viral infectivity to more than in min [ table ]. [ ] various reagents such as ethanol and % formalin ( . % formaldehyde) usually used in cytopathology processing at initial stages as fixative have virucidal activity. [ ] based on the aforementioned, sars-cov- in any specimen processed with routine fixatives in cytopathology should be inactivated [ table ]. [ , ] e data presented thus far suggest that the susceptibility to infection follows the bell curve, with an increase in the severity of the disease with the higher mortality in members of the population over years. [ , ] is observation should be considered while organizing cytology laboratory services, namely, the potential effect on the personnel associated with various cytology services in regard to their potential for sars-cov- exposure. a summary of recommendations for procedure specific protocols in routine cytopathology is enumerated in table . ese guidelines are recommended in addition to the who basic protective measures at a personal level [ table ]. [ ] e virus survives on various plastic/metal surfaces, cardboards, disposable gowns, and paper surfaces for a significant amount of time [ table ]. [ , , , , ] to avoid dissemination of the virus, it is recommended that on-site adequacy services should be canceled or postponed during the pandemic. in case a particular procedure is deemed necessary to be performed, the on-site personnel should follow the guidelines generated based on the information discussed in this review and summarized in table . notes from the field: a novel coronavirus genome identified in a cluster of pneumonia cases wuhan a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster transmission of sars and mers coronaviruses and influenza virus in healthcare settings: e possible role of dry surface contamination severe acute respiratory syndrome coronavirus on hospital surfaces human coronaviruses: insights into environmental resistance and its influence on the development of new antiseptic strategies antiseptic stewardship: biocide resistance and clinical implications early transmission dynamics in wuhan, china, of novel coronavirus infected pneumonia molecular and serological investigation of -ncov infected patients: implication of multiple shedding routes detection of sars-cov- in different types of clinical specimens centers for disease control and prevention. fact sheet for healthcare providers. -ncov real-time rt-pcr diagnostic panel coronavirus false test results: with the push to screen come questions of accuracy reports of "false negatives" could complicate efforts to control the spread of covid- . e mercury news correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases false negative: covid- testing's catch- standard precautions for all patient care basic protective measures against the new coronavirus coronavirus disinfection in histopathology aerosol and surface stability of sars-cov- as compared with sars-cov- we can pick up the covid- by touching surfaces contaminated with the new coronavirus, but it is only just becoming clear how long the virus can survive outside the human body how long can the virus that causes covid- live on surfaces? persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents inactivation of surrogate coronaviruses on hard surfaces by health care germicides efficacy of various disinfectants against sars coronavirus virucidal activity of world health organizationrecommended formulations against enveloped viruses, including zika, ebola, and emerging coronaviruses stability and inactivation of sars coronavirus virucidal efficacy of physico-chemical treatments against coronaviruses and parvoviruses of laboratory animals canine coronavirus inactivation with physical and chemical agents chemistry and art of cell-block making a detailed review of various historical options with recent advances (review) ese charts show who is most vulnerable to covid- charts that explain the coronavirus pandemic centers for disease control and prevention. interim laboratory biosafety guidelines for handling and processing specimens associated with coronavirus disease (covid- ) centers for disease control and prevention. recognizing the biosafety level interim infection prevention and control recommendations for patients with suspected or confirmed coronavirus disease (covid- ) in healthcare settings biological safety cabinet (bsc): types and working mechanism an nhk experiment found that microdroplets emitted while sneezing and coughing and during conversations stay in the air for longer than normal droplets, potentially posing a uniquely dangerous risk for coronavirus infection. shot in cooperation with shin nippon air technologies barrier enclosure during endotracheal intubation a study quantifying the hand-to-face contact rate and its potential application to predicting respiratory tract infection face touching: a frequent habit that has implications for hand hygiene routine air drying of all the smears prepared during fine needle aspiration and intraoperative cytology studies: an opportunity to practice a unified protocol, offering the flexibility of choosing variety of staining methods e authors also thank "webpage capture -archive-today" https://archive.is/ to archive some of the web references for permanent archival so that their original links may be inactive in the future. original url: https://blog.wellcomeopenresearch. org/ / / /newcollection-ensures-immediate-access-to-the-latest-wellcome-fundedresearch-in-the-global-fight-against-covid- / long term url: https://archive.is/wip/vtsph e authors declare that they have no competing interests. all authors of this article declare that we qualify for authorship as defined by icmje http://www.icmje. org/#author. each author has participated sufficiently in the work and takes public responsibility for appropriate portions of the content of this article. vs conceived the idea, conducted literature review, and wrote the manuscript. all (vs, nf, and ll) critically reviewed the article. all authors read and approved the final manuscript. key: cord- -bxm yxjm authors: zeng, yawen; pu, xiaoying; du, juan; yang, xiaomeng; li, xia; mandal, md. siddikun nabi; yang, tao; yang, jiazhen title: molecular mechanism of functional ingredients in barley to combat human chronic diseases date: - - journal: oxid med cell longev doi: . / / sha: doc_id: cord_uid: bxm yxjm barley plays an important role in health and civilization of human migration from africa to asia, later to eurasia. we demonstrated the systematic mechanism of functional ingredients in barley to combat chronic diseases, based on pubmed, cnki, and isi web of science databases from to . barley and its extracts are rich in ingredients to combat more than chronic diseases, which include the similar and different chronic diseases between grains and grass, due to the major molecular mechanism of six functional ingredients of barley grass (gaba, flavonoids, sod, k-ca, vitamins, and tryptophan) and grains (β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, and resistant starch). the antioxidant activity of barley grass and grain has the same and different functional components. these results support findings that barley grain and its grass are the best functional food, promoting ancient babylonian and egyptian civilizations, and further show the depending functional ingredients for diet from pliocene hominids in africa and neanderthals in europe to modern humans in the world. this review paper not only reveals the formation and action mechanism of barley diet overcoming human chronic diseases, but also provides scientific basis for the development of health products and drugs for the prevention and treatment of human chronic diseases. global cost of five chronic diseases (diabetes, cardiovascular disease, mental illness, chronic respiratory disease, and cancer) treatment to reach $ trillion from to [ ] . the intake of high sodium with low whole grains and fruits was the top most dietary risk factors for deaths and disability-adjusted life years globally and in many countries [ ] . diabetes in production regions of polished rice with high glycemic index (gi ≥ ) caused the biggest reduction in health-adjusted life expectancy at birth in regions in countries from to [ ] . the micronutrients deficiencies at the highest risk are fe, zn, and vitamins (v b , v b , v b , and v c ) [ ] . the outbreak of human chronic disease is due to taste pursuit that changes a healthy diet, i.e., the ancients switched from brown rice (gi ≤ , high k and high micronutrients) and barley (gi ≤ ) or its grass flour (k/na ≥ ) as staple foods to modern polished rice (gi ≥ ) and wheat white flour (gi ≥ ) with low and low micronutrients as staple foods [ , ] . barley grass is not only the best functional food for cell nutrition and detoxification in human body but also the most abundant bioactive ingredients for lots of health-promoting effects [ , ] . it can combat more than chronic diseases due to gaba, flavonoids, sod, k-ca, vitamins, and trypto-phan mechanism in barley grass ( figure ) [ ] . the sustaining major foods+barley grass powder can achieve the who's intake target of low sodium (< g) with high potassium (> . g) every day [ ] . more than functional ingredients in barley grass can combat over chronic diseases, and functional ingredients in barley grains may prevent chronic diseases [ ] . barley enhanced the sterols accumulation through ltp gene action that take part in the abiotic stress reaction of mediating intracellular lipid transport [ ] . barley straw ( . ~ . g/l) for phenolic acid in degradation inhibited the alga (m. aeruginosa) blooms of aquatic eutrophication by cell shrinkage of metabolic activity and chlorophyll a fluorescence decay [ , ] . therefore, barley grass powder plays an important role for solving human chronic diseases. barley grains have the highest functional value (low gi with high β-glucans and resistant starch) and antioxidant properties among cereal crops. the soluble fiber β-glucans is a group of polysaccharides found in barley, oats, mushrooms, yeasts, and seaweed [ ] . hulless barley variety zangqing has a . -gb sequence with , genes in seven chromosomes [ ] ; three hvcslf genes take part in ( , ; , )-β-glucan synthesis [ ] . qingke (hulless barley) is a major food for tibetan people and an important livestock feed in the qinghai-tibetan plateau, which has lots of gene family related to stress reactions [ ] , especially different antioxidant capacities due to some polysaccharide and phytochemical compositions [ ] . regular daily consumption of whole barley flour can prevent chronic diseases, especially diabetes, colonic cancer, hyperlipidemia, high blood pressure, and gallstones [ ] . although barley grains have played an important role in health effects of human being, health contribution and different major mechanisms from barley grass for preventive human chronic diseases and functional ingredients in barley grains are unclear. [ , , ] resistant starch (%) whole grains : ± : . ~ . [ ] [ ] [ ] arabinoxylan (%) endosperms : ± : . ~ . [ ] barley bran : ± : . ~ . [ , ] grains flour : ± : . ~ . [ , ] polyphenols (mg/ g) whole grains : ± : . ~ . [ , ] barley bran : ± : . ~ . [ ] grains flour : ± : . ~ . [ ] phenolic acids (mg/ g) whole grains : ± : . ~ . [ ] total flavones (mg/ g) whole grains : ± : . ~ . [ , , ] flavonoids (mg/ g) whole grains : ± : . ~ . [ ] catechin (mg/ g) whole grains : ± : . ~ . [ , ] quercetin (mg/ g) purple grains : ± : . ~ . [ , ] kaempferol (mg/ g) whole grains : ± : . ~ . [ , ] myricetin (mg/ g) whole grains : ± : ~ . [ ] total alkaloid (mg/ g) whole grains : ± : . ~ . [ ] total anthocyanin (mg/ g) whole grains : ± : . ~ . [ ] barley bran : ± : ~ . [ ] refined flours : ± : . ~ . [ ] proanthocyanidin (mg/ g) whole grains : ± : . ~ . [ ] total tocols (mg/ g) whole grains : ± : . ~ . [ , , , ] antioxidant activity (%) whole grains : ± : . ~ . [ ] gaba (mg/ g) whole grains : ± : . ~ . [ ] protein % whole grains : ± : . ~ . [ ] folates (mg/ g) whole grains : ± : . ~ . [ , ] phytosterols (mg/ g) whole grains : ± : . ~ . [ ] p (mg/kg) whole grains , : ± , : ~ [ ] k (mg/kg) whole grains , : ± , : ~ [ ] ca (mg/kg) whole grains : ± : . ~ . [ ] mg (mg/kg) whole grains , : ± : . ~ . [ ] fe (mg/kg) whole grains : ± : . ~ . [ ] zn (mg/kg) whole grains : ± : . ~ . [ ] cu (mg/kg) whole grains : ± : . ~ . [ ] mn (mg/kg) whole grains : ± : . ~ . [ ] na (mg/kg) whole grains : ± : . ~ . [ ] s (mg/kg) whole grains , : ± : . ~ . [ ] oxidative medicine and cellular longevity and metal chelating activity . - . %) than that of plains ( ~ m altitude), but the soluble β-glucan and arabinoxylan content ranged from . % to . % and . % to . %, respectively [ ] . . . β-glucan. β-glucan in barley is the most abundant group of polysaccharides in cell wall. the molecular weight of β-d-glucan in hulless barley grains is . kda, which composes of ( → )-and ( → )-glucopyranosyl residues, especially its trisaccharide and tetrasaccharide accounted for . % of total cellulosyl units [ ] . β-glucan content (%) in naked, malt, black, waxy-naked, and blue barley is . , . , . , . , and . , respectively, especially waxy-naked barley flour has the highest extraction rate ( . %); however, gi in vitro starch digestibility was lowered by adding β-glucan [ ] . the fatty acid derived flavouring substance (dodecanoic acid, octyl butanoate, ethyl decanoate, and decyl acetate) in beer has important role in the aggregation behavior of barley β-glucan [ ] . the β-glucan concentrations in the six hulless barley grains varied from . % to . %, among shorts ( . ~ . %)>bran ( . ~ . %)>flour ( . ~ . %) [ ] . the total β-glucan contents in the nine hulless barley are . ~ . % in bran and > . ~ . % in refined flour [ ] . barley. phenolic compounds have the antioxidant, anti-inflammatory, and antitumor potentials [ ] . there are the most abundant polyphenols in barley grains, especially p-hydroxybenzoic ( . %), pcoumaric ( . %), and ferulic acids ( . %) [ ] . the most abundant polyphenol in barley extract include free polyphenols ( : ± : mg/ g) and bound polyphenols ( : ± : mg/ g), especially ferulic acid ( . mg/ g) and >procyanidin b ( . mg/ g) [ ] . phenolic acids such as ferulic acid and p-coumaric acid were . mg/ g and . mg/ g in barley grains and . mg/ g and . mg/ g in malt, respectively [ ] . the extraction polyphenols yield of barley lactobacillus fermented solution of % ethanol concentration was . %, main components (mg/ g) including rutin ( . ), vanillic acid ( . ), ferulic acid ( . ), coumaric acid ( . ), gallic acid ( . ), protocatechuic acid ( . ), and p-coumaric acid ( . ) [ ] . compared to the raw barley extract, the protein, total phenols, and βglucan of fermented barley extract with lactobacillus plantarum dy- can significantly increase to . %, . mg/g, and . %, respectively [ ] . there were larger genetic variations in the contents of total polyphenol ( : ± : mg/ g), total flavonoid content ( : ± : mg/ g), and antioxidant activity ( : ± : %) among the barley genotypes; however, major qtls between bpb- and bpb- control phenolic compounds in tibetan wild barley, especially the udp-glycosyltransferase gene with biosynthesis of flavonoid glycosides was colocated with bpb- [ ] . barley. nacl stress increased the phenolic compounds (vanillic acid, p-coumaric acid, ferulic acid, and sinapic acid) accumulation and synthesis by upregulating the gene expression of phenylalanine ammonia lyase, cinnamic acid -hydroxylase, -coumarate coenzyme a ligase, p-coumaric acid -hdroxylase, and caffeic acid/ -hydroxyferulic acid o-methyltransferase of germinated hulless barley [ ] . the blue hulless barley grains have larger variation on phenolic compounds and antioxidant activity, such as the free, bound, and total phenolic acids varied between . ~ . , . ~ . , and . ~ . mg/ g, respectively, where the major phenolic compounds include quercetin, rutin, naringenin, hesperidin, (+)-catechin, gallic acid, benzoic acid, syringic acid, and -coumaric acid [ ] . the anthocyanin and total phenolic contents in hulless barley grains are higher for high altitude, and the contents in its refined flours were . ~ . mg/ g and . ~ . mg fae/ g and in its bran were . ~ . mg/ g and . ~ . mg fae/ g, respectively [ ] . whole grain hulless barley had high contents of total phenolic ( . mg/ g), total pentosan ( . g/ g), and orac values ( : ± : mol/ g) [ ] . the bran extract of hulless barley rich in phenolic acids on the nε-carboxymethyllysine formation during processing biscuits, which can reduce glycation and benefiting health [ ] . total polyphenols ( . mg/ g) and proanthocyanidins ( . anthocyanin for human health belongs to flavonoids, which is a secondary metabolite that plants adapt to harsh environments. ant gene ( hl) with a bhlh domain control purple grain and ant gene control red leaf sheath and pericarp in barley, r r -myb (ant )+bhlh (ant ) complex promotes the synthesis by affecting expression of the anthocyanin biosynthesis structural genes (f ′ h) and ans genes [ ] . the hl alleles from barley purple pericarp synthesis the peonidin- glucoside [ ] . the anthocyanin synthesis hvmyc gene is the major variant factor for blue aleurone of barley [ ] . barley anthocyanins take part in the amino acid biosynthesis, carbon metabolism, phenylpropanoid biosynthesis, and metabolic pathways [ ] . flavonoid ′ -hydroxylase (f ′ h) and flavonoid ′ , ′ -hydroxylase (f ′ ′ h)-coding genes take part in anthocyanin synthesis in barley [ ] . the anthocyanin bran-rich fractions of yellow ( . mg/ g, anthocyanins) and purple barley ( . mg/ g, anthocyanins) are times higher than that of the whole grain flours ( . and . mg/ g), especially cyanidin -glucoside, delphinidin -glucoside, petunidin -glucoside, delphinidin -rutinoside, and cyanidin chloride [ ] . arabinoxylan in barley is the second highest cell wall polysaccharide [ ] . arabinoxylan in barley plays an important role in quality traits of malt and beer product [ ] ; however, arabinoxylan arabinofuranohydrolase i can be used as novel enzyme products in the beer industry [ ] . the starch degradation for seedling relies on cell wall degradation, where the iminosugar , dideoxy- , -imino-l-arabinitol inhibits dextrinase and arabinoxylan arabinofuranohydrolase but permits rapid diffusion of αand β-amylase [ ] . arabinoxylan contents in barley grains range from . % to . % [ ] where it ranged from . mg/ g to . mg/ g at an average value of . mg/ g in barley endosperm in spring -row barley; its two qtls include glycosyltransferases and glycoside hydrolases [ ] . arabinoxylan accounts for % of total polysaccharide [ ] and ~ % of total monosaccharide in barley husk [ ] . arabinoxylan contents in hulless barley are in bran . %>in shorts . %>in flour . % [ ] which differ from the report of moza et al. [ ] (in bran . ~ . %>in flour . ~ . %). . . phytosterols. phytosterols in plant membrane are similar in structure to cholesterol [ ] . higher phytosterols are found in the outer layers of barley grains and ranged between . mg/ g and . mg/ g, among which β-sitosterol is : ± : mg/ g and campesterol is : ± : mg/ g. the other phytosterols include stigmasterol ( . mg/ g), brassicasterol, δ -avenasterol, stigmastanol, stigmastadienol, and other minor sterols (δ -and δ -avenasterols, δ -stigmastenol, and stigmastadienol: : ± : mg/ g) [ ] . . vitamin e is the major lipid-soluble antioxidant for human health, which has eight different stereoisomers [ , ] by three chiral centers in tocopherols from barley. spring barley has higher α-tocotrienol content in four tocols (β-tocotrienol, α-tocotrienol, β-tocopherol, and α-tocopherol) [ ] . [ ] . a tocochromanol in barley grains ranged from . to . mg/ g, but which is much higher than in oat ( . mg/ g) and triticum ( . mg/ g) [ ] . tocochromanols content in barley is % in pericarp, > % in endosperm, and > % in germ; about % of the tocochromanols were tocotrienols, and tocopherols in germ ( %) was higher than that in pericarp ( %) [ ] . the hulless barley especially with waxy, double waxy and tercel cultivars have the highest tocols content. tocol in whole grain was . mg/ g to . mg/ g, and in pearling flour was . mg/ g to . mg/ g; however, the ratios of total tocotrienols to total tocopherols ranged from . to . [ ] . the highest content of tocols ( . ~ . mg/ g) and vitamin e concentrations ( . ~ . mg/ g) was found in the waxy barley, especially in the hulless waxy washonubet (tocols . mg/ g and α-tocotrienols isomer . mg/ g) [ ] . . . resistant starch. resistant starch (rs) can prevent dietrelated chronic diseases such as diabetes and colon cancer. rs in hulless barley grains is related with b-type granules and the amylopectin f-iii fraction; however, sequential rate of enzymatic hydrolysis in diets is waxy>normal>high amylose barley [ ] . rs of spring barley cultivars approved and popularized during the past years in oxidative medicine and cellular longevity europe, in which rs content ranged from < % to > % [ ] . rs content of unprocessed grains of high-amylose, normal, and waxy barley is : ± : %, : ± : %, and : ± : %, respectively, but slowly digestible starch in unprocessed grains of normal ( : ± : %)>high-amylose ( : ± : %)>waxy barley ( : ± : %) and rapidly digestible starch in normal ( : ± : %)<high-amylose ( : ± : %)<waxy barley ( : ± : %) [ ] . the rs content (mg/ g) of accessions barley grains was : ± : %, with a highest content up to . % [ ] . snps (i.e., th exon g( )-to-t and fifth exon c( )-to-t) in three exons play different roles on the expression of the waxy transcript, granule-bound starch synthase i (gbss i), protein, amylose, and starch properties of hulless barley [ ] . the chronology of rs contents in different barley diets is ground pearled barley ( . %)>pearled barley flakes ( . %)>whole pearled barley ( . %)>cut barley . . gaba and linoleic acid. gaba increased α-amylase gene expression by treating barley aleurone with exogenous gaba, especially α-amylase activity began to rise after about h and reached a peak at h [ ] . the gaba content (mg/ g) in accessions of barley grains is : ± : mg/ g, the highest up to : : ± : mg/ g [ ] . the gaba has a very important role in mediating nacl stress phenolic compounds accumulation in germinated hulless barley [ ] . linoleic acid content increased from . % to . % and oil from . to . %, while oleic content decreased from . % to . % and palmitic acid from . % to . % during barley malting process [ ] . . . phytases. hydrolyze phytate in barley associated the bioavailable nutrient elements (p, fe, and zn), which exists as a single gene (paphy_a) in barley, but as two or three homeologous copies in wheat [ ] . the improvement of hvpaphy_a transformed barley showed phytase activity increases up to -fold in green leaves, -fold in grains, and -fold in dry straw [ ] . barley grains for preventive chronic diseases . . . antidiabetic properties. diabetes is a chronic metabolic disease with high mortality rates; therefore, search for novel natural inhibitors has gained much attention [ ] . major antidiabetic elements in barley are β-glucan, phenolic compounds (phenolic acids and flavonoids), phytosterols, tocols, arabinoxylan, and resistant starch ( table ) . oxidative stress not only leads to insulin resistance, impaired glucose tolerance, b-cell dysfunction, ultimately diabetes but also can treat diabetes and obesity by phytochemicals (phenolic acids, flavonoids, phytosterols, and tocols) in barley [ ] . chronic consumption of foods with high β-glucans in barley can improve insulin resistance and lower the postprandial glucose response and increase satiety [ ] . the β-glucan in hulless barley reduced the insulin resistance, arterial sclerosis, serum glucose, and serum lipid in high-fat mouse [ ] . high phenolic content ( . mg/ g) and low rapidly digested starch ( . %) make barley muffin to modulate glycemic response [ ] . the hypoglycemic effect of ethanol extract polysaccharide from barley malt is better for decreased fasting plasma glucose of the diabetes mice than that of water extract [ ] . the boiled barley kernels evening meal can facilitate glucose regulation, increase the release of glucagon-like peptide- , and reduce energy intake and fasting serum free fatty acids, mediated through gut microbial fermentation of the indigestible carbohydrates [ ] . the glycemic index (gi = : ) of all-wheat bread is higher than that (gi = : ) of % wheat+ % barley flour ( . % β-glucan) [ ] . gi for barley with . % β-glucan and oat tempe are and , respectively [ ] . the hulless barley can reduce postprandial glucose and improved insulin sensitivity by amino acid and biogenic amine profiles [ ] . . . . antiobesity. major antiobesity components in barley are β-glucan, resistant starch, polyphenols, dietary fiber, arabinoxylan, tocols, and phytosterols ( table ) . β-glucan in barley significantly treats obesity that reduced low-density lipoprotein, total cholesterol, and serum p-cresyl sulfate levels and increased flow-mediated dilation [ , ] . barley β-glucan can prevent visceral fat (≥ cm ) obesity and increase faecal scores, but decreased nutrient digestibility and antiobesity [ , ] . rs and β-glucans as well as soluble arabinoxylan were utilized mainly in the caecum, especially rs shifted the utilization of other polysaccharides to more distal parts of the colon of pigs [ ] . obesity and insulin resistance associated with bile acid changes and lower dietary fiber (β-glucan) in barley diet [ ] . the aqueous extract of fermented barley has antiobesity effects due to β-glucan and phenolic acids (vanillic acid and ferulic acid) [ ] . β-glucans in black and blue hulless barley for preventive obesity were very higher than that of white one, based on its molecular weights, particle sizes, viscosities, binding capacities (fat, cholesterol, and bile acid), and inhibiting activities on pancreatic lipase [ ] . the polyphenols extracted in black hulless barley show notable decreases in total cholesterol, low-density lipoprotein cholesterol, and atherosclerosis, but significant increase in high-density lipoprotein cholesterol levels [ ] . barley β-glucan can reduce low-density lipoprotein cholesterol and non-highdensity lipoprotein cholesterol as well as alters the gut microbiota for preventing cardiovascular disease (see table ) [ ] . oxidative stress and inflammation are two important factors of atherosclerosis, and polysaccharide extracts with antioxidation and anti-inflammation of hulless barley prevent cardiovascular diseases [ ] . some other functional components of barley have been associated with oxidative medicine and cellular longevity cardiovascular health, such as polyphenols, phytosterols, lignans, tocols, and folate [ ] . major anticancer elements in barley are β-glucan, phenolics, arabinoxylan, phytosterols, lignan, and resistant starch ( table ). functional ingredients of barley with antioxidative and immunomodulatory activities are associated with anticancer effects [ ] . barley with high dietary fiber (β-glucan) has an important role for the prevention of colon cancer and cardiovascular diseases [ ] ; low molecular weight β-d-glucan can enhance antioxidant and antiproliferative activities [ ] . aqueous extract of fermented barley can induce subcutaneous transplantation tumor apoptosis that can be used for a nutrient supplement in the treatment of human colon cancer [ ] . β-glucans in hulless barley has anticancer activities in vitro, but its anti-inflammatory activities increased as their molecular weights decreased [ ] . the bound phenolics in dehulled hulless barley have excellent antioxidant and antiproliferative effects to human liver cancer cells [ ] . a water soluble polysaccharide (glucose : xylose : arabinose : rhamnose = . : . : . : . ) from hulless barley can inhibit colon cancer, which induce ht- apoptosis through ros-jnk and nf-κb-regulated caspase pathways [ ] . . . . antioxidation. antioxidants are compounds that remove reactive oxygen species from cells, which play a dual role in aggravating and preventing diseases [ ] . major antioxidants in barley are phenolic compounds (phenolic acids, flavonoids, and anthocyanin), tocols (vitamin e), polysaccharide (arabinoxylan), dietary fiber, and phytic acid ( table ) . antioxidant effects of polyphenols in barley are flavanols>flavonols (quercetin)>hydroxycinnamic acids (ferulic, caffeic, and coumaric acids) [ ] . malt has higher phenolic content (sinapinic acid and epicatechin) than its barley grains, which plays a key role on antioxidant stability of beer [ ] . the antioxidant activity for anthocyanin in barley bran was markedly higher than that of whole grains flour [ ] . five of the seven associations in barley were with markers near genes associated with the tocochromanol (vitamin e with the most powerful antioxidants) pathway [ ] . overexpression of homogentisate geranylgeranyl transferase for barley enhanced the tocotrienol levels (δ-, β-, and γ-tocotrienol - %) and antioxidant capacity (radical scavenging activity - %) in barley seeds [ ] . the antioxidant effects of dietary fiber in hulless barley bran were associated with total phenolic concentration, which had the dpph ( , -diphenyl- -picrylhydrazyl radical , -diphenyl- -( , , -trinitrophenyl)-hydrazyl) radicalscavenging activity and ferric-reducing antioxidant power oxidative medicine and cellular longevity [ ] . gaba induces the accumulation of proline and total phenolics and enhances the antioxidant system in germinated hulless barley under nacl stress [ ] . the chapatti quality score reduced by % and its phenolic concentration increased from . to . mg/ g, while biscuit spread factor reduced by % and its β-glucan concentration increased from . to . % as well as phenolic content increased from . to . mg/ g after blending of % hulless barley flour, especially markedly increased antioxidant activity [ ] . . . . anti-inflammation. major anti-inflammatory ingredients in barley are β-glucans, lignans, vanillic acid, arabinoxylan, and so on ( table ) . endothelial cell adhesion molecules were identified as an early step in inflammation and atherogenesis; barley β-glucans not only have a maximum anti-inflammatory activity at mw~ : × , especially the inhibition of tnf-α-induced expression of vascular cell adhesion molecule was stronger than that of oat β-glucans, but also have a higher ratio of -o-β-cellobiosyl-d-glucose to -o-β-cellotriosyl-d-glucose oligomers in the polymeric chains [ ] . the molecular weights of β-glucans in hulless barley increased that add inhibitory abilities on α-amylase and pancreatic lipase, but the antiinflammatory abilities decreased, especially the low intrinsic viscosity and high solubility of β-glucans acid hydrolysis for min might contribute to its higher anti-inflammatory activity, which significantly affected their bioactivities (e.g., anticancer), which was beneficial for a better understanding of their structure-function relationships [ ] . the fermented barley extracts (vanillic acid) downregulate glucose consumption and reducing proinflammatory cytokine secretion [ ] . the anti-inflammatory property of malt and wholegrain barley is due to the formation of short chain fatty acid (scfa) and changes in microbiota composition [ ] . . . . immunomodulation. major immunomodulatory substances in barley are β-glucans, arabinoxylan, and so on ( table ). the immunomodulatory activity of barley β-glucans insolubility associated with its particle size, granule conformation, and particulate homogeneity. all β-glucan fractions can induce more cytokines in bone marrowderived dendritic cells than their oat equivalents; however, the insolubility of β-glucan affects its immunomodulatory activity, which is related to its particle size, particle configuration, and particle uniformity [ ] . a water-soluble polysaccharide (bp- , molecular weight : × da) from hulless barley can improve the immune ability of immunosuppressive mice through increasing the serum levels of il- , tnfα, and ifn-γ, such as bp- ( mg/kg and mg/kg) can not only significantly increase the number of bone marrow cells and peripheral blood white blood cells, as well as enhance the production of il- , tnf-α, ifn-γ, igg, and igm in the spleen and serum levels for improving the immune function, but also promote the proliferation and phagocytosis activity of macrophages as well as repair the damage induced by ctx in the spleen cells of immunosuppressive mice [ ] . table ), which shows a % survival rate after cardiac ischemia ( min)/reperfusion ( min) injury, reduces left ventricular anion superoxide production ( %) and infarct size ( %), and increases the capillary ( %) and arteriolar density ( %) and vegf expression ( . %) of hearts in mice [ ] . the products of barley grains can reduce the cardiometabolic risk and regulate the blood glucose and appetite hormones in - h after intake; however, its mechanisms are gut fermentation of indigestible carbohydrates [ ] . . . . hypocholesterolaemic effects. cholesterol is a synthesis lipid in the body [ ] . dietary β-glucan of hulless barley reduces the plasma ldl cholesterol content (see table ) by promoting the excretion of faecal lipids and regulating the activities of -hydroxy- -methyl glutaryl-coenzyme a oxidative medicine and cellular longevity reductase and cholesterol -αhydroxylase in hypercholesterolaemic rats [ ] . barley bran % and % in diet to the hypercholesterolaemic rats improved the level of lipids, lactate dehydrogenase, liver enzymes, and creatine kinase-mb [ ] . whole grain hulless barley has hypocholesterolaemic effects by promoting bile acid synthesis and reabsorption, controlling cholesterol synthesis and accumulation in peripheral tissue, decreasing the expression of -hydroxy- methylglutaryl coenzyme a reductase, while increasing the hepatic expressions of amp-activated protein kinase α, cholesterol α-hydroxylase, ldl receptor, liver x receptor, and pparα [ ] . . . . blood pressure regulation. higher consumption of barley β-glucan is associated with lower systolic and diastolic blood pressure (see table ), i.e., diets rich in β-glucans reduce systolic blood pressure by . mmhg ( % ci . to . mmhg) and diastolic blood pressure by . mmhg ( % ci . to . mmhg) for a median difference in β-glucans of g [ ] . the consumption of high molecular weight barley β-glucan can reduce blood pressure [ ] . . . . bowel health improvement. gastrointestinal tract disease is a major global health problem. β-glucan in hulless barley has protective effects to the gastrointestinal tract (see table ) [ ] . the dietary fiber in hulless barley improved indices of bowel health compared with refined cereal foods, especially himalaya possesses high amylose and resistant starch due to lacking activity of a key enzyme responsible for starch synthesis; however, consumption of himalaya foods resulted in % higher faecal weight, a lowering of faecal ph from . to . , a % higher faecal concentration, a % higher excretion of butyrate, a % higher faecal total scfa excretion, and a % lower faecal p-cresol concentration [ ] . fermented barley extract ( mg/ g) can act as a promising laxative agent to cure spastic constipation [ ] . butyric acid for improving the colonic health is produced by degradation of barley dietary fiber by microbiota [ ] . . . . gastroprotective effects. β-glucan from hulless barley can mitigate the gastric lesions and gastric mucosal damage as well as gastric oxidative stress injury through decreasing the level of malondialdehyde [ ] . the oral administration of fermented barley extract had strong gastroprotective effects through strengthening antioxidant defense system and anti-inflammatory effects, as well as decreasing lipid peroxidation and cat activity by increasing the gsh levels and sod activity in the body, and the mg/kg dose of fermented barley extract was similar gastroprotective as the mg/kg dose of omeprazole, which indicates that this dosage can be used for patients suffering from different levels of gastric damages [ ] . . . . reduce chronic kidney disease. barley β-glucans is associated with a saccharolytic shift in the gut microbiota metabolism by a reduction of pcs toxin blood levels and an increase of scfa production at colonic site, which can reduce the microbial-derived uremic toxin and cardiovascular complications in end-stage renal disease (see table ), especially chronic kidney disease [ ] . the total cholesterol and triglycerides were reduced, and hdl cholesterol increased in % and % barley intervention in breakfast diet; however, barley in the diet of stage chronic kidney disease patients has significantly improved the nutritional status and renal functions [ ] . . . . improve metabolic syndrome. barley β-glucan can improve postprandial glucose response and cholesterol levels as well as the metabolic syndrome based on individual gut microbiota composition (see table ) [ ] . tibetan hulless barley can reduce insulin resistance, dyslipidemia, and body weight gain, which can diminish the prevalence of metabolic syndrome induced by high-fat-sucrose diets, i.e., rats fed with tibetan hulless barley can increase the assessment of insulin resistance scores (body weight, abdominal fat deposition, liver weight, liver fat deposition, triglyceride, fasting blood glucose, and serum fasting insulin) and decrease lowdensity lipoprotein cholesterol levels compared to rats fed with a basal diet [ ] . . . . hepatoprotective effect. β-glucans in barley can decrease fatty liver in diabetes with obesity (see table ) [ ] . the free phenolic extract in barley added the hepatic levels of antioxidant enzymes [ ] . whole grain hulless barley had significantly lower liver lipid levels (total phenolic and pentosan) [ ] . barley sprout extract protects liver cells under oxidative stress by activating nrf and adding glutathione synthesis, especially against alcohol-induced liver injury, as it inhibits glutathione depletion and hepatic lipid accumulation, reduces serum biochemical markers of liver injury, and inhibits inflammatory responses [ ] . . . . wound healing acceleration. nowadays, β-glucans represent effective topical agents for the treatment of chronic wounds and burns due to the activation of the immune and cutaneous cells (see table ), which increase wound repair by enhancing the infiltration of macrophages and promote tissue granulation, collagen deposition, and reepithelialization based on inducing the proliferation and migration of keratinocytes and fibroblasts through specific receptors such as dectin- , cr , or tlrs [ ] . barley β-glucan in vivo promotes the wound closure in mouse skin by promoting the migration and proliferation of human dermis fibroblasts [ ] . . . . heart failure prevention. barley β-d-glucan is a natural activator of mnsod expression, which can prevent heart failure (see table ) [ ] . the food and drug administration has made a health claim between β-glucan and reduced risk of coronary heart disease, diabetes, and heart-related problems [ ] . barley products can prevent and reduce the risk of coronary heart disease, which is associated with the constituents like β-glucan, phenolics, tocols, linoleic acid, and folate [ , ] . . . . atopic dermatitis alleviation. the allergen produced by barley and the protein expressed in insect cells induce the same amount of ifn-γ and il- in pbmc from vaccinated horses (see table ) [ ] . fermented barley extract oxidative medicine and cellular longevity p reduced skin lesions by inhibiting inflammatory cytokines [ ] , but gaba alleviated atopic dermatitis by suppressing serum immunoglobulin e and splenocyte interleukin production [ ] . barley intake reduces the risk of total stroke by significantly increasing the medium and small particle sizes of high-density lipoprotein cholesterol (see table ) [ ] . increasing the expression of antioxidant genes in the liver and the regulation of nrf played a role in the regulation of metabolic diseases in stroke-prone spontaneously hypertensive rats consuming a fermented barley extract p diet [ ] . . . . allergic rhinitis alleviation. ma-al-shaeer formulation based on barley can treat for allergic rhinitis, especially reduced the nasal congestion, post nasal drip, and headache (see table ) [ ] . fermented barley extract alleviated allergic rhinitis in ova-sensitized mice by regulating cytokines (ifn-γ or il- ) related to chronic inflammation [ ] . in addition, the common edible whole-barley flour can reduce the risk of hyperlipidemia and cholelithiasis [ ] , antiaging [ ] as well as increase body strength and increase the content of linoleic acid and linolenic acid in pigs, cattle, sheep, and geese. in a word, there are more than kinds health effects for barley grain preventive chronic diseases according to the summary of current retrieval literature. the descriptions of some literatures for barley preventive chronic diseases (antidiabetes, antiobesity, anticancer, antioxidation, anti-inflammation, hypocholesterolaemic effects, blood pressure regulation, cardioprotection, immunomodulation, improve gastrointestinal, hepatoprotection, bowel health, cardiovascular disease prevention, atopic dermatitis alleviation, wound healing acceleration, heart failure prevention, and so on) are relatively sufficient, but some literatures for barley preventive chronic diseases (antiaging, reduce cholelithiasis, and increase body strength) are relatively less. these results fully demonstrate the health contribution relationship between barley functional food and human chronic disease prevention. the efficacy of preventing chronic diseases is related to barley genotype and its location, composition, extraction, and compatibility, and with the in-depth study of barley grain in prevention and treatment of human chronic diseases, the novel mechanisms for the prevention of chronic diseases may be ascertained as well as its putative role, either at major or minor level, would be further validated. grains for preventive chronic disease . . . β-glucans mechanism. β-glucans can be used as candidates for the medication in the treatment of human chronic diseases [ ] . β-glucans have many bioactivities including antidiabetes; anticancer; antiobesity; anti-inflammation; immunomodulation; cardioprotection; lower cholesterol and lower blood pressure; improve bowel health, gastroprotection, and hepatoprotection; reduce chronic kidney disease and metabolic syndrome; prevent the risk of heart and cardiovascular diseases; and accelerate wound healing activities (figure , tables and ) [ , , , , , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the major mechanisms of β-glucans of barley involve in the prevention of chronic diseases are as follows: β-glucans can interact with intestinal lipids and bile salt to reduce cholesterol levels and subsequently prevent diabetes, hypertension, cardiovascular disease, and metabolic syndrome [ ] . barley β-glucans not only control appetite and improve insulin sensitivity by gut hormone secretion via microbiota produced scfa [ ] due to its high molecular weight and high viscosity but also increase their antigen ability and enhances the proinflammatory cytokines, which can be degraded by macrophages and natural killer cells mediating its cellular cytotoxicity opsonized tumor cells [ ] . β-glucans of barley are the major regulators of adipogenesis, especially markedly downregulated the target genes in the adipose tissue including adipocyte fatty acid-binding protein, lipoprotein lipase, uncoupling protein- , and glucose transporter in t -l cells [ ] and also have the effect of inhibiting the α-amylase and pancreatic lipase [ ] . barley β-glucan can reduce blood pressure and cardiovascular diseases that alters the composition of gut microbiota, decrease body mass index, waist circumference, and triglyceride levels [ ] and also reduce the systemic inflammatory profile, prevent alveolar bone loss, and improve βcell function in diabetic animals [ ] . barley β-glucans can not only regulate immune responses and connect innate and adaptive immunity [ ] but also have cardioprotective mechanism of promoted angiogenesis through endothelial upregulation of the vascular growth factor [ ] . the hypocholesterolaemic effect of β-glucan in barley is due to the increased bile acid synthesis [ ] and improved bowel health by inhibited feed intake and increased cecal fermentation [ ] . barley β-glucan not only has the gastroprotective effects by increasing the sod and cat activity, decreasing the gastric ulcer index, and increasing prostaglandin e and nitric oxide in laboratory rodents [ ] but also affects lipid metabolism and scfa production, lowering microbes in patients with metabolic syndrome [ ] and improving chronic kidney disease by reducing the microbial-derived uremic toxin. chronic consumption of barley β-glucans can decrease fatty liver by increasing small intestinal contents viscosity and improving glucose, lower glycated hemoglobin and relative kidney weights [ ] , strengthen the angiogenic ability of ros-exposed endothelial cells for preventive heart disease [ ] , and accelerate the wound closure by promoting the migration and proliferation of human dermal fibroblasts [ ] . barley polyphenols have lots of bioactivities including antidiabetes, antiobesity, anticancer, antioxidant, anti-inflammation, hepatoprotection, and prevention of cardiovascular and heart diseases ( figure , tables and ). barley lignan as natural polyphenols has anticancer, antioxidant, anti-inflammation properties, and preventive role for cardiovascular diseases. anthocyanin belongs to flavonoids, and flavonoids belongs to polyphenols. black, purple, and blue barley grains have gained much attention recently because their anthocyanins have anticancer, glycemic and body weight regulation, antioxidation, anti-inflammation, neuroprotection, hypolipidemia, retinal protection, hepatoprotection, and antiaging effects [ , ] . several mechanisms of barley polyphenols for preventing chronic diseases have been documented so far. zhang et al. [ ] has verified the molecular mechanism of insulin resistance by fermented barley extract vanillic acid through regulating mir- expression. the polyphenols with antiobesity from black hulless barley has strong superoxide radical, hydroxyl radical and , -diphenyl- -picrylhydrazyl radicalscavenging activity, ferric reducing antioxidant power, and moderate metal ion-chelating activity [ ] . an efficacious antiproliferation capacity in caco- cells of black barley malt free extract was predicted due to its phenolic constituents which have cellular antioxidant and oxygen radical absorbance as well as peroxyl radical scavenging activities, dpph, and abts radical scavenging assays [ ] . black barley sprouting stimulates the phenolic biosynthesis by upregulating proline-associated pentose phosphate pathway to support structure of sprouts with antioxidant capacity [ ] . total phenolic and pentosan in hulless barley grains have antioxidant activity by downregulated expression of heat shock protein and phosphatidylethanolamine binding protein but upregulated expression of enoyl-coenzyme a hydratase and peroxiredoxin [ ] . the total polyphenol (flavonoid) content and the , -diphenyl- -picrylhydrazyl and abts radical scavenging abilities increased as the barley added to the food mixture [ ] . lignan (-)- (s)-hydroxymatairesinol inhibited tumor necrosis factor-α stimulated endothelial inflammation by inhibiting nf-κb activation and upregulating nrf antioxidant element signaling pathway [ ] . lignans in barley have high anti-inflammatory abilities in endothelial cells by reducing nuclear factor-κb and extracellular signal as well as regulating kinase phosphorylation [ ] . the polyphenols such as (+)-catechin, protocatechuate, and quercetin in barley not only have hepatoprotective effect [ ] but also prevent coronary heart disease by reducing oxidativeinduced tissue damage through modulating intracellular signaling pathways [ ] . polyphenols play an important role in alleviating cardiovascular diseases due to their antiradical scavenging abilities [ ] . barley arabinoxylan has a lot of health benefits, which include antidiabetes, antiobesity, anticancer, lowering cholesterol, immunomodulation, antioxidant, cardiovascular diseases prevention, and so on (figure , tables and ). arabinoxylan in barley is the most abundant polysaccharide that has the capacity of lowering cholesterol and glucose as well as antioxidant activities [ ] . the major mechanisms of barley arabinoxylan for preventing chronic diseases are as follows: arabinoxylan can improve urinary metabolites associated with diabetes by improvement of carbohydrate and lipid as well as amino acid metabolism [ ] . the antioxidant and antiobesity as well as immunomodulation of arabinoxylans associated with prebiotic effects and short-chain fatty acids production by interaction of gut microbiota and arabinoxylans [ ] . arabinoxylan rice bran can increase anticancer effects in the older population by increased nk activity [ ] . the arabinoxylan in barley with the immunomodulatory activity consisted of a xylan backbone with acetate, arabinose, galactose, glucuronic acid, and -o-methylglucuronic acid [ ] . the arabinoxylan diet led to a lower postprandial blood for glucose-dependent insulinotropic polypeptide response, especially fat oxidation has an important role in the antiobesity and in the prevention of cardiovascular diseases [ ] . barley polysaccharide prevent cardiovascular diseases by the vasodilatory effect of controlling angiotensin-converting enzyme production [ ] . tables and ). barley grains contain phytosterols that can esterify to fatty acids, phenolic acids, steryl glucosides, or acylated steryl glycosides [ ] . phytosterols significantly inhibited the ability of oxysterols to activate the liver x receptors transcription in modulating cancer cell behavior [ ] , which are thought to influence multiple processes related to cancer, such as carcinogen production, cancercell growth, angiogenesis, invasion, metasis, and cancer-cell apoptosis [ ] . for the prevention of cardiovascular diseases, barley phytosterols can compete with cholesterol for micelle formation, inhibiting cholesterol absorption in intestine and lowering cholesterol in central nervous system of the brain [ , ] . the effect of plant sterols on neurodegenerative diseases is due to its passage through the blood-brain barrier, modulating cholesterol metabolism and inflammation in the central nervous system process in the brain, which involve low-density-lipoprotein, apolipoprotein e, and scavenger receptor class b type [ ] . the lowering of cholesterol and prevention of cardiovascular disease are due to the fact that the phytosterol structure in the barley membrane is similar to the configuration of different cholesterol [ ] , but functionally similar to precursors in phytohormone synthesis, but lowering the blood concentration of cholesterol is beneficial to reduce the risk of cardiovascular disease. extremely rich in content in barley embryos. barley tocols are the best in cereals due to a high concentration and favorable distribution of eight active vitamers [ ] . barley tocols have anticancer, antiobesity, and antioxidant effects and can lower cholesterol level, reduce the risk of stroke, and prevent cardiovascular and heart diseases ( figure , tables and ) [ , ] . tocotrienols can suppress various cancers (breast, lung, ovary, prostate, liver, brain, colon, myeloma, and pancreas) by its molecular mechanisms of cellular proliferation, apoptosis, angiogenesis, metastasis, and inflammation [ ] , which are associated with human intake of whole grains, especially barley and rye. tocotrienol is a functional food of obesity and diabetes by regulating adipogenesis and increase apoptosis of adipocytes and improve glucose homeostasis through oxidative medicine and cellular longevity suppression of inflammation and oxidative stress [ ] . tocol is one of the most powerful antioxidants that has the ability to interact with polyunsaturated acyl groups and scavenge lipid peroxyl radicals and quench reactive oxygen species, thus protecting fatty acids from lipid peroxidation [ ] . all barley pitas had the greatest antioxidant and vitamin e levels from barley malt flour [ ] . the antioxidant properties of barley tocols due to its ability to inhibit lipid peroxidation in biological membranes induce the immune system, promote apoptosis induction, and reduce the risk factors of cardiovascular diseases and stroke by atherosclerotic blockages in the carotid artery [ ] . barley rs has many immune properties like antidiabetes, antiobesity, anticancer, and so on ( figure ,table ). ceramide can promote lipid storage, impaired glucose utilization, and inhibited enzyme dihydroceramide desaturase , which can treat hepatic steatosis and metabolic disorders [ ] . antidiabetes of rs can be increased by suppressing amylopectin synthesis through silencing of starch branching enzymes in barley [ ] . t cell development and gut iga production suppress host lipid absorption by modulating cd expression [ ] to achieve the effect of antiobesity of barley rs. gbss i is mainly responsible for amylose synthesis whereas sss i and sbe ii for amylopectin synthesis in amyloplasts [ ] . barley with high β-glucan and moderate rs may benefit hyperglycemia-impaired lipid metabolism [ ] . the blending of barley starch citrate with resistant starch iv up to % can produce noodles of acceptable quality and numerous health benefits [ ] . early hominids used fruits/vegetables and leaves rich in polyphenols and k-ca as well as vitamins as staple foods to increase the dependence of the human body on these functional ingredients (see figure ). diet played an important role in early hominids evolution [ ] , but no reports have been delivered to date for diet in coevolution of human chronic diseases. miocene ( . ~ . ma) apes had a variety of foods that included folivory, soft-fruit eating, and hard-object feeding [ ] . the diet of pliocene ( . ~ . ma) and early pleistocene ( . ~ . ma) hominids in africa was mainly fruits and leaves of c plants (trees, bushes, shrubs; . ~ . ma) which was gradually transformed into grass (c plant/tropical grasses and sedges) and hard-object (seeds and nuts) ( . ~ . ma) [ ] [ ] [ ] , which was due to low availability of fruits in dry and active glacier ( . ~ . ma) as well as migration to warm grasslands. ethiopia's pliocene lucy is one of the oldest and most complete fossils in hominid bones, her death due to fall out vertically and live on tall tree [ ] . early hominids and australopithecines inhabited forests and savannas for collinearity oxidative medicine and cellular longevity found between tasty fruits (fructose/sucrose, quinine, and tannins) and primate sensory perception, which offered evidence of the two-direction evolutionary trend determining taste sensitivity [ ] . our ape ancestors possessed a digestive dehydrogenase enzyme capable of metabolizing ethanol about ma that they began using fruits fermentation from the forest floor [ ] . there were differences in the proportion of meat and vegetables between the early hominids australopithecus and paranthropus; paranthropus ate more hard food than australopithecus [ ] . early hominid australopithecus africanus, like chimpanzees, are dominated by fruit, leaves, and carbon- -enriched foods about ma [ ] . the worldwide daily consumption of fruits and vegetables as well as tea has become the main tool for prevention of cardiovascular disease, stroke, cancer and diabetes due to their polyphenols modulate tau hyperphosphorylation and beta amyloid aggregation [ ] . the anthocyanins and polyphenols for major functional ingredients in blueberry played a key role in preventing chronic diseases [ ] . the organopolysulfides and quercetin for major functional ingredients in allium genus played a key role in preventing chronic diseases [ ] . baobab was cultivated from seeds from countries in east and west africa, its leaves had the highest vitamin b content ( : ± : mg/ g) from senegal, adult leaves provided the highest ca content ( . %) and young leaves with the highest ca and k content of nankoun in burkina faso [ ] . the diets of early hominids related with five center of crop origin (mediterranean, middle east, central asia, indo-burma, and china-korea); however, the rich food structure maintained the survival and development of early hominids who lacked survival competition and migration could not improve their intelligence. therefore, polyphenols and k-ca as well as vitamins ingredients in fruits/vegetables and leaves as well as grass/seeds (such as gramineae and its ancestor species of barley) for preventive chronic disease are the results of long-term dependence for diet from pliocene hominids in africa to modern human beings ( figure ). ingredients for neanderthals. neanderthals used mushrooms and nuts rich in polysaccharide and phytosterols as well as linoleic acid as staple foods to increase the dependence of the human body on these functional ingredients (see figure ). neanderthals as well as early homo sapiens show high dietary variability in mediterranean evergreen habitats, but less diet in high latitude steppe or coniferous forests [ ] . the steppe-like neanderthals of belgium feed on the meat of the woolly rhinoceros and wild sheep, while the neanderthals of the spanish forest feature root feed on mushrooms and pine nuts [ ] ; neanderthals in northern spain roasted vegetables and used medicinal plants about . ~ . ma [ ] . neanderthals ate meat (high chloroprostol) and plants ( β-sitosterol) as staple foods [ ] . plant foods of neanderthals from iraq and belgium had the typical modern human diets, which include palms, date, legumes, and grass seeds [ ] . medicinal functions (major polysaccharide) in medicinal mushrooms and fungi can prevent and treat more than chronic diseases [ ] ; however, β-glucans in barley preventive chronic diseases. the total polysaccharide content in morchella sp. reached up to . % of dried biomass in a mixture of : of wheat grains and potato peels [ ] . β-glucans are group of polysaccharides found in mushrooms, yeasts, seaweed, barley, and oats [ ] . macrofungal β-glucans are major β- , and β- , -glycosidic bonds, which have immunomodulatory, anticancer, and antioxidant properties; total β-glucan content varied from . % in a. bisporus to . % in t. rutilans [ ] . phytosterols are diet ingredients found in an array of nuts, seeds, and vegetables which have anticancer activities via their interactions with the plasma cell membrane [ ] . oil palm (elaeis guineensis jacq.) is one of the highest oil-yield crops in the world; however, palm oil is the largest variety of plant oil produced, consumed ( %), and internationally traded in the world, rich in linoleic acid ( %) that is associated with egfad gene [ ] . ~ % carbohydrate diets (especially whole-grain breads, vegetables, and nuts) had minimal risk of mortality [ ] . fruits, vegetables, and whole grains were indispensable among four top diets (mediterranean diet with five best ranked first, dash diet with lower blood pressure, flexitarian diet with lose weight and mind diet with brain health). the diets of neanderthals was not correlated with the complete center of crop origin, poor diet (meats, mushrooms, and pine nuts) and lack of migration lead to extinction ( figure ). these results reveal the importance of whole grain and vegetables or fruits in human health; however, lack of three categories of food suggesting the cause of the neanderthal destruction. therefore, polysaccharide (βglucan) and phytosterols as well as linoleic acid in mushrooms and nuts as well as palm oil for preventive chronic disease are the results of long-term dependence for diet from neanderthals in europe to modern human beings ( figure ). ingredients for homo sapiens. homo sapiens not only used grass and seeds rich in gaba and enzymes as well as resistant starch as staple foods to increase the unique dependence of the human body on these functional ingredients (see figure ) but also inherited the staple foods of early hominids and neanderthals. feeding and diet played key roles in human evolution, especially homo sapiens have a relative masticatory structure similar to that of other primates [ ] . homo sapiens moved from africa into the middle east about ka, according to fossils at skhul and qafzeh caves in israel [ ] . the aba-mirna- in belladonna with highly homologous to homo sapiens mirna can target and downregulate human brain-enriched transcription factor (znf- ) and gene expression in the human central nervous system [ ] . for homo sapiens, foragers have greater complexity than farmers or pastoralists; meanwhile, the old world foragers had significantly higher anisotropy values than new world foragers, but similarity between hard food foragers and hard food farmers [ ] . the meat diet abuse by a herbivorous homo sapiens can lead to atherosclerosis [ ] . the diets of african homo sapiens associated with center of crop origin in ethiopia, their migration along eight center of crop origin changed the fate of mankind [ ] . variation in cranial robusticity from geographical homo sapiens associated with cranial shape, size, climate, and neutral genetic distances; however, cranial robusticity may be an adaptation to cold and harsh environments as well as masticatory differences in diet [ ] , especially higher gaba (such as barley grass . mg/ g) grass diets in crop suiting environmental extremes improved intelligence [ ] . gaba contents in picked tea leaves under anoxic treatments at h and h are : ± : and : ± : mg/kg (fresh weight) [ ] . gaba a receptors modulate vigilance, emotions, cognition, and muscle tension, and they are the targets of anxiety-reducing and sedative-hypnotic benzodiazepines and some general anesthetics [ ] . shisa regulates gaba ar trafficking, function, and pharmacology, especially modulates benzodiazepine action in the brain [ ] . barley can be grown in four seasons (spring, summer, autumn, and winter) at , - , m in yunnan province of china which may be associated with harboring enzymes in barley grass [ ] . the diet high in sodium and low four diets (whole grains, fruits, vegetables, nuts, and seeds) were major dietary risk factors for deaths and disability-adjusted life-years globally and in many countries [ ] ; however, the whole grain is the manifestation of resistant starch type i surrounded by protein matrix and bran layer for making the starch unavailable for enzymes. the least chronic disease of ancient humans due to replacing the salt with seasoning crops, such as onions, ginger, garlic, coriander, pepper, chili, and so on [ ] . the coevolution of the preventive human chronic diseases are related to major diets of vavilov's eight crop origin centers (ethiopia, mediterranean, middle east, central asia, indo-burma, china-korea, mexico-guatemala, and peru-ecuador-bolivia) [ ] . human chronic diseases are related with six dietary structures (fruits/vegetables, young grass/barley grass, carnivorous, cereals crop, polished rice/wheat flour, and polished rice/-wheat+grass powder), but polished rice/wheat+barley grass powder is the most major healthy dietary guidelines for modern humans; therefore, it is necessary to unravel coevolutionary mechanism between preventive chronic diseases and human diet for functional foods [ ] . these results support homo sapiens used grass/seeds (rich in gaba and enzymes as well as resistant starch), fruits/vegetables and leaves (rich in polyphenols and k-ca as well as vitamins), mushrooms, and nuts (rich in polysaccharide and phytosterols as well as linoleic acid) as staple foods to increase the dependence of the human body on these functional ingredients. barley is the oldest and more important cereal crop with the utmost dietary fiber in the world; its malt, as a functional food, is not only the largest beer raw material in the world but also one of the most commonly used chinese herbal medicines [ ] . our review point out that barley grass has antidiabetic, anticancer, antidepressant, antioxidant, fatigue, anti-inflammatory, hypolipidemic, antigout, calcium supplementary, and antiacne/detoxifying effects; promotes sleep; regulates blood pressure; enhances immunity; protects liver; reduces hyperuricemia; alleviates atopic dermatitis; improves cognition, constipation, gastrointestinal function; and prevents hypoxia, cardiovascular diseases, and so on [ ] . barley grass powder is known to play a pivotal role in prevention of chronic diseases that involves six molecular mechanism of gaba, flavonoids, sod, k-ca, vitamins, and tryptophan [ ] ; however, barley grains play key roles in prevention of chronic diseases that involves six molecular mechanism of β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, and resistant starch. modern humans had originated in the progeniture of african homo sapiens with cognitive hominin [ ] . the staple foods of modern human are the synthesis of homo sapiens that inherited early hominids and neanderthals, which carry the neanderthal dna due to interbreeding between homo sapiens and neanderthal took place in the middle east. human flt ligand isolated from transgenic barley seeds is a glycoprotein including α( , )-fucose and α( , )xylose, which showed expression of human growth factor in barley grains with active protein [ ] . the peptide ll- is a component of the human innate immune system, it accumulated . mg/kg in the barley grains [ ] . human fgf- gene was fused with barley α-amylase signal peptide dna sequence and expressed in transgenic salvia miltiorrhiza plants driven by s promoter; however, recombinant fgf- in leaves was ng/ fresh weight [ ] . therefore, functional ingredients in barley grass and grains are essential for the health contribution of modern human (homo sapiens), neanderthals, and early hominids staple food to prevent and treat human chronic diseases. barley has health beneficial properties and was part of the modern hominid diet; thus, it has evolved in its functional ingredients and contributed to a reduced risk of diseases. it is amazing that barley grains and malt as well as grass powder can prevent or treat more than human chronic diseases. we think the major scientific basis for that is as follows: first, barley prevent over humans chronic diseases which associated with the similar origin and evolution center of barley and human beings: ethiopia and morocco in africa are top choices for cradle of modern humans homo sapiens and miocene hominoids as well as are the centers of origin for functional barley ( figure ) [ , , ] . ethiopia, morocco, fertile crescent, and tibet of china have been proposed as centers of barley origin and the primary habitat of wild barley (figure ) [ ] . wild barley is a selfing annual grass of predominantly morocco and irano-turanian and israel-jordan in arid desert or salt environments, the cold region in tibet of china and ethiopia, which has accumulated abundant functional ingredients for drought, salt, and cold resistances. the earliest modern human originate from ethiopia and morocco are dated to~ ka and~ ka (figure ) , respectively [ ] . the earliest human occupied high-altitude habitats in the andes and the tibetan plateau, especially late pleistocene humans adapted to the severe environments of these glaciated above , -meter elevation oxidative medicine and cellular longevity in the bale mountains of ethiopia ( figure ) [ ] . neanderthals in modern-day iraq and belgium ate grasses, cooked barley grains, and others. second, barley grass powder plays a key role in the promotion of human intelligence in the early stage: the incremental evolution of globular braincase associated with diets of brain health from ethiopia and middle east as well as from mediterranean center of crop origin, especially the highest gaba in crop diets suiting environmental extremes improved intelligence. brain development is a self-reinforcing process in which brain cells proliferate, differentiate, migrate, and connect functional neural circuit, especially primate-specific features of gabaergic interneuron development [ ] on the basis of gaba content in diet. survival depends on the selection of behaviors adaptive for environment; however, stimulation of dorsal raphe gaba neurons promoted movement in negative but not positive environments to promote environment-specific adaptive behaviors of serotonin [ ] . barley is a major crop in many developed countries [ ] ; gaba in barley grass suiting environmental extremes (cold, arid, and salt) can significantly increase, which can improve cognition and prevent chronic diseases [ ] . the average content of gaba in cultivars that we bred is . mg/ g which is . fold higher than that of other barley crops around the world [ ] . third, healthy effects of functional ingredients of barley grass and grains are the sum of staple foods for early hominids and neanderthals as well as homo sapiens: early hominids used fruits/vegetables and leaves rich in polyphenols (flavonoids) and k-ca as well as vitamins (tocols); neanderthals used mushrooms and nuts rich in polysaccharide (β-glucans and arabinoxylan) and phytosterols as well as linoleic acid; homo sapiens used grass and seeds rich in gaba and enzymes (sod) as well as resistant starch; modern human used barley grass rich in gaba, flavonoids, sod, k-ca, vitamins and tryptophan; however, barley grains rich in β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, resistant starch, and so on. therefore, barley played an important role in solving the problem of depending functional ingredients of homo sapiens and hominids as well as neanderthals, especially food safety in the process of migration and evolution from ancient humans to modern people. migration. food shortages and survival struggles caused by climate change were the causes of early human evolution of suiting environmental extremes from africa to asia and later to eurasia (figure ) [ , ] . barley is not only the most widely used cereal crop with comprehensive utilization of forage, materials for intoxicating liquor, functional food, stable food, ornamental weaving, and chinese medicines but also is the crop with the strongest resistance to stress (drought, cold, and salt) for the highest content of functional components, especially the growth period of barley varies from to days, which can be grown in four seasons in the world or at , ~ , m in yunnan province of china. these excellent characteristics become the best food for human migration. interestingly, there is a striking similarity between the human migration route and barley translocation/evolution route (see figure ). in [ ] . the spread of farming peoples of eurasia from the near east ( . ka), with movements both westward and eastward, especially ancestor of modern south asians is a mixture between early holocene populations of iran and south asia; however, yamnaya in the bronze age of europe moved both westward and eastward from north of the black sea [ ] . discovery of different crushing apparatuses in mountains of iran revealed that people were grinding wheat and barley about , years ago [ ] . these results support the healthy food contribution between human migration and barley translocation/evolution, especially climate change increased functional ingredients in barley for preventive chronic diseases. civilization. this point of view reveals the evolution of human skull morphology on the basis of the hybridization between h. sapiens and other hominin species in morocco at , years ago, all of which are descendants of the african homo sapiens population [ ] . all living people in europe and asia carry the same amount of neanderthal dna due to the interbreeding between homo sapiens and neanderthal that took place in the middle east [ ] . fertile crescent is the concentrated area of wild barley [ ] and the distribution area of ancient babylonian civilization and ancient egyptian civilization, among which jerusalem is the holy place of judaism, christianity, and islam ( figure ). barley is one of the oldest crops used by ancient farmers, its cultivation has been optimized by modern humans in the ancient era for less shattering, higher yield and better grains; however, the sharp awn of barley has become an important guarantee for the most resistant birds trouble and largescale farming civilization in cereal crops. climate change stimulated agricultural innovation and exchange across asia between , and , years ago; sorghum and millet made their way from china to central asia; wheat and barley moved from central asia to the far east and became a staple food in the north of china at . ka, which exchanges across central and high-altitude asia coalesced to form the silk road ( . ka~ . ka) and grand canal for traffic great artery of north-south in ancient china ( . ka) [ ] . tibetan barley (qingke) is derived from eastern domesticated barley, north pakistan, india, and nepal between , and , years ago, which supports a feral or hybridization origin for tibetan weedy barley [ ] . the rise of barley against stress (drought, cold, salt, and bird) to staple food has increased functional ingredients (especially gaba) in diet to prevent chronic diseases and promoted human civilization. gaba-mediated inhibitory interneurons control memory-encoding ca neurons; nucleus incertus (ni) establishes gabaergic inhibitory synapses on interneurons; ni gabaergic cells can regulate hippocampusdependent episodic memory formation bidirectionally, and its dysfunction may contribute to anxiety-like syndromes [ ] . cities and words and metallurgy as well as complex ceremonial buildings are the four standards of world civilization; fertile crescent for one of centers of barley origin is closely related to ancient babylonian and ancient egyptian civilizations, especially ancient babylonian civilizations have the earliest human civilization (agriculture ka, cities ~ ka, metallurgy ~ ka, words . ka, calendar ka, and systematic religion ka) in the world; however, ancient egyptian civilizations has the earliest empire (mathematics . ka, geometry ka, and writing tool ka). the ancestral blocks of the domesticated barley genomes were descended from all over the fertile crescent, especially levantine (western) and zagros (eastern) clusters of the origin of agriculture for nine wild barley populations, i.e., carmel and galilee, golan heights, hula valley and galilee, judean desert and jordan valley, lower mesopotamia, negev mountains, north levant, sharon, coastal plain and judean lowlands, and upper mesopotamia [ ] . the neolithic crops facilitated the early agricultural establishment; the barley evolution followed the agricultural development in the near east [ ] . for humans from hunter gathering to agriculture about ka of the levant in near east, barley was a founder crop for converting the brittle floral axis of the wild-type into a tough and nonbrittle spike, which made a major contribution to the emergence of early agrarian societies [ ] . for ancient indian civilization, the earliest known farming cultures in south asia emerged in the hills of balochistan in pakistan about . ka; however, seminomadic peoples domesticated wheat, barley, sheep, goat, and cattle [ ] . for ancient chinese civilization, the earliest beer recipe in china included broomcorn, millet, barley, job's tears, and tubers around , y ago, which may have motivated the initial translocation of barley from the western eurasia into the central plain of china [ ] . diseases. western medicine solves the issue of human organs, and traditional chinese medicine solves the issue of human body system; however, functional food is to address the problem of human cells. barley grains and its grass not only are the best functional food that provides nutrition and eliminates toxins from cells in human beings, which are rich in ingredients to combat more than chronic diseases but also have all the nutrients needed for cell nutrition and detoxification, which is the result of the long-term coevolution of the dietary structure of ancient apes for plants and early homo sapiens with the staple food of barley. interestingly, the types of prevention and treatment of human chronic diseases by key functional components in barley grain were in order: β-glucans ( )>polyphenols ( )>arabinoxylan ( ) = tocols ( )>phytosterols ( )>resistant starch ( ), but gaba ( )>flavonoids ( )>sod ( )>k-ca ( ) = vitamins ( )>tryptophan ( ) in barley grains. the key functional components of barley grains and its grass not only play an important role in the prevention and treatment of more than chronic human diseases but also interact with other nutritional functional components to provide the possibility to solve hundreds of human diseases caused by cell undernutrition and their detoxification disorders. the unique theory and practice system of the whole regulation improve the immunity of chinese medicine to prevent and treat human diseases. with advances in science and technology, barley will also find many behavioral mechanisms that combat human diseases, such as barley malt has been used in many prescriptions for the prevention and treatment of covid- in lots of provinces in china, which includes jiangxi, guangdong, gansu, guizhou, and jiangsu province in china [ ] . dietary restriction is an activator of prolongevity molecular pathways based on an escape from costs incurred under nutrient-rich conditions [ ] . chronic disease deaths due to heredity, environment, and lifestyle are as high as million, accounting for more than % of all deaths worldwide [ ] . these results support that human has only new cell disease theory, which are made up of sixty million cells, more than , diseases are due to cell nutritional deficiencies and detoxification disorder caused by the disease [ ] . ingredients of barley were preserved in the chinese crop genebank. the detection of functional ingredients in grains and grass powder of more than , accessions barley gremplasm especially combinated with high-density snp markers can not only reveal the molecular mechanism of functional ingredients and their molecular breeding and gene editing techniques but also make great contributions for improving human health by increasing functional ingredients in barley grains and grass powder. ingredients in barley. some excellent barley varieties have very high functional ingredients both in grains and grass powder, which are the result of adaptation to high and low temperature, drought and waterlogging, long and short sunshine, and day and night changes caused by altitude, latitude, and seasonal differences. first of all is the breeding of high functional ingredients in barley grains. we have bred some functional barley by crossbreeding between the highest functional ingredients germplasm (β-glucan > . %, resistant starch > %, arabinoxylan > %, or polyphenols > . %) and cultivated barley at low temperature and drought as well as high altitude, such as yunke and zangqing with high β-glucan, yungongmai , and yungongmai with high vitamin c. secondly is the breeding of high functional ingredients in barley grass. we have bred some functional barley by crossbreeding between the highest functional ingredients germplasm (gaba > . %, k > . %) and cultivated barley at low temperature and drought as well as high altitude. the average content of gaba in cultivars that we bred is . mg/ g which is . fold higher than that of other barley crops around the world, especially barley grass powder of yungong brand contains times gaba ( . mg/ g) and times ca as well as times k than those of polished rice [ ] . in addition, the functional ingredients of barley grain and grass powder can also be greatly improved by gene editing technology, which needs to be further studied and enriched in the future. functional ingredients of barley. functional ingredients of barley grain and its grass powder have larger variation due to latitude, altitude, season, light, temperature, water, day, and night. all the high-altitude ( , ~ , m) hulless barley can increase higher functional ingredient content than that of plains ( ~ m altitude) [ ] . therefore, it is necessary to promote the functional components of the barley grain and its grass powder under the best ecological conditions, and we also make a useful exploration of the functional components at different altitudes and in different seasons. in addition, the functional ingredients of different parts of barley grain (see table ) and the functional ingredients of different grass cutting stages were different. barley is the oldest and the richest functional food among global cereals. its grains are rich in β-glucan; polyphenols (phenolic acids, flavonoids, and anthocyanins), polysaccharide (arabinoxylan), phytosterols (β-sitosterol, campesterol), tocols (β-tocotrienol, α-tocotrienol, β-tocopherol, α-tocopherol), resistant starch, alkaloid, gaba, folates, linoleic acid, phytate, and so on. this review paper summarizes the obvious efficacy of barley grains that includes antidiabetes, antiobesity, anticancer, antioxidants, anti-inflammation, immunomodulation, cardioprotection, gastroprotection, and hepatoprotection properties, and also, barley grains can lower blood pressure; prevent cardiovascular diseases; optimize cholesterol; improve bowel health and metabolic syndrome; prevent heart disease; reduce chronic kidney disease; decrease stroke; alleviate allergic rhinitis and atopic dermatitis; and accelerate wound healing activities. barley grains, grass, straw, husk, bran, and fine powder are rich in ingredients and food structure to defeat chronic diseases during human migration, especially molecular mechanisms of six functional ingredients barley grass (gaba, flavonoids, sod, k-ca, vitamins, and tryptophan) and grains (β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, and resistant starch) involve to combat more than chronic diseases. these results suggest that barley plays an important role in a healthy diet and in the promotion of early human intelligence. in particular, the healthy effects of functional components of barley grains and grass are the result of longterm continuous evolution of early hominids (fruits/vegetables and leaves rich in polyphenols, k-ca, and vitamins), neanderthals (mushrooms and nuts rich in polysaccharides, phytosterols, and linoleic acids), and homo sapiens (grasses and seeds rich in gaba, enzymes, and resistant starch), which associate with modern humans originating in the progenitor of african homo sapiens with cognitive hominin, especially after interbreeding between homo sapiens and neanderthals that took place in the middle east. the migration route from africa to asia and then to eurasia is basically consistent with the origin and spread of barley and its domestication path, which indirectly supports that barley against stress (drought, cold, and salt) enriched with functional ingredients prevented chronic disease from ancient humans to modern people. fertile crescent is the concentrated area of wild barley and the distribution area of ancient babylonian civilization and ancient egyptian civilization, among which jerusalem is the holy place of judaism, christianity, and islam. these results indirectly support this great contribution of barley for promoting world civilization. the polyphenols in fruits/leaves and polysaccharide in mushrooms/nuts as well as gaba in grass/seeds for prevention of chronic disease are associated with depending functional ingredients for diet from pliocene hominids in africa to modern humans. ethiopia and morocco in africa are top choices for cradle of modern humans homo sapiens and miocene hominoids as well as are the centers of origin for functional barley. food shortages and survival struggles caused by climate change were the causes of early human evolution associated with gaba in the barley grass increased oxidative medicine and cellular longevity sharply under environmental extremes from africa to asia and later to eurasia, especially gaba in crop diets suiting environmental extremes improved intelligence. these results support findings that barley and its grass may be the best functional food crop, especially barley prevents over human chronic diseases based on six functional ingredients of barley grass and grains due to three aspects of the scientific basis, i.e., the similar origin and evolution center of barley and human, the promotion of human intelligence in the early stage, and the sum of staple foods for early hominids and neanderthals as well as homo sapiens. we put forward the strategy of increasing the functional ingredients of barley grain and its grass powder that is as follows: ( ) exploration of excellent germplasm with high functional ingredients in barley; ( ) breeding excellent cultivars with high functional ingredients in barley; ( ) optimization of ecological conditions for high functional ingredients of barley. in addition, the functional ingredients of different parts of barley grain and the functional ingredients of different grass cutting stages are different. although therapeutic mechanisms of functional ingredients in barley grains and grass powder for prevention of human chronic diseases seem a very complicated task, and functional food for therapeutic interventions opens up new ways, it is necessary to find further scientific evidence that demonstrates the health effects of functional ingredients of barley and their extracts from barley on the treatment of chronic diseases. barley is one of the most exciting potential natural sources for the development of functional foods and new drugs with improved efficiency and safety. although we have found some relationship of origin and migration between human and barley, especially preventive role of barley for chronic diseases of human beings, it is necessary to conduct more systemic studies to unravel coevolutionary interconnection mechanism between chronic diseases prevention and human diet for barley functional foods. unfortunately, so far there, is no evidence provided of barley evolving as part of evolutionary consumption. barley plays an important role in promoting the development of functional food and has a potential underlying molecular mechanism and formation as well as action mechanism, which is worthy of further study. this review can be used as a starting point for novel nutraceuticals and functional foods and drugs for barley to improve the prognosis of chronic diseases. the authors declare that they have no conflict of interests whatsoever to declare. the global economic burden of non-communicable diseases health effects of dietary risks in countries, - : a 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analysis of functional ingredients in barley grains from different regions between southwest china and icarda nutrition analysis and food process of barley the research and publication of this article was funded by china agriculture research system (cars- - a, cars- - b) and yunnan provincial expert grass-roots scientific research workstation. key: cord- -n ylgqfu authors: giri, rajanish; bhardwaj, taniya; shegane, meenakshi; gehi, bhuvaneshwari r.; kumar, prateek; gadhave, kundlik; oldfield, christopher j.; uversky, vladimir n. title: when darkness becomes a ray of light in the dark times: understanding the covid- via the comparative analysis of the dark proteomes of sars-cov- , human sars and bat sars-like coronaviruses date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: n ylgqfu recently emerged coronavirus designated as sars-cov- (also known as novel coronavirus ( -ncov) or wuhan coronavirus) is a causative agent of coronavirus disease (covid- ), which is rapidly spreading throughout the world now. more than , , cases of sars-cov- infection and more than , covid- -associated mortalities have been reported worldwide till the writing of this article, and these numbers are increasing every passing hour. world health organization (who) has declared the sars-cov- spread as a global public health emergency and admitted that the covid- is a pandemic now. the multiple sequence alignment data correlated with the already published reports on the sars-cov- evolution and indicated that this virus is closely related to the bat severe acute respiratory syndrome-like coronavirus (bat sars-like cov) and the well-studied human sars coronavirus (sars cov). the disordered regions in viral proteins are associated with the viral infectivity and pathogenicity. therefore, in this study, we have exploited a set of complementary computational approaches to examine the dark proteomes of sars-cov- , bat sars-like, and human sars covs by analysing the prevalence of intrinsic disorder in their proteins. according to our findings, sars-cov- proteome contains very significant levels of structural order. in fact, except for nucleocapsid, nsp , and orf , the vast majority of sars-cov- proteins are mostly ordered proteins containing less intrinsically disordered protein regions (idprs). however, idprs found in sars-cov- proteins are functionally important. for example, cleavage sites in its replicase ab polyprotein are found to be highly disordered, and almost all sars-cov- proteins were shown to contain molecular recognition features (morfs), which are intrinsic disorder-based protein-protein interaction sites that are commonly utilized by proteins for interaction with specific partners. the results of our extensive investigation of the dark side of the sars-cov- proteome will have important implications for the structural and non-structural biology of sars or sars-like coronaviruses. significance the infection caused by a novel coronavirus (sars-cov- ) that causes severe respiratory disease with pneumonia-like symptoms in humans is responsible for the current covid- pandemic. no in-depth information on structures and functions of sars-cov- proteins is currently available in the public domain, and no effective anti-viral drugs and/or vaccines are designed for the treatment of this infection. our study provides the first comparative analysis of the order- and disorder-based features of the sars-cov- proteome relative to human sars and bat cov that may be useful for structure-based drug discovery. intrinsically disordered proteins (idps) and intrinsically disordered protein regions (idprs)), in order to better understand an interplay between the ordered and disordered components of the proteome. in classical structure-function-paradigm, it is believed that a unique, stable, and well-defined -dimensional structure is a prerequisite for a protein to accomplish its unique biological function. although this notion dominated scientific minds for over the hundred years, eventually an idea of the presence of functional intrinsic disorder in proteins came to the attention of the structural biologists. according to this "heretic" viewpoint, a noticeable amount of biologically active proteins (of protein regions) fail to fold into the well-defined structures and instead remain disordered, existing as highly dynamic ensembles of rapidly interconverting conformations under the physiological conditions. these proteins and protein regions are known now as intrinsically disordered proteins (idps) and intrinsically disordered protein regions (idprs), respectively. the propensity of being functional intrinsically disordered proteins (similar to the propensity of forming unique biologically active structures of ordered proteins) is determined by the amino acid sequences [ ] [ ] [ ] . idps exhibit their biological functions in numerous biological processes commonly associated with cellular signalling, gene regulation, and control by interacting with their physiological partners [ ] [ ] [ ] [ ] [ ] . these functions of idps and idps are regulated by their protein-protein, protein-rna, protein-dna interactions [ , ] . molecular recognition features (morfs) are the regions in idps implicated in regulation of idps function by protein-protein interactions and serve as the primary stage in molecular recognition. zhang and colleagues have reported the genomic sequence of sars-cov- with genbank accession number nc_ having , nucleotides. the virus was isolated from the bronchoalveolar lavage fluid of a patient, went through a circle or renaming, from novel wuhan seafood market pneumonia virus to deadly wuhan coronavirus, to the novel coronavirus ( -ncov) or the wuhan- novel coronavirus (wuhan- -ncov, and was eventually named sars-cov- by the who [ ] . it is known that the idps/idprs are present in all three kingdoms of life, and viral proteins often contain unstructured regions that have been strongly correlated with their virulence [ ] [ ] [ ] [ ] . in this report, we investigated the disordered side of the sars-cov- proteome using a complementary set of computational approaches to check the prevalence of idprs in various sars-cov- proteins and to shed some light on their disorder-related functions. we also have comprehensively analyzed idprs among the closely related viruses, such as human sars cov and bat sars-like cov. furthermore, we have also identified protein functions related to protein-protein interactions, rna binding, and dna binding from all three viruses. since these three viruses are closely related, our study provides important means for a better understanding of the sequence and structural peculiarities of their evolution. we believe that this study will help the structural and non-structural biologists to design and perform experiments for a more in-depth understanding of this virus and its pathogenicity. this also will have long-term implications for developing new drugs or vaccines against this currently unpreventable infection. sequence retrieval and multiple sequence alignment. the protein sequences of bat cov (sars-like) and human sars cov were retrieved from uniprot (uniprot ids for individual proteins are listed in table ). the translated sequences of sars-cov- proteins [genbank database [ ] (accession id: nc_ . )] were obtained from genbank. we used these sequences for performing multiple sequence alignment (msa) and predicting the idprs. we have used clustal omega [ ] for protein sequence alignment and esprit . [ ] for constructing the aligned images. for the prediction of the intrinsic disorder predisposition of cov proteomes, we have used multiple predictors, such as members of the pondr ® (predictor of natural disordered regions) family including pondr ® vls [ ] , pondr ® vl [ ] , pondr ® fit [ ] , and pondr ® vlxt [ ] , as well as the iupred platform for predicting long (≥ residues) and short idprs (< residues) [ ] . these computational tools predict residues/regions, which do not have the tendency to form an ordered structure. residues with disorder scores exceeding the threshold value of . are considered as intrinsically disordered residues, whereas residues with the predicted disorder scores between . and . are considered flexible. complete predicted percent of intrinsic disorder (ppid) in a query protein was calculated for every protein of all the three viruses from outputs of six predictors. the detailed methodology has been given in our previous reports [ , ] . and disopred [ ] . the protein residues with anchor, morfpred, and disopred score above the threshold value of . and morfchibi_web score above the threshold value of . are considered morf regions. idprs facilitate interactions with rnas and dnas and regulates many cellular functions [ ] . thus, for predicting the dna binding residues in cov proteins, we have used two online servers: drnapred [ ] and disordpbind [ ] . for rna binding residues, we used pprint (prediction of protein rna-interaction) [ ] and disordpbind [ ] . the mean values of the predicted percentage of intrinsic disorder scores (mean ppids), that were obtained by averaging the predicted disorder scores from six disorder predictors (supplementary table - ) for each protein of sars-cov- as well as human sars, and bat cov are represented in table . * these sequences are based on genome annotations conducted by wu et al. [ ] . proteins and their ppids are coloured to reflect their disorder status (ordered -blue, moderately disorderedpink, highly disordered -red) figures a, b , and c are d-disorder plots generated for sars-cov- , human sars and bat cov proteins, respectively, and represent the ppid pondr-fit vs. ppid mean plots. based on their predicted levels of intrinsic disorder, proteins can be classified as highly ordered (ppid < %), moderately disordered ( % ≤ ppid < %) and highly disordered (ppid ≥ %) [ ] . from the data in table , figures a, b , and c, as well as the ppid based classification, we conclude that the nucleocapsid protein from all three strains of coronavirus possesses the highest percentage of the disorder and is classified as highly disordered protein. orf b protein in bat cov, orf protein in sars-cov- , human sars, and bat cov, and orf b protein in human sars and sars cov belong to the class of moderately disordered proteins. while the structured proteins, namely, spike glycoprotein (s), an envelope protein (e) and membrane protein (m) as well as accessory proteins orf a, orf a, orf (orf a and orf b in case of human sars) of all three strains of coronavirus are ordered proteins. orf and orf proteins also belong to the class of ordered proteins. in ch-cdf plot of the proteins of (d) sars-cov- (e) human sars and (f) bat cov, the y coordinate of each protein spot signifies distance of corresponding protein from the boundary in ch plot and the x coordinate value corresponds to the average distance of the cdf curve for the respective protein from the cdf boundary. in order to further investigate the nature of the disorder in proteins of sars-cov- , human sars, and bat cov, we utilized the combined ch-cdf tool that uses the outputs of two binary classifiers of disorder, charge hydropathy (ch) plot and cumulative distribution function (cdf) plot. this helped in retrieving more detailed characterization of the global disorder predisposition of the query proteins and their classification according to the disorder "favours". the ch plot is a linear classifier that differentiates between proteins that are predisposed to possess extended disordered conformations that include random coils and premolten globules from proteins that have compact conformations (ordered proteins and molten globule-like proteins). the other binary predictor, cdf is a nonlinear classifier that uses the pondr ® vlxt scores to discriminate ordered globular proteins from all disordered conformations, which include native molten globules, pre-molten globules, and random coils. the ch-cdf plot can be divided into four quadrants: q (bottom right quadrant) is an area of ch-cdf phase space that is expected to include ordered proteins; q (bottom left quadrant) includes proteins predicted to be disordered by cdf and compact by ch (i.e., native molten globules and hybrid proteins containing high levels of both ordered and disordered regions); q (top left quadrant) contains proteins that are predicted to be disordered by both ch and cdf analysis (i.e., highly disordered proteins with the extended disorder); and q (top right quadrant) possesses proteins disordered according to ch but ordered according to cdf analysis [ ] . figures d, e and f represent the ch-cdf analysis of proteins of sars-cov- , human sars, and bat cov and show that all the proteins are located within the two quadrants q and q . the ch-cdf analysis leads to the conclusion that all proteins of sars-cov- , human sars, and bat cov are ordered except nucleocapsid protein, which is predicted to be disordered by cdf but ordered by ch and hence lies in q . molecular recognition features (morfs) are short interaction-prone disordered regions found within idps/idprs that commence a disorder-to-order transition upon binding to their partners [ , ] . these regions are important for protein-protein interactions and may initiate an early step in molecular recognition [ ] . in this study, we have analyzed and compared morfs (protein-binding regions) in sars-cov- with human sars and bat cov. the results of this analysis are summarized in table , which clearly shows that most of the sars-cov- proteins contain at least one morf, indicating that disorder does play an important role in the functionality of these viral proteins. in addition to protein-protein interactions/protein-binding functions, idps and idrs also mediates functions by facilitating their interactions with nucleotides such as dna and rna [ , ] . therefore, we have used a combination of two different online servers for locating protein residues that are showing the propensity to bind with dna as well as rna. nucleotide-binding residues in proteins of three studied coronaviruses are listed in supplementary coronaviruses encode four structural proteins, namely, spike (s), envelope (e) glycoprotein, membrane (m), and nucleocapsid (n) proteins, which are translated from the last ~ kb nucleotides and form the outer cover of the covs, encapsulating their single-stranded genomic rna. s protein is a large multifunctional protein forming the exterior of the cov particles [ , ] . it forms surface homotrimers and contains two distinct ectodomain regions known as s and s . in some covs, the s protein is actually cleaved into these subunits, which are joined non-covalently, whereas an additional proteolytic cleavage within the n-terminal part of the s subunit that takes place upon virus endocytosis generates spike proteins s '. subunit s initiates viral infection by binding to the host cell receptors, s acts as a class i viral fusion protein that mediates fusion of the virion and cellular membranes and thereby promotes the viral entry into the host cells, whereas s ' serves as a viral fusion peptide [ , ] . spike binds to the virion m protein through its c-terminal transmembrane region [ ] . belonging to a class i viral fusion protein, s protein binds to specific surface receptor angiotensin-converting enzyme (ace ) on host cell plasma membrane through its n-terminal receptor-binding domain (rbd) and mediates viral entry into host cells [ ] . the s protein consists of an n-terminal signal peptide, a long extracellular domain, a singlepass transmembrane domain, and a short intracellular domain [ ] . a . Å resolution structure (pdb id: acc) of s protein from human sars complexed with its host binding partner ace has been obtained by cryo-electron microscopy (cryo-em the biophysical analysis reported in previous study has also revealed that the s protein from sars-cov- has a higher binding affinity to ace than s protein from human sars [ ] . which has been calculated by averaging the disorder scores from all six predictors is represented by a short-dot line (sky-blue line) in the graph. the light sky-blue shadow region signifies the mean error distribution. the residues missing in the pdb structure or the residues for which pdb structure is unavailable are represented by the grey-coloured area in the corresponding graphs. (e) aligned disorder profiles generated for spike glycoprotein from sars-cov- (black line), human sars (red line), and bat cov (green line) based on the outputs of the pondr ® vsl . msa analysis among all three coronaviruses demonstrates that s protein of sars-cov- has a . % sequence identity with bat cov and . % identity with human sars (supplementary figure s a) . all three s proteins are found to have a conserved c-terminal region. however, the n-terminal regions of s proteins display noticeable differences. given that there is significant sequence variation rbd located at the n-terminal region of s protein, this might be the reason behind variation in its virulence and its receptor-mediated binding and entry into the host cell. according to our intrinsic disorder propensity analysis, s protein from all three covs analysed in this study are highly structured, as their predicted disorder propensity lies below % ( table ). in fact, the mean ppid scores of sars-cov- , human sars cov, and bat cov are calculated to be . %, . %, and . %, respectively. figures b, c , and d represent the intrinsic disorder profiles of s proteins from sars-cov- , human sars and bat cov obtained from six disorder predictors. finally, figure e shows aligned disorder profiles of s proteins from these covs and illustrates remarkable similarity in their disorder propensity, especially in the c-terminal region. it is of interest to map known functional regions of s proteins to their corresponding disorder profiles. the maturation of s protein requires specific posttranslational modification (ptm), proteolytic cleavage that happens at two stages. first, host cell furin or another cellular protease nicks the s precursor to generate s and s proteins, whereas the second cleavage that takes place after the viral attachment to host cell receptors leads to the release of a fusion peptide generating the s ' subunit. in human sars cov, the first and second cleavage site is located at residues r and r , respectively, whereas in bat cov, the corresponding cleavage sites are residues r and r . as it follows from figure , these cleavage sites are located within the idprs. in human sars cov s protein, fusion peptide (residues - ) is located within a flexible region, is characterized by the mean disorder score of . ± . . similarly, in bat cov s protein, fusion peptide (residues - ) has a mean disorder score of . ± . . s protein contains two heptad repeat regions that form coiledcoil structure during viral and target cell membrane fusion, assuming a trimer-of-hairpins structure needed for the functional positioning of the fusion peptide. in human sars cov s protein, heptad repeat regions are formed by residues - and - , which have mean disorder scores of . ± . and . ± . , respectively. the analogous situation is observed for the s protein from bat cov, where these heptad repeat regions are positioned at residues - ( . ± . ) and - ( . ± . ). another functional region found in s proteins is the receptor-binding domain (residues - and - in human sars cov and bat cov, respectively) containing a receptor-binding motif responsible for interaction with human ace . in human s protein of human sars cov this motif (residues - ) is not only characterized by structural flexibility, possessing a mean disorder score of . ± . , but also contains a disordered region (residues - ). since s protein is known as spike glycoprotein, it contains numerous glycosylation sites. due to rather close similarity of disorder profiles of s proteins analysed here, we can assume that all the aforementioned indications of the functional importance of disorder and flexible regions in s proteins from sars cov and bat cov are also applicable to sars-cov- s protein. finally, table shows that s protein from sars-cov- contain one morf region at its cterminal (residues - ) by morfchibi_web, two morf regions ((residues - ) & (residues - )) by morfpred, and one morf region at n-terminal (residues - ) by disopred . these results indicating that intrinsic disorder is important for its interaction with binding partners. interestingly, the n-terminal region of s protein (residues - ) from all three viruses are observed to be a disorder-based protein binding region by two predictors (morfpred and disopred ). n-terminal morf displays its role in viral interaction with host receptor and c-terminal morf displays its role in m protein interaction and viral assembly. moreover, morf region mainly lies in the n-and c-terminal regions suggesting a possible role during cleavage as well. in addition to protein-binding regions, s protein also shows many nucleotide-binding residues. tables , , and shows that numerous rna binding residues predicted by pprint in all three viruses and a single rna binding residue were predicted by disordpbind in human sars. further, drnapred and disordpbind predicted the presence of many dna binding residues in s protein of all three viruses. these results signify the role of s protein functions related to molecular recognition (protein-protein interaction, rna binding, and dna binding) such as interaction with host cell membrane and further viral infection. therefore, identified idps/idprs and residues/regions from s protein crucial for molecular recognition can be targeted for disorder-based drug discovery. envelope (e) protein is a small, multifunctional inner membrane protein that plays an important role in the assembly and morphogenesis of virions in the cell [ ] [ ] [ ] . e protein consists of two ectodomains associated with n-and c-terminal regions, and a transmembrane domain. it homo-oligomerize to form pentameric membrane destabilizing transmembrane (tm) hairpins to form a pore necessary for its ion channel activity [ ] . figure a shows the nmr-structure (pdb id: mm ) of human sars envelope glycoprotein of - residues [ ] . msa results illustrate ( figure b ) that this protein is highly conserved, with only three amino acid substitutions in e protein of sars-cov- conferring its % sequence similarity with human sars and bat cov. bat cov shares % sequence identity with human sars. mean ppid calculated for sars-cov- , human sars, and bat cov e proteins are . %, . %, and . % respectively ( table ) . the e protein is found to have a reasonably well-predicted structure. our predictions suggest that the residues of n-and c-terminals are displaying a higher tendency for the disorder. the last hydrophilic residues (residues - ) have been reported to adopt a random-coil conformation with and without the addition of lipid membranes [ ] . literature suggests that the last four amino acids of the c-terminal region of e protein containing a pzd-binding motif are involved in protein-protein interactions with a tight junction protein pals . our results support literature as we identified long n-terminal region of approximately residues long as disorder-based protein binding region in all three viruses (see table , supplementary table and ). pals is involved in maintaining the polarity of epithelial cells in mammals [ ] . respective graphs in figures c, d , and e show the predicted intrinsic disorder profiles for e proteins of sars-cov- , human sars, and bat cov. we speculate that the disordered region content may be facilitating the interactions with other proteins as well. in agreement with this hypothesis, table shows that in e protein from sars-cov- , the c-terminal domain serves as protein-binding region. we found that the residues from - is a long morf in e proteins of all three viruses as predicted by morfchibi_web ( table , supplementary table and ). as aforementioned, these randomly-coiled binding-residues at c-terminus may gain structure while assisting the protein-protein interaction mediated by e protein. one more morf region (residues [ ] [ ] [ ] [ ] [ ] in the transmembrane domain was observed by disopred in the e protein of all three viruses. since these residues are the part of ion channel, we speculate that these residues do specific interactions and may be guiding the specifi functions of ion channel activity. few rna binding residues by pprint and disordpbind and several dna binding residues by drnapred are predicted for e protein in all three viruses. assembly by interacting with the nucleocapsid (n) and e proteins [ ] [ ] [ ] . protein m interacts specifically with a short viral packaging signal containing coronavirus rna in the absence of n protein, thereby highlighting an important nucleocapsid-independent viral rna packaging mechanism inside the host cells [ ] . it gains high-mannose n-glycans in er, which are subsequently modified into complex n-glycans in the golgi complex. glycosylation of m protein is observed to be not essential for virion fusion in cell culture [ , ] . cryo-em and tomography data indicate that m forms two distinct conformations, a compact m protein having high flexibility and low spike density, and an elongated m protein having a rigid structure and narrow range of membrane curvature [ ] . some regions of m glycoproteins might serve as important dominant immunogens. although no structural information is available for the full-length m protein as of yet, a short peptide of the membrane glycoprotein (residues - ) from human sars cov was co-crystallized with a complex between a- alpha chain of the hla class i histocompatibility antigen and β microglobulin (pdb id: i g) [ ] . figure a shows that within this complex, the cocrystallized m protein region exists in an extended conformation. m protein of sars-cov- has a sequence similarity of . % with bat cov and . % with human sars m proteins ( figure b ). our analysis revealed that the intrinsic disorder levels in m proteins of sars-cov- , human sars cov, and bat cov are relatively low since these proteins show the ppid values of . %, . %, and . % respectively. this is in line with the previous publication by goh et al. on human sars hku where they found the mean ppid of % using additional predictors such as topidp and foldindex along with the predictors used in our study [ ] . figures c, d , and e represent per-residue disorder profiles generated for m proteins of sars-cov- , human sars cov, and bat cov and show that with the exception to their n-and c-terminal regions, these proteins are mostly ordered. the last residues of mers-cov m protein are important for intracellular trafficking and contains a determinant that localizes it into the golgi network [ ] . our results in table illustrates that the disordered c-tail of the m protein is predicted to have disorder based protein-binding region and therefore can serve as a binding site for its specific partner required for its localization inside the host cell. a long morf region (residues - ) at the c-terminal of m protein in all three viruses were observed by morfchibi_web. two morf regions (one at n-terminus (residues - ) and one at c-terminus (residues - )) was observed by disopred in human sars and bat cov. however, single morf (residues - ) observed in sars-cov- by disopred . morfpred also predicts a short morf at c-terminus of sars-cov- (residues - ), human sars (residues - ), and bat cov (residues - ) ( table , supplementary tables and ) . furthermore, the m protein from all three viruses displays strong tendency to bind with rna (as predicted by pprint and disordpbind) and dna (as predicted by drnapred and disordpbind) (see supplementary tables , , and ). our understanding on m protein of covs (idps and morf at c-terminus and molecular recognition) elucidates its crucial role in interaction with the n and e proteins for viral assembly. nucleocapsid (n) protein: nucleocapsid (n) protein is one of the major viral proteins playing several significant roles in transcription, and virion assembly of coronaviruses [ ] . it binds to viral genomic rna forming a ribonucleoprotein core required for the rna encapsidation during viral particle assembly [ ] . sars-cov virus-like particles (vlps) formation has been reported to depend upon either m and e proteins or m and n proteins. for the effective production and release of vlps, co-expression of e or n proteins with m protein is necessary [ ] . n protein of human sars consists of two structural domains, the n-terminal rna-binding domain (ntd: - residues) and the c-terminal dimerization domain (ctd: - residues) with a disordered patch in between these domains. n protein has been demonstrated to bind viral rna using both ntd and ctd [ ] . figure a displays the nmr solution structure of the ntd of human sars cov nucleocapsid protein ( - residues) (pdb id: ssk) [ ] . figure a shows an x-ray crystal structure of the ctd of human sars cov nucleocapsid protein ( - residues) (pdb id: gib) [ ] . a model of the domain organization of the n-protein from sars-cov- is shown in figure b . the amino acid-long n protein of sars-cov- shows a percentage identity of . % with n protein of bat cov n protein and . % with human sars n protein (supplementary figure s b) . our analysis revealed that the n proteins of coronaviruses contain the highest levels of intrinsic disorder (see figure and table ). in fact, n proteins from sars-cov- human sars cov, and bat cov are characterized by the mean ppid of . %, . %, and . %, respectively. in accordance with the previously evaluated intrinsic disorder predisposition [ ] , n protein is highly disordered in all three sars viruses analysed in this study ( table ) . graphs in figures c, d , and e depict the disorder profiles of sars-cov- , human sars cov, and bat cov nucleocapsid proteins and show that their n-and c-terminal regions are completely disordered, and all three proteins also contain the central unstructured segment. as expected, the intrinsic disorder predisposition of the n protein of sars-cov- is remarkably similar to that for the n protein of human sars cov as reported in a previous study [ ] . this is further supported by figure f , where pondr ® vsl -generated disorder profiles of these three proteins are overlapped to show almost complete coincidence of their major disorder-related features. it is clear that in n-proteins, the n-and c-termini and a log central segment are completely disordered. figure c shows that in the n protein from sars-cov- , residues - , - , - , - , and - are found to be disordered. many of these residues are lying within the ntd and ctd regions, and which, due to their structural plasticity, were not crystallized in human sars cov n protein. sars-cov- has a disordered segment from - residues while human sars has predicted to have an unstructured segment from - residues. overall, all three n proteins are found to be highly disordered. the n protein from human sars cov has one phosphorylation site (residue s ) and several regions with compositional biases, such as ser-rich (residues - ), poly-leu, poly-gln, and ploy-lys (residues - , - , and - ), all predicted to be disordered. similarly, in n protein from bat cov, s is phosphorylated, and this protein has ser-rich, poly-leu, and ploy-lys regions (residues - , - , and - , respectively), all of which are disordered. it has been reported to interact using the central disordered region with m protein, hnrnp a , and self n-n interaction [ ] [ ] [ ] . the middle flexible region is also responsible for its rna-binding activity [ ] . deletion of - residues, - residues, - residues of n abolishes its multimerization, rnabinding capacity, and hnrnp a interactions respectively. supplementary table and , and table shows that n protein is heavily decorated with numerous morfs, suggesting that this protein is a promiscuous binder. long disorder-based protein bonding regions at nand c-terminus of n protein of all three viruses were observed by all four predictors (morfchibi_web, anchor, morfpred, and disopred ). indeed, this is the single protein where we found many morfs as compared with the other structural, non-structural and accessory proteins of covs. the morfs present in these regions may mediate the abovementioned interactions of n proteins. figure a represents another important disorderrelated functional feature of the n protein. in fact, the ctd homodimer shown there is characterized by highly intertwined morphology, which is typically a result of bindinginduced folding [ ] [ ] [ ] , indicating that a very significant part of ctd gains structure during dimerization. we identified numerous rna binding residues in all three viruses using pprint server. this finding supports the function of n protein as it interacts with genomic rna for a ribonucleoprotein core formation which is crucial step for rna encapsidation during viral particle assembly. in addition, drnapred and disordpbind predicts multiple dna binding residues for n protein in sars-cov- , human sars, and bat cov. the long flexible (idprs) regions at n and c-terminus of sars-cov- have long protein-binding as well as nucleotide-binding regions that may have important role in its interaction with viral rna. these flexible regions can be targeted to inhibit interaction of n protein with viral genomic rna. literature suggests that some viral proteins are translated from the genes interspersed in between the genes of structural proteins. these proteins are known as accessory proteins, and many of them are proposed to be involved in viral pathogenesis [ ] . proteins orf a and orf b. orf a is a multifunctional protein with the molecular weight of ~ kda that has been found to localize in different organelles inside the host cells. also referred to as u , x , and orf , the gene for this protein is present between the s and e genes of the sars-cov genome [ ] [ ] [ ] . the homo-tetrameric complex of orf a has been demonstrated to form a potassium-ion channel on the host cell plasma membrane [ ] . it performs a major function during virion assembly by co-localizing with e, m, and s viral proteins [ , ] . orf b protein can be found in the cytoplasm, nucleolus, and outer membrane of mitochondria of the host cells [ , ] . in huh cells, its over-expression has been linked with the activation of ap- via erk and jnk pathways [ ] . transfection of orf b-egfp leads to cell growth arrest at the g /g phase of vero, , and cos- cells [ ] . orf a induces apoptosis via caspase / directed mitochondrial-mediated pathways, while orf b is reported to affect only the caspase -related pathways [ , ] . on performing msa, results of which are shown in figure d , we found that orf a protein from sars-cov- is slightly evolutionary closer to the orf a of bat cov ( . %) than to the orf a of human sars cov ( . %). graphs in figures a, b , and c depict the propensity for disorder in orf a proteins of novel sars-cov- , human sars cov, and bat cov (sars-like), respectively. mean ppids in these orf a proteins are . % (sars-cov- ), . % (human sars), and . % (bat cov (sars-like)). orf a of sars cov- shows protein-binding regions at its n-terminus (by morfchibi_web (residues - ), morfpred (residues - ), and disopred (residues [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) and at c-terminus (by morfchibi_web (residues - ) and morfpred (residues - )) ( table ) . similarly, orf a of human sars and bat cov also shows morfs at n-and c-terminus with the help of morfchibi_web and morfpred (supplementary tables and ). these protein-binding regions in orf a may have role in its co-localization with e, m, and s viral proteins. apart from morfs, it also displays several nucleotide-binding residues in all three viruses (see supplementary tables , , and ). in fact, this represents maximum number of rna and dna binding residues as compared with all other accessory proteins. these results indicate that the idps/idprs of this protein could be utilized in molecular recognition (protein-protein, protein-rna, and protein-dna interaction). according to the intrinsic disorder predisposition analysis of orf b proteins, their mean ppid values in sars-cov- , human sars cov, and bat cov are %, . %, and . % respectively, as represented in figures a, b , and c. msa results ( figure d ) demonstrate that orf b of sars-cov- is not closer to orf b protein of human sars and orf b protein of bat-cov, having a sequence similarity of only . % and . %, respectively. as we can see in table , there is not a single morf found in orf b of sars-cov- . however, for human sars we identified three morfs (residues - , - , and - ) and for bat cov one morf at n-terminus (residues - ) by morfchibi_web server. protein orf . orf is a short coronavirus protein with just residues. also known as p , this membrane-associated protein serves as an interferon (ifn) antagonist [ ] . it downregulates the ifn pathway by blocking a nuclear import protein, karyopherin α . using its c-terminal residues, orf disrupts karyopherin import complex in the cytosol and, therefore, hampers the movement of transcription factors like stat into the nucleus [ , ] . it contains a ysel motif near its c-terminal region, which functions in protein internalization from the plasma membrane into the endosomal vesicles [ ] . another study has also demonstrated the presence of orf in endosomal/lysosomal compartments [ , ] . msa results demonstrate that (figure d) , sars-cov- orf is closer to orf protein of human sars cov, having a sequence similarity of . % than to the orf of bat cov (sars-like) ( . %). novel sars-cov- orf is predicted to be the second most disordered structural protein, with ppid of . %, and with especially disordered cterminal region. our analysis of the intrinsic disorder predisposition using six predictors revealed the mean ppid in orf proteins of sars-cov- , human sars, and bat cov to be . %, . %, and . %, respectively ( table ) . graphs in figures a, b and c illustrate that orf proteins from all three studied coronaviruses are expected to be moderately disordered proteins with the high disorder content in their c-terminal regions. these disordered regions are important for the biological activities of orf . as aforementioned, this hydrophilic region contains lysosomal targeting motif (ysel) and diacidic motif (ddee) responsible for binding and recognition during translocation [ ] . however, the n-terminal region does not contain a noticeable disorder. the - residues of the n-terminal region of human sars cov orf was shown to be α-helical and embedded in the membrane, although orf is not a transmembrane protein [ ] . a long morf region ((residues - in sars-cov- ), (residues - in human sars), and (residues - in bat cov)) is also present at cterminus of orf proteins which are tabulated in table , and supplementary tables and . no predictor other than morfchibi_web has located morfs in this protein. supplementary table , , and shows nucleotide-binding residues in orf of all three viruses. it represents very few rna binding residues by pprint and few dna binding residues by drnapred. orf a and orf b proteins. alternatively called u , orf a is a type i transmembrane protein [ , ] . it has been proven to localize in er, golgi, and peri-nuclear space. the presence of a krkte motif near the c-terminal region is needed for importing this protein from the er to the golgi apparatus [ , ] . orf a contributes to viral pathogenesis by activating the release of pro-inflammatory cytokines and chemokines, such as il- and rantes [ , ] . in another study, overexpression of bcl-xl in t cells blocked the orf a mediated apoptosis [ ] . on the other hand, orf b is an integral membrane protein that has been shown to localize in the golgi complex [ , ] . the same reports also confirm the role of orf b as an accessory as well as a structural protein in sars-cov virion [ , ] . figure d represents the . Å x-ray crystal structure of the - fragment of the orf a from human sars cov (pdb id: xak) and demonstrates the compact seven-stranded topology of this protein, which is similar to that of the ig-superfamily members [ ] . importantly, in this crystal structure, residues - constituted the region with missing electron density, indicating high structural flexibility of this segment. in line with this hypothesis, the nmr solution structure of the - fragment of the orf a from human sars cov (pdb id: yo ) showed that residues - are highly disordered [ ] . at the domain level, the structure of the orf a protein includes a signal peptide, a luminal domain, a transmembrane domain, and a short cytoplasmic tail at the c-terminus [ , ] . we found that -residue-long orf a protein of sars-cov- shares . % and . % sequence identity with orf a proteins of bat cov and human sars cov, respectively ( figure e) . on the other hand, the orf b of sars-cov- is found to be closer to orf b of human sars than to orf b of bat cov, showing sequence identities of . % and . %, respectively (see figure d ). as can be observed from table , our disorder predisposition analyses resulted in the overall ppid for orf a proteins of . % for sars-cov- , . % for bat cov and . % for human sars cov. mean ppids estimated for orf b proteins are . % for sars-cov- , . % for bat cov and . % human sars cov. figures a, b , and c represent the residues predisposed for disorder in orf a proteins of sars-cov- , human sars cov, and bat cov, respectively. table shows that orf a protein is expected to have several morfs indicating the potential involvement of this protein in disorder-dependent proteinprotein interactions. at the n-terminus, we observed one morf region (residues - ) with the help of disopred in all three viruses. in addition to protein binding regions, orf a also contains several rna and dna binding residues. analysis also represents, orf b proteins from all three viruses have low disorder content, likewise, they are not predicted to contain any morf by any of the predictors used in this study ( table , supplementary table and ). although the orf b does not contain protein-binding regions, it was found to contain nucleotide (rns and dna) binding regions in the protein figures a, b , and c depict the residues predisposed for disorder in orf b proteins of sars-cov- , human sars cov, and bat cov, respectively. according to our analysis, both proteins in all three studied coronaviruses have a mostly ordered structure. proteins orf a and orf b. in animals and isolates from early human infections, the orf gene codes for a single orf protein. however, in late infections, more specifically, at middle and late stages, a nucleotide deletion in the orf gene led to the formation of two distinct proteins, orf a and orf b containing and residues respectively [ , ] . both proteins have conformations different from that of the longer orf protein. it has been reported that overexpression of orf b resulted in the downregulation of e protein while the proteins orf a and orf /orf ab have no effect on the expression of protein e. also, orf /orf ab was found to interact very strongly with proteins s, orf a, and orf a. orf a interacts with s and e proteins, whereas orf b protein interacts with e, m, orf a and orf a proteins [ ] . the disorder-based protein binding regions of this protein identified in this study may have important role in interaction with other proteins. orf protein found in early sars-cov- isolates having residues and according to our analysis, it shares a . % sequence identity with orf protein of bat cov ( figure c) . furthermore, figures a and b show that there is no intrinsic disorder in both orf proteins from sars-cov- and bat cov. therefore, these two proteins predicted to be completely structured having a mean ppid of . %. in orf a and orf b proteins of the human sars, the predicted disorder is estimated to be . % and . %, respectively (table ) . graphs in figures a and b illustrate the presence of some disorder near the n-and c-terminals of orf a and orf b proteins. table shows the identified morf regions in orf of sars-cov- . it shows three morf regions (residues - , - , and - ) by morfchibi_web and one morf region (residues - ) by disopred . in human sars, the n-terminus of both orf a (residues - ) and orf b (residues - ) was found to be morf by morfchibi_web server (supplementary table ) . further, four proteinbinding regions (residues - , - , - , and - ) were identified by morfchibi_web server in bat cov (supplementary table ). apart from protein-binding, orf of sars-cov- , orf a and orf b of human sars, and orf of bat cov also comprise several nucleotide-binding residues (see supplementary table , , and ). this protein is expressed from an alternative orf within the n gene through a leaky ribosome binding process [ ] . inside the host cells, orf b enters the nucleus, which is a cell cycle-independent process and represents a passive entry. this protein was shown to interact with a nuclear export protein receptor exportin (crm ), using which it translocate out of the nucleus [ ] . our morfs analysis shows the presence of disorderbased protein binding regions in orf b protein which may have role in its interaction with crm and further translocation outside the nucleus. a . Å resolution crystal structure of orf b protein from human sars cov (pdb id: cme) shows the presence of a dimeric tent-like -structure along with the central hydrophobic amino acids ( figure d) . the published structure has the highly polarized distribution of charges, with positively charged residues on the one side of the tent and negatively charged on the other [ ] . based on the sequence availability of accession id nc_ . , the translated protein sequence of orf b is not reported for the sars-cov- as of yet. however, based on the report by wu and colleagues [ ] , the sequences of the sars-cov- are already annotated. therefore, we took the corresponding amino acid sequences from that study and conducted the intrinsic disorder analysis. according to the msa, results shown in figure e , orf b protein from sars-cov- shares . % identity with human sars and . % identity with bat cov. our idp analysis ( table ) shows that orf b from human sars is a moderately unstructured protein with a mean ppid estimated to . %. as depicted in figure a , b, and c, disorder mainly lies near the n-terminal end - residues and - residues near the central region with a well-ordered inner core of human sars orf b protein. the x-ray crystal structure of orf b has a missing electron density of the first residues and - residues near the central region. this indicates that the corresponding regions are disordered, which are difficult to crystallize due to their highly dynamic structural organization. sars-cov- orf b protein with a mean ppid of . % has an n-terminal ( - residues) predicted disordered segment. orf b of bat cov is shown to have an intrinsic disorder content of . %, comparatively lower than that of the human sars orf b protein. morfs lies in the n-terminal region of orf b proteins ( table , supplementary table and ). in the absence of other viral proteins, its first residues have been demonstrated to induce membranous structures similar to dmvs [ ] . the available crystal structure also has the missing electron density in the n-terminal region suggests that these flexible amino acids are likely to interact with host lipids. the first - residues of sars-cov are identified as disorder-based protein binding region that may have role in its interaction with host lipids and formation of dmvs. supplementary tables , , and represents nucleotide-binding residues for orf b of sars-cov- , human sars, and bat cov. the newly emerged sars-cov- has an orf protein of amino acids. orf of sars-cov- has a % sequence similarity with orf of bat cov strain bat-sl-covzc [ ] . however, we did not conduct the disorder analysis for orf from the bat-sl-covzc strain, since all our studies reported here are related to a different strain of bat cov (reviewed strain hku - ). therefore, we report here only the results of disorder analysis for the orf protein from sars-cov- , according to which this protein has a mean ppid of . % (see also figure for disorder profile of orf ). this protein contains a morf from - residues at its n-terminus as predicted by morfchibi_web. further, we found its tendency to nucleotides and found the presence of few rna binding sites, however, it does not contain dna binding residues. protein orf . this is a -amino-acid-long uncharacterized protein of unknown function, which is present in human sars and bat cov. in sars-cov- , orf is a -amino-acidlong protein. according to the msa, orf of sars-cov- has . % identity with human-sars and . % identity with bat cov as represented in figure d . we have performed the intrinsic disorder analysis to see the peculiarities of the distribution of disorder predisposition in this protein. figures a, b, and c show the resulting disorder profiles of orf of sars-cov- , human sars cov, and bat cov. although these proteins have calculated mean ppid values of . %, . %, and . % respectively, figure shows that they have flexible n-and c-terminal regions. this protein can use intrinsic disorder or structural flexibility for protein-protein interactions since it possesses morfs. it mainly contains morfs at n-and c-terminal regions as tabulated in (table , supplementary table and ). it was also found to contain several rna and dna binding residues (supplementary table , , and ) . these results indicating its vital role in protein function related to molecular recognition such as protein-protein, protein-rna, and protein-dna interaction. in coronaviruses, due to ribosomal leakage during translation, two-third of the rna genome is processed into two polyproteins: (i) replicase polyprotein a and (ii) replicase polyprotein ab. both contain non-structural proteins (nsp - ) in addition to different proteins required for viral replication and pathogenesis. replicase polyprotein a contains an additional nsp protein of amino acids, the function of which is not investigated yet. the longer replicase polyprotein ab of amino acids accommodates five other non-structural proteins (nsp - ) [ ] . these proteins assist in er membrane-induced vesicle formation, which acts as sites for replication and transcription. in addition to this, non-structural proteins work as proteases, helicases, and mrna capping and methylation enzymes, crucial for virus survival and replication inside host cells [ , ] . global analysis of intrinsic disorder in the replicase polyprotein ab table represents the ppid mean scores of non-structural proteins (nsps) derived from the replicase polyprotein ab in sars-cov- , human sars cov, and bat cov. these values were obtained by combining the results from six disorder predictors (see supplementary table s -s ) . figures a, b , and c represent the d-disorder plots of the nsps coded by orf ab in sars-cov- , human sars cov, and bat cov, respectively. based on the mean ppid scores in table , figures a, b, c , and taking into ppid based classification [ ] , we conclude that none of the nsps in sars-cov- , human sars cov, and bat cov are highly disordered. the highest disorder was observed for nsp proteins in all three coronaviruses. both nsp and nsp are moderately disordered proteins ( % ≤ ppid ≤ %). we also observed that nsp , nsp , nsp , nsp , nsp , nsp , nsp , nsp , and nsp have less than % disordered residues and hence, belong to the category of mostly ordered proteins. other non-structural proteins, namely, nsp , nsp , nsp , and nsp have negligible levels of disorder (ppid < %), which tells us that these are highly structured proteins. the ch-cdf analysis of the nsps from sars-cov- , human sars and bat cov have been represented in figures d, e , and f respectively. it was observed that all the nsps of the three coronaviruses are located within the quadrant q of the ch-cdf phase space, indicating that all the nsps are predicted to be mostly ordered. replicase polyprotein ab. the longer replicase polyprotein ab is a , amino acid-long polypeptide, which contains non-structural proteins listed in table . nsp , nsp , and nsp are cleaved using a viral papain-like proteinase (nsp /pl-pro), while the rest of nsps are cleaved by another viral c-like proteinase, nsp / cl-pro. we mapped the cleavage sites of the replicase ab polyprotein from human sars cov to the disorder profile of this polyprotein. figure represents the results of this analysis by showing zoomed-in regions surrounding all the cleavage sites with few residues spanning at both terminals. interestingly, we observed that all the cleavage sites are largely disordered, suggesting that intrinsic disorder may have a crucial role in the maturation of individual non-structural proteins. as the nsps of human sars cov are evolutionary close to the nsps of sars-cov- , we hypothesize that the cleavage sites in the sars-cov- replicase ab polyprotein are also intrinsically disordered or flexible. to shed more light on other implications of idprs, the structural and functional properties of nsps and their predicted idprs are thoroughly described below. this protein acts as a host translation inhibitor as it binds to the s subunit of the ribosome and blocks the translation of cap-dependent mrnas as well as mrnas that uses the internal ribosome entry site (ires) [ ] . figure d shows the nmr solution structure (pdb id: gdt) of human sars nsp protein ( - residues), whereas residues - were not included in this structural analysis [ ] . sars-cov- nsp shares . % and . % sequence identity with nsp s of human sars cov and bat cov, respectively. its n-terminal region is found to be more conserved than the rest of the protein sequence ( figure e ). mean ppids of nsp s from sars-cov- , human sars cov, and bat cov are . %, . %, and . %, respectively. figure a , b, and c represent the graphs of predicted per-residue intrinsic disorder propensity of these nsp s. according to the analysis, the following regions are predicted to be disordered: sars-cov- (residues - and - ), human sars cov (residues - and - ), and bat cov (residues - and - ). nmr solution structure of nsp from human sars revealed the presence of two unstructured segments near the n-terminal ( - residues) and c-terminal ( - residues) regions [ ] . the disordered region residues) at c-terminus is important for nsp expression [ ] . based on sequence homology with human sars cov nsp , the predicted disordered c-terminal region of sars-cov- nsp may play a critical role in its expression. alanine mutants at k and h in the cterminal region of nsp protein is reported to abolish its binding with the s subunit of the host ribosome [ ] . in conjunction with this data, several morfs are present in the unstructured segments of nsp proteins. these regions are tabulated in table , and supplementary tables and . this protein functions by disrupting the host survival pathway via interaction with the host proteins prohibitin- and prohibitin- [ ] . reverse genetic deletion in the coding sequence of nsp of the sars virus attenuated little viral growth and replication and allowed the recovery of mutant virulent viruses. this indicates the dispensable nature of the nsp protein for sars viruses [ ] . the sequence identity of the nsp protein from sars-cov- with nsp s of human sars cov and bat cov amounts to . % and . %, respectively (supplementary figure s a) . we have estimated the mean ppids of nsp s of sars-cov- , human sars cov, and bat cov to be . %, . %, and . % respectively (see table ). the per-residues predisposition for the intrinsic disorder of nsp s from sars-cov- , human sars cov, and bat cov are depicted in figures a, b , and c. according to this analysis, the following regions in nsp proteins are predicted to be disordered, residues - (sars-cov- ), residues - (human sars), and residues - (bat cov). as listed in table , and supplementary tables and , human sars cov does not contain morf while sars-cov- and bat cov have an n-terminally located morf region predicted by morfchibi_web. nsp is an almost , -residue-long viral papain-like protease (plp) that affects the phosphorylation and activation of irf and therefore antagonizes the ifn pathway [ ] . it was also demonstrated that nsp works by stabilizing nf-inhibitor further blocking the nf-pathway [ ] . figure d represents the . Å resolution x-ray crystal structure of the catalytic core of nsp protein from human sars cov (pdb id: fe ), which was obtained by andrew and colleagues [ ] . this structure consists of the residues - of nsp . the structure revealed folds similar to a deubiquitinating enzyme in-vitro deubiquitinating activity of which was found to be efficiently high [ ] . nsp protein of sars-cov- contains several substituted residues throughout the protein. it is equally close with both nsp proteins of human sars and bat cov sharing respective . % and . % identity (supplementary figure s b) . according to our results, the mean ppids of nsp proteins of sars-cov- , human sars, and bat cov are . %, . %, and . % respectively ( table ) . graphs in figures a, b , and c portray the tendency of nsp proteins of sars-cov- , human sars, and bat cov for the intrinsic disorder. nsp proteins of all three studied sars viruses were found to be highly structured and characterized by rather similar disorder profiles. this is further supported by figure e , where pondr ® vsl -generated disorder profiles of these three proteins are overlapped to show almost complete coincidence of their major disorder-related features. according to the mean disorder analysis (see figures a, b, and c) , nsp proteins are predicted to have the following idprs, sars-cov- ( - , - , - ), human sars ( - , - , - ) and bat cov ( - , - , - ) . the first residues in nsp represent a ubiquitin-like globular fold while - residues form the flexible acidic domain rich in glutamic acid. it is thought to bind and ubiquitinate viral e protein using the n-terminal acidic domain [ , ] . this unstructured segment has many morfs predicted by anchor and morfpred servers which may facilitate the protein-protein interaction ( table ) . interestingly, nsp of all three viruses was found with highest number of rnabinding residues (supplementary tables , , and ) . nsp has been reported to induce the formation of the double-membrane vesicles (dmvs) with the co-expression of full-length nsp and nsp proteins for optimal replication inside host cells [ ] [ ] [ ] . it localizes itself in ermembrane, when expressed alone but is demonstrated to be present in replication units in infected cells. it was observed that nsp protein contains a tetraspanning transmembrane region having its n-and c-terminals in the cytosol [ ] . no crystal or nmr solution structure is reported for this protein as of yet. nsp protein of sars-cov- has multiple substitutions near the n-terminal region and has a quite conserved c-terminus (supplementary figure s c) . it is found to be closer to nsp of bat cov ( . % identity) than to human sars nsp ( %). mean ppids of nsp s from sars-cov- , human sars, and bat cov are estimated to be . %, . %, and . % respectively. the low level of intrinsic disorder is further illustrated by figures a, b , and c. with ppids around zero, nsp were classified as highly structured proteins, which, however, contain some flexible regions. likewise, table shows the presence of only nand c-terminal morfs which possibly assist in cleavage of nsp protein from long polyproteins a and ab. also referred to as cl-pro, nsp works as a protease that cleaves the replicase polyproteins ( a and ab) at major sites [ , ] . x-ray crystal structure with . Å resolution (pdb id: c o) obtained for human sars cov nsp is shown in figure d . here, cl-protease is bound to a phenyl-beta-alanyl (s, r)-n-declin type inhibitor. another crystal structure resolved to . Å revealed a chymotrypsin-like fold and a conserved substrate-binding site connected to a novel α-helical fold [ ] . recently, the x-ray crystal structure (resolution . Å) was solved for the sars-cov- nsp in complex with an inhibitor n (pdb id: lu ) ( figure e ). nsp protein is found to be highly conserved in all three studied cov viruses. sars-cov- nsp shares a . % sequence identity with human sars nsp and . % with nsp of bat cov (supplementary figure s d) . therefore, it not surprising that our analysis demonstrated the identical mean ppid values of . % for nsp s from sars-cov- , human sars, and bat cov ( table ) . the predicted per-residue intrinsic disorder propensity of sars-cov- , human sars, and bat cov nsp s are presented in figures a, b , and c, respectively. as the graphs depict, nsp s have several flexible regions and n-terminally idpr of six residues. due to the low flexibility of this protein, a single morf predicted by morfchibi_web is present in the n-terminal region (residues - ) in nsp s of all three viruses (table , supplementary tables and ) . further, the identified nucleotide-binding residues in nsp of all three viruses are tabulated in supplementary tables , , and . non-structural protein (nsp ). nsp protein is involved in blocking er-induced autophagosome/autolysosome vesicle formation that functions in restricting viral production inside host cells. it induces autophagy by activating the omegasome pathway, which is normally utilized by cells in response to starvation. sars nsp leads to the generation of small autophagosome vesicles thereby limiting their expansion [ ] . nsp of sars-cov- is equally close to nsp s from both human sars and bat cov, having a sequence identity of . % ( figure d ). according to our analysis, mean ppids for nsp s are calculated to be . %, . %, and . % for sars-cov- , human sars cov, and bat cov, respectively. figures a, b, and c show the corresponding graphs of intrinsic disorder tendency of nsp s from sars-cov- , human sars cov, and bat cov and demonstrate that these proteins are highly ordered and show low flexibility. as it is a membrane protein, nsp proteins are predicted to have only a single morf near the nterminal region (residues - in sars-cov- , residues - in human sars, and residues - in bat cov) by the disopred server (table , supplementary tables and ) . the role of these protein-binding regions for the induction of autophagy is need to be elucidated. nsp and ) . the ~ kda nsp helps in primaseindependent de novo initiation of viral rna replication by forming a hexadecameric ring-like structure with nsp protein [ , ] . both non-structural proteins and contribute molecules to the ring-structured multimeric viral rna polymerase. site-directed mutagenesis in nsp revealed a d/exd/e motif essential for the in vitro catalysis [ ] . figure d depicts the . Å resolution electron microscopy-based structure (pdb id: nur) of the rdrp-nsp -nsp complex bound to the nsp . the structure identified conserved neutral nsp and nsp binding sites overlapping with finger and thumb domains on nsp of the virus [ ] . we found that nsp of sars-cov- share % sequence identity with nsp of bat cov and . % with nsp from human sars (figure e) , while sars-cov- nsp is closer to nsp of human sars ( . %) than to nsp of bat cov ( . %) ( figure d ). due to the high levels of sequence identity, mean ppids of all nsp s were found to be identical and equal to . %. both sars-cov- and human sars nsp proteins were calculated to have a mean ppid of . % and, for nsp of bat cov mean disorder is predicted to be . %. figures a, b , and c display the intrinsic disorder profiles for nsp s, whereas figures a, b , and c represent the predicted intrinsic disorder propensity of nsp s. as our analysis suggests, nsp s might have a well-predicted structure, while nsp s are moderately disordered. nsp s are predicted to have a long idpr (residues - ) in both sars-cov- and human sars, and a bit shorter idpr in bat cov (residues . furthermore, sars-cov nsp using its n-terminus residues (v , c , v , and v ) forms a hydrophobic core with nsp residues (m , m , l , m , and l ). additionally, h-bonding takes place between nsp q and nsp t residues [ ] . these amino acids are the part of morfs predicted in nsp and nsp proteins. the results are tabulated in both supplementary tables , , and ). nsp protein is a single-stranded rna-binding protein [ ] . it might protect rna from nucleases by binding and stabilizing viral nucleic acids during replication or transcription [ ] . our results on nucleotide-binding tendency of nsp shows the presence of several rna binding and few dna binding residues in nsp of sars-cov- , human sars, and bat cov (supplementary tables , , and ) . presumed to evolve from a protease, nsp forms a dimer using its gxxxg motif [ , ] . figure d shows a . Å crystal structure of the homodimer of human sars nsp (pdb id: qz ) that identified a unique and previously unreported for other proteins, oligosaccharide/oligonucleotide fold-like fold [ ] . here, each monomer contains a coneshaped β-barrel and a c-terminal α-helix arranged into a compact domain [ ] . nsp of sars-cov- is equally similar to nsp s from both human sars and bat cov, having a percentage identity of . %. the difference in three amino acids at , and positions accounts for these similarity scores ( figure e ). as calculated, the mean ppids of nsp s of sars-cov- , human sars cov, and bat cov are . %, . %, and . % respectively. figures a, b , and c depict the predicted intrinsic disorder propensity in the nsp protein from sars-cov- , human sars, and bat cov. according to our analysis, all three nsp s are rather structured but contain flexible regions. nsp contains conserved residues (r , k , y , r , r , f , k , y , f , k , r , and r ) of positively charged side chains suitable for binding with the negatively charged phosphate backbone of rna and aromatic side-chain amino acids providing stacking interactions [ ] . these residues are a part of multiple disorder-based binding sites predicted by morfchibi_web server ( table , supplementary table and ) . nsp performs several functions for sars-cov. it forms a complex with nsp for dsrna hydrolysis in ′ to ′ direction and activates its exonuclease activity [ ] . it also stimulates the methyltransferase (mtase) activity of nsp required during rna-cap formation after replication [ ] . figure d represents the x-ray crystal structure of the nsp /nsp complex (pdb id: c t) [ ] . in agreement with the results of previous biochemical experimental studies, the structure identified important interactions with the exon (exonuclease domain) of nsp without affecting its n -mtase activity [ , ] . sars-cov- nsp protein is quite conserved having a . % sequence identity with nsp of human sars and . % with nsp of bat cov (figure e) . mean ppids of all three studied nsp proteins are found to be . %. figures a, b , and c represent disorder profiles of nsp s and signify the lack of long idprs but presence flexible regions in these proteins. furthermore, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] was predicted by morfpred server. interestingly, the sars-cov nsp residues f , f , and v form van der waals interactions with many of the nsp amino acids [ ] and one residue (f ) is located in morf region which we have identified. furthermore, many nucleotide-binding residues which are found in all three viruses (supplementary table , , and ) and above-mentioned residues are not found to interact with dna/rna. in coronaviruses, nsp is an rna-dependent rna polymerase (rdrp). it carries out both primer-independent and primer-dependent synthesis of viral rna with mn + as its metallic co-factor and viral nsp and as protein co-factors [ ] . as aforementioned, a . Å resolution structure of human sars nsp in association with nsp and nsp proteins (pdb id: nur) has been reported using electron microscopy ( figure d ). nsp has a polymerase domain similar to "right hand", finger domain ( - , - residues), palm domain ( - , - residues) and a thumb domain ( - ) [ ] . sars-cov- nsp protein has a highly conserved c-terminal region (supplementary figure s e ). it is found to share a . % sequence identity with human sars nsp and . % with bat cov nsp . mean ppid values for all three nsp s are estimated to be . % (table ) . figures a, b , and c show that although these proteins are mostly ordered, they have multiple flexible regions. as rdrp protein is observed to be mostly structured, significant morfs in disordered regions are not found ( table , supplementary table and ). nsp functions as a viral helicase and unwinds dsdna/dsrna in ' to ' direction [ ] . recombinant viral helicase expressed in e.coli rosetta strain was reported to unwind ~ bp per second [ ] . figure d represents a . Å x-ray crystal structure of human sars nsp (pdb id: jyt) [ ] . this helicase contains a - loop on a domain, which is primarily responsible for its unwinding activity. furthermore, the study revealed an important interaction of nsp with nsp that further enhances its helicase activity [ ] . the -amino-acid-long nsp of sars-cov- is almost completely conserved, as it shares . % with nsp of humans sars and . % with nsp of bat cov (supplementary figure s f) . in accordance with our results, the mean ppids of all three nsp proteins are estimated to be . %. figures a, b , and c show that nsp s contain multiple flexible regions but do not possess significant disorder. as expected, being a low disorder protein nsp does not contain any morf region and not a single bindingregion is located by any server used in all three viruses ( table , supplementary table and ). it has many nucleotide-binding residues (rna and dna) which are tabulated in supplementary tables , , and . nsp is a multifunctional viral protein that acts as an exoribonuclease (exon) and methyltransferase (n -mtase) in sars coronaviruses. it's ' to ' exonuclease activity lies in the conserved dedd residues related to exonuclease superfamily [ ] . its guanine-n methyltransferase activity depends upon the s-adenosyl-lmethionine (adomet) as a cofactor [ ] . as aforementioned, nsp requires nsp for activating its exon and n -mtase activity inside the host cells. figure d depicts the . Å crystal structure of human sars nsp /nsp complex (pdb id: c t), where amino acids - form the exon domain and - residues form the n -mtase domain of nsp . a loop (residues - ) is essential for its n -mtase activity [ ] . figure s g) . mean ppid values for nsp s from sars-cov- and human sars is calculated to be . %, while the nsp from bat cov has a mean ppid . %. predicted per-residue intrinsic disorder propensity of nsp s from sars-cov- , human sars, and bat cov is represented in figures a, b , and c, respectively. as can be observed from these plots and corresponding ppid values, all nsp s are found to be highly structured. likewise, table shows nsp contains two protein binding regions (residues - and - ) predicted by the morfpred server in all three viruses. as shown in supplementary tables , , and , the orf represents multiple nucleotide-binding residues. nsp is a uridylate-specific rna endonuclease (nendou) that creates a ′- ′ cyclic phosphates after cleavage. its endonuclease activity depends upon mn + ions as co-factors. conserved in nidovirus, it acts as an important genetic marker due to its absence in other rna viruses [ ] . figure d represents a . Å crystal structure of uridylate-specific nsp (pdb id: h ) that was deduced by bruno and colleagues using x-ray diffraction [ ] . the monomeric nsp has three domains: nterminal domain ( - residues) formed by a three anti-parallel -strands and two α-helices packed together; a middle domain residues) that contains an α-helix connected via a -amino-acid-long coil to an ordered region containing two α-helices and five -strands; and a c-terminal domain ( - residues) consisting of two anti-parallel three -strand sheets on each side of a central α-helical core [ ] . the nsp is found to be quite conserved across human sars and bat covs. sars-cov- nsp shares an . % sequence identity with nsp of human sars and . % with nsp of bat cov (supplementary figure s h) . calculated mean ppids of nsp s from sars-cov- , human sars, and bat cov are . %, . %, and . %, respectively. similar to many other non-structural proteins of coronaviruses, nsp s from sars-cov- , human sars, and bat cov are predicted to possess multiple flexible regions but contain virtually no idprs (see figures a, b, and c) . similarly, no significant disorderbinding regions are predicted in nsp proteins ( table ) . sars-cov- contain one morf (residues - ) predicted by morfpred server. human sars do not have a single morf while bat cov possesses two very short binding regions (supplementary table and ). supplementary table , , and depicts the presence of many rna binding residues and few dna binding residues in nsp of all three viruses. nsp protein is another mtase domain-containing protein. as methylation of coronavirus mrnas occurs in steps, three proteins nsp , nsp , and nsp acts one after another. the first event requires the initiation trigger from nsp protein, after which nsp methylates capped mrnas forming cap- ( me) gpppa-rnas. nsp protein, along with its co-activator protein nsp , acts on cap- ( me) gpppa-rnas to give rise to final cap- ( me)gpppa( 'ome)-rnas [ , ] . a Å x-ray crystal structure of the human sars nsp -nsp complex is depicted in figure d (pdb id: r ) [ ] . the structure consists of a characteristic fold present in class i mtase family comprising of α-helices and loops surrounding a seven-stranded β-sheet [ ] . nsp protein of sars-cov- is found to be equally similar to nsp s from human sars and bat cov ( . %) (supplementary figure s i) . mean ppids for nsp s from sars-cov- , human sars, and bat cov are . %, . %, and . %, respectively. in line with these ppids values, figures a, b, and c show that nsp s are mostly ordered proteins containing several flexible regions. correspondingly, no significant morfs are present in this protein ( table , supplementary table and ). a single morf (residues [ ] [ ] [ ] [ ] [ ] [ ] were found with the help of morfpred in all three viruses. further, several rnabinding and few dna-binding residues are also identified (supplementary table , , and ). replicase polyprotein a. since replicase polyprotein a contains non-structural proteins - identical to those found in replicase polyprotein ab, we did not perform their disorder analysis separately. however, replicase polyprotein a has one additional non-structural protein designated as nsp . nsp is a small uncharacterized protein cleaved from the replicase polyprotein a. this small protein with unknown function requires experimental insights to further characterize this protein. the intrinsic disorder predicting software used in this study requires amino acid sequences, which are at least -residue long. therefore, because of their short sequences (just residues) nsp s from all three studied coronaviruses were not checked for the intrinsic disorder, disorder-based protein binding regions, and nucleotide-binding residues. based on the msa outputs, nsp from sars-cov- was found to have a sequence identity of . % with nsp s from human sars and bat cov (figure ). the emergence of new viruses and associated deaths around the globe represent one of the major concerns of modern times. despite its pandemic nature, there is very little information available in the public domain regarding the structures and functions of sars-cov- proteins. based on its similarity with human sars cov and bat cov, the published reports have suggested the functions of sars-cov- proteins. in this study, we utilized information available on sars-cov- genome and translated proteome from genbank, and carried out a comprehensive computational analysis of the prevalence of the intrinsic disorder in sars-cov- proteins. additionally, a comparison was also made with proteins from close relatives of sars-cov- from the same group of beta coronaviruses, human sars cov and bat cov. our analysis revealed that in these three covs, the n proteins are highly disordered, possessing the ppid values of more than %. these viruses also have several moderately disordered proteins, such as nsp , orf , and orf b. although other proteins have shown lower disorder content, almost all of them contain at least some idprs, and all cov proteins analysed in this study definitely have multiple flexible regions. importantly, our study provides novel information on presence of intrinsic disorder at the cleavage sites of the replicase ab polyprotein of covs. this observation confirms the crucial role of idprs in maturation of individual proteins. we also established that many of these proteins contain disorder-based binding motifs. since idps/idprs might undergo structural transition upon association with their physiological partners, our study generates important grounds for better understanding of the functionality of these proteins, their interactions with other viral proteins, as well as interaction with host proteins in different physiological conditions. this will also guide structural biologists to carry out a structure-based analysis of sars-cov- proteome to explore the path for the development of new drugs and vaccines. the periodical outbreaks of pathogens worldwide always remind the lack of suitable drugs or vaccines for proper cure or treatment. in , nearly deaths were reported due to the sars outbreak in more than countries. but this time, the outbreak of wuhan's novel coronavirus (sars-cov- ) has quickly surpassed this number, indicating more causalities soon. the lack of accurate information and ignorance of primary symptoms are major reasons, which cause many infection cases. although efficient transmission from human to human is confirmed, the actual reasons for fast sars-cov- spread are still unknown, but some assumptions were made by researchers and chinese authorities. the fast spread of sars-cov- , covid- pandemic, and associated introduction of quarantine also have made major impacts on economy and education worldwide due to several restrictions, such as limited transportation, restrained or frozen traveling, halted attendance of mass events, the introduction of distant teaching and learning, etc. due to advancements in sequencing techniques, the full genome sequence of sars-cov- was made available in a few days of the first infection report from wuhan, china. however, massive subsequent research needs to be done to identify the actual cause of sars-cov- infectivity and to design suitable treatment in the coming future. certain possibilities can be explored with the available information. the mutational pressure study on this virus will be very interesting to see if this virus transforms from bat sars to human sars to sars-cov- . more in-depth experimental studies using molecular and cell biology techniques to establish structurefunction relationships are required for a better understanding of the functioning of sars-cov- proteins. additionally, based on the sequence homology and information on proteinprotein interactions, the associated viral and host proteins should be explored, for finding means suitable for limiting replication, maturation, and ultimately pathogenesis of this virus. although structural biology techniques (so-called rational drug design) can be used in drug development utilizing high throughput screening of compounds virtually or experimentally, the applicability of these techniques is limited by the presence of intrinsic disorder in target proteins. therefore, the thorough disorder analysis of three coronaviruses conducted in this study will help structural biologists to rationally design experiments keeping this information in mind. authors contribution: rg: conception and design, interpretation of data, writing, and review of the manuscript, and study supervision. vnu: conception and design, acquisition and interpretation of data, writing, and review of the manuscript. ms, tb, pk, brg, kg: acquisition and interpretation of data, writing of the manuscript. table . evaluation of intrinsic disorder in non-structural proteins of bat cov. table : predicted morf residues in human sars proteins. supplementary table : predicted morf residues in bat cov proteins. supplementary table : predicted nucleotide-binding residues in sars-cov- proteins. supplementary table : predicted nucleotide-binding residues in human sars proteins. supplementary table : predicted nucleotide-binding residues in bat cov proteins. supplementary figures s . multiple sequence alignment of structural proteins of all three studied coronaviruses are generated using clustal omega. the aligned images are created using esprit . . figure s a . msa of sars-cov- , human sars, and bat cov spike glycoproteins. figure s b . msa of sars-cov- , human sars, and bat cov nucleoproteins. supplementary figure s . multiple sequence alignment of non-structural proteins of all three studied coronaviruses are generated using clustal omega. the aligned images are created using esprit . . figure s a . msa of sars-cov- , human sars, and bat cov nsp proteins. figure s b . msa of sars-cov- , human sars, and bat cov nsp proteins. figure s c . msa of sars-cov- , human sars, and bat cov nsp proteins. figure s d . msa of sars-cov- , 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syndrome coronavirus nonstructural protein nsp /nsp exoribonuclease complex in vitro reconstitution of sars-coronavirus mrna cap methylation structural basis and functional analysis of the sars coronavirus nsp -nsp complex biochemical characterization of a recombinant sars coronavirus nsp rna-dependent rna polymerase capable of copying viral rna templates mechanism of nucleic acid unwinding by sars-cov helicase delicate structural coordination of the severe acute respiratory syndrome coronavirus nsp upon atp hydrolysis discovery of an rna virus '-> ' exoribonuclease that is critically involved in coronavirus rna synthesis major genetic marker of nidoviruses encodes a replicative endoribonuclease crystal structure and mechanistic determinants of sars coronavirus nonstructural protein define an endoribonuclease family coronavirus nonstructural protein is a cap- binding enzyme possessing (nucleoside- 'o)-methyltransferase activity biochemical and structural insights into the mechanisms of sars coronavirus rna ribose '-o-methylation by nsp /nsp protein complex key: cord- - rd o authors: wong, mun-teng; chen, steve s-l title: emerging roles of interferon-stimulated genes in the innate immune response to hepatitis c virus infection date: - - journal: cell mol immunol doi: . /cmi. . sha: doc_id: cord_uid: rd o infection with hepatitis c virus (hcv), a major viral cause of chronic liver disease, frequently progresses to steatosis and cirrhosis, which can lead to hepatocellular carcinoma. hcv infection strongly induces host responses, such as the activation of the unfolded protein response, autophagy and the innate immune response. upon hcv infection, the host induces the interferon (ifn)-mediated frontline defense to limit virus replication. conversely, hcv employs diverse strategies to escape host innate immune surveillance. type i ifn elicits its antiviral actions by inducing a wide array of ifn-stimulated genes (isgs). nevertheless, the mechanisms by which these isgs participate in ifn-mediated anti-hcv actions remain largely unknown. in this review, we first outline the signaling pathways known to be involved in the production of type i ifn and isgs and the tactics that hcv uses to subvert innate immunity. then, we summarize the effector mechanisms of scaffold isgs known to modulate ifn function in hcv replication. we also highlight the potential functions of emerging isgs, which were identified from genome-wide sirna screens, in hcv replication. finally, we discuss the functions of several cellular determinants critical for regulating host immunity in hcv replication. this review will provide a basis for understanding the complexity and functionality of the pleiotropic ifn system in hcv infection. elucidation of the specificity and the mode of action of these emerging isgs will also help to identify novel cellular targets against which effective hcv therapeutics can be developed. hepatitis c virus (hcv) infects more than million people worldwide and represents a heavy burden to global health, with the highest prevalence rates found in africa and the eastern mediterranean. acute hcv infection is asymptomatic, and in % of infected individuals, the virus persists and progresses to chronic liver diseases, including fibrosis, steatosis, cirrhosis and hepatocellular carcinoma. , furthermore, hcv is a major cause of type i mixed cryoglobulinemia, which occurs in % of patients. using the hcv genotype a isolate japanese fulminant hepatitis- genome-based cell culture-derived infectious hcv (hcvcc), zhong et al. demonstrated that hcv and cells coevolve in vitro during chronically persistent infection, which involves the selection of viral mutants with increased infectivity and cells with resistance to viral entry and/or rna replication. in this coevolution process, hcv exhibits multifaceted interactions with the host cells, and these cellular stress responses subsequently affect virus replication. for instance, infection with hcvcc or expression of the japanese fulminant hepatitis- genome has been shown to trigger cytopathic effects, endoplasmic reticulum (er) stress, the unfolded protein response (upr), autophagy and the innate immune response. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the competition between this virus and host cells, viral infection often triggers a first-line host defense through the production of type i interferon (ifn), which is a broadly acting antiviral cytokine, and inflammatory cytokines. these cytokines confer an antiviral state on the host cells, thereby interfering with viral replication. , with the ability to enhance the immune response for virus clearance or to inhibit viral replication, ifn-based therapies have been used to treat hcvinfected patients for over two decades. to guard against viral infection, the host cell has developed multiple restriction strategies to limit viral infection. the expression of many of these restriction factors is subject to transcriptional regulation by ifn. , upon infection by viruses such as hcv, viral rna is first sensed by cellular pattern recognition receptors (prrs), and the prr-mediated recruitment of adaptor proteins and the activation of downstream signaling lead to ifn production. , , after binding to its receptor (ifnar) complex present on the cell surface, ifn triggers the janus kinase (jak)/signal transducer and activator of transcription (stat) pathway to drive the synthesis of over ifn-stimulated genes (isgs), which block virus replication at different phases of the virus replication cycle. [ ] [ ] [ ] [ ] these isgs are usually not synthesized at the basal state, but are induced to express and mediate the antiviral effector functions of ifn upon viral infection or ifn treatment. in response, viruses have developed elaborate strategies to escape the ifn antiviral system by blocking the expression or antiviral functions of ifn. , , therefore, the host and hcv maintain a homeostatic state, allowing tightly restricted viral replication without killing the host. , [ ] [ ] [ ] recent studies on genome-wide sirna screens have also added new candidates to this growing list of anti-hcv isgs. these findings highlight the complexity and pleiotropic roles of ifn and its induced isgs in modulating innate immunity and virus replication. nevertheless, the complete spectrum of isgs and their functionality in suppressing hcv replication have yet to be defined. , in this review, we focus on the molecular aspects of the type i ifn system and its effector mechanisms in modulating hcv replication. first, we briefly discuss the signaling triggered by the retinoic acid-inducible gene -like receptor (rlr) and the toll-like receptor (tlr), which leads to type i ifn synthesis and ifn-mediated signaling pathway activation, resulting in the expression of a variety of effector isgs. we also summarize the strategies that hcv uses to escape ifn antiviral surveillance. additionally, we highlight what is currently known regarding the pivotal isgs in viral infections, with an emphasis on their anti-hcv activities, and the emerging isgs identified from recent genome-wide sirna screens in relation to anti-hcv activities. finally, we discuss the potential functions of several critical cellular factors, such as high-mobility group box (hmgb ) and immunity-related gtpase family m (irgm), and cellular pathways, such as upr and autophagy, during hcv infection. although these cellular determinants are not stimulated by ifn, these factors critically control the host immune response. therefore, these determinants may also play crucial roles in modifying hcv replication. this review provides a perspective for a better understanding of the anti-hcv mechanisms of ifn, isgs and several critical cellular determinants known to contribute to the regulation of innate immunity. the gathered information not only provides a clearer picture for the specificity, functionality and complexity of the ifn system and its effector mechanisms in the control of hcv infection, but also helps to identify novel cellular targets against which efficacious therapeutic strategies can be developed. clinically, the identification of new isgs will also help to optimize the current ifn-based therapy and to provide a basis for more accurate predictions of ifn treatment outcomes. hcv is an enveloped, positive-sense, single-stranded rna virus classified within the genus hepacivirus in the flaviviridae family. currently, hcv isolates are classified into seven major genotypes, i.e., genotypes through , and an array of subtypes. hcv genotypes differ by %- % in genome sequence, whereas subtypes within each genotype can differ by least %. genotype is the most prevalent ( %), followed by genotype ( %); genotypes , and (cumulatively approximately %); and genotype (less than %). different genotypes exhibit distinct geographic distributions. genotype predominates in america and europe, genotype in japan, genotype in asia, genotype in africa and middle east and genotype in southeast asia. hcv is transmitted via blood transfusion, intravenous drug abuse, unsafe therapeutic injection, liver transplantation and other risk factors. the combination of pegylated ifn-a and ribavirin is the standard therapy for hcv infection. however, this treatment is associated with side effects, and the efficacy of this regimen varies among genotypes, limiting the success rate of this treatment. compared with genotype , infection with genotypes a and b results in more severe liver disease and low responsiveness to ifn therapy. seventy-one percent of patients with genotype infection respond to ifn therapy, whereas only % of genotype a and % of genotype b show a response. patients infected with genotype generally show higher sustained virological responses to ifn therapy than genotype infected patients, whereas genotype -infected patients show a lower sustained virological response compared with genotype -infected patients. the heterogeneity of hcv genotypes also translates to differences in the manifestation of liver disease. for example, hepatic steatosis is most common in patients infected with genotype and is attributed to its core protein. recently, the use of active direct-acting antiviral molecules to block hcv infection has led to substantial improvements in sustained virological response rates in genotype -infected patients. however, the use of these drugs may allow selection of resistant variants if direct-acting antiviral monotherapy is adopted, and a high relapse rate occurs after direct-acting antiviral treatment is discontinued. the approximately . -kb hcv genome contains a single open reading frame flanked by untranslated regions (utrs) at its and ends (figure ). the internal ribosome entry site (ires) located in the -utr directs cap-independent translation, whereas the -utr contains sequences critical for viral replication and translation. the -utr (positioned at nucleotides - of the hcv genome) contains a poly(u/uc) (pu/uc) tract located at nucleotide positions - , which was identified as an hcv pathogen-associated molecular pattern (pamp) that triggers rlr-mediated type i ifn production ( figure ). translation of the hcv genomic rna produces a single polyprotein of approximately amino acids, which is further processed by cellular and viral proteases to yield the structural proteins core, e and e and the non-structural (ns) proteins p , ns , ns , ns a, ns b, ns a and ns b (figure ). ns proteins participate in different phases of the viral replication cycle (figure ). for example, ns to ns b proteins are important for rna replication. jones et al. showed that p and ns are required for entry and assembly of the virus. other researchers have also reported that ns , - ns / a and ns a , are involved in virion assembly and production. hepatocytes are the primary target cells for hcv replication. upon infection, the virus particle circulating in the blood biochemically resembles the very low-density lipoprotein, which is rich in apolipoprotein (apo) e and apob. , first, the apolipoprotein-associated lipoviral particle (lvp) attaches to glycosaminoglycan and low-density lipoprotein receptor and then interacts with cluster of differentiation (cd ) and scavenger receptor class b number (figure ). the lvp is subsequently translocated to the tight junction of hepatocytes where the lvp binds to the tight junction proteins claudin- and occludin followed by internalization of the hcv particle via ph-dependent endocytosis, which occurs on the plasma membrane ( figure ). in addition to these receptors, cell surface molecules, such as epidermal growth factor receptor, ephrin receptor a and niemann-pick c -like l cholesterol uptake receptor, are also essential for virus internalization. subsequent to internalization, the acidic ph in the endosome triggers fusion of the viral envelope with the endosomal membrane, allowing the release of the viral genome into the cytoplasm ( figure ). hcv genomic translation occurs at the rough er, and hcv rna replicates in an er-derived or er-associated lipid-rich environment termed the membranous web ( figure ). all hcv ns proteins except for ns are involved in viral rna replication. the ns proteins are colocalized with the replicating viral rna on a light density, detergent-resistant cytoplasmic membrane structure termed a 'lipid raft'. lipid droplets (lds), which comprise a neutral lipid core with a single phospholipid layer, serve as energy storage sites and reservoirs of neutral lipids in adipose tissue and hepatocytes. lds are indispensable for viral rna replication and infectious virus formation. during the initial stage of virus assembly, hcv core protein interacts with lds, and the viral replication complex is also directed to lds in an ns a-and core-dependent manner, allowing encapsidation of the viral rna by the core protein and assembly of the nucleocapsid ( figure ). , additionally, the interaction of ns and ns a with actin and tubulin in the microtubule network mediates translocation of the hcv replication complex to lds. the late stage of virus assembly, which occurs in the lumen of the er, involves the acquisition of a lipid envelope, the embed- assembly and budding liver cell figure hcv replication cycle. as shown, the hcv lvp is coated with apob and apoe, which are marked by light green and light blue stripes, respectively, on its surface. the lvp attaches to srb and to cd and further interacts with the tight junction protein claudin- and with occludin. virus entry into cell proceeds through receptor-mediated endocytosis at the cell surface. subsequent to internalization, the viral envelope fuses with the endosomal membrane under acidic ph, and the viral genome is uncoated and released into the cytoplasm. to dissect these two events, internalization and fusion are conventionally depicted as two seemingly separate steps in the cytoplasm. viral rna is translated at the er to produce the polyprotein, which is subsequently processed into mature structural and non-structural proteins. viral non-structural proteins, in conjunction with host factors, form a membranous web where viral rna replication occurs. hcv particle assembly most likely initiates near the er and ld where core protein and viral rna accumulate. finally, hcv particles are secreted into the extracellular milieu via the secretory pathway. viral replication and assembly occur in the proximity of lds and in lipid raft microdomains, which are shown in the inserted dashed rectangle. apo, apolipoprotein; cd , cluster of differentiation ; er, endoplasmic reticulum; hcv, hepatitis c virus; ld, lipid droplet; lvp, lipoviral particle. ding of e and e into the envelope and the formation of the nascent virion ( figure ). then, the nascent virus particles associate with apob, apoe and other very low-density lipoprotein lipids to form lvps. finally, lvps are released from cells through the very low-density lipoprotein secretion pathway or the endosome secretory pathway ( figure ). similar to the ns proteins, p plays numerous crucial functions in virion assembly and egress. p , an integral, oligomeric membrane protein consisting of amino acids, is grouped into the family of viroporins that form membrane pores or channels. in functioning as an ion channel, p modulates membrane permeability to facilitate virus entry by promoting virus uncoating and to enhance assembly or release. p conducts ions across the membrane, and this channel activity can be abrogated by the drug amantadine and iminosugar derivatives. , during maturation and egress, the ion channel activity of p maintains the ph gradients within the secretory pathway and thereby stabilizes the hcv particle. in addition, p has been shown to be necessary for capsid assembly and envelopment because mutations in p result in the accumulation of incompletely assembled capsids that are unable to encapsidate viral rna. the ifn systems constitute the first-line defense mechanism against viral infection in humans. based on their antiviral properties, ifns are grouped into three classes: type i, ii and iii ifns. in humans, type i ifns comprise a large group of molecules encoded by multiple genes, mainly ifn-a and ifn-b, and other genes such as ifn-e, ifn-k and ifn-v. ifn-a and ifn-b combat viruses directly by inhibiting virus replication or indirectly by inducing the innate immune response. most cell types can elicit a type i ifn response by activating the tlr, rlr and jak-stat pathways. , type ii ifn contains only one member, ifn-c. unlike type i ifns, which are elicited as a direct response to viral infection, ifn-c is secreted by natural killer cells and mitogenically activated t cells. ifn-c exerts potent anti-hcv activity in vitro and mediates antiviral t-cell responses. it has also been reported that ifn-c inhibits hcv infection by downregulating claudin- and cd . type iii ifns consist of three members, termed ifn-l , ifn-l and ifn-l or il- (l ) and il- a/b (l / ). as with type i ifns, viral infection also directly activates type iii ifns. however, the antiviral properties and the mechanisms of action of type iii ifns remain unknown. type iii ifns can be secreted by many cell types, but their receptors show a limited tissue distribution. hcv infection results in type iii ifn induction predominantly in the human liver. despite modulation by the irf and nf-kb pathways for induction of type iii and type i ifns, these two systems upregulate distinct subsets of isgs with different kinetics of induction. during hcv infection, cells produce type i ifn to counteract viral infection, to modulate viral replication and to activate natural killer cells, dendritic cells and kupffer cells. recognition of pamps by prrs, including tlrs and rlrs, triggers ifn synthesis and ifn-mediated cascade signaling pathways, leading to the production of type i ifn and a wide range of isgs to mediate ifn antiviral activity ( figure ). , [ ] [ ] [ ] upon virus infection, tlrs and rlrs operate through different signaling pathways, depending on the nature of the viral signals. tlrs are expressed and localized in the intracellular compartment, similar to endosomes, or on the cell surface. , unlike rlrs, tlrs potentially detect viral double-stranded rna (dsrna) released by cells into the extracellular milieu ( figure ). three types of tlrs, i.e., tlr , tlr and tlr , are involved in the recognition of virus infections. tlr detects the dsrna formed during the replication of positive-stranded rna viruses, whereas tlr recognizes the urine-rich ribonucleotide region of rna, and tlr senses dna pamp motifs encoding cpg dinucleotides. upon binding to a pamp, tlr dimerizes and initiates the binding of its cytosolic toll-il- receptor to the adaptor protein toll-il- receptor domain-containing adaptor inducing ifn-b (trif), resulting in the association of tlr with trif ( figure ). , , this interaction leads to the recruitment of tumor necrosis factor (tnf) receptor-associated factor (traf) , traf and the traf family member nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) activator-binding kinase (tbk ), resulting in the phosphorylation and activation of ifn regulatory factor (irf) by tbk and by inhibitor of kappa b (ikb) kinase-related kinase (ikk)e. , after phosphorylation, the irf protein dimerizes and is translocated into the nucleus to form an enhanceosome complex with nf-kb and other transcription factors, thereby inducing the expression of target genes, such as ifns. moreover, the binding of viral dsrna to tlrs also activates nf-kb activity and pro-inflammatory cytokine synthesis through the interaction of trif with receptor-interacting protein- . tlr and tlr bind to myeloid differentiation pro-inflammatory response (myd ) and activate il- receptor-associated kinase (irak) and traf , followed by the activation of ikka/b, which in turn activates nf-kb through phosphorylation, polyubiquitination and proteasomal degradation of its associated inhibitor ikba. migration of nf-kb into the nucleus results in ifn production ( figure ). the rlr receptors consist of rig-i, melanoma differentiation-associated protein (mda ) and laboratory of genetics and physiology- . , , rig-i recognizes the hcv replication intermediate dsrna within hours of infection, which triggers the downstream signaling before the viral protein is extensively synthesized (figure ). rig-i senses the short, non-self dsrnas with -triphosphates, whereas rnas lacking -ppp, such as picornaviruses, are recognized by mda . both rig-i and mda contain two n-terminal caspase activator and recruitment domains (cards). the recognition of dsrna by rig-i is dependent upon the atp-driven translocase activity of cards and helicases, and binding to dsrna induces conformational changes in rig-i that facilitate its oligomerization and translocation from the cytosol to the mitochondrial surface. , on the mitochondria, rig-i engages via its tandem cards with the card of its downstream effector, mitochondria antiviral signaling protein (mavs), which is also termed ifn-b promoter stimulator- , virus-induced signaling adaptor or card adaptor inducing ifn-b ( figure ). , the chaperone protein - - e and the ring finger domaincontaining e ubiquitin (ub) ligase triple motif-containing protein (trim) also participate in this process. trim mediates the ubiquitination of rig-i at position lys- , which is important for mavs binding and for ifn production. the interaction between rig-i and mavs promotes the formation of a signaling complex on the mitochondrial surface that recruits and activates the downstream classical ikk complex, ikka/ikkb, and two non-classical ikk-related kinases, tbk and ikke. , activation of tbk and ikke leads to the phosphorylation, dimerization and nuclear translocation of the transcription factor irf ( figure ). traf , traf and mitogen-activator protein kinase/extracellular signal-regulated kinase (erk) kinase (mekk ) are also recruited to mavs to activate nf-kb. the canonical ikka and ikkb induce nf-kb-dependent gene transcription via phosphorylation, polyubiquitination and proteasomal degradation of ikba, thereby resulting in the release and nuclear migration of nf-kb. nf-kb activation involves the interaction of card with b-cell lymphoma/leukemia protein. activated nf-kb and irf are translocated into the nucleus to form an enhanceosome, thereby stimulating the expression of ifn and inflammatory cytokines with the help of other cellular factors, such as activating transcription protein and c-jun. then, secreted ifn binds to ifnar on the cell surface and triggers the jak-stat signaling pathway ( figure ). , following ifnar receptor binding, tyrosine kinase- and jak are activated and phosphorylate stat and stat to form a heterodimer, which subsequently recruits irf to form the transcription factor ifn-stimulated gene factor (isgf ). then, isgf is translocated into the nucleus, binds to ifnsensitive responsive element (isre) and transactivates the expression of various isgs, such as - oligoadenylatesynthetase ( - oas)/ , -linked oligoadenylate ( - a)-dependent, latent endoribonuclease (rnase l), dsrna-dependent protein kinase r (pkr), and irf ( figure ). acute hcv infections can be spontaneously cleared in some infected individuals, suggesting that the innate immunity induced by hcv pamp sensing can control acute viral infection. , however, % of acutely infected people are not effectively cleared of hcv infection, and these patients may further develop chronic infection, suggesting that hcv has developed strategies to escape or to counteract the host immune response, leading to the emergence of resistance to ifn therapy. in this regard, several hcv proteins have been shown to block host antiviral responses, resulting in progression to chronic hcv infection ( figure ). , , obtaining further data regarding hcv evasion of host innate immunity will certainly improve ifn-based therapy outcomes. core protein hcv core protein is involved in the formation of the viral nucleocapsid and modulates many cellular functions, including transcription and signal transduction. expression of the full-length hcv genome or core protein downregulates ifn signaling by depressing stat tyrosine phosphorylation, which then blocks stat heterodimerization with stat and inhibits ifn signal transduction and isg expression ( figure ) . in addition, expression of core protein induces synthesis of suppressor of cytokine signaling (socs ) in hepg cells. socs is an important repressor of the jak-stat pathway due to its ability to inhibit stat phosphorylation ( figure ). , thus, hcv core protein induces socs and suppresses ifn-mediated isg expression. - socs expression is upregulated in chronically hcv-infected patients who are ifn non-responders compared with responders. core protein expression has also been demonstrated to inhibit irf synthesis, transcriptionally repressing several isgs, such as il- , il- and pkr. many viruses use molecular mimicry as an important immune evasion strategy to promote virus survival and persistence. viruses express proteins that are structurally similar to host defense proteins, and these viral proteins can act as immune modulators. hcv employs this molecular mimicry strategy to resist type i ifn through its e envelope protein. , e comprises a -amino acid sequence identical to eukaryotic initiation factor a (eif a) and pkr. this domain operates to prevent pkr-dependent phosphorylation of eif a and repression of protein synthesis, thus possessing an ability to resist type i ifn treatment ( figure ). , ns / a the hcv ns / a protease is not only responsible for the maturation of ns proteins, viral rna replication and virion morphogenesis but is also important for suppressing the host antiviral system. , , , , the ns / a complex is anchored to the intracellular membrane through the ns a transmembrane domain and the amphipathic a-helix at the ns n-terminus. all these domains facilitate cleavage of their two cellular targets, mavs and trif, which act as key players in type i ifn production ( figure ). , mavs is an essential antiviral signaling protein in the rlr system and, therefore, is an ideal target for viral immune evasion. ns a serves as the primary membrane subcellular targeting subunit to escort ns / a to mavs. ns / a binds to mavs on mitochondria and cleaves mavs at cys- , resulting in the dislocation of the n-terminal portion of mavs from mitochondria and the suppression of ifn production ( figure ). the hydrophobic amino acid stretch in the ns amphipathic a-helix is also required for controlling rig-i signaling. cleavage of mavs and reduction of ifn levels have been observed in chronically hcv-infected patients. thus, ns / a-mediated cleavage of mavs rig-i signaling impairs ifn synthesis. additionally, the ns / a protease also cleaves trif, an adaptor protein linking tlr to kinases responsible for activating irf and nf-kb ( figure ). , cleavage of trif interferes with poly(i:c)-activated tlr signaling and irf and nf-kb activation, thereby limiting the expression of multiple host defense genes and enhancing hcv persistence. stimulator of interferon gene (sting), which is also known as mediator of irf activation (mita), is a -kda protein mainly localized to the er. in response to dsdna transfection or dna virus infection, sting plays a crucial role in the activation of transcription pathways, essential for effective innate immune signaling. upon dsdna stimulation, sting polymerizes and translocates from the er to a cytoplasmic punctate structure where the sting polymer provides a platform to connect tbk with irf , which phosphorylates irf , thereby triggering downstream signaling. in viral infection, ns b from yellow fever virus (yfv) blocks the induction of the ifn production pathway through an interaction with sting. ns b from dengue virus (denv) acts as a protease to cleave sting, thereby shutting down ifn signaling. in hcv infection, ns b interacts with and sequesters sting on the er to inhibit the association of sting with tbk, suppressing ifn signaling ( figure ). , therefore, targeting sting to inhibit innate immunity might be beneficial for virus survival. the mature hcv ns a is present as two phosphoproteins, the hypophosphorylated p and hyperphosphorylated p . , ns a phosphorylation occurs at multiple serine residues, such as serine , and upstream of the ifn sensitivity determining region (isdr) of ns a, which spans residues - (based on genotype b hcv-j strain), and these serine residues are important for hyperphosphorylation. , the functional and locational significance of ns a p and p remains unclear; however, maintenance of these two forms at a specific ratio is critical for hcv replication. functions as a pleiotropic protein that modulates the host environment to favor virus replication and persistence. additionally, ns a binds to myd , which is a major adaptor molecule in the tlr pathway, and inhibits the recruitment of irak to myd , attenuating tlr signaling and impairing cytokine production. a sequence within the ns a isdr, which spans residues - , was shown to be responsible for interaction with the death domain of myd in macrophage cells. pkr is an ifn-induced gene product that is activated by binding to dsrnas commonly produced during viral replication. ns a rescues hcv replication in ifn-treated cells and inhibits ifn antiviral activity by binding to pkr and blocking pkr autophosphorylation and eif a phosphorylation. , ns a expression is sufficient to rescue the replication of an ifn-sensitive virus. the interaction of pkr with ns a requires the isdr that overlaps a broader pkr-binding region, residues - , and results in the inhibition of pkr activation and resistance to ifn in hcv-expressing cells. , consistent with this mechanism, mutations in or deletion of isdr correlate with sensitivity to ifn-a-mediated antiviral activity. , , moreover, meta-analysis and long-term follow-up support the association of this isdr region with the outcome of ifn therapy. this region, which encompasses a genetically flexible domain that allows mutations to occur, is the key site of adaptation to ifn therapy and influences the fitness of hcv rna replication. in contrast, other studies suggest that the inhibitory effect of ns a on ifn may be independent of pkr. ns a increases expression of il- , also known as chemokine cxcl , by upregulating the il- promoter, which in turn, inhibits ifn antiviral activity and facilitates virus infection. , in a cell culture model, il- -positive cells are associated with chronic hcv infection, and il- removal mitigates hcv replication. importantly, the serum level of il- is elevated in chronic hepatitis c patients compared with control individuals or is higher in ifn non-responders relative to responders. these observations suggest that ns a expression increases il- production, which somehow perturbs the ifn antiviral pathway. moreover, ns a impedes the - oas/rnase l system to inhibit ifn signaling. the - oas/rnase l antiviral pathway is present in virtually every cell. this pathway involves the activation of a latent endoribonuclease and degrades hcv mrna with a dsrna structure during replication. ns a physically binds to - oas through amino acid residues - of ns a. a single point mutation at amino acid of ns a affects the ns a and - oas binding and the antiviral activity of the - oas/rnase l system. thus, ns a inhibits ifn antiviral activity in an isdr-independent manner. moreover, ifn-resistant strains, such as genotypes a and b, have fewer rnase l recognition sites in their genomes than the ifn-sensitive strains, such as genotypes and , providing a means for ifn-resistant strains to escape from nucleotylic cleavage. apoptosis also plays a key role in the host defense system by restricting viral spread and persistence. blocking apoptosis could be critical for the establishment of life-long persistence in the host organism. ns a was shown to block the activation of caspase and to inhibit proteolytic cleavage of the death substrate poly(adp-ribose) polymerase in tnf-a-induced cells. adenovirus infection in ns a-transgenic mice downregulates and upregulates the expression of t-box transcription factor and trans-acting t cell-specific transcription factor , respectively, resulting in lower ifn-c expression and a delay in virus clearance. furthermore, stable expression of ns a in the human hepatoma cell line huh decreases sensitivity to tnf-a-mediated apoptosis, and activation of caspase- , and by tnf-a is inhibited in ns a-expressing cells. thus, ns a protects cells from tnf-a-mediated apoptotic death. hcv-induced er stress hcv protein expression can induce an er stress response and lead to calcium release from the er, which in turn activates the cyclic amp responsive element-binding protein that binds to the cyclic amp responsive element in the promoter of protein phosphatase a (pp a), resulting in upregulation of pp a. expressed in essentially all cell types, pp a is a serine/threonine phosphatase that is involved in multiple cellular processes, such as the cell cycle, signal transduction and stress response. , increased expression of pp a has been observed in a cell line inducibly overexpressing hcv protein, in liver extracts from hcv transgenic mice and in liver biopsies from patients with chronic hepatitis c. duong et al. showed that upregulation of pp a by hcv can inhibit the enzymatic activity of protein arginine methyltransferase (prmt ), which leads to decreased methylation of stat . hypomethylated stat is more prone to bind to protein inhibitor of activated stat and inhibits stat dimerization, resulting in impaired nuclear translocation into the nucleus, binding of stat to the isre, and isg production. thus, hcv-induced pp a activation disrupts the ifna-induced antiviral state, leading to hcv evasion of innate immunity. these authors also showed that prmt can methylate hcv ns at arginine , resulting in the inhibition of ns helicase activity. therefore, hcv-mediated pp a upregulation enhances ns helicase activity by inhibiting prmt enzymatic activity, which in turn facilitates virus replication. human genomes encode hundreds of isgs, and the first isg, k, was discovered more than years ago. synthesis of some isgs can be triggered by viral infection without ifn production. some isg products can directly regulate cellular processes, such as protein synthesis and cell growth, survival and apoptosis, whereas others may modify the ifn antiviral activity against invading viruses. the gene products of isgs can target many steps in the hcv replication cycle to limit viral replication. many pamp receptors and their subsequent sig-naling partners are also isgs. isgs expressed at the basal level provide antiviral surveillance before ifn activation or therapy; however, their levels markedly increase after ifn production. in the innate immune response to virus infection, viral rna acts not only as a inducer of the production of ifn and its effector functions but also as a substrate and product for cellular enzymes, such as pkr and - oas/rnase l. the inverse correlation between the upregulated expression of isgs, such as oas-like (oasl), isg and viperin, in liver biopsies from hcv-infected patients and infected hepatocytes and decreased viral rna levels suggest the anti-hcv activities of these isgs. in this section, we will highlight the involvement of isgs that are critical for modulating innate immunity in hcv replication, and the potential functions of these isgs, as outlined in table . rig-i rig-i, which is encoded by the ddx gene and which belongs to the dexd/h box rna helicase (ddx) family, is a key player in the defense against invasion by many rna viruses. rig-i senses the intracellular viral components and initiates antiviral responses to stimulate ifn production (table ). in turn, ifn activates the transcription of rig-i, hence forming a positive feedback loop for amplifying antiviral signals. studies have shown that rig-i is essential for eliciting an immune response against vesicular stomatitis virus (vsv), sendai virus, newcastle disease virus (ndv) and hepatitis c virus. , , knockout of the rig-i gene in mouse embryonic fibroblasts severely limits type i ifn production and isg activation, thereby potentiating viral replication; conversely, overexpression of rig-i restricts viral replication. in addition, upregulation of rig-i gene expression has been observed in ifn-treated human dendritic cells, suggesting that rig-i serves as an isg. rig-i contains two tandem cards at its n-terminal region, with a repressor domain in its c-terminal region. the card is responsible for downstream signaling and activation of type i ifn after recognition of non-self rna, whereas the repressor domain is essential for the autoregulation and recognition of viral rnas. without viral stimulation, the card interacts with the helicase domain, placing rig-i in an auto-inhibitory state and disabling signal transduction. upon binding to viral rna, rig-i undergoes conformational changes that expose the card, allowing rig-i to be ubiquitinated. rig-i is ubiquitinated by two different ligases, trim and ring finger protein . trim ubiquitinates rig-i at lysine to mediate the antiviral response, whereas ubiquitination by ring finger protein regulates the degradation of rig-i by the proteosome, thereby downregulating rig-i-mediated signaling. ubiquitination by trim induces rig-i to form a tetramer, promoting the cards of rig-i to engage with the cards of mavs. this results in the accumulation of mavs on the mitochondrial membrane and the activation of ikk and tbk , which, in turn, activates the transcription of nf-kb, irf and irf to promote ifn production. moreover, ubiquitination by trim also prevents cards from interacting with the helicase domain to reinstate the auto-inhibitory state. ubiquitination at lysine is crucial for rig-i function because a mutation at this residue renders rig-i unable to bind to mavs, thus abrogating downstream signaling. ddx is also a dexd/h box helicase whose function remains unclear. ddx slightly resembles the yeast ski protein, which is a cofactor of the rna exosome required for controlling host rna quality. ddx , which is the human homolog of yeast ski, and the rna exosome exhibit antiviral activity against monkey leukemia virus and sindbis virus (sinv). ddx expression is upregulated during infections of measles virus (mev) and hcv. , the ddx mrna level is robustly upregulated in human fetal liver cells within - h after hcv infection, providing a means to initiate the antiviral mechanism. unlike rig-i, ddx does not contain the cards to interact with mavs. after viral infection, ddx is induced and binds to rig-i as well as mda and laboratory of genetics and physiology- and promotes the binding of rig-i to dsrna. , ddx is essential for type i ifn expression during dna virus infection and is induced to suppress viral replication in a rlr-dependent manner (table ). ddx knockdown reduces the expression of type i ifn after hcv, hiv and yfv infections. irf irf was first identified as a transcriptional activator of the ifn-a/b gene. in unstimulated cells, irf is expressed at a low level; however, its expression is increased by the induction of ifn-a/b, tnf-a, il- and viral infection. nevertheless, the precise pathway leading to irf activation by virus infection remains elusive. irf activation may proceed through a pkrdependent pathway after virus infection. pkr indirectly phosphorylates irf and activates its dna-binding properties. thus, activated irf regulates the promoter function of ifna/b promoter and acts as a modulator of many isgs by binding to the isre in the promoter region, thereby regulating viral replication (table ) . , irf controls the ifn antiviral response by affecting a set of isgs, such as irf and irf . irf cooperates with irf and irf to regulate cellular antiviral genes, such as ifn-a/b. hcv infection increases the level of irf , which may affect other irf pathways and isg expression, thereby leading to a reduction in viral replication. irf overexpression induces an antiviral state that affects various viruses, including ndv, vsv and hcv. , the expression level of irf is reduced in cells harboring hcv subgenomic replicons (sgrs), whereas irf overexpression in these cells increases the isre activity and attenuates hcv replication. additionally, hcv infection mediates irf expression, thus affecting the intracellular level of hcv rna. however, hcv may evade the irf anti-hcv effect through core-mediated suppression of irf synthesis. irf is an essential transcription factor for the induction of ifn-a/b and isgs. all of the elements of ifn responses, either innate or adaptive immunity, are regulated by irf . irf is constitutively expressed in certain cells, such as macrophage and plasmacytoid dendritic cells, priming these cells for rapid ifn production. during infection, ifn-a/b binds to its receptor and activates the jak-stat pathway, resulting in irf expression. then, irf is phosphorylated, forms a heterodimer with irf and is translocated into the nucleus. in the nucleus, the irf -irf heterodimer binds to the irf elements in the promoter region of ifn-a genes, leading to enhanced expression of the ifn-a subtype and a diverse range of isgs (table ) . , in turn, these events increase the abundance of rig-i and viral pamp signaling components, whereas sustained signaling serves to amplify ifn production. , moreover, irf induces expression of other isgs without activating ifn signaling. thus, irf -mediated transcriptional cascades serve as an intrinsic antiviral mechanism allowing rapid isg expression before ifn production. irf plays an important role in eliminating hcv infection. sirna knockdown of irf decreases ifn-a production and increases the hcv titer. , mice lacking irf show rapidly lethal infection by west nile virus (wnv) and high virus burdens. irf deficiency represses the induction and accumulation of ifn-a, thus favoring wnv replication. although hcv seems to suppress the basal expression of irf , tlr stimulation activates irf and suppresses hcv replication. this observation suggests that hcv may only partially inhibit irf activity in hcv-expressing cells. pkr pkr, which is also known as eif ak , is a serine/threonine kinase that phosphorylates eif a in response to virus infection. this ifn-inducible kinase has two distinct activities: autophosphorylation, resulting in its activation, and phosphorylation and inactivation of eif a. through phosphorylation events, pkr mediates the inhibition of translation initiation of both cellular and viral mrna. [ ] [ ] [ ] it is well documented that the anti-hcv activity of pkr occurs through its translational control (table ) . [ ] [ ] [ ] however, viruses have evolved elaborate strategies to counteract the detrimental effects of pkr. hcv ires activity has been shown to be resistant to pkr activation in cells harboring hcv sgr and in the hcv infection model (table ) . [ ] [ ] [ ] mechanistically, viruses may use their proteins to impede the dsrna-dependent pathway in various ways, such as sequestering dsrna, inhibiting pkr activation, producing pkr pseudosubstrates, activating antagonist phosphatases and degrading pkr. as indicated above, hcv employs ns a and e to antagonize pkr function, resulting in resistance to ifn and a blockade of the pkr-mediated inhibition of viral protein synthesis (table ) . analogous to alphaviruses sinv and semliki forest virus, hcv can activate pkr and eif a phosphorylation to enhance its own viral protein translation (table ) . , compared with other previously studied dsrnas, domains iii-iv of the hcv ires were shown to bind to the n-terminal dsrnabinding domain of pkr, leading to increased pkr autophosphorylation and activation. additionally, cap-dependent but not hcv ires-mediated translation is inhibited by pkr and eif a phosphorylation. these results indicate that while escaping the deleterious effects of pkr activation, hcv can employ its structured ires to direct its own protein translation. karamichali et al. demonstrated that activated pkr or silencing pkr upregulates or downregulates hcv ires activity (table ) . these authors further showed that the inhibitory effect of ns a on ires-dependent translation occurs through pkr inactivation. in contrast, hcv can translate its viral protein via a bacterial-like pathway that uses eif b, which is an analog of bacterial if , and eif , instead of eif a and its gtpase-activating protein eif , as the initiation factor (table ) . the use of eif a-independent translation initiation provides an alternative tactic for hcv translation when eif a is inactivated by phosphorylation under stress conditions. many lines of evidence have revealed that hcv-mediated phosphorylation and activation of pkr, in turn, inhibit its downstream target, eif a, and attenuate the expression of host cellular proteins, including isgs, without any inhibitory effects on viral ires-mediated viral protein translation (table ) . , , pkr knockdown in hcv-infected cells restores isg expression and enhances the antiviral effect of ifn. these results demonstrate that hcv escapes ifn antiviral activity by promoting the phosphorylation of pkr and inhibiting the production of antiviral isg proteins, thus providing an interesting pathway for the virus to evade the ifn antiviral response. furthermore, accumulating evidence has revealed that pkr and eif a participate in the formation of stress granules (sgs). sgs are large, dynamic structures between to nm in size, that form in the cytoplasm when cells undergo extracellular stresses, including viral infections. sg formation is important for the posttranscriptional regulation of gene expression. sgs contain stalled translation pre-initiation complexes, including cellular mrnas, translational initiation factors, the small subunit of the ribosome and many cellular rna binding proteins, such as t-cell-restricted intracellular antigen (tia- ), the homologous tia- -related protein tiar and rasgap-sh domain binding protein (g bp ), involved in regulating mrna functions. [ ] [ ] [ ] [ ] many viruses, including hcv, can modulate sg assembly and co-opt sgs to promote their own protein synthesis. , hcv induces sg formation via eif a phosphorylation (table ) . , consistent with this notion, upregulation of the regulatory subunit of protein phosphatase that dephosphorylates eif a and growth arrest dna damage-inducible protein , inhibits sg formation. these results indicate the importance of eif a phosphorylation in hcv-induced sg formation. moreover, garaigorta et al. demonstrated that hcvinduced sg formation is ifn-and pkr-dependent and is inversely correlated with the induction of isg proteins, such as myxovirus resistance gene a (mxa) and ub-like (ubl)specific protease (usp ), in hcv-infected cells without affecting the mrna levels of these isgs. furthermore, the sg proteins tia- , tiar and g bp have been shown to play a critical role in hcv replication and infectious virus production. in support of this finding, g bp was also reported to be essential for hcv rna replication, presumably through its relocalization to lds or its interaction with ns b. , the results of garaigorta et al. demonstrated that hcv hijacks pkr phosphorylation-triggered sg formation to downregulate the translation of antiviral isgs, thereby promoting viral rna replication, virus assembly and egression. oas and rnase l upon sensing and activation by the pamp of viral dsrna, certain ifn-stimulated - oas proteins can synthesize - a from atp. after binding to - a short oligoadenylates, a ubiquitous, latent endonuclease, rnase l, is activated through dimerization and degrades either cellular or viral rnas, resulting in the inhibition of protein synthesis, cellular apoptosis and impaired virus propagation. , , therefore, the oas/ rnase l pathway represents a critical arm of ifn's antiviral effector mechanism against many viruses, including hcv. depending on the specific rna substrates and the extent of enzymatic activity, rnase l can block different types of viruses through different mechanisms, such as apoptosis, or through the 'suppressor of virus rna' derived from cellular or viral rna. nevertheless, some members of the oas family can exert antiviral activity independent of rnase l. the oas system has been reported to exert anti-hcv effects through the rnase l pathway. the ua and uu dinucleotides within loops of predicted stem-loop structures in the viral rna is prone to cleavage by rnase l (table ) . additionally, the sensitivity of hcv infection to ifn therapy correlates with the efficiency of rnase l-mediated viral rna cleavage. the anti-hcv activity of oas p and oas p in the oas family occurs in an rnase l-dependent fashion (table ) . hcv replication is suppressed in hcvcc-infected huh cells co-cultured with hepatic stellate cells (lx- ) treated with ppp-dsrna or incubated with conditioned medium from lx cells stimulated with ppp-dsrna. in these hcvccinfected cells, the expression of oas- and mxa is upregulated. the two different domains in oas-like a (oasla), a major isoform in human liver that is induced by hcv, contribute to the antiviral activity. the n-terminal oas homology domain, which lacks the cleavage activity, impairs cell proliferation as well as viral replication, whereas the c-terminal ublike domain impedes hcv replication without affecting cell growth (table ). the ifn-stimulated gene kda protein (isg ) has emerged as a second ifn-regulated rnase that inhibits rna virus replication. , isg , along with the closely related isg l and isg l , belongs to the yeast rna exonuclease homolog subfamily within the deddh exonuclease family and members of this superfamily possess both rnase and dnase activities. the - exonuclease activity of isg demonstrates a greater preference for single-stranded rna than for single-stranded dna. isg overexpression restricts infection by encephalomyocarditis virus, vsv, influenza virus (infv), human immunodeficiency virus (hiv), yfv, picornavirus and hcv. , isg has been reported to impair hcv genotype b sgr replication in hek cells (table ) . in addition, isg can hinder genotype a viral rna replication either in sgr or hcvcc infection, and its anti-hcv effect is not shared with isg l and isg l (table ) . apart from degrading viral rna through its - exonuclease activity, , the anti-hcv mechanism of isg in hcv replication remains poorly understood despite its possible action on cellular factors. adar rna-specific adenosine deaminase (adar) is constitutively expressed in normal cells as an inactive form. however, viral infection triggers the two mammalian adar genes, adar and adar , to express two active proteins, adar and adar . adar catalyzes adenosine to inosine editing in rnas that possess double-stranded structures. , because i is recognized as guanosine by rna polymerase, a to i editing causes nucleotide substitution as well as dsrna destabilization because of the reduced stability of i:u mismatch base pair compared with the normal base pair. , the rna editing ability of adar affects many biological processes, including viral replication and persistence, apoptosis, ion channel function and the posttranscriptional modification of genes. , only the adar transcription level is induced by ifn treatment and by pathogen infections. in addition, adar , but not adar , affects the stability of hcv replicon rna (table ) . in hcv sgr replication, ifn-a treatment decreases viral rna replication and concomitantly increases adar expression, suggesting that adar possesses an antiviral activity in the hcv rna replicon. adar knockdown conversely increases the hcv replicon rna. loss of hcv rna by adar may be due to several reasons. first, an i base-specific rnase might target mutated viral rna. second, the mutated rna might lead to insufficient replication and genome instability. third, the cellular mrna involved in viral replication may also be targeted by adar . thus, the rna editing ability of adar negatively affects hcv rna replication, representing a potent strategy in anti-hcv therapy. in sharp contrast, the replication of hepatitis delta virus (hdv) benefits from adar editing. the editing of hdv rna by adar converts the uag stop codon to a uig tryptophan codon, allowing the synthesis of a larger hdv antigen. without viral rna editing, the hdv genome cannot be packaged into a virion. nonetheless, adar overexpression increases rna editing but decreases hdv replication. ifn-inducible transmembrane protein (ifitm) family ifitm family members, including ifitm , ifitm and ifitm , inhibit, in an ifitm-specific manner, the replication of diverse pathogenic membrane-enveloped viruses, including marburg virus and ebola (ebov) filoviruses; severe acute respiratory coronavirus; hiv; rift valley fever virus (rvfv); respiratory syncytial virus; reovirus; flaviviruses, including denv and wnv; and hcv. [ ] [ ] [ ] [ ] [ ] [ ] in contrast, ifitms show no inhibitory effects on entry of amphotropic mouse leukemia virus, machupo virus, lassa virus and lymphocytic choriomeningitis virus. ifitms are topologically located at different intracellular membrane compartments. ifitm and ifitm , which are type ii transmembrane proteins, are primarily localized to endosomes and lysosomes, , whereas ifitm also localizes to the cell periphery. , ifit interacts with tbk , irf and other ifitm members and enhances ifn signaling. , lipid raft membranes, which are enriched in cholesterol and sphingolipids, play vital roles in cellular pathways and in virus entry, assembly and budding. , vesicle-associated membrane protein-associated protein a (vapa) and oxysterolbinding protein (osbp) modulate the intracellular trafficking and de novo synthesis of cholesterol. vapa interacts with osbp to regulate the transfer of cholesterol from the er to other organelles. , the regulation of intracellular cholesterol homeostasis, particularly in the endosomal compartment, is critical for the entry of viruses such as ebov and marburg viruses. ifitms have been demonstrated to interfere with virus infection by blocking virus-endosome fusion (table ) , , presumably through the modification of cellular membrane properties, such as fluidity and spontaneous curvature. , , amini-bavil-olyaee et al. demonstrated that the interaction of ifitm with vapa antagonizes the association of vapa with osbp, thereby inducing the accumulation of cholesterol in multivesicular bodies and in late endosomes. the disruption of intracellular cholesterol homeostasis subsequently impairs the membrane fusion of intraluminal virion-containing vesicles and endosomes, resulting in a block of vsv release into the cytosol. using immortalized human hepatocytes and huh infection models, raychoudhuri demonstrated that ifitm expression inhibits hcv replication but not at virus entry. later, wilkins et al. identified that ifitm is a hepatocyte tight junction protein whose antiviral action occurs through modification of the interactions of the hcv coreceptors cd and occludin, thereby inhibiting hcv entry (table ) . this study represents an interesting mode of antiviral innate immunity; an isg can exert its anti-hcv action by disrupting viral coreceptor associations. the ifn-induced protein with tetratricopeptide repeats (ifits) family represents a class of isgs featured by their unique helix-turn-helix motifs, known as tetratricopeptide repeats. ifits mediate a broad range of protein-protein interactions; in particular, the tetratricopeptide repeat motif is critical for modulating protein translational initiation and transport, cell proliferation and migration, virus replication, and antiviral signaling. [ ] [ ] [ ] [ ] proteins in the ifit family have been linked to ifn antiviral functions, including those against wnv and lymphocytic choriomeningitis virus. ifit plays an important role in modulating innate immunity by bridging tbk to mavs on mitochondria as ifit expression facilitates the association of its tetratricopeptide repeat motif with the n terminus of tbk , thereby enhancing irf -mediated gene expression. ifit , which is also known as isg , belongs to a family that also contains other stress-induced, structurally related proteins, p , p and p , in humans. ifit acts as a negativefeedback regulator for sendai virus-triggered induction of type ifn antiviral signaling transduction, presumably through its interaction with the adapter protein sting and through disruption of the normal association between sting/mita and mavs or tbk . moreover, ifit / preferentially targets mutants of poxvirus, coronavirus, and wnv that lack -o methylation in their viral rna cap, thereby rendering these mutant viruses unable to replicate. this study addresses the mechanism by which -o methylation of the cap of viral rna renders viruses insensitive to ifit-mediated host innate antiviral activity. wang et al. demonstrated that ifit mediates its ifn antiviral activity and blocks hcv rna replication, presumably by targeting an eif -dependent step in viral ires-mediated translation (table ) . in immortalized human hepatocyte and huh infection models, raychoudhuri documented that ifit expression inhibits hcv replication by suppressing hcv ires-mediated transcription. conversely, ifit knockdown facilitates hcv replication. these results suggest that ifit restricts hcv infection primarily at the viral translation/replication site. protein posttranslational modifications by ub and ubl modifiers not only play important roles in numerous cellular processes, such as protein localization, interaction, activity and degradation, signal transduction, vesicular trafficking and dna damage repair, [ ] [ ] [ ] but also modulate pathogen-host interactions, such as the viral replication cycle and the host antiviral response. - isg , which was the first ubl protein modifier identified, is post-translationally attached via its c terminus to the lysine residues of isgs and to hundreds of target proteins involved in different pathways. , similar to its ub homolog, isg is linked to proteins via a tightly regulated process known as 'isgylation', and the activating e (ube l), conjugating e (ubch ), and ligating e (ceb ) enzymes catalyze these sequential events. , isg , together with its conjugation e ligase (ceb ) and its deconjugation enzyme usp , are in the same isg /usp ubl pathway. isgylation modulates signal transduction pathways and host antiviral responses. isg exerts its modulatory roles by inhibiting virus release, isoylating viral proteins, or modifying host proteins. isg targets many cellular proteins, including jak , stat and many isgs. three antiviral effector molecules, irf , rig-i and pkr, are also modified by isgylation. activated irf is stabilized by isgylation and therefore, positively regulates type i ifn signaling. , the isg conjugation-mediated reduction of the non-isgylated rig-i correlates with the reduced ndv-triggered ifn response. additionally, viral rna-independent pkr activation requires the isgylation of pkr. isg expression enhances ifn-mediated antiviral activity against many viruses, including hiv and sinv. overexpressing isg in ifn-a/b receptor knockout mice decreases sinv replication and protects the mice from sinv-induced lethality. isg / mice are more susceptible to infection by many rna and dna viruses, such as infv and herpes simplex virus (hsv) type , and the protection effect of isg from sinv infection is dependent on isgylation. lu et al. demonstrated that induction of isg expression in ndv-infected cells counteracts the ub-mediated degradation of irf and enhances the ndv-mediated host innate antiviral response. their findings revealed a feedback mechanism of isg in enhanced antiviral immunity. despite functioning as an antiviral molecule, isgylation of the antiviral rig-i enzyme inhibits ifn signaling in mouse embryonic fibroblast cells. using the genotype a j /japanese fulminant hepatitis- chimeric hcv infectious model, chen et al. unexpectedly found that isg acts as a pro-hcv regulator because increased isg /isgylation facilitates hcv production, whereas blocking isgylation decreases virus production (table ) . moreover, knockdown of ube l, the e activating enzyme, inhibits hcv replication, particularly hcv egress, without affecting ifn-mediated isg expression in hcv-infected cells. using the hcv-huh . .cd infection system, arnaud et al. dissected the acute ifn response to hcv infection into early, pkr, and late, rig-i, phases. hcv infection rapidly induces the expression of many irf -dependent genes, including isg , through a pkr-dependent mechanism before the rig-i phase, which recruits mavs. then, isg induction blocks hcv rna-mediated rig-i activation by inhibiting rig-i ubiquitination, thereby negatively controlling the rig-i/mavs pathway. these studies illustrate that hcv may exploit isg to antagonize host innate immunity and to promote viral replication. the deconjugation of usp from its target proteins is catalyzed by usp (mouse ortholog ubp ). usp can function in both isg -dependent and isg -independent modes. usp was shown to bind to ifnar and attenuate the jak-stat pathway, thereby negatively regulating ifn signaling (table ) . reduced usp expression results in increased antiviral activity against many viruses, such as sinv, hepatitis b virus and vsv, in usp knockout mice. , [ ] [ ] [ ] usp knockdown is concomitant with increased cellular protein isgylation, prolonged stat tyrosine phosphorylation and enhanced isg expression, thus greatly enhancing the anti-hcv potency of ifn. all these studies suggest that usp disruption can impede its negative regulatory effect on ifn signaling, resulting in sustained jak-stat activity and antiviral activity. consistent with these observations, murray et al. demonstrated that ifn-a signaling and isg induction were greatly increased when ups was knocked down in both hcv sgr-and hcvcc-infected huh cells. however, usp knockdown did not have a significant effect on anti-hcv activity. these observations suggest a slight dependency of ifn-mediated antiviral activity on usp activity. additionally, usp upregulation is predictive of a nonsustainable viral response to ifn treatment. , , the expression levels of ups and isg increase in liver biopsy specimens from chronically hcv-infected patients who do not respond to ifn-based therapy, inferring that hcv hijacks the isg /usp pathway to evade the antiviral immune response and to facilitate its replication (table ) . , this observation also explains, at least partially, the failure of ifn-based treatments in non-responders, although non-responders express higher levels of isgs, particularly isg , compared with ifn responders. , taken together, these findings demonstrate that usp is an attractive target for the development of anti-hcv therapeutics. viperin, which stands for virus inhibitory protein, endoplasmic reticulum-associated, ifn-inducible, plays crucial roles in virus replication, signaling and the immune response. , the viperin protein sequence is highly conserved, and all viperin homologs contain three functional domains: the amphipathic, n-terminal domain, which mediates er and ld association; the central cxxxcxxc motif, which is functionally important for fe-s cluster formation; and the highly conserved c-terminal domain, which is essential for antiviral activity. [ ] [ ] [ ] in addition to type i, type ii and type iii ifns, dsdna and dsrna analogs, bacteria, lipopolysaccharide, poly(i:c) and a broad spectrum of dna and rna viruses can induce viperin expression. , , viperin expression regulates many cellular functions, such as forming lds and reducing membrane fluidity. viperin possesses antiviral activity against diverse families of dna and rna viruses, including infv, hiv, sinv, the flaviviruses japanese encephalitis virus, denv and wnv, and the hepacivirus hcv (table ) . , , viperin functions in different ways to defend against virus infections. for instance, viperin alters membrane fluidity by interacting with farnesyl diphosphate synthase, which is an enzyme essential for isoprenoid biosynthesis, thus disrupting the formation of lipid rafts, the sites of infv budding, leading to interference with virus release from the cell surface. the induction of viperin into hiv- -infected cells disrupts lipid rafts, causing viperin redistribution to cd compartments, where hiv- buds in human macrophages. the radical s-adenosyl-methionine enzymatic activity of viperin is required for the inhibition of hiv production. in cells infected with japanese encephalitis virus, the antiviral function of viperin is attenuated due to its degradation by the proteasome-mediated protein degradation system. in contrast, viperin enhances human cytomegalovirus infection through its interaction with the viral mitochondrial inhibitor of apoptosis vmia protein, resulting in viperin relocalization from the er to mitochondria. in mitochondria, viperin interacts with the mitochondrial trifunctional protein and reduces cellular atp generation, resulting in the disruption of the actin cytoskeleton and enhancement of the virus infection. viperin is upregulated in huh cells transfected with either poly(i:c) or hcv rna, and transient expression of viperin in hcv sgr replicating cells significantly decreases hcv replication. the putative radical s-adenosyl-methionine enzymatic activity of viperin is required for this anti-hcv activity. helbig et al. further demonstrated that the restriction of hcvcc replication by viperin depends on both the n-terminal amphipathic a-helix and the c-terminal domain. the anti-hcv function of viperin coincides with its binding to ns a at the ld interface, whereas ns a normally associates with the human homolog of the -kda vesicleassociated membrane protein-associated protein (hvap- ), which is a pro-viral cellular factor, at the viral replication complex. the interaction of hcv ns a with hvap- was previously shown to be critical for the formation of the viral replication complex. therefore, the association between viperin and hvap- requires both of their c-terminal domains, which then disturbs the interaction of hvap- with ns a and inhibits hcv replication (table ) . together, these findings imply that viperin hinders viral rna replication by perturbing the interaction between hvap- and ns a. by conducting bioinformatic analyses of murine bone marrowderived macrophages, liu et al. showed that cholesterol- -hydroxylase (ch h), which is an ifn-a-and ifn-c-stimulated isg, can mitigate the replication of many membraneenveloped viruses, including hiv, vsv, hsv and murine c-herpesvirus, and many pathogenic viruses, such as rvfv, ebov, russian spring-summer encephalitis virus and nipah virus in vitro and in vivo. these viruses contain different structural characteristics in their fusion proteins. for instance, hiv and ebov contain class i fusion peptides, rvfv and russian spring-summer encephalitis virus harbor class ii peptides, and vsv and hsv belong to class iii fusion proteins. , the broadly antiviral action of the ch h gene product is mediated by the ability of its enzymatic product, hydroxycholesterol, to inhibit ph-dependent and ph-independent membrane fusion between cells and viruses, as typified by vsv and hiv, respectively (table ) . this study not only demonstrates that ifn can confer an antiviral state to host and/ or target cells by inducing a natural oxysterol inhibitor but also suggests that modification of membrane oxysterols can be used as a potential antiviral approach. determining whether this broad antiviral isg can block hcv-mediated membrane fusion would be interesting. several genome-wide sirna screens were recently performed to identify isgs or ifn-mediated effector genes (iegs) that mediate ifn antiviral functions. these studies have identified many new isgs or iegs and have revealed interesting features of the actions of isgs. using an overexpression screen approach, schoggins et al. demonstrated that each virus exhibits a unique but partially overlapping profile of antiviral isg expression. the expression levels of isgs may vary depending on viral infection or on the time, dose, or cell type used for ifn treatment. in hcv infection, higher expression levels of unique isgs were found to correlate with a reduction in the hcv viral load. schoggins et al. further observed that multiple isg genes could target each viral species with a range of inhibitory activities. a set of effectors, including irf , c orf (also known as mb d ), heparanase, rig-i, mda and ifitm , exert broad antiviral activities against different viruses, including hcv, yfv, wnv, chikungunya virus, venezuelan equine encephalitis virus and hiv- . however, other effectors, such as ddx , ifn-inducible proteins l and , ifitm , mapk kinasekinase , moloney leukemia virus , nicotinamidephosphoribosyltransferase, oasl, receptor transporter protein , three prime repair exonuclease and protein unc- homolog b, display species-specific antiviral effector functions. these results also demonstrated that different isgs can exert additive antiviral effects on virus replication. remarkably, several isgs, such as adar, family with sequence similarity , member c, lymphocyte antigen e and mucolipin- , can enhance the replication of certain viruses. certainly, further characterizing how these isgs antagonize ifn-mediated antiviral functions and determining which steps of virus replication are targeted by these isgs are important. these findings indicate the complexity of the type i ifn-mediated innate immune response in virus replication. performing a sirna-based 'gain of function' screen, metz et al. identified several new anti-hcv isgs in addition to those previously reported anti-hcv isgs. this study demonstrated that both ifn-a and ifn-c can upregulate the expression of several isgs, including ifit , trim , phospholipid scramblase and inducible nitric oxide synthase . these isgs possess anti-hcv activity, although the precise roles of these isgs in hcv replication are not understood. this study also reported a substantial overlap in antiviral innate immune responses triggered by either cytokine. however, some isgs are more specifically induced by ifn-a or by ifn-c. for instance, phospholipid scramblase and nitric oxide synthase primarily function as ifn-c-mediated anti-hcv effectors. moreover, different isgs function additively or synergistically to interfere with hcv infection, indicating that the combinatorial and concerted actions of multiple effectors mediate repression of hcv replication. in addition to the signaling molecules involved in the ifn/ jak-stat/isgpathway, the majority of genes identified by fusco et al. are not transcriptionally activated by ifn. in contrast to the notion that isgs target specific virus replication steps, some of these genes can exert ifn-mediated antiviral effects at multiple steps of the hcv replication cycle. for instance, dipeptidyl-peptidase /cd /adenosine deaminase complexing protein blocks virus entry, initial rna replication, and amplified translation. myst histone acetyltransferase inhibits hcv entry, translation, rna replication and virion release, and protein phosphatase , catalytic subunit, b isoform (ppp cb) impairs virus entry, initial rna replication and subsequent translation. taken together, these findings reveal that these ifn-insensitive iegs, together with isgs, constitute the host cellular genes mediating the antiviral activity of ifn against viral replication. a functional genomic screen has shown that several new genes comprising the u /u .u tri-small nuclear ribonucleoprotein (snrnp) possess the ability to mediate ifn antiviral activity. u /u .u tri-snrnp is the major component of human spliceosome complexes involved in mrna processing. this genomic screen demonstrated that squamous cell carcinoma antigen recognized by t cells (sart ) is a u / u .u tri-snrnp-specific factor required for ifn-a-mediated anti-hcv activity, although sart is not induced by ifna. the anti-hcv activity of sart acts by regulating the expression of isgs, such as mxa, oas and pkr, either in the presence or absence of exogenous ifn-a. this genetic screen links an unappreciated role of rna processing to the control of antiviral immunity. in this section, we discuss recent findings regarding the roles of several cellular factors and/or machinery involved in the immune response in modulating hcv replication. although these determinants are not directly induced or activated by ifn, knowledge of their interplay with the host immune response will help to elucidate their effects on hcv infection. the functions of these cellular determinants in hcv infection are summarized in table . ikka hcv can co-opt an intrinsic innate pathway and hijack cellular lipid metabolism to facilitate its assembly. ikka was initially identified as a critical factor for hcv replication in a genomewide rna interference screen. subsequently, hcv infection was shown to activate ikka through the interaction of the viral genome -utr with dead box polypeptide , x-linked (ddx x). ikka translocates into the nucleus and induces the cbp/p -mediated expression of lipogenic genes, including sterol regulatory element-binding proteins, followed by the promotion of core-mediated ld formation and the enhancement of hcv assembly ( table ). dansako demonstrated that upon hcv expression, class a scavenger receptor type (msr ) expressed on the plasma membrane of infected and adjacent uninfected cells can bind to dsrna released from infected cells and mediate its endocytosis and transport to endosomes where the dsrna is sensed by tlr and initiates a local antiviral ifn response to restrict hcv replication. the msr -mediated binding, transport, and release of dsrna at the acidified endosome requires a stretch of conserved basic residues within the c terminus of the collagen superfamily domain of msr . therefore, msr acts as a key element for the tlr -mediated prr, thereby rendering both infected and uninfected hepatocytes refractory to hcv replication (table ) . hmgb , which is an abundant nuclear protein that mediates activation of host immune responses and inflammation, represents a prototype damage-associated molecular pattern that participates in the pathogenesis of diverse pathogens. , hmgb is passively released by cell injury or ischemia without pathogen invasion, but is actively secreted from stimulated immune cells, such as natural killer cells, macrophages and mature dendritic cells. many types of tlrs, such as tlr , tlr and tlr , can act as receptors for hmgb . the production of reactive oxygen species can mediate translocation from the nucleus to the cytoplasm and the subsequent release of hmgb . , interestingly, it has been shown that hcv core and ns a can trigger oxidative stress in infected cells. [ ] [ ] [ ] jung et al. demonstrated that hcv infection causes the nuclear-to-cytoplasmic translocation of hmgb and its release into the extracellular milieu. tlr acts as a major component of the receptor complex that recognizes lipopolysaccharide lps and plays a role in the production of pro-inflammatory cytokines and antiviral ifns via signaling myd and the tlr adapter protein trif. jung et al. also demonstrated that hmgb interacts with tlr to activate ifn signaling (table ). because hmgb is present at higher levels in the sera of patients with chronic hepatitis and cirrhosis compared with those detected in control individuals, the results of jung et al. may help to elucidate the potential inhibitory action of hmgb in hcv propagation in chronically hcv-infected patients. autophagy autophagy is a conserved 'self-eating' process that engulfs and delivers cytoplasmic cargos and invading pathogens within double-or multiple-membrane autophagosomal structures to lysosomes for degradation. [ ] [ ] [ ] [ ] the purpose of autophagic induction is to maintain cellular homeostasis in the host when the host undergoes extracellular or intracellular stresses. autophagy plays pivotal roles in the stress response, nutrient deprivation, damaged organelles, unfolded protein aggregation, intracellular quality control and cell death. [ ] [ ] [ ] [ ] the autophagic process requires two ubl conjugation complexes: autophagy-related gene (atg) -atg -atg l and microtubule-associated protein light chain -phosphatidylethanolamine. , autophagy has emerged as an immune regulator that commands the innate and adaptive immune responses against intracellular viruses. [ ] [ ] [ ] [ ] [ ] autophagy also participates in the modulation of virus-host interactions. in contrast, viruses can subvert the host autophagic pathway to potentiate their own growth. , analogously, hcv is able to subvert the host autophagic machinery and enhance viral growth, including rna replication, translation of the incoming viral rna genome and the release of infectious viruses ( table ) . two laboratories have independently demonstrated that hcv can activate autophagy via er stress-mediated induction of the upr and that upr-autophagy is required for hcv replication. , hcv ns , ns b, ns a and ns b have also been implicated in the induction of autophagy. , huang et al. showed that hcv induces er stress and inhibits the akttuberous sclerosis-mtor complex signaling pathway, resulting in autophagy activation. in contrast, shrivastava et al. demonstrated that hcv induces autophagy by stimulating beclin mrna expression and by activating mtor signaling, which may enhance hepatocyte growth. ke and chen demonstrated that in the context of hcv infection or without hcv infection, activation of the upr and autophagy downregulates innate immunity; in contrast, disruption of the upr and autophagy upregulates innate immunity. these results demonstrate that hcv hijacks upr and autophagy to stimulate viral rna replication by suppressing immune antiviral immunity. the upr-autophagy pathway represents a unique mode of reversible control in the innate immunity capacity in target cells. , subsequently, shrivastava et al. found that beclin or atg gene silencing in genotype a h strain hcv-infected immortalized human hepatocyte upregulates ifn signaling and isg expression, which are concurrent with apoptotic cell death. together, the results from these two groups suggest that autophagy may protect hcv-infected cells from the damage caused by excessive ifn antiviral stimulation, thereby promoting hcv rna replication. furthermore, a specific mode of autophagy, termed 'mitophagy', was recently reported to play a critical role in hcv replication and in the elimination of damaged mitochondria in infected cells in a parkin-dependent manner. knockdown of parkin and pink gene expression suppresses viral rna replication (table ). these results suggest a critical role for mitophagy in hcv replication. nevertheless, the molecular basis for the roles of autophagy and mitophagy in suppressing innate antiviral immunity in hcv infection has yet to be investigated. a recent study has demonstrated that many different families of rna viruses can target the autophagy network to promote viral growth. among these targets is irgm, which modulates autophagy by interacting with many autophagy-associated proteins, such as atg , atg and light chain c. strikingly, irgm knockdown impairs autophagy induced by many viruses, such as hcv, mev and hiv- , resulting in mitigated viral replication (table ). moreover, the c protein of mev, ns of hcv, and nef of hiv- were shown to induce autophagy by interacting with irgm. these results suggest that rna viruses have evolved to use a common strategy to target a critical molecule in autophagy to benefit their growth. microrna is a class of endogenous small non-coding rnas that bind to the -utr of target mrnas to control gene expression. micrornas also participate in innate and adaptive immunity response by binding to their complementarily mrnas and regulating the expression and translation of their target genes. for example, mir- regulates the host antiviral immune response by promoting type i ifn, whereas mir- enhances mrna degradation. mir- was shown to be upregulated in liver samples from hepatocellular carcinoma patients and in hcv-infected cells. during hcv infection, mir- expression is activated by the pkce/jnk/cjun and pkca/erk/cfos pathways. cjun and cfos form the ap- protein, which binds to the mir- promoter and activates mir- expression. mir- upregulation was shown to suppress the expression of myd and irak , which are two genes involved in the tlr signaling cascade, thereby repressing the production of type i ifn and isg and promoting hcv replication ( table ) . these results indicate that hcv usurps mir- to enhance its replication. likewise, mir- also increases the production of hiv, vsv and enterovirus by suppressing type i ifn production. the mechanisms by which viruses and cells coevolve and the tactics each party employs to establish the dynamic equilibrium are emerging as a fascinating area in hcv-host interaction research. previous studies that aimed to understand the hcv cell coevolution process have revealed several interesting aspects of virus-host cell interactions, such as er stress, upr, autophagy and innate antiviral immunity responses in hcv replication. , , further determining how the virus-cell interplay subsequently reshapes the host defense mechanisms and how virus replication is modulated in response to these cellular stresses will be important for gaining a complete understanding of the molecular basis of the hcv-host interaction in the pathogenesis of hcv infection. viral infection can trigger the ifn-mediated frontline host defense mechanism, including the production of a wide range of isgs to limit virus replication. many studies have also hitherto demonstrated that some of the identified isgs can exert broad antiviral activities against a diverse spectrum of viruses, whereas other isgs may have virus type-specific functions. the majority of studied isgs mediate ifn antiviral activities, acting as negative regulators in virus replication. strikingly, some isgs function as negative modifiers in the innate immune response, thereby promoting virus replication. nevertheless, the modes of action of most of the isgs remain unclear. although most identified isgs target individual steps of virus replication, some isgs seem to act at multiple stages of the virus replication cycle. determining the mechanisms by which these isgs function at different steps of the virus replication cycle would be interesting. current studies have indicated that different types of ifns may substantially overlap in mediating their innate immune response by activating the same set of isgs. however, the induction of some isgs may be unique to only one type of ifn, indicating the specificity in the induction of these isgs by ifns. clearly, different isgs can additively or synergistically suppress hcv replication, suggesting that inhibiting hcv replication depends on the combinatorial effects of individual isgs induced by ifn under the specific context of hcv infection. therefore, ifn-mediated suppression of hcv replication is not caused by a single isg but rather by the concerted actions of multiple isgs. moreover, gene expression profiling of hepatocytes from chronically hcv-infected patients treated with ifn has consistently shown differences between ifn-responders and ifnnon-responders. for instance, the levels of specific isgs, such as isg and usp , and viral sensors, such as rig-i, mda and laboratory of genetics and physiology- , are upregulated in patients with non-sustained virological responses compared with patients with sustained virological responses. , therefore, profiling gene expression for cytoplasmic viral sensors and related regulators involved in the innate antiviral immune response can identify new isgs that can be used as markers for predicting the clinical outcome of ifn therapy. in conclusion, the emergence of complexity in the highly pleiotropic type i ifn system in hcv infection reveals that the host has evolved to erect multiple checkpoints for anti-hcv innate immune surveillance to ensure that hcv is under tight control at all times, even when a single effector fails to confer antiviral activity, without drastically downgrading the overall efficacy of the ifn treatment. therefore, further deciphering which isgs and/or iegs are induced by ifns upon hcv infection and the specificity and action of these isgs and 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this study was supported by research grants from the ministry of science and technology ( - -b- - -my ) and academia sinica, taipei. this manuscript was edited for the english language by american journal experts (aje). attribution-noncommercial-noderivs . unported license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/ . / key: cord- - bb sp authors: halim, ashraf abdel; alsayed, badr; embarak, sameh; yaseen, taha; dabbous, salwa title: clinical characteristics and outcome of icu admitted mers corona virus infected patients date: - - journal: egyptian journal of chest diseases and tuberculosis doi: . /j.ejcdt. . . sha: doc_id: cord_uid: bb sp abstract middle east respiratory syndrome (mers) is a novel respiratory illness firstly reported in saudi arabia in . it is caused by a new corona virus, called mers corona virus (mers-cov). most people who have mers-cov infection developed severe acute respiratory illness. aim of the work this work is done to determine the clinical characteristics and the outcome of intensive care unit (icu) admitted patients with confirmed mers-cov infection. patients and methods this study included laboratory confirmed mers corona virus infected patients who were admitted into icu. it included ( . %) males and ( . %) females. the mean age was . ± . years. diagnosis was done by real-time reverse transcription polymerase chain reaction (rrt-pcr) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. clinical characteristics, co-morbidities and outcome were reported for all subjects. results the main symptoms among the included patients were: fever ( . %), cough ( . %), dyspnea ( . %), sore throat ( %), runny nose ( %), sputum ( %), headache ( . %), myalgia ( . %), chest pain ( . %), hemoptysis ( . %), nausea and vomiting ( . %), abdominal pain ( . %) and diarrhea ( . %). the presence of abdominal symptoms is significantly (p < . ) associated with bad prognosis. out of the included patients, patients ( . %) survived and patients ( . %) expired. there was a statistically significant difference in the duration of symptoms before hospitalization, mechanical ventilation and icu and total hospital stay between the expired group and survivors (p < . ). current smoking and smoking severity were statistically significantly (p < . ) higher in the expired group compared to survivors. also, there was a statistically (p < . ) significant positive correlation between mortality and smoking severity (r = . ). most of the expired patients presented with bilateral pulmonary infiltrates or unilateral infiltrates, but most of the survivors presented with normal radiology or increased bronchovascular markings, and this difference in the results was statistically highly significant (p < . ). there were statistically highly significant (p < . ) differences in the mean values of apache ii score ( . ± . vs . ± . ), sofa score ( . ± . vs . ± . ) and cpis ( . ± . vs . ± . ) between the expired group and survivors respectively. also, there was a statistically significant decrease in ph, pao , o saturation and hco (p < . ) among the expired group in comparison with the survivors, but no statistical difference regarding paco (p > . ). there was a statistically significant positive correlation between mortality and old age (r = . ), obesity (r = . ), diabetes mellitus (r = . ), renal failure (r = . ), chronic heart diseases ( . ), copd (r = . ), malignancy (r = . ), kidney transplantation (r = . ) and liver cirrhosis (r = . ) (p < . ). there was a statistically (p < . ) positive correlation between the number of associated co-morbidities and mortality (r = . ). conclusions most mers corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. the presence of abdominal symptoms may indicate bad prognosis. prolonged duration of symptoms before patients’ hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, copd, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of icu admitted mers corona virus infected patients. organization reported the first case of pneumonia caused by mers-cov in saudi arabia. all cases of mers have been linked to countries in and near the arabian peninsula. this virus has spread from ill people to others through close contact, such as caring for or living with an infected person. also, contact with the camels may be a potential source. however, there is no evidence of sustained spread in community settings. most mers patients developed a severe acute respiratory illness [ , ] . this work is done to determine the clinical characteristics and the outcome of icu admitted patients with confirmed mers-cov infection. this study included laboratory confirmed mers corona virus patients who were admitted into icu. consent was taken from the patients or their relatives. all studied cases were subjected to: ( ) full medical history. ( ) thorough clinical examination. ( ) calculation of body mass index (bmi): a bmi of > is considered obese according to who [ ] . ( ) routine laboratory investigations (complete blood count, kidney and liver functions, and blood sugar testing). ( ) radiological assessment: chest x-ray (posteroanterior and lateral views) and computed tomography (ct). ( ) arterial blood gases, including; ph, pao , sao , paco and hco . ( ) acute physiology and chronic health evaluation ii (apache ii) score, the sequential organ failure assessment score (sofa) and the clinical pulmonary infection score (cpis). ( ) throat swab (eurotubo, deltalab, rubı´, barcelona, spain), sputum, tracheal aspirate or bronchoalveolar lavage specimens were taken and stored at °c, and transported within h to the reference laboratories, where they were subjected to real-time reverse-tran scriptase-polymerase-chain-reaction (rrt-pcr) assays to test for mers-cov (altona diagnostics gmbh, hamburg, germany). for all patients, the results of rrt-pcr tests were confirmed by measuring cyclethreshold values for viral load [ ] . the statistical analysis was performed with the statistical package for the social sciences, version for windows (spss inc., chicago, il, usa). chi-square test was used to measure association. pearson's correlation test was used to measure correlation. values of p < . were considered statistically significant. in this study, there were ( . %) males and ( . %) females. the mean age was . ± . years. the main symptoms among the included patients were: fever ( . %), cough ( . %), dyspnea ( . %), sore throat ( %), runny nose ( %), sputum ( %), headache ( . %), myalgia ( . %), chest pain ( . %), hemoptysis ( . %), nausea and vomiting ( . %), abdominal pain ( . %), abdominal pain ( . %) and diarrhea ( . %). the mean duration of symptoms before seeking medical advice was . ± . days (table ) . the chest radiological findings among the included patients were as follows: ( %) normal chest radiology, ( . %) increased bronchovascular markings, ( . %) unilateral infiltrates, and ( . %) diffuse bilateral infiltrates. pneumothorax occurred in ( . %) ventilated patients, for of them it was unilateral and for other patients bilateral ( table ) . regarding the abgs findings in our study: mean ph: . ± . , mean pao : . ± . mmhg, mean o saturation: . ± . , mean paco : . ± . mmhg, and mean hco . ± . meq/l ( table ). the mean apache ii score was . ± . , the mean sofa score was . ± . and mean cpis was . ± . (table ) . regarding the presence of co-morbidity, ( . %) patients out of the included patients were associated with one or more co-morbidities and ( . %) patients were without co-morbidity ( table ) . all patients were admitted in icu and a mean duration of icu stay was . ± . days. regarding mechanical ventilation need, there were ( . %) ventilated patients and ( . %) patients without mechanical ventilation. for ventilated patients the mean duration of mechanical ventilation was . ± . days. the mean total hospital stay was ± . days ( table ) . according to the outcome of patients included in this study, there were two groups: ( ) the survivor group that included patients ( . %) with a mean age of . ± . years. ( ) the expired group that included patients ( . %) with a mean age of . ± . years. there was a highly statistically significant (p < . ) difference between the expired group and the survivors regarding the duration of mechanical ventilation (expired: . ± . , survivors: . ± . ), icu stay (expired: . ± . , survivors: . ± . ) and total hospital stay (expired: . ± . , survivors: . ± . ) ( table ) . in this study, in spite of a statistically significant (p < . ) increase in occurrence of corona virus infection among males in comparison with females ( . % males and . % females), there was no statistically significant difference (p > . ) in the mortality among them ( % among males and . % among females) (p > . ) ( table ) . current smoking and smoking severity were statistically significantly (p < . ) higher in the expired group compared to the survivors. non smoking and ex smoking were statistically significantly (p < . ) higher among survivors in comparison with the expired group. also, there was a statistically (p < . ) significant positive correlation between mortality and smoking severity (r = . ) ( table ) . in this study, there were no statistically significant differences between the expired and survivors groups as regards fever, cough, dyspnea, runny nose, sore throat, chest pain, hemoptysis, myalgia or headache (p > . ), but there was a statistically significant increase in frequency of nausea, vomiting and diarrhea among the expired group than that among the survivors (p < . ). also, there was a statistically significant increase in the duration of the symptoms before the hospitalization among the expired group than that among the survivors (p < . ) ( table ). there was a statistically significant (p < . ) positive correlation (r = . ) between mortality and prolonged duration of illness before hospitalization. our study showed that most of the expired patients presented with bilateral pulmonary infiltrates or unilateral infiltrates, but most of the survivors presented with normal radiology or increased bronchovascular markings, and this difference in the results was statistically highly significant (p < . ) ( table ) . there were statistically highly significant (p < . ) differences in the mean values of apache ii score ( . ± . vs . ± . ), sofa score ( . ± . vs . ± . ) and table ) . this study found a statistically significant decrease in ph, pao , o saturation and hco (p < . ) among the expired group in comparison with the survivors, but no statistical difference regarding paco (p > . ) ( table ) . as regards the correlation between mortality and specific co-morbidities, there was a statistically significant positive correlation between mortality and old age (r = . , smoking (r = . ), obesity (r = . ), diabetes mellitus (r = . ), renal failure (r = . ), chronic heart diseases ( . ), copd (r = . ), malignancy (r = . ), kidney transplantation (r = . ) and liver cirrhosis (r = . ) (p < . ), (table ) . there was a statistical (p < . ) positive correlation between the number of associated comorbidities and mortality (r = . ). in this study, there were ( . %) males and ( . %) females. this sex distribution showed a significant (p < . ) increase in the occurrence of corona virus infection among males, but there was no statistically significant difference (p > . ) in the mortality among them ( % among males and . % among females). our results agree with other workers' results [ , ] . the increased occurrence of corona virus infection among males can be explained by excess movement of the males through community with more exposure to the infected patients. in this study, there was high mortality ( patients ( . %) out of ) among corona virus infected patients and there was a statistically significant (p < . ) increase in mortality in middle and old aged patients in comparison to young patients (mean ages: . ± . among the expired group vs . ± . among survivors). also, there was a statistically significant (p < . ) positive correlation (r = . ) between age and mortality. other researchers found similar results [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this high mortality rate among corona virus infected old patients is explained by the high virulence of the new virus in the absence of previous immunity and already compromised immune system among old peoples. the most common symptoms among the included patients were: fever ( . %), cough ( . %), dyspnea ( . %), sore throat ( %), runny nose ( %), and sputum ( %). there were statistically significant differences between the expired and survivors groups regards fever, cough, dyspnea, runny nose, sore throat, chest pain, hemoptysis, myalgia or headache (p > . ), but the frequency of nausea, vomiting and diarrhea were statistically significant higher among the expired group in comparison to the survivors (p < . ). these findings could be explained by the fact that the presence of nausea, vomiting and diarrhoea leads to dehydration (due to poor oral intake and fluid loss) with its detrimental effects. the duration of symptoms before hospitalization, icu stay, mechanical ventilation and hospital stay in our studied patients were statistically significantly prolonged in the expired group compared to the survivors. also, there was a statistically significant (p < . ) positive correlation (r = . ) between mortality and prolonged duration of illness before hospitalization. other investigators found similar results [ ] [ ] [ ] [ ] . these results can be explained by a delay in diagnosis and management of corona infected patients' result in deterioration in their health status with more need for icu admission, mechanical ventilation, increased total hospital stay and poor outcome. also, al-tawfiq et al. [ ] found that corona patients in comparison with controls were more likely to be admitted to the intensive care unit ( % vs %; or, . ; p = . ) and to have a high mortality rate ( % vs %; or, . ; p < . ). the chest radiological findings among the included patients were as follows: ( %) normal chest radiology, ( . %) increased bronchovascular markings, ( . %) unilateral infiltrates, and ( . %) diffuse bilateral infiltrates. pneumothorax occurred in ( . %) ventilated patients, for of them it was unilateral and for other patients bilateral. most of the expired patients presented with bilateral pulmonary infiltrates or unilateral infiltrates, but most of the survivors presented with normal radiology or increased bronchovascular markings, and this difference in the results was statistically highly significant (p < . ). our findings agree with other investigators who detected similar results [ ] [ ] [ ] ] . these extensive radiological shadows are denoting extensive lung pathology with its detrimental effects on pulmonary gas exchange and general condition of the patients with more need for mechanical ventilation and are associated with bad prognosis. this study found a statistically significant decrease in ph, pao , o saturation and hco (p < . ) among the expired group in comparison to the survivors, but no statistical difference regarding paco (p > . ). our results are in accordance with that of reyes et al. [ ] who reported that one of the independent variables that were associated with high mortality was hypoxemia among sars patients caused by a corona virus. current smoking and smoking severity were statistically significantly (p < . ) higher in the expired group compared to the survivors. non smoking and ex smoking were statistically significantly (p < . ) higher among the survivors in comparison with the expired group. also, there was statistically (p < . ) significant positive correlation between mortality and smoking severity (r = . ). these results are in agreement with previous data that showed that smoking is a major determinant of morbidity and mortality in respiratory tract infection, especially in populations that smoke heavily [ ] [ ] [ ] . cigarette smoking is associated with a variety of alterations in cellular and humoral immune system functions. these alterations include a decreased level of circulating immunoglobulins, a depression of antibody responses to certain antigens, a decrease in cd + lymphocyte counts, an increase in cd + lymphocyte counts, depressed phagocyte activity and a decreased release of pro inflammatory cytokines. [ ] . smoking increases cd + lymphocytes in the cell mediated system and suppresses the host defense against infections [ ] . there was a statistically significant (p < . ) positive correlation between mortality and obesity (r = . ). several studies indicate that morbid obesity may be an independent risk factor for complications and mortality from mers corona virus infection [ ] [ ] [ ] [ ] . there are some explanations for these results. obesity can impede pulmonary function (reduced functional residual capacity and expiratory volume). a subsequent ventilation-perfusion abnormality may decrease ventilatory reserve and predispose the obese to respiratory failure after even mild pulmonary challenges [ , ] . obstructive sleep apnea is present in % of obese persons and is associated with systemic hypertension, pulmonary hypertension, and cor pulmonale [ , ] . obese persons are at an increased risk of developing pulmonary emboli and aspiration pneumonia. morbid obesity is associated with complications in the intensive care unit including prolonged stay, prolonged ventilation and death [ , ] . in addition to its effects on pulmonary function, obesity is frequently, but not always associated with diabetes, hypertension, hyperlipidemia, cardiovascular disease and high overall mortality [ ] . our work found a statistically significant positive correlation between mortality and diabetes mellitus (r = . ), renal failure (r = . ), chronic heart diseases ( . ), copd (r = . ), malignancy (r = . ), kidney transplantation (r = . ) and liver cirrhosis (r = . ) (p < . ), (table ) . there was a statistically (p < . ) positive correla-tion between the number of associated co-morbidities and mortality (r = . ). our results are in accordance with other workers' results [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . also, arabi et al. [ ] concluded that people with dm, renal failure, and chronic lung disease and immuno-compromised persons are considered to be at a high risk of severe disease from mers cov infection. also, our results are in agreement with previous data showing that patients who are immuno-compromised as a result of chronic diseases, malignancy, receiving treatment related to solid organ transplants and dm are at a high risk of respiratory tract infection-related complications including mortality [ , ] . immuno-compromised patients have an increased attention because of the documentation of prolonged viral shedding in critically ill patients, with the subsequent emergence of resistance to neuraminidase inhibitor drugs [ ] . renal failure population has many factors such as overwhelming uremia, neutrophil dysfunction, malnutrition, trace elements' deficiencies, iron overload, impaired glucose metabolism, and hyperparathyroidism, and the use of immunosuppressive drugs to treat and control underlying diseases leads to increased morbidity and mortality of infection [ ] . casqueiro et al. [ ] concluded that the infectious diseases are more frequent and/or serious in patients with dm, which potentially increases their morbidity and mortality due to many factors as a deficiency of the c component in dm, this reduction of c is probably associated with polymorphonuclear dysfunction and reduced cytokine response decrease secretion of inflammatory cytokines as interleukin- (il- ) and il- decreased mobilization of polymorphonuclear leukocytes, chemotaxis and phagocytic activity. cd t-lymphocytes and their response to antigens are impaired. most mers corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. the presence of abdominal symptoms may indicate bad prognosis. prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. also, old 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patients infectious complications in chronic kidney disease infections in patients with diabetes mellitus: a review of pathogenesis key: cord- -ulsz d authors: grabia, monika; markiewicz-Żukowska, renata; puścion-jakubik, anna; bielecka, joanna; nowakowski, patryk; gromkowska-kępka, krystyna; mielcarek, konrad; socha, katarzyna title: the nutritional and health effects of the covid- pandemic on patients with diabetes mellitus date: - - journal: nutrients doi: . /nu sha: doc_id: cord_uid: ulsz d covid- related restrictions aimed at curbing the spread of the coronavirus result in changes in daily routines and physical activity which can have a negative effect on eating and health habits. the aim of the study was to assess the impact of the covid- pandemic on patients with diabetes and their nutrition and health behaviours. a survey conducted in july included individuals with type (n = ) and (n = ) diabetes mellitus from poland. to assess nutritional and health behaviours, an online questionnaire covering basic information, anthropometric data, and details regarding physical activity, eating, and hygiene habits was used. almost % of all respondents with type and diabetes mellitus (dm) stated that their disease self-management had significantly improved. over % of all participants declared that they had started eating more nutritious and regular meals during the covid- pandemic. enhanced hygiene, in particular, during the period, a statistically significant increase in hand sanitiser use was reported by respondents ( % vs. %, p < . ). the study demonstrated that the pandemic had a significant impact on the behaviour of patients with dm. improved disease self-management and making healthy, informed food and hygiene choices were observed. since the emergence of sars cov- , a new coronavirus known as severe acute respiratory syndrome coronavirus- , at the end of , the related disease called covid- has spread rapidly around the world [ ]. from march , a "cordon sanitaire" was formed around poland and on march the state of epidemic including home confinement was introduced in the country. at the time of writing, the peak number ( ) of daily cases in poland occurred on june . at the time of commencing the study, there had already been , infections and deaths in poland [ ] . from july to july , the period when the survey was conducted, the following regulations were in force in the country: nose-and-mouth coverings in confined public areas; closure of primary and secondary schools, and institutions of higher education; food and drink establishments were operational with enhanced sanitary measures in place (nose-and-mouth coverings required when not at the table, disinfecting tables, keeping a minimum distance of . m between patrons); gyms and swimming pools were reopened on june ; individuals were allowed to socialise indoors in small groups; in most places, access to specialist medical care was provided at outpatient clinics [ ] . diabetes mellitus (dm), a metabolic disorder of various etiologies, is characterised by chronic hyperglycaemia and disturbances in insulin secretion or its activity, or both. type diabetes mellitus (t dm) is an insulin-dependent, multifactorial autoimmune disease which results in degradation of the beta cells of islets of langerhans, which causes impaired insulin production and secretion [ ] . patients with t dm require intensive treatment involving administration of exogenous insulin in the form of multiple daily injections or as continuous subcutaneous infusion of insulin using personal insulin pumps [ ] . type diabetes mellitus (t dm) is described as a condition of insulin resistance with relative insulin deficiency, commonly caused by qualitative and quantitative secretory defects [ ] . initial treatment of this type of diabetes involves administration of medication to achieve glycaemic stability. ultimately, many patients require insulin therapy because of progressive failure of the beta cells and development of complications [ ] . regulations imposed to curb the transmission of covid- are likely to have an impact on daily routines, including exercise and eating habits. for people with diabetes mellitus, exercise is an integral part of their disease management [ , ] . patients may also experience increased mental stress caused by the unpredictability of the situation and as a result of social isolation [ ] . this may lead to excessive consumption of products rich in simple carbohydrates which can alleviate stress, since their ingestion stimulates the production of serotonin and enhances the mood. consuming such products is associated with an increased risk of obesity and complications of covid- [ ] . a lack of physical activity, poor food choices, and heightened psychological stress may have a detrimental effect on the immune system, which may not produce a proper response when exposed to the new virus [ ] . according to a report by the centers for disease control and prevention, individuals with t dm and t dm may be at an increased risk for severe illness from covid- due to susceptibility to lung infection, which is a consequence of dm-related metabolic disturbances and immunosuppression [ , ] . an in vitro study demonstrated that chronic hyperglycaemia changed the innate immune system, thereby acting on chemotaxis, phagocytosis, but also on bactericidal activity of neutrophils and macrophages [ ] . according to the american diabetes association (ada), currently available data regarding covid- is not comprehensive enough to show whether individuals with dm, particularly well-controlled dm, are more prone to developing the disease as compared with the general population. however, if patients are not metabolically balanced, they may experience more considerable blood glucose fluctuations which can cause a number of diabetes-related complications. these complications may make patients with dm more susceptible to contracting covid- and other viral infections due to the body's limited ability to fight them [ ] . the aim of the present study was to assess the impact of the covid- pandemic on the nutritional and health behaviours of patients with dm. the study, which collected data via an online survey, was conducted among polish patients with dm, with a median age of years (lower to upper quartile, - years old) between july and july via private facebook groups of polish diabetes societies. the main study inclusion criterion was completion of the survey section regarding diabetes, which was a prerequisite for completing the remaining sections. responses from individuals residing abroad, women with gestational diabetes, and individuals in quarantine were rejected. each participant was informed of the anonymity and confidentiality of the survey and its purpose. each respondent was allowed to complete the survey only once and exit it at any time, which would result in unsaved responses. participants confirmed their voluntary consent for study participation by completing the survey. they could not provide names and personal data. parents of young children completed the questionnaire on their behalf. the study was conducted in full compliance with national regulations (consent of bioethical commission of the medical university of bialystok no. r-i- / / ) and the declaration of helsinki. the questionnaire (see appendix a) consisted of three sections. the first section contained questions regarding type of diabetes mellitus the participant suffered from, their gender, age, body height and weight, level of education and place of residence. anthropometric measurements were self-reported. the body mass index (bmi) is a measure used to determine nutritional status. it was calculated using the following formula: body weight in kg divided by height in meters squared. in paediatric patients (under years of age), bmi was interpreted in relation to norms contained in clinical growth charts. the th, th, and th centiles correspond to the limits of underweight, overweight, and obesity, respectively [ ] . for adults, the following who approved standards were applied: underweight (below . kg/m ), normal ( . - . kg/m ), overweight ( . - . kg/m ) and obese (above . kg/m ) [ ] . the second section contained questions relating to disease duration, type of treatment received, and result of the hba c test performed within three months of questionnaire completion. the last section contained questions regarding physical activity, eating behaviours, and hygiene habits such as stress level, daily screen time, and sleep routine. as for physical activity, respondents could indicate whether and how the type of activity they participated in had changed. the activities included dancing, fitness, swimming, running, gym, cycling, gymnastics, and walking. the frequency of exercise sessions could be described by respondents as "i don't exercise", " - times per week", " - times per week", " and more times per week". assessment of changes in eating habits was based on the consumption of the following products: coffee, convenience food, dairy products, delivery meals, eggs, energy drinks, fresh bread, fresh fish, fresh vegetables, frozen fish, grain products, homemade bread, nuts, red meat, salty snacks, sweet beverages, sweet snacks, water, and white meat. as for hand washing and sanitiser use, the respondents could indicate the following situations: "after coming home", "after using the toilet", "before cooking", "after contact with animals", "after leaving public transport", and "after leaving shops". the number of hours the respondents spent sleeping fell into the following categories: "under - h", " - h", or "over h". as for time spent in front of the computer or tv, the respondents could select from the following: "less than h a day", " - h a day", " - h a day", or " or more hours a day". stress levels could be classified as follows: "low", "medium", "high", and "very high". the questionnaire was based on previously published work of other authors with modifications reflecting the situation under investigation and study cohort [ , ] . furthermore, questionnaires originally published in foreign languages were translated into polish and assessed by a polish native speaker to exclude bias in interpretation. the questionnaire was pretested on a small sample of respondents from the target population to allow for subsequent eradication of formal and substantive errors. statistical analysis was performed using statistica software (version ; statsoft inc., krakow, poland). normal distribution of the studied variables was checked using the shapiro-wilk test. the mann-whitney u test was used when data was not symmetrically distributed. relationships between qualitative features (e.g., periods before and during the covid- pandemic) were evaluated using the chi-square independence test. in justified cases, yates' correction was used. prior to conducting the survey, a minimum sample size was calculated, which was used for estimating of the number of people who should be tested in order for intended results to be obtained with a specified confidence level (α = . ) and a maximum error ( %). the p-value < . was considered to be statistically significant. due to the possibility of confounding between variables, additional characteristics of the results for all items in which it occurred were included in the supplementary material. the results section contains outcomes relating to all study participants (adults and children together). due to the fact that / of respondents with t dm were children, we presented the results obtained in this group of patients in a separate subsection. the outcomes obtained, after the exclusion of children under years of age from the study group, were consistent with the results for the entire study cohort. characteristics of the study cohort are presented in table . the majority of respondents were individuals with type diabetes mellitus ( %) and women ( %). among survey participants, % of patients with t dm used personal insulin pumps, while % used insulin pens. as for patients with t dm, % used insulin pens and % oral drugs. none of the participants reported simultaneous use of insulin injections and oral medication. among the completed questionnaires, % were filled in by parents of children with t dm. a normal bmi was observed in % of the study cohort, overweight in %, obesity in %, and underweight in %. the majority of study participants ( %) had a university degree, % of respondents were unemployed, % worked in the office, % worked from home, and the remainder were students ( %). there were no participants who were quarantined prior to questionnaire completion. over half ( %) of the respondents lived with their parents, % lived with a partner, and the remaining % resided alone. the study participants were asked if their disease self-management had improved during the covid- pandemic. among the participants, % individuals with t dm declared that their disease control had deteriorated, % stated that it had improved, while % did not report any changes. as for body weight, % of study participants reported an increase in body weight during the pandemic, % reported an increase of ≤ kg, and % reported > kg. only % of patients with dm stated that their body weight did not change. the remaining % of patients reduced their body weight, % by ≤ kg and % by > kg. prior to the covid- pandemic, % of study participants did not engage in any physical activity, % participated in physical activity - times a week, % - times a week, and % over times a week as compared with the period during the pandemic ( %, %, %, and %, respectively). there were statistically (p < . ) significant differences among the above variables (see table ). figure shows the type of physical activity chosen before and during the covid- pandemic. a statistically significant increase in walking was demonstrated ( % vs. %, p < . ) while a statistically significant decrease in participation in gymnastics, swimming, and dancing (p < . each), gym and fitness classes (p < . each) was observed. additional characteristics between type of disease and gender are presented in supplementary table s ). nutrients , , x for peer review of abbreviations: type diabetes mellitus (t dm), type diabetes mellitus (t dm). differences between "before" and "during" the covid- period were evaluated by the chi-square test (* p < . ). differences between the type of physical activity "before" and "during" the covid- pandemic. differences between "before" and "during" the covid- pandemic were evaluated by the chi-square test (* p < . and ** p < . ). when asked if they started eating more healthily during the pandemic, % of respondents declared improvements in their dietary habits. survey results demonstrated that % of respondents had started eating more regular meals, in particular main meals. the same total percentage of the study cohort declared that they had started preparing their own meals (see supplementary table s ). differences between the type of physical activity "before" and "during" the covid- pandemic. differences between "before" and "during" the covid- pandemic were evaluated by the chi-square test (* p < . and ** p < . ). ( ) % ( ) % ( ) % ( ) city (≥ k inhabitants) % ( ) % ( ) % ( ) % ( ) % ( ) % ( ) up to years % ( ) % ( ) % ( ) % ( ) % ( ) % ( ) hba c (%) d . ( . - . ) . ( . - . ) . ( . - . ) values are expressed as median, lower, and upper quartile (me (q -q )) or percentage and number of respondents (% (n)). abbreviations: type diabetes mellitus (t dm), type diabetes mellitus (t dm). differences between "before" and "during" the covid- period were evaluated by the chi-square test (* p < . ). when asked if they started eating more healthily during the pandemic, % of respondents declared improvements in their dietary habits. survey results demonstrated that % of respondents had started eating more regular meals, in particular main meals. the same total percentage of the study cohort declared that they had started preparing their own meals (see supplementary table s ). the results revealed that % of respondents consumed one to two meals per day before the pandemic as compared with % during the pandemic, % vs. % consumed three to four meals a day, and % vs. % had more than five meals a day. no statistical significance was demonstrated between the pre-covid- period and the period during the pandemic for any of the above variables, in any of the study participants. more than % of respondents admitted that frequency of snacking between meals increased during the pandemic. consumption of selected food products during the covid- pandemic is presented in figure and the breakdown by the type of diabetes and gender is included in supplementary table s . the most marked increase in intake was revealed for the following products: water ( %), fresh fruit ( %), vegetables ( %), and grain products ( %). the most substantial decrease in consumption was recorded for the following products: fast food ( %), convenience food ( %), salty snacks ( %), delivery meals ( %), red meat ( %), and sweet snacks ( %). nutrients , , x for peer review of ( %), vegetables ( %), and grain products ( %). the most substantial decrease in consumption was recorded for the following products: fast food ( %), convenience food ( %), salty snacks ( %), delivery meals ( %), red meat ( %), and sweet snacks ( %). the study revealed a statistically significant increase in hand sanitiser use during the covid- pandemic (p < . ). prior to the pandemic, % of study participants never used hand sanitisers, % used them sometimes, while % used them very often. use of sanitising solutions increased during the study revealed a statistically significant increase in hand sanitiser use during the covid- pandemic (p < . ). prior to the pandemic, % of study participants never used hand sanitisers, % used them sometimes, while % used them very often. use of sanitising solutions increased during the pandemic, i.e., % of respondents declared that they used them very often, while % reported using them sometimes. a statistically significant relationship between frequent hand washing/antibacterial agent use before and during the covid- pandemic was found (figure ). the biggest, statistically significant differences were observed in hand washing after leaving shops ( % vs. %, p < . ), public transport ( % vs. %, p < . ), and after returning home ( % vs. %, p < . ). additional characteristics regarding the type of disease and gender are presented in supplementary table s ). nutrients , , x for peer review of figure . frequency of hand washing/antibacterial agent use before and during the covid- pandemic. differences between "before" and "during" the covid- period were evaluated by the chi-square test (* p < . ). a statistically (p < . ) significant dependence between the period before the start of the covid- pandemic and the time during the pandemic was demonstrated in the number of hours spent in front of the tv or computer. prior to home confinement caused by covid- , only % of study participants spent more than h per day in front of the tv or computer, while % spent - h, % spent - h, and the remainder ( %) had less than h of screen time (during the pandemic it was %, %, %, and %, respectively). the dependence between the period before the covid- pandemic and that during the pandemic, and the number of hours of sleep was also statistically significant (p < . ), i.e., % of respondents declared sleeping more than h per day prior to the pandemic, with the figure reaching % during the pandemic. the percentage of study participants sleeping less than the recommended number of hours decreased, i.e., % vs. % of respondents slept between and h per day, and % vs. % slept less than h per day. statistically significant (p < . ) differences were observed in stress levels before, at the beginning of the covid- pandemic, and at the time of completing the questionnaire. average stress levels increased at the start of the pandemic and returned to nearly pre-pandemic levels at the time of survey completion, as shown in figure . detailed characteristics of the above variables (screen time, sleep routine, and stress levels) in relation to the type of diabetes and gender are presented in supplementary tables s -s . differences between "before" and "during" the covid- period were evaluated by the chi-square test (* p < . ). a statistically (p < . ) significant dependence between the period before the start of the covid- pandemic and the time during the pandemic was demonstrated in the number of hours spent in front of the tv or computer. prior to home confinement caused by covid- , only % of study participants spent more than h per day in front of the tv or computer, while % spent - h, % spent - h, and the remainder ( %) had less than h of screen time (during the pandemic it was %, %, %, and %, respectively). the dependence between the period before the covid- pandemic and that during the pandemic, and the number of hours of sleep was also statistically significant (p < . ), i.e., % of respondents declared sleeping more than h per day prior to the pandemic, with the figure reaching % during the pandemic. the percentage of study participants sleeping less than the recommended number of hours decreased, i.e., % vs. % of respondents slept between and h per day, and % vs. % slept less than h per day. statistically significant (p < . ) differences were observed in stress levels before, at the beginning of the covid- pandemic, and at the time of completing the questionnaire. average stress levels increased at the start of the pandemic and returned to nearly pre-pandemic levels at the time of survey completion, as shown in figure . detailed characteristics of the above variables (screen time, sleep routine, and stress levels) in relation to the type of diabetes and gender are presented in supplementary tables s -s . in children under years of age (n = ), statistically significant (p < . ) differences were found between the frequency of physical activity before and during the pandemic (see supplementary table s ). the percentage of young respondents who practiced gymnastics ( % vs. %, p < . ), swimming ( % vs. %, p < . ), and running ( % vs. %, p > . ) decreased. however, participation in walking ( % vs. %, p < . ) and cycling ( % vs. %, p > . ) increased. prior to the pandemic, over % of surveyed children consumed one to two meals per day while the remainder had five or more meals. during the covid- pandemic, more than % of respondents consumed three to four meals, % had five or more meals while fewer than % reported having one to two meals per day. survey results demonstrated a statistically significant relationship between the number of meals consumed per day before the pandemic and the number of meals eaten during the pandemic (p < . ) (see supplementary table s ) . study results revealed several improvements in dietary habits of the youngest respondents during the pandemic (see supplementary table s ). over % of children reported drinking increased amounts of water during the pandemic, whereas % declared that their intake of grain products, fresh bread, fruit, and vegetables had increased. additionally, % of all respondents reported a higher intake of dairy products, while % declared increased consumption of eggs. it is worth noting that the intake of fresh fish also grew, i.e., % of children reported increased consumption. additionally, decreased consumption of red meat, fast food, and snacks (both salty and sweet) was observed. a statistically significant increase in hand sanitiser use by the youngest respondents during the covid- pandemic (p < . ) was observed. prior to the pandemic, % of all study participants never used hand sanitisers, % used them sometimes, while % used them very often. use of sanitising solutions increased during the pandemic, i.e., % of all respondents declared that they used them very often while % reported using them sometimes. a statistically significant relationship between frequent hand washing/antibacterial agent use before and during the covid- pandemic was found (see supplementary table s ). the biggest statistically significant differences were observed in hand washing after leaving shops ( % vs. %, p < . ) and public transport ( % vs. %, p < . ). statistically significant differences were found when screen time of the youngest respondents was analysed (p < . . survey results demonstrated that prior to the pandemic, the majority of in children under years of age (n = ), statistically significant (p < . ) differences were found between the frequency of physical activity before and during the pandemic (see supplementary table s ). the percentage of young respondents who practiced gymnastics ( % vs. %, p < . ), swimming ( % vs. %, p < . ), and running ( % vs. %, p > . ) decreased. however, participation in walking ( % vs. %, p < . ) and cycling ( % vs. %, p > . ) increased. prior to the pandemic, over % of surveyed children consumed one to two meals per day while the remainder had five or more meals. during the covid- pandemic, more than % of respondents consumed three to four meals, % had five or more meals while fewer than % reported having one to two meals per day. survey results demonstrated a statistically significant relationship between the number of meals consumed per day before the pandemic and the number of meals eaten during the pandemic (p < . ) (see supplementary table s ) . study results revealed several improvements in dietary habits of the youngest respondents during the pandemic (see supplementary table s ). over % of children reported drinking increased amounts of water during the pandemic, whereas % declared that their intake of grain products, fresh bread, fruit, and vegetables had increased. additionally, % of all respondents reported a higher intake of dairy products, while % declared increased consumption of eggs. it is worth noting that the intake of fresh fish also grew, i.e., % of children reported increased consumption. additionally, decreased consumption of red meat, fast food, and snacks (both salty and sweet) was observed. a statistically significant increase in hand sanitiser use by the youngest respondents during the covid- pandemic (p < . ) was observed. prior to the pandemic, % of all study participants never used hand sanitisers, % used them sometimes, while % used them very often. use of sanitising solutions increased during the pandemic, i.e., % of all respondents declared that they used them very often while % reported using them sometimes. a statistically significant relationship between frequent hand washing/antibacterial agent use before and during the covid- pandemic was found (see supplementary table s ). the biggest statistically significant differences were observed in hand washing after leaving shops ( % vs. %, p < . ) and public transport ( % vs. %, p < . ). statistically significant differences were found when screen time of the youngest respondents was analysed (p < . . survey results demonstrated that prior to the pandemic, the majority of young respondents ( %) spent - h a day in front of the tv or computer, almost one fifth had less than h of screen time a day, whereas only % spent or more hours watching tv or using electronic devices. during the covid- pandemic, all respondents declared spending more than h a day in front of the tv or computer, % - h, % - h, and % more than h. there was a statistically significant (p < . ) increase in the number of hours devoted to sleep, i.e., % of children slept for up to h and % for more than h per day before the covid- pandemic as opposed to % and %, respectively, during the pandemic. survey results revealed that prior to the pandemic, more than one third of the youngest respondents suffered moderate or high levels of stress. at the start of the pandemic, the majority of participants experienced moderate levels of stress, while over % suffered high stress levels. at the time of questionnaire completion, fewer than % of all respondents declared suffering very high levels of stress and over % of all children evaluated their stress levels as low. comparison of periods before, at the beginning of the covid- pandemic, and the time of completing the questionnaire revealed statistically significant (p < . ) relationships (see supplementary table s ). the present study demonstrated that the pandemic had a significant impact on the nutritional and health behaviour of patients with dm. frequency of the consumption of both recommended and non-recommended products changed. the covid- pandemic also contributed to more frequent hand washing and increased use of antibacterial agents. due to limited access to medical care, some patients with dm may have experienced difficulty managing their disease. however, the study showed that the majority of respondents were metabolically balanced (median of hba c . % to . %). only % felt that they were less able to control their disease. it is worth noting that % of all surveyed patients started to monitor their disease more rigorously and over % improved their diet by eating more regular, nutrient-dense meals. a negative effect of the covid- pandemic and related government-imposed restrictions on movement was limited outdoor activity. as compared with the pre-covid- period, the study demonstrated an increase in the percentage of individuals not practicing any physical activity ( % vs. %) and those exercising one to two times per week ( % vs. %). the number of people who took up walking increased one and a half times ( % vs. %). this can probably be explained by the fact that from april , residents of poland were allowed to leave home only in the following circumstances: commuting to and from work, voluntary involvement in the fight against the covid- pandemic, and to address matters necessary for everyday living. outdoor activity was to be kept to a minimum with only walks allowed, as public parks, boulevards and playgrounds were closed. at the time the survey was conducted, the gradual process of relaxing restrictions started. swimming pools and gyms re-opened a month before the commencement of the study but were not extensively patronised [ ] . when the results of our investigation were compared with those of an italian study by renzo et al., a similar decrease in the frequency (three to four times per week) of physical activity before and during the lockdown in italy was noted ( . % vs. . %, our study % vs. %). the authors also observed a decline in interest in sports such as fitness classes, running, and gym workout [ ] . nachimuthu et al. published a brief survey conducted among indian patients with dm which revealed that % of respondents monitored their diet regularly and engaged in physical activity at home [ ] . the present study revealed that the percentage of individuals consuming five or more meals increased during the covid- pandemic ( % vs. %). an increase in the number of meals consumed ( % vs. %) was also observed by ammar et al. who investigated eating habits of healthy people in different countries [ ] . the results obtained by scarmozzino also confirmed that home confinement caused by covid- resulted in increased food consumption for around % of respondents [ ] . the problem with body weight management experienced by almost % of participants of our study may have been caused by a higher number of meals consumed per day, and thus increased calorie intake. a polish study, conducted during the lockdown, found that individuals with a higher bmi, particularly obese people, were at a heightened risk of adverse dietary changes (increased food consumption and snacking) [ ] . a study by renzo et al. investigated whether there were differences in the consumption of selected products among italians. as in our study, they observed an increase in the consumption of certain foods such as grain products, hot beverages, eggs, dairy products, fresh bread, white meat, and fresh vegetables [ ] . scarmozzino also observed an increase in consumption of fresh vegetables [ ] . several eating patterns emerged among the youngest participants of our study. on the one hand, over % of the surveyed children declared drinking increased amounts of water and nearly half reported that their consumption of grain products, dairy products, fruit, and vegetables had increased. almost a third of the surveyed children declared a higher consumption of fresh fish. on the other hand, decreased consumption of red meat, fast food, as well as snacks (both salty and sweet) was also reported. white meat and dairy products are beneficial foods which contribute to the prevention of t dm [ , ] . increased consumption of fruits and vegetables is associated with a reduced risk of t dm not only because they contain dietary fibre, essential vitamins and minerals, but also due to the antioxidant and anti-inflammatory effects of their components which include vitamins b and c, carotenoids, and polyphenols [ , ] . similar to the results of our study, a decrease in the consumption of salty snacks, sweet beverages, and delivery food was observed in an italian survey [ ] . a high intake of such products may contribute to unstable diabetes. in patients with dm, a high salt intake may carry a risk of microalbuminuria, particularly in overweight individuals. sodium retention and blood volume in dm can cause the progression of diabetic microangiopathy [ , ] . excessive sugar consumption may accelerate the development of t dm and sugar-sweetened drinks (ssbs) can be particularly harmful to children genetically predisposed to t dm [ ] . high consumption of fructose, ssbs, and high-fructose corn syrup contributes to an epidemic of insulin resistance, visceral obesity, and t dm [ , ] . bleich and wang studied consumption patterns of sugar-sweetened beverages among adult americans with t dm. they observed high consumption of these products in young adults and in low-income individuals [ ] . the glycated haemoglobin (hba c) reflects mean glycaemia over the period of approximately three months and is a useful retrospective marker of blood glucose levels as there is an association between blood glucose levels and mean glycaemia, and the risk of developing chronic diabetic complications [ ] . a healthy lifestyle involving consumption of regular, nutrient-dense meals prepared at home with quality ingredients brings many beneficial effects, in particular a reduction in hba c level. poor metabolic control is associated with frequent dining out, particularly in fast food establishments, and consumption of high-fat products and snacks between meals [ , ] . our study demonstrated a statistically significant increase in the use of sanitising agents and frequency of hand washing, particularly after coming home and leaving shops or public transport. however, a surprising finding of the study was that the percentage of people sanitising their hands before preparing food decreased. this can be explained by the fact that sanitising solutions are provided at shop entrances and their use is monitored, whereas being home gave individuals a sense of security, and therefore the need for using an antibacterial agent was removed. mental stress activates neuroendocrine processes that affect blood glucose levels by releasing cortisol, endorphins, and growth hormone [ ] . this is of adaptive importance for a healthy body but in patients with dm, post-stress hyperglycaemia can exacerbate the disease. moreover, negative emotions can reduce motivation to adhere to the prescribed treatment and to follow dietary recommendations, which can contribute to poor glycaemic control and increase susceptibility to infections [ ] [ ] [ ] . the results of our study revealed that at the beginning of the covid- pandemic, stress levels in patients with dm increased sharply (high % vs. %, very high % vs. %). at the time the survey was conducted, stress levels were starting to return to pre-pandemic levels. this may have been related to an increase in respondents' awareness that taking care of their health helps to strengthen the immune system through proper nutrition and compliance with hygiene rules. our study has several limitations. the study was retrospective and allowed us to only estimate the impact of the covid- pandemic, some respondents could not fully or accurately recall information they were required to provide. body weight and height were not measured by a qualified individual, they were self-reported and may not have been accurate. the male subgroup was not sufficiently representative, although it is a common problem in voluntary research. this study was conducted among the inhabitants of one country (poland). performing such a study on populations of other countries would allow for a more comprehensive understanding of eating habits and hygiene behaviours of patients with dm. another limitation was the fact that changes in eating habits were estimated and not correlated with data regarding the sale of particular product groups. the study could only collect data via an online questionnaire since no unauthorised persons were allowed to enter healthcare facilities due to pandemic-related restrictions. the aim of the investigation was to describe eating and health habits that occurred during the covid- pandemic, and therefore these results should not be interpreted in the context of long-term effects. despite the fact that the sample size was small (n = ), the power of the test reached %, with a confidence level (α = . ). to our knowledge, this is the first nutritional behaviour study conducted among patients with dm during the covid- pandemic. a significant effect of the pandemic on the behaviour of patients with dm was observed. the surveyed patients reported improved disease self-management and making healthy, informed food choices and hygiene habits. the success of nutritional therapy in patients with dm depends on the selection of appropriate food products by individuals, and therefore short reports in the form of questionnaires regarding patients' nutritional behaviours and their adherence to the recommended dietary regimen should be part of routine nutritional assessment performed by healthcare providers. the results reported in the present study should be used to promote public health during the covid- pandemic. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : characteristics of the subgroups, type of physical activity before and during the covid- pandemic, table s : characteristics of the subgroups, healthy/regularity of meal consumption during the covid- pandemic, table s : characteristics of the subgroups, variation in food intake during the covid- pandemic, table s : characteristics of the subgroups, frequency of hand washing/antibacterial agent use before and during the covid- pandemic, table s : characteristics of the subgroups, duration of screen time before and during the covid- pandemic, table s : table s . characteristics of the subgroups, sleep length before and during the covid- pandemic, table s : characteristics of the subgroups, stress level distribution before, at the beginning of the pandemic and at the time of survey completion, table s : frequency of physical activity before and during the covid- pandemic in children population, table s : number of meals per day in children before and during the covid- pandemic, table s : variation in food intake during the covid- pandemic in children population, table s : frequency of hand washing/antibacterial agent use before and during the covid- pandemic in children population, table s : stress level distribution before, at the beginning of the pandemic and at the time of survey completion in children population. main meals/yes, most meals q have you started cooking more meals yourself? no/yes q has the frequency of eating certain foods changed during the pandemic? i eat more often/ i eat less often/ no change/ i don'tever eatthese products • salty snacks (crisps, crackers, bread sticks, etc.) • sweet snacks (cakes, cookies, chocolate bars) meat (beef, pork) • white meat (chicken, turkey) • fresh fish • frozen fish • eggs • dairy products (milk, yoghurt, cottage cheese) products (rice, pasta etc epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study coronavirus outbreak map (sars-cov- ) polish ministry of health. current rules and restrictions definition, diagnosis and classification of diabetes mellitus and its complications. part : diagnosis and classification of diabetes mellitus. provisional report of a who consultation natural history, and prognosis diabetes in covid- : prevalence, pathophysiology, prognosis and practical considerations physical activity/exercise and diabetes: a position statement of the american diabetes association eating habits and lifestyle changes during covid- lockdown: an italian survey relationship between diabetes and respiratory diseases-clinical and therapeutic aspects neutrophil and lymphocyte function in patients with diabetes mellitus how covid- impacts people with diabetes the height-, weight-, and bmi-for-age of polish school-aged children and adolescents relative to international and local growth references obesity: preventing and managing the global epidemic effects of covid- home confinement on eating behaviour and physical activity: results of the eclb-covid international online survey coping with diabetes during the covid - lockdown in india: results of an online pilot survey covid- and the subsequent lockdown modified dietary habits of almost half the population in an italian sample dietary choices and habits during covid- lockdown: experience from poland dietary protein consumption and the risk of type diabetes: a dose-response meta-analysis of prospective studies meat consumption, diabetes, and its complications effect of increasing fruit and vegetable intake by dietary intervention on nutritional biomarkers and attitudes to dietary change: a randomised trial fruit and vegetable intake and type diabetes: epic-interact prospective study and meta-analysis the twin white herrings: salt and sugar +sugar intake is associated with progression from islet autoimmunity to type diabetes: the diabetes autoimmunity study in the young consumption of sugar-sweetened beverages is associated with components of the metabolic syndrome in adolescents consumption of sugar-sweetened beverages among adults with type diabetes use of glycated haemoglobin (hba c) in the diagnosis of diabetes mellitus: abbreviated report of a who consultation eating behavior among type diabetic patients: a poorly recognized aspect in a poorly controlled disease food habits are related to glycemic control among people with type diabetes mellitus the relationship between stress and diabetes mellitus empirically derived patterns of perceived stress among youth with type diabetes and relationships to metabolic control disease-related distress, self-care and clinical outcomes among low-income patients with diabetes this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank the participants of the study. the authors declare no conflict of interest. key: cord- -m spgpyu authors: liu, ying; song, jia-wei; lin, jian-yu; miao, ran; zhong, jiu-chang title: roles of microrna- in cardiovascular fibrosis and related diseases date: - - journal: cardiovasc toxicol doi: . /s - - - sha: doc_id: cord_uid: m spgpyu fibrotic diseases cause annually more than , deaths worldwide, where of the majority accounts for cardiovascular fibrosis, which is characterized by endothelial dysfunction, myocardial stiffening and reduced dispensability. micrornas (mirs), small noncoding rnas, play critical roles in cardiovascular dysfunction and related disorders. intriguingly, there is a critical link among mir- , cardiovascular fibrosis, sirtuin (sirt ) and angiotensin-converting enzyme (ace ), which was recently identified as a coreceptor for sars-cov and a negative regulator of the rennin-angiotensin system. mir- overexpression appears to exacerbate the angiotensin ii-mediated loss of autophagy and increased inflammation, apoptosis, extracellular matrix deposition, cardiovascular fibrosis and dysfunction by modulating the sirt -elabela-ace , lgr -β-catenin, tgfβ-ctgf and pten-pi k-akt signaling pathways. more importantly, the inhibition of mir- has proautophagic, antioxidant, anti-inflammatory, anti-apoptotic and antifibrotic effects. clinical and experimental studies clearly demonstrate that mir- functions as a crucial hallmark of fibrogenesis, cardiovascular injury and dysfunction. additionally, the mir- level is related to the severity of hypertension, atherosclerosis, atrial fibrillation, acute myocardial infarction and heart failure, and mir- expression is a risk factor for these diseases. the mir- level has emerged as an early-warning biomarker cardiovascular fibrosis, and targeting mir- is a novel therapeutic approach against progression of cardiovascular dysfunction. therefore, an increased understanding of the cardiovascular roles of mir- will help the development of effective interventions. this review summarizes the biogenesis of mir- ; regulatory effects and underlying mechanisms of mir- on cardiovascular fibrosis and related diseases; and its function as a potential specific biomarker for cardiovascular dysfunction. cardiovascular fibrosis, a vital cause of heart failure (hf), refers to the development of scar tissue in the injured heart and blood vessels due to an aberrant wound-healing response to injury or insult, and arises from enhanced inflammatory processes, uncontrolled cell proliferation and excessive oxidative stress [ ] [ ] [ ] . established fibrosis is a hallmark of chronic disease progression and a key contributor to cardiovascular stiffness and injury of the heart and vessels, ultimately leading to hf and vascular dysfunction [ ] [ ] [ ] [ ] . since wound healing and fibrotic responses to myocardial and vascular injury are multifactorial processes, current therapies that specifically target factors that contribute to only cardiovascular disease pathogenesis offer limited overall anti-fibrotic efficacy [ ] . fibrosis-induced cardiovascular remodeling and injury are growing problems for improving strategies to prevent the development of fibrosis and reduce the mortality of hf [ ] [ ] [ ] . micrornas (mirs) are non-protein-encoding rnas with a length of - bases, that degrade mrna or bind the ′ noncoding regions (ucrs) of their target genes with incomplete complementarity and regulate gene expression at the [ , , ] . mir- may be a sensitive and strong predictor of cardiovascular injury and failure in patients with fibrosis. although the biological functions of mir- are still largely unknown, mir- has been shown to regulate cardiovascular inflammation, autophagy, apoptosis, oxidative stress, fibrosis and dysfunction [ , , [ ] [ ] [ ] . mir- exerts different and even opposing regulatory effects on the cardiovascular system and binds its target genes to control the levels of pro-inflammatory and fibrotic factors [ , [ ] [ ] [ ] . notably, mir- functions as a risk biomarker of cardiovascular fibrosis and appears to be a direct participant in the development of cardiovascular diseases, including hypertension, atherosclerosis, hf, myocardial infarction (mi) and atrial fibrillation ( fig. ) [ , [ ] [ ] [ ] [ ] [ ] . in this review, we highlight the developments and latest advances concerning the biogenesis and biological effects of mir- signaling and the regulatory roles of mir- in cardiovascular fibrosis and dysfunction. mir- is located on chromosome , from which the ~ . kb pre-mir- is generated, after which pre-mir- is processed to mature mir- , which moves from the nucleus to the cytoplasm [ ] . the promoter region of mir- is highly conserved and located approximately kb upstream of the conserved stem-loop sequences in mir- , which include large nuclear receptor factor-binding sequences, such as the tata-box and ccaat-box [ ] . notably, sirtuin (sirt ), a mir- target gene, is important regulator of cardiovascular fibrosis, remodeling and dysfunction [ ] . intriguingly, sirt , sirt , apelin (apln), apelin receptor (aplnr) and forkhead box o (foxo ) were identified as mir- target genes (fig. ) . the predicted interactions between mir- and the abovementioned targets were analyzed through the mirs target gene prediction website (https ://www.micro rna.org). both mir- - p and mir- - p are widely expressed in endothelial cells (ecs), cardiomyocytes (cms), cardiac fibroblasts (cfs), adipocytes, vascular smooth muscle cells (vsmcs) and adventitial fibroblasts (afs) in the cardiovascular system (table ; fig. ) [ , , , ] . recently, administration of a mir- inhibitor was shown to reduce the key transforming growth factor-β (tgf-β)-induced fibrotic signaling pathway, promotes collagen synthesis and stimulates fibrinogenesis, consequently resulting in the accumulation of fibroblasts and extracellular matrix (ecm), in hypertension and cardiovascular diseases [ , , , ] . in our previous study, we observed cardiovascular fibrosis, systolic dysfunction, cardiovascular remodeling and dysfunction in rats after transverse aortic constriction (tac) or angiotensin ii (ang ii) stimulation, and these central roles of mir- in hf, hypertension, mi, atherosclerosis and atrial fibrillation. mir- has been shown to promote apoptosis, inflammation, fibrosis, pathological hypertrophy and remodeling in the cardiovascular system; decrease the lvef, lvfs and cardiac contractility; and increase nt-probnp and ros generation, leading to arrhythmia and cardiovascular dysfunction. there-fore, mir- can cause cardiovascular fibrosis and heart dysfunction, ultimately resulting in hypertension, atherosclerosis, mi and hf. mi myocardial infarction, hf heart failure, ros reactive oxygen species, lvfs left ventricular fractional shortening, lvef left ventricular ejection fraction effects were associated with increased levels of mir- - p, and reduced levels of sirt , elabela (ela), angiotensin-converting enzyme (ace ) and phosphorylated adenosine '-monophosphate-activated protein kinase (ampk) ( table ) [ , , ] . in uric acid-treated hk- tubular epithelial cells, treatment with mir- mimics markedly increased nlrp inflammasome activation by increasing the levels of brca / brca -containing complex (table ) [ ] . intriguingly, mir- - p was found to accelerate ischemic reperfusion (i/r) injury-induced renal damage, through increased fibrosis, inflammation, and apoptosis, and promote renal dysfunction [ ] . in a rat model of renal i/r injury, supplementation with a mir- inhibitor prevented apoptosis and reactive oxygen species (ros) generation through modulating phosphatase and tensin homolog (pten)/ phosphoinositide -kinase (pi k)/ akt pathway activity (table ; fig. ) [ ] . conversely, levels of the proinflammatory cytokines tumor necrosis factor-α (tnf-α) and the number of macrophages (f / + cd b + ) were reduced in mir- knockout (ko) mice, suggesting that mir- deficiency inhibited inflammation and tissue damage [ ] . in l cells pretreated with oleic acid, treatment with a mir- inhibitor mitigated lipid accumulation and inflammation through repressing the tlr /myeloid differentiation primary response gene (myd )/ nuclear factor-кb (nf-кb) p pathway (table ) [ ] . mir- levels were elevated in a mouse model of lung fibrosis, and this increase was linked with enhanced inflammation and pulmonary vascular remodeling [ ] . importantly, mir- functions as a predictive fig. schematic diagram of the activities, and target genes of mir- and potential mir- -binding sites in these target genes. a schematic diagram showing the mechanism of mir- . b apln, aplnr, sirt , sirt and foxo were identified as mir- target genes by the use of the publicly available bioinformatics tool microcosm targets and microrna.org. the predicted interactions between mir- and the abovementioned targets were shown and analyzed with a mir target gene prediction website (https ://www. micro rna.org). c the sequence of mir- is highly conserved in humans, mice and rats. foxo forkhead box o , sirt sirtuin , sirt sirtuin , apln apelin, aplnr apelin receptor rat afs pretreated with ang ii mir- inhibitor ↓tgfβ /ctgf ↓lgr /β-catenin ↓apoptosis, cellular migration [ ] patients with hf and ckd __ ↑mir- levels ↑tgf-β ↑renal fibrosis [ ] renal tubular epithelial hk- cells mir- mimics ↑nlrp inflammasome ↑inflammation [ ] sd rats with renal ischemia-reperfusion mir- inhibitor ↑pten/pi k/akt ↓ros production [ ] mir- ko mice __ ↑tnf-α ↑f / + cd b + [ ] l cells pretreated by oleic acid mir- inhibitor ↓tlr /myd ↓nf-кb p [ ] ang ii induced hypertensive mice __ ↑mir- , bach- , mcp- ↓ho- ↑collagen deposition and fibrosis [ ] nrvms mir- mimics ↓foxo ↑calcineurin, anp, bnp ↑hypertrophy, remodeling [ ] younger patients with hypertension __ ↑mir- levels ↑endothelial dysfunction ↓cat- [ ] patients with bav __ ↑ mir- ,tgf-β , vegf levels ↑valvular dysfunction ↑apoptosis; ecm remodeling [ ] patients with hf __ ↑mir- , nt-probnp levels ↑heart dysfunction [ ] ligation of lad in sd rats anti-mir- ↓infarct size ↑lvfs; lvef [ ] ogd-induced h c cells mir- mimics ↑akt/gsk- β/β-catenin ↓apoptosis, epicardial expansion ↓epithelial-mesenchy ↑cardiac dysfunction [ ] ogd-induced h c cells mir- inhibitor ↑jnk/p mapk ↓mtor; apoptosis ↑cell viability [ ] mice with atrial fibrillation mir- inhibitor ↑ bcl-x ↓ caspase- ; p-erk / [ ] marker of fibrosis and appears to stimulate the inflammation and oxidative stress in the heart, blood vessels and liver [ , , , , ] . mir- has been found to increased levels of the fibrotic factors collagen α , collagen α and tgf-β and the proinflammatory factor monocyte chemoattractant protein- (mcp- ) [ ] . collectively, these results reveal that mir- - p plays critical roles in the controlling cardiovascular fibrosis, inflammation, apoptosis, myocardial remodeling and dysfunction by activating the tgfβ -ctgf, myd /nf-кb and pten/pi k/akt signaling pathways (fig. ). cardiovascular fibrosis contributes to cardiac or arterial stiffening, endothelial dysfunction, wall thickening, reduced dispensability, and coronary atherosclerotic stenosis, leading to hemodynamic damage, the destruction of cardiovascular tissues and hf [ , [ ] [ ] [ ] [ ] . a variety lgr leucine-rich repeat-containing g protein-coupled receptor , ros reactive oxygen species, mtor mammalian target of rapamycin, ecm extracellular matrix, ampk adenosine '-monophosphateactivated protein kinase, gata gata binding protein , sirt sirtuin , ela elabela, erk extracellular signal-regulated kinase, xiap x-linked inhibitor of apoptosis protein, foxo forkhead box o , bach- btb and cnc homology , ho- heme oxygenase , mcp- monocyte chemotactic protein of alterations in the interstitial myocardial collagen network, macrophages, cms, cfs, ecs and vascular cells are closely linked with fibrillar alteration in the heart. at the molecular and cellular levels, cardiovascular fibrosis is linked with reduced nitric oxide release, increased ros generation, the activation of transcription factors, the stimulation of proinflammatory and profibrotic pathways, increased collagen deposition and ecm remodeling [ , , [ ] [ ] [ ] [ ] . inflammation and oxidative stress seem to be positively associated with remodeling processes, ultimately contributing to cardiac dilation and dysfunction [ , ] . mirs participate in regulating cell proliferation, migration, differentiation, and apoptosis and function as crucial determinants and biomarkers of cardiovascular fibrosis [ , , , , ] . in particular, mir- has been implicated in the progression of fibrosis and acts as a circulatory biomarker in hypertension and hf [ , , , , [ ] [ ] [ ] [ ] [ ] . we previously demonstrated that mir- overexpression exacerbated the loss of autophagy and increased cellular migration, apoptosis, extracellular matrix deposition mediated by angiotensin ii by modulating the sirt -ela-ace , leucine-rich repeat-containing g-protein-coupled receptors (lgr )-β-catenin, and tgfβ-ctgf signaling pathways (table ; fig. ) [ , ] , indicating that mir- inhibition is a promising therapeutic strategy for cardiac fibrosis and dysfunction. surprisingly, the levels of circulating exosomal mir- were positively associated with cardiac dysfunction in patients with hf with a reduced left ventricle (lv) ejection fraction (ef) and elevated levels of nt-probnp [ ] . a recent study demonstrated that mir- was highly expressed in cultured h c cells under hypoxia/reoxygenation, and overexpression of mir- by recombinant adeno-associated infection significantly augmented the apoptosis of h c cells [ ] . in contrast, inhibition of mir- attenuated pathological cardiac remodeling and decreased cell apoptosis in myocardial ischemic injury [ ] . in cultured neonatal rat ventricular myocytes (nrvms), overexpression of mir- by mir- mimics increased the size of cms and hypertrophic gene expression, but downregulated the expression of anti-hypertrophic genes upon ang ii stimulation (table ) [ ] . intriguingly, bioinformatics analysis and luciferase reporter assays revealed that mir- directly targets foxo (fig. ) . moreover, mir- was found to decrease foxo levels but promote activation of the calcineurin signaling pathway, implying that mir- accelerates the development of cardiovascular hypertrophy and remodeling via modulation of the foxo -calcineurin pathway (table ; fig. ) [ ] . thus, mir- inhibition might be a promising field for the therapeutic approach in cardiovascular fibrosis and related diseases. hypertension is a complex cardiovascular syndrome, while its pathogenesis is closely related to abnormalities in mirs and the renin-angiotensin system (ras) [ , , , , , [ ] [ ] [ ] [ ] . mir- plays a key role in the regulation of cardiovascular fibrosis and endothelial function during hypertension (table ) [ , , , , ] . intriguingly, stimulation with ang ii markedly increased the expression of mir- in nrvms [ ] . our previous study demonstrated that administration of a mir- inhibitor effectively prevented the loss of autophagy and increased cellular proliferation, migration, apoptosis and cardiovascular fibrosis induced by ang ii via modulation of the sirt -ela-ace , lgr /β-catenin and tgfβ -ctgf-nfat signaling pathways (table ; fig. ) [ , ] . intriguingly, mir- was identified as an independent predictor of renovascular hypertension linked with increased collagen deposition and cardiovascular fibrosis. administration of a mir- mimic led to a marked increase in btb and cnc homology transcription factor (bach- ) expression in mesangial cells [ ] . in contrast, inhibition of mir- blunted expression of the proinflammatory factors bach- and mcp- and the profibrotic factor collagen α in aged mice with ang ii-induced hypertension (table ; fig. ) [ ] . these data suggested a crosstalk between mir- and the ras in the control of fibrosis, inflammation and remodeling during hypertension. mir- has been implicated in occurrence and development of hypertension and hf [ , , , , [ ] [ ] [ ] . elevated levels of plasma mir- were found in patients with essential hypertension, especially younger patients with hypertension [ ] . overexpression of mir- resulted in endothelial dysfunction by suppressing the expression of cationic amino acid transporter , which destroyed the endothelial structure, increasing risk factors for cardiovascular dysfunction during hypertension (table ; fig. ) [ ] . mir- levels were also significantly upregulated in patients with hypertension, indicating the role of mir- as predictive hazard factor in cardiovascular homeostasis and disorder [ , , [ ] [ ] [ ] . mir- plays a critical role in the development of the bicuspid aortic valve (bav) and aortic dilation by promoting the apoptosis of ecs and cardiovascular remodeling via the activation of tgf-β-vegf signaling pathways (table ; fig. ) [ ] . intriguingly, bioinformatics analysis and luciferase reporter assays revealed that mir- directly targets sirt and attenuates the level of sirt (fig. ) [ ] . our previous work revealed that sirt overexpression strikingly promotes cardiac levels of pampkα and ace and decreases fibrotic gene levels of ctgf, tgfβ , collagen i and collagen iii, leading to alleviation of ang ii-induced pathological hypertrophy, myocardial fibrosis, cardiac ultrastructural injury and dysfunction in hypertensive rats [ ] . mir- , which is expressed in a variety of cardiovascular system cell types, can regulate gene expression at the posttranscriptional level and plays an important role in the occurrence and development of cardiovascular diseases (fig. ) . furthermore, mir- was implicated in cardiorenal injury and vascular remodeling in spontaneously hypertensive in african green monkey and rat models of renovascular hypertension by regulating lipoprotein metabolism and cholesterol homeostasis [ , ] . taken together, these findings show that the inhibition of mir- exerts protective effects against cardiovascular fibrosis and dysfunction during hypertension. atherosclerosis is a chronic inflammatory vascular disease characterized with abnormal cholesterol metabolism, an abnormal inflammatory response, endothelial dysfunction, apoptosis and vsmc proliferation [ , , ] . mir- is a well-known marker of cardiovascular events and a good predictor of atherosclerosis. mir- levels were shown to be upregulated in patients with atherosclerotic lesions, and serum mir- levels were positively correlated with atherosclerotic severity [ , , ] . in fact, mir- promotes proinflammatory factors and oxidant injury in the liver and cardiovascular system [ , , ] . thus, potential usage of mir- antagonism in treatment of inflammatory injury may represent a novel therapeutic approach for atherosclerosis. moreover, apoptosis in ecs is the basis of pathophysiology of atherosclerosis [ , , ] . in apolipoprotein e deficient mice fed a high-fat diet, mir- expression was increased in aortic ecs. in addition, mir- inhibitor strikingly suppressed ox-ldl-induced apoptosis of human aortic ecs, suggesting the anti-apoptotic role of mir- inhibition under pro-atherogenic conditions [ ] . further evidence revealed that the x-linked inhibitor-of-apoptosis protein (xiap) is directly targeted and suppressed by mir- in ecs. more importantly, xiap knockdown diminished the pro-apoptotic effect of mir- , indicating that xiap is a prominent target that mediates mir- -mediated regulation of ec apoptosis (fig. ) [ ] . notably, mir- has been identified as a possible risk factor for recurrent cardiovascular events in acs and cerebral ischemic events [ ] . mir- was found to mainly originate from circulating ecs and monocytes and was increased in patients with ami compared with patients with unstable angina [ ] . thus, mir- may serve as a positive regulator of ec apoptosis induced by atherogenic factors, suggesting its use as a noninvasive diagnostic biomarker and potential treatment for therapeutic interventions for atherosclerosis, acs and other atherosclerotic heart diseases. myocardial fibrosis and remodeling are the key causes of hf. physiological, pharmacological and clinical studies have demonstrated that abnormal mirs and activation of the ras are important mediators of the progression of hf, which is the end stage of cardiovascular disease and leads to systolic and/or diastolic dysfunction in the heart [ , , , , ] . these effects include blood stagnation in the venous system and insufficient perfusion in arteries in the cardiovascular system. myocardial fibrosis was associated with higher long-term mortality in patients with hf [ , ] . recently, we demonstrated that ang ii triggered increased apoptosis and reduced of sirt , ela and ace levels in rat afs, which were exacerbated by the overexpression of mir- - p via mir- - p mimic but were prevented by administration of mir- - p inhibitor [ ] . notably, mir- mimics significantly reversed the ela-mediated beneficial effects on apoptosis and autophagy imbalance in rat afs, accompanied with decreased ampk phosphorylation and elevated mtor phosphorylation level (table ; fig. ) [ ] . in addition, pretreatment with apelin- inhibited ang ii-induced cardiac fibrosis in rats with hf by blocking collagen production and activating the tgf-β-ctgf signaling pathway [ , ] . importantly, the upregulation of ace mediated by sirt and apelin is a key mechanism by which the ras, cardiovascular fibrosis and hf are inhibited [ , , , ] . ace is a negative receptor of ras and a coreceptor for severe respiratory syndrome coronavirus (sars-cov ), which causes entry for the novel coronavirus disease (covid- ) pneumonia [ , ] . the major contributor to progressively worsened systemic manifestations of covid- was due to imbalance of the ang-( - )/ang ii through a loss of functional tissue ace , leading to severe inflammatory storm [ ] . in pre-clinical models and clinical populations, recombinant human ace use in patients with hf led to improvement of pathological hypertrophy, fibrosis, inflammation, oxidant injury and heart dysfunction, reflecting ace -mediated cardiac protective impacts [ , , , ] . the apelin/ace pathway has been implicated in hypertension and hf, making it a promising therapeutic target [ , , , , ] . recently, mir- - p was shown to be increased in a rat model of postinfarction hf, and the proapoptotic role of mir- - p was revealed [ ] . furthermore, in addition to its role as a clinical risk factor, mir- has been implicated as a prognostic biomarker and increases nt-probnp level. an elevated level of mir- was significantly associated with right ventricular dysfunction and could predict all-cause and cardiovascular mortality, especially improving hf risk stratification of patients with reduced lvef (table. ) [ ] . however, lower fluctuating serum mir- - p levels were observed in patients with acute hf, indicating that mir- - p may be linked with acute cardiac decompensation through treatment courses [ ] . thus, mir- may be an independent risk factor for the development of chronic and acute hf. circulating mir- - p is considered a promising novel diagnostic biomarker for patients with acute mi [ , [ ] [ ] [ ] [ ] , as levels of mir- - p have been found to be obviously increased in patients with acute mi [ ] [ ] [ ] [ ] . circulating mir- - p expression was increased in ami patients at , , , and h after mi compared to non-ami controls and displayed a trend similar to that of ctni levels concentrations in ami patients [ ] . ischemic cell death leads to the formation of damaged tissue in the heart, which is replaced with a fibrotic scar produced by fibroblasts and myofibroblasts, eventually resulting in scarring, cardiac fibrosis, and remodeling [ , [ ] [ ] [ ] [ ] . fibrosis during mi can be classified as reactive or replacement fibrosis. importantly, reactive fibrosis at the infarct border zone and the remote uninjured myocardium, leads to chamber compliance alteration, increased ventricular stiffness and compromised cardiac output [ , ] . replacement fibrosis refers to scar formation, a critical process for preventing bursting of the ventricular wall after ischemic insult during mi [ , ] . in addition, an elevated ratio between circulating mir- - p/mir- b is a specific early prognostic biomarker in acute mi [ ] . therefore, mir- is emerging as a prognostic biomarker of infarct size and cardiac dysfunction in mi. overexpression of mir- remarkably attenuated lycium barbarum polysaccharide (lbp)-mediated protective activity in hypoxia-injured h c cells, while the inhibition of mir- accelerated lbp-mediated protective activity [ ] . surprisingly, the lbp-induced activation of the phosphorylated extracellular signal-regulated kinase (erk) and ampk signaling pathways was attenuated by mir- overexpression and accelerated by mir- suppression [ ] . an in vivo investigation revealed that, the administration of anti-mir- decreased infarct size and improved cardiac function in mi rats associated with improvements to the lvef and left ventricular fractional shortening, indicating that mir- inhibition may have potential for the prevention and treatment of acute mi (table ; fig. ) [ ] . overexpression of mir- by mir- mimics inhibited cellular viability and promoted the apoptosis of cms, while mir- knockdown promoted cell viability and inhibited apoptosis [ ] . mir- - p significantly reversed the beneficial effects of salvianolate on cardiac injury in rats with increased bax and caspases- expression and decreased of bcl- expression [ ] . the inhibition of mir- participates in the regulation of cm injury during oxygen and nutrient deprivation in acute mi through the activation of akt/mtor signaling and inactivation of the jnk/p mapk pathway. in contrast, the activation of mir- prevents cell survival and promotes cellular apoptosis during myocardial ischemia/reperfusion. furthermore, mir- has been confirmed to be involved in cardiac repair, impairing epicardial expansion, and epithelial-mesenchymal transition through activation of the akt/ gsk- β/β-catenin signaling pathway, eventually resulting in a rapid decline in cardiac function (table ; fig. ) [ , ] . mir- was highly expressed in the border zone of the ischemic myocardium in acute mi pigs without ventricular fibrillation compared with pigs in a sham group. mir- was decreased in platelets and increased in pbmcs from the acute mi pigs [ ] . these results suggest that mir- inhibition in controlling cm proliferation, apoptosis and cardiac dysfunction in mi. mir- has been shown to predict the risk of atrial fibrillation [ , ] . atrial fibrillation is associated with atrial structural, electrical, and contractile remodeling and atrial fibrosis, which is denoted by the deposition of ecm. fibrous scarring and interstitial fibrosis in the heart decrease cardiac contractility, subsequently interfering with the normal electrical function of the heart, resulting to occurrence and development of arrhythmia [ , ] . surprisingly, compared with those in mice in sham-operated and control groups, mir- levels were significantly increased in male c bl/ mice with atrial fibrillation, and this increase was significantly decreased by transfection with the mir- inhibitor [ ] . cm viability was increased and the cm apoptosis rate was significantly decreased following mir- transfection. in addition, mir- inhibition upregulated expression of the anti-apoptotic protein bcl-x and downregulated expression of the pro-apoptotic protein caspase- and the phosphorylation of erk / (table ; fig. ) [ ] , that implicating mir- in the molecular mechanisms underlying the proliferation and apoptosis of cms in atrial fibrillation. mir- appears to be a direct participant in the deteriorating cardiovascular system by promoting inflammation, oxidative stress, apoptosis and ecm deposition in various cardiovascular diseases. clinical and experimental studies clearly support the physiological and pathophysiological roles of mir- in cardiovascular fibrosis and dysfunction. mir- overexpression exacerbates the loss of autophagy and increased inflammation, apoptosis, extracellular matrix deposition, cardiovascular fibrosis and dysfunction mediated by ang ii. most importantly, the inhibition of mir- can act as antifibrotic, anti-apoptotic, anti-inflammatory, antioxidant and proautophagic functions. targeting the mir- has emerged as a prewarning biomarker and novel therapeutic approach against progression of cardiovascular fibrosis and related diseases and an increased understanding of cardiovascular actions of the mir- will help to develop effective interventions. microrna- aggravates angiotensin ii-mediated apoptosis and autophagy imbalance in rat aortic adventitial fibroblasts via the modulation of sirt -elabela-ace signaling the elabela-apj axis: a promising therapeutic target for heart failure targeting the nlrp inflammasome to treat cardiovascular fibrosis fibrosis in chronic liver disease: an update on diagnostic and treatment modalities cholesterol impairs hepatocyte lysosomal function causing m polarization of macrophages via exosomal mir- - p ascending aortic adventitial remodeling and fibrosis are ameliorated with apelin- in rats after tac via suppression of the mirna- and lgr -β-catenin signaling the liver-specific micro-rna mir- : biology and therapeutic potential integration of microrna mir- in hepatic circadian gene expression the effect of renal dysfunction and haemodialysis on circulating liver specific mir- pharmacological interventions for heart failure in people with chronic kidney disease gender differences in hypertension lncrna anril promotes nlrp inflammasome activation in uric acid nephropathy through mir- - p/brcc axis mir- regulates cell apoptosis and ros by targeting dj- in renal ischemic reperfusion injury rat models injured liver-released mirna- elicits acute pulmonary inflammation via activating alveolar macrophage tlr signaling pathway the mir- inhibition alleviates lipid accumulation and inflammation in nafld cell model mrnas and mir-nas in whole blood associated with lung hyperplasia, fibrosis, and bronchiolo-alveolar adenoma and adenocarcinoma after multi-walled carbon nanotube inhalation exposure in mice hypertension exaggerates renovascular resistance via mir- -associated stress response in aging microrna- promotes cardiomyocyte hypertrophy via targeting foxo the circulating level of mir- is a potential risk factor for endothelial dysfunction in young patients with essential hypertension specific circulating microrna signature of bicuspid aortic valve disease liver-specific microrna- as prognostic biomarker in patients with chronic systolic heart failure resident fibroblast expansion during cardiac growth and remodeling salvianolate blocks apoptosis during myocardial infarction by downregulating mir- - p lncrna malat knockdown alleviates oxygen-glucose deprivation and reperfusion induced cardiomyocyte apoptotic death by regulating mir- upregulation of mir- is associated with cardiomyocyte apoptosis in atrial fibrillation myocardial interstitial fibrosis in heart failure: biological and translational perspectives cardiac fibroblasts and cardiac fibrosis: precise role of exosomes cardiac fibroblast diversity vascular fibrosis in aging and hypertension: molecular mechanisms and clinical implications antigen-based nano-immunotherapy controls parasite persistence, inflammatory and oxidative stress, and cardiac fibrosis, the hallmarks of chronic chagas cardiomyopathy, in a mouse model of trypanosoma cruzi infection myocardial fibrosis: biomedical research from bench to bedside the ace /apelin signaling, micrornas, and hypertension endothe-lial dysfunction in diabetes and hypertension: role of micrornas and long non-coding rnas micrornas in the development of left ventricular remodeling and postmyocardial infarction heart failure mir- marks the differences between subcutaneous and visceral adiposetissuesand associates with the outcome of bariatric surgery exosomal micro-rna- mediates obesity-related cardiomyopathy through suppressing mitochondrial adp-ribosylation factor-like downregulation of mir- attenuates hypoxia/ reoxygenation (h/r)-induced myocardial cell apoptosis by upregulating gata- the diagnostic values of circulating mirnas for hypertension and bioinformatics analysis angiotensin converting enzyme : sars-cov- receptor and regulator of the renin-angiotensin system-celebrating the th anniversary of the discovery of ace non-coding rna in the pathogenesis, progression and treatment of hypertension differential expression of hypertensionassociated micrornas in the plasma of patients with white coat hypertension therapeutic potential of blood flow mimetic compounds in preventing endothelial dysfunction and atherosclerosis reciprocal regulation between sirt and mir- controls liver metabolism and predicts hepatocarcinoma prognosis the sirtuin prevents angiotensin ii-mediated myocardial fibrosis and injury by targeting ampk-ace signaling signature microrna expression profile is associated with spontaneous hypertension in african green monkey correlations between interleukin- expression and hypertensive kidney injury in a rat model of renovascular hypertension roles of growth differentiation factor in atherosclerosis and coronary artery disease association of circulating micro-rna- with presence and severity of atherosclerotic lesions microrna- promotes endothelial cell apoptosis by targeting xiap: therapeutic implication for atherosclerosis micrornas as prognostic markers in acute coronary syndrome patients-a systematic review elevated plasma mirna- , - - p, - , - , and - during early period of acute coronary syndrome are derived from peripheral blood mononuclear cells role of the ace /angiotensin - axis of the renin-angiotensin system in heart failure pyr-apelin- is a negative regulator of angiotensin ii-mediated adverse myocardial remodeling and dysfunction dynamic changes of serum microrna- - p through therapeutic courses indicates amelioration of acute liver injury accompanied by acute cardiac decompensation differential microrna expression and regulation in the rat model of post-infarction heart failure circulating mir- - p as a potential novel biomarker for diagnosis of acute myocardial infarction circulating mir- - p/mir- b ratio is a specific early prognostic biomarker in acute myocardial infarction circulating mir- - p and mir- - p are promising novel biomarkers for diagnosis of acute myocardial infarction specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart lycium barbarum polysaccharides protects h c cells from hypoxia-induced injury by down-regulation of mir- microrna- regulates caspase- and promotes the apoptosis of mouse cardiomyocytes integrins and integrin-related proteins in cardiac fibrosis long non-coding rna uca relieves cardiomyocytes h c injury aroused by oxygen-glucose deprivation via declining mir- conflict of interest all the authors declare that they have no conflict of interest. key: cord- -o hvuwvq authors: harvey, david j. title: analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: an update for – date: - - journal: mass spectrom rev doi: . /mas. sha: doc_id: cord_uid: o hvuwvq this review is the sixth update of the original article published in on the application of maldi mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of . general aspects such as theory of the maldi process, matrices, derivatization, maldi imaging, arrays and fragmentation are covered in the first part of the review and applications to various structural typed constitutes the remainder. the main groups of compound that are discussed in this section are oligo and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals. many of these applications are presented in tabular form. also discussed are medical and industrial applications of the technique, studies of enzyme reactions and applications to chemical synthesis. © wiley periodicals, inc. mass spec rev : – , . this review is a continuation of the six earlier ones in this series (harvey, (harvey, , (harvey, , (harvey, , (harvey, , (harvey, , on the application of matrix-assisted laser desorption/ionization mass spectrometry (maldi) mass spectrometry (ms) to the analysis of carbohydrates and glycoconjugates. it is intended to bring the coverage of the literature to the end of . although the intention is to be a comprehensive as possible, there is an increasing tendency to publish maldi data in supplementary information. because of the ever-increasing number of papers and journals, it has not been possible to check all supplementary information and, consequently, papers that do not refer to maldi in the main text may well have been omitted. also omitted are papers that simply report the mass of glycoproteins and those concerned with nucleotides and nucleosides: the latter compounds, although containing carbohydrates are considered to be different types of compound. maldi continues to be a major technique for the analysis of carbohydrates; figure shows the year-by-year increase in papers reporting use of the technique for the period - . because the review is designed to complement the earlier work, structural formulae, etc. that were presented earlier are not repeated. however, a citation to the structure in the earlier works is indicated by its number with a prefix designating the review containing the structure (i.e., /x refers to structure x in the first review and /x to a structure in the second). a large number of books and review articles directly concerned with, or including maldi analysis of carbohydrates and glycoconjugates, have been published during the review period. those of a general nature are listed in table ; those concerned with specific carbohydrate types are listed in the appropriate sections. details of the maldi process are still not fully understood and several investigators have attempted to obtain greater understanding. although not all of these studied have involved carbohydrates, they are included here because maldi processes for different compounds are likely to be similar. one of the commonly accepted models for the formation of analyte ions in maldi-ms assumes a primary ionization of the matrix, for example, by photoionization leading, among other things, to stable protonated and deprotonated matrix ions. peptide and protein ions are then envisaged as being formed by secondary proton transfer reactions in the expanding matrix plume. this model has been checked experimentally by hillenkamp et al. ( ) by comparing the yield of positive to negative ions of three peptides (bradykinin, angiotensin i and fibrinopeptide a) and six matrices a-cyano- -hydroxycinnamic acid (chca, / ), , dihydroxybenzoic acid (dhb, / ), -azo- -thiothymine (att, / ), -nitroaniline ( -na, / ), -amino- -nitro- -picoline (anp, ) and -aminoquinoline ( -aq, ), differing in gas-phase basicity by about kj/mol. it was shown that the observed ion yields cannot be explained by any single and consistent set of parameters such as gas-phase basicity or acidity of the analyte and matrix. it was concluded that the existing simple model needs be modified to fully explain the experimental findings. liu et al. ( a) have used synchronized dual-polarity maldi ms to demonstrate incoherent production of positive and negative matrix ions. in both positive and negative ion modes, matrix ions were found to appear from thin, homoge-neous dhb matrix films at different threshold laser fluences. the presence of singly charged molecular matrix ions suggests that the existence of dhb ion-pairs may not be a prerequisite in the maldi process. photoelectrons induced by the laser excitation may assist the production of negative dhb ions, as shown in experiments conducted with stainless steel and glass substrates. at high laser fluences, the relative yield of positive and negative matrix ions remained constant when homogeneous matrix films were used, but the yield fluctuated significantly with inhomogeneous crystal morphology. this result was also inconsistent with the hypothesis that matrix ion-pairs are essential primary ions. thus, results from both low and high laser fluences suggest that the production of positive and negative matrix ions in maldi may occur via independent pathways. the same authors have examined the initial ionization reaction in maldi based on the appearance of photoelectrons. the threshold laser fluence for the ejection of photoelectrons from dhb, sinapinic acid ( / ) and , , trihydroxyacetophenone (thap, / ) on stainless steel targets was found to be . , . , and . mj/cm , respectively. these values were considerably lower than those for maldi ions, indicating that the electron detachment probably precedes other ionization reactions. the stainless steel target was thought to general review with a section on glycolipids - glycan analysis by mass spectrometry short review, maldi and esi, applications to n-linked glycans (sekiya & iida, ) deciphering carbohydrate structures by ion mobility ms short general review of glycomics and ion mobility. applications to flavonoids, gags and glycoproteins modern maldi-tof mass spectrometry development of tof mass spectrometers since the introduction of maldi (vestal, ) maldi mass spectrometry of carbohydrates short general review (in chinese) - play an insignificant role in the production of photoelectrons because suspended dhb produced a photoelectron signal similar to dhb on the surface. in addition, decreasing the dhb thickness on the target reduced the photoelectron intensity. for crystalline dhb and sinapinic acid, the photoelectron intensity was found to increase with the laser fluence (nitrogen laser at nm) in less than a second order relationship, suggesting considerable reductions of ionization potentials in comparison with free molecules. according to ab initio calculations, the ionization potential of dhb clusters was found to reduce as the cluster size increased from monomer to octamer. the paper discusses the impact of these abundant electrons on ion production in maldi. the earlier rate equation model for maldi ion formation and reaction (knochenmuss, (knochenmuss, , , has been extended to include positive and negative ions of both matrix and analyte (knochenmuss, ) . the resulting positive/negative ratios of secondary analyte ions show that a recent static equilibrium approach is not adequate for quantitative analysis of maldi experiments. although the ion ratios remain close to unity whenever the reaction free energies are at least moderately favorable, deviations from this condition result in unequal ratios of oppositely charged ions and show once again that the dynamic aspects of maldi cannot be neglected. molecular dynamics simulations of maldi have been performed to investigate laser pulse width and fluence effects on primary and secondary ionization process. at the same fluence, short ( or psec) pulses were found to give much higher initial pressures and ion concentrations than longer ones ( ns). these differences were found not to persist because the system relaxes towards local thermal equilibrium on a nanosecond timescale. higher fluences were found to accentuate the initial disparities. axial velocities of ions and neutrals were found to span a wide range and to be fluence-dependent. the total ion yield was found to be only weakly dependent on the pulse width and to be consistent with experimental estimates. secondary reactions of matrix cations with analyte neutrals were efficient even though analyte ions were ablated in clusters of matrix (knochenmuss & zhigilei, ) . lai et al. ( ) have employed transition state theory for modeling the desorption of surface ions, assuming chemical and thermal equilibrium in the solid state prior to desorption. the method was different from the use of conventional models that assume chemical equilibrium in the gas phase. this solid-state thermodynamic interpretation was used to examine the desorption of thap and of an angiotensin i/thap mixture. it successfully described the changes in ion yield with the effective temperature under various laser fluence and initial temperature conditions. the analysis also revealed the key role played by ion concentration in the modeling used to provide the best fit of the model to observations. divergence of the ion beam with laser fluence was also examined using an imaging detection method and the signal saturation normally seen at high fluence was appropriately reduced by ion focusing. simplified but deceptive theoretical interpretations were obtained when the analysis was conducted without adequate calibration of the instrument bias. the laser plume produced by several ionic liquid matrices has been studied by a post-ionization approach in which the neutrals in the ablation plume were ionized with a second laser pulse. it was found that after the initial event that produced the ions, a second, time-delayed, ablation event occurred in which the plume contained only neutral molecules. the presence of these neutral molecules was explained by a reflected-shockwave model in which the shockwave emerging from the laser ablation is reflected from the sample plate behind the sample. it was assumed that when the shockwave arrived at the sample surface it caused a second ablation of the neutral molecules (hellwig et al., ) . the nm multiphoton dissociation and ionization of , -dihydroxyacetophenone (dhap, / ) has been studied (dyakov et al., ) . the experimental results indicate that photoionization that occurs in the gas phase after dhap vaporizes from the solid phase may not play an important role in the maldi process. a combined ir-maldi ion source with an electrospray ionization (esi) emitter for post desorption ionization has been described (sampson, murray, & muddiman, ) . the source produced multiply charged ions from proteins but singly charged ions from carbohydrates (o-glycans cleaved from mucin (muc) were tested) and avoided the fragmentation produced by some other techniques. ir-maldi ms with a laser emission in the mm wavelength range, which utilized energy absorption at the co double-bond stretch region, has been investigated for analysis of several types of biomolecule. the softness of ir-maldi ms was evident in the negative ion mode where abundant [mÀh] À ions were obtained for acidic biomolecules with sulfate, phosphate, or carboxylate groups. better sensitivity was obtained than with ultraviolet (uv) maldi ms. furthermore, there was no substantial loss of sialic acid due to the prompt fragmentation from sialylated glycoconjugates such as gangliosides. such loss is a common problem with maldi analysis of sialylated carbohydrates. the technique was used in conjunction with a potent new matrix, oxamide ( ), resulting in the use of low laser fluence, and removing one of the limitations of maldi ms for biomolecular analysis of uv-maldisensitive molecules (tajiri, takeuchi, & wada, a) . tu and gross ( ) have reviewed methods for miniaturizing sample spots for maldi analysis. topics include minimizing sample dispersion by target modification, the use of hydrophobic materials as maldi-plate surfaces, the use of microdispensing devices such as piezoelectric dispensers and the use of droplet charging by induction or polarization. a simple device for maldi sample preparation, based on the spraying of matrix/sample solution through a stainless steel sieve, has been used for the preparation of maldi samples of peptides, polysaccharides (pss) and high molecular weight (mw) proteins (cristoni et al., ) . the spectra obtained by laser irradiation of the resulting microspots exhibit resolution and sensitivity higher than those achieved by the commonly employed dried droplets method. furthermore, the target surface was more homogeneous than those obtained by the dried droplet method. chca and super-dhb (dhb þ -oh, -ome-benzoic acid) (s-dhb) matrices were used and applications were to insulin and dextran oligomers. ultrasound produced by a simple piezoelectric device has been used as an alternative method for soft ionization of biomolecules. cavitation was proposed as the major mechanism producing the ions and the technique was applied to carbohydrates, proteins and fatty acids. however, although an abundant ion, said to be the [mþh] þ from the high-mannose glycan man glcnac ( / ), was obtained in the presence of dhb, thap, or sinapinic acid, neither the mass nor the stated elemental composition were consistent with this structure . the recent availability of commercial ion mobility instruments offers another dimension to carbohydrate analysis by providing the ability to separate by molecular shape and offering the possibility of rapid isomer differentiation. however, resolutions on current instruments are comparatively poor and do not match those that can be obtained with high performance liquid chromatography (hplc). a comparison of three types of ion mobility ms (field asymmetric waveform ion mobility (faims), drift tube and traveling wave ion mobility spectrometry (twims)) for separation of chiral molecules with applications to monosaccharides has been reported (enders & mclean, ) . data are best reported as rotationally averaged collisional cross-sections. these can be obtained directly with drift tubes and indirectly with the twims instruments. obtaining such measurements with faims instruments, however, is more challenging. collisional-cross sections have been measured for a large number of biologically relevant molecules including oligonucleotides (n ¼ ), carbohydrates (n ¼ ), lipids (n ¼ ), and peptides (n ¼ ). collisional cross sections increased with mass but were found to be different for each molecular type in the order oligonucleotides < carbohydrates < peptides < lipids. the specific correlations were best described by logarithmic regressions. thus, the technique was able to separate compounds of different structures but with the same or similar mws. in addition, some separation of compounds with the same mass within a particular class was possible. the latter point was demonstrated by separations of isobaric oligonucleotides, which were interpreted by molecular dynamics simulations mclean, ) . have used ion mobility to extract carbohydrate ions from incubation mixtures obtained from protein-n-glycosidase (pngase) f release of n-glycans. glycoproteins such as ribonuclease b (rnaseb) were first digested with trypsin followed by pngase f and then analyzed directly by ion mobility ms with a waters synapt instrument. the carbohydrate ions showed different mobilities from peptide and other contaminating ions allowing them to be extracted directly from the crude mixtures. both esi and maldi ion sources were used; the maldi ion source was better at ionizing the larger carbohydrates and did not suffer from the problem of producing both [mþh] þ and [mþna] þ ions that were seen in the esi source. the technique would appear to have great potential for rapid glycan analysis, particularly as the synapt instrument also offers the ability to fragment the isolated ions. hossain and limbach ( ) have reviewed matrices used for maldi analysis of several compound classed including carbohydrates. the review covers common matrices such as dhb and chca and less common systems such as liquid matrices and carbon nanotubes ( references). the use of ionic liquid matrices has also been included in a review by liu et al. ( e) although the bulk of this review discusses the use of ionic liquids for sample preparation. the thermal stability of several commonly used crystalline matrices, , -dhb, thap, chca, sinapinic acid, nor-harmane ( / ) and harmane ( / ) has been studied by heating them at their melting point and characterizing the remaining material by a variety of spectroscopic and chromatographic techniques. in general, all compounds, except for chca and sinapinic acid, remained unchanged after fusion. chca showed loss of co , yielding a trans-/cis- -hydroxyphenylacrylonitrile ( ) mixture. sinapinic acid showed mainly cis-to-trans thermal isomerization and, with very poor yield, loss of co (tarzi, nonami, & erra-balsells, ) . h-pteridine- , -dione (lumazine, ) has been described as a good matrix for phospholipids where the presence of cationcontaining compounds suppresses signals from neutral compounds. phosphatidyinositol was reported to give a signal that was an order of magnitude higher than that obtained from dhb (calvano, carulli, & palmisano, ) . a number of papers have reported the use of various nanoparticles as matrices. unlike traditional chemical matrices, these materials generally produced little or no signal in the low mass region, thus making them ideal for analysis of small carbohydrates. titanium dioxide micro-and nano-particles, prepared by hydrolysis of ti butoxide and maintained in aqueous solution, have been evaluated as matrices for the detection of several small molecules. most of the reported applications were to lipids but the nanoparticles were also applied to flavonols/anthocyanins and their glycosides in rose petals. the spectra showed ions from many more small molecules than did spectra recorded from dhb, particularly in the region of the dhb matrix ions. one advantage of the nanoparticles is that their small size (average of about nm) facilitates their penetration into tissue for in situ imaging (lorkiewicz & yappert, ) . titanium dioxide (tio ) has also be used by kawasaki, okamura, and arakawa ( ) for ionization of several types of compound, including carbohydrates, with a similar absence of low mass matrix ions. in a study of the most suitable crystalline form, the anatase-type tio , was shown to be the best. nanoparticles of diamond, tio , titanium silicon oxide, barium strontium titanium oxide, and silver have been examined for their potential as maldi matrices for direct laser desorption/ionization of carbohydrates, especially fructans, from plant tissue. two sample preparation methods including solventassisted and solvent-free (dry) deposition were compared. all examined nanoparticles except ag were found to desorb/ionize standard sucrose ( ) and fructans in both positive and in negative ion mode. in positive ion mode, sugars gave [mþna] þ and/or [mþk] þ ions depending on the ionic composition of the sample spot, and [mÀh] À ions in negative mode. ag nanoparticles yielded good signals only for non-salt doped samples that were measured in the negative ion mode. when used to study fructans in plant tissues all the nanoparticles except ag could desorb/ionize carbohydrates in both ion modes. nanoparticles gave similar limit of detection (lod) for standard fructan triose ( -kestose, ) in the positive ion mode and better lods in the negative ion mode than those given by the common crystalline organic maldi matrices such as dhb, nor-harmane or carbon nanotubes. although lower signal-to-noise ratio (s/n) signals were obtained from the tissues with solvent-free matrix deposition than when solvent was used, the reproducibility averaged over all sample was more uniform (gholipour et al., ) . maldi detection at the level of several hundred zmoles has been achieved by the addition of gold nanoparticles (aunps) with a diameter of several tens of nanometers into a sample solution (shibamoto et al., ) . n-acetyl-tetraose fmol/ml gave a strong signal in the presence of nm aunp ( .  particles). the mechanism appears to be related to surface plasmon (sp) excitation of the aunps. citrate-capped aunps have been shown to act as matrices for the determination of several types of biomolecule, including carbohydrates such as starch, dextrins, and glycosphingolipids (gsls) in the presence of high concentrations of salt (wuhrer, koeleman, & deelder, b) . with the gsls, however, some loss of sialic acid ( / ) was found. a combination of immobilized silica and dhb on iron oxide magnetic nanoparticles has been shown to give a clean background and to be appropriate for the analysis of a range of small molecules, including glycolipids . the ratio between sio and dhb was found to affect the surface immobilization of dhb on the nanoparticle, critically controlling the ionization efficiency and background. enhancements of molecular ion signal over those produced from dhb alone were noted and high quality product-ion spectra were obtained. fullerene ( )-derivatized silica (pore size nm) also appears to be good for small molecules including monosaccharides (szabo et al., b) . multifunctional zro nanoparticles and zro -sio nanorods have been successfully used as matrices for cyclodextrins in atmospheric pressure and vacuum maldi. the lod for cyclodextrins was found to be . - fmol and the matrices were used to analyze cyclodextrins in urine samples (kailasa & wu, ) . manganese dioxide nanoparticles have been used to ionize ginsenosides with the advantage that the spectra lacked matrix ions in the low mass region allowing low-mass postsource decay (psd) ions to be clearly visible. the technique was named nanoparticle-assisted laser desorption/ionization (nano-paldi) (sahashi, osaka, & taira, ) . single-crystalline euf hexagonal microdisks with hollow interiors have been prepared as background free matrices. the long-lived excited state of europium ions is capable of transferring energy to the molecules allowing the microdisks to act as matrices. they were successfully used for analysis of small peptides, amino acids and hydroxypropyl b-cyclodextrin ( ) (chen et al., d) . another new matrix with low background is mesoporous silica, sba- , functionalized with quinoline . the material was obtained by using calcined . compared with dhb and sba- itself, the modified material demonstrated several advantages in the analysis of small molecules such as less background interference ions, high homogeneity, and better reproducibility. detection limits in the region of pmol were reported. in a more traditional approach for reducing low-mass matrix ions, fujita et al. ( ) have used b-cyclodextrin ( / ) mixed with thap or , -dhap. the latter compounds, in particular, formed inclusion complexes with the cyclodextrin as demonstrated by the lack of a similar effect when the corresponding linear carbohydrate, maltoheptaose ( ) was used as a co-matrix. a method for recording negative ion spectra that is suitable for small molecules in that it produces no matrix-related ions in the low mass region uses the proton sponge, , -bis(dimethylamino)naphthalene (dman, ) as a solution in ethanol as the matrix. this compound has very high proton affinity and can take up protons from even weak acids to form deprotonated anions. moreover, dman in solution has a strong uv absorption band in the region - nm, fully compliant with frequencies of nitrogen and neodymium-doped yttrium aluminium garnet (laser) (nd:yag) uv lasers. the matrix was found to be suitable for a range of small molecules including fatty acids, carbohydrates and prostaglandins (shroff & svato, ; shroff & svatos, ; . the authors proposed to name the technique as matrix-assisted ionization/laser desorption, abbreviated as maild, ms . n(ch ) (ch ) n , -bis(dimethylamino)naphthalene, zhang et al. ( i) have reported a new method for the analysis of small molecules by using matrices such as metalphthalocyanines (mpc, ) which form matrix-analyte adducts and shift the molecular ions into a high and interference-free mass range. the mass of the target analyte was calculated by subtracting the mass of mpc from the mass of the mpc-analyte adduct. the mpcs themselves were also detectable and could serve as internal standards. various mpcs with aromatic or aliphatic groups and different metal centers were explored. aluminum-phthalocyanines (alpcs), gallium-phthalocyanines (gapcs), and indiumphthalocyanines (inpcs) were found to be efficient matrices for a large number of compound types and formed mpc-analyte adducts in either the positive or negative ion mode. the detection limits varied from to fmol, depending on analyte. mass spectrometry reviews doi . /mas graphene has also been reported to be a good matrix for low mw compounds with essentially no ions from the matrix (dong et al., ) . colloidal graphite has also been used for atmospheric pressure maldi and has been shown capable of producing both [mþh] þ and [mÀh] À from many types of small molecules, including glycosylated flavonoids (perdian, schieffer, & houk, ) . two reviews on ionic liquids, including their use as maldi matrices have been published (soukup-hein, warnke, & armstrong, ; sun & armstrong, ) in an extensive study to find new liquid matrices matrices were tested and new ionic liquids were prepared (crank & armstrong, ) . both the anionic and cationic moieties were varied systematically to find a matrix with the best physical properties, analyte signal intensity and widest mass detection range. the developed matrices showed a wide mass detection range (< , da to > , da) for proteins and peptides with greater s/n ratios than solid matrices and could effectively ionize proteins of large mass without disrupting noncovalent interactions between monomers. it was found that both the proton affinity and pk a of the cation have a large effect on the ability of the matrices ionize the analyte. the matrices could be used to detect carbohydrates with fewer degradation products than solid matrices. n,n-diisopropylethylammonium a-cyano- -hydroxycinnamate and n-isopropyl-nmethyl-t-butylammonium a-cyano- -hydroxycinnamate were the best matrices for proteins and peptides, while n,n-diisopropylethylammonium a-cyano- -hydroxycinnamate and n,ndiisopropylethylammonium ferulate were found to be the best matrices for carbohydrates. another novel ionic liquid matrix has been made from the , , , -tetramethylguanidinium ( ) salt of thap and found to be well suited for the maldi analysis of glycopeptides and glycans, particularly as it appeared to overcome the well-known ionization suppression of carbohydrates in the presence of peptides. for example, even glycopeptides containing short peptide backbones and large carbohydrate moieties gave high signal intensities when using this matrix even though they did not produce spectra directly from thap. in a second experiment, glycans were released with pngase f from total tryptic digests derived from glycoproteins and analyzed by maldiquadrupole ion trap (qit)-time-of-flight (tof). when using the liquid matrix, it was possible to detect the glycans with high intensities in the presence of the tryptic peptides, whereas, once again, glycan ionization was completely suppressed when measured with thap alone. the extent of metastable decay of glycopeptides was also found to be reduced when using the liquid matrix (ullmer & rizzi, ) . ionic liquid matrices have shown considerable advantages over conventional matrices for maldi-ms analysis of polysulfated carbohydrates such as heparin and heparin sulfate. these compounds are not easily analyzed by uv-maldi ms analysis because of the thermal lability of their o-and n-so moieties. two new ionic liquid matrices based on the combination of -( -hydroxyphenylazo)benzoic acid (haba, / ) with , , , -tetramethylguanidine or spermine ( / ) have been successfully applied to the analysis of these compounds (przybylski et al., b) . these matrices gave improved signalto-noise ratios as well as a decrease of fragmentation and desulfation. sulfated oligosaccharides were detected with higher sensitivity than with the usual crystalline matrices, and their intact sulfated ions [mna] À were easily observed. maldi-ms characterization of challenging analytes such as heparin octasaccharide carrying eight o and four n-sulfo groups, and heparin octadecasulfated dodecasaccharide was also successfully analyzed. in a paper published in , gomenez et al. ( ) reported that dhb, with vacuum drying to improve target spot homogeneity, was a better matrix than sinapinic acid for obtaining reliable molecular mass values of intact glycoproteins because it prevented sugar fragmentation. in a follow-up to this work, the group have investigated the use of liquid matrices prepared from dhb and sinapinic acid with the aim of avoiding the vacuum drying step (giménez et al., ) . the best results were obtained with a variety of glycoproteins such as bovine a acid glycoprotein, bovine fetuin and human transferrin (tf) from matrices prepared by adding an equimolar amount of butylamine ( . ml) to , ml of a mm solution of sinapinic acid or dhb in meoh. the mixture was vortexed and sonicated for min, evaporated to approximately ml with air, and finally reconstituted with ml of etoh or mecn, for dhb and sinapinic acid mixtures, respectively. furthermore, it was noted that both matrices gave the same masses for the tested glycoproteins that agreed with literature values, suggesting that no fragmentation of the carbohydrate moieties had occurred. a matrix consisting of chca and aniline ( ) has also proved to be successful for a number of different compounds, including raffinose ( ) (calvano, carulli, & palmisano, ) . although it was noted that this matrix gave a stronger signal than chca alone, the latter matrix is not very efficient in ionizing carbohydrates. a comparative investigation of several matrices for analysis of the small carbohydrates, glucose (glc, / ) and sucrose ( ) has been performed by yang et al. ( c) . of the matrices studied, sodiated dhb, carbon nanotubes, an ionic liquid matrix of dhb-pyridine, a binary matrix of dhb-aminopyrazine ( / ), zinc oxide nanoparticles and aunps, the best sensitivity was provided by the carbon nanotubes. the detection limit was pmol. both carbon nanotubes and the ionic liquid matrix exhibited the highest reproducibility. tzeng, zhu, and chang ( ) have investigated doping various maldi matrices with alkali metal hydroxides. it was found that for neutral underivatized oligosaccharides, the addition of % naoh to dhb caused partial alkaline degradation by glycosidic cleavages upon laser desorption. the effect intensified when nonacidic compounds such as thap or -amino- -mercapto- , , -thiadiazole (amt, / ) were used as the matrix. the degradation allowed identification of the reducing end residue of the analyte and facilitated its structural characterization by psd tof-ms. use of matrices consisting of lioh and thap or amt led to the production of singly as well as multiple lithiated ions. multiple lithiation appeared to occur with carbohydrates containing free -oh groups. up to li atoms were found to be incorporated for maltoheptaose, b-cyclodextrin, and dextran . such a "lithium tagging" technique makes it possible to differentiate positional isomers of milk-neutral oligosaccharides, lacto-n-difucohexaose i and ii (lndfh-i ( ) and lndfh-ii, ( )), without the need of chemical derivatization or tandem ms analysis. lndfh-ii, = glcnac, = galactose, = fucose, = glucose (for details, see (harvey, et al., c) choi and lee ( ) have studied the ionization efficiencies of maltooligosaccharides (degree of polymerization, dp - ) with the cations na þ , li þ , and k þ in salts containing tfa À , cl À , and oh À with dhb as the matrix. the signal strength rose with the number of glucose units with sodium consistently giving the most intense signals. the nature of the anion also affected the ionization with the tfa À salts generally being the most effective. a sample preparation method developed by nishikaze and amano ( ) has been compared with the conventional drieddroplet or ethanol (etoh) recrystallization methods and reported to give superior results in terms of ion yield and signalto-noise ratio. the method, named the "reverse thin layer method" consists of first, complete drying of the oligosaccharide solution on the maldi target plate and then deposition of the matrix dissolved in a small amount of meoh. using this method, a relatively homogeneous matrix crystal was generated and higher yields of both positive and negative ions were obtained. the authors comment that the method could be applied to various other matrices including both solid and liquid matrices. a method for direct archaebacterial glycolipid and lipid analysis by maldi ms in intact membranes, without prior extraction/separation steps, has been developed by angelini et al. ( ) . the purple membrane isolated from the extremely halophilic archaeon halobacterium salinarum was used as a model, lyophilized and ground with -aminoacridine ( -aa, / ). the mixture was crushed in a mechanical die press to form a thin pellet, small pieces of which were attached directly to the maldi target. in parallel, solution spectra of individual phospholipids and glycolipids, were analyzed by maldi-tof/ ms using -aa as the matrix. results show that -aa is a suitable matrix for the conventional maldi-tof/ms analysis of lipid extracts from archaeal microorganisms, as well as for fast and reliable direct dry lipid analysis of lyophilized membranes. a new technique termed matrix-free material-enhanced laser desorption/ionization mass spectrometry (mf-meldi-ms) has been described which employs a single compound prepared by immobilizing bradykinin on silica gel coupled to -( triethoxysilylpropylureido)azobenzene ( ) for both the maldi matrix and a stationary phase for thin-layer chromatography (tlc). the technique was applied to the analysis of carbohydrate reference standards such as glucose, sucrose, raffinose and plant extracts from quercus robur (oak). carbohydrates formed [mþna] þ and abundant [mþk] þ ions. the meldi material adsorbs the laser energy sufficiently for desorption and ionization and delivered excellent results in respect to signal-to-noise (s/n) ratio (s/n ratio: > / ) and sensitivity with a lod in the low ng/ml range. for preparation of the tlc plates, the modified silica gel was suspended in methanol, acetone, acetonitrile or acetonitrile/water ( : ) for min. about - ml of the suspension was applied in narrow channels cut into a stainless steel target in the form of a thin layer and dried at room temperature. the sample was placed on this layer for subsequent tlc in n-buoh/acetone/acetic acid/ h o ( : : : ) as mobile phase (qureshi et al., ) maldi imaging has seen many developments during the review period and several reviews and related articles have been published; details are in table . a mass spectrometer with ion mobility separation capability (waters synapt) has been used by snel and fuller ( ) to produce high spatial resolution images of glucosylceramide ( ) in the spleens of mouse models of gaucher disease. the matrix (chca) was applied to the tissue sections with an airbrush. for data acquisition, the laser was continually fired at one position until no more ions were observed and then the sample was moved by mm (laser diameter about mm). ions were generated from only the un-irradiated surface at each of these positions achieving an effective spacing of mm. at this spacing, it was possible to visualize macrophages enriched in glucosylceramide which could be distinguished from other cell types in the spleen. current maldi mass spectrometric imaging (msi) spatial resolution is typically limited by the diameter of the laser spot-size, which is usually between and mm, covering an area equivalent to tens of mammalian cells. perdian and have addressed the problem of acquiring high resolution imaging with high resolution ms on an orbitrap mass spectrometer. at a spatial resolution of mm, the orbitrap mass analyzer is able to image a ,  , mm sample area in - min with one scan per step, depending on the resolution. if the spatial resolution is increased to mm, the same size sample area will take more than - hr to complete the experiment, a time that is not practical in the normal laboratory. however, because the orbitrap also has a linear ion trap (it) analyzer, this was utilized, together with a two-motion plate movement to reduce the time while maintaining the resolution. thus, for every pixel position on the target, the laser spot was moved in a spiral fashion such that both orbitrap and ms/ms data were acquired. with a fast nd:yag laser the data acquisition time was decreased by - % compared to that from orbitrap-only scans; however, % or more time could be saved for higher mass resolution and with a higher repetition rate laser. using this approach, a high spatial resolution of mm was maintained at it imaging, while orbitrap spectra were acquired at a lower spatial resolution, - mm, all with far less data acquisition time. furthermore, various ms imaging methods were developed by interspersing ms/ms and ms fragmentation n times (ms n ) it scans during orbitrap scans to provide more analytical information. the method was applied to several flavonol glycosides from an arabidopsis flower petal in which ms/ms, ms n , it, and orbitrap images were all acquired in a single data acquisition. spectra were acquired in negative mode and no matrix was required. for uv-absorbing compounds such as flavonoids and their glycosides, a matrix is not necessary for imaging as demonstrated by imaging at the single cell level of secondary metabolites in arabidopsis thaliana and hypericum species (hölscher et al., ) . the highest spatial resolution achieved, mm, was much higher than that achieved by commonly used maldi) imaging protocols. a new matrix-free technique called nanostructure-initiator mass spectrometry (nims) has been developed for the analysis e c n e r e f e r s n o i t a t i c s t n e t n o c t c e j b u s general review short article, mainly applications (murayama et al., ) general review methods and applications to peptides and lipids (including glycolipids) general review short general review (award lecture) general review from origins to state of the art (francese et al., a) concise review of mass spectrometry imaging (sugiura et al., b) and tissue imaging of carbohydrates and steroids (patti et al., ) . analysis was accomplished by spray depositing nacl or agno with a fused-silica picotip emitter onto a porous silicon surface to provide a uniform layer rich with cationization agents prior to desorption of the fluorinated polymer initiator. upon laser irradiation, carbohydrates produced [mþna] þ ions whereas steroids formed [mþag] þ . for glucose, a plot of concentration against the c/ c ratio was linear with a correlation coefficient r of . over the range - mm. carbohydrates and steroids could be detected down to the -amol and -fmol levels, respectively. the ability of the method to perform tissue imaging was demonstrated by imaging the distribution of sucrose in a gerbera jamesonii flower stem and the distribution of cholesterol ( ) in a mouse brain. the flower stem and brain sections were placed directly on the ion-coated nims surface without further preparation and analyzed directly. no deposition of a matrix onto the sample surface was needed. a similar matrix-free method, termed nano-assisted laser desorption-ionization (naldi) ms for tissue imaging has been demonstrated by vidová et al. ( ) . commercially available nano-structured surfaces were used as substrates for imprinting tissue sections. the lithographic transfers were washed and the lipids were imaged by laser desorption mass spectrometry (ldms). the naldi imaging of lipid transfers was compared with standard maldi imaging of matrix-coated (chca) tissue sections and the resulting images were found to be of the same quality with no spatial information being lost due to the imprinting process. naldi imaging was reported to be faster due to the absence of the time-consuming matrix deposition step, and the naldi mass spectra were less complex and easier to interpret than maldi spectra. the method was applied to the analysis of phospholipids, gsls and glycerophospholipids in mouse kidney slices. naldi ms was able to identify the same lipid species as maldi and was reported to provide better distinction between kidney and adrenal gland tissues based on the lipid analysis. miura et al. ( a) have developed a maldi imaging system that is claimed to be able to image from single cells in thin tissue sections. an indium tin oxide-coated glass slide marked with a mm-wide mesh work to enabled matching of optical and ms images was used for mounting tissue sections. a suspension of hela cells in culture medium was mounted onto the slide and incubated for hr at ˚c. cells were then washed with phosphate-buffered saline (pbs) and the appearance of single cells adhering to the glass was observed by optical imaging. the single cell-adhered glass slide was then mounted onto maldi sample plate with a parallel experiment involving brain tissue slices and the matrix, -aa, was applied with an airbrush. several metabolite peaks were detected at the position of the single cell. adenosine triphosphate (atp, ; m/z . , identified by comparison with a standard sample) was identified with a good signal-to-noise ratio. peaks were also obtained from other metabolites such as fructose- , -bisphosphate ( ) a major hurdle for imaging gangliosides in tissue using ms is that sialic acid residues can be dissociated in the ionization process. chan et al. ( a) have investigated the ionic liquid matrix chca/ -methylimidazol ( / ) ( : w/w) and have found that it produces excellent sensitivity for ganglioside detection without significant loss of sialic acid residues. the matrix was applied to the tissue samples with an airbrush; the method best adapted to handling a mixed matrix whose components have different volatilities. image analyses of mouse brain tissue sections demonstrated that the n-fatty acyl chains of gangliosides were differentially distributed in mouse hippocampal regions. gangliosides with n-c acyl chains were enriched in the ca region, while gangliosides with n-c acyl chain were enriched in dentate gyrus. colsch and woods ( ) have developed a method for imaging sialylated gsls in mouse brain. the total glycolipid profile was obtained by maldi-tof following solvent extraction and then individual species were mapped from frozen brain slices by maldi using a linear tof/tof system in negative ion mode. the matrix, which consisted of saturated , dihydroxyacetophenone (dhap, / ) dissolved in % ethanol with the addition of ammonium sulfate ( mm) and . % of heptafluorobutyric acid (hfba) was applied with a chip- chemical inkjet printer with a piezoelectric head. twenty-eight nanoliters of matrix were deposited per spot with the distance between two spots of mm. the ammonium sulfate in the matrix mixture limited the formation of salt adducts, while the addition of hfba increased the stability of dhap in the vacuum and reduced its rapid sublimation. some sialic acid loss was noted, particularly with gd and gt , which were detected as the ganglioside ( the results showed differences in gsl localization in several brain regions depending on the sialic acids and the ceramide (cer). imaging of lipids, including gsls, has been reported by landgraf et al. ( ) using a hybrid linear it/orbitrap mass spectrometer that allowed the acquisition of ms/ms spectra. a dramatic improvement in mass spectral resolution and a decrease in background were observed from lipids distributed within nerve tissue when compared with results obtained from fragmentation within the linear it. the dhb matrix was applied to the dried tissue samples with an epson inkjet printer and the maldi ion source was operated at a pressure of about mtorr. the technique was used to image lipids from rat spinal cord sections. goto-inoue et al. ( ) have imaged the glycolipid, seminolipid ( ) in mouse testis. this sulfated glycolipid comprises more than % of the glycolipid in mammalian testis and disruption of the gene catalyzing transfer of galactose (gal) results in male infertility due to the arrest of spermatogenesis. different molecular species are defined by fatty acid composition. tissue imaging was performed from thaw-mounted tissue slices that had been sprayed with dhb with an airbrush. it was found that the major molecule (c : -alkyl-c : -acyl) was expressed throughout the tubules: some (c : -alkyl-c : acyl and c : -alkyl-c : -acyl) were predominantly expressed in spermatocytes and the other (c : -alkyl-c : acyl) was specifically expressed in spermatids and spermatozoa. this is the first report to show the cell-specific localization of each molecular species of seminolipid during testicular maturation. experimental details for performing imaging of glycolipids using the airbrush application method for applying dhb, as used by this group, has been published separately (goto-inoue, hayasaka, & setou, a) . the distribution and localization of gsls present in mouse brain sections have also been investigated using nanoparticleassisted laser desorption/ionization imaging ms. aunps modified with alkylamine were used as a new matrix to maximize the detection of gsls. the mouse brain was frozen in liquid nitrogen, and serial sections were thaw-mounted onto indiumtin oxide (ito)-coated glass slides. a thin layer of aunps or dhb matrix was applied to the surface with an airbrush. ims analyses were performed by raster-scanning in the x-axis with a scan pitch of mm in the y-axis. sulfatides and gangliosides were detected in mouse brain sections with the new matrix whereas it was difficult to detect them using dhb (goto-inoue et al., c ). an oscillating capillary nebulizer (ocn) was also used by chen et al. ( g) for analysis of sphingolipids in tissue slices in tay-sachs/sandhoff disease. in addition to the above-mentioned matrix-free methods, jung et al. ( ) have reported imaging of cellulose in poplar (populus deltoids) stem using more traditional techniques. analysis of microcrystalline cellulose was performed first to determine the best matrix. dhb gave much stronger signals than matrices such as chca or sinapinic acid and was applied to poplar cellulose with an ocn. ions at m/z , , , , and , (dp , , and ) were monitored with a voyager de str instrument and produced images that closely resembled the optical image. taira et al. ( a) , on the other hand used an airbrush with chca to image ginsenosides in cross-sections of panax ginseng root and used ms/ms to obtain detailed structural information. ginsenosides were found to be located more in the cortex and periderm than in the medulla and that they were at greater concentration in the root tip than in the center of the root. several carbohydrates including hexoses (hex) and d-fructose -phosphate have been imaged in eggplant (solanum melongena, also known as aubergine) fruits from dhb although the paper was mainly concerned with imaging of g-aminobutyric acid (gaba, ) (goto-inoue, setou, & zaima, b) . li, bohn, and sweedler ( h) have compared two ms imaging methods, maldi and sims, for glycan detection in the stems of the perennial grass miscanthus  giganteus. several methods of sample preparation were investigated for maldi. a thin ( nm) gold coating provided high quality signals of oligosaccharide-related ions and dhb also showed high efficiency for ion production. on the other hand, chca produced only very weak spectra, consistent with its use as a stand-alone matrix for carbohydrates. direct laser ablation of untreated sections gave high resolution images, although coating the sections with a nanometer thick layer of gold greatly enhanced the quality of the sims images. two reviews describing derivatization reactions have been published (busch, ; ruhaak et al., b) , the second ( references) is more comprehensive and deals specifically with n-linked carbohydrates. these derivatives are most often used for introducing fluorescent tags for chromatographic detection but also find use in ms. -aminobenzamide ( -ab, / ) and -aminopyridine ( -ap, / ), favored by japanese investigators, are the derivatives most frequently encountered; use of the latter derivatives has been reviewed by hase ( ) . several new derivatives (or labels) have been reported. thus, -amino- -naphthalenesulfonic acid (ansa, ) has been used to derivatize n-glycans by reductive amination for capillary electrophoresis (ce), hplc, and maldi-tof analysis (briggs et al., ) . the derivative was reported to give superior resolution in both ce and hplc analysis to the wellused -aminopyrene- , , -trisulphonic acid (apts) derivatives and in maldi-tof analysis, the negative charge enabled both neutral and acidic glycans to be examined simultaneously. -amino- -ethylcarbazole ( / ) (mou et al., ) , another new derivatizing agent, has been reported to increase sensitivity of ms detection. -picoline-borane ( ) has been proposed as an alternative to toxic sodium cyanoborohydride for the reductive amination reaction (ruhaak et al., a) . pabst et al. ( ) have compared the preparation and performance of different labels for n-glycan analysis. several cleanup procedures were developed for cleaning these derivatives before analysis, of which hydrophilic interaction solidphase extraction (spe) appeared to be the most widely used. however, the cleanup was laborious and a better method was sought. simple addition of acetone resulted in the formation of a precipitate, which turned out to be the labeled oligosaccharide. a single addition and removal of acetone reduced the amount of reagent to approximately . % (measured with -ab). two further extractions of the pellet with acetone reduced the excess of amine reagent by at least as much as clean-up with a cyano-spe cartridge. in addition, reduction of the schiff base of -aplabeled glycans proceeded faster and/or required less reagent when the samples were pre-purified before the addition of reducing agent. acetone extraction was successfully applied to many other labels such as -ab and -aminobenzoic acid ( -aa) ( / ). with respect to the performance of the individual labels, procainamide ( ) emerged as more sensitive than -aa for normal-phase hplc, but its chromatographic performance was not convincing. -ap gave the lowest retention on reversedphase and graphitic carbon columns and, thus, appeared to be most suitable for glycan fractionation by multidimensional hplc. most glycan derivatives performed better than native carbohydrates in maldi and esi ms, but the sensitivity gain was small and hardly sufficient to compensate for sample loss during preparation. amano et al. ( a) have labeled oligosaccharides with a pyrene derivative in order to acquire negative ion maldi-qit-tofms n spectra. the oligosaccharides were reacted with pyrene butanoic acid hydrazine ( / ) and then reduced with nabh and cleaned with a small c column. the derivatives gave intense and stable molecular ions in both positive and negative ([mÀh] À ions) modes and as little as fmol of pyrene-labeled oligosaccharides, such as monofucosyllacto-nhexaose ( ) gave sufficient signals for analysis. although some fucose loss by in-and post-source occurred in positive ion mode, this loss was significantly less in negative mode. a method, termed glycan reductive isotope labeling (gril) has been introduced for glycan quantitation. free glycans or those released from glycoproteins, were derivatized by reductive amination with either [ c ]aniline or [ c ] aniline. these dual-labeled aniline-tagged glycans could be recovered by reversed-phase chromatography and could be quantified by uv absorbance or ms. one labeled variety was used as the reference standard against which the test glycan, labeled with the other isotope was measured. unlike previously reported isotopically coded reagents for glycans, the derivatives did not contain deuterium, which can sometimes be chromatographically resolved. the technique allowed linear relative quantitation of glycans over a -fold concentration range and could accurately quantify sub-picomole levels of released glycans (xia et al., ) . on-target derivatization with the matrix -aminoquinoline ( / ) has yielded schiff bases which could be measured directly in positive and negative ion mode from one single spot. the optimal conditions were mg/ml of -aq in a mecnwater mixture ( : v/v) with . % of an inorganic acid to give a ph of . in negative ion mode, spectra from chloride adducts of the derivatives were acquired from fmol of oligosaccharide. furthermore, psd fragmentation in positive and negative ion mode was enhanced, providing information on oligosaccharide sequence, linkage, and branching. the method was applied to trifucosyllacto-n-hexaose ( ) and trifucosyl-para-lacto-n-hexaose ( ), two isomers occurring in human breast milk samples, which were easily identified and distinguished (rohmer et al., ) . tfplnh, (symbols as defined for structures and . anomericity not specified) reductive amination derivatization has also been exploited in other areas, combined with their use in ms. binding of sugar chains to proteins, viruses and cells is conveniently monitored by the surface plasmon resonance (spr). key to this method is the use of linker compounds for immobilization of the sugar chains to the gold-coated spr chip. sato et al. ( a) have developed a novel linker molecule, named "f-mono," which combines a linker with a fluorescent moiety to allow high sensitivity monitoring of the glycans. since the molecule ( ) contains a , -diaminopyridyl group and a thioctic acid group, conjugation with sugar chains can be achieved by the reductive amination reaction. the mass spectra of thee compounds contained a peak da higher than the molecular ion due to the reduction of the thioctic acid moiety providing a convenient method for identifying the glycans even using unfractionated crude samples. immobilization of the derivatives onto gold-coated chips, and their subsequent spr analyses were successively accomplished. use of hydrazone formation removes the need for a reductive step to stabilize the derivative as required by schiff base formation from primary amines. experimental details for synthesis of the phenylhydrazone derivatives discussed in earlier reviews (lattova & perreault, a,b; lattova, perreault, & krokhin, ) have recently been reported in an edition of methods in molecular biology devoted to glycomics . small oligosaccharides and n-glycans from chicken ovalbumin have been converted into their biotin derivatives by incubating them with biotinamidocaproyl hydrazide (bach, ) (kapková, ) . the derivatives imparted improved mass spectral sensitivity over that of the free glycans and, because the labeling reagent contained a biotin handle, it allowed the interaction between lectins and biotin-derivatized oligosaccharides to be investigated. fragmentation of the n-linked glycans was dominated by y and b/y-type glycosidic ions. han et al. ( a) have used a new substituted hydrazine, -( -cyanophenyl)- -piperidinecarbohydrazide ( ) and produced a derivative that increased detection sensitivity by about -fold over that from the underivatized glycan. the observation of [mþna] þ ions rather than the expected [mþh] þ species suggested that the sensitivity increase was the result of increased hydrophobicity. maldi analysis employed dhb and super-dhb; recrystallization of the super-dhb sample with acetonitrile substantially improved the signal-to-noise ratio and reproducibility. girard's t reagent ( / ), with its constitutive cationic charge, has been used in quantitative measurements, as described below and for measuring a-galactose-containing n-glycans in porcine pig corneal endothelial cells and keratocytes (kim et al., c) . because of the in-built charge, signal strengths from glycans of different structures were thought to be equal. this is not necessarily the case for formation of [mþna] þ ions. rather than reacting the reducing-terminal aldehydes of carbohydrates with amines or hydrazines, the reverse reaction can be used if the glycan is first converted into its glycosylamine ( ). in fact, this type of reaction can be used directly on pngase f-released n-glycans because these are released in this form. chemical formation of glycosylamines generally utilizes the kochetkov reaction in which the glycan is treated with an excess of ammonium carbonate. unfortunately, this reaction is slow and can take up to five days for completion. to overcome this problem, liu, salas-solano, and gennaro ( h) have used microwave assistance to speed the reaction up to as little as min. following amidation the glycans were reacted with tris-( , , -trimethoxyphenyl)phosphonium acetic acid n-hydroxysuccinimide ester ( ) to introduce a permanent charge on the glycan and the investigators were able to detect derivatized maltoheptaose at fmol/ml by maldi-tof ms using dhb and chca matrices. glycans from rnaseb, chicken ovalbumin and asialofetuin were also detected at high sensitivity. a potential problem arises from possible cleavage of the reducingterminal n-acetylglucosamine (glcnac) residue as such a reaction has recently been reported as a by-product of the kochetkov reaction when, for example, ammonium bicarbonate at ˚c was used (murase & kajihara, several carbohydrates, including maltoheptose, blood type b antigen, pullulan and the glucan of ganoderma lucidum have been converted into their naphthimidazole (naim) derivatives ( ) in high yields by the iodine-promoted oxidative condensation (scheme ; lin et al., b) . the reaction took about hr to go to completion giving derivatized carbohydrates that gave enhanced maldi signals ([mþh] þ ions) compared with those from the free carbohydrates or their -ab derivatives with dhb or thap as matrices. less than pmol of carbohydrate could be detected. furthermore, the derivatives could easily be hydrolyzed to the parent glycans. a further series of such derivatives has been synthesized by condensation with diamines such as substituted benzene- , -diamine ( ) in order to increase hydrophobicity and detection sensitivity (lin et al., c) . using maltotriose ( , n ¼ ), derivatives with pyrimidine- , diamine ( ), pyridine- , -diamine ( ) and , , -oxadiazole- , -diamine ( ) b. derivatives of other sites permethylation has long been used in carbohydrate analysis, most frequently for linkage determination by gas chromatography/mass spectrometry (gc/ms) following hydrolysis and acetylation (permethylated alditols acetates). however, there appears to be an increasing trend to employ the reaction for maldi analysis. the advantage of permethylation is that it increases sensitivity and several investigators employing its use appear to detect larger glycans, particularly n-glycans, than by the use of underivatized glycans. however, sample clean-up of the highly basic reaction mixtures can be a problem with, in some cases, losses offsetting any gain in sensitivity. introduction of solid-phase permethylation has improved the situation. experimental details of the solid phase permethylation method (methyl iodide on sodium hydroxide beads) (kang, mechref, & novotny, ; kang et al., ) that is capable of permethylating very small amounts of carbohydrate have been published in methods in molecular biology (mechref, kang, & novotny, a) . on the negative side, it has been reported that permethylation can lead to loss of o-linked acetyl groups from certain sialic acids (liu & afonso, ). an extension of the solid-phase method to allow sulfated glycans to be analyzed by maldi-tof ms has been developed (lei, mechref, & novotny, ) . sulfated glycan samples were permethylated followed by methanolytic cleavage of the sulfate groups. the desulfated, permethylated glycans were then subjected to another permethylation step using deuteromethyl iodide to label the hydroxyl groups that were exposed by removal of the sulfates. the number of attached sulfate groups could be calculated from the mass-shift caused by the presence of the deuterium label and the position of the sulfate substitution could be determined by collision-induced dissociation (decomposition) (cid). the method was validated with linear standard glycans and used to identify sulfated n-glycans released from bovine thyroid-stimulating hormone (btsh). yu et al. ( c) have shown that it is possible to permethylate sulfated glycans with methyl iodide and sodium hydroxide (ciucanu and kerek method) (ciucanu & kerek, ) , without loss of the sulfated glycans. the trick is to avoid the normal chloroform/water partition stage, which results in much of the sulfated material (unmethylated) partitioning into the aqueous phase. instead, clean-up employed c reversedphase spe cartridges and microtips self-packed with nh -beads. the methylation reaction was capable of methylating phosphates but not sulfates, allowing them to be readily identified. formation of methyl esters by reaction of the sodium salt with methyl iodide has frequently been used to stabilize sialic acids to maldi analysis by eliminating the labile proton on the acid group. an alternative procedure for methyl ester formation that provides information on the sialic acid linkage directly from the maldi spectrum has been published by wheeler, domann, and harvey ( ) (fig. ) . the sugars were desalted, dried, dissolved in methanol and treated with -( , -dimethoxy- , , triazin- -yl)- -ethylmorpholinium chloride (dmt-mm, / ). after removal of the solvent, the products were transferred directly to the maldi target and examined from dhb. however, for the analysis of small amounts of n-glycans derived from biological sources, the method benefited from an additional clean-up stage involving the use of a nafion membrane. unlike the reaction with methyl iodide with the sodium salt that produced a single peak from each sialylated glycan, irrespective of the linkage, the reaction with dmt-mm produced different derivatives from a ! -and a ! -linked sialic acids. the a ! -linked sialic acids produced only methyl esters whereas a ! -linked sialic acids were converted into their lactones providing a da difference in mass, thus enabling the linkage to be determined directly from the maldi-tof spectrum (fig. ) . negative ion cid mass spectra of these neutralized glycans provided information, in many cases, on the antenna of n-linked glycans to which the variously linked sialic acids were attached. in an application of the method to the glycoprotein carcinoembryonic antigen-related cell adhesion molecule (ceacam ), it was shown that both a- ! -and a- ! linked sialic acids were present (harvey, baruah, & scanlan, a) . -( , -dimethoxy- , , -triazil- -yl)- -methylmorpholinium chloride in the presence of ammonium chloride, converts sialylated glycans into amides with the same linkage-specific reactivity difference. thus, the a ! -linked sialylated glycans yield lactones, as above, whereas the a ! -linked compounds form amides. have used this reaction to examine blood serum glycoproteins but their technique differed from the above methyl ester formation in that the glycans were permethylated after reaction with dmt-mm. this reaction formed the methyl ester from the a ! -linked compounds as the result of the opening of the lactone ring, whereas the amide that was derived from the a ! -linked compounds was converted into the corresponding dimethylamide with a da increase in mass over that of the methyl ester. -( , -dimethoxy- , , -triazil- -yl)- -methylmorpholinium chloride can also be used to form substituted amides directly and has been used by endo et al. ( ) to link the fluorescent derivative -( -pyridylamino)ethylamine (paea, ) to the carboxy group of sialic acids in sialo-oligosaccharides and gangliosides. the derivative gave good hplc and tlc sensitivity and possessed the following advantages for maldi analysis: suppression of preferential cleavage of neu ac; enhancement of molecular-related ion intensities; simplification of ms spectra; and finally, since paea-amidation did not cleave the linkage between sugar and aglycon, allowed maldi-tof-ms and ms/ ms analyses to reveal the complete structure of the molecule. -( , -dimethoxy- , , -triazil- -yl)- -methylmorpholinium chloride has also been used to form amides from hexuronic acids (hexas) in an investigation of the n-linked glycosylation of structural subunit rvh of rapana venosa hemocyanin . as well as containing the rather unsusal (for n-glycans) hexuronic acid, the glycans also contained naturally methylated hexoses and internal fucose residues. gil et al. ( ) have stabilized sialic acids by formation of amides with acetohydrazide under mild acidic conditions in the presence of -ethyl- -( -dimethylaminopropyl) carbodiimide hydrochloride (edc, ) . glycoproteins were first reduced and alkylated and then the sialic acids were amidated. glycans were released with pngase f and a permanent charge was attached to the reducing terminus by further reaction with girard's t reagent. alternatively, derivatization with -aa was used and the products were examined both by hplc and maldi-tof ms. the amidation reaction was performed on the glycoprotein because acetohydrazide would also have reacted with the aldehyde function of the released glycan, precluding derivatization with an amine more suited to the detection method used for monitoring the glycans. the method was applied to the analysis of n-glycans from transgenic pig-derived human factor ix. another method for synthesis of methylamides has been reported by liu et al. ( i) . sialylated glycans were reacted with methylamine in the presence of ( -azabenzotriazol- yloxy)trispyrrolidinophosphonium hexafluorophosphate (pyaop, ) and n-methylmorpholine ( ) for min at room temperature. after methylamidation, sialylated glycans were analyzed by maldi-tof and esi ms without loss of the sialic acid moiety. both a ! -and a ! -linked sialic acids were quantitatively converted to their methylamides. this method was validated with n-glycans released from the well-characterized glycoproteins, fetuin, human a -acid glycoprotein, and bovine a -acid glycoprotein and was used to study n-glycans from serum glycoproteins from human, mouse, and rat. both neu ac and oacetylated analogues were stable under maldi conditions. glycopeptides tend to produce weaker signals than nonglycosylated peptides and it is frequently difficult to observe their molecular ions in samples rich in unglycosylated peptides. have developed a highly sensitive on-plate pyrene derivatization method using -pyrenyldiazomethane ( ) for acquisition of maldi ms n spectra of glycopeptides in amounts of less than fmol. unusually, the pyrene groups were easily released from glycopeptides during ionization when dhb was used as a matrix. thus, most ions were observed in their underivatized form. at the same time, pyrene derivatization was found to reduce the ionization of peptides and to produce signals from the glycopeptides that were strong enough for acquisition of ms n spectra that contained ions from both glycan and peptide. when the liquid matrix, aq-chca, was used, the sialic acid linkages of the pyrene sialylated glycopeptides were found to be stable because of inefficient release of the pyrene group allowing ms n spectra of the intact glycans to be obtained. the method was used to examine glycopeptides from ng of prostate specific antigen. ohara et al. ( ) have developed a method for analysis of sulfated carbohydrates by forming complexes with -(pyren- ylmethyl)guanidine (pmg, ) from a matrix consisting of this compound and p-nitroaniline ( / ). two types of sulfated carbohydrate were examined, chondroitin sulfate ( ) and carrageenan ( ). the pmg complexed with the sulfate group and was eliminated under maldi conditions such that the molecules produced a ladder of peaks separated by masses corresponding to losses of each complexed sulfate (mass difference u). in positive ion mode, the molecular ions from the chondroitin sulfates contained one more pmg molecule than the number of sulfate groups. one pmg group was presumed to bind to a carboxylate group. in negative mode, one less pmg molecule was bound. carrageenans showed a slightly different pattern in that the number of pmg molecules found in the positive ion spectra equaled the number of sulfates. sialic acids are classically analyzed by hplc after formation of fluorescent , -diamino- , -methylenedioxybenzene (dmb, ) derivatives. these derivatives require an a-keto acid group in the sialic acid. galuska et al. ( b) have developed a method for specifically measuring nucleotide-activated sialic acids in the presence of unactivated acids by first reducing the keto group that is present only in the non-activated acids. under the conditions of the derivatization reaction, the nucleotide group was removed leaving, in the case of the activated acids, only, an intact a-keto acid group that reacted with the dmb reagent. subsequent analysis was by hplc and maldi-tof. maldi ms is used extensively in analyses with glycan arrays as summarized in recent reviews (see table ). table lists glycans that have been used in array construction. most of the above work has been with carbohydrate preparations prior to array construction. however, maldi ms has also been used to interrogate arrays directly. as an example, a new type of glycan array covalently or non-covalently attached to aluminium oxide-coated glass slides has been developed for studies of enzymatic reactions and protein binding . the array was prepared by tagging glycans with a polyfluorinated hydrocarbon ( ) tail and spotting them robotically onto the aluminium oxide-coated slide surface which contained a layer of polyfluorinated hydrocarbon terminated with phosphonate. after incubation and washing, the noncovalent array was characterized by maldi-tof at a low laser energy without addition of matrix. a cellotetraose (d-glc-(b-( ! )-d-glc) -b-( ! )-d-glc) array was developed to study the activity and specificity of different cellulases and to differentiate the exo-and endoglucanase activities. compared to the preparation of glycan arrays on glass slides and other surfaces, this method using phosphonic acid reacting with aluminium oxide-coated was said to be more direct, convenient and effective and represented a new platform for the highthroughput analysis of protein-glycan interactions. polyfluorinated hydrocarbon tag, in another application, a ligand affinity capture (lac) method for detection of biotinylated biomolecules has been developed by jørgensen, juul-madsen, and stagsted ( ) . metal-coated glass slides were treated with amino-silane and derivatized with biotin followed by avidin. biotinylated biomolecules could then be captured and detected in the low femtomole to low picomole range by maldi-tof ms using chca in a dried droplet method. the technique was used to detect biotinylated lipopolysaccharide (lps) and its binding to the antagonist polymyxin b. the a-mannose-specific lectin concanavalin a (con a) has been bound to polydopamine-modified gold, indium, and iridium surfaces and its activity was demonstrated with rnase b using spr spectroscopy. surface-maldi-tof ms experiments revealed that the affinity of the immobilized con a depended on the oligosaccharide structure and composition. thus con a was found to bind certain man -( / ), man -( / ), and man -glcnac rnase b glycoforms more strongly than man -and man -glcnac glycoforms (morris, peterson, & tarlov, ) . concern has frequently been expressed about the ability of maldi-tof ms to provide quantitative information. fortunately, this concern appears to be unjustified as two recent studies have shown. thus, a systematic study of widely different glycopeptides was performed by thaysen-andersen, mysling, and højrup ( ) to determine the relationship between the relative abundances of the individual glycoforms and the maldi-tof ms signal strength. glycopeptides derived from glycoproteins containing neutral glycans (rnaseb, igg, and ovalbumin) were profiled and yielded excellent and reproducible quantitation (correlation coefficient r ¼ . , n ¼ ) when evaluated against a normal-phase hplc -ab glycan profile. similarly, precise quantitation was observed for various forms of n-glycans (free, permethylated, and fluorescence-labeled) using ms. in addition, three different sialo-glycopeptides from fetuin were site-specifically profiled, and good correlation between peak intensities and relative abundances was found with only a minor loss of sialic acids (r ¼ . , n ¼ ). for glycopeptide purification, a range of hydrophilic and graphite materials packed in microcolumn format was evaluated and proved capable of desalting without loss of quantitative information. thus, maldi-tof ms signal strength of glycopeptides has been found to accurately reflect the relative quantities of glycoforms, enabling rapid and sensitive site-specific glycoprofiling of n-glycan populations. the second study relates to the concern that has often been expressed over possible losses of fucose residues when glycans are ionized by maldi. tajiri, kadoya, and wada ( b) have recently assessed possible fucose loss and found it to be insignificant. fucose (fuc) is known, however, to migrate within [mþh] þ ions particularly when these are derivatized by reductive amination. experiments on fucose loss were conducted with fucosylated glycopeptides from tf and haptoglobin. studies with increasing collision energy on the [mþh] þ ions showed that the major fragmentation was cleavage at glcnac residues. biantennary glycans containing a , / -antenna fucose or a , -core fucose showed different fragmentation behaviors in experiments. stability was dependent on peptide backbone sequences. cleavage of the glcnacb ! man linkage occurred at a slightly lower activation energy than for the core fucosylated (cf) species, while the linkage of a , core fucose was more stable than that of antenna a , / fucose. however, these fragmentations only occurred at relatively high collision energy. consequently, the authors concluded that quantitation of fucosylated glycans by ms of glycopeptides, without collisional activation, was justified. the fucosylation levels calculated from the signal intensities in nanospray ionization and uv maldi mass spectra were essentially the same. the mass spectrometric profiling of glycopeptides from tf from patients with congenital disorders of glycosylation (cdg-ia and cdg-iic) demonstrated that the elevation or reduction of fucosylation in pathological conditions can be reliably determined by ms of glycopeptides. in spite of these reassurancies, it is possible that for mixtures of compounds, complex suppression effects may degrade quantitative results. consequently some investigators prefer lc/ms methods because the lc step removes, or minimizes, the effect that other compounds in a mixture have on the ionization of the target compounds. instability of sialylated glycans under maldi conditions complicates quantification and errors can possibly also be introduced by unequal ionization of glycans in mixtures. to overcome these problems with n-glycans from a therapeutic glycoprotein from a chinese hamster ovary (cho) cell line, jang et al. ( a) first formed methyl esters of the sialic acids to stabilize them and then converted the glycans to their girard's t derivatives. these derivatives have a constitutive positive charge, thus overcoming the problem of unequal ionization. percentages of sialylated glycans were measured at . and . % in two cell lines. the results were comparable to those obtained by np-hplc combined with fluorescence detection using -ab or -ap derivatization. girard's t derivatives have also been used by kim et al. ( a) to quantify glycans released from gsls originating from miniature pig endothelia and islet cells. a method using a deoxyribonucleic acid (dna) sequencer has been described for the quantitative analysis of plant nglycans released with pngase a or f and derivatized with apts ( / ). maldi-tof analysis was used to confirm structures with the aid of exoglycosidase digestions (lee et al., i) . a method for the quantification of fructo-oligosaccharides using maldi tof ms with dhb as the matrix, has been developed with the fructan, raftilose, a partially hydrolyzed inulin with a degree of polymeration - as the test compound (onofrejová, farková, & preisler, ) . nystose ( / ), which is chemically identical to the raftilose tetramer, was used as the internal standard. two mathematical approaches, conventional calculations and artificial neural networks (ann), were compared for data processing. the conventional method relied on the assumption that a constant oligomer dispersion profile will change after the addition of the internal standard. on the other hand, ann was found to compensate for a non-linear maldi response and variations in the oligomer dispersion profile with raftilose concentration. as a result, the application of ann led to lower quantification errors and excellent day-to-day repeatability compared to the conventional data analysis. this reproducibility was satisfactory for ms quantification of raftilose in the range of - pg with errors below %. the method was applied to measurements of the content of raftilose in dietary cream and it was stressed that no special optimization of the maldi process was carried out. maldi-tof ms with dhb, thap or p-nitroaniline ( / ) has been used to determine the concentrations of the unsaturated disaccharide from chondroitin sulfate ( ) obtained by enzymatic digestion of native chondroitin sulfate with chondroitin abc lyase. the signal-to-noise (s/n) ratio in the spectrum was used as a quantitative measure: amounts of chondroitin sulfate (measured as the disaccharide) down to at least fmol could be detected and there was a direct correlation between the s/n ratio and the amount of chondroitin sulfate between about and pmol although the curve was sigmoidal. the influence of different parameters such as the matrix, the applied laser intensity and different methods of data analysis were also tested. advantages and drawbacks of this approach are critically (song et al., d) bacillus anthracis tetrasaccharide with thiol linker maldi for attachment to a maleimide functionalized microarray to study of carbohydrate-antibody interactions (oberli et al., ) glycodendrimers with n group terminating in α-man, β-glcnac or β-gal tof immobilized on an acetylenyl-terminated gold substrate via click chemistry high-mannose glycans -oxime linked tof used to probe binding to malectin muc glycopeptides tof synthesis on an amine-reactive hydrogelcoated microarray glass surface. to detect autoantibodies in breast cancer correction: (blixt et al., ) n-glycan-asn-fmoc conjugates from chicken ovalbumin, bovine fetuin, and horseradish peroxidase (hrp) tof/tof printed onto commercially available nhydroxysuccinimide (nhs) -activated glass slides after deprotection. glycans interrogated using plant lectins and antibodies in sera from mice infected with schistosoma mansoni (song et al., e) n-glycan clusters tof (dhb) biantennary and high-mannose n-glycans linked to non-reducing terminus of man glcnac core, plus biotin (song et al., f) thiol-terminated nonamannoside tof coupling of a thiol-terminated mannoside to maleimide-functionalized glass surfaces derived from γ -aminopropyl silane slides (dietrich et al., ) various oligosaccharides derivatized with -( aminoethyl)aniline by reductive amination reagent has amine groups at both ends allowing the modified carbohydrates to be covalently attached to an amino-reactive nhs-activated glass surface by formation of stable amide bonds (seo et al., ) discussed in the paper. finally, the method was validated by the determination of the chondroitin sulfate content in samples of known concentration as well as in enzymatically digested bovine nasal cartilage and compared with two further established methods of chondroitin sulfate determination (the carbazole and alcian blue methods) . positive ion fragmentation of carbohydrates is fairly well understood with two types of cleavage, glycosidic cleavage that occurs between the sugar rings and involve hydrogen migration and cross-ring cleavages that involve the rupture of two of the bonds forming the rings. glycosidic cleavages revealing sequence information predominate in positive ion spectra whereas negative ion spectra frequently contain very abundant cross-ring product ions that provide information on linkage. the nomenclature introduced by domon and costello ( ) is universally used to describe the ions. the stabilities of glycosyl bond linkages in various carbohydrates have been investigated by computational calculations to find general rules of fragmentation of sodiated oligosaccharides . the calculations revealed that a-glucose, a-galactose, b-mannose, a-fucose, b-glcnac and b-galnac linkages were cleaved more easily than b-glucose, b-galactose, and a-mannose linkages because the transition states of the former were stabilized by the anomeric effect. the - linkage was more stable than the others whereas the sialyl bond was the most labile of all the linkages investigated. comparison of activation energies and binding affinities to the sodium cation revealed an increase in activation energy in proportion to the increment in binding affinity. the calculated stabilities of glycosyl bonds were: a-man (mana ! , or man ) > a-neunac (neunaca ! or ); this result was close to the experimentally deduced trend. in-source decay frequently accompanies ionization of permethylated glycans. although the presence of fragments can lead to complex spectra, they can also be used to obtain pseudo-ms spectra. smargiasso and de pauw ( ) have investigated matrices and conditions for optimal production of such ions and have concluded that dhb was the most versatile matrix; the presence or absence of isd fragments could be controlled, depending on the location of the laser shots. spectra obtained from the center of dhb targets produced mainly [mþna] þ ions that did not yield isd fragments, whereas spectra from the crystals surrounding the target yielded mainly [mþh] þ ions that fragmented readily. -aa ( / ), on the other hand, formed homogeneous matrix spots and did not induce isd. soltwisch and dreisewerd ( ) have noted that collisional cooling in an orthogonal tof mass spectrometer stabilizes fragment ions that are generated in-source and that by varying the buffer gas pressure, production of isd and post-source (psd)-type ions could be varied. under high-pressure conditions, isd-type fragments of o-linked glycosylated peptides were generated that retain the glycan. psd fragments, on the other hand, showed partial loss of the glycan from y-type peptide fragments. the detailed positive ion psd and isd fragmentation of deprotonated d-ribose ( / ) and d-fructose ( / ) has been studied with the aid of the isotopically labeled analogues, [ - c]-d-ribose, [ - c]-d-ribose and [c- - h]-ribose (bald et al., ) . the fragmentation was compared with fragmentation through dissociative electron attachment (dea). fragmentations of deprotonated monosaccharides formed in the maldi process, as well as their transient molecular anions formed upon electron attachment, were characterized by loss of different numbers of h o and ch o units. two different fragmentation pathways leading to cross-ring cleavage were identified. metastable decay of deprotonated d-ribose proceeded either via an x-type cleavage yielding fragment anions at m/z ¼ , , and (dominant ion, c h o ), or via an a-type cleavage resulting in m/z ¼ , and . this result is in contrast to previous cid studies where only a-type cross-ring cleavage was proposed. it was found that the heavier fragment anions at m/z ¼ and m/z ¼ generated via metastable decay exclusively arise from an x-type cleavage whereas the smaller fragment anions at m/z ¼ and arise predominantly from an a-type cleavage. a fast and early metastable cross-ring cleavage of deprotonated d-ribose observed in isd was dominated by x-type cleavage leading mainly to m/z ¼ and ; the latter ion is not the same as that found in psd. for dea of d-ribose, a sequential dissociation was identified that included metastable decay of the dehydrogenated molecular anion leading to m/z ¼ . the most dominant fragment ions in dea were due to faster decompositions occurring within several hundred nanoseconds (as in isd) and, thus, sequential reactions including an initial dehydrogenation could be excluded. several oligosaccharides (aldoses) and oligosaccharide alditols derived from agaroses, kand i-carrageenans obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides, have been used as model compounds to study prompt (isd) and psd fragmentation. sulfated alditols gave [mÀh] À ions in negative-ion mode together with prompt fragment ions produced mainly by desulfation. sulfated aldoses gave mainly prompt fragmentation ions (c-cleavages and desulfation). non-sulfated aldoses and alditols only gave ions ([mþna] þ ) in positive ion mode with no prompt fragmentation. the aldoses yielded cross-ring fragmentation in the psd mode. several different matrices (dhb, norharmane ( / ), ferulic acid ( / ) and the ionic liquid matrices dhb/acid-n-butylamine and ferulic acid/n-butylamine) were investigated; the best results were obtained with dhb and nor-harmane (fatema et al., ) . cid on tof/tof and magnetic sector instruments have been compared with several types of biomolecule . the sector instruments produce high-energy collisions ( - kev) yielding more structural information from many compounds than instruments producing only low energy ( kev) collisions. the case with different tof/tof instruments is less clear-cut because the collision energy is spread over a wide range. fewer differences were seen with carbohydrates than with some other compound types because most fragmentations (except formation of x-type cross-ring cleavages) occur at low energies. high-energy fragmentation in positive ion mode generally enhanced the relative abundance of cross-ring cleavage fragments, particularly x-type ions and has been used with hplc (offline) to provide a powerful method for glycoprotein analysis . full experimental details are given in the paper. experimental details for obtaining infrared multiphoton dissociation (irmpd) spectra from carbohydrates have been described . the technique offers the advantage that, because both parent and product ions can absorb ir photons, the spectra can be richer in information than those obtained by cid. in the same publication, the authors discuss sustained off-resonance irradiation-collision-induced dissociation (sori-cid) implemented with a maldi-ft-icr mass spectrometer which produced similar spectra to irmpd. experimental details are described. sori-cid with maldi-ft-icr has also been applied to muc-type o-glycans . the nm photodissociation of n-linked glycopeptides has been investigated in a modified maldi tof/fof instrument by irradiating the ions within the collision cell. singly charged glycopeptide ions from horseradish peroxidase (hrp) yielded abundant peptide and glycan fragments. the peptide fragments included a series of x-, y-, v-, and w-ions with the glycan remaining intact. these ions provide information about the peptide sequence and the glycosylation site. the glycan fragmented to give both glycosidic fragments and abundant cross-ring fragments that were not observed in low-energy cid spectra. doubly charged glycopeptides generated by nanospray in a linear it mass spectrometer also yielded peptide and glycan fragments. however, the former were dominated by low-energy fragments such as b-and y-type ions while the glycan was primarily cleaved at glycosidic bonds (zhang & reilly, ). maldi-lift-tof-ms/ms and esi tandem mass spectrometry (esi-it-ms/ms n ) have been used for the characterization of free oligosialic acids and those derivatized with dmb, as well as partially o-acetylated derivatives. electrospray required the acids to be lactonized but the larger lactones could only be detected by maldi-tof. the fragmentation spectra were dominated by simultaneous cleavage of glycosidic linkages and the corresponding lactone ring, whereas classical cross-ring fragments were of minor abundance. however, the combined use of the two different types of fragmentation analysis allowed a sensitive and detailed characterization of both short-and longchained species. furthermore, oxidation of the nonreducing end sugar moiety enabled sequence determination and localization of acetylated and nonacetylated sialic acid residues (galuska et al., a) . the effect of the pressure of cooling gas in the ion source of an orthogonal-tof ms has a strong influence on the extent of analyte ion fragmentation. soltwisch et al. ( ) have investi-gated the effect of this parameter on peptide and oligosaccharide fragmentation using substance p and the milk sugar, lnfp-ii ( ), respectively in both uv-and/or ir-maldi. a range of pressures, from . to . mbar was used in combination with seven different buffer gases (he, ne, ar, n , co , ch , and isobutane). the influence of the ion extraction voltage on the analyte fragmentation was also investigated for a selected set of gas parameters. fragment ions exhibited characteristic fragment yield-pressure dependencies that were classified into three groups. for lnfp-ii, the yield of molecular ions rose with pressure until at the higher pressures, it was similar to that from substance p. the authors speculated that the consistently lower ion yields reported from oligosaccharides could be attributed to the generally low pressures used when recording their spectra. with respect to fragmentation, glycosidic fragment ions (termed type i) ions dominated the spectra at low pressure but their relative abundance fell dramatically as the pressure rose. the ions resembled species that are also found in maldi-psd ms. the abundance of type ii ions, which resembled typical isd fragments, and consisted mainly of cross-ring products, rose with pressure, probably because of a reduction in the secondary fragmentation process that resulted in loss of fucose. a few other ions, termed type iii ions did not show such dramatic changes with pressure. comparing the yields of fragmentation for the different buffer gases revealed a correlation between their internal degrees of freedom and their collisional cooling efficiency. changing the buffer gas pressure and/or extraction field provided an easy means to influence analyte ion fragmentation and to switch from the primary production of one type of fragment species to another. hexose rearrangements of protonated molecules of n-glycopeptides and reductively aminated n-glycans have been observed by maldi-tof/tof-ms/ms and esi-it-ms/ms (wuhrer, koeleman, & deelder, b) . fragmentation of proton adducts of -ab and -aa-labeled high-mannose n-glycans from rnaseb resulted in transfer of one to five hex residues to the fluorescently labeled innermost glcnac residue. glycopeptides from various biological sources containing high-mannose glycans were likewise shown to undergo hex rearrangement reactions, resulting in migration of one or two hex moieties to the chitobiose core. tryptic fc-glycopeptides from igg peptides containing biantennary n-glycans were also shown to undergo hex rearrangements. such rearrangement products can cause major problems with the interpretation of unknown glycans but, fortunately, do not appear to occur from [mþna] þ ions, the major ions in maldi spectra of most neutral carbohydrates. the use of computer software for analyzing carbohydrate spectra is not as advanced as that for proteins; nevertheless, many investigators have developed software for spectral interpretation, composition calculations, and have constructed databases, usually for specific glycan types. much of this software is applicable to spectra generated from the majority of common ion sources. a good source of information is the book bioinformatics for glycobiology and glycomics: an introduction by the late claus-wilhelm von der lieth, luetteke, and frank ( ) . in addition, various tools for glycomics and available databases are covered in a comprehensive review by frank and schloissnig ( ) . the simplest of these algorithms calculates compositions from m/z values. although such software is extremely valuable, a composition is not a structure and such programs should not be used as the basis of labeling spectra unless the proposed structures are confirmed by suitable techniques. one such tool reported in the review period is called lipid and calculates molecular compositions for glycerophospholipids, gsls, fatty acids and small oligosaccharides from m/z values entered as single values or as mass lists. the user-extendable software is a microsoft excel add-in developed using visual basic for applications and is compatible with all versions of ms excel since ms excel (hübner, crone, & lindner, ) . kronewitter et al. ( ) have constructed a library of possible n-glycan masses by successive dismantling of tetraantennary hybrid and high-mannose glycans. these calculations gave the possible masses that would be expected in a glycan mixture. three hundred thirty one distinct neutral compositions were obtained but many of these will represent several isomeric glycans. the smallest mass difference that was observed was . da. however, many of the masses coincided with isotope peaks from ions of different compositions meaning that, without deisotoping, a resolution of at least , would be needed to resolve all peaks. the theoretical masses were matched against measured masses from n-glycans released from human serum glycoproteins and discrete compositions were detected. in a similar way, an in silico glycan database of possible nglycan compositions has been constructed by addition of known monosaccharide residues, such as those in a neu ac-gal-glcnac antenna, to the common trimannosyl chitobiose core. the derived masses were then matched to the experimental mass and the software, named glyquest, predicted compositions and possible structures. next, it calculated possible glycosidic fragments from the proposed structures, matched these to the experimental mass spectrum and constructed a spectrum labeled with the proposed structures (gao, ) . the software could also be applied to glycans containing fluorescent labels such as -ab but was not applicable to glycopeptides with unknown modifications. however, as with much of the software developed for this work, the detailed structural details such as the linkage of each monosaccharide are not available and the software must be regarded as a guide to the total structure. a similar "branchand-bound" algorithm developed by peltoniemi, joenväärä, and renkonen ( ) starts with the trimannosyl-chitobiose core and then constructs n-type glycans in an iterative process until the target carbohydrate composition is reached. the algorithm identified several glycans from tf and human serum samples. glycospectrumscan is a web-based tool that identifies the glycoheterogeneity on a peptide from mass spectrometric data. two experimental data sets are required as inputs: ( ) oligosaccharide compositions of the n-and/or o-linked glycans present in the sample and ( ) in silico derived peptide masses of proteolytically digested proteins with a potential number of nand/or o-glycosylation sites. glycospectrumscan uses ms rather than ms/ms data, to identify glycopeptides and determine the relative distribution of n-and o-glycoforms at each site. it can be used to assign monosaccharide compositions on glycopeptides with either single or multiple glycosylation sites. the algorithm allows the input of raw mass data, including multiply charged ions, making it applicable for both esi and maldi data from all mass spectrometer platforms. low resolution data from, for example, its are heavily smoothed to yield the average mass whereas data from high resolution instruments receive a milder smooth and deisotoping to give the monoisotopic mass. the software was used to characterize the n-and o-linked glycopeptides from human secretory iga (siga), consisting of secretory component ( n-linked sites), iga ( n-linked, o-linked sites), iga ( n-linked sites) and the j-chain ( n-linked site). glycospectrumscan is freely available at www.glycospectrumscan.org (deshpande et al., ) . prediction of glycosylation sites is another area of software development. a program that predicts n-and o-glycosylation sites based on local information, general protein information, sub-cellular localization and binding specificity of glycosyltransferases has been developed and was claimed to be about % accurate (sasaki, nagamine, & sakakibara, a) . however, as with all predictive programs that are not % accurate, results should only be taken as a guide for designing appropriate location experiments. software that attempts to predict structures from spectra is possibly the most active area in computer applications. simglycan is one such tool (apte & meitei, ) . the software accepts raw or standard experimental ms data files, matches them with its own database of theoretical fragments and generates a list of probable candidate structures. each structure is scored to reflect how closely it matches the experimental data. the software also predicts novel glycan structures by drawing a glycan and mapping it onto the experimental spectrum. other biological information is also available for easy reference. the program can be downloaded from http://www.premierbiosoft. com/glycan/index.html. another software platform for carbohydrate assignment is sysbioware, developed by vakhrushev, dadimov, and peter-katalinić ( ) and designed to work directly from raw ms data. the data are first imported into the spectrum browser, baseline corrected and denoized. peak detection is based on shape matching and the software detects monoisotopic m/z values and charge states. a biological filter is used during compositional analysis of the monoisotopic ions. the software was successfully tested with human urine. sysbioware, a software platform developed for ms data evaluation in glycomics, has been applied to the interpretation of spectra from human serum gsls. the masses of predicted ions arising from cleavages in the glycan and the ceramide moieties were calculated, thus enabling structural characterization of both entities. the calculated masses were then used to match with those in the spectra for structural identification . böcker, kehr, and rasche böcker ( ) have presented an algorithm for calculating glycan structures from tandem mass spectra. twenty-four spectra (of [mþh] þ ions) of -ap-labeled n-glycans obtained from batroxobin (from bothrops moojeni venom) were used as test compounds, the spectra were measured with a tof/tof instrument with a maldi ion source and the algorithm rapidly predicted possible topologies. biological restraints needed to be used to limit the predictions to reasonable structures. goldberg et al. ( ) have compared three algorithms, "max subgraph," "parsimony," and "randomwalk" that make inferences about glycan synthesis from biological knowledge for their ability to assign structures from single-ms spectra from a variety of tissues and organisms, containing more than , manually annotated peaks. max subgraph behaved better than the other two but only uniquely assigned the correct structure to about half of the peaks in out of the spectra. a computer model that predicts n-linked glycan profiles based on cellular enzyme activities has been developed (krambeck et al., ) . the paper describes the expansion of a previously developed detailed model for n-linked glycosylation (krambeck & betenbaugh, ) with the further application to analyze maldi-tof mass spectra of human n-glycans. the glycosylation reaction network is automatically generated by the model, based on the reaction specificities of the glycosylation enzymes and allows prediction of the complete glycan profile and its abundances for any set of assumed enzyme concentrations and reaction rate parameters. a predicted mass spectrum of model-calculated glycan profiles is obtained and enzyme concentrations are adjusted to bring the theoretically calculated mass spectrum into agreement with that obtained experimentally. the result is a complete characterization of a measured glycan mass spectrum containing hundreds of masses in terms of the activities of enzymes. in addition, a complete annotation of the mass spectrum in terms of glycan structure is produced, including the proportions of isomers within each peak. the method was applied to mass spectrometric data obtained from normal human monocytes and monocytic leukemia (thp ) cells. a kinetic model originally developed for the prediction of peptide cid spectra has been extended to predict the cid spectra of n-glycopeptides. the model was trained with cid spectra obtained with an ion trap and was able to predict cid spectra with excellent accuracy in ion intensities for n-glycopeptides up to , da in mass. the program is said to be capable of predicting up to common glycoforms including high-mannose, hybrid and complex n-glycans with two to four antennae (zhang & shah, ) . spencer et al. ( ) have devised a computational approach to predict the fine structure and patterns of domain organization of heparan sulfate (hs). analysis uses chemical composition data obtained after complete and partial enzymatic digestion of mixtures of hs chains and produces populations of theoretical hs chains with structures that meet both biosynthesis and enzyme degradation rules. the program was used to analyze hs from various cell types and good agreement was found between experimental data and computer predictions. glycoviewer (http://www.systemsbiology.org.au/glycoviewer) is a web-based tool that can visualize, summarize, and compare sets of glycan structures. it takes as its input a list of glycan structures in international union of pure and applied chemistry (iupac) format or glycan structures constructed with a sugar structure builder. the output is a graphic, which summarizes all salient features of the glycans according to features such as the nature and length of any chains and the types of terminal epitopes. the tool can summarize several hundred glycan structures in a single figure. the report contains an example of use of the tool for analysis of normal and disease associated glycans from the human glycoproteome (joshi et al., ) . several glycan databases are available. the kyoto encyclopedia of genes and genomes (kegg) glycan databases contain useful information on glycan structures and metabolic pathways (hashimoto & kanehisa, ) and data mining the protein data bank for glycol-related data using the glycosciences. de internet portal has been discussed in methods in molecular biology (lütteke & von der lieth, ) . ito et al. ( a) have synthesized n-and o-linked glycan libraries (named glycan mass spectral database, gmdb) and constructed a library of their positive ion ms , ms and ms fragmentation spectra. n-glycans were in the form of their -ap derivatives whereas oglycans that were released by b-elimination were not. the library was said to be accessible on-line at http://riodbdev.ibase. aist.go.jp/rcmg/glycodb/ms (however, attempts by the reviewer to connect to the site have failed.) it can be searched either by mw of glycan composition in terms of isobaric monosaccharides and instructions on how to use the software are given in the paper. although such databases and tools for glycomics are readily available on the web, these have, until now, been isolated. this unfavorable situation has been discussed (von der lieth, ) and has been largely overcome by ranzinger et al. ( ) who have developed glycomedb, a meta-database for public carbohydrate sequences. at the time of publication ( ) it contained , unique structures in glycoct (www. glycome-db.org) encoding, referencing more than , external records from , different taxonomic sources. a user-friendly, web-based graphical interface has been developed which allows taxonomic and structural data to be entered and searched. the structural search possibilities include substructure search, similarity search, and maximum common substructure. a novel search refinement mechanism allows the assembly of complex queries. with glycomedb, it is now possible to use a single portal to access all digitally encoded, public structural data in glycomics and to perform complex queries with the help of a web-based user interface. a list of databases is given in aoki-kinoshita ( ). n-and o-glycan structures are usually depicted with small cartoons in which each constituent monosaccharide is shown by a symbol. unfortunately, there is no consensus on which symbol to use for any particular monosaccharide with most investigators preferring to develop their own system. several years ago, the consortium for functional glycomics (cfg) attempted to redress the problem with the introduction of a system that has since been adopted by several laboratories. unfortunately, this system has several major drawbacks: (a) it does not diagrammatically show linkage or anomericity and (b) it uses color to differentiate hexoses, thus causing problems when structures are printed in black and white and making the system difficult to use with pen or pencil on paper. a new system that overcomes these problems has recently been introduced (harvey et al., c) . monosaccharides are shown as shapes with various additions to indicate functional groups (e.g. an inclusive dot to indicate a deoxy-sugar and a filled shape to code for an n-acetyl sugar). linkage is shown by the angle of the lines linking the sugar symbols and anomericity is shown by the type of line (full for a b-bond and broken for an a-link). examples can be seen below. although color is not used to define monosaccharides, the cfg colors can be used with the oxford symbols. unfortunately, color was omitted from the table of symbols in the original article but was published later as an erratum (harvey et al., b) . the article received comments from the authors of the cfg system (varki et al., ) and discussion is continuing. this scheme and others are compared in a review by frank and schloissnig ( ) . two tools for displaying n-glycans found in the mammalian cho cell line have been developed (mcdonald et al., ) . both take as input the -digit identifier devised by krambeck and betenbaugh ( ) that uniquely defines each structure assuming the existence of the trimannosyl-chitobiose core. the first of these tools, glycoform, is designed to display a single structure from an identifier entered by the user. the display is updated in real time, using symbols for the sugar residues, or in text-only form. the two symbol sets discussed above are used, the symbols and layout devised by the cfg http://www. functionalglycomics.org/static/consortium/nomenclature.shtml or the alternative "oxford" system used by glycobase, a relation database of -ab labeled n-glycans (campbell et al., ) . however, although glycoform can display structures using the oxford system, unfortunately, it does not display the correct linkage information that is inherent to the full oxford system. in addition, glycoform can display the name of the glycan as used by glycobase. glycobase formalism does not yet handle nacetyllactosamine (naclac, ) repeating units and is, therefore, currently limited to structures with one gal residue per branch. structures can be added to a library, which is recorded in a preference file and loaded automatically. individual structures can be saved as image files either portable network graphics (png), jpeg or windows bitmap (bmp) formats. the second program, glycologue, reads a file containing columnar data of nine-digit codes, which can be displayed on-screen and printed at high resolution. both programs, for windows, mac os x and linux x gtk can be downloaded from http://www.boxer.tcd. ie/gf. a major problem with the analysis of monosaccharides or small oligosaccharides by maldi is the presence of very abundant ions from matrices such as dhb in the low mass region of the spectra. various methods for overcoming this disadvantage are discussed above. nevertheless, maldi with conventional matrices has produced results and the technique works well for the larger oligosaccharides. thus, oligosaccharides from dextran, alginate, hyaluronan and chondroitin sulfate have been characterized by maldi-tof ms directly from a tlc plate after soaking it in the dhb matrix. the plate had a metal backing to ensure electrical contact. the tlc solvent system was n-butanol/formic acid/water ( : : , v/v/v). it was found that the high content of formic acid caused few problems but was responsible for partial formylation of glycosaminoglycans (gags) and minor n-acetyl loss from hyaluronan and chondroitin sulfate . a comparative study of maldi and a new technique, electrospray droplet impact secondary ion mass spectrometry (edi/sims) has been applied directly to fruits such as bananas, apples, grapes and strawberries. the major constituents, fructose ( / ), glucose ( / ), sucrose ( ) and organic acids gave abundant [m þ k] þ ions positive mode and cf coo À adducts in negative mode (the cf coo À ions came from cf cooh in the esi spray. these negative ion spectra were almost free of background ions. maldi from dhb, on the other hand, although producing positive ions gave virtually no ionization in negative ion mode (asakawa and hiraoka, ) . reviews of the analysis of polysaccharides are listed in table . large polysaccharides need to be hydrolyzed, often enzymatically but sometimes chemically, to smaller fragments before they are amenable to maldi analysis. one chemical method involves the selective cleavage of the rhap-( ! )-a-galap linkage in rhamnogalacturonans. enzymic cleavage of this linkage is often ineffective, especially in highly branched rhamnogalacturonans but deng et al. ( ) have developed an improved chemical fragmentation method based on belimination of esterified -linked galacturonic acid (galpa, ) residues that overcomes the problem. at least % of the carboxyl groups of the galpa residues in a. thaliana seed mucilage were converted to methyl or hydroxypropyl esters and b-elimination was found to be more extensive with hydroxypropyl-esterified than with methyl-esterified rhamnogalacturonans. the non-reducing -deoxy-b-l-threo-hex- -enepyranosyluronic acid ( ) residue formed by the b-elimination reaction was removed by treatment with aqueous n-bromosuccinimide. this method was used to fragment the branched rhamnogalacturonan from peppergrass seed mucilage with product characterization by maldi-tof ms, glycosyl residue composition analysis, and and d nmr spectroscopy. the results showed that the most abundant low-mw fragments contained a backbone rhamnose ( / ) residue substituted at o- with a single side-chain, and suggest that peppergrass seed mucilage rhamnogalacturonans is composed mainly of the another chemical method for analysis of rhamnogalacturonan ii makes use of mild acid hydrolysis to hydrolyze the acidlabile monosaccharides -deoxy-d-manno-oct- -ulosonic acid (kdo, / ), -deoxy-d-lyxo- -heptulosonic acid (dha) and apiose (api, ) at the branchpoint between the sidechains and the oligogalacturonide backbone to release short polysaccharide chains that were analyzed by esi and maldi-tof ms (séveno et al., ) . the method was optimized using citrus pectin and then applied to other plant species. experimental details for examination of extracellular polysaccharides (epss) from plants following digestion with a variety of endoglycosiodases and maldi-tof analysis from dhb has been reported by günl, gille, and pauly ( ) . other examples are listed in tables (plants) and (bacteria). ionization efficiencies of cyclodextrins and corresponding linear compounds (maltohexaose and maltoheptaose) have been compared together with differences in the ionization efficiencies of aand b-cyclodextrins ( / ) . alkali metal salts of li þ , na þ , k þ , and cs þ were used as the cationizing agents to enhance the ionization efficiency. relative ion intensities of the cyclodextrins were much larger than those of the linear carbohydrates and the difference showed an increasing trend with the size of the alkali metal cation. b-cyclodextrin had higher ionization efficiency than a-cyclodextrin ( / ) and the difference increased with increasing size of the alkali metal cation. the ionization efficiency was also found to be affected by the counter anions. the higher ionization efficiencies of cyclodextrins were explained with the number of coordination sites and the binding energies. analysis of milk oligosaccharides appears to be receiving increasing attention. reviews of mass spectrometric methods for their analysis have been published by niñonuevo and lebrilla ( ) , kolarich and packer ( ) and urashima et al. ( ) . wu et al. ( b) have developed an annotated library of neutral human milk oligosaccharides with characterization by hplc, maldi-ft-icr ms and exoglycosidase digestion. pyrene labeling (amano et al., a) has been used by amano et al. ( b) to enable neutral carbohydrates from human milk to be observed by negative ion maldi-tof ms n . the neutral oligosaccharides from the milk of a woman (blood type a, le bþ ) were obtained by anion-exchange column chromatography after the removal of lipids and proteins. further fractionation was performed by means of aleuria aurantia lectin-sepharose column chromatography and reversed-phase hplc after labeling. twenty-two oligosaccharides with decaose cores were identified and, of these had novel structures. locascio et al. ( ) have published a method for measuring the consumption of human milk oligosaccharides by strains of bifidobacteria. oligosaccharides were quantified with deuterated and reduced oligosaccharide standards that were added after bacterial growth and results were processed with inhouse software called glycolyzer after removal of contributions from c isotopes. high growth was found for bifidobacterium longum biovar infantis strains, which consumed nearly all available substrates, while other bifidobacterial strains showed low or only moderate growth ability. other examples of the use of maldi analysis of milk glycans are listed in table . glycoproteins and their attached n-and o-glycans is possibly the largest group of compounds that have been analyzed by maldi, catalyzed largely by developments in the biotechnology industry. analysis of these compounds has been reviewed by many authors (table ) . n-glycans are normally attached to an asparagine residue in a asn-(xxx)ser-(thr) consensus sequence where xxx is any amino acid (xxx) except proline however, asn-linked n-glycans have recently been found at the . - . % level on a non-consensus amino acid sequence (tvswn sgal) in the c h domain of human antibodies and on igg (valliere-douglass et al., b). many investigators have published methods for glycoprotein enrichment. solid-phase glycan/glycoprotein capturing methods have become popular in recent years and some of these have been highlighted in an article by . (kooy et al., ) upis ceramboides (alaskan beetle) xylomannan first identification of a nonprotein anti-freeze compound (walters et al., ) commercial chitosan oligosaccharides analysis of commercial samples and preparation of sample with glcnac - not stated but has been found in cryptococcus neoformans human macrophage activation shown to be triggered by chitotriosidase-mediated chitin and chitosan degradation (gorzelanny et al., ) format (not all items present): maldi method (matrix), compounds run (derivative), other methods. enrichment strategies for glycopeptides based on lectinaffinity chromatography and polysaccharide hydrophilic affinity physicochemical chromatography have been discussed by ito, hayama, and hirabayashi ( b) . the combined use of these techniques effectively removes non-glycosylated peptides. the ability of boronic acids to form cyclic derivatives with the cis-dihydroxy groups present in most glycans has been extensively used. thus, -aminophenylboronic (apb) acid ( / )functionalized beads, mesoporous silica, and nanodiamonds have been developed to enrich glycosylated peptides and proteins but the direct immobilization of the apb group was found to be insufficient to suppress nonspecific adsorption/ adhesion. consequently jang et al. ( b) have designed a selfassembled monolayer (sam)-based plate, which contained a spacer group such as oligo(ethylene glycol) to reduce the nonspecific adsorption/adhesion, for direct detection of glycoproteins after affinity-capture (or enrichment) on the plate. the utility of the plate was demonstrated with model glycoproteins such as ribonuclease g and tf. a two-stage glycopeptide enrichment technique using boronate-functionalized beads has been developed by chen et al. ( f) . samples were incubated with the functionalized magnetic beads in slightly alkaline conditions at room temperature for about hr with gentle shaking. the beads were washed and the enriched glycoproteins/peptides were eluted under acid conditions and dried in a speed-vac evaporator. the glycoproteins were then either dissolved in mm ammonium bicarbonate and digested by lys-c overnight or separated by sodium dodecyl sulfate (sds)-polyacrylamide gel electrophoresis (page) and the resulting gel-bands were digested in-gel by lys-c overnight. the compounds were then ready for a second enrichment. alternatively, digestions could be carried out with trypsin. analysis was by maldi-tof. boronic acid functionalized nanoparticles have also been used to concentrate antibodies by capturing the carbohydrates attached to the fc region of igg . chalagalla and sun ( ) have prepared a boronic acid-containing polymer capped with biotin ( ) for linkage to a magnetic bead and used the product for glycan capture and lin et al. ( f) have constructed magnetic nanoparticles with immobilized apb acid for glycoprotein capture. "sno @poly(hema-co-st-co-vpba)" core-shell nanoparticles containing boronic acid groups have been prepared by of copolymerization between hydroxyethyl methacrylate grafted ( ) on sno nanoparticles, styrene, and -vinylphenylboronic acid (vpba, ). they have been used to extract tryptic peptides from hrp, bovine asilotf and human serum glycoproteins. analysis was by maldi-ms/ ms using an axima qit instrument (sheng, xia, & yan, survey epo and of several analytical methods including maldi, chromatography (reichel & gmeiner, ) application of proteomics in biomarker discovery mainly proteins and glycoproteins. a novel boronic acid functionalized mesoporous silica, which holds the attractive features of high surface area and large porosity has also been used to concentrate glycopeptides. in comparison to direct (traditional) analysis, this method was stated to enabled two orders of magnitude improvement in the detection limit of glycopeptides irrespective of the nature of the attached glycans . the same group has also synthesized boronic acid functionalized core-satellite composite nanoparticles that possess both the superparamagnetic properties of magnetic nanoparticles and the surface chemistry of aunps. glycoproteins or glycopeptides could be obtained in high yield by use of a magnet. the composite nanoparticles were used to enrich glycosylated proteins from human colorectal cancer tissues for identification of n-glycosylation sites. in all, unique glycosylation sites mapped to different glycoproteins were identified, of which sites ( . %) were new. boronic acid functionalized gold-coated si wafers have been used as maldi plates to isolate and enrich glycopeptides . this method was claimed to be beneficial for several reasons. thus, solution transfer and eluting steps required in conventional enrichment strategies were not needed, thereby reducing sample loss. secondly, the lower limits of detection of glycopeptides were said to have been increased by two orders of magnitude. thirdly, non-specific bindings were not detected even when non-glycopeptides were times more concentrated than glycopeptides. furthermore, glycopeptides could be detected in the presence of mm ammonium bicarbonate or the physiological buffer, pbs. in a related method (tang et al., a; yao et al., ) , aunps were first spotted and sintered onto a stainless steel plate, then modified with -mercaptophenylboronic acid ( ) to provide a porous substrate with a large surface for capturing glycopeptides from peptide mixtures. the captured peptides were then analyzed by maldi-tof ms simply by deposition of a dhb matrix. the technique enabled sample enrichment, washing and detection steps to be fulfilled on a single maldi target plate. well-characterized glycoproteins, such as hrp and asialofetuin, were employed as standards to investigate the enrichment efficiency. fe o @c@au magnetic microspheres functionalized with -mercaptophenylboronic acid have been synthesized by the same group (qi et al., ) and successfully used for enrichment of glycoproteins and glycopeptides. a polyfunctional device has been constructed from macroporous silica foam (mosf) containing boronic acid (bmosf) or amino groups (nh -mosf) and used to immobilize enzymes such as trypsin and selectively enrich glycopeptides. use of the device considerably speeded up hydrolysis times as demonstrated with glycopeptides from hrp (qian et al., ) . tang et al. ( a) have immobilized the lectin con a on apb acid-functionalized magnetic nanoparticles using methyl a-d-mannopyranoside as a linker. the selective capturing ability of the con a-modified nanoparticles was tested using standard glycoproteins and cell lysate of human hepatocelluar carcinoma cell line , . regeneration of the protein-immobilized nanoparticles could easily be performed by utilizing the reversible binding between the boronic acid and the sugar. cona has also been used in conjunction with hollow fiber flow field-flow fractionation (hf ) to preconcentrate high mannose type nlinked glycoproteins from bacterial lysates as exemplified by glycoproteins from streptococcus pyogenes . the specificity of datura stramonium agglutinin (dsa) for triand tetra-antennary glycans has been utilized to enrich human liver glycoproteins containing these larger glycans which were then separated and identified by sds-page followed by maldi-tof analysis (sun et al., b) . the performance of chromatographic columns consisting of agarose-bead-bound -aminophenyl boronic acid, agarosebound wheat-germ agglutinin (wga) or a mixture of both compounds (boronic acid lectin affinity chromatography, blac) has been evaluated for glycoprotein enrichment using the model proteins of rnaseb and trypsin inhibitor in the presence of the non-glycosylated proteins, myoglobin (neutral) and lysozyme (basic) over a wide temperature range ( - ˚c). the results showed that glycoaffinity micropartitioning at ˚c provided the highest recovery rate for glycoprotein enrichment. a large amount of lysozyme was present in the elution fractions of the -aminophenyl boronic acid-containing micropartitioning columns due to an ion-exchange mechanism occurring between the positively charged protein and the negatively charged stationary phase. at ˚c, nonspecific interactions with the agarose carrier prevailed, evidenced by the presence of myoglobin in the eluate (olajos et al., ) . a novel boronate affinity monolith, poly-( -acrylamidophenylboronic acid-co-ethylene dimethacrylate) ( ) has been prepared in mm capillaries by a one-step in situ polymerization procedure . the monolith was used to separate glycopeptides from peptides produced from hrp and to separate this glycosylated protein from non-glycosylated bovine serum albumin (bsa). the maldi-tof spectrum of the hrp peptides showed little evidence of the presence of glycopeptides before passage through the capillary but revealed abundant glycopeptide ions after treatment. mass spectrometry reviews doi . /mas b. other solid-phase methods titanium dioxide (tio ) microspheres, synthesized by a sol-gel method, have a high affinity for the acid groups of sialic acids and peptides. they have successfully been used for simultaneous enrichment of glycopeptides and phosphopeptides from, for example bovine rnaseb and human igg . detection was by esi but the method would be equally applicable to maldi-tof analysis. mysling et al. ( ) have used zic-hilic in a microcolumn format for spe and glycoprotein enrichment involving trifluoroacetic acid (tfa) ion pairing to increase the hydrophilicity difference between glycopeptides and non-glycosylated peptides. three mobile phases were investigated: % formic acid, . % tfa and % tfa all containing % acetonitrile and experiments were conducted on single glycoproteins, a five-glycoprotein mixture and depleted plasma. the presence of tfa, particularly at the % level, in the mobile phase significantly improved the glycopeptide enrichment ( . -fold) as evaluated by maldi-tof ms and rp-lc-esi-ms/ms. four types of hydrazine functionalized carboxyl and epoxysilanized magnetic particles (hfmp) have been developed by sun et al. ( a) for isolation of glycopeptides. particles prepared by adipic dihydrazide functionalization from carboxylsilanized magnetic particles yielded the maximum capture capacity. the method was verified by successful isolation of all formerly glycosylated peptides from standard glycoproteins (fetuin, rnaseb, and human serum albumin (hsa)) and by identification of their glycosylation sites. c. other techniques maldi-tof ms has been used by carvalho et al. ( ) to characterize the aand b-subunit of recombinant and pituitary glycoprotein hormones that have been separated by a new method of incubating the glycoproteins overnight with acetic acid ( . - . m) at ˚c. a. use of mass spectrometry to detect glycosylation of proteins a simple method to detect glycosylation is to measure the mass of a glycoprotein and then to repeat the measurement after incubation with pngase f to remove the n-glycans. the method has been used to detect the presence of n-glycans in recombinant bovine cd expressed in pichia pastoris (muller-steffner et al., ) . the molar masses of non-glycosylated ( , da) and penta-glycosylated thermomyces lanuginosus lipase ( , ) as measured by maldi-tof ms have confirmed glycosylation and shown that each glycan-moiety adds approximately , da to the molar masses (pinholt et al., ) . in another example, cu/zn superoxide dismutase monomer was determined to have a mass of , da before deglycosylation and , da afterwards giving a mass for the glycan of about , (nedeva et al., ) . the difference between the sequence mass ( , da) and measured mass of about , da of the peroxidase from royal palm tree (roystonea regia) together with the fact that the amino-acid sequence includes possible n-glycosylation sites, suggests heavy glycosylation. glycosylation sites were identified, in this case, by n-terminal sequencing and maldi-tof-ms analysis of tryptic peptides . b. mass spectrometric detection of glycoforms of intact glycoproteins although resolution of glycoforms by maldi-tof ms is generally inferior to that obtained by esi, glycoproteins with masses in the region of kda, containing only a limited number of glycoforms have been resolved successfully as illustrated by the resolution of four glycoforms of antithrombin in a study of altered glycosylation causing antithrombin deficiency . glycans appeared to be sialylated biantennary (residue mass , da). ogawa et al. ( ) have observed resolution of glycoforms of stereum purpureum endopolygalacturonase i produced in p. pastoris ( . kda). three main ion peaks corresponding to the protein with the high-mannose glycans man - glcnac together with some minor unresolved ions were observed. c. detection of glycosylation sites and site occupancy the most common method for detecting site occupancy is to utilize the conversion of the asn to which the n-glycans are attached to aspartic acid (asp) when the glycans are released with pngase f. the increase by one mass unit is readily detected by ms. the method has been used, for example to confirm occupancy of six of the seven potential n-linked glycosylation sites in the envelope glycoprotein gp and three of the four potential sites in the gp protein of the yellow head virus from the penaeus monodon shrimp (soowannayan et al., ) . periodate oxidation and hydrazide capture on a solid support have been used by lewandrowski and sickmann ( ) to study glycosylation sites in human platelet proteins. the bound glycoproteins were sufficiently stable to allow washing, following which the proteins were hydrolyzed. glycopeptides remained bound to the solid support through the glycan moiety from where they were released with pngase f and the glycosylation site was identified by means of the asn to asp conversion. a new method using tandem o stable isotope labeling (tosil) to quantify the n-glycosylation site occupancy has been reported (liu et al., k) . glycoproteins were digested with trypsin and pngase f in the presence of either h o or h o. three o atoms were introduced into n-glycosylated peptides, two at the carboxyl terminus of all peptides and the third at the n-glycosylation site. the samples were mixed to give pairs of ions in the resulting maldi or esi spectra. a unique mass shift of da was produced by n-glycosylated peptide with a single glycosylation site, whereas non-glycosylated peptides produced an ion pair spaced by only da. intensity ratios could be used to monitor site occupancy in various physiological and disease conditions. the method yielded good linearity within a -fold dynamic range with the correlation coefficient r > . . the standard deviation (sd) ranged from . to . , for four glycopeptides from two model glycoproteins. the method was used to monitor glycoproteins in the sera from a patient with ovarian cancer and healthy individuals. eighty-six n-glycosylation sites were quantified and n-glycosylation levels of glycopeptides showed significant changes. a similar o-labeling technique was used by alvarez-manilla ( b) to identify n-glycosylation sites in con-a-extracted glycopeptides from pluripotent murine embryonic stem cells. glycopeptides rather than glycoproteins were extracted from tryptic digests to avoid false positives produced from non-glycosylated proteins that were bound to extracted glycoproteins if no digestion had been performed. segu et al. ( b) have published a method for detecting sites occupied by glycans carrying a fucose residue attached to the core. the method made use of the endoglycosidase m which, like pngase f has a broad spectrum of activity with the exceptions that (a) it is not active on core-fucosylated glycans and (b) it shows reduced activity with larger glycans. the second exception was overcome by conducting incubations in the presence of sialidase, b-galactosidase and b-n-acetylhexosaminidase, which reduced the size of the glycans. then, the results were compared with the products of digestions performed with pngase f allowing the core-fucosylated sites to be determined. analyses were by lc/ms but the technique would be equally applicable to maldi-tof analysis. d. analysis of glycopeptides because many glycoproteins are too large and heavily glycosylated for direct analysis by ms, much work is performed on derived glycopeptides, most commonly tryptic glycopeptides. tryptic cleavage of glycoproteins is frequently hindered by steric hindrance imposed by the glycans but improvements can be made by the use of heat to increase the rate of proteolysis. segu, hammad, and mechref ( a) have used microwaveassisted enzymatic digestion to achieve higher sequence coverage of several model glycoproteins such as fetuin, tf, and fibrinogen. efficient digestion was achieved in min at an optimum temperature of ˚c; there was no apparent loss or partial cleavage of the glycans. signals from glycopeptides are often weak or absent from the spectra of mixed peptides and glycopeptides, a situation that can be improved by fractionation of the two compound classes. wohlgemuth et al. ( ) have investigated several techniques and have shown that hydrophilic interaction chromatography (hilic) chromatography with zic-hilic and tskgel amide- are very specific at capturing glycopeptides from mixtures. sialylated glycopeptides could also be enriched with tio . capture using a hydrazide column resulted in lower recovery and involved a more complex enrichment scheme. a new material for glycopeptide concentration, termed "click maltose," has been synthesized by linking the alkynyl-derivatized maltose chain to the azide derivatized silica gel through click chemistry. unlike the rigid structure of sepharose, the saccharide chain of click maltose exhibits a certain amount of flexibility, which provides a sufficient number of hydroxyl groups for the effective formation of hydrogen bonds with the glycans attached to glycopeptides. the material was used to isolate glycopeptides from igg, rnaseb, and agp . cellulose columns have also been used for concentration of glycopeptides (snovida et al., ) . a method for detecting core-fucosylated (cf) glycoproteins for screening purposes has been reported by jia et al. ( ) . after igg depletion, fucosylated plasma proteins were enriched by use of lens culinaris lectin and the bound glycoproteins were digested by trypsin. these compounds were enriched by use of a , da cut-off filter, a procedure that also combines de-salting and concentration. the recovered glycopeptides were then treated with endoglycosidase f , which specifically cleaves the glycosidic bond between the two proximal (core) glcnac residues and leaves the fucosyl-glcnac residues attached to the peptides. four standard glycoproteins, apo-tf, fetuin, rhepo, and rnaseb, was used to illustrate the method. in addition, a part of the untreated tryptic peptides was treated with pngase f in order to locate the glycosylation site by the asn to asp conversion. products were detected by maldi-tof. in a related method using an ion trap, a neutral loss scan for fucose ( da) was also used to detect fucosylation. the methods were applied to the detection of fucosylated glycoproteins in the plasma of healthy subjects and subjects with hepatocellular carcinoma. over fucosylated glycoproteins and attachment sites were identified, and over , mass spectra of cf glycopeptide were analyzed. a method termed the sulfate emerging method has been described for specifically extracting sulfated glycopeptides (toyoda, narimatsu, & kameyama, ) from mixtures. the method overcomes the often negative contribution from other charged groups in the molecules. to accentuate the negative charge on the sulfate group, basic amino acids were eliminated and carboxylic acids were neutralized as follows: the protein was first hydrolyzed with trypsin and then the positively charged c-terminal lysines (lyss) and arginines (args) were eliminated by incubation with carboxypeptidase b. the negative charges of the carboxylic acid groups on the peptides were then neutralized by chemical modification with acetohydrazide using the recently reported quantitative modification of carboxyl groups in sialic acid using this reagent and -ethyl- -( -dimethylaminopropyl) carbodiimide hydrochloride ( ). the sulfated glycopeptides in the mixture were then captured by anion exchange resin with a basic buffer (ph . ) in which protonation of histidine residues was suppressed. finally, the sulfated glycopeptides were eluted from the anion exchange resin by increasing the ionic strength of the elution buffer for analysis by ms. rather than trypsin, pronase has been used as a nonselective enzyme to reduce the protein to a single amino acid (asn) or short peptide attached to n-glycans. these compounds are generally smaller than those obtained from o-linked glycopeptides, probably because o-glycans lie closer to the peptide backbone than n-glycans and protect the polypeptide from enzymatic digestion. n-glycans usually rise above the peptide backbone exposing the polypeptide to enzymatic digestion. dodds et al. ( ) have described immobilized pronase which retains its activity after repeated use for at least weeks. use of negative ion detection has been reported by nwosu et al. ( ) as providing distinct advantages over detection in positive ion mode for the detection of glycopeptides produced by pronase digestion. analysis in positive ion mode, although most commonly used for glycopeptide characterization, is hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides carrying sialic acids. furthermore, cid spectra of glycopeptides in the positive ion mode predominantly yields glycan fragments with minimal information on the peptide moiety. in the study by nwosu et al., which employed bovine lactoferrin for detection of n-glycosylation and k-casein for o-glycosylation, potential n-linked glycopeptides were detected in the positive ion mode whereas potential n-linked glycopeptides were detected in negative ion mode. analysis of k-casein, which contained mainly sialylated glycans, yielded improved results in negative mode. experimental details for peptide mass fingerprinting and identification of glycosylation sites have been published by wilson, simpson, and cooper-liddell ( ) . unlike the case with released glycans, where a relatively low mass accuracy measurement is usually sufficient to determine the composition in terms of the constituent monosaccharides, the situation is very different for measurements of glycopeptides. desaire and hua ( ) have examined the accuracy required and have concluded that in only a few cases can the mass accuracy provided by most commercial instruments be sufficient to unambiguously assign compositions to all glycopeptides in a mixture. e. n-glycan release once glycosylation sites have been determined, detailed structural analysis of the attached glycans is more conveniently carried out after releasing the glycans from the protein or peptide. both chemical and enzymatic methods are available but although, in the past, chemical release with hydrazine was popular, most investigators now prefer enzymatic methods. i. enzymatic release pngase f. peptide n-glycosidase f (pngase f), an amidase, is the most popular enzyme for cleaving n-glycans from their asn linkage site. it shows a broad range of substrate specificity with the exception that it does not release glycans bearing a fucose residue attached to position of the reducingterminal glcnac; in these cases, pngase a is appropriate. pngase f cleaves the entire glycans, which are released as the corresponding glycosylamines. these compounds rapidly hydrolyze to the glycan with retention of the reducing terminus. this method is, thus, distinctly advantageous to techniques such as b-elimination, popular with o-glycans (see below) because this site can conveniently be used to attached tags for fluorescence or other detection methods. wang et al. ( i) have recently found that the activity of the enzyme towards denatured glycoproteins can be enhanced by removal of the nterminal h helix from the enzyme. bereman et al. ( b) have studied methods for optimizing the release of glycans with this enzyme. dialysis of plasma prior to incubation was found to have little or no effect. however, microwave-assisted glycan release was found to be beneficial; min at ˚c with approximately w was found to give optimal results. surprisingly, no protease digestion was required as needed with standard incubation methods, and it was found that an -hr incubation with no detergent (np ) led to the greatest ion abundance of glycans from plasma glycoproteins. data could be obtained in less than day from raw plasma samples utilizing microwave irradiation. pngase f-glycan release from human serum glycoproteins has been achieved in min by using a constant microwave power of w, giving a temperature of ˚c (kronewitter et al., ) . in this study, the glycans were recovered by spe using a robotic liquid handler and examined by maldi with an ft-icr instrument from dhb. replicate analysis gave coefficients of variation of less than . . the standard protocol for n-glycan release from glycoproteins requires relatively long deglycosylation times (from several hours to, usually, overnight) and relatively high enzyme concentration (from : to : enzyme/substrate ratio). szabo, guttman, and karger ( a) have used a high-pressure method, both to reduce the reaction time and the amount of enzyme required. thus, a pressure-cycling device was use to cycle the pressure from atmospheric to as high as kpsi. greater than % release of the asn-linked glycans from bovine rnaseb, human tf, and polyclonal human immunoglobulin was achieved in only a few minutes using as low as : , enzyme: substrate molar ratio. a reactor with immobilized pngase f on a monolithic polymer support in a capillary has been developed that allows fast and efficient release of n-linked glycans. performance was determined with rnaseb, chicken ovalbumin, and human igg with detection by maldi-tof ms. the optimized reactor was integrated into a multidimensional system comprising on-line glycan release and hydrophilic interaction liquid chromatography (lc) followed by esi-tof ms detection. using this system, human igg was deglycosylated at room temperature in . min to an extent similar to that achieved with the soluble enzyme after hr at ˚c (krenkova, lacher, & svec, ) . immobilization of pngase f on detonation nanodiamonds has resulted in glycan release from glycoproteins in less than min . the method, using trypsin immobilization, also gave good results and proved to be better for proteolysis than the use of commercial immobilized trypsin beads. artefacts associated with pngase release of n-glycans. for solution release of glycans, the glycoprotein is usually denatured by reduction and alkylation or by use of detergents or other compounds such as urea. in general, low concentrations of urea (< m) do not usually cause irreversible protein denaturation. indeed, pngasef itself is stable in . m urea at ˚c for hr and still possesses about % activity in m urea. however, other glycoproteins appear more susceptible. for example, analysis by sds-page and maldi-tof ms have revealed that additional . kda of glycans can be released by pngase f if the deglycosylation is conducted in m urea suggesting that urea treatment exposes a glycosylation site that was previously inaccessible to pngasef . use of urea, however, can cause problems with the released glycans because it has been reported to compete with water for hydrolysis of the initially formed glycosylamines with the formation of a urea complex (omtvedt et al., ) . another artefact that has been found in glycans released with pngase f involves the reaction of the glycosylamines with h s to form the glycan-sh analogue. the h s arises from dithiothreitol (dtt, / ), a reagent used for protein alkylation and present in some commercial preparations of the reagent. the consequence of this reaction is an increase in da giving the impression, from a simple mass measurement, that the glycan has an additional oxygen atom. addition of da can also be observed in maldi spectra as a consequence of the formation of [mþk] þ rather than [mþna] þ ions, but this possibility can be excluded by formation of [mþcs] þ ions whereupon the da mass increase will still be present. the reaction with h s was first noted with glycans from human igg and negative ion ms/ms of the artefactual products located the da to the reducing-terminal glcnac residue. the negative ion fragmentation pattern was the same as that expected for a glycan with a hexose attached to the -position of the residue rather than fucose (deoxy-hex) that was actually the case. this result emphasizes how careful one must be, not only in deducing compositions from glycan masses but also in interpreting their ms/ms spectra . another artefact of the pngase f release step, detectable by ce but not by ms has been identified as the product of epimerization of the terminal glcnac residue to n-acetylmannosamine (mannac, ) under the slightly basic conditions usually employed in the release reaction. reducing the ph to . effectively removed the by-product (liu, salas-solano, & gennaro, h) . other endoglycosidases. another popular enzyme for n-glycan release is endo h which cleaves the chitobiose core of high-mannose and hybrid glycans but not complex ones, leaving a glcnac residue, with any linked fucose, attached to the protein. pace et al. ( ) have made use of this property to release and identify minor glycans in igg without interference from the more abundant complex glycans. endo f has a similar specificity and has been used by, for example, voutilainen et al. ( ) to detect glycosylation in talaromyces emersonii cellobiohydrolase cel a produced in the yeast saccharomyces cerevisiae. ammonium hydroxide/carbonate-based chemical deglycosylation and pngase a enzymatic release have been compared for glycan release from a plantibody produced in tobacco plants (triguero et al., ) . although both methods gave similar profiles as evaluated by hplc of -ab derivatives, the main drawback of the chemical release method was that it induced degradation of a , -fucosylated n-glycans. ii. extraction and purification of released glycans. cleanup of samples prior to ms is crucial to obtaining good spectra. many methods are in use; porous graphatized carbon (buser et al., ) is popular and we have found nafion membranes (börnsen, mohr, & widmer, ) to be convenient at removing both salts and some hydrophobic compounds. avoiding the introduction of contaminants is also important. disposable plasticware such as plastic test tubes that are normally used to process samples have been shown to be a major source of contamination. the contaminants, which produce ions, mainly prompt fragments across the entire mass range to about m/z , , originate from polymers that are used to protect the plastic against oxygen or uv light degradation. such compounds are hindered amine light stabilizers (halss) used in modern polyolefin (polypropylene, polyethylene) stabilization. the polymeric agent: poly-(n-b-hydroxyethyl- , , , -tetramethyl- -hydroxy-piperidinyl succinate, ), known as tinuvin- or lowilite , has been found to leach from laboratory polypropylene or polyethylene plastic test tubes into solvents used for sample preparation. . ml plastic tubes were found to be the major source of the contamination but the authors of the paper found that this could be minimized by using large solvent volumes of, for example, matrix solution (sachon et al., ) . amano and nishimura ( ) have used two kinds of hydrazide-functionalized glycobeads, termed glycoblot h and glycoblot abc of which the latter carries an additional fluorescent probe, to extract pngase f-released n-glycans from solution. sialic acids were then converted into methyl esters using the -methyl- -p-tolyltriazene (mtt, / ) reagent de-scribed by miura et al. ( ) and the products were examined by maldi-tof ms after release of the glycans from the beads by mild acid hydrolysis. the method was used to examine human serum glycoproteins for cancer biomarkers. full experimental details of the extraction and derivatization procedure are given in the paper. enrichment of serum and cellular glycoproteins with glycoblot h beads has also been used for o-glycan analysis . glycans were released from human milk osteopontin and urinary muc glycoproteins with ammonium carbamate and the method was proposed as ideal for identification of biomarkers. a method for sequentially enriching sulfated glycans by strong anion-exchange chromatography according to their degree of sulfation has been described by lei, novotny, and mechref ( ) . the method is based on modifying the binding ability of strong anion-exchange material with different sodium acetate concentrations, thus enabling selective binding and a subsequent elution of different glycans according to their degree of sulfation. before this enrichment, the negative charge on any sialic acid was eliminated by permethylation. the method was initially optimized using sulfated oligosaccharide standards and then used to examine the sulfated n-glycans from btsh, a glycoprotein possessing mono-and disulfated n-glycans. f. analysis of released glycans ahn et al. ( ) have obtained good resolution of n-glycans as their -ab derivatives using hilic columns packed with . mm sorbent. glycans were released from rnase b with pngase f and extracted using a microelution hilic spe well plate. the labeled glycans were also extracted from the preparative reagents using the same plate and their integrity was checked by maldi-tof ms. in another technique, guillard et al. ( ) have experimented with optimizing a linear ion trap instrument for automated measurement of permethylated n-glycans in serum. glycans were released with pngase f, cleaned with graphitized carbon and permethylated with the sodium hydroxide method. dhb, although the favored matrix for carbohydrates, failed to give the necessary reproducibility because of the large crystal size. chca, on the other hand, proved to be satisfactory. full experimental details for analysis of o-and n-linked glycans have been published by several authors (azadi & heiss, ; morelle et al., a; north et al., b) . g. total methods for glycoprotein structure there have been many reports of methods for total glycoprotein analysis of which the following are representative. a small-scale method for n-glycan release and analysis from plants used to produce recombinant glycoproteins has been described . concentration, protease digestion and deglycosylation are carried out in a single concentrator unit mass spectrometry reviews doi . /mas without the need for intermittent purification. this approach minimized adsorptive losses and facilitated handling. the plant protein was concentrated in a unit with a kda cutoff and after buffer exchange, pepsin digestion was carried out in the concentrator overnight. deglycosylation was carried out with pngase a for hr. released n-glycans were purified using reversed-phase and cation exchange chromatography in micro-columns and analyzed by maldi-tof ms without derivatization. a chip-based reversed-phase lc/ms method for n-glycan analysis suitable for biomarker discovery has been developed by . n-glycans were released from bovine fetuin as a model glycoprotein and human serum glycoproteins with pngase f and reduced to alditols with an ammonia-borane complex. the glycans were then permethylated in dimethylformamide to avoid artefacts in ms measurements and their structures were checked by maldi-tof measurements. reversed-phase microfluidic lc of the permethylated n-linked oligosaccharide alditols was then performed and was shown to resolve some closely related structures. optimized lc gradients, together with nanospray ms were then used with human serum samples to distinguish breast cancer patients from control individuals. a previously established two-dimensional hplc technique has been adapted as a hplc-maldi ms method for n-glycan analysis by gillmeister et al. ( ) . glycans were released from glycoproteins with pngase f purified with graphitized carbon and fluorescently labeled with -ap. the labeled glycans were analyzed on a -mm reversed phase (rp) hplc column and spotted onto a maldi-tof ms plate together with the dhb matrix using an automated plate spotter. the method gave a -fold reduction in the required amounts of starting protein compared with the earlier procedure. the entire process could be carried out in - days for a large number of samples as compared to - weeks per sample for previous two-dimensional hplc methods. the modified method was verified by identifying n-glycans from an igg antibody from human sera samples and applied to analysis of tissue plasminogen activator (tpa) from cho cell cultures under varying culture conditions. kim et al. ( b) have released glycans on a polyvinylidine difluoride (pvdf) membrane with pngase f and cleaned them with graphitized carbon contained in a -well plate before converting them into girard's t derivatives to introduce a constitutive cationic charge for quantification. analysis was by maldi-tof ms and the robust method was used to profile n-glycans from ovarian cancer patients using as little as ml of serum. a high-throughput method for the analysis of human plasma glycomes using a -channel multiplexed capillary gel electrophoresis (cge) dna sequencer with laser-induced fluorescence detection (cge-lif) system has been described with maldi-tof ms used to provide structural information (ruhaak et al., c) . glycans were released from plasma glycoproteins in a -well plate using pngase f and converted into apts derivatives with the help of -picoline borane ( ) as the reducing agent. analysis by cge-lif using the dna sequencer allowed samples to be analyzed in only . hr (the experimental time was longer because of two overnight incubations). the method was applied to a study of glycosylation patterns during first, second, and third trimesters of pregnancy, as well as weeks, months, and months postpartum. although analysis of glycoproteins carrying neutral glycans is now routine, analysis of glycoproteins with sialylated glycans is more difficult. hao, ren, and xie ( ) have approached the problem by first performing a tryptic digestion to give peptides and glycopeptides. peptide mass fingerprinting was performed on the peptides in order to identify the protein. the glycopeptides, separated by hilic chromatography, were examined by maldi-tof ms and ms/ms and treated with pngase f to release the glycans, which, together with the resulting peptides were again examined by ms. the asn to asp conversion in the peptide fraction enabled the glycosylation site to be identified. finally, the glycans were desialylated with dilute hcl and again analyzed by ms. the technique was applied to glycoproteins from human serum separated by -d electrophoresis and the differences in n-glycosylation were successfully determined for a -antitrypsin between different gel spots. in another method, sialylated glycoproteins have been selectively periodate-oxidized, captured on hydrazide beads, trypsinized and released by acid hydrolysis of the sialic acid glycosidic bonds. mass spectrometric fragment analysis allowed identification of glycan structures and additional fragmentation of deglycosylated ions yielded peptide sequence information which allowed glycan attachment sits to be identified together with identification of the protein. using this method, the investigators identified n-linked and o-linked glycosylation sites on glycoproteins from human cerebrospinal fluid . related to this method is one developed by klement et al. ( ) for enrichment of o-glcnac-modified proteins. glycoproteins were again oxidized with periodate and captured by hydrazide resin capture. rather than release of the peptide enzymatically, the glycopeptide was released by hydroxylamine treatment which also converted the aldehyde groups of the oxidized glycan to oximes. the open nature of the carbohydrate ring, following oxidation, lead to the production of characteristic fragment ions facilitating both glycopeptide identification and site attachment. the method was applied to analysis of a-crystallin a and the drosophila proteaosome. h. comparisons of methods for n-glycan analysis a comparative study of three techniques, maldi-tof, sds-page and cge-on-a-chip, for measuring the mws of large glycoproteins has been reported by müller et al. ( b) . it was found that all three techniques were capable of determining the mw of all high mw (glyco)proteins tested. the non-commercial cge-on-a-chip assay allowed electrophoretic separation of proteins in the mw range from kda to mda. mw assignment was limited to kda in the case of sds-page but with the proper matrix (thap for most glycoproteins, sinapinic acid for a -macroglobulin) and sample preparation, analysis with a standard maldi-tof-ms provided accurate mws for all high mw proteins up to mda. three methods for n-glycan characterization, namely maldi-ms of glycopeptides from tryptic digestion, negativeion esi-ms/ms of released n-glycans, and normal-phase hplc of fluorescently labeled glycans, in combination with exoglycosidase sequencing, have been evaluated for glycan identification using monoclonal antibodies expressed in tobacco plants as model compounds (triguero et al., ) . the ms methods identified the major glycans, but the hplc method was found to be the best for identification and relative quantitation. negative-mode esi-ms/ms easily provided direct identification of features such as the linkage position of the fucose residue linked to the inner core glcnac residue. grey et al. ( ) have developed a high-performance ion exchange chromatographic method for n-glycan analysis and have shown that it gives very similar results to analysis by maldi-tof. a series of standard glycans was examined and the method was extended to the analysis of n-glycans released from igg . an inter-laboratory study involving eleven uk laboratories using their routine glycan analysis procedures looked at reproducibility on glycan profiling from n-glycans released before the study from four glycoproteins, human and bovine agp, bovine pancreatic rnaseb and human serum immunoglobulin g (higg). data interpretation focused on the relative amounts of different glycan structures present, the degree of sialylation, galactosylation profiles, fucosylation, and bisecting glcnac content and the number of glycan components identified. all laboratories found high levels of sialylation for human and bovine agp, but varying amounts of di-, tri-, and tetra-antennary glycans. values obtained from mass spectrometric and chromatographic methods clustered separately. the proportion of the major man glcnac from rnaseb was between % and %. proportions of fucosylated and bisected glcnac glycans from higg were between % and % and % and %, respectively. mass spectrometric approaches consistently identified more glycan species, especially when both n-glycoylneuraminic acid (neu gc) and neu ac were present (thobhani et al., ) . a recent test of the ability of several laboratories to identify n-glycans released with pngase f from a mixture of four glycoproteins, asialo-fetuin, chicken ovalbumin and both human and bovine agp has also yielded some alarming results (orlando, leymarie, & keck, ) . although of the laboratories detected the presence of fucosylated complex nglycans, of them incorrectly located the fucose to the core glcnac of human agp rather than to an antenna. all nine of the labs using ms (not ms ) misidentified the site and all five of laboratories relying on software to identify the site also reported it incorrectly. although the ionic charge was not specified, it is assumed to be positive because it would be impossible to make this mistake with negative ion fragmentation. features such as the position of fucose residues produce diagnostic cross-ring fragments whose mass depends on the location of the fucose residue . such mis-identifications are particularly worrying because serum agp is elevated in inflammatory disease and is of potential use as a biomarker. many laboratories using this potential biomarker in serum also report the structure incorrectly. another problem in the survey arose with n-glycoylneuraminic acid ( / ), present in the bovine version of a glycoprotein. this carbohydrate is antigenic and is of concern to pharmaceutical companies producing pharmaceuticals in organisms that utilize this sialic acid. in the survey, eight of the laboratories, including seven of the eight participating industrial laboratories failed to detect its presence. all of the four laboratories that used a fluorescence tag, failed to detect this sialic acid. slightly better results were obtained by laboratories using maldi; of ten labs that used this technique, only two failed to detect this sialic acid. most laboratories that successfully detected this carbohydrate, permethylated their samples, which, of course, would stabilize it to maldi conditions. with mixtures containing different quantities of glycans, no lab correctly detected either three or four of the changes, one lab identified two of the four changes, seven labs identified one change but the other labs failed to identify any changes correctly. clearly, current analytical methods leave much to be desired. i. applications of maldi to the detailed structural determination of n-linked glycans a large number of reports have appeared on the applications of the above techniques to analysis of n-glycans from specific glycoproteins. these are summarized in tables and . other examples can be found in the tables on medical applications of maldi ms (table ) and biopharmaceuticals (table ) . some of the more unusual structures to be discovered are tetraantennary glycans with poly-n-lactosamine extensions with up to nine fucose residues in human seminal plasma , a man glcnac glycan with a bisecting glcnac residue ( ) (buser et al., ) ; a man glcnac -type glycan with two bisecting glcnacs ( ) proposed from dictyostelium discoideum feasley et al., ) and an unusual glycan with internal fucose and glucuronic acid (glca) from rapana venosa hemocyanin (velkova et al., ) . however, in the latter case, the structure was based on evidence from only one positive ion ms/ms spectrum and is open to alternative interpretations. symbols as defined for structures and plus = mannose j. miscellaneous studies among other studies, maldi-tof ms has been used to confirm the glycan compositions of several well-known glycoproteins in a study showing that glycosylation protects proteins against free radicals generated from toxic xenobiotics (martínek et al., ) the rice-derived recombinant human transferrin (rhtf) has been shown to be non-n-glycosylated by maldi and pngase f enzyme digestion . structural determination (highmannose, with bisect on branching mannose and antenna) (continued) (continued) sodium hydroxide followed by reduction to prevent a peeling reaction is the most common method but has the disadvantage of eliminating the reducing terminus, thus preventing tagging with fluorescent or other tags. some investigators have, thus, investigated the use of milder bases with the aim of avoiding the reductive stage and retaining the reducing terminus. zheng, guo, and cai ( ) have compared ammonia, methylamine and dimethylamine at ˚c for hr for release of n-acetylgalactosamine (galnac) from a small glycopeptide. b-elimination with dimethylamine and methylamine resulted in the conversion of the glycopeptide to . % of the dimethylamine derivative at m/z . and . % of the methylamine derivative at m/z . , respectively. however, the incubation of the glycopeptide with ammonia only resulted in % production of the product. the authors concluded that elimination with dimethylamine was the most efficient for release the o-linked glycans. release with methylamine was used by sun et al. ( b) to determine the glycosylation sites in human protein c inhibitor by the . mass increment introduced by the reaction. release with ammonia has been investigated in detail by yu et al. ( a) for o-glycan chains with b , -linked cores. in contrast to b , -linkages of the n-glycan-type, which were shown to be stable under the ammonium-based alkaline conditions, the b , -linkage was found to be labile and to give considerable peeling . the results indicated that complete prevention of peeling under nonreducing alkalicatalyzed hydrolysis conditions remains difficult. the yields of o-and n-glycans from bovine fetuin released by conventional means (pngase f and reductive b-elimination with naoh) were found to be greater. it was concluded that great care should be taken when employing such non-reducing alkaline conditions in glycomic analysis and in obtaining some o-glycans for functional studies. because the hydroxide ion appears to cause the unfavorable peeling reactions, miura et al. ( b) have investigated the use of the ammonium salt, ammonium carbamate for glycan release. the efficiency of release with ammonium carbamate was compared with a common conventional procedure, namely saturated ammonium carbonate/aqueous ammonia with bovine submaxillary mucin (bsm) as the test compound. release with ammonium carbamate did not exhibit significant loss of glcnac-b , (neu aca , )galnac or glcnacb , (neu gca , )galnac structural determination (highmannose, bisected high-mannose glycans). development associated with alteration of fucosylated glycans structural determination (highmannose, hybrid, complex glycans). poly-fucosylation human (ht- m colon cancer cells) pngase f, tof, glycans (per-me) pattern of n-glycosylation serves as a recognition signal for galectin- . high-man, hybrid, bi-, tri-tetra-antennary. (stechly et al., ) human (cytolytic t lymphocytes) pngase f, tof/tof (dhb), glycans (per-me) loss of effector function shown to be accompanied by major changes in nand o-glycosylation (antonopoulos et al., ) marmota monax (woodchuck with liver cancer), gal-α-( → )-gal in mouse epidermis but galnac-β-( → )-glcnac in human. high-mannose, hybrid complex (uematsu et al., ) pngase a pngase f, tof/tof (dhb/ % plants engineered to produce lewis x epitopes. (paucimannosidic glycans) mass spectrometry reviews doi . /mas and the profile of the major o-glycans was similar to that obtained following conventional reductive amination with naoh/nabh . on the other hand, glycans obtained by treating bsm with ammonium carbonate/ % aqueous ammonia and analyzed by maldi-tof ms showed a significant increase of the disaccharide components, neu aca , galnac and neu -gca , galnac, suggesting the presence of a peeling reaction. use of ammonium carbamate, thus, appears to produce efficient release without concomitant peeling. the release was performed by addition of powdered ammonium carbamate and incubation for hr at ˚c. yamada et al. ( ) have used a recently developed automated release apparatus using lithium hydroxide to obtain o-glycans from leukemia and epithelial cancer cells. because these cells usually contain free glycans, the investigators first reduced these with sodium borohydride and then labeled the released glycans with -aa in order to avoid interference. a new release method reported by goetz, novotny, and mechref ( a) combined enzymatic and chemical techniques and used b-elimination to cleave glycans from serine (ser) and threonine (thr) but not asn. the method involved first a nonspecific proteolysis with pronase, followed by solid-phase hydrazine, tof (dhb), glycans ( -ap) structural determination. high-man, hybrid, bi-, tri-, tetra-antennary complex (sumiyoshi et al., ) rabbit and chicken (erythrocytes) tof, glycans ( -ap) to study alterations in receptor-binding properties of h -type swine influenza viruses in embryonated chicken eggs. high-man, hybrid, bi-, tri-antennary (takemae et al., ) rat ( structural determination (highmannose, hybrid, complex glycans) girard's t for quantitation (kim, et al., c) sus domestica (respiratory epithelial cells) pngase f, tof, tof/tof (dhb), glycans (per-me) influenza virus shown to utilise α → linked neu ac to infect cells permethylation with sodium hydroxide. the basic sodium hydroxide caused the glycans to be released by b-elimination and these were immediately permethylated. this combination of the enzymatic and chemical procedures was reported to give a substantial improvement in sensitivity and analytical reproducibility over existing methods by minimizing sample losses. moreover, the approach was reported to extend the cleavage protocols to large glycoproteins where small oligosaccharides were not accessible to conventional chemical treatment. the method was developed with fetuin and used to identify new o-glycans from bile salt-stimulated lipase (bssl). maniatis, zhou, and reinhold ( ) have released o-glycans with aqueous dimethylamine in the presence of sodium borohydride by use of a microwave oven. the release was performed at ˚c and, for a heptapeptide carrying a glcnac group attached to thr, was complete in min. the reaction also labeled the site of detachment with a dimethylamino group. use of a : mixture of dimethylamine and [ h ] nh produced doublets in the peptide mass spectrum separated by six units, allowing the glycosylation sites to be readily identified. n-glycans are frequently removed from glycoproteins before o-glycan removal by b-elimination. however stone et al. ( ) have reported improved recovery of o-glycans from murine tissues without prior n-glycan removal. kbh and koh were used to remove the o-glycans and possible low levels of concomitantly released n-glycans were tolerated. ii. use of hydrazine. hydrazine has also been used to release these glycans with a new gas-phase method using anhydrous hydrazine being evaluated by goso, tsubokawa, and ishihara ( ) with muc-type oligosaccharides from porcine gastric mucin (pgm) and bovine fetuin. released glycans were examined by hplc and maldi-tof as -aa derivatives. glycans obtained by the treatment with hydrazine at ˚c for hr resembled those obtained by b-elimination, except for the additional disaccharide fractions derived from the core side of the oligosaccharides by further degradation. the other degraded products derived from the core side could not be derivatized by -aa, therefore, were not visible by fluorescence detection. release of the glycans was incomplete after hr but almost complete liberation was achieved by extending the treatment to hr. however, degradation increased. in this case, the addition of barium oxide to the reaction vessel decreased the degree of further degradation. results similar to pgm were obtained from bovine fetuin, but with less degradation. application of this method to the analysis of rat gastric mucin (rgm) showed that rgm has a large oligosaccharide portion on the core side. iii. other methods. a new method for o-glycan removal for study of the residual deglycosylated protein has been reported . desialylated glycoproteins whose sugar chains consisted of gal-galnac, were immobilized on alkali-stable, reversed-phase poros beads and treated with periodate to oxidize the cis-glycol groups in the gal residue. the resulting aldehydes were then susceptible to b-elimination under mild (nh ) basic conditions. the remaining galnac residue, which now contained a cis-glycol group, was oxidized with further treatment with periodate and removed with base. although the number of cycles required depended on the number of gal-galnac repeats, large core -type glycans that usually only have gal attached to the -position of the core galnac, could be deglycosylated in only two steps. o-linked glycosylation often occurs in muc-type domains that are heavily and heterogeneously glycosylated. several strategies to determine the heterogeneity of these domains have recently been investigated with four glucanases secreted in large quantities from trichoderma reesei, all of which contained heavily o-glycosylated muc-like linker regions, being used as models. the strategies involved monosaccharide compositional analysis and identification of the released glycans by hpaecpulsed amperometric detection (pad) and carbon-lc esi-ms/ ms. glycosylated peptides were generated by different protease digestions (trypsin, papain, asp-n, pretaq) and enriched by hilic microcolumns. the complex o-glycan heterogeneity was determined by maldi-ms and esi-ms, but the dense o-glycosylation in the muc-type domains conferred high resistance to protease cleavage. etd-ms/ms of the glycopeptide-enriched protease digests was unsuccessful for the assignment of o-glycosylation at individual sites within the muctype domains but allowed several previously unknown o-linked sites outside the defined linker region to be found on two of the four glucanases (christiansen et al., ) . iv. comparison of methods. three samples of iga isolated from the serum of patients with multiple myeloma have been distributed on behalf of the human proteome organization human disease glycomics/proteome initiative to laboratories for comparative analysis of their o-glycans. a range of techniques was used; the two strategies that yielded the best data were direct positive ion ms analysis of permethylated glycans and lc-ms analysis of native reduced glycans in negative ion mode. the studies reinforced the pre-eminent performance of ms techniques for o-glycan profiling (wada et al., b) . several reviews and general analytical methods have been reported; these are listed in table . loss of sulfate is a major problem in the maldi analysis of these compounds but this can be avoided by use of desorption esi (przybylski et al., a) . little use appears to have been made of the peptide binding method for stabilization of sulfate groups in these compounds as reported by juhasz and biemann ( ) . one report concerns the identification of a pentasulfated hexasaccharide responsible for binding to the growth factor pleiotrophin . the compound was complexed with (arg-gly) and identified by maldi-tof from dhb. in another study , a pentasulfated hexasaccharide with a novel structure (d , hexaa - galnac( s)b - idoa( -s)a - galnac( s)b - idoa( s)a - galnac( s)) has been isolated from the chondroitinase ac-i digest of shark skin. again, (arg-gly) was used as the complexing agent. bultel et al. ( ) have developed a method for analysis of heparin oligosaccharides by using controlled nitrous acid degradation followed by high-performance anion exchange chromatography (hpaec) separation and uv-maldi-tof analysis. the use of three different matrices, dhb, chca, and nor-harmane were investigated but only dhb and nor-harmane were needed to assign the position of sulfate groups. dhb allowed the molecular ion to be detected in nearly all cases and gave fragments arising from the loss of sulfate groups. nor-harmane, in contrast, produced mainly fragments. in all cases, ions retaining the sulfate groups were observed making these fragments essential for assigning the sulfated positions of each residue. while nor-harmane was not able to produce enough analyte desorption/ionization, fragments useful for structural assignment were produced by the addition of butylammonium formate to the dhb matrix. a method for analysis of gpi anchors on the "proteomic" scale has been described . partially purified proteins were separated by sds-page and then blotted onto a pvdf membrane. following identification of the protein, the gpi anchor was analyzed by three methods. first, the compound was hydrolyzed with hcl in the presence of [ , , , , , - h ]myo-inositol and the hydrolysate was analyzed as trimethylsilyl (tms) derivatives by gc/ms. next, the phosphate bonds were cleaved and the carbohydrate structure was elucidated by electrospray or maldi-tof ms. finally, the diacylglycerol-attached myo-inositol moiety was detached by nitrous acid deamination and analyzed by negative ion electrospray. bütikofer et al. ( ) have studied lipid remodeling of gpi glycoconjugates in procyclic-form trypanosomes and shown that, in trypanosoma congolense, the steady-state lipids consist of lyso-(acyl)phosphatidylinositol (pi, ), deacyl-pi and deacyl-(acyl)pi species, where (acyl) indicates an acyl group attached to the inositol moiety. maldi-qit-tof in negative ion mode from thap was used to analyze the pi species after deamination with nitrous acid. structural determination (core , core ). samples from geographically remote labs. similar glycans (babu, et al., ) human, (enterocytelike ht- cells) β-elimination, tof, glycans (per-me), gc/ms structural determination (core ) (morelle, et al., b) human ( absence of fucose on core glycans impairs baba-mediated adhesion to gastric mucosa (magalhães et al., ) (continued) although many bacteria produce s-layer proteins with glycosylation, qazi et al. ( ) have used maldi-tof ms to show that those from clostridium difficile are not glycosylated. this topic has been reviewed by zhang et al. ( f) and capote and sanchez ( ) . maldi is used mainly to determine the extent of glycation of intact proteins or to determine the sites of attachment of the covalent glycans. a. specific methods for glycated peptides amadori peptides have been enriched with boronate affinity tips for measurement by maldi-tof/ms. the tips showed the highest binding efficiency for glucose at ph . employing ammonium chloride/ammonia buffer with ionic strength of mm. the bound constituents were released by sorbitol ( / ) or formic acid. using sorbitol for elution required desalting prior to analysis. of three different sorbents tested: fullerenederivatized silica, ziptips (c ), and c silica, fullerenederivatized silica and ziptips showed the same performance with respect to the numbers of glycated peptides and gave better performance than c silica. fewer glycated peptides were detected by lc-ms/ms than by maldi . a novel fullerene(c )-derivatized silica material has been compared with octadecyl(c ) and triaconthyl(c )-silicas for their ability to recover peptides from digests of hsa and fibrinogen. c -and particularly the c ( nm)-spe materials were found to be the two most effective. after glycation the digests of fibrinogen and hsa were also separated. this new method made the detection of a considerably higher number of glycated peptides possible compared to the unfractionated digests and the use of boronate affinity chromatography in the case of fibrinogen. for hsa, new sites of glycation at lys and arg residues were found . a mass spectrometric method for screening large tandem mass spectrometric (ms/ms) datasets for protein glycation with glucose ( / ), lactose ( ) and maltose ( ) has been developed (montgomery, tanaka, & belgacem, ) . control experiments using a standard peptide containing a single glycation site led to the discovery of characteristic neutral loss fragmentation patterns in ms/ms analysis for glucose, lactose and maltose condensed with peptide. for glucose glycation, neutral losses of , , and da were observed in accordance with previously published reports. the neutral loss patterns for lactose and maltose were found to be identical, with characteristic losses of , , , and da. these signature losses were observed irrespective of the maldi mass spectrometer used and were valid in both tof-tof and qit-tof instruments. these neutral loss signatures were then applied to elucidation of modified peptides from a complex hsa digest glycated with each of the proposed sugars. screening of these large datasets o-labeled digests indicated that lyss and also had significant degrees of modification. the modifications that occurred at these sites were variations of fructosyl-lys and advanced glycation end products (ages) which included -alkyl- formyl- , -glycoyl-pyrole ( / ) and pyrraline ( / ). the fragmentation behavior of the peptide ac-paapaa-papaektpv-oh (human histone h . , positions - ) glycated via its lys residues to adp-ribose has been studied by fedorova, frolov, and hoffmann ( a) . under maldi conditions, the adp-ribosyl group was cleaved, almost completely at the pyrophosphate bond by isd and psd. however, this cleavage was very weak in esi-ms. the remaining phospho-ribosyl group was stable, providing a direct and reliable identification of the glycation site via the b-and yion series. as well as being associated with health problems, in for example, diabetes, protein glycation is important in the food industry in, for example, the browning of food during cooking. the reaction is also being used to attach carbohydrates to proteins to improve technological and biological functionalities. in relation to this latter use, corzo-martnez et al. ( ) have used maldi-tof ms to study the reaction between b-lactoglobulin and the sugars galactose and tagatose ( ) and found that the reaction can be competitively moderated with pyridoxamine ( ). other reports of the use of maldi to study glycation of specific proteins are summarized in table . typical structures consist of glcnac-murnac ( ) disaccharides cross-linked by short peptides. they are usually analyzed as muropeptides following enzymatic digestion. reports of the use of maldi to study peptidoglycans and muropeptides are summarized in table . this is a very large group of compounds but most work involving maldi has been concentrated on the lps from bacteria and the gsls. work, mainly involving structural determination, on the many other types of glycolipids found in bacteria and similar organisms, is summarized in table . a general review on analysis of microbial glycopolymers has been published by , and fuchs and schiller ( ) phenol/chloroform/petroleum ether and the molecules are frequently split into their component parts by mild acid hydrolysis for further structural analysis. dephosphorylation and deacylation are also common. reviews on the structural investigation of bacterial lps by ms and ms/ms have been published by banoub et al. ( ) and by grice and wilson ( ) . lipid a from coxiella burnetii, the causative agent of q fever has been discussed by toman, skultety, and ihnatko ( ) and a more general review of the core region and lipid a of lps has been published by holst and molinaro ( ) . the first structures of lps to be elucidated from cyanobacteria has been reported by snyder et al. ( ) . two strains of marine water-soluble proteins glucose l-tof ( , -dhap) use of maldi-tof to study glycation during malting synechococcus, wh and cc , were used and were shown to have very simple structures without the complex o-chain found in most proteobacteria. the lps ( ) of these cyanobacteria did not contain phosphate, heptose (hep) or kdo ( / ) but instead possessed -linked glucose as their main saccharide component, with low levels of glcn and galacturonic acid (gala). maldi-tof ms of the intact minimal core lps revealed triacylated and tetraacylated structures with a heterogeneous mixture of both hydroxylated and nonhydroxylated fatty acids connected to the di-glcn backbone. in contrast to enteric lipid a, which can be liberated from lps by mild acid hydrolysis, lipid a from these organisms could be produced only by two novel procedures: triethylamine-assisted periodate oxidation and acetolysis. unique caryophyllose (a- , -dideoxy- -c-(d-altro- , , , tetrahydroxyhexyl)-d-xylo-hexopyranose, )-containing cell wall glycolipids have been identified in lps from mycobacterium marinum (rombouts et al., ) . maldi-tof spectra were obtained from dhb and esi-ms/ms was used to elucidate the glycan sequence. b. lipid a sample preparation has been shown to be critical for assessing the true composition of these compounds. in a comparative study, kawasaki ( ) have shown that maldi-tof ms analysis of lipid a prepared using a commercial "tri-reagent"based procedure with a -chloro- -mercaptobenzothiazole (cmbt) ( / ) matrix gave the best results for compounds with a phosphoethanolamine (petn) modification. in contrast, the analysis of lipid a prepared using an lps extraction kit-based procedure with dhb was preferable for the detection of an aminoarabinose modification. for isolation of lipid a from yersinia enterocolitica, pérez-gutiérrez et al. ( ) used an ammonium hydroxide-isobutyric acid method. lyophilized crude cells were incubated with isobutyric acid and ammonium hydroxide ( : , v/v) at ˚c for hr, washed twice with methanol and the insoluble lipid a was solubilized in chloroform-methanol-water ( : . : . , v/v/v). analysis was by maldi-tof ms from dhb in negative ion mode because of the presence of two phosphate groups. the lipid as a contained both four or six fatty acyl chains whose (boniface et al., ) ratio changed with growth temperature. a similar method has been used by march et al. ( ) to study lipid a from acinetobacter baumannii. the molecules were found to have from four to seven acyl groups. a microwave-assisted method for obtaining lipid a from helicobacter pylori has been developed by zhou et al. ( a) . lyophilized cells were suspended in sodium acetate buffer (ph . ) containing proteinase k and subjected to microwave irritation at w for min at ˚c and then kept for hr at c . after centrifugation and washing, the dried supernatant was examined by maldi-tof/tof from cmbt. the reliability of the technique was demonstrated by analysis of the lipid a from bacterial cells of different h. pylori strains. the phosphorylation and acylation patterns could be elucidated using material from a single colony. furthermore, the investigators found unusual heptaacyl lipid a species present in low abundance in h. pylori mutant that have not been previously reported. the study was claimed to provide the first characterization by ms of the lipid a component from a single bacterial colony. the mass spectrometric behavior of lipid a is highly dependent on both the matrix and phosphorylation patterns. zhou et al. ( a) have investigated the effects of different matrices and co-matrices using lipid a from escherichia coli o as a model system. good results were obtained with cmbt with added edta ( / ) ammonium salt as the matrix. this matrix system was found to enhance the sensitivity of the detection of diphosphorylated lipid a by more than -fold and, in addition, provided tolerance to high concentrations of sds and to both sodium and calcium chlorides at mm concentrations. the method was evaluated for analysis of lipid a with different phosphorylation patterns and from different bacteria, including h. pylori, salmonella enterica serovar riogrande, and francisella novicida. an lc/ms-based assay has been developed for the quantitation of aminosugars, including glcn ( ), galactosamine (galn, ) and aminoarabinose (aran, ) together with ethanolamine (etn), present in lipid a and has been applied to the analysis of lipid a isolated from several biosynthetic and regulatory mutants of s. enterica serovar typhimurium and francisella tularensis subspecies novicida characterized by maldi-tof. lipid a was treated with tfa to liberate and deacetylate individual aminosugars and mass tagged with -aminoquinolyl-n-hydroxysuccinimidyl carbamate ( ), which reacts with primary and secondary amines. the derivatives were separated using rp-chromatography and analyzed with a quadrupole ms to detect quantities as small as fmol. galn was detected only in francisella and aran only in salmonella, while glcn was detected in lipid a samples from both species (kalhorn et al., ) use of grazing-incidence x-ray scattering and monte carlo simulation to investigate physics of bacterial survival against cationic antimicrobial peptides . (continued) c. core oligosaccharide during an analysis of the permethylated derivative of the core oligosaccharide from aeromonas hydrophila, prepared by the hakamori method, ions appearing at mass units higher than the [mþh] þ ions were observed. these ions were determined to be dimethylsulfoxide (dmso) covalent addition products resulting from the michael addition of the dimsyl anion on the c- -c- double bond of a , -anhydro kdo ( / ) residue followed by an addition of a proton on the double bond. corresponding ions were also seen in methylations performed using the naoh technique and represent the first characterization of these addition products (sioud et al., ) . typical pre-maldi techniques for the analysis of these compounds include separation by tlc and cleavage of the cer portion so that the glycan can be analyzed without the heterogeniety produced by the lipid. reviews on the analysis of gsls are summarised in table . a. analysis of intact compounds stübiger et al. ( ) have separated lipids, including gsls by high-performance tlc, stained them with coomassie blue and analyzed them either directly from the tlc plates or, after their removal, with an appropriate solvent. thap ( / ) was used as the matrix with acetone as the solvent for the on-plate analysis because its high volatility minimized sample spreading. a method for structural profiling of individual gsls in a single thin-layer chromatogram by multiple sequential immunodetection has been developed by souady et al. ( ) . structures of the antibody-detected gsls were determined by direct coupling of tlc with ir-maldi after treatment of the tlc plate with glycerol. this combined technique was used to demonstrate structural gsl profiling of crude lipid extracts from human hepatocellular cancer. a new method for analysis of gsls involves selective ozonolysis of the c-c double bond in the ceramide moiety of biological samples and subsequent enrichment of the generated gsl aldehydes by chemical ligation using aminooxy-functionalized gold nanoparticles (aognp). the gsl-bound nanoparticles were removed by ultrafiltration and the gsls were analyzed by maldi-tof and -tof/tof ms from dhb. the method was used for structural profiling of mouse brain gangliosides such as gm , gd a/gd b, and gt b for adult or gd in the case for the embryonic mouse. because the saturated acyl groups remained intact, the spectra provided information on both the carbohydrate and fatty acyl moieties (nagahori, abe, & nishimura, ) . the direct structural characterization of microbial gsl receptors by use of the tlc overlay assay combined with ir-maldi-o-tof-ms has been described (müsken et al., ) . glycan mixtures were separated by tlc in three parallel lanes. one lane was stained with orcinol and a second was overlayed with gsl-specific bacteria. the bound microbes were detected with primary antibodies against bacterial surface proteins and the relevant gsls were detected in the third lane by ir-maldi-tof. the combined method worked on the microgram scale of gsl mixtures and was successfully applied to the compositional analysis of globo-series neutral gsls recognized by p-fimbriated e. coli bacteria, used as model microorganisms for infection of the human urinary tract. (niedziela et al., b) incubation of botulinum neurotoxin serotype d with the gsl, gt b has produced a complex (mw , ) that was detected intact by maldi-tof ms from sinapinic acid and provided evidence that the toxin attacks neurons in a ganglioside-dependent manner (strotmeier et al., ) . a method for generation of novel fluorocarbon derivatives from gsls has been described by li et al. ( f) . the derivatives had high affinity for fluorocarbon phases allowing them to be recovered from biological matrices by fluorous solid phase extraction (f-spe). sphingolipid ceramide n-deacylase was used to remove the fatty acid from the ceramide moiety, after which the fluorocarbon-rich substituent (f-tag, ) was linked to the free amine. finally, the molecules were permethylated for ms analysis and the method was used to examine a crude ganglioside mixture extracted from bovine brain. in addition, the flourous tag was used in a microarray format to fix f-tagged gm ganglioside to a fluorous glass surface, with the glycan intact and available for interaction with a fluorescent derivative of cholera toxin b chain. cheng et al. ( a) have used -aa ( / ) as a matrix for quantitative analysis of sulfatides in biological samples. the matrix was said to promote selective ionization of sulfatides in negative ion mode with a detection limit in the high attomole range. experimental details have been published for highperformance tlc separation of glycolipids followed by blotting to a pvdf membrane in a technique termed far-eastern blot with analysis by maldi-tof ms (taki et al., ) . b. studies on the glycan moiety following removal of the ceramide for studies of the carbohydrate portion of these molecules, lipid heterogeniety is frequently reduced by removing the ceramide with enzymes such as rhodococcal endoglycoceramidase or leech ceramide glycanase. li et al. ( e) have described the preparation of the intact oligosaccharides from gm (neuacggose cer) and gbose cer as examples to show the use of ceramide glycanase and have optimized the specificity and detergent requirements of rhodococcal endoglycoceramidase for the release of glycan chains from various gsls. a novel method of detecting -gala series gsls (those possessing an r-galb - galb - -cer, group) has been reported (ishibashi et al., ) . the method used the specificity of endogalactosylceramidase, an enzyme that is capable of hydrolyzing -gala series gsls to produce intact oligosaccharides and ceramides but which also catalyzes transglycosylation reactions. in the latter reaction, the enzyme transferred oligosaccharides from the gsls to acceptors such as fluorescent -alkanols. in this application, -nitro- , , -benzoxadiazole pentanol (nbd-pentanol, ) was used as an acceptor. the fluorescent products, nbd-pentanol-conjugated- -gala oligosaccharides, were separated and detected by tlc or hplc with a fluorescent detector and characterized by maldi-tof ms. the method could also be applied to glycoglycerolipids and digalactosyldiacylglycerol and was successfully applied to detect -gala series gsls in the fungus, rhizopus oryzae and the parasite, taenia crassiceps. other applications of maldi to the analysis of these compounds is summarised in table . rhamnolipids are glycolipids of the type produced by pseudomonas spp. maldi-tof ms approaches have been developed for high-throughput screening of naturally occurring (fungus), mycelia tof (chca), psd ident. of novel neogala-series glycosphingolipids with terminal man and glc (tani et al., ) human (mesenchymal stem cells from bone marrow) macrobdella decora endoglyco-ceramidase, r-tof/tof, ft-icr (dhb), glycans (per-me) structural determination (heiskanen, et al., ) human (colonic adenocarcinoma cell lines) ceramide glycanase, tof/tof, q-tof (dhb), glycans (per-me) enhanced expression of β -gal-t activity induces in vivo synthesis of extended type chains on lactosyl-cer human (umbilical vein endothelial cells) tof (chca) acyl mainly : . may act as biomarker for inflammation, gsl = gb (okuda et al., ) human (umbilical vein endothelial cells) tof (chca) structural determination and dynamics of globotetraosylceramide under tnf-α stimulation (okuda, et al., ) human (kidney and colon) tof identification of gsls that bind shiga toxin from e. coli human (embryonic stem cells) switching of the core structures of gsls from globo-and lacto-to ganglio-series on cell differentiation miniature pig (endothelial and islet cells) ceramide glycanase, r-tof (dhb), esi (-ve), glycans (per-me and girard's t) structural determination. identification of neu gc epitopes ) (krambeck, et al., ) mouse (thymus) tof/tof (dhb), esi structural determination and natural killer t cell development (li et al., f) mouse (liver and serum) tof (-ve) acute kidney injury down-regulates gene expression of hepatic cerebroside sulfotransferase. quant. by maldi-tof. (zhang et al., h) mouse ( use of -aminoacridine ( -aa) as matrix, structural determination -phospholipids, gal-cer-sulfate, gb trichoderma viride (fungus) l-tof ( -nh - -mecoumarin) ident. of phosphocholine-containing glycosyl inositol phosphoceramides respectively. the method was validated by compositional analysis using gc/ms, fractionation by rp-hplc and analysis by and d nmr applications of maldi to the analysis of other glycolipids are listed in table . although much work has been published on glycosides during the review period, maldi appears to occupy a relatively minor position with fast atom bombardment (fab) and, particularly esi being the preferred techniques. most work has been on the identification of glycosides from various plant sources using a variety of techniques such as nuclear magnetic resonance (nmr), uv and ir spectrometry. applications of maldi to the analysis of glycosides and other natural products are summarised in table . several investigators have reported that the main fragmentation pathways of flavonoids are apparently independent of the ionization mode (esi, atmospheric pressure chemical ionization (apci), or maldi) or the types of analyzers used to acquire the spectra (triq, it, or qtof) as reported in a review of the structural characterization of flavonoid glycosides by multistage ms (vukics and guttman, ) . maldi-tof (dhb) was used by bankefors, nord, and kenne ( ) to examine saponins from quillaja saponaria bark extracts in connection with the development of a multidimensional method using hplc and esi-it ms n for profiling complex mixtures of natural products. increasing use of maldi has been made in the characterization and detection of disease and in the identification of biomarkers. some of the glycan biomarkers reported for, for example, cancer, however, appear to be more associated with glycopro-teins involved in inflammation and are, thus, secondary to this disease. blomme et al. ( ) have noted that "although individual liver diseases have their own specific markers, the same modifications, hyperfucosylation, increased branching and a bisecting glcnac, seem to continuously reappear in all liver diseases." increases in fucosylated triantennary glycans from agp is a case in point. several reviews have appeared and are summarised in table . practical details for the characterization by maldi-tof and esi-ms of n-linked glycosylation on recombinant glycoproteins produced in p. pastoris and for detecting potential cancer biomarkers in various cell lines and sera from patients have been published. bereman, williams, and muddiman ( a) have developed a nano-lc linear trap quadrupole (ltq) orbitrap method for analysis of released n-glycans and compared the spectra with those obtained by maldi-ft-icr. whereas the maldi spectra showed much loss of sialic acids from the sialylated glycans, the orbitrap spectra showed no decomposition. the method was applied to glycans released from plasma glycoproteins in benign gynecologic tumors and from epithelial ovarian cancer patients. one of the biantennary glycans found to be down-regulated in the cancer patients was a fucosylated biantennary glycan in which the fucose was unusually shown attached to a gal residue rather than to the core as determined by ms/ms. the compounds were ionized as [mþh] þ species suggesting that this might be an erroneous structure and the result of an internal rearrangement that is known to occur under these conditions. a detailed statistical analysis has been performed on eight data sets of n-glycans released from serum glycoproteins from prostate, breast and ovarian cancer patients (barkauskas et al., ) and measured by maldi-ft-icr ms. significant differences between control and cancer groups were found in all eight datasets. two structurally related compounds were found to be significantly different between control and cancer groups in all three types of cancer. these compounds had compositions of hex -hexnac -fuc and hex -hexnac -fuc and were probably from igg rather than being produced by the cancer cells. narimatsu et al. ( a) have developed a high-throughput method for detecting cancer biomarkers in early stages of the disease. briefly, the method consisted of the extraction of tissue mrnas and measurement of the expression by quantitative realtime polymerase chain reaction (pcr). the results suggested that different glycan structures were synthesized in different cell lines. secreted proteins from the same cancer cells were collected from serum-free culture and then applied to a lectin microarray to select lectin(s) that showed differential binding to glycoproteins secreted from each cancer cell line. after selection of a specific lectin, isotope-coded glycosylation sitespecific tagging (igot) was used to identify core proteins that carry an epitope bound by a specific lectin. each candidate biomarker was immunoprecipitated from serum using commercially available antibodies and their glycan structures were profiled by lectin microarray, and finally determined by ms n technology with measurements by maldi-qit-tof ms. other applications are listed in table . α-glc( - )-α-hep-( - )-α-kdo p-( - )-β-glcn p-( - )-α-glcn p arthrobacter globiformis and a. scleromae diglycosyl glycerol ft-icr (dhb), nmr structural determination. mannose and galactose (paściak et al., b) aspergillus fumigatus glycoinositolphospho-ceramides tof the mita gene shown to be required for mannosylation (kotz et al., ) hymenobacter sp. carotenoids maldi identification of '-methyl and '-xylosyl derivatives of 'hydroxyflexixanthin (klassen et al., ) leishmania major glycoinositolphospholipids tof (chca), esi-ms n demonstration of a udpglucose independent udpgalactose salvage pathway (lamerz et al., ) lipoteichoic acid tof (dhb) identification of two enzyme systems involved in biosynthesis phosphatidyl-myoinositol mannosides (pims) glycopeptidolipids shown to mask pims in cell wall (rhoades et al., ) there is currently great interest in the production of therapeutic antibodies and maldi-tof ms is frequently used in the analysis of their attached glycans. reviews have been published on methods for the production and ms analysis of igg (huhn et al., ) therapeutic antibodies (beck et al., ; del val, kontoravdi, & nagy, ; higgins, ; zhang, pan, & chen, i ) and on the humanization of recombinant glycoproteins expressed in insect cells (tomiya, ) . practical details for characterization of antibody glycans have been published by several investigators janin-bussat et al., a ,bjanin-bussat et al., a . a discussion, with experimental details, of methods based on blot detection with glycan-specific probes, ms of released glycans and lc/ms detection of glycopeptides with the aim of determining whether, how and where plant-derived biopharmaceuticals are glycosylated has also been published . several new methods have been reported. thus: a highthroughput method for monitoring igg glycosylation using a well plate format with iggs purified from ml of human plasma has been developed by selman et al. ( ) . iggs were extracted using immobilized protein a, cleaved with trypsin and the resulting glycopeptides were purified by reversed-phase or hydrophilic interaction spe. glycopeptides were analyzed by intermediate pressure maldi-fticr-ms using both dhb and chca, both of which produced signals from sialylated as well as nonsialylated glycopeptides. the method showed an interday variation of below % for the six major glycoforms of both igg and igg and was found to be suitable for isotype-specific high throughput igg glycosylation profiling from human plasma. the method was applied to the igg glycosylation of human samples. two lectin-affinity chromatography techniques, con a and lens culinaris agglutinin, have been used to enrich, by removal of high-mannose glycans, the nonfucosylated n-glycans from igg with product detection by maldi-tof following pngase f digestion (tojo et al., ) . prien et al. ( ) have used a stable isotopically labeled derivative for rapid glycan screening of biotherapeutics. glycans were labeled with either [ c ]-or c ]- -aa for both maldi-tof or lc-ms analysis. the -aa label provided high sensitivity detection in negative ion mode and the mass separation of six units between the isotopically labeled variants eliminated problems arising from isotopic overlap. pegylation of proteins is frequently used to prolong the serum half-life time of recombinant proteins but their very high mws put many of them outside the mass range of commercial maldi-tof systems using conventional secondary electron multiplier (sem) detectors. seyfried et al. ( ) have investigated the use of a high mass (hm) detector combined with a maldi linear tof ms system for the detection of pegylated (glyco)proteins in the range of - kda. the system consisted of a shimadzu axima cfrþ instrument equipped with both a conventional detector and additionally, with an inline moveable hm ion conversion detector (icd hm , from covalx). spectra were run from sinapinic acid in the linear positive ion mode and were obtained from small (interferon a a), middle (hsa) and high (coagulation factor viii and von willebrand factor (vwf), both heavily glycosylated proteins) molecular mass proteins. the particular challenge was the heterogeneity of the (glyco)proteins in the high mw range in combination with heterogeneity added by the pegylation, nevertheless, the performance of maldi linear tof ms was found to be superior to that of other methods. although the sem was able to obtain information about protein pegylation in the mass range up to kda (e.g., pegylated hsa), the hm system was crucial for detection of ions from the larger compounds, the masses of which sometimes exceeded . mda. detection of these compounds was impossible with the standard sem. the particular challenge for the analysis was the heterogeneity of the (glyco)proteins in the high mw range in combination with additional pegylation, which even introduced more heterogeneity and was more challenging for interpretation. nevertheless, the performance of maldi linear tof ms with both detector systems in terms mw and heterogeneity determination depending on the m/z range was superior to the other methods. isolation and characterization of rhamnolipid-producing bacterial strains from a biodiesel facility (rooney et al., ) proposed as a member of a new genus and species (golyshina et al., ) aplysia kurodai (sea hare) glycosaminoglycans tof, ms/ms structural determination (yoon et al., a) arthrobacter crystallopoietes n- α ,α -trehalose tof, esi, nmr first report that trehalose can be produced from maltose in this bacterium beverage from fermented plant (gülcemal et al., ) radix puerariae (kudzu) tof identification from cultivated kudzu root (nguyen et al., b) red wine procyanidins and anthocyanins tof (indole- -acrylic acid) phenolic extracts shown to induce nitric oxide (no)mediated vasoprotectivity (auger et al., ) salmo salar ( a novel method for the determination of aminoglycoside antibiotics used surface-assisted laser desorption/ionization mass spectrometry (saldi ms) with the aid of silver-coated gold nanoparticles (au@agnps) capped by anionic citrate. these nanoparticles were used both as concentrating agents and as matrices in saldi ms. the lods at signal-to-noise ratio of were , , , , and nm for paromomycin, kanamycin a, neomycin, gentamicin and apramycin respectively. the lods of the first four of these antibiotics in plasma samples were , , , and nm respectively. recoveries of the antibiotics from plasma were about % (wang et al., h) . further examples of the use of maldi ms in the analysis of therapeutics are given in table . applications in this section mainly involve the use of maldi to investigate products of newly isolated enzymes. these are summarised in table . other studies are aimed at elucidating enzyme activity as illustrated by the development of a method to determine the cleavage site in small oligomannoses that has been developed by hekmat et al. ( ) . enzymatic cleavages were performed in o-labeled water, conditions that introduced o into the anomeric position of the cleaved glycans. thus, measurements by maldi-tof could determine if a product arose from the non-reducing end of the original oligomannose by its incorporation of the label. the addition of a small amount of various ionic liquids has been found to modify the activity and regioselectivity of different immobilized preparations of rhizomucor miehei lipase that catalyzes the hydrolysis of hexa-o-acetyl lactal in aqueous media (filice, guisan, & palomo, ) . the activity of the enzyme glcnac-transferase vb, which transfers glcnac to the -position of the -antenna in n-glycans has been compared with that of gnt-v. one unusual product found after hr was the addition of glcnac to the -position of both antennae (alvarez-manilla et al., a). relevant reviews on carbohydrate synthesis are summarised in table principal component analysis, shows glycobiological differences between normal and cancer. hybrid, complex glycans (goetz et al., b) breast cancer cd hc clycoprotein pngase f, tof/tof (dhb), complex glycans (per-me) identification of the glycoprotein that binds to galmbp (fragment of mannose-binding protein) (powlesland et al., ) breast cancer serum trypsin, tof/tof (dhb), glycopeptides expression of helix pomatia lectin binding glycoproteins in women with breast cancer in relationship to their blood group (welinder et al., ) breast cancer cells β-elimination (nh carbamate), r-tof/tof (dhb), glycans, ms/ms use of new release method using glycoblotting and ammonium carbamate for β-elimination, core o-glycans breast cancer serum set of glycans identified that can be used as biomarkers (hammoud et al., ) (continued) changes in glycans . three n-glycans sufficient for detection with % accuracy hepatocellular carcinoma serum glycoproteins tof/tof (dhb), glycans (per-me) analysis using hierarchical clustering analysis. potential glycan markers identified. man glcnac , bi-, tri-antennary (tang et al., c) hepatocellular carcinoma serum and liver glycoproteins of rats pngase f, r-tof (dhb), glycans (per-me) increase in core-α- , fucosylated glycoproteins. possible biomarker hepatocellular carcinoma suggests shiga toxin as potential therapeutic agent (distler et al., ) enhanced expression of monosialylated triantennary glycans in cancer (yoon et al., b) stomach cancer mkn cells, serum automated β-elim. (matsuno et al., ) , tof (dhb), glycans (phenylhydrazones) mkn cells found to express characteristic trisialopolylactosamine-type glycans. reduced triantennary complex and increased biantennary glycans at asn- in patients table and general reactions in table . methods to change the glycosylation of a glycoprotein are common for recombinant antibiotic production as outlined above. a general method for producing homogeneous glycoproteins with eukaryotic n-glycosylation has been reported and involves the transfer of the campylobacter jejuni glycosylation machinery to e. coli and production of glycosylated proteins with the key glcnac-asn linkage. the bacterial glycans were then trimmed and remodeled in vitro by enzymatic transglycosylation to give a eukaryotic-type n-glycosylation (schwarz et al., ) . a method for immobilization of unstable membrane-bound enzymes to a commercially available sepharose support for glycan synthesis has been published ito et al. ( c) . it involves modification of the protein c-terminus and a transpeptidase reaction by staphylococcus aureus sortase a (srta) has been developed. recombinant human b , -galactosyltranseferase or recombinant h. pylori a , -fucosyltransferases were bound with simple aliphatic amino groups displayed on the surface of the solid support and were shown to have the required glycosyltransferase activity. as with previous reviews in this series, two types of compound appear to be particularly suited to maldi-tof analysis; namely glycodendrimers and carbohydrate/protein complexes. lysosomal proteins in mouse model in contrast to previous assumptions, cho cells shown to be capable of adding antigenic α-gal residues to n-glycans (bosques et al., ) α -antitrypsin (in human age hn cells) tof to characterize novel human cell line (northoff et al., ) α -proteinase inhibitor (in aspergillus niger) trypsin, pngase f, βelimination, tof/tof (dhb), glycans (per-me), gc/ms production, purification, and characterization, glycans = high-mannose (large antennae) (chill et al., ) anti-cd igg s, anti-cd igg rituximab mutant (s d/s a i e), (human) removal of fucosylated antibody ingredients from therapeutics shown to elicit high antibody-dependent cellular cytotoxicity in blood by two mechanisms anti-egfrxanti-cd bispecific igg ( model for comparison of release and glycan analysis methods (triguero, et al., ) centellosides (in centella asiatica plant cell cultures) tof to obtain a more efficient production system. α-amyrin, converted into centellosides by c. asiatica cells introduction of bisecting glcnac suppresses → -fucosylation and xylose attachment to form paucimannosidic glycans (karg, et al., ) α-l-iduronidase (human in seeds of brassica napus and nicotiana tabacum) tof (sinapinic), glycoprotein. gc/ms of monosaccharides attached carboxy-terminal er-retention motif, sekdel, reduces xyl and fuc in n-glycans but has little effect on enzyme activity (galpin et al., ) iga and iga (in murine melanoma cells) pngase f, l-, r-tof (dhb, thap), glycans structural determination. hybrid, bi-, tri-, tetra-antennary complex. igg ( use of glycosidase inhibitor to produce igg with man glcnac . effect on xtal structure and effector functions (crispin et al., a) igg (mouse in tobacco by cells) hydrazine, tof (dhb), glycans ( -ap) use of an er retention signal (kdel) increased high-mannose glycans but did not eliminate paucimannosidic glycans igg (human ij in silkworm larva hemolymph) pngase f, tof, glycans ( -ap) produced mg per kg of larvae with recovery of . %. paucimannosidic glycans igg (in cho cells) pngase f, tof, glycans production of non-fucosylated glycans by mutation (glycans -high-man, biantennary complex) (von horsten et al., ) igg (in cho cells) pngase f, tof chromatographic method to enrich nonfucosylated glycans (tojo, et al., ) igg ( effect of culture medium components on product yield. glycans: g f, g f (paul et al., b) a. synthesis of multivalent carbohydrates, dendrimers, and glycoclusters an extensive review of dendrimers (astruc, # ) , with a section on glycodendrimers, illustrates the breadth of this subject. masses of the larger compounds are frequently in the range - kda. the largest compound, based on a polyglycerol scaffold contained an estimated mannose residues but the authors had difficulty obtaining a maldi spectrum because of the high mw. syntheses frequently involve huisgen-type click chemistry because high-yield reactions are essential when so many carbohydrate molecules have to be attached. table lists papers reporting syntheses of glycodendrimers and similar compounds. among the largest of these compounds to have been reported during the review period has involved conjugation of lpsderived oligosaccharides to diphtheria toxin crm protein in an attempt to develop a vaccine against invasive meningococcal disease. conjugates with a mass of kda were analyzed by maldi-tof from sinapinic acid, the most widely used matrix for compounds of this type. conjugates can be characterized by maldi-tof ms but for large molecules the resolving power of most instruments is insufficient to distinguish each product and only a broad peak is observed. whereas the center of the peak represents the mean copy number of ligands per protein, information on the dispersity of the sample is usually neglected. patel et al. ( ) have produced a mathematical approach for calculating dispersity. by simply measuring the width at half maximum of the broad peaks that usually arise from carbohydrate-protein complexes and from the unmodified proteins, they were able to calculate the product distribution variance. furthermore, since the area between ae s equates to % of the total, m ae s represents the range within which % of adducts exist, this gives a direct measure of dispersity. as one example of the type of work involved with this type of compound, the glycosylation sites of o-specific polysaccharide of vibrio cholerae o , serotype ogawa linked to bsa via squaric acid chemistry have been determined by maldi-tof/ tof ms (from chca). the spectra showed the presence of hapten-bsa neoglycoconjugates with different hapten-bsa ratios ( . , . , and . ). sites of glycation were determined by comparison of the masses of the peptides resulting from the digestion of the bsa glycoconjugates and bsa itself using tandem ms/ms with a high-collision energy cell. the spectra improved secretion of molecular chaperone-assisted igg. no alterations in n-glycans (dojima, et al., ) interferon-γ, (human in cho cells) tof, glycans (per-me) study of intracellular glycosylation activities. effects of nucleotide sugar precursor feeding (wong et al., b) interferon ( enzyme, which usually fucosylates core of n-glycans, shown to fucosylated chitooligosaccharides (ihara et al., a) galactofuranosyltransferase glft (recombinant in m. tuberculosis h rv) l-, r-tof (chca) polymer length shown to be controlled by a template-independent polymerase (may et al., ) β-d-galactoside-α , -gal-transferase and β-d-galactoside-β , -galtransferase from pigeon two novel enzymes catalyzing the formation of galα → galβ →-galβ - glcnac galnac transferase (human) tof/tof (dhb) adds galnac to several unnatural peptides (yoshimura et al., ) galnac transferase (human) tof (dhb) enzyme found to have a unique galnac-o-ser/thr-binding site (perrine et al., ) galnac transferase (human) tof found in testis and brain (peng et identification of gal-transferase adding gal to core fuc. (titz et al., ) gnt-i fuzed to maltose binding protein from nicotiana tabacum tof/tof recombinant expression and characterization (dohi et al., ) gnt-v and gnt-vb (recombinant human. incubations in t cells) maldi enzymes transfer glcnac to position of -antennae in n-glycans. comparisons of enzymes (alvarez-manilla, et al., a) gpi-modifying β - -glcnac transferase from trypanosoma brucei enzyme characterization (izquierdo et al., ) glycosidases penicillium canescens tof (dhb) isolation and biochemical characterization, (burtseva et al., ) cellulase cel a from trichoderma reesei tof/tof (dhb, thap), esi-ms/ms enzyme shown to catalyze hydrolysis of glucosidic bonds adjacent to mono-substituted anhydroglucose units (enebro et al., b) chitinase-a from cycas revolute (cycad) tof (dhb) biochemical characterization, cdna isolation, and posttranslational modification (taira et al., b) chitinase-a and b from serratia marcescens strain bjl expressed in e. coli top effects on inhibition efficacy of allosamidin, a general competitive inhibitor of family chitinases. (zakariassen et al., ) chitinase and n-acetylhexosaminidase from verticillium fungicola tof (dhb) effect of ph on the production of enzymes in submerged cultures cellulomonas fimi tof (dhb) use of mutations to affect mannose binding (hekmat, et al., ) exocellulase cel b from thermobifida fusca tof proposal of a novel hydrolysis mechanism involving proton-transfer er glucosidase ii from rat liver tof found to be a broad specificity hexosidase (miyagawa et al., ) family gh α-mannosidase from streptococcus pyogenes tof, high-man glycans (per-me) characterization. α → mannosidase (suits et al., ) β-galactosidases from arabidopsis subfamily a tof comparative characterization (gantulga et al., ) (continued) β-galactosidases and from tomato tof enzymatic activity and substrate specifcity (ishimaru et al., ) β-galactosidases (bgac protein) from streptococcus pneumoniae specific for galβ - glcnac moiety (jeong et al., ) α-glucosidase from aspergillus niger r-tof (dhb) monitoring hydrolysis and transglycosylation activity (shimba et al., ) α-glucosidase from aspergillus niger tof (thap) activity towards dextrin and starch (ota et al., ) β-glucuronidase from aspergillus niger tof isolation and substrate specificity for transglycosylation (kiryu et al., ) shown to be involved in the maintenance of chronic infections (domenech et al., ) cholinephospho-transferase from mycoplasma fermentans tof/tof (dhb) molecular cloning and expression of enzyme involved in glycol-glycerophospholipid biosynthesis hemicellulase from chrysosporium lucknowense c tof investigation of the fungus for hemicellulase production (hinz et al., ) showed the presence of three conjugation sites on lys residues , , and , assumed to be the most accessible. the identification of y-series product ions was found to be useful for sequencing of various peptides and the a-and b-product ions confirmed the sequence of the conjugated peptides . other examples are listed in table . maldi-tof analysis, combined with ir spectroscopy has demonstrated covalent modifications of chitin with silk-like proteins in the formation of shells in the mollusc mytilus galloprovincialis (weiss et al., ) . maldi-tof ms has also been used as a standard against which to evaluate and optimize a fluorophore-assisted carbohydrate electrophoresis (face) method for analysis of pectic oligosaccharides (sun et al., a) (article in chinese). koroleva et al. ( ) have used maldi-tof ms to identify all the major glucosinolates that accumulate in s-cells of arabidopsis leaves and flower stalks. cell sap was diluted in ml methanol and mixed : with chca before being spotted onto the maldi target plate. the analysis was performed in negative-ion mode using an ultraflex tof/tof instrument. finally, mun, rho, and kim ( ) have used maldi to confirm the molecular sizes of commercial cycloamyloses and patsos et al. ( ) have used it to detect aryl glycans in cells treated with inhibitors of o-glycan processing. maldi continues to be a major technique for carbohydrate analysis although electrospray is more widely used. a major advantage of maldi is that is gives a cleaner profile of glycan mixtures because of the absence of multiple charging. new techniques, such as ion mobility have emerged to complement both maldi and electrospray but there is still much scope for improvement in carbohydrate analysis. surveys during the review period have highlighted the fact that many laboratories still make mistakes when assigning structures. much of this can be attributed to assumptions made between a simple mass (urch et al., ) s-adenosyl-l-methionine dependent methyltransferase from haloferax volcanii tof (chca) enzyme shown to methylate n-linked pentasaccharide tlmk from strepto-alloteichus hindustanus ft-icr functional characterization in tallysomycin biosynthetic pathway (wang et al., g) trehalose synthase from thermotoga maritima dsm tof molecular cloning and characterization (ryu et al., ) udp-arabinopyranose mutase from rice tof (sinapinic), glycoprotein arg residue shown to be reversibly glycosylated with single glycosyl residue; residue needed for activity (konishi et al., b) (gao et al., b) aminated xyloglucan (from tamarind) tof (dhb) physico-chemical properties of aminated xyloglucan extracted from tamarind seed (simi & abraham, c) bridged c-furanosides tof intramolecular nucleophilic attack of bzo group in a triflated cyclooctenol (jürs & thiem, ) -deoxynojirimycins with dansyl capped n-substituents tof as probes for morbus gaucher affected cell lines (fröhlich et al., ) , -dibromo- -methyl- phenylphospholane -oxide tof as novel anticancer agent (yamada et al., b) synthesis of polysaccharide intracellular adhesins using an acid reversion reaction of n-acetylglucosamine in hf-pyridine (leung et al., ) amino-bridged oligosaccharide mimetics r-tof (dhb), fab synthesis using reductive amination. glycomimetic target structures as potential ligands for the receptor protein nkr p of natural killer cells (neumann & thiem, ) amylose chains tof from starch by action of phosphorylase. preparation of a-type crystals (montesanti et al., ) bi-fluorescently-labeled maltooligosaccharides tof for amylase assays (oka et al., a) bis-hydrazides tof for conjugation with proteins etc. (adak et al., ) blood group tetrasaccharides b (types , and ) tof -aminopropyl glycosides of tetrasaccharides synthesised using acetylated galα( → )-(fucα( → ))gal trichloroacetimidate as a glycosyl donor (korchagina et al., ) , -branched arabinogalactan-type tetraand hexa-saccharides tof for investigation of monoclonal antibodies raised against arabinogalactan proteins from pressed juice of echinacea purpurea. carboxymethylated cyclosophoraose tof as a novel chiral additive for the stereoisomeric separation of flavonoids by ce (jeon et al., ) chitooligosaccharides tof by the enzymatic hydrolysis of chitosan ) chitooligosaccharides tof by the enzymatic hydrolysis of chitosan (xu et al., a) chitooligosaccharides tof/tof (dhb) glycans (amac) characterization of family chitosanase from streptomyces coelicolor a ( ) and use for degradation of chitosans chitooligosaccharides tof to study the antibacterial activity against bifidobacteria (Šimůnek et al., ) chitosan and chitooligosaccharides adsorption properties for uranium (paper in chinese) (jiang et al., b) chitosans tof synthesis from lobster chitin by naoh deacetylation and enzymatic hydrolysis. to protect crops from main pathogens (falcón et al., ) chito-tetrasaccharide β- , -glcnac-β- , -glcn repeat tof (dhb) by condensation of two disaccharides deacetylated chitohexaose tof hydrolysis of chitosan. could limit cell proliferation of breast cancer cells (xiong et al., ) -deoxy cyclic and linear oligosaccharides tof (dhb) synthesis by oligomerization of monomers (paul et al., a) β-d-fructopyranosyl-( → )-d-glucopyranose tof synthesis from d-glucose and d-fructose by thermal treatment (yamamori et al., ) galactofuranose oligomers tof/tof (chca) to probe mechanism by which polymer length is controlled in mycobacteria (splain & kiessling, ) galactomanno oligosaccharides tof from hydrolysis of guar gum by βmannosidase (paper in chinese) (zhao, et al., b) galactooligosaccharides ft-ms, gc/ms synthesis by acid hydrolysis of the polysaccharides from nerium indicum mill α-glucan pentasaccharide from aconitum carmichaeli use of chiral-auxiliary-mediated , -cisglycosylations for the solid-supported synthesis (boltje et al., ) glucan with α -( → ) linkages and α -( → ) and α -( → ) branch points produced from glucan of leuconostoc mesenteroides nrrl b- by microwave assisted hydrolysis (majumder et al., ) glucosylated raffinose tof (dhb) use of glucansucrase (alternansucrase) for synthesis glc-glc, gal-gal, gal-glc, gal-gal disaccharides tof (dhb) -member library containing all regioisomers. solid-phase synthesis (Ágoston et al., b) linear isomaltooligosaccharides (dp - ) tof one-step synthesis using synthetic fusion enzyme of dextransucrase and dextranase (kim et al., e) macrocyclic oligosaccharides tof copper(i)-catalyzed huisgen's , -dipolar cycloaddition of alkyne and azide provides size-defined macrocyclic carbohydrates (muthana et al., ) mannose-capped disaccharide with a thiol terminus to provide a tethered sugar for attaching to gold nanoparticles to mimic carbohydrateinvolved cell-surface interactions (continued) chemical stereochemically controlled synthesis (pastore et al., a) nigerose-containing oligosaccharide tof/tof (chca) escherichia coli non-glycosidically linked pseudodisaccharides tof (thap) thioethers, sulfoxides, sulfones, ethers, selenoethers, and their binding to lectins oligosaccharides from red seaweed polysaccharides tof (chca), esi efficient conversion of galactans into cglycosyl aldehydes (ducatti et al., ) oligosaccharide mimics of sialyl lewis a trisaccharide. neu ac and fuc replaced with hso and d-ara respectivly (jakab et al., ) pentasaccharide anticoagulant (idraparinux) maldi compound is fully o-sulfated, o-me mimic of antithrombin iii binding domain of heparin ) n-quaternary chitosan derivatives tof synthesis, characterization and antibacterial activity (rúnarsson et al., ) sialylated lactosides tof (dhb), per-me for coupling to bsa by huisgen reaction. as glycocongugate antigen (mosley et al., ) sialyllacto-n-tetraose and sialyllacto-n-neotetraose tof use of α - and α - -sialylated lactosaminide precursors obtained by enzymatic procedures with glycosylations employing triflic acid/n-iodosuccinimide (schmidt & thiem, ) sucrose-based guanidinecontaining g molecular transporters tof show different patterns of intracellular localization depending on the nature of the linker chains as well as the fluorescent dyes (lee et al., l) sulfated oligofucosides maldi synthesis of sulfated octyl tetra-to octaoligofucosides with different sulfation patterns (zong et al., a) α to achieve steric instead of electrostatic stabilization. two-step synthesis (kloser & gray, ) cyclic β-glucan r-tof/tof (chca) synthesis using laminarinase a glycosynthase mutant from the basidiomycete phanerochaete chrysosporium (vasur et al., ) dextrin-hydroxyethylmethacrylate and dextrinvinyl acrylate hydrogels tof/tof (dhb) for the determination of biocompatibility and biodegradability in mice (moreira et al., ) epoxy-starch derivatives tof (dhb) synthesis by epoxidation of allylated starch (huijbrechts et al., ) poly-n-acetyllactosamine tof (dhb, chca) chemo-enzymatic synthesis. characterization for cgl -galectin-mediated binding of ecm glycoproteins to biomaterial surfaces (sauerzapfe et al., ) triblock co-oligomers of tri-o-methylated and unmodified cello-oligosaccharides: tof (dhb) synthesis and structure-solubility relationships (kamitakahara & nakatsubo, ) xylan-based polysaccharides tof (dhb) amino-modified low mw xylan reacted with unmodified xylan and cellodextrins ) glycosaminoglycans and related compounds n-acetyl-heparosan oligosaccharides tof digestion of n-acetyl-heparosan with heparitinase. for study of enzymology (sugiura et al., a) , -desulfated heparin maldi for control of inflammation by inhibition of e-selectin (lakshmi et al., ) heparan sulfate oligosaccharides tof/tof (dhb), esi use of modular building blocks for preparation of a library of oligosaccharides used to probe the structural features of hs for inhibiting the protease, bace- (arungundram et al., ) heparan sulfate oligosaccharides tof synthesis and inhibition of endothelial cell functions essential for angiogenesis (cole et al., ) hyaluronan tof for nmr study of chemical proton exchange over the β( → ) glycosidic linkage (nestor et al., ) hyaluronic acid decasaccharide maldi chemical synthesis using preactivation-based chemoselective glycosylation strategy hyaluronic acid subunit and fully protected oligomers maldi tetra-, hexa-and octa-saccharides. multigram synthesis (virlouvet et al., ) isosteric sulfonate analogues of at-iii binding domain of heparin tof (thap) d-glca-and l-idoa-containing disaccharide. related to antithrombin-binding pentasaccharide of heparin. one sulfate ester replaced by na sulfonato-me moiety bisected octasaccharide maldi chemical synthesis (wang et al., e ) galβ( → )[neuacα( → )] glcnacβ( → )man motif tof chemical synthesis as molecular probe (bao et al., ) glucuronyl oligosaccharides tof, glycans ( -ap) synthesis of glucuronyl and sulfoglucuronyl oligosaccharides from hnk- glycoprotein (yagi et al., ) high-mannose glycans tof (dhb) related to hiv gp . one-pot catalytic glycosidation/fmoc removal (pastore et al., b) high-mannose glycans with glc units tof (dhb) study of the conformational properties of the glc man unit (mackeen et al., ) high-mannose glycansmethotrexate derivatives tof identification of the recognition motif of the glycoprotein-folding sensor enzyme, udp-glc: glycoprotein glucosyltransferase biantennary and high-mannose n-glycans linked to non-reducing terminus of man glcnac core, plus biotin for arrays n-glycan library tof (dhb), glycans ( -ap) enzymatic construction of library for building ms database phosphorylated highmannose glycans tof high-mannose glycans from ribonuclease. incubation with recombinant glcnac phosphotransferase. (bohnsack et al., ) (continued) (song, et al., f) sialic acid containing complex-type n-glycan tof new solid-phase synthesis. stereoselective βmannosylation, microfluidic α-sialylation and glycosylation of n-phf acetimidate on jandajel resin thiol-terminated nonamannoside tof for synthesis of microarrays (dietrich, et al., ) various complex glycans tof development of hek t expression system for human glycosyltransferases enzymatic construction of library for building ms database glycosylated amino acid derived from psgl- tof use of trichoroacetimidate donors and thioglycosyl acceptors (vohra et al., ) kl- antigen tof/tof (dhb) library of glycans to determine binding to anti-muc antibody aglycoristocetin derivatives containing hydrophobic side chain-substituted cyclobutenedione. synthesis and anti-influenza activity androgenic gland hormone of the woodlouse, (armadillidium vulgare) tof with various glycans. showed that thermodynamically most stable form not most active: result of disulfide pairing (katayama et al., b) antifreeze glycopeptide analogues tof (dhb, chca) microwave-enhanced synthesis and functional studies (heggemann et al., ) β-hfsh with chitobiose units at the natural linkage sites tof use of the sinaÿ radical glycosidation for simultaneous installation of biantennary chains and glycal chemistry to construct the tetrasaccharide core (nagorny et al., ) β-sheet-rich protein plus glcnac at various positions tof effect of glcnac position on protein folding kinetics and thermodynamics (price et al., a) biantennary n-glycans plus peptide tof/tof (dhb) solid-phase peptide synthesis. glycans linked -nh-co-ch -s-peptide (murase et al., ) bivalent glycopeptide tof mannosides linked with squaric acid (lindhorst et al., a) cd glycopeptide antigens tof (dhb, chca) chemo-enzymatic synthesis of glycopeptide with n-and o-linked glycans (huang et al., c ) c-glycosyl β -and β/β peptides maldi solution-phase synthesis. - amino acids (inaba et al., ) c-linked antifreeze glycoprotein analogues tof (dhb) effect of the length of the amide-containing side chain between the carbohydrate moiety and the polypeptide backbone influences ice recrystallization inhibition (tam et al., ) c-mannosylated peptides tof (dhb, chca) peptides shown to interact with hsc to modulate its signaling in raw . cells ) cyclic antifreeze glycopeptides tof exhibited antifreeze activity by forming hexagonal-bipyramidal ice crystals (hachisu et al., ) cyclic neoglycopeptides maldi (chca) for binding to adjacent protein binding sites in wheat germ agglutinin (schwefel et al., ) dihydrofolate reductase tof glycans = glcnac, lactose, maltotriose analysis as tryptic peptides. for study of effects of glycosylation on stability (tey et al., ) mass spectrometry reviews doi . /mas microwave-assisted synthesis. ( )-carboxyfluorescein shown to be stable fmoc-threonine bearing a core- glycan tof as building block for psgl- via fmocassisted solid-phase peptide synthesis (krishnamurthy et al., ) o-fucosylated epidermal growth factor-like repeat of mouse notch- receptor chemical synthesis and studies on folding (hiruma-shimizu et al., ) galactosylated naringinase tof (sinapinic) modification of glycosylation to effect deglycosylation of rhamnosylated drugs (garnier et al., ) gal-β- galnac-α-lys tof immunogen design for tumor t antigen immuno-targeting (sendra et al., ) glycopolypeptide-based cholera toxin inhibitors maldi effect of peptide charge and glycan linker length on activity (maheshwari et al., ) glycopeptide carrying tetra-n-ac-lactosamine containing core decasaccharide tof (dhb) solid-phase synthesis (ueki et al., ) glycosaminoglycan-protein linkage tetraosyl peptide moieties of betaglycan tof to investigate structures that best serve as a hexosamine acceptor for enzymatic glycosyl transfer (tamura et al., ) glycosulfopeptides from nterminus of human endoglycan tof/tof (thap) containing tyrosine sulfate residues and sialyl lewis x in core o-glycans and bind to human l-selectin (leppänen et al., ) glycosylated peptoids tof by on-resin click (huisgen reaction) glycoconjugation (norgren et al., ) glycosylated cell-penetrating peptide (r /w ): ac-rrwwrrwrr-nh tof one, two, or three galactose(s), with or without a spacer introduced via a triazole link (dutot et al., ) grgdy grafted to chitosan tof (chca) linked with sulfosuccinimidyl- -[ '-azido- 'nitropheny-lamino]hexanoate as drug carrier n-glycosylated insulin l-tof (dhb) addition of three glcnac residues at nh groups on peptide. use of endo m to transfer glycan to one of them. (tomabechi et al., a) n-glycoproteins carrying intact natural n-glycans tof/tof (dhb) enzymatic synthesis of biantennary glycans (huang et al., b) glycosylated analogues of glucagon-like peptide tof, lc-ms to improve proteolytic stability and blood glucose-lowering activity. sugars = glcnac, lacnac, sialyl lacnac synth. by chemoenzymatic approaches (ueda et al., b) glycosylated neurotensin analogues tof containing o-linked β-melibiose and α-tf antigen or β-lactose units linked by a peg spacer. exhibit subpicomolar anticonvulsant potency in model of epilepsy (lee et al., f) glycosylated tetracontapeptide with acidlabile sialyl-t n antigens maldi (dhb) -residue glycopeptide-α-thioester and residue glycopeptide with cys at n-terminal. solid phase synthesis; coupled by cys ncl ser . o-glycopeptide mimetics tof (dhb, chca) aziridine ring opening as regio-and stereoselective access to o-glycosyl amino acids and their transformation into oglycopeptide mimetics (schäfer et al., ) heptasaccharide from campylobacter jejuni plus acra - tof/tof (chca) development of nmr method for d structural determination of glycoproteins using enzymatically synthesised glycoprotein (slynko et al., ) (continued) -oh-proline oligomers q-tof (dhb) compounds form very stable polyproline ii helices (owens et al., ) iga-hinge peptide tof to study effect of glycosylation on cis/trans isomerization of prolines (by nmr) man - glcnac -asn-n- c r-tof (dhb) for micro-method for determining precise oligosaccharidic specificity of mannosebinding lectins (debray et al., ) mannosylated lysine derivative tof bivalent carbohydrate branching unit. suitable for solid-phase peptide synthesis chemical synthesis, for enzymatic study in dictyostelium (see table ,o-glycans) (wang, et al., k) s-linked glycopeptides maldi thioglycosylated building blocks prepared from per-ac sugars via glycosyl iodides in one-pot fashion and used in sub-monomer solid phase strategy (comegna & de riccardis, ) synthesis using using the newly developed n-troc-protected gm and galn intermediates (komori et al., ) anchor with n-terminal cys maldi general method for producing proteins containing a natural gpi anchor using expressed protein ligation (schumacher et al., ) anchor with unsaturated lipid chains tof to investigate mechanism of gpi anchoring (swarts & guo, bearing pyrazolines, isoxazolines, and dihydropyrimidine- ( h)-thiones. as antibiotics (el-sayed et al., ) hydroquinone fructoside l-tof synthesis using leuconostoc mesenteroides levansucrase hydroquinone glucoside l-tof (dhb) synthesis using leuconostoc mesenteroides levansucrase lobatoside e-related triterpene glycoside maldi gold(i)-catalyzed glycosylation with glycosyl ortho-alkynylbenzoates as donors man-α-( → )-man-o-octyl analogues maldi synthesis and evaluation as potential substrates and inhibitors of a ppm-dependent α-( → )-mannosyltransferase involved in lam/lm biosynthesis (tam & lowary, ) mannosylated pyreneperfluoroalkyl lipid maldi to study multivalent binding on the lateral phase separation of adhesive lipids (liem et al., ) mannosyl glycolipids with perfluoroalkyl membrane anchors maldi to assess the cluster glycoside effect during the binding of concanavalin a to mannosylated artificial lipid rafts (noble et al., ) mesogenic azobenzenes tethered to sugar alcohols (gretskaya & mikhalyov, ) cholesterol plus linear glucose tof (dhb) synthesis and gelling properties , -dipalmitoyl- -(npalmitoyl- '-amino- '-deoxyα-d-glucosyl)-sn-glycerol tof glycoglycerolipid of a marine alga with a high inhibitor activity against human myt kinase. synthesis starting from α-me-glcp enzymatic synthesis from , dipalmitoylglycerol in one pot reaction hydroquinone galactosides l-tof (dhb) a potential skin whitening agent. synthesis by reaction of lactase with lactose as donor. fluorescently labelled synthetic ionophore synthesises in steps with % yield (coppola et al., ) phosphatidyl-myo-inositol mannosides tof to study mannosyltransferase corynebacterium glutamicum pimb' (batt et al., ) puerarin-cycloamylose inclusion complex l-tof (dhb) enzymatic synthesis using -αglucanotransferase and maltogenic amylase (choi et al., ) sialic cinnamoyl-α-cyclodextrin tof initiates polymerization of δ-valerolactone (osaki et al., ) cinnamoyl α-cyclodextrin tof cds self-organize to give different supramolecular complexes in aqueous solutions (tomimasu et al., ) (continued) synthesized via hydrosilylation reaction under thermal or uv-activated polymerization (tian et al., ) cd-based telluronic acid plus mn(iii)meso-tetra[ -( adamantyl methyl ketone)- pyridyl] porphyrin maldi as artificial enzyme with superoxide dismutase and glutathione peroxidase activities (yu et al., d) cyclodextrin dimers tof one homo-dimer of β-cd and two heterodimers of α-cd and β-cd as hydrolases (ikeda et al., ) cyclodextrin dimers and trimers tof/tof, esi bridged cds with links at different positions formed supramolecular adducts with shapespecific ligands (aime et al., ) β-cyclodextrin cyclic-nitrone conjugate maldi with superoxide radical anion and dodecyl chain for membrane insertion α-, βand γ-cyclodextrinesters (acrylate, pent- -enoate and undec- -enoate) synthesis with nitrophenol esters (nielsen et al., a) β-cd plus hydroquinone-αglycoside tof synthesis and doxorubicin-inclusion abilities (oda et al., ) β-cd monoalkyn tof (dhb) for conjugation to long-chain thiols for selfassembled monolayer prep. (dubacheva et al., ) cyclodextrin-polyester polymers tof ring-opening polymerization -heating cyclic esters and cds (harada, ) γ-cds possessing an azido group and a triisopropylbenzenesulfonyl group as useful synthetic and authentic intermediates for unsymmetrically functionalized derivatives (himeno et al., ) β-cd-ended linear poly(nisopropylacrylamide) (β-cd-pnipam) tof (thap, dctb) self-assembly of pnipam-based amphiphiles formed by inclusion complexation (zou et al., ) β-cd with octadecyl-linked perylene bisimide maldi self-assembled amphiphilic perylene-cd conjugate for vapor sensing of organic amines (jiang et al., a) [ -deoxy- -( -h- , , triazol- -yl)(me) -( -omebi-ph- '-yloxy) hexanoyl]-βcyclodextrin tof (dhb) synthesis, liquid-crystalline properties, and supramolecular organization for controlled release application. (adrian et al., ) inclusion complexes of β-cd with bipyridine guests tof with , '-vinyl-enedipyridine, , 'vinylenedipyridine, -( -pyridyl)- -( pyridyl)ethylene, , '-ethylene-dipyridine, , '-dithiodipyridine, and , 'dithiodipyridine insulated molecular wire with highly conductive πconjugated polymer core tof rod-like -ph-c=c-ph-core coated with α-cds (terao et al., b) ionic-liquid-functionalized βcyclodextrin tof as bonded chiral stationary phases for hplc (zhou et al., b ) linear α-cyclodextrin oligomers tof controlled synthesis using copper-catalyzed huisgen , -dipolar cycloaddition (rawal et al., ) oligothiophene derivatives bearing β-cyclodextrin tof (dhb) t-β-cd and t-β-cd , with bithiophene and terthiophene with β-cd at each end form supramolecular assemblies in aqueous solns. (sakamoto et al., b) perylene bisimide-bridged bis-(permethyl-β-cds) ft-icr as solid-state fluorescence sensor for vapor detection perylene-bridged bis(βcyclodextrin) ft-icr (kitano et al., ) glycodynamers maldi dynamic polymers bearing oligosaccharide residues such as glc obtained from α-cd (ruff et al., ) membranes (cross-linked) tof (dhb) based on acrylated cyclodextrins and polyethylene glycol dimethacrylates (rölling et al., ) multivalent galactotrehaloses tof α,β-gt isomers converted into vinyl monomers then radical copoly-merization with -acrylamidophemyl-β-glc or β-glcnac) with acrylamide (miyachi et al., b) pentafluorostyrene copolymers with glucose tof synthesis by nitroxide-mediated polymerization and "click" chemistry (becer et al., ) polydioxanone with a protected monosaccharide end-group l-tof (dithranol) ring opening polymerization of p-dioxanone using a protected monosaccharide ( , ; , -di-o-isopropylidene-α-d-galactopyranose) /al(oipr) initiator system (sugih et al., ) polyfluorene derivative with mol% , , benzothiadiazole plus α-man on an amine-reactive hydrogel-coated microarray glass surface. to detect autoantibodies in breast cancer correction: (blixt, et al., ) nanofibres tof nanofibers formed through π· · ·π stacking of the complexes of glucosyl-c -salicyl-imine and phenylalanine (continued) for study of carbohydrate-protein interaction oligosaccharides from plants tof (dhb) for construction of microarray to screen for plant transglycosidases activity (kosík, et al., ) -sulfo-n-acetyl-dglucosamine containing, on gold tof to study mechanism of amyloidosis of amyloid β peptides (fukuda et al., b) miscellaneous β-d-allopyranoside-grafted ru(ii) complex tof synthesis and both acid-base and dnabinding properties azo-sugar nucleotides plus many alkynes tof (dhb) click chemistry (huisgen reaction) to produce inhibitors of glycosyltransferases -deoxy- -nitropiperidinoses maldi synthesis from a protected galactooctopyranose (collin & vasella, ) , -di-f- , -di-me- -bora- a, a-diaza-sindacene- propionic acid derivative of man glcnac . (bodipy) for development of fluorescence assay (haga et al., ) carbohydrate-functionalized salphen-metal complexes tof complexes with peripheral glucose and galactose substituents. self-assembled supramolecular structures were produced (hui et al., ) dioxolane-type ( 'anthracenyl)methylene acetals tof synthesis, regioselective hydrogenolysis, partial hydrogenation, conformational study (jakab et al., ) (kim et al., f) amino-sugars tof (chca), esi deprotection method for the , , trichloroethoxycarbonyl (troc) group using tetrabutylammonium fluoride , -anhydrosaccharides tof use of mol % of aubr to catalyse glycosylations of -oh-propargyl/me mono-, diand tri-saccharides (thadke & hotha, ) colored carbohydrates tof addition of a fmoc analogue protecting group based on guaiazulene (aumüller & lindhorst, ) (bindschädler et al., ) glycoamino acid building blocks tof use of staudinger ligation glycopeptides tof use of pyruvoyl as a novel protecting group for solid-phase synthesis (katayam et al., ) gpi-anchored proteins tof sortase a-catalyzed transpeptidation of gpi derivatives for chemoenzymatic synthesis (wu et al., f) heptasaccharide asparagine building block tof (dhb) use of one-pot catalytic glycosidation/fmoc removal (mezzato & unverzagt, ) homolinear α( → )-linked octamannosyl thioglycosides maldi imidazolium cation-tagged mannosyl fluoride and thiomannoside using block couplings. efficient alternate approach for oligosaccharide synthesis (yerneni et al., ) homooligosaccharides tof (dhb) (per-ac, per-me) base-promoted glycosylation of unprotected glycosyl fluorides (steinmann et al., ) lipophilic thioglycosides tof use of heavy lipophilic tag to assist biphasic liquid-liquid separation (encinas & chiara, ) macrocyclic neoglycoconjugates tof (chca) macrocyclization of linear d-galacto- heptulopyranose-containing oligoketosides by intramolecular glycosidation and ring-closing metathesis mucin-type oglycopeptides and oligosaccharides tof (dhb, thap, chca) using transglycosylation and reverse-hydrolysis activities of bifidobacterium endo-α-nacetylgalactosaminidase neu gc-containing glycans tof sialylation with n-glycolylneuraminyl phosphite (hanashima et al., ) n-linked glycopeptides tof condensation of glycosylamines with asn. demonstrated with man glcnac . (chen & tolbert, ) measurement and structure, particularly when structures are selected directly from databases. at present, no one mass spectrometric technique can identify all structural features of carbohydrates. sialylated and sulfated glycans still remain a problem although sialylated glycans can be handled after suitable derivatization, particularly by permethylation or simply by methyl ester formation. permethylation appears to be becoming more popular, particularly for quantification and maldi. the next few years are expected to bring many developments, particularly in instrumentation and ionization techniques, that will possibly address some of the problems with carbohydrate analysis highlighted above. -ab -aminobenzamide -aa -aminobenzoic acid -aa -aminoacridine aboe aminobenzoic acid octyl ester aeab -amino-n-( -aminoethyl)-benzamide age advanced glycation end products agp a -acid glycoprotein alpcs aluminum-phthalocyanines a-tf a-thomsen-freidenreich antigen all allose amac aminoacridone amt -amino- -mercapto- , , -thiadiazole ann artificial neural networks anp -amino- -nitro- -picoline ansa -amino- -naphthalenesulfonic acid -ap -aminopyridine apb aminophenylboronic apci atmospheric pressure chemical ionization api apiose apts -aminopyrene- , , -trisulphonic acid aq aminoquinoline ( -or -) use of genetic engineering to produce glycoproteins in e. coli which are then enzymatically remodelled (see text) (schwarz, et al., ) o-sulfated trisaccharyl glycopeptide tof (dhb) solid-phase synthesis. use of new benzyl protection method. (kawahira et al., ) oligosaccharides tof (dhb) rate acceleration on stereoselectivity and velocity of o-glycosylation reactions (ishiwata et al., ) pseudooligosaccharides maldi use of cross-metathesis reaction between sugarolefins, followed by intramolecular cyclization (ronchi et al., ) s-linked glycoconjugates tof, esi by-thiyl glycosylation of olefinic proteins (floyd et al., ) sialylated glycans tof use of koenigs-knorr reaction (with ag co ) (pazynina et al., ) thioglycosides r-tof/tof solvent-free synthesis by use of ball-milling (patil & kartha, ) trisaccharide libraries tof use of linker-tagged building blocks imobilized on a soluble polymeric support (elsayed, ) acceptor specificity in transglycosylation reactions using endo-m l-tof (dhb), esi (tomabechi et al., b ) acetolysis of -deoxyhexosemethyl glycosides. role of sugar configuration tof (dhb) (cirillo et al., ) acid-catalyzed hydrolysis of β- , -glucan, including cellobiose and crystalline cellulose with so h-bearing amorphous carbon tof (suganuma et al., ) allyloxycarbonyl group removal provides a practical orthogonal protective strategy for carbohydrates tof (zong et al., ) amadori ketoses synthesis in microwave field via mo vi -catalyzed stereospecific isomerization of -c-branched sugars bearing azido function tof (hricovíniová, (camponovo et al., ) benzhydrylamine-lysine capped with to mono-, di-and tri-α-d-manp residues tof reactive n-oh-succinimide esters to ensure complete reaction of dendrimer amines (g mass = , da) (greatrex et al., ) , -bis( , -bis( -( -( azidoethoxy)-ethoxy)ethyl)fluorenyl)benzo-thiadiazole with mannose residues tof (dhb) as an intelligent energy transfer pair for label-free visual detection of concanavalin a boltorn h (commercial polymer) with gal-cer groups tof binds hiv- gp optically pure fullerodendron formed by diastereoselective diels-alder reaction calixarene-based glycocluster oligonucleotide with galactose residues tof click chemistry. triazole-tethered glycoclusters with arrangements. affinities towards pa-il and rca with dna-based glycoarray. (moni et al., ) carbosilane with , or sialyl-α-( → )-lactose residues tof for anti-influenza properties carbosilane with , , or neu ac residues tof, fab, esi influenza neuraminidase inhibitors. dendrimers uniformly functionalized with thioglycoside-type sialic acid moieties resistant to neuraminidases (sakamoto et al., a) carbosilane with or mannose residues tof (dithranol) hydrosilylation of allyl tetra-ac-man with carbosilane dendrimers containing monohydrosilane end groups and the subsequent deacetylation (ortega et al., ) carbosilane with sialyl-nacetyllactosamine groups tof combined chemical and enzymatic synthesis (matsuoka et al., ) cyclic α-( → )-octaglucoside with from - mannose residues tof oxidation of vic-diols, reductive amination with -aminoethylmannoside β-cyclodextrin with mannose or glucose residues tof (dhb) heteroglycoclusters. antennae each with two mannose and one glucose. for lectin binding (gómez-garcía et al., ) β-cyclodextrin with dextran tof (chca) synthesis by click chemistry using huisgen reaction (nielsen et al., b) cyclopeptide with mannose residues tof synthesis by click chemistry and molecular recognition study by surface plasmon resonance (chen et al., c) dihydroxy-benzamide based with - mannose, galactose, lactose, glucose or glcnac groups tof (chca) octa-dendrimers. for screening of lectins for multivalency effects. click chemistry (pera et al., b) diphenyldisulfide with glucose residues and others without sugars tof, gc/ms use as catalyst to convert allyl alcohols into carbonyl compounds (tsuboi et al., ) cysteine with or (from dicysteine) mannose residues tof as inhibitors of type fimbriae mediated bacterial adhesion (schierholt et al., ) ethylene glycols with , or galactose,lactose, maltose or lacnac groups ferrocene with one or two glucose, mannose or lactose residues tof/tof as electrochemical probes for molecular recognition studies (casas-solvas et al., b) polyglycerol substituted phenylboronic acid tof dendrimer formed adducts with, fru, d-(+)-gal, d-(+)-glc, d-(+)-man, and me-α-d-man, by removal of four h o (hashidzume & zimmerman, ) fullerene. sugar balls with iminoglucose residues tof synthesis by click chemistry. glucosidase inhibition shown with resulting iminosugar balls (compain et al., ) fullerene. sugar balls with glucose or galactose residues tof, esi, fab residues on arms, synthesis by click chemistry gd-diethylenetri-aminepentaacetic acid with glucose residues tof synthesis, in vitro and in vivo studies of gd-dtpa-xda-d -glc(oh) complex as a new potential mri contrast agent (ozaki et al., a) hydroxy benzenes and naphthalenes with , or mannose residues tof synthesis by click chemistry (rajakumar et al., ) l-lysine plus gd chelates and galactose residues tof/tof as liver imaging probes (luo et al., ) tetramer of glycodecapeptide from muc with sialyl t n antigen as vaccine candidate (keil et al., ) peptide/tris-oh-methylmethylamine. galactose or mannose residues attached to β-cd containing doxorubicin tof for uptake studies in the human hepatocellular carcinoma cell line hepg (bernardes et al., ) peptide/tris-oh-me-methylamine/ [ru(bipy) ] + with galactose or mannose residues maldi to study lectin interactions by monitoring change in fluorescence quantum yield of ru(ii). (kikkeri et al., a) phloroglucinol, triazyne, tetrachlorosilane, pentaerythritol, myo-inositol with , , or clinked sialic acids tof synthesis by huisgen cycloaddition of azide and alkyne (click chemistry). to explore sialic acid binding to cell surfaces (papin et al., ) phthalocyanine with α-galp residues tof potential application as photosensitizers in photodynamic therapy (soares et al., ) pentaerythritol, methyl α-dglucopyranoside, d-glucose, and dmannitol with , or mannose tof (dhb) synthesis by click chemistry, measurement of binding affinities (sattin et al., ) poly-glycerol with mannose residues tof maldi poor because of high mw (up to kda) (kizhakkedathu et al., ) poly-lysine with biantennary nglycans tof synthesis by click chemistry. effect of sialic acid linkage on in vivo dynamics. mass around kda (tanaka et al., b) poly-lysine on tris-( -ethylamino)amine with aulfated cellobiose groups tof (sdhb) synthesized by sulfation of polylysinedendritic cellobiose (prepared from cellobiose and polylysine dendrimer generation ) (han et al., b) porphyrin with lactose glycans tof synthesis by huisgen click cycloaddition of azide and alkyne (okada et al., b) porphyrin with galactose or lactose maldi synthesis by click chemistry. activity on carcinogenic hep cells activators of natural killer lymphocytes (renaudet et al., ) ruthenium(ii) bipyridine with or mannose or galactose residues tof as probes to study lectin-carbohydrate interactions and to measure mono and oligo-saccharide concentrations electrochemically (kikkeri et al., b) ruthenium porphyrins with glucose residues tof water-soluble catalyst for carbenoid transfer reactions tetrabenzo-porphyrins with glucose residues tof to improve targeting of cancer cells (ménard et al., ) tetraphenyl-ethylene with lactose or sialyl-lactose residues tof/tof synthesis by click chemistry. as fluorescent probes for detection of influenza virus tetraphenyl-ethylene with or mannose residues tof (dhb) for fluorescence turn-on sensing of lectins based on aggregation-induced emission (sanji et al., ) dihydroxybenzamide base with galabiose residues tof (chca) for isolation of pathogenic streptococcus suis bacteria (pera et al., a) , , -tris-( -propynyloxy)-benzene with β-cyclodextrin rings tof (dhb) synthesis by microwave-assisted click chemistry as fluorescent tripod detection system for pesticides (mallard-favier et al., ) trimesic acid and others with , or phosphocholine residue related to glycol-sphingolipid from earthworm pheretima hilgendorfi tof for enhanced immune responses when compared to their monovalent counterparts various amino-alkyl with , or lactose residues tof (chca) synthesis from carbamate-linked lactose ) zinc(ii) phthalocyanines with glc, gal or cellobiose residues tof (dhb) chemical synthesis as photosensitizer in photodynamic therapy (iqbal et al., a ) zinc(ii) phthalocyanines with glc, gal, cellobiose or maltose residues tof (dhb, chca) eight residues. for photodynamic therapy (iqbal et al., ) zinc(ii) naphthalocyanines with glucose residues tof as photosensitizer in photodynamic therapy (iqbal et al., b ) zinc(ii) naphthalocyanine with glucose or galactose residues tof, esi ex post glycoconjugation (berthold et al., ) free amino group on lps used for site-specific conjugation. towards bivalent immunogens (grandjean et al., ) -amino- -deoxy-larabinose (ara n) maleimideactivated bsa potent immunogen (müller et al., a) n-acyl-modified sialylated glycans hsa tof squaric acid chemistry. as inhibitors of adenoviruses causing epidemic keratoconjunctivitis biantennary gal, -keto-gal + fluorescent probe single-chain antibody tof method for drug targeting using antibodies galactose bsa tof new photoinduced thiol-ene coupling galactose hsa tof two step synthesis of gal as optical imaging agent for peritoneal carcinomatosis (regino et al., ) ginsenoside rg bsa tof (sinapinic) generation and characterization of monoclonal antibody to ginsenoside rg (joo et al., ) α-glc, α-man, β-gal, α- to study role on antigen in immune system interaction (tefsen et al., ) heparin tetra-saccharide complement factor h tof (sinapinic, chca) study of interaction between protein and tetrasaccharide by cross-linking (blaum et al., ) (hirata et al., ) α-mannosides (bi-and penta-) bsa tof immunogenicity and induction of candidacidal activity methyl glyoxal (advanced glycation end product, polysialic acid insulin maldi shown to increase insulin lifetime in vivo (bezuglov et al., ) serogroup pneumococcal oligosaccharides bsa seldi-tof, fab synthetic carbohydrate conjugates express epitopes found in native capsular polysaccharides (parameswar et al., ) tn antigen hsa maldi to study effects of hapten density on induced antibody repertoire tumor-associated muc glycopeptides to study multivalent binding to human α-defensin (hd ) (lehrer et al., ) faims field asymmetric waveform ion mobility spectrometry fc fragment (crystallisable) region of igg fmoc -fluorenylmethoxycarbonyl fru fructose f-spe fluorous solid phase extraction ft fourier transform fuc fucose g (g , g ) biantennary glycans with , ( or ) galactose residues g ca coumaric , , , ,-tetra-methylguanidine gaba gamma-aminobutyric acid gags glycosaminoglycans gal galactose gala galacturonic acid galn galactosamine galnac n-acetylgalactosamine g ca coumaric , , , ,-tetra-methylguanidine (liquid matrix) gapcs gallium-phthalocyanines gc/ms gas chromatography/mass spectrometry production of chitooligosaccharides and their potential applications in medicine synthesis of biotinylated sialoside to probe cd -ligand interactions nanofibers formed through p…p stacking of the complexes of glucosyl-c -salicyl-imine and phenylalanine: characterization by microscopy, modeling by molecular mechanics, and interaction by a-helical and b-sheet proteins bishydrazide glycoconjugates for lectin recognition and capture of bacterial pathogens the applicability of enzymes in cellulose ether analysis electron impact ion fragmentation pathways of peracetylated c-glycoside ketones derived from cyclic , -diketones inclusion complexes of gcyclodextrin and carboxyl-modified g-cyclodextrin with c : synthesis, characterization and controlled release application via microgels biochemical characterization of two xylanases from yeast pseudozyma hubeiensis producing only xylooligosaccharides synthesis of -o-glycosylated , -anhydro-d-fructose and of , -anhydro-d-tagatose from a common intermediate , -o-isopropylidene-d-fructose solid-phase random glycosylation comparative solution and solid-phase glycosylations toward a disaccharide library mutational and functional analysis of large in a novel cho glycosylation mutant separation of -aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with . mm sorbent depolymerization of sodium alginate under hydrothermal conditions cell wall b-( , )-glucan of saccharomyces cerevisiae. structural characterization and in situ synthesis new cyclodextrin dimers and trimers capable of forming supramolecular adducts with shape-specific ligands development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage reflection colour changes in cholesteric liquid crystals after the addition and photochemical isomerization of mesogenic azobenzenes tethered to sugar alcohols preparative enzymatic synthesis of polyprenyl-pyrophosphoryl-n-acetylglucosamine, an essential lipid intermediate for the biosynthesis of various bacterial cell envelope polymers plant cell wall proteomics: mass spectrometry data, a trove for research on protein structure/function relationships introducing capillary electrophoresis with laser-induced fluorescence detection (ce-lif) for the characterization of konjac glucomannan oligosaccharides and their in vitro fermentation behavior introducing capillary electrophoresis with laser-induced fluorescence (ce-lif) as a potential analysis and quantification tool for galactooligosaccharides extracted from complex food matrices nmr spectral mapping of lipid a molecular patterns affected by interaction with the innate immune receptor cd bioconjugation of d-glucuronic acid sodium salt to well-defined primary amine-containing homopolymers and block copolymers glycomic analysis of sialic acid linkages in glycans derived from blood serum glycoproteins chip-based reversed-phase liquid chromatography-mass spectrometry of permethylated n-linked glycans: a potential methodology for cancerbiomarker discovery plasticity of xyloglucan composition in bean (phaseolus vulgaris)-cultured cells during habituation and dehabituation to lethal concentrations of dichlobenil comparison of the substrate specificities and catalytic properties of the sister n-acetylglucosaminyltransferases, gnt-vand gnt-vb (ix) glycoproteomic analysis of embryonic stem cells: identification of potential glycobiomarkers using lectin affinity chromatography of glycopeptides negative-ion maldi-qit-tofms n for structural determination of fucosylated and sialylated oligosaccharides labeled with a pyrene derivative structural determination by negative-ion maldi-qit-tofms n after pyrene derivatization of variously fucosylated oligosaccharides with branched decaose cores from human milk derivatization with -pyrenyldiazomethane enhances ionization of glycopeptides but not peptides in matrix-assisted laser desorption/ionization mass spectrometry large-scale glycomics for discovering cancer-associated n-glycans by integrating glycoblotting and mass spectrometry a concise review of mass spectrometry imaging a glycomics approach to the discovery of potential cancer biomarkers determination of glycosylation sites and site-specific heterogeneity in glycoproteins glycomics and disease markers structural analysis of a fucoidan from the brown alga fucus evanescens by maldi-tof and tandem esi mass spectrometry structural analysis of a highly sulfated fucan from the brown alga laminaria cichorioides by tandem maldi and esi mass spectrometry n-glycan targeted gene delivery to the dendritic cell sign receptor oxazole-modified glycopeptides that target arthritis-associated class ii mhc aq and dr proteins glycocluster design for improved avidity and selectivity in blocking human lectin/plant toxin binding to glycoproteins and cells carbamate-linked lactose: design of clusters and evidence for selectivity to block binding of human lectins to (neo)glycoproteins with increasing degree of branching and to tumor cells maldi-tof/ms analysis of archaebacterial lipids in lyophilized membranes dry-mixed with -aminoacridine structural characterization of cell wall polysaccharides from two plant species endemic to central africa, fleurya aestuans and phragmenthera capitata disease-associated glycosylated molecular variants of human c-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and indian visceral leishmaniasis the n domain of human angiotensin-i-converting enzyme: the role of n-glycosylation and the crystal structure in complex with an n domain-specific phosphinic inhibitor, rxp loss of effector function of human cytolytic t lymphocytes is accompanied by major alterations in n-and oglycosylation glycome informatics: methods and applications a-carba-glycopyranoside primers: potential building blocks for biocombinatorial synthesis of glycosphingolipid analogues identification, characterization and immunogenicity of an o-antigen capsular polysaccharide of francisella tularensis characterization of oligomeric xylan structures from corn fiber resistant to pretreatment and simultaneous saccharification and fermentation dense-shell glycodendrimers: uv/vis and electron, paramagnetic resonance study of metal ion complexation bioinformatics in glycomics: glycan characterization with mass spectrometric data using simglycan tm functional identification of the proteus mirabilis core lipopolysaccharide biosynthesis genes three enzymatic steps required for the galactosamine incorporation into core lipopolysaccharide redox proteomics of fat globules unveils broad protein lactosylation and compositional changes in milk samples subjected to various technological procedures lactosomes: structural and compositional classification of unique nanometer-sized protein lipid particles of human milk the pleurotus ostreatus hydrophobin vmh and its interaction with glucans comparison of the water-soluble carbohydrate composition and fructan structures of agave tequilana plants of different ages synthesis, conformation, and biological characterization of a sugar derivative of morphine that is a potent, long-lasting, and nontolerant antinociceptive modular synthesis of heparan sulfate oligosaccharides for structure-activity relationship studies direct profiling of saccharides, organic acids and anthocyanins in fruits using electrospray droplet impact/secondary ion mass spectrometry syntheses of mucin-type o-glycopeptides and oligosaccharides using transglycosylation and reverse-hydrolysis activities of bifidobacterium endo-a-nacetylgalactosaminidase recovery of water-soluble compounds from ganoderma lucidum by hydrothermal treatment binding and cellular activation studies reveal that toll-like receptor can differentially recognize peptidoglycan from gram-positive and gram-negative bacteria grafting of aminated oligogalacturonans onto douglas fir barks. a new analysis of carbohydrates and glycoconjugates & route for the enhancement of their lead (ii) binding capacities defining criteria for oligomannose immunogens for hiv using icosahedral virus capsid scaffolds dendrimers designed for functions: from physical, photophysical, and supramolecular properties to applications in sensing, catalysis, molecular electronics, photonics, and nanomedicine the red wine extract-induced activation of endothelial nitric oxide synthase is mediated by a great variety of polyphenolic compounds coloring carbohydrates: investigation of azulene derivatives as blue protecting groups differences in the sialylation patterns of membrane stress proteins in chemical carcinogen-induced tumors developed in balb/c and il- a deficient mice mass spectrometry of n-linked glycans structural characterisation of neutrophil glycans by ultra sensitive mass spectrometric glycomics methodology structural characterization of non-reducing oligosaccharide produced by arthrobacter crystallopoietes n- fragmentation of deprotonated d-ribose and d-fructose in maldi-comparison with dissociative electron attachment multidimensional profiling of components in complex mixtures of natural products for metabolic analysis, proof of concept: application to quillaja saponins structural investigation of bacterial lipopolysaccharides by mass spectrometry and tandem mass spectrometry probe design and synthesis of galb( ! )[neuaca( ! )]glcnacb( ! )man motif of n-glycan glycoprofiling bifidobacterial consumption of galacto-oligosaccharides by mass spectrometry reveals strain-specific, preferential consumption of glycans n-glycosylation of plant recombinant pharmaceuticals synthesis of thiourea-tethered cglycosyl amino acids via isothiocyanate-amine coupling permeate from cheese whey ultrafiltration is a source of milk oligosaccharides detecting glycan cancer biomarkers in serum samples using maldi ft-icr mass spectrometry data quantitative analysis of glycation sites on human serum albumin using o/ olabeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry characterization of wrss and wrss , new second-generation virg(icsa)-based shigella sonnei vaccine candidates with the potential for reduced reactogenicity mass spectrometry in the characterization of human genetic n-glycosylation defects combined treatment of human mcf- breast carcinoma with antibody, cationic lipid and hyaluronic acid using ex vivo assays ex vivo assays of cem cells cultured and treated in the three dimensional cultures glycan analysis and influenza a virus infection of primary swine respiratory epithelial cells: the importance of neuaca - glycans acceptor substrate discrimination in phosphatidyl-myo-inositol mannoside synthesis. structural and mutational analysis of mannosyl transferase corynebacterium glutamicum pimb synthesis and antimicrobial evaluation of amphiphilic neamine derivatives clicking pentafluorostyrene copolymers: synthesis, nanoprecipitation, and glycosylation trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and fc-fusion proteins chemical structure of bacteriovorax stolpii lipid a core chirality based tailoring of the liquid crystalline properties of supermolecular tetrapedes development of a nanolc ltq orbitrap mass spectrometric method for profiling glycans derived from plasma from healthy, benign tumor control, and epithelial ovarian cancer patients development of a robust and high throughput method for profiling n-linked glycans derived from plasma glycoproteins by nanolc-fticr mass spectrometry genomic and biochemical analysis of n-glycosylation in the mushroom-forming basidiomycete schizophyllum commune the structurally similar, penta-acylated lipopolysaccharides of porphyromonas gingivalis and bacteroides elicit strikingly different innate immune responses design, synthesis and biological evaluation of carbohydrate-functionalized cyclodextrins and liposomes for hepatocyte-specific targeting ex post glycoconjugation of phthalocyanines modification of recombinant proteins by covalent polysialation illustrated with the example of human insulin influence of protein molecular mass on the glycation historical overview of glycoanalysis synthesis of differentially protected glucosamine building blocks and their evaluation as glycosylating agents de novo synthesis of differentially protected l-iduronic acid glycosylating agents chemical analysis of flavonoid constituents of the seagrass halophila stipulacea: first finding of malonylated derivatives in marine phanerogams substantial spatial flexibility and hydrogen bonding within the catalysis exerted by cyclodextrin artificial glycosidases lysine and arginine side chains in glycosaminoglycanprotein complexes investigated by nmr, cross-linking, and mass spectrometry: a case study of the factor h-heparin interaction glycomic analysis of n-linked carbohydrate epitopes from cd of mouse brain o-glycosylation pattern of cd from mouse brain a high-throughput oglycopeptide discovery platform for seromic profiling a high-throughput oglycopeptide discovery platform for seromic profiling-correction alteration of protein glycosylation in liver diseases investigating the molecular basis for the virulence of escherichia coli k by nuclear magnetic resonance analysis of the capsule polysaccharide monitoring of malting process by characterization of glycation of barley protein z determination of glycan structure from tandem mass spectra use of fullerene-, octadecyl-, and triaconthyl silica for solid phase extraction of tryptic peptides obtained from unmodified and in vitro glycated human serum albumin and fibrinogen site specific conjugation of fluoroprobes to the remodeled fc n-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection innovative approach for producing injectable, biodegradable materials using chitooligosaccharides and green chemistry reciprocal principle of molecular recognition in supramolecular chromatography-highly selective analytical separation of cyclodextrin congeners on a silica-bonded [ ]fullerene stationary phase cation-independent mannose -phosphate receptor. a composite of distinct phosphomannosyl binding sites synthesis of lacdinac-terminated glycoconjugates by mutant galactosyltransferase-a way to new glycodrugs and materials new , -difluoro- a, a-diaza-sindacene (bodipy)-labeled sphingolipids for membrane studies solid-phase synthesis of a pentavalent galnac-containing glycopeptide (tn antigen) representing the nephropathy-associated iga hinge region chiral-auxiliary-mediated , -cis-glycosylations for the solid-supported synthesis of a biologically important branched a-glucan multimeric bivalent immunogens from recombinant tetanus toxin hc fragment, synthetic hexasaccharides, and a glycopeptide adjuvant the elucidation of the structure of thermotoga maritima peptidoglycan reveals two novel types of cross-link a m b family metallopeptidase of helicobacter pylori required for cell shape, pole formation and virulence lysosomal storage of oligosaccharide and glycosphingolipid in imino sugar treated cells multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of hiv- ion exchange and purification of carbohydrates on a nafion(r) membrane as a new sample pretreatment for matrix-assisted laser desorption-ionization mass spectrometry chinese hamster ovary cells can produce galactose-a- , -galactose antigens on proteins characterization of atll, a bifunctional autolysin of staphylococcus lugdunensis with nacetylglucosaminidase and n-acetylmuramoyl-l-alanine amidase activities dimeric architecture of the hendra virus attachment glycoprotein: evidence for a conserved mode of assembly unusual molecular architecture of the machupo virus attachment glycoprotein synthesis of polymerizable cyclodextrin derivatives for use in adhesion-promoting monomer formulations physicochemical properties of microbial glycopolymers an analytical system for the characterization of highly heterogeneous mixtures of n-linked oligosaccharides site directed processing: role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type o-glycan core high content phenotypic cell-based visual screen identifies mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling strategies for analysis of the glycosylation of proteins: current status and future perspectives characterization of irx and irx -like reveals an essential role in glucuronoxylan biosynthesis in arabidopsis uv-maldi-tof mass spectrometry analysis of heparin oligosaccharides obtained by nitrous acid controlled degradation and high performance anion exchange chromatography distribution of o-glycosylhydrolases in marine fungi of the sea of japan and the sea of okhotsk: characterization of exocellular n-acetyl-b-d-glucosaminidase of the marine fungus penicillium canescens derivatization in mass spectrometry identification, characterization, and biosynthesis of a novel n-glycan modification in the fruiting body of the basidiomycete coprinopsis cinerea lipid remodelling of glycosylphosphatidylinositol (gpi) glycoconjugates in procyclic form trypanosomes: biosynthesis and processing of gpis revisited caenorhabditis elegans n-glycan core b-galactoside confers sensitivity towards nematotoxic fungal galectin cgl novel mannosidase inhibitors probe glycoprotein degradation pathways in cells chitosan oligosaccharides modulate the supramolecular conformation and the biological activity of oligogalacturonides in arabidopsis efficient synthesis of a -deoxytalose tetrasaccharide related to the antigenic o-polysaccharide produced by aggregatibacter actinomycetemcomitans serotype c aniline/a-cyano- -hydroxycinnamic acid is a highly versatile ionic liquid for matrix-assisted laser desorption/ionization mass spectrometry h-pteridine- , -dione (lumazine): a new maldi matrix for complex (phospho)lipid mixtures analysis characterization of acp, a peptidoglycan hydrolase of clostridium perfringens with nacetylglucosaminidase activity that is implicated in cell separation and stress-induced autolysis glycobase and autogu: tools for hplc-based glycan analysis carrageenans: biological properties, chemical modifications and structural analysis-a review click" glycodendrimers containing , and modified xylopyranoside termini the plasma von willebrand factor o-glycome comprises a surprising variety of structures including abh antigens and disialosyl motifs analytical progress for protein glycosylation in china strategies for proteomic analysis of nonenzymatically glycated proteins preparation of saturated and unsaturated fatty acid hydrazides and long chain c-glycoside ketohydrazones glycoproteome study in myocardial lesions serum by integrated mass spectrometry approach: preliminary insights changes of serum-associated fucosylated glycoproteins and changes in glycosylation of iga in human cirrhosis efficient isolation of the subunits of recombinant and pituitary glycoprotein hormones ferrocene-cyclodextrin conjugates: synthesis, supramolecular behavior, and use as electrochemical sensors ferrocene-carbohydrate conjugates as electrochemical probes for molecular recognition studies characterization of main anthocyanins extracted from pericarp blue corn by maldi-tof ms highly glycosylated human alpha interferon: an insight into a new therapeutic candidate the protease-associated domain and c-terminal extension are required for zymogen processing, sorting within the secretory pathway, and activity of tomato subtilase (slsbt ) design and creativity in synthesis of multivalent neoglycoconjugates -deoxy-dmanno-octulosonic acid (kdo) hydrolase identified in francisella tularensis, helicobacter pylori, and legionella pneumophila synthesis and characterization of biotin chainend functionalized boronic acid-containing polymer (boropolymer) as functional glyco-affinity macroligand maldi mass spectrometry imaging of gangliosides in mouse brain using ionic liquid matrix das inhibits h n influenza virus infection of human lung tissues synthesis, characterization, and self-assembled nanofibers of carbohydrate-functionalized mono-and di( , : , -terpyridinyl)arenes developmental regulation of oligosialylation in zebrafish glycan array on aluminum oxide-coated glass slides through phosphonate chemistry mannose receptor interacts with fc receptors and is critical for the development of crescentic glomerulonephritis in mice efficient synthesis of idraparinux, the anticoagulant pentasaccharide the efficient total synthesis of bisglycosyl apigenin from naringenin: a greener way a mixed cyclodextrinbiphenyl thermotropic liquid crystal: synthesis, liquid-crystalline properties, and supramolecular organization boronate affinity monolith for highly selective enrichment of glycopeptides and glycoproteins application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms) in preparation of chitosan oligosaccharides (cos) with degree of polymerization (dp) - containing well-distributed acetyl groups study of on-resin convergent synthesis of n-linked glycopeptides containing a large high mannose n-linked oligosaccharide facile synthesis of cyclopeptide-centered multivalent glycoclusters with 'click chemistry' and molecular recognition study by surface plasmon resonance glycosite analysis in glycoproteomics by mass spectrometry combination of matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry for the analysis of intact glycopeptides from horseradish peroxidase an introduction to sphingolipid metabolism and analysis by new technologies one-pipeline approach achieving glycoprotein identification and obtaining intact glycopeptide information by tandem mass spectrometry imaging maldi mass spectrometry of sphingolipids using an oscillating capillary nebulizer matrix application system single-crystalline euf hollow hexagonal microdisks: synthesis and application as a background-free matrix for maldi-tof-ms analysis of small molecules and polyethylene glycols selective desorption/ ionization of sulfatides by maldi-ms facilitated using -aminoacridine as matrix tethered derivatives of d-glucose and pentacyclic triterpenes for homo/heterobivalent inhibition of glycogen phosphorylase total synthesis of a furostan saponin, timosaponin bii subcompartment localization of the side chain xyloglucan-synthesizing enzymes within golgi stacks of tobacco suspension-cultured cells carbohydrates as recognition receptors in biosensing applications expression system for human glycosyltransferases and its application production, purification, and characterization of human a proteinase inhibitor from aspergillus niger direct amidation of aldoses and decarboxylative amidation of a-keto acids: an efficient conjugation method for unprotected carbohydrate molecules novel succinylated and large-sized osmoregulated periplasmic glucans of pseudomonas syringae pv. syringae chiral separation of hesperetin and hesperetin-o-glycoside in capillary electrophoresis using microbial b- , -glucans structural modification and characterization of rice starch treated by thermus aquaticus -a-glucanotransferase enzymatic biosynthesis of a puerarincycloamylose inclusion complex by -aglucanotransferase and maltogenic amylase analysis of mixture of maltooligoses using maldi-tofms: influence of cationizing agent types comparison of ionization behaviors of ring and linear carbohydrates in maldi-tofms challenges of determining o-glycopeptide heterogeneity: a fungal glucanase model system mutational analysis of bacillus megaterium qm b cortex-lytic enzymes mass spectrometric imaging for biomedical tissue analysis synthesis of muc peptide and glycopeptide dendrimers synthesis and biological evaluation of multivalent carbohydrate ligands obtained by click assembly of pseudo-rotaxanes pseudomonas aeruginosa exploits lipid a and muropeptides modification as a strategy to lower innate immunity during cystic fibrosis lung infection straightforward synthesis of novel akt inhibitors based on a glucose scaffold the role of sugar configuration in the acetolysis of -deoxyhexose methyl glycosides synthesis of a b-glcn-( ! )-murnac building block en route to n-deacetylated peptidoglycan fragments a urea-linked glucosamine dimer as a building block for the synthesis of linear and cyclic neosaccharides a simple and rapid method for the permethylation of carbohydrates trypanosoma brucei amp-activated kinase subunit homologs influence surface molecule expression porphyromonas gingivalis resistance to polymyxin b is determined by the lipid a -phosphatase, pgn_ a new archaeal b-glycosidase from sulfolobus solfataricus. seeding a novel retaining b-glycan-specific glycoside hydrolase family along with the human non-lysosomal glucosylceramidase cba b-glycosyl azides as substrates for a-glycosynthases: preparation of efficient a-l-fucosynthases synthetic heparan sulfate oligosaccharides inhibit endothelial cell functions essential for angiogenesis towards the synthesis of -deoxy- -nitropiperidinoses synthesis and evaluation of s-and c( )-substituted analogues of lincomycin localization and imaging of sialylated glycosphingolipids in brain tissue sections by maldi mass spectrometry an efficient modular approach for the assembly of s-linked glycopeptoids glycosidase inhibition with fullerene iminosugar balls: a dramatic multivalent effect modification of gastric mucin oligosaccharide expression in rhesus macaques after infection with helicobacter pylori identification of novel contributions to high-affinity glycoprotein-receptor interactions using engineered ligands synthesis and conformational analysis of a novel carbohydrate-fused bis-crown ether: crown-cyplos design, synthesis and characterisation of a fluorescently labelled cyplos ionophore application of liquid chromatography-tandem mass spectrometry for the characterization of galactosylated and tagatosylated b-lactoglobulin peptides derived from in vitro gastrointestinal digestion effect of glycation on the gastrointestinal digestibility and immunoreactivity of bovine b-lactoglobulin role of pyridoxamine in the formation of the amadori/heyns compounds and aggregates during the glycation of b-lactoglobulin with galactose and tagatose acceptor products of alternansucrase with gentiobiose. production of novel oligosaccharides for food and feed and elimination of bitterness glucosylation of raffinose via alternansucrase acceptor reactions investigating the candidacy of lps-based glycoconjugates to prevent invasive meningococcal disease: chemical strategies to prepare glycoconjugates with good carbohydrate loading investigating the candidacy of lps-based glycoconjugates to prevent invasive meningococcal disease: immunology of glycoconjugates with high carbohydrate loading towards a second generation of ionic liquid matrices (ilms) for maldi-ms of peptides, proteins, and carbohydrates sinorhizobium fredii hh cgs mutants are unable to nodulate determinate-and indeterminate nodule-forming legumes and overproduce an altered eps carbohydrate and domain architecture of an immature antibody glycoform exhibiting enhanced effector functions a human embryonic kidney t cell line mutated at the golgi a-mannosidase ii locus sieve-based device for maldi sample preparation. ii. instrumental parameterization a link between the assembly of flagella and lipooligosaccharide of the gram-negative bacterium campylobacter jejuni nonglycosidically linked pseudodisaccharides: thioethers, sulfoxides, sulfones, ethers, selenoethers, and their binding to lectins stable expression of a human-like sialylated recombinant thyrotropin in a chinese hamster ovary cell line expressing a , -sialyltransferase impaired lysosomal trimming of n-linked oligosaccharides leads to hyperglycosylation of native lysosomal proteins in mice with a-mannosidosis biochemical and immunocytological characterizations of arabidopsis thaliana pollen tube cell wall engineering of n. benthamiana l. plants for production of n-acetylgalactosamine-glycosylated proteins-towards development of a plant-based platform for production of protein therapeutics with mucin type o-glycosylation xylan-based nanoparticles: prodrugs for ibuprofen release towards unnatural xylan based polysaccharides: reductive amination as a tool to access highly engineered carbohydrates a simple micromethod for determining precise oligosaccharidic specificity of mannose-binding lectins functional and morphological adaptation to peptidoglycan precursor alteration in lactococcus lactis towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns molecular characterization of a putative sucrose: fructan -fructosyltransferase ( -sft) of the cold-resistant patagonian grass bromus pictus associated with fructan accumulation under low temperatures biomarkers and diagnosis of congenital disorders of glycosylation improved procedures for the selective chemical fragmentation of rhamnogalacturonans when can glycopeptides be assigned based solely on high-resolution mass spectrometry data? mmps promotes glycopeptidolipids biosynthesis and export in mycobacterium smegmatis glyco-spectrum-scan: fishing glycopeptides from ms spectra of protease digests of human colostrum siga common sialylated glycan in actinobacillus suis the major surface carbohydrates of the echinococcus granulosus cyst: mucin-type o-glycans decorated by novel galactosebased structures surface analytical characterization of carbohydrate microarrays shiga toxin receptor gb cer/ cd : tumor-association and promising therapeutic target in pancreas and colon cancer synthesis of some quaternary n-( , -anhydro- -deoxy-d,l-ribitol- -yl)ammonium salts a barley cellulose synthase-like cslh gene mediates ( , ; , )-b-d-glucan synthesis in transgenic arabidopsis analytical performance of immobilized pronase for glycopeptide footprinting and implications for surpassing reductionist glycoproteomics recombinant expression and characterization of n-acetylglucosaminyltransferase i derived from nicotiana tabacum comparison of the n-linked glycosylation of human b , -n-acetylglucosaminyltransferase expressed in insect cells and silkworm larvae improved secretion of molecular chaperone-assisted human igg in silkworm, and no alterations in their n-linked glycan structures structure of glycosylated cu/znsuperoxide dismutase from kluyveromyces yeast nbimcc glycan structures and antiviral effect of the structural subunit rvh of rapana hemocyanin baca, an abc transporter involved in maintenance of chronic murine infections with mycobacterium tuberculosis a systematic nomenclature for carbohydrate fragmentations in fab-ms/ms spectra of glycoconjugates transformation of linear oligoketosides into macrocyclic neoglycoconjugates a new ligation strategy for peptide and protein glycosylation: photoinduced thiol-ene coupling graphene as a novel matrix for the analysis of small molecules by maldi-tof ms envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens clinical collection and protein properties of expressed prostatic secretions as a source for biomarkers of prostatic disease mycobacterium marinum mmar- , a predicted transmembrane acyltransferase, is essential for the presence of the mannose cap on lipoarabinomannan electrochemically controlled adsorption of fc-functionalized polymers on b-cd-modified selfassembled monolayers production of carbohydrate building blocks from red seaweed polysaccharides. efficient conversion of galactans into c-glycosyl aldehydes polysaccharide mimicry of the epitope of the broadly neutralizing anti-hiv antibody, g , induces enhanced antibody responses to self oligomannose glycans glycosylated cell-penetrating peptides and their conjugates to a proapoptotic peptide: preparation by click chemistry and cell viability studies nm multiphoton dissociation and ionization of , -dihydroxyacetophenone lack of complex n-glycans on hiv- envelope glycoproteins preserves protein conformation and entry function prebiotic oligosaccharides: in vitro evidence for gastrointestinal epithelial transfer and immunomodulatory properties c-furyl glycosides, ii: synthesis and antimicrobial evaluation of c-furyl glycosides bearing pyrazolines, isoxazolines, and , -dihydropyrimidine- ( h)-thiones structural characterization of bordetella parapertussis lipid a polysaccharide pharmacokinetics: amphotericin b arabinogalactan conjugate -a drug delivery system or a new pharmaceutical entity hexosamine template. a platform for modulating gene expression and for sugar-based drug discovery combinatorial syntheses of trisaccharide libraries on a soluble polymeric support lipophilic thioglycosides for the solution-phase synthesis of oligosaccharides using biphasic liquid-liquid separation chiral and structural analysis of biomolecules using mass spectrometry and ion mobility-mass spectrometry fluorescent labeling of a carboxyl group of sialic acid for maldi-ms analysis of sialyloligosaccharides and ganglioside liquid chromatography combined with mass spectrometry for the investigation of endoglucanase selectivity on carboxymethyl cellulose investigation of endoglucanase selectivity on carboxymethyl cellulose by mass spectrometric techniques cellulosic graft copolymer: poly(methyl methacrylate) with cellulose side chains radially oriented cellulose triacetate chains on gold nanoparticles amphiphilic carbohydrate-phthalocyanine conjugates obtained by glycosylation or by azide-alkyne click reaction different and new nod factors produced by rhizobium tropici ciat following na þ stress identification of the polyketide synthase involved in the biosynthesis of the surface-exposed lipooligosaccharides in mycobacteria bioanalysis of recombinant proteins and antibodies by mass spectrometry lectin and carbohydrate microarrays: new high-throughput methods for glycoprotein, carbohydrate-binding protein and carbohydrate-active enzyme analysis chitosans as bioactive macromolecules to protect economically relevant crops from their main pathogens serum n-glycome biomarker for monitoring development of dena-induced hepatocellular carcinoma in rat secondary cell wall formation in cryptococcus neoformans as a rescue mechanism against acid-induced autolysis identification and quantification of protein posttranslational modifications matrixassisted laser desorption/ionization time-of-flight (maldi-tof) mass spectrometry analysis of oligosaccharides and oligosaccharide alditols obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides glycopeptidome of a heavily n-glycosylated cell surface glycoprotein of dictyostelium implicated in cell adhesion fragmentation behavior of amadori-peptides obtained by non-enzymatic glycosylation of lysine residues with adp-ribose in tandem mass spectrometry effect of , ; , -b-d-glucan and the products of its enzymatic transformation on the formation of germs of buckwheat fagopyrum esculentum mönch synthesis of , -branched arabinogalactan-type tetra-and hexasaccharides for characterization of monoclonal antibodies phosphoglucomutase of yersinia pestis is required for autoaggregation and polymyxin b resistance mass spectrometric investigation of molecular variability of grass pollen group allergens cyclodextrin aldehydes are oxidase mimics simultaneous glycoproteomics on the basis of structure using ion mobility-mass spectrometry characterizing ion mobility-mass spectrometry conformation space for the analysis of complex biological samples effect of ionic liquids as additives in the catalytic properties of different immobilized preparations of rhizomucor miehei lipase in the hydrolysis of peracetylated lactal synthetic pseudopterosin analogues: a novel class of antiinflammatory drug candidates purification, characterization and in vivo studies of salmon heparin thieme chemistry journal awardees-where are they now? synthesis of the marine glycolipid dioctadecanoyl discoside thiyl glycosylation of olefinic proteins: s-linked glycoconjugate synthesis synthesis of the lewis b pentasaccharide and a hsa-conjugate thereof maldi mass spectrometry imaging, from its origins up to today: the state of the art detection of honeybee venom in envenomed tissues by direct maldi msi bioinformatics and molecular modeling in glycobiology expression of rat b( , )-n-acetylglucosaminyltransferase iii in nicotiana tabacum remodels the plantspecific n-glycosylation glycoprotein expression in human milk during lactation -deoxynojirimycins with dansyl capped n-substituents as probes for morbus gaucher affected cell lines new secoiridoid glucosides from ligustrum lucidum induce erk and creb phosphorylation in cultured cortical neurons application of maldi-tof mass spectrometry in lipidomics an update of maldi-tof mass spectrometry in lipid research maldi-tof-ms directly combined with tlc: a review of the current state isolation and structural analysis in vivo of newly synthesized fructooligosaccharides in onion bulbs tissues (allium cepa l.) during storage maldi mass spectrometry using , , -trihydroxyacetophenone and , -dihydroxyacetophenone with cyclodextrins: suppression of matrix-related ions in low-molecular-weight region change in glycosylation pattern with extension of endoplasmic reticulum retention signal sequence of mouse antibody produced by suspensioncultured tobacco by cells chemical characterization of the oligosaccharides in bactrian camel (camelus bactrianus) milk and colostrum dendritic sugar-microarrays by click chemistry aggregation of alzheimer amyloid b peptide ( - ) on the multivalent sulfonated sugar interface scope and limitations of imidazolium-based ionic liquids as room temperature glycosylation promoters the carboxy-terminal erretention motif, sekdel, influences the n-linked glycosylation of recombinant human a-l-iduronidase but has little effect on enzyme activity in seeds of brassica napus and nicotiana tabacum mass spectrometric fragmentation analysis of oligosialic and polysialic acids quantification of nucleotide-activated sialic acids by a combination of reduction and fluorescent labeling synaptic cell adhesion molecule syncam is a target for polysialylation in postnatal mouse brain comparative characterization of the arabidopsis subfamily a b-galactosidases preparation and gelling properties of sugar-contained low-molecular-mass gelators: combination of cholesterol and linear glucose generation of asparagine-linked glycan structure databases and their use the first total synthesis of -o-b-dglucopyranosyl- -o-a-l-rhamnopyranosyl apigenin via a hexanoyl ester-based protection strategy efficient synthesis of -amido-n -acetyl- -deoxyneuraminic acid and its application to the c- modification of sialic acids endothelial galectin- binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation lectin-directed enzyme activated prodrug therapy (leapt): synthesis and evaluation of rhamnosecapped prodrugs characterization of a new b( - )-glucan branching activity of aspergillus fumigatus enhanced production and partial characterization of an extracellular polysaccharide from newly isolated azotobacter sp. ssb phase diagrams of monoacylated amide-linked disaccharide glycolipids diamond, titanium dioxide, titanium silicon oxide, and barium strontium titanium oxide nanoparticles as matrixes for direct matrix-assisted laser desorption/ionization mass spectrometry analysis of carbohydrates in plant tissues synthesis of a tetrasaccharide corresponding to the teichoic acid from the cell wall of streptomyces sp. vkm ac- synthesis of the hexasaccharide repeating unit corresponding to the cell wall lipopolysaccharide of azospirillum irakense kbc sialic acids in different leishmania sp., its correlation with nitric oxide resistance and host responses -o-acetylated sialic acids enhance entry of virulent leishmania donovani promastigotes into macrophages high throughput quantification of n-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time-offlight mass spectrometry gdp-d-mannose , -epimerase (gme) plays a key role at the intersection of ascorbate and non-cellulosic cell-wall biosynthesis in tomato an hplc-maldi ms method for n-glycan analyses using smaller size samples: application to monitor glycan modulation by medium conditions towards a reliable molecular mass determination of intact glycoproteins by matrixassisted laser desorption/ionization time-of-flight mass spectrometry ionic liquid matrices for maldi-tof-ms analysis of intact glycoproteins synthesis of pegylated lactose analogs for inhibition studies on t. cruzi trans-sialidase glycosylation site-specific analysis of clade c hiv- envelope proteins enzymatic/chemical release of o-glycans allowing ms analysis at high sensitivity glycomic profiling of invasive and non-invasive breast cancer cells assessment of chemoselective neoglycosylation methods using chlorambucil as a model glycan family analysis for deducing n-glycan topology from single ms detection of hepatocellular carcinoma using glycomic analysis discovery of rifampicin as a new anti-glycating compound by matrix-assisted laser desorption/ionization mass spectrometry-based insulin glycation assay first total synthesis of , -dipalmitoyl- -(n-palmitoyl- -amino- -deoxy-a-dglucosyl)-sn-glycerol-a glycoglycerolipid of a marine alga with a high inhibitor activity against human myt -kinase acidiplasma aeolicum gen. nov., sp. nov., a euryarchaeon of the family ferroplasmaceae isolated from a hydrothermal pool, and transfer of ferroplasma cupricumulans to acidiplasma cupricumulans comb. nov synthesis of polyhydroxy [n]-polyurethanes derived from a carbohydrate precursor characterization of n-linked glycosylation on recombinant glycoproteins produced in pichia pastoris using esi-ms and maldi-tof dna-templated homo-and heterodimerization of peptide nucleic acid encoded oligosaccharides that mimick the carbohydrate epitope of hiv human macrophage activation triggered by chitotriosidase-mediated chitin and chitosan degradation evaluation of conditions for release of mucin-type oligosaccharides from glycoproteins by hydrazine gas treatment imaging mass spectrometry of glycolipids visualization of spatial distribution of g-aminobutyric acid in eggplant (solanum melongena) by matrix-assisted laser desorption/ionization imaging mass spectrometry the specific localization of seminolipid molecular species on mouse testis during testicular maturation revealed by imaging mass spectrometry the detection of glycosphingolipids in brain tissue sections by imaging mass spectrometry using gold nanoparticles practical heavy fluorous tag for carbohydrate synthesis with minimal chromatographic purification production and nglycosylation of recombinant human cell adhesion molecule l from insect cells using the stable expression system. effect of dimethyl sulfoxide glyco-sams by 'dual click': thiourea-bridged glyco-oeg azides for cycloaddition on surfaces mass spectrometry in the elucidation of the glycoproteome of bacterial pathogens investigation towards bivalent chemically defined glycoconjugate immunogens prepared from acid-detoxified lipopolysaccharide of vibrio cholerae o , serotype inaba the synthesis and immune stimulating action of mannose-capped lysine-based dendrimers some patterns in dimer ii formation in bodipy-fl-labeled lipids bodipy-labeled ganglioside probes for membrane and biological studies development of a high performance anion exchange chromatography analysis for mapping of oligosaccharides analytical approaches towards the structural characterization of microbial wall glycopolymers differential effect of plasma or erythrocyte age-ligands of rage on expression of transcripts for receptor isoforms biosynthesis of a new udpsugar, udp- -acetamido- -deoxyxylose, in the human pathogen bacillus cereus subspecies cytotoxis nvh - effective enlargement of fluorescence resonance energy transfer of poly-porphyrin mediated by b-cyclodextrin dimers total synthesis of the heptasaccharide repeating unit of the iron-binding exopolysaccharide secreted by klebsiella oxytoca bas- mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry oxidative modifications in glycated insulin new insights into the early steps of phosphatidylinositol mannoside biosynthesis in mycobacteria. pimb' is an essential enzyme of mycobacterium smegmatis fatty acyl structures of mycobacterium tuberculosis sulfoglycolipid govern t cell response automated measurement of permethylated serum n-glycans by maldi-linear ion trap mass spectrometry monoterpenoid glucoindole alkaloids and iridoids from pterocephalus pinardii production, refining, structural characterization and fermentability of rice husk xylooligosaccharides oligo mass profiling (olimp) of extracellular polysaccharides facile synthesis of three bidesmosidic oleanolic acid saponins with strong inhibitory activity on pancreatic lipase sortase-catalyzed peptide-glycosylphosphatidylinositol analogue ligation one-pot synthesis of cyclic antifreeze glycopeptides syntheses of glycoclusters containing a phosphocholine residue related to a glycosphingolipid from the earthworm pheretima hilgendorfi establishment of a real-time analytical method for free oligosaccharide transport from the er to the cytosol purification, characterization and molecular cloning of a novel endo-b-n-acetylglucosaminidase from the basidiomycete, flammulina velutipes comparative glycomic profiling in esophageal adenocarcinoma derivatization and analysis of oligosaccharides by matrix-assisted laser desorption/ionization time of-flight mass spectrometry sulfated oligosaccharide cluster with polylysine core scaffold as a new anti-hiv dendrimer lipophilic b-cyclodextrin cyclic-nitrone conjugate: synthesis and spin trapping studies sialylation using n-glycolylneuraminyl phosphite donors to synthesize neu gc-containing glycans chemical de-oglycosylation of glycoproteins for application in lc-based proteomics secondary acylation of vibrio cholerae lipopolysaccharide requires phosphorylation of kdo rapid characterization of n-linked glycans from secreted and gel-purified monoclonal antibodies using maldi-tof mass spectrometry synthesis of porphyrin-carbohydrate conjugates using "click" chemistry and their preliminary evaluation in human hep cells an improved protocol for n-glycosylation analysis of gel-separated sialylated glycoproteins by maldi-tof/ tof artificial polymerases and molecular chaperones interaction of nectarin with a fungal protein triggers a microbial surveillance and defense mechanism in nectar n-glycans on the link domain of human hare/stabilin- are needed for hyaluronan binding to purified ecto-domain, but not for cellular endocytosis of hyaluronan analytical characterization of monoclonal antibodies: linking structure to function identification of rhodococcus equi lipids recognized by host cytotoxic t lymphocytes matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update covering the period - analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update covering the period analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update for electrospray and maldi mass spectrometry: fundamentals, instrumentation, practicalities, and biological applications analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update for the period analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update for identification of by-products formed during the release of n-glycans with protein n-glycosidase f in the presence of dithiothreitol application of negative ion ms/ ms to the identification of n-glycans released from carcinoembryonic antigen cell adhesion molecule (ceacam ) structural and quantitative analysis of n-linked glycans by maldi and negative ion nanospray mass spectrometry proposal for a standard system for drawing structural diagrams of n-and o-linked carbohydrates and related compounds fragmentation of negative ions from n-linked carbohydrates, part . fragmentation of complex glycans lacking substitution on the -antenna identification of high-mannose and multiantennary complextype n-linked glycans containing a-galactose epitopes from nurse shark igm heavy chain o-glycoside biomarker of apolipoprotein c : responsiveness to obesity, bariatric surgery, and therapy with metformin, to chronic or severe liver disease and to mortality in severe sepsis and graft vs host disease pyridylamination as a means of analyzing complex sugar chains switching the selectivity of a polyglycerol dendrimer monomolecularly imprinted with d-(À)-fructose kegg glycan for integrated analysis of pathways, genes and glycan structures sphingolipidomics: methods for the comprehensive analysis of sphingolipids antifreeze glycopeptide analogues: microwaveenhanced synthesis and functional studies degradation of chitosans with a family chitosanase from streptomyces coelicolor a ( ) glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage rational engineering of mannosyl binding in the distal glycone subsites of cellulomonas fimi endo-b- , -mannanase: mannosyl binding promoted at subsite - and demoted at subsite - uv-laser ablation of ionic liquid matrices structure analysis of n-glycoproteins toward synthesis of the isosteric sulfonate analogues of the at-iii binding domain of heparin plant immunity induced by oligogalacturonides alters root growth in a process involving flavonoid accumulation in arabidopsis thaliana conversion of a-amyrin into centellosides by plant cell cultures of centella asiatica carbohydrate analysis throughout the development of a protein therapeutic positive and negative analyte ion yield in matrix-assisted laser desorption/ionization revisited g-cyclodextrins possessing an azido group and a triisopropylbenzenesulfonyl group as useful synthetic and authentic intermediates for unsymmetrically functionalized derivatives hemicellulase production in chrysosporium lucknowense c genetically engineered mannosylated-human serum albumin as a versatile carrier for liverselective therapeutics free oligosaccharides to monitor glycoprotein endoplasmic reticulumassociated degradation in saccharomyces cerevisiae chemical synthesis, folding, and structural insights into o-fucosylated epidermal growth factor-like repeat of mouse notch- receptor a water-soluble ruthenium glycosylated porphyrin catalyst for carbenoid transfer reactions in aqueous media with applications in bioconjugation reactions synthesis and self-assembled nanostructures of novel chiral amphiphilic liquid crystals containing b-d-galactopyranoside end-groups synthetic antitumor vaccines from tetanus toxoid conjugates of muc glycopeptides with the thomsen-friedenreich antigen and a fluorine-substituted analogue matrix-free uv-laser desorption/ionization (ldi) mass spectrometric imaging at the singlecell level: distribution of secondary metabolites of arabidopsis thaliana and hypericum species core region and lipid a components of lipopolysaccharides matrix-assisted laser desorption/ionization mass spectrometry (maldi-ms) application in carbohydrate analysis glycoproteomic analysis of wgabound glycoprotein biomarkers in sera from patients with lung adenocarcinoma synthesis, characterization and in vitro pharmacology of novel pregabalin derivatives an efficient approach to the discovery of potent inhibitors against glycosyltransferases electrospray and maldi mass spectrometry: fundamentals, instrumentation, practicalities, and biological applications design, synthesis, and immunochemical characterization of a chimeric glycopeptide corresponding to the shigella flexneri y opolysaccharide and its peptide mimic mdwnmhaa antiproliferative cardenolide glycosides of elaeodendron alluaudianum from the madagascar rainforest the influence of microwave irradiation on stereospecific mo(vi)-catalyzed transformation of deoxysugars a new approach to amadori ketoses via mo vicatalyzed stereospecific isomerization of -c-branched sugars bearing azido function in a microwave field substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase hvxet from barley (hordeum vulgare l.) determination of n-glycosylation site and glycan structures of pectin methylesterase in jelly fig (ficus awkeotsang) achenes the composition of polysaccharides in primary walls of litchi chinensis sonn xyloglucans of monocotyledons have diverse structures the effect of acid dextrinisation on enzyme-resistant starch content in extruded maize starch new oligosaccharides prepared by acid hydrolysis of the polysaccharides from nerium indicum mill and their anti-angiogenesis activities lectin microarray widely applicable deprotection method of , , -trichloroethoxycarbonyl (troc) group using tetrabutylammonium fluoride hydrolytically stable bioactive synthetic glycopeptide homo-and copolymers by combination of nca polymerization and click reaction chemoenzymatic synthesis and lectin array characterization of a class of n-glycan clusters expeditious chemoenzymatic synthesis of cd glycopeptide antigens arthrobacter endo-b-n-acetylglucosaminidase shows transglycosylation activity on complex-type n-glycan oxazolines: one-pot conversion of ribonuclease b to sialylated ribonuclease c glycosynthases enable a highly efficient chemoenzymatic synthesis of n-glycoproteins carrying intact natural n-glycans synthesis of oleanolic acid saponins mimicking components of chinese folk medicine di wu lipid-a software tool for automated assignment of lipids in mass spectra a lipid profile typifies the beijing strains of mycobacterium tuberculosis. identification of a mutation responsible for a modification of the structures of phthiocerol dimycocerosates and phenolic glycolipids igg glycosylation analysis supramolecular assembly of carbohydrate-functionalized salphen-metal complexes synthesis and application of epoxy starch derivatives an echinococcus multilocularis coproantigen is a surface glycoprotein with unique o-gycosylation glycoproteomics in neurodegenerative diseases structural elucidation of a novel b. cenocepacia et- lipooligosaccharide isolated from a cystic fibrosis patient after lung transplantation against the rules: a marine bacterium, loktanella rosea, possesses a unique lipopolysaccharide first structural characterization of burkholderia vietnamiensis lipooligosaccharide from cystic fibrosis-associated lung transplantation strains fucosylation of chitooligosaccharides by human a , -fucosyltransferase requires a nonreducing terminal chitotriose unit as a minimal structure c-mannosylated peptides derived from the thrombospondin type repeat interact with hsc to modulate its signaling in raw . cells two mechanisms of the enhanced antibody-dependent cellular cytotoxicity (adcc) efficacy of non-fucosylated therapeutic antibodies in human blood production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons homotropic cooperativity of cyclodextrin dimer as an artificial hydrolase glycomimicry: display of the gm sugar epitope on escherichia coli and salmonella enterica sv typhimurium enzymatic conversion of diacetylated analysis of carbohydrates and glycoconjugates & sophoroselipid into acetylated glucoselipid: surface-active properties of novel bolaform biosurfactants preparation and conformational analysis of c-glycosyl b -and b/b -peptides the multiplicity of n-glycan structures of novine milk kda lactophorin (milk glycam- ) synthesis of an octasubstituted galactose zinc(ii) phthalocyanine anomerically glycosylated zinc(ii) naphthalocyanines synthesis of octaglycosylated zinc(ii) phthalocyanines. synthesis sesquiterpene coumarins from ferula gumosa transglycosylation-based fluorescent labeling of -gala series glycolipids by endogalactosylceramidase molecular cloning and expression of a novel cholinephosphotransferase involved in glycoglycerophospholipid biosynthesis of mycoplasma fermentans enzymatic synthesis of mycoplasma fermentans specific glycoglycerophospholipid from , -dipalmitoylglycerol isolation and structural determination of reducing fructooligosaccharides newly produced in stored edible burdock antiallergic potential of oligomannose-coated liposome-entrapped cry j as immunotherapy for japanese cedar pollinosis in mice enzymatic activity and substrate specifcity of recombinant tomato b-galactosidases and development of highly efficient and stereocontrolled o-glycosylation methodologies and its application to the construction of bacterial glycans effects of frozen conditions on stereoselectivity and velocity of o-glycosylation reactions specificity analysis of lectins and antibodies using remodeled glycoproteins production, purification and structural characterization of an exopolysaccharide produced by a probiotic lactobacillus plantarum mtcc in vitro and in vivo enzymatic syntheses and mass spectrometric database for nglycans and o-glycans strategy for glycoproteomics: identification of glyco-alteration using multiple glycan profiling tools enrichment strategies for glycopeptides photooxidative mineralization of microorganisms-produced glycolipid biosurfactants by a titania-mediated advanced oxidation process highly oriented recombinant glycosyltransferases: sitespecific immobilization of unstable membrane proteins by using staphylococcus aureus sortase a a first total synthesis of ganglioside hlg- identification of a glycosylphosphatidylinositol anchormodifying b - n-acetylglucosaminyl transferase in trypanosoma brucei direct profiling of tissue lipids by maldi-tofms glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the o-ps of vibrio cholerae o , serotype ogawa, and bsa revealed by matrix-assisted laser desorption-ionization tandem mass spectrometry contribution of porphyromonas gingivalis lipopolysaccharide to periodontitis synthesis of new sulfonic acid-containing oligosaccharide mimetics of sialyl lewis a synthesis, regioselective hydrogenolysis, partial hydrogenation, and conformational study of dioxane and dioxolanetype ( -anthracenyl)methylene acetals of sugars mass spectrometric quantification of neutral and sialylated n-glycans from a recombinant therapeutic glycoprotein produced in the two chinese hamster ovary cell lines method development for direct detection of glycoproteins on aminophenylboronic acid functionalized self-assembled monolayers by matrix-assisted laser desorption/ionization mass spectrometry antibody glycans characterization structural characterization of antibodies by mass spectrometry mucin-lectin interactions assessed by flow cytometry mucin-type o-glycosylation-putting the pieces together carboxymethylated cyclosophoraose as a novel chiral additive for the stereoisomeric separation of some flavonoids by capillary electrophoresis characterization of the streptococcus pneumoniae bgac protein as a novel surface b-galactosidase with specific hydrolysis activity for the galb - glcnac moiety of oligosaccharides characterization of n-linked protein glycosylation in helicobacter pullorum a strategy for precise and large scale identification of core fucosylated glycoproteins self-assembly of amphiphilic perylenecyclodextrin conjugate and vapor sensing for organic amines structures of chitosan and chitooligosaccharides and their adsorption toward radionuclide uranium a high-throughput purification of monoclonal antibodies from glycoengineered pichia pastoris a bifunctional enzyme in a single gene catalyzes the incorporation of glcn into the aeromonas core lipopolysaccharide genetics and proteomics of aeromonas salmonicida lipopolysaccharide core biosynthesis selective monitoring of rutin and quercetin based on a novel multi-wall carbon nanotube-coated glassy carbon electrode modified with microbial carbohydrates a-cyclosophorohexadecaose and succinoglycan monomer m utilizing the o-antigen lipopolysaccharide biosynthesis pathway in escherichia coli to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach design, synthesis, and evaluation of n-acyl modified sialic acids as inhibitors of adenoviruses causing epidemic keratoconjunctivitis natural phosphoryl and acyl variants of lipid a from neisseria meningitidis strain i differentially induce tumor necrosis factor-a in human monocytes profiles of structural heterogeneity in native lipooligosaccharides of neisseria and cytokine induction golgi targeting of drosophila melanogaster b galnactb requires a dhhc protein family-related protein as a pilot strong igg antibody responses to borrelia burgdorferi glycolipids in patients with lyme arthritis, a late manifestation of the infection generation and characterization of monoclonal antibody to ginsenoside rg a novel, simple and sensitive ligand affinity capture method for detecting molecular interactions by maldi mass spectrometry glycoviewer: a tool for visual summary and comparative analysis of the glycome mass spectrometric molecular-weight determination of highly acidic compounds of biological significance via their complexes with basic polypeptides enzymatic synthesis of salicin glycosides through transglycosylation catalyzed by amylosucrases from deinococcus geothermalis and neisseria polysaccharea direct analysis of cellulose in poplar stem by matrix-assisted laser desorption/ionization imaging mass spectrometry superior in vivo efficacy of afucosylated trastuzumab in the treatment of her -amplified breast cancer benzyl ethers as nucleophiles: from hydroxy cyclooctanes toward bridged c-furanosides multifunctional zro nanoparticles and zro -sio nanorods for improved maldi-ms analysis of cyclodextrins, peptides, and phosphoproteins a synthetic vaccine consisting of a tumor-associated sialyl-tn-muc tandem-repeat glycopeptide and tetanus toxoid: induction of a strong and highly selective immune response fully synthetic vaccines consisting of tumor-associated muc glycopeptides and a lipopeptide ligand of the toll-like receptor ghrelin-like peptide with fatty acid modification and o-glycosylation in the red stingray, dasyatis akajei synthesis of glycopeptides reactivity of rare sugar d-allose during glycation of human serum albumin arabidopsis thaliana alg mutant synthesizes immature oligosaccharides in the er and accumulates unique n-glycans two arabidopsis thaliana golgi a-mannosidase i enzymes are responsible for plant n-glycan maturation a sensitive liquid chromatography/mass spectrometry-based assay for quantitation of amino-containing moieties in lipid a selfpoisoning of mycobacterium tuberculosis by targeting glge in an aglucan pathway definitive evidence that a single n-glycan among three glycans on inducible costimulator is required for proper protein trafficking and ligand binding aglj adds the first sugar of the nlinked pentasaccharide decorating the haloferax volcanii s-layer glycoprotein aba-and bab-triblock cooligomers of tri-o-methylated and unmodified cello-oligosaccharides: syntheses and structure-solubility relationship lectin-based enrichment method for glycoproteomics using hollow fiber flow field-flow fractionation: application to streptococcus pyogenes synthesis and characterization of hydroquinone fructoside using leuconostoc mesenteroides levansucrase high-throughput solid-phase permethylation of glycans prior to mass spectrometry solid-phase permethylation of glycans for mass spectrometric analysis antibacterial activity of a disaccharide isolated from streptomyces sp. strain jj against xanthomonas sp insight into the regulation of glycan synthesis in drosophila chaoptin based on mass spectrometry a tandem mass spectrometric approach to the identification of o-glycosylated glargine glycoforms in active pharmaceutical ingredient expressed in pichia pastoris mass spectrometric analysis of carbohydrates labeled with a biotinylated tag localization of the attachment site of oligoglucans to mesorhizobium loti hambi murein synthesis of a heptasaccharide fragment of the mannan from candida guilliermondii cell wall and its conjugate with bsa synthesis of , -branched oligomannoside fragments of the mannan from candida albicans cell wall corresponding to the antigenic factor reduction of n-linked xylose and fucose by expression of rat b , -n-acetylglucosaminyltransferase iii in tobacco by- cells depends on golgi enzyme localization domain and genetic elements used for expression a small-scale method for the preparation of plant n-linked glycans from soluble proteins for analysis by maldi-tof mass spectrometry analyses of biologically active steroids: antitumor active osw- and cardiotonic marinobufotoxin, by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry structural analogues of diosgenyl saponins: synthesis and anticancer activity pyruvoyl, a novel amino protecting group on the solid phase peptide synthesis and the peptide condensation reaction chemical synthesis of mouse pro-opiomelanocortin( - ) by azido-protected glycopeptide ligation via the thioester method correct disulfide pairing is required for the biological activity of crustacean androgenic gland hormone (agh): synthetic studies of agh loss of udp-galnac: polypeptide n-acetylgalactosaminyltransferase and reduced oglycosylation in colon carcinoma cells selected for hepatic metastasis development of tetraphenylethylene-based fluorescent oligosaccharide probes for detection of influenza virus further structural study of the xyloglucanase-derived eggplant xyloglucan oligo-saccharides initiation of methylglucose lipopolysaccharide biosynthesis in mycobacteria synthesis of a chito-tetrasaccharide b- , -glcnac-b- , -glcn repeating unit a novel polycondensation method for the synthesis of a two-faced b- , -glucan solid-phase synthesis of o-sulfated glycopeptide by the benzylprotected glycan strategy salmonella enterica serovar typhimurium lipopolysaccharide deacylation enhances its intracellular growth within macrophages influence of crystalline forms of titania on desorption/ionization efficiency in titania-based surfaceassisted laser desorption/ionization mass spectrometry alternative procedures for analysis of lipid a modification with phosphoethanolamine or aminoarabinose recognition of endogenous ligands by ctype lectins: interaction of serum mannan-binding protein with tumorassociated oligosaccharide epitopes highly fucosylated n-glycan ligands for mannan-binding protein expressed specifically on cd (dppvi) isolated from a human colorectal carcinoma cell line structural analysis of two trisaccharides isolated from fermented beverage of plant extract phosphorylase-catalyzed n-formyl-a-glucosaminylation of maltooligosaccharides gold-catalyzed glycosidations: unusual cleavage of the interglycosidic bond while studying the armed/ disarmed effect of propargyl glycosides dendrimers of vaccines consisting of tumor-associated glycopeptide antigens and t cell epitope peptides bacillus anthracis surfacelayer proteins assemble by binding to the secondary cell wall polysaccharide in a manner that requires csab and tago sialic acids acquired by pseudomonas aeruginosa are involved in reduced complement deposition and siglec mediated hostcell recognition mass spectrometric analysis of sulfated n-and oglycans steroidal monoglycosides from the far eastern starfish hippasteria kurilensis and hypothetic pathways of polyhydroxysteroid biosynthesis in starfish two new steroid glycosides from the far east starfish hippasteria kurilensis lectin biosensing using digital analysis of ru(ii)-glycodendrimers ru(ii) glycodendrimers as probes to study lectin-carbohydrate interactions and electrochemically measure monosaccharide and oligosaccharide concentrations enzymatic synthesis and characterization of hydroquinone galactoside using kluyveromyces lactis lactase click synthesis of estradiol-cyclodextrin conjugates as cell compartment selective estrogens grandidentatin isomer from bark of suwon poplar (populus alba l.  populus glandulosa uyeki) biotransformation of mulberroside a from morus alba results in enhancement of tyrosinase inhibition glycosylation pattern of humanized igg-like bispecific antibody produced by recombinant cho cells mass spectrometric analysis of the glycosphingolipid-derived glycans from miniature pig endothelial cells and islets: identification of neugc epitope in pig islets rapid and high-throughput analysis of n-glycans from ovarian cancer serum using a -well plate platform identification of a-gal and non-gal epitopes in pig corneal endothelial cells and keratocytes by using mass spectrometry qualitative and quantitative comparison of n-glycans between pig endothelial and islet cells by highperformance liquid chromatography and mass spectrometry-based strategy construction of a fusion enzyme of dextransucrase and dextranase: application for onestep synthesis of isomalto-oligosaccharides enzymatic synthesis of alkyl glucosides using leuconostoc mesenteroides dextransucrase alteration in the glycan pattern of pilin in a nonmotile mutant of synechocystis sp. pcc suppression of b-n-acetylglucosaminidase in the n-glycosylation pathway for complex glycoprotein formation in drosophila s cells carbohydrate moieties contribute significantly to the physicochemical properties of french bean s globulin phaseolin structural characterization of multibranched oligosaccharides from seal milk by a combination of off-line high-performance liquid chromatographymatrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and sequential exoglycosidase digestion phenylethyl glycosides from digitalis lanata acceptor and substrate specificity of b-glucuronidase with transglycosylation activity from aspergillus niger kinetic study on the binding of lectin to mannose residues in a polymer brush high molecular weight polyglycerol-based multivalent mannose conjugates -methyl and -xylosyl derivatives of -hydroxyflexixanthin are major carotenoids of hymenobacter species modifications of human total serum n-glycome during liver fibrosis-cirrhosis, is it all about immunoglobulins? immunoglobulins are the major glycoproteins involved in the modifications of total serum n-glycome in cirrhotic patients enrichment of o-glcnac modified proteins by the periodate oxidation-hydrazide resin capture approach surface grafting of cellulose nanocrystals with poly(ethylene oxide) in aqueous media a quantitative model of ultraviolet matrix-assisted laser desorption/ionization a quantitative model of ultraviolet matrix-assisted laser desorption/ionization including analyte ion generation a bipolar rate equation model of maldi primary and secondary ionization processes, with application to positive/ negative analyte ion ratios and suppression effects electrospray and maldi mass spectrometry: fundamentals, instrumemtation, practialities and biological applications molecular dynamics simulations of maldi: laser fluence and pulse width dependence of plume characteristics and consequences for matrix and analyte ionization enzymatic polymer synthesis: an opportunity for green polymer chemistry synthetic studies on the carbohydrate moiety of the antigen from the parasite echinococcus multilocularis mass spectrometry for the analysis of milk oligosaccharides amphiphilic block copolymers based on cyclodextrin host-guest complexes via raftpolymerization in aqueous solution synthesis and conformational analysis of (a-d-galactosyl)phenylmethane and a-,b-difluoromethane analogues: interactions with the plant lectin viscumin structures of aldouronic acids liberated from kenaf xylan by endoxylanases from streptomyces sp study on systematizing the synthesis of the a-series ganglioside glycans gt a, gd a, and gm using the newly developed n-troc-protected gm and galn intermediates characterization of sialylated and fucosylated glycopeptides of b -glycoprotein i by a combination of hilic lc and maldi ms/ms glycopeptide profiling of beta- -glycoprotein i by mass spectrometry reveals attenuated sialylation in patients with antiphospholipid syndrome application of deep sea yeast-production of biosurfactants biosurfactant-producing yeast isolated from calyptogena soyoae (deep-sea cold-seep clam) in the deep sea an arginyl residue in rice udp-arabinopyranose mutase is required for catalytic activity and autoglycosylation structural identification of the main ellagitannins of a boysenberry (rubus loganbaccus  baileyanus britt.) extract by lc-esi-ms/ms, maldi-tof-ms and nmr spectroscopy quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis block synthesis of blood group tetrasaccharides b (types , and ) advances in the separation, sensitive detection, and characterization of heparin and heparan sulfate glucosinolateaccumulating s-cells in arabidopsis leaves and flower stalks undergo programmed cell death at early stages of differentiation polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts synthesis of a neoglycoconjugate containing a chlamydophila psittaci-specific branched kdo trisaccharide epitope the mita gene of aspergillus fumigatus is required for mannosylation of inositol-phosphorylceramide, but is dispensable for pathogenicity sulfated oligosaccharides: new targets for drug development stability of ( )-carboxyfluorescein in microwave-assisted synthesis of fluoresceinlabelled o-dimannosylated peptides triterpene glycosides from agrostemma gracilis porphyrin-cyclodextrin conjugates as a nanosystem for versatile drug delivery and multimodal cancer therapy a mathematical model of n-linked glycosylation a mathematical model to derive n-glycan structures and cellular enzyme activities from mass spectrometric data glycosyl azides: novel substrates for enzymatic transglycosylations multidimensional system enabling deglycosylation of proteins using a capillary reactor with peptide-nglycosidase f immobilized on a porous polymer monolith and hydrophilic interaction liquid chromatography-mass spectrometry of glycans glycomic analysis: an array of technologies synthesis of an fmoc-threonine bearing core- glycan: a building block for psgl- via fmoc-assisted solid-phase peptide synthesis the development of retrosynthetic glycan libraries to profile and classify the human serum n-linked glycome human serum processing and analysis methods for rapid and reproducible n-glycan mass profiling shigella sonnei oligosaccharide-protein conjugates immunochemical studies of shigella flexneri a and , and shigella dysenteriae type o-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates a,a-trehalose-based polyacetals and macrocyclic acetals application of spectroscopic methods for structural analysis of chitin and chitosan the molecular basis of inhibition of golgi a-mannosidase ii by mannostatin a glycomics and proteomics analyses of mouse uterine luminal fluid revealed a predominance of lewis y and x epitopes on specific protein carriers productionof sophorolipid biosurfactants by multiple species of the starmerella (candida) bombicola yeast clade four new triterpenes from anchusa azurea var. azurea sialyltransferases of marine bacteria efficiently utilize glycosphingolipid substrates dethreading of deoxyribonucleotides through a-cyclodextrin efficient guest inclusion by b-cyclodextrin attached to the ends of dna oligomers upon hybridization to various dna conjugates determination of sialic acid and gangliosides in biological samples and dairy products: a review solid-phase thermodynamic interpretation of ion desorption in matrix-assisted laser desorption/ionization controlled release of , -desulfated heparin exerts its anti-inflammatory activity by effectively inhibiting e-selectin deletion of udp-glucose pyrophosphorylase reveals a udp-glucose independent udp-galactose salvage pathway in leishmania major imaging of lipids in spinal cord using intermediate pressure matrix-assisted laser desorption-linear ion trap/orbitrap ms utilization of the linear mode of maldi-tof mass spectrometry in the study of glycation during the malting process profiling of n-linked oligosaccharides using phenylhydrazine derivatization and mass spectrometry labelling saccharides with phenylhydrazine for electrospray and matrix-assisted laser desorption-ionization mass spectrometry matrix-assisted laser desorption/ ionization tandem mass spectrometry and post-source decay fragmentation study of phenylhydrazones of n-linked oligosaccharides from ovalbumin method for investigation of oligosaccharides using phenylhydrazine derivatization n-glycomic changes in human breast carcinoma mcf- and t-lymphoblastoid cells after treatment with herceptin and herceptin/lipoplex mass spectrometric study of n-glycans from serum of woodchucks with liver cancer chemical characterization, antiproliferative and antiadhesive properties of polysaccharides extracted from pleurotus pulmonarius mycelium and fruiting bodies polymerizable vancomycin derivatives for bactericidal biomaterial surface modification: structure-function evaluation the chap domain of cse functions as an endopeptidase that acts at mature septa to promote streptococcus thermophilus cell separation biorecognition of chemically modified bovine serum albumin with lactose prepared under different conditions conjugates of bovine serum albumin with chitin oligosaccharides prepared through the maillard reaction characterization of -a-glucanotransferase from synechocystis sp. pcc and its application to various corn starches effects of differential glycosylation of glycodelins on lymphocyte survival targeted molecular imaging of vegf receptors overexpressed in ischemic microvasculature using chitosan-dc conjugates the poplar gt e and gt f glycosyltransferases are functional orthologs of arabidopsis parvus involved in glucuronoxylan biosynthesis the f h glycosyltransferase is a functional paralog of fra involved in glucuronoxylan biosynthesis in arabidopsis down-regulation of pogt c expression in poplar results in a reduced glucuronoxylan content and an increased wood digestibility by cellulase the arabidopsis family gt glycosyltransferases form two functionally nonredundant groups essential for the elongation of glucuronoxylan backbone glycosylated neurotensin analogues exhibit sub-picomolar anticonvulsant potency in a pharmacoresistant model of epilepsy techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein isolation and tandem mass fragmentations of an anti-inflammatory compound from aralia elata high-throughput quantitative analysis of plant nglycan using a dna sequencer synthesis of selenium nanowires morphologically directed by shinorhizobial oligosaccharides core o-glycan synthase suppresses tumor formation and metastasis of prostate carcinoma pc and lncap cells through down-regulation of a b integrin complex core o-glycan structure is essential for the cell surface expression of sucrase isomaltase and dipeptidyl peptidase-iv during intestinal cell differentiation synthesis and cellular uptake properties of guanidine-containing molecular transporters built on the sucrose scaffold tracing the development of structural elucidation of nglycans synthesis of undecaprenyl pyrophosphate-linked glycans as donor substrates for bacterial protein nglycosylation deficiency of dol-p-man synthase subunit dpm bridges the congenital disorders of glycosylation with the dystroglycanopathies n-glycosylation patterns of hemolymph glycoproteins from biomphalaria glabrata strains expressing different susceptibility to schistosoma mansoni infection multivalent binding of carbohydrates by the human a-defensin, hd structural analysis of sulfated glycans by sequential double-permethylation using methyl iodide and deuteromethyl iodide sequential enrichment of sulfated glycans by strong anion-exchange chromatography prior to mass spectrometric measurements a new allergen from ragweed (ambrosia artemisiifolia) with homology to art v from mugwort an unusual galactofuranose lipopolysaccharide that ensures the intracellular survival of toxin-producing bacteria in their fungal host human l-selectin preferentially binds synthetic glycosulfopeptides modeled after endoglycan and containing tyrosine sulfate residues and sialyl lewis x in core o-glycans efficient synthesis and protein conjugation of b-( - )-d-n-acetylglucosamine oligosaccharides from the polysaccharide intercellular adhesin steroid compounds from two pacific starfish of the genus evasterias n-glycosylation site analysis of human platelet proteins by hydrazide affinity capturing and lc-ms/ ms phosphoethanolamine substitution of lipid a and resistance of neisseria gonorrhoeae to cationic antimicrobial peptides and complement-mediated killing by normal human serum infrared multiphoton dissociation mass spectrometry for structural elucidation of oligosaccharides collision-induced dissociation tandem mass spectrometry for structural elucidation of glycans pancreatic cancer serum detection using a lectin/ glyco-antibody array method structure of pleiotrophin-and hepatocyte growth factor-binding sulfated hexasaccharide determined by biochemical and computational approaches overexpression and topology of bacterial oligosaccharyltransferase pglb effects of hapten density on the induced antibody repertoire advances in the glycosylation of milk protein light-switchable janus [ ] rotaxanes based on a-cyclodextrin derivatives bearing two recognition sites linked with oligo(ethylene glycol) maldi-tof-ms analysis of small molecules using modified mesoporous material sba- as assisted matrix preparation of homogenous oligosaccharide chains from glycosphingolipids mass spectrometry of fluorocarbon-labeled glycosphingolipids gold(i)-catalyzed glycosylation with glycosyl ortho-alkynylbenzoates as donors: general scope and application in the synthesis of a cyclic triterpene saponin immunologic glycosphingolipidomics and nkt cell development in mouse thymus comparison of sample pre-treatments for laser desorption ionization and secondary ion mass spectrometry imaging of miscanthus  giganteus glycan array: a powerful tool for glycomics studies switching of the core structures of glycosphingolipids from globo-and lacto-to ganglio-series upon human embryonic stem cell differentiation the effect of multivalent binding on the lateral phase separation of adhesive lipids -n, -o-carbonyl- , , -tri-o-chloroacetyl-protected sialyl donor for the stereoselective synthesis of a-( ! )-tetrasialic acid enhanced expression of b -galactosyltransferase activity is sufficient to induce in vivo synthesis of extended type chains on lactosylceramides of selected human colonic carcinoma cell lines a new naphthimidazole derivative for saccharide labeling with enhanced sensitivity in mass spectrometry detection i -catalyzed oxidative condensation of aldoses with diamines: synthesis of aldo-naphthimidazoles for carbohydrate analysis in vivo protection provided by a synthetic new alpha-galactosyl ceramide analog against bacterial and viral infections in murine models fabrication of oriented antibody-conjugated magnetic nanoprobes and their immunoaffinity application characterization of alginate-like exopolysaccharides isolated from aerobic granular sludge in pilot-plant synthesis of magnetic nanoparticles with immobilized aminophenylboronic acid for selective capture of glycoproteins the grignard reaction of cyclodextrin- -aldehydes revisited: a study of the stereoselectivity upon addition of organometallic reagents to aldehydes and ketones a bivalent glycopeptide to target two putative carbohydrate binding sites on fimh en route to photoaffinity labeling of the bacterial lectin fimh muropeptide rescue in bacillus subtilis involves sequential hydrolysis by b-n-acetylglucosaminidase and n-acetylmuramyl-lalanine amidase incoherent production reactions of positive and negative ions in matrix-assisted laser desorption/ionization initial ionization reaction in matrix-assisted laser desorption/ionization isolation and identification of two novel sds-resistant secreted chitinases from aeromonas schubertii norclerodane diterpenoids from rhizomes of dioscorea bulbifera an apple oligogalactan prevents against inflammation and carcinogenesis by targeting lps/tlr /nf-kb pathway in a mouse model of colitis-associated colon cancer phosphoryl moieties of lipid a from neisseria meningitidis and n. gonorrhoeae lipooligosaccharides play an important role in activation of both myd -and trif-dependent tlr -md- signaling pathways efficient synthesis of flaccidoside ii, a bioactive component of chinese folk medicine di wu concise synthesis of two natural triterpenoid saponins, oleanolic acid derivatives isolated from the roots of pulsatilla chinensis ionic liquids in sample preparation albizosides d and e, two new cytotoxic triterpene saponins from albizia chinensis cytotoxic oleanane triterpene saponins from albizia chinensis electrospray ionization mass spectrometry as a critical tool for revealing new properties of snake venom phospholipase a f-labeled galacto and pegylated rgd dimers for pet imaging of a v b integrin expression alteration of n-glycome in diethylnitrosamine-induced hepatocellular carcinoma mice: a noninvasive monitoring tool for liver cancer is permethylation strategy always applicable to protein n-glycosylation study? a case study on the o-acetylation of sialic acid in fish serum glycans microwave-assisted kochetkov amination followed by permanent charge derivatization: a facile strategy for glycomics methylamidation for sialoglycomics by maldi-ms: a facile derivatization strategy for both a , -and a , -linked sialic acids investigation of sample preparation artifacts formed during the enzymatic release of n-linked glycans prior to analysis by capillary electrophoresis supramolecular assembly of perylene bisimide with b-cyclodextrin grafts as a solid-state fluorescence sensor for vapor detection elevation of sulfatides in ovarian cancer: an integrated transcriptomic and lipidomic analysis including tissue-imaging mass spectrometry tandem o stable isotope labeling for quantification of n-glycoproteome plant-expressed recombinant mountain cedar allergen jun a is allergenic and has limited pectate lyase activity modular approach to triazole-linked , -a-d-oligomannosides to the discovery of inhibitors of mycobacterium tuberculosis cell wall synthetase glycosylation of b subunits regulates gaba a receptor biogenesis and channel gating lipidomic analysis of porcine olfactory epithelial membranes and cilia a versatile and scalable strategy for glycoprofiling bifidobacterial consumption of human milk oligosaccharides the egmur xyloglucan galactosyltransferase from eucalyptus grandis complements the mur cell wall phenotype in arabidopsis thaliana morphological, toxicological and molecular characterization of a benthic nodularia isolated from atlantic estuarine environments enthalpic studies of xyloglucan-cellulose interactions titania microparticles and nanoparticles as matrixes for in vitro and in situ analysis of small molecules by maldi-ms reactivity-based onepot synthesis of immunosuppressive glycolipids from the caribbean sponge plakortis simplex chemical synthesis of a hyaluronic acid decasaccharide a yeast glycoprotein shows highaffinity binding to the broadly neutralizing human immunodeficiency virus antibody g and inhibits gp interactions with g and dc-sign fatal outcome due to deficiency of subunit of the conserved oligomeric golgi complex leading to a new type of congenital disorders of glycosylation two kdo-heptose regions identified in hafnia alvei lipopolysaccharide: the complete core structure and serological screening of different hafnia o serotypes structural analysis of the lipid a isolated from hafnia alvei and pcm lipopolysaccharides functional l-lysine dendritic macromolecules as liver-imaging probes structural characterization of n-linked oligosaccharides of defibrase from agikistrodon acutus by sequential exoglycosidase digestion and maldi-tof mass spectrometry studying a cell division amidase using defined peptidoglycan substrates data mining the pdb for glyco-related data facile preparation of fluorescent neoglycoproteins using p-nitrophenyl anthranilate as a heterobifunctional linker synthesis of the pentasaccharide repeating unit of escherichia coli o antigen a b-d-allopyranoside-grafted ru(ii) complex: synthesis and acid-base and dna-binding properties new phosphane based on a b-cyclodextrin, exhibiting a solventtunable conformation, and its catalytic properties structural analysis of the o-specific polysaccharide isolated from plesiomonas shigelloides o lipopolysaccharide the conformational properties of the glc man unit suggest conformational biasing within the chaperone-assisted glycoprotein folding pathway euscaphinin, a new ellagitannin dimer from euscaphis japonica (thunb.) kanitz study of lysozyme glycation reaction by mass spectrometry and nmr spectroscopy fut -null mice display an altered glycosylation profile and impaired babamediated helicobacter pylori adhesion to gastric mucosa aglp is a s-adenosyl-l-methionine-dependent methyltransferase that participates in the n-glycosylation pathway of haloferax volcanii manipulation of electrostatic and saccharide linker interactions in the design of efficient glycopolypeptide-based cholera toxin inhibitors chemical synthesis and proinflammatory responses of monophosphoryl lipid a adjuvant candidates glucooligosaccharides production from glucan of leuconostoc mesenteroides nrrl b- by microwave assisted hydrolysis isorhizochalin: a minor unprecedented bipolar sphingolipid of stereodivergent biogenesis from the rhizochalina incrustata development of continuous flow type hydrothermal reactor for hemicellulose fraction recovery from corncob phenolic compounds from bursera simaruba sarg. bark: phytochemical investigation and quantitative analysis by tandem mass spectrometry iodine-hexamethyldisilane (hmds)-mediated anomerization of peracetylated , -trans-linked alkyl and aryl glycosides a simple, mild, and regioselective method for the benzylation of carbohydrate derivatives promoted by silver carbonate efficient synthesis of a fluorescent tripod detection system for pesticides by microwaveassisted click chemistry highly efficient deletion of fut in cho cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies rapid de-o-glycosylation concomitant with peptide labeling using microwave radiation and an alkyl amine base dissection of host cell signal transduction during acinetobacter baumannii-triggered inflammatory response consumption of human milk oligosaccharides by gutrelated microbes analysis of protein posttranslational modifications using mass spectrometry interactions of lipopolysaccharide and polymyxin studied by nmr spectroscopy amino-acetone-bridged cyclodextrins-artificial alcohol oxidases a systematic approach to protein glycosylation analysis: a path through the maze flavonoids with prolyl oligopeptidase inhibitory activity isolated from scutellaria racemosa pers glyconanoparticles: polyvalent tools to study carbohydrate-based interactions glycosylation protects proteins against free radicals generated from toxic xenobiotics gold manno-glyconanoparticles: multivalent systems to block hiv- gp binding to the lectin dc-sign antithrombin murcia (k e) causing antithrombin deficiency: a possible role for altered glycosylation a series of -o-trifluoromethylsulfonyl-dmannopyranosides as precursors for concomitant f-labeling and glycosylation by click chemistry synthesis and delivery activity of new cationic cholesteryl glucosides mutational deglycosylation of the fc portion of immunoglobulin g causes osulfation of tyrosine adjacently preceding the originally glycosylated site biochemical and immunological characterization of the plantderived candidate human immunodeficiency virus type mucosal vaccine ctb-mpr - supported molecular matrix electrophoresis: a new tool for characterization of glycoproteins development of an apparatus for rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in chondroitin sulfate-type proteoglycans synthesis of sialyllactosamine clusters using carbosilane as core scaffolds by means of chemical and enzymatic approaches novel synthesis of functional mucin glycopeptides containing both n-and o-glycans functional neoglycopeptides: synthesis and characterization of a new class of muc glycoprotein models having core -based o-glycan and complex-type n-glycan chains a tethering mechanism for length control in a processive carbohydrate polymerization structural analysis of arabinoxylans isolated from ball-milled switchgrass biomass extracellular monoenzyme deglycosylation system of -o-linked flavonoid brutinosides and its disaccharide transglycosylation activity from stilbella fimetaria biodistribution and excretion of monosaccharidealbumin conjugates measured with in vivo near-infrared fluorescence imaging glycoform and glycologue: two software applications for the rapid construction and display of n-glycans from mammalian sources site-directed mutagenesis to probe catalysis by a thermobifida fusca b- , -glucanase (lam a) the mass-mobility correlation redux: the conformational landscape of anhydrous biomolecules glycomic analysis by capillary electrophoresis-mass spectrometry solid-phase permethylation for glycomic analysis high-sensitivity analytical approaches to the analysis of n-glycans quantitative serum glycomics of esophageal adenocarcinoma and other esophageal disease onsets the effect of glycation on foam and structural properties of b-lactoglobulin structural characterization of glycans on omega- , a major schistosoma mansoni egg glycoprotein that drives th responses proteomic scale high-sensitivity analyses of gpi membrane anchors glycotyping of trypanosoma brucei variant surface glycoprotein mitat studies on the boronation of methyl-b-d-cellobioside-a cellulose model assessment of heat treatment of dairy products by synthesis of tetraglucosyl-and tetrapolyamine-tetrabenzoporphyrin conjugates for an application in pdt synthesis of an fmoc-asn-heptasaccharide building block and its application to chemoenzymatic glycopeptide synthesis fluorescent bodipylabelled gm gangliosides designed for exploring lipid membrane properties and specific membrane-target interactions helicobacter pylori binding to new glycans based on n-acetyllactosamine transcriptomic analysis of arabidopsis developing stems: a close-up on cell wall genes glycoforms of human endothelial cd that bind l-selectin carry sulfated sialyl lewis x capped o-and n-glycans chemical structure analysis of starch and cellulose derivatives genetic and structural characterization of l lipooligosaccharide from neisseria meningitidis serogroup a strains the n-glycolyl form of mouse sialyl lewis x is recognized by selectins but not by heca- and fh antibodies that were raised against human cells -(d-glucopyranosyl- -deoxy- -iminomethyl)- -hydroxybenzene as chemosensor for aromatic amino acids by switch-on fluorescence ultrahighly sensitive in situ metabolomic imaging for visualizing spatiotemporal metabolic behaviors rapid and simple solid-phase esterification of sialic acid residues for quantitative glycomics by mass spectrometry glycoblotting-assisted o-glycomics: ammonium carbamate allows for highly efficient o-glycan release from glycoproteins synthesis and absolute structures of mycoplasma pneumoniae bglyceroglycolipid antigens multivalent galacto-trehaloses: design, synthesis, and biological evaluation under the concept of carbohydrate modules promiscuous activity of er glucosidase ii discovered through donor specificity analysis of uggt novel rhamnosyltransferase involved in biosynthesis of serovar -specific glycopeptidolipid from mycobacterium avium complex systematic synthesis and inhibitory activity of haloacetamidyl oligosaccharide derivatives toward cytoplasmic peptide: n-glycanase structural and functional characterization of recombinant human serum transferrin secreted from pichia pastoris the cytotoxic activity of linum grandiflorum leaves new acylated flavone and cyanogenic glycosides from linum grandiflorum synthesis of novel gluco-and galacto-functionalized platinum complexes design of triazoletethered glycoclusters exhibiting three different spatial arrangements and comparative study of their affinities towards pa-il and rca by using a dna-based glycoarray capsule polysaccharide conjugate vaccine against diarrheal disease caused by campylobacter jejuni a-type crystals from dilute solutions of short amylose chains the n-glycosylation of classical swine fever virus e glycoprotein extracellular domain expressed in the milk of goat n-glycosylation pattern of e glycoprotein from classical swine fever virus glycation pattern of peptides condensed with maltose, lactose and glucose determined by ultraviolet matrix-assisted laser desorption/ionization tandem mass spectrometry regulated and aberrant glycosylation modulate cardiac electrical signaling synthesis of hyperbranched b-galceramide-containing dendritic polymers that bind hiv- rgp in vivo biocompatibility and biodegradability of dextrin-based hydrogels analysis of glycosylation and other post-translational modifications by mass spectrometry analysis of n-and olinked glycans from glycoproteins using maldi-tof mass spectrometry glycosylation pattern of brush borderassociated glycoproteins in enterocyte-like cells: involvement of complex-type n-glycans in apical trafficking anthocyanin components and mechanism for color development in blue veronica flowers biochemical characterization of plasma-derived tissue factor pathway inhibitor: post-translational modification of free, full-length form with particular reference to the sugar chain isolation of basidiomycetous yeast pseudozyma tsukubaensis and production of glycolipid biosurfactant, a diastereomer type of mannosylerythritol lipid-b production of glycolipid biosurfactants, mannosylerythritol lipids, by a smut fungus, nbrc production of a novel glycolipid biosurfactant, mannosylmannitol lipid, by pseudozyma parantarctica and its interfacial properties coupling of planar chromatography to mass spectrometry selective binding of rnase b glycoforms by polydopamine-immobilized concanavalin a glycosylated cu/zn-superoxide dismutase from kluyveromyces yeast, determined by mass spectrometry chemoenzymatic synthesis of conjugatable oligosialic acids analysis of recombinant cd glycans by maldi-tof-ms reveals prevalence of sialyl-t antigen separation and identification of oligosaccharides labeled with -amino- -ethylcarbazole using high performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry o-acetylation of peptidoglycan in gramnegative bacteria. identification and characterization of peptidoglycan o-acetyltransferase in neisseria gonorrhoeae identification of the n-glycosylation sites on recombinant bovine cd expressed in pichia pastoris: their impact on enzyme stability and catalytic activity efficient synthesis of -amino- -deoxy-l-arabinose and spacerequipped -amino- -deoxy-l-arabinopyranosides by transglycosylation reactions molecular weight determination of high molecular mass (glyco)proteins using cge-on-a-chip study of inclusion complexes of cycloamylose with surfactants by isothermal titration calorimetry analysis of n-and o-linked protein glycosylation in children with prader-willi syndrome supramolecular nanocycles comprising b-cyclodextrin-click-ferrocene units: rings of rings of rings unique cleavage of -acetamido- -deoxy-dglucose from the reducing end of biantennary complex type oligosaccharides efficient and systematic synthesis of a small glycoconjugate library having human complex type oligosaccharides imaging mass spectrometry: principle and application application of thin-layer chromatography/infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry to structural analysis of bacteria-binding glycosphingolipids selected by affinity detection chemoenzymatic synthesis of a new class of macrocyclic oligosaccharides advances on the compositional analysis of glycosphingolipids combining thin-layer chromatography with mass spectrometry shiga toxins, glycosphingolipid diversity, and endothelial cell injury heparin/heparan sulfate -o-sulfatase from flavobacterium heparinum. integrated structural and biochemical investigation of enzyme active site and substrate specificity heparin/heparan sulfate n-sulfamidase from flavobacterium heparinum. structural and biochemical investigation of catalytic nitrogen-sulfur bond cleavage utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics anti-influenza virus activity and structure-activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains structural and functional glycosphingolipidomics by glycoblotting with an aminooxy-functionalized gold nanoparticle synthesis of urea tethered glycosylated amino acids and glycopeptides mediated by dppa employing na-fmoc-asp/glu- -oxazolidinones toward fully synthetic homogeneous b-human follicle-stimulating hormone (b-hfsh) with a biantennary n-linked dodecasaccharide. synthesis of b-hfsh with chitobiose units at the natural linkage sites experiments on the synthesis of carotenoid glycosides glycomic analyses of glycoproteins in bile and serum during rat hepatocarcinogenesis transferrin receptordependent cytotoxicity of artemisinin-transferrin conjugates on prostate cancer cells and induction of apoptosis synthesis, structural analysis and application of novel acarbose fructoside using levansucrase extraction and characterization of native heteroxylans from delignified corn stover and aspen a strategy for discovery of cancer glyco-biomarkers in serum using newly developed technologies for glycoproteomics effect of glycosylation on cis/trans isomerization of prolines in iga -hinge peptide proteomic approaches to study structure, functions and toxicity of legume seeds lectins. perspectives for the assessment of food quality and safety engineering host cell lines to reduce terminal sialylation of secreted antibodies purification and partial characterization of cu/ zn superoxide dismutase from kluyveromyces marxianus yeast experimental evidence of chemical exchange over the b( ! ) glycosidic linkage and hydrogen bonding involving hydroxy protons in hyaluronan oligosaccharides by nmr spectroscopy synthesis of amino-bridged oligosaccharide mimetics production of chitin oligosaccharides with different molecular weights and their antioxidant effect in raw . cells genetic analysis of genes involved in synthesis of modified -amino- , -dideoxyglucose in flagellin of pseudomonas syringae pv. tabaci identification of a naturally-occurring -[a-d-glucopyranosyl-( - )-b-d-glucopyranosyl] daidzein from cultivated kudzu root novel rhamnolipid biosurfactants produced by a polycyclic aromatic hydrocarbon-degrading bacterium pseudomonas aeruginosa strain ny novel o-antigen of hafnia alvei pcm lipopolysaccharide with a teichoic acid-like structure structures of two novel, serologically nonrelated core oligosaccharides of yokenella regensburgei lipopolysaccharides differing only by a single hexose substitution syntheses and characterisation of novel cyclodextrin vinyl derivatives from cyclodextrin-nitrophenol-derivatives use of b-cyclodextrins to control the structure of water-soluble copolymers with hydrophobic parts facile synthesis of b-cyclodextrin-dextran polymers by "click" chemistry fullerene sugar balls gold nanoparticle arrangement on viral particles through carbohydrate recognition: a non-cross-linking approach to optical virus detection enrichment of glycopeptides for glycan structure and attachment site identification characterization of the quantitative relationship between signalto-noise (s/n) ratio and sample amount on-target by maldi-tof ms: determination of chondroitin sulfate subsequent to enzymatic digestion differently complex oligosaccharides can be easily identified by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry directly from a standard thin-layer chromatography plate identification of o-glycosylated decapeptides within the muc repeat domain as potential mhc class i (a ) binding epitopes mass spectrometric methods for analysis of oligosaccharides in human milk preparation and characterization of branched bcyclodextrins having a-l-fucopyranose and a study of their functions reverse thin layer method for enhanced ion yield of oligosaccharides in matrix-assisted laser desorption/ionization assessing the cluster glycoside effect during the binding of concanavalin a to mannosylated artificial lipid rafts systematic screens of a candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity onresin click-glycoconjugation of peptoids mass spectrometry in the analysis of n-linked and o-linked glycans glycomics profiling of chinese hamster ovary cell glycosylation mutants reveals n-glycans of a novel size and complexity mass spectrometric analysis of mutant mice characterization of the novel human age hn cell line for production of recombinant proteins production and characterization of monoclonal antibodies specific to lactotriaosylceramide repairing faulty genes by aminoglycosides: development of new derivatives of geneticin (g ) with enhanced suppression of diseases-causing nonsense mutations development of novel aminoglycoside (nb ) with reduced toxicity and enhanced suppression of disease-causing premature stop mutations enhanced detection and identification of glycopeptides in negative ion mode mass spectrometry structural characterization of an oligosaccharide made by neisseria sicca electrospray and maldi mass spectrometry: fundamentals, instrumentation, practicalities and biological applications microanalysis of plant cell wall polysaccharides molecular analysis of carbohydrate-antibody interactions: case study using a bacillus anthracis tetrasaccharide endo-b-n-acetylglucosaminidasecatalyzed polymerization of b-glcp-( - )-glcpnac oxazoline: a revisit to enzymatic transglycosylation syntheses and doxorubicininclusion abilities of b-cyclodextrin derivatives with a hydroquinone a-glycoside residue attached at the primary side synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat a , -sialyltransferase expressed in bmnpv bacmid-injected silkworm larvae expression, purification, and analyses of glycosylation and disulfide bonds of stereum purpureum endopolygalacturonase i in pichia pastoris chemically responsive supramolecular assemblies of pyrene-b-cyclodextrin dimer matrix-assisted laser desorption/ionization mass spectrometric analysis of polysulfated-derived oligosaccharides using pyrenemethylguanidine structual analysis of neutral and acidic xylooligosaccharides from hardwood kraft pulp, and their utilization by intestinal bacteria in vitro structural study of the n-glycans of intercellular adhesion molecule- (telencephalin) an essential epitope of anti-muc monoclonal antibody kl- revealed by focused glycopeptide library simple and conveniently accessible bi-fluorescence-labeled substrates for amylases syntheses and biological evaluations of carbosilane dendrimers uniformly functionalized with sialyl a ( ! ) lactose moieties as inhibitors for human influenza viruses characterization of endoplasmic reticulum-localized udp-dgalactose: hydroxyproline o-galactosyltransferase using synthetic peptide substrates in arabidopsis structural analysis of three novel trisaccharides isolated from the fermented beverage of plant extracts novel fructopyranose oligosaccharides isolated from fermented beverage of plant extract convenient approach to access octa-glycosylated porphyrins via "click chemistry n-glycosylation engineering of lepidopteran insect cells by the introduction of the b , -n-acetylglucosaminyltransferase iii gene efficient substitution reaction from cysteine to the serine residue of glycosylated polypeptide: repetitive peptide segment ligation strategy and the synthesis of glycosylated tetracontapeptide having acid labile sialyl-tn antigens structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under tnf-a stimulation boronic acid lectin affinity chromatography (blac). . temperature dependence of glycoprotein isolation and enrichment physical mechanisms of bacterial survival revealed by combined grazing-incidence x-ray scattering and monte carlo simulation use of ftir and mass spectrometry for characterization of glycated caseins artefacts formed by addition of urea to n-linked glycans released with peptide-n-glycosidase f for analysis by mass spectrometry okicamelliaside, an extraordinarily potent antidegranulation glucoside isolated from leaves of camellia japonica quantification of fructooligosaccharides based on the evaluation of oligomer ratios using an artificial neural network quantitative glycomics results of the glycoprotein research group's (gprg) study on quantitative glycoprotein analysis click multivalent heterogeneous neoglycoconjugates-modular synthesis and evaluation of their binding affinities globular carbosilane dendrimers with mannose groups at the periphery: synthesis, characterization and toxicity in dendritic cells biosynthesis and structure of the burkholderia cenocepacia k - lipopolysaccharide core oligosaccharide. truncation of the core oligosaccharide leads to increased binding and sensitivity to polymyxin b switching of polymerization activity of cinnamoyl-a-cyclodextrin action of a-dglucosidase from aspergillus niger towards dextrin and starch contiguous ogalactosylation of (r)-hydroxy-l-proline residues forms very stable polyproline ii helices microarrays with varying carbohydrate density reveal distinct subpopulations of serum antibodies synthesis, in vitro and in vivo studies of gd-dtpa-xda-d -glc(oh) complex as a new potential mri contrast agent structural characterization and hypoglycemic effects of arabinogalactan-protein from the tuberous cortex of the white-skinned sweet potato (ipomoea batatas l.) comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method characterization of minor n-linked glycans on antibodies using endo h release and maldi-mass spectrometry methods in molecular biology, glycomics: methods and protocols the caenorhabditis elegans bus- mutant reveals a new class of o-glycans affecting bacterial resistance surfactant-assisted lipopolysaccharide conjugation employing a cyanopyridinium agent and its application to a competitive assay multifaceted approaches including neoglycolipid oligosaccharide microarrays to ligand discovery for malectin glycoproteomic profile in wine: a 'sweet' molecular renaissance mass spectrometry based targeted protein quantification: methods and applications convergent synthesis of a common pentasaccharide-repeating unit corresponding to the o-specific polysaccharide of escherichia coli o : k , o : k , and o : k synthesis of penta-and hexasaccharide fragments corresponding to the o-antigen of escherichia coli o analysis of the human seminal plasma glycome reveals the presence of immunomodulatory carbohydrate functional groups fast access to robust c-sialoside multimers synthesis, conjugation, and immunological evaluation of the analysis of carbohydrates and glycoconjugates & serogroup pneumococcal oligosaccharides human igg expression in silkworm larval hemolymph using bmnpv bacmids and its n-linked glycan structure the synthesis and spectral properties of an encapsulated aminoazobenzene dye o-glcnacylation disrupts glyceraldehyde- -phosphate dehydrogenase homo-tetramer formation and mediates its nuclear translocation immunochemical studies of trehalose-containing major glycolipid from tsukamurella pulmonis structural characterization of the major glycolipids from arthrobacter globiformis and arthrobacter scleromae bioconjugation and detection of lactosamine moiety using a , -galactosyltransferase mutants that transfer c -modified galactose with a chemical handle muropeptides trigger distinct activation profiles in macrophages and dendritic cells rapid assembly of gp oligosaccharide moieties via one-pot glycosidation-deprotection sequences one-pot catalytic glycosidation/fmoc removal-an iterable sequence for straightforward assembly of oligosaccharides related to hiv gp analysis of the dispersity in carbohydrate loading of synthetic glycoproteins using maldi-tof mass spectrometry solvent-free synthesis of thioglycosides by ball milling synthesis of mycobacterial triacylated phosphatidylinositol dimannoside containing an acyl lipid chain at -o of inositol o-glycan inhibitors generate arylglycans, induce apoptosis and lead to growth inhibition in colorectal cancer cell lines detection of carbohydrates and steroids by cation-enhanced nanostructure-initiator mass spectrometry (nims) for biofluid analysis and tissue imaging protein glycosylation analysis with capillary-based electromigrative separation techniques glycoprotein analysis using protein microarrays and mass spectrometry synthesis of -deoxy cyclic and linear oligosaccharides by oligomerization of monomers maintaining product titer while replacing undefined components in a cho culture system model a-mannoside conjugates: immunogenicity and induction of candidacidal activity hydrodynamic properties of cyclodextrin molecules in dilute solutions koenigs-knorr glycosylation with neuraminic acid derivatives synthesis of the core structure of the lipoteichoic acid of streptococcus pneumoniae de novo glycan structure search with the cid ms/ms spectra of native n-glycopeptides identification of a novel human udp-galnac transferase with unique catalytic activity and expression profile molecular engineering of exocytic vesicle traffic enhances the productivity of chinese hamster ovary cells the vesicletrafficking protein munc b increases the secretory capacity of mammalian cells detection of pathogenic streptococcus suis bacteria using magnetic glycoparticles rapid screening of lectins for multivalency effects with a glycodendrimer microarray imaging ms methodology for more chemical information in less data acquisition time utilizing a hybrid linear ion trap-orbitrap mass spectrometer atmospheric pressure laser desorption/ionization of plant metabolites and plant tissue using colloidal graphite click multivalent homogeneous neoglycoconjugates-synthesis and evaluation of their binding affinities role of lipid a acylation in yersinia enterocolitica virulence glycopeptide-preferring polypeptide galnac transferase (ppgalnac t ), involved in mucin-type o-glycosylation, has a unique galnac-o-ser/thr-binding site in its catalytic domain not found in ppgalnac t or t steroidal glycosides from the leaves of ruscus colchicus: isolation and structural elucidation based on a preliminary liquid chromatography-electrospray ionization tandem mass spectrometry profiling off-line coupling of microcolumn separations to desorption mass spectrometry selective discrimination of cyclodextrin diols using cyclic sulfates d-hplc and maldi-tof/tof analysis of barley proteins glycated during brewing synthesis of cationic derivatives of quil a and the preparation of cationic immunestimulating complexes (iscoms) cardenolides from pergularia tomentosa display cytotoxic activity resulting from their potent inhibition of na þ /k þ -atpase structural determination of the o-chain polysaccharide from the haloalkaliphilic halomonas alkaliantarctica bacterium strain crss structure of the core region from the lipopolysaccharide of plesiomonas shigelloides strain - (serotype o ) structural characterization of the core region from the lipopolysaccharide of the haloalkaliphilic bacterium halomonas alkaliantarctica strain crss the complete structure of the core of the lps from plesiomonas shigelloides - and the identification of its o-antigen biological repeating unit enhancement of repair of radiation induced dna strand breaks in human cells by ganoderma mushroom polysaccharides structural investigation of an exopolysaccharide substituted with a lactyl ether group produced by raoultella terrigena ez- - isolated in the chernobyl exclusion zone a protein important for antimicrobial peptide resistance, ydei/omda, is in the periplasm and interacts with ompd/nmpc role of e-cadherin n-glycosylation profile in a mammary tumor model influence of glycosylation on the adsorption of thermomyces lanuginosus lipase to hydrophobic and hydrophilic surfaces diazo transfer-click reaction route to new, lipophilic teicoplanin and ristocetin aglycon derivatives with high antibacterial and anti-influenza virus activity: an aggregation and receptor binding study high-energy collision induced dissociation of biomolecules: maldi-tof/rtof mass spectrometry in comparison to tandem sector mass spectrometry polymer structure of commercial hydrolyzable tannins by matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry design, synthesis, and evaluation of novel fluoroquinolone-aminoglycoside hybrid antibiotics cycloartane-type glycosides from astragalus amblolepis triterpenoid saponins from astragalus wiedemannianus fischer mechanisms involved during the ultrasonically induced depolymerization of chitosan: characterization and control oligonucleotide carbohydrate-centered galactosyl cluster conjugates synthesized by click and phosphoramidite chemistries substrate recognition and hydrolysis by a fungal xyloglucan-specific family hydrolase the structural elucidation of glycosaminoglycans chemical modification and biological evaluation of new semisynthetic derivatives of , -didehydronystatin a (s hp), a genetically engineered antifungal polyene macrolide context-dependent effects of asparagine glycosylation on pin ww folding kinetics and thermodynamics maldi-tof mass spectrometry of naturally occurring mixtures of monorhamnolipids and dirhamnolipids functionalized c-glycoside ketohydrazones: carbohydrate derivatives that retain the ring integrity of the terminal reducing sugar mass spectrometric-based stable isotopic -aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics desorption electrospray ionization mass spectrometry of glycosaminoglycans and their protein noncovalent complex haba-based ionic liquid matrices for uv-maldi-ms analysis of heparin and heparan sulfate oligosaccharides mannose-substituted conjugated polyelectrolyte and oligomer as an intelligent energy transfer pair for label-free visual detection of concanavalin a development of a ce-ms method to analyze components of the potential biomarker vascular endothelial growth factor lipopeptides produced by a soil bacillus megaterium strain mass spectrometric analysis of the s-layer proteins from clostridium difficile demonstrates the absence of glycosylation facile synthesis of mercaptophenylboronic acid-functionalized core-shell structure fe o @c@au magnetic microspheres for selective enrichment of glycopeptides and glycoproteins a smart glycol-directed nanodevice from rationally designed macroporous materials secondary disorders of glycosylation in inborn errors of fructose metabolism online coupling of thin layer chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: synthesis and application of a new material for the identification of carbohydrates by thin layer chromatography/matrix free material enhanced laser desorption/ionization mass spectrometry acylated triterpenoidal saponins and cytokinins from gleditsia aquatica click chemistry approach for the synthesis of water-soluble glycodendrimer with triazole as building unit synthesis and evaluation of n-acetyl- -amino- -deoxy-a-d-galactosyl- -thio- -oxaceramide, a new analogue of a-d-galactosyl ceramide mass spectrometry for pectin structure analysis multiple site-specific in vitro labeling of single-chain antibody the effect of ph on the production of chitinolytic enzymes of verticillium fungicola in submerged cultures glycome-db. org: a portal for querying across the digital world of carbohydrate sequences iodine-sodium cyanoborohydride-mediated reductive ring opening of , -o-benzylidene acetals of hexopyranosides glycosylation patterns of hiv- gp depend on the type of expressing cells and affect antibody recognition the glycosylation of myeloperoxidase the glycosylation and characterization of the candidate gc macrophage activating factor controlled synthesis of linear acyclodextrin oligomers using copper-catalyzed huisgen , -dipolar cycloaddition two-step synthesis of galactosylated human serum albumin as a targeted optical imaging agent for peritoneal carcinomatosis handbook of experimental pharmacology , doping in sports synthesis of multivalent glycoconjugates containing the immunoactive lelte peptide: effect of glycosylation on cellular activation and natural killing by human peripheral blood mononuclear cells golgi function and dysfunction in the first cog -deficient cdg type ii patient mycobacterium abscessus glycopeptidolipids mask underlying cell wall phosphatidyl-myo-inositol mannosides blocking induction of human macrophage tnf-a by preventing interaction with tlr capsule anchoring in bacillus anthracis occurs by a transpeptidation reaction that is inhibited by capsidin synthesis of acceptor substrate analogs for the study of glycosyltransferases involved in the second step of the biosynthesis of o-antigen repeating units identification of n-linked carbohydrates from severe acute respiratory syndrome (sars) spike glycoprotein identification of n-glycans from ebola virus glycoproteins by matrix-assisted laser desorption/ionisation time-of-flight and negative ion electrospray tandem mass spectrometry synthesis, characterization, and immunogenicity in mice of shigella sonnei o-specific oligosaccharide-core-protein conjugates -aminoquinoline acting as matrix and derivatizing agent for maldi ms analysis of oligosaccharides cross-linked membranes based on acrylated cyclodextrins and polyethylene glycol dimethacrylates for aromatic/aliphatic separation mycobacterium marinum lipooligosaccharides are unique caryophyllose-containing cell wall glycolipids that inhibit tumor necrosis factor-a secretion in macrophages structural analysis of an unusual bioactive n-acylated lipo-oligosaccharide los-iv in mycobacterium marinum exploiting the cross-metathesis reaction in the synthesis of pseudo-oligosaccharides isolation and characterization of rhamnolipid-producing bacterial strains from a biodiesel facility characterisation of haptoglobin in the blood plasma of harbour seals (phoca vitulina) n-glycan moieties of the crustacean egg yolk protein and their glycosylation sites synthesis of lewis x epitopes on plant n-glycans glycodynamers: dynamic polymers bearing oligosaccharides residues -generation, structure, physicochemical, component exchange, and lectin binding properties oligosaccharide analysis by graphitized carbon liquid chromatography-mass spectrometry -picoline-borane: a non-toxic reducing agent for oligosaccharide labeling by reductive amination glycan labeling strategies and their use in identification and quantification optimized workflow for preparation of apts-labeled n-glycans allowing high-throughput analysis of human plasma glycomes using -channel multiplexed cge-lif decreased levels of bisecting glcnac glycoforms of igg are associated with human longevity antibacterial activity of n-quaternary chitosan derivatives: synthesis, characterization and structure activity relationship (sar) investigations the presence of monoglucosylated n -glycan is important for the structural stability of storage protein, arylphorin molecular cloning and characterization of trehalose synthase from thermotoga maritima dsm : syntheses of trehalose disaccharide analogues and ndp-glucoses maldi tof-tof characterization of a light stabilizer polymer contaminant from polypropylene or polyethylene plastic test tubes quantification of soyasaponins i and bg in italian lentil seeds by solid-phase extraction (spe) and high-performance liquid chromatography-mass spectrometry (hplc-ms) nutrition analysis by nanoparticle-assisted laser desorption/ionisation mass spectrometry isolation and characterization of antibodies against three consecutive tn-antigen clusters from a phage library displaying human single-chain variable fragments o-glcnac modification of the extracellular domain of notch receptors systematic syntheses of influenza neuraminidase inhibitors: a series of carbosilane dendrimers uniformly functionalized with thioglycoside-type sialic acid moieties supramolecular assemblies of oligothiophene derivatives bearing b-cyclodextrin h- , , -triazol- -yl thiodigalactoside derivatives as high affinity galectin- inhibitors synthesis and structural characterization of sialic acid-glutamic acid hybrid foldamers as conformational surrogates of a- , -linked polysialic acid the remarkable stability of chimeric, sialic acid derived a/d-peptides in human blood plasma lack of a-xylosidase activity in arabidopsis alters xyloglucan composition and results in growth defects intact and top-down characterization of biomolecules and direct analysis using infrared matrix-assisted laser desorption electrospray ionization coupled to ft-icr mass spectrometry triumfettosterol id and triumfettosaponin, a new (fatty acyl)-substituted steroid and a triterpenoid 'dimer' bis(b-d-glucopyranosyl) ester from the leaves of wild triumfetta cordifolia a. rich. (tiliaceae) fluorescence turn-on sensing of lectins with mannose-substituted tetraphenylethenes based on aggregation-induced emission synthesis of tri-and pentasaccharide fragments corresponding to the o-antigen of shigella boydii type biorecognition of escherichia coli k adhesin for glycated porcine albumin synthesis of a single-molecule l-rhamnose-containing three-component vaccine and evaluation of antigenicity in the presence of anti-lrhamnose antibodies glycosylation of liver acute-phase proteins in pancreatic cancer and chronic pancreatitis support vector machine prediction of n-and o-glycosylation sites using whole sequence information and subcellular localization silkworm expression and sugar profiling of human immune cell surface receptor, kir dl high levels of e -pha-reactive oligosaccharides: potential as marker for cells with characteristics of hepatic progenitor cells highsensitivity analysis of naturally occurring sugar chains, using a novel fluorescent linker molecule di-tert-butylsilylenedirected a-selective synthesis of p-nitrophenyl t-antigen analogues analysis of the human cancer glycome identifies a novel group of tumor-associated n-acetylglucosamine glycan antigens the n-glycome of human embryonic stem cells inhibition of dc-signmediated hiv infection by a linear trimannoside mimic in a tetravalent presentation chemo-enzymatic synthesis of poly-n-acetyllactosamine (poly-lacnac) structures and their characterization for cgl -galectin-mediated binding of ecm glycoproteins to biomaterial surfaces aziridine ring opening as regio-and stereoselective access to o-glycosyl amino acids and their transformation into o-glycopeptide mimetics identification of a polyprenylphosphomannosyl synthase involved in the synthesis of mycobacterial mannosides reductive amination of the lysine n eamino group leads to a bivalent glyco-amino acid building block suited for spps utilizing staudinger ligation for the synthesis of glycoamino acid building blocks and other glycomimetics cysteinebased mannoside glycoclusters: synthetic routes and antiadhesive properties development of dictyostelium discoideum is associated with alteration of fucosylated n-glycan structures tlc/ hptlc with direct mass spectrometric detection: a review of the progress achieved in the last years o-glycosylation modulates proprotein convertase activation of angiopoietin-like protein . possible role of polypeptide galnac-transferase- in regulation of concentrations of plasma lipids inhibition binding studies of glycodendrimer/lectin interactions using surface plasmon resonance self-assembling carbohydrate-functionalized oligothiophenes chemical synthesis using enzymatically generated building units for construction of the human milk pentasaccharides sialyllacto-n-tetraose and sialyllacto-n-neotetraose epimer hydroxypropyl-substituted b-cyclodextrins: influence of degree of substitution on the thermodynamics of complexation with tauroconjugated and glycoconjugated bile salts site-specific analysis of nlinked oligosaccharides of recombinant lysosomal arylsulfatase a produced in different cell lines synthesis of a gpi anchor module suitable for protein post-translational modification a combined method for producing homogeneous glycoproteins with eukaryotic n-glycosylation structural basis of multivalent binding to wheat germ agglutinin mass spectrometric characterization of the surface-associated kda lipoprotein jlpa as a glycosylated antigen in strains of campylobacter jejuni probing the lactose gm carbohydrate-carbohydrate interaction with glycodendrimers identification of n-glycosylation in prolyl endoprotease from aspergillus niger and evaluation of the enzyme for its possible application in proteomics rapid and efficient glycoprotein identification through microwave-assisted enzymatic digestion assigning nglycosylation sites of glycoproteins using lc/msms in conjunction with endo-m/exoglycosidase mixture glycan analysis by mass spectrometry immunoglobulin g glycopeptide profiling by matrixassisted laser desorption ionization fourier transform ion cyclotron resonance mass spectrometry glycan bioengineering in immunogen design for tumor t antigen immunotargeting characterisation of cell wall polysaccharides from okra (abelmoschus esculentus (l.) moench) okra pectin contains an unusual substitution of its rhamnosyl residues with acetyl and alpha-linked galactosyl groups synthesis and characterization of hydroquinone glucoside using leuconostoc mesenteroides dextransucrase lipoprotein lipase and hydrofluoric acid deactivate both bacterial lipoproteins and lipoteichoic acids, but platelet-activating factor-acetylhydrolase degrades only lipoteichoic acids a functional carbohydrate chip platform for analysis of carbohydrate-protein interaction structural and immunological characterization of the n-glycans from the major yellow jacket allergen ves v : the n-glycan structures are needed for the human antibody recognition identification of glycosyltransferase family members as xylosyltransferases acting on o-glucosylated notch epidermal growth factor repeats developments and applications of mass microscopy characterization of a putative -deoxy-d-manno- -octulosonic acid (kdo) transferase gene from arabidopsis thaliana structural characterisation of the pectic polysaccharide rhamnogalacturonan ii using an acidic fingerprinting methodology maldi linear tof mass spectrometry of pegylated (glyco) proteins synthetic glycosides of ent-caurene series containing substituents with benzyl, phenoxyl, and uracyl fragments sno @poly(hema-co-st-co-vpba) core-shell nanoparticles designed for selectively enriching glycopeptides followed by maldi-ms analysis mass spectrometry based analysis of protein o-glycosylation synthesis and characterization of watersoluble conjugated glycopolymer for fluorescent sensing of concanavalin a laser desorption ionization mass spectrometry by using surface plasmon excitation on gold nanoparticle the n-linked oligosaccharide at fcgriiia asn- : an inhibitory element for high fcgriiia binding affinity to igg glycoforms lacking core fucosylation monitoring the hydrolysis and transglycosylation activity of aglucosidase from aspergillus niger by nuclear magnetic resonance spectroscopy and mass spectrometry n-linked glycan analysis of glycoproteins secreted from rice cell suspension cultures under sugar starvation characterization of receptor proteins using affinity cross-linking with biotinylated ligands proton sponge: a novel and versatile maldi matrix for the analysis of metabolites using mass spectrometry , -bis(dimethylamino)naphthalene: a novel superbasic matrix for matrix-assisted laser desorption/ionization timeof-flight mass spectrometric analysis of fatty acids acid-base-driven matrixassisted mass spectrometry for targeted metabolomics phenolic compounds in the leaves of populus ussuriensis and their antioxidant activities phenolic compounds from populus davidiana wood the structure of the carbohydrate backbone of the lipooligosaccharide from the halophilic bacterium arcobacter halophilus the structure of the carbohydrate backbone of the lipooligosaccharide from an alkaliphilic halomonas sp delineation of the roles of fadd , fadd and fadd in the biosynthesis of phthiocerol dimycocerosates and related compounds in mycobacterium tuberculosis biodegradable biocompatible xyloglucan films for various applications transparent xyloglucan-chitosan complex hydrogels for different applications physico chemical properties of aminated tamarind xyloglucan fractionation of sugar beet pulp by introducing ion-exchange groups the antimicrobial action of low-molar-mass chitosan, chitosan derivatives and chitooligosaccharides on bifidobacteria structural details and composition of trichomonas vaginalis lipophosphoglycan in relevance to the epithelial immune function determination of distinctive carbohydrate signatures obtained from the aeromonas hydrophila (chemotype ii) core oligosaccharide pinpointing the presence of the -o-phosphorylated -o-linked kdo reducing end group using electrospray ionization quadrupole orthogonal time-offlight mass spectrometry and tandem mass spectrometry biosynthetic origin of the galactosamine substituent of arabinogalactan in mycobacterium tuberculosis aftd, a novel essential arabinofuranosyltransferase from mycobacteria nmr structure determination of a segmentally labeled glycoprotein using in vitro glycosylation the role of peptides and proteins in melanoidin formation optimization of matrix conditions for the control of maldi in-source decay of permethylated glycans high-spatial resolution matrix-assisted laser desorption ionization imaging analysis of glucosylceramide in spleen sections from a mouse model of gaucher disease a simple cellulose column procedure for selective enrichment of glycopeptides and characterization by nano lc coupled with electron-transfer and high-energy collisional-dissociation tandem mass spectrometry structure of compositionally simple lipopolysaccharide from marine synechococcus synthesis of water-soluble phthalocyanines bearing four or eight d-galactose units discrimination of isobaric leucine and isoleucine residues and analysis of post-translational modifications in peptides by maldi in-source decay mass spectrometry combined with collisional cooling effect of gas pressure and gas type on the fragmentation of peptide and oligosaccharide ions generated in an elevated pressure uv/ir-maldi ion source coupled to an orthogonal time-of-flight mass spectrometer formation and structure elucidation of n-( , , -tri-o-acetyl-b-d-glucopyranosyl)-n -acetylthiourea disaccharide-modified liposomes and their in vitro intracellular uptake carbohydrate arrays: recent developments in fabrication and detection methods with applications discovery of the first series of small molecule h n entry inhibitors transglycosylation properties of maltodextrin glucosidase (malz) from escherichia coli and its application for synthesis of a nigerose-containing oligosaccharide fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays novel fluorescent glycan microarray strategy reveals ligands for galectins generation of a natural glycan microarray using -fluorenylmethyl chloroformate (fmoccl) as a cleavable fluorescent tag glycan microarray analysis of ptype lectins reveals distinct phosphomannose glycan recognition development and application of mass spectrometric methods for the analysis of progranulin n-glycosylation francisella tularensis blue-gray phase variation involves structural modifications of lipopolysaccharide oantigen, core and lipid a and affects intramacrophage survival and vaccine efficacy glycosylation of gp and gp envelope proteins of yellow head virus of penaeus monodon shrimp structural profiling of individual glycosphingolipids in a single thin-layer chromatogram by multiple sequential immunodetection matched with direct ir-maldi-o-tof mass spectrometry software utilities for the interpretation of mass spectrometric data of glycoconjugates: application to glycosphingolipids of human serum ionic liquids in analytical chemistry isolation of b-mannanase from cocos nucifera linn haustorium and its application in the depolymerization of b-( , )-linked d-mannans n-glycan trimming by glucosidase ii is essential for arabidopsis development immuno-maldi-tof ms: new perspectives for clinical applications of mass spectrometry a computational approach for deciphering the organization of glycosaminoglycans synthesis of galactofuranose-based acceptor substrates for the study of the carbohydrate polymerase glft analysis of n-glycans in embryonated chicken egg chorioallantoic and amniotic cells responsible for binding and adaptation of human and avian influenza viruses the mechanism of boron mobility in wheat and canola phloem removal of the outer kdo from helicobacter pylori lipopolysaccharide and its impact on the bacterial surface galectin- -regulated delivery of glycoproteins to the brush border membrane of enterocyte-like cells characterization of an immunodominant cancer-specific o-glycopeptide epitope in murine podoplanin (ots ) formation of homooligosaccharides using base-promoted glycosylation of unprotected glycosyl fluorides glycosyltransferase function in core -type protein o-glycosylation xyloglucan endotransglycosylases (xets) from germinating nasturtium (tropaeolum majus) seeds: isolation and characterization of the major form botulinum neurotoxin serotype d attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner the chain length of lignan macromolecule from flaxseed hulls is determined by the incorporation of coumaric acid glucosides and ferulic acid glucosides analysis of human plasma lipids and soybean lecithin by means of high-performance thin-layer chromatography and matrix-assisted laser desorption/ionization mass spectrometry chemoenzymatic synthesis of a glycolipid library and elucidation of the antigenic epitope for construction of a vaccine against lyme disease acylated cholesteryl galactosides are specific antigens of borrelia causing lyme disease and frequently induce antibodies in late stages of disease detailed n-glycan analysis of mannose receptor purified from murine spleen indicates tissue specific sialylation synthesis and acid catalysis of cellulose-derived carbon-based solid acid experimental studies on the ring opening polymerization of p-dioxanone using an al(o i pr) -monosaccharide initiator system glucuronyltransferase activity of kfic from escherichia coli strain k requires association of kfia. kfic and kfia are essential enzymes for production of k polysaccharide, n-acetylheparosan current imaging mass spectrometry for metabolite molecules imaging mass spectrometry for visualization of drug and endogenous metabolite distribution: toward in situ pharmacometabolomes imaging mass spectrometry protocols for mass microscopy structure and kinetic investigation of streptococcus pyogenes family gh a-mannosidase strategic glycan elution map for the production of human-type n-linked oligosaccharides: the case of hen egg yolk and white comprehensive analysis of n-linked oligosaccharides from eggs of the family phasianidae pectic oligosaccharide analysis by fluorophore-assisted carbohydrate electrophoresis ionic liquids in analytical chemistry dsa affinity glycoproteome of human liver tissue isolation of n-linked glycopeptides by hydrazine-functionalized magnetic particles further insight into the roles of the glycans attached to human blood protein c inhibitor synthesis of -petn-a-d-galpnac-( ! )-b-d-galp-( ! )-b-d-glcpnac-( ! )-b-d-galp-( ! )-b-d-glcp, a haemophilus influenzae lipopolysacharide structure, and biotin and protein conjugates thereof computationally and experimentally derived general rules for fragmentation of various glycosyl bonds in sodium adduct oligosaccharides molecular cloning of pigeon udp-galactose: b-d-galactoside a , -galactosyltransferase and udp-galactose: b-dgalactoside b , -galactosyltransferase, two novel enzymes catalyzing the formation of gal-a - gal-b - gal-b - glcnac sequence structural analysis of n-glycans from gull egg white glycoproteins and egg yolk igg synthesis of a glycosylphosphatidylinositol anchor bearing unsaturated lipid chains rapid release of n-linked glycans from glycoproteins by pressure-cycling technology laser desorption/ionization mass spectrometric analysis of small molecules using fullerene-derivatized silica as energy-absorbing material synthesis of three regioisomers of the pentasaccharide part of the skp glycoprotein of dictyostelium discoideum b-glycosidation of sterically hindered alcohols an aeromonas caviae genomic island is required for both oantigen lipopolysaccharide biosynthesis and flagellin glycosylation new triterpene glycoside from cyclamen adzharicum tubers mycobacterium leprae phenolglycolipid- expressed by engineered m. bovis bcg modulates early interaction with human phagocytes defects in flagellin glycosylation affect the virulence of pseudomonas syringae pv. tabaci mass spectrometric imaging of ginsenosides localization in panax ginseng root thermosensitive hydrogels composed of cyclodextrin pseudorotaxanes. role of [ ]pseudorotaxane in the gel formation hydrogels composed of organic amphiphiles and a-cyclodextrin: supramolecular networks of their pseudorotaxanes in aqueous media a plant class v chitinase from a cycad (cycas revoluta): biochemical characterization, cdna isolation, and posttranslational modification distinct features of matrix-assisted mm infrared laser desorption/ionization mass spectrometry in biomolecular analysis dissociation profile of protonated fucosyl glycopeptides and quantitation of fucosylation levels of glycoproteins by mass spectrometry loose-fit polyrotaxane composed of g-cyclodextrin and single poly(ethyelene glycol) chain: making room in g-cd cavity for additional inclusion complexation optically pure fullerodendron formed by diastereoselective dielse-alder reaction physiological and glycomic characterization of n-acetylglucosaminyltransferase-iva and -ivb double deficient mice synthesis and ring-opening polymerizations of novel s-glycooxazolines enrichment of amadori products derived from the nonenzymatic glycation of proteins using microscale boronate affinity chromatography alterations in receptor-binding properties of swine influenza viruses of the h subtype after isolation in embryonated chicken eggs tlc blot (far-eastern blot) and its applications epimeric and amino disaccharide analogs as probes of an a-( - )-mannosyltransferase involved in mycobacterial lipoarabinomannan biosynthesis solution conformation of c-linked antifreeze glycoprotein analogues and modulation of ice recrystallization application of proteomics in biomarker discovery: a primer for the clinician synthesis of the glycosaminoglycan-protein linkage tetraosyl peptide moieties of betaglycan, which serve as a hexosamine acceptor for enzymatic glycosyl transfer baca is indispensable for successful mesorhizobium-astragalus symbiosis synthesis of a sialic acid containing complex-type n-glycan on a solid support a combined p-azaelectrocyclization/ staudinger approach to protein and cell engineering: noninvasive tumor targeting by n-glycan-engineered lymphocytes noninvasive imaging of dendrimer-type n-glycan clusters: in vivo dynamics dependence on oligosaccharide structure synthesis of non-natural xyloglucans by polycondensation of , -dimethoxy- , , -triazin- -yl oligosaccharide monomers catalyzed by endo-b- , -glucanase novel dialkoxytriazine-type glycosyl donors for cellulase-catalysed lactosylation on-plate-selective enrichment of glycopeptides using boronic acid-modified gold nanoparticles for direct maldi-qit-tof ms analysis concanavalin a-immobilized magnetic nanoparticles for selective enrichment of glycoproteins and application to glycoproteomics in hepatocelluar carcinoma cell line biomaterials from sugars: ring-opening polymerization of a carbohydrate lactone synthesis of a monophosphoryl derivative of escherichia coli lipid a and its efficient coupling to a tumor-associated carbohydrate antigen identification of n-glycan serum markers associated with hepatocellular carcinoma from mass spectrometry data novel neogala-series glycosphingolipids with terminal mannose and glucose residues from hirsutella rhossiliensis, an aureobasidin aresistant ascomycete fungus functional characterization of tlmh in streptoalloteichus hindustanus e - atcc unveiling new insight into tallysomycin biosynthesis and affording a novel bleomycin analog the effect of temperature on the stability of compounds used as uv-maldi-ms matrix: , -dihydroxybenzoic acid, , , -trihydroxyacetophenone, a-cyano- -hydroxycinnamic acid, , -dimethoxy- -hydroxycinnamic acid, nor-harmane and harmane towards an integrated proteomic and glycomic approach to finding cancer biomarkers chemoenzymatic synthesis of multivalent neoglycoconjugates carrying the helminth glycan antigen ldnf fragment-based development of triazole-substituted o-galactosyl aldoximes with fragmentinduced affinity and selectivity for galectin- tempo oxidation of gelatinized potato starch results in acid resistant blocks of glucuronic acid moieties molecular sieves provoke multiple substitutions in the enzymatic synthesis of fructose oligosaccharide-lauryl esters synthesis of organic-soluble conjugated polyrotaxanes by polymerization of linked rotaxanes insulated molecular wire with highly conductive p-conjugated polymer core reaction of the antitumor antibiotic olivomycin i with aryl diazonium salts. synthesis, cytotoxic and antiretroviral potency of -aryldiazenyl- -odeglycosyl derivatives of olivomycin i highly site-selective stability increases by glycosylation of dihydrofolate reductase gold-catalyzed glycosidations: synthesis of , -anhydro saccharides site-specific glycoprofiling of n-linked glycopeptides using maldi-tof ms: strong correlation between signal strength and glycoform quantities purification and characterization of a new recombinant factor viii (n ) identification and quantification of n-linked oligosaccharides released from glycoproteins: an inter-laboratory study determination of nlinked sialyl-sugar chains in the lungs of domestic cats and dogs in thailand susceptible to the highly pathogenic avian influenza virus (h n ) cyclodextrin-based hyperbranched polymers: molecule design, synthesis and characterization glycoproteomics: past, present and future molecular basis for galactosylation of core fucose residues in invertebrates. identification of caenorhabditis elegans n-glycan core a , -fucoside b , -galactosyltransferase galt- as a member of a novel glycosyltransferase family glycoproteomic analysis of abnormal n-glycosylation on the kappa chain of cryocrystalglobulin in a patient of multiple myeloma solvolytic depolymerization of chondroitin and dermatan sulfates a chromatographic approach for elevating the antibody-dependent cellular cytotoxicity of antibody composites structural characterization and surface-active properties of a succinoyl trehalose lipid produced by rhodococcus sp. sd- chemo-enzymatic synthesis of glycosylated insulin using a glcnac tag acceptor specificity in the transglycosylation reaction using endo-m coxiella burnetii glycomics and proteomics-tools for linking structure to function social self-sorting: alternating supramolecular oligomer consisting of isomers humanization of recombinant glycoproteins expressed in insect cells inactivation of mycobacterium tuberculosis mannosyltransferase pimb reduces the cell wall lipoarabinomannan and lipomannan content and increases the rate of bacterial-induced human macrophage cell death the recognition motif of the glycoprotein-folding sensor enzyme udp-glc: glycoprotein glucosyltransferase enrichment method of sulfated glycopeptides by a sulfate emerging and ion exchange chromatography enzymatic solubilization of brewers' spent grain by combined action of carbohydrases and peptidases poly(n-vinylpyrrolidone) bearing covalently attached cyclodextrin via click-chemistry: synthesis, characterization, and complexation behavior with phenolphthalein chemical and enzymatic n-glycan release comparison for n-glycan profiling of monoclonal antibodies expressed in plants analysis of carbohydrates on proteins by offline normal-phase liquid chromatography maldi-tof/tof-ms/ ms carbohydrate structural analysis of wheat flour arabinogalactan protein the fine structure of neisseria meningitidis lipooligosaccharide from the m strain and three of its variants dihydrobenzoic acid modified nanoparticle as a maldi-tof ms matrix for soft ionization and structure determination of small molecules with diverse structures photoinduced isomerization of allyl alcohols to carbonyl compounds using dendrimer disulfide as catalyst miniaturizing sample spots for matrix-assisted laser desorption/ionization mass spectrometry glycomics profiling of heparan sulfate structure and activity alkali-hydroxide-doped matrices for structural characterization of neutral underivatized oligosaccharides by maldi time-of-flight mass spectrometry phosphocholinecontaining glycosyl inositol-phosphoceramides from trichoderma viride induce defense responses in cultured rice cells targeted serum glycoproteomics for the discovery of lung cancer-associated glycosylation disorders using lectin-coupled pro-teinchip arrays chemoenzymatic synthesis of glycosylated glucagon-like peptide : effect of glycosylation on proteolytic resistance and in vivo blood glucoselowering activity daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis solid-phase synthesis of glycopeptide carrying a tetra-n-acetyllactosamine-containing core decasaccharide glycosylation specific for adhesion molecules in epidermis and its receptor revealed by glycoform-focused reverse genomics isolation and identification of arabinose mycolates of cell wall skeleton (cws) derived from mycobacterium bovis bcg tokyo (smp- ) synthesis, cell-surface binding, and cellular uptake of fluorescently labeled glucose-dna conjugates with different carbohydrate presentation use of a novel ionic liquid matrix for maldi-ms analysis of glycopeptides and glycans out of total tryptic digests efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline milk oligosaccharides structural and functional characterization of a putative polysaccharide deacetylase of the human parasite encephalitozoon cuniculi glycosyl conjugates of biotinylated diaminopyridine applied for study of carbohydrate-to-carbohydrate interaction an oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides deciphering carbohydrate structures by ims-ms. applications to biological features related to carbohydrate chemistry and biology software platform for high-throughput glycomics preparation and properties of polyurethanes based on castor oil chemically modified with yucca starch glycoside synthesis and characterization of neurostatin-related compounds with high inhibitory activity of glioma growth o-fucosylation of an antibody light chain: characterization of a modification occurring on an igg molecule asparagine-linked oligosaccharides present on a non-consensus amino acid sequence in the c h domain of human antibodies biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (tafi) glycosylation pattern of mature dimeric leukocyte and recombinant monomeric myeloperoxidase. glycosylation is required for optimal enzymatic activity n-linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human igg immunoglobulin g galactosylation and sialylation are associated with pregnancyinduced improvement of rheumatoid arthritis and the postpartum flare: results from a large prospective cohort study structural and functional analysis of glycosphingolipids of schistosoma mansoni elucidation of molecular diversity and body distribution of saponins in the sea cucumber holothuria forskali (echinodermata) by mass spectrometry qualitative and quantitative saponin contents in five sea cucumbers from the indian ocean localization of secondary metabolites in marine invertebrates: contribution of maldi msi for the study of saponins in cuvierian tubules of h. forskali elevated sulfatide levels in neurons cause lethal audiogenic seizures in mice rhizobium leguminosarum biovar viciae , deficient in -hydroxyoctacosanoate-modified lipopolysaccharide, is impaired in desiccation tolerance, biofilm formation and motility glycome profiling using modern glycomics technology: technical aspects and applications symbol nomenclature for glycan representation synthesis of cyclic b-glucan using laminarinase a glycosynthase mutant from the basidiomycete phanerochaete chrysosporium purification and nglycosylation analysis of melanoma antigen dopachrome tautomerase rapana venosa hemocyanin with antiviral activity stable isotope-enhanced two-and threedimensional diffusion ordered c nmr spectroscopy (sie-dosy c nmr) modern maldi time-of-flight mass spectrometry orthogonal activation of propargyl and n-pentenyl glycosides and , -orthoesters laser desorption-ionization of lipid transfers: tissue mass spectrometry imaging without maldi matrix characterization of branched polysaccharides using multiple-detection size separation techniques multigram synthesis of a hyaluronic acid subunit and synthesis of fully protected oligomers finding new posttranslational modifications in salivary proline-rich proteins enzymatic glycosylations on arrays rapid assembly of oligosaccharides: a highly convergent strategy for the assembly of a glycosylated amino acid derived from psgl- databases and informatics for glycobiology and glycomics production of nonfucosylated antibodies by co-expression of heterologous gdp- -deoxy-d-lyxo- -hexulose reductase characterization of the acidic n-linked glycans of the zona pellucida of prepuberal pigs by a mass spectrometric approach pectin, a versatile polysaccharide present in plant cell walls expression of talaromyces emersonii cellobiohydrolase cel a in saccharomyces cerevisiae and rational mutagenesis to improve its thermostability and activity structural characterization of flavonoid glycosides by multi-stage mass spectrometry the absence of an identifiable single catalytic base residue in thermobifida fusca exocellulase cel b quantitation of saccharide compositions of o-glycans by mass spectrometry of glycopeptides and its application to rheumatoid arthritis comparison of methods for profiling o-glycosylation. human proteome organisation human disease glycomics/proteome initiative multi-institutional study of iga fluorinated glycosyl amino acids for mucin-like glycopeptide antigen analogues a nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant alaskan beetle upis ceramboides glycans on influenza hemagglutinin affect receptor binding and immune response synthesis of a mannose-capped disaccharide with a thiol terminus steroidal saponins from the rhizomes of polygonatum odoratum oxidative stress-induced peptidoglycan deacetylase in helicobacter pylori convenient synthesis of an n-glycan octasaccharide of the bisecting type matrix-assisted laser desorption/ionization mass spectrometry (maldi-ms). application in carbohydrate analysis molecular aggregation behavior of perylene-bridged bis(b-cyclodextrin) and its electronic interactions upon selective binding with aromatic guests functional characterization of tlmk unveiling unstable carbinolamide intermediates in the tallysomycin biosynthetic pathway silver-coated gold nanoparticles as concentrating probes and matrices for surface-assisted laser desorption/ionization mass spectrometric analysis of aminoglycosides conversion of squid pen by pseudomonas aeruginosa k fermentation for the production of nacetyl chitooligosaccharides and biofertilizers n-terminal deletion of peptide: n-glycanase results in enhanced deglycosylation activity a new chalcone glycoside, a new tetrahydrofuranoid lignan, and antioxidative constituents from the stems and leaves of viburnum propinquum cosmc is an essential chaperone for correct protein o-glycosylation role of a cytoplasmic dual-function glycosyltransferase in o regulation of development in dictyostelium crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (roystonea regia) two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in listeria monocytogenes n-glycan analysis of recombinant l-selectin reveals sulfated galnac and galnac-galnac motifs glycosylation of the phosphate binding protein, psts, in streptomyces coelicolor by a pathway that resembles protein o-mannosylation in eukaryotes immobilization of enzyme on detonation nanodiamond for highly efficient proteolysis comparative glycoproteomics: approaches and applications covalent modification of chitin with silk-derivatives acts as an amphiphilic self-organizing template in nacre biomineralisation expression of helix pomatia lectin binding glycoproteins in women with breast cancer in relationship to their blood group phenotypes analysis of site-specific glycosylation of renal and hepatic g-glutamyl transpeptidase from normal human tissue release and characterization of single side chains of white cabbage pectin and their complement-fixing activity characterization of wbpb, wbpe, and wbpd and reconstitution of a pathway for the biosynthesis of udp- , -diacetamido- , -dideoxy-d-mannuronic acid in pseudomonas aeruginosa maldi-tof ms and ce-lif fingerprinting of plant cell wall polysaccharide digests as a screening tool for arabidopsis cell wall mutants branched arabino-oligosaccharides isolated from sugar beet arabinan derivatization of sialic acids for stabilization in matrix-assisted laser desorption/ionization mass spectrometry and concomitant differentiation of a( - ) and a( - ) isomers glycomic characterization of prostate-specific antigen and prostatic acid phosphatase in prostate cancer and benign disease seminal plasma fluids synthesis of bifunctional peptide derivatives based on a b-cyclodextrin core with drug delivery potential glycoprotein production for structure analysis with stable, glycosylation mutant cho cell lines established by fluorescence-activated cell sorting triple recognition of b-dna by a neomycin-hoechst -pyrene conjugate introductory glycosylation analysis using sds-page and peptide mass fingerprinting synthesis and cytotoxic activity of g pamam-nh dendrimer-modified digoxin and proscillaridin a conjugates in breast cancer cells quantitative site-specific analysis of protein glycosylation by lc-ms using different glycopeptide-enrichment strategies profiling of n-glycosylation gene expression in cho cell fed-batch cultures an investigation of intracellular glycosylation activities in cho cells: effects of nucleotide sugar precursor feeding vitro bacterial polysaccharide biosynthesis: defining the functions of wzy and wzz ultrasound ionization of biomolecules new development of glycan arrays gold nanoparticles as assisted matrices for the detection of biomolecules in a high-salt solution through laser desorption/ionization mass spectrometry development of an annotated library of neutral human milk oligosaccharides synthesis of monomeric and dimeric repeating units of the zwitterionic type capsular polysaccharide from streptococcus pneumoniae chemoenzymatic synthesis of glycosylphosphatidylinositol-anchored glycopeptides synthesis of mangiferin, isomangiferin, and homomangiferin sortase a-catalyzed transpeptidation of glycosylphosphatidylinositol derivatives for chemoenzymatic synthesis of gpi-anchored proteins synthesis of talosin a and b, two bioactive isoflavonoid glycosides direct labelling of peptides with -[ f]fluoro- -deoxy-d-glucose ([ f]fdg) structural glycomics using hydrophilic interaction chromatography (hilic) with mass spectrometry hexose rearrangements upon fragmentation of n-glycopeptides and reductively aminated nglycans ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry glycan reductive isotope labeling for quantitative glycomics core -derived o-glycans are essential for intestinal mucus barrier function rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies preparation of chitooligosaccharides by the enzymatic hydrolysis of chitosan potent angiogenic inhibition effects of deacetylated chitohexaose separated from chitooligosaccharides and its mechanism of action in vitro synthesis of well-defined -arm and -arm poly(nisopropylacrylamide) star polymers with b-cyclodextrin cores via click chemistry and their thermal phase transition behavior in aqueous solution chitooligosaccharides protect human embryonic hepatocytes against oxidative stress nduced by hydrogen peroxide on-plate enrichment of glycopeptides by using boronic acid functionalized gold-coated si wafer highly specific enrichment of glycopeptides using boronic acid-functionalized mesoporous silica n-glycosylation profiling of turtle egg yolk: expression of galabiose structure development and application of high performance liquid chromatography map of glucuronyl n-glycans comparative studies on the structural features of o-glycans between leukemia and epithelial cell lines hyphenated technique for releasing and maldi ms analysis of oglycans in mucin-type glycoprotein samples preparation and characterization of phospha sugar analogues, , -dibromo- -methyl- -phenylphospholane -oxide derivatives, as novel anticancer agents synthesis of b-d-fructopyranosyl-( ! )-d-glucopyranose from d-glucose and d-fructose by a thermal treatment switching from altro-a-cyclodextrin dimer to pseudo[ ]rotaxane dimer through tumbling a molecular reel: shuttling of a rotor by tumbling of a macrocycle selective enrichment of glycopeptides/phosphopeptides using porous titania microspheres synthesis of di-and tetrasaccharide containing -deoxytalose from the o-antigenic polysaccharide of b. pseudomallei strain b mechanism of mild acid hydrolysis of galactan polysaccharides with highly ordered disaccharide repeats leading to a complete series of exclusively oddnumbered oligosaccharides heterogeneous components of chitosans glucopyranoside-incorporated n-heterocyclic carbene complexes of silver(i) and palladium(ii): efficient water-soluble suzuki-miyaura coupling palladium(ii) catalysts detection of small neutral carbohydrates using various supporting materials in laser desorption/onization mass spectrometry synthesis of a novel class of glycocluster with a cyclic a-( ! )-octaglucoside as a scaffold and their binding abilities to concanavalin a isolation and structural characterization of a polysaccharide fcap from the fruit of cornus officinalis expression, glycoform characterization, and antibody-binding of hiv- v glycopeptide domain fused with human igg -fc synthesis of kaempferol -o-( , -di-o-e-p-coumaroyl)-b-d-glucopyranoside, efficient glycosylation of flavonol -oh with glycosyl odlkynylbenzoates as donors total synthesis and structural revision of tmgchitotriomycin, a specific inhibitor of insect and fungal b-n-acetylglucosaminidases chemoselective glycosylation of carboxylic acid with glycosyl ortho-hexynylbenzoates as donors homogeneous synthesis of grgdy grafted chitosan on hydroxyl groups by photochemical reaction for improved cell adhesion facile synthesis of -mercaptophenylboronic acid functionalized gold nanoparticles for selective enrichment of glycopeptides new triterpenoid saponins from the roots of gypsophila paniculata l the crystal structure of a xyloglucan-specific endo-b- , -glucanase from geotrichum sp. m xyloglucanase reveals a key amino acid residue for substrate specificity imidazolium cation supported solution-phase assembly of homolinear a( ! )-linked octamannoside: an efficient alternate approach for oligosaccharide synthesis growth phasedependent expression of proteins with decreased plant-specific nglycans and immunogenicity in tobacco by cells oxidation of the primary hydroxyl group of galactose of galactaosyl ceramide analogue by chemical method-precursors for the synthesis of labeled conjugates developmental changes in glycolipids and synchronized expression of nutrient transporters in the mouse small intestine differences in n-glycan structures found on recombinant iga and iga produced in murine myeloma and cho cell lines extraction of glycosaminoglycan from sea hare, aplysia kurodai, and its functional properties . structural properties of purified glycosaminoglycan synthesis of dopamine and l-dopaa-glycosides by reaction with cyclomaltohexaose catalyzed by cyclomaltodextrin glucanyltransferase n-glycosylation status of b-haptoglobin in sera of patients with prostate cancer vs. benign prostate diseases unexpected tolerance of glycosylation by udp-galnac:polypeptide a-n-acetylgalactosaminyltransferase revealed by electron capture dissociation mass spectrometry: carbohydrate as potential protective groups modification-specific proteomics in plant biology effect and limitation of excess ammonium on the release of o-glycans in reducing forms from glycoproteins under mild alkaline conditions for glycomic and functional analysis hydrophilic interaction chromatography based enrichment of glycopeptides by using click maltose: a matrix with high selectivity and glycosylation heterogeneity coverage construction and transmembrane dissociation behavior of supramolecular assembly of quinolinocyclodextrin with porphyrin metabolic labeling of glycoconjugates with photocrosslinking sugars identification of blood group a/ a-leb/y and b/b-leb/y active glycotopes co-expressed on the oglycans isolated from two distinct human ovarian cyst fluids a supramolecular bifunctional artificial enzyme with superoxide dismutase and glutathione peroxidase activities enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans synthesis and c nmr spectroscopy of model compounds for the microstructure analysis of poly(vinyl glycoside)s production of anti-carbohydrate antibodies by phage display technologies. potential impairment of cell growth as a result of endogenous expression precise structure of acidic polysaccharide present in salvia hydrogels n-glycosylation in archaea: on the coordinated actions of haloferax volcanii aglf and aglm on-line separations combined with ms for analysis of glycosaminoglycans mass spectrometry and glycomics matrix-assisted laser desorption/ionization imaging mass spectrometry effect of enzyme processivity on the efficacy of a competitive chitinase inhibitor catalytic properties of endo- , -b-d-glucanase from the vietnamese edible mussel perna viridis cerebrocostomandibular-like syndrome and a mutation in the conserved oligomeric golgi complex, subunit a new mutation in cog extends the spectrum of cog subunit deficiencies clickable lipids: azido and alkynyl fatty acids and triacylglycerols expression, purification, and characterization of recombinant human transferrin from rice structural characterization and anti-fatigue activity of polysaccharides from the roots of morinda officinalis a modified synthesis and serological evaluation of neoglycoproteins containing the natural disaccharide of pgl-i from mycobacterium leprae synthesis of novel aminoglycosides via allylic azide rearrangement for investigating the significance of -amino group development of a plate-based scintillation proximity assay for the mycobacterial aftb enzyme involved in cell wall arabinan biosynthesis transient expression and purification of chimeric heavy chain antibodies extracting both peptide sequence and glycan structural information by nm photodissociation of n-linked glycopeptides synthesis of neu ac-gal-functionalized gold glyconanoparticles total synthesis of apigenin- -yl- -o-(pcoumaroyl)-b-d-glucopyranoside recent developments of nanoparticle-based enrichment methods for mass spectrometric analysis in proteomics specific enrichment methods for glycoproteome research boronic acid functionalized core-satellite composite nanoparticles for advanced enrichment of glycopeptides and glycoproteins condensed tannins from mangrove species kandelia candel and rhizophora mangle and their antioxidant activity lew , encoding a putative a- , -mannosyltransferase (alg ) in n-linked glycoprotein, plays vital roles in cell-wall biosynthesis and the abiotic stress response in arabidopsis thaliana a perspective on the maillard reaction and the analysis of protein glycation by mass spectrometry: probing the pathogenesis of chronic disease a novel strategy for maldi-tof ms analysis of small molecules steroidal saponins from the rhizomes of paris delavayi acute kidney injury induced by proteinoverload nephropathy down-regulates gene expression of hepatic cerebroside sulfotransferase in mice, resulting in reduction of liver and serum sulfatides prediction of collision-induced dissociation spectra of common n-glycopeptides for glycoform identification mass spectrometry for structural characterization of therapeutic antibodies analysis of protein glycosylation and phosphorylation using liquid phase separation, protein microarray technology, and mass spectrometry preparation and characterization of galacto-mannan-oligosaccharides hydrolyzed from guar gum by bmannanase inclusion behavior of bcyclodextrin with bipyridine molecules: factors governing host-guest inclusion geometries combination of b-elimination and liquid chromatography/quadrupole time-of-flight mass spectrometry for the determination of o-glycosylation sites study of matrix additives for sensitive analysis of lipid a by matrix-assisted laser desorption ionization mass spectrometry microwaveassisted sample preparation for rapid and sensitive analysis of h. pylori lipid a applicable to a single colony bc , a duf -containing and golgi-located type ii membrane protein, is required for cell-wall biosynthesis in rice synthesis of ionic liquids functionalized b-cyclodextrin-bonded chiral stationary phases and their applications in high-performance liquid chromatography rapid identification of gallotannins from chinese galls by matrix-assisted laser desorption/ ionization time-of-flight quadrupole ion trap mass spectrometry engineered nanoparticle surfaces for improved mass spectrometric analysis convenient synthesis of sulfated oligofucosides synthesis of a mannose hexasaccharide related to the cell wall mannan of candida dubliniensis and trychophyton mentagrophytes synthesis of the -deoxytalose-containing tri-and hexasaccharides of the o-antigen polysaccharide from mesorhizobium huakuii ifo t highly efficient removal of allyloxycarbonyl (alloc) function provides a practical orthogonal protective strategy for carbohydrates dual reversible self-assembly of pnipam-based amphiphiles formed by inclusion complexation cytotoxic triterpenoid saponins acetylated with monoterpenoid acid from albizia julibrissin key: cord- - rqnu bu authors: nan title: th international symposium on intensive care and emergency medicine: brussels, belgium. - march date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: rqnu bu nan the relationship between systemic glycocalyx degradation markers and regional glycocalyx thickness in non-septic critically ill patients is unclear. conjunctival sidestream dark fieldimaging for the purpose of glycocalyx thickness estimation has never been performed. we aimed to investigate whether changes in glycocalyx thickness in conjunctival and sublingual mucosa are associated with global glycocalyx shedding markers. methods: in this single-centre prospective observational study, using techniques for direct in-vivo observation of the microcirculation, we performed a single measurement of glycocalyx thickness in both ocular conjunctiva and sublingual mucosa in mixed cardio surgical (n= ) and neurocritical patients (n= ) and compared these data with age-matched healthy controls (n= ). in addition we measured systemic syndecan- levels results: in the sublingual and conjunctival region we observed a significant increase of the perfused boundary region (pbr) in both neuro critical and cardiac surgical icu patients, compared to controls ( . ], p< , ). we detected a weak correlation between syndecan- and sublingual pbr(r= . , p= . ) but no correlations between global glycocalyx damage markers and conjuctival glycocalyx thickness. conclusions: conjunctival glycocalyx thickness evaluation using sdf videomicroscopy is suitable and is impaired in non-septic icu patients but only measurements in sublingual mucosa are correlating with systemic glycocalyx shedding markers. global glycocalyx damage is more severe in cardiac comparing to neuro critical patients. introduction: endothelial dysfunction plays a major role in the sepsis related organ dysfunction, and is featured by vascular leakage. amp-activated protein kinase (ampk) is known to regulate actin cytoskeleton organization and interendothelial junctions (iejs), contributing to endothelial barrier integrity. we have already demonstrated its role in defence against sepsis induced hyperpermeability [ ] , but the underlying mechanisms remain unknown. this project aims to identify molecular targets involved in the beneficial action of ampk against endothelial barrier dysfunction. methods: experiments have been performed in human microvascular dermal endothelial cells. α ampk activity has been modulated via the use of a specific sirna or treatment by two pharmacological ampk activators (aicar, ). we have investigated the effect of this modulation on the expression/phosphorylation of connexin (cx ) and heat shock protein (hsp ), two proteins playing a key role in maintenance of iejs and actin dynamics respectively. results: we show that α ampk is required to sustain the level of cx expression as it was drastically reduced in cells transfected with a sirna targeting specifically α ampk. regarding hsp , its expression level was not affected by α ampk deletion. however, both ampk activators increased its phosphorylation on ser , in a α ampkdependent manner, while they had no effect on cx . our results also reveal that hsp phosphorylation concurred with the appearance of actin stress fibers at the periphery of cells, suggesting a beneficial role for phsp as well as f-actin stress fibers in vascular barrier function through reinforcing the endothelial tethering. conclusions: our work identifies the regulation of cx expression and hsp phosphorylation as potential protective responses underlying the beneficial action of ampk against endothelial barrier dysfunction. ampk could consequently represent a new therapeutic target during sepsis. introduction: sepsis induced cardiomyopathy (sic) is a serious condition during sepsis with a mortality rate up to % ( ) . sic is clinically manifested with left ventricle impaired contractility ( ) . melusin is a muscle-specific protein involved in sustaining cardiomyocyte survival thorough the activation of akt signaling pathways ( ) . pi k-akt signaling pathway plays a pivotal role in regulating calcium channel activity ( ) . we hypothesized that melusin overexpression could exert a protective effect on cardiac function during septic injury. methods: animals were treated with an intraperitoneal injection of lipopolysaccharide (lps) at mg/kg. sv strain knockout mice (ko) for melusin gene and fvb strain with cardiac-specific overexpression (ov) of melusin were compared. each group was studied together with a control group (wt). hemocardiac parameters were studied at hour and hours through echocardiography. another cohort of animals was sacrificed hours after mg/kg lps treatment and cardiac tissues and blood sample were harvested for wb analysis to quantify the expression of akt, p-akt and cacna c and elisa analysis for troponin levels. results: sv wt, ko melusin and fvb wt mice groups, fractional shortening (fs) was significantly impaired after lps challenge and was associated with compensatory tachycardia (fig. ) . fvb ov mice group didn't show decrease in fs. consistent with the increased akt phosphorylation observed in ov mice, the expression of cacna c was also significantly higher both at basal levels and after lps treatment in ov mice compared to wt mice (fig. ) . troponin levels didn't differ between mice groups after lps treatment conclusion: melusin has protective role in lps induced cardiomyopathy, likely through akt phosphorylation controlling the cacna c protein density. introduction: liver dysfunction is frequent in sepsis, but its pathophysiology remains incompletely understood. since altered liver function has also been described in icu patients without sepsis [ , ] , the influence of sepsis may be overestimated. we hypothesized that sedation and prolonged mechanical ventilation after abdominal surgery is associated with impaired liver function independent of sepsis. methods: sedated and mechanically ventilated pigs underwent abdominal surgery for regional hemodynamic monitoring and were subsequently randomized to fecal peritonitis and controls, respectively (n= , each), followed by h observation. indocyanine green (icg) retention rate minutes after injection of . mg/kg icg (icg r ) was determined at baseline, and , and h after sepsis induction (si), and at the same time points in controls. concurrent with icg r , plasma volume, total hepatic perfusion (ultrasound transit time), and bilirubin and liver enzymes were measured. anova for non-parametric repeated measurements was performed in both groups separately. results: icg r increased over time without significant differences between groups (table ). there was a parallel increase in bilirubin in septic but not control animals. the other measured parameters were similar in both groups at the end of the experiment. conclusion: liver function was impaired under sedation and prolonged mechanical ventilation after abdominal surgery, even in animals without sepsis. the underlying reasons should be further explored. introduction: previous work has shown the cytoprotective properties of antithrombin-affinity depleted heparin (aadh), by neutralization of cytotoxic extracellular histones [ ] , major mediators of death in sepsis [ , ] . aadh was produced from clinical grade heparin, resulting in preparations that have lost > , % of their anticoagulant activity. to gain insight into the mechanisms and the basic pharmacological aspects of aadh protective properties, we performed a systematic analysis of how aadh is tolerated in mice and ascertained its effects in three different in vivo models of inflammation and infection. methods: dose ranging studies, short term and medium term, were performed in c bl/ mice. the effects of i.v. administration of extracellular histones in the presence or absence of aadh were assessed in mice. we further analysed the effect of aadh in models of concanavalin a-and mrsa-mediated lethality. in all studies we assessed clinical signs, lab parameters and histology. results: aadh was well tolerated in both short term and intermediate term (till days) experiments in mice, in the absence of any signs of tissue bleeding. aadh was able to revert the cytotoxic properties of i.v. administered histones. in a concanavalin a mediated model of sterile inflammation, we confirmed that aadh has protective properties that counteract the cytotoxic effects of extracellular histones. in an in vivo lethal mrsa model, for the first time, aadh was shown to induce a survivalbenefit. conclusions: we conclude that aadh contributes to the overall increased survival by means of neutralization of extracellular histones and represents a promising product for further development into a drug for the treatment of inflammatory diseases and sepsis. introduction: urokinase (uk) and tissue plasminogen activator (tpa) mediate thrombolytic actions by activating endogenous plasminogen. thrombomodulin (tm) complexes with thrombin to activate protein c and thrombin activatable fibrinolysis inhibitor (tafi). activated protein c (apc) modulates coagulation by digesting factors v and viii and activates fibrinolysis by decreasing pai- functionality. methods: the purpose of this study is to compare the effects of rtm and apc on urokinase and tpa mediated thrombolysis utilizing thromboelastography. results: native whole blood was activated using a diluted intrinsic activator (aptt reagent, triniclot). the modulation of thrombolysis by tpa and uk (abbott, chicago, usa) was studied by supplementing these agents to whole blood and monitoring teg profiles. apc (haematologic technologies, vt, usa) and rtm (asahi kasai pharma, tokyo, japan) were supplemented to the activated blood at . - . ug/ml. the modulation of tpa and uk induced thrombolysis by apc and rtm was studied in terms of thromboelastograph patterns. the effect of both apc and rtm on plasma based systems supplemented with tpa was also investigated. patients treated with antibiotic therapy were eligible for inclusion. the plausibility of infection (definite, probable, possible, none) was determined based on the centers for diseases control (cdc) criteria. patients with sepsis (definite/probable/possible infection and a sofa score increase of >= ) were screened for death within days and secondary infections h to days after icu admission, using the cdc criteria. hla-dra and cd mrna expressions were determined by reverse transcription quantitative pcr. results: among icu admissions, a blood sample for rna analysis was collected in cases. two hundred fifty-seven patients met the inclusion criteria and provided written informed consent. sepsis was noted in patients. the sepsis patients experienced death in cases ( %), secondary infection in cases ( %), and death and/or secondary infection in cases ( %). table shows the results of hla-dra and cd expression related to death and secondary infections. conclusions: the mrna expression of hla-dra on icu admission was significantly decreased in patients with sepsis who died or contracted secondary infections within days. cd expression was not significantly decreased in patients with negative outcome. introduction: acid-base disturbances are common in patients with infection admitted to the intensive care unit (icu). more attention is given to hyperlactatemia in this patient population as a prognostic factor, although other acid-base disturbances may also have an impact on patient outcomes. our objective is to describe the acid-base profile of this patient population and determine the association between different acid-base abnormalities and icu mortality. methods: retrospective cohort of patients admitted with infection to an intensive care unit. patients were stratified according to ph (< . ; . - . ; > . ) and, then, according to the standard base excess (sbe) (< - ; - -+ ; > + ). in each of these strata and the whole population, the proportions of acid-base disturbances were quantified during the first hours of icu admission. to assess the association between acid-base disturbances and outcome, a logistic regression model was fit, adjusting for age, sex and saps score. results: patients were analysed. ( %) patients were acidemic and ( %) presented with a normal ph. metabolic acidosis (as assessed by sbe) was observed in all subgroups, regardless of ph levels (ph < ). lactic acidosis was observed in % of the whole population; sig (strong ion gap) acidosis, in %; sid (hyperchloremic) acidosis, in %; metabolic alkalosis, in %; and respiratory acidosis, in % of the patients. introduction: sepsis-induced brain dysfunction has been neglected until recently due to the absence of specific clinical or biological markers. there is increasing evidence that sepsis may pose substantial risks for long term cognitive impairment. methods: to find out clinical and inflammatory factors associated with acute sepsis-induced brain dysfunction (sibd) serum levels of cytokines, complement breakdown products and neurodegeneration markers were measured by elisa in sera of sibd patients and healthy controls. association between these biological markers and cognitive test results was investigated. results: sibd patients showed significantly increased il- , il- , il- and c d levels and decreased tnf-α, il- , c a and ic b levels than healthy controls. no significant alteration was observed in neuronal loss and neurodegeneration marker (neuron specific enolase (nse), amyloid β, tau) levels. increased il- β, il- , il- , il- , tnf-α and decreased c d, c a and ic b levels were associated with septic shock, coma and mortality. transient mild cognitive impairment was observed in of patients who underwent neuropsychological assessment. cognitive dysfunction and neuronal loss were associated with increased duration of septic shock and delirium but not baseline serum levels of inflammation and neurodegeneration markers. conclusions: increased cytokine levels, decreased complement activity and increased neuronal loss are indicators of poor prognosis and adverse events in sibd. cognitive dysfunction and neuronal destruction in sibd do not seem to be associated with systemic inflammation factors and alzheimer disease-type neurodegeneration but rather with increased duration of neuronal dysfunction and enhanced exposure of the brain to sepsisinducing pathogens. introduction: high levels of some aromatic microbial metabolites (amm) in serum are related to the severity and mortality of critically ill patients [ ] . several studies have discussed the imbalance and loss of the diversity of gut microbiota but there are practically no data on the gut microbial metabolites in critical conditions, only a little -in healthy people [ , ] . the aim of this work is to analyze the connection between serum and fecal levels of amm in icu patients. methods: simultaneously serum and fecal samples (sfs) from icu patients with nosocomial pneumonia (group i), sfs from icu neurorehabilitation patients (group ii) and sfs from healthy people were taken for gc/ms analyses. the following amm were measured: phenylpropionic (phpa), phenyllactic (phla), p-hydroxybenzoic (p-hba), p-hydroxyphenyllactic (p-hphla), p-hydroxyphenylacetic (hphaa), p-hydroxyphenylpropionic (p-hphpa) and homovanillic (hva) acids. data were presented as medians with interquartile range (ir, - %) using statistica . results: the sum of the level of most relevant metabolites ( amm) -phla, p-hphla, p-hphaa, and hva -in serum samples from group i and group ii were equal to . ( . - . ) μ m and . ( . - . ) μ m, respectively, and were higher than in healthy people - . ( . - . ) μ m (p< . ). we suppose the presence of the correlation of amm profile in blood and intestine. particularly, sfs of healthy people are characterized by the prevalence of phpa; amm are not detected in feces of non-survivors but only hva dominates in their serum in the absence of other (fig. ) . conclusions: the amm profiles in gut and serum are interrelated; amm in serum probably reflect the violation and loss of biodiversity of the gut microbiota in critically ill patients. introduction: since nitrogen oxide (no) is an essential component of the immune system, the dynamics of plasma no concentration was studied in order to predict the development of sepsis [ , ] . methods: with the permission of the ethics committee included the full-term newborns with respiratory diseases on a ventilator, retrospectively divided into two groups (i, n= -sepsis - days; ii, n= without sepsis), at , - , days was studied by elisa the plasma concentration of no, nos- , nos- , adma (multilabel coulter victor- , finland). to select points "cut-off" used the method of roc-lines. results: the statistical power of the study was . % (α< . ). at admission in patients of groups i and ii decrease the concentration of no and increased adma in plasma (p< . ) relative to healthy newborns. after - days, relatively in patients of groups introduction: sepsis-associated disseminated intravascular coagulation (sac) is associated with decreased platelet counts and formation. the widespread activation of platelets contribute to vascular occlusions, fibrin deposition, multi-organ dysfunction, contributing to a two-fold increase in mortality. the purpose was to measure markers of platelet function in the plasma of patients with clinically established sac and to determine association to disease severity and outcome. methods: plasma samples from adult intensive care unit (icu) patients with sepsis and suspected sac were collected at baseline and on days and . dic scores were calculated using platelet count, d-dimer, inr, and fibrinogen. patients were categorized as having no dic, non-overt dic, or overt dic. plasma levels of cd l, von willebrand factor (vwf), platelet factor- (pf- ), and microparticles (mp) were quantified using commercially available elisa methods. results: markers of platelet activation were significantly elevated in patients with sepsis alone and with suspected dic compared to normal healthy individuals on icu day (p< . ). levels of platelet-associated biomarkers were compared between survivors and non-survivors. pf- was significantly decreased in non-survivors compared to survivors (p = . ). patients were stratified based on platelet count and levels of markers were compared between groups. cd l, vwf, pf , and mp showed significant variation based on platelet count, with all markers exhibiting stepwise elevation with increasing platelet count. conclusions: markers of platelet activation were significantly elevated in patients with sac compared to healthy individuals. pf levels showed significant difference based on dic score or mortality, and differentiated the non-survivors compared to survivors. cd l, vwf, pf , and mp showed significant association with platelet count, increasing in a stepwise manner with increases in platelet count (table ) . prognostic value of mean platelet volume in septic patients: a prospective study a chaari king hamad university hospital, bussaiteen, bahrain critical care , (suppl ):p introduction: mean platelet volume (mpv) has been reported as a valuable marker of inflammatory diseases. the aim of the current study is to assess the prognostic value of mpv in septic patients. methods: prospective study including all patients admitted to the intensive care unit (icu) with sepsis or septic shock. demographic, clinical and laboratory data were collected. the mpv was checked on admission and on day . two groups were compared: survivors and non-survivors. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] days in survivors and . [ . - ] days in non-survivors (p= . ). conclusions: the decrease of the platelet count but not the increase of the mpv was associated with increased mortality in critically-ill septic patients. endotoxin activity assay levels measured within hours after icu admission affected patients' severity assessments a kodaira , t ikeda , s ono , s suda , t nagura tokyo medical university, tokyo, japan, introduction: sepsis profoundly alters immune homeostasis by inducing first a systemic pro-inflammatory, then an anti-inflammatory state. we evaluate the prognostic value of ex vivo lipopolysaccharide (lps) stimulation of whole blood in septic patients, at day and after intensive care unit (icu) admission. methods: this prospective cohort study included patients with severe sepsis or septic shock admitted to a surgical icu of a university hospital. blood was drawn on day and day , and stimulated ex vivo with lps for hours. tumor necrosis factor alpha (tnf), interleukin (il) , il and il were measured. twenty-three healthy adults served as controls. outcomes were ventilator and icu-free days, sofa score at day and , and need for dialysis during the course of sepsis. results: forty-nine patients were included (mean age ± years). the blood of septic patients was less responsive to ex vivo stimulation with lps than that of healthy controls, as demonstrated by lower tnf, il , il and il release ( fig. ). at day , patients above the th percentile of il release had significantly fewer ventilator and icu-free days than those in the lower th percentile (fig. ) . in contrast, patients in whom il release increased between day and day had significantly lower sofa scores at day and and need for dialysis, and more icu-free days than patients in whom il release decreased (table ) . conclusions: greater lps-stimulated il release in septic patients at day was associated with poorer clinical outcomes and may reflect the severity of the forthcoming immunoparalysis. however, an increase in il release between day and day was associated with favorable outcomes, perhaps signaling immune restoration. introduction: hyperthermic intraperitoneal chemotherapy with cytoreductive surgery (hipec-crs) is a curative treatment modality for peritoneal carcinomatosis. extensive debulking surgery, peritoneal stripping and multiple visceral resections followed by intraperitoneal installation of heated high-dose chemotherapeutic agents, a process leads to a 'high-inflammatory' syndrome. serum procalcitonin (pct), a biomarker for bacterial sepsis, in the heightened inflammatory state after hipec-crs might be of limited utility. our aim is to determine the trends of pct in the early postoperative phase of hipec-crs and to identify trends in patients with and without bacterial sepsis methods: in a case-control design, we reviewed all patients undergoing hipec-crs over a -month period ( ) ( ) ( ) . patients were divided into groups based on whether they developed bacterial sepsis in the first days after surgery (infected v/s non-infected). summary data are expressed as medians and ranges. two-tailed nonparametric tests were performed and considered significant at p values of less than . results: patients' data was analyzed. infections developed in % ( patients) with escherichia coli as the predominant pathogen isolated ( % isolates). pct levels (ngm/ml) were elevated postoperatively in both infected and non-infected patients; day infected . (iqr . introduction: early outcome in cardiac surgery has been an area of growing interest where the given risks raise several predictive models for assessment of postoperative outcome [ ] . procacitonin (pct) emerges as a possible predictive tool in cardiothoracic intensive care unit (cticu).we aim at testing the predictive power of pct for early morbidity, prolonged ventilation, icu and hospital stay, in patients developing early fever after cardiac surgery methods: a retrospective descriptive study done in tertiary cardiac center, enrolling patients who stayed for more than hours post-operatively in the cticu risk stratification included additive euro score and pct immunoluminometricaly prior to surgery and every hours in response to onset of fever. results: we screened consecutive patients who underwent open heart cardiac, of which patients were enrolled in the study. patients were divided into two groups based on the level of pct, those with value > ng/ml (group ) and those with level < ng/ml (group ). patients in group as compared to group , over the postoperative course was associated with prolonged icu stay (p= . ), length of mechanical ventilation (p= . ), length of hospitalization (p= . ), acute kidney injury (p= . ) and culture positivity (p= . ). multivariate analysis showed that pct > ng/ml was was significantly associated with positive cultures. (p= . ) conclusions: a rise of serum pct carries the signals of early icu morbidity and lengths of ventilation, icu stay and hospital stay methods: patients aged ( - ) days ( - days) underwent cardiac surgery with cardiopulmonary bypass for severe congenital heart disease. in the dynamics levels of pct, mr-proadm, ct-proavp and mr-proanp were measured before surgery and on the , , and days after the operation with the kryptor compact plus analyzer. data are presented as medians with interquartile range. the mann-whitney u-test was used to compare the data. values of p < . were statistically significant. results: patients ( %) required alv for more than hours. in this group statistically significant higher levels of pct, mr-proadm and mr-proanp were found throughout the period ( table ). the level of ct-proavp had increased to statistical significance since the day after the operation. patients were in the icu for more than hours. in this group statistically significant higher levels of pct, mr-proadm were found throughout the whole period ( table ). the higher level of mr-proanp was statistically significant on the st and th days after surgery, mr-proanp had a tendency of increasing values on nd and rd days. ct-proavp increased to statistical significance since the nd day after the operation and persisted throughout the studied period. conclusions: pct, mr-proadm and mr-proanp can be used as predictors of prolonged alv for children of the first year of life after cardiac surgery with cardiopulmonary bypass. the level of ct-proavp can be considered since the day after surgery. pct and mr-proadm may be used to predict the los in the icu. mr-proanp and ct-proavp can be considered since the and days after surgery respectively. introduction: early prediction of the risk of death among patients admitted at the emergency department (ed) remains an unmet need. the prognostic performance of hbp that is secreted by neutrophils was prospectively validated in a series of sequential ed admissions. methods: hbp and elements of qsofa were analyzed prospectively in serial ed admissions (main reasons for admission: acute abdominal pain . %; fever . %; vomiting/diarrhea . %; dyspnea . %; neurologic signs . %; non-specific complaints . %; most patients admitted for more than one reasons). upon ed admission patients were scored as low-risk, intermediate-risk and high-risk at the discretion of the physician. hbp was measured in blood samples upon admission by an enzyme immunosorbent assay. results: hbp was significantly greater among patients who died very early (fig. ). in five out of six of patients dying early hbp was greater than ng/ml. we combined hbp more than ng/ml and the presence of one sign of qsofa into a new score; this had . % sensitivity to predict -day mortality. the respective sensitivity of two signs of qsofa was . % (p: . ). the use of this new score allowed better stratification of patients originally considered at the triage as low-risk into high-risk (fig. ) . conclusions: we propose hbp more than ng/ml and one qsofa sign as an early score for -day mortality at the ed. introduction: despite of our growing knowledge in pathophysiology of septic shock still remain one of the most important factors of hospital mortality. it is thought that early diagnosis and treatment at early stage of septic shock would decrease its mortality. there have been on-going studies in recent years which research the usability of heparin binding protein (hbp) in early diagnosis of sepsis [ ] . to seek the usability of c-reactive protein (c-rp), procalcitonin (pct) and hbp biomarker combination in early diagnosis of septic shock. methods: patients, who have the diagnosis of septic shock, that are expected to stay in intensive care unit more than hours, and aged between - are included in the study. data are collected from the patients' blood samples that are drawn on admission, on the th hour, and on the day of discharge or death. results: it has been found in our study that, best "cut-off" value ng/ml, specificity . and sensitivity . for hbp. compared with other biomarkers, hbp was the best predictor of progression to organ dysfunction (area under the receiver operating characteristic curve (auc) = . ). conclusions: although there have been many biomarkers for early diagnose of septic shock, c-rp and pct are the most common used markers in nowadays' clinical practice. the usability of hbp in early diagnosis of sepsis is still being researched. we concluded that pct, c-rp and hbp biomarker combination is usable to diagnose septic shock at the end of our study. introduction: reduced adamts- and increased von willebrand factor (vwf)/adamts- ratio have been observed in sepsis and are associated with the severity of the disease [ , ] . however, their change during the septic episode and in the event of a change in the clinical status of the septic patients has not been investigated. the aim of the study was to assess the variation of these hemostatic parameters in critically ill patients during the course of a septic episode. methods: we monitored septic patients admitted in the intensive care unit (icu). improved (group a) while deteriorated (group b). we assessed vwf, adamts- and the vwf/adamts- ratio on admission in icu (time point ) and at the time of a change in patients' clinical condition (remission or deterioration, time point ). results: in group a, adamts- and the vwf/adamts- ratio did not significantly change ( . ± . vs . ± . conclusions: hemostatic disorders, as assessed by vwf and adamts- levels were detected in septic patients, while their changes differed according to the evolution of the septic episode. adamts- changes may be associated with outcome. methods: adult patients with at least one sign of qsofa and infection or acute pancreatitis or after operation were prospectively followed-up. blood was sampled the first hours; those with hiv infection, neutropenia and multiple injuries were excluded. sepsis was diagnosed using the sepsis- criteria. soluble urokinase plasminogen activator receptor (supar) was measured by an enzyme immunoassay. results: sixty patients were classified with sepsis using the sepsis- definitions. presence of at least two signs of qsofa had . % sensitivity, . % specificity, . % positive predictive value and . % negative predictive value for the diagnosis of sepsis. the integration of qsofa signs and supar improved the diagnostic performance ( fig. ) . conclusions: conclusions two signs of qsofa have significant positive prognostic value for sepsis but low sensitivity. this is improved after integration with supar. the intelligence- study is supported by the european commission through the seventh framework programme (fp ) hemospec. introduction: sepsis is a frequent reason for admission in the emergency department (ed) and its prognostic mainly relies on early diagnosis. in addition, no validated prognostic tool is currently available. therefore, identification of patients at high risk of worsening in the ed is key. the triage objective was to assess the prognostic value of a blood marker panel to predict early clinical worsening of patients admitted in the ed with suspected sepsis. methods: triage was a prospective, multicenter ( sites in france and belgium) study on biological samples conducted in partnership with biomerieux s.a. patients admitted in the ed with suspected or confirmed community-acquired infection for less than h were included. exclusion criteria were: admission in the ed for more than hours, septic shock at admission, immunodepression, sepsis syndrome days prior to admission. the protocol included clinical and biological time points (h , h , h , h , d ). patients were classified in groups at admission (infection, sepsis, severe sepsis) and divided into evolution/prognosis groups depending on worsening or not from their initial condition to severe sepsis or septic shock and sofa score's evolution. the evolution criteria were centrally evaluated by an independent adjudication committee of sepsis experts including emergency physicians and intensivists. patients were followed up to day for mortality. results: the study duration was years with patients included ( excluded). the centralized analysis is in progress to select the combination of biomarkers with the best prognostic performance comparing both evolution/prognosis groups. currently, patients have been classified as worsening and some results will be available in . conclusions: triage is the largest prospective multicenter study assessing the prognostic value of a panel of blood markers in eds which could help identification of septic patient at risk of worsening at time of admission in the ed and develop specific management. introduction: immune status characterization in intensive care unit (icu) patients presents a major challenge due to the heterogeneity of response. in this study, the filmarray® system was used with customized gene assays to assess the immune profile of critically-ill icu patients compared to healthy volunteers; from within the realism cohort. methods: a customized filmarray® pouch containing assays was designed; target and reference genes. detection and semiquantification of assays from whole blood collected in paxgene tubes occurs in the device within hour. a total of subjects from the realism cohort were tested in duplicates: trauma, septic shock and surgery patients, along with healthy volunteers. the patients' selection was based on hla-dr expression on monocytes, and pha-(phytohaemagglutinin) stimulated t-cell proliferation assay, to have various immune profiles. results: quantification cycle values of the target genes were normalized by the geometrical mean of reference genes to account for the different cell counts among specimens. the number of the cd + cells and hla-dr, determined by flow cytometry, showed good correlation to cd d and cd gene expression, respectively. seven genes showed significant differences in expression levels between the healthy volunteers and patient groups: cd d, cd , ctla & cx cr were down-regulated, while il- , il rn and s a were up-regulated in the patient populations. the use of relative quantitative difference of some markers was able to distinguish and introduction: early, rapid diagnosis is integral to the efficient effective treatment of sepsis; however, there is no gold standard for diagnosis, and biochemical surrogates are of limited and controversial utility. the cytovale system measures biophysical properties of cells by imaging thousands of single cells per second as they are hydrodynamically stretched in a microfluidic channel. this platform has been shown to measure dozens of mechanical, morphological, and cell surface biomarkers of wbc activation simultaneously [ , ] . in this study, we show the performance of the cytovale system in measuring biophysical markers for sepsis detection in the emergency department (ed). methods: we conducted an irb-approved prospective cohort study of emergency department (ed) patients with + sirs criteria and evidence of organ dysfunction. patients were included for analysis. blood samples for the cytovale assay were collected in the ed, and the diagnosis of sepsis was adjudicated by blinded clinician review of the medical record. captured imaging data were analyzed using computer vision to quantify mechanical parameters per cell, and a logistic model was trained to discriminate patients who had sepsis from those who did not. results: we found substantial biophysical differences between cells from septic and non-septic patients as observed at both the single cell level (fig. ) and when looking at the overall leukocyte populations (fig. ) . a multiparameter classification algorithm to discriminate septic from non-septic patients based on biophysical markers currently yields a sensitivity of % with a negative predictive value of %. conclusions: in patients presenting to the ed with of sirs criteria and evidence of organ dysfunction, the cytovale system provides a potentially viable means for the early diagnosis of sepsis via the quantification of biophysical properties of leukocytes. oxidative stress and other biomarkers to predict the presence of sepsis in icu patients v tsolaki, m karapetsa, g ganeli, e zakynthinos icu, larissa, greece critical care , (suppl ):p introduction: early identification of sepsis adds a survival benefit in icu patients. several biomarkers have been evaluated, yet an optimal marker is still lacking [ ] . methods: we prospectively determined oxidative status in patients admitted in a general intensive care unit of the university hospital of larisa. oxidative status was determined measuring the novel static (sorp) and capacity (corp) oxidation-reduction potential markers. other biomarkers (bnp, presepsin, crp) were measured, and the discriminative properties for the detection of sepsis were evaluated. results: oxidative status was evaluated in a hundred and fifty two consecutive patients. patients with severe sepsis and septic shock had significantly higher sorp values than patients without sepsis ( introduction: c-reactive protein (crp), is reported to be an effective marker for the assessment of vascular inflammation activity and acute coronary events prediction [ ] .we hypothesized that preoperative crp elevation is related to the occurrence of postoperative adverse cardiovascular outcomes. methods: we prospectively included patients scheduled to undergo different vascular surgeries from december to september . we assessed demographic data, comorbidities, revised cardiac risk index (rcri) and biomarkers (crp, cardiac troponin high sensitive ths, creatinine and urea) in the preoperative period. we also noted type and duration of surgery, intraoperative blood loss, icu stay and mortality. we evaluated crp as a predictive marker of major cardiovascular events defined as chest pain, ths elevation, electrocardiogram changes, arrhythmia, pulmonary embolism, stroke occuring within postoperative months. results: during our study, patients were scheduled to undergo vascular surgeries. from the patients, % developed adverse cardiac events (table ) . we showed the predictive value of crp in major cardiovascular event in a roc analysis (fig. ) . the cuttoff value of cpr was giving % of sensitivity and % of specificity. conclusions: our study pointed out that crp preoperative elevation could have a very strong predictive value of post-operative cardiovascular events in vascular surgery, this is in line with results showed by previous studies [ ] . introduction: elderly are particularly susceptible to bacterial infections and sepsis, and they comprise an increasing proportion of intensive care unit (icu) admissions. our aim was to evaluate the impact of age on critically ill infected patients. methods: we performed a post-hoc analysis of all infected patients admitted to icu enrolled in a -year prospective, observational, multicenter study involving icus. patients aged < , - and >= years were compared (group a, b, and c). multidrug-resistance (mdr) was defined as acquired non-susceptibility to at least one agent within three or more antimicrobial categories. results: of the patients analyzed, ( . %) were infected on icu admission. of these, ( %) belonged to group a, ( %) to group b and ( %) to group c. group c were more dependent, had higher saps ii and charlson scores (p< . ). icu and hospital length of stay did not differ between groups. microorganism isolation and bacteremia were higher in group b ( % and %, respectively) than groups a ( % and %, respectively) and c ( % and %, respectively; p< . ). septic shock was present in % of patients and was more frequent in groups b ( %) and c ( %) than group a ( %). the most common sources of infections were respiratory and intra-abdominal. isolation of gram-negative bacteria was significantly increased in group b and c (p= . ). the most common isolated bacteria were escherichia coli ( %), staphylococcus aureus ( %) and pseudomonas aeruginosa ( %) for all groups. in total, isolates ( %) corresponded to mdr bacteria, of which % were staphylococcus aureus. age was not a risk factor for infection by mdr. all-cause mortality in icu and hospital was: % and %; % and %; % and % -respectively for groups a, b, and c (p < . ). conclusions: old patients ( - years) were more prone to present with bacteremia, which could account for the increased severity of sepsis and higher all-cause mortality. age was not a risk factor for mdr infection. introduction: the rapid identification of pathogens using patient samples is crucial. delays in this can potentially have serious implications for patients and infection prevention/control [ ] . the aim of this project was to identify the number of microbiology samples sent, the number rejected and reasons for rejection, with the intention to reduce such instances. methods: data was collected retrospectively on icu admissions from january-june to a university hospital in the uk. patients were identified and data collected using the intensive care national audit and research centre (icnarc) database and from electronic patient records. data collected included: demographics, length of stay, microbiology samples sent and details on the rejected samples. results: patients were identified with a total of (median: samples/patient) samples sent to microbiology. were rejected ( %). ( %) patients had at least sample rejected. the median number of samples rejected per patient was (range: - ). the fig. (abstract p ). the area under the curve for crp elevation is . most common samples rejected were urine ( %), blood ( %), faeces ( %) and sputum ( %). ( %) of the samples were resent for testing (median day; range - ). reasons for sample rejection are shown in table . most rejections occurred within -hours of admission ( fig. ) . conclusions: this study confirms a high number of samples are sent to microbiology. although a few are rejected, overall this represents a large number, with most occurring during the first days of admission. reasons for sample rejection are remedial through improved training and vigilance. a bespoke guide to sample collection for microbiology coupled with a training program for healthcare professionals has been introduced with the aim to reduce sample rejections from % to . %. introduction: careful hand hygiene of health-care workers (hcws) is recommended to reduce transmission of pathogenic microorganisms to patients [ ] . mobile phones are commonly used during work shifts and may act as vehicles of pathogens [ , ] . the purpose of this study was to assess the colonization rate of icu hcws' mobile phones before and after work shifts. methods: prospective observational study conducted in an academic, tertiary-level icu. hcws (including medical and nursing staff) had their mobile phones sampled for microbiology before and after work shifts on different days. samples were taken with eswab in a standardized modality and seeded on columbia agar plus % sheep blood. a semiquantitative growth evaluation was performed at and hours after incubation at °c. results: fifty hcws participated in the study ( % of department staff). one hundred swabs were taken from mobile phones. fortythree hcws ( %) reported a habitual use of their phones during the work shift, and of them ( . %) usually kept their mobiles in the uniform pocket. all phones ( %) were positive for bacteria. the most frequently isolated bacteria were coagulase negative staphylococcus, bacillus sp. and mrsa ( %, %, %, respectively). no patient admitted to the icu during the study period was positive for bacteria found of hcws' mobile phones. no difference in bacteria types and burden was found between the beginning and the end of work shifts. conclusions: hcws' mobile phones are always colonized mainly by flora resident on hcw's hands, even before the work shift and irrespective of the microbiological patients' flora. further studies are warranted to investigate the role of mobile phones' bacterial colonization in the icu setting and to determine whether routine cleaning of hcws' mobile phones may reduce the rate of infection transmission in critical patients. methods: sixty samples were collected from aicu (n= ), picu (n= ) and or (n= ) during august to september . samples were randomly selected and taken at the end of the hcws duty with a sterile swab covering all mp surfaces. the inoculation was made into blood sheep and eosyn methilene blue agar for culture. isolated bacteria were identified according to standard microbiological techniques. antibiotic sensitivity testing was performed using disc diffusion method. results: overall mp bacterial colonization rate was %. main results are detailed in table . most common non pathogenic bacteria was staphylococcus epidermidis n= ( %). isolated pathogenic bacteria conclusions: we found high rates of mp colonization with pathogenic bacteria. an educational program is necessary to reduce the contamination and transmission of these high risk microorganisms. introduction: the objective of this study was to evaluate the variability in the dynamics and levels of airborne contamination within a hospital intensive care unit in order to establish an improved understanding of the extent to which airborne bioburden contributes to cross-infection of patients. microorganisms from the respiratory tract or skin can become airborne by coughing, sneezing and periods of increased activity such as bed changes and staff rounds. current knowledge of the clinical microflora is limited however it is estimated that - % of nosocomial infections are transmitted via air. methods: environmental air monitoring was conducted in glasgow royal infirmary icu, in the open ward and in patient isolation rooms. a sieve impactor air sampler was used to collect l air samples every minutes over hour ( : - : h) and hour ( : - : h) periods. samples were collected, room activity logged and the bacterial contamination levels were recorded as cfu/m of air. results: a high degree of variability in levels of airborne contamination was observed over the course of a hour day and a period in a hospital icu. counts ranged from - cfu/m over hours in an isolation room occupied for days by a patient with c. difficile infection. contamination levels were found to be lowest during the night and in unoccupied rooms, with an average value of cfu/m . peaks in airborne contamination showed a direct relation to increased room activity. conclusions: this study demonstrates the degree of airborne contamination that can occur in an icu over a hour period. numerous factors were found to contribute to microbial air contamination and consideration should be given to potential improved infection control strategies and decontamination technologies which could be deployed within the clinical environment to reduce the airborne contamination levels, with the ultimate aim of reducing healthcareassociated infections from environmental sources. new practice of fixing the venous catheter of the jugular on the thorax and its impact on the infection f goldstein, c carius, a coscia quintad'or, rio de janeiro, brazil critical care , (suppl ):p introduction: central line-associated bloodstream infection (clabsi) is an important concern in the icu, mainly in those with a high density of use of central venous catheter. any measures that may have an impact on the reduction of clabsi are important in reducing morbidity and mortality of hospitalized patients. therefore we present a retrospective study comparing the fixation site (neck vs. thorax) of the catheters implanted in the jugular vein, guided by ultrasonography and evaluating its impact on the incidence of clabsi. the purpose of our study was to identify if there is any positive impact on the reduction of clabsi when the catheter is fixated on the thorax. methods: a retrospective unicentric study comparing the infection rates between the year of , when the traditional technique of catheter fixation on the neck was used, and , when % of the catheters were fixated on the thoracic region. the criteria for clabsi were defined by the infection commission of quintad`or hospital and the data on clabsi were provided by the same commission. during this period there were no changes in the team of our unit and the patient's profile was the same. no deep vein catheter impregnated with antibiotics were used in the patients included in the study. the comparison used fisheŕs test as a tool. all the patients hospitalized in the intensive care unit with indication of the central venous catheter of short permanence in the internal jugular vein were included. patients with the central venous catheter of short permanence in other topographies, patients with hemodialysis catheter or with picc were excluded. results: during the year of , internal jugular vein catheters were installed in our unit using the traditional technique, fixing the catheter on the neck. in this period, cases of clabsi were detected. on the other hand, in the year of , internal jugular vein catheters were installed in the same unit, all of them, using the thorax as the point of fixation. although the number of catheters installed this year was higher, there was no case of clabsi. it appears that this position, provides a better fixation of the catheter, avoiding that the bandage gets uncovered. conclusions: during the year of , though there were more patients using deep vein catheters of short permanence, we had less clabsi events on our unity compared to the year of . fisher's exact test identified a p-value of this association of . . fixation of the internal jugular vein catheter in the thorax seems to contribute to the prevention of clabsi. further prospective and randomized studies are required to evaluate the contribution of fixation of the jugular vein catheter in the thorax in the clabsi prevention. introduction: the oral cavity of a patient who has been hospitalized presents a different flora from normal healthy people. after h hours of hospital stay, the flora presents a bigger number of microorganisms that can be responsible for secondary infections, like pneumonia, because of their growth and proliferation. the objective of our study was to assess the dental plaque index on patients on admission to an intensive care unit, and reassess days later, to evaluate the efficacy of oral hygiene. methods: prospective, descriptive and observational study in an intensive care unit of the chp. demographic, admission motive, hospital length of stay, feeding protocol, respiratory support need and oral hygiene protocol data was collected. the greene & vermillion simplified oral hygiene index (iho-s) was used as the assessment tool on the first h and on th day. results: patients were evaluated, of which were excluded for not meeting the minimal dentition. patients had a mean age of , ± , years, , % were males and most of medical and surgical scope ( , % each). mean hospital length of stay was , ± , days. the majority of patients were sedated ( %), under ventilator support ( , %) and with enteric nutritional support, under nasogastric tube feeding. initial iho-s score was , ± , , rising to , ± , (p< , ) days later. conclusions: various studies have proven the importance of a good oral hygiene to avoid bacterial growth and reduce the risk for nosocomial infections. in this study, we've observed a significant worsening of oral hygiene one week after admission. although this could be unimportant for a one week staying patient, it could indicate an increased risk for nosocomial infections for longer staying patients, which could benefit from a more efficient oral hygiene protocol. positive pocket cultures and infection risk after cardiac electronic device implantation-a retrospective observational single-center cohort study p pekić methods: we performed a retrospective observational single-center cohort study on patients who received de novo implantation of pacemaker, cardioverter-defibrillator or cardiac resynchronization therapy device in a two-year period. each patient was implanted using standard aseptic procedure according to local protocol and antibiotic (cefazolin) prophylaxis before the procedure. pocket aspirate was taken after irrigating the wound with normal saline just before device placement. results: we analyzed patients ( . % male, . % female). the most often implanted device was a ddd pacemaker followed by a vvi pacemaker. mean length of hospital stay was . ± . days. there were ( . %) positive cultures with overall ( . %) clinically apparent infections which required prolonged iv antibiotics, removal of device and reimplantation after infection resolution. in regard to microbiology, s. epidermidis ( . %) and coagulase negative staphylococcus ( . %) were the most often finding which is in contrast to the cultures described in the literature. the only statistically significant risk factor for positive pocket culture was male sex and presence of a urinary catheter. invasive vascular devices, previous intrahospital infection, and diabetes were not found to increase the likelihood of positive pocket culture. conclusions: positive pocket cultures after cied implant are a frequent finding mostly due to contamination and colonisation. the risk factors for such a finding differ from the usual and expected clinical circumstances. our results are consistent with those in the literature. it turns out that the most important preventive measure in cied implantation is strict aseptic procedure. introduction: intensive care patients are in constant risk of contamination due to suppression of their immune system, use of invasive procedures and medical equipment and health associated infections (hai). chlorhexidine gluconate (chg) is an antiseptic and disinfectant product. in medical research it has been found that daily chg bathing is affective in reducing levels of skin and central line related infections (climo, ) . it is also referred to in the recommendations of the ministry of health "prevention of septicemia due to central lines" ( ). methods: unit guide lines for patient dry bathing were written in may and thereafter began the implementation and instruction of nursing staff. quality control was inspected by observation. there was a phase questioner that included several categories such as: preparation of the chg solution, staff protection actions, infusions and surgical wound dressings, bathing performance and documentation. results: a gradual rise of %was observed in theperformance ofdry bathing according to the unit guidelines conclusions: % of observed dry baths where performed according to the guide lines. points for improvement: correct care of infusions and surgical wound dressing and verify use of separate wipes for each body part. next we will examine the correlation between the use of dry baths and theextent of infections in the unit. dry baths are nowconsidered an integralpart of the daily nursing routine. they have no substantial costs, help prevent complications from infection and add to the patient's safety. introduction: despite reductions in mortality reported with sdd, concerns about bacterial resistance and alteration of microbiome limit use. a retrospective observational study was conducted into the effect of local sdd protocols on vap rates and resistance patterns. over a -year period, regimens were used dependent on drug availability and hospital antibiotic stewardship concerns. the study was designed to review practice and identify any risks of partial implementation. methods: patients ventilated on a general intensive care were identified via clinical information systems. three periods were reviewed for adherence to sdd protocols, pre sdd (jan -feb ), full (july -sept ) and partial (july -sept ). high-risk patients during both sdd periods also received iv antibiotics for hours. patients admitted with pneumonia or tuberculosis were excluded from vap analysis. remaining patients' records were reviewed and the clinical pulmonary infection score (cpis) calculated for each ventilated day to identify vap rates. positive respiratory microbiological results for all patients admitted to the icu during each time period were reviewed to assess for wider changes in local resistance patterns. results: protocol adherence was assessed in patients during the full sdd period and during the partial ( table ). the number of patients included for analysis of vap rates during each period was pre sdd, during full sdd and during partial sdd. there were no significant changes in resistance patterns or clostridium difficule rates (table ) . conclusions: compliance with the available enteral antibiotics was reasonable but with iv antibiotics was poor. it is accepted that alterations and non-adherence to protocols risk development of resistant bacterial strains. within our unit no decrease in vap rates was seen but reassuringly no increased rates of extended bacterial resistance were identified during the treatment periods. introduction: arterial catheters are commonly used in intensive care units (icu) and are among the most frequently manipulated vascular access devices. our aim was to evaluate the rate of arterial catheterrelated bloodstream infection and colonization. methods: this was a -month, prospective and monocentric cohort study, performed in a multipurpose icu. all arterial catheters, inserted in or presented to the icu, were cultured and assessed for colonization or catheter-related bloodstream infection (crbi). results: we enrolled patients ( . % males, average age ± years, saps ± ) of whom a total of arterial catheters were analyzed for a total of catheter-days. radial arterial catheters were inserted in . % (n= ), femoral arterial catheters in . % (n= ) and other arterial catheters in . % (n= ). signs of dysfunction were found in . % and . %, respectively. radial arterial catheters colonization (n= ) and crbi (n= ) occurred at a rate of . and . / catheter-days. femoral arterial catheters colonization (n= ) and crbi (n= ) occurred at a rate of . and . / catheter-days, respectively. mean catheter time insertion was significantly higher in colonized catheters/crbi ( ± days; % ci: - ) when compared to arterial catheters with negative cultures ( ± days; % ci: - ); p = . ). colonized lines showed acinetobacter baumannii (n= ), staphylococcus epidermidis (n= ), enterococcus spp (n= ) and pseudomonas aeruginosa (n= ). crbi were caused by staphylococcus epidermidis (n= ) and staphylococcus haemolyticus (n= ). conclusions: the incidence of radial arterial catheters colonization and crbi were lower than reported rates in literature. colonization and crbi rates were higher in femoral catheters. femoral catheters showed dysfunction more frequently. prolonged catheterization was associated with colonization and crbi. a multimodality approach to decreasing icu infections by hydrogen peroxide, silver cations and compartmentalization and applying acinetobacter as infection marker introduction: nosocomial infections at the intensive care unit (icu) represent a substantial health threat [ , ] . icu infections are mainly attributed to the extended hospital delay which results in high morbidities and mortalities. methods: a cross sectional study was conducted at the intensive care unit, aseer central hospital, saudi arabia over months period ( ) ( ) . the intervention program included the application of mist of hydrogen peroxide and silver cations, physical separation and compartmentalization of the intensive care unit. the glosair™ system was used to deliver a mist of hydrogen peroxide and silver cations. hydrogen peroxide is an oxidizing agent, which kills microorganisms. results: a total of strains of acinetobacter species were identified from the patients over the months period (fig. ) . the mean infection rates decreased from . in the first three months of the program to in the last three month after continuous. conclusions: the program using the three procedures offered a significant decrease in infections at the icu as measured by acinetobacter count, which is one of the most hazardous nosocomial pathogens. introduction: the efficacy of ß lactam antibiotics is related to the time above mic. continuous or extended infusions can be used to increase the time above mic, especially in patients with normal or increased drug clearance. administering antibiotics by continuous infusion is not a new concept. a review in looks at the outcomes of continuous infusions [ ] . more recently an improvement in mortality has been demonstrated [ ] . our perception was that uptake of this low cost intervention was not common, so we undertook a survey to determine how commonly continuous infusions are used in england. methods: a telephone survey of all intensive care units in england was undertaken. questions included: -are you using continuous or extended antibiotic infusions? -which antibiotics are you using for continuous or extended infusions? -if not currently using has it been considered? data was collected over a week in june . results: there was an % response rate. ( . %) of the units continuously infuse some antibiotics, however . % of those only infuse vancomycin and not ß lactams. only of the total responders ( . %) infuse antibiotics other than vancomycin (i.e. ß lactams). conclusions: the theoretical advantage of continuous infusion of ß lactam antibiotics has been described for over years. there is now evidence that this may improve survival. despite this, uptake in england has been slow. introduction: infections contribute to a significant proportion of morbidity and mortality worldwide. while many infections are successfully managed with antimicrobial therapy, rates of antimicrobial resistance (amr) are increasing. certain patient populations such as those admitted to intensive care units (icu) are at high risk. methods: we conducted a retrospective, observational study of all icu patients at a tertiary referral hospital in rwanda from january through december we collected data on diagnosis, icu length of stay, mortality and hospital length of stay, as well as microorganism, site of culture, amr and antibiotics prescribe. results: overall, patients were admitted to the icu. most patients were admitted from the main operating theater (n= , %).the most common admitting diagnoses were sepsis (n= , %), head trauma (n= , %). a total of samples were collected from patients. the samples were from blood (n= , %), tracheal aspirate (n= , %),. the most common organisms isolated were klebsiella (n= , %), acinetobacter (n= , %), e.coli (n= , %), proteus (n= , %), citrobacter (n= , %), s aureus (n= , %), pseudomonas (n= , %), and other (n= , %). of klebsiella isolates, % and % were resistant to ceftriaxone and cefotaxime, respectively. of e.coli isolates, % and % were resistant to ceftriaxone and cefotaxime, respectively. all acinetobacter isolates were resistant to ceftriaxone and cefotaxime. conclusions: there is an alarming rate of antimicrobial resistance to commonly used antibiotics in the icu. expanding antibiotic options and strengthening antimicrobial stewardship are critical for patient care. the last three days g latten , p stassen zuyderland mc, sittard-geleen, netherlands, introduction: this study provides an overview of the prehospital course of patients with a (suspected) infection in the emergency department (ed). most research on serious infections and sepsis has focused on the hospital environment, while potentially most delay, and therefore possibly the best opportunity to improve treatment, lies in the prehospital setting. methods: patients were included in this prospective observational study during a week period in . all patients aged years or older with a suspected or proven infection were included. prehospital, ed and outcomes were registered. results: in total, patients visited the ed during the study period, of whom ( . %) patients had a (suspected) infection. (fig. ) median duration of symptoms before ed visit was days (iqr - days), with . % of patients using antibiotics before arrival in the ed. most patients ( %) had been referred by a general practicioner (gp), while . % of patients had visited their gp previously during the current disease episode. twenty-two patients ( . %) experienced an adverse outcome (icu admission and/or -day all-cause mortality): these patients were less often referred by a general practicioner (gp) ( . vs. . %, p= . ) and were considered more urgent both by ems and in the ed. conclusions: the prehospital phase of patients with an infection provides a window of opportunity for improvement of care. patients become ill days before the ed visit and . % already visited their gp previously during the current disease episode, while . % is currently using antibiotics. future research should focus on quality improvement programs in the prehospital setting, targeting patients and/or primary care professionals. introduction: worldwide, the prevalence of tetanus has decreased.-however, even if progress has been made in the combat to eradicate tetanus it may be a cause of admission to intensive care.the objectives of our study are to determine epidemiological,clinical and prognostic characteristics for severe tetanus in our unit. methods: we conducted a retrospective study in the medical intensive care unit of ibn rushd hospital in casablanca in morocco from to .we studied the epidemiological,clinical and prognostic characteristics of the patients who were admitted for severe tetanus. results: the incidence of severe tetanus was . % affecting male in %. . % were aged between and years old. in . % there were a integumentary portal of entry. contractures were present in %of the cases. at intensive care unit admission, . % of the patients were sedated. the anti-tetanus vaccination was never updated. according to the dakar score . % of the patients were listed dakar , . % dakar and . % dakar . for the mollaret score, the crude form was found in . %, the acute generalized form was found in . % and the severe form in . % of the cases.mechanical ventilation was necessary in . %. diazepam and baclofen were used in . %, phenobarbital in . % and propofol in . %. a serotherapy was used for all the patients and a preliminary vaccination dose for . %. all the patients received antibiotics, penicillin g . % and metronidazole . %. the mortality was . %. the length of intensive care stay was significantly higher. the need for an intubation,its duration and the occurrence of autonomic dysfunction have significantly influenced the mortality. conclusions: to improve the prognosis in these serious forms of tetanus,it is highly important to identify the warning signs and refer patients in intensive care for early and appropriate management in intensive care. introduction: bloodstream infections (bsis) are associated with increased mortality in the icu. the aim of the study was to evaluate the epidemiology and resistance patterns during the period to . methods: bacteria and fungi isolated from the blood of patients hospitalized in a mixed icu during the study period were retrospectively analyzed. sensitivity testing was performed with disk diffusion (kirby-bauer) and microscan walkaway plus for minimal inhibitory concentrations. results: during the study period patients were hospitalized in the icu. bsis were diagnosed in cases ( . %). the isolated microorganisms were acinetobacter baumannii ( %), klebsiella pneumoniae ( %), other enterobacteriaceae ( %), pseudomonas aeruginosa ( %), stenotrophomonas maltophilia ( %), enterococci ( %), staphylococci ( %) and candida spp. ( %). of the a. baumannii isolates, % were resistant to carbapenems, . % to colistin, and % to tigecycline. of the k. pneumoniae isolates % were resistant to carbapenems, % to colistin, and . % to tigecycline. of the p. aeruginosa species % were resistant to carbapenems and they were all susceptible to colistin. the rate of resistance to vancomycin was % for the e. faecium isolates, . % for the e. faecalis, while the resistance to methicillin of the coagulase negative staphylococci was %. the most commonly isolate species of candida was c. albicans. conclusions: multi-drug resistant isolates, especially a. baumannii and enterobacteriaceae, are a serious problem in our icu. gram positive bacteria are less common, but the resistance of enterococci to vancomycin is significant. antibiotic stewardship and infection control measures should be applied in a more strict way. nosocomial sinusitis in intensive care unit patients i titov introduction: nosocomial sinusitis (ns) is a complication of critically ill patients which develops - h after admission and is mostly linked but not limited to such invasive procedures as nasotracheal intubation and nasogastric tube placement. ns is often overlooked as a source of pyrexia of unknown origin, meningeal manifestations, sepsis and ventilator associated pneumonia in icu patients. ct scanning and sinus puncture are used to confirm the inflammatory process and identify the pathogen behind it. methods: a retrospective case study of . icu patients for a period of - was performed. we have analysed data from the ct scans of paranasal sinuses and bacteriological findings of samples obtained from sinus puncture. results: ( . %) patients were suspected of ns on the - th day of stay in the icu. the ct scan confirmed pathological changes in patients ( . %). hemisinusitis was detected in patients ( . %) and pansinusitis in patients ( . %). there was also an isolated case of maxillary sinusitis in patient ( . %). the pathogenic culture was identified only in ( %) samples, . % of which revealed isolated bacteria and . % a polymicrobial association. gram positive bacteria were detected in . % of cases and gram negative in . %. most cases revealed multiple antibiotic resistance. conclusions: . ns has proved to be largely caused by gram negative bacteria and polymicrobial associations. the use of broad spectrum antibiotics in icu may justify the presence of sterile cultures. .early identification of risk patients in icu as well as the use of screening ct scan may benefit timely diagnosis and adequate treatment of patients. .preventive considerations include: patient's bed head elevation, the use of oral gastric tube in sedated and coma patients on ventilation, nasotracheal intubation only if indicated, removal of nasogastric tube at night, proper hygiene. conclusions: only of , tb patients ( %) required critical care intervention (table ) . those admitted to icu were older and more likely to have pulmonary, cns, miliary or abdominal tb (table ) . mortality was high despite critical care input in a unit familiar with managing tb, and hour access to infectious diseases advice within the trust, likely due to overwhelming organ dysfunction, patient frailty and advanced tb infection. rates of drug resistant tb were low and comparable to uk-wide rates over that period ( % mono-drug resistant, % mdr) thus less likely a contributory factor to the majority of deaths. short term antibiotics prevent early vap in patients treated with mild therapeutic hypothermia after cardiac arrest t daix , a cariou , f meziani , pf dequin , c guitton , n deye , g plantefève , jp quenot , a desachy , t kamel , s bedon-carte , jl diehl , n chudeau , e karam , f renon-carron , a hernandez padilla , p vignon , a le gouge introduction: patients treated with mild therapeutic hypothermia after cardiac arrests with shockable rhythm are at high risk of ventilator-associated pneumonia (vap) [ ] . despite retrospective trials suggesting a benefit of short-term ( h) antibiotics in this setting [ ] , it is not recommended. the primary objective was to demonstrate that systematic antibiotic prophylaxis can reduce incidence of early vap (< days). the impact on incidence of late vap and on day mortality was also assessed. methods: multicenter, placebo-controlled, double-blinded, randomized trial. icu patients > years, mechanically ventilated after out-of-hospital resuscitated cardiac arrest related to initial shockable rhythm and treated with mild therapeutic hypothermia were included. moribund patients and those requiring extracorporeal life supports, with ongoing antibiotic therapy, known chronic colonization with multiresistant bacteria or known allergy to beta-lactam antibiotics were excluded. either iv injection of amoxicillin-clavulanic acid ( g/ mg) or placebo was administered times a day for days. all pulmonary infections were recorded and blindly confirmed by an adjudication committee. results: in intention to treat analysis, patients were analyzed, (treatment group n= ; mean age . ± . years, sex ratio= , sofa score . ± . ). global characteristics of cardiac arrest were similar (no flow= . min vs . min, low-flow= . min vs . min). vap were confirmed incl. early vap, in treatment group vs in placebo group (hr= . ; ic %=[ . ; . ]) (fig. ) . occurrence of late vap ( % vs . %) and day mortality ( . % vs . %) was not affected by the study procedure. conclusions: short-term antibiotic prophylaxis significantly decreases incidence of early vap in patients treated with mild therapeutic hypothermia after out-of-hospital cardiac arrest related to shockable rhythm and should be recommended. introduction: antibiotics are the most commonly prescribed drugs in icu.in the era of antibiotic resistance it is difficult to choose antibiotics during septic episode.the choice antibiotics mainly depends on clinical diagnosis,culture sensitivity and local flora. whether severity of illness really maters is not well known. to study antibiotic prescription pattern and whether the choice of antibiotic varies according to hemodynamic stability in patients admitted in icu.to study of microbiological isolates and their variability according to hamodynamic stability in icu patients. methods: all icu patients of more than years age who received antibiotics and where cultures had been sent were included in the study.patients discharged against medical advice and where treatment had been withdrawn were excluded in this study. this prospective observational study was conducted between july to march .patients were divided into stable and unstable group according to hemodynamic parameter and usage of antibiotics and microbiological isolated were correlated. icu mortality and length of stay were correlated between hemodynamically stable and unstable group. results: sepsis episode were analysed. mean age was years, male predominant, and average apache iv score was (sd ). we had patients in unstable group of which % patients got discharged and % of patients got discharged in stable group. antibiotic combination therapy was used more in hemodynamically unstsble patients(p . ). blbli was used more in stable group. drug resistance in microbiological isolates did not reveal any statistically significant difference among stable or unstable group. conclusions: there is a tendency to administer combination antibiotics in sicker group of patients with hemodynamic instability. prevalence of microbial flora did not show any statistical difference. outcome is worse in hemodynamically unstable patients. the clinical significance of candida score in critically ill patients with candida infection h al-dorzi , r khan , t aldabbagh , a toledo , s al johani , a almutairi , s khalil , f siddiqui , y arabi king abdulaziz medical city, riyadh, saudi arabia, msd, riyadh, saudi arabia, king saud bin abdulaziz university for health sciences, riyadh, saudi arabia critical care , (suppl ):p introduction: candida score (cs) is used to identify patients with invasive candidiasis in the icu, but its clinical use has not become widespread. our objective was to evaluate the clinical significance of cs in a mixed population of icu patients. methods: this was a prospective observational study of critically ill patients who had candida species growth during their stay in any of six different icus of a tertiary-care center. two intensivists classified patients as having candida colonization or invasive candidiasis according to predefined criteria. cs was calculated for each patient on the day of candida species growth as follows: . see text for description point for parenteral nutrition + point for surgery + point for multifocal candida colonization + points for severe sepsis. the receiver operating characteristic (roc) curve was plotted to assess cs ability to discriminate between invasive candidiasis and candida colonization. results: cs was . ± . in patients with candida colonization (n= ) and . ± . in those with invasive candidiasis (n= ) (p< . ). however, only . % of invasive candidiasis cases had cs >= (compared with . % of candida colonization cases; p< . ). the roc curve (fig. ) showed that cs had fair ability to discriminate between invasive candidiasis and candida colonization (area under the curve . , % confidence interval . to . ; p< . ). in patients with invasive candidiasis, cs was similar in hospital survivors and nonsurvivors ( . ± . and . ± . , respectively; p= . ). cs did not discriminate between survivors and nonsurvivors (area under the roc curve . , % confidence interval . to . ; p< . ). conclusions: cs was higher in patients with invasive candidiasis than those with candida colonization. however, its ability to discriminate between these patients was only fair. cs was not associated with hospital mortality. poor reliability of creatinine clearance estimates in predicting fluconazole exposure in liver transplant patients m lugano, p cojutti, f pea asuiud, udine, italy critical care , (suppl ):p introduction: invasive candidiasis (ic) is a frequent complication in liver transplant (lt) recipients, especially during the first - months after lt. fluconazole is a triazole antifungal used for prophylaxis and treatment of ic. due to its renal elimination, dose adjustments are usually based on estimated creatinine clearance (ecrcl). however, the reliability of ecrcl in predicting fluconazole clearance has never been investigated in this population. the aim of this study was to conduct a population pharmacokinetic (poppk) analysis in a cohort of lt patients who underwent therapeutic drug monitoring (tdm) in order to find out which covariates may influence fluconazole pharmacokinetics (pks). methods: this retrospective study included lt patients who were admitted to the intensive care unit of our university hospital between december and may , and who were treated with intravenous fluconazole in the first months after lt. tdm of fluconazole was performed with the intent of attaining the efficacy pharmacodynamic target (auc h/mic > . ). the tested covariates were: age, gender, ckd-epi ecrcl, time from lt, serum albumin and transaminases, saps ii score. poppk was carried out with pmetrics software. results: nineteen patients (mean±sd age, weight and serum creatinine of ± . years, ± . kg, . ± . mg/dl, respectively) with a total of fluconazole trough plasma concentrations were included in the poppk analysis. mean±sd fluconazole distribution volume (vd) and clearance (cl) were . ± . l and . ± . l/h. age and time from lt were the only clinical covariates significantly correlated with fluconazole vd and cl, respectively. conversely, ckd-epi eclcr was unable to predict fluconazole cl. conclusions: ckd-epi eclcr is unreliable in predicting fluconazole exposure in lt recipients. consistently, in this population adaptation of fluconazole dose should be based on measured crcl, and tdm may be helpful in optimizing drug exposure. outcomes of a candidiasis screening protocol in a medical icu m boujelbèn , i fathallah , h kallel , d sakis , m tobich , s habacha , n ben salah , m bouchekoua , s trabelsi , s khaled , n kouraichi introduction: the aim is to determine the incidence, characteristics and risk factors of invasive candidiasis (ic) in critically ill patients by using a weekly screening protocol. methods: a months' prospective study was conducted in a -bed micu. the candidiasis screening consisted of the culture of plastic swabs (from different body sites), urine and respiratory tract samples.it was conducted upon admission and on weekly basis for all the patients. decision to treat was based on clinical and microbiological features. results: patients were included. the colonization rate with candida spp was . %(n= ). screening samples were collected with a positivity rate at . %(n= ). table describes the isolated candida species by site. antifungal resistance was tested in ( %) species. the resistance rate to fluconazole was . %(n= ). the antifungal resistance of candida albicans is detailed in table . ( . %) patients presented an ic with a mean age and mean saps ii at . ± years and ± . respectively. ( %) presented acute renal failure upon admission. . % (n= ) of the patients needed mechanical ventilation. the median length of stay was days [ . - . ] and the mortality rate was . %(n= ). the mean sofa score upon infection was . ± . . the candida score was >= . and the colonization index was >= . in fig. (abstract p ). roc curve for candida score discrimintaing between invasive candidiasis and candida colonization . %(n= ) and . %(n= ) of the patients respectively. only one patient had a positive blood culture. mannan antigen and anti-mannan antibodies were screened only in five patients with a positivity rate at %(n= ). the most isolated species was: candida albicans . %(n= ). multivariate analysis showed that prior use of imipinem more than days was a risk factor for ic (or= . , ci [ . ; . ], p= . ). conclusions: this study showed the ecology and epidemiology of candida species in our micu with an increased ic rate and high mortality. prior imipinem use was a risk factor for ic. introduction: icu-acquired infection is as high as . episodes per patient-days in lower-middle income countries like india (who). almost three times higher than in high-income countries [ ] . candida infection is the rd most commonly acquired nosocomial infection in india burdening the debilitated patient with longer icu stay [ ] . there are no definite guidelines on whether & when to start antifungal treatment, specific to india where ifi risk is high and diagnostic facilities are limited. currently, the intensivists across india are using antifungals, according to their clinical experience and selective application of international guidelines leading to non-uniformity of patient outcomes. in an endeavour to synchronize anti-fungal therapy and educate intensivists from small cities of india, intensivists and infectious disease specialist of international repute were approached to design a module on 'invasive fungal infections -when to start anti-fungals in icu [ fig. ]. the ifi in india was summarised into a compact hour session for dissemination of knowledge using idsa as a reference guideline. intensivists from across india were trained on the module by our faculty. the module was rolled out to intensivists and pulmonologists focussing particularly on the tier- & tier - cities where avenues for learning are limited [ fig. introduction: trichosporon species are fungi found in nature and human normal flora but they can be an opportunistic pathogen, introduction: this study assessed whether empiric combination antibiotic therapy directed against gram-negative bacteria is associated with lower intensive care unit (icu) mortality compared to single antibiotic therapy. methods: retrospective cohort study on prospectively collected data conducted in the icu of a tertiary care hospital in india between july to march . all consecutive infection episodes treated with empiric antibiotic therapy and with subsequent positive culture for gram-negative bacteria were included. primary and secondary outcomes were all cause icu mortality and icu length of stay (los). outcomes were compared between infection episodes treated with single vs.combination antibiotic therapy. results: of total episodes of gram-negative infections . % received combination-antibiotic therapy. baseline demographic and clinical characteristics between single vs. combination therapy groups were similar (mean age: p= . ; sex: p= . ; mean apache iv score: p= . ). overall icu mortality did not significantly differ between single and combination antibiotic groups ( . % vs. %; p= . ). in single antibiotic group, icu mortality was significantly higher for antibiotic-resistant compared to antibiotic-sensitive bacteria ( . % vs. . %, p= . ). in combination group, significantly lower icu mortality was noted if bacteria was sensitive to even one antibiotic compared to pan-resistant bacteria ( . % vs. . %, p= . ). icu los was similar between antibiotic-sensitive bacteria and antibiotic-resistant bacteria, both in single and combination therapy groups (single, antibiotic-sensitive vs. antibiotic-resistant: mean los±sd . ± . vs. . ± days; p= . ; combination, antibioticsensitive vs. antibiotic-resistant: . ± . vs. . days; p= . ). conclusions: irrespective of the number of antibiotics prescribed as empiric therapy, outcome of patients solely depends on the sensitivity pattern of the bacteria isolated. pharmacokinetics of trimethoprim and sulfametrole in critically ill patients on continuous haemofiltration r welte , j hotter , t gasperetti , r beyer , r introduction: the combination of trimethoprim and sulfametrole (tmp-smt, rokiprim®) is active against multi-drug resistant bacteria and pneumocystis jirovecii. in critically ill patients undergoing continuous veno-venous haemofiltration (cvvh), however, its use is limited because of lacking pharmacokinetic data. methods: pharmacokinetics of both drugs were determined after standard doses in patients on cvvh and in critically ill patients with approximately normal renal function. quantification of tmp and smt was done by high pressure liquid chromatography (hplc) and uv detection after pre-purification by solid phase extraction. the total clearance (cltot) was estimated from arterial plasma levels and the haemofilter clearance (clhf) from plasma and ultrafiltrate concentrations. results: six patients on cvvh ( after the first dose, at steady state) and nine patients off cvvh have been enrolled ( after first dose, at steady state). after a single dose, cltot of smt was . ( . - . , median [range]) and . ( . - . ) l/h on and off cvvh, respectively. at steady state, we observed a cltot of . ( . - . ) and . ( . - . ) l/h, respectively, on and off cvvh. steady state trough levels (cmin) of smt amounted to - mg/l in patients on cvvh and - in patients off cvvh. cltot of tmp was . ( . - . ) l/h on cvvh and . ( . - . ) l/h off cvvh after the first dose. at steady state, its cltot amounted to . ( . - . ) and . ( . - . ) l/h on and off cvvh, respectively. cmin was - mg/l on cvvh and - mg/l in patients off cvvh. clhf accounted for - % of cltot of smt and - % of cltot tmp. conclusions: exposure to both antimicrobial agents is highly variable, but comparable in patients on and off cvvh. as considerable amounts of smt and tmp are eliminated by cvvh, no excessive accumulation appears to take place during treatment with standard doses. the positive impact of meropenem stewardship intervention at a brazilian intensive care unit w freitas introduction: loss of colistin as a clinical option has profound public health implications. widespread use of colistin in agriculture and humans has seen the emergence of mcr- mediated resistance amongst south african patients [ ] . we sought to describe the trends of colistin minimum inhibitory concentrations (mic) over two years using data collected by smart. methods: smart monitors the in vitro susceptibility of clinical aerobic and facultative gram-negative bacterial isolates to selected antimicrobials of importance, enabling longitudinal analyses to determine changes over time. the dataset comprised bacterial isolates from four different south african private pathology laboratories and one public sector pathology laboratory from - . the methods used in the study have been described elsewhere [ ] . isolate proportions between years were compared using the chisquared test with yates' continuity correction. ( ) ( ) ( ) ( ) days]; patients underwent renal replacement therapy. the median treatment duration (iqr) was ( - ) days. in . % of cases, antibiotic-therapy therapy combination (phosphomycin and colistin) was chosen. all the patients experienced a clinical response by / hours from the ceftazidime/avibactam commencing. in / bacteraemic patients negativization of blood culture occurred by hours as well as of the rectal swab in / patients. a (b) recurred and a second treatment was given. / ( . %) patients survived, whereas death was caused by multi-organ failure. the susceptibility test of strains showed sensitivity to ceftazidime/avibactam, whereas % of resistance to carbapenems, quinolones and iii/iv generation cephalosporin, tigecycline and piperacillin/tazobactam; . % of susceptibility to fosfomycin and colistin; (v) less than % of suceptibility to aminoglicosides. conclusions: the strains of kp-cp were susceptible to ceftazidimeavibactam despite the high carbapenem-resistance recorded in our icu, because od rare identification of kp-cp vim/ndl +. the preliminary data seems to confirm the efficacy and clinical utility of this antibiotic for the critically ill patients. introduction: multidrug resistant bacteria (mdr) are an increasing problem on intensive care units. lung infections caused by acinetobacter baumannii are frequently difficult to treat. phages have regained attention as treatment option for bacterial infections due to their specificity and effectivity in lysis. the aim of this preclinical study was to determine efficacy and safety of a novel phage preparation in mice. methods: mice were transnasally infected with a mdr a. baumannii strain [ ] and hours later treated intratracheally with a specific phage or solvent. phage acibel [ ] was produced as suspension including efficient depletion of endotoxins. at defined time points, clinical parameters, bacterial burden in lung and bronchoalveolar lavage fluid (balf) and cell influx were determined. further, lung permeability and cytokine release were quantified and histopathological examination was performed. results: mice treated with phages recovered faster from infectionassociated hypothermia. hours after infection, phage treatment led to a reduction in bacterial loads in lungs and balf. in addition, lung permeability and cytokine production were reduced in phagetreated mice. histopathological examination of the lungs showed less spreading of bacteria to the periphery in phage-treated mice, whereas cellular recruitment into the lung was unaffected. no adverse effects were observed. conclusions: for the first time a highly purified phage against a. baumannii was successfully used in vivo. the current preclinical data support the concept of a phage-based therapy against pulmonary a. baumannii infections. introduction: vap is common in critically ill patients and associated with high morbidity and mortality, especially when caused by antibiotic resistant bacteria. recently, phage therapy has emerged as a promising non-antibiotic based treatment of antibiotic resistant bacterial infections. however, proof-of-concept experimental and clinical studies are missing before its wider use in clinical medicine. the goal of this experimental study was to compare the efficacy of phage therapy versus antibiotics for the treatment of mrsa in a rat model of vap. methods: four hours after intubation and protective ventilation, rats were inoculated via the endotracheal tube with - x cfu (ld ) of the mrsa clinical isolate aw . the animals were subsequently extubated. two hours after bacterial challenge, rats were randomised to receive intravenously either teicoplanin (n= ), a cocktail of lytic anti-s. aureus bacteriophages (n= ) or combination of both (n= ). animals served as control (no treatment). survival by hours was the primary outcome. secondary outcomes were bacterial count in lungs, spleen and blood. kaplan-meier estimates of survival were done and multiple comparisons of survival rates performed using the holm-sidak method. results: treatment with either phages, antibiotics or combination of both significantly increased survival ( %, %, % respectively, compared to % survival for controls, p< . ). there were no statistical differences in survival rates between either forms of treatment ( fig. ) . treatments hinder the systemic extension of the infection into the blood and spleen without impacting bacterial counts within the lungs, but the numbers are too small to perform statistical tests (table ) introduction: the aim of the study was comparative evaluation of the clinical and microbiological efficacy of combination of amikacin thru nebuliser aeroneb pro and standard antimicrobal therapy (amtcomb) with standard antimicrobal therapy (amtst) in treatment of ventilator-associated pneumonia (vap) and ventilator-associated tracheobronchitis (vat) caused by multi-drug resistant gram-negative bacteria. methods: in prospective two-center study with retrospective control included patients with vap and vat. in amtst group (retrospective, n= ) we used combination of meropenem g every h iv as continuous infusion, cefoperazon/sulbactam g every h iv as continuous infusion and amikacin g iv every h. in amtcomb group (prospective, n= ) we used combination of amtst and amikacin inhalation mg every h thru nebuliser aeroneb pro. results: in amtcomb clinical cure rate was %, while in amtst . % (p< . ), clinical pulmonary infection score (cpis) on day was ( - ) points in amtst and ( - ) points in amtcomb (p< . ). recurrence of vap/vat was . % in amtst and . % in amtcomb (p= . ). on day infectious agent titer in tracheal aspirate was ( - ) cfu/ml in amtst group, while (no growth- ) cfu/ml in amtcomb (p= . ). microbiological eradication observed in patients in amtcomb vs in patient in amtst and microbiological persistance observed in patients in amtcomb vs patients in amtst (p= . ). in amtcomb on rd day sputum was less purulent (p= . ). amikacin nebulisation didn't led to deterioration of organ dysfunction: on day there was no difference in platelet count, creatinine and bilirubin levels as compared to day (p= . ; p= . , p= . , respectively). conclusions: addition of amikacin inhalation mg every h thru aeroneb pro nebuliser in patients with vap and vat was more efficacious than intravenous standard antimicrobal treatment with comparable safety profile. introduction: the aim of the study was to assess the effectiveness of inhaled colistin (ic) as an adjunct to systemic antibiotics in the treatment of ventilator-associated pneumonia (vap). methods: icu patients with vap were enrolled in this observational study. resolution of vap was assessed as primary endpoint; eradication of pathogens in sputum, weaning time, duration of icu stay and mortality were assessed as secondary outcomes. patients were split into groups: gr. (n = ) -addition of ic to systemic antibiotics without changing the basic regimen; gr. (n = ) -change in systemic antibiotics according to sensitivity. groups were comparable. ic was administered in a dose of million iu tid (xselia pharmaceuticals aps, denmark). statistical analysis was performed using statistica . (m, σ, newman-keuls test; p < . ). results: vap resolution rate was % in gr. (vs. % in gr. , p = . ); eradication of pathogens from sputum by the th day. treatment was achieved in % of gr. and % in the gr. (n = ) (p> . ); in gr. weaning from ventilation was possible earlier than in gr. - . ± . days. in gr. vs. . ± . days. in gr. (p = . ); in gr. duration of icu stay was shorter than in gr. - . ± . days vs. . ± . days. in gr. (p = . ). no mortality differences were detected. conclusions: administration of inhaled colistin million iu tid is effective as an adjunct to systemic antibiotics in the treatment of vap. this modified treatment promotes a more rapid resolution of vap, earlier weaning from ventilator, reduction of the duration of icu stay, with no impact on mortality. the addition of ic to systemic antibiotics should be considered as second-line regimen in vap patients. factors associated with no de-escalation of empirical antimicrobial therapy in icu settings with high rate of multi-drug resistant bacteria c routsi introduction: de-escalation is recommended in the management of antimicrobial therapy in icu patients [ ] . however, this strategy has not been adequately evaluated in the presence of increased prevalence of multidrug-resistant (mdr) bacteria. the aim of this study was to identify factors associated with no de-escalation in icus with high rate of mdr bacteria [ ] . methods: prospective, multicenter study conducted in greek icus over a -year period. patients with laboratory confirmed infections were included. sofa score on admission, on septic episode and thereafter every h over days, infection site(s), culture results, antimicrobial therapy, and mortality were recorded. only the first septic episode was analyzed. in order to assess the factors associated with no de-escalation, a multivariate analysis was performed. results: a total of patients (admission sofa score ± ) were analyzed. % of those had septic episode on icu admission; % patients had an icu-acquired. de-escalation was applied to ( %) patients whereas it was not feasible in patients ( %) due to the recovery of mdr pathogens or it was not applied, although the microbiology results allowed it, in patients ( %). septic shock on the day of septic episode was present in % and % of patients with and without de-escalation, respectively, p= . ). compared to no de-escalation, de-escalation strategy was associated with a shorter duration of shock ( ± vs. ± days, p< . ) and all-cause mortality ( . % vs. . %, p< . ). multivariate analysis showed that the variables associated with no de-escalation were: a deteriorating clinical course as indicated by an increasing sofa score (or . , p< . ) and a lack of de-escalation possibility due to recovery of mdr pathogens (or . , p= . ). conclusions: deteriorating clinical course and mdr pathogens are independently associated with no de-escalation strategy in critically ill patients. conclusions: the qsofa scale in the prognosis of sepsis does not differ significantly from the sirs criteria, but in the prognosis of mortality is significantly better than sirs. qsofa significantly worse in the prognosis of sepsis and death than the sofa scale. the international task force of sepsis- introduced the quick sequential failure assessment (qsofa) score to supersede the systemic inflammatory response syndrome (sirs) score as the screen tool for sepsis. the objective of this study is to prospectively access the diagnostic value of qsofa and sirs among patients with infection in general wards. methods: a prospective cohort study conducted in ten general wards of a tertiary teaching hospital. for a half-year period, consecutive patients who were admitted with infection or developed infection during hospital stay were included. demographic data and all variables for qsofa, sirs and sofa scores were collected. we recorded daily qsofa, sirs and sofa scores until hospital discharge, death, or day , whichever occurred earlier. the primary outcome was sepsis at days. discrimination was assessed using the area under the receiver operating characteristic curve (auroc) and sensitivities or specificities with a conventional cutoff value of . results: of patients (median age, years [iqr, - ]; male, [ %]; most common diagnosis pneumonia, [ %]) who were identified with infection in general wards, ( %) developed sepsis at a median of (iqr, - ) day, patients ( %) and patients ( %) met qsofa and sirs criteria at a median of (iqr, - ) and (iqr, - ) day, respectively. the qsofa performed better than sirs in diagnosing sepsis, with an auroc of . ( % ci, . - . ) vs . ( % ci, . - . ). with a conventional cutoff value of , qsofa had lower sensitivity ( % [ % ci, %- %] vs. % [ % ci, %- %], p < . ) and higher specificity ( % [ % ci, %- %] vs. % [ % ci, %- %], p < . ) than sirs (table ) . conclusions: among patients with infection in general wards, the use of qsofa resulted in greater diagnostic accuracy for sepsis than sirs during hospitalization. qsofa and sirs scores can predict the occurrence of sepsis with high specificity and high sensitivity, respectively. prognostic accuracy of quick sequential organ failure assessment (qsofa) score for mortality: systematic review and meta-analysis introduction: the purpose of this study was to summarize the evidence assessing the qsofa [ ] , calculated in admission of the patient in emergency department (ed) or intensive care unit (icu), as a predictor of mortality. the hypothesis was that this tool had a good prediction performance. methods: systematic review and meta-analysis of studies assessing qsofa as prediction tool for mortality found on pubmed, ovid, embase, scopus and ebsco database from inception until november . the primary outcomes were mortality (icu mortality, inhospital mortality, and -day mortality). studies reporting sensitivity and specificity of the qsofa making it possible to create a x table were included. the diagnostic odds ratio (lndor) was summarized following the approach of dersimonian and laird using the software r ('mada' package). the summary roc curve was created using the reistma model (bivariate model). the revman software was used to organize the data. results: the search strategy yielded citations. of unique citations, met the inclusion criteria ( , patients). the sensitivity and specificity from each study are shown in fig. . the meta-analysis of the dor was . ( % confidence interval (ci): . - . ) and of the lndor was . ( % ic: . - . ) (fig. ) . the pooled area under the summary receiver operating characteristic (sroc) curve was . . the summary estimative of the sensitivity was . and the false positive rate was . , by bivariate diagnostic random-effects metaanalysis. the chi-square goodness of fit test rejects the assumption of homogeneity, and the fit of the model for heterogeneity was better (p-value = . ). conclusions: the qsofa has a poor performance to predict mortality in patients admitted to the ed or icu. introduction: sepsis and septic shock patients are the most common cause of death in intensive care units. [ ] the aim of this study is to quantify the relationship between hours sequential organ failure assessment (sofa) scores change and in-hospital mortality as a treatment outcome in sepsis and septic shock patients. introduction: an outreach team, akin to a rapid response team, is made up of healthcare professionals assembled together for quick and effective reviews in managing of rapidly deteriorating or gravely deteriorated patients [ ] . this study aimed to look at the variety of patient referrals in terms of their severity, patient dynamics, reasons for referral and their subsequent dispositions. methods: patient records were randomly reviewed retrospectively from july to october . data were collated in an excel spreadsheet for comparison and then sorted in accordance with the clinical questions and percentages calculated. results: from the referrals, the severity criteria was done by calculating the national early warning score (news). it was found that % patients had a score of - , % had a score of - , and % scored more or equal to . % of patients were in the age range - years old. % referrals came from the emergency department (ed) where a consultant was involved in the decision of the referral; of this, % were referred during office hours of am to pm where there was greater manpower to aid management. % referrals came from inpatients on the general wards; % were done during office hours. % of referrals were transferred to ic/hd upon review; % were not, from whom died and were later admitted after procedures ( %) or because they deteriorated further ( %). for reasons for referrals and disposition decisions, see fig. . conclusions: despite having no set criteria for outreach team referrals, the accuracy rate was nearly % admissions to ic/hd based on clinician concerns. there was only % re-admission rate having been re-reviewed when the patients had not been deemed suitable for ic/hd admission initially. therefore referrals were done accurately and safely with the protocol of clinician referral openness directly to ic consultants. introduction: prompt recognition of patient deterioration allows early initiation of medical intervention with reduction in morbidity and mortality. this digital era provides an opportunity to harness the power of machine learning algorithms to process and analyze big data, automatically acquired from the electronic medical records. the results can be implemented in real-time. intensix (netanya, israel) has developed a novel predictive model that detects early signs of patient deterioration and alerts physicians. in this study we prospectively validated the ability of the model to detect patient deterioration in real time. methods: the model was developed and validated using a retrospective cohort of consecutive patients admitted to the intensive care unit in the tel-aviv sourasky medical centera tertiary care facility in israel, between january and december . in this study, we tested model performance in real time, on a cohort of patients admitted to the same icu between june and august . significant events that lead to major interventions (e.g. intubation, initiation of treatment for sepsis or shock, etc.) were tagged upon medical case review by a senior intensivist, blinded to model alerts. these tags were then compared with model alerts. [ ] [ ] [ ] [ ] . reviews occurred despite 'low news' (fig. ) . rrt review led to cc admission in ( . %) cases; median [iqr] news [ ] [ ] [ ] [ ] [ ] [ ] . probability of admission increased with higher news (fig. ), however admissions had 'low news'. of these were excluded due to high news trigger in the preceding hrs or post-operative status. the remaining ( . %) represented genuine low news cases; age [ - ], % male, admission apache ii [ - ] and day sofa [ ] [ ] [ ] [ ] [ ] . admission source was emergency department %, medical %, surgical %. diagnoses are shown in table . no low news patients with sepsis were qsofa positive. cc length of stay was [ ] [ ] [ ] [ ] days and icu mortality was . %. conclusions: a high proportion of rrt activity occurs at low levels of abnormal physiology. despite an association between news and cc admission, news fails to trigger for approximately one in ten admitted cases. clinical concern remains an important component of the escalation of acutely ill patients. meanwhile, novel markers of deterioration should be sought and validated. introduction: although rapid response systems are known to reduce in-hospital cardiac arrest rate, their effect on mortality remains debated. the rapid response call (rrc) is a system designed to escalate care to a specialised team in response to the detection of patient deterioration. there are diurnal variations in hospital staffing levels that can influence the performance of rapid response systems and patient outcomes. the objective of this study was to examine the relationship between the time of rrc activations and patient outcome. methods: review of retrospectively collected, linked clinical and administrative datasets, at a private hospital during a -month period. all patients with medical emergency team activation were included. rapid response calls occurring between : - : were defined as 'out of hours'. results: between january and october there were rrc. the trigger for rrcs activation was nurse concern ( ; . %), modified early warning score ( ; . %) and cardiac arrest ( ; . %). rrcs were "out of hours" being the main activation trigger a modified warning score > . "out of hours" patients had higher icu admissions ( . % versus %) and were more likely to have an inhospital cardiopulmonary arrest (or= . , p< . ). conclusions: the diurnal timing of rrcs appears to have significant implications for patient outcomes. out of hours calls are associated to a poorer outcome. this finding has implications for staffing and resource allocation. and septic shock) and severe sepsis (incl. septic shock) using icd- codes coded as primary and secondary discharge diagnoses and procedural ops codes. we assessed incidences and discharge disposition incl. mortality. results: incidences, mortalities and discharge disposition comparing and and the mean annual increase in incidence rates are reported in tables and . conclusions: the annual increase in standardized sepsis incidence rates is greater than in infections, but similar to the increase in infectious disease patients with organ dysfunction, which are less prone to coding incentives than sepsis codes. an increasing number of patients is discharged to nursing homes and hospice. given the alarming increase in sepsis cases and deaths, this analysis confirms sepsis as a key priority for health care systems. introduction: patients with urgent admissions to the hospital on weekends may be subjected to a higher risk of worse outcomes, which may be due to differences in compliance to established processes. because delays to antibiotic administration is an important measure of sepsis protocol efficiency and has been associated to worse outcomes, we aimed to assess the association of the weekend effect (admissions on weekend) with timing to antibiotic administration. methods: patients included in the sepsis protocol in the emergency department (ed) of hospital sao rafael, from january to july were retrospectively evaluated. sepsis protocol is supposed to be activated to every patient with a suspected sepsis diagnosis in the ed. we evaluated the association of weekend (saturday or sunday) admission with timing to antibiotic administration. introduction: current sepsis guidelines emphasize resuscitation of hypotension to a mean arterial pressure (map) of at least mmhg [ ] . a map less than mmhg appears to be associated with poor outcomes in postoperative patients in the intensive care unit (icu) [ ] . however, extent of hypotension in critically ill septic patients during icu stay and its relationship with adverse outcomes is poorly defined. we determined the magnitude of hypotension in icu patients with a diagnosis of sepsis and its association with major complications. conclusions: reduced mortality may be supposed to be correlated to a quicker recovery of organ damage sepsis related. pcrts should be warranted in the future to corroborate these preliminary data. introduction: the pd- /pd-l immune checkpoint pathway is involved in sepsis-associated immunopathy. we assessed the safety of anti-pd-l (bms- , bristol-myers squibb) and its effect on immune biomarkers and exploratory clinical outcomes in participants with sepsis-associated immunopathy. methods: participants with sepsis/septic shock and absolute lymphocyte count <= cells/μ l received bms- i.v. ( - mg; n= ) or placebo (pbo; n= ) + standard of care and were followed for d. primary endpoints were death and adverse events (aes); secondary endpoints were monocyte (m)hla-dr levels and clinical outcomes. methods: this observational study was performed using a prospective, multi-center registry of septic shock. we compared the -day mortality between patients who were excluded from the new definition (defined as < mmol/l after volume resuscitation) and those who were not (lactate level >= mmol/l after volume resuscitation), from among a cohort of patients with refractory hypotension, and requiring the use of vasopressors. results: of patients with refractory hypotension, requiring the use of vasopressors, had elevated lactate levels, while did not have elevated lactate levels (neither initially nor after volume resuscitation), and ( . %) had elevated lactate levels initially, which normalized after fluid resuscitation (fig. ). thus, these patients were excluded by the new definition of septic shock. significantly lower -day mortality was observed in these patients than in those who had not been excluded ( . % vs . %, p= . ). conclusions: it seems reasonable for septic shock to be defined by the lactate levels after volume resuscitation, however due to small sample size further large scale study is needed. results: significant downregulation (p< . ) of about pro-and anti-inflammatory cytokines, including il- , ip- , tnf-a, mip- a, mip- ß, il- , was documented. ifn-g effect on macrophages and dendritic cells was inhibited at the level of phosphorylated stat . ifn-ginduced expression of cxcl and cxcl in macrophages was reduced. patients treated in vivo with higher dosages of apoptotic cells had lower cytokine/chemokine levels compared to those treated with lower levels, and in inverse correlation to agvhd staging. in vitro binding of apoptotic cells to lps was documented. conclusions: the cytokine storm is significantly modified towards homeostasis following apoptotic cell treatment. the mechanism is multifactorial and was shown to include tam receptor triggering, nfkb inhibition, and lps binding. these results together with previous studies showing significantly higher murine survival in sepsis models of lps and cecal ligation puncture suggest that apoptotic cells may be used to treat patients with sepsis. a multicenter clinical trial in septic patients is planned in . moreover, the urine output significantly increased in survival group. conclusions: the present study suggests that cytokine-oriented critical care using pmma-chdf might be effective the treatment of sepsis and ards, particularly,in the treatment of ards associated with aspiration pneumonia in elderly patients. the polymyxin b immobilized fiber column direct hemoperfusion has an effect for septic shock but has no effect on sepsis: a cohort study and propensity-matched analysis k hoshino introduction: overwhelming cytokine release often referred to as "cytokine storm" is a common feature of septic shock, resulting in multiple organ dysfunction and early death. attenuating this cytokine storm early by eliminating cytokines may have some pathophysiological rationale. our aim was to investigate the effects of extracorporeal cytokine removal (cytosorb) therapy on organ dysfunction and inflammatory response within the first hours from the onset of septic shock. methods: patients with: sepsis of medical origin, on mechanical ventilation, noradrenaline > mg/min, procalcitonin > ng/ml and no need for renal replacement therapy, were randomized into cytosorb and control groups. cytosorb therapy lasted for hours. in addition to detailed clinical data collection, blood samples were taken to determine il- , il- ra, il- , il- , il- , tnf-α, pct, crp levels. introduction: blind pericardiocentesis leading to low success rate and high complication rates such as ventricular wall or oesophageal perforations, pneumothorax or upper abdominal organ injury.real time needle visualisation is allowing us to avoid this major complication [ ] . methods: we presented cases of acute traumatic cardiac tamponade secondary to severe chest injury. both patients presented with haemodynamic instability and echocardiographic features of pericardial tamponade. pericardiocentesis under ultrasound guidance at left parasternal area with needle directed from medial to lateral technique were performed (fig. ) . real time needle tip visualisation done throughout the procedure (fig. a) . needle placement in pericardial space was confirmed with agitated saline and guidewire visualisation (fig. b) . pigtail catheter was inserted and blood was aspirated until the patient were haemodynamically improved. repeated ultrasound was done to confirm the absence of ultrasonographic features of tamponade and complications. results: we demonstrated a successful real time needle visualisation ultrasound guided pericardiocentesis in cases acute traumatic pericardial tamponade. procedural time (time from needle piercing the skin to time needle entering the pericardium) in both cases were less than minute. post procedural ultrasound confirmed no major complications. conclusions: the real time needle visualisation using ultrasound was important to reduce major complications during pericardiocentesis. the safety of the highly invasive procedure can be improved with real time needle visualisation. osman a et al. eur j emerg med (in press), introduction: diagnosis of cardiac tamponade post continuous-flow left ventricle assist devices (cf-lvads) is challenging due to missing pulsatility. recent case study of sublingually microcirculation with incident dark-field imaging (idf) provide a new improved imaging for clinical assessment of cardiac tamponade in a patient with cf-lvad. we sought to examine the changes in microvascular flow index (mfi) as a sign of cardiac tamponade following lvad implantation. methods: off-site quantitative analysis of sublingual microcirculation clips with automated vascular analyses software (ava; microvision medical©), and the velocity distributions followed during admission till discharge in patients with end-stage heart failure treated with cf-lvad complicated by cardiac tamponade. results: eleven out of thirty lvad implantations, males, mean age ± years, april to january , (( heart mate (hm ) and heartmate ii (hm ii) (thoratec corp., ca)), were complicated by rethoracotomy due to early postoperative cardiac tamponade within week. there sublingual microcirculation was examined by a novel incident dark-field imaging (idf) before and daily post-lvad implantation. pre-lvad microcirculation was typical for heart failure, characterized by slowly, sludging movement of red blood cells (rbcs), (fig. a arrows) . directly after implantation, a normal microcirculatory flow was seen with a high rbcs velocity (fig. b) . on the day of tamponade the patients were stable except for severe failure of microcirculation as reflected by drop in mfi (fig. c ) and congestion in venules (* in fig. c ). in out of patients there was a significant drop in mfi before tamponade was clinically recognized (p< . ). shortly after rethoracotomy a quick restoration of microcirculatory flow has been found. conclusions: sublingual microcirculation imaging is a simple and sensitive non-invasive tool in early detection of cardiac tamponade. survey on the use of cardiovascular drugs in shock (ucards) - results: a total of physicians responded. as detailed in table , the respondents think that dobutamine is first-line inotrope to increase cardiac pump function (n= , %) and should be started when signs of hypoperfusion or hyperlactatemia despite adequate use of fluids and vasopressors in the context of low left ventricular ejection fraction are present (n= , %). the most accepted target was an adequate cardiac output (n= , %). the combination of noradrenaline and dobutamine was preferred to single treatment with adrenaline mainly due to possibility to titrate individually (n= , %). the main reason for adding another inotrope was to use synergistic effects of two different mechanisms of action (n= , %). according to respondents, phosphodiesterase-inhibitors should be used in the treatment of predominant right heart failure because of prominent vasodilatory effect on the pulmonary circulation (n= , %). they also believe levosimendan is the only inotrope that does not increase myocardial oxygen demand (n= , %). vasodilators are used in cardiogenic shock to decrease left ventricular afterload (n= , %). there is no experience or no opinion about the use of ß-blockers in shock states (n= , %). conclusions: this web-based survey provided latest trends on inotrope use in shock states which showed considerable diversity among respondents in opinions about its use. introduction: recent literature data clearly indicated that in patients with shock the resuscitation of macro-circulation often does not match with microcirculation and tissue perfusion improvement. unfortunately, the bed-side assessment of regional perfusion remains difficult, particulary in critically ill patients. in the last years thermography has been used in different medical fields but no studies have been performed on the use of this technique in critically ill patients. the aim of this study was to evaluate whether thermography is feasible and may provide useful data during resuscitation of patients with septic shock. methods: in patients with septic shock we collected central systemic temperature and infrared images (flir-t digital camera) of limbs at , , and hours after shock occurrence. thermal pattern distribution of the limbs was obtained by a specific analysis of the images (thermacam™researcher p). a systemic to peripheral temperature gradient called "Δ systemic-limb temperature" was calculated for each single temperature data collected. results: macrocirculatory and perfusion parameters improved in all the patients throughout the study period: mean values of noradrenaline dose decreased from . to . γ/kg/min, mean map increased from to mmhg and mean blood lactate decreased from . to . mmol/l. the "Δ systemic-limb temperature" pattern showed an heterogenous time course in the patients with a mean overall increase at and hours (fig. ) . conclusions: as expected, the regional data obtained by thermography did not match with macrocirculatory and systemic perfusion parameters. the significance and the relationship between treatments and data observed will be investigated by appropriate studies. regional differences in the treatment of refractory septic shockan analysis of the athos- data introduction: vasodilatory shock is a common syndrome with high mortality. despite established care protocols, regional differences in treatment remain. we sought to characterize these differences using data from the recently published athos- study [ ] . methods: individual patient data were analyzed at baseline and at h for regional differences in demographics, clinical characteristics, and treatment patterns, and grouped according to four geographical areas: the united states (us), canada (ca), europe (eu) and australasia (au). p-values were calculated by kruskal-wallis tests for continuous data and chi-square tests for categorical data. subsequent temporal analysis compared changes in the treatment of shock, indexed by changes in patient acuity level. results: regional differences existed with respect to bmi (p= . ), albumin (p< . ), cvp (p= . ), meld score (p= . ), apache ii score (p= . ) and sofa score (p= . ). baseline norepinephrine (ne) and ne equivalent doses were significantly higher in eu (p< . and p= . , respectively), and utilization of vasopressin was correspondingly lower (p< . ). at baseline, stress dose steroids were utilized to a greater extent in the us and ca (p= . ). temporal analysis revealed differences in the utilization of vasopressin and steroids with changes in patient acuity: in eu, increasing acuity was associated with a lower utilization of vasopressin, and in ca, increased acuity was associated with a lower utilization of steroids. steroid utilization was higher with increased level of acuity in au and the us. conclusions: significant differences in the treatment of vasodilitory shock exist globally, with important implications: (a) there are introduction: levosimendan is a calcium sensitizer and katp-channel opener exerting sustained hemodynamic and symptomatic effects. in the past fifteen years, levosimendan has been used in clinical practice also to stabilize at-risk patients undergoing cardiac surgery. recently, the three randomized, placebo-controlled, multicenter studies licorn [ ] , cheetah [ ] and levo-cts [ ] have been testing the peri-operative use of levosimendan in patients with compromised cardiac ventricular function. over smaller trials conducted in the past [ ] suggested beneficial outcomes with levosimendan in peri-operative settings. in contrast, the latest three studies were neutral or inconclusive. we aim to understand the reasons for such dissimilarity. methods: we re-analyzed the results of the latest trials in the light of the previous literature to find sub-settings in which levosimendan can be demonstrated harmful or beneficious. results: none of the three latest studies raised any safety concern, which is consistent with the findings of the previous smaller studies. in levo-cts, mortality was significantly lower in the levosimendan arm than in the placebo arm in the subgroup of isolated cabg patients ( fig. ) [ ] . the trend towards both hemodynamic and long term mortality benefits is maintained in recent meta-analyses [ , ] including the three larger recent studies. conclusions: despite the fact that the null hypothesis could not be ruled out in the recent trials, we conclude that levosimendan can still results: patients were included in levosimendan group and in control group. in the whole population, weaning failure incidence and mortality was comparable between the groups (respectively % vs %, pr , and % vs %, pr= , ). higher assistance duration, longer stay under mechanical ventilation and longer duration of stay in critical care unit were observed in levosimendan group. in the post-cardiotomy sub-group (table ) , weaning failure was lower in levosimendan group ( % vs %, pr , ) and levosimendan was an independent protective factor from weaning failure (or , , pr , ). positive impact of levosimendan may be explained in part by his calcium sensitizer effect and by facilitating recovery of myocardial calcium homeostasis in postcardiotomy cardiac stunning. conclusions: levosimendan failed to reduce the incidence of ecmo weaning failure, except for post-cardiotomy population. renal outcomes of vasopressin and its analogues in distributive shock: a systematic review and meta-analysis of randomized trials introduction: venous return (vr) is driven by the difference between mean systemic filling pressure (msfp) and right atrial pressure (rap) and determines the maximum ecmo flow. msfp depends on stressed volume and vascular compliance. it can be modified by absolute blood volume changes and shifts between stressed and unstressed volume. norepinephrine (ne) may increase stressed volume by constriction of venous capacitance and at the same time increase the resistance to systemic flow. we therefore studied the effects of ne on msfp, maximum ecmo flow and the ecmo pressure head (map-rap). methods: msfp was measured with blood volume at euvolemia and ne to ( . , . and . μg/kg/h) in a closed-chest porcine va-ecmo model (n= , central cannulation with left atrial vent and avshunt) in ventricular fibrillation. the responses of rap and vr (measured as ecmo flow, qecmo) were studied at variable pump speeds including maximum possible speed without clinically apparent vessel collapse at constant airway pressure. results: the ecmo pump speed and qecmo showed a strictly linear relationship (r . to . , range over all conditions) despite increased pressure head, indicating that the maximum qecmo was determined by vr alone. ne led to both increases in msfp and qecmo in a dose dependent way, indicating a rightward shift in the vr plot ( fig. ) via recruitment of stressed from unstressed volume ( table , fig. ). this resulted in an increased msfp during ne despite decreased absolute blood volume ( . ± . l vs. . ± . l, p= . ). the reduced blood volume was associated with hemoconcentration suggesting plasma leakage. conclusions: ne shifts the vr curve to the right, allowing a higher maximum ecmo flow. the ne induced increase in msfp results from recruitment of unstressed volume to stressed volume, which may be modified by changes in vascular compliance. the effects on pump afterload were not limiting. introduction: to locate vessels for percutaneous central venous catheterizations, it may be helpful to apply not only real-time ultrasound (us) guidance but also us-assistance vein prelocation. the aim of this study was to evaluate the superiority of two us methods compared to surface landmark methods by reviewing randomized control trials (rcts). methods: as updating an earlier systematic review [ ] , we searched pubmed and central in november . we included rcts which compared the failure rates of internal jugular or femoral venous cannulations among ) real-time us guidance, ) us-assistance vein prelocation and ) surface landmark methods. a frequentist network meta-analysis was conducted using the netmeta package on r. results: out of citations, rcts ( patients) were eligible. the number of studies comparing outcomes between real-time us guidance vs. surface landmark methods, us-assistance vein prelocation vs surface landmark methods and real-time us guidance vs us-assistance vein prelocation was , and . regarding cannulation failure rate, network meta-analysis in a fix-effect model showed that a p-score was lower in the real-time us guidance than us-assistance vein prelocation ( . vs. . ), by reference to surface landmark methods, and also regarding arterial punctures, a p-score was lower in the real-time us guidance than us-assistance vein prelocation ( . vs. . ). conclusions: based on the present network meta-analysis of rcts, pscores of cannulation failure and arterial puncture were lower in the real-time us guidance, suggesting that the us-assistance vein prelocation is superior than the real-time us guidance, both of which achieve lower rates of failure and arterial puncture compared to the landmark methods. we speculates that the inferiority of real-time guidance is associated with difficulties in manipulating the needle together with an echo probe in targeting relatively smaller veins in children. introduction: we present a case report of 'shoshin beriberi' in a young female who was 'fussy with food' that developed an acutely progressive metabolic acidosis and multi-organ failure requiring intensive care support. methods: our patient was a -year-old british woman who presented to the emergency department (ed) with a ten-day history of diarrhea, vomiting and increasing fatigue. she had a past medical history of gastroparesis, polycystic ovary syndrome (on metformin), laparoscopic cholecystectomy and hysteropexy. she lived with her husband and two children who had viral gastroenteritis two weeks previously. results: the patient had a metabolic acidosis (ph . ) with raised lactate (> ) on initial blood gas in the ed. a . % sodium bicarbonate infusion and hemofiltration were commenced overnight. the patient's ph and lactate remained static with an increasing work of breathing over this period. by morning she developed flash pulmonary oedema and hypotension, the first signs of acute cardiac failure. an echocardiogram displayed severely impaired left ventricular function with ejection fraction of %. the patient was intubated and inotropic support was commenced. it was thought that a micronutrient deficiency may have caused a rapid onset cardiac failure. pabrinex (containing ml of thiamine hydrochloride) was commenced and within hours the patient's metabolic acidosis markedly improved ( fig. ). complete reversal of the cardiac failure occurred over hours. conclusions: shoshin is a rare clinical manifestation of thiamine deficiency [ ] . it is an important differential diagnosis to bear in mind after excluding more common aetiologies of heart failure. especially in this case as our patient had no obvious risk factors at the time of presentation. we suggest empiric use of thiamine should be considered in treatment algorithms for young patients presenting with acute cardiac failure. the pateint had provided informed consent for publication. introduction: takotsubo syndrome (ts) is known to be an acute transient cardiac condition accompanied with acute heart failure. ts is often triggered by critical illness but that has been rarely studied in icu practice.therefore, it is known, that the use of catecholamines can directly induce ts, worsen lvot obstruction, and delay spontaneous recovery in ts patients, it is nearly impossible to avoid their administration in critically ill [ ] . methods: we have analyzed medical records from patients with ts, that were revealed during year in our hospital. ts was defined due to mayo criteria, including transient regional wall motion abnormalities, mildly elevated troponin level and no signs of obstructive cad on coronary angiography. results: out of patients who developed ts in icu or iccu, hemodynamic instability occurred in acute phase of ts in ( %) cases. ( %) of patients were admitted to icu in due to septic shock ( patients), major bleeding ( ), cerebral mass lesion ( ) and ards ( ) and required treatment with catecholamines. general mortality rate in ts patients was ( %), and ( %) in critically ill ts patients. mean duration of noradrenalin infusion was , days, dobutamine infusion , days. patients with ts needed more icu resources and longer icu-stay. mortality rate was higher in ts patients ( %) vs the icu-population ( %), p = . . conclusions: ts seems to be an often cause of lv dysfunction and acute heart failure in critically ill. it seems that ts could be a predictor of worse prognosis in critically ill patients. although catecholamine administration may worsen the patient prognosis and induce further ahf in critically ill patients it rearely can be avoided. introduction: previous studies on readmission following lvad implantation have focused on hospital readmission after dismissal from the index hospitalization. since there are very little data existing, the purpose of this study was to examine intensive care unit (icu) readmission in patients during their initial hospitalization for lvad implantation to determine reasons for, factors associated with, and mortality following icu readmission. methods: this was a retrospective, single center, cohort study in an academic tertiary referral center. all patients at our institution undergoing first time lvad implantation from february to march were included. patients dismissed from the icu who then required icu readmission prior to hospital dismissal were compared to those not requiring icu readmission prior to hospital dismissal. results: among lvad patients, ( . %) required icu readmission. the most common reasons for admission were bleeding and respiratory failure (fig. ) . factors found to be significantly associated with icu readmission were preoperative hemoglobin level of less than g/dl, preoperative estimated glomerular filtration rate < ml/min/ . m , preoperative atrial fibrillation, preoperative dialysis, longer cardiopulmonary bypass times, and higher intraoperative allogeneic blood transfusion requirements. mortality at year was . % in patients requiring icu readmission vs. . % in those not requiring icu readmission (age-adjusted or= . , % ci . to . , p= . ). conclusions: icu readmission following lvad implantation occurred relatively frequently and was associated with significant one-year mortality. these data can be used to identify lvad patients at risk for icu readmission and implement practice changes to mitigate icu readmission. future larger and prospective studies are warranted. atrial fibrillation and infection among acute patients in the emergency department: a multicentre cohort study of prevalence and prognosis t graversgaard odense university hospital, odense, denmark critical care , (suppl ):p introduction: patients with infection presenting with atrial fibrillation (af) are frequent in emergency departments (ed). this combination is probably related to a poor prognosis compared to lone af or infection, but existing data are scarce. aim: to describe the prevalence and prognosis for af and infection individually and concomitantly in an ed setting. introduction: its afterload reducing effects make peep the treatment of choice for cardiogenic pulmonary edema. studies indicate that peep may lower coronary blood flow. its effects on left ventricular contractility is unclear. most of the surrogate measures for cardiac contractility are dependent on afterload and contractility assessment under peep may therefore be biased. we have investigated cardiac contractility under peep with the endsystolic pressure volume relationship (espvr) as a load-independent measure of contractility. methods: patients scheduled for coronary angiography were ventilated with cpap and a full face mask at three levels of peep ( , and cmh o) in random order. structural cardiac pathologies were excluded with echocardiography. at every peep level, left ventricular pressure volume loops (millar conductance catheter with inca system, leycom, netherlands) were obtained. the endsystolic elastance was derived from a pv-loop family under preload reduction with an amplatzer sizing balloon in the inferior caval vein. all participants gave written informed consent. the study was approved by the bernese ethics committee. results: women and men with an age ± years were studied. ejection fraction was ± % at baseline. mean espvr at peep levels of , and were . ± . , . ± . and . ± . mmhg/ml (p = . , repeated measurements anova). dp/dt and ejection fraction did not differ between the peep levels (p= . and . ). conclusions: moderate levels of peep did not influence endsystolic elastance. higher peep and patients in cardiogenic shock should be investigated. introduction: we sought to assess the feasibility of d volumetric analysis with transthoracic echocardiography in critically ill patients. we choose a cohort typical of icu where accurate volumetric analysis is important: hypoxic, mechanically ventilated patients. d analysis is enticing in simplicity and wealth of data available. it is accurate in cardiology patients [ ] but has not been assessed in the icu. methods: patients were imaged within hours of admission. inclusion criteria: adult, hypoxic (p:f < ), mechanically ventilated, doppler stroke volume (sv) assessment possible. echocardiography: seimens sc real-time volumetric analysis with standard b-mode and doppler assessment. images unacceptable if > segments unable to be seen in volumetric planes. d left ventricle (lv) and right ventricle (rv) analysis with tomtec imaging and seimens acuson respectively and compared to doppler derived sv. % limit of agreement considered clinically acceptable [ ] . imaging was optimised for volumetric analysis ( - vols/sec). results: patients, in sinus, in af. no significant difference seen between doppler vs d simpson's biplane, d lv or d rv sv estimation. feasibility, sv values and bias are reported in table and fig. . limit of agreement for corrected doppler vs lv d sv = - % to %; rv d sv = - . % to . %. conclusions: d lv and rv volumetric analysis is feasible in majority of patients requiring mechanical ventilation, however lacks agreement with doppler derived stroke volume assessment. although images may appear sufficient, the semi-automated software appears to underestimate stroke volume. further larger studies using thermodilution are warranted. introduction: body position changes such as leg raising are used to determine fluid responsiveness. we hypothesized that the trendelenburg position increases resistance to venous return. together with abolishment of the hepatic vascular waterfall, this may limit the increase in regional blood flow. methods: inferior vena cava (ivc), portal vein (pv), hepatic, superior mesenteric (sma) and carotid artery blood flows and arterial, right atrial (ra) and hepatic (hv) and portal venous blood pressures were measured in anesthetized and mechanically ventilated pigs in supine and °trendelenburg positions. all hemodynamic parameters were measured during end-expiration at cmh o peep, and at inspiratory hold with increasing airway pressures (awp) of , , and cmh o, respectively. paired t test was used to compare pressures and flows in different positions during end-expiration. repeated measures anova was performed to evaluate the effects of awp on hemodynamic parameters. results: trendelenburg position significantly increased ra, hv and pv blood pressures at end-expiration, while qpv and qsma remained unchanged, qha increased and qivc showed a trend to decrease (table ). in both positions, all blood flows decreased with increasing awp, and the difference between ppv and qsma became smaller, indicating splanchnic blood pooling ( table ). in the trendelenburg position, splanchnic blood pooling was less severe compared to supine position. conclusions: trendelenburg position tended to decrease venous return from inferior vena cava. further increases in rap by augmenting awp led to a decrease in all flows and signs of abolished hepatic vascular waterfall. passive manoeuvers to assess fluid responsiveness evoke complex hemodynamic reactions which are not fully understood. introduction: despite of preventive measures, the incidence of deep venous thrombosis (dvt) in icu patients is estimated to range from - %. while clinical diagnostics is unreliable, ultrasound compression test (uct) has proven to be a highly sensitive and specific modality for the recognition of lower extremity dvt [ ] . delegating this competence to icu nurses can increase uct availability and enable preventive dvt screening. therefore, we decided to conduct a clinical study to evaluate the sensitivity and specificity of uct performed by general icu nurse in icu patients compared to an investigation by icu physician certified in ultrasound. methods: prior to the study, each nurse participating in the study completed one-hour training in uct and examined patients under supervision. then, icu patients without known dvt underwent uct in the femoral and popliteal region of both lower extremities performed by trained general icu nurse. on the same day, the examination was repeated by an icu physician. the results of the examinations of each patient were blinded to each other for both investigators until both tests were performed. in case of a positive test, the nurse immediately reported the result to the icu physician. the sensitivity and specificity of the test performed by general nurse was calculated in comparison with the examination by a specialist. results: a total of patients were examined. both lower extremities were examined in all patients. the prevalence of dvt of , % has been found. the overall sensitivity of the examination performed by general nurse was . %, the specificity % with negative predictive value of . %, positive predictive value of % and accuracy of . %. the results of our study have shown that general icu nurses are able to perform bedside screening of dvt by compression ultrasound test with a high degree of reliability after a brief training. methods: a cytosorb® (cytosorbents, new jersey, usa) ha device was inserted within the cpb circuit in ten patients undergoing elective cardiac surgery. one hour after cpb onset, the activity of coagulation factors (antithrombin (at), von willebrand factor (vwf), factors ii, v, viii, ix, xi, and xii) were measured before and after the device. pre and post device measurements were compared using student ttest, a p value < . was considered statistically significant. results: patients' mean age was . ± . years, % were female, the mean euroscore ii was . ± . . procedures were: coronary artery bypass graft (cabg) ( / ), aortic root replacement ( / ) and cabg combined with aortic valve replacement ( / ). mean cpb duration was . ± . min. pre and post ha measurements of coagulation factors activity are presented in fig. . post-device at and fii activity was significantly lower (respectively from . to . , p= . and from . to . , p= . ) compared to predevice measurement. there was no statistically significant difference between pre-and post-ha measurements for all other coagulation parameters conclusions: pre and post ha cytosorb® measurements for coagulation factor activity were not different except for a small decrease in at and fii activity. this might be related with intra-device consumption or adsorption. further analyses accounting for cpb fluid balance, the entire study population and timepoints are pending. introduction: the aim of this study is to evaluate changes in hemodynamics and microvascular perfusion during extracorporeal blood purification with cytosorb in patients with septic shock requiring renal replacement therapy. methods: eight adult patients with septic shock requiring continuous renal replacement therapy for acute renal failure were enrolled and underwent a -hour treatment with the emodasorption cartridge cytosorb. measurements were taken at baseline before starting cytosorb, after h (t ) and h (t ) and included: blood gases, macrohemodynamic parameters (picco ), vasopressor and inotropic dose, plasma levels of cytokines (interleukin [il]- , il , il , il , tumor necrosis factor alpha) and parameters of microvascular density and perfusion (sublingual sidestream dark field videomicroscopy). procalcitonin was measured at baseline and after h of treatment. results: a non-significant decrease in plasma levels of cytokines was observed over time. hemodynamic parameters and vasopressor requirement remained stable. the microvascular flow index increased significantly at t , total vessel density and perfused vessel density increased at t and t ( introduction: objective renal replacement therapy (rrt) with the oxiris filter is used in sepsis septic shock with aki, but few clinical studies compare the adsorbing effect of oxiris filter on the inflammatory mediators to rrt. the aim of this study is -to confirm whether oxiris decreases cytokines and procalcitonin in sepsis septic shock. -this effect is superior to rrt. -this translates in a better cardio renal response. methods: a coohort study and a propensity-matched analysis included patients admitted to three intensive care (aurelia hospital, european hospital, tor vergata -rome) with a diagnosis of septic shock. patients were submitted to rrt with oxiris filter and patients to rrt.il , procalcitonin, the cardiorenal indices and sofa score were compared before (t ) and at the end of the treatments (t ). all data are expressed as mean±sd. anova one way was used to compare the changes of the variables in the time. p< . was considered statistically significant. results: of patients submitted to rrt with the oxiris filter could be matched to septic patients who received rrt. il and procalcitonin decreased in the oxiris group (p< . ) but not in the rrt group.-map increased (p< . ) and noradrenaline dosage decreased in oxiris group (p< . ), but non in rrt group. also pao /fio ratio, diuresis, sofa improved only in the in the oxiris group (p< . ). conclusions: in sepsis/septic shock patients with aki, il and procalcitonin decrease more in the oxirs group then in the rrt group.this is associated with an improvement of the cardio -renal function and the clinical condition.the study confirms that rrt with oxiris filter may be useful in sepsis/septic shock when other convective/diffusive techinques fail. introduction: advos (hepa wash gmbh, munich, germany) is a recently developed ce-certified albumin-based hemodialysis procedure for the treatment of critically ill patients. in addition to the removal of water-soluble and albumin-bound substances, acid-base imbalances can be corrected thanks to an automatically regulated dialysate ph ranging . to . . methods: patients treated with the advos procedure between in the department of intensive care medicine of the university medical center hamburg-eppendorf were retrospectively analyzed. overall treatments in critically ill patients (mean sofa score ) were evaluated. additionally, subgroup analysis for hyperbilirubinemia, respiratory acidosis and non-respiratory acidosis were conducted. results: severe hyperbilirubinemia (> mg/dl) was present in treatments, while and treatments were performed to treat respiratory (paco > mmhg) and non-respiratory (paco < mmhg) acidosis (ph< . ), respectively. mean treatment duration was h. advos procedure was able to correct acidosis and reduce bilirubin, bun and creatinine levels significantly. the subgroup analysis shows an average bilirubin reduction of % per advos multi treatment in the hyperbilirubinemia group ( . mg/dl vs . mg/dl, p< . ). moreover, ph ( . vs. . , p< . ) and paco ( . vs. . mmhg, p< . ) were corrected in the respiratory acidosis group, while in the non-respiratory acidosis group, an improvement in ph ( . vs. . , p< . ), hco ( . vs. . , p= . ) and base excess (- . vs. - . , p= . ) could be observed. there were no treatment-related adverse events during therapy. conclusions: advos is a safe and effective hemodialysis procedure, which is able to remove water soluble and protein bound markers and correct severe acidosis in critically ill patients. score for timely prescribing (stop) renal replacement therapy in intensive care unit -preliminary study of a mneumonic approach introduction: the moment of initiation of renal replacement therapy (rrt) in critically ill patients and a reason for debate, without having objective criteria that indicate it. the objective of this study was to propose a score to help identify the ideal time for the initiation of rrt, and if there is correlation between this score and intensive care unit length of stay and mortality. methods: patients admitted to the intensive care unit, > -yearsold, to whom rrt were indicated by the intensivist. the study protocol was approved by the hospital das forças armadas ethical committe, and written informed consent was obtained from all patients. the stop was assigned according to the presence or not of each of the items (fig. ). they were classified into groups a and b according to fig. , and the group change was recorded. results: patients admitted to icu in the period, excluded for limitation of therapeutic efforts. were admitted to the study, with the mean age of . years; , % males (n= ). distribution among the groups: a (n= , . %), a ( , . %), a ( , . %), b ( , . %), b ( , . %) e b (no patients). there were statistically significant correlation between group change and mortality (p . ), and between the stop and nephrologist agreement (p . ). there was no correlation between stop value and icu los (p , ) or stop and mortality (p . ). conclusions: the stop value is correlated with hemodialysis indication agreement between intensivists and nephrologists, and not correlated with icu los or mortality. the group change was correlated to increased mortality, in the study population. the significance of stop as a tool in determining the moment of initiation of renal replacement therapy remains a work in progress. introduction: liver transplant (lt) in patients with renal dysfunction presents intraoperative challenges and portends postoperative morbidity. continuous renal replacement therapy (crrt) is increasingly used for intraoperative support; however, there is a paucity of data to support this practice. methods: pilot randomized open-label controlled trial in adults receiving cadaveric lt with a modification of end-stage liver disease (meld) score >= and preoperative acute kidney injury (kdigo stage ) and/or estimated glomerular filtration rate < ml/min/ . m . patients were randomized to intraoperative crrt (icrrt) or standard of care. primary endpoints were feasibility and adverse events. secondary endpoints were changes in intraoperative fluid balance, complications, and hospital mortality. analysis was intention-to-treat. results: sixty patients were enrolled, ( %) were randomized ( to icrrt; to control). mean (sd) was age ( ) years, meld was ( ), % (n= ) had cirrhosis; % (n= ) received preoperative rrt; and % (n= ) were transplanted from icu. one patient allocated to icrrt did not receive lt. seven ( %) allocated to control crossed over intraoperatively icrrt ( ( ) min, with only interruptions (all due to access). icrrt fluid removal was . l (range - . ). fluid balance was . l ( . ) for icrrt vs. . l ( . ) for control (p= . ). postoperative crrt was similar ( % vs. %, p= . ). there were no differences in reexploration (p= . ), mechanical ventilation time (p= . ), reintubation (p= . ), sepsis (p= . ), or mortality (p= . ). conclusions: in this pilot trial of high acuity lt patients, icrrt was feasible and safe. these data will inform the design of a large trial to define the role of icrrt during lt. clinicaltrials.gov: nct . the uptake of citrate anticoagulation for continuous renal replacement therapy in intensive care units across the introduction: the purpose of this descriptive study is to report the trend of citrate anticoagulation uptake, used for continuous renal replacement therapy (crrt), in intensive care units (icus) across the united kingdom (uk). citrate anticoagulation has been used in the uk since , but its uptake since then is unknown [ ] . methods: a survey questionnaire targeted pharmacists working in uk adult icus providing crrt. invitations to participate were distributed utilising the united kingdom clinical pharmacy association online forum as a platform for access. survey administration was by self-completion and submissions were accessible over a total of six weeks. basic demographic data, icu specifications, the citrate system in use and implementation details were sought. a descriptive statistical analysis ensued. results: responses were received of which were analysed after duplication removal. trusts, encompassing a total units, in the uk confirmed use of citrate anticoagulation for crrt. units reported a mean of days to implement a citrate system (range to days). prismaflex® (baxter) and multifiltrate (fresenius) were reported as the most commonly used citrate systems; ( . %) and ( . %) units respectively. conclusions: there are icus in the uk [ ] . we conclude that a minimum of units ( %) use citrate anticoagulation for crrt in uk critical care centres. citrate systems of anticoagulation are becoming an increasing popular choice for regional anticoagulation, falling in line with international guidance [ ] . these guidelines were introduced in which corresponds to increase national uptake. introduction: patients requiring renal replacement therapy (rrt) whilst on significant doses of vasoactive medications have often been deemed unsuitable to undergo ultrafiltration (uf). however with better understanding of the pathophysiology of renal injury [ ] in intensive care patients we hypothesise that vasopressor/inotrope requirement will not significantly increase with uf or with a more negative fluid balance (fb). methods: data was retrospectively collected in a general icu/hdu of adult patients requiring acute rrt for acute kidney injury. patients on chronic dialysis were excluded. percentage change in vasopressor index and mean arterial pressure were combined to form the combined percentage change (cpc) which we used as an index of patient stability. results: patients were assessed undergoing a total of rrt sessions. the mean age was with females and males. mean fb for the hours from start of rrt was + mls (range - to + mls). using a model to correct for significant covariates and plotting hour fb against cpc we found no significant effect of fb on stability p= . (fig. ). mean uf volume was mls (range - mls). there was a non linear relationship between uf and stability with moderate volumes improving but larger volumes worsening stability (fig. ). this did not reach statistical significance (p= . ) so may be due to chance but is likely due to a lack of power. conclusions: fluid balance has no effect on cardiovascular stability during rrt in our cohort but there may be a varying effect of uf depending on volume. introduction: exposure of blood to a foreign surface such as a continuous renal replacement therapy (crrt) filter could lead to activation of platelets (plt) and fibrinogen (fib) trapping. thrombocytopenia has been reported in adults on crrt but data in pediatrics are scarce. our institution uses regional citrate anticoagulation (rca) as standard of care with prefilter hemodilution and hf filters (polysulfone, surface area (sa) . m ) regardless of patients' (pts) age and size. as filter sa is relatively larger in younger pts, we aimed to investigate the impact of crrt filter change on hemostasis parameters in infants on crrt in up to first three filter changes. methods: retrospective chart review results: patients < kg were included, age . ( . - ) months, weight . + . kg, with filters. metabolic disease was the most common principal diagnosis ( / , %), liver failure (lf) was the most common comorbidity ( / , %). all patients received prefilter continuous venovenous hemodiafiltration with minimum dose of ml/ . m /h. thrombocytopenia was common at crrt start ( / , %). plts decreased in % filter changes ( / ) by + % (pre vs post plt ( - ) vs ( - ), p< . ). fibrinogen also decreased from ( - ) to ( - ), p< . ; there was no change in ptt, pt, or inr values before and after filter changes. bleeding events were seen in / ( %) of pts ( / of lf pts vs / others, p= . ), but were not more common in pts who had decrease in plts or fib with filter changes ( % with drop in plts vs % without, p= . ; % with drop in fib vs % without, p= . ). conclusions: thrombocytopenia is common in infants on crrt. further decreases in plt and fibrinogen can be seen in with crrt filter changes if the filters are relatively large compared to patient size. bleeding events seems more related to underlying comorbidity, and less to changes in hemostatis parameters observed with filter change but would need to be confirmed with further studies. intensive monitoring of post filter ionized calcium concentrations during cvvhd with regional citrate anticoagulation: is it still required? introduction: the aim of the present study was to evaluate the role of postfilter calcium concentrations (pfca) in terms of safety and efficacy in large retrospective cohort of patients treated with cvvhd and regional citrate anticoagulation. methods: retrospective, observational study at a university hospital with icus. all patients treated with rca-crrt were included in the study. results: among patients treated with rca-cvvh pfca at the start of the cvvhd was available in pts. the pfca concentrations were in target range ( . - . mmol/l) in the majority of patients ( %), whereas % and % of patients had the pfca below or above the target range, respectively. in the further h of cvvhd treatment the propotion of patients with targeted pfca increased to % and remained stable. at the start of the rca-cvvhd there was a significant but weak correlation between the pfca and ionized systemic ca (ica) with a spearman rank-order correlation coefficient (rho) of . (p < . ). the coefficient of variation of pfca concentraions was significantly higher if compared to the coefficient of variation of ica concentration. using per protocol adaptations the incidence of a severe hypocalcemia (< . mmol/l) was low and present only at first hours of therapy: % and % of patients with pfca below the target range and . % and . % of patients with pfca in target range, at h and h respectively (p< . ). there was no correlation between pfca concentrations and filter lifetime. the results of the present study support the previous reports about higher measurements variation of pfca compared to systemic ica ( ). nevertheless due to the weak correlation of ica and pfca as well as a low number of patients with a severe metabolic complication, the results of our study question the necessity of intensive pfca monitoring during rca-crrt. present results need to be validated in further trials. introduction: in critically ill patients, occurrence of pain is frequent and usually correlates with worse outcomes, such as prolonged icu length of stay (los) and mechanical ventilation. in this regard, pain leads to sympathetic activation, inflammatory mediators and therefore, potentially to organic dysfunction. the aim of this study is to evaluate the relationship between acute pain in critically ill patients and their association with acute kidney injury (aki). methods: retrospective cohort with adults patients admitted between june and june , from the icu of hospital sírio libanês hospital in sao paulo (brazil). main exclusion criteria were: length of stay < h, coma and previous aki. the predictor pain was obtained through daily electronic records according to numerical verbal scale ( - ). the outcome was defined as serum creatinine elevation equal to or greater than . mg/dl and/or greater than % increase at any time after the first hours in the icu. the multivariate analysis was performed by binary logistic regression through distinct groups of early or late predictive factors in relation to aki. results: after the exclusion of patients, the incidence of pain with numerical verbal scale equal to or greater than points was . %. the outcome occurred in . % of the cohort. in the binary regression, using the more early predictive factors, sex and pain presented independent relation with the outcome -adjusted or . ( . - . ) and . ( . - . ), respectively (p < . ). in the analysis conclusions: poor management of icu pain is associated to worse outcomes, including increased risk to aki. the search for a better pain management strategy in the icu scenario should therefore be reinforced. introduction: acute kidney injury (aki) is a common complication in hospitalised patients, strongly associated with adverse outcomes [ ] . a lack of baseline incidence and outcome data limits our ability to assess local strategies aimed at improving aki care. methods: in an audit in three linked inner london hospitals we interrogated our electronic patient data warehouse (cerner millennium power insight electronic data warehouse) with a specially written query to identify cases of aki, defined by kdigo creatinine criteria, in patients aged over y admitted for > h during january to june . we excluded palliative care and obstetric patients. in the absence of premorbid baseline (median - d pre-admission) the admission creatinine value was used. end stage renal disease (esrd) and primary sepsis diagnosis was obtained from icd coding. results: of admissions, we excluded with pre-existing esrd (hospital mortality . %) and with fewer than one creatinine result who could not be assigned aki status (mortality . %). of the remaining there were with aki ( . %), with mortality increasing from no aki group ( . %), to aki stage ( . %), and a further increase to aki stages - ( . %) (p< . ) ( table ) . patients with aki were older (p< . ), more likely to be medical than surgical (p< . ), more likely to have a primary sepsis diagnosis (p< . ) and had higher baseline creatinine (median vs p< . ). no known baseline was found in . % of patients with aki, but their mortality did not significantly differ to those with a baseline ( . % vs . %, p= . ). conclusions: an electronic query identified the local burden of aki and it's associated hospital-mortality; such baseline data is essential to assess the effect of quality improvement interventions in aki prevention and care. introduction: acute kidney injury (aki) is a common condition in critically ill patients [ , ] . loop diuretics are generally used as first line treatment. however, controlled trials show controversial results. we ought to search systematically and realize a metaanalysis on the matter. methods: an electronic search of randomized clinical trials in adult patient treated with diuretics for aki compared with standard treatment or a control group was conducted. the primary objective of the analysis was to assess recovery of renal function. secondary endpoints included time to recovery of renal function, need for renal replacement therapy (rrt), mortality in the intensive care unit (icu) and complications. introduction: increased venous pressure is one of the mechanism leading to acute kidney injury (aki) after cardiac surgery. portal flow pulsatility and discontinuous intra-renal venous flow are potential ultrasound markers of the impact of venous hypertension on organs. the main objective of this study was to describe these signs after cardiac surgery and to determine if they are associated with aki. methods: this single center prospective cohort study (nct ) recruited adult patients able to give consent. ultrasound studies were performed before cardiac surgery and repeated on post-operative day (pod) , , and . abnormal portal and renal venous flow patterns are defined in fig. . the association between the studied markers and the risk of new onset of aki in the following hours period following an assessment was tested using logistic regression with a % confidence interval. clinical variables associated with the detection of the signs were tested using generalized estimating equation models. this study was approved by the local ethics committee. results: during the study period, patients were included. the presence of the studied ultrasound signs is presented in fig. . during the week following cardiac surgery, patients ( . %) developed aki, most often on pod ( . %). the detection of portal flow pulsatility and severe alterations in renal venous flow (pattern ) at icu admission (pod ) were associated with aki in the subsequent hours period and was independently associated with aki in multivariable models including euroscore ii and baseline creatinine ( table ). the variables associated with the detection of abnormal portal and renal patterns were associated with lower perfusion pressure, higher nt-pro-bnp and inferior vena cava measurements (table ) . conclusions: abnormal portal and intra-renal venous patterns are associated with early aki after cardiac surgery. these doppler features must be further studied as potential treatment targets to personalize management. introduction: acute kidney injury (aki) is very prevalent after cardiac surgery in children, and associated with poor outcomes [ ] . the present study is a preplanned sub-analysis of a prospective blinded observational study on the clinical value of the foresight nearinfrared spectroscopy (nirs) monitor [ ] . the purpose of this subanalysis was to develop a clinical prediction model for severe aki (saki) in the first week of picu stay. methods: saki was defined as serum creatinine (scr) >/= times the baseline, or urine output < . ml/kg/h for >/= h. predictive models were built using multivariable logistic regression. data collected during surgery, upon picu admission, as well as monitoring and lab data until h before saki onset, were used as predictors. relevant predictors with a univariate association with saki, were included in the models. accuracy of the models was tested using bootstraps, by auroc and decision curves. results: children were enrolled, admitted to the picu of the leuven university hospitals after cardiac surgery, between october and november . patients were excluded. children ( . %) developed saki in the first week of picu stay. a multivariate model with admission parameters (maximum lactate during surgery, duration of cpb, baseline scr, rachs and pim scores), and postoperative measurements (average heart rate, average blood pressure, hemoglobin, lactate), was most predictive for saki ( fig. ) . conclusions: the risk of saki in children after congenital cardiac surgery could be predicted with high accuracy. future models will also include medication data. these models will be compared against and combined with nirs oximetry data to investigate the independent and added predictive value of the foresight monitor. introduction: acute kidney injury (aki) occurs in over % of the patients in the intensive care unit (icu). the predominantly ethiology of aki is septic shock, the most common diagnosis in the icu. aki significantly increases the risk of both morbidity and mortality [ ] . methods: icu patients with septic shock was studied within hrs from admission. patients after cardiac surgery served as control group. all patients were sedated and mechanically ventilated. renal blood flow (rbf) and glomerular filtration rate (gfr) were obtained by the infusion clearance of paraaminohippuric acid (pah) and by extraction of cr-ethylenediamine ( cr-edta). n-acetyl-β -d-glucosaminidase (nag), was measured. results: rbf was % lower, renal vascular resistance % higher and the relation of rbf to cardiac index was % lower in patients with septic shock compared to the control group. gfr ( %, p= . ) and renal oxygen delivery (rdo ) ( %) where both significantly lower in the study group (table ) . there was no difference between the groups in renal oxygen consumption (rvo ) but renal oxygen delivery was almost % lower in septic shock patients. renal oxygen extraction was significantly higher in the study group than in the control group. in the study group, nag was . ± . units/mikromol creatinine more, i.e times the value in patients undergoing cardiac surgery [ ] . conclusions: sepsis related aki is caused by a renal afferent vasoconstriction resulting in a reduced rbf and lowered rdo in combination with an anchanged rvo , this results in a renal oxygen supply/ demand mismatch. introduction: the primary aim was to determine if the addition of daily creatine kinase (ck) measurement was usefully guiding decision making in intensive care units within greater glasgow and clyde. methods: after a change to the daily blood ordering schedule to include ck, a retrospective audit was carried out covering a -month period within intensive care units. all patients with ck > units/ litre were included. basic demographics, apache score and admitting diagnosis were recorded. utility of ck was assessed by determining the associated diagnosis and whether the diagnosis was first considered (diagnostic trigger) due to ck level, clinical suspicion or haematuria. additionally, it was determined if and what actions had been taken based on the raised ck and associated diagnoses. results: data was collected from / / to / / . patients were captured with ck > units/litre from an average combined admission rate of patients/month [ ] . total male patients ( . %) and female ( . %). age range to years (mean . ). apache score range to (mean . ) with estimated mean mortality of . %. patients ( . %) had associated diagnoses with elevated ck including: burns ( . %), compartment syndrome ( . %), myocardial infarction ( . %), myositis/myocarditis ( . %), neuroleptic malignant syndrome ( . %), rhabdomyolysis ( . %), serotonin syndrome ( . %), surgical procedure ( . %). as outlined in fig. the diagnostic trigger was the routine ck measurement in patients ( . %), prior clinical suspicion ( . %), haematuria ( . %) and unclear in ( . %). action was the correlation analysis showed the egfrs from every formula could all to some extent reflect the glomerular function or gfr accurately. the gfr (scys) formula was a quickly and accurate method for estimating gfr and may apply clinically in critically ill patients. perioperative chloride levels and acute kidney injury after liver transplantation: a retrospective observational study s choi introduction: the risk of developing acute kidney injury (aki) after liver transplantation in the immediate postoperative period ranges between to %. most studies in critically ill and surgical patients evaluated the link between chloride-rich resuscitation fluids, not serum chloride levels, and the incidence of aki. the association between preoperative chloride level or difference in perioperative chloride levels and the incidence of postoperative aki after liver transplantation were evaluated. methods: adult patients (>= years old) who underwent liver transplantation at seoul national university hospital between and were included in the retrospective analysis. the difference between preoperative serum chloride level and the immediate postoperative serum chloride level was defined as intraoperative chloride loading. postoperative aki within days of liver transplantation was diagnosed according to the rifle criteria. patients were divided into normochloremia group ( - meq/l), hypochloremia group (< meq/l), or hyperchloremia group (> meq/l) according to their preoperative chloride level. intraoperative chloride loading was defined as the difference between preoperative serum chloride level and immediate postoperative serum chloride level. . ) compared to patients with preoperative normochloremia. meld scores > and age > years were also associated with increased risk of aki. intraoperative chloride loading was not a significant risk factor for aki after liver transplantation. conclusions: preoperative hyperchloremia and hypochloremia were both associated with an increased risk of developing aki in the immediate postoperative period after liver transplantation. introduction: perioperative acute kidney injury (aki) is associated with significant morbidity and mortality [ ] . certain urinary biochemical parameters seem to have a standardized behavior during aki development and may act as surrogates of decreased glomerular filtration rate (gfr) aiding in early aki diagnosis [ ] . aim of this prospective observational study was the evaluation of urinary biochemical parameters as early indicators of aki in a cohort of major surgery patients. methods: patients were studied. aki was defined according to akin criteria within hrs after surgery [ ] . at pre-defined time points (preoperatively, recovery room [rr] and on postoperative days [pod] to ) simultaneous serum and urine samples were analyzed additional studies must confirm these findings and reevaluate these simple parameters as potential aki monitoring tools. urinary liver-type fatty acid-binding protein is the novel biomarker for diagnosis of acute kidney injury secondary to sepsis t komuro, t ota shonan kamakura general hospital, kamakura, kanagawa, japan critical care , (suppl ):p introduction: acute kidney injury (aki) is the predictor of poor prognosis for the patient with sepsis and septic shock. several diagnostic criteria for aki is used on clinical settings, but useful biomarker is not known yet. urinary liver-type fatty acid-binding protein(l-fabp) is associated with kidney function and aki [ ] , but that is not still discussed about aki secondary to sepsis. thus, we conducted the study of the association between urine l-fabp and aki with secondary to sepsis. (fig. ) . the cut-off line of l-fabp was . μg/g cr. conclusions: l-fabp can be the novel biomarker for diagnosis of aki. further investigation need for diagnostic value of l-fabp and usefulness of early intervention for aki used by l-fabp. introduction: biotransformation of -hydroxyvitamin d to active , (oh) d occurs primarily in the kidney. our aim was to explore whether this process was altered in patients with acute kidney injury (aki). methods: consecutive patients admitted to critical care at a tertiary hospital were recruited. the aki group comprised patients with kdigo stage ii or stage iii aki; the non-aki group were patients requiring cardiovascular or respiratory support, but with no aki. vitamin d metabolite concentrations were measured on days , and . statistical analysis included comparison between groups at each time point, and longitudinal profiles of vitamin d metabolites. results: interim analysis of participants ( % of the recruitment target) showed that , (oh) d concentrations were significantly lower in patients with aki at day and day . considering longitudinal changes, -hydroxyvitamin d profiles were not different between the groups ( fig. ) but there was a trend towards a longitudinal increase in , (oh) d in patients without aki, which was not seen in aki patients (fig. ) . conclusions: interim analysis indicates significant differences in concentrations of , (oh) d, but not (oh)d, in critically ill patients with aki. recruitment is ongoing and further results are awaited. introduction: acute renal failure affects from % to % of patients in the intensive care units (icus) and it is associated with excess mortality. hydratation is a useful preventive measure but it is often controindicated in critically ill patients who, on the contrary, often benefit by a strictly conservative strategy of fluid management. fenoldopam, a selective dopamine -receptor agonist, increases renal blood flow and glomerular filtration rate by vasodilating selectively the afferent arteriole of renal glomerulus. the aim of our study is to compare renal effects of fenoldopam and placebo in critically ill patients undergoing a restrictive fluid management. methods: we enrolled patients admitted to our icu. patients were assigned by randomization to study groups: fenoldopam (n= ) and placebo (n= ). fenoldopam was infused continuously at , mcg/kg/ min and equivalent volume for placebo during a period of seven days. creatinine, cystatin c and creatinine clearance were daily measured as markers of renal function. the incidence of aki according to rifle criteria (risk, injury, failure, loss, end stage kidney disease) was also calculated. results: patients with a negative fluid balance at the end of the week (~- ml, p= , ) were included in the analysis, in the placebo group and in the fenoldopam group. there were not significant differences in the trend of creatinine, creatinine clearance, cystatin c and in the incidence of aki between the groups during the week of infusion. conclusions: a continuous infusion of fenoldopam at , mcg/kg/ min does not improve renal function and does not prevent aki in critically ill patients undergoing a strictly conservative strategy of fluid management. introduction: this study aims to evaluate the efficacy of a protocol implemented for dysphagia risk factors [ ] in hospitalized patients in a cicu (coronary intensive care unit). methods: patients hospitalized in the cicu of a medium-sized hospital in presidente prudente, sp, brazil, were subjected to a survey that screened for dysphagia during the period from january of to september of . patients with at least one risk factor for dysphagia were evaluated by a phonoaudiologist and are the subject of this study. the information was statistically analyzed using epi info, version . . . software. considering significant p < . two-tailed, for logistic regressions multivariate estimated in the sample. results: for this study patients were selected, of which . % were male and the mean age was . ± . years. a higher incidence of dysphagia was observed among patients who had at least one of the following risk factors: stroke (odds ratio . p< . ); brain tumor (or . p= . ); chronic obstructive pulmonary disease (copd) (or . p= . ); degenerative diseases (or . p< . ); lower level of consciousness (or . p< . ); ataxic respiration (or . p< . ); aspiration pneumonia (or . p< . ); orotracheal intubation > h (or . p< . ); tracheostomy (or . p< . ); airway secretion (or . p< . ); nasoenteral tube (or . p< . ); gastrostomy (or . p= . ). there was no statistical significance for age > , traumatic brain injury, oropharyngeal surgery and unfavorable dentition. four factors appeared less than times and could not be analyzed (chagas disease, human immunodeficiency virus (hiv), orofacial burn and excess saliva). conclusions: we concluded that the dysphagia triage protocol insertion was effective to identify dysphagic patients and can be used as an additional tool in the intensive care risk management. physiological bases of this age old concept, more recently applied to endotracheal intubation, have never been confirmed by current methods. we therefore decided to study the effects of an apnea oxygenation period under hfnc oxygen therapy by means of a novel modelization of the respiratory system. methods: firstly, an airway model was built with anatomical, physical and physiological attributes similar to that of a healthy subject (fig. ) . this system reproduces the physiological evolution of intrapulmonary gases during apnea by progressively increasing co levels after having cut off previous o supplies (fio %). secondly, the effects of a hfnc apnea oxygenation of l/min with an fio of % were analyzed by collecting intrapulmonary gas samples at regular intervals (fig. ) . results: after minute of apnea oxygenation, intrapulmonary oxygen levels remain stable at %. after minutes, oxygen fraction reaches %, and increases up to % in minutes. regarding co levels, no significant modifications were observed. conclusions: a novel experimental and physiological model of the respiratory system has been developed and confirms the existence of an alveolar oxygen supply as well as the lack of a co washout during hfnc apnea oxygenation. however, these effects are only observed after a delay of about . to minutes. therefore, the clinical interests of this technique to reduce apnea-induced desaturation during intubation of a hypoxemic patient in the icu seem limited without adequate preoxygenation. combination of both preoxygenation and apnea oxygenation by hfnc can most likely explain positive results observed in other clinical studies. effect of % nebulized lignocaine versus % nebulized lignocaine for awake fibreoptic nasotracheal intubation in maxillofacial injuries in emergency department h abbas, l kumar king george's medical university,lucknow,india, lucknow, india critical care , (suppl ):p introduction: topical lignocaine is most commonly used pharmacological agent for anaesthetizing upper airway during fibreoptic bronchoscopy. we compare the effectiveness of two different concentrations, % lignocaine and % lignocaine, in nebulised form for airway anaesthesia during awake fibreoptic nasotracheal intubation in terms of patient's comfort and optimal intubating conditions, intubation time. methods: institutional ethics committee approved the study and written informed consent obtained; patients of either sex, between - years age with anticipated difficult airway planned for intubation were included for this study. patients were randomly allocated into two groups (a and b) based on sealed envelope method; patients and observers were blinded by using prefilled syringes of lignocaine.one group was nebulized with ml of % lignocaine(group a) and other with ml of % lignocaine(group b) in coded syringes via ultrasonic nebuliser for minutes followed by inj midazolam . mg/kg iv and inj fentanyl microgram/kg iv just before the procedure. the fibreoptic broncoscope was introduced via nostril and the other nostril was used for oxygen insufflation ( - l/min). the fibroscope was introduced through the glottic opening and visualising tracheal rings and carina.the endotracheal tube railroaded over the fiberscope and cuff inflated. results: the primary outcome measure was patient's comfort during awake fibreoptic nasotracheal intubation. the mean patient comfort puchner scale score of group a was . ± . and of group b was . ± . . the mean value of puchner scale of group b was significantly higher.the mean procedural time of group b was significantly higher ( . %) as compared to group a (p< . ). the no of intubations attempts did not differ between the two groups. conclusions: % nebulised lidocaine provided adequate airway anaesthesia and optimal intubating conditions, patient comfort, stable hemodynamics. introduction: this systematic review and meta-analysis aims to investigate whether video laryngoscopy (vl) improves the success of orotracheal intubation, when compared with direct laryngoscopy (dl). methods: a systematic search of pubmed, embase, and central databases was performed to identify studies comparing vl and dl for emergency orotracheal intubations outside the operating room. the primary outcome was rate of first pass intubation. subgroup analyses by location, device used, clinician experience, and clinical scenario were performed. the secondary outcome was rate of complications. results: the search identified studies with , emergency intubations. there was no overall difference in first-pass intubation with vl compared to dl. subgroup analysis showed first-pass intubations were increased with vl in the intensive care unit (icu) ( . ( . - . ); p< . ), but not in the emergency department or pre-hospital setting. rate of first-pass intubations were similar with glidescope® and dl, but improved with the cmac® ( . ( . - . ); p= . ). there was greater first-pass intubation with vl than dl among novice/trainee clinicians (or= . ( . - . ); p< . ), but not among experienced clinicians or paramedics/nurses. there was no difference in first-pass intubation with vl and dl during cardiopulmonary resuscitation or trauma. vl was associated with fewer oesophageal intubations than dl (or= . ( . - . ); p= . ), but more arterial hypotension (or= . ( . - . ); p= . ). conclusions: in summary, compared to dl, vl is associated with greater first-pass emergency intubation in the icu and among less experienced clinicians. vl is associated with reduced oesophageal intubations but a greater incidence of arterial hypotension. compared success rate between direct laryngoscope and video laryngoscope for emergency intubation, in emergency department: randomized control trial p sanguanwit, n laowattana ramathibodi hospital, bangkok, thailand critical care , (suppl ):p introduction: video laryngoscope was used as an alternative to intubate in the emergency room, designed for tracheal intubation more success [ , ] . methods: we performed a prospective randomized controlled trial study of patients who had sign of respiratory failure or met indication for intubation from july to june . patients were randomly by snose technique; assigned to video laryngoscope first or direct laryngoscope first. we collect the demographics, difficult intubation predictor, rapid sequence intubation, attempt, cormack-lehane view and immediate complication. primary outcome was first attempt success rate of intubation. results: first attempt success rate of video laryngoscope was . % trend to better than direct laryngoscope was . %, (p= . ), good glottic view (cormack-lehane view - ) of video laryngoscope was . % better than direct laryngoscope . %, and statistically significant (p= . ), no statistical significant in immediate serious complication between direct laryngoscope or video laryngoscope. conclusions: compared to the success rate between using video laryngoscope or direct laryngoscope for intubation, video laryngoscope trend to better success rate, and better glottic view. -year cohort of prehospital intubations and rescue airway techniques by helicopter emergency medical service physicians: a retrospective database study p de jong, c slagt, n hoogerwerf radboudumc, nijmegen, netherlands critical care , (suppl ):p introduction: in the netherlands the pre-hospital helicopter emergency medical service (hems) is physician based and an adjunct to ambulance services. all four hems stations together cover / specialist medical care in the netherlands. in many dispatches the added value is airway related [ ] . as part of our quality control cycle, all airway related procedures were analysed. high quality airway management is characterized by high overall and first pass endotracheal intubation (eti) success [ ] . methods: the hems database was analysed for all patients in whom prehospital advanced airway management was performed in the period - . balloon/mask ventilation, supraglottic airway (sga) devices, total intubation attempts, cormack & lehane (c&l) intubation grades, successful eti, primary and rescue surgical airway procedures and professional background were reviewed. results: in the -year period, there were dispatch calls. in total patients were treated in the prehospital setting by our hems. of those, required a secured airway. eti was successful in of ( . %). in the remaining patients ( fig. ) an alternative airway was needed. rescue surgical airway was performed in . %, . % received a rescue sga, rescue balloon/mask ventilation was applied in . % of cases, was allowed to regain spontaneous ventilation and in . % of patients all airway management failed. hems physicians, ambulance paramedics, hems paramedics and others (e.g. german emergency physicians) had eti first pass success rates of . %, . %, . % and . % respectively (fig. ) . difficult laryngoscopy (no epiglottis visible) was reported in . % of patients (table ) . conclusions: our data show that airway management performed by a physician based hems operation is safe and has a high overall eti success rate of . %. the total success rate is accompanied by a high first pass eti success rate. introduction: incidences associated with endotracheal tubes are frequent during mechanical ventilation (mv) of intensive care unit (icu) patients and can be associated with poor outcomes for patients and detrimental effects on health care facilities. here, we aimed to identify factors associated with event occurrence due to unsafe management of endotracheal tubes (e-umet). methods: a retrospective observational study was conducted in three icus: one surgical icu, one stroke icu, and one emergency department, at a tertiary hospital in japan from april to march . patients requiring mv and oral intubation during their icu stay were included. the primary finding was the incidence rate of e-umet (biting, unplanned extubations, and/or displacement of the endotracheal tube). the patients were divided into two groups: with or without e-umet. to investigate e-umet, potential factors possibly related to its occurrence were obtained from electronic medical records. we conducted univariable and multivariable analyses to investigate e-umet factors. results: of patients, e-umet occurred in ( . %). the mean and standard deviation for age and acute physiology and chronic health evaluation (apache) ii score were ( ) and ( ), respectively. according to a multivariate logistic-regression analysis, significant risk factors associated with e-umet included patients of neurosurgery (odds ratio (or) . ; % ci, . - . ; p= . ), sedative administration (or . ; % ci, . - . ; p< . ), and higher richmond agitation-sedation scale (rass) scores (or . ; % ci, . - . ; p< . ). the use of a restraint (or . ; % ci, . - . ; p= . ) was an independent factor associated with a lower probability of e-umet. conclusions: this study suggests that risk factors associated with e-umet include neurosurgery, higher rass scores, and the administration of sedatives. patients with these factors and longer oral intubation periods might require extra care. introduction: the use of nasal high flow (nhf) as a respiratory support therapy post-extubation has become increasingly more common. nhf has been shown to be non-inferior to niv and reduces escalation needs compared to conventional oxygen therapy. clinical outcomes using nhf in patients with type ii respiratory failure (rf) is less well understood. our aim was to determine if nhf can be used successfully when extubating type ii rf patients compared to type i rf. methods: we conducted a retrospective observational study on the use of nhf as an extubation respiratory support in (n= ) consecutive patients in icu over a -month period. primary outcome was the need for escalation in therapy (niv, intubation and palliation) post extubation. patients were categorised as high risk if they scored >= from: age>= years, bmi>= and >= medical comorbidity. results: analysis was conducted on all fifty-six (n= ) patients. type i rf group was composed of (n= ) patients with a mean age of . (±sd) years. type ii rf group had (n= ) patients with a mean age of . (±sd) years. in type i rf patients ( %) were successfully extubated with nhf compared to patients ( . %) in type ii. in type ii rf the outcomes were more variable with a greater requirement for niv. of these patients % required niv, . % required intubation and . % received nhf therapy for palliation. a higher average bmi ( . vs . kg/m ) was found in unsuccessfully vs successfully extubated patients in type ii rf. in type i rf escalation of therapy was equally distributed with % in each category. conclusions: the use of nhf for respiratory support post-extubation may become standard practice for type i rf in critical care settings. our data suggests that nhf can be used but with caution in type ii rf and clinicians should risk stratify patients to identify those at risk of re-intubation and post-extubation respiratory failure. introduction: pathogenesis of ventilator-associated pneumonia (vap) relies on colonization and microaspiration. oral topical decontamination reduced the vap incidence from to % [ ] . the persistence of antiseptic effect in the oral cavity is questionable; we hypothesize that continuous oral antiseptic infusion may offer a better decontamination. aim of the work: we developed endotracheal tube that allows continuous oral infusion of chlorhexidine (chx), and we want to test the technique versus the conventional on bacterial colonization. (provisional patent: ) methods: a two identical bio models for the upper airways were manufactured by ( dx diagnostics, usa) to adapt the modified and the ordinary endotracheal tubes (ett). the two techniques tested were using six hourly disinfection with chx (group a) versus disinfection through the hours infusion technique (group b). five microorganisms plus mixed bacteria were used and each was tested for five times. normal saline was used constantly to irrigate the biomodels and ten ml aliquot was collected by the procedure end. culturing of the aliquots from decanted broth pre and post disinfection was performed. the time to apply chx by practitioner was also compared. results: there was a trend towards lower bacterial growth in group a in experiments which reach statistical significance only with pseudomonas aeruginosa (p= . ). in one experiment the growth was lower in group b (fig. ) . additionally there was time saving advantage in group b ( ± . versus ± . min, p= . ). conclusions: the novel technique got at least non inferior results, plus time saving advantage. these results may warrant future clinical trial. monitoring airways non invasive online analysing different particle flow from the airways is never done before. in the present study we use a new technology for airway monitoring using mass spectrometric analysis of particle flow and their size distribution (pexa particles in expired air). the exhaled particles are collected onto a substrate and possible for subsequent chemical analysis for biomarkers. our hypothesis was that by analysing the particle flow online, we could optimise the mechanical ventilation. our hypothesis was that a small particle flow would probably be more gentle for the lung than a large particle flow when the lung is squeezed out and the majority of all small airways are open. methods: in the present study we analyse the particle flow from the airways in vivo, post mortem and during ex vivo lung perfusion using different ventilation modes; volume controlled ventilation (vcv) and pressure controlled ventilation (pcv) comparing small tidal volumes( ) versus big tidal volumes( ) at different peep (positive end-expiratory pressure) and after distribution of different drugs in six domestic pigs. results: we found that vcv resulted in a significant lower particle flow than pcv in vivo but in ex vivo settings the opposite was found (fig. ). in both in vivo and ex vivo settings we found that big tidal volume resulted in a larger particle flow than small tidal volumes.air. the opening and the closure of the small airways reflect the particle flow from the airways. we found that different ventilation modes resulted in different particle flow from the airways. we believe this technology will be useful for monitoring mechanical ventilated patients to optimise ventilation and preserve the lung quality and has a high potential to detect new biomarkers in exhaled air. introduction: malaria is a common problem in underdeveloped countries, with an estimated mortality of more than one million people per year. pulmonary involvement is one of the most serious manifestations of plasmodium falciparum malaria. non-invasive ventilation (niv) decreases muscular works and improves gas exchange by recruitment of hypoventilated alveolus. in this context, we analyze the impact of the use of non-invasive ventilation in malaria with pulmonary dysfunction. methods: it's a retrospective cohort study. we analyzed electronic records of patients who were diagnosed with malaria, with acute respiratory failure, who underwent respiratory therapy with niv between - within the intensive care unit (icu). the study variables were: icu mortality, length of hospital stay, niv time and outcome groups. statistical analysis was performed with the pearson correlation coefficient, with significance level of p < . . the statistics were performed using the bioestat . program. results: thirty-one patients were included in the study. four results were analyzed according to table and fig. . % of the patients were discharged from the hospital. pearson's correlation coefficient analysis showed statistical significance in the group (niv/discharge) in the analysis of patients hospitalized versus niv ( % ci = . to . <(p) = . ). conclusions: the use of niv was positive in patients using this resource as first-line treatment of malaria in the fight against respiratory decompensation, with improvement of symptoms. introduction: cpap is used to improve oxygenation in patient with arf. we aimed to determine non-inferiority (ni) of helmet cpap to facemask in arf based on physiological (heart rate (hr) and respiratory rate (rr)) and blood gas parameters (pao and paco ). we also compared patients' perception in dyspnea improvement after cpap using dyspnea scale (visual analogue scale (vas)) and likert score. methods: we randomized patients to helmet (n= ) and facemask (n= ) with . % of arf was due to acute pulmonary edema. cpap was applied for minutes. patients' physiological and blood gas parameters were recorded before and after intervention. patients then marked on dyspnea scale and likert score. ni of helmet would be declared if confidence interval (ci) of mean difference between groups (helmet's mean minus facemask's mean) in improving physiological, blood gas parameters and dyspnea scale was no worse than predetermined non-inferiority margin (nim). secondary outcome was to compare incidence of discomfort and mucosal dryness between groups. methods: this is a single center retrospective study performed in the icu of tel aviv medical center, israel, a tertiary academic referral hospital. using the electronic medical record system and intensix predictive critical care system for analysis, all patients admitted to the icu between . and . were assessed. respiratory deterioration in mv patients was defined as acute adjustment of fio increase > % or peep increase > cmh o that persisted for at least hours. the primary outcome was icu mortality. secondary outcome was length of icu stay (los). a chi square test for trends was used for the significance of mortality data and a one way anova test for los. results: mv patients were admitted to the icu with an overall mortality of . %. mortality and los were tripled in patients who experienced at least one respiratory deterioration when compared to no events ( . % vs. . %, p< . and . vs. . days, p< . respectively) (fig. ) . increased events of respiratory deteriorations showed significant trend of increased mortality (p< . ). conclusions: in mv patients, a single respiratory deterioration event carries a times higher mortality rate and length of stay (los). any additional event further increases both parameters. association of lung ultrasound score with mortality in mechanically ventilated patients j taculod, jt sahagun, y tan, v ong, k see national university hospital singapore, singapore, singapore critical care , (suppl ):p introduction: lung ultrasound is an important part of the evaluation of critically ill patients. it has been shown to predict recruitability in acute respiratory distress syndrome. however, little is known about the application of lung ultrasound in predicting mortality in mechanically ventilated patients. methods: observational study of mechanically ventilated patients admitted to the medical intensive care unit (icu) of a tertiary hospital (national university hospital, singapore) in and . only the first icu admissions of these patients were studied. lung ultrasound was done at six points per hemithorax and scored according to soummer (crit care med ): normal aeration = ; multiple, well-defined b lines = ; multiple coalescent b lines = ; lung consolidation = . the lung ultrasound (lus) score was calculated as the sum of points (score range - ). we analysed the association of lus score with icu/hospital mortality, using logistic regression, adjusted for age and acute physiology and chronic health evaluation (apache) ii score. results: patients were included (age . ± . years; female [ . %]; apache ii . ± . ; sepsis diagnosis [ . %]). icu and hospital mortality were . % and . % respectively. lus score was associated with increased icu (or . , % ci . - . , p= . ) and hospital (or . , % ci . - . , p= . ) mortality, adjusted for age and apache ii score. conclusions: lus score was associated with increased icu/hospital mortality and may be useful for risk stratification of mechanically ventilated patients admitted to icu. introduction: ventilator asynchrony results in morbidities and mortality. the aim of this study was to explore whether and how physicians used patient-ventilator interactions(pvi) to set mechanical ventilators(mv) in thailand. methods: thai physicians treating mv patients were asked to respond to questionnaires distributed in conferences and to e-mails sent. types of asynchronies encountered and frequency of mv adjustment guided by pvi were evaluated. in addition, correlations between physician's knowledge and )confidence to manage asynchronies and )their experience were analyzed. results: two hundred and eleven physicians answered the questionnaires. most of them were medical residents and icu specialists. % of them set and adjusted mv by asynchrony guidance and the majority used waveform analysis to more than a half of their patients. the most and the least common asynchronies encountered were double triggering and reverse triggering, respectively, while the most difficult-to-manage and the most easily managed asynchronies were periodic/?a b show $ #?>unstable breathing and flow starvation, respectively. lack of confidence and knowledge of pvi were the major reasons of physicians who did not perform asynchrony assessment. for knowledge evaluation, more than % of physicians incorrectly managed asynchrony. chest and icu fellows had the greatest skills in waveform interpretation and asynchrony management with the mean score of . from the total , compared with specialist( . ), medical residents( . ), internists( . ) and general practitioner( . ). there were poor correlations between years' experience in mv management and the skill in waveform interpretation (r = . , p= . ) and between physician's confidence in pvi management and the clinical skill (r = . , p< . ) conclusions: the majority of thai physicians realized the importance of pvi, but the skill in asynchrony management was moderate. intensive programs should be provided to improve their clinical performance. methods: six deeply anesthetized swine underwent tracheostomy, thoracostomy and experimental plef with ml/kg of radiopaque saline randomly instilled into either pleural space. animals were ventilated at vt= ml/kg, frequency= bpm, i/e= : , peep= cmh o, and fio = . . quantitative lung computed tomographic (ct) analysis of regional aeration and global frc measurements by nitrogen wash-in/wash-out technique were performed in each of these randomly applied positions: semi-fowler's (inclined °from horizontal in the sagittal plane); prone, supine, and lateral positions with dependent plef and non-dependent plef (fig. ) . results: no significant differences in frc were observed among the horizontal positions, either at baseline (p= . ) or with plef (p= . ) ( fig. a) . however, component sector total gas volume in each phase of the tidal cycle were different within all studied positions with and without plef (p=<. ). compared to other positions, prone and lateral position with non-dependent plef had a more homogenous vt distribution among quadrants (p=. , fig. b ). supine was associated with most dependent collapse (fig. c ) and greatest tendency for tidal recruitment ( % vs~ %, p= . , fig. d ). conclusions: changes in body position in the setting of effusioncaused chest asymmetry markedly affected the internal distributions of gas volume, collapse, ventilation, and tidal recruitment, even when commonly used global frc measurements provided little indication of these important positional changes. of the respondents, % were affiliated with multidisciplinary icus, % with thoracic and/or cardiac icus and % with neuro-icus. most respondents ( %) had completed their specialist training. overall, arterial oxygen tension (pao ) was the preferred parameter for the evaluation of oxygenation (fig. ). the proportions of doctors' preferences for increasing, decreasing or not changing an fio of . in two (out of six) patient categories at different pao levels are presented in table and table . conclusions: this is the largest survey of the preferred oxygenation targets among icu doctors. pao seems to be the preferred parameter for evaluating oxygenation. the characterisation of pao target levels in various clinical scenarios provide valuable information for future clinical trials on oxygenation targets in critically ill icu patients. introduction: sonographic assessment of diaphragmatic excursion and muscle thickening fraction have been suggested to evaluate diaphragm function during weaning trial [ ] . the purpose of this study is to compare these two parameters to predict extubation success. methods: this prospective study was carried out during months from march to november . we enrolled patients who were mechanically ventilated for more than h and met all criteria for extubation. the non inclusion criteria were: age < years, history of neuromuscular disease or severe chronic respiratory failure. we excluded subjects who needed reintubation for upper airway obstruction, neurological or hemodynamic alteration. the scenario involves a patient expected to receive mechanical ventilation for at least hours in the icu. all proportions are percentages of respondents with % confidence intervals. *p < . for comparisons of proportions of "no change" versus adjacent lower pao level (mcnemar's test) introduction: ventilator induced diaphragmatic dysfunction is known to be a contributor to weaning failure. some data suggest that assisted ventilation might protect from diaphragmatic thinning. aims of this study are to evaluate, by ultrasound (us), the change in diaphragm thickness and thickening in patients undergoing controlled and assisted mechanical ventilation (mv) and clinical factors associated with this change. methods: we enrolled patients who underwent either controlled mv (cmv) for cumulative hours or hours of pressure support (psv) if ventilation was expected to last for at least days. patients < years old, with neuromuscular diseases, phrenic nerve injury, abdominal vacuum dressing system and poor acoustic window were excluded. diaphragm thickness and thickening were measured with us as described by goligher and clinical data were collected every hours until icu discharge. results: we enrolled patients, were excluded because they had less than measurements and for low quality images, leaving patients for analysis. as expected, during cmv diaphragm thickening was almost absent and significantly lower than during psv (p< , ). diaphragm thickness did not reduce significantly during cmv (p= . ), but during psv significantly increased (p< . ) (fig. , where "day " represents the first day of psv). during cmv, in / patients diaphragm thickness showed a >= % reduction. they had a significantly higher fraction of days spent in cmv (p= . ) and longer neuromuscular blocking drugs (nbds) infusion (p= . ). during psv, / patients showed an increase in diaphragm thickness >= %. duration of hospital stay was significantly lower for these patients (p . ). differences between the two groups are reported in table . conclusions: longer time spent in cmv and with nbds infusion seems associated with a decrease in diaphragm thickness. assisted ventilation promotes an increase in diaphragm thickness, associated with a reduction in the length of hospitalization. prediction of intrinsic positive end-expiratory pressure using diaphragmatic electrical activity in neutrally-triggered and pneumatically-triggered pressure support f xia nanjing zhongda hospital, southeast university, nanjing, china critical care , (suppl ):p introduction: intrinsic positive end-expiratory pressure (peepi) may substantially increase the inspiratory effort during assisted mechanical ventilation. our purpose of the study was to assess whether electrical activity of the diaphragm (eadi) can be reliably used to estimate peepi in patients undergoing conventional pneumaticallycontrolled pressure support (psp) ventilation and neutrally-controlled introduction: atelectasis develops in critically ill obese patients submitted to mechanical ventilation. the pressure exerted by the abdominal weight on the diaphragm causes maldistribution of ventilation with increased pleural pressure and diminished response to peep. our objective was to analyze the effects of peep in the distribution of ventilation in obese and non-obese patients according to bmi (obese >= kg/m , or non-obese: to . kg/m ), using electrical impedance tomography (eit). methods: we assessed the regional distribution of ventilation of surgical and clinical patients submitted to a decremental peep itration monitored by eit. we calculated the percent ventilation to the nondependent (anterior) lung regions at the highest and lowest peep applied. the highest compliance of respiratory system was consistently observed at intermediate values of peep (between those extreme values), indicating that the highest peep caused pulmonary overdistension, whereas the lowest peep likely caused dependent lung collapse results: were enrolled patients, with non-obese patients ( , ± kg/m ) and obese patients ( . ± . kg/m ). all patients presented progressively decreased ventilation to dependent (posterior) lung regions when peep was lowered (p< . ). obese patients consistently presented higher ventilation to the anterior lung zones (when compared no nonobese), fig. introduction: lung protective ventilation is the mainstay of mechanical ventilation in critically ill patients [ ] . extracorporeal co removal (ecco r) can enhance such strategies [ ] and has been shown to be effective in low flow circuits based on renal replacement platforms [ , , ] . we show the results of a pilot study using a membrane lung in combination with a hemofilter based on a conventional renal replacement platform (prismalung™) in mechanically ventilated hypercapnic patients requiring renal replacement therapy (nct ). methods: the system incorporates a membrane lung ( . m ) in a conventional renal replacement circuit downstream of the hemofilter. mechanically ventilated patients requiring renal replacement therapy were included in the study. patients had to be hypercapnic at inclusion under protective ventilation. changes in blood gases were recorded after implementation of the extracorporeal circuit. thereafter ventilation was intended to be decreased per protocol until baseline paco was reestablished and changes in vt and pplat were recorded. data from patients were included in the final analysis. results: the system achieved an average co removal rate of . ± . ml/min which corresponded to a paco decrease from . ± . to . ± . mmhg (p< . ) and a ph increase from . ± . to . ± . (p< . ) [ fig. ]. after adaption of ventilator settings we recorded a decrease in vt from . ± . to . ± . ml/kg (p< . ) and a reduction of pplat from . ± . to . ± . cmh o (p< . ). these effects were even more pronounced in the "per protocol" analysis [ fig. ]. conclusions: low flow ecco r in combination with renal replacement therapy provides partial co removal at a rate of over ml/min can significantly reduce invasiveness of mechanical ventilation in hypercapnic patients. introduction: in ecco r-crrt, efficiency of co removal is higher positioning the oxygenator (oxy) up-stream than down-stream the haemofilter due to higher blood flow (bf) [ ] . we tested whether this effect was due to lower pre-filter pressure (pfp). methods: ecco r-crrt circuit was tested in-vitro (n= ) with the following settings: l bovine blood; bf ml/min; oxy . m (euroset); cvvh post mode; substitution flow ml/h; uf rate function off; . m haemofilter (diapact®, b.braun avitum); sweep air flow . l/min. pfp was evaluated at baseline, , and hours. co extraction was measured at bf of , and ml/min. sweep air flow/blood ratio was : . co was add to obtain paco of mmhg. co removal rate calculation ( ): co removal rate = (co ecco r inlet-co ecco r outlet)* blood flow (eq. ) co molar volume at °c [l/mol] = ; solubility of co at °c = . mmol/(l*mmhg); hco i = inlet hco concentration [mmol/l]; hco o = outlet hco concentration [mmol/l]; pi co = inlet co partial pressure [mmhg]; poco = outlet co partial pressure [mmhg] equation becomes: co removal rate= x ((hco i + . x pico ) -(hco o + . x poco )) x blood flow (eq. ) results: bf of ml/min was always reached with the up-stream configuration. bf was reduced to ml/min with the down-stream configuration due to high pfp alarm (table ). co removal increased to . ± . to . ± . , and . ± . ml/min, at bf of , and ml/ min (p< . ). conclusions: bf of ml/min can be reached only with the upstream configuration due to lower circuit pfps. bf directly correlates to co removal efficiency. we may speculate that simultaneous use of crrt and lf-ecco r and activation of the uf rate function with the down-stream setting may further increase pfp thus forcing to more enhanced reduction of bf and less effective co -removal. introduction: we describe the use of a novel low-flow ecco r-crrt device (prismalung-prismaflex, baxter healtcare gambro lundia-ab-lund, sweden) for simultaneous lung-renal support. methods: a retrospective review of patients submitted to prismalung-prismaflex due to aki associated to hypercapnic acidosis during the period may -august at prato hospital icu was performed. data collected were: demographic, physiologic, complications, outcome. data were presented as mean ± ds; anova test was used to compare changes of parameters over time; significance was set at p< , . results: we identified patients (mean age ± yr, mean sofa ± ). causes of hypercapnia were moderate ards (n= ) and ae-copd (n= ). in all patients a fr double lumen cannula was positioned and ml/min blood-flow with lt oxygen sweep-gas-flow was maintained; iv-heparin aiming to double aptt was used. haemo-diafiltration (effluent flow ml/kg/hour) was delivered. in all cases prismalung-prismaflex improved respiratory and metabolic parameters (figs. and ) without any complications. all patients survived to the treatment, nevertheless patients ( ae-copd; ards) died during icu stay due to irreversible cardiac complications. in ards cases: patients were successfully weaned from imv, mean duration of the treatment was ± hours, mean duration of imv after ecco r-crrt was ± days. in ae-copd cases: intubation was avoided in patients at risk of niv failure, patients were successfully weaning from imv, mean duration of the treatment was ± hours, mean duration of imv after ecco r-crrt was , ± , days. fig. (abstract p ) . minutes after implementation of the combined renal replacement and ecco r circuit a moderate decrease in paco (- . mmhg) corresponding to a slightly higher ph ( . ) was observed conclusions: the use of prismalung-prismaflex has been safe and effective: it may be argued that it could be due to the low-blood-flow used. the positive results of this preliminary study may constitute the rational for the design of a larger randomized control trial. systemic il- production and spontaneous breathing trial (sbt) outcome: the effect of sepsis introduction: spontaneous breathing trial (sbt), a routine procedure during ventilator weaning, entails cardiopulmonary distress, which is higher in patients failing the trial. an intense inflammatory response, expressed by increased levels of pro-inflammatory cytokines, is activated during sbt. sepsis, a common condition in icu patients, has been associated with increased levels of the pro-inflammatory cytokine il- . il- produced among others by skeletal muscles, has been associated with severe muscle wasting and maybe by icu acquired weakness. we hypothesised that il- increases during sbt, more evidently in sbt failures. we anticipate this response to be more pronounced in formerly septic patients fulfilling the criteria for sbt. methods: sbts of -min duration were performed and classified as sbt failure or success. blood samples were drawn before, at the end of the sbt and hours later. serum il- levels and other inflammatory mediators, commonly associated with distress, were determined and correlated with sbt outcome. subgroup analysis between septic and non-septic patients was performed. )kg/m ) were monitored for . ± . hours. apneas were identified ranging from - s (fig. a) . apneas were observed in % of patients, suggesting low predictability of respiratory insufficiency. the average mv was ± . %mvpred, as patients were often sleeping or mildly sedated. we assessed the effects of each apnea on the temporally associated mv (fig. b) . while apneas ranging in length from - s decrease mv by as much as %, their effect over min is < %. on a min time scale, even s apneas led to lowmv just % of the time (fig. c) . conclusions: while apneas were ubiquitous, they seldom led to lowmv over clinically relevant time scales. large compensatory breaths following an apnea generally restored mv to near pre-apnea levels. nonetheless, some apneas can become dangerous when ignored, as when subsequent sedation decreases compensatory breath size. rvm data provide a better metric of respiratory competence, driving better assessment of patient risk and individualization of care. introduction: diffuse alveolar hemorrhage (dah) is an acute lifethreatening event and recurrent episodes of dah may result in irreversible interstitial fibrosis. identifying the underlying cause is often challenging but is needed for optimal treatment. lung biopsy is often performed in the diagnostic evaluation of patients with suspected dah. however, the role of lung biopsy in this clinical context is unclear. hence, we sought to identify the spectrum of histopathologic findings and underlying causes in patients with dah who underwent lung biopsy, surgical or transbronchial. methods: we identified patients who underwent surgical lung biopsy (n = ) or bronchoscopic biopsy (n = ) in the evaluation of dah over a -year period from to . we extracted relevant clinical pathologic and laboratory data. results: the median age in our cohort was years with % females. serologic evaluation was positive in % of patients (n= ). most common histopathologic findings on surgical lung biopsy included alveolar hemorrhage (ah) with capillaritis in patients of whom six had necrotizing capillaritis, followed by ah without capillaritis in patients. the most common histopathologic finding on bronchoscopic lung biopsy was ah without vasculitis/capillaritis in patients, followed by ah with capillaritis in patients. there were no procedure related complications or mortality observed with either method of lung biopsy. the clinico-pathologic diagnoses in these patients are shown in tables and . conclusions: in patients with dah undergoing lung biopsy alveolar hemorrhage without capillaritis was found to be the most common histopathologic finding followed by pulmonary capillaritis. these histopathologic findings contributed to the final clinico-pathologic diagnoses of granulomatous polyangiitis and microscopic polyangiitis in a substantial portion of cases. future studies are needed to ascertain the benefits vs. risks of lung biopsy in patients with suspected dah. note that, an apnea of -sec will (by definition) drive mv over a -sec window down to , but will only decrease mv over a -sec window down to~ % mvpred and to less than % over a -min window. (c) likelihood of an apnea of specific length to decrease mv below the low mv cutoff over various time windows. note that a single -sec apnea has just a % chance to decrease mv below % in a -sec window and less than % chance to decrease mv below the cutoff over a -min window. even -sec apneas have just % chance of decreasing sustained mv over a -min window below the % mvpred cutoff ( ) granulomatosis polyangitis ( ) ah without capillaritis ( ) antiphospholipid syndrome ( ) microscopic polyangitis ( ) ah with diffuse alveolar damage( ) microscopic polyangitis ( ) ah with pulmonary vascular changes( ) pulmonary hypertension( ) introduction: assessing the sensitivity of the peripheral chemoreflex (spcr), we can predict the likelihood of developing respiratory and cardiovascular disorders. spcr is one of the markers of disease progression and good prognostic marker [ ] . disturbed respiratory mechanics can make it difficult to evaluate. breath-holding test may be helpful in such situation, the results of this test are inversely correlated with peripheral receptor sensitivity to carbon dioxide in healthy people [ ] .the aim of the study was to compare the breath-holding test to single-breath carbon dioxide test in the evaluation of the sensitivity of the peripheral chemoreflex in subjects with copd. methods: the study involved patients with copd with fev /fvc < % of predicted, all participants were divided into two groups depending of disease severity (gold classification, ). in group (mild-to-moderate copd, n= ) all patients had fev >= % and in group (severe-to-very severe copd, n= ) all patients had fev < %. breath-holding test was performed in the morning before breakfast: voluntary breath-holding duration was assessed three times, with min intervals [ ] . a mean value of the duration of the three samples was calculated. the single-breath carbon dioxide test [ ] was performed the next day. the study was approved by the local ethics committee. all subjects provided signed informed consent to both tests. and january . the data was collected from the hospital electronic and paper notes, and data collected was mortality rate, apa-che ii score, icnarc score, type of respiratory support received and whether there was documentation of advanced decisions in case of acute deterioration. results: there were patients admitted to the icu with acute respiratory failure as a complication of pulmonary fibrosis. the median apache ii score was and icnarc standardised mortality ratio was . . nine patients died on icu ( %) and hospital mortality was ten ( %). eight patients ( %) received high flow nasal oxygen, six ( %) received non-invasive ventilation, and two ( %) received invasive ventilation. the median time to death was . days. of patients for whom paper notes were available, no patient had any documented ceiling of care or end of life decisions. conclusions: our study confirmed a very high mortality in this cohort of patients, supporting national guidance that invasive respiratory support has limited value. we advise that frank discussion with patients and their families should happen early after diagnosis, such that end of life plans are already in place in the event of acute deteriorations. introduction: arf is common in critically ill patients. we compared diaphragm contractile activity in medical and surgical patients admitted to icu with a diagnosis of arf. methods: adult medical and major abdominal laparotomic surgical patients admitted to a general icu with a diagnosis of arf were enrolled. arf was defined as a pao /fio ratio<= mmhg/% and need for mechanical ventilation (mv) for at least hours. diaphragmatic ultrasound was realized bedside when the patient was stable and able to perform a trial of spontaneous breathing. a convex probe was placed in right midaxillary line ( th- th intercostal space) to evaluate right hemidiaphragm. diaphragmatic respiratory excursion and thickening were evaluated in m-mode on consecutive breaths and thickening fraction (tf) was calculated. antropometric, respiratory and hemodynamic parameters, saps , sofa score, duration of mv, need for tracheotomy and timing, septic state and site of infection, superinfections, icu and inhospital length of stay (los) and outcome were recorded. patients with trauma and neuromuscular disorders were excluded. p< . was considered significant. results: we enrolled patients: % medical and % surgical, without differences for age, sex, bmi, saps , sofa score, sepsis and superinfections. moderate arf was prevalent in both groups. during diaphragmatic examination, no differences were recorder for respiratory rate, hemodynamic state and fluid balance. surgical patients showed a lower but not significant diaphragm excursion ( . vs . cm), instead tf was significantly reduced ( vs %,p< . ). no differences emerged on duration of mv, but tracheotomy were higher in medical ones ( vs %,p< . ). icu and inhospital los do not differ between medical and surgical patients and mortality rate was respectively % and %. conclusions: in arf, surgical patients showed a lower diaphragm contractility compared to medical ones, maybe due to the combination of anesthetic and surgical effects, but with no influence on outcome. (fig. ) . the slope of the regression line for pes/paw plots was consistently higher for slow compressions ( . ± . ), as compared to fast ones ( . ± . ). a good agreement between Δ pes and Δ paw (fig. ) was found during slow maneuvers, but not during the fast ones. conclusions: slow chest compressions must be used when checking position/inflation of esophageal balloon. the fast maneuver produces hysteresis and underestimation of Δ pes (but not in direct Δ ppl). pes monitoring at high respiratory rates may be problematic. methods: consecutive comatose post cardiac arrest patients were ventilated with volume assist ventilation ( ml/kg ibw, peep cm h o) using elisa eit (lowenstein medical, ge). orogastric tube (nutrivent, sidam, it) was inserted, and eit vest (swisstom ag, ch) was applied in all patients. measurements were performed min after admission and after hrs (fig. ) . optimal peep was defined as lower inflection point using pv curve (pv), positive ptpeep (ptp) and optimal regional stretch/silent spaces (eit) results: methods to determine peep using pv, ptp and eit were comparable in non obese patients (p=ns introduction: the driving pressure of respiratory system (dp) reflects the extent of lung stretch during tidal breathing, and has been associated with mortality in ards patients during controlled mechanical ventilation [ ] . aim of this study was to examine dp during assisted ventilation, and examine if and when high dp occurs in patients in assisted ventilation with pav+. methods: critically ill patients hospitalized in the icu of the university hospital of heraklion, on mechanical ventilation in pav+ mode were studied. continuous recordings of all ventilator parameters were obtained for up to three days using a dedicated software. dp was calculated from the pav+ computed compliance (c) [ ] , and the measured exhaled tidal volume (vt, dp=vt/c). periods of sustained dp above cmh o were identified, and ventilation and clinical variables were evaluated. results: sixty-two patients and hrs of ventilation were analyzed. in half of the patients, dp was lower than cmh o in % of the recording period, while high-dp (> cmh o) more than % of the total time was observed in % of patients. icu non-survivors had more time with high dp than survivors (p= . ). periods of sustained high-dp (> cmh o for > h) were observed in patients. level of assist, minute ventilation, and respiratory rate were not different between the periods of high dp and the complete recordings, while vt was higher and c was lower during the high-dp period compared to the complete recording. the median compliance was below ml/ cmh o during the high-dp period, and above ml/cmh o during the complete recording. conclusions: high dp is not common, but does occur during assisted ventilation, predominantly when compliance is below ml/cmh o, and may be associated with adverse outcome. table summarizes the percent of monitored time with reported data for the two devices. figure depicts mv decrease following propofol and cannula dislodgement fol- fig. (abstract p ) . bland-altman analysis demonstrated that cvp-derived Δppl and Δpes were correlated significantly lowing a jaw thrust. table ) . negative (a-et) pco was strongly associated with good outcome and were significantly associated with overall survival (fig. ) conclusions: in conclusion, the negative arterial to end-tidal co pressure gradient may predict patient survival in some subgroups. introduction: ards may result from various diseases and is characterized by diffuse alveolar injury, lung edema formation, neutrophil-derived inflammation and surfactant dysfunction. various biomarkers have been studied in diagnostics and prognostication of ards. the purpose of the study was to measure the expression of proinflammatory mediators like il- and tnf, a cellular receptor with a role in innate immunity(tlr- ),and a biomarker of fibrogenesis (mmp- ) in different phases of ards patients. methods: we studied patients admitted to our icu with diagnosis of ards during the month of january . six ml of blood were prospectively collected at two times: during the acute phase and in a sub-acute phase before icu discharge. blood samples were centrifuged to obtain the platelet-rich plasma and plasmatic rna (crna) was isolated from platelets.il- , tnf, tlr- and mmp- expression in crna was determined by the droplet digital™ pcr as copies/ml. results: all patient showed a decrease in il- , tnf, tlr and mmp- levels after the acute phase of ards (fig. ) . patient and were affected by influenza a virus (h n ), patient was admitted for pneumococcal pneumonia and patient was affected by legionella. adequate ethiologic treatment was promptly started in patients with bacterial infection. mean duration of mechanical ventilation was . days. all patient survived icu stay and were discharged from hospital. conclusions: il- , tnf, tlr- and mmp- expression detected by extracted platelets rna, may be a novel tool useful for clinicians indicating persistent inflammation with resulting progressive alveolar fibrosis and impaired lung function. more data are necessary to understand the real clinical significance of this biomarkers and their role in fibroproliferation and progression of ards. introduction: although mesenchymal stem cells (mscs) transplantation has been shown to promote lung respiration in acute lung injury (ali) in vivo, its overall restorative capacity appears to be restricted mainly because of low engraftment in the injured lung. ang ii are upregulated in the injured lung. our previous study showed that ang ii increased mscs migration in an angiotensin ii type receptor (at r)dependent manner [ ] . the objective of our study was to determine whether overexpression of at r in mscs augments their cell migration and engraftment after systemic injection in ali mice. methods: a human at r expressing lentiviral vector was constructed and introduced into human bone marrow mscs. we also downregulated at r mrna expression using a lentivirus vector carrying at r shrna to transduce mscs. the effect of at r regulation on migration of mscs was examined in vitro. a mouse model of lipopolysaccharide (lps) induce ali was used to investigate the engraftment of at r-regulated mscs and the therapeutic potential in vivo. results: overexpression of at r dramatically increased ang ii-enhanced human bone marrow msc migration in vitro. moreover, msc-at r accumulated in the damaged lung tissue at significantly higher levels than control mscs h and h after systematic msc transplantation in ali mice. furthermore, msc-at r-injected ali mice exhibited a significant reduction of pulmonary vascular permeability and improved the lung histopathology and had additional anti-inflammatory effects. in contrast, there were less lung engraftment in msc-shat r-injected ali mice compared with msc-shcontrol after transplantation. thus, msc-shat r-injected group exhibited a significant increase of pulmonary vascular permeability and resulted in a deteriorative lung inflammation. conclusions: our results demonstrate that overexpression of at r enhance the migration and lung engraftment of mscs in ali mice and may provide a new therapeutic strategy for the injured lung. introduction: reorganization of endothelial barrier complex is critical for increased endothelial permeability implicated in the pathogenesis of acute respiratory distress syndrome. we have previously shown hepatocyte growth factor (hgf) reduced lipopolysaccharide (lps)-induced endothelial barrier dysfunction. however, the mechanism of hgf in endothelial barrier regulation remains to be unclear. methods: recombinant murine hgf with or without mtor inhibitor rapamycin were introduced on mouse pulmonary microvascular endothelial cells (pmvecs) barrier dysfunction stimulated by lps. then, endothelial permeability, adherent junction protein (occludin), endothelial injury factors (endothelin- and von willebrand factor), cell proliferation and mtor signaling associated proteins were tested. results: our study demonstrated that hgf decreased lps-induced endothelial permeability and endothelial cell injury factors, and attenuated occludin expression, cell proliferation and mtor pathway activation. conclusions: our findings highlight activation akt/mtor/stat- pathway provides novel mechanistic insights into hgf protective regulation of lps-induced endothelial permeability dysfunction. introduction: mechanical ventilation (mv) is a life-saving intervention for critically ill patients, but may also exacerbate pre-existing lung injury, a process termed ventilator-induced lung injury (vili). interestingly, we fig. (abstract p ) . fluorescein isothiocyanate-dextran or fluorescein isothiocyanate-bsa analysis of the effect of hgf on pmvecs permeability fig. (abstract p ) . western blot analysis of hgf on mtor signaling pathway discovered that the severity of vili is modulated by the circadian rhythm (cr). in this study, we are exploring the role of the myeloid bmal , a core clock component, in vili. methods: we employed mice lacking bmal in myeloid cells (lyzmcre-bmal -/-) and lyzmcre mice as controls. at circadian time (ct) or ct , mice were subjected to high tidal volume mv to induce vili. lung compliance, pulmonary permeability, neutrophil recruitment, and markers of pulmonary inflammation were analyzed to quantify vili. to assess neutrophil inflammatory responses in vitro, myeloid cells from bone marrow of wt and bmal -deficient animals were isolated at dawn zt (zeitgeber time ) and dusk (zt ), incubated with dcfh-da and stimulated for min with pma or pbs. neutrophil activation (ly g/cd b expression) and ros production (dcfh-da/ly g+ cells) were quantified. results: injurious ventilation of control mice at ct led to a significant worsening of oxygenation, decrease of pulmonary compliance, and increased mortality compared to ct . lyzmcre-bmal -/-mice did not exhibit any significant differences when subjected to mv at ct or ct . mortality in lyzmcre-bmal -/-mice after vili was significantly reduced compared to lyzmcre controls (ct ). neutrophils isolated from control mice at zt showed a significantly higher level of activation and increased ros production after pma-stimulation compared to zt . ros production of lyzmcre-bmal -/-neutrophils did not differ from zt to zt . conclusions: the lack of the clock gene bmal in myeloid cells leads to increased survival after injurious ventilation and to loss of circadian variations in neutrophil ros production. this suggests that the internal clock in myeloid cells is an important modulator of vili severity. introduction: hemodynamic resuscitation by means of fluids and norepinephrine (ne) is currently considered as a cornerstone of the initial treatment of septic shock. however, there is growing concern about the side effects of this treatment. the aim of this study was to assess the relationship between the hemodynamic resuscitation and the development of the ards. methods: new zealand rabbits. animals received placebo (sham= ) or lipopolysaccharide (lps) with or without (edx-r, n= ; edx-nr, n= ) hemodynamic resuscitation (fluids: ml/kg of ringer's lactate; and later ne infusion titrated up to achieve theirs initial arterial pressure). animals were monitored with an indwelling arterial catheter and an esophageal doppler. respiratory mechanics were continuously monitored from a sidestream spirometry. pulmonary edema was analyzed by the ratio between lung wet and lung dry weight (w/d), and the histopathological findings. results: sham group did not show any hemodynamic or respiratory changes. the administration of the lps aimed at increasing cardiac output and arterial hypotension. in the lps-nr group, animals remained hypotensive until the end of experiment. infusion of fluids in lps-r group increased cardiac output without changing arterial blood pressure, while the norepinephrine reversed arterial hypotension. compared to the lps-nr group, the lps-r group had more alveolar neutrophils and pneumocytes with atypical nuclei, thicker alveolar wall, non-aerated pulmonary areas and less lymphocyte infiltrating the interstitial tissue. in addition, the airway pressure increased more in the group lps-r, and the w/d, although slightly higher in the lps-r, did not show significant differences. conclusions: in this model of experimental septic shock resuscitation with fluid bolus and norepinephrine increased cardiac output and normalized blood pressure but worsened lung damage. obese patients have been excluded from most of the clinical trials testing the effects of peep in ards. we hypothesized that in morbidly obese patients the massive load of the abdomen/chest further increases lung collapse thus aggravating the severity of respiratory failure due to ards. methods: we performed a clinical crossover study to investigate the contribution of lung collapse to the severity of respiratory failure in ards obese patients and to determine the specific contribution of titrated peep levels and lung recruitment to changes in lung morphology, mechanics and gas exchange. patients were studied at the peep (peepicu) levels selected at our institution and at peep levels establishing a positive end-expiratory transpulmonary pressure (peepinc) and at peep levels determining the lowest lung elastance during a decremental peep (peepdec) trial following rm. results: thirteen patients were studied. at peepicu end-expiratory transpulmonary pressure was negative, lung elastance was increased and hypoxemia was present (table ) . regardless the titration technique there was no difference in the peep level obtained. at peepinc level endexpiratory lung volume increased, lung elastance decreased thus improving oxygenation. setting peep according to a peepdec trial after a rm further improved lung elastance and oxygenation. at peedec level after a rm lung collapse and overdistension were minimized (fig. ) . all patients maintained titrated peep levels up to hours without complications. conclusions: in severely obese patients with ards, setting peep according to a peepinc trial or peepdec trial following a rm identifies the same level of optimal peep. the improvement of lung mechanics, lung morphology and oxygenation at peepdec after a rm suggests that lungs of obese ards patients are highly recruitable and benefit from a rm and high peep strategy. introduction: lung protective ventilation (lpv) strategies, principally focused around the use of tidal volumes < ml/kg predicted body weight (pbw) remains an enduring standard of care for ventilated patients. however, implementation of and compliance with lpv is highly variable. we used 'nudge'-based interventions to assess if these can improve lpv. methods: ventilation data analysis over years ( hours in patients) showed patients had been ventilated with a median tidal volume of . ml/kg pbw with a significant proportion receiving over ml/kg pbw (fig. ) , an effect more pronounced in female patients and those with higher bmi. interventions: ) creation of a software tool to easily identify and monitor patients receiving tidal volumes that were too high for their pbw ) attached laminated reference guides to each ventilator to calculate pbw ) presentation, opportunistic education and verbal prompts to relevant clinical care staff regarding importance of lpv and use of pbw rather than actual body weight ) incorporating checking of tidal volumes on a daily ward rounds from junior clinical members results: we collected hourly ventilation data of the patients over a -week period ( hours in patients) following our interventions. there was, overall a statistically significant reduction tidal volume (p< . ). there was improvement in the ventilation of male patients (p< . ) but female patients endured higher tidal volumes. there was a mixed picture in different bmi grades. conclusions: reducing tidal volumes in mechanically ventilated patients can be done through a mix of behavioural and educational interventions, as well as using technological shortcuts. this helps to reduce the effort on the part of clinical staff to adhere to best practices, and ultimately improve patient outcomes. introduction: lung protective ventilation (lpv) using a tidal volume (vt) of - ml/kg ideal body weight (ibw) is recommended in the intensive care unit and theatres to reduce the incidence of pulmonary complications. the aim of this audit was to assess the extent to which lpv is used in theatres in a busy district general hospital and to implement measures to promote adherence to the recommendations. methods: anaesthetists completed questionnaires for all patients undergoing general anaesthesia at northwick park hospital over week. demographics, actual body weight (abw), height, american society of anesthesiologists (asa) score, and procedural information were recorded. ventilatory parameters included the ventilation mode, vt, and positive end expiratory pressure (peep (fig. ) . significantly more females ( %) received vt >= ml/kg than males ( %) (p< . ) (fig. ) . vt was independent of age, asa, bmi, ventilation mode, speciality, and patient position. conclusions: over half of the patients received vt >= ml/kg ibw. females were more likely to be over ventilated. a likely contributing factor is the disparity between abw and ibw in this cohort. we organised staff teaching and constructed ibw charts with the appropriate corresponding tidal volumes to be displayed in all theatres to promote the use of lpv. results: there were significant differences in ards incidence between groups: ards developed in . % of protective mv groups vs. . % of standard mv group (p= . , fisher's exact test). vap patients ventilated in a protective mode presented with lower duration of mv ( . ± . days) and icu stay( . ± . days) than patients with standard mv ( . ± . and . ± . days). there were significant differences in mortality rates between patient groups: . % in protective mv and . % in standard mv (p= . , fisher's exact test). conclusions: protective mv prevents the development of ards in vap septic patients. introduction: reduction of tidal volumes (tv) below ml/kg associated with low driving pressure (dp) might improve lung protection in patients with acute respiratory distress syndrome (ards). the current study tests the combination of coaxial double lumen endotracheal tube (to reduce instrumental dead-space) and moderately respiratory rate (rr) (< bpm) to maintain co at clinically acceptable levels while using ultraprotective tv. the objective is to considerably reduce dp, which has been preconized as an index more strongly associated with survival than tv, per se, methods: juvenile pigs were anesthetized, intubated and mechanically ventilated. severe lung injury (p/f< ) was induced using a double-hit model: repeated surfactant wash-out followed by injurious mechanical ventilation using low positive end-expiratory pressure and high dp (~ cmh o) for hours. then vts of , , and ml/kg were used in random sequence for min each, both using a standard and coaxial endotracheal tube. at each vt level, rr was adjusted to achieve paco = mmhg but not exceeding bpm. lung functional parameters and blood gas analysis were measured at each vt level. statistical analysis was performed using mixed linear model. results: coaxial endotracheal tube, but not the conventional tube, allowed decreasing vt to and ml/kg, while keeping paco at approximately mmhg and rr< bpm, reducing dp of . cmh o and . cmh o, respectively, compared to the conventional vt of ml/kg (fig. ) . conclusions: in this ards model, coaxial tube ventilation associated with moderately high rr allowed ultraprotective ventilation (vt= ml/kg) and reduced dp levels, maintaining paco at acceptable levels. this strategy might have a significant impact on mortality of severe ards patients. the table shows oxygenation and respiratory mechanics. figure : echocardiographically measured right heart function. conclusions: in morbidly obese mechanically ventilated patients with ards an increase in peep by cmh o (from . ± . cmh o to . ± . cmh o) did not impair right heart function, but improved respiratory mechanics and oxygenation. introduction: mechanical ventilation can, while being lifesaving, also cause injury to the lungs. the lung injury is caused by high pressures and mechanical forces but also by inflammatory processes which are not fully understood [ ] . heparin binding protein (hbp) released by activated granulocytes has been indicated as a possible mediator of increased vascular permeability in the lung injury associated with trauma and sepsis [ , ] . we wanted to investigate if hbp levels were increased in bronco alveolar lavage (bal) fluid or plasma in a pig model of ventilator induced lung injury. methods: anaesthetized pigs were surfactant depleted by saline lavage and randomized to receive ventilation with either tidal volumes of ml/kg with a peep of cm h o (controls, n= ) or ml/kg with a peep of cm h o (ventilator induced lung injury (vili) group, n= ). plasma and bal samples of hbp were taken at , , , and hours (fig. ) . results: characteristics of pigs by study group are shown in table . plasma levels of hbp did not differ significantly between pigs in the control and vili group at any time of sampling. hbp levels in bal fluid were significantly higher in the vili group after (p= . ), (p= . ), (p< . ) and (p= . ) hours of ventilation (fig. ) . conclusions: in a model of ventilator induced lung injury in pigs, levels of heparin binding protein in bal fluid increased significantly over time compared to controls. plasma levels however did not differ significantly between groups. (fig. ) . conclusions: this meta-analysis concluded that corticosteroid treatment in ards provided no benefit in decreasing mortality. in addition, this treatment was not associated with increasing risk of nosocomial infection. (fig. ) . the change in the pao /fio ratio was significant [rr( %ci)= . ( . - . ), p= . ] (fig. ) . finally, trial sequential analysis and grade indicated lack of firm evidence for a beneficial effect. conclusions: surfactant administration may improve oxygenation but has not been shown to improve mortality for adult ards patients. large rigorous randomized trials are needed to explore the effect of surfactant to adult ards patients. moderate to severe acute respiratory distress syndrome in a population of primarily non-sedated patients, an observational cohort study l bentsen, t strøm, p introduction: extracorporeal carbon-dioxide removal (ecco r) might allow ultraprotective mechanical ventilation with lower tidal volume (vt) (< ml/kg predicted body weight), plateau (pplat) (< cmh o) and driving pressures to limit ventilator-induced lung injury. this study was undertaken to assess the feasibility and safety of ecco r managed with a renal replacement therapy (rrt) platform to enable ultraprotective ventilation of patients with mild-to-moderate ards. methods: patients with mild (n= ) or moderate (n= ) ards were included. vt was gradually lowered from to , . and ml/kg, and peep adjusted to reach <=pplat<= cm h o. stand-alone ecco r (prismalung, no hemofilter associated with the rrt platform) was initiated when arterial paco increased by > % from its initial value. ventilation parameters (vt, rr, peep), respiratory system compliance, pplat and driving pressure, arterial blood gases, and ecco r-system characteristics were collected during at least hours of ultraprotective ventilation. complications, day- mortality, need for adjuvant therapies, and data on weaning off ecco r and mechanical ventilation were also recorded. results: while vt was reduced from to ml/kg and pplat kept < cmh o, peep was significantly increased from . ± . at baseline to . ± . cm h o, and the driving pressure was significantly reduced from . ± . to . ± . cm h o (both p< . ). the pao / fio ratio and respiratory-system compliance were not modified after vt reduction. mild respiratory acidosis occurred, with mean ph decreasing from . ± . to . ± . from baseline to -ml/kg vt. mean extracorporeal blood flow, sweep-gas flow and co removal were ± ml/min, ± . l/min and ± ml/min, respectively. mean treatment duration was ± hours. day- mortality was %. introduction: there is no consensus on the management of anticoagulation during extracorporeal membrane oxygenation (ecmo). ecmo is currently burdened by a high rate of hemostatic complications, possibly associated with inadequate monitoring of heparin anticoagulation. this study aims to assess the safety and feasibility of an anticoagulation protocol for patients undergoing ecmo based on thromboelastography (teg) as opposed to an activated partial thromboplastin time (aptt)-based protocol. methods: we performed a multicenter, randomized, controlled trial in two academic tertiary care centers. adult patients with acute respiratory failure treated with veno-venous ecmo were randomized to manage heparin anticoagulation using a teg-based protocol (target - minutes of the r parameter, teg group), or a standard of care aptt-based protocol (target . - of aptt ratio, aptt group). primary outcomes were safety and feasibility of the study protocol. results: forty-two patients were enrolled, were randomized to the teg group and to the aptt group. duration of ecmo was similar in the two groups ( ( - ) days in the teg group and ( - ) days in the aptt group, p= . ). heparin dosing was lower in the teg group compared to the aptt group ( . ( . - . ) iu/kg/h versus . ( . - . ) iu/kg/h respectively, p= . ). safety parameters, assessed as number of hemorrhagic or thrombotic events and transfusions given, were not different between the two study groups. as for the feasibility, the teg-based protocol triggered heparin infusion rate adjustments more frequently (p< . ) and results were less frequently in the target range compared to the aptt-based protocol (p< . ). number of prescribed teg or aptt controls (according to study groups) and protocol violations were not different between the study groups. conclusions: teg can be safely used to guide anticoagulation management during ecmo. its use was associated with the administration of lower heparin doses compared to a standard of care apttbased protocol. methods: single-center retrospective study of patients (n= ; ± . years; % males) undergoing vv-ecmo for severe ards. the acp-score ( - ) was calculated immediately before ecmo initiation and at ecmo-day , -day and -day , as follows: pneumonia as cause of ards - point; driving pressure >= cmh o - point; pao /fio ratio < mmhg - point; paco >= mmhg - point. results: longer duration of mechanical ventilation before vv-ecmo was associated with higher acp-scores. patients with higher acp-scores before vv-ecmo also presented longer total duration of mechanical ventilation and hospital stay. after vv-ecmo initiation, acp-scores significantly decreased from . ± . to . ± . , . ± . and . ± . at ecmo-day , -day and -day , respectively. at ecmo-day , patients with higher acp-scores ( - ) presented increased hospital mortality when compared with patients with lower acp-scores ( - ): . vs. . %, respectively (p= . ). at ecmo-day , high driving pressures and low pao /fio ratios were the acp-score determinants that significantly associated with increased hospital mortality. conclusions: in severe ards, vv-ecmo support allowed a significant and sustained acp-score reduction in most patients. this was achieved by artificial lung correction of low pao /fio , hypercapnia and elevated driving pressures. after an initial period of vv-ecmo support, patients with higher acp-scores present higher mortality rates. our results suggest that on-going adjustment of ecmo and ventilation parameters is necessary to maximize outcome. introduction: we sought to use mechanical power to describe "lung rest" in patients with acute respiratory distress syndrome (ards) supported with extracorporeal membrane oxygenation (ecmo). mechanical power describes work done by the ventilator on the patient's respiratory system over time. this concept unifies tidal volume, rate, and total pressure delivered during the ventilatory cycle into a discrete value that may be useful to guide ventilatory support. we hypothesized that initiation of ecmo led to decreased mechanical power delivered to the patient. methods: we reviewed the charts of the three medical intensive care unit patients at our institution supported with ecmo for severe ards. we collected data on plateau pressure, driving pressure, and mechanical power before initiating ecmo, then at < hours, hours, and hours after. we calculated the mechanical power delivered by the ventilator to the patient in joules per minute as . x respiratory rate x tidal volume x (peak pressure -½ x driving pressure) [ ] . results: all patients were alive at discharge and at days. mean pao /fio at ecmo initiation was ± , mean plateau pressure was ± cm water. all patients received neuromuscular blockade at initiation of ecmo. following ecmo initiation, mechanical power decreased by an average of %± % initially, by %± % at hours, and by %± % at hours (fig. ) . by comparison, driving pressure changed by an average value of - . ± . , - . ± . , and - . ± . cm water over those same intervals. average plateau pressure changed by - . ± . , - . ± . , and - . ± . cm water during the same time period (fig. ) . conclusions: in our limited case series, mechanical power decreased significantly following initiation of ecmo in patients with severe ards. we suggest mechanical power may be more useful than changes in driving pressure or plateau pressure when pursuing "lung rest" during ecmo. introduction: it is not clear whether acute respiratory distress syndrome (ards) is independently associated with mortality after controlling for underlying risk factor and baseline severity of illness. we attempted to assess the attributable mortality of ards by performing a systematic review and meta-analysis. methods: we systematically searched pubmed, embase, scopus and reference lists to identify observational studies reporting mortality rates of critically ill patients with and without ards. all included studies were matched for underlying risk factor. primary outcomes were all-cause in hospital-mortality and short-term mortality (combined day-mortality and intensive care unit-mortality). we calculated pooled risk ratios (rr) and % confidence intervals (ci) with a random-effects model. our meta-analysis was registered with prospero. results: of the initially retrieved articles, studies ( cohorts) involving patients were included. the underlying risk factor was sepsis, trauma and other in , and cohorts, respectively. in-hospital mortality was higher in patients with versus without ards ( cohorts; patients; rr . , % ci . - . ; p< . ). we saw a numerically stronger association between ards and inhospital mortality in trauma (rr . , % ci . - . ; p< . ) than sepsis (rr . , % ci . - . ; p= . ). short-term mortality was higher in patients with versus without ards ( cohorts; patients; rr . , % ci . - . ; p= . ). ards was independently associated with mortality in approximately half of the cohorts which controlled for baseline severity of illness using a multivariable analysis. conclusions: the accumulated evidence suggests that ards is independently associated with mortality after controlling for underlying risk factor; the association is stronger for trauma than septic patients. evidence is mixed as to whether ards is independently associated with mortality after controlling for baseline severity of illness. introduction: evidence is mixed as to whether acute respiratory distress syndrome (ards) is independently associated with mortality after controlling for baseline severity of illness, particularly in patients with sepsis. methods: this was an observational study comparing mortality rates of septic patients with and without ards. subjects for the present study were enrolled in ongoing prospective cohorts of critically ill patients hospitalized in medical intensive care unit (icu) in the united states or south korea. ards was defined using the berlin definition for cases after and the american-european consensus conference definition for cases before . sepsis was defined using the sepsis- definition. baseline severity of illness was assessed using a modified sequential organ failure assessment (sofa) after exclusion of the respiratory component. the primary outcome was inhospital mortality. results: of the critically ill patients enrolled in the cohorts, ( . %) had sepsis and comprised the population of the present study. of the septic patients, ( . %) had ards. patients with versus without ards had higher sofa score; both total (median vs ; p< . ) and modified ( vs ; p< . ). the unadjusted mortality of septic patients with ards was higher than septic patients without ards ( . % vs . %; p< . ). after controlling for baseline modified sofa score, both moderate and severe ards remained significant predictors for in-hospital mortality [odds ratio (or) . ; % confidence intervals (ci) . - . ; p< . and or . ; % ci . - . ; p< . , respectively]. in contrast, after controlling for baseline modified sofa score, mild ards was not associated with in-hospital mortality (or . ; % ci . - . ; p= . ). conclusions: among critically ill patients with sepsis, moderate and severe, but not mild, ards are associated with mortality after controlling for baseline severity of illness. a multicenter study on the inter-rater reliability of heart score among emergency physicians from three italian emergency departments introduction: previous studies suggested that the heart (based on history, ecg, age, risk factors, troponin) score could be a valid tool to manage the patients with chest pain at the emergency department (fig. ). our hypothesis was that there could be heterogeneity in the assignment, because of the history and ecg parameters. for this reason, our objective was to test the heart reliability. there are no published studies on this topic. methods: this is a multicenter retrospective study conducted in italian eds between march and october using clinical scenarios. twenty emergency physicians were included, provided that they had undergone a course on heart score. we used scenarios from a medical database with each scenario including demographic and clinical characteristics. each participant assigned scores to the scenarios using the heart. we tested the measure of interrater agreement using the kappa-statistic, the confidence intervals are bias corrected. a p-value of < . was used to define statistical significance. results: the participants' assignment is shown in fig. . the overall inter-rater reliability was good: kappa = . (ci %; . - . ); with a good agreement between the low and high class of risk but a moderate reliability in the medium class: kappa= . , . and . . we have not found differences of inter-rater reliability among the senior (more than yrs in ed) and junior physicians: kappa= . (ci %; . - . ) and . (ci %; . - - ).the heart score showed the worse value of inter-rater reliability in the history and ecg parameters : k inter = . (ci %; . - . ) and . (ci %; . - . ). conclusions: the heart showed a good inter-rater reliability but a fair agreement in the history parameter. the clinical experience doesn't influence the agreement in the assignment. the main limit of this study lies in using scenarios rather than real patients. introduction: the aim of the experiment was to study the efficacy of preconditioning, based on changes in inspiratory oxygen fraction on endothelial function in a model of myocardial ischemia/reperfusion injury in conditions of cardiopulmonary bypass (cpb). methods: the prospective study included rabbits divided into four equal groups: hypoxic preconditioning; hyperoxic preconditioning (hyperp); hypoxic-hyperoxic preconditioning (hhp); control group. animals were anesthetized and mechanically ventilated. we provided preconditioning, then started cpb, and then induced acute myocardial infarction by ligation of left anterior descending artery. after minutes of ischemia we performed minutes of reperfusion. we investigated endothelial function markers (endothelin- (et- ), asimmetric dimethylarginine (adma), nitric oxide metabolites) at stages before ischemia (after preconditioning in study groups), after ischemia and after reperfusion. results: the level of et- after the stage of ischemia increased in all groups, a significant difference was between hhp and control group (p= . ), then et- increased even more after the stage of reperfusion (p= . hhp vs control group). the concentration of nitrite decreased after the stages of ischemia and reperfusion in comparison with the baseline in all groups. however, the level of nitrite after all types of preconditioning was higher than in the control group (p= . ; . ; . ). the total concentration of nitric oxide metabolites in the study groups was higher than in the control group: before ischemia (after preconditioning) p= . ; after ischemia p= . ; after reperfusion, p= . . concentration of adma was lower in the hhp comparing with the control group at the stages after ischemia (p= . ) and after reperfusion (p= . ). conclusions: hyperp and hhp maintain endothelial function: the balance of nitric oxide metabolites and the reduction of et- hyperproduction in a model of myocardial ischemia/reperfusion injury in conditions of cpb. upscaling hemodynamic and brain monitoring during major cancer surgery: a before-after comparison study introduction: hemodynamic and brain monitoring are used in many high-risk surgical patients without well-defined indications and objectives. in order to rationalize both hemodynamic and anesthesia management, we implemented monitoring guidelines for patients undergoing major cancer surgery. methods: early , and for all eligible patients, we started to recommend (standard operating procedure, sop) cardiac output, central venous oxygen saturation, and depth of anesthesia monitoring with specific targets (map > mmhg, svv < %, ci > . l/min/ m , scvo > %, < bis < ). eligibility criteria were pelvic or abdominal cancer surgery expected to last > hours in adult patients. pre-, intra-, and post-operative data were collected from our electronic medical record (emr) database and compared before (from march to august ) and after (from march to august ) the sop implementation. results: a total of patients were studied, before and after the sop implementation. the two groups were comparable in terms of age, asa score, duration and type of surgery, the surgical possum score was higher after than before ( vs , p= . ). the use of cardiac output, scvo and bis monitoring increased from to %, to %, and to %, respectively (all p values < . ). intraoperative fluid volumes decreased ( . vs . ml/kg/h, p= . ), whereas the use of inotropes increased ( vs %, p= . ). the rate of postoperative delirium ( vs %, p= . ) and urinary track infection ( vs %, p= . ) decreased, as well as the median hospital length of stay ( . vs . days, p= . ). conclusions: in patients undergoing major surgery for cancer, despite an increase in surgical risk, the implementation of guidelines with predefined targets for hemodynamic and brain monitoring was associated with a significant improvement in postoperative outcome. introduction: tissue perfusion and oxygen delivery is low in patients with severe preeclampsia, which would explain multiple organ failure and death in these patients. the aim of this study was to determine the relationship between the base deficit and the risk of adverse maternal and perinatal outcomes. methods: retrospective multicenter cohort study included pregnant patients with severe preeclampsia admitted to six intensive care units at tertiary referral centers during a ten years period in colombia. clinical information was gathered from hospital medical records. the correlation of base deficit with adverse maternal outcomes was evaluated using logistic regression analysis. outcomes were maternal death, acute kidney injury, hellp syndrome, transfusion, eclampsia and extreme neonatal morbidity. results: patients were included in the study, we found a total of ( , %) maternal deaths, the median calculated base deficit obtained was - . meq/l. patients with base deficit greater than - . meq/l had significantly higher mortality rates or . (ci . - . ) p , . this group of patients was also associated with a higher probability of developing a class hellp syndrome or . (ci . - . ) p , . a more mild alteration in the base deficit (greater than - . meq/l) was related to the appearance of kidney injury or . (ci . - . ) p . y complete hellp or . (ci . - . ) p . . conclusions: base deficit is related to worse outcomes in patients with severe preeclampsia. according to our results, a cut-off point greater than - meq/l, there is a higher risk of death and worse outcomes such as class hellp syndrome. comparison of two different laser speckle contrast imaging devices to assess skin microcirculatory blood flow g guven, y ince, oi soliman, s akin, c ince erasmus mc, university medical center rotterdam, rotterdam, netherlands critical care , (suppl ):p introduction: laser speckle contrast imaging (lsci) is a common, non-contact and practical method used to assess blood flow of tissue surfaces. we have lack of knowledge about comparability of different lsci devices due to the arbitrary units (au) used to define blood flux. we sought to examine the linearity between skin blood flux, recorded using two different lsci devices. methods: we performed post-occlusive reactive hyperemia test (porh) on the arm and measured blood flux on the hand using two different lsci devices (moor instruments, devon, uk and perimed ab, järfälla, sweden). all volunteers were measured at baseline, during occlusion and after release of occlusion during the hyperemia phase. the third finger and fourth finger nail were selected for recording blood flux and au were used to express values. results: fifteen healthy, non-smoker male volunteers participated in this study. an excellent correlation was found between the two lsci devices (finger: r : . , p< . & finger nail: r : . , p< . ). data were also assessed in terms of the variability at different stages of the porh test (fig. a-d) . correlation of devices was still high at baseline, first minute of occlusion and in the post-occlusion hyperemia phase. however, in the period between minute after start of the occlusion and the beginning of the hyperemia, correlation was lower for the whole finger (r : . , p= . ) and correlation was lost for fingernail (r : . , p= . ) between the two devices. conclusions: skin blood flux measured with two different lsci devices are linearly correlated with each other. however care should be taken when assessing patients with low blood flux such as occurs during shock and ischemic organs. introduction: the aim of this study was to evaluate the effects of hyperoxia and mild hypoxia on microcirculatory perfusion in a rat model. methods: spontaneously breathing anesthetized (isoflurane) male wistar rats (n= ) were equipped with arterial (left carotid) and venous (right jugular) cannulae and tracheotomy. rats were randomized in groups: normoxiainspired oxygen fraction (fio ) of . ; hyperoxia -fio ; mild hypoxia -fio . . the following measurements were taken hourly for hours: blood gases, mean arterial pressure (map), stroke volume index (svi) and heart rate (echocardiography), skeletal muscle microvascular density (sidestream dark field videomicroscopy). results: at hour, arterial o tension was ± mmhg in normoxia, ± mmhg in hyperoxia, ± mmhg in mild hypoxia (p< . ). hyperoxia induced an increase in map (from ± to ± mmhg at h, p< . ) and a decrease in svi (from . ± . to . ± . ml/kg at h, p< . ), while in mild hypoxia map tended to decrease and svi tended to increase (p> . ). microvascular density decreased in hyperoxia and increased in mild hypoxia (fig. ) . conclusions: in anesthetized rats, microvascular density decreased with hyperoxia and increased with mild hypoxia. introduction: the imbalance between oxygen (o ) delivery and o requirement in patients with sepsis can be assessed by central venous oxygen saturation (scvo ). the low or high scvo may indicate cellular hypoxia or inability to utilize the o . this study aims to determine the relationship between high scvo and mortality in patients with sepsis. methods: a retrospective observational cohort study was done by collecting data (i.e., baseline characteristics, severity of infection and vasopressors) from medical records of >= -year-old patients with sepsis and st scvo measurement within hours of sepsis, who were admitted in a university hospital between and . the patients were stratified by st scvo level (< %, - %, > %) and apache-ii score (<= , > ). the primary outcome was inhospital mortality. results: among patients, those with high scvo ( . %) and low scvo ( . %) were associated with adjusted hazard ratios for death of . ( . - . , p= . ) and . ( . - . , p= . ), respectively, while those with normal scvo ( . %) as control. when the patients were stratified by scvo level and apache-ii score, using patients with normal scvo and low apache-ii score as control, those with high scvo and low apache-ii score, and those with low scvo and low apache-ii score had adjusted hazard ratios of . ( . - . , p= . ) and . ( . - . , p= . ). for those with normal, high and low scvo , and high apache-ii score had adjusted hazard ratios of . ( . - . , p= . ), . ( . - . , p= . ), and . ( . - . , p= . ), respectively. conclusions: the scvo > % with apache-ii score > , but not only scvo > %, is independently related to increased mortality in patients with sepsis. introduction: serum lactic acid levels and scvo are useful predictive parameters for patients with sepsis. however, little is known the differences in the impact of lactate levels and scvo on the prognosis of septic patients. in this study, we investigated these differences by analysing septic patients' characteristics and prognosis. methods: this study is a post hoc analysis of data obtained from a multicentre, prospective, randomized controlled trial, which compared two fluid management strategies for septic patients requiring mechanical ventilation. we categorised patients into the following four groups: scvo >= % and lactic acid levels < mmol/l (hh group); scvo >= % and lactic acid levels < mmol/l (hl group); scvo < % and lactic acid levels >= mmol/l (lh group) and scvo < % and lactic acid levels < mmol/l (ll group). sofa score, saps ii score, lactic acid levels, scvo and bnp were evaluated. primary outcome was -day mortality, whereas secondary outcomes were the duration of mechanical ventilation, administration of crrt, duration of catecholamine therapy and length of icu stay. results: in total, patients were included: hh group (n = ), hl group (n = ), lh group (n = ) and ll group (n = ). no significant differences were observed in terms of patient characteristics. further, -day mortality was % in the lh group, . % in the hh group, % in the ll group and % in the hl group, and there was no significant difference in terms of mortality among the groups. furthermore, there were no significant differences in terms of secondary outcomes. on multivariate analysis using the hl group as reference, the odds ratios for -day mortality in the lh, hh and ll groups were . ( %ci, . - . ), . ( %ci, . - . ) and . ( %ci, . - . ), respectively. conclusions: because -day mortality was higher in the hh group than in the ll group, serum lactic acid levels may have bigger impact on the prognosis of septic patients. introduction: in septic shock endothelial damage can lead to failure of microcirculation and low microcirculatory oxygen saturation. in the skin this is seen as mottling and can be quantified using hyper fig. (abstract p ) . changes in microvascular density spectral imaging. there is insufficient data about associations between skin oxygenation, severity of illness, biomarkers of endothelial damage and mortality in patients with septic shock. methods: this single centre observational study was performed in consecutive intensive care patients with septic shock. within hours of admission hyper spectral imaging of knee area skin was performed and blood was sampled for assay of biomarkers of endothelial cell damage (plasminogen activator inhibitor - (pai- ), soluble intercellular adhesion molecule (sicam- ), soluble vascular cell adhesion molecule (svcam- ), thrombomodulin, angiopoetin- ). nonlinear fitting of optical density spectra was used to calculate relative skin oxy/deoxy hemoglobin concentration and obtain oxygen saturation. the association between skin oxygen saturation, biomarkers, sepsis severity (apache ii, sofa) and -day mortality was analyzed. results: the median (iqr) age of patients was years ( to ), and % were males. the median sofa and apache ii scores were ( to ) and ( to ) and -day mortality rate was %. patients ( %) had mottling. there was a relationship between skin oxygenation, plasma biomarkers (thrombomodulin and svcam- ) and sepsis severity assessed by sofa and apache ii scores, p < . . using logistic regression analysis, skin oxygenation and biomarker concentrations were not associated with -day mortality rate. conclusions: in our cohort of patients with septic shock, skin oxygenation and biomarkers of endothelial injury were strongly associated with initial severity of sepsis but poorly predictive of -day mortality. comparison between ultrasound guided technique and digital palpation technique for radial artery cannulation in adult patients: a meta-analysis of randomized controlled trials s maitra, s bhattacharjee, d baidya all india institute of medical sciences, new delhi, new delhi, india critical care , (suppl ):p introduction: possible advantages and risks associated with ultrasound guided radial artery cannulation in-comparison to digital palpation guided method in adult patients are not fully known. previous meta-analyses included both adult and pediatric patients and long axis in-plane technique and short axis out of plane technique in the same analysis, which may have incurred biases [ , ] . methods: pubmed and cochrane central register of controlled trials (central) were searched (from to th november ) to identify prospective randomized controlled trials in adult patients where dimensional ultrasound guided radial artery catheterization has been compared with digital palpation guided technique. for continuous variables, a mean difference was computed at the study level, and a weighted standardized mean difference (smd) was computed in order to pool the results across all studies. for binary outcomes, the pooled odds ratio (or) with % confidence interval ( % ci) was calculated using the inverse variance method. results: data of patients from studies have been included in this meta-analysis. overall cannulation success rate was similar between short axis out of plane technique and digital palpation [p= . ; fig. ] and long axis in-plane technique with digital palpation. ultrasound guided long axis in-plane approach and short axis out of plane approach provides better first attempt success rate of radial artery cannulation in comparison to digital palpation [p= . and p= . respectively; fig. ]. no difference was seen in time to cannulate between long axis and short axis technique with palpation technique. conclusions: usg guided radial artery cannulation may increase the first attempt success rate but not the over all cannulation success when compared to digital palpation technique. introduction: ultrasound guidance may improve the success rate of vascular cannulation. there is lack of data regarding the utility of usg guided arterial cannulation in critically ill patients in shock. we aim to compare the impact of using real time ultrasound guidance versus palpation method in achieving arterial catheterization in critically ill patients in hypotension. methods: a single center, prospective, randomized trial was performed among critically ill patients aged > years, with hypotension (or requiring vasopressor infusion) and on not previous cannulated radial arteries. patients were randomized in a ratio of : to the ultrasound group or palpation group. under aseptic precautions, arterial puncture was performed using appropriate sized leader cath (vygon, ecquen, france), under real time usg guidance using short-axis out-of-plane view with bevel down. data were recorded and compared between two groups. the unpaired student's t-test or mann-whitney u test were used for continuous variables, and the uncorrected chi-squared or fisher's exact test were used for proportions. results: a total of patients with hypotensive shock requiring radial artery catheterization were randomized into palpation (n = ) and ultrasound (n = ) groups. first pass success rate was significantly higher in ultrasound group as compared to palpation group ( % vs %, p< . ). cannulation time was significantly shorter in ultrasound group ( . vs . ,p< . ). early complications were significantly higher in palpation group compared to ultrasound group ( . % vs . %, p< . ). conclusions: in critically ill patients with hypotension (or requiring vasopressors), ultrasound guidance improved first pass success rate, shortened the cannulation time and reduced the rate of early complications in radial artery catheterizations. relationship between inferior vena cava diameter and variability with mean arterial pressure and respiratory effort b kalin, k inci, g gursel gazi university school of medicine, ankara, turkey critical care , (suppl ):p introduction: there is no consensus on the use of vena cava inferior (ivc) diameter and variability in the assessment of fluid response (fr) in spontaneously breathing icu patients. influence from respiratory effort, experience requirement and measurement problems are reasons for not being preferred. the aim of the study is to investigate the relationship between ivc diameter, variability and spontaneous breathing effort and hypotension measured by ultrasonography in spontaneously breathing intensive care patients methods: the maximum and minimum diameters of the ivc were measured and the collapsibility index (ci) was calculated. measurements were made in d mode on cineloop recordings. diaphragm thickening ratio was used as a measure of respiratory effort. correlations between respiratory effort criteria with ivc minimum diameter and ci were calculated by pearson's correlation coefficient. ivc measurement criterias, such as inspiratory diameter of < cm, %, %, % of the ci were compared with chi square test in hypotensive and non-hypotensive patients. we took two mean arterial pressure threshold for hypotension as and mmhg for this calculation. results: patients were included in the study. for both hypotensive threshold values, there was no significant difference in the rates of hypotensive and non-hypotensive patients with and without a minimum ivc diameter of cm below. even there was no significant relationship between the ci higher than %, % and % and hypotension (p> . ). in spontaneously breathing patients, a significant correlation was found between respiratory effort and ivc ci and ivc diameter < cm conclusions: at the end of the study, there was a correlation between spontaneous breathing effort ivc diameter and ci in the intubed patients. additionally the result that ivc ci is not different even between hypotensive and non-hypotensive patients suggests that this method should be used with caution in predicting fr. introduction: fluid responsiveness in icu patients can be assessed using changes in pulse rate and blood pressure following administration of a fluid bolus, assisted if necessary by cardiac output (co) monitors such as the lidcoplus. this uses pulse contour analysis to estimate stroke volume (sv), with > % change in sv following a fluid challenge (fc) signifying overall benefit. there is no evidence that the use of co monitoring improves patient outcomes and it is unclear if it improves clinical decision making. methods: a lidcoplus monitor was set up with the screen covered. a ml fc was administered over minutes. the heart rate, systolic and mean arterial pressures were recorded before and after the fc. the clinician administering the fc was asked to decide if the patient was fluid responsive. following this decision, the sv change was revealed and the clinician asked again to assess fluid responsiveness. results: forty-five fluid challenges were studied. use of the lidco changed the decision made on occasions (fig. ) . in three patients ( %), this change in decision was appropriate and either corrected a misinterpretation of the haemodynamic data or represented a patient whose only marker of fluid responsiveness was a sv change. in four patients ( %), the lidco changed the decision inappropriately from a correct interpretation of the haemodynamic data. in six patients ( %) the sv change was ignored when it should have changed the initial decision. in the remaining patients ( %) the decision made with the haemodynamic data was in agreement with the sv change and unchanged by revealing the lidco data. conclusions: the use of lidco monitoring only appropriately changed the decision made with information from basic haemodynamic monitoring in % of patients. this augmentation of decision making was only seen in patients whose basic haemodynamic parameters did not respond to fluid. it changed a correct decision inappropriately in %. overall, no improvement in the assessment of fluid responsiveness was seen. introduction: there are accumulating evidences suggesting that intraoperative blood pressure affects postoperative outcome including myocardial injury, acute kidney injury, stroke, and mortality. in a patient undergoing laryngeal microsurgery (lms), blood pressure usually rises sharply due to the stimulation on the larynx. since pulse transit time (ptt) has been reported to reflect arterial blood pressure fairly well, it has possibility to be a marker for blood pressure which reflects beat-to-beat changes in blood pressure and is less invasive than arterial catheterization. methods: intraoperative noninvasive blood pressure (nibp), electrocardiogram (ecg), and photoplethysmogram (ppg) of patients undergoing lms were recorded simultaneously. ptt was defined as a time interval between the r-wave peak on ecg and the point which the maximal rising slope appears on the ppg. the mean ptt values for one minute before and after the increase in blood pressure due to the stimulation on larynx were compared. parameters of ppg such as width, height, maximal slope, minimal slope, and area were also compared. then, correlation between blood pressure and each variable was calculated. results: as the larynx was stimulated by lms, nibps have surged (systolic blood pressure, . p< . ) significantly in most of the patients. systolic blood pressure and ptt were inversely correlated (r = - . , p < . ). minimum slope of ppg also showed good negative correlation with systolic blood pressure (r = - . , p < . ). conclusions: ppt showed good correlation with systolic blood pressure and may have potential to be used as noninvasive continuous blood pressure monitor during a surgery in which blood pressure changes abruptly. introduction: aim of this prospective randomized pilot study was to investigate influence of intra operative restrictive volume approach and post operative lung ultrasound (lus)on prevention and early detection of postoperative interstitial syndrome development methods: cardiac patients who underwent non cardiac surgical procedure were randomly assigned for: group a-liberal volume approach or for group b-combination of restrictive intra operative volume approach and small dose of norepinephrine. all patients post operatively received <= . ml/kg/h fluids, mostly crystalloids. lus was performed before surgical procedure and hours after their admission in icu together with arterial blood gases measurements. the ultrasound characteristic of interstitial syndrome was development of b profile results: before surgery all patients had a profile. twenty for hours later in a group significantly higher number of patients / ( . %) vs / ( . %) in b group,had b profile (p< . ).at the same time there were no significant difference between the groups in amount of patients with pao /fio ratio <= ( patients with positive b lines from a group vs patients from group b).(p> . ) conclusions: intra operative fluid restriction is efficient in prevention of post operative cardiogenic pulmonary edema development. lus is a simple non invasive method for early detection of interstitial syndrome even before development of signs of respiratory deterioration. introduction: the peak rate of left ventricular (lv) pressure (dp/dtmax) has been classically used as a marker of lv systolic function. since measuring lv dp/dtmax requires lv catheterization, other surrogates have been proposed using the peripheral arterial waveform. the aim of this study was to test the performance of lv and arterial (aortic and femoral) dp/dtmax for assessing lv systolic function against the gold-standard (the slope of the end-systolic pressure-volume relationship, emax) during different cardiac loading and contractile conditions. methods: experimental study in pigs. lv pressure-volume data was obtained with a conductance catheter and peripheral pressures were measured via a fluid-filled catheter into the aortic, femoral, and radial arteries. emax was calculated during a transient occlusion of the inferior vena cava. the experimental protocol consisted in three consecutive stages with two opposite interventions each: changes in afterload (phenylephrine and nitroprusside), preload (bleeding and fluid bolus), and contractility (esmolol and dobutamine) (fig. ) . measurements were obtained before and after each hemodynamic intervention. results: emax variations and lv, aortic, femoral and radial dp/dtmax changes throughout the study are shown in fig. . all peripheral artery-derived dp/dtmax underestimated lv dp/dtmax. percentage changes in lv and femoral ddp/dtmax were tightly correlated (r = . ; p< . ). both lv and femoral dp/dtmax were affected by preload changes during fluid infusion. all peripheral dp/dtmax estimations allow to detect lv systolic function changes according to emax during isolated variations in contractility. conclusions: femoral and lv dp/dtmax accurately reflected emax changes, although both were affected by preload changes during fluid administration. fig. (abstract p ) . emax, lv dp/dtmax and aortic, femoral and radial dp/dtmax changes. (table , fig. ). concordance was < % and radial loa was ±< °for all devices; mean polar bias was < °for ft only (table , fig. ) . conclusions: cs, ft and pa are not interchangeable with tptd, because of inaccuracy [ ] . when considering limitations they may be used for trending. introduction: about years ago, the german physiologist pflüger stated that the cardio-respiratory system fulfils its physiological task by guaranteeing cellular oxygen supply and removing waste products of cellular metabolism. methods: the study was performed in early postoperative period after major abdominal surgery in patients. the physical condition of patients corresponded to class of asa. the median age was . ( . - . ) years. duration of the surgery was , ( , - , ) hours. surgery was performed under combined epidural anesthesia with mechanical ventilation. the study was conducted in the following stages: -admission from operating room; -in - hours; - - hours; - - hours; -after - hours after the surgery. results: depend on rate of oxygen extraction index (ero ) groups were revealed: group (n= )low ero (< %) followed by recovery to normal levels to stage - (ero = - %), group (n= )normal level ero ( %) in all the stages, group (n= )high levels ero (> %) with recovery to normal levels to stage , group (n= )high ero (> %) in all the stages. oxygen extraction index at admission to icu after surgery can be normal ( . % of patients), reduced ( . % of patients) or high ( . % of patients). when oxygen extraction ratio is reduced metabolic recovery occurs classically after - hours; when ero is elevated -after hours. core temperature improvement is connected with the restoration of oxygen homeostasis. so, under normal and reduced ero even mild central hypothermia after surgery were not observed, and at an elevated ero moderate hypothermia after surgery was observed with only to - hours post-surgery restoration. conclusions: maintaining an adequate tissue oxygenation is the cornerstone of metabolic response and postoperative recovery in patient after major abdominal surgery. (fig. ) . patients with cso < %time above %h had an odds ratio of hospital survival of . ( %ci . - . , p= . ) (fig. ) . conclusions: cerebral oxygen desaturation below % was significantly associated with outcome in patients undergoing vaecmo. in patients with cso < %time above h%, prognosis was especially poor. prospective trials are needed to evaluate if cso is a viable target for therapeutic interventions. introduction: during the second consensus meeting on microcirculatory analysis the exploration of novel parameters related to physiological function of the microcirculation was proposed. capillary hematocrit (chct) is a direct measure of capillary hemodilution, a potential mechanism of microcirculatory dysfunction in states of shock. our hypothesis was that by application of advanced computer vision (i) chct can be reliably measured in given capillaries, and (ii) change in chct reflects capillary hemodilution induced by cardiopulmonary bypass (cpb). methods: in patients undergoing coronary artery bypass surgery sublingual capillary microscopy videos were recorded before and during cpb primed with hes / . . per-capillary chct was estimated as the product of the number of red blood cells (rbc) and an assumed volume of nl, divided by the capillary volume including plasma gaps. rbc number was assessed by manual counting in the first frame of a given video clip, as well as using a novel advanced computer vision algorithm employing blob detection to calculate the mean per-capillary rbc number in all frames of a given video clip (fig. ) . results: capillaries were analyzed, within a total of and frames using manual and algorithmic analysis. a good correlation was found between both methods for chct (r= . , p< . , fig. ). cpb initiation resulted in an decrease in chct from (mean±sem) . ± . to . ± . , p< . and . ± . to . ± . , p= . in manual and algorithm. conclusions: accurate measurement of chct is possible using advanced computer vision, and it reflects hemodilution induced by initiation of cpb. chct further is a determinant of capillary delivery of oxygen. combined with the assessment of functional capillary volume, blood flow velocity, and capillary hemoglobin saturation, chct may enable direct optical quantification of capillary delivery of oxygen as an integrated functional parameter of the microcirculation. fig. (abstract p ) . prognosis of patients with cso < %time above %h was poor fig. (abstract p ) . detection of single erythrocytes using a novel advanced computer vision algorithm in a representative capillary ribbon extracted from a video frame of the sublingual microcirculation fig. (abstract p ) . the area under cso < % was significantly lower in survivors introduction: cardiac function is known to be impacted by sepsis. passive leg raise (plr) is an effective method to predict fluid responsiveness (fr) or cardiac response to preload expansion. preload functional status and trending cardiac output may identify patient phenotypes with varying cardiac reserve, dysfunction and outcome. methods: patient data were analyzed from a currently enrolling prospective randomized controlled study, evaluating the incidence of fr in critically ill patients with sepsis or septic shock (fresh study, nct ). patients randomized to plr guided resuscitation were classified as plr+ (fluid responsive/preload dependent) if stroke volume (sv) increased >= % when measured with a non-invasive bioreactance device (starling sv, cheetah medical). patients were categorized into different phenotypic cohorts based on changing physiology exhibited on plr and trending cardiac output over the initial hours of therapy. results: a total of plr assessments were performed in patients. overall, % ( / ) of assessments indicated a patient was plr+ after receiving initial resuscitation fluid of~ l. most patients ( %) demonstrated a dynamic physiology with changing plr status occurring > time over hours. there were no differences among the groups with respect to age, gender, or qsofa score (fig. ) . patients in group exhibited a significantly decreased icu stay ( . hours) compared to group ( . hours, p= . ) (fig. ) . patients in group exhibited significantly increased echo evidence of lv/rv cardiac dysfunction ( %), compared to group ( %, p= . ) ( table ) . patients in group exhibited % evidence of echo based lv/rv cardiac dysfunction. conclusions: physiological based resuscitation phenotypes identify significantly different patient groups. patients who are initially not plr+, but then become plr+ with no improved co are significantly more likely to have confirmed lv/rv dysfunction and a significantly longer icu stay. introduction: accurate measurement of a patient's intravascular volume status remains an unsolved clinical problem in the icu setting. in particular, septic and cardio-renal patients often receive volume challenges or diuresis, respectively, with little appreciation of baseline bv or the resulting response. this can lead to volume overload and/or depletion and associated increases in morbidity, mortality and hospital length of stay. methods: we tested the performance of a novel, rapid, minimally invasive technique capable of measuring pv, bv and glomerular filtration rate (mgfr) in human subjects. the method consists of a single iv injection of a large ( kda) carboxymethyl dextran conjugated to a rhodamine-derived dye and a small ( kda) carboxymethyl dextran conjugated to fluorescein. plasma and blood volumes were quantified minutes following the injection of the dye based on the indicatordilution principle. results: this phase b study included normal subjects, chronic kidney disease (ckd) stage iii and ckd stage iv subjects. pv and bv varied according to weight and body surface area, with pv ranging from to mls, and both were stable for greater than six hours with repeated measurements. there was excellent agreement ( fig. ) with nadler's formula for pv in normal subjects. a hour repeat dose measurement in healthy subjects showed pv variability of less than +/- %. following an intravenous bolus of ml % albumin solution the mean +/-(sd) measured increase in pv was . ml +/- . ml post infusion (fig. ) . conclusions: this novel bedside approach allowed for rapid and accurate determination of pv, bv, mgfr (data not shown) and dynamic monitoring following clinical maneuvers such as fluid administration, with a high level of safety, accuracy and reproducibility. this approach should assist the intensivist especially with volume administration and removal in septic and cardiorenal patients. introduction: accumulating evidence shows that fluid overload is independently associated with adverse outcome in children and adults with acute lung injury. fluid restriction initiated early in the disease process may prove beneficial, potentially by diminishing the formation of interstitial edema. the main goal of this study was to determine the short-term biophysical effects of intravenous (iv) fluid restriction during acute lung injury in relation to age. methods: infant ( - weeks) and adult ( - months) wistar rats were mechanically ventilated (mv) hours after intratracheal inoculation with lipopolysaccharide to model acute lung injury. both age groups were randomized to either a normal or restrictive iv fluid regimen during hours of mv. thereafter the rats were sacrificed and studied for markers of interstitial edema formation (wet-dry weight ratios), lung permeability (total protein and alpha- macroglobulin (a m) in bronchoalveolar lavage; bal) and local inflammation (cell counts and cytokines in bal). results: restrictive fluid therapy was not associated with worsening of hemodynamic indices during the period of mv in either infant or adult rats. however, as compared to the normal fluid regimen, restrictive fluid therapy led to lower wet-dry weight ratios of the lungs and kidneys in adult rats (p < . ), but not in infants (figs. and ). no difference was found in total protein and a m in bal between the two fluid regimens in both age groups. also, neutrophil influx in the lungs did not differ between fluid regimens in both age categories, nor did the influx of inflammatory cytokines il- and mip- in bal fluid. conclusions: there is an age-dependent effect of early fluid restriction on the formation of interstitial edema in local and distant organs in the disease process of acute lung injury. further investigation of the effects of fluid therapies in experimental models may help steering towards better treatment in critically ill patients. . ) . in a multivariate analysis fb was independently associated with: group c (p< . ), a history of diabetes (p= . ), the acute physiology and chronic health evaluation iii score (< . ) and the duration of aortic-cross clamp (p< . ). the main findings of this study substantiated the hypothesis that the introduction of continuous fb-tracking throughout the entire care process, is associated with a significant reduction in the administration of fluids in post-cardiac surgery patients, independent of differences in their baseline characteristics. demonstrating that certain organizational changes can influence medical behavior beyond the scope of teaching and instruction, and therefore serves to provide awareness to the current issue known as 'knowledge-to-care gap'. using a protocol for fluid resuscitation: how well is it followed? introduction: positive fluid balance in icu patients has been correlated with worse outcomes [ ] . consequently, we developed a protocol to guide fluid resuscitation. the protocol was introduced in and mandates that fluid responsiveness is assessed when administering fluid boluses. once a patient becomes fluid unresponsive, no further resuscitation fluid should be administered. to assess responsiveness, the protocol advises the use of haemodynamic data such as heart rate and blood pressure as well as the change in stroke volume (sv) measured by a lidcoplus monitor. after years of use and a rolling education program this protocol was felt to be well ingrained in our unit culture. we then assessed how well it was being followed. methods: staff performing fluid challenges were asked to fill out a form recording the haemodynamic and sv data measured before and after a fluid challenge. they were also asked to record their interpretation of just the haemodynamic data and then this data combined with the sv data. results: forty five forms were completed. the protocol was not followed on occasions ( %). four patients who should have been assessed as responsive were deemed to be unresponsive. six patients who should have been assessed as unresponsive were assessed as being responsive. the remaining deviations from the protocol represent misinterpretation of the haemodynamic data but correct use of the sv data to reach a correct final assessment. conclusions: despite being a longstanding ingrained practice in our icu, this review suggests that the protocol for fluid resuscitation is being followed incorrectly approximately a third of the time. this could result in inappropriate under or over administration of iv fluid. we plan to review the educational programme and raise awareness of the protocol to try and improve future compliance. introduction: understanding the effects of therapeutics on the left ventricular (lv) loading conditions is of utmost importance in critically ill patients. the effective arterial elastance (ea=esp/sv, where esp is aortic end-systolic pressure and sv stroke volume) is a lumped parameter of arterial load that has been proposed as an index of lv afterload. we aimed at comparing the effects of fluid administration on esp (i.e., the lv afterload in the pressure-volume phase-plane according to the classic "cardiocentric" framework) and on ea. methods: in mechanically ventilated patients, we recorded ea from the femoral peripheral systolic arterial pressure sap (ea=( . ×femoral sap)/sv) before and after the infusion of -ml of saline. patients in whom fluid administration induced an increase in cardiac index (picco- ) >= % were defined as "responders". introduction: the respiratory variations of the inferior vena cava (ivc) diameter in mechanically ventilated patients with preload responsiveness could be explain by a higher compliance of the ivc and/or higher respiratory variations of the ivc backward pressure, i.e., the central venous pressure (cvp).we aimed at determining the respective weight of these two phenomena. methods: in mechanically ventilated patients, haemodynamic, respiratory and the intra-abdominal pressure (iap) signals were continuously computerised. cvp, iap and the ivc diameter (transthoracic echocardiography) were recorded during end-inspiratory and endexpiratory occlusions, before and after the infusion of -ml of saline. patients in whom fluid administration induced an increase in cardiac index (picco- ) >= % were defined as "responders". the respiratory variations of the ivc diameter, cvp and iap were calculated as (end-inspiratory -end-expiratory values)/mean value. the compliance of the ivc was estimated by the ratio between (end-expiratoryend-inspiratory) values of ivc diameter and cvp. results: fluid administration increased cardiac index by more than % in patients. the respiratory variations of the ivc diameter predicted fluid responsiveness (area under the roc curve: . ( %ci: . - . ), p< . ). before fluid administration, the compliance of the ivc was not different between responders and non-responders ( . ± . vs. . ± . mm/mmhg, p= . ), whereas the respiratory variations of the cvp were higher in responders than in nonresponders ( ± vs. ± %, p= . ). the respiratory variations of the ivc diameter were associated with the respiratory variations of cvp (r= . , p= . ) but not of iap (r=- . , p= . ). conclusions: the respiratory variations of the ivc diameter rather depend on the respiratory variations of the cvp than on the ivc compliance. the iap seems to not be involved in the respiratory variations of the ivc diameter. hours and gedi measured at the same time was examined. since the dataset used in this study consists of repeated measurement data, the analysis used the general linear mixed effect model (glmm). the multivariate analysis adjusted with age, cr, and cardiac index was also conducted. results: of the patients with the total bnp measurements conducted for times and gedi measurements for times, the median of age and saps were (iqr - ) and (iqr - ), and the hospital mortality rate was %. the univariable analysis and the multivariable analysis using glmm respectively found statistically significant differences, with regression coefficient at . %ci . - . (p= . ), and . %ci . - . (p< . ). conclusions: while a positive correlation between gedi and bnp was statistically identified, its effect may be minor in clinical terms, and its significant clinical difference remains unclear. introduction: fluids are a cornerstone of the management of critically ill patients who are at risk of multiple organ dysfunction syndrome. however positive fluid balance (fb) is associated with worse morbidity and mortality in this population, so fluid administration needs to be carefully titrated and the nutritional support products must be taken in consideration. objective: evaluate the impact of nutritional support in the fluid balance in a intensive care unit methods: observational prospective study, conducted in eleven portuguese icus of nine general hospitals. patients with years of age or older were eligible if they were ventilated and had a length of stay (los) in icu greater than days. demographic data, fluid balance along type of nutritional support used in the first days and were collected from the selected patients. results: patients were enrolled, . % were male, the median age - ± ( - ), icu los - . ± . days, mortality rate of . % ( ). % of patients were admitted for medical reasons, . % had normal weight, the remaining patients were either overweight or obese. the average daily fb in the eight days was ± ml, being the maximum at day with + ml, slowly trending down reaching a neutral balance at day and reaching - ml at day . in the first days the majority of the intake is due to resuscitation driven fluids, however the nutritional support contribution rises as the days passes, reaching % at day and % at day ( fig. ) . regarding the administration route, the enteral route was responsible to , % of fluids at day compared to , % of parenteral route. the nutritional support is an factor to take into account regarding fluid balance in intensive care units. in this study after the th day the nutritional support, it was responsible for more than % of the total volume that was delivered to the patient and with an higher impact with the increase in los results: we included patients with mean age years, % male, apache ± , saps ii ± , sofa in admission ± , mechanical ventilation %, continuous renal replacement techniques %. the mean total volume administered during the first days was ± l with a mean dcb of ± l and a mean fluid accumulation of % ± . regarding fluid accumulation: % have < %, % between - % and . % > %. th-day mortality and icu mortality were % i % respectively. during the first week, the percentage of fluid accumulation was significantly higher in non-survivors than in survivors ( . ± . l vs. . ± . l, p . ) (fig. ) . cumulative survival was significantly lower (logrank = . , p= . ) in patients with > % of volume gain since the th day (fig. ) . > % volume gain in the th day is a independently associated variable to mortality after adjusting by age, apache and haemodialysis (or = . ; ci % . - . ; p = . ) ( table ) . conclusions: in septic shock patients, fluid overload more than % since -day of evolution is associated with a higher th-day mortality. its early detection may influence the prognosis and survival. introduction: sepsis is defined as a life-threatening organ dysfunction due to a deregulated host response to infection [ ] . fluid infusion is one of the cornerstones of sepsis resuscitation therapies. one of the major adverse effects reported is fluid overload (fo). the objective of this study was to assess influence of fo on sofa score changes from day to day . methods: this study is a retrospective, multicenter, epidemiologic data analysis. it was performed in three french icus. all adult patients admitted for septic shock, caused by peritonitis or pneumonia and mechanically ventilated, were enrolled. delta sofa score was defined as the sofa score measured on admission minus sofa score measured on day . results: patients met the inclusion criteria of the study. fo occurs in about % of the patients. cumulative fluid balance at day was greater in the fo group ( . versus . ml, p < . ) ( table ) . delta sofa score was higher in the no fo group than in the fo group ( . versus . , p = . ) (fig. ). there was a stepwise decrease of delta sofa score when duration of fluid overload was greater (p = . ) (fig. ) . in linear modelling, association between fo status and delta sofa score was confirmed with an adjusted rr of . [ . - . ] (p = . ) ( table ) . conclusions: ) fo patients had more prolonged multi-organ failure during septic shock; ) the longer the fo is the longer the more multi-organ failure last. , (t ) and (t ) minutes later. cardiovascular parameters were also measured at above time points. biomarker change from baseline (fold-change), indexed to hemoglobin, was compared between groups using mixed effects models (bonferroni-holm corrected p< . ). results: minor differences in measures of shock between groups after fluid administration resolved by t . cryst showed increased fold-change in hyaluronan compared to other groups at t (fwb p= . , hes p< . , gelo p< . ), t (fwb p< . ) and t (fwb p< . ) (fig. ) . gelo had increased fold-change in hyaluronan compared to other groups at t (hes p= . ), t (fwb p< . ) and t (fwb p< . , cryst p= . ), as did fwb at t (hes p= . ). cryst showed increased fold-change in il compared to other groups at t (hes p< . , gelo p= . ), t (hes p= . , gelo p= . ,), t (hes and gelo p< . ) and t (hes and gelo p< . ) (fig. ) , of il at t (gelo p= . ), and of kc at shock (fwb p= . , gelo p= . ), t (fwb p= . , gelo p= . ), and t (gelo p= . ). conclusions: rapid large-volume crystalloid given for hemorrhagic shock was associated with increased hyaluronan, a biomarker of endothelial glycocalyx damage, and inflammation, including increased il , il and kc. introduction: a bi-center randomized controlled trial has recently been published that investigates the impact of the type of fluid (crystalloid versus colloid) on patient outcome following major surgery [ ] . the study used a closed-loop fluid delivery system to eliminate the clinician bias when determining when to deliver fluids. the goal of the current analysis is to compare the immediate hemodynamic response to ml fluid boluses of either a crystalloid or a colloid solution. methods: patient consent was obtained prior to transferring the data from [ ] to edwards lifesciences for further post-hoc analysis. the percent change in stroke volume (dsv) following each ml bolus was tabulated and cross-referenced to the type of fluid. the responder rate and the dsv cumulative distribution function (cdf) were determined for each type of fluid administered. a responder was defined as a dsv >= % for a ml fluid challenge. the mean dsv was compared between the two groups using a student t-test. results: from the datasets reported in [ ] , were used in the analysis. descriptive statistics are summarized in table and the cdfs are plotted in fig. . more crystalloid boluses were administered. in both groups, the responder rate was around %. mean dsv was not significantly different between groups (p = . ). we observed similar responder rates and cdfs with the two fluid types, suggesting that the immediate hemodynamic response to ml fluid boluses is independent from the fluid type. we therefore hypothesized that it is the longer intra-vascular persistence of the colloid that explain the lower number of boluses required to achieve the hemodynamic endpoints targeted in the clinical study [ ] . fig. (abstract p ) . cumulative distribution functions of delta stroke volume for crystalloid and colloid fluid boluses the reduction projected to an average annual saving of , usd ( introduction: colloids are widely used for volume resuscitation. among synthetic colloids, hydroxyethyl starch (hes) is commonly administered. in cardiac surgery, priming of the cardiopulmonary bypass (cpb) circuit with colloids minimizes resuscitation volume and results in less pulmonary fluid accumulation. however, the use of hes has been associated with a higher incidence of renal damage and a higher occurrence of coagulopathy. the aim of this study was to investigate the effect of low dose ( - ml/kg) hes % ( / , ) in cpb pump priming on fluid balance, blood loss, transfusion requirement and occurrence of acute kidney injury. methods: in a pre-post design, data from patients undergoing cardiac surgery with cpb were analyzed. in patients, priming solution consisted of ml balanced crystalloids, ml mannitol %, tranexamic acid g and i.e. heparin. for the other patients, ml of the crystalloids were replaced with hes % ( / . ), the other components were the same. patients were matched : with propensity score method. the primary endpoint was intraoperative fluid balance. secondary endpoints were perioperative blood loss, transfusion requirement and the occurrence of acute kidney injury. results: in total, patients were analyzed. the hes group showed less positive fluid balance than the crystalloid group (p< . ). there was no difference in intraoperative blood loss (p= . ) and transfusion requirement (p= . ). the occurrence of acute kidney injury was not significantly different between the two groups (p= , ). conclusions: low-dose administration of - ml/kg hes % ( / . ) to cpb pump priming decreased intraoperative fluid accumulation without increasing perioperative blood loss and transfusion requirement. there was no effect on the incidence of acute kidney injury. priming cpb pumps with a low-dose of hes % ( / . ) is an important component for a restrictive volume strategy and might safely be used in patients with preexisting renal dysfunction. introduction: most crystalloid solutions used in critically ill patients have a greater chloride (cl) concentration than plasma, which may be detrimental. replacing some cl with bicarbonate (hc ) reduces cl, but may increase partial pressure of carbon dioxide (pc ) in blood. such an increase in pc may be harmful [ ] . the main objective was to determine if a hco balanced fluid resulted in increased paco compared to a conventional balanced fluid. methods: single center randomized controlled trial in an adult icu, comparing balanced fluid (sodium,na= mmol/l, chloride,cl= mmol/l, hco = mmol/l) vs conventional fluid (na= mmol/l, cl= mmol/l, hc <= mmol/l). university ethics committee approval:m . we used the absolute difference between the pco and mmhg as a comparison for the fluid groups. betweengroup comparisons of pc from d -d was done by repeated measures anova. a p value < . was considered significant. results: patients were allocated to the conventional group and to the balanced group. at baseline the groups were well matched (p> . ) for age, weight, gender, severity of illness and organ support. there were no significant differences in pc between the two fluid groups, overall or at d , d or d . the balanced group showed a significant improvement in egfr (scr), between d and d (p= . ) while the conventional group exhibited a significant decline (p= . ). there were no significant differences between the groups with respect to fluid requirements, number of positive blood cultures, icu renal replacement utilization, icu length of stay, icu mortality and day mortality. conclusions: the use of a balanced fluid did not result in an increase in pco and appears to be safe. a beneficial effect on renal function was observed. introduction: the effects of crystalloids and colloids on macro-and microcirculation is controversial. our aim was to compare their effects on microcirculation during free flap surgery when management was guided by detailed hemodynamic assessment. methods: patients undergoing maxillo-facial tumour resection and free flap reconstruction were randomized into a crystalloid (ringerfundin, rf, n= ) and a colloid ( % hydroxyethyl starch, hes, n= ) groups. cardiac index (ci), stroke volume (svi) and pulse pressure variation (ppv) were continuously monitored by a non-calibrated device (pulsioflex -pulsion, maquet). central venous oxygen saturation (scvo ), venous-to-arterial pco -gap (dco ), lactate levels and hourly urine output was also measured, and a multimodal, individualized approach based algorhithm was applied [ ] . microcirculation was assessed by laser doppler flowmetry (periflux ldpm, perimed jarfalla, sweden). measurements were performed at baseline and from the start of reperfusion hourly for hours. for statistical analysis, two-way rm anova was used. results: there was no difference between the groups regarding age, sex, length of surgery (whole population: ± min). patients in the rf-group required significantly more fluid in total (rf: ± , hes: ± ml, p= . ). both groups remained hemodynamically stable (ci, svi, ppv, scvo , dco , lactate and urine output) throughout the study. there was no difference between the rf-, and hes-groups in the laser doppler measurements neither on the control site nor in the flap (fig. ) . conclusions: we found that when hemodynamic management is guided by a multimodal assessment and stability is maintained, there was no difference between crystalloids and colloids in macrocirculation and microcirculatory perfusion. introduction: our aim is to evaluate the impact of crystalloid fluids on immune cells. intensive care unit (icu) patients' inflammatory status can switch from an early pro-inflammatory to a late anti-inflammatory phase, which favors infections. they can receive different crystalloids, either normal saline (ns), ringer's lactate (rl) or plasma-lyte (pl). high chloride concentration present in ns has been associated with various complications [ ] , whereas high doses of nacl have inflammatory effects on immune cells [ ] . however, the immune consequences of crystalloids in humans are ill-defined. methods: using our comprehensive immunemonitoring platform, we assessed the immunological phenotype of peripheral blood mononuclear cells (pbmc) in humans. healthy subjects received a liter of ns, rl and pl. blood samples were taken before and h later. pbmc phenotypes were assessed by flow cytometry and cytokine concentrations were measured by a multiplex assay. off-pump cardiac surgery patients were also randomized to receive either ns, rl or pl during surgery and their stay in the icu. blood samples were drawn at various time-points. all leucocytes were analyzed in a similar fashion. we are still recruiting. results: study of healthy subject's pbmc suggested that rl reduced classical monocytes, whereas ns increased lymphocyte activation and il- and mip- b levels. in cardiac surgery patients, our preliminary results suggested that rl and pl reduced classical monocytes and increased non-classical monocytes compared to ns. neutrophils were also affected differently by crystalloids, where ns seemed to activate them more. conclusions: our results suggest that crystalloids have different immune consequences. a better understanding of their immune modulation will lead to personalization of their use according to the inflammatory status of patients to restore their immune homeostasis. this randomised controlled open-label pilot study included patients presenting to an emergency department with suspected infection requiring a fluid bolus. patients received either a single bolus of ml/kg of . % nacl (isotonic group) or ml/kg of % nacl (hypertonic group). blood biomarker concentrations of glycocalyx shedding (syndecan- , hyaluronan), endothelial activation (sicam- , svcam- ) and inflammation (interleukin- , - , - , ngal, resistin) were measured at t (before fluid) and hour (t ), hours (t ) and - hours (t ) later. changes in biomarker concentrations were compared between study groups using mixed regression models, with fold-change from t reported. differences in fluid volumes were compared using the wilcoxon rank sum test. significance was set at p< . . results: syndecan- concentration in the isotonic group decreased from t to t (fold-change . , % ci . - . ), which was significantly different to the hypertonic group (fold-change . , % ci . - . )(p= . )( table ) . interleukin- concentration decreased in the isotonic group from t to t (fold-change . , % ci . - . ), which was significantly different to the hypertonic group (fold-change . , % ci . - . )(p= . ). otherwise, there were no significant differences in change over time between groups for measured biomarkers. total fluid volume administered between t and t was significantly higher in the isotonic group (p< . ) ( fig. ) but not different for subsequent time periods. conclusions: biomarkers of glycocalyx shedding, endothelial activation and inflammation were not different between patients receiving either . % or % saline. also, % nacl did not reduce administration of additional fluids. introduction: acute changes in pco are buffered by non-carbonic weak acids (atot), i.e., albumin, phosphates and hemoglobin. aim of the study was to describe acid-base variations induced by in-vitro pco changes in critically ill patients' blood and isolated plasma, compare them with healthy controls and quantify the contribution of different buffers. methods: blood samples were collected from patients admitted to the icu and controls. blood and isolated plasma were tonometered at and % of co in air. electrolytes, ph, blood gases, albumin, hemoglobin and phosphates were measured. the strong ion difference (sid) was calculated [ ] and non-carbonic buffer power was defined as β=-Δhco -/Δph [ ] . t-tests and linear regression were used for analysis. results: seven patients and controls were studied. hemoglobin, hematocrit and albumin were lower in patients (p< . ), while sid and phosphates were similar. pco changed from ± to ± mmhg, causing different blood ph variations in patients and controls ( . ± . vs. . ± . , p= . ). patients had lower blood and plasma β ( ± vs. ± , p< . and ± vs. ± , p= . , respectively). figure shows changes in [hco -] and sid induced in blood by pco variations. in both populations, ± % of [hco -] change was due to sid variations, while only ± % to changes in atot dissociation. a significant correlation between hematocrit and Δsid was observed in the whole study population (fig. ) . conclusions: the β of icu patients was lower, likely due to reduced albumin and hemoglobin concentrations. similar pco increases caused therefore greater ph variations in this population. electrolyte shifts, likely deriving from red blood cells [ ] , were the major buffer system in our in-vitro model of acute respiratory acidosis. introduction: there is an increasing trend in the incidence of aneurysmal subarachnoid haemorrhage in hong kong and the disease carries high morbidity and mortality rate. electrolyte disturbance is one of the known complications of sah and the outcomes associated with this are not fully understood. the objective of this retrospective local study is to evaluate the pattern of electrolyte disturbances in patients with sah and their impact on the prognostic functional outcome. methods: patients with spontaneous aneurysmal sah who were admitted to icu at pamela youde nethersole eastern hospital, hong kong between st january and st december were included into this retrospective local study. collected data include demographic details, comorbidities, serum electrolyte levels (sodium and potassium) from day to of admission into icu, radiographic intensity of haemorrhage using fisher scale and the clinical grading of sah using wfns. prognosis of these patients was estimated using the glasgow outcome scale at months after initial insult (fig. ) . results: a total of patients were included in this study. the mean age was , with the majority of patients being female ( . %). the most common aneurysm location was in anterior communicating artery, though poor outcomes were shown significant in patients with posterior circulation aneurysms. whilst early-onset hyponatremia was not correlated with poor outcome, late-onset hyponatremia was associated with better outcome. logistic regression analysis identified independent predictors of poor outcome (table ) . patients who underwent interventional radiological procedure treatment was shown to have better outcome. conclusions: hypernatremia after sah is associated with poor outcome. there does not appear to be significant evidence that hyponatremia has an effect on short-term mortality or certain outcome measures such as gos, and its longer-term effects are not well characterized. fig. note logarithmic transformation of los data). we found a statistically significant difference between the two groups when comparing the length of stay (p < . ). conclusions: dean et al demonstrated no significant difference in the mean length of stay using the same definitions of hypo and eunatraemia as in this study [ ] . even though our data appears to contradict their findings, regarding the statistical significance seen, we feel that this is not significant clinically, given the very similar median times for los between the two groups; the unbalanced design may contribute to the statistical significance. fig. (abstract p ) . length of stay between the two groups (note logarithmic scale for los) fig. (abstract p ). gos at months group consisted of patients with mean age . (sd . ) years and mean sodium . (sd . ) mmol/l with a median los of . (iqr . - . ) days. we found no statistically significant difference (p = . ) between the two groups when comparing the length of stay (fig. ) . conclusions: darmon et al demonstrated prognostic consequences of an admission sodium greater than , eliciting hypernatraemia as a factor independently associated with -day mortality [ ] . in contrast, our study suggests that hypernatraemia (as defined) is not associated with the length of stay, however this result is limited by the unbalanced design of this small study. introduction: our aim is to determine whether auscultation for bowel sounds helps in clinical decision making in icu patients with ileus. ileus can be the consequence of an operation, a side effect of drugs or the result of an obstruction requiring direct operative correction. although auscultation for bowel sounds is routinely performed in the icu and a well-established part of the physical examination in patients with suspected ileus, its clinical value remains largely unstudied. methods: a literature search of pubmed, embase and cochrane was performed to study the diagnostic value of auscultation for bowel sounds. results: auditory characteristics (tinkling, high pitched and rushes) were highly variable in postoperative ileus, mechanical ileus and healthy volunteers. the inter-observer variability for the assessment of the quantity, volume and pitch of bowel sounds was high, with a moderate interobserver agreement for discerning postoperative ileus, bowel obstruction and normal bowel sounds (kappa value . ). the intra-observer reliability of duplicated recordings for distinguishing between patients with normal bowels, obstructed bowels or postoperative ileus was % [ ] . no clear relation between bowel sounds and intestinal transit was found (table ) . sensitivity and positive predictive value were low: respectively % and % in healthy volunteers, % and % in obstructive ileus, and % and % in postoperative ileus ( table ) . conclusions: auscultation with the aim to differentiate normal from pathological bowel sounds is not useful in clinical practice. the low sensitivity and low positive predictive value together with a poor inter-and intra-observer agreement demonstrate the inaccuracy of utilizing bowel sounds for clinical decision-making. given the lack of evidence and standardization of auscultation, the critically ill patient is more likely to benefit from abdominal imaging. introduction: stress ulcer prophylaxis has become a standard of care in intensive care unit (icu). however, it has been proposed that enteral nutrition (en) could play preventive role for gastrointestinal bleeding and some studies revealed no added benefit of acid suppressive drugs to patients on en. based on these backgrounds, we use proton pump inhibitor (ppi) as stress ulcer prophylaxis during starvation period, and discontinue it within hours after commencing meals or en. the aim of this study is to evaluate the applicability of our protocol by reviewing the incidence of upper gastrointestinal bleeding (ugib) in our icu. methods: we conducted a retrospective observational study. all consecutive patients admitted to our icu between april and march were reviewed. patients who had ugib within hours after admission, had previous total gastrectomy, or underwent upper gastrointestinal surgery were excluded. the primary outcome was the incidence of overt or clinically important ugib, and the secondary outcome was protocol adherence. we presented descriptive data as number (percentage) and median (interquartile range). results: a total of patients were included. of those, ( . %) were male, median age was ( - ), and median sofa score was ( ) ( ) ( ) ( ) ( ) ( ) ( ) . of all patients, ( . %) had overt bleeding, and ( . %) had clinically important bleeding. both patients who introduction: patients requiring operative procedures admitted under non-surgical specialties typically experience delays in treatment and fail to meet peri-operative standards with regards to the timing of operative intervention. patients admitted from medicine requiring an emergency laparotomy have an increased mortality when compared to those patients admitted from surgery ( . % v . %) [ ] . methods: we undertook a retrospective case note review of patients requiring a non-elective laparotomy at our hospital during a sixmonth period in . patients were identified using the emergency theatre booking system. data were gathered on admission details, peri-operative care and post-operative stay. results: two main investigators reviewed patients to standardise data extraction. six patients presenting with inflammatory bowel disease were excluded from analysis. most patients ( . %) were admitted through the emergency department; ( . %) of whom were initially admitted under medicine, with only . % of these reviewed by a senior clinician prior to admission (table ). there was no statistically significant difference in mortality between the medicine and surgery groups. there was a trend to increased length of stay in icu and in hospital in the medical group (table ) . conclusions: lack of senior decision making may have a direct impact on patient care due to the inappropriate streaming of patients to medicine. the increased mean length of stay in those patients admitted to medicine may reflect a delay in surgical intervention and therefore a prolonged recovery period. we are introducing an acute abdominal pain screening and immediate action tool to improve identification of these high-risk patients and early involvement of senior decision makers. introduction: biomarkers reflecting the extent of surgical tissue trauma should be investigated in an effort to predict and prevent postoperative complications. the aim of the present study was to investigate blood concentrations of selected alarmins in patients after colorectal surgery in comparison to healthy individuals. the secondary aim was to analyze the relationship between alarmins and inflammatory biomarkers during early postoperative period. methods: the prospective, single-center, observational study consisted of non-surgical (ns) group (n= ) and surgical (s) group (n= ) undergoing colorectal surgery. serum levels of selected alarmins (s a and s a ) and inflammatory biomarkers (leukocytes; c-reactive protein, crp; interleukin- , il- ) were analyzed. results: proteins s a an s a had significantly higher serum values in the s-group during all three days after the surgery. the multidimensional model taking into account age, sex, weight, group and days revealed significant differences between study groups for both proteins s a and s a (p< . , p= . , respectively). biomarkers (leukocytes, crp, and il- ) showed significant differences between study subgroups (p< . , p< . , and p< . , respectively). in s-group, moderate positive correlations were found between s a and all biomarkers: leukocytes (r= . ), crp (r= . ), and il- (r= . ). s a had moderate positive correlation with leukocytes (r= . ). levels of s a also positively correlated with intensive care unit and hospital length of stay (r= . , r= . , respectively) conclusions: protein s a might be considered as early biomarker of first wave of immune activation elicited by surgical injury after colorectal surgery. the increase of the alarmins is reflected by the elevation of routine inflammatory biomarkers. introduction: critical illness-induced liver test abnormalities are associated with complications and death in adult icu patients, but remain poorly characterized in the pediatric icu (picu). in the pepanic rct, delaying initiation of parenteral nutrition to beyond day (late pn) was clinically superior to providing pn within h (early pn), but resulted in a higher rise in bilirubin. we aimed to document prevalence and prognostic value of abnormal liver tests and the impact of withholding early pn in the picu. methods: we performed a preplanned secondary analysis of of the pepanic patients aged days to years, as neonatal jaundice was considered a confounder. plasma concentrations of total bilirubin, alt, ast, γ gt, alp were measured systematically during picu stay. analyses were adjusted for baseline characteristics including severity of illness. results: during the first picu days, the prevalence of cholestasis (> mg/dl bilirubin) ranged between . %- . % and of hypoxic hepatitis (>= -fold uln for alt and ast) between . %- . %, both unaffected by the use of pn. throughout the first week in picu plasma bilirubin concentrations were higher in late pn patients (p< . ), but became comparable to early pn patients as soon as pn was started on day . plasma concentrations of γ gt, alp, alt and ast were unaffected by pn. high day plasma concentrations of γ gt, alt and ast (p<= . ), but not alp, were independent risk factors for picu mortality. day plasma bilirubin concentrations displayed a ushaped association with picu mortality, with higher mortality associated with bilirubin concentrations < . mg/dl and > . mg/dl (p<= . ). conclusions: in conclusion, overt cholestasis and hypoxic hepatitis were rare and unrelated to nutritional strategy. however, accepting a large macronutrient deficit during week increased plasma bilirubin. a mild elevation of bilirubin on the first picu-day was associated with lower risk of death and may represent an adaptive stress response rather than true cholestasis. positive fluid balance is an independent risk factor for intensive care unit mortality in patients with acute-on-chronic liver failure introduction: muscle wasting is a common consequence of disuse and inflammation during admission to intensive care with critical illness. limb muscles are known to decrease in size during critical illness, but less is known about muscles of the trunk. in this study, we tracked how psoas muscle area changes at multiple levels, in a group of patients with acute severe pancreatitis. methods: paired computed tomography (ct) scans were obtained from patients admitted to the royal liverpool university hospital's icu with acute severe pancreatitis. the first scan was within days of admission, and the second took place between to days later. for each scan, three slices were identified: the top and bottom plates of l , and the mid-point of l vertebral body. on each slice, the cross sectional area (csa) of the left and right psoas muscle was calculated using imagej. the difference and percentage change in csa between both scans was calculated. white cell counts and c-reactive protein results were obtained, with peak levels correlated against change in muscle size. results: combined csa of the left and right psoas muscle increased from top to bottom plates and was positively correlated with height (r= . , p< . mid l level)) and weight (r= . , p= . , mid l level) at all three levels. at all three levels, there were significant losses of csa between the two scans (see table ). crp was moderately correlated with percentage change in csa (r= - . , p= . ). increasing weight on admission was associated with greater percentage losses in csa (r= - . , p< . ). wcc did not correlate with change in size. in critically ill patients with acute severe pancreatitis, there are significant losses in both psoas muscles throughout the l level. further prospective studies are required to determine if inflammatory markers and cytokines have a role in these losses, and to determine the functional effects of these losses. introduction: the evidence for penta-therapy for hyperlipidemic severe acute pancreatitis (hl-sap) is anecdotal. the purpose of our study is to evaluate the efficacy of penta-therapy for hl-sap in a retrospective study. methods: retrospective study between january and december in a hospital intensive care unit.hl-sap patients were assigned to conventional treatment alone (the control group) or conventional treatment with the experimental protocol (the penta-therapy group) consists of blood purification, antihyperlipidemic agents, lowmolecular-weight heparin, insulin, covering the whole abdomen with pixiao (a traditional chinese medicine).serum triglyceride, serum calcium, apache ii score, sofa score, ranson score, ct severity index, and other serum biomarkers were evaluated. the hospital length of stay, local complications, systematic complications, rate of recurrence, overall mortality, and operation rate were considered clinical outcomes. results: hl-sap patients received conventional treatment alone (the control group) and patients underwent penta-therapy combined with conventional treatment (the penta-therapy group). serum amylase, serum triglyceride, white blood cell count, c -reactive protein, and blood sugar were significantly reduced, while serum calcium was significantly increased with penta-therapy. the changes in serum amylase, serum calcium were significantly different between the penta-therapy and control group on th day after the initiation of treatment. the reduction in serum triglyceride in the pentatherapy group on the second day and th day were greater than the control group. patients in the penta-therapy group had a significantly shorter length of hospital stay. conclusions: this study suggests that the addition of penta-therapy to conventional treatment for hl-sap may be superior to conventional treatment alone for improvement of serum biomarkers and clinical outcomes. average energy expenditure (ee) for all patients was ± kcal/kg (mean ± sd). there was no difference in the average ee between the patients who survived and those who died: ± and ± kcal/ kg (mean ± sd) respectively (p > . ). however, there was a negative correlation between ee and saps score in the non-survivors groupcorrelation coefficient - . , p < . . the energy deficit (computed by subtracting caloric intake from ee measurement) was similar among survivors and non-survivors, . ± vs . ± kcal/kg, respectively (mean ± sd) (p > . ). the patients who survived had received ± kcal/kg while those who died - ± kcal/kg (mean ± sd) (p > . ). the provision of protein was also similar for both groups: . ± . g/kg for survivors and ± . g/kg for nonsurvivors (mean ± sd) (p > . ). there was no statistically significant correlation between provision of calories and protein and outcomes such as length of hospital and icu stay or duration of mechanical ventilation. conclusions: average energy expenditure in critically ill patients with acute severe pancreatitis roughly equals to aspen estimation of kcal/kg and does not differ among survivors and non-survivors. outcomes such as survival, length of hospital and icu stay and duration of mechanical ventilation were unaffected by caloric nor protein provision in this sample. introduction: disturbances in gastrointestinal motility are common in critically ill patients receiving enteral nutrition. slow gastric emptying (ge) is the leading cause of enteral feeding intolerance (efi), which compromises nutritional status and is associated with increased morbidity and mortality. this phase a study evaluated the efficacy, safety and tolerability of acute tak- (previously td- ), a selective agonist of the hydroxytryptamine receptor ( ht ), compared with metoclopramide in critically ill patients with efi. methods: this was a double-blinded, double-dummy study conducted in mechanically ventilated patients with efi (> ml gastric residual volume) randomized to receive either intervention (tak- . mg over hour and . % saline ml injection qid) or control ( . % saline over hour and metoclopramide mg injection qid). within hour of the first dose, patients received a test meal of ml ensure® and ge was measured using scintigraphy. primary objectives were to evaluate the safety and tolerability of tak- and its effect on ge (% retention at mins) vs control. results: a total of patients (intervention, n = ; control, n = ) were studied. the median ages were and years in these groups, respectively. post-treatment, a -fold greater number of patients had normal gastric retention (< % at mins) in the intervention group vs the control group ( vs ; fig. ). in the intervention and control groups, (table ) . no aes led to treatment discontinuation. conclusions: a greater proportion of patients receiving tak- had normal gastric retention after a single dose compared with those receiving metoclopramide. treatment with tak- was not associated with an increase in aes. these results support further evaluation of tak- in critically ill patients with efi. method to assess gastric emptying in the fed state in enterally tube fed patients: comparison of the paracetamol absorption test to scintigraphy j james introduction: the paracetamol absorption test (pat) is the most common and practical approach for assessing gastric emptying (ge) in critically ill patients. however, current methods require that paracetamol be administered to an empty stomach, removing gastric contents and depriving patients of feeding for several hours. the objective of this study was to develop methods to assess gastric emptying in these patients without interrupting feeding. methods: gastric emptying was assessed in the fed state using pat and scintigraphy in healthy volunteers. paracetamol g in ml was ingested immediately before consumption of a test meal of ml ensure plus containing kcal, . g protein, and . g fat plus mbq of mtc-dpta as a scintigraphic agent. comparisons were made between paracetamol absorption and the time to % and % gastric emptying by scintigraphy at baseline and after administration of ulimorelin μg/ kg, a prokinetic agent known to enhance gastric emptying. blood samples for paracetamol were collected for up to h post administration. values for normal gastric emptying were based on the % confidence intervals for pk parameters. sensitivity and specificity were assessed by receiver operating characteristic (roc) analysis before and after treatment. results: the pat correlated with scintigraphy and pk parameters for normal emptying were determined. cmax and auc were the most sensitive and specific parameters for assessing ge with lowest variability and areas under the roc curve of . and . , respectively. a h sampling period appeared sufficient to distinguish normal from abnormal emptying. conclusions: the pat can be used to distinguish normal versus abnormal ge in the fed state. under the conditions used, patients can receive up to ml enteral feeding over a h test period ( ml/hr). this method can be used to distinguish normal from abnormal gastric emptying in enterally tube fed patients without interrupting feedings. introduction: for mechanically ventillated critically ill patients, the effect of full feeding on mortality is stil controversial. we aimed to investigate the relationship of energy intakes with -day mortality, and nutritional risk status influenced this relationship. methods: this prospective observational study was conducted among adult patients admitted to icu and required invasive mechanical ventilation (imv) for more than h. data on baseline characteristics and the modified nutritional risk in critically ill [mnutric] score was collected on day . energy intake and nutritional adequacy was recorded daily until death, discarge or until twelfth evaluable days. patients were divided into groups: a)received < % of prescribed energy b) received >= % of prescribed energy. results: patients ( % male, mean age . ± . years, mean body mass index . ± . kg/m , mean mnutricscore . ± . ) were included. in the univariate analysis, mnutrİc score was associated with -day mortality. in the multivariable logistic regregression analysis, mnutric score(odds ratio, or . , ci . - . , p < o.oo ) was associated with -day mortality. nutritional adequacy was assessed, median nutritional adequacy was . ( . - . ). in patients with high mnutrİc score ( - ), received >= % of prescribed energy was associated with a lower predicted -day mortality; this was not observed in patients with low mnutrİc score ( - ). conclusions: nearly % of imv required patients admitted to icu were at nutritional risk, mnutrİc score is associated with -day mortality. energy adequacy of >= % of prescribed amounts were associated with decreased mortality in patients with a high mnutrİc score. results: patients included in the study were asa iv. four patients died in the first few days after surgery ( ÷ days). mean length of stay in icu was . ± . days. univariate analysis showed a correlation between hypoalbuminemia and the onset of mof (p = . ); reduction of the lymphocyte count and risk of mof (p = . ). sofa score showed a significant correlation with occurrence of pneumonia (p = . ) and mof (p = . ). including the -day mortality among confounders, albumin and lymphocyte count were the strongest predictors of mof. length of stay in icu and ventilation days did not have statistical significance. bmi showed no predictive value of any outcome. conclusions: our sample was poor but results of our study seem to indicate malnutrition as an independent risk factor for elderly patients undergoing emergency surgery. early multidisciplinairy screening of dysphagia at admission to the emergency departmenta pilot study d melgaard, l sørensen, d sandager, a christensen, a jørgensen, m ludwig, p leutscher north denmark regional hospital, hjørring, denmark critical care , (suppl ):p introduction: dysphagia increase the risk of aspiration pneumonia, malnutrition, dehydration and death. this combined with the fact that patients with dysphagia have a longer stay in the hospital makes early prognosis and appropriate treatment important. knowledge about effect of early dysphagia screening is limited. the aim of this study is to examine the prevalence of dysphagia in the emergency department (ed) population. methods: this study included consecutively hospitalized patients in days from pm- pm at the ed of north denmark regional hospital. the screening took place within hours of admission. inclusion criteria were any of the following: age ≥ years, neurological disorders, alcoholism, copd, pneumonia, dyspnoea, diabetes or unexplained weight loss. a nurse screened patients with a water test and with signs of dysphagia tested by an occupational therapist with the v-vst and the meof-ii. results: of eligible patients ( % male, median age years) ( %) were screened. it was impossible to screen patients ( %) to limited time and patients ( %) due to poor health condition and patients ( %) declined participation. the prevalence of dysphagia in the study population was % ( patients). results from the water test were confirmed with v-vst and meof-ii. in patients with lung related diseases or circulatory diseases was the prevalence respectively % and %. patients, not screened due to poor health condition, were tested during hospitalisation and the prevalence of dysphagia was % in this group of patients. conclusions: the prevalence in ed patients was %. patients transferred to other departments due to poor health condition had a prevalence of %. it is possible to screen patients in the ed. the water test is a useful screening tool in an acute setting. introduction: to improve protein and energy delivery in a nutrition delivery bundle was introduced to a level icu. greater protein and energy intake is associated with improved outcomes in the critically ill [ ] [ ] [ ] [ ] , but only % of prescribed protein and energy is delivered in icus worldwide [ , ] . methods: percentage of target protein and energy delivery was measured via participation in the international nutrition survey (ins) before and after a "nutrition delivery bundle" was introduced by the icu dietitian. the nutrition delivery bundle involved all stakeholders in icu nutrition care (fig. ) and included the following quality improvement measures: increased icu dietetic staffing, update of icu enteral feeding protocol with staff education, use of higher protein formulations, earlier patient nutrition assessment, daily calculation of percentage nutrition delivery, increased nutrition communication through more regular discussion of patient care with medical team, expansion of choice of nasojejunal tube available, monthly reporting of key nutrition performance indicators, improved resources for cover dietitian(s) when icu dietitian on leave (fig. ) . results: prior to a nutrition delivery bundle being introduced the mater misericordiae university hospital (mmuh) icu achieved % of protein and % of energy targets over the first admission days of consecutive mechanically ventilated patients in icu > hrs enrolled in the international nutrition survey. this increased to % of protein and % of energy targets in (table ) . conclusions: a % improvement in protein and energy delivery to critically ill patients was seen after the introduction of a dietitian-led nutrition delivery bundle. introduction: the critically ill polytrauma patient with sepsis presents with variable energetic necessities characterized by a proinflammatory, pro-oxidative and hypermetabolic status. one of the challenges the icu doctor faces is adapting the nutritional therapy based on the individual needs of each patient. through this paper we wish to highlight the trend of energy needs in the case of critically ill polytrauma patients with sepsis by using non-invasive monitoring of respiratory gases based on indirect calorimetry (ge healthcare, helsinki, finland). methods: this is a prospective observational study carried out in the anesthesia and intensive care unit "casa austria", emergency county hospital "pius brinzeu", timisoara, romania. we monitored vo , vco , energy demand (ed), and specific clinical and paraclinical data. we measured energy demand values monitored by direct calorimetry with values calculated based on standard formulas. results: values have been recorded in the study. the mean vo was . ± . ml/min/kg, the mean vco was . ± . ml/min/kg. in regard with energy demand, the mean ed obtained through direct calorimetry was . ± . kcal/day, and those obtained by using mathematic formulas were . ± kcal/day (p < . ). moreover, statistically significant differences have been observed regarding the mean difference between energy demand determined using indirect calorimetry and that determined mathematically, respectively between the enteral and parenteral administered ed. conclusions: continuous monitoring of the energy demand in critically ill patients with sepsis can bring important benefits in regard with the clinical prognosis of these patients through the individualization and adaption of intensive therapy for each patient. introduction: cachexia is defined as a complex metabolic syndrome associated with underlying illness, characterized by loss of muscle with or without loss of fat. in cancer cachexia, reduction in muscle size has been demonstrated to be an independent risk factor for mortality. loss of muscle in icu patients is rapid and extensive and is also associated with mortality risk, but methods to measure muscle mass in these patients are lacking. surrogate methods (dexa, ct, ultrasound, total body water) do not measure muscle mass directly methods: the d -creatine (d -cr) dilution method takes advantage of the fact that % of cr is found in muscle and that muscle mass can be assessed by cr pool size. cr is transported into muscle against a concentration gradient and irreversibly converted to creatinine (crn), which is excreted in urine. a single oral dose of d -cr is transported to skeletal muscle, and measurement of d -crn enrichment in a spot urine sample provides an accurate estimate of skeletal muscle mass. results: the method has been validated in preclinical and clinical studies; in a large longitudinal observation study in older men, d -cr muscle mass was strongly associated with habitual walking speed, risk of falls, and incident mobility limitation; dexa failed to show these relationships. the d -cr method is being used in a nicu study to measure changes in muscle mass in neonates (gates foundation grant). further, this method has been incorporated into a trial assessing the treatment effects of a ghrelin agonist in icu patients with enteral feeding intolerance (nct ). in this trial, the d -cr dose is delivered intravenously and a spot urine sample is collected at baseline and postdose. conclusions: the d -cr method provides a non-invasive, accurate way to assess therapeutic agents that may mitigate the loss of skeletal muscle mass; it is of particular utility in clinical settings where changes in muscle mass are consequential, such as muscle loss during an icu admission. introduction: vitamin c, an enzyme cofactor and antioxidant, could hasten the resolution of inflammation, which affects most intensive care unit (icu) patients. while many observational studies have demonstrated that critical illness is associated with low levels of vitamin c, randomized controlled trials (rcts) of high-dose vitamin c, alone or in combination with other antioxidants, yielded contradicting results. the purpose of this systematic review and meta-analysis is to evaluate the clinical effects of vitamin c when administered to various populations of icu patients. methods: eligible trials: rcts comparing vitamin c, by enteral or parenteral routes, to placebo in icu patients. data collection and analysis: we searched medline, embase, and the cochrane central register of controlled trials. after assessing eligibility, data was abstracted in duplicate by two independent reviewers. overall mortality was the primary outcome; secondary outcomes were infections, icu length of stay (los), hospital los, and ventilator days. pre-specified subgroup analyses were conducted to identify more beneficial treatment effects. results: pooling rcts (n= ) reporting mortality, vitamin c was not associated with a lower risk of mortality (risk ratio [rr]: . , % confidence interval [ci]: . - . , p= . , i = %). in a subgroup analysis, trials of lower quality (n= ) were associated with a reduction in mortality (rr . , % ci . , . , p= . ), whereas high quality trials (n= ) were not. no statistical difference existed between subgroups (p= . ). in addition, no effect was found on infections, icu or hospital length of stay, and ventilator days. conclusions: current evidence does not support the hypothesis that vitamin c supplementation improves clinical outcomes of icu patients. introduction: the protein intake for patients who met adequacy for energy was assessed within our cardiothoracic intensive care. nutritional support should aim to provide at least % of calorie requirements to achieve nutritional adequacy with suggested protein requirements of . - g/kg/day [ ] . guidelines highlight the difficulty achieving the correct protein:energy ratio from nutritional support to meet this target especially in the obese population. methods: the audit was registered with clinical governance. data was collected prospectively from patients requiring tube feeding for three or more days from january -october (table ). data included type and volume of feed and calories from other sources. patients who met adequacy for energy (fig. ) introduction: patients admitted to the intensive care unit (icu) are usually at high risk of malnutrition [ , ] . the purpose of our study was to compare the accuracy of nutric score, nrs and sga in predicting los-icu, los-hosp and in-hospital mortality. methods: a total of consecutive patients admitted between march to june in a mixed (medical/surgical) icu were assessed on day of admission using the three screening tools to classify them into high-risk and low-risk of malnutrition. day apache scores and demographic data were recorded. los-icu, los-hosp inhospital mortality and secondary outcomes studied were need for supplemental nutritional support, need for ventilation and need for dialysis in high-risk and low-risk patients by each nutrition assessment tool. results: of the patients studied, ( . %) were males and ( . %) were females. . % males and . % females were found to be at a high risk of malnutrition by at least one of the scores. the mean apache score for patients at high risk (using any one screening tool) was . (sd . ) and . for the low risk group (sd . ; p < . ). the nrs and sga demonstrated statistically significant correlation(p= . ) for length of icu stay for both the high risk and low risk group whereas only the nrs correlated significantly for the length of hospital stay(p= . ). mortality was significantly higher in high risk patients identified using all scores. conclusions: there was a wide difference in the percent of patients identified as high-risk using each of the scores. introduction: nitrogen balance (nb) may be an important tool in the nutritional management of critically ill patients. cancer patients present a special challenge regarding nutrition, due to its peculiar characteristics related to neoplasia and adjuvant treatments. objectives: to evaluate nb in patients with solid cancer in the postoperative period in the icu, analyzing the correlation between nb and the mortality outcome in the icu. methods: retrospective cohort study. we evaluated adult patients (> years) admitted to the icus of two different hospitals, with diagnosis of current cancer in postoperative period (elective or emergency surgeries). patients were excluded if the diagnosis of cancer was not confirmed. nb (measured through analysis of dietary protein intake subtracted from -hour urinary urea plus an estimate of nonurinary losses) was calculated on the st, rd and th icu day. nb was measured only while the patient was in the icu. results: during the study period, patients were included (mean age . , mean apache . , . % male). admission apache ii and abdominal-site surgery were predictors of mortality. the nb of all patients was negative on the st icu day. in the patients who survived, nb of the rd and th day remained stable (negative), whereas in patients who died nb was more positive (fig. ) . there was no difference in the amount of protein ingested on the st day between survivors and deceased patients. conclusions: among adult patients with solid cancer in the postoperative period in the icu, nb was persistently negative in the survivors between st and th icu day, but among the patients who died nb tended to be more positive on the rd day. nb monitoring could allow a more adequate individualization of nutritional management in this group of patients. fig. (abstract p ) . nitrogen balance in st, rd and th icu day introduction: nutritional therapy plays an important role in the treatment of critically ill patients. caloric and protein goals are defined, and artificial nutrition tailored to the targets which are related to outcome [ ] . questions rise about the mean caloric and protein needs of patients, once discharged from icu, and the evolution of body weight, and nutritional adequacy. the aim is to know the ratios between caloric needs and intake of patients with a minimum stay at icu of days. methods: after evaluation of critically ill patients, patients were prospectively followed during their entire hospitalization. data concerning nutritional needs, prescriptions and delivery were collected from the electronic medical file. nutritional calculations of oral intake were done by nubel. ratios were made during the entire stay and body weight was followed up. results: in female and male patients, median age . years (range - year), estimated body weight of . ± kg and actual body weight of . ± kg, a mean caloric need of ± kcal/ day and an effective delivery of ± kcal/day was observed. body weight increased in two patients and decreased in ( %). in ten out of twelve patients, underfeeding was present. one patient with a caloric need of kcal/day received a mean caloric load of kcal/day ( . %). conclusions: the overall observed evolution in body weight was negative in most of the patients. nutritional adequacy was low after icu discharge and never reached target. introduction: severe burn injury can create a rapid-onset, sustained proinflammatory condition that can severely impair all major organs. this massive systemic response has been documented clinically by associated biomarker measurements including dramatic elevations in cytokines such as il- . the severity of multi-organ injury and subsequent development of other systemic complications in burn patients have been well-correlated with il- levels, including the increased risk of sepsis/multi-organ failure and associated morbidity and mortality. considering that estrogen is a powerful and easy to use anti-inflammatory agent, an experimental burn model was created to test the potential value of parenteral β-estradiol (e ) as a feasible and inexpensive early intervention to mitigate the the profound pro-inflammatory response associated with severe thermal injury. methods: male rats (n = ) were assigned randomly into three groups: ) controls/no burn (n = ); ) burn/placebo (n = ); and ) burn/e (n = ). burned rats received a % °tbsa dorsal burn, fluid resuscitation and one dose of e or placebo ( . mg/kg intra-peritoneal) minutes post-burn. eight animals from each of the two burn groups (burn/placebo and burn/e ) were sacrificed at minutes (sham group at days only), with four each of the two burn groups sacrificed at days. tissue samples from major organs and serum were obtained and analyzed by elisa for il- at each of these intervals. results: in the burned rats, β-estradiol decreased the organ levels of il- significantly as measured at both early ( min.) and late ( day) phases post-burn (figs. & . also, sham animal levels were comparable to the estradiol group, conclusions: experimentally, a single, early post-burn dose of estrogen significantly mitigates the associated detrimental inflammatory response in all major organs up to days. in turn, this may present a promising potential therapy to decrease the widespread multipleorgan dysfunction seen in severe burn injury patients. early, single-dose estrogen increases levels of brain-derived neurotrophic factor (bdnf), a neurotrophin for neuronal survival and neurogenesis following indirect brain inflammation caused by severe torso burns introduction: prior studies have found that patients with severe burns may suffer significant neurocognitive changes. while frequently attributed to psycho-social issues, we have found a substantial, rapid and sustained ( min - day) increase in rat brain inflammatory markers (for example, il- ) following remote torso burns that is blunted by a single post-burn dose of estrogen. brain-derived neurotrophic factor (bdnf), one of the most active neurotrophins, protects existing neurons and encourages the growth and differentiation of new neurons and synapses. as estrogens not only blunt inflammation but also exert an influence on cns growth factors, we hypothesized that β-estradiol (e ) might affect levels of bdnf in the post-burn rat brain. methods: male rats (n = ) were assigned randomly into three groups: controls/no burn (n = ); burn/placebo (n = ); and burn/e (n = ). burned rats received a % °tbsa dorsal burn, fluid resuscitation and one dose of e or placebo ( . mg/ kg intraperitoneally) minutes post-burn. eight animals from each of the two burn groups (burn/placebo and burn/e ) were sacrificed at hours and at days, respectively (sham group at days only), with four each of the two burn groups sacrificed at days. brain tissue samples were analyzed by elisa for bdnf. results: mean levels of bdnf were significantly elevated within hours and continued to increase up to days post-injury in burned animals receiving the β-estradiol (> pcg/mg) as compared with the placebo-treated burned animals (< pg/mg) and controls (< . pcg/mg). see fig. . conclusions: early, single-dose estrogen administration following remote severe burn injury significantly elevated levels of bdnf in brain tissue. this finding may represent an extremely novel and important pathway to enhance both neuroprotection and neuroregeneration in burn patients. the value of cortisol in patients with the infection and multiple organ dysfunction. s tachyla, a marochkov mogilev regional hospital, mogilev, belarus critical care , (suppl ):p introduction: hormones changes in patients with infection and multiple organ dysfunction is a topic that hasn't been adequately studied. goal of study: to establish the value of cortisol in patients with infection and multiple organ dysfunction. methods: after approval the ethics committee of the mogilev regional hospital a prospective observational study was performed. the study included patients aged to years. all patients were hospitalized in the intensive care unit with the infection and multiple organ dysfunction. patients with endocrine diseases and receiving glucocorticoids were excluded. cortisol levels were measured on admission and during the course of treatment by radioimmunoassay. in group l (n = ) patients had a low levels of cortisol, in the m group (n = ) -normal cortisol, in group h (n = ) -high cortisol. results: cortisol level was in l-group . ( . , . ) nmol/l, in mgroup . ( . ; . ) nmol/l, in h-group . ( , ; . ) nmol/l. it is found that the mortality was higher in the groups l - . % (p = . ) and h - . % (p = . ), than in the m-group - . %. the mgroup odds ratio equals . at % confidence interval . - . when compared with the h-group. in the m-group in survivors patients (n = ) showed a decrease cortisol with ( . , . ) nmol/l to . ( . , . ) nmol/l (p = . ). while the no survivors patients (n = ) showed increase cortisol with ( . , . ) nmol/l to . ( ; ) nmol/l (p = . ). thus itself cortisol level is not a marker of mortality. receiver operating curve analysis for cortisol was performed: area under the curve equals . at % confidence interval of . - . (p = . ), sensitivity . %, specificity . %. conclusions: in patients with infection and multiple organ dysfunction may be observed disorders in cortisol levels. these disorders require correction to prevent the increased mortality. introduction: the hypothalamic-pituitary-adrenal (hpa) axis is a key regulator of critical illness. cortisol and adreno-corticotrophic hormone (acth) are pulsatile, which emerges from the feed forwardfeedback of the two hormones [ ] . different genes are activated by continuous or pulsatile activation of the glucocorticoid receptor, even when the total amount is the same [ ] . we aimed to characterise the acth and cortisol profiles of patients who were critically ill after cardiac surgery and assess the impact of inflammatory mediators on serum cortisol concentrations. methods: patients with > organ system failure, > days after cardiac surgery were recruited. total cortisol was assayed every min, acth every hour and il , il , il , il , il , tnf-α every hours. cortisol binding globulin (cbg) was assayed at and hrs. the relationship between cortisol and the inflammatory mediators was quantified in individual patients using a mixed regression model. results: all profiles showed pulsatility of both cortisol and acth and there was concordance between the two hormones (see fig. ). one patient died after hours (see fig. ). this patient lost pulsatility and concordance of cortisol and acth. mean cbg was . μ g/ml at the start of sampling and . μ g/ml at the end. there was an association between il (p= . ), il (p< . ), il (p= . ) and serum cortisol levels. there was no association between the other mediators and cortisol. conclusions: cortisol and acth are both pulsatile in critical illness. because pulsatility emerges from the interaction between the two hormones[ ]the premise of a 'disconnect' between the pituitary and adrenal gland is refuted. il , il and il may have roles in the control of cortisol during critical illness. introduction: elevation in plasma cortisol is a vital response to sepsis and partially brought about by reduced cortisol breakdown in which bile acids (bas) may play a role. vice versa, cortisol can also upregulate bas. we hypothesized a central role for the hepatic glucocorticoid receptor (hgr) in cortisol and ba homeostasis and in survival from sepsis. methods: in a mouse model of sepsis, we documented hgr expression and investigated the impact of hepatocyte-specific shrnaknockdown of gr on markers of corticosterone (cort), ba and glucose homeostasis, inflammation and survival. we also compared hgr expression in human septic icu and elective surgery patients. results: in mice, sepsis reduced hgr expression with % (p= . ), elevated plasma cort, bas and glucose and suppressed a-ringreductases. also in human patients, sepsis reduced hgr expression (p< . ), further suppressed by treatment with steroids (p= . ). in septic mice, further and sustained hgr-inhibition increased mortality from % to % (p< . ). at h, hgr-inhibition prevented the rise in total plasma cort, but did not affect a-ring-reductases expression. however, it further reduced cort binding proteins, resulting in elevated free cort equal to septic mice without modified hgr. after days of hgr-inhibition in sepsis, total and free cort were comparable to septic mice without modified hgr, now explained by further reduced a-ring-reductase expression, possibly driven by higher hepatic ba content. hgr-inhibition blunted the hyperglycemic sepsis response without causing hypoglycemia, markedly increased hepatic and circulating inflammation markers and caused liver destruction (p< . ), the severity of which explained increased mortality. conclusions: in conclusion, sepsis partially suppressed hgr expression, which appears to upregulate free cort availability via lowered cort binding proteins and a-ring-reductases. however, further sustained hgr suppression evoked lethal excessive liver and systemic inflammation, independent of cort availability. introduction: cortisol levels have been found to be increased in sepsis patients, and high cortisol levels have been correlated with increased mortality. the purpose of this project is to assess the association of plasma cortisol levels with severity of coagulopathy in a population of patients with sepsis and clinically confirmed dic. methods: citrated, de-identified plasma samples were collected from adults with sepsis and suspected dic at the time of icu admission. platelet count was determined as part of standard clinical practice. pt/inr and fibrinogen were measured using standard techniques on the acl-elite coagulation analyzer. cortisol, d-dimer, pai- , cd l, nlrp , and microparticles were measured using commercially available elisa kits and were performed. dic score was calculated using isth scoring algorithm. results: cortisol showed significant variation based on dic status (kruskal-wallis anova, p < . ). patients with non-overt dic and overt dic exhibited significantly elevated cortisol levels compared to healthy controls (p < . for both groups). cortisol levels showed dic based variations. patients with sepsis and overt dic had elevated cortisol compared to patients with sepsis and no dic (p = . ) (fig. ) . correlations were evaluated between cortisol and hemostatic markers platelets, fibrinogen, inr, d-dimer, and pai- as well as with the inflammatory marker, nlrp and the platelet markers cd l and microparticles. cortisol conclusions: cortisol showed a significant association with hemostatic status in a population of patients with sepsis and welldefined coagulopathy. cortisol levels were significantly elevated in patients with overt or non-overt dic compared to healthy individuals and in patients with overt dic compared to those with sepsis without dic. introduction: in most cases presenting with hypoglycemia in emergency departments (eds), the etiology of the hypoglycemia is almost identified. however, about % of cases, the etiology of hypoglycemia cannot be determined. methods: this is a -year prospective observational study. a total of patients were transported to our ed with hypoglycemia. after the investigation, a rapid acth loading test (synthetic - acth μg iv.) was performed on patients with unexplained hypoglycemia; i.e., μg acth was administered intravenously and blood specimens were collected before loading, at min and min after acth administration. we adopted a peak serum cortisol level < μg/dl or a delta cortisol of < μ g/dl for the diagnosis of adrenal insufficiency. results: among the patients, of ( . %) were using antidiabetic drugs, ( . %) were using hypoglycemia-relevant drugs, ( . %) suffered from digestive absorption failure including malnutrition, ( . %) had been consuming alcohol, ( . %) suffered from malignancy, and ( . %) suffered from insulin autoimmune syndrome. initially, an etiology was unknown in of ( . %) patients. rapid acth test revealed the adrenal insufficiency in ( . %) among them. administration of hydrocortisone in adrenal insufficiency patients promptly improved hypoglycemia. in those patients, serum sodium level was lower (na; vs. meq/l, p< . ) and serum potassium level was higher (k; . vs. . meq/l, p< . ) than in the other hypoglycemic patients, respectively. there was no significant difference in baseline plasma glucose level on ed between the groups of patients ( vs. mg/dl, p= . ). conclusions: the probability of adrenal insufficiency was much greater than that of the better-known insulinoma as a cause of hypoglycemia. when protracted hypoglycemia of unknown etiology is recognized, we recommend that the patient is checked for adrenal function using the rapid acth loading test. introduction: sepsis caused have showed serious alternations of thyroid hormones releasing, causing a nonthyroidal illness syndrome. the aim of the study was to measure thyroid hormone levels in septic patients and analyse its relation with clinical state and outcome. methods: prospective study in a cohort of consecutive septic patients. we studied thyrotropin (tsh), free triiodothyronine fraction (ft ) and free thyroxin fraction (ft ) serum levels, apache ii and sofa score. statistical analysis was performed using spss . . results: we analysed episodes of sepsis ( %) and septic shock (ssh) ( %), the median age of the patients was (inter-quartile range, . - ) years; the main sources of infection were: respiratory tract ( %) and intra-abdomen ( %); . % had medical diseases. apache ii score was [ - ], sofa score was [ . - ] and day mortality was . %. our data shown . % with low levels of tsh (< . uui/ml), . % had low levels of ft (< . ng/dl) and . % low levels of ft (< pg/ml). the tsh ( . vs. . uui/ml) and ft ( . vs . pg/ml) concentration of ssh group were significantly lower than those of sepsis group, whereas ft ( . vs . ng/dl) it was not statistically significantly. correlation of ft to apa-che ii (r = − . , p = . ) and sofa score (r = − . , p = . ). the profile of death patients were men ( . %, n = ), with significantly older ( vs. years; p= , ), as well as clinical severity scores, apache ii ( . vs. . ; p< . ) and sofa ( . vs . ; p< , ). non-survivors had significantly lower tsh . vs. . uui/ ml; p= . , and ft . vs. . pg/ml, p= . , however ft did not show statistical significance . vs. . ng/dl, p=ns. conclusions: conclusions: most of our septic patients present an altered thyroid function. our data suggest that tsh and specially ft may be used as a marker of disease severity and a mortality predictor. observational study to evaluate short and long-term bone metabolism alteration in critical patients. introduction: reduction of bone mineral density and/or muscle mass can be short and long-term complications in critical patients admitted in intensive care unit (icu). the study aims to evaluate, during a -month period, the following parameters: ) the alterations of bone metabolism and quantitative and qualitative parameters of bone tissue, ) the proportion of subjects with bone fragility, and ) the identification of specific risk factors. methods: an observational-longitudinal monocentric study is being conducted in adult patients hospitalized in icu. the evaluations performed at baseline, and month visits include analysis of biochemical and instrumental exams. results: a specific clinical-care pathway was created between bone metabolic diseases unit and icu, in order to perform specific anamnestic collection, biochemical analysis of bone metabolism, and instrumental exams. patients were enrolled and evaluated at the baseline visit. biochemical exams, performed within hours of hospitalization, showed that % (n: ) of subjects had a deficit of ohvitamind < ng/dl, associated with normal corrected serum calcium levels and of these % (n: ) had high pth levels. bone alkaline phosphatase was increased in % (n: ) of patients. conclusions: critical patients are "fragile" subjects, which should be monitored with a short and long-term follow-up. the creation of a clinical pathway that includes specialists of bone metabolism may be a virtuous way to identify patients who report bone mass loss and increased fracture risk. this study will allow to implement the knowledge regarding specific risk factors of bone fragility and the most appropriate therapeutic choices as prevention and treatment. a retrospective analysis of predictors for length of intensive care stay for patients admitted with diabetic ketoacidosis a fung, tl samuels, ae myers, pg morgan east surrey hospital, redhill, uk critical care , (suppl ):p introduction: diabetic ketoacidosis (dka) is one of the most common metabolic causes of admission to the intensive care unit (icu). the incidence of dka is quoted as between . - episodes per patients with diabetes mellitus (dm) [ ] . we aim to establish the factors that affect length of stay (los) on icu. methods: we undertook an analysis of patients admitted to icu over the last years with a primary diagnosis of dka. we assessed whether there was an association between the following factors and an increased length of icu stay: age, gender, body mass index (bmi), systolic blood pressure, heart rate, sodium, potassium, haemoglobin and ph. these factors were assessed using multiple linear backward stepwise regression. results: overall, admissions were identified over the time period from the ward watcher database. the median los was . days (iqr . - . ). our analysis demonstrated that length of icu stay (alpha level < . ) was significantly associated with bmi, low systolic blood pressure, and the presence of hyponatraemia or hypernatraemia. conclusions: we found the variables that affect the los for patients presenting to our unit with dka are bmi, elow systolic bp, low sodium and high sodium. we intend to extend this work to include survival analysis with the same subgroup of patients. maximal glycemic gap is the best glycemic variability index correlated to icu mortality in medical critically ill patients t issarawattna, r bhurayanontachai prince of songkla university, songkla, thailand critical care , (suppl ):p introduction: several evidences shown a correlation of glycemic variability (gv) and icu mortality. however, there have been no report of the correlation between various parameters of gv and mortality in medical icu patients. the aim was to determine the correlation between various parameters of gv and medical icu mortality, as well as, to identify the best gv index to predict icu mortality. methods: a retrospective chart review was then conducted in medical icu at songklanagarind hospital. the patient characteristics, causes of admission, apache ii, blood glucose within the first hours of icu admission and icu mortality were recorded. glycemic variability parameters including maximal glycemic gap, standard deviation, coefficient of variation and j-index of blood glucose were calculated. the correlation of those gv index to icu mortality was determined. the roc and auroc of each gv index were then compare to identify the best gv index to predict icu mortality. results: of patients, patients ( . %) were survived ( table ). all gv indexes were significantly higher in non-survival group (p < . ) ( table ). maximal glycemic gap was independently correlated to icu mortality and give a highest auroc compared to others gv. (maximal glycemic gap auroc . ( %ci . - . vs. coefficient of variation auroc . ( %ci . - . ) vs standard deviation auroc . ( %ci . - . ) vs j-index auroc . ( %ci . - . ), (p< . ) (fig. ) . conclusions: maximal glycemic gap independently correlated to icu mortality and was the best gv to predict icu mortality in medical critically ill patients. reliability of capillary blood glucose measurement for diabetic patients in emergency department h ben turkia, s souissi, a souayeh, i chermiti, f riahi, r jebri, b chatbri, m chkir regional hospital of ben arous, ben arous, tunisia critical care , (suppl ):p introduction: acute glycemic disorders should be early diagnosed and treated in emergency department (ed), especially hypoglycemia. can capillary blood glucose (cg) replace plasmatic glucose (pg). the objective of this study was to compare capillary blood glucose with venous blood glucose methods: patients with type diabetes were included. we realize a capillary blood glucose with a glucose meter (acu-check active-roche) and a concomitant determination of venous blood glucose with laboratory machine (synchrony cx delta system beckman coulter). a correlation study (pearson correlation) between the two measurements was evaluated and linear fitting equation was established. the concordance was checked with bland and altman method. results: during the months of the study, patients were included. the average age was +/- years old, with a sex ratio = . majority of patients ( %,n= ) had type diabetes and % was treated with insulin. we found an excellent correlation between the two techniques with a pearson correlation coefficient r= . .topredict the pg from cg, we can use this equation: pg(g/l)= . cg(g/l)+ . (r = . ; p= . ). we noticed a good concordance between the two techniques especially in case of hypoglycemia and moderate hyperglycemia (fig. ) . however, releases were noted with a pg higher than g/l. conclusions: in ed, the measurement of capillary glucose can exempt from venous blood glucose especially in case of hypoglycemia and moderate hyperglycemia. is frequently found in critically ill patients in icu, especially patients who are treated for a long time. this study aims to analyse the comparison between length of stay and dvt incidents in critically ill patients. methods: a cross-sectional study was employed. we include all patients who were years or older and were treated in icu of dr soetomo public hospital for at least days. data were collected from june until june . the patients were examined with sonosite usg to look for any thrombosis in iliac, femoral, popliteal, and tibial veins and well's criteria were also taken. results: thirty patients were included in this study. this study shows that length of stay is not the only risk factor for dvt in patients treated in icu. in our data, we found out that the length of treatment did not significantly cause dvt. other risk factors such as age and comorbidities in patients who are risk factors may support the incidence of dvt events. the diagnosis of dvt is enforced using an ultrasound performed by an expert in the use of ultrasound to locate thrombus in a vein. conclusions: length of treatment is not a significant risk factor for dvt. several other factors still need to be investigated in order for dvt events to be detected early and prevented. [ ] was used to retrospectively study trends and outcomes of cancer patients admitted to the icu between and . logistic regression analysis was performed to assess predictors of -day and -year mortality. results: out of , icu admissions, , hemato-oncological, , oncological and patients with both a hematologic and solid malignancy were analyzed. hematologic patients had higher critical illness scores, while oncological patients had similar apache-iii and sofa-scores. in the univariate analysis, cancer was strongly associated with mortality (or . , table ). over the -year study period, -day mortality of cancer patients decreased by % (fig. ) . this trend persisted after adjustment for covariates, with cancer patients having significantly higher mortality (or= . , %ci: . , . ). between and , the adjusted -day mortality decreased by % every year. over the decade, -year mortality decreased by %. having cancer was the strongest individual predictor of -year mortality in the multivariate model (or= . , %ci: . , . ) (fig. ) . conclusions: between and , the number of cancer patients admitted to the icu increased steadily and significantly, while longitudinal clinical severity scores remained overall unchanged. although hematological and oncological patients had higher mortality rates than patients without cancer, both -day and -year mortality decreased significantly over the study period. introduction: sepsis was redefined in with the introduction of an increase in sequential organ failure assessment Δsofa) score of >= and the quicksofa (qsofa) as screening tools for sepsisrelated mortality. however, the implementation of these criteria into clinical practice has been controversial and the applicability for hematological patients is unclear. methods: we therefore studied the diagnostic accuracy of different sepsis criteria for sepsis and mortality according to definition criteria in a retrospective analysis of hematological patients in an academic tertiary care hospital. patient characteristics and variables were collected in icuand non-icu patients to determine the systemic inflammatory response syndrome (sirs), Δsofa and qsofa. by applying the definition of sepsis as "life-threatening organ dysfunction caused by a dysregulated host response to infection" [ ] as reference, the scores were evaluated. in patients with sepsis who died, / were sirs-negative, / Δsofa-negative and / qsofa-negative ( fig. and table ). conclusions: in conclusion, these findings suggest that criteria proposed in the sepsis- definition might have limitations as screening fig. (abstract p ) . results of the logistic regression analysis for (a) -day and (b) -year mortality. all covariates were statistically significant except for white race in the -year mortality model. ***p-value< - , **p-value< . , *p-value< . fig. (abstract p ) . longitudinal change in -day mortality for cancer patients (yes) compared with controls (no) over the -year study period. mortality in the cancer group decreased from % to % (- %), while mortality in the control group decreased from to % (- %). enoxaparin pharmacokinetics in patients with augmented renal clearance, preliminary results of a single center study introduction: augmented renal clearance (arc) has being described in some groups of critically ill patients. the aim was to investigate the impact of arc on the pharmacokinetics of enoxaparin. methods: this is a prospective study in a surgical and medical intensive care unit (icu) carried out from august to november . patients < years old, under prophylactic treatment with enoxaparin and normal plasma creatinine, were included. anti-xa activity was measured at second day under treatment. creatinine clearance was calculated from urine sample collected during -hours. arc was defined by a creatinine clearance >= ml/min/ . m . results: thirteen patients aged years old (± . ) were included. six patients developed arc and of them were in therapeutic range. seven patients did not develop arc and of them were in therapeutic range. there was no differences between the two groups in achieving therapeutic range (fisher test, p= . ). we did not observe thromboembolic events. conclusions: we found no relationship between arc and therapeutic failure in patients under prophylactic treatment with enoxaparin. introduction: this study reviewed argatroban use in patients in a tertiary hospital critical care unit. argatroban is a direct thrombin inhibitor approved for use in proven or suspected heparin-induced thrombocytopenia (hit) in patients with renal dysfunction. methods: this was a retrospective cohort study in a medical and surgical icu in a tertiary teaching hospital. data was collected for adult patients treated with argatroban for proven or suspected hit april-august , excluding patients requiring ecmo. we scored patients using the t score and compared this to an elisa immunoassay optical density score which quantifies the pf /h antibody level. also noted was use of continuous haemodialysis and organ failure using the sequential organ failure assessment (sofa), scoring >= defines failure. results: patients were treated with argatroban for proven or suspected hit. / patients had a positive elisa. there was no relationship between t score and elisa optical density (fig. ) . infusions were commenced at either the manufacturer recommended dose of μg/kg/min or a reduced dose of . μg/kg/min. patients receiving the reduced dose had a median of organs failing compared to in the standard regimen. the time taken to the first aptr in range was longer with the reduced dose regimen, however, the time to a stable aptr was less (table ). in patients the dose of argatroban never stabilised. died and was very sensitive to argatroban and required cessation of the infusion for interventions. in the reduced regimen group, there were episodes of bleeding, minor pr bleed in a patient with organs failure and upper gi bleed. conclusions: in this population of icu patients the t score did not correlate with the elisa optical density score, as found previously. patients with multi-organ failure mostly received the reduced starting dose. however, the bleeding events were still confined to this group. this correlates with previous studies that organ dysfunction necessitates a dose reduction for argatroban. results: the mean age in our study group was ± years. the effects of tpe on standard coagulation were increased aptt ( ± to ± s, p= . ) and decreased fibrinogen levels ( ± to ± mg/dl, p= . ). a non-significant decrease in platelet count was observed ( ± to ± /mm , p= . ). on rotem parameters tpe was associated with increased ct in extem ( ± to ± s, p= . ) and intem ( ± to ± s, p= . ) and increased maxvt on extem ( ± to ± s, p= . ) and intem ( ± to ± s, p= . ). all other rotem parameters changed non-significantly. the decrease observed in fibrinogen levels was not associated with a decrease in fibtem mcf ( ± to ± mm, p= . ). conclusions: our results demonstrate that tpe is associated with minimum changes in clot kinetics initiation that do not result in either pro-or anti-coagulant changes. therefore, tpe with fresh frozen plasma can be safely used in normal subjects. introduction: acutely ill patients are prone to critical illness anaemia, a multifactorial condition with potential contribution of iatrogenic anaemia defined as lowered hb due to large/frequent venepunctions. decline in hb is most pronounced in the first days of icu stay. it correlates with the need for rbc transfusion, but the impact on patient outcome is uncertain. the aim of this study was to determine impact of phlebotomy on change in hb (Δhb), and correlation of Δhb with need for transfusion, presence of central venous catheter (cvc) and patient outcome. conclusions: critical illness anaemia is an unexplained phenomenon. impact of phlebotomy is hard to unequivocally determine since there are many confounders. the change in hb levels during icu stay correlates with the need for transfusion that could cause immunomodulation and potentially adverse outcome. every effort should be made to maintain adequate hb levels and lower the risk of iatrogenic anemia. introduction: anemia is prevalent in critically ill traumatic brain injury (tbi) patients and red blood cell (rbc) transfusions are often required. over the years, restrictive transfusion strategies have been advocated in the general critically ill population. however, considerable uncertainty exists regarding optimal transfusion thresholds in critically ill tbi patients due to the susceptibility of the injured brain to hypoxemic damages. methods: we conducted an electronic self-administered survey targeting all intensivists and neurosurgeons from canada, australia and the united kingdom working caring for tbi patients. the questionnaire was developed using a structured process of domains/items generation and reduction with a panel of experts. it was validated for clinical sensibility, reliability and content validity. results: the response rate was . % ( / ). when presented with a scenario of a young patient with severe tbi, a wide range of transfusion practices was noted among respondents, with % favoring rbc transfusion at a hemoglobin level of g/dl or less in the acute phase of care, while % would use this trigger in the plateau phase. multiple trauma, neuromonitoring data, hemorrhagic shock and planned surgeries were the most important factors thought to influence the need for transfusion. the level of evidence was the main reason mentioned to explain the uncertainty regarding rbc transfusion strategies. conclusions: in critically ill tbi patients, transfusion practices and hemoglobin thresholds for transfusion are said to be influenced by patients' characteristics and the use of neuromonitoring in critical care physicians and neurosurgeons from canada, australia and the uk. equipoise regarding optimal transfusion strategy is manifest, mainly attributed to lack of clear evidences and clinical guidelines ( -year) . no significant associations were found between ffp:rbc ratio and mortality rates. patients with higher apache ii score received more platelet transfusions and mortality rates were higher in those who received platelets:rbc ratio > . on multivariate analysis, higher apa-che ii score was an independent predictor of increased mortality. conclusions: the compliance with the recommended : : ratio of blood products was poor. there was no association between transfusion ratios and mortality after adjusting for apache ii score. introduction: the lack of evidence-based medicine supporting the transfusion decision is illustrated by the wide range of blood product use during first-time coronary artery bypass grafting (cabg). use of red blood cells (rbc) ranges from to percent, while the use of platelets range from to [ ] . approximately percent of cabg patients suffer abnormal bleeding, with platelet dysfunction thought to be the most common culprit [ ] . methods: the objective of this study was to evaluate the use of allogeneic blood and blood products among patients undergoing first-time cabg over the past years. the first patients who underwent cabg (on-pump and off-pump) from st of march each year were included for analysis. the percentage of patients receiving rbc, fresh frozen plasma (ffp), platelet and cryoprecipitate during the first hours intra-and postoperatively were analysed. linear regression analysis was performed in each group. results: our analysis shows that the use of rbc decreased over the last years, in contrast to the use of the other investigated products. (see fig. ) the increase of platelets was the most pronounced with a direction coefficient of . and had the least variability (r = . ). (see fig. ) the decrease in rbc was less obvious than the rise in platelet use (direction coefficient of . ) and had a higher variability (r = . ). the consumption of ffp and cryoprecipitate stayed constant (direction coefficient of . and . respectively). the higher incidence of semi-urgent cabg in recent years, which involves continuation of anti-platelet therapy until the day before surgery, can be an explanation for our observed increased use of platelets. the observed decrease in rbc transfusion over the past years might be due to rising awareness of complications associated with red cell transfusion. introduction: red blood cells (rbc) transfusion is frequently required in cardiac surgery and is associated with increased morbidity and mortality rates. the aim of this study is to identify predictors of rbc transfusion for patients undergoing cardiac surgery, emphasizing the use of bioelectrical impedance analysis (bia). methods: this was a retrospective study of patients who underwent elective cardiac surgery between years and in a tertiary reference center. patients' demographic and clinical variables, preoperative bia measurements and postoperative data were analyzed. the univariate and multivariate logistic regression analyses were used to identify the predictors of postoperative rbc transfusion. all of the calculations were performed with ibm spss v. . introduction: red blood cells (rbc) transfusion is a common intervention in cardiac surgery and is associated with higher mortality rates and predisposes serious adverse events. the aim of this study was to determine whether red blood cells (rbc) transfusion is linked to long-term results after cardiac surgery. methods: this observational retrospective study included all of the patients who underwent any of the sts defined elective cardiac surgery types from to . we evaluated - year all-cause mortality rates and secondary postoperative outcomes defined by the sts risk prediction model. patients were categorized according to whether they received rbc transfusions postoperatively; long-term results were compared using cox-regression analysis and kaplan-meier method. introduction: transfusion of packed red cells (prcs) is an important treatment option for patients requiring intensive care but, like all treatments, it is not without risk. these patients, although may be more sensitive to anaemia, are also at increased risk of transfusionrelated complications. we conducted an audit of blood prescribing and administering practices in our intensive care unit. methods: audit proformas were placed in blood prescribing forms for a -month period. all transfusions of prcs were logged over this time, and transfusion triggers, post-transfusion haemoglobin (hb) and whether hb was checked between units was recorded, in addition to other supplementary information. results: over a -month period, transfusion events were recorded, with an average age of the transfused patients of years old (range - years). % of transfusion events were for low hb, % for bleeding and in % of cases the indication was not documented. for patients transfused for a low hb, the mean transfusion trigger was g/l (range: g/l - g/l). only % had a transfusion trigger of g/l or less, and a further % who were transfused for a low hb had a hb of g/l or more. % of transfusion events involved transfusing or more units and, in only % of these cases the hb was checked between units. excluding the two bleeding patients, the mean increase in hb following a single unit transfusion was . g/l (range g/l - g/l), whilst in patients transfused two units, the average increase in hb was g/l per unit transfused (range g/l - . g/l), suggesting single unit transfusions may have greater hb yields. conclusions: our audit demonstrated variability in transfusion triggers and progress needed with administering practices when transfusing multiple units of blood in the non-bleeding patient. we have since implemented measures to meet guidelines in both prescribing prcs with restrictive triggers and in the administration and assessment of hb between units, and will be re-auditing. introduction: there is a perceived increased risk of bleeding in cirrhosis patients undergoing invasive procedures. this lead to a high rate of empirical prophylactic transfusion, which has been associated to increased complications and cost. the best strategy to guide transfusion in these patients remains unclear. our aim was to compare three strategies to guide blood component transfusion prior to central venous catheterization (cvc) in critically ill cirrhosis patients. methods: single center, randomized, double-blinded, controlled clinical trial conducted in brazil [ ] . all cirrhosis patients admitted to the icu with indication for a cvc were eligible. participants were randomized : : to three transfusion strategies based on: ( ) standard coagulation tests (sct), ( ) rotational thromboelastometry (rotem) and ( ) restrictive. the primary outcome was proportion of transfusion of any blood component prior to cvc. secondary outcomes were incidence of major and minor bleeding, icu length of stay (los), and -day mortality. analysis was intention-to-treat. results: participants ( in each group) were enrolled between september and december . most were male ( . %) and listed for liver transplantation. the study ended after reaching efficacy in first interim analysis. there was no significant difference in baseline characteristics among groups. regarding primary endpoint, there was ( . %), ( . %), and ( . %) events in sct, rotem and restrictive groups, respectively (p < . ). there was no difference between sct and rotem groups (p > . ). overall -day mortality was . % and was similar between groups. icu los did not differ between groups. there was no major bleeding. overall minor bleeding occurred in . % with no difference between groups. conclusions: a restrictive strategy is safe and effective in reducing the need of blood component transfusion prior to cvc in critically ill cirrhosis patients. a rotem-based strategy was no different from transfusion guided by sct. introduction: desmopressin (ddavp) is a vasopressin analogue which improves platelet function. its general use as a haemostatic agent is still controversial. the aim of study was to evaluate the effect of prophylactic desmopressin in blood coagulation in patients undergoing heart valve surgery. methods: prospective, randomized, double-blind clinical trial performed at the heart institute of the university of são paulo. a total of adult patients undergoing heart valve surgery were enrolled from february to november . immediately after cardiopulmonary bypass weaning and heparin reversal, patients were randomized in ratio : to intervention group: ddavp ( . μg/kg) or control group. blood samples were drawn at three different times, at baseline (t ), hours (t ) and hours (t ) after study medication. blood coagulation and perioperative bleeding were analysed using laboratorial tests and thromboelastometry, chest tube drainage and requirement of allogenic transfusion within hours. results: a total of patients were allocated to intervention and to control group. blood levels of factor viii at t ( . conclusions: prophylactic use of desmopressin in heart valve surgery does not influence coagulation and thromboelastometric parameters. identifying the impact of hemostatic resuscitation on development of multiple organ failure using factor analysis: results from a randomized trial using first-line coagulation factor concentrates or fresh-frozen plasma in major trauma (retic study) p innerhofer introduction: to clarify how hemostatic resuscitation affects occurrence of multiple organ failure. methods: analysis of secondary endpoints of the retic study [ ] (coagulation factors, activated protein c (apc), thrombin generation, rotem parameters, syndecan- , thrombomodulin (tm) and d-dimer) measured at randomization, and after patients had received ffp or coagulation factor concentrates (cfc) at admission to icu, and hours thereafter. we used factor analysis to reduce the highly interrelated variables to a few main underlying factors and analysed their relation to mof before and after hemostatic therapy. results: the factors concentration, clot and hypoperfusion representing trauma-induced coagulopathy (table ) were comparable between groups at baseline (fig. ) and only high hypoperfusionscore predicted mof, while after therapy a low clot-score also predicted mof. only the changes of the clot-score independently affected occurrence of mof (p= . , adjusted or . , ci . - . ), while changes of concentration (p= . , adjusted or . , ci . - . ) and hypoperfusion (p= . , adjusted or . , ci . - . ) did not. a lower clot-score occurred after ffp transfusion than use of cfc, mainly through persistent thrombocytopenia (platelet count r - ffp vs cfc p< . ) (fig. ) . the higher concentration-score after ffp did not affect mof and ffp had no beneficial effect on fibrinolysis, syndecan- , tm or apc. conclusions: hemostatic resuscitation should augment the factor clot, which is feasible with early fibrinogen administration but not with ffp. the found platelet-saving effect of early fibrinogen administration is important as platelets play a major role in inflammation and transfusion of platelets did not correct thrombocytopenia. introduction: the trauma induced coagulopathy clinical score (ticcs) was developed to be calculable on the site of injury with the objective to discriminate between trauma patients with or without the need for damage control resuscitation (dcr) and thus transfusion [ ] . this early alert could then be translated to in-hospital parameters at patient arrival. base excess (be) and ultrasound (fast) are known to be predictive parameters for emergent transfusion. we emphasize that adding this two parameters to the ticcs could improve its predictability. methods: a retrospective study was conducted in the university hospital of liège. based on the available data in the register (from january st to december st ), the ticcs was calculated for every patient. be and fast results were recorded and points were added to the ticcs according to the ticcs.be definition (+ points if be < - and + points in case of a positive fast). emergent transfusion was defined as the use of at least one blood product in the resuscitation room. the capacity of the ticcs, the ticcs.be and the trauma associated severe hemorrhage (tash) to predict emergent transfusion were assessed. results: a total of patients were included in the analysis. ( %) needed emergent transfusion. the probability for emergent transfusion grows with the ticcs.be value (fig. ) . positive predictive values (ppv) and negative predictive values (npv) of the three scores are displayed in table . conclusions: our results confirm that be and fast results are relevant parameters that can be added to the ticcs for better prediction of the need for emergent transfusion after trauma. fig. (abstract p ) . probability for emergent transfusion with ticcs.be values. fig. (abstract p ) . boxplots show available measurements of extrinsically activated clot firmness at min (exa ), fibrin polymerization at min (fiba ) and platelet count at baseline (r ) and after therapy at admission to icu, and hours thereafter (r to r ) for the cfc (blue, n= ) and the ffp (yellow, n= ) group as well as for patients without (white, n= ) and with (grey, n= ) multiple organ failure. table ) for the cfc (blue, n= ) and the ffp (yellow, n= ) group, as well as for patients without (white, n= ) and with (grey, n= ) multiple organ failure. each factor is given at the measurement time point baseline (r ) and following haemostatic resuscitation at admission to icu, and hours thereafter (r to r ). introduction: the management of the critically ill polytrauma patient is complex and is often a challenge for the intensive care team. the objectives of this study is to analyze the oxidative stress expression in polytrauma cases as well as to evaluate the impact of antioxidant therapy on outcomes. methods: this prospective study was carried out in the clinic for anaesthesia and intensive care "casa austria", form the "pius brînzeu" emergency county hospital, timisoara, romania, with the approval of the hospital's ethics committee. clinicaltrials.gov identifier nct . the patients' selection criteria included an injury severity score (iss) of or higher, and age of or higher. patients were eligible for the study. they were divided in two groups, group a (antioxidant free, control, n= ), and group b (antioxidant therapy, study group, n= ). the antioxidant therapy consisted in continuous iv administration of mg/ h of vitamin c until discharge from icu. the patients included in the study presented with similar characteristics, and no statistically significant differences were shown between group a and b regarding age (p > . ), sex (p > . ), iss upon admission (p > . ), percentage of patients admitted in the icu more than hour post-trauma (p > . ), and associated trauma (p > . ). among patients in group b statistically significant differences were identified regarding the incidence of sepsis (p < . ), multiple organ dysfunction syndrome (p < . ), mechanical ventilation time (p < . ), and mortality (p < . ). no statistically significant differences were shown regarding the time spent in the icu (p > . ). conclusions: following this study we can state that the administration of substances with a strong antioxidant character has positive influences on the outcome of critically ill patients, decreasing the incidence of secondary pathologies as well as mortality rates. icc increased by . %, icd increased by . %, slightly increased ma, and ircl was nearly in the normal range. conclusions: rapid and accurate diagnosis of the coagulation system by lpteg method at different stages of traumatic disease allows for more accurate selection and adjustment of the therapy, which allows improving the prognosis of the disease. introduction: evidence for tranexamic acid (txa) in the pharmacologic management of trauma is largely derived from data in adults [ ] . guidance on the use of txa in pediatric patients comes from studies evaluating its use in cardiac and orthopedic surgery. there is minimal data describing txa safety and efficacy in pediatric trauma. the purpose of this study is to describe the use of txa in the management of pediatric trauma and evaluate efficacy and safety endpoints. methods: this retrospective, observational analysis of pediatric trauma admissions at hennepin county medical center from august to november compares patients who did and did not receive txa. the primary endpoint is survival to hospital discharge. secondary endpoints include surgical intervention, transfusion requirements, length of stay, thrombosis, and txa dose administered. results: there were patients [<=] years old identified for inclusion using a massive transfusion protocol order. twenty patients ( %) received txa. baseline characteristics and results are presented as median (iqr) unless otherwise specified, with statistical significance defined as p < . . patients receiving txa were more likely to be older, but there was no difference in injury type or injury severity score (iss) at baseline (table ) . there was no difference in survival to discharge, need for surgical intervention, or thrombosis (table ) . patients who did not receive txa had numerically higher transfusion requirements and longer length of stay, but these did not reach significance. conclusions: txa was utilized in % of pediatric trauma admissions at a single level i trauma center, more commonly in older patients. though limited by observational design, we found patients receiving txa had no difference in mortality or thrombosis. introduction: the risk of venous thromboembolism (vte) in trauma is greatly increased and one of the leading causes of morbidity and mortality after an accident [ ] . prophylactic measures to prevent vte primarily consist of anticoagulants. in instances in which anticoagulation is contraindicated or inadequate, inferior vena cava (ivc) filters can be used [ ] . however, insertion of ivc filter as a prophylactic measure is controversial as filter-related complications are well documented and increase with treatment time [ ] . the objectives of our study were to evaluate ivc filter insertion indications and filter related complications in pelvic trauma patients. methods: patients with pelvic fractures were operated during the study period / / - / / . all patients who received ivc filter during the period were included into analysis. relevant data was collected from electronic patient journal. results: thirty four patients received retrievable filters during the study period ( males and females) ( table ) . median age of patients was years (range, - ). the predominant indication ( %) was prophylactic insertion. the median indwell time was days (range - days). despite ivc filter insertion one patient experienced lung embolism and another -dvt. in eleven cases ivc filters were tried to be removed at the treating hospital. in two cases filter extraction was unsuccessful and in another two cases filters were left in place due to ivc thrombosis. conclusions: majority of ivc filters were inserted outside guidelines [ ] and proportion of prophylactic indications is significantly higher ( % vs %) than seen in registry studies [ ] . filter related complications were observed in % of patients. more restrictive approach to prophylactic ivc insertion should be exercised. the impact of preinjury antiplatelet and anticoagulant pharmacotherapy on outcomes in patients with major trauma admitted to intensive care unit ( conclusions: patients on preinjury anticoagulants and antiplatelet agents showed an increased mortality; this may be the result of the greater incidence of bleeding, the older age and more comorbidities in this groups. is enzymatic debridement better in critically burned patients? introduction: early debridement of burned tissue reduces infection rate, icu stay and mortality. the use of proteolytic enzymes such as bromelain allows a faster, more effective and selective debridement of denatured tissue, preserving and exposing healthy tissues, reducing debridement times compared to standard of care. methods: retrospective observational study performed in the critical burn unit (march to september ) including patients > years old with a total body surface area (tbsa) burned > % and < %, or > years old with a tbsa burned > %, who underwent enzymatic debridement. mean and standard deviation were used for normal quantitative variables and median and interquartile range in the opposite case. qualitative variables were presented by absolute and relative frequencies. results: mean age was . ± . years old, % males, apache ii (ri - ), absi (ri - ). median tbsa burned was % (ri - %), % (ri - ) were deep dermal or full thickness. time until debridement was hours (ri - ). . % (n= ) had incomplete debridement after first application, % (n= ) received regional anesthesia, % (n= ) didn't need blood transfusion. % of patients who didn't have vasopressors prior debridement, needed the use of it with a mean dose of , mcg/kg/min. % of patients with vasopressors prior treatment, required an increase of dose by a mean of . mcg/kg/min. median icu stay was days. mortality was %. conclusions: topical bromelain allows a fast start of tissue debridement with a low rate of failure. the need for fasciotomy and blood transfusion was very low. topical treatment involved a fast and simultaneous debridement of the tbsa burned, generating an inflammatory response that in some cases required vasopressors. . . / ). the bche activity was measured by using point-ofcare-test system (securetec detektions-systeme ag, neubiberg, germany). levels of the routine inflammation biomarkers, i.e. c-reactive protein (crp) and the white blood cell count (wbcc), were measured during the initial treatment period. measurements were performed at the admission, followed by , and -hour time points. injury severity score (iss) was used to assess the trauma severity. results: the observed reduction in the bche activity was in accordance with the change in the crp concentration and the wbcc. the bche activity measured at the hospital admission negatively correlated with the length of the icu stay in patients with polytrauma (r = - . , spearman's rank correlation coefficient). conclusions: the bche activity might be used as an early indicator for the magnitude of the systemic inflammation following polytrauma. moreover, the bche activity, measured at the hospital admission, might predict the patient outcome and therefore prove useful in early identification of the high-risk patients. pharmacological interventions for agitation in traumatic brain injury: a systematic review introduction: among tbi complications, agitation is a frequent behavioural problem [ ] . agitation causes potential harm to patients and caregivers, interferes with treatments, leads to unnecessary chemical and physical restraints, increases hospital length of stay, delays rehabilitation, and impedes functional independence. pharmacological treatments are often considered for agitation management following tbi. however, the benefit and safety of these agents in tbi patients as well as their differential effects and interactions are uncertain. methods: the major databases and the grey literature were searched. we included all randomized controlled, quasi-experimental, and observational studies with control groups. the population of interest was all patients, including children and adults, who have suffered a tbi. studies in which agitation was the presenting symptom or one of the presenting symptoms, studies where agitation was not the presenting symptom but was measured as an outcome variable and studies assessing the safety of these pharmacological interventions in tbi patients were included. results: we identified references with our search strategy. two authors screened after removal of duplicates. after searching the grey literature and secondary databases, a total of potential articles were identified. eleven studies in which agitation or an associated behavior was the presenting symptom, studies where agitation was not the presenting symptom but was measured as an outcome variable, and studies assessing the safety of these pharmacological interventions were identified. overall, the quality of studies was weak. in studies directly addressing agitation, pindolol and propranolol may reduce assaults and agitation episodes. amantadine and olanzapine may reduce aggression, whereas valproic acid may reduce agitated behavior. conclusions: there is weak evidence to support the use of pharmacological agents for the management of agitation in tbi. impact of decompressive craniectomy on neurological functional outcome in critically ill adult patients with severe traumatic brain injury: a systematic review and meta-analysis p bonaventure, ja jamous, f lauzier, r zarychanski, c francoeur, a turgeon chu de québec -université laval, québec, canada critical care , (suppl ):p introduction: severe traumatic brain injury is associated with high mortality and functional disability. several interventions are commonly used to control the intracranial pressure to prevent secondary cerebral injuries. among them, decompressive craniectomy (dc) is widely performed; however, its impact on functional outcome is still under debate. our objective was to assess the efficacy and safety of this procedure in adult patients with severe traumatic brain injury. methods: we systematically searched in medline, embase, cen-tral, web of science, conference proceedings and databases of ongoing trials for eligible trials. we included randomized controlled trials of adult patients with severe traumatic brain injury, comparing dc to any other intervention. our primary outcome was the neurological function based on the glasgow outcome scale. secondary outcomes were mortality, intensive care unit (icu) and hospital length of stay, intracranial pressure control, and complications. two reviewers independently screened trials for inclusion and extracted data using a standardized form. we used random effect models to conduct our analyses and the i index to assess heterogeneity. results: we identified citations, from which we included trials for a total of patients. we observed no impact on the [ ] . univariate logistic regression analyses were performed to identify predictors associated with the decision for icp monitoring. results: a total of adult patients were included (tables and ). the risk of poor outcome estimated by the impact model was associated to the decision to monitor icp (fig. ) . icp was more often monitored in patients with severe tbi, with one dilated pupil at admission and positive ct findings (in particular, high marshall scores). conclusions: according to our results, the clinician follows a multifactorial reasoning: the main determinants for the decision to monitor icp are gcs, pupils' abnormalities and, above all, ct findings. future studies will be needed to clarify specific indications for the clinicians in the identification of patients who would benefit from invasive monitoring. trajectories of early secondary insults after traumatic brain injury: a new approach to evaluate impact on outcome. introduction: secondary insults (si) occur frequently after traumatic brain injury (tbi). their presence is associated with a worse outcome. we examined the early trajectories of hypotension (sbp< mmhg), hypoxia (spo < %) and pupillary abnormalities from the prehospital settings to the emergency department (ed), and their relationship with -months outcome. methods: in this retrospective, observational study we included all tbi patients admitted to our neuro intensive care unit (nicu) from january to december . we defined the trajectories of si: -"sustained" if present on the scene of accident and at hospital admission, -"resolved" if present on the scene but resolved in ed, -"new event" if absent on the scene and present in ed, -"none" if no insults were recorded. we investigated the association of si trajectories with -months dichotomized outcome (glasgow outcome scale (gos); favorable= - ; unfavorable= - ). results: patients were enrolled in the final analysis. hypoxia and hypotension were related with unfavourable outcome when introduction: guidelines for management of pediatric traumatic brain injury recommend maintaining intracranial pressure (icp) < mmhg [ ] . use of . % sodium chloride (nacl) is considered safe and effective for management of icp in adults, but evidence for concentrations > % in pediatrics is limited. this study will describe the safety and efficacy of . % nacl in reducing icp among pediatric patients. methods: this retrospective study evaluated patients <= years old who received . % nacl and had continuous icp monitoring. cerebral perfusion pressure (cpp), mean arterial pressure (map), icp, and brain tissue oxygenation (pbto ) were recorded hourly and were compared to baseline for hours after each dose. safety outcomes included peak serum sodium, peak serum chloride, and the incidence of stage acute kidney injury (aki) (serum creatinine elevation >= . mg/dl or >= %) [ ] . results: between august and july , eligible pediatric patients received doses of . % nacl; doses were included in the analysis of perfusion parameters. mean age was . +/- years ( months to years), and the median initial glasgow coma scale score was . subjects received a median of four . % nacl boluses, with a mean dose of . +/- . ml/kg. significantly lower icp and higher cpp (p< . ) were observed at all post-treatment time points (fig. ) ; pbto was also significantly increased during of the hours recorded (p< . ). there was no difference in map. peak post-treatment serum sodium and chloride were +/- meq/l and +/- meq/l, respectively (fig. ) . stage aki was observed in . % of patients, and in-hospital mortality was . %. conclusions: our data suggests that . % nacl is a safe and effective therapy for elevated icp in pediatric patients. methods: we performed a prospective study in adult patients with mild head trauma (gcs and ) qualified for acquisition of urgent head ct scan. the clinical symptoms potentially related to intracranial lesion including abnormal vitals, vomiting, headache, persistent dizziness were recorded. ons as well as head ct were then performed. all ons examinations were executed by an experienced sonographer to eliminating interrater bias. head ct findings were dichotomized as positive or negative finding for ich based on formal radiology reports. the patients' disposition including admission, surgery and safe discharge were followed. results: patients were enrolled for the survey. patients had at least one symptom related to potential intracranial lesion ( . %). the mean onsd was ± mm. patients were found to have ich and underwent neurosurgery thereafter. no significant difference of onsd was found between the groups with and without ich, as well as the group receiving surgery or conservative treatment. with introducing a conventional mm threshold of onsd, the sensitivity, specificity, ppv and npv was . , . , . and . , respectively. while incorporating occurrance of at least one positive clinical symptom with the onsd measurement greater than mm as a composite threshold, the sensitivity, specificity, ppv and npv was . , . , . and . , respectively. conclusions: the diagnostic value of ons in mild head trauma is defective. nevertheless, with the supplemental aid of recognition of clinical symptoms relevant to potential intracranial lesion, the overall accuracy would improve. a correlation between ykl- concentrations in cerebrospinal fluid and marshall classification in traumatic brain injurypreliminary results g pavlov , m kazakova , p timonov , k simitchiev , c stefanov , v sarafian medical university -plovdiv, plovdiv, bulgaria, university of plovdiv, plovdiv, bulgaria critical care , (suppl ):p introduction: establishment of prognostic models in traumatic brain injury (tbi) would improve the classification based on predictive risks and will better define treatment options [ ] . in recent years, one of the most intensively studied glycoprotein is ykl- . it is expressed as a consequence of broad spectrum of inflammatory and malignant diseases [ ] . this is study aimed to investigate the level of ykl- in tbi patients and its relationship with several clinical models. methods: we determined plasma and cerebrospinal fluid (csf) ykl- levels in six ( ) patientson the th and th hour after the tbi. each patient was examined by physical and instrumental methods for somatic and neurological status, clinical assessment and prognostic scales (gcs, marshall classification, apache iii). routine haematological and biochemical tests were also performed. as control served the csf of age-matched suddenly deceased healthy individuals (n = ), which was examined post mortem for ykl- levels. results: we found no significant difference between plasma ykl- levels till th and th in all patients (mean difference ± sd: ± ng/ml ) and calculated cerebral autoregulation (ar) as correlation coefficients (pearson) for each ih wave. z-ratios were divided according to binary ar outcome and correlation calculated with intracranial pressure before, during and after the ih waves. results: our preliminary analysis demonstrated a negative correlation between intracranial pressure and z-ratio in the grouped ih waves with preserved ar, but no correlation in the grouped ih waves with impaired ar (table and fig. ). this indicates a decrease in power in the eeg low frequencies ( - hz) and/or an increase in the eeg high frequencies ( - hz) for increased values of intracranial pressure when ar is preserved. conclusions: features of ih waves differ depending on the ability of the injured brain to autoregulate cerebral blood flow. these features might include different signature of eeg frequency changes. the causative links and clinical significance of the different eeg patterns remain unexplored and might represent a signature of neurovascular coupling. introduction: targeted temperature management of patients who have suffered a traumatic brain injury is often used in the hope of preventing further insult to the brain; however, there is no uniform approach to managing temperature either locally, nationally or internationally, and maintenance of goal temperature in this patient population is often challenging due to hypothalamic injury. we sought to evaluate the feasibility and safety of an esophageal heat transfer device (ensoetm, attune medical, chicago, il) to perform temperature management of patients suffering from traumatic brain injury. methods: this was an irb-approved prospective study of patients undergoing temperature management after traumatic brain injury. patients were treated with an esophageal heat transfer device connected to an external heater-cooler, and maintained at target temperature for at least hours. patient temperature obtained via foley catheter was recorded hourly, and the deviation from goal temperature during treatment reported. results: a total of patients were treated from august to may . temperature targets during treatment ranged from . to . degrees c. maintenance of target temperature was successful, with % of readings within +/- degrees c of target, and % of readings within +/- . degrees c of target. one patient developed a small hydrothorax, not attributed to device use. all patients survived to discharge from the icu, with median cpc of (range to ). conclusions: targeted temperature management of patients with traumatic brain injury using an esophageal heat transfer device was feasible and safe, providing a tight maintenance of goal temperature in this challenging patient population. introduction: traumatic brain injury (tbi) represents a serious problem in europe. it still is the principal cause of death in us and europe. every year in italy people on , suffers of tbi and on , dies. disability and incapacity from tbi provides "strong ethicals, medicals, social and health economy imperative to motivate a concerted effort to improve treatment and preventions" methods: our hospital is the hub for modena's county for tbi and we took part in the past year on european project creactive (collaborative researce on acute traumatic brain injury in intensive care medicine in europa) as branch of italian group giviti (gruppo italiano per la valutazione degli interventi in terapia intensiva). our study concerned about patients with tbi dismissed from icu that "personally" or by the family will accepted the consensus to be included in our follow up conducted after months from the dismissal. we collected clinical data from the admission to the dismissale and measured impact of tbi on all day life with gos-e and qolibri-os using telephonical interview. results: we collected data about patients, answered to the telephonical follow-up and only compilated the qolibri-os. we found out that patients admitted with lower gcs has worst outcome in terms of quality of life. it also appears that anisocoria during icu staying represents an odds ratio for death and is connected with worst quality of life after months from the dismissal (tables & ) . inability to re-start a normal work-activity appeared to be the most important factor on the perception that our patient have of their new lives. conclusions: anisocoria seems to be an indicator of severe brain damage. gcs, despite it's simplicity, still represent the best and easiest way to score tbi. work impairment appear to be the most important disability to determine subjective perception of quality of life after tbi, so efforts have to be made to improve work rehabilitation after the dismissal from hospital. introduction: hyperventilation (hv) reduces elevated intracranial pressure (icp) by changing autoregulatory functions connected to cerebrovascular co reactivity. criticism to hv is due to the possibility of developing cerebral ischemia and tissue hypoxia because of hypocapnia-induced vasoconstriction. we aimed to investigate the potential adverse effects of moderate hv of short duration in the acute phase in patients with severe traumatic brain injury (tbi), using concomitant monitoring of cerebral metabolism, continuous brain tissue oxygen tension (pbro ), and cerebral hemodynamic with transcranial color-coded duplex sonography (tccd). methods: a prospective trial was conducted between may and may at the university hospital of zurich. adults (> years), with non-penetrating tbi, first gcs < , icp-monitoring, pbro and/or microdialysis (md)-probes were included within hours after injury. data collection and tccd measurements took place at baseline (a), at the begin of moderate hv (paco - . kpa) (c), after minutes of moderate hv (paco - . kpa) (d), and after return to baseline (e) (fig. ) . repeated measures anova was used to compare variables at the different time points followed by post hoc analysis with bonferroni adjustment as appropriate. p-value < . was considered significant. results: eleven patients were included ( % males, mean age ± years). first gcs was ( - : median and interquartile range). data concerning paco , icp, pbro , mean flow velocity (mfv) in the middle cerebral artery, and md values are presented in table . during hv, icp and mfv decreased significantly. pbro presented a trend of reduction. glucose, lactate and pyruvate did not change significantly ( table ) . conclusions: short episodes of moderate hv have a potent effect on the cerebral blood flow, as assessed by tccd, reduce icp and pbro , and do not induce significant changes in cerebral metabolism. outcome of pediatric patients six months after moderate to severe tbi -results of creactkids study from three picu in israel paco arterial partial pressure of co , cpp cerebral perfusion pressure (mmhg), icp intracranial pressure (mmhg), pbro brain tissue oxygen tension (mmhg), mfv mean flow velocity in the middle cerebral artery introduction: delirium is a major cause of complications in postoperative patient in icu. risk factors for delirium include poor cerebral hemodynamics and peri-operative cerebral desaturations. intraoperative target cerebral oximetry monitoring may decrease the incidence of postoperative delirium in elective major abdominal surgery patients. methods: a single-blinded, randomised controlled trial in patients undergo elective major abdominal surgery who received postoperative care in surgical icu with age more than years were randomised into two groups. the intervention group was received intra-operative target cerebral oxygen monitoring using cerebral oximetry whereas the control group was not. delirium was assessed in both group at , , hour postoperatively. other risk factors for delirium, mechanical ventilator day, length of icu stay, length of hospital stay and post-operative complication were recorded. results: from august -march , patients who met the criteria were randomised to patients in intervention group and patients in control group. overall incidence of delirium was . % (intervention . % vs control . %, p= . ). baseline cerebral oxygen in intervention group was . ± . %. desaturation below % from baseline was found in from patients ( . %) and was the only significant risk factor associated with delirium (p=. , odd ratio . ). there was no significant different in mechanical ventilator day, icu length of stay, hospital length of stay and postoperative complication between both groups. there was no complication associated with application of the cerebral oximetry probe in the intervention group. conclusions: from this preliminary report can not demonstrated the significant different of intra-operative target cerebral oxygen monitoring by using cerebral oximetry in prevention of delirium. however the reduction of cerebral oxygen more than % from baseline in intervention group showed significantly associated with delirium postoperatively. the set score as a predictor of icu length of stay and the need for tracheotomy in stroke patients who need mechanical ventilation introduction: set score was initially developed as an in-house screening tool based on tracheotomy predictors identified in several retrospective studies. it combined the categories of neurological function, neurological lesions, and general organ function/ procedure, and weighed by allocation of certain point values [ ] . in our study it was very interesting to us to find a tool to judge application of early tracheotomy, and as we have a good culprit number from stroke cases so we decided to try to apply this score in our icu after discussion with the inventor of this score. methods: stroke patients were prospectively included in the study as they were ventilated or were very little potential for ventilation and assessed by the stroke-related early tracheotomy score (set score, table ) within the first h of admission (table ) . endpoints were length of stay and ventilation time (vt) after doing early tracheotomy. we examined the correlation of these variables with the set score using standard analytical methods. results: the set score with a value cutoff point of had a significant effect on decision of making tracheotomy and hence decreasing ventilation time and length of stay in icu, which affected outcome (figs. & ) . conclusions: all efforts must be exhausted in neuro intensie care to decrease the secondary changes of brain injury after stroke,early tracheotomy is a good tool to decrease length of stay in icu and ventilation time in these patients.inventing a tool to judge these decisions of doing tracheotomy was a challenge. set score proved to be valuable.further multi center trials are needed. fig. (abstract p ) . specificity for the cutoff point of set score. cut point of is the best to predict tracheostomy with sensitivity of . % and specificity of . %. cut point of is the best to predict early tracheostomy with sensitivity of . % and specificity of . %. since no patients had score so the previous analysis that consider cut-point of should remain the same but just change the number in the text to contraindication for pharmacological vte prophylaxis ( . %). overall, ncc patients were more likely to receive mechanical ( . % icu days) than pharmacological vte prophylaxis ( . % icu days), however pharmacologic was more likely among younger patients with lower apache ii scores. guideline concordant care varied by recommendation; lower for pharmacological and higher for mechanical vte prophylaxis. conclusions: ncc patients uncommonly receive guideline concordant pharmacological vte prophylaxis. collectively, our findings suggest that current vte prophylaxis prescribing practices may reflect uncertainty around risks associated with vte prophylaxis among ncc patients. methods: we retrospectively analysed prospectively collected data from consecutive ich patients that received dvt prophylaxis in a tertiary hospital. he was defined as an increase of > ml measured using the abc/ method or the semiautomatic software based volumetric approach. using multivariate analysis, we analysed risk factors including early dvt prophylaxis for he> h, hospital mortality and poor -month functional outcome ( m modified rankin score> ). results: patients presented with a median gcs of (iqr - ), hematoma volume of ml (iqr - ) and were y old (iqr - ). % received early dvt prophylaxis, % late dvt prophylaxis and % had unclear bleeding onset. hematoma volume was smaller in the early dvt prophylaxis group with . ml (iqr - . ) vs . ml (iqr - ) in the late prophylaxis group (p= . ) without any other significant differences in disease severity. delayed he (n= / , . %) was associated with higher initial hematoma volume (p= . ) and lower thrombocyte count (p= . ) but not with early dvt prophylaxis (p= . ) in a multivariate analysis adjusted for known risk factors. early dvt prophylaxis was not independently associated with m outcome. conclusions: although limited by the retrospective design, our data suggest that early dvt prophylaxis (< h) may be safe in patients presenting with primary ich, which supports the recommendations given by the neurocritical care society. introduction: there is a paucity of literature describing the relationship between clevidipine and its impact on intracranial pressure (icp). the safety of clevidipine in patients with intracranial hemorrhage is often extrapolated from studies using nicardipine, which has demonstrated a neutral effect on icp [ ] . the objective of this study was to determine if there was a relationship between clevidipine initiation and changes to cerebral hemodynamic parameters. methods: this study was a retrospective analysis of adults admitted to hennepin county medical center between july and july . individuals were included if they had intracranial bleeding and icp data recorded prior to initiation of a clevidipine infusion. baseline demographic data was collected, including age, gender, type of injury, and initial glasgow coma score (gcs introduction: aneurysmal subarachnoid hemorrhage (sah) is an acute cerebrovascular event with high mortality and is an important cause of neurologic disability among survivors. many complications in the course of sah, such as hydrocephalus, also play a role in the poor outcome. the aim of the study was to describe the characteristics of patients with sah admitted to the icu to evaluate the factors associated with outcome. methods: this study was conducted in two reference centersone in rio de janeiro and one in porto alegre. from july to september , every adult patient admitted to the icu with aneurysmal sah was enrolled in the study. data were collected prospectively during hospital stay. the primary endpoint was mortality and dichotomized functional outcome (poor outcome defined as glasgow outcome scale to ) at hospital discharge and months. dichotomous variables were analyzed using two-tailed fisher's exact test. results: a total of patients were included. demographic characteristics are presented in table . frequency of clinical and neurological complications are presented in table . in univariate analysis, factors most frequently seen in patients with unfavorable outcome were seizure ( % vs %, p= . ), hydrocephalus ( % vs %, p< . ), meningitis ( % vs %, p= . ), rebleeding ( % vs %, p= . ), vasospasm ( % vs %, p= . ), pneumonia ( % vs %, p< . ), sepsis/septic shock ( % vs %, p< . ), postsurgical neurological deterioration ( % vs %, p= . ) and delayed cerebral ischemia ( % vs %, p< . ). at months, out of patients with follow-up, % had poor outcome. conclusions: sah is associated with high morbidity. both neurological complications as clinical complications were associated with unfavorable outcomes. therapeutic interventions to prevent those may have an impact on clinical outcomes. introduction: brain tissue hypoxia (brain tissue oxygen tension, pbto < mmhg) is common after subarachnoid hemorrhage (sah) and associated with poor outcome. recent data suggest that brain oxygen optimization is feasible and may reduce the time with brain tissue hypoxia to % in patients with severe traumatic brain injury [ ] . little is known about the effectiveness of protocolized treatment approaches in poor-grade sah patients. methods: we present a retrospective analysis of prospectively collected data of poor-grade sah patients admitted to tertiary care centers where pbto < mmhg was treated using an institutional protocol. treatment options were left to the discretion of the treating neuro-intensivists including augmentation of cerebral perfusion pressure (cpp) using vasopressors if necessary, treatment of anemia and targeting normocapnia, euvolemia and normothermia. the dataset used for analysis was based on routine blood gas analysis for hemoglobin data matched to hourly averaged data of continuous cpp, pbto , temperature and cerebral microdialysis (cmd) samples over the first days of admission. results: patients were admitted with a gcs of (iqr - ) and were (iqr - ) years old. overall incidence of brain tissue hypoxia was %. during this time we identified associated episodes of cpp< mmhg ( %), hyperglycolysis (cmd-lacta-te> mmol/l, cmd-pyruvate> μmol/l; %), pco < mmhg ( %), metabolic distress (cmd-lactate-to-pyruvate-ratio> ; %), pao < mmhg ( %), hb< g/dl ( %), and temperature> . °c ( %) (fig. ). of these variables only hyperglycolysis was significantly more common ( %) during episodes of normal pbto ( % of episodes). conclusions: underutilization of ivt despite the overwhelming evidence that support the effectiveness of such therapy can be partly attributed to the fear of hemorrhagic complications. this fear is not justified by current data. the estrangement of the emergency medicine community regarding ivt and the domination of stroke experts in decision making is also a barrier. regional wall motion abnormalities and reduced global longitudinal strain is common in patients with subarachnoid hemorrhage and associated with markedly elevated troponin k dalla sahlgrenska university hospital, gothenburg, sweden critical care , (suppl ):p introduction: stress-induced cardiomyopathy after subarachnoid hemorrhage (sah) is a life-threating condition associated with poor outcome. regional wall motion abnormalities (rwma) is a frequent finding, however, assessment of rwma is known to be difficult. in the present study we hypothesized that global and regional longitudinal strain (gls and rls) assessed with speckle tracking echocardiography can detect myocardial dysfunction indicated by increased levels of the cardiac biomarker troponin (tnt). methods: this prospective study comprised patients with sah. the tnt was followed daily from the admission up to days postadmission and elevated tnt was defined as > ng/l. a transthoracic echocardiography examination was performed within hours after the hospitalization. the peak gls was determined using the three apical projections and presented as the mean of the segments. reduced gls was defined as > - % and reduced rls was considered present when segmental strain was > - % in > adjacent segments. introduction: deviations from strict eligibility criteria for intravenous thrombolysis (ivt) in ischemic strokes regarding either license contraindications to alteplase or relative contraindications to thrombolysis have been reported in international literature, with conflicting results on patients' outcome.the aim of our study was to evaluate safety and efficacy for patients receiving ivt outside standard inclusion criteria. methods: retrospective analysis of our department's thrombolysis database.we compared patients with strict protocol adherence (strict protocol group) [mean age years and national institutes of health stroke scale (nihss) at admission /range - ] and patients with protocol deviations (off-label group) [mean age years and nihss at admission /range - ],in particular patients > years old, patients with mild stroke-nihss< ,and with symptom-to-needle time - . hours ( patients had deviations). results: patients in the off-label group were older but had no difference in baseline severity scores (sapsii, nihss). they had no statistically significant difference on short-term (nihss at days, need for critical care support, primary adverse event) and long-term (mortality,functional outcome at months) outcome measures when compared to standard protocol patients. conclusions: in accordance with international literature,off-label thrombolysis is save and equally effective to standard protocol thrombolysis.thrombolysis strict protocol needs expansion of inclusion criteria. introduction: most scales (gcs,nihss) don't consider the pathway of secondary acute brain failure (sabf). neuron-specific-enolase (nse) could be usefull in diagnostic and treatment pts. with sabf [ , ] . methods: prospective study incl. pts. with abf. pts. were identical in condition, age and comorbidies. in main group, nse examed and choline alfoscerate was used, pts. was divided into subgroups ia (n= ) with acute ischemic stroke(ais) and ib (n= ) pts. with posthypoxic encephalopathy. the control group (n= ) pts. with ais treated by loc.protocol № . clinical, laboratory, and imaging variables were fully compared. pts. examed by abcde algorithm, gcs and nihss. brain ct, carotid doppler performed. considering criteria:nse(days , , ), neurological status, length of stay in icu (icu los). "ss- . "was used. results: the baseline nse was higher and correlated to nihss ( . ± . , ÷ = . ) in all pts. in ia, ib sbgroups nse decreased for - days vs. control group - days (÷ = . ) and correlated with regression neurological deficit. icu los in main group was . ± . days vs. control group . ± . days. sensitivity and specificity of nse as a marker of brain injury in pts. with ais were and % and in posthypoxic pts. were and %, respectively, which showed nse as eligible diagnostic criterion of posthypoxic cerebral edema. in ia (ais) pts. and ib (posthypoxic edema) were confirmed by increasing nse in fold and -fold respectively more vs. pts. who had only brain ct at first day. nse also correlated with regression neurological deficit and improving of the neurological status. although, two pts. in iib group died with nse - ìg/ml conclusions: . nse is an effective marker of the severity of damages even in the sabf, and shoved efficacy efficacy of treatment. . negative outcome can be in pts. with sabf and more -fold increasing nse and increasing up to - ìg/ml is a mortality predictor. . we included nse in local protocols p n-terminal pro-brain natriuretic peptide as a bio-marker of the acute brain injury introduction: the detection of biomarkers levels facilitates an early diagnosis of brain tissues damage, allows assessing the prognosis of the disease and its outcome, and performs the monitoring of the patient treatment. methods: we studied patients ( m, f.). st group comprised patients with severe brain trauma: asurvivors with good outcome (on glasgow outcome scale groups i-ii) (n= ), bdead or severely disabled (on glasgow outcome scale groups iii-v) (n= ). nd group comprises patients with intracranial and sub-arachnoid hemorrhages: assignment to groups a (n= ), b (n= ) was done using the same criteria as group . rd group comprises patients operated in conjunction with brain tumor. assignment to groups a (n= ) and b (n= ) was done using the same criteria as groups and . we tested the level of n-terminal pro-brain natriuretic peptide in blood ( - pg/ml) between st and rd days after severe brain injury and then every - days for the total duration of days. results: : statistical analysis failed to demonstrate noticeable difference in the level of ntprobnp between groups , , . we detected the differences between subgroups (p< . ). patients from groups a, a, a (n= ) ntprobnp level stayed below pg/ml in cases ( %), in the cases ( %) the level was above pg/ml, but by - th day decreased to the normal values. for patients in subgroups b, b, b (n= ) in cases ( %) ntprobnp level was above pg/ml at least once, in cases ( %) level stayed below pg/ ml but remain high for the entire duration of the study without significant decrease. conclusions: all the patients with acute brain injury show the increased level of ntprobnp above normal values, irrespective of ethiology of injury. in case when ntprobnp level increases above pg/ml and/or does not decrease to the normal values it is possible to predict a negative outcome. introduction: cerebrovascular and coronary artery diseases share many of the same risk factors [ ] . cardiac mortality accounts for % of deaths and is the second commonest cause of death in the acute stroke population, second only to neurologic deaths as a direct result of the incident stroke. methods: this is a prospective observational study from july to april done on adult patients (groupi: pts acute ischemic strokes & group ii: pts as control) in kafr-elsheikh general hospital icu. inclusion criteria: all patients with acute ischemic stroke while exclusion criteria: patients with heart or renal failure/sepsis&septic shock/ischemic heart disease or hemorrhagic stroke,full clinical examination&labs including admission quantitative serum cardiac troponin i elisa immunoassay,ecg, d echocardiography&ct brain on day & ,alberta stroke program (asp) early ct (aspect) to predict neurological outcomes and mortality in patient with acute ischemic stroke within days so survivors vs non-survivors in group were divided to g a & g b respectively. results: dyslipidemia, hypertension, diabetes mellitus were significant comorbidities in all ischemic stroke pts.tlc, urea, inr and troponin were significantly higher in case group vs control group.gcs was found to be lower in non-survivors at day &at rd day follow up while aspect was significantly lower only at rd day follow up.troponin level was significantly higher in non-survivors g b, it was also higher in patents who developed convulsion later during their icu stay& it was significantly inversely correlated to gcs and asp. troponin had sensitivity % and specificity % (roc curve analysis) conclusions: troponin level was predictor for mortality in patient with acute ischemic stroke.it is well correlated to gcs and asp on admission.it was a predictor for occurence of convulsions later in icu stay. introduction: based on examination and treatment of hyperkinetic disorder in patients with uws and mcs, we supposed that hyperkinesis manifesting the formation of the generator of pathologically enhanced excitation in cerebral cortex, basal ganglia, which subsequently causes the formation of hyperkinesis. halogencontaining anesthetic sevoflurane had a good clinical effect in patients with prolonged impairment of consciousness. methods: the study included patients with uws ( -hypoxia, -encephalitis) and patients with mcs ( -hypoxia, -encephalitis). hyperkinetic disorder presenting as permanent myoclonus of arms and legs, face. all patients were performed head mri and eeg (before, during and after anesthesia), crs-r assessment, patients -[ f]-fgd pet. initial anesthesia: propofol - mg/kg, rocuronium bromide (esmeron) , mg/kg, fentanyl - mg/kg and clonidine (clophelin) . - . mg/kg. maintenance of anesthesia is carried out due to the following scheme: inhalation anesthesia using sevoflurane ( . - . vol%, mac . - . ). additionally, during the nd - th hours of medical anesthesia was prescribed the intravenous injection using ketamine - mg/kg/hr. the anesthesia is used during hours. the patients were nurtured by balanced mixtures through nasogastric tube. after hours the patients were gradually transferred to the autonomous breathing. the control clinical and instrumental studies to evaluate the therapy effectiveness (eeg, crs-r) were performed. results: in patients ( mcs, uws) was observed the hyperkinetic disorder regression as decrease of hyperkinesis manifestation, patients didn't have a significant dynamics. conclusions: the artificially formed "pharmacological dominant" (using sevoflurane and ketamine) may decrease the activity of pathological system of the brain, which clinically presented as significant decrease of hyperkinesis manifestation in out patients. -year experience of using benzodiazepines in predicting outcomes and targeted treatment of patients in unresponsive wakefulness syndrome (uws). introduction: we accepted a hypothesis that in some patients uws is a consequence of a pathologic system (ps), that limits the brain functional activity. identification of a ps allow to predict consciousness recovery. eeg registration under benzodiazepines test (bt) has become the method of ps identifying in uws patients. methods: we examined uws patients ( -traumatic, -non traumatic). crs scales assessment, eeg with bt, mri of brain were performed for all patients. the midazolamum was administered iv . mg/kg,.in - min after bzd was recorded eeg for min. the test was considered to be positive if against the background of bzd eeg pattern restructuring was observed: the low-amplitude eeg activity was rebuilt with the advent of alpha-and beta-spectrum.in patients with slowwave activity of theta-and delta-spectrum appeared stable fast forms, and in patients with baseline polymorphic eeg pattern was recorded prevalence of alpha activity and (or) the alpha rhythm. in order to confirm the correlation between the bzd effect and eeg pattern restructuring, flumazenil was administrated at rate of . mg every to minutes until the original eeg pattern has been registered again. results: the bt was true positive (recovery consciousness in - month later) in traumatic and non traumatic patients. true negative (permanent uws month later) in traumatic and non traumatic patients. false positive - traumatic, non traumatic. false negative traumatic, non traumatic patients. sensitivity bt to vs/uws = . % sensitivity to mcs = . % conclusions: our data confirmed the correctness of hypothesis that a ps limits the activity of the brain in patients in a uws. we proposed diagnostic method of a ps activity and suppression. apparently, bzd are the drugs of first stage examination choice in the treatment of uws patients. early identification of sepsis-associated encephalopathy with eeg is not associated with short-term cognitive dysfunction introduction: septic-associated encephalopathy (sae) affects approximately % of septic patients. recent studies showed sae is associated with short-term mortality and long-term cognitive disability. however, diagnosis of sae is one of exclusion and its association with short-term cognitive deficit is uncertain. the aim of this study is to evaluate the sensitivity of clinical examination in detecting sae. the association between sae and short-term cognitive impairment is also assessed. methods: prospective observational study enrolling adult septic patients admitted to a mixed icu. exclusion criteria were: encephalopathy from another cause, history of psychiatric/neurologic disease, cardiac surgery. all patients received continuous eeg monitoring and were assessed for sae for up to days after inclusion. we performed a comprehensive consciousness assessment twice daily during the icu (gcs; full outline of unresponsiveness, four; coma recovery scale-revised, crs-r; reaction level scale , rls ; confusion assessment method for the icu, cam-icu). we defined altered brain function as gcs< , no correlation between cognitive function at hospital discharge and severity of eeg alteration was found. conclusions: eeg was more sensitive than clinical assessment in detecting sae. altered eeg was not associated with short-term cognitive function. analysis of the training needs in italian centers that use brain ultrasound in their daily practices: a descriptive, multicenter study r aspide introduction: as mission of siaarti neuroanesthesia and neuroicu group of study, we are mapping out the brain ultrasound training needs in our centers. although brain ultrasound is widely used to study the intracranial vessels and other issues, it is still not clear the homogeneity of the skills required in both neuro and general icu in italy. the aim of this study is to explore the use of us-tcd and validate a collection of criterea which would prove useful in any future national wide survey. methods: starting from sept. the seven center involved (bologna, catania, pisa, verona, bergamo, cesena, roma) collected clinical and sonographic data, basing on a crf of twenty criteria such as: kind of hospital and icu, number of beds and neuro-patients/year, the physicians specialization trained to perform us-tcd, the kind of us doppler device used and the kind of training course followed. as a second step, data were analyzed by coordination team, as third step, during annual siaarti conference, these centers had a deep discussion on these selected items, further modifying and adapting the content of the items. results: the result is a ready list of items, an available tool for all the participant centers, that are going to start with an internal test survey for a final validation. conclusions: there is more than one path to train a physician on brain us in italy and there are new possible applications, even outside of the neuro sub-speciality. from the preliminary discussion, it is clear that in italy we have a inhomogeneous frame of training and use. this group of study believes that among the anesthesiologists/intensivists, it is possible to find a useful number of physicians interested in training on this topic. the main aim is the production of a validated criterea collection, available for eventually future national survey, useful to help map out the real national training needs in italy on us brain. perinatal neurosurgical admissions to intensive care c nestor, r hollingsworth, k sweeney, r dwyer beaumont hospital, dublin, ireland critical care , (suppl ):p introduction: beaumont hospital is the neurosurgical centre for ireland serving a population of . million. we present data on all perinatal patients who required icu admission for neurosurgical conditions over an year period. our data presents an insight into the incidence and outcome of neurosurgical conditions during pregnancy methods: searching our database identified pregnant and recently pregnant patients admitted to icu with neurosurgical conditions. patient data was collected retrospectively by review of charts and of an electronic database. a further pregnant patients were admitted for neurosurgical intervention but did not require critical care. results: intracranial haemorrhage was the most common diagnosis ( subarachnoid haemorrhage and had intra-cerebral haemorrhage). patients presented with intracranial tumours and patient had a traumatic brain injury. patient was admitted post spinal tumour resection. patient was referred with an ischemic stroke after iatrogenic injury to the carotid and vertebral artery. the requirement for organ support in this cohort of patients was high; % required ventilation and % inotropes. patients underwent neurosurgical intervention & medical treatment. maternal deaths occurred at & weeks gestation. the modified rankin score (mrs) on discharge from hospital was <= for of the surviving patients (median= ). of the pregnancies (all singleton) there were foetal deaths. patient miscarried spontaneously at weeks, had a medical termination of pregnancy at weeks to facilitate chemotherapy and foetus died after maternal death at weeks. the remaining patients delivered normal babies. conclusions: neurosurgical disease requiring icu admission during pregnancy is rare; our data suggest an incidence of case per million population. maternal outcomes were mixed with more than half having a mrs> on discharge. foetal outcomes were good with only one miscarriage and good neurological outcome in all surviving infants. stepwise multivariable analyses that included interaction between time of day and arrest location were performed in a stepwise manner. results: prehospital als (adjusted or, . ; %ci, . - . ) but not good-quality of bystander-performed ccs ( . , . - . ) was associated with sustained return of circulation (rosc). neither provison of good-quality ccs nor prehospital als was a major factor associated with on-month survival. however, good-quality of bystanderperformed ccs ( . , . - . ) in addition to shockable rhythm ( . ; . - . ) and bystander-witnessed ohca ( . ; . - . ) were associated with higher chances of neurologically favourable one-year survival, whereas prehospital als ( . ; . - . ) and elderly ohca ( . ; . - . ) were associated with lower chances of the survival (fig. ) . the impact of good quality ccs on survival were preserved in bystander-witnessed ohcas with shockable initial rhythm. noncentral region (adjusted or for good-quality, . ; %ci, . - . ), lack of bls training experience ( . ; . - . ), elderly-only rescuers ( . ; . - . ), cc initiation following dispatcher-assisted cardiopulmonary resuscitation ( . ; . - . ), and female-only rescuer ( . ; . - . ) were associated with poor-quality ccs. cc quality in athome ohcas remained low throughout the day, whereas that in outof-home ohcas decreased during night-time. conclusions: provision of good-quality ccs before ems arrival but not prehospital als was essential for neurologically favourable survival. new protocol for start of chest compressions before definitive cardiac arrest improved survival from out-of-hospital cardiac arrest witnessed by emergency medical service introduction: healthcare providers including emergency medical service (ems) personnel usually confirm absence of carotid pulse before starting chest compressions. at the end of , ishikawa medical control council implemented new criteria for start of chest compressions encouraging ems to start chest compressions when carotid pulse was week and/or < /min in comatose adult patient with respiratory arrest or agonal breathing. methods: data were prospectively collected for out-of-hospital cardiac and respiratory arrests during the period of - . definitive cardiac arrest was recorded when loss of carotid pulse was confirmed by pulse checks performed every min after the early start of chest compressions. the effect of early chest compressions on the proportions of definitive cardiac arrest was analysed in cases with respiratory arrest and circulatory depression in initial patient evaluation. before/after comparison of neurologically favourable -y survival was performed in cases with ems-witnessed ohca. results: the early start of chest compressions did not significantly prevent definitive cardiac arrest that followed respiratory arrest with circulatory depression in the initial patient evaluation (fig. ) . time interval between start of chest compressions and definitive cardiac arrest confirmation (median; iqr) was ; . - min. the survival rate of all ems-witnessed ohcas after the implementation of new criteria was significantly higher than that before the implementation: adjusted or; % ci, . ; . - . (fig. ) . no complications related to early chest compressions were reported during the study period. conclusions: start of chest compressions before definitive cardiac arrest improved survival from out-of-hospital cardiac arrest witnessed by emergency medical service. healthcare providers including ems personnel should be encouraged to provide chest compressions on cases with respiratory arrest and severe cardiovascular depression. introduction: our study sought to determine if there is a difference in time to tracheal intubation between direct and video laryngoscopy during cardiac compressions. guidelines suggest no more than seconds should be taken to perform intubation to minimise any delay in compressions [ , ] . it is unclear if use of video laryngoscopes results in faster intubation times during cardiac arrest. methods: observational trial involving emergency, anaesthesia and intensive care doctors. participants' baseline data obtained by questionnaire. resusci-anne™ manikin with airway trainer™ head [laerdal] with grade airway was utilised. participants intubated the manikin times, once with each of: macintosh size blade, c-mac video laryngoscope (karl storz, germany) with size blade and portable mcgrath mac enhanced video laryngoscope (medtronic, usa) with size blade. order of laryngoscopes was randomised by computer generated sequence. continuous cardiac compressions were performed throughout attempts. results: total participants. there was a statistically significant difference in time to intubation between the devices using friedman test (p< . ). wilcox signed-rank test demonstrated time to intubation with videolaryngoscopy was longer, c-mac (p= . ) and mcgrath (p= . ) compared with direct laryngoscopy. there was no significant difference between the two videolaryngoscopes (p = . ). when controlled for participants level of seniority and previous experience with device, direct laryngoscopy was still significantly faster than c-mac (p = . ) and mcgrath (p = . ) conclusions: our study showed a disadvantage of video laryngoscopy during cardiac compressions. faster intubation times with direct laryngoscopy could result in less pause in compressions and decrease periods without perfusion. direct laryngoscopy is an appropriate first choice for tracheal intubation during cardiac arrest. introduction: the aim of this study was to describe the coronary angiographic findings in relation to specific ecg changes and comorbidity in survivors after cardiac arrest. methods: a retrospective cohort study of out-of-hospital cardiac arrest patients with data retrieved between - from national registries in sweden. unconscious patients with coronary angiography performed within days after return of spontaneous circulation and available ecg were included (fig. ) . results: after exclusion, patients were analyzed (fig. ) , (table ) . ( %) were women and mean age were years. patients without st-elevation were separated into groups with specified ecg changes and comorbidities. differences were observed in the incidence of any significant stenosis, total occlusion and pci performed, between the specified ecg changes, as well as between the comorbidity groups ( introduction: fewer women after return of spontaneous circulation from out-of-hospital cardiac arrest (ohca) are undergoing coronary angiography (cag) with possible percutaneous coronary intervention (pci). the aim was to investigate gender differences in comorbidity, cag findings and outcome after ohca in comatose patients with a shockable first ecg rhythm. methods: a retrospective cohort study of out-of-hospital cardiac arrest patients with data retrieved between - from national registries in sweden (fig. ) . results: there was no difference in age or comorbidity except for men having more ischemic heart disease, . vs . % (p= . ). rates of previous myocardial infarction did not differ, . vs . %. no difference was seen in rates of ecg indicating prompt cag according to guidelines. still, more men underwent cag but no difference in numbers of cag leading to pci was seen (table ) . furthermore, in patients with st elevation or lbbb, no gender difference in cag and subsequent pci was found. men had lower rates of normal cag findings but more triple vessel and left main coronary artery disease ( table ) . year survival did not differ, . vs . %. conclusions: our study suggests, that despite no gender differences in rate of ecg findings indicating a prompt cag, men seems to have a more severe coronary artery disease while women have more frequently normal angiograms. however, this did not influence year survival. introduction: the circadian clock influences a number of cardiovascular physiological processes. a time-of-day variation in infarct size has recently been shown in patients with st segment elevation myocardial infarction. however, there is no clinical evidence of circadian variation in patients with out-of-hospital cardiac arrest (ohca) of cardiac etiology. methods: we performed retrospective analysis using data from japan's nationwide ohca registry from january through december , which includes all ohca patients presented with ventricular fibrillation as first documented rhythm, and consequently confirmed cardiac etiology. in order to eliminate the night and weekend effects, we enrolled only patients suffered ohca in the morning we conduct a retrospective cohort study focusing on the association between ohca outcome and icu bed availability. the ohca data was acquired from a regional emergency operation center, and the icu bed information was obtained from a regional sur it exceeds physiological levels in order to avoid insufficient oxygenation [ ] . hyperoxia has been associated with increased in-hospital mortality, though uncertainty remains about this association. multiwave pulse co-oximetry has safely been studied intraoperatively as a guide to monitor hyper-and hypoxia by calculating an oxygen reserve index (ori) which could add information to pulse oximetry measures when spo is > % [ ] . methods: this is a monocentric prospective study including patients with successful resuscitation following ohca. the aim of our study is to evaluate the feasibility and assess the availability of novel non invasive oxygen and hemodynamic variables. collected data principally concern blood oxygen and circulation such as ori, spo , total hb, perfusion index and pulse rates. recording is ideally started at time of rosc. results: we monitored consecutive patients for a total time of . min during transport from ohca place to the er. spo signal was present for . % of transport time.oxygen reserve index signal was present for . % of the total transport time. pleth variability index (pvi) signal was present . % of the total transport time. sphb signal was present . % of total time from rosc to hospital. the confidence interval for each variable is given in fig. . conclusions: our pilot study shows that noninvasive measurements of hyperoxia, fluid responsiveness and hemoglobin are readily available from the prehospital phase of post-rosc care allowing for early tailored and goal directed interventions. increase in sofa score was associated with € ( % ci - €) increase in the cost per day alive in the first months after ca. the sofa score is a good indicator of disease severity but the overlap between outcome groups does not allow its use for early prognostication in ca patients. the association of sofa and its sub-scores with -month outcome and healthcare costs highlights that in addition to neurologic damage the full spectrum of multiple organ failure affects the survival and morbidity of ca patients. public opinion on cardiopulmonary resuscitation decision and outcome in out-of-hospital cardiac arrest patientsquestionnaire study ty li introduction: metabolomics is a novel approach that can characterize small molecules (metabolites) and has the potential to explore genotype-phenotype and genotype-environment interactions, delivering an accurate snapshot of the subject's metabolic status. in this context, the aim of metabolomics is to improve early diagnosis, classification, and prediction over the development of a pathological condition. to this end, metabolomics have not been used in the characterisation of cardiac arrest (ca), cardiopulmonary resuscitation (cpr) and return of spontaneous resuscitation (rosc). the aim of the present study was to explore whether metabolomics can characterize the ca versus rosc in a swine model of ventricular fibrillation (vf). methods: ten animals were intubated and instrumented and vf was induced with the use of a cadmium battery. vf was left untreated for min and the animals were then resuscitated according to the guidelines. defibrillation was attempted in all animals. venous blood was drown at baseline, min, min, min during untreated ca and finally at min, min, h, h after rosc in order to determine the metabolomic profile during ca and during the early post-resuscitation period. rosc was defined as the presence of an organized cardiac rhythm with a mean arterial pressure of at least mmhg for > min. blood was centrifuged and serum was analysed by high resolution h-nmr spectroscopy. nmr spectral data were submitted to multivariate discriminant analysis. results: eight animals survived the experiment and were included in the analysis. metabolites upregulated in the immediate rosc versus ca were succinate, hypoxanthine, choline and lactate. metabolites upregulated in the hour rosc versus ca were ornithine and alanine. the measured phases are shown in fig. introduction: early outcome prognostication in successfully resuscitated out-of-hospital cardiac arrest (ohca) patients remains challenging. prediction models supporting the early decision to continue with full supportive treatment could be of major interest following ohca. we constructed prognostic models able to predict good neurologic outcome within hours after icu admission. methods: upon icu admission, targeted temperature management at °c, hemodynamic and neuromonitoring (cerebral oxygen saturation measured with near-infrared spectroscopy and bispectral index (bis)) was initiated. prediction models for good neurologic outcome at days post-ca were constructed at hour , , and after admission using variables easily collectable and known to be predictive for outcome. after multiple imputation, variables were selected using the elastic-net method. each imputed dataset was divided into training and validation sets ( % and % of patients, respectively). cut-off probabilities yielding a sensitivity above % were determined and performance of all logistic regression models was assessed using misclassification rates. introduction: in many venues, ems crews limit on-scene care for pediatric out-hospital cardiac arrest (pohca), attempting treatment during transport. hypothesizing that neuro-intact survival can be improved by prioritizing on-site care, strategies were effected to expedite on-scene drug delivery and intubation (with controlled ventilation). methods: from / / to / / , data for pohca cases were collected. in , new training prioritized on-site resuscitation (phase i) using expedited drug delivery and intubation with controlled ventilation (~ breaths/min). training included psychological and skills-enhancing tools to boost confidence in providing on-scene care. in , drugs were prepared while responding (phase ii). american heart association guidelines were used throughout and no other modifications were made. neuro-intact survival in - was compared to phase i & ii outcomes. results: over the . -years, ems faced consecutive pohca cases. the great majority presented in asystole throughout. in those resuscitated, mean time from on-scene arrival to the st epinephrine infusion fell from . min ( - ) to . min (phase i) and . min (phase ii). by , it was min. for resuscitated patients and . min. for all patients. intubation and intraosseous insertion occurred more frequently in phase i/ii, but there were no significant differences in age, sex, etiology, response times, bystander cpr or drug sequencing. neuro-intact survival improved significantly from / in - to . % ( / ) in phase i and . % ( / ) in phase ii (p < . ; -tailed fisher's exact test) (fig. ) . conclusions: although historically-controlled, the sudden appearance of neuro-intact survivors following a renewed focus on rapid on-site care was profound, immediate and sustained. beyond skillsenhancing strategies, physiologically-driven techniques and supportive encouragement from leadership, pre-arrival psychological and clinical tools were also likely contributors to the observed outcomes. fig. (abstract p ) . effecting neurologically-intact survival for children with out-of-hospital cardiac arrest p improved outcomes with a bundled resuscitation technique to enhance venous return out of the brain and into the heart during cardiopulmonary resuscitation pe pepe , ka scheppke , pm antevy , d millstone , c coyle , c prusansky , s garay , jc moore introduction: lowering intracranial pressure to improve brain perfusion during cpr has become a focus for our team. combined with devices that enhance venous return out of the brain and into the thorax during cpr, outcomes have improved using head/chest elevation in the laboratory (fig. ) . this study's purpose was to confirm the safety/clinical feasibility of this new approach involving mechanical cpr at an angle. methods: , consecutive out-of-hospital cardiac arrest (oohca) cases (all rhythms) were studied for . years ( / / to / / ) in an expansive, socio-economically-diverse u.s. county (pop. . mill). in , ems crews used the lucas© and impedance threshold (itd) devices on such patients, but, after april , they also: ) applied o and deferred +-pressure ventilation several min; ) raised the backboard~ °; and ) solidified a pit-crew approach to expedite lucas© placement. neuro-intact survival was not recorded until , so resuscitation by ems to hospital admission was used for consistency. quarterly reports were run to identify any periodic variations or incremental effects during protocol transition (quarter , ). results: no problems were observed with head/torso-up positioning (n= , ), but rates of resuscitation rose steadily during the transition period with an ensuing sustained doubling (fig. ) over the ensuing years when compared to those studied (n= ) prior to the head-up approach (mean . %; range - % vs. . %, range - %; p < . ). outcomes improved across subgroups. response intervals, indications for attempting cpr and bystander cpr rates were unchanged. resuscitation rates in - remained proportional to neuro-intact survival. conclusions: the head/torso-up cpr bundle was not only feasible, but also associated with an immediate, steady rise in resuscitation rates during the transition phase with a sustained doubling of resuscitation rates, making a compelling case that this bundled technique may improve oohca outcomes in future clinical trials. introduction: cardiac arrest (ca) often requires intensive care unit (icu) treatment, which is costly. while there are plenty of data regarding post-ca outcomes, knowledge of cardiac arrest associated healthcare costs is virtually non-existent. methods: we performed a single-center registry-based study to determine expenditure data for icu-treated ca patients between and . healthcare cost evaluation included costs from the initial hospital treatment, rehabilitation costs and social security costs up to one year post-ca. we calculated mean healthcare costs for one year survivors and for hospital survivors who died within the first year after cardiac arrest. we calculated effective costs per independent survivor (ecpis) as an indicator of cost-effectiveness. we adjusted all costs according to consumer price index (cpi) in finland as of . all costs are presented as euros (€). results: we identified , ca patients eligible for the analyses. at one year after ca % of the patients were alive and % were alive and independent in daily activities. one year survival stratified by cardiac arrest location group was % for out-ofhospital ca patients, % for in-hospital ca patients and % for in-icu ca patients. for the whole study population, mean healthcare costs were , € per patient. healthcare costs for hospital survivors were , € per patient and for hospital non-survivors , € per patient. healthcare costs for those who survived to hospital discharge but died within the first year were , € per patient, while for one year survivors they were , € per patient. healthcare costs stratified by ca location are presented in fig. . mean ecpis were , €. conclusions: for icu-treated cardiac arrest patients, the mean ecpis were close to , €. the best prognosis and the lowest costs were observed for out-of-hospital ca patients. introduction: in lithuania the incidence of out-of-hospital cardiac arrest (ohca) is unknown, as there is no official coding for ohca as a cause of death in the national death registry. kaunas emergency medical service (ems) underwent major stepwise changes since , including implementation of medical priority dispatch system and dispatcher-assisted cpr instructions. we sought to describe the epidemiology and outcomes from ohca in kaunas, the second largest lithuanian city. methods: the incidence, demographics and outcomes of patients who were treated for an ohca between st january and st december in kaunas ems, serving a population of almost . million, were collected and are reported in accordance with utstein recommendations. results: in total, ohca cases of ems treated cardiac arrests were reported ( per . of resident population). the mean age was . (sd= . ) years and . % were male. % ohca cases occurred at home and . % were witnessed by either ems or a bystander. in non-ems witnessed cases, . % received bystander cpr, whilst public access defibrillation was not used. medical dispatcher identified ohca in . % of all cases and provided over-the-phone cpr instructions in . % of them. average ems response time ( % fractile) was min. cardiac aetiology was the leading cause of cardiac arrest ( . %). the initial rhythm was shockable (vf or pvt) in % and non-shockable (asystole or emd) in . % of all cases. return of spontaneous circulation (rosc) at hospital transfer was evident in . % and survival to hospital discharge was . %. conclusions: rosc and survival to hospital discharge in kaunas were similar to those reported in united kingdom in [ ] . routine ohca data collection and analysis will allow us to track the efficiency of service improvements and should become a standard practice in all lithuanian regions. outcomes of patients admitted to intensive care following cardiac arrest j mcloughlin, e landymore, p morgan east surrey hospital, surrey, uk critical care , (suppl ):p introduction: patients who have return of spontaneous circulation following a cardiac arrest are haemodynamically unstable and require critical care input. outcomes are often poor, with unadjusted survival to hospital discharge at . %, following an in hospital cardiac arrest [ ] . the aim of the study was to assess the survival of patients admitted to intensive care following a cardiac arrest, reviewing whether age and gender impacted on their outcome. methods: the inarc database for a general intensive care unit (icu) at a district general hospital was reviewed. since , patients were admitted following a cardiac arrest (both in and out of hospital). their age, gender and survival to icu discharge and overall hospital discharge were recorded. results: female patients and male patients of varying ages were admitted to our icu following a cardiac arrest. the mortality for both genders increased with increasing age. overall survival to the time of icu discharge following a cardiac arrest was similar for both females ( . %) and males ( . %). figures (female) and (male) below show the number of patients who survived or died on icu discharge, by age and gender. mortality rates increased when reviewing hospital outcome, as some patients died following discharge to the ward. conclusions: overall mortality in our icu following a cardiac arrest at any age is at least %, with the general trend appearing to rise with increasing age. more male patients were admitted to icu following a cardiac arrest than female, with similar survival rates for both male and female patients. more research could be undertaken to assess whether survival rates following a cardiac arrest have improved since and also to compare outcomes following either an in or out of hospital arrest. introduction: raw simplified eeg tracings obtained by a bispectral index (bis) device significantly correlate with standard eeg [ ] . this study aimed to investigate whether simplified bis eeg tracings can predict poor neurologic outcome after cardiac arrest (ca). methods: bilateral bis monitoring (bis vistatm, aspect medical systems, inc. norwood, usa) was started following icu admission. six, , , , and hrs after targeted temperature management (ttm) at °c was started, raw simplified bis eeg tracings were extracted and reviewed by two neurophysiologists for the presence of burst suppression, cerebral inactivity and epileptic activity. at days post-ca, neurologic outcome was determined using the cpc scale, where a cpc - and cpc - corresponded to good and poor neurologic outcome, respectively. results: of the enrolled ca-patients enrolled, had good and poor neurologic outcome. with a positive predictive value (ppv) of . and a negative predictive value (npv) of . , epileptic activity within - hrs predicted a cpc - with the highest accuracy. epileptic activity within time frames - hrs and - hrs showed a ppv for poor outcome of . and . , respectively. cerebral inactivity within - hrs had a poor predictive power (ppv= . , npv= . ). in contrast, cerebral inactivity between - hrs predicted a cpc - with a ppv of . and a npv of . . the pattern with the worst predictive power at any time point was burst suppression with a ppv of . , . and . at - hrs, at - hrs and at - hrs, respectively. conclusions: based on simplified eeg derived from a bis device, both the presence of epileptic activity at any time as well as cerebral inactivity after the end of ttm can be used to assist with poor outcome prognostication in successfully resuscitated ca patients. the helicopter as first response tool -rio de janeiro fire department experience. (interquartile range= ) min, followed by tih with flights ( %) and median time of (iqr= ) min, and ( %) were neo missions with median time of (iqr= ) min. total time of aircraft usage was higher for tih ( %), followed by neo ( %). evam was the most frequent mission, however it accounted for % of aircraft utilization time, where most victims had traumatic brain injury (tbi) followed by other traumatic injuries ( and cases respectively). tbi victims were predominantly males ( %) with a median age of (iqr= ) years. most commonly, tbi is a consequence of transportation accident ( %), where a motorcycle was involved in %, car collision in % and pedestrian run over % of the cases. conclusions: goa utilizes the air ambulance helicopter as a first response tool in % of total missions, where respect for the trauma golden hour is paramount. traumatic brain injury is the most prevalent diagnosis at the scene of event. therefore, goa training and equipment must be tailored to meet this demand, which translates in stabilization of critical patients outside hospital environment with limited resources. introduction: the intra-hospital transport of critical patients is associated with adverse events and worse outcomes. the objective of this study was to evaluate the safety profile of intrahospital transport after the creation of a specific group for this purpose. methods: evaluated all the transports of critical patients from october to september , in a large hospital, after the creation of a group consisting of intensive care physician, nurse and physiotherapist. clinical and non-clinical complications related to the transport and outcome of the patients were evaluated. results: a total of , transports were performed, . % of the male patients and . % of the patients being hospitalized. . % were under mechanical ventilation and . % under vasoactive drugs. at the time of transport, . % were clinically stable. during transport, . % presented clinical complications, being more frequent hemodynamic instability ( patients) and respiratory failure ( patients). non-clinical complications occurred in patients ( . %), and communication failures were responsible for . % of the occurrences. in cases ( . %) there was worsening of the clinical conditions during transportation, and in only one case this worsening resulted in an increase in the length of stay in the icu and in the hospital, with no correlation with deaths. conclusions: the implantation of a group specialized in critical patients to carry out in-hospital transport made the process safer with complications rates lower than literature and guarantee better quality of care. clinical profile of patients admitted to icu due to acute poisoning mp benitez moreno , e curiel balsera , mc martínez gonzález , s jimenez jimenez intensive care unit, hospital regional universitario carlos haya, malaga, spain; hospital regional universitario carlos haya, málaga, spain critical care , (suppl ):p introduction: patients suffering from acute intoxication, whether voluntarily for autolytic or accidental purposes, often require life support in intensive care units. methods: retrospective observational study of all patients admitted for acute intoxication who required admission to the icu of the regional hospital of malaga between january and august , older than years with admission to the icu for intoxication of any kind. we study patient characteristics in terms of age, sex and medical history, type of toxicity, severity and evolution in our unit. results: we found cases of patients who required admission to the icu due to acute intoxication, of which . % were women. the average age was . (standard deviation . ). the average stay in icu was . (standard deviation . ). . % of patients had a psychiatric history. as other background highlights, . % were addicted to illegal drugs and % were hypertensive. most patients took more than one toxic . % and intoxication was voluntary in . % versus accidental in . % of cases. the toxic was known in %. the most used benzodiazepines in . % of the total. the main cause of admission to the icu was due to neurological deterioration in of the cases registered and mechanical ventilation was necessary in patients. the maximum time in mechanical ventilation was days. the infection occurred in . %, with the majority being respiratory infection. the . % died in icu. the hospital stay presented an average of . days. conclusions: the profile of a patient admitted to the icu due to acute intoxication is that of a woman of middle age and psychiatric history, with voluntary intoxication of several toxic substances and requiring mechanical ventilation for a low level of consciousness for an average of days. the survival is very high and it would be necessary to analyze the possible relapses of these patients. mushroom that break hearts: a case report e karakoc, k demirtas, s ekemen, a ayyildiz, b yelken eskisehir osmangazi university, eskisehir, turkey critical care , (suppl ):p a introduction: because of the high mortality and morbidity mushroom poisoning is a significiant medical emergency [ ] . amanita phalloides (a. phalloides) is responsible for the % of the mortality in adults caused by mushroom poisoning. it causes damage in liver, kidneys and rarely pancreas, causing encephalopathic coma, disseminated intravascular coagulation, hemorrhage, hypovolemic shock and death but its effect on cardiac functions has not been established yet. there are three main groups of toxins;phallotoxins, virotoxins and amatoxins;amatoxin is the common responsible toxin from the fatality. we aimed to present a -year-old woman poisoned by mushroom complicated with hepatic,renal and cardiac toxicity methods: patient with nausea and vomiting started hours after mushroom eating,creatine kinase mb . ng/ml and cardiac troponin i . ng/ml her blood urea nitrogen, creatinine levels and liver enzymes were higher than upper limits in lab tests (table ) ; she was admitted to icu, treated for acute renal failure by hemodialysis.plasmapheresis was applied against potent mushroom toxins. at .day in icu, hypoxemia and severe swelling resistant to ultrafiltration was evaluated as a global left ventricular hypokinesia with ejection fraction(ef) %, end-diastolic diameter of . cm, and systolic pulmonary artery pressure (spap) of mmhg. oxygen was administrated to treatment.urine output improved at .day, three more plasmapheresis sessions were performed. hypoxemia was recovered,liver enzymes and creatinin levels decreased results: at control ef measured was %, end-diastolic diameter of . cm, spap of mmhg.than at the .day patient discharged from the icu.after a year follow up assessment she has no complaints conclusions: one of the major problems for amanita poisoning is diagnosis. patients who had mushroom poisoning should also be evaluated especially in terms of cardiac dysfunction with clinic signs, ecg, cardiac enzyme tests and eco introduction: the characterization of clinical and/or biological variables found in the emergency room predictive of a secondary admission in icu would help to improve the identification of patients at risk of aggravation in order to avoid the associated consequences, such as, an increased mortality and increased hospital stay. methods: this is a retrospective monocentric study of years with patients admitted secondarily to a medical icu within hours of admission to the general wards from the emergency department in the pitié-salpêtrière hospital in paris. each case was matched to controls. different variables were collected in the emergency room. results: patients, of whom were cases and controls were studied. pneumonia is the diagnosis the most frequent in cases followed by sepsis (in and %, respectively). conclusions: the risk of being admitted secondarily to intensive care is higher if patients consult for dyspnea or fever, if they are old smokers, if they have a high igs score, if an arterial blood gas is requested and if an icu medical advice is taken. the meds score under and being an active smoker seems to be protects for the unexpected transfer. introduction: managing the special needs of patients who present with agitation or psychosis can pose a greater challenge to an already busy emergency department as their symptoms can escalate rapidly. traditional antipsychotics used in the ed, such as haloperidol or ziprasidone often do not fully relieve patient's symptoms and may require administration of repeat doses or additional medications such as benzodiazepines to achieve effective results. this can induce excess sedation which can lead to longer length of stay in the ed and requires additional time at the bedside by the ed physicians and staff to manage these patients. adasuve® is an antipsychotic drug that works in a single-use device providing an aerosol form of loxapine that is rapidly absorbed by the lungs which may offer faster symptom relief, allowing subsequent earlier psychiatric evaluation and disposition. methods: to test this hypothesis, data including time of physician assignment and time physician documented discharge disposition and number of hours physician was assigned to the patients was retrospectively collected from patients who arrived to the emergency department presenting with agitation or psychosis that received adasuve or other types of antipsychotic medication such as ziprasidone, haloperidol and benzodiazepines or a combination of the three. results: we found that physicians who administered adasuve spent an average of . hours assigned to their patient compared to . hours when the physician administered any other type of antipsychotic medication. this resulted in a significant . -hour difference (p < . ) between the two groups. conclusions: in conclusion, less time spent assigned to a patient that received adasuve can be attributed to faster symptom relief which allowed the physicians to complete their psychological evaluations and develop dispositions more rapidly than with patients that received other antipsychotic agents. clinical work in language-discordant emergency department introduction: emergency residents are particularly vulnerable to sleep deprivation due to persistent conflicts between work schedule and the biological clock. recent approaches to address fatiguerelated risk mainly focused on reducing work hours and ensuring sufficient recuperation time. such approach has demonstrated its limits due to growing emergency rooms visits and emergency residents' shortage. dawson & mcculloch ( ) introduced the notion of proofing as a complementary approach to manage fatigue-related risk [ ] . fatigue proofing strategies (fps) aim to reduce the likelihood a fatigued operator will make an error, in contrast of reduction strategies (frs) aiming to reduce the likelihood a fatigued operator is working. most formal risk control systems do not encompass the notion of proofing and fps typically develop as informal practices. in this study, we aim to ) identify informal reduction and proofing strategies used by residents and ) to investigate how they relate to fatigue-related risk indicators. methods: first, we organized focus-group with a total of residents in order to identify informal strategies used to manage fatigue-related risk. second, we designed a questionnaire assessing the frequency of use of each reported strategy. introduction: this randomized controlled study assessed the impact of a -hour intravenous medication safety simulation-based learning (sbl) on self-efficacy, stress, knowledge and skills of nursing students. medication administration error is a worldwide concern [ ], that has been linked with a lack of knowledge and skills in safe medication administration among new graduate and student nurses [ ] [ ] [ ] . preventing medication errors could therefore involve training through simulation. methods: participants (n= ) were randomly assigned either to the control group (cg, n= ) or the experimental group (eg, n= ). while cg and eg both had a traditional clinical internship, eg beneficiated in addition the -hour sbl, using standardized patients in the context of an intensive care unit. the two groups were assessed twice: at t and t (four weeks later), through an objective structured clinical examination (osce) and questionnaires. two blinded experts rated the students osce with an evaluation grid. results: mean participants age was , . there were no statistically differences between groups at t . compared to the cg ( %), the eg increased its self-efficacy (+ . %) with a significantly difference (p< . ) at t . the sbl conducted to a greater increase of knowledge and skills in the eg (respectively + %, + %) than in the cg (respectively + % and + %), with a statistically significant difference (p< . ). conclusions: results reinforce the interest of a short sbl using standardized patients to improve medication administration. clinical impact of these observations requires further evaluation to determine potential transfer in clinical settings and retention over time. introduction: medication errors occur frequently in the intensive care unit (icu) and during care transitions. medication reconciliation by a pharmacist could be useful to prevent such errors. therefore, the aim of this study was to determine the effect of medication reconciliation at the icu. methods: a prospective -month intervention study with a pre-and post-phase was performed in haga teaching hospital ( ) and erasmus university medical center ( ). the intervention consisted of medication reconciliation by pharmacists at icu admission and discharge. the severity of potential harm of the medication transfer errors (mte) (pade= ; . ; . ; . ; . ) was scored. primary outcome measures were the proportions of patients with >= mte at icu admission and icu discharge. secondary outcome measures were the proportions of patients with a pade score >= . , the severity of the pades and a cost-benefit analysis. odds ratio and % confidence intervals were calculated. results: table shows patient characteristics. figure shows the primary outcome measures (oradj admission = . [ % ci . - . ] and oradj discharge = . [ % ci . - . ]). the proportion of patients with a pade >= . at icu admission reduced from . % to . % and after icu discharge from . % to . %. the pade reduction resulted in a potential net cost benefit of € per patient. conclusions: medication reconciliation by pharmacists at icu transfers is an effective safety intervention, leading to a significant decrease in the number of errors and a cost effective reduction of potential adverse drug events. introduction: in intensive care unit, administration of numerous drugs in icu patients via a central venous catheter provide a high risk of drugs incompatibilities. it has been reported in experimental studies [ ] that particles issued of drug incompatibilities could induce thrombogenesis, microcirculation impairment and inflammatory response which could aggravate the occurrence of organ dysfunctions [ ] . the objective of this study was to evaluate the occurrence of particles by reproducing in vitro the intravenous system and the drugs combination used in icu for patients suffering either septic shock or acute respiratory distress syndrome (ards). methods: first, we registered during a period of months the most common central venous catheter system used in patients admitted for septic shock or ards in three university hospital in lille. the second part of the study was to reproduce in vitro the previous infusion system in order to quantify the amount of particles generated during a simulated period of hours infusion. the egress of the iv line was connected to a dynamic particle counter qicpic analyser (sympatec inc ; clausthal zellerfeld, germany) (fig. ) . results: the most common intravenous system observed was a three lumen central catheter. the proximal lumen was dedicated for vasoactive agents, the medial lumen for sedation and the distal lumen for the other drugs infused continuously and discontinuously..among the drugs infused via the distal lumen of the central venous catheter, introduction: insufficient identification of possible organ donors in the icu is one of the main factors contributing to the loss of donors after brain death [ ] . up to % of potential donors might not be identified [ ] . the aim of this study was to evaluate how active search of possible brain dead donors affect the potential deceased donor pool. methods: the strategy implemented at university hospital with specialized icus from december to october and data compared to the matching period of the previous year. donor coordinator visited all icus every day and selected patients who met possible brain dead donor criteria: ) gcs <= ; ) severe brain injury. all data registered in original color coded follow-up system according to the patient status. results: a total of patients were identified as possible donors. there was no significant difference of potential donor numbers in study period comparing to previous year ( vs ). main causes of brain death remain intracranial hemorrhage and subarachnoid hemorrhage. the length of hospital stay of potential donors was significantly longer in study period comparing to previous year ( ± . vs . ± . , p= . ). there was no significant difference of donor's demographic data, conversion rates to actual donor or frequency of family refusals and medical contraindications. conclusions: active search of brain dead donors neither increased total number of potential donors nor increased conversion rates and did not change a donor profile in our donor center. longer observational period and more sophisticated follow-up system might be required. a fast hug bid a day keeps the patient ok! e sousa, t leonor, r pinho centro hospitalar de entre douro e vouga, santa maria da feira, portugal critical care , (suppl ):p introduction: regardless the underlying diagnose, providing meticulous supportive care is essential to critically ill patients management. in , vincent jl introduced the fast hug (feeding, analgesia, sedation, thromboembolic prophylaxis, head of bed elevation, ulcer prevention, glucose control) mnemonic for recalling what he considered the key issues to review in daily clinical practice. our intensive care unit (icu) decided to add bid (bowel regimen; indwelling catheter removal; de-escalation of antibiotics) indicators following some published data. since , the adequate use of this mnemonic became an instrument for quality of care evaluation. objectives for each variable were designed; regular annual audits done. the present study aims to audit the use of this mnemonic in a portuguese tertiary hospital icu, in . methods: a prospective observational study was performed. admissions in icu staying at least one h min and h min period, during the first six months of were included. all mnemonic variables were recovered from icu medical record database, as well as demographics, severity scores and clinical information. data was analyzed with microsoft office excel software. results: we included admissions. the predictable global fast hug bid assessment was entries [one per each full day ( h - h ) in the unit, per patient]. the mnemonic was used in about % of the opportunities. the target thresholds were considered as achieved in % of entries (concordance equal or superior to %). looking to individual variables, the best performance was achieved in h and u; worse performance was seen in s. the daily use of this mnemonic aims to revisit important intervention sectors in critical patient. applying the "plan-do-check-act" policy, this study allowed us to identify growth opportunities, reviewing or creating protocols, adopting more frequent training measures and seeking to take this model to other hospital areas. impact of incidents and adverse events in intensive care unit and its characteristics on outcomes e siqueira, l taniguchi, j vieira junior hospital sírio libanês, sao paulo, brazil critical care , (suppl ):p introduction: critically ill patients are usually exposed to adverse events (ae) due to acuity and complexity of care. ae might potentially result in disability or death, and increase in length of stay. our aim was to assess the incidents and ae in a general intensive care unit (icu). methods: this is a prospective cohort study conducted in a private tertiary hospital (hospital sírio-libanês) in são paulo, brazil. all consecutive patients who were admitted to the icu and all incidents and ae reported in the study period were evaluated. univariate and multivariate analysis were used to identify risk factors associated with hospital mortality. results: between may to november we studied patients and reported incidents and ae. overall, patients ( %) experienced some incident or ae during icu stay. we found higher severity of illness (saps of versus ; p< . ), mechanical ventilation (mv), use of vascular lines, drains and catheters, physical restraints, delirium and also an increased length of icu ( vs days; p< . ) and hospital stay ( vs days; p< . ) and hospital mortality ( % vs %; p< . ) among patients who experienced any incident or ae. independent risk factors for hospital mortality in our logistic model were: higher saps , mv and at least one adverse event during the icu stay. mortality was higher among patients who experienced late ae (> hours after icu admission) compared to patients who experienced early ae ( % vs %; p< . ). saps , sofa and mv were predictors of moderate and/or severe ae and a negative correlation between these events and icu occupancy rate was found. conclusions: patients who experienced incident or adverse event during icu stay had poorer outcome. ae, mainly moderate or severe, mv and severity of illness were independent risk factors to mortality. there was a negative correlation between moderate or severe adverse event and icu occupancy rate. monte carlo modelling of patient flow can aid complex intensive care bed and workforce capacity planning. introduction: models for icu populations based on the queuing model use arrival rate, length of stay, and bed number [ , ] . these models lack the complexity of specialised icus with different admission types, and patient subpopulations. results: > % of patients reported satisfaction on all areas except noise, patient facilities for hand hygiene and being informed about timing of operations. staff survey results revealed confusion regarding the interventions that are provided. baseline capacity for new patients was %, bed occupancy varied between and per day (overflow to recovery) with overall capacity at . % and mean length of stay (los) was . days (sd= . , n= , =range - ). following intervention, the los was reduced to . days (sd= . , n= , range - ). new patient capacity was increased to % with a bed occupancy range - . introduction: in clinical practice, when harm or potential harm occurs to patients, this can adversely impact upon the morale of staff involved and thereby affect clinical care delivered to subsequent patients. the personal narratives behind clinical incidents contain learning opportunities and individuals involved may reflect on the course of events and make changes to their practice to avoid recurrence. the aim of this study was to evaluate whether sessions enabling trainees to discuss their mistakes in a confidential environment improved trainee morale and safe clinical practice in an anaesthetic trainee cohort. methods: we conducted a survey amongst anaesthetic trainees in a london teaching hospital before and after a monthly, hour long, confidential, semi-structured, trainee lead "confession session" was introduced. results: initial results demonstrated that % of respondents (n= ) had made a mistake resulting in patient harm with % of these individuals describing negative feelings about themselves as a consequence. additionally, % of respondents had made a mistake causing a near miss, with % of these describing negative feelings as a result. of note, only % of respondents felt comfortable discussing errors with more senior colleagues, whilst % felt comfortable discussing errors with their peers. a follow-up survey identified that % of respondents (n= ) agreed that the session had the potential to improve clinical practice and trainee morale with % agreeing that their own clinical practice had improved from attending the sessions. conclusions: clinical mistakes leading to harm and "near misses" are common and provide opportunities to improve care. this trainee lead "confession session" appears to improve trainee morale and may improve patient care by encouraging trainees to engage in a process that seeks to understand error through sharing stories in a non-judgmental setting. funnel plots for quality control of the swiss icu -minimal data set introduction: a clinical database should be representative of the labelled population and guarantee completeness and accuracy of collected data. without explicit permission of the patients, swiss laws regarding data protection do not allow external audits based on periodic checks of random samples, supposed to give a general pattern of accuracy. to test alternative methods for quality control we introduced the principles of statistical process control to derive funnel plots from the swiss icu -minimal data set (mdsi). the mdsi from all certified adult swiss icus ( and ) was subjected to quality assessment (completeness and accuracy). for the analysis of accuracy, a list of logical rules and cross-checks was developed as e.g. range of saps ii according to age. errors were classified in coding errors (e.g. nems score > points) or implausible data (nems without basic monitoring). we also checked for icus producing significantly more errors -outliers -(> mean ± standard deviations [sd] or > . % confidence interval [ci] of an adapted version of the funnel plots, which allows the presence of trends depending of the icu's size. results: a total of ' patient mdsi ( items/patient; items for trauma patients) from the certified icus.were investigated. we detected ' patients ( . %) with an overall sum of coding errors and ' implausible situations. implausible situations related to supposedly inaccurate definitions (diagnostic and patient's provenance prior to icu admission) and discrepancies in the logical rules between diagnostics and treatments. figure is an example for imprecise coding of the diagnostic: icus declared having treated - % of their patients without a defined diagnosis. conclusions: accuracy of data in mdsi needs further improvement. funnel plots may be useful for meaningful interpretation of data quality and permit to identify icus disproportionately generating inaccurate and/or implausible data. introduction: lung cancer is the leading cause of intensive care unit (icu) admission in patients with the advanced solid tumors. this study was aimed to elucidate the clinical factors associated with icu mortality of advanced lung cancer patients and the effect of intensivist's contribution on their clinical outcomes. methods: we included patients with advanced lung cancer including non-small cell lung cancer (nsclc) with stage iiib or iv and small cell lung cancer (sclc) with extensive stage who admitted to icu from to . multivariate logistic regression analysis was performed to find the variables associated with icu mortality and in-hospital mortality. we applied autoregressive integrated moving average (arima) for time-series analysis of the intenvention of intensivists. results: among total patients with advanced lung cancer, patients ( . %) were admitted icu before introduction of organized intensive care at , and ( . %) were admitted after (fig. ) . the leading cause of admission was the respiratory failure ( . %) and cancer-related event ( . %) in terms of intensivist's and oncologist's perspective. before and after , the -day icu mortality rate was . % and . % (p = . ), and the hospital mortality rate changed from . % to . % (p = . ) (fig. ) introduction: decisions when to refer and to admit patients to the intensive care unit (icu) care are very challenging. demand typically exceeds supply in icu beds, which results in a constant need for evaluation of the processes involved in icu referral and admission with a view to optimising resource allocation and patient outcomes. the aim of this study was to evaluate the theoretical impact of a newly designed triage tool for icu referrals on a cohort of patients referred to icu (fig. ) . methods: we reviewed all patients consecutively referred to our icu, whether admitted or not, in february . demographics, referring speciality, role of the referrer, comorbidities, the presence of advanced disease or terminal illness, the presence of acute organ failure, dnr status, reason for not admitting, and icu mortality were recorded. a retrospective analysis of icu referrals using a pilot triage tool was carried out independently by three authors. results: forty-six patients were referred to our icu over the study period. of these, ( %) were admitted. patients were declined icu if their admission was deemed unnecessary ( %), futile ( %), or were transferred due to bed shortage ( %). of the patients referred, ( %) had an advanced disease or a terminal illness. of those, ( %) were admitted, dnr status was unclear in ( %), family was involved in ( %) and their icu mortality was %. by analysing retrospectively these referrals with the aid of a triage tool, we propose that the overall referrals could have decreased from to ( % percentage difference). dnr status and family involvement would have been clarified in all patients with advanced disease or terminal illness before icu referral. kappa score for inter-rater agreement was . . conclusions: adopting a triage tool for icu referrals could reduce the overall proportion of inappropriate referrals and admissions. end-of-life discussion would also be proactively clarified prior to icu admission. introduction: intensive care unit (icu) admission triage occurs frequently worldwide and often involves decisions with high subjectivity, possibly leading to potentially inappropriate icu admissions. in this study, we evaluated the effect of implementing a decision-aid tool for icu triage on icu admission decisions. methods: urgent icu referrals before (may, to november, , phase ) and after (november, to may, , phase ) the implementation of a decision-aid tool were prospectively evaluated. our primary outcome was the proportion of potentially inappropriate icu referrals (defined as priority b or patients, as described by the or society of critical care medicine [sccm] guidelines) that were admitted to the icu in hours following referral. we conducted multivariate analyses to adjust for potential confounders, and evaluated the interaction between phase and triage priorities to assess for differential effects in each priority strata. results: of urgent icu referrals, ( %), ( %), ( %), ( %) and ( %) were categorized as priorities b, a, , and (sccm ) or ( . %), ( . %), ( %), ( %) and ( %) were categorized as priorities , , , and (sccm ), respectively. overall, ( %) patients were admitted to the icu in hours following referral. the implementation of the decision-aid tool was associated with a reduction of admission of potentially inappropriate icu referrals [adjor ( % ci) = . ( . - . ), p = . ] (fig. ) . there was no difference on hospital mortality for the overall cohort between phase and phase . conclusions: the implementation of a decision-aid tool for icu triage was associated with a reduction of potentially inappropriate icu admissions. introduction: the aim was analyze the icu bed rotation pattern, the epidemiological characteristics of patients and to correlate them with prognostic score after software implementation methods: this is an epidemiological and retrospective study. data were collected between june and november , using epimed® monitor software, applied in an adult icu of a public hospital in bahia/brazil. authorization for collection and use of data was granted by the institution. all patients hospitalized in the period were included regardless of other exclusion criteria. results: during the period evaluated, there were . new hospitalizations, men ( . %) and women ( . %). . % ( ) were in the age group of to years, followed by . % of the patients ( ), who were between and . the mean duration of hospitalization in our unit was approximately , days. during the period covered, . exits occurred: patients ( . %) were introduction: early debriefing after stressful events holds great value in reflection on both an individual and team-based level. our objective was to implement routine structured debriefing sessions for doctors working in intensive care in order to optimise learning and develop strategies to improve practice. methods: % of junior doctors (n= , pre-implementation questionnaire) on the intensive care unit expressed a need for regular debriefing sessions to discuss challenging and complex cases. weekly sessions were implemented and structured using the sharp performance tool [ ] . key learning points were collected and added to a debrief list to track progress and assimilate learning. informal feedback was obtained on a weekly basis with formal feedback assessed following one month of implementation. results: min sessions occurred on a weekly basis supported by a consultant intensivist. desired outcomes included assessment of team performance, identification of key learning points and psychological support. following one month, % doctors involved felt that debriefing sessions were important and should continue. % felt that they left every session with a key learning point applicable to future clinical practice. common themes in perceived benefits included improved team communication and creation of an open environment to address concerns. conclusions: working in intensive care exposes doctors to challenging and stressful situations. implementation of a regular structured debrief session provides an opportunity for clinicians to address concerns, consolidate learning and develop strategies to improve clinical practice. nurse staffing patterns, outcomes and efficiency in resource use in the context of icus with a "low-intensity" nurse staffing: a multicenter study in brazilian icus m soares introduction: studies investigating nurse staffing and outcomes were often conducted in high-income countries with low bed/nurse ratios. our objective was to investigate the association between nurse staffing patterns, outcomes and resource use in brazilian icus. methods: retrospective cohort study in , ( % medical) patients admitted to medical-surgical icus during - . we retrieved patients' data from an icu registry (epimed monitor system) and surveyed participating icus about characteristics related to icu organization. we used multilevel logistic regression analysis to identify factors associated with hospital mortality. we evaluated efficiency in resource use using standardized mortality rates (smr) and resource use (sru) based on saps . results: saps score was ( - ) points and hospital mortality was . %. intensivists were present / in % icus. median bed/ nurse ratio was . ( . - . ) and at least the chief nurse was boardcertified in critical care (bccc) in % icus. bed/nurse technicians ratio was . ( . - . ). adjusting for relevant characteristics at patientlevel (age, admission type, sofa, performance status, comorbidities, hospital days before icu) and icu-level (hospital type, checklist use, / intensivist, protocols), bed/nurse ratio was not associated with mortality [or= . ( % ci, . - . )]. however, mortality was lower in icus with at least the chief nurse bccc [or= . ( . - . )]. in multivariate analysis, bed/nurse ratios <= [or= . ( . - . )] and having the chief nurse bccc [or= . ( . - . )] were associated with higher efficiency. conclusions: in a "low intensity" nurse staffing scenario, bed/nurse ratios were not associated with mortality. however, having at least the nurse chief bccc was associated with higher survival. moreover, bed/nurse ratios <= and presence of chief nurse bccc were associated with higher efficiency in resource use. methods: a systematic search on the value of acute non-physician provider on the icu was conducted. the methodological quality of the included studies was rated using the newcastle ottawa scale (nos). the agreement between the reviewers was assessed with cohen's kappa. results: in total studies were identified. twenty comparative cohort studies were identified which compared non-physicians with either residents or fellows. all studies comprised adult intensive care. most of the included studies were moderate to good quality. a random effects meta-analysis from all studies regarding length of stay and mortality showed no differences between non-physicians and physicians, although there was a trend to better survival when implementing acute non-physician providers in the icu (figs. & ) . mean difference for length of stay on the icu was . ( % ci - . - . ; i = %) and for in hospital - . ( % ci = - . - . ; i = %); while the odds ratio for icu mortality was . ( % ci = . - . ; i = %) and for hospital mortality . ( % ci . - . ; i = ). conclusions: the acute care non-physician provider in the icu seems a promising clinician on the icu with regard to quality and continuity of care. whether they also can reduce mortality remains to be determined by designing studies, which adequately measure the contribution of the non-physician providers in icu care overall and per task. their role in europe remains to be elucidated. burnout and depression in icu staff members n bahgat menoufia university hospital, shibin elkom, egypt critical care , (suppl ):p introduction: family and success in work are the most important sources of person satisfaction in life, chronic prolonged exposure to stressful high workload in intensive care units (icu), create a bad psychological state named burnout syndrome in which person is depressed, exhausted and thinks to leave job. in this study we made a survey on icus staff members in egypt menoufia university hospital to explore and find risk factors increase depression and burnout among nurses and doctor. methods: questionnaires were given to all intensive care staff for estimating the prevalence and associated risk factors of burnout using maslach burnout inventory (mbi) with its three subscales emotional exhaustion (ee), lack of accomplishment (la), and depersonalization (dp). depressive symptoms using the beck depression inventory scale. blood sample was taken for assessing depression biomarkers including il- , tumor necrosis factor (tnf)-alpha, and coenzyme q (coq ), which appears to be one of the most reliable peripheral biomarkers. results: participants were respond in our survey from icu members the response rate was . %, the depression symptoms found increased in nurses more than physicians in icu with more desire to leave the job. there was strong correlation between the degree of depression symptoms and decrease percent of personal accomplishment. impaired personal relationships at work and increased night shifts were major risk factors of burnout syndrome. levels of the proinflammatory cytokine (il and tnf alpha) were elevated in members who recorded sever degree of depression score with decrease in concentration of co-enzyme q . conclusions: the health workers in icu had high liability for depression and burnout syndrome. the risk factors differ between nurses and doctors. il , co-enzyme q and tnf alpha concentrations had god correlation with degree of severity of symptoms. impact of a tailored multicomponent program to reduce discomfort in the icu on post-traumatic stress disorder: a casecontrol study p kalfon , m alessandrini , m boucekine , m geantot , s renoult , s deparis-dusautois , o mimoz , j amour , e azoulay , c martin , t sharshar , m garrouste-orgeas , k baumstarck , p auquier introduction: reducing discomfort during the icu stay should be beneficial on long-term outcomes. the aim of this study was to assess the impact of the implementation of a tailored multicomponent program to reduce discomfort in the icu [ ] on the occurrence of posttraumatic stress disorder (ptsd) months after discharge from the icu. methods: design: case-control study; the cases were patients hospitalized in the icus which implemented the tailored multicomponent program; the controls were patients hospitalized in the icus which did not implement the program. exposition: the tailored multicomponent program consisted of assessment of icu-related self-perceived discomforts by using the iprea questionnaire, immediate and monthly feedback to healthcare teams, and tailored site-targeted measures under control of a duo of local champions. general procedure: eligible patients were recalled months after the icu stay. data collection: sociodemographics, clinical data related to the icu stay, discomfort's levels assessed the day of discharge from the icu, life situation (home/care center), pstd (ies-r) and anxiety-depression symptoms (hads) months after the icu discharge. results: from the eligible cases and eligible controls, cases and controls were included (reason for exclusion: deaths after discharge from the icu, lost to follow-up, patient refusal, cognitive incapacity). a total of . % of the cases and . % of the controls presented certain symptoms of ptsd at months (p= . ). after adjustment for age, gender, iprea score, mccabe score, presence of invasive devices during the icu stay and considering anxietydepression symptoms at months, cases are less likely to have ptsd symptoms than controls. conclusions: our tailored multicomponent program for discomfort reduction in the icu can reduce long-term outcomes as ptsd. diffusion of such a program should be enhanced in the icus paving the way for a new strategy in care management. introduction: cognitive dysfunction is a major factor leading to disability and poor quality of life in icu survivors. in order to identify patients at risk for developing cognitive dysfunction due to critical illness or icu treatment, one has to discriminate between patients with pre-existing cognitive dysfunction and those developing new cognitive dysfunction or worsening of cognitive function during icu treatment. we investigated the incidence of pre-existing cognitive dysfunction in icu patients using the informant questionnaire on cognitive decline in the elderly (iqcode) and its relation with delirium during icu treatment. methods: patients relatives were asked to fill in the iqcode on admission. an overall score on cognitive dysfunction was calculated by the average of the score on each item of the questionnaire. the incidence of delirium was based on the cam-icu score. statistical analysis was performed using the fisher's exact test. p-values of less then . were deemed significant. results: in total consecutive patients admitted to our icu were analyzed, of whom . % (n= ) showed decline in cognitive function prior to icu admission. cognitive function was divided in four groups; no change . % (n= ), slight decline . % (n= ), moderate decline . % (n= ) and severe decline . % (n= ) (fig. ). incidence of delirium is shown in fig. . patients with moderate to severe cognitive dysfunction showed significant more delirium during icu treatment than patients with no change in cognition ( . % and . % respectively, (p= . )). conclusions: almost half of the patients admitted to the icu have cognitive dysfunction prior to icu admission. to assess ones cognitive function after icu treatment one has to take in to account the patients pre-existing cognitive functioning. patients with a moderate to severe pre-existing cognitive dysfunction develop significantly more delirium during icu treatment. introduction: our aim was to identify and analyse patients treated for pocd admitted to a thoracics/urology intensive care unit at university college london, uk. pocd is rising in the ageing high-risk surgical patient. early identification of those at risk and timely intervention could help reduce associated morbidity and mortality [ ] . methods: we identified patients treated with haloperidol, midazolam, lorazepam, olanzapine, clonidine or chlordiazepoxide from our electronic data system. these pharmacological interventions were used as surrogate markers of primarily hyperactive pocd, acknowledging other forms of delirium may be unaccounted for. of admissions ( . %) were shortlisted from august to july . patients were excluded if the drugs had been used for other indications. prevalence of known pocd risk factors were then detailed. on these data we performed a cluster analysis using r. results: of the patients ( . %) suitable for analysis, the mean age was . patients underwent elective procedures. were male and were female. % patients had thoracic surgery. the mean pain score in the first hours post-op was . (sd= . ), (with = no pain, = very severe pain). % had evidence of poor sleep and % evidence of anxiety. in the hours prior to evidence of pocd, the mean pain score remained . (sd= . ), % had evidence of poor sleep and % had evidence of anxiety. % of our population was septic during their itu admission. conclusions: our analysis demonstrates pocd is highly prevalent in male patients over undergoing thoracic procedures. we will now develop a pocd pathway targeting improved postoperative management of pain, sleep, anxiety and infection in this patient population. introduction: our objective was to determine the feasibility of employing family-administered tools to detect delirium in the critically ill. the use of family-administered delirium detection tools has not been assessed in the icu where patients are critically ill and frequently intubated. family members may be able to detect changes in patient cognition and behavior from pre-illness levels earlier than unfamiliar providers. these tools may be a valuable diagnostic adjunct in the icu. methods: consecutive patients and family members (dyads) in the largest adult icu in calgary, canada were recruited (aug. -sept. , ). inclusion criteria were: patients with a richmond agitation sedation scale (rass) >=- ; no primary brain injury and glasgow coma scale score of < ; ability to provide informed consent (patient/ surrogate); and remain in icu for hours. data were collected for up to days. family-administered delirium assessments were completed once daily (family confusion assessment method & sour seven). to assess feasibility, we assessed proportion of eligible patients and percent family member enrollment. barriers to enrollment were categorized. results: of admitted patients with family, ( %) met inclusion criteria and ( %) dyads consented. % of admitted patients did not have family and were thus ineligible. % of enrolled dyads assessed delirium at least once, with a median of (of total) assessments. the most common reason for non-enrollment was refusal by the family, who commonly reported feeling overwhelmed by the icu environment. barriers with nursing staff were encountered, including not providing access to patients and patient exclusion. conclusions: these data suggest that employing family-administered delirium detection tools in the icu is feasible for a subset of the population. future studies will validate the use of these tools in the icu, decrease modifiable barriers to enrollment, and test strategies to overcome attitudinal barriers towards employing these tools. introduction: psychological impact of critical illness and icu stay on patients can be severe and frequently results in acute distress as well as psychological morbidity after discharge [ ] . however, the stressful experience in icu and its influence on patient recovery, remain relatively understudied. we assessed patients in icu for acute distress and psychological symptoms with validated tools. methods: we conducted an observational study in a group of awake icu adult patients admitted in a tertiary centre for at least hours, from january until october , with mixed diagnosis on admission. we collected demographic factors, saps ii at admission, mechanical ventilation, day of sedation, history of psychopathological disorder. un-sedated and alert, critical care patients were assessed with tools such as intensive care delirium screening checklist (icdsc), hospital anxiety and depression scale (hads) and intensive care psychological assessment tool (ipat). results: patients were recruited, (mean age . ± . years, . % males). saps ii at admission was . ± . , . % was mechanically ventilated (mean duration . ± ), mean duration of sedation was . ± . days and a rate of . % had an history of psychopatological disorder. . % of the sample had clinical delirium (icsdc> ) and was not assessed with others tools, . % had subclinical delirium (icsdc <= ). regarding psychological outcomes, . % (mean score . ± . ) reported a score (>= ) on hads that indicates a possible diagnosis of anxiety and . % (mean score . ± . ) of depression. a rate of . % reported a score >= on ipat suggesting an acute distress. conclusions: the study's key finding was that acute psychological distress was high in awake icu patients. further work is needed to determine the efficacy of early psychological interventions to reduce the incidence of acute distress and psychological outcomes after icu stay. introduction: a high percentage of polytrauma patients require surgery within the first hours to stabilize primary traumatic injuries. one of the main intraoperative complications in this type of patients is due to hemodynamic instability [ ] . thus, it is necessary to implement multimodal monitoring involving both hemodynamic monitoring and monitoring of general anesthesia. the objectives of this study were to identify the possible implications of entropy monitoring on hemodynamic stability in critically ill polytrauma patients. methods: prospective observational study, deployed in the clinic of anesthesia and intensive care, emergency county hospital "pius brinzeu" timisoara, romania. clinicaltrials.gov identifier. there were two groups, group a (n = ), in which the depth of hypnosis was monitored through entropy (ge healthcare, helsinki, finland) and group b (n = ). results: the incidence of hypotension and tachycardia episodes was statistically significantly lower in group a, unlike the control group (p < . ). moreover, a statistically significant (p < . ) consumption of inhaled anesthetic agent was recorded in group a compared with group b. consumption of vasopressor was also lower in group a (p < . , difference between means . ± . , % confidence interval . - . ) conclusions: deploying monitoring for the depth of hypnosis in general anesthesia using entropy can significantly increase the hemodynamic stability of critically ill polytrauma patients. introduction: the use of methadone as a potent analgesic has been gaining ground in the intensive care setting, such as where it is possible to properly select the group of patients who will benefit from the drug, as well as monitoring of possible complications. the objective of this study is to evaluate the safety of the use of methadone in critically ill patients in a large hospital. methods: a retrospective analysis of all patients who used methadone in a neurological intensive care unit for a period of four months and the results were evaluated. results: in the four-month period, patients used methadone during intensive care. % of the patients were male, with a medical age of . ± . years. the main indication for the use of the medication was for analgesia in patients who were weaned from mechanical ventilation. the mean time of use was . days. in all cases evaluated, analgesia was effective, with methadone being used alone or in combination with other drugs, according to an institutional protocol. among the complications found, patients presented hypotension ( %); presented bradycardia ( %); presented constipation ( %); had excessive sedation ( %) and had other complications. all complications were reversible. patients of the studied population died, however, without correlation with the use of methadone. conclusions: the use of methadone, in the studied group, was effective in the control of analgesia, with no impact on patient safety when used in a monitored way. introduction: renal colic is a common disorder which presents with dramatic acute pain. providing rapid relief, using effective pain control medications is the clinical priority to treat the patients. this study aims to compare the effect of iv ketorolac versus morphine in releasing renal colic pain by measuring pain severity and duration and also the need for additional doses. methods: we performed a clinical pilot cohort study from during on patients with the clinical diagnosis of renal colic who recruited from the emergency department (ed) of rasool-e-akram hospital and firoozgar hospital. participants who were candidate to receive either morphine or ketorolac were divided into two groups who received either mg ketorolac iv or mg morphine. the pain was evaluated using the visual analog scale (vas) at four time points: before drug injection (vas- ), minutes (vas- ), minutes (vas- ), and minutes (vas- ) after injection. in cases when the pain was not controlled with the first injection of drug beyond minutes; additional doses (rescue) were injected. statistical analyses were performed using spss . results: one-hundred-fifty patients treated with morphine and ones with ketorolac were studiedthe group treated with morphine scored on average . before the injection, which was roughly . points higher than ketorolac. morphine reduced patients' vas scores more intensely (median: , iqr: versus median: , iqr: ; p value< . ). in general, patients treated with morphine were more likely to need a second (rescue) dose, when compared to ketorolac group ( . % vs %, p value= . ). conclusions: morphine is a better option for pain release in cases of renal colic. ketorolac released the pain to an acceptable level; but, because of its slower action time, we recommend it in cases with moderate than severe pains. effect of analgesics on cardiovascular and hormonal response to operative trauma d loncar stojiljkovic, mp stojiljkovic sgh, , serbia critical care , (suppl ):p introduction: objective of this study was to compare the effects of two analgesic regimens, one opioid and one non-opioid, on cardiovascular and hormonal reaction of patients undergoing elective surgery under general endotracheal anaesthesia. methods: a total of elderly patients, asa , scheduled for elective abdominal surgery were assigned to receive on induction a single dose of either fentanyl ( . mg, + . mg) or a fix combination of etodolac and carbamazepine (novocomb, dose mg+ mg iv bolus). haemodynamic parameters and concentrations of prolactin cortisol and growth hormone (gh) [ ] were determined at critical points and h after operation. results: both fentanyl and novocomb blocked the hypertensivetachycardic response to surgical trauma. cortisol was a more appropriate endocrine marker of stress than prolactin or gh since fentanyl as an opioid analgesic increased secretion of prolactin [ ] , while carbamazepine from novocomb did the same with gh [ ] (figs. & ) . conclusions: cortisol plasma concentration correlates positively with cardiovascular parameters in patients undergoing elective abdominal surgery who received fentanyl or novocomb as intraoperative analgesic. its suppression is better marker of analgesia than prolactin and gh. volatile anaesthetic consumption and recovery times after long term inhalative sedation using the mirus system -an automated delivery system for isoflurane, sevoflurane and desflurane introduction: the new mirus system as well as the anaconda uses a reflector to conserve volatile anaesthetics (va) [ , ] . both systems can be paired with icu ventilators, but mirus features an automated control of end-tidal va concentrations (etva). we compare feasibility and recovery times for inhalational long term sedation with isoflurane (iso), sevoflurane (sevo) or desflurane (des). methods: asa ii-iv patients undergoing elective or emergency surgery under general anaesthesia were included. patients were randomized into three equal groups iso, sevo and des. the mirus system was started with a targeted etva of . mac. we used the puritan bennett icu ventilator and performed a spontaneous breathing trial. if successful, the target concentration was set to mac and recovery times measured. results: patients were comparable in demographics, tidal volume, respiratory rate and sedation time (total h: iso ± h; sevo ± h; des ± h; p= . ). in all patients, a mac of . was reached. conclusions: mirus could automatically control end-tidal va concentrations in ventilated and spontaneously breathing patients. the recovery times are only prolonged in the iso group and could be shortened by removing the reflector. the higher etva required for a . mac using des and sevo were associated with an increased va consumption. introduction: intranasal analgesia is increasingly used in order to relieve pain in the emergency department. this non-invasive approach avoids discomfort, stress and risks related to the parenteral route of administration. the objective is to compare intranasal (in) fentanyl versus any parenteral opioid (intravenous, subcutaneous, intramuscular) for the effectiveness of acute pain relief in an adult population. methods: the systematic review was registered in prospero (crd ). the research of articles was conducted through embase, central, and medline databases. randomized clinical trials comparing the effectiveness of in fentanyl to any parenteral opioid for acute pain relief (<= days) in an adult population (>= years old) were considered for inclusion. studies on breakthrough cancer pain were excluded. two different reviewers extracted data and analyzed the quality of the selected articles. the main outcome was the difference between pain levels before and after analgesia. the effect size was approximated using the inverse of variance of standardized mean differences, based on a random-effect model. heterogeneity was quantified using a test of i . results are presented with % confidence interval. results: eight randomized clinical trials with cohorts and a total of patients were selected ( in fentanyl vs control group). selected articles contained a low risk of bias. there is no significant difference between the average levels of pain before and after analgesia comparing the two groups (smd . [ic % - . à . ], p= . ; i = %) (fig. ) . conclusions: in fentanyl is as effective as other parenteral opioid to relieve pain during the first hour of treatment. introduction: the aim of this study is to underline the importance of sedation protocol when performing the pegj procedure in advanced parkinson's disease (pd) patients. research about the use of sedation in endoscopy is getting more and more widespread as to answer to the increasing grade of complexity and duration of endoscopic procedures as to offer comfort to the patient in terms of analgesia, tolerability, and amnesia. sedation is also a way to assure quality and safety examination and to improve its outcome [ ] . methods: this observational retrospective study includes pd patients scheduled for pegj procedure (fig. ) in order to start therapy with duodopa gel. we propose an anesthetic technique (table ) to support pegj with both local anesthesia and moderate sedation so as to provide analgesia and patient's comfort. this technique ensures mean duration of pegj procedure was '± '. mean stay time in recovery room '± '. compared to our old experience, we collected lack of patient's discomfort, anxiety, and memory, high procedure compliance and improvement of the quality of procedure without use of opioids. conclusions: based on our experience, we consider this sedation protocol effective for different reasons: to relieve or abolish patient's discomfort, anxiety, and memory, to ensure compliance with the procedure, to ensure patient's analgesia and patient's safety and, finally to assure procedure's quality and rapid discharge. anyways, a multicentric study should be done to test our protocol. introduction: studies have shown that icu survivors exhibit longterm neurocognitive impairment and perceived reduction in quality of life after icu discharge, but studies examining sleep architecture and sleep disordered breathing (sdb) in icu survivors after icu discharge are scanty. the aim of our study was to assess sleep architecture and sbd in icu survivors. methods: icu survivors were screened for eligibility. inclusion criteria were: age - yrs, mechanical ventilation >= hours, gcs of at the time of hospital discharge. patients with a history of sbd, chronic neuromuscular disorders, chronic restrictive lung disease, congestive heart failure and respiratory failure at hospital discharge were excluded. patients were evaluated within one week after hospital discharge and months later. at both visits patients completed health related quality of life questionnaires (sf and epworth sleepiness scale), underwent a physical examination, lung function tests including maximum inspiratory and expiratory mouth pressures, and an overnight full polysomnography (psg). results: sleep quality at days of hospital discharge is poor, characterized by severe disruption of sleep architecture and excessive sdb, mainly of obstructive type which in % of patients was classified as moderate or severe. although at six months after hospital discharge sleep quality remained relatively poor, significant improvement in n stage and ahi was observed, with more patients to be classified as normal or mild sdb. both at hospital discharge and months later quality of life was reduced but there was no relationship between the health related quality of life and sleep disturbances. conclusions: icu survivors experience significant deterioration in their quality of life status with minor improvement months later and a variety of sleep disturbances that seems to start getting better months later. introduction: disrupted sleep in critically ill patients may be associated with delirium, prolonged stay in icu and increased mortality. polysomnography (psg), the criterion standard method of sleep monitoring, is challenging in icu due to interpretation difficulties, as the patterns defined by the standard classification for scoring sleep are absent in many critically ill patients. the aim of this study was to investigate if the presence of atypical patterns in critically ill patients' psg is associated with poor outcome measured by -days mortality in conscious critically ill patients on mechanical ventilation. methods: psgs (median duration hours) recorded in conscious critically ill mechanically ventilated patients were scored by an expert in sleep medicine blinded to patient characteristics. standard sleep scoring classification was used if possible. otherwise, modified classification for scoring sleep in critically ill patients proposed by watson et al. was applied [ ] . the association of sleep patterns (normal or atypical) and micro-sleep phenomena (sleep spindles and kcomplexes) with days mortality was assessed using weibull model by calculation of hazard ratios (hr). results: hr analysis showed twice as high mortality risk in case of atypical sleep compared to normal sleep; this was however not significant (hr . ; % ci . - . ; p= . ). the presence of sleep spindles in psg significantly reduced mortality risk to / (hr . ; % ci . - . ; p= . ). the presence of k-complexes in psg reduced mortality risk to ½, though not significantly (hr . ; % ci . - . ; p= . ). conclusions: the absence of normal sleep characteristics in psg in conscious critically ill patients on mechanical ventilation is associated with poor short-term outcome. antipsychotics (aps) prescribing in critically ill delirious patients, the reported versus the perceived practice e almehairi , g davies , d taylor introduction: aps are the most commonly prescribed drugs in hyperactive/mixed delirium and agitation in critical care (cc) [ ] . yet evidence in cc is scant, there are known adverse effects (ade) and prescription is out with the european license. meticulous observation of ap selection, prescribing and safety, alongside delirium assessment/plan is essential to gain new knowledge and patients. when accompanied by prescribing clinicians perspective of delirium ap treatment results are more interpretable. we conducted a two-part single centre cohort study that aimed to describe/compare real to perceived delirium assessment/plan, aps prescribing and safety in cc adult patients at gstt. methods: part : a prospective survey, of cc prescribing clinician's beliefs and attitudes to delirium assessment/plan, aps prescribing and safety over previous months. part : a meticulous audit of aps prescribing and safety and delirium/agitation assessment and plan, over period of months. results: part survey. of prescribers ( . %) completed survey. % of reported using aps to treat delirium, with % selecting atypical aps as first option. part audit. there were admissions to cc. aps were prescribed in . % ( prescription), . % ( prescription) were in delirium/agitation patients (table ) . survey (vs.) audit: in the survey % reported daily delirium screening whereas only . % undertook daily screening in audit (fig. ) . higher quetiapine and lower iv haloperidol maximum daily dose were prescribed in audit in comparison with survey reported doses ( table ) . lead ecg was used to monitor ap ade. in survey % reported assessing ecg once or more daily. audit revealed only % actually did so (fig. ) . conclusions: authors believe perceived vs actual can identify key areas for quality improvement (qi). major differences were in delirium assessment/plan and safety monitoring sedation practices in turkish icus, the aim was to provide knowledge on this matter. methods: an electronic survey form was generated with google forms. first part of the form included questions about demographics, and choices and routines of sedation administration. this part mostly contained multiple choice questions, which more than one choice could be indicated. second part was comprised of some statements to investigate the attitudes of physicians, which were indicated on a five-point likert scale. the link for the survey was posted to all email addresses registered in the turkish society of intensive care member database. results: of members, ( %) completed the survey form. demographics are given in table . sedation was generally applied by the physicians ( %). the indications were mechanical ventilation ( %), agitation ( %), seizures ( %), anxiety ( %), delirium ( %). drug choices of the respondents are shown in fig. . sedation level was evaluated daily by % of respondents, mostly using ramsay scale ( %). daily established sedation level was indicated in . %, and daily interruption of sedation was indicated in . % answers. sedation protocol was not used in . % of the answers. analgesics applied commonly, while % routinely evaluated pain and visual analogue scale (vas) was the preferred method in % of the answers. . % of physicians indicated routine use of neuromuscular blockers. in . % answers routine evaluation for delirium was indicated, mostly using cam-icu.when the knowledge of sccm guideline pain, agitation and delirium management, % indicated a positive answer.the respondents indicated their opinion for some comments on sedation, the answers are shown in the table . conclusions: it may be concluded sedation practices may need to be improved by increasing awareness on novel concepts in this area. fig. (abstract p ) . the prediction-corrected vpc plots for dexmedetomidine pk. the vpc plots show the simulation-based % confidence intervals around the th, th, and th percentiles of the pk data in the form of blue ( th) and gray ( th and th) areas. the corresponding percentiles from the prediction corrected observed data are plotted in black color methods: a prospective multinational cohort study was performed in icus in sweden, denmark and the netherlands. all adult patients with an icu stay >= hours were screened for inclusion. primary outcome was psychological problems three months after discharge from the icu, assessed with the questionnaires hospital anxiety and depression scale (hads) and post-traumatic stress symptoms checklist- (ptss- ). a subscale score > in the hads and a score > in the ptss- part b indicate clinically significant symptoms of depression, anxiety and pts and was considered an adverse outcome. we collected data on known risk factors for psychological problems post-icu. univariable and multivariable logistic regression modelling of risk factors was performed in order to create an instrument to be used bedside, predicting individual risk for adverse psychological outcome. results: patients were included and ( %) returned follow-up questionnaires. % of patients scored above the predefined cut-offs having symptoms of depression, anxiety or pts. age, lack of social support, depressive symptoms and traumatic memories at discharge remained significant after multivariable modelling and constituted the screening instrument ( table ) . the predictive value of the instrument was fairly good with an area under the receiver operating characteristics curve (auroc) of % (fig. ) . we developed an instrument to be used at icu discharge, predicting individual patients' risk for psychological problems three months post-icu. the instrument can be used as a screening tool for icu follow-up and enable early rehabilitation. improving the patients hospitalization experience in an intensive care unit by contact with nature w yacov , y polishuk , a geal-dor , g yosef-hay kaplan medical center, rehovot, israel; kaplan medical center, rehovot, israel critical care , (suppl ):p introduction: the intensive care unit is characterized by a noisy and threatening work environment using multi tecnologic equipment.the staff works very intensively caring for very complicated and unstable patients.whilst caring for the patients physical needs one must not forget the patients mentally needs.the improvement of the patients hospitalization experience by changing the environment improves the mood and responsiveness to treatment gives hope for healing to the patient and family. methods: a quality questionare with open questions relating to the subjective sensory experience of the patients and their families. the patients were transferred to the "sun balcony" for a period of - minutes having their families alongside. music was transmitted and the patients were offered food and drinks if their condition allowed. results: the patients reported a significant improvement of hospilizaton experience following their exposure to the "nature environment". patients described the sensory experience as a positive, pleasant, quiet and relaxing experience. the contact with the sun, wind, sky and grass and being outside on the "sun balcony" allows a disconnection from the threatening icu environment. conclusions: the "sun balcony" gave the patients a sense of hope and wish for healing. mobilizing complicated patients to the "sun balcony" is a big challenge which requires planning and preparation by the staff. yet by the proactive and creative thinking of the staff the patients are tranferred to the "sun balcony" to give them encouragement, a feeling of well being and hope for recovering. this intervention is costless and a routine procedure in the intensive care unit. introduction: long-term psychological outcomes of patients(pts) discharged from icu represent an emergent relevant matter of concern.systematic reviews refer prevalence of %- % for anxiety, %- % for depression and %- % for posttraumatic symptoms in ards patients.the onset of psychiatric symptoms after discharge, might be associated with patient's competence to process memories related with hospitalization and with memories. methods: we selected ards pts in icu of a tertiary centre (jan -dec ) at least hour, for months follow-up and pts for months follow-up after discharge. the psychopathological assessment was performed using scale as: impact event scale-revised (ies-r), hospital anxiety and depression scale (hads), icu memory tool (icu-mt). results: mean age was . ± . at months follow-up and , ± , at months. ptsd symptoms was fund respectively in % and . % pts at and months; anxiety symptoms % and . % of pts;depression symptoms in % and . %. significant correlations were fund between psychopathology at months and memories of icu: hads anxiety with delusion memories (r . ,p< . ); hads depression with factual (r . ,p< . ), feeling (r . ,p< . ) and delusion memories (r= . ,p< . ); feeling (r . ,p< . ). at months significant correlations was fund between hads anxiety and feeling memories (r . ,p< . ); ies-r and factual (r . ,p< . ), feeling (r . ,p< . ) and delusion memories (r . ,p< . ). the results of the study confirmed the importance of assessing psychopathology after discharge from icu. the onset of these symptoms appeared to be mediated by specific traumatic memories related with icu hospitalization. the main clinical recommendation emerging from this study is to investigate psychiatric history and develop psychological strategies to manage frightening or delusional experiences during icu stay. introduction: the aging of the population is a fact. the subgroup of very old (>= years (ys)) is the one that increases the most rapidly. intensive care unit (icu) admission of these patients is an ongoing discussion worldwide. our icu has designed the voolcano aiming its characterization and reviewing outcomes, to find some predictive indicators. the purpose of this first analysis is to evaluate specifically the group of very old patients (volds) admitted to a tertiary portuguese hospital icu. methods: retrospective observational study was preformed, included all volds admitted in icu during years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . demographic data, admission diagnosis, severity scores, charlson comorbidity index, length of stay and outcomes were considered. data analysis used spss software. results: we found a total of admissions. the median age was . ys with iqr ; mostly male with medical admission diagnosis (sepsis and respiratory failure due to infection). there was a median acute physiology and chronic health evaluation ii of (iqr ) and simplified acute physiology score ii of (iqr ). median charlson comorbidity index was . (iqr ). median length of stay was . days (iqr . ). concerning outcomes, we found intra-icu mortality of %; intra-hospital after icu discharge mortality of % and mortality after hospital discharge of %. identified as predictors of intra-hospital mortality the use of mechanical ventilation (p < . ), urgent surgical admission or medical admission versus schedule surgical admission (p < . ) and the absence of oncologic disease (p = . ). on multivariate analysis, only mechanical ventilation (p = . , hr . , % c.i. . - . ) and urgent surgical admission versus schedule surgical admission (p = . , hr . , % c.i. . - . ) remain significant. conclusions: recognizing the need to understand what is the biologic|funcional age (opposed to chronologic age) would be beneficial in the selection of volds to icu admission. organ failure and return to work after intensive care s riddersholm , s christensen , k kragholm , cf christiansen , bs rasmussen aalborg university hospital, aalborg, denmark; aarhus univeristy hsopital, aarhus, denmark critical care , (suppl ):p introduction: organ failure is associated with an unfavorable prognosis. nevertheless, the association with capability to return to work remains unclear. therefore, we investigated the association between organ support therapy as a proxy for organ failure and return to work in a nationwide cohort of icu survivors. methods: we linked danish registry-data on icu-and hospitalsurvivors working prior to hospital admission during - , - years of age, with an icu length of stay > hours and not previously treated with dialysis, to data on return to work. we reported cumulative incidences (chance) of return to work with death as competing risk, and compared rate of return to work in adjusted cox regression-models by number of organ support therapies including renal replacement therapy, cardiovascular support and mechanical ventilation and stratified on primary hospital-admission diagnosis. results: of , patients - years of age, % ( , ) survived to hospital discharge (tables and ). among these, the chance of return to work was . % ( % ci [ . - . ]) within two years (fig. (fig. ) . when stratified an increasing number of organ support was associated with a decreased chance of return to work among patients with infection, respiratory failure or trauma but not among patients with neoplasms or endocrine, gastrointestinal and cardiovascular diagnoses. introduction: mortality rates among people with moderate to severe learning disabilities (ld) are times higher than in the general population [ ] [ ] [ ] . this study was designed to examine critical care admissions with learning disabilities in terms of mortality, demographics and reason for admission. methods: data was retrieved for adult patients (> years old) between sept and . the ward watcher database for icus within surrey and sussex healthcare nhs trust was interrogated using search words including, learning disability, cerebral palsy, down's syndrome and autism. results: there were episodes ( . % of all admissions) of patients admitted with ld. % of the ld patients had more than admission. respiratory is the most common system affected ( %). logistic regression suggests survival is highest in those with a neurological reason for admission (p= . ). proportionally ld patients were young compared to the total population (fig. ) . we found that mortality appears to increase rapidly in those over years of age and overall mortality is greater in those with ld (fig. ) . conclusions: from april all uk trusts will be required to complete a detailed review for patients with ld who die whilst in hospital care. this follows mencap's report 'death by indifference' which exposed deficiencies in the care of people with lds who died whilst in nhs care and the subsequent confidential inquiry into premature deaths of people with learning disabilities. in our population, ld patients have an earlier death than the general population and the overall mortality from critical illness is greater. a multidisciplinary approach at the emergency department to admit potential organ donors for introduction: the aim of the present study is to improve the recognition of potential organ donors by implementing a multidisciplinary approach for organ donation at the emergency department (ed) [ ] . methods: in a prospective intervention study, we implemented this approach in six hospitals in the netherlands. when the decision to withdraw life sustaining treatment was made at the ed in patients with a devastating brain injury without contra indications for organ donation, an intensive care unit (icu) admission for end-of-life care was considered. every icu admission for end-of-life care was evaluated. interviews were conducted with emergency physicians, neurologists and icu physicians according to a standardized questionnaire. this interview focused on medical decisions that were made and difficulties arising during hospitalization. results: from january to november data were collected on the number of patients admitted to the ed with acute brain injury. in total, potential organ donors were admitted to the icu for end-of-life care. donation was either requested in the ed ( %), icu ( %), neurology department ( %), or donation was not requested ( %). out of donation requests, families ( %) consented to donation. this led to successful organ transplantations. in four of these patients family consent was obtained to intubate them solely for the purpose of organ donation. the most important points raised during the interviews were: explaining the non-therapeutic icu admission to the family, the location where donation should be requested (ed/icu) and utility of icu resources. conclusions: a close collaboration between the ed, neurology department and icu is necessary and achievable in order not to miss potential organ donors in patients with acute brain injury with a futile prognosis in the ed. introduction: there is a relationships between intensive care patients losing the ability to speak and negative emotions [ ] . nursing care is challenging when patients are unable to verbalise and factors like pain and comfort are misjudged.. our intensive care unit has introduced a communication tool intelligaze grid which enables patients with primary motoric disorders to communicate their needs. a quality improvement study reviewed the methods of communication and interactions that our nurses use for patients who are ventilated. the objective of the study was to promote areas of improvement with communication in the icu. methods: we used a mixed-methods qualitative and quantitative study to evaluate the communication tools used by our nursing staff to interact with ventilated patients. a convenient data sample for all nurses working on particular dates was collected which is % of the nursing workforce. the study has been approved as a quality assurance project by the human research ethics committee of nepean hospital. results: sixty registered nurses ( %) participated in the study. the most common communication tool used with patients was closed yes/no questions( %), followed by hand gestures( %), magnetic writing board( . %), lip reading( . %) and alphabet board( . %). the descriptive analysis identified challenges were levels of sedation, weakness, non-english speaking patients and delirium. a significant finding was that only % of nurses identified the patients message being understood and % acknowledged listening as effective communication. conclusions: communication is a vital aspect of icu nursing and is achieved through dialogue and specialised skills. the study concluded that icu nurses find it difficult to communicate effectively with ventilated patients. the introduction of intelligaze grid has improved patient communication and promotes holistic nursing care. p withdrawn introduction: substantial variability in eolp occurs around the world [ ] . differences in eolp were previously reported in europe in the ethicus i study [ ] . methods: icus worldwide were invited to participate through their country societies. consecutive admitted icu patients who died or had treatments limitations during a month period from . . to . . were prospectively studied. regions included north, central and southern europe (ne, ce, se), north and latin america (na, la), asia (as), australia (au) and africa (af). previous eolp definitions were used [ ] . results: icus in countries participated enrolling , patients. figure shows differences in eolp by region and figure in patient competency by region. conclusions: worldwide differences included more cpr in af, la, and se and less cpr in ne, au and na. there was more withdrawing (wd) in ne and au and less wd in la and af. more patients were competent in au and ne and less were competent in af, se and la. introduction: the decision of end-of-life care in the icu is very tough issue because the law, ethics, traditions and futility should be concerned involving family's will. especially, stop or withdraw therapy is a quite difficult operation in japan because of our traditions. recently there are few legal issues due to some guidelines. our hypothesis is some difference over time exists in thoughts about end-of-life care in the icu. the purpose of this study is to know changing methods: a questionnaire survey, which consists of questions with optional answers related to the thoughts of participants about end-of-life care of hopeless or brain death patients, was performed to nurses and doctors in our icu. the questions were; whether accept to withdraw therapy or not and with family's will, whether positive or not to donate of organs from brain death patient, necessary of icu care for brain death patient, feel guilty and stress for doing stop or withdraw therapy. the optional answer has gradations from 'yes' to 'no' for all questions. it was guaranteed to be anonymous for them in the data analysis. we conducted entirely same survey in . the answers between in and in were fig. (abstract p ) . patient mental compentency by region kidney disease: improving global outcomes acute kidney injury working group references nice clinical guidelines: idiopathic pulmonary fibrosis in adults: diagnosis and management references . zambon et al annual update in inten care references references references damage control management in the polytrauma patient crash- trial collaborators guidelines for the management of severe traumatic brain injury references references references . soar et al; european guidelines for resuscitation we acquired the confirmed date of death from the finnish population register centre database and gross -month healthcare costs from the hospital billing records and the database of the finnish social insurance institution. results: a total of patients were included in the study and were alive at months. median (interquartile range, iqr) -hour sofa score was ( - ) in -month survivors and ( - ) in non-survivors. the sofa score had an area under receiver operating characteristic curve of . ( % ci . - . ) for predicting -month mortality. in multivariate regression model with age and gender, sofa score had an odds ratio, or ( % confidence interval, ci) of . ( . - . ) for predicting -month mortality. all except cardiovascular sub-score also had p predictive factors for secondary icu admission within hours after hospitalization in a medical wards from the emergency room m cancella de abreu hôpital saint antoine p acquired neuromuscular weakness in eldery patients with femoral bone fracture, could we decrease the incidence? d pavelescu, i grintescu, l mirea emergency hospital floreasca p adasuve enables quicker dispositions of acute psychiatric patients in the emergency department k hesse , e kulstad , k netti , d rochford isi web of science and clinicaltrials.gov. data extraction: eligible studies were case reports and randomised controlled trials (rcts) that evaluated the effects of drug incompatibilities in critically-ill patients on morbidity or mortality as primary or secondary outcomes, or adverse events. two investigators independently reviewed the eligibility of the study from abstracts or manuscript data. data synthesis: twelve articles met the selection criteria (fig. ). the six articles reporting rcts concern only four rcts. rcts were single-centre studies comparing infusion with or without filter. two of them included adult patients. the others included pediatric and neonatal intensive care unit patients. primary endpoints were systemic inflammatory response syndrome (sirs), organ failure, overall complication rate, bacteremia, sepsis, phlebitis and length of stay. results: the results are mixed with one rct reporting a reduction in sirs, organ failure and overall complication rate, two studies in disagreement over the occurrence of sepsis and one study reporting no impact on length of stay. the six articles on case reports show different drug incompatibility situations european directorate for the quality of medicines & healthcare of the council of europe. guide to the quality and safety of organs for transplantation p current status and problems of organ transplantation before and after the enactment of the revised organ transplant law in p morale: introducing the anaesthetic trainee confession session results: of total patients admitted during study period, were eligible for study; . % were males and ( %) patients were transferred during after-hour. mean age of two groups (daytime vs. after-hour . ± . vs. . ± . years) was similar(p= . ) methods: retrospective analysis of prospectively collected data between october to february of a tertiary care icu in india. patient data collected on all consecutive icu admissions. primary and secondary outcomes were icu los and hospital mortality respectively. icu patients payer status were categorized as self-paid, corporate (paid-fully or partially-by-employer), and insurance (paid-fully or partially-by-third-party-payer). all analyses were adjusted for illness severity and icu support (vasopressor use, mechanical or noninvasive ventilation, blood transfusion). results: of patients admitted during study period significantly higher number of patients received icu support in self-paid and corporate groups compared to insured group ( . %and . % vs. . %; p= . ) braden scale is predictive of mortality in critically ill patients, independent of its efficiency as a predictive tool of pressure ulcer risk d becker , tc tozo discharged and died ( . %). the turnover rate of the icu was . . the occupancy rate calculated during the period was . %. there were only readmissions ( . %) within hours of admission. regarding the hospital evolution of these patients we had exits in this period, ( . %) were discharge and ( . %) were deaths, of these, ( . %) were after discharge from the icu. the mean saps score was . (ranging from to ). the probability of death, according to the standard equation was . % and the adjusted for latin america of . %. conclusions: the icu has a high occupancy rate and rotation turnover, as well as a higher mortality than predicted by the score. these indicators show the great population demand that we have and alert to the impact on the sustainability of the unit and patient safety methods: research/ethics approvals were obtained. surveys, interviews, round tables, targeted delphi exercises and non-participant observation were conducted across four adult critical care units, involving professionals. these methods were used to describe the baseline 'paper-based' workflow/inter-professional communication systems; and semi-quantitative quality improvement measures. secondly, critical care services worldwide were visited to generate a database of experience, lessons and models of optimised informatics delivery. results: key challenges at baseline in relation to workflow/communication information transfer between different healthcare professionals site visits revealed the importance of human resources; lead time technology advances; the prioritisation of nursing workflow and pharmacy medicines/prescribing database creation/testing and the importance of the hardware interface and ergonomics. improvements included patient safety/experience p work-related stress amongst doctors and nurses in intensive care, a&e, acute medicine, anaesthetics and surgery i lever *, h nawimana introduction: work-related stress is associated with anxiety, depression, days off-work, errors and 'near misses' [ ]. our objective was to references . kerr et al p pre-existing cognitive dysfunction in critically ill patients and the incidence of delirium during icu treatment p validation of the sos-pd scale for assessment of pediatric delirium: a multicenter study e ista , b van beusekom children's hospital, rotterdam, netherlands; umc groningen -beatrix children's hospital p introduction: delirium in critically ill children has gained attention in the last few years and the incidence seems higher than anticipated before. the sophia observation withdrawal symptoms-pediatric delirium (sos-pd) was developed to combine assessment of delirium with iatrogenic withdrawal syndrome, two conditions with overlapping symptoms. the current study evaluates the measurement properties of the pd component (pd-scale) of the sos-pd scale. methods: in a multicenter prospective observational study in four dutch picus, patients aged months to years and admitted for more than hours were included. these patients were assessed with the pd-scale three times a day. criterion validity was established: if the pd total score was or higher the child psychiatrist was consulted to confirm the diagnosis of pd using the diagnostic and statistical manual-iv criteria as the "gold standard". the child psychiatrist was blinded to outcomes of the pd-scale. in addition, the child psychiatrist assessed a randomly selected group of patients to establish false-negatives the pediatric delirium scale had an overall sensitivity of . % and a specificity of . % for a cut off score of points. the positive predictive and the negative predictive value were respectively, . % and . %. the icc of paired nurse-researcher observations was . ( % ci . - . ). in total patients were diagnosed with delirium during the picu stay. conclusions: the pd scale shows a good validity for early screening of pd. so, the pd scale is a valid and reliable tool for nurses to assess delirium in critically ill children p frequency, risk factors and symptomatology of iatrogenic withdrawal from opioids and benzodiazepines in critically ill neonates, children and adults: a systematic review of clinical trials ma duceppe , m perreault we also examined the grey literature. we included studies reporting frequency, risk factors or symptomatology of iatrogenic withdrawal of opioids, benzodiazepines (or both) in critically ill patients. we considered all study designs except case reports and case series. pairs of reviewers independently abstracted data and evaluated methodological quality using the cochrane collaboration tool, newcastle-ottawa or quadas- . pros-pero (registration number: crd ). results: we identified unique citations through database search and full-texts were assessed for eligibility. thirty-three studies were included; the majority were observational and only a few included adults proportion of perfused small vessels at to p use of methadone in critically ill patients p the use of intranasal fentanyl versus parenteral opioid for acute pain relief in adults: systematic review and meta-analysis p sleep disorders in icu survivors c alexopoulou, a proklou p impact of dexmedetomidine on the duration of invasive mechanical ventilation in pediatric intensive care patients -dexped trial m genest peri-operative dexmedetomidine in high risk cardiac surgerymulticentre randomized double blind placebo controlled pilot trial y shehabi we compared vasopressors, inotropes, pacing and cardiac complications for safety and severe acute kidney injury (aki), dialysis and death (major adverse kidney events make) for efficacy. methods: adults patients undergoing cardiac surgery [combined (valve + coronary bypass) or complex] or with preoperative glomerular filtration rate (egfr) < mls/min/ . m were included. salvage or transplant surgery, dialysis, egfr < mls/min/ . m and those on extracorporeal support were excluded. dex ( . ug/kg/hr) was started at induction of anaesthesia and continued up to hours after surgery. equivalent volume of saline was given to control group. standard intra and post-operative care was provided. results: we randomized patients in the dex group and in the placebo (pgp). the mean(sd) age . ( . ) and egfr . ( . ) in all patients. no significant differences at baseline. in the dex, . % underwent complex surgery vs . % pts in the pgp. the mean(sd) bypass time and aortic clamp was comparable ( ) and ( ) min. the vasopressor requirements methods: nine tracheostomized copd patients ready to be weaned from ventilation were enrolled.for each patient, the sleep architecture was studied by polisomnography (sleep profiler-advanced brain monitoring) performing recordings:basal registration, continuos infusion of propofol or dexmetomidine from pm to am. rass target was - /- . results: the mean dose was . mg/kg/h for propofol and . mcg/ kg/h for dexmedetomidine.quantitative analysis showed, a statistically significant longer total sleep time (tst) and less sleep fragmentation (awakenings/hour) using dexmedetomidine. qualitative analysis showed non statistical differences between the two regimens: longer n and n stage with propofol and a longer n and rem phase with dexmetedomidine. furthermore, a reduced number of dosage adjustment was needed during dexmedetomidine sedation methods: a -year prospective observational cohort analysis was performed. all patients consecutively admitted to the medical or surgical icu or burn unit of a university hospital with an icu-los of >= days were included. qol was assessed at baseline (bl) and at months (m), year (y) and lt (median . years (iqr . - . )) after icu discharge with eq- d and sf- surveys. at lt, questions about daily life were added. in subanalysis, we compared groups (g and g ) based on median icu-los. results: patients ( % men) with a median age of , an apa-che ii score of and a sofa score of at icu admission were included. patients ( . %) were lost to follow-up. median icu-los in the cohort, g and g was (iqr - ), (iqr - ) and days (iqr - ) respectively. during icu stay, g had significantly more and longer need for any type of organ supportive therapy (p< . ) and had higher maximum sofa scores (p< . ). icu, hospital, m, y and lt-mortality rates in the cohort were , , , and % respectively. these rates were similar in g and g and the outcomes were assessed by telephone interviews at month after discharge. factors associated with readmission and post icu mortality are presented as odds ratios. results: during the study period, elderly patients were discharged alive. the follow up was possible for ( . %) patients. predictors of one-month readmission in univariate analysis were coronary disease (p= . ), sapsii (p= . ) and decline in functional status (p= . ). in multivariate analysis ) were the independent predictors of early readmission. mortality rate at month was . %. risk factors of onemonth mortality in univariate analysis were sapsii (p= . ), heart rate at discharge (p= . ), world health organization(who) performance status at discharge (p= . ) and decline in functional status (p= . ). in multivariate analysis p= . ), decline in functional status (or, . ; %ci comorbidities don't have an important impact on short term outcome after critical illness, which is most strongly predicted by severity of illness and physiological reserve at discharge. p characteristics and outcome of elderly patients in intensive care unit i coelho health inequalities & people with learning disabilities in the uk: emerson & baines cipold p comparison of home and clinic follow-up visits after hospital discharge for post-icu patients: a cross-sectional study r rosa , c robinson , p berto , p cardoso , l biason in a post-icu follow-up service which is reference for tertiary hospitals in southern brazil. post-icu patients with a icu stay > h (for medical and emergency surgical icu admissions) or > h (for elective surgical icu admissions) who were discharged alive from the hospital were invited by telephone to participate in a clinic-based multidisciplinary appointment months after icu discharge. home visits were offered to patients who claimed impossibility to attend the clinic appointment due to the severity of their disabilities graph of total mortality in ld vs all patients fig. (abstract p ). graph of admission age in ld vs all patients references p evaluation of family satisfaction instrument in multicultural middle eastern critical care units a p breaking bad news in the emergency department: a randomized controlled study of a training using role-play simulation i bragard , jc servotte , i van cauwenberghe p introduction: this is a randomized controlled study aiming to assess the impact of an e-learning and a -hour role-play training in breaking bad news (bbn) each assessment included a video-recorded role-play with two actors playing the role of relatives, and questionnaires. two blinded experts rated the videos. results: out of participants, % were trainees and % were anaesthesia residents. eg (n= ) and cg (n= ) were not different at baseline on the several variables. there were significant group and time interaction effects. only eg increased their selfefficacy p deficits of end-of-life care (eolc) perceptions among physicians in intensive care units managed by anesthesiologists in germany m weiss , a michalsen , a toenjes p ethicus end-of-life practices (eolp) in worldwide intensive care units (icus)-the ethicus ii study a avidan p multidisciplinary team perceptions about terminal extubation in a teaching hospital in brazil s p changing thoughts about end-of life care in the icu; results of a survey the feel guilty for withdraw therapy in nurses was also significantly decreased in years ( % vs. %, p< . ). conclusions: some of end-of-life thoughts in the icu were shown differences in nurses compared with years ago introduction: workload resulting from in-flight emergencies has not been quantitatively analysed in the literature. for hospitals local to major airports, this may have significant financial implications. methods: review was carried out of all cases admitted to east surrey hospital from gatwick airport over a year period beginning in . data were collected by interrogating the icnarc database. demographics, presenting pathology and length of stay for each patient were recorded. in addition, the cost of care for patients admitted during was calculated using recent median figures for intensive care admission (local ccg rates). results: since , patients were admitted from gatwick airport. this was approximately % of our critical care admissions. the mean (sd) age was . ( . ) years, and the median [iqr] length of stay [ . - . ] days. around % of these patients were non-uk or eu nationals and therefore not entitled to nhs care. reasons for admission included cardiac ( . %), respiratory ( . %), central nervous system ( . %), and gastrointestinal issues ( %). during , patients were admitted resulting in a total of . patient days in critical care. the total cost attributable to this group of patients was calculated to be £ , . conclusions: there is a substantial additional financial burden on hospitals that regularly receive admissions from major airports simply due to their geographical location. there is no additional funding available for providing this service. the pattern of presenting conditions in our population is similar to that seen in previous reports describing inflight emergencies [ ]. given the increasing accessibility of air travel and the economic pressures on healthcare providers, further analysis of the financial impact of this patient group on certain hospitals would be welcome. methods: we developed a monte-carlo simulation [ ] with separate referral rates for emergency, elective, and ventilated patients. bed occupancy is classified according to admission type with a conversion to prolonged ventilated stays at a rate of % [ ]. we used data from our neurointensive care unit to complete the parameters required for the model e.g. beds and , referrals/day. outcome measures were bed occupancy, and failed admissions. we tested two scenarios: increased referral rate ( . /day), and increasing to beds. results: the model simulated our intensive care where we have a high occupancy rate. increasing referral rate led to a consumed icu and an increase in failed admissions (fig. ) . lastly, increasing bed numbers eased pressures with fewer failed admissions. conclusions: we recommend a personalised icu monte-carlo population model for specialist units for a more accurate representation of icu bed occupancy. these icu specific models should be more useful for predicting staff, bed and financial requirements in specialist units where healthcare resources are changing e.g. increasing geographical referral radius. conclusions: better patient flow increased occupancy and standards. staff education and clear protocols are needed to improve patient booking and efficiency. assess stress levels and causes of stress among doctors and nurses at university hospital lewisham and queen elizabeth hospital woolwich. we surveyed staff using uk health and safety executive's management standards (hsems), a -question validated tool which identifies stressful work conditions requiring intervention. methods: we conducted an anonymous survey of doctors and nurses working in intensive care, accident and emergency (a&e), acute medicine, anaesthetics and surgery over six weeks. results were analysed using the hsems analysis tool and broken down into seven areas: job demands, managers' support, peer support, relationships, role, level of control and possibility of change. each area was scored from - ( represents lowest stress). we compared the trust's results against national standards. results: healthcare professionals completed the survey. intensive care had the lowest stress levels and scored above average in all areas (n= , mean . , s.d. . ). this was followed by a&e (n= , mean . , s.d. . ), anaesthetics (n= , mean . , s.d. . ), surgery (n= , mean . , s.d. . ) and acute medicine (n= , mean . , s.d. . ) which had the highest stress levels. when compared to hsems targets peer support exceeded national standards. however, there is a clear need for improvement in staff's ability to control and change their working environment. conclusions: stress levels on intensive care were reassuringly low when compared to other departments as well as national standards. we identified areas that need improvement and with the support of hospital management we will initiate hsems-validated measures to reduce stress. p tools for sepsis-associated mortality in hematological patients and should be studied in larger cohorts.conclusions: our results present clinical data of protocolized pbto -targeted therapy and show that there is room for further optimization. a larger cohort with predefined interventions is needed to proof the effect on longterm outcome after sah. impact of phone cpr on rosc outcome a giugni , s gherardi , l giuntoli introduction: early cardiopulmonary resuscitation (cpr) improves survival in out-of-hospital cardiac arrest, and phone-cpr instructions can increase the number of victims receiving cpr before emergency medical service (ems) arrival. little is known about the impact of cpr phone instructions on the outcome of patients (pts) with return of spontaneous circulation (rosc). the target of this study is to investigate the impact of phone instructions on mortality, and on neurological outcomes of patients who survived an out-of-hospital cardiac arrest. methods: we enrolled pts admitted to icu after rosc following out-of-hospital-cardiac-arrest, from / / to / / ; pts younger than , in-hospital cardiac-arrest-victims, pts who underwent cardiac arrest in health facilities, and missing data records were excluded. written informed consensus was obtained for all pts during follow up. data about comorbility, mortality, neurologic outcome, cpr timing according to utstein style, complications in icu, metabolic state on er admission, were collected. study population was divided into two groups for statistical analysis: pts with immediate cpr guided by phone instructions (phone-cpr group) and those who did not underwent immediate cpr by laic bystanders. data were extracted from icu, ems databases and registered ems phone calls. results: pts met study criteria. phone cpr were given in cases, % of the whole study population. results are summarized in tables and conclusions: phone-cpr significantly reduced cpr-free interval. it correlates with a significative increase in shockable rhythms on ems arrival. there is no significative reduction in mortality and in disability, even if a decrease trend can be observed. phone-cpr seem to be a promising, effective and easy to use tool to improve survival and disability in rosc, and should be widely applied. early hemodynamic complications in cardiac arrest patients-a substudy of the tth- study j hästbacka introduction: our aim was to determine the incidence and severity of hemodynamic complications during therapeutic hypothermia and analyze whether these complications can be predicted from data available on admission. methods: this is a substudy of the tth- study, where cardiac arrest (ca) patients were randomized to receive therapeutic hypothermia treatment for either or h [ ] . hypotension within four days from admission was recorded and defined as mild, moderate, severe or circulatory failure. arrhythmias were recorded and classified as mild, moderate or severe. we calculated the incidence and distribution of severity of the events. we used multivariate logistic regression analysis to test association of admission data with any hypotension or any arrhythmia. results: of all patients, . % had hypotension which was mild in . %, moderate in . %, severe in . % cases. . % had circulatory failure. an arrhythmia was present in % of patients. of these, . % were mild, % moderate and . % severe. bradycardia (n= ), new ca (n= ) and circulatory shock (n= ) were hemodynamic reasons for preterm rewarming. in multivariate analysis age (p= . , or . ) and admission map (p= . , or . ) were significantly associated with hypotensive complications. only use of mechanical compressions was significantly associated with risk for arrhythmia (p= . , or . ). conclusions: hypotension and arrhythmias were frequent in cardiac arrest patients during days - from admission, but mostly mild or moderate in severity. age and admission map were associated with hypotension. only the use of mechanical compressions was independently (negatively) associated with arrhythmias. introduction: in a retrospective study from the pittsburgh clinic, which analyzed survival data from patients admitted to a hospital with a cardiac arrest outside the hospital, it was found that patients with opioid overdose showed significant improvements in neurological status when discharged from the hospital compared with patients who did not receive opioids [elmer j. et al., ] . methods: after local ethic committee approval case-records of patients with cardiac arrest and subsequent resuscitation for the period - in the clinic of traumatology and orthopedics in astana were analyzed. criteria for inclusion in the study were hospital cardiac arrest, trauma to the musculoskeletal system. results: out of case-records, ( . %) patients with out-ofhospital cardiac arrest were excluded. among all hospital stops of blood circulation, we found only successful cpr ( . %). among the patients who were successfully resuscitated, groups were identified: i - patients ( %) who received ketamine or/and opioids before the blood circulation stopped ( - minutes); ii - patients ( %) who did not receive these medicines. the mean age in group i of patients was . ± . years, in group ii - . ± . years (p> . ). patients of the second group had an average life expectancy of . ± . days, with a maximum postresuscitation life of days. patients of the first group were in the hospital for . ± . days (p < . ), with a maximum period of days. in the first group, the final neurologic evaluation according to the glasgow scale was . ± . points, while in the second group it was . ± . points (p < . ). conclusions: a retrospective analysis revealed a better survival and neurological outcome in patients who received ketamine or/and opioids before circulatory arrest.introduction: the revised organ transplant law was enacted in japan in . under the revised law, it is now possible to donate organs with the consent of the family even if the intention of the potential donor is unknown. organs from brain-dead children under the age of can also be donated. methods: the aim of this study was to assess how to provide prompt transplant medical care and improve the donor's condition. this was achieved by clarifying the problems encountered in the process leading to brain-dead organ transplantation at our institute before and after the enactment of the revised organ transplant law. there were cases of organ donation at our institute from january to june . among them, the background factors of cases leading to organ donation were examined. results: the causes of the brain-dead condition were cerebrovascular disease (n = ; subarachnoid hemorrhage, intracerebral hemorrhage), trauma (n = ), suffocation (n = ), cardiopulmonary arrest on arrival (n = ), suicide by hanging (n = ), cardiomyopathy (n = ), and lethal arrhythmia (n = ). the organs donated for transplantation were kidneys, eyes, lungs, livers, hearts, and tissues (i.e., heart valve, bone, and skin). the time lapses were as follows. the number of days from informed consent to family acceptance was . days before the enactment of the revised organ transplant law and . days after the revision. the number of days from informed consent to organ removal was . days before the revision and . days after the revision. even after the enactment of the revised organ transplant law in japan, it still takes about days from informed consent to organ removal, with no current initiatives to shorten the time to organ removal. conclusions: although years have passed since the enactment of the revised organ transplant law in japan, there are still administrative and management problems that need to be addressed to achieve optimal organ transplantation. the financial impact of proximity to a major airport on one critical care unit introduction: in septic patients, increased plasma levels of cell-free hemoglobin (free-hb) are associated with a reduction of perfused vessel density (pvd) of sublingual microcirculation and to adverse outcomes caused by hemoprotein-mediated lipid peroxidation. recent studies show that acetaminophen protects from damage due to lipid peroxidation in sepsis [ ] . the aim of this study is to detect changes in sublingual microcirculation after the infusion of a standard dose of acetaminophen in febrile septic patients. methods: prospective observational study on adult septic patients admitted to our intensive care unit. pre-infusion (t ), minutes (t ) and hours (t ) after the end of the infusion of acetaminophen, sublingual microcirculation was assessed with incident dark field illumination imaging; vital signs, plasma levels of acetaminophen and free-hb were assessed. results: preliminary descriptive analysis on the first patients shows a median sequential organ failure assessment (sofa) score of (interquartile range iqr - ) and baseline temperature of , c°( iqr . - °c). an increase of the proportion of perfused vessels (ppv) was evident both at t and t ( introduction: in common sedation is required during mri for adult uncommunicative patients or those with different psychiatric disorders [ ] . although it can be challenging to obtain the deep sedation level required to prevent the patient's movement while maintaining respiratory and hemodynamic stability. limited access to the patient may pose a safety risk during mri. objectives: to compare efficacy and safety of dexmedetomidine sedation versus propofol during mri in adults.methods: this prospective randomized study was conducted at department of anesthesiology and intensive care at postgraduate institute of bogomolets national medical university (kyiv, ukraine) during - . uncommunicative conscious patients with acute ischemic stroke were included in the study and randomly allocated to groupsdexmedetomidine (d) and propofol (p). the sedation goal was the same in the both group (rass to - ). patients in group d receive dexmedetomidine infusion in dose . conclusions: in this prospective randomized study dexmedetomidine comparing to propofol was associated with higher sedation quality and lower incidence of complication during acute ischemic stroke patients sedation for mri. the usefulness of dexmedetomidine after lung transplantation in intensive care unit. introduction: dexmedetomidine (dex) showed some advantages in the sedation of patients in intensive care unit (icu) [ ] . other studies described efficacity of dex in icu delirium [ ] . the aim of this study was to evaluate the efficacity and safety of dex after lung transplantation in icu. methods: we conducted a prospective monocentric study in our surgical icu between november at november . in the first part of the study (november at november ), lung recipients did not received dex; in the second part of the study dex was used for the sedation in mechanically ventilated patients after lung transplantation. we compared the duration of mechanical ventilation in the two groups and the occurence of adverse effects. results: in total lung recipients were enrolled. there was no difference between the two groups in demographic data, one or double-lung transplants, the cause of lung transplantation and the use of epidural infusion. in the dex group, mechanical ventilation support was hours versus . hours in the other group (p= . ). there was no difference between delirium in the two groups ( / , p= . ). the occurence of adverse events like hypotension and bradycardia was significantly higher in the dex group ( / for hypotension, p= . ; / for bradycardia, p= . ). conclusions: the use of dex after lung transplantation in icu was not more efficience for the mechanical ventilator weaning. lung recipients delirium was significantly the same in the two groups. the most notable effect was the occurence of bradycardia and hypotension in the dex group.introduction: dexped evaluated the impact of a prolonged exposure (>= hours) to dexmedetomidine on the duration of invasive mechanical ventilation (imv), length of picu and hospital stay and use of other sedative agents. methods: dexped is a retrospective cohort study that included patients aged to years, admitted to the picu of the montreal children's hospital between november st and april th , requiring imv and sedative agents for >= hours. patients exposed to dexmedetomidine during imv (n= ) were compared to non exposed patients (n= ) using a propensity score analysis ( : ratio). , and received more opioids and benzodiazepines. however, a secondary analysis redefining exposure as initiation of dexmedetomidine within the first hours from intubation suggested that exposure was associated with a greater short-term probability of extubation, although this study was not powered to perform this analysis. conclusions: dexmedetomidine was associated with a longer duration of imv. however, the association was inversed when patients received dexmedetomidine as a primary sedative agent. it is uncertain whether this difference of associations is due to immortal time bias or clinical features. timing of initiation of dexmedetomidine in relationship to other sedatives may impact patient outcomes and should be considered in the planning of future trials. is an α -agonist which has been increasingly used for analgosedation. despite of many papers published, there are still only a few concerning the pk of the drug given as long-term infusion in icu patients. the aim of this study was to characterize the population pharmacokinetics of dex and to investigate the potential benefits of individualization of drug dosing based on patient characteristics in the heterogeneous group of medical and surgical patients staying in icu. methods: all the subjects were sedated according to modified ramsay sedation score of - . blood samples for dex assay were collected on every day of the infusion and at the selected time points after its termination. the dex concentrations in the plasma were measured using lc-ms/ms method. the following covariates were examined to influence dex pk: age, sex, body weight, patients' organ function (sofa score), catecholamines and infusion duration. non-linear mixed-effects modelling in nonmem (version . . , icon development solutions, ellicott city, md, usa) was used to analyse the observed data. results: concentration-time profiles of dex were obtained from adult patients ( table ). the dex pk was best described by a twocompartment model (fig. ) . the typical values of pk parameters were estimated as l for the volume of the central compartment, . l for the volume of the peripheral compartment, . l/h ( . ml/min/kg for a kg patient) for systemic clearance and . l/h for the distribution clearance. those values are consistent with literature findings. we were unable to show any significant relationship between collected covariates and dex pk. conclusions: this study does not provide sufficient evidence to support the individualization of dex dosing based on age, sex, body weight, sofa, and infusion duration. seems to reduce the wakefulness time and the sleep fragmentation but, while we haven't found differences in sleep architecture using dexmedetomidine or propofol. introduction: the early mobilization program during intensive care hospitalization presents numerous benefits related to the outcome of the patient. the objective of this study is to evaluate the safety of the implementation of an early mobilization protocol within the first hours of admission and its impact on high functional status of the icu. methods: retrospective study, from march to may , evaluating patients admitted to the neurological icu, assessing the hemodynamic, respiratory and neurological variables in patients submitted to the early mobilization program, consisting of progressive therapeutic activities, including sedestation and orthostatism assisted on the board and evaluated the impact on the functional status/degree of high muscle strength of the icu. results: from march to may , , patients were admitted to a neurological intensive care unit, of whom , were included in the early mobilization program. the mean age of the patients was . years, with saps of . points (estimated mortality risk of . %) and real mortality of . %. during the program, % presented clinical instability, which was promptly reversed in all situations. ninety-one percent of the patients presented maintenance or gain of muscle strength/functional status. conclusions: the application of an early mobilization program within hours of patient admission was shown to be safe, positively influencing the rehabilitation of neurological patients. introduction: given the worldwide rapidly aging of the population, the demand of critical care for elderly is increasing. data on short -term outcomes of elderly patients after icu discharge are sparse. the objective of our study was to assess short term outcomes of elderly after icu discharge and their potential risk factors.introduction: patients aged years or older presently account for approximately - % [ ] of all intensive care unit (icu) admissions in europe. the major challenge nowadays is to admit those elderly patients who will benefit from icu treatment. the objective of this study is to describe the characteristics and outcomes of patients >= years old admitted to the icu. methods: retrospective observational study of all patients aged >= years admitted for > h in . demographic data, admission diagnosis, apache ii and saps ii scores, use of icu resources and mortality were collected. results: patients ( %) were included, with a mean age of , . female gender was more prevalent ( . %). mean length of stay was , days with mean saps ii and apache ii scores of , and , respectively. the most prevalent type of admission was medical, , % (n= ) and from these the main reasons for admission were respiratory disease (n= ; , %) and sepsis (n= ; %). icu mortality rate was , % (n= ), whereas -month mortality was , % (n= ).survival rate was often related with cardiovascular ( [ , %], p<. ) and respiratory diseases ( [ %], p=. ), whereas nonsurvivors were admitted due to sepsis and neurologic causes. mortality rate was independent from the mean length of stay, noninvasive ventilation and renal replacement therapy, but dependent for previously comorbidities. mechanical ventilation was an independent predictive factor of icu mortality (p<. ) and -month mortality (p=. ). conclusions: nearly % of patients aged >= years were discharged alive from icu, and less than % survived months after icu admission.our study revealed a better prognosis for admissions due cardiovascular and respiratory diseases. efforts should be done to identify earlier septic and neurological patients that benefit icu treatment, and reevaluate the critical patient pathway, in this special population.conclusions: more than % of icu-survivors returned to work. overall, the chance of return to work within two years was independent of the number of organ support therapies in patients with at least one organ support therapy. however, in subgroups, the chance of return to work decreased with increasing number of organ-support therapies. factors associated with non-return to work among general icu survivors: a multicenter prospective cohort study r rosa introduction: critical care patients may develop long-term health problems associated to their illness or icu treatments, which may affect their work capacity. unfortunately, studies evaluating the impact of critical illness on work-related outcomes are scarce.therefore, we aimed to investigate factors associated with non-return to work among icu survivors. methods: a prospective cohort study involving icu survivors of brazilian tertiary hospitals was conducted from may to august . patients with a icu stay > h (for medical and emergency surgical icu admissions) or > h (for elective surgical icu admissions) who were discharged alive from the hospital were evaluated through a structured telephone interview months after discharge from the icu. a stepwise multivariate poisson regression analysis adjusted by age, gender and years of education was used to evaluate the association of sociodemographic-and icu-related variables with nonreturn to work. results: in total icu survivors completed the -month follow-up. of these, ( %) were working before icu admission. only of patients ( %) returned to work within the first months after discharge from the icu. percentage of risk of death at icu admission (relative risk [rr], . ; % confidence interval [ci], . - . ), decrease in physical functional status in relation to the pre-icu period measured by barthel index (rr, . ; % ci, . - . ), not having a introduction: the aim of this study was to assess the accuracy of physician's prediction of hospital mortality in critically ill patients in an intensive care unit (icu) scarcity setting. methods: prospective cohort of acutely ill patients referred for icu admission in an academic, tertiary hospital in brazil. physicians' prognosis and other variables were recorded at the moment of icu referral. results: there were analyzed referrals. physician's prognosis was associated to hospital mortality. there were ( . %), ( . %) and ( . %) deaths in the groups ascribed a prognosis of survival without disabilities, survival with severe disabilities or no survival, respectively (p< . ) (fig. ) . sensitivity was %, specificity was % and the area under the roc curve was . for prediction of mortality. after multivariable analysis, severity of illness, performance status and icu admission were associated to an increased likelihood of incorrect classification, while worse predicted prognosis was associated to a lower chance of incorrect classification. physician's level of expertise had no effect on predictive ability. conclusions: physician's prediction was associated to hospital mortality, but overall accuracy was poor, mainly due to low sensitivity to detect mortality risk. icu admission was associated to increased incorrect classification, but there was no effect of physician's expertise on predictive ability. what are physicians in doubt about? an interview study in a neuro-intensive care unit. introduction: inescapable prognostic uncertainty, lack of decisionmaking capacity, risk of death or disability and long recovery trajectories complicate decision-making after traumatic brain injury. methods: to elicit experienced physicians' perspective we interviewed neurosurgeons, intensive care-and rehabilitation physicians from oslo university hospital about being in doubt about whether to offer, continue, limit or withdraw life-sustaining treatment and on how such cases were approached. interviews were audiotaped and transcribed verbatim, coded and analysed using systematic text condensation by a clinician (ar) and a medical ethicist (rf). results: the difficulty of decision-making when there is prognostic uncertainty was acknowledged, leading to adaptive approaches; willingness to change and adjust plans along the way. to have access to different opinions within the physician group was seen as constructive. time-critical decisions were based on team discussions and physician's discretion. none-time critical decisions were reached through a process of creating common ground between the medical team and family. themes physicians where in doubt about or expressed different opinions towards: ) appropriate aggressiveness of treatment in a given situation. ) if and when to initiate discussions on appropriateness of treatment. some believed that even addressing the issue in young patients or if small improvements were seen was inappropriate due to the possibility of late recovery. physicians questioned the value of previously expressed patient's wishes in this context. ) optimal timing and type of decisions. the need for nuanced individualized plans was recognized. to have a plan as opposed to just "wait and see" was seen as especially important in medical unstable patients. conclusions: physicians expressed different views on appropriateness and optimal timing of level of care discussions and decisions in traumatic brain injury. a need for a more structured approach was exposed. fig. (abstract p ) . association of physician's prognosis with hospital mortality (p< . ).introduction: this cross sectional study was designed to investigate the level of family satisfaction in intensive care units in a tertiary hospital in the united arab emirates (uae), which is a multicultural society methods: family members of patients who were admitted to intensive care unit for more than hours or over were included in the study. families were approached with a validated fs -icu family satisfaction survey questionnaire [ ] . one hundred questionnaires were collected over a period of months from january to march in our pediatric medical surgical and cardiac, adult cardiac and adult medical/surgical intensive care units. results: the overall level of satisfaction rate was comparable to other high-income and developed countries with total satisfaction score, medical care score and decision making score of . ± . , . ± . , and . ± . respectively (table ) . conclusions: this is the very first study from the uae demonstrating a high level of patient family satisfaction in both adult and pediatric intensive care units. this study also highlighted areas where further improvement needed to occur.introduction: in order to apprehend the structural aspects and current practice of end-of-life care (eolc) in german intensive care units (icus) managed by anesthesiologists, a survey was conducted to explore implementation and relevance of these items. methods: in november , all members of the german society of anesthesiology and intensive care medicine (dgai) and the association of german anesthesiologists (bda) were asked to participate in an online survey to rate items. answers were grouped into three categories: category reflecting high implementation rate and high relevance, category low implementation and minor relevance, and category low implementation and high relevance. results: five-hundred and forty-one anesthesiologists responded, representing just over / of anesthesiology departments running icu's. the survey revealed new insights into current practice, barriers, perceived importance, relevance, and deficits of eolc decisions. only four items reached >= % agreement as being frequently performed, and items were rated "very" or "more important". items attributed to category , to category , and to category , representing a profound discrepancy between current practice and attributed importance. items characterizing the most urgent need for improvement (category ) referred to desirable quality of life, patient outcome data, preparation of health care directives and interdisciplinary discussion, advanced care planning, distinct aspects of changing goals of care, standard operating procedures, implementation of practical instructions, continuing eolc education, and inclusion of nursing staff and families in the process. conclusions: the survey generated awareness about deficits in eolc matters in critical care. consequently, already available eolc tools have been made available through the website of the german society of anesthesiology and intensive care medicine (dgai): http:// www.ak-intensivmedizin.de/arbeitsforen.html.introduction: this study evaluated differences in eolp after years in european icus that also participated in the ethicus i study [ ] . methods: all previous ethicus i centers were invited to participate in the ethicus ii study. consecutive admitted icu patients who died or had treatment limitations during a month period from . . to . . were prospectively studied. previous eolp and region definitions were used [ ] . eolp in the different regions of the ethicus i study [ ] were compared to the same icus in the ethicus ii study. results: of the original icus participated again in this study. figure shows the differences in eolp by region. figure notes differences in patient mental competency at the time of decision, information about patient's wishes and patient discussions in both ethicus studies. conclusions: changes included less cpr (especially in the south) with more withholding and withdrawing therapies. there was a greater number of competent patients with discussions and knowledge of their wishes.introduction: palliative extubation is performed in patients with terminal ilnesses in which mechanical ventilation might prolong suffering. even though the procedure involves nurses, respiratory therapists and doctors, some professionals feel unconfortable performing a palliative extubation. the concept of withdrawing life support can be easily confounded with euthanasia, specially in low income countries, where there is usually less education on palliative care. methods: a questionary containing open ended questions concerning a hypotetical case of intracerebral hemorrhage and prolonged coma, with potential indication for palliative extubation was applied to members of an emergency department intensive care unit staff ( doctors, nurses, respiratory therapists (rt). results: more than half of the professionals ( %) had never participated in a palliative extubation. four professionals ( %) believed palliative extubation is euthanasia. when asked about their own preferences in such a situation, only two ( %) would like to be tracheostomized. symptoms anticipated by most professionals were dyspnea and respiratory secretions. four ( %) would feel very uncomfortable performing palliative extubation because they either felt to be killing the patient or unable to manage symptoms conclusions: most professionals in this tertiary emergency intensive care unit never witnessed a palliative extubation. however, most of believe this procedure is beneficial. some still cannot understand the difference between palliative extubation and euthanasia. education in palliative care and withdrawal of life support can be helpful to clear concepts and relieve moral distress in the team. key: cord- -t x gknd authors: nan title: abstract presentations from the aabb annual meeting san diego, ca ctober ‐ , date: - - journal: transfusion doi: . /trf. sha: doc_id: cord_uid: t x gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks , , , and following index donations from donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by months. urine and saliva detection decreased significantly after weeks and was undetectable by months. of donors who were enrolled in the acute pre-seroconversion stage of infection % ( / ) developed multiple zikv related symptoms week post index donation, compared to only % ( / ) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* , yuelong guo , ritchard g. cable , joseph e. kiss , michael paul busch , grier page , stacy endres-dighe , steve kleinman , simone glynn , alan mast and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) . american red cross, rti international, american red cross blood services, blood systems inc., blood systems research institute, university of british columbia, nih/ nhlbi, blood research institute, nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over , donors were enrolled from us blood centers for ferritin testing. the study population was enriched for racial minorities [ african-american (aa), asian (as), hispanic (hisp)] and for "super donors" ( , who had completed donations in two years without low hemoglobin deferral). the minority donors and the remaining non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin < ng/ml) and low ferritin (lf, ferritin < ng/ml). results/findings: across all subjects, % had ais and % had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed % % decreased risk for ais compared to nhw, while hisp donors had % higher risk. daily use of exogenous iron reduced risk for lf and ais by to %, respectively, while the estimated benefit from less-than-daily use was lower ( to % protection). regular use of antacids was associated with a % or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by % - %, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean ( sd) *p < . compared to batf / , ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to % of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d and d of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration ( % on d vs % on d of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized ( %) and macrophage-depleted ( %) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than % at h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at h posttransfusion. at h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* , amanda l richards and krystalyn e hudson . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod otii mice are predisposed to have autoreactive cd t cells. study design/method: four cohorts of hodxotii f mice ( - mice/ cohort) were bled monthly for months to assess for autoab production. peripheral rbcs were stained with anti-complement (c ) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd and ter to assess for the presence of rbc progenitors. statistical analysis between hod otii autoab vs. hod otii autoabvs. hod -otii was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd t cells were not deleted in the thymus of hod otii mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod otii . however, as they aged, - % of hod otii were positive for rbc autoab by months. thereafter, $ % of the autoab mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in of the cohorts, - % of autoab mice were female. hod otii autoab mice also had enlarged spleens compared to hod otii autoaband hod -otii mice ( . g vs. . g and . g, resp., p< . ). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd hi ter inter ) were observed in the spleens of hod otii autoab mice but not in hod otii autoaband hod -otii ( . % vs. . % and . % resp., p< . ). moreover, autoab and c deposition were found ( . - % and - %, resp.) on ter rbcs in all of the hod otii autoab mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b -a a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards , christopher a tormey and krystalyn e hudson* . background/case studies: red blood cell (rbc) alloimmunization occurs in up to % of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c bl/ (b ) mice were treated with pbs, or anti-ly g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at - hours post-transfusion. anti-hod alloantibody generation was assessed days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n ), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n ). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls ( / experiments, p< . ). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l ), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p< . ); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b -a b cxcr pd and ccr expressions characterize responders to rbc immunization benoît vingert* , , , marie tamagne , , , sadaf pakdaman , , , anoosha habibi , , , philippe bierling , , , , , rachid djoudi and france pirenne , , , . efs ile de france, laboratory of excellence gr-ex, imrb u -eq , ap-hp, universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd t cells have a th profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd t cells with a cxcr pd phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr pd hi profile, with a differentiated expression of ccr . ccr is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr and cxcr can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr pd lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr pd cells were compared in groups of transfused sickle cell patients : alloimmunized (n ) and non-alloimmunized patients (n ). the analysis was also performed in non-transfused healthy controls (n ). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr pd hi subpopulation expression was identical between transfused groups and controls. ccr and cxcr expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr expression was very strong independently of the expression of pd . in the aim to determine the help of the circulating cxcr pd cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr pd subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr pd cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr on circulating cxcr pd cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr pd profile and the ccr expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* , ashley bennett , kathryn girard-pierce , connie arthur , amanda mener , patty zerra , christopher a tormey , jeanne hendrickson and sean stowell . emory university, yale-new haven hospital, yale university, emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod kel). to examine the role of cd t cells, pic/kel primed b recipients were cd t cell depleted prior to transfusion. in addition, b recipients were adoptively transferred with cd t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < . ); pic/kel primed recipients transfused with (hod kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < . ) and transfer of cd t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < . ). conclusion: these results demonstrate that cd t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c bl/ ) mice or oti mice, whose cd t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after , , , , and hours and on days - . results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after hours and peaking at hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is - % versus > % in wt recipients at hours (p< . ), whereas transfusion of wt platelets into either oti or wt recipients is approximately % at hours after transfusion. adoptive transfer of oti cd t cells into wt mice recapitulates the effect, with significant mova platelet clearance at hours compared to wt platelet clearance (p< . ). conclusion: this work extends the ability of cd t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c bl/ mouse blood and treated with uv r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv r treated wbc-rich prp, or uv r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c bl/ donor blood. a second transfusion of untreated wbc-rich c bl/ prp was given weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p< . ) or necrotic (p< . ) wbcs, but not those given uv r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c bl/ wbcs were reduced in recipients of either uv r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so ) of venous blood is generally assumed to be around - % as measured from a central venous line. however, a recent investigation of so levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so distribution (mean . % . % [yoshida et al. ; blood transfusion , ] . the present study was undertaken to determine the distribution of so in lr-rcc produced at a medium-size blood center using a novel non-invasive so probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so levels. study design/method: the so from units of lr-rcc were examined on five consecutive days representing % of the collected units during the period at a regional blood center where all the units were processed at room temperature within hours of blood collection. so was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a u ; pendar technologies, cambridge ma). in addition to so , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n ) from human volunteers were stored in as- under normoxic, hyperoxic, or hypoxic conditions for up to days (so ranging from < to > %) prior to uhplc-ms metabolomics analyses in presence of c, n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so carried out non-invasively at a blood center yielded a similar wide distribution as previous study from units of lr-rcc procured and sampled invasively within hours after blood collection [yoshida ibid]. the shape of the so distribution appeared near normal with the mean of . % . %, median . %, range < % to > % and inter-quartile range (iqr) of . %- . %. male donors showed higher so compared to female donors (p< . ). no correlations were observed between so levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so levels ameliorate the energy and oxidative metabolic lesion. lower so levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so levels was observed from lr-rcc manufactured at a blood center using -hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a -fold increase in the absolute lymphocyte count (ko . . x /l vs. wt . . x /l, p . ), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko . . fl vs. wt . . fl, p . ). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd foxp regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd foxp tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd foxp tregs derived exosomes and their functions involved in cd tregs mediated immune-modulation were seldom reported. study design/method: cd t cells were freshly purified from pbmcs, cultured with anti-cd /cd antibody packaged beads and il- , and then polarized with tgf-b and rapamycin into cd foxp treg cells. the harvest cells were co-cultured with cd /cd beads stimulating cd cd effector cells in the transwell plate. the supernatant derived from cd tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd , cd , tsg and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir- , let- b, let- d were measured by qpcr. the precipitated exosomes were further purified by cd immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd treg cells could suppress the proliferation of effector cells with a small decline (p> . ), which means some non-contact factors involved in the cd treg mediated immune modulation. a total number of . . / cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle - nm in diameter ( . . nm by nta). cd and cd were expressed on these background/case studies: regulatory t cells (tregs), containing cd and cd subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd foxp regulatory t cells (ntregs) in inflammation conditions (including instability of foxp , conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd regulatory t cells in inflammation and transfusion. study design/method: human cd tregs were induced with tgf-b and rapamycin from cd t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd tregs when encountering with inflammation were test by foxp expression, th and th cells conversion in inflammations conditions (il tgf-b il il and il tgf-b il b il ) on day , day and day . in vivo, cd tregs were transfused into cia mice and then their survival in mice and foxp express were evaluated to reveal the stability of cd tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd tregs when induced factor tgf-b and rapamycin were removed by testing the foxp expression on day , day and day . results/finding: ex vivo induced human cd treg were foxp ( . . %) and did not secret il a (both in supernatant and % of cells). foxp express in cd tregs were maintained after induced factor tgf-b and rapamycin were removed on day , day and day . in vitro, foxp , il and ifn-c expression has no significant difference when compared with controlled tregs on day , day and day and did not secret il a when encounter with inflammation conditions (il tgf-b il il and il tgf-b il b il ). in vivo, cd treg cells were transfused into cia mice on the peak of disease onset ( days after the first collagen immunization, has inflammation condition in vivo) to test cd tregs survival. cd tregs were found in cia mice foot ( . . %), blood ( . . %) and spleen cells ( . . %) hours after transfusion and their % of foxp were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd tregs are stable in inflammation and transfusion and can maintain foxp expression when induced factor were removed, these make cd treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd /cd /cd expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p s k and its downstreanm proteins, especially the protein s , which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s related protein translation inhibition. xiaoyun fu* , , mikayla anderson and james c zimring , . bloodworksnw research institute, university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day (one day past their expiration). bioactive lipids including common fatty acids, oxylipins, and lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in stored rbc units. for example, arachidonic acid (aa) ranges from . - . mm, linoleic acid (la) ( . - mm), dihomo-c-linolenic acid (dgla) ( . - . mm), eicosapentaenoic acid (epa) ( . - . mm), docosahexaenoic acid (dha) ( . - . mm), and alpha-linolenic acid (ala) ( . - . mm). ten oxylipins including hetes, hodes, and dihomes, and lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of analytes quantified, showed a significant difference in concentration among different blood types by one-way anova testing (fdr< . ). the ab rh blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh ). the fold increase of o rh-/o rh among pufas ranges from . to . , suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna- was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna- targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps ( nm) surrounded by a thick fluorescent silica shell ( nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r rnaprobe . and r dnaprobe . ) with mirna- concentration, down to a -nm limit of detection. hybridization assays in % human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in % human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. . . a . . . . a . . . . a . . total atp,lm/ghb . . a . . . . a . . . . a . . extracellular lactate,mm a extracellular glucose,mm a a a extracellular na ,mm a extracellular k ,mm a a a a p< . , paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n ) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by % ( / , ) compared to the previous year without pathogen inactivation ( / , , p . , chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased ( / , vs / , ). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count ( . x ) than unaffected controls ( . x ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above . x . in vitro quality of single dose amotosalen/uva treated platelets in % plasma/ % pas- after days of storage crystal stanley , marguerite kelher , nero evero , melissa vongoetz , betsy donnelly and anna erickson* . belle bonfils memorial blood center, university of colorado, cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas- to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct ) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas- and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in % plasma/ % pas- , collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, . . platelets in ml, were collected on the trima apheresis platform in % plasma/ % pas- . a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n , were ml (t) and ml (c) with doses of . . (t) and . . (c). all pc were stored under the same conditions and evaluated on day and day for physical/metabolic characteristics. results/finding: on days and all t and c pc had ph c ! . . the dose recovery for t was % %. on day , t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table ) . conclusion: trima pc in % plasma/ % pas- treated with the inter-cept blood system for platelets using the sv set and stored for days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan , , yang yu , li-ping sun , shu-fang wang , rui wang , lei-ying zhang and deqing wang* . the department of blood transfusion, the pla general hospital, the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d , d and d . we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in % final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. levels of k and lactic acid (la) were tested using automatic biochemical analyzer. k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. treated hips-cms with d ssrbcs, d k and cell culture media for h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: d ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d ssrbcs stop beating, but beating patterns restored at h. hips-cms treated with d ssrbcs stop beating, and beating patterns did not restored at h. levels of k and la in ssrbcs changed most obviously. only d k solution made hips-cms stop beating and can restore in h; d k, d k and la solution did not influence the beating pattern in at the end of the treatment for h, hips-cms treated with d ssrbcs show obvious shrinkage. at the end of the treatments for h, cells treated with d k and d ssrbcs both show obvious shrinkage, the shrinkage in d ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. gene expression array results show a total of genes were differentially expressed in d ssrbcs group compared with naive group. there was no consistent separation within the d k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. further study should be applied to signal pathways on ssrbcs induced cytotoxicity. large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as- as compared to sagm. the presence of citrate in as- seems to be necessary to prevent hemolysis of thawed cells. during storage in as- , atp and , -dpg levels rapidly decline. recently developed additive solutions like pag m and as- have shown to better maintain , -dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag c in which the mannitol of pag m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at - c in pag c. study design/method: leukoreduced rcc (n ) in pag c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at - c. on day , rccs were glycerolized using acp (haemonetics v r , braintree, ma) to a final concentration of % (w/v), frozen and stored for at least two weeks at - c. after thawing and deglycerolization using acp , rcc were resuspended in pag c. during storage at - c, stability (hemolysis), atp and , -dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n ) resuspended in or sagm (n ). results/finding: pre-freeze storage in pag c resulted in increased , -dpg levels at day as compared to storage in sagm, resp. . . mmol/g hb and . . mmol/g hb. hemolysis during post-thaw storage in pag c remained below . % for days and was comparable with storage in as- . in sagm, hemolysis remained below . % for days. during the first weeks of post-thaw storage in pag c, both atp and , -dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag c showed significantly higher atp and , -dpg levels compared to as- or sagm. while in sagm and as- , , -dpg levels were undetectable after days post-thaw storage, in pag c, , -dpg levels only decreased to . lmol/g hb after days of storage. conclusion: pre-freeze storage in pag c resulted in increased , -dpg levels. as compared to as- , post-thaw storage in pag c showed comparable hemolysis while atp and , -dpg levels were much better maintained. based on a maximum allowed hemolysis of . % and an atp content of > . mmol/g hb, thawed rcc can be stored at - c for days in pag c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold ( c, c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n - ) in % iso using a trima or in % int using an amicus and stored for days at rt and c. samples were tested on day (baseline, bl), , , and of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at % hct. labeled blood was perfused through microfluidic channels (fluxion) coated with ug/ml type- collagen at s - shear rate. images were acquired every sec for min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p< . . results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day of storage compared to bl (bl: . . %, rt: . . %; p< . ). c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day (p . ) and compared to c-int by day (p< . ). conclusion: our work suggests that c storage of plts collected with a trima ap system in iso for up to days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at c. these results are surprising since both c-int and c-iso have been shown to express similar levels of cd p, pac- , and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table . comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after -week storage between unirradiated and irradiated groups (n ) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone ( mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin ( mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th cells and increase in cd regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th ,th ,tfh and tfr cd subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* , sarah kloss , sara crew and sandra j nance . american red cross, american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from plasma and serum clinical specimens. group contained a single hpa alloantibody specificity with or without hla antibodies (n ). group included specimens with hla antibodies alone and group consisted of patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa- , hpa- , hpa- , hpa- , hpa- , gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is % concordance observed for hpa- a, hpa- b and hpa- b antibodies. the pak-plus assay had difficulty discriminating hpa- b from hpa- a antibody when hpa- a antibody was present ( false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa- a when compared to hpa- b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within % of the cut-off for pakplus and < . adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut overexpression on cell motility. results/findings: fut overexpression increases both ley expression and cell migration, while fut knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac . as tumor promoter, hdac becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n ) and stored at c for up to days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means sem, and paired student's t-tests were used to determine statistical significance (p< . ). results/finding: on day , p-selectin levels were significantly higher in pre than bl (p . ). mirasol treatment caused a significant increase in pac- expression compared to pre (pre: . . %, post: . . %; p . ), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post- samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after day storage at c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day c-stored pas plts followed by incubation ( minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* , arthur p. bode , anne s hale , michael stanton , mark johnson and g. michael fitzpatrick . cellphire, bodevet, inc, cellphire, background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to , , and . % of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control ( -day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to % of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of % and % of the tcpc produced a significant decrease in blood loss. the lcps at % and % tcpc were as effective in mitigating blood loss as -day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to % of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of % and % of the tcpc reduced blood loss. these results suggest a starting dose above . % tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as % tcpc had similar efficacy signals as % tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso c. the study on pcr-ssp technique for the genotyping of cd - del.ac mutation and the genetic polymorphism of cd - del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd (platelet glycoprotein iv, scarb ) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd deficiency in china. cd gene mutation is the main reason that leads to cd deficiency. cd - del.ac (frameshift at aa ) mutation is one of the cd mutations that causes cd deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in pediatric patients who received both doses of the mmr vaccine at and months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are ! % for all mmr components. results/finding: table shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged . years ( . to . years). thirteen patients ( %) were chronically transfused at the time of serology. twenty-three patients ( %) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper , , franklyn cladis , richard saladino , darrell triulzi , barbara a gaines and mark yazer* . university of pittsburgh, children's hospital of pittsburgh of upmc, institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients ! years old and ! kg with evidence of hemorrhagic shock were eligible to receive up to cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (< ) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately months, trauma patients received wb: group o and group a recipients, % male, median (iqr) age was ( . - ) and % blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of ( - ) and % mortality rate. the median (iqr) quantity of wb transfused to group o recipients was . ( . - . ) ml/kg versus . ( - ) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean standard deviation haptoglobin concentrations for non-group o recipients was . . mg/dl on day , . . mg/dl on day , and . . mg/dl on day ; the corresponding haptoglobin concentrations for group o recipients were . . mg/dl, . . mg/dl, and . . mg/dl, respectively (p> . for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n ) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n ). the mean standard deviation platelet volume administered was cc for whole blood recipients versus cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) with anti-human igg only, and a positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a minute c incubation, followed by automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer and the breast milk showed anti-d with a titer between and . the patient had a consistent plasma anti-d titer of . the patient's mother chose to stop breast feeding after weeks, and the patient's hemoglobin was improved at and weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that % of c scd patients from the west indies and west and central africa are partial c and at ( %) risk for alloimmunization to the c antigen through transfusion of c rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce( g, t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce( - )-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of patients with genotype/rh phenotype data available, ( . %) were c antigen positive serologically. the allele frequency of rhce*ce( g, t) was . . in total, ( . %) patients possessed rhce*ce( g, t) in the absence of conventional c gene in trans. of the c antigen positive patients, individuals ( . %) were predicted to be partial c based on four molecular profiles [rhce*ce( g, t)/rhce*ce: ; rhce*ce( g, t)/rh*ce: ; rhce*ce( g, t)/rh*ce( g): ; rhce*ce( g, t)/rh*ce( g, t): ]. in these partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after transfusion exposures ( c-antigen negative units; mean: , range: - ), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce( g, t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* , megan l nguyen , melanie c proctor , david e krysztof , gregory a foster , erin k sash , sandy s dickson , joua yang , jeffrey m linnen , kui gao , jaye p brodsky and susan l stramer . american red cross, grifols diagnostic solutions inc., grifols diagnostic solutions, inc, quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on / / (fl, ga, sc, ms, al). following revised guidance on / / , testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on / / . travel history questions were discontinued on / / . confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of / / , , , donations were tested including , ( %) in , mps. no reactive donations were identified by mp-nat. of the , , id-nat donations, were initial reactive (ir) of which ( %) confirmed positive (cp) by subsequent testing (cp rate of : , ; positive predictive value of %; specificity of . %). five ( %) cp donations were id-nat repeat reactive (rr); ( %) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and in fl, of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from to days prior to donation. two donors with a travel risk reported clinical symptoms; cp donors ( %) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than copies (c)/ml to about Ê c/ml. at the time of writing, the longest period of detection in rbcs was days vs. days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from ir and all rr donors, ranging from to c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of were prepared by diluting nat plasma : and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n ) were sorted into categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm /high vl; igm /low vl. results/finding: of , donations collected april -december , were reactive for zikv rna. igm-index donations had higher vls (mean . x vs . x iu/ml) and higher proportions of simulated mp-detectable results ( % vs %) than igm donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm donations increased (table ) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r / systems lisa lee pate* , phillip c williamson , michael paul busch , susan rossmann , scott jones , ann butcher , john duncan , jean stanley and susan a galel . roche molecular systems, inc., creative testing solutions, blood systems research institute, gulf coast regional blood center -sugar land, qualtex laboratories background/case studies: in february , the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march , and testing of puerto rico donations began on april , . as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august , the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april , -february , using the investigational cobasv r zika for use on the cobas v r / systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a : dilution to simulate mini-pool testing. results/findings: a total of , , blood donations were screened using cobasv r zika. of ir donations, were repeat reactive (rr), non-rr and had no repeat testing. of the rr donations, were positive by altnat; of these were igm positive. all altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the rr donors that were not igm positive on index, enrolled in follow-up and all seroconverted. of non-rr donations, were altnat negative and is pending supplemental testing. / donors were igm positive on index. donors were igm negative on index; / enrolled in follow-up; remained igm negative and was gm inconclusive. of donations without repeat testing results, met criteria for positive ( was altnat positive, igm negative and altnat negative, igm positive). donation is pending additional testing. altogether, / ir donations met the criteria for true positive on the index donation. / ( %) true positive donations were reactive when retested in a simulated minipool. / were igm positive. conclusion: . % of the , , donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* , marc germain , gilles delage , maria esther lopes and yves gr egoire . hemorio, hemaquebec, h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia ( ) , and in brazil ( / ), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since % of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january st , through november, th , , from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: days, with % of the values lower than days); % of infected donors with symptoms lasting days; . donation/donor/year for wb and . for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x ( -proportion of refused donors) x ( proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain : , , for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from donations). the residual volume of mp plasma, . - . ml, is routinely discarded. beginning in april each blood center saved $ mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april - ) mp were combined into pools of donations; thereafter mp were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and % confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity ( % limit of detection < copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first months of samples is complete for , mp, comprised of , donations collected from april to october , . a total of pools were positive, with detected between april-june . the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over . % of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over . % of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in , zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* , sze sze chua , mars stone , michael paul busch and ai leen ang . health sciences authority, blood services group, blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on august . the numbers rose rapidly to cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since october . zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using blinded frozen samples consisting of replicates of half log dilutions of the who international standard for zikv and replicates of negative controls prepared by bsri. probit analysis was performed to determine the % and % limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a member blinded zikv reference panel from the usa-fda. results/finding: a total of , donations were screened from october to march , with false positive case and zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of . x copies/ml. zika igm was negative in the index donation sample but present in the -day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be . copies/ml at % lod and . copies/ml at % lod. the procleix zikv assay detected rna in out of patient samples and provided . % agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of in , donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types , and express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to reactivity on initial gel testing. if genotyping demonstrated weak d types , or , the intent was to manage the patient as rhd-positive. if weak d types , or were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in to weeks. results/finding: rhdgenotyping was performed on patient samples over months. of these patient samples, ( %) were weak d types or . the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types and patients have not received transfusions at this institution since they were genotyped. four of obstetric weak d types and patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this month study period serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using fda approved anti-d reagents. when reactivity with all reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of months we tested rhd-negative blood donors. there were ( . %) partial-d, weak d ( . %), and ( . %) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs - _ deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these donors showed that rhd-negative recipients received rbcs from of these donors. five of these recipients underwent antibody screening after an average follow-up period of months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which % of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d " with reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october to march , we performed routine d typing (neo, immucor) on obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of using at least antibody. solid phase and manual testing used the series and series reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh ) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all samples. two of ( . %) were d with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs - c, rs ; ivs c, rs ; and ivs a, rs ). two ( . %) were d and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four ( . %) patients had rhd alleles with known potential to make anti-d (rhd*dol , rhd*dar . , and with weak d type . ). one had weak d type , which has uncertain susceptibility to alloimmunization and one was weak d type , which has not yet been associated with anti-d. interestingly, two ( . %) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type is a variant of the rhd protein that comprises an amino acid substitution located in the th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c. g>c which is the first nucleotide of the exon of the rhd gene and thus could be implicated in exon skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v (agilent) and the nextseq platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of patients previously characterized by beadchip technology. interestingly, out of carry the c. - c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last patients, one has been previously characterized as rhd weak type carrying the c. g>c (p.gly ala). independently, sanger sequencing on unrelated rhd weak type samples pinpoint to a linkage disequilibrium between c. g>c (exac, maf . ) and the c. - c>t (exac, maf . ). in silico analysis of both mutation located close to the splice acceptor site of the exon does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon , mutant rhd c. g>c, mutant rhd c. - c>t and double rhd mutants c. g>c plus c. - c>t, we showed no influence on skipping of exon due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type between ala (transmembrane helix ) and val (transmembrane helix ) hampering membrane insertion. conclusion: the c. - c>t variation is always associated in cis with the missense mutation c. g>c on the allele rhd weak type . the c. - c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type red blood cells is due to the substitution of alanine at amino acid position to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has transmembrane (tm) and tilted ureapore a-helices, a long extracellular connector segment, and cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p. . we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and - aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, ) . results/finding: seven snmvs located within amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all at the exofacial ends (p.a t, p.w r, p.v d) are jk-weak; the two jkneg exceptions p.g e and p.g e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v m, p.e k, p.v i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the jk-neg variants are within aa (p. -p. ) of jk a/b at p. . none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n s and p.s p are adjacent to p. f and p. l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc a gene, which encodes the urea transporter ut-b . the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in . in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc a gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within aa from tm a-helix end v i, a t, w r, w r, g e, g e, v d* cytoplasmic n-terminal v m, g s, e k, l p in membrane tm and urea-pore a-helices r w, r q, g d, i t, a v, l r, a t ‡, a a §, l f, n s, s p, t m / * second nucleotide variant in this allele is synonymous (p.p p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a mbp region in q . - . with an lod score of . . using deep sequencing, we identified a potential deleterious mutation in the znf gene, which deletes bp resulting in loss of an entire zing finger domain. the identical del -znf mutation is present in all affected individuals, and is absent from all controls tested (n> ). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf del . none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc a gene, is a urea transporter that has been associated with renal function, we found that people with the znf del in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf , prevalent in southern spain due to a founder mutation, leads to ut-b dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-( -ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis( -ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at days and year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the pools included group a, group o and group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than - c within hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day (pool), day , and year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day , day , and year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp . ppm; mehp . ppm; deht . ppm; and meht . ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than % of its initial value. plasma stored in deht bags had an average plasticizer content % lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at g to separate rbcs from platelet-rich plasma (prp). prp was diluted -fold in pipes-saline with . mm pge and centrifuged at g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of - % and , - , platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted : (spdp %) with plasma from a patient with type vw disease (t vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of s - for seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was % (spdp/ffp > . ). results/finding: six batches of spdp/ffp were evaluated using subjects. there was no statistical difference between the spdp/ffp pairs (p . ). the mean ratio of spdp/ffp was . with a % ci of . - . . comparing spdp vs. spdp %, there was no difference (median ratio . , range: . - . ) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was % greater than in samples reconstituted with ffp. the lower limit of the th % ci is a difference of %, which is less than the a priori determined margin of noninferiority of %. even with % dilution with t vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* , qiyong peter liu , grantham c. peltier , ryan c. carney , ashley s. taylor , colby s. mcintosh , james a. bynum and andrew p cap . u.s. army institute of surgical research, velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: ( ) ffp; ( ) ffp with mm glycine; ( ) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; ( ) spdp pretreated with glycine-hcl ( mm); and ( ) spdp pretreated with glycine-hcl:glycine ( mm: mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood ( % hct with platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < . ). fibrin polymerization density was slightly diminished in rspdp vs. ffp ( . vs. . o.d., p < . ), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < . ). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < . ) and an additional twofold in pretreated spdps vs. rspdp (p < . ). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < . ). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples ( . % surface coverage vs. . - . %, p < . ). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* , anita tuip-de boer , ruqayyah almizraq , jason p. acker , philip j. norris , jennifer a muszynski and nicole juffermans . academic medical center, university of alberta, canadian blood services, blood systems research institute, nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from donors (blood type a or b). supernatants were prepared after - (fresh) and - days of storage (stored) for measurement of thrombin generation and ev analysis. a type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to % stretch using a cellstretcher. control cells were not stretched. after hours, il- and il- production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il- and il- production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p< . ) . incubation of stretched cells with stored wbf products resulted in higher il- production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by different methods from individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after - days (fresh) and - days (expiry). monocytes were co-cultured in media plus % rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table ) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin- production was higher after exposure to fresh wbf ( % control, p . ) or wbd at expiry ( % control, p . ). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* , alessandro tocchio , anita howell , kaushik sridhar , jason p. acker and utkan demirci . stanford university, canadian blood services, centre for innovation, background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of - g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at , , , , and days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from volunteers with four different age and sex categories (male, - years; male, > years; female, - years; female, > years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young ( . g/ml) and older female donors ( . g/ml) (p < . ). moreover, rbcs from young males ( . g/ml) were significantly less dense compared to rbcs profiled from older female donors ( . g/ml) (p < . ). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il- (pg/ml) il- (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p< . ). substantial correlations were also found between orp and free hemoglobin (p< . ) and orp and free heme (p< . ). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections vs (p< . ). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than days compared to rbcs stored for days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of to days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to days storage duration -reference group), medium age (at least rbc of - days storage), and oldest (at least rbc greater than days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every days, and b) a finer partition using cut-points every days. results/finding: , patients receiving , rbcs were included in the analysis. exposure to rbcs stored for more than days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for days or less) after adjusting for several fixed and time-dependent potential confounders (hr . ; % ci: . , . ; p . ). exposure to blood stored for at most - days yielded a similar hazard ratio (hr . ; % ci: . , . ; p . ). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than days compared to exclusive exposure to rbcs stored days or less was not significant (hr . ; % ci . , . ; p . ). the confidence intervals around the hazard ratios for the other -day intervals all include . similar findings were obtained with partitioning exposure data into day intervals where exposure to rbcs stored for - days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for - days (hr . ; % ci . , . ; p . ). the confidence intervals around the hazard ratios for the other -day intervals all include . conclusion: individuals exposed to rbcs stored for more than days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* , cynthia walser , tatsuro yoshida , andrew dunham and pedro cabrales . university of california san diego, new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o saturation < %) stored rbcs, or anaerobic/hypercapnic (o saturation < % and pco (@ c) $ mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as- after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either ) conventional; ) anaerobic; or ) anaerobic/hypercapnic conditions. rats ( - g) were hemorrhaged to % of blood volume, held in hypovolemia for minutes, and resuscitated to recover blood pressure to % pre-hemorrhage with prbc stored for either or weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. ( %) neg patient showed short term response and ( %) patients showed progressive disease. at the neg group standard eval ( %) patient showed response and ( %) had progressive disease. ( %) neg patient had long term response compared to ( %) pos patients. at the pos short term eval ( %) patients showed response and ( %) patients had progressive disease. at the pos group standard eval, ( %) patients showed response and ( %) patients had progressive disease. overall, ( %) pos patients responded compared to ( %) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd neutralizing substance could play a role in treatment response. alternatively, reduced cd expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a -hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate % replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of . m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* , laurie sutor , , germ an leparc , marjorie doty and william crews . carter bloodcare, ut southwestern medical center, oneblood background/case studies: anti-cd drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with . m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and . m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual ( th edition). each of the plasma aliquots was further separated into aliquots and stored at - c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. ( ) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than with the untreated or dtt-treated cells during the study. conclusion: long term storage of . m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque- ), pooled, suspended in cryopreservation media ( % dmso; : ) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips ( c, % co , h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o ) rbcs sensitized with either anti-d (positive control), anti-scianna- (sc ) or anti-anwj or lipopolysaccharide stimulated for h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/ monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed . % viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il- b, il- , il- , mip-a (p < . ), mip-b and gro (p < . ) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc -and anwj-sensitised rbcs resulted in a pi of . % and . . % respectively vs anti-d sensitized rbcs (pi: . %). a weak ( ) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi> %). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in patients, involving antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all patients had - positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients and typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c by hea precise-type. ega-treated rbcs gave reactions with the same anti-c reagent. patient rbcs gave variable reactivity (vw- ) with bio-rad seraclone and ortho bioclone anti-c. patient rbcs gave reactivity with all anti-c reagents when incubated for the maximum incubation time allowed. patient rbcs were jk(a ) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients and tested s with bio-rad seraclone anti-s ( - ), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat rbcs but not all manufacturers include reagent limitations regarding testing of dat rbcs. we describe cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and cases with false positive tests with anti-s (n ) and anti-jk a (n ) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with - positive dat and supports testing to dissociate igg from rbcs strongly dat before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/ opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r : prob(ab|al-loexp), so that prob(ab) prob(ab|alloexp)*prob(alloexp) and r ; rewriting prob(alloexp) prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar months before ( / - / ) and months after ( / - / ) was selected; for state b, a similar months before ( / - / ) and months after ( / - / ) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (< months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a -and -fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from to (state a) and to , (state b), which annualized, represents a potential gain of , (state a) and , (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from - , there were donors identified who had changed their gender from their birth gender; female donors changed their gender to male and male donors changed their gender to female. there were ( %) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to . ml/kg or % of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the % limit. variable volume scales [vvs] can be programmed to vary unit volume (up to ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by ml at ebvs < . l in donors ! yo, but increase by - ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the mos. before a mo. phased implementation of the vvs, and the subsequent mos. multivariable analysis [mva] by -mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods & , continued during impl and post-impl periods & , returning to the baseline rate in post-impl periods & (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods & . the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of . ml during post-impl periods & from the temporally matched baseline & pre-impl period . conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* and yves gr egoire . hema-quebec, h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, . the vaccine is produced with live and attenuated yfv, which can circulate for at least weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received , blood donors candidates; from those, , were accepted as a blood donor, after medical interview. the deferral rate was . %. at the same period of the year , there were , prospective donors, and blood donations. the deferral rate was . %. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, , additional donors, compared to same dates. that represents a . % increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from . % in to . % in . conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of ! . x is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: , apheresis collections from centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of . x for single (s), . x for double (d), and . x for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or % plasma) assuming i) a minimum dose (allowing for production loss) of . x for s and . x for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as %) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc rbc plt/p plt plt/rbc/p plt/rbc plt plt/rbc plt/p #donations citrate exposure (mls) - study design/method: a randomized ( : ), placebo-controlled, single blind, subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into cohorts, receiving increasing doses, ranging from / , - / of the lowest effective dose found in the above rabbit model. cohorts and received the / th dose, but cohort received two / th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for hrs post infusion and followed for up to days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of aes: were treatment emergent (teae), of which were treatment-related ( thrombosomes and control). all teaes were mild or moderate in severity. in cohorts and , / thrombosomes subjects had treatment related adverse events. one cohort subject developed an upper respiratory infection and elevated wbcs within hours post infusion, which resolved by hours, and an elevated d-dimer at hours post infusion, which resolved by day . this subject also had an elevation of prothrombin fragment at baseline, which increased post transfusion and peaked at hours with resolution by day . one cohort subject developed non-specific t-wave changes at and hours following her nd infusion that resolved by day without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort subject developed an igg platelet autoantibody on days - , which was undetectable on days - ; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days - , and negative on days - . background/case studies: cryopreservation of platelets (plts) could extend the shelf life from - days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with % dmso and stored at c. after thawing, the unit was reconstituted in thawed ffp spiked with either lm puromycin (pm) or nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after , and hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x- containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by -fold during hour storage. immunoblot analyses of the plts showed a -and -fold increase in pm incorporation after and hours of storage, respectively. massspectrometry revealed unique proteins that were synthesized after hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac , rap and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac , rap and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in , the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r - cooler with units of ffp, units of rbcs, and unit of whole blood. three to ml of platelets were collected via syringe from each unit at min (before storage in cooler or refrigerator) and after . , , , , and hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac- binding) were measured by coulter counter, channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p< . deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c -a h molecular sieving: beyond genotyping ghazala hashmi , reinhard klemm and michael seul* , . biomolecular analytics, immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi http://bit.ly/ ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, * * samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for sensitized sickle cell anemia ("sca") patients (tb in cas-tro , http://bit.ly/ oplxhr, excluding le and e(variant) and assuming request per patient), presenting with up to allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only = plate holding * candidate units from actual black donors, followed by profiling of samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for of requests ( . %), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining pooled samples produced additional assignments to a second set of requests, for a total of assignments from only wells. in another scenario, sieving of a full plate of * samples, produced $ assignments for two successive batches of requests from sca patients, a yield exceeding . x. sieving alone typically fills - % of requests of moderate complexity ( ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez , monica kalvelage , ghazala hashmi* and michael seul . biomolecular analytics, lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou http://bit. ly/ ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including at the rhce locus) that encode mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag " (e.g. e ,c ,e ,c ) to specific combinations of "ag " (e.g. c e k fya and c e jsa ) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the c samples, that were also v and vs and, among the e samples, that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select specific "ee" pools of which were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b ( pools), co a|b ( ) and others. conclusion: molecularsieving of a single -well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c , e and jsa . these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam (landsteiner-wiener) and ackr (duffy). for longer genes, such as abo of > kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of > kb each was used for all physically confirmed ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v . . was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least clades representing clusters of to alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors ) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons - , and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors . a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos (b galnt ) cells, and cell-surface expression of fors antigen was immunologically monitored with a monoclonal anti-fors antibody. results/findings: we found that met thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon or of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met thr/ ser or exon / deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt genes is reminiscent of common ancestral origin of alpha , -gal(nac) transferase genes. the finding that at can synthesize fors implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of , cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period / / - / / . abo genotyping targeting specific snps for groups a, a , b, o , and o and, if needed, gene sequencing was conducted in cases with indeterminate results, and in cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two ( . %) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in % the predicted abo phenotype was a rh neg (table a ). the predominant donor race was caucasian ( %). four cbu with abo discrepancy were also evaluated by genotyping (table b) . in of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r / systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: , fresh and , frozen edta plasma samples from american red cross donors, collected from february - , were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of , valid results, a total of donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a -year old male in indiana, a -year old male in california, and a -year old female in kentucky. all donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna ( iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as a, the california donation genotype b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was % ( % exact ci: . % to %). conclusion: based on the confirmed-positive donations of , tested, the hev prevalence was . % ( % exact ci: . % to . %) with a detection rate of : , ( % ci, : - : , ). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than % of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using % confidence intervals. this analysis contains data from / / - / / . results/findings: among , , donations reported ( . % from firsttime and . % from repeat donors), there were respectively , and cp results for hbv, hcv and hiv with corresponding rates of . , . and . per , (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of : , : and . : for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv . vs . ; hcv . vs . ; hiv . vs . ). in general, higher rates for all markers were seen among minority donors, those in the - -year age group (also - year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when -month periods were compared. conclusion: data from major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* , melanie c proctor , deanna self , monique portugal , adrian gurrola , laura tonnetti , sonia bakkour , cheryl lobo , michael paul busch , susan l stramer and jeffrey m linnen . grifols diagnostic solutions inc., american red cross, blood systems research institute, new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening , unlinked whole blood donations collected from august th to april th in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of . results/finding: the procleix babesia assay detected all four babesia species with a % lod ranging from . - . copies/ml. the preliminary % lod in parasites/ml ranged from . - . p/ml for b. microti (n ), from . - . p/ml for b. duncani (n ), and from . - . p/ml for b. divergens (n ). of the , donations screened, initial reactive and confirmed positive donations were identified for specificity of . % ( %ci: . - . %). of the confirmed positive specimens, were reactive by both ifa and pcr, by ifa only and by pcr only. all confirmed positive samples were reactive in lysate pools of . donors of reactive donations resided in ct ( ), nj ( ), nh ( ) and me ( ) for an overall incidence of : , , and : , in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* , whitney r steele , ed p notari , james haynes , roger y dodd and susan l stramer . american red cross, american red cross (retired) background/case studies: from - , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from - . study design/methods: prevalence was calculated in -year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of . , . and . days for hbv, hcv and hiv, respectively. linear regressions were calculated with p< . (*) as significant. results/findings: from / / - / / , there were more than million donations from , , donors ( . % female, % first-time (ft), . % caucasian). there were significant decreases in donation prevalence for hbv and hcv (p . and . ), but no significant decrease in hiv during the years (see table for f and r values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p . and . ). prevalent ft donors were significantly more likely to be male ( . % -hbv, . % -hcv, . % -hiv; p< . ). incidence for all agents declined (significant only for hbv; p . ). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last -year period ( in - vs. in - ) . hcv incident donors in - were more likely to be male ( . % vs . % in - , p< . ) and were younger ( . % vs. . % in - < years, p . ). overall, incident donors were more likely to be caucasian males (p< . ). rrs for all agents decreased over time with rrs in - of in , , ; in , , ; and in , , for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the -year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in , mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as- rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with lm amustaline, and incubated for hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, > . log , or > . log pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was > . log , or > . log pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts- inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* , , hollie m reeves , and robert w maitta , . case western reserve university school of medicine, university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts activity of < % and high inhibitor ( . - ). mean age of cohort . years (range - ). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean . x /l, range - x /l) and low a-ipc (mean . x /l, range . - . x /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab mg/m ( patients) and cyclophosphamide mg/m (one patient). tpe continued until platelet count reached x /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of . days [range - days]) when they achieved a three-fold increase in a-ipc from baseline (mean . x /l, range . - . x /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean . x /l, range - x /l) and a-ipc (mean . x /l, range - . x /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of . days (range - days) mean platelet count was . x /l (range x /l) and mean a-ipc . x /l (range . - . x /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of . days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* , michelle n stram , joan sevcik , alesia kaplan , and joseph e. kiss , . department of pathology, university of pittsburgh medical center, blood systems inc., university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within - hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january , to november , was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts- activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the ttp patients identified, were excluded due to missing temporal data for important variables. the majority ( %) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients ( %) had a prior history of ttp and % had severe adamts deficiency on admission. the median time from tpe request to initiation was . hours (interquartile range: . - . hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table ) . treatment was not started within an -hour window in patients; the median time to cv access was significantly longer in these patients ( . vs . hours, p< . ). two of these patients had a prior history of ttp and only four patients had severe adamts- deficiency. the majority (more than %) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table ) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus - hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi- a transfusion vol. supplement s hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, through january, , we performed cytapheresis (cy) treatments (txs) for pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. pts ( %) had cml-at and received leukapheresis (lp) txs; pts ( %) had et and received thrombocytapheresis (tc) txs. cml-at pts presented with median wbc x /l (range - x /l), of which % had blast percent > % or blast count > x /l. median age was years ( - years); % were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. % of cml-at pts had no sxs of lks; % pts had sxs of either cns or pulm lks ( sxs), and % pts had sxs of both cns and pulm lks ( sxs). et pts presented with median platelet (plt) count of: x /l ( - x /l)and % pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was years ( - years); % pts were male. results/finding: all pts received a course of cy tx with following objectives: ) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and ) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) < x /l for cml-at pts, and plt ct < x /l for symptomatic et pts and < x /l for asymptomatic et pts. cml-at pts received median of lp txs (mean . txs/pt; range - txs). et pts underwent median of tc txs (mean . txs/pt; - txs). outcomes were evaluated by percentage of pts who: ) reached wbc (or plt ct) tx goal, and ) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved > % reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, % pts improved, % pts stabilized; and % pts worsened. in et cohort, % improved, % stabilized, and % were unchanged. for cml-at pts, median final wbc ct x /l (range - x /l); % pts received ind chemo. for et pts, median final plt ct x /l ( - x /l); % pts had resolution of thrombotic a transfusion vol. supplement s symptoms. % of cml-at pts and % of et pts expired within - days after course of cy tx. of expired pts, pts had both blast crisis and sxs of cns/ pulm lks; pt had intracranial hemorrhage or cva; and pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median - txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used % albumin or % albumin- % normal saline ( / ) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered ( % albumin vs / ), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where % albumin was used versus those that used / . covariates included were fluid types, age and gender. odds ratios (or) and % confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < . . results/finding: during the study period, procedures were documented for subjects ( % female), age range - years, of which , ( . %) received / . the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with % albumin had a significantly lower risk of having either event than by using / , [p . , or (ci): . ( . , . )] , and also had a significantly lower risk of causing hypotension [p . , or (ci): . ( . , . )] in addition to a lower risk of causing citrate toxicity [p . , or (ci): . ( . , . )]. age had a significant effect on having a hypotensive event [p . , or (ci): . ( . , . )] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a year period and compared it to published literature. study design/method: we conducted a -year retrospective study of ta procedures performed and aes were classified according to criteria described in table . during the study period, ta were performed using cobe spectra (software versions . and . ) and since the spectra optia apheresis system (version . ). literature search was conducted for data published on aes associated with ta. four studies from us and non-us studies (canada, europe and japan) were analyzed. trend for ae rates from - was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was . % ( of , procedures) during year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher ( . %, p< . ) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho . , p . ) over the years and significant down trend of moderate and severe aes with a spearman rho of - . (p . ) and - . (p . ) respectively. there were no fatalities during the study period. majority of aes were grade i ( %) and grade ii ( %): / ( . %) procedures were not completed due to aes. comparison of aes [ . % ( / , )] to both european [ . % (n , , / , ) ] and other us studies [ . % (n , / , )] showed a statistically significant difference (p< . ). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table ) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether platelet donors with a donation activity of up to platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* , jordy jurgens , jacoline buchner-doeven , joris roelofs , philip spinella , jennifer a muszynski , carel goslings and nicole juffermans . academic medical center, washington university school of medicine, nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells ( days old) and platelets ( days old) by washing. plasma was filtered through a . um filter. rats ($ grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $ % of their estimated blood volume, which was calculated to be ml/kg. hemorrhage continued until a mean arterial pressure of mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of ml/kg of blood products in a : : ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april , which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and -day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january and july (n ). because of missing data on patient characteristics, patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january to march , n ) and after (april to july , n ) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of baseline variable, generating pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common ( . % vs . %, p< . ) and ventilation time was longer ( h vs h, p . ) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group ( ml vs ml, p . ; and ml vs ml, p< . , respectively). however, -day mortality was not statistically different between the groups ( . % vs . %, p . ). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april , www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over , joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy . length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of -factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: -factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in . marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p . ) and ptt (p . ) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p . ) and plasma (p . ) after off-label use was significantly greater than on-label use. cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were . times (p . ) more with cell saver or anh, and . (p . ) times more with cpb. post-pcc thromboses were identified in cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to days. the transfusion service medical director reviews the case and gives final approval. we observed only patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight ( ) patients were in-patients continuously until delivery. five patients were discharged prior to delivery- moved to another state, was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was days (range - ). six ( ) patients delivered within days of approval. after approval, the mean number of additional specimens per patient was . (range, - ). no patient required transfusion prior to delivery. five patients received transfusion of at least rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only patients delivering within days of approval for extended specimens, patients avoided collection of at least specimen each, and patients avoided at least collections each. since new antibodies are not detectable for at least days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* , , jan m. binnekade , benjamin nota , pieter r tuinman , kirsten van de groep , olaf l cremer , janneke horn , marcus j schultz , robin van bruggen and nicole p juffermans . academic medical center, sanquin research and landsteiner laboratory, vu university medical center, university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in tertiary icus in the netherlands comparing patients who developed ai during icu stay with control groups: non-anemic patients with sepsis, non-anemic patients without sepsis, and patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron ( . vs. . mmol/l, p< . ) and transferrin saturation ( vs. %, p< . ), and low ferritin ( vs. mg/l, p< . ). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately . x cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to %, whilst leaving % of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than % of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately % of the cd fraction post separation and freeze thaw (table ) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table ) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd cd ) and early projenitor cells expressing oct and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: . routine recovery of the wcf at levels higher than current methods, independent of volume. . higher percentage recoveries of all cell types tested than can be achieved with existing methods. . markedly higher post-thaw recovery of viable nucleated cells than any current methodology. . almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving > . x lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd cell target of . x /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf plerixafor (g pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< . considered significant. results/finding: no alc and alc collections occurred among the patients. fenwal amicus was used for % of the no alc and % of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was hodgkin's and non-hodgkin's lymphoma (no alc); hodgkin's and non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc . , lymph . x /l) than alc (wbc . , lymph . x /l). equivalent whole blood (corrected for ac) was processed for no alc ( . l) and alc ( . l). for alc group, extra collections beyond cd target were: days: %, day: %, days: %, days: %, and days: %. significantly more patients were mobilized with g pl in no alc group (n ) than alc group (n ) and collections in alc group had mobilization discontinued after cd cell target reached. there was no significant difference in g ( . x lymph) compared to g pl mobilized collections ( . x lymph); both were significantly higher than the collections where mobilization had been discontinued ( . x lymph). days to wbc engraftment ( . no alc vs . alc) and platelet engraftment ( . no alc vs . alc) were not significantly different. median number of collections for no alc ( ) and alc ( ) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the . x lymph/kg or even the . x lymph/kg targets. implementation of a lymph target increased patients obtaining . x lymph/kg from % to %. only % had < . x lymph/ kg. discontinuation of mobilization once cd cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* , and nicolas pineault , . canadian blood services, university ottawa, canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost hours at room temperature (rt) as long as units are cryopreserved by -hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n ) were split with one half processed immediately (baseline - hours) and the second after hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd cells and cd cell (n ). primary nsg mice were transplanted with a ucb cell dose that contained a total of , annexinv neg viable cd cells. the latter was done to avoid any bias towards one group or another. short term platelets ( vs. hplt/ml, p . ) and leucocytes ( . % vs. . % hcd , p< . ) engraftment at -weeks were significantly reduced in stored mice vs. baseline (n ), and similar results were observed long-term at -weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated months post-transplant. strikingly, the frequency of human cd bm cells was -fold greater in baseline vs. stored mice (p< . , n ). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured -weeks post-transplants were reduced by % in unit , and by % in unit . conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($ mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to %, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of sec). total lymphocyte recovery was % and monocyte concentration was reduced %. furthermore, in a two-pass process platelets were reduced by %. in a -fold parallel system we tested rbc separation from plasma and achieved % separation at ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from clinical protocols from - . an infusion reaction was defined as any symptom from the time of nk cell infusion up to hours afterwards. a severe reaction was defined as any symptom with grade or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r . . . two major endpoints of interest were: ) infusion reaction with any symptom and ) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of nk cell infusions. there were ( %) patients with an infusion reaction of any symptom and there were ( %) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median . , p . ) and those with severe reaction (median . , p . ). infusion rate (ml/min/kg) was also similar among those with any reaction (median . , p . ) and those with severe reaction (median . , p . respectively). incubation of nk cell product overnight in il- vs il- had similar reaction rates for those with any symptom ( % had reaction with il- , % had reaction with il- , p . ) and those with severe reaction ( % had severe reaction with il- , % had severe reaction with il- , p . ). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median . x ) versus those without (median . x , p . ). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan , maryanne c herzig* , barbara a christy , james a. bynum and andrew p cap . us army institute of surgical research, u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at - . mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at - , cells/ well and cultured in well plates for - h in their respective medias. on day , mscs were washed, resuspended in pbmc media and incubated with or without , freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, - lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by h, with > fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was . %. inter-assay variation of msc preps run under identical conditions was . %. inhibition of pbmc proliferation was graded from - % over the range msc concentrations therefore an ec of msc cell number resulting in % suppression of pbmc could be determined for each msc prep. this ec however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute % or more of the us blood supply. differences between donors - years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged - were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the / academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin < ng/ml) and low ferritin (lf, ferritin < ng/ml) were estimated for , , and - yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of donors contributed donations. donors were evenly split by gender, % were ft donors, and % were - yo. ft and rpt - yo donors had on average lower ferritin values at enrollment (p<. ), and a greater percentage were iron-depleted than donors - yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged - have sharply higher risk for iron depletion than donors - yo. odds for lf were to times greater in the younger donors, and for ais were -to fold higher. preliminary statistical models indicate yo donors may have greater risk for lf than or yo by to percentage points, controlling for other factors (p . ). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in - yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on / / by a large blood collector. testing was performed on successful - y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < ng/ml in females (f) and < ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations ( months for f and months for m) and counseled to take - mg of elemental iron daily for days. for m and f, a ferritin < ng/ml indicated absent iron stores (ais) and < ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! ng/ml in f and ! ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior months. an appreciable number of donors with no rbc donations in the prior months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* , joan williams , michelle humphries , nancy haubert , ben reynolds , michael phillips , randall spizman , ralph r vassallo , hany kamel , sally caglioti , german leparc , and phillip c williamson . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. study design/methods: over , serum samples from donors aged , and years were analyzed for ferritin levels using the beckman coulter au instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among , adolescents (ages - ; . % female) who donated blood from to . study design/method: abnormal risk factor levels were defined as hba c ! . %, sbp/dbp ! / mm hg and tc ! mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, , ( . %) adolescents had at least one abnormal risk factor ( . % of males, . % of females). of these, , adolescents had isolated abnormal risk factors, and , adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as ! . g/dl for men and ! . g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from blood services worldwide and complete data were available for blood services. deferral percentages for low hb varied from . % to . % among male donors and . % to . % among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with % lower hb deferral rates in men ( % confidence interval [ci] % to %) and % lower rates in women ( %ci % to %). iron supplementation was associated to % lower hb deferral rates among women ( %ci % to %) but there was no evidence of such an effect among men (p . ). each one-week increase in minimum donation intervals resulted in % lower hb deferral rates among women ( %ci % to %) but not among men (p . ). at the % level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: . % of all candidates for wb donation were deferred in continental france in . deferral was significantly more frequent in women ( . %) than in men ( . %), due to anemia in . % of deferred women and . % of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from to weeks. analysis (table) identified main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the previous years. conclusion: the main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, days stored or days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, ng/kg). blood was sampled every hours up to hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from . e (iqr . e - . e ) /ml in the fresh product to . e (iqr . e - . e /ml; p< . ) in the stored product (p < . ), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: cases of taco and matched controls were enrolled from , transfused patients who received , blood components from may until july . taco incidence was case per patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation ( % vs. %; p < . ), experienced longer intensive care ( vs. days; p . ) and hospital length of stay following transfusion ( vs. days; p< . ), and had higher mortality ( % vs. %; p . ). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from to days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july and to routinely extend ap outdate to day since february . this study reports a month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july -january , ap underwent rt on day . day and units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day had a second rt performed. from february -january , ap underwent rt on day with routine outdate extension to days by performing a second rt on day and a third rt on day , as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type ) or repeat rt positive with negative confirmatory culture (type ). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july , , ap were entered into inventory. of these, , ( %) were transfused prior to rt testing. the remaining ( %) underwent rt on day or day . of these ( . %) were rt positive ( type fp, returned to inventory; type fp, discarded), leaving a total available inventory of units tested by rt. of these, ( % of original inventory) were transfused before the end of day and the remaining ( % of original inventory) reached a day outdate. a total of ( % of original inventory) were transfused on day or day . of these, underwent a second rt on day ( rt positives; fp type one and fp type ) and underwent a third rt on day (no positive results). a total of ( % of original inventory) outdated on day . of these, underwent a second rt on day (no positive results). conclusion: to date we have performed rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted % to only %. a total of ap have been tested twice by rt ( on day and ; on day and ) with ( . %) positive results, both of which were deemed fp by repeat testing or culture. a total of units have been tested times (day , day and day ) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with to million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in . contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between - , parasites/ml. each parasite concentration in wb was tested x . an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at c for up to days; platelets were stored at c (rt) under agitation for days and plasma was frozen at - c. aliquots for culture were removed weekly from rbcs, daily from platelets and after days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at c for detection of live parasites for up to weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at c, rbcs from all units spiked with , parasites/ml were positive for up to days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with parasites/ml were positive for up to days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with , and parasites/ml were positive up to days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at c for up to weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* , marion lanteri and larry corash . cerus corporation, scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom ( - ), french ( - , swiss ( - ), and belgium( - hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately . million dlvbc-screened were issued with a day outdate after release into inventory days after collection, and $ . million amotosalen/uva-treated pc were released into inventory on day or , with a to day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored > . million conventional, non-dlvbc-screened pc and recorded str and fatalities. concurrently, zero definite and possible str were reported with , amotosalen/uva-treated pc, significantly fewer than with conventional pc (table ) ( . str per million vs. . per million, p< . ). one definite, possible, undetermined/indeterminate non-fatal str and contaminated "near miss" pc were reported with . million dlvbc-screened pc between and , for a reduced falsenegative rate compared with the prior five years ( . str per million vs. . per million, p < . ). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october for all platelets received at our institution. at time of receipt at the blood bank (day post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at c for three days. results/finding: a total of / , ( . %) platelet products were successfully cultured ( / [ . %] and / [ . %] in october and march respectively). over the -month period, two true positive cultures were obtained (incidence of in platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us . per product tested. the cost per averted case was $us , . conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january and december . the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the -year study period, a total of transfusions reactions were reported, of which were bcptr ( . % of transfusion reactions). of the bcptr, ( %) were associated with apheresis platelets, ( %) with red blood cells, and ( %) with plasma. recipient diagnoses spanned hematologic/oncology (n ), renal (n ), cardiac (n ), autoimmune (n ), and obstetrics (n ). an organism was identified in both the blood product and recipient in ( %) cases; in ( %) cases an organism was grown in the blood product but not the recipient; and in ( %) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in of the cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever ( %), chills ( %), nausea and vomiting ( %), pain ( %) and dyspnea ( %). blood pressure (bp) decreased in %, increased in %; % of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie , jenna lebedev , linda kapp , xiaohong wang , meghan delaney , lay see er and james c zimring* . bloodworksnw research institute, bloodworks nw, university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg -igg ), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all known variants. study design/methods: the heavy and light chain variable regions of an anti-k monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg , igg , and igg had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table ). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg or for any of the monoclonal ahgs tested. monoclonal anti-igg had a blindspot for igg - , due to the shorter hinge region on igg - . no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* , yves dominique pastore and maryse st-louis . chu sainte-justine, hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since , our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of , scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september , ( %) patients had been transfused and had antibodies with known blood group antigen specificity: anti-c, anti-e ( ), anti-hrb, anti-fya, anti-jka, anti-jkb ( ), anti-s, anti-m, anti-sc , anti-leb ( ). seventeen patients ( . %) were either d or partial d. rhce results showed that patients expressed a normal c antigen and expressed partial c. as for e antigen, had a normal antigen, bore a partial antigen and were weakly expressed. fy(a b ) phenotype was found in ( %) patients. a total of genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos , emilia sippert , mayra dorigan de macedo , sheila fatima perecin menegati and lilian castilho* , . hemocentro unicamp, university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa- a, il b- t cytokine polymorphisms, rhag g>a and hla-drb * alleles may predict a good responder phenotype (sippert et al, transfusion ) and that rhag a and hla-drb* alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included non-alloimmunized patients with scd, homozygous for hbs, receiving a range of - rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa- g>a, il b- c>t) and the rhag g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among non-alloimmunized patients, were homozygous or compound heterozygous for rh variant alleles. from those, had rhag a and/or hla-drb* alleles and at least one cytokine polymorphism (tnfa- a or ilb - t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and were confirmed by sequencing. the third sample was found to be rhce*cevs. ,rhce*cebi on sequencing (predicted phenotype v ,vs ). the samples were typed as v (or ce s ) and vs (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[ g]in samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising % of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies ( % of patients) bound young and old rbcs with no apparent prejudice. band- is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band- is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band- to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band- aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band- tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing type i and type ii patients, we found that type i is characterized by percollv r fractions (similar to healthy storage-matched controls) but increased band- tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by - percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band- tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band- suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* , burak bahar , jeanne hendrickson , krystalyn e hudson and christopher a tormey . yale-new haven hospital, yale university, background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam abstract algorithm, e value x - , word size , gap costs: existence exten-sion ). search results were restricted to bacteria and fungi, with a selective threshold of > % identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b - . , r . & p . ); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of alloimmunized patients reviewed, were culture-positive. of these, % of the anti-c/c group ( of patients) and % of the anti-k group ( of patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from - . %. overall, . % ( of patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of > % sequence identity. while . % ( of ) patients reviewed had positive cultures for klebsiella species, . % of these ( of patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed tegs performed on patients undergoing cv surgeries at our institution from jan to dec , . no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at minutes (min) all within reference range), "hypocoagulable" (r> min, k> min, a< degrees, ma< mm) and "hypercoagulable" (r< min, k< min, a> degrees, ma> mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of tegs analyzed, patients ( %) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients ( % vs. %, p . ). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused ( % vs. %, p . ). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused ( % versus %, p . ). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from liver transplants, performed from to in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, olts were performed. a total of patients was submitted to cs. the median age was years (range - yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in , % of the patients. the average meld score was , , and it was slightly higher in the cs group ( , vs , , p< , ) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was ml and mean reinfused blood volume was ml. allogeneic blood transfusion was required in , % patients in the cs group, compared to , % patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group ( , units vs , units, p< , background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july -december was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp ( and products, respectively) however, obp wastage occurred more frequently in the month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations ( versus products). this is skewed by one month when products were wasted due to expiration of product on the floor. cooler-related issues ( ) and products dwelling too long out of a controlled environment ( ) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were . %, . %, and . %, respectively, with a total exsanguination protocol waste rate of . %. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p . ). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d ) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d- (table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* , edward smith , thomas brown , foeks jeremy , metcalf suzanne , james johnson , peter davis , karafa sw badjie and abba zubair . department of laboratory medicine and pathology, transfusion medicine, mayo clinic, department of anesthesia, mayo clinic background/case studies: our institution performs an average of solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused o( ) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the o( ) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in % of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a y.o. female patient should not have received o( ) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with admissions during the hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight ( kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o( ) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o( ) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately , surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from - %, whereas transfusion rates for vaginal and robotic pfd surgeries range from . - . % and . - . %, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately % of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may -may in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p< . . results/finding: we identified patients for analysis, of whom ( . %) had a preoperative t&s ordered. two ( . %) of these patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine ( . %) of the patients required a second abo/rh specimen per hospital protocol; ( . %) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table ) . no abo/rh discrepancies were identified. one patient received unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution ( . % vs. %, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n ) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of . g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs- i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), units were subjected to the volume reduction while recording the time needed to process all units. this was performed twice for a total of units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of % (range %- %). in units between and days (n ), the estimated mean residual k was . meq (range . to . ). in the two mock mtp trials, the time to complete the procedure was approximately minutes and we estimate an additional - minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal ( ) and caesarian ( ) births. uc collections were divided into segments to test conditions. segment explants were placed on . % gelatin-coated gridded tissue culture plates ( explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the remaining tissue segments were soaked in (ab/am) saline solution for hr and hrs at c, respectively. tissue segments were frozen in cryo bags with a proprietary % dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process or times the total blood volume (bv) of the patient, up to a maximum of liters (l) per procedure, to obtain peripheral blood cd stem cells. as a consequence, a patient often would need to spend hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd pre-count and cd yield, normalized per liter of blood processed, was derived utilizing the patient's cd pre-count, the patient's weight in kilograms (kg), and the target cd dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd stem cells. the initial equation was modified to add an additional % to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in patients, representing both allogeneic and autologous donors, the average blood volume processed was . l. the range was . l - . l. the target dose was achieved in all patients. our previous practice for these patients would have required, assuming a standard bv procedure, processing an average of up to l per patient, with a range of - l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd yield. the result was a high correlation between these two ratios (r . ), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r . , confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses ( - ) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june and march . patients (n ) evaluated were diagnosed with malignant lymphoma (n ), multiple myeloma (n ) and primary amyloidosis (n ) and were mobilized according to standard protocols. collection cd cellularity target was established ! x e /kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by > years old, previous fludarabine, lenalidomide, or bendamustine treatments or ! previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v . . results/finding: the media (range) general collection parameters were: cd (day ) . /ml ( . - . /ml), blood volume processed ml ( - ml) and . ( - . ) exchanged volemias. seventeen patients were considered bad mobilizers, needed plerixafor and had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p . ]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n ), fever (n ) and flu syndrome; all grade ]. two patients could not undergo hematopoietic stem cell transplantation due low cd cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd ) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with % fetal bovine serum. bmsc and amsc at passage - were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses ( . /ml, /ml and . /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par ) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within min and hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: seconds, versus low, medium and high doses of amsc ( , , and seconds), and bmsc ( , , . seconds), p< . ), clot formation time (cft, p< . ) and increased alpha angle (p< . ) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par . no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* , neil bagamasbad , reynold dilag , melissa nasser , nicole bauer , jennifer wheeler and mary berg . department of pathology, university of colorado -anschutz medical campus, department of medicine, division of hematology, university of colorado hospital, scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from collection procedures using the mnc protocol and collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd )-positive (cd ) throughput, cd collection efficiency (ce%), platelet loss a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included and allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd throughput was significantly higher in the cmnc group than the mnc group. the cd ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient # , originally typed as an a , had bone marrow donor and cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient # is now typing as type o. patient # was originally typed as a and received a bone marrow transplant from a type b donor. patient # is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient # and patient # indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with % fetal bovine serum under either normoxia ( % o ) or hypoxia ( . % o ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd /cd and cd were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc ( . /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by %, but depressed the growth of amsc by % at day in comparison to normoxia. both bmsc and amsc equally expressed cd and cd but not cd under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: (control), versus (bmsc), and (amsc) seconds) by natem. hypoxia also significantly shortened ct ( (bmsc), (amsc) seconds, p< . as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd cells. study design/method: cryopreserved cd cells from healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef -alpha-yfp lentivirus ( . % concentration) and media (x-vivo- , human serum albumin(hsa), ng/ml each of cytokines (scf, tpo and flt -l) over days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of % dmso, % pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd cd -cd ra -cd cd f cells) phenotyping and cfu assays were done following first thaw (pt ), post-transduction (ptxn) and second cryopreservation-thaw (pt ). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd %, cfus were similar before and after pt . hscs ranged from to cells/ cd cells in the pt -tr arm compared to a range of to / cd cells after pt . viability, % cd and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt (table) . conclusion: dec of mpb human cd cells decreases tnc recovery, but has minimal effects on cd cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd % in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of % hydroxyethyl starch, % human serum albumin and % dmso at final concentration. pbsc were cryopreserved by direct immersion on - c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of consecutive days of neutrophil count > . x /l and platelet count > x /l after days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd was below x cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd on the day of the collection versus collected cd per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd was calculated. final laboratory count of cd per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss software. results/findings: among patients collecting hpc for autologous transplantation, , % needed only one day of hpc harvesting, while , % needed two days and , % needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was - , %. after comparing predicted values with cd collected in the final product, we found a very strong correlation of . (p< . ) for patients and a strong correlation . for healthy donors (p< . ). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; ! % cd , cd , cd and % cd , cd , cd , cd , hla-dr) study design/method: umbilical cord tissue (n ) was washed, blood vessels removed, cut into . - mm pieces, and washed twice in saline. fresh tissue was immersed in . % saline for same day culture, while frozen tissue was cryopreserved for at least hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a cm tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures > % confluence. all cells were tested on an msc flow panel at passage just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of days (fresh . , frozen . ), and days (total) for the msc's to reach passage (fresh . , frozen . ). all cells were ready for flow analysis in approximately weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p . ), or their growth rates (p > . for all). flow cytometry showed average ! % for positive markers and % negative markers. there was no statistical difference between fresh and frozen flow result (p > . ). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of up to years ( to ) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with % concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a c water bath and . ml aliquots were diluted at a : proportion with % human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using -aad marker through flow cytometric analysis. results/finding: ucb storage period was . years (mean) and cell recovery was . % (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p . ). post-thaw cell viability of . % (mean) showed no statistically significant correlation with unit storage period (p . ). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln vapor in a dmso-based cryoprotectant for yrs. ( . . ; n ). units were rapidly thawed and rinsed in dpbs, then pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a x grid pattern in msc-supportive medium and incubated for days, after which the tissue was discarded and media exchanged. cells were isolated on the th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a % success rate. cells were positive for the msc markers cd , cd , and cd ( . . %, . . %, and . . %, respectively) and negative for the hematopoietic markers cd / ( . . %). passage and passage doubling times were . . days and . . days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical a transfusion vol. supplement s research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd yield prediction algorithm ines bojanic* , nelly besson , ivana vidovic and branka golubic cepulic . department of transfusion medicine and transplantation biology, university hospital centre zagreb, terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version ) in adult and pediatric lvl. a prediction algorithm for cd cell yield was also tested. study design/method: we evaluated retrospectively lvl performed in adult patients, and lvl in pediatric patients treated in uhc zagreb from march till september . mobilization regimen combined chemotherapy and filgrastim; poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio : ). in patients weighting kg (n ), a rbc prime was performed. cd , lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd cell count and cd cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd values to real cd yield. results are presented as median (iqr). results/finding: in both groups, cd , ly and mo ces were high. target cd dose was successfully reached in procedure in ( , %)adults and in ( . %) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in ( . %) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd cells and cd cells collected/ blood volume was observed in both groups (r . and . in adults and children respectively, p< . ) suggesting cd yield could be predicted based on precd cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd yield and observed cd yield (r . and . in adults and children respectively, p< . ) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of . ( . - . )l of blood in adult procedures, and . ( . - . )l in pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd , ly and mo ce were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* , aniko barta , arpad batai , zoltan csukly , zita farkas , laszlo gopcsa , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) times per case weekly at a dose of million cells/kg. clinical response was assessed days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all patients had received cycles of msctreatment ( dose per cycle). the median age was years old ( - ) with a male/female ratio of : . distribution of the original malignancies (n): acute myeloid leukemia: ; acute lymphoblastic leukemia: ; myelofibrosis: ; myelodysplastic syndrome: ; multiple myeloma: ; t-cell lymphoma: . nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median rd day ( - ). the involved organs were skin ( ), gut ( ), skin and gut combined ( ) and even lung in cases. the median time of msc's first infusion was days after the stem cell transplantation (hsct) and ( - ) days after the first episode of gvhd. of the cycles of msc-treatment led to complete remission ( . %) and resulted inpartial remission ( . %). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with % overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is million cd cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the million cells/kg goal. the ideal minimum cd cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from patients to evaluate the predictive value of the cd cells/ml level. data was collected over months from every patient who underwent a stem cell collection. four patients were allogenic donors and were autologous donors. the patients' weight, diagnosis, and pre-procedure cd cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd cells collected were recorded. the collection efficiency and the cd cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd cells/ml and post-procedure cd cells/kg (r . ). any patient who had a pre-procedure cd cells/ml count of or greater had a collection of at least million cells/kg. any patient who had a pre-procedure cd cells/ml count of or less collected less than conclusion: the pre-procedure cd cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd cells/kg level. to confidently know that a patient will be able to produce the desired million cells/kg, a pre-procedure cd cells/ml count of at least should be obtained. for any patient with a count below , they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between and cd cells/ml should be conducted. heidi elmoazzen , antonio giulivi , michael halpenny* , lisa martin , donna perron , chris bredeson , lin yang , locksley mcgann , paul birch and jason p. acker . canadian blood services, ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso ( % final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd , viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in % dmso and . % hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of . days for anc with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current % dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using % dmso and . % hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from % to %. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is . mg/kg, therefore patients weighing > kg would require a second vial, thus doubling the drug cost. in we implemented a policy of capping plerixafor at mg for patients weighing > kg. this retrospective study compares the mobilization of patients > kg who received capped doses ( ) ( ) ( ) ( ) , with historical control patients ( - ) who received full or uncapped doses. study design/method: patients weighing > kg with crcl > ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of and consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd /cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at mg for patients > kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (< yrs) have a higher pre-apheresis %cd level than any of the other groups, reaching statistical significance when comparing the %cd pre-apheresis between the youngest group (< yrs) and the oldest group (> yrs). hispanic donors show statistically similar %cd pre-apheresis levels over all age groups. moreover, the hispanic older age group (> yrs) had a statistically higher %cd pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last years was analyzed to determine the th percentile, median and th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc ( x cell / ml) and three low wbc ( x cells / ml) concentrations, each at high ( ml), low ( ml) and median ( ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax (pericell protocol, cs. . kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and -aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting -aad viability of % [range - ]% and a hematocrit of % [ - ] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was % [ - ] with a % [- - ] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of [ , ] % and a change in -aad viability of [ , ] % from the input product. the method was found to have a cv of . %. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating a transfusion clinical assessment. in the first phase, cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd -aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to hours post thaw. the second in vivo phase included use of an infusion pump for consecutive autologous patients, with comparison of infusion and transplant outcomes to previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than % within hour, while cd cell viability remained stable up to hours post thaw. small aggregates appeared after hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were . . and . . , respectively (p-value . ). platelet days to engraftment for pump and drip were . . and . . , respectively (p-value . ). infusion rates were slightly higher for the pump group. for control patients, required transfer of products to syringes due to slow infusion rate and others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the zikv ineligible cbus was: caucasian %, asian %, black/aa %, and multi-race %. racial distribution of all clinical cbu donors was caucasian %, asian %, black/aa %, and multi-race %, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: % of all ineligible cbus and % of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete , -diphosphoglycerate ( , -dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring , -dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n ), which were stored in sagm for days, to act as untreated controls. the remainder of each unit ($ ml) underwent treatment with the rejuvenation solution ( ml, minutes at o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros . pc were centrifuged at g in a sorvall rc c centrifuge (sorvall, usa) for min. the combination cryoprotectant dmso dextran (cryosure dex , germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at degrees c (barkey plasmatherm) for min. cpc osmolality was measured with an osmomat osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru u ). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso dextran (cryosure dex ) , as a cryoprotectant, to obtain a final concentration of % dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m were instrumental in automating that phase. pc to be frozen had an osmolality of no less than mosm/l. prp and ppp were frozen at a cooling rate of - c/min and stored at - in the chest freezer for up to months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru u ). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to mosm/l. freeze-thaw recovery of platelets was % or more of the original population. defrosted pc were stored at - with continuous gentle stirring from a helmer platelet agitator for no longer than hours before transfusion. it took no more than min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day and day evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day . a total of lrap units were tested over a -month period: were cultured and rapid tested on day ; were rapid tested on day . the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were true negatives (tn) and false positive (fp) on day when tested by bact/alert, with tns on day . bactx testing results showed tns on day and tns on day . testing using the pgd kit showed tns on day ; and tns and fps on day . fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in % plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day and day during the night shift to be accomplished without additional staffing and allows to extend outdate to day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for year jos lorinser , pieter f van der meer , hans van der heiden and dirk de korte* . department of product and process development, sanquin blood bank, mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers ( ml) is unknown. if these products can be stored at - c it will be feasible to store this product in -star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at - c or <- to - c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <- c for - months and controlled thawing, six different sera were used to fill a large number of mini ( ll) containers, which were refrozen and stored at either - c or <- c. during storage at months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <- c. growth factors tested were pdgf-aa&ab/bb, tgf-ß / / , vegf, a transfusion vol. supplement s egf, fgf . the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß were the most abundant growth factors, on average , resp. ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average ng/ml. tgf-ß , egf and vegf were detected at relatively low values, resp. ng/ml, . ng/ml and . ng/ml. average levels of fgf and tgf-ß were close to detection limit (< . ng/ml). the controls stored at <- c showed for all growth factors close to % of the initial values in samples at t (moment of filling mini containers). for serum stored at <- c for up to months, most factors showed less than % decrease, except for pdgf-aa and tgf-ß , showing % resp. % lower values. for serum stored at - c the values for tgf-ß , egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß showed a decrease of resp. , and %. conclusion: human serum eye drops can be stored in the new micro dose device at - c ( -star household freezers) or <- c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at - c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond year. ruqayyah almizraq* , heather inglis , phillip norris , , jennifer a muszynski , nicole juffermans , jelena holovati and jason p. acker , . university of alberta, blood systems research institute, university of california, san francisco, nationwide children's hospital, academic medical center, canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n per method). residual platelets and white blood cells (wbcs) were measured on day using flow cytometer (fc). on storage day and , number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day , apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p< . , wbd p< . ) and wbf (vs: apheresis p< . , wbd p< . ) methods. while rcf units yielded the lowest count of platelet-evs (cd a ) on day and , the highest number of platelet-evs were in apheresis (day ) and in wbd (day ). similarly, there was significant difference among methods in the number of wbc-evs (cd , cd , cd , cd , cd b ) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day vs day in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< nm) was greater than large evs (! nm) in all of the products on day and , and the highest level of evs < nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p< . ). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at centigrade maryanne c herzig* , crystal lafleur , chriselda g fedyk , sherrill j. slichter and andrew p cap . us army institute of surgical research, u.s. army institute of surgical research, university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least weeks of storage at c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for , , , or days after collection. units were stored for days without agitation. units stored for , or days were agitated during storage with a model hybridization incubator at c set for end over end rotation at - rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at - c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai- ) as another fibrinolytic measure; and complement activation markers c a, c d, c a and c b- . data was analyzed by one way repeated measure anova. results/finding: only % of the platelets were recovered in units stored for days without agitation. these levels did not meet fda requirements of . x platelets per wb unit. subsequently, wb was agitated and platelet recovery was - %. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t (day of collection) and t , , , or measurements. significant elevations of pai- and scd l indicate activation of platelets and inhibition of fibrinolysis (p< . ). activated complement peptides c a, c a, and c d were all elevated over time (p< . ) while sc d- was not. however, only c a and c d levels at t were above normal reference ranges at . and . times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at c for - days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc d- reported, wb showed elevation of c a, a and c d and not sc d- . complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* and christian todd . cerus corporation, community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly . x . x . x . x rcf . x . x . x . x apheresis . x . x . x . x wbd . x . x . x . x platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following collection targets: . x in ml, . x in ml, . x in ml, and . x in ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of . x or . x was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: % of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was . . conclusion: it is possible to treat % of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward % while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* , andrey skripchenko , fei xu , ying li , stephen j wagner , pamela h whitley and jaroslav g vostal . fda/cber/ obrr/dbcd/lch, american red cross holland laboratory, american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature ( - o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts ( hrs ct: hr o c [tc]). autologous apheresis plts stored for -days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n ) and the same non-labeled plts were also infused into mice (n ). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts < % were considered background. results/finding: the mean recoveries of infused plts were . . % for rt, . . % for tc and . . % for ct in humans. in mice, mean recoveries of the same plts were . . % for rt, . . % for ct and . . for ct (mean sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $ % and ct was $ % of rt. in mice tc was $ % and ct was $ % of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to % for rt plts. human tc plts had % auc while ct plts had % auc compared to rt plts in humans. in comparison, the same tc plts had % auc and ct plts had % auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are . and . , respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* and geeta paranjape , . coastal bend blood center, carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g with the compomaster net software for data management. implementation was planned for a november go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g s and compomaster in june . training and validations were successfully completed and a full launch occurred mid-march . device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december . validation was completed and signed off in march of . manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g system. data points were collected from units bi and units ai. results/finding: upon initial implementation, staff training and use, the compomat g was found to be easy. plt weight spread was reduced from an average of gm to an average of gm. actual plt weights were reduced from an average of gm to gm, resulting in an average increase in recovered plasma of . ml per unit. plt count on average increased from a count of to ( /mm ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by . % after implementation of the compomat g and our plt concentrations increased on average by %. we were able to consistently produce a smaller volume plt (average gm), which gave us . ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell , angela hill , brandie dennis and jason p. acker* , . canadian blood services, centre for innovation, canadian blood services, university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: abo/rh matched lr sagm rccs were pooled and split to produce large ( ml) and small ( ml) rccs. the rccs were stored to d and glycerolized manually by mixing ml of glycerol with the rcc in a ml freezing bag. units were frozen at - c for ! h before being removed from frozen storage and thawed in a c water bath. large rccs and small rccs were deglycerolized using the organization's current procedure on the cobe cell processor prior to re-suspension in . % saline, . % dextrose. the remaining rccs were transferred into a l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of %, and deglycerolized in a ml centrifuge bowl on the acp- with re-suspension in as- . rbc quality was tested at h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p . , acp : p . ) and lower cell recovery (cobe: p . , acp : p< . ) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe had higher hemolysis (p< . ) and supernatant potassium (p . ) than did small volume rccs. large cobe rccs had higher hematocrits (p . ), hemoglobin (p . ), and recovery (p . ) than did large acp- rccs. however, all cobe rccs had higher (p< . ) hemolysis ( . . %) levels than did acp- rccs ( . . %). cobe rccs failed to meet regulatory hemolysis standards of . %. conclusion: addition of a ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as- and storage for h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp- cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using . % saline and centrifugation and the semi-automated washing method (sam) using the cobe blood cell processor. study design/method: in this study, units of single donor platelets were evaluated ( washed using the mm and washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas . results/finding: table shows that the average platelet recovery for the sam ( %) was significantly higher compared to the mm ( %). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took - minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas- ) for input platelet doses of . to . platelets in to ml of to % plasma and - % pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas- containing doses of . to . platelets in a volume of to ml. study design/methods: apheresis pcs (amicus v r ) were collected in % plasma and % pas- . one study was performed at the nominal dose ( . - . x platelets), volume ( - ml) in % pas/ % plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition ( . - . x platelets in - ml) using either single or pooled donations. input pcs (n ) were treated with the intercept ts set by the end of day post collection; the incubation time in the compound adsorption device (cad) container ranged from to hours and the intercept treated pcs were stored in containers (n ). day and post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas- treated in the intercept ts set demonstrated acceptable in vitro function (table ). all intercept treated pcs had ph( c) ! . . platelet dose and volume recovery post-treatment ranged from % to % and % to %, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing % plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n ). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n ). numerous in-vitro quality markers (plt concentration, atp, po , pco , ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days , , and for apheresis pcs, and on days , , and for wb-derived pcs. two flow cytometry assays were used to evaluate cd p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion vol. supplement s results/finding: platelet recovery was % and % for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells ( % (test), vs. . % . (ctl) on day ) and a higher rate of cd p expression than control pc units ( % (test), vs. % (ctl)) on day ). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* , lorraine blagg , christi e marshall , herman woodson , sean erony , krishna patel and eric gehrie . the johns hopkins hospital, johns hopkins hospital transfusion medicine dept, johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day . as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day . the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of lrap were tested. lraps initially tested negative by bactx, while lraps initially tested positive by bactx. all initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was . %. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only platelet units. the expected rate of bacterial contamination of platelets is less than per units. the . % initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as- and cp d/as- rbc alan d. gray* , matt landrigan , pamela whitley , michael wellington , sherrie sawyer , shalene hanley , emily rondeau , louise herschel , neeta rugg , patricia a.r. brunker , shawnagay nestheide , jose cancelas-perez , larry dumont and zbigniew m. szczepiorkowski . and , -dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for > hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood ( - ml) was collected and processed at sites into leukocyte-reduced rbc (a total of n cpd/ as- and n cp d/as- ). ml of rejuvenation solution (citra labs) was added to each rbc on day (d- ), incubated for minutes with agitation at c water bath (helmer dh ), washed (haemonetics acp ), and stored in as- at - oc for days (d- through d- ). in vitro recovery (%) was calculated and hemolysis, atp, and , -dpg were determined on day , d- , d- after rejuvenation and washing (postrjv), d- , d- , d- , and d- . all units were cultured on d- postrjv and on d- , and then concentrated by centrifugation on d- . results/finding: in vitro rbc recoveries were . % and . % (as- and as- , respectively) and no bacterial growth was observed. hemolysis on d- was maintained < % in / ( %) as- units and / ( . %) as- units. all as- and as- units ( %) had hemolysis < % following concentration by centrifugation. morphology score was reduced to % (as- ) and % (as- ) by d- , restored after rejuvenation ( %, %, respectively) and maintained through d- (> %). atp was restored and maintained above fresh levels after rejuvenation. , -dpg was restored above fresh levels and was maintained ! % of fresh levels through d- . all values were significantly different compared to d- except as noted (p< . , paired ttest) ( table ) . conclusion: rejuvenation of stored rbc restores atp and , -dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d- when compared to nonrejuvenated rbc on d- . this study is funded by zimmer biomet. storage > hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante , jason p. acker* , and jelena holovati . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during -day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and , -dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome rejuvesol-treated (l r). the prbcs were incubated for h at c with hepes-nacl (sham), liposomes (dopc:chol, : mol%, mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and , -dpg at day hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control ( . . %): l ( . . %, p . ), r ( . . %, p . ), l r ( . . %, p . ). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r ( . . , p . ) and l r ( . . , p . ) treatments compared to s ( . . ) but not l ( . . , p . ). rbc rigidity (kei) increased in all treatments compared to sham ( . . ): l ( . . , p . ), r ( . . , p . ) and r l ( . . , p . ). aggregation amplitude was significantly increased by r treatment only ( . . au vs. . . au, p . ). atp levels were significantly higher in all treatments compared to sham ( . . mmol/g hb): l ( . . mmol/g hb, p . ), r ( . . mmol/g hb, p . ), l r ( . . mmol/g hb, p . ). the levels of , -dpg were no longer detectable in s and l treatments at day . the combined treatment was comparable to r ( . . mmol/g hb vs. . . mmol/g hb, p . ). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer , eric ducas , patricia landry , nathalie dussault , jacques bernier , danny brouard* and anne maltais . h ema-qu ebec, institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the -ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < c) of one to six -ml whole blood units (wbu) within h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between c and c for h under extreme external conditions (- c to c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for h at - c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, -ml wb bags were filled with ml saline . % at t c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (- c) and summer ( c) conditions. shipping boxes were filled with either one or six bags (n ). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under c in . . h and maintain their internal temperature between c and c for h with final values ranging between . c and . c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the c threshold value in . . h and the bags' internal temperatures were within the acceptable range for h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than - c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* , christopher c. c silliman , beth shaz , marguerite kelher and claudia s. cohn . new york blood center, bonfils blood center, department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either % donor plasma (n ) or % pas- / % donor plasma (n ). within hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd ligand (scd l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c ( / products) compared to plasma platelets ( / products); however, the hla-antibody screen-positive supernatants of pas- a transfusion vol. supplement s abstract c platelets had fewer hla specificities ( specificities) compared to those of the plasma platelets ( specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd l levels were increased in the supernatant of pas-c compared to plasma platelets (table ) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd l and not bioactive lipids. although scd l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below x wbc in us and x wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n ) and leukoreduced (lr) rbc units (n ) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of wbc/ul. the spiked samples of , . , , , and wbc/ul were prepared from the source sample of wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations ( , . , , , , wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples ( , , , wbc/ul). tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of samples (plt and rbc) were run on both analyzers, repeated for days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt . , slope . ), (r-rbc . , slope . ). the %diff-plt at , , wbc/ul were . , . and , respectively. the %diff-rbc at , , wbc/ul were . , . and . , respectively. the average total testing time was similar on both instruments; min for the facsvia and min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated % ( of min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* and mickey koh . department of laboratory medicine and pathology, university of minnesota, st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of completed and partially completed surveys were received. results/findings: responses came from countries, but the majority of responses came from the united states (us). of the respondents, % reported aprp use in their hospital. aprp was used predominantly for outpatients, though > % of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by - mds; however, hospitals had > mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures ( %); however, respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, ) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n ) under blood bank conditions at day (d ), at day (d ), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from . - . % at d to . - . % at d . rejuvenation markedly reduced this storage-induced spherocytic shift ( . - . %) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell , angela hill , tracey turner , april xu , brandie dennis and jason p. acker* , . canadian blood services, centre for innovation, canadian blood services, university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units ( ) than in rcf units ( ), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as- additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: small rcf ( - ml) and large wbf ( - ml) rccs were stored for d before being glycerolized and frozen at - c for ! h. large rccs whose red cell mass exceeded the capacity of the ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a c water bath, deglycerolized and re-suspended in as- . rccs were stored d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p< . ) hematocrit, specific gravity, hemoglobin per unit, supernatant k and na concentration, deformability (ei max ), and higher (p< . ) recovery than did large wbf units. no significant differences in hemolysis, atp, , -dpg, p , rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table ) , however of large wbf units had rbc recoveries < % due to pre-glycerolization volume reduction, and of the small rcf units had hemoglobin values < g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above % and the hemoglobin failure rate would be below % of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. . . . . . . . . elongation index ( pa) . . . . . . . . this study is funded by zimmer biomet. (hasan ) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two ( ) rbc units (leukocyte-reduced), cpd/as- or cp d/as- , on day , day , and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o /g hb) and total releasable oxygen (tro) of the unit (ml o ). orc was determined by assessing the change in % o saturation from mm hg po (e.g., lung) to mm hg po (e.g., venous blood) multiplied by . ml o /g hb (li ). a simulated baseline pretransfusion vo of ml o /min was estimated using the day orc and assuming a g/dl transfusion trigger with a cardiac output of l/min and l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day restored orc and tro to levels greater than day ( table ) . orc of the rejuvenated unit was . . times and . . times greater than rbc on day and day , respectively (p< . ). vo increased after a simulated single unit transfusion of rbc (day , day , and pw) by . %, . %, and . % over the pre transfusion vo , respectively (p< . ). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release . times the volume of o compared to standard, untreated rbcs stored for days. inferior oxygen delivery to tissues (vo max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for days vs days which seem dependent on genetic variability and storage time (bennett-guerrero ). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* , jay srinivasan , gustaaf de ridder , alan d. gray , matt landrigan , keaton charles stoner , angela crabtree , jessica poisson and ian welsby . duke university school of medicine, duke health pathology, citra labs, a zimmer biomet company, zimmer biomet, duke university, department of pathology, durham veterans affairs medical center, duke university hospital, duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted , -diphosphoglycerate ( , -dpg). the loss of , -dpg increases the oxygen affinity of hemoglobin, resulting in lower p (partial pressure of oxygen at % hemoglobin saturation). decreased p may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and , -dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a , leukoreduced prbc stored in as- were obtained from our local blood center. after days of storage, units were divided into separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution ( ml) was added to the cr group, and all groups were then stored for another days at - c. on day of storage, the sr group was incubated for hour at c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps ) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a . . results/finding: significant differences in p were noticed between all groups (table ) . ei, ps , and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p ) seen over days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc ( ml) units stored in as- , obtained from a regional blood donor center at expiration ( days), were passed by gravity through sorbent-devices containing ml of multifunctional polymer bead, at a flow rate of ml/min. supernatants were analyzed for k removal as well as free hb, antibodies and cytokines ( -plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k ] from . . to . . meq/l; equivalent to an . % reduction. free hb was reduced by . % from . . to . . mg/ml. antibodies, specifically igg, iga, and igm decreased from . . to . . mg/ml ( . %), . . to . . mg/ml ( . %), and . . to . . mg/ml ( . %), respectively. inflammatory cytokines were significantly reduced, specifically: ip- from . . to . . pg/ml ( . %), mip- b from . . to . . pg/ml ( . %), and pdgf from . . to . pg/ml ( . %). filtration had no significant impact on cell surface markers of rbc viability (< . % decrease) or sensitivity to osmotic changes. values listed represent mean sem (p < . for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency ( . %) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in apheresis and whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and , -dpg profiles to fresh levels. the objective was to compare % hemoglobin-oxygen saturation (p ) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was . . %. hemolysis (%) was similar on day before and after dry-air incubation with the rejuvenation solution ( . . % vs . . %). percent hemolysis (%) decreased after washing ( . . %) and was maintained below < % for all units during storage for hr ( . . %). average atp and , -dpg were restored above the average fresh values. the morphology score decreased $ % by day , which was restored to near fresh values following rejuvenation and washing and storage hr ( . % and . %, respectively). rbc oxygen affinity, as assessed by p , was restored above fresh values. all values were significantly different compared to day (p< . , paired t-test) ( table ) . conclusion: rbc morphology was restored to near fresh and average atp, , -dpg, and p were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and , -dpg were maintained during storage hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d tetrahydrocannabinol (thc) and -oh-d -tetrahydrocannabinol ( -oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and -oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and -oh-thc to produce samples for lc-ms/ms analysis. lc used a c column. post-column detection by ms/ms used positive ion electrospray with q :q ion pairs of m/z . : . (internal standard (is), d -thc), m/ z . : . (thc), and m/z . : . ( -oh-thc). quantitative results for thc and -oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from - ng/ ml for both thc and -oh-thc. limits of quantitation, defined as standard deviations above background, were . ng/ml for thc and ng/ml for -oh-thc. results/finding: a total of donor plasma samples were tested for thc and -oh-thc. no samples tested positive for either thc or -oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a % probability of one or more positives at a prevalence of . % positive samples, and a % probability of one or more positives at a prevalence of . % positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than % among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than hours for post-exposure detection of thc and/or -oh-thc in plasma. conclusion: testing of donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than %. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* , ruqayyah almizraq , daniel millar and jason p. acker . university of british columbia, university of alberta, lightintegra technology inc., canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days , , and of storage. one rcf rcc was tested on days , , , , and and six ml aliquots were stored in parallel and tested on days , , and . all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days , and (p< . ) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the aliquots were consistent at each time point but statistically higher than in the original rcc on and after day of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow % screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n ) and paired ffp aliquots were stored for - days at - c and - c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! % levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c esterase and alpha -proteniase inhibitors). the level of factor xiii in odp was slightly lower, about % of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i ii and ddimer) and complement (c a and c a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion vol. supplement s abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to % in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to minutes. in addition, donations with collection times between and minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from - minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n ) were prepared from one - minutes bc and ml of autologous plasma in a ml pvc-dehp container. as a reference, spc from donations with collection times of < minutes were prepared (n ). in addition, pc were prepared from bc, of which at least bc were from - minutes donations (n ). after pooling of the bc, ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for days at c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume ( vs. ml) and platelet content ( vs. x ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day , ph( c): . . vs . . , other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day and/or in / pc (possibly because sometimes ab incompatibility was accepted). on day , plt showed low cd p expression ( . . %) and phosphatidylserine exposure (annexin v binding, . . %). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from - minutes whole blood donations had a normal composition and showed good in vitro quality during day storage. to substantiate that the exclusion of - minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* , jessie miller , ranee marie wannarka-farlinger , sandra bryant , scott a hammel , sherry stern and camille van buskirk . mayo clinic, mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as- rbcs, irradiated and non-irradiated, were selected for the study. the units varied in age, ranging from to days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< . ) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p . ). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after days of storage pei lun karen lim* , erma sofia sumardi , isamar eduardo ancheta , susan lim , christina yip , lip kun tan and shir ying lee . national university hospital singapore, national university hospital, national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within hours of processing and stored at - c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for days and kept at to c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than iu/dl. study design/method: randomly selected units of ffp (n ) were measured for fviii concentration based on clotting assay (stav r -deficient a transfusion vol. supplement s viii diagnostica stago). fviii levels were measured at five time points: prefreezing, , , and hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at to c for minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration ( hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at to c for days for subsequent testing. results/finding: results obtained were listed in table . units to were not tested for fviii at post thaw- hour due to operational issues. the overall fviii concentration decreased at an average of % from pre-freezing to post thaw hour. after further storage of tp post thaw- hour and - hour, residual fviii level remain to be above iu/dl except unit which had a lower initial fviii concentration. at post thaw- hour, out of units tested had residual fviii activity within the pre-set standard of iu/ dl. the average decline from -hour post-thaw to -hour, -hour and hour post-thaw was . %, . % and . % respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least iu/dl of fviii. typically patients with factor levels below iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi , ayman mohamad sabri , ali abdullah alajeafi , ashwaq hasan alhekri , saleem bin mahfouz , ali hasan alkhodari , rawya saeed shealy , marcus picard-maureau* and hussain bana almalki . king abdulaziz hospital and oncology center, cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in % plasma over a day storage period and the new "test" pathogen-reduced, pooled (pools of ) prp pc in % plasma over a day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of leucoreduced test pcs were assessed at day of storage and compared to leucoreduced control pc at day of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day ; the process was completed by day post-collection. samples were taken daily for quality analysis from test and control pc until day and day , respectively. for bacterial spiking, additional pc were spiked with each receiving ml of mcfarland ($ . x cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was . % . , the total average platelet loss at day was . % . . the average platelet loss in the control units at day was . % . . the average ph of the test units at day was . . and in the same range as the control pc, ph . . . glucose concentration in test pc at day ( . . mmol/l) was lower than in the day control units ( . . mmol/l). lactate levels increased during the course of storage; lactate levels at days and were outside the range of the assay (> mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* , jay srinivasan , jessica poisson and ian welsby . duke university, duke university school of medicine, duke university hospital, duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified proteins in cryo; of the most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf ( . mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial ( Á lm) filtration. cryoprecipitate mini-pools ( ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di( -ethylhexyl) phthalate (dehp)] adsorption device and a Á lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table ) . kit ensured bacterial sterility (table ) and most importantly, final product was free of hbv, hcv and hiv (table ) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase- , thereby blocking synthesis of thromboxane a from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n ) were prepared from a nsaid-bc and ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for days at c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n ) were investigated as a reference. values are expressed as mean sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume ( vs. ml) and plt content ( vs. x ) were similar in both groups. on day , both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day was significant higher in a subset of donors who had used ibuprofen (n ). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin ( , - , p< . ), diclofenac ( , - ) and naproxen ( , - , p< . ), compared to normal controls ( , . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (< hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, ] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type diabetes (t d). because of the strong rise of people with t d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t d, but accepted as donor. study design/method: twelve whole blood donors with t d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for days at c and sampled on day , or and . the diabetic marker hba c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 'good' (ph day > . ) and 'poor' (ph day < . ) storing spc were selected and analysed in more detail. results/finding: donors were of age year and primarily men ( %). donors with t d had a higher mean bmi ( . . vs. . . kg/m ) and higher hba c than controls. the spc of both groups had the same volume ( vs ml) and plt content ( vs x ) but on day glucose concentration was higher in the diabetic group ( . . vs . . mm, p< . ). on day , the average in vitro quality was comparable in both groups (data not shown). when combining a transfusion vol. supplement s the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed ( . . vs . . mmol/ day/ plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc- ) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day ('poor': . . vs 'good': . . , p< . ). conclusion: bc from donors with t d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table ). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* , , katharine a downes , , hollie m reeves , and robert w maitta , . case western reserve university school of medicine, university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy month old unvaccinated girl presented with history of diarrhea for days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: x /l, platelets: x /l, bun: mg/dl, creatinine: . mg/dl, lactate dehydrogenase u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to x /l, adamts sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation ( x /l and a-ipc of . x /l). two consecutive tpe were completed which resulted in a platelet count decrease to x /l and a-ipc of . x /l. a-ipc ratio was . below the ratio of which has been reported for ttp patients. similarly a-ipc count was not below x /l threshold reported in setting of ttp with severe adamts deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o :h toxin. testing of c , c , factor h, factor h autoantibody, factor i and factor b were normal. adamts activity was %. patient was treated for the infection and platelet count improved within days to x /l, with resolution of her renal failure: bun: mg/dl, creatinine: . mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts activity. many patients with severe autoantibody-mediated adamts deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts activity < %) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value - days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column ), increased peak a-ipc value (results shown as percent increase, column ), delayed a-ipc peak, and delayed plt recovery (table ) . moreover, recurrent episodes required more procedures compared to initial presentation (table ) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february to march on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version . after taking consent from the donors. the target collection of each procedure was a dose of x platelets in - ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at consecutive donations within days were considered. data was analyzed by stata . within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the donors, repeated the plateletpheresis within a week (group i) and underwent nd plateletpheresis within - days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the groups (p . ). though above the eligibility cutoff of . lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at nd plateletpheresis in group ii donors. there were donors who presented to us for the rd time for plateletpheresis with a mean gap between st and rd plateletpheresis being days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p . ). plateletpheresis through all the cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type- receptor antibody (at rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at r ab are reviewed. results/findings: case : the patient is a currently -year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age , and a second deceased donor transplant due to a rejection of the transplanted kidney at age . three years post-transplant, her creatinine (cr) started to rise from . to . mg/dl and a biopsy showed banff criteria grade amr, grade a t-cell mediated rejection (tcmr) and grade interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at rab was identified at > u/ ml (high: > u/ml, intermediate: - u/ml, negative: < u/ml). she received tpe treatments every other day and started losartan. after a course of tpe, at rab decreased to u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to . ml/dl. in one month, her at rab increased again to > u/ml, therefore, she received more tpe treatments with a decrease in her at rab to u/ml. although at rab level increased slightly to u/ml after months, her cr has been stable at . - . ml/dl. case : the patient is a -year-old mean /-se - . /- . % * . % /- . %* * p< . a female with malignant hypertension who received a deceased donor kidney transplant at age . her cr started to rise weeks post-transplant from . to . mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at rab level at u/ml. she received tpe procedures every other day and at rab decreased to u/ml with a decrease of cr to . mg/dl and improved arteriopathy in histology. because her at rab level slightly increased to u/ml over the next weeks, she started weekly tpe treatment. after weekly tpe, tpe treatment was stopped because her at rab level remained relatively unchanged. her cr has been stable at around . ml/dl to date. conclusion: we present kidney transplant recipients who received tpe treatments for high at rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at rab levels; however, weekly tpe had no effect on reducing at rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < %], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a year old man (patient b) with a medical history of hypothyroidism (on synthroid for years), end stage renal disease and non-ischemic cardiomyopathy (ef of - %) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a - . plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within hours of the procedure completion. their total t , t and free t levels trended to normal or near normal range within hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy - weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* , laura martínez molina , cristina muniesa montserrat , octavio servitje bedate , silvia cosano navarro and maria isabel gonz alez medina . banc de sang i teixits, dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) , as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category . since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of patients diagnosed with ss and compare them in their first evaluation once the th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( -methoxypsoralen, -mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from . to total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in / patients) and with online system (therakos) just in . main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is ' % (partial remission . % and complete remission . % with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : u/l) requiring transfusions, mild thrombocytopenia ( x /l), acute kidney injury (bun mg/dl, creatinine . mg/dl). by the third hospitalization day hgb improved to g/dl, however with worsening thrombocytopenia ( x /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days - ). platelet count and a-ipc improved to x /l and . x /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count ( x /l) and a-ipc . x /l. these dynamics did not resemble those which had been described for ttp patients with adamts deficiency. adamts obtained prior to tpe initiation was resulted at this time and was %. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc ( . x /l) that preceded platelet count increase to x /l three days later when patient was discharged. other laboratory values at this time were ldh of u/l, hgb: . g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts deficiency eiman hussein* and jun teruya . department of clinical pathology, cairo university, texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts . since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts , the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts . the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january through march were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts activity of less than %. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts deficiency. eight patients ( %) were associated with suspected bacterial infection. four of the patients ( %) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than kilograms using a single apheresis procedure. study design/method: in october and june , two children with possible leukemia were submitted to tl procedure. they were and months old, and weighted , and , kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, % hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin ( ml of acd-a and , units of heparin), at a blood to anticoagulant ratio of : . a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were . and . / mm . the formula "collection pump flow , x inlet flow x preapheresis wbc count" was used with the goal of removing up to x leukocytes/ml. a single leukapheresis procedure was performed with total blood volume processed per patient. immediately after the -hour procedures, wbc count were . and . wbc/mm , and -hour post tl, wbc count were respectively . and . /mm . net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since . we report the data from the year . study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of , apheresis procedures were performed at hospitals. therapeutic plasmapheresis was the most frequent procedure ( . %) followed by autologous peripheral blood stem cell (pbsc) collection ( . %), allogeneic pbsc collection ( . %), donor leukapheresis ( . %), and therapeutic leukapheresis ( . %). cobe spectra ( . %) and amicus ( . %) were the most widely distributed instruments. centrifugation was the dominant technique ( . %) for therapeutic plasmapheresis. detailed information was given for , therapeutic plasmapheresis procedures performed on patients (some items were not completely filled out). spectra optia ( . %) and cobe spectra ( . %) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently ( . %) as the replacement fluid followed by % albumin ( . %), % albumin ( . %), and % albumin ffp ( . %). most of the procedures were performed for plasma volume ( . %). acd ( . %) and heparin ( . %) were used for anticoagulation. central venous catheter ( . %) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation ( . %), antibody mediated rejection in renal transplantation ( . %), thrombotic microangiopathy ( . %), desensitization for abo compatible renal transplantation ( . %), neuromyelitis optica spectrum disorders ( . %), and hyperviscosity in monoclonal gammopathies ( . %). adverse reactions were observed in . % of the procedures. allergic reaction ( . %), hypocalcemic symptom ( . %), and hypotension ( . %) were frequently reported. therapeutic effect was achieved in . % of the patients. our apheresis registry has been well run for years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride > - mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $ deaths/ , cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of pts who were diagnosed with hp from january, through january, , and referred for immunotherapy evaluation. / ( %) pts received conventional therapy (ct) and pe (pe group), and / ( %) pts received ct alone (ct group). mean age was years (range - ), and % were female. baseline mean triglyceride level (normal < mg/dl) for pe group was , mg/dl ( , - , ) versus , mg/dl ( , - , ) for ct group. baseline mean lipase level (normal < u/l) for pe group was , u/l ( - , ) versus u/l ( - , ) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving - medications. / ( %) of pe group and / ( %) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. / ( %) of pe group and / ( %) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of . pe treatments (txs) (median of , range - daily txs) using % albumin; / ( %) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were < - mg/dl and lipase < - u/l ( . - . x upper limit of normal). mean triglyceride levels after pe txs were , mg/dl ( - , ) for pe group (mean decrease %); mean triglyceride levels after additional hours of ongoing ct were , mg/dl ( - , ) for ct group (mean decrease %). while the pe group achieved a greater mean decrease in triglyceride levels after pe txs (compared to the ct group after hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p> . ). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a -year-old man with a chronic history of hypertriglyceridemia > mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa guidelines, in a patient unresponsive to optimal medical management. asfa guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely % albumin for exchange fluid ( % albumin procedures) or partial plasma replacement ( - units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while were performed with % albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* , , metha apiwattanakul , sompis santipong , jutaluk jaipian , jettawan siriaksorn and ponlapat rojnuckarin . chulalongkorn university, king chulalongkorn memorial hospital, prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june through february were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a % solution. before using, it was diluted to a % albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of tpes in patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was , (range , - , ) ml. although the corrected calcium level was low (< mg/dl) in . % ( / ) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in patients. the first patient had events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was . % ( / ). in , the incidence of tpe adverse effects was . % ( / ) when commercial albumin was used. the difference was not statistically different (p . ). median serum albumin levels pre-tpe and post-tpe were . ( . - . ) and . ( . - . ) g/dl. the increase in serum albumin after tpe was statistically significant (p< . ). eighty-two percent of pre-tpe serum albumin levels were lower than . g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether outpatients who underwent a total of la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer ( %) and ovarian cancer ( %). based on differences in the study protocols la was performed either one-time ( %), two-times ( %) or three-times ( %), with an interval of at least weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, out of patients ( %) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in % of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was . x wbc consisting of . x mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was % lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg -related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to , iu/mls (ref. range < ) and anti-cyclic citrulline peptide antibody was elevated to , units (ref. range < ) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were , and mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was . . peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be . centipoise (cp) at admission (ref. range . - . ). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated - total plasma volume; replacement fluid - % albumin and normal saline in a : ratio; replacement fluid volume: % of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to . cp. serum igg, igm and iga levels decreased to , and mg/dl respectively. her rf had decreased to , iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan -dec ). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with % human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between to years age (m: f; : ) underwent tpe procedures with an average of . per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three ( %) patients had only visual symptoms, ( %) had both visual as well as muscular symptoms whereas ( %) patients had muscular symptoms only. three ( %) out of the seven tested, were positive for aqp -igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in patients with grade- , in patient, and by grade- , in seven. adverse events were observed in % ( / ) of the procedures with allergic reactions to replacement fluid as most common event (n- ) followed by hypotension (n- ). follow up was available in % ( / ) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from . to . for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version . as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version . . the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to / / , plt collections were performed on nine trima accel machines operating with version . . upgrading and validating all nine machines to version . occurred from / / to / / . the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version . ( / / to / / ) was compared to version . ( / / to / / ). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version . and . , adjusting for multiple visits per donor, with significance defined as p-value < . . results/findings: following the upgrade to version . , staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version . of the trima accel showed a statistically significant increase in possible leukocyte contamination from % to % of collections as compared with version . . both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version . . conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version . software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version . currently does not provide added value over version . for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of ml % albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with acute episodes requiring prolong hospital admission of approximately months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of mg/dl, lipase u/l, glucose mg/dl, bicarbonate mmol/l, anion gap . ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day after admission. results/finding: the patient tg decreased by % ( mg/dl) with medical therapy, followed by additional % ( mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below mg/dl at days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from to , a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected , cases whose ferritin levels have been monitored more than twice with an interval of detection in - days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over lg/l. and the upper limit was set to be lg/ l in male and lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: times, to times, to times, to times and more than times. the high frequency (more than times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were , donors included in the study, of which , were male ( . %) and were female ( . %). the mean ferritin was . lg/l in male ( % ci: . - . lg/l) and . lg/l in female ( % ci: . - . lg/l). the result of anova indicates that the group with the highest frequency (more than times) has the significant lowest ferritin level (p< . ). the average change of ferritin if donation over times would up to . and . lg/l in younger and elder y/o male and and lg/l in female. and then for high frequency (half a year more than times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over times in $ days) was reduced from . lg/l in the first period to . lg/l in the third period ( period $ days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* , mary townsend and lizabeth rosenbaum . university of new mexico hospital, blood systems, inc., blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* , william korzun , teresa nadder , susan roseff and elizabeth ripley . virginia commonwealth university, virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for % of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a -hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from to could be a result of a change in the blood drive timing of the training schedule of that location. in , basic trainees at site a were scheduled at day of . in january , the blood drive date changed to day of . the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from to of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in . these observations support the hypothesis that the increase in hemoglobin deferrals in resulted from the implementation of the male hemoglobin standard change from . to . g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional % of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* , eva alonso , oscar bascuñana , monica romero , teresa vich , elena castaño , laura carbonell , eva palomas , saray almerge , francesc carpio and xavier curia . banc de sang i teixits, institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january to december we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, donors came to give blood, of these, ( %) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t but his hemoglobin levels were lower than our selection criteria. of the donors selected for donation ( . %) had sci lower than t and t . adverse reactions to donation ( . %) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of , automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an month period from january to november . the same information was assembled for the automated bp process for the month period of january to november . the automated bp process implemented in mid-december ; so the december data for both and has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < . . results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p . ). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < . ) with the automated bp while and reactions remained non-significantly lower (p . ). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below . g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < . . results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under . . statistically more visits with hgb less than . g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under . was . % higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from january to march . after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of donors who has donated blood in the blood bank's main branch were used as the baseline for this study. % of donors (n ) accepted automatic appointment booking, whereas some donors (n ) were not comfortable with it. % of those who declined still preferred walk-ins (n ) based on their own time schedule, the rest decided that variable situations (n ), donation frequency (n ) and choice of preferred donation locations (n ) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was %. a comparison was made and found that this study shown a significant increase of acceptance rate by %. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. ) , . . poisson distribution, normal distribution, logistic distribution, lognormal distribution a transfusion (p> . ) in donor and reference populations except in younger ( - yrs) male donors (p< . ; donor . %, reference . %). mean donor sbp, dbp, and pulse were . mmhg, . . mmhg, and . . bpm, respectively. screening blood pressure levels consistent with hypertension ( . % male; . % female) in the - year donor group, significantly (p< . ) higher than the reference population ( . % male; . % female). no differences were observed in the - year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in - year old females. developing blood donor educational materials gay wehrli* , susan rossmann , louis m. katz and dan a waxman . university of virginia health system, gulf coast regional blood center -sugar land, americas blood centers, indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, . page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table . results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level ( th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from to after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from . % to . % in and , respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from . % in to % in . moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately . million doses of ap transfused within . billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since . firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than times and had not donated for more than days or less than times with an interval of more than days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by . times from to and the doses of ap increased by . times from to within years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into groups: those who donated ap once, those who donated - times, - times, - times, and those who donated more than times, respectively. it was found that the number of permanent ap donors who donated ap more than times was only ( . %), but they denoted a total of doses of ap ( . %) from to . conclusion: aps increased at a rapid and steady pace in wuhan blood center from to , which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at sites on consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer < ng/ml and zpp levels > umol/mol heme) at hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp . % (r . ) at first and . % (r . ) at second donations. at first donation when compared to fs hb, only . % (r . ) of variation could be explained by variation in fs zpp, . % (r . ) by ven zpp and . % (r . ) by ven fer. at second donation, when compared to fs hb, only % (r . ) of variation could be explained by variation in fs zpp, . % (r . ) by ven zpp and . % (r . ) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p< . ) suggesting strong evidence against correlation. % ( ) responded to the survey of which % ( ) reported not feeling well after donation. it should be noted that noted that % ( ) female study participants reported feeling unwell after the first donation and had ferritin levels below ng/ml but the zpp levels were less than umol/mol heme. of the % ( ) male participants that reported not feeling well none had ferritin levels below ng/ml nor ven or fs zpp levels above umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from . to . gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from blood centers over two intervals, july-dec. and july-dec. (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male m, female f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab . , chicago il). p <. was considered significant. results/findings: data were provided by of centers invited, representing , , and , , wb donations and , and , ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from . % to . % in the two intervals among aggregated donation attempts (p<. ), and for m ap from . to . % (p<. ). the mean "by center" deferral rates (table) were similar to that and significant (p<. ). mean by center hb deferral rates among f donations during the two intervals were . and . % (p . ) for wb, . and . % (p . ) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only centers could provide specific high vs. low vs. irregular pulse deferrals; provided only a summary (i.e total pulse deferrals), and could provide none. for bp, provided detail (high vs. low), summary and none. p deferrals increased in the successive intervals among f wb donors from a center mean of . to . % (p . ) and for m wb donors from . to . % (p . ). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba c levels among those with rbs > mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from st march to st march . total of , blood donors were tested for rbs. those with rbs > mg/dl were further tested for hba c by gold standard hplc method using variant ii biorad. blood donors with > mg/dl rbs and hba c > . % were advised to consult a physician for further evaluation. results/findings: of the , donors tested, ( . %) donors showed a rbs of > mg/dl. forty two ( . %) were males and ( . %) females with a mean age of . years ( - years). of these, ( . %) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining , ( . %) of them had a family history of dm. of these donors, donors did not give a consent for testing for hba c. among the donors tested for hba c levels, ( . %) had hba c > . %. all the donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is . % ( of donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* , hwee huang tan and ai leen ang . health sciences authority blood services group, health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from . to . g/dl last may . the current minimum acceptable hemoglobin for male donors in singapore is . g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels ( . - . ) and in donors with hemoglobin g/dl and above. study design/method: during a month period, serum ferritin testing was performed on regular male whole blood and regular male apheresis donors who made at least donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin . - . ) group b (whole blood with hemoglobin ! , group c (apheresis with hemoglobin . - . ) and group d (apheresis with hemoglobin ! ). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below ug/l is considered low and levels below < ug/l are considered having absent iron stores. results/findings: . % of donors in the study have ferritin levels below ug/l. there were more donors with low ferritin in group a compared to group b, % and % respectively (p< . ). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, % and % respectively (p . ). ferritin results for the groups can be seen in table . conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or . % have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin . - . g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to . g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of % male and % female students in the age group of - years. only % of the students have heard about voluntary blood donation and % of the students have given blood once in their lifetime and among them % are blood donors at the moment. % of the participants believed that there is a specific reason why they don't donate blood and % believed that there is a risk involved for the donors, when donating blood. % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. ( ); miscellaneous effects were reported in courses. side effects led to interruption of supplementation in instances. ferritin levels (mgt sd) at entry into the program and at the last visit were . and . . mg/l in participants, vs . . and . . mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took ! % of the tablets. ferritin levels< mg/l were found in , % of participants and . % of controls. deferral for low hemoglobin was below % in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only % of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under cfr . and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § . to make a collection under this provision if the requirements set forth in § . (a)( ) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in , an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of - ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached , of whom ( %) are c y homozygotes. without active recruitment, accrual rate is about per quarter, with % of subjects qualifying as allogeneic donors. the mean current age is . years, % male, % caucasian. the majority of hh donors ( of an active cohort of ) are in the maintenance phase of therapy with an average of . donations/year and a % deferral rate. over the last years, hh donors contributed approximately - % of the hospital's allogeneic blood supply, averaging whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided - % of blood for in vitro research at our institution with an average of wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over years. since / / , with an increase in male hgb deferral threshold to g/dl, there has been only hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm- non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm- non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm- non-invasive occlusion spectroscopy device. over a span of days, eligible blood donors, both male and female, were first screened by the nbm- non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the blood donors for the performance of hb measurement on the sysmex hematology analyzer within - hours of collecting the venous samples. results/finding: the sd of the difference between the nbm- non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed . g/dl. the hb measurements obtained from the nbm- and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be . g/dl. the precision of the nbm- yielded a co-efficient of variation of . g/dl and a standard deviation of . g/dl. conclusion: the operators found the nbm- easy to install, maintain, and operate with minimal training. the nbm- non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder , ravi reddy , dhuly chowdhury , don brambilla and edward l. murphy* . sanbs, rti international, ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in and followed them for one year. within days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used -point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. ) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included , first-time black donors with median age and female predominance ( %). within one year, , donors ( %) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable ( strongly agree to strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) . , % ci . - . ), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or . , % ci . - . ). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or . , % ci . - . ) and "i wasn't treated well by the <blood center> staff" (or . , % ci . - . ). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or . , % ci . - . ). a secondary analysis treating the likert scales as -level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard , ramya ghantasala , obhijit d hazarika , nicole leonard , cori a polonski , zachary b wunrow , michelle heleba , jan k carney and mark k fung* . university of vermont larner college of medicine, american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" ( . %) and "traveling is a time for me to relax." ( . %). of the respondents who travel in the summer, very few reported donating while traveling ( . %). summer donation rates between summertime travelers ( . %) and non-travelers ( . %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website ( . %) and phone ( . %). willingness to use a regional blood donation smartphone app was highest among respondents ages of to ( - %) and lowest among ages and older ( - %). of respondents with no prior knowledge of summer seasonal shortages ( %), / rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv , and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin- (il- ) and il . others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent hard spins at rpm for minutes with separation after each spin on a compomatev r g . plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers ( m [ %]: [ %] f); median age years (range - ) donated a unit ( ml) blood from which buffy coats (average volume ml) were processed. the buffy coat process was previously validated on wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures and . all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by % within months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for days. results/findings: there were completed responses, of which ( %) indicated that their hospital had an msbos and ( %) did not. the majority of hospitals without an msbos were academic centers ( / , %) from oceania ( / , %) or europe ( / , %), had between - beds ( / , %); the majority of these hospitals transfused between , - , rbcs ( / , %) per year. / ( %) are going to implement an msbos in . of those with an msbos, the majority / ( %) were from north america. the majority were academic hospitals ( / , %) with - beds ( / , %) that transfused ! , rbc units per year ( / , %) offering a wide range of surgical services. on average there were procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of % of the procedures listed, a pre-operative type and screen for %, crossmatching rbc units for %, and for % of procedures a different recommendation was made. most ( / , %) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only / ( %) of msbos' were created solely by using procedure-specific data, and most ( / , %) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually ( / , %), and the hospital transfusion committee is often ( / , %) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents ( %) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, % of respondents felt that it was regularly used by all surgeons and anesthesiologists; % felt that it was not used at all at their hospital, % did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, % of the hospitals currently without one indicated that it would be implemented in suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a -bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see , hospital admissions and nearly , emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in was approximately $ . m. in nov. , an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $ . m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns ( and ), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee . development of evidence based transfusion triggers. . education on evidence based transfusion triggers across multiple campuses, specialties and resident programs . clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to " " unit instead of " " units. . updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in , we were still able to reduced blood product expenditures by $ , when compared to . conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* , anna w rains and christopher t clark . university of tennessee graduate school of medicine, univeristy of tennessee medical center, univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for week post-delivery, with cost of $ . per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc system. by using the blood volume values, and assigning a value of . ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in , there was a total of , infants delivered at our facility. out of all the deliveries, ( %) infants were transferred to the nicu. of those infants, % received at least one red blood cell transfusion and % received at least one platelet transfusion. of the infants transferred to the nicu, ( %) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to % (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from . % all the way up to . %. in those infants, the birth weight ranged from - grams, and the gestational age ranged from weeks to weeks and days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* , shannon davis , suzan new , vaishali patel and oren guttman . university of texas southwestern medical center, ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable : communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to % of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july ; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of randomized controlled trials ( , conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* and stephanie rogers . dignity health st joseph's medical center, dignity health background/case studies: over million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels > . g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of . g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of . g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to hospitals including cme presentations, online physician and nursing educational videos, communication tools including infographics and " is the new " buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb > . g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy to fytd , there was a % reduction in prbc units transfused to patients with hgb > . g/dl, starting at a baseline of % down to %. this represents an fy annualized savings of $ . m, from a baseline of units per , patients days down to an average units and approximately , fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels > . g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm - . ) reduced rbc units and two studies decreased the percentage of patients transfused (or . ). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or . ) and rbc units transfused (sdm - . ). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a- method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was g/dl/ % or g/dl/ %. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately % of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was units/ month. after the first months of buc activation the mean number of units was units/month a reduction of units/month or % of nonsurgical blood use (p . by t-test). non-surgical rbc use now represents approximately % of the total rbc use hospital wide a % reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital - month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital ( beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance ( q vs. q ): present and completed consents ( vs. %), present and completed nursing flow sheets ( vs. %), transfusion thresholds supported ( vs. %), discharge instructions provided ( vs. %); ( q vs. q ) vital sign compliance ( % vs. %). jwp increased from to ( / - / ). cost savings were realized by decreased utilization and implementation of bpa. (table - q ) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately - trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only percent of coolers fully used. it also consumed valuable staff time as technologists typically made - trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated - hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains - units of group o rhd negative, units of o rhd positive, and units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in , the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june -october were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a -month period, , cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table below. overall, ( %) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." ( ), "active bleeding" ( ), "platelet count of . . ." ( ), and "downtrend" ( ), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, ( %) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years - . study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of , g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey ( questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of complete responses ( %). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, % with physicians, and of these, % reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only % responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of - g/dl ( %), platelet count of - , ( %), and inr of greater than . ( %). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per , inhabitants may vary folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies ( . % and . %), for the remaining strategies weights varied between . % and . %. we estimate that , patients would be eligible for pbm strategies in one year time horizon, resulting in premature death avoided ( . % reduction) corresponding to a gain of approximately , life years and a reduction of , ( . %) disability adjusted life years (daly) relative to the current clinical practice. a decrease of , in-hospital days is expected mainly due to a . % reduction in hospital length of stay and a . % reduction in -day readmission rate. in this population the overall transfusion rate could decrease to . % from the current . % ( . % reduction) implying , blood transfusion avoided and , red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted < ml of rbcs, all cases with ml transfused were captured and only . % of the time > ml were used. if - ml rbcs were predicted to be transfused, > ml were used % of the time. if predicted usage was > ml, % of the time it exceeded ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for > ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than . is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a month period (jan to mar ). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the month period, there were patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of units of blood were requested. units were crossmatched, of which units were sent to the operating theatre (ot). only . % of blood issued to ot were transfused (n ) while the rest were unutilized. the observed ct ratio was . . conclusion: although only % of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of ! . , with almost % of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study patients who received tranexamic acid (txa) (study group) and patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < . g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were unique alert encounters. of these, ( . %) led to a crossmatch being ordered while ( . %) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds ( . g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds ( . or . g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at academic hospitals with data-derived msbos. study design/method: the hospitals were in groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if % of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if - % of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if ! % of the patients had been transfused. data were collected at each center over a month period between january to march and included a maximum of cases per hospital during that one month to ensure equal representation between centers results/finding: between these centers there were a total of cases analyzed. some of the more frequently performed surgeries included orthopedics ( % of cases), general surgery ( %) and cardiac surgery ( %). there were t&s ordered for these cases, of which were positive for antibodies on the day of surgery. of all the t&s ordered, % were ordered in accord with the msbos recommendation, % were ordered when the msbos did not recommend one, and in . % a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio . for prenatal hemoglobin (hgb) - . ). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of % among obstetric patients. study design/method: we studied a sample of anemic (hgb< . g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of women were enrolled, with median age (interquartile range - ) years, median gravida / para and median gestational age weeks. mean hgb before referral was . g/dl and most were already taking oral iron therapy. a total of women were hiv positive with mean cd lymphocytes counts of cells/ul; ( %) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and ( %) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in ( %) of women. there was concurrent chronic disease (n ), infection (n ), vitamin b deficiency (n ) and antenatal hemorrhage (n ); had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a month old boy presented to our institution after a month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige ( ku/l; rr: - . ). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp , while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp cd lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $ . %. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of . % over the last years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from to , with a peak of . % (range . %- . %). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for mtps. results/finding: in , mtp was activated times. in cases the patient did not receive any blood product and in cases plasma was already available at the time of rbc allocation/issue. this left cases to evaluate. the median time to plasma availability was minutes (range minutes - minutes). the mean plasma wastage for mtp activations was % (range - %). of the cha replies, were using thawed plasma and their wastage was </ %. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of minutes. our implementation began with thawing a single unit of plasma for "level neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are . %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated cases of gtx in pediatric patients with age from months to years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of granulocytes donations from to were collected from health donors ( male/ female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, mcg/kg/dose (donors up to kg, maximum of mcg) and oral dexamethasone ( mg), h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with gy. results/finding s: granulocytes number increased times from basal levels and the median of cells collected were . x (range . - . ). in general donors reported no significant adverse reactions. from donations, only in was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table . adverse reactions for patients were minor and not frequent ( in gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* , elena kurnikova and pavel trakhtman . russian institute for paediatric haematology, oncology and immunology, instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period / - / . donors underwent granulocyte mobilization with g-csf in a dose - mg/kg s.c. - hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed granulocytes collections in donors ( f, m), ages to . the mean increment of wbc after stimulation was , x /l ( , - , x /l; f- , x /l; m- . x /l); in the age groups - years (n ) and over years (n ), the mean increment of wbc was respectively , x /l ( , - , x /l) and , x /l ( , - x /l). the mean volume of treated blood was , a , ) . the mean number of total wbc in the product was , x /l ( . - x / l; f- , x /l, m- , x /l). only three donors ( , %) had apheresisrelated adverse effects the analysis included granulocyte transfusions in cases of infectious complications in patients. the mean number of transfusions was . ( - ). each patient received transfusions from mean donors ( - ); the granulocyte transfusions started at the mean days ( - ) after infection; the mean dose of wbc on the transfusion was . x /kg ( - . x /kg). wbc increment was able to estimate after transfusions. wbc increment was recorded after transfusions ( . %); the mean increment was , x /l ( , - , x /l). efficacy was determined by the number of patients who survived an infection episode and amounted to . %. in the cases of severe sepsis, the efficacy of transfusions was . %. the frequency of post-transfusion reactions was %, (n ): febrile nonhemolytic reactions , % (n ); pulmonary complications , % (n ); the anti-hla antibodies formation , % (n ) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp hemolytic disease of the newborn due to anti-rh keegan barry-holson* and jennifer andrews . stanford university, dept of pathology, stanford university, dept of pathology & pediatrics background/case studies: anti-rh is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g p > mother with a negative st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at hours of life because he was found to have anemia (hemoglobin . g/dl), severe hyperbilirubinemia (total bilirubin (t bili) . mg/dl), reticulocytosis ( %) and a positive direct antiglobulin test (igg ). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of . mg/dl on day of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin . g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* , cyril jacquot , valli criss , philippe p pary , jay greenberg , naomi lc luban and edward cc wong . children's national medical center, quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as - %. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of . g/dl/ . %. wbc counts ( x /l) were mildly elevated and platelet counts ( x /l) were within normal limits. her history was notable for upper respiratory tract infection days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total . mg/dl, direct . mg/dl), normal ldh ( u/l), and undetectable haptoglobin (< mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin ( - reactivity) with positive autocontrol. dat was positive for anti-igg and negative for c despite a positive cold antibody screen. the patient weighed . kg with an estimated total blood volume of ml. she initially received simple transfusions totaling ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of %, utilizing the central venous catheter. no adverse events took place over the course of the hour exchange. her one hour post-exchange hb was . g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin ( ). after initiation of steroid therapy (methylprednisolone, mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels ( mg/dl). at a subsequent follow-up visit months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a or gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, . /- . ml/ second) or with a mechanical syringe pump (slowly, ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the g catheter, the mean change in hct was - . /- . % with the one-way valve and . /- . % without (p< . ). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p< . ). during rapid manual transfusion with a g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse . ml when using a one-way valve (change in hct versus time: r - . , p< . ) compared to a significantly different (p . ) slight increase in hemolysis for samples that took less time to transfuse . ml when not using a one-way valve (change in hct versus time: r . , p . ). correlations between time and hemolysis were similar, but insignificant using g with washed rbcs and the g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in . % of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge . study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at weeks gestation with passive anti-d and an anti-ge titer of . she was d-and ge:- ,- , by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge . the birth hemoglobin (hgb) was . g/dl, reticulocyte (retic) was . %, bilirubin (bili) was . mg/dl; the infant was discharged. on day of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb . g/dl, retic . %, and bili . mg/dl. ge -blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh . obstetrics had to authorize maternal blood donation due to her hgb of . g/dl. maternal blood collection and rbc washing was expedited and the infant received ml of maternal rbcs within hours, at which time his hgb was . g/dl. post-transfusion hgb was . g/dl. one week later, the infant was symptomatic with hgb . g/dl, retic . %, bili . mg/dl. a nd aliquot of ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb . g/dl, retic . %, anti-ge titer , and needed another transfusion. the maternal blood stored for just weeks had hemolyzed necessitating a nd maternal donation for baby's rd transfusion. at weeks, the infant's anti-ge titer was , hgb . g/dl, retic . %; no transfusion was necessary. at weeks of life, hgb was . g/dl, retic was . %, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge hdfn. molecular analysis revealed that the mother was homozygous ge -negative ge* .- , the father had homozygous wild type ge* , and the infant was heterozygous ge* /ge* .- . conclusion: the infant had hdfn due to antibodies to the high prevalence ge antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge . hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results ( ) supporting a hemoglobin trigger of g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in , , rbc orders occurred and the top three patient groups were: % in congenital heart disease patients, % in hematology/oncology patients and % in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was . g/dl as measured in the hours prior to rbc order placement. in , ffp orders occurred and the top three patient groups were: % in neonates in the nicu, % in congenital heart disease patients and % in pediatric intensive care patients. average inr of every patient was . as measured in the hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates ( - months of age), infants (> - months of age) and children (> months- years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after months, pr-plt represented % of our platelet inventory (average daily plt inventory: units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among transfused neonates ( - m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased , dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of pediatric patients receiving prbc transfusion over a month period were retrospectively reviewed. a total of patients were identified as receiving allogeneic prbc transfusion. patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was . g/dl with post-transfusion hgb rising to . g/dl. the mean prbc volume transfused was . ml using a dose of ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of - ml/kg in a kg patient, for instance, would translate into full prbc units (about ml) in an average size adult. the current standard dose of - ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a month-old, previously healthy female infant who presented to the hospital with a -week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of . g/dl and . %, respectively, platelets of , , and a reticulocyte count of . %. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of . mg/dl with a direct fraction of . mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens ( total) were non-detectable. the patient was started on a -day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c . an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her -day hospital course, the patient received rbc transfusions on the day of admission and several rbc transfusions thereafter (see table ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day . her hemoglobin rose to . g/dl on hospital day and increased to . g/dl on hospital day . at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to . g/dl on day after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is or . platelet and leukocyte immunohematology, testing and genetics table . of pairs, pairs were complete match ( / ), pairs were partial match ( / ), pairs were complete mismatch ( / ). the matching rate of hla-dpb in our study is %. conclusion: the matching rate of hla-dpb in / hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china ( ) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts . therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a year old male with a -year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts < % and inhibitor of . . on day of admission platelet count was x /l which decreased within five days to x /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were x /l and x /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to . x /l achieving the ratio of previously shown to be diagnostic of ttp. on day his a-ipc and platelet counts had improved to . x /l and x /l respectively. absence of anti-pf antibodies ruled out heparin-induced thrombocytopenia at this time. on day he had an unexpected decrease in both a-ipc and platelet count to . x /l and x /l respectively, worsening by day to . x /l and x /l respectively despite daily tpe. patient received additional tpes that failed to improve a-ipc or platelets which on day were . x /l and x /l respectively. a-ipc had remained at this level for days suggesting that the observed decrease was irreversible. adamts activity remained < % low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa- a, hpa- a, and hpa- a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa- a, hpa- a, and hpa- a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p and gi , specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa- a, anti-hpa- a, anti-hpa- a and anti-hpa- b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa- aa, hpa- ab and hpa- aa) were collected and reacted with anti-hpa- a, anti-hpa- a, anti-hpa- a and anti-hpa- b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex . the hpa- a serum was diluted to serial dilutions (from neat to / ) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa- a, hpa- a, hpa- a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( . vs . ), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa- a, anti-hpa- a, anti-hpa- a. furthermore, because the platelet was hpa- aa, the hpa- b serum did not react with the coupled beads with mfi was comparable to negative control ( . vs . ). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa- a was / ( . iu/ml) and / ( . iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa- a, hpa- a and hpa- a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in % of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from january to december . clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c , c bp, thbd, dgke, cfhr , cfhr , cfhr and cfhr ) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of patients tested, pathogenic mutations were detected in % ( / ) and vus in % ( / ). % ( / ) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and ( %) had vus. % ( / ) of patients had primary ahus; of these, % ( / ) had pathogenic mutations and % ( / ) had vus. % ( / ) of patients had secondary tma; of these, % ( / ) had pathogenic mutations and % ( / ) had vus. in patients with pathogenic mutations, % ( / ) were children, . % ( / ) had a positive family history of ahus and % ( / ) had recurrent disease. patients with primary ahus had a significantly lower age at presentation ( vs. yrs; p-value: . ) and a higher proportion of pathogenic mutations ( % vs. % p-value: . ) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl and sysmex xn compared with a flow cytometric method farshid ezligini , kjersti roen eriksen , annette vetlesen , thomas larsen titze and geir hetland* , . oslo university hospital, university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion ). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl (horiba abx, montpelier, france) and xn sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x /l sd were , (<) , (<) and for counting by sysmex toa, pentraxl , and the gallios flow cytometer, respectively. sysmex count was the very lowest a transfusion vol. supplement s abstract ( . % less than for flow cytometry), but all plt counts were significantly different (p< . ), although least so ( . %) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing , arishma lata , roland russnak , zachary antovich , heather dunckley and thierry viard* . new zealand blood service, linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of reactions that identify both variants of relevant snps located within hpa genes (hpa- through hpa- , and hpa ). genomic dna purified from blood samples, previously genotyped for hpa- ,- ,- ,- ,- and - by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were % concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late and to date has tested dna samples from blood donors ( donors were tested in duplicate). concordance between the sample replicates was %. there were occasions where the assay had to be repeated, giving a repeat rate of . %. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa- ( . %) and hpa- ( . %) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd antigen deficiency expression in jiangsu chinese han population qing chen* , jianyu xiao and chengyin huang . jiangsu province blood center, jiangsu province blood center background/case studies: cd has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd deficiency varies widely among different ethnic populations, with the frequency of - % in asians and . % of african americans, respectively. however, there is little information on the molecular basis of individuals with cd deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd expression levels and to determine the molecular basis of cd deficiency on the platelet surface of the han population in jiangsu region. cd expression levels on platelets were detected by flow cytometry among blood donors in jiangsu region. donors without cd antigen expression on their platelet surface were further to be determined the expression of cd antigen on their peripheral blood monocyte cells. the coding exons of cd gene and adjacent introns were amplified and sequenced in cd deficient individuals. results/finding: among these blood donors, cd -deficient and cd -expression individuals were . % ( / ) and . % ( / ), respectively. the frequencies of type i and type ii cd deficiency among the study population were . % ( / ) and . % ( / ), respectively. among individual with platelet cd expression, according to mean fluorescence intensity (mfi) value, , and individuals showed low, moderate and high expression levels of cd , respectively, and their mfis were . . , . . and . . (p< . ), respectively. the type i cd deficiency individual were heterozygous for - a>g and - c>g, respectively. among type ii cd deficiency individuals, two harbored a t insertion at position in exon which caused frameshift at codon ; one has a t>c exchange at position in exon which resulted in a tryptophan to arginine substitution at codon ; one has a a insertion before the th bp of the start codon atg in the promoter region; one were heterozygous for t>c and t>g, respectively. conclusion: platelet cd surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd deficiency was higher than that in type i. the study findings indicated that the frequency of cd deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd -deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd -deficient individuals widely varies among ethnic groups, with % to % in japanese, % in sub-saharan africans, . % in african americans, and . % in caucasians. although some studies of cd deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from unrelated platelet-apheresis donors in the eastern china. the expression of cd antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd and peanti-cd ). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd -nagtive phenotype. for those donors with cd -negative platelets, cd antigen expression on monocytes was analyzed further to distinguish between cd type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china ( ) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd in all samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd phenotypes using the (mean sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd deficiency on platelet, in which one sample was cd negative both on platelet and monocyte. the frequency of cd type i and type ii deficiency in the eastern chinese donors was . % and . %, respectively. the average mfi of cd deficiency samples was significantly lower than cd positive samples ( . . vs . . , p< . ). conclusion: the frequency of platelet cd deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd -deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd antibody. background/case studies: cd (gpiv, chromosome q . ) is an kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd deficiency (cd -n) is observed in - % of africans (t g) & is classified as either type i (cd -n plt, cd -n mono) or type ii (cd -n plt, cd mono). an acquired type ii cd -n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type cd -n individuals can develop anti-cd alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an year-old, group o african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin . g/dl & plt count k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, - % blasts & a complex karyotype with del( )(q q ) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < . hla antibody testing was negative (class i panel reactive antibody (pra) %). the patient was plt-xm-incompatible with most donors ( / ). a trial of group o, plt-xm-compatible plts was unsuccessful (cci ). subsequent testing for plt-specific alloantibodies identified anti-cd . fc-phenotyping showed no cd on patient's mono or plt, consistent with type i cd -n. preliminary dna results show that the patient is heterozygous for t g. because cd -n apheresis plt were unavailable from blood suppliers, the patient's children & grandson were screened as possible donors: all showed normal cd expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day ) demonstrated new class i alloantibodies (pra %) in response to transfusion ( apheresis plts, rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd -n & severe plt refractoriness in the setting of new mds, and q-chromosomal abnormalities. the absence of cd on plt & mono support congenital type cd -n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel , kristopher fernandez* , eric senaldi , pascal george , michael seul and ghazala hashmi . biomolecular analytics, central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur http://bit.ly/ q heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $ adult donors who had made ! donations in the previous months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for , , , , , , , , , and for hla class (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/ pdplf ) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a * : chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are , hla alleles documented according to the imgt / hla sequence database in janury , and more than % of them were identified in the last years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a * : allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a * : : : , but nucleotide substitution in exon , by nt c-a (codon acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a year-old pregnant woman typed as ab , who delivered a baby affected by severe hdfn. the newborn was typed as b and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted with the four monoclonal anti-d used (igm clones p x and rum and the blends clones th ms and d d ) and were typed as c-c e-e . an anti-d was identified in her serum. molecular analysis showed the c>t and a>c in exon , the snp t>c changes in exon and the t>g nucleotide change in exon . the set of snps found is similar to the molecular background of dol , except for a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of immuno hematological tests ( abo/d grouping (including newborn samples), extended erythrocytic phenotype, antibody screening, antibody identification, dat) and crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in . % of the abo/d tests (n ), , % of the antibody screening tests (n ), , % of the antibody identification tests (n ) and % of the dat tests (n ). there were discrepancies ( abo/d for the same patient, for antibody screening and antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than min). detection threshold of the d antibody was assessed at . ng/ml ( . ui/ml) whereas the french recommendations are ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a year old g p presented during her fifth pregnancy with anti-kpb with an initial titer measured of . by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams ( . moms) peaking at weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was . mg/dl with . g/dl hemoglobin. the baby typed as o positive, kp (b ) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by weeks of age. his hemoglobin recovered to . g/dl with an indirect bilirubin of . mg/dl at weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c d-specific dat may be too insensitive to detect low, but significant levels of c d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a " " reaction corresponds to about molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c d have been published. however, these are mainly designed to quantify the fraction of rbcs with c d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c d. study design/method: ten microliters (ul) of : (after documenting experimentally that this amount ensured maximum binding of anti-c d) mouse monoclonal anti-human anti-c d (abcam, clone c ) were added to ul of a . % rbc suspension. after incubation for minutes at c, samples were washed x , and ul of : diluted anti-mouse-f(ab) -pe (ro , dako) were added. after incubation at c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a rbcs stained with levels ( fold dilution, : - : ) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c d, edta-stabilized samples from healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c d (values ranging from to , abc) with level of -serum dilution (used to sensitize a rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r . , p < . ). compared with dc-screening , the sensitivity of the flow cytometric assay was superior. it detected c d sensitization at least dilution steps further. the median normal level of rbc-bound c d was abc (range - abc, n ). the assay enabled demonstration of specific c d-sensitization in the patient; the level of rbc-bound c d in the sample was significantly elevated ( , abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt gene. in humans, the abo and gbgt genetic loci are located on chromosome q , and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors ) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt genes have been constructed of vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion vol. supplement s the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c , e-, e and k -. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d , c-, e-, partial c , partial e . the probable rhce genotype, rhce*ce-jal/rhce*ce g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type allele associated with the skipping of exon in a pregnant woman and her newborn carolina bonet bub* , maria giselda aravechia , thiago costa , marilia sirianni , eduardo bastos , leandro santos , lilian castilho and jos e kutner . hospital israelita albert einstein, hemocentro unicamp background/case studies: rhd*weak d type is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion ) the c. g>c change (p.g a), which characterizes the rhd*weak d type allele is a splicing variant that induces skipping of the whole rhd exon . we report an altered splicing in the rhd*weak d type allele associated with the skipping of exon in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with commercially available monoclonal anti-d reagents: blended igm/igg (clones th- /ms- ), igm (clones ms and p x ) and igg (ms ) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d w c-c e e . the samples showed weak hemagglutination reactions ( / ) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon in both dna samples. sequencing showed the c. g>c change and the intronic c. - t>a and c. - t>c substitutions, which are associated to the rhd*weak d type allele. conclusion: our results showed that c. g>c associated with c. - t>a and c. - t>c variations had probably a functional impact on splicing inducing exclusion of exon in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type . background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than % of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with , patients and studies from other regions (brazil, egypt and france) with patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was to years and for the other countries to years. available data from us studies included a total of alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens ( . %, . %, . %, . %, . % and . % respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of . % ( . to . , % ci) vs. . % for non-us studies ( . - . , % ci) (p . ) and a transfusion vol. supplement s more alloantibodies per transfused patient ( . vs. . , p . ). similarly, the number of alloantibodies per transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort ( Á vs. . , p Á ). average number of rbc units transfused per patient in the us was also higher compared to data from france ( vs. , p . ). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* , melissa grohotolsky , lisa deblass , bala carver and kip kuttner . health network laboratories, miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: year old white mennonite female g ,p presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at . although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g (r g r) cells. both rhce*c and rhd genes encode ser which determines g expression. rare rhd variant antigens lacking ser are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e c e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r r , r'r, r g r and rr cells. this patient is predicted to be r r (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is a transfusion vol. supplement s excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* , christa voliva , kathy fletcher , heather vaught and tracie ingle . indiana university health, indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology ; : - ) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were > dat positive, were weakly dat positive and were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman . - . ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: bb, bg and bp. the ph of all eluates ranged from . - . with the highest percentage of eluates at a ph of . ( %). sixteen of the eluates tested yielded the same results in both automation platforms (concordance of %). four eluates with different results are summarized in table . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results ( ) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in led to the discovery of human blood groups. in the abo system > alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor ( units over years) who is actually a w . study design/methods: donations were tested with the pk instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns , and and exons and . specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table . tests with anti-a, -a , -b anti-a,b were negative as were the a cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table . the significant changes were found in exons and . in exon there was a nucleotide (nt) deletion of g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly ala. mutations in exon included a nt substitution causing a pro -leu change and a nt deletion c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o. . /abo*aw. ] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a and b cells. this abo discrepancy was caused by the presence of anti-a in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer > : . (schmidt, nacarrow et al. ) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. a transfusion vol. supplement s extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: edta blood tubes collected from random blood donors were used to extract dna from microliters of whole blood on day , and days post collection. blood samples were stored at - c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a /a using a nanodrop (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result ) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the samples, samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from . to . ng/ul. all readings with the exception of ( . ng/ ul) had concentrations > ng/ul. interestingly, the one that was < ng/ ul on day , yielded > ng/ul on day and post collection. over the next months, sets of samples were extracted and tested by hea. eighty-three ( . %) failed extraction and ( . %) failed hea. none of the samples that failed extraction were or days post collection; none of those that failed hea were days post collection; . % were > < days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas , carlos villa , rachel davis-rauser* , helen carpenter and vrunda patel . university of pennsylvania, hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in . communications suggest all patients receiving therapy would have a positive antibody screen because cd is a common antigen expressed on red blood cells. currently, patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at : dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for negative patients was observed up to a : dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a y/o g p at approximately weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [ , ] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m n-s s phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency > . ), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [ , ] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [ ] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays a transfusion vol. supplement s that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in dna samples from chronically transfused patients with scd, in patients with thalassemia and in dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in levels: ( ) rh and k matching; ( ) extended matching and ( ) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for % of our thalassemic and scd patients at level , % for scd patients and % for patients with thalassemia at level and % for patients with scd and % for patients with thalassemia at level . the patients were transfused with a median of . rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to - % with c e k matching and < % with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube ( drops plasma, minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to minutes and specimen volume to drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of clinically significant antibodies were detected using sprca technique, as well as warm autoantibodies and nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being % specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions ( versus ), it identified more clinically significant antibodies ( ) than liss ( ). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd on rbcs patricia a arndt* , anthony salazar and regina m. leger . american red cross blood services, long beach memorial medical center background/case studies: monoclonal anti-cd , e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or aminoethylisothiouronium bromide (aet). chapuy et al described ( ) and validated ( ) ), and % aet (ph . ) as per the aabb technical manual, th ed. these treated and untreated rbcs were stored in alsevers at c and tested on days , , , and by two methods: ) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from total dara patients were tested with reactivity - ), and ) flow cytometry using phycoerythrin (pe)labeled anti-cd . rbcs were also tested on days and or with a serum containing anti-k by peg iat. results/findings: the . m dtt in ph . pbs had a final ph of . and the ph of the commercial . m dtt was . . results are in table ; flow cytometry results from days , and (data not shown) were similar to those from days and . rbcs treated with . m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd by flow cytometry for up to days after treatment. rbcs treated with . m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening ( - %) of reactivity with pe anti-cd . background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon and ) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr ) in gestational week . the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of pregnant women were included. nip rhd was positive in / ( %), negative in / ( %) and inconclusive in / ( . %). compared to the postnatal rhd type, / ( . %) of nip rhd results were false positive (fp) and / ( . %) were false negative. in / ( %) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n ) at gestational week was . ( -and -percentiles: . - . ). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p , ). the fraction of ccfdna was calculated for randomly selected nip rhd true positive cases. median ccfdna ratio was . (the distribution had a highly positive skew, -and -percentiles: . - . ). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r , ; p . ). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of , suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only in , donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, lan-rbc units were transfused over days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day , the patient had symptomatic anemia with hemoglobin (hb) . g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of . g/dl was maintained for days. the antibody screen was negative on day post-transfusion, but strongly panreactive on day , with a positive dat (igg , c ) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from . g/dl on day to . g/dl by day with no bleeding identified, and increase in total bilirubin and ldh (peak . mg/dl and u/l on day ) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day with good response (hb . g/dl). the patient remained stable and was discharged to a skilled nursing facility days later. conclusion: transfusion of lan rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* , andrea gerner , lynne stewart , carol sostok , mollie bell and gregory r halverson . st. elizabeth healthcare, hoxworth blood center background/case studies: anti-f was first described in by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the th antigen assigned to the rh blood group system (isbt rh ). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused unit o-rbcs. two weeks later the patient received an additional o-rbc. within days the hgb had decreased from . to . g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused r r (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c e f phenotype. the rh phenotype and as was repeated on a sample collected days later. the c typing was micro positive, mixed field only after minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post hour hgb increment from the receipt of a standard unit of blood should be near g/dl (or % hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a . g/dl increase, and the second unit was only . g/dl. the last transfusion of units increased by only . g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of r r units was nearly complete. in a case from , ohto and kariyone (transf. ; vol , no. ) reported a cr Ásurvival study of f rbcs in a patient with anti-f. they showed that the initital survival of f cells was fairly normal, however, after days, there was a sudden increase of red cell destruction, and by day all f cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with . m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to days following the dtt treatment of rrbc. reactions were graded using standard serological grading of (negative) to (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to days. this suggests that dtttreated reagent red cells can be stored for at least days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with . m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract - c, and observed for hemolysis (none was seen) for up to days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted - with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* , monique scott , garcia curtis , ellice wong , alexa j siddon and christopher a tormey . yale-new haven hospital, va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately % to % of persons of african descent is characterized by neutrophil count of < . x /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p< . . results/finding: subjects who were clinically identified as having probable ben included patients (mean age . ; all self-identified as african-american; / were male) and controls included patients (mean age . ; self-identified as african american; ( / male). all of the cases ( %) diagnosed with ben had fy(a-b-) phenotype. mean anc ( . x /ul) and wbc counts ( . x /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p . and . , respectively) compared with controls (mean anc . x /ul ; mean wbc count . x /ul). there was no significant difference in mean platelet counts ( x /ul vs x /ul; p . ) or mean hemoglobin levels ( . g/dl vs . g/dl; p . ) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih- had % concordance for all blood grouping assays. for ahg assays, the ih- detected an anti-jka e, anti-fya warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih- identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih- with anti-igg,-c d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih- analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih- is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of tests were performed on , adult patient samples and donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in . % of samples tested. there were discrepancies, all antibody screening ( false positives, failure to detect a very weak prophylactic anti d and positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing - group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* , kathleen bensing , michael schanen , cindy piefer , randall w. velliquette , christine lomas-francis and connie m. westhoff . immunohematology reference laboratory, versiti/bloodcenter of wisconsin, immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new / / / cecf / / / rhce*ce or rhce*ce compound heterozygotes ce g ce g or ce c, g or ces or ceti / / / ce g ce c, g or c, g / / / ce g ces or cemo or ceek or ceek(var) or cern / / / ce c, g ce c, g or cemo or ceti / / / ce c, g/ce g/ g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi / / / total / / / a transfusion vol. supplement s reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd / and rd / , and a licensed comparator anti-e (p gd ms ), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n ) or edta blood from donors (n ) and were tested using a manual tube method or on a pk automated platform. a score ( ) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table ) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r r , r r , r r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: were rhce*ce that were in trans to rhce*ce; were various rhce*ce plus rhce*ce c compound heterozygotes; were rhce*ce or rhce*ce homozygotes; were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with rhce*cear/rhce*ce c compound heterozygote, and with rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and of rhce*cecf homozygotes were detected using the comparator reagent. rd / and rd / failed with and e variants, respectively (table ) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd / clone. none of the reagents detected e antigen variant expressed on example of rhce*cehar/rhce*ce. conclusion: rd / and rd / anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of blood group genes associated with the expression of blood group antigens from blood group systems. we used the illumina's hiseq / system to perform next generation sequencing first on sureselect-enriched genes from dna reference samples with average target design coverage of . %, and then on haloplex-enriched genes from dna reference samples with average target design coverage above . %. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for blood group genetic variants in these genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads ( . %) were mapped to the target regions relative to the sureselect reads ( . %). the mean sequence coverage depth of the targeted bases was around x for sureselect method and x for haloplex method. some exons, such as rhd exons and , , rhce exon , ermap exons and , cd exons and , cr gene (most exons) and gypb exon , are consistently covered with less than x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than % concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than % blood group genetic variants in selected genes. evidence rhce*cehar does not encode for rh (hr b ) antigen debra j bailey* , trina horn , paul mansfield , najmi qazi , pamela nickle , jessica keller , margaret a keller and jan r hamilton . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in and has a phenotype of c e c e w f w , g , hr w , hr , hr s , rh: , rh: with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh: (hr b ) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c. c>g and rhd c. c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d c e c e . her plasma contained an alloanti-s and an antibody that reacted strongly with all random e k s reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d and dc red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e s k red cells homozygous for the rhd*diiia-ce( - )-d, rhce*-ce c, g, t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce c, g, t/ rhd*diiia-ce( - )-d, rhce*ce c, g, t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd* , rhce*cehar and rhd*diiia-ce( - )-d, rhce*ce c, g, t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr ( of sources) and hr b ( of sources) phenotype. conclusion: the rhd*diiia-ce( - )-d, rhce*ce c, g, t haplotype is one of the rh haplotypes expressed by the original hr b individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd* , rhce*cehar and rhd*diiia-ce( - )-d, rhce*ce c, g, t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between / / and / / . all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately years, patients had htla established at least once by titration studies. serological investigations on a total of samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on samples was successful in rule out in % of cases. in an additional patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only % of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for % of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c. c>t (p.thr met). the dau allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion , : ) recently summarized serologic characteristics and associated anti-d alloimmunization for dau family alleles. we investigated two samples with the c. c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample was from a yo multiracial female. her rbcs reacted s at immediate spin (is) and in iat with immucor gammaclone and series and , and mi at is and in iat with ortho bioclone anti-d. rbcs did not react with of (lhm / & / ) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c. c>t characteristic of dau. rhd sequencing confirmed c. c>t and identified two adjacent changes, c. g>t and c. g>t (c. _ delinstt), in exon encoding p.gly leu. sample rbcs reacted w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series and , and quotient albaclone blend and alpha anti-d. papain treated rbcs were s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c. c>t. sequencing confirmed c. c>t and found a new c. c>t change (p.ser -leu) in exon . the c. t has not been reported, but c. g encodes a stop codon (p.ser stop) in japanese (vox sang , : ). conclusion: we report two new alleles: rhd with c. _ delinstt (p.gly leu) and rhd with c. c>t (p.ser leu), both also carrying the c. c>t (p.thr met) characteristic of the african dau cluster. d antigen associated with p. leu is a partial d antigen with a novel epitope pattern. the p. leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by / commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to . the number and diversity of alleles in the dau cluster supports that the c. c>t change is a major ancestral african background allele (wagner et al, blood , : ). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september to present day we got samples of repeated blood donors who are known to be d negative, c positive and/or e negative from blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon , exon and exon in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon to confirm the existence of c. g>a and c. t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the sample, we identified cases ( . %) of total rhd deletion, cases ( . %) of rhd-ce-d hybrid, and cases ( . %) of rhd variant. of rhd variant were determined to be asian type del with c. g>a variation. cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n ) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon and exon of the abo gene were amplified and sequenced. the abo*b . allele was confirmed by pcr-rflp analysis. results/finding: among subjects with b or ab phenotypes, were genotyped as abo*b . . the abo*b . group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage ( . . ) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other subjects with b or ab subjects, genotyped as abo*b . (n ), abo*bw. (n ), abo*bw. (n ), abo*bw. (n ) and abo*bw. (n ), displayed flow patterns differed from the abo*b . group. the abo*bw. , abo*bw. and abo*b . subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (< % in abo*bw. and abo*bw. subjects and < % in abo*b . subject). both abo*bw. and abo*bw. displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b . genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout , the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in % (n ) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in % (n ), % (n ), and % (n ) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n ), anti-jka (n ), anti-k (n ), anti-jkb (n ), both anti-e and anti-c (n ) (see table ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly in cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study volunteers in the gambela region (nct ). the whole ackr gene was amplified in one reaction covering , base pairs (bp). this primary amplicon was re-amplified using nested primers covering nucleotides. nucleotide sequence was obtained by sequencing reactions and manually annotated using ncbi refseq ng_ . . the sequencing covered bp of both exons, bp of intron, bp of '-flanking region, bp of '-utr, bp of '-utr and bp of '-flanking region and encompassed all the variations present in dbsnp and nhlbi esp databases. results/finding: among the samples, a total of snps, including one novel snp in '-utr were observed. snps occurred in the exons, in 'and 'flanking region, in '-utr and in the intron. all individuals carried the snp indicative of the common fy: phenotype; while individuals were homozygous and was heterozygous for the gata box mutation. no splice site mutation was detected. as individuals were observed as being homozygous or heterozygous for snp, we could unambiguously assign distinct alleles. in the remaining individuals with or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than . kb of the ackr gene and identified at least different alleles. the present study found that the vast majority of alleles ( / ) in the gambela population as defined by snps, were similar to the clinically relevant fy* n. allele, which in turn is defined by only snps at positions c. - t>c and c. g>a. out of the remaining alleles, were similar to fy* with the fy(b ) phenotype and was similar to fy* w. with the fy x phenotype. the high frequency of fy* n. ( %) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique ( %- %). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce( - )-d is the most common hybrid and is found in african blacks. it arose by conversion of exons - of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample (male) and sample (multiracial female), both c c e e (presumed r r ), presented with weaker than expected d typing; is and / at iat. rhd beadchip identified the common african rhd*diiia-ce( - )-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c. c>g and c. g>t (heterozygous) was also detected. as rhce*ce with g and t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons and replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce( - )-d were found in both samples. sample (scd male), d c e c e , by rh beadchip had one conventional rhd and rhd*diii type , and rhce*ce g/ces. as rhd*diiia type has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce( c) exons and had replaced those exons in the common hybrid rhd*diiia-ce( - )-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky and ky . we report two different and novel complex rh rearrangements: two samples thought to be r r had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia( - )-ce. in kind, a sample genotyped as diii type rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce c( - )-diiia( )-ces( - )-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples and have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a -year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at g/l. her pregnancy history was not provided. she had received units of packed red blood cells (rbcs) in the past including unit within the last months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r r , r r , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse polymorphisms which determine antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a ) and lu(b ). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of antigens encoded by the kel gene, is organized into exons. there are approximately kel alleles associated with a kell null phenotype (k ) in which no kell antigens are expressed, and alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons , , , and and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k kp(a-b ) js(a-b ). kel-cdna sequence analysis was performed and detected a single transcript species with c. c, c. c, t, and missing the sequences corresponding to exons , and . amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel* allele. this donor was presumed to have a k phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel* m. . here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a and a cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c. deleted g, characteristic of o alleles, c. t, characteristic of a and some uncommon o alleles, and c. a and c. a, characteristic of b alleles. genomic sequencing of exons and confirmed the presence of an o allele, abo*o del g, t, t), and the presence of a b allele ( g, g, t, a, a, c, and a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* , tatiane vendrame , janaína muniz , diana gazito , afonso cortez , lilian castilho and flavia latini . associa, associac¸ão beneficente de coleta de sangue, hemocentro de são jos e do rio preto, hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the -bp deletion in smim in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting and in samples with reactivity of . dna was isolated from peripheral blood and smim was sequenced. results/finding: from donor samples studied, were serologically vel negative by gel-iat but positive by adsorption-elution, presented a reaction and the remained samples showed a reactivity of . genotyping results showed that the samples with negative results and of samples that presented reaction were heterozygous for the bp deletion and presented the a allele rs in homozygous status. from the of remaining samples with reactivity of , ( %) had the a allele of rs and ( . %) had the a allele of rs . in contrast, in the samples with stronger reactions we found the a allele of rs in ( . %) samples and the a allele of rs in ( . %) samples. conclusion: the molecular changes rs and rs are located in intron distancing nucleotides. this study reinforces the association of the a allele of rs with reduction of vel expression and suggests the involvement of a new rs change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as (rh negative), or ! on the neo or ! on the echo (rh positive) for both series and series anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series and series anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in of ( . %) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in of ( %) of samples. of ( . %) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. of ( %) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining of ( . %) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but < on the galileoneo or positive but < on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* , tatiane vendrame , janaína muniz , rosangela person , lilian castilho , afonso cortez and flavia latini . associa, associac¸ão beneficente de coleta de sangue, hemocentro de são jos e do rio preto, hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of samples were included in the study, being previously genotyped as rhd*dar . , rhd*dar . and rhd*dau . the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from - corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon . rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that of samples ( dar . , dar . and dau ) had rhd genes, were phenotyped as c e-c e and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in dar . samples showed the rhce*-cear/ce s genotype, in dar . samples the rhce*cevs. /ce s genotype and in the dau sample the rhce*ce s /ce genotype. table describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized f antibody (hu f -g ) that binds human cd has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu f -g (anti-cd ) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a year old female with progressive follicular lymphoma who was enrolled in phase b/ trial of hu f g in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a year old male with refractory diffuse large b cell lymphoma enrolled in hu f -g clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing ( with anti-a, non-reactive with anti-b) and the reverse typing ( with both a cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase ( to ), at liss- c ( to ), at liss-polyspecific ahg (m ), and at peg-anti-igg (m to ). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at o c ahg phase. the abo typing of the second case performed after anti-cd administration showed a discrepancy between the forward ( with anti-a) and the reverse ( with both a and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is ( ), at c in liss ( ), and liss-polyspecific ahg (m ). the dat and autocontrol were negative. his genotype was determined to be a /o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o. . / o. . , consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n ) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt and a galt. papain-treated patient rbcs were used to screen donor plasmas (n ) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with polyclonal anti-a,b and a monoclonal anti-b (clone g / ) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors or nor antigens. the patient was le(a-b ) and thus a secretor. a positive crossmatch was seen with % of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type or ) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $ years earlier. the reactions are likely due to uptake of recipient-derived ble b (type ) antigen (isbt no. ), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b ). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of . shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive ( ) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from / / to / / . reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of , columns run as part of antibody screens, , ( . %) columns generated "?" results. assuming seconds of technologist time per "?", we estimate that . hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table ) . in cases, all three columns were visually negative but the analyzer reported reactivity with of cells. all cases had mts-gel tm antibody identification panels performed, of also had a mts-gel tm ficin panel. the yield for the panels performed was two routine panels with weak reactivity against hla cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; were negative. one patient newly demonstrated anti-jka. fifty percent ( / ) of visually negative but analyzer positive samples were tested with gel card lot number , % ( / ) with lot , and % ( / ) with lot . conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from patients ( cross-match samples, units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in / patients. further testing was performed in / . eight were tested for the presence of antibodies at c and confirmed in / . rouleaux formation was observed in / patients, / had reactivity detectable at c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* , sandra nance , david moolten and p. dayand borge . ( ): - ). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this . year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from random allogeneic and autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing . % bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* , anna burgos , virginia lew , sunitha vege , susan veneman , christopher j gresens , jonathan hughes and connie m. westhoff . immunohematology and genomics laboratory, new york blood center, blood centers of the pacific, bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons - , and long range sequencing of exon - ( . kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with commercially available monoclonal anti-d reagents: blended igm/igg (clones th- / ms- ), igm (clones ms and p x ) and igg (ms ) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m n-individuals (meyer et al. br j haematol. ; : - ) . the st a allele, also described as gyp* , is a hybrid gypb-gypatranscript with the crossover in intron . we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a years old pregnant, african american female g p was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a like phenotype. genetic testing did not support the serological findings of a subgroup and a new abo allele, abo* c that has never been reported in correlation with an a like subgroup was detected study design/method: the patient rbcs were typed with anti-a (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a antibody work up was performed using three different lots of a cells and three lots of a cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and c for min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo* c found in the mother. the previously reported abo* a allele encoded an aspartic acid to asparagine change at position p. consistent with an a weak phenotype. also, at least five other alleles encoding an a phenoytpe consisted of polymorphisms at positions c. through c. , giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a / aweak phenotype is encoded by the variant allele abo* c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since ; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n ) were processed with the automated biorobot m robot using the magattract dna mini m extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of . ng/ml and . respectively. in the second phase of the study (n ), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was - ng/ll and the recommended purity was an absorbance ratio of . - . (a / ) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was % concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also % concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano , izaskun apraiz , maría azcarate , miguel angel vesga , montserrat rubia , mercedes piedrabuena , fernando puente , barbera veldhuisen , ellen van der schoot and m onica l opez* . progenika biopharma, a grifols company, centro vasco de transfusi on y tejidos humanos, banco de sangre y tejidos de arag on, sanquin blood supply research background/case studies: it is well established that weak d , and phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type , rhd*weak d type , rhd*weak d type , rhd deletion, rhd*pseudogene and rhd*diiia-ce( - )-d and itgb gene: hpa a and hpa b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa- blood group typing. study design/method: a cohort of previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications / /ce for a ivd product of list a (! % clinical samples, > % neonatal specimens and ! % weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n , %). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa- predicted phenotype were used for comparison. transfusion results/finding: no system failure, % call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a % concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was %. the following id rhd xt predicted phenotype results were obtained: d negative (n ), no amplification variant (n ), weak d type (n ), weak d type heterozygous (n ), weak d type (n ), weak d type heterozygous (n ), weak d type (n ), weak d type heterozygous (n ), weak d types , or not detected (n ). regarding hpa- blood group, the predicted phenotype results obtained by id rhd xt were % concordant with bds results: hpa- a positive (n ) and hpa- a negative (n ), hpa- b positive (n ) and hpa- b negative (n ). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types ( % specificity and % sensitivity for d antigen, hpa- a and hpa- b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value < . (clinical analysis) or < x - (gwa) was considered statistically significant. results/finding: of the cohort patients, ( . %) transfused subjects were included with alloimmunized children < years ( . % of ) and alloimmunized adults ( . % of ). in multivariable logistic regression models, age (or . , p . , for age compared to - ), gender (or . , p . , for female compared to male), transfusion history (or . , p< . , for transfusions compared to - ), site, hemolysis (or . , p . , for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or . , p< . ) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from newborns less than , gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on sample pairs. dat test was negative on sample pairs and two were positive. there was % concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on placental blood samples and heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < , g birth weight newborns. o-( . %), ab ( . %), b-( . %), and a-( . %). among the tested donors, . % were d positive with r r being the most common rh phenotype. in the kell blood group system, . % of the donors were k positive, while the k antigen was found to be . %. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a b ) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a b ) and m n-s s at % and . % respectively. the le a and le b alleles were seen in . % and . % of donors respectively, while lu b -phenotype was found in . % of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a b ) and lu(a-b-) were . % , . % and . % respectively, while the m n-s-s-and m-n s-s-phenotypes were not found. the frequency of the p antigen was found to be at . % similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph . and the supernatant was discharged. a dilution buffer containing % human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with ll of washed red cells. the cell suspensions were incubated for min at c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional min at c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for - nm. events were recorded at a frequency of cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* , mark yazer , nancy m. dunbar and biomedical excellence for safer transfusion (best) collaborative . university of vermont medical center, university of pittsburgh, dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of . study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a : dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a -year and -year period, respectively. one center provided plasma and wb testing data for a -year period. results/findings: in total there were group a plasma units tested and ( . %) had a high titer anti-b. the range of high titer group a plasma units between these three centers was . %- . %. of the wb units tested, ( . %) units had a high titer; / ( . %) of the units had a high titer anti-a, / ( . %) had a high titer anti-b, and / ( %) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with . m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd on reagent rbcs and render them free from plasma anti-cd drug interference. procedures for the preparation of . m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on . m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of . m dtt, was adjusted to ph . , ph . , ph . using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n ) were treated with the . m dtt solutions in parallel by mixing : ratio of packed rbcs to . m dtt solution followed by incubation at c. for up to minutes during treatment, the expression of k antigens was measured every minutes by tube method using different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every minutes for each ph level. the reduction in average scores between different phs were also calculated at every minutes to measure the impact of . m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by ! ) after minutes of dtt treatment at ph . ; minutes at ph . and minutes at ph . . complete loss of k expression was seen after minutes of dtt treatment at ph . ; minutes at ph . and minutes at ph . . the reactivity patterns of k antigen tested with sources of anti-k correlate with each other. the reductions in average scores were seen between to minutes range of dtt treatment time when ph . was raised to ph . ; to minutes range when ph . was raised to ph . ; and to minutes range when ph . was raised to ph . . conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to minutes and/or beyond minutes of incubation. the ph of the . m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of . m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n , ) drawn between july , and june , . these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, , patients were found to possess clinically significant red cell antibodies for an overall incidence of . percent. the three most commonly encountered antibodies were anti-d (n ) . %, anti-m (n ) . %, and anti-e (n ) with a frequency of . %. a total of ( . %) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with instances ( . %) followed by anti-e and anti-c with ( . %), and anti-c, anti-e with ( . %). of the multiple antibodies identified, ( . %) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with or . % of the total. the west had ( . %), the midwest ( . %) and the northeast with ( . %). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of . and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the . thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p . ). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* , lindsy rich , sherry stern , sharon wangen and camille van buskirk . mayo clinic, mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was to days. results/finding: of the ntd specimens from the immucor neo, resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all results, it was determined that there was no reactivity and a valid result was present. the other samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the absc specimens that were resulted out as positive on the immucor neo, specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: - with ms (n ), - s with ms (n ), no reaction with ms , dgc , p x (n ). samples tested with a polyclonal anti-c showed a - reactivity. d c e c e cases tested with a polyclonal and monoclonal anti-e (ms , ms , ms , ms ) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c. g>a mutation in exon , predicted to encode the p.gly ser substitution. for apparent r r donors, a f-negative type allowed the prediction of a rhce*ce a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce a allele (c and e in cis) in all samples. d c e c e individuals were reactive s with the original source of anti-rh , slightly weaker when compared to rhce*ce a/rhce*ce rbc samples available from our cryobank ( ). conclusion: our results confirm that the c. g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh ). the locr reactivity appears to be rather similar when coded by rhce*ce a or rhce*ce a alleles. this was quite an unexpected finding, since the p.gly ser substitution is close to the critical amino-acid for c/c expression (p.pro ser). none of our cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce a was reported to code for a partial c (rh:- ), we consider that rhce*ce a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes , or . the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d , , or genotypes. study design/methods: between / and / samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types , , or genotypes, but a transfusion vol. supplement s had evidence of rhd genetic sequences in exon and/or intron in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons - to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients ( %) followed by transfusion patients ( %); % had no clinical indication provided. samples ( %) were found to be weak d type , , or ( , , and samples, respectively). samples ( %) appear to be genetically rhd negative. genetic sequencing was performed on samples; had rhd genetic variants that were not weak d types , , or (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples ( %) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types - . of the samples that had evidence of an rhd gene and did not carry the known weak d types - polymorphisms, ( %) of were found to have other rhd variants, and ( %) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types - variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel , curr opin hematol : ) , that individuals with weak d types , , and are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report months experience with rhd genotyping on samples referred with discrepant or weak d typing investigated from january to april . study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for samples ( . % caucasian, . % african american/african, . % multiracial, . % hispanic, % asian, and . % other). results/finding: rhd genotyping identified weak d types , , and in / ( %) and alleles known to encode partial d phenotypes in / ( . %) (table) . uncommon or rare weak d alleles including types , , , , , , (n ), , , , , and were found in ( . %). the partial d alleles found were diverse, but the largest number included partial rhd*d . (n ) and *dar ( conclusion: in a multiracial cohort of individuals with weaker than expected d typing % were due to weak d types , , or and would not be considered at risk of clinical significant anti-d, but for % there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp rhd* . allele causes discrepant genotyping results for rhce small c sabine scholz* , sandra schneider , sabrina k€ onig , susanne helmig and vicky van sandt . inno-train diagnostik gmbh, rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c ( c) and c ( t) is caused by the snp on position on the rhce gene. the rhd* . allele (also known as rhd cat vii type ) carries the snp t>c on the rhd gene and additionally the snp t>c. this rhd* . allele has been described to partially express rhc on the d polypeptide (faas, transfusion, ) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, ) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles ( t>c, t>c) confirming a rhd* . and one rhd* allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series and anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series and series , and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c. a>g change in exon encoding an amino acid change p.met val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c. a>g (p.met val) change in exon . several snps, deletions, and insertions have been reported with changes in exon . phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c. a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* , juan merayo-rodriguez , christopher lough and nancy eckert . lifesouth community blood centers, life south community blood centers, lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is % in most populations and greater than % in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is . / . and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal (k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to . / . the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient was a yo female, c e c e , whose rbcs reacted by echo and by neo with anti-d , and '?' with anti-d . testing with d and d by tube gave and w on initial spin (is) respectively and by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and w with immucor gammaclone anti-d, and all were at iat. rbcs did not react with two (lhm / & / ) of anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c. c>a change encoding p.his gln. patient was a yo pregnant female, c e c e , whose rbc were w at is and at iat with immucor series and and gammaclone, and moderately reactive, is and iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm / & / ) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon from c. to c. encoding amino acid changes p.ile leu and ser asn. conclusion: we found two previously reported rare alleles: rhd with a c. c>a (p.his gln), previously found in france (lefloch et al. genbank ku ), and rhd*dar with part of exon replaced by rhce, reported in sub-sahara africa (granier et al. transfusion : ) designated rhd*dar(ce :v v-s n) with an allele frequency of . to . . blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu f -g is a human monoclonal igg antibody recognizing cd that is in clinical trials to treat hematologic or solid malignancies. cd is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd is thought to enhance phagocytosis and promote anti-tumor responses. cd is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n ) from patients were tested over the course of month treatment. plasma was tested at immediate spin (is) and by iat with r r , rr, d--, rh mod and rh null rbcs, as cd expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg ) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd was observed in plasma as soon as hour post infusion. plasma reacted to at is and with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker ( and ) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was . in contrast, iat reactivity using gamma-clone anti-igg was only w to , and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd titer was at is and peg iat with gamma-clone anti-igg, but was ! with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n ) were reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after x allo-adsorption with papain treated rr cells, but in some samples low level (micro- ) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu f -g anti-cd therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu f -g is igg . reactivity was observed in all phases and with all test methods. cd is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg , can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in . in august , she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was with untreated and with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:- etc. had been excluded, k-phenotyping revealed a k -phenotype. a total of silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel* n. with c. t encoding p. ter (reported in an individual from austria in ). there are two known k -patients in our country, both homozygous for c. t. the daughter was a c. t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k -donors are available in our country. with the help of the isbt rare donor working party, a k o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* , kshitij srivastava , houda romdhane , saloua jemni yacoub and willy albert flegel . nih, dtm/cc/nih, regional blood transfusion center sousse, regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, a transfusion vol. supplement s since . the tunisian population has the largest known prevalence of weak d type . alleles, occurring in of rh haplotypes, compared to in , or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type . in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of , random blood donors were serologically screened for the d antigen using routine techniques. samples with weak reactivity were tested with a panel of monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of discrepant samples ( . %) were observed and expressed the serologic weak d phenotype. among them, carried an allele of the weak d type cluster ( . %), of which samples ( . %) showed the weak d type . allele. only sample each was found for the weak d types , and and the dvii, while samples showed the consensus rhd sequence. no mutation in any of the rhd exons was detected in another samples. the molecular analysis of the rhce gene showed that out of samples with serologic weak d phenotype ( . %) had a variant rhce allele and the most common associations were: weak d type . linked to rhce*cevs. . ; weak d type . . with cear; and weak d type . to rhce*cevs. , while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost % of the weak d phenotypes in tunisia were caused by alleles of the weak d type cluster, of which % represented the weak d type . allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type . in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type . phenotype. there is a possibility that the rhce*cevs. . allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* , heather simmons , christine lomas-francis , gayane shakarian , sunitha vege , lauren hutelmyer , sandra nance , jessica poisson , nicholas bandarenko and connie m. westhoff . duke university hospital, immunohematology and genomics laboratory, new york blood center, arc pennjersey, american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a , year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with . m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c , k , fy(a ),s ]. reactivity was detected to a titer of ; it was not removed by prewarm technique or by x peg alloadsorption. the adsorbed plasma reacted with . m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k , k , js(b ), kp(a b ) and sc: , . her plasma reacted with k o , mcleod, sc: , rbc samples and dtt-treated sc: rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused aliquots of crossmatch incompatible kp(b ), s rbcs. her post-transfusion dat was with anti-igg, with anti-c d. the eluate reacted with all rbc samples except kp(b ) sample. she tolerated additional aliquots from phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k , k , kp(a b ), js(a b ) and sc: , , discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c. a>t (p.glu val) (kel* . ) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a ). sc sequencing found heterozygosity for a '- g>a change (rs , to % prevalence) and conventional sc* , predicting sc: , , . kel and sc results on the mother were kel* /kel* . , heterozygous for the sc change '- g>a, and her rbcs typed k k kp(a b ), sc , ula , consistent with dna predictions. plasma collected months later was nonreactive at rt and in peg iat. her rbcs were dat and now typed k , kp(a b ), ul(a ) sc and sc ,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel* . homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc: , rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b ). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a -year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs - a)genotype, associated with a jkb null phenotype. anti jk was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a ) and jk(b ) . genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk . however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms a transfusion vol. supplement s (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a b-); kp(a b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon revealed a g>a mutation, fy *n. , known to silence fya. sequencing of kel exons - exposed a silent polymorphism in exon , g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy *n. mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly % of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have genetic variants not previously reported in those of african descent. only was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient . study design/methods: patient was a year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at years of age. anti-b changed from undetectable/weak to strong at the age of years. patient was a month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was / . both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient with - reactions up to years of age. thereafter, abo typing showed mainly strong anti-b. patient had / anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for children on long-term tpn. patient had absent/weak anti-b since birth up to years of age, then developed strong anti-b with no change in feeding regiment and medications. patient had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* and elizabeth hart . brigham and women's faulkner hospital, university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title , cfr part . (a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in . a total of samples were evaluated. each abid was subcategorized; ( ) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and ( ) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of abids were performed on new patient samples. of the new abid samples, ( %) had microscopic absc results. for the previously known antibody patients, there were which accounted for % of the total abids performed. when reviewing the total abid workups, a total of ( %) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the new antibody samples were: conclusion: a total of % of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: ( ) discontinue the use of the microscope, ( ) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or ( ) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than % in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a -yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a -year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only ( - ), and negative with anti-c b, c d reagent. the antibody showed a peak gel-igg iat titer of . results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w ), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from to %, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody anti-p anti-m anti-sd a anti-le b anti-jk a anti-k anti-e anti-c results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of % (type ), % (type ), % (type ), % (type ) and % other than , , or . further investigation was conducted to determine the molecular identity of the «others». out of samples, ( %) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon or both exons and . a surprising amount of samples were discovered to be normal rhd. conclusion: along with sandler et al. ( ) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types , , and obtained in serological weak d, years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was , %. no trali happened in the period. prophylaxis were used in % of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p < . ). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) ( . % vs . %, p < . ) or aes with a non-allergic type inflammation etiology ( . % vs . %, p < . ) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes ( . % vs . %, p . ) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for , , , and weeks were . %, . %, . % and . %, respectively (p . , logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week ( . %) and longer than a week ( . %) (p < . ). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found patients from - , who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these cases, only of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as out of transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was . g/dl but declined to . g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days and with poor responses. on day , routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from . mg/dl to . mg/dl (reference . - . mg/dl), and lactate dehydrogenase was above reportable linearity, > u/l (reference - u/l). testing revealed additional anti-e, anti-jkb, dat c , plasma free hb . mg/dl (reference - . mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b ), one of which was also e . one volume tpe was performed to remove free hb on days , , and using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at . mg/dl on day , decreased to . mg/dl before discharge on day results/findings: twenty three cases were identified, of which had medical records available for analysis. ten ( %) patients were male, the mean age was . years (range - years), ( %) had an underlying hematologic malignancy or bone marrow disorder, and ( %) had a history of coronary artery disease (cad). the implicated units included ( %) red blood cells and ( %) platelets; ( %) patients received a single unit, and ( %) received two or more within the previous hours; the mean volume transfused was . ml (range - ml). the mean time to onset of chest pain was . minutes (sd minutes), with % of patients presenting within . hours and % within hours of starting the transfusion. chest pain was present as the only symptom in % of the cases, and for the other cases the accompanying symptoms included dyspnea ( %), fever ( %), back pain ( %), and hypo-and hypertension ( %). a post-transfusion chest x-ray was performed in % of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in % of cases and showed no findings to suggest acute ischemia. three ( %) patients had a minimal increase in their troponin levels, although had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen ( %) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than minutes in the majority of patients ( %). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts . based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, degree, or anti-human globulin phase. check cells were found to be . these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o to a ) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a -year-old o man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha- antitrypsin deficiency who presented for olt (donor o ). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving units of o rbcs and units of o plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a plasma were transfused to wash out anti-a antibody prior to transfusion of a rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for hours post-transplant. the patient's total estimated blood loss was > l. he received a total of units of rbcs (including a ), units of plasma (including a ), units of cryoprecipitate, and units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o . on postoperative day (pod) , a ts showed predominantly a rbcs with trace o rbcs, as well as very low anti-a igm and igg titers (table ) . he received two additional o rbc units ( each on pod and pod ) with increasing o rbcs on ts and rising anti-a titers. his blood type was unequivocally o by pod . the patient showed recovery of liver synthetic function on pod (factor activity %) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod , the patient had reverted to o with recovery of anti-a titers. at months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* , alfred mingo , maria isabel gonzalez , antoni mena and juan pedro benitez . bst, at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h ) and an oncology center (h ). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of transfusion activity in both h and h shows pse and bca, out of and respectively, since the tss deployment in . retrospective analysis and classification of security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h , nm related to pse accounted for . % of all, being the mistake in concordance between patient identification and prescription order the most frequent ( . %). the nm detected in bcas were . % of all and mostly ( . %) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are . % of all and mostly ( %) the systems detects a not assigned bracelet. for h , nm related to pse accounted for the . % of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent ( . %). the nm detected in bcas accounts for . % of all and in . % occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are . % of all and in . % of them the blood components were assigned to another patient. ( , , , , , , , , , , , , , ) were analyzed via a commercially available elisa. comparison of adequate response to ppv , defined as ! mcg/ml for > serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in patients (alloimmunized, ); pre-and post-vaccine titers were available for patients (alloimmunized, ). of the patients, were on chronic transfusions, were on hydroxyurea, were surgical splenectomized, patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv in the previous years; / also reported previous history -valent sp conjugate vaccine within the last years. baseline pre-vaccination titers (n ) showed no difference between alloimmunized and non-alloimmunized patients (all p-values > . ). in the group with pre-and post-vaccination (n ) titers available, out of ( %) non alloimmunized patients had an adequate response versus out of in the alloimmunized group ( %, p ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from to ). patient harm events recorded within the veritas system from january to july were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of tres per month and per year were found over five years. % of tres are associated with pre-bb activities, % occur within bb, and % are post-bb events. sample collection and handling represent % of total tres. most tres ( %) were reported by bb staff, % were reported by non-bb staff. patient harm analysis revealed an average of four level (near miss), three level (no known harm), and . level (patient harm) per month. no deaths related to tre were detected over the seven month january to july period. patient harm was associated with tres occurring in the bb ( %) and post-bb ( %). these events were reported externally ( %) and by bb staff ( %). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january , and march , was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by and added to the number of individual whole blood plts; apheresis plasma units were multiplied by and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was . the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the -hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions ( [ /l vs. [ /l, p . ). there were no significant differences in the frequency of effective therapeutic ( % vs. %, p . ) and prophylactic ( % vs. %, p . ) transfusions between the prcs and gypcs. we did not find significant differences between prcs and gypcs in cci after prophylactic ( . . vs. . . ) and therapeutic ( . . vs. . . ) transfusions, in cc after prophylactic ( . . vs. . . ) and therapeutic ( . . vs. . .) transfusions. there were no significant differences between prcs and gypcs also in ma after prophylactic ( . . vs. . , p . ) and therapeutic ( . . vs. . . , p . ) pc transfusions. reduction of the severity of bleeds was obtained in ( %) of the cases after prpc transfusions and in ( %) of cases after gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, and cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in . it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were units administered using the emergency transfusion process in the months before the change was implemented. it was found that / ( %) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month ( components administered), / ( %) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: ) a stable anemic inpatient, ) a stable anemic inpatient to be discharged, and ) an asymptomatic post-operative inpatient. results/finding: we identified canadian tm experts: ( . %) provided a response and most had a primary place of practice in a laboratory setting ( / ; . %). for a stable, non-bleeding, anemic inpatient, . % of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with . % generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period ( - hours), a repeat hemoglobin > hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse ( . %) compared to an inpatient potentially symptomatic due to anemia ( . %). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium- ( cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n ) were divided into two ml aliquots, which were labeled with selected concentrations of s-nhsbiotin ( and lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of . . %). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that nm excitation of phycoerythrin (pe)-sa and high laser power ( mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with lg/ml of biotin resulted in $ , mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, % ci) of in , ( . %). the lld for rbc labeled with biotin at lg/ ml was $ in million ( . %). biotinylation was not associated with increased levels of hemolysis ( . . % before labeling versus . . % after labeling; p . ) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes ( ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer- (hboc- ) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: ( ) wb, ( ) wb % hboc volume (model of two units in an adult), ( ) wb % fdp, and ( ) wb % hboc % fdp. samples ( )-( ) simulated autoresuscitation by adding % plasmalyte to - . susceptibility to lysis was tested with ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of %, %, %, and % volume replacement with hboc and/or fdp, with or without prior % plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb hboc, and wb hboc fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb hboc in autodilution simulation (mean lysis . % vs. . %; p<. ). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc ( %) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even % hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc- , there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in / and / . we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of patients, were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery ( %). pcc was given for warfarin reversal in % of cases. a subset of patients received plasma within hours prior to pcc ( %) or hours after ( %). pcc was most frequently ordered in the or/perioperative service ( %). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in , several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; ) simplify and expedite the process; ) improve communication and expectations to decrease tat; ) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood ( units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of er episodes were received. the average tat from order to delivery at the bedside was reduced by % ( . minutes compared to minutes previously), while the compliance rate for er orders and physician documentation was % ( / ), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february -february . mt was defined as the transfusion of ! rbc units in a -hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at days. results/findings: thirty mts occurred during a one year period. a total of , blood products were transfused during that time period. gender distribution was / ( %) males, and the average age of all patients was with a range of to years of age. surgical patients accounted for / ( . %) mts, and / ( . %) were critical care patients. tumor categories included carcinomas ( / ), sarcomas ( / ), leukemias ( / ) and lymphoma ( / ). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer ( / ) was the most common disease seen followed by sacral chordoma ( / ). mtps were activated in only / ( . %) cases. thirty-day survival was seen in / ( . %) patients. only of mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage ( / ) or perisplenic hematoma ( / ). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a -year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a -year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a -year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* , , rebecca ross , christopher a tormey , , and amit gokhale , . yale university school of medicine, yale-new haven hospital, va connecticut healthcare background/case studies: daratumumab (dara) is a igg monoclonal antibody therapy that specifically targets cd , a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, subjects were identified for analysis. their mean age was . years, with male and female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of % ( / ) prior to administration of dara. of these patients, were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these ( %; / ) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september , through april , were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in , , unique women receiving a total of , units of red cells (rc) in , transfusion episodes were identified. their median age was (range - ). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in ( . %) and ( . %). ( . %) transfusion episodes were associated with the use of units or more rc. as a result, ( . %) episodes resulted in a post transfusion hb ! g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given (< . %) and ( . %) women respectively. upon discharge, ( . %), ( . %) and , ( . %) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to ( . %) women. conclusion: in the present study, it is observed that . % transfusion episodes were given at hb ! g/dl. a substantial number of episodes ( . %) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! g/dl). for iron replenishment and bleeding control, up to . % transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes ( . - . x per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with gy and transfused over - hours within hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* , lee grabner , brenda herdman , robert fallis , amin kabani and charles musuka . canadian blood services, kenora rainy-river regional laboratory program, diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between and before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and % were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels ( . g/dl; p . ); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units ( . vs . g/dl; . vs. . g/dl; both p . ). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients ( . g/dl vs. . g/dl; p< . ). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p . ). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p . and p . , respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than % of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered times in total, affecting patients and users. stratified by location, the majority of triggers occurred in the perioperative areas ( times) and the liver icu ( times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin % iv solution, human albumin % iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* , , mahmut akgul , , hollie m reeves , , robert w maitta , , marcie pokorny , anne capetillo and katharine a downes , . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a -month retrospective study (january-july ) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was - mg/dl with critical value of mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were adult (> years) orders reviewed by tms out of which were approved. of the approved orders, ( . %) were in agreement with tms's estimated dose. of ( . %) orders that were not in agreement with the tms's estimate, ( %) were underestimated and ( %) were overestimated. seventeen of orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of . mg/dl (range . to mg/dl) and a median excess of . mg/dl (range . to mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was mg/dl above target, which is significantly higher with intervention than without (which could have been mg/dl below the target; p< . ). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention ( vs. . mg/dl, p . ). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention ( vs. - . mg/dl, p< . ). seven of ( ) ( ) orders were for critically low fibrinogen (< mg/dl) and of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were and units ( . % and %) i.e. and pools and the most frequent orders in the disagreement group were , , and units ( %, %, % and %). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was . and posttransfusion was . . only % of patients had their inr corrected to . , while % had no change, or had increased inr. (table) . the majority ( %) of patients received units of plasma. the mean plasma dose was ml/kg. there were transfusion reactions reported, non-hemolytic and transfusion associated circulatory overload reactions in which required admission to the icu. two patients experienced bleeding during ir procedures (tips) and developed a hematoma (tunneled central line). the median of inr correction in this study was . with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is . . randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. of the patients experienced bleeding complications indicating that inr of . may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately % of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population ( %) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately % of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july , as follows: units of group a plasma and units of group ab plasma is provided for the massive transfusion protocol (mtp) along with units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed mtps at our institution between july and march . twenty patients ( . %) were transfused with incompatible group a plasma ( group ab and group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april , our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in , bonfils immunohematology reference lab (irl) sent out approximately special platelets for patients with hla antibodies. by , hla platelet orders increased dramatically and the irl sent out over special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over , donors in the database with historical hla typing. however, only approximately of those donors actively donate. in the denver area, one of the most common hla types is a a b b . only of the , donors have this type ( . %). therefore, to fill an hla platelet order request for a common hla type, only donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a a b b , there is only out of , donors ( . %) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a for example, all of the a positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in , approximately % of these special order platelets were pra matched and the remaining % were hla matched by donor recruitment. by , approximately % of special platelets sent are pra matched. this change resulted in a . fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains packed red blood cells (prbcs), fresh frozen plasma (ffp) units, and plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august to march mtps were activated at ucm, of which orders could be traced to the origin: on inpatient floor (including icus), in the operating rooms, in the emergency department, in labor and delivery, and in other procedure rooms. of the prbcs that were issued, were transfused ( % utilized); of the units of ffp that were issued, were transfused ( % utilized); of the platelet packs that were issued, were transfused ( % utilized); of the units of cryoprecipitate that were issued, were transfused ( % utilized). since march , the time of first product issue after the initiation of an mtp has also been tracked. of the events that fall within this time period, ( %), had the first product issued in minutes or less. another ( %) were issued between - minutes, resulting in over % of patients being issued their first blood product within the first minutes. only of ( %) events had an initial time greater than minutes and none were greater than minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($ - %). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* , shailesh macwan , arline stein , jane fischman , nancy nikolis , matthew bank , lennart logdberg , alexander indrikovs , sherry shariatmadar and vishesh chhibber . north shore university hospital, northwell health background/case studies: massive bleeding is generally defined as any patient who requires blood volume replacement within hours and/or receives transfusion of greater than or equal to units in one hour with a transfusion vol. supplement s ongoing bleeding. our mtp was officially implemented in in preparation for an initial verification as a level trauma center by acs. our mtp has the following packages: st pack has a ratio of : : (rbcs, plasma & platelets) and subsequent packs a ratio of : : . our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march to add cryo and pcc at a defined point in the mtp (cryo is included in the rd pack and pcc in the th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received > rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had patients who received > rbc in and . mtp had been activated for all patients and all patients received between . to unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt > seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of mg/dl (table ) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of > rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's -day and -day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective -year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of published cases ( reports) of ta-pls, ( reports) were stem cell and ( reports) were organ transplants. all ( %) stem cell transplants and ( %) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the reports of stem cell ta-pls, actively screened for antibodies in the immediate post-transplant period, and of the reports of organ ta-pls, actively screened for antibodies. these screens detected cases of stem cell ta-pls before hemolysis became apparent and cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* , rosario mallari , marc de asis , elaine shu , jonathan hughes and tho pham , . stanford university, stanford health care, stanford blood center, bloodsource background/case studies: mur antigen is present in - % of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received - rbc units every - weeks to maintain a hemoglobin (hb) level of g/dl. the patient remained stable for months when his hb level acutely dropped to . g/dl. the antibody screen remained negative for an additional months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for of the rbc units he received. units were from an asian donor, and a unit transfused days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of , donors at a hospital-associated blood center located in a region where asians comprise approximately % of the population. results/finding: . % of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for of ( . %) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over % of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* , princess maynie , carol chandler , shelia garret and pampee young . vanderbilt, vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [ ] [ ] [ ] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of samples have been analyzed (table) , rh and non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being . (range - ) times higher. the average fold change for rhd/c/e antibody titers were . , whereas the average fold change for non rh titers was . (range - ). the range for anti d titers was particularly variable, - , whereas for c/e, it was - . the overall reproducibility/precision of the automated analyzer was $ %. to correlate the a transfusion vol. supplement s increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $ times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of . units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to % of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately % of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix . % suspension of a and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, group a, and group b. results/finding: of the whole blood edta samples tested, ( group o and group b) exceeded a high titer threshold of . when the pas samples of these donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of , a % decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the ( %) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these hospitals had a lower overall rate of wastage including their own donations than the other hospitals that did not collect in-house plt. the other hospitals had wastage rates ranging between - %. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* , , christopher j gresens , anne capetillo , hollie m reeves , and katharine a downes , . case western reserve university school of medicine, university hospitals cleveland medical center, bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of < . %. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a -year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure ( / mm hg to / mm hg) followed by hematuria ( ml). chills and rigors resolved; blood pressure stabilized after min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of (igm) and , (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* , leila patricia de sousa fontenele , isabel nagle reis , carolina bonet bub , araci sakashita , raffael zamper , cristiane nakazawa , tatiane almeida omura paula , patricia silva batista , marcio dias almeida , fernanda loureiro de andrade orsi and jose mauro kutner . hospital israelita albert einstein, hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of cases of olt performed between and in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of consecutive patients submitted to liver transplantation between and were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or , - % ci: , - , , p: , ), absence of hcc (or , - % ci: , - , , p: , ), cirrhosis of any cause (or , - % ci , - , -p: , ), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or , %ic , - , ) and retransplantation due to primary non function of the graft (or , %ci , - , , , p: , ) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a /b in group o platelet products. charles k. childers* , mark destree , ashley rose and theresa nester , . madigan army medical center, bloodworks northwest, bloodworks nw, dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a /b in group o platelet products is presented from a large regional blood center collected over - months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in ml edta sample tubes. a single : dilution of plasma was prepared using a hamilton microlab series dilutor using . ml saline diluent and . ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at rpm for seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of is used, approximately % of group o apheresis platelets will have a high titer, most commonly with anti-a . less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their day outdate. after months of testing pspp units and verifying that the products rarely had a high titer ( . %), the blood center stopped performing this testing for pspp units. rh ) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to % (from % on the previous day). his hematocrit did not increase ( %), and over the ensuing hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of % while reducing the number of a rbcs in his circulation by approximately %. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes - days for antibodies to develop and they are short-lived ( - weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the calendar year from hospitals. an additional hospital* provided data for august-december . rbc transfusions in patients < year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/ years was also determined. results/findings: see table . the fraction of all transfused rbcs that were oneg ranged from - % (row f). the percentage of oneg rbcs transfused to oneg patients ranged from - % (row g); thus, non-oneg patients received - % of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients ( %- %; row i). overall use of oneg rbcs could have been reduced by %- % if opos units had been given to all oneg patients >/ years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* , nehad mohammed , marwa aly and nashwa fahmy . national blood transfusion services, nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on multitransfused patients who received blood transfusion chronically at our central blood center. they were thalassemia patients ( bthalassemia patients, one patients with a thalassemia), sickle cell anemia patients and immune hemolytic anemia patients ( auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). oncology patients, chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: out of ( %) alloimmunized patients and out of ( %) non alloimunized patients(p< . ) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june . screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified articles for data abstraction, where articles were transfusion guidelines. there were guidelines ( %) that made a recommendation, for a single unit and for multiple unit transfusion strategy (table ) . review b identified retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or . , % ci . - . ), although heterogeneity was high (i %). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december and february was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the months, units of platelets were transfused to recipients. over the months, a median of units was given to each patient with a range of to . the overall distribution of products used was % plasma, % pr, % pas f and % pas c. thirty percent of patients (n ) received all of their products on a single day. single units were given to patients while , and received , , and units respectively. the distribution by product type was % plasma, % pr, % pas c and % pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the month period (p . ). the distribution by service was different for the groups receiving multiple units. for single units the distribution was % hematologic malignancy, % infusion clinic (nos), % solid tumor medicine, % surgery, and % pediatrics. for those receiving multiple units the distribution was % surgery and % each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of . . conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* , , yvemarie b.o. somsen , maike e. van hezel , marleen straat , robin van bruggen and nicole p juffermans . sanquin research and landsteiner laboratory, academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains mg of iron and % of the rbcs are cleared by macrophages within hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in icu patients who received one rbc transfusion, different iron parameters were measured before and hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il- levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion ( . vs. . mmol/l, p . ). also, the transfusion had no effect on transferrin saturation ( vs. %, p . ), ferritin ( . vs. . mg/l, p . ) and il- levels ( . vs. . pg/ml, p . ). hepcidin levels increased in these icu patients after rbc transfusion ( vs ng/ml, p . ). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (- . vs. . % change, p . ). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il- or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since and pathogen reduced platelets have been available since . in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between and . during this time pas c, pas f and pr went from % to % of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in patients received an average of . units/recipient/month and in the average was . units/recipient/month. the intervening data points for , , and were . , . , and . respectively. the year average was . . the slope of the graph for all points was y - . . . the two sample t-test showed that the plt/recipient/month from to was not statistically different with a p value of . . conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that % to % develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least k/ul. the analysis revealed that the mean of . k/ul (n ) had a percent lower bound confidence interval platelet increment of k/ul (p< . ) results/findings: (median range [ - ]) hla matched leucoreduced irradiated sda platelets were transfused to ( m/ f) patients, median age years (range - ). / ( %) patients showing broad alloimmunization to hla class i/class ii antigens. / ( %) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority / ( %) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); / ( %) female patients had prior exposure via pregnancy and / ( %) had a history of hsct. ( %) platelets were abo identical-platelet increment median k/ul (range - to ), ( %) were abo compatible -platelet increment median of k/ul (range - to ) and ( %) were abo incompatible with platelet increments median k/ul ( range - to ). platelet counts were performed within hours in ( %) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* , renee leblanc , dongfu xie , alice cabe and yanyun wu . overlake hospital, bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about for years (from to ) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than . % of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a -year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of . g/dl (baseline - g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was . g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin ( . to . mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to . g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october to march . patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were rbc products transfused to waiha patients. twenty-three ( . %) patients received at least incompatible unit. the mean age was . years (range - yrs) with % women. ethnic composition was % african-american, % caucasian, and % patients of mixed/other ethnicity. one hundred fourteen ( %) of these products were released as li products and ( %) were compatible. ninetythree ( . %) of the li product transfusions had a post-tfn hct change of < % whereas only ( %) of the compatible product transfusions resulted in a post-tfn hct change of < % (p . , v ( ), exact methods). the mean hct increase in the compatible group was . % per unit vs. a slightly lesser per-unit increase of . % in the li group (p . , t-test, -tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for units. units that were incompatible had a lower mean post-tfn hct rise compared to all other li units ( . % vs. . %); however, this difference was not statistically significant (p . ). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected % per unit more frequently than it was for waiha patients who received compatible products ( . % vs. %). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first -hours from mt onset) was calculated with % and . % control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were mt cases from hospitals ( tertiarylevel, smaller/medium sized acute-care and specialist women's). number of mt cases per hospital ranged from to . patient median age was years (iqr , ), % were male and % required admission to intensive care. the most common clinical groups were cardiac surgery ( % cases), trauma ( %) and gastrointestinal hemorrhage ( %); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the tertiary-level hospitals was % (range % to %) and / ( %) were within the % control limit. cb that required ! rbcs within -hours of mt onset occurred in % of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals ( % versus %; p . ). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of bcs and ml of platelet additive solution iii (pasiii) were used to produce pcs (n ). pcs were stored on a flatbed agitator ( cycles/min) in a temperature-controlled cabinet at c for - days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of mm hg was applied. using clamps, a flow velocity of - ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted - %, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration ( . - . x /l) and number (> x /unit). simulated transfusion had no effect on the percentages of cd p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* , , joan sevcik and joseph e. kiss , . university of pittsburgh, blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution : is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient and and (control). results/finding: all patients were ab blood type. patient , a year old female with recurrent adamts deficient ttp, received courses of tpe (total tpe procedures) for relapse and exacerbation. ten out of procedures were performed with ab and a plasma (average ml of a plasma or % of total plasma volume for tpe procedures). patient , a year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of tpe procedures. four out of procedures were performed with ab and a plasma (average . ml of a plasma or % of total plasma volume for tpe procedures). patient , a year old female with adamts deficient ttp who served as a control, received a total of procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between patients. the trends of hemolysis laboratory data for patient and were comparable with patient . all patients had negative dat. only patient received rbc transfusions. all patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to % of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd negative platelet unit is not available for a patient with anti-cd antibodies sameer khatri* , charles harmon , brian r curtis and chisa yamada . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd ) is one of the identified plt surface ags and deficiency is rare, but found in asians ( - %), sub-saharan africans ( - %) and also in some people from mediterranean descent. two types of cd deficiency have been described. type deficiency is the complete lack of cd on both plts and monocyte-macrophages whereas type deficiency lacks cd on plts with variable expression ( - %) on monocytemacrophages. transfusing plts in a patient with cd deficiency is challenging given the rarity of cd negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than units of apheresis plt units over a week period without any significant increase in plt count. cross-match compatible plt unit found in of units and hla matched units were tried without success. at that point, a cd ab was identified in the serum and the patient's type cd deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was % due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd- negative (but blood type different and hla a transfusion vol. supplement s unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd antibody positive reactivity in serial dilutions has reduced from : to : dilutions and his hla class i pra has decreased to %. he is currently receiving apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to . k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec , , blood donations were screened for b microti by immunetics elisa. of those, ( . %) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between . % and . %. no patient babesia transmission has been reported since implementing this test, but we only had documented babesia ttd cases from - . donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of positive test results, lookback investigations were initiated representing % of positive donations. lookbacks were only performed when there was a donation within months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to % were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented . % loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only . % of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in > % of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for blood donors, were positive with an average load of . x copies/ml of plasma, a median value of . copies/ml of plasma, ranging from to . x copies/ml. pre-transplant viral loads were similar. for transplant candidates, were positive with an average of . x copies/ml of plasma, a median value of copies/ml of plasma, ranging from to . x copies/ml. post-transplant viral loads were remarkably different. for transplant recipients, all were positive with an average of . x copies/ml of plasma, a median value of . x copies/ml of plasma, ranging from to . x copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around - copies were present in > % of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt ( ) . post-ivig: all plt ( ) . post-ivig: cd -negative plt from relative ( ) . post-ivig: single donor apheresis ( ) . post-ivig: cross-match compatible ( ) . post-ivig: flow cross-match compatible plt ( ) . a transfusion vol. supplement s detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv ( / / / ), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows % of specificity, with no false positive results on the control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of tcid /ml for denv- , denv- and chikv and of tcid /ml for denv- , denv- and zikv. finally, the first results obtained on denv( ), zikv( ) and chikv( ) clinical samples show %, % and % correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of , , and were prepared from plasma or serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from : to : ( -fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of or higher and was not eliminated by the addition of a blocking step. pools of or samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of or samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $ cfu/ml of organisms associated with platelet contamination and incubated at room temperature for - hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher ( log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc , were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta d and virtuo the organism was recovered % . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc . however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of ml lrap demonstrated % recovery when loaded onto the virtuo and d ( table ) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus - , and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in , a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as- rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and hrs after amustaline addition, respectively, for titration by plaque assay on vero cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, > . log or log /ml of rrv was achieved, with > . log or > . log /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; - ml), large volume (lv; - ml) and dual storage containers (ds; - ml) designed to treat platelet doses between . and . x . the new triple storage (ts) set was designed to expand the dose range to . x and the maximum volume to ml, generating either or doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or % plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in % plasma/ % pas or % plasma with a final volume of $ ml and a dose of - platelets. these conditions represent inactivation using the lowest amotosalen concentration ( mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of - logs in respiratory secretions of mers patients, and with lower genomic titers of - logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log reduction/ml (log /ml) %plasma/ % pas e .coli . <- . > . e. cloacae . <- . > . k. pneumoniae . < . > . s. aureus . <- . > . blue tongue virus . <- . > . bovine viral diarrhea virus . <- . > . adenovirus- . <- . > . %plasma k. pneumoniae . - . > . s. aureus . <- . > . adenovirus- . <- . > . n a transfusion vol. supplement s abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged times up to days, assessing the infectious titer and genomic titer every rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of ! . log infectious titer. no viral replication was observed after days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* , jen-wei chen , chi-ling chen , sheng-nan lu and pei-jer chen . department of research, head office, taiwan blood services foundation, graduate institute of clinical medicine, college of medicine, national taiwan university, division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by , and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since ) and -sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since ) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during - and - , respectively. age-standardized prevalence and its % confidence interval ( % ci) were calculated with adjustment of who world standard population - . for the incidence study, donors who have donated blood two or more times during - and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its % confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from . per , donors ( % ci: . - . ) to . per , ( % ci: . - . ) during - , and the agestandardized prevalence was also decreased from . per , donors ( % ci: . - . ) to . per , ( % ci: . - . ). the agestandardized prevalence of anti-hcv was generally higher in female donors before , but it was significantly higher in male donors at (p-value . ). a total of , hcv rna positive cases, . % of them were anti-hcv negative, identified from , first-time donors during - , and the crude and age-standardized prevalence of hcv rna was . per , ( % ci: . - . ) and . per , ( % ci: . - . ), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value < . ), but no significant difference was found after age standardization (p value . ). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend< . ). in the incidence study, a total of new hcv rna positive cases, . % of them were anti-hcv negative, found from , , donors followed for , , person-years. the incidence of hcv rna was . per , person-years ( % ci: . - . ), and no significant difference was observed between both genders (p-value . ) and between age groups (p for trend . ). conclusion: the prevalence of hcv infection has been dramatically decreased by . % during - . it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* , sahar muhmmad and dalia el dewi . national blood transfusion services, azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is - days, hiv from to days, and hbv from - days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from to , the total number of donor samples to be screened is , the age of the donors ranged from to years, and they were of both sexes (m: f : ).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v ) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of nat yield donations among ( . %) seronegative donors. among these nat yields cases, ( . %) were reactive for hbv, ( . %) were reactive for hcv and ( . %) were reactive for hiv- . we stratified the age of the donors into groups; group a ( - years), group b ( - years) and group c ( - years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p . ; with % confidence interval (ci) . - . & p . ; with % ci . - . respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p . ; with % ci . - . ). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p . ; with % ci . - . ). nat-hcv; did not differ significantly between the three groups (p . ; with % ci - . to . between groups a and b & p . ; with % ci - . to . between groups a and c & p . ; with % ci - . to . between groups b and c). nat-hiv; did not also differ significantly between the three groups (p . ; with % ci - . to . between groups a and b & p . ; with % ci - . to . between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p . ; with % ci . to . ). nat hbv was significantly higher in males (p . ; with % ci . - . ), but the prevalence of either hcv or hiv did not differ significantly between males and females (p . ; with % ci - . - . & p . ; with % ci - . - . ; respectively). conclusion: in this study the nat yield of in assumes more significance when one considers the fact that single donation is used for generating components that can be used by recipients. hence, in effect the nat yield becomes times that is, in . saving recipients from tti out of ( . %) is indeed very significant. results/finding: of the , donors who were tested by our donor center, , ( . %) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at : titer. the screening elia s/co of this donor was . . both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were - transfusion transmitted babesia cases per year from - (table ). in the months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed ( in , donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of years of additional nat testing at blood bank, dmch, ludhiana from september to december . results/finding: results . % ( of ) units were initially nat reactive. these units were further tested, of which . % were discriminated ( hiv, hcv, hbv and co-infections). the remaining . % ( ) were repeat non-reactive and . % ( ) could not be discriminated. overall, nat yield rate was one in , whereas virus-specific nat yield rates were one in , for hiv, one in for hcv, one in for hbv and one in , for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past years has increased the screening sensitivities to check viral load and prevented transmission of probable transfusion transmitted viral infections. assuming % component preparation it saved transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu , , wei mao , tao he , yashan yang , , zhan gao , , chunhong zhang , hongmei liao , jingxing wang , and miao he* , . institute of blood transfusion, chinese academy of medical sciences & peking union medical college, sichuan blood safety and blood substitute international science and technology cooperation base, chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from , voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as e - . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: . gb raw data with , , reads were generated in the dna library. meanwhile, . gb raw data with , , reads were generated in the rna library. after cleaning the human background, reads from bacteria, reads from viruses, and reads from parasites were identified (table ) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table ) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of february to february . thrombocyte concentrates are prepared out of single donation units by the buffycoat method. results/finding: over the period platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* , germ an leparc , phillip c williamson , lani palmer , ben reynolds , maria noedel and lindsey houghton . creative testing solutions, oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february , recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march , . with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february , and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february , , the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in , who reported $ million new cases worldwide, resulting in > , deaths. malaria prevalence is highest in sub-saharan africa, home to % of all infections accounting for % of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of . mm amustaline and mm glutathione (gsh) and a h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with % fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching > % parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at > . log or > . log /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from to days using an fda cleared rapid test (rt). in february , our hospital based transfusion service implemented the use of rt on day , and to routinely extend ap shelf life to days. prior to this, we tested aps by rt on day and transfused day or day units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of day platelets. study design/methods: data were obtained for two -month study periods: october -september (pre-implementation) and february -january (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table . the number of ap transfusions increased by % post-implementation, comparable to a % increase in inpatient admissions and an % increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different ( . pre; . post, p . ). the number of rts performed increased by %. the percentage of transfused units tested at least once by rt prior to transfusion increased by % (p< . ). the outdate rate decreased from % to % (p< . ). ad-hoc ordering decreased from % to % (p< . ). conclusion: use of an approved rt for routine ap outdate extension to day was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* , vito scalia , carla osiowy , michael carpenter , anton andonov and margaret fearon . canadian blood services, public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low ( . and . per , donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of units. hcv nat was in place since (using minipools of ) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at - o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the ' ntr-e and ns b regions. sanger sequencing of these regions represents approximately % of the genome. results/findings: all confirmed positive donations were whole blood donations. there were hbv positive donations. of these, had tested hbv nat positive. genotypes were type a, b, c, d and e. there were samples hbv nat negative but hbsag positive ( were anti-hbc reactive). of these, could not be sequenced and one was genotype a (also anti-hbc reactive). there were samples considered hcv positive. of these, samples were hcv nat positive. genotypes were type a, b, c, b and a. there were also samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert d microbial detection system (bta d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert d (bta d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels ( - cfu/ml) of bacterial species commonly associated with platelet contamination, and replicates ( per instrument) of ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta d and the other into virtuo and incubated until signaled positive by the instruments or for up to days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august . this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a % voluntary donor program since and is the only center in the country that has achieved this goal. results/findings: a total of , blood donations were studied from august to december . in the rbdc, where only voluntary blood donations are recruited, the prevalence was per , donations for hiv (ic % - : , ); per , for hbv (ic % - : , ) and per , for hcv (ic % - : , ). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was per , donations for hiv (ic % - : , ); per , for hbv (ic % - : , ) and per , for hcv (ic % - : , ). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of : , for hbv; : , for hiv and : , for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s system. hbv s region was also sequenced. results/finding: obi were found in the , donations. in the viral loads assay, samples were negative and samples' viral loads were lower iu/ml. the mean viral loads was . . (log ) iu/ml in other samples,while the mean viral loads with hbsag /hbv dna samples was . . (log ) iu/ml. samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype ( . %) and the other was hbv c genotype( . %). compared the samples with hbsag /hbv dna ,we found two obi samples carrying with t>c mutation, which could cause an amino acid s f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag /hbv dna , and some unique variation was identified in the obi individuals. a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat th days, month, months& months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may , we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may to dec was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s platform using pools of serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, seronegative donations were screened by nat and a total of hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group - years old showed a large proportion, who accounted for % of reported infections. most of the hbv dna cases (about . %) reached senior high school education. the average hbsag dna positive rate was . % ( / ). incidence among apheresis donors in this period for hbsag dna were . / . these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf with day stability at c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control ( ml) and test components ( ml ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within hr and wbd pf within hr. cryo was manufactured according to site sops and frozen at - c (test ml, control ml ). test and control cryos were thawed at c, and characterized immediately post thaw (t ), and after d storage at c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over d at c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through d storage at c. conclusion: pr cryo can be processed from plasma sources, including pf , and stored at rt for days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf with stability over days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* , ed bakker , anton van weert , jane bryant , mark paradowski , lynne fleischmann , mirjana sarac , george chen , george schlauder and gregg williams . abbott diagnostics, sanquin diagnostics, abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects - million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over days using a panel of positive and negative samples. sensitivity was evaluated on presumed antibody positive specimens and specificity was evaluated on random blood donor samples. results/finding: precision was % cv or less for positive samples over days. the overall specificity in a blood donor population was . % ( / ). sensitivity was . % for presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* , anton vanweert , ed bakker , jane bryant , mark paradowski , lynn martin , daniela kaleve , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, abbott gmbh & co. kg, abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over days. sensitivity was evaluated using known positive samples, commercially available seroconversion panels, the who standard, hbsag mutants, and hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for presumed positive samples. sensitivity was % for all genotypes. % of the mutants were detected vs % for the comparator assay. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. analytical sensitivity ranged from . to . iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* , ed bakker , anton van weert , jane bryant , mark paradowski , kevin callear , susan sullivan , george chen , george schlauder and gregg williams . abbott diagnostics, sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types and (anti-hiv- / ). blood centers require very high throughput anti-hiv- / assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv- / antibodies and hiv- p antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv- / antibodies and hiv- p antigen was evaluated on the abbott alinity s system. precision was assessed over days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv- , hiv- and hiv group o antibodies and hiv- p antigen. seroconversion sensitivity was evaluated with commercial seroconversion panels. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for presumed antibody positive samples comprised of hiv- , hiv- and hiv- groups o, n, p, crf and urf samples. also, sensitivity was % for antigen positive viral isolate samples comprised of hiv- , hiv- and hiv- groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* , anton vanweert , ed bakker , jane bryant , mark paradowski , joyce siregar , angela vockel , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, abbott gmbh & co. kg, abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who st international standard. seroconversion sensitivity was evaluated using commercial seroconversion panels. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from . to . iu/ml. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* , anton vanweert , ed bakker , mark paradowski , jane bryant , tuan bui , joyce siregar , george chen , george schlauder and gregg williams . abbott laboratories, sanguin diagnostics, abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over days using htlv i and htlv ii positive samples. specificity was evaluated using , blood donor specimens from europe and diagnostic samples obtained from the united states. sensitivity was evaluated using preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot . . results/finding: imprecision was less than . % for positive samples over days. clinical sensitivity was . % ( / ) on preselected htlv i and htlv ii positive samples. the specificity was . % ( , / , ) on a blood donor population and . % ( / ) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez , michel garcia* , fernando palomino and guillermo orjuela-falla . national blood bank colombian red cross, universidad del rosario, fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than . ; abbott architect i sr) underwent supplemental testing by immunoblot (either chiron riba hcv . sia or hcv blot . test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: - . , - . , > . band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in . % ( / ) of samples, indeterminate in . % ( / ) and were positive in . % ( / ). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group - . ( . %) compared with the - . ( . %). in samples with indeterminate results, ns _ was the most frequent band detected ( , %). in contrast, the most frequent band in the group of positive results was core ( , %). only one sample from the indeterminate group ( . %) had a strong band intensity ( ), compared with samples from the positive group ( . %). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group ( - . ) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (> ). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns _ and ns _ cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* , ivanka mihaljevic , manuela miletic , miljana stojic vidovic , irena jukic , jane bryant , mark paradowski , angela vockel , george chen , gregg williams and george schlauder . abbott laboratories, croatian institute of transfusion medicine, abbott gmbh & co. kg, abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over days using positive samples. specificity was evaluated on samples obtained from , blood and plasmapheresis donors from the united states and europe and diagnostic samples obtained from the united states. sensitivity was evaluated using preselected positive samples. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than . % cv for positive samples over days. clinical sensitivity was . % ( / ) on preselected syphilis positive samples. the specificity was . % ( , / , ) for blood donor specimens and . % ( / ) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* , ed bakker , anton vanweert , jane bryant , mark paradowski , tuan bui , lynn martin , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over days evaluating positive samples. sensitivity was evaluated using preselected positive samples and seroconversion panels. specificity was evaluated on samples obtained from , blood and plasmapheresis donors from the united states and europe and diagnostic samples obtained from the united states. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than . % cv for positive samples over days. overall clinical sensitivity was % on preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying more bleeds than the comparator assay. the specificity was . % ( , / , ) for blood donor specimens and . % ( / ) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of , was declared as the public health emergency of international concern by who in feb . in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where . % of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, . % of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately : to : . thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of lm and assayed after illumination with visible light from both sides for , , and min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was . log % tissue culture infectious dose (tcid )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for min, min or min and the losses of the infectivity were further demonstrated by repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial min of treatment whereby ct-value jumped from . (control) to . (mbpt for min) (table ) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* , margaret fearon , susan l stramer , megan l nguyen , france bernier , sheila o'brien , vito scalia , sakina smith , yves gr egoire and boris hogema . h ema-qu ebec, canadian blood services, american red cross, sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in , canadian blood donors in . in a subset of , donor samples the seroprevalence was . %. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately , canadian whole blood donors including , from canadian blood services (cbs) and , from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all a transfusion vol. supplement s donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test ( % lod . iu/ml, % ci . - . ) for use on the cobas v r / system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous months are destroyed. recipients will be traced in the event of any products transfused in the previous months. results/finding: as of april , , of , ( , cbs, , hq) tested samples with valid results have been found hev-nat reactive: donors have been confirmed by further testing to date. confirmation is pending in donor. of the donors, were from quebec, and one each from nova scotia and alberta ( male, female). ages ranged from to years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: ate pork (including who ate pork liver), ate shellfish, ate venison, and drank well water. one donor had no identifiable risk factor. viral loads ranged from to iu/ml, of which were < , were - , and were > iu/ml; were anti-hev igm positive and anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around / . the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about to million cases and to million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august . both thin and thick glass stained blood smears of blood samples with giemsa was examined using microscope. results/finding: of the donated blood samples, ( . %) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p> . ), majority of the blood donors that tested positive belonged to blood group o ( . %). the prevalence of malaria parasite in the study was . %. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert d (bta d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life ( , and days after collection). study design/method: pooled lrap were seeded with low levels of organisms commonly associated with platelet contamination at , and days post collection. the seeded lrap were inoculated into bpa and bpn bottles on different days (not consecutive) alternating between teams of people each. seeded bottles were loaded into a virtuo and a bta d and incubated until declared positive or negative (up to days). additionally, bpa and bpn bottles inoculated with ml of unseeded lrap were tested on the virtuo and the bta d ( and bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of . % for the virtuo and . % for the bta d. the virtuo demonstrated an average improved ttd of . hours, when compared to the bta d in the presence of ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for days barring continued zikv testing and nonreactive results. a total of , donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low ( . %). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn , felicia santa maria , yvette girard , peter bringmann , marion lanteri* and adonis stassinopoulos . microbiology department, cerus corporation, scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in . the rapidly increasing number of infections in brazil, with hundreds of fatalities since december , is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate d-yfv using amotosalen (s- ) and uva light prt of platelet components (pc). study design/method: pc in %pas (n ) or % plasma (n ) were spiked with high titers of d-yfv and treated with s- /uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were . . log pfu/ml for pc in % plasma and . log pfu/ml for pc in % plasma while titers in post-prt samples were <- . . log pfu/ml for pc in % plasma and <- . log pfu/ml for pc in % plasma. inactivation to the limit of detection of > . . log or inactivation of > . . log pfu/ml was achieved for pc in % plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples ( hiv- antibody and hiv- p antigen, and htlv-i antibody and htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were . for hcv, hbc, syphilis, cmv igg, and chagas, . for hiv ag/ab and htlv i/ii, and . for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were . % (antibody sample) and . % (antigen sample) for hiv ag/ab combo; . % (htlv i antibody sample) and . % (htlv ii antibody sample); - . % for anti-hcv, - . % for chagas, - . % for hbsag, - . % for anti-hbc, - . % for syphilis, and - . % for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of ng/ml. robustness of the abbott prism methods to biotin interference c fischer , r schneider , w leonard , m cobb , g schlauder , g williams , m zuske m janulis* . transfusion medicine, abbott diagnostics, wiesbaden, germany, add diagnostics, transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from to colony forming units (cfus)/bag (i.e. . to . cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of - cfu/ml over the days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, hours (day ) after collection a sampling volume of spiked platelets ( . - cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate ( . %). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann , frank tolksdorf , wiebke handke , thomas h. m€ uller and axel seltsam* . german red cross blood service nstob, maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from buffy coats using the additive solution ssp (macopharma) with a residual plasma content of %. for inactivation kinetics, pcs (n ) were spiked with bacteria to a final concentration of approx. colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately . cfu/ml. bacteria were allowed to grow for h in the pcs at c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log reduction factors ranged from to for enterobacter cloacae ( . . , pei-b-p- ), pseudomonas fluorescens ( . . , pei-b-p- ), staphylococcus aureus ( . . , pei-b-p- ), and streptococcus bovis ( . . , pei-b-p- ). pcs (n for each species) spiked with these different bacteria species were efficiently sterilized ( out of ). treated pcs remained sterile during storage for days, while bacteria in non-treated pcs grew to high titers of - cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july , to july , was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than . were considered significant. results/finding: a total of , consecutive blood donors were screened between and . the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was . %, . %, . % and . % respectively. the hiv-hbv co-infection was higher among blood donors ( . %) followed by hbv-hcv co-infection whish accounts about ( . %). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of - . in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was , . ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a month old infant patricia davenport* , geeta paranjape and laurie sutor , . carter bloodcare, ut southwestern medical center background/case studies: in at a large pediatric hospital, a month old infant was supported for days by extracorporeal membrane oxygenation (ecmo). over this time blood products were transfused. about days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of units of red blood cells. approximately weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of : . the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* , andrea j linscott and donny dumani . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from - , % of transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a -year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day of hospitalization showed no growth after five days. on day , the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the ml unit had been aliquoted via sterile connecting device days prior for a pediatric patient. all ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was . c. within minutes of starting the transfusion, the patient's temperature increased to . c and subsequently reached a maximum of . c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at - c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of determinations of a low positive control in run. inter-assay variability was determined by testing at least representative negative production pool samples, at least low positive sample (about s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of . and were . and . respectively. the hbsag assay detection limit was . iu/ ml for source plasma and . iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at - c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between : , to : , , depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than % for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than %. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on donors ( - days after the index donation) - donors in the follow up study and tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all donors. denv antibodies were negative in donations and equivocal in . our initial reactive rate is higher than that reported to date for the procleix zikv tma of per , [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since . over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late , for which the program consisted of types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june , bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with ml of plasma, vs. ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, ; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation # : updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation # : implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over , donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on - year olds and premenopausal women (ages - ) donors. on average, - year olds donate . times a year and premenopausal women donate . times a year. if both of these groups were limited to donating once a year, a total of , donations from - year olds and , donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin # - , the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for - year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a . hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: % ( / ) of the simulation group students improved their post-test scores and had an average likert scale rating of . (very good). % ( / ) of hybrid group students improved their post-test scores and had an average likert scale rating of . (very good). % ( / ) of online only students improved their post-test scores and had an average likert scale rating of . (good). the average changes in scores were statistically significant within all training groups (p value < . ). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p< . ) and the hybrid group (p< . ). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in . all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july through january were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a -month period, cases of complex antibody identification workups ( %), transfusion reactions ( %), consultations for blood component utilization ( %), and deviations from standard operating procedures and massive transfusion protocols ( %) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $ , of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* , david lancaster , dianne geary , robert scott , anh thu nguyen , adam garcia , raina shankar , leslie buchanan and tho pham . stanford health care, stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to ( ) streamline the ordering process to accurately reflect inventory status and transfusion practices and ( ) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a -month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a -month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: ( ) the ratio of units transfused per week to the number stocked (t:s), ( ) the number of products ordered as stat, and ( ) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. lines of code were written for both programs, including class modules and distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were . , . , and . before the pilot period compared with . , . , and . during the pilot period. these differences did not reach statistical significance (p . ). we also monitored the number of stat ordered products before and during the pilot period, which were and stat units per week, respectively (p . ). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were and units, respectively (p . ). an estimated hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to . fte and $ , per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* , yulia lin , troy thompson , allison collins and sheena scheuermann . ontario regional blood coordinating network, sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that of the province's hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in indicated that % plan to implement or already have implemented the ptqip and % of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in . publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* , rana hajjeh and cees th. smit sibinga . world health organization regional office for the eastern mediterranean, international quality management (iqm) consulting background/case studies: more than million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between and ; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may in tunisia. results/finding: we found publications on disaster from five countries in the region and publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries ( . %) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated - % of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. ( , , and for - ) . the number of collections per registered trt donor varied significantly, ranging from to therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from . to . between and . conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from through . it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of . after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a % response rate (n ). of these, . % have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. . % of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although . % of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only % of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate % ( units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in , apheresis red cells (arc) represented . % of total red cell collections at our center. hae mcs ln was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs operators first and then operators new to apheresis with a training goal of % of mobile staff. validation of the alyx began / / and took approximately days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs machines were removed from service / / . alyx go-live occurred / / . additional operator training continued through september . results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $ . each providing an estimated annual savings of $ , . conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately . % and decrease our kit costs by %. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october -march . background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! g/l) and for females (minimum interdonation interval increased from to days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october , changes in rebooking of donation appointments in december and culminating with eprogesa criteria changes on march , . both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by . percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from . % in the weeks pre-implementation to . % in the weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were . % in september, . % in october/november, and . % from december to march, . conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past years, , blood products, derived from , procedures, were distributed to different investigators in over laboratories. whole blood was the most common product ( . %), followed by unmanipulated mononuclear cell collections ( . %), and elutriated monocytes or lymphocytes ( . %). less common requests included platelets ( . %), plasma ( . %) and granulocytes ( . %). adverse donor reactions were infrequent ( . % of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable % collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in % plasma. the corresponding pathogen reduction system used for the study has kits with different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than . x , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than x /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of x / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of . x and platelet concentration of x /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of . x and . x at the volume of mls and platelet yield of . x , . x , and . x at the volume of mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* and steve cihura . bonfils blood center, bbc / bsi background/case studies: in , the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated devices with the following criteria in mind: ) device disposable costs, ) reagents/controls/quality control, ) objective hgb/hct measurement, ) portability and durability for a mobile environment, ) ease of use, ) donor experience, ) battery life, ) validation requirements plans, ) blood center suitability, and ) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february with a targeted implementation date of july . after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately %. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to . % in and . % in . during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in , the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may . conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* , ayumi araki , hiromi sanyoshi , hiromi kanai , hiroya kikuchi , katsushi tsukada and kazuhide mure . hokkaido red cross blood center, japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from individuals who donated platelets during the -month period between february and august , and data from the donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the men ( . %) and of the women ( . %) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the donors in the vvr group was . . %, which was significantly higher than the . . % in the non-vvr group. at a maximum dbf threshold of %, sensitivity for discriminating between vvr and non-vvr donors was . % and accuracy was . %. when % dbf was used as the alert level, alerts were issued for donors, including in the vvr group. therefore sensitivity for predicting vvr was . % and specificity was . %. mean time from alert to diagnosis in the vvr group was . . minutes, and accuracy of the alert was . %. some of the vvr could not be predicted even the value of maximum dbf exceeded %. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* , , salam abdus and shabrina shah . inova blood donor services, inova fairfax medical campus, inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every to weeks from december to march . blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of patients, females and males, who underwent a total of rbcx from october to march , using an average number of rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of healthy volunteers were collected before blood donation and after blood donation immediately, day, week, weeks, and weeks among men and weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component ( c ) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at weeks among men and weeks among women, while c significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover week after blood donated and reached their levels before blood donated within weeks among men and weeks among women. conclusion: the biomarkers mutually changed over the course of weeks among men and weeks among women. donating ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low % split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate percent and increased concurrent plasma collections by percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october . after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in and , a total number , rns and rns completed the questionnaires, giving a response rate of . % and . % respectively. the overall mean score in was . points (range to ). the mean score in was . points (range to ). the percentage of rns having perfect scores of increased from . % in to . % in . table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; purposively selected blood prescribing clinicians and nurses from hospitals in countries of the human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at . level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r . ; p . ; df ) and accessibility (r . ; p . ; df ) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f( , ) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, junior faculty co-investigators from west coast institutions each had months to create a minute powerpoint presentation on a fundamental tm topic, after which other members had months to review and edit. therefore, each member created and reviewed presentations (three total steps). during each step, members wrote multiple-choice questions for those particular topics. in the end, each topic would have quiz questions to assess learning. at completion, evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: ) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; ) pre and post-lecture question validated examination (best collaborative) to assess learning; ) resident in-service examination trends specific to tm. results/finding: six presentations were developed as of the abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables and . abo leaders pre-test data could not be obtained for institution b, and trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsÕ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the minute vignette performance and the minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean %) when compared to the pre-test scores (mean %) ttest p< . . during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and % reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between . to . (in the range of - ). nps varies between and . according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased - from . to . . conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* , anne eder , beth a. dy and mary o'neill . american red cross, georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about in , apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. , a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to , hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the months before and after launching the educational outreach. results/finding: the web based course was completed by more than participants; were physicians. based on a review of the evaluations, the course was highly valued with % of participants rating it excellent or very good. the blood center physicians gave over presentations to hospital customers. reporting of suspected strs in increased by % compared to the prior year. the increased reporting came from specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required - minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, .) subscription-based e-learning utilizes - minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june , with a new equipment module assigned each month for the following five months. the series concluded in december with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of . on a -point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with positive and negative comment. level : learning the average score of users increased % from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of . and a t-stat value of . . level : results while equipment-related errors decreased by % after training, there is not enough data to demonstrate a statistical significance. conclusion: our level and evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october , a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n ) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was %. of those, % endorse that fbc creates a climate of respect within our transfusion practice, % believe it has improved communication between work units, and % feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only % responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: students of undergraduate semester and students of semester participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with % of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, / / from - am. there were attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, / / from - am. there were attendees, including regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) % as patients with fcr % may benefit from delaying the procedure for performance in the future. validation process included ( ) a deming regression to globally assess the predicted vs. actual results and ( ) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| %. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during days ( academic hours) the trainees can attend lectures, discuss the methodical materials, participate in seminars, interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, with the group capacity a transfusion vol. supplement s of up to people the number of medical specialists who have attended the training is nearly . results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of - ; being least satisfied/comfortable and being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of total technologists, technologists took the pre esd survey and technologists took the one year post esd implementation survey. table shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, ( . %) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were unplanned sop deviations; in the year after esd implementation there were only deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from to . the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in % plasma is broken up in to two days. on day platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for - hours. on day products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day and day . in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: of employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was . for the two month training period the daily average rose to . conclusion: our "flip the switch" training plan for implementing prt platelets in % plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly % of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as % of an activity. in we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of we performed a supply inventory on all departments to plan future purchases and control residual stocks. in , we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter (q / ) with last quarter (q / ). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q / blood bags were used compared to in q / , demonstrating an activity " . %. price negotiation resulted in . % readjustment. both indicated an estimated cost " . % with a possible impact of over us$ , . we have identified a real cost # . % in q / , representing an overall # . % and us$ , . (r$ , . ) savings. conclusion: economy had deteriorated in our country in with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* , candace williams , carmen meyer , paul lamonby , anne cleverley and silke milbradt-pohan . bio-rad laboratories, diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih- tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v , statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in . in the - nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on % individual and % for the other four levels ( % for each). the bonus (%) is calculated based on the iis as follows; category a: % (iis > %), category b: % (iis: -< %), category c: % (iis: -< %) and category d: % (iis < %). on the pilot implementation, the individual scores for staff ranged from % to %. the iis were % to %. the number of staff in each bonus categories were , % (category a) and , % (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* , deborah r fludd and sandra j nance . background/case studies: rare donors are defined as a blood type occurring in less than in people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in , there were , active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the ( . % of total active donors) returned contact cards alerting ardp of changes in calendar year , ( . %) were donors moving from one ardp facility to another, ( . %) were donors no longer eligible to donate, and an additional ( . %) were address changes. other changes were ( . %) reactivated donors and ( . %) donors who we were notified were deceased, or did not want to be listed in the ardp. in , new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in was ( ), which would be . % of the new donors submitted. conclusion: with nearly a % response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in , donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* , barbara j bachman , mike leamy , susan olson and candace williams . university of florida college of medicine, bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih- (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two ( ) runs of one to six ( ) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing , types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih- and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v , and statistical significance was assessed using the paired t-test, with p values of < . considered significant. regardless of quality or speed metrics evaluated, the ih- demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < . ). ih- process steps and time studies addressed in the table below did not account for the ih- reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih- ( % reduction, a difference of hours/year). conclusion: this study verified the ih- provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* , kimberly monnin , barbara j bachman , kyla warren , susan olson and candace williams . clinical pathology labs, bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih- tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of separate test runs of or samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately , type & screens (t&s). the workflow patterns observed were then repeated on the ih- and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v , and statistical significance was assessed using the paired t-test, with p values of < . considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih- demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < . ). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih- (difference of hours/year). conclusion: this study verified the ih- provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* , candace williams , carmen meyer , paul lamonby , anne cleverley and silke milbradt-pohan . bio-rad laboratories, diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih- tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih- and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih- with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v . statistical significance was assessed using the paired t-test, with p values of < . considered significant. results/findings: using automated result verification, only . % out of , samples evaluated for abo/rh testing would require visual verification, resulting in a % reduction in operator touchpoints (p < . ) and a labor saving of minutes ( : hh:mm) for abo/rh testing. for , antibody screens, automatic validation of results would result in . % reduction in operator touchpoints (p < . ) and a labor savings of minutes ( : hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by % annually and peaked at % in mid . to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an week period in late . hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than day shelf life remaining. units with tie tags attached were in hospital inventories for up to months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was % of the hospitals surveyed. mean percent o neg rbc gross sales for a month period before, during, and after the survey was . %, . % and . %, respectively. mean percent o neg net sales during the -month survey fell to . % compared to an average of . % in the months prior. during the -month survey period o neg rbc monthly return rate increased to an average of . % compared to an average of . % in the months prior. for the months after the survey the average o neg rbc return rate further increased to . % while mean percent o neg rbc net sales trended slightly upward to . %. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* , jeremie gachelin , veronique ollivier , thibaut mutin , xavier telot , benoit ho tin noe and sandra sanfilippo . aenitis technologies, hôpital bichat, inserm u background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac ) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than % (p< . ) and a purity of platelets close to . %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over . blood donations for an area with more than million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in , orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in , when we moved from this equipment to atreus c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in we changed to atreus c (terumo bct) and finally in , we moved to reveos system (terumo bct). since the changes in , our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in , bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during and . conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of - patients and an average, round-trip travel time of approximately -minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about % of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november , the hb has significantly improved the turnaround time of rbc issue -from -minutes to less than -seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately rbc per month out of the window for non-surgical patients. this has been reduced to approximately rbc per month, a % average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in , they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the st rescue squadron ( rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the rqs master sergeant in january . we asked what rqs's order and delivery expectations were. he said sporadic use and the blood order would be rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the rqs needs. staff was trained based on data from january meeting. we contacted the rqs in september to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the rqs association with a civilian blood center. based on his field experiences, he changed the blood order from to rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and rqs performed a mock run on october , and we felt prepared for any future events. results/finding: on november , , the rqs was deployed to a civilian aeromedical evacuation. we anticipated a rbc order. the actual order was rbcs and ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at : am by the rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november th , we established a maximum blood order of rbc and liquid plasma, noting future orders may request fewer products, yet meet the preferred rbc; plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table ). the highest mean fib concentration was mg/donor unit; lowest mean fib concentration was mg/donor unit. all sites had a mean fib concentration at least mg/donor unit above the fda minimum requirement of mg/donor unit. fifteen of blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; manufactured pooled donor cryoprecipitate. most froze plasma in a - c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every minutes for a hour period or until the temperature exceeded c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached > c in minutes as shown in table . the control thermometer recorded temperatures maintained at - c for the entire hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at - c for more than minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: % conventional (c-pc), % pr-pc, and mix of % c-pc/ % pr-pc. the model predicts a modest ($ %) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january . a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august , inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january . a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from to (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in , zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within weeks (sept. phase ) and nationwide within weeks (nov. phase ). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools ( -donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab had % and lab had % of clients requiring universal zkv testing. we evaluated a -month test result upload performance period to determine the impact of zkv test implementation. results/finding: during , lab upload time performance ranged from % to . % from january to july; upload time performance fell between august through november, returning to . % performance in december. lab upload time performance ranged from . % to . % january to august. performance fell september through december . % - . %. lab experienced a low of % upload time performance during phase when there was a rapid implementation; % clients required zkv nat. improved performance was observed during phase , with a % increase in zkv clients. for lab : phase experienced a modest decline of upload performance ranging from . % to . % with . % of clients implementing zkv nat. performance was . % in phase , when an additional . % of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* and colleen a. aronson . advocate lutheran general hospital, acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in of the hospital transfusion services. the sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the sites which implemented the verax pgd test perform testing on all day and day platelets to be issued for transfusion. this abstract summarizes the data collected for the first weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day , day , and those that were tested twice. inventory reports were reviewed for platelets issued on day or day that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february ( week of performing the test), . % of all platelets issued by the participating transfusion services were day or day platelets. in march that number dropped to . %. it is expected that this number will level off at some percentage at or below . % with further data collection. in february . % of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day or day ) will likely level off at some number at or below . % due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, of the sites performing testing are level trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed %. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n ). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days , , and of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was ( . . )%, relative change rate of hsr was ( . . )%, the residual leukocytes were ( . . ) . the ph, hsr, and the cd p expression of pooled platelet concentrates before and after filtering were ( . . ) vs ( . . ), ( . . )% vs ( . . )% and ( . . ) % vs ( . . )%. there is significant change for wbc after filtering (p< . ). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table . conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb - ) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of ml and ml. two users then evaluated the ss tubing segment types with ml or ml samples for a total of data points. samples were collected into the attached ml or ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january , a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by %. the platelet outdate rates dropped after implementing the platelet inventory tool from % ( units) to % ( units); a % decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of fte to . fte, lowering fte by %. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* , jaclyn mckay , jennifer curnes and rowena punzalan , . bloodcenter of wisconsin, children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: ( ) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, ( ) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within -hours after collection. this tight hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight -hour completion time constraint for cryo production (capacity expansion). in particular, during the th -quarter of , a blood processing region was able to process about more cryo units/month (an increase of %) at a slightly lower collection cost (cost avoidance), resulting in an approximately % reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the -the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after -day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from to days in the usa using an fda cleared rapid test (rt). in august , our hospital based transfusion service began using a rt on day and to routinely extend ap shelf life to days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of -day ap. study design/methods: data were obtained for two study periods: september -february (pre-implementation) and september -february (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from -day to -day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by % post-implementation while inpatient admissions and surgical volume increased by % and %, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by % post-implementation and the outdate rate decreased from % to % (p< . ). ad-hoc ordering was not statistically different between study periods (p . ). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different ( . and . , respectively, p . ). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years ( ) ( ) ( ) ( ) ( ) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was , , and units respectively. the mean number of discarded rbc units of the five years of the study exceeds % of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below mg/dl, and so the rejection threshold for lipaemia is level equal to or more than mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including rbcs, plasma products and apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from mg/dl to mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* , , rebecca ross , debra mraz , anne baker , zenna neal , melanie champion and edward l. snyder , . johns hopkins university school of medicine, yale university, yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, pr-sdp were transfused at our hospital (out of a total of platelets transfused). after months of scale-up, pr-sdp were approximately % of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($ mg per g) to the content in pr-sdp (< ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september , the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from donors was collected in k edta tubes. plt concentrations were determined at the qc department using the coulter act diff hematology analyzer (beckman coulter). sample tubes were stored at - c and measured at , and hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd ( ml; n ) or dpd units ( ml; n ). plt pools were stored at - c under mild agitation for seven days except for dpd, which were split in two -ml bags after h. samples were taken on days and . ph, po and pco , hypotonic shock response (hsr), extent of shape change (esc), cd p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at h ( pltx /l), h ( pltx /l) and h ( pltx /l) postdonation. dpd can be stored in the same collection bag for h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits ( - pltx / l) before splitting. on day , lactate and pco concentrations increased, and po decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march . there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted % increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* , colleen vincent and patricia kopko . university of california -san diego, american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only - % of double platelet collections meet requirements for pathogen reduction treatment. blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: , apheresis platelets annually), which includes two hospitals ( inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week ) and the bone marrow transplant (bmt) ward (week ). an e-mail communication explained the change to all physicians and nurses. in phase , we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase , we expanded usage to include the inpatient bmt ward. in phase , we lifted all restrictions so prp could be used throughout the health system, with the goal to reach % prp within months. results/finding: in phase (weeks - ), we requested prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of prp weekly (range - ), and prp constituted % of platelet transfusions in the cancer center. in week , excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase . in phase (weeks - ), we formally expanded issuing of prp to include the inpatient bmt ward and requested prp products weekly. our blood supplier provided an average of prp weekly (range - ), and prp constituted % of platelet transfusions in the phased-in areas. in phase (weeks - ), we began issuing prp throughout the health system. our supplier provided an average of prp weekly (range - ), and prp constituted % of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table ) resulting in a cost savings of $ k. an additional cost savings of approximately $ k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as a transfusion vol. supplement s we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* , james r stubbs , scott a hammel and manish gandhi . mayo clinic, mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing % pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of . , . , and . x may end up below a . in the final storage bag and would need a post-processing sample to ensure the product met criteria at ! . x platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a . yield but ended with a yield below . . these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe- d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that of results were . or . x / mcl with the exception being the wbc failure with a count of . . further monitoring of the wbc counts discovered a result of . which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of . will be tested on the adam to confirm a leukoreduced product. we also discovered of ( %) of the . preprocessing yields products ended with a post processing yield < . . we decided to increase the yields requiring post processing samples to include the . . conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of ! . x platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from / - / were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need ) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in ; the revised prediction method (estimated clinical need ) was then evaluated retrospectively using the same data sources covering / - / and then compared to the prior method. results/finding: the average number of platelets transfused from / - / was . u/d with a standard deviation of . u/d; the predicted amount was . u/d. the difference between the predicted amount and the number of units used was - . u/d. % days ( d/month) were under-predicted (average: u/d). % of days ( ) were under-predicted by ! u (average: u; max: u ( x)). the average number of platelets used from / - / was . u/d with a standard deviation of . u/d; the predicted amount was . u/d. the difference between the predicted and units used was a . u/d. % days ( d/ month) were under-predicted (average: . u/d). one day ( %) over this period was under-predicted ! u ( u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages ( % a %), reduced the number of days under-predicted ( % a %), and decreased the discrepancy on those under-predicted days ( . u a . u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* , maria noedel , nancy haubert , kenneth hudson , larry morgan , robert shaw , tracy fickett , jamie jue , valerie winkelman , sally caglioti , german leparc , and phillip c williamson . background/case studies: on / / , fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within weeks; nationwide in weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on of manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for clients within weeks, and an additional clients within weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the -week period but overcome during the -week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* , hala samuel boules , fatemah saleh al matroud , rabab hussien ali dashti , hanan alawadhi and reem al radwan . kuwait central blood bank, kuwait central blood bank, kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of rbcs baby units were randomly chosen to be traced to their final deposition from the year till . half of them ( units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb ( units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is % efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (< days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march . results/findings: a total of products were tested. fifteen units ( %) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all by the vendor and none were true positives by re-culture. of the units that were successfully tested, were tested again on day for use on day ( %). there were platelets transfused ( %) and expired after day ( %). the cost to test the products including controls was $ , and our calculated cost to produce products would be $ , . if we had needed to import products to meet needs, the cost would be roughly $ , without shipping costs which are estimated at $ , . . we averaged expired platelet products per month (range - ) before verax testing and (range - ) after implementation. conclusion: using verax point-of-care testing saved platelet products from discard. the cost savings were $ , . from importing and $ , from producing a replacement for those products. the average discard rate per month went from to after verax implementation. extending platelet shelf life to days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for data, units were investigated without the use of secure texting. of these, units were identified as preventable wastage, and wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: ) product not released after procedure/ or when patient stabilized ( ) ) product returned outside of appropriate temperature range ( ) ) clinician unaware product was assigned ( ). thus far in , wastage records have identified units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, responses thanked the transfusion service for the information, and in instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: . % dmso, plas-a, % hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) . %dmso: plas-a : %hsa : : . plas-a and hsa are kept at room temperature ( - c, rt) and refrigerated at c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point . c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total tests. at least minutes cooling after dmso, before adding the next reagent. see table: ( ) after directly adding . % dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. ( ) in tube, autologous plasma first, dmso next, powder-like precipitates. ( ) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. ( ) & ( ) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at c. ( )&( ) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the c group is slightly milder/slower than rt group. so hsa should not be added first. ( )&( ) trace of hsa(< ml) was mixed into the plas-a bag ( ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive -month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from . % to . %. with an annual platelet usage of approximately , units, this reduction equates to approximately units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $ , to $ , , per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* , russell thorsen , rosaline ma , antonio g insigne , amy decourten , florence panganiban , patricia mckean , cyril jacquot , sara bakhtary and ashok nambiar . ucsf health, children's national medical center background/case studies: plasma (ffp, pf , pf rt ) stored at - c outdates hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (< months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited ( hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk and richard gammon* . oneblood, oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average - minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average - minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from % to %). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp- using the low glycerol ( %) freezing method and frozen at - c within six days of collection. thawing occurred in a c water bath in the following order: o positive and o negative on january ; o positive and o negative on february ; and o negative on february . deglycerolization occurred on site using the acp- with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $ - and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $ per unit to produce and distribute. drbcs have a shorter shelf life, days versus the days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp- 's and deglycerolize four units at a time. in january and february , it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c izekial butler* , karen leighton , scott jones and rachel beddard . qualtex laboratories, biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c instrument. precision of the new assay parameters was determined by testing replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from iu/ml to iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of . to . iu/ml. stability of samples was determined by testing samples stored at - c and - c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from . to . . so, precision was acceptable since the %cv for all samples tested was %. the mean values for the samples tested in the accuracy study were all % of the expected value which was much lower than the acceptance criteria which was % of the expected value. the linearity of the assay was acceptable with a r ! . %. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to days at - c and up to month at - c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for days at - c and stored up to one month at - c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, ) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april , to march , . local research ethics board approval was obtained. results/finding: patients received ivig for itp at smh over the study period for a total of unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding ( , %), pre-operative or antepartum care ( , %), a platelet count of less than and contraindication to corticosteroids ( , %). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between - x /l. patients received ivig for a likely diagnosis itp while a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. / ( %) of infusions consisted of g/kg over days; the remainder of infusions consisted of g/kg. of those who received g/kg, of patients ( %) had evidence of partial remission after a first g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to staff members in early february of . twenty-two technologists responded for a % response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, % indicated that workspace size was insufficient and % that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* , neil bangs and kimberly sanford . vcu health system, virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from / / to / / to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (< hours, > hours < hours), and if a second sample was required. results/finding: a total of t&s orders were initiated from the ed in this time period. ( . %) patients were not subsequently transfused any type of blood product within hours of tsd and ( . %) patients were not subsequently transfused any type of blood product within hours of tsd. a total of ( . %) patients required a second sample. of these patients requiring a second sample, ( . %) were not subsequently transfused any type of blood product within hours of tsd and ( %) were not subsequently transfused any type of blood product within hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within hours of tsd amounted to an estimated $ , in unnecessary patient charges and approximately . nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* , debra berry , yunchuan delores mo and gay wehrli . university of virginia health system, children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* , elizabeth halperin , sharon breining and mona papari . acl laboratories/ advocate hospitals, advocate health care, itxm background/case studies: a large midwest hospital system with level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and months of data were evaluated that contained events. results/finding: there was an equal number of events that were initiated in the ed and the or ( ). male patients were involved % of the time and % of time the patients expired. trauma of some type was the majority of the cause but . % of the cases involved gi bleed and only . % were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average . with the post hgb average of . . ratios of : for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of : . it was found that the rbc: plasma was . : , rbc: plt was . : and rbc to cryo was . : . use of txa was only . % and cacl was utilized in . % of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* , jody a barna , donald e ulinski and nancy m. dunbar . dartmouth hitchcock medical center, dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july , we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august and november (pre-implementation) , newly identified clinically significant antibodies were resulted for patients compared to patients between the months of august and november (post-implementation). the tat for allergy alert entry for both periods is shown in table . we observed that % of allergy comments were performed within hours in the post-implementation period versus only % pre-intervention (p . ). using the new process, nearly all of the alerts were entered into the emr within hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* , shailesh macwan , nancy nikolis , arline stein , janelle richardson , manju bagu , lennart logdberg , alexander indrikovs , vishesh chhibber and sherry shariatmadar . north shore university hospital, northwell health background/case studies: our institution is a tertiary care facility performing over cardiovascular surgeries (cvs) in , an increase of % after the healthcare system cvs integration in . transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december , the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after reported events in q that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: . open discussions and collaboration between blood bank and cvs nursing teams . mapping the process using flowcharts for additional blood orders from cvs. . identify bottlenecks and brainstorm solutions. . a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. . the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. . follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period / / - / / the blood bank has received verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* , tracie ingle and heather vaught . indiana university health, indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter - and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with - o, d negative red blood cells over days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series , anti-d series , a cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion ; : - who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for % and % of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february -june as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june -october . results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table ) . this led to a reduction in observed overall sli ( . . days vs . . days, p< . ) and odr ( . % vs . %). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. ( )) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january and december thirty-five ( ) notifications were sent to physicians using epic letters and of those, fourteen ( ) responded to the epic notification and five ( ) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* , denden benabdessadek , annu george and alexandra jimenez . westchester medical center, new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of orders were reviewed. approximately, % of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases ( %), but of the issued products, all were returned to the blood bank in % of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> type and screens (t&s) per day] blood banks , respectively. our laboratory which serves a large -bed multispecialty academic hospital and receives - t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of . . and . . minutes , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was % longer and had a larger standard deviation (s.d.) than the published trial result of . . . transfusion vol. supplement s abstract during phase i visionv r performed panels. during phase ii visionv r performed of the visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* , john roback , ronald arkin , michael bartlett , robert geiger and jaxk reeves . university of georgia, emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over , emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a % target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of participants responded to the survey ( . % response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n- - df) of the null hypothesis that the mean vs. the alternative that the true mean is> . overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on run cycles. ct was comprised of metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc ( : ), xm ( : ), cb ( : ) and dat ( : ). by implementing the future state, an average $ . min. lt and vt is saved on each sample loaded for ts equating to a % labor reduction over the current state. a % improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a % lt reduction. on average, a min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $ type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing -column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on patient samples for abo/d typing and antibody screening; of which at least had a positive antibody screen. out of the , had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which were d(-) and were d( ). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the patient and donor samples tested ( % concordance). all samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave % sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for samples at different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* , craig fletcher and peter millward . beaumont hospital, beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short -day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october and quality data was reviewed from august to december . the collected data was then analyzed using descriptive statistical methods. results/finding: data from indicates platelet wastage comprised % of total received platelets and % of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units months preimplementation of the report was units, compared to units months post-implementation and units months post-implementation. wastage rates improved from % (wasted yearly platelets/total received yearly platelet units) in , the year of report implementation, to post-implementation rates of % in and % in (see table) . importantly, this occurred despite a greater than % increase in platelet inventory between and and resulted in cost savings of over $ , in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* , lisa marie button , lori scanlan-hanson , karen koch , janet finley , deepi goyal and camille van buskirk . mayo clinic-rochester, mayo clinic, mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in and were / (events/ed transfusions - ). study design/method: electronic ordering for the ed was implemented march st . any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients < kg and pediatric patients > kg. a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for years (april -march ), and during that time there was instance of blood being ordered for an unintended patient . % ( / ). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in , / ( . %) units were transfused in the ed but not charted in the patient's medical record. in , / ( . %) transfusions were not charted. however, in , the first full year of electronic transfusion order capability, only / ( . %) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level trauma center, with over inpatient beds and over operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option (verbal) for verbal orders and coolers set up; and option (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by % with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* , steven zibrat , geoffrey wool and angela treml . university of chicago medicine, university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in , blood bank accounted for % of all rejected clinical laboratory samples, yet comprised only % of total laboratory volume; % of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was . %. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september . results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of . %, three times the national average of . %. under the new system, rejected blood bank samples decreased from an average of % to % of all rejected laboratory samples, a % decrease. implementation of the new process produced a net savings of $ . k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured residual products for suspected str. the tat for final culture result entry into the patient's emr was - days (mean days, sd ). in the postimplementation period, we cultured residual products for suspected str. the tat for final culture result entry into the patient's emr was - days (mean days, sd ; p . ). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* , marcus holme , johnathan bakst , gunta musa and angela treml . university of chicago medicine, university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an minute turnaround time (tat). in april of , the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased %. tat analysis of a representative one week sampling per month showed an increase in outliers from per month to per month. average monthly tys samples performed is . these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was / / . results/finding: the average number of outliers decreased % from per month to . further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $ , for fiscal year . conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from - days with a mean of . days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of - days with a mean of days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* and randy levine . northwell health, lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, units were transfused on the oncology floor with units ( %) requiring irradiation and only of those units ( %) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, additional irradiated units were issued ( / ; %). post-intervention, units were transfused on the oncology floor with units ( %) requiring irradiation and all of those units ( %) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus of the ( %) total units were ordered as irradiated. again, additional irradiated units were issued ( / ; %) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* , charlotte van dyke , dina garza van hoose and rachel beddard . biobridge global, south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january , to april , , , total collections were flagged for additional qc by our trima accels and haemonetics instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in , the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: ) risk assessment, ) quality control plan and ) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* , rachel jug , kimberly ingersoll , nicholas bandarenko , nicole guinn and jessica poisson . duke health pathology, duke university hospital, duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced : : transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july -december . data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the : : ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* , nancy nikolis , linda benison , ruthmire thelusca , renee liberty , sherry shariatmadar , alexander indrikovs and vishesh chhibber . north shore university hospital, northwell health background/case studies: our blood bank (bb) processes approximately , specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june , a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed / . the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of . the implementation of this new process has led to a decrease in the number of unacceptable specimens up to % quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from % to %, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* , , morgan rockwell , joseph hagan , jun teruya , and shiu-ki hui . texas children's hospital, baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within hours pre and post transfusion were evaluated. patients ( - years) receiving prophylactic ptx from july to december admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < ml/kg for patients < kg and one apheresis unit (au) for patients > kg; therefore, patients were separated into groups: < kg and > kg. a significant proportion of orders for both < kg and > kg did not meet patient platelet threshold criteria (p< . ). conclusion: ptx threshold above ir for both groups were ( kg) and % (> kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than % of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p> . ) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only . % of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated ( x ) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately patients ( x ) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts ( . %) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by % with the communication category average rpn having the greatest reduction of %. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* , karen king and joseph sweeney . rhode island hospital, lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different ( minutes for the provuev r and for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over minutes are shown in the table. the results show a reduction in tat by minutes with a % reduction of tat greater than minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* , , lorna orengo , monique scott and christopher a tormey , , . yale university school of medicine, yale-new haven hospital, va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as in , blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as % prior to interventions, but may potentially be reduced to as little as . %. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of < %, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged . %. reasons for specimen rejection were divided into groups: ) hemolysis, ) blood bank witness collection form errors, ) quantity not sufficient, abstract ) duplicate sample, and ) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table ) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a . % rejection rate with only rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s system christopher shahan* , christina dejesus , mosi mccall , fallon hampton , tangi herring , judy davis , anjali patel , sonya gomillion and bonnie maltby . qualtex laboratories, qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual - hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently % of whole blood donor testing turnaround time delays are due to issues and failed runs on the s system and we have technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s system. study design/method: the number of technician related failed runs on the s system were tracked from / / thru / / . a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s system. after counter measures were implemented, the number of technician related failed runs decreased from to . failures per week, which was a % decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by %. patients who were transfused for pre-transfusion hgb > g/dl with resulting post-transfusion hgb > g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: patient charts were reviewed. were excluded for bleeding and cardiovascular instability. / ( . %) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors ( . % vs . %, p . ) and anemia of chronic disease ( . % vs . %, p . ) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb . g/dl vs . g/dl, p< . ). inappropriately transfused patients also had higher median post-transfusion hemoglobin ( . g/dl vs . g/dl, p< . ). moreover, lab evalutions revealed association with lower folate levels (median . nmol/l vs . nmol/l, p . ). / ( . %) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. / providers were responsible for . % of all inappropriate transfusions. / appropriately-transfused patients experienced an fnhtr. deaths unrelated to transfusion occurred ( in appropriate, in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* , willem martin smid and ashley john duits . red cross blood bank foundation, sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* , dee dee cassidy , jed b gorlin , and nancy l van buren , . hennepin county medical center, innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant or more minutes may be required for transit of units often released in less than minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july . data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january -june , and post change included august -december . july data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of rbc/month in coolers. post change this dropped to rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of rbc/month were returned ( %). post change, the average rbc/ month returned was ( %), this represents an absolute % reduction in number of returned products. each rbc dispensed and returned takes approximately minutes to complete paperwork and transport, therefore this change saved an average of minutes per month. it was also noted that the average rbc/month transfused was for baseline and post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of - minutes (estimated) was reduced to - minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over patent-applications have been filed related to "transfusion medicine" and over related to "transfusion alarm", during the last years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an / inch color cmos sensor, providing effectively . mp, a . mm lens, ir-cut, day/night minimum illumination . lux/f and viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood a transfusion vol. supplement s abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h. , video frame rate (fps) - /s, refresh rate hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/ . /b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p p is provided. typical v power-supply, sized x x mm and weighing g. client software is required. the ir range is - m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a gb sd-card. pan/tilt-horizontal o and pan/tilt-vertical o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot ( cfu), multishot cfu or highdose k organism preparations at a low level (< cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < days and the fungal organisms in < days. the overall agreement was . % in bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* , diane schafer , debra brown , jesse cox , scott koepsell and sara shunkwiler . nebraska medicine, the nebraska medical center, university of nebraska medical center background/case studies: anticipating the implementation of the new ( th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september , ), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling in months post implementation compared to in the months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late ' s and in early , intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january , to june , . data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: % were female and % were less than weeks of gestational age. none had co-existing g pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn ( %) and rhesus-hdn ( %). antibodies most often implicated in rh-hdn were anti-d ( / ), anti-d and anti-c ( / ) and anti-c ( / ). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was g/kg (range from , g/kg to , g/kg). neonates received one to four ivig administrations. table shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, required an et for rh-hdn and for abo-hdn. forty-five ( %) patients needed top-up transfusions during hospitalisation and until three months of age: with abo-hdn and with rh-hdn. the mean number of transfusions was three (range: to ). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over % case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a -part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate ( each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, georgia institute of technology ap reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to a) all adult males (am), b) women of non-childbearing age (wncba), and c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to a) women of childbearing age (wcba) and b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed %) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap saving apheresis platelets through use of verax point of care testing jennifer rhamy* and rebecca wride . st. mary's regional blood donor center, st. mary's regional medical center background/case studies: our rural hospital-based blood center serves hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between and per day in ), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson and jun teruya , . texas children's hospital, baylor college of medicine ap vision titers --easier or problematic? (table ) . results/findings: post intercept, t had volumes of - ml, with % hemoglobin (hb) recovery. t had -fold less extracellular protein than c. after days of storage t had higher atp and na than c while lactate and hemolysis were lower. hct, ph, k and glucose were equivalent between t and c on d . d hemolysis for t was . - . %, while for c it was . - . %. t and c atp was > mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table ) . hematocrit (hct, %) . . * . . . . . . hemoglobin (g/unit) not measured hemolysis (%) . . * . . . . * . . ph ( c) . . * . . . . . . total atp (mmol/g hb) . . * . . . . * . . k (mm) . . * . . . total tested total plts issued feb mar totals table: . resident reports to the intranet "drop box" increased from . % to . % to %, each over month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from . per day or , dpmo to . per day or , dpmo. this is a statistically significant difference since the p-value calculated was . . conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately , specimens per month. since , the requirement of having a second blood type on record was met by: . utilizing the historical blood type and the current specimen, or . having second type performed on same specimen by different technologist, and . each type and screen specimen signed by staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards th edition, # . . . a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april , the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding: . there was a minor adjustment period with more phone calls made to blood bank to explain the process. . there was minimal impact on turn around times for release of components. . aborh retype workload decreased from to ( % to % of t&s volume) per month. . unnecessary blood draws minimized, improving patient experience. . no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as to %. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our bed hospital, a retrospective chart review was performed ( / / - / / ) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< grade" or "> grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples ( %) were > titer results higher, while the majority was titer results different ( %). the cost analysis is summarized in table . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated % decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc. samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another samples were measured with fc in ucc maribor. results/finding: samples ( rbc, plt, ffp-all leucocyte depleted and non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from , - , ; plt from , - , ; ffp from , - , ; and for non-leucocyte depleted ffp from , - , (table ) . samples ( rbc, plt, ffp -all leucocyte depleted and nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from , ; plt from , , ffp from , ; and for non-leucocyte depleted ffp from , - , (table ) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than x /unit for leucocyte depleted or x / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control. key: cord- -ezrn cva authors: nan title: physicians – poster session date: - - journal: bone marrow transplant doi: . /bmt. . sha: doc_id: cord_uid: ezrn cva nan hematopoietic stem cell transplant unit, hematology department, hospital universitario de donostia, donostia/san sebastián and informatics and automatics department, university of salamanca, spain the immature platelet fraction (% ipf) is a relatively new parameter that measures young (reticulated) platelets in peripheral blood (pb). ips rise as bone-marrow (bm) production of platelets increases. several clinical utilities of the %ipf have been already proved, as the treatment response monitoring in aplastic anemia or immune thrombocytopenic purpura. in this study, we aimed to found if ip measurement might be useful during the grafting phase of hsct. this study includes patients who underwent allo-hsct in our center during the last . years. were male ( %) and female ( %). median age was years (range: - ). baseline diseases were: acute leukemias ( ), lymphoproliferative disorders ( ), myelodysplastic syndromes ( ), chronic myeloproliferative diseases ( ), multiple myeloma ( ) and bone marrow failures ( ) . donor was unrelated in cases, and related in (including haplo-identical). conditioning regimen was: busulphan-based ( ), melphalan-based ( ), tbi-based ( ) and others ( ) . progenitors source was pb in , and bm in . platelet count, %ipf and absolute ip count (aipc) from day + to the day of stable graft were analyzed. . % patients reached plat ⩾ /mcl at day + , . % at day + and . % at day + . median first day of plat ⩾ /mcl was day + (range: . median %ipf was . % (range: - . ), . % (range: - . ) and . % (range: - . ) at days + , + and + , respectively. median aipc was /mcl (range: - ), (range: - ) and (range: - ) at days + , + and + , respectively. among the time points analyzed, aipc at day + showed the best positive correlation with platelets counts at day + (r = . ). interestingly, patients with lower aipc at day + showed a delayed platelet graft (see table ). contrarily, patients with higher aipc at day + had an earlier platelet graft. absolute immature platelet count before the graft seems to predict the precocity of the platelet graft for the majority of patients undergoing allo-hsct. this finding might help physicians for the patient management (anticipation of hospital discharge and so on). disclosure of conflict of interest: none. [p ] p analysis of genetic polymorphism for cardiovascular diseases (cvd) in placental and maternal blood in hypertension and hypercholesterolemia c khalil , a azar and a ibrahim , reviva stem cell research and application center, lebanese university, middle east institute of health hospital and faculty of medical sciences, lebanese university, lebanon cardiovascular diseases are the world's leading cause of death representing % of the total global mortality. the genetic polymorphism of the cvd genes, especially the ace: angiotensin converting enzyme gene risky alleles (ins/del) which are associated with a high and inappropriate level of ace can be considered as a genetic model in the development of hypertension and its complications in cvd. we evaluated the mutation impact of the cvd genes in the lebanese population, based on samples derived from placental blood (pb) and samples derived from peripheral blood of postpartum mothers. adult females (age ⩽ years) were divided (n = per group) into group (normotensive, normocholesterolemia: nn), and group (hypertension, hypercholesterolemia: hh). buffy coat were extracted from the pb. all tests on pb and maternal blood were done by using the test strip assay to identify the most relevant genetic variations to estimate the risk for cvd. the presence of a double mutation (ins+/del+) related to the ace gene in the hh group was %. the presence of a single mutation (ins − /del+) was only associated to the hh by %. (ins − /del − ) was absent in % of the pb and nn. despite the presence of double mutation ins/del for cvd in maternal blood, pb was free of this mutation. therefore, beyond genetic mutations, other factors can play a major role in the occurrence of cvd. disclosure of conflict of interest: none. s b e mt automated red blood cell depletion in abo incompatible grafts in the pediatric setting c del fante, l scudeller, s recupero, g viarengo, f compagno, m zecca and c perotti fondazione irccs policlinico san matteo red blood cell (rbc) depletion by apheresis is employed to reduce the rbc content from abo major or bidirectional mismatch bone marrow (bm) grafts mainly to avoid severe haemolysis . rbc depletion results in a significant volume reduction (due to both rbc and plasma depletion) and buffy coat concentration . . in pediatric setting, both rbc depletion and volume reduction before transplantation or cryopreservation can avoid fluid overload and renal impairment, especially in low/very low body weight recipients. the aim of this study was to evaluate the quality of the graft and immediate post infusion complications in rbc depleted bm in major and minor abo mismatch recipients using an automated device. patients and methods: bm aspirates for transplantation in pediatric setting were processed at our centre using the spectra optia (terumo bct) automated device. the initial collection preference was set at level and then was adjusted in order to maintain a haematocrit of % (colorgram) in the collection bag. flow speed was set at ml/min for cycles. mean recipients' body weight was kg (range: - ). pre and post procedure bm bag volume, hct%, mononuclear cells (mncs) count, (including b and t lymphocytes), cd + cell and cell viability were calculated. moreover, post procedure rbc volume and procedure time were registered. on the patient's side, post infusion complications (renal impairment, fluid overload, fever and haemolitic reactions) and time to engraftment were evaluated. results: a total of rbc depletion procedures were consecutively performed on bm grafts ( major and minor abo incompatibility, mud and related donors). data about pre and post procedure graft composition are reported in table . mean time to engraftment for pmn was . days (range: - ) and for plt was . (range: - ). pre and post-procedure cell viability were always %. mean procedure time was . minutes (range: - ). no bacterial or fungal contamination was detected. no infusion complications were recorded. one graft failure was observed. conclusions the spectra optia automated system is efficient in rbc depletion of abo mismatched grafts, permitting an effective volume reduction and an excellent mncs and cd + cell recovery in pediatric setting. automated rbc depletion may be proposed in low/very low body weight recipients both in abo major and minor incompatibility setting to minimize graft infusion side effects. building up a stem cell transplantation program in an emergent country, in the public setting, with limited economic resources, is not an easy work to do. international cooperation may be essential for the development of the program, in training, technological support and implementation of international guidelines. after years, we show an experience of international cooperation between a highly developed center in france (institut paoli calmettes, marseille) and the stem cell transplantation department of hospital maciel, a public assistance service in montevideo, uruguay. fourteen persons between doctors and nurses have been trained in france in stem cell collection and processing, patient's clinical handling, nursing, outpatients care and quality management. french missions of experts have been also received in hospital maciel every year since for in situ human resources training. in last years we developed a program for optimizing transplant results and reducing transplant related mortality (trm), based on several measures: improvement of patients selection, applying the sorror comorbidy index; adjustment of conditioning regimen doses, in order to reduce toxicity; development of a program to improve interaction with the intensive care unit; protocolization of the standard proceedings treatments; and initiating a program of quality and safety at the national institute of quality of uruguay inacal. adult patients have been treated with autologous (asct) ( ) or allogeneic (allosct) ( ) sct, with hematological malignancies. different modalities of allosct have been included progressively, becoming the only center accredited by the national regulation authorities (fnr) to perform unrelated donor sct and the haploidentical donor sct. this increased the proportion of allogeneic transplants from the historical % until % in last years. regarding patients health coverage, % comes from the private assistance system and % from the public health system. the major indications are lymphoid malignancies and acute leukemia, for asct and allosct, respectively, showing the same trend than cibmtr. three-year overall survival (os) for acute myeloid leukemia after allosct is %. considering asct for diffuse large b cell lymphoma, years os after autologous sct is % and % for chemo sensitive and resistant disease, respectively. threeyears os after asct for hodgkin disease is and % for sensitive and resistant disease, respectively. asct in multiple myeloma shows an os of and % at and years, respectively. in trm, results during the last years (after the described strategy) are shown in figure . the development of the-program of continuous improvement in quality-and the impact of results was locally recognized by two annual prices from inacal in (bronze) and (silver) in the category ‛commitment to public service.' a successful mirna- and the level of proangiogenic cytokines: angiopoietin- (angpt ), matrix metalloproteinase- (mmp- ) and vascular endothelial growth factor (vegf) in patients with lymphoproliferative malignancies prior to autologous hematopoietic stem cell transplantation (hsct) and in early posttransplant period. twenty-four patients were enrolled to the study ( f, m). the median (me) age was years. the investigated group consisted of multiple myeloma and lymphoma patients. the plasma samples were collected on time points: before chemotherapy-‛bc', on the day of hsct -‛ ', days after hsct-‛+ ' and days after hsct-‛+ . ' the cytokines were evaluated using elisa method, while mirna levels were estimated by qpcr method. the wilcoxon matched-pairs test was used to compare groups of dependent continuous variables: mirna's relative quantification (rq) levels or cytokines expression at two different time points. spearman rank correlation coefficient (r) was used to compare independent variables. we observed continuous decline of cytokines and mirnas level after conditioning treatment. the deepest decrease of expression was marked on ‛+ ' day ( table ) . we noticed a positive correlation between mirna- , mirna- cells in pbsc product. among the autologous transplanted patients between march and october , we have selected according to diagnosis, conditioning regimen and number of infused bags of cryopreserved pbsc. this group included females and males with median age of (range: . most of them, ( . %), had multiple myeloma (mm), ( . %) had non-hodgkin's lymphoma (nhl) and ( . %) had hodgkin's disease (hd). after harvesting, cd + cell and leukocyte number in pbsc product were enumerated on flow cytometer and blood cell counter, respectively. pbsc were cryopreserved with % dymethil sulfoxide (dmso) and cell viability was measured with trypan blue exclusion test before and after adding dmso, and as well after thawing in water bath on °c. as a conditioning regimen for the mm patients, melphalan was used and for the nhl and hd patients we used beam regimen. all received one bag of cryopreserved pbsc and pegfilgrastim mg on the first or the second post-transplant day. time to hematopoietic recovery was measured; for neutrophils . × /l, leukocytes × /l and platelets × /l with at least days without platelet transfusion. the median number of total leucocytes infused was . × /l (range: . - . × /l) of which cd + cells were - . × /kg of patient's body mass (median . × /kg). pre-freezing cell viability before and after adding dmso was with a median of % ( . - ) and , % ( . - ), respectively, and post-thaw viability . % ( . - ) . the average time to engraftment was . days ( - ) for neutrophils, days ( - ) for leucocytes and . days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) for platelets. our results confirmed the known correlation between the number of infused cd + cells and engraftment of neutrophils (po . ), leukocytes (p o . ) and platelets (p = . ). we found inverse correlation between the infused leukocytes and cell viability with dmso (p = . ) and after thawing (p = . ). no correlation was found between prefreezing and post-thaw viability with hematopoietic recovery, and also between the cd + number and these viabilities. no differences were found considering patients' age, gender, diagnosis, conditioning regimen or day of applying pegfilgrastim. we can indirectly infer good survival of cd + cells and higher sensitivity of other nucleated cells to preparation of pbsc product. trypan blue exclusion assay, due to its inability to distinguish type of stained cells, is not relevant for cd + cells survival determination. disclosure of conflict of interest: none. chronic granulomatous disease (cgd) is a kind of primary immunodeficiency disorder of phagocytic cells which resulting in failure to kill a defined spectrum of bacteria and fungi and in concomitant chronic granulomatous inflammation. allogeneic hematopoietic stem cell transplantation is the only treatment proved to be potentially curative in cgd. unrelated umbilical cord blood (ucb) is increasingly used as an alternative to bone marrow. methods: unrelated ucbt was performed consecutive cgd children at our center between and . median age was . months (range: - months), median body weight was . kg (range: - kg). all patients received myeloablative conditioning regimen consisting of busulfan, fludarabine, cytarabine, cyclophosphamide and g-csf. all patients received tacrolimus as prophylaxis for graft-versus-host disease (gvhd). median nucleated cells were . × /kg (range: . - . × /kg), and median cd + cells were . × /kg (range: . - . × /kg). median follow-up time was . months (range: - months) results: of patients engrafted. median time to neutrophil engraftment was days, and median time to platelet engraftment was . days. / patients were alive, and / had full donor engraftment. overall survival rate was . %. disease-free survival was . %. of patients had grades iii-iv acute gvhd. no patients developed chronic gvhd. only one patient died from multi-organ failure related to adenovirus infection. conclusion: unrelated ucbt should be considered as potential curative methods in children with cgd. myeloablative conditioning regimen has improved the engraftments of the ucb. disclosure of conflict of interest: none. reduced muscular mass and excess visceral fat in patients undergoing hsct are associated with higher mortality, longer hospitalization, longer use of immunosuppressive drugs, graftversus-host disease (gvhd) and comorbidities leading to shorter survival time. a recent study of patients undergoing allogeneic hsct showed that occurrence of enlarged areas of visceral and peripheral fat is inversely associated with the disease-free interval after the transplant. reduced muscle mass has also been associated with higher prevalence of chronic gvhd and low rates of success following allogeneic hsct. objectives: to investigate whether amount muscle mass and muscle strength (ms) as well as the amount of visceral fat (vf) of patients undergoing hsct would influence the duration of the engraftment time (en). we evaluated hsct patients (⩾ years) at hospital israelita albert einstein, são paulo, brazil, on their first day of hospitalization, before hsct. the thickness of the right femoral quadriceps muscle (rfq), measured at cm from the top edge of the patella was measured using ultrasound (us) in b-mode. the dominant upper limb strength of the patients was evaluated by the hand grip test. the vf was measured in the abdominal region, by the thickness of the fat layer between the linea alba and the anterior wall of the aorta. most patients were women ( %) with a mean age of years (± years) and % of our patients were elderly (⩾ years). the haploidentical ( %) was the predominant hsct, autologous ( %) and allogeneic ( %). most patients were overweight, with body mass index (bmi) of kg/m (± kg/m ). the average time en was days (± days). rfq was . cm (± . cm), ms was kgf (± . kgf) and the vf was . cm (± . cm). patients with lower rfq had a longer engraftment time that was statistically significant as the negative correlation between rfq and en was rs = . , p o . ), independent of the age and the hsct type as analyzed by linear regression. no significant correlation between vf or ms with en was found. in this cohort of patients we found that longer engraftment times were significantly correlated to reduced muscle mass but no positive or negative correlation was found with superior limb muscular force or with the amount of visceral fat. disclosure of conflict of interest: none. hematopoietic stem cell transplant unit, hematology department and pharmacy department. university hospital of donostia. donostia/san sebastiań introduction: lymphocytes are the cells responsible for the cellular and humoral immunity and, consequently, critical for hematological patients. the aim of this study was to analyze the eventual conexion between lymphocyte recovery and survival (srv) after allogeneic hematopoietic stem cell transplantation (allo-hsct). patients and methods: we retrospectively analyzed data from consecutive patients who underwent allo-transplants in our unit. in total, patients were male ( . %) and female ( . %). median age was years old (range: . baseline disease was: acute leukemia ( . %), lymphoma ( . %), myelodysplastic syndrome ( . %), chronic myelogenous leukemia ( . %), multiple myeloma ( %), aplastic anemia ( . %), chronic lymphocytic leukemia ( . %) and others ( . %). . % of allo-hscts were from an unrelated donor, and . % from a family donor ( % of them haplo-identical). the sc source was pbsc in . %, and bm in , %. a variety of conditioning regimens were employed, including: busulphan-based ( . %), melphalan based ( . %), tbi-based ( . %) and others ( . %). evolution of absolute lymphocyte counts (alc) and subpopulations during the first year after allo-hsct were analyzed. results: as shown in table , alc decreased abruptly during conditioning therapy and recovered up to baseline at days + and + ; at day + median alc had clearly improved compared with admission values. median cd + cells were lower than /mcl in two thirds of pts at day + and in only one third at day + . as shown in table , we found a significant link between alc at day + and srv, as well as between cd + cells at day + and srv. in our series, immunity recovery was a late event for the majority of patients undergoing allo-hsct. in addition, in our experience, the precocity and quality of the alc and cd + recovery was clearly linked with long-term survival. disclosure of conflict of interest: none. although there is experimental evidence suggesting the presence of a common mesoderm cell as origin of both hematopoietic (hsc) and mesenchymal progenitor cells (msc) in an animal model, it is still controversial if durable engraftment of native donor-derived mscs without ex vivo treatment can occur in the recipient of allogeneic hsct. to assess the presence of donor-derived msc following hsct. between july and july , a total of recipients of hsct were analyzed for hsc and msc chimerism. eighteen patients received bm grafts ( %), patients had peripheral blood as stem cell rescue ( %) and finally patients had a cord blood transplantation ( %). patients received myeloablative ( %) or reduced intensity conditioning ( %) for malignant ( %) or nonmalignant disease ( %). bm aspirate cells were plated and expanded in α-mem with % human platelet lysate at cells/cm . after - days, nonadherent cells were removed, while the adherent cells were expanded until they reached confluence. after weeks we quantified msc precursors as colony forming unit fibroblast (cfu-f). finally the amplified sequences were resolved by capillary electrophoresis ( ruo genetic analyzer, applied biosystems) and analyzed by comparing genotypes of bmt recipell detachment, nuclear dna was extracted (dneasy blood and tissue kit-applied biosystems) and specific polymorphic tandemly repeated regions (strs) were amplified by means of the polymerase chain reaction(pcr) following the specific manufacturers' instructions. (ampfℓstr identifile kit, applied biosystems following hsct (hsc and msc) to those of donors. we cultured whole bm aspirates from patients following hsct with a median time of day (range: - ). cfu-f/ × growth was observed in a majority of bm the prevalence of human pegivirus in recipients of allogeneic hematopoietic stem cell olga koroleva , e parovichnikova , l kuzmina , m drokov , v vasilyeva , z konova , ekaterina mikhalcova , d dubnyak , n popova , tamara romanova , d tikhomirov , t tupoleva and v savchenko bone marrow transplant department, national research center for hematology and virology department, national research center for hematology human pegivirus (hpgv; previously named as gb virus c/hepatitis g virus) was discovered more than years ago. it is an rna virus referred within the genus pegivirus of the family flaviviridae. hpgv rna is found in liver, spleen, bone marrow and peripheral blood mononuclear cell, including t-and b-lymphocytes, nk-cells and monocytes. despite of the fact that it is a molecular structure, mechanism of replication and transmission routes are very well understood but the clinical significance of hpgv is still not determined. recipients of allogeneic hematopoietic stem cell have a high risk infection of hpgv. it is known, that hpgv is a nonpathogenic virus, however, it may play a role in immunocompromised individuals. to investigate the frequency of occurrence of hpgv and its clinical significance in recipients of allogeneic hematopoietic stem cell. blood samples were obtained from patients who underwent allogeneic hematopoietic stem cell transplantation (allo-hsct): all n = , aml n = , mpn n = , cll n = , mm n = , lpd n = , aa n = , mds n = . a median of age was years ( - years) . forty five patients were males and patients were females. conditioning regimen was ric in cases, mac in . bone marrow as a graft source was used in , pbsc- . all patients received multiple transfusions of blood components at the previous stages of treatment. hpgv rna had been assayed by polymerase chain reaction real time (rt-pcr) on plasma samples before started pre-transplantation conditioning. despite the diagnosis incidence of hpgv was high . % (rna-hpgv was positively in patients). patients with piercings and tattoos had incidence of hpgv in % that was not statistically significant (p- . ). hpgv is known as nonhepatotropic virus. in our study there was also no statistical reliability of specific changes in liver function test such as elevating the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin due to the rna-hpgv. liver enlargement was also not statistically significant according to ultrasound scan results in patients infected with hpgv. we also analyzed the co-infection with hepatitis b and c virus. results are presented in table . coinfection was not statistically significant. however, only one patient with hepatitis c was coinfected hpgv. leukocytes recovery median was days ( - ). thrombocytes recovery median was ( - ). the presence of rna-hpgv did not affect the recovery of peripheral blood cells in patients after allo-hsct. according to our study the frequency of hpgv infection in recipients of allogeneic bone marrow was quite high ( . %), and it did not depend on the presence of any other hepatotropic viruses. clinical significance of hpgv infection in recipients of allogeneic hematopoietic stem cell has not been revealed, it is possible due to the short follow-up. it needs further clinical research. disclosure of conflict of interest: none. quantification of cd + recent thymic emigrants and t cell receptor excision circles (trecs) in umbilical cord blood transplanted patients v devlia , , j gridlestone , m raymond , s tulpule , d tewari , , r hough , c navarrete , a madrigal , , b shaw , , r danby and a saudemont , anthony nolan research institute, london, uk; ucl cancer institute, london, uk; nhsbt colindale, london, uk and ucl, london, uk reconstitution of t lymphocytes is a limiting factor in the regeneration of an effective immune system in adult patients following hematopoietic stem cell transplantation. cd (pecam- ) is a transmembrane glycoprotein expressed on naive t-cells that have recently emigrated from the thymus into the periphery. in peripheral blood, cd + t lymphocytes also contain high numbers of t-cell receptor excision circles (trecs); excision loops of dna excised during t-cell receptor gene rearrangement during t cell maturation within the thymus ( ) ( ) ( ) . however, quantification and correlation of cd and trec has not been formally investigated in patients following umbilical cord blood (cb) transplantation. quantification of cd and trecs post cb transplant will provide an insight into the immune reconstitution of t cells from the thymus. we therefore sought to measure cd and trecs in patients after cb transplant and assess whether these markers provided evidence of thymic recovery. we followed adult patients (median age . years) who underwent cb transplant in the uk. patient samples were collected , , , , and days post transplant. using flow cytometry, we determined absolute counts of cd +cd +cd ra+ and cd +cd +cd ra+, and quantified the copy numbers of trec genes in peripheral blood mononuclear cells (pbmcs) via real time pcr. results: at the six time points, the number of samples collected were the following: , , , , and . in all of the samples, the overall median number of cd +cd +cd ra+ was cells/μl (range: - cells/μl). the median level of cd +cd +cd ra+ cells increases from to cells/μl from day to day . absolute counts of cd +cd +cd ra+ at all of the six time points is -fold lower compared to healthy controls (median: cells/μl, range: - cells/μl). the overall median number of cd +cd +cd ra+ cells is cells/μl (range: - cells/μl). there is an increase in the median number of cd +cd +cd ra+ cells between days and posttransplant from to cells/μl. however, the absolute median counts of cd +cd +cd ra+ cells in patients are twofold lower, years post transplant, compared to healthy controls (median: cells/μl, range: - cells/μl). in the majority of the patient samples throughout all time points the trec gene copy numbers were undetected (n = ). in a few patient samples (n = ) trec gene copy numbers were quantified but with this limited sample size no correlations can be made between the absolute counts and trec gene copy numbers. our data suggests that cord blood transplant patients within the uk have reduced levels of cd +cd introduction: common variable immunodeficiency (cvid) is a highly heterogeneous group of primary immunodeficiency characterized by defective antibody production, recurrent infections, lymphoproliferation and autoimmunity. autosomal recessive mutations in lrba, encoding lps-responsive beigelike anchor protein were first described as a cause of cvid-like disease in . although hsct is accepted as a standard treatment modality for long-term resolution of severe primary immunodeficiencies, its role is less established in patients with lrba deficiency. patients and methods: whole exome sequencing of patient's genomic dna obtained prior to the hsct revealed a homozygous deletion in lrba (c. delt:p. c fs). immunological analyses including serum immunoglobulin levels, flow cytometry analyses of lymphocyte subsets, cytotoxicity/proliferation assays, vaccine responses were studied at several time points throughout the disease course, prior to and after hsct. a -year-old boy, born to consanguineous healthy parents of turkish origin became symptomatic at the age of months. he hospitalized several times due to recurrent pulmonary infections. he developed pancytopenia, lymphadenopathy, hepatosplenomegaly and autoimmunity (autoimmune hemolytic anemia and thyroiditis) with low serum immunoglobulin levels at the age of . as a result, he received several courses of steroid and prophylactic immunoglobulin and wide-spectrum antibiotics. over time he manifested growth failure and diagnosed with ibd-like colitis. due to the cumulating severe cvid-related complications, a hsct was performed at the age of years with the bone marrow stem cells from his hla identical brother after a conditioning regimen including fludarabine, busulfan and atg. severe intractable colitis with hypoalbuminemia continued till the engraftment despite vigorous fluid-electrolyte replacement therapy and accompanied with severe episodes of acute gastrointestinal bleeding. after the achievement of full donor chimerism, diarrhea episodes resolved. he received three doses of abatasept because of persistent cytopenia thinking about unresolved immune dysregulation. he is in complete remission at -year post-hsct with no signs of graft versus host disease. allogeneic hsct should be considered in patients with lrba deficiency prior to the development of disease-related severe cumulative manifestations. disclosure of conflict of interest: none. inflammatory bowel disease (ibd) is a chronic disorder of the gastrointestinal tract. very early onset ibd (veo-ibd) represents those severe children with disease onset occurring before -years-old. interleukin- receptors (il- ra, il- rb) mutation are considered to be one of the very important genes for veo-ibd. currently variant treatment, such as steroid medication, immunosuppressive agents and biological agents could not get complete remission. allogeneic hematopoietic stem cell transplantation (allo-hsct) was reported to induce remission in those with veo-ibd. we performed unrelated umbilical cord blood transplantation (ucbt) in five consecutive children with veo-ibd due to il- receptor mutation between and . median age of five children was months (range: - months), and median body weight was kg (range: . - . kg). all patients received reduced intensity conditioning (ric) regimen consisting of busulfan, fludarabine and cytarabine. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. most patients ( %) received a or hla alleles-mismatched cord unit. median nucleated cells of the cord blood were . × /kg (range: . - . × /kg), and median cd + cells were . × /kg (range: . - . × /kg). median follow-up time was months (range: - months). all patients engrafted, median time of neutrophil engraftment was days, and median time of platelet engraftment was days. four of five patients were alive with continuous donor engraftment, and achieved complete clinical remissions. colonoscopy at months after transplantation in two children revealed the mucosa healing. two children had grade iii acute graft-versushost disease (gvhd). one child developed severe chronic gvhd of both lungs and died of ards at months after transplantation. it is the first clinical trial that unrelated ucbt was performed in veo-ibd children in china. our data should unrelated ucbt with ric should be considered as a potentially curative therapeutic option in children with veo-ibd. disclosure of conflict of interest: none. patients with refractory primary induction failure and resistant relapse are poor candidates for hematopoetic stem cell transplantation (hsct) . additional attempts at remission induction with various combinations of chemotherapy will unlikely improve the outcome and will contribute to excess toxicity. a major goal of sct has been to develop strategies to reduce the risk of gvhd while maintaining or enhancing gvl. tcrαβ+/cd +lymphocytes depletion is a technology of graft manipulation with a potential to increase gvl effect and improve gvhd control and immune reconstitution in this group of patients. a total of pts with refractory aml (primary induction failure (n = ), refractory relapse (n = )), female/ male, median age . years ( . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , underwent allogeneic sct between may and august , median fu . years ( . - . ). pts were transplanted from haploidentical donors and from mud. all pts had active disease (ad) at the moment of sct and received treosulfanbased high-intensity conditioning regimen. three regimens of gvhd prophylaxis were used. regimen (n = ): atgam mg/kg with (n = ) or without (n = ) post-transplant tacro/ mtx; regimen (n = ): thymoglobulin mg/kg, rituximab mg/m and post-transplant bortezomib on day+ ,+ (n = ); regimen (n = ): tocilizumab mg/kg on day- and post-transplant bortezomib (n = ), pts receive additional abatacept mg/kg on day+ , + , + , + . tcrαβ+/cd +-depletion of sct with clinimacs technology was implemented in all cases. the median dose of infused cd + cells was × /kg (range: . - ), tcra/b- × /kg (range: - ). all engrafted pts received additional post-transplant courses of low-dose chemotherapy, including hypomethylating agents and dli. primary engraftment was achieved in of pts(three pts had disease progression, one died at the moment of engraftment), the median time to neutrophil and platelet recovery was days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . early mortality within days was . % (one pt with aml had acute lung injury after engraftment on day + ), . -years ptrm- . % ( %ci: . - ) . there were no allergic or infusion-related adverse events associated with tocilizumab or abatacept. ci of gvhd grades ii-iv and iii-iv was . % ( % ci: - ), and . % ( %ci: . - ), respectively. ci of cgvhd was % ( % ci: - ). ci of acute gvhd was lower in a group with prophylaxis regimen without serotherapy: % ( % ci: - ) vs . % ( % ci: - ) in atg group. no correlation between graft composition, donor type with the incidence of agvhd and cgvhd was noted at . years ppfs (event = death or relapse or progression) was % ( % ci: - ), . -years pos - % ( % ci: - ). median time of fu for survivors is . years (range: . [ ] [ ] . we confirm that the depletion of tcrαβ +/cd +lymphocytes from the graft ensures high engraftment rate and low transplant-related mortality in pediatric pts with refractory aml. we suggest that tocilizumab and abatacept can be safety administered to children with acute leukemia in the context of treosulfan-based conditioning regimens. long-term follow-up will demonstrate if the gvhd prophylaxis without serotherapy and combined administration of tocilizumab, abatacept and bortezomib post-tcrαβ+/cd +depleted grafting will improve gvl effects without extensive gvhd-related morbidity and mortality in pts with refractory aml. disclosure of conflict of interest: none. the jacie experience at the university hospital of amiens l marie-noelle , w brigitte , f isabelle , g bérengère , h muriel , h anne , v elsa , m jean-pierre and c amandine lacassagne; oncopôle, chu amiens picardie; hématologie clinique et thérapie cellulaire-chu amiens picardie; oncopôle-chu amiens picardie and the jacie (joint accreditation committee of isct and ebmt) accreditation aims to improve the management of patients benefiting from autologous or allogenic hematopoietic stem cell (hsc) transplants. usually, candidates' centers for jacie accreditation have already existing clinical activity when they have willingness to comply with jacie standards. here, we present our new experience in the implementation of jacie quality process, at the same time as allograft clinical activity. implementing process autograft clinical activity existed at amiens hospital, since , but in lack of center cellular therapy laboratory and hsc collection that were outsourced. the collection activity for autologous transplant was set up in , the cellular therapy laboratory in november and then the allogenic transplant was started in july . as early as march , we set up a steering committee with hematological clinicians, managers of each sector, a transplant coordinator nurse, the head of the processing laboratory and a part-time quality engineer recruited part-time. each actor had to become familiar with standards to obtain information from accredited centers in order to evaluate objectives and their prioritization. steering committee decided on deadlines and established a roadmap including the following: the list of jacie required standard operating procedures and their writing; assignment of the tasks for each actors in order to evaluate, writing and approval each document; organization of documents diffusion; information to all staff on the approach; creation of feedback committee for adverse events management; establishment of morbidity and mortality review; formalization of initial and continuing training for medical and paramedical staff; and organization of cross audits with external teams. at the same time, we assessed requirements for starting activity: training for medical and paramedical staff; training for the transplant coordinator nurse; circuits for taking care of donors; the organization of the in-patient department; the organization of follow-up of post-transplant patients; and authorizations of the national regulation agencies for processing facility on manipulations and cellular qualifications and for regulatory collection and transplant. allogenic transplant clinical activity started in july . accreditations jacie visit occurred on and june and our center has been officially accredited since march . neither quality approach, nor clinical activities were easy to implement. medical and paramedical staff had to get acquainted with a new organization and restrictions. despite difficulties, implementing jacie quality process, concomitantly with allograft activity allowed to create a true team dynamics with a common reflection on the means to be implemented. moreover, quality approach has assured us best ensure to care graft patients. the result is true satisfaction, which be credited to all. disclosure of conflict of interest: none. previously published p three-dimensional co-culture of peripheral blood monocytes supports and expands functional hematopoietic stem/progenitor cell without immobilization y xu , x li , b wang , w shan , h chen , s liu , r tie , y long , s cai , h xu , x yu and h huang bone marrow transplantation center, the first affiliated hospital, school of medicine, zhejiang university very low numbers of circulating hematopoietic stem/progenitor cells (chspcs) are found in normal human peripheral blood (pb) without mobilization. here, we developed a three dimension co-culture system to seize and expansion chspcs from pb monocytes without mobilization. flow cytometry analysis was carried out to identify chspc phenotypes. multipotential properties of chspcs were determined using colonyforming unit assay in methylcellulose and reconstitution ability in the compromised animals. the critical regulation mechanism underlying chspcs was identified with transcriptome analysis based on next-generation sequencing technology at total or single cell levels. loose cobble stone colonies (lcs), round or vessel-like compact colonies (rccs or vccs) were presented in three dimension co-culture system after about weeks. the colonies lasted for at least six passages with no obvious apoptosis sign, and expanded more than~ fold during the period. we studied the niche-mediated regulation mechanism of chspc fate at molecular level compared to the conventional method of two dimension culture. furthermore, chspcs were capable of forming all types of hematopoietic colonies, including cfu-gemm, and especially held short term engraftment capacity for compromised nogs by radiotherapy. transcriptome analysis by deepsage identified genes significantly associated with regulating the function of chspcs. figure : the cellular morphology in three dimension culture system for peripheral blood monocytes without mobilization during the culture for - weeks. figure : short transplantable potential analysis of chspcs. figure : (a) static of differentially expressed genes between three-and two-dimensional culture systems for peripheral blood cells. (b) go functional analysis classifies those genes by biological process, cellular component and molecular function. (c) the significant differences between the molecular phenotypes of three-and two-dimension chspcs indicating that chspcs from three dimension culture hold stem properties. our system may provide a more ideal and balanced approach which not only seizes circulating chspcs, promotes selfrenewal and expansion of chspcs, but also holds phenotypic and functional attributes of chspcs. . to wash or not to wash? comparison of neutrophil and platelet engraftment after infusion of cryopreserved autologous stem cells before and after the implementation of bedside thawing am halldorsdottir , s atladottir , m thorsteinsdottir, na arnason , g runarsson , t jonsson , oe sigurjonsson , and s reykdal the blood bank, landspítali, the national university hospital of iceland; department of hematology, landspítali, the national university hospital of iceland and school of science and engineering, reykjavik university cryopreserved autologous peripheral blood stem cell (pbsc) grafts are widely used after high-dose chemotherapy in the treatment of patients with myeloma or lymphoma. prior to infusion, cryopreserved grafts can be thawed at the bedside, or thawed and washed at the cell therapy laboratory. at our institution the practice of routine washing of stem cell grafts in the laboratory was discontinued in april and bedside thawing implemented instead. this was done to minimize the time thawed cells are exposed to toxic dmso. this study was performed at a single center, at landspítali-the national university hospital of iceland, which is the only transplant center in iceland. autologous pbsc transplants have been performed in iceland since . the study compares outcome for two groups of patients, who received either; (a) thawed and washed autologous pbsc cell grafts from january to [p ] april , or (b) autologous pbsc grafts thawed at the bedside from april to november . the following outcomes were compared; days to neutrophil engraftment (absolute neutrophil count (anc) . per μl), and platelet engraftment ( and × e /l). data on mean cd + cell content/kg of the infused grafts, measured prior to cryopreservation, were also compared. all patients have received premedication with solucortef, clemastine and ondansetron prior to infusion of the graft. from january to april a total of patients received thawed and washed autologous pbsc grafts, and between april and november patients received autologous pbsc grafts thawed at the bedside. majority of the patients were diagnosed with either multiple myeloma or related disorders (n = ) or lymphoma (n = ) whereas the remaining patients (n = ) had miscellaneous diagnoses. days to engraftment and the dose of cd + cells infused are compared in table . there was no significant difference in the mean cd content of infused autologous stem cells in the two groups ( . vs . × e cd +cells/kg, p = . ). there was also no difference in the mean number of days to engraftment of neutrophils ( . vs . days, p = . ), platelets at days ( . vs . days, p = . ) or platelets at days ( . vs . days, p = . ) after transplant. one hundred day mortality was comparable in the two groups or . %. additional data on transfusion requirements, infections and use of granulocyte-colony stimulating factor will be presented. [p ] there was no difference in neutrophil or platelet engraftment after changing the autologous stem cell graft thawing procedure from post-thaw washing in the laboratory to bedside thawing. bedside thawing of stem cells is a safe procedure that results in acceptable cellular engraftment. disclosure of conflict of interest: none. the procedure of autologous hematopoietic stem cell (hsc) transplantation requires cryopreservation of hscs. addition of dmso (dimethyl sulfoxide) is necessary to secure the viability of such cells, but this cryoprotectant causes adverse reaction during infusion into patient. the concentrations of dmso in cryopreservation mixture vary strongly between different transplant centers. usually, the hscs are stored in mixtures containing % dmso, however, many centers successfully use lower concentrations. the main aim of the study was to evaluate the clinical impact of different dmso concentrations in cryopreservation mixture ( %, . %, %) on reconstitution of hematopoiesis after autologous hsc transplantation. the project was approved by the local bioethics committee. written informed consent obtained from all of patients. the study is registered to clinicaltrials.gov (identifier: nct ). between january and july , consecutive patients with hematological malignancies or solid tumors, referred for autologous hsc transplantation, were recruited in the study. the patients were randomly assigned to one of three study arms ( patients each). hscs obtained by leukapheresis were cryopreserved in three concentrations of dmso: %, . %, %, respectively. study groups did not differ significantly with regard to the diagnosis (mostly mm, nhl or hl), age or conditioning regimen (chemo-or radiotherapybased). all patients received granulocyte-colony stimulating factor (g-csf, filgrastim) starting from day + after transplantation to support neutrophil recovery. in case of patients, the transplantation was cancelled due to progression or other medical reasons. four patients died shortly after transplantation, due to refractory infections. data for patients were subjected to statistical analysis. the viability of nucleated cells on the day of transplantation was similar in all groups (median %, range: - % for % dmso group; %, range: - % for . % dmso; %, range: - % for % dmso; p = . ). the dose of transplanted cd + cells was comparable in all group: (median . × /kg of recipient body weight for % dmso, . × /kg for . % dmso and . × for % dmso, p = . ). the median time to leukocyte recovery, defined as the first day with wbc count exceeding . × /l was days in all groups (ranges: - for % dmso; - for . % dmso; and - for % dmso; p = . ). similar results were obtained in case of neutrophil recovery-the median day, when the anc exceeded . × /l, was in all arms (ranges: - ; - and - , respectively; p = . ). the day when the platelets level were greater than or equal to × /l (sustained without transfusion within days) was similar in all groups: medians were days in %, . % and % dmso (ranges: - ; - ; - ; p = . ). no serious adverse effects were observed during hscs infusion and during h after transplantation. reduction of dmso concentration from in cryoprotective mixture % to . % and % has no negative impact on cell viability during cryopreservation and engraftment after auto-hsc transplantation. disclosure of conflict of interest: none. a real-world cost-effectiveness analysis demonstrates that introducing plerixafor to improve mobilization in multiple myeloma patients who behave as poor mobilizers is cost-effective considering the whole mobilization and transplant procedure r touzani , , a-m stoppa plerixafor, a cxcr -antagonist, is efficient to improve cd + cell mobilization and collection in candidates for autologous transplantation who behave as poor-mobilizers. the cost of the drug is however of concern. published medico-economics studies were mostly conducted in the us, and few including detailed and comprehensive micro-costing of the collection and transplantation process; conclusions may thus not apply to european countries where cost structures are different. to compare costs and effectiveness of plerixafor-free and plerixafor-replete management strategies for multiple myeloma patients who behaved as poor-mobilizers after adequate administration of a standard rhg-csf mobilization regimen. sixty patients diagnosed with multiple myeloma were consecutively identified during years - , immediately before and after ema granted marketing authorization for plerixafor. poor-mobilizers were defined as having circulating cd + cell counts below /μl. plerixafor was introduced or not as a result of the attending physician's decision, reflecting progressive changes in medical practices over this transitional period. the historical and study groups were matched over four criteria: disease stage at diagnosis, age, gender and number of chemotherapy treatments received before mobilization. two cost-effectiveness analyses (cea) were conducted; the primary cea looked at the criterion ‛collecting at least × cd + cells'; a secondary cea looked at the criterion ‛successful autologous transplant administered'. detailed micro-costing evaluations ( figures) did not or did include transplantation costs for the first and second cea, respectively. the two groups were similar in terms of age, sex distribution, disease characteristics or previous treatments. / and / patients proceeded to high-dose melphalan and autologous transplantation in the study and historical groups, respectively. there was a trend to a higher number of collected cd + cells in the control group; however, the proportion of patients who met the minimal target number of × collected cd + cells/kg was identical ( / ). length of hospitalization, times to neutrophil and platelet recoveries, numbers of prbc and platelet transfusions were identical in the two groups. mobilization and collection costs per patients were more important in the plerixafor group that in the historical group ( . vs . €, p o . ), and proportionally higher in patients who received plerixafor as part of a remobilization treatment rather than pre-emptively ( . vs . €, respectively). the main cea concluded to a . € increase in costs for the same number of patients achieving a minimal target number of × collected cd + cells/kg. the second cea found a decrease in the cost of transplant, with . € in the study group vs . € in the historical group (ns). in total, the . € increase for the complete procedure cost ( . € per successfully autografted patient in the study group vs . € in the historical group) was not statistically different. cost-effectiveness arguments should not been used against the administration of plerixafor in multiple myeloma patients in the european context. future prospective researches looking at patients reported outcome criteria and labour organization in apheresis facilities are needed. disclosure of conflict of interest: this work was supported by a grant from sanofi s.a.; cc: research support, honorarium & hospitality from sanofi s.a. administration of plerixafor for peripheral blood cd + stem cell content of o × /l for autologous stem cell mobilization leads to decreased apheresis days and increased total yield m kamdar , s abebe , gr gonzalez fontal, l gates , a hammes , d abbott , j gutman , b haverkos , d sherbenou and c smith division of hematology and transplantation and department of biostatistics and informatics, university of colorado, denver, colorado, usa autologous stem cell transplantation (asct) is an effective treatment for lymphoma and plasma cell neoplasm (pcn) (multiple myeloma and amyloidosis). granulocyte-colony stimulating factor (g-csf) is the most commonly used upfront mobilizing agent with plerixafor-based higher cost approaches reserved for poor/unsuccessful mobilizers. several mobilization algorithms utilizing g-csf and plerixafor have been published however the most efficient and cost effective strategy is yet to be determined. most transplant centers administer plerixafor for peripheral blood (pb) cd + stem cell content of o × /l on day (d) of g-csf mobilization. at the university of colorado (uch) we changed our programmatic approach in and administered plerixafor for pb cd + count of o × /l on d of g-csf mobilization. in this study we evaluate the impact of this novel mobilization algorithm on apheresis days and total stem cell yield. patients (pts) with lymphoma and pcn who underwent asct at uch until / received plerixafor if pb cd + cells on d of g-csf mobilization was o × /l. based on our institutional review of poor/unsuccessful mobilizers and using logistic regression analysis this algorithm was revised in / . in the new algorithm all pts received plerixafor if pb cd + cells on d of gcsf mobilization was o × /l. demographics were compared between pts with lymphoma and pcn before (group : / - / ) and after (group : / - / ) the new algorithm was implemented. the primary goal of this analysis was to assess the total days of apheresis and total stem cell yield between the two groups. we also sought to analyze days to wbc engraftment and platelet engraftment. a total of pts were included in this analysis. group consisted of pts ( pts had lymphoma and pts had pcn). group consisted of pts ( pts had lymphoma and pts had pcn). we found that there was a significant increase in total yield (p = . ) in group as compared to group . on further disease subtype assessment we noted that pts with pcn in group had a significant increase in total yield (p = . ). in lymphoma pts on univariate analysis group showed a significant decrease in apheresis days ( . days, p = . , % ci: (− . , − . )). on multivariate analysis there was still a marginally significant decrease in group ( . days, p = . , % ci: (− . , − . )) compared to group . in pcn pts on univariate analysis group showed a significant decrease in apheresis days ( . days, p = . , % ci: (− . , − . )). on multivariate analysis group continued to show a significant decrease in apheresis days ( . days, p = . , % ci: (− . , − . ) compared to group . we found no significant difference between the two groups in days to neutrophil engraftment and platelet engraftment. our analysis showed that a mobilization algorithm of administering plerixafor for a pb cd + stem cell count of o × /l on d of g-csf mobilization led to a decrease of roughly . days in the lymphoma cohort and a significant decrease of . days in the plasma cell neoplasm cohort. we also noted a significantly increased total yield of stem cell collection in group . overall our programmatic approach led to decreased chair-time for apheresis and better resource utilization. pharmacoeconomic impact of this approach will be updated at the meeting. disclosure of conflict of interest: mk: speakers bureau, seattle genetics; remaining authors declare no conflict of interest. administration of stem cell boosts (scbs) from the original donor offers a therapeutic option. we report on pediatric patients with pgf who received a total of boosts with cd + selected peripheral blood stem cells (pbsc) after transplantation from matched unrelated (n = ) or mismatched related (n = ) donors. median time between hsct and infusion of the scbs was days ( - ). boosts contained a median number of . × cd + progenitor cells/kg body weight (range: . - . × ) with a median number of /kg (range: - ) residual cd + t cells. within weeks after application, a significant increase in median neutrophil counts ( vs /mm , po . ) and a decrease in erythrocytes and thrombocytes transfusion requirement (median frequencies and vs , p o . and o . ), were observed, and . % of the patients resolved one or two of their initial cytopenias whereas . % had a complete hematological response. additionally median lymphocyte counts for cd +, cd +cd +, cd + and cd + increased . fold, . fold, . fold and . fold, respectively. the rate of de novo acute gvhd grade i-iii was only % and resolved completely after treatment. no gvhd iv or chronic gvhd occurred. patients who showed a response to scb displayed a trend toward better overall survival (os) (p = . ). administration of cd + selected scbs from alternative donors is a safe and effective procedure. we hypothesize that the cd + progenitor boosts may have an enhancing effect on maturation of committed lymphoid precursors already present in the host or generate another wave of thymic seeding with accelerated t-cell differentiation process in the absence of any immune suppression. further studies are warranted to better define the impact on immune reconstitution and survival. disclosure of conflict of interest: none. plerixafor plus granulocyte-colony stimulating factor (g-csf) has been shown to mobilize more cd + cells than g-csf alone for autologous hematopoietic stem cell transplantation (hsct). however, there are few studies that analyze the impact of this strategy in engraftment. the aim of our study is to compare mobilization and engraftment between patients who received a combination of plerixafor plus g-csf and patients (pts) who mobilized with g-csf alone. a retrospective casecontrol analysis was performed in pts with myeloma who mobilized with plerixafor plus g-csf (group p/g-csf) and was compared with matched for sex and age controls who mobilized with g-csf alone (group g-csf). all pts underwent hsct between and . mobilization with g-csf at dose of μg/kg/day was used in all pts. the aphaeresis was scheduled on day + . plerixafor ( . mg/kg) was added if the number of cd +cells on day + was o /μl for × cd +/kg requested (or o /μl for × cd +/kg), or if the number of cd +cells collected in the first apheresis was o % of cd + requested. conditioning and supportive care were similar in both groups. in p/g-csf group, were male and female. median age was . years (range: - ). in group g-csf, were men and female. median age was . years (range: - years). there were no differences between both groups. disease status at time of mobilization was different between groups (p = . ). in p/g-csf group: ( . %) pts were in complete remission (cr), ( . %) very good partial responses (vgpr), ( . %) partial response (pr) and ( . %) had no response to treatment. in g-csf group: ( . %) pts had reached cr, ( . %) vgpr and the remainder in pr. sixteen ( . %) pts in p/g-csf group had received ⩾ lines of treatment vs ( . %) pts in g-csf group (p = . ). no difference was seen on mean day-dose of g-csf ( μg/kg/ h in p/g-csf group vs μg/kg/ h) (p = . ). there was no difference on cd +/kg requested ( / pts in p/g-csf were requested × /kg vs / in g-csf group) (p = . ). p/g-csf group needed more apheresis sessions, ( . %) pts required ⩾ sessions against ( . %) pts in group g-csf (p o . ). we obtained enough cd + cells to carry out hsct in all patients, although mean number of cd + cells obtained in p/g-csf group was lower than in g-csf group ( . × /kg vs . × /kg, respectively) (p o . ). also, mean number of cd + infused in p/g-csf group was lower ( . × /kg vs . × /kg) (po . ). however, engraftment results were similar in both groups, as represented in table . patients who required mobilization with plerixafor plus g-csf got an engraftment as good as patients who do not require the combination despite of worse baseline parameters. given that the number of cd + infused in the p/g-csf group has been lower than g-csf group, these results might suggest that the different composition of graft cell with plerixafor plus g-csf mobilization, described in some studies, could impact on engraftment outcomes. high-dose chemotherapy following autologous hematopoietic stem-cell transplantation (autohsct) is an effective method of treatment both recurrent and primary refractory lymphoma patients. however, some patients have mobilization failure (‛poor mobilizers') with inadequate collection of peripheral blood stem cell (pbsc). aim: to evaluate the efficacy and factors influencing pbsc mobilization and collection for the autohsct in patients with lymphomas. thirty patients were included in this study: -with hodgkin lymphoma, -with non-hodgkin lymphoma, -with multiple myeloma; women and men of them. the median age of patients was years ( - years). the mobilization of pbsc with only colony-stimulating factors (csf) was carried out for patients, chemotherapy (cyclophosphamide, etoposide) in combination with csf-for patients. only one patient had plerixafor mobilization. the concentration of cd + in peripheral blood (pb) was studied on the day of the intended cytapheresis. cytapheresis was commenced when cd + concentration had been greater than . × cells/ml. twenty-four patients ( %) from had collection of pbsc. the collection was not performed in six patients ( %) because the concentration of cd + in pb on the day of the intended cytapheresis was lower than . × cells/ml. there was no possibility to use plerixafor in these cases for economic reasons. the median concentration of cd + in pb on the first day of the intended cytapheresis in the group of patients that had cytapheresis was . × cells/ml whereas in the group of failed- . × cells/ml (p o . ). fifty-nine tests of cd + in pb were done. distribution and test results by days from the first day of the intended cytapheresis are presented in table . the total number of the cytapheresis was . the majority of patients had procedure of pbsc collection (n = ), patients had procedures and only had . the last patient had had two previous failed cytapheresis procedures and the adding of plerixafor helped him to collect necessary number of cells. the median of cd + cells on patient's kilo was . × cells/kg. sex, age, mobilizing regimen, previous radiation therapy, the count of lines of chemotherapy before autohsct were not significantly associated with poor pbsc mobilization and collection. only tumor response before autohsct (complete/ partial response or stabilization) was significantly associated with cd + cell count in the product of cytapheresis. patients with complete or partial response had significantly better cd + count. [p ] disclosure of conflict of interest: none. factors associated with failure in mobilization of peripheral blood hematopoietic progenitor cells in autologous transplantation je dulon-tarqui, bl acosta-maldonado, l rivera-fong, sa sánchez-guerrero, jf zazueta-pozos, ja padilla-ortega, wj ladines-castro and lm valero-saldaña high dose therapy followed by autologous stem cell transplantation (asct) obtained from peripheral blood is currently the standard model for treatment consolidation in various hematologic malignancies. a global incidence of - % of failure to mobilization is reported, and some factors associated with poor mobilizers in hodgkin's lymphoma (hl), non-hodgkin's lymphoma (nhl) and multiple myeloma (mm) when the yield in peripheral blood stem cells (pbsc) collection is unsatisfactory, the effects for the recipient can be serious. the donor's age, gender, body surface area (bsa), processed blood volume and the method of g-csf dose calculation may affect the cd + yield. as g-csf has a low distribution volume in the peripheral blood (pb), it might be appropriate to calculate the doses by using the bsa instead of per kg body weight. consecutive allogeneic pbsc donations performed in healthy donors at the karolinska university hospital in stockholm were included. a complete medical history, physical examination, electrocardiogram, chest x-ray and laboratory testing were done before pbsc donation. relevant data for analysis were collected from the institutional quality database for a retrospective review. the total blood volume was calculated using the formula by nadler et al. the bsa was calculated using the formula by du bois and du bois. the concentration of cd + cells in the pb and the processed volume of blood were significantly correlated to cd + cells yield (po . and po . , respectively, see table ). the g-csf dose per m was significantly correlated to the concentration of cd + cells in the pb (p = . ) and in the product (p = . , see table ). smaller bsa (p o . ) and less processed volume (p o . ) were found among female donors, who were given lesser g-csf dose per m (p o . ) and showed lower yield compared to men (po . ). however, multivariate analysis of the yield showed that only the concentration of cd + cells in the pb and the processed volume remained independent significant (see table ). [p ] in this study, we found the concentration of cd + cells in the pb and the processed volume of blood to be independent predictors of yield. we recommend to get a high concentration of cd + cells in the pb, and to process adjusted volumes of blood when needed. an evaluation if the calculation of g-csf dose per m is more appropriate than per kg body weight should be done in future studies. autologous stem cell transplantation (asct) has been widely used in the treatment of hematological malignancies over the last two decades. despite its broad use, some characteristics that might influence engraftment have not been exhaustively investigated, particularly graft purity with respect to contamination by platelets (plts) and white blood cells (wbc). here we report collection characteristics and engraftment kinetics of a single center consecutive series of asct. we retrospectively collected clinical records of patients who underwent leucapheresis procedures (la; followed or not by asct) and data on asct at our institution over years ( - ) ( table ). the impact on engraftment kinetics of conditioning chemotherapies, amount of infused cd + cells and wbc/plts graft contamination were analyzed. absolute neutrophil count (anc) engraftment was defined as the duration of neutropenia (from day to the first of consecutive days of anc /μl post asct). regarding cd + cell collection, no impact of mobilizing regimens and wbc count during la was observed. on the other hand, we observed a difference in the number of total cd + cells collected among different diagnoses: the median overall collection was . ( . - . ) × /kg cd + cells for nhl patients, . ( . - . ) × /kg for mm patients, . ( . - . ) × /kg for hl patients and . ( . - . ) × /kg for aml patients) (p = . ). considering cd + cells/kg harvested on the first day of la, . % of nhl and hl, . % of mm patients and % of aml patients harvested ⩾ × /kg cd + cells. of note, among aml patients, . % collected o . × /kg. the differences were statistically significant (p = . ). moreover, an inverse correlation between collected cd + cells and age was shown (p = . ). anc recovery after asct was not influenced by conditioning regimen whereas diagnosis impacted on the duration of neutropenia (aml patients displayed a longer aplasia, po . ). we observed that the median days with anco /μl were , and in patients who received . × /kg, . - . × /kg and o . × /kg cd + cells, respectively (po . ). furthermore, the same finding was observed considering the duration of thrombocytopenia (median number of days with plts o /μl: , and in patients who received . × /kg, . - . × /kg and o . × cd + cells, p o . ). looking at the apheresis product, we analyzed the impact of harvest contaminating wbc and plts on engraftment kinetics. notably, when the asct collection contained × /μl wbc, anc engraftment (days with anc o /μl) lasted longer (median days ) compared to patients who received a graft with lower wbc count (po . ). a faster anc engraftment was also observed in patients receiving harvests with plts levels × /μl compared to those who infused a collection bag with plts o × /μl (p = . ). herein, we confirmed that the disease and the amount of infused cd + cells significantly influence time of anc and plts engraftment; furthermore, we observed for the first time that quality and purity of the graft have a substantial impact on engraftment kinetics. a combination of chemotherapy with growth factor is a commonly used strategy for hematopoietic stem cell (hsc) mobilization. the collection of timely and adequate numbers of hscs is a prerequisite for proceeding to transplantation. a variety of mobilization strategies are currently used. the knowledge of efficacy, safety and predictability of different hsc mobilization strategies might help blood and marrow transplantation (bmt) programs to effectively schedule patients for mobilization. given the many variables associated with the mobilization of hsc, collecting an adequate stem cell dose in a timely and effective manner is an art and science. factors that might affect the process includes type of disease and mobilization protocol, financial clearance, availability of chemotherapy beds, scheduling various diagnostic procedures and transplant urgency. to evaluate the effectiveness and related coordination efforts of ‛just-in-time' strategy of hsc mobilization and collection, we performed a retrospective study comparing all patients in whom peripheral hsc mobilization was attempted at khcc from january through november . data collected included the disease type, mobilization protocol, days to and number of collections, cd + cell dose, calendar of the mobilization and collection. the records of a total of mobilizations were reviewed. were of healthy allogeneic donors, and the remaining were of patients undergoing autologous transplantation. table depicts the overall summary of number of days and collection procedures per each protocol. detailed mobilization kinetics per disease type and mobilization protocol were also captured and evaluated. [p ] s [p ] detailed analysis of mobilization kinetics comparing different mobilization strategies aids in prediction of number of days of mobilization and anticipated number of collections. this helps in proactively scheduling patients based on collection predictability. a seamless communication through a shared calendar between key parties, primarily bmt physicians and nurse coordinators, bmt and flow cytometry laboratories and chemotherapy unit can be achieved. autologous stem cell transplantation is still a standard of care in the treatment of multiple myeloma. lenalidomide-based regimens are commonly used in both transplant-ineligible as well as -eligible patients. prolonged lenalidomide-exposure is known to affect mobilization of cd + cells, although the basic mechanisms are poorly understood. limited prospectively collected data is available on the effect of lenalidomide in the capacity to mobilize cd + cells for transplantation as well as graft cellular composition and post-transplant hematological recovery compared to the lenalidomide-naive patients. this prospective study included newly diagnosed myeloma patients who received mobilization with low-dose cyclophosphamide + g-csf, were successfully apheresed and transplanted before the end of . twenty-six patients had received a median of three cycles of lenalidomide-based induction ( %), whereas patients were lenalidomide-naive and served as the control group. both baseline characteristics and collection targets were similar between the groups. cd + mobilization and apheresis yields were analyzed and compared between the groups. blood graft cellular composition was analyzed from the thawed cryopreserved samples with a flow cytometry. graft function was evaluated by collecting engraftment data as well as by total blood counts at day + and at , , and months after post-transplant. the patients in the lenalidomide group had both lower median peak b-cd + counts and about % lower cd + yields of the first apheresis but without statistical significance ( table ). the median number apheresis was significantly higher in the lenalidomide arm ( . vs . , p = . ). the number of cd +cd +cd -, cd +cd +, cd +cd + cells and nk cells in the cryopreserved grafts were comparable between the arms. time to neutrophil engraftment was days in the both groups. the median time to platelet engraftment was d in the lenalidomide group and d in the control group. hematological recovery was comparable between the groups within months post-transplant. lenalidomide-based induction therapy seems to have an impact in the number of apheresis needed, but not in the total yield of cd + cells in the graft. neither the graft cellular composition nor posttransplant recovery in myeloma patients was affected by the limited duration of lenalidomide used before mobilization and collection of blood grafts. between september and november . siemens hematek system was used for luc count. luc numbers and percentage was measured before leukapheresis. we used pearson test for the correlation and roc curve for cut off value. patients' characteristics were shown in table- . there was not a correlation between luc number and mobilized cd positive stem cell number. but luc percentage was positively correlated with mobilized stem cell number (p: . ). a count of × /kg collected stem cells are optimal for autologous stem cell transplantation. we found % luc percentage as a cut-off value for prediction of collecting optimal number of stem cells with % sensitivity and % specificity. as expected luc percentage was negatively correlated with white blood cell count. there was no correlation between mobilized cd positive stem cell number and age. both luc percentage and mobilized cd positive stem cell number did not differ with underlying disease. we found only one study in the literature that evaluated luc percentage as a tool for the prediction of successful stem cell collection. they found that baseline luc numbers negatively correlated with stem cell mobilization in healthy donors ( ) . but we measure luc on apheresis day and found a positive correlation between luc percentage and stem cell mobilization. and we found a cut-off value for optimal stem cell mobilization with acceptable sensitivity and specificity. in our study we demonstrate that luc percentage measurement on apheresis day may be a very simple and cheap tool for the prediction of optimal stem cell mobilization. the spectra optia (so) apheresis system performs a wide range of therapeutic procedures, including peripheral blood stem cell (pbsc) collection in mobilized donors and patients (pts). the device was studied to evaluate the cellular composition of pbscs harvested in pts with multiple myeloma (mm), non hodgkin's lymphoma (nhl) and hodgkin's lymphoma (hl) planed for autologous peripheral stem cell transplantation (apbsct), and to optimize the collection of pbscs using the cd + precount and collection efficiency (ce ) of apheresis device which is calculated as follows: ce = total cd + cells collected × /kg; cd + precount/ μl × blood processed (liters). the blood volume processed is calculated as follows: desired cd + × /kg × recipient weight (kg): ce × cd + precount/μl in our study enrolled pts undergoing pbsc mobilization and planed for apbsct. we evaluated so system's mononuclear cell (mnc) collection performance, with respect to cd + cells and mnc collection efficiency, platelet reduction pre to post apheresis, and product purity in view of using prediction algorithms to optimize the procedure and predict the cd + yield, blood volume processed and platelets loss. we also evaluated neutrophil and platelet recovery in pts who underwent apbsct. results: between / / and / / , pts underwent pbsc harvesting by so device. median age was years ( - ). there were females and males. diagnosis was mm in pts, hl in pts and nhl in pts. the number of ahereses procedures was . mobilization consisted in g-csf alone in pts, chemotherapy and g-csf in pts, and g-csf + cxcr inhibitor in one patient. median count of cd + cells pre-collection was /μl ( . - ) . median total blood volume processed was . l ( . - . ). median count of cd + cells collected was . × /kg ( - . ). median mnc collection efficacy was % .median cd + cell collection efficacy was . % ( - %). median platelet reduction pre to post apheresis was % ( - %). median product hematocrit and granulocytes product was % ( - ) and % ( - ), respectively. twenty-six of the pts underwent myeloablative high dose chemotherapy followed by apbsct which was performed for mm in pts, hl in pts, and nhl in pts. the median count of cd + cells infused was . × / kg ( . - . ). all the pts received g-csf post-apsct until neutrophil recovery. the median day for neutrophil recovery was ( ) ( ) ( ) ( ) ( ) ( ) ( ) . median duration of severe neutropenia (anc o . × /l) was days ( - ). the median day for platelet recovery was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . median duration of severe thrombocytopenia (platelets o × /l) was . days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . conclusion: the study results confirm that the so apheresis system's mnc collection protocol is safe and effective. the neutrophils and platelets recovery in pts auto-transplanted was not inferior compared to historical controls. in addition, this system help to use prediction algorithms for whole blood processing to achieve a desirable and optimal yield based on cd + precounts and ce of the apheresis device. disclosure of conflict of interest: none. peripheral blood stem cell apheresis in small children is difficult! aa hedayati-asl , m emam-jome, p dinarooni, v fallah, a mehrvar and r zangooei in low-weight children with cancer and healthy donor children, peripheral blood progenitor cells (pbpcs) have largely replaced bone marrow as source of autologous and allogeneic stem cells in part because of their relatively easy collection. however, there is a concern regarding medical, psychosocial and technical difficulties in small children. we retrospectively analyzed peripheral blood stem cell apheresis in collections. patients were with cancer ( patients = neuroblastoma, patients = retinoblastoma, patients = germ cell tumor, patient = hepatoblastoma, patient = wilm's tumor) and healthy children donors. the study was conducted between and . peripheral stem cell apheresis was performed in the mahak cancer children's hospital in a nice room for children where the patients stayed with their families. patients s were not routinely sedated. pbpc were collected by a cobe spectra cell separator (cobe, denver, co, usa). harvesting was performed after days mobilization. mean body weight was . kg (range: - kg) for a median age of years (range: months- years). mean duration of harvesting was min (range: - min). mean volume of stem cell collection was ml (range: - ml). the mean number of total nucleated cells collected was . × /kg (range: . - . × /kg recipients). no side effects occurred. children didn't require an additional haematopoietic progenitor mobilization or additional apheresis in other day. pbsc collection was without transfusion in healthy donor children. pbsc collection may be difficult in small children owing to the large volume apheresis compared to the child's weight. various problems, such as metabolic or haemodynamic disorders may be were seen. peripheral stem cell harvest can be performed in low-weight children under safe and effective conditions even when systematic priming by blood is avoided. processing with increase of blood volume may to increase in the yield by recruiting progenitor cells. disclosure of conflict of interest: none. peripheral blood stem cell collection in low body weight children: a single centre experience g del principe*, g leone, s lazzaro, a meschini, k feri, p marchitelli, d carasso, f locatelli and m montanari department of pediatric hematology/oncology and transfusion medicine, irccs bambino gesù children's hospital, rome, italy pbsc became preferred source for autologous transplantation because of easier collection and faster engraftment. however apheresis for low body weight children ( o . kg) is affected by some issues: venous access, extracorporeal volume, metabolic and hemodynamic complications, citrate toxicity, so is crucial to standardize harvesting procedure both maximizing stem cells collection and reducing adverse events. a dual lumen central venous catheter was used to obtain a minimal blood flow of - ml /min and pbsc collection was performed with spectra optia mnc v . apheresis system, starting with cd + cell ⩾ μl in peripheral blood. the priming of extracorporeal circuit was made with compatible, irradiated, leucodepleted packed red cell to avoid hypovolemic state. citrate dextrose solution a(acd-a), with a ratio of : to whole blood, and a bolus of heparin ui/kg were used as anticoagulants. all patients, treated without sedation, were monitored by ecg, pulse oximetry and non invasive blood pressure; electrolytes panel (na, k, ca) and act (activated coagulation time) were assessed at the beginning, minutes after and then every hour during apheresis. hypocalcemia was managed by mg calcium gluconate slow infusion. we report our experience of pbsc collection in low body weight children ( o . kg) treated in our apheresis department between january and november . a total of pbsc collections were performed in children ( m/ f, median age months, median weight . kg) affected by medulloblastoma (n = ), germ cell tumor (n = ), neuroblastoma (n = ), retinoblastoma (n = ), brain cancer (n = ). total blood volume processed ranged from . to . tbv (median . ) and median count of cd + collected was . × /kg(range: . - ). all procedures were performed with a median duration time of minutes (range: - min) and no serious adverse events occurred. in our experience pbsc collection is safe and feasible also in low body weight children using a tailored apheresis procedure. disclosure of conflict of interest: none. plerixafor on demand in the first or in the second attempt of cd mobilization j romejko-jarosinska, e paszkiewicz-kozik, l targonski, m szymanski, z pojda and j walewski high-dose chemotherapy and autologous hematopoietic cell transplantation (auto-hct) is a recommended strategy for patients with relapsed, refractory or high risk lymphoma. mobilization failure of cd + cells after granulocyte colonystimulating factor (g-scf) with or without chemotherapy is a factor limiting patient access to this potentially curative procedure. the use of plerixafor with g-csf may improve cd + cell harvest in poor mobilizing patients. we evaluated the clinical effectiveness of plerixafor and g-csf ± chemotherapy administered on demand in the first and second attempt of mobilization in lymphoma or myeloma patients who were eligible for auto-hct. we evaluated data on consecutive patients with hodgkin lymphoma ( ), dlbcl ( ), mantle cell lymphoma ( ) , myeloma ( ) and other lymphoma subtypes ( ) who were mobilized with plerixafor between january and october . median (range) age of patients was ( - ). patients received a median of ( - ) chemotherapy lines. radiotherapy was applied in patients. all patients received g-csf ( μg/kg/day) ± chemotherapy and plerixafor ( μg/kg/day) on demand in the absence of increase in the number of cd + cells in peripheral blood above /μl on the day of the scheduled apheresis (within days following the chemotherapy and after at least days of g-csf). plerixafor was given to patients in the first attempt of mobilization and to patients during the second mobilization. the mobilization was considered effective if the harvest cell dose was × /kg cd or more. after plerixafor administration circulating cd + cells increased to /μl in patients ( %) and in patients ( %) in the first and in the second mobilization, respectively (p = . ). the cd + cell collection was performed in / patients ( %): in / ( %) patients in the first and in / patients ( %) during the second mobilization cycle. the median number of apheresis was (range: - ), for both mobilizing cycles. the median (range) cd cell dose collected in the first and second cycle was . (range: . - . ) × /kg and . (range: - . ) × /kg, respectively (p = . ). the harvest was successful in / patients ( %) in the first and in / patients ( %) in the second cycle (p = . ). three patients ( %) who failed the collection with plerixafor in the first attempt, succeeded in the second cycle. additional second mobilization with plerixafor was successful in five patients ( %) who failed the first mobilization. in total, / ( %) and / ( %) of patients given plerixafor in the first or in the second mobilizing cycle harvested at least a minimum cd cell dose for auto-hct (p = . ). these results show that plerixafor administered on demand is an effective rescue strategy for poor mobilizing patients. each mobilization cycle with plerixafor resulted in the increase of circulating cd cell count. successful harvest is more frequent if plerixafor is administered in the first than in the second mobilization attempt. the evaluation of the prognostic factors for mobilizing failure with plerixafor is necessary to identify the poor mobilizers precisely. disclosure of conflict of interest: jr-j, ep-k, lt and jw: sanofi (travel grants); ms and zp: none; jw: lecture, honoraria cryopreserved stem cell grafts are still widely used both in the autologous or allogeneic settings. cryopreserved grafts can be thawed at the bedside or thawed and washed at the cell therapy laboratory. we recently reported that post-thaw washing did not impair hematopoietic engraftment, in a cohort of autologous transplanted patients receiving either unwashed or washed grafts (calmels b et al, bone marrow transplant. ). post-thaw washing can be implemented using various methods such as manual centrifugation, automated centrifuge-based (sepax , biosafe) or spinningmembrane devices such as lovo (fresenius kabi). we here report a step by step implementation of the lovo biomedical device (bmd) for washing thawed stem cell grafts. having defined a washing program, we aim to compare this protocol to our routine process, using the sepax bmd. we took advantage of apheresis products intended for destruction and cryopreserved in identical bags; after dry-thawing (plasmatherm, barkey), bags were connected to the sepax or to the lovo bmd, diluted volume to volume with + - °c % hydroxyethylstarch / . (voluven, fresenius kabi) and processed using the smartwash program (sepax ) or a cycles standard wash protocol on lovo (a cycle referring to one pass through the spinning membrane). the lovo settings were customized for this application: reduction retentate pump rate ml/min, desired inlet pcv %, and automated volume to volume dilution. after processing, cd and cd absolute counts and viability were evaluated by single platform flow cytometry (stem-kit, beckman coulter) and dmso was quantified by capillary zone electrophoresis (p/ace, beckman coulter). post-wash data show comparable cd + cell recovery, viability and effective dmso depletion. we conclude that lovo enables high efficiency dmso depletion while preserving optimal cd viability and recovery. comparison with sepax , a widely used automated centrifugebased device, reveals comparable efficiency. moreover, the length of the procedure when using the lovo does not significantly delay the process as compared to bedside thawing. we are currently evaluating lovo for the processing of multiple bags and higher cell contents, due to its ability to concentrate large volumes of cells suspension. post-thaw washing using automated cell processing systems have thus to be preferred over bedside thawing, since they provide multiple benefits including a short processing time, efficient dmso and cell debris removal, precise determination of infused cd + cell dose, and improved cellular stability. [p ] disclosure of conflict of interest: none. using bone marrow (bm) as the graft source results in lower graft-versus-host disease incidence, which is particularity important in haploidentical (haplo) stem cell transplantations (sct). nonetheless achieving adequate cd + cell count might be complicated in cases of donor-recipient weight differences. priming with g-csf may partly solve this problem. also there are reports of immunomodulatory effect of bm priming. in the retrospective study we have evaluate the effect of priming on stem cell yield and the outcomes of sct. patients and methods: patients with primed bm graft were matched in the ratio : to non-primed grafts. the criteria for matching were type of the donor, age of the recipient, underlying disease and disease status at the time of sct. priming was performed with three injections of filgrastim - mcg/kg daily for days prior to bm harvesting. median recipient age was years (range: - ). % of patients received the graft from haplo donor, % from matched related donor (mrd). % had acute lymphoblastic leukemia, % had acute myeloid leukemia, % had aplastic anemia, % had other malignancies. % were classified as salvage patients. % received myeloablative conditioning, % received reduced intensity. post-transplantation cyclophosphamide (ptcy) was used as graft-versus-host disease prophylaxis in % of patients. results: the yield of cd + × cells /kg of recipient weight was only non-significantly higher in the priming group: . ± . vs . ± . , p = . . the yield of cd + cells per kg of donor weight was also not different: . ± . vs . ± . , p = . . there was no difference in the incidence of primary graft failure ( % vs %, p = . ). median time to neutrophil ( vs days, p = . ) and platelet ( vs days, p = . ) engraftment was shorter in nonpriming group. there was no differences between priming and non-priming groups in the incidence of acute grade ii-iv gvhd ( % vs %, p = . ), moderate and severe chronic gvhd ( % vs %, p = . ), -year non-relapse mortality ( % vs %, p = . ), relapse incidence ( % vs %, p = . ), overall survival ( % vs %, p = . ), event-free survival ( % vs %, p = . ) and gvhd-relapsefree survival ( % vs %, p = . ). conclusions: priming of the bone marrow with reported schedule did not result in higher cd + cell yield and was not associated with any differences in the outcomes of sct. nonetheless, these results should be interpreted with caution, because our study included large proportion of pediatric patients, patients with active disease and ptcy as gvhd prophylaxis, and they may not translate to the other groups of patients. disclosure of conflict of interest: none. priming with granulocyte-colony stimulating factor preserves the contents and abundant ifn-γ production capacity of γδ t cells z bian , q fu , m huo , xj huang and j liu peking university people's hospital the increasing evidences indicate that removal of αβ t-cell and b-cell from grafts was efficient and reproducible in allogeneic hematopoietic stem cell transplantation (allohsct). γδ t cell is one of the functional subpopulations preserved by this graft manipulation and supposed to play a role in improving the transplant outcomes. thus, comprehensive understanding the subsets and functional capacities of γδ t cells in graft becomes important. although there is increased attention paid on this special t-lymphocyte subpopulation, the contents and cytokine production capacities of peripheral γδ t cells before and after granulocyte-colony stimulating factor (g-csf) mobilization for allohsct have not been reported. peripheral blood (pb) before g-csf treatment, g-csf-primed pb and bone marrow (bm) grafts were obtained from healthy donors. the proportions of total γδ t cells and various γδ t-cell subsets were detected by flow cytometry. furthermore, effects of g-csf on the contents and cytokines production by γδ t-cell subsets were also determined. the percentages of most γδ t-cell subsets including cd +, cd -, vδ +, vδ +cd +, vδ +cd -, vδ +, vδ +cd +, vδ +cd -, and non-vδ /δ were preserved in the g-csf-primed pb grafts compared with those before g-csf mobilization. interestingly, we found that peripheral γδ t cells and various subsets all predominantly expressed ifn-γ in response to stimulation. this abundant ifn-γ production capacity of peripheral γδ t cells were maintained after g-csf treatment. in contrast, production of il- by γδ t cell and its subsets were decreased in the same context. priming with g-csf preserved the contents and abundant ifn-γ production capacity of γδ t cells. our data suggests a reasonable role of γδ t cells in preventing from allohsct associated complications and may help establish an effective γδ t cell-based immunotherapeutic approach to improve the overall survival of allohsct. disclosure of conflict of interest: none. processing of hematopoietic stem cells grafts: towards automation of cryopreservation/thawing steps a-l chateau , j gaude , c malenfant , a autret , c lemarie , c chabannon and b calmels centre de thérapie cellulaire-institut paoli-calmettes and unité de biostatistiques-institut paoli-calmettes autologous hematopoietic stem cells (hsc) support is still widely used to allow for high-dose chemotherapy in the context of myeloma and lymphoma treatment. in the autologous setting, mobilized aphereses are systematically cryopreserved. currently, cryopreservation and subsequent thawing rely on manual and largely operator-dependent processes such as manual addition of dmso for cryopreservation or thawing in standard water baths. these operations are thus hampered by significant intra-and inter-facility variability and have to be replaced whenever possible with automated and harmonized processes. the aim of our study was to evaluate a recent, versatile device: smartmax (biosafe, eysins, switzerland), based on the peltier-seebeck effect, for its ability to automatically add the dmso-containing solution to the cell product and to thaw hsc bags. we thus compared three different cryopreservation/thawing protocols ( figure ). we first evaluated the use of the smartmax at the thawing step by comparing cryopreserved apheresis products thawed using our routine device: the plasmatherm (barkey), an automated dry-thawing device that contains water (protocol a), with products thawed with the smartmax (protocol b); after thawing, all products were washed using the smartwash program of the sepax (biosafe). we then evaluated the smartmax for its ability to automatically add the dmso solution: autologous grafts were processed with the smartmax, both for cryopreservation and thawing (protocol c); we compared these ‛fully automated processes' to apheresis processed with protocol b. absolute cd + and cd + cell counts and viability were measured before cryopreservation and after washing using single platform flow cytometry. for all three protocols, the quality of the collected product was comparable in terms of median cd + cell and neutrophil contents. when comparing protocols a and b, viable cd + cell recovery after thawing and washing was slightly lower in the smartmax group ( %) as compared to the plasmatherm group ( %, p = . ). when comparing protocols b and c, viable cd + recovery was comparable (p = . ) when the cryopreservation solution was automatically added by the smartmax ( %), as compared to the manual technique ( %). these preliminary data need to be validated on larger numbers of procedures, however suggest that smartmax use can safely be substituted both to the manual addition of the cryoprotectant and to the traditional thawing step in water baths; potential advantages include complete water removal from sensitive clean rooms and gmp environments. full automation of previously manual and operatordependent technical processes will ultimately allow for improved standardization and reproducibility across cell processing facilities. [p ] disclosure of conflict of interest: none. reduced efficacy of mobilisation using gdp compared to ive a hunter, w merrison, am martin, k hodgson, f miall, r moore and r lewin university hospitals of leicester, nhs trust the use of ive ± rituximab for relapsed/refractory disease in lymphoma is well established. stem cell mobilisation using g-csf post ive administration has been the standard of care in our unit for years. recent interest in cisplatnin-based treatments has seen a change in practice with the use of gdp ± rituximab increasingly common. we have assessed the success of stem cell mobilisation post gdp and compared it to ive using g-csf. patients were eligible for augmentation with plerixafor if their peripheral blood cd levels were between - × cells/l at the time of collection. from sept to oct patients with progressive or relapsed lymphoma underwent stem cell collection. patients characteristics: dlbcl, follicular and t-cell nhl. had a median age ( - years). received gdp, ive. overall % patients failed to mobilise a sufficient cd cell dose to proceed to hdt. all the patients who received ive mobilised successfully but / ( %) patients receiving gdp failed to mobilise. of the patients who did mobilise the average cd collection was higher in the patients who received ive . ( . - . ) and the number of apheresis procedures was lower, median ( - ) compared to . ( . - . ) and ( ), respectively, in the gdp group. patients in the gdp group who failed to mobilise were not eligible for plerixafor because cd levels were below × /l. taking age into account the median age in the ive group was higher ( - ) than the gdp group ( - ) and the lines of previous therapy were not different. patients who had successful stem cell collections went on to receive hdt with leam and all patients engrafted. in this small collection of patients we have experienced a higher failure of mobilisation post a cisplatnin-based protocol compared both to our historical controls pre plerixafor usage (data not shown) but also to current patients. further investigation is needed to ascertain the impact of cisplatnin on stem cell mobilisation and its impact of treatment strategies. disclosure of conflict of interest: none. single centre experience of zarziotm biosimilar granulocyte-colony stimulating factor (gcsf) for the mobilisation of healthy donors demonstrates good leukapheresis yields and safety profile at month median follow-up jg taylor , , t seddon , k alizadeh , c agrawal , l kempster , jg gribben , and sg agrawal , centre for haemato-oncology, barts cancer institute, dept. haemato-oncology, st bartholomew's hospital, london, uk and experimental pathology, blizard institute, queen mary university of london, uk biosimilars have led to significant improvements in the affordability of growth factors such as granulocyte-colony stimulating factor (gcsf). data has shown similar performance and efficiency to parent drugs but concern has been raised about their use in healthy donors due to lack of data examining adverse effects in this setting. we conducted a retrospective analysis investigating mobilisation and adverse effects in healthy sibling donors of adults undergoing an allogeneic haematopoietic stem cell transplant at st bartholomew's hospital from to . harvest data were gathered from hospital records. adverse effects data were gathered from hospital records and telephone follow up. % of donors were male with a median age at harvest of ( - ). all donors were mobilised using zarziotm biosimilar gcsf at a dose of μg/kg/day. median number of apheresis required was ( ) ( ) ( ) . median cd + cell count was . × /kg bodyweight ( . - . ) with × cd +/μl ( - ) in peripheral blood. the target cd + count ( × /kg) was achieved in % of donors and an adequate yield ( - × /kg) in %. in four donors ( %), the harvest was deemed to have been unsuccessful as the cd + count was o × /kg. the patients with donor harvest yields o × /kg proceeded to transplant; all four patients engrafted and one patient had mixed chimerism at day but was fully donor by day . median cd + cell count was . × /kg bodyweight ( . - . ) . median days to neutrophil engraftment ( . × /l) was . median days to platelet engraftment ( × /l) was ( - ) with one patient never engrafting. forty ( %) of donors were contacted at a median of months ( - ) post mobilisation to establish incidence of adverse effects. three donors were uncontactable as they had moved overseas. eight donors were not contacted to avoid distress as their sibling had died since transplant. among contacted donors . % reported side effects including bone and lower back pain controlled with analgesia, constipation and low mood. other side effects included chest pain which was considered to be musculoskeletal in origin on day of gcsf administration associated with taking an increased dose due to patient error (n = ) and abdominal contractions like labour while receiving gcsf (n = ). three ( . %) reported side effects lasting beyond one month post mobilisation: lower back pain lasting months (n = ), fatigue of months duration (n = ), and cough of months duration (n = ). our data demonstrates good mobilisation using μg/kg/day zarziotm biosimilar gcsf without significant adverse effects at years median follow up. this supports its ongoing use for the mobilisation of healthy donors. disclosure of conflict of interest: sga has received honoraria from sandoz and grant support from sandoz and amgen. stem cell mobilization in poor mobilizers with multiple myeloma (mm) or non-hodgkin lymphoma (nhl) before and after introduction of plerixafor: single center comparative analysis using a cost-efficient single fixed-dose schedule r wäsch , c greil , c kiote-schmidt , s hildenbeutel , k kühbach , r bosse , j duyster and m engelhardt department of hematology, oncology and stem cell transplantation, university medical center, freiburg, germany collection of hematopoietic stem cells (hsc) from the peripheral blood (pb) is routinely conducted prior to highdose chemotherapy and autologous transplantation. despite safety and efficiency of current apheresis procedures including mobilizing chemotherapy and granulocyte colony-stimulating factor (g-csf), there is a significant rate of mobilization failures due to different patient-dependent factors necessitating additional agents like plerixafor. while plerixafor is approved for patients with mm or nhl based on prospective studies using steady state mobilization with g-csf − /+ plerixafor, prospective studies using chemo-mobilization are lacking. here we compared the outcome of poor mobilizer from the pre-plerixafor era with poor mobilizers who received additional plerixafor in a real world analysis. we analyzed consecutive patients with mm or nhl who were mobilized at our academic center between and and received plerixafor, because they were expected to be poor mobilizers, due to . low counts of cd + cells in pb samples prior to apheresis, . after a first apheresis day with insufficient yield or . as a rescue strategy after insufficient harvest with previous mobilizing chemotherapy (greil c,…engelhardt m, wäsch r. leukemia & lymphoma , in press). we examined cd + cell counts in pb and in apheresis products to identify those patients who were able to collect a sufficient cd + cell count for transplantation after application of plerixafor. we compared these data with consecutive poor mobilizers from the pre-plerixafor era, who were mobilized between and without plerixafor. the median pb cd +/μl count at first apheresis was significantly higher after the first dose of plerixafor when compared to the pre-plerixafor group with . vs . (p o . ). accordingly, the median collected cd + cells/d (× /kg bw) and total cd + cells (× /kg bw) were significantly increased with . vs . (p o . ) and . vs . (p o . ), respectively. the rate of × cd + cells/kg bw in first apheresis (%) increased from % in the pre-plerixafor era group to % after the first dose of plerixafor in the plerixafor group. consistently, the successful transplantation rate increased from % in the preplerixafor group to % in the plerixafor group. successful stem cell mobilization could be achieved with only a single fixed-dose of plerixafor in % of poor mobilizers as previously reported by our group. the addition of plerixafor to chemomobilization in poor mobilizers with mm or nhl significantly increased pb cd +/μl counts, apheresis yields and transplantation rates when compared to poor mobilizers from the pre-plerixafor era. these favorable apheresis results can be obtained using our cost-efficient, single fixed-dose plerixafor schedule in the majority of the patients leading to a % transplantation rate in poor mobilizer. disclosure of conflict of interest: rw received research funding, advisory and speaker's honoraria from sanofi-aventis. high-dose chemotherapy followed by autologous peripheral blood stem cells transplantation (pbsct) is the standard of treatment for patients with hematological malignancies. recombinant granulocyte colony-stimulating factors (g-csfs) are widely used alone or in combination with chemotherapy, in order to mobilize patient's stem cells (cd +) for autologous and allogeneic peripheral blood stem cells transplantation. aim: the aim of our study was to compare effectiveness and safety of different biosimilar products of filgrastim used in autologous pbsc mobilization in patients with hematological malignancies. our retrospective analysis included patients ( women and men) with median age years ( range: ,who underwent the procedure of autologous pbsct in years - in the haematology, blood neoplasms, and bone marrow transplantation clinic of medical university in wrocław. there were three different biosimilar products of filgrastim used: tevagrastim (teva) in patients, nivestim (hospira) in patients and zarzio (sandoz) in patients. ( %) patients were diagnosed with plasma cell neoplasms, ( %) with hodgkin's and non-hodgkin's lymphomas, ( %) patients had acute myeloid leukemia and ( %) had other hematological malignancies. statistical analysis was conducted using statistica (statsoft polska) statistical software. for quantitative variables arithmetic means and standard deviations were calculated for the estimated parameters in the studied groups. distribution of variables was tested using w-shapiro-wilk test. p . ). there were also small variations in the mean number of leukapheresis necessary to obtain the minimum cd + cell count: . in zarzio group, . in nivestim group and . in tevagrastim group. however, there were no difference between biosimilar g-csfs. the highest rate of successful mobilizations (defined as × /kg cd + cells collected) was observed in . % patients received zarzio, in . % received nivestim and in . % patients received tevagrastim. the safety profile was comparable between the biosimilar g-csf and included bone pain in ( %) patients and headache in ( %) patients. the results are shown in table . all three used biosimilar g-csfs demonstrated similar efficacy and safety in stem cell mobilization in patients with hematological malignancies. therefore, it seems that all the analyzed products can be used interchangeably. presented observations should be verified with wider prospective research. [p ] disclosure of conflict of interest: none. use of g-csf stimulation of bmt donors might prove to be beneficial in many respects, improving tnc yield but also through immunomodulatory effect on donor t cell function and apcs . we analyzed outcomes of consecutive patients receiving bone marrow transplants from hla-haploidentical related donors that received g-csf stimulation prior to harvest. fourteen patients received hla-haploidentical bmt with pt-cy between / and / . five donors were siblings, children, mothers and father. donors received g-csf at the dose of mcg/kg bw sc. on days − , − and before bm collection. twelve patients received nonmyeloablative conditioning according to baltimore protocol , while two patients received myeloablative conditioning (bucy). along with post-transplantation cyclophosphamide, all patients received tacrolimus and mmf form day + , as described earlier . median age was years (range: - ), female and male patients. eight patients had aml, cml, mh and one all. ten of them were in remission, while mh patients were in pr, and aml patients had residual disease as evident by immunophenotyping. median number of infused tnc was . × /kg bw (range: . - . ); cd + cells . × /kg bw (range: - . ) and cd + cells . × /kg bw (range: . - . ). median follow up was days (range: - ). eleven patients engrafted ( %), one patient had primary rejection, one had overt disease relapse at day + and one patient died in aplasia due to sepsis. median day to neutrophil recovery (anc . × /l) was (range: - ), median days to platelet recovery (plt × /l) was (range: - ). in all patients mmf was discontinued at d + . two patients developed acute gvhd in our cohort ( %), one after receiving dli for falling chimerism at day + . one patient ( %) developed chronic gvhd, after having received dli due to disease progression. at the time of analysis patients are evaluable; patients had disease relapse/progression ( %), patients are alive and in remission. one patient died due to sepsis in aplasia (accounting for % non-relapse mortality). one patient that rejected the graft was transplanted again from the same donor, using myeloablative conditioning and peripheral stem cells as graft source and engrafted. overall survival median is . years, with significantly shorter survival if patient was not in complete remission at time of transplant (p o . ). even though the experience with g-csf mobilized bm graft in the hlahaploidentical setting with pt-cy is relatively small, in our series it has been beneficial in terms of tnc yield. also, the incidence of acute and chronic gvhd in our patients has been low, particularly agvhd with one case developing only after dli. whether the observation is the result of limited number of patients, or it reflects the immunomodulatory effect of g-csf on bm graft as previously suggested remains to be seen as further studies are warranted. autologous transplantation of haematopoietic stem cells (ahsct) is usually perceived as a fully standardized and safe procedure; however, a minority of patients experience a delayed engraftment and seldom even an engraftment failure, possibly related to a poor quality of the graft. therefore the current policy in many centers is aimed to increase the target dose of collected cd + cells up to an ‛optimal' level of × /kg. plerixafor was introduced in the clinical practice to maximize the mobilization of hsc, in order to collect an optimal number of cd + cells in a limited number of collections also in poor and slow mobilisers. we carried out a retrospective analysis of our case series aimed to individuate mobilization predictors optimize the ‛on demand' use of plerixafor. we analyzed patients who underwent mobilization with cyclophophamide ( g/sqm) and filgrastim mcg/kg from + in our unit from and . diagnosis were multiple myeloma (mm) ( . %), non-hodgkin lymphoma (nhl) ( . %), hodgkin lymphoma (hdg) ( . %) and ( . %) autoimmune disease (ms . %; ssc . %). median age (range) was years ; male/female ratio / . circulating cd + cell count was started at white blood cells (wbc) recovery, which was defined as the first day when their count exceeded × /l. the primary goal was to identify at wbc recovery one or more factors predicting a suboptimal mobilization, which was defined as the failure to exceed cd +/mcl circulating cells in the day after the wbc recovery. patients were excluded from this analysis if ) showed a cd + count /mcl at wbc recovery (very good mobilizers) and/or ) had received plerixafor and/or ) did not proceed to another cd + count the day after wbc recovery. binary logistic regression was used to obtain the factors that increased the odds for an optimal mobilization. overall out ( . %) patients were shown as very good mobilisers as their cd + count exceeded /mcl at wbc recovery. on the remaining , were excluded for the lack of a second assessment and for the lack of data. among the remaining patients, the threshold of cd +/mcl cells on the second day was reached by ( . %) of patients (group a) while the remaining ( . %) failed the goal (group b). median (range) wbc × /l and cd +/mcl counts in group a and b at wbc recovery were . ( - . ) and . ( . - . ) and . ( - ) and . ( - ), respectively, with a statistically significant differences among group (mann-whitney u test with p = . and p = . , respectively). wbc (or = . ; % ci: . - . ) and cd +/mcl (or = . ; % ci: . - . ) in first day count, but not gender, disease category and time from mobilization chemotherapy to first cd + count, were predictors of optimal mobilization. combining these two predictors we found that wbc/cd + ratio has a sensitivity of . % with an auc . in roc analysis. assessment of circulating wbc, cd + and their ratio at wbc recovery in a chemo-based mobilization strategy can predict sub-optimal mobilization of hsc and support the decision of adding plerixafor. these data will be prospectively validated in a broader set of patients. disclosure of conflict of interest: none. human platelet lysate (hpl) is rich in growth factors (gf) and nutritive elements and represents a powerful xeno-free alternative to fetal bovine serum (fbs) notably for mesenchymal stem cell (hmsc) proliferation. however, there is a large variability in hpl preparations (various sources, use of different and non-standardized production protocols, with variable and limited number of donors), resulting in discrepancies in product quality, low management of product safety and poor batch-to-batch standardization. we describe here the development and the characterization of a standardized hpl prepared from outdated transfusional grade screened normal human donor platelet concentrates (pcs), manufactured on an industrial scale (batch size of donors) and following a highly qualified process (clean room, trained operators, validated aseptic filtration). pcs were frozen at − °c and thawed at + °c to lyse platelets. cell debris were removed by centrifugation and the supernatant (hpl) was recovered. clinical grade l batches of aseptic filtered hpl were characterized. first, we showed that hpl prepared from a limited number of donors displayed a variability in terms of gf contents. on the contrary, we observed a robust standardization between industrial batches of hpl ( donors) in terms of gf contents (bfgf, egf, vegf, pdgf-ab, tgf-beta and igf- ), biochemical analyses (total proteins, albumin, fibrinogen, vitamins and iron) and efficacy on bone marrow (bm)-hmsc proliferation. secondly, we compared expansion and functional characteristics of bm-hmscs grown in clinical grade hpl vs msc-screened fbs batches. we showed a reproducible increase in cell growth kinetics using hpl, a maintenance of bm-hmsc clonogenic potential and membrane marker expression (with however a strong overexpression of cd ). we observed a similar adipogenic and osteogenic differentiation potential and finally that immunosuppressive properties of bm-hmscs (inhibition of t-cell proliferation) cultivated in parallel in both conditions also remained identical. finally, we demonstrated the stability over time of hpl stored at − °c and − °c. in conclusion, we demonstrated the feasibility to use a standardized, characterized, efficient and clinical grade hpl for research and cell therapy applications. disclosure of conflict of interest: sv, se, lc, pb, tb, al, fg and bd are employees of macopharma. previously published p alpha/beta t cell depleted donor lymphocyte infusion m karakukcu, e Ünal, l kaynar, s Özcan, g tezcan karasu and mb acar the main objective of this project is to improve a safe and efficient new donor lymphocyte infusion (dli) with depletion of αβ+ t cells which cause graft versus host disease (gvhd), and enrichment of anti-leukemic γδ+ t cells, nk cells and dendritic cells to build an effective and permanent anti-tumor effects for patients relapsed hematological cancers after allogeneic hematopoietic stem cell (hsc) transplantation who have blasts and mixed chimerism. this study is conducted with collaboration of erciyes university pediatric and adult hsct units, and bahcesehir university, medical park hospital pediatric hsct unit. the tcr αβ+ t cell depleted dli product that is used in the study was collected and separated at erciyes university apheresis unit. the cell contents obtained for tcr αβ+ t cell depleted dli used for patients were cd cells were reduced to . - . × cells/kg, γδ+ t cells were reduced to . - . × cells/kg, αβ+ t cells were reduced by . %, and were obtained at - cells/kg. a total of patients ( female, male) were included in the study, consisting of an adult and children. nine patients had hematological malignancies. five patients were referred for all, three for aml, one for mds and one for griscelli syndrome. efficiency: the clinical response to the αβ+ t cell depleted dli treatment was achieved in / patients ( %). in these patients, although the increase of chimerism was limited in patients, no recurrence was occurred. one of the two patients who previously responded to the treatment but experience of decreasing chimerism had relapsed after months, and months later. one of these two patients died after relapse. the other was managed by the second transplant. the most important objective of this study was to show that αβ + t cell depleted dli treatment is reliable. none of the patient showed severe gvhd except one patient with mild grade ii gvhd. despite the presence of severe gvhd after hsct in two patients, reactivation for gvhd was not observed after treatment with αβ+ t cell depleted dli. none of the patients had a bone marrow aplasia. as a result, αβ + t cell depleted dli treatment seems to be highly safe, and effective in selected patients. disclosure of conflict of interest: none. hematopoetic stem cell transplantation (hsct) is associated with several potentially lethal complications; for example, relapse of the malignant disease, graft rejection, infectious complications and graft versus host disease (gvhd). higher levels of cd + cells in the graft have clearly been associated with increased risk of gvhd, but also superior gvl effect and less infectious complications. to tackle post-transplant complications such as graft failure and relapse, donor lymphocyte infusion (dli) have successfully been used for decades but with an associated risk of gvhd. to decrease the risk of gvhd but still use facilitating cells in the cell product we performed αβ depletion of grafts for use as stem cell booster after allogeneic hsct to treat infections or poor immune reconstitution. in this study, patients were infused post-hsct with αβ t-cell depleted grafts. the indication for infusion of αβ t-cell depleted graft in all patients was poor immune reconstitution with associated infectious complications. for all patients, the original hsct donor was used for the αβ t-cell depleted boost. to characterize the αβ-depleted stem cell grafts, samples were stained for various cellular subsets and analyzed by flow cytometry. we could show a median log depletion of αβ cells of . and a median yield of γδ t-cells (%) of . . the median cd + cell dose (× /kg) was . . all patients were alive months after infusion. after year only one patient succumbed. despite that the majority of patients suffered from agvhd grade or before infusion of αβ t-cell depleted graft none showed increased symptoms afterwards. in more than % of the patients there was a increase in granulocytes, thrombocytes and white blood cells months after infusion. in conclusion, we describe the use of αβ t-cell depleted grafts as stem cell booster in patients suffering infectious complications due to graft failure after hsct with encouraging results. disclosure of conflict of interest: none. delayed engraftment or graft failure still remains a concern in bone marrow transplantation (bmt). graft composition may predict engraftment after infusion. this study aims to determine which quality control parameters used for the characterization of bone marrow grafts are the most predictive in order to minimize the risk of engraftment delay or graft failure. we conducted a multicenter retrospective study in pediatric patients who underwent first allogenic bmt at two centers in barcelona (catalonia, spain) between and . quantitative variables considered for the study were: total nucleated cells (tnc), mononucleated cells (mnc), cd + cells, cd + cells and granulocyte-monocyte (gm) colonies enumeration. qualitative variables considered for the study were viability assessed by flow cytometry and clonogenic efficiency of the cd + cells (clonegm) which is the ratio between gm colonies and cd + cells. patients were included (median age (range) were years old ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ). the median tnc(range) was . e /kg ( . - . e /kg) and . e /kg ( . - . e /kg) for mononuclear cells (mnc). on the other hand, the median (range) cd + cell dose was . e /kg ( . - . e /kg) and t-cell dose (cd +) was . e /kg ( . - . e /kg). the median (range) colonyforming unit granulocyte macrophage (gm/kg) dose was . e /kg ( . - . e /kg). the median (range) of cd + cell viability, was % ( - %) and the median(range) of the clonogenic potential of cd + cells (clonegm) was . % ( . - . %). the median (range) of engraftment was days for neutrophils and ( - ) days for platelets. patient was considered as primary graft failure. in the univariate analysis, cd + (p = . ) and mnc (p = . ) cell dose predicted a faster neutrophil engraftment and female donor a slower neutrophil and platelet engraftment (p = . and p = . ). cell viability also correlated to a better platelet engraftment (p = . ). in the multivariate analysis we observed a trend for a faster neutrophil recovery according cd + cells infused. again, female donor was associated with slower engraftment. in order to establish a safety threshold, we did a quartile analysis of cd dose and found . e /kg (quartile ) discriminates a faster neutrophil engraftment [median days vs days for those with higher cd + cells (p = . )]. in conclusion, we found an association between mnc and cd + cell dose and time to engraft, and established a safety threshold of . e cd +/kg. also, bm grafts from female donors were associated with slower engraftment. no other qualitative parameters were predictive of engraftment. disclosure of conflict of interest: none. plasma cell myeloma (pcm) is currently treated with chemotherapy and autologous stem cell transplantation (asct) but relapse rates remain high. adoptive transfer of mature haploidentical natural killer (nk) cells is a promising approach to provide pcm patients with highly immunocompetent effector cells with anti-myeloma function early post transplantation. here we report on the current clinical phase i/ii trial of multiple preemptive infusions of good manufacturing practice (gmp) expanded nk cells to pcm patients (clinicaltrials.gov nct ). ten pcm patients were recruited (seven males, three females, median age: y). all patients received four cycles of vtd chemotherapy (reaching cr: × , vgpr: × and pr: × ) before high dose therapy with melphalan mg/m and asct. after successful stem cell mobilization and cryopreservation of patients' stem cells after the third vtd cycle, nk cells from haploidentical family donors were purified from unstimulated leukapheresis by t cell depletion and nk cell selection using clinimacs. highly pure nk cells (mean: . × cells) were obtained with a minimal t cell contamination corresponding to a . log t cell depletion. nk cells were expanded ex vivo for days in gmp-medium containing autologous irradiated feeder and interleukin- and - . nk cell numbers increased -fold (range: - -fold). in three nk cell products t cell contents were × cells/kg body weight (bw: × above limit of clinical trial) and were successfully reduced by °cd -depletion to . × cells/kg bw. nk cell products were cryopreserved in escalating doses ( . × , . × and rest as multiple doses of maximal . × cells/ kg bw). the pcm patients received - × expanded nk cells (median: . × cells/kg bw, range: . - . × cells/ kg bw) as - infusions (median. dlis). the nk-dlis were administered between day and after asct and were well tolerated without any acute adverse events. no signs of acute or chronic graft-versus-host disease were observed in any of the patients after a total of nk-dlis. engraftment occurred between days - (median: days). infused donor nk cells were monitored by short-tandem repeats pcr. donor nk cells were detected in peripheral blood one and h post infusion (% donor nk of enriched blood nk cells: mean: %, range: - %, and mean: %, range: - %, respectively) indicating significant nk cell survival in recipients in the absence of il- support in vivo. clinical responses at last follow-up compared to a retrospective cohort of matched control patients will be presented. these results demonstrate the feasibility of large-scale gmp expansion and safety and immunotherapy with third-party leukemia-specific t cells (leuk-sts) represents an attractive approach for acute leukemia (al) patients lacking a fully matched donor or relapsing after allogeneic hematopoietic cell transplantation (hct). its application however, is limited by the demand for high numbers of antigen presenting cells (apcs), capable to produce clinically relevant numbers of leuk-sts. low volume, non-transplantable cord blood units (cbus) could theoretically serve as an easily accessible source to generate high numbers of dendritic cells (dcs) and subsequently leuk-sts, providing also the advantage of reduced alloreactivity, even in cases of partial matching. our goal was to generate clinically relevant doses of leuk-sts targeting al-related antigens, the wilms tumor protein (wt ) and the preferentially expressed antigen in melanoma (prame), through the exploitation of non-transplantable cbus. to generate dcs, immunomagnetically enriched cd + cells from cbus ⩽ ml were cultured in g-rex devices in the presence of scf, gm-csf and il- . dcs matured by toll-like receptor ligand and / were immunophenotypically characterized by flow cytometry (fcm). secreted cytokines were measured with elisa. matured dcs were activated with a peptide-mix of wt and prame and used as apcs to repeatedly stimulate naive t-cells (derived from the cd fraction of the same cbu). the phenotype and the specificity of generated leuk-sts were determined by fcm and ifn-γ/ elispot, respectively. starting from mean . × ± . × cd + cells, from non-usable cbus, we generated . × (range: . - . × ) myeloid dcs (cd +/cd c+: . ± . %) in days (fold change~ . ). the produced cells highly expressed maturation markers (cd +/cd +: ± %; cd c+/hla-dr+: ± %) and secreted high levels of th cytokines (Ιl- : ± pg/ml; il- : . ± . × pg/ml, tnf-α: ± pg/ml) and low levels of the th -cytokine, il- . the average number of cd -cell-derived leuk-sts after week-culture was . ± . × (~ logs above clinical doses). the produced cells were enriched in cd + polyclonal cells ( ± %), comprising of cd + ( ± %) and predominantly cd + cells ( ± %), expressing effector memory (cd ra − /cd l − : . ± %) and effector memory ra markers (temra: cd ra+/cd l − : ± %), while containing insignificant numbers of cd +/cd +cells ( ± . %). specificity was seen after the second stimulation at the earliest and was increasing after each stimulation [mean spot forming cells (sfc)/ × cells at second, third, fourth stimulation: ± ; ± ; ± ; respectively]. in particular, produced cells were highly specific for both targeted antigens (prame: ± , wt : ± ), while they expressed low the programmed cell death protein- (cd +/pd- +: ± %), implicating absence of cell exhaustion after repeated stimulations. we report a paradigm of ‛circular economy' in science, by the exploitation of non-usable cbus, towards scalable generation of cb-cd +-cell-derived dcs and cb-cd -cellderived leuk-sts from the same cbu and establishment of leuk-sts banks. whether similarly produced leuk-sts could significantly advance the treatment of al or leukemic relapse after hct, will be ultimately determined in vivo. disclosure of conflict of interest: none. comparison of two different methods to generate antifungal-specific t-cells under pre-clinical-scale conditions r geyeregger , s tischer , invasive infections with aspergillus fumigatus constitute a major cause of morbidity and mortality in immunocompromised patients after haematopoietic stem cell transplantation. although adoptive immunotherapies against viral pathogens are already in phase i/ii trials, clinical-grade methods for the generation of aspergillus-specific t-cells (asp-t-cells) from healthy transplant donors or even related or unrelated thirdparty donors are still under development. in this study, two different strategies interferon-gamma (ifn-g) cytokine capture system (ccs) vs short-term in vitro expansion (ste) were performed from the same healthy volunteers in order to evaluate the most suitable approaches for the in-time generation of clinical applicable asp-t-cells. pbmcs from leukapheresis of healthy donors (n = ) were first prepared in hannover for the ifn-g-ccs and then sent to vienna to prepare the ste. all donors belong to the allocell registry (www.allocell.com) of hannover medical school and the frequency of asp-t-cells was pretested by high-throughput ifn-g elispot assay. for the ifn-g-ccs, × cells were stimulated for h with gmp-conform aspergillus lysate followed by magnetic selection of ifn-g-producing t cells. cells were characterized for phenotype and function by multicolour flow cytometry. for the ste, × cells were cultured in g-rex devices and stimulated for days with either the aspergillus lysate alone or with pooled overlapping pepmixes (catb, crf , f , gel , pmp , shmt and sot) and il- . to further characterize the final cell products, multicolour flow cytometry, ifn-g elispot and ifn-g/granzyme b flurospot analyses were performed. ifn-g-ccs: frequency of ifn-g positive asp-t-cells pre-magnetic enrichment ranged between . and . %. recently we defined t-cell donors as eligible if ⩾ . % specific ifn-g+ t cells are detectable. the purity of asp-t-cells among cd + cells, obtained from three donors after magnetic selection was in mean % ± (range: - %). the absolute number of selected ifn-g+ cd + t-cells was ± . this could be approximately multiplied by a factor of , if × pbmcs are used for the generation of clinically applicable t cells using the ccs and the prodigy device. ste: after days, asp-t-cells (n = ) showed highly specific activity against the lysate (in mean ± spot forming colonies (sfc)/ cells) and pooled pepmixes (in mean ± sfc/ cells). in both methods (lysate vs pooled pepmixes), predominantly cd + t-cells were expanded ( % ± . vs % ± . of cd +) compared to cd + t-cells ( . % ± , vs . % ± . ). interestingly, whereas after ste, cd + t-cells include mainly central memory t-cells (mean %; cd l+cd ra − ), cd + t-cells include mainly effector memory t-cells ( %; cd l − cd ra − ). generated t cells were highly functional and cytotoxic as determined by the secretion of effector molecules granzyme b and ifn-g. based on the purity of up to % after the ifn-g-ccs and the high number of sfc received after ste with lysate and pepmixes, both methods seem to be suitable for clinicalscale productions. for patients who are in need for high asp-tcell numbers the application of first in-time ccs-purified asp-t-cells followed by the administration of ste cells might be a promising way to boost antigen-specific t-cell response. disclosure of conflict of interest: none. complete computerization of cell therapy product files (‛zero paper') in the qap software o christéle , r catherine , r aline , k mathias , m lavinia , d vincent , m jean-pierre and l marie-noelle , hematologie clinique et thérapie cellulaire-chu amiens picardie, simedia-ver, hématologie clinique et thérapie cellulaire-chu amiens picardie, simédia-ver, direction système informatique-chu amiens picardie, hématologie clinique et thérapie cellulaire, lacassagne and the computerized management of cell therapy products (ctp) is an obligation for processing laboratories to meet regulatory requirements. the software used is often independent of institutional systems in view of the specificity of cellular therapies and do not always allow the implementation of the ‛zero paper' policies that are being put in place. we report here our experience with the qap software (quality assurance partner) developed by the company simédia (www. qap .com) in open source (mit license) allowing the management of fully computerized ctp files. the qap software has been developed to ensure the traceability of ctp for both preparation and quality control by combining the product preparation environment (personnel, premises, reagents, consumables, equipment). initially, with the help of the company simédia, we parameterized the software in accordance with our procedures for the preparation and quality control of ctp. we built a file that we printed out for archiving on paper. it soon seemed necessary to reverse this mode of operation to add to the software the documents papers to obtain a file completely computerized and to avoid paper archiving. the close collaboration between the cell therapy laboratory staff, the software referent within the information system department of the amiens hospital and the company simédia enabled: set up a document backup server sufficiently proportionate in memory. have simedia carry out the necessary developments so that all documents can be integrated into the software, set up a coherent working circuit, organize the registration of documents, put in place a rigorous verification of the mandatory elements of the file. the reflection on the computer file made it possible to evolve the software to widen its use to all documents of management of the laboratory: maintenance of equipment, control of premises, housekeeping, staff training, quality control of automatons, reagents and consumables, process, reception, distribution. rigorous formalization was mandatory to ensure that the record was organized in a uniform manner. an intermediate paper record is still necessary for a period of about month: from the programming of the graft to the final validity of the injected product. this folder consists only of transient elements that cannot be integrated into the qap software immediately. the transition from the paper file to a computer file took place in several stages, calling into question our functioning. the difficulties of this implementation are of several natures: the heterogeneity of the documents components a cell therapy product file, the impossibility of benefiting from an interface between all computer software used on the hospital, the psychological barrier prompting us to keep a paper copy, work habits, the guarantee of computer backup quality as well as its verification. but the complete computerization of the ctp file has the following advantages: easy and secure accessibility of information, resolution problems archiving paper files, a single backup media folder. disclosure of conflict of interest: none. conditioned media from allogenic mesenchymal stem cell culture (msc-cm) enhances wound healing in an allogenic d skin model moyasasr al-shaibani, x wang , p lovat, a tulah and a dickinson newcastle university migration of the epidermal layer towards the wound centre is an important step in the healing process. full thickness in vitro skin models can be used to investigate epidermal migration towards an injury site. since wound healing therapies often require allogenic transplantation of primary keratinocytes, an allogenic d skin model was developed to investigate epidermal migration. the effect of mesenchymal stem cell conditioned media (msc-cm) was assessed for wound healing using this in vitro human d skin model. human mscs were derived from human hip joints, and characterised using standard protocols. at % confluence, msc secretions were collected in serum free medium and referred to as msc-cm which were then analysed for protein content using elisa. fully humanised allogenic d skin models were developed (n = ) and a mm punch was induced into each model followed by daily treatment with msc-cm to investigate the migration of the epidermal layer towards the punch centre over the dermal layer at different time points ( week, weeks, and weeks). intact and wounded models were characterised structurally by haematoxylin/eosin (h&e) staining and immunofluorescence (if) was used to validate the dermal and epidermal biomarkers such as collagen (col ), cytokeratin (k ), keratin (k ), loricrin and involucrin. mscs were characterised as stipulated by the international society for cell therapy, that is, fibroblast like cells with the ability to differentiate into tri-lineages (adipocyte, chondroblast and osteoblast). phenotypically, over % of the cells were able to express phenotypic markers for variant stem cells such as cd , cd and cd . over % of the cells were negative for the expression of cd , cd , cd , cd and hla-dr (p = . ). msc-cm contained different concentrations of a variety of growth factors such as keratinocyte growth factor (kgf), hepatocyte growth factor (hgf), platelet-derived growth factor (pdgf), stromal-derived factor- (sdf- ) and macrophage stimulating protein- (msp- ). h&e staining showed that the models had distinct dermal and epidermal layers similar to that of real skin. additionally, if showed that the models expressed dermal and epidermal biomarkers, for example, col , k , k , loricrin and involucrin. after treatment with msc-cm, the epidermal multilayers of the punched models started to migrate towards the punch centre and covered the whole punched area after weeks of treatment with recovered expression of the epidermal biomarkers, for example, k , k , loricrin and involucrin. a fully humanised allogenic d skin model is a useful tool to mimic the in vivo environment and evaluate the wound healing process. it could also be used as a screening method to test candidate wound healing drugs. allogenic keratinocytes could be used as a cellular sheet to cover the wound area with the ability to migrate towards the wound centre and promote wound healing. a possible explanation for promoting epidermal migration at the injury site is that msc-cm contains cytokines which accelerate cell migration such as kfg, sdf- and msp- , in addition to other cytokines which promote both migration and proliferation of epidermal cells, for example, hgf and pdgf. disclosure of conflict of interest: none. before each freezing and after each thawing, a quality control is performed including a minima: (i) cd + quantification; (ii) estimation of the percentage of hsc cd + viability, via aminoactinomycin-d ( -aad) staining and (iii) evaluation of hsc functional ability to form colony ‛cfu-gm' (colony forming unit-granulocyte macrophage). apoptosis, or programmed cell death, involves complex pathways in part the path fas-fas ligand (fasl), mitochondrial components and caspase enzymes. the involvement of apoptosis dependent on caspases activation pathway in hsc cd + after thawing remains unknown. here, we assess the extent of apoptosis caspase-dependent before and after cryoconservation of hsc cd +, using a fluorescent labeled inhibitor of caspases ‛flica. ' we tested the induction of apoptosis caspasedependent, before and after hsc cd + cryoconservation from patients with different hematological malignances: multiple myeloma (n = ), lymphoma (n = ). caspases pathway activation status was evaluated by flow cytometry, using a fluorescent labelled inhibitor of caspases ‛flica' staining test, in hsc cd +, lymphocytes cd +, monocytes cd + and natural killer cells cd +. in order to assess cell viability, cells were stained in parallel with -aad. we determined positive cells %, that is, showing caspase activation in viable cells (flica+ cells), before and after cryoconservation. caspase pathway activation level was then correlated with hsc functional ability to form colony ‛cfu-gm,' and day's number of clinical aplasia. in our cohort, we showed a significant caspases pathway activation, with . % cd + flica+ cells after thawing, compared with the . % described in fresh cd + cells (p o . ). moreover, caspases pathway was significantly activated in thawing cd +, cd + and cd + cells: flica+ cells % in thawing cells were, respectively, . %, . % and . % vs %, . % and o % in fresh cells. we also report a significant increase of apoptosis caspasedependent in lymphoma patients ( . % of cd + flica+) in comparison to myeloma patients studied ( . % of cd + flica+) (po . ). in contrast, no correlation has been established between observed caspases pathway activation and hsc cd + capacity to form cfu-gm, or still day's number of clinical aplasia. our results show substantial cell death, induced by the increase in caspases pathway activation, secondary to the thawing process, and across all study cell types. this advance of apoptosis caspase-dependent may affect the immune response quality during recipient aplasia, without detecting a clinical impact. moreover, caspases pathway activation through cd + and cd + subpopulations could modify the therapeutic result of donor lymphocytes infusion dli, though yet untested. thawing process in autologous graft induces apoptosis caspase-dependent in all apheresis product cells, particularly in hsc cd +, without clinical impact in graft fate. disclosure of conflict of interest: none. donor-derived nk cell infusion combined with hla halpoidentcial blood stem cell transplantation to decrease leukemia relapse for high risk acute myeloid leukemia patients b wu, y huang, j xu, y he, jxm zhang*, z wu* hematology department, zhujiang hospital of southern medical university, guangzhou, china *shenzheng hank biologoical engineering co.ltd. hla halpoidentcial blood stem cell transplantation have solved the donor deficiency for patient who need to treat by transplantation. the high relapse of leukemia especially for high risk patient post transplantation affect the outcome of haploidentical stem cell transplantation. natural killer (nk) cells are part of the innate immune system and play a scavenger role to detect targets marked by ‛missing self' induced by viral infection or malignant transformation. infusion nk cells into receipt prior to stem cell transplantation could decrease the gvhd in mouse bone marrow transplantation model. in an effort to decrease the leukemia relapse and gvhd after halpoidentical stem cell transplantation for high risk acute myeloid leukemia patients, we evaluated the addition of donor-derived nk killer cells before halpoidentical stem cell transplantation in high risk acute myeloid leukemia patient. here we report interim results for five patients enrolled last year. five high risk acute patients received halpoidentcial stem cell transplantation combined with donor-derived nk cells infusion. all patients received an fbca conditioning regimen, which consisted offludarabine ( mg/m /day, intravenous) on days − to − , busulfan ( . mg/kg/day, intravenous) on days − to − , cyclophosphamide( mg/kg/day, intravenous) on days − to − and rabbit antilymphocyte globulin (atg . mg/kg/day, intravenous) on days − to − . donor-derived nk cells were infused into patient prior to stem cell transplantation. gvhd prophylaxis was a combination of cyclosporine a (csa) and short term methotrexate. five high risk patients ( patients with aml m cr , patient with aml m nr, patient with aml m cr and patient with aml m cr ) enrolled from jan to nov. ; the donors are parents and sibling. hla were mismatched between donor and patients. median cd + dose infused was . /kg (range: . - /kg) and the nk cell dose infused was × /kg ( . - . × /kg). all five patients got hematology recovery and achieved hematology cr. only one patient occurred grade ii agvhd post transplantation and controlled by methylprednisolone. at a median time of months (range: - months) post peripheral blood stem cell transplantation, the incidence of acute gvhd grade ii is % ( / ) . no chronic gvhd observed. four patients are still cr and survival with event free survival with median year follow up. one patient with aml m who had not achieved remission before transplant relapsed after months and got cr with second nk infusion and still survival. nk infusion prior to transplantation was found to be safe and feasible. there was no increase acute gvhd or chronic gvhd risk. there was a trend towards increased -year survival for high risk leukemia patient. the potential benefit on overall survival remains to be further evaluated with additional patient enrollment and longer follow up. however, given the favorable safety profile of nk cells, future strategies to enhance efficacy such as repeat dosing or modification of nk cells are worth potential exploration. disclosure of conflict of interest: none. donor lymphocyte infusion (dli) is a therapeutic option in the treatment or prevention of relapse after allogeneic stem cell transplantation (allohsct).of note, the risk of graft-versus-host disease (gvhd) associated with the graft-versus-tumor (gvt) effect may be influenced by the level of hla disparity between donor and recipient. data on use of dli after unmanipulated haploidentical hsct (haplohsct) with post-transplant cyclophosphamide (pt-cy) are still currently limited. we report patients (pts) receiving dli between and for prevention or treatment of relapse after haplohsct. seven pts were given haplodli doses, as treatment for relapsed disease (n = ) or as preventive therapy of relapse for high risk disease (n = ). four pts had acute myeloid leukemia (aml), had acute lymphoblastic leukemia and lymphomas - hodgkin (hl) and non-hodgkin dlbcl. pt had intermediate risk disease features, adverse risk and pts had refractory disease at time of haplohsct. pts had a previous hsct ( allogeneic and autologous). of the pts received a ric regimen and the source of stem cells was peripheral blood s (n = ) and bone marrow (n = ). gvhd prophylaxis was cyclosporine and mycophenolate mofetil (mmf), atg and pt-cy. median follow-up after haplohsct was (range: months. median time to neutrophil and platelet ( g/l) recovery were and days, respectively. after haplohsct, pts developed acute gvhd (agvhd) of grade i (n = ) or ii (n = ), at a median of days after haplohsct. the median time from haplohsct to first dli was days (range: - ). all pts had full donor chimerism at time of dli. before dli pts relapsed at a median time of days (range: - ), of whom pts had aml and received salvage chemotherapy and pt with hl being treated by dli alone. of the relapsed pts, showed progressive disease after first dli dose and achieved a sustained cr (with duration of cr of and months at last follow-up). the remaining pts were given dli in cr, in case (of aml) associated with azacytidine. pts received dli dose and pts were given dli injections with escalating doses. the first dose of dli was × cd /kg in pts, × in pt and × in pts. the pts who received dli doses (lymphomas) were given: ( ) × - × - × ; ( ) × for doses followed by dose of × . four pts developed chronic gvhd (cgvhd, %) in a median time of days (range: - ) after dli ( of them had presented previously agvhd grade i-ii). cgvhd was limited in case, moderate in pt and severe in pts. of these pts presented features of an overlap syndrome (acute/chronic gvhd) with signs of agvhd de grade i,ii and iii in pt each. involved organs were skin/mucosal (n = ), liver (n = ), gastrointestinal tract (n = ), lung (n = ) and joints (n = ). all patients experiencing gvhd after dli were treated by systemic corticotherapy, extracorporeal photopheresis and cyclosporine or weekly low dose methotrexate. median follow-up after first dli was months (range: . none of the pts receiving prophylactic dli relapsed during the follow-up period. pts died, of relapse and of severe cgvhd. pts were in cr at last follow-up, with no signs of gvhd and with limited cgvhd. despite the limited cohort, dli after haplohsct appears to be a therapeutic option in high risk pts allowing enhancement of gvt in the setting of haplohsct with post-cy infusion. disclosure of conflict of interest: none. previously published p early and sequential ctla ig primed donor lymphocyte infusions (dli) following post-transplantation cyclophosphamide (ptcy)-based haploidentical pbsc transplantation for advanced hematological malignancies promote proliferation of mature natural killer (nk) cells with cytotoxic potential and markedly reduces relapse-risk without increase in gvhd sr jaiswal, s zaman, p bhakuni, s bansal, s deb, s bhargava and s chakrbarti we have earlier shown that cd enriched cell infusion following ptcy resulted in rapid proliferation of mature nk cells with attenuation of gvhd and early use of prophylactic g-csf mobilized dli resulted in improved disease-free survival. ctla ig has been shown to be effective in attenuating t cell activation and induce transplantation tolerance in preclinical models. it has recently been employed to induce transplantation tolerance and reduce early alloreactivity in patients with nonmalignant disorders undergoing ptcy-based haploidentical hsct. nk cells on the other hand are resistant to ctla ig and in fact might demonstrate better anti-tumour effect in presence of ctla ig as cd is a putative activation receptor. to explore this phenomenon, we employed sequential ctla ig primed dli following ptcy-based haploidentical hsct in patients with relapsed/refractory hematological malignancies. patients ( - years; aml- , all- , nhl- ) received abatacept (ctla ig) as a part of gvhd prophylaxis at mg/kg on day − followed by pbsc and sequentially on days + , + and + followed h later by dli of × cd cells/kg containing . - × /kg cd + cells. ptcy was administered on days + and + with cyclosporine from day + to day + and subsequent rapid tapering. the immune reconstitution of the study group (ctla ig-dli) was compared with the cohort of patients with both malignant and nonmalignant diseases who received abatacept but not dli (n = ; ctla ig group) and those receiving cd enriched donor cell infusion on day + (n = ; nki group). results: there were no acute infusion related toxicities. all patients engrafted at a median of days ( - days). the incidences of acute and chronic gvhd (all mild) were % and %, respectively. three patients reactivated cmv and there was only one non-relapse mortality ( . %). only patients relapsed ( . %) with a disease-free survival of . % at year. these cells had greater expression of cd a compared to normal healthy donors. the recovery of cd +, cd + + and cd + − cells were similar in the ctla ig-dli and nki groups at days , and post-transplant and this was significantly higher than the ctla ig group ( figure ). in contrast to ctla ig group, nk cells recovered at day + with predominantly cd dim cd + phenotype with significant population of cells expressing kir+nkg a phenotype in both ctla ig and nki groups with higher expression of cd a. interestingly, the patients who relapsed had attenuated recovery of cd + + cells at and days( /μl and cells/μl) without cd a expression, in contrast to the rapid and sustained recovery of this population of nk cells in those not experiencing relapse (cd + + cells /μl and /μl). however, the recovery of tregs was prompt and sustained in the comparator groups, which remained low in the ctla ig-dli group until day + . there were no differences in the recovery of other t cell subsets between the three groups. the study demonstrates the unique ability of ctla ig to augment nk cell proliferation, maturation and cytotoxicity and reduce relapse with attenuation of t cell activation and gvhd in the context of the early use of ctla ig primed dli following ptcy-based haploidentical hsct without ex vivo selection or expansion. we hope this novel strategy might offer less expensive and yet a viable alternative in the field of nk cell therapy. [p ] disclosure of conflict of interest: none. enhanced cytotoxicity of γδ-cytokine induced killer cells against hematologic malignancies n bloom, s eldror, s caspi, s teihuman,h vernitsky, e jacoby, b bielorai and a toren cik cells are ex vivo expanded by scheduled addition of anti-cd mabs and a cytokine cocktail that contains ifn-γ, il- or il- . cells represent an in vitro generated heterogeneous population consisting of different effector cells-cd poscd pos, cd negcd pos and cd poscd neg-t cells that mainly ( %) express α/β t-cell receptor (tcr) s and to a lesser extent (o %), tcr γδ phenotype. these nklike t cells product show a dual functional activity, retaining their original t cell specificity and nk cytotoxic capacity via marked up regulation of the nk cell receptor, nkg d. pre and clinical studies showed that the optimal cytotoxic effect of cik cells against different malignancies (target cells) is achieved at : e:t ratio, which means high numbers of αβ t-cells that might increase the risk of gvhd. here we produced ciks from αβ tcr depleted cellular products (defined as γδcik) and tested their phenotype expression and in vitro cytotoxic activity against hematological malignancies. fresh apheresis products were processed using the clinimacs depletion reagent, according to manufacturer instructions. target product was cultured with rpmi supplemented with % fcs and ex vivo expanded by scheduled addition of cytokine cocktail that contains ifn-γ ( iu/ml), anti-cd mabs ( ng/ml) and iu/ml il- . the cells were cultured for days. cytotoxic activity of the γδcik was evaluated against various target hematological malignant cell lines (k , reh, jurkat, and u ). after days, the αβ depleted cik cultures resulted in . % γδ t-cells ( folds expansion) compared to . % of γδ t-cells immediately after depletion, and compared to only . % in non-selected cik cells. the percentage of αβ t cells in γδcik cell cultures started from . % (immediately after depletion) to . % compared to . % αβ t cells were found in non-selected cik cells cultures. γδcik cells produced robust cytotoxic activity at a : e:t ratio against reh cells ( . ± . %), jurkat cells ( ± . %); u ( . ± . %) and k ( . ± . %), compared to nonmanipulated cik cell activity against the same targets ( ± . %; . ± . %; . ± . %; . ± . %, respectively). we found higher degranulation capacity of γδcik cells compared to non-selected cik cells against reh ( . ± . % vs . ± . %), jurkat ( . ± . % vs . ± . %), u ( . ± . % vs . ± . %) and k ( . ± . centre de thérapie cellulaire, institut paoli-calmettes; unité de transplantation et de thérapie cellulaire, institut paoli-calmettes; centre d'immunologie de marseille-luminy and laboratoire d'immunomonitoring, institut paoli-calmettes during the past years, the major improvements in the field of allogeneic hematopoietic stem cell transplantation (hsct) (reduced intensity conditioning regimen, high level hla typing, alternative donors, gvhd prophylaxis…) significantly extended the feasibility of this procedure. in contrast, disease recurrence after hsct remains a main issue. thus, many post-hsct prophylactic interventions are under investigation. unmanipulated donor lymphocyte infusion (dli) remains one of the most frequently used post-hsct treatment, but its potential benefit in increasing gvl effect may be counterbalanced by the induction of gvhd. in this setting, the use of adoptive transfer of ex vivo enriched and activated nk cell infusions from the same donor (dli-nk) may induce gvl effect without causing gvhd. we therefore report on a single-center phase clinical trial (nct ) evaluating the safety of ex vivo activated allogeneic nk cells infused between days and after hsct. the aim was to determine the maximum tolerated dose (mtd) of ex vivo highly purified and activated dli-nk after matched related donor hsct. the schedule plan a first phase of + dose escalation method using dose levels ( . e /kg, . e /kg and . e /kg). grade - secondary adverse events according to nctci classification and severe gvhd occurring within days after dli-nk were considered as dose-limiting toxicities (dlt). a second step allowed enrolling patients at the mtd. over a period of . years, patients with various hematological malignancies (aml, all, hl, nhl, mds) were infused with activated nk cells at a median time of days (range: - ) post-hsct. apheresis products were collected from the hsc donor, cd -depleted and cd -selected by immunomagnetic separation using clinimacs. selected nk cells were cultured for days in medium supplemented with % fetal calf serum in the presence of u/ml of il- in air-permeable cell culture bags. after immunomagnetic separation, cd enriched products had a median cd + cell purity of % (range: - ) and viability of % ( - ). after il- activation, the median cd + cell dose was . × e /kg ( . - . ) , with a viability of % ( - ) and a residual cd + cell content of . × /kg ( - . × /kg). all release criteria to be met were fulfilled for the preparations infused : viability %, negative microbiological testing, cd + cell count ⩾ × /kg, and cd + cell content o × /kg. standardized quality controls were employed at all steps of the manufacturing process, adding a level of consistency to the product testing before release. activated-nk cells were well tolerated in all patients, with no occurrence of dlt. thus, mtd was not reached. two patients presented with a moderate chronic gvhd, both of them during cyclosporine a dose reduction. relapse occurred in patients with aml. one patient died from idiopathic pneumoniae, without evidence of relapse, gvhd or infectious disease. with a median follow up of months ( - ), year os was % ( figure ). therefore, infusion of highly purified, activated-nk cells of donor origin as a substitute to standard dli does not induce gvhd nor other side effects after hsct: the demonstration that modulation of nk cell activity can achieve disease control after hsct deserves to be investigated in larger trials. [p ] disclosure of conflict of interest: none. feasibility, safety, rapid production and efficacy of institution-produced cd car staff were trained on site for collection, processing and cryopreservation by regional experts. a total of units were collected and processed as part of the initial validation of the project. ucb units were processed on either axp or sepax systems, and all cryopreserved in bioarchive (an automated, robotic cryopreservation system that can archive up to units). the characteristics of which as well as the post processing data are depicted in table . [p ] we shared a successful story of establishing the first public cord blood bank in jordan. the first units collected showed excellent sterility, viability, collection volume and total nucleated cells. a very good recovery of both nucleated and cd + cells were obtained using axp and sepax cell separation systems. the process of validation of equipments and methodology is complete. we anticipate moving to permeant facility of the cord blood bank in the new expansion in early . we look forward for steady progress in ucb recruitments, hla typing, cryopreservation and adherence to netcord-fact standards as well as participation in international registries. functionally active ifn-gamma secreting cmv pp specific t cell therapy as an alternative for clinically urgent cmv related diseases n kim, y-s nam , k-i im , j-y lim, y-w jeon , y song and s-g cho the catholic university of korea, seoul cytomegalovirus (cmv) related diseases are a serious cause of morbidity and mortality following hematopoietic stem cell transplantation (hsct). it has been reported over the last two decades that cmv-specific cytotoxic lymphocytes (cmv-ctls) can provide long-term cmv-specific immunity without major side effects as an alternative to antiviral drugs. however, its application has been limited by prolonged manufacturing process of cell therapy. in this study, we apply the ifn-γ cytokine capture system (ccs) using the fully automated clinimacs prodigy device to rapidly produce cmv-ctls that may be applicable in clinically urgent cmv-related diseases. five validation runs were performed using apheresis samples from randomly selected cmv-seropositive healthy blood donors. then, clinimacs prodigy automatically performed successive processes including antigen stimulation, anti-ifn-γ labelling, magnetic enrichment, and elution which took~ h. the original apheresis samples consisted of . % ifn-γ secreting cd + t cells in response to cmv pp antigen (cd +ifn-γ+ cells) which were mainly cd ra+cd l+ naive t cells. following ifn-γ enrichment, the target fraction contained . % cd +ifn-γ+ cells with reduction in naive t cells and the selection of cd ra − cd l − and cd ra +cd l − memory t cells. furthermore, extended culture of these isolated cells revealed functional activity including efficient proliferation, sustained antigen-specific ifn-γ secretion and cytotoxicity effect against pp pulsed target cells. therefore, we suggest ifn-γ ccs by clinimacs prodigy as a simple and robust approach to produce cmv-ctls, which may be highly feasible and applicable in clinically urgent cmvrelated diseases. disclosure of conflict of interest: none. in vitro generation of tumor antigen-specific t cells from patient and healthy donor stem cells s bonte , s snauwaert , g goetgeluk , b vandekerckhove and t kerre hematology, ghent university hospital, ghent, belgium and department of clinical chemistry, microbiology and immunology, ghent university, ghent, belgium acute myeloid leukemia remains a therapeutical challenge, as many patients relapse after chemotherapy. allogeneic stem cell transplantation is in most of these patients the only option for cure, but carries a high risk of morbidity and mortality and a suitable donor may be lacking. recently, advances are being made in the field of t cell immunotherapy. the classical protocol, in which peripheral blood lymphocytes (pbl) are transduced with a tumor antigen-specific t cell receptor (tcr), can generate t cells with low and possibly hazardous specificities (due to mispairing of the endogenous and introduced tcr α and β chains). therefore, we have developed a novel protocol in which we generate tumor antigen-specific t cells from cd + hematopoietic stem cells. we have already succeeded in generating large numbers of tumor-specific, naive and resting t cells that only carry the introduced tcr, starting from postnatal thymus and cord blood cd + cells. now we are optimizing this protocol for clinically more relevant samples, such as mobilized peripheral blood from healthy stem cell donors and from patients in remission after chemotherapy and/ or other treatments, and leukapheresis samples from patients at diagnosis. in our protocol, cd + cells were isolated from hla-a + fresh patient and healthy donor samples and cultured on op -dl in the presence of scf, flt l and il- , until t cell commitment. subsequently, the cells were transduced with a tumor antigen-specific tcr and again co-cultured until cd + cd + double positive cells were abundantly present. at that point, agonist peptide was added, which induces maturation. finally, cells were polyclonally expanded on feeder cells. for hla-a negative samples, cd + cd + double positive cells were co-cultured with a cell line (t pulsed with the agonist peptide or a cell line with endogenous expression of the agonist peptide) which can present the agonist peptide to the maturing t cells. using the above protocol, we were able to generate tumor antigen-specific t cells from out of healthy donor samples, / sample from a patient in remission and / samples from patients at diagnosis, who were all hla-a +. for most samples, multiple rounds of agonist peptide stimulation were necessary to obtain further maturation. in contrast, generation of mature t cells from cd + cd + double positive cells in postnatal thymus or cord blood co-cultures, requires only round of agonist peptide stimulation. for the hla-a negative samples, we were able to generate an adequate cd + cd + double positive population from / healthy donor sample, / samples from patients in remission and / sample from a patient at diagnosis. agonist selection using a cell line seems inefficient as cd is not upregulated and cells did not mature to cd + or cd + single positive mature t cells. we are currently co-culturing more samples using our protocol. furthermore, we are investigating the effect of freezing and thawing on the in vitro t cell generation process (cell numbers and efficiency). finally, we are also working on optimizing the protocol for generation of tumor antigen-specific t cells from hla-a negative patient and healthy donor samples. disclosure of conflict of interest: none. increase of polyspecific immune responses against leukemia-associated-antigens (laa) and reduction of regulatory cytotoxic t-cell (ctl) responses against malignant cells play a major role in maintaining remission and prolonging overall survival in patients with hematologic malignancies after allogeneic stem cell transplantation (allo-sct) and/or donor lymphocyte infusions (dli). graft versus leukemia (gvl) effects after allogeneic stem cell transplantation and/or dli are considered to be t cell-mediated. many groups described specific t-cell responses against several leukemia associated antigens (laa) in different hematological malignancies. however, t cell responses after allo-sct and dli are not well characterized. in this study, we analyzed laa-specific t cell responses after allo-sct and dli. to this end, we assessed the frequency and diversity of laa-specific cd + t cells using elispot analysis and tetramer assays in patients ( patients (pts) with acute myeloid leukemia, pts with chronic myeloid leukemia, pts with multiple myeloma and pts with chronic lymphatic leukemia) before and after dli. epitopes derived from prame, npm mut, rhamm, wt- and other laa were tested. moreover, the frequency of regulatory t (treg) cells was measured and the course of cytokine profiles before and after dli was analyzed. these immunological findings were correlated to the clinical course in the respective patients. in elispot and tetramer assays, an increase in frequency and diversity of laaspecific t cells was observed in all patients. importantly, there was a significant increase from a median of to laa-derived t cell epitopes (p = . ) in clinical responders (r) when compared to non-responders (nr). these positive results in r vs nr where s confirmed by tetramer-based flow cytometry assays, where an increase in frequency from . to . % in the r group of laaspecific t cell/all cd + t cells was observed. interestingly, the frequency of tregs in clinical responders decreased significantly from a median . % to . % (p = . ) while the frequency of tregs kept stable over time in non-responding patients. t cell subset analysis did not reveal significant differences before vs after dli administration. in cytokine assays using elisa for the detection of more than cytokines before and after dli we found a shift towards proinflammatory and t cell stimulating cytokines. taken immunologic surveillance of leukemia is employed for the prevention and treatment of relapse post allohsct. to augment this effect donor lymphocytes are infused (dli) in patients at risk. this procedure is associated with a high risk of agvhd and we believe that this route of administration may not make the direct contact between infused cells and blasts the optimal one. to address these issues, we started delivering donor lymphocytes directly to the bone marrow cavity (ib-dli) in patients post allohsct at relapse. three with aml and one with cll, all relapsed post allohsct: allosib: -year-old female aml patient (relapsed years post hsct), -year-old aml male q del (relapsed in years, traumatic brain injury), -year-old male aml flt itd+ received mud hsct (relapsed months) and -year-old cll male, tp del, ebv reactivation (progressed years). two patients ( % and % blasts in the marrow) received ib-dli up-front and two others due to higher proportions of leukemic cells received either flag (aml case) or anti-cd moab (cll case) followed by ib-dli. tcr clonotyping revealed in all patients the presence of the prevailing oligoclonal response on the polyclonal background (characteristic for each individual) which was identified in the marrow and in the blood. however, in two out of patients a distinct oligoclonal peak was seen at first in the marrow and then in the blood. microarray analysis of the transcriptome in the marrows of patients who received three ib-dli courses revealed in all patients preferential use of genes associated with lymphocyte or lymphocyte activation pathways. the patients who responded favorably (cr or pr) clustered with the transcriptomes of normal individuals, but those who failed to respond clustered separately. ib-dli was safe and not associated with gvhd. selective accumulation of cd +cd + as well as the presence of a distinct oligoclonal peak in the marrow suggest that tcrbeta clonotypes may be private to leukemia cells recognition. the response may result in cr or pr and the patients were in a good physical shape during the treatment, which makes it possible to deliver the salvage chemotherapy if required. broad spectrum antibiotics were started. after the orthopedic consultation, the fourth finger was amputated and amputation from the left ankle was recommended. a stem cell transplantation option was offered to patients and their relatives as one of the therapeutic approaches. upon acceptance by patient, μgr/kg of colony stimulating agent was started to patient. when the stem cell was /μl, the stem cells were collected. the obtained stem cell product was injected intra-lesionally (picture b). granulation tissue began to develop from the second week in the foot floor of the patient. after from th week, the necrotic tissue was disappeared and the granulation tissue was appeared. at weeks, % of the lesion healed. at th week, there was normal tissue instead of necrotic tissue on plantar surface at left leg (picture c). this case report suggests that diabetic foot/ulcer can be healed with intralesional application of stem cells in patients with diabetes mellitus. [p ] disclosure of conflict of interest: none. and third (n = ) cell infusions were cryopreserved. cells were infused following conventional chemotherapy (ia, mec, hdac) in cases ( %), chemotherapy plus hypomethylating agents in cases ( %) and hypomethylating agents alone in cases ( azacytidine, decytabine; %). the procedure was well tolerated, with mild and transient ‛haploimmunostorm syndrome' (fever %, rash %, diarrhea %). only the two patients with cmml received corticosteroid. one patient suffered early infusional reaction that was resolved with support treatment. none of the patients showed acute or chronic gvhd or persistent donor engraftment in chimerism tests. four patients had bacterial infections, but no other significant invasive fungal or viral infections were observed. all aml/raeb patients treated achieved complete remission with microhct treatment ( ; %). only one patient, with cmml, died during microhct induction ( %). four patients relapsed at , , and months after the infusion; two of them achieved a second sustained complete remission with another micro-hct from a different donor (one of them had developed anti-hla antibodies). as described in figure , median overall survival is months and overall survival at years is %. microhct is a well tolerated procedure in elderly aml/mds patients who are not candidates to allogeneic hct. infectious complications are insignificant and the remission rates are very encouraging in very high risk cases, with no evidence of gvhd. patients can undergo a second microhct from a different donor. in addition to the experience by ai et al, we have also shown that microhct can be safely administered following a hypomethylant agent course instead of conventional chemotherapy. a large, international, randomized clinical trial will address the safety and efficacy of microhct for elderly aml/ mds patients (nct ). [p ] disclosure of conflict of interest: none. wilms tumor protein (wt ) is expressed in a variety of solid tumors and is found in more than % of patients with acute myeloid leukemia which makes it an attractive target for immunotherapy. previously it was shown that t cells recognizing wt are suitable for adoptive t-cell therapy by increasing the graft versus leukemia effect. however, the efficiency of this therapeutic strategy is still limited due to the low precursor frequency and specificity of wt -specific t cells in the peripheral blood of healthy donors. the ubiquitous antioxidant inducible enzyme heme oxygenase- (ho- ) and its products have immunomodulatory effects, which render it as a potential target for the modification of t-cell responses. recently, we found that inhibition of ho- enzyme activity via tin-mesoporphyrin (snmp) results in activation and proliferation of antiviral t cells from healthy donors. in this study we aimed ( ) to identify the mechanism of ho- modification in the generation of wt -specific t cells and ( ) to develop strategies for the sufficient generation of wt specific t cells from healthy donors to augment effective t-cell immunity in leukemia patients and to broaden the applicability of adoptive t-cell therapy to the majority of patients. the frequency of wt -specific t cells in peripheral blood of healthy donors (n = ) was examined before and after snmp treatment via ifn-γ elispot using the wt -overlapping peptide pool (ppwt ). enrichment efficiency of wt -specific t cells after ho- inhibition was verified in response to ppwt and the hla-a* : -restricted wt peptides (vldfappga, wt ) and (rmfpnapyl, wt ) by ifn-g secretion assay and expression analysis of the t-cell activation marker cd . phenotypic and functional characterization of wt specific t cells were further assessed by multicolor flow cytometry, luminex assays and elisa with respect to t-cell subsets, cytotoxicity, proliferative capacity and secretion of effector molecules. in % of donors we found specific t cells against ppwt by ifn-γ elispot ( spots/ . pbmcs). the frequency of wt -specific t cells in these donors could be increased fivefold after inhibition of the enzymatic activity of ho- via snmp. to assess the possibility that ho- modulation might be clinically applicable in conformity with good manufacturing practice, enrichment of snmp-treated wt -s specific t cells was evaluated based on ifn-g secretion and cd expression. compared to snmp-untreated cells there was a . -fold higher response of ho- modified wt -specific t cells pre-enrichment and an up to -fold higher enrichment efficacy, while snmp treatment did not affected the t-cell functionality. in conclusion, modification of the enzymatic activity of ho- resulted in a more effective generation of functionally active wt -specific t cells suitable for adoptive t-cell therapy. this makes ho- a promising therapeutic target to boost antigen-specific t-cell responses for treatment we recently developed and characterized a standardized and clinical grade human platelet lysate (hpl) that constitutes an advantageous substitute for fetal bovine serum (fbs) for human mesenchymal stem cell (hmsc) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issue. because of the progressive use of pathogen reduced (pr) labile blood components, we evaluated the impact of the novel procedure theraflex uv-platelets for pathogen reduction on hpl quality (growth factors content) and efficacy (as a medium supplement for hmsc expansion). this technology is based on short-wave ultraviolet light (uv-c) and has the main advantage not to need the addition of any photosensitizing additive compounds (that might secondary interfere with hmscs). we applied theraflex uv-platelets procedure on fresh platelet concentrates (pcs) suspended in platelet additive solution and prepared hpl from these treated pcs. we compared the quality of pr-hpl with the corresponding non-pr ones, in terms of growth factor contents. then, we evaluated the efficacy of pr-hpl, in comparison with hpl and msc-screened fbs. we performed large scale culture of hmscs during passages and evaluated the proliferation of cells and the maintenance of their properties: profile of membrane marker expression, clonogenic potential, immunosuppressive properties (inhibition of t-cell proliferation) and potential to differentiate in adipocytes and osteoblasts. we showed no impact on the content in growth factors tested (egf, bfgf, pdgf-ab, vegf and igf) and a significant decrease in tgf-b (− %, n = , p o . ). a large scale culture of hmscs during passages showed that hpl or pr-hpl at % triggered comparable hmsc proliferation than fbs at % plus bfgf (n = ). moreover, after proliferation of hmscs in hpl or pr-hpl containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties (inhibition of t-cell proliferation) were maintained, in comparison with hmscs cultured in fbs conditions. the potential to differentiate in adipogenic lineage of hmscs cultured in parallel in the conditions, evaluated using oil red o and nile red stainings and the measurement of triglyceride accumulation, remained quantitatively identical. we also showed that the potential to differentiate in osteoblasts (quantified using alizarin red s and von kossa stainings and alp activity measurement) of hmscs grown in hpl or pr-hpl was not impaired, in comparison with fbs. in conclusion, we demonstrated the feasibility to use uv-c treatment to subsequently obtain pathogen reduced hpl, while preserving its optimal quality and efficacy for hmsc expansion for cell therapy applications. although it is still not used widely in clinical practice. in this paper, we demonstrated a case of ada-scid who received hsct as an adolescent from matched unrelated donor (mudd) after termination of her peg-ada treatment due to severe intractable thrombocytopenia induced by peg-ada. patient showed good engraftment and incremental clinical improvement. her post transplantation course was complicated with multiple complications including: grade i gut gvhd as well as hemorrhagic cystitis (btk related) and ebv infection, additionally, she developed several cns complaints like headache, vomiting and dizziness which were found to be due to increased intracranial pressure with multiple enhancing cerebral lesions found on brain imaging. further investigations for the brain lesions confirmed the diagnosis of malignant diffuse large b cell lymphoma (dlbcl) involving the brain. the lymphoma was highly suggested to be originated from donor cells giving the timing relationship between transplant and establishment of the diagnosis. this lymphoma was successfully treated with full recovery and good final immune reconstitution but with lack of b cell engraftment and need for monthly ivig. we conclude that, peg-ada can rarely induce thrombocytopenia in an autoimmune manner by forming antibodies against platelets and good recovery of thrombocytopenia can be achieved after discontinuation of peg-ada. hsct can be considered as modality of treatment even in older patients with scid due to ada deficiency keeping in mind high possibility of complications including, autoimmunity and malignancy. disclosure of conflict of interest: none. ( ) ( ) ( ) ( ) . based on the pre and post-apheresis cd + cell counts, the collection efficiency of the apheresis amicus device was median . % ( - ) and of the comtec median % ( - ). in mm the apheretic collections were started on median day ( - ), while in lymphoma patients, due to chemotherapy, the day of apheresis start was ( - ). after cryopreservation and thawing, viability ( -aad, bd) was median . % ( - ). with these cell products, up to now we engrafted patients following high-dose chemotherapy ( mm autografted after mel , hl and nhl autografted after beam). engraftment was prompt and stable in all with anc . and . × /l on median day ( - ) and . ( - ), respectively, and with platelet count and × /l on median day ( - ) and . ( - ), respectively. these results are similar to those obtained by most experienced centers in europe and us, and confirm the fact that autologous transplantation may be implemented also in developing countries when appropriate technology and application of standard procedures are employed. with this experience our center is also developing allogeneic transplantation, and the initial results in thalassemia will be reported in a separate abstract. disclosure of conflict of interest: none. extracorporeal photopheresis (ecp) is a safe and effective immunoregulatory therapy for steroid-refractory graft-versushost disease (gvhd) but its mechanism of action is poorly understood. ecp is a non-immunosupressive therapy whose modulating mechanism is thought to result in an increase in t-regs in the patient and in inversion of the cd /cd ratio at the end of treatment. in this study, we evaluated the effect of ecp on t cell response in a cohort of steroid-refractory gvhd patients. from november to november , patients ( con acute gvhd and with chronic gvhd) treated with ecp in our unit, were retrospectively evaluated. patient characteristics are shown in table . we performed an ‛off-line' system ecp using a cell separator (spectra optia, teruno bct) for the cmn apheresis; after -methoxypsoralen was added, the product was photoinactivated in the ultraviolet a irradiator (uvamatic-g , macopharma). ecp procedures were performed for two consecutive days, initially weekly (agvhd), or every two weeks (cgvhd) and afterwards monthly according to clinical response. anthracycline-induced cardiotoxicity (aic) is irreversible, which has limited the use of this anthracycline in cancer chemotherapy. to explore the therapeutic effect and its possible mechanism of bone marrow derived mesenchymal stem cells (bmscs) on cardiac damage induced by anthracyclines in a rat model. study selects sd rats aged - weeks to isolate and culture bmscs, and flow cytometry was used for phenotypic identification of bmscs. female sd rats were first randomly divided into groups: the sham control, bmscs control, . mg/kg daunorubicin (dnr), dnr with bmscs, dnr with dexrazoxane (dzr), dnr with bmscs and dzr. left ventricular (lv) function before, during and after chemotherapy were assessed by echocardiography. at the end of weeks, animals were euthanized and organs were collected in % buffered formalin for histopathology using hematoxylin and eosin staining and immunohistochemical analysis was used to identify the cellular subpopulations that infiltrate the cardiac tissues. after the construction of microrna- (mir- )modified bmscs with lentiviral vector, sd rats were randomly assigned into groups: the normal control, the empty vector control, dnr, dnr with bmscs, dnr with mir- -modified bmscs. the density of new blood vessels of rats in each group was detected by immunohistochemical method. mir- , bcl- , bax and vegf mrna expressions were detected by qrt-pcr. bcl- , bax and vegf, cx , troponin t and bnp protein expressions were detected by western blotting. all procedures performed in studies involving animals were in accordance with the ethical standards of the institutional. an animal model of drug-induced cardiomyopathy was built in the dnr treated rats.lv ejection fraction (lvef) and lv fractional shortening (lvfs) were significantly decreased compared to that of the sham control (p o . ), and the signs of the myocyte injury (myocytolysis, vacuolization and disruption) in paralleled with the inflammatory infiltrates, marked by cd and hla-dr, were observed in the dnr group, while bmscs alone or synergistic with dzr facilitate the anthracycline-induced lv dysfunction returning to the baseline values and the recovery of myocarditis (p o . ). in the mir- -modified bmscs transplant group, mir- expression, cell migration and proliferation ability were higher than that in the bmscs and empty vector groups (p o . ). the cardiac regenerative capacity of bmscs following significant myocardial injury were further enhanced by mir- compared to that of the dnr group and the control groups (all po . ), revealed by the significantly higher density of new blood vessels and upregulation of vegf expressions, during which the pro-apoptotic protein bax were down-regulated and the anti-apoptotic protein bcl- function were upregulated in the mir- overexpression group compared to that with the bmscs, dnr group and the control groups (all po . ). western blotting demonstrated that the expression of c × were significantly decreased, while expressions of troponin t and bnp were significantly increased in the mir- overexpression group in contrast to that with the dnr group (all p o . ). these results showed that bmscs could reverse cardiac damage induced by anthracycline, and the cardioprotective efficacy was further enhanced by mirna- -mediated regulation of apoptosis and angiogenesis. disclosure of conflict of interest: none. effective adoptive t cell therapy against cancer is dependent on long-lived tumor-specific stem cell-like t cells with the ability to self-renew and differentiate into potent effector cells. however, current protocols for ex vivo generation of tumorspecific cd + t cells result in terminally differentiated effector t cells. it was found that minor histocompatability antigen (miha)-specific cd + t cells with an early memory-like phenotype and long-lived memory transcription profile could be expanded from naive precursors using akt-inhibitor viii . importantly, these akt-inhibited tumor-specific cd + t cells showed a superior expansion capacity and anti-tumor effect multiple myeloma bearing mice. for the clinical exploitation of ex vivo generated akt-inhibited tumor-specific cd + t cells, we tested the effect of potential clinical grade akt-inhibitors azd , gdc , gsk , gsk , mk and triciribine in polyclonal stimulations, allogeneic mixed lymphocyte reactions (mlr), and antigen-specific t cell assays. polyclonal stimulation with anti-cd /cd beads on cd naive t cells was used for a first screening of the akt-inhibitors. for all inhibitors, a dose dependent effect on the naiveassociated receptors ccr , cd l and cxcr was observed. this had limited effect on viability, activation and proliferation except for triciribine, which was therefore excluded for further assays. moreover, in the mlr, treatment of naive cd + t cells with remaining akt-inhibitors resulted in a dose dependent effect associated with higher ccr , cd l, cxcr and cd expression. furthermore, the akt-inhibited cd + t cell products showed a - fold increased expansion capacity upon restimulation in vitro. when expanding miha-specific cd + t cells from the naive repertoire in the presence of one of the akt-inhibitors, the miha-specific cd + t cells showed a more early memory phenotype compared to controls. this was displayed in higher levels of the naive-associated receptor cd l ( figure ). in addition, these miha-specific cd + t cells were shown to be functional, as antigen-specific restimultation resulted in degranulation (cd a) and ifn-γ production. based on this ifn-γ production, the akt-inhibited antigenspecific cd + t cells can be selected using the cytokine capture assay (miltenyi, for enriched infusion in patients suffering from hematological malignancies. using aktinhibition in the generation of tumor-reactive t cells results in a more early memory tumor-specific cd + t cell product. this adoptive immunotherapy product retains superior proliferation capacity upon infusion, and its potential selfrenewal capacity could result in a long-term anti-tumor effect in patients suffering from a hematological malignancy. chimeric antigen receptors (cars) are composed of an extracellular domain-derived from a tumour-reactive monoclonal antibody, linked to one or more signalling endodomains. in early clinical trials, cd car-t cells have demonstrated impressive anti-tumour activity against different b-cell malignancies, including chronic lymphocytic leukaemia, acute lymphoblastic leukaemia (all) and non-hodgkin lymphoma. conventional alpha-beta car-t cells are however hla-restricted and could cause graft-versus-host disease (gvhd) when used across major mismatches, as expected in the highly anticipated setting of off-the-shelf car-t cells from third-party donors. besides being non-hla restricted, gammadelta t cells possess intrinsic anti-tumour reactivity, making them attractive effectors for next-generation car-t cell therapies. so far, however, attempts at exploiting gammadelta t cells in patients have been largely disappointing, possibly because of sub-optimal ex vivo culture conditions. the aim of our study was to optimise the generation of gammadelta car-t cells and to test their anti-tumour potency both in vitro and in vivo. starting from peripheral blood mononuclear cells of healthy donors, we stimulated gamma-delta t cells with zoledronate and il- /il- , and transduced them with retroviral vectors encoding for cd cars carrying either cd .z or - bb.z signalling endodomains. we assessed antitumour activity in vitro by measuring killing, secondary expansion and cytokine production after co-culturing gamma-delta car-t cells with different cd + all cell lines, and in vivo in nsg previously engrafted with a b-all semi-cell line. although allogeneic hematopoietic stem cell transplantation (allosct) is a curative option to treat hematologic malignancies, disease recurrence remains a concern in the setting of high risk diseases. thus, post allosct therapeutic strategies are needed to treat and/or prevent disease progression. in this setting, donor lymphocytes infusion (dli) is an option as post allosct immunotherapy aiming to enhance graft versus leukemia (gvl) effect. although dli may induce persistent remission, graft versus host disease (gvhd) is a potential complication following dli. because of the suspected higher incidence of gvhd in the presence of hla mismatches, few series focused on dli following haploidentical stem cell transplantation (haplosct) so far. we therefore report our experience of dli following haplosct using post-transplantation cyclophosphamide (pt-cy) platform. we included in this single center study all consecutive adult patients with hematological malignancies who received dli after haplosct with pt-cy as part of gvhd prophylaxis from to (n = ). conditioning regimens were non-myeloablative (low dose tbi-based) or with reduced toxicity (various dose of busulfan according to disease and patient characteristics). ciclosporine a and mycophenolate mofetil were given as additional gvhd prophylaxis in all cases. dli were given at escalating doses, expressed as cd +cells/kg, without gvhd prophylaxis, and ranged from × our study suggests that dli following haplosct with pt-cy is feasible. gvhd is frequent but with a relatively low incidence of severe forms. no response rate was achieved in the context of hematological relapse, underlining that preemptive or prophylactic strategy might be preferred. indeed, the overall good outcome in patients receiving prophylactic dli is promising taking into account the poor prognostic of the diseases indicated for alternative donor transplantation. further prospective studies are needed in specific disease settings to assess the benefit for using such post allohsct immune-intervention. [p ] disclosure of conflict of interest: none. dual specific cytokine-induced killer cell therapy as a treatment option for life-threatening ptld-a case report of the frankfurt experience l-m pfeffermann infection with epstein-barr virus (ebv) is a frequent complication after allogeneic hematopoietic stem cell transplantation (hsct) and besides relapse remains a significant cause of morbidity. prolonged immunosuppression or delayed t-cell recovery may favor ebv reactivation after transplantation, which under these circumstances can lead to life-threatening lymphoproliferative disease (ptld). consensus is lacking on the optimal treatment of ptld. adoptive immunotherapies with both anti-tumor capacity and restored virus-specific cellular immunity may represent optimal treatment options especially when considered in the context of ptld. in this case report we applied in vitro activated t-cells namely cytokineinduced killer (cik) cells with dual specific cytotoxic capacities transferring both anti-cancer potential and donor t-cell memory against ebv infection for the treatment of ebvassociated ptld which progressed to highly proliferative large b cell lymphoma during delayed t-cell recovery after allogeneic hsct. the reported patient had received an allogeneic hsct for secondary myelodysplastic syndrome following acute myeloid leukemia, and due to delayed t-cell recovery had developed ebv-related ptld two months after transplantation. treatment with rituximab, conventional ebvspecific t-cells and wildtype cik cells failed, therefore the patient was offered ebv-specific cik cells on a compassionate use basis. ebv-specific cik cells were generated from peripheral blood mononuclear donor cells. cells were activated and expanded in the presence of ifn-γ, il- , anti-cd antibody and il- . on day and of culture an ebv peptide pool was added for additional priming. follow-up analysis included in vitro and in vivo monitoring of ebv-specific cik cells. with above mentioned protocol we were able to generate cik cells containing × cd + ebv-specific t-cells/kg body weight of the patient. infusion of ebv-specific cik cells resulted in rapid clearance of plasma ebv dna level and sustained disappearance of large (vol. cm ) ptld-malignant lymphoma. during one-year follow-up analysis we were able to detect ebv-specific cik cells (cd + and cd + ) in vivo by flow cytometry using specific mhcdextramers. facs-monitoring of the patient´s blood revealed besides cd bright t-cells also an increasing cd dim t-cell population with a remarkable percentage of t emra cells within this compartment (up to %) indicating virus-specific t-cells. no cytokine release syndrome appeared after ebv-specific cik cell treatment, but cytokine secretion patterns, analyzing serum of the patient, reflected cytotoxic and anti-virus capacity provided by this treatment. cytotoxic potential, as well as t h cell differentiation and function offered by ebvspecific cik cell treatment were further confirmed by in vitro analysis. ebv-specific cik cells revealed an . s hematologic disease. the global survival ratio in the follow-up was . % (with . %, . % and . % survivals in , and months, respectively). the variables significantly associated with greater survival were: type of gvhd (cgvhd), number of affected organs (an organ had to be moderately or severely affected to be included in this category) and steroid dependence as the main reason to initiate ecp (see figure ). there was a trend towards significance for the degree of gvhd and cutaneous involvement to be factors associated to enhanced survival ratios. extracorporeal photopheresis is a safe treatment option for patients with gvhd, generating a response and decreasing immunosuppression in an important percentage of them. the presence of cgvhd rather than agvhd, a lower severity degree of the condition, having a lower number of affected organs, skin as main affected organ and steroid dependence as the reason to start the ecp treatment were all factors associated with greater survival in our sample. disclosure of conflict of interest: none. tx) . we report the case of a patient refractory to chemotherapy treated with ibrutinib as debulking therapy before allo-tx. in june , a years old woman was diagnosed with mcl. the staging performed by whole body ct scan, colonoscopy, egds and bone marrow (bm) biopsy was conclusive for stage iva with bulky lymph node over and below the diaphragm, bm, enteric and peripheral blood (pb) localization. the planned treatment included cycles of r-chop, cycle of high dose (hd) cy, cycles of hd arac and autologous sct. after completion of hd cy, the restaging showed progressive disease, with a thyroid involvement and histologic switch in a blastoid variant. disease continued to progress even after cycles hd arac, so we tried to control the disease with r-bendamustine ( mg/mq on days - of -d cycle), but after the first cycle the neck circumference increased. we shift to lenalidomide ( mg on days - of -d cycle) without any response after two cycles. we excluded patient from autologous sct programme because of chemo-refractoriness and we searched matched unrelated donor because no hla identical sibling was available. we started a therapy with ibrutinib ( mg/die on days - of d cycle). after the first cycle we observed a rapid response with decrease of neck size and the disappearance of superficial lymphnode; we performed cycles of ibrutinib, and we reached a good partial remission with lymphnode of max cm, and a bm and pb involvement of %. meantime an unrelated donor with / hla matching was identified, so in december we performed allo-tx with reduced-intensity conditioning (thiotepa mg/kg-fludarabine mg/m -melphalan mg/m ) and cyclosporine and short term methotrexate as gvhd prophylaxis. engraftment was at day + . in the first days after allo tx she experienced a clostridium enteritis, transient cmv reactivation and acute gastrointestinal gvhd on day + with rapid response to steroid therapy. main complication happened on day , when sudden fever and stupor, progressive to coma, occurred; subsequently pneumococcal encephalitis was diagnosed, with positive csf microbiological exam and two signal alteration in the right cerebral hemisphere at mri. the patient was treated with ampicillin and ceftriaxone with a favourable outcome. the mcl revaluations performed at , and months showed complete remission with disappearance of all pathological lymph node and pb involvement. currently the patient is at year post asct, she is enrolled in a rehabilitation program and mcl is in complete remission. our experience seems to indicate that ibrutinib is safe and can be used as bridge to allo-tx therapy in refractory mcl. we will investigate side effects of this platform of therapy, and, given the early occurrence of pneumococcal infection, we will consider to perform capsulated bacteria vaccination before allo-tx. disclosure of conflict of interest: none. [p ] pt was diagnosed with c-all in and received a mud-hsct (tbi gy, cyclosphosphamide) in / due to persistend mrd. following early rel / , cycles of blina led to mrd+ cr, for which a nd hsct from a haploidentical family donor (busulfan, thiotepa, fludarabine) was performed in / . molcr lasted months and rel was treated with cycles of weekly io followed by one dli ( x - cd + cells/kg), resulting in mrd+ cr and complete donor chimerism. five weeks after the last io cycle, the pt was admitted with ascites, hyperbilirubinemia and reduced general condition. vod was suspected, but diagnostic paracentesis revealed malignant ascites demonstrating fatal progressive disease. conclusion. our data suggest that the sequential use of io and dli is feasible even for heavily pretreated patients with r/r all after hsct and can induce molecular remissions. we observed an unusual case of late onset, severe vod responding to defibrotide and one all relapse manifesting itself with ascites in our patients. we therefore suggest close monitoring of liver function tests in the setting of this therapy and extensive diagnostic work-up for any developing liver abnormalities or ascites. disclosure of conflict of interest: ng: advisory board (pfizer, amgen); research support (amgen); gb: honoraria (amgen). [p ] p while allogeneic hematopoietic stem cell transplantation from matched related and unrelated donors has become a standard of care treatment for patients with hematological malignancies, transplantations from mismatched or haploidentical family donors remain challenging. currently t-replete and t-deplete transplantation strategies are applied aiming to improve the outcome after haploidentical transplantation. despite high rates of relapse many centers regard post-transplant cyclophosphamide, a t-replete strategy, as a standard of care approach. we have developed a t-depleted transplant approach where donor lymphocytes selectively depleted of alloreactive t-cells (atir ) using th , arhodamide-like dye, are infused after cd -selected haploidentical hsct, to overcome the challenges of infectious complications, gvhd and relapse. in phase i (cr-air- ) we have demonstrated safe infusion of these lymphocytes at doses up to × viable t-cells/kg. recently, we reported a promising -year grfs was % from a phase ii trial (ash ),that is awaiting final results soon. here, we introduce a randomized, multicenter phase study (cr-air- ), where patients with acute myeloid leukemia (aml), acute lymphoblastic leukemia (all) or myelodysplastic syndrome (mds) are planned to undergo a haploidentical hsct with either a t-cell depleted graft and adjunctive treatment with atir , or with a t-cell repleted graft and use of posttransplant cyclophosphamide. inclusion and exclusion criteria are listed in table . all patients will undergo myeloablative conditioning consisting of either tbi ( gy) or melphalan/ busulfan, in combination with thiotepa and fludarabine. patients in the atir study group will receive atg ( . mg/ kg once daily for days) during conditioning and atir infusion at a dose of × viable t-cells/kg is given between and days after the hsct. patients in the ptcy group will receive cyclophosphamide ( /mg/kg) on day and (or ) with subsequent use of immune suppression up to months post-hsct. the primary endpoint of the study is gvhd-free, relapse-free survival (grfs). grfs is defined as time from randomization until grade iii/iv acute graft-versus-host disease (gvhd), chronic gvhd requiring systemic immunosuppressive treatment, disease relapse, or death, whichever occurs first. this endpoint captures both safety and efficacy. additional secondary endpoints are overall survival (os), progression-free survival (pfs), relapse-related mortality (rrm) and transplantrelated mortality (trm). patients are planned to be randomized in study centers in europe and north america. a number of - sites are planned to participate in the study. enrolment is expected to continue until mid- with initial results being available first half . results of this study will determine which transplant regimen provides most clinical benefit in haploidentical donor transplantation, with the promise of an effective regimen without the use of post-transplant immune suppression. disclosure of conflict of interest: jr is an employee of kiadis pharma. multipotent mesenchymal stromal cells (mscs) are used for prevention and treatment of graft versus host disease after allogeneic hematopoietic stem cells transplantation due to their immunomodulatory properties. mscs fate in vivo after infusion is unknown. the aim of this study was to analyze the changes in mscs and allogeneic lymphocytes properties when co-cultured in vitro to simulate their interactions in vivo. the bone marrow from donors ( male and female aged - years, median years) was used. mscs were cocultured with allogeneic lymphocytes in a ratio of about : for days and their basic properties were analyzed over time. lymphocytes were activated by adding to the culture medium mg/ml of pha (pha-lymphocytes). some mscs were treated for h with u/ml ifnγ (γmscs). determination of gene expression levels was performed by reverse transcription polymerase chain reaction in real time (modification of the taq-man) and of antigen expression on mscs and lymphocytes by flow cytometry. significant reduction in the proportion of viable cells was observed in mscs co-cultured with pha-lymphocytes. in γmsc co-cultured with pha-lymphocytes no reduction in the proportion of living cells was revealed. this indicates the sensitization of mscs by ifnγ to factors secreted by pha-lymphocytes. in mscs co-cultured with pha-lymphocytes and lymphocytes mean fluorescent signal intensity level (mfi) of cd gradually decreased. ifnγ treatment and co-cultivation with lymphocytes led to significant increase of hla-dr mfi on mscs. co-cultivation with lymphocytes increase the hla-dr mfi on mscs much stronger than ifnγ treatment. relative expression level (rel) of ido gene increased dramatically in both mscs and γmscs when co-cultured with lymphocytes. at a day in γmscs rel of ido increased fold, and then gradually declined. in mscs cocultured with lymphocytes il- rel increased almost -fold and then decreased -fold at the fourth day. the csf rel in γmscs showed twofold increases, upon incubation mscs and γmscs with lymphocytes csf rel increased fourfold and sevenfold, respectively. co-culture of msc and γmscs with lymphocytes led to decrease in the proportion of cd , cd , cd , hla-dr, and pd- positive cells (both cd and cd ) after one day, compared with pha-lymphocytes without mscs. proportion of cd +, hla-dr+ and pd- + cells also decreased after days of co-culturing with msc or γmscs (compared with pha-lymphocytes without mscs), but anyway number of activated cells increased . - folds compared with first day. number of cd + lymphocytes after days of co-culturing with mscs or γmscs did not vary significantly from control and decreased in comparison with first day. main inhibition of activated lymphocytes by co-culturing with mscs occurs during the first day of their interaction, and then the inhibition became less effective, moreover in mscs decreased the rel of the main immunomodulating factors, and most of them were eliminated. mscs treatment with ifnγ resulted in improved survival and resistance of these cells to lymphocytes action. the results indicate that the effect of mscs injected intravenously to patients is limited to several days. disclosure of conflict of interest: none. autologous adipose-derived mesenchymal stem cells (admscs) embedded in platelet-rich fibrin (prf) promote healing in different types of wounds. by avoiding the needlerelated complications, prf-embedded autologous admscs graft provides a new effective stem cell-based therapeutic strategy for wound healing. adult male (age ⩽ yo) were equally divided (n = per group) into group (prf only), and group (prf+admscs). regular dressing (without any agent) was used for both groups with a frequency of changing every days. rpf with or without admscs was patched on the wound (maximum surface area cm ). all patients were followed up until complete healing. a complete healing was noted in both groups; however, the healing in group was very slow (after weeks), compared to a quicker one in group (after weeks). control of the moisture was very well noted in group , less in group . group showed a lot of exudates on the wound; less exudate in group were noted. infections were absent. both groups had a colonized wound. signs of inflammation were very well noted in group ; no signs of inflammation in group . admscs embedded in prf offered rapid wound healing responses then prf alone. keywords: mesenchymal stem cells, platelet-rich fibrin, engineered tissue wound healing disclosure of conflict of interest: none. stem cell source p additionally cryopreserved g-csf primed pbsc can substitute the second transplantation for the patients with acute leukemia who lately relapsed after hematopoietic stem cell transplantation y lee , j moon , ih lee and s sohn department of hematology, kyungpook national university hospital; department of hematology,kyungpook national university hospital and kyungpook national university hospital although allogeneic hematopoietic cell transplantation (allo-hct) is a potentially curative therapy for acute leukemia, survival outcomes of the patients relapsed after transplantation remains poor with high early mortality and a small percentage of second remission. this study evaluated the efficacy of dli using g-csf primed pbsc additionally cryopreserved for the patients who relapsed after allo-hct. we retrospectively reviewed the medical records of the patients who received allo-hct for acute myelogenous leukemia (aml), myelodysplastic syndrome (mds), and acute lymphoblastic leukemia (all) between and in kyungpook national university hospital. among the patients who had relapsed after allo-hct, the patients received dli using the additionally harvested cells. at the time of harvest for the first hct, collecting targeted pbscs (greater than × /kg cd + cells) allowed us to cryopreserve surplus pbscs, including cd + cells with dimethylsulfoxide in a nitrogen tank. then, we analyzed the efficacy of dli for the patients who were classified into early relapse or late relapse group by the median time of relapse after transplant. the median age at transplant was . years (range - years) and male was patients ( . %). primary diseases for allo-hct were aml/mds (n = , %) and all (n = , %). one hundred forty three patients ( . %) were in cr (complete remission), ( %) in further cr, and ( . %) in relapsed and refractory status. one hundred seventy one patients ( . %) received myeloablative conditioning regimen. the median dose of cd + cell was . × /kg (range: ~ . × /kg). almost % of patients achieved the neutrophil engraftment with a median time of days (range: - days). the -year overall survival (os), relapse free survival (rfs), non-relapse mortality (nrm) and graft-versus-host disease (gvhd)-free/relapse-free survival (grfs) since hct was . ± . %, . ± . %, . ± . %, and . ± . %, respectively. there was no significant difference according to s the infused cd + cell dose (lower o × /kg vs higher ⩾ × /kg). the incidence of chronic gvhd was more frequent in higher cd + group ( . % vs . %, p = . ). median time from hct to relapse was days (range: ~ days). after relapse, patients ( . %) were treated with salvage chemotherapy, patients ( . %) with second allo-hct, and patients ( . %) with dli. the median number of cd +t cell was . × /kg (range: . ~ . × /kg). fourteen patients ( . %) achieved dli induced cr, patients progressed, and patients were not evaluable for response. dli induced acute gvhd was observed in patients ( . %) and chronic gvhd developed in patients ( . %). in late relapse group, the -year os since post-transplant relapse was significantly higher in dli group than non-dli group (figure , . ± . % and . ± . %, p = . ) but, early relapse group had no difference. the patients treated with dli showed significantly survival benefit in late relapse group (median days vs days, p = . ). the incidence of dli-induced gvhd does not differ between two groups. dli for the patients who lately relapsed after allo-hct can be a feasible and an effective option in terms of response, donor convenience and it's cost. in the late relapse group, g-csf primed dli may replace second transplantation. disclosure of conflict of interest: none. cord blood transplantation- years of experience c alves , f amado , f bordalo , s ferreira , s lopes , c pinho , t rodrigues , l antunes and s roncon serviço de imuno-hemoterapia; serviço de terapia celular and registo oncológico do norte, instituto português de oncologia do porto francisco gentil, epe allogeneic haematopoietic stem cell transplantation (hsct) is a well-established treatment for patients with malignant and non-malignant haematological disorders. cord blood transplantation (cbt) has extended the availability of hsct to patients that would not otherwise be eligible for this curative approach because of the lack of human leucocyte antigen (hla) identical donor. the aim of this retrospective study was to analyse and characterize years of cbt activity in our institution ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . we examined patient electronic files and created a database in excel to register cord blood unit (cbu) parameters and patient characteristics. after thawing, cbu was washed/diluted with validated procedures; the cellular content was evaluated by immunophenotyping and followed ishage recommendations; sterility was assessed by bacterial/fungal cultures, viability by trypan blue exclusion assay and functionality by clonogenic capacity. the transfusion of blood products after transplant was quantified and the hematological recovery (hr) established using cibmtr criteria. correlation between continuous variables was assessed with spearman coefficient. overall survival (os) was determined according to cellular content and hla-disparity by the kaplan-meier estimator. survival between groups was compared using the log-rank test. a total of cbu were administered to patients (table ) : / female, the main diagnosis was acute leukaemia ( / ). a sibling cbu was used for patients; the unrelated were imported from europe ( %), usa ( %) and oceania ( %). hla-matching was / , / , / and / for , , and patients, respectively; % were abo-identical. after thawing, % were washed and presented no microbial growth. the majority of patients were submitted to a bussulfan-based myeloablative conditioning regimen; graft versus host disease prophylaxis was performed with a calmodulin inhibitor+mycophenolate mofetil. complete and mixed chimerism was verified in % and % of patients; % had graft failure; the rest were unknown results ( %). at the moment, we reported patients alive ( in complete remission, with evidence of disease relapse) and dead at a median of ( . - ) months after cbt; the most frequent cause ( %) was recurrence of the initial clinical condition. the correlation between nucleated cells (nc) and cd + cells per kg/hr (p = . ; p = . ) and number of cd + cells per kg/os (p = . ) was not statistically significant. however, the engraftment and os was associated with hla-mismatch (p = . ; p = . ) and os was related to nc per kg (p = . ). our clinical results suggest that despite increased hla disparity, ucb offers promising results. ucbt is feasible in patients when the unit contains a high number of cells. there are several strategies for the future, related to cbu expansion and homing techniques, nurturing procedures, selection of optimal cbu unit and enhancement of immune recovery, in order to improve the application of cbu. s received sirolimus and mmf. median time to neutrophil and platelet engraftment was days ( - ) and days ( - ), respectively. one patient died from parainfluenza pneumonia (d ), one patient from ptld (d ), one patient from late pulmonary vod (d ), and one patients from relapse (d ). with a median follow up for survivors of months, one year survival is %. three patients had grade - gvhd and none of the survivors have chronic gvhd. though unrelated donor chimerism was dominant early after transplant and contributed to early count recovery, definitive engraftment was dominated by ucb chimerism in all but one patient. conclusion: among older adult patients with hematological malignancies,~ % lack haplo-identical relatives. for these patients, double or single ucb transplant is challenging because of delayed engraftment. cd selected partially matched grafts from unrelated donors hasten hematopoietic recovery and are over time outcompeted by ucb grafts which provide robust hematopoiesis with low risk of chronic gvhd. the combination of mismatched unrelated hematopoietic progenitors and ucb grafts provides an attractive alternative for older patients lacking hla-idental donors or haploidentical relatives. in planned trials, mismatched donors may be selected based on kir type to further enhance gvl effects. disclosure of conflict of interest: partially supported by miltenyi biotec. haploidentical stem cell transplantation (haplo-sct) is an attractive option for patients who do not have an hlamatched donor. historically it has been associated with high rates of graft rejection, relapse and low incidence of graft versus host disease (gvhd). to decrease these issues we have considered the use of primed bone marrow as stem cell source, early withdrawal of immunosuppressive therapy and the use of donor lymphocytes infusions (dli) in haplo-sct with high-dose post-transplantation cyclophosphamide (ptcy) as main gvhd prophylaxis. to analyse our incidence of acute and chronic gvhd and overall survival (os) in patients with haplo-sct with short course of inmunosupressive therapy. we retrospectively analyzed a cohort of patients who underwent haplo-sct with primed bone marrow as stem cell source, between years and in our centre. gvhd prophylaxis consisted in ptcy ( mgr/kg on days + and + ) and tacrolimus plus mycophenolate (mmf) from day + as recommended by baltimore group. mmf was stopped on day + . tacrolimus was tapered off from day + with withdrawal on day + in patients without gvhd or with active disease. dli were considered if mixed chimerism, relapse or disease progression appeared. the characteristics of the patients are shown in table . results: there was no primary graft failure. eight of patients ( . %) developed agvhd (grade ii-iv) and it was severe (grade iii-iv) in patients ( . %). cutaneous agvhd location was the most common presentation ( patients ( %)) and it was associated with intestinal gvhd in patients. twenty two patients were evaluable for cgvhd. thirteen patients ( %) developed chronic gvhd that was mild, moderate and severe in: ( . %), ( . %) and ( %) patients, respectively. the median time of onset cgvhd was months (range: - ) and it was related with previous withdrawal of the immunosuppression in ( . %) patients, tapered off immunosuppression in ( . %) patients and dli in ( %) patients. systemic treatment was required in / patients but only patients were treated with high doses of steroids ( mg/kg/day). the median days of is therapy in patients who developed gvhd was days (range: - ). dli were used in ( %) patients because of: relapse/disease progression in ( . %) and secondary graft failure in ( . %). two patients achieved complete remission and patients developed cghvd. the median number of dli per patients were ( - ) with a median cd +cell of × /kg (range: × - × /kg). with a median follow-up of months (range: - ), the estimated os at and years after haplo-sct were % and %, respectively. at the moment of this study patients ( . %) were alive, patients in complete remission, in partial response and in progression. eleven ( . %) patients died due to: disease ( ), infections ( ), pleuropericarditis ( ), hepatic veno-occlusive disease ( ) and refractory gvhd. five patients ( . %) are without is therapy and without gvhd symptoms. in our experience, early withdrawal of immunosuppression following haplo-sct with primed bone marrow and posttransplantation cyclophosphamide facilitates the development of chronic gvhd and can decrease the relapses in patients with high-risk hematological malignancies. it is necessary more follow up and more studies to confirm this preliminary results. [p ] disclosure of conflict of interest: none. s myeloid malignancy (n = except patient with saa) received fludarabine (flu)/busulfan ± tbi gy, while lymphoid malignancies (n = ) received flu/tbi gy or cy/ tbi gy. all patients received g-csf-mobilized t-cell replete pbsc from a haplo donor. gvhd prophylaxis was ptcy mg/ kg on day + /+ , tacrolimus (d+ to + ), and mycophenolate (d+ to ). the median duration of follow up of surviving patients is months. median age was ( - ) years, patients ( %) were male, ( %) were african american, and patients ( %) had comorbidity index (hct-ci) ⩾ . all patients had hematological malignancy (except patient with saa) including patients ( %) not in cr. disease risk index was high/very high in ( %) and intermediate in ( %). on the day of transplant, patients ( %) did not receive steroid premedication ( = no-steroid group), while patients ( %) received mg of methylprednisolone minutes prior to pbsc product infusion ( = yes-steroid group). all the following outcomes are described in the ‛no-steroid' vs ‛yessteroid' group, respectively. cumulative rate (ci) of anc engraftment (⩾ /μl) on day + was % ( % ci - %) and % ( % ci - ) (p = . ). ci of platelet engraftment (⩾ /μl) on day + was % ( % ci - %) and % ( % ci - %) (p = . ). primary engraftment failure was observed in patients; in yes-steroid and in no-steroid. no primary engraftment failure was observed with myeloablative tbi ( - gy) (n = ). ci of agvhd gii-iv (day+ ) was % ( % ci: - %) and % ( % ci: - %) (p = . ). ci cgvhd ( year) was % ( % ci - %) and % ( % ci - %) (p = . ). ci of relapse ( year) was % ( % ci - %) and % ( % ci - %) (p = . ). ci of non-relapse mortality (nrm) ( year) was % ( % ci - %) and % ( % ci - %) (p = . ). post-infusion noninfectious fever (d to + ) was observed in / ( %) and / patients ( %). median tmax was f and f (p = . ). only patient in the no-steroid group developed life-threatening cytokine release syndrome and survived. no difference of viral reactivation was noted between groups. cmv reactivation occurred in ( %) and patients ( %), bk reactivation in % (in both groups), hhv in % and %, ebv in % and %. the -month overall survival was % ( % ci - %) and % ( % ci - %) (p = . ). the -month disease-free survival was % ( % ci - %) and % ( - %) (p = . ). t-cell replete haplo pbsc transplant is effective therapy for patients with high-risk hematological malignancies. high-dose steroid premedication with pbsc infusion neither influences transplant outcome nor prevents post-infusion febrile reaction. disclosure of conflict of interest: as discloses grant support (american porphyria foundation), consultation (medpace inc), research support (astellas and fate therapeutics), honoraria (alxion, and spectrum), and royalty for licensing of intellectual property (incysus biomedical). intrabone transplant of unwashed cb in hematological malignancies: engraftment and safety f giglio , s marktel , r greco , m morelli , mt lupo-stanghellini , e xue , l lazzari , m marcatti , m zambelli , c parisi , r milani , s piemontese , a assanelli , c corti , m bernardi , f ciceri and j peccatori unit of hematology and bone marrow transplantation, irccs san raffaele scientific institute, milano, italy and immunohematology and transfusion medicine unit, irccs san raffaele scientific institute, milano, italy cord blood transplant (cbt) in adult patients (pts) is limited by the risk of graft failure or delayed engraftment due to low cell counts. to improve the capacity and speed to engraft, intrabone (ib) cbt technique has been investigated, showing high rate of engraftment and low acute gvhd, also when compared with double cb transplant. cb units washing procedure has been suggested to remove dmso toxic potential effect. we report our experience in adult pts with hematological malignancies receiving ib unwashed cb in an attempt to reduce the loss of progenitor cells and the risks associated with cell-washing procedure. between and we performed allogeneic hematopoietic stem cell transplant (hsct) using unwashed cbu as a source and infusing them ib. all pts were adult and suffering from hematological malignancies. this population was characterized by very high-risk and advanced phase disease. all pts received a cb hsct because of unavailability of sibling or matched unrelated donors. eleven pts received a treosulfanbased myeloablative conditioning regimen and a sirolimusbased ghvd prophylaxis; four pts received busulfan-based myeloablative conditioning and a cyclosporine-based ghvd prophylaxis. cb units were thawed and diluted with albumindextran solution immediately before the transplant. this ‛nowash' dilution was implemented to reduce product manipulation that may results in cell loss. furthermore, graft manipulation risks potential contamination, requires increased technologist time, and delays time to infusion. the ib infusion was performed under local anesthesia and with short conscious sedation, at bedsite in the bmt ward. the infusion was preformed monolaterally or bilaterally according to the volume to be infused. starting from a % dmso concentration in the cb units before the dilution, the graft products contained a median of . % dmso (range: . - . ) at ib-hsct. the median cd + cells infused were . × /kg b.w. (range: . - . ). the median mono-nucleated cells were . × /kg b.w. (range: . - . ). the median cd + t-cells were . × /kg b.w. (range: - . ). the median infused volume was ml (range: - ). no procedure-related adverse events were observed, nor related to the ib technique, neither to the sedation. of the transplanted pts, were evaluable for engraftment ( patient rejected the graft and patients died before day + because of severe infections). all achieved anc . × /l after a median of days (range: - ) and achieved plt × /l at a median of days (range: - ). three patients developed grade iii-iv acute gvhd grade. according to extreme heterogeneity of the population no correlations with relapse incidence and diseasefree survival could be evaluated. ib infusion of unwashed cb is feasible, safe, easy to perform. no adverse events related to the procedure were documented. no dmso toxicity was documented. engraftment was obtained in all evaluable pts. our data confirm that direct ib cbt overcomes the problem of graft failure even when low numbers of cb cells were transplanted, thus leading to the possibility of using of this technique in a large number of adult pts, for whom this approach represents the sole possibility of long-term survival. the ‛no wash' cb dilution can also help the implementation of ib transplant thanks to the easier graft manipulation. [p ] disclosure of conflict of interest: none. lower incidence of cgvhd after cord blood transplantation for hematological malignancies in comparison with hematopoietic stem cell transplantation from other donors: years' experience in a single institute m yoshino, m obiki, m osakie, s ikeno, t sato, m nasashima, y kagaya, n kawashima, t morishita, y ozawa and k miyamura department of hematology, japanese red cross nagoya first hospital the outcome of cord blood transplantation (cbt) for hematologic malignancy was investigated. however the incidence of gvhd is not accurately known. the goal of this study was to compare the outcome of cbt with allogenic hematopoietic stem cell transplantation (allo-hsct) from other sources, mainly unrelated bone marrow (urbm). patients' characteristics: patients who underwent allo-hsct, between and in our hospital were retrospectively analyzed. donor sources were cord blood cell (n = ), urbm (n = ), hla matched sibling bone marrow (sibling bm) (n = ), and hla matched sibling peripheral blood stem cell (sibling pbsc) (n = ). in cbt, the median age was . ( - ), and the diagnosis included aml ( ), all ( ), mds ( ), cml ( ) and other ( ). the disease risk was good in and poor in . disease risk was slightly higher in comparison with other sources. prophylaxis of acute gvhd was tacrolimus, short-term methotrexate ( ), cyclosporine, short-term methotrexate ( ) and others ( ) . the -year overall survival (os) rate after cbt ( cbt ( - . %, engraftment failure . %, acute gvhd . %), relapse . % and other . %. in cbt cases, engraftment failure after allo-sct was observed in cases ( . %) which is higher than that among urbmt ( . %), out of . cbt cases underwent the second allo-hsct and patients achieved engraftment and patients were alive at days after allo-hsct. of them survived at years after allo-hsct. our results suggest that the outcome of cbt has improved in recent years. moreover, cbt has an advantage in the least cumulative incidence of acute/chronic gvhd. cbt may well create the best outcome in the future. disclosure of conflict of interest: none. chronic active epstein-barr virus (ebv) infection is a major type of ebv-associated t/nk-cell lymphoproliferative disorders (lpd) in childhood. however, young adults rarely develop chronic active ebv infection (caebv), and shows more aggressive features than that of childhood. umbilical cord blood transplant (ucbt) is a possible treatment option for caebv patients who have no hla-matched donor, but there is little information available about the efficacy and safety of ucbt for adult patient with caebv. we analyzed six adult patients with caebv who underwent a single-unit ucbt between and at our institute (including a case reported in [ ] ). the diagnosis of caebv was made according to the criteria proposed in [ ] ; persistent infectious mononucleosis (im)-like symptom and detection of increased ebv genomes in peripheral blood mononuclear cells (pbmc). ebv-dna load was measured using real-time quantitative pcr. median patient age at diagnosis was ( - ) years. target cells of ebv-infection were cd +t cells (n = ) or nk cells (n = ). median ebv-dna load was . × copies per microgram of dna in pbmc (range: . × - . × ) at the diagnosis. all patients were given prednisolone and cyclosporine, and then etoposide (n = ) or combination chemotherapy (n = ) before transplant. ebv load has slightly decreased to a median of . × copies (range: . × - . × ), but disease status was active at ucbt in all. median time from the diagnosis to ucbt was days (range: - ). one patient received total body irradiation (tbi) gy + cyclophosphamide (cy) mg/kg + cytosine arabinoside g/m , and the other five patients received fludarabine (flu) + melphalan (lpam) - mg/m or cy mg/kg with tbi gy before ucbt. umbilical cord blood (ucb) was / hla-matched to the recipients. median number of infused ucb cd + cells was . × /kg (range: . - . × ). gvhd prophylaxis was consisted of tacrolimus + methotrexate or mycophenolate mofetil. neutrophil engraftment and complete donor chimerism were achieved in four patients, but two of them developed secondary graft failure (gf) early after engraftment. the other two patients developed primary gf. second ucbt was successfully performed in the patients with gf a median of . days (range: - ) after the first ucbt. ebv genomes in pbmc became undetectable immediately after ucbt. at a median follow-up of days (range: - ), ebv-dna was undetectable or very low, and im-like symptoms were resolved in all cases. however, at - months after ucbt, two patients developed ebv+ b-cell lpd derived from donor cells, that was successfully treated with rituximab therapy. this study suggested that ucbt could eradicate ebv-infected cd + t cell-or nk cell-clones. ucbt can be a treatment option for adults with caebv. rituximab monotherapy was effective for post-transplant lpd from donor b cells. however, a high incidence of gf was observed in patients receiving reduced-s intensity conditioning of flu/lpam or cy /low-dose tbi. further studies are needed to find more optimal regimens for stable engraftment of ucb in adult patients with caebv. there is an increased incidence of ab incompatibility- - %, in allogeneic hematopoietic stem cell transplantation (allohsct) in patients who are russian citizens as a result of the variability of genetic polymorphism in the multi-ethnic population and a significant number of unrelated donors from international bone marrow registries. ab incompatibility in different types of allohsct may be an additional aggravating factor for the development of immunological complications and decrease effectiveness of treatment, but the data is still controversial [ ] . from may to december in raisa gorbacheva memorial institute for children oncology, hematology and transplantation patients with leukemia, malignancies and hereditary diseases were included to the study, who were performed hsct: allogeneic unrelated - ( %); allogeneic related- ( . %); haploidentical - ( . %); umbilical cord blood in patients ( . %). age was - , median- years. patients were predominantly with acute myeloid leukemia- % (n = ), acute lymphoblastic leukemia- % (n = ) and chronic myeloid leukemia - % (n = ). results: in . % of cases (n = ) АВ incompatibility was determined: major- . % (n = ); minor- . % (n = ); combined- . % (n = ). АВ incompatibility in allohsct did not influence overall survival (p = . ) and frequency of acute graft versus host disease (gvhd) (p = . ). also there was no difference in overall survival depending on combination of condition regimen and ab incompatibility: reduced intensity (ric) or myeloablative (mac) (p = . ). an increased frequency of acute gvhd was observed in ric and АВ incompatibility ( . %) compared to mac ( . %, p = . ). ab incompatibility was not a major factor (log worth . ) which influenced the fact and speed of donor's transplant engraftment in comparison to level of hlacompatibility ( . ), hematopoietic stem cell source ( . ) and type of hsct. but the presence of major ab incompatibility increase the period of erythroid recovery (p = . ) as reflected in the higher amount of blood transfusions. complications caused by ab incompatibility were identified in . % of all cases (n = ) including acute and delayed hemolysis, partial red cell aplasia and immune thrombocytopenia. conclusion. the presence of АВ incompatibility is not a limiting factor to perform allohsct, however, it demands high quality prophylaxis and accurate transfusion therapy depending on ab incompatibility type to prevent immune complications. keywords: allogeneic hsct, ab -incompatibility. poor graft function or graft failure have become common indications for infusion of immune-selected cd + cells (‛boost') or second unprocessed allo-hsct, creating the need for remobilization of the same related or unrelated. we retrospectively compared the results of two consecutive cycles of rh-g-csf treatment and peripheral blood progenitor cell collections in related donors cared for at our institution between and . mobilization consisted of the administration of rh-g-csf at a dose of μg/kg per day injected in the evening, and apheresis was started in the morning of the fifth day after the fourth dose of rh-g-csf. collection was performed with a spectra or spectra optia cell separator (terumo bct). eleven out of were haplo-mismatched donors and were hla matched donors. four donors were re-collected because of recipient graft failure and because of poor graft function; in the latter situation, immunomagnetic selection of cd + cells was performed on the collected cell product prior to infusion into the recipient, using the clinimacs medical device, as previously published. median donor age was years (range: - ) at time of first donation, median weight kg ( - ) and bmi ( - ). median delay between mobilizations and was days ( - ). interestingly, the median delay between collections was days ( - ) in the haplomismatched setting and ( - ) in the matched setting. median number of circulating cd + cells/μl after the first injections of rhg-csf was vs at the first and second mobilization cycles (po . , table ). seven out of donors ( %) requested more than one apheresis session to obtain the target number of collected cd + cells during the first cycle, as compared to out of ( %) for the second cycle: this is largely due to the higher target of cd + cells for the second collection, expecting that the median cd recovery after immunomagnetic selection is % in our experience. our study shows that a second cycle of mobilized peripheral blood progenitor cell collection from related donors is associated with a significant reduction in response to hematopoietic growth factors and mobilization capacity. this information allows planning the number of aphereses at the second cycle-and subsequently the number of immunoselection procedures to be carried out-taking into account the higher cd + cell dose target needed for subsequent immunomagnetic selection. cmv reactivation remains one of the main complications after allogeneic stem cell transplantation (hsct), requiring antiviral therapy, causing myelosuppression, prolonged hospitalization, higher treatment costs and mortality. cmv seronegative donors are recommended for cmv seronegative recipients. however data about donor selection for cmv positive (cmv-pos) recipients is not conclusive. some studies showed that selecting cmv-pos donors for cmv-pos patients might be beneficial. cmv-seropositivity is very high in lithuania among healthy blood and bone marrow donors ( %) and even higher among hsct recipients (up to %), so donor selection for cmv-pos recipients is an object of interest. retrospective analysis of cmv reactivations in cmv-pos allogeneic hsct recipients (transplanted during - year in vilnius university hospital) who survived at least months post hsct was performed. data about cmv reactivation frequency, time post hsct, duration, maximal cmv dna copy number at each reactivation collected. cmv reactivation was considered when cmv dna copies detected /ml in patient's blood. statistical analysis conducted using sas . ; student's tests for statistical significance; kaplan-meier methods for overall survival. among allogeneic hsct recipients ( . %) were cmv-pos. cmv-pos allo-hsct recipients were further analysed. of them received graft from cmv-pos (pos/pos group) and -from cmv-seronegative donors (pos/neg group). more patients in pos/neg group experienced cmv reactivation in first months post hsct in comparison to pos/pos group ( . % vs . %, p o . ). pos/neg group patients had more cmv reactivations ( . vs times in months post transplant period, po . ), reactivations were diagnosed earlier post transplant ( . vs days post hsct, p o . ), had longer duration ( . vs . days, po . ) and larger maximal cmv dna copy number ( , vs . copy/ml, p o . ) in comparison to pos/pos group patients. pos/pos group patients showed tendency for better survival than pos/neg group patients, however did not reach statistical significance. in a univariate analysis only hla mis-match and donor cmv seronegativity were factors statistically significantly associated with cmv reactivation. donor cmv serostatus is significant factor selecting donor for allo-hsct recipients. according to our findings, selecting cmv-pos donor for cmv-pos recipient may reduce cmv reactivation frequency and duration. disclosure of conflict of interest: none. selection of the best hsc donor when a matched donor is not available is still a matter of debate, and reports in pediatric population are scarce. this is a retrospective study conducted by brazilian society of hsct (sbtmo), including centers, aimed to compare matched unrelated (matched-urd), mismatched unrelated (mm-urd) and unrelated cord blood (ucb) hsct. all or aml/mds patients o y/o who have received first unrelated hsct between - were included. hla -digit typing was available for urd; for ucb, hla class-i -digit typing. overall survival (os), and cumulative incidence (ci) of agvhd, cgvhd, nrm and relapse were analyzed. on an unplanned analysis, we fitted a lognormal bayesian survival model with random effects, imputing the probabilities of ucb matching at loci. a total of patients were included ( matched-urd, mm-urd and ucb). median age was . y/o. most patients had all ( %). proportion of early disease in matched-urd was higher ( %, against and %). matched-urd were / -digit hla matched (except one, for whom dq was not available), while most of mm-urd ( %) were / hla matched. ucb were loci ( %) or -loci typed ( %). based on previous report on extending -loci hla typing to -loci ucb, we estimated that ucb were - mismatched at loci, had mismatches and had or more mismatches. conditioning regimens were mainly myeloablative, tbi-( %) or bu-( %) based. grafts were in vivo t-cell depleted in % of the patients, not balanced between groups (p = . ). with median follow-up of . years, y os was %, % and % for matched-urd, mm-urd and ucb, respectively (p = . , log rank test). for matched-urd, mm-urd and ucb, ci of grades iii-iv agvhd at months were %, % and % (p = ns); moderate/severe y-cgvhd, %, % and % (po . ); y-relapse, %, % and % (p = ns); and y-nrm, %, % and % (p = . ). we found out that primary graft failure occurred in ( %) of ucb, compared to % in mm-urd and % in matched-urd. when ucb matching probabilities at loci were imputed and analyzed in a bayesian model (controlled for age, gender, disease status and diagnosis, and t-cell depletion), survival was inferior in ucb with + mismatches ( . -times lower median survival, ci . - . ), but not with up to mismatches ( . -time higher median survival, ci . - . ). of note, in vivo t-cell depletion marginally impaired survival ( . -times decrease, ci . - . ). discussion: in our population, overall survival achieved with mm-urd was not different to / matched-urd, despite higher incidence of moderate/ severe cgvhd. on the other hand, survival with ucb was significantly lower. recent report have shown excellent os with ucb compared to / hla-matched urd. ucb cohort inferior results may have been due to hla disparity degree, since survival in ucb with up to mismatches (out of ) was not worse. one limitation of our study is that tnc and cd from ucb units were not available, impairing primary graft failure analysis. we have also found that in vivo t-cell depletion might have a detrimental effect on survival and should be studied further in prospective trials. in conclusion, mm-urd, especially hla / , is a suitable option when a fully hla / matched-urd is not available. ucb matched at least / may also be a good option. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is a proven treatment for patients with high risk or relapsed hematological malignancy. the probability of having a hla matched family donor is about %. in populations with high consanguinity rates, the probability of having non-sibling matched family donor(mfd) is much higher. to explore the impact of msd vs non-sibling mfd on outcome of hsct recipients, we undertook a single center retrospective analysis of pediatric patients transplanted with the diagnosis of hematological malignancy at our center in the last five years. a retrospective cohort from to current included pediatric patients with hematological malignancies transplanted from family donors, of which were from msd and from non-sibling mfd. hla matched family donors were identified by high resolution allelic typing and were matched of hla loci. diseases were all (n = ), aml (n = ), mds (n = ), jmml (n = ), kml (n = ), nhl (n = ) and hodgkin's disease (n = ). conditioning regimens were tbi or busulphan-based myeloablative in all patients. the median age of the patients was . years (range: month- . years). although peripheral stem cell seemed to be used more commonly in non-sibling mfd recipients ( % vs %), the difference was not statistically significant. the median follow up time for alive patients months ( - months) . two year overall survival and leukemia free survival did not differ between patients with transplantations from msd or nonsibling mfd ( % ± . vs ± . , p = . ) similarly, leukemia free survival was not different between msd and non-sibling mfd transplants ( . % ± . vs . % ± . , respectively). the incidences of grade ii-iv acute gvhd in msd and nonsibling mfd transplants were % and % , respectively. the incidences of relapses were % in msd transplants and % in mfd transplants and the difference was not significant (p = . ). these data show that the results of hsct from nonsibling mfd is comparable to hsct of msd in children with hematological malignancy. our data emphasize the need for extended high resolution family typing for patients in regions where there is high rate of consanguinity. disclosure of conflict of interest: none. [p ] p donor-recipient rh incompatibility is a risk factor for mortality after pediatric matched related allogeneic hematopoietic stem cell transplantation k ghanem , n hariss , z merabi, n kreidieh , n tarek, r saab , s muwakkit , h el-solh and m abboud american university of beirut, department of pediatrics and adolescent medicine and american university of beirut, optimal donor selection is critical to achieve the best outcome after allogeneic hematopoietic stem cell transplantation (allo-hsct). there is no consensus regarding the effect of donor-recipient rh incompatibility on survival after matchedrelated donor (mrd) allo-hsct in children and adolescents. this abstract aims to study this effect in a single-institution cohort over a period of years. this is a retrospective chart review for all patients aged o years who underwent allo-hsct at the american university of beirut medical center between august and june . a total of patients with a median age of years (range: . - years) underwent allo-hsct from mrd for the following diseases: leukemia (n = ), bone marrow failure (n = ), thalassemia (n = ), scid (n = ), metabolic diseases (n = ) and lymphoma (n = ). the stem cell source was bone marrow for patients ( %) and mobilized peripheral blood stem cells for patients ( %). the grafts contained a median of . × /kg total cd cells. tbi was used in patients ( %). all but patients achieved sustained neutrophil and platelet engraftment. after a median follow-up of months (range: - months), the -year overall survival rate was % ( % ci: - %). by multivariate analysis using cox proportional hazard regression model looking at the following factors for overall mortality: diagnosis, recipient's age, donor's age, the use of tbi, stem cell source, cd count, donor-recipient abo incompatibility, donorrecipient rh incompatibility, and donor-recipient sex-mismatch, the only statistically significant risk factor for mortality was donor-recipient rh incompatibility (hr: . , p: . ). this risk was not statistically significant when looking at transplantrelated mortality (hr: . , p: ) and relapse-related mortality for malignant diseases (hr: , p: . ). there was no association between the incidence of acute or chronic gvhd and rh incompatibility. donor-recipient rh incompatibility was associated with an increased risk of mortality in children and adolescents undergoing mrd allo-hsct. further studies with larger number of patients are needed to confirm this finding. disclosure of conflict of interest: none. effect of iron or vitamin b deficiencies on in vitro colony forming capacity of peripheral blood-derived hematopoietic stem cells in children ny Özbek, mm zabun, y köksal and m Özgüner iron deficiency (id), id anemia (ida)and vitamin b deficiency (vit-b d) are common disorders in developing countries. in urgent situations, children with these disorders could be donors before treatment. in this study, we investigated capacity of peripheral blood-derived hematopoietic stem cells to develop colony-forming units (cfu) in children with id and vit-b , in vitro. patients and methods: we included children (age months- years) in the study in groups: children with id (n = ); children with ida (n = ); children with vit-b d (n = ); children with both id and vit-b d (i/ vit-b d; n = ); and control children (n = ) who has normal peripheral blood findings, and normal ferritin and vit-b levels. from each child complete blood counts (cbc), and levels of ferritin, vit-b , and cero-reactive protein (crp) have been obtained. who criteria, adjusted for age and sex, have been used for definition of anemia, id, ida and vit-b d. four ml peripheral blood drawn into tubes with edta has been used for cfu analysis. mononuclear cell suspension ( . × cell/ml), obtained from peripheral blood by ficoll-hypaque density gradient separation method, has been cultured in dishes containing semi-solid agar culture medium (methocult, h classic, stem cell technologies, canada) in appropriate conditions. after weeks, number of cfu colonies [burst forming erythroid (bfu-e); colony forming unitgranulocyte macrophage (cfu-gm); colony forming unitgranulocyte-erythrocyte-monocyte-megakaryocyte (cfu-gemm)] have been investigated by an inverted microscope. results: statistical analysis showed no difference between groups for age, sex, crp levels, and cfu-e, cfu-gm and cfu-gemm numbers. however, expected differences between groups were present concerning mean values of hemoglobin, ferritin and vit-b levels, mean corpuscular volume (mcv), and red cell distribution width (rdw) ( table ) . discussion: this study shows in vitro proliferation capacity of peripheral stem cells has not been influenced by id, ida, vit-b d, or i/vit-b d. our results may indicate normal grafting ability of peripheral stem cells obtained from donors with iron, vit-b or i/vit-b deficiencies for hematopoietic stem cell transplantation. however, in vivo analysis should also be performed in order to reach a definite conclusion. [p ] disclosure of conflict of interest: none. efficiency of day compared to day stem cell mobilization in allogeneic donors h al-gaithi , s al-mamar , m al-huneini , d dennison , s al-kindi , k al-farsi and m al-khabori hematology residency training program, oman medical specialty board; hematology department, sultan qaboos university hospital granulocyte colony stimulating factor (g-csf) given for - days is commonly used for mobilization of allogeneic stem cell donors. the optimal days of g-csf administration is still debatable. the primary objective of this study is to compare the yield of stem cell mobilization, assessed using cd + cell count, between day and day . secondary objectives include the assessment of the impact of donor's age, weight, mean corpuscular volume and blood group on the difference in the cd + cell count. in this retrospective study we included all allogeneic stem cell donors mobilized with g-csf for days from january till october in the bone marrow transplantation unit at sultan qaboos university hospital. of donor records reviewed, were with available data and selected for the study. descriptive and analytical statistics were performed using stata . . we included donors with median age and weight of years and kg, respectively. the median day wbc and cd + cell count were . × /l and × /l respectively; while the median day wbc and cd + cell count were . × /l and × /l, respectively, (figure) with a statistically significant difference from day (p o . ). in the multivariable model, there were no significant impact of donor's age (p = . ), weight (p = . ), height (p = . ) and mean corpuscular volume (p = . ) on the difference in cd + cell yield. however, donor's blood group ab predicated a significantly higher difference (p = . ). six days of g-csf mobilization achieves higher cd + cell count than days in allogeneic stem cell donors especially in donors with blood group ab. however, cd + cell count on day is high enough to allow for successful mobilization. appropriately designed prospective trial is needed to confirm these results. disclosure of conflict of interest: none. there are known differences between individuals on an unrelated hsc donor register who decide to proceed with verification typing (vt) vs those who choose not to. in the anthony nolan registry, white british donors are more than twice as likely as other ethnic groups to continue with testing at vt (or . ; po . (unpublished data)). the purpose of this study was to explore differences in key characteristics between white british donors and british donors from other ethnic groups with a view to developing interventions to reduce vt stage attrition. study recruitment occurred april -may . all donors not proceeding at vt were invited to participate, and a stratified random sample of those proceeding at vt were recruited to meet pre-determined targets for each ethnic group. data were collected via structured interview (telephone or online). broad categories of participant characteristics were assessed: demographic, culturally related, psychosocial, and donation-related. measures were previously validated scales with established psychometric properties either created for, or used in other donation-related settings. for analyses donors were divided into two groups based on ethnicity: white british (wb), and non-white british (nwb). results: wb donors and nwb donors completed interviews donors proceeding at vt were more likely than their counterparts to participate in the study ( % vs %, p o . ). mean donor age was . with no difference between ethnic groups and % of donors in both groups were female. nwb were statistically more likely to have completed higher education, and have a stronger religious affiliation. in contrast they were less likely to be blood or organ donors. nwb also described greater mistrust of the medical system and of hsc allocation. nwb donors were more likely to have joined the register at a recruitment event (p= . ) or a place of worship (p= . ), while wb donors were more likely to have joined online (p= . ). wb donors reported significantly higher scores regarding feeling well informed about donation both at the point of joining, and at the point of vt and were more likely to remember joining the register and the two donation methods. this study highlights important differences in demographics, culturally related variables and donor interaction with the register between white british donors and donors from other ethnic backgrounds. given the higher rate of vt attrition in nwb donors, these findings could be used to tailor interactions/information given to donors on the register to ensure their priorities are addressed. disclosure of conflict of interest: none. data on mismatched family donor transplants for myelofibrosis are scarce due to the risk of poor engraftment, gvhd and exclusion from trials. outcomes from such transplants performed between and reported to the ebmt are presented. sixty-nine patients, median age ( - ) years; ( %) male, ( %) had primary, ( %) had secondary myelofibrosis ( from et, from prv and others) and unknown ( %). jak v f was mutated in / . karnofsky performance status was % in %; median time from diagnosis to allograft was . (range: . - ) months. the donors were predominantly male ( %), median age ( - ) years, hla mismatched at locus in ( %) and or more loci in ( %). donor-recipient serology was cmv − / − in ( %) ± in ( %), − /+ in ( %) and +/+ in ( %) missing ( %) . bone marrow was used in ( %) and peripheral blood in ( %). the median total nucleated cell count (tnc) was . × /kg (range: . - × /kg) (n = ). the median cd + cell dose was . × /kg [p ] s (range: . - . × /kg)(n = ). patients. conditioning was myeloablative in ( %) and ric in ( %). predominant conditioning regimes were fludarabine, busulphan, atg (fbatg) and thiotepa, busulphan, fludarabine (tbf n = ). tbi was administered in ( %) and t cell depletion in vivo in ( %) and ex vivo in ( %) patients. gvhd prophylaxis varied with post transplant cyclophosphamide administered in / ( %) and atg in / patients ( %).neutrophil engraftment occurred in ( %) patients at a median of days (range: - ). primary graft failure ensued in ( %) and secondary graft failure in ( %) patients at a median of (range: . - ) months. eleven patients had a second allograft at a median interval of ( - ) months. responses to the first allograft censoring for a second allograft, data available in patients, showed that complete remission was achieved in patients ( %), ( %) were never in cr and ( %) were not evaluable. relapse occurred in ( %) of patients at a median interval of ( . - . ) months. the cumulative incidence (ci) of grade ii-iv acute gvhd (agvhd)was % ( % ci - %) and for grade iii-iv agvhd at was % ( % ci - %). data for chronic gvhd (cgvhd) was valid in patients of whom % developed cgvhd. the ci of cgvhd at years was % ( % ci - %):ci of limited cgvhd was % ( % ci - %) whereas the ci of extensive cgvhd was % ( % ci - %). median follow-up was ( % ci - ) months. the and year os was % ( % ci - %) and was % ( % ci - %). the and year rfs was % ( % ci - %) and % ( % ci - %). the -year ci of relapse was % ( % ci - %). the year nrm was % ( % ci - %), which increased to % ( % ci - %) at years. thirty patients died due to infection ( , %), gvhd ( , %), organ damage or failure ( , %), relapse/disease progression ( , %) and secondary malignancy or ptld ( , %) unknown . there was no significant effect (univariate analysis) of recipient or donor gender, degree of hla mismatch, cmv matching, primary or secondary mf, chronic vs advanced disease at transplant, conditioning intensity or regimen, gvhd prophylaxis with atg or post transplant cyclophosphamide or stem cell source on overall survival. the data are encouraging for patients with myelofibrosis, with engraftment, pfs and os being attained with limited severe chronic gvhd from family mismatched donors. disclosure of conflict of interest: none for all other authors, fc consulting with molmed. feasibility of salvage second allogeneic stem cell transplantation for disease relapse or graft failure: a single centre experience g battipaglia , , d salvatore , , r dulery , f giannotti , f malard , e brissot , s sestili , f isnard , s lapusan , a-c mamez despite high rates of toxicity and mortality, a second salvage allogeneic stem cell transplantation (second allohsct) might be an option to consider in patients experiencing disease relapse or graft failure after first allohsct. we retrospectively analyzed outcomes after second allohsct in a cohort of patients ( males and females) transplanted either for disease relapse (group , n = ) or graft failure (group , n = ) between and in a single centre in france. median age at second allohsct was (range: - ) years. diagnoses were acute myeloid leukemia (group : n = ; group : n = ), acute lymphoblastic leukemia (group : n = ; group : n = ), myelodysplastic syndrome (group : n = ; group : n = ), myeloproliferative neoplasm (group : n = ; group : n = ), bone marrow failure (group : n = ; group : n = ). median time from first allohsct to second allohsct was (range: . - ) months in group and . (range: - ) months in group . graft source for the second allohsct were: haploidentical bone marrow (group : n = ; group : n = ), haploidentical pbscs (group : n = ; group : n = ), cord blood (group : n = ; group : n = ), matched unrelated pbsc (group : n = ; group : n = ). at time of second allohsct, patients were in cr and presented active disease in group . conditioning regimen was myeloablative in patients (group : n = ; group : n = ), reduced intensity (ric) in cases (group : n = ; group : n = ). a sequential schema consisting of a combination of thiotepa, etoposide and cyclophosphamide followed by a fludarabine and busulfanbased ric was used in out of patients with active disease in group . sixteen patients received atg as part of the conditioning regimen for second allohsct (group : n = ; group : n = ). all but one patient engrafted, at a median time of (range: - ) days. cumulative incidence of acute and chronic gvhd were ± % and ± %, respectively, - ) months, non-relapse-mortality (nrm) and relapse incidence (ri) were ± % and ± %, respectively, while disease-free (dfs) and overall survival (os) were ± % and ± %, respectively, for the entire cohort. in all, patients died of infections (n = ), hematological disease (n = ), gvhd (n = ), hemorrhage (n = ) and for unknown causes (n = ). main outcomes of patients in group were: ri ± %, nrm ± %, agvhd ± %, cgvhd ± %, dfs ± %, os ± %, respectively. main outcomes of patients in group were: ri ± %, nrm ± %, dfs ± %, os ± %, agvhd ± %, cgvhd ± %. historically, a second allohsct was hampered by significant morbidity and mortality. however, the advent of reduced-toxicity conditioning regimens and improved supportive care allowed to significantly improve the results of patients receiving a second allohsct as suggested from the above results. therefore, a second allohsct could be considered as an option to rescue a certain number of patients experiencing disease relapse or graft failure, for which prognosis is very poor. decision is to be discussed on a case-by-case basis. disclosure of conflict of interest: none. haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide for patients with high-risk hematologic malignancies am carella department of oncology and hematology, irccs casa sollievo della sofferenza, san giovanni rotondo allogenic hematopoietic stem cell transplantation (sct) has been increasingly used for treatment of adult with high risk hematologic malignancies. for patients lacking an hlamatched related or unrelated donor, unmanipulated haploidentical (haplo)-sct is a potential alternative. haploidentical transplantation performed with post-transplantation cyclophosphamide (ptcy)-based graft-versus-host disease (gvhd) prophylaxis has been associated with favorable outcomes for patients with acute leukemia and lymphomas we analyzed outcomes of patients with hematologic malignancies who received t-cell-replete haematopoietic stem cells and posttransplantation cyclophosphamide after myeloablative or nonmyeloablative hla-haploidentical donor transplantation. the median age was years ( - ); twelve patients were in first remission (cr ), in second remission (cr ) and had an active disease. ). the diagnosis was acute leukemia (n = ), myelodisplastic syndrome (n = ), hodgkin disease (n = ) non hodgkin lymphoma (n = ) and multiple myeloma (n = ). median follow-up was days. stem cell source was bone marrow (bm) for patients, and peripheral blood (pb) for . myeloablative conditioning (mac) was used in patients and reduced intensity regimen (ric) in patients. thirty one patients were first grafts, the others underwent previous autologous sct (n = ) or mud (n = ). gvhd prophylaxis s consisted in pt-cy on days + and + , cyclosporine (from day + ), and mycophenolate (from day + ). the median day for neutrophil engraftment was day + ( - ). no graft failure was observed. chimerism was evaluable in patient; on day + all patients had % donor chimerism on marrow cells median follow-up was days. the cumulative incidence of acute gvhd grade ii-iv was %, grade iii-iv % and chronic gvhd %. one-and -years os was . % and . %, respectively. with a median follow-up for the surviving patients of days ( - ), the cumulative incidence of transplant-related mortality (trm) is %, and the relapserelated death is %. thus, we demonstrate excellent rates of engraftment, gvhd, and trm in adult patients treated with haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide. this approach is a widely available, safe, and feasible option for adult patients with high risk hematologic malignancies, including those with a prior history of myeloablative bmt and/or those with co-morbidities or organ dysfunction, also for patients with active disease at the time of transplant. disclosure of conflict of interest: none. it has recently been shown that t-replete allogeneichematopoietic stem-cell transplantation (allo-hsct) from a haploidentical donor (haplo-id) could be a valid option when a matched donor is not available. unfortunately, the worldwide donor registries comprise mainly donors of caucasian origin and patients of non-caucasian origin have a much lower chance of finding a matched unrelated donor (mud). the lengthy period of international search when required and the financial burden of this process are considered as additional significant limitations. at the american university of beirut medical center (aubmc) in lebanon, we started the mud program in and haplo-id hsct program in . we report here our experience in this two groups of patients. patients and methods: we have transplanted patients from a haplo-id donor since and compare their outcome with the patients transplanted from a mud since . the patients and transplant characteristics are listed in the table . the groups were comparable except for conditioning. patients in haplo-id group received two days of posttransplant high-dose cyclophosphamide (pt-hdcy) followed by cyclosporine a (csa) and mycophenolate-mofetil while patients in the mud group received pre-transplant antithymocyte-globulins and csa starting on day- . all patients engrafted in the mud group, while one patient did not engraft in the haplo-id group, the patient had refractory all transplanted with progressive disease, and died on day + . the median of anc /mm was days ( - ) vs days ( - ) in the haplo-id and mud groups, respectively. fourteen patients from the haplo-id group developed grade acute graft-versus-host disease (agvhd) vs one after mud-hsct. two patients haplo-id group developed limited cgvhd and none after mud grafts. six patients relapsed in the haplo-id group vs three patients in the mud group. two and three patients died from non-relapse mortality in the haplo-id and mud group, respectively. at the last follow-up, patients are still alive in the haplo group vs patients in mud group and all of them are in cr. we conclude that t-replete haplo-id hsct followed by pt-hd cy is associated with promising results or at least comparable to patients transplanted from mud. haplo-id hsct seemed to be safe and feasible in patients with high risk hematological malignancies. finally, because of the obvious advantage in rapidly finding a donor ( haplo transplants in three years vs mud transplants in years), development of haplo-id hsct is warranted to satisfy the regional needs. [p ] disclosure of conflict of interest: none. haploidentical hematopoietic stem cell transplantation (haplo-hsct) using t-cell-replete (tcr) grafts and posttransplantation cyclophosphamide (ptcy) provides a curative approach for patients with high-risk mds/aml lacking a conventional hla-matched donor. in children and adults haplo-hsct using ptcy as gvhd prophylaxis seems to be safe with low treatment related morbidity and mortality (trm). however, few data are available for elderly patients with advanced disease. we retrospectively analyzed the outcome of patients with mds (n = )/aml (n = ) age - years (median age years; patients - years, patients ⩾ years; male), who underwent tcr haplo-hsct with high-dose ptcy at our institution between january and november . disease was active in patients while had achieved cr. patients failed previous allo-hsct. pretransplantation risk factors were scored using the hematopoietic cell transplantation-specific comorbidity index (hct-ci) which was ⩾ in patients (median hct-ci = , range: - ). a sequential therapeutic concept using either flamsa (n = ) or clofarabine (n = ) as cytoreduction was used prior to reduced intensity conditioning (ric) in all but patients. ric consisted of fludarabine/cyclophosphamide combined with either melphalan (n = ), busulfan (n = ) or gy tbi (n = ). post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mmf in all patients. % received a bone marrow graft. one graft rejection occurred. neutrophil and platelet engraftment was achieved in % and % of evaluable patients, respectively at a median of ( - ) and ( - ) days. acute gvhd grade i-iii occurred in % of the patients whereas no grade iv agvhd was observed. chronic gvhd presented in %. it was most frequently assessed as mild to moderate ( pts). only patients developed severe cgvhd; no gvhd related death was observed. cmv reactivated in of patients at risk, one patient developed cmv disease (pneumonia). no ebv reactivation or ptld occurred. one-year trm was %. / ( %) patients relapsed, three within the first days after haplo-hsct. at a median follow up of months (range: - months) estimated one-and two-year overall survival (os) was / %, respectively. when stratified by age, estimated one-and two-year os was / % in patients o years and / % in patients ⩾ years (p = . /p = . ). one-and two-year progression-free survival (pfs) was / %, respectively. stratified by age estimated one-and two-year pfs was / % in patients o years and / % in the elderly (p = . /p = . ). unmanipulated haploidentical allografting using ptcy-based gvhd prophylaxis in high-risk mds and aml patients aged over years is safe and well tolerated resulting in acceptable trm. a remarkable survival outcome can be achieved in elderly high-risk aml/mds patients with significant comorbidities. disclosure of conflict of interest: none. key performance indicators to assess the quality of a collection facility: experience of a single center s roncon*, c pinho , f bordalo , s lopes , s ferreira and f amado allogeneic hematopoietic stem cell transplantation (allo-hsct) has evolved into an effective immunotherapy for the treatment of a variety of disorders. when patients do not have a familiar matching donor, transplant centers (tc) search for an unrelated and volunteer donor. this one must be previously evaluated by the collection center (cc) to donate peripheral blood stem cells (pbsc) or bone marrow (bm); lymphocytes can also be asked after allo-hsct. this work aims to evaluate our performance as cc, ensuring donor safety, quality of cell therapy products (ctp) and the accomplishment of tc requirements. we retrospectively analyzed all the requests of ctp collections sent by the portuguese registry from to . countries of destination, number and type of ctp were determined. we established eight key performance indicators (kpi) classified into four categories: response time; product quality; satisfaction of patients and donors; and on-site donor motivation. the intended target was defined by the mean result obtained in the first half of (excluding kpi- ). written comments from donor center (dc) and tc were received by email or written in the local notebook. the donor's answers were obtained through a survey given on the collection day. a total of requests were assessed: pbsc, bm, lymphocytes and cancellations; % were sent to europe ( / to portugal), % to america and % to oceania; / were withdrawn by tc ( patients died, presented progressive disease and had a better hlamatched donor) and / by dc ( donors not cleared and refused). the results obtained with kpi- , - , - and - exceeded the intended target (table ) . after the first kpi- results, we verified a positive evolution. we took an average of days of delay in sending donor clearance. however, there is no holdup in the ctp delivery, as demonstrated by kpi- . regarding kpi- it is important to notice that % of ctp with a cell number less than requested were bm and lymphocytes; when pbsc was considered separately, the result increased ( % vs %). analyzing kpi- , % (n = / ) of the contaminated ctp were bm. concerning kpi- , - acknowledgments and - commitment, we recognize that our initial targets were too ambitious ( %). the kpi- shows a low number of complaints (n = ): one due to a misreading of the request and three to communication failures; all were properly examined and rectified. a good general status was guaranteed in almost all the donors (kpi- ). the decrease of kpi- is due to the fact that one donor refused to proceed after three postponements of the collection date by tc. table -key performance indicators of the quality of our activity as a cc. the overall good level of our results reflects an extremely professional performance as a cc. we consider that these kpi should be continuously monitored with the purpose of earlier detect any deviation of the stated goals and assess the progress against settled strategies. we further suggest the establishment of universal indicators in order to standardize [p ] practices, share expertise and improve the quality of services and products provided to patients and donors. two year later, the patient had a genoidentical allogenic stem cell transplant (from the bone marrow stem cell of his sister, who was years old, hla compatible). he had a reduced intensity conditioning, associating busulfan, fludarbine and antilymphocyte serum. months from the asct, he was in complete remission with % donor chimerism. years after the asct, the patient presented a progressive thrombocytopenia without any other peripheral causes. the bone marrow aspiration initially showed a refactory cytopenia with multilineage dysplasia. the patient was followed up during months, and then a second bone marrow aspiration has shown a refractory anemia with excess blasts raeb . a cytogenetic study has every time demonstrated a female karyotype ( ,xx) on mitoses out is , and chimerism was % donor. the diagnosis of the myelodsplastic syndrome of the donor cells was approved. the patient was treated by azacitidine ( mg/m , from j to j , j = j ). after cycles, the patient was in complete hematologic response (normalization of the platelet count) and a partial bone marrow response (normalization of the blasts rate but persistence of the signs of dysplasia). he received more cycles, and presented hematologic relapse (reemerging of thrombopenia). a phenoidentical allogenic stem cell transplantation was suggested. conclusion the occurrence of mds on the donor cells is rare. these anomalies are secondary to intrinsic factors (of donor) or extrinsic factors )of the transplant recipient). the treatment is not definitely determined. disclosure of conflict of interest: none. nk-cell alloreactivity based on kir/ligand mismatch in the donor vs recipient direction provides better graft-versustumor effect in patients with active hematological malignancies undergoing allogeneic t-replete haploidentical transplantation followed by post-transplant cyclophosphamide a wanquet , , s bramanti , , s harbi , s fürst , f legrand , c faucher , a granata , p-j weiller , , c picard , b calmels , c lemarie , c chabannon , , , l castagna , , d blaise , , and r devillier , haplo-sct have been developed in the past years with very interesting results in high risk patients. gvhd prophylaxis using post-transplant cyclophosphamide (pt-cy) recently allowed extending the use of unmanipulated haplo-sct. it was shown that nk alloreactivity, triggered by donor-recipient inhibitory kir gene-gene mismatches, could lead to better outcomes and survival in the setting of in t-cell-depleted haplo-sct. however, few data is available on the impact of kir-ligand mismatch on the outcome after t-replete haplo-sct with pt-cy. we thus assessed the impact of nk alloreactivity on the outcome of patients who received haplo-sct followed by pt-cy. we retrospectively collected the data from patients from two centers who were treated for various high risk hematological diseases and underwent a haplo-sct with pt-cy from december to december . we assessed the kir-binding epitope in hla-c and hla-b molecules for all patients, and we predicted nk cell alloreactivity in the donor vs recipient direction via the immune polymorphism database kir ligand calculator, based on the kir-ligand mismatch between donors and patients. because disease status at the time of haplo-sct is one of the most important predictor of outcome, we separately analyzed two cohorts of patients: those transplanted in complete remission (cr group) and those transplanted with active disease (no cr group). using a multivariate cox model (adjusted by disease type, age and conditioning), we therefore evaluated the impact of nk alloreactivity on outcome in both cr and no cr groups. we analyzed patients with a median age of y ( - ). they were mostly transplanted for lymphoma (n = , %) or aml/ mds (n = , %). patients mostly received a tbi-based nonmyeloablative conditioning regimen (n = , %) and pbsc as graft source (n = , %). eighty one and patients were transplanted in cr and in no cr, respectively. nk alloreactivity was found in / cr patients ( %) and / no cr patients ( %). with a median follow up of months ( - ), cr patients had a significantly better outcome than those in the no cr group ( -year pfs % vs %, respectively, p o . ). in no cr patients, multivariate analysis showed that nk alloreactivity was significantly associated with reduced the risk of relapse (hr = . , p = . , figure a ) with no increase of both acute (hr = . , p = . ) and chronic gvhd (hr = . , p = . ), and nrm (hr = . , p = . ). this led to significantly better pfs (hr = . , p = . , figure b ) and a trend for better os (hr = . , p = . ). in contrast, in cr patients, we found no difference in outcome according to nk alloreactivity for all end points (acute gvhd: hr = . , p = . ; chronic gvhd: hr = . , p = . , nrm: hr = . , p = . , relapse: hr = . , p = . , figure c ; pfs: hr = . , p = . , figure d ; os: hr = . , p = . ). our results suggest that nk alloreactivity provides better disease control with no increase of gvhd, especially in patients transplanted with active disease. thus, donor selection should rely on the prediction of nk alloreactivity. this may contribute to improve outcome of these patients with high risk of relapse after transplantation, underlining the need of a specific strategy of donor search, and the promising perspective of early post-transplant nk-cell-based immunotherapy. haploidentical bone marrow transplantation (haplo-bmt) with post-transplant cyclophosphamide (pt-cy) is being increasingly used, in the last five years, for patients lacking a suitable hla-matched donor. genoa study (eudract number: - - ) provides for a modified gvhd prophylaxis platform compared to the original baltimora protocol. aim of the study: in this study we assessed outcomes in consecutive patients transplanted from a haploidentical donor for haematological malignancies. all patients received a uniform gvhd prophylaxis: cyclosporine (csa) starting on day , mycophenolate (mmf) starting on day + , and post transplant cyclophosphamide (pt-cy) mg/kg, on days + and + . all patients received a myeloablative conditioning consisting of thiotepa, fludarabine, busulfan (three doses n = or two doses n = ), or tbi, fludarabine (n = ). the median age was years ( - ); at transplant ( %) patients were in remission of disease (cr and cr ), and had an active disease ( %); all patients were first grafts. the diagnosis were acute myeloid leukemia (n = ), myelodisplastic syndrome (n = ), acute lymphoblastic leukemia (n = ), myelofibrosis and myeloproliferative diseases (n = ), non hodgkin lymphoma (n = ), chronic lymphocytic leukemia (n = ) and multiple myeloma (n = ). the median follow up was days (range: - days). the median infused mononucleated cells was . × e /kg (range: . - . ). seven patients died before engraftment, and ( %) had autologous recovery: ( %) after conditioning with doses of busulfan. full-donor chimerism on day + was reached in ( %) patients. the median day for neutrophil engraftment was day + (range: - days). the cumulative incidence of grade ii-iv and iii-iv acute gvhd (agvhd) was % (n = ) and % (n = ), respectively. two years cumulative incidence of moderate-severe chronic gvhd (cgvhd) was % (n = ).sixty one ( %) patients experienced haemorragic cystitis. at years the cumulative incidence of non relapse mortality (nrm), relapse and relapse related death was % (n = ), % (n = ) and % (n = ), respectively. causes of death were infections (n = ), hemorrhage (n = ), gvhd (n = ), secondary neoplasia (n = ) and relapse (n = ). at years of follow up overall survival and disease free survival was . % and %, respectively. at the same time overall survival rate was % for patients in remission and % for patients with active disease at transplant(p o . ). in conclusion, a modified pt-cy as gvhd prophylaxis and ma conditioning regimen followed by haploidentical bmt results in a low risk of agvhd and cgvhd and encouraging rates of trm and dfs. disclosure of conflict of interest: none. the italian bone marrow donor registry (ibmdr), in collaboration with admo (associazione donatori di midollo osseo) since has implemented, as part of the donor enrollment strategy, public enrollment events (pe). our donor center (dc) has taken part to those events since the first years. one or more clinician (or trained biologist) has been present to pe to inform the potential donors, evaluate the candidates and supervise the collection of biological fluids. all the local permission where obtained. aim: aim of this study was to compare the compliance of the donor enrolled in pe with donors enrolled at our dc institutional site. we prospectively evaluated all the donors recalled for further evaluation and/or for requalification in the years and at our dc itmi . we defined possible results for the call: ‛success' (the donor was eligible and accepted to be evaluated, or only temporarily ineligible) ‛not eligible' (the donor was definitively ineligible) and ‛consent denied'. results: a total of donors were called back in the years and ( not found). eightyfour recalled donors had been enrolled after . among them ( . %) had been enrolled at the dc and during pe ( . %). the two populations were not different for age at the call, age at enrollment and gender (table ) . [p ] when evaluating the probability of obtaining a "success", no significant difference was found between the two populations: . % vs. . % (chi square p= . ). no significant difference was also found for the "not eligible" and the "consent denied" categories. of note, when we turned to the whole donor population we had called back (median age , range - ), the probability of "success" and "consent denied" were not related to donor age, and time from enrollment to recall, whereas donor ineligibility was (spearman test p= . and . ). public events with the presence of an adequate trained medical team represent a valid option for the enrollment of new unrelated donors. disclosure of conflict of interest: none. the search for hematopoietic stem cell unrelated donors in patients with malignant hemopathies with not-sibling matched family donor: the experience of a center a pérez , r goterris , m gómez , s blanco , a segado , c arbona , jch boluda , m poch and c solano hematology department, hospital clínico universitario, valencia unfortunately, as few as - % of patients will have an hlaidentical matched sibling donor available for hematopoietic stem cell (hst) donation. the search for an unrelated donor (urd) (adult or cord blood) is often the best option for those patients lacking a suitable matched donor. below we describe the experience with the search for an unrelated donor in our center. between september and march the search for urd was activated for patients. the median age of the patients was years (range: . - ), % were under years and % were males. acute myeloid leukemia (n = ), acute lymphoblastic leukemia (n = ), non-hodgkin's lymphoma (n = ), chronic/prolymphocytic lymphocytic leukemia (n = ), hodgkin's lymphoma ((n = ), multiple myeloma (n = ), chronic myeloid leukemia (n = ), philadelphianegative myeloproliferative neoplasms (n = ), myelodysplastic syndrome (n = ), aplastic anemia/paroxysmal nocturnal hemoglobinuria (n = ), others (n = ). the disease status in hematological malignancies was: first cr (n = ), second cr (n = ), pr (n = ) and refractoriness (n = ). the donor type requested at the activation of the search was an adult (n = ), umbilical cord blood (n = ) and two options (n = ). results: a compatible donor was found in patients ( % of the series) after a median of days (range: - ) from the activation of the search. the degree of adult donor compatibility (not available in cases) was: complete hla identity ( / : n = , / : n = ); an hla difference ( / : n = , / : n = ); lower degree of compatibility (n = ). the degree of umbilical cord blood compatibility: identity ⩾ / (n = ). a total of patients ( %) were transplanted, from adult donor and from umbilical cord blood. the median time between the activation of the search and the hst transplantation was months (range: . - ), being . months for acute leukemia and . months for other pathologies, and between the location of the donor and the hst transplantation days (range: - ), being days for umbilical cord blood and days for an adult donor. there were cancellations of the urd search ( % of the total) for the following reasons: clinical status of the patient (n = ), performing a haploidentical transplant (n = ), transplant center does not consider (n = ), norms of the registry (n = ) and loss of indication of transplantation (n = ). the median time from the beginning of the search to its cancellation was . months (range: . - ). at the time of analysis, the median follow-up of the patients is months. the survival of the series in the years is % and % for patients transplanted from urd. % of the searches activated in our center allowed the localization of a urd with an adequate degree of hla compatibility. however, only % of the patients for whom the search was activated were finally transplanted. the most frequent cause of cancellation of the procedure was the clinical deterioration of the patient. disclosure of conflict of interest: none. the leukemic transformation of otherwise healthy donor stem cells provides a useful in vivo model to study the mechanisms involved in leukemogenesis. we report two cases of donor cell-derived haematological malignancy in which wholeexome sequencing (wes) was performed in bone marrow (bm) samples from recipient at different times after allogeneic hematopoietic stem cell transplantation (allo-hsct) in order to study the dynamics of emergence of mutations that precede the development of donor cell leukemia (dcl) and donor cell myelodysplastic syndrome (dc-mds). case : a -year-old female diagnosed with lymphoblastic leukemia-b t( ; ), who developed acute myeloid leukemia (aml) with normal karyotype, npm +of donor origin months after unrelated cord blood transplantation (ucbt). case : a -year-old male diagnosed with mantle cell lymphoma, who developed mds ,xx,- ,del( )(p ) of donor origin, months after allogeneic bm transplantation from his hla-identical brother. the donor also developed mds several months later. wes (sureselect-xt human-exon mb) was performed by next generation sequencing (hiseq) on donor stem cells (scs) infused as well as on bm samples from recipient after allo-hsct. the exome of donor scs and bm samples, from case , were aligned to the human reference genome (grch /hg ) and donor scs and bm samples were aligned to grch / hg in the second case. in both cases non-synonymous variants in the coding regions or synonymous variants in splice regions of genes related to leukemia were selected. in addition, bm samples were matched to their scs and to prior bm samples to identify the acquired variants. variants meeting such criteria were evaluated with functional predictor software's (sift, polyphen and mutation taster). wes analysis revealed progressive emergence of multiple somatic mutations probably related to the development of leukemia in bone marrow samples post allo-hsct ( figure ). both scs showed alterations that may be involved in leukemogenesis. (case : sh b and case : kmt c, kmt a, arhgap and monosomy ). somatic mutations, acquired over time, fall into genes that play well-established roles in signalling pathways (ras-mapk, pre-mrna splicing factor, apoptosis, dna doublestrand break repair, dna replication and so on). mutations in leukemic subclones that disappear after chemotherapy were indentified, as well as the acquisition of new mutations in resistant subclones. we propose a possible model of leukemogenesis in these cases ( figure ). the present study reveals a process of sequential clonal expansions, promoted by the acquisition of additional somatic mutations in donor hematopoietic cells. detection of heritable or acquired gene mutations in donor associated with predisposition to haematological malignancies could have clinical implications for the patients undergoing to allo-hsct. although the cause of donor cell-derived haematological malignancy onset seems to be multifactorial, the infusion of a scu with pre-leukemic potential in a context of residual toxicity in recipient as a result of pre-transplant chemotherapy, a post-transplant environment characterized by a decreased immune surveillance may well have played role in these cases. the study of a greater number of dcl cases by next generation sequencing could help to understand this process and to detect new mutations involved in the emergence of aml. disclosure of conflict of interest: none. the impact of donor and recipient sex in allogeneic stem cell transplantation-single center experience (cic ) y petrov , p ganeva , g arnaudov , s lozenov , y davidkova , v stoeva , i tonev , m guenova and g mihaylov national hospital for active treatment of hematological diseases allogeneic hematopoietic stem cell transplantation (hsct) has been one of the most effective therapeutic modalities for patients with hematological malignancies and bone marrow failure syndromes. optimal donor selection is one of the key factors to enhance the success rate of this procedure. we [p ] s retrospectively investigated whether and how donor-recipient sex affects transplantation outcomes of patients transplanted between and in our center. the median age of the patients was years (range: - ). thirty-nine of the patients ( %) received a pbsc from a hla-identical sibling, and patients ( . %) received pbsc from matched unrelated donor. forty-six percent were male recipients with male donors (m-m), . % were female recipients with male donors (m-f), . % male recipients with female donors (f-m), and . % female recipients with female donors (f-f). we performed a crosstab analysis and χ tests to observe whether the donor sex affects our study population. patients with male donor had superior overall survival and progression-free survival compared to those with female donor ( . % vs . % p = . for os, and . % vs . % p = . for pfs; cramer`s v = . ). we further investigated how the disparity of the donor in the four groups (m-m, m-f, f-m and f-f) affects the os, pfs and nrm. the f-m group had a worse overall and progression-free survival comparing the other groups ( % -year os and % pfs; p o . ).this group had % relative increase in the non-relapse mortality compared with m-m group (p = . ). for m-m group there was a % relative increase in the subdistribution hazard of nrm compared with m-f group (p = . ). the f-f group and m-f group had similar subdistribution hazard of nrm ( % vs % p = . ). the incidence of acute gvhd and chronic gvhd for the groups was: % and % (m-m), % and % (m-f), % and % (f-m), % and % for the (f-f) group. the appearance of either acute or chronic gvhd did not show statistical significance regarding the os and pfs in the groups (p = . ). we examined the effect of donor-recipient sex incompatibility on the outcome of hsct in out center. our results showed inferior os and pfs for f-m group and a higher incidence of nrm compared with other groups. these effects might be associated with allogeneic immune responses against h-y antigens. key words: stem cell transplantation, donor sex, recipient sex, overall and progression-free survival [p ] disclosure of conflict of interest: none. from to , % of the patients affected by hematological malignancy searching for an unrelated donor through the italian registry successfully identified a suitable donor. this proportion increases up to % when searching for a cord blood unit was considered, corresponding to total transplant efficiency of %. from april , the rome transplant network adopted a unique policy for the identification of a potential alternative donor, following a hierarchical selection that considered as first choice a volunteer unrelated donor, secondly a cord blood unit and last a haploidentical related donor. before starting the unrelated donor search, a preliminary query through the bone marrow donor worldwide database was performed for all the patients referred to the rome transplant network. based on the low resolution hla typing (a, b and drb ) it was possible to arbitrary assign a good or poor score that might predict the identification of a full matched ( / a, b, c and drb ) donor. therefore, aims of the present study were to assess the utility of the preliminary query and the impact of the use of high resolution hla typing since the starting of donor search on the timing for the unrelated donor identification. moreover, the final aim was of comparing donor identification and transplant efficiency between the national registry, that considers only the unrelated donor and the rome transplant network, whose policy includes also haploidentical donor as third choice in the donor search process. at rome transplant network % out of adult patients met criteria of a good preliminary query corresponding to a matched unrelated donor identification in % of cases vs only . % for patients with poor preliminary query. our policy led to % and %, respectively, of alternative donor identification and transplant efficiency, significantly higher than the corresponding data of % (p = . ) and % (p o . ) reported by the national registry. moreover, the median duration of search process for mud identification has been significantly reduced by the use of hr hla typing patient at the start of the formal search activation from (range: - ) to (range: - ) days at ibmdr (po . ) and from to days ( - ) at rtn (po . ). in conclusion, the preliminary query represents a useful tool to address the search towards the best donor choice and to perform transplant in adequate time. moreover, the timing of donor identification has been significantly reduced with the use of high resolution typing at the start of donor search. a search and selection donor policy should be basically established and should include the haploidentical donor to improve the transplant efficiency. disclosure of conflict of interest: none. the long term prognosis of elderly acute myeloid leukemia (aml) patients remains poor. advances in the uses of alternative donors and reduced intensity conditioning regimens have extended the use of allogeneic hematopoietic stem cell transplantation (hsct) to a wider number of patients. however, few studies have reported data on the efficacy of hsct from alternative donors in elderly aml patients. we retrospectively analyzed the transplantation outcome in consecutive elderly aml patients aged years who received hsct ( hsct ( - at the catholic blood and marrow transplantation center. donor types were autologous (n = ) or hla matched related (mrd, n = ), unrelated (mud, n = ), or haploidentical (n = ). for graft-versus-host disease (gvhd) prophylaxis, methotrexate and cyclosporine (mrd) or tacrolimus (mud/haploidentical donor) were used. mud and haploidentical donors were given antithymocyte globulin. the median age was years, with patients ( %) years. intermediate-or adverse cytogenetic risk was observed in % of patients. with a median follow-up of . months, overall survival (os) and disease-free survival (dfs) at years after transplantation were % and % for autologous, % and % for mrd, % and % for mud, and % and % for haploidentical hsct, respectively. the -year relapse was significantly higher for autologous hsct compared to allogeneic hsct ( % vs %, p = . ), while it was similar among allogeneic donors: mrd, %; mud, %; haploidentical, % (p = . ). the -year non-relapse mortality (nrm) for mud ( %) or haploidentical donor ( %) hsct was comparable to that of autologous hsct ( %), while it was relatively higher for mrd hsct ( %, p = . ). of the patients receiving allogeneic hsct, the -year cumulative incidence of moderate to severe chronic gvhd was significantly increased for mrd ( %) compared to alternative donor hsct ( %, p = . ). in multivariate analysis, patient age (hr . , % ci . - . , p = . ) and donor type (hr . % ci . - . , p = . for mud; hr . , % ci . - . , p = . for mrd compared to haploidentical donor) were significantly associated with the cumulative incidence of moderate to severe chronic gvhd, while female-to-male hsct showed a borderline significance (hr . , % ci . - . , p = . ). incidence of acute gvhd was similar according to donor type. in the multivariate analysis for nrm, patient age (hr . , % ci . - . , p = . ), mrd (hr . , % ci . - . , p = . ), and hematopoietic cell transplantation-comorbidity index high risk (hr . , % ci . - . , p = . ) were significantly associated. in conclusion, our results showed significantly higher relapse rate for elderly aml patients receiving autologous hsct compared to allogeneic hsct, responsible for the lower survival rate in autologous hsct. we observed that nrm rate for mud and haploidentical donors for elderly aml patients were lower than expected and similar to autologous hsct. relatively higher incidence of nrm for mrd hsct seemed responsible for the low long term dfs. these results suggest a need for strengthening of gvhd prophylaxis in mrd hsct for elderly aml patients. our results suggest a potential role of alternative donor hsct to improve long term survival rates in elderly patients with aml. disclosure of conflict of interest: none. for patients with saa, transplantation from an unrelated donor (ud) is usually considered after failure of at least one course of immunosuppression. this strategy is based on a relatively high risk of complications for ud transplant recipients, such as graft rejection, graft-versus-host disease (gvhd) and infections. however, the outcome of unrelated donor transplants has significantly improved in recent years, due to better donor selection, conditioning regimen optimization and better supportive care. the authors describe results from patients with saa who receive unrelated allogeneic transplants in a single reference institution from to . data was retrieved from the center databasis and there were females and males. median age was years old . median total number of cells infused was . × /kg. % of the patients have received more than transfusions previously. conditioning regimen were: cy + tbi ± atg in ( %) patients, bu mg/kg+ cy + atg in ( %), and fludarabine + cy+atg in ( %), fludarabine, cy+tbi in ( %) patients. stem cell source was marrow in %, cord blood in % and peripheral blood in % of patients. transplants were full matched in ( %) patients, had one mismatch (out of ) in ( %) and mismatches in ( %) patients. engraftment was complete as evaluated by donor chimerism at day and post transplant in patients ( %), partial in ( %) and graft failure was observed in ( %) patients. acute gvhd grade ii-iv was seen in patients ( %) and nih moderate to severe chronic gvhd was seen in ( %) patients. median overall survival was days ( - ) and estimated years overall survival was %. risk factors for survival identified were: hla mismatch and stem cell sources other than marrow. unrelated transplants are a feasible salvage therapy for patients with saa refractory to immunosuppression, being hla compatibility and marrow stem cell source factors with a positive impact on survival. disclosure of conflict of interest: none. use of haploidentical stem cell transplantation continues to increase, the european society for blood and marrow transplant activity survey report jr passweg , h baldomero , p bader c bonini , rf duarte , c dufour , , a gennery , n kröger , j kuball , f lanza , s montoto , a nagler , ja snowden , j styczynski and m mohty for the european society for blood and marrow transplantation (ebmt) hematopoietic stem cell transplantation (hsct) is an established procedure for many acquired and congenital disorders of the hematopoietic system, including disorders of the immune system, and as enzyme replacement in metabolic disorders. the annual activity survey of the ebmt describes the status of hsct in europe and affiliated countries and has become an instrument used to observe trends and to monitor changes in technology use. teams were invited to report their transplant activity for by indication, stem cell source and donor type using a single paged survey. a record number of ' hsct in ' patients ( ' allogeneic ( %), ' autologous ( %)) were reported by centers in countries in . trends include continued growth in transplant activity during the period and , with the highest percentage increase seen in middle income countries (allo %, auto %), and the lowest in very high income countries (allo %, auto %), for both allogeneic and autologous hsct. in contrast the absolute growth is highest in the very high income countries (growth allo rates transplants per × inhabitants, auto rates for very high income countries; allo rates , auto rates for middle income). main indications for hsct were myeloid malignancies ' ( %; % allogeneic); lymphoid malignancies ' ( %; % allogeneic); solid tumors; ' ( %; % allogeneic); and non-malignant disorders; ' ( %; % allogeneic). remarkable is a decreasing use of allogeneic hsct in cll from patients in to in and is most likely due to the development of potentially very effective cll drugs. use of haploidentical donors for allogeneic hsct continues to increase ' in ; a % increase since . the highest growth is seen in myeloid malignancies ' , with lymphoid malignancies , nonmalignant disorders and others. in aml, haploidentical hsct increases similarly for patients with both advanced disease and those in cr . both marrow and peripheral blood is used as stem cell source for haploidentical hsct with higher numbers reported for the latter. this year's activity survey shows continued increase in the use of haploidentical hsct across europe within the main indication groups and cell source. it reflects in a timely manner current trends in stem cell transplantation and is an essential tool for health care planning and health policy makers. human bone marrow mesenchymal stromal cells derived exosomes (hbmmdes) are small membrane vesicles secreted from mesenchymal stromal cells that may serve as a vehicle for protein, mrna and microrna (mirna) transfer to distant cells; affecting gene expression, proliferation, and differentiation of the recipient cells. therefore, mdes may possess some of the immunoregulatory properties of their parental cells. in the present study we aim to explore the immunomodulatory function of mdes and understand the molecular mechanisms enabling it. for this purpose, we co-cultured hbmmdes with activated human lymphocytes. using ultracentrifugation, hbmmdes were isolated from expanded human bone marrow derived mesenchymal stromal cells (hbmmscs). using em and zeta sizer, particles were shown to be in the range of - nm. pha activated human peripheral blood lymphocytes (pbls), r- /il activated b cells and anti cd /cd activated t cells were co cultured with purified mdes. cell proliferation was tested using thymidine incorporation assay. we found that exosomes derived from × to × mscs exhibited a dose-dependent inhibition of lymphocyte proliferation. exosomes derived from × mesenchymal stromal cells co cultured with pha activated pbls, activated b cells and activated t cells showed proliferation inhibition of %( p ⩽ . ), . % (p ⩽ . ) and . % (p ⩽ . ), respectively. in order to understand the molecular mechanism behind the immunomodulatory effect of mdes, we have profiled mde's mir content using illumina hiseq platform and we are currently profiling co cultured activated lymphocytes mrna content using next-generation sequencing system, illumina. preliminary results demonstrate some higher abundance of specific mscs derived mirs in the mdes. hbmmscs have been shown to serve as immune modulators in patients with acute and chronic graft versus host (gvhd). in the future, mdes may provide an alternative therapy for gvhd. compared with bmmscs, mdes are more stable, have no risk of aneuploidity or ectopic proliferation and have less probability of immune rejection. additional studies are needed to explore the applicability of mdes to serve as modulators of the immune response. disclosure of conflict of interest: none. graft-versus-host disease (gvhd) is the major complication after allogenic haematopoietic stem-cell transplantation s (hsct). extra virgin olive oil (evoo) is a source of phenolic compounds such as glycoside oleuropein, hydroxytyrosol and tyrosol. olive oil polyphenols have shown antioxidant, immunomodulatory, antiproliferative, anti-apoptotic and antiinflammatory properties that might be useful in the prophylaxis and treatment of gvhd. polyphenolic extract (pe) of evoo was obtained by the method described by vazquez roncero et al. with some modifications. briefly, fifty grams of evoo (oleoestepa, seville, spain) was extracted with methanol/water ( : , vol/vol, ml ). the mixture of evoo, methanol and water was decanted and the methanolic extract was concentrated and lyophilized. then, the effect of pe in cell viability and activation of t lymphocytes from healthy donor's buffy coats either resting or activated with anticd plus anticd was analyzed by flow cytometry after staining with aad, anexin-v and cd . proliferation assays were performed with pkh and the quantification of il- , il- , il- , il- , tnf-α and ifn-γ cytokines in cell culture supernants with bd cytometric bead array (cba). signaling pathways were analyzed by western blot. finally, in a mouse model of acute gvhd (c bl/ in balb/c), mice were randomized into two experimental diet groups: standard diet ( s harlan laboratories) and standard diet ( s harlan laboratories) supplemented with ppm of pe obtained of evoo. the severity of gvhd was assessed by a scoring system described by cooke et al. that incorporates five clinical parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity. pe did not affect t cell viability. by contrast, pe decreased t-cell activation and proliferation of t-lymphocytes stimulated with anticd plus anticd . in addition, there was a decreased production of th (ifnγ, il- and tnf) and th cytokines (il- , il- and il- ) in the presence of pe. regarding the signaling pathways analyzed, pe inhibited phosphorylation of akt and nuclear translocation of nfkb in activated t cells. in the mouse model of acute gvhd, animals which received the pe supplemented diet had an increased survival as compared to mice receiving a standard diet. also, gvhd incidence was significantly lower among mice receiving the pe supplemented diet as assessed by both the presence of gvhd signs as well as pathological examination. polyphenols obtained from evoo are an important immunomodulatory agent capable to reduce the proliferation and activation of activated t cells and the production of proinflammatory cytokines. in a mouse model of acute gvhd, pe supplemented diet reduced the incidence and severity of the disease and increased the survival of mice. disclosure of conflict of interest: none. graft-versus-host disease (gvhd) is a leading cause of postallogeneic haematopoietic stem cell transplantation (hsct) morbidity and mortality ( ) . extracorporeal photopheresis (ecp) is an alternative therapeutic strategy that appears to act in an immunomodulatory fashion, potentially involving regulatory t lymphocytes and dendritic cells in patients who are refractory to steroids. dendritic cells (dcs) are the most important antigen-presenting cells, playing a pivotal role in t-cell function and in the link between innate and adaptive immunity. moreover, dcs are also critical mediators of immune tolerance and energy. they can be divided into two major subsets, plasmacytoid dcs (pdcs) and myeloid dcs (mdcs) which have distinct functions. pdcs play a pivotal role in peripheral tolerance through generation of regulatory t (treg). on the other side mdcs promote, as well as pdcs, th and th /tr responses ( - ). our study was performed to understand the mechanism of action involved in immunomodulatory effect of ecp. as the modulation of dcs and tregs number and function ( , ) may be a central mechanism of ecp in maintaining self-tolerance, down-regulating immune responses, and limiting inflammation ( ). eight patients affected by gvhd were included in this pilot study. in ecp apheresed mononuclear cells are exposed to methoxypsoralen and uva radiation. after this photoactivation, which induces dna damage and apoptosis, the cells exposed are re-infused into the patient inducing an immunomodulatory effect. all patients or their legal guardians gave their consent for this study. a sample of peripheral blood (pb) (basal condition), a sample of apheresis pre-uva photoactivation (pre-pa) and a sample of photoactivated apheresis (pa) were collected at the first day of ecp and every week for the first month of treatment. circulating dcs, mdcs (cd / -cd +cd +), pdcs (cd / -cd +cd +) and tregs (cd +cd +foxp +) were directly enumerated and phenotypically characterized. the assays were performed at day+ ,+ , + ,+ ,+ data are expressed as mean ± s.d. of absolute number of cells/μl. at day + there were no differences in the absolute number of both mdcs and pdcs between pre-pa and pa. consequently there were no differences between pb and pa. from day + till + we observed an increase of these two cellular populations at every date of treatment. comparing the basal pb of day + vs day + we observed an increment of % and %, respectively for mdcs and pdcs (mdc from cell/μl to cell/μl; pdc from cell/μl to cell/μl). comparing the basal pb of day + vs day + we observed an increment of % of tregs (from cell/μl to cell/μl) while we observed a median increment of % calculated between pre-pa and pa of each day of treatment from day to day + . no firm conclusions can be drawn from a clinical point of view, however a biological effect has certainly highlighted. in particular no substantial differences in basal pb mdc or pdc emerged during the first month of treatment while a significant increase of mdc and pdc can be observed since day + following uva photoactivation. regarding tregs we observed an increment of % of tregs between pb from day + to day+ and a median increment of % calculated between pre-pa and pa of each day of treatment. disclosure of conflict of interest: none. [p ] p impact of th cells on xenogeneic graft-versus-host disease l delens, s servais , g ehx , l vrancken , g fransolet , c gregoire , m hannon , s dubois , c daulne , f baron and y beguin giga i : hematology, university of liege acute graft-versus-host disease (gvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation. its pathophysiology is complex and not yet fully understood. in particular, the impact of th cells on murine acute gvhd has yielded conflicting results, while demonstration of increased levels of th cells at the site of acute gvhd provided only indirect evidence of their involvement in humans. here, we assessed the potential implication of th cells in a humanized mouse model of xenogeneic gvhd (x-gvhd). methods: x-gvhd was induced by infusing human peripheral blood mononuclear cells (pbmcs) into nod-scid il- rγnull (nsg) mice given . gy total body irradiation day prior transplantation. th cells were generated by culturing naive cd + t cells with anti-cd /anti-cd coated beads under th -skewing cytokines (tgf-β , il -β, il- , il- , il- , neutralizing anti-il- and anti-ifnγ antibodies) in hypernatremic conditions (nacl mm). results: after days of culture, a median of . % of il- a+ cells was obtained. we confirmed the expression of il- a, rorc and il- r by these cells by rt-qpcr. we next assessed the co-injection of human pbmcs ( . ) with in vitro differentiated cells under th skewing conditions ( × ) (co-injection group, n = ), in comparison with the injection of pbmcs alone ( × cells, pbmcs group, n = ). we observed higher x-gvhd score (p %) of cells expressing both il- a+ and ifnγ+ cells (th / th -like phenotype) among cd + il- a+ cells while coinjected mice had higher blood concentration of il- a (p = . ) than pbmc mice. these results demonstrate that addition of th cells worsened x-gvhd confirming their role in acute gvhd pathogenesis. disclosure of conflict of interest: none. although survival from allogeneic stem cell transplantation (hsct) has significantly improved, acute graft-versus-host disease (gvhd) remains a major cause of death. intestinal dysbiosis has been associated with acute gastrointestinal gvhd and poor outcome after hsct. we reported a correlation between microbiota (gm) composition and short chain fatty acid (scfa) production and gvhd in transplanted children. to assess how the metabolic pathways of gm change during transplantation and identify modulators of immune response, we perform first longitudinal metagenomic analysis in children undergoing hsct. patients (pts) ( male; mean age: y) with hematologic malignancies ( all, aml), who received busulphan-based myeloablative conditioning and t-cell replete bone marrow graft were enrolled. pts were prospectively enrolled in a protocol with at least specimens fecal samples collected: one before and two after hsct, in order to build a proper trajectory. gvhd prophylaxis was cyclosporine for pts receiving a matched related donor and cyclosporine, short-term mtx and atg for pts receiving a matched unrelated donor. non-gvhd and gvhd patients had similar exposures to antibiotics during the stool collection. of these pts, % developed gvhd within the first days. we applied shotgun metagenome sequencing to total fecal dna from samples collected. functionalities were assigned by reads mapping at different levels of the kegg database. relative abundance was calculated and statistical analysis was performed. according to our findings, core functional profiles were overall conserved through the time-points in all patients ( figure a ), in contrast to the phylogenetic profiles behavior, this finding confirming the overall redundancy of gut microbiome core functionalities. analyzing the single metabolic pathways in subjects who developed gvhd, we found in the pre-hsct period a higher relative abundance of nucleobasis (purine and pyrimidine) metabolism (p o . ) and branched-chain amino acids biosynthesis (p o . ). functions related to the production of branched-chain amino acids are involved in the biosynthesis of the cell wall of gram-negative bacteria, microorganisms including subgroups with well know opportunistic pro-inflammatory. in addition, post-hsct samples of gvhd patients showed a lower abundance of genes involved in polysaccharides metabolism, as glycan biosynthesis and glycosaminoglycan degradation (p o . ) ( figure b ). glycosaminoglycan degradation activity gets bacteria able to survive during extreme situations, as fasting using mucus polysaccharides as energy source, contributing to maintain a mutualistic composition of gm and scfa production by the saccharolytic functions of the endogenous mucus polysaccharides. this study detects functional peculiarities in the gm of non-gvhd pts. the gut metagenome configuration of non-gvhd patients is structured to derive scfa after hsct. the production of these metabolites promotes peripheral regulatory t-cell generation , potentially explaining the protective role of gm from gvhd. although intestinal epithelial cells (iecs) are crucial regulators of barrier function and immune homeostasis, they also facilitate inflammation in exaggerate responses to proinflammatory mediators by pretransplant conditioning regimen, which plays a critical role in amplifying graft-versus-host disease (gvhd). thus inhibition of the converting to pathogenic iecs by conditioning may represent a novel approach to inhibit gvhd. aryl hydrocarbon receptor (ahr) is the ligandactivated transcription factor which has the ability to mediate the biochemical, metabolic, and toxic effects of environmental chemicals. recently, it has been demonstrated that ahr is an important regulator of cell development, differentiation, and function of both innate and adaptive immune cells. the ability of ahr is induced by respond to endogenous ligands generated from the host cell, diet, and microbiota. here, we investigated the regulatory role of ahr in iecs under inflammatory responses and its therapeutic activity for modulation of gvhd. ahr and cyp a expression in mouse iecs were determined by real-time pcr. mouse iecs were pretreated with endogenous ahr ligands l-kynurenine (l-kyn, mm) or pbs for h and then stimulated with lps or il- b for h. cytokine levels were measured using the mouse flex-set cytokine bead array or real-time pcr. b d f (h- b/d) recipients were administrated l-kyn daily by i.p. injection for days. then the recipients were lethally irradiated and transplanted with × tcd-bm plus × t cells from b (h- b) donor. mice were monitored every other day for survival and clinical score. colons were collected and stained with hematoxylin and eosin (h&e) for histopathological scoring. we found that ahr was constitutively expressed in the mouse iecs. cyp a (an ahr target gene) was significantly increased by treatment of l-kyn under un-stimulatory condition. we further observed that l-kyn completely abrogated il- β-mediated il- or lps-mediated tnf-a expression in iecs. administration of bdf recipient mice with l-kyn before transplantation significantly reduced the lethality and severity of gvhd. histopathology clearly revealed that treatment of l-kyn inhibited intestinal gvhd. our results demonstrate that ) ahr is constitutively expressed in iecs, ) treatment of endogenous ligand l-kyn induce ahr activation in the steady status, ) ahr activation blocks conversation of the epithelial cells into pathogenic cell type, and ) pre-administration of ahr ligand reduces gvhd. our study suggests that activation of ahr pathway in iecs before allogeneic hematopoietic stem cell transplantation (hsct) is a possible strategy to reduce intestinal gvhd. disclosure of conflict of interest: none. [p ] s relating with acute graft-versus-host disease (gvhd) after allogeneic hematopoietic stem cell transplantation (allo-hsct), protecting endothelial cells (ecs) from damage may be a potent prophylaxis and therapeutic strategy of acute gvhd (agvhd). conventional agvhd therapies may cause many adverse side effects because of their multiple targets. therefore, we explored the therapeutic efficacy of simvastatin, a lipid-lowering drug, which has been demonstrated endothelial protection. our previous clinical observation has found patients with agvhd had lower angiopoietin- (ang- ) level at day but higher ang- level at day than those without agvhd. in this study, we explored changes in ang- and ang- expression in an agvhd mouse model and determined whether simvastatin prevents gvhd through regulating ang- and ang- expression. we preincubated ea.hy ecs with simvastatin ( mmol/l) h before stimulated with tnf-a, then ang- and ang- concentration in the cell supernatant was measured by elisa. ang- and ang- mrna and protein level of treated and untreated cells were examined simultaneously. in vitro simvastatin increased ang- production and release but conversely inhibited ang- release from ea.hy ecs. donor mice spleen cells were injected along with bone marrow cells into recipient mice after lethal irradiation to induce agvhd. simvastatin was administered orally once daily to mice ( mg/kg) for days after allo-hsct and started − day after allo-hsct. then mice survival time was monitored and organ damage was evaluated. the plasma level of ang- and ang- was measured by elisa, expressions of ang- and ang- in aortic endothelium were assessed by immunohistochemistry. simvastatin improved the survival and attenuated the histopathological gvhd grades of agvhd mice. plasma levels of ang- were significantly decreased, while plasma levels of ang- obviously increased in agvhd mice after transplantation. simvastatin reduced plasma levels of ang- , elevated the plasma levels of ang- as well as the aortic endothelial levels of ang- and ang- . in summary, simvastatin represents a novel approach to combat gvhd by increasing ang- production while suppressing ang- release to stabilize endothelial cells. there is a growing evidence of safety and efficacy of posttransplantation cyclophosphamide (ptcy) in stem cell transplantations (sct) from different donors and graft sources. still the optimal combination of immunosuppressive agents with ptcy should be elucidated for different types of scts. we report the -year update of the prospective nct single-center trial that evaluated risk-adapted graft-versushost disease (gvhd) prophylaxis with ptcy in related, unrelated and haploidentical scts. adult patients (median age y.o., range: - ) with hematologic malignancies, including aml ( . %), all ( . %), cml ( . %), mds ( %), and lymphomas ( . %), were enrolled in the study. % of patients were classified as salvage. % received the graft from matched related (mrd), % from matched/mismatched unrelated (mud/mmud), and % from haploidedntical (haplo) donor. % received bone marrow graft (bm) and %peripheral blood stem cell (pbsc) graft. . % had myeloablative conditioning and . %-reduced-intensity conditioning. gvhd prophylaxis for matched bm grafts consisted of single-agent ptcy mg/kg days+ ,+ , for matched pbsc graft-ptcy+ tacrolimus+ mycophenolate mofetil (mmf) mg/kg days - , and for any mismatched graft-ptcy+ tacrolimus+ mmf mg/kg days - . median follow-up was months (range: - ). grade ii-iv ( % vs % vs %, p = . ) and grade iii-iv acute gvhd ( % vs % vs %, [p ] p = . ) were not different in mrd, mud/mmud and haplo groups, respectively. moderate and severe chronic gvhd was infrequent in all groups with slightly lower incidence after mud/mmud graft: and % vs % vs %, p = . . nonrelapse mortality (nrm) was not different after mrd, mud/ mmud and haplo sct ( % vs % vs %, respectively, p = . ), while relapse incidence was higher after mrd and haplo grafts: ( % vs % vs %, p = . ). -year overall survival (os), event-free-survival (efs), and gvhd-relapse free survival (gfrs) were % vs % vs % (p = . ); % vs % vs % (p = . ); % vs % vs % (p = . ) for mrd, mud/mmud and haplo groups, respectively. in the multivariate analysis only disease risk index (hr . %ci . - . , p = . ), severe sepsis (hr . %ci . - . , p = . ) and chronic gvhd (hr . %ci . - . , p = . ) were predictive for efs, while type of donor was not a significant factor (hr . %ci . - . , p = . ) (figure ). the incidences of complications were: hemorrhagic cystitis- %, sepsis- %, severe sepsis- %, invasive mycosis- %, cmv reactivation- %, veno-occlusive disease- . %, transplant-associated microangiopathy- . %, grade - liver toxicity- %, grade - kidney toxicity- %. more than one third of patients experienced poor graft function during days after sct, and in % of them cmv, hhv and bk virus reactivations were identified as the cause. the reported risk adapted strategy alleviates the risk of gvhd and nrm after mmud and haplo grafts. the observed differences in the relapse incidence, os and efs were predominantly due to unbalanced disease risks in the groups. the relapse of underlying malignancy with this prophylaxis still significantly influences the outcome. substantial number of patients experience poor graft function, which doesn't translate into nrm. disclosure of conflict of interest: none. a high migratory capacity of donor t-cells in response to the lymph node homing receptor ccr increases the incidence and severity of graft-versus-host disease vg garcía de soria , ip sainz , e jiménez , a arriero , c fernández-arandojo , c cuesta , b colom , a marcos , a rosendo and cecilia muñoz calleja department of hematology hospital universitario de la princesa and department of inmunology. hospital universitario de la princesa graft-versus-host disease (gvhd) pathogenesis involves migration of the donor t-cells into the secondary lymphoid organs (slo) in the recipient, which is steered by two homing molecules: cd l and ccr . therefore we investigated whether the migratory capacity of donor t-cells is associated with gvhd. this single center prospective study included donor-recipient pairs. in vitro chemotaxis assays of the lymphocytes of the apheresis product were performed in parallel with the analysis of cd l and ccr by flow cytometry. the potential of activation of ccr + t-cells was assessed through ex vivo activation assays with peripheral blood monuclear cells (pbmc) from healthy donors using anti-cd and anti-cd mabs. the migratory index to the ccr ligands, ccl and ccl , was higher in t-cells from donors whose recipients will develop gvhd. these data indicated that the migratory capacity of the donor t-cells is clearly related to the development of gvhd. this prompted us to study the relationship between gvhd and the expression of two of the most relevant molecules in the trafficking of lymphocytes towards slo, cd l and ccr ,as a subrogate index of the migratory potential of t-cells. consequently, we quantified the numbers of cd l+ and ccr + t-cells in the graft. the initial transversal analysis of our data revealed that the percentage of cd l+ lymphocytes in the apheresis product was very low compared to healthy lymphocytes. the analysis also confirmed that cd l undergoes plasma membrane shedding after g-csf mobilization thus making it a non-valid biomarker. the analysis of ccr molecule revealed that the acute gvhd group received higher percentage of cd +ccr + t-cells, whereas chronic gvhd patients were transplanted with higher percentage of cd +ccr + t-cells compared to the non gvhd group. these results were confirmed when patients were subdivided into degrees of severity. a multivariate analysis was performed to investigate the real value of ccr to predict the development and severity of gvhd, and confirmed that ccr expression is a risk factor for the development of gvhd. thus, the percentage of ccr +cd + t-cells increases the probability of developing acute gvhd (or = . , c.i ( %) = . - . , p = . ) and suffering a higher degree (or = . , c.i ( %) = . - . , p = . ). similarly, the or of the percentage of ccr +cd + t-cells was . (c.i ( %) = . - . , p = . ) and . (c.i ( %) = . - . , p = . ) for the development of chronic gvhd and its degrees, respectively. finally, to study the potential of activation of ccr + t-cells, we carried out ex vivo activation assays with pbmc from healthy donors using anti-cd and anti-cd mabs and the expression of cd l on cd + t-cells and of cd on cd + t-cells as markers of activation, demonstrating that ccr + t-cells exhibited higher potential of activation than ccr -t-cells. to our knowledge this is the first analysis of the influence of the migratory capacity of the donor t-cells on clinical outcome following allogeneic hsct. our data show that ccr could be considered a subrogate biomarker of the migratory capacity of the donor lymphocytes for predicting the risk of suffering gvhd. based on the previous findings, we propose that the selective depletion of ccr expressing cells could be an effective preventive therapy for gvhd. disclosure of conflict of interest: none. previously published p a single center research for outcome in patients receiving imatinib for steroid-refractory chronic gvhd after allogeneic stem cell transplantation l ni, y luo, y tan, y hu, y zhao, j shi and h huang despite of major progress in allogeneic stem cell transplantation over the last decades, steroid-refractory chronic graftversus-host disease (sr-cgvhd) remains a leading cause of late morbidity and mortality. pre-clinical evidence confirms cgvhd has antibodies activating the platelet-derived growth factor receptor (pdgf-r) pathway. since this pathway can be inhibited by imatinib, we performed a study including patients with sr-cgvhd given imatinib at a dose of mg per day. all patients with a median age of years (range: - ) underwent allogeneic hematopoietic stem cell transplantation in our single center between and , and chronic gvhd occurred at a median time of months (range: - ) after transplantation. patients had active cgvhd with measurable involvement of skin, lung or other districts and had previously failed in first-line immunosuppressive therapy. the major organs involved were lung (n = ), skin (n = ) and mouth (n = ), including cases involving both lung and skin, cases involving or more organs. according to the national institutes of health (nih) criteria and nih global severity, patients were evaluated as severe cgvhd, and the other three were moderate. meanwhile, the nih working group had updated its recommendations for overall responses, consisting of complete remission (cr), partial remission (pr), and lack of response (unchanged, mixed response, progression). cr was defined as resolution of all manifestations in each organ or site, and pr was defined as improvement in at least organ or site without progression in any other organ. after months treatment, patients receiving sufficient dose of imatinib revealed overall response rate (orr) at . %, and orr remained unchanged at months assessment, but with cr rate increased to . %. two patients couldn't meet the response of cr or pr were considered as a lack of response, including one evaluated as unchanged and one mixed response because of pr in lung accompanied by progression in eyes. with a median follow-up of months, patients were alive, with a year estimated overall survival was . %. patients eventually died of pneumonia. except patient discontinued imatinib because of grade toxicity as gastrointestinal discomfort at the first month, no one had imatinib-related grade to toxicity. this study suggests that imatinib is a promising and better tolerated treatment for patients with sr-cgvhd. disclosure of conflict of interest: none. acute graft-versus-host-disease (agvhd) is a major complication after allogenic hematopoietic transplantation (allo-sct). in recent years, a number of tissue-specific proteins have been described as biomarkers that could contribute to anticipate and/or diagnose this complication earlier and more accurately. reg α (regenerating-islet-derived- -alpha) has been directly related to gastrointestinal (gi) agvhd. our objective was to analyze plasma levels of reg α at days + and + in patients who underwent unmanipulated haploidentical transplantation with reduced conditioning regimen (haplo-ric), and to correlate the results with the development of agvhd. we retrospectively analyzed consecutive patients ( - ) who underwent haplo-ric with post-transplant cyclophosfamide (days + , + ), mmf and csa as gvhd prophylaxis. seven cases were excluded due to early death (before day + ) and cases due lack plasma sample. characteristics of the patients included in the analysis are described in table . reg α detection was performed by elisa (mbl international corp, woburn, ma) according to manufacturer's instructions on μl of plasma obtained at day + and + . the association of the incidence of agvhd with known clinical variables and plasma reg a levels were performed by cox regression and mann-whitney u-test, respectively. the determination of the best cut-off of reg α levels to stratify patients with gi agvhd was performed with roc curves. the stadistical program used was r v . . . the cumulative incidence of grade ii-iv and grade iii-iv agvhd was % and %, respectively. characteristics of agvhd are shown in table . no association was found between agvhd and usual clinical variables (stem cells source, age, sex, conditioning regimen, donor/recipient sex and number of infused cd + cells), and with plasma reg α levels at day + .plasma reg α levels at day + were higher in patients who devolved gi agvhd compared to patients who did not showed gi agvhd (median [p ] s and range: ( - ) vs ( - ) pg/ml, p = . , figure ).the best cut-off selected on day + was pg/ml (s %, e %). patients with levels higher than pg/ml at day + had a significantly higher incidence of gi agvhd grade ii-iv (hr . , p = . , figure ). plasma levels of reg α at day + after haplo-ric correlated with the occurrence of gi agvhd grade ii-iv. therefore, plasma levels of reg α could be use for the prediction and/or diagnosis of gi agvhd. disclosure of conflict of interest: none. anti-fibrotic treatment with pirfenidone in patients with gvhd-associated bronchiolitis obliterans syndrome ke hostettler, s gerull, g nair , j passweg , m tamm and j halter hematology, university hospital basel, switzerland and pneumology, university hospital basel, switzerland prognosis of lung gvhd remains poor due to progressive decrease of lung function and repeated infections. pirfenidone exhibits anti-fibrotic effects and has been shown to reduce disease progression in patients with idiopathic pulmonary fibrosis. five patients with established bos (nih criteria ) and stable or deteriorating lung function under standard immunosuppressive treatment without active infection were treated with pirfenidone ( mg/d) in addition to their current therapy. clinical assessments and pulmonary function tests were performed every three months. five patients ( m, f), median age y (range: - y) that were diagnosed with bos at a median time of . months post-transplant started pirfenidone at a median time of months ( - ) after diagnosis of bos. two patients are currently still under treatment after and days. two patients had to stop treatment due to financial reasons after and days of therapy. one patient never reached more than % of the planned dose due to gastro-intestinal symptoms and was excluded from further analysis. at the start of treatment median fev was . l ( . - . ); . % predicted (range: - %) and median fvc . l ( . - . ); - % predicted. median fev trajectory was − . % predicted/ month during median months before start of pirfenidone (median − ml/month) and + . % predicted/month (+ . ml/month) during treatment with pirfenidon. the treatment was well tolerated except in one patient with gastrointestinal complaints, no phototoxic reactions or serious drugrelated adverse events occurred. in our small number of patients pirfenidone was rather well tolerated and generally safe. the observed, albeit small trend in change of fev trajectory justifies further studies of anti-fibrotic therapy as a new therapeutic option in bos after allogeneic hsct. disclosure of conflict of interest: none. anti-thymocyte globulin has been widely used for the prevention of severe graft versus host disease in patients undergoing hsct from unrelated donor. however, the optimal dose remains to be defined. in samsung medical center (seoul, korea), institutional strategy for the atg use has been changed since april , and we hypothesized that the incidence of chronic gvhd may differ by atg strategy. before april , atg . mg/kg was routinely used in allogeneic hsct from unrelated donor, whereas, the dose of atg was escalated to . mg/kg since april . in this study, a total of patients who underwent allogeneic hsct from matched or unmatched unrelated donor between jan and dec were retrospectively analyzed. peripheral blood was used as the source of stem cells in all patients. after a median follow up of . months, the cumulative incidence of moderate to severe chronic gvhd was . % ( % confidential interval [ci], . to . ) in the low-atg group and . % ( % ci, . to . ) in the non-atg group (p = . ). the rate of year overall survival (os) was not significantly different between the groups ( . % in low-atg group vs . % in high-atg group, p = . ), as was the rate of disease free survival (dfs) ( . % in non-atg group vs . % in atg group, p = . ) and cumulative incidence of relapse (cir) ( . % in non-atg group vs . % in atg group, p = . ). in allogeneic hsct from unrelated donor, larger atg dose ( . mg/kg) did not reduce the incidence of chronic gvhd when compared to lower atg dose ( . mg/kg). disclosure of conflict of interest: none. allogeneic hsct provides a curative chance for patients with hematological fatal disease. however, substantial risks remain for morbidity and mortality caused by disease relapse and graft-versus-host disease. in samsung medical center (seoul, korea), institutional strategy for the atg use has been changed since april , and we hypothesized that the incidence of chronic gvhd may differ by atg strategy. before april , atg was not routinely used in matched sibling donor (msd) transplantation, whereas, atg mg/kg has incorporated into hsct process in transplantation from msd thereafter. in this study, a total of patients who underwent allogeneic hsct from msd between jan and dec were retrospectively analyzed. peripheral blood was used as the source of stem cells in all patients. after a median follow up of . months, the cumulative incidence of moderate to severe chronic gvhd was . % ( % confidential interval [ci], . to . ) in the atg group and . % ( % ci, . to . ) in the non-atg group (p = . ). the rate of -year overall survival (os) was not significantly different between the groups ( . % in non-atg group vs . % in atg group, p = . ), as was the rate of disease free survival (dfs) ( . % in non-atg group vs . % in atg group, p = . ) and cumulative incidence of relapse (cir) ( . % in non-atg group vs . % in atg group, p = . ). in allogeneic hsct from msd, atg use was significantly associated with less occurrence of chronic gvhd, but not linked to increasing risk of relapse, with showing similar os and dfs between atg and non-atg group. disclosure of conflict of interest: none. long-term follow-up from the prospective randomized phase iii multicenter trial comparing a standard gvhd prophylaxis with cyclosporine a and methotrexate with or without additional pretransplant atlg (grafalon, previously atg-fresenius s) (given mg/kg/day, days − to − ) in unrelated donor hematopoietic cell transplantation after myeloablative conditioning resulted in a significant reduction of acute and chronic gvhd without compromising relapse rate and survival [ , , ] . here we report on a subsequent prospective non interventional observational study evaluating the outcome of patients receiving atlg in unrelated donor transplantation in day to day clinical practice without the selective measures of a clinical trial (german clinical trials register drks ). thirteen transplant centers included patients with haematological malignancies (median age years, iqr - years, range: - years) in early (n = , %), intermediate (n = ; %) or advanced (n = ; %) disease status receiving marrow (n = ) or pbsc (n = ) from / matched ( ; %) or mismatched ( ; %) unrelated donors (n = related) after myeloablative (n = , %) or ric (n = , %) conditioning. gvhd prophylaxis consisted of calcineurin inhibitors, mainly csa (n = , %) with mtx or mmf and atlg. different dosing regimens were allowed according to current practise of centers. median total atlg dose was mg/kg (iqr - mg/kg, range: - mg/kg). median follow-up was months (range: - months). as compared to patients in our randomized phase iii multicenter trial [ , , ] , patients in this study were older; advanced disease status, / match, pbsc transplantation were more frequent, and given median atlg dose was lower. acute and chronic gvhd, nrm, relapse risk, dfs and os at one year were similar to the results obtained in our randomized trial: incidence of°ii-iv agvhd: %, iii-iv agvhd: %; moderate/severe cgvhd: %; nrm: %; risk of relapse: %; relapse mortality: %; os: %. the experience in day to day clinical practice confirms the results shown in our randomized trial, namely the gvhd protective effect of atlg without compromising nrm or relapse rates. baseline calprotectin as a predictor for acute gastrointestinal graft versus host disease (gvhd)-a prospective study n schmidlin , a holbro , , jp halter , d heim , l infanti , , a plattner , r plattner , c rothen , a buser , , c bucher and jr passweg division of hematology, university hospital basel, switzerland; blood transfusion center, swiss red cross, basel, switzerland and rothen medical laboratories, basel, switzerland graft versus host disease (gvhd) is a major complication after allogeneic stem cell transplantation. so far there is no good validated predictor for the incidence and severity of gvhd. fecal calprotectin (cpt) is a protein in leukocytes with antibacterial properties. it has been shown to be elevated in acute gastrointestinal gvhd. additionally, cpt may be predictive for treatment response. the aim of the current prospective study was to investigate the role of baseline cpt in predicting incidence and severity of intestinal gvhd. in this prospective study conducted at the university hospital basel, switzerland, we included all adult patients undergoing hsct. the institutional review board approved the study. data were collected prospectively. cpt was measured twice before conditioning and at transplantation. fecal samples for cpt were obtained before conditioning and on the day of transplantation and assessed twice by standard elisa. between march and april a total of patients ( % males, patients with both baseline and transplant cpt values) were included. patient, disease and transplant characteristics are described in table . median age at transplant was years (range: - years). most patients had myeloid neoplasia and % received myeloablative conditioning. gvhd prophylaxis consisted mainly of cyclosporine containing regimens ( %). cpt levels ranged from to μg/g both at baseline (median: μg/g) and at transplantation (median: μg/g), with a good consistency between the two measurements performed (internal quality control). on the other hand, cpt did not correlate with c-reactive protein. the two measurements were taken in median days apart, depending on the conditioning regimen. eighty-five patients had an increase of at least μg/g between baseline and transplantation. overall ( . %) patients developed acute intestinal gvhd (grade : ; grade : ; grade : , and grade : patients, respectively). cpt both at baseline and at transplantation was not predictive for the incidence of gvhd, acute intestinal gvhd, and for acute intestinal gvhd grade - ( figure) . additionally, we did not find a significant association between cpt levels and the above mentioned endpoints for patients showing an increase of cpt of at least μg/g between baseline and transplantation. in the current prospective study, we didn't find any correlation between baseline cpt values and the incidence and severity of gvhd and intestinal gvhd. further studies identifying early markers and predictors of gvhd are urgently needed. [p ] disclosure of conflict of interest: none. calcinuerin inhibitor (ci) free graft-versus-host disease (gvhd) prophylaxis: its effects on resource utilization, renal function, and the cost of care m muilenburg , k cole, m abidi , , s williams and as al-homsi , spectrum health blood and marrow transplantation and michigan state university, college of human medicine effective gvhd prevention following allogeneic hematopoietic stem cell transplantation (ahsct) is vital to reducing transplant morbidity and mortality and improving overall outcomes. several strategies are currently utilized for gvhd prophylaxis including mtx, mmf, cis, post-transplant cyclophosphamide (cy), and proteasome inhibitors. recently, we described the results of a phase i-ii trial of cyclophosphamide (cy) and bortezomib (bor) where patients (pts) received cy ( mg/kg) on days (d) + & + and bor on d & + . the incidences of grade ii-iv and grade iii-iv acute gvhd were % and %. the incidence of chronic gvhd was %. in addition to gvhd, there are other factors that affect patients' quality of life and cost of care and that should be considered. it is well documented that cis have an unfavorable toxicity profile. this includes nephrotoxicity and electrolyte disturbances. furthermore, the cis need serial level monitoring. thus, we endeavored to compare the effects of cybor combination against ci-based regimens by focusing on electrolyte requirements, specifically mg, and renal function. we also sought to better understand financial considerations surrounding the need for ci drug level monitoring. sixteen pts were randomly selected from the cybor group and patients from an internal control group of patients who received mmf and cyclosporine or tacrolimus following reduced-intensity ahsct. the groups were well matched in regards to age, sex, disease status, pam score, and baseline renal function. on each pt, mg results from d to + were compiled. based on institutional protocol, a mg replacement value was assigned as well as the corresponding drug and infusion charges. next, the number of immunosuppressant (is) trough levels from d to + was tallied and the internal lab charges calculated. to compare renal function, gfr was calculated at baseline, d , and d + . χ tests and wilcoxon rank square tests were used to analyze the data. for the cybor group, median mg value was . mg/dl (iqr . ) vs . ( . ) in the control group (po . ). cybor pts required a median of grams ( ) vs grams ( ) in the control group (p = . ). the cost of mg replacement and infusion was significant (p = . ) ( table ) . for is checks, drug levels were checked a mean of . times per patient in the cybor group compared to . times in the control group (po . ), which also translates to significant savings ( table ) . considering these costs, the cybor group saved~$ . for gfr, cybor pts and control pt had reduced gfr at baseline. on d + , cybor pts had better renal function in comparison to the control group (p = . ) ( figure ). in summary, cybor significantly reduced the use of resources post-transplant and thereby the associated cost related to mg replacement and need for drug level monitoring. furthermore, cybor preserved renal function at d + . these findings could also impact patient's quality of life. although our cost analysis was restricted to certain aspects of care and did not take into account other factors, it highlights specific important benefits of ci-free gvhd prophylaxis and supplicates further study. a formal prospective comparison of cost and qol is warranted. . related donor n = and unrelated donor n = (compatibility / n = ), with conditioning regimen: myeloablative n = and non-myeloablative n = . median interval between transplantation and diagnosis of cgvhd of months( . - . ); and between cgvhd diagnosis and sativex months ( . - . ), with a median of prior treatment lines ( - ). at the time of beginning, the cgvhd was extensive in all patients, severe cgvhd n = and moderate cghvd n = . all patients except one had cutaneous involvement (n = with sclerodermal features). in addition, other organs were affected: digestive n = , pulmonary n = , hepatic n = , ocular n = , oral n = , genital n = and muscular n = . drug was started because of pulmonary affectation in patients and due to sclerodermal/muscular involvement in patients. concomitant therapies during treatment were: topical cutaneous treatment n = , topical ocular treatment n = , pulmonary n = , sirolimus n = , tacrolimus n = , oral corticosteroids n = , extracorporeal photopheresis s n = , ruxolitinib n = , imatinib n = , mesenchymal stem cells n = . the mean dose were three puff/day ( ) ( ) ( ) ( ) , with good tolerance, only two discontinuations of treatment because of adverse effects. median time of treatment days ( to ). at the time of the analysis patients were still under treatment. responses mainly occurred within the first days, s with a median time of duration of days ( in ). responses after two months of treatment were: partial organ response, mixed responses, unchanged and organ progressions; at th day ( / ) only two patients maintained their responses (one pr and one mixed response). it must be pointed out that one patient who reached pr with sativex in monotherapy maintain response after months of treatment. in addition, cramps were resolved in patients. sativex appears to be an effective treatment option in patients with chronic gvhd, particularly in those having cramps, sclerodermal features and pulmonary affectation. as seen in multiple sclerosis context, the main issue with its use is the loss of response in the long-term follow up. the median dose is inferior to the one described in ms, leaving the question if higher doses can deepen the response. these results should be confirmed in prospective trials. disclosure of conflict of interest: none. several risk factors associated with acute and chronic graftversus-host-disease (gvhd) have been identified in multiple studies. most commonly associated factors are human leukocyte antigen (hla), mismatch between recipient and donor, as well as several other characteristics such as age, conditioning regimen and prior acute gvhd. objective: the aim of this study was to evaluate the characteristics of acute and chronic gvhd in patients who underwent an allogenic hematopoetic stem cell transplantation (hsct), identify differences in the profile risk factors for acute and chronic gvhd and their impact in post-transplant morbidity and mortality. this retrospective study included mexican adult patients who received an allogenic hsct between january and march , at instituto nacional de cancerologia. we analyzed patients with a median age of years ( - ), from which, % were male patients. among the participants with hematologic malignancies, were previously diagnosed with acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, lymphoblastic lymphoma and with myelodysplastic syndrome. because bone marrow transplants are not performed at this institution, all transplants were from peripheral blood stem cell harvest. acute gvhd prophylaxis consisted in a triple immunosuppressive drug regimen for all patients. . % of the patients had high risk disease prior to hsct. myeloablative conditioning represented % of the applied regimens, which consisted of iv busulfan in . % of the cases. . % of patients, were transplanted within months from diagnosis. the cumulative incidence of acute gvhd at days was . % ( patients). patients with acute gvhd had % grade a, % grade b and % grade c, according to the ibmtr grading system. patients had skin involvement, with grade - acute gvhd in % of the cases, patients developed liver involvement and patients had gastrointestinal tract disease. % of the patients developed chronic gvhd, from which, % were classified as severe, . % as moderate and . % as mild. % of the patients who developed chronic gvhd had a single organ involvement, while . % had or more organs/sites. prior acute gvhd was associated with de development of chronic gvhd. the multivariate analysis identified hla unrelated donor as the only risk factor associated with the development of acute gvhd (hr, . ; % ci, . - . , p = . ). the overall survival at years was of % poor patients who developed acute gvhd and of % for those who didn´t (p = . ). our analysis showed that the incidence of acute and chronic gvhd at our center is lower than the reported at other centers, but we were not able to identify risk factors usually associated with the development of gvhd, perhaps due to the small population that we evaluated. graft versus host disease (gvhd) remains one of the main obstacles to broader application of allogeneic stem cell transplantation (sct). despite the routine use of prophylactic therapies, chronic gvhd (cgvhd) occurs in to % of patients undergoing allogeneic sct. ciclosporin a (csa) remains the backbone for gvhd prophylaxis in both myeloablative (mac) and reduced intensity conditioning (ric) sct. however, in a significant proportion of patients, csa causes important side effects and needs to be discontinued. in this study we have evaluated the impact of substituting csa for mycophenolate mofetil (mmf) as immunosuppression (is), on the incidence of cgvhd. we have compared the outcome of consecutive patients that underwent allogeneic sct from march to november at the bmt unit of the hammersmith hospital and received csa as part of the gvhd prophylaxis. of them, patients ( %) remained on csa prophylaxis for the duration of the planned post sct immunosuppression period and patients ( %) required a switch to mmf before day + . the reason for changing the is was nephrotoxicity in the majority of cases (n = , %), neurological toxicity (n = , %), disease relapse (n = , %), intolerance (n = , %) or not determined (n = , %) . we excluded from the analysis those patients whose is was changed due to the presence of acute gvhd. both groups had similar patient and transplant characteristics (see table ). [p ] however, distribution according to diagnosis showed a predominance of aml ( %) in patients that remained on csa and mds ( %) for those that switched to mmf. the mean survival rate of the entire cohort was . days (± ) . the mean survival of each group was: csa . days (± . ) and mmf . (± . ). this difference in survival reached statistical significance (p: . ). we graded cgvhd using the nih scoring system as mild, moderate and severe. out the patients that continued with csa, . % (n = ) had no cgvhd; . % (n = ) had mild cgvhd; . % (n = ) had moderate and . % (n = ) had severe cgvhd. in patients that switched to mmf . % (n = ) did not develop any cgvhd; . % (n = ) developed mild; . % (n = ) moderate cgvhd and . % (n = ) developed severe cgvhd. (p: . ). the cumulative incidence of any cgvhd at years post sct was % for the csa/mmf group and % for the csa only group (p = . ). csa is one of the standards of care for gvhd prophylaxis in both ric and mac sct. in our cohort of patients, those who remained on csa had a better overall survival and a reduced incidence of chronic gvhd compared with those patients that stopped csa and replaced it by mmf. csa toxicity should be prevented to avoid gvhd-related complications. disclosure of conflict of interest: none. although the outcome of allogeneic stem cell transplantation (sct) from an unrelated donor (ud) has considerably improved over the recent years, graft versus host disease (gvhd) still represents a severe and potentially lethal complication. in vivo t-cell depletion with anti-thymocyte globulin (atg) has been shown to significantly decrease the risk of both acute and chronic gvhd without compromising survival, however the optimal dose has not been defined yet. aim of present retrospective study was to evaluate the impact of two different doses of rabbit atg (thymoglobulin) on gvhd incidence, infectious complications and outcome of patients undergoing sct from ud. between february and september , patients received thymoglobulin mg/kg (atg- group) and received thymoglobulin mg/kg (atg- group) in addition to cyclosporin and short course mtx as gvhd prophylaxis. the two groups were comparable regarding sex, age, diagnosis and disease phase at transplant, comorbidity index, stem cell source and antimicrobial prophylaxis. conditioning treatment was myeloablative in % of atg- group patients and in % of atg- group patients. donor and recipient pairs were / hla matched in % of the cases of the atg- group and in % of the cases of the atg- group (p . ). netrophil engraftment occurred in ( %) patients at a median of days post transplant (range: - days); six patients ( in the atg- group and in the atg- group) died before engraftment. overall, patients ( %) developed grade ii-iv acute gvhd, without significant differences between the two groups (atg- % and atg- %, p . ). similarly, chronic gvhd was not significantly different between the two groups: moderate to severe chronic gvhd occurred in % of the patients in the atg- group and in % of the patients in the atg- group (p . ). univariate logistic regression analysis didn't show any significant differences between the two groups respect the incidence of bacteremia, invasive fungal infections acute and chronic gvhd. with a median follow-up of . months, patients ( %) are alive, in complete remission and after disease relapse. transplant related mortality was superimposable in the two groups (atg- % vs atg- %). kaplan-meier estimates of overall survival and event free survival were % and %, respectively, without statistically significant differences between the two groups and between hla matched and hla mismatched sct. the results of our study suggest that different doses of atg tailored on hla compatibility might be effective for preventing gvhd with any detrimental effect on overall survival and incidence of infectious complications. a prospective randomized study is mandatory to confirm our preliminary results. disclosure of conflict of interest: none. c-reactive protein levels at acute gvhd diagnosis predict steroid-resistance, treatment related mortality and overall survival after allogeneic hematopoietic stem cell transplantation l minculescu , ls friis , bt kornblit , sl petersen , i schjødt , ns andersen and h sengeløv department of hematology, rigshospitalet, copenhagen, denmark acute graft versus host disease (agvhd) remains an excessive cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (hsct). primary treatment consists of high-dose corticosteroids, but a small group of patients are steroid-resistant and their prognosis is especially poor. a predictor of patients at risk of steroid-failure would aid the decision of additional immunosuppressive treatment at an early stage. there is experimental evidence that co-existing inflammation aggravates agvhd. since c-reactive protein (crp) is a systemic inflammatory marker, we aimed to investigate whether crp levels at agvhd diagnosis could predict the risk of failing first-line therapy and developing steroid-resistance. we retrospectively studied patients transplanted between and , table . acute gvhd was diagnosed in patients, of whom had grade ii-iv. crp, total white blood cell-, lymphocyte-and neutrophil counts were available for all patients at the time of agvhd diagnosis. according to local protocol, patients with failed response to high-dose steroid ( mg/kg) were treated with the tumor necrosis factor (tnf) alpha inhibitor infliximab and categorized as steroid-resistant. of grade ii-iv agvhd patients ( %) developed steroid resistant disease. crp levels at diagnosis among these where between o and mg/l. crp levels where significantly higher in patients who developed steroid resistance compared to patients responding to high-dose corticosteroids, p = . , hr . ( % ci . , . ). this translated into significantly increased transplant-related mortality (trm) and decreased overall survival in patients with high crp levels, figure . total white blood cell-, lymphocyte-and neutrophil counts were not associated with steroid resistance in agvhd patients. cxcr is chemokine receptor expressed on activated t lymphocytes, in particular on th cells, nk cells, dendritic cells, and subsets of epithelial and endothelial cells. cxcr ligands attract th cells into inflamed tissues and concomitantly block the migration of th cells. furthermore, inhibitory functional autoantibodies against cxcr occur in humans which play an important role in cxcr -dependent immune regulation. in addition, cxcr regulates endothelial cell homeostasis. there are two variants of cxcr : cxcr -a and cxcr -b. overexpression of cxcr -a on endothelial cells is associated with an increase in cell survival, whereas overexpression of cxcr -b dramatically reduced dna synthesis and up-regulated apoptotic endothelial death. here we have studied if a dysfunctional cxcr axis might be involved in gvhd pathogenesis and could link endothelial and t cell pathology in acute gvhd. we assessed concentrations of the cxcr ligands cxcl , cxcl and cxcl as well as anti-cxcr autoantibodies in patients with high grade ( ) ( ) acute intestinal gvhd for whom serum was available at gvhd onset. furthermore, anti-cxcr autoantibodies and cxcl levels were measured in sera stored before conditioning therapy. all variables were tested for influence on post-gvhd survival using cause-specific cox regression analysis. at gvhd onset, we observed a strong inter-correlation of cxcr ligands, but no correlation with anti-cxcr auto-antibodies. compared with pre-conditioning probes, cxcl levels strongly increased (median to pg/ml, p o . ), whereas anti- these results suggest crp levels at diagnosis as a valid predictor of developing steroid resistant disease in agvhd grade ii-iv and survival in allogeneic hematopoietic transplant recipients. [p ] s cxcr decreased (median . to . u/ml, po . ). anti-cxcr levels before conditioning and at gvhd onset correlated (coeff. . , p o . ), whereas cxcl levels did not. in multivariable analyses, low anti-cxcr and high cxcl measured at disease onset were strongest predictors of survival after acute gvhd. notably, high levels of the proinflammatory chemokine cxcl were particularly prognostic of an adverse outcome of gvhd in the presence of a high endothelial risk as assessed by the previously published easix score, while high anti-cxcr levels were most protective in patients with low easix score (that is, low endothelial risk). a score based on cxcl , anti-cxcr , and easix allowed an effective prediction of acute gvhd outcome ranging from mortality % (high cxcl + high easix) to mortality o % (low cxcl , low easix, high anti-cxcr . our data suggest a strong role for the cxcr axis in the pathology of acute high grade gvhd. the opposing effects of cxcl and anti-cxcr indicate a functional, attenuating role for these auto-antibodies. the overall prognostic impact of the immunemodulating cxcr axis appears to depend on the underlying integrity of the patients' endothelial homeostasis. despite some progress in acute lymphoblastic leukemia (all) treatment including modern chemotherapy modalities, monoclonal antibodies and newer tyrosine kinase inhibitors (tki) for ph positive cases, the final success is still difficult to reach. allogeneic hematopoietic stem cells transplantation (allohsct) has remained an essential approach in attempts to cure all. tki routinely used for all ph(+) pre-and post-transplant treatment are also described as an alternative and adjunctive approach for chronic gvhd especially with fibrotic features due to their antifibrotic activity targeting the platelet-derived growth factor receptor (pdgfr) pathways. in this study we have tried to estimate the potential influence of pretransplant tki treatment on gvhd occurrence comparing all ph(+) and all ph(− ) cases treated with allohsct. a cohort of all patients consisted of all ph( − ) and all ph(+) cases treated with allohsct was retrospectively analyzed. all patients were transplanted from sibling or unrelated donor (no haploidentical procedures were included). all ph(+) patients achieved pretransplant treatment with imatinib and chemotherapy, and ph( − ) patients with chemotherapy alone. the median age in ph( − ) and ph(+) group was vs (p = . ), the percentage of hla mismatched transplantations - ,. vs . (p = . ), the percentage of acute gvhd cases- . vs . (p = . ) and extensive chronic gvhd cases- . vs . , respectively. there were no significant difference between groups in patients sex (f/m- / vs / respectively), ric/mac conditioning, unrelated/sibling donor, donors age, bm/pbpc transplantation, number of cd cells and chronic gvhd incidence. all patients received cyclosporine-and methotrexate-based gvhd prophylaxis. gvhd occurrence was analyzed in subgroups as previously described: all ph( − ) and all ph(+). as mentioned above the incidence of acute gvhd was higher in ph(+) group (higher number of hla mismatched transplantations in this group) but the incidence of extensive chronic gvhd was higher in ph (-) group. cox proportional hazard model analysis revealed death risk caused by gvhd higher in ph negative group (hazard ratio = . ; ci % = . - . ; p = . ). the analysis of competing events was performed to estimate the probability of death caused by gvhd vs other complications (transplant related mortality, infections and relapse). the impact of conditioning was not significant on gvhd related deaths vs other complications (p = . vs . , respectively- figure ). the same results were achieved with donor cmv status (p = . vs . - figure ). we have not found any significant difference either in gvhd or other complications related deaths taking into account patient s sex/age, donor sex/age, patients cmv status, number of cd cells transplanted. on the other hand, the influence of agvhd and chgvhd on deaths related to other complications was not significant (p = . vs . ). cumulative probability of overall survival was higher in ph(+) group but the difference was not significant. the impact of pretransplant treatment with imatinib on gvhd occurrence has not been estimated so far. we are aware of our results to be preliminary and variety of data is still to be evaluated. however our results, if confirmed, may suggest the influence of imatinib on decreasing the extensiveness of chronic gvhd. disclosure of conflict of interest: none. seta-tsukinowa, otsu, - , japan; department of pathology and laboratory medicine, emory university hospital, atlanta, ga, usa; department of biostatistics and bioinformatics, rollins school of public health, emory university, atlanta, ga, usa; pathology and pediatrics, emory university school of medicine, atlanta, ga, usa, aflac cancer center and blood disorders service, children's healthcare of atlanta, atlanta, ga, usa and bloodworks northwest research institute, seattle, wa, usa more than % of allogeneic hematopoietic stem cell transplant (allo-hsct) patients receive red blood cell (rbc) and platelet (plt) transfusions in the peritransplant period. preclinical models indicate that rbc and plt transfusions trigger inflammation, raising the question of whether such transfusions are associated with development of severe acute graft-versus-host disease (grade iii/iv agvhd) and mortality in allo-hsct recipients. we conducted a retrospective analysis of rbc and plt transfusions, agvhd incidence, and mortality among consecutive adult patients receiving non-t celldepleted allogeneic bone marrow ( %) or g-csf-mobilized blood stem cell grafts ( %). common underlying diseases were acute myeloblastic leukemia ( %), myelodysplastic syndrome ( %), and acute lymphoblastic leukemia ( %) . underlying disease risk was ranked as low ( %), intermediate ( %) or high ( %). allografts were obtained from / hlamatched sibling donors ( %), unrelated donors ( %), or from donors mismatched at - hla alleles ( %). graft sources were bone marrow ( %) or mobilized pbsc ( %). the cumulative incidences of grade iii-iv agvhd and mortality prior to day without developing grade iii/iv agvhd were estimated using the cumulative incidence function and a cox proportional hazards regression model. covariates included in multivariable analysis was limited to baseline covariates associated with grade iii/iv agvhd at the p median number of rbc or plt transfusions ( figure ). univariate analysis showed a lower hematocrit on admission (median of rbc units transfused (p = . ) were significantly associated with the risk of developing grade iii/iv agvhd, while a longer time to neutrophil engraftment was inversely associated with grade iii/iv agvhd ( ⩾ median of days, hr . , p = . ). multivariate cox regression analysis showed only larger numbers of rbc units transfused and hla mismatch independently associated with severe agvhd (p = . and p = . , respectively), while underlying disease risk and larger numbers of transfused rbc units were independently associated with overall survival in a multivariate analysis that excluded agvhd grade. overall mortality rate was lowest for the group with fewer rbc and plt transfusions ( %), and greatest for the group with more rbc and plt transfusions ( %). groups that received more rbc units had higher rates of mortality due to gvhd, while patients who received more plt transfusions and fewer rbc transfusions had greater mortality from relapse ( figure ). these data support the hypothesis that peritransplant rbc transfusions are associated with the risk of developing severe agvhd and worse overall survival following allo-hsct. prospective studies are warranted to whether rbc transfusions promote t-cell activation and inflammation in allo-hsct recipients, leading to increased severe agvhd. disclosure of conflict of interest: none. early high umbilical cord blood cd chimerism associated with acute gvhd at time of onset in haplo-cord transplantation h choe , j hsu , s mayer , u gergis , a phillips , t shore and k van besien department of hematology/oncology, weill cornell medicine, new york, ny , usa introduction: haplo-cord transplantation is a combined haploidentical and cord blood transplant that allows for more rapid engraftment by the haplo with eventual loss of the haplo graft upon engraftment of the cord. haplo-cord transplants are associated with an approximate - % incidence of agvhd. reported, using chimerism assessments at approximately day after transplant for aml and mds, that lower umbilical cord blood (ucb) chimerism in the cd or cd lineages were associated with increased rates of relapse. we did not find a statistically significant association between day chimerism and risk for acute gvhd.( ) here we report our analysis of chimerisms at the onset of agvhd. patients and methods we retrospectively reviewed all patients who underwent haplocord sct for all hematologic malignancies between july and march . ucb for haplo-cord transplants were selected based on hla-typing and cell count. grafts were matched for at least of hla loci by the standard criteria and contained a minimum cell count of × nucleated cells per kilogram (kg) of the recipient's body weight before freezing. the haploidentical donor was a relative in the large majority of cases. we identified patients evaluable for agvhd (onset before day ) without preceding relapse or early death. of the total patients, patients were diagnosed with agvhd of any stage and grade. fractionated chimerisms including cd and cd components were routinely sent to evaluate for engraftment of the recipient vs haplo vs ucb. chimerism data was collected for both agvhd and no agvhd patients. the two-sided student's t-test was used to compare the agvhd cohort to the no agvhd cohort. chimerisms collected on patients with agvhd were within median ± days of onset of agvhd. the median time to onset of agvhd was days (range: - days). the median post-transplant chimerism recorded for comparison with the no agvhd patients was days. the agvhd cohort had significantly lower cd recipient (p = . ) and higher cd ucb engraftment (p = . ). all other fractions, including the cd chimerisms, were not significantly different between the two cohorts. the agvhd vs no agvhd cohorts were further compared by degree of hla mismatch ( - out of vs - out of ). the frequency of agvhd was similar in the - out of ( / , %) and the - out of ( / , %) groups. within these subgroups, cd ucb chimerism was higher for those with agvhd (p = . and p = . , respectively). conclusion the onset of agvhd in haplo-cord transplantation is associated with a significantly higher cd ucb chimerism and lower cd recipient chimerism. higher ucb chimerism may indicate that full ucb chimerism poses a higher risk of agvhd development. vice-versa persistent recipient chimerism may protect from acute gvhd. il- is a pleiotropic cytokine with both pro-and antiinflammatory properties (scheller ). the proinflammatory properties are mediated through trans-signaling that depends on the soluble il- receptor. il- trans-signaling is involved in several autoimmune diseases and in regulation of tissue regeneration of the gi-tract. specific snps in the il- receptor have been associated with increased baseline crp levels, severity of autoimmune diseases and response to interleukin- inhibition in rheumatoid arthritis. so fare little is known about the role of trans-signaling in graft-versus-host-disease (gvhd). in this study we investigated how specific snps in the il- receptor influence pretransplant levels of crp, il- sil- r and the risk of grade ii-iv acute gvhd in allogeneic stem cell transplantation (asct) in patients with family donor. dna was available for patients ( male, female median age , range: - ) and donors, that underwent asct with a matched related donor ( sibling) at haukeland university hospital in the period - . the majority received conditioning with either bycy ( ) or flubu ( ) and only patients were transplanted with tbi-based conditioning. four different snps in the il- r gene (rs , rs , rs , rs ) were chosen on the basis of (i) documented or suspected roles in autoimmune disorders; and (ii) allele frequency between . - . and r o . between the different snps. genotyping was done using kaspar assays with viia instrument (life technologies). the overall genotype call rate was %. no departures (p-values o . ) from hardy-weinberg equilibrium were observed. pretransplant serum levels of il- , sil- r and were analyzed with bio-plex kits (bio-rad, hercules, usa). both serum and dna analyses were performed in duplicates. patients being homozygous for the rs minor allele had significantly higher pretransplant serum sil r levels but lower crp levels compared with patients homozygous for the major allele. the overall incidence of agvhd requiring high-dose steroid treatment (grade ii gastrointestinal, grade iii-iv liver and skin) in the cohort was %. when analyzing the conventional clinical and laboratory parameters only transplantation with a non-sibling donor was associated with increased risk of agvhd (p-value o . hr , confidence interval . - . ). the presence of the rs in donor or recipient was associate with a significant increase in the rate of aghvd (p-value . hr . confidence interval . - . ). the snp rs (p-value . hr . , confidence interval . - . ) was also significant in an adjusted model including both donor type and rs . none of the evaluated snps were associated with an increase in early or late trm and did not influence os either. this study suggests that snps in the il- r influence pretransplant biochemical characteristics and clinical outcomes after asct. future studies investigating the effect of il- inhibition as gvhd prophylaxis or treatment should include analyses of il- receptor snps to investigate their possible influence on treatment outcomes. graft-versus-host disease (gvhd) continues to be the major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant (allo-hsct). the prophylaxis scheme varies according to the center and the country. in ours institution we use triple-prophylaxis based on cyclosporin a (cya), metrotexate (mtx), and mycophenolate mofetil (mmf). this scheme has been used for more than one decade in asian centers where it has proven adequate effective and safe to prevent gvhd. we evaluated patients undergoing allogeneic hematopoietic stem cell transplantation treated at the national cancer institute from january to december . the triple-prophylaxis scheme consists in cya (adjusted serum levels, mtx ( mg/m days + , + , + , + ) and we evaluated different doses of mmf, one of them includes mg bid × days and the other has high doses ( mg/kg bid × days), as gvhd prophylaxis. the response characteristic was analyzed using the pearson test, fisher's exact test on categorical variables and student's t-test, mann-whitney u on continuous tests. kaplan-meier method was used to estimate the probabilities of os, sle with the differences compared by the log-rank test. we analyzed patients with median of age of years (range: - ), % male gender, all were transplant with peripheral blood progenitor cells as a source. . % were acute lymphoid leukemia and . % acute myeloid leukemia, . % chronic myeloid leukemia, . % myelodysplastic syndromes, . % aplastic anemias, . % non-hodgkin's lymphomas and . % hodgkin's lymphomas. myeloablative conditioning was used in % (bucy, cfm-gat) and % reduce intense conditioning (flubu, flucy, flucy-gat), . % related hla compatibility. mmf mg twice daily (bid) for days (group ) and of mmf mg/kg bid for days (group ), in the group the . % developed febrile neutropenia vs . % in group (p = . ). the frequency of gvhd was . % group vs . % group (p = . ), chronic gvhd was . % vs . % respectively (p = . ). at the moment of analysis . % vs . % were free of disease (p = . ). there no difference at -year overall survival was % (group ) vs % (group ) (p = . ), neither free-survival disease (p = . ). the mmf regimen shows noninferiority scheme for gvhd. the low doses and for shorter administration did not show differences in the incidence and severity of acute or chronic gvhd, os, dfs compared to the mmf regimen at days with high doses. the high doses shows higher incidence of febrile neutropenia, but there were no differences in documented infections. disclosure of conflict of interest: none. a protein-losing enteropathy can develop due to conditioning regimen related gut toxicity and can cause albumin decline during peritransplant period in allogeneic stem cell transplantation (allohct). damaged intestinal mucosal barrier results in alloactivation of donor t cells and this situation is considered a primary event in the pathogenesis of acute graft-versus-host disease (agvhd). peritransplant albumin decline, as a result of conditioning regimen related protein-losing enteropathy, may predict agvhd ( ) . in this retrospective study we tested this hypothesis. we evaluated patients who received allohct between and . albumin decline from the day of conditioning initiation until its nadir in the first weeks of post-transplant period was calculated as delta albumin. acute gvhd was proven by biopsy in all patients. chi square and mann-whitney test were used for statistical analysis. patients' characteristics were shown in table- . acute gvhd was developed in patients and severe agvhd was developed in patients. delta albumin was not different between agvhd patients and no agvhd patients. delta albumin was not related with severe agvhd. delta albumin was not different between patients who received myeloablative and reduced intensity conditioning regimens. when we used a cutoff value of . gr/dl for delta albumin, we could not find a relation between delta albumin and development of both agvhd and severe agvhd. we repeated the analysis for acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) patients who receive myeloablative conditioning regimen and we found the same results, there was no difference between agvhd patients and no agvhd patients in terms of delta albumin. there was a number of studies that used albumin as a predictive and prognostic marker in the setting of agvhd. but albumin may decrease in patients due to many reasons like malnutrition, proteinuria, enteropathy, liver disease or being negative acute phase reactant. because of albumin value can show variability between patients, albumin decline may be a more objective criterion. rashidi et al. showed that . gr/dl decline in albumin may be a predictor of severe agvhd in patients who was diagnosed with aml and mds and received myeloablative conditioning regimen. we repeat this analysis in our mds and aml (n = ) patients but we couldn't find this relation. when we evaluated all our patients, again there was no relation between delta albumin and development of both agvhd and severe agvhd. in conclusion, our study did not support rashidi et al.'s findings. because serum albumin level shows variability due to many reasons, it is hard to use albumin as a predictor of agvhd. sclerodermatous chronic graft-versus-host disease (scl-cgvhd) in its severe manifestation affects the patient quality of life and, due to complex pathomechanism, does not respond to standard immunosuppressive therapy-calcineurin inhibitors (cni) with corticosteroid. methotrexate (mtx) and rituximab appeared to be effective in some patients but the novel strategies, including extracorporeal photopheresis (ecp), imatinib, m-tor inhibitors (for example, sirolimus) and ruxolitinib seem to become the real breakthrough. we retrospectively analysed data of patients with scl-cgvhd, who underwent allogeneic hematopoietic cell transplantation (hct) between - in transplant centres. the study group consisted of patients with haematological malignancies and one with aplastic anaemia, female and male, with the median age ( - ). donors' median age was , with predominance of matched sibling donors ( donors) and even distribution of the donors' gender. in patients ( %) acute gvhd (agvhd) was diagnosed with skin involvement observed in ones. acute gvhd directly progressed to cgvhd in cases. in patients ( %) cgvhd developed ‛de novo' and in cases cgvhd was induced by dli. median time from hct to cgvhd diagnosis was months and to scl-cgvhd diagnosis- months. seven patients ( %) were scored as moderate cgvhd and patients ( %) as severe cgvhd according to nih- cgvhd activity classification. in patients sclerotic features had superficial form and in ones deep sclerosis was observed. chronic gvhd manifestation in other organs includes: mouth ( %), joints and fascia ( %), liver ( %), eyes ( %), gi tract ( %) and lungs ( %). patients were treated with ecp and/or sirolimus and /or imatinib with % response rate (complete-cr, partial-pr or minimal-mr). median duration of ecp therapy, sirolimus and imatinib treatment was months ( - ), months ( - ), and months ( - ), respectively. sirolimus was added more likely ( patients) as the first in case of suboptimal response to ecp after median weeks and in patients was subsequently replaced by imatinib with no favourable outcome in cases. in patients imatinib was initially used in combination with ecp therapy, leading to pr or mr. mtx without novel therapies was effective in patients with limited skin involvement, patients responded to mtx plus imatinib and patient to mtx plus sirolimus. two patients, after failure of other therapies, have been receiving ruxolitinib with improvement. only patient ( %) were nonresponsive to ecp (progressive or stable disease), patients ( %) to sirolimus and patients ( %) to imatinib. toxicity incidence was equally observed in case of sirolimus and imatinib and lead to the therapy discontinuation in altogether patients. infectious complications were observed in patients ( %). ecp confirms to be the most effective therapeutic strategy in severe forms of scl-cgvhd with favourable safety profile. imatinib and sirolimus, targeting different fibrotic pathways, both play important role in nonresponsive patients, improving the outcome in ecp and non-ecp group. in case of limited access to ecp, mtx remains to be beneficial in combination therapy of moderate scl-cgvhd and an alternative to cni. disclosure of conflict of interest: none. post-transplant morbidity and mortality are majorly determined by gvl effect counter-balanced by gvhd. treatment with systemic steroids represents the first-line therapy for gvhd, but is associated with increased incidence of infection and relapse. ecp can reduce the extent of gvhd while preserving anti-virus/-tumor activity. to elucidate this clinical phenomenon on an immunological level, we correlated clinical data with immunological findings in patients under ecp treatment. nine patients with acute gvhd (agvhd) of the gut ii-iv suffering from severe diarrhea were treated by ecp in addition to triple-drug immunosuppressive therapy. furthermore, patients with chronic gvhd (cgvhd) of the skin or lung despite triple-drug received ecp treatment. patients were evaluated according to their individual response and clinical condition. phenotypical analysis of different cellular subsets of patients and healthy donors was performed by multicolor flow cytometry. functional properties of virus-specific cd + t and nk cells were evaluated by inf-γ-elispot and cr-release assay. about patients were treated by ecp in this study. however, two agvhd and two cgvhd patients had to be withdrawn from ecp treatment after a few ecp cycles due to pancytopenia or poor clinical condition. for patients with agvhd up to ecp cycles were needed for response. all patients achieving a complete response (cr) were still alive year after initiating ecp therapy. overall response, that is, cr or partial response (pr) according to nih criteria, was obtained in of patients with agvhd ( . %) including cr in of ( . %). out of cgvhd patients ( . %) reached pr, and ( . %) remained stable under ecp treatment. after year, overall survival (os) was % for agvhd patients responding to ecp, while only % for non-responders. os for cgvhd patients was %. during intensive ecp treatment for patients with agvhd of the gut, the average stool volume and frequency decreased and consistency changed from loose to formed stool. steroids could be tapered down to a mean of % of the initial dosage. cgvhd patients were stabilized under ecp treatment and steroid dosage could be reduced to a mean of %. clinically responding patients showed increased numbers of regulatory cells including mdscs, foxp +cd + and foxp +cd +cd + tregs, as well as cd − cd − cd + t, vδ + t cells and regulatory b lymphocytes. furthermore, loss of cd l expression on effector cells like cd + te, cd + te, nk and nkt cells was observed under ecp treatment. interestingly, ecp treatment did not dramatically influence the frequency of cd +cd +cd + t, γδ t cells and nkt cells, which possess anti-virus/-tumor function. elispot and cr-release assays revealed stable anti-viral activity of cd + t cells as well as functional cytotoxicity of nk cells. moreover, cd + t, cd + tem, cd l+cd + temra, cd +cd − nk and cd brightcd − nk cells could serve as reliable biomarkers for prediction of response to ecp. conclusion: ecp treatment might stabilize or even improve clinical situation of patients suffering from gvhd. in clinically responding patients an immunomodulation was observed in terms of increasing numbers of regulatory cells with loss of migratory capacity of effector cells while anti-virus/-leukemia t-cell function was preserved. disclosure of conflict of interest: none. extracorporeal photopheresis affects dendritic cells by reducing total numbers and blunting cytokine production in patients with graft versus host disease tj altmann , , m bickerton , am flinn , u cytlak , p milne , s pagan , m collin , , v bigley , and ar gennery , institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom and newcastle upon tyne hospitals nhs foundation trust, newcastle upon tyne, uk graft versus host disease (gvhd) and concomitant immunosuppression is a leading cause of morbidity and mortality post hematopoietic stem cell transplantation (hsct). the pathophysiology of gvhd is complex, involving presentation of histo-incompatible antigen by activated recipient dendritic cells (dcs), activation and proliferation of donor t cells and resultant tissue damage. extracorporeal photopheresis (ecp) is a second-line treatment for steroid refractory or dependent gvhd that facilitates the reduction of immunosuppression. ecp's mechanism of action is unclear and is likely to be multifaceted. apoptosis of lymphocytes, induction of a th favoured environment and increased numbers of regulatory lymphocytes have been implicated . although ecp has been shown to modify the function of in vitro monocyte-derived dcs , its effect on primary (non monocyte-derived) dcs has not been studied. our aim was to determine whether ecp had immediate or long-term affects on primary dc numbers or function. we enumerated monocyte and dc subsets (cdc , cdc myeloid dcs and plasmacytoid dcs) in whole blood before, during and after ecp cycles, and developed a novel dc function assay, suitable for use on clinical samples. four adults with immunosuppression withdrawal gvhd and four children with acute gvhd, received ecp during the study. all received ciclosporin gvhd prophylaxis and corticosteroid treatment at onset of gvhd. children received ⩾ dose of infliximab prior to starting ecp. adults received two ecp treatments (one cycle) every weeks and children received two ecp treatments (one cycle) weekly. whole blood was taken before and after each cycle of ecp. trucount flow cytometry analysis of whole blood was used to enumerate mononuclear leukocytes. to assess function, peripheral blood mononuclear cells were isolated by density centrifugation and stimulated with toll-like receptor agonists. cell-specific cytokine production was then analyzed by flow cytometry. samples were compared to healthy controls and pre-ecp samples. median time to first ecp treatment from gvhd diagnosis was . days. no gvhd flares were experienced during study period. ( ) adult had a cycle of treatment delayed due to intercurrent pneumonia. numbers of cdc , pdcs and classical monocytes were significantly reduced after each ecp treatment in the adult group. dc numbers followed the same trend after ecp in the paediatric group but were not significantly different before and after ecp. this is perhaps due to initial lower dc count compared to adults in the children before the first ecp cycle. functional analysis showed a reduction in cytokine production in dcs and monocytes in both groups over the course of ecp treatment. our data support a cell-intrinsic effect of ecp on monocytes and dcs, with numerical and functional consequences. this may contribute to the beneficial effect of ecp both through reduction of inflammatory effector function and through modulation of interactions with other immune cells. correlation with immunosuppression withdrawal and clinical events during treatment may provide further insight into the role of monocytes and dcs in gvhd and ecp, which may aid in the development of novel targeted therapies for gvhd. extracorporeal photopheresis as early second-line treatment for patients with steroid-dependent or refractory acute graft-versus-host disease: a single-centre experience i sakellari , i batsis , e gavriilaki, a-k panteliadou, a lazaridou, k leontopoulos, d mallouri, a bouinta, v constantinou, e yannaki, c smias and a anagnostopoulos department of hematology bmt unit, g. papanicolaou hospital, thessaloniki, greece acute graft-versus-host disease (agvhd) remains a severe complication of allogeneic haematopoietic cell transplantation (allohct). corticosteroids as the backbone of initial therapy for agvhd result in varied complete responses ( - %). traditional secondary treatments lead to profound immunosuppression without improved survival. on the basis of our experience in chronic gvhd, we aimed to prospectively assess the role of extracorporeal photopheresis (ecp) as early secondline treatment in steroid-dependent and refractory agvhd. we enrolled consecutive patients with steroid-dependent or refractory grade (gr) ii-iv agvhd post allohct from january to august . all patients with unrelated or haploidentical donors received thymoglobulin (atg) mg/kg as prophylaxis. post-transplant gvhd prophylaxis included cyclosporine-methotrexate in myeloablative and cyclosporine-mycophenolate mofetil in reduced toxicity or intensity regimens. ecp was commenced after assessment of response to days of steroid treatment according to our protocol: two sessions per week for month, one session per weeks for months, evaluation of response and one session per month for months. we studied patients, aged ( - ), post allohct with myeloablative ( ), reduced toxicity ( ) and intensity ( ) conditioning, from sibling ( ), matched ( ) or one locus mismatched ( ) volunteer unrelated and haploidentical ( ) donors. disease risk index was high ( ), intermediate ( ) and low ( ). acute gvhd was observed at day + ( - ) in patients, late onset at + ( - ) in patients and induced at + post donor lymphocyte infusion in a relapsed aml patient. skin, intestine and liver involvement was evident in patients, skin and intestine in and skin only in patients. nine patients ( with grii, with griii agvhd) were steroid-dependent and ( with griii, with griv) steroidrefractory. atg was administered simultaneously with ecp initiation in six refractory patients that further developed ebv reactivation (p = . ) treated pre-emptively with rituximab. ecp was commenced at day + for ( - ) sessions. the majority of patients ( / ) presented partial ( ), very good ( ) or complete ( ) response to ecp. with . ( . - ) months of follow-up, immunosuppression was reduced in / and ceased in patient. clinically significant bacterial infections were found in patients, fungal in , cmv and ebv reactivation in and , respectively, and other viral in patients. cumulative incidence (ci) of chronic gvhd was . at year. one-year ci of agvhd-related mortality was %. one-year overall survival (os) was % and significantly increased in steroid-dependent vs refractory patients ( % vs %, p = . ). reduction of immunosuppression (p = . ) and response to ecp (p = . ) were associated with improved os, irrespectively of other factors. our data indicate that ecp should be considered early in the course of steroiddependent or refractory agvhd, before irreversible end organ damage has been established. optimal timing of intervention, frequency, duration and tapering schedule of ecp remain important unanswered questions. [p ] disclosure of conflict of interest: none. extracorporeal photopheresis for treatment of chronic graft versus host disease m lanska, a zavrelova, j radocha and p zak faculty of medicine, th department of internal medicine-hematology, university hospital hradec kralove, czech republic allogeneic stem cell transplantation represents a curative approach to many hematologic disorders. graft versus host disease (gvhd) is a complication with significant morbidity, mortality and decreased quality of life. extracorporeal photopheresis (ecp) represents possible treatment approach. mononuclear cells (mnc) collected by apheresis are photosensibilized with -methoxypsoralenem ex vivo, irradiated with uva and transfused back to the patient. aim of the study: evaluation of patients treated with ecp for gvhd at our center. thirteen patients ( females and males, median age years) were treated with ecp. about patients (pts) had matched sibling donor and patient had unrelated donor. about pts had sclerodermic form of gvhd, had concomitant pulmonary gvhd, had pulmonary gvhd alone. one pts had mild, seven moderate and five severe gvhd according to nih. about patients were treated with steroids. mnc separation was prepared on cobe spectra and spectra optia (terumo bct, usa). -methoxypsoralen was added, irradiation was done on macogenic g (macopharma, mouvaux, france). about procedures in pts were performed (median procedures, - procedures). the schema was as follows: ecp on consecutive days every - weeks first months with subsequent increase in interval. median follow up was months. in sclerosing form two pts reached cr, six pts pr, one is stable and one patients progressed. in pulmonary gvhd one reached cr, two partial improvement, one is stable. seven pts are still alive, six died (two due to relapse, one secondary malignancy and three infections). it was possible to withdraw steroids in pts. adverse events were clinically negligible. ecp is an effective treatment for chronic gvhd. especially sclerodermic form responds to ecp very well. it is safe and well tolerated procedure with minimal toxicity. supported by prvouk p- . disclosure of conflict of interest: none. allogeneic haematopoietic stem cell transplantation (hsct) is a potentially curative treatment option for children with a variety of haematological, oncological and immunological diseases. graft versus host disease (gvhd) represents a major cause of post-transplantation mortality and morbidity affecting multiple organs including skin, gut, liver and lungs. gvhd is considered a succession of inflammation and donor t-cell activation initiated by translocation of gastro-intestinal microorganisms through impaired mucosal barriers after chemotherapeutic conditioning and/or infection. diagnosis of gvhd is based on clinical symptoms and histological findings, necessitating invasive and potentially harmful procedures including endoscopy and biopsy. as yet, no non-invasive markers are available for diagnosis or treatment monitoring in children with gvhd. faecal calprotectin (fc) reflects intestinal mucosal inflammation of any origin. in the setting of allogeneic hsct in adults, fc has shown to be a marker for acute (steroid-resistant) gvhd. we aimed to evaluate the feasibility of prospective fc measurement as a non-invasive marker for diagnosis and treatment in children with gvhd. a prospective, observational, single centre study was started in july . by december , paediatric allogeneic hsct patients (age - years) were included after informed consent. faecal samples were collected from weeks before to months after hsct. fc levels were measured by elia, according to manufacturer's instructions. clinical symptoms were prospectively evaluated and managed according to local guidelines. if gvhd was suspected on clinical grounds, histological confirmation was obtained. first-line therapy for gvhd consisted of corticosteroids. in case of steroid-resistant disease, more advanced immune modulation was applied. a total of five patients developed histologically confirmed gvhd: acute gvhd of skin and gut (n = , one patient with steroidresistant disease), acute gvhd of skin only (n = ); chronic gvhd of lung only (n = ) and acute gvhd of skin followed by chronic oromucosal gvhd (n = ). without exception and regardless of gut involvement, gvhd occurrence was accompanied by rises in fc levels to values μg/g (range: - μg/g). fc levels correlated with clinical and histological grading. moreover, adequate response to therapy was consistently reflected by return of fc levels to values o μg/g. sensitivity of fc levels to diagnose gvhd was poor due to increased fc levels in patients with posttransplant complications other than gvhd such as viral reactivation and pulmonary or gastro-intestinal infections. fc levels reflect gvhd occurrence and correlate with clinical and histological grading in paediatric allogeneic hsct patients. fc levels increase in case of gvhd regardless of gut involvement, supporting a central role for (subclinical) intestinal inflammation in gvhd initiation. although, in this interim analysis, fc lacks sensitivity to diagnose gvhd, fc may serve as a noninvasive marker for monitoring therapy response and, thereby, reduce the need for repeated invasive procedures including endoscopy and biopsy. acute graft-versus-host disease (agvhd) is a major complication of allogeneic hematopoietic cell transplantation (hct), and glucocorticoids are typically used as first-line treatment. the aim of our study was to evaluate the effect of first-line ecp +/ − steroid therapy in order to reduce the incidence of infections and toxicity. from december to january , of pts ( %), were diagnosed with agvhd grade ⩾ following allosct. pts were treated with ecp +/ − steroid as first-line therapy. about ( %) pts were treated with ecp only and ( %) with ecp + steroid - mg/kg/day. we compare this cohort with an historical group of patients, transplanted between and , who were treated with steroid only for grade - agvhd (n = out of ). the two cohorts were well balanced in terms of median age (p = . ), disease type (p = . ), disease status (p = . ), graft source (p = . ), conditioning regimen (p = . ) and hct-ci (p = . ). there were more female patients (p = . ) and more haploidentical transplant (haplo-sct) (p = . ) in the cohort treated with ecp+/ − steroid. ecp was performed using the offline technique, and was started as soon as possible with a treatment schedule consisting of four rounds of two procedures per week, three rounds of two procedures every other week and finally two procedures every month. steroid was tapered as soon as possible after starting ecp. the clinical response was evaluated at day + . median follow-up for alive patients was months for ecp group and months for control group. there was no difference in terms of median time of agvhd onset ( vs days) and number of pts with grade or - agvhd ( figure ). ecp was started after a median of ( - ) days from agvhd diagnosis. every patient underwent a median of ( - ) ecp procedures, during a median time of months. on day after starting agvhd treatment with ecp+/ − steroid, pts ( %) achieved cr or pr, pts did not respond and experienced agvhd relapse to front-line therapy. one year cumulative incidence (ci) of agvhd relapsed/refractory was . % for ecp+/ − steroid. these percentages were not different from the cohort receiving steroid alone. ci of moderate-severe cgvhd was lower in the ecp group, probably due to the higher frequency of haplo-sct with pt-cy in the ecp group. about days after agvhd onset, ci of infection ( % vs %), especially cmv reactivation ( % vs %), was lower in the ecp group, but was not statistical significant. ecp allowed a faster taper of steroid: ( - ) vs days ( - ) (p o . ). overall survival, progression-free survival, non-relapse mortality and ci of relapse rates did not differ in the two groups. in multivariate analysis, visceral involvement by agvhd was associated with an increased risk of failure to front-line therapy (hr: . ; range: . - ; p = . ). this observational study suggests that the overall response rate of ecp +/ − steroids is similar to steroid alone for front-line treatment of grade - agvhd, but is potentially associated with lower incidence of infection, and in particular of cmv reactivation. a prospective phase clinical trial is warranted to address whether augmentation with ecp may be beneficial for agvhd frontline treatment. [p ] a -year-old girl with neuroblastoma received autologous stem cell transplant (asct), followed by antibiotic prophylaxis and filgrastim. her transplant preparative regimen consisted of busulfan and melphalan. engraftment of neutrophil took place on day after asct. twentieth day after asct, she experienced nausea and diarrhea. there was neither skin rash nor elevation in liver enzymes. the diarrhea continued to worsen day by day and reached to a daily volume of ml/ m . infectious studies for stool and blood including testing for influenza a and b, parainfluenza, adenovirus, epstein-barr virus, amebiasis, cryptosporidium parvum, cytomegalovirus, clostridium difficile, salmonella, campylobacter, yersinia and shigella were all negative. colonoscopy and endoscopy were performed by an experienced pediatric gastroenterologist and findings were suspicious for severe graft versus host disease (gvhd). colonoscopy and rectoscopy revealed severe inflammatory changes, friability and patchy dark exudates on the mucosa of rectum. endoscopy revealed erosions, ulcers in the esophagus and a pale mucosal surface with reticulated submucosal vessels accompanied with erosion and erythema in the antrum of stomach. grade gvhd was confirmed by pathologic analysis that revealed diffuse crypt dropout and mucosal erosion on rectal mucosal biopsy. mucosal erosions, apoptosis of epithelial cells and small lymphocytic infiltration of the lamina propria were found on duodenal biopsy. after these results, we started methylprednisolone intravenously at a dosage of mg/kg/day. on the fourth day of treatment we increased the dosage to mg/kg/day and added cyclosporine to treatment. because of unresponsiveness to treatment we decided to administer thirdparty mesenchymal stem cells (msc) ( × cd +/cd + cells per kg). these were given intravenously at day + asct as single infusion. the second dose was given at day + . within days after first application of mscs, the frequency of diarrhea decreased to one-third. at day + after second dose of mscs, the patient's stool became nearly normal. we tapered the steroids first and stopped cyclosporine at + th days after asct. discussion and conclusion: to our knowledge, this is the second case report of spontaneous severe autologous gvhd in a child with a solid tumor malignancy. regarding the pathogenesis of autologous graft-versus-host disease, there may have multiple causes for the loss of tolerance to self because of disrupted immune system. alteration of t regulatory cells by previous chemotherapy may be key point. endogenous cells that survive conditioning and assist in post-transplant maintenance of self-tolerance may be affected. microchimerism due to maternal cells transmitted during fetal development and persisting throughout adult life has also been postulated as a cause. however it is not very clear for factors that may contribute to the pathogenesis of this rare disease. autologous gvhd has the potential to cause critical illness in the hematopoietic stem cell transplantation patient population. in patients with multiple myeloma some experts report pathologically verified gastrointestinal gvhd as high as %. responses to steroids are variable. however, a significant proportion improve dramatically after early therapeutic intervention. so clinicians and pathologists should be aware in suspecting and recognizing gvhd in patients with diarrhea to guide therapy as soon as possible. disclosure of conflict of interest: none. haplosct patients did not receive additional gvhd prophylaxis aside from the ex vivo t-cell depletion (tcd) with clinimacs system. of the bud bmts out of patients engrafted, % of which had gvhd. the % who did not have any gvhd, relapsed. eighty one percent had agvhd, of which majority ( %) were grade ( table ) three patients did not have agvhd ( / mud and two cord blood grafts), but developed cgvhd. one of the patients who had grade agvhd died and one still has intermittent cgvhd years post bmt. there were % who had cgvhd. currently, all of our haplosct receive a cd /cd ra tcd grafts (n = ). the depletion techniques for the others were either cd tcd, cd / cd ra /tcrab tcd+cd +/cd ra tcd; tcrab tcd. all patients who received haplosct engrafted and % had agvhd ( % grade ) ( table ). we noted that some of the patients presented with nonclassical agvhd signs (upper gut gvhd, oral gvhd, blood). there were no patients who presented with grade agvhd. cgvhd in the cohort was %. of note, % of patients did not have any gvhd and did not receive any form of immunosuppression post bmt. only eight patients received further immunosuppression for agvhd, median duration (range: - ) days. at the time of this report, there are four patients with agvhd still receiving immunosuppression all o days and all on tapering doses. haplosct using ex vivo tcd techniques has a lower risk of gvhd with comparable, if not superior outcome to bud. the degree and duration of immunosuppression is also much less. this may translate to earlier immune reconstitution and less viral reactivation. [p ] disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (allo-hsct) offers a potential cure for several hematological diseases, but it is burdened by severe life-threatening complications, being gvhd the major cause of morbility and mortality. recently, more have been understood of the physio-pathologic relationships between endothelium and graft-versus-host disease (gvhd), showing that vascular endothelium is an early phase target of gvhd. in recent years, the direct count of circulating endothelial cells (cec) has emerged as a valuable biomarker of endothelial damage in a variety of disorders. however, due to their rareness and complex phenotype, different published techniques have showed variable degrees of uncertainty, reporting a wide range of cec values in healthy subjects. by means of the commercially available rare cell isolation platform cellsearch system, for cec identification and count, we correlated cec count changes to gvhd onset and response to treatment in allo-hsct patients. cec were analysed in allo-hsct patients ( aml, all, hd, nhl, cll, mds, cms, mm, saa) at the following time points: t (pre-conditioning), t (pre-transplant), t (engraftment), t (day+ or onset of gvhd), t ( week after steroid treatment). the median cec/ml at t was (range: - ), in comparison to a value of (range: - ) in healthy controls (p %: or . , % ci . - . ; p = . ). we confirm that cec count represent a valid biomarker to monitor endothelial damage in patients undergoing allo-hsct and can be a valuable tool in supporting the diagnostic definition of gvhd and in monitoring responsiveness to treatment. moreover, the use of the cellsearch system can be crucial in order to move routinely cec monitoring into clinical practice of allo-hsct. reference clinicaltrials.gov nct . disclosure of conflict of interest: this research was conducted with the support of the investigator-initiated study program of janssen diagnostics, llc to ca. kb is employee of janssen diagnostics. results of hla mismatched unrelated donor (mmud) hematopoietic cell transplants (hct) are worse than results of fully matched hct due to higher risk of gvhd, infection and graft failure. atg during conditioning reduces incidence of gvhd but can increase risk of infection and relapse. high doses posttransplant cyclophosphamide ( × mg/kg) prevent gvhd in haploidentical hct. we initiated this approach instead of atg in hct from one alelle or antigen mismatched unrelated ( / )mmud-hct in . here we present outcome of patients (cy-group) transplanted between and , comparing it with outcome of patients transplanted between and from / mmud with atg-f (fresenius) mg/kg given during conditioning. patients in cy-group ( males, females) were transplanted from mmud mismatched for hla ( a- , b- , c- , dr- ). about patients had aml, mds, cll, cml, all and mps. med. age of patients was years ( - ). about patients received myeloablative (flu- mg/m + iv bu . mg/kg) and nonmyeloablative(flu- mg/m+mel - mg/kg +-tt mg/kg) conditioning. about patients received pbpc and bm as a graft. graft versus host prophylaxis consisted of cyclophosphamide ( mg/kg aibw) on d+ and + , cyclosporine a from d and mmf from d+ . all patients received antibacterial, antifungal, hsv and pcp prophylaxis. historical control (atg) group consisted of patients ( males, females), med. age y ( - ) who had mmud-hct for aml- , mds- , nhl- , mf- , all- , cll- , cml- , h.d- , saa- , mps- and mm- . there were mismatches for a, for b, for c and for dr. myeloblative conditioning was used in and nonmyeloablative in patients. all patients received atg-f mg/kg × given d- and- . cyclosporin was initiated d- and mmf d- . all patients received anti-infectious prophylaxis as described previously. three of patients from cy group died so far. two of them due to relapse and one due to toxicity and infection during aplasia. five patients relapsed . two achieved cr after dli and one is alive in relapse expecting second hct. about patients are alive, of them in cr. eight patients experienced agvhd (gr.i- , gr.ii- ,gr.iii- , gr. iv- ) and eight developed clinically mild cgvhd). about patients from atg group are alive - m (med. m) posthct. about patients died - m postransplant (med. m) due to vod, gvhd, infections and relapse. -day mortality is % ( / ) in cy group and % ( / ) in atg group. one year mortality is % ( / ) in cy and % ( / ) in atg group. patients from cy group have % probability of os at months posthct vs % from atg group. cyclophosphamide × mg/kg instead of atg fresenius( mg/kg) for gvhd prophylaxis reduced -day and -year mortality, and improved probability of m os significantly in our cohort of patients. this approach seems to be safe and effective in / mmud-hct. disclosure of conflict of interest: none. high transplanted cd + cells are not associated with beneficial effect on graft-versus-host disease-free, relapse-free survival (grfs) after allogeneic hematopoietic cell transplantation y lee and ih lee department of hematology, kyungpook national university hospital and inhee lee the success of allogeneic hematopoietic cell transplantation (allo-hct) is comprehensively assessed by individual comorbidity, relapse, graft-versus-host disease (gvhd) and death. besides, inconsistent results have been reported regarding the dose of cd + cells. in the current study we have addressed the issue of the potential effect of stem cell dose on the of gvhd-free/relapse-free (grfs) associated with cd + cells doses. we retrospectively reviewed the medical records of the patients who received allo-hct for acute myelogenous leukemia (aml), myelodysplastic syndrome (mds) and acute lymphoblastic leukemia (all) between and in kyungpook national university hospital. the grfs included grade - acute gvhd, systemic therapy-requiring chronic gvhd, relapse or death. the patients were reclassified into two groups according to the targeted cd + cell doses ( × per kg) by knuh protocol. a lower cd + group (n = , . %), patients who underwent allo-hct with cd + cell dose o × per kg; and a higher cd + group (n = , . %) patients who underwent allo-hct with cd + cell ⩾ × per kg. the median age at transplant was . years (range: - years) and male was patients ( . %). primary diseases for allo-hct were aml/mds (n = , %) and all (n = , %). one hundred forty-three patients ( . %) were in cr (complete remission), ( %) in further cr and ( . %) in relapsed and refractory status. one hundred seventy-one patients ( . %) received myeloablative conditioning regimen. gvhd prophylaxis consisted of methotrexate and cyclosporine a or mtx and tacrolimus. the median dose of cd + cell was . × per kg (range: . - × per kg) in lower cd + group and . × per kg (range: . - . × per kg) in higher cd + group. there was no significant difference in neutrophil, platelet engraftment between two groups. the incidence of chronic gvhd was more frequent in higher cd + group ( . % vs . %, p = . ). the median follow-up duration was . months, with a range of . - . months. the -year overall survival (os), relapse free survival (rfs), nonrelapse mortality (nrm) and graft-versus-host disease (gvhd)free/relapse-free survival (grfs) since hct was . ± . %, . ± . %, . ± . % and . ± . %, respectively. there was no significant difference according to the infused cd + cell dose (figure ). the relapse rate was not proportionally affected by the cell dose ( . % vs . %, p = . ). and there was no significant correlation between the number of cd + and cd + cells infused (spearman correlation coefficient: p = . ). in a univariate analysis, patients transplanted with the higher cd + cell doses and higher cd + cell doses had no increased grfs (p = . and p = . ). an independent factor associated with worse grfs was risk status at transplant (hr = . , % ci: . - . , p = . ). these results suggest that careful assessing the cd + and cd + graft content and tailoring the cell dose infused may help in reducing cgvhd risk without negative impact on grfs. a large and prospective study in a homogenous population will be needed to confirm the effect of stem cell dose. disclosure of conflict of interest: none. imatinib associated with extracorporeal photopheresis can fully reverse severe sclerotic-type lesions in patients with chronic graft-versus-host disease: the lille university hospital experience l magro , j gauthier, b catteau , l mannone , a lionet , v coiteux and i yakoub-agha lille university hospital and nice university hospital severe sclerotic-type chronic graft-versus-host disease (cgvhd) is difficult to reverse and can dramatically alter the quality of life of patients after allogeneic hematopoietic cell transplantation (allo-hct). imatinib or extracorporeal photopheresis (ecp) used separately yield sustained responses in s only about % of patients with steroid-refractory cgvhd. given their respective modest efficacy we hypothesized that the combination of imatinib with ecp could lead to higher response rates. we are reporting here on seven patients with severe steroid-refractory sclerotic-type cgvhd treated at our institution using this combination. we retrospectively analysed all patients treated at our institution (n = ) with the combination of imatinib with ecp for severe steroidrefractory scgvhd. imatinib was started at mg/day and increased to mg/day if well tolerated. the cellex closed system was used for ecp. ecp was initiated twice weekly during weeks. after this « induction » period, ecp sessions were scheduled less frequently according to the response to treatment. additional immunosuppressants were tapered gradually in responding patients. initial grading and response evaluation was determined according to the nih criteria. steroid-refractoriness was defined as progression of gvhd on high-dose steroids (⩾ mg/kg) or progression during corticosteroid tapering. patient characteristics are displayed in table . patients received an allo-hct between may and april . median age at allo-hct was (range: - ). a variety of myeloablative (n = ) and non-myeloablative conditioning regimens were used (n = ). antithymocyte globulin was used before allo-hct in one patient. gvhd prophylaxis consisted of ciclosporine and methotrexate in six patients. one patient received tacrolimus and methotrexate. five patients had prior history of acute gvhd. nih global severity grade was severe in all patients (n = ) due to severe sclerotic features. the median number of previous therapies was (range: - ). all patients were steroid-refractory. after a median follow-up of months (range: - months) the overall response rate was %. the complete response rate was %. median time on ecp associated with imatinib was months (range: - months). median time to best response was months (range: - months). corticosteroids could be discontinued in all patients after a median time of months (range: - months). patients # , # and # received maintenance therapy with ecp upon discontinuation of imatinib. in four patients, both ecp and imatinib led to complete response and could be discontinued after , , and months for patients # , # , # and # , respectively. patient # and # passed away after due to a myocardial infarction and the development of a solid tumour, respectively. patient # was off therapy while patient # remained on maintenance with ecp. both remained in complete response. patient # remained in response during months before progression of cgvhd while on imatinib and ecp. none of our patients experienced adverse events related to either imatinib or ecp. despite the limited number of patients in this report, we observed that the combination of imatinib and ecp can lead to complete and sustained reversal of severe steroid-refractory sclerotic-type cgvhd. these encouraging results should be confirmed in a larger cohort. disclosure of conflict of interest: lm: therakos (honorarium). allogeneic hematopoietic cell transplantation (allo-hsct) is an established treatment modality that is potentially curative for many patients (pts) with acute myeloid leukemia (aml). aml itself is the most common indication for pts undergoing hsct nowadays. for pts with high-risk disease, allo-hsct is, perhaps, the most effective curative treatment and is considered the standard post-remission therapy in first complete remission (first cr). this is a retrospective study to analyze those variables which were associated with patients' overall survival (os) after allo-hsct. the study population consisted of pts who were diagnosed of aml from january to july at the hospital universitario central asturias, and submitted to allo-hsct in first cr. risk status based on validated cytogenetics and molecular abnormalities following recommendations of european leukemianet was performed. sixteen ( . %) were male. median age was years old (range: - ). clinical characteristics at transplantation are represented in table . median follow-up was months ( - ). considering the donor type, os at year was higher in pts receiving sd ( . %) compared to % in those who received urd (p = . ). regarding graft source, os at year was . % who received pbsc compared to % in pts receiving bmsc (p = . ). gender also showed significant association with os, which was higher among men, os at year was %, compared to . % for women) (p = . ). the presence of minimal residual disease (mrd) detected using multiparametric flow cytometry was performed prospectively after induction and consolidation, and before transplantation. thirteen pts had negative mrd before transplantation. median os was greater in pts with negative mrd before transplantation compared to the group with positive mrd ( vs months, respectively) (p = . ). this difference did not reach statistical significance probably because the low number of the sample. thirteen pts developed agvhd. only ( . %) pts receiving sd developed agvhd compared to ( %) pts among those who had an urd; however this association was not statistically significant (p = . ). also, we observed higher incidence of agvhd in bmsc group ( pts; %) whereas only ( . %) in pbsc group developed agvhd. this tender did not reach significant association (p = . ). one year os was . % in pts who developed agvhd and . % who did not (p = . ). all factors that had a significant influence on pts survival were included in a multivariate analysis (cox regression model): graft source, donor type, pts gender and agvhd development. developing agvhd kept an independent association with mortality (or . , % ci . - . , po . ) and male gender also persisted as an independent protective factor (or . , % ci . - . , p = . ). in our series, agvhd has shown a significant and independent association with os over other parameters such as graft source, type of donor or mrd before transplantation. identifying reliable predictors for agvhd development, controlling well known risk factors for this disease, as well as improving management of immunosupressors should still be the key to potentiate longer os in our patients. larger studies are needed to confirm our results. acute and chronic graft-versus-host diseases (gvhd) are associated with increased morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-hsct). older patients undergoing allo-hsct may experience a high degree of transplant-related complications and this concern has historically limited the use of allo-hsct for some older patients. in many studies, age has been shown to be a negative prognostic factor for survival and associated with higher transplant-related mortality (trm). however, in others, age was not shown to be a significant factor if appropriate adjustments for other comorbidities are incorporated in the analyses. there are very few studies that evaluated the relationship between patient's age, the presence of gvhd and long-term transplantation outcome. the aim of this study is to evaluate the impact of age in patients who develop acute and/or chronic gvhd after allo-hsct for hematological malignancies on the trm incidence. we included in the study patients with hematological malignancies who received allo-hsct and were followed in our center between january and january . for the purpose of this study, only patients who developed grade ii-iv acute gvhd and/or limited or extensive chronic gvhd where considered for analysis (n = ). patients were split into three homogeneous groups according to age at transplantation taking into consideration the underlying disease, type of conditioning and disease response at transplantation. group (younger) included patients aged o years (n = ), group (intermediate) included patients aged between and years (n = ) and group (older) included patients older than years (n = ). gvhd evolution over time was followed as well as the cumulative incidence of trm was calculated in case of acute or chronic gvhd in each group. thirty seven percent of grade ii gvhd occurred in the younger group (n = ), % (n = ) in the intermediate group and % (n = ) in the older group; majority ( %) resolved in the younger group as well as in and % in the latter two groups, respectively, while trm rates at year were %, % and %, respectively, sdhr = . , p = . . among patients who had acute gvhd grade ii, , and % in the three respective groups developed chronic gvhd later. grade iii-iv gvhd occurred in % (n = ) in the younger group, % (n = ) in the intermediate group and % (n = ) in the older group; with a respective resolution in %, % and % of patients and were associated with comparable trm rates at year of %, % and %, respectively, p = . . among patients who had acute gvhd grade iii-iv, , and % in the three respective groups developed chronic gvhd later. de novo chronic gvhd was observed with a higher rate in the intermediate and in the older group (table) while patients with extensive chronic gvhd older than years had significantly higher trm at years ( %) compared to % in those younger than years, sdhr = . , p = . . patients who develop acute gvhd grade iii-iv could incur over % of trm at year independently of age. resolution of acute gvhd grade ii was significantly better in younger patients while older patients with grade ii acute gvhd or with extensive gvhd had higher mortality compared to younger ones. in addition to an adapted prophylaxis, a better preemptive gvhd strategy should be warranted in older patients. [p ] disclosure of conflict of interest: none. in vivo effects of nilotinib on lymphocyte subpopulation and function following allogeneic stem cell transplantation e marinelli busilacchi , a costantini , j olivieri , n viola , s coluzzi , e pirro , g mancini chronic graft versus host disease (cgvhd) is a major complication of allogeneic stem cell transplantation and is characterized by frequent multiorgan involvement resembling autoimmune diseases; its pathogenesis is still incompletely defined and a standard treatment is lacking. donor-derived cd + and cd + t lymphocytes have been considered the main effector cells mediating cgvhd pathogenesis; however, recent studies suggest that b cells might also play an important role. in vitro data indicate that tyrosine kinase inhibitors (tkis) such as imatinib and nilotinib affects both innate and adaptive immune response by interacting with different cell populations (t cells, b cells, dendritic cells, mast cells and macrophages). we sought to evaluate the impact of different doses of nilotinib on the distribution and function of lymphocyte subpopulations. we analyzed samples obtained from patient with steroiddependent/refractory cgvhd enrolled in a phase - study with nilotinib in steroid-refractory cgvhd (nct ): triplets of patient were treated with escalating doses starting from mg/die ( ), mg/die ( ), up to mg/die ( ) . blood and plasma were collected at baseline and at day and of therapy. trough plasma nilotinib concentrations had been previously determined by hplc (abstract c , haematologica: evaluation of nilotinib safety in patients with steroid-refractory chronic graft-versus-host disease: a phase i-ii gitmo study). peripheral blood mononuclear cells were isolated by density gradient centrifugation using ficoll biocoll. six color flow cytometry analysis (facs canto ii) was performed using conjugated antibodies (anti-cd , cd , cd , cd , cd , cd ). inflammatory cytokine analysis was performed on plasma samples according to the instruction of bioplex pro human cytokine plex assay (bio-rad). statistical analysis was performed by -tailed student's t-test; differences were considered statistically significant for po . . flow cytometry analysis showed that nilotinib did not exert any significant impact neither on the proportion of t lymphocytes subpopulation (cd +cd + t helper, cd +cd +cd + t regulatory, cd +cd − t cytotoxic), nor on b lymphocytes and nk cells. on the contrary, a statistically significant and dose-independent decrease of pro-inflammatory and th- cytokine production was observed ( figure ): reduction of il (po . ), il (po . ) and ifnγ (po . ) were already significant after days; decreases of il (po . ) and tnfα (po . ) become significant after days. interestingly, after days of therapy, among the patients enrolled (according to the itt criteria) ten patients showed cgvhd improvement and the other five remained stable. this study shows that therapeutic doses of nilotinib can reduce plasma levels of inflammatory cytokines without affecting the proportions of lymphocyte subpopulations. these findings correlate with clinical response and suggest that besides the previously demonstrated anti-fibrotic effects, nilotinib has also potent anti-inflammatory and immune regulatory properties, supporting its role in patients with cgvhd. disclosure of conflict of interest: none. [p ] previously published p infectious gastro-enteritis after allogeneic hematopoietic transplantation after reduced intensity conditioning (allo-ric): incidence and possible role in gastro-intestinal acute gvhd i garcía-cadenas , r martino , a esquirol , a bosch , n rabella , s saavedra , c muñoz , j briones , s brunet and j sierra hematology and microbiology departments and hospital de la santa creu i sant pau, autonomous university of barcelona, spain enterotoxigenic c. difficile-associated associated disease or infection (cdi) is a common cause of diarrhea after hematopoietic stem cell transplantation (sct). recent studies have suggested the relationship of cdi with gastro-intestinal (gi) graft-versus-host disease (gvhd). the possible role of other types of infectious gastro-enterocolitis (g-ec) in gvhd development has not been studied. as a prior investigation to a national prospective observational study on this issue, we conducted a single-center retrospective analysis including all adult patients who received an allo-ric sct between january and march . the aim was describing the cause(s) (if known), timing and outcomes of recipients with possible g-ec (defined as new onset acute diarrhea grade ⩾ ) in the first year after sct. of the patients studied (median age: years, % male, % aml or mds as underlying disease), ( %) had a total of episodes of acute diarrhea, with ( %) developing more than one event. these acute diarrheas occurred at a median of days (range: - ) after sct. overall, a g-ec causing pathogen was identified in of stool specimens ( %) and included: cdi ( ), c. jejuni ( ), rotavirus ( ), adenovirus ( ), norovirus ( ), b. hominis ( ), s. stercoralis, g. lamblia, a. caviae, salmonella enterica and cryptococcus (one in each case). most postransplant diarrheas ( / ; %) occurred during the weeks after infusion and were attributable to mucosal damage caused by the ric (negative microbial screening and no evidence of gvhd).the rate of infectious g-ec among the diarrheas occurring after day + was % ( / ). the overall incidences of enteric infection were . % ( % ci: . - . ) and . % ( % ci: . - . ) at + and + months after sct, respectively. all the infected patients had mild to moderate disease, and no deaths were attributable to this complication. there were no differences in year-os and nrm between the infected and uninfected patients ( % vs %, p = . and % vs %, p = . , respectively). in univariate analysis age o years, prior sct, donor type, atg administration and prior grade - agvhd were associated with development of infectious gastro-enteritis. in multivariate analysis, unrelated donor and grade - agvhd were the only factors significantly associated with gastrointestinal infection (hr . ; % ci: . - . , p = . and hr . , % ci . - . , p = . ; respectively). acute gvhd occurred in % of patients (n = ), with a median onset of days (range: - ). the cumulative incidences of - acute gvhd at days and months post-sct were % ( % ci: . - %) and . % ( % ci: . - %), respectively, and there was a trend toward a higher risk of - gvhd in the group of patients with an enteric pathogen ( . % vs % at year, p = . ). more importantly, an enteric infection occurred just before the onset or aggravation of gvhd in / infected patients in our study ( %) at a median interval of days after the infection (range: - ). in summary, our results confirm that enteric infections are a common complication after allo-ric, representing at least % of the episodes of acute diarrhea during the first year post-sct. a possible interplay between infectious g-ec and gvhd was observed in this study. disclosure of conflict of interest: none. long-term efficacy of extracorporeal photopheresis in chronic graft versus host disease m nygaard , t karlsmark , n smedegaard andersen , i schjødt , s lykke petersen , l smidstrup friis , b kornblit and h sengeløv department of dermatology, bispebjerg hospital, copenhagen, denmark and department of hematology, rigshospitalet, copenhagen, denmark chronic graft versus host disease (cgvhd) activity is known to fluctuate over time, so we evaluated cgvhd continuously throughout the extracorporeal photopheresis (ecp) treatment course and after stopping ecp. patients with at least year follow-up, who were treated with ecp at department of dermatology, bispebjerg hospital between and were evaluated. a single investigator retrospectively evaluated response to ecp monthly for months, every months until years and every months until years. prednisolone doses were recorded every months. responses were defined as complete remission (cr) if no symptoms of cgvhd were present, partial remission (pr) as improvement in cgvhd or stationary cgvhd with more than % reduction in prednisolone, no change (nc) as no difference in symptom burden and o % reduction in prednisolone. progressive disease (pd) was defined as worsening of symptoms with unchanged or intensified immunosuppressive medication. ecp was performed with therakos uvar xts or cellex. there were evaluable patients with moderate (n = ) or severe (n = ) steroid-refractory, dependent or -intolerant cgvhd. the median age was years (range: - ) and there were females and males. conditioning regimen was myeloablative (n = ) and non-myeloablative (n = ). seventeen had related donors and had unrelated. stem cell source was peripheral blood (n = ), bone marrow (n = ) or umbilical cord blood (n = ). number of organs affected by cgvhd was one (n = ), two (n = ), three (n = ), four (n = ) or five (n = ) and involved organs were skin (n = ), eyes (n = ), mouth (n = ), lungs (n = ), genitals (n = ), liver (n = ), musculoskeletal system (n = ) or gastrointestinal tract (n = ). time from diagnosis of cgvhd to first ecp was median days (range: - ) and time from referral to ecp and the first ecp procedure was median days (range: - ). at the time of the first ecp procedure patients were also treated with prednisolone (n = ), sirolimus (n = ), calcineurininhibitor (n = ), mycophenolate mofetil (n = ), imatinib (n = ), methotrexate (n = ) or rituximab (n = ). one patient received no immunosuppression. total number of ecp cycles was median (range: - ). responses over time are shown in figure . overall response to ecp was seen in ( %) of the patients. most responses were seen after more than months ecp treatment. in univariate analysis of possible baseline predictors of response, no significant associations were found. prednisolone dose was significantly reduced at every months after start of ecp (p o . ). additional cgvhd treatment was administered to ( %) patients during ecp treatment (sirolimus n = , calcineurininhibitor n = , uva n = , methotrexate n = , rituximab n = , mycophenolate mofetil n = ). about ( %) patients had more than additional treatment. prednisolone dose was increased at least once in ( %) patients during ecp treatment. overall survival at years was %. follow up was median days (range: - ). more than half the patients with cgvhd ( %) improve overall after treatment with ecp, but flares in cgvhd activity still occur. prednisolone dose is significantly reduced at all time points after starting ecp, but short term increased doses or additional immunosuppression was necessary in more than one-third of the patients. larger prospective studies with long-term end points are warranted. disclosure of conflict of interest: marietta nygaard has received a travel grant and speaker's fee from therakos/ malinckrodt. chronic graft versus host disease (cgvhd) remains a major cause of morbidity and mortality after hematopoietic stem cells transplantation despite the improvement of the immunosuppressive prophylaxis. skin, buccal, lacrymal and hepatic disorders are the most frequent. sclerotic gvhd remains a severe form and often refractory to standard treatment lines such as corticosteroids and calcineurin inhibitors. the antifibrotic activity of imatinib by the inhibition of pdgf-r and tgfb-β pathways has been used in the treatment of refractory gvhc with sclerotic features and systemic scleroderma. here, we report the results of imatinib treatment in patients (pts) with refractory cgvhd. over a period of years (january -december ), pts received allogeneic stem cells transplantation from related donors, of whom were treated with imatinib for refractory cgvhd: pts for malignant diseases ( cml, aml, nhl) and pts for aplastic anemia. the median age is years ( - ), the sex ratio m/f: . . conditioning regimen used with chemotherapy alone: myeloablative ( pts with gvhd prophylaxis combining ciclosporin and methotrexate), reduced intensity ( pts with prophylaxis combining ciclosporin-mycophenolate mofetil). all pts received peripheral blood stem cell transplant with an average of cd cell count: . × /kg ( . - . ). the median duration of the cgvhd is months ( - ). the firstline treatment consisted of the combination of steroidsciclosporin with or without mycophelonate mofetil. imatinib was administered to these pts after median treatment duration of months ( - ) for moderate ( pts) and severe ( pts) cgvhd according to the nih classification. treatment with imatinib, at doses ranging from to mg/d, was introduced in the second line for all pts. the evaluation is conducted in october after a median follow-up of months ( - ). tolerance was good except in a one pt with severe thrombocytopenia that led to a transient cessation of treatment. after months, analysis of pts who received imatinib according to couriel criteria and nih criteria: complete remission (cr): pt ( %), partial remission (pr): pts ( %), stable disease (sd): pts, failure: pts ( %). a long-term evaluation performed after a mean duration of treatment months ( - ) finds similar results with a cr: pts ( %), pr: pts ( %), sd: pts ( %) and failure: pts ( %). corticosteroids were tapered or discontinued in pts (cr or pr). at october , pts ( %) were alive and pts ( %) died of severe infections. treatment with imatinib seems to be a good therapeutic option in the treatment of cgvhd in its moderate or severe form refractory to a minimum of two immunosuppressive agents according to the nih criteria as shown by our results in terms of response and survival with good tolerance. disclosure of conflict of interest: none. atg significantly reduces the risk of cgvhd both in unrelated and in hla identical sibling. the finke's study randomised pts undergoing an allogeneic unrelated stem cell transplant (sct) after a myeloablative regimen to receive or not mg/kg atg-grafalon reporting a significant reduction of cgvhd without increase of relapse and no os and dfs effect. however a successive study didn't confirm those results (significant reduction of acute and chronic gvhd but poorer survival mainly due to higher relapse probability in the atg arm). the conflicting data reported on urd sct have several explanations, one is about the dose and the timing of atg. the timing of atg infusion has been demonstrated to be crucial for cb transplant : an earlier administration is still active in preventing gvhd while ensuring engraftment and low hampering of immune reconstitution. here we report a large ( sct) retrospective monocentric analysis on low atg doses (and - mg/kg for bm according to the degree of hla matching and mg/kg for all pbsc sct) given early (from day − to − ). pts in the study were aml (n = , %), all (n = , %), hr mds (n = , %), cr (n = , %) cr or (n = , %), active disease (n = , %) for al; median age was (range: - ). myeloablative conditioning were bu-cy (n = , %), bu-flu (n = , %), edx-tbi (n = , %), other (n = , %); pbsc was used in % (n = ); sct were performed between and at the bologna transplant center. sct were performed from hla / identical urd (n = , %), or from / (n = , %), / (n = , %) and o / (n = , %). median follow up was months. overall, grade - agvhd was %, grade - agvhd %; cumulative incidence (ci) of cgvhd of any severity was %, for moderatesevere cgvhd %. ci of relapse and nrm was % and %, respectively. the -year overall and disease-free survival were % ( % ci: - %) and % ( % ci: - %). the gvhd (agvhd grade - and moderate-severe cgvhd) and relapse free survival (grfs) of the entire population ( figure ) was % at years ( % ci: - %). restricting the analysis to patients in cr - , we found that cgvhd (any severity), gfrs and os at years were %, % and %, respectively. comparing transplants with / urd to mismatched ones ( / or less) we found a trend for increased mod/sev cgvhd in pts undergoing transplant with mismatched urd (shr . , % ci: . - . , p = . ); agvhd grade - and cgvhd overall were not significantly increased; relapse incidence according to hla mismatches resulted % and % in / and ⩽ / , respectively; grfs was % in / and % in ⩽ / . the data reported show that low and early administration of atg is able to effectively prevent acute and chronic gvhd without increasing relapse thus ensuring really convincing grfs, even for o / matched urd transplants. graft versus host disease (gvhd) is one of serious complications in patients after allogeneic hematopoietic stem cell transplantation. the application of mesenchymal stem cells (msc) represents a promising method for the treatment of severe steroid refractory gvhd. we present the data from an interim analysis of clinical trial, within which we applied msc in patients with acute or chronic gvhd after allogeneic transplantation. the diagnoses included aml ( pts), mds ( pts), all ( pts), cll/nhl ( pts), mpn ( pts). the patients underwent sibling hla-compatible ( ), haploidentical ( ), unrelated hla-compatible ( ) or hla-mismatched ( ) transplants. the median interval between the transplantation and msc was months ( - ). the indications for msc infusion were steroid resistant acute gvhd ( pts), steroid-dependent gvhd (agvhd pts, cgvhd pts) or chronic gvhd with the need for long-term immunosuppression and corticosteroid intolerance ( pts). msc were applied as a single infusion at a median dose of . ( . - . ) × /kg. response to treatment was assessed on day , , and . the severity of gvhd prior to msc was graded as clinical stage ( - ) in acute and stage ( - ) in chronic gvhd, respectively. the median dose of corticosteroids was . ( . - . ) mg/kg/day in agvhd and s . ( . - . ) mg/kg/day in cgvhd patients. on day + the partial response (pr) was achieved in % of patients with agvhd, the stabilisation of gvhd (sd) was found in % of patients with cgvhd. the dose of corticosteroids was reduced in most patients with agvhd (to % of the starting dose; - %), while the early reduction was possible only in % of cgvhd patients. on day+ only patients were evaluable. the agvhd patients ( pts) achieved a significant clinical response: pr, cr and dose of corticosteroids was reduced in all of them (to %; - %). the minor responses were achieved in cgvhd patient ( pts.) with pr and sd. however the dose of corticosteroid was reduced in % of these cases (to % of the initial dose; - %). a total of patients died because of infectious complications. most of them ( / ) were agvhd patients who expired early up to day + . there were observed no side effects of msc application neither during the infusion nor later during the follow-up of ( - ) months. the analysis of lymphocyte reconstitution revealed the changes of kinetics of some subsets as compared to the day + benchmark. the b-lymphocyte count tended to decrease in % of patients from chronic gvhd subgroup (vs % in agvhd). conversely nk cells declined in most agvhd patients ( % vs % in cgvhd). also the pro-inflammatory th cell was affected especially in agvhd (decrease in % pts vs % pts in cgvhd). the counts of myeloid/plasmocytoid dendritic cell increased in %/ % agvhd and %/ % ccvhd patients. the screening testing of cytokines (raybiotech, cytokines, pts, day + to + ) revealed changes of some analytes after msc infusion, including a decrease of proinflammatory cytokines such as ifn-γ, tnf-α, il- . our experience with the treatment of gvhd using msc confirmed the safety of this immunotherapy. the favourable clinical effect with reduction of severity of gvhd and steroids dose was observed, especially in patients with acute form of gvhd. methotrexate day + omission is not associated with higher incidence of acute graft-versus-host-disease mm rivera franco, e leon rodriguez and a campos castro allogeneic hematopoietic stem cell transplantation (allo-hsct) remains a high-risk procedure due to its related morbimortality, limiting the broader application of this important treatment modality. despite extensive research over the years, acute graft-versus-host-disease (agvhd) affects the majority of patients undergoing allo-hsct, and up to % will develop clinically significant grades (ii). over the years, several methods for gvhd prophylaxis have been implemented, including immunosuppresive agents. methotrexate (mtx) is one of the earliest drugs used for gvhd prophylaxis. frequently, a short course of intravenous methotrexate (given on days + , + , + and + after hsct) is combined with a -month tapered course of cyclosporine. there is no consensus on which drugs and schedules for prevention of gvhd are best and clinical practice varies by institution. further, it is not clear whether omission of the day + dose of mtx has a negative effect on outcome in terms of morbidity. to describe the frequency of acute and chronic gvhd, mucositis and engraftment in patients receiving methotrexate (plus csa) as prophylaxis, omitting day + . ninety-five consecutive patients who underwent allo-hsct from to september , and received mtx as immunosuppressive prophylaxis were included. all patients received three doses of mtx, always excluding day + . mtx was administered iv, either mg/m day + , + , + or mg/m day + , and mg/m during days + and + . we included patients ( % male). the most frequent underlying diseases were aplastic anemia ( %) and acute lymphocytic leukemia ( %). ninety-nine percent of patients had a matched related donor. forty patients ( %) had gender disparity with their donor, and % presented abo incompatibility (major in %). most of the patients received myeloablative conditioning regimens (n = , %). the median of cd + infused cells were × (range: . - . ). the median neutrophil and platelet engraftment was ( - ) and (range: - ) days, respectively. from all the cohort, only patients ( %) developed acute gvhd ( % grades i-ii) ( figure ). thirty patients ( %) developed chronic gvhd, which was limited in %. most of the patients, % (n = ), presented acute toxicity after the conditioning regimen, from which % (n = ) corresponded to superior mucositis ( % grade i-ii and % grade iii-iv. the -year overall survival was % and the -year relapse free survival was %. our results showed a low incidence of acute gvhd, mostly grades i-ii, and similar survivals compared to previously reported studies, proposing that the administration of day + mtx as gvhd prophylaxis is not mandatory, however, prospective studies might be necessary to test our results. [p ] disclosure of conflict of interest: none. outcome of refractory graft versus host disease (gvhd) treated with extracorporeal photopheresis (ecp) as second line: a single-center experience j cornago navascues, b aguado bueno, av arriero garcía, edc jimenez barral, i vicuña andres and a alegre amor hematology department, university hospital la princesa, madrid, spain gvhd is a common and, sometimes, life-threatening entity related to hematopoietic stem cell transplantation (hsct). steroids remain the first-line therapy but they are not always enough to control it, or their side effects are simply unacceptable. both acute and chronic gvhd are responsible of impairment occurred in different organs that can lead to increase morbidity and mortality in our patients. different options are available as second line, but it is a well known fact that ecp, due to its inmunomodulatory mechanism, yields satisfactory response rates and presents excellent safety profile. from may to october , patients with steroid-dependent or refractory gvhd have been treated in our centre with ecp. we have performed ecp procedures with the therakos cellex device, an integrated 'on line' system. the transplant was from a sibling donor in cases and from an unrelated donor. the median of cd + infused was . × cd /kg. eight patients ( . %) presented agvhd, ( . %) cgvhd and finally, ( . %) had an overlap gvhd syndrome. most of patients ( . %) with agvhd had a severe intestinal involvement as the main manifestation of the disease. however, all patients with cgvhd had a multiorgan involvement with a median of four organs affected, being skin, mouth, eyes and lungs the most common implicated. ten patients in our series have died, for gvhd complications or infections and due to relapse of aml. as firstline treatment they all received steroids and cyclosporine or mycophenolate mofetil. median ecp per patient has been ( - ). ecp procedures were performed for consecutive days, in initial phase weekly (in those with agvhd), or every weeks (cgvhd) and then monthly according to clinical response, evaluated by clinical assessment and reduction in immunosuppression. about % of patients with agvhd had a significant clinical response to ecp so that steroid doses could be tapered and even in . % of them withdrawal was possible. in the cgvhd group overall response rate (orr) to ecp was . %. in % of these patients steroids could be suspended after a median of . ecp procedures. all patients who responded to ecp in cgvhd are still alive. independently of gvhd type, . % of patients responded to ecp and % of them even could stop steroid therapy. those who had no response are dead. in cgvhd, . % of patients remain alive, in contrast with agvhd or overlap syndrome patients whom survival is around %. about adverse events, % of patients did not present any complication associated with ecp. complications were mostly related to central venous catheter, with cases of bacteremia and thrombosis, easily recovered. in our experience, ecp is effective as second line treatment in gvhd, obtaining the best results in the chronic gvhd group. in fact, cgvhd patients with a good clinical response to ecp, specially when steroid doses can be tapered, have the better outcomes and longer survival. the tolerance to the procedure is excellent without severe adverse events. more experience is required to determine the best scheme of ecp and its role as prophylactic treatment. mesenchymal stromal cells (mscs) possess immunomodulatory properties and may play important roles in graft-versushost disease (gvhd) and engraftment. this study examined co-transplantation of mscs and hscs (hematopoietic stem cells). we investigated co-administration of ex vivo expanded mscs along with hla-identical sibling-matched hscs in β thalassemia major patients. we recruited patients from january to january in our study. all participants received cyclophosphamide-based or fludarabine-based conditioning regimens and short-course methotrexate and cyclosporine as gvhd prophylaxis. mscs were administered intravenously ( . - . × /kg) into patients (n = ) h before infusion of hscs. the outcomes were then compared to those of patients transplanted with hscs alone. the median follow-up in the msc and non-msc group was . and . years, respectively. median time to wbc engraftment . × /l was . days (range: - days) in both groups (p-value = . ) and median time to platelet engraftment × /l was . days (range: - days) in the msc group, while it was . days (range: - days) in the non-msc group (p-value = . ). fifty-six percent of patients had acute gvhd in the msc group compared to the non-msc group where . % developed acute gvhd (p-value = . ). meanwhile, chronic gvhd was % in the msc group and % in the non-msc group (p-value = . ). although the incidence of acute and chronic gvhd was lower in co-transplantation of hscs and mscs, no statistically significant difference was noted between the two groups. three-year overall survival rate was % and % in the msc and non-msc group, respectively (p-value = . ). three-year thalassemia-free survival rate was % in the msc group and % in the non-mscs group, showing no statistically significant difference (p-value = . ). the -year rejection incidence in the msc and non-msc group was % and %, respectively (p-value = . ). there was no statistically significant difference between the two groups in terms of -year transplant-related mortality (pvalue = . ). this study indicates that co-transplantation of hla-identical sibling hscs with mscs does not inflict harm on bone marrow transplantation procedure and seems to be safe and secure. on the other hand, differences between the two groups in acute and chronic gvhd, engraftment, overall survival, thalassemia-free survival and rejection incidence did not reach statistical significance. therefore, despite the immunomodulatory activity of mscs and their role in gvhd amelioration and engraftment improvement resulted from in vitro studies, their efficacy in the clinical setting has not been conclusively proven which indicates further multicenter randomized clinical trials are required. keywords: β-thalassemia major, co-transplantation of mesenchymal and hematopoietic stem cells, engraftment, graft-versus-host disease. hematology-oncology and stem cell transplantation research center, tehran university of medical sciences, shariati hospital, tehran, iran. disclosure of conflict of interest: none. a number of studies were published with contradictory results comparing tacrolimus (tac) and cyclosporine a (csa) for graftversus-host disease (gvhd) prophylaxis, but there are only few that accounted for pharmacokinetic (pk) parameters. in this retrospective study we have identified pk parameters that affected gvhd incidence and incorporated them in the s multivariate comparison of tac-and csa-based prophylaxis. the retrospective study included consecutive patients with csa and consecutive patients with tac prophylaxis. % were grafted from matched related donor (mrd) and % from unrelated donor (ud). about % received busulfanbased myeloablative conditioning (mac) and % reducedintensity conditioning (ric). second agent for gvhd prophylaxis was short-course methotrexate (mtx) - mg/m on days + , , , in % of patients and mycophenolate mofetil mg/kg days − to + in %. unrelated graft recipients also received antithymocyte globulin (atgam, pfizer, ny, usa) mg/kg. the pk parameters analyzed were mean and median concentrations, pk variability parameters and number of concentrations below the targeted limit (nlow) within , and days after hsct. for tac the highest predictive value for acute gvhd was observed for median concentration during first days (auc = . ), and for absolute skewness (auc = . ) of concentration data. for csa parameters with highest predictive value were median concentration (auc = . ) and variability coefficient (auc = . ) during first days. nlow was also a significant parameter for tac current gvhd prevention regimens are partially effective, delay immune reconstitution, impair graft versus tumor effect and are cumbersome to use. therefore, there is a pressing need to develop innovative approaches for the prevention of gvhd. we completed a phase i-ii study employing a calcineurin and mtor inhibitor-free regimen based on a combination of post-transplant cyclophosphamide and bortezomib (cybor) in patients receiving fludarabine and busulfanbased reduced-intensity conditioning followed by peripheral blood, matched related or unrelated transplant. patients receiving grafts from unrelated donors also received ratg. we reported that the regimen was feasible and safe and yielded promising outcomes. ( , ) herein, we compare the results to those of a quasi-contemporaneous matched group of patients receiving a calcineurin-based gvhd prophylaxis. the experimental and control groups were well-matched in terms of age, sex, donor type, disease status, renal function and pam score. the cybor group (n = ) was treated during a timeframe spanning from to and the control group (n = ) from to . gvhd prophylaxis for the control group was mmf and csa (n = ) or tacrolimus (n = ). both groups received supportive care according to standard institutional protocols. median follow-up for the cybor group was . months as opposed to . for the control group. median times to neutrophil engraftment for the cybor and control groups were days ( - ) and ( - ), respectively (p = . ). two patients from the cybor arm died before achieving platelet engraftment. for the remaining s patients, median time to platelet engraftment was days ( - ). for the control group, five patients never dropped their platelet count below × /l. for the remaining patients, median time to platelet engraftment was days ( - ) (p = . ). there was no primary or secondary graft failure in either of the two groups. the incidences of acute grade ii-iv and grade iii-iv for the cybor group were . and . %. for the control group, the incidences were (p = . ) and % (p = . ). the incidence of chronic gvhd for the cybor and control groups were % and . %, respectively (p = . ). treatment-related mortality was . % and % for the cybor and control groups, respectively (p = . ). the incidences of cmv, ebv and bk reactivation for the cybor group were . %, . % and . %, respectively. for the control group, the incidences were . % (p = . ), . % (p = . ), % (p = . ). the -year progression free survival and overall survival were . % and . % for cybor group and . % and . % for the control group ( figure ). the year gvhd and disease-free survival (grfs) were . % and %, respectively. despite the limitations of our study that include its size and its design and the delayed neutrophil and platelet engraftment associated with the cybor regimen in comparison to calcineurin-based prophylaxis, our data confirm the promising outcomes previously reported with the cybor combination and reaffirm the need for a large randomized study comparing cybor to a standard calcineurin-based regimen. clostridium difficile infection (cdi) causing enterocolitis may represent a serious clinical problem in patients undergoing allogeneic hematopoietic cell transplantation (allo hct). the reported prevalence varies substantially among heterogeneous patient cohorts. although cdi has been proposed as a risk factor for the development of gastrointestinal (gi) acute graft-versus-host-disease (agvhd), limited knowledge on the prevalence of cdi, occurrence of gi agvhd in cdi patients, relapse incidence and mortality of cdi patients in large patient cohorts is available. the aim of this analysis was to study the implications of cdi in a homogenous cohort of patients with either aml or mds undergoing allo hct. at our center all patients undergo stool test once a week for clostridium difficile antigen while in aplasia until discharge, irrespective of clinical symptoms for enterocolitis. patients with positive stool antigen tests (that is, toxin test) in the absence of clinical symptoms were referred to as cd+, in contrast to patients without a positive test and without clinical symptoms which were referred to as cd − . we retrospectively analyzed the data of a total of n = patients with either aml or mds undergoing allo hct in our institution between and . overall survival (os) was measured from allo hct to the date of death or last follow-up. after hsct, relapse and nonrelapse mortality were considered as competing events. eventprobabilities were calculated according to kaplan-meier for os and using competing event statistics for the cumulative incidence of relapse (cir), non-relapse mortality (nrm) and agvhd. % confidence intervals (ci) were provided for major endpoints. statistical analyses were performed using the r environment for statistical computing version . . (r core team , vienna, austria, www.r-project.org). from a total of n = patients with either aml or mds who underwent allo hct, we identified n = ( %) who were cd − , n = ( %) who were cd+, and n = ( %) who had cdi. interestingly, n = ( %) of patients with cdi were diagnosed having gi agvhd as compared to n = ( %) of patients who were cd+ and compared to n = ( %) of patients who were cd − , p = . . the three groups harbored no differences when comparing incidences of liver and skin agvhd or chronic gvhd, respectively. when dissecting gi agvhd according to ctcae criteria, only n = ( %) of cdi patients vs n = ( %) of cd+ patients, and n = ( %) of cd − patients had grade - gi agvhd, p = . . with regard to os and trm, no statistical differences were observed between the three groups. the cir was % for patients with cdi, % for cd+ patients and % for cd − patients, p = . , respectively. this analysis represents the largest published analysis of clostridium difficile in patients with aml or mds who underwent allo hct. the prevalence of cdi in this patient cohort was %. patients with cdi developed significantly more often gi agvhd as compared to patients who were either cd+ or cd − , respectively. however, this did not translate into differences in os or trm. disclosure of conflict of interest: friedrich stölzel has received research funding from astellas. the majority of studies on cytokines in allogeneic hsct were performed with classical gvhd prophylaxis, consisting of nonspecific immunosuppressive agents. with this type of prophylaxis almost in all studies published, higher levels of proinflammatory cytokines are associated with development of acute gvhd, while lower levels indicate the success of immunosuppressive agents in abrogation of alloreactive response. currently, there is no data, whether the dynamics of cytokines after ptcy is similar to the situation of classical gvhd prophylaxis. out of adult patients transplanted at first state medical university with ptcy between and we have identified cases with acute gvhd and plasma samples available. these patients were matched in the ratio : to patients who did not develop acute gvhd. the study group was comprised of adult patients with hematological malignancies who underwent hsct. all patients received ptcy-based gvhd prophylaxis. five plasma biomarkers were studied by elisa: il- a, il- , il- , tnf-α and ifn-γ. blood samples were obtained from patients on days − , , + , + . the fifth time point varied between day + and + to represent the sample after engraftment, but before onset of acute gvhd. about ( %) out of gvhd patients had a grade i, ( %) grade ii, ( %) grade iii agvhd, ( %) patients developed multiorgan agvhd. about patients ( . %) had chronic gvhd. there was no difference between gvhd + and gvhd − groups in any of the clinical parameters. the median of engraftment for all patients was ( - : range). the median agvhd was days ( - : range). neither of the cytokine levels was significantly different in patients with agvhd grades i − iv and without gvhd. however, for patients with agvhd grade ii − iv we found that low levels of il- on day + ( . ± . vs . ± . pg/ml, p = . ) and ifn-γ on day + - ( . ± . vs . ± . pg/ml, p = . ) were associated with increased risk of gvhd. the roc analysis was performed to determine the cut off values for il- - . pg/ml (auc = . ) and ifn-γ - . pg/ml (auc = . ). the incidence of agvhd grade ii-iv was significantly higher in patients with levels of cytokines lower than cut off ( % vs . %, p = . and . %, p = . for il- and ifn-γ, respectively). the same pattern was observed for patients with agvhd grade iii-iv. low levels of il- ( . ± . vs . ± . pg/ml, p = . ) and ifn-γ ( . ± . vs . ± . pg/ml, p = . ) on day + were especially predictive. the cut off values for il- was . pg/ml (auc = . ) and for ifn-γ- . pg/ml (auc = . ). the incidence of agvhd grade ii-iv was also significantly higher in patients with levels of cytokines lower than cut off (p = . and p = . for il- and ifn-γ, respectively). for chronic gvhd only higher level of il- at day + ( . ± . vs . ± . pg/ml, p = . for patient with and without gvhd, respectively) was significantly predictive. in this pilot trial we have demonstrated that dynamics of cytokines after gvhd prophylaxis with ptcy may be different from conventional one, and well-known predictive biomarkers might not work after ptcy. further large prospective trials are warranted to elucidate reliable biomarkers for gvhd after this type of prophylaxis. disclosure of conflict of interest: none. graft versus host disease (gvhd) remains one of the main obstacles to broader application of allogeneic transplantation. gvhd prevention and treatment techniques are poorly standardized. the st-line treatment of newly diagnosed chronic (c) gvhd is corticosteroid. there is no standard ndline treatment for cgvhd. approximately - % of patients (pts) with cgvhd require secondary treatment within y after initial systemic treatment. recently the jak / inhibitor ruxolitinib emerged as an efficacious treatment for corticosteroid-refractory (sr) acute and c-gvhd with a % of sr-cgvhd patients reporting a long lasting immunosuppression-free complete response. the current study seeks to analyse the efficacy and safety of ruxolitinib in highly pre-treated sr-cgvhd pts in our centre. ruxolitinib treatment was given off label after provision of an informed signed consent and in the absence of alternative therapeutic options including clinical trials. we analysed data prospectively collected at our long-term follow-up clinic between and . a written consent was given by pts allowing the use of medical records for research in accordance with the declaration of helsinki. overall pts (median age y-range: - years; mean karnofsky score %) with sr-cgvhd were treated s with ruxolitinib. median time from transplant was months (range: - ). ruxolitinib was initiated at a starting dose of mg twice daily-median time on ruxolitinib months (range: - )- / pts increased the dose up to mg twice daily. four pts had a classic and an overlap sr-cgvhd. all of them had skin sclerodermatous involvement and / joint and fascia involvement with significant decrease of range of motion and limitation of adl. all pts were previously treated with several lines of immunosuppression ( - ) including high-dose prednisone in st line ( / ), rapamycin ( / ), tk-inhibitor imatinib ( / ), extracorporeal photopheresis ( / ). all pts were pre-screened for risk of infection and regularly checked on a fortnightly basis. all pts were under active prophylaxis according to recommendation for gvhd pts and ruxolitinib therapy. after a cumulative follow-up of days we reported only one serious adverse event represented by a cmv pneumonia requiring hospitalization with complete recovery. early time point evaluation ( / pts evaluable) at + month underlined how all pts were reporting subjective improvement at the patient global ratings according to nih . data were confirmed at the health care provider global ratings. month evaluation ( / ) confirmed meaningful responses (partial responses / ) according to nih , with both patient and health care provider global ratings improvement and concomitant enhancement in lee skin symptoms score and sf- health-related qol. at last followup no evidence of myelosuppression, infections, pml, nonmelanoma skin cancer was registered. considering the concomitant treatment (with reference to azoles and rapamycin or cyclosporine) no cases of toxicity due to drug-druginteraction was reported. ruxolitinib is well tolerated in highly pre-treated sr-cgvhd. its safety profile seems to be reassuring. the efficacy data observed also at this early time point is preliminary but promising in this subset of pts with a long history (⩾ lines) of treatment for cgvhd. confirmatory study in a larger number of patients is underway on a multicentre basis. disclosure of conflict of interest: none. severe acute enteral graft-versus-host-disease (gvhd) is a lifethreatening complication of allogeneic bone marrow transplantation. in case of resistance to corticosteroids as the firstline treatment severe enteral gvhd harbors a high morbidity and mortality. retrospective analyses indicate efficacy of the jak / -inhibitor ruxolitinib in the treatment of acute or chronic gvhd in adults, but experience in paediatric patients is limited. here, we report a small cohort of paediatric patients with stage steroid-refractory gvhd of the gut who received ruxolitinib as salvage therapy within a multimodal immunosuppressive regimen. we retrospectively analysed four patients aged - years with severe, steroid-resistant acute gvhd of the gut who were treated with ruxolitinib in our institution. all patients were transplanted for non-malignant haematologic disorders, graft source was × mmud, × mud, × msd. the conditioning regimen consisted of treosulfan, fludarabine and thiotepa. serotherapy with thymoglobuline was administered in all patients transplanted from unrelated donors. all patients received mtx and cyclosporine as gvhdprophylaxis. gvhd was staged according to the glucksberg-scale. ruxolitinib was added to the immunosuppressive regimen when acute stage gvhd was reached and became resistant to treatment with methylprednisolone ( mg/kg/day) as well as infliximab and mycophenolate (mmf) as second-line immunosuppressants. acute stage enteral gvhd developed at a median of days after transplant ( - days) and ruxolitinib was started at a median of days post-transplant ( - days). the starting dose varied between mg/day and mg/day, that is, . - . mg/kg/day, taking into account the expectedly low bioavailability of the oral drug during severe diarrhea. upon improvement of gvhd symptoms and/ or increasing side effects the dose was gradually tapered and ruxolitinib was discontinued after a median of treatmentdays ( - days) . after addition of ruxolitinib to the immunosuppressive regimen, the symptoms of acute gut gvhd gradually improved in all four patients with decreasing abdominal pain and stool volumes. immunosuppression with steroids and mmf could slowly be tapered. all patients are alive after a median follow-up of days ( - days) from diagnosis of acute stage gut gvhd. the most prominent side effect attributable to ruxolitinib was thrombocytopenia with a nadir in platelet counts after days of ruxolitinib treatment in / patients. platelets recovered within weeks after ruxolitinib was discontinued. neutropenia was observed in one patient with anc dropping o . /nl after days of ruxolitinib treatment. mild to moderate elevation of liver transaminases was observed in all four patients during ruxolitinib treatment. one patient developed imminent acute renal failure, another patient showed symptoms of hemolytic uraemic syndrome. however, due to the multimodal treatment of these critically ill patients, these complications could not clearly be attributed to ruxolitinib. ruxolitinib is potentially beneficial in severe acute enteral gvhd in children refractory to corticosteroids as well as second-line immunosuppressants. however, randomized trials are warranted to verify safety and efficacy of ruxolitinib in this patient cohort. disclosure of conflict of interest: none. steroid refractory acute gvhd is a major cause of mortality after allogeneic stem cell transplantation. until date, no agent or treatment strategy has demonstrated superior efficacy in this patient group. the dose and duration of steroid treatment is associated with several short and long-term side effects, therefore concepts facilitating rapid steroid taper may be beneficial. both ruxolitinib and ecp have been reported to be effective in treatment of steroid refractory (sr) agvhd. we analyzed data from consecutive adult patients who received ruxolitinib for sr agvhd between march and august in our institution overall, patients (male n = ; female n = ) with a median age of years (range: - ) were included. donors for allogeneic sct were msd (n = ), mud (n = ) and mmud (n = ). median time to gvhd onset after stem cell transplantation was days (range: - days). about patients had agvhd grade iii or iv (all with gi involvement), while patients had skin grade involvement. sr agvhd was diagnosed if agvhd manifestations were progressive after days or persistent and without improvement after days or no partial remission after days of treatment with mg/kg bw of systemic steroids. patients received additional ecp (n = ), if response to ruxolitinib was lacking or slow (n = ) or instead of ruxolitinib due to cytopenias (n = ). ruxolitinib was first-line treatment for sragvhd in patients ( %). median initial dose of ruxolitinib was mg (range: - mg) twice daily. steroids were tapered and stopped, even if agvhd was still active. primary end point was non-relapse mortality at months. secondary end point was response on day after initiation of ruxolitinib. response occurred relatively slowly, resulting in a day overall response rate of % (cr = , pr = ). however, a total nine patients ( %) attained a complete response (cr), five with ruxolitinib alone and four others in combination with ecp. about patients ( %) required dose reduction or interruption of ruxolitinib mainly due to cytopenias. after a median follow-up of days, patients are alive. causes of death were relapse of malignant disease (n = ), gvhd (n = ), infections (n = ) and other (n = ). median survival from diagnosis of sr agvhd was days for non-responders and days for responders ( figure , p = . ). in univariate analysis, non-response was associated with higher risk of nonrelapse mortality (rr; . , % ci: . - . , p = . ). ruxolitinib and ecp are two effective promising treatment options, which may be complementary in patients with sr agvhd. cytopenia is the most frequent side effect of ruxolitinib while infections remain the major cause of death. [p ] disclosure of conflict of interest: ayuk-therakos: honoraria; kröger: novartis: honoraria, research funding. steroid-refractory acute graft-versus-host disease (sr-agvhd) is associated with a dismal outcome. janus kinase (jak) / signaling has been shown to be instrumental in multiple steps leading to inflammation and tissue damage in gvhd. jak / inhibitor ruxolitinib was studied in the treatment of sr-gvhd by zeiser et al. (leukemia ) , and the overall response rate was reported to be . %. we have now studied ruxolitinib in the treatment of six adult patients with steroid-refractory, grade iii-iv, intestinal agvhd. all the patients were male. the median age of the patients was (range: - ) years. three of the patients were transplanted for aml, one for all, mds and mm each. all the patients had been given a myeloablative conditioning treatment (cytbi , treosulfan+fludarabine ). two patients had a sibling donor and four a matched unrelated donor. the graft was from peripheral blood in all the patients. gvhd prophylaxis consisted of cyclosporine and a short course of methotrexate, and in addition antithymocyte globulin in the unrelated donor setting and methylprednisolone in one sibling recipient. agvhd of the intestine manifested on days + , + , + , + , + and + with diarrhea. in two patients it was preceded by agvhd of the skin by and days, respectively. gi-biopsy showed acute gvhd of grade iii and of grade iv in three patients each. treatment of intestinal gvhd was started with methylprednisolone mg/ kg/day, tapering the dose to and mg/kg after doses each. gastroduodenoscopy and colonoscopy were performed at the onset of symptoms indicating intestinal gvhd. biopsy confirmed the diagnosis in all cases. because the diarrhea continued in spite of methylprednisolone treatment, ruxolitinib was started , , , , and days from the first day of diarrhea. the dose of ruxolitinib was mg × per day orally. four patients showed a clear response to ruxolinitib, normalization of bowel function, after , , and days from the start of ruxolitinib treatment. the healing of the intestinal lesions was verified by biopsy. two of these patients had received extracorporeal photopheresis simultaneously. two patients did not benefit from ruxolinitib treatment. one of them had continuous infectious complications and therefore ruxolitinib was only started after days from the start of diarrhea. the other patient died of fulminant diarrhea after weeks of ruxolitinib treatment. cmv reactivation was detected in three of the responders, and two of them had also polyoma virus cystitis. one patient developed a pulmonary aspergilloma, which is under control with drugs. corticosteroid-resistant gastrointestinal acute gvhd was treated in six patients, out of whom four showed a good response. disclosure of conflict of interest: none. although methotrexate (mtx) is commonly used in the prophylaxis of graft-versus-host disease (gvhd) after allogeneic hematopoietic stem cell transplantation (allo-hsct), some small studies have also reported its use in the treatment of chronic gvhd. the aim of this study was to evaluate the efficacy and safety profile of low-dose mtx for treatment of sclerodermatous chronic gvhd (sgvhd) after the failure of first and second line treatments. we retrospectively evaluated adult patients who received low-dose mtx as salvage treatment of sgvhd during the period elapsed between june and june in a tertiary referral university hospital in spain. there were ( %) males and ( %) females. the median age was years (range: - ). all had received an allo-hsct for hematologic malignancies. the median time from allo-hsct to sgvhd was days (range: - ). thirteen patients ( %) had presented previous acute skin gvdh. superficial skin lesions mimicking lichen planus (lichenoid gvhd) were diagnosed in ( %) patients, while lesions resembling lichen sclerosus, morphea or fasciitis (sgvhd) where seen in all ( %) patients. the total body surface area was affected by more than % in patients ( %). besides the skin, other organs/tissues involved were the eyes ( %), mouth ( %), nails ( %), lungs ( %), liver ( %) and gastrointestinal tract ( %). treatment lines prior to mtx administration were: prednisone (pdn) in patients ( %), phototherapy (pht) in ( %), cyclosporine (cya) in ( %), mycophenolate mofetil (mfm) in ( %), pht + pdn + cya in ( %), pdn + mfm + cya in ( %), extracorporeal photopheresis + pdn in ( %). the median time from sgvhd onset to mtx treatment was days (range: - days). mtx was administered subcutaneously in patients ( %) and orally in patients ( %). median dose of mtx was . mg/week (range: . - . mg/week) and median length of treatment was weeks (range: - weeks). in two patients ( %) early withdrawal of mtx occurred (one due to early death secondary to septic shock and other due to rapid disease progression). mtx-related toxicity occurred in three patients ( %): megaloblastic anemia, asymptomatic increase of liver enzymes and mucositis, respectively. response to mtx was evaluated in the patients ( %) s who did not suffer early mtx discontinuation. seventeen patients ( %) presented a partial response; of them, two are still under mtx treatment for and weeks, respectively. fourteen patients ( %) received pdn concomitantly to mtx (median dose mg/day, range: - ); year after mtx treatment, only four patients were receiving pdn (median dose mg/day, range - ). seven patients have finished mtx treatment without reappearance of symptoms, receiving only topical treatment with emollients, tacrolimus or corticoids for short periods. in four patients ( %) sgvhd progressed despite mtx administration. our data suggest that mtx is a safe, inexpensive and effective alternative for refractory sgvhd. its potential used in earlier phases of sgvhd deserves further investigation. disclosure of conflict of interest: none. severe chronic gvhd has a major influence on late morbidity and mortality after hematopoietic stem cells transplantation (hsct). ecp is a good approach to treat refractory-gvhd: leucocytes are obtained from peripheral blood by apheresis, incubated with -mop, irradiated and then infused to the patient where they undergo apoptosis and induce tolerance. it is a promising alternative that reduces doses of immunosuppressive therapy and their side effects in the treatment of gvhd. this study shows its efficacy in persistent refractory cgvhd. the procedure was applied to three patients (pts) aged , and years (two aml and one cml), sex ratio ( m/ f) who underwent allogeneic-hsct with myeloablative conditioning regimen based on chemotherapy alone from a peripheral blood stem cells with cd levels: . , . and . × /kg respectively. prophylaxis of gvhd combined ciclosporin and methotrexate in short cycle. severe extensive cgvhd (according to nih criteria) was observed in the three cases after an average delay of . months ( - ) with involvement of - organs (mouth, eyes, skin, liver, joints and lungs). all pts are refractory to three lines of immunosuppressive agents (ciclosporin-corticosteroids, mmf and imatinib), with an average of thrusts/pt ( - ) over an average period of months ( - ). ecp was performed under the open system or dissociated system (macopharma) for two sessions per week for weeks, one session per week for weeks, one session every weeks for weeks and one session per month for months. after a median period of months ( - ), an average of sessions/pt ( - ) was performed. in terms of tolerance, a red blood cell transfusion was required in one pt, spontaneously resolved lymphopenia was observed in another pt, and a poor venous approach led to the pause of a central catheterization in one pt. the month and -month evaluation according to the couriel response criteria shows a partial response observed as of the first month with net improvement especially on skin sclerotic features and joints retractions initially refractory to all therapeutic lines. this allowed gradual reduction doses of corticosteroids. pce is recommended in the curative treatment for refractory chronic gvhd from the second line. this encouraging study on a small series shows its efficacy in persistent and late refractory forms. it is nevertheless necessary to evaluate it on a larger number of pts. disclosure of conflict of interest: none. successful treatment of steroid-refractory acute gastrointestinal graft-versus-host-disease by fecal microbiota transplantation p neumeister steroid-refractory acute gastrointestinal (gi) graft-versus-host disease (agvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation (allo-hsct) associated with a high mortality rate. loss of intestinal bacterial diversity is thought to be associated with severity of gi-agvhd and an impaired intestinal microbiota with reduced diversity is an independent predictive factor for mortality. fecal microbiota transplantation (fmt) is the application of a fecal suspension derived from a healthy donor into a patient's gi tract. it has been successfully applied in recurrent clostridium difficile associated diarrhea including patients who underwent allo-hsct. we report the complete resolution of lower gi-agvhd following colonoscopic fmts in three patients that had been refractory to - lines of immunosuppressive therapies. microbiota analysis by s rdna before fmts revealed a severely depleted microbiota in all patients. donors (different persons for each patient) were healthy adult subjects. repetitive ( - ) colonoscopic fmts were necessary to permanently establish the donor's microbiome. all patients responded clinically by reduction/normalisation of stoll volumes, stopping total parenteral nutrition and tapering of steroids. a possible causative relationship of fmt in the reversal of severe intestinal dysbiosis and subsequent resolution of gi-agvhd can therefore be hypothesized. the establishment of donors' microbiota and increase in bacterial richness was associated with disease control. no immediate procedure-related infections or other side effects were observed. besides restoration of an initially severely reduced microbial richness by fmts, response of gi-agvhd was sustained despite reduction and discontinuation of concomitant immunosuppressive treatments. restoration of dysbiosis by fmt might represent a promising novel therapeutic approach for refractory lower gi-agvhd. disclosure of conflict of interest: none. tear film proteomics reveals important differences between patients with chronic ocular gvhd and healthy controls k plattner , n gerber-hollbach , s moes , p jenoe , d goldblum and j halter ophthalmology, university hospital basel, ch- basel and proteomics core facility of the biozentrum, university of basel, ch- , basel chronic gvhd frequently involves the eyes, leads to important decrease of quality of life and may threaten visual capacity. sicca syndrome is one of the hallmark of ocular cgvhd. analysis of tear protein composition may help to identify biomarkers for early diagnosis and prognosis of ocular cgvhd. tear fluid of patients with ocular cgvhd were compared with healthy individuals in this single center study. results of the first patients are reported here. tryptic digests from schirmer strips were analyzed on an orbitrap mass analyzer. clinical examinations included slit lamp examination, fluorescein staining, schirmer test, break-up-time (but) and a quality of life questionnaire (osdi). outcome measures were differences and consistency of proteins in human tear fluid s between patients with ocular gvhd and healthy controls. statistical analysis was performed by one sample wilcoxon-tests, p-values o . was considered significant. ten patients (eight males, two females) with a median age of years (range: - ) were analyzed. all underwent pbsct, eight from an unrelated donor. cgvhd overall score was moderate in three and severe in seven. eye organ score was in six and in four patients. all patients had more than one organ manifestation of cgvhd. eight were under systemic immunosuppressive therapy at the time of analysis, two had topical treatments only. in total different proteins were detected in tears analyzed. compared to controls, were differentially expressed in ocular cgvhd. expression was highly significantly different in proteins. compared to controls, expression of proteins was at least -fold increased, representing different categories. among them, more than % of all proteins belong to one of three categories: cytoskeletal proteins, nucleic acid binding or structural proteins. albumin, cluster or keratin (keratin type i-iii) and cluster of pyruvate kinase were most highly overexpressed. expression of proteins was decreased to o to %, belonging to different protein classes. half of them belong to defense/immunity proteins, enzyme modulators, hydrolases, nucleic acid binding and carrier proteins. expression of lactotransferrin, proline-rich protein and prolactin-inducible protein was most profoundly decreased. compared to healthy controls, a high number of protein is found to be differentially expressed in tears in ocular cgvhd. among them high expressions are observed for proteins that may indicate disturbed integrity of ocular surface and leakage of conjunctival capillaries. most profoundly decreased proteins include proteins with important functions in host defense and immunomodulation. more detailed pathway analysis is necessary to identify biomarkers for ocular cgvhd. disclosure of conflict of interest: none. steroid-dependent chronic gvhd after allogeneic peripheral blood stem cell transplantation is a great problem. nonresponders to corticosteroid therapy are at high risk of mortality. we hypothesized that such patients could benefit from treatment strategy using in patients with primary severe autoimmune diseases like multiple sclerosis and crohn's disease. patient z., y.o. was diagnosed in july with myelomonocytic leukemia (jmml). initially he was treated with low-dose of cytarabine and epigenetic agents. in september , jmml progression was observed with leukocytosis, thrombocytopenia, splenomegaly. bone marrow aspirate showed . % monocytes and . % blast cells. splenectomy was performed in november due to refractoriness to blood components transfusions. in december unmanipulated haploidentical peripheral blood stem cell transplantation from mother with . × /kg cd + and . × /kg cd + was performed. the conditioning regimen was myeloablative including melphalan mg/m day − and treosulfan mg/m days − , − , − . no organ toxicity grade was observed. gvhd prophylaxis consisted of hatg mg/kg on days − , − , − , + , i.v. tacrolimus from d − and mmf mg/kg from d . engrafted was fast and prompt ( % donor) with wbc . × /l on d + , plts × /l on d + . acute gvhd of stage ii was observed in early posttransplant period and treated with steroids and tnf-α inhibitor (infliximab). patient also received five procedures of ecp. all attempts of immunosupression tapering failed and the patient was staying on high dose of tacrolimus, mmf and courses of steroids till october . in october , gvhd stage ii flare with blood eosinophilia occurred after another attempt of steroids withdrawal. clinical examination showed that the patient was in complete remission with full donor chimerism. mild response of gvhd to steroids was observed. in april and may patient received two doses of rituximab mg/m with no significant response. in order to restore naive immune system first course of chemotherapy with cyclophosphamide mg/m was performed in the end of may . no toxicity grade was observed. the patient recovered wbc . × /l on d + , plts × /l on d + . in the phase of hematological recovery he was mobilized with g-csf and two leukaphereses of pbscs were performed. in june our patient was transplanted with previously collected . × cd +/kg following nonmyeloablative regimen including cyclophosphamide mg/m on day − and fludarabine mg/kg, on days − , − , − . second dose of cyclophosphamide was not administered because of severe hyponatriemia with seizures due to the cpm administration. no other significant toxicity was observed. the patient did not require either blood product or i.v. antibiotics. doses of tacrolimus and mmf were picked on months late and no more steroids were given. the patient is well in cr with no signs of gvhd for months. we speculate that pbsc collection from patients under massive immunosupression underwent allogeneic transplant is difficult but feasible. the nonmyeloablative regimens in such group of patients could be well tolerated and ensure the restoration of naive recipient immune system. this option could be discussed as an attractive alternative for treating resistant gvhd in steroid resistant patients. disclosure of conflict of interest: none. severe acute gi-gvhd is a serious early complication of allotransplants, and still remains a clinical diagnosis.( ) endoscopic biopsies provide the best supportive evidence, but are invasive and morbid in patients who are already medically compromised. f-fdg pet/ct may be able to stratify patients who require endoscopy and biopsy. to evaluate the performance of fdg pet/ct in differentiating moderate to severe gi-gvhd from no or mild disease in pediatric patients with suspected gi-gvhd. retrospective chart review of all paediatric allo-transplant patients referred for f-fdg pet/ct with suspected gi-gvhd from to . clinical follow-up, endoscopy and biopsy findings were correlated with f-fdg pet/ct. regional suv parameters were extracted by placing rois around stomach, duodenum, distal ilium, caecum, ascending, transverse, descending colon, recto-sigmoid colon and rectum. regional, and average large and small bowel suv data were statistically compared between patients with no or mild git-gvhd vs moderate to severe disease. the clinical and biopsy-supported diagnosis of acute gi-gvhd was taken as the true positive diagnosis for acute gi-gvhd. roc curves were generated for whole bowel suvmax values. about scans in patients, median age of years ( mths to y), were performed at a median of days post bmt. there were stage , stage - and with no acute gi-gvhd. transverse colon suvmax was significantly higher in the stage - gi-gvhd compared to no or stage disease (mann-whitney-u-test p o . ). there was a non-significant trend for average large bowel suvmax to be higher in the stage - group than the no or stage disease group (mean suvmax . compared to . , p = . ). a cut off whole bowel suvmax . had a sensitivity of % and specificity of % for detecting moderate to severe gi-gvhd. f-fdg pet/ct is a feasible and potentially useful non-invasive tool in the diagnosis and monitoring of therapeutic efficacy in acute gi-gvhd ( ) . large bowel suvmax may be higher in patients with stage - gi-gvhd, and transverse colon suvmax could have the ability to differentiate children with no or stage gi-gvhd from those with stage - disease. a negative fdg-pet/ct could serve as a criteria to avoid invasive endoscopic procedures and observe for the persistence of gastrointestinal symptoms before subjecting these patients to an imageguided biopsy. in patients too unwell for endoscopy, suvmax (roc curve specificity %) and a high suvmax in the transverse colon could serve as supportive evidence for moderate to severe acute gvhd, in the absence of biopsy findings. a major advantage of a pre-endoscopic f-fdg pet/ ct is to guide the procedurals to sample areas with the best diagnostic yield. prospective controlled studies are needed. oral mucosal progenitor cells (omlp-pcs) possess immunomodulatory and antibacterial properties, suggestive of their in vivo function in healthy tissue and their potential contribution to scarless wound healing in the buccal mucosa ( , ). our aim was to establish whether the function of oral stromal progenitors is impaired in chronic graft versus host disease (cgvhd) and restored with response to treatment. a patient with grade oral cgvhd was treated with systemic thalidomide for weeks ( mg/day). punch biopsies of buccal mucosa were taken before and after treatment. oral progenitor cells were isolated and expanded in vitro. numbers of progenitors was assessed using colony forming unitfibroblast (cfu-f) assays. stem cell markers (cd , cd , cd , cd , cd , cd , hla i and ii) were evaluated by flow cytometry. wound healing and antibacterial potential were assessed using a collagen gel lattice assay and bacterial cocultures as previously described ( , ) . secreted levels of relevant cyto-and chemokines associated with wound healing were assessed by elisa. significant clinical improvement with reduced inflammation in the oral mucosa and healing of ulcers was seen after weeks of thalidomide treatment, with continued improvement after weeks. cell surface expression of cd and cd on omlp-pcs was elevated postthalidomide; markers correlated with stemness and angiogenesis in mesenchymal stromal cells. this correlated with a restoration of wound healing potential and antibacterial function after thalidomide treatment ( figure ). figure : antibacterial testing demonstrated a loss of antibacterial function against (a) gram positive and (b) gram negative micro-organisms in the cgvhd omlp-pcs that could be completely or partially restored to levels comparable with healthy controls after thalidomide treatment. *p ⩽ . , **p ⩽ . , ***p ⩽ . . we demonstrate, for the first time a correlation between clinical improvement of oral cgvhd with thalidomide treatment and restoration of endogenous progenitor cell function. this study highlights the importance of a dysfunctional oral mucosal stroma in the pathogenesis of cgvhd. further studies should focus on the role of the stroma in promoting cgvhd and the precise mechanisms by which thalidomide is able to restore its functions. chronic graft-versus-host disease (cgvhd) is a major cause of late morbidity and treatment-related mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). cgvhd is driven by a th biased t-cell mediated alloreactive immune response that leads to chronic inflammation and fibrosis in various organs. thymic stromal lymphopoietin (tslp) is an epithelial cell-derived cytokine that mainly affects myeloid cells. upon stimulation with tslp, dendritic cells are polarized towards a dc phenotype driving th biased immune response. we hypothesized that tslp is involved in the pathogenesis of cgvhd and that elevated levels of tslp post-transplant may lead to an increased risk of cgvhd. in the present study, we measured plasma tslp levels during hsct to study associations with clinical outcomes including cgvhd. about adult patients undergoing myeloablative hsct at rigshospitalet, denmark, from to were included. diagnoses included aml (n = ), all (n = ), myelodysplastic syndrome (n = ), other malignancies (n = ) and anemias (n = ). donors were either hla matching siblings (n = ) or mud (n = ). grafts were either bmsc (n = ) or pbsc (n = ). conditioning included tbi (n = ) or high-dose chemotherapy alone (n = ). plasma tslp was measured by elisa (abcam) before transplantation, at the day of transplantation and at day + , + , + and + post-hsct. monocytes were counted daily, and t, b and nk cells were measured at day + and + using flow cytometry. about ( %) patients engrafted; acute gvhd grade - was seen in ( %) patients, and ( %) patients developed severe cgvhd. oas was . %, trm . % and relapse rate . %. median plasma tslp levels increased from before conditioning ( pg/ml) to reach a peak at day + ( pg/ ml, p = . ), followed by a gradual decline. the plasma levels of tslp at day + were positively correlated with same-day monocyte counts (ρ = . , p = . ). approximately half of the patients (n = ) experienced an overall rise in tslp from baseline ( pg/ml) to day + ( pg/ml). this increase in tslp was not significantly associated with any transplantrelated baseline characteristics. however, patients, who had an increase in tslp levels from baseline to day + , had a significantly higher risk of extensive cgvhd compared to those in whom tslp levels at day + were similar or below baseline levels (cumulative incidence of cgvhd: % (increased tslp at day + ) vs % (normal/low tslp at day + ), p = . ). development of cgvhd was also associated with the nucleated cell dose infused (p = . ) and transplant using pbsc (p = . ). tslp plasma levels were not associated with acute gvhd, oas, trm, relapse rate or numbers of t cell, b cell or nk cells posttransplant. we have found that increased levels of tslp from baseline to day + were associated with an increased risk of extensive cgvhd. this association may be due to the ability of tslp to polarize the immune system toward a th response. importantly, the increase in plasma tslp levels was not associated with any transplant-related characteristics suggesting that tslp may be an independent predictor of cgvhd. these findings indicate that anti-tslp treatment may be a new approach to fight severe cgvhd. disclosure of conflict of interest: none. thymopoiesis following hct: a retrospective review comparing interventions for agvhd in a paediatric cohort c roberts , am flinn , ma slatter , , r skinner , h robson , j lawrence , j guest and ar gennery , institute of cellular medicine, newcastle university and great north children's hospital, newcastle-upon-tyne, uk acute graft-versus-host disease (agvhd) is a life threatening complication of allogeneic haematopoietic cell transplantation (hct), treated with topical and/or systemic corticosteroids. in steroid-refractory agvhd extracorporeal photopheresis (ecp) can be effective. ecp exposes apheresed mononuclear cells to -methoxypsoralen and ultra-violet radiation. systemic corticosteroids and agvhd are damaging to thymic tissue. delayed immune reconstitution, especially of the t lymphocyte compartment, is associated with increased morbidity and mortality. therefore, management strategies must be effective in treating agvhd but endeavour to minimise resulting thymic damage. we compare the effect of topical steroid therapy, corticosteroids and ecp on thymic reconstitution following hct in paediatric patients. statistical analysis was performed using the kruskal-wallis test. about paediatric allogeneic hcts were performed between june and april , at the great north children's hospital, newcastle for malignant and non-malignant disease. we reviewed computerised records to categorise patients into four groups: no agvhd, mild agvhd treated with topical steroid, agvhd treated with systemic steroid, agvhd treated with ecp. laboratory data were reviewed to provide values of naive (cd + and cd − )cd ra+cd + t-lymphocytes at , , and months post-hct. values for thymic output for the ecp group were additionally recorded at , , and months during ecp. excluded were patients with no available data, those with o months follow-up, those with chronic gvhd, recipient of hct or received dli post-hct. about patients were included, ( . %) had no agvhd, ( . %) had agvhd treated topically or systemically, ( . %) had agvhd and received ecp. for analysis, the group treated with steroids were divided into those treated with topical therapy and those given systemic steroids. the median values of all groups at each time point ( , , and months) are shown ( figure ). there was a significant difference between the rate of thymopoiesis (measured by the addition of cd + and cd − cd +cd + cells) between all groups (no agvhd, agvhd treated with topical or systemic steroids, and agvhd treated with ecp) at , , and months post-transplant (p = . , p o . , p o . , p = . respectively). further analysis excluded those treated with ecp (so including the no gvhd (n = ), topical treatment (n = ) and systemic steroid treatment group (n = )). at each time point p = . , p = . , p = . and p = . , respectively, demonstrating a statistically significant difference in time to thymopoiesis between those that had developed agvhd and those that had not. acute graft-versus-host disease (agvhd) is a significant cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. which despite first line treatment is well-established (esteroids), second line is not well defined. evaluate the results with different second line treatment used and the risk factors associated with of sr-agvhd. we review the clinical records of consecutive patients undergoing allogenic hsct from to in our hospital. about % presented agvhd. sr-agvhd was defined as progression after days, no clinical change in days or incomplete response after days of treatment. about patients ( %) met s criteria for sr-agvhd. there were no significant differences between both groups (sr-agvhd vs no sr-agvhd) respect to age (recipient/donor), unrelated donor, prophylaxis of gvhd, cd lymphocyte and cd cell. the median time between transplantation and agvhd diagnosis was days ( - ). patients who did not respond on fifth day of steroid treatment have an % rate mortality vs % on no sr-agvhd group (p = . ). sr-agvhd: patients presented sr-agvhd and this was related to: hla mismatch ( % sr-agvhd vs % no sr-agvhd, p = . ), male recipient/female donor ( % sr-agvhd vs % no sr-agvhd, p = . ) and advanced underlying disease ( % sr-agvhd vs % no sr-agvhd, p = . ). second line: basiliximab ( . %); extracorporeal photopheresis (ep) ( . %), timoglobulin ( . %) and others therapies ( . %). two patients ( %) obtained complete response (cr) and patients ( %) partial response (pr). global response (cr, pr) after second line (mainly basiliximab) showed better overall survival (p = . ). third line: basiliximab ( . %); ep ( . %), mesenchymal cells (msc) ( . %), ruxolitinib ( , %) and others ( . %). ruxolitinib improve gvhd cutaneous and hepatic but not intestinal. the best results were achieved with ep ( cr, pr) and basiliximab/msc ( pr, respectively). only patients who achieved cr survived. fourth line: msc ( %)/ ruxolitinib ( %) does not improve the prognosis. no serious adverse effects were observed with msc therapy, basiliximab and ep. about % of patients showed cmv reactivation with basiliximab. about patients died ( %), patients with early mortality ( o months) due to refractory agvhd ( %) or secondary infections ( %). overall survival at months and year was ± % and %, respectively. in multivariate analysis the main factor for trm was the steroid-refractory vs steroidsensitive (hr . , % ci . - . ; p = . ) and was unfavorable the association of hepatic and intestinal agvhd (hr . , % ci . - . ; p = . ) no sr-agvhd: patients. trm- was % (n = ), mainly due to infection ( %). trm- year was % (n = ), mainly by gvhd ( %) and infections ( %). median follow-up of months, os- months and year were ± %/ ± %, respectively. trm was associated with not obtained cr/pr after second line (p = . ), no cr after third line (p = . ) and relapse of gvhd despite achieving cr initially (p = . ). in our series only the patients that obtained cr/pr after second-line or cr after third-line improved os. the best results in sr-agvhd were obtained with basiliximab and extracorporeal photopheresis. trm was associated with relapse of gvhd and advanced disease to the transplant. randomized clinical trials are needed to assess different treatment modalities for sr-agvhd. [p ] disclosure of conflict of interest: none. extracorporeal photopheresis (ecp) is a therapy for steroidrefractory chronic graft versus host disease (cgvhd). therapeutic response to ecp has been linked with a progressive increase in circulating granulocytic myeloid-derived suppressor cells (g-mdsc) in acute gvhd, but not in cgvhd . low density neutrophils (ldn) phenotypically resembling g-mdsc (putative g-mdsc) show marked flux in cgvhd patients receiving ecp, and a reduction in their frequency is associated with a sustained therapeutic response to ecp . recent data has identified lectin-type oxidized ldl receptor- (lox- ) as a specific marker of ldn with t-cell (tc) suppressive activity . using this marker we have conducted a cross-sectional study to assess whether putative g-mdscs in this patient cohort have suppressive activity. about patients with steroid refractory or steroid-dependent cgvhd (mean treatment duration of months) receiving ecp and healthy controls were recruited. patients had gvhd affecting skin ( / ), liver ( / ) and gut ( / ). pbmc were isolated and immunophenotyped by flow cytometry for markers of g-mdscs (cd − ve , cd , cd b, hla-dr − ve , cd int ) and lox- expression. suppressive function was determined by measuring the inhibition of proliferation of anti-cd /cd -activated purified cd tc from healthy donors by -day co-culture with g-mdscs from patients. statistical analysis was conducted using graphpad . ecp patients had substantially greater frequencies of circulating putative g-mdsc than healthy controls (p o . ; median: % and iqr %- % vs . % and iqr . %- . %, respectively). while there were substantially greater frequencies of circulating lox- + cells in pbmc from ecp patients than healthy controls (p o . ; median: . % and iqr . %- % vs . % and iqr . %- . %, respectively), these were mainly the minority population within the putative g-mdsc fraction with no significant difference between ecp patients and healthy controls in the proportion of lox- + cells ( % ± % vs % ± %, respectively). ecp had no significant effect on circulating putative g-mdsc frequency measured before and the day after treatment (median: . % and iqr %- % vs % and iqr %- %; n = , respectively) nor on lox- frequency (median: % and iqr . %- % vs . % and iqr . %- . %; n = , respectively). at a tc:g-mdsc ratio of : , isolated g-mdscs from ecp patients suppressed cd tc proliferation (mean ± sd: % ± %; n = ). however, the potency of suppression was highly variable (min-max: - %). the pattern of lox- expression suggests that only a subset of putative g-mdscs in ecp patients are suppressive and may explain why suppressive function in this cell fraction is so highly variable. however, the relatively high frequency of lox- cells in this patient cohort might contribute to immunosuppression resulting in increased susceptibility to opportunistic infections. according to the revised eortc/msg criterion, patients were diagnosed with cns-ifd. among those patients without cns-ifd ( patients), cns-ifd were matched in a : ratio for analyzing the risk factors of cns-ifd. and among ( . %) patients who occurred pulmonary ifd without cns involvement, patients were selected as control group for analyzing the risk factors associated with involvement of cns in pulmonary ifd. we selected the control group using a : ratio matched-pair method with the variates of ( ) age; ( ) sex; ( ) underlying disease. we retrospectively reviewed patients complicated with cns-ifd after hsct in our single center during a years period. most patients received haploidentical stem cell transplantation. the median onset time of cns-ifd was ( - ) after hsct, and most ( . %) of them have prior pulmonary ifd. the most frequent pathogen was aspergillus, while no crypoccosis and candidas were found. the most common clinical presentation was space-occupying symptoms and signs. brain abscess were the most common imaging finding. prior pulmonary ifd (po . , hr . ( % ci, . - . )) was the only risk factors associated with occurrence of cns-ifd. while poor response at weeks (p = . , hr . ( % confidence interval: . - . )) was the only risk factor predicting the involvement of cns in pulmonary ifd. the response (complete and partial response) at weeks and last follow-up was . % and . %, respectively. the overall survival was . % at the last follow-up with a median ( - ) days after transplantation. in conclusion, patients with pulmonary ifd had higher risk of cns-ifd, especially in those with poor response after weeks of treatment. and the prognosis of cns-ifd was very poor after hsct. disclosure of conflict of interest: none. adv may cause severe infections in hsct recipients, especially from unrelated donors or cord blood particularly in pediatrics. disseminated infections usually occur after digestive reactivations. at mo.post-hsct, the incidence of adv digestive infection and viremia in pediatric hsct is about % and %, respectively. therapeutic strategies to control adv infections are limited to the use of infusion of cidofovir (cdv) or ex vivo anti-adv selected cytotoxic lymphocytes (ctl). however cdv is not labeled for adv treatment, presents a renal toxicity and has shown limited efficacy. specific-ctl remain difficult to produce. brincidofovir (cmx , bcv, chimerix, usa) is an orally-available lipid conjugate of the nucleotide analog cdv that has demonstrated broad clinical antiviral activity against double-stranded dna viruses (that is, herpes-, adeno-, orthopox-and polyomaviruses. the drug has an increased bioavailability compared to cdv and has shown encouraging results. we report here the results obtained with this compound in patients treated in six centers from january . there were pts ( m/ f), median age at hsct: mo. ( - ). hsct indication was all in nine, pid in six, aml in two, fa in two and ibmf in one. donor was / or / mud in four and six pts, respectively; haplo-identical familial donor in ; / or / unrelated cb in two and three pts, respectively; msd in one. stem cell source was bm for pts, cb in and pbsc in . two pts underwent a second hsct. cond' regimen were mac in pts. all pts received either ex vivo or in vivo t-cell depletion. three pts presented with adv-disseminated disease, seven pts with blood + other site (throat, urine or stools) adv infection, three with adv-related gut disease, three with blood infection and three with gut infection. the remaining patient received bcv for jc viremia with fever. median time for virus infection diagnosis was d post-hsct (range: d- to d+ ). about pts experienced other viral infection episodes after hsct (cmv: ; ebv: ; bk: ; hsv: ; hhv : ; influenzae: ( ). about pts received - injections of cdv prior to bcv treatment. one pt received specific-adv ctl before bcv without efficacy. the reason to switch from cdv to bcv was uncontrolled adv infection (n = ) or cdvinduced renal failure (n = ). two additional pts experienced renal impairment after cdv. about pts received - lines of immunosuppressive therapies (including ecp) in addition to calcineurin inhibitor at time of bcv therapy due to grade iii and iv acute gvhd in seven and seven pts, respectively. median adv load at time of bcv initiation was . log copies/ ml (range: - ) in blood and log copies/ml ( . - . ) in stools. median duration for bcv therapy was weeks (range: - ). about seven pts with blood adv infection or disseminated disease experienced adv disappearance as well as four pts with gut disease or infection. three of them experienced adv infection relapse and received thereafter cdv, bcv or advspecific ctl. five pts presented with grade - diarrhea during bcv treatment. about were alive at end point where seven died from septis (n = ), multi-organ failure (n = ), gvhd (n = ) and adv disseminated infection (n = ). adenovirus infections occur often in immunocompromized pts receiving concomitant nephrotoxic drugs that may avoid cdv use.bcv appears as efficient therapy against adenovirus infection in such pediatric pts since here out of pts where alive after adv infection and bcv treatment. in this study we retrospectively analyzed cmv reactivation determined by pcr and response to pre-emptive therapy in patients receiving an haplo (n = , %) comparing them with a control group of non haplohsct ( mrd and mud) (n = , %). median age was years (range: - ), for haplohsct and for control group. conditioning regimen was myeloabaltive (mac) in . % and reduced intensity (ric) in . %. haplohsct characteristics: haplo conditioning was fludarabine ( mg/m or mg/m × days in ric or ma regimen) and busulfan ( . mg/kg × in ric or days in ma) ( . % mac, and . % ric). cyclophosphamide-post was used for gvhd prophylaxis in %. median of days to reach more than × granulocytes and more than × platelets were ( - ) and ( - ), respectively. incidence of acute gvhd was % (grade i-ii . %, and iii-iv . %), with two steroid-refractory cases. cmv reactivation: . % of haplohsct patients presented cmv reactivation, vs . % in control group (p = . ). median number of cmv reactivation episodes was in both groups. median time to cmv pcr detection was days ( - ) and ( - ) in haplohsct and control group respectively (p = . ). average maximum cmv iu by pcr was . in haplo vs . in the control group (p = . ). first antiviral pre-emptive therapy (valganciclovir in . %) was effective in % in haplohsct vs % in control group (p = . ). main reason for antiviral treatment switch was failure in cmv iu reduction, and foscarnet was the most used therapy in refractory cases. twenty patients developed cmv disease ( in haplo and in control group) (gi disease % and pulmonary disease % in both groups). in a multivariate cox-regression model, receiving an haplohsct, serological cmv status (positive patient/negative donor), mac regimen and development of acute gvhd grade i/ii or grade ii/iv were variables associated with a higher risk of cmv reactivation. based on these results, haplohsct is associated with a higher cmv reactivation compared to non-haplohsct, despite a lower incidence of all other risk factors as agvhd or mac in the haplo group. although it is not statistically significant, response to pre-emptive therapy is higher in haplohcst and no differences in cmv disease were observed. disclosure of conflict of interest: none. although a number of patients with hiv infection and hematological disease have successfully undergone allogeneic hsct together with combination anti-retroviral therapy (cart), short and long-term outcomes remain not well known. we report the spanish experience treating hiv-infected adult patients with high-risk hematological malignancies with allogeneic hsct. we retrospectively reviewed hiv-positive patients who received allogeneic hsct in three institutions in spain within geth (grupo español de trasplante hematopoyético y terapia cellular). seventeen patients have been transplanted between and . median age was ( - ), % male. diagnosis and transplant characteristics are summarized in table . cumulative incidence of neutrophil and platelet engraftment were % at days (median days), and % at days (median days), respectively. with a median follow-up of months ( - ), os and efs were %. trm was % at months and % at months. grade ii-iv agvhd rate was %, and moderate/severe cgvhd rate was %. all patients received cart. two patients showed severe toxicity related to interaction of immunosuppressive s drugs and protease inhibitors. about % of patients showed infectious complications. viral infections were the most frequent cause: cmv ( ), bk ( ), adv ( ), hhv- ( ), hcv ( ), hhv- ( ), parainfluenza ( ). two patients had invasive aspergillosis and one patient presented disseminated tuberculosis. causes of death were: relapse ( ), infection ( ), gvhd ( ) and toxicity ( ). all surviving patients showed undetectable hiv load after hsct. allogeneic hsct is an effective therapy for high-risk haematological malignancies in patients with hiv infection, and long-term hiv suppression with cart is feasible. however, interactions between immunosuppressive agents and anti-retroviral drugs, high rates of significant gvhd, and frequent infectious complications account for a complex procedure in this population. selected hiv-infected patients with hematologic malignancies should be considered for allo-hsct when indicated, in experienced centers. disclosure of conflict of interest: none. clostridium difficile infection (cdi) is one of the most common causes of nosocomial infectious diarrhea in europe and usa, which results in high morbidity and mortality among hospitalized patients. allogenic hematopoietic stem cell transplant (hsct) recipients remain at high risk for cdi. incidence rate ranges from to %. numerous risk factors including acute graft-versus-host disease (agvhd), hla matching status, conditioning-intensity, use of total body irradiation (tbi) may play an important role in the course of cdi in these patients. the aim of this study was to evaluate the prevalence of cdi in children, and to assess the influence of such factors as gender, age, diagnosis, hla matching status, conditioning-intensity, use tbi-containing regimen, source of graft (bone marrow/bm/ vs peripheral blood/pb/)or agvhd on course, duration of treatment and outcome in children undergoing hsct. between and a total of hscts were performed in five polish pediatric transplant centers, including allogeneic and autologous. all patients were followed up to months post hsct. we analyzed retrospectively episodes of cdi infection in the group of children. twenty-one of children were diagnosed with hematological malignancies: acute lymphoblastic leukemia (all), acute myeloblastic leukemia (aml), myelodysplastic syndrome (mds), two were diagnosed with severe aplastic anemia (saa), one with chronic granulomatous disease (cgd) and of them-with solid tumors. the median age was . years (range: . - . years). majority of patients underwent myeloablative conditioning protocol ( / ). in allogeneic setting / patients underwent mud-hsct, / pts msd-hsct and one patient was given a haploidentical pbsct. in this series, in out of cases bm was a transplant source, and pb in out of . cdi was defined as having diarrhea that tested positive for c. difficile via pcr, cytotoxin assay, or dual enzyme immunoassays. kruskal-wallis test, wilcoxon test and χ -test were used to estimate the influence of risk factors on severity of disease, duration of treatment and outcome. we observed episodes of cdi ( . %) in hsct recipients: in allotransplant recipients ( . % of all transplants) and in autotransplant recipients ( . % of all auto-hsct). nine patients responded to therapy with metronidazole, seven patients responded to vancomycin alone, and in two patients rifaximine was administered. six children required adding second drug: vancomycin or metronidazole, five patients were not given any medications. there was no significant correlation between such factors as diagnosis, gender, age, conditioning regimen, hla matching, agvhd and severity of disease, and duration of treatment. recurrence rate was difficult to assess due to lack of data. we observed three deaths. one of them was connected with cdi. there was one -year-old boy with saa (mud-pbsct, hla / ) with no agvhd. the other two deaths were due to progression of s disease. cdi occurred in nearly % of pediatric patients undergoing hsct, surprisingly often in autologous hsct too ( . %). almost all patients experienced mild cdi with adequate response to antibiotic therapy. cdi is a rare cause of death among transplant recipients. disclosure of conflict of interest: none. antifungal prophylaxis in high-risk paediatric patients with haematological malignancies: a monocentric experience k perruccio, i capolsini , a carotti , n albi , l pitzurra , a velardi and m caniglia pediatric haematology oncology section; haematology and clinical immunology section; transfusion medicine and the choice of antifungal prophylaxis in high risk paediatric haematological patients (according to the latest ecil /seifem guidelines) remains an open question. a recent retrospective survey from associazione italiana ematologia oncologia pediatrica (aieop) showed that, in these patient categories, the only variable which significantly impacted on invasive fungal infection (ifi) occurrence was the presence or not of antifungal prophylaxis at the ifi onset (unpublished data). from january , in our pediatric hematology oncology unit, allogeneic hematopoietic stem cell transplantations (hsct) were performed (median age: years; range: months- years), mainly for acute leukaemia (median follow-up: months; range: - ). patients received liposomal amphotericine b (n = ), micafungin (n = ), or fluconazole (n = ) as primary antifungal prophylaxis until neutrophil recovery ( × /l). seven patients developed acute gvhd ( %) which evolved in gvhd in ( %). as outpatients, they continued with posaconazole (n = ), voriconazole (n = ), or micafungin (n = ) until cd +t-cell recovery ( /cmm) or gvhd immune suppressive prophylaxis/treatment withdrawn. during the last year, according to ecil /seifem guidelines , we administered primary antifungal prophylaxis also to / high risk (hr) acute leukaemia patients. two patients with aml were treated with posaconazole, four patients with hr-all received micafungin, four relapsed all patients received micafungin (n = ), or liposomal amphotericine b (n = ), or posaconazole (n = ). one aml patient was then transplanted; all relapsed patients are waiting for transplant. no differences were observed in terms of breakthrough proven/probable (pp)-ifi incidence, according to antifungal prophylaxis in the various patient groups. in particular, in the early phase, we observed a pp-ifi incidence of % in both treatment arms (micafungin vs liposomal amphotericine b, p = ns). in the late phase, we observed case of pp-ifi who were receiving posaconazole as prophylaxis. overall survival (os) was %, with % mortality rate. in hr leukaemia patient group, we observed pp-ifi in the only two patients who were not receiving any antifungal prophylaxis at the ifi onset. antifungal prophylaxis is strongly recommended in paediatric patients with haematological malignancies who are at high risk of ifi. the choice of antifungal drug depends on the treatment phase, drug interactions (particularly for azoles), patient compliance and clinical conditions which interfere with intestinal absorption. in our experience, as no differences were observed in term of efficacy, micafungin resulted the best choice in terms of tolerability, toxicity, compliance and cost saving. antifungal prophylaxis with micafungin and bridging to inhaled liposomal amphotericin b after engraftment in patients undergoing allogeneic hematopoietic stem cell transplantation d rivera* , , c de ramon , a avendaño , j carrillo , d caballero , l lopez , i ormazabal , a navarro , a martin micafungin is an effective antifungal for prophylaxis, active against candida spp. including those resistant to other antifungals (c. glabrata and c. krusei) and also active against aspergillus spp. guidelines focused on antifungal prophylaxis, recommend its use during preengraftment and early postengraftment period in allogeneic hematopoietic stem cell transplant (allo-hsct) recipients. moreover, its profile of low drug interactions and side effects, makes it a suitable alternative for patients who need concomitant treatments, present hepatic insufficiency and in those who do not tolerate oral drug administration. the addition of inhaled liposomal amphotericin b (lamb) after engraftment, provides an alternative way to effectively prevent mold infections, that are acquired mainly by inhalation. inhaled lamb has good tolerance with absence of drug interactions and low toxicity. the aim of this study was to describe the experience in the hsct unit of the university hospital of salamanca with micafungin and lamb as primary prophylaxis in patients undergoing allo-hsct with reduced intensity conditioning (ric) and graft-versus-host disease (gvhd) prophylaxis with tacrolimus and sirolimus. thus evaluating efficacy and tolerability in our population. retrospective observational study from january to august , including all adult patients undergoing allo-hsct with ric and gvhd prophylaxis with tacrolimus and sirolimus, in whom an azole derivative is not indicated, due to drug interactions. therefore received prophylaxis with micafungin during the preengraftment period and bridging to lamb after engraftment at discharge, and continuing it during the first days post-transplant. data from patients from our hsct unit. ten ( . %) patients who had invasive fungal infection before undergoing allo-hsct, and ( . %) patients who received prophylaxis with drugs other than micafungin-lamb were excluded. underlying disease was grouped by leukemia in ( . %) patients, lymphoma in ( . %), myelodysplastic syndromes in ( %), multiple myeloma in ( . %) and other diseases in ( . %) patients. eighty patients underwent peripheral blood allo-hsct, of whom were related donor in ( %) patients and unrelated donor in ( %). prophylaxis with micafungin in ( . %) patients, dose of mg per day, with a mean of days (± days) with postengraftment bridging with lamb mg weekly, continuing it during the days posttransplant. days of neutropenia during preengraftment, o days in ( . %) patients, - days in ( . %), more than days in ( . %). during follow-up there were three cases ( . %), two catheter related candida infection, and one esophageal candidiasis. there were no reported aspergillosis cases (possible, probable or proven), according to the european organization for research and treatment of cancer (eortc) criteria. finally, prophylaxis with micafungin and inhaled lamb, was considered an effective and safe strategy in ( . %) patients, with no side effects reported. according to our experience with micafungin and the addition of inhaled liposomal amphotericin b, the results indicate that, this is an appropriate alternative for antifungal prophylaxis, in patients undergoing allo-hsct, because of their efficacy, few side effects and drug interactions. disclosure of conflict of interest: none. recipients of allogeneic hematopoietic cell transplantation (allohct) are at high risk of developing invasive fungal infections (ifi). in the early phase (o days) after allohct, the use of antimold prophylaxis has been generalized, although there is no consensus on the best therapeutic strategy. the use of nebulized liposomal amphotericin b and fluconazole has been shown to be effective, safe and associated with low economic costs in lung transplantation . however, the use of this prophylactic strategy in the early phase of allohct setting has not been evaluated. we included all consecutive patients who received their first allohct in our center from january to august and who underwent antifungal prophylaxis according to the prospective ambineb protocol (nebulized liposomal amphotericin b mg administered three times per week as loading dose and once per week and fluconazole mg per day until day + ). patients with a previous ifi were excluded. patients with graft-versus-host disease (gvhd) receiving high dose corticosteroids were allowed to be changed to voriconazole or posaconazole at physician's discretion. the primary objective of the study was the incidence of ifi at day + . the secondary objectives were to assess adherence and toxicity of the ambineb protocol. only cases with proven or probable ifi according to eortc-msg criteria were considered. a multidisciplinary team of experts in hematology, infectious diseases, microbiology and radiology prospectively evaluated and categorized each case. we included patients with a median age of years (range: - ) and a median follow-up for survivor of months (range: - ). patients received allohct mainly for acute leukemia ( %), non-hodgkin lymphoma ( %) and myelodysplastic syndrome ( %) . patients received allohct from hla unrelated ( %) or related donors ( %) mostly using a reduced intensity conditioning ( %). graft-versushost disease (gvhd) prophylaxis was performed with calcineurin inhibitors mainly in combination with sirolimus ( %) or methotrexate ( %). after the comprehensive review, only one case of proven or probable ifi at day + was diagnosed. prophylaxis with ambineb was completed in patients ( %) while ( %) stopped the treatment. the most frequent causes of discontinuation were possible ifi ( %), gvhd ( %), admission in intensive care unit ( %) and toxicity ( %) (figure) . ninety-four patients ( %) did not have adverse advents associated with ambineb. eight patients presented organ toxicity which was at least partially attributed to ambineb, including gastrointestinal symptoms (n = ), liver function test abnormalities related to fluconazole (n = ) and cough (n = ). of the patients who discontinued ambineb, ( %) were switched to other antifungal drugs including (echinocandins ( , %) posaconazole ( , %), voriconazole ( , %) or others ( , %)). overall survival and non-relapse mortality of all patients at median follow-up were % ( % ci - ) and % ( % ci - ), respectively. the combination of nebulized ambisome and fluconazole is effective in preventing ifi in the early phase of allo-hct and is associated with high adherence and low toxicity. neutropenia-related infections is a common complication of apsct in patients (pts) with mm and dlbcl. our study aims are to: ( ) assess antibacterial susceptibility patterns of isolated organisms, to guide antibiotic prophylaxis ( ) identify the epidemiology of bacteremia with susceptibility patterns to direct empiric therapy of febrile neutropenia ( ) assess the interval between the occurrence of neutropenia and the isolation of resistant bacteremia to identify the best timing to start prophylaxis ( ) identify contributing factors for the development of bacteremia and mortality. our retrospective study included adult pts who underwent apsct for mm and dlbcl between and . we recorded the following: age, gender, basic illness, comorbidities, number of cd + cells infused, a central venous catheter, duration of neutropenia, diarrhea and mucositis, mechanical ventilation, positive bacterial cultures with susceptibility profiles and history of broad-spectrum antibiotic intake for more than h for the past months on hospital setting, and mortality. statistical analysis was carried out using spss (version ). about isolates were obtained: urine ( . %), blood ( . %), sputum ( %), wound ( %), venous catheter ( %) and stool ( %). gram-negative (gn) species were predominant ( . %) with e. coli ( . %), klebsiella (k) ( %) and pseudomonas (pseudo) ( %). isolates sensitive to third generation cephalosporins ( gc) represented % of the enterobacteriaceae (entero) including % in e. coli and % in k. all entero isolated were susceptible to carbapenems (carba), pipercillin/ tazobactam (pip/taz), amikacin and ciprofloxacin (cipro). all pseudo (n = ) and acinetobacter (n = ) isolates were susceptible to carba, pip/taz, amikacin, cipro, colistin and tigecycline. as for gram-positive (gp) bacteria ( . %), coagulase negative staphylococci (cns) were predominant ( %) . oxacillin susceptibility reached % and two isolates methicillin resistant s. aureus were identified. all gp were susceptible to glycopeptides. a total of bacterial isolates were identified ( episodes of bacteremia) from pts. gn were predominant ( . %) with e. coli being most common ( %). all gn were susceptible to gc, carba, pip/taz, amikacin and cipro. as for gp ( . %), cns predominated ( %) including % oxacillin-susceptible causing seven episodes of bacteremia with six central line-associated. no glycopeptide resistance was identified. none of the clinical features and pts' characteristics reached statistical significance as risk factor for bacteremia. however, the need for mechanical ventilation and mortality were higher in bacteremic vs non-bacteremic pts ( . % vs %, p = . , and . % vs %, p = . , respectively). all bacteremic episodes occurred after developing neutropenia (median = . days, range: - ) except for one case of clabsi caused by e. coli occurring day before neutropenia. pip/taz was prescribed in % of bacteremia episodes followed by quinolones ( %) and carbapenems ( %). no previous use of third and fourth generation cephalosporins was observed. we recommend quinolone prophylaxis in apsct pts. for empiric therapy, antibiotics recommended by international guidelines including, cefepime and pip/taz still fit. thus, we could spare the use of carba and other last-resort antibiotics to other conditions. we also recommend continuous surveillance of resistance. disclosure of conflict of interest: none. fever is an almost universal complication in patients undergoing autologous stem cell transplantation (asct), however, microbiological documentation is only achieved in - % of such febrile episodes (fe). this low diagnostic efficiency makes epidemiological assessment in transplant units difficult, and may lead to a suboptimal empirical treatment. we have studied the utility of strict blood culture (bc) extraction as a mechanism to improve microbiological documentation of fe in these patients. we conducted a retrospective study over consecutive asct performed in our centre between june and may ( year). about patients were male and female, with age between and years (mean . ). diagnosis was hodgkin lymphoma, non hodgkin lymphoma and multiple myeloma. ascts were performed in reverse isolation conditions, in rooms equipped with hepa and pall filters. prophylaxis against herpes virus and p. jirovecii with acyclovir and pentamidine was used. no prophylaxis against gram negative bacteria or filamentous fungi was performed. bc were extracted at the beginning of every fe and every - h if fever persisted (or more frequently, following clinical criteria). blood samples from intravascular devices and peripheral blood were collected in two bactec bottles each (for aerobic and anaerobic microorganisms). complementary diagnostic techniques and empiric antibiotic therapy were performed following our institution's guidelines. fe were classified in microbiologically documented infections (mdi), clinically documented infection (cdi) and fever of unknown origin (fuo) following his criteria. fe were studied (average . fe per patient, . days of fever duration per fe). about % of fe were classified as mdi, % as cdi and % as fuo. mdis were cause by gram positive bacteria ( %), gram negative bacteria ( %) and polymicrobial infections ( %). no viral or fungal infections were observed. an average of . bc per fe and . per patient were extracted. the proportion of fe classified as mdi was related to the number of blood cultures extracted during the episode. only % of fe with three or less bc extracted were classified as mdi, % if - bc were extracted, and % if - bc were extracted (p o . ). no significant difference in proportion of mdi classified fe between the extraction of or more bc and - . all patients were discarded in good clinical conditions. according to our experience, a strict - blood culture extraction is related to a high rate of microbiological documentation of febrile episodes. moreover, we have not observed the rise in gram negative bacteria reported by other studies and gram positive cocci persist as the main infection cause in our centre. candida which has been traditionally related to duration of neutropenia, emerges as a pathogen beyond the aplastic period in allogeneic haematopoietic cell transplantation (allohct). in the setting of alternative transplants and aggressive immunosuppressive therapy, these infections are a challenging problem. there is scarcity of data regarding the significance of breakthrough candidaemia in allohct. to that end, we aimed to determine the incidence, clinical and microbiological characteristics and outcome of candidaemia in allohct recipients. we studied consecutive allohct recipients from january to june . blood cultures were obtained from peripheral vein or central venous catheters (cvcs) routinely and on febrile patients. well-known risk factors for candidaemia were studied: neutropenia, type of transplant, moderate to severe graft-versus-host disease (gvhd) and coexisting infections. among allohct recipients, we identified seven patients with candidaemia: five post matched unrelated (four myeloablative and one reduced intensity conditioning) and two post haploidentical transplant. in median time of . ( . - ) months, episodes of candidaemia were noted, despite antifungal prophylaxis with echinocandins or azoles. infections with non-albicans candida spp. occurred more frequently ( / ) and c. parapsilosis was the predominant microorganism ( / ). other species were isolated: c. famata ( ), c. krusei ( ) and c. haemulonii ( ). all candida spp. isolates were phenotypically susceptible to antifungal agents already administered to patients. there was no resistance to echinocandins indicated by minimum inhibitory concentrations (mics). all patients had severe acute or late-onset gvhd with intestinal involvement and cvcs prior to candidaemia. although cvcs were removed in / and patients were treated with echinocandins, new cvcs were re-contaminated in / with the same or other species. all patients presented well known risk factors for candidaemia (use of broad spectrum antibiotics due to severe bacterial infections, total parenteral nutrition due malnutrition, long-term high-dose corticosteroids and other immunosuppression), but no neutropenia. one patient survived, whereas five patients succumbed to gvhd and multi-organ failure and one patient to sepsis due to bacteremia. candidaemia was observed in non-neutropenic patients with agvhd and cvcs on antifungal prophylaxis, despite difficulties in diagnosis due to poor sensitivity of blood cultures. the epidemiology of candidaemia has changed in the last decade and its risk is more diverse and complex. the irreversible intestinal gvhd lesions might be the main source of candida in patients receiving antifungal prophylaxis. our data show that candidemia remains an important issue in profoundly immunosuppressed patients contributing to excessive morbidity. our aim was to compare the rate of neutropenic sepsis, defined as fever of o c and a neutrophil count of o . × /l, before and after the introduction of ciprofloxacin prophylaxis. one hundred and eight adult patients, of which had acute myeloid leukaemia, had acute lymphoblastic leukaemia, had lymphoma and had other haematological malignancies, were identified through our admission database. of these patients, received oral ciprofloxacin during their neutropenic phase. the median duration of neutropenia was days in both the no-prophylaxis and ciprofloxacin groups. there was a significant reduction in the rate of neutropenic sepsis from . % ( / ) in the no-prophylaxis group to . % ( / ) in the ciprofloxacin group (p = . ). prolonged infection, suggested by the use of broad-spectrum antibiotic treatment for more than days, was more common in the group which did not receive prior ciprofloxacin prophylaxis ( . % vs . %, p = . ). rate of intensive care admission ( . % vs . %, p = . ) was also reduced by the use of ciprofloxacin. however, there was no significant difference in the length of stay (mean of vs days, p = . ), or in the -day infection-related readmission rate ( . % vs . %, p = . ) between the two groups. in terms of the cause of neutropenic sepsis, escherichia coli, klebsiella pneumoniae and pseudomonas aeruginosa were the most common bacteria isolated from cultures in the no-prophylaxis group. eighty percent of these organisms showed sensitivity to ciprofloxacin. in the ciprofloxacin group, staphylococcus epidermidis was the most frequently found bacteria. with regards to treatment related adverse effects, none of the patients who received ciprofloxacin prophylaxis developed clostridium difficile diarrhoea. in conclusion, ciprofloxacin is still an effective antibacterial prophylaxis during neutropenia following allogeneic stem cell transplantation. clinicians should have a high suspicion of a gram-positive infection in patients who develop neutropenic sepsis on ciprofloxacin prophylaxis. disclosure of conflict of interest: none. s hematopoietic stem cell transplantation (haplohsct) to cure leukemia, malignancy and some inherited diseases, different additional reasons interfere microbiota metabolism and integrity. among them are radiation and chemotherapy, mucositis, infection and graft versus host disease (gvhd). the curative mechanism of fmt is based on the ability of donor intestinal microbiota to substitute and to provide all necessary functions of altered patient's microbiota. three patients ( , and years old) after haplohsct, who observed pseudomembranous colitis (toxin b-positive) as gvhd of intestine outcome, were enrolled to the study and performed fmt. relatives (mother, father and brother) were used as microbiota donors. donor and patient examination have included routine clinical and biochemistry laboratory data, microbiota cultural methods, pcr of most common intestinal microorganisms. additional for patient-level of fecal calprotectin by elisa was tested, identification of drug resistant bacteria and histology of intestine were made. patient's preparation for fmt included-probiotic (inulin) administration h prior procedure, discontinuation of all antibiotics h prior procedure and antiemetics ( ht agonist), prokinetics and proton pump inhibitor. delivery of donor's microbiota was performed in two consecutive steps under total intravenous anesthesia: with esophagogastroduodenoscopy-to the duodenum; with colonoscopy-to the caecum. all patients observed complete clinical response in - days after fmt (table ). in days we have revealed significant quantitative and qualitative changes in microbiota composition, which was matched to donor's microbiota. in days after fmt we identify microbiota changes in oropharynx and urogenital tract similar to donor microbiota. this leads to substitution of multidrug resistant klebsiella pneumoniae strains by drug sensitive microorganisms and helps to treat severe infection complications after haplohsct. platelet aphereses were carried out in donors ( males and females with median age . years) using haemonetics instrument with simultaneous leucoreduction. quantitative detection of cmv, ebv and hhv- dna was performed by multiplex real-time quantitative pcr kit (interlabservice, russia) in donors' whole pb, plasma and platelet aphereses at the time of platelet collections. viral load in hsct recipients was monitored weekly after hsct and days before hsct by the same pcr kit. lower limit of detection (llod) of the applied kit for all viruses was copies/ml. in specimens of platelet donors we additionally performed ultra-sensitive pcr with llod copies/ml. only one patient ( . %) was cmv-positive by pcr prior to hsct. cmv reactivation after hsct ⩾ copies/ml was noted in whole pb of patients ( . %) with median time of days (range: - ). donor source in cmv-reactivated patients was as follows ( mud, msd, haplo). cmv viremia ⩾ copies per ml was detected in seven patients ( . %). cmv disease was found in five cases ( . %). none of patients were positive by pcr for ebv or hhv- prior to hsct. ebv reactivation ⩾ copies/ml was found in six cases ( . %), ⩾ copies/ml in ( . %) with median time of days (range: - ). no signs of ptld or other ebv-dependent clinical symptoms were observed. hhv- level after hsct ⩾ copies/ml was detected in patients ( . %), ⩾ copies/ml in ones ( . %) with median time of reactivation days (range: - ). hhv- disease was observed in one patient. none of platelet donors were cmv-positive in plasma, whole blood or platelet aphereses products. ebv ⩾ copies/ml was detected in whole pb specimens of five platelet donors ( . %). application of ultra-sensitive pcr revealed low level of ebv-viremia in additional pb cases with median ebv level copies/ml (range: - ). none of platelet donors have any clinical signs of ebv disease. there is no any ebv-positive case among platelet concentrate specimens. in two cases low levels of hhv- was found in a whole pb ( and copies/ ml). none of hhv- -positive case was observed among plasma and platelet concentrate specimens. despite high incidence of cmv, ebv and hhv- reactivation after hsct in pediatric patients we could not show that source of viral reactivation was contamination of platelet apheresis products by donorderived herpes viruses. disclosure of conflict of interest: none. conventional respiratory virus (crv) infections are known to be major causes of morbi-mortality after stem cell transplantation (sct) due to the increased risk of progression to lower respiratory tract infection (lrti) in this setting. risk of developing severe lrti is mostly related to factors specific to the patient and the underlying disease, although the intrinsic virulence of crvs may also determine their outcomes. we conducted a single-center retrospective study including all adult sct recipients who had crv disease (defined as patients with symptomatic respiratory disease and crv identification) during a -year period ( - ) with the main objective of evaluating epidemiological changes over time and their association with infection outcomes. during the study period episodes of crv disease were diagnosed in patients (median age: years, % male, % aml or mds as baseline disease). patients ( %) received an allogeneic-sct (allo-sct) ( % had a prior sct) and ( %) an autologous sct. crv disease was diagnosed at a median of days after sct (range: - ), with cases ( %) occurring before day + . during the infectious episode of allo-sct recipients ( %) had active gvhd and ( %) were under s prednisone (pdn) mg/kg. most of the patients ( %) had symptoms compatible with an upper respiratory tract infection (urti), with of them ( %) progressing to a lrti, while ( %) had a lrti only. hospital admission was required in episodes ( %) with a median duration of hospitalization of days (range: - ), % required supplemental oxygen, % were transferred to the intensive care unit and % required mechanical ventilation. the most commonly identified pathogens over time are shown in figure . twenty-four cases ( %) had concomitant bacterial or fungal infections. influenza a virus was the most frequent crv detected ( episodes, %) followed by human respiratory syncytial virus ( episodes ( %) and human parainfluenza virus type ( episodes, %). during the flu pandemic, only of the crv infections diagnosed in sct recipients ( %) were associated to influenza a virus h n . antiviral treatment was started in episodes ( %), antibiotics in % and combined therapy in % during a median of days (range: - ). the rv resolved in cases ( %) at a median of days ( - ) from onset, with crv being considered the leading cause of death in only patients ( % of all cases and / ( %) in those with a lrti). predictors of severe crv infection (including icu admission, need for supplemental oxygen, need for mechanical ventilation requirement or death) in multivariate analysis were lymphocyte count o cells/μl (hr: . , % ci: - , p = . ) and co-infection with other pathogens (hr: . , ci %: . - , p = . ). no specific crv nor period post-sct of the infection influenced the risk of severe infection. our results confirm that crv infections are a frequent cause of morbidity after sct with a high need for hospital-based care. temporal changes in the principal circulating crvs has been identified during the -year study period, with influenza a virus being the most common. profound lymphocytopenia and presence of co-pathogens are associated with infection severity. [p ] disclosure of conflict of interest: none. the consecutive hsct performed from to are being analysed retrospectively. out of them ( %) performed in hepa filter room and ( %) in non-hepa filter room, criterion was purely financial to make this decision. / ( %) were allogeneic and / ( %) were autologous hscts. blood cultures both bacterial and fungal were taken at onset of fever and with every change of antibiotics till patient became afebrile. chest x-ray and if required hrct chest was done for all patients who had respiratory complaints. we did not use antibacterial prophylaxis; however, antifungal prophylaxis was administered along with conditioning; and at the onset of fever systemic antibiotics were started. antifungal agents were added if fever persisted for days pre empatively. extremely well trained nurses were looking after both the groups. all treatment protocols, antibiotic/antifungal policies were same in both the groups. median time for neutrophil engraftment was days in hepa filter room and days in non-hepa filter room. total / ( %) patients did not engraft till days. out of them / ( %) were in hepa filter room and / ( . %) in non hepa filter room. blood cultures were positive in total / ( %) patients, were positive for bacterial and for fungal organisms. in hepa filter hsct / ( %) were positive and in non-hepa filter hsct were / ( %) positive. total / ( %) patients developed pneumonia, out of them / ( %) were in hepa filter and / ( %) hsct were in non-hepa filter room. statistically not significant. no central venous access cather issues or infections were documented in any groups gr - agvhd : hepa rooms / ( . %),non hepa rooms / ( . %) :was not statistically significant the -day mortality was / ( %), / ( %) patients were from hepa filter rooms and / ( %) were from non-hepa filter rooms. cost : average cost of allogeneic hsct in hepa room : usd . average cost of allogeneic hsct in non hepa room: usd . average cost of auto hsct in hepa room: usd . average cost of auto hsct in non hepa room : usd . incidence of blood culture positivity & incidence of pneumonia was not different. these are two very important issues in outcome of hsct. agvhd incidence did not depend on the room type. these are significant findings from this study. results were slightly better in hepa filter rooms compare to non-hepa filter rooms, which was statistically insignificant. our study had few confounding factors hence we could not be concluded that hepa-filtered rooms are not necessary. nevertheless, our experience suggests that availability of dedicated hepa units with special air-handling equipment should not be considered a critical and essential precondition for providing allogeneic hsct to patients even in developing world with financial constraints. these would otherwise succumb to potentially curable hematological illnesses with background of financial constraints and wait list of hepa filter rooms. early hsct in a clean patient in non hepa rooms is extremely cost effective with comparable outcomes. nursing care, experience of the team, experience in hsct program & well established protocols are more important in outcome of hscts. disclosure of conflict of interest: none. we present five cases of cytomegalovirus (cmv) pneumonitis occurring in patients after recent allogeneic stem cell transplantation (allohsct). these cases were complicated by an organising pneumonia (during the recovery period) with a predominantly central peribronchial pattern. all patients presented with evidence of active cmv pneumonitis which was treated successfully with anti-viral therapy but was followed by persistent severe dyspnoea, cough and hypoxia. high resolution computed tomography (hrct) imaging showed widespread central peribronchial consolidation with traction bronchiectasis. in most cases there was a marked clinical and physiological improvement after treatment with systemic corticosteroids. however, in all patients the lung function remained abnormal and in some cases imaging revealed a fibrosing lung disease. these cases represent a previously undescribed central peribronchial pattern of organising pneumonia complicating cmv pneumonitis that can result in chronic lung damage. disclosure of conflict of interest: none. cytomegalovirus reactivation in pediatric acute leukemia after stem cell transplantation has an effect on relapse and survival in aml but not in b-precursor all j-s kühl , l sparkuhl and s voigt department of pediatric oncology/hematology/sct, charité universitätsmedizin berlin, berlin, germany several studies have indicated better survival after stem cell transplantation (sct) for acute leukemias, especially acute myeloid leukemia (aml), in case of cytomegalovirus (cmv) reactivation. here, we investigated if cmv reactivation had an impact on survival after sct for aml or acute lymphoid leukemia (all) in children. pediatric allogeneic stem cell transplant recipients from our institution who received myeloablative conditioning were included. transplant indications included aml, t-all and b-precursor all. cmv reactivation was correlated with relapse, mortality as well as acute graft-versus-host disease (gvhd) and was analyzed by fisher's exact test or χ -test (if n ). from the patients included, were transplanted for aml ( %), for t-all ( %), and for b-precursor all ( %). mortality and relapse rates ( - % and - %, respectively), cmv reactivation rates ( - %) as well as numbers of negative cmv serology status ( - %) of donor and recipient were comparable between different acute leukemias. when patients were analyzed altogether, cmv reactivation had no effect on relapse rates or mortality. however, a tendency towards fewer relapses after cmv reactivation was observed in aml patients (no relapse ( %) with cmv reactivation vs relapse cases ( %) without cmv reactivation; p = . ). in those leukemia patients capable of reactivating cmv (that is, donor or recipient cmv seropositive prior to sct), cmv reactivation had a protective effect on relapse rates in aml (no relapse ( %) with cmv reactivation vs relapse cases ( %) without cmv reactivation; p = . ). a similar tendency could be seen in t-all whereas no effect in patients with b-precursor all was documented. numbers of acute gvhd cases grade i between aml and t-all with or without cmv reactivation were similar. different effects of cmv on relapse rates and mortality in aml vs b-precursor all were noticed in patients who were either not capable of cmv reactivation or who did reactivate cmv post sct. in aml patients, there were no relapses ( %) and deaths ( %) in contrast to relapse cases ( %) and deaths ( %) in children with b-precursor all (p = . and p = . , resp.). latently cmv infected aml patients without documented cmv reactivation after sct have a significant worse prognosis compared with all other aml patients. this is also likely to be the case in patients with t-all, however, patient numbers in our cohort were too few. the protective effect of cmv reactivation in aml and possibly t-all does not appear to be gvh-related since the rate of relevant acute gvhd cases was comparable. cmv reactivation after sct for b-precursor all lacks significance. disclosure of conflict of interest: none. infections are among the most frequent and relevant complications of hematopoietic stem cell transplantation (hsct). little is known about the role of dental foci for the prevalence of infections in hsct. dental status was prospectively evaluated in all patients at our center before undergoing hsct. a total of different patients before undergoing hsct ( allogeneic and autologous), with a median age of years (range: - years) were evaluated. for evaluation a panoramic x-ray evaluation was performed. dental findings included the status of third molars and root fillings as well as caries, periodontitis, destructed teeth and apical bone loss. as non-dental parameters we used age, sex, type and status of central venous line, mucositis, and type of transplantation. these were correlated with neutropenic fever, bacteremia and pneumonia in a bivariate manner before a multivariate analysis was performed. no correlation of initial dental status to neutropenic fever, bacteremia or pneumonia was found. however, bacteremia and suspected infection of central venous lines was a significant predictor of neutropenic fever. in conclusion, dental surgery should only be performed prior to hsct if urgently required and limited to those individuals with overt infection. disclosure of conflict of interest: none. [p ] early experience with clinimacs prodigy ccs method in generation of virus-specific t-cells for pediatric patients with severe viral infections after hematopoietic stem cell transplantation k kallay viral reactivation especially in children is a frequent complication of allogeneic hematopoietic stem cell transplantation. most of these episodes can effectively be controlled by an antiviral or antibody therapy; in refractory cases a novel virusspecific t-cell therapy could be a promising management option. in our pediatric cohort of allogeneic transplantation during year patients fulfilled criteria for virus-specific t-cell therapy ( boys, girls, median age of ( . - ) years). six patients were transplanted because of hematological malignancies and for inborn errors. donor distribution was the following: matched unrelated, sibling and haploidentical donor. in cases bone marrow, cases peripheral blood and case cord blood was used as a stem cell source. the underlying viral illness was cmv in , ebv in and adenovirus in case, while more than one virus was detected in cases (cmv+adenovirus cases, cmv+ebv cases). viral diseases necessitating a t-cell therapy were cmv pneumonitis and colitis, adenovirus enteritis and cystitis and ptld. patients initially received cidofovir for adenovirus, rituximab for ebv and a combination of gancyclovir and foscarnet for cmv infections. the indication for t-cell therapy was progressive viral disease in of the cases and uncontrollable viral load in case. the procedure was performed on a median of ( - ) day post transplant. donors were st degree relatives in cases, nd degree relatives in cases and an unrelated person in case, the best hla match was haploidentical. the median age of the donors was ( - ) years. cells were produced by the clinimacs prodigy cytokine capture system (ccs) method after mononuclear leukapheresis. the system produced a median of . ( . - ) times /kg cd + and a median of . ( - . ) times /kg cd + interferon producing cells while the non-interferon producing cells were far below gvhd limit with a median of . ( . - . ) times /kg cd + and a median of . ( . - . ) times /kg cd + cells. the t-cell products were administered uneventfully in all but one case. we observed a manageable cytokine storm in one patient. glucocorticoid treatment was ongoing due to acute gvhd in children; however we could manage to keep the steroid dose below mg/kg in all cases. eight patients became completely asymptomatic, while also cleared the virus. we experienced decreasing viral load in all cases, the first negative viral results were achieved on a median day of ( - ). six patients are alive without viral illness or sequale, and complete viral dna clearance in peripheral blood with a median follow up of ( - ) days. one patient with cmv pneumonitis improved during the first week but deteriorated on the second week and died of respiratory insufficiency despite of mechanical ventilation. in cases the viral illness improved or cleared, but the patients died of invasive aspergillosis. no cases of gvhd, rejection, organ toxicity or recurrent infection were noticed. virus-specific t-cell therapy produced by the clinimacs prodigy ccs is a feasible, fast, safe and effective way to control resistant viral diseases after pediatric hematopoietic stem cell transplantation. this treatment can be implemented within a week in most cases. in order to define the appropriate place of this approach for patients with viral reactivations more data should be collected. disclosure of conflict of interest: none. central venous catheter (cvc) is essential for the treatment of recipients of stem-cell transplant. it is usually placed for the administration of conditioning regimen, stem cell infusion, intravenous antibiotics, immunoglobulins, electrolyte and nutritional support and blood concentrates. this patient group is at high risk for catheter-related bloodstream infections that can result in substantial morbidity and mortality. the neutropenia secondary to the conditioning regimen determines the risk of catheter-related infections, which may serve as an entry into the blood circulation, leading to bacteremia, fungemia, and consequently to septic shock and death. the risks of infection and the spectrum of infectious syndromes differ according to the type of transplant, conditioning regimen, type of implant of stem cells and therapies used after the procedure. gram-positive bacteria, particularly coagulase-negative staphylococcus spp, remain the leading cause of catheter-related bloodstream infection, although an increase in gram-negative bacteria as the causative agent has been noted. aim of the study: to evaluate the impact of the early cvc removal on the frequency of febrile episodes and infections in our group of patients. during a years period we have treated patients with hematologic neoplasm with high-dose chemotherapy and stem cells transplantation. patients were treated in sterile room conditioned with hepa filtration. in every patient was introduced double-lumen cvc ( subclavia, jugular, and femoral). % were febrile ( % fuo), catheter-related infection was present in %, while positive culture from cvc was present in %. the most frequent isolated bacteria from cvc were gram positive-staphylococcus coagulasa negative. the catheter was removed on the day of discharge. trm is . %. from january to november we have transplanted additional patients. to aim to decrease infection related mortality we perform strategy to remove cvc on day + after stem cell transplantation. the febrile episodes decreased on % ( / ), there were no early post-transplant mortality due to infection. early removal of the cvc and adequate handling from the nursing staff is essential for outcome of this patient population in regard of infective complications efficient prevention, early diagnosis, and effective treatment of catheter related infection are essential to providing the best care to these patients and can minimized morbidity and mortality. disclosure of conflict of interest: none. fever in patients with agranulocytosis during autologous hematopoietic stem cell transplantation (autohsct) can be associated with non-infectious causes due to g-csf, vancomycin, engraftment syndrome. in this case biochemical markers, such as presepsin (psp), procalcitonin (pct) and c-reactive protein (c-rp), can help in differential diagnosis of fever of infectious and non-infectious genesis. psp, pct and c-rp were assessed on the day of admission to the hospital (da), on d+ , on d+ , on d+ and on the day of discharge (dd). if patients developed neutropenic fever (nf), the markers were assessed at the beginning of the fever, h after, then on the second, third, fourth days after. if patients developed nf immediate empirical antibiotic therapy (at) was implemented with meropenem. in cases of ineffective st line ab, nd line at was added or totally changed. there were patients included in the study: patients with hodgkin lymphoma, with non hodgkin's lymphoma, with multiple myeloma, out of patients there were women and men. the median age was years ( - years). the conditioning regimens were cbv, beeac or hd melphalan. patients developed infectious complications (ic): of them had sepsis and others-nf. the median of nf development was . days. depending on the efficiency of at therapy patients were divided into two groups: group : patients that have had effective at (they 've had fast clinical response and they haven't needed to change medicine (n = )); group : patients that have had ineffective st line at, they haven't had response to st line at and they've needed to change another at (n = )). there were significant differences in psp levels on the third day after ab had been admitted: . pg/ml in group and . pg/ml in group (p = . ). similar differences between the analyzed groups were observed on the fourth day: . and . pg/ml, respectively (p = . ). pct and c-rp didn't show any significant changes between group and on each day of the study (table ) . disclosure of conflict of interest: none. enterovirus related immune reconstitution inflammatory syndrome (iris) following haploidentical stem cell transplantation in an mhc class ii deficient child r shah , s waugh , k foong ng , z nademi , t flood , m abinun , s hambleton , a gennery , m slatter and a cant paediatric immunology and bmt, great north children's hospital, newcastle upon tyne, uk and department of virology, great north children's hospital, newcastle upon tyne, uk immune reconstitution inflammatory syndrome (iris) has been described after hsct in association with fungal, viral and bcg infections. we describe a case of post-hsct iris associated with enterovirus infection. case: a girl with mhc ii deficiency (rfxp c. mutation) underwent treosulfan/fludarabine/ thiotepa/atg conditioned tcrαβcd + depleted haploidentical hsct at . years of age. pre-transplant work up did not reveal any viral or fungal infections except norovirus in stool. cyclosporine (csa) was given as gvhd prophylaxis. neutrophil and platelet engraftment occurred on d+ and d+ , respectively. on d+ , her stool was tested positive for enterovirus (taqman pcr), however; she was asymptomatic. the child started having fevers and irritability from d+ which persisted despite the use of antimicrobials. no evidence of fungal or bacterial infection was found. enterovirus pcr in blood was found positive on d+ (cycle threshold value, ct . ) and further typing showed it to be echovirus . at this time, symptoms progressed with diarrhoea, developmental regression and signs of radiculopathy. mri (brain and spine) was normal and csf showed pleocytosis ( wbc/mcl- % lymphocytes, protein . g/l) with positive enterovirus pcr (ct ). subsequently, immunoglobulin prophylaxis was increased to . g/kg bi-weekly, and with supportive measures, the patient slowly recovered. blood enterovirus pcr remained positive. with no evidence of gvhd, csa was tapered off by day+ and child was discharged on d+ on a bi-weekly ivig replacement. she presented days later with signs of raised intracranial pressure. mri showed hydrocephalus, and vp shunt was placed and broad spectrum antibiotics administered. csf showed wbc o /mcl, protein . g/l and enterovirus positive. methylprednisolone mg/kg/day was started suspecting iris. in subsequent csf testing days later, enterovirus was negative. enterovirus pcr remained positive in blood during this period. patient's clinical deterioration correlated with a rise in cd /cd counts and c reactive protein with clearance of enterovirus from csf, blood and stool ( figure ). subsequently, the child showed gradual but marked improvement and discharged home. discussion: the clinical features of index case fits into criteria for iris . markedly raised crp suggests high il- levels without any bacterial or fungal pathogens being isolated. in addition, iris occurs at the site of prior active infection (brain in index case) and viral clearance and clinical recovery demonstrated with the continuation of steroids. the incidence of enterovirus infection in hsct recipients is around % . iris, in this case, had a temporal correlation with discontinuation of csa, and it has been shown that discontinuation of immunosuppression is associated with higher risk of iris. a high index of suspicion for iris is necessary during immune recovery post-hsct especially when immunosuppression is being tapered in a patient with pre-existing infection. aggressive antiviral treatment (when available) and judicious immunosuppression are the keys to managing iris complications. posttransplantation lymphoproliferative disease (ptld) is a significant cause of morbidity and mortality in allogeneic stem cell transplant patients. identifying high risk patients, routine pcr screening, early diagnosis and therapy are crucial for successful management. patients and methods primary objectives of this study were to describe epidemiology of ebv associated ptld and to assess risk factors in our paediatric cohort. additionally, role of immunoglobulin (ig) levels as a possible diagnostic/prognostic marker was analyzed. between january and june , allogeneic transplantations were performed in pediatric patients ( boys and girls) at our center. median age was . years ( . - ). underlying diseases were hematological malignancies ( %), nonmalignant hematological conditions ( %), immunodeficiencies ( %) and others ( %). stem cell source was bone marrow ( %), peripheral blood ( %) and cord blood ( %). donors were unrelated ( %), sibling ( %), haploidentical ( %) or other matched family donors ( %). routine ebv pcr screening and ig level detection were performed weekly. rituximab prophylaxis was given only in nine cases. results ebv dnaemia was found in / patients ( . %), while ptld was diagnosed in / patients ( . %). all ptld cases were related to ebv infection, median of highest viral load was copies/ml ( - ). diagnosis was confirmed by biopsy in / cases, further five fulfilled criteria of probable ptld (positive pcr with appropriate clinical symptoms). ptlds occurred at a median of day + ( - ) after transplantation. all patients received rituximab treatment along with a reduction of immunosuppressive therapy. four patients died of ptld (mortality %), all confirmed by autopsy. a higher incidence of male gender ( / ; . % vs . %), bone marrow graft ( / ; . % vs . %), hematological malignancy ( / ; . % vs . %) and second transplantation ( / ; . % vs %) could be detected among ptld patients when compared to the non-ptld group. elevated igg, iga or igm levels were observed in / patients. nine out of had positive ebv pcr testing, eight of them developed ptld. five of the ptld patients had monoclonal or biclonal immunoglobulin elevation, two of them died. in cases, elevated ig level preceded the positive ebv pcr results by at least week. conclusion: at our centre incidence and mortality of ptld was similar to published data. we observed a tendency that a higher representation of male gender, hematological malignancy, bone marrow graft and second transplantation could be confirmed in the ptld group however due to small number of patients, a correlation and statistical significance could not be calculated. elevation of immunoglobulin levels do not seem to be specific for ptld but in selected cases it could predict ebv disease earlier than pcr testing. disclosure of conflict of interest: none. autologous peripheral hematopoietic stem-cell transplantation is a procedure of a stem cell rescue with patients' own previously collected hematopoietic stem cells, after myelotoxic therapy. the purpose of stem cell reinfusion is to ensure adequate recovery of hematopoiesis, shorten the period of profound neutropenia and to reduce the risk of infections. the transplantation itself carries a moderate risk for infection but some patients have higher risk due to the nature of underlying disease, earlier treatment and in case of severe mucositis. for these reasons, all treated patients are in isolated clean rooms and receive ciprofloxacin, fluconazole and acyclovir prophylaxis. in the . -year period, autologous transplantations were performed. the patients were - years old, with median of . years. of all transplanted patients, or . % had multiple myeloma, or . % had lymphoma and or . % had acute myeloid leukemia. all of the patients received pegfilgrastim mg on the first or the second posttransplant day. febrile neutropenia (ne o . × /l) was reported if patient's temperature was above . °c in one measurement or above °c in two consecutive measurements. these patients were treated empirically with piperacillin/tazobactam . g four times a day with the addition of vancomycin in the case of severe mucositis or pulmonary infiltrates. in all cases blood and urine cultures were performed, as well as testing for seasonal flu. time to neutrophil recovery (ne . × /l) was - days, with a median of days, and average of . days. febrile neutropenia was reported in patients ( . %) and in ( . %) patient's samples pathogen was isolated. gramm negative bacteria caused sepsis in . % of patients. we had to change empirical therapy according to antibiogram in . % patients. in month follow-up period, there were two ( . %) infection related deaths. our data on incidence of infections is consistent with literature data but large number of papers show satisfactory results of safety of patients discharged from hospital immediately after the autologous stem cell transplantation and who were treated at home during the phase of profound neutropenia. there is still an ongoing debate whether it is possible to conduct this procedure in such manner in our health system. disclosure of conflict of interest: none. fluconazole was equal to mold-active drugs in preventing early invasive fungal disease after allogeneic stem cell transplantation regardless of transplantation type y sun, j hu, h huang, j chen, j-y li and x-j huang there are still controversies that whether mold-active drugs is better than fluconazole in preventing invasive fungal disease (ifd) after allogeneic stem cell transplantation (hsct). we hypothesis that the optimal prophylaxis might be different in patients with different risk profile, such as in different time period after hsct or received alternative donor transplantation. in the prospective china assessment of antifungal therapy in haematological disease (caesar) study database, out of patients received primary antifungal prophylaxis were analyzed. the ifd incidence of different time period after transplantation (early, late and very late) and survival were compared among different drug groups. in patients with fluconazole, itraconazole, voriconazole or micafungin prophylaxis, the overall incidence of ifd after transplantation were . %, . %, . % and . %, respectively (p = . ). however, there is no difference in early ifd (o days post hsct) among groups of patients. the risk factors associated with occurrence of ifd were neutropenia duration days (po . , or . ( . - . )), adult (p = . , or . ( . - . )) and alternative donor (unrelated donor or haploidentical donor) transplantation (p = . , or . ( . - . )). in the sub-group analysis with only alternative donor (unrelated donor and haploidentical donor), it also demonstrated that fluconazole is equal to other mold-active drugs in preventing early ifd. patients received fluconazole prophylaxis has even better overall survival. the overall survival in patients with fluconazole, itraconazole, voriconazole or micafungin prophylaxis were . %, . %, . % and . %, respectively (p = . ). our current [p ] study suggests that fluconazole is equal to mold-active drugs to prevent early ifd in hsct patients, even in high-risk patients received transplantation from alternative donors. however, further prospective randomized study was warranted to confirm this conclusion. disclosure of conflict of interest: none. ( . %) received autologous hsct and ( . %) allogeneic hsct. sixty-five out of patients ( . %) were affected by different haematological diseases: by lymphoma, by multiple myeloma, by chronic lymphocytic leukaemia and by others diseases including mastocytosis, amyloidosis and essential thrombocythemia. hbv reactivation prophylaxis prescribed was entecavir for hbsag+ inactive carrier patients and prolonged lamivudine (lmv) course for ( %) patients. in patients ( . %) lmv prophylaxis was withdrawn - months after the end of immunosuppressive therapy. eight out of patients ( . %) experienced hbv reactivation: of them during lmv treatment and then they were switched to entecavir or tenofovir therapy, patients reactivate hbv after lmv interruption ( . %). in these patients reactivation was observed after an average time of months (range: - ) after discontinuation of lmv prophylaxis. median duration of prophylaxis was months (range: - ) after the end of immuno-suppression. three out of patients ( %) underwent allogeneic hsct and patients ( %) received rituximab. one out of ( %) seroreverted in hbsag positive and hbsab negative status, with hbv-dna ui/ml (table ) . two patients out of ( %) experienced hbv-dna detection below ui/ml. disclosure of conflict of interest: none. [p ] [p ] the severity is measured on grades (grade : microscopic hematuria to grado : clots cause urinary tract obstruction). the treatment is based on support measures: hyperhydration, continuous bladder irrigation, instillation of topical agents and in severe cases must be performed a cystoscopy for clot evacuation. in the case of the presence of poliomavirus virus (bk virus) the use of cidofovir had been demonstrated in vitro studies to have activity against bk virus. we performed allogenic transplants of which are haploidentical from to october . we realized a retrospective case study to analyses the experienced in the management of hc. results: of a total of allogenic transplants realized, developed a hc: patient received an identical hla transplant and the patients remaining haploidentical allotrasplant. all cases were male, with an age range of - years. the status of the disease was: were in complete remission and had visible disease. of the patients received cyclophosphamide as immunosuppressive therapy and all patients received cyclosporine and mofetil micofenolate also. the onset of the symptomatology was between day and day post transplant and the range of duration was from to days. the four patients precised continuous bladder irrigation but because of the poor response they received instillation of hialuronic acid ( doses). two patients required the use of cidofovir ( doses). one of the four patients required urinary tract catheterization because of hydronephrosis and renal impairment. in our review we confirmed that this entity is more frequent in the haploidentical transplant and bkv is the most prevalent cause in the late hc. -the three patients received doses of cidofovir ( mg/kg) without probenecid and had a good response. -three patients present acute renal failure associated to hc. the four patients needed bladder instillations with saline but they had poor response and received at least doses of hyaluronic acid. disclosure of conflict of interest: none. hsv infection in allo-hsct setting is mostly reactivation of latent virus. hsv disease commonly presents as mucocutaneous lesions of the oral cavity. however some patients develop serious fatal visceral dissemination. prophylactic use of acyclovir has markedly reduced the incidence of hsv disease during the period of neutropenia after allo-hsct. in this study, our aim is to demonstrate the incidence, clinical outcome and risk factors for hsv disease in adult allo-hsct. between and , patients who underwent allo-hsct in our center were included to the study. all hsct candidates and donors were tested for hsv- / immunoglobulin g (igg) antibodies prior to transplantation. all patients received acyclovir prophylaxis (related transplants mg tid, unrelated transplants mg tid) during conditioning and after allo-hsct up to months. chlorhexidine oral solution as well as bioadherent oral protective gels was used for oral hygiene. all patients were followed for symptoms of reactivation. hsv / igg seropositivity was detected in recipients ( %) and donors ( %). the distribution of hsv status was as follows: recipient and donor seropositive in ( %), recipient and donor seronegative in ( %), recipient seropositive and donor seronegative in ( %), recipient seronegative and donor seropositive in ( %) transplants. the median age of the patients was (range: - ), patients were male ( %) and ( %) had malign disease. the stem cell source was peripheral blood in ( %) patients and ( %) received grafts from related donors. sixty four patients ( %) received myeloablative conditioning regimen. the most common graft-vs-host disease (gvhd) prophylaxis administered was cyclosporine (csa) and methotrexate (mtx) in patients ( %). acute graft vs host disease was detected in patients ( %).four patients from seropositive patients ( %) had hsv reactivation, the patient characteristics are given in the table. all patients had hsv reactivation within month of allo-hsct except one patient had symptoms at sixth month posttransplant when he suffered from oral gvhd. all patients s and donors were seropositive prior to allo-hsct and responded well to antiviral treatment. the incidence of hsv reactivation in allo-hsct was detected as % which is lower to previous studies. successful primary prophylaxis and oral hygiene might reduce the incidence. all patients were responded to antiviral treatment and no visceral dissemination was detected. disclosure of conflict of interest: none. patients who have received hematopoietic stem cell transplantation (hsct) may suffer, to some extent, losses in humoral and cell immunity against antigens to which they had been previously exposed naturally (infection caused by wild microorganisms) or artificially (through vaccination). the conditioning regimen for hsct replaces the patient's immune system and involves the loss of previous immunity. this study analyzed patients included in the vaccination program for hsct recipients in the salamanca health care complex during the period - . we assessed the serological status prior to hsct for the following immunopreventable diseases (hepatitis b, hepatitis a, varicella), and the study after hsct also included measles, rubella and parotitis, prior to their inclusion in the hsct vaccination program. the study included patients, . % of which (n = ) were men. . % of the patients (n = ) were allogeneic hsct recipients with an average age of ± years, and . % ( ) were autologous hsct recipients with an average age of ± years. prior to hsct, % of the patients showed immunity against hepatitis b (hbv antibodies ui/l), . % against hepatitis a (positive for hav igg) and % against varicella (positive for varicella igg). no statistically significant differences were observed regarding this variable hepatitis b anti-hbs ui/l, hepatitis a, igg positive, varicella igg positive, measles igg positive, rubella igg positive, parotitis igg positive. table compares the serological status before and after transplantation. in the pre-transplant serological study we observed that less than half of the patients are protected against hepatitis b, while over % of them are protected against hepatitis a and varicella. regarding the diseases in which we know the serological status before and after transplantation (hepatitis a, hepatitis b and varicella), we observed that most patients maintain immunity. in the case of rubella, measles and parotitis we only have access to the serological status after transplantation, and we observed that parotitis is the disease with the lowest seroprotection. therefore, vaccination would be indicated, just as in the case of hepatitis b. the clinical results support the need to adapt the vaccination schedule to the immunological status of the patients after hsct individually. disclosure of conflict of interest: none. impact of cumulative steroid dose on infectious diseases after allogeneic hematopoietic cell transplantation m watanabe , j kanda, t kitano, t kondou, k yamashita and a takaori-kondo after allogenic hematopoietic cell transplantation (hct), highdose steroids are used to treat transplantation-related complications such as graft-versus-host disease (gvhd). however, the use of high-dose steroids is associated with an elevated risk of infectious diseases. information on the association between cumulative steroid dose and infectious diseases after hct is scarce. a total of patients who underwent their first hct in kyoto university hospital from to and survived at least days after transplantation were included in this study. we analyzed the association between cumulative steroid dose used within days after transplantation and the occurrence of infectious diseases, including invasive fungal infection (possible/probable/proven cases), cytomegalovirus (cmv) antigenemia, and bacteremia through days after transplantation. sixty-three patients received transplantation from a related donor, received unrelated bone marrow grafts, and received unrelated cord blood units. their median age was (range: - ) years and median day of neutrophil engraftment after transplantation was . patients were categorized into groups according to their cumulative steroid dose within days: no steroid administration (n = ), low-dose cumulative steroid administration under mg of prednisolone in total (n = ), and high-dose cumulative steroid administration over mg of prednisolone in total (n = ). reasons for steroid administration were treatment for gvhd in patients, engraftment syndrome in , and other reasons including lung complications in . the rate of invasive fungal infection was % ( possible cases with pneumonia and proven case of candida blood stream infection) and we found no apparent association between fungal infection and steroid use regardless of dose. cmv antigenemia was diagnosed in %, % and % of patients in the groups respectively, and both low-dose and high-dose steroid groups were significantly associated with a high risk of cmv antigenemia (low-dose group, adjusted hazard ratio (ahr), . , p = . ; high-dose group, ahr . , p = . ). bacteremia was diagnosed in . %, % and % of patients in the groups, respectively. high-dose steroid use was a risk factor for bacteremia (ahr . , p = . ). seven patients died from infection (fungal, ; viral, ; bacterial, ). two of three bacterial infection-related deaths occurred in the highdose steroid group, although the number of events was too small to analyze. our data confirmed that steroid administration is itself a risk factor for cmv antigenemia and close observation to detect cmv antigenemia is mandatory for patients using steroids regardless of its cumulative dose. high-dose cumulative steroid use is a risk factor for bacteremia. contrary to our expectations, steroid administration showed no apparent association with invasive fungal infection in our study, perhaps because of its generally low incidence in our hospital. disclosure of conflict of interest: none. impact of different t-cell depletion techniques on the incidence of infectious complications after allogenic hematopoietic stem cell transplantation k aikaterini , s federico , d-l vu , e boely , c dantin , a pradier , y tirefort , a-c mamez , o tsopra , c stephan , y beauverd , e roosnek , s masouridi-levrat , c van delden , y chalandon department of oncology, hematology unit, university hospital of geneva and department of medicine specializations, infectious diseases, university hospital of geneva t-cell depletion (tcd), obtained by either in vivo antithymocyte globulin (atg) administration or ex vivo depletion, is a well-established strategy for graft-versus-host-disease (gvhd) prevention after allogeneic hematopoietic stem cell transplantation (hsct) - . however, the prolonged lymphopenia associated with tcd can result in increased incidence of disease relapse and infections. although many studies investigated the impact of tcd on disease relapse - , little is known about the impact of tcd strategies on the incidence of infectious complications after allogeneic hsct. we retrospectively evaluated the incidence of infectious complications in consecutive patients who underwent allogeneic hsct at our center from september to december . patients received tcd grafts obtained by in vivo atg administration as part of the conditioning regimen (atg group). patients received partially tcd grafts obtained through incubation with alemtuzumab in vitro washed before infusion followed on day + by an add-back of donor t cd + cells (ptcd group). patients received grafts tcd by both methods combined. patients did not receive any form of tcd (no-tcd group). cumulative incidence estimates of infectious complications were calculated and compared using the gray test. given the increased risk of infection associated with gvhd and its treatments, gvhd or death from other causes were defined as competitive events in the analysis. we didn't observe any significant difference in the -year cumulative incidence of bacterial infections in patients receiving tcd by atg ( % ( % ci - . %)) ptcd ( . % ( % ci . - . %)) or both ( % ( % ci . - . %)) compared with patients receiving no tcd ( . % ( % ci . - . %)). similarly, the -year cumulative incidence of viral infections or reactivations was comparable in patients receiving no-tcd grafts ( . % ( % ci . - . %)) compared with patients receiving tcd grafts (atg: . % ( % ci . - . %); ptcd: . % ( % ci . - . %); atg+ptcd: . % ( % ci . - . %)). finally, no significant impact of tcd was observed on -year cumulative incidence of fungal (no-tcd: . % ( % ci . - . %); atg: . % ( % ci . - . %); ptcd: . % ( % ci . - . %); atg+ptcd: . % ( % ci . - . %)) and parasitic (no-tcd: . % ( % ci . - %); atg: % ( % ci . - . %); ptcd: . % ( % ci . - . %); atg +ptcd: . % ( . - . %)) infections. image/graph: -year cumulative incidence estimates of infectious complications depending on the tcd strategy employed. the results of our retrospective analysis indicate that the cumulative incidence of bacterial, viral, fungal and parasitic infectious diseases are similar in patients receiving tcd grafts compared to those receiving no-tcd graft, suggesting a favorable toxicity profile of different tcd strategy in respect of infections. these results should be confirmed by similar analysis in large scale, prospective clinical trials assessing the potential benefits of tcd on transplantation outcomes. patients with aml were considered eligible for hsct, died before transplantation. patients ( %) underwent transplantation from hla-identical sibling, ( %) from haploidentical family donor and ( %) from matched unrelated donor, while patients ( %) received unrelated cord blood cells. twenty ( %) out of eligible patients have had an ifi episode before transplant: were proven, probable and possible; ( ( %) pneumonia, ( %) gastroenteritis, ( %) sinusitis, ( %) candida sepsis, ( %) meningitis and ( %) cutaneous abscess were registered). five ( %) out of patients with a previous ifi and ( %) out of without previous ifi did not receive hsct (or . % ci . - . , fisher test p: . ). the majority ( %) of patients with a previous ifi waited hsct more than months from the date of eligibility in comparison with those without a previous ifi ( % vs %; or . , % ci . - . , p-value . fisher test) overall a post transplant ifi episode was diagnosed in ( %) of transplanted patient; ( %) had a relapse of a past ifi vs ( %) of the patients without a previous ifi who had a new episode. (or . , % ci . - . , p-value . yates test).a higher number of patients with ifi ( out of , %) respect to those without a previous ifi ( out of , %) died in a median time of days(range: - ) after hsct. furthermore, those who had a previous ifi had a lower median survival ( days (range: - )) compared to patients without a previous ifi ( days period (range: - )) (student's t-test p: . )). a previous ifi episode in the pre transplant period slows and limits the accessibility to hsct, and is significantly associated with an increased mortality. disclosure of conflict of interest: none. s delayed immune reconstitution has been described for haploidentical hematopoietic stem cell transplantation (hsct) compared to conventional hsct, nevertheless the incidence of invasive aspergillosis infections (iai) in haploidentical sct and the efficacy of primary prophylaxis are not well defined. our objective is to describe the incidence, risk factors and mortality of iai in our patients, using as prophylaxis micafungin during the conditioning and neutropenia period, switched to posaconazole or voriconazole when oral intake is feasible. we retrospectively analyzed consecutive patients from to who received haploidentical grafts: unmanipulated for adults, tcrab depleted in children and cd ra depleted in children. the stem cell source was peripheral blood in all cases. adults ( - yo) were treated for aml/mds (n = ), all (n = ) and lymphoma (n = ). children ( mo- yo) were treated for aml (n = ), all (n = ), aplastic anemia (n = ) and immunodeficiencies (n = ). conditioning regimen was bu-flu-cy (n = , adults), thio-bu-flu (n = , adults), flu-mel-thio for all pediatric patients; atg was used in children and tli in children. median follow up was months ( - ) for adults and months ( - ) for children. we used eortc criteria for iai and analyzed probable or definite as cases. there were events of iai, with a bimodal presentation: events ( . %) during neutropenia period and ( . %) after months of hsct ( figure ). five of them were probable and one definite (aspergillus niger). site of infection was mainly pulmonar; cns was suspected in two adult patients and skin was proven in one adult patient. all patients at the late period had chronic gvhd at diagnosis. one patient had primary graft failure. severe cmv disease (hepatitis and colitis) was present in one adult. mortality related to iai was high ( / ), patients died at a median of days. figure . iai patients characteristics the global incidence of iai in haploidentical hsct is similar to conventional hcst. primary prophylaxis with micafungin switching to oral triazole is successful ( . %) during the early period. late cases ( . %) had clearly known risk factors (chronic gvhd, steroids and cmv), and primary prophylaxis had been modified due to toxicity or interactions. iai mortality in our patients is very high ( %) despite effort in prophylaxis, diagnosis and treatment. visceral intractable abdominal pain prior to skin lesions from herpes zoster can be misdiagnosed as gvhd post stem cell transplantation which may lead to initial increase in immunosuppression and hence high mortality if we don't suspect. case report and literature review through pubmed results: year-old male with relapsed all post mud pbsct ( / ) transplant in following cy tbi atg conditioning presented at day + with intractable diffuse abdominal pain with constipation. no history of nausea, vomiting or skin rash. on physical examination his abdomen was soft, diffuse tenderness but no rigidity, muscle guarding and rebound tenderness. laboratory tests including liver function test, amylase, lipase were normal. usg abdomen and mri abdomen showed no abnormalities, except for presence of fecolith. during the stay his pain worsened needing morphine infusion, pca and later ketamine. he had previous history of acute gut gvhd controlled on budesonide and cyclosporine which was later being weaned once his symptoms were controlled. in view of previous history of gvhd, gi consultation was sought and he underwent ugi endoscopy and biopsy which was non-significant. on day of his admission he developed a pustular skin lesion on thigh and scrapping from that showed vzv and his blood pcr was also positive, he was started on intravenous acyclovir. his lesions improved and crusted and his abdominal pain subsided after h of acyclovir and was discharged on oral acyclovir after days of intravenous therapy. review of literature illustrated in table . severe abdominal pain in patients who received an allogeneic stem cell transplant has a broad differential. here we describe a case of vzv presenting with intractable abdominal pain needing opioids. because of the poor prognosis and life-threatening nature of disseminated vzv disease, it should be considered and included in the patient's workup. intravesical cidofovir ( mg/kg, diluted in ml sterile water) was once weekly applied until symptom control for min. via a transurethral catheter, i.v. cidofovir was initiated if no symptom control was achieved after local applications. in patients with hc or a lavage catheter was added. bkv cystitis (dysuria (n = ) or dysuria combined with hematuria (n = )) developed in out of transplants ( %). median age was years, % were female and % received a mismatch transplantation after mac or ric conditioning regimens. in % of bkv cystitis cases also cmv reactivation within the first days could be detected. % had acute gvhd ii°-iv°at the onset of bkv cystitis and % received steroid medication. the median time to symptom occurrence was day + after hsct (iqr - : - ). patients ( with dysuria and one either hc °and °) didn´t require therapy due to self limiting symptoms. ( %) of treated patients showed only dysuria, ( %) hc °, ( %) hc °, ( %) hc °and ( %) hc °. the first patient was treated with i.v. cidofovir twice and symptoms relieved. all the following patients were exposed to intravesical cidofovir as st line therapy. patients ( %) achieved a complete remission with a median of intravesical procedures (range: - ). patient showed symptom improvement and all patients didn´t require further therapy. patients had to be switched to i.v. application due to bladder spasms during intravesical application (n = ) or to insufficient symptom control (n = ). out of these responded to i.v. treatment, whereas patient receiving nd transplant didn´t respond at all. in patients with spontaneous symptom relieve the median bkv concentration at the time of symptom onset was log lower compared to those requiring antiviral therapy. local therapy reduced bkv viruria by log. pain during cidofovir instillation in % of patients was the only significant side effect of local therapy compared to creatinine increases by % in . % of i.v. treated patients. intravesical treatment of symptomatic bkv cystitis with cidofovir ( mg/kg) is safe and effective with an % symptom improvement rate and no systemic side effects. in patients without sufficient symptom or bleeding control i.v. cidofovir is still an option, which however induces significant renal toxicity. we therefore recommend intravesical cidofovir as st line therapy in case of dysuria or hematuria induced by bkv after hsct. disclosure of conflict of interest: none. haemorrhagic cystitis is a recognised complication of stem cell transplant (sct), with a reported incidence of - % of cases ( ) . the majority of cases are associated with bk polyomavirus (bkv), and less often adenovirus and cytomegalovirus. there are a lack of high quality studies on the optimal prevention and management of haemorrhagic cystitis. treatment options are restricted by conditioning toxicity, immunosuppression and other co-morbidities such as renal impairment. cidofovir has an inhibitory effect on bkv replication and has been used extensively in the treatment of haemorrhagic cystitis. however, severe nephrotoxicity limits routine intravenous use in sct patients. alternative options include using low dose intravenous cidofovir or intravesical administration. we conducted a retrospective case review of post sct patients presenting with bk virus associated haemorrhagic cystitis in our institution between january and november . we identified patients in total ( male, female). the indications for stem cell transplant were as follows: severe aplastic anaemia high risk aml relapsed aml relapsed all onset of symptoms (haematuria and painful micturition) ranged from day − to day + , and the time to resolution of symptoms varied from days to days. four of the patients were treated with intravesical cidofovir only, with the number of doses required varying from to . one patient received combination treatment with both intravenous ( doses), and intravesical cidofovir ( doses). all patients had a good clinical response with complete resolution of symptoms and no major complications. however, the level of bk virus in the urine did not always correlate with clinical response. some of the patients did not tolerate urethral catheterisation and required a general anaesthetic for the placement of the urethral catheter; patient required a supra-pubic catheter. currently out of patients are alive and well; patients died from causes not related to bk virus associated haemorrhagic cystitis. our experience shows that intravesical administration of cidofovir is a safe and effective option for the treatment of bk virus associated haemorrhagic cystitis. an allogeneic stem cell graft from a cytomegalovirus (cmv) seronegative donor puts recipients at high risk of cmv reactivation which can lead to cmv disease and mortality. based on the immunogenicity of cmv phosphoprotein (cmvpp ) we initiated a clinical phase i trial with a novel vaccine designed by our group: a cmvpp -derived peptide in water-in-oil emulsion (montanide) plus administration of granulocyte-macrophage colony stimulating factor. ten patients received four vaccines s.c. at a biweekly interval after allogeneic stem cell transplantation. we monitored the patients for their clinical outcome and cmvpp antigenemia. multi-color flow cytometry test were performed to assess cmvspecific cd + and gamma-delta t cells. novel neutralizing anti-cmv antibody assays were established and correlated to clinical parameters. findings: in general, patients tolerated the peptide vaccination well, no drug-related adverse events others than rash or induration at the site of injection were detected. seven of nine patients with cmvpp antigenemia cleared the cmv after four vaccinations and were hitherto free from antigenemia. two patients with cmv reactivation showed persisting cmv antigenemia. one of these two refractory patients received additional four injections and remained hitherto free from cmv antigen. another patient obtained a prophylactic vaccination and did not develop antigenemia. an up to six-fold increase in frequency of both cmv-specific cd + t cells or vdelta -gamma-delta t cells was detected in five patients. moreover, titers of neutralizing antibodies increased in four patients up to -fold over the time of vaccination. humoral and cellular immune responses correlated with clearance of the cmv load. cmvpp peptide vaccination was safe and well tolerated in patients after allogeneic stem cell transplantation at high risk for cmv reactivation. the vaccine showed encouraging immunological and clinical results. a prophylaxis study using the vaccine in solid-organ transplant patients is ongoing. disclosure of conflict of interest: none. sporopachydermia cereana is a rare yeast found in necrotic cactus tissue, predominantly in the americas. infection in humans has only been reported in neutropenic patients with fatal course, either directly from the pathogen or other complications of immunosuppression. treatment is complicated by difficulties in pathogen-identification with conventional diagnostic techniques and by resistance to echinocandins. here we present a patient with acute myeloid leukemia (aml) and s. cereana infection. this is the first patient who was successfully treated with antifungal therapy and who survived s. cereana infection. case presentation we present the case of a -year-old female patient who was diagnosed with normal karyotype aml with dnmt a and idh mutations in december . she achieved complete remission after two cycles induction chemotherapy. during the nd induction cycle the patient developed persistent fever in neutropenia despite broad-spectrum antibiotics and the replacement of prophylactic fluconazole to caspofungin. blood cultures showed growth of s. cereana, shown to be sensitive to azoles (mic fluconazole o mg/l, mic voriconazole o . mg/l) as well as amphotericin b (mic o . mg/l), but resistant to caspofungin (mic mg/l). following the susceptibility profile the treatment was changed first to liposomal amphotericin b, and with the availability of mic results to voriconazole. metastatic fungal infection (that is, endocarditis, endophthalmitis, hepatosplenic candidiasis) was excluded. after regeneration of peripheral blood values the treatment was switched to oral voriconazole. a ct scan of the chest and abdomen prior to allo-hsct after weeks of treatment with voriconazole revealed new multiple necrotic mesenteric lymph nodes. an ultrasound-guided biopsy of a node revealed no growth on fungal cultures, a grocott stain revealed no hyphae or spores. a panfungal pcr of an its (internal transcribed spacer) fragment revealed fungal dna, which could be confirmed as s. cereana. at this time the level of voriconazole in serum was found to be sub-therapeutic ( . mg/l), and the dosage was increased accordingly. subsequent ct scans and weeks later revealed a regression of the affected abdominal lymph nodes. in the further course non-myeloablative conditioning with fludarabine and busulfan prior to allo-hsct using pbsc from her hla-matched brother was performed. under prophylaxis with cyclosporine, methotrexate and antithymocyte globulins (atg) graft-versus-host disease (gvhd) remained absent. the allo-hsct was performed under voriconazole treatment with no further complications and the patient engrafted at day . the treatment was changed to fluconazole mg daily before discharge. due to the complete radiological regression of the infection in follow-up scans and excellent general condition of the patient months after hsct, fluconazole was discontinued. the patient remains in morphological complete remission months after hsct and has a % donor chimerism. the first published case of survival of infection with s. cereana exemplifies the continual progress made in treating infections in the severely immunocompromised patient. diagnosis via its sequence-analysis seems reliable but a high index of suspicion is required for neutropenic patients who do not respond well to standard antimycotic therapy. the increased availability of the technology may lead to more frequent diagnoses in the future. disclosure of conflict of interest: none. neutropenic enterocolitis (ne) is a clinical syndrome characterized by fever and abdominal pain in patients who received chemotherapy for hematological malignancies and who treated with stem cell transplantation (sct). the aim of this study was to determine the incidence, risk factors and outcome of ne after autologous sct (auto-sct). we retrospectively evaluated patients with non-hodgkin lymphoma (nhl), hodgkin lymphoma (hl) and multiple myeloma (mm) who underwent auto-sct between january and december in our center. patients with lymphoma were conditioned with carmustine, etoposide, cytarabine, melphalan (beam) or thiotepa, etoposide, cytarabine, cyclophosphamide, melphalan (tecam). patients with multiple myeloma were treated with melphalan as conditioning. diagnosis of ne was established in case of neutropenic fever, abdominal pain or diarrhea, and bowel wall thickening mm on abdominal ultrasonography. febrile neutropenia was seen in ( %) patients of all. the median time from transplantation to neutropenia was . days (range: - days). ne occurred in ( . %) in all neutropenic patients. the median time to ne after auto-sct was days (range: - days). the median neutrophil engraftment time was . days (range: - days). abdominal pain was seen in all patients with ne. twenty one patients ( %) had diarrhea. ileus was seen in ( . %) patient and septic shock was developed in ( . %) patients. five ( . %) of patients had bloodstream infection. klebsiella pneumoniae in , pseudomonas aeruginosa in , escherichia coli in , staphylococcus aureus in and coagulasenegative staphylococcus in patient were documented in patient's blood stream. early diagnosis was made by abdominal ultrasonography in all patients at a day of median days (range: - ). twenty ( %) patients were resolved completely with good supportive care and proper antibiotherapy. two ( %) patients died of septic shock and ileus. ne is a rare but serious complication in patients underwent high dose chemotherapy followed by auto-sct. gramnegative bacteria are the main causative pathogens. abdominal ultrasonography is the simple, cheap, fast diagnostic and noninvasive procedure that allows the early diagnosis and effective treatment. disclosure of conflict of interest: none. [p ] neutrophil transfusions in the treatment of neutropenic patients submitted to allogeneic hsct: possible role on graft failure s giammarco, p chiusolo , l laurenti , f sorà , n piccirillo , l teofili and s sica hematology department, università cattolica del sacro cuore and hematology departement, università cattolica del sacro cuore granulocyte transfusions (gtx) from g-csf-stimulated donors have been shown to increase the absolute neutrophil count (anc) before expected haematopoietic recovery in neutropenic patients after chemotherapy or haemopoietic sct. thus gt offers a therapeutic option along with antimicrobial agents and growth factors to improve clinical outcome of neutropenic patients with severe infections. the primary limitations of gt include low component cell dose and leukocyte incompatibility. the transfusion of g-csf-mobilized, hla-matched granulocyte components resulted in sustained anc increments, but the efficacy of this procedure has not been established by convincing randomized control trials. aim: we focused our attention on gt in the setting of allogeneic hsct, in particular on the feasibility and safety of this procedure on the rate of engraftment. between and our centre performed allogeneic hsct. we analyze data from transplanted patients receiving gt at some point during their disease. indication for gt was severe sepsis mainly due to mdr gram-bacteria. patients received a median of gt ( - ), in different phase: patients during induction therapy, during hsct, at diagnosis and during hsct and after hsct. patients' characteristics are summarized in table . median cd + cells dose was . × /kg (range: . - ). donor source was in patients g-csf mobilized peripheral blood, bone marrow and cord blood. median neutrophil recovery ( /mmc) was days and platelet recovery ( / mmc) was days. sepsis were documented in pts and pts developed fuo. relapse was documented in pts ( %). twenty-two pts are still alive and in complete remission ( %), death occurred in pts: due to trm and the remaining for disease relapse. graft failure occurred in of the pts submitted to hsct. among the patients ( %) who experienced graft failure, six ( %) received gt before hsct, because of sepsis during the induction therapy, and the remaining after hsct, during aplasia period. in the remaining group ( pts) not receiving gt, only ( %) graft failure were observed. thus a statistically difference (p = . fisher's exact test) increase in the rate of graft failure was detected in patients receiving gt. the role of gt in the treatment of infections in neutropenic patients remain still unclear for several reasons including the lack of clinical trials convincingly and consistently demonstrating efficacy, by availability of gt donors and by center's experience. gt has been successfully used in our center in patients with severe sepsis from mdr gram-bacteria during severe neutropenia but an increase number of graft failure has been registered in patients subsequently receiving hsct. alloimmunization to hla antigens in patients receiving gt might lead to an excess of graft failure requiring hla antibodies detection and attempt to reduce titer prior to hcst and maximizing stem cell dose. disclosure of conflict of interest: none. viridans streptococci are microorganisms frequently isolated from blood cultures of patients undergoing myeloablative allogeneic hematopoietic cell transplantation (allohct). poor dentition status has been associated with an increased risk of streptococcal bacteremia in the immediate post-allohct neutropenic period. the objective of this study was to evaluate the impact of oral health status on bacteremia risk in a cohort of patients undergoing therapy for acute myeloid leukemia (aml). a retrospective study was conducted in patients with aml treated at dana-farber/brigham and women's cancer center (df/bwcc) from to . there was no formal dental assessment prior to aml induction therapy. all patients underwent protocol directed pre-allohct dental evaluation that included a standardized examination, comprehensive dental radiographs, and detailed treatment planning guidelines. poor oral health status was defined as presence of acute or chronic odontogenic infection, and it was assumed that oral health status at the time of induction therapy was the same as the pre-allohct evaluation findings. oral health status at the time of allohct was determined by the completion of required dental treatment. positive blood cultures were recorded from aml induction to day + post allohct. organisms that caused bacteremia were classified as 'of possible oral source' by a blinded microbiologist. two-sided fisher's exact test was used to compare the oral health status of the entire cohort to patients with blood cultures of potential oral source. from january to january , patients with aml underwent myeloablative allohct at df/bwcc and were s followed through today + , and of these, patients met the inclusion criteria and were included in the cohort. the median age was years (range: - ) and there was similar distribution of genders. the most common aml induction regimen was daunorubicin and cytarabine ( / ; %) and of those that received consolidation therapy ( / ; %), almost all patients were treated with cytarabine. nearly all patients ( / ; %) received cyclophosphamide and total body irradiation for allohct conditioning and the majority of patients ( / , %) received tacrolimus/methotrexate (n = ) or tacrolimus/sirolimus (n = ) for gvhd prophylaxis. over half of patients ( / , %) experienced mucositis during their course of therapy for aml. pre-allohct dental evaluations were completed in / ( %) of patients. of the / ( %) patients identified as having poor oral health status, / ( %) completed all required dental treatment prior to allohct. bacteremias occurred in / ( %) patients, and / ( %) had positive blood cultures of potential oral source. of the patients with positive blood cultures of potential oral source, / ( %) patient developed bacteremia during induction and / ( %) patients developed bacteremia during allohct. of the / ( %) patients identified as having poor oral health status, one patient ( / ; %) had a positive blood culture with a bacteria of potential oral source during induction/consolidation (p = . ). oral health status was not associated with risk of bacteremia of potential oral source at either aml induction/consolidation or allohct. risk of such bacteremia in the setting of myeloablative allohct may be related more to overall gastrointestinal translocation. disclosure of conflict of interest: none. is one of the main alternatives to trimethoprimsulfamethoxazole (tmp-smx) for prophylaxis of pneumocystis pneumonia (pcp)(maertens et al. jac ). ato is less effective than tmp-smx to prevent pcp but the reasons of this lower efficacy are not well understood. ato acts on pneumocystis, plasmodia and toxoplasma species by inhibiting mitochondrial pyrimidine biosynthesis. ato is highly lipophilic and its absorption in volunteers is improved by a fatty meal. there is a wide inter-individual variability in bioavailability and many drug interferences. the aim of this study was to assess the plasma concentrations of ato in patients under pcp prophylaxis with ato oral suspension and explore the factors which might impact its bioavailability. all adult patients receiving ato for pcp prophylaxis in the hematology and clinical immunology wards between may and september were included in the study. the prescribed dose was mg of oral suspension twice a day. blood samples were collected around h after the evening dose (cmin) and - h after the morning dose (cmax). plasma was immediately separated after each sample and frozen at − °c until proceeding to the assay. ato plasma levels were measured by uv-high-performance liquid chromatography. clinical and biological data, exact timing and modalities of intake (during a meal or not), and concomitant medications were collected. cmin and cmax results are presented as median (iqr - %) and compared by mann-whitney u-test or signed rank test when appropriate. patients: a total of measurements were performed in patients (allogeneic hsct patients: ; hematology non-transplanted patients: ; hiv-infected patients: ). the mean age (range) was years ( - ), the m/f ratio was / . only two patients were neutropenic. the median cmin was . μg/ml ( . - . ) and the median cmax was . μg/ml ( . - . ). thirteen of the ( %) patients had a cmin. disclosure of conflict of interest: none. presepsin as a marker of infectious complications during high-dose chemotherapy following autologous hematopoietic stem cell transplantation in lymphoma patients y dubinina, v sarzhevskiy and v melnichenko national pirogov medical surgical center lymphoma patients, who undergo high-dose chemotherapy following autologous hematopoietic stem cell transplantation (autohsct), are at high risk of developing infectious complica- tions (ic). mortality from ic during the transplantation, according to various data ranges from to %. thus the development of models of early prognosis of ic during autohsct has become more urgent. it's reasonable to include the dynamics of biochemical markers of inflammation in these models. presepsin (psp), procalcitonin (pct) and c-reactive protein (c-rp) were assessed on the day of admission to the hospital (da), on d+ , d+ , d+ and on the day of discharge (dd). if patients developed neutropenic fever (nf), the markers were assessed at the beginning of the fever, h after, then on the second, third, fourth days after. there were patients included in the study: patients with hodgkin lymphoma, with non-hodgkin's lymphoma, with multiple myeloma, out of patients there were women and . the median age was years ( - ). the conditioning regimens were cbv, beeac or hd melphalan. depending on the presence of ic, the patients were divided into groups: group patients without infectious complications (n = ), group patients with the development of infectious complications (n = ). the median of the nf development was . days. patients from group had no microorganism growth in blood stream, either in repeated studies. gram+ flora was detected in patients, patient had gram-, patients had mixed flora and patient had pneumocystis jirovecii infection with respiratory insufficiency grade . significant differences in psp level between groups and were determined on d+ , on d+ and the dd after autohsct. considering the median day of the nf appearance ( . days), it's supposed both the prognostic value (differences on d+ , that is, days before the clinical manifestation of infection) and the diagnostic value of psp (differences on d+ and on the dd) ( table , graph ). [p ] disclosure of conflict of interest: none. hc is often a serious complication and occurs in % of allo-hsct recipients. early bleeding is usually the result of chemotherapy toxicity however late occurring hc is multifactorial. bk virus infection has been shown to be related with hc. most studies demonstrate bk virus at the time of bleeding therefore not allowing the risk imposed by asymptomatic infection to be estimated. in this study, our aim is to show the effect of risk factors as well as pre-transplant bk viral load in asymptomatic recipients on development of hc in allo-hsct. between and , we prospectively evaluated allo-hsct. in order to detect the bk viral load, we performed quantitative bk virus pcr (altona diagnostics, germany) from blood samples at days , , and after allo-hsct. informed consents were obtained from all participants. bk virus pcr was considered positive if any number of copies were detected above the analytical sensitivity of the tests. the patients were monitored for signs and symptoms of hs. the risk factors for the development of hs were evaluated by univariate and multivariate analysis. p o . was considered statistically significant. the median age of the group was (range: - ), of the patients ( %) were aged . male to female ratio was . ( / ). fifty two patients ( %) had diagnosis of malign hematological disease. stem cell source was peripheral blood in ( %), bone marrow in ( %) allo-hsct. patients received stem cells from related donors ( %) vs ( %) unrelated or haplo donors. myeloablative conditioning was administered in patients ( %). forty-four of the conditioning regimens ( %) included cyclophosphamide. hc was diagnosed in patients ( %) at a mean of days (range: - ), early hc was detected in of patients ( %). the frequency of bk viremia and number of viral copies are given in detail in table. the frequency of bk viremia increases during transplantation in relation to clinical hc ( %, %, %, %; p = . ). acute graft vs host disease (agvhd) was diagnosed in patients ( %) at a median time of posttransplant day : grade i-ii gastrointestinal/skin/liver in ( %), grade iii-iv gastrointestinal/ skin/liver in patients ( %). the most common gvhd prophylaxis preferred was cyclosporine and methotrexate in patients ( %). in univariate and multivariate analysis (age , sex, diagnosis, stem cell source, donor type, conditioning regimen, agvhd, cy administration, bk virus pcr at days , , ) bk virus titer positivity at day , , (p = . , p o . , p o . ), myeloablative conditioning (p = . ), the presence of agvhd after day (p = . ) and conditioning regimen that includes cyclophosphamide (p = . ) are found to be related with increased risk of hs. patients with hc and clots were treated with continuous bladder irrigation as well as of patients with bk viremia received cidofovir and six of them responded to treatment ( %). our study showed that, bk titer positivity, myeloablative conditioning, presence of agvhd, cyclophosphamide containing conditioning are associated with hc. detection of bk viremia in later transplant period is more sensitive for clinically proven hc. prophylactic treatment might be considered in patients with asymptomatic bk viremia in pretransplant period. [p ] disclosure of conflict of interest: none. this project has been granted by ankara university scientific research committee numbered as b . high-dose chemotherapy with peripheral blood progenitor cell (pbpc) collection followed by a myeloablative conditioning and autologous stem cell transplantation (asct) is considered the standard of care of relapsed/refractory non hodgkin/hodgkin lymphoma (nhl/hl). a widely adopted conditioning regimen is the combination of carmustine etoposide cytarabine and melphalan (beam), whose feasibility and efficacy has been largely demonstrated. high dose fotemustine plus etoposide, cytarabine and melphalan (feam) has in some cases replaced beam conditioning. neutropenic enterocolitis (nec) is a life threatening complication of patients (pts) treated with chemotherapy (cht) with mortality rate up to %. it's a clinical syndrome in neutropenic patients (pts) characterized by abdominal pain (ap), fever (f) and diarrhoea (d). ultrasound (us) was used to evaluate bowel-wall thickening (bwt), and mm is considered diagnostic of nec. early diagnosis is crucial to start conservative medical management (cmm), which appears the optimal strategy for most cases. objective: . to evaluate if nec incidence and outcome differs in beam vs feam and . to evaluate prospectively if bed-side-us (bus) can detect early signs of nec and guide a prompt treatment (cmm or surgical) in order to reduce mortality. in the last years all pts with nhl/hl admitted in our bmt unit wards at university of pisa (italy), undergoing asct were prospectively enrolled. abdominal us was performed, baseline before treatment, and as only one symptom (or a combination) appeared within h from onset: f and/or d and/or ap in cht-related neutropenic pts. pts were conditioned with beam and pts with feam. nec was diagnosed in n = / feam and in n = / beam patients. incidence was % and % respectively, without a statistically significant difference (p = . ). two pts died/ in feam arm ( . %) and pts/ in beam arm ( . %), without a statistically significant difference (p = . ). at time of diagnosis (dx) symptoms were: f+ap+d %, f+d %, f+ap %, ap+d %,d %,ap %. f alone was never present at diagnosis of nec. at dx, f was absent in / nec episodes ( %). all pts were treated promptly as bus allowed diagnosis with cmm except one pts who underwent surgery, guided by us features, during neutropenia. the likelihood of nec dx in a discriminant st model (bayes theorem) for pts with bwt and ap = . %, ap+d = . %, ap+d+f = %, ap+f = . %, d+f = %. bus allowed to detect early signs of nec and to start prompt treatment in this life threatening complication, of nhl/hl pts undergoing asct. this is a prospective study thus the true incidence of nec in nhl/hl undergoing asct should not be underestimated. there is not a statistically significant difference in incidence and outcome of nec in pts conditioned with beam in respect to feam. with bus pts do not live the isolation room. fever is not a condition sine qua non for nec diagnosis. early diagnosis allows most of pts to be treated with cmm. images of bus and ct were superimposable with lower costs, and less radiation exposure. a low mortality rate in pts with a - % chance of developing this life threatening complication suggests that a prompt bus in neutropenic patients as just one symptom presents allows to make early diagnosis of this life threatening complication and guide prompt treatment (conservative or surgical), reducing mortality. disclosure of conflict of interest: none. quantiferon-cmv in the evaluation of cmv-specific immunity after autologous and allogeneic hsct j moreno , ltesta , l zanetti , l serra , b pereira , m souza , a carolina souza , mp souza , vr colturato and cm machado , hsct program, amaral carvalho foundation and virology laboratory, institute of tropical medicine, university of são paulo cytomegalovirus (cmv) is a major cause of morbidity and mortality after allogeneic hsct. the same is not observed in autologous hsct recipients who do not need to receive immunosuppression after transplantation. in the present study, we compared the reconstitution of cmv-specific immunity in autologous and allogeneic hsct recipients. patients were invited to participate in the study and signed the informed consent. cmv surveillance with the antigenemia (ag) test (cmv brite, biotest, germany) was done weekly in the first months of transplant in allogeneic hsct recipients. preemptive ganciclovir therapy was initiated whenever a positive antigenemia was detected. the presence or absence of cmvimmunity was determined by a commercial interferon (inf) gamma release assay (quantiferon cmv, qiagen) before hsct and monthly thereafter up to d+ . from january to october , hsct recipients ( auto and allo) were included in the study. ag was positive in ( %) of the allohsct recipients at a median of (range: - ) days. ag recurrences occurred at a median of . ( - ) days, in of the pts ( . %) who had at least one episode of positive ag. hsct recipients were included in the analysis of qtf-cmv. in the pre-hsct sample, qtf-cmv was reactive in of the allohsct ( . %) and in of the autohsct ( . %). significantly less allo hsct recipients recovered cmvimmunity at day + ( . %) and day+ ( . %) in comparison with autohsct ( % and %, respectively, p o . ). up to day + , all autohsct have recovered cmvimmunity, in comparison to % of the allohsct recipients (p = . , figure ). the qtf-cmv test performed at d+ , d + and d+ did not predict the risk of cmv reactivation in the following month. similarly, the test did not anticipate the risk of ag recurrences: % of the hsct recipients with undetermined or non-reactive qtf-cmv test at d+ had ag recurrence after this period, in comparison with % of the patients with a reactive result (p = . ). in the present study, the qtf-cmv test alone could not predict the risk of cmv reactivation or recurrences. [p ] disclosure of conflict of interest: qiagen. recovery of vδ + γδ t cells is critical to epstein-barr virus reactivation after haploidentical hematopoietic stem cell transplantation j liu, z bian, q fu, l xu, x zhang, y wang and x-j huang peking university people's hospital, peking university institute of hematology, beijing, china epstein-barr virus (ebv) reactivation and its related disease are life-threatening complications in patients undergone haploidentical hematopoietic stem cell transplantation (haplohsct). our previous studies found that impaired cd − cd − t-cell recovery correlated to the increased occurrence of ebv infection after haplohsct. γδt cells make up - % of cd − cd − t cells in the peripheral blood of healthy donors. expansion of vδ + γδt t cells after hsct has been reported and this subset could respond against autologous ebv-lcl in vitro. selective activation and expansion of vγ vδ -t cell could inhibit ebv-lpd development in humanized mice. however, the association of γδ t-cell recovery with ebv reactivation after allohsct remains unknown. this is a prospective cohort study including consecutive patients who were diagnosed as hematological malignancy and underwent haplohsct. recovery of t lymphocyte and a panel of subsets, including cd +, cd +, cd +, cd -cd -, tcrαβ+, tcrγδ+, vδ +, and vδ + t cells, were determined by flowcytometry at , , , days after haplohsct. all recipients and donors were tested negative for ebv dna in the peripheral blood before transplantation. recipients were monitored weekly for ebv dna load until day after transplantation. recipients with peripheral blood plasma ebv dna load copies/ml at least on two consecutive occasions were diagnosed as ebv reactivation (ebv +). ebv − cohort generally represents patients whose ebv dna loado copies/ml in peripheral blood. within days after haplohsct, of ( . %) recipients were diagnosed as ebv reactivation. compared to recipients with negative ebv dna load, the counts of cd +, cd +, and tcrαβ+ t cells were not statistically different in the ebv+ cohort from to days after haplohsct. in contrast, recoveries of cd + and cd -cd -t cells in ebv+ patients were significantly hampered at days after transplantation (p = . and p = . , respectively). although the tcrγδ+ t-cell counts were also decreased at and days in the ebv+ cohort, the comparisons did not reach the statistical significance (p = . and p = . , respectively). notably, recoveries of vδ + γδ t cells at , and days were continuously delayed in recipients with ebv reactivation (p = . , p = . and p = . , respectively). whereas the counts of vδ + γδ t cells were similar between the two groups from to days in this context. in this prospective and large cohort study, we showed that the occurrence of epstein-barr virus (ebv) reactivation was associated with the hampered recovery of vδ + rather than vδ + γδ t cells after haplohsct. our findings will help explore γδt subset-dependent therapeutic strategies to control the serious complications due to ebv infection post transplantation and improve the overall survival of haplohsct recipients. disclosure of conflict of interest: none. in particular, bloodstream infection (bsi)is a frequent complication in the pre-engraftment phase with an impact on the morbidity and mortality of these patients. objectives: to analyze the incidence of bsi in patients undergoing hsct in our center, and to identify predisposing factors for the development of bsi in pre-engraftment phase patients after hsct. fifty-one consecutive patients undergoing hsct were analyzed retrospectively in our center during the period of july and june . the characteristics of the sample are shown in table . we have reported all the bsi between day and day after stem cell infusion. . % ( patients) received antibacterial prophylaxis with ciprofloxacin, five patients with broad spectrum antibiotics and five did not received any drug. the average days of fever have been . days ( - days). a total of blood cultures has been collected ( . per patient). there have been bsi ( . % of the patients) with ( . %) of cases caused by gram-negative organism ( escherichia coli, klebsiella pneumoniae, acinetobacter baumanii, proteus vulgaris and delftia acidovorans) and ( . %) by gram-positives ( enterococcus faecium, enterococcus faecalis, staphylococcus epidermidis, streptococcus mitis and streptococcus viridians group). one patient presented different episodes of bsi, two patients independent episodes and the rest eight, only one microorganism isolated. we have identified two bsi by extended-spectrum betalactamases (esbl-producing organism) and one isolation of carbapenem-resistant gram-negative bacteria. the rate of quinolone-resistant is % in all the sample. in univariate analysis, several factors like presence of comorbidities, presence of severe mucosits, type of catheter and antibacterial prophylaxis modality don't increased the risk to develop bsi (p . ). the place where the procedure is performed does not influence the development of bsi. although the presence of previous infections is not a risk factor, hospitalization for infection in the days before hsct does influence the development of bsi with statistical significance (po . ). the crude mortality rate of the sample has been very low ( %), with only one death related to bloodstream infection. bsi are a common relative complication in the patient undergoing hsct but with an extremely low mortality in our sample. hospitalization for infection in the days before hsct does influence the development of bsi. it is important to note that outpatient model and conventional rooms don't increased the incidence of bsi. although the use of quinolones in prophylaxis does not result in an increase in infections caused by multiresistant micro-organisms (esbl and carbapenemias) with acceptable resistance rates ( %), it also does not reduce the incidence of bsi in our sample. according to our analysis, his routine employment still throws light and shadows. [p ] disclosure of conflict of interest: none. septic episodes with multiple bacterial strains during antithymocyte globulin (atg) therapy for conditioning for allogeneic stem cell transplantation under rifaximin gut decontamination d markel , c schultze-florey , t brockmeyer , v panagiota , c lück , a schwarzer , m beck , e dammann , a ganser , g beutel and m eder recent evidence demonstrates the importance of the enteric microbiome for the development of gastrointestinal graftversus-host disease (gvhd) and mortality after allogeneic stem cell transplantation (sct) ( , ) . accordingly, the usage of the non-absorbed rifamycin derivate rifaximin for gut decontamination has been reported to preserve the intestinal microbiota composition with a positive effect on overall survival in a single centre retrospective analysis ( ). we here report severe septicaemia requiring therapy at the intensive care unit (icu) during atg application for conditioning in three patients with rifaximin used as single agent for gut decontamination within months. after changing our gut decontamination from a chinolon-metronidazole regimen to rifaximin, three cases of severe septicaemia by gram-negative and gram-positive bacteria during atg treatment occurred within months. patient # was a -year-old woman with tmds/aml after breast cancer conditioned according to the flamsa-bu protocol. the second (# ) and third (# ) patient were and -year-old males with a complex karyotype secondary aml after omf and relapsed inv( ) aml with meningeosis leucaemica, respectively. patients # and # were treated with flamsa-bu and flamsa-tbi, respectively. all patients received rabbit atg (atg fresenius/grafalon) at a dose of × mg/kg body weight and rifaximin ( × mg) for gut decontamination. patient # developed severe escherichia coli and pseudomonas aeruginosa septicaemia on day − of the conditioning regimen and had to be transferred to the icu with septic cardiomyopathy for therapy with vasopressants and levosimendan. in patient # escherichia coli, klebsiella oxytoca, staphylococcus hemolyticus and staphylococcus epidermidis were simultaneously detected in blood cultures at day − . the patient was transferred to the icu and treated with vasopressants for septic shock. patient # developed septic shock due to klebsiella pneumoniae and enterobacter cloacae on day − under atg therapy. mechanical ventilation and vasopressor therapy were required. fortunately, all three patients survived and completely recovered without any sepsis related disabilities under escalated anti-infective and intensive care therapy. all were discharged from the hospital in the outpatient clinics. interestingly, all isolated gram-negative pathogens were found to be sensible for a chinolon based gut decontamination. the reasons for these septic complications under atg therapy are not exactly understood but raise a note of caution on the use of rifaximin as single agent gut decontaminant during atg application in conditioning for allogeneic sct. infections with mycobacterium genavense were described for the first time in . since then, several cases have been reported, but almost exclusively in patients with aids. most patients who underwent hsct have insufficient cellular immunity. here we report a mycobacterium genavense infection in a patient mimicking a lymphoma-relapse after hsct. a year-old female patient was diagnosed in july with stage ivb alk-negative anaplastic t-cell-lymphoma with cervical, retro-/supraclavicular, mediastinal, axillary and retroperitoneal lymphadenopathy as well as pulmonary manifestation. two chemotherapy treatment lines and autologous stem cell transplantation resulted in a partial remission. to improve remission prior to hsct the patient received courses of brentuximab-vedotin. after conditioning therapy with fludarabine, busulfan, cyclophosphamide and atg, hsct from a hla compatible unrelated donor was performed in april . a pet-ct-scan in november confirmed complete remission. after hsct the patient remained lymphocytopenic with cell count of cd + cellso /μl. after acute stage iii gastrointestinal graft-versus-host disease (gvhd) low dose immunosuppressive therapy was maintained due to mild chronic gvhd of the liver and the upper gastrointestinal tract. beginning in june the patient experienced increasing fatigue, general weakness, loss of appetite, nausea, night sweating and fever. abdominal ultrasound, urine and blood culture as well as ct scans revealed no focus of infection. different lines of empirical antibiotic therapy resulted only in short term improvement. several blood culture tests remained sterile. a fdg-pet-ct scan showed a paraaortal and parailiacal lymphadenopathy with a high fdg uptake (suv between . and . ), highly suggestive of lymphoma relapse. endoscopic evaluations revealed two polypoid lesions in the bulbus duodeni. histology of duodenal biopsies revealed a massive accumulation of weakly pas-positive bacilli. pcr analysis confirmed an infection with mycobacterium genavense. despite several attempts mycobacteria were not recoverable on solid media even by long term culture. treatment was started with rifampicin, ethambutol, ciprofloxacin and clarithromycin. lymph node manifestation responded to therapy with decreasing fdg-uptake (suv . ) in a control fdg-pet-ct scan months later. after months treatment was terminated due to therapy refractory nausea. lymphocytopenia was persisting with cd + cellso /μl. six weeks after stopping the antibiotic therapy, symptoms as fever and weakness reappeared. duodenal biopsy could not confirm persistent mycobacterial infection. fdg-positive intraabdominal lymph nodes (suv . ) and spleen (suv . ) were detected in a control fdg-pet-ct-scan. five lymphnodes were surgically removed. immunohistology detected histiocytic cell proliferation with no sign of lymphoma relapse. pcr confirmed the presence of mycobacteria-dna. consequently, antibiotic treatment was resumed. mycobacterium genavense can present with all the symptoms of a lymphoma relapse and should be considered in immune compromised patients. reliable diagnosis can only be obtained from lymph node biopsies and/or endoscopic evaluation. treatment has to be accompanied by restoring cellular immunity and should only be stopped after pcr-negative biopsies. disclosure of conflict of interest: none. stratification of patients with multiple myeloma and lymphoma undergoing autologous hematopoietic stem cell transplantation in term of antifungal prophylaxis r moghnieh, s khaldieh, l awad, d abdallah, n droubi, a youssef, a mougharbel, t jisr and a ibrahiim makassed university hospital, beirut, lebanon autologous hematopoietic stem cell transplantation (ahsct) is at intermediate risk for invasive fungal infections (ifi). the recommendations of international scientific societies are not homologous regarding prophylaxis against ifi in patients (pts) undergoing ahsct. the primary end point was to assess risk factors for the need of empiric/preemptive antifungal therapy in ahsct recipients, and to extrapolate to the subgroup of pts that requires antifungal prophylaxis in our population of ahsct pts. the secondary endpoint was to determine the fungal species distribution infecting or colonizing the pts. our study included adult pts ( yo) who underwent ahsct for lymphoma and multiple myeloma (mm) between and . all febrile neutropenic pts are being managed according to the infectious diseases society of america (idsa) guidelines regarding the use of antimicrobial agents in neutropenic pts with cancer. eligible pts were divided into two groups: those who received empirical antifungal therapy and those who did not need it. we recorded demographic and baseline clinical characteristics including: age, gender, comorbidities, stage, disease status at ahsct, high-dose therapy regimen, the presence of mucositis and its grade, the number of cd + cells transfused, the presence of central line or portacath, the need for mechanical ventilation, the presence of diarrhea, the duration of neutropenia, and the presence of bloodstream infections. pts who had lung infiltrates suggestive of ifi were analyzed separately. the causative fungal pathogens and colonizers were analyzed. univariate and multivariate analysis of potential risk factors to assess further significance was performed using spss. patients were included. pts ( %) had lymphoma and pts ( %) had mm. the need of empiric antifungal therapy was statistically more significant in lymphoma than mm pts (po . ).the presence of mucositis grade ⩾ showed a statistical significance for the need of antifungal therapy (p = . ). in the lymphoma group, remission status (pr vs cr) was not a significant factor for the need of empiric antifungal therapy (p = . ).the presence of mucositis grade ⩾ was at the limit of significance ( p = . ). in the mm group, remission status (pr vs cr) did not affect the need of empiric antifungal therapy (p = ). however, mucositis grade ⩾ was found to be a significant risk factor for the need of empiric antifungal therapy (p = . ). following factors: the number of cd + cells transfused, the presence of central line and portacath, the need for mechanical ventilation, the presence of diarrhea, the duration of neutropenia, and bloodstream infections did not show any significance for the need of antifungal prophylaxis in both groups. all recovered fungal isolates (n = ) were not from deep seated tissues biopsies or blood, and were identified as candida albicans in with lymphoma, and in with mm. they reflected the candida ecology in this pts series rather than deep seated fungal infections. we suggest to give antifungal prophylaxis to all lymphoma pts because of the higher need of empirical antifungal therapy, and give antifungal prophylaxis to mm pts having a predisposition for severe mucositis. fluconazole is the antifungal of choice for prophylaxis since all the fungal isolates were candida albicans. keywords: autologous hematopoietic stem cell transplantation, antifungal prophylaxis. disclosure of conflict of interest: none. a -year-old previously fit woman from a rural area of eastern europe was admitted to the hospital for severe aplastic anemia. steroids, csa, antinfective prophylaxis and supportive therapy were administered without response; therefore rabbit atg was then administered, with minor response; the year later, she underwent allogeneic-hsct (mud / , ric: tbi, cyclophosphamide and fludarabine; gvhd prophylaxis: atg, csa, mtx). several days after transplantation she developed left migraine with ipsilateral back-eye pain. brain mri and ct showed a diffuse opacification of paranasal sinuses, mainly in the sphenoid sinus. the symptoms gradually improved with a specific treatment. the patient achieved a quick and complete haematological recovery and she was discharged. at follow-up visits she complained a flare of the migraine, with a left-sided headache that did not improve with nsaids. the headache gradually intensified until vision in the left eye became blurred with conjunctival injection. after consultation with ophthalmologist, for suspected toxoplasma retinitis, administration of intravitreal steroids and clindamicine was begun with partial benefit. however days after (d + ) she was admitted in hospital because of worsening headache, irradiated in the occipital area, and weakness in the right hemibody. tests on csf were negative for neurotropic pathogens. an mri showed a complete occlusion of the intracranial tract of left internal carotid artery, with likely infectious material localized in the left lateral cerebral fissure. a chest tc showed a nodule with initial excavation in the right superior pulmonary lobe. for suspected tuberculosis she started antitubercular therapy. despite a second lumbar puncture confirmed pleocytosis compatible with acute purulent meningitis, microbiological research for bacteria, fungi and bk were negative. so antitubercular and antitoxoplasma therapy were stopped and the patient underwent surgical biopsy within the sphenoid sinus. pathological examination of the biopsy specimens showed acute and chronic inflammation of the respiratory mucosa, periodic acid. schiff and grocott staining ( figure ) highlighted several septate fungal hyphae. cultural analysis revealed colonies of scedosporium apiospermum so the patient started targeted voriconazole intravenous therapy. nevertheless, days later, she developed aphasia and right hemiparesis. a brain angio-mri confirmed the appearance of new lesions compatible with infectious localizations associated to an increased defect of left internal carotid artery vascularisation and complete left choroid detachment. after weeks of voriconazole a significant clinical improvement have been observed and she was discharged, continuing oral antifungal therapy with voriconazole. at the last follow-up she achieved a complete resolution of neurologic symptoms, with permanent left eye blindness. months later (d + ) she was asymptomatic, with normal haematological and neurological conditions and was able to stop the antifungal therapy. this case-report confirms that the risk of invasive fungal infection (ifi) is relevant in patients receiving hsct for aa, probably due to the prolonged neutropenia and association of other risk factors such as the immunosuppressive therapy and the iron overload. in this very poor prognosis infection, the early diagnosis of cns ifi remains challenging, but the administration of voriconazole was extremely effective. disclosure of conflict of interest: none. in this study, we aim to present the seroprevalence of ebv and incidence of posttranplant lymphoproliferative disease as well as to evaluate the relation with gvhd. between and , the ebv serology of patients that underwent allogeneic hematopoietic stem cell transplantation and their donors were evaluated in the study. ebv ig g (vca-igg, ebna ig g, ea-igg) and igm (vca-igm) antibodies were detected by chemolluminesance method (abbott, abd). all patients were followed for reactivation. ebv igg seropositivity was detected in patients ( %) and donors ( . %). there was no statistically difference in related vs unrelated transplants in seropositivity. the median age of the patients was (range: , patients were male ( %) and ( %) had malign disease. the stem cell source was peripheral blood in ( %) patients and ( %) received grafts from related donors. myeloablative conditioning regimen was received by of patients ( %) (table) . all patients received acyclovir prophylaxis (related transplants mg tid, unrelated transplants mg tid) during and after allo-hsct up to months. twenty six-yearold pretransplant ebv seropositive aplastic anemia patient had ebv ig m positivity after months of allo-hsct and developed lymphoproliferative disease. he was in complete remission after courses of rituximab and methylprednisolone. three patients were ebv igm seropositive in th, th and th months of allo-hsct and received symptomatic treatment. acute gvhd was detected in patients ( %) whereas patients ( %) had chronic gvhd. acute gvhd and chronic gvhd incidences were similar in comparison of donor ebv seropositive vs seronegative status ( % vs %, p = . ; % vs %, p = . ). ebv seropositivity was detected in . % of patients. the donor ebv serology was not related with acute or chronic gvhd. [p ] disclosure of conflict of interest: none. the umc utrecht pediatric experience with brincidofovir after allo hsct ca lindemans, m bierings and jj boelens pediatric blood and marrow program, dept. of pediatrics, university medical center utrecht, the netherlands viral reactivation with dna viruses form a considerable complication of allogeneic hematopoietic stem cell transplantation (hsct). there are little effective antiviral therapies and most have considerable toxicity. especially for adenovirus, there is no satisfactory therapeutic option. recently a new oral antiviral agent, the cidofovir prodrug brincidofovir became available to european patients only on the basis of urgent medical need and after a case by case approval by the health authorities. the aim was to describe our single center experience with brincidofovir in the pediatric allogeneic hsct setting. in the umc utrecht, pediatric patients receive t-replete bone marrow or unrelated cord blood (ucb) as the donor source after mostly myeloablative conditioning regimens (+ serotherapy in unrelated-hct). as gvhd prophylaxis patients receive cyclosporine a (csa) and mtx for bone marrow, csa and prednisone for ucb. patients are by standard weekly monitored for the presence of adenovirus, ebv, cmv en hhv viremia by rt pcrs in the plasma. extensive immune reconstitution measurements are performed every weeks. since , patients that developed viral reactivation with adenovirus, or a combination of other dna viruses (cmv, bk or hhv ) were offered brincidofovir if the viremia was progressive or in the context of poor immune reconstitution. brincidofovir was given in suspension ( mg/ml) at the dose of mg/kg biw, or mg biw for larger children. de drug was discontinued when the viral load was below detection level. in total, six pediatric patients (age range: - ) received brincidofovir ( patients tablets, the suspension). four received it for adenovirus reactivation, a th patient for cmv and bk and a th patient for cmv en hhv . the median day post-hsct of the first administration was days post hsct (range: − to ), the median day post detection of viral reactivation days . the median duration of administration was days ( - ) with two patients being discontinued because of death. in no patient the drug was discontinued due to toxicity issues. the patients that died had multi-organ failure due to a combination of severe agvhd and multiple infectious issues. the patients were discontinued when the viral load was low and when they had cd counts of at least /μl. none of the four alive patients reactivated after the drug was discontinued. urgent medical need administration of brincidofovir is feasible. in our limited series we found the drug was well tolerated. disclosure of conflict of interest: i am a medical consultant for brincidofovir (chimerix). reactivation of herpes simplex virus (hsv- ) or varicellazoster virus (vzv) occurs frequently after allogeneic stem cell transplantation (asct). here, we report three unusual cases, two with reactivation of hsv- and one with vzv. patients and methods: patient (pt) ( -year-old, male) was allografted for high risk acute lymphoblastic leukemia in first complete remission after conditioning with total body irradiation ( gy) and etoposide ( mg/kg). graft-versus-host disease (gvhd) prophylaxis was performed using cyclosporine a, short course methotrexate and anti t-lymphocyte globulin (atg). pts ( year-old, female) and ( -year-old, male) were allografted for acute myeloid leukemia in second and first complete remission, respectively. conditioning regimens used were flamsa-ric in pt and fludarabine/busulfan in pt . in both cases, gvhd prophylaxis consisted of cyclosporine a, mycophenolate mofetil, and atg. pts and had already experienced hsv- -positive oral mucositis following induction chemotherapy and had successfully been treated with acyclovir. both developed hsv- -positive oral mucositis again after asct. in both cases, initial therapy with acyclovir i.v. at a dose of up to mg/kg t.i.d. was ineffective. to explore the mechanism leading to clinical acyclovir resistance, the thymidine kinase genes of both viral strains were sequenced. pt presented with severe abdominal pain and nausea months after asct. in this case, acyclovir prophylaxis post asct had been stopped months before due to side effects. moreover, low dose prednisolone therapy was necessary for chronic gvhd. the hsv- -strain from pt showed a single base pair deletion in the region from nucleotide position to of the thymidine kinase gene (which consists of a guanosine repeat). in pt a single base pair insertion in the same region was found. both genetic alterations lead to a loss of enzyme activity and acyclovir resistance. in both pts treatment was changed to foscarnet which led to rapid improvement. in the case of pt , multiple mucosal erosions were found on endoscopy of the esophagus. in these vzv dna was detected by polymerase chain reaction (pcr). only days later, a vesicular skin eruption developed, which did not follow a dermatomal distribution. again, in the vesicular fluid vzv dna was detected by pcr. in this patient, acyclovir ( mg/kg i.v., t.i.d.) resulted in rapid improvement. reactivation of hsv- and vzv after asct is a frequent finding. usually, hsv- strains respond well to acyclovir. in some cases, resistance can develop, especially in patients that had been treated with acyclovir before. acyclovir resistance of hsv- caused by mutations in the thymidine kinase gene can be overcome by treatment with foscarnet which directly inhibits the viral dna polymerase. disseminated vzv reactivations after asct have been described. clinical presentation can be misleading, for example, beginning with severe abdominal pain that precedes the vesicular eruption by several days. disclosure of conflict of interest: none. toxoplasmosis is a rare but severe complication after hematopoietic stem cell transplantation (hsct) ( ) . it can involve the central nervous system alone or can manifest as a disseminated disease. in the paediatric population the mortality rate is high and sequelae are often severe. new diagnostic tools, such as the pcr assay, may allow for rapid diagnosis and preemptive therapy ( , ) . we retrospectively analysed all children who underwent allogeneic hsct in our centre between january and december . patients lost to follow up before day + were excluded. patients and donors were tested before transplant in order to assess their immunological status against t. gondii. a total of allo-hsct were analysed. before transplant, . % of recipients (r) were toxo-igg positive and . % were toxo-igg negative. among donors (d), serology was available only for / : % were toxo-igg positive, % were toxo-igg negative. we found a high number of not tested donors ( . %, / ) which included, in most cases, mud from foreign registries. the group at higher risk for toxoplasmosis, d − /r+, included . % pairs, whereas d − /r − were . %, d+/r-were . % and d +/r+ were . %. in our series the cumulative incidence of toxoplasmosis disease was . %, with cases out of transplants. two of them (case and ) had cerebral toxoplasmosis, one (case ) had disseminated toxoplasmosis and case had toxoplasmic chorioretinitis. mortality rate was %: two patients died because of multiorgan failure and disseminated toxoplasmosis respectively. in no case localized cerebral toxoplasmosis was the main cause of death. no complications were seen in surviving patients. all patients who developed toxoplasmosis were toxo-igg positive before hsct and three of them were transplanted from a toxoplasma igg negative donor (fourth donor not tested). in the two fatal cases the interferon-gamma releasing assay (igra) never became positive, confirming the absence of specific cellular immunity. toxoplasmosis disease can affect hsct outcome in paediatric recipients and pre-hsct seropositivity is the most important risk factor for toxoplasma disease in the post transplant period. in our cohort seroprevalence was higher than expected, probably due to the high number of patients coming from eastern europe. in order to reduce the burden of toxoplasmosis disease in our population we decided to implement a real-time pcr screening protocol for d − /r+ pairs, to provide rapid diagnosis and early therapy. all positive recipients with a seronegative donor will undergo real-time pcr screening starting on the day of stem cells infusion, and regularly until cd + t cell recovery. in the future we will analyse the impact of this strategy in this particular subset of immunocompromised patients. treatment with brincidofovir for adenovirus disease in pediatric hematopoietic transplants introduction adenovirus may cause serious morbidity and mortality after allogeneic hematopoietic transplants in children. severe lymphopenia is the main risk factor associated with progression to disseminated and often fatal disease. treatment with unlicensed cidofovir is based on monitoring of plasma viral load by pcr. however, cidofovir is only moderately effective at controlling adenovirus and it is associated with significant renal toxicity. brincidofovir is a lipid conjugate of cidofovir. it has a good oral bioavailability and achieves higher intracellular levels of active drug than cidofovir with a better safety profile. it is a potent inhibitor of viral dna synthesis so it could be indicated in immunocompromised patients with adenovirus disease. patients and methods we present three children of , and years old diagnosed of acute lymphoblastic leukemia (all) in nd complete remission (the first two patients) and severe aplastic anemia the last one. there were girls and boy. they underwent a peripheral blood hematopoietic stem cell transplantation using αβ/cd depletion with a haploidentical donor in the two patients with all and cd ra depletion with a matched unrelated donor in the other patient. patients that underwent haploidentical transplants developed early acute graft versus host disease grade iii with gut and skin involvement so immunosuppressive treatment with corticoids was started. they developed severe lymphopenia ( o / mm ). in the first month after transplant an adenovirus disease was diagnosed in the three patients from the weekly monitoring of plasma viral load by pcr. adenovirus was also tested in stools, urine and respiratory sample. in all patients adenovirus was also detected in urine sample. in one of them adenovirus was detected in nasal exudate too and in the other the virus was isolated in stools and in a skin biopsy. results: all of them were initially treated with cidofovir with poor results. foscarnet and gancyclovir was also used without improvement. finally they started a treatment by compassionate use with oral brincidofovir twice a week. with the first dose of brincidofovir plasma viral load started to go down until its complete disappearance. brincidofovir tolerance was good with only mild and limited diarrhea in two cases in the day they were taking brincidofovir. two of the three patients were alive without signs of adenovirus disease. in the other patient blood adenovirus load by pcr decreased below /ml, but remain high in urine. she died of respiratory failure due to pulmonary graft versus host disease. conclusion brincidofovir may be a promising therapeutic option for the treatment of severe adenovirus disease in immunocompromised patients with a good toxicity profile. disclosure of conflict of interest: none. table . all patients were transplanted with pbsc for haematological malignancy, and s received reduced intensity conditioning (ric) regimens with in vivo t-cell depletion. the proportion of patients with baseline and post-vaccination hi titres ⩾ : were . and % for a(h n )pdm , . % at both time points for a (h n ), and . and % for b/phuket. pre and postvaccination geometric mean titres gmt) were higher by mn than hi for a(h n )pdm and a(h n ), but lower for b/ phuket (p = . ). no post-vaccination seroconversions were detected by hi, while a single seroconversion to a(h n ) pdm was detected by mn in a patient vaccinated at - months. the mn assay did not detect any additional low-titre seroresponses (negative to detectable titre) below hi threshold. none of patient age, lymphocyte count, days from transplant to vaccination, donor type, and gvhd or ist at vaccination correlated with baseline or post-vaccination titres by either assay. response to iiv was virtually absent throughout the first year post-hsct, with a single seroconversion to a(h n )pdm detected by mn but not hi, although the sample size was small and half of patients were vaccinated at - months. there is a clear need for a novel, immunogenic seasonal iiv and/or novel vaccination regimens in this population. vaccination of recipients' relatives and close contacts, and hsct healthcare workers should be strongly encouraged. pre-and post-transplant iron overload (io) has been associated with considerable long-term morbidity and mortality in pts undergoing transplantation. classically, management of io in the post-allo-hsct setting has been based in the performance of therapeutic phlebotomies (tp), which are inconvenient for the patient and are often not feasible due to ongoing anemia. we recently published the first prospective study of deferasirox in adult allo-hsct pts with io (vallejo, et al. haematologica ). in this retrospective analysis, we analyzed the real-life management of io in the post-allotransplant setting. this study includes the last pts with a minimum follow-up of weeks, who underwent allo-hsct in our center (october -october ). pts were male ( . %) and female ( . %). median age was years (range: - ). baseline diseases were: aml ( . %), lymphoproliferative disorders ( . %), mds ( . %), all ( . %), chronic myeloproliferative diseases ( . %), mm ( . %), and bm failures ( . %). donor was unrelated in cases ( %; of them hla mismatched), and related in ( %; of them haplo-identical). conditioning regimen was: busulphan-based ( . %), melphalan-based ( . %), tbi-based ( . %), and others ( . %). progenitors source was pb in ( . %), and bm in ( . %). pre-hsct: pts had been transfused with a median of prbc (range: - ), and their median serum ferritin (sf) was ng/ml (range: - ). day + post-hsct: pts had died, and pts had not reached that day yet, so pts were evaluable. they had been transfused with a median of prbc (range: - ), and their median serum ferritin (sf) was ng/ml (range: - ). % pts had sf superior to ng/ml. liver mri (by sir method) to assess liver iron concentration (lic) was performed in pts at day + . seven pts ( . %) had no io (lic - mg/g), pts ( . %) had moderate io (lic . - . mg/g), and pts ( . %) had severe io (lic superior to . mg/g). median lic was . mg/g (range: . - . ). among the cases with history of more than prbc transfused and sf higher than ng/ml at day + , ( . %) were proved to have liver io by mri; the other pt had io in spleen. pts started some kind of therapy to treat the io: pts with severe io initiated a tp program and pts ( out of with moderate io, and out of with severe io) initiated chelation therapy with deferasirox. the drug was started at low dose ( . - mg/kg/ day), and was increased if tolerated up to a maximum of mg/kg/day. of note, the majority of pts were also taken a number of medications (immunosuppressants, statins, antimicrobials, etc). of those pts ( . %) did not tolerate the drug, and were changed to tp. for more details, see the table. ( ) the combination of the history of prbc transfusions and serum ferritin levels was, in the majority of cases, enough to assess the io in the post-allo-hsct setting. ( ) liver mri (by sir method) helped to assess io in doubtful cases. ( ) deferasirox, initiated at low doses and increased if tolerated, was safe and its use helped to avoid the need of therapeutic phlebotomies for the majority of patients. this study reproduces, in a real-life setting, our previous findings in a prospective clinical assay. [p ] disclosure of conflict of interest: none. a case-control study of risk factors of primary graft failure with a focus on associated early-onset severe infections v alcazer , a conrad , f-e nicolini , s ducastelle-lepretre , f barraco , x thomas graft failure (gf) is a rare but devastating event after allogeneic haematopoietic stem cell transplantation (ahsct), exposing the recipient to disease relapse, drawbacks of marrow aplasia, infections and death. the aim of this study was to analyse the risk factors associated with graft failure after ahsct, with a specific focus on early-onset severe infections (esi). we conducted a retrospective, observational, single-centre, matched case-control ( : ) study among adult s ahsct recipients transplanted at the haematology department of our institution between and , with a subsequent follow-up of months. engraftment was assessed at day+ post-ahsct. gf cases were classified as primary gf (pgf), defined as failure to achieve donor-derived absolute neutrophil count (anc) ⩾ . × /l or lasting more than consecutive days without evidence of disease relapse and early-secondary gf (esgf), referring to the loss by day post-ahsct of a previously functioning graft associated without evidence of disease relapse. each case was matched with two controls according to underlying haematological disease, hla matching, stem cell source, intensity of conditioning and temporal proximity of ahsct. demographics, haematological and graft characteristics as well as esi report were retrieved. esi were classified in invasive fungal infections, viral infections (cmv, ebv, hhv- , other viruses), toxoplasmosis and severe sepsis of bacterial origin. during the study period, ahsct were performed at our center. seventeen ( . %) gf cases were identified, of which pgf and esgf, and were matched with controls. in the descriptive analysis, gf and control populations did not significantly differ when considering demographics, haematological characteristics and hematopoietic stem cell source. regarding pretransplantation status and graft characteristics, only disease status (progressive disease) and cell dose (both cd + and cd + cells number/ kg) were associated with graft failure. the proportion of patients with ⩾ esi before day was significantly higher in cases than in controls ( / vs / , p = . ), with an overall number of esi events of and among cases and controls, respectively. five cases had ⩾ concurrent esi. the median time from ahsct to the first esi event for gf cases was days (interquartile range (iqr), - ) vs (iqr, - ) days for controls (p = . ). in the gf setting, the most prevalent infections were herpesviridae infections (n = including hhv- n = , ebv n = , cmv n = ), probable ifi (n = ), severe sepsis of documented bacterial origin (n = ), toxoplasmosis (n = ) among whom one patient developed haemophagocytic syndrome. when further analysing subsets of esi using logistic regression, only toxoplasmosis was a significant risk factor for gf (p = . ). death related to an infection was proven for gf patients vs control patients (p = . ). the overall survival probability at months was significantly lower in the gf setting than in control patients (hr = . ( % ci . − . ), p = . ). the survival rates at months were . % and . % for gf and control patients, respectively. at our center, graft failure is statistically associated with early-onset severe infections, and already known graft characteristics such as cell dose and disease status. however, our study would need more power to increase its significance. disclosure of conflict of interest: none. allogeneic stem cell transplantation (asct) is a curative option for hematological disorders, especially malignancies. in immunosuppressed women after asct, the progression from cervical dysplasia to invasive carcinoma is accelerated, and cervical cancer is likely a more aggressive disease. therefore, follow-up protocols after asct should include regular gynecologic evaluation with papanicolaou (pap) smears. we retrospectively evaluated pap smears in women who underwent asct and searched the risk factors for abnormal cervical cytology. the median age at transplantation was . years (range: - years). the most frequent indication for asct was leukemia ( %), and % of the patients received a transplant from a sibling hla-matched donor. stem cell source was peripheral blood in all patients. myeloablative conditioning regimen was used in % of patients. cyclophosphamide, busulfan and fludarabin were used in ( %), ( %) and ( %) patients, respectively. acute graft versus host disease (gvhd) occurred in patients ( %) and chronic gvhd in patients ( %). secondary cancer ( breast cancer) was reported in only one patient at months after asct. the follow-up time was months (range: - months). after asct, benign and abnormal pap smears were found in ( %) and ( %) women, respectively. the median time between asct and development of abnormal cytology was months (range: - months). four ( %) women had at least one smear with atypical squamous cells of unknown significance (asc-us), one ( %) had a low-grade squamous intraepithelial lesion (lsil), one ( %) had atypical squamous cells/high-grade lesion (asc-h) and one ( %) had asc-us and asc-h. one ( %) patient had malign smear. two patients with asc-h showed high-grade atypia mimicking cancer but had a negative follow-up. patient who had malign smear died because of aorta dissection. cervical biopsy showed cervical intraepithelial neoplasia (cin) i in ( %) women who had asc-us or asc-h. one patient was hpv-positive. we did not find any relationship between cervical cytological abnormality and clinical factors. after asct, patients are high risk for abnormal cervical cytology and secondary gynecological cancer. regular surveillance of patients is the most important factor for decreasing the risk of developing cervical and other secondary cancers. gynecologic examinations and cervical cytological testing after asct allows early diagnosis and effective management of cervical abnormalities. disclosure of conflict of interest: none. kidney dysfunction is a frequent complication of allogeneic stem cell transplantation (sct) and contributes to the morbidity and mortality of the procedure. incidence of severe acute kidney injury (aki) in patients undergoing nonmyeloablative allogeneic sct for malignant diseases ranges from to %. lymphoma patients are often heavily pretreated through both chemotherapy and autologous sct and may be at increased risk of developing kidney injury. we performed a retrospective analysis of consecutive patients with lymphoma undergoing nonmyeloablative allogeneic sct between and (table ) . acute kidney injury (aki) within days of allogeneic sct was diagnosed and staged according to rifle-criteria, and severe aki was defined as rifle stage i-e ( doubling of creatinine or % decrease of egfr). chronic kidney disease was defined as an estimated glomerular filtration rate (egfr) o ml/min/ . m year after allogeneic sct. we performed multivariate logistic regression to evaluate potential risk factors for severe aki. severe aki developed in patients ( . %). reduced overall survival was observed in these patients, although not statistically significant. no significant associations were seen with age at transplantation, baseline kidney function or prior autologous sct. severe aki was associated with acute graft versus host disease (gvhd) (or . , p = . ) and the use of an unrelated donor (or . , p = . ). chronic kidney disease was observed in ( . %) of patients alive after year. we report a substantially higher incidence of severe aki after nonmyeloablative allogeneic sct for lymphoma than has been reported for other malignancies. acute gvhd and unrelated donor stem cell s source were associated with severe aki, while prior autologous sct, age and baseline kidney function were not. [p ] disclosure of conflict of interest: none. patients with acute myeloid leukemia who are treated with conventional chemotherapy still have a substantial risk of relapse. we, therefore, retrospectively analyzed data to investigate the effects and some risk factors of allogeneic hematopoietic stem cell transplantation in relapsed and refractory acute myeloid leukemia patients, and to provide some suggestion for the clinical treatment. a total of refractory and relapsed acute myeloid leukemia patients receiving allogeneic hematopoietic stem cell transplantation in our center between february and december were retrospectively analyzed, including patients in no-remission (nr) and patients in second complete remission (cr ) at the time of transplant. the median age was years (range: - ). conditioning was myeloablative using cyclophosphamide, busulfan and total-body irradiation (bu/cy, n = ; tbi/cy, n = ), and others were underwent nonmyeloablative stem cell transplantation. patients had successful engraftment. acute-gvhd and chronic-gvhd appeared in and patients. the year overall survival (os), relapse rate and disease-free survival (dfs) of the cases was ± . %, . ± . % and . ± . %, respectively. the -year dfs were higher for patients in cr patients ( . ± . %) than in nr patients ( . ± . %), and the relapse rate in nr group and cr group were . ± . % and . ± . % respectively. there was no significant difference in treatment-related mortality compared cr group with nr group. sex, age, related-donor graft were not independent factors affecting os, dfs and relapse rate. it is concluded that allo-hsct is an effective salvage therapy for patients with refractory and relapsed aml. non-remission before transplant and severe agvhd are high risk factors of poor prognosis for allo-hsct. patients in cr group who accept reinduction chemotherapy before transplantation have better prognosis than those in nr. the overall outcome seems related to the disease status. hsct during refractory and relapsed can achieve long-term survival in selected patients with individual therapy. disclosure of conflict of interest: none. the incidence of most hematologic malignancies increases with age. aging is related with a greater prevalence of impaired functional status and comorbidities. although cure of malignant and non-malignant hematological diseases is potentially possible with allo-hsct, it could lead to significant transplant-related mortality. decision making about referral to allo-hsct in older adults is a challenging task. in this study we aim to present our geriatric allo-hscts. from to , [p ] patients (age ) underwent allo-hsct in our center included to this retrospective study. pre-transplant status as well as posttransplant toxicities, complications and outcomes were determined. the age distribution of the group: patients was aged and o , patients was aged and o , patient was years old. the median age of donors was (range: - ). the pre-transplant patients' characteristics are given in the table. remission was achieved in twenty-three ( %) patients. twenty-six patients ( %) had neutrophil engraftment ( . × /l) at a median day of (range: - ) and platelet engraftment ( × /l) at a median day of (range: - ). post-transplant complications are detailed in the table. acute graft vs host disease (gvhd) was occurred in patients ( %) and chronic gvhd in patients ( %). eight patients ( %) were diagnosed with a relapse and year relapse-free survival was %. the -year and -year os were detected as % and %. the most common reason for mortality was sepsis. the -year os was higher in patients who had reduced intensity conditioning regimen and remission status pre-transplant however they were not statistically significant ( % vs %, p = . ; % vs %, p = . ) (figure) . since increasing number of older patients being diagnosed with hematologic malignancies, this trend of increasing number of allo-hsct will continue. tolerability and effectiveness are lesser, toxicity is higher in older adults. although study population is relatively small, reduced-intensity conditioning and pre-transplant remission status may be related to better survival. comprehensive geriatric assessment may be considered prior to allo-hsct for global evaluation. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (asct) is a procedure with high morbidity and mortality ( - %) requiring a complex hospital infrastructure. improved support measures and development of homecare units has allowed that asct at-home programs may be possible. our center has launched a pioneering program in our country in patients with asct to perform at home the following of aplasia, control of immunosuppressive therapy (ist) and intravenous support from the d+ of asct until the engraftment and independent ambulatory patient. to evaluate the patient safety, we compared the group of patients at-home (asct-op) with a cohort of asct 'in patient' with similar characteristics (asct-ip). asct patients between january and october at the hospital clinic of barcelona. patients performed asct-op and had an asct-ip. all patients received conditioning (myeloablative-mac-or reduce intensity-ric-) in the hospital with fludarabine mg/m (d - ) and busulphan . mg/kg ( - doses), prophylaxis of gvhd was performed with tacrolimus/mycophenolate (mmf) in asct-op group and cyclosporine(csa) and methotrexate (mtx) or mmf in asct-ip group. in all patients, the infectious prophylaxis was conventional (levofloxacin, fluconazole and acyclovir). moreover, the asct-op group received prophylaxis with ceftriaxone g intravenous (iv) once daily and liposomal amphotericin b inhaled mg twice a week during neutropenia. the asct-op group from d+ received a nurse visit once daily and physician visits twice a week in the hospital. baseline characteristics were analyzed those related to toxicity and patient outcomes. the median age (range) was years ( - ), male/female / ; ( % male). the source of the progenitors was peripheral blood in all cases and analysis of the results detailed in the table: disclosure of conflict of interest: none. an increase in rdw-sd after allogeneic hematopoietic transplantation is associated with a poor prognosis s leotta, a cupri, a di marco, a spadaro, l scalise, g sapienza, mg camuglia, g avola, g moschetti and g milone istituto oncologico del mediterraneo red cell distribution width (rdw), is an erythrocyte index influenced by stress erythropoiesis, inflammation and antioxidants. rdw predict mortality in sepsis, chronic kidney diseases and in cardiovascular disease. no data are available on rdw after hematopoietic transplantation. in a retrospective study we collected data on changes of rdw-sd in a group of patients who received allogeneic hematopoietic transplantation. fortyeight patients were affected by acute leukemia, by lymphoma, by mm, and by other diagnosis. rdw was studied at baseline and monthly for the first months. a subset of patients were studied prospectively for clinical and laboratory signs of microangiopathy. at baseline before the transplant a rdw-sd higher than normal upper limit was observed in % of allogeneic candidates. a high co-morbidity score (htc-ci score - ) at the pre-transplant screening was a factor associated to high rdw-sd (χ p = . ). a value of rdw-sd higher than normal range, at baseline, was not associated to any other factors, such as age, diagnosis, phase of the disease, previous transplantation, c-reactive protein, bilirubin, creatinine and arterial hypertension. early after allogeneic transplant we noticed at day + a significant reduction of rdw-sd but subsequently (at day + ) the proportion of patients showing an abnormal rdw-sd increased to %. an abnormal rdw-sd at s day + was registered in % of allogeneic transplant patients who presented an acute gvhd while in only % of patients who did not presented during the first months an acute gvhd (χ p = . ). in allogeneic transplantation group, patient who, at day + , had a rdw-sd higher than normal value had a inferior outcome in respect to patients having a rdw-sd within normal ranges (os was % vs %; logrank: p = . ;), (ci of trm: % vs %).these two groups were not significantly different for pretransplant features in the subset of patients studied prospectively, abnormal rdw-sd was associated to presence of schystocytes in pb (chi test: . ) and patients having ⩾ % schystocytes had a median rdw-sd of (iqr ) vs a median rdw-sd of (iqr . ) in patients who did not show schystocytes in pb (mann-whitney u-test p = . ). rdw-sd was significantly correlated also to serum triglycerides (r = + . , p = . ) and to red blood cell mean corpuscular volume (r = + . , p = . ). abnormal rdw-sd is frequent after allogeneic transplantation. abnormal rdw-sd is associated to acute gvhd and its value obtained at day + marks a group of patients with poor prognosis because of high trm. this simple parameter warrant further studies to determine its clinical usefulness in monitoring of patients suffering acute-gvhd and in diagnosis and monitoring transplant associated microangiopathy. [p ] disclosure of conflict of interest: none. sickle cell disease (scd) poses a lot of psychological burden for the patient and the caregiver. it also poses a significant financial burden over the family. ohaeri et al. developed a point questionnaire to asses sickle cell disease burden called as sickle cell disease burden index (scdbi) and its impact on caregiver's quality of life (qol). we used this questionnaire to assess the impact of hematopoietic stem cell transplant (hsct) on caregiver's qol. point questionnaire was sent to set of parents whose child underwent hsct between january and june . scdbi contained questions in various domains ( :family finances, :family interactions, :routine family activity and :parental coping ability). answers were graded on a score of - ( :never occurred and :occurred regularly or had a severe impact on the family). the results were interpreted in two headings a. family finances and interactions ( : no impact; - : insignificant impact; - : moderate impact; - : severe impact) and b. routine family activity and parental coping ability ( : no impact; - : insignificant impact; - : moderate impact; - : severe impact). all these domains were assessed before and after hsct. ten parents replied with duly filled questionnaire. mean age at hsct was . years (range: - ), m/f: / . all were symptomatic for months before hsct with % having more than hospital admissions. majority of parents were from middle class with median family income of usd per annum (range - usd). median score for family finances and interactions (a) before hsct was (range: [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] which decreased to (range: - ) after hsct. median score for routine family activities and parental coping ability (b) before hsct was (range: which decreased to (range: - ) after hsct. our results suggest that before hsct there was a moderate impact on family finances and interactions which reduced to no impact after hsct. similarly there was severe impact on family activities and parental coping ability before hsct which changed to no impact after hsct. our study suggests that hsct not only improves the qol of the child but also of the caregivers. chronic graft versus host disease (cgvhd) is a late complication of allogenic hematopoietic stem cell transplantation (hsct) that affects many tissues and organs and manifests with polymorphic clinical features similar to autoimmune diseases. poorly understood pathophysiological mechanisms are implicated in inflammation and tissue fibrosis which is a hallmark of cgvhd. the affection of lachrymal glands is frequent and contributes to ocular manifestations presenting as dry eye syndrome. autologous serum eye drops (aesds) are used topically to facilitate tissue healing and ease the symptoms in a variety of ocular diagnosis. it is unclear if the serum of a patient with cgvhd is suitable for remedy preparation and if the transplanted patient himself can meet the criteria for autologous donation. aim is to show the safety, feasibility and efficacy of autologous serum preparations in ocular lesions after allogenic hsct. donors should meet criteria for autologous blood donation (infectious disease status, complete blood count hgb g/l, hct %, adequate venous access). aesds are prepared from ml of autologous blood left to clot, irradiated and centrifuged to separate serum which is diluted with saline in : ratio or : if requested. product is dispensed into . ml ampules, stored at − °c and a -month supply is released to the patient after receiving negative results of sterility testing. in period from to . in the aesds program patients ( female, male) with ocular symptoms were included. all met required predonation criteria. of collections performed, one failed due to venous access problem and one product had to be discarded due to hemolysis. cgvdh global nih score of the patients at start of the program was: severe, moderate, mild and not scored. all patients presented with moderate to severe dry eye symptoms. in ( %) patients aesds alleviated dry eye symptoms. in ( %) out of patients referred to aesd program, more than autologous blood collections were performed (range: - ) and aesds were used regularly through period of - months, which points to the beneficial effect of the long-term use of the serum. three patients dropped out because aesds showed no advantage compared to commercial lubricant eye drops preparations. one patient dropped out because of a venous access problem, patients had disease s progression and needed other therapies: cases of amniotic membrane application of which continued with aesds to facilitate the healing effect. one patient was recently included and the effect of aesd is still evaluated. autologous donations in cgvhd patients are feasible, safe and autologous serum preparations can help relieve symptoms of dry eyes. it needs to be further elucidated specifically in which patients and at what point of the disease course the effect of the aesds is the most beneficial to make optimal use of these preparations. disclosure of conflict of interest: none. idiopathic pneumonia syndrome (ips) is a non-infectious pulmonary complication with diffuse lung injury that develops in - % of patients who undergo hematopoietic cell transplantation (hct) and the mortality rate remains high at % . the major aim of this study was to identify prognostic biomarkers for ips and establish positive and negative predictive values (ppv and npv) of ips. in a case-control study, we compared patients with ips with available samples (transplanted between and at fhcrc) with hct control recipients who did not require bronchoscopic examination and who did not grow any bacterial or fungal blood cultures. for each subject, plasma samples at day post hct and onset of ips or matched time points for controls were analyzed. the 'onset sample' for controls was the sample closest to day (median day of onset for patients with ips). we measured six proteins by elisa: suppressor of tumorigenicity (st ), tumor necrosis factor receptor (tnfr ), interleukin- (il- ), lymphocyte vessel endothelial receptor (lyve)- , endothelial protein c receptor (epcr), and herpes virus entry mediator (hvem). multivariable logistic regression models were used to evaluate the association of each protein with ips vs controls. cytokine cutoff values that maximized discrimination between ips and controls were identified using receiver operating characteristic (roc) analysis. ppv and npv of ips were calculated using the identified cytokine cutoffs across a range of hypothetical ips prevalence values ( - %) day weighted kaplan-meier survival curves were estimated for high/low cytokine subgroups. similarly, a weighted log-rank test was used to evaluate p-values. a multivariable logistic regression model including six cytokines showed that st and il- were significantly important markers to identify ips at the onset (table ) . st value at day post hct was significantly associated with occurrence of ips and il- had a marginal association. predictive values for ips by a plausible percentage of the actual hct population (up to %) are shown in figure . of the six proteins, st showed the highest ppv both at onset and day post hct followed by tnfr , and il- . npv were high in all the markers. to analyze whether st and il- at day after hct can predict survival following ips, we dichotomized the patients into cytokine high and low groups (cutoff level: st , ng/ml; il- , pg/ml) and compared survival after downweighting the observations to represent a plausible percentage of the actual population (ips prevalence, %). day survival rate were significantly lower in st high value group than in st low value group ( % vs %, p = . ). similarly, il- high value was associated with high mortality (day survival rate, % vs %, p = . ). st , il- , and tnfr were good prognostic markers for occurrence ips. especially, st and il- at day after hct can be a predictor for both ips occurrence and survival following ips. these results require validation in an independent prospective hct population. body composition parameters are sensitive nutritional indicators that influence response to treatment and mortality in cancer patients. research is not conclusive on the changes in muscle attenuation and adipose tissue areas in the stem cell transplantation (sct) phases. objective is to assess the changes in adipose tissues, skeletal muscle index (smi) and waist circumference (wc) among stem cell recipients in the peri-transplantation phase. study design: institutional review board approved this retrospective study with adult patients (age years) having b and t lymphoma who underwent sct. each patient was imaged by pet/ct scan pre-sct and months post transplantation. a cross sectional image was analyzed at the level of the l to calculate total adipose tissue (tat), visceral adipose tissue (vat), intra-muscular fat (imf), smi and wc. data was analyzed by gender since body composition parameters differed significantly between the two categories in the literature. the study sample consisted of patients (mean age: . ± . years, ( %) males, ( . %) autologous sct, median overall survival in months: . in males and . in females). death was observed in ( . %) males and ( . %) female. patient characteristics were similar for males and females except for weights (kg) and body mass index (kg/m ): . and . vs . and . in males and females respectively. changes from pre-sct to months post sct revealed that tat, vat, smi and wc decreased with mean differences of ± . cm , . ± . , . ± . cm /m and . ± . cm, respectively in males (po . ). in females, tat and wc significantly decreased with mean differences of . ± cm and . ± . cm, respectively (po . ). in females, vat and smi decreased clinically but did not reach clinical significance. in multivariate analysis, no significant associations were shown with mortality and progression rates. this study fills a research gap by providing data on the evolution of body composition parameters in the peri-transplantation phase. tat, vat, smi and wc decrease months post transplantation. future studies should evaluate the associations of these parameters with major outcomes on larger sample sizes. [p ] disclosure of conflict of interest: none. . patients received tbicontaining preparative regimen. all these patients were exposed to calcineurin inhibitors for prevention and treatment of gvhd. patients suffered from cgvhd-grade moderate or severe. all patients required systemic corticosteroids, because of gvhd ( pts) or during basic treatment of lymphoma ( pts). all patients had deficient states of vitamin d initially and required replacement. all of them, except for patients, had balanced adrenal insufficiencies and patients had balanced hypothyroidism. all women had premature ovarian failure ( received hrt). according to measurements of bone mineral density (bmd), low bone mass was detected in patients; osteopenia ( pts), osteoporosis ( pts). bone loss of femoral neck ( -osteopenia, -osteoporosis) occurred more often than lumbar vertebral ( -osteopenia, -osteoporosis) or radius ( osteopenia, -osteoporosis). presence of avascular necrosis of bone (avn), confirmed by mri, was detected in patients and the most common site of involvement was the femoral head(all patients), knee( pts) and shoulder( pts). one of the first symptoms of avn was pain and functional limitation. all patients required intensive analgesic treatment, usually nsaids and patientsfentanyl. fractures occurred in patients. the femoral neck ( pts) and thoracic or lumbar vertebral ( pts) were two most common fracture sites. all patients were qualified for surgery; patients required hip replacement, patients still awaited to perform surgery or were disqualified because of severe, skin cgvhd. bone complications may occur in about % of allo-hct survivors (including % patients with gvhd, and up to % patients with severe or moderate cgvhd) within first years after allotransplantation. bone loss, particularly at the femoral head, is the most common complication. avascular necrosis usually requires surgical intervention because of fractures. exposure to higher doses of corticosteroids (during treatment of gvhd) increases risk of bone complications. early diagnosis by mri and dxa may help to detect bone complications. ( %) and (e) ( %). a total of ( %) pts failed to meet these criteria but remained alive on day and ( %) died before day . the overall survival (figure ) for the pts was % at years with an overall mortality in icu of % ( / ) compared to % ( / ) for those who did not meet our criteria. the overall survival for pts that met our criteria at fifth day and were discharged to the haematology ward (n = ), was % at years. in this study, % of patients survived their icu admission. patients could be stratified according to the reason for admission and given an individualized -day trial: those who met our criteria for successful icu trial ( %) had a low icu mortality ( %) and those who were subsequently discharged home had a overall survival of % at years. this study raises the possibility of offering a short-term icu stay to oncohematologic patients and perhaps allows for the ceiling of intensive care for those who fail these criteria. [p ] disclosure of conflict of interest: none. in contrast, only . % of patients without cgvhd showed a thromboembolic complication in the later time course, with one patient showing an additional thrombotic risk factor. in multivariate analysis cgvhd was an independent risk factor for thromboembolic complications after hsct. . % of patients with thrombosis before hsct showed one afterwards. thrombosis before hsct was not found as risk factor for thromboembolic complication after hsct. our retrospective analysis showed an increased risk for thromboembolic complications after allogeneic hsct, with substantial higher risk in patients with chronic gvhd ( . %). in ongoing studies we currently investigate a vascular screening procedure with additional biomarkers according to inflammation and endothelial damage in patients with cgvhd prospectively. we hope to identify patients at risk for thromboembolism and prevent future complications on an individualized basis. disclosure of conflict of interest: none. ( ). pts with cns involvement received intrathecal therapy with cytarabine and in one case additional cns irradiation was applied. / pts died after a median time of mts (range: - mts) due to resistant systemic relapse, infectious complications or extensive graft-versus-host disease following allohsct. patient remains alive and disease-free at + mts following secondary allohsct. conclusions: our data indicate that em disease following allohsct affects a significant proportion of pts with aml. sites of em relapses vary widely among the pts with skin and cns being frequently involved. an aggressive approach combined of local and systemic therapy including secondary allohsct may produce favorable response in a small proportion of pts, however, overall prognosis for pts with isolated em relapses still remains poor. due to the lack of effective treatment strategies, there is a need for novel approaches to manage isolated em relapses after allohsct. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct)-associated thrombotic microangiopathy (tma) is a multifactorial complication, and has variable incidence in study populations due to different diagnostic criteria. aim: our aim was to identify pediatric patients with hsct associated tma using different diagnostic tma criteria published in literature and to compare the various groups for tma parameters and outcomes. we enrolled pediatric patients who underwent allogeneic hsct using treosulfan based or reduced intensity conditioning therapy. different tma diagnostic criteria, the bmt ctn toxicity committee consensus definition ( ), the overall thrombotic microangiopathy grouping ( ), the diagnostic criteria created by city of hope ( ) and the criteria proposed by jodele et al. ( ) were used to startify the patients. we determined and registered the following tma activity markers: presence or development of increased ldh and decreased haptoglobin levels, new onset anemia, thrombocytopenia, fragmentocytes, coombs test, kidney function, proteinuria, hypertension and terminal complement complex (sc b- ). complement pathway activities, components and sc b- were measured during early hsct period. two/ ( ), / ( ), / ( ) and / ( ) subjects met the different tma diagnostic criteria according to the four different systems on day and ( ) and on median ( ), ( ), ( ) post-hsct days. all of the / patients who were defined with the first three criteria, met the forth definition. due to normal haptoglobin levels and kidney function, / patients fulfilled only the forth criteria. tma coexisted with acute graft-versus-host disease in / cases ( / vs / ; p o . ). patients who met any of the different tma diagnostic criteria had higher sc b- level on day ( vs ng/ml; p = . ) compared to those without. all of the / subjects defined with tma had elevated sc b- ( ng/ml) level during the early hsct period. two patient died before day after hsct, out of which one patient met all of the four tma diagnostic criteria. after a median . ( . - . ) year follow-up time, overall survival was / . / patients with tma survived, compared to / patients without tma. relapse related mortality was the most common cause of death (n = / , po . ), while tma was not a significant cause of mortality after reduced toxicity conditioning therapy. hsct-associated tma has a variable and complex pathophysiology. using the different diagnostic criteria may influence the incidence and the time of diagnosis of this transplant-related complication. monitoring all of the published tma activity parameters, including complement terminal pathway activation marker, may help to guide physicians to recognise tma after hsct. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) is associated with a risk of non-relapse mortality (nrm). it's important to assess the risk of complications and mortality before the hsct. some indexes quantify the impact of patients' comorbidities on hsct outcome. the most frequency used is the hct comorbidity index (hct-ci) and the european group for blood and marrow transplantation score (ebmts). this study tried to determine which of the two indexes best predicts the outcome in a series of patients submitted to hsct in a single center. between and , hsct were performed in our center. a total of hsct have been analyzed (we excluded patients o years (yr), nd hsct, haploidentical donors and hsct for specific diseases with very low number ( o %) of hsct performed: aplastic anemia, cll, prolymphocytic leukemia, mycosis fungoides, sezary syndrome, dendritic cell neoplasia, plasma cell leukemia and poems syndrome). the hct-ci and ebmts were calculated retrospectively (yr - ) and prospectively (yr - ). overall survival (os), relapse incidence (ri) and nrm were analyzed in the overall series and separately according to the type of hsct: autologous hsct (auto-hsct) or allogeneic hsct (allo-hsct). male: ( %) patients. median age: yr (range: - ). diseases: aml ( %), all ( %), mm ( %), nhl ( %), hl ( %), mds ( %), cmpd ( %). disease status: st complete remission (cr) or st chronic phase ( %), ⩾ second cr ( %), first partial remission (pr) ( %), ⩾ second pr ( %), no response ( %) and without previous treatment ( %). auto-hsct in patients ( %) and allo-hsct in ( %) patients. related and unrelated donor were ( %) and ( %), respectively. the conditioning regimen was standard in ( %) cases and reduced intensity in ( %). hct-ci and ebmts grouped - , and ⩾ were ( %), ( %), ( %) and ( %), ( %), ( %) in auto-hcct and ( %), ( %), ( %) and ( %), ( %), ( %) in allo-hsct, respectively. median follow-up was . yr ( . ; . ) for the overall series, . yr ( . ; . ) for auto-hsct and . yr ( . ; . ) for allo-hsct. significant differences in os and nrm were found according to the ebmts in patients submitted to auto-hsct. one-yr-os and -yr-os were % ( % ci: %; %) and % ( % ci: %; %), respectively, in patients with ebmts - , vs % ( % ci: %; %) and % ( % ci: %; %), respectively, in patients with ebmts ⩾ (p = . ). one-yr-nrm and -yr-nrm were % ( % ci: %; %) in patients with ebmts - , vs % ( % ci: %; %) in patients with ebmts ⩾ (p = . ). no significant differences were observed for ri according to ebmts in patients submitted to auto-hsct. no significant differences in os, ri and nrm were observed according to ebmts in patients submitted to allo-hsct. no significant differences regarding os, ri or nrm were found when the hct-ci was assessed. in our series, only the ebmts was predictive of os and nrm in patients submitted to auto-hsct. failure to find statistically significant differences for the hct-ci and for ebmts in allo-hsct recipients could be due to an insufficient number of patients or to a partial retrospective collection of data. infertility is common after hct predominantly as a result of the chemoradiotherapy used in conditioning. nonetheless, some patients do retain or recover fertility. newer reduced intensity regimens may be less gonadatoxic. in addition, patients are increasingly encouraged to store gametes, or embryos before transplant. we sent questionnaires to ebmt centers requesting retrospective details of number of pregnancies and pregnancy outcome for all patients treated between - . centers responded from countries detailing patients who became pregnant/partners conceived. the most frequent underlying diagnoses were acquired bone marrow failure (n = , f) aml (n = , f), hd (n = , f), cml (n = , f), all (n = , f) and b nhl (n = , f). other diagnoses included mds, mps, solid tumours, autoimmune disease, cll, t-nhl, haemoglobinopathy. of females (f), ( %) involved assisted reproductive techniques (art). f had tbi ( seven o gy) of which ( %) had art. f had reduced intensity conditioning of whom ( %) had art. f were specified as having standard conditioning of whom ( %) had art. f had allogeneic ( art, %) and f had autologous transplants ( art, %). of men (m) whose partners conceived, ( %) had art. m received tbi of which ( %) had art. where specified, had reduced intensity hct ( art, %) and had standard conditioning ( art, %) . had had allogeneic hct ( art) and autologous ( art). men had reduced intensity transplants. men received tbi (two o gy) of whom ( %) had art compared to men without tbi, ( %)of whom had art. data on return of menstruation was available for . indicated yes and ( %) had art. indicated amenorrhoea of whom ( %) had art. specifying number of children had live births (lb) and ( %) patients had more than one child after hct. lb occurred in female patients ( art, %) and lb were in partners of male patients ( art, %). the median gestational age for female patients was weeks (range: - ) and the median birth weight was kg (range: . - . ). there were / congenital anomalies. the median follow up of the offspring was y (range: - ). developmental problems were indicated for / (fine motor skills) and learning difficulties in / (adhd). in partners of male patients the median gestational age for offspring was weeks (range: - ). the median birth weight for offspring was kg (range: . - . ). congenital malformations occurred in / . one infant died of pulmonary infection. in women, several methods of assisted conception were used including hormone stimulation, ivf, cryopreserved embryos, donor embryos and cryopreserved ovarian tissue. the most frequent method was use of donor embryos ( / ) in which a minimum of attempts led to lb. the median number of attempts was (range: [ ] [ ] [ ] [ ] [ ] . art were frequently used in this group of posttransplant patients particularly in male patients vs female, tbi vs non-tbi, amenorrhoeic vs menstruating women, standard conditioning vs ric. in patients who conceive after hct, successful pregnancy leading to healthy offspring is the likely outcome. disclosure of conflict of interest: none. reduction of trm after sct was observed over the transplant periods and supportive care with danaparoid was found to be significantly effective to reduce trm. therefore, prophylactic administration of danaparoid is considered to be a reasonable option to improve the transplant outcomes for children. [p ] disclosure of conflict of interest: none. the attainment of transfusion independence after transplant is sometimes hampered by a combination of factors, ranging from infections to the need of combined therapy for clinical complications, as well as control of gvhd. iron overload is frequently observed in hematological patients before and after hematopoietic stem cell transplantation (hsct). whereas several reports have focused on iron overload before transplant, up to now, this is the only report that show full recovery of hematopoiesis and correlate this to deferasirox chelation performed on this particular subset of patients. we report on patients, transplanted for hematological diseases ( acute leukemia, aplastic anemia, multiple myeloma) heavily transfused before transplant that, considering the iron overload, were treated with deferasirox after hsct. before starting deferasirox, the patients were fully engrafted and in complete remission, although transfusion dependent, and with incomplete hematological reconstitution after allogeneic hsct. patients were selected according to the following inclusion criteria: ( ) transfused pre-transplant with more than rbc units; ( ) incomplete hematological recovery; ( ) transfusion-dependence; ( ) serum ferritin ng/ml; ( ) normal creatinine value. the workup for other aetiologies resulted negative. all patients received an initial dose of deferasirox mg/kg/day, later adjusted according to side effects. all patients experienced an increase in hemoglobin levels, with a reduction in the frequency of rbc transfusions, followed by transfusion independence (median time: days from the first dose of deferasirox). in addition, it was promptly (median time: days) associated with hematological improvement, with sustained values and no further platelet support or growth factors administration. no relevant modifications with immunosuppressive or myelosuppressive drugs were made during deferasirox treatment. deferasirox was well tolerated. basing on our results, we think that deferasirox determined stimulatory, and/or depressive effects on hematopoiesis after allo-hsct. this clinical experience raises the possibility of a potential additive benefit on hematopoiesis after transplant following iron chelation therapy with oral deferasirox. further long term studies, in larger cohorts of patients are needed to confirm these data and to design an efficient strategy to reduce iron loading after transplant. disclosure of conflict of interest: none. supported in part by ail pesaro onlus. ( ) hypertension (n = ). all five patients had normal adamts levels and negative testing for shiga toxin. complement mutation genetic studies were obtained for four patients including genes (n = ) and genes (n = ) and were all negative. testing for complement pathway including c b- were obtained for patients and were normal. all five patients were treated with eculizumab with induction treatment at mg weekly × doses, followed by one dose of mg on the fifth week, and mg every weeks thereafter. patients had a recovery of hemoglobin and platelets and a rise in haptoglobin and a normalization of ldh within - weeks from the start of eculizumab. eculizumab was discontinued for of the patients without recurrence of their tma; they are now - months since the discontinuation of eculizumab. in summary, there is a subacute syndrome of thrombotic s microangiopathy that can occur late post transplant. this syndrome appears to be complement mediated as shown by its response to a terminal complement inhibitor. it also appears to be transient without recurrence following treatment discontinuation. disclosure of conflict of interest: none. transplant associated microangiopathy (tam) is a very severe complication occurring after allogeneic bone marrow transplantation (bmt), burdened by a high case-fatality rate. it is characterized by abnormal complement activation, triggered by various agents (calcineurin inhibitors, acute gvhd, infections) with subsequent endothelial damage. in the literature, cases of mutations in recipient complement genes are described, but none in donor dna. here we describe for the first time patients affected by tam, carrying mutations in donor complement genes. in our lab, we studied patients affected by tam; they were screened for cfh autoantibodies, adamts function and variants and macro-rearrangements in cfh (and related), cfi, cfb, cd , c , dgke, thbd genes and at-risk haplotype (cfh-h and mcpggaac) by means of next-generation sequencing (ngs) and multiplex ligationdependent probe amplification analysis (salsa mlpa p armd mix- ; mrc holland). ngs was used to sequence dna by haloplex kit (agilent) on a miseq (illumina) platform with -fold coverage of every target base. the bioinformatic analysis was performed using sophia genetics and the pathogenicity was assessed by means of in silico predictions (polyphen , sift, mutationtaster, aligngvgd). all of the predicted pathogenetic variants were confirmed using sanger sequencing. the same genetic screening was extended also to donor dna in all cases. the screening for known causes of tam revealed mutations in recipient complement genes in one case; no mutations were found neither in recipient nor in donor dna in two cases; instead, donor genetic alterations were found in patients whose characteristics are summarized in table s donor hematopoietic cells. in the three cases presented, tam was relatively delayed with respect to hsct, in particular in two cases ( months) and this timing is compatible with the concept of reticulo-endothelial 're-population' by donor cells of monocytic lineage, responsible for the production of regulatory proteins of the alternative pathway of the complement. we also underline the response to anti-c inhibition in the patients who were treated with eculizumab; this fact further supports the hypothesis that the disease was related to complement dysregulation. we therefore suggest that both the recipient and the donor should be screened for complement gene mutations, so that more cases could be identified and the pathogenesis of tam could be further clarified. among these we observed one autologous engraftment, one death due to septic shock before engraftment and two primary gf. we used a desensitization treatment based on plasma exchange procedures, intravenous immunoglobulin ( g/kg) and rituximab ( mg/sm) in patients. one of these patients (aml, haploidentical donor) had dsa against hla-b (mfi ). she experienced primary gf with increasing titles of dsa (maximum mfi ); so, on day , a second transplant from the same donor was performed after a desensitization treatment. a progressive decrease in dsa was documented (up to mfi ⩽ ). on day patient achieved neutrophil count over /μl and on day platelet count over /μl. the second patient (mds, haploidentical donor), instead, received a desensitization procedure before the first transplant. she had dsa against hla-a (mfi ), and after desensitization dsa levels decreased and reached . on day patient achieved neutrophil count over /μl and on day platelet count over /μl. dsa were detected in / of usct candidates ( %) and / of haplosct candidates ( %) and they were associated with failure to obtain allogeneic engrafment in cases. desensitization treatment achieved dsa clearance and engraftment in the patients in which it was performed, underlining the potential benefit of this procedure in the setting of hsct with dsa that has to be validated by prospective and controlled studies. disclosure of conflict of interest: none. early complications and late effects and quality of life at myeloma multiplex patients z trajkovska-anchevska, a pivkova, s genadieva-stavrich, l chadievski, z stojanoski, l chevreska and b georgievski university hematology clinic, skopje, macedonia the subject of this research is the quality of life at patients with myeloma multiplex at diagnosis and during therapy within - months. the research aims to analyze patients to be able to continue activities which will contribute for improving their quality of life as a priority task placed before the patient, his family, health institutions and social environment. this research was conducted at the university clinic for hematology skopje in the period from june to march . it covers patients infected with multiple myeloma, diagnosed and treated during this period. a total of patients analyzed, using the eortc qlq c ver. . standardized questionnaires for hr quality of life that analyzed the physical, cognitive, emotional, personal and social functions related to the patients. it also analyzed and general health and quality of life. analysis of physical functioning at diagnosis is . during treatment . , significantly improved. personal functioning at patients at the diagnosis is . , during therapy − . . analyzing emotional functioning in patients at diagnosis is . , during the therapy over . significantly improved. in examining the cognitive functioning is also a significant difference at diagnosis . , during treatment . . social functioning of the patients was . at the diagnosis; during the treatment grow to . . significant improvement was notices in these patients' symptoms like fatigue, nausea and vomiting, pain, dyspnea, insomnia, loss of appetite, constipation and diarrhea. the analysis of the financial difficulties of patients at diagnosis is . and . during treatment, meaning no significant difference in the time given. the analysis of the overall health and quality of life at patient has a value of . , and during therapy . . quality of life at patients with myeloma multiplex that makes the research group was significantly improved as a result of on time diagnosis and treatment with modern medicaments and the role of social worker with the application of certain social skills, continuous counseling, guidance and education for their reintegration in the community. installing the quality of life as a separate category and investigating the factors that affect its expression in the daily functioning of the patients within the changed framework of action, as like this example for malignant disease. the needs of clearly defined interactions patient illness and treatment, quality of life and specifying the segments where it can effectively act and improve in order to achieve positive progression towards improving the qualitative features of this category is a clear and primary objective that must be inserted into the current approaches to monitoring patients with malignant hematological diseases. acute graft-versus-host disease (agvhd) is a common and severe complication after allogeneic stem cell transplantation. since the current first-line treatment is based on treatment with systemic glucocorticoids (gc), steroid-induced hyperglycaemia develops frequently in patients with (agvhd) potentially impacting on their outcome. we performed a retrospective analysis on patients who received systemic gc for agvhd and thoroughly investigated the consequences of aberrant glucose metabolism. in particular, we focused on glucose parameters early after initiation of gc. with a median of (range: - ) blood glucose measurements during gc treatment, increasing mean, median and maximum glucose levels as well as the need for insulin treatment were associated with decreased overall survival (os) in simple and multiple survival analysis. early hyperglycaemia, as defined by mean blood glucose levels mg/dl during the first days of gc therapy, was also found to be highly associated with adverse outcome: in multivariate analysis, the hazard ratio (hr) for death was . ( % ci . - . , p = . ) in patients with early hyperglycaemia. while the risk of death due to relapse was not increased, the hr for death due to non-relapse mortality was . ( % ci . - . , p = . ) in a competing risk analysis. a score based on early hyperglycaemia and non-response to gc within days allowed the identification of three risk groups: patients with both risk factors had an inferior os at years of . % as compared to . % in patients with none. patients with one risk factor had a -year os rate of . % (p = . for trend). in this retrospective study, we identified early hyperglycaemia after gc initiation as a prominent factor predicting increased nonrelapse mortality in agvhd patients. in addition, a score based on early hyperglycaemia and lack of response to gc was highly predictive for overall survival in these patients. disclosure of conflict of interest: none. early toxicity because of infectious complications not relapse is the main cause of death after allogeneic transplantation in aplasia for patients with refractory or relapsed acute myeloid leukemia high-dose cytarabin was given in / pts with induction failure. the search for a stem cell donor was started immediately after results of high-risk cytogenetic, no achievement of bone marrow aplasia on day of induction therapy, or immediately after diagnosis of relapse. four patients had a related / donor, for patients a / matched unrelated donor was identified and patients received a transplant from a / unrelated donor. the interval between diagnosis of primary disease or relapse and tx was ( - ) months (mo) for both groups . in patients melphalan ( - mg/m ) was used to induce an aplasia before starting conditioning therapy. the interval between melphalan and conditioning therapy was ( - ) days. three pts started the conditioning therapy while in aplasia after previous chemotherapy. the conditioning therapy was of reduced intensity in all pts. and consisted of treosulfan ( g/m )/fludarabin(flu) in pts, tbi( gy)/flu in pts and busulfan( m/(kg)/flu in pts, respectively.atg was given to all pts with an unrelated donor. most pts ( / ) had a severe neutropenia with a median of . /nl ( . - . ) before starting melphalan because of refractory leukemia. after a median follow-up of ( - ) mo pts ( %) were alive without relapse. ( %) pts died because of a relapse after a median of ( - ) mo. the nonrelapse mortality was % ( / pts). most of these pts ( / , %) died because of infectious complications early after transplantation (med ; - mo). in pts graft versus host disease was the main cause of mortality. in this retrospective 'real-life' analysis, we showed that an early allogeneic transplantation is feasible for patients with primary refractory or relapsed aml. a reduced intensity conditioning after induction of aplasia with melphalan offers a chance of long-term relapse-free survival for about % of patients with an otherwise dismal prognosis. nrm is high, especially because of infectious complications early after transplantation, probably related to the long period of severe neutropenia. therefore, the focus has to be set on early recognition and intervention of infectious complications. disclosure of conflict of interest: none. recent evidence supports the effector role of complement activation in several types of thrombotic microangiopathythe atypical hemolytic uremic syndrome (ahus) as well as the transplantation-associated thrombotic microangiopathy (ta-tma). the blockade of the terminal complement complex formation by anti-c monoclonal antibody eculizumab provides an effective treatment option in severe and devastating cases of ta-tma. the experience with the use of eculizumab in this indication is slowly accumulating in the hsct community, however the published data originate from small case series or uncontrolled trials and sharing of emerging real-life observations may be valued. on case reports of two pediatric patients treated with eculizumab for ta-tma with very detailed followup of multiple complement parameters, including terminal complex sc b- and eculizumab drug levels we would like to demonstrate: ( ) achieving therapeutic levels of eculizumab ( μg/ml) may be unsuccessful even with initially intensified dosing interval. furthermore, we documented rapid eculizumab clearance from circulation which allowed only for short periods ( o h) of efficient drug levels during the weekly dosing. ( ) we did not observe tightly correlated sc b- and eculizumab levels within the dosing intervals; however the long-term sc b- formation suppression was achieved concomitantly with improved eculizumab levels and slowed drug clearance. ( ) classical complement pathway activity assay (ch ) may not reliably substitute for therapeutic efficiency monitoring in case of hypocomplementaemia due to protein losses (profound diarrhea, proteinuria, gi bleeding, catabolism). this holds true also for the alternative pathway activity which remained low during treatment in both patients. ( ) mycotic infections may represent serious therapy related risks in eculizumab treatment after hsct (both patients achieved control of complement activation after multiple doses of eculizumab, however suffered fatal infections subsequently). besides, we observed a significant increase in c a concentrations correlated with clinical onset of infection which invites for further investigation of this complement cascade product as early indicator of mycotic infection. in conclusion, we would like to highlight the great added value of timely available complement assay results, including sc b- and especially eculizumab drug level values-to be used together with detailed clinical parameters for directing effectively these highly personalized (and also costly) treatments. [p ] disclosure of conflict of interest: none. table . no difference in terms of drug-related adverse events was observed in the three patient cohorts with no reported serious adverse events. similar results were obtained performing two separate sub-analysis only for lymphoma or myeloma patients. despite the limitations due to the non-randomized nature of the study, from our data on a large cohort of patients s with a long-term follow-up biosimilar filgrastim (zarzio®) could be considered substantially equivalent in terms of efficacy and safety to lenograstim (myelostim®) and peg-filgrastim (neulasta®), when used for hematological recovery and febrile neutropenia prophylaxis after asct in adult patients with hematologic malignancies. disclosure of conflict of interest: none. we studied all adults who underwent allo-hsct during a -month period ( january to november ) in our center. a total of pts ( . %) received epag for pfg with thrombocytopenia. three pts were male, and three female. median age was years ( - ). the baseline diagnoses were: alm ( ), mds-raeb ( ), idiopathic myelofibrosis ( ), aa ( ), and cll ( ). three transplants were from family donor (all of them haplo-identical), and from unrelated donor (the three of them hla / ). sc source was pb in cases, and bm in . epag was started at mg/day and escalated each weeks to , and mg if platelet count was o × /l. we analysed the platelets, anc, and hgb at epag initiation and days after being with the maximum dose. median time between allo-hsct and eltrombopag initiation was days ( - ). median maximum dose used of epag was mg/day ( - ). median platelets, anc and hgb before starting treatment were × /l ( - ), × /l ( . - . ) and . g/dl ( . - . ), respectively. five patients ( %) were severely thrombocytopenic (platelet count ⩽ × /l), ( %) were anemic (hbg o g/dl), and ( %) were neutropenic (anc o . × /l). median platelets, anc and hgb at day + of maximum dose were: × /l ( - ), . × /l ( . - . ) and . g/dl ( . - . ), respectively. the thrombocytopenic pts ( %) responded to epag, with increases of , , , and × /l in the platelet count. three anemic pts ( %) responded and achieved increases of hgb of . , . and . g/dl. finally, the neutropenic pts ( . %) responded and achieved increases of anc of and × /l. at the moment of the analysis close, pts are at a median of + . months post-hsct ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and all but one (who died from a septic shock) are alive and outpatient. this survival is striking for subjects who develop a complication with such a high expected mortality as pfg. pgf is a life-threatening complication, relatively frequent after alternative donor hsct, whose treatment has been very disappointed. we report our experience in pts who developed pgf during the last years. epag induced responses in platelets in all pts of the studied group. bilineal and trilineal responses were also seen. in our opinion, prospective studies are warranted in order to confirm epag as a new efficient treatment of post-hsct poor graft function. disclosure of conflict of interest: none. s ing patients who developed es with an equal number of patients who did not between january and november . we analyzed variables such as cd + cells per kg infused, use of granulocyte colony-stimulating factor (csf-g) and engraftment day. analytical data, including baseline and maximum determination of serum glutamic oxaloacetic transaminase (got) and glutamic pyruvic transaminase (gpt), c-reactive protein (crp) and procalcitonin (pct), as well as clinical data fever, weight gain, digestive and respiratory symptoms, pulmonary infiltrates were analyzed. sixty-eight patients were women. median age was years old (range: - ). patients were conditioned with beam ( %), melphalan mg/m ( %) and bcnu-tt ( %). nineteen patients developed es in our series, which correspond to eight percent of all asct. case and control groups were matched according to age, sex, diagnosis and conditioning regimen. the most prevalent baseline disease in the group with es was myeloma ( . %), followed by mantle cell lymphoma ( . %). all patients who developed es had fever, % skin rash, % respiratory symptoms, % pulmonary infiltrate an % digestive symptoms. a summary of the comparison of data analyzed in subgroups is shown in table . we found significant difference in the percentage of weight gain (p = . ), increase of tgo (p = . ), increase of tgp (p = . ) and increase the number of cd + cells per kg infused (p = . ), we found an inverse correlation between the number of cd + cells per kg infused and incidence of es. however, in terms of post-transplant csf therapy (p = . ) and crp and pct valor (p = . and p = . , respectively) we did not find significantly difference to develop es. in our series, weight gain and tgo and tgp rise were risk factors for es development. therefore, we should be aware of es in patients who develop fever, elevated liver enzymes and weight gain during graft phase. we did not find a significant difference in crp and pct suggested in other studies. further studies are required to better characterize risk factors of es development. busulphan-based ( ), melphalan-based ( ), beam ( ), tbi-based ( ), and others ( ). weight at hospital discharge was significantly lower than at admission ( . % in allo-hsct, and . % in auto-hsct). weight at day + was also significantly decreased compared with the admission ( . % in allo-hsct, and . % in auto-hsct). weight at day + was lower than the ideal for their sex and height in the allo-hsct setting. contrarily, among the patients undergoing auto-hsct, the weight at day + remained higher than the ideal for their sex and height in a high proportion of cases. regarding serum albumin, it was significantly decreased at discharge ( % in allo-hsct, and . % in auto-hsct), but recovered values similar to admission at day + . in the auto-hsct setting, prealbumin levels were significantly reduced at discharge ( %), and in lower proportion at day + ( %), compared with admission values. in the allo-hsct patients, prealbumin levels were significantly reduced at discharge ( %), but had been recovered at day + , compared with admission values. disclosure of conflict of interest: none. recently, blood and marrow transplant clinical trials network has proposed a composite endpoint: gvhd-free, relapse-free survival (grfs) for hsct outcomes. this endpoint includes as event: iii-iv acute gvhd (agvhd), relapse, death or chronic gvhd (cgvhd) requiring systemic treatment. in the last embt annual meeting a redefinition of this endpoint was proposed changing cgvhd event from those patients with cgvhd requiring systemic treatment (the original one) to those with just severe cgvhd (the redefined one). we retrospectively analysed patients consecutively transplanted ( - ) excluding non-malignant diseases, second allo-sct and those o years old age. we had generated two composite endpoints: in both iii-iv agvhd, relapse or death were considerated events but we defined grfs as the one with cgvhd event including those who required systemic treatment (as the original one) and in grfs just those with severe cgvhd (the ebmt redefined one). the median age was years ( - ) and % ( ) were males. other characteristics of patients are resumed in table . with a median follow up for patients alive of months , the median estimated survival in months and the % at + year and + years was: months, % and % overall survival (os); months, % and % event free survival (efs); months, % and % grfs ; months, % and % grfs . ( %) and ( %) hadn't any event in grfs and in grfs , respectively. in grfs , event's incidence was: ( %) for iii-iv agvhd, ( %) for cgvhd, ( %) for relapse and ( %) for death; in grfs was ( %), ( %), ( %) and ( %), respectively. considering those patients with cgvhd as event in grfs , of them hadn't the event as cgvhd at the same time in grfs (since they had cgvhd requiring systemic treatment but not severe cgvhd). for these patients, the alternative event in grfs was: without any event, relapsed and died. in the multivariate, the factors associated with better outcomes were: in grfs early ebmt stage (p o . with early as reference; intermediate p factor with more impact in both, but it is interesting to point it out that haploidentical donor had an advantage in grfs . these results are being validated in a large series and the definitive results will be available at the moment of the meeting congress. [p ] disclosure of conflict of interest: none. steroid refractory acute graft-versus-host disease (gvhd) remains a major complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). affected patients have a very poor prognosis. gvhd has been associated with transplant-associated thrombotic microangiopathy (ta-tam). endothelial damage mediated by radiation, viral reactivation, drug exposure or alloreactivity results in exposure of subendothelial collagen, activation of coagulation and small vessel occlusion to a degree that results in organ failure. complement is thought to be a major mediator of endothelial damage. although a consensus exists about the exceedingly high morbidity and mortality of ta-tam and diagnostic criteria have been converging to a consensus, no biomarkers to diagnose tam and predict outcome have been established. we hypothesize that a ta-tma, related to dysregulation of the alternative complement pathway correlates with organ damage. a retrospective analysis of consecutive patients with hematological malignancies receiving an allo-hsct at the university hospital basel in the period from to was performed. data on the occurrence, risk factors and outcome of patients with ta-tma and the correlation with acute gvhd was collected. available biopsies of organs suspected to be affected by tam and/or gvhd will be performed. routine bone marrow biopsies for histological, immunohistochemical signs of ta-tam and complement activation will be analyzed. serum samples will be used to characterize markers of complement activation using plasma levels of c b- and c b- deposition in tissues biopsies. patients (aml n = ; all n = ; mds/mpn n = ; lymphoid neoplasm n = ; plasma cell disorder n = ; bone marrow failure n = ) underwent myeloablative (n = ) and non-myeloablative (n = ) allo-hsct at a median age of years (range: - years). forty-eight ( . %) patients matched the established diagnostic criteria for tam (increased ldh, platelet count o g/l or o % of normal baseline, schistocytes per high power field, creatinine increase). the median time to onset of tam was days post-transplant (range: - days). subjects with ta-tam had significantly higher -year nonrelapse mortality compared to those without ( . % vs . %, p o . ). grades - agvhd and cytomegalovirus viremia were independent risk factors for ta-tam, and serum ldh level u/l as well as arterial hypertension were early signs of ta-tma occurrence. patients with clinically relevant agvhd (⩾ grade ) had more ta-tam than patients without agvhd ( % vs %; po . ). tam correlated with agvhd severity; the higher the agvhd grade, the more the patients who suffered from tam. allo-hsct recipients with grades - agvhd or cytomegalovirus viremia should be closely monitored for the presence of ta-tma. at the meeting first results of histological, immunohistochemical and complement activation analyses will be presented. disclosure of conflict of interest: none. hemorrhagic cystitis (hc) after stem cell transplantation (sct) can cause significant morbidity and prolonged hospitalization. early bleeding occurs almost exclusively when using cyclophosphamide (cy) ( - % of cases), while late onset hc are classically attributed to bkv infection, and occurs up to % of patients (pts) receiving myeloablative haplo-sct who had positive bk viruria ( , ). we retrospectively studied hc cases among pts submitted to haplo-hsct in our department. thirty-eight pts receiving an haplo-sct with post-transplant cy (pt-cy) were included (table ) . prophylaxis for cy included hyperhydratation ( l/m of . % saline) and mesna administration ( mg for each mg of cy/daily divided into three doses). hematuria was graded as follows: grade i, microscopic; grade ii, macroscopic; grade iii, with clots; and grade iv, leading to urinary retention or requiring surgical intervention ( ). pts with hc and clots were treated with continuous bladder irrigation. twenty-three pts ( . %) developed hc at a median of . days post-sct (range: - ). clinical severity was grade i in cases ( . %), grade ii in cases ( . %), grade iii in cases ( . %) and grade iv in cases ( . %). at the onset of hc diagnosis, bk viruria was investigated in / pts. five pts ( . %) had bkv negative (bkv − ) hc and pts ( . %) bkv positive (bkv+) hc. bkv-hc occurred after a median of days (range: - ) while bkv+ hc after . days (range: - ), respectively (p = . ). among bkv+ pts, received iv cidofovir mg/kg once a week for weeks and then once every weeks. median number of administrations was (range: - ). oral probenecid was given at the dose of g h before and g and h after cidofovir administration. two pts obtained a complete response (cr) after and days, respectively, one patient reached a partial response after days and one pt failed to obtain a response. no pts developed renal toxicity during treatment. one pt received ganciclovir for concurrent cmv viremia and bkv+ hc resolved in days. three patients did not receive any treatment for mild or asymptomatic cystitis. all of them achieved remission after a median of days from the onset (range: - .) among bkv-hc, pts obtained spontaneous resolution after a median of days (range: - ), while two pts died early after sct. finally, among pts for whom bk viruria was not available, a remission was reached in of them after a median of . days (range: - ), while pts died early after sct. in our cohort of pts, hc occurrence was of . % and bkv was responsible for the . % of cases. contrary to its high incidence, hc showed a relative benign course, with an overall remission rate of . %, regardless of treatment. finally, we found a trend for a longer interval between sct and hc onset in pts with bkv+ hc, as compared to cy-related hc (p = . ). sos is a rare and serious complication of hematopoietic stem cell transplantation (hsct). it is diagnosed using the modified baltimore criteria of hyperbilirubinemia, weight gain or ascites % over baseline, hepatomegaly or right upper quadrant pain of liver origin. only defibrotide has been approved for the treatment of veno-oclusive disease. hdmp has been described as effective sos therapy in a few case series ( , ). we describe our experience of treating adult sos using hdmp. objective is to retrospectively analyze the treatment efficacy and overall survival of patients diagnosed with sos after hsct and treated with hdmp. we used vilnius university hospital data base to identify patients diagnosed with sos under baltimore criteria and treated with hdmp over - period. patient demographics, transplant and clinical data, response, survival (kaplan-meier survival analysis) and hdmp infusion related complications were analyzed. we identified patients ( males) of whom had had allogeneic hsct ( reduced intensity conditioning) and one had received a double autologous hsct. sos was diagnosed on the median day + (+ to + days). the median bilirubin value was . μmol/l ( . - μmol/l). all patients had liver enlargement of median mm ( - mm) on ultrasound. two patients had normal bilirubin values but displayed the remaining signs and symptoms of sos at diagnosis. patients received intravenous methylprednisolone mg/m every h for days. none received defibrotide. seven ( %) patients responded on median day + (+ to + days) after the start of hdmp. four responded by decrease in serum bilirubin by % and resolution of symptoms without the need of further treatment. the remaining three responders received maintenance treatment after one course of hdmp with reduced doses of methylprednisolone until resolution of symptoms. four patients failed to respond and died of multiorgan failure on median day + (+ to + ). the median observation time was months ( - months). the median overall survival for the sos group was months (range: - ) and it was months among the responders. no adverse reactions related to hdmp infusion were observed. hdmp therapy in adult sos results in clinically relevant response rate. further prospective trials are required to assess hdmp efficacy in comparison to defibrotide or as add on therapy. prevalence of hypertension (ht) in general pediatric population is~ %, while in children treated with hematopoietic stem cell transplantation (hsct) it is up to %. we assessed factors contributing to the development of ht in children treated with hsct and usefulness of ambulatory blood pressure monitoring (abpm) in this population of patients. the study included children ( boys, girls; mean age . years) treated with hsct for neoplasms (n = ; %) or non-neoplastic disorders (n = ; %). control group included children ( boys, girls; mean age years). abpm measurements (spacelab device) were performed before hsct and after a mean of months after hsct (in of the children). blood samples were collected from children treated with hsct and all controls. total rna extraction was performed and microarray analysis was conducted using genechip human gene . st arrays (affymetrix). in patients after hsct no antihypertensive treatment was used. mean systolic blood pressures (sbp) before and after hsct did not differ significantly from the control group. mean diastolic blood pressures (dbp) before and after hsct were . ± . mm hg and . ± . mm hg, respectively, and mean dbp percentiles were . ± . and . ± . , respectively; the differences between the study group and the control group were significantly higher before hsct. mean -hour arterial pressure (map) percentiles were . ± . and . ± . , respectively; the differences between the study group and the control group were significantly higher before hsct. before hsct and after the procedure, the european society of hypertension criteria for high normal blood pressure (bp) and ht were fulfilled in %/ % patients and %/ % patients, respectively. nocturnal bp decrease o % was found in %/ % patients and % nocturnal bp decrease in %/ % patients, respectively. in the control group o % nocturnal bp decrease was found in % of children and % nocturnal bp decrease in % of children. when the groups of patients before and after hsct were compared, highly significant differences were found in gene expression levels for mthfr ( in children referred for hsct a trend towards higher bp values was seen. in children assessed months after hsct more abnormalities in nocturnal bp measurements were seen, which may be a predictor of ht. in children treated with hsct significant differences in the expression of ht-related genes were found. abpm was useful in bp monitoring in children treated with hsct. hypothyroidism may complicate of allogeneic hematopoietic stem cell transplantation (allo-hsct); risk factors are analysed. we studied patients with aml who underwent an allo-hsct between and with different conditioning regimens (myeloablative, reduced-intensity, chemotherapybased, total body irradiation-based). thyroid stimulating hormone (tsh) and free thyroxin levels (ft ) were available in patients before and after allo-hsct. median age at transplantation (n = ) was years (iqr - ), ( . %) were female and overall mortality was . % (n = ) ( table ) ursodeoxycholic acid (udca) has been shown to have a protective effect in the liver complications after allogeneic stem cell transplantation (allo-sct), but it also has other immunomodulatory effects; it has been described also a potential benefice as graft-versus-host disease (gvhd) protection. we retrospectively analysed patients consecutively transplanted between - excluding second allo-sct and those o years old. we analysed the differences between those with and without prophylactic udca using spps v . results: the median age was years ( - ) and % ( ) were males. other patient characteristics are resumed in table objective of study was to evaluate the impact of disease status on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-hsct) in the treatment of patients with refractory and relapsed acute lymphoblastic leukemia(all). patients with refractory and relapsed all, including cases in advanced stage (nonremission, nr) and cases in more than or equal to second complete remission(⩾ cr ), received allo-hsct after myeloablative conditioning regimen in our department. results: patients engrafted successfully. the transplantation-related mortality (trm) rate of nr and ⩾ cr was . % vs . % (p = . ). the incidence of agvhd was . % vs . % (p = . ), including . % vs . % (p = . ) with mild (grade i-ii) and . % vs . % (p = . ) with severe (grade iii-iv) agvhd. the incidence of cgvhd was similar also( . % vs . %, p = . ). with a median follow-up of ( . - . ) months, the cumulative relapse rate of nr and ⩾ cr was % vs . %(p = . ), respectively. the estimated year overall survival (os) and year leukemia-free survival (lfs) rate were . % vs . % (p = . ) and . % vs . % (p = . ), respectively. multivariate analysis results showed that cgvhd was independent favorable risk factor for os and lfs of r/r all. for relapsed patients, os was significantly better with first cr duration months and time to transplant ⩽ months. alio-hsct is an effective salvage treatment option for patients with refractory and relapsed all. our retrospective analysis showed that r/r all with different status prior transplant had similar outcome post transplantation. disclosure of conflict of interest: none. the deleterious effect of intensive care unit (icu) admission during hematopoietic stem cell transplantation (sct) on patient survival is well established. however, it is unknown whether admission into the icu during the chemotherapy for the underlying disease has any impact on survival after sct. we reviewed patients who had received a first sct between the years and in our institution, and we compared the overall survival (os), relapse incidence (ri) and non-relapse mortality (nrm) between patients who required icu admission during the chemotherapy prior to the sct (icu group) with matched patients ( : ) who did not need it (no-icu group). sixty-six patients were included, of them in the icu group and in the no-icu group. as shown in table , the main clinic-biologic variables and the sct procedure were comparable between the patient groups. the causes of icu admission for the icu group patients were: ( %) respiratory failure, ( %) septic shock, ( %) neurological disturbance, ( %) post-surgery and ( %) tumor lysis syndrome. seventeen patients ( %) needed mechanical ventilation. the median time between icu admission and the sct procedure was days (range: - ), and the median days of icu stay were . . with a median follow-up after sct of . years ( . - . ) for the icu group and . ( . - . ) for the no-icu group, the year os (ic %) probabilities were % ( - %) and % ( - %) in the icu and no-icu patients (p = . ), the -yr probabilities of relapse were % ( - %) and % ( - %)(p = . ) and the -yr probabilities of nrm were % ( - %) and % ( - %)(p = . ), respectively. there were no differences in either os, ri or nrm between icu and no-icu in the allogeneic or autologous subgroups considered separately. at the moment of the study, s ( %) of icu and ( %) of no-icu group had died. the causes of death in the icu group were: relapse in ( %), infection in ( %), gvhd in ( %) and gvhd plus infection in ( %). the causes of death in the no-icu group were: relapse in ( . %), infection in ( . %), gvhd in ( . %), gvhd plus infection in ( . %) and veno-occlusive disease and secondary malignancy, one each ( . %). in this series, admission to the icu before sct did not have an impact on outcomes after sct. these results suggest that these patients benefit from this treatment as much as the other patients, without expecting worse outcomes as a result of a previous icu admission. supported in part by grants rd / / (rticc, fejer), pi / , instituto carlos iii, and sgr (gre), spain. disclosure of conflict of interest: none. autologous stem cell transplant (asct) is a well established treatment for several haematologic and non haematologic malignancies, either as front-line or rescue therapy. however it is associated with toxicity and complications which might lead to treatment-related mortality (trm). although decrease in trm has been reported, data about the precise reduction and detailed analysis of causes of mortality throughout years are scanty. the aim of this study was to evaluate early trm and its causes in patients who underwent an asct in a single center along the last three decades. data of all consecutive adults ( years old) asct recipients were prospectively collected at a single center from december to august and then retrospectively analysed. trm was defined as mortality happened into the days post asct or during conditioning treatment due to any cause except relapse or progression of main diagnosis. demographic characteristics, diagnosis, conditioning regimen and cause of death were analysed. data were compared for two periods: from december to december and from january to august . a total of patients were included, median age was years ( - ) and . % were male. diagnoses were: lymphoma (n = ), multiple myeloma (mm) (n = ), acute myeloid leukaemia (aml) (n = ), amyloidosis (al) (n = ), acute lymphoblastic leukaemia (all) (n = ), solid tumours (including breast cancer and germ-cell tumours) (n = ), chronic myeloid leukemia (cml) (n = ), thrombotic thrombocytopenic purpura (ttp) (n = ) and autoimmune disease (n = ). the most frequent indication for asct was lymphoma ( . %) and mm ( . %). twenty patients died within d from asct (trm). demographic characteristics and causes of death for this patients are shown in table . the cumulative incidence of trm at day + was . % ( % ci . - . ). comparing both periods, trm cumulative incidence was . % ( % ci . - . ) in first period ( - ) vs . % ( % ci . - . ) in last period ( - ) po . . (figure ). according to main diagnosis trm cumulative incidence was higher in patients diagnosed with solid tumour . % ( % ci . - . ) and al . % ( % ci . - . ) followed by acute leukaemia (aml/all) . % ( % ci . - . ), mm . % ( % ci . - . ) and lymphoma . % ( % ci . - . ) p o . (figure ). sepsis ( %) was the main cause of death in both periods of time, and the only one cause of death in the last period. the second cause was sinusoidal obstruction syndrome (sos/vod) ( %), which only appeared in the first period. this study shows a low incidence of trm in asct recipients, with a significant decrease in the last period ( - ), despite the higher risk in some groups of patients such as those with amyloidosis and solid tumours. in our experience, infection is the main cause of early death in asct recipients and sos/vod has disappeared in last years as a cause of early transplant related mortality. disclosure of conflict of interest: none. incidence and risk factors for hepatic sinusoidal obstruction syndrome after allogeneic hematopoietic stem cell transplantation: a retrospective multicenter study of turkish hematology research and education group (threg) hepatic sinusoidal obstruction syndrome (hsos) is a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). the mean incidence of hsos was found to be . % ( - %) in the literature. we examined the incidence and risk factors for hsos after allo-sct. eight centers from turkey were enrolled in the study. we retrospectively evaluated the medical records of patients who were treated with allo-sct between january and december . a baltimore criterion was used for assessment of hsos. two hundred eighty three ( %) of patients who were treated with prophylaxis with defibrotide alone or one or more of the n-acetylcysteine, diuretics and heparin used defibrotide ( - mg/kg/day). the study included patients ( males/ females) with median age of ( - ) years. the demographic and clinical characteristics of patients were summarized in table . the incidence of hsos was . % ( ). prophylaxis for hsos was used in ( . %) of patients, who developed hsos. defibrotide as prophylaxis was received by of ( %) of patients. hsos developed in a median of ( - ) days after stem cell infusion. seventy-five ( . %) of patients who developed hsos had infection at the time of diagnosis. forty-five of them had ascites, had hepatomegaly and, had weight gain. seventy-two ( . %) of patients with hsos were treated with defibrotide after diagnosis. the median time of starting defibrotide in these patients was ( - ) days. thirty-six ( . %) of patients with hsos recovered completely and forty-seven ( . %) of them died as a result of multi organ failure. the incidence of hsosrelated mortality in allo-hsct cohort was found to be . %. in univariate analysis, statistically significant associations were not found between hsos incidence and age/sex of recipient, type of conditioning regimen, stem cell source and type of gvhd prophylaxis. on the other hand primary diagnosis of myelofibrosis, donor type, engraftment status and prophylaxis for hsos were significantly associated with hsos development. hsos prophylaxis was significantly decreased hsosassociated mortality (p = . ). hsos still remains a serious life-threatening complication of allo-sct. although the incidence is low, hsos is associated with increased -day nonrelapse mortality. hsos prophylaxis especially with defibrotide, seems to reduce hsos associated mortality in high risk patients. [p ] disclosure of conflict of interest: none. hemorrhagic cystitis (hc) is a serious complication occurring after allogenic hematopoietic stem cell transplantation (hsct) more frequent on haploidentical (haplo) hsct, with an incidence of - % associated mainly with the effect of cytotoxic agents such as cyclophosphamyde (cy). the conditioning regimen, bkpyv infection and graft versus host disease have an implication in the incidence. other authors related the reactivation of cmv and a previous transplantation as risk factors to hc development . with this study we aim to describe the hc incidence and risk factors in all haplo-hsct performed in the canary islands. we analyzed all consecutive haplo-hsct from family donors performed at our hospital between and . the conditioning regimen used for the transplant was the hopkins haplo protocol with high dose cy ( mg/kg on days and ) posttransplantation (ptcy). we used as hc prophylaxis intense hydratation on the cy administration day and the following h (using bladder wash only in patient with cardiac dysfunction) and perfused mesna at % of cy dose beginning min before the cy administration on pts and at % of the last dose at , and h on all pts. we used spss v. to determine the cumulative incidence (ci) of hc. we performed haplo-hsct, of which were males ( was transplanted times) and were women. the mean age was (range: - ). the pts presented the following diagnosis: aml ( ), all ( ), eh ( ), nhl ( ), am ( ). % of pts received the haplo-hsct in remission, % with refractory disease and % of pts did not receive previous treatment. pts developed hc ( . % ci at day + ) (figure a ) with a median time from haplo-hsct to onset of days (range: - ), ( %) was grade i, ( %) grade ii and ( %) grade iv. the grade i case did not received the mesna infusion like most of the other pts. no pts died due to hc and all cases resolved without sequelae. pts received cy pre-and post-transplant and only pts received ptcy. the ci at day + for the pts with ptcy was . % and for cy preand post-transplant . % (figure b) . we found no statistically significant difference on the ci of hc between these two groups. the development of hc was related to cy in patient, who suffered from this complication on the second and third haplo-hsct. for the rest of the pts (after day + ) the hc was related to bkpyv infection, as a consequence of the immunosuppression state of the patient, we also observed all these pts had positive serum viral load for cmv. the incidence of hc associated to post-hsct high cy dose in our series is % lower than other ones. most of them on grade or and without mortality associated. the risk of hc is high, particularly in the setting of highly pre-treated patients (especially those undergoing a nd transplant). the development of hc after day + is evidently associated to bkpyv as a contributing factor for continuous inflammation and cmv reactivation (as an immunosuppression marker). in our study, hc did not have an impact on mortality of high-risk patients after haplo-hsct. the hc remains frequent with a high morbidity in particular when it is severe, often causing prolonged hospitalization and resource use. we need further studies to recognize the at-risk population early. [p ] ta-tam is not a rare post-transplant complication and it is potentially fatal. in survivors, it was often associated with longterm morbidity and chronic organ damage, mostly to the kidney with poor renal prognosis. our retrospective study showed ta-tam associated risk factors included t reg haplo hsct as the incidence was highest in this group, tbi-based conditioning or tbi based conditioning plus cyclophosphamide. although acute gvhd and infection were associated with ta-tam in retrospective studies, no association emerged between acute gvhd or infection preceding diagnosis in our series of patients. in order to prevent ta-tam we need to understand its underlying biological mechanism so we are investigating its pathogenesis by means of cytokine assays, histology and murine models. disclosure of conflict of interest: none. mortality in children requiring invasive mechanical ventilation (imv) after allogeneic hematopoietic stem cell transplantation (hsct) is known to be high. little is known about the longterm outcome of those who survive imv. we therefore reviewed the medical records of children who survived s imv after they had received a hsct between and in the two pediatric hsct centers in the netherlands. retrospective multi-center cohort study in two university hospitals that perform all pediatric hscts in the netherlands. long-term survival of hsct recipients who had received imv was assessed. health status was reviewed more in detail for those who were still alive years after discharge from the pediatric intensive care unit (picu). in the absence of standardized use of quality of life questionnaires, health status was expressed as the number of affected domains (cardio-respiratory, motor and miscellaneous, regardless of the degree of dysfunction) and level of education. health status was categorized as follows: no health problems when all four domains were normal; mild health problem when there was an abnormal score in one of the four domains; moderate health problems when there was an abnormal score in two domains; severe health problem when there were abnormal scores in three or all four domains. between january and december , patients underwent a hsct in the two study centers together. a total of hsct recipients received imv within year after hsct ( % of all hsct recipients). median time between hsct and picu admission was days (iqr - days). the most common indication to start imv was respiratory failure ( %). median duration of imv was days (iqr - days). patients ( %) died during their picu admission. of the patients who were discharged alive from picu, patients were still alive years after picu discharge ( % of those who survived picu admission). health status of these long-term survivors was assessed in december by hospital database review, using the most recent hospital contact. follow-up time varied from to years (median . years) after picu discharge. two patients ( %) had no health impairment, eight patients ( %) had mild health problems, five patients ( %) had moderate health problems, and nine patients ( %) had severe health related problems. very little is known about long-term mortality and morbidity of hsct recipients who survived imv. survival of picu treatment in pediatric hsct recipients is limited. however, long-term outcome of those who survive picu treatment seems promising: a considerable proportion of them still is alive years later without obvious sequelae. this is the first study which assessed long-term outcome of imv after hsct. further studies in this population are urgently required to counsel parents and to optimize quality of life outcomes in these children. disclosure of conflict of interest: none. long-term surveillance data support lack of increase in mortality or cancer risk in brincidofovir clinical trial participants m morrison, k fitzgerald , t brundage, a harrison and wg nichols chimerix brincidofovir (bcv) is an orally bioavailable lipid conjugate of cidofovir (cdv), with broad-spectrum activity against doublestranded dna viruses, including cytomegalovirus (cmv), adenoviruses (adv), polyomaviruses (bk and jc viruses), and orthopoxviruses. bcv is being evaluated for prevention of cmv and other dna viruses in high-risk hematopoietic cell transplant (hct) recipients, and for the treatment of serious adv infection. bcv is also being developed for the treatment of smallpox under the us fda's animal efficacy rule. because bcv, cdv, and other marketed nucleoside analogs are reported to be carcinogenic in rodents, a registry was established to evaluate the long-term safety of bcv in subjects who have participated in bcv clinical studies. to date, the registry includes prior participants from study (a placebo (pbo)-controlled study of bcv for cmv prevention) and study (a single-arm study of bcv for adv treatment). subjects are encouraged to consent for long-term follow-up in the registry following participation in bcv clinical studies. registry participants are followed at -month intervals for a minimum of years from the time of completion of the parent study. development of malignancies (new or relapsed), lifethreatening or fatal adverse events (aes) assessed as potentially related to bcv, and subjects' vital status are collected. a total of subjects were enrolled in the parent studies ( bcv and pbo from study , bcv from study ). of these, are enrolled in the registry as of october ( bcv and pbo from study , bcv from study ). bcv recipients in the registry are % male, % white, with a median age of ( o - ) years, similar to the bcv recipients in the parent studies. the median duration of follow-up is ( - ) months, with % of subjects continuing in follow-up at the time of analysis. all-cause mortality from the time of first dose in the parent study through current registry follow-up is % for bcv vs % for pbo (p = . ) in study , and % for bcv in study . all-cause mortality in the registry since completion of the parent study is % bcv vs % pbo for subjects from study (p = . ) and % bcv for subjects from study (figure ) . the incidence of a new malignancy in registry subjects from study is % bcv vs % pbo (p = . ), and the incidence of relapsed primary malignancy is % bcv vs % pbo (p = . ). in registry subjects from study , % developed a new malignancy and % had a relapse of the primary malignancy. no bcv-related life-threatening or fatal aes have been reported to date in the registry. registry data collected to date support no increase in late mortality or increased risk for carcinogenicity in patients treated with bcv. long-term surveillance for cancer risk and other drivers of mortality is important for novel compounds undergoing development in hct and other immunocompromised patient populations, with high background risk for these outcomes. [p ] disclosure of conflict of interest: all authors of this abstract are employees and stockholders of chimerix. hematopoietic stem cell transplantation (hsct) is a medical procedure that allows the cure of many paediatric diseases. it has been described an increased risk of new malignancies in this population and it represents an important cause of late mortality. we analyzed the late evolution of patients submitted at pediatric age to hematopoietic transplantation (hsct) (allogeneic or autologous) in santa creu i sant pau hospital between and . a total of hsct was analyzed. it has been calculated the cumulative incidence of secondary malignancies at years of follow-up. it has been done univariate and multivariate analysis of risk factors through χ -test and binary logistic regression method (odds s ratio, or). it has been studied the relative risk (rr) for new malignancies through comparison of observed cases in our cohort with the expected cases in the general population. we observed cases of secondary malignancies with a cumulative incidence of % at years, % at years and % at years of follow-up. the risk was higher of expected in general population for each tumor type and in the different range of age, being the rr for malignancies in our cohort of . at years of follow-up. solid tumors were the most prevalent malignancies ( out of cases). the median time of latency from hsct to diagnosis of malignancy was years ( - years) . the thyroid tumors were the later ones and hematologic malignancies the earliest to be developed. chronic graft versus host disease was a statistically significant risk factor in univariate (or = ; p = . ) and multivariate analysis (or = . ; p = . ). total body irradiation of conditioning was a significant risk factor only in multivariate analysis (or = . ; p = . ). previous radiotherapy was a significant risk factor only in univariate analysis (or = . ; p = . ). mortality was % ( out of ) between patients with a new malignancy and it was the cause of death for all the cases. we observed an incidence of secondary malignancies after hsct of . % that is significantly higher compared to the expected in the general population (p = . ). the factors that have been related to an increased risk were chronic gvhd, tbi and previous radiotherapy. microalbuminuria defined, as urinary albumin: creatinine ratio (acr) of - mg albumin/g creatinine is a marker of endothelial dysfunction and inflammation. in general populations albuminuria predicts development of chronic kidney disease (ckd) and cardiovascular disease ( ). in the general population microalbuminuria is associated with the metabolic syndrome ( ) . in patients with hypertension, diabetes and the critically ill, it is a marker of adverse events and poor outcomes. following hematopoietic cell transplant (hct), microalbuminuria at day was associated with a four-fold increased risk of chronic kidney disease (ckd) at year in a single centre study; macroalbuminuria at day was associated with a . -fold increased risk of non-relapse mortality ( ) . international guidelines for adult and children survivors of hct recommend that proteinuria is assessed at least annually ( , ) . there is a paucity of data on the prevalence and implications of micro and macroalbuminuria in long-term survivors ( years) of adult hct, however. this was a single-centre retrospective study conducted in patients attending a dedicated clinic for long-term (minimum years) survivors of hct. we investigated prevalence of albuminuria and its association with renal disease, cardiac disease and the metabolic syndrome. of patients, were treated for acute leukemia, for aplastic anaemia and for cml. the median follow up time was years (range: - years) and the median age at follow up was years (range: - years). for / urinalysis was normal (group a) and ( %) had microalbuminuria (group b). none had macroalbuminuria. group b were significantly more likely to have ckd grade - (egfr o ) compared to those in group a (p = . ). group b patients were significantly more likely to have diabetes or impaired glucose tolerance / ( %) vs / ( %) in group a (p = . ). group b patients were also significantly more likely to have dyslipidaemia (p = . ) with / ( %) affected vs / ( %) in group a. cardiac disease and hypertension were more frequent in group b, / ( %) and / ( %), respectively vs group a / ( %) and / ( %) but these data were not statistically significant. the more features of the metabolic syndrome present, however, (elevated hba c, /glucose, dyslipidaemia, hypertension) the more likely a patient was to have microalbuminuria (p = . ). our data demonstrates that microalbuminuria is a frequent finding in long term survivors of hct. patients with microalbuminuria are more likely to have ckd grade or below. they are also more likely to have diabetes and dyslipidaemia. as this was a retrospective study we are not in a position to comment on whether microalbuminuria is predictive of the development of renal disease, metabolic syndrome or cardiovascular disease in this group of patients. this warrants further study as intervention, for example with ace inhibitors, may have the potential to reduce morbidity. the purpose of the study is the improvement of transplantation techniques and supportive care lead to an increasing number of long-term survivors after allogeneic hematopoietic stem cell transplantation (ahsct). recipients of ahsct have a higher prevalence of cardiovascular risk factors. ambulatory blood pressure measurement (abpm) is the 'gold standard' to diagnose arterial hypertension (ht). the prevalence and treatment control of ht by abpm is unknown in ahsct patients (pts). this prospective single center study at university hospital basel included all pts ⩾ year after ahsct in complete hematological remission during annual follow-up consultation. office blood pressure (obp) was measured on both arms after minutes rest. abpm by noninvasive continuous bp monitoring (pulse transit time method) was performed on the same day. ht was defined as obp ⩾ / mm hg, mean systolic bp ⩾ mm hg on abpm s (bp ) and/or current use of antihypertensive drugs. pts ( % female) were included with median age of years (range: - ) and years (range: - ) after transplantation. ( %) pts received total body irradiation-based conditioning, ( %) pts had chronic graft-versus-host disease, and ( %) required immunosuppression. mean bmi (kg/m ) (± sd) was ± , with ( %) pts . twenty-seven ( . %) pts were current smokers. fourty-three ( %) pts had chronic kidney disease (egfr o ml/min/ . m ) and ( %) diabetes. ( %) pts were on antihypertensive drugs consisting of ace/at-ii-inhibitors in ( %), calciumchannel blockers in ( %), beta-blockers in ( %) and diuretics in ( %) pts. thirty-nine ( %) pts were on ⩾ drugs. among our cohort ( %) pts were normotensive without antihypertensive treatment (mean age ± years, % female and mean bp (systolic/ diastolic bp) ± / ± mm hg). ( %) pts were hypertensive and/or on antihypertensive treatment. untreated ht was diagnosed in ( %) pts (mean age ± years, % female and mean bp of ± / ± mm hg), including ( %) with white-coat hypertension and ( %) masked hypertension (normal obp, high abpm). in the group of pts with current antihypertensive medication / ( %) were controlled (mean age ± years, % female, and mean bp ± / ± mm hg) whereas / ( %) were hypertensive on abpm (mean age ± years, % female, mean bp ± / ± mm hg). thirty-four ( %) pts with uncontrolled ht were already hypertensive at obp. although long-term survivors after ahsct are known to be at elevated cardiovascular risk, diagnosis of arterial hypertension was missed in every fifth patient. the proportion of controlled hypertension is poor with only %. disclosure of conflict of interest: none. myasthenia gravis (mg) is a rare complication of allogeneic stem cell transplantation (sct) and is often associated with graft-versus-host disease (gvhd). we report a -year-old man who presented with oculobulbar and neck weakness months after an unrelated donor, allogeneic sct for chronic myeloid leukaemia (cml). he was diagnosed in with chronic phase cml. this responded poorly to tyrosine kinase inhibitors (tkis) and he was found to carry the t i mutation with additional monosomy . he underwent a fully hla matched unrelated donor sct with y -anti cd targeted radiotherapy, fludarabine, melphalan and alemtuzumab conditioning. he had grade cutaneous gvhd on ciclosporin withdrawal but no other significant gvhd. he has an immune mediated neutropenia since months post sct and has reduced immune reconstitution as demonstrated by a sub-normal absolute cd level. he remains on pneumocystis prophylaxis and has not experienced increased infection. chemotherapeutic agents have a cytotoxic effect on the oral mucosa and is a major problem following cancer treatment. cooling the oral mucosa in conjunction with chemotherapy infusion, using ice chips, is known to reduce the severity of oral mucositis ( , ) . although effective, ice chips are perceived as uncomfortable. the aim of the present study was to determine the optimal cooling temperature to prevent adverse effect of chemotherapeutic agents using tissue engineered oral mucosal models (teom). teom were incubated at °c, °c, °c or °c for min followed by exposure to μg/ml of -fu for h (control models were incubated at °c). teom were then washed and further incubated for h at °c co . cell viability and inflammatory cytokine production (il- and tnf-α) were measured using (prestoblue) and (elisa), respectively this study demonstrates an increased capacity to restore cell viability with decreasing temperature (figure a ). teom treated with -fu further showed an increased secretion of the pro-inflammatory cytokines tnf-α and il- at all temperatures compared to un-treated controls. for il- , secretion increased markedly when cells were incubated with μg/ml -fu at °c and °c compared to cells incubated with medium alone at °c (figure b) . for tnf-α, secretion was significantly higher (p o . ) in cells treated with μg/ml fu at °c compared to untreated mucosal models and mucosal models treated with μg/ml fu but incubated at °c (figure c ). teom models incubated at °c has an increased capacity to restore cell viability following exposure to -fu. incubation at °c further reduces the release of pro-inflammatory cytokine compared to those incubated at °c. ( ) and one received fludarabine and cyclophosphamide. all patients received campath- h as part of the conditioning regimen. stem cell source: peripheral blood stem cells patients and bm. comorbidity was assessed using the haematopoietic cell transplantation co-morbidity index (hct-ci), with patients ( %) having no co-morbidities, ( %) a co-morbidity index of - and ( %) had a score ⩾ . follow up of survivors ranged from to months (median: months). at the specified end point patients had relapsed ( %) with an actuarial -year relapse rate of %. there were deaths ( %). relapse ( ) was the main cause of death with transplant related mortality of % ( ) at day , % ( ) at months and % ( ) at year. the actuarial os at years was %, with a -year dfs of %. of the surviving relapsed patients all received chemotherapy and donor lymphocyte infusions resulting in effective recovery of remission, showing the utility of this approach. in terms of co-morbidity, actuarial survival rates were % in those with an hct-ci index of , % with an index of - and % with an index ⩾ . the results of this retrospective study indicate that allosct using reduced intensity conditioning regimens can be an effective treatment strategy for older patients with high risk myeloid malignancies including those with significant co-morbidities. relapse remains the main cause of treatment failure and strategies to reduce relapse risk are required. patients that relapse post allosct may respond to further treatment such as azacytidine or intensive chemotherapy and donor lymphocyte infusions. ( ) whether patient-related variables were associated with disagreement. this is a secondary analysis of a cross-sectional multicenter study where patients and clinicians completed an identical qol questionnaire (fact-bmt) at day . clinical and demographic variables as well as anxiety and depression (hads) were collected. agreement was analyzed with the intraclass coefficient correlation (icc). rates of under-and over-estimation were calculated. logistic regression models identified predictors of disagreement. we analyzed pairs of questionnaires, filled in by patients and clinicians. patients' median age was years, ( %) were men, and ( %) received an allogeneic hct. clinicians' median age was years, were men and had worked on the transplant field for a median of years (range: - ). agreement on qol was moderate (icc = ). exploratory analyses revealed that agreement for emotional (icc = ) and social (icc = ) wellbeing was poor, whereas it was moderate for physical (icc = ), functional (icc = ) and bmt concerns (icc = ). patients' wellbeing was overestimated in - % of the categories of wellbeing parameters, and underestimated in - %. patient-related variables explained - % of the variance on disagreement across scales. specifically, anxiety contributed to disagreement in all subscales, except in social wellbeing, where non-significant univariate associations were observed (p . ). type of transplant (allogeneic vs autologous), performance status, and graft-versus-host disease were not associated with disagreement (p . ). patients and clinicians agreement on qol is suboptimal, particularly on emotional and social wellbeing. patients' wellbeing cannot be estimated from other sources than themselves. these results highlight the unmet needs of hct recipients with respect to qol-related issues; an outcome that must be addressed by hct programs since their wellbeing is as important as survival endpoints. disclosure of conflict of interest: none. . we wanted to test the function and the safety of picc device as alternative to standard cvc in patients submitted to autologous stem cell transplantation (abmt). the primary end point of the study was to individuate the cause leading to the failure of picc (its removal or the needing of another cvc during the abmt procedure). secondary end points were the correct function of the device and its praticity. twenty patients submitted to abmt for multiple myeloma ( ) or lymphoma ( ) experienced a double lumen picc device ( ) or a single lumen ( ) if the patient already carried a permanent single lumen cvc such as hickman or port-a-cath. we excluded from this experience patients with high risk of life-threatening situation or high risk of intensive care already before abmt. picc devices were placed from a specialistic nurse team by ultrasound identification of a deep venous vessel in upper arms. melphalan or ceam were the standard conditioning regimens employed in myeloma and lymphoma abmt respectively. we considered a failure all the causes leading to the removal of picc or requiring another cvc before the end of the transplant procedure. at last we collected nurses and clinicians opinions about the picc functionality. no complication has been recorded in positioning phase. / patients maintained the picc device for all the time of transplant procedure. only one patient needed to remove the device for infection. the opinion of nurses and clinicians about the picc device was a significatively slower speed of infusion and resistance to the flow; in fact, / patients needed an infusional pump. the idraulic resistance of the catheter was particular evident against cellular fluids (stem cells suspension, transfusions of blood and platelets). for this reason picc seems to be less indicated in patients requiring many endovenous infusions (nurses' opinion). the rate of infection of picc devices seems to be lower compared to cvc, but the number of cases tested in this experience is too limited for definitive conclusions about it. for other aspects picc is similar to other cvcs. picc seems to be a valid alternative to standard cvc in patients who do not require intensive care, and in particular in patients with low intensity abmt who do not present a high number of endovenous infusions. maybe picc is less burdened of infections respect to normal cvc. this fact, summed to the lower risk during the positioning of the device, leads to consider the use of this device in abmt setting for standard risk patients. disclosure of conflict of interest: none. there are only few algorithms for the selection of hlamismatched unrelated donors, when no fully matched donor is available. indirect recognition of hla-mismatches can be predicted using the model of 'predicted indirectly recognizable hla epitopes' (pirche). the pirche model is a recently developed computer-based strategy, which classifies hladerived epitopes that are potentially presented by patientdonor shared hla-molecules. we performed a multicenter retrospective study evaluating the impact of pirche on outcome after allogeneic stem cell transplantation from hla / matched unrelated donors. the study cohort included adult patients who had undergone allogeneic stem cell transplantation for aml or mds. pirche scores were computed for recipients of hla / matched unrelated donor transplants ( / mud) using a web-based tool. primary endpoint was overall survival at years. patients with a / mud were divided into groups according to the sum of pirche i+ii values (pirche score). eighty-five ( ) patients had a pirche score of (no pirche detected), a pirche score . km estimate of year os was higher for / mud with pirche score = compared to pirche score : % ( % ci: - %) vs % ( % ci - %), p = . . os was similar for / mud with pirche score = and / mud ( % vs %). cox regression analysis revealed poorer os for pirche scores (rr . , % ci: . - . , p = . ). cumulative incidence of nrm at years was lower for / mud with pirche score = compared to pirche score ( % vs %, p = . ). multivariate cox regression analysis revealed poorer nrm for pirche score (rr . , % ci: . - . , p = . ). cumulative incidence of agvhd grade - at months was not significantly different for / mud with pirche score compared to pirche score ( % vs %, p = . ). cumulative incidence of cgvhd at years was lower for / mud with pirche score compared to pirche score ( % vs %, p = . . our findings require confirmation, ideally in a large prospective cohort study. if validated, the pirche model would allow selection of permissible hla-mismatches that may be associated with an improved transplant outcome in terms of reduced nrm and better os. [p ] disclosure of conflict of interest: none. this study was supported by a research grant from pirche-ag to the university medical center, hamburg-eppendorf. pretransplant liver dysfunction has been recognized as a risk factor for complications and mortality after allogeneic hematopoietic cell transplantation (allo-hct). however, there is no consensus on the optimal way to evaluate liver function in hct candidates. transient elastography (te) is a noninvasive method for diagnosing liver damage and cirrhosis. while elastography is widely used in the setting of viral hepatitis, its possible role in allo-hct recipients has not been deeply evaluated. patients receiving allo-hct in our center from may are scheduled to receive pretransplant evaluation by a hepatologist under a prospective protocol. the evaluation includes a hepatologist consultation, liver function and infectious serology tests and te. all patients receive ursodiol from hct admission to day + . this study constitutes the first evaluation of the ongoing protocol for patients receiving their first allo-hct from may to august . sixty patients received a first allo-hct during the study period. sixteen patients did not undergo hepatologist evaluation due to timing issues (n = ), unstable medical condition (n = ) or other reasons (n = ). finally, patients received pretransplant evaluation by a hepatologist under the current protocol and constitute the study population. median age at transplantation was years (range: - ). most patients received a transplant for acute leukemia (n = , %) or non-hodgkin's lymphoma (n = , %) mainly from hla matched unrelated donors (n = , %). thirty-two patients received reduced-toxicity regimens ( %). graft-versus-host disease (gvhd) prophylaxis consisted of tacrolimus in combination with another agent. median follow-up for survivors of months (range: - ). median elastography was . kpa (range: . - . ). considering the hct-ci categories on hepatic dysfunction, , and patients scored , and points, respectively. there were two cases of veno-oclusive disease (vod). overall survival and non-relapse mortality of all patients at median follow-up were % ( % ci - ) and % ( % ci - ), respectively. in the univariate analysis, median elastography was not associated with a higher risk of nrm (p-value = . ), os (p-value = . ) or hepatic chronic gvhd (p-value = . ). the two patients with vod had normal pre-hct transaminase levels and te. this first analysis of an ongoing protocol with universal pre-hct evaluation of hepatic function indicates that increased values of transient elastography are not associated with higher nrm or lower os after the procedure. further studies including a larger number of patients are needed in order to clarify the possible role of elastography in the hct setting. disclosure of conflict of interest: none. allogeneic hematopoetic stem cell transplantation (hsct) remains associated with a high morbidity and mortality in spite of advances in hsct management. specifically, pulmonary complications account for a substantial proportion of deaths within the first days after hsct. therefore, identification of lung dysfunction and additional comorbidities are crucial for preventive strategies in hsct. given the inconsistent association of pretransplant lung function s parameters on mortality after hsct and the significant changes in hsct care over the last decades, the aim of our study was to assess the effect of pulmonary function and comorbid conditions on mortality in patients undergoing hsct for hematological disorders. we retrieved relevant clinical data of all consecutive patients at the hematology division of the basel university hospital with a transplant for hematological disorders between and . we examined the lung function at baseline and , and months after hsct-including the s forced expiratory volume (fev % of predicted), fev /vcmax and diffusing capacity for carbon monoxide (dlco, adjusted for hemoglobin concentration). in addition, we assessed pretransplant conditions such as age, sex, karnofsky performance status (kps), donor type and various risk scores in hsct (hematopoietic cell transplantation comorbidity index (hct-ci), european society for blood and marrow transplantation (ebmt), revised pretransplant assessment of mortality score (pam)). using uni-and multivariate cox proportional-hazards regression analysis, we evaluated patient-and transplant-related risk factors for all-cause mortality by including the following categorical candidate variables: fev (⩾ % vs - % vs o % of predicted), kps ( o % vs ⩾ %), age ( o vs ⩾ years), conditioning intensity and donor type (matched-related vs mismatchedrelated vs matched-unrelated vs mismatched-unrelated). within the study period, patients with predominantly acute leukemia ( %) or lymphoproliferative disorders ( %) underwent myeloablative (n = ) and non-myeloablative hepatic veno-occlusive disease/sinusoidal obstruction syndrome (vod/sos) is a potentially life-threatening complication of conditioning for hematopoietic stem cell transplantations (hsct). recombinant thrombomodulin (rtm) is a new drug for treating disseminated intravascular coagulation (dic) and is an endothelial anticoagulant cofactor that promotes the thrombin-mediated formation of activated protein c (apc). rtm has been used to treat vod/sos, but its ability to prevent vod/sos has not been established. we evaluated the cases of pediatric hematology and oncology patients ( ( %) acute myeloid leukemia, ( %) acute lymphoblastic leukemia, and ( %) neuroblastoma patients, and ( %) patient each with myelodysplastic syndrome, rhabdomyosarcoma, hemophagocytic syndrome (hlh), and wiskott-aldrich syndrome) who underwent hsct at our institution between and and had ≧ risk factors for vod/sos. these risk factors included previous treatment with gemtuzumab ozogamicin (go), receiving hsct, undergoing conditioning with busulfan (bu), and being diagnosed with hlh. the patients who received hsct after (n = ; rtm group) were treated with rtm as a prophylaxis against vod/sos ( u/kg per day for days; from days to ) together with ursodeoxycholic acid (urso) and low-molecular-weight heparin (lmwh), and the others (n = ; control group) were only treated with urso and lmwh. the incidence of vod/sos was evaluated, and various coagulation parameters and markers of endothelial injury (plasminogen activator inhibitor type (pai- ) and apc) were measured in both groups. the patients' median age was (range: - ) years, and ( %) were male. clinical characteristics, including vod/sos risk factors, were wellmatched in both groups. the risk factors possessed by the patients included receiving hsct ( / , %), previous go treatment ( / , %), conditioning with bu ( / , %), and a diagnosis of hlh ( / , %). although vod/sos occurred by post-hsct day + in ( %) patients in the control group, vod/sos was not seen in the rtm group. two of the former patients ( : previous treatment with go, : a diagnosis of hlh) suffered severe vod/sos, and (a diagnosis of hlh) died of the condition. no grade / adverse events involving bleeding or severe organ damage were reported in the rtm group. interestingly, the mean peak value of pai- and apc (markers of endothelial injury) were significantly lower in the rtm group (table ) . [p ] disclosure of conflict of interest: none. protective effect of early human cytomegalovirus reactivation on relapse of myeloproliferative disorders after allogeneic hematopoietic stem cell transplantation z peric , , j wilson , n durakovic , , l desnica , a ostojic , vv rezo , v marekovic , , r serventi-seiwerth and r vrhovac , school of medicine, university of zagreb, zagreb, croatia and university hospital centre, zagreb, zagreb, croatia there have been conflicting results regarding the association between early cytomegalovirus (cmv) reactivation and decreased incidence of relapse after allogeneic hematopoietic stem cell transplantation (allo-hsct). this prompted us to retrospectively evaluate the potential impact of cmv reactivation on transplantation outcomes in a study population of consecutive adult patients who underwent allo-hsct in our institution and were treated and followed in a homogenous manner. patients were monitored for cmv reactivation once weekly for the first days after allo-hsct. monitoring was done with a real time qpcr with lower limit of detection of genome copies per ml of blood. when cmv viremia was detected, all patients were treated with intravenous ganciclovir or oral valganciclovir untill two consecutive negative qpcr assays. univariate and multivariable proportional hazards models using the fine and gray approach were considered to evaluate the variables for relapse, treating death as competing event. between and , male and female patients underwent allo-hsct at a median age of years (range: - ). among them, most patients were treated for myeloid malignancies ( aml, mds and mpn with cml, mf and cmml), while the rest had lymphoproliferative disorders ( all, nhl, mm, mh and cll) and one patient had aplastic anemia. the donors were unrelated in cases, related in patients and haploidentical in patients. most of the patients ( %) received peripheral blood stem cells after a reduced-intensity conditioning regimen ( %). with a median follow-up of months, early cmv reactivation occured in % patients at a median of days after transplantation and did not affect relapse incidence in patients with lymphoproliferative disorders. on the contrary, the cumulative incidence (ci) of hematologic relapse in patients with myeloproliferative disorders (aml and mpn) at months after allo-hsct was % ( % ci, - %) in patients without, opposed to % ( % ci, - %) in patients with cmv reactivation (p = . ). however, cmv reactivation did not significantly affect (p = . ) overall survival between patients with ( %; % ci - %) and without cmv reactivation ( %, % ci - %). a striking and previously unreported correlation between cmv reactivation and relapse was found in patients with mpn; the ci of relapse was % ( % ci, - %) in patients without, opposed to only % ( % ci, - %) in patients with cmv reactivation (p = . ). a substantial and independent reduction of the relapse risk in myeloproliferative disorders (aml+mpn) associated with early cmv reactivation was confirmed by multivariate analysis using time-dependent covariate functions for high-risk disease, use of atg, chronic graft-versus-host disease (hazard ratio . ; % ci, . - . , p = . ), and cmv reactivation (hazard ratio . ; % ci, . - . , p = . ). in summary, this report supports an independent role of cmv reactivation on relapse in patients with myeloproliferative disorders. to our knowledge, we are the first to show a significant reduction of relapse incidence in patients with mpn, even though our findings are based on a relatively small number of patients. however, this putative virus-versus-myeloproliferation effect definitely warrants further research. [p ] disclosure of conflict of interest: none. final result of fact-bmt is score ranged - point (the higher the score, the better qol). for qualitative assessment of donor-recipient relationship, the adult sibling relationship questionnaire (asqr) in polish version was used. the asrq-s consists of items which are spread over eight scales designed to investigate three factors: warmth, conflict and rivalry. the questionnaires were given to both subgroups, donors and recipients of msd-hsct and the results were compared to each other. the overall result of the fact-bmt questionnaire was . ± . points, which means that the examined group generally described their qol as 'quite good'. the best results were found in functional well-being ( . ± . ), while the worst in emotional well-being ( . ± . ) dimension. statistically, the qol score was not influenced by age at hsct (p = . ), current age (p = . ) or gender (p = . ) of the respondents. the recipients scored highest on warm factor ( . ± . ), while donor respondents scored slightly higher rivarly ( . ± . ) than warm ( . ± . ). the second dimension scored by recipients was rivarly ( . ± . ). conflict scores were lowest, although donor respondents scored higher on these than recipient respondents ( . ± . in donors vs . ± . in recipients). statistical analysis revealed that the being a donor or recipient of msd-hsct determines the level of rivarly in the sibling relationship (p = . ) with no impact on warm and conflict dimension. health-related qol in transplanted patients is quite good. sibling donor-recipient relationship is unbalanced with recipient respondents being more likely to assess a warm relationship, while rivalry was more likely to be present among donor. further multicenter studies based on larger cohort of patients are necessary to assess sibling relationship after transplantation life experience. disclosure of conflict of interest: the authors have nothing to disclose. this work was supported by grant from poznan university of medical sciences ( - - - ). rate of re-admission in patients undergoing allogeneic transplants from identical siblings, unrelated donors or haploidentical donors f sora , s sica, l laurenti, p chiusolo, s giammarco, i innocenti, e metafuni, a corbingi and a bacigalupo department of hematology, catholic university of rome hla identical siblings (sib), unrelated (ud) and family hla haploidentical donors (haplo) are currently being used for patients undergoing an allogeneic transplant (hsct) for hematologic disorders. gvhd prophylaxis is usually different, and is commonly based on a calcineurin inhibitor (cni) and methotrexate (mtx) with or without atg for sibs and uds, wheres post-transplant cyclophosphamide (pt-cy)+a cni and mycophenolate (mmf) is used for haplos. we will refer as sib, ud, haplo platform, the combination of a given donor and a given gvhd prophylaxis. the outcome of these three different platforms is usually measured in terms of gvhd, non relapse mortality (nrm) and survival. days of admission and readmissions are important in terms of morbidity, but also of costs, and are usually not reported. aim of the study: assess the duration of the first admission and the incidence of a new re-admissions, in the first days after the transplant. we retrospectively analyzed patients from to . sixtyone received peripheral blood stem cell graft from an ud, and gvhd prophylaxis with cya+mtx+atg; received a peripheral stem cell graft from a sib and gvhd prophylaxis with cya +mtx; patients received bone marrow hsct from haplorelated donor and pt-cy+cya+mtmf. patients characteristics are shown in table . relapses were excluded from the readmission analysis. the median time from the transplantation to discharge was days for ud, for haplo and days for sib: there was no significant difference between haplo vs ud (p = . ), whereas the admission of both haplo and ud was longer than sibs (po . ). first readmission. fiftyone patient out of required of a new admission for complications after tranplant ( out of after mud ( %), out of ( %) using a sibling donor and out of using an haploidentical donor ( %)). there were significantly more re-admissions in the ud vs sib group ( . ) and a trend for more ud readmissions vs haplo (p = . ); siblings had the lowest number of readmissions. time to neutrophil engraftment was comparable in haplo vs ud patients (p = . ) and in sib vs ud (p = . ); the time was longer in haplo vs sibs (p o . ). the reason to re-admitted the patients in the hospital after tranplantation was fever in out of ( %) new admissions in ud setting, out of ( %) in sib and out of ( %) in haplo; acute gvhd was the cause for re-admission in out of ( %) ud, out of ( %) sib and none in haplo. the other causes for re-admission in the hospital were hemorragic cistitis, thoracic or abdominal pain. second re-admission. of hospitalization is registered in out of patients in ud ( for aghvd and fever), out of ( %) in sib ( episodes of fever) and out of ( %) patients in haplo ( for fever and progressive disease). also for second episodes, ud grafts had significantly more admissions compared to haplo and sibs. third re-admission was recorded only in ud patients ( out of - %). this study shows a comparable duration of admission for transplant for haplo and ud patients, both significantly longer than sib grafts. the number of re-admissions is comparable in haplo vs sibs and there is a trend for lower number of re-admission as compared to uds. we interpret this outcomes with caution given the relatively small sample size and heterogeneous disease population included. future studies need to confirme our results. disclosure of conflict of interest: none. prolonged thrombocytopenia (pt) is frequent event after allogeneic haematopoietic stem cell transplantation (hsct), especically in haploidentical transplantation, which could be up to % according to our previous report. pt has significant negative impact on long-term outcomes, mainly due to increased non-relapse mortality. however, there are no efficious treatment. in this study, we report the preliminary results of recombinant human thrombopoietin (rhtpo) in treating this kind of patients. from . to . , patients were enrolled under the following inclusion criterion: ( ) diagnosed with dpe or sfpr after allogeneic stem cell transplantion; ( ) no sign of minimal residual disease or recurrence of hematological malignancy; ( ) not using other tpo receptor agonist or il- within month of enrollment. pt include delayled platelet engraftment (dpe) and secondary failure of platelet recovery (sfpr). the former was defined as failure to achieve platelet counts ⩾ /μl for consecutive without transfusion until days after transplantation, while the latter was defined as a decline in platelet counts below /μl for consecutive days, or requiring transfusion support after achieving sustained counts without transfusions for consecutive days after hsct. the prescription of rhtpo was iu once daily for days, or if patients achieve platelet ⩾ /μl for consecutive days with a duration o days. response was defined as success of achieve platelet counts ⩾ /μl for consecutive days. the response time was defined as the first day achieve response from the start of prescription. the primary end point was response rate, and the secondary end point was reponse time. a total of patients were enrolled, including males and females. the median age was ( - ) years. all patients received haploidentical transplantation. among these patients, patients were dpe and were sfpr. all patients received a -day prescription. the overall response rate was % ( out of ) in the overall population, while % ( out of ) in dpe and . % ( out of ) in sfpr, respectively. among the patients with response, the median response time was ( - ) days from the first dose of rhtpo. after weeks of the last dose of rhtpo, none of the responsed patient lose response. since the followup time is too short, the impact of relapse, gvhd were not reported. this single-arm preliminary result suggest that rhtpo could be a efficious method to manage pt after stem cell transplantation. however, these result need further confirmation. disclosure of conflict of interest: none. reproductive health in long-term female survivors after allogeneic hematopoietic stem cell transplantation z peric , , a samardzic , n durakovic , , d tina , , l desnica , r serventi-seiwerth and r vrhovac , school of medicine, university of zagreb, zagreb, croatia and university hospital centre zagreb, zagreb, croatia most female recipients of allogeneic hematopoietic stem cell transplantation (allo-hsct) suffer from premature menopause, infertility and endocrine imbalance owing to gonadal damage from myeloablative conditioning. in order to evaluate ovarian recovery and long-term endocrine complications in our institution, we performed a retrospective study of female patients who received a myeloablative allo-hsct during their reproductive age. we identified female patients who underwent myeloablative allo-hsct in our institution between and and were still alive with available follow-up at the time of this study. among them, patients accepted to participate and responded to a query designed for this s purpose. the median age of our patients at transplantation was years (range: - years). they were interviewed at a median of years (range: - years) post allo-hsct. the majority of patients were transplanted for a myeloid malignancy ( acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndromes and chronic myelofibrosis), while patients had aplastic anemia and had acute lymphoblastic leukemia. all patients received bone marrow transplant from a hla-matched related donor after a myeloablative conditioning. conditioning regimen consisted of cyclophosphamide with or without total body irradiation (tbi) or in combination with busulfan. only patients ( %) resumed a normal menstrual cycle after allo-hsct, without the need for hormonal replacement therapy (hrt). all these patients were transplanted for aplastic anemia and none of them received tbi in the conditioning regimen. eight patients ( %) remained amenorrheic indefinitely and never started hrt, even though most of these women were transplanted under the age of years. % of these patients were diagnosed with osteoporosis later in life. the remaining patients ( %) started hrt at a median of months after allo-hsct (range: - months). however, only seven patients on hrt ( %) resumed regular menstrual cycle. a median duration of hrt therapy was years (range: - years). none of the women receiving long-term hrt had severe cardiovascular complications or breast cancer. finally, five women gave birth to eight healthy children in our study population. three unassisted pregnancies were observed in two female patients after spontaneous recovery of ovarian function (both patients with aplastic anemia). the remaining two patients restored ovarian function with the use of hrt and gave birth after an assisted pregnancy (one woman gave birth to triplets after an in vitro fertilization (ivf), while other became pregnant with a donated oocyte). in spite of the fact that almost all women who undergo allo-hsct develop an ovarian failure, spontaneous recovery is sometimes possible, particularly following conditioning regimen without tbi. in patients without spontaneous recovery, hrt should be initiated promptly to prevent the early and late unwanted effects related to estrogen deficiency. moreover, recovery of normal ovarian function and even a viable pregnancy is a realistic possibility in patients placed on hrt, particularly with the use of potential therapeutic interventions as ivf or oocyte cryopreservation. it is therefore crucial to provide adapted pre-transplant counselling and recommendations for regular post-transplant follow-up in female patients who undergo allo-hsct. disclosure of conflict of interest: none. transplant-associated thrombotic microangiopathy (ta-tma) is a multifactorial disorder caused by systemic vascular endothelial injury leading to end-organ damage often involving the kidney. ta-tma occurs in up to % of patients undergoing hsct, and may be associated with poor outcome. although pathogenesis has not been fully clarified, activation of the complement system has been suggested to play a central role, and eculizumab, a monoclonal antibody (mab) that mediates terminal complement blockade, has shown therapeutic benefit in cases unresponsive to immunosuppression modulation. we report the case of a pediatric allogeneic hsct recipient with severe ta-tma, who did not tolerate treatment with eculizumab, now successfully treated with oms , a novel human mab targeted to the mannan-binding lectin-associated serine protease- (masp- ), a molecule central to the activation of the lectin pathway of complement. a -year-old girl received an allogeneic hsct from a hla-compatible unrelated donor for the treatment of diamond-blackfan anemia. at month + of the posttransplant course, she developed progressive deterioration of renal function, microhematuria and serositis, that prompted the cyclosporine discontinuation. from month + , the patient experienced progressive trilinear cytopenia, elevated ldh, schistocytes, undetectable haptoglobin, hypertension, increased serum creatinine, nephrotic range proteinuria, and serositis, and a diagnosis of ta-tma was established. laboratory investigations documented no abnormalities in the patient but identified a stop-codon heterozygous variant in cfhr c. _ dupaa (p.glu lysfs* ) in the donor's dna. the patient was initially treated with eculizumab, but she developed acute pulmonary edema soon after eculizumab administration as the consequence of a possible reaction to the drug which had to be discontinued. the patient was subsequently treated with plasma exchange, with only limited benefit. upon ta-tma relapse at month + , eculizumab was re-administered at lower doses, but she developed a new episode of acute pulmonary edema, preventing further eculizumab continuation. renal function progressively deteriorated and she was started on hemodialysis, reaching a times weekly regimen. the patient received oms , kindly provided on a compassionate use basis by omeros corporation, seattle, usa, starting with an iv dosing schedule. she did not experience any adverse events, and was able to tolerate the treatment well. at months from oms initiation, she has shown improvement in ldh and haptoglobin levels, and, more importantly, her creatinine levels have normalized, allowing for complete discontinuation of hemodialysis and partial outpatient management. anti-masp- mab oms is a promising new option for the treatment of ta-tma occurring after hsct, and seems to have a safe profile also in the pediatric/adolescent setting. disclosure of conflict of interest: none. severe cytokine release syndrome after t-cell replete haploidentical transplantation with post-transplant cyclophosphamide is associated with increased death rate d taurino , j mariotti , b sarina , l morabito , s bramanti , c carlo-stella , a santoro and l castagna bone marrow unit, humanitas cancer center, istituto clinico humanitas, rozzano, italy and hematology department, humanitas cancer center, istituto clinico humanitas, rozzano, italy haploidentical stem cell transplant (haplo-sct) represents a potential curative strategy for several hematological malignancies. haplo-sct may represent an alternative option when a hla matched-identical sibling (hlaid) or a matched unrelated donor (mud) is not available. the syndrome of systemic inflammation, characterized by fevers, vascular leak, hypotension, and respiratory and renal insufficiency, in the context of elevated inflammatory markers and cytokine levels was previously described as cytokine-release syndrome (crs) . recent publications have elicited the occurrence of crs after haploidentical transplant, especially after peripheral blood stem cell graft, and its high-related mortality - . here we report the experience of our institution with crs after haplo-sct. between march and october , we treated patients with haplo-sct with a graft source represented by peripheral blood stem cells. we monitored the occurrence of crs symptoms and utilize a previously described grading system , starting from day , up to day after transplant. severe crs is defined as grade or higher because it requires aggressive interventions and is characterized by oxygen requirement ⩾ %, l nasal cannula, hypotension requiring high dose or multiple vasopressors, grade renal toxicity or grade transaminitis. other characteristics comprise newonset altered mental status without other explanation and new cardiomyopathy without wall motion abnormality. results: out of patients experienced fever between day and day post transplant with most episodes ( patients) occurring between day and day . on day after transplant, patients had grade , grade and grade crs, respectively. by day post haplo-sct, patients had crs grade , grade and grade . overall, the incidence of crs any grade was % ( % ci - %). year after transplant patients died because of non-relapse related side effects. with a median follow-up for alive subjects of months, -year overall survival (os) was % ( % ci: - %). -year os was % for patients with a crs on day (p = . ). conclusions: crs represent an important complication after haplo-sct. crs score on day after hst apparently correlates with long-term survival. better strategies need to be implemented for an early detection of severe crs in order to develop effective treatments, such as tocilzumab, for this important side effect. further studies are ongoing at our institution in order to correlate post-haplo crs with graft composition, laboratory parameters and immunereconstitution. hematopoietic cell transplantation (hct) is associated with significant morbidity that impairs survivor's sexual functioning. however, few studies have specifically addressed it. thus, we examined ( ) sexual functioning during the first year post hct, ( ) differences between allogeneic and autologous hct, and ( ) whether demographic, clinical and psychological variables were associated with sexual functioning. this is a prospective multicenter study assessing patients before hct, at day , and . sexual functioning was assessed with the changes in sexual functioning questionnaire, which yields a total score, along with scores for the dimensions of frequency, pleasure, orgasm, desire and arousal. anxiety and depression (hads) were also collected. we included consecutive hct recipients: ( %) were men, with a median age of years (range: - ), ( %) received an allogeneic hct and ( %) an autologous hct. sexual functioning was significantly affected: % of the sample reported impairment at pre-hct, % at day , % at day and % at day . mixed model analysis indicated that sexual functioning was not associated with time from hct (p = . ) or hct type (p = . ). however, there was an interaction between these two variables (p = . ), particularly at day , since sexual functioning had improved among autologous survivors and worsened among allogeneic survivors leading to nonsignificant differences between hct type (p = . ). frequency of sexual functioning improved during the study period (po . ), and no differences were observed between hct type (p = . ). again, there was a borderline interaction between post-hct time and hct type (p = . ), since autologous survivors reached higher frequencies than allogeneic survivors, with significant differences at day (p = . ). pleasure significantly improved during the study period (p = . ), without observing differences between hct groups (p = . ). again, however, autologous survivors reported significant improvements in pleasure at day (p o . ) and a trend at day (p = . ) when compared with allogeneic survivors. orgasm did not improve during the study period (p = . ), and no differences were obtained between hct groups (p = . ). allogeneic survivors had higher orgasm scores at pre-hct (p = . ), which worsened during the study period, particularly at day (p = . ). in contrast, autologous survivors reported improvements in orgasm by day . non significant results were obtained in the sphere of sexual desire and arousal (p . ). bivariate analyses indicated that women, older age and depression were associated with impaired sexual functioning at all assessed time-points (p o . ). chronic graft-versus-host disease (gvhd) was associated with worse sexual functioning at day (p = . ) and (p = . ). no differences were obtained when considering diagnosis, having received previous hct, intensity of the conditioning regimen and whether patients lived with a partner (p . ). stepwise multivariate regression analyses indicated that gender (p = . ) and extensive chronic gvhd (p = . ) predicted for worse sexual functioning at day . sexual functioning should be routinely assessed and considered for eventual targeted intervention in both hct populations, particularly during the first year post transplant. additional clinical efforts should focus on patients more vulnerable to impaired sexual functioning. disclosure of conflict of interest: none. significant improvement of qol by using atg as part of the conditioning regimen followed by hla-identical peripheral stem cell transplantation in acute leukemia patients. results from a prospective, randomized phase iii study (atg family study) b francesca , s carlos , w christine , s mariarosaria , p massimo , s carmine , m giuseppe , b wolfgang , cm angelo , p francesca , m nicola cgvhd is a major complication after allogeneic sct. we previously demonstrated that the addition of anti-tlymphocyte globulin (atlg neovii, formerly atg-fresenius) to a myeloablative preparing regimen followed by peripheralblood sct from an hla-identical sibling for pts with acute leukemia resulted in a significant reduction of cgvhd, without increasing the risk of relapse or infection. the study protocol included quality of life (qol) questionnaires (eortc qlq- and hdc ) before and after sct (day+ , , and mos). the qlq-c includes a global qol scale, five functional scales (physical, role, emotional, cognitive and social function) and nine symptom scales (fatigue, nausea-vomiting, pain, dyspnea, insomnia, appetite loss, constipation, diarrhea and financial problems). the qlq-hdc includes six multi-item scales and eight single items that describe impairment through highdose treatment. mixed models for repeated measures (mmrm) and linear mixed models (lmm) were used to analyze the time courses and the slopes of the outcomes depending on treatment arm (atg vs non atg), age, country, sex, and cgvhd. (clinicaltrials.gov: nct ). pts with a qol form returned decreased by visit ( % pre-sct, % at days and % at mos after sct). forty-nine percent in the atg and % in non atg arm provided any qol forms after sct. return of any post-sct qol forms by country was % for germany, % for italy and % for spain. pts with cgvhd were more likely to return qol questionnaires ( % vs % w/out cgvhd) while neither age nor sex were closely associated with qol form return. the majority of subscales of the qlq- indicated an average improvement of qol and reduction of symptoms over time, notably in the atg group. in an mmrm model controlling for country, age, sex and cgvhd, pts treated with atg showed significantly more pronounced improvement of global health status/qol over time compared to non-atlg (p = . ), with a treatment group difference of . ± . points (marginal mean ± sem) at day and increasing to . ± . points at month favoring atg. significant superiority of atg (po . ) was also observed for four of the five functional scales as well as for several symptom scales scores including appetite loss, insomnia, nausea-vomiting and dyspnea. for the qlq-hdc , significant treatment effects favoring atg were observed for gi side effects and impact on family. lmm analyses of qol by country indicate that patients from italy generally gave more favorable ratings for all functional scales and lower scores for most symptom scales than those from germany while the time courses and slopes were similar for most scales. these results underline the importance of the habits and cultural environment which are distinctive of each country. males and females showed similar qol ratings at pre-and post-sct. patients up to years tended to provide more favorable functional ratings and less severe symptom scores than older patients and also showed more pronounced improvements of qol. pts receiving atg in a randomized study have significantly less cgvhd and improved grfs, resulting in an improved qol regarding global health status and most functional scales. notably, we also observed a significant difference in qol assessment between pts from germany and italy. oral mucositis (om) is a well-known side effect of high-dose chemotherapy and radiotherapy in hematological patients, which influences the health-related quality of life (hrqol) of the affected patients. the purpose of this study is to demonstrate the impact of om on hrqol in stem cell transplanted patients in routine care. prospective, noninterventional single-center observational study was performed at a german university hospital. inpatient allogenic and autologous stem cell transplant patients ⩾ years with high-dose chemotherapy. om was assessed with the who oral toxicity scale, pain using the numeric rating scale (nrs) and the performance status with the ecog score. hrqol was captured with the eortc qlq-c and the qlq-oh questionnaires ( days before hematopoietic stem cell transplantation (hsct); days after hsct; days after hsct). statistical significance was assumed p o . . a total of patients ( autologous and allogenic) was included from august to december . a total of ( %) patients developed om. of these patients, suffered from grade , from grade , from grade and from grade om. three days before hsct, the mean qol of all patients was %, the mean qlq-c summary score . % and the mean oral health related quality of life . %. most of the patients suffered from om around day after hsct. after days, quality of life (qol) was higher in patients with no om ( . %) than in patients with om ( . %). the qlq-c summary score was significantly (p = . ) lower in patients affected by om ( . %) than in patients who did not develop an om ( . %). om affected patients had significantly more limitations in emotional (no om . %; om . %; p = . ) and cognitive functioning (no om . %; om . %; p = . ) and in fatigue (no om . %; om %; p = . ), pain (no om . %; om %; p = . ) and insomnia (no om . %; om %; p = . ), they had a significantly higher rate of problems. oral health-related quality of life was significantly (p = . ) lower in patients who were affected by om ( . %) compared to patients who did not develop an om ( . %) and patients with an om had significantly more problems with a sore mouth (no om . %; om . %; p = . ), sticky saliva (no om . %; om . %; p = . ) and sensitive mouth (no om . %; om . %; p = . ). after days, qol was higher in patients with no om ( . %) compared to patients with om ( . %). patients with no development of om had a higher but not significant physical functioning, cognitive functioning and social functioning. patients affected by om had higher levels of fatigue and pain and more often suffered from a sore mouth. oral health-related quality of life was higher in patients without om ( %) compared to patients with om ( . %). comparing all assessed days patients with om had higher scores on the nrs increasing with a higher grade of om (mean nrs score grade ; - . , grade ; - . ), the ecog index was higher in om affected patients during episodes with om (mean ecog score- . ) compared to episodes without om (mean ecog score- . ). om has a major impact on the hrqol, health-related symptoms and functionality. in the future, there has to be a higher awareness from clinicians and patients of the prevention, assessment und causes of om. more research has to be initiated to ease the symptomatology and to improve patients' quality of life. disclosure of conflict of interest: none. according to ebmt data, chronic gvhd (cgvhd) occurs in - % of all patients after allogenic hematopoietic stem cell transplantation (allo-hsct). pulmonary cgvhd is the most severe form. but it is very unpredictable to use due to the fact that many factors can affect it (breath-dependent; need experience not only from physician but from patients also and so on). here we show that routine software-based image analysis algorithm can provide data that highly correlated with pft results and have excellent sensitivity and specificity in pulmonary cgvhd diagnosis. we blindly analyzed ct scans (made without additional expiration) in allo-hsct patients at different time points. all scans were performed on ct scanner aquilion , toshiba, japan. according to hounsfield units (hu) definition, − hu ('air') have approximate density at g/ml; hu ('water') have approximate density at g/ml. the analysis of ct scans (heart, vessels and bronchi were excluded from analysis) was based on automated software conversion (image-analysis algorithm providing by multivox software, msu, moscow, russia) of each ct-image pixel from hu to density units (g/ml). pft were performed using standard procedures at same as ct scans time points (spirolab iii, italy). all patients with hematological malignancies (acute leukemia- , aplastic anemia- , chronic myeloid leukemia- , t-cell lymphoma- , chronic myeloproliferative disorder- , myelodysplastic syndrome- ) were transplanted in national research center for hematology between and . median of age was . years (range: - years). eight patients were males, -females. seventeen received reduced-intensity and -myeloablative conditioning regimen. graft from match unrelated donor (mud) were used in cases, 'mismatch' mud- , match related donor (mrd)- , 'mismatch' mrd- . median follow-up is . months. we analyzed lung tissue experimental density in patients before and after allo-hsct at different time points. median of lung tissue experimental density were . (interquartile range (iqr), . - . ), . (iqr, . - . ) and . (iqr, . - . ) for patients before allo-hsct, after allo-hsct with cgvhd (except pulmonary cgvhd) and with pulmonary cgvhd, respectively. mann-whitney u test was used to reveal significant differences between these groups (see figure ). also, we found strong correlation between pft and experimental density (spearman's correlation coefficient r = . ) (see figure ). forty-five ct scans of patients with pulmonary cgvhd and ct scans of patients without pulmonary cgvhd at the time of ct scan as control subjects were included in roc analysis to assess the clinical values of our model. we generated an roc curve and found that the area under the curve (auc) was . ( % ci, . - . ) (p o . ) (figure ). standard ct scan is presented as easy to perform, breath-independent, standardized and wide spread method for every patient after allo-hsct. it can be performed many times during all their post-hsct life. ct scan with a simple software analysis allows to select a group with high probability of pulmonary cgvhd and who can be suspected of cgvhd development by this method with sensitivity- % and specificity- . %. disclosure of conflict of interest: none. the choice of effectiveness criteria affects conclusions of economic evaluation of newer allogeneic bone marrow transplantation modalities :example based on a randomised multicenter trial comparing two reduced intensity conditioning regimen (flu-bu-atg) vs (flu-tbi) for matched related allo-sct s le corroller*, anne-gaelle , c siani , , r tabrizi a re-evaluation of the per-diem hospitalization cost was performed in and included the utilization of hospital technical facilities and a more precise estimation of overheads costs. we performed three separated cost-effectiveness analysis, using, respectively, pfs, os and qaly as end point. when using pfs as effectiveness, relapse costs were not included. weighting coefficients for the cost per qaly analysis came from the literature. at years, os and pfs were % and %, respectively, and did not statistically differ between groups. the mean total cost per patient was not statistically different between groups ( € for fba vs € for ftbi, ns). using pfs as end point, the icer of fba compared to ftbi is € per year of pfs gained. using os, the icer became non-statistically significant, signifying that when handling uncertainty, no difference in term of cost-effectiveness was observed between fba and ftbi with os as end point. using s qaly, the icer was statistically ns again, showing no advantage in terms of cost per qaly of one conditioning regimen over the other. this result was obtained both considering three weighted health states (dfs, progression and death) and four weighted health states (dfs without gvhd, dfs with gvhd, progression and death) for the qaly calculation. using os and qaly, the two conditioning regimens were not different in terms of cost-effectiveness, while fba may be considered as more cost-effective using pfs as effectiveness criterion. using intermediary end points allows economic evaluation to be available earlier in the life cycle of an innovation. however, it implies strong hypotheses about the predictive value of the pfs over the os. longer period evaluation and qaly may reverse preliminary results. this situation is likely to exist in the hematology setting where alternatives between chances of cure and toxicities of treatment are often observed. research about allogeneic sct modalities is archetypical of such situations and decisions makers should be aware of the necessity of further economic re-evaluation along the development and diffusion process of innovative treatments. disclosure of conflict of interest: none. the impact of corticosteroids prophylaxis for the engraftment syndrome incidence during autologous stem cell transplantation in multiple myeloma and amyloidosis the es is a complication of asct characterized by an inflammatory response during peripheral blood recovery. the standard treatment is based on corticosteroid therapy. the incidence of es after asct increases in chemotherapy lowtreated patients such as those with multiple myeloma (mm) and amyloidosis (al).moreover, the es is associated with the use of g-csf after infusion of stem cells. therefore, our bmt team does not use g-csf since in this population reducing the incidence and severity of es. therefore, it makes sense to use low-dose prednisone to prevent this complication. in this study, we compared two consecutive cohorts of patients with mm/al that performed an asct while evaluating the corticosteroids prophylaxis (cp) in the es incidence and its effect on other clinical variables. we included patients with mm (n = ; %) and al (n = ; %) that performed an asct between january and november in a single institution. the median age (range) was . ( . - . ) years. during the procedure, all patients received melphalan as conditioning chemotherapy and none received g-csf. fortyseven patients ( %) received intravenous methylprednisolone or oral prednisone . mg/kg/day from day + until reaching a neutrophil count ⩾ per mm for consecutive days (cs group), and ( %) patients did not receive corticosteroids (noncs group). the characteristics of patients in both groups (age, gender, status performance and previous treatment were similar (p . )). the cs group, received higher doses of cd + than the noncs group ( . × /kg vs . × /kg, respectively, p = . ). the median (range) days of neutropenia ( o per mm ) was ( - ) days. es was diagnosed in ( %) patients. fifty-seven ( %) patients had fever, showing infectious focus or microbiological isolation in ( %) cases, whereas the incidence of grade iii-iv oral mucositis and relevant gastrointestinal toxicity was % and . %, respectively. the complete analysis between groups (cs versus noncs) for the whole series and in the mm/al subgroups is detailed in table . the administration of corticosteroids as prophylaxis seems to reduce the incidence of es in the overall series or in the analysis for the subgroups (mm and al) without increasing infection. [p ] disclosure of conflict of interest: none. chronic gvhd is a condition that might occur after allo-hsct and has been proved to impair long-term survival and quality of life of patients. graft failure is also a major potential complication for patients undergoing transplant for an aplastic anemia/bone marrow failure (bmf). partial in vivo t-cell depletion, employing anti-thymocyte globulin (atg) during conditioning, has been proved to successfully prevent the mentioned potentially life-threatening complications in highrisk patients. however, the possibility of developing epstein-barr virus (ebv)-induced post-transplant lymphoproliferative disorders (ptlp) has been a limiting factor to use atg. this study includes the last pts with a minimum follow-up of days, who underwent allo-hsct in our center (november -august ). a total of pts were male and female. median age was years (range: - ). baseline diseases were: acute leukemias ( ), lymphoproliferative disorders ( ), myelodysplastic syndromes ( ), chronic myeloproliferative diseases ( ), multiple myeloma ( ) and bone marrow failures ( ) . donor was unrelated in cases, and related in (including haplo-identical). conditioning regimen was: busulphan-based ( ), melphalan-based ( ), tbi-based ( ) and others ( ). progenitors source was pb in and bm in . patient/donor ebv pre-transplant serology was: +/+ in cases, +/ − in and − /+ in . rabbit atg (thymoglobuline) was employed in cases: at . - mg/kg (urd transplants) (low dose), and cases at . mg/kg (all of them pts with bmf) (intermediate dose). family donor (including haplo-identical) transplants of those pts with diagnosis different from bmf ( cases) did not receive atg. systematic monitoring of ebv using quantitative pcr was employed. ebv reactivation was considered when dnaemia was superior to copies per ml. a total of pts presented ebv reactivation: / ( %) in cases without atg, / ( . %) in cases with low-dose atg and / ( %) in cases with intermediate-dose atg. median time of reactivation was the day + (range: + to + ). there was one single case of ebv-induced ptld which belonged to the intermediate-dose atg group. all cases (including the one with ptlp) were successfully treated with rituximab at mg/m /week. median number of doses employed were (range: [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . mortality due to ebv was % in our series. limited donor availability in the form of either matchedrelated or unrelated donors drew attention to haplo-hct. donors of haplo-hct shares an exact haplotype with the recipient but is mismatched for hla genes on the unshared haplotype. most studies have shown promising results in terms of graft success and survival. in this study our aim is to present the early and late outcome of our haplo-hct patients. between and , we retrospectively evaluated haplo-hct in terms of post-transplant outcome, survival and complications who diagnosed and followed in our center. the median age of patients was (range: - ), ( %) of them were male recipients. the patient characteristics were given in table . thirteen patients ( %) had pre-transplant active disease. neutrophil and platelet engraftment was achieved in patients ( %) at a median day of (range: - ) and (range: - ). eight of patients ( %) died within month after transplant because of sepsis without achieving engraftment. haplo-hct is the second transplant in four of patients ( %): patient relapsed after full-matched related transplant, patient relapsed after / matched unrelated transplant, patient had engraftment failure after full-matched unrelated transplant, patient underwent haplo-hct in another center, followed in remission for years and relapsed. acute graft vs host disase (agvhd) was diagnosed in patients ( %), whereas chronic gvhd in patients ( %). four patients were relapsed ( %) during follow-up with median rfs of months. three patient had bk virus-positive hemorrhagic cystitis ( %). the distribution of infections is shown in figure, viral infections were detected later than fungal and bacterial infections. previous history of invasive pulmoner aspergillosis was detected in of the patients ( %) ( of them were re-transplanted) and received secondary prophylaxis. overall survival (os) of months and year were % and %, respectively. the choice between alternative graft sources depends on the urgency of the transplant on each institutional preference. higher complication and infection rates in addition to decreased survival compared with previous studies since our patient population consisted of refractory patients with comorbidities. preferable patient profiles undergoing haplo-hct may have better outcomes. disclosure of conflict of interest: none. the third month risk factor score: detection of disease at day + of allogeneic stem cell transplantation is the most important risk factor of worse prognosis m celis , c fernández , l yáñez , , a bermúdez , , a insunza , m colorado , m lópez-duarte , i romón , s garcía-Ávila , a cabero , a casado , m sánchez-escamilla , c richard and e conde , hematology department, hospital universitario marqués de valdecilla and university of cantabria before allogeneic stem cell transplant (sct), several index can provide prognostic information (ebmt risk score and hcti score). however, there is scarce data for the impact of the procedure during the first days of transplant, in which opportunistic infections and the acute graft versus host disease (gvhd) can induce harmful effects. our purpose is to create a risk factor score, measured at day + post sct, to give information about the prognosis of the patient. we retrospectively analyzed seven clinical (disease, fungal and cmv infection, acute gvhd, treatment with corticosteroids, karnosfsky status and length of hospitalization) and eight analytical (related to immune status, liver and lung function, nutritional status, iron overload and platelet count) risk factors in patients who underwent sct in our center between and and were alive at day + . data were collected as categorical variables and compared by χ -test. significant variables (p o . ) were evaluated in a multivariate logistic regression model. those who maintained statistical significance were then assigned a point value calculated with their β-coefficient. summation of the points resulted in a weighted risk score. median age was years (range: - ) and were males ( . %). the most frequent disease was aml, patients ( . %). the conditioning regimen was myeloablative in patients ( %) and bone marrow was the principal stem cell source ( %). donor was mrd in ( . %), mud in ( . %) and mmd in ( . %). the median followup was months (range: - ). the univariant model identified five prognostic variables: detection of disease by molecular, cytogenetic or flow cytometry asses in leukemias, myelodisplastic syndrome and multiple myeloma or image (ct scan ± pet) in lymphoma, dose of corticosteroids ⩾ . mg/kg/ day, ferritin ng/ml, albumin o . g/dl and platelet o per mm . table shows variables evaluated. in the multivariate model, the detection of disease (hr . , % ci . - . , p ng/ml (hr . , % ci . - . , p = . ), and platelet o per mm (hr . , % ci . - . , p = . ) were associated with higher risk of death and according with their-coefficient , and points were, respectively, assigned. the third month risk score (tmrs) was calculated in all patients and they were stratified into three groups: low risk of death (a, - points), intermediate risk (b, points) and high risk (c, ⩾ points). at years post sct, the estimated overall survival according with the tmrs was . % ± . in group a, . % ± . in group b and . % ± . in group c, po . . although the harmful effect of the first months of transplant can impact in the survival, the detection of disease at day + is the most determinant risk factor of death. this fact gives us the need of transplant in the best response and, in those who cannot, to plan promptly rescue strategies. the next objective is to confirm our risk score in a validation group. disclosure of conflict of interest: none. recombinant human soluble thrombomoduline alpha (rhtm) is a novel anticoagulant agent and approved for disseminated intravascular coagulation in japan. the aim of the study is to evaluate the therapeutic potential of rhtm for sinusoidal obstructive syndrome/hepatic veno-occlusive disease (sos/ vod). we retrospectively studied times of allogeneic hematopoietic cell transplantation in toranomon hospital from june to june . we extracted the patients who used rhtm for dic and satisfied the diagnostic criteria of sos/ vod around the same time, because the use of rhtm for sos/ vod alone is off-label. data on the patients who used rhtm for days within days after transplantation were analyzed. the patients who were already treated with rhtm before the emergence of the first symptom or sign of sos/vod, and who started rhtm over days after the emergence of the first symptom or sign of sos/vod, were excluded from the [p ] analysis. to diagnose classical sos/vod (⩽ days after transplantation), we used two classical criteria of the modified seattle and the baltimore. for late-onset sos/vod ( day of transplantation), we used the criteria of ebmt. we defined as severe sos/vod, if the patients had renal (cr ⩾ times of baseline), respiratory (spo ⩽ % or the need for positive pressure) or central nervous system failure until weeks after the diagnosis of sos/vod. complete response (cr) was defined as the resolution of all the symptoms and the signs in sos/vod diagnostic criteria. a total of patients were extracted. the median age was years (range: - ) and patients ( %) was male. donor cell sources were ucb (n = ) and ubm (n = ). most of the prophylaxis regimen was the combination of ursodeoxycholic acid and dalteparin in patients ( %). classical sos/vod was diagnosed in ( %) and patients ( %) by the criteria of the modified seattle and the baltimore at the median day of (range: - ) and (range: - ), respectively. twenty-eight patients ( %) were diagnosed as late-onset sos/vod at the median day of (range: - ). severe sos/vod developed in patients ( %) (renal, n = ; respiratory, n = ; central nervous system, n = ). the elevation of transaminase was observed in patients ( %). the median interval from the emergence of the first symptom or signs of sos/vod to rhtm administration was days (range: - ). the median duration of rhtm use was days (range: - ). rhtm was used alone in patients ( %), in combination with dalteparin in ( %), with atiii in ( %), with dalteparin and atiii in ( %), with atiii and pge in ( %), and with pge in ( %). corticosteroid was used concomitantly in patients ( %). finally, patients achieved cr of sos/vod. the cumulative incidence of cr of sos/vod was . % at year after the administration of rhtm ( % confidence interval, . - . %). the median interval from the administration of rhtm to cr of sos/vod was days (range: - ). at year after transplantation, overall survival was . % ( % confidence interval, . - . %). from the administration of rhtm to weeks after the cessation of rhtm, hemorrhagic adverse events were observed. seven out of events were at grade - , and out of events were fatal (intra-abdominal in , gastrointestinal in , lung in and brain in ). we concluded that rhtm had a therapeutic potential for sos/vod. disclosure of conflict of interest: none. thrombopoietin receptor agonists for delayed and prolonged clinically-relevant severe thrombocytopenia after allogeneic hematopoietic stem cell transplantation v bosch vilaseca , i garcía cadenas , e roldán , s novelli , r martino , p barba , a esquirol, l díaz polo , g orti , d valcárcel and j sierra hematology department, hospital de sant pau, barcelona, spain and hematology department, hospital de la vall d'hebron, barcelona, spain persistent thrombocytopenia is a common complication after allogeneic stem cell transplantation (allosct), which dramatically increases the patients' dependence on hospital-based healthcare. thrombopoietin receptor agonists (tpoa) increase platelet counts in other clinical settings; however, the experience regarding their use after allosct is limited. we retrospectively evaluated tpoa efficacy in consecutive adult allosct recipients who received tpoa as a compassionate use for severe thrombocytopenia post-engraftment. five patients ( %) had primary and prolonged failure of platelet recovery, while had secondary thrombocytopenia: in seven of these cases, gvhd and/or a viral infection were the 'attributed' cause, while three were classified as post-allosct itp. all patients were dependent on platelet transfusions (median: times per week, range - ), with severe bleeding episodes in nine cases ( %) before tpoa onset. tpoa was started at a median of days after allosct (range: - ). romiplostim was used in ( %) cases. the median starting dose was μg/kg once a week (range: - μg/kg), while the final dose identified as most beneficial was μg/kg (range - μg/kg). eltrombopag was used in cases ( %), with an initial dose of mg daily; while the final doses were and mg daily. overall, / patients responded to tpoa therapy (defined as a stable platelet recovery to ⩾ /μl without transfusion support). the -day cumulative incidence of successful platelet recovery to ⩾ /μl and ⩾ /μl was % ( % ci, - %) and % ( % ci, - %), respectively, which were reached at a median of and days from start of therapy. five of the patients ( %) with severe bleeding at onset responded to tpoa ( of them without further hemorrhages) at a median of days (range: - ). at a median follow-up of days from start of therapy, three patients who responded continue tpoa treatment, while four other responders were able to discontinue it without recurrence of thrombocytopenia. among these patients, s the median total duration of treatment was days (range: - ). one patient lost his response within months after tpoa onset when he developed thrombotic microangiopathy associated with progressive gvhd. the remaining responder experienced disease relapse on day + after allosct. among the non-responders, had leukemia relapse during tpoa treatment, switched from romiplostim to eltrombopag without success and the remaining cases had active severe infections at tpoa onset ( hemorrhagic cystitis and cmv colitis) or non-controllable intestinal bleeding due to progressive gvhd. tpoa were well tolerated, with only patients showing adverse events (grade liver toxicity and grade fatigue), which did not lead to any change in therapy. six patients ( %) underwent follow-up bone marrow biopsies that did not display any increase in marrow fibrosis, including the patient who had myelofibrosis prior to allosct. although six patients in the study had active gvhd when tpoa was started, no patients showed worsening of gvhd. our results support the safety and efficacy of tpoa for the treatment of persistent thrombocytopenia in allosct recipients. further studies should compare the efficacy of romiplostim and eltrombopag and identify surrogate clinical and laboratory variables that are predictive of response to one (or both) of these tpoa. disclosure of conflict of interest: none. . clinical response in both groups was defined as improvement of organ function (no neurological residues; normalization of kidney function) and independence of red blood cell and platelet transfusions. results: the median time of ta-tma onset was . months ( . - . ) after hsct. thirty-five of patients ( %) were under treatment with calcineurin-inhibitors or sirolimus at the time the ta-tma occurred. in all cases, the immunosuppressive drug was stopped promptly. in patients, classical treatment was the primary therapy with a response rate of % (including four patients who switched to ec), whereas the response rate to ec treatment was significantly higher with % (p = . ). all patients receiving ec showed sufficient blockade of the terminal complement pathway after the second ec application (ch o %). despite the increased response rate for ec therapy, there was no difference seen between these two groups according to overall survival in weeks: classical treatment ( % ci - . ) vs ec treatment . ( % ci . - . ) p = . . the main cause of death differed significantly between this two treatment approaches with a therapy-related mortality due to infection with % in the ec group during tma therapy and none seen in the classical treatment group (p = . ). progressive gvhd was identified as an adverse prognostic factor in both groups (p = . and p = . ). conclusion: in our analysis, we show that ec shows a significant higher response rate in severe ta-tma patients compared to the classical treatment approach. however, in both groups the outcome remained very poor. since most patients presented with advanced, severe ta-tma, especially in the ec group, we hypothesize that earlier diagnosis and treatment of ta-tma and more effective prevention and treatment of infections will improve the outcome of patients with this complication. however, randomized studies are essential for comparison of these two treatment strategies to identify patient groups that benefit from a treatment with ec. disclosure of conflict of interest: none. tocilizumab as an effective treatment in cytokine release syndrome as an early peri-transplant complications in patients subjected to allogeneic stem cell transplantationproinfammatory/autoimmune patient/donor hla haplotype life-threatening early allogeneic hsct complication risk factor hypothesis m-g patrycja , , p-j beata , , s marcin , k ksenia , s-k agnieszka and sb aleksander , bone marrow transplantation unit, department of haematology, krakow university hospital and jagiellonian university collegium medicum cytokine release syndrome (crs) is classical complication of car t cells therapy, but also can be connected with early peritransplant complications in patients subjected to allogeneic stem cell transplantation. it can be connected with atg infusion, but also with inflammatory response during periengraftmetnt period (pre-engraftment syndrome and engraftment syndrome) and septic infections. severity of these complication can differ depending on patient's performance status and therapeutic options from just observation and vigilance to mechanical ventilation need. we would like to present small patient series (n = ) subjected to msd (n = ) and mud (n = ) with early transplant-related complications treated with combination of steroids (dexamethason) and tocilizumab. in two of them, tocilizumab was used after second dose of atg. both patients present hypotonia with decreased urine output, prompt increase of creatinine level and presence of acute inflammatory parameters crp, beta microglonulin and procalcitonin level, fluid retention and decreased oxygen saturation. in another one patient, these symptoms were connected with pbsc infusion from unrelated donor. in later two patients, we observe almost the same clinical presentation in preengraftment phase. in every of patients infection was ruled out-blood cultures were negative. all these patients were treated with tocilizumab in a single dose of mg/kg. in all patients, we observed prompt response-normalization of clinical state, renal function, oxygen saturation and decrease of inflammatory factors-crp, procalcitonin and beta microglobuline. discussion: crs is a rare complication connected with early phase of allogeneic stem cell transplantation. there were no results of treatment with steroids, reduction of a dose of cyclosporine a according to decreased renal function, but all patient completely/fully recovered after single dose tocilizumab treatment. all our patients were subjected to reduced intensity protocols, what might be a risk factor to develop crs because non complete depletion of the patient origin monocytes/macrophages active population. we also analyzed other factor connected with crs in early peritransplant period finding possible connection with s proinflammatory hla phenotype. it was obvious in the patient one our patients with peri-engraftment phase crs-he was diagnosed previously with rheumatoid arthritis b pos, dr . in three of five, we have found sle predisposition in hla phenotype (drb * /dqb * or drb * / dqb * ), in later one-ra associated hla antigen drb .these patients were analyzed correlating with historical cohort of additional five patients with mortal and another three with very severe early peri-transplant complications and in all we have found the same 'sle or ra hla phenotype'. because small number of analyzed patients and documented high frequency of these haplotype in population, this is still an opened question is proinflammatory/autoimmune hla phenotype connected pathogenically with predisposition to develop severe transplant complications and are we able to treat all these patients with combination of steroids with tocilizumab. further analysis is needed. disclosure of conflict of interest: none. transplant-associated thrombotic microangiopathy (ta-tma) is a severe complication post haematopoietic cell transplantation (hct) leading to high mortality rates. however, outstanding questions regarding its diagnosis, pathophysiology and treatment remain in the literature. recent studies suggest evidence of complement activation, implicating that complement inhibition may be an effective alternative treatment strategy in refractory patients. therefore, we hypothesized that increased complement activation can be detected in ta-tma patients using a functional assay, the modified ham test. we enrolled consecutive patients with ta-tma according to the international working group criteria from january to june . as controls, we studied patients with graft-versushost-disease (gvhd). complement activation was detected using the modified ham test, a cell proliferation assay based on the susceptibility of a pnh-like cell line to complement activated serum. normal human serum was used as a negative control and lipopolysaccharides(lps)-incubated normal serum as a positive control. all samples were tested in triplicates and twice. we studied ta-tma patients transplanted from unrelated / matched ( ) or / mis-matched ( ) donors, identical ( ) and haploidentical ( ) siblings. all patients presented severe acute and/or chronic gvhd. ta-tma presented at median + ( - ) day post-transplant. in the control group, we studied two patients with steroidsensitive grii and two with steroid-refractory griv acute gvhd. we were able to detect significantly increased complement activation in the serum of ta-tma compared to gvhd patients (p = . ). based on previous studies and present controls, percentage of non-viable cells higher than % was considered a positive modified ham test, indicating increased complement activation in four ta-tma patients. regarding treatment outcomes, two patients with a negative modified ham test responded to cyclosporine cessation and steroid administration. plasma infusion with/without plasma exchange was initiated in seven patients. however, only three of them responded to second-line treatment. the modified ham test result was significantly increased in refractory patients (p = . ). the terminal complement inhibitor eculizumab was administered in one refractory patient with a positive modified ham test and renal failure at presentation. despite delayed initiation ( days post ta-tma diagnosis), response was observed after three doses of eculizumab including evidence of reduced hemolysis, schistocytosis and transfusion needs. however, the patient succumbed to complications of end-stage renal disease ( days post ta-tma diagnosis). among ta-tma patients, succumbed at a median + ( - ) day to transplant-associated complications, related to gvhd and infections from multi-resistant pathogens. ta-tma is associated with increased morbidity, mortality and severe complications, including gvhd. unlike gvhd, increased complement activation was observed in a significant portion of ta-tma patients. complement inhibition seems an encouraging therapeutic option in these patients. given the lack of robust functional assays for complement activation, the modified ham test may be useful for early recognition of patients that would benefit from complement inhibition. . this proposal includes, along with the 'classical sos' (cases diagnosed before day + ), the new type 'late onset sos' (cases diagnosed afterwards). new ebmt criteria for severy grading classify cases of sos into four grades (mild, moderate, severe, and very severe). the aim of this retrospective study is to analyze the cases of severe/very severe, both classical and late onset sos, occurred in our unit during the most recent period of time. we studied the last pts, with a minimum follow-up of days, who underwent allo-hsct in our center (november -august ). pts were male and female. median age was years (range: - ). baseline diseases were: acute leukemias ( ), lymphoproliferative disorders ( ), myelodysplastic syndromes ( ), chronic myeloproliferative diseases ( ), multiple myeloma ( ), and bone marrow failures ( ) . donor was unrelated in cases, and related in (including haplo-identical). conditioning regimen was: busulphan-based ( ), melphalan-based ( ), tbi-based ( ) , and others ( ). all patient received prophylactic [p ] s ursodeoxycholic acid. progenitors source was pb in , and bm in . five patients developed severe/very severe sos ( % incidence); were classical (at days + , + and + ), and were late onset (at days + and + ) (see table ). four cases had received conditioning with a busulphan (iv)-based regimen (doses from . to . mg/kg), and one case with tbi plus cyclofosfamide at high doses. all cases presented with right upper quadrant pain, jaundice, ascites, weight gain, hiperbilirrubinemia, and renal function impairment. all but one had increased transaminases. the five cases were treated with defibrotide, in spite of which all of them died. considering that overall day + mortality was %, severe/ very severe sos was the most important cause of death of the series. [p ] although milder forms of sos might resolves within weeks, the most severe forms are still associated with a very high mortality rate. prophylaxis with defibrotide (the drug currently licensed for treatment) for high-risk patients has not been sufficiently studied yet. therefore, a high index of suspicion, early detection and early therapy are the only ways to try to reduce mortality due to sos in the hsct setting. disclosure of conflict of interest: this research has been performed entirely with public financial support. the royal marsden hospital, sutton, uk; anthony nolan research institute, london, uk and university college london, london, uk secondary poor graft function (spgf) complicates up to % allogeneic hcts, and is associated with increased mortality and poor quality of life due to recurrent infections and the need for ongoing blood product support. potential interventions include a second allograft using further conditioning, however many patients with spgf have a reduced performance status and are at an increased risk of complications from this procedure. unconditioned haematopoeitic progenitor cell (hpc) top-ups are associated with a high risk of gvhd if unmanipulated cellular products are used. cd + selection offers an attractive alternative, but incurs a loss of up to % hpcs and is an expensive procedure, unavailable to many centers internationally. alemtuzumab, a monoclonal anti-cd antibody, is routinely used in allogeneic transplant conditioning in the uk to prevent gvhd. we report the results of a retrospective study examining the efficacy of alemtuzumab conditioned hpc top-ups for spgf. data pertaining to patients who had undergone a second infusion of hpcs from their original donor were identified from our hospital-specific promise database. those who met the criteria of spgf defined as ⩾ of hemoglobin × /l without support. patients ( pediatric, adult) who underwent initial allogeneic transplants for malignancy ( ) or bone marrow failure ( ) received an alemtuzumab conditioned hpc top-up for spgf at our center - . the diagnosis of spgf was made at a median . months post allograft (range - ) with trilineage cytopenias in patients and bilineage cytopenias in patients. all patients had received transplants from / ( patients) or / ( patients) matched unrelated donors. the median interval between initial transplant and top-up was days (range - ), and a median cd dose of . × /kg recipient weight (range . - . ) was infused. % patients achieved haematological improvement (hi) at a median days post-top-up (range - ), with the only failure to achieve hi seen in the patient who had received the lowest cd dose ( . × /kg). one patient developed grade i agvhd post top-up but no grade ii-iv agvhd was observed. year os was % and year os % following hpc top-up. deaths occurred due to infection at , and months post top-up, and one due to relapse of a prior non-haematological malignancy. patients had an aplastic or hypocellular bm trephine pre-top up, which was repeated at days post topup in patients, of whom had a normocellular bm trephine, while remained hypocellular. alemtuzumab conditioned hpc top-up appears an effective intervention for spgf with results comparable to those of cd selected top-ups, and therefore represents a feasible alternative. larger studies are needed to exclude complications including viral reactivation and to investigate immune reconstitution following this procedure. disclosure of conflict of interest: none. high dose chemotherapy (hdt) followed by autologous stem cell transplantation (asct) has shown to improve outcome in patients with relapsed/refractory diffuse large b cell lymphoma (dlbcl). in the rituximab era, the benefit of asct has been debatable as prior study (coral study) has shown that patients who received r-chop as induction chemotherapy & responded to salvage chemotherapy had a poorer outcome following asct compared with those who received chop alone. in addition, it remains unclear whether addition of rituximab to standard high dose beam regimen provides any additional benefit. we retrospectively analyzed dlbcl patients receiving high dose beam (n = ) or rituximab +beam (r-beam) (n = ) followed by asct for relapsed/ refractory dlbcl since . all patients who received chop (n = ) ± rituximab (n = ) as first line therapy and who received ⩽ lines of salvage chemotherapy before asct were analyzed. rituximab was given at the dose of mg/m on day + and + of asct. twenty-two ( %) patients in beam group and all the patients ( %) in r-beam group received rituximab-based salvage chemotherapy prior to asct. the year overall survival (os) was % and event-free survival (os) was % for the whole cohort. r-chop induced patients did not fare any worst after asct than chop induced patients ( year os vs %; p = . ). there was a trend towards better survival in patients with pre-transplant disease free interval (dfi) months compared to those with dfi /μl) time was days and days, respectively. median platelet recovery ( /μl) time was days and days, respectively (p = . ). ten year os ( % r-beam vs % s beam, p = . ) and efs ( % r-beam vs % beam, p = . ) were also comparable between both groups. hdt with beam and asct remains beneficial for patients with relapsed/ refractory dlbcl. it should be offered to all patients who respond to salvage chemotherapy with the expectation that they fare no worse than patients who do not receive rituximab in the induction chemotherapy. addition of rituximab following the standard beam for hdt and asct does not compromise haematopoietic recovery, but does not result in improved outcome in our study. prior use of rituximab during first-line or salvage therapy in most of the patients of r-beam group might have negated the beneficial effect of r-beam over beam. ( ) . in this study, we aimed to develop a cns targeted chemotherapy regimen, which has lower toxicity and higher complete remission rates, in combination therapy. eight patients with secondary cns lymphoma (scnsl) and two with primary cns lymphoma (pcnsl), followed between the years and , were included in the study, retrospectively. the patients were histologically diagnosed with biopsy and underwent autologous stem cell transplantation (apkht). all patients were treated with r-idaram/ rt (radiotherapy)/subsequently autologous stem cell transplantation (apsct) with r-beam protocol. the r-idaram regime consists of the following substances: rituximab mg/m , cc/h infusion, day ; cytosine arabinoside . gr/m i.v., h infusion, days and ; dexamethasone mg, h infusion, days , and ; idarubicin mg/m i.v., min infusion, days and ; methotrexate gr/m ( gr/m at years old-patients), h infusion, day ; and cytosine arabinoside mg plus methotrexate mg, intrathecally, days and . the patients included seven males and three females. the median age was years (range: - ). six scnsl patients were diagnosed in the application and two of them were diagnosed during r-chop chemotherapy (ct) protocol. five patients ( %) were stage ivb, and the others ( %) were stage iiib at diagnosis. after two or three chemotherapy cycles, patients were mobilized with growth factor support and median . cells per kg (range: - ) stem cells were collected. then, at a dose of - cgy cranial rt was administered for days. after the third cycle of r/idaram, the state of remission was evaluated by cranial mri and lumbar puncture (lp). all patients achieved complete remission. neutrophil engraftment occurred at a median of days (range: - ) and platelet engraftment occurred at a median days (range: - ). after apkht, three patients relapsed and died at the fourth, ninth, and thirteenth months. grade i-ii manageable neurological toxicity occurred in two patients. the median follow-up time was (range: - ) months. the five-year overallsurvival (os) was %. serious signs of infection were not observed in patients during transplantation. in pcnsl and scnsl, a standard treatment regimen has not yet been found. apsct with r-beam following modified r/idaram/rt is a curative and applicable therapeutic regimen with low toxicity, which can provide high rates of long-term survival and disease-free survival. despite the advent of novel therapies, autologous hematopoietic stem cell transplantation (ahsct) following melphalan (m)-based conditioning remains the standard of care for patients with multiple myeloma who are eligible. still, the majority of patients experience disease progression and ultimately succumb to their disease. we hypothesize that integrating novel agents in the conditioning is feasible and safe and may increase complete remission rates and overall survival. we completed a phase i, dose escalation study of carfilzomib (c) added to a backbone of bendamustine (b) and melphalan. all patients received a fixed dose ( mg/m ) of c on days (d) − , − , − , − , − and − . in addition, patients were conditioned as described in table . due to dose-limiting toxicity in cohort , the study was amended after the first patients. subsequently, the dose of m was reduced to mg/m and the d + dose of c was omitted, per oversight of a data safety monitoring board. fifteen patients were enrolled, males and females. median age was years ( - ). performance status was ⩾ % (kps) in all patients. per the international staging system (iss), patients had stage i disease, had stage ii, had stage iii, and had unknown staging. three patients had high-risk cytogenetics: with t( ; ) and with deletion p. four patients had undergone a prior ahsct. disease status at enrollment was stable disease (sd) (n = ), partial response (pr) (n = ), or very good partial response (vgpr) (n = ). median cd + cell dose infused was . × /kg ( . − . × ). median follow-up was . months ( . - . ). all fifteen patients are evaluable s for engraftment. median time to neutrophil engraftment was d ( - ). one patient died before achieving platelet engraftment. for the remaining patients, median time to platelet engraftment was d ( - ) . non-hematologic toxicities included grade acute mucositis (n = ), lower gi complications (n = ), electrolyte disturbances (n = ), transaminase elevation (n = ) renal insufficiency (n = ), atrial fibrillation (n = ), hypoxia (n = ), prolongation of the qtc interval (n = ), and grade acute sepsis (n = ), including death (cohort ) on d + . eight patients went on to receive maintenance therapy: with bortezomib, with lenalidomide, and with lenalidomide, dexamethasone, and c. posttransplant disease status was assessed per protocol by spep, spif, serum free light chains, and light chain ratio. twelve patients were evaluable on d + . two patients had sd, had vgpr, and had complete response (cr). eight patients were evaluable on d + . two patients had progressive disease, had pr, had vgpr, and had cr. the combination of cbm prior to ahsct appears feasible, with manageable toxicities, at the doses described in cohort b. a prolonged follow-up and a phase ii study are warranted to determine response rates and long-term outcomes. disclosure of conflict of interest: none. beam (carmustine, etoposide, cytarabine, melphalan) is the most frequently used high-dose chemotherapy regimen for patients with lymphoma referred for autologous hematopoietic cell transplantation (autohct). in recent years a novel conditioning protocol containing bendamustine instead of carmustine (beeam) has been proposed in order to potentially increase the efficacy. so far, however data on its safety are limited. the aim of this study was to retrospectively compare the safety profile of beam and beeam based on single center experience. consecutive patients with lymphoma treated with beam and patients treated with beeam between year and were included in the analysis. the median age was ( - ) years and ( - ) years, respectively (p = ns). clinical characteristics of both groups were comparable. patients with hodgkin's lymphoma constituted % in the beam group and % in the beeam. among those with non-hodgkin lymphoma the diagnosis of dlbcl predominated. beam treatment consisted of carmustine mg/m on day − , etoposide mg/m /d on days − to − , cytarabine mg/m /d on days − to − , and melphalan mg/m on day − . in the beeam regimen carmustine was substituted by bendamustine administered on days − , − at the total dose of mg/m i.v. peripheral blood was used as a source of stem cells. cd + cell dose was . ( . - . ) × /kg in the beam group and . ( − . ) × /kg in the beeam group (p = ns). time to engraftment and the rates of adverse events up to day + after autohct were the study endpoints. all patients engrafted in both study groups. median time to neutrophil . × recovery was ( - ) days after beam and ( - ) days after beeam (p = . ). median time to achieve platelet count × was ( - ) days and ( - ) days, respectively (p = . ). two patients died without progression before day + in the beam group, both due to bacterial infections. no early deaths were reported in the beeam group. the rates of grade or adverse events were comparable (see: table ). administration of bendamustine instead of carmustin as part of conditioning does not affect engraftment as well as toxicity profile of the regimen. therefore beeam may be safely used in patients with lymphoma undergoing autohct. its efficacy requires evaluation in prospective studies focused on homogenous patient populations. [p ] disclosure of conflict of interest: none. the baltimore group reported a low dose tbi-based nonmyeloablative conditioning regimen followed by t cell replete bone marrow, with post-transplantation cyclophosphamide (pt-cy) to control gvhd and graft rejection. based on the fact that in our facility conventional low dose tbi was not available, we wanted to explore whether tmi/tli could be a potential substitute the aims of our study was to explore if tmi/tli can be considered an effective substitute of tbi in terms of os, pfs and nrm. retrospective analysis was applied in cases of haploidentical hsct from april to october . all patients underwent baltimore conditioning associating fludarabine ( mg/m /day) day − to − , cy ( . mg/kg/day) on days − and − , and tbi gy in patients and tmi/tli gy in patient at day − . unmanipulated bone marrow graft was infused at day . postgrafting immunosuppression consisted of cy ( mg/kg/day) on day + and + , and mycophenolate mofetil for days, and tacrolimus or cyclosporine. no differences between the two groups was observed in term of age, gender diagnosis, disease status and donor type. % of patients engrafted in both arm ( / and / ). in tbi cohort vs tmi/tli cohort, the median time to anc /μl and platelet recovery /μl was not different ( and days vs and . days, p = . and . , respectively). in all tmi/tli evaluable patients, full chimerism was observed at days + . after a median followup of months in tmi/tli cohort and months in tbi arm, -year nrm was . % and . % (p = . ), respectively. the years os and pfs were not statistically different in the two groups % vs . %, p = . and . % vs . %, p . , respectively). the -year relapse incidence was % in tmi/tli group and . % in tbi group, p = . . no difference in incidence of both agvhd and cgvhd was observed between the two groups. this retrospective analysis suggests that tmi/ tli could be considered an effective substitute of low dose tbi, with a sufficient degree of immunesuppression of recipient, allowing engraftment and full chimerism. the gvhd both acute and chronic as well as the -y nrm were not different. disclosure of conflict of interest: none. comparison of the beeam conditioning regimen and the beam conditioning regimen in the autologous transplantation for hl and nhl s lozenov , p ganeva , y petrov , g arnaudov and g mihaylov the beam has established itself as a standard of care conditioning regimen in the autologous lymphoma hsct setting for most transplant centres in europe. yet however various other regimens are being compared with it in order to achieved better safety profile, better os and dfs, in order to improve results with chemoresistant and unfavourable patients. one such regimen is beeam (bendamustine, etoposide, cytarabine, melphalan).we aimed to compare the efficacy of the beam and beeam conditioning regimens and to compare their myelotoxicity profile. we evaluated retrospectively adult patients (mean age . with sd . ), receiving auto-hsct at the national specialized hospital for active treatment of hematological diseases in sofia, bulgaria for relapsed/refractory hl or nhl (of them mh - , dlbcl - , pmbcl - , fl - , lbl - , ptcl-nos - , aitl - , alcl - , mcl - , mzl - ) for the period from . . to . . with a follow-up of patients up to . . . ninety-two of the patients received the beam (as previously described -bcnu mg/m i.v. day − , etoposide mg/m i.v. days − to − , cytarabine mg/m i.v. days − to − , and melphalan mg/m i.v. day − ) regimen and received beeam regimen (bendamustine on days − and − ( mg/m ); cytarabine, mg/m intravenously daily, from day − to day − ; etoposide, mg/m intravenously daily, from day − to day − ; and melphalan, mg/m intravenously on day − ). the overall survival at the second and third years of follow-up (os- , os- ) and dfs at the third year, the cr rates and the average time periods to hematological recovery, were compared. the os at and years, respectively, was . % and . %, for beeam and . % and % for beam, the dfs at years was . % for beeam and . % for beam, provided that the differences did not have statistical significance (p . for os and p . for the dfs). the cr rate was . % in the beeam group versus % in the beam group. from the patients who received autologous hsct in stable disease or progression pre-transplant status (chemoresistnat patients), . % of the patients receiving beeam achieved cr at the first post-transplant evaluation versus . % respectively for the beam group. the mean time to hematological recovery for neutrophils was . ± . days (beeam) versus . ± . days (beam) and . ± . days (beeam) versus . ± . days (beam) for platelets. beeam appears to be a non-inferior alternative conditioning regimen to the standard beam, it shows a trend towards higher myelotoxicity, but also a trend towards better response rates in chemoresistant patients. [p ] disclosure of conflict of interest: none. autologous hematopoietic stem cell transplantation (asct) is widely used as a consolidation therapy in aggressive non-hodgkin's lymphoma (nhl) and recurrent or refractory classic hodgkin's lymphoma (hl). in mexico, the use of carmustine (bcnu) in the conditioning regimen of these patients is limited due to the lack of access to the drug and its high costs. this study aims to compare results in terms of toxicity, disease-free and overall survival between a group of patients treated with the standard regimen beam and another group treated with a scheme in which carmustine was replaced by cisplatin (peam regimen). a comparison of two groups with lymphoma was performed and the clinical aspects of cisplatin mg/m d . the characteristics were well balanced between the two groups. the mean time for neutrophil grafting ( per mm ) was significantly slower with beam than with peam ( vs days, p = . ), hospitalization time was longer with beam compared to peam ( vs , p = . ). on the other hand, proportion of patients who require red blood cell s transfusion was significantly higher in beam group ( %) versus peam group ( %) (po . ), but total amount of platelet transfusion did not differ between groups. about the toxicity, beam patients had significantly more frequent incidence and severity of nausea/vomiting ( % vs . %) and diarrhea ( . % vs %) compared to peam (p o . ). no significantly differences were observed in incidence of mucositis (p = . ). at the moment of the analyses, % of patient of the peam group were in complete response versus % of the patients treated with beam, but it did not represent a significant difference. disease-free survival and -year overall survival in the peam vs beam scheme were similar with % vs % (p = . ) and % vs % (p = . ) respectively but with less toxicity using the peam scheme. peam regiment is not inferior scheme compared with beam, because it shows similar outcomes in disease-free survival and overall survival. additionally, peam is a well-tolerated regime and beam scheme was associated with greater gastrointestinal toxicity such as nausea, vomiting and diarrhea, also greater hematology toxicity such as more requirement of red blood cell transfusion. [p ] disclosure of conflict of interest: none. cumulative busulfan exposure is associated with relapse following busulfan and cyclophosphamide myeloablative allogeneic stem cell transplantation for acute myeloid leukaemia e wong, d kliman , m chau , j szer , c nath , p shaw , d ritchie , d gottlieb and a bajel westmead hospital, new south wales, australia and royal melbourne hospital, victoria, australia the optimal busulfan exposure to reduce disease relapse in adult patients with acute myeloid leukaemia (aml) undergoing busulfan/cyclophosphamide myeloablative allogeneic stem cell transplant (allosct) is poorly defined. we retrospectively analysed busulphan pharmacokinetics (pk) and outcomes of patients who underwent busulfan/cyclophosphamide conditioned allosct for aml from to . busulfan was administered intravenously over days ( . mg/ kg/d for days followed by . mg/kg for days). peripheral blood was obtained for busulfan pk after the first dose. subsequent doses of busulfan were decreased if daily busulfan exposure (area under the curve; auc) was anticipated to exceed μm per min/day. cyclophosphamide was dosed at mg/kg. the primary outcome was the cumulative incidence of relapse (cir) accounting for non-relapse mortality (nrm) as a competing risk. independent variables analysed included age, sex, cytogenetic risk group, disease risk index (dri), donor type, stem cell source, t-cell depletion, and cumulative busulfan auc (cumauc) calculated as previously described. (figure ) . t-cell depletion was also associated with increased cir (hr . ; p = . ). patient age, sex, cytogenetic risk, dri and graft type were not significantly associated with cir. on multivariate analysis, cumauc μm per min remained significantly and independently associated with lower cir (hr . ; p = . ). cumauc was not associated with nrm, rfs, os, or the incidence of acute or chronic gvhd. figure . cumulative incidence of relapse in patients stratified by total busulfan exposure. [p ] cumulative busulfan exposure μm per min is independently associated with reduced relapse following busulfan/cyclophosphamide allosct for adults with aml. these findings support further evaluation of the optimal busulfan exposure to reduce aml relapse in a prospective clinical trial, whereby patients could be randomised to target cumauc μmol per min versus standard practice. hematopoietic stem cell transplant with busulfan and cyclophosphamide (bucy) based conditioning has a relatively high incidence of liver toxicity and sinusoidal obstruction syndrome (sos). busulfan and cyclophosphamide metabolites share the same glutathione conjugation in the liver metabolism. a small number of studies addressed different sequence of both drugs bucy vs cybu during conditioning. differences in liver toxicity, sos, transplant related mortality (trm), relapse incidence (ri) and overall survival (os) were reported favoring cybu conditioning. we decided to address the above issues at the umc ljubljana, slovenia. this was a retrospective study following patients with myeloid malignancies (aml, mds, mpn) with bucy (n = ) and cybu (n = ) conditioning through a three year period in a single institution. primary endpoint was detecting difference in liver toxicity by measuring levels of liver enzymes. secondary endpoints were incidence of sos, difference in trm, ri and os. patients characteristics between groups at the time of the transplant did not differ significantly. we observed significantly higher liver toxicity through elevated bilirubin and alt in the bucy . % than cybu . % patient group (picture ). the highest probability of liver toxicity was around d in the bucy group and in the second week after the transplant in the cybu group. the incidence of sos, trm and ri were comparable between the groups. there was no difference in os between the patient groups during the -month follow-up. bucy conditioning for hematopoietic stem cell transplant causes higher incidence of liver toxicity compared to cybu conditioning. there is no difference in sos frequency, trm, ri and os between bucy and cybu conditioning. prospective controlled comparison would be needed for further study of the subject. disclosure of conflict of interest: none. early monocyte recovery is associated with better overall survival after busulfan containing myeloablative conditioning allogeneic hematopoietic cell transplantation in patients with acute myeloid leukemia a lojko-dankowska the outcomes of allogeneic hematopoietic cell transplantation (allohct) in acute myeloid leukemia (aml) depend on different patient-, disease-and transplant related factors, including the dose and combination of agents used for conditioning. the aim of the study was to analyze the outcomes of allohct in patients with intermediate or high risk aml according to disease risk index (dri) who received myeloablative conditioning consisted of intravenous busulfan ( . - . mg/kg) combined with cyclophosphamide ( mg/ kg) or fludarabine ( mg/m ) between and in our institution. the published data indicate that the combination of busulfan (bu) and fludarabine (flu) seems to have more favorable toxicity profile than combination of bu and cyclophosphamide (cy), so bucy regimen has been substituted with buflu as the myeloablative conditioning for aml patients in our institution practice since . we evaluated the influence of type of regimen on transplant outcomes along with the impact of other potential prognostic factors, including age of patient, dri, donor type, hla and gender mismatches, stem cells source, and lymphocyte and monocyte recovery. the study group consisted of aml patients, median age years (range: - ), classified as intermediate (n = ) or high (n = ) risk according to the dri, who were conditioned with bucy (n = ) or buflu (n = ) followed by allohct from hla identical sibling (n = ) or - / matched unrelated donor (n = ). the stem cell were collected from peripheral blood (n = ) or bone marrow (n = ). gvhd prophylaxis consisted of calcineurin inhibitor combined with mtx plus atg in allohct from unrelated donors. engraftment was observed in all patients. the median time to neutrophil count ( . g/l) and platelet count ( g/l) recovery was shorter after buflu in comparison with bucy ( days vs days; p = . and days vs days; p o . ), however peripheral blood stem cells were used more often after buflu regimen than after bucy ( % vs %, p /mm on+ day after transplant ( -year os % vs %, p = . ) and intermediate vs high dri ( -year os % vs %, p = . ). in multivariate analysis higher amc after allohct remained the only independent favorable prognostic factor for os (rr . ( % ci . - . ), p = . ). our results suggest that early monocyte recovery after myeloablative bu containing conditioning allohct is significant favorable predictor of outcome. in our experience both bucy and buflu myeloablative regimens result in similar long-term survival after allohct in aml patients. [p ] disclosure of conflict of interest: none. the use of t-cell depletion as part of the conditioning protocol has the potential to improve the tolerability of allogeneic stem cell transplantation (hsct) through the reduction in graft versus host disease (gvhd). despite the wide spread adoption of this practice in many parts of the uk and europe, definitive recommendations regarding the most appropriate dose remain elusive. previous experience by our group with mg of alemtuzumab combined with fludarabine and busulfan based conditioning demonstrated good long-term outcomes with low rates of gvhd. however, due to concerns of high relapse risk especially in patients with high-risk myelodsypastic syndrome and acute myeloid leukaemia, we instituted a policy change in to reduce the dose of alemtuzumab in the conditioning protocol from a total of - mg. we conducted a retrospective analysis of all consecutive patients undergoing reduced intensity unrelated allogenic stem cell transplantation with fludarabine ( mg/m ), busulfan ( . mg/kg iv or . mg/kg iv) and alemtuzumab (fb c or fb c, respectively) conditioning for neoplastic myeloid disorders between and . patients were subsequently analysed in two cohorts; those receiving mg of alemtuzumab (n = ) and those receiving mg of alemtuzumab (n = ). apart from a decreased proportion of females in the mg alemtuzumab group, the cohort was balanced across the different dose levels ( table ). the longterm overall survival (os) of the entire cohort was good with a year os of %. no significant differences in overall outcomes across the two groups were observed with a year os of % in the mg group vs % in the mg group (p = . ). cumulative incidence of relapse (cir) and nonrelapse mortality (nrm) was % and % and % and % in the mg and mg groups, respectively. interestingly, age had a significant effect on nrm in the mg ( % age o , % age - and % age p = . ), but not in the mg group ( % age o , % age - and % age p = . ). the effect on relapse rate was not significant in either group (p = . and p = . , respectively). this retrospective analysis did not demonstrate an overall improvement in transplant outcomes with dose de-escalation of alemtuzumab from to mg. in particular, we did not see the anticipated improvement in relapse rate in this cohort. notably older patients seem to tolerate the mg dose better due to the lower nrm. prospective trials with accompanying translational work are required to determine the optimal dosing and schedule for this group of patients. disclosure of conflict of interest: none. bendamustine was given at mg/m /d for the first pts then mg/m /d for the subsequent pts and finally at mg/m /d for the remaining pts ( pts). among the beam group, % had non-hodgkin's lymphoma (nhl) and % hodgkin's lymphoma (hl) compared to % and %, respectively, in the beeam group (p = . ). hhv- detection was performed by pcr for symptomatic pts (fever, rash or prolonged cytopenia). patients were housed in single bedrooms with air filtration and received the same supportive care. median age was ( - ) and ( - ) in the beam and beeam groups respectively and median of previous chemotherapy regimens was (range: - ). fifty two out of patients were male ( / in the beam group and / in the beeam group). pts were in cr ( . % vs . %) or pr ( . % vs . %) at time of transplant. there was no difference in terms of hematologic recovery (median = days (range: - )), blood and platelets transfusion, mucositis toxicity. there was no statistical difference in the incidence of acute renal failure when comparing the two groups. however, there was a very striking difference when considering the highest dose of bendamustine when compared as well to the two others doses of bendamustine (po . ) as to the beam group (p = . ). additionally, we also observed a high incidence of symptomatic hhv- infections ( . % vs . %, p o . ), digestive toxicity ( . % vs %, p = . ) and a longer hospitalization duration ( days (range: - ) vs days (range: - ), p = . ) for patients in the beeam group overall. with a median follow up of . and . months for beam and beeam respectively, overall survival ( % vs %), transplant related mortality ( % vs %) and event free survival ( % vs %) were comparable. overall, beeam regimen was associated with longer duration of hospitalization, higher rate of digestive toxicity and increased risk of symptomatic hhv- infection as compared to the beam regimen. in addition, higher doses of bendamustine ( mg/m /d for two consecutive days) were associated with unacceptable high rate of acute renal toxicity. with a still short follow-up, the absence of benefit on disease control together with higher short term toxicity does not allow to recommend the use of beam instead of classical beam. should it be used, we suggest that pts should be carefully monitored for renal toxicity and for hhv- infection in case of symptoms. disclosure of conflict of interest: none. high-dose treosulfan and melphalan for consolidation therapy in high-risk ewing sarcoma me abate, a paioli, a longhi, m cesari, e palmerini and s ferrari musculoskeletal department, rizzoli orthopaedic institutes, bologna, italy common toxicities observed after high dose chemotherapy with busulfan and melphalan for high risk ewing sarcoma (es) are generally well managed by current supportive care but some patients can develop severe complications. treosulfan is an alkylating agent that has recently been used as a substitute of busulfan to prevent potential serious complications related to busulfan. medical records of es patients undergoing autologous peripheral blood stem cell (apbsc) transplantation after intravenous treosulfan (treo) and melphalan (mel) from / / to / / were analyzed with regard to toxicity and outcome. patients were included into the study if they were eligible for the protocols activated in our institution for es and presented reasons that did predict potential complications related to busulfan, such as previous radiotherapy on axial skeleton/pelvis or coexistence of high risk of epilepsy. as consolidation treatment patients received intravenous treo g/m over days and mel mg/sqm with support of apbsc transplant and use of granulocyte colony stimulating factor. in those patients with lung metastases total lung irradiation was performed at least months after treomel. frequency of toxicity for treomel was recorded with at least months of follow-up and was evaluated according to nci ctg common toxicity criteria. the median age at diagnosis of patients receiving treomel was years (range - years), males and females. patients had localized disease at diagnosis with poor radiological or histological response to standard chemotherapy; one patient had lung metastases at diagnosis and one patient had relapsed disease with lung metastases. before receiving treomel the primitive tumour underwent radiation therapy in cases ( pelvis, cervical vertebra, sacrum), surgical resection in one case(tibia) and surgical resection plus radiation therapy in one case (fibula). patients showed eeg abnormalities at high risk of developing epilepsy. the median number of infused cd + cells was . × /kg (range . - . ). febrile neutropenia occurred in / patients and lasted one day in patients and days in patients. median time to granulocyte engraftment was days (range - days); median time to platelet engraftment was days (range - days). only one patient needed red blood cells transfusions; patients needed platelet transfusion and patients needed platelet transfusions. none developed grade - stomatitis or grade - [p ] hematuria or grade - liver toxicity. surprisingly, a patient became pregnant after year and months from transplantation. with a median follow-up of months (range - months) patients are alive in complete remission, one patient is alive with relapsed disease and one patient died for disease progression. these results, related to a limited cohort of patients, confirm the lower toxicity observed for treosulfan with respect to busulfan. although more data are needed to clarify the role of treosulfan in es, the impact of potential severe complications observed with busulfan, including infertility, should suggest its replacement with treosulfan in selected cases. disclosure of conflict of interest: none. immunoadsorption procedures prior to haploidentical allogeneic pbsct could prevent graft failure in patients with hematological malignancies displaying anti-donorspecific hla antibodies donor-specific anti-hla antibodies (dsa) have been shown to be associated with a high risk of rejection in solid organ transplantation and with graft failure (gf) in allogeneic hematopoietic stem cell transplantation. a combination of anti-cd (rituximab), plasma exchange (pe), and ivig in patients with additional buffy coat infusion in among them prevented graft loss in all patients that became c q negative before sct. we addressed the question whether immunoadsorption in combination with rituximab can also be applied in patients with dsa to prevent graft failure in haploidentical pbsct. four patients with acute myelocytic leukemia or myeloma in second complete remission were enrolled. the presence of dsa was determined by luminex at pre bmt checking. immunoadsorption was performed with polyclonal sheep anti-human igg adsorbers (miltenyi biotec gmbh, germany) on life apheresis system. in addition all patients received rituximab mg/kg bw in a single dose. patients were conditioned with a reduced intensity regimen comprising tbi gy, cyclophosphamide mg/kg, and fludarabin mg/m . all patients received cyclophosphamide post bmt (ptcy) mg/kg. non-t-cell depleted pbsct were transfused in a sequential manner in doses each. the data of the patients and treatments is summarized in table . two patients had a normal hematopoietic reconstitution and are alive at + and + months post-transplantation, one with hepatic gvhd; chimerism was % in peripheral blood on last follow up. one patient died following a graft failure. by a combination of rituximab and repeated immunoadsorption prior to allogeneic pbsct the titer of dsa could be lowered sufficiently to enable engraftment. ia turned out to be a safe procedure without relevant clinical side effects. hematopoietic reconstitution was in the normal range in of evaluable patients. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative option for patients with beta thalassemia major. however, the availability of hla-matched related donor remains the main obstacle for allogenic hsct. although, a few studies have been reported, experience with hla matched unrelated donors is limited. we present the result of children with beta thalassemia major who received allogeneic hsct from hlamatched unrelated donors with using a novel conditioning regimen. we retrospectively assessed unrelated hsct in children with beta thalassemia major. all patients received busulphan (bu) based myeloablative conditioning regimen. busulphan was used according to weight adjusted doses. in addition, all patients received fludarabine mg/m in days, cylophosphamide mg/kg in days, thiotepa mg/kg in one day and atg mg/kg in days. cyclosporin-a and mtx were used for graft versus host disease (gvhd) prophylaxis. donor chimerism was evaluated in the peripheral blood on days + , + and + . the median age of the patients was . years (range month- year). two of the patients were grouped in class i and rest of them were class ii. the median serum ferritin level was . ng/ml (range, - ). all of s the donors were matched / with high-resolution hla typing in gvhd direction but three of them / with graft failure direction. twenty-three of them received bm (median tnc: . x /kg) and pbsc (median mnc: . x /kg) with median cd + cell number . x /kg. the median neutrophil and platelet engraftment days were and days in pbsc and and days in bm group, respectively. grade i-iv acute gvhd was observed in patients ( %) and only one experienced limited chronic gvhd with only skin involvement. mild to moderate vod was seen in patients ( %) and treated with defibrotide successfully. all patients except one are alive with full donor chimerism (between - %) with a median months (range - months) follow-up. one patient died because of cmv pneumonia. these data show that the results of hsct from unrelated donors in selected low risk thalassemia patients may be comparable to hsct of matched sibling donors. however, it needs further studies with long term follow up and larger study population. disclosure of conflict of interest: none. table . data about cytogenetic risk of group patients were available only in individuals. differences between groups were analyzed by t-student and chi square tests. survival was analyzed by kaplan-meier method and differences in survival between groups were evaluated by log rank test. no differences were found between groups regarding gender, sc source, disease status at sct, type of donor and number of cd + cells infused. patients in group were significantly older (median age for groups and : vs , p = . ). gvhd prophylaxis protocols included atg in a higher frequency in group . no differences between groups and were observed in neutrophils recovery (median days to anc /μl: vs respectively, p = . ) and platelets recovery (median days to platelets /μl: vs respectively, p = . ). patients in group required more red cell transfusions (median packed rbc: vs , p = . ). no differences were observed regarding platelets transfusion requirements or length of hospitalization. post-sct os was significantly better in group ( years-os group : %; group : %; p = . ) (figure ). there were no significantly differences between groups regarding frequency of mucositis, diffuse alveolar haemorrage, sepsis, acute and chronic gvhd. vod was more frequent in group ( / vs / , p = . ). trm mortality was higher in group ( / vs / ), being this difference no statistically significant (p = . ). as it was reported by others, the use of fludarabine-based conditioning regimen was associated with a significantly better post-sct os and a reduced frequency of vod in aml patients. reduction in trm and differences in the frequency of described complications are not statistically significant probably due to the small size of this sample. since march , given the limited availability of melphalan, we administer the beac regimen (carmustine, etoposide, cytarabine, and cyclophosphamide), instead of the gold standard conditioning regimen beam, followed by autologous haematopoietic cell transplantation (ahct) in relapsed or refractory hodgkin (hl) and non-hodgkin (nhl) lymphoma patients. the primary goal of this analysis was to assess the immediate related toxicity of this alternative regimen. we used beac (carmustine mg/m , etoposide mg/m , cytarabine mg/m , and cyclophosphamide mg/kg) in consecutive lymphoma patients ( hl, nhl) who underwent ahct for relapsed or refractory disease. the median age of the patients was . years ( - ). they all received peripheral stem cell grafts with a median cd + cell dose of . × /kg cells ( . - . ). disease status post salvage treatment (at ahct) was complete remission (cr) in , partial remission (pr) in and progressive disease in . the disease was chemosensitive to salvage therapy in / patients. median follow up was days ( - ). toxicity was assessed according to the who toxicity scale grading. all patients engrafted successfully. median time for engraftment was day + (d+ ) for neutrophils ( /mm ) and d+ for platelets ( /mm , without transfusion within the previous days). patients were hospitalized for a median of days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . no treatment-related mortality occurred. two patients died due to disease progression (both nhl patients, on d+ and d+ ). toxicity assessment until d+ is presented in table : moreover, no hemorrhagic cystitis or macroscopic hematuria, and no cardiac events were encountered. febrile neutropenia was recorded in and bacteremia in patients ( gram+, gram-, / related to central venous catheter), with fever ≤ grade in all cases. during d+ - two patients presented fever of unknown origin, and patients had upper or lower respiratory infections, with no other adverse events being recorded. in terms of disease best response within months post ahct ( / patients evaluated), patients achieved or sustained cr, pr ( of these patients eventually died due to disease progression), relapsed and succumbed due to disease progression (no response). according to our preliminary results, the early toxicity profile of beac is very low, the regimen is easily tolerable for the patients, and without any treatment-related mortality. its use as an alternative conditioning regimen in ahct for lymphoma patients seems feasible. further investigation including more patients and comparative analysis to other conditioning regimens are warranted for reliable conclusions on the toxicity and efficacy of beac. disclosure of conflict of interest: none. veino-occlusive disease (vod) is a potentially fatal adverse event caused by intravenous (iv) busulfan used in bone marrow transplantation (bmt) conditioning. the objective of this study was to identify determinants of vod in children treated by iv busulfan. this was a retrospective analysis of data collected in children from two bmt centers over years. vod was diagnosed according to modified seattle criteria. individual pharmacokinetic data, including busulfan area under the concentration-time curve (auc) and maximal concentration (cmax) were estimated in all children by using a validated bayesian approach. we examined the relationships between the occurrence of vod and available data in a learning (n = patients) and validation set (n = patients) obtained by random splitting. logistic regression was used as a continuous statistical model. in addition, we used classification and regression tree (cart) analysis, a machine learning and binary partitioning technique, to identify determinants of vod and their optimal cut-off values. the predictive performance of variables within both models was assessed by these results are compared with historical data from our service using beam as conditioning followed by auto-sct in lymphoma patients. nine patients were enrolled to receive neam: mitoxantrone mg/m day - to - , etoposide mg/m every hours and cytarabine mg/m every hours day - to - , and melphalan mg/m day - , followed by auto-sct. the median age was years ( - ); five non-hodgkin lymphomas (nhl) and four hodgkin lymphomas (lh). six patients were in partial remission (pr), two in complete remission (cr), and one with progressive disease at time of auto-sct. neam patients were compared with a historical control group of patients receiving beam regimen (n = ). differences between groups were analyzed by t-student and χ -tests. median cd + cells infused in neam and beam groups was . × ⁶/kg ( . - . ) and . × ⁶/ kg ( . - . ), respectively (p = . ). the median time to neutrophil recovery ( /μl) was days ( - ) and days ( - ) (p = . ) and median time for platelets recovery ( /μl) was and days ( - ) and days ( - ) (p = . ) respectively, for neam and beam patients. median duration of hospitalization was days ( - ) with neam and days ( - ) with beam (p = . ). among neam patients, % had one or more febrile episodes during neutropenia. no case of grade iii or iv mucositis was described. there was no transplant-related mortality (trm: %) associated with the use of neam regimen. at the present, all neam patients are alive, two of them in relapse ( %). due the difficulties in obtaining carmustine in our region, neam can be considered as a feasible alternative to beam. however, despite the sample was small enough to draw conclusions, we find that neam presents prolonged aplasia of significant value, we are currently exploring conditioning regimens followed by auto-sct in hodgkin's and non-hodgkin's lymphomas based on bendamustine, etoposide, cytarabine and melphalan. disclosure of conflict of interest: none. at present, decision-making about conditioning regimens for allogeneic hsct is based on patient's and donor's features, and disease characteristics. during the last years, terms as 'myeloablative/non-myeloablative/reduced-intensity' have been frequently employed in a confusing and unequal way among the different centers. knowing the expected intensity and myeloablative effect from each regimen is very useful and constitutes the aim of this analysis. we have analysed the severe neutropenia (anc o per mcl), and thrombocytopenia durations (platelets o per mcl), the need for platelet concentrates transfusion and the duration of the inpatient period of the allo-hsct carried out during the last four years in our centre. these data are reported according to the conditioning regimen used and to the type of transplant performed. then, they are compared among them in order to stablish intensity ranks. results: population characteristics are described in table : conventional intensive regimens (bu-cy , cy-bu , tbi-cy) reported more days of severe neutropenia and greater need of platelet concentrates transfusion. the regimens with less days of severe neutropenia, less need of platelet concentrates transfusion and fewer days of admission were flu-bu and flu-bu . allotransplants carried out with stem cells from bm presented more days of severe neutropenia and longer hospital stay. similar platelet transfusion need was reported. haplo-identical allotransplants reported more days of severe thrombocytopenia, but were not asociated to longer neutropenia or longer hospital stay than the others. these data are described in detail in table . flu-bu: the number ( , or ) expresses the doses of busulphan administered at . mg/kg/day. pc: platelet concretates data is expressed in medians. within the analysed conditioning regimens, an intensity rank is stablished regarding the myelosupression induced (in descending order): conventional intensive regimens (cy-bu , bu-cy , tbi-cy), flu-mel, flu-bu , flu-bu and flu-bu . all of them induced severe neutropenia and thrombocytopenia, and for that reason they must be considered myeloablative regimens. disclosure of conflict of interest: none. myeloablative allogeneic stem cell transplant for aml and mds: the impact of advanced age in the outcome m sánchez-escamilla* , , s garcía-Ávila , l yáñez san segundo , , ma bermudez rodriguez , , mm colorado araujo , , a casado diez , m celis alvarez , a cabero martinez , c fernandez martinez , a insunza garminde , , c richard espiga , and e conde garcía , allogeneic hematopoietic stem cell transplantation (allo-hsct) is the only curative option in high risk myeloid hematological malignancies. myeloablative conditioning (mac) regimen has been proven to be effective in the control of high risk diseases in advanced age patients. objective: the aim of this study was to analyze the efficacy of myeloablative allo-hsct in two cohorts of patients considering their age at transplant. we also analyzed the incidence of acute and chronic graft versus host disease (gvhd) and procedure related outcomes who underwent to myeloablative allo-hsct were retrospectively analyzed. the median age was years (iqr - ). both groups were divided regarding their age at allo-hsct [group , age ⩾ years (n = ) and group , age o years (n = )]. patient´s characteristics are shown in picture . data were collected as either continuous data and compared by two-tailed unpaired t-test or mann-whitney test, or as categorical variables and compared by chi-square. the procedure related outcomes were analyzed with the kaplan-meier test. the incidence of acute gvhd grade ii-iv was similar in both groups ( . % in group and . % in group , p = . ). the mean day to acute gvhd (grade ii-iv) development was days in group and days in group . the most involved organs in both groups were skin (group : . % and group : . % [p = . ]) and gut (group : . % and group : . % [p = . ]). at day + post-transplant patients were alive and evaluable for chronic gvhd. the incidence of cgvhd development was similar between group and ( . % versus . %, respectively, p = . ). however, severe grade s of cgvhd was high in group patients ( . % versus . %). with a median follow up of months (iqr, - ) the probability of os was significantly low (p = . ) in group ( . % +- . ) compared with group ( . % ± . ). pfs was also significantly low (p = . ) in group ( . % +- . ) compared with group ( . % ± . ). trm at months was higher in group compared with group ( . % versus . %). mortality due to relapses was also higher in group ( . % versus . %). most of the patients died during the first month. comparing both groups at this time ( months post-transplant), trm was higher in group compared with group ( . % versus . %). deaths due to relapse were also higher in this group ( . % versus . %). in our series, myeloablative conditioning regimen provides good survival rates and disease control in high risk hematopoietic diseases, however in patients aged ⩾ years confers high toxicity. it may be necessary to evaluate other strategies in this group of patients. disclosure of conflict of interest: none. allogenic hematopoietic cell transplantation (hct) is reserved for a group of high risk multiple myeloma (mm) patients having relapsed after high-dose melphalan and autologous transplantation. in general, reduced-intensity conditioning (ric) regimen are applied in this patient group with the aim of reducing transplant related mortality (trm). however, relapse of disease remains a major challenge after allogeneic hct. to address this issue, we added radioimmunotherapy (rit) to a conventional ric regimen. we have used a rhenium anti cd antibody in combination with a ric conditioning regimen. this ß-emitter leads to a so called 'cross-fire' effect allowing for bone marrow doses of gy and in parallel may target cd on myeloma cells. we hypothesized that this strategy may decrease the incidence of relapse. so far, we have treated nine patients with high risk relapsed multiple myeloma. all patients had received one (n = ), two (n = ) or three (n = ) high-dose regimens and autologous hct. conditioning therapy was flu/mel (n = ), flu/ bu (n = ) or flu/treo (n = ). flu/cy and gy tbi were applied before haploidentical transplantation. patients received g-csf mobilized pbsc from unrelated (n = ) or haploidentical (n = ) s donors. either tacrolimus/ methotrexate/ bortezomib or cyclosporine a/ methotrexate were used for gvhd prophylaxis. early extramedullary toxicity was limited. neutrophil and platelet engraftment was timely and complete in time in seven of nine cases. all patients achieved full donor chimerism around day fifteen after hct. severe acute graft-versus-host-disease (gvhd grade iii-iv) occurred in two patients and was lethal in both cases. two patients have experienced extramedullary relapse, one of them in the central nervous system and the other in the soft tissue. in two patients, a transplantation-associated thrombotic microangiopathy (ta-tma) was diagnosed. four patients are alive and in complete remission. we conclude that the combination of a ric regimen with a rhenium anti-cd radioimmunotherapy is save and feasible. the incidence of gvhd, ta-tma and extramedullary relapse will be monitored closely and will be presented in a larger patient cohort. disclosure of conflict of interest: none. the sirolimus/tacrolimus (sir/tac) combination has been associated with a better outcome after allogeneic hematopoietic stem cell transplantation (allo-hsct) when compared with conventional prophylaxis for graft vs host disease (gvhd) as cyclosporine/methotrexate in the true reduced-intensity conditioning (ric) setting but not in the myeloablative setting. in moderate-intensity regimens as thiotepa/busulphan/fludarabine (tbf), the sir/tac combination has not been evaluated. from january to december , all consecutive ric-allo-hsct recipients who received sir/tac combination to prevent gvhd in three spanish institutions were included in the study. the reduced-toxicity regimens used in this study where: (a) intravenous busulphan ( . mg/kg) and fludarabine mg/ m (bf), or (b) thiotepa days - and - and mg/kg if yrs old or mg/kg if o yrs old) on days - and - added to the bf regimen (tbf). the gvhd prophylaxis with sir/tac was given as detailed elsewhere (cutler c blood ) and was consistent within the center. the outcomes of the procedure according to the conditioning regimen were analyzed. overall, patients were included: tbf and patients in the bf group, with a median follow-up of months (range - ) and no difference in the median age ( vs years old). there were more males ( % vs %, p = . ) and more female donors to male recipients ( % vs %, p = . ) and more patients with lymphoid diseases and previous asct in the tbf group ( % vs %, p = . ), whereas there were more unrelated donors in the bf group ( % vs %, p = . ). other baseline characteristics were balanced between the groups (table ) . sir/tac prophylaxis had to be discontinued in % and % patients in the tbf and bf groups, respectively. toxicity was the main reason for discontinuation in the tbf group. the most frequent toxicities were renal injury (tbf % and bf %) and neurologic impairments (tbf %, bf %). in the bf group, the main reason of discontinuation was relapse or a mixed chimera. patients who received tbf presented higher incidence of extensive chronic gvhd ( % vs %, p = . ), higher nrm at days ( % vs %) and at years ( % vs %, p = . ). there were no differences in os ( years) between both groups ( ± . % vs ± . %, p = . ) (figure) . there were no differences regarding to acute gvhd - ( % vs %, p = . ), acute gvhd - ( % vs %, p = . ), or relapse (up to years, % vs %, p = . ) between the groups, either. the combination of sir/tac as gvhd prophylaxis was associated with higher incidence of chronic gvhd and nrm in patients receiving conditioning regimen with tbf compared to those receiving bf. there were no differences in os between both groups. [p ] cr complete remission, pr partial remission, nr non remission, hct-ci hematopoyetic cell transplant comorbility index. disclosure of conflict of interest: none. reduced-intensity conditioning regimen with fractionated total body irradiation of gy and cyclophosphamide mg/kg for allogeneic hematopoietic stem cell transplant is well tolerated and offers a potential disease control as treatment of acute leukemia and lymphoproliferative disorders m adler, t girinsky , s koscielny, g ferini, s wittnebel, s mayeur, c chahine, m vanghele, s pilorge, c castilla-llorente and j-h bourhis the use of reduced-intensity conditioning regimens (ric) before allo-hsct is widely extended since it preserves the graft-versus-leukemia effect but reduces treatment related mortality. however, there exist different ric regimens with diverse outcomes and the choice of the ric regimen relies on the type of disease treated, experience of the center and previous therapies. this is a retrospective study of patients treated in our institution within / and / . the ric regimen consisted of fractionated total body irradiation (ftbi) of gy administered in consecutive days ( gy/day) and cyclophosphamide mg/kg given in days ( mg/kg/day). post-transplant immunosuppression consisted of csa started the day before allo-hsct and short mtx on days , and after transplantation. for patients receiving transplant from unrelated donors, anti-thymocyte globulin at a dose of mg/ kg ( . mg/kg/day for days at day - and - ) was used as part of the immunosuppressant therapy. patients (median age: years: range: - years) were included. the median hct-ci was . (range: - ). primary disease was multiple myeloma (mm) in ( %), al/mds in ( %), cll in ( %), nhl in cases ( %) . patients ( %) received transplant from matched related donors, ( %) from matched unrelated donors and ( %) from mismatched unrelated donors. female to male mismatch incidence was % (n = ). most of the patients (n = ) received a peripheral blood graft. patient received a second allogeneic transplant. mm patients were transplanted in a "tandem" autologous-allogeneic hsct program in cases. the median number of chemotherapy lines prior to transplant was . in cll, . in mm and . in nhl. patients ( %) engrafted by day post transplant. neutrophil engraftment occured at a median of days (range: - days) and platelet engraftment at a median of . days (range: - days). full donor chimerism was observed in out of patients ( %) having survived by day . primary graft rejection was observed in patients. treatment related toxicities consisted of grade / mucositis in patients ( %), grade (range: - ) cardiac toxicity in patients ( %), grade (range: - ) hemorrhagic complications in patients ( %) including cases of hemorrhagic cystitis and secondary malignancies in patients, this within a median follow-up of . years. infectious complications during aplasia included fever of unknown origin (n = ), bacteremia (n = ) with cases of bacteremia with severe sepsis and cases of infections defined by bacterial foci. incidence of agvhd was % with cases of grade / refractory agvhd. cgvhd occurred in pts ( %). the non-relapse mortality (nrm) at days was % including cases of septic shock, case of acute cardiac toxicity and case of agvhd. the nrm at year was %. -year survival rates were % in al, % in cll and % in nhl with extended survival benefit. in al patients, the relapse incidence was % comprising patients who progressed during conditioning. the -year survival rate in mm patients was %. in mm patients who were in complete response prior to transplant, median overall survival was . years. the used ric regimen resulted in durable donor engraftment with an acceptable toxicity profile permitting efficient disease control in the described cohort. disclosure of conflict of interest: none. graft manipulation using selective depletion of αβ-t cells provides a source for haploidentical hematopoietic stem cell transplantation (haplo-hsct) enriched in effector cells. initial reports demonstrated safety and rapid immune reconstitution using this method, in malignant and non-malignant disorders using several proposed conditioning regimens. no specific considerations were given to hematologic malignancies. we reviewed a total of twenty seven pediatric patients who underwent haplo-hsct using αβ-t cell depletion between - in a single tertiary referral center. we report the results of procedures performed in eighteen patients transplanted for malignancies. twenty two haplo-hsct were performed in eighteen patients. the indication for hsct was acute leukemia in sixteen (all = , aml = ) and neuroblastoma in two. median age at hsct was . years. six patients had failed a prior hsct, and the remainder had no matched donor. the initial reduced-toxicity conditioning regimen consisted of melphalan, fludarabine, thiotepa and atg, and resulted in a high rate of graft rejections ( of ). thus, a totalbody irradiation (tbi)-based regimen was implemented, with prompt engraftment in all the patients. we observed rapid neutrophil and platelet engraftment kinetics (median time to engraft, days and . days, respectively). significant treatment related complications were all due to graft failure in patients receiving reduced-toxicity conditioning, with two infection-related mortalities in the presence of prolonged neutropenia. none of the patients developed hepatic sinusoidal-obstruction syndrome, and no grade - acute graft-versus-host disease (gvhd) or chronic gvhd were observed with either regimen. importantly, the majority of patients with acute leukemia were free of immunosuppression in the first days post hsct. the -year actuarial event-free and overall survival of the entire cohort were % and % respectively, with results for tbi-based conditioned patients being % and %. overall, we demonstrated that a tbibased conditioning for haplo-hsct using αβ-t cell depletion for malignant diseases resulted in acceptable outcomes in these high-risk patients without increased toxicity. disclosure of conflict of interest: none. high-dose chemotherapy conditioning regimens followed by autologous stem cell transplantation (auto-sct) generally provide good results in relapsed and refractory lymphomas. we evaluated the efficacy and safety of tecam regimen as conditioning with autologous stem cell support in patients with relapsed/refractory lymphomas. thirty-two ( patients were refractory, patients were relapse and one frontline treated) patients ( m, f) with lymphoma at various stages (stage ii, %; stage iii, %; stage iv, %) who underwent asct were included in this retrospective study. the median age at transplantation was . years (range, - years). the diagnosis were as follows: diffuse large b-cell non-hodgkin lyphoma (nhl), hodgkin lymphoma (hl), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma and t-cell nhl. all patients received tecam as conditioning regimen that consist of thiotepa ( mg/m × days), etoposide ( mg/m × days), cyclophosphamide ( mg/ kg × day), ara-c ( mg/m × days) and melphalan ( mg/m × days). median cd (+) cells were . × /kg (range; . - . × /kg) which were infused at day , followed by recombinant human granulocyte colonystimulating factor (rhug-csf) at a dose of μg/kg/day. the median time between mobilization and auto-sct was months (range; - months). the median time to recovery of absolute neutrophil and platelet counts independent of transfusion were (range; - ) and (range; - ) days, respectively. the median stay in hospital was days (range, - days). bacterial, sitomegalovirus and invasive fungal infection were detected in ( %), ( %) and ( %) patients, respectively in first days of auto-sct. three s patients ( . %) died from transplant-related complications. the overall response rate was % ( cr, . %; pr, . %) after auto-sct. relapse developed in patients during median follow-up period of . months (range; - months) after auto-sct. the -year estimated dfs ( figure ) and os were % and %, respectively. no statistical significance was observed for os and pfs in terms of gender, patient age ( o and ⩾ years) and nhl and hl lymphoma group (p ⩾ . ). the tecam regimen for auto-sct in lymphoma seems to provide encouraging results in terms of response and its good tolerance with acceptable toxicity. [p ] disclosure of conflict of interest: none. allogeneic hematopoietic cell transplantation (allosct) is the only curative treatment for myelofibrosis. however, its widespread use is limited by early non-relapse mortality (nrm). the optimal modalities of the conditioning regimen are a major unmet clinical need. in an attempt to reduce early nrm, we used a tbf conditioning regimen (thiotepa, busulfan (bu), fludarabine (flu) and antithymocyte globulin (atg)). our aim was to reduce nrm and improve engraftment by using such tbf conditioning. thirty consecutive patients with a median age of years (range, - ) who underwent allosct for primary (n = ) or secondary (n = ) myelofibrosis were included. according to the refined dynamic international prognostic scoring system (dipss-plus), patients were stratified as intermediate- (n = ), intermediate- (n = ), and high (n = ) risk. five patients had blast transformation. ruxolitinib was given to patients ( %) prior to allosct. graft source was pbscs in patients ( %) and bm in patients ( %). donors were matched related (mrd, n = ), unrelated (n = ) and haploidentical (n = ). conditioning regimen was tbf in patients ( %). in our historical cohort patients ( %) received fb (flu, bu, atg). in addition, patients received a 'tec-ric' sequential conditioning (thiotepa, etoposide, cyclophosphamide, and after days rest, flu, bu and atg) for blast transformation (n = ) or refractory proliferative myelofibrosis (n = ). gvhd prophylaxis consisted of cyclosporine (csa) and mycophenolate mofetil in patients ( %), csa and short course methotrexate in patients ( %) with abo mismatch and csa alone in patient ( %) with mrd. high dose posttransplant cy (pt-cy) was added in haplo cases. no significant difference was observed between tbf, fb and tec ric patients in terms of age, gender, karnofsky score, comorbidity index, number of previous treatment line, history of ruxolitinib administration and source of stem cells. median follow-up was months (range, - ). two tbf patients died of septic shock before engraftment at day + and + after allosct, respectively. one fb patient died of graft failure at day + post allosct. median time to neutrophils and platelets ( g/ l) recovery was days (range, - ) and days (range, - ) with tbf, days (range, - ) and days (range, - ) with fb, and days (range, - ) and days (range, - ) with tec ric. grade ii-iv acute gvhd occurred in . % of tbf patients, . % of fb patients, and % of tec ric patients (p = . ). moderate chronic gvhd developed in / evaluable tbf, / fb and / tec ric patients. no severe forms of chronic gvhd were observed. at last follow-up, patient relapsed, died and are still alive. main causes of death were disease progression (n = ), infection (n = ) and gvhd (n = ). nrm at years was . % in tbf patients, % in fb patients, and % in tec ric patients. the -year os were . % in tbf patients, . % in fb patients, % in tec ric patients, respectively. cd +-selected stem cell boost without further conditioning allowed to patients for poor graft function, with significant hematological improvement in patients. tbf conditioning regimen seems to be efficient in allosct for patients with myelofibrosis and compares favorably with previously published fb regimens. these preliminary results give a rationale to support a prospective evaluation of this platform. disclosure of conflict of interest: none. we proposed here to compare the outcome of patients receiving either thymoglobuline (atg), a rabbit anti-human thymocyte immunoglobulin or campath, a recombinant dna-derived humanized monoclonal antibody directed against cd . campath and atg are both commonly used as in vivo tcd before hsct, respectively in united kingdom and france but very few comparing data are available. all consecutive patients with acute myeloblastic leukemia (aml), myelodysplastic syndrome (mds) or myeloproliferative neoplasia (mpn) who received a reduced intensity hsct from an unrelated donor transplanted between and were included in this study. a propensity score was used to identify and control potential confounding to relate the treatment group to the outcomes. in the matched sample, cox regression model was used to describe the association between treatment and outcomes. patients have been included. all patients received fludarabine and busulfan with either atg (n = ) or campath (n = ). patients treated by atg received cyclosporine plus mycophenolate mofetil or methotrexate and patients treated by campath received cyclosporine alone as gvhd prophylaxis. comparing patient and transplant characteristics, atg patients were older ( vs years), had less often aml ( vs %), had higher disease risk (adverse dri: vs %; poor cytogenetics: vs %; high cibmtr score: vs %), were less often in complete remission at time of transplant ( vs %) and were transplanted less often from a mismatched hla donor ( vs %). cumulative incidence of sustained engraftment was in % and % campath and atg patients. time to neutrophil engraftment was longer in atg patients ( vs days). acute gvhd ii to iv rate were higher after atg ( % vs %) as well as chronic extensive gvhd ( % vs %). relapse rate was higher after campath ( % vs %). disease-free survival (dfs) was higher after atg ( vs %) and the gvhd-free relapse free survival (grfs) was similar ( % vs %). according to the prognostic factors for outcome, a propensity score was developed selecting patients from the original cohort. the estimation of tcd effect was than studied. relapse risk was higher in patients treated by campath while there is a non-significant advantage for atg in dfs (table ) . [p ] tcd with atg or campath gives similar os, dfs and grfs. severe acute or chronic gvhd is lowered by campath but the higher relapse risk counterbalances the potential benefit of campath finally given similar os. nevertheless, lower risk disease patient might benefit from campath while higher risk patients might benefit from atg. disclosure of conflict of interest: none. high-dose chemotherapy (hdt) with autologous stem cell transplantation is the standard of care for relapsed/refractory (rr) or high grade non-hodgkin-lymphoma (nhl) and hodgkin-lymphoma (hl) . the standard hdt in autologous stem cell transplantation (asct) for lymphoma is carmustinebased hdt using a combination of carmustine, etoposide, cytarabine and melphalan (beam); this standard conditioning programme is used by most groups worldwide . we have designed novels hdt regimens in which carmustine was substituited by an equal dose of fotemustine (feam) or thiotepa (team) and we compared these two hdt regimens in terms of engraftment times, toxicity, tolerability and frequency of relapse after asct. from february to september we consider a total of relapsed/refractory patients affected by hl and nhl respectively hl and nhl with different grade of initial disease (grade i-iv) and different response to prior treatments. the all other drugs were administered according to a standard beam regimen . after a day of rest, autologous peripheral blood progenitor cells were infused on day , followed by s.c. g-csf ( mg/kg) from day of asct until consecutive days when the ancs were × /l . the primary objectives of the study were to assess the feasibility and safety of the feam and team regimens in terms of acute toxicity, grade of mucositis, hemopoietic engraftment and relapse after asct. acute toxicity include chemotherapy-induced nausea and vomiting, diarrhea, hepatotoxicity, nephrotoxicity and infection complication. in all patients cd + cells were collected from peripheral blood and the median number of infused cells per patient was . × e /kg. the median time of engraftment was days for neutrophil recovery (n × /l) and days for plt recovery ( × /l). acute toxicity occurred in total patients ( . %), mucositis grade - occurred in patients ( % of cases). frequency of relapse in all cases was . %. feam conditioning regimen was used in cases showing a median time of neytrophil recovery of days and a median time of plt recovery of days. acute toxicity occurred in of these cases ( . %), mucositis grade - occurred in patients ( . % of cases). frequency of relapse in feam group of patients was . %. team conditioning regimen was used in cases showing a median time of neytrophil recovery of days and a median time of plt recovery of days. acute toxicity occurred in of these cases ( . %), mucositis grade - occurred in patients ( . % of cases). frequency of relapse in team group of patients was %. relapse/progression of lymphoma and conditioning regimen toxicities remain limitations to treatment success. the two novels hdt regimens feam and team are safe and feasible and show similar engraftment times, tolerability and frequency of relapse. maybe the team regimen shows toxicity slightly higher than feam regimen but longer follow-up is needed to evaluate fully its efficacy and long-term safety. disclosure of conflict of interest: none. treosulfan is a prodrug of a bifunctional alkylating cytotoxic agent. there are few reports regarding toxicological side effects of treosulfan-based conditioning prior to hsct. here we report on incidence of early potential treosulfan-related toxicity in patients treated with treosulfan-based conditioning before hsct. treosulfan was given at a dose of g/m /d for days in combination with fludarabin mg/m /d for days prior to hsct. most patients (n = ) had a haematological malignancy, while patients had a non-malignant disorder as hsct indication. an hla-a, -b and -dr matched unrelated donor (mud) was used in cases, patients had a hla-identical sibling donor and received an hla-a, -b or -dr allele mismatched unrelated donor. as graft versus host-disease (gvhd) prophylaxis, most patients (n = ) received cyclosporine and methotrexate. patients medical records were scrutinized retrospectively to collect laboratory tests (aspartate aminotransferase (ast), alanine aminotransferase (alt), creatinine) before hsct and then weekly until weeks after hsct. levels of ast and alt were significantly increased week after hsct compared to before hsct. however, only a few patients had transaminase levels over or times the upper normal level (unl) levels decreased sharply after the first week. most of the cases with high levels of ast/alt at one week had normal or close to normal levels before hsct. creatinine levels increased after week but no patient had levels ⩾ × unl. clinical features of all oral mucositis (om) were recorded using the world health organization (who) scoring system. most patients ( %) had no or very limited (grade i) om, % had grade ii and % had grade iii or iv of om. according to our toxicological results this is low-toxic protocol. however, all patients became neutropenic, % already at the time of graft infusion, indicating that the protocol has a myelo-toxic effect comparable to conventional mac protocols. all patients engrafted, except three patients who died very early. median time to neutrophil and platelet engraftment was (range - ) and days ( - ), retrospectively, which is significantly later when compared to engraftment data for other ric protocols used at our centre (data not shown). median duration of neutropenia (o . × /l) was days , comparable to what is expected after conventional mac conditioning. secondary graft failure (gf) occurred in ( . %) patients, all having a nonmalignant disorder and / having a urd. non-relapse mortality (nrm) was . % ( % ci . - . %) at days and . % ( . - . %) at one year after hsct. causes of death within one year after hsct were: relapse , epstein-barr virus associated posttransplant lymphoproliferative disease (ptld) , other infections , organ failure , gvhd , hemophagocytic lymphohistiocytosis (hlh) . other infections occurring within days after hsct were cytomegalovirus (cmv) reactivation ( %), invasive fungal infection ( . %) and blood stream infection ( %). veno-occlusive disease of the liver or sinusoidal obstruction syndrome (vod/sos) occurred in one patient and haemorrhagic cystitis in two patients. this study shows that early regimen-related toxicity after hsct was low despite similar marrow toxicities compared to mac regimens. disclosure of conflict of interest: none. allogeneic stem cell transplantation from haploidentical donors (haplosct) is an increasingly adopted option for patients (pts) with high-risk hematological malignancies. in our institution, we previously described a platform for unmanipulated peripheral blood stem cell (pbsc) haplosct using a calcineurin-free gvhd prophylaxis with sirolimus, micophenolate and anti-human t-lymphocyte immunoglobulin (atg) after conditioning with treosulfan and fludarabine (trramm; peccatori et al., leukemia ) . as an attempt to decrease relapse rate, especially in advanced-phase diseases, we designed a new phase ii prospective clinical trial intensifying conditioning regimen with the addition of gy total-body irradiation (tbi) (trramm gy; eudract# - - ). we report results on pts. pts affected by aml (n = ), other myeloid (n = ) and lymphoid (n = ) malignancies were prospectively enrolled from may to june . median pts age was y (range - ). revised disease risk index (r-dri) was low or intermediate in pts, high in pts and very-high in pts. twenty-five pts had previously received an allogeneic stem-cell transplantation with a median time from st to nd sct of months . median hct-comorbidity index by sorror et al. was ( ) ( ) ( ) ( ) ( ) ( ) . pts received a myeloablative conditioning regimen consisting of treosulfan ( g/m /d from - to - ), fludarabine ( mg/m /d from − to − ) and tbi gy (fractionated in doses, from − to ). source of stem cells were unmanipulated g-csf-mobilized pbsc from haploidentical donors. gvhd prophylaxis consisted of atg-fresenius (grafalon, neovii) mg/kg/d from − to − , rituximab mg/m on − , mycophenolate mofetil mg/ kg from − to + and sirolimus (target concentration - ng/ ml) from − . median infused cd + and cd + cell doses were . × ⁶/kg and . × ⁸/kg, respectively. median follow-up for survivors was months ( - ). neutrophil engraftment occurred in % of pts with a median of d ( - ), platelet engraftment was reached in % of pts with a median of d ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the -d cumulative incidence (ci) of grade ⩾ acute gvhd (agvhd) was ± % and of grade ⩾ agvhd ± %; the years ci of chronic gvhd was ± %. the ci of transplant-related mortality (trm) at y and y were ± % and ± %, respectively. the ci of relapse at y and y were ± % and ± %, respectively, with a median time to relapse of d . interestingly, we did not observe any extramedullary relapse; loss of mismatched hla-haplotype occurred in % of relapses. among pts who were in active disease at time of haplosct and who were evaluable, % achieved complete remission (cr) and full donor chimerism at day+ . the y and y probabilities of disease-free survival (dfs) were ± % and ± %, respectively. at y, % of pts are alive, disease-free and immunosuppression-free minimal residual disease, tolerance, chimerism and immune reconstitution the number of human leukocyte antigen (hla)-mismatched hematopoietic cell transplantation (hct), including cord blood transplantation, has been increasing. hla-flow method can discriminate mismatched hla antigens between donor and recipient by using flow cytometry, and can evaluate minimal residual disease (mrd) or chimerism after hla-mismatched hct. by developing more simple methodology, hla-flow might be more widely applicable. we have developed modified -colorbased hla-flow method. the aim of this study is to evaluate the utility of the -color-based hla-flow for monitoring of mrd and chimerism after hla-mismatched hct in children. from june to november , serial monitoring of mrd or chimerism by the -color-based hla-flow was performed in twelve patients undergoing hla-mismatched hct ( tests). nucleated cells obtained from bone marrow were stained by immunofluorescent antibodies against hla antigens mismatched between donors and recipients. these cells were also stained by immunofluorescent antibodies against surface antigens such as cd , cd , cd , cd and cd /cd for determining lineage of the cells. these surface antigens were also used as a marker of leukemic blasts in the mrd study. we used -color-based flow cytometry (facs-navious) and the data were analyzed with flow jo. erythroblasts and dead cells were excluded from the analysis. in each study, at least cells were analyzed. for mrd analysis, we concurrently tested real-time quantitative polymerase chain reaction (pcr) of peripheral wt mrna or leukemia-specific fusion genes. pcr of polymorphic short tandem repeats or fluorescent in situ hybridization of x/y chromosomes was concurrently tested for chimerism study. age of patients ranged from to years. donor sources included bone marrow (n = ) and cord blood (n = ). for mrd monitoring of acute leukemia (n = ), the -color-based hla-flow could detect mrd in three patients. five patients have not experienced relapse. no discordance with other mrd markers was observed in these patients. hla-flow could not separate donor-derived cells from recipient-derived ones in one patient receiving bone marrow transplantation. as for chimerism testing (n = ), the -colorbased hla-flow could successfully evaluate quantitative lineagespecific chimerism in all patients. there is no discrepancy between hla-flow and other methods. we could complete evaluation of the -color-based hla-flow within two days in all tests. the -color-based hla-flow is a simple, quick and useful method for the quantitative evaluation of mrd and lineagespecific chimerism after hla-mismatched hct in children, irrespective of donor sources. it is thought that our method is applicable in all institutions owing -color-based flow cytometry. acknowledgement: we thank drs. nobukazu watanabe, eri watanabe and natsuko sato (university of tokyo) for their technical advices. we also thank drs. tomoko okunushi (chiba university), hidefumi hiramatsu and katsutsugu umeda (kyoto university) for their care of patients involved in this study. disclosure of conflict of interest: none. survival (pfs), cumulative incidence of relapse (cir) and acute/ chronic gvhd incidence in aml patients (pts) submitted to allo-hsct at our institution between january and december . this retrospective study evaluated aml pts submitted to allo-hsct from matched sibling donors (msd) and matched unrelated donors (mud) who provided bone marrow (bm) or peripheral blood as stem cell grafts.ir was evaluated at , and days post-transplant in all pts. cmv-dna copies were determined in peripheral blood by quantitative pcr twice weekly in the first days post-transplant and subsequently once weekly. cmvi/r was analyzed as a timedependent covariate.effect of cmvi/r on os and pfs was estimated by cox proportional hazard model. cir and gvhd incidence were analized with a competing risk approach, considering death from any cause as a competing event. effect of cmvi/r on cir and gvhd were evaluated by fine & gray model. median age at allo-hsct was . years ( . - . ). in our population % of donors were seropositive for a previous cmv infection and pts transplanted from these donors showed a significantly lower cumulative incidence of cmvi/r than pts transplanted with seronegative ones (shr = . , %,ci: . our study demonstrates that cmvi/r influences the success of allo-hsct by determining a better ir characterized by a higher cd + cell number that might exert an immune protective control on disease outcome by improving os,pfs and cir with no effect on gvhd. another factor of utmost importance to achieve this same goal might be constituted by the significantly increased nk cell number six months after allo-hsct. to assess the dynamics of molecular response to treatment in aml adult patients with concomitant flt and npm mutations. this retrospective single center studystudy was approved by the institutional review board of american university of beirut medical center. twelve consecutive newly diagnosed (n = ) or relapsed (n = ) aml patients received idarubicin/cytarabine induction and one or two consolidation (s) ( table ) . seven patients received allogeneic stem cell transplant (allo-sct) and had haploidentical-sct (hap-lo_sct); all followed by post-transplant sorafenib maintenance. median follow-up was . ( - ) months. all transplanted patients remain alive and disease free.flt mutation was tested on dna using a qualitative method with a sensitivity of . %. npm- mutation was tested on cdna using a qualitative or a quantitative rt-pcr with a sensitivity of . % and . ncn respectively. patients were tested at diagnosis, after induction, after each consolidation, before and s at days , and after allo-sct for kinetics of npm and flt molecular response. after induction, flt became negative in all tested patients (n = ). after first consolidation, flt- was not tested in patients who had a negative result after induction, was negative in patients including the patients who were not tested after induction, whereas a molecular relapse was noted in one patient who developed a hematological relapse and rapidly died. another patient died after the third consolidation, in complete remission, due to septic shock. no molecular positivity for flt- was noted later on, whether after second consolidation or post-transplant. conversely, npm- mutation became negative in out of tested patients after induction, in additional patient after first consolidation and in additional patients after sct, mostly after starting sorafenib. npm- mrd value remained elevated in out of patients with quantitative assessment at diagnosis and post induction (figure ). flt become negative early after induction while npm negativity lags behind. persistent npm- mrd does not seem to predict post-transplant outcome and may indeed become negative after sorafenib. these results need confirmation in larger studies. disclosure of conflict of interest: none. in allogeneic stem cell transplantation (allo-sct), an early detection of the transplant outcomes such as overall survival (os), event-free survival (efs), cumulative incidence of relapse (cir) and non-relapse mortality (nrm) is fundamental regarding the use in time of additional therapy after sct. therefore, we investigated the association between early immune reconstitution (ir) on day + after allo-sct and outcomes in children suffering from acute leukemia or myelodysplastic syndrome (mds). this study collected data from allo-sct from january until december in our institution. the median survival follow-up was months. indications of allo-sct were all (n = , %), aml (n = , %) and mds (n = , %). the median age was years (range, . - ). patients were transplanted in cr (n = , %) and pr/nr (n = , %). patients included in the study received st sct (n = , %), nd sct (n = , %) or sct (n = , %). grafts were from sibling (msd; n = , %), matched unrelated (mud; n = , %), haploidentical (n = , %) or mismatched unrelated (mmud; n = , %). conditioning regimens were tbi-based (n = , %) or chemo-based (n = , %). stem cells were from bone marrow (n = , %) or peripheral blood (n = , %). we analyzed the absolute count of lymphocytes (alc), monocytes, cd + t cells, cd +cd + t-helper cells, cd +cd + cytotoxic t-cells, cd -cd + natural killer (nk) cells and cd + b cells assessed on day ± after sct. we used the percentiles of the lymphocyte subsets of the same cohort to categorize the samples throughout the study. patients with alc over the th percentile of alc (alc o cells/μl) had a . -fold increased hazard ratio (hr) to develop relapse (p = . ). nk cell counts on day after sct were strong associated with os, efs, cir and nrm. patients with nk cell count over the th percentile (nk . cells/μl) had increased hr for os (hr = . , p = . ) and efs (hr = . , p = . ) compared to patients with nk count under the th percentile. patients with nk cells over the th percentile (nk o . cells/μl) had a hr = . (p = . ) for relapse and hr = . (p = . ) for nrm compared to patients with nk cell count under the th percentile. monocyte cell count on day correlated with os, efs and cir. patients with cd + cells count under the th percentile of cd + (cd + o cells/μl) has an increased hazard ratio for os, efs and relapse compared to patients with cd + cell counts over the th percentile. no association between absolute cell count of cd +, cd +, cd + and cd + on day + after allo-sct and any outcomes either os, efs, cir or nrm was found. the study confirms the strong association between early ir and outcomes after allo-sct in children. our study suggests that especially nk cell and monocyte cell count on day + may have significant prognostic implications. our findings suggest that the cells count of alc, nk cells and monocytes on day + after allo-sct could be useful to predict outcomes after allo-sct and should be taken into account in considering alternative treatment. disclosure of conflict of interest: none. early immune reconstitution (eir) has proven to be a significant determinant for the outcome of allogeneic hematopoietic stem cell transplantation. in the setting of unmanipulated haploidentical transplantation (haplo-hsct), some groups have identified the absolute leukocyte count on day + (alc ) as an independent prognostic factor in terms of overall survival (os), disease free survival (dfs) and infectious mortality (im). the aim of this study was to evaluate the impact of eir on os, dfs and im among patients who underwent haplo-hsct with postransplant cyclophosphamide (ptcy) at our institution. from july to april , haplo-sct were performed at our institution. threedied before day after haplo-sct, and patients had missing data. conditioning regimen consisted of fludarabine, cyclophosphamide and busulfan. twenty-nine patients received a reduced intensity conditioning regimen ( - days of busulfan) while a myeloablative regimen ( - days of busulfan). gvhd prophylaxis comprised ptcy, cyclosporine and mycophenolate mofetil. patients were assessed for eir by means of alc , cd + t lymphocyte count on + (cd ), nk lymphocyte count on + (nk) and nk cd bright percentage on + (cd br ). we analyzed pts, with a median follow-up of months ( - ). the median age of the pts was (range - ), %men. diagnosis were: aml( %), hl ( %)non-hl ( %), all ( %),mds( %), cml( %), others( %). % were in complete remission at the time of transplant, % in partial remission and % had overt disease. in terms of infectious complications, cmv reactivation was documented in % of the pts, % developed a proven invasive fungal infection and % suffered from bk+hemorrhagic cystitis. median os and dfs were ( - ) and months ( - ), respectively. im rate was % at the end of follow up. median follow-up was months ( - ). roc curves were used to determine the optimal cut-off values for each of the studied parameters: cells/μl for alc , cells/μl for cd , cells/μl for nk and % for cd br were chosen. pts with alc ⩾ /μl had better os (p = . ) and dfs (p = . ), than those with alc o /μl. median os and dfs were months vs not reached (nr) and months vs nr, respectively. pts with cd br ⩾ % had better os (p = . ) than those with lower values. median os was months vs nr; however no difference was seen in terms of dfs. we didn´t observe statistically significant differences in os or dfs, among pts with different levels of cd and nk on + . cumulative incidence of im was significantly lower in pts with an alc ⩾ (p = . ), pts with cd ⩾ /μl (p = . ) and pts with nk ⩾ (p = . ); patients with cd br ⩾ % showed tendency to have lower cumulative incidence of infectious mortality (p = . , non-significant). cumulative incidence of relapse was not affected by alc , cd , nk or cd br . our study supports the independent prognostic value of early immune reconstitution after unmanipulated haploidentical transplantation with ptcy, especially in terms of lower infectious mortality. os and dfs were better among patients with alc ⩾ cells/μl. pts with cd br ⩾ % also showed better os. no correlation was found between cd or nk on + with os or dfs. cumulative incidence of infectious mortality was affected by alc , cd and nk on + ; while cd br seems to have less impact. [p ] disclosure of conflict of interest: none. an early absolute lymphocyte count (alc) recovery after autologous stem cell transplantation (asct) for hematologic malignancies has been related with an improved transplant outcome due to a faster autologous immune restoration. in this retrospective study we analyze post-transplant survival of non hodgkin lymphoma (nhl) patients and its relation with alc at day + post-asct. we analyzed consecutive adult nhl patients who underwent asct at the hematology and sct department of hospital maciel (montevideo, uruguay). only individuals with at least months post-transplant follow up were included. all patients received beam (bcnu, etoposide, cytarabine and melphalan) conditioning regimen followed by peripheral blood stem cells previously collected by apheresis. median cd + cell dose was . × e /kg ( . - . ). median alc at day + was /μl. patients were divided into two groups: alc at day + inferior than /ul (group ) and alc at day + superior or equal than /ul (group ). differences between groups were analyzed using t-student and chi-square tests, with statistical significance determined at p o . . disease free survival (dfs) and overall survival (os) were analyzed by kaplan meier method. differences in survival between groups were determined by log-rank test. no differences were observed between groups regarding gender, histology, disease status at transplant and cell dose. patients in group were older and more heavily pretreated. neutrophils and platelets engraftment were significantly faster in group (table ) . after a median follow up of months, disease-free survival (dfs) and overall survival (os) were superior in group . median dfs was months and not reached (p = . ) and os was months and not reached (p = . ) in groups and respectively (figure ). an early alc recovery after asct was associated with a superior dfs and os in nhl patients. individuals with alc major or equal than /ul had also a shorter time to neutrophils and platelets recovery and a shorter hospital stay. in this study, cd + cell dose does not seems to be a determinant factor for lymphocyte recovery. the load of previous treatment may influence lymphocyte recovery after asct. these results support the association between early post-asct lymphocyte recovery and improved survival in nlh patients. [p ] disclosure of conflict of interest: none. t cell depletion (tcd) reduces the risk of graft versus host disease (gvhd) but also the graft versus leukaemia (gvl) effect, thus increasing the risk of relapse. donor lymphocyte infusions (dli) can be given to boost donor chimerism, with the intention of enhancing the gvl effect. it is not currently known whether giving dli based on bone marrow chimerism (bmc) influences survival, or whether certain groups of patients benefit more from dli than other groups. in addition, it is not known whether the overall aim of achieving % bmc associates with improved survival. we investigated whether day (d ) bmc was predictive of survival, and whether giving dli based on this result was associated with improved overall survival. data were retrospectively collected from case notes and laboratory reports for patients who underwent allogeneic stem cell transplant (allosct) for aml or mds at the northern centre for bone marrow transplantation between and . patients who died prior to d were excluded from the analysis. of the patients analysed (aml , mds ), % were male and % female. the median age was years (range - ). conditioning was with flu/bu/ alemtuzumab ( ), flu/mel/alemtuzumab ( ), cy/tbi alemtuzumab ( ), flamsa tbi/bu atg/alemtuzumab ( ), other ( ) . ( %) received a graft from an unrelated donor, ( %) a matched sibling donor and ( %) another source. ( %) received mobilised pbscs, ( %) bone marrow and ( %) cord blood. statistics were performed using graphpad prism. p values were calculated using the chi square test and taking po . to determine significance. bmc was divided into groups %, - % and o %. % bmc at d was associated with a significant increase in year overall survival (os) ( . % vs . % and . % for - % and o %, respectively, p o . ). patients with a d bmc o % had a year os of o % (with relapse the cause of death in %). in patients whose d bmc was o %, there was a significant improvement in year os seen in those who received dli ( . % survival at years vs % with no dli, po . ) (figure : os by d bm chimerism (with and without dli). attainment of % bmc at a subsequent time point also significantly improved survival in those with a d bmc of - % ( . % year survival vs . % who never attained %, p o . ) and o % ( % year survival vs . %, p o . ). we found d bmc to be predictive of os in this population. in addition, dli was associated with an improvement in os, especially in patients whose bmc at d was o %. there was also a statistically significant improvement in os seen in patients who subsequently attained a % bmc, where it was o % at d . the objectives of this analysis were to examine the optimal alc recovery cutoff utilizing receiver operator characteristics (roc) analysis and to examine infused allograft characteristics associated with early alc recovery. after due irb approval, patients (pts) with aml and all who underwent hct at our institution between - were identified. pts with t-cell depletion or maintenance post hct were excluded. data were collected retrospectively from the patient's records. cellular contents of infused products (cd , cd , tnc, mnc, alc and amc) in addition to alc post hct were analyzed and optimal cutoff, if present, was established using roc analysis for the end point of relapse. time to end point analysis was computed using the kaplan-meier with log ranks. for competing events, cumulative incidence was computed using grey's model. univariable and multivariable analyses were performed using cox proportional hazard regression. a total of pts met the inclusion criteria and were analyzed. optimal alc cutoff by roc analysis was established to be on day + (d ) with alc . × /l and was subsequently defined as early lymphocyte recovery (erl). pts with alc ⩽ . × /l were deemed to have delayed lymphocyte recovery (dlr). patients were subsequently stratified accordingly and patient, disease and transplant related factors were well balanced between the groups. median follow up of the entire cohort was ( - . ) months. graft characteristics: roc analysis established optimal cellular cutoff, if present to predict elr. pts in the elr group were more likely to receive cd × /kg o ( . ), cd × /kg ( . ) and alc . × /kg (p = . ). we did not find a significant threshold for other allograft variables i.e. (tnc, mnc or amc). post hct outcomes: at years, corresponding cumulative incidence of relapse (cir) and non-relapse mortality (nrm) was . % vs . % (p = . ) and . % vs . % (p = . ), for elr and dlr cohorts, respectively. there was a trend towards improved progression free survival (pfs) and overall survival (os) in favor of elr vs dlr at . % vs . % (p = . ) and . % vs . % (p = . ), respectively. median time to neutrophil and platelet engraftment was and days, respectively for both groups. incidence of acute graft vs host disease (agvhd) was similar (p = . ); however, chronic gvhd (cgvhd) was more prevalent in the elr group at % vs %, respectively (p = . ). on s multivariable analysis for relapse, elr retained its prognostic significance with hr . ( . - . ; p = . ). cgvhd and first complete remission (cr ) at the time of hct were also protective factors from relapse in multivariable analysis. we observed that elr is an independent predictor for relapse in patients receiving allogeneic hct for acute leukemia with a trend towards improved os. this is possibly related to higher incidence of cgvhd. elr was influenced by infused allograft characteristics particularly cd count. given the sample size and retrospective nature of the analysis, these important observations should be examined prospectively. disclosure of conflict of interest: none. allosct is the only curative option for the treatment of hematological disorders with depression of hematopoiesis and primary immunodeficiencies.non-myeloablative conditioning (mac) regimens lead to long persistence of mixed chimaerism (mc) in the majority of patients. purpose: to estimate the relationship between type of hematopoietic chimaerism and appearance of gvhd in patients with non-malignant diseases after allosct eleven patients ( boys and girls) with median age of years (range - ) were included in the current study. among them there were patients with severe aplastic anemia (saa), with fancony anemia (fa), with thalassemia, with nijmengen syndrome, treated in our center from to . donors' sources were as follows: siblings in cases, mud ( / ) in ones. in cases bone marrow aspirate were used, in mobilized peripheral blood hematopoietic stem cells. conditioning regimens included fludarabin, cyclophosphamide and horse atg for saa patients, in fa and nijmengen syndrome patients this scheme was augmented by low-dose busulfan. in thalassemia patient we used mac with busulfan, fludarabin and horse atg. in majority of case gvhd prophylaxis consisted of tacrolimus and methotrexate combination. when allosct was performed form mud patients were additionally administered with mycophenolate mofetil. median of follow-up period was mo (range - ). quantitative evaluation of chimerism was done by multiplex amplification of str loci with subsequent fragment analysis using «cordis plus» kit («gordiz llc», russia). we analyzed whole bone marrow and peripheral blood together with cd + and cd + lymphocyte subpopulations. presence of ⩾ % donors' hematopoietic cells was considered as complete donor chimerism (cc), less than % was considered as mc. all patients engrafted in time and all of them are alive at the time of current analysis. there were no severe life-threatening complications, infections or graft rejections. only patients achieved cc at day + . at day + only these patients stayed in cc. at this time point mc was mainly revealed in cd + lymphocytes. in year after hsct proportion of cc patients enlarged to % ( patients did not achieved this time point). there is no any correlation between time of engraftment and chimerism value at day + , either between the dose of transplanted cd + cells and chimerism level ((p . in both cases). severe gvhd was noted only in female patients with cc at day + . in the first case it was acute gvhd grade iii after hsct from mud, in the second case extensive form of chronic gvhd in year after hsct from sibling was observed. there are no other cases of grade iii-iv acute gvhd in the observed cohort of patients. localized form of chronic gvhd [p ] was revealed in ( %) patients. in other patients there were no signs of chronic gvhd. despite limited number of observations we assumed that fast achievement of cc corresponds to severe gvhd. and vice versa, long persistence of mc prevented emergence of gvhd. however our findings need to be confirmed in a larger group of patients and preferably in a multicenter setting. disclosure of conflict of interest: none. interest of quantitative assessment of hematopoietic chimerism by real-time quantitative polymerase chain reaction after hematopoietic cell transplantation for hematological malignancies: a retrospective analysis on adult patients from rennes university hospital g laure , c mathilde , b marc , n stanilsas , d charles , l christine , a mehdi , lb laetitia, s gilbert , l thierry and r virginie department of hematology, chu pontchaillou, rennes; immunogenetics and histocompatibility laboratory, etablissement français du sang, rennes and etablissement français du sang-bretagne chimerism (percentage of recipient versus donor-derived blood cells) is used to document engraftment after hematopoietic stem cells transplantation (hsct). detection of persistant host cells, - as well as an increase in recipient cells chimerism has been associated with impaired dfs and os. quantitative real time pcr (qrt-pcr) is a highly sensitive, reproducible method, which can detect very low levels of recipient cells. the aim of this study was to evaluate the prognostic impact of early chimerism days (d ) and days (d )) after hsct and the meaning of detection of an increase of chimerism, even at low levels, during follow-up. adult patients who underwent hsct in rennes between and were included in this retrospective study. all chimerism analyses were done with qrt-pcr using whole blood sample. complete chimerism (cc) was defined by less than . % recipient cells detected. with a median follow-up was days, patients relapsed with a median time of . days after hsct. both d and d mixed chimerism (mc) ( . % recipient cells detected) were associated with an increased relapse risk (p = . and p o . respectively) compared to patients with cc in univariate analysis. however, when looking at subgroups analysis, d and d mc vs cc was significantly associated with increased relapse risk in this cohort for myeloid diseases (p = . and p o . ) but not for lymphoid diseases (p = . and p = . ). no difference in os was observed (p = . and p = . ). more important, detection of an increased of mc (imc) was associated with an increased relapse risk in univariate and multivariate analysis (or = . [ . ; . ], or = . [ . ; . ]), (po . ), as well as impaired os (p = . ) and dfs (p o . ). among the patients with aml and at least chimerism analysis available, only relapsed without imc detected but the patients' last available chimerism analysis was , and days before relapse respectively. median levels of recipient cells detected was . %. altogether, these results indicate that serial analyses of chimerism with qrt-pcr are a useful tool for post-transplant monitoring and might help identify patients at highest risk to relapse after transplant, especially in myeloid disease. monitoring frequency is critical in order to obtain the highest clinical impact, and the timing of monitoring as well as the safety and type of pre-emptive interventions still need to be explored. considering the kinetics of the disease, frequent analysis in myeloid pathology might improve the detection of impending relapse. although an approximately -log higher sensitivity of quantitative pcr (qpcr) compared to short tandem repeat (str) has been documented in different studies, the latter remains the standard procedure for hc assessment to date. we hypothesized that qpcr could be superior to str for monitoring the molecular kinetics of donor cell engraftment, response to donor lymphocyte infusions (dli) and development of relapse post-hct. we analyzed patients (pts) who underwent mainly / hla-matched unrelated hct mostly for acute myeloid or lymphatic leukemia at the university hospital essen between and . transplant conditioning was mostly myeloablative and gvhd prophylaxis was by cyclosporin a and methotrexate without anti-thymocyte globulin (atg). median follow-up of pts was days (d) ( - ). cytomegalovirus (cmv) reactivation in the first d posttransplant was measured by pp (n = ) or pcr (n = ). a total of retrospective genomic dna samples from peripheral blood (pb; n = ) or bone marrow (n = ) collected between d and d post-transplant were available for hc analysis in parallel by str (mentype chimera, biotype) and qpcr (alleleseq, abbott). threshold for hc positivity in qpcr was set at . % following published protocols. concordance in hc analysis between qpcr and str was found in / ( . %) samples, with all discordant cases positive in qpcr but negative in str. engraftment could be assessed in samples drawn at d -d from pts without relapse in the first months post-hct. these samples showed concordant negative or positive qpcr and str results reflective of full donor engraftment or persistent mixed chimerism (pmc) in and pts, respectively. in pts, qpcr but not str documented delayed conversion to full donor chimerism until d . in the remaining pts, positive results in qpcr but not str during early engraftment were observed exclusively in bm, in particular those drawn before d post-hct. qpcr but not str was also able to document the kinetics of conversion to full donor chimerism which took d and d in pts receiving dli for treatment of pmc and relapse, respectively. informative relapses could be assessed in samples drawn at least d before onset. / bm and / pb were positive in qpcr, compared to / bm and / pb in str, with a sensitivity of / ( %) and / ( . %) relapses, respectively. consistent with previous reports on a protective effect of early cmv reactivation on relapse in gvhd prophylaxis regimens without atg, relapse occurred in / ( %) pts who experienced cmv reactivation in the first d post-transplant, compared to / ( %) pts who did not. no apparent associations were observed between early cmv reactivation and engraftment kinetics post-hct. hc assessment by qpcr is highly concordant with str, but markedly superior for molecular monitoring of engraftment kinetics and relapse. positive qpcr results in bm should be interpreted with caution during early engraftment, while both bm and pb were highly informative for relapse in our series. these results advocate the feasibility and clinical utility of qpcr for post-hct hc monitoring in routine use. disclosure of conflict of interest: the commercial assays for qpcr chimerism analysis were provided by abbott molecular free of charge. tyrosine kinase inhibitor (tki) has become the standard of care in patients (pts) with chronic myeloid leukemia (cml) and an unavoidable tool in the combined therapy for pts with philadelphia chromosome positive (ph+) acute lymphoblastic leukemia (all). nevertheless, allogeneic stem cell transplantation (hsct) remains the standard therapy of all ph+ and of cml pts failing st line therapy with tki, with failure or insufficient response or intolerance or mutations resistant to nd generation tki, or in the advanced phase at diagnosis. in the past decade the feasibility and safety of post-hsct imatinib administration as prophylactic or therapeutic strategy was confirmed. second and rd generation tki administration after hsct is under investigation. here we are reporting our experience in post-hsct treatment with the rd generation tki ponatinib in pts treated between and at our institution. pts data and information were collected from institutional database and chapters revision. a written consent was given by pts allowing the use of medical records for research in accordance with the declaration of helsinki. pts and diseases features are reported in table . pre-transplant treatment for the all ph+ patient consisted of chemotherapy combined with dasatinib, followed by a st mud hsct and dasatinib in maintenance. the patient relapsed year after hsct with documentation of mutation v l. ponatinib was introduced as salvage treatment to bridge nd haplo hsct. pre-transplant treatment for the cml patients consisted of tki therapy with combination of chemotherapy in case of uncontrolled progression of disease. two pts received a st mud hsct but relapsed respectively months and years later. ponatinib was introduced as salvage treatment to bridge nd haplo hsct. four pts received ponatinib mg daily before the last hsct: one patient achieved sustained major molecular response, pts obtained transient response. all pts were presenting nd generation tki resistant mutations (table ) . ponatinib was started at a median of days after hsct (range, - ) as salvage treatment in overt relapse ( cases), prophylaxis ( case) or preemptive therapy ( case). acute gvhd was diagnosed in pts before ponatinib administration, of them also experienced chronic gvhd. no new cases of gvhd were observed after initiation of ponatinib. immunosuppressive treatment and azoles treatment were discontinued before ponatinib in all but one patient who was under combined treatment for chronic gvhd: therapeutic drug monitoring was closely performed without evidence of drug-drug interaction. pts were regularly evaluated for toxicities and monitored for cardiovascular events. no serious adverse events were reported in our experience: we administered ponatinib at a median maximum dosage of mg daily (range, - mg), for a median of weeks (range, - weeks). at last evaluation one patient maintained the status of molecularly undetectable leukemia (follow-up post hsct: months) and one major molecular response (follow-up post hsct months). three patients who received therapeutic ponatinib in overt relapse did not respond and died for progressive disease. ponatinib is safe and well tolerated as bridge to hsct and to maintain disease control after transplant. prophylaxis targeted therapy and preemptive therapy with ponatinib may lead to the reduction of disease relapse for high-risk ph+ leukemia. disclosure of conflict of interest: none. at nuh singapore we have adopted the cd ra depletion to ameliorate graft versus host disease. materials and methods: we have transplanted leukemia patients with cd depleted hsct followed by cd ra depleted donor lymphocyte infusion. no additional gvhd prophylaxis or gcsf was used. results: % patients achieved primary engraftment. median time for neutrophil engraftment ( /μl without gcsf) was days (range - days), platelet engraftment ( ) was days (range to days). immune reconstitution was rapid with median cd and cd cell counts /μl at day . by day median cd count was /μl (range - /μl). no patient developed grade iv acute gvhd. there was a significantly reduced incidence of invasive viral infections as compared to conventional transplants. importantly, all patients achieved complete remission (cr) on day + and remained in cr for longer time as compared to conventional transplants. conclusion: our preliminary data suggests that rapid immune-reconstitution of nk cells and t cells with this strategy correlates with reduced infection related mortality without loss of graft versus leukemia effects. disclosure of conflict of interest: none. the use of post-transplantation high-dose cyclophosphamide (pt-cy) has overcome the need for extensive depletion of t lymphocytes from haploidentical donor grafts, which traditionally resulted in severe and prolonged immunosuppression. it is therefore relevant to investigate the degree and the tempo of immune reconstitution after t cell replete haploidentical stem cell transplantation (haplo-sct) by use of pt-cy. we prospectively monitored cellular immunity in consecutive adult patients (male/female: / ), who underwent haplo-sct with pt-cy for myeloid (n = ) or lymphoid (n = ) malignancies. the median age at transplant was (range, - ) years. the conditioning regimen was myeloablative in , reduced-intensity in , and non-myeloablative in case. the source of the graft was peripheral blood (n = ) or bone marrow (n = ). in addition to pt-cy, graft-versus-host disease (gvhd) prophylaxis included tacrolimus and mycophenolate mofetil. absolute counts of cd +cd +, cd +cd +, cd + and cd +cd + cells were measured by flow cytometry at , , , , , and months post transplant. the median doses of infused cd + and cd + cells were . (range, . - . ) × /kg and . (range, . - ) × /kg, respectively. neutrophil engraftment ( /ul) was achieved at a median of (range, - ) days, whereas platelet engraftment ( /ul) was observed at a median of (range, - ) days. seven patients developed acute gvhd (grade i/ii: , grade iii: ). chronic gvhd occurred in patients, and was extensive in the of them. cytomegalovirus infection was detected in / cases at a median interval of (range, - ) days post transplant. two patients were administered rituximab for epstein-barr virus reactivation at months, whereas one patient developed bk virus-associated hemorrhagic cystitis at . months following haplo-sct. there was death due to gvhd and infection at months post transplant. at a median follow-up of (range, - ) months, / patients remain alive and disease-free. the absolute counts of t and b cells were extremely low early post transplant, while nk cells recovered from the first month (mean count, /μl). the number of cd + t cells started to increase beyond the first month, and exceeded lower normal limit at months (mean count, /μl). cd + t cells remained in general low ( o /μl) for the first months, increased moderately by months (mean count, /μl), and approached lower normal values at months (mean count, /μl) [ figure ]. of note, cd +cd ra+ naïve t cells remained significantly impaired (absolute count range, - /μl) in all patients in which they were assessed beyond the st year from transplant. cd + b cells were suppressed for the entire first trimester (mean count at months, /μl), but increased rapidly between and months (mean counts, /μl and /μl, respectively). in haplo-sct with pt-cy, reconstitution of cellular immunity can be achieved at adequate levels by - months following transplant. the observed deficit in the recovery of naïve t-helper cells may be related to a possible effect of pt-cy on thymopoiesis and warrants further investigation. [p ] disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) is a curative therapy for patients with sickle cell disease (scd). hemoglobin a in scd ameliorates the manifestations of the disease and this could be achieved with stable mixed chimerism after a reduced intensity hsct. this study aims to estimate the proportion of patients who develop mixed chimerism after hsct for scd and to characterize its progression in patients who develop it. this is a retrospective cohort study conducted at sultan qaboos university hospital (squh) bone marrow transplant unit in oman. we included all patients with scd who received hsct over the course of years between may to may . patients who received second hsct were excluded. short tandem repeat polymerase chain reaction was used for chimerism assessment. mixed chimerism was defined as − % chimerism at months from hsct. the data was analyzed by r program . . . χ or student t-test were used to assess the impact of acute graft versus host disease (agvhd) prophylaxis, age at transplantation, gender, red blood cell antigen alloimmunization, preparative regimen, and ferritin on the development of mixed chimerism. we included eligible patients. the median follow-up time after hsct was months (interquartile range: . − . months). the mean age at transplant was . years (standard deviation: . ). fifty-nine percent of patients were male. most patients had s/s genotype ( %), followed by s/beta-thalassemia mutation ( %). the indications for bmt were: stroke in %, acute chest syndrome (acs) in %, recurrent vaso-occlusive crisis (voc) in %, stroke and acs in %, acs and voc in %, orbital compression syndrome in %, stroke and moyamoya disease in %, and moyamoya disease in %. the two most frequently used preparative regimens were busulfan/fludarabine/atg in % and thiotepa/treosulfan/fludarabine in %. twenty-five percent of patients developed mixed chimerism at six months after hsct. on follow up of patients with mixed chimerism, % rejected the graft, % developed complete chimerism, and % continued to be in mixed chimerism. preparative regimen and the development of agvhd were statistically significant predictors of mixed chimerism at months (p values: . and . respectively). age at transplant, gender, red blood cell antigen alloimmunization, and ferritin were not statistically significant predictors of the mixed chimerism (p . ). the study confirmed that mixed chimerism can commonly be achieved in patients with scd after hsct and in majority, it remains stable on long-term follow-up. reduced intensity preparative regimen and lack of agvhd predicts the development of mixed chimerism. larger prospective studies are needed to confirm these results. disclosure of conflict of interest: none. there was a majority of male patients ( %). the median hbf level was % ( - ), median monocyte count was . /l (range: - . ), median platelet count was . /l ( - ), median marrow blasts was % ( - ) above patients who were explored by marrow karyotype, % of them had a monosomy . mutations in ptpn were detected in patients. fifty patients ( %) were treated with the bu/cy/mel regimen, whereas patients ( %) received the bu/flu/mel regimen. at years, the overall survival (os) was % ( % ci: - ). nineteen and patients developed vod after bu/cy/ mel and bu/flu/mel conditioning regimen, respectively. the cumulative incidence of agvhd - was % ( % ci: - ). the -year cumulative incidence of relapse and non-relapse mortality was % ( % ci: - ) and % ( % ci: - ), respectively. the median delay of relapse was days (range - ). among relapsing patients, were transplanted twice and one underwent hsct. in multivariate analysis, female donor to male recipient sex-mismatch, cmv status, total body irradiation and ras-double mutation/other additional mutation predicted poorer outcomes. the bu/flu/mel conditioning regimen was associated with a decreased risk of relapse. however, there was no statistical difference for os between the two main preparative regimens, bu/cy/mel vs bu/ flu/mel. our results show that allogeneic hsct may cure approximately % of patients with jmml and are similar to the best results published by other groups. relapse represents the main cause of treatment failure and a second hsct should be proposed. despite a decreased risk of relapse with the bu/ flu/mel regimen, there was no statistical difference in terms of os between the two main conditioning regimens, bu/flu/mel vs bu/cy/mel. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (ahsct) is the only curative treatment modality for the majority of pediatric patients with myelodysplastic syndrome (mds) and myeloproliferative neoplasms (mpn). the purpose of this study is to evaluate overall (os) and failure (relapse or death from any cause) free survivals (ffs), non-relapse mortality (nrm) and relapse incidence in children who underwent ahsct for mds or mpn in a single center from turkey. we retrospectively analyzed ahsct carried out in patients (median age: . years; range: . - ; males). thirty four had primary mds and had secondary mds. according to the modified who mds and mpn classification, had refractory cytopenia (rc), , refractory anemia with excess blast (raeb), , refractory anemia with excess blast in transformation (raeb-t) and , juvenile myelomonocytic leukemia (jmml). amongst patients with secondary mds, had been treated for acute myeloid leukemia, had been treated for non-hodgkin's lymphoma and had been treated for acute lymphoblastic leukemia, retinoblastoma and osteosarcoma, each, previously. donors were related in transplantation ( haploidentical transplantation) and the stem cell resources were bone marrow (n = ), peripheral blood (n = ), cord blood (n = ) and bone marrow +peripheral blood (n = ). three-year ffs and os for patients with mds were % and . %, respectively; and for patients with jmml, % and %, respectively. crude incidence of nrm and relapse for entire group were % and %, respectively. ahsct offers durable ffs and os for a significant group of pediatric patients with mds and mpn. less toxic conditioning regimens could result in better results in some patients. disclosure of conflict of interest: none. allogeneic stem cell transplantation in children with autism z antonella, g alessandra, ma beatriz and r vanderson bone marrow transplantation unit, hospital sirio-libanes, são paulo, brazil autism spectrum disorders (asd) are severe heterogeneous neurodevelopmental abnormalities characterized by dysfunctions in social interactions and communication skills, restricted interests, repetitive, and stereotypic verbal and non-verbal behaviors. the etiology of asd remains unknown, but recent studies suggest a possible association with altered immune responses and asd. inflammation in the brain and central nervous system has been reported with microglia activation and increased cytokine production in postmortem brain specimens of individuals with asd. other studies have established a correlation between asd and family history of autoimmune diseases, associations with mhc complex haplotypes, and abnormal levels of various inflammatory cytokines and immunological markers in the blood. the paracrine, secretome, and immunomodulatory effects of stem cells would appear to be the likely mechanisms of application for asd therapeutics. we describe two cases of patients with asd who underwent hsct for acute lymphoblastic leukemia (all) and whose symptoms were markedly decreased like an improvement of social interaction, communication, and behaviors. the first patient is an -year-old girl with asd who was diagnosis with ph-positive all in october (at the end of treatment, bcr-abl remained positive). she underwent a matched sibling hsct in march . the conditioning regimen was total body irradiation (tbi) and cyclophosphamide. during the -month follow-up period, we observed improvement in social interaction, communication, and behaviors (according to the childhood autism rating scale-cars). the second case is a -year-old boy with asd, asperger syndrome, who was diagnosis with all in september . he presented with bone marrow and testicular relapse in may and underwent a matched unrelated hsct in november . the conditioning regimen used was etoposide, atg and tbi. during the -month follow-up period, we observed improvement in social interaction, communication, and behaviors (according to cars). there is no treatment for asd thus every effort to minimize the symptoms are valuable. in both cases, social interaction was significantly increased, and the aggressive behaviors decreased. clinical cases have reported responses in autistic children receiving cord blood cd + cells. several incurable neurological disorders have shown benefits with cellular therapy. thus, autism should be explored as an indication. clinical studies are an immediate need to fully explore its potential in autism. disclosure of conflict of interest: none. conditioning is the initial phase of hematopoietic stem cell transplantation, based on high dose chemotherapy, combined or not with total body irradiation, aiming to eradicate the disease and prepare the environment of the bone marrow for the new cells. conditioning regimens can be characterized as myeloablative or non-myeloablative. during the period of conditioning and immunological reconstitution, signs and symptoms of the gastrointestinal tract are frequent, negatively influencing oral food intake, and may require the use of complementary nutritional therapies, aiming at an adequate caloric intake with the objective of avoiding decreasing in the nutritional status. the study aims to describe the association between the regiment intensity and the nutritional aspects during hospitalization of children and adolescents undergoing allogeneic hematopoietic stem cell transplantation (hsct) at a tertiary hospital. a retrospective study with medical records of patients undergoing allogeneic hsct, aged between and years of age (incomplete) between january and december . data were collected (regimen intensity, clinical signs of mucositis and nutritional therapies used) during the hospitalization and analyzed by the relative risk (rr). sixty-three patients were evaluated, being % male, with a median age of years. nineteen types of conditioning protocols were used. of these, % were high intensive regimen and % were low intensive regimen. the four most applied ( % of cases) were bucy (busulfan + cyclophosphamide) with and without atg (thymoglobulin), as well as cytb (cyclophosphamide+total body irradiation), also with and without atg. mucositis were observed in % of patients, being % grade and grade . the association was positive when analyzed the regimen intensity (myeloablative) with mucositis (rr = . ( . - . )) as well with the use of parenteral nutrition (rr = . ( . - . )). patients showed high prevalence of mucositis during hospitalization decreasing food intake, being necessary to use the parenteral nutrition. myeloablative regimen needed more nutritional therapy intervention when compared to non-mieloablative regiment. results demonstrate that an appropriate nutritional screening tool considering these aspects could help to intervene earlier maintaining an adequate nutritional status. autoimmune cytopenia (aic) is a potentially serious complication of hematopoietic stem cell transplantation (sct). autoimmune hemolytic anemia (aiha) is the most common aic, followed by immune thrombocytopenic purpura and autoimmune neutropenia. aic after sct is considered difficult to treat and associated with high morbidity and mortality. the aim of this cohort study is to evaluate incidence, outcome, potential risk factors and current treatment strategies and to explore the immune dysregulation predisposing to aic. the ebmt-promise database was accessed to identify all pediatric scts between and complicated by aic at our center. potential risk factors (i.e., age, gender, diagnosis, donor type, stem cell source, conditioning regimen) for aic after sct were assessed using univariate and multivariate cox regression analysis. in addition, we summarized treatment decisions of all aic patients. a nested matched case-control study was performed to search for possible biomarkers for aic. of consecutive scts, were complicated by the development of aic (cumulative incidence . %) at a median of months post-sct (figure) . aiha was the most common aic ( %), followed by combinations of two or more aics (evans syndrome, %). non-malignant disease, young age, alemtuzumab serotherapy pre-sct, non-tbi based conditioning regimen and cmv reactivation were associated with aic in univariate analyses. using multivariate cox regression analysis, non-malignant disease (hr . , p = . ), alemtuzumab use (hr . , p = . ) and cmv reactivation (hr . , p = . ) were independently associated with aic (figure) . for patients with cmv reactivation, diagnosis of aic was made at a median of months (iqr [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) after detection of maximum viral load. in our nested case-control analysis, serum levels of individual anti-and proinflammatory, and regulatory cytokines did not differ significantly between patients and controls. however, the cytokine profile of aic patients appeared to skew towards a more pronounced th response, compared to controls. firstline treatment, usually with prednisone and/or ivig, or a waitand-see approach led to resolution of aic in ( %) cases. second and subsequent-line therapies, often in combination with continuation of other treatments, consisted of rituximab (n = ), bortezomib (n = ) or sirolimus (n = ) and eventually led to resolution of aic in %, % and % of cases, respectively. overall survival of aic patients was %. in this retrospective cohort study, we identified cmv reactivation post-sct, alemtuzumab use and non-malignant disease as independently associated clinical risk factors for the development of aic. treatment with first-line therapy was mostly insufficient. for patients with severe aic, rituximab, bortezomib or sirolimus can be regarded as promising step-up therapies. figure (bkv) may cause polyomavirus-associated nephropathy or polyoma virus-associated hemorrhagic cystitis in bone marrow-transplant patients.we present patients with bk polyoma virus (bkv) ascociated hemorrhagic cystitis and patients with bk polyoma virus associated hemorrhagic cystitis and nephritis. between and , patients received an allogeneic bmt at acıbadem adana hospital pediatric bone marrow transplantation unit. patients occurred bkv associated hemorrhagic cystitis and nephritis. bkv was detected in the urine analysis and blood by pcr (polymerase chain reaction) in all patients. we presented patients with bkv infection, age ranging from to with a average of . years. they underwent allogeneic bmt due to thalassemia major ( patients), aplastic anemia ( patients) and acute lymphoblastic leukemia ( patients). the patients were treated with hydration, continuous bladder irrigation, ciprofloxacin, and weekly intravesical hyaluronic acid instillation for four weeks, and cidofovir. fourteen patients showed complete resolution of hematuria. one patient with refractory above these therapy also received hyperbaric oxygen and recombinant factor viia (rfviia, novoseven; novo nordisk,bagsvaerd, denmark). hemodialysis was performed in two patients who developed renal failure due to nephritis. bkv is ubiquitously present in the general population. reactivation of infection occurs under conditions of immunosuppression, particularly hsct or renal transplantation, and causes late-onset hc. bkv the management of bkv cystitis and nephritis sometimes may be very difficult and refractory all treatments, we presented our experience of bkv infection and management in transplanted patients in our center. patients with high-risk hematologic malignancies (hrhm) are among those in the highest risk group for developing invasive fungal disease (ifd), especially mold infections. allogenic hematopoietic stem cell transplantation (alsct), acute myeloid leukemia (aml), refractory and relapsed acute lymphoblastic leukemia (all), myelodysplastic syndromes and chronic extensive graft-versus-host disease are considered hrhm. ifd are a major cause of morbidity and mortality in these patients, however, the optimal strategy for antifungal p s prophylaxis in this population is not well defined yet. we performed a retrospective, observational study to investigate documented ifd during antifungal prophylaxis in children with hrhm who were admitted in our unit between and . demographic and clinical data were collected from patient's electronic medical records. all patients were treated with prophylactic voriconazole (vcz) according to our local practice. oral administration was preferred when available. vcz therapeutic drug monitoring (tdm) was not available in our center until june . breakthrough ifd was defined as occurrence of a proven or probable ifd according to eortc/ msg criteria while on vcz prophylaxis (⩾ days of treatment) or within days after discontinuation of prophylaxis. during the study period, hrhm patients were treated with prophylactic vcz in our unit. patients out of developed a breakthrough ifd. patient's demographic characteristics, main diagnosis and treatment are collected in table . initial and maintenance vcz doses are adjusted by weight in all patients except in patient- (adjusted according to vcz plasma level). adherence and tolerance to treatment was excellent in all patients. disclosure of conflict of interest: none. ( ) stable mixed chimerism (smc) when fluctuations of ac were o %; and ( ) mixed progressive chimerism (pmc) when ac were ⩾ %. - hscts performed in patients (pts) were included: children with a median age of . yrs (iqr . - . yrs) at diagnosis and . yrs (iqr - yrs) at hsct received one hsct ( . %), pts two hsct ( . %), and pts three hscts. primary diagnosis were bone marrow failures in pts ( . %), primary immunodeficiencies in ( . %), inborn errors of metabolism in ( . %) and haemoglobinopathies in ( . %). the donor was match related in ( %) procedures, match unrelated in ( %), and haploidentical in ( %); stem cell source was bone marrow in ( %), peripheral blood in ( %) and cord blood in ( %). conditioning regimen (cr) included busulfan in hscts ( . %), treosulfan in ( . %), while hscts ( . %) were conditioned with reduced intensity crs (including low dose of tbi in ); pt did not received cr. gvhd prophylaxis was based on csa/mtx (or mmf) in association with atg ( ) or alentuzumab ( ) ; recipients of tcrαβ/cd depleted haploidentical graft did not received post transplant immunosuppression. engraftment was observed in hscts ( after st , after nd and after rd hsct) after a median of day (iqr - days). acute gvhd occurred in hscts at risk ( %), and it was severe (gr. iii-iv) in ( %), chronic gvhd in ( %). at last follow-up (median . yrs), ( %) pts were alive, while pts are dead for infections (n = ), vod (n = ), c-gvhd (n = ) and vascular event (n = ). figure reported the evolution of chimerism over time. in our experience in children with non malignant disease, cc increased from % to % at subsequent analyses. % of pts with mc at st evaluation became cc, % remained smc, % evolved in pmc, and % rejected. only pts with cc at first time point rejected the graft. this study highlight the extreme variability of chimerism in the early post transplant course of children with non malignant disease and confirmed the relevance of performing serial analysis to monitor and, if necessary, improve graft function. naive t-cells identified by cd ra expression are believed to cause graft-versus-host-disease (gvhd), while cd ra-t-cells are memory cells that provide anti-infection and anti-tumoral effects. depleting cd ra+ naïve cells and retaining memory t-cells in the graft is a novel approach to haploidentical hsct for children. children with high risk leukemia ( aml, all) received cd ra-depleted haploidentical hsct following non-myeloablative conditioning. cell-selection performed on g-csf-mobilized peripheral blood. two cellular products obtained using clinimacs device, infused to each patient: a cd selection and a cd ra depletion from the cd negative fraction. product infused contained a median of . (range . - . ) x /kg cd + cells and a median of . (range . - ) × /kg of cd + cells in the cd -selected s graft. the second product was the cd ra depletion, cd ra +/kg was a median of . × /kg (range - × /kg) and a median . (range . - . ) depletion log of cd ra + cells. median dose of cd ro+ cells (memory t-cells) infused was (range . - ) × /kg. seventeen patients achieved neutrophil engraftment at median of days (range - ) post-transplant. one patient could not achieve engraftment, died at day + due to sepsis. two patients presented secondary graft failure (day + and + ), both received a second hsct. three patients developed agvhd grade ii with gastrointestinal tract involvement, all steroids responsive. three patients presented clinical features of cgvhd. patients have an extensive skin involvement, with hepatic findings in one and pulmonary affection in other, at day + , + and + post. ten of patients ( . %) remain alive in remission with median follow-up (range - ) days post-transplant. eight patients died, due progression at day + , + , + ( presented positive minimal residual disease at hsct), due to infectious complications (days + , + , + , + ) and due to cardiogenic shock at day + . four patients relapsed, of them died afterward with progressive disease. t-cells led immune recovery, achieved values higher than , , and cells/mcl at day , , and respectively. most of t cells were cd +cd ra-(median of , and × /mm respectively on day + , + and + ) and cd +cd ra-t cells (median of , and × /mm respectively on day + , + and + ), while cd + ra+ and cd + ra+ cells remained low recapitulating the cd ra depleted graft composition. six patients presented cytomegalovirus reactivation, one progressed to cmv disease. five patients with hhv- encephalitis. probable aspergillosis in patient (aml-m with secondary graft failure) at day + after second hcst. two cases of toxoplasmosis ( cns, pulmonary). cd ra-depleted haplo-hct showed acceptable tolerability with rapid and sustained engraftment as well as a full donor chimerism, minimal risk of acute gvhd and accelerated inmunologic reconstitution. to note the high incidence of hhv- encephalitis seen. disclosure of conflict of interest: none. collection of peripheral blood stem cells in teenager sibling donors: a single center experience c carvalho , f amado, f bordalo, s ferreira, s lopes, c pinho and s roncon serviço de terapia celular, instituto português de oncologia do porto francisco gentil, epe human leukocyte antigen (hla) compatibility is important in allogeneic haematopoietic stem cell transplantation in order to reduce post-transplant complications; however, siblings only present a % chance of hla-match with the patient. the well-known advantages and the low risk of complications associated to peripheral blood stem cells (pbsc) collected by apheresis made this procedure the first option in teenagers. the aim of this retrospective study was to analyse and characterize the paediatric sibling pbsc donor population assuring safety during the collection procedure, providing a high-quality product and accomplishing patient needs. we consulted the clinical files of donors under years old since - ; a database in excel ® was created to register population characteristics, collection parameters and graft requirements. the informed consent was obtained from parents before procedure. the leukapheresis were performed with a cobe spectra system; since , we use a spectra optia apheresis system. the donor/patient weight ratio (proposed by styczynski et al.) was determined for each pair. the collection was programed based on clinical and analytical donor's features as well as transplant requirements. the analytical assays were done by a certified laboratory. we performed pbsc apheresis in healthy donors, females and males ( table ) . all of them started on the th day after mobilization with granulocyte colony-stimulating factor (g-csf) administered subcutaneously, bidaily. the weight ratio was o in eight situations. most of donations were performed by peripheral vein; a central venous catheter (cvc) was placed into a femoral vein in six adolescents. a median of ( ) ( ) ( ) blood volemias were processed during ( - ) minutes; the anticoagulation used was citrate+heparin (ratio : ). in general, one-collection day was enough to obtain the number of cd + cells required; six donors had to perform a nd collection. in cases, we cryopreserved the exceeding cells after graft infusion. the procedure was well tolerated, with only adverse reactions registered (one hematoma in the puncture local; one paraesthesia due to hypocalcaemia induced by citrate). no blood products were used after the procedure or needed for the priming of the extracorporeal circuit. so far, no serious long-term adverse events were observed. table . median (range) of donors and leukapheresis products data. our long lasting experience shows that pbsc collection in the teenage population is safe and feasible, allowing us to obtain a high-quality product for the patients. there were no adverse events associated with the g-csf mobilization or cvc placement which is different from the experience of other groups. we recognize that leukapheresis by peripheral vein is a lengthy procedure but no complaint was reported to the collection team. [p ] disclosure of conflict of interest: none. correspondence between clinical and hystological grading of gastro-intestinal grading acute graft versus host disease in children m faraci , a rizzo , p gandullia , s arrigo , , a barabino , e lanino , s giardino and c coccia hematopoetic stem cell transplant unit, institute g gaslini, genoa, italy; pediatric department, institute g gaslini, genoa, italy; gastroenterology and digestive endoscopy unit, institute g gaslini, genoa, italy; gastroenterology and digestive endoscopy unit, institute g gaslini, genova and department of pathology, institute g gaslini, genoa, italy diagnosis of gastro-intestinal acute graft versus host disease (gi-agvhd) is frequently confirmed by apoptosis findings on mucosal biopsies. aims of this single center retrospective study is to evaluate the correlation between clinical and histological grading of gi-agvhd in children undergoing allogeneic haematopoietic stem cell transplantation (allo-hsct), and to describe histological findings obtained by gi endoscopies in order to evaluate usefulness in the diagnosis of gi-agvhd. allo-hscts were performed in our department between january and december . gi biopsies were performed in pts ( . %) because of suspected gi-agvhd. pts were transplanted for malignant ( . %) and for not malignant diseases. the median age at hsct was . years ( . - . ). pts ( %) received myeloablative and ( %) reduced intensity conditioning regimen. pts ( . %) received an unrelated donor (ud), pts a related donor (rd) ( . %), and an haploidentical donor ( . %). at onset of diarrhea, microbiological examinations of stool were performed and pcr research for cmv, adenovirus, hhv , ebv were evaluated in blood and in mucosal biopsies. mucosal biopsies were obtained with esophago-gastro-duodenoscopy in pts ( . %),esophago-gastro-duodeno-colonscopy in ( . %), pancolonscopy in ( . %), flexible sigmoidoscopy in ( . %), and rectal suction biopsy in pts ( . %). all mucosal biopsies, except in case of rectal suction, were obtained under sedation. the interval between mucosal biopsies and onset of gi acute symptoms was days (from − to days). biopsies were taken from different sites in the gi tract, were stained using hematoxylin-eosin and evaluated using histological grading of agvhd. in these pts the maximum grade of agvhd was: grade in one ( %), grade in ( %), and grade in pts ( %). at time of histological evaluation, diarrhea was the most common gi symptom ( . %); children had also cutaneous agvhd and hepatic agvhd. pcr-cmv was positive in mucosal biopsies obtained with pancolonscopy, pcr-adenovirus in other obtained with upper and pancolonscopy, pcr-hhv in rectal biopsies, and pcr-ebv in one with upper and pancoloscopy. the comparison between clinical and histology grading of gi-agvhd is shown in table . mucosal biopsies were positive in / pts evaluated with esophago-gastroduodenoscopy ( %) (grade agvhd), in / pts undergone esophago-gastro-duodeno-colonscopy (grade in and grade in ), in / ( %) who received a pancolonscopy (grade in , grade in , grade in ), and / ( %) of rectal biopsy obtained by sigmoidoscopy or rectal suction biopsy (grade in , grade in , grade in , and grade in ). one patient developed duodenal intraparietal hematoma after upper endoscopy. in our experience, we did not demonstrated a overall correlation between clinical and histological grading of agvhd showing that hystological examinations underestimated the grade mild or moderate of agvhd. we confirmed , that rectal biopsies represent to be more effective and safe diagnostic method for the confirm of diagnosis of gi-agi. during the past few decades, hematopoietic cell transplantation (hct) as a treatment modality for primary immunodeficiencies (pid) has undergone remarkable advancement mainly due to better availability of alternate donors resulting in increase in not just matched unrelated donor (mud) but also increased haploidentical (haplo) and cord blood transplants (cbt). additionally, refinement of the conditioning regimens and better graft versus host prophylaxis have presumably led to better survival outcomes. however, a literature gap is identified in evaluation of these outcomes in general with respect to donor and conditioning regimens. we conducted a systematic review by performing a comprehensive search of the pubmed and ovid library from its inception to august . mesh terms included 'primary immunodeficiency (immunodeficiencies)', 'stem cell transplant', 'bone marrow transplant' and 'hematopoietic cell transplant'. all pid studies which used hct as a treatment modality were included. experimental cellular therapies were excluded. both cellular immunodeficiencies (e.g. scid, was, a-t), and innate immunity disorders (e.g. ifngr, cgd) were included in the search. reviews, case reports, meta-analysis and non-english language articles were excluded from our electronic search. publication bias was excluded by performing a methodological search of unpublished conference abstracts from the annual meetings of cis, aspho, asbmt, ebmt, and siop from to . the data were analyzed considering the outcomes -overall survival and gvhd. studies fulfilled the strict selection criteria for the electronic search comprising of pid patients. in majority of the hcts, a myeloablative conditioning regimen (mac) was utilized ( % of the evaluable) but a shift towards more reduced intensity conditioning (ric) was observed in the later years. cbts were identified. % of patients developed some degree of acute gvhd, whereas chronic gvhd was identified in % of the patients. total number of haplos was . overall survival was found to be % post-hct. a meta-analysis could not be performed due to the heterogeneity of both the predictor variable data (combined stem cell sources were also used for hct) and due to the extremely small number of the patients when categorized in subgroups (e.g. for omenn syndrome, rag deficiencies). this is the largest study of hcts in pid, and we observe that alternate donor hcts have increased significantly over the past decade for the treatment of pid. while the incidence of chronic gvhd was low, acute gvhd still remains a problem in about a third of the pid patients transplanted. disclosure of conflict of interest: none. hepatic veno-occlusive disease (vod) is a common and serious complication of hemotopoietic stem cell transplantation (hsct) in children. we aimed to assess prospectively the use of prophylactic defibrotide in pediatric patients undergoing hsct. in this study, patients who underwent hsct were given defibrotide prophylaxis as mg/kg per day in four divided intravenous infusions over h, starting on the same day as the pretransplantation conditioning regimen. the mean duration of use of defibrotide is days as a prophylaxis. in this study, patients were recruited, male patients and female patients, with the average of . years, range - ; % infants, % children and % adolescent. there were patients with thalassemia major, patients with leukemia, patients with aplastic anemia, one patient with diamond blackfan anemia, two patients with congenitale dyserythropoetic anemia, one patient with osteopetrosis, four patients with famial hemophagocytic lymphohistiocytosis, two patienrs with severe immune deficiency and one patient with kostman syndrome. all transplants were allogeneic. no serious side effects were seen. in eight patients developed clinical vod (seattle criteria). in these patients, defibrotide dose was increased to a treatment dose of - mg/kg per day. one infant patient with kostman syndrome died due to hepatic and pulmonary veno-occlusive disease. after months of follow up, patients who developed vod are being well and no patient have transplant related complications. hepatic veno-occlusive disease, which is caused by hepatocyte and sinusoidal vessel endothelium damage, can ocur early after hsct, and in its severe form, may lead to liver faillure, hepatorenal syndrome, portal hypertension, and eventually death from multiorgan faillure. in this prospective study, we demonstrated that the use of defibrotide is safe and effective in preventing and treating vod in pediatric patients at high risk. immune reconstitution (ir) is critical for clinical outcome after allogeneic hematopoietic stem cell transplantation (hsct). host and proceeding-related factors affect the ir dynamics and survival. isolated ir parameters are commonly correlated and proposed to predict clinical outcomes after hsct, but these approaches only confer prognostic value at single time points or for single markers. we aim to demonstrate an appropriate methodology to assess the capability of combined serial measurements of lymphocyte subsets to reflect the impact of infections on ir after paediatric hsct. retrospective data of patients receiving a first hsct for any indication with any cell source in the paediatric hsct program from to were included. to characterize the kinetics of immune reconstitution, cd +, cd +, cd + t-cells, b-cells, nk-cells and their naive and memory subsets were measured and analysed at various time points at years post-hsct to stablish a joint model for the evolution of cell subpopulations. slope per month (cellular increase or decrease) of each lymphocyte subsets were calculated and compared with clinical outcomes and cumulative risk of infections. a total of children (range from - y.o. median y.o.) were included, with cb (n = ) pb (n = ) and bm (n = ) as cell sources. the cumulative incidences after early period were % for viral infections (ebv %, cmv %, bk %, adv %) and % for bacterial infections. data on ir were available for %, of the diseasefree survivors. in a exploratory multivariate analysis we detected mainly differences in cd +, cd +cd ro+ memory and nk cells at year after hsct, with dependent tendency according on the cell source and hla compatibility. analysis of the slope tendency patterns were stablished for the analysis of the impact of infections in the ir. delay in cd + and cd +ra+ appearance (mean slope/m = − . % and − . % respectively) remarks the ir profile for bacterial infections, and delayed in nk, cd and cd ro+ (− . %, − . %, − % respectively) for overall viral infections. additional correlations allow differences in ebv (cd +ra+ high mean slope/m = . %), cmv (delayed in cd ro+ slope/m = %), and bk infection (cd +ra + plus cd ro+ and nk high mean slope/m = . %, % %). understanding the dynamics of reconstitution by integrating information from the monitoring of lymphocyte subpopulations allows the establishment of kinetic profiles that may help to evaluate the risk of infections and adjust infection prophylaxis in the follow-up of transplanted patients. mortality rate in hsct patients admitted to intensive care unit (icu) is still as high as % to %. this rate increases when respiratory complications progress to acute respiratory failure (arf) requiring mechanical ventilation (mv). the aim of this study was to determine the feasibility and effectiveness of early continuous positive airway pressure (cpap) delivered in a pediatric hematology-oncology ward to prevent occurrence of arf requiring mv. we retrospectively analysed children treated with cpap in our pediatric hematology-oncology ward between october and october . thirty-two patients received cpap delivered with helmet during the study period. data were available for patients, males and females, median age years [range - ]. eighteen patients underwent allogenic hsct: from sibling donor, from matched unrelated donor, from haploidentical family donor, from cord blood unit. seven patients had a malignant disease: all, aml, ewing sarcoma. infectious pneumonia was the main cause of arf in / patients ( . %): viral pneumonitis ( rhinovirus, parainfluenzae virus, respiratory syncitial virus and cmv). five patients had proven/ probable invasive fungal infection according to eortc criteria ( aspergillosis and mucormycoses). other causative agents were pneumocystis jiroveci ( ), bacillus of calmette and guerein ( ), toxoplasma gondii ( ) and st. mitis ( ) . non infective causes of arf were acute transfusion related lung injury ( ), hemorragic alveolitis ( ), cryptogenic organizing pneumonia ( ), tumor lysis syndrome ( ), and alveolar oedema due to renal failure ( ). according to chest imaging, / patients ( %) presented with pulmonary consolidations, while % had both interstitial infiltrates and pulmonary consolidations. at baseline median neutrophil count was . × /μl (range - . × /μl), mean heart rate bpm, pulsiossimetry saturation in room air %. h-cpap was applied in / patient with a curative aim, in / patients as palliative support to reduce respiratory distress. median positive pressure delivered was cmh o ( - cmh o), median fio was % ( - %). h-cpap was applied for a median of days . no patient failed h-cpap because of agitation or adverse events (skin breakdown, conjunctivitis, gastric distension or epistaxis). ten patients were transferred to icu ( . %), / because of hsct complications. median icu stay was . days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . only patients required mechanical ventilation, in cases associated to ecmo. nether psao in room air (p . ci %) nor cpap level (p . ci %) correlated with the need of icu admission. patients requiring higher fio during cpap demonstrated a not statistically significant trend to higher icu admission rate (p = . ).there was a higher rate of mv in patients with higher cpap fio level (p = . ). mv prolonged icu stay (p . ). cumulative mortality was . % ( / ); only patient died in icu ( %), because of post hsct parainfluenza virus pneumonitis requiring ecmo. helmet cpap delivery in pediatric hsct ward is feasible and safe, both for curative and palliative aim. if applied early, cpap could reduce icu admission rate for mv and icu mortality. veno occlusive disease (vod) and graft versus host disease (gvhd) are both dreadful complications of hematopoietic stem cell transplantation (hsct). although they have different clinical signs, it is suggested that they share similar pathophysiological pathway. defibrotide is used in the treatment of vod for a long time but it is very less known about its effect on gvhd. in this study, we analyzed a 'high risk' patient population in pediatric hsct to show the effect of defibrotide on acute gvhd. between june -august totally 'high risk' pediatric allogenic hsct procedures were enrolled in this study. 'high risk' definition involves busulphan/ melphalan usage in conditioning regimen, second myeloablative hsct, pre-existing liver disease, allogenic hsct for leukemia with second relapse and diagnosis of hemophagocytic lymphohistiocytosis (hlh) or osteopetrosis. defibrotide prophylaxis group (n = ) received mg/kg/day per day and continued for at least days after transplantation. the control group (n = ) received only continuous infusion of low-dose heparin until days after transplantation. for the comparison between groups, the fisher's exact test was used. all analyses were performed using spss and p-value of . was considered statistically significant. we analyzed totally hsct procedures with different diagnosis; beta thalassemia major, leukemia, hlh, primary immunodeficiencies, osteopetrosis, fanconi aplastic anemia (faa), myelodysplastic syndrome, neuroblastoma, congenital amegakaryocytic thrombocytopenia, krabbe disease, aplastic anemia, hypereosinophilic syndrome and sickle cell disease. all the procedures meet the 'high risk' definition; most of them (n = ) have busulphan for conditioning, also there are hlh and osteopetrosis patients, neuroblastoma patients had the second myeloablative regimen, faa and aplastic anemia patients had pre-existing liver disease, and of the leukemias had beyond second relapse. the mean age was . years old ( . - . ), hsct performed from match sibling donor (msd) ( %), hsct from match family donor (mfd) ( %), hsct from match unrelated donor (mud) ( %) and hsct from haploidentical mother ( %). we especially focused on gvhd and vod. totally vod cases ( %) in these hsct procedures were detected. only two of them detected in the prophlaxis group ( %) and cases in the control group ( %). there were cmv reactivation cases detected in hsct procedures ( %). in the prophlaxis group there were cases ( %) and in the control group cases ( %). we detected acute grade i-iv gvhd cases in hsct procedures ( %). only of them were in the prophlaxis group ( %) and cases were in control group ( %). the prophlaxis group's agvhd ratio was significantly lower than the control group (p = . ). defibrotide for vod prophylaxis is confirmed by several studies, but its benefits for agvhd is not clear. in this study, we show the significant effect of defibrotide on agvhd. we speculate that the protective effect of defibrotide on both vod and agvhd could be explained by the similar pathophysiology of these complications. we need larger studies on the pathophysiological pathways, then we could invent more effective interventions. disclosure of conflict of interest: none. conventional extracorporeal photopheresis (ecp) has proven efficient for the treatment of graft-versus-host-disease (gvhd) but is limited to patients with sufficient body weight. a mini buffy coat ecp (mini-ecp) 'off line' technique that allows treatment of small children has been developed, using various methods for mononuclear cell (mnc) separation from whole blood. we present treatment of low body weight child with mini-ecp 'off line' technique using sepax system for mononuclear cell (mnc) selection and macogenic irradiator. a toddler with juvenile myelomonocytic leukemia (jmml) developed acute gvhd, after a matched unrelated stem cell transplantation (sct) performed at the age of months. acute gvhd of the skin occurred three months after sct and responded to high dose steroids, but recurred six months after sct (biopsy of the skin confirmed acute gvhd) together with gvhd of the liver. because of the resistance to steroids and cyclosporine, mini-ecp was introduced as therapy. the patient weighed kilograms. blood was collected from tunneled central venous catheter, and collected volume was replaced with saline infusion. the cord blood collection bag (macopharma, france), which contains ml citrate phosphate dextrose (cpd) anticoagulant solution was used for whole blood collection. whole blood was processed using sepax system separator (biosafe, switzerland), and final volume of buffy coat was set on ml. extracted buffy coat was transferred into the uv-a illumination eva bag (macopharma, france) and diluted with saline solution up to ml. methoxypsoralen (gerot, austria) was injected directly into the uv-a illumination bag, and cells were irradiated by the uv-a illumination device macogenic (macopharma, france). irradiated cells and autologous residual blood were reinfused back to the patient. during the whole procedure patient's vital signs were monitored. ecp procedures were performed times per week for weeks, followed by times per week at weeks intervals for months. in month period mini-ecp procedures were performed. median of collected whole blood was ml (range - ). median of total nucleated cell (tnc), and mononuclear cell recovery after sepax separation were . % (range . - ), and . % (range - ), respectively. median of hematocrit in final irradiated product was % (range . - %). patient was reinfused with median of . (range . - . ) tnc × /kg bw, and median of . (range . - . ) lymphocyte × /kg bw. after one month of ecp together with steroids and cyclosporine, gvhd of the skin improved, and the steroids were gradually weaned, with no recurrence. gvhd of the liver showed no improvement, and other therapies had to be introduced, but without steroids. for the first time in croatia, mini-ecp was performed in a child with gvhd, in whom conventional ecp could not be used because of insufficient body weight. mnc separation using automated closed system sepax separator has proven efficient and safe. mini-ecp treatment was continued for three months, without technical difficulties. positive effect was experienced concerning the skin gvhd, but not the liver gvhd. after the first experience in our country, in future we plan to use this technique for low-weight patients or patients with contraindications for apheresis, which are in need of second-or third-line therapy for gvhd. disclosure of conflict of interest: none. gonadal failure represents one of the late effects of haematopoietic cell transplantation (hsct) with a negative impact on quality of life in young patients (pts) undergoing hsct , . the aim of this retrospective multicentre ebmt study was to assess gonadal function in untreated pts undergoing allogeneic hsct between to years (yrs) of age, after a preparative regimen with busulphan (bu) or treosulfan (treo). eighty-seven pts ( females, males) were reported from out of contacted ebmt centers: / ( %) received allogeneic hsct during pre-pubertal and / ( %) in pubertal phase. of the pts, ( . %) received bu in myeloablative dose [ pre-pubertal, (median age of . yrs) and pubertal, (median age of . yrs)] and pts ( . %) received treo ( in pre-pubertal and in pubertal period). underlying diseases were primary immunodeficiency ( . %), chronic myeloid leukemia ( . %), myelodisplastic syndrome ( . %), familial haemophagocytic lymphohistiocytosis ( . %) and shwachman-diamond syndrome ( . %). / of prepubertal pts ( %) developed spontaneous puberty ( . % in the bu group and % in treo group). / ( %) females undergoing hsct during puberty completed their pubertal development ( . % in bu group and % in treo group). none of females ( / ) with bu during pre-pubertal phase developed spontaneous menarche (sm), while . %( / ) of females who received bu in pubertal period had sm. all females (n = ) treated with treo during pubertal phase had sm ( %). for both conditioning regimens, the . % ( / ) s of females treated during the puberty experienced sm. among the remaining females (for pts the information is missing) who did not developed sm, received hrt . yrs after hsct and of them had ovarian recovery after a median of . yrs from hsct ( . - . ). the median age at last follow up was . and . yrs in bu and treo pre-pubertal group, and . and . yrs in bu and treo pubertal group respectively. in the pubertal group, females ( . %) are still receiving hormonal replacement therapy (hrt) ( in the bu group and in treo group). pts ( . %) had spontaneous pregnancy. no problems in newborns are reported. sperm analysis was performed in . % of pubertal pts ( / ) of males, and % (n = / treated with bu) were azoospermic (data regarding pts were missing). the sperm analysis was repeated in half of the males. until now no paternity was reported. in this experience, the pubertal development in pts who received treo (n = ) was normal, and in the bu group the majority of females ( %) had normal puberty. the rate of sm is higher ( %) in females after treo than bu ( %). the hrt is ongoing at last follow-up in % of females treated with bu and in % of those who received treo. our data suggests that treo may have a better outcome than bu in young girls receiving allogeneic hsct and larger studies are warranted. male patients require longer follow-up. prevention in patients transplanted from partially matched donors. we report the single centre experience in haploidentical sct. in years - in the department of pediatric bmt, oncology and hematology at wroclaw medical university, children underwent sct from partially matched, haploidentical parental donors. graft manipulation in patients consisted of cd sel, in patients the cd immunomagnetic depletion (tcd-cd ) was performed, and in -tcr alpha-beta depletion (tcd-ab). we analysed the impact of type of manipulation procedure, conditioning regimen, demographic factors, and kir genotype on survival and probability of neutrophil recovery. the probability of engraftment and neutrophil recovery was % vs % in cd sel group (p = ns). probability of year overall survival in the tcd group was similar to the cd sel group ( % vs %, p = ns). in the tcd patients, neither use of busulfan vs treosulfan, nor kir genotype, nor donor sex had noticeable impact on sct result and survival. patients transplanted after tcd due to non-malignant disease had higher survival probability, than those with malignancies ( % vs %, p = . ). the trm in tcd patients was reduced in comparison to cd sel ( vs %, p = . ). the trm after tcd resulted mostly from severe viral infections in tcd-cd patients. in / tcd patients spontaneous acute, skin (stage ) gvhd was diagnosed and successfully treated. two patients received unmanipulated donor lymphocyte infusions (dli) and developed severe acute steroid-resistant (grade ) gvhd, in one of them with fatal outcome. tcd methods are superior to cd sel due to significant reduction in treatment related mortality. haploidentical sct after tcd can result in durable engraftment, but warrants intensive post-transplant monitoring for infectious complications and cautious approach to dli therapy. disclosure of conflict of interest: none. median of days for neutrophils in both groups, for platelets ( in ptcy, in αβ+cd +/cd +depleted, p . ). donor chimerism was complete in patients ( . %). in αβ +cd +/cd +depleted group, patient rejected ( . %: primary and secondary reject, , , and days after haplo, respectively) and were rescued with a second transplant. seven patients ( %) developed acute (a-) gvhd in ptcy group (grade - in ; grade - in ), compared to one ( . %: grade ) in αβ+cd +depleted group (p . ). among patients at risk, out of in ptcy group developed chronic (c-) gvhd ( . %: score- , overlap, score- ), compared to / patients in αβ+cd +/cd +depleted group (p . ). the cumulative risk of cmv-reactivation was % and % in ptcy and αβ+cd +/cd +depleted groups, respectively (p . ). t-cell reconstitution was significantly different in the two groups,with a median absolute number of cd + +cd -and γδ+cd + higher in αβ+cd +/cd +depleted group on day + (p . ) and a median number of cd +cd + higher in ptcy group on day+ (p . ). length of hospitalization was shorter in the αβ+cd +/cd +depleted group, with a median time from haplo to discharge of days compared to days in the ptcy group (p . ). some children have not donor and an urgent need to proceed to transplantation because of disease status. we reviewed the role of haploidentical transplantation in children and report our single center experience. ten children were transplanted from haploidentical family members donors (median age: . years). we performed alfa beta t cell depleted transplantation in three patients and unmanipulated bone marrow transplantation with posttransplant cyclophosphamide in seven patients. the diagnosis were eight high risk leukemias (three all and five aml) and two severe aplastic anemia. patients were myeloablative conditioned with cyclophosphamide, fludarabine and total body irradiation in aplastic anemia received alfa beta t cell depleted grafts with a median cd cell dose of . × /kg (range: . - . ) and busulphan, cyclophosphamide in high risk leukemias received unmanipulated bone marrow grafts with posttransplant cyclophosphamide in rd and th day of posttransplant with a median cd cell dose of . × /kg (range: . - . ). median follow up of our patients months. six of patients are alive and in disease free follow up. four patients were relapsed and dead median . months of transplantation. the rate of relapse was % for leukemia patients in remission and % for patients with active disease. myeloablative conditioning regimen followed by haploidentical bone marrow transplantation with posttransplant cyclophosphamide may be an option in high risk leukemia patients need urgent transplantation because of desease status who have not donor. table . all patients received hd-cy mg/kg on d+ and d + . cyclosporine a mg/kg/d i.v., then mg/kg/d p.o. adjusting for blood levels - ng/ml and mycophenolate mofetil mg/kg times daily po were started on d+ . mmf was discontinuated on d+ , csa-after d+ . all pts received anti-microbial prophylaxis for bacteria, fungal, herpes infection and pneumocystis according to institutional practices. analysis for donor chimerism and mrd were performed at d+ , + , + , + . pts, donors and stem-cell harvest characteristics are described in table . pts had high risk hematological malignancies, and relapsed after auto-sct neuroblastoma (hr-nb). pt was transplanted in st cr (aml m ) and others in nd cr. pts had full engraftment (neutrophil engraftment at , and days). pt (hr-nb) was concerned as a primary failure for achieving neutrophil and platelet engraftment by d+ , despite of complete donor chimerism in bone marrow. he was transplanted additionally with the same donor at d+ after st transplant. pt died before engraftment at d+ (fulminant ps. aeruginosa-sepsis). pts remain alive in cr ( ndcr-aml and st cr-m aml) with follow-up of and days ( / / ) without cgvhd with complete donor chimerism. pts relapsed after d+ ( were transplanted in nd cr-flt aml and nd cr-nb) and died. pt died because of infectious complication at d+ (transplanted in d cr-all). / pts had grade acute gvhd. hla-haploidentical hsct with post-transplant t-cell in vivo repletion grafts by using hd-cy is feasible and effective in children with hr-haematological malignancies. [p ] who were match unrelated donor. thalassemic reconstitution occurred in three patients. acute graft-versus-host disease (gvhd) of grade ii-iv occurred in % and chronic gvhd in %. acute and chronic gvhd were seen more frequently in patients with class - compared to class . mortality rate was also higher in these groups. seven patients died. one patient died on post-transplant day due to intracranial bleeding. the other patients with chronic gvhd died between and days, on average days post-transplant. these data suggest that allogeneic bmt remains an important treatment option for children with beta-thalassaemia major, particularly when compliance with iron chelation is poor. the society to support children suffering from cancer, also known as mahak, was set up in as a non-governmental and non-profit organization. in the past two decades, the organization has attracted a vast public support and fulfilled a great part of its mission which is to support children with cancer, reduce the child mortality rate and create an appropriate environment that empowers families who have children with cancer. pediatric stem cell transplant also is used to treat many types of conditions affecting children and adolescent, including cancer and certain hematologic, immune reconstitution inflammatory syndrome (iris) is a clinical condition emerging after immune recovery of an immunocompromised status, mostly after the initiation antiretroviral therapy (art) in human immunodeficiency virus (hiv) infected patients, but also in several other settings, such as the recovery from the severe combined immunodeficiency (scid) status after hematopoietic stem cell transplantation (hsct). herein, we report a patient transplanted for scid who developed iris for two times, namely shortly after transplantation and after donor lymphocyte infusion (dli) ( table ) in our patient, t cells passing from the donor probably contributed to the immediate post-transplant increase in the size of granulomas. this inflammatory response waned after the institution of immunosuppressive and methylprednisolone therapy. however, immunosuppressives were stopped due to lowered chimerism at follow-up, and the inflammatory response re-appeared after administering stem cell support containing a large amount of t cell from the donor for dli purpose. although the mechanism by which dli results in clinical responses is unclear, it is presumed to be a t cellmediated process. several studies have been performed to strengthen our understanding of the immunopathogenesis of iris. while some of those studies put forth t cell-associated causes, others implicated cytokines and non-t cells. the reaction that developed in our patient is suggestive of t cellassociated causes. immune reconstitution inflammatory syndrome remains a poorly understood entity. the dli procedure in our case provides a unique clue supporting a t cell mediated process. pediatric transplant teams need to be s aware of the previous iris phenomenon of bcg-adenitis while making the decision of dlis. [p ] disclosure of conflict of interest: none. pediatric patients treated with a hematopoietic stem cell transplantation (hsct) often suffer from late side effects caused by the treatment. the aim of this study is to investigate the late effects of a hsct on dental development. in addition, patients and parents awareness on this topic was investigated. young adults treated and followed at the ghent university hospital who were under the age of y at the time of hsct were examined clinically and radiographically (planmeca promax d). transplants ( autologous/ allogeneic) were done for malignant disease in pts. eight patients received a hsct for a non-malignant disease. twelve patients underwent a conditioning regimen with total body irradiation (tbi), patients with busulfan and patients with other chemotherapeutic agents. patients were o y, patients were - years and patients were years at hsct. every patient was evaluated on dental agenesis, microdontia and rootcrown ratio. patients and their parents were asked about their knowledge and interest for dental screening at the follow-up clinic using a questionnaire. overall, the prevalence of agenesis and microdontia of one or more dental elements is respectively . % and . % in our study population. . % of patients have a strongly aberrant root-crown ratio of at least one element. patients treated o years of age show significantly more microdontia ( . %; po . ) as well as agenesis ( . %; p o . ) compared to patients treated at an older age. the first premolar of the mandible is the most vulnerable element for agenesis as well as for microdontia. more microdontia is found in patients treated with a busulfan conditioning regimen compared to the other conditioning regimens ( . % versus %). patients older than years, treated with busulfan have statististically more microdontia compared to patients y treated with tbi conditioning regimen (p = . ). there was no difference of the conditioning regimens on agenesis nor on root-crown ratio. almost all patients/parents find it important to receive information about the dental late effects of a hsct and are interested in dental screening at the follow-up clinic. treatment with hsct has an explicit negative impact on dental development. the degree of this effect depends on age at hsct and used conditioningregimen. dental follow-up of these patients is essential and should be incorporated in the follow-up program. disclosure of conflict of interest: none. importance of body composition in the outcome of hematopoietic stem cell transplantation in elderly patients l koch , n hamerschlak , r garcia , c prado , c silva and a pereira hospital israelita albert einstein the loss of muscular mass is a well recognized cause of the decline in muscle strength and functionality that accompany the aging process. in , irwin rosenberg proposed the term 'sarcopenia' to describe the decline in muscle mass associated with aging. changes in body composition after hematopoietic stem cell transplantation (hsct) have been the subject of previous studies. immunosuppressive therapy and corticosteroids are known to alter skeletal muscle metabolism. infections and graft-versus-host disease (gvhd) that can occur after hsct may also affect body weight and composition. therefore, both the treatment and complications after hsct exert large negative effects on lean muscle mass, especially in elderly patients. patients with hematologic malignancies are usually well nourished before undergoing hsct. objective: the aim of this study is to determine in an elderly population whether parameters of body composition could be correlated to outcomes after hsct. we performed a retrospective longitudinal study through review of medical records of patients ⩾ years old undergoing hsct at hospital israelita albert s einstein, from to , that were subject to tomography scans (cts) in a period ranging from days before and days after hsct. table . there were no differences between groups with respect to age, gender, diagnosis, stage of disease, and source of stem cells. in ly patients, the quantity of peripheral cd + cell dose (× /kg) infused was different between groups (group ly-ct the incidence of nf was significantly higher in group mm-g ( ( . %) vs ( . %); p = . ). no differences were observed in the incidence and severity of mucositis, first day and duration of fever, documented bacterial infections or readmission rate between mm patients groups. this study suggests that in at home asct, the use of piperacillintazobactam prophylaxis significantly reduces the incidence of neutropenic fever and hospital readmission in patients with ly, and also that no administration of g-csf in mm patients reduces significantly the incidence of neutropenic fever. disclosure of conflict of interest: none. [p ] allogeneic stem cell transplantation (sct) has been recognized as a curative treatment for patients with wiskott-aldrich syndrome (was). in sct for was, myeloaberative conditioning (mac) has been indicated to avoid a mixed chimera. however, risk factors for a mixed chimera in patients with was who have received sct have not been evaluated. here, we analyzed the outcomes of sct and risk factors for a mixed chimera in patients with was who underwent sct in japan since . we reviewed medical records of consecutive was patients who received sct since january who were registered with the japan society for hematopoietic cell transplantation. the age of the patients at transplantation ranged from months to years, and the mean age was . years. the origin of the stem cells was related bone marrow (bm) or peripheral blood stem cells (pbsc), unrelated bm or pbsc, and unrelated cord blood (cb) for , and patients, respectively. a preparative conditioning regimen consisting of mac was provided to patients, and reduced-intensity conditioning was provided to patients. fifty-one patients received prophylaxis against graft-versus-host disease (gvhd) with cyclosporine in combination with methotrexate (mtx) or a steroid, and patients received tacrolimus (tac) with mtx or a steroid. chimerism analysis had been performed in patients. neutrophil engraftment was achieved in patients ( . %). the engraftment rate was significantly higher in patients who received tac for gvhd prophylaxis, (p = . ) overall survival rate was significantly higher in patients with complete chimerism than in patients with mixed chimerism ( . ± . % and . ± . %, respectively, p = . ), though there was no significant difference in stem cell sources. using multivariate analysis, the rate of complete chimerism in patients who received mac including cyclophosphamide (cy) at more than mg/kg was significantly higher (p = . ) than the other conditioning. not only patients with mixed chimerism but also patients with complete chimerism were complicated with auto-immune diseases. in this study, achievement of complete chimerism after sct was important for survival in patients with was. we found that patients who underwent mac including cy at more than mg/kg had a higher rate of complete chimerism. we also found a higher neutrophil engraftment rate in patients who received tac for gvhd prophylaxis. thus, mac including cy at more than mg/kg and tac for gvhd prophylaxis are optimal conditions of sct for patients with was. disclosure of conflict of interest: none. adenosine deaminase (ada) deficiency is an inherited autosomal recessive immunodeficiency which represents about - % of scid. since we diagnosed patients affected by ada-scid: underwent hematopoietic stem cell transplantation (hsct), were treated with replacement therapy with peg-ada and received gene therapy; patients died before or after treatment. maternal t lymphocyte engraftment is frequently detected in scid patients, but this is never been found in ada deficient patients. a -months-old italian girl, from non-consanguineous parents, presented to our hospital with a history of frequent bronchiolitis associated with dermatitis, mycosis, hypogammaglobulinaemia, marked lymphopenia (t cells cd , /mmc; cd /cd , /mmc; cd / cd , /mmc, b cells . /mmc, and nk cells, /mmc) and in vitro absence of proliferative response to pha. level of immunoglobulins was almost normal (igg mg/dl, iga mg/dl, igm mg/dl). high levels of toxic metabolites were found: axp, . micromol/ml rbc; daxp, . micromol/ml rbc; %daxp, . . ada activity in rbc lysates was abnormally high for scid-ada ( . u/g hb). molecular analysis confirmed diagnosis: the sequencing of exon revealed two mutations: a missense mutation previously reported called p.ser leu (c. c t) and a new missense mutation defined p. leu pro (c. t c). t-cells str analysis of patient showed . % maternal t lymphocytes engraftment never reported before in ada-scid patients. the girl was transferred to the isolated bmt unit and the respiratory symptoms progressively improvement. replacement therapy with peg-ada was started immediately at a dose of u/kg/twice per week. ig therapy was started at a dose of mg/kg every two weeks. after three months of treatment patient showed an increase in t cells count (cd , /mmc), and a decrease of toxic metabolites: axp, . micromol/ml rbc; daxp, . micromol/ml rbc; %daxp, . maternal t-cell engraftment persists, despite a good response to the peg-ada therapy. the last examination before hsct reveals maternal t-cell engraftment of . %. patient underwent hsct from mud hlaidentical donor after a myelo-ablative reduced intensity conditioning regimen protocol d ebmt/esid guidelines. the number of infused cd + cells was . × /kg and . × cd /kg. she is actually at day+ post hsct, is doing well and shows % engraftment of donor cells. disclosure of conflict of interest: none. graft versus host disease (gvhd) is a frequent complication in patients undergoing haematopoietic stem cell transplantation. while the exact pathophysiology of gvhd is not known, the gut microbiome has been implicated in its development since it was shown that total gut decontamination (tgd) decreases the incidence of gvhd. with this study we aim to get insight into the diversity of the gut microbiota before, during and after total gut decontamination in comparison with selective gut decontamination (sgd). secondly, we want to identify changes in microbiota composition that relate to the occurrence of graft-versus-host disease. for this prospective cohort study we recruited children (o y) that were eligible for a stem cell transplantation at the leiden university medical center between january and december . of these, % (n = ) received tgd (consisting of piperacillin/ tazobactam and oral amphotericin b), whereas the other % (n = ) received selective gut decontamination with polymyxin /neomycin and oral amphotericin b. in total, fecal samples were collected, weekly during admission for the stem cell transplantation and monthly thereafter up to months after transplantation. also samples were collected from family stem cell donors as healthy controls. samples were processed within hours and stored in the - freezer, after which s v amplicon sequencing (illumina hiseq, rapid mode, bp read length) was applied. data analysis (taxonomy and shannon diversity) was primarily done using qiime (ref). compared to microbiota diversity in stem cell donors (mean shannon index (si) . ), we observed an overall lower mean si during tgd ( . ) and slightly higher mean si during sgd ( . ). microbiota diversity months after sgd ( . ) was similar to diversity during sgd ( . ), while diversity months after tgd ( . ) was higher than during tgd ( . ). further analysis of repopulation dynamics demonstrated no differences in repopulation duration after both decontamination strategies. however, we did observe differences in the type of bacteria that repopulated, with bacteroidales being more prominent in sgd and lactobacillales more prominent in tgd patients. actinomycetales (genus rothia) was exclusively present in tgd patients during decontamination. also, the clostridiales (blautia, lachnospiraceae and peptostreptococcaceae) were bacteria that appeared after the decontamination period. four patients ( %) in this cohort developed gvhd grade or more. in these patients we did observe individual compositional changes of the gut microbiota at the time of ghvd diagnosis, e.g very low diversity or dominance of enterobacteriales. considerable microbiota diversity is observed in patients that received tgd. different repopulation dynamics were observed between tgd and sgd. no common pattern was found in the gvhd cases. disclosure of conflict of interest: none. minimal residual disease (mrd) pre-and post-hct for children with aml is highly predictive of event-free survival: a pediatric blood and marrow transplant consortium study d jacobsohn johns hopkins all children's hospital, children's hospital los angeles, usc keck school of medicine multicenter data regarding the significance of mrd in children with aml pre-and early post-hct are lacking. we hypothesized that pre-and post-hct mrd assessments using wt pcr combined with multi-dimensional flow cytometry (mdf) would be predictive of disease relapse and event-free survival (efs) at -yrs post-hct. subjects were o yrs with aml in morphologic cr undergoing ma allogeneic hct. stem cell sources included bm, pbsc, or cb. bm and pb samples were collected at time points: baseline ( o weeks prior to preparative regimen); day+ (± days); and day+ (± days). bm samples were analyzed for both wt expression and mdf mrd (single reference lab using a 'difference from normal' approach without access to diagnostic phenotype); pb samples were analyzed for wt only. mdf detection limit was . %; however, we required that independent analysts certify that the abnormal population was aml. in addition, sorted mrd+ cells were tested for chimerism. wt positivity was defined as ⩾ copies for bm and ⩾ copies for pb. results were not available to the treating clinician. subjects were enrolled at centers in us and canada. enrolled subjects did not undergo hct and were excluded for progression prior to hct or other ineligibility. in eligible subjects, -yr efs and os were % and %, respectively. the -yr ci of relapse and trm were % and %, respectively. mdf identified subjects pre-hct having . - % residual disease. the -yr relapse rate in subjects with +mrd by mdf pre-hct was % vs % ( - %) in those who were negative. -yr dfs and os were % and % ( - %) for positive mdf pre-hct, and % ( - %) and % ( - %) for negative mdf. pre-hct mdf sensitivity for -yr dfs was %; specificity was %. mdf mrd at days and were similarly predictive of outcome. sorted mrd+ cells from post-hct samples were all noted to be of recipient origin. pb wt had no correlation with dfs or relapse; bm wt at day+ correlated with -yr os ( % ( - %) low/neg vs % ( - %) high). other wt cutoffs studied demonstrated no correlation with outcomes. figure : relapse probability by flow cytometry mrd at time points. mdf mrd pre-hct and at days + and + was significantly associated with lower efs and os in children with aml undergoing hct. mdf is specific but not sensitive, as many negative mdf patients relapsed. our goal was to define a reproducible assay that did not depend on having the initial aml profile. this would facilitate multi-institutional studies aimed at decreasing relapse. given that constraint, we were able to detect clear mdf mrd in a small percentage of patients that was highly predictive and can be used in trials. wt level was not predictive in this multi-institutional trial. the sensitivity of flow was significantly affected by not having the initial flow available. future attempts to improve sensitivity should include initial flow and/or test higher channel flow or molecular pcr techniques. in addition, we confirmed that mrd + cells obtained by cell sorting post-hct were of recipient origin. future testing of 'suspicious' sorted cells by fish, molecular, or comparative genomic hybridization could possibly increase mfd sensitivity. novel cellular or targeted therapies should be tested in clinical trials to improve outcomes in patients with mfd mrd noted either pre-or post-hct. [p ] disclosure of conflict of interest: none. novel mutations were identified with ngs and low intensity of conditional regimen succeeded in children with fanconi anemia who received allo-hsct s hu , h hou, j lu, p xiao, x bian, h liu, y hu, j ling, l li, l kong, z zhai and y yao children's hospital of soochow university to explore the possiblity of applying next-generation sequencing (ngs) to diagnose the disease of fanconi anemia (fa) and evaluate the efficiency and safety of low intensity conditional regimen on children with fa receiving allogenic hematopoietic stem cells transplantation (allo-hsct). five patients initially suspected as severe aplastic anemia were diagnosed as fa by the method of next-generation sequencing (ngs)-based genetic diagnosis panel. one patient received hla-identical sibling donor hematopoietic stem cell transplantation (mrd), three patients underwent unrelated donor matched (ud) hsct, and one patient received unrelated cord blood transplantation (ucb). the conditional regimen consisted of either cgy tbi or . - . mg/kg of busulfan with - mg/kg of cyclophosphamide. meanwhile, atg at mg/kg and fludarabin at - mg/m were included as well. cyclosporin or tacrolimus as well as mycophenolate mofetil (mmf) were used for the prophylaxis of graft versus host disease (gvhd). engraftment of neutrophil and platelet and complications followed transplantation such as infection, gvhd, and hemorrhagic cystitis (hc) were observed. of cases diagnosed as fa by ngs, only case showed the abnormality of chromosome fragility test which has been regarded as golden criteria in the diagnosis of fa. meanwhile, we found novel mutations in cases of fa which enriched chinese national database with data of rare diseases by ngs. the counts of mononuclear cells (mnc) were ( . - . ) × /kg for non-ucb and . × /kg for ucb. the counts of cd + were ( . - . ) × /kg for non-ucb and . × /kg for ucb. all cases succeeded in allo-hsct with the low intensity of conditional regimen. the median time for neutrophils engraftment was days (range ~ days), median time to platelets (plt) engraftment was days (range ~ days). one case occurred with grade i of agvhd, cases with hemorrhagic cystitis. after transplantation, all patients were monitored the copies of ebv-dna and cmv-dna of whole blood, and five case with ebv positive and cases with cmv positive. no patient suffered of ebv or cmv disease. the hepatic veno-occlusive disease (vod) and hc were observed in fa patients after transplantation. ngs showed much more specific and facilitated for the diagnosis of fa. low intensity of conditional regimen is efficient and safe which should be recommended for the treatment of fa patients. disclosure of conflict of interest: none. outcome of alternate donor stem cell transplantation in children: an indian experience sp yadav , , n rastogi , , d thakkar , , s kohli , , s nivargi , , r misra and s katewa in india due to lack of donor registries and cord blood banks very few alternate donor stem cell transplants (sct) are performed. haploidentical sct has become feasible with availability of post-transplant cyclophosphamide (ptcy) technique. here we present our experience of setting up alternate donor program for sct for children in india and report the outcomes of the same. we collected data retrospectively of all children who underwent alternate donor sct during jan to dec in two centres. a total of sct were performed for children; median age years ( - years) and were boys and girls. of these, underwent haploidentical ( ptcy and tcr alpha-beta/cd depleted), matched unrelated donors (mud) and unrelated cord blood (ucb) sct. the diagnosis was: primary immunodeficiency- , thalassemia major- , sickle cell disease- , inherited bone marrow failure- , acquired aplastic anemia- , acute lymphoblastic leukemia- , acute myeloid leukemia- , neuroblastoma- , ewings sarcoma- & leukodystrophy- . the conditioning was with fludarabine, cyclophosphamide and total body irradiation backbone in children (thiotepa added in ), fludarabine and treosulfan in , fludarabine and busulfan in , busulfan and cyclophosphamide in . serotherapy was part of conditioning, rabbit anti-thymoglobulin . mg/kg in and campath mg/kg in . graft vs host disease (gvhd) prophylaxis was ptcy along with tacrolimus and mycophenolate mofetil in patients ( haploidentical, mud & ucb) and ex-vivo tcr alpha-beta depletion in and cyclosporine and methotrexate in . all were transplanted after a signed informed consent. a median of million of cd cells/kg was infused (range - million/kg).graft source was peripheral blood in and bone marrow in and ucb in . five children died before engraftment. the remaining had neutrophil engraftment by median of days (range - ) and platelet engraftment by median of days (range - ). chimerism at day+ was available in cases; of them had full donor hematopoiesis. one had mixed chimerism and fully recipient. four children underwent a second haploidentical sct after rejection of which are alive and disease free. the median follow-up of remaining patients is months (range - ); the cumulative incidence of graft versus host disease (gvhd) acute and chronic extensive was % and % respectively. grade-iii-iv acute gvhd was seen in patients. a total of patients have died (sepsis- , stroke- , gvhd- , vod- , encephalitis- and progressive disease- ). among encephalitis deaths, one child had undergone ucb with ptcy and another tcr alpha-beta depleted second sct.; both had bk virus in the csf.there were / deaths in haploidentical (ptcy- / & tcr alpha-beta- / ), / in mud and / in ucb sct. overall survival is % and disease free survival is % at last follow up. alternate donor sct is an acceptable curative option for children lacking a matched sibling donor. haploidentical donor sct is more feasible in the setting of lack of donor registries having indian ethnicity donor. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) from an unrelated donor (ud) is largely used for pediatric patients with all in second complete remission (cr) lacking an hlaidentical sibling. in this study, we retrospectively analyzed outcome of patients (pts) given ud-hsct in centers affiliated to the associazione italiana di ematologia ed oncologia pediatrica (aieop) network between and . three hundred fifty-six pts with all in second cr experiencing either bone marrow (bm), isolated extramedullary or combined relapse were included in the study; were males and females, median age at hsct being . years (range . - ). bm, peripheral blood (pb) and cord blood (cb) were the stem cell source in %, % and % pts, respectively. all children received a myeloablative conditioning regimen, either tbi-( pts) or chemotherapy-based ( pts). as gvhd prophylaxis, the combination of cyclosporine a, short-term mtx and atg was employed in most pts. according to the berlin-frankfurt-munster (bfm) classification of first leukemia recurrence, % and % of pts were assigned to the s +s and s +s groups, respectively. level of pre-hsct minimal residual disease (mrd), measured within days before hsct through flow cytometry (fcm) in the laboratory of padova, is available in children; more data will be presented during the s meeting. with a medium follow-up of . years (range . - ), the overall survival (os) was %, while the event-free survival (efs) was %. the cumulative incidence of transplant-related mortality (trm) and leukemia recurrence were % and %, respectively. the efs probability for children transplanted in the time period [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and - was %, % and %, respectively (p = ns). patients who received a tbi-based conditioning regimen had a significantly better outcome in comparison to children who received chemotherapy-based treatment, efs being % and %, respectively (p = . ). efs of pts belonging to s +s and s +s groups was % and % respectively (p = . ). the difference in efs is largely explained by an increased incidence of leukemia recurrence in s +s ( %) compared to s +s pts ( %) (p = . ). efs of pts who experienced grade ii acute gvhd was %, while that of pts with either absent/grade i acute gvhd or grade iii-iv acute gvhd was % and %, respectively (p = . ). pts with limited chronic gvhd had a better efs as compared to those with either extensive or absent chronic gvhd ( %, % and %, respectively; p = . ). the choice of stem cell source (bm, pbsc, cb) did not influence the probability of efs, which was %, %, % respectively (p = ns). importantly, among pts with evaluable mrd before hsct (n ), the group with detectable levels . % (n ), respectively % and % (p = . ). conclusions. outcome of children with nd cr all who underwent transplant from an ud is significantly influenced by the presence of tbi in the conditioning regimen, limited severity of acute and chronic gvhd and bfm classification at time of st relapse. notably, mrd level before transplant, namely with a cut-off of . %, influences efs. disclosure of conflict of interest: none. the median mononuclear cell dose was . × /kg. the median time to reach absolute neutrophil count . × /l was days, and the median time to platelet count × was days. grade and grade mucositis was seen in % of our patients. transplant-related mortality at days not occurred. only three patients relapsed , and months after transplant (mean . m.). with a median follow-up of months ( - months) after transplant the event free survival were %. only one patient had death, two years after transplantation. no significant different between cbv group vs ceam group in engraftment day. high-dose therapy with stem cell rescue can lead to durable remissions in children and adolescents with advanced hd. future investigations should focus on strategies designed to decrease relapse after auto-transplantation, particularly in patients at high risk for relapse. our analysis suggests that these regimens (ceam, cbv) are feasible in pediatric patients with acceptable engraftment and toxicity. pbsc collection may be difficult in small children owing to the large volume apheresis compared to the child's weight. various problems, such as metabolic or haemodynamic disorders may be were seen. peripheral stem cell harvest can be performed in lowweight children under safe and effective conditions even when systematic priming by blood is avoided. processing with increase of blood volume may to increase in the yield by recruiting progenitor cells. disclosure of conflict of interest: none. outcomes of children with hemophagocytic lymphohistiocytosis given allogeneic hematopoietic stem cell transplantation in italy allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment for patients with familial hemophagocytic lymphohistiocytiosis (hlh) or relapsed/ refractory hlh. we analyzed outcomes of a cohort of patients ( m, f) with hlh given hsct between and . median age at hsct was years (range . - ). genetic testing was performed for / patients ( %). mutation of prf was found in patients ( %), of unc d in ( %), of stxbp in ( %), of rab a in ( %), of sh d a in ( %), of birc in ( %) and of lyst in patient ( %). no known gene abnormality was found in patients who had recurrent/ refractory hlh. central nervous system (cns) involvement at diagnosis was recorded for patients ( %) and was present in of them ( %). the primary endpoint was event-free survival (efs), defined as the probability of being alive and in continuous complete remission (cr) at last follow-up. in order to determine efs, death from any cause, relapse or graft failure were considered events. ninety-five patients received one transplant, while received more than one hsct, because of rejection in patients or disease relapse in (preceded by rejection in case): hsct were performed in cases, while and hsct were performed in case each. donor for first transplant was an hla-matched sibling for patients ( %), an unrelated donor for patients ( %) and a partially matched family donor for patients ( %). conditioning regimen was busulfan-based for patients ( %), treosulfan-based for patients ( %) and fludarabine-based for patients ( %). the -year probability of overall and efs were % and % respectively (fig. ) . twenty-six ( %) patients died due to transplant-related causes, while ( %) and ( %) patients experienced graft rejection and/or relapse, respectively (see also fig. ). twelve out of children ( %) given a nd hsct after graft failure/relapse are alive and disease-free. active disease at hsct was not statistically associated with adverse outcomes, while patients had a trend for a worse outcome if the interval between diagnosis and hsct was months. patients transplanted from partiallymatched family donors (pmfd) had a significantly worse efs ( %) than recipients of a matched family donor transplant ( %) or a matched unrelated donor allograft ( %, po . ). the main reason for the dismal efs of pmfd recipients was graft rejection, which, however, was largely rescued by a nd hsct. patients given peripheral blood stem cell transplantation had a lower efs probability ( %) as compared to bone marrow ( %) or cord blood recipients ( %, p = . ). children given hsct o o/u months from diagnosis had a better efs as compared to those transplanted months from diagnosis ( % vs %, p = . ). in multivariate analysis, only the use of a pmfd predicted a worse efs probability (relative risk: . , p = . ). these data suggest that in patients with hlh allogeneic hsct is able to cure / of patients. haploidentical hsct in patients with hlh is currently associated with unsatisfactory rate of engraftment, new approaches being needed to ameliorate this outcome. active disease does not preclude the chance of benefiting from transplantation, which should be ideally performed within months from diagnosis. [p ] defibrotide shows efficacy in the prevention of sinusoidal obstruction syndrome (sos) after allogeneic hematopoietic stem cell transplantation: a retrospective study on patients. disclosure of conflict of interest: none. standard gvhd prophylaxis regimens impair the graft-versustumor (gvt) effect, delay immune reconstitution and are associated with high rate of infections. high-dose posttransplantation cyclophosphamide (ptcy) targets alloreactive donor t cells proliferating early after bmt, promotes regulatory t cell and permits rapid immune reconstitution. in this pilot trial we evaluate the safety and effects of ptcy in unmanipulated haploidentical and matched unrelated transplantation (mud) in pediatric patients with all. fifteen pediatric patients with high risk all underwent unmanipulated allogeneic bone marrow (bm) (n = ) or peripheral blood stem cell (pbsc) (n = ) transplantation followed by ptcy between april and march with a median follow-up of months ( - ). eight patients were transplanted from haploidentical donors and from mud. the median age was . years (range . - ) and were in complete remission (cr) at the moment of bmt. in patients this was a second bmt. all pts. received myeloablative conditioning regimen (treosulfan-based n = , tbi based n = ) and ptcy on day + , + , posttransplant prophylaxis consisted of tacrolimus from day + (n = ), tacrolimus/mmf (n = ), atg (rabbit, thymoglobuline) at mg/kg without other posttransplant prophylaxis(n = , both from mud). primary engraftment was achieved in % of pts., the median time to neutrophil recovery was days and to platelet recovery was ( - ) days. all pts. had full donor chimerism on day + . causes of death included viral infections (n = ); gvhd and viral infection (n = ). cumulative incidence (ci) of acute gvhd grade ⩾ ii was % ( % ci: - ), grade iii-iv- . % ( % ci: . - ) and chronic gvhd- . % ( % ci: . - . ). two-year event-free survival (efs) and overall survival (os) were . % ( % ci: . - ) and were equal. median time of follow-up for survivors is years (range . - . ). we demonstrate that unmanipulated hsct and posttransplantation cyclophosphamide allows for high rate of engraftment with acceptable transplant-related mortality in pediatric patients with all. all major outcomes were equivalent between transplantation from unrelated and haploidentical donor. gvhd prophylaxis including ptcy was effective. event-free survival was high despite chemotherapybased conditioning in most patients. disclosure of conflict of interest: none. serotherapy with atg is frequently used in allogeneic hsct to prevent gvhd and rejection. however, the choice of the two most frequently used rabbit atg brands depends on country, disease protocol, national recommendations and/or physician's preference. atg-genzyme (atg-g, thymoglobulin) is prepared by immunizing rabbits with human thymocytes, whereas rabbits are immunized with a jurkat cell line for production of atg-fresenius (atg-f, recently named as antihuman t-lymphocyte immunoglobulin atlg, grafalon, noveii biotech). the recommended dose of both brands differs a factor - . we have previously reported the pharmacokinetics/ pharmacodynamics (pkpd) of atg-g in a large cohort of pediatric hsct recipients and concluded that the clearance of the active component of atg, which is the portion of atg binding to lymphocytes, had a major impact on immune recovery post-hsct, while total atg did not. both atg brands have frequently been compared according to disease outcome, without detailed analysis of composition and clearance of the active components. in the present study, we compared clearance of the active component and immune recovery after atg-g and atg-f, respectively. the serum concentrations of total and active atg were measured longitudinally after hsct in children ( atg-g, atg-f), transplanted with bm or pbsc of unrelated donors for all or aml between january and june in leiden (n = ) or copenhagen (n = ). atg-g treated patients received a total dose of - mg/kg and atg-f was given at a total dose of - mg/kg in both cohorts administration was divided over - days. serum samples (pre-conditioning, day of hsct, + ; + ; + ; + and + weeks and + and + months after hsct) were analyzed by elisa for total atg and by quantitative flow cytometry on hut cells for active (lymphocyte binding) atg. lymphocyte (sub-)populations were analyzed at + , + and + months post-hsct by flow cytometry. as reference group for immune recovery, children transplanted for all or aml with an hla-identical donor and not receiving serotherapy were included. the median serum concentration of total atg at the day of hsct was times higher for atg-f (atg-g μg/ml, atg-f μg/ ml; figure a) as the result of the higher dose of atg-f given. the active atg concentration was twice as high for atg-f (atg-g . au/ml, atg-f . au/ml figure b ). three weeks later at the expected time of engraftment, the total atg concentration was decreased with the same factor for both atg brands (atg-g from to μg/ml, factor . ; atg-f from to μg/ml, factor . ). however, the active atg concentration showed a much faster decline for atg-f (atg-g from . to . iu/ml, factor . ; atg-f from . to . iu/ml, factor ). correspondingly, the number of cd t-cells at month post-hsct was higher after atg-f than after atg-g (atg-g, atg-f and no-serotherapy , and cells/μl, respectively. figure c) . this is the first study to compare the pkpd of total and active atg-genzyme and atg-fresenius. active atg-f showed a much faster clearance than atg-g, which was associated with a significantly faster cd t-cell recovery at month post hsct. thus, atg-f is not only quantitatively but also qualitatively very different from atg-g, which will clearly impact hsct outcomes. reduced toxicity myeloablative conditioning regimen in pediatric hematologic malignancies not associated with improved outcomes s chaudhury , , i helenowski , r duerst , , wt tse , , m kletzel , , j schneiderman , and d jacobsohn ann and robert h. lurie children's hospital of chicago; northwestern university feinberg school of medicine, chicago and children's national health system, washington dc allogeneic (allo) hematopoietic cell transplantation (hct) is the only curative potential therapy in refractory and relapsed pediatric leukemias. poor outcomes in allo hct are associated with treatment-related mortality (trm), mostly due to regimen-related toxicities (rrt) and graft-versus-host disease (gvhd) after myeloablative conditionings (mac), but high relapse rate with reduced-intensity or nonmyeloablative regimens. to improve trm, without compromising conditioning intensity, we prospectively explored the feasibility and efficacy of a mac but reduced-toxicity conditioning (rtc) regimen, consisting of fludarabine mg/m /d (given first) × d, daily busulfan dosed to target an auc of microm*min/d × , ratg . mg/kg/d × and cgy of total body irradiation in patients (table ) with hematologic malignancies. gvhd prophylaxis was cyclosporine and mmf. all patients tolerated the rtc well, with no graft failures. rrt included moderate mucositis ( %), infections (bacterial %, viral reactivation %, fungal %) and cases of venoocclusive disease (vod). cumulative incidence d ⩾ gr acute gvhd was % ( % confidence interval [ci], - ), extensive chronic gvhd was . % ( % ci, . - ). mortality at days was . % ( % ci - ), due to infections with agvhd and vod. with a median follow-up of . y (range, . - ), the cumulative incidences of relapse at years was % ( % ci, - ). mortality due to severe agvhd was %. overall survival (os) and progression-free survivals (pfs) for year was % ( % ci, - ), and % ( % ci, - ) respectively. on univariate analysis there was no association of outcomes with donor type, graft source, disease or busulfan exposure except significantly higher cgvhd in unrelated donors, agvhd severity with peripheral blood. in summary, the use of the myeloablative rtc resulted in comparable trm, with high relapse rate was high, including in those developing chronic gvhd. this suggested a less robust graft-versusleukemia effect resulting in poor pfs and os. nonetheless, this regimen may be used as a lower-trm platform to combine with other strategies, intensive disease monitoring pre and post hct, addition of post hct maintenance therapy in combination with marrow as the stem cell source to decrease relapse or gvhd. specific immune response to vaccinations decline after hematopoetic stem cell transplantion (hsct). re-vaccination of all hsct recipients is recommended in all guidelines but bcg vaccination is not recommended due to safety concerns after hsct. mycobacterium tuberculosis can cause severe disease in children including meningitis and milliary tuberculosis (tb). the bacille-carmette-guerin (bcg) is a liveattenuated vaccine with documented efficacy against milliary disease and meningitis. routine vaccination of all infants residing in countries with high tb incidence is recommended by world health organization. however, there is no data in literature regarding its safety in post hsct setting. here, we report children who underwent matched related allogeneic hsct at ankara university pediatric bone marrow transplant (bmt) unit and received bcg -months post-transplant. all patients were free of graft versus host disease (gvhd) and immunosuppressive therapy (ist) and had negative ppd skin test prior to vaccination. none of the recipients developed local or disseminated tuberculosis as a complication of bcg with a median follow up of years. we conclude that the bcg vaccine is safe in the post hsct period when administered at least months out of transplant to a selected group of patients who are free of gvhd and ist. disclosure of conflict of interest: none. single centre experience of harvesting bone marrow from donors o years of age r raj, r uppuluri , d subburaj , d jayaraman , k mullanfiroze , v swaminathan and l vaidhyanathan department of paediatric blood and marrow transplantation, apollo speciality hospital harvesting bone marrow for allogeneic marrow transplantation from donors o kg presents special challenges. we present data on sibling donors from our institution between and . the mean age was months with a range between months to months. children less than one year accounted for % of our donors with the youngest being months of age and the smallest donor weighed . kg. all aspirations were performed from iliac crests and all donors were given general anaesthesia by a paediatric anaesthetist. irradiated blood was transfused in % of the donors during the procedure. the volume of marrow obtained ranged from to a maximum of ml/kg donor weight. the product contained an average cd count of . × /kg recipient weight with a range from . to × /kg. only on one occasion was a second harvest needed, where the donor weighed kg and recipient kg with major blood group incompatibility requiring red cell reduction. the yield of cd cells per ml of bone marrow was on average % higher than children above years of age. all recipients showed brisk engraftment in weeks. none of these donors experienced major difficulties following the aspiration procedure. thus, very young children may safely donate marrow for allogeneic transplantation and the yield of stem cells obtained is substantial. this data is particularly relevant in transplantation for haemoglobinopathies like thalassaemia major and sickle cell anaemia, where families are being counselled about a target of kg for the donor in order to plan transplantation. disclosure of conflict of interest: none. sinusoidal obstruction syndrome-veno-occlusive disease in pediatric patients given either autologous or allogeneic hematopoietic stem cell transplantation (hsct). a retrospective study of the aieop-sct (italian haematology-oncology association-stem cell transplantation) group m faraci , r luksch, e calore , f saglio , a prete , mc menconi , v trevisan , g de simone , v tintori , s cesaro , s santarone , mg orofino , e lanino , m zecca and a bertaina sinusoidal obstruction syndrome (sos), known as venoocclusive disease (vod), is a potentially life threatening complication that can develop after hsct. although sos progressively resolves within few weeks in most patients, the severe forms result associated with multi-organ dysfunction and high mortality rate ( %). aim of this survey is to evaluate incidence and management of sos in a large cohort of children receiving either allogeneic or autologous hsct. we retrospectively reviewed pediatric hscts performed in ( %) out of aieop affiliated centers, between january and april . new ebmt criteria have been used for the diagnosis of sos (serum total bilirubin ⩾ mg/dl and of the following criteria: painful hepatomegaly, weight gain %, and ascites) and for the classification of severity grading. , among a total number of hsct procedures ( autologous and allogeneic), we identified ( . %) patients with sos. this complication occurred in and cases after autologous and allogeneic hsct, respectively. fiftytwo pts ( %) received iv busulphan (bu) at myeloablative dose, ( %) oral bu, while ( %) were treated with different conditioning regimen. the median time of sos occurrence was days after hsct. details about prophylaxis and therapy are reported in figure . out of the children, ( %) fulfilled all sos-ebmt criteria. bilirubin ⩾ mg/dl, gain of weight %, ascites, and painful hepatomegaly did not occurred in , , and patients, respectively. thrombocytopenia was present in pts ( %), thickening of gallbladder in ( %) and abnormalities of coagulation parameters in ( %). according to sos ebmt severity grading, levels of transaminases were mild in pts ( %), moderate in ( %), severe in ( . %), and very severe in ( . %). notably, creatinine was mild in pts ( %), while ( . %), ( . %), and ( %) children showed moderate, severe and very severe grade of renal failure. thirty-three pts ( %) had respiratory failure, and ( %) of them experienced right pleural effusion. six out of the patients who developed acute kidney injury, required dialysis. severe encephalopathy occurred in pts ( . %) and ( %) out of the pts evaluated, were admitted in intensive care unit. as therapy of sos, pts received defibrotideâ (df); the dosage was mg/ kg/day in % of them. the median duration of df treatment was . days (range - ). thirty-three ( %) pts received methylprednisolone (median dose of mg/kg). fifteen pts ( . %) died due to mof ( in moderate, in severe, and in very severe group) at a median time of days from sos diagnosis (range - gg). our multicenter survey showed that, at least in our experience, there is a significant variability in the management approaches to sos/vod in children, while, diagnostic evaluations are more homogeneous. interestingly, in our cohort, the increase of bilirubin may be an absent criteria, while thrombocytopenia and abnormalities of coagulation parameters are more frequent. as expected, mof occurred mostly in patients experiencing severe sos. df represents first strategy to treat sos in the majority of patients, even if steroids and ursodeoxycholic acid are still used. the hyper-ige syndromes are characterized by marked elevations in plasma ige levels and eosinophilia with impairment in t cells which clinically results in combined immune deficiency. dock deficiency, the autosomal recessive form, brings about allergic/atopic manifestations and unusual susceptibility to infections with herpesvirus family members (herpes simplex virus, human papilloma virus) and molluscum contagiosum. symptoms in patients with dock deficiency typically emerge during childhood, and the majority results in death because of infections and malignancy by the third decade. hematopoietic stem cell transplantation (hsct) is now considered a standard of care for dock deficiency when an appropriate donor is available. in this study, we present the unrelated hsct results of children with dock mutation. the demographic and clinical data of the patients with transplantations studied are shown in table . hsct was administered between august and august at bahçeşehir university medical park antalya hospital and the clinical data of the hscts are presented in table . all patients were transplanted from unrelated donors with bone marrow, except one with cord blood. the cord blood transplantation´s regimen was non-myeloablative which resulted with rejection. despite existence of serious morbid problems before transplantation, all the patients engrafted successfully. majority of the complications mentioned in the table were improved and they are in the follow-up in an outpatient basis. discussion dock deficiency has high mortality, and hsct should be considered as early as possible before development of significant organ damage. despite myeloablative conditioning and high morbidity before the transplantation, survival was very good in our patients. myeloablative and nonmyeloablative transplants have been performed from related and unrelated donors and have reported successful results even without the preparative regimen. in our center, all transplants performed from unrelated donors by myeloablative regimen have been successful but have resulted in transplant rejection with cord blood transplantation after nonmyeloablative regimen. in all of our patients, stable full chimerism has been detected, however mixed chimerism have also been shown to be useful in several reports. whether hsct also cures the autoimmune complications and reduces the risk of cancers is as yet undetermined. however, a myeloablative conditioning regimen followed by allogeneic hematopoietic stem cell transplantation from unrelated donors in dock deficiency results in improvement of the clinical phenotype with a low incidence of regimen-related toxicity. disclosure of conflict of interest: none. successful bone marrow transplantation after myeloablative conditioning in a child with ipex syndrome b kuşkonmaz , d ayvaz , mh abur , fv okur , g karagüzel , f orhan , İ tezcan and du Çetinkaya immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome is a rare disorder. although most patients present in infancy with a clinical triad of intractable diarrhea, insulin-dependent diabetes, and eczematous dermatitis, some patients present with severe food allergies and other autoimmune manifestations. the disease is caused by mutations in the forkhead box p (foxp ) gene, a transcription factor that is essential for the development and function of regulatory t (treg) cells. this cells plays an essential role in controlling immune responses and preventing autoimmunity. patients usually die in the first years of life without treatment. the only effective cure is hematopoietic stem cell transplantation (hsct). here we report a patient with ipex syndrome who underwent hsct after myeloablative conditioning. months of age boy with the history of diarrhea, insulin-dependent diabetes, eczematous dermatitis, pneumonia, coombs positive hemolytic anemia, referred to our hospital for investigation of immunodeficiency. on admission physical examination showed eczematous skin rash, submandibular lymphadenopathy, hepatosplenomegaly. before hsct the patients treated with immunosuppressive agents including methylprednisolone, mycophenolate mofetil and monthly intravenous immunoglobulin. complete blood count revealed anemia (hb: . g/dl), and eosinophilia ( /mm ). serum immunoglobulins were: ig g: mg/dl ( - ), igm: mg/dl ( - ), iga: . mg/dl ( - ), ige : iu/ml. lymphocyte subset analysis showed cd %, cd %, cd %, cd + %, cd %. foxp gene analysis showed c. _ delaag mutation. at the age of year, patient underwent hsct from his hla matched sibling. myeloablative conditioning regimen including busulfan ( . mg/kg) and fludarabine ( mg/m ) was given to the patient. cyclosporine a and methotrexate (day + , day + , day + ) were used as graft versus host disease prophylaxis. bone marrow was used as the stem cell source and the number of cd + cells was . × /kg. neutrophil and platelet engraftment were achieved on day + and + [p ] s respectively. acute and chronic gvhd were not observed, but patient developed veno-occlusive disease treated with defibrotide, sepsis treated with broad spectrum antibiotics. chimerism analysis showed % donor profile at the third month of hsct. after hsct, autoimmune hemolytic anemia, eczematous dermatitis, food allergies, diarrhea and type diabetes resolved completely within two months after hsct. now the patient is in good clinical condition without any symptoms months after hsct. early hsct provides better outcome in patients with ipex, before the organ damage due to autoimmunity and/or adverse effects of immunosuppressive therapy. myeloablative conditioning is associated with substantial transplantation-related mortality whereas nonmyeloablative conditioning carries an increased risk of rejection because of dysregulated effector t-cell function. in this patients, myeloablative conditioning was preferred because of the risk of rejection. although the required levels of donor chimerism and conditioning intensity are unknown, engraftment of donor treg cells seems to be sufficient to control the disease. the patient is well without any symptoms of ipex after hsct with full donor chimerism. disclosure of conflict of interest: none. interferon gamma receptor deficiency (ifnr ) is a rare autosomal recessive immune deficiency disorder associated with very poor outcome secondary to severe and disseminated mycobacterial infections. hematopoietic stem cell transplantation (hsct) has been proposed as a curative option. however, hsct for these patients is particularly difficult owing to a high rate of graft rejection. the use of a non t-cell depleted transplant from an hla-identical sibling and fully myeloablative conditioning regimen has been shown to have improved outcomes. we report the first successful hsct with a t-depleted haplo-identical donor, performed in a girl with severe ifnr deficiency. we reviewed the medical chart of a -year-old hispanic girl with ifnr deficiency who was diagnosed at birth, since her brother had previously been diagnosed with the same complete ifnr deficiency. they were found to have a novel mutation variant detected at c. - g t. as expected with this disorder, she developed disseminated infection with mycobacterium abscessus infection at months of age and was subsequently found to have mycobacterium abscessus osteomyelitis. she was treated with multiple antibiotics including: amikacin, linezolid, meropenem and clarithromycin while tigecycline was added a few weeks prior to admission for hsct. she was continued on this therapy until day + following which antimicrobials were gradually weaned off. she was enrolled on the bp- trial, a multicenter, prospective phase i-ii trial (enrolling both malignant and non-malignant diseases) evaluating αβtcr +/cd + depleted haplo-transplantation followed by administration of bpx- t cells containing the ic suicide gene, (clinicaltrials.gov nct ). her conditioning regimen included busulfan ( mg/kg/day for days) and cyclophosphamide ( mg/kg/day for days). fludaragbne, tli ( cgy) . gvh prophylaxis with atg/rituximab. the patient received a graft with: tnc- . × cells/kg, cd + cells- × cells/kg, and αβtcr+ t cell content of . × cells/kg. as per protocol, since the αβ tcr+ t cells in the product was below threshold of × cells/kg, she did not receive any post-transplant immune suppression. bone marrow recovery occurred at day + with anc /mm and platelet recovery at day + . full engraftment with % donor chimerism based on cytogenetic analysis was observed at day + after transplantation and has remained stable. she is currently months post-transplant, and has done well without major complications and or signs of mycobacterial infection. there is limited data in patients receiving hsct for ifnr deficiency with very poor outcomes either relating to graft failure, transplant complications and progressive mycobacterial infection. to our knowledge, this is the first patient with ifnr deficiency transplanted successfully with a haploidentical donor and alive without any active mycobacterial infection. this report suggests that using a highly immunopotent graft depleted of only αβtcr+ t cells while retaining other immune effectors might offer a potential strategy to engraft these high risk patients using haplo-identical donors thereby allowing access to virtually all patients in need. disclosure of conflict of interest: none. tandem autologous stem cell transplantations for high risk pediatric embryonal central nervous system tumors: a single center experience k rosenfeld , r dvir , s constantini, j roth , s edelman , a tal , d levin , m manisterski , s achituv and r elhasid , department of pediatric hematology-oncology, tel aviv medical center; department of pediatric neurosurgery, tel aviv medical center and sackler faculty of medicine, tel aviv university pediatric embryonal central nervous system tumors are highly malignant tumors, which tend to disseminate through the cerebrospinal fluid to the brain and spinal cord and include: medulloblastoma, pinealoblastoma and primitive neuroectodermal tumors (pnets). the recommended treatment for these tumors is a complete surgical excision, craniospinal radiation and chemotherapy. the use of high dose chemotherapy with tandem autologous hematopoietic stem cell transplantation (hsct) has been advocated for high risk patients, and infants who could not be irradiated. between july and november , pediatric patients ( males, females) suffering from high risk medulloblastoma, pnet or pinealoblastoma underwent tandem autologous hsct. they were treated according to two protocols: group a consisted of ten patients with median age of . years (range . - . years) received the st jude sjmb protocol, while group b consisted of six patients with median age of . years (range . - . years) who received the children's oncology group -acns protocol. all patients engrafted with median time for neutrophil engraftment of days (range - days) and for platelets engraftment ( ) of days (range - days). median follow-up was . years (range week- years). neurological toxicity: two group a patients had convulsions episodes, one occurred during infusion of cryopreserved stem cells, and the other was a result of progressive disease during the last course of hsct. gastrointestinal toxicity: seven patients required total parenteral nutrition due to mucositis. diarrhea occurred in seven patients, two of them were diagnosed with rota virus and two with clostridium difficile. infectious complications: all patients suffered from at least one episode of neutropenic fever which was treated with broad spectrum antibiotics. there were documented bacteremia in patients. ( klebsiella pneumonia, proteus mirabilis, staphylococcus aureus, streptococcus viridans and staphylococcus epidermidis). metabolic complications: four patients in group a developed reversible syndrome of inappropriate anti-diuretic hormone secretion (siadh) during chemotherapy, and all group a patients developed hypomagnezemia. four patients died, one due to progressive disease, one due to early relapse months post treatment, one due to late relapse years post treatment and one due to sepsis months post treatment. another patient relapsed . years s post treatment, underwent surgery and radiotherapy and is now years post therapy. late effects: four group a patients developed endocrinological sequelae at a median of months (range - months) and require hormone replacement therapy. tandem autologous hsct is a feasible treatment for pediatric high risk embryonal tumors, with good engraftment and acceptable toxicities using sjmb and acns protocols, with overall survival of %. long follow-up is needed in order to diagnose and treat late effects. disclosure of conflict of interest: none. the diagnostic role of liver stiffness measurement in predicting hepatic veno-occlusive disease (vod) in pediatric hematopoietic stem cell transplantation (hsct) k kleinschmidt , f ravaioli , r rondelli , g marasco , r masetti , a prete , a colecchia , d festi and a pession pediatric oncology and hematology unit, department of pediatrics, university of bologna, sant 'orsola-malpighi hospital and department of medical and surgical sciences, university of bologna vod is a potentially life-threatening complication associated with hsct in which immediate therapeutic action is crucial for patients' outcome. liver stiffness measurement (lsm) using fibroscan represents a non-invasive method to detect the grade of liver fibrosis and portal hypertension as in case of vod. to evaluate the predictive potential of lsm in pediatric patients (pts) at risk for developing vod, a prospective, ongoing, single-center study has been performed at the university hospital of bologna. lsm was performed by using the fibroscan device, which consists of a . mhz ultrasound transducer probe that transmits low-frequency vibrations ( hz) to the liver tissue. the propagation velocity is proportional to the stiffness (elasticity) of tissue. lsm will obtain pathological high values ( . kpa) when the tissue is altered like in liver fibrosis, or post-sinusoidal portal hypertension. from november -september , pediatric pts ( male, female), aged - years (mean . ), affected by hemato-oncologic disease, eligible to allogeneic ( ) or autologous ( ) sct conditioned with busulfan-based chemotherapy, were enrolled. pts were scheduled for study examinations with lsm: at t (baseline) before chemotherapy, t (day - after sct), t (day - ) and t (day - ). the diagnosis of vod was defined according to modified seattle/baltimore criteria. twenty-five pts were enrolled in the protocol, of which were evaluable for the study (pts characteristics table ). out of pts ( %) developed vod. the cumulative incidence (se) of vod in our setting was % ( . ). baseline lsm values on t of all pts were normal ( . kpa at t (p = . ) and t (p = . ). from our observations, an anticipating pattern of pathological lsm in presence of clinical and laboratory parameters within normal ranges in patients who develop vod can be derived. preliminary data indicate a high predictive potential of lsm in the diagnosis of vod, however the number of cases is not sufficiently representative to draw definitive conclusions. to optimize the predictive potential of the method, more frequent (daily) measurement in the critical time frame are currently investigated. [p ] all= acute lymphoblastic leukemia, aml= acute myeloid leukemia, bu= busulfan, treo=treosulfan, fluda= fludarabine. disclosure of conflict of interest: none. [p ] the exact role of extra-corporeal photopheresis in children with gvhd: an unanswered question ss anak, h bilgen , , , , , , , y yaman, et saribeyoglu, k ozdilli, v hazar, m elli, am kokrek, h hizli and k payalan ecp continues to be a controversial treatment, probably due to the mechanism of action not being identified, the varying photopheresis procedures and treatment schedules, and the difficulty of conducting trials on relatively rare diseases with involvement of clinically heterogeneous organs. ecp was performed in our pediatric transplant center to patients mean age of years ( - ) diagnosed to have all ( pts), thalassemia ( pts), aplastic anemia ( ), blacfan diamond ( ), refractory hodgkin disease ( ) following our internal protocol for ecp sessions. five of the patients had mud, had hla id sibling transplants. chronic gvhd was diagnosed in of the patients had acute gvhd. skin was involved in all the patients, liver in of the patients, lung in , gut in and mucous membranes in patients. the ecp treatment consisted essentially of three steps: ( ) collection of mncs from the patient, ( ) processing of mnc buffy coat, and ( ) return of mncs to the patient. collection was performed using a cell separator (haemonetics mcs plus), processing two blood volumes. our protocol provides for a maximum final mnc volume to be collected at ml, with a hematocrit (hct) value below %. the maximum procedure time was set at min. the mncs collected were adjusted to a constant volume of ml by the addition of saline and ml of -mop in aqueous solution, to always obtain a final concentration of the drug of ng/ml. the diluted buffy coat was transferred into a special uv-a-permeable bag (pit-kit medtech solutions), and uv-a radiation at j/cm was performed (uva-pit irradiator). the photoactivated mncs were returned to the patient within minutes using a blood transfusion set. during ecp procedure, patients' vital signs were monitored. anticoagulation consisted in acidcitrate-dextrose formula a set at a variable ratio ( : - : ) according to the patient's characteristics (clinical conditions, body weight, coagulation values) and platelet (plt) count. prophylaxis of hypocalcemia consisted of the administration of calcium gluconate ( ml diluted in - ml saline) every to minutes. all procedure related side effects were recorded. during the reinfusion and postreinfusion phases, the patients were monitored for fever, chills, headache, rash, erythema, urticaria, itching and edema. no serious complication was detected. all the patients had also steroids, had concurrent mesenchimal stem cells. ecp was applied on consecutive days every - weeks which is continued for approximately months followed by a maintenance schedule tapered to an every -to -weeks. the mean session cycle was ( - ) between february to november . the most commonly involved organ was the skin which demonstrated a response rate of %, followed by liver ( %), lung ( %), gut ( %) and mucous membranes ( %) the concurrent immunosuppression could be reduced during ecp therapy, and no increase in opportunistic infections was detected. / patient died after a relapse, / are alive with chronic mild gvhd. however, despite our good response rates, our understanding of ecp remains limited. patients who suffer from acute and chronic gvhd have limited treatment options. ecp remains an important therapeutic option. future basic, translational, and clinical research studies will provide a better understanding of its mechanism of action and optimize its therapeutic potential. disclosure of conflict of interest: none. tolerability and responses to ex vivo il activated nk cells from haploidentical parental donors in paediatric patients with refractory leukaemia/lymphoma pl tan prognosis for patients with refractory leukaemia/lymphoma ineligible for transplants and those who relapse posttransplant is poor. in adult settings, adoptive transfers of ex vivo il activated natural killer ('ank') cells from nk alloreactive donors, especially for nk sensitive cancers, has been successful in bridging patients to curative transplants.( ) this approach has not been reported in paediatric patients. we report our experience in consecutive patients, of median age (range, - ) years, with refractory leukaemia/ lymphoma (aml, ; all, ; mixed phenotype acute leukaemia, ; lymphoma, ) who received treatments with 'ank' from haploidentical parental donors on institutional protocol, between aug and . parents/legal guardians/patients provided informed consents as per institutional guidelines for donors and patients procedures. donor lymphocytes harvested at steady state were cd depleted followed by overnight culture in il before being infused into patients lymphodepleted with fludarabine and cyclophosphamide. additional rituximab were given to patients and another received tbi gy. subcutaneous il injections at doses - mu/m /dose started on d- and were planned for doses, as tolerated. nk alloreactive donors (kir-ligand mismatch) and kir b/x genotype were available to all except patients. two patients were treated for post-transplant relapse; of whom also received 'ank' pre-transplant; other patients had failed best conventional therapy including cd /cd bispecific t cell engager (blinatumomab) in . lymphodepletion was well tolerated. a median tnc and cd + dose of . (range, . to ) × /kg and . (range, . - ) × /kg, respectively were administered. cytokine release syndrome (crs) was observed in of treatments ( grade , grade , grade ). the patient with dock deficiency, disseminated ebv+ cerebral lymphoma had grade crs and robust tumour lysis syndrome but succumbed to neurotoxicity. of the treatments, there were responses, including the given posttransplant. excluding the treatments given post-transplant and non-responders, median peak donor chimerism was % (range, - %) occurring at a median of (range, - ) days. five patients ( responders, non-responder) proceeded to transplants at a median of (range, - ) days after 'ank.' responders had longer survival time compared to nonresponders (median vs days). two responders ( %) achieved sustained minimal residual disease (mrd) remission after transplants and are alive and days from 'ank.' five eventually died of their primary leukaemia/lymphoma; from crs. our preliminary experience in a small cohort of paediatric patients with refractory leukaemia/ lymphoma showed that adoptive transfers of ex vivo il activated nk cells from haploidentical parental donors were tolerable; with responses seen in % of patients; and % achieving prolonged mrd remissions after transplants. patients with cerebral diseases might be at increased risks of neurotoxicity with this approach; and care must be taken in patient selection and the design of the lymphodepletion therapy. alternative donor choices are limited in multi-racial, multiethnic societies with small families such as singapore. unrelated cord blood transplant provides a feasible alternative to patients lacking adult stem cell donors in children with primary immunodeficiency diseases. method: we describe our experience using unrelated cord blood transplant (ucbt) for children with pid from august to november . during this period we performed hsct for children with pid: with unrelated cord blood ( %); with msd and i mud. out of cases of ucbt there were severe combined immunodeficency (scid), chronic granulomatous disease (gcd), hyperigm syndrome and wiskott aldrich syndrome (was). the median age of transplant was . months (range . to . months). all presented with multiple infections ranging from disseminated bcg infection to parainfluenza /rsv /rotavirus infection to pseudomonas sepsis, staphylococcal endocarditis to pulmonary aspergillosis for scid. hyper igm presented with pnemocystitis carini pneumonia while cgd conditions presented with perianal abscess and fungal pneumonia. the child with was had life threatening git bleeding and a hemorrrhage trachaebronchial cast removed after a failed initial extubation for gastroscopy. conditioning regimes consisted of reduced intensive (fludarabine based) conditioning regime for scid and myeloablative regime for the rest. the median tnc dose was . × ( )/kg (range . to . ) and median cd + cells dose was . × ( )/kg (range to . ). results: all engrafted well except for one graft failure in cgd. he refused nd transplant and died . years post transplant from fungal pneumonia. median engraftment time for neutrophil was days (range to ) and platelet was days (range to days). grade skin aghvd occurred in one patient while another patient died of agvhd of liver and lungs. chronic gvhd was found skin and liver in one patient. trm was % (due to agvhd). median follow up was days (ranged to ). overall years survival was %. post-transplant complication with life threatening puemonitis was not uncommon. one patient developed biopsy -proven idiopathic interstitial pneumonitis and required ecmo for one month. he received immunosupressive drugs including methylprednisolone, infliximab, oral imatinib (tk inhibitor), azithromycin and nebulised becotide. he was weaned off oxygen after - months. conclusion: our limited experience showed unrelated cord blood is good source of stem cell for transplant in pid in a multiracial population. one case of graft failure was likely due too low cell dose cd +cells dose × ( )/kg. the expertise in icu has enabled us to support several patients who presented with infective pneumonia pre-transplant and post -transplant. with better technology like alpha/beta depletion haploidentical transplant may be a better option to achieve engraftment earlier so as to avoid stormy post-transplant infections seen in unrelated cord blood setting. disclosure of conflict of interest: none. in spite of these recommendations, literature from developing countries suggest that pbscs are used more and more frequently without compromising the transplant results, as they seem to be preferred graft source for donors in many countries incl. poland. therefore we analyzed the efficacy of mud-hsct in children with saa transplanted in our centre. clinical data of saa and pnh children and adolescents ( boys and girls), who underwent mud-hsct between october and july were retrospectively analyzed. the median age was . years (range . - years) according to the graft source, the patients were divided into pbsct group ( patients) and bm group ( patients). four patients required second mud transplant due to graft rejection. overall survival for all patients was %. estimated -year overall survival (os) was not statistically different between pbsct group and bm group [( % vs % ) p = . ]. there was no significant difference in os between group who had ist before transplant and the group, who had an upfront transplant as a first line of therapy [ % vs %, p = . ].the time to neutrophil and platelet engraftment was statistically longer in bm group than in pbsc group [(anc vs days, plt vs days, respectively) p = . ]. the incidence of grade iii-iv acute graft-versus-host disease (gvhd) in pbsct group was similar to that in bm group [ % ( / ) vs % ( / )]. the incidence of chronic gvhd in pbsct group was similar to that in bmt group [ % ( / ) vs % ( / )]. other transplantrelated complications like heart failure, central nervous bleeding, incidence of infections were comparable within the two regimens. there were deaths in the whole group. the main reason of death were infectious complications or multiorgan failure (mof) in severely pretransfused patients in this historical cohort of patients. unrelated donor pbsct in children and adolescents with saa seems to be not inferior to unrelated donor bmt. the incidence of chronic gvhd was surprisingly low in saa recipients of mud pbsc. increased morbidity and mortality due to infections was due to individual poor clinical situation of patients before transplant (i.e. fungal infections, contamination with resistant bacteria, prolonged neutropenia). disclosure of conflict of interest: none. dyskeratosis congenita (dc) is characterized by the clinical triad of reticular skin pigmentation, nail dystrophy, and oral leukoplakia. the majority of patients with dc develop bone marrow failure (bmf), which is the main cause of death in dc patients. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment for bmf associated with dc. transplant-related morbidity/mortality is common, especially after myeloablative conditioning regimens. hsct has been introduced into the management of dc, which has had remarkable clinical results. we report our experience in children with dc who underwent allogeneic transplantation at a single medical center. patients received a fludarabine-based reduced intensity conditioning (ric), and the graft source was unrelated peripheral blood stem cells. median age at the time of hsct was . years (range, - years). the numbers of infused mononuclear cells and cd + cells were . ± . × /kg and . ± . × /kg, respectively. the median time of neutrophil and platelet recovery were . days (range, - days) and . days (range, - days). two patients experienced grade ii-iii acute graftversus-host disease (gvhd), and chronic gvhd was only observed in one patient. all four patients remained alive and transfusion independent at the median follow-up of . months (range, - months). correction of previously existing physical defects was observed in two patients. unrelated peripheral blood hsct can be a curative option for dc. ric based on the type of disease is important to s achieve successful hsct. a larger sample size and extended follow-up of this rare patient population are needed to determine whether the changes in therapy will improve longterm survival. disclosure of conflict of interest: none. autosomal recessive hyper-ige syndrome due to dock mutation is a combined primary immunodeficiency, characterized by severe eczema, recurrent infections, and susceptibility to autoimmunity, malignancy, and multiple allergies, in addition to unusual high serum ige level. dock patients tend to have a progressive severe clinical course with mostly fatal outcome during second to third decade of life without hematopoietic stem cell transplantation (hsct). in our center we have a large number of dock patients. during a period of years ( - ), we transplanted patients with documented dock- mutation confirmed by molecular genetics. one patient did not receive any conditioning because of poor clinical condition and he died from severe cutaneous and gut gvhd and another patient received cbt with bu/flu with zero engraftment. the rest of the patients received hsct from hla full matched donor with chemoablation with bu/cy for all with % lymphoid and myeloid engraftment (str). among those patients who received chemoablation, gvhd developed in patients mostly grade i and ii. in addition patients died: one died of severe gvhd and the other two died of sepsis. for dock patients we highly recommend early hsct if fully matched donor is available to prevent the high mortality associated with the disease. alloreactivity triggered by interactions between killer cell immunoglobulin-like receptors (kir) and natural killer (nk) cells plays a role in graft-versus-tumor (gvt) effects after hematopoietic stem cell transplantations (sct). in particular, kir-ligand mismatching between the donor and recipient might promote nk cell alloreactivity after unrelated cord blood transplantations (ucbt) in adult patients with acute myeloid leukemia (aml). recently, it has been suggested that allogeneic nk cells could be the effector cells that mediate gvt effects after mismatched allogeneic transplants for refractory childhood solid tumors. however, there are few reports about the efficacy of kir-ligand mismatched sct in pediatric cases. here, we report the excellent outcomes of kirligand mismatched cbt (kir-cbt) in pediatric patients with refractory malignant disease. we evaluated the cases of pediatric hematology and oncology patients [ ( %) aml, ( %) myelodysplastic syndrome [mds] , and ( %) neuroblastoma [nbl] patients] who underwent kir-cbt between and at our institution. among the aml cases, one involved refractory disease (induction failure), and the other three involved relapsed aml (one patient relapsed after the st sct because of q-). all nbl patients underwent kir-cbt followed by auto-peripheral blood stem cell transplantation (pbsct) because of stage disease. the mds patient underwent kir-cbt because of refractory anemia with excess blasts. kir mismatching was defined as incompatibility between the donor kir and recipient kir ligand, and only inhibitory kir that interacted with human leukocyte antigen (hla)-bw , -c , or -c group ligands were considered. the median age of the patients was (range - ) years. all of the aml patients were in complete remission (cr) at the time of the hsct (cr = one case, cr = cases). the mds patient was in a non-cr state, and all of the nbl patients were in their st cr at the time of the hsct. the aml patients received total body irradiation (tbi)-based conditioning ( gy tbi and mg/kg cyclophosphamide [cy]), and the mds patient received busulfan (bu)-based conditioning ( . mg/kg bu and mg/kg cy). the nbl patients received reducedintensity conditioning regimens ( mg/m fludarabine, mg/m l-pam, and gy tbi). the cb exhibited hla - locus mismatches (dna typing), including at least one inhibitory kir gene mismatch. the prophylaxis for graftversus-host disease (gvhd) consisted of tacrolimus and shortterm methotrexate. anti-thymocyte globulin (atg) was not used as a gvhd prophylaxis in any case. after the median follow-up period of months (range: - months), all patients were alive, and none of them had relapsed after the kir-cbt. although grade ii-iv gvhd was observed in patients ( %), it was controlled with prednisolone. chronic gvhd was not seen in any case. the present findings suggested that nk cell alloreactivity plays a role in preventing childhood myeloid leukemia and nbl relapse after kir-cbt. although our results are limited, this report provides novel data to support further investigations into the use of kir-cbt for the treatment of pediatric refractory malignant disease. disclosure of conflict of interest: none. impact of fcm-based minimal residual disease on transplant outcomes in patients with aml in hematological complete remission t oka, j kanda , k ohmori , m hishizawa , t kitano , t kondo , k yamashita it is reported that the presence of minimal residual disease (mrd) before hematopoietic stem cell transplantation (hsct) is associated with poor overall survival in patients with acute myelogenous leukemia (aml) in hematological complete remission (cr). we retrospectively analyzed the association between flowcytometry (fcm)-based detection of mrd and transplant outcomes. we included adult patients with aml in hematological cr, who underwent their first allogeneic hsct between april and may at kyoto university hospital. mrd of bone marrow before hsct was measured using fcm. to search for target antigens to detect mrd, threecolor fcm analyses were performed using a differential panel for every disease and patient, which allowed us to detect ⩾ . % of mrd. of the patients (median age: . , range: - ), patients were included in the mrd-negative group (mrd o . %), whereas were included in the mrd-positive group (mrd ⩾ . %). in the latter group, patients were included in the mrd-low group (mrd o . %), and were included in the mrd-high group (mrd ⩾ . %). there was no significant difference in the patient background between the mrd-negative and mrd-positive groups. the -year overall survival rates for the mrd-negative, mrd-low, and mrd-high groups were %, %, and %, respectively (p = . , figure ). in a multiple regression analysis, the mrd-high group was significantly associated with higher overall mortality than the mrd-negative group (mrd-low vs mrd-negative, hazard ration [hr] . , p = . ; mrd-high vs mrd-negative, hr . , po . ). the -year relapse rates for the mrdnegative, mrd-low, and mrd-high groups were %, , and %, respectively (p o . ). there were no significant differences in non-relapse mortality among the three groups. the analysis of fcm-based detection of mrd revealed that an mrd positivity of ⩾ . % was significantly associated with high risk of relapse and death even in patients with aml with hematological cr. the stronger consolidation or conditioning therapy before hsct based on mrd could improve transplant outcomes in these patients. [p ] disclosure of conflict of interest: none. post-transplant cyclophosphamide (ptcy) and megadose t cell depleted (tcd) haplohsct for tolerance induction f aversa , e bachar-lustig , n or-geva , y zlotnikov klionsky , l prezioso , s bonomini , a monti , i manfra , c schifano , s pratissoli , f lohr , r lamanna , v sgobba , n giuliani and y reisner hematology and bmt unit, university hospital of parma, italy; department of immunology, weizmann institute of science, rehovot, israel; neurology department, stanford school of medicine, stanford, california; radiotherapy unit, university hospital of modena, italy; radiotherapy unit, university hospital of modena, italy; genetic unit, university hospital of parma, italy and hematology and bmt unit, university hospital of parma, italy the use of ptcy is associated with reduced risk for gvhd in t cell replete nma haplo-hsct; however, this intervention is still not sufficiently safe to justify treatment of non-malignant diseases or as a platform for organ transplantation. experimental data: in a total of mice, we showed that combining the power of megadose tcd hsct with high dose ptcy (fig. a) , enables marked and durable chimerism following nma conditioning, while each modality alone was ineffective (figure a) . chimerism included all myeloid and lymphoid lineages, and lda analysis of alloreactive t cells revealed specific immune tolerance towards donor stimulators (fig. b) , also associated with acceptance of donor but not rd party skin. clinical trial: a similar protocol was developed for clinical use. the first patient, a yr old male with high-risk multiple myeloma in cr after autohsct, received megadose ( . x cd + cells/kg) cd /cd depleted ( . x cd +t cells/kg) haploidentical pbpcs after atg, fludarabine and gy single frcation tbi. ptcy was given to control both hvg and gvh reactions (fig. c) . hematopoietic engraftment was achieved at day + with over % donor type chimerism during the first months in the myeloid and b cell lineages. t cells during this period were predominantly of host type ( - % donor type), gradually increasing to - % at - months post transplant (fig. d) . the patient overcame cmv and subsequently ebv reactivation without any treatment (fig. e- g) . dextramer facs analysis revealed that cmv and ebv specific cd t cells were exclusively of host origin (fig. f- h) . at + months, cr and normal free light chain ratio were confirmed. the second patient, a year-old male with high risk heavily pretreated multiple myeloma (tandem auto-hsct, yr maintenance with lenalidomide, salvage therapy with vd) received a similar hsct ( . x cd + cells/kg, . x cd +t cells/kg). despite transient engraftment ( % donor cell on day + ), graft failure with autologous recovery ( . % donortype chimerism) was documented on day + . this may be due to the extended treatment ( yrs) with lenalidomide, but rejection cannot be excluded. after months, this patient tolerated a second haplo-hsct (different donor) after myeloablative conditioning (atg, treosulfan, thiotepa and fludarabine) and alfa/beta tcr/cd -depleted pbpcs. at month follow up, he shows no sign of gvhd, good immunological reconstitution, excellent quality of life, and remains in complete remission. collectively, our murine proof of concept data supported by clinical experience in the first high risk mm patient. the marked level of host t cells persisting over the first year after hsct can provide anti-viral immune protection until thymus-derived donor t cells are generated. avoiding additional post transplant immune suppression ensures a robust anti-viral immunity and a graft vs tumor effect. the rejection experienced by the nd patient, although corrected by a nd myeloablative tcd hsct, indicates that the conditioning must be fine-tuned to optimize engraftment in every patient. we are therefore testing, increasing tbi from gy to gy. further studies will determine the efficacy of this approach in elderly mm patients, in non-malignant hematopoietic diseases, or as a prelude for organ transplantation and cell therapy. over the last decade the addition of alemtuzumab to fludarabine-based reduced intensity conditioning regimen is common practice in the unrelated donor allograft setting. in recent years, however, its use has extended to reduced intensity hla-identical sibling donor allografts with the aim of providing an additional prophylaxis against gvhd. it is difficult to assess though whether this practice has any negative influence in the relapse rate or whether it has any net benefit or disadvantage in terms of overall survival. in this retrospective study we have analysed a historical cohort of patients [ males, females, mean age . ( - )] who s received a ric fully matched unrelated donor ( patients) or sibling donor ( ) hsct as consolidation treatment for hr aml in transplant centres in uk and greece. the conditioning regimen included fludarabine in all cases, together with melphalan and alemtuzumab( patients), busulphan and campath ( patients), busulphan and thiotepa ( patient), melphalan ( patients), busulphan with and without atg ( patients) total body irradiation ( cgy, patients). in total, patients received alemtuzumab ( mud mg alemtuzumab and sibling donor hsct recipients mg alemtuzumab) and patients ( mud and sibling donor hsct recipient) received atg with patients receiving t replete allografts. gvhd prophylaxis was ciclosporin for patients receiving alemtuzumab based or atg based regimen and ciclosporin with low dose methotrexate for t-replete allografts. the median follow up was . months (range - months).all but four patients were transplanted in cr overall, patients receiving conditioning without alemtuzumab suffered more frequent (po . ) and more severe (po . ) acute gvhd. this group, however, had a significantly (po . ) lower relapse rate. the overall survival remained unaffected. the subgroup of patients receiving allografts from mud had a clear benefit in terms of a lower incidence (p o . ) and severity (p o . ) of acute gvhd: none of the patientsreceiving alemtuzumab experienced grade iv agvhd, but up to / patients not receiving alemtuzumab suffered severe grade iv gvhd. however, the use of campath was associated with a significantly higher rate of relapse or progression of the aml (po . ), so that none of the mud recipients not having campath relapsed, while / patients having alemtuzumab relapsed. although none of these factors had a net impact on survival, there was a nonsignificant (p = . ) trend towards a higher survival in patients who received alemtuzumab. in the sibling donor allograft setting, alemtuzumab had no significant impact on the incidence of acute gvhd, relapse or survival. finally, in diseases where cytogenetic or molecular markers of high risk were available, our results showed a better overall survival (po . ) in ric alemtuzumab conditioning undergoing fully matched unrelated donor hsct, probably as a result of the protection against graft versus host disease while maintaining graft versus leukaemia effect. overall, alemtuzumab is a highly protective agent against agvhd in mud hsct recipients while it maintains the graft versus leukaemia effect.however it did not show any clear benefit of its use in the identical sibling donor setting. larger prospective studies are required in order to determine the need for this agent in this particular setting. disclosure of conflict of interest: none. blastic plasmacytoid dendritic cell neoplasm (bpdcn) is a rare disease which constitutes o % of all hematologic neoplasms annually. majority of bpdcn present with diverse skin involvement prior to leukemic dissemination, whereas a minority (~ %) have systemic involvement at diagnosis. there are no established therapies for bpdcn and most pts receive acute leukemia, myeloid or lymphoblastic, induction regimens; but responses are short-lived and prognosis is poor upon relapse. allogeneic hematopoietic cell transplantation (allo-hct) is offered to bpdcn cases based on small retrospective or registry case series. we retrospectively analyzed outcomes of bpdcn pts who received an allo-hct at transplant centers in the usa. a total of pts were eligible for analysis ( table ). the primary endpoint was overall survival (os). twenty patients (m = , %), median age of ( - ) yrs, received an allo-hct from a matched related (n = , %), matched unrelated (n = , %), mismatched-unrelated (n = , %), umbilical cord (n = , %) or haploidentical (n = , %) donor using myeloablative (mac) (n = , %) or reducedintensity (ric) (n = , %) conditioning. fifteen pts received hyper-cvad as pre-allograft therapy (front-line = , salvage = ). the majority (n = , %) were allografted in cr . median f/u for survivors was . ( . - . ) months. median time-to-neutrophil and platelet engraftments were ( - ) days and ( - ) days, respectively. five pts never dropped s platelet counts below /μl. three pts (mac = , ric = ) relapsed at , , and months, respectively. all relapsed with marrow involvement ( had also skin involved). mean os was . ( . - . ) months. one-year and -year os were % ( % ci = - %) and % ( % ci = - %), respectively. there was no difference in -year os when comparing mac versus. ric (hr = . ( % ci = . , . ), p = . ). median time to onset of acute gvhd was ( - ) days; grade ii-iv acute gvhd occured in cases. chronic gvhd was seen in cases (mild = , mod/severe = ). allo-hct is an effective therapy for bpdcn resulting in durable remissions. encouraging outcomes observed in this analysis may be explained by offering allo-hct early in the disease course and in the setting of complete remission. larger studies are needed to better understand risk factors for relapse to develop post-transplant strategies to improve outcomes. disclosure of conflict of interest: none. a risk-factor analysis for overall survival in patients with acute leukemia that relapse following t-replete haploidentical transplantation: on behalf of the acute leukemia working party of the european society for blood and marrow transplantation s piemontese , , m labopin , , f ciceri , , c schmid , , a ruggeri , , w arcese , z gulbas , y koc , j tischer , b bruno , w depei , d blaise , d beelen , g ehninger , a boumendil , , m houhou , , m mohty , and a nagler , relapse of acute leukemia is the leading cause of transplantation failure with devastating results. relapse post t-replete haploidentical transplantations (haplo-sct) is not well characterized. the objective of this study was to identify riskfactors for overall survival in patients with al that relapsed after a haplo-sct. from to , haplo-sct were performed in ebmt centers as first allogeneic transplantations for adults with acute leukemia. out of patients for whom we were able to receive updated data, relapsed and were included in this analysis. median follow-up among survivors was months after haplo-sct ( - ) and . months ( - ) after relapse. median time from haplo-sct to relapse was months ( d- m). diagnosis was acute myeloid leukemia (aml) in % and acute lymphoblastic leukemia (all) in % of the patients, respectively .fifty-two ( %) patients were transplanted in first complete remission (cr ), ( %) in cr or cr , while ( %) were transplanted in active disease. ric regimen was used in ( %) patients and ( %) received bone marrow as stem cell source. post-transplant cyclophosphamide (pt-cy) was used for graft-versus-host disease (gvhd) prophylaxis in patients ( %). fifty-two ( %) of the patients who relapsed post haplo-sct experienced previously acute gvhd and ( %) chronic gvhd post transplantation. treatment of relapse varied and included: none in ( %), ist withdrawal only in ( %), chemotherapy (ct) only in ( %), tyrosine-kinase inhibitor (tki) only in'( %), tki and ct in ( %), dli only in ( %), subsequent transplant in ( %), ct and dli in ( %), ct and subsequent transplant in ( %), tki ct and subsequent transplant in ( . %), dli and subsequent transplant in ( . %) patients. donors for second allogeneic transplant were unrelated (n = ), haploidentical (n = ) and cord blood (n = ). second transplant was performed in cr for patients and in relapse for patients. only patients who received a second haplo were alive at and months post second transplant. the majority of patients who received dli were in relapse at time of dli ( %), and % achieved cr after dli. os y after dli was %, patients being alive at a median time of mo ( - ) post dli. overall, the one-year overall survival (os) following relapse was % ( % ci: . - . ). in univariate analysis disease status at haplo-sct (cr vs active disease), cytogenetics (good/intermediate vs poor) and median time from haplo-sct to relapse ( or o . months) were associated to a higher os at one year after relapse: % (p = . ), % (p = . ) and % (p o - ), respectively. in multivariate analysis complete remission at haplo-sct (p = . ; hr . ; ci: . - . ) and time from haplo-sct to relapse higher than . months (p = . ; hr . ; ci: . - . ) were risk factors for a higher os after relapse. in the patients transplanted in cr and relapsing more than month after haplo, and y os were respectively % and %. these findings suggest that similar to other transplantation setting os for acute leukemia that relapse post haplo-sct is dismal. disease status at transplant and time from transplant to relapse are the two important prognostic factors that can predict somewhat better survival. indication for second transplant should be carefully evaluated. integrations with novel therapies are in unmet need to prevent and treat relapse post haplo-sct. disclosure of conflict of interest: none. patients (pts) with aml who relapse after autologous stem cell transplantation (asct) have a dismal outcome but some can be rescued with an allogeneic transplantation (allohsct). yet, available evidence presently stems from analyses of limited patient numbers. we decided to analyze the ebmt registry to evaluate the outcome and determine the prognostic factors in a large series of such pts. the ebmt registry was screened for adult pts with de novo aml (non-apl) who received an allograft in cr or first relapse ( - ) after being autografted in cr . pts receiving ex vivo t cell depletion (tcd) were included only if they received a haploidentical allohsct. inclusion criteria were met by pts ( % female, median age [range - ] years). median time from asct to relapse was (range . - , iqr . - . ) months. at allohsct, pts were in st relapse ( %) or cr ( %). donors were matched sibling ( %), unrelated ( %), haploidentical ( %), or cord blood ( %), respectively. conditioning was myeloablative in % and reduced intensity in % of the pts, respectively. the median follow up was months (range o - months). at years post allograft (figure), leukemia free survival ( - ] of the pts. all factors significantly associated with ⩾ endpoint in univariate analysis were entered in a multivariate cox regression model (table ) . ri was lower in pts transplanted in cr rather than in relapse ( . % vs . %; hr . , p = . ) and in pts who relapsed later ( months, median value) as opposed to those who relapsed early post asct ( . % vs . %; hr (per month) . , p o - ). ri was lower in pts transplanted with an unrelated donor (ud) in comparison to those transplanted from a matched sibling donor ( . % vs . %; msd, hr: . , p o - ). patient age, poor cytogenetics, transplantation in relapse, previous tbi for asct, myeloablative conditioning (mac) vs reduced intensity (ric) and ud, haplo or cbt vs msd all significantly increased nrm. lfs was significantly better in pts with good risk ( . %) than in pts with intermediate risk or poor risk cytogenetics ( %; hr . , p = . ) or in pts who relapsed late (per month: hr . , p = . ) post asct. lfs was worse in pts who previously had received tbi ( % vs %; hr = . ; po - ). the same prognostic factors were significant for os. haploidentical (hr . , p = - ) and cord blood (hr . , p = . ) transplants resulted in lower os than those from msd. finally, date of transplant significantly influenced os which was higher in pts transplanted after january vs those allografted before; . % vs . %, hr (per year) . , p = . ). about one third of adult patients with aml who relapse post asct can be rescued with an allogeneic transplantation, especially if the duration of persisting cr post asct is long and no tbi was received in the past. transplantation from an msd while in cr rather than at relapse offers the best outcome. disclosure of conflict of interest: none. high incidences of graft-versus-host disease (gvhd) and relapse have seriously impeded the widespread application of haploidentical hematopoietic stem cell transplantation (haplo-hsct) for high-risk acute leukemia lacking conventional hla-matched donors. one hundred and ten high-risk acute leukemia patients underwent haplo-hsct with idarubicin (ida) intensified conditioning regimen (ida intensified bucy for acute myelocytic leukemia (aml) and ida intensified tbi-cy for acute lymphoblastic leukemia (all)). for donor-recipient hla / or / transplant, we separately administered a total of mg/kg or mg/kg antithymocyte globulin (atg) and basiliximab for gvhd prophylaxis. all enrolled patients were observed longitudinally until death or lost to follow-up. the -day cumulative incidences of Ⅱ-Ⅳ and Ⅲ-Ⅳ agvhd for all patients were . %, . %, respectively. the -year cumulative incidence of extensive cgvhd was . %. the relapse rate was . %. the -year probability of overall survival (os) reached . %. the patients in non-complete remission (nr) showed significantly higher relapse and worse survival than complete remission (cr) minimal residual disease (mrd) (-) and cr mrd (+) patients. however, the relapse, y-os and disease-free survival (dfs) of cr mrd (-) did not differ from cr mrd (+) patients, indicating our intensified transplant technique could overcome the poor prognosis of mrd. for whatever aml or all patients, the relapse rates, agvhd, cgvhd and the estimated -year os and dfs between two atg group were equivalent, except that all patients in atg mg/kg experienced higher relapse ( . % vs . %, p = . ). although the incidence of cytomegalovirus (cmv) reactivation in atg mg/kg and mg/kg was . %, . %, the average episodes of cmv reactivation were remarkably [p ] higher in mg/kg. our ida intensified haplo-hsct technique could improve the outcome of high-risk acute leukemia and could be recommended as a good alternate for patients lacking hla-matched sibling donors. diagnosed secondary aml were randomized : to cpx- or standard + therapy. cpx- induction was units/m on days , , (first induction) and days , (reinduction); + first induction was cytarabine mg/m /day × days and daunorubicin mg/m on days , , , and reinduction was cytarabine mg/m /day × days and daunorubicin mg/ m on days , . a dynamic allocation procedure stratified patients by age group ( - or - years) for each study arm. patients with complete response (cr) or cr with incomplete platelet or neutrophil recovery were considered for allogeneic hct, based on institutional criteria. overall survival (os) landmarked at the time of hct was assessed. a total of patients were enrolled on the induction trial. . patient and aml characteristics in the hct age subgroups were generally similar between arms. in both age subgroups of patients receiving hct, median os was longer in the cpx- arm than in the + arm (table ). in the - group, serious adverse events (saes) prior to hct in the cpx- and + arms occurred in % and % of patients, respectively; in the - group, in % and %, respectively. the most common sae was febrile neutropenia (cpx- , . %; + , . %), occurring in all age groups. relapse after allogeneic haematopoietic stem cell transplant (allo-hsct) for acute myeloid leukaemia (aml) and myelodysplastic syndrome (mds) remains the main cause of treatment failure. it is associated with dismal prognosis and short survival. proposed salvage strategies are tapering of immunosuppressive therapy, re-induction with chemotherapy and consolidation with donor lymphocyte infusion (dli) or second allo-hsct, although, results remain disappointing. azacitidine (aza) and dli has proved to be an effective and well-tolerated outpatient approach in this setting, and results in at least temporary disease control in the majority of patients, thus, representing a valuable alternative to current treatments. between january and november , patients with relapsed aml or mds after allo-hsct were treated with subcutaneous aza mg/m days - every days and escalating doses of dli if feasible at manchester royal infirmary, uk. aza was continued until cr or disease progression. patients characteristics: median age (range - ) years, % males, diagnoses were aml (n = ) and mds (n = ). five ( %) patients had either monosomal or complex karyotype. fifty percent of patients were in cr before transplant, . % in cr , . % had a partial response and % did not receive any chemotherapy before the transplant. fifteen out of received fludarabine-base reduced intensity conditioning regimen and all but one had a t-cell depleted graft. at relapse % had mixed donor chimerism. median time to relapse was . (range - ) months after allo-hsct. with a median follow up of . (range - ) months a median of (range - ) courses of aza were administered and median of (range - ) dli were infused. doses of dli were administered starting at . x /kg and escalating by log . aza and dli infusions were well tolerated; only two patients withdrew due to intolerance. seven patients were admitted at least once due to infections ( %) or progressive disease. only two patients developed mild gvhd grade . complete remission was achieved in . % patients and stable disease in %. patients in cr had full donor chimerism. median overall survival for patients in cr was months compared to months for those who did not respond (p = . ). patients with more than % blasts on bone marrow at time of relapse after allo-hsct had a worse outcome than those with less than % blasts ( months and months respectively, p = . ). no differences were seen when compared time to relapse ( o months vs ⩾ months) s and outcome, or disease and overall response, although numbers in this series are small. image/graph: overall survival following azacitidine and dli, patients in complete remission, stable disease and disease progression. azacitidine and dli can provide long term remissions in patients with relapsed aml/mds post allo-hsct with low toxicity. lower disease burden at relapse carries better outcomes. low rates of gvhd are seen following azacitidine and dli most likely showing the immunomodulatory effect of azacitidine described by other groups. acute myeloid leukemia (aml) is a frequent complication in patients affected by telomere maintenance disorders ('telomeropathies') such as dyskeratosis congenita (dkc). treatment of aml in dkc patients by chemotherapy and hematopoietic stem cell transplantation is characterized by frequent remission failure, high organ toxicity and poor outcome. a -yearold patient with aml was admitted to our hospital in december . he had been treated with cycles beacopp for hodgkin´s lymphoma (hl) in . on admission, the patient presented clinical signs of premature aging with hair greying and lack of fully recovered hair growth after chemotherapy (cx) for hl. flow-fish analysis revealed tl below the % percentile within leucocytes in line with the suspected diagnosis of telomeropathy. retrospective tl analysis by confocal q-fish from bm at hl diagnosis confirmed short tl before the start of any chemotherapy. he received standard aml induction cx ( + ), but follow-up revealed persistence of aml. salvage cx with flag-ida was applied resulting in partial remission with only weak regeneration of normal hematopoiesis. the patient received an allogeneic stem cell transplantation (asct) after conditioning with mg/m melphalan and fludarabin from his hlamatched brother whose tl was found to be normal. after asct, he developed sinusoidal obstructive syndrome and progressive liver failure treated with defibrotide and he was admitted to icu for sepsis. leucocyte count showed sufficient engraftment on day ; however, liver function recovered only partially. during critical care treatment, the patient showed cardiomyopathy, renal failure and extensive wound healing problems without epithelial proliferation indicative of severe replicative exhaustion. finally, he died due to sepsis with acute liver failure on day after asct. aml arising from dkc is a rare event with substantial impact on patients´prognosis. therapy remains challenging due to poor bm function and high risk of organ toxicity, especially liver failure and lung fibrosis. dose reduction of alkylating agents and avoidance of total body irradiation are necessary in conditioning prior to asct in patients with dkc and aml, however no clear data or recommendations exist for the management of these patients. tl screening can help to identify patients with suspected dkc related bm failure or aml and to identify family donors without telomeropathy. physicians should be aware of possible dkc related aml, especially in familial cases of aml or bone marrow failure, impaired or prolonged recovery following cytoreductive treatment or coincidence of solid (e.g. oral cavity carcinomas) and hematological malignancies. disclosure of conflict of interest: none. chronic graft-versus-host disease and donor lymphocyte infusions in patients with non-de novo acute myeloid leukemia or advanced myelodysplastic syndromes after allogeneic stem cell transplantation pg hemmati , k pfeifer , lg vuong , cf jehn , p le coutre , b dörken and r arnold medizinische klinik mit schwerpunkt hämatologie, onkologie und tumorimmunologie, charité-universitätsmedizin berlin, campus virchow-klinikum, berlin, deutschland aml with myelodysplasia-related changes and therapy-related aml (taml), collectively termed secondary aml (saml) in daily clinical routine, represent distinct subgroups in the revised who classification of myeloid neoplasm and leukemias. as compared to de novo-aml, saml is associated with a poor survival when using conventional chemotherapy approaches. this is mainly due to unfavorable cytogenetics, older age and/or the presence of comorbidities as well as poor response to induction therapy. furthermore, cumulative organ toxicity resulting from treatment of the antecedent solid malignancy in patients with therapy-related disease has to be taken into account. allogeneic stem cell transplantation (allosct) represents the only option to achieve long-term disease control and definitive cure. we retrospectively analyzed patients with saml or advanced mds (eb- according to who) transplanted at our center between and . at the time of allosct, patients ( %) were in complete hematologic remission (chr), whereas patients ( %) had active disease. cytogenetic risk was categorized according to the swog/ecog classification and was favorable (n = ; %), intermediate (n = ; %), unfavorable (n = ; %), or unknown/undetermined (n = ; %). standard myeloablative conditioning (mac) using gy total body irradiation (tbi) and cyclophosphamide was used in patients ( %), whereas fludarabin/busulfan/atg-based reduced intensity conditioning (ric) was applied in patients ( %). grafts were from related (n = ; %) or unrelated (matched: n = ; % or mismatched: n = ; %) donors. the median follow-up of the surviving patients was ( - ) months. a graft failure occurred in / patients ( %). at last day of follow-up / patients ( %) were alive and in chr. relapse occurred in / patients ( %) after a median interval of . (range: . - ) months. cause of death were either relapse or nrm (gvhd and/or infections) in / patients ( %) or / patients ( %). at , , , and years after allosct overall survival (os) or disease-free survival (dfs) of the entire cohort was %, %, %, and % or %, %, %, and %, respectively. at the same time points, the cumulative incidence of relapse (ci-r) or non-relapse mortality (ci-nrm) was %, %, %, and % or %, %, %, and %, respectively. extensive uni-und multivariate analyses revealed a number of factors associated with inferior outcome, e.g. poor-risk cytogenetics, the presence of taml, advanced age, reduced physical performance, and comorbidities, whereas donor type (unrelated versus unrelated), and remission status had no significant impact on overall outcome. furthermore, the development of gvhd, especially the presence of cgvhd, and the use of donor-lymphocyte infusions (dli), either in a prophylactic or pre-emptive setting, were identified as independent predictors for a reduced relapse incidence, which in turn, led to an improved os and dfs. our results indicate that allosct represents an important treatment option for patients with saml. however, a relapse rate of % at months prompts the development of novel approaches to prevent early disease recurrence. strategies to augment the graft-versus-leukemia (gvl) effect of allosct may help to improve the results. disclosure of conflict of interest: none. myeloid sarcoma (ms) is a rare hematologic myeloid neoplasm that can involve any site of the body. it can occur as an exclusively extramedullary form or it can be associated with an acute myeloid leukemia (aml), a chronic myeloproliferative neoplasm (mpn) or a myelodysplastic syndrome (mds) at onset or at relapse. the rarity of ms does not enable prospective clinical trials and therefore a specific multicenter register can be useful for the clinical and biological studies of this rare disease. we report the clinical characteristics and outcome of histologically confirmed ms, diagnosed and treated in italian hematological centers in the last years. the patient's median age was years. there were / de novo extramedullary ms, / de novo aml-related ms and / were secondary aml-related ms. the most common extramedullary anatomic sites of disease were: skin, lymph nodes and soft tissues. forty-three patients ( %) underwent a program of intensive chemotherapy including flai, hdac-ida, hypercvad and mec schemes, with a cr rate of % ( / ). twenty-two ( %) patients underwent allogeneic sct, from a mud, from an hla-identical sibling donor and from an haploidentical donor. the median os of the whole population ( pts) was . months. the os probability at , and years was %, % and %, respectively. the os was better in patients that underwent an intensive therapeutic program (median os: months vs months). among the intensively treated patients, in univariate analysis, the os was better in young patients (p = . ), in patients that underwent allo-sct (p = . ) and in patients that achieved a cr during treatment (p = . ), and was worse in pts with secondary aml-related ms (p = . ). age, response to intensive chemotherapy and allo-sct were the only three variables that significantly influenced dfs (p = . , p = . and p = . , respectively). in multivariable analysis, allo-sct and response to intensive chemotherapy remained significant in predicting a better os (p = . and p = . , respectively), and response to intensive chemotherapy was the only significant variable in predicting dfs (p = . ). after allo-sct we observe a survival advantage in patients who achieved a pre-transplant cr (p = . ) and in those who developed a chronic gvhd (p = . ). patients with ms, both with de novo and secondary forms, still have a very unfavorable outcome and require an intensive therapeutic program, that includes allo-sct, whenever possible. the outcome after allo-sct is positively influenced by the development of chronic gvhd suggesting a graft versus ms effect. disclosure of conflict of interest: none. relapse of acute lymphoblastic leukemia (all) after allogeneic stem cell transplantation (sct) is associated with poor prognosis. blinatumomab may enhance the efficacy of donor lymphocyte infusions (dli) in this specific situation but data on the concurrent use of dli and blinatumomab are sparse. the patient presented here was diagnosed with standard risk pre-b-all (presence of t( ; ); bcr-abl and cd negative) at the age of . during treatment according to the german multicenter all-study group (gmall) protocol he presented with molecular relapse and months after initial diagnosis he received a tbi-based myeloablative sct from an unrelated hla-identical ( / ) donor. post sct he was negative for minimal residual disease (mrd) with % donor engraftment. given the high relapse risk he received prophylactic dli without occurrence of graft-versus-host disease (gvhd). one year after st sct he presented with an extramedullary (testes) and molecular relapse. after remission induction resulting in negative mrd he received a nd sct from an alternative, hlaidentical ( / ) donor after reduced intensity conditioning. this again resulted in negative mrd with % donor chimerism without any gvhd. six months after nd sct he presented with bone marrow relapse. we decided on the concurrent use of blinatumomab and dli. the first cycle of blinatumomab was initiated at standard dose including dose escalation without relevant toxicities. on day of the nd cycle, i. e. in the infusion-free interval before the rd cycle the patient received the first dli at x cd /kg. no toxicities or gvhd occurred. the rd cycle of blinatumomab was initiated and a second dli at . x cd /kg was applied on day of the rd cycle. on day of the rd cycle, i. e. day after nd dli the patient presented with signs of overlap gvhd (mouth, skin) and topical steroids were started. upon progression of clinical gvhd systemic steroids were initiated with immediate response. steroids were rapidly tapered and a th cycle of blinatumomab was started. gvhd did not recur. current staging after the th cycle blinatumomab, i.e. on day + after nd sct and months after initiation of blinatumomab treatment revealed complete remission with negative mrd, % donor chimerism and no signs of extramedullary relapse. counts of cd -cells at that time point were /μl. no relevant infections or relevant blinatumomab-associated toxicities were present during the entire course after the nd sct. in this case concurrent treatment of blinatumomab and dli resulted in the longest disease-free interval for our patient compared to preceding chemotherapy or dli alone. together with the small number of reported cases (ueda et al.) this supports the concept of concurrent blinatumomab and dli as an effective post sct treatment. the objective of the study is to evaluate the clinical efficacy and safety of decitabine (dac) in combination with haag regimen [homoharringtonine (hht), cytarabine (ara-c), doxorubicin (acla) and recombinant human granulocyte colony stimulating factor (g-csf)] for advanced patients with acute myeloid leukemia (aml). thirty-six patients with advanced aml receiving dac combined with haag chemotherapy in our center from december to august were enrolled in this study. eighteen of them were refractory or relapsed aml, and another patients were those who didn't achieve complete remission (cr) after a course of induction chemotherapy. the therapeutic responses, side effects and longtime survival were retrospectively analyzed. after a course of treatment, the rate of cr and partial response (pr) was . % ( / ) and . % ( / ) respectively, while the overall response rate (orr) was . % ( / ) in the cohort. for the patients with refractory or relapse aml, cr was . % ( / ), pr was . % ( / ), and orr was . % ( / ). while for the other not getting cr after a course of induction chemotherapy, cr was . % ( / ), pr was . % ( / ), and orr was . % ( / ). grade hematological toxicities were observed in all patients, and . % cases experienced infection. and all non hematological side effects were mild and well-tolerated. with a median follow-up of . ( . ~ . ) months, the -year overall survival (os) rate was . %, . % for the refractory or relapsed aml patients, and . % for those not achieving cr after a course of induction chemotherapy. the difference was significantly (p = . ). conclusion dac combined with haag regimen is safe and effective salvage treatment for advanced stage aml patients. disclosure of conflict of interest: none. aml patients harboring flt -itd mutation are associated with decreased survival compared to patients without flt -itd mutation. nevertheless, whether flt-itd mutation also has negative impact on the post-transplant survival is less clear. for flt -itd mutated aml, a decreased leukemia-free survival (lfs) after allogeneic hsct was observed in ebmt analysis but not cibmtr. in this study, unlike studies of ebmt or cibmtr which only pre-specified populations of patients were analyzed (cr in ebmt, cr +cr in cibmtr), we examined the prognostic impact of flt -itd mutation on post-transplant outcome of "all" the adult aml patients reported to taiwan bone marrow transplant registry (tbmtr). tbmtr is a research collaboration affiliated to the taiwan society of blood and bone marrow transplantation. it comprises all the transplantation centers in taiwan that contribute detailed data on hsct. adults aged ⩾ years with a diagnosis of aml and with known flt-itd mutation status in the registry were included. patient characteristics and transplant outcome following allogeneic hsct for flt -itd mutated and nonmutated aml were compared. kaplan-meier estimates were used to calculate the probability of lfs and overall survival (os). multivariable analyses for lfs and os were performed using cox proportional hazards model. patients who met the eligibility criteria were enrolled for analysis. the median follow-up of survivors was months. of the patients, ( . %) were positive and ( . %) were negative for flt -itd mutation. flt -mutated patients had significantly more transplantation at cr ( . %), shorter time interval between diagnosis and hsct ( . months), and higher wbc count at diagnosis ( . × /l) comparing to patients without flt mutation ( . % at cr , . months from diagnosis to hsct, and . × /l wbc count at diagnosis). significant more flt mutated patients had intermediate-risk ( . %) and normal ( . %) karyotype at diagnosis. the age, donor type, stem cell source, conditioning regimen, and atg use were not significant different between flt -mutated and non-mutated patients. of the whole population, flt mutation status did not negatively impact the transplant outcome ( years os for flt mutated and non-mutated patients: . % vs %, log rank p = . ; years lfs for flt mutated and non-mutated patients: . % vs . %, log rank p = . ). when different pre-transplant conditions (cr , subsequent cr, and no cr) were analyzed separately, flt -itd mutation status is still not a significant prognostic factor of os and lfs for patients in cr (equally good) and no cr (equally bad). however, for patients in subsequent cr, flt -itd mutation is the only significant factor predicting poor os and lfs in multi-variable analysis (median os and lfs for flt mutated and nonmutated patients: vs days, log rank p = . ; vs days, log rank po . respectively). the incidence of non-relapse mortality, grade / acute gvhd and extensive chronic gvhd is comparable between flt -mutated and nonmutated patients. flt -itd mutation is a significant and strong predictor of poor survival for aml patients in subsequent cr at hsct. for flt -itd non-mutated aml, a sizable portion of patients can have disease free survival after allogeneic hsct at subsequent cr. however, allogeneic hsct at cr should be strongly recommended for flt -itd mutated aml. [p ] disclosure of conflict of interest: none. allogeneic stem cell transplantation (asct) is a curative strategy in acute myeloblastic leukemia (aml) and myelodysplastic syndrome (mds). however, relapse keeps being the main cause of treatment failure. extramedullary relapse (er) is a rare event and its management is not well standardized. we retrospectively analyzed patients who received asct from to and developed er in our centre. we performed a descriptive study to analyze characteristic of these patients, post-relapse treatment and survival. statistic analysis was performed using spss v. . we found a total of patients with er, one of them with er after consecutive asct, so we analyzed cases of er. patient and transplant characteristics are summarized in table . at day + , % of patients were in complete response (cr). er occurred after a median of ( - ) months post-asct. eleven patients ( %) presented with a bone marrow relapse concomitant with the er. er affected central nervous system (cns) in patients ( . %), bone in patients ( %), skin or soft tissue in patients ( . %), mama in patients ( . %), ocular globe in patients ( . %) and teste in patients ( . %). two of them presented with multiple sites affected. between the patients who developed cns relapse, of them had received intrathecal prophylaxis. regarding post-er management, immune modulation was conducted in patients (immunosupression tapering in , donor lymphocyte infusions in and both strategies in ). all patients except one received systemic treatment (salvage chemotherapy in , azacitidine in , low dose arac in and atra in patient with a promyelocytic leukemia). together with systemic treatment, received radiotherapy and intrathecal therapy was used in all patients with cns involvement. response: out patients treated, ( . %) achieved cr and ( . %) progressed. two responding patients received a nd asct. after a median follow-up of months ( - ), patients are alive and disease free, with an estimated overall survival of % at years. patients receiving salvage chemotherapy followed or not by a nd asct experienced a significantly better os than those receiving other therapies (median os vs months; p = . ). patients with bone marrow involvement at relapse show a worse prognosis (median os vs months; p = . ) although not statistically significant due to small number of patients (image ). ten patients died due to disease progression. er must be considered in patients receiving an asct in case of organ symptoms. patients can be rescued with salvage chemotherapy followed or not by a nd asct achieving good results in terms of long term os. it seems that involvement of bone marrow at relapse confers a worse prognosis, what should be confirmed in a larger series of patients. [p ] disclosure of conflict of interest: none. flag-ida regimen as bridge therapy to allotransplant in refractory/relapsed aml patients: a single-center experience c pasciolla, m delia, d pastore, p carluccio, a ricco, a russo rossi, a mestice, f albano and g specchia university of bari, italy although treatment outcome in acute myeloid leukemia (aml) adult patient has improved over the past decade, relapse still occurs in up to - % of cases. furthermore, - % of patients fail to achieve complete remission (cr) because of treatment-resistance. the management of primary refractory and/or relapsed disease remainschallenging for clinicians. in our study, we reviewed the outcome of refractory and/or relapsed aml patients who underwent salvage therapy with the flag-ida regimen between and at our institution. the study aim was to determine the efficacy of the flag-ida regimen in order to clarify which variables (who ps, ldh, bone marrow, peripheral blood blasts and platelets counts, white blood cells (wbc), pmn, molecular-cytogentic risk, duration of response and relapsed or refractory disease), present before starting flag-ida treatment, might have an impact both on cr and on os. we analyzed consecutive adult patients ( males, females; median age years, range - ) with newly diagnosed acute myeloid leukemia refractory to standard induction regimens or relapsed after cr, who received the flag-ida protocol as salvage therapy between january and december . sixty-eight of the patients ( %) were in first relapse, forty-seven patients ( %) were refractory to conventional chemotherapy. median wbc count before salvage therapy was . x /l (range . - ). median bone marrow and peripheral blasts counts were and %, respectively; median platelets count was x e /μl. according to the fab classification, patients had m , m , m , m , m , m , had biphenotype acute leukemia. according to molecular-cytogenetic risk stratification ( %), ( %) and ( %) patients belonged to poor, intermediate and good risk group, respectively. sixty-nine of patients ( %) achieved complete remission (cr); forty-seven %) patients were refractory to the salvage therapy. in multivariable analysis, variables with positive impact on response rate were lower wbc counts (o e /μl, p = . ), higher platlets counts ( x e /μl, p = . ), molecular-cytogenetic risk (p = . ), duration of response in relapsed aml (p = . ) and relapsed rather than primary refractory disease (p = . ), respectively. median os was months (m). cox regression analysis confirmed that both higher platlets counts, p = . ( ( x e /μl) vs m (o x e ul), log rank, p = . ) and relapsed disease, p = . ( (relapsed) vs m (refractory), gehan-breslow, p = . ) correlated with better survival. of note, molecular-cytogenetic risk evaluated before starting treatment was associated with cr, while no correlation was found with os. our data seem to confirm the value of flag-ida in relapsed amland may suggest its best usage as bridge-therapy in patients awaiting allotransplantation. disclosure of conflict of interest: none. s leukemia relapse is the major cause of death in patients received allogeneic hematopoietic stem cell transplantation (allo-hsct). the precise etiological mechanisms of leukemia relapse remain unclear. both leukemia cells themselves and hematogenesis micro-environment are involved in the relapse event. in our previous study, we reported a case of donor derived relapse of acute myeloid leukemia (aml) after allo-hsct. the patient and his donor-sister both harbored a germline mutation(c. - dup) in cebpa gene. donor hematopoietic cells transformed to aml by developing two somatic cebpa mutations ( dupc and - dup) in the patient's microenvironment. hence we suspect that - dup mutation of cebpa gene may altered hematopoiesis microenvironment and increased the survival of aml cells. to conform our hypothesis, we transfected mesenchyme stem cells (mscs) with cebpa - dup or wide type and took vector as control. aml cell line hl cells were co-cultured with transfected mscs and then treated with ng/ml doxorubicin. apoptosis and cell cycle were detected at day . mscs protected hl cells from toxicity of doxorubicin. this protection was enhanced by overexpression of cebpa - dup . apoptosis rates of hl cells in group of msc-vector and msc-cebpa - dup were . ± . % vs . ± . % (p＜ . ). a larger part of hl cells remains quiescent with s higher rate of g /g phase in msc-cebpa - dup group, which may reduce the sensibility of hl cells to doxorubicin. to explore mechanisms involved in the alteration of microenvironment, we performed rna sequence with each group of mscs. we found that col a , col a and col a were upregulated in msc-cebpa - dup group compared with msc-cebpa wt group (col a :cebpa wt vs cebpa - dup was . vs . , p = . e- ; col a :cebpa wt vs cebpa - dup was . vs . , p = . e- ; col a : cebpa wt vs cebpa - dup was . vs . , p = . e- ). furthermore, we found that ddit and herpud genes, which were important factors in cellular unfolded protein response(upr) and to topologically incorrect protein, failed to augment in cebpa - dup group (ddit : vector vs cebpa wt the cure rate of childhood acute lymphoblastic leukemia (all) has improved considerably and approaches % today. however, the outcomes of patients who suffer from leukemic relapse remain unsatisfactory. despite the high cure rate of children and adolescents with all a subgroup of patients benefit from allogeneic hsct. allo hsct remains the standard treatment for intermediate/high risk aml patients. patients, all = and aml = age to years with median age years, m/f = / (m/f all = / , aml = / ) underwent sct in our hospital (from to ). fifty-eight patients transplanted allo hsct and pt aml auto hsct. conditioning regimens consisted of busulfan (iv) +cyclophosphamide for allo and cyclophosphamide + vp +cytarabine for auto hsct. peripheral blood (pb) was the source of progenitor cells in patients, bone marrow (bm) in patients and cord blood in one patient. in allo hsct, patient transplanted / matched and patients / matched. gvhd prophylaxis regimen was cyclosporine + mtx. all patients engrafted. in allogeneic pbsct all patients' median time to absolute neutrophil count (anc) . × /l was days, and the median time to platelet count × was days vs and days in allo bm all patients. in allogeneic pbsct aml patients median time to anc . × /l was days, and the median time to platelet count × was days. (all patients with aml transplanted with pb). at present pts are alive ( all, aml) and pts died due to ards, vod, hemorrhagic stroke, sepsis and relapse. trm was % at days. median time of death after transplantation was days in all and in aml. in allo pbsct all patients hospitalization period were days vs in allo bm all patients. acute gvhd appeared in % pts. chronic gvhd appeared in % pts. with a median follow-up of months ( - months) after transplant the event-free survival were % and four years overall survival % in all patients. a median follow-up of . months ( - months) after transplant the event-free survival were % and three years overall survival % in aml patients. hematopoietic stem cell transplantation can lead to durable remissions in children and adolescents with leukemia and increase in survival of children. pbsct in childhood all was consistent with significant faster anc and platelet recovery in allogeneic pbsct, hospitalization was shorter. longer follow-up is required to evaluate fully efficacy and long-term results. disclosure of conflict of interest: none. group hla-c /c subtypes were defined as previously practiced. median age of patients was , % of them were male. allo-hscts were performed from % unrelated donors vs % related donors. remission status was detected in % of patients whereas % had active disease pre-transplant. stem cell sources were as follows: % peripheral blood, % bone marrow, % cord blood, % bone marrow plus peripheral blood. the most frequent fab subtype was aml-m . patients were grouped by hla-c status: % c /c homozygote, % c /c heterozygote and % c /c homozygote. the frequency of hla c donor/recipient mismatch allo-hscts was %. relapse was detected in % of patients. the relapse risk was significantly lower in c /c homozygote patients compared to c /c homozygotes ( % vs %, p = . ). lfs was similar between c /c homozygote group and c /c homozygote group (p = . ) (figure ). in multivariate analysis (age, sex, remission status, related/ unrelated transplant, aml subtype), lfs was increased by pre-transplant remission status (p year). there was no difference detected between -years os in c /c homozygote group and other groups ( % vs %, p = . ) (figure ) . when similar analysis were repeated with donor hla type results were not significant. our results confirm two earlier published reports on aml and all. even in the absence of kir genotyping, hla group c has a protective effect. if hla matched donor is not possible a donor-recipient hla-c mismatch favoring c to c may be preferable. disclosure of conflict of interest: none. immunomodulatory kits do not induce aml-blasts' proliferation ex vivo: ipo- is an appropriate and reliable marker to detect and quantify proliferating blasts c plett, dc amberger, a rabe, d deen, z stankova, a hirn, y vokac, j-o werner, j schmohl, d krämer, a rank, c schmid and h schmetzer aml-blasts can be converted to dcleu by immunomodulatory 'kits' (ex vivo). t-cells' energy can be overcome after stimulation with dc/dcleu and results in antileukemic reactivity. a potential induction of blast-proliferation (e.g. by immunomodulatory kit-application in vivo) in aml-pts has to be excluded. kits containing combinations of gm-csf with - additional factors (pge- / , picibanil, ifnα, tnfα, calciumionophore) were studied with respect to the generation of dc/dcleu from blasts, mediation of antileukemic reactivity (after dc/dcleu-stimulation) and with respect to their potential to induce blast-proliferation in a whole blood (wb) culture-system. we studied different markers (ipo- , ki- , cd ) and quantified blast proliferation before/after culture. we correlated findings with (ex vivo) antileukemic functionality, with disease-entities and the course of the disease. dc-generation: we could generate dc/dcleu regularly from wb culture from aml-pts. detection of blast proliferation: Ø . % (range - %) of uncultured blasts expressed ipo- , . % ( - %) cd , . % ( - %) ki- . induction of blast proliferation: pooling all results we found lowest amounts of proliferating blasts after culture with kit i (gm-csf+picibanil, % ± . ), kit k (gm-csf+pge , . % ± . ), kit m (gm-csf+pge , . % ± . ). amounts of proliferating blasts were lower compared to uncultured cells. highest expression of proliferating blasts was found with ipo- followed by cd and ki- .we found few individual aml-samples with increased blast-proliferation after ex vivo kit-culture. antileukemic activity: t-cells stimulated with dcleu (generated with kits) improved antileukemic activity. correlations between blast-proliferation and antileukemic activity will be presented. clinical correlations: pts with bad (vs good) cytogenetic risk were characterized by higher proportions of proliferating blasts in uncultured blasts; in some pts with iron-deficiency anemia (ida) proportions of cd +unculured blasts were lower than of ipo- /ki- + blasts. ipo- is a stable marker to be used to quantify proliferating blasts in aml-pts. cd is also a good marker, although not suitable for some pts with ida, ki- is no reliable marker for every given pt. subtypes of pts correlated with proportions of proliferating blasts. in general kit treatment of blasts did only exceptionally induce blast proliferation ex vivo. in general lowest risk for blast proliferation was seen after culture with kit i, k and m. t-cellstimulation with dc/dcleu generated after kit-treatment resulted regularly in antileukemic reactivity. we conclude that an in vivo treatment of aml-pts with kits i, k or m might be safe (no induction of blast proliferation). disclosure of conflict of interest: none. the occurrence of additional cytogenetic abnormalities (acas) is common in philadelphia chromosome-positive acute lymphoblastic leukemia (ph+ all), but is of unknown significance in the tyrosine kinase inhibitor era. recent study [aldoss et al., ] has revealed the acas appear to have a significant deleterious effect on outcomes post-hsct in adult ph+ all patients only. we retrospectively analyzed data from adult and pediatric patients with ph+ all who had undergone allogeneic hematopoietic stem cell transplantation (allo-hsct) at our university between and . among patients with ph+ all, patients had available data on conventional cytogenetics before allo-hsct. all patients and transplant characteristics are listed in table i . thirty-three of patients ( %) had isolated t( ; ). acas were revealed in / ( %) pts, including / ( %) pts with ⩾ cytogenetic abnormalities (with complex karyotype, ck). the median follow-up was ( - ) days. overall survival (os) and event free survival (efs) were % ( % ci - ) and % ( % ci - ) at years, respectively. in univariate analysis, prognostic factors associated with increased os and efs were donor type (match related/match unrelated vs haploidentical; p = . for both), the disease status at transplant (cr vs beyond cr ; p = . , only for efs), acas (aca-vs aca+; p = . , only for os) and, especially, the complex karyotype (ck-vs ck+; p = . , only for os) (figure ). multivariate analysis showed that the independent prognostic factors for os and efs remained the complex karyotype (hr- . , % ci, . - . ; p = . ) and disease status at transplant (hr- . , % ci, . - . ; p = . ), respectively. the study demonstrates the acas and disease status at allo-hsct to be independent prognostic factors not only for adult, but for pediatric ph+ all patients too. up to % of newly diagnosed acute myeloid leukemia (aml) patients (pts) present initially with hyperleukocytosis consequently placing them at increased risk for morbidity and mortality during induction. , whereas early publications have indicated that hyperleukocytosis is an adverse prognostic factor associated with poor long term outcome, it is currently unknown whether hyperleukocytosis still retains prognostic value for aml patients undergoing allogeneic stem cell transplantation. furthermore, it is unknown whether hyperleukocytosis retains prognostic value when modern molecular markers such as flt and npm are accounted for. we hypothesized that hyperleukocytosis at initial diagnosis is still an independent adverse prognostic factor influencing long term outcome in aml pts undergoing allogeneic stem cell transplantation. we performed a retrospective analysis using the multicenter registry of the acute leukemia working party (alwp) of the european society for blood and marrow transplantation (ebmt). pts included in the analysis were over years of age, with de-novo non-m aml, a presenting white blood cell count of over k, with an hla matched related or unrelated donor, transplanted between and . clinical outcome indices of hyperleukocyotosis pts namely, non-relapse mortality (nrm), graft versus host disease (gvhd), relapse incidence (ri), leukemia free survival (lfs), overall survival (os) and gvhd-free/relapse-free survival (grfs) were compared to a cohort of pts without presenting leukocytosis. multivariate analyses were used to assess whether hyperleukocytosis was independently associated with ri, nrm, os, lfs, and grfs. age, gender, number of chemotherapy inductions, cytogenetics, donor type, fms-like tyrosine kinase- (flt ) status, nucleophosmin (npm ) status, and conditioning intensity were covariates for regression modeling. a cohort of pts with hyperleukocytosis ( patients with wbc over k and less than k, and patients with wbc over k) was compared to pts without hyperleukocytosis. pts with hyperleukocytosis were younger, had an increased rate of favorable risk cytogenetics, were more likely to be flt and npm mutated, and had an increased rate of myeloablative conditioning. on univariate analysis pts with hyperleukocytosis had an increased rate of ri ( % vs . %, p = . ), and decreased incidence of grfs ( . % vs . %, p = . ). in multivariate regression analysis, hyperleukocytosis was significantly associated with increased ri (hazard ratio [hr] of . , % confidence interval [ci], . - . ; p = . ), s poorer lfs (hr of . , % ci, . - . ; p = . ), decreased grfs (hr of . , % ci, . - . ; p = . ), and poorer os (figure ) (hr of . , % ci, . - . ; p = . ). image/graph hyperleukocytosis at initial presentation retains a significant prognostic role for aml patients undergoing allogeneic stem cell transplantation even in the current era of advanced molecular prognostication. outcome after hematopoietic stem cell transplantation for philadelphia-positive aml: relatively favorable outcome in patients allografted in first complete response; a survey from the acute leukemia working party of the european society for blood and marrow transplantation (ebmt) v lazarevic , m labopin , w deipei , i yakoub-agha , a huynh , p ljungman , n schaap , d blaise , jj cornelissen , n maillard , p pioltelli , t gedde-dahl , s lenhoff , m houhou , j esteve , m mohty and a nagler , aml with t( ; ) and bcr-abl rearrangement (ph-pos aml) is a very rare aml subtype, recognized as a new provisional entity in the recent who classification. the role of stem cell transplantation (sct) in the era of abl tyrosine-kinase inhibitors (tkis) is mostly unknown. we analyzed long-term outcome in patients ⩾ years after allogeneic or autologous sct performed between - in ebmt centers responding to a designated survey. patients with blast crisis cml and philadelphia-positive all were excluded. primary end-point was os. secondary end-points were nrm, acute gvhd, chronic gvhd, lfs, ri, and the effect of tki on outcome. patients (median age, years, range: - ; males and females) with ph-pos aml undergoing sct (allogeneic, ; autologous, ) were identified. median wbc count at diagnosis was x /l ( . - ) and % had splenomegaly (data missing on patients). translocation t( ; ) was the sole abnormality in patients ( . %). the majority of the patients received one or two courses of chemotherapy before transplant and % attained cr after one course. the majority ( %) received a tki (mostly imatinib, / ) before transplant, with a median period of exposition of days (iqr - ), while ( %) received tki after the transplant either as maintenance (n = ) or treatment for relapse (n = ). at time of transplant, patients were in complete response (cr - , including all autosct; cr - ) and the remaining patients were allografted in advanced phase. among patients with available information, achieved a mrd negative status at transplant (ratio bcr-abl/abl o ).regarding allosct, conditioning regimen was myeloablative (mac) in / ( %) patients o years, while in patients years received a reduced intensity regimen (ric) and mac. cell source was peripheral blood stem cells in and bone marrow in allogeneic transplants. the donor was a hla matched sibling (msd) in cases and unrelated (ud) in , amongst whom were / and were / hla matched, respectively. in the patients undergoing autologous sct the majority received busulfanbased conditioning (n = ) and peripheral stem cells (n = ). median follow-up was . . %, . %, . %, and . %, respectively. by the univariate analysis, age ( o vs ⩾ ) was associated with ri ( -yr: . vs %), lfs ( -yr: . vs . %, and grfs ( -yr: . vs . %), whereas mrd-negative status before allosct was associated with an improved grfs ( . vs . %). in the patients autografted, ri, nrm, lfs and os at -year was % ( . - . ), %, % ( . - . ), and . % ( - ), respectively. outcome of patients with ph-pos aml who received allosct in cr in recent years was relatively favorable, especially among younger patients, probably reflecting the beneficial effect of tki. disclosure of conflict of interest: none. outcome of allografting for aml-cr is equivalent across the bsbmt and ebmt and is associated with encouraging os and dfs across all age groups j byrne, j perry , k kirkland , r pearce and g jackson bsbmt and newcastle university and freeman hospital, newcastle relapsed aml has a very poor prognosis with a high mortality, even if a second cr is achieved. the only curative treatment is with an allogeneic hsct but allografts for aml in cr are considered to have a worse outcome compared to those performed in cr , especially in older patients for whom this therapy may not be considered. the bsbmt undertook a bench-marking study analysing the outcomes for all patients allografted for aml-cr from to . the uk outcomes from paediatric and adult patients were compared to non-uk patients transplanted for the same indication reported to the ebmt during the same period. allogeneic transplants for aml-cr represent an important part of any allograft program and numbers referred for allograft were stable between - in both programs. the median age of patients was . yrs and . years in the bsbmt and ebmt cohorts respectively, with % and % of patients aged o years and % and % aged years in the groups. the length of first remission was missing in many of the ebmt registrations so time from diagnosis to transplant was used as a surrogate for this and was similar in both cohorts ( m and m respectively). similarly the presence of comorbidities was poorly reported in both databases but was similar. the bsbmt cohort included fewer patients undergoing ric conditioning protocols ( % vs %), fewer sibling transplants ( % vs %) and more pbsc allografts ( % vs %). transplant related morbidity and mortality were similar across the two cohorts (bsbmt v ebmt) with rates of severe acute gvhd (grade iii and iv) % v %, limited chronic gvhd % v %, relapse at year % v % and death in cr at year % v %. chronic gvhd (both limited and extensive) appeared more common in the bsbmt cohort ( % v %) although reporting of cgvhd was more comprehensive and complete within the bsbmt registry and may be under-reported in the ebmt registry. outcomes were excellent in both cohorts with outstanding rates of leukaemia free survival in this high risk cohort at day (bsbmt v ebmt) % v %, at year % v %, at years % v % and at years % v %. although os and lfs was significantly shorter in patients aged years, at % and % the results in this high risk age group are acceptable and warrant its continued use. multivariate analysis of the combined cohorts showed that age at transplant ( o yrs/ - / yrs), time from diagnosis to transplant and the presence of agvhd were important factors affecting dfs. risk factors for relapse included the type of conditioning used, presence of agvhd and time from diagnosis to transplant, whereas those for trm included age, agvhd, source of stem cells and time to transplant. there was no significant difference in outcomes between the bsbmt and ebmt for this indication. outcomes for patients allografted for aml-cr are excellent and appear superior to those reported for patients not undergoing an allograft in both the bsbmt and ebmt cohorts. the os and dfs observed are comparable to those reported for allografts in aml-cr and, although this study has not considered outcomes for patients who did not achieve a nd cr, it nevertheless supports the practice of risk stratification of aml patients such that only high risk patients are offered an allograft in cr with the remainder being offered an allograft as salvage after relapse. disclosure of conflict of interest: none. to evaluate the efficacy and safety of intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-hsct) for atl. a total of evaluable atl patients treated at our center from to are retrospectively analyzed. the htlv-i proviral dna load in peripheral blood mononuclear cells using pcr assay was developed, in comparison with htlv- tax protein expression measured by western-blot, to confirm the diagnoses and monitor the disease control. patients were male and patients were female. median age was . (range - ) years. all obtained patient samples were subjected to flow cytometric examination and karyotype analysis. patients received chemotherapy as the induction therapy while quit at the time of diagnosis, with da-edoch regimen while with other regimens such as chop, vcap and amp. da-edoch regimen is a variation of dose-adjusted epoch regimen with the replacement of prednisone ( mg/m per day) by dexamethasone ( mg/m per day). before the conventional regimens bucy followed by prophylaxis donor lymphocyte infusion, both received a course of salvage chemotherapy including fludarabine and cytarabine for days registered on http://clinicaltrials.gov (nct ). the gvhd prophylaxis consisted of atg, csa and mtx. the patient characteristics, therapeutic effect and survival data were collected. all patients came from the coastal area in the south-east of china. subtype classification of these atll were acute, lymphoma and chronic type. the main manifestations were characterized with cutaneous and respiratory involvement, hepatosplenomegaly, lymphadenopathy and the laboratory abnormalities as leukocytosis with atl cells, hypercalcemia and elevated serum ldh. typical morphological characterisitic of "flower cells" were observed in . % cases and most of the atll cells are cd +cd -. chromosomal abnormalities were detected in cases. all patients who didn't receive da-edoch regimen died of disease progress, while among patients with da-edoch regimen, achieved cr, pr and died. with a median follow-up of . ( - . ) months, patients respond to da-edoch are still alive. patients in cr achieved successful engraftment with complete donor chimerism in one month post haplo-identical transplant. both were received prophylaxis donor lymphocyte infusion and the immunosuppressive agents were abruptly discontinued for induction of a graft-verus-atl (gvl) effect. they keep remain alive and disease free longer than years so far without severe graftversus-host (gvhd), and the htlv- proviral dna became undetectable after allo-hsct. conclusion: it shows great promise of da-edoch regimen followed by allo-hsct to the long-term cure of atl with apparent clearance of the virus. haplo-identical transplantation can be an alternative option for the atl patients without increasing non-relapse mortality. [p ] disclosure of conflict of interest: none. in the absence of an hla-matched related donor or a good matched unrelated donor in time, haploidentical stem cell transplantation (haplo-sct) is an option for patients requiring an allogeneic hematopoietic stem cell transplant. substantial progress has been made in the last two decades with a dramatic improvement in patient outcomes, with some groups reporting preliminary beneficial effects similar to the ones in hla matched unrelated donor and cord blood transplant. several strategies have been adopted through the years for the procedure. the two strategies used in haplo-sct are ex vivo t-cell depletion and t-cell replete transplantation. the latter can be done with a combination of immunosupressive drugs ( beijing approach) or with post-transplantation highdose cyclophosphamide (post-cy). due to of its lower cost, feasibility and practicality, post-cy has become the most often used platform for haplo-sct in the majority of allogeneic transplantation units worldwide. we analyzed our experience in haplo-sct, since the first one in march to the last one that has just been done in october . we collected all complications reported, also mortality related to treatment and to the disease. we analyzed the overall survival (os). transplants were treated, with different sct indications, the most common being acute myeloblastic leukemia (n = , %), the rest of indications are exposed in figure . all our patients, independent of the conditioning receive post-cy as t cell depletion measure and stem cells were collected from peripheral blood. age at the time of transplant was . years, % were males, % females, the rest of patient characteristics are listed in table . in our series the treatment related mortality (trm) was low with only patient ( %) that died before the day + . as complications, we reported % of hemorrhagic cystitis, % of sinusoidal obstruction syndrome, % reports systemic inflammatory response syndrome, % of citomegalovirus (cmv) reactivation. neutrophils graft is . days (r = - ) and the platelets graft is . days (r = - ). in our series we haven't reported any case of graft failure, one of the transplantes the patient had antihla antibodies, this was treated with a plasmapheresis previous the stem cell infusion, and was infused with a high number of cd + cells ( × /kg), no graft problems, and has had no complications since then with the graft. the os for the whole group is months, with a median not reach at months, with patient's dead at time of analysis. two patients had a relapse after the haplo-sct ( %), both of them received lymphocyte donor infusion, sadly, neither of them responded. the haplo-sct procedure is been adopted by many centers for high risk hematology malignancies, mainly because the fast availability of donors, and because of the preliminary results that have been reported place it as good as the unrelated donor or cord unit transplant. our center is getting experience in these types of transplants, and our results reflect similar outcomes as larger studies. with longer follow ups we will be able to keep the trend of good results both in procedure safety and disease efficacy. os, toxicity and trm are expected for these high risk malignancies. in overall, the haplo-sct seems a reasonable technique that is reflecting in our short series, the results being reported in studies worldwide at bigger scale. disclosure of conflict of interest: none. complex karyotype, the presence of flt- itd and losses of genetic material in chromosome , are all considered high risk markers in aml. patients bearing these abnormalities could undergo a myeloablative allogeneic transplantation in cr whenever this is possible, although this could significatly reduce the chances for cure in older patients due to increased transplant related mortality. ric allografts could be performed in older patients in order to overcome the deleterious effects of these individual abnormalities but its effect still remains controversial in the high risk group. between and , a total patients [ males, females, mean age . ( - )] received a ric allograft ( from fully matched unrelated donors, and from an identical sibling) for high risk aml in transplant centres. high risk disease was classified according to their response to treatment, the presence of complex karyotype, the presence of individual cytogenetic/molecular abnormalities or a combination of these. in particular, patients presented one single karyotype abnormality and presented three or more. ten patients presented alterations of chromosome and patients presented flt- itr. the conditioning regimes included fludarabine in all cases, together with melphalan and campath ( patients), busulphan and campath ( patients), busulphan and thiotepa ( patient), melphalan ( patients), busulphan with and without atg ( patients) total body irradiation ( cgy, patients). graft versus host disease prophylaxis was ciclosporin for patients receiving alemtuzumab or atg but for patients who had a t-replete allograft ciclosporin and low dose methotrexate was the preferred prophylaxis. all but four patients were transplanted in cr patients were followed-up for a mean . months (range - ). thirty-one ( %) patients remain alive. the causes of death ( cases) include relapse or progression of the original disease ( ), transplant-related causes ( ) and unknown in cases. the influence of the genetic abnormalities on survival was analysed, showing there were no significant differences between patients with normal karyotype, single chromosomal abnormalities and two or more abnormalities. likewise, the kaplan-meier survival analysis of patients bearing flt- itd was not significantly different to the rest of the cohort (p = . ; / died, with only one case being related to relapse). patients bearing chromosome abnormalities (with or without other chromosomal aberrations) had a comparable, not significantly (p = . ) different survival to the rest of the cohort: / patients bearing this abnormality died although, most interestingly, none of these deaths were related to relapse. our results indicate ric allografts can provide an adequate consolidating effect in hr aml with complex karyotype, alteration of chromosome or flt- itr, yielding clinical results that are comparable to those obtained in patients with aml allografted for other indications. this is particularly important as these alterations are more frequent in patients whose age prevents them from having myeloablative grafts. disclosure of conflict of interest: none. early detection of inapparent replicative human cytomegalovirus (hcmv) infection together with its preemptive antiviral treatment has led to a marked reduction of life-threatening hcmv disease after allogeneic hematopoietic stem cell transplantation (allosct). we first reported in a retrospectively performed study that hcmv reactivation is associated with a reduced risk for relapse in patients with aml after transplant. now, we evaluate the impact of early hcmv replication on the risk for leukemic relapse in patients with aml after t cell depleted transplantation in a prospectively performed observational study (registration trial drks ). between january and march we enrolled patients with aml in this trial who were consecutively transplanted at the university hospital of essen. patients received a myeloablative regimen (tbi based conditioning n = , chemotherapy based conditioning n = ) and patients a ric (n = chemotherapy based regimen). patients were transplanted in .cr (n = ), .cr (n = ) or more progressive disease stages (n = ) from hla-identical sibling donors (n = ) or hlaidentical unrelated donors (urd) (n = ) or mismatched unrelated donors (n = ). patients who received a second transplant were excluded from the study. the median age of patients was years (range - ) and that of the donors years (range - ). gvhd prophylaxis was performed with mtx and csa, or csa and mmf with (n = ) or without atg (n = ) ( - mg total dose). the incidence of acute gvhd grade - was statistically not different in both groups ( % versus %). hcmv-reactivation (hcmv-r) detected by pcr occurred between and days (median days) after allo sct. only patients surviving day after transplant were included in the study for estimation of relapse incidence (cir) or overall survival (os). hcmv status of recipients (r) or donors (d) were in % r-/d-, % r-/ d+; % d+/r-and % r+/d+. patients with a documented hcmv-r had a cir at -year after transplant of only % as compared to % in patients without a hcmv-r (p = . ). estimates for -year os were in favor for patients with hcmv-r ( % for patients with hcmv-r versus only % for patients without hcmv-r), but this did not achieve statistical significance. non-relapse mortality was greater in patients with hcmv reactivation ( % versus %, ns) a substantial and independent reduction of relapse risk associated with early hcmv replication was confirmed by multivariate analysis including competitive factors as unfavorable cytogenetics according to eln, advanced disease stages of aml, hla-identical donor versus mismatched donor, sibling versus unrelated donor, presence of acute gvhd grades ii-iv, chronic gvhd, and hcmv-r [(hazard ratio: . , % ci: . - . , p o . ) for occurrence of hcmv-r]. the final result of this first prospective performed study confirms an independent advantageous effect of early hcmv replication on the leukemic relapse risk in patients with aml after transplant. disclosure of conflict of interest: none. . primary engraftment of wbc and platelets was achieved in pts, one patient (pt) died at day + after hsct, and one had a secondary graft failure. a median time to wbc engraftment was days ( - ), to platelets - days ( - ). cumulative incidence (ci) of acute gvhd (agvhd) grade ⩾ ii was % ( % ci: - ): from haplo- %( - ), from mud % ( - ),p = . . ci of agvhd iii %( % ci: - ): haplo vs mud - % ( % ci: - ) vs %( % ci: - ), respectively,p = . . ci of chronic gvhd (cgvhd)- % ( % ci: - ). ci of agvhd was significantly lower in a group with regimen ( - ) of gvhd prophylaxis: % ( % ci: - ) vs %( % ci: - ), po . . regimen was also effective in prevention of cgvhd: ci at year after hsct was % vs %, p = . . Сi of trm was %( % ci: - ): haplo- %( - ), mud - % ( - ). early mortality (before + -day) was relatively low (n = ): pt died from bacterial sepsis; two pts died due to adv infection. thirteen pts died after days: pts relapsed and died due to complications of second hsct; bacterial sepsis in pt and viral infection (adv and cmv) in pts ( with extensive chronic gvhd). ci of relapse was % ( % ci: - ) at year: from haplo- % ( % ci: - ), from mud- % ( % ci: - ),p = . . event-free survival (efs) at years was % ( % ci: - ): haplo - %( % ci: - ), mud - % ( % ci: - ), p = . . os was % ( % ci: - ) at years: haplo- % ( % ci: - ), mud - % ( % ci: - ), p = . . relapse-gvhd-free survival at years was different among recipients of haplo and mud hsct: %( % ci: - ) vs % ( % ci: , p = . . we confirm that the depletion of tcrαβ+/cd + depletion from the graft ensures high engraftment rate and acceptable transplant-related mortality in pediatric aml pts. there is a trend towards better efs for haploidentical transplantation. gvhd prophylaxis including ratg/rituximab/bortezomib improves gvhd control s in recipients of tcrαβ+/cd +depleted grafts in comparison to hatg/tacro/mtx apparently without loss of anti-leukemic activity. disclosure of conflict of interest: none. results of t-cell depleted haploidentical stem cell transplantation in adults with acute leukemia improve with time: a study from the acute leukemia working party of the european society of blood and marrow transplantation (ebmt) s simona , , m labopin , a ruggeri , , , a velardi , f ciceri , j maertens , l kanz , f aversa , d bron , d bunjes , m mohty , and a nagler , t cell-depleted (tcd) transplants from haploidentical donors are increasingly used in the absence of a hla full-matched donor for patients (pts) with high risk acute leukemia (al). progress has been made in optimization of conditioning regimens and post-transplant cellular therapy to potentiate the graft-versus-leukemia effect with no graft-versus-host disease (gvhd). however, relapse incidence (ri) and non relapse mortality (nrm) remain the main obstacles for pts outcomes. we report adults with de novo al, given a tcd haplo from to . to analyze the effect of transplant period on tcd haplo outcomes, pts were analyzed in two separate groups: - (n = ) and - (n = ). tcd haplo were performed in ebmt centers. median follow-up was months with no difference according to transplant periods. median age was different between groups, being and years respectively (p = . ). the majority of pts had acute myeloid leukemia (aml) ( % vs %, p = . ) and disease status at haplo was first complete remission (cr ) in % and % of pts respectively (p = . ). pts transplanted before had more frequently a karnofsky performance status o % ( % vs %, p = . ). conditioning was myeloablative in % and % of tcd haplo before and after (p = . ), mainly based on fludarabine(flu)-tbi, flu-melphalan-thiotepa or cyclophosphamide-tbi. as for ric it was flu-melphalan-thiotepa, flu-tbi or cyclophosphamide-tbi. the cumulative incidence (ci) of neutrophil engraftment, grade ii-iv acute gvhd and chronic gvhd were not different according to transplant period, being % and %, p = . ; % and %, p = . ; % and %, p = . , respectively. in the whole population -year ri and nrm were % and %, with no difference before and after ( % vs %, p = . ; % vs %, p = . , respectively). ri was % before versus % after . the main cause of nrm was infection, with no difference over time ( % vs %, p = . ). in multivariate analysis, disease status was the only factor associated with ri (hr . , % ci . - . , p = . ). tcd haplo after (hr . , % ci . - . , p = . ), younger age (hr . , % ci . - . , p = . ) and ric (hr . , % ci . - . , p = . ) were independently associated with lower nrm. -year os was % with a marked improvement for tcd haplo performed after ( % vs %, p = . ), while lfs and grfs were % and %, respectively. according to disease status, -year lfs, os and grfs were higher for pts transplanted in cr ( % vs %, p o . ; % vs %, p = . ; % vs %, respectively p = . ). in multivariate analysis, tcd to further establish the role of haplosct in high-risk aml, we performed a retrospective matched-pair comparison of hlamatched sct vs haplosct/ptcy in two german centers. highrisk aml was defined by any of the following criteria: refractory or relapsed aml, secondary aml, or genetic aberrations classified as intermediate-high or adverse accordingly to the eln classification. all consecutive adults, who fulfilled ⩾ of these criteria before either hla-matched or haplosct/ptcy were included (n = ). recipients of haplosct were pair-matched with patients receiving a matched donor sct. matching variables were ( ) stage at sct, ( ) genetic subgroups accordingly to eln, and ( ) age (± y). patients (pts) undergoing haplosct/ptcy could be successfully pairmatched (p = . for stage and genetic subgroup, . for age) with recipients of matched sct ( family, unrelated sct). within the entire cohort, median patient age was y ( - ). at start of conditioning, % of patients were in cr, % had refractory, % had relapsed, and % had untreated disease. genetics were favorable ( %), intermediate i ( %), intermediate ii ( %) and unfavorable ( %). hla-matched sct was uniformly performed following flamsa-ric. recipients of haplosct/ptcy ( %) received cytoreductive chemotherapy with flamsa (n = ) or clofarabine (n = ) before ric was started. median follow-up among survivors was months. overall cr rate at d+ was %, patients suffered from refractory disease or early death, (n = each). overall-survival (os) for the entire cohort was %/ . % at y/ y from sct. the corresponding y/ y leukemia-free survival (lfs) was . %/ . %. median time to engraftment was . and . days after matched and haplosct, respectively (p = . ). with respect to outcome, no difference was observed between the two groups: os at y/ y was . %/ . % after matched sct, and . %/ . % after haplosct/ptcy (p = , , figure ). lfs at y/ y was . %/ . % within the hla-matched group and . %/ . % within the haploidentical group (p = , ). recipients of haplosct showed a higher incidence of agvhd ⩾ ii°( % vs %, p = . ), as well as a trend towards increased y-nrm ( % vs %, p = . ), whereas y-relapse rates were comparable ( % after haplosct/ptcy vs % after matched sct, p = . ). relapse was the most frequent cause of death in both cohorts, main causes of nrm were gvhd and infections (no difference between the two groups). allogeneic sct following sequential conditioning can achieve excellent results in high-risk aml. in our study, results after haploidentical transplantation were comparable to those obtained after hla-matched sct. hence, haplosct/ptcy following sequential conditioning can be considered as a reasonable option for patients with high-riaml. [p ] disclosure of conflict of interest: none. a significant proportion of patients with acute myeloid leukemia ( aml) will either be refractory to initial chemotherapy or will suffer refractory relapse. the role of allogeneic transplantation (hct) in active disease is contentious. there is a growing body of literature that sequential chemotherapy, pioneered by the german flamsa regimen, followed by ric hct is a safe and efficacious modality in these patients, and there have been numerous modifications of this regimen, especially as amsacrine is not widely available. fludarabine, cytarabine and etoposide (vp ) (flav) have been reported as an an effective salvage regimen. here we report on single center outcomes of a variation of the flamsa regimen, substituting amsacrine for etoposide with mainly myeloablative conditioning. patients were consented for a clinical protocol if they had aml that was refractory to cycles of chemotherapy, or cycle and considered to be at risk of complications of a second cycle, and if they had a matched related donor. patients with myelodysplasia received flav if they had high or very high risk cytogenetics. cytoreductive chemotherapy consisted of fludarabine mg/m / day × days, cytarabine g/m /day × days, etoposide mg/m /day × days, commenced simultaneously. after days of rest, conditioning chemotherapy consisted of fludarabine mg/m × days and and iv busulfan . mg/ m q hours; the number of busulfan doses varied between - , depending on patient comorbidity. ten patients ( %) received myeloablative doses of busulfan. patient received doses of atg at . mg/m /day on day - and - . patients received gcsf mobilized peripheral blood hematopoietic cells. post-transplant gvhd prophylaxis was csa and mmf. csa was tapered from day+ and stopped at day + in the absence of gvhd. mmf was discontinued between day + and day + . donor lymphocyte infusions were collected for planned prophylactic dli. thirteen patients received a flav-sct between march and october . the median age was ( - ); male:female ratio was( : ). patients ( %) had aml and ( %) pts had mds. all patients had detectable disease prior to flav. the median time for plt engraftment was days ( - ). the median time for anc engraftment was days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . cytogenetic cr rate on a day - bone marrow was %, and morphological cr was %. patients ( %) developed veno-occlusive disease. acute gvhd grade ii-iv occured in pts ( %). ( %) patients developed chronic gvhd. death was due to disease relapse in ( %) and nrm in ( . %) patients, resulting from h n pneumonia. patients ( %) received dli for post transplant relapse, and one of these is in molecular remission. at a median follow up of . months post transplant ( . - m), year dfs was %. the year and year os was % (± %) (figure ). our experience, consistent with published data, demonstrates that for patients with active aml refractory to chemotherapy, transplantation is an effective modality of disease control and may be the only curative therapy in a significant proportion. etoposide appears to be a suitable alternative to amsacrine. our patients tolerated busulfan at myeloablative doses, and this may be required for adequate disease control. our report is limited by small numbers and relatively short follow-up, but is encouraging enough for us to continue offering flav hct for these high-risk patients. [p ] disclosure of conflict of interest: none. sequential high-dose chemotherapy reinduction followed by haploidentical transplantation in acute leukemias l brunello , , cm dellacasa , l giaccone , , e audisio , d ferrero , , s d'ardia , b allione , s aydin outcomes after t-cell replete haploidentical stem cell transplantation (haplo hsct) have been encouraging and haplo hsct has become an alternative option for patients without a hla-identical related or unrelated donor. , as previously published, the sequential use of intensive chemotherapy and allogeneic transplantation represents a possible approach to the treatment of high-risk acute myeloid leukemia (aml). , between and , acute leukemia (al) patients received sequential therapy (s.t.) consisting of high-dose chemotherapy reinduction and haplo hsct during the chemotherapy-induced neutropenia. median age at transplant was years (range: - ); median number of previous therapy lines was (range: - ) and / ( %) patients had received a previous allogeneic hsct. twelve and out of patients had de-novo aml and secondary aml, respectively; furthermore two patients presented blastic crises of chronic myeloid leukemia. all patients had active disease at the time of s.t. and median marrow blast count before reinduction was % (range: - %). hematopoietic cell transplantation comorbidity index was ≥ in / patients ( %). all patients received high-dose cytarabine (≳ g/sqm) containing regimens as reinduction therapy. the conditioning regimen was started after a median of days from the end of reinduction (range: - ). sixty-eight percent of patients ( / ) were conditioned with a myeloablative regimen (thiotepa tot. mg/kg, busulfan tot. . mg/kg, fludarabine tot. mg/ sqm), while / ( %) patients received a non-myeloablative conditioning. bone marrow was used as stem cell source in / ( %) patients. graft-versus-host disease (gvhd) prophylaxis consisted of post-transplant cyclophosphamide with calcineurine inhibitors and mycophenolic acid. all patients engrafted but one, who was rescued with a second haplo-hsct with peripheral blood stem cells from the same donor. median day of neutrophil recovery was day + (range: - ). median follow-up of survivors was . years (range: . - . ); -year overall and event-free survivals were % and %, while -year relapse incidence and non relapse mortality were % and %, respectively. overall cumulative incidences of acute and chronic gvhd were % and % at day + and + . among patients who developed gvhd, grade iii-iv acute gvhd and moderate-severe chronic gvhd were observed. at . years post haplo-hsct, % of patients are alive and disease free. in our cohort of heavily pre-treated and high-risk patients, s.t. with a myeloablative conditioning was safely used to reduce leukemic burden pre-transplant and enhance graft-versus-leukemia effects. only the prompt availability of a haploidentical donor allowed to implement this treatment modality. though small the patient cohort, our findings suggest that transplant-related toxicity was acceptable and early relapse was the major treatment-failure. however, long-term survival and disease-free rates of % in these very poor prognosis patients are highly encouraging. in elderly patients with acute lymphoblastic leukemia (all) and kinase activating lesions allogeneic stem cell transplantation (allo-sct) is considered to be the only curative option. however, high risk of relapse and non-relapse mortality (nrm) often withholds elderly patients with existing comorbidities from definitive therapy. even though, age alone as the most important eligibility criterion for allo-sct has become less important. a -year old patient with a medical history of adipositas, hypothyreosis, arterial hypertension, hypercholesterolemia and coronary artery disease with stent implantation was diagnosed with common-b-cell-all based on immunophenotype. cytogenetic analysis showed two coexisting clones with an abnormal karyotype of ,xx; t( ; )(q ;q ), + [ ] and ,xx; t( ; )(q ;q ),der( )( ; )(p ;q ) [ ] and no evidence of bcr-abl positive disease using fluorescence in situ hybridization (fish) technique. rt-pcr and sequencing of the fusion transcript was performed to validate the rcsd -abl t( ; )(q ;q ) fusion between exon of rcsd and exon of abl . western analysis of phosphorylated abl and its downstream target crkl was performed to investigate the in vivo activity of dasatinib. clinical monitoring of minimal residual disease (mrd) levels has been performed via rt-pcr of the rcsd -abl fusion transcript followed by nested pcr of the amplicon to detect early relapse or mrd. as positive control the plasmid pcr . -topo_rcsd -abl ( bp) was synthesized encoding rcsd -abl amplicon with the fusion site. our patient was enrolled on gmall elderly ( / ) and treated accordingly. thereby, no sustained remission could be achieved. flag-ida re-induction and study treatment with an oral pi k/mtor inhibitor remained futile. cytogenetics and verification of the rcsd -abl fusion gene prompted salvage treatment with the tyrosine kinase inhibitor (tki) dasatinib as single agent. the in vivo activity of dasatinib was highlighted by a decrease of rcsd -abl amplicon and inhibition of phosphorylated abl and its downstream target crkl was shown. clofarabine and cyclophosphamide complemented treatment and mrd negative remission was achieved due to administration of the bi-specific t-cell engager blinatumomab. consolidation with allo-sct was performed. ongoing remission has been achieved for more than months now. we demonstrate that monotherapy with tki like dasatinib is effective in refractory rcsd -abl positive all. to the best of our knowledge, this is the first elderly patient with rcsd -abl positive all with a sustained and ongoing complete remission. thereby, we suggest allo-sct after successful tki even for elderly patients with existing comorbidities and uncommon cytogenetics. relapse is the most important cause of failure of allogeneic hematopoietic stem cell transplantation (hsct) for flt -itdpositive acute myeloid leukemia (aml). in those cases, treatment with flt tyrosine kinase inhibitors (tki) constitutes a promising clinical approach to induce remission without conventional chemotherapy. forty-eight-year-old woman was diagnosed of aml secondary to myelodisplastic syndrome with npm mutation and internal tandem duplications of the flt gene (flt -itd). after achieving complete remission (cr), she received a sibling allogenic-hsct. four months later, aml relapsed at the medullary level, without central nervous system (cns) involvement, treated with conventional chemotherapy and donor lymphocytes infusions (dli). she achieved second cr and developed chronic graft-versus-host disease (cgvhd). nine months later, she suffered the first extramedullary relapse, at the mammary, cutaneous and probably pericardial levels. there was not medullary involvement. disappearance of the lesions at all levels was achieved with conventional chemotherapy and radiotherapy, and full donor chimerism. eight months later, she referred atypical precordial pain irradiated to the back. cardiac mri was performed in which several masses were visualized in a pericardial sac up to cm in diameter (dec- ). bm was maintained in cr. in study of pericardial fluid, infiltration by leukemic flt positive cells was observed. the patient was not considered candidate for further systemic chemotherapy nor radiotherapy treatment. then, treatment with the flt sorafenib inhibitor was started, by compassionate use, at dosage of mg/ h, which maintains after one year. after first month with sorafenib, pericardial lesions decreased considerably, ranging from cm in diameter to . cm (jan- ). in the subsequent ct controls, progressive decrease of the lesions has been observed and no new lesions have appeared in other locations. in the last ct (oct- ) pericardial thickening is almost non-existent, without new lesions. after one year of treatment, she maintains cr at medullary and extramedullary levels images. in our patient, treatment with sorafenib has achieved sustained control of extramedullary disease, which had escaped the mechanisms of action of conventional chemotherapy, allotransplant and dli. further studies are needed to corroborate the efficacy of flt inhibitors in the control of aml extramedullary disease and in the treatment of relapses after allo-hsct. all pts with tbi-based regimen received rabbit atg. tcrαβ+/cd + depletion of hsct with clinimacs technology was implemented in all cases according to manufacturer's recommendations. the median dose of cd + cells in transplant was × /kg (range: . - . ), tcrα/β- × /kg (range: . - ). primary engraftment was achieved in of pts. ( pts died before engraftment, one received second hsct), the median time to neutrophil and platelet recovery was and days, respectively. early ( day) mortality was %( % ci: - ), -year ptrm- % ( % ci: - ). the three early deaths were due to bacterial sepsis (n = ) and viral infections(n = ), seven late: viral infection in pts (adv = , adv+cmv = , cmv = ), bacterial sepsis in pts and rhinocerebral mucormycosis in pt, all late deaths were associated with cgvhd and prolonged corticosteroid therapy. ci of acute gvhd grades ii-iv and iii-iv was . % ( % ci: . - ), and % ( % ci: . - ), respectively. ci of cgvhd was . % ( % ci: . - ). regimen was more effective in prevention of agvhd ii-iv: ci at year after hsct was % vs , % in regimen , p = . and in cgvhd % vs . %, p = . . ci of relapse at years was % ( %ci: . - . ). two-year pefs(event = death or relapse) was . % ( % ci: - ), -year pos- % ( % ci: - ). in patients, who received tbi-based conditioning pefs was % ( % ci: - ), as compared with treosulfan-based % ( % ci: - ), p = . . median time of follow-up for survivors was . years (range: . - . ). we confirm that the depletion of tcr-alpha/ beta and cd lymphocytes from the graft ensures high engraftment rate and acceptable transplant-related mortality in pediatric all patients. viral infections and leukemia relapse await further improvement of control. all major outcomes were equivalent between transplantation from unrelated and haplo donor. disclosure of conflict of interest: none. the prognosis of patients (pts) with relapsed/refractory acute lymphoblastic leukemia (all), especially after allogeneic hematopoietic stem cell transplantation (allo-hsct), is very poor. therapeutic options for these pts are limited. blinatumomab is a bispecific t-cell engager (bite) antibody construct with dual specificity for cd and cd . bite therapy may help to overcome the resistance to chemotherapy (ct) with minimal toxicity, and may be a bridge to allo-hsct. we analyzed data of pts from hematologic centers in russian federation with relapsed cd positive all, who received bite. the median age was (range: - ) years, ( %) pts o yrs, ( %) pts ≥ yrs. the diagnosis was all b-i (egil) subtype in pts, b-ii-in , b-iii-in pts, and patient had mixed phenotype leukemia (m (fab) and b-all). three ( %) pts had philadelphia positive (ph+) all. in pts it was the first relapse of all, in -second, in -third. thirty pts had isolated bone marrow relapse, pts-combined relapse (bone marrow and extramedullary sites). the bone marrow blast infiltration was o % in pts, % in pts. disease relapse was revealed after ct in pts ( ( %) pts received allo-hsct after the therapy of relapse), after allo-hsct ( -from related, -from unrelated, -from haploidentical donors)-in pts ( pts received second allo-hsct after the therapy). in pts with posttransplant relapse donor lymphocyte infusion (dli) was used in combination with bite. every patient received from to cycles (median ) of bite. complete remission (cr) was achieved in ( %) pts (in ( %) pts it was minimal residual negative remission): in ( %) pts with all relapse after ct, in ( %) pts-after allo-hsct. pts with less than % blasts in bone-marrow at baseline experienced substantially higher response rates compared with patients with % blasts or higher ( % ( / ) vs % ( / )). response rates were similar although the number of relapse- % ( / ) in first relapse, % ( / ) in second relapse and % ( / ) in third relapse. pts with posttransplant all relapse who received bite in combination with dli had higher response rate than pts, who received bite as monotherapy: . % ( / ) vs % ( / ), respectively. one-year os was % ( % ci - %). one-year dfs was % ( % ci - %). grade ≥ hematological toxicity (neutropenia, thrombocytopenia) was observed in ( %) pts, grade ≥ liver toxicityin ( %) pts. five patients ( %) developed toxic neuropathy during the therapy. cytokine release syndrome occurred in ( %) pts. one patient after allo-hsct (but not after dli) developed grade i agvhd. there were no fatal treatment related toxicity. tree ( %) pts who responded to bite had relapse. eighteen ( %) pts died: pts-of disease relapse/ progression. the treatment of relapsed/refractory all with bite is effective and has acceptable toxicity. we demonstrated high efficacy in therapy of posttransplant all relapses, especially when bite was combined with dli, perhaps, due to additional immunological effect of the transplant. disclosure of conflict of interest: none. the mechanism of sorafenib anti-leukaemic effect seen in aml patients relapsing post allohsct involves the augmentation of alloreactivity which includes infiltration of the affected marrow by cd + cells having pd- receptor which presence characterize lymphocytes with antitumour potential multikinase inhibitor (mki) sorafenib is clinically active in acute myeloid leukaemia (aml) patients, especially in those with flt itd who receive allohsct as a part of their primary treatment. to throw some light on the mechanism of this antileukemic post-transplant sorafenib effect we studied the fate of two patients (flt itd-positive, npm -positive) who relapsed ( -year-old male) and ( -year-old female) days post-transplant and their salvage treatment included sorafenib. the multikinase inhibitor ( mg twice daily) was given either together with flag or da + . the response was prompt. the patients became, after completion of the chemotherapy, leukaemia free. both patients continued the sorafenib ( mg twice daily) treatment together with the aml standard maintenance chemotherapy (female case) or without any chemotherapy (male patient, substantial comorbidity and liver toxicity). ( ) . the patients responded well to the therapy and were free of leukaemia for and + months after initiation of the mki treatment (flt itd negative, % chimerism documented in the blood and in the marrow). ( ) . in both patients, and months on sorafenib, skin lesions appeared either in the context of cgvhd, which progressed to a life-threatening level in a male patient or as a photodermatitis-like cheek eruption. histopathology revealed the presence of severe cd + cells infiltration in affected tissues in both patients. ( ) . cd positive lymphocytes colonized the marrow of both patients. these marrows infiltrating cells co-expressed cd (pd- receptor) in proportions which were higher than those seen in the blood ( . % ± . % vs. . % ± . %, p = . ). a similar observation was made for cd +cd + cells ( . % ± . % vs. . ± . %, po . ). . transcriptome analysis of the marrow cells, which addressed the genes involved in lymphocyte activation, revealed the presence of proinflammatory profile which included a higher expression of tlr and il- . ( ) sorafenib given with or without moderate chemotherapy was effective in two patients in maintaining the anti-leukaemic effect of salvage chemotherapy. ( ) this was associated with the presence of alloreactivity (affected tissues infiltration with cd + cells) clinically seen as a severe fatal cgvhd aggravated by sorafenib treatment associated unwanted effects in one cases and with rather mild skin lesions appearing later during the treatment. the outcome of elder patients with acute myeloid leukemia or high risk myelodysplastic syndrome treated with allogeneic hematopoietic stem cell transplantation ch tsai , , division of hematology, department of internal medicine, national taiwan university hospital; tai-cheng stem cell therapy center, national taiwan university and genome and systems biology degree program, national taiwan university allogeneic hematopoietic stem cell transplantation (hsct) is a curative-intent treatment for patients with high-risk hematologic diseases, including acute myeloid leukemia (aml) and myelodysplastic syndrome (mds). both the incidences of aml and mds increase with age and patients elder than years of age were traditionally excluded from hsct because of high possibilities of therapy related morbidity/mortality. recently, with the introduction of reduced intensity conditioning regimens and the improvement of hsct care, more and more elder patients could undergo hsct for consolidation or salvage purposes. however, literature regarding the treatment outcome of elder patients receiving hsct is scarce. patients diagnosed as aml or high risk mds aged equal or more than years were recruited consecutively at national taiwan university hospital. the high risk mds was defined to include myelodysplastic syndrome with excess of blasts- and according to the world health organization (who) criteria. the cytogenetic risk stratification was based on original medical research council classification. from to , a total of patients were enrolled consecutively. the median age was . (range: - ) years and the gender distribution was even. among them, ( . %) patients had high risk mds, ( %) had de novo aml, ( . %) had secondary aml, and three ( . %) had therapy related aml. at diagnosis, four ( . %) patients had extramedullary disease. nine ( . %) had unfavorable-risk cytogenetics, ( . %) had either unfavorable-risk cytogenetics or intermediate-risk cytogenetics but with flt -itd mutations. regarding the pre-hsct disease status, nine patients had the first complete remission (cr), had the second cr, and patients were treatment naive or had refractory disease. the graft-versushost-disease(gvhd)-free/relapse-free survival (grfs) in which events include grade - acute gvhd, chronic gvhd with moderate severity according to cibmtr criteria, relapse, or death of any cause. with median follow-up of months (range: . - . ), the median overall survival (os) for all patients was . months, the relapse-free survival (rfs) was . months, and the grfs was . months. in univariate analysis for os and rfs, high-risk mds was a favorable prognostic factor but secondary or therapy related disease (p = . for os and . for rfs, respectively), unfavorablerisk cytogenetics or intermediate-risk cytogenetics but with s flt -itd mutations (p = . and . , respectively), pre-hsct refractory disease (p = . and . , respectively), and grade - acute gvhd (p = . and . , respectively) were unfavorable prognostic factors. however, for grfs, only unfavorable-risk cytogenetics or intermediate-risk cytogenetics but with flt -itd (p = . ) and pre-hsct refractory disease (p = . ) were unfavorable prognostic factors. in multivariate cox proportional hazards regression analysis for os and rfs, grade - acute gvhd was a significant unfavorable risk factor; for gfrs, pre-hsct refractory disease status was a significant unfavorable risk factor. our results showed that the choice of hsct should not solely based on the age factor and pre-hsct disease status. incorporating cytogenetics and genetic mutation status could risk-stratify elder patients with hsct. further prospective trials are warranted to validate these findings. disclosure of conflict of interest: none. children affected with acute lymphoblastic t cell leukaemia (t-all) and relapse after allogeneic stem cell transplantation (sct) have limited treatment options and a poor prognosis. immune checkpoint inhibitors targeting the programmed death (pd- ) receptor pathway may enhance the graftversus-leukaemia (gvl) effect by blockade of inhibitory signals to t cells mediated by its ligand pd-l . we report a -year old girl with refractory t-all after allogeneic sct, who was treated off-label with the pd- inhibitor pembrolizumab. the girl was diagnosed with t-all ( . g/l wbc, % bone marrow infiltration, cns negative, t ( ; )) and underwent hlahaploidentical bone marrow transplantation from her mother with post-transplant cyclophosphamide since she failed to achieve molecular remission despite an intensified chemotherapeutic regimen. on day post sct, she had a % donor chimerism and decreasing minimal residual disease (mrd) marker (minimal × − ). days post sct she had a molecular relapse with an mrd of × − and a subsequent morphological relapse as well as mixed donor chimerism. further treatment regimens included chemotherapy, intrathecal therapy and four donor lymphocyte infusions (dlis). initially, she displayed a good morphological response to dlis but the leukaemic burden eventually remained stable with an mrd of × − . considering . % pd- expression on cd + t cells in the patient's bone marrow and the encouraging data in other hematologic malignancies an off-label therapy with the pd- inhibitor pembrolizumab - was initiated. the patient and her parents gave informed consent and she received a single dose of pembrolizumab at . mg/kg days after sct. one week after administration of pembrolizumab, the patient developed acute gvhd grade iv of the skin, mucosa, liver, lung, central nervous system and eyes. she had a severe generalized inflammatory reaction with high inflammatory markers, increased hepatic transaminases and lymphocytic infiltration of the liver, cerebrospinal fluid and bronchoalveolar lavage fluid. magnetic resonance imaging (mri) of the brain revealed periventricular white matter lesions and hyperintensities of basal ganglia and bilateral temporal lobe consistent with autoimmune encephalitis. treatment with high-dose corticosteroids, cyclosporine and the anti-interleukin receptor antibody tocilizumab slightly improved her clinical condition. her mrd value significantly decreased to × − two weeks after administration and she achieved a % donor chimerism in bone marrow. despite this promising response her medical condition deteriorated and the severe inflammatory reaction caused fatal multi-organ failure. this is to our knowledge the first report on a remarkable and fast response to pd- inhibition in a patient with pediatric t-all refractory to multiple lines of therapy including allogeneic sct. this case illustrates the potential risk of checkpoint inhibitors to trigger severe gvhd that is not responsive to steroids. induction of inflammatory gvl responses without causing severe gvhd by therapeutic checkpoint inhibition needs to be addressed in future clinical trials. in recent years, there is a remarkable trend in the use of haploidentical-related hematopoietic cell transplantations (haplo-hct) in patients who do not have a hla matched related or unrelated donor. here, we report our single-center experience, in patients who underwent haplo-hct for acute leukemia. between and seventeen consecutive adult patients, seven males and ten females, median age years (range: - years) with high-risk acute leukemia underwent unmanipulated, bm or pbsc transplantation from an haploidentical family donor. eleven patients transplanted for acute myeloid leukemia ( in cr , in cr , in minimal active disease after cr , second trasplant in cr , transformed mds in cr , aml secondary to myelofibrosis in cr ), for acute lymphoblastic leukemia ( in cr , in active disease) and mastcell leukemia (secondary to aml) in active disease. sixteen patients received myeloablative conditioning, and reduced intensity, respectively. in five patients stem cells source was bm, in were g-csf mobilized pbsc. the median infused cd + cell dose was . × (range: . × - . × ). conditioning regimens were: bu-flu-mac (n = ), tbf-mac (n = ), tbf-ric (n = ) the regimens for gvhd prophylaxis were: ptcy as sole gvhd prophylaxis (n = ), mtx-csa-atg (n = ), methylpred-atg-tacrolimus (n = ), atg-csa-mtx-mmf (n = ). sustained trilineage engraftment occurred in patients ( %), two patients died of transplantation-related complications before day after transplantation without myeloid recovery. for patients receiving bm or pbsc grafts, the median time to neutrophils recovery was days (range: - ), and , platelets recovery was days (range: - ). / patients ( . %) and / ( . %) had ii-iv and iii-iv grade of acute gvhd, respectively.the incidence of grade ii-iv cgvhd was %. after a median follow-up of months, / patients ( . %), out patients transplanted in cr , are alive and disease free at , , , months (inclusing the patient transplanted for aml after imf). the -year probability of overall and progression-free survival was % ( % ci, . - . %) and . % ( % ci, . - . %), respectively. causes of death were: sepsis (n = ), fatal agvhd (n = ), pneumonia (n = ), toxicity (n = ), progression (n = ) and relapse (n = ). in our experience unmanipulated bm or pbsc transplantation from haploidentical family donor is feasible approach with high engraftment rates and acceptable trm ( %) and rate of grade iii-iv agvhd, associated with durable remission in a proportion of patients with high-risk acute leukemia, specially in patients with aml transplanted in first remission. it is generally recognized that allogeneic hematopoietic stem cell transplantation (allo-hsct) should not be administrated to patients with severe aplastic anemia (saa) or very severe aplastic anemia (vsaa), when they got active infection. however, without neutrophil, severe infection is usually difficult to control and even fatal. under these circumstances, rapid recovery of neutrophil by allo-hsct might be an alternative to control infection. from january to december , there were young patients received allo-hsct for saa or vsaa at shanghai children's medical center in china. among them, patients ( males and females) with a median age of . years (range: . - . years) received allo-hsct with refractory active infections. refractory active infection was defined as persistent neutropenic fever with nonresponse to standard doses of broad-spectrum antibacterial agents and antifungal agents for more than three weeks, with or without definite focus of infection. prior to allo-hsct, four patients had persistent fever of unknown origin, patients with singlesite infection, and patients with multiple-site infections. sites of infection included lung, sinus, cellular tissue, peritoneum, liver, spleen and skin. the conditioning regimen consisted of fludarabine, cyclophosphamide and rabbit-antithymocyte globulin with or without total body irradiation (tbi) ( - gy). twelve patients were transplanted from mismatched unrelated donors, from matched sibling donors, and from haploidentical donors. sixteen patients received g-csf mobilized peripheral blood stem cells, three patients g-csf mobilized peripheral blood stem cells plus g-csf primed bone marrow stem cells, two patients bone marrow stem cells, and patient umbilical cord blood stem cells. a median of . × /kg mononuclear cells with . × /kg cd + cells were transfused, except the patient who underwent ucbt with a total of . × /kg mononuclear cells and . × /kg cd + cells transfused. eighteen patients achieved recovery of neutrophil and finally control of infections, including one patient who suffered primary graft failure and had autologous marrow recovery. three patients died of infection and one patients died of acute renal failure before recovery of neutrophil. one patient died of pneumonia months after allo-hsct. one patient become thrombocytopenia after allo-hsct. the other patients are all disease-free. there were five patients developing grade i-ii acute gvhd, and patient grade iii-iv acute gvhd. all were cured at last. three patients had localized chronic gvhd and one patient had extensive chronic gvhd. with a median of years follow-up, the overall survival rate and disease-free survival rate are . % ± . % and . % ± %, respectively. allo-hsct could be a feasible way to control infection for children with saa or vsaa in the present of refractory active infections. disclosure of conflict of interest: none. paroxysmal nocturnal hemoglobinuria (pnh) may present hemolysis isolated (classical pnh) or associated with aplastic anemia (aa; aa/pnh syndrome). while classical pnh patients require anti-complement treatment (eculizumab), the treatment of aa/pnh patients should target their underlying aa by immunosuppression (ist), or even bone marrow transplantation (bmt). however, in a few patients clinically meaningful aa and hemolysis may be concomitant, eventually justifying both ist and eculizumab. to date there is no standard treatment for s this rare condition. amongst a large cohort of pnh patients (between and ) at our reference centers, st. louis hospital (paris) and federico ii university (naples), we retrospectively assessed characteristics and outcomes of patients diagnosed with aa/pnh who received intensive ist during or immediately before ( - months) eculizumab treatment. nine patients were identified. eight patients fulfilled the criteria of severe aa, and one had an immunemediated isolated agranulocytosis. since no patient had a hla-matched related donor for bmt, all patients received intensive ist according to institutional guidelines. six out of patients were already on eculizumab treatment at the moment of starting intensive ist (concomitant treatment) whereas patients received ist in the - months (median time of months) before the introduction of anti-complement therapy (sequential treatment). for all patients already on treatment, eculizumab was not discontinued to minimize the risk of rebound intravascular hemolysis and thrombotic complications. eculizumab was administered at the standard dose of mg fortnightly in all but one patient, who needed an increased dose ( mg) because of pharmacokinetic breakthrough. six patients ( aa and agranulocytosis), including the three undergoing a sequential treatment, received standard ist with horse-antithymocyte globulin (h-atg, mg/kg for four consecutive days) combined with cyclosporine a (csa). the remaining three aa patients received alemtuzumab ( - - - mg subcutaneously in four consecutive days) and csa within the prospective trial nct ; one of these patients a few months later also received a second ist course with rabbit-atg ( . mg/kg for five consecutive days) and csa. all the patients completed the scheduled treatment without any side effect, including infusion-related reactions. lymphocyte depletion (o /μl) was observed in all patients irrespective of sustained therapeutic complement blockade. all the patients were available for response assessment at months. among the six patients receiving a concomitant treatment we observed one partial response (pr) and two complete responses (cr), whereas the three remain patients were non-responders (nr). of them one was rescued with an unrelated bmt, while two remained on eculizumab treatment. one of the cr relapsed at years showing clonal evolution and finally died. all the other patients are alive, keeping their hematological response. patients receiving a sequential therapy were one in pr and two in cr months after introduction of eculizumab. in conclusion, for patients diagnosed with severe aa/pnh syndrome intensive ist and eculizumab treatment, can be safely delivered either concurrently or sequentially, with an overall response rate of nearly %. this is the first systematic description of the management of severe aa in hemolytic pnh patients receiving eculizumab treatment. disclosure of conflict of interest: none. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in aa, ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in rcc, and ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) years in rcmd. sixty-five patients underwent bmt from hla-matched (related , unrelated ) and from hla-mismatched (related , unrelated ) donors. conditioning regimens were used as follows; cyclophosphamide (cy)+antithymocyte globulin (atg) ± total body irradiation (tbi) (n = ), fludarabine (flu)+cy ± atg ± tbi (n = ), and flu +melphalan (mel) ± atg ± tbi (n = ). all patients got engraftment after bmt. however, late graft failure was found in patients with rcc, and with rcmd, but none with aa. out of patients who developed late graft failure, patients used flu+cy ± atg ± tbi, used cy ± atg ± tbi, and used flu +mel ± atg ± tbi for conditioning regimens. five-year cumulative incidence (ci) of graft failure was higher in rcmd ( ± . %) than in aa ( %) and rcc ( ± . %), significantly (po . ). five-year ci of graft failure tended to be higher in flu regimen ( ± . %) than in cy+atg ± tbi regimen ( ± . %), but not significant (p = . ). five-year ci of graft failure did not differ between with ( ± . %) or without tbi ( ± . %) (p = . ). multivariate analysis revealed that the morphological classification was a significant risk factor for graft failure (po . ). five-year failure free survival rate ( ± %) in rcmd was significantly lower than in aa ( ± . %) and rcc ( ± . %) (p = . ). graft failure, second malignancy, and death were considered as failure events. one patient with aa died of infection, four with rcc died of infection (n = ), bleeding (n = ) and myocarditis (n = ), and one with rcmd died of infection. five-year overall survival rates were not different among groups (aa, ± . %; rcc, ± . %; rcmd, ± . %) (p = . ). high incidence of graft failure in rcmd may be due to higher bm cellularity than in aa and rcc. the optimal conditioning regimen of bmt should be established for children with abmf based on the bm cellularity and morphological classification. disclosure of conflict of interest: none. recent studies have suggested inferior outcome of patients treated with rabbit atg (thymoglobulin, sanofi) as compared to horse atg (atgam,pfizer or lymphoglobulin, genzyme), and a higher early mortality with rabbit atg has been suggested to explain this difference. aim: to assess early mortality, response rates at , and months and long term outcome, in a large cohort of aa pts, treated in europe or asia with rabbit atg and cyclosporin, as first line treatment. eligible for this study were pts with aa, treated with thymoglobulin between and in europe (n = ) or asia (n = ). median year of treatment, was : characteristics were comparable : median age and years, interval diagnosis treatment ( and days) and severity of the disease ( % and % with vsaa). early mortality was analyzed for all patients.long term outcome was also analyzed for pts for who response data (no, pr, cr) were available. mortality o days was . % and . %, respectively, in the time period - and - (p = . ). in these time periods, early mortality for patients aged - , was reduced from . % to . % and for patients over , from % to %. overall response was recorded in patients. at months the cumulative incidence of response was comparable in the time periods: % vs %, and at year, % vs % (p = . ). response rates at months were age dependent: %, %, %, % respectively in patients aged - , - , - , (p = . ). when non responders at months were reevaluated at year, % had responded, % were non responders, % had died, and % had received other treatment. responses at months, were %, %, %, in pts with very severe, severe and non severe aa (p = . ). the actuarial year survival for the entire population was %, and %, when pts were censored as surviving at transplant. actuarial year survival in univariate analysis was as follows: % vs % for day responders vs non responders (p o . ), % vs % for males versus females (p = . ); %, %, %, % in pts aged - , - , - , years (p o . ); %, %, % in pts with neutrophils o . × /l, - × /l and . × /l (p o . ); %, %, % for pts with an interval diagnosis-treatment of o days, - days or days (p = . ). finally pts treated had a year survival superior to pts treated before ( % vs %, p = . ). survival at years, in the recent period ( - ), was % for pts aged - and % for pts over years. in multivariate cox analysis the following variables remained independent predictors of survival: patient age, year of treatment, severity of the disease, interval diagnosis treatment, and gender. thymoglobulin +csa is effective and safe in patients with aa. the outcome is mainly age dependent. the inrerval between diagnosis and treatment remains a strong predictor: the earlier the better. for pts o years old current early mortality. . for pts years of age, current early mortality is higher ( %), response rate ( %) and year survival ( %) are lower. . the actuarial year survival for the enire population was %. survival at years has improved from % (o o/u ) to % ( - ), especially for pts over years ( % vs %, p = . ). [p ] s disclosure of conflict of interest: we thank centers for providing up date follow up of their patients . this study was supported by a grant of sanofi-genzyme. both of the patients have extremely low anc o . × /l, reto . %, plt o × /l. both was given antibiotic treatment with carbapenem, vancomycin/linezolid, voriconazole or amphotericin b liposome and got no response. no pathogenic bacteria or fungus was found from either of the patients. both of them had no full sibling or matched unrelated donor and had their father as their haploidentical donor. bone marrow combined with peripheral blood stem cell (pbsc) was adopted. conditioning: fludarabine days − through − , ( mg/m × ), intravenous busulfan ( . mg/kg q h) on days − to − . gvhd prophylaxis: high-dose cyclophosphamide mg/kg on days + and + , mmf and tacrolimus since days + . rabbit anti-thymocyte globulin (thymoglobulin) . mg/kg on days − to − . stable neutrophil engraftment (anc . × /l) occurred on day + and day+ respectively. platelet achieved × /l on day + and day + , respectively. both transplant course was complicated by febrile neutropenia without detected etiology, while both children have no fever since the first day anc . × /l.the facial swelling was resolved in both patients except for palatal fistula and fistula of maxillary sinus as the sequela of severe nasosinusitis. no acute or chronic gvhd. case had hemorrhagic cystitis on day + which last for about days, and suspected thrombotic microangiopathy (tma) with hypertension, thrombocytopenia, elevated ldh and creatine on day + which was resolved soon after discontinue of tacrolimus. case had delayed engraftment of platelets and herpes simplex virus encephalitis on days + which was cured by ganciclovir and high dose intravenous immunoglobulin. now they are and months post-hsct respectively and are doing well with % chimerism and no gvhd. alternative donor hsct may be considered as the first line salvage therapy for patients of vsaa with extremely low anc and active infection. fast reconstruction of neutrophil helped to control the infection. hallo-identical hsct make sure nearly every patients can find a donor. ptcy is proved to be efficient and safety in gvhd prophylaxis and facilitating engraftment in these two challenging cases. disclosure of conflict of interest: none. long-term outcome of patients with severe aplastic anemia receiving allogeneic hematopoietic cell transplantation using nonmyeloablative conditioning with fludarabine and low dose total-body irradiation l cheryl xiu qi , l yeh ching , p michelle li mei , t lip kun , h william ying khee , g yeow tee , t patrick huat chye and k liang piu department of haematology-oncology, national university cancer institute, singapore and department of haematology, singapore general hospital, singapore allogenic haematopoeitic stem cell transplant (ahct) offers the best prospect of cure in patients with severe aplastic anaemia (saa). the use of myeloablative hct is however limited by the toxicity of preparative regimens, the lack of matched sibling donors, transplant related mortality and graft rejection. the introduction of non-myeloablative (nm) conditioning offers the possibility of extending this potentially curative treatment to patients in whom ahct was previously contradindicated. in , we reported the outcome of patients with saa who have received ahct using nonmyeloablative conditioning comprising of days of fludarabine at mg/m and total body irradiation at gy (flu + tbi gy). here, we report a longer follow-up, with additional patients who had recevied ahct with this regimen. fourteen patients with a median age of years old (range: - years old) received filgastrim-mobilised peripheral blood stem cell transplant from either hla identical sibilings (n = ) or matched unrelated donor (n = ) after receiving nm conditioning consisting of flu + tbi gy. the first two patients received cyclosporine (cya) and mycophenolate mofetil (mmf) for the post-transplant immunosuppessive therapy. the remainining patients received cya, mmf and a short course of methotrexate (mtx) for additional graft-versus-host dsease (gvhd) prophylaxis. results all patients achieved prompt engraftment. the median time for engraftment of neutrophils ( . × /l) and platelets ( × ) were days (range: - days) and days (range: - days), respectively. chimerism analysis on day and subsequently showed % donor cells in all patients except , who developed secondary graft failure at months and required salvage hct. none of the patients experienced grade and above regimenrelated toxicity. five patients developed grade ii-iv acute gvhd and patients developed limited chronic gvhd. with a median follow-up of . years (range: . - . years), the estimated overall survival and event free survival were % and % respectively. the two patients who did not receive mtx developed acute gvhd of the liver and succumbed to infective complications. the remaining patients who had received triple immunosuppressive therapy were well, with limited chronic gvhd seen only in . our results suggest that the nm conditioning regimen comprising of flu + tbi gy provides sufficient immunosuppression to allow prompt and stable engraftment with minimal regimen-related toxicity. it is an attractive option for patients with saa who require ahct but are at increased risk of regimen-related complications from more intensive cyclophosphamide-based regimens. disclosure of conflict of interest: none. paroxysmal nocturnal hemoglobinuria (pnh) is a rare acquired clonal disorder of hematopoietic cells characterized by the triad of hemolytic anemia, cytopenias and high risk of venous thrombosis. due to the rarity of the disease, most reported data derive from multicenter studies. we describe the natural history of the disease in a -year (yrs) long single center series of pnh patients (pts). we performed a retrospective analysis of consecutive pts followed at our center from to . since , the diagnosis was made by ham test; starting from , flow cytometry (fc) analysis was used to diagnose new pts and to confirm pnh in pts previously diagnosed by the ham test. at diagnosis, pts had classic pnh, aplastic pnh and intermediate form. the cumulative incidences of thrombosis, cytopenia and clonal neoplasm were %, % and %, respectively. except for pt with aplasia, no severe infections were diagnosed, nor renal failures or pulmonary hypertention. the yrs overall survival (os) was %. a nonsignificant better os was associated to the absence of thrombotic events ( % vs %) and to a diagnosis made during the last decade ( % vs % vs %).up to the treatment options were supportive care or allogenic bone marrow transplantation. since , eculizumab was used in transfusion-dependent patients and/or in case of a thrombotic history. overall, pts were transfusion-independent for the entire period of the illness, were transfusion-dependent and/or had thrombotic events( pts). six of the latter pts never received eculizumab but only transfusion support ( pts) or allogeneic bone marrow transplant ( pts), while pts received eculizumab (the first pts were included in the phase iii triumph and shepherd trials).considering the increased risk of meningococcus infection for pts on eculizumab, vaccination with conjugated anti-meningococcus serotypes acwy was employed and, since , conjugated antimeningococcus serotype b was added. overall, pts treated with eculizumab became transfusion-independent and four remained transfusion-dependent. no thrombotic event was observed after eculizumab, even if pts had recurrent thromboembolisms prior to receiving the drug. no severe infection was documented. one patient developed extravascular hemolysis and receive a successfully selective splenic artery embolization. the yrs os in the eculizumab group was %.no pnh-associated death occurred. our study confirms that thrombosis is a major complication in pnh pts not receiving eculizumab, influencing os. the better os in the last decade is probably due to the use of eculizumab and to lack of thrombotic events. in particular, for pts on eculizumab the year os was %, even though half of the pts had thromboembolism and diagnosis made prior to the last decade. although kidney failure and lung hypertension have been reported, we did not observe these complications in our long follow-up case series. we can assume that the availability of a dedicated emergency room at our center allows to perform, promptly, hyper-hydration or transfusion support in case of hemoglobinuria crisis, reducing the risk or organ damage. no infections have been observed after eculizumab, probably due to the vaccination program schedule recommended in the literature, plus the addition of conjugated anti-meningococcus serotype b. however, shared guidelines are needed. disclosure of conflict of interest: none. mortality following hsct in saa pts over the age of is reported to be in the order of %, without taking in to account long term sequelae such as chronic gvhd, known to be more frequent in older patients. this has prompted international guidelines to recommend first line immunosuppressive therapy above years of age. the question is whether this is still true in . the aim of the study is to assess whether trm in saa patients grafted - is reduced,as compared to the era - . we used the wpsaa ebmt registry, and identified pts aged years or more, with acquired saa, grafted between and . we divided pts in transplant eras: - (n = ) and - (n = ). in the more recent period ( - ) pts were older ( vs year, p o . ), were more often grafted from alternative donors (alt) ( % vs %, po . ), with a greater use of bm ( % vs %, p o . ), and with a longer interval dx-tx ( vs days, p . ), and more often received a fludarabine containing regimen ( % vs %, p o . ). the os year of pts grafted in - was %, compared with % for pts grafted - (p = . ). in multivariate analysis, including the interval diagnosis transplant, patient's age, donor type, stem cell source and conditioning regimen, the lack of improved survival in - was confirmed (p = . ). a very strong age effect was shown both in univariate and multivariate analysis: survival of pts aged - years, - years and years, was respectively %, %, % (p o . ) and this was confirmed in multivariate analysis. the conditioning regimen, also proved to be a significant predictor, with improved survival for alt transplants receiving flu containing regimens ( % vs %, po . ). in general pts receiving either cy or a flu containing regimen, did significantly better than pts receiving other preparative regimens ( % vs %, p = . ). the use of a sibling donor (sib) did not prove to predict survival in multivariate analysis. pts receiving campath in the conditioning,did significantly better than pts not receiving campath ( % vs % po . ); similarly survival of patients with atg was superior % vs % compared to patients not receiving atg (p o . ). when pts receiving either campath or atg (n = ) were compared to patients not receiving either (n = ), the difference in survival was % vs % (p o . ), and this was significant also in multivariate analysis. combined primary and secondary graft failure was reduced from % to % in the two time periods (p = . ), acute gvhd grade ii-iv was reduced from % to % (p = . ) and chronic gvhd was also reduced from % to % (p = . ) infections remain the leading cause of death in both transplant eras ( % and % respectively), followed by gvhd ( % and %) and graft failure ( % and %), whereas ptld have been reduced from % to . %. hsct in pts with acquired saa aged and over, continues to carry a significant risk of trm also in - , ranging from % in younger pts ( - ) to % in older pts ( years). survival is predicted in multivariate analysis, by two crucial predictors: patients' age and the use of either campath or atg,the latter giving a % survival advantage over no campath/atg. alt and sib donors produce similar survival. this study gives further support to current guidelines, suggesting first line therapy with atg+csa, in pts over the age of . [p ] disclosure of conflict of interest: none. autoimmune diseases p allogeneic haematopoetic stem cell transplantation as curative therapy for early-onset, refractory crohn's disease e groene , p bufler , k krohn , s immler , g marckmann , t feuchtinger , s koletzko and m albert dr. von hauner university children's hospital and institute of ethics, history and theory of medicine, lmu results of a recent randomized trial suggest that autologous hsct is an option in adult patients with severe, therapyrefractory crohn's disease (cd) with an associated mortality risk of %. however, relapse of the disease is frequent ( ). in contrast allogeneic hsct has resulted in long-term cure of cd in affected patients transplanted because of haematological malignancy ( ). we report a year old girl who was diagnosed with severe cd at age seven (paris classification l , l a, b ). neither next generation sequencing nor immunological work up identified a monogenetic cause of cd. progressive chronic inflammation manifesting ubiquitously in the gastrointestinal tract resulted in severe complications, such as perianal fistulas with rectal stenosis, intestinal abscesses, dysphagia, severe weight loss, failure to thrive, delayed puberty and the need for ileostomy and long-term exclusive enteral nutrition via tube feeding. despite multiple lines of therapy, including repeated nutritional therapy, steroids, immunosuppressants (methotrexate, azathioprine) and biologicals (infliximab, adalimumab, certolizumab) a lasting remission could not be achieved resulting in poor quality of life. after careful risk/benefit assessment including ethical counselling allogeneic hsct was offered. she underwent allogeneic hsct from a matched ( / ) unrelated bone marrow donor ( . × /kg total nuclear cells). conditioning was performed according to a protocol successfully applied in adolescents with chronic granulomatous disease ( ) with alemtuzumab ( × . mg/kg/d), targeted busulfan (tauc ng × h/ml) and fludarabine ( × mg/m ). cyclosporine a and mycophenolate mofetil were used as gvhd prophylaxis. neutrophil and platelet engraftment were observed on days + and + , respectively. the post hsct course was complicated by grade i acute skin gvhd treated with topical steroids and toxic megacolon secondary to scarring stenosis on both ends of the unused colon on day + requiring surgery and a colostomy. at months post hsct the patient is well, off immunosuppressive medication, without gvhd and exhibiting % donor chimerism. the cd is in complete clinical and histological remission as proven by endoscopy and biopsies. stoma reversal with restitution of intestinal continuity is planned for the next months. refractory cd can lead to life-threatening complications and severely reduced quality of life. although long-term outcome in our patient will need to be carefully assessed, allogeneic hsct may offer a curative therapy in children and young adults with severe cd, even in the absence of an identified monogenetic cause. current ebmt recommendations include consideration of ahsct in exceptional circumstances for patients with severe refractory cd. the only randomised trial of ahsct in cd (astic) confirmed substantial short-term benefits but failed to meet its primary year endpoint. to further clarify the longterm safety and efficacy of ahsct in cd we performed a retrospective analysis of patients undergoing ahsct for cd outside the astic trial using the ebmt registry. patients were identified from the ebmt registry. all adult patients undergoing ahsct for a primary diagnosis of cd from to were eligible for inclusion. patients who were enrolled in the astic trial were excluded. from a total of patients (across centres) on the ebmt registry, data were obtained from patients transplanted in centres in countries. median patient age was yrs (range: - ) and % were female. median age at first diagnosis of cd was yrs (range: - ). patients were heavily pre-treated, having failed or been intolerant to a median of previous lines of therapy (range: - ). % had received experimental therapy prior to auto-hsct. % of patients had undergone at least operation. the median time from first diagnosis of cd to auto-hsct was . years (range: . - . ). all patients received peripheral blood stem cells following conditioning with cyclophosphamide mg/kg and % received anti-thymocyte globulin (atg). the median cd + dose infused was . (range: . - . ) × /kg. twelve percent of patients underwent cd + selection. neutrophil and platelet engraftment occurred at a median of day (range: - ) and day (range: - ), respectively. sixety-one percent received post transplant g-csf. median length of follow-up following auto-hsct was months (range: - ). at days post auto-hsct, % of patients were in clinical remission (cr), defined as no abdominal pain and normal stool frequency. a further % experienced significant improvement, defined as improvement in abdominal pain and stool frequency. for % there was no appreciable change in disease and in % the disease worsened compared to baseline. at year post auto-hsct, % were in cr, % were improved, % were unchanged and % had worsened. at last follow-up, % were in cr, % were improved, % were unchanged and % had worsened. overall % restarted medical therapy post auto-hsct and % required further surgery. overall % developed an infection requiring treatment post auto-hsct ( % bacterial, % viral). ebv and cmv reactivation occurred in % and % respectively and herpes zoster occurred in %. a secondary autoimmune disease developed in %, most commonly thyroid disease ( %). malignancy developed in %, of which skin cancer accounted for % of cases. one patient died at days post auto-hsct due to cmv infection, sepsis and multiorgan failure. this large retrospective series further supports the safety and efficacy of ahsct in a population with severe and treatment-refractory crohn's disease, % of patients experienced complete remission or significant improvement in cd symptoms with long-term follow-up. trm observed was similar to ahsct for other indications. in summary, ahsct appears to be an extremely promising therapy for severe refractory cd. further follow up of astic patients and future randomised trials are warranted. disclosure of conflict of interest: none. memory stem t cells (tscm) are long living self-renewing memory t cells with long-term persistence capacity, which play a relevant role in immunological memory and protection against infectious diseases and cancer , , , , , . the aim of this work is to investigate the potential role of tscm as a reservoir of arthritogenic t cells in rheumatoid arthritis (ra). we analysed the dynamics of circulating tscm (here identified as cd ra+ cd l+ cd + t cells) and other memory t-cell subpopulations by multiparametric -color flow cytometry in patients with active ra and in of them also during treatment with anti-tnfα biological agents (etanercept). to analyse cytokine productions, functional assays were performed stimulating peripheral blood mononuclear cells (pbmcs) with pma/ionomycin and brefeldin a. after the stimulation, cells were stained for surface markers, fixed and stained for intracellular cytokines. we traced circulating antigen specific cd + t cells for the vimentine-derived citrullinated peptide (vimcit) savracitssvpgvr , in hla-drb × : ra patients before and during the anti-tnfα treatment using custom mhc class ii tetramers. viral antigen specific cd + t cells were traced using mhc class i dextramers. age-matched healthy donors (hds) were used as control for all the experiments. we found a significant expansion of cd + tscm in patients with active ra both in terms of frequency and absolute counts. notably, cd + tscm significantly contracted upon anti-tnfα treatment, suggesting a role of tnfα in tscm accumulation. in contrast to cd +t cells, cd compartment did not show significative alterations compared to (hds). furthermore, cd +tscm in ra patients displayed an enrichment in the th phenotype, largely implied in autoimmune disorders, while the other t cell subpopulation were not enriched in the th phenotype. at the antigen specific level, we were able to trace in hla-drb × : patients antigen specific cd + t cells, comprising tscm, specific for the vimentin-derived citrullinated peptide. of notice, citrullinated vimentin specific cd + t cells, including tscm, contracted during anti-tnfα administration, while viral-specific cd + t cells (ebvbhrf- ) and antiviral cd specificities (cmvpp , flump, ebvbmlf- ) were not affected by etanercept administration, thus suggesting a possible role of cd + tscm as reservoir of arthritogenic autoreactive t cells. overall, our results suggest that tscm, by representing a long-term reservoir of undesired specificities, might play a non redundant role in sustaining ra and possibly other t cell mediated disorders, thus representing novel biomarkers as well as therapeutic targets. ongoing experiments will characterize the tcr repertoire on sorted tscm and cd + memory subsets in order to identify a possible oligoclonality in tscm repertoire. in conclusion, the analysis of tscm dynamics in autoimmune disorders could have relevant clinical implications as new biomarkers and for devising innovative therapeutic strategies. ebv and cmv reactivation following autologous haematopoietic stem cell transplantation (hsct) for autoimmune neurological diseases resolves spontaneously and rarely requires treatment c mapplebeck , b sharrack , h kaur , y ezaydi , h jessop , l pickersgill , l scott , m raza and ja snowden departments of haematology, neurology and virology, sheffield teaching hospitals nhs foundation trust, sheffield, uk autologous haematopoietic stem cell transplantation (hsct) for severe autoimmune diseases involves immunosuppressive conditioning regimens and current guidelines recommend monitoring for viral reactivation of cytomegalovirus (cmv) and epstein barr virus (ebv) (snowden et al ) . however, the incidence, degree and management of viral reactivation are not established. we performed a retrospective observational service evaluation study of all patients receiving cyclophosphamide mg/kg + rabbit anti-thymocyte globulin mg/kg (atg, thymoglobulin) followed by autologous hsct for various autoimmune neurological diseases between and at our centre. data collected included the baseline serological status of the patient prior to transplant and serial blood pcr quantitation (copies/ml). if ebv and cmv reactivation occurred details of further management was collected and descriptive statistics were used to summarise outcomes. twenty-three patients received autologous hsct between january and october ; patients with multiple sclerosis (ms), with chronic inflammatory demyelinating polyneuropathy (cidp) and with stiff person syndrome. twenty-two patients had positive ebv igg serology prior to transplant and patient had an equivocal result. seventeen patients had evidence of ebv reactivation and a further patient had ebv dna detected post-transplant but with less than copies/ml. the average time to peak ebv pcr was . (range: - ) days post-transplant and a range: in ebv pcr peak level from to copies/ml. the patients who had ebv pcr results of over copies/ml had ct scans of chest, abdomen and pelvis performed which did not demonstrate significant lymphadenopathy or hepatosplenomegaly. in all patients monitored for a detectable ebv reactivation, the ebv pcr spontaneously began to fall within months (average days, range: - days) post-transplant and no specific treatment was required. one patient had late ebv reactivation of copies/ml at months post-hsct associated with chronic tonsillitis and tonsillectomy specimens showed follicular hyperplasia without evidence of post-transplant lymphoproliferative disorder (ptld) and ebv pcr levels normalised without other treatment. ( %) patients had positive cmv igg serology prior to transplant and one patient had an equivocal result. only of patients had a significant reactivation of cmv with copies/ml at days post-transplant, successfully treated with intravenous immunoglobulins and valganciclovir. two other patients had low level cmv reactivation with and copies/ml, respectively which resolved spontaneously without treatment. ebv reactivation in patients with neurological autoimmune disease undergoing autologous hsct is common and usually resolves spontaneously without treatment. asymptomatic cmv reactivation occurs in approximately % of patients in this setting and may require treatment. autologous hematopoietic stem cell transplantation (hsct) has been utilised for the treatment of severe multiple sclerosis (ms). it results in significant improvement of neurological function, although patients can experience exacerbations of ms-related symptoms during the procedure. we reviewed patients with ms who underwent stem cell mobilisation and collection from march to november . the median age was years ( - ). nine patients ( %) were male. the interval from diagnosis to hsct was . months (range: . - . ). patients ( %) had relapsing-remitting (rrms), six patients ( %) secondaryprogressive (spms) and two patients ( %) primary-progressive (ppms) multiple sclerosis. only patients ( %) had not received any prior treatment, whereas patients ( %) received two prior treatments, three patients ( %) received three treatments and two patients ( %) received four treatments. the median expanded disability status scale (edss) score was (range: - ). peripheral blood stem cells were mobilised with cyclophosphamide (cy) g/m on day + and daily gcsf ( μg/kg subcutaneously) from day + until the completion of the harvest. hsct was performed at a median of days after mobilisation (range: - ). the conditioning regimen consisted of cy ( mg/kg/day from day − to − ) and atg ( mg/kg/day from day − to − ). exacerbation of ms symptoms was defined as the appearance of new or worsening of old symptoms for at least h duration in a previously stable ( weeks) patient. of the total cohort, patients ( %) underwent mobilisation with cy+gcsf uneventfully. only two patients ( %) had an exacerbation of ms requiring hospital admission after collection (one with fatigue and increase of spasticity, other with worsening weakness). no patient required hospital admission during the mobilisation procedure. the median cd + cell dose was . × /kg (range: . - ). the median number of apheresis was ( - ). a total of patients have undergone hsct at the time of this analysis. during transplant a total of patients ( %) experienced an exacerbation of ms. of these, % (n = ) before day and % (n = ) between day + and + . symptoms of exacerbation were: muscle spasms in patients ( %), weakness and reduced power of limbs in patients ( %), increase instability and tremor in two patients ( %) and one patient ( %) with worsening of neuropathic pain. only three patients ( %) received treatment with methylprednisolone for ms exacerbation and symptoms had fully resolved by discharge in all patients. other transplant complications included neutropenic fever in all, invasive fungal infection in , fluid overload in ( %) and atg related complications in ( %) such as fever (n = ) and pericarditis/serositis (n = ). the median time to neutrophil engraftment was days ( - ) and the median duration of hospital admission was days ( - ). exacerbation of ms symptoms is common during hsct and can also occur during mobilisation. in our hands, after cy and gcsf mobilisation only two patients ( %) developed an exacerbation of ms symptoms compared with patients ( %) after ct and atg conditioned hsct. it is possible that the addition of atg to cy triggers an immunological response involved in this transient deterioration of the ms symptoms. further studies are required to confirm this hypothesis. disclosure of conflict of interest: none. inflammatory immune response syndrome (iris) is a noninfectious worsening of neurological condition during immune recovery and has been documented to occur in hiv and in multiple sclerosis following alemtuzumab. the manifestation of iris includes headache, nausea, weakness, neurologic deficits, and mri enhancing lesion. we report three cases of iris after autologous non-myeloablative hematopoietic stem cell transplantation (hsct) in patients for which the transplant indication was an inflammatory neurologic disease: neuromyelitis optica (nmo), chronic relapsing inflammatory optic neuritis (crion), and multiple sclerosis (ms). mobilization was with cyclophosphamide gr/m and gcsf. conditioned regimen was mg/kg cyclophosphamide ( mg/kg/d) and . mg/kg ratg (thymoglobulin). the conditioning regimen for nmo and crion also included mg rituximab. case . a years old african-american female with systemic lupus erythematosus (sle) and nmo was discharged day and readmitted on day for fever, headache, progressive altered mental status with dysarthria and legs. brain mri had numerous t /flair hyperintense and enhancing lesions in the subcortical and periventricular white matter. a lumbar puncture was negative for infection including jcv. complete recovery occurred after treatment included high dose of steroids and plasmapheresis. case . a years old female with crion experienced blindness, weakness and slurring of speech three months post hsct. mri showed a large enhancing brain stem lesion. lumbar puncture was jcv negative. complete recovery occurred after solumedrol and rituximab. mri months later demonstrated complete resolution of the enhancement with return of vision to baseline. case . a ten year-old boy diagnosed with paediatric ms developed hemichorea seven days after hsc reinfusion. brain mri revealed a gadolinium-enhancing lesion in the contralateral basal ganglia. lumbar puncture was negative for infection including jc virus. symptoms resolved spontaneously after seventeen days. the appearance of new neurologic symptoms and mri enhancing lesions early after autologous hsct is unexpected and may be related to lymphocytes in the graft, immune recovery post engraftment, and or persistent auto-antibodies. it is mandatory to perform a lumbar puncture to exclude the possibility of infections including progressive multifocal leukoencelopathy (pml) due to jcv. the timing of presentation, the negativity of jc viral load, and the complete recovery with or without immune suppression suggest the hypothesis of iris, as an epiphenomenon of the immune reconstitution following autologous hsct for neurologic diseases disclosure of conflict of interest: none. hematopoietic stem cell transplantation is the effective method of therapy for cns autoimmune disorders in children. long-term outcomes and late effects estimation required. the aim of the study is to estimate long-term outcomes and late effects at children underwent auto-hsct for multiple sclerosis (ms) and allo-hsct for neuromyelitis optica (nmo). twelve pts. with ms and pts. with nmo were included to the analysis. ms pts. gender: female - % (n = ), male - % (n = allogeneic haematopoietic stem cell transplantation (hsct) remains the sole curative option for patients with myelofibrosis (mf). although a spectrum of conditioning regimens has been used, the optimal preparative treatment before hsct remains to be defined. we did a phase ii randomized study at transplant centers in italy with the aim of comparing the reduced-intensity conditioning (ric) fludarabine-busulfan (fb) (conventional arm), that had been already tested in the prospective ebmt study ( ) with the ric fludarabine-thiotepa (ft) (experimental arm), that has been widely used in italy in the last two decades ( ) . eligible to this study were patients with primary mf or a mf subsequent to a previous essential thrombocytemia or polycyhemia vera, an age ≥ ≤ years, a karnofsky performance status , a comorbidity index o o/u and with at least one of the following unfavorable prognostic factors: anemia (hb o g/dl), leukocitosis ( × /l), circulating blasts % or constitutional symptoms. patients were randomized to receive intravenous busulfan . mg/kg for doses or thiotepa mg/kg for two doses associated to fludarabine mg/m for impact of pre-transplant ruxolitinib in myelofibrosis patients on outcome after allogeneic stem cell transplantation syed abd kadir, sharifah shahnaz, author , , zabelina, tatjana, co-author , christopeit, maximilian, co-author , wulf, gerald, co-author , wagner, eva, co-author , bornhaeuser, martin, co-author , schroeder, thomas, co-author , crysandt, martina, co-author , mayer, karin tina, co-author , stelljes, matthias, co-author , badbaran, anita, co-author , wolschke, christine, co-author , ayuk ayuketang, francis, co-author , triviai, ioanna, co-author , wolf, dominik, co-author ruxolitinib (rux) is the first approved drug for treatment of myelofibrosis. because spleen size and constitutional symptoms may influence outcome after allogeneic stem cell transplantation (asct), rux is recommended before stem cell transplantation in order to reduce therapy-related morbidity and mortality and improve outcome (ebmt/eln recommendation, leukemia ) the aim of this retrospective study was to evaluate the impact of pretreatment with rux in comparison to transplantation of rux-naïve mf patients with regard to outcome after asct. we included myelofibrosis patients (pts) with a median age of years (range: - ) who received asct between and from related (n = ), matched (n = ) or mismatched (n = ) unrelated donor. all patients received busulfan-based reduced intensity conditioning. while pts ( %) did not receive rux, pts ( %) received rux at any time point prior to asct. the median daily dose of rux was mg (range: - mg) and the median duration of treatment was days (range: - days). in pts rux was stopped before stem cell transplantation because of no response or loss of response, while in pts rux was given until start of conditioning. gvhd prophylaxis consisted of cni plus short course mtx or mmf and anti-lymphocyte globulin. according to dynamic ipss (dipss) (n = ) the patients were either low (n = ), intermediate- (n = ), intermediat- (n = ), or high risk (n = ). as the median follow up was shorter for patients treated with rux ( vs months, po . ). primary graft failure was seen in pts in the rux and three in the non-rux group. the median leukocyte engraftment was days (range: - ) in the ruxolitinib and days (range: - ) in the non rux group (p = . ). the incidence of acute gvhd grade i to iv was significantly lower in the rux group ( % vs %, p = . ), while agvhd grade ii-iv ( % vs %, p = . ) and grade iii/iv ( % vs %, p = . ), did not differ significantly. the ci of nrm at year was % ( % ci: - %) for the rux group and % ( % ci: - %) for the non-rux group (p = . ), and the ci of relapse at years was % ( % ci: - %) vs % ( %ci: - %, p = . ). the years rfs and os was % ( %ci: - %) and % ( %ci: - ) for the rux group and % ( % ci: - %) and % ( % ci: - %) for the non-rux group (p = . and p = . , respectively). within the rux group (n = ), pts responded to rux (more than % spleen size reduction), while pts did not respond or lost response prior to stem cell transplantation. here, no significant difference could be seen between the responding and non-responding group for nrm ( % vs %, p = . ), relapse ( % vs %, p = . ), rfs ( % vs %, p = . ) and os ( % vs %, p = . ). in a multivariate analysis including rux treatment as variable there was a non-significant trend in favor for in the tyrosine kinase inhibitor (tki) era, allogeneic haemopoietic stem cell transplantation (allo-hsct) has become the later-line therapy but still remains the only known curative treatment for chronic myeloid leukemia (cml). since the introduction of tki in our centre in , the trend of allo-hsct among our cml cohort has changed over time. the purpose of this study is to examine hsct outcomes of our cml cohort who was either tki naïve or has received tki therapy prior hsct. between may and december , cml patients in our center received allo-hsct with % were tki naïve. the time of diagnosis to transplant was significant shorter among the tki naive group as compared to those received tki prior hsct ( . ± . months versus . ± . months, respectively). there were no gender different ( % males) but the median age at hsct was younger among tki naïve group, . years (range: - years) versus . years (range: - years) respectively. malays remained majority ethnic group but the percentage was reduced among patients received tki prior hsct ( . % versus . % respectively). the disease phase at hsct was significant different whereby majority of tki naïve group was in first chronic phase (cp ) ( . %) as compare to patients with prior tki exposure ( . %). all the patients in the tki naïve group received hla-matched related siblings donor (mrd) with . % marrow stem cell source whereas only . % of patients who have prior tki exposure received mrd with . % were from peripheral blood stem cell (pbsc). all patients in the tki naïve group but only . % among patients who have prior tki exposure received full myeloablative conditioning regimen. there was slower neutrophil and platelet engraftment ( . ± . days versus . ± . days and . ± . days versus . ± . days respectively) among tki naive group. at june , the -year overall survival (os) of cml at all disease status was % in tki naive group versus % for patients who have prior tki exposure and transplanted in more advance disease stage. in general, patients in cp have the best os. there was higher incident of grade to acute graft-versus-host-disease (gvhd) among the tki naïve group ( . % versus . %, respectively) likely due to intensity of conditioning regimen with no significant different in chronic gvhd incident. similarly, there was higher relapse rate among tki naive patients ( . % versus . %, respectively) as upfront post transplant tki was not routinely given to this group of patients in the past. further multivariate analysis to ascertain predictors of transplant outcome among this cohort of patients included disease status, donor-recipient gender combination, ethnic difference will be presented. in conclusion, despite emergent of effective and potent next generation tki, hsct still has it role as curative modality for patients who failed tki. as showed in our data, the transplant outcome is excellent for patients who remain in cp at the time of hsct and it is important to identify patients earlier, before disease progression, especially young patients, in order to optimize transplantation outcomes. disclosure of conflict of interest: none. the purpose of this analysis was to provide -year follow-up of the gcllsg cll x trial which aimed at evaluating reducedintensity conditioning (ric) hsct in patients with poor-risk cll. the cll x trial included patients (median age ( - ) years), of whom patients were allografted with blood stem cells from related ( %) or unrelated donors ( %) using fludarabine-alkylator-based ric regimens. % had refractory cll at hsct, and % had a tp deletion and/or mutation. the -year follow-up of the trial including the observation that genetic risk factors such as tp lesions and sf b and notch mutations had no prognostic impact has been previously reported. survival and relapse information was requested for all patients who underwent hsct within the cll x trial in german centres (the canadian centre was unavailable for follow-up) and were alive at the -year followup. results: follow-up information was received for / patients ( %) alive at the -year follow-up. of these, patients had died ( cll, chronic gvhd, secondary cancer), and had experienced disease recurrence. with a median follow-up of survivors of . ( . - . ) years, -year nrm, relapse incidence (rel), event-free survival (efs), and overall survival (os) of all patients allografted was %, %, % and %, respectively, without significant effects of tp lesions on outcome. absence of minimal residual disease (mrd) at the -month landmark post hsct was highly predictive for a reduced relapse risk, in particular if mrd eradication occurred only after immunosuppression withdrawal, suggesting of effective graft-versus-leukemia activity (gvl; -year rel %). in the patients who were alive and event-free years post allohct, nrm, rel, efs, and os years after this landmark (or years after transplant) was . %, %, %, and % with a median follow-up of . years ( . - . ) after the -year landmark. notably, no relapse event occurred beyond years post hsct. of those who remained event-free beyond years, all patients who were available for mrd assessment at their most recent follow-up were mrdnegative. altogether of the allografted patients had cll recurrence after transplant; between and , and from onwards. whilst the median survival of those patients who relapsed during the earlier period was months, all patients with late relapse are currently alive - (median ) months after the event. conclusions: long-term observation of patients allografted in the cll x trial confirms that ric hsct can provide gvl-mediated sustained disease control in a sizable proportion of patients with poor-risk cll independent of the tp status. patients who are in mrdnegative remission one year after hsct have an % probability of remaining disease-free at least for years. however, late relapses do occur but may benefit from strategies involving innovative pathway inhibitors. hallek: consultancy and speakers bureau for pharmacyclics, llc and an abbvie company; speakers bureau for janssen; m. kneba: consultancy, honoraria, travel grants and research funding from gilead and roche; consultancy, honoraria and travel grants from abbvie and janssen-cilag; research funding from amgen; travel grants from glaxo-smithkline; p.dreger: consultancy for roche and janssen; consultancy and speakers bureau for novartis and gilead. no evidence for an increased gvhd risk associated with post-transplant idelalisib given for relapse of chronic lymphocytic leukemia or lymphoma: first results of a survey by the ebmt chronic malignancy and lymphoma working parties p dreger , , a boumendil, l koster , c scheid idelalisib is a kinase inhibitor (ki) approved for the treatment of cll and follicular lymphoma (fl). idelalisib has a specific adverse effect profile including immune-mediated inflammatory conditions such as colitis and pneumonitis, raising concern about the safety of this ki if administered for treatment of malignancy recurrence after allogeneic hematopoietic cell transplantation (allohct). the purpose of this ongoing study is to provide information on the safety and efficacy of idelalisib in this setting. we included in this study adult patients who had been registered with the ebmt for an allohct for cll or lymphoma and who received idelalisib for treating disease relapse or persistence at any time after transplant as indicated by participating investigators upon request by the ebmt study office in leiden. baseline patient, disease, and transplant data were collected from med-a forms. centers were requested to provide additional treatment and follow-up information. as of november , , a total of patients have been registered, of whom a full dataset as required for this analysis was available for patients (cll , fl , diffuse large b-cell lymphoma (dlbcl) , peripheral t-cell lymphoma , unspecified ) who had undergone allohct between july and april . all patients except one were male. median age at transplantation was ( - ) years and the median interval from diagnosis to allohct was . ( . - . ) years. prior to allohct, patients ( cll and lymphoma) had received an autohct and two other patients had been exposed to ki (idelalsib , ibrutinib ). disease status at allohct was sensitive in % of the patients. conditioning was reduced-intensity in % of the transplants and included in vivo t cell depletion in the majority of cases ( %). donors were identical siblings in % with pbsc being the stem cell source in all cases. the interval between hct and idelalisib commencement was ( - ) months in the cll group but only ( - ) months in the lymphoma group. prior to idelalisib, grade ii-iv acute gvhd and chronic gvhd had been observed in % and % of the patients, but was still active at the time of idelalisib commencement in only two cases ( %) . four patients with cll had already failed ibrutinib given for post-hct relapse prior to idelalisib. the median time on idelalisib until documented withdrawal or event (progression, retreatment, death) was ( - ) days. after start of idelalisib, one patient developed grade acute gvhd and subsequently chronic gvhd, however, in this patient idelalisib was started as early as days after transplant. efficacy of idelalisib in this high-risk patient sample was limited with only one pr in the cll group (stable disease , progressive disease , not available ; lymphoma not available), translating into a median event-free survival after start of idelalisib of days. five patients with cll underwent a subsequent treatment with an alternate ki (ibrutinib , venetoclax ). altogether, there were five deaths, all due to diease progression (cll , lymphoma ). median overall survival was days for the whole sample and not reached for cll. this preliminary data does not support concerns about the safety of idelalisib in the post allohct setting. updated results of this ongoing study will be presented at the meeting. tested patients ( %) achieved a ccyr and at least a mmolr, respectively. the response to transplant by day assessment correlated significantly with the disease status before transplant. a higher percentage of patients who experienced cytogenetic response before transplant experienced molecular response post-transplant ( %) compared with those who did not ( %; p = . ). for the entire group, the -year cumulative incidence (ci) of acute gvhd grade ii-iv and grade iii-iv were % and %, respectively; -year ci of extensive chronic gvhd was %. there was no significant difference in the ci of severe acute or chronic gvhd between donor types. the ci of nrm at days and year was % and %, respectively. the ci of cytogenetic and molecular relapse at years was % and %, respectively. overall the -year os, pfs and gvhd-free, relapse-free survival (grfs) were %, %, and %, respectively. in multivariable analysis for grfs, transplant in cp and the use of haploidentical donor significantly associated with better grfs. the -year grfs of patients in cp , ap and bp before transplant was %, % and %, respectively (p = . ). ( figure a ) patients receiving a haploidentical donor had a better -year grfs when compared with hla matched transplants ( % vs %, p = . ). ( figure b ) for pfs, transplantation in cp , using a haploidentical donor and mac regimen associated with better pfs while age, cytogenetic and molecular response before transplant did not predict survival. ahsct is curative for a proportion of patients with advanced cml. pfs and grfs are favorably influenced by percentage of bm blasts and donor type, with haploidentical donor having at least as good outcomes as hla matched donors, while molecular and cytogenetic response before transplant do not appear to correlate with survival posttransplant disclosure of conflict of interest: none. allogeneic stem cell transplantation (sct) has been considered as the treatment of choice for younger patients (pts) with high-risk chronic lymphocytic leukemia (cll). role of allogeneic sct in era of novel drugs is widely discussed. here we present our results after sequential use of chemotherapy and reduced-intensity conditioning (ric) in cohort of high-risk cll pts. high-risk cll was defined by one of the following: disease refractory to purine analogs, short response or early relapse (within months) after purine analog combination treatment, and/or progressive disease with unfavorable genetic abnormalities (del [ p]/tp mutation). we analyzed pts with high-risk cll undergoing chemotherapy and ric sct in our centre from august to june . the median of pretransplant lines were (range: - ), novel drugs (idelalisib, ibrutinib) were used in % of pts ( / ). fludarabine ( mg/m ) and cytarabine ( g/m ) for days (fc) were used for cytoreduction in all pts. after days of rest, ric consisting of gy tbi, anti-thymocyte globulin - mg/ kg/day for days, and cyclophosphamide - mg/kg/day for days followed. median age of pts was years (range: five-year overall (os) and relapse-free survival (rfs) was % and %. ci for cgvhd in pts surviving more than months post-hct was % after years and % after years. in a multivariate cox-regression model the occurrence of cgvhd independently improved os (p = . , hr . ; % ci . - . %) as well as rfs (po . , hr . ; % ci . - . ). high risk dipss plus score demonstrated significant inferior survival compared to intermediate- (os p = . ; rfs p = . ), int- (os p = . ; rfs p = . ) and low risk (os p = . ; rfs p = . ) which could be confirmed in multivariate analysis for os (p = . , hr . ; % ci . - . ) and rfs (p o . , hr . ; % ci . - . ). rfs additionally was improved by splenomegaly (n = ) vs. normal spleen size (n = ) at time of hct (p = . , hr . ; % ci . - . ). ruxolitinib (n = ) or none (n = ) pre-treatment compared to other drug therapy (n = ) resulted in improved os (p = . ) and rfs (p = . ) and was an independent factor for rfs in multivariate analysis (p = . , hr . ; % ci . - . ). non-relapse mortality (nmr) was significantly determined by high-risk dipss plus score (p = . ) or dipss high and int- (p = . ). relapse incidence was significantly lower in pts with splenomegaly compared to asplenic or normalspleensized pts prior to hct (p = . ). our data point out that pre-therapy and dipss or dipss plus score are relevant pre-transplant outcome factors while chronic gvhd is the most important independent hct-related factor. furthermore, splenomegaly at hct reduces risk of relapse and therefore improves rfs. [p ] disclosure of conflict of interest: none. thalassaemia major affects new babies in india each year and haematopoietic stem cell transplantation offers the only chance of cure. we present data on children with thalassaemia major aged between months and years using a uniform conditioning regimen consisting of thiotepa mg/kg, treosulphan gm/m and fludarabine mg/m . equine antithymocyte globulin at a dose of mg/kg was added to children who were undergoing transplantation from an unrelated donor source. there were eight deaths before engraftment due to sepsis or bleeding and two related to graft versus host disease. all patients showed complete chimerism on day . however, in children there was an acute drop in donor chimerism between day and post transplantation. immunosuppression was abruptly stopped when donor chimerism dropped below % in all children. seven children responded well and re-established complete chimerism with this measure. seven children progressed to develop complete graft loss. donor lymphocyte infusion (dli) in the form of small aliquots of peripheral whole blood from the donor was administered in seven children. dli was used in a graded fashion every weeks starting from × /kg of cd , followed by × /kg and × /kg. all of them continued to maintain their graft with these interventions. drop in chimerism was seen particularly in children less than years at the time of transplantation comprising out of children. older children with lucarelli class iii were also prone to rejection in our earlier series and this complication has now been eliminated with pre-transplant immunosuppression and hypertransfusion. children above the age of years were more prone to graft versus host disease and required on average months of immunosuppression. treosulphan based protocol has been equally well tolerated by all age groups, all lucarelli classes of children with thalassaemia major and different donor sources. the transplant related mortality and graft rejection rates have been low at . % and . %, respectively. however, children less than years need to be monitored carefully during the first months of transplantation as early withdrawal of immunosuppression can prevent graft rejection resulting in excellent outcomes. disclosure of conflict of interest: none. institute of cellular medicine, newcastle university, newcastle-upon-tyne, uk hemophagocytic lymphohistiocytosis (hlh; hemophagocytic syndrome) is a rare syndrome of potentially fatal, uncontrolled hyperinflammation. allogeneic stem cell transplantation (allosct) is indicated in familial, recurrent or progressive hlh. additional recommendations include central nervous system involvement and unknown triggering factor. while data for allosct outcome are available for the pediatric setting, information for adults is very limited. the aim of this study was to retrospectively analyze the information from the ebmt databases about adult hlh patients who underwent allogeneic stem cell transplantation. we obtained data of adult (≥ years of age) patients transplanted due to hlh. additionally, an hlh-oriented questionnaire was sent to the clinical centers, with responses received so far. median age at transplantation was (range: - ). there was a slight male predominance / ( %). the majority of patients were reported with secondary hlh / ( %), the familial disease was reported in / ( %) patients. in two patients triggering factor was attributed to malignancy. the majority of patients received stem cells obtained from the peripheral blood ( / ; %) while for the remaining ones it was bone marrow. reduced intensity conditioning was used since in / ( %) of patients. thirteen ( %) patients received tbi. donor choice was: matched unrelated ( %), mismatched unrelated ( %), identical sibling ( %). engraftment was observed in / ( %). the cumulative incidence of acute graft versus host disease (gvhd) at days was % ( % ci - %). the cumulative incidence of chronic gvhd at year after allosct was % ( % ci - %) and increased to % at years ( % ci - %). the -year probability of overall survival is shown in fig. . the median survival time was months. the -year os was % ( % ci - %). for patients who survived until months, this proportion was more favorable with an os of % ( % ci - %) at years after transplantation. among patients with observation times longer than months, only one patient died (in the th month after allosct due to relapse which occurred in the th month. after months no more relapses of hlh were recorded-the cumulative incidence reached %. the non-relapse mortality reached % after months. the familial disease was associated with a better prognosis than secondary hlh (p = . ). unlike the pediatric population, where reduced intensity conditioning (ric) was associated with higher survival, in adult patients there was no difference between the conditioning types. data form the questionnaires confirm clinical picture typical for hlh at the diagnosis: fever in / ( %), splenomegaly in / ( %), hemophagocytosis in / ( %) and hyperferritinemia with median concentration of ng/ml (range: - ). image fig. overall survival after allogeneic stem cell transplantation for adult hlh patients until months ( % confidence intervals are shown in grey). the number of patients at risk is indicated below the time axis at the corresponding time points. to our knowledge, this is the largest group of adult patients with hlh who underwent allogeneic stem cell transplantation. relatively low relapse incidence shows that allosct can effectively cure hlh. patients who survive the first period after this procedure can expect a long disease-free survival. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative option for children suffering from various life-threatening inherited non-malignant diseases with best results using hla-identical family donor. hsct from unrelated or mismatched family donors is associated with increased risk of agvhd and graft rejection.use of post-transplantation cyclophosphamide (ptcy) with or without additional immunosuppression has been shown to be effective prophylaxis against gvhd in patients with hematological malignancies. there are limited reports of hsct using pt cy for patients with non-malignant disorders. we retrospectively analyzed results of hsct in patients with life-threatening non-malignant diseases using ptcy-based gvhd prophylaxis. patients characteristics are presented in table . thirteen patients ( . %) were transplanted upfront, patients ( , %) were rescued after primary or secondary graft failure after first hsct. donors were hla-matched (n = ) or mismatched ( - / ) (n = ) unrelated, haploidentical (n = ) or hla-identical family (n = ). bone marrow was used as graft source in ( . %) patients and peripheral blood stem cell in ( . %). median cd +/kg recipient weight- . × ( . - . ), cd +/kg- . × ( . - . ). the conditioning regimen was myeloablative in patients (conventional- , reduced toxicity- ), reduced intensity- . the gvhd prophylaxis consisted of a combination of ptcy at dose of mg/kg on days + and + with calcineurin inhibitors (tacrolimus- pts, cyclosporine a- pts) or sirolimus ( pt) and mmf ( pts) starting on day + . all but one patients received also serotherapy with rabbit ( pts) or horse atg ( pst) and rituximab ( pts). with a median follow-up of months (range: - ), the kaplan-meier estimates of os − . %. one patient with thalassemia died before engraftment on day+ from severe vod. / pts ( %) achieved engraftment. the median time for neutrophil and platelet engraftment was ( - ) and days ( - ), respectively. primary graft failure was observed in patients ( was successfully retransplanted from another haploidenticle donor, was not eligible for a second transplantation, but alive). at last follow up, ( %) patients had full donor chimerism, ( %) had stable mixed chimerism without signs of disease progression. one patien with wiscott-aldrich syndrome had secondary graft failure with progressive loss of donor chimerism and were successfully rescued with second haploidentical transplant from the same donor. of engrafted patients, agvhd ii-iv was seen in ( . %) patients. one patient developed grade ii (gut stage ii) agvhd, which resolved with systemic steroids. severe (griii-iv) agvhd was observed in pts after second hsct, both had calcineurin and mtor-inhibitors induced toxicity leading to discontinuation of this drugs, but responded on combined (steroids and ruxolitinib) therapy. one patient with was developed grade iii gvhd (gut stage ) after severe cmv-colitis and died on day from multiple organ failure (suspected tma). one patient developed extensive chronic gvhd of kidney (minimal change [p ] disease) after tapering of immunosupression. one patient with hurler syndrome had seizures, died on day+ from multiple organ dysfunction syndrome. conclusion: ptcy is a promising option for agvhd prophylaxis in patient with non-malignant disease, lacking an hla-matched family donor. disclosure of conflict of interest: none. an exploratory, open-label study to evaluate the safety and feasibility of atir , a t-lymphocyte enriched leukocyte preparation depleted ex vivo of host alloreactive t-cells (using photodynamic treatment), as adjuvant treatment to a t-cell depleted haploidentical hematopoietic stem cell transplantation in patients with beta-thalassemia major c selim , w rob , l sarah , f josu de la previous studies demonstrated that donor lymphocytes, selectively depleted of alloreactive t-cells (atir), could be given safely in adult patients receiving a haploidentical hsct. in patients a single dose of atir, at doses up to × viable t-cells/kg, was given and no grade iii/iv acute gvhd has been reported. this confirms the efficacy of the elimination method of allo-reactive t-cells and attributes to its beneficial safety profile. in an ongoing phase study, cr-air- (nct ), infusion of atir at days post-hsct results in a reduction of transplant-related mortality (trm) and improvement of overall survival and event-free survival. adjunctive treatment with donor lymphocytes in patients receiving a t-cell depleted, haploidentical hsct for nonmalignant diseases such as beta thalassemia major, could provide early immunological support and better immune reconstitution in the absence of gvhd. in an open-label, multicenter phase study (cr-bd- ; eudract - - ), patients age ≥ years and ≤ years with beta thalassemia major will undergo a haploidentical hsct with adjunctive administration of atir . patients will receive a t-cell depleted graft (cd -selected, or cd /cd depleted, or tcr-αβ depleted, depending on the experience of the study center) from a related, haploidentical donor, patient conditioning will be myeloablative following standard practices at the study center. atir infusion at a dose of × viable t-cells/kg is given between and days after the hsct. to assess safety, patients will be evaluated for the occurrence of dose limiting toxicity (dlt), defined as acute gvhd grade iii/iv within days post hsct. efficacy will be primarily evaluated by transfusion-free survival (tfs), occurrence of severe infections, and time to t-cell reconstitution, taking into account hematologic and sustained engraftment. all patients will be closely monitored for cmv, ebv and adenovirus titers, with initiation of pre-emptive treatment upon rising blood titers. regulatory authorities in the united kingdom and germany have approved this clinical study protocol. enrolment of the study is expected to continue during , with first report of safety of atir to be expected first half . disclosure of conflict of interest: j. rovers is employee of kiadis pharma, sponsor of the study. sickle cell disease (scd) can be cured with haematopoietic cell transplantation (hct), yet progress in the practice and research of hct for scd has not come without risks and uncertainty. the information and decisions that families and physicians encounter in this field are complex and hanging. in this hermeneutic study, we analyze the case of one family who advocated for hcts for two of their four children knowing the potential risks. these experiences have had a profound impact on both the family and the medical team. this study was conducted through the research method of hermeneutic phenomenology. hermeneutic inquiry is described as the practice and theory of interpretation and understanding in human contexts and aims to make sense of the particulars of these contexts and arrive at deeper understandings. data collection: in-depth interviews were conducted with the mother of the family, the hct nurse coordinator, and the hct physician. the interviews were audiotaped and transcribed verbatim. the transcribed interviews were later reviewed by the physician, who then wrote an additional reflection. this work culminated in approximately pages of single spaced data in textual form. in hermeneutics, interpretation takes place through a careful reading and re-reading of the data, looking for statements and instances that resonate with the researcher. initial individual interpretations of researchers are then raised to another level of interpretive analysis in the research team's communal attention to the data. particular criteria guide the analysis: agreement, coherence, comprehensiveness, potential, and penetration. the following excerpts and interpretations are provided as examples of the analysis, with names changed for confidentiality. "being heard" arose repeatedly in this family's experience, including at the time of their request for a transplant without meeting the traditional criteria for hct. they persisted in their belief that their children would benefit from hct. "they gave us options to see if there was a chance for a transplant...how life would look…. and then we figured…a transplant for him was better at the time…worth the risks…. and you wouldn't even know. he plays basketball now, he plays sports, he's active and he can exercise and run. i never had any regrets because i felt it was better and the most important thing is his organs were really intact; none of the organs were destroyed…so i think it's the right decision we made" (mother). "this family has changed my career, and my life as a result. they challenged my practice and way of thinking. they did so in a considerate way, out of a duty to advocate for their children. we worked through the tension of different viewpoints with respect and all of us grew in the process. at least i can say our team did. i certainly did... i am humbled by their trust and respect…i am grateful to them" (physician). patients and providers are deeply impacted by their interactions. dr. robert buckman stated that it was the individual case that changed his practice always. he claimed he could not walk into a new patient's room without his practice being forever changed. in presenting this hermaneutic analysis, we aim to remind ourselves of the opportunity for growth that can result from reflection on this sacred patientprovider relationship. disclosure of conflict of interest: none. defibrotide (df) prophylaxis and adjustment of busulfan schedule to prevent veno-occlusive disease and thrombotic microangiopathy in an infant with a membrane cofactor protein (mcp) gene mutation and metachromatic leukodystrophy undergoing hematopoietic stem cell gene therapy (hsc-gt) v calbi , , f fumagalli , , , r penati , g consiglieri , m migliavacca , , d redaelli , s acquati , v attanasio , r chiesa , f ferrua , , f barzaghi , , m cicalese , , a assanelli , , p silvani , s tedeschi, r arora , a soman , f ciotti , m sarzana , g antonioli , , c baldoli , s martino , gl ardissino , mg natali sora , l naldini , , f ciceri , , a aiuti , , and me bernardo hepatic veno-occlusive disease (vod) and thrombotic microangiopathy (tma) are life-threatening complications that can occur after hsc transplantation. expert consensus guidelines support use of df for treatment and prophylaxis of vod due to its ability to restore thrombo-fibrinolytic balance and protect endothelial cells. presymptomatic monozygous twins affected by late infantile metachromatic leukodystrophy (mld) underwent investigational hsc-gt after conditioning with busulfan. no comorbidities were evident at baseline. the dose of transduced cd + cells was similar in both patients ( . × cd +/kg for patient and . × cd +/kg for patient ). patient (p ): at months of age, received conditioning with iv busulfan mg/m /dose for doses (target auc mg × h/l). on day (d) + after gt, he developed severe vod and was treated with diuretics, fresh frozen plasma, paracentesis and df. on d+ schistocytes in peripheral blood, marked proteinuria, complement factor consumption, and increases ldh and bilirubin were observed. the patient's condition worsened, with reduced urine output and generalized oedema with pleural effusion. stool, urine and blood cultures were negative and adamts activity was %; anti-complement factor h (cfh) antibodies (ab) were positive ( ui/ml). these findings led to the diagnosis of atypical hemolytic uremic syndrome (ahus; a form of tma) and eculizumab ( mg/weekly dose) was started on d + . patient subsequently developed pulmonary oedema and needed non-invasive ventilation. molecular analysis revealed a heterozygous deletion of cfhr -r and ala val mutation in the mcp gene, a defect previously shown to be associated with ahus. due to the presence of ab anti-cfh and antiplatelet, weekly doses of rituximab ( mg/m ) were administered. after treatment, p progressively improved although he showed prolonged severe anaemia and thrombocytopenia and bone marrow (bm) hypoplasia, secondary bleeding which required reinfusion of unmanipulated autologous bm cells on d + . nine months after hsct-gt p has shown good hematopoietic recovery, stable engraftment of the transduced hscs, no signs of renal damage or complement activation, albeit with neurodevelopmental delay. patient (p ): given his twin history and genetics, this month old infant was considered at increased risk of vod, so prophylaxis with df ( mg/kg/d) was administered from d- to d+ and the busulfan conditioning was modified by adjusting the auc to a lower target ( mg/kg/dose for doses; target auc . mg × h/l). the child had a good clinical recovery and didn't develop signs of vod or tma after hsc-gt. on d+ and + , respectively, anti-cfh and anti-platelet ab were positive. considering the history of the twin, weekly doses of rituximab were administered. p is currently months after gt with persistent engraftment of transduced hscs and no signs of tma. data from this case-control report of monozygous twins diagnosed with mld, and subsequently shown to also harbor mutations in complement regulator gene, suggest that df prophylaxis and busulfan adjustment may have helped prevent systemic microangiopathic damage in the second twin. patients with rare disease may have mutations in genes in addition to those that cause their disease. patients at risk of post-transplant tma following hsc-gt for genetic diseases may require tailored df prophylaxis and treatment. disclosure of conflict of interest: a. aiuti is the principal investigator of the tiget-mld clinical trial of gene therapy. the mld gene therapy was licensed to glaxosmithkline (gsk) in and gsk became the financial sponsor of the trial. all authors declare no other competing interests. hematopoietic stem cell transplantation (hsct) using an optimized conditioning regimen is essential for the longterm survival of patients with inherited bone marrow failure syndromes (ibmfs). we report hsct in children with fanconi anemia (fa, n = ), diamond-blackfan anemia (dba, n = ), dyskeratosis congenita (dc, n = ) and shwachman-diamond syndrome (sds, n = ) from a single hsct center. the graft source was peripheral blood stem cells (n = ) or cord blood stem cells (n = ). fa, dc and sds patients received reduced-intensity conditioning, while dba patients had myeloablative conditioning. the median numbers of infused mononuclear cells and cd + cells were . × /kg and . × /kg, respectively. the median time for neutrophil and platelet recovery was and days, respectively. there was one primary graft failure. after median follow up years the overall survival was %. the incidence of grade ii-iii acute and chronic graft versus host disease (gvhd) was % and % respectively. in a multivariate analysis, the type of conditioning regimen was the only factor identified as significantly associated with grade ii-iii acute gvhd (p = . ). we conclude that hsct can be a curative option for patients with ibmfs. disease specific conditioning regimen was important to disease the transplant-related mortality. [p ] disclosure of conflict of interest: none. homozygous sickle cell disease (scd) patients suffering from end-stage renal disease (esrd) show a variable outcome after kidney transplantation as underlying disease can cause poor allograft survival and disease-specific problems. we present a case of a -year old patient with severe scd and esrd who underwent haploidentical bone marrow transplantation (bmt) with consecutive living kidney transplantion (lkt). the patient suffered from multiple complications of scd including stroke with secondary hemorrhage, symptomatic epilepsy, esrd and uncontrolled hypertension. the patient had been on hydroxyurea without success and required regular blood transfusion due to severe renal anemia. the rationale for bmt was uncontrollable iron overload. a reduced intensity conditioning regimen was used with (fludarabine, cyclophosphamide and gy of tbi, dose-adjusted to esrd). graft-versus-host disease (gvhd) prophylaxis consisted of post-transplant high-dose cyclophosphamide, cyclosporine a (cya) and mycophenolate mofetil (mmf). the donor was her -year old mother with hbs trait, the stem cell source was bone marrow, the cell dose . × nucleated cells/kg. during conditioning daily hemodialysis was performed to keep drug levels stable. neutrophil engraftment occurred on day + , chimerism at day + was %. hbs increased from . % pre-hsct to . % months after hsct. hemoglobin values increased from g/l pre-hsct to g/l post-hsct and reticulocytes from g/l to g/l. erythropoietin levels increased from . iu/l pre-hsct to iu/l months after hsct. during the follow-up, the patient did not show any sign of acute gvhd or vaso-occlusive crisis, hemolysis or sickling. relevant complications were disease-related (therapy resistant hypertension and epileptic seizure due to former brain damage). on day + a lkt from the same donor was performed. the initial immunosuppressive treatment with mmf was continued, cya was switched to tacrolimus and steroids were added for months. the post-transplant period was uneventful. currently, months after haploidentical bmt and months after lkt there are no signs of gvhd, the blood chimerism is %, the kidney allograft function is very good (gfr ml/min/ . m ) and immunosuppression is withdrawn. iron overload is being corrected by regular phlebotomies. the patient no longer requires antihypertensive medication and there is evidence of vascular remodeling. this is the first report of a successful haploidentical bmt followed by kidney transplantation from the same donor in a patient with scd. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) can cure non-malignant diseases, such as primary immune deficiency (pid), severe aplastic anemia (saa) and osteopetrosis (op). in the absence of a well-matched donor, transplantation from a haplo-identical donor maybe considered. post-transplant cyclophosphamide (ptcy) is a new strategy derived from the treatment of malignant diseases in adults that has been little studied in high-risk pediatric nonmalignant diseases. fifteen children ( . years, range: . - . ) underwent hsct in the pediatric immunology and hematology unit of necker hospital, paris, between december and september . these children were suffering from op (n = ), saa (n = ), hemophagocytic lymphohistiocytosis (hlh) (n = ), immunodysregulation polyendocrinopathy enteropathy x-linked (ipex) syndrome (n = ), combined immune deficiency (n = ) and leukocyte adhesion molecule deficiency (n = ). three patients received a low-intensity conditioning regimen (cr) (based on fludarabine, cyclophosphamide, and total body irradiation) whereas the other received myeloablative cr (based on busulfan auc targeted and fludarabine). fourteen patients received serotherapy before hsct. post-transplant cyclophosphamide ( mg/kg/ day) was given on d and d and graft versus host disease (gvhd) prophylaxis with cyclosporine and mycophenolate mofetil was initiated on d . the transplanted stem cells were obtained from bone marrow in all cases. engraftment with full donor chimerism was observed in patients. the median cd + cell dose was . × cells/kg body weight (range: . - . × ). neutrophils recovered after a median of days (range: - ), and overall survival (os) was % after a median follow-up of year (range: . - . ). three patients died due to graft failure (n = ) or infectious complications related to gvhd (n = ). grade ii acute gvhd occurred in of the patients displaying engraftment ( %), and chronic gvhd and/or autoimmune complications were observed in four patients ( %). viral complications were frequent, occurring in patients ( %) with cmv infection (n = ) /disease (n = ), adenovirus disease (n = ) and bk virus cystitis (n = ). haploidentical transplant with ptcy is feasible in high-risk patients with non-malignant diseases. chronic gvhd and autoimmunity were more frequent than for more conventional approaches in such patients. infection rates were high. disclosure of conflict of interest: none. sickle cell disease (scd) remains associated with high risks of morbidity and early death. even best of supportive care fails to improve quality of life. hematopoietic stem cell transplant (hsct) can be considered for selected group of patients. in long run it is not just economical but also substantially improves quality of life (qol). we report our experience with hsct for scd from india. seventy three consecutive patients suffering from scd who underwent hsct between january and november were included in the study. fifty two underwent matched sibling donor (msd), matched family donor (mfd), matched unrelated ( / or / ), cord blood transplant cbt ( matched sibling cord blood and matched unrelated) and patient underwent haploidentical transplant. different conditioning regimens were used and so was the graft versus host disease prophylaxis depending on institutional protocols as depicted in table . a total of patients underwent sct. the median age was years ( months- years). m/f ratio was / . majority of patients were either from african union or oman. all patients suffered from one or other severe symptoms making them eligible for sct. graft source was bone marrow (bm) in with median cd count of . x /kg ( . - . ), peripheral blood (pb) in with median cd count of . x /kg ( . - . ), cord blood in with median cd count of . x /kg ( . - . ) and combined bm & pb in with median cd count of . x /kg ( . - . ). of the patients, are alive and disease free with lansky/karnofsky scores of . there were deaths ( msd/mfd/mud; haploidentical and matched unrelated cbt). four patients rejected the graft ( haploidentical and msd/mfd/mud). at the last follow up, the probabilities of survival, scd-free survival, and transplantrelated mortality were %, . %, and %, respectively. outcome of hsct in scd has improved significantly. with better conditioning regimens, improved supportive care, the outcome of alternative donor transplant and adult scd has improved and matches sibling donor transplant. hsct should be strongly considered as a curative modality for selected patients suffering from scd. disclosure of conflict of interest: none. s staff jointly defined more than local standard operating procedures. patients with low-risk characteristics (age ≤ years, liver size ≤ cm below costal margin) and a hla matched sibling donor were considered eligible in this initial phase of activity. a downstaging protocol with hydroxyurea and deferoxamine or deferasirox was adopted. conditioning regimen included iv busulfan and cyclophosphamide. gvhd and rejection prophylaxis included atg from day − to − and csa, mtx and methylprednisolone. gcs-f primed bone marrow was chosen as stem cell source. the first allogeneic hsct of the whole iraq was performed in a child with thalassemia at hiwa hospital in october . up to now, patients ( females, male) underwent hsct; median age at transplantation was years; median infused tnc . × /kg, cd + . × /kg. all of them engrafted. no major complication were observed. one of them developed grade ii agvhd (skin only) which resolved after increasing the dose of steroids. a huge number of patients with low-risk thalassemia are now in the waiting list and some of them have already started downstaging having planned hsct in a short time. a matched sibling transplant program in children with thalassemia is feasible and safe in kurdistan. such a program can provide many advantages: far less psychosocial and financial burden for the families and significant saving for the government. the estimated costs of performing locally hsct are much less than in the countries where patients were previously referred. the continuation of cooperation is of paramount relevance for further implementing the activity and extending the transplant accessibility to patients with other hemato-oncological disorders of childhood. disclosure of conflict of interest: none. long term follow-up after reduced-intensity conditioning and stem cell transplantation for thalassemia major r rihani, a natsheh, sm abu, e khattab, r najjar, f sheab, s sharma, n hussein, a tbakhi and m sarhan bone marrow and stem transplantation program-king hussein cancer center, amman, jordan hematopoietic stem cell transplantation (hsct) is the only curative treatment for thalassemia major (tm). reducedintensity conditioning (ric) before hsct for high risk tm patients results in fewer complications, when compared with myeloablative regimens. one hundred and three tm patients received hscts from an hla-identical related donor at king hussein cancer center, between january and november . of those, were high risk tm ( %) who received ric hscts. in this report, we describe follow-up beyond years (median, ; . - months) post ric hscts. forty-four class ii-iii patients ( %) were identified ( % with hepatitis c); with a median age of ( . - ) years. females accounted for % (n = ). conditioning regimen consisted of oral busulfan mg/kg, fludarabine mg/m ,tli cgy and atg followed by pbsct. gvhd prophylaxis consisted of mmf and csa. median infused stem cell dose was . × /kg. all patients attained neutrophil and platelet engraftment (median, . and . days, respectively). persistent mixed donor or full donor chimerism were observed in . % (n = ) and . % (n = ), respectively. immune-suppressive therapy for gvhd treatment was required in ( . %) patients (agvhd, n = ; cgvhd, n = ). moreover, veno-occlusive disease occurred in patients ( %) that resolved completely. secondary graft failure was noted in ( %) patients. the -year overall survival was %, while the -year probability of thalassemia-free survival was . %. other factors evaluated include: growth parameters, endocrine and other organ functions, in addition to functional status. this report confirms the safety and efficacy of ric regimens in hscts for high risk tm patients. those regimens are associated with excellent engraftment and sustained mixed donor chimerism; and lead to excellent thalassemia-free and overall survival rates. [p ] disclosure of conflict of interest: none. in-time hsct for pts. with hurler syndrome (hs) can significantly improve the results. long-term follow-up and late effects estimation required to prepare a special observation and rehabilitation programs. aim. to analyze our experience with hsct for hs in the field of special observation and rehabilitation programs. forty hsct during the - were performed for pts. with hs. median age at the diagnosis was months ( - months), at hsct- months ( - months). bm used in . % (n = ), pbsc- . % (n = ), cb- . % (n = ). mac conditioning was used for hsct, ric-for . ric regimen: flu+mel+atg, mac: bu/treo +flu+thio/mel (bu was used in early ) and atg +rituximab (in case of mud hsct). all pts. with ric received mud hsct, pts. with mac mud- pts., mrd- pts. pts. received csa/tacro-based gvhd prophylaxis. mmf/mtx was additionally added in all cases. in ric hsct immunomagnetic Сd /Сd + depletion of pbsc (by clinimacs) was used. a special observation protocol including somatic and neurocognitive estimation was developed. all pts. engrafted with full donor chimerism on d+ . median of engraftment day- ( - days). thirty three pts. survived. reasons of death-mac: infections- pts., ric: trali- pt., agvhd- pt. trm improved, over the years, with improving of supportive care and donor selection as well as pre-transplant screening. no early severe toxicity revealed. pulmonary infection episodes was registered in % of pts. in our study. gvhd: grade iideveloped pts., grade iii-iv- pts. (after ric), local cgvhd- pts. (ric). no extensive cgvhd. pts. rejected (mac and ric rejection rate was same). at median follow up of months ( - months), the estimated years pos was %. best response correlated with early hsct (and better status before hsct) and higher level of aidu after. late effects estimation showed that . % (n = ) of patients experienced late effects: cardio-vascular- pts., skeletal- pts., endocrine- pts. all pts. with cardio-vascular effects received mac. skeletal effects affected patients of older age, pts. transplanted in younger age do not have such effects. median period of late effects arising after hsct was month ( - months). only pts. experienced serious pulmonary late effects (infections), all episodes was before . no pts. in our study have progressive retinal degeneration. % of pts. improved in the neurosensory component and all pts. improved in neurocognitive status and development after hsct. best response correlated with neurocognitive rehabilitation based on unique computer model used by our group in russian national rehabilitation center "russkoe pole." in-time hsct is an effective and safe way to stop neurodegenerative process for pts. with hs. both mac and ric regimens can be used with the same effectiveness. mac regimens associated with bigger number of cardiovascular late effects. long-time follow-up showed that these patients require the special observational protocol including estimation of cardio-vascular, skeletal, endocrine and neurocognitive risks. better neurocognitive response correlated with intensive rehabilitation using computer model. russian joint study showed effective cooperation for treatment pts. with hs in the national setting. disclosure of conflict of interest: none. little is known about pathogenesis of solid tumors after hsct but, intensive cytotoxic conditioning therapy with defective dna repair of persisting stem cells/stromal cells, viral infection, and immunosuppression may play a role. / patients with solid tumors had a melphalan-based conditioning. melphalan was linked to sarcoma and lung cancer in animal model. there are few data linking parotid mec to infection by cmv and hhv which can remain dormant in the salivary glands. both affected patients had hhv during the transplant period. p and p had a family history of solid tumor pointing to a possible genetic factor. whilst secondary malignancy post-hsct for patients with malignant disorders is well recognised, non-ptld malignancy post-hsct for pid has not previously been reported. a larger study is needed to evaluate incidence and risks. allogeneic hsct is a treatment of choice for the bone marrow failure in patients with sds. hsct from unrelated or mismatched family donors is associated with higher morbidity and mortality compared with matched sibling. combined pgd and hla antigen testing is a possible option to preselect a compatible donor for an affected sibling requiring hsct. we describe a case report demonstrating first successful hsct for years girl with sds by using preimplantation genetic diagnosis and hla matching. diagnosis of sds was suspected at m.o., based on clinical features, family history, laboratory studies. at m.o., bone marrow (bm) aspiration revealed hypocellular marrow with signs of dysplasia and expansion of blasts ( . % blasts). the sanger sequencing of sbds gene showed c. - ta ct and c. + t c mutations. the patient had recurrent infections, including bilateral pneumonia caused by phaeohyphomyces, bloodstream infection, cmvdisease. due to the lack of matched related or unrelated donors, hsct with ric (flu, mel, atg) from haploidentical father was performed at months of age. after the st allo-hsct, engraftment was achieved on d+ , initial str study showed full donor chimerism. post-transplant period was complicated with severe cmv-infection and signs of secondary hlh. at d+ , graft rejection was registered. the girl became dependent on regular rbc and platelet transfusions, bm examination revealed hypocellularity with moderate signs of myelodysplasia without elevated blast count. due to lack of available hla-compatible donors, an option of in vitro fertilization (ivf) with preimplantation selection of a normal hla-matched embryo was considered. after controlled ovarian hyperstimulation embryos were hla-compatible and healthy (first, wild-type; second, heterozygous for sbds gene mutation c. - ta ct). hence, the only unaffected hla-identical embryo was transferred resulting into full-term pregnancy. at the age of . years after st hsct, the nd s transplant was performed with a combination of cb and bm as a source of hematopoietic stem cells. the donors' age was years a reduced toxicity conditioning regimen (rtc) based on flu mg/m , treo g/m , thiotepa mg/kg with serotherapy (thymoglobuline . mg/kg) was used. because of neurotoxicity, arterial hypertension, since d+ csa was changed to sirolimus +mmf for gvhd prophylaxis. the total number of infused nc was . × /kg; cd +, . × /kg; cd +, . × /kg. engraftment was achieved on d+ . any signs of gvhd, severe infectious or toxic complications were not observed. eight months later, the patient is alive, has full donor chimerism in bm and is not transfusion-dependent. in the absence of hla-identical donor, ivf with preliminary pgd and hla-typing could be a chance for matched donor to cure patients with non-malignant genetic diseases. in case of low cord blood cellularity, a combination of cb and bm from the same sibling could be used. our experience showed a successful engraftment of sds patient and stable donor chimerism after second hsct of cb and bm from pgd-selected sibling with rtc. disclosure of conflict of interest: none. the safety and efficacy of familial haploidentical (fhi) stem cell transplantation utilizing cd enrichment and cd addback in patients with high risk sickle cell disease (scd) ( figure a ). probability of yr efs is . % (ci : - %) ( figure b ). immune cell reconstitution has been robust and similar to rtc and msd allosct in scd (table ). there have been deaths, vod, steroid refractory agvhd and cgvhd. mac followed by fhi utilizing cd enrichment and t-cell addback in patients with high-risk scd is safe, tolerable and results in long-term donor chimerism and absence of scd symptoms or complications. a larger cohort and follow-up will be required to confirm these preliminary findings. disclosure of conflict of interest: none. supported by r fd - a . [p ] s lymphoma p a clinical prognostic index for assessing patients aged being considered for high-dose therapy and autologous stem-cell transplant in relapsed or refractory high-grade non-hodgkin lymphoma d edwards , k kirkland , r pearce, s robinson and g cook bsbmt patients with relapsed high-grade nhl or disease refractory to first-line therapy can still be cured with high-dose therapy and autologous stem cell transplant if they respond to salvage chemotherapy. this aggressive algorithm is accepted in younger patients but is less well established in the elderly. age has a negative predictive score in the international prognostic index (ipi) and there are concerns that the outcomes of hdt in these patients are significantly worse. deciding which older patients will benefit from hdt is challenging and there are no established predictive tools to guide physicians. we present a clinical prognostic index derived from information readily available at the time a patient is being assessed for asct the bsbmt audited the outcomes for uk patients aged transplanted between - (n = ) and benchmarked against the european bone marrow transplant (ebmt) database for the same period (n = ). the primary outcome was progression-free survival (pfs) but data was also analysed for overall survival (os), relapse rate (rr) and non-relapse mortality (nrm). we included all patients with a diagnosis of high grade nhl and the following demographic features were also analysed: age at diagnosis; age at transplant; m/f; year of transplant; cr/not cr at transplant; no. of prior therapies; no. of cells infused; clinical staging; karnofsky status at transplant; histology; ipi at diagnosis; mobilising regime and conditioning regime. candidate prognostic indices were factors achieving significance in univariate and multivariate analyses of the main outcomes by regression analysis. the best prognostic index was selected based on the bsbmt dataset and then applied to the rest of the ebmt dataset (the validation dataset). there were no significant differences in patient characteristics between the uk and non-uk groups nor in outcomes of pfs, os, rr or nrm. (figure ). in both univariate and multivariate analysis the following features were associated with a significantly worse outcome for pfs, os, rr and nrm : age , karnofsky score. disclosure of conflict of interest: none. underwent an allo-sct at our center after a treosulfan-based conditioning regimen. eleven pts received a mrd, pts a mud, and pts a haplo unmanipulated pbsc allo-sct. at allo-sct pts were in cr, pts were in pr, and pts had sd/pd. hct-ci was evaluable for pts, had a score ≥ . the backbone conditioning regimen consisted of treosulfan g/m from day − to − , and fludarabine mg/m from day − to − ; twenty-five pts were treated with this reduced toxicity conditioning (rtc) regimen. intensification with other alkylating agent (melphalan, thiotepa, or cyclophosphamide) or radiotherapy ( gy total dose) was applied on the remaining pts (myeloablative conditioning, mac). gvhd prophylaxis was based on cyclosporine a and methotrexate ( pts) or rapamycin and mycophenolate mofetil ( pts), plus anti-thymocyte globulin or post-transplant cyclophosphamide accordingly to donor type. median numbers of infused cd +/kg and cd +/kg were . × (range: . - . ) and . × (range: . - . ), respectively. median follow-up was months (range: - ). thirty-nine pts were evaluable for engraftment; median time to neutrophil ≥ . × /l was days (range: - ), and days (range: - ) to platelet ≥ × /l. treosulfan conditioning provided a cr in and pts respectively in pr and sd/pd at transplant. no graft failure was observed. one and years overall survival (os) was . % and . %, respectively. progression free survival (pfs) and gvhd-free/relapse-free survival (grfs) were respectively . % and . % at year, . % and % at years. one and years relapse/progression incidence (ri) was . % and . %, respectively. transplant related mortality (trm) was . % at days, . % at year and for the entire follow-up. the -day cumulative incidence (ci) of agvhd grade ≥ was . %; ci of moderate to severe cgvhd was . % at years. the outcome of pts in cr at years was significantly better compared to that of pts with active disease in terms of both os ( . % vs . %, p o . ), pfs ( . % vs %, p o . ), grfs ( . % vs . %, p o . ), and ri ( . % vs %, p o . ). no statistical differences in os, pfs, and ri were found when pts were stratified according to donor type and [p ] the use of rtc or mac regimen. at last follow-up, patients are alive and disease free; of them obtained a durable cr using chemotherapy and/or dli for disease progression after allo-sct. treosulfan-based conditioning regimen is effective and well-tolerated in patients with advanced b-nhl undetgoing allo-sct. disclosure of conflict of interest: none. systemic anaplastic large cell lymphoma (salcl) is a very infrequent well-defined histological entity that comprises around % of all t-cell non-hodgkin lymphoma. in the absence of prospective clinical trials, autologous stem cell transplantation (autosct) is considered the standard of care as consolidation therapy after first line therapy for those patients not expressing the alk protein (alk neg salcl) and for patients with relapsed disease. the objective of this retrospective analysis was to analyse the long-term outcome of patients diagnosed with salcl and being treated with autosct during the course of the disease, making special emphasis on the potential impact of the administration of brentuximab vedotin (bv). eligible for this study were patients years or above with salcl who underwent autosct between to and were reported to the ebmt. baseline patient, disease, and transplant data were collected from ebmt med-a standard forms. centers with potentially eligible patients were contacted to provide additional treatment and follow-up information including a written histopathology report for central review. seventy-nine patients ( males) with a median age at diagnosis of years (range: - ) and at transplantation of years were included in the final analysis. thirty-nine patients were alk negative, alk positive and in patients expression of alk protein was unknown. at diagnosis, patients ( %) presented with advanced stage and ( %), with b symptoms. sixty-three patients ( %) received - lines of therapy before autosct. ten patients were treated with bv at some point before autosct; two patients as second line therapy, three as third line, one as fourth line and four as fifth line therapy. the median number of bv doses was (range: [ ] [ ] [ ] [ ] [ ] [ ] . the median time between diagnosis and transplantation was months (range: . most patients had chemosensitive disease at autosct [ patients ( %)] and in all but patients peripheral blood was used as the source of stem cells. conditioning regimen consisted on beam / beam-like protocols in patients ( %). all patients engrafted. with a median follow up for surviving patients of months (range: - ), patients are alive ( %), patients died ( %) and patients ( %) are lost for follow up. disease relapse after transplantation was the most frequent cause of death after the procedure. cumulative incidence of non-relapse mortality for the whole series was % ( % ci, . - ) at days, year and years. cumulative incidence of relapse was % ( % ci - ) and % ( %ci - ) at and years, respectively. and years progression free survival (pfs) was % ( % ci - ) and % ( % ci - ), respectively and and -years overall survival (os) was % ( % ci - ) and % ( % ci - ), respectively. there were no significant differences in any of the outcomes between bv treated and non-treated patients. autosct results in a promising pfs and os in patients with salcl. the potential impact of the administration of bv as salvage strategy before the procedure needs to be further elucidated. disclosure of conflict of interest: none. coeliac disease (cd) is a t-cell immune-mediated enteropathy to dietary gluten, characterized by small bowel villous atrophy resulting in malabsortion. the enteropathy is reversible with a gluten-free diet (gfd), however symptoms and signs which persist year are defined as refractory coeliac disease (rcd). rcd is divided into type i and ii, depending on absence/ presence respectively of clonal intra-epithelial t-lymphocytes (iels) with an aberrant phenotype (cytcd pos, membranous cd , cd and cd neg). rcdii patients have a year survival of . , plts ) was successful at a median of . (range: - ) days and no transplant-related mortality occurred. all patients achieved a clinical complete remission, with normalization of nutritional indices at days, but persistently abnormal iels and clonal t-cells on duodenal biopsy. with a median follow-up of . (range: - ) months, patients remain in clinical remission, patient relapsed with rcd and no patient progressed to eatl. chemotherapy and asct is a safe and effective strategy for the treatment of rcd offering the possibility of sustained clinical responses. clonal tcr in duodenal biopsy/blood and iel flow cytometry form part of the patient evaluation prior to the chemotherapy/asct program. most patients with hodgkin lymphoma (hl) are cured with conventional chemotherapy. however, approximately % of patients relapse after primary treatment. for those, high-dose chemotherapy (hdc) followed by autologous stem cell transplantation (asct) is the standard of care. fifty seven adult patients with relapsed or refractory hl submitted to asct between and were reviewed. variables examined were sex, age, ann arbor stage (i-ii vs iii-iv), b symptoms, bulky disease, extranodal involvement, nodal areas involved (≥ vs vs ≤ months) and response to the treatment prior to asct. log-rank test was used to compare differences in survival for each factor. patients median age was ( - ) years at diagnosis. ann arbor stage iii-iv in ( %) patients, b symptoms in ( %), extranodal involvement in ( %) and bulky disease in ( %). all patients were treated according to the abvd protocol in first line. indications for asct were relapsed disease (n = , . %) and lack of complete response (cr) or progressive disease with st line treatment (n = , . %). there were a median of ( - ) treatment-lines before asct (protocols eshap, ice, beacoop, gvd and others). the disease was chemosensitive in % cases: cr in and partial response (pr) in patients prior to asct. refractory disease (rd) in % (n = ). in . % patients, the hematopoietic cells mobilization was performed under stimulation with granulocyte-colony stimulating factor in hematologic recovery after the cycle of nd line chemotherapy, and most of which required ( - ) apheresis. conditioning regimens were beam ( %) and gmb ( %). the median time to hematologic recovery was days ( - ) for neutrophils /ul and days ( - ) for platelets , / ul. three months after asct, thirty-nine ( . %) patients had cr, one ( . %) patient maintained pr and ( . %) patients had disease progression. status unknown in patients and four ( %) patients died. relapse rate % (n = / ). with a median follow-up time after asct of ( - ) months, median disease-free survival (dfs) was ( - ) months and overall survival (os) was ( - ) months. there were deaths ( . %), four ( %) related to early infectious complications of asct, two ( . %) due to late infectious complications, eleven ( . %) due to disease progression and ( . %) in context of secondary acute myeloid leukemia. response to the treatment prior to asct was the only factor with survival influence. the dfs and os differed significantly in chemosensitive disease compared with rd (dfs mean: vs months,p = . , os mean: vs months, p = . ). the response to salvage treatment prior to asct is the main prognostic factor for survival after asct. prognosis remains poor in patients with rd or early and disseminated relapses. for these patients, the therapeutic approach should include intensive treatment with tandem hdc and stem cell transplantation, allogeneic transplant or early consolidation with brentuximab-vedotin after asct. hodgkin's lymphoma (hl), although considered a curable neoplasm in adults, could be associated with a very poor prognosis when refractory to primary induction therapy or when it relapses within months from an autologous stem cells transplant (auto-sct). the optimal treatment of patients with heavily pretreated/refractory hl is controversial. brentuximab vedotin (bv) is an active single agent in this context; unfortunately, there are no well established therapies when patients fail to respond or progress after bv. encouraging results were recently described with checkpoint inhibitors. similarly, data pertaining to efficacy of bendamustine (benda) shows encouraging activity in various refractory lymphomas. we included in this study adult patients with hl who relapsed post auto-sct and were refractory to or progressed after salvage bv and were treated with benda as salvage therapy with an intention to proceed with an allo-sct. this study was [p ] conducted in two major centers in lebanon, the american university of beirut medical center (aubmc) and makassed university hospital. we identified eligible cases. the primary study endpoint was objective response rate (orr). the secondary endpoint evaluated successful rate of bridging into an allo-sct. the median follow-up times from auto-sct and from benda salvage were ( - ) and ( - ) months, respectively. the median age of patients was years ( - ). all patients had bv as salvage therapy post auto-sct, and all of them progressed after a median of ( - ) cycles. clinical characteristics are outlined in table . patients received a median of cycles ( - ) of benda. the treatment was well tolerated, with rather infrequent adverse events and transient and manageable toxicities. the orr was %, in of patients, with % obtaining a complete response. eventually, of proceeded to allo-sct using a matched related donor, and the remaining patients are planned for allo-sct. only one patient died from disease progression after months post allo-sct. two of patients who progressed following benda received salvage therapy with nivolumab and are being planned for haplo-identical transplant while the third one is being planned for therapy with nivolumab. from the initiation of benda, the median duration of response for the patients was months ( - ); all these patients had maintained a continuous response at the last follow-up examination. conclusion: notwithstanding the limitations associated with our analysis, namely a small sample size and its retrospective nature, these results suggest a role for bendamustine in post bv failures. these findings also provide the basis to evaluate the concept of benda as a bridge to allo-sct in a large prospective study. [p ] disclosure of conflict of interest: none. brentuximab vedotin for relapsed or refractory hodgkin lymphoma, single center experience king faisal specialist hospital and research center, riyadh, kingdom of saudi arabia ms rauf , i maghfoor , a badran , mn zahir and s akhtar hodgkin lymphoma (hl) patients with relapsed or progressive disease after high dose chemotherapy (hdc) and auto-sct have limited curative options. fda granted approval of brentuximab vedotin (bv) for the treatment of hl and anaplastic large cell lymphoma (alcl) patients who fail auto-sct or have had at least prior multiagent chemotherapy regimens and are not candidates for auto-sct. we are reporting single center experience of bv usage in this "approved" setting. medical records were reviewed to collect required data. kaplan-meier (km) method was used to calculate overall survival (os) and progression free survival (pfs) from date of first dose of bv. from - , patients received bv. / had hl ( classic hl-nodular sclerosis, hl-mixed cellularity) and alcl. / ( %) pts were primary refractory or had early relapse after initial treatment. / ( %) pts received bv were refractory to the last treatment. all the baseline characteristics of patients are mentioned in table . median bv cycles administered were ( - ). overall response rate (orr) was % ( patients): cr in ( %), pr in ( %) ( / were primary refractory or early relapsed). median pfs for whole group was months ( % ci, . - . ). km estimated -year os was % and year was %, median os has not been reached yet. for patients who responded, pfs at months was % ( % ci, %- %), median pfs not reached. for / patients with progressive disease (pd) or non responders after bv, median pfs was only months ( % ci, . - . ). there was no difference in os between patients with responders and non responders. median os has not yet been reached in either group as mentioned in survival curves. at the median follow up of months (range: - months) patients are alive, patients are alive without disease, patients received consolidation bone morrow transplant ( auto-sct and allo-sct). patients completed courses and achieved cr. rest of patients who are alive without disease; they had pd on bv but achieved cr with other treatments. patients are alive with disease; patient is on bv and are on another treatment. patients have died, because of pneumonia while being on bv and due to pd. / patients who received bv, achieved cr after failing all previous treatments and are in cr. peripheral sensory neuropathy developed in patients; one required dose reduction. patient stopped treatment due to pulmonary toxicity. we are reporting largest single center data from middle east which confirms that bv as a single agent is effective and safe. overall response rate is lower as compare to pivotal trial but cr rate is comparable to other reported case series. this analysis also concludes that bv can be used as bridge to transplant in patients who don't respond salvage chemotherapy. disclosure of conflict of interest: none. was used to diagnose hiv infections. cox proportional hazards models were used to evaluate risk factors of overall mortality. fifty-six patients with nhl ( . %) and patients with mm ( . %) were positive for hiv antibody. in patients with nhl, overall survival was significantly lower in the hiv-infected patients than in the hiv-negative patients [ - year overall survival: hiv-infected patients, % ( % confidence interval, %- %) vs. hiv-negative patients, % ( % confidence interval, %- %), p o . )]. in a multivariate analysis, hiv infection was significantly associated with an increased risk of mortality (hazard ratio . , p o . ), and this effect was consistent regardless of transplant year. on the other hand, overall survival in patients with mm was similar between the groups [ % ( % confidence interval, %- %) vs. % ( % confidence interval, %- %), p = . ]. previous studies in europe and the united states showed comparable survival rates between hiv-infected and hiv-negative patients with nhl. however, our study showed that hiv infection was associated with a higher risk of mortality in patients with nhl in japan. suppression of t cell-mediated immunity or hiv related diseases might affect transplant outcomes in japanese patients. [p ] disclosure of conflict of interest: none. while beam and beac regimens (bcnu, etoposide, cytosar in both regimens and melphalan or cytoxan, respectively) are commonly used as conditioning high-dose therapy (hdt) in patients with non-hodgkin lymphoma (nhl), there have been few reports comparing these regimens. a retrospective analysis found the superiority of beam over beac in terms of overall survival (os) and event-free survival (efs). toxicities were similar, except that beam was associated with more frequent lower gastrointestinal (gi) mucositis. other studies reported that these regimens had similar efficacy and outcome. recently, a concern regarding cardiotoxicity of beac has risen. the current study aimed to compare efficacy and toxicity of beac and beam as consolidation hdt in young patients with mantle cell lymphoma (mcl) undergoing autologous stem cell transplantation (asct). this is a retrospective analysis of outcomes in mcl patients who received hdt with beam or beac followed by asct at bone marrow transplant centers in israel. os, disease-(dfs) and progressionfree survival (pfs) and regimen toxicity were compared. seventy seven mcl patients who were diagnosed between - / and received consolidation with beac or beam were included in the analysis. forty nine patients were treated with beam and patients-with beac. no significant differences between the groups were revealed in terms of age, sex, the mantle cell lymphoma international prognostic index (mipi) risk score, induction protocol and% of patients transplanted in first complete response (cr ) (mean age yrs in beam vs yrs in beac group; % of patients in beam group had mipi risk score - vs % in beac group; % of patients in beam group were transplanted in cr vs % in beac group). the amount of infused cd cells was significantly higher in the beam group (median cd cells/ kg: . in beam vs . in beac groups; p = . ); the number of days to platelet engraftment was significantly greater in the beac group (median days in beam vs days in beac group; p = . ). there were no differences in the number of blood transfusions or hospitalization days between the groups. the rate of grade - upper mucositis was significantly higher in the beam group ( % in beam vs % in beac group; p = . ); no other differences in toxicity (grade - lower mucositis, pulmonary congestion, infections) were observed between the regimens. non-relapse mortality by day posttransplant was % in both groups. a median follow-up was (range: - ) months. the -yr dfs in beam and beac groups was % and %, respectively (p = . ). there was no difference in the -yr os between the groups ( % in beam and % in beac group; p = . ). there was a trend to improved dfs and os in patients transplanted in cr receiving beam (p = . , figure) . in multivariate analysis, low-to-intermediate mipi and transplant in cr were found to significantly increase pfs (p = . and. , respectively), while the hdt regimen did not affect pfs. beac and beam hdt regimens followed by asct had similar efficacy in mcl patients. there was a trend to improved dfs and os in patients transplanted in cr and treated with beam vs beac. the toxicity profile was similar in both groups, except a significantly higher rate of grade - upper gi mucositis. [p ] disclosure of conflict of interest: none. early or refractory relapsed ( o year) diffuse large b-cell lymphoma has a very poor prognosis especially for those not responding to salvage chemotherapy. allogeneic stem cell transplantation is potentially curative. even though this is less likely in those not responding or having frank progression pretransplantation. methods: at our institution we identified all patients with aggressive b-cell lymphoma (diffuse-large b-cell lymphoma and blastoid mantle cell lymphoma) who were refractory or progressive to salvage chemotherapy with r-dhap and who had peripheral blood stem cells ( × cd +/kg body weight) collected after the st or nd cycle. after high-dose melphalane and autologous stem cell transplantation patients had a partial and a complete remission. patient died due to neutropenic infection, patients died due to progressive disease leading to a transplant related mortality of . %. median progression-free survival after autologous transplantation was . months. proceeded to allogeneic stem cell transplantation. patients had a matched related sibling, had a matched unrelated donor and had a mismatched unrelated donor. transplant related mortality was % in this heavily pretreated population. -year overall survival of all patients intended for treatment is %. one of these patients with relapsed mediastinal lymphoma after allogeneic transplantation was cured by salvage radiotherapy and is in long-term remission ( years). conclusions: salvage high-dose melphalane and autologous peripheral blood stem cell transplantation for diffuse large b-cell lymphoma as a bridge to allogeneic transplantation is potentially curative for a minor fraction of these patients. however, the remission rate of % ( % pr, % cr) and progression-free survival of . months after high-dose melphalane and autologous stem cell transplantation provides a window of opportunity to use new drugs and cellular therapies in these poor prognosis patients. high dose chemotherapy and autologous stem cell transplantation is the treatment of choice for patients with relapsed refractory hodgkin lymphoma. several factors including number of chemotherapy lines received before conditioning, time of relapse and remission status before transplantation can predict survival and pfs in patients undergoing autologous stem cell transplantation. in , we reported on a patients who underwent high dose chemotherapy followed by autologous stem cell transplantation from to . all patients with relapsed or refractory hodgkin lymphoma in the period of - , who underwent high dose chemotherapy followed by autologous transplantation were retrospectively analyzed. the main outcomes of the study were complete remission (cr) at day , overall survival (os) and relapse-free survival (rfs). the impact of the following variables on os and rfs: (a) disease status at the time of transplant, (b) number of chemotherapy lines prior to conditioning and (c) time of relapse months and (d) age. a total of patients were identified. the median age was year. there were . % females and . % males. complete remission (cr) was achieved in . % of patients and . % with chemotherapy sensitive disease at the time of transplantation. prior to conditioning regimen, . % received two chemotherapy lines, and . % received more than two lines. % relapsed in less than months and % relapsed more than months after completion of therapy cr at day was . %. the median os for the whole group was . months; the median rfs was , months. the number of chemotherapy lines significantly impacted os and efs. cr status before conditioning, favorably influenced os and efs with a trend toward better os in favor of those who underwent abmt while in complete remission. the time of relapse and the age did not affect survival outcomes. [p ] the outcome of patients with relapsed or refractory hodgkin lymphoma is favorable and the number of chemotherapy lines received before conditioning is the only factor that had a statistically significant impact on os and efs. since the identification of human immunodeficiency virus (hiv), a clear association between hiv and specific malignancies has been recognized. high-grade b cell lymphomas are the most common malignancy complicating hiv infection and one of three aids defining malignancies. diffuse large b cell lymphoma (dlbcl) accounts for % of cases. before , lymphomas were the cause of % of all deaths attributable to aids. after the introduction of highly active antiretroviral therapy (haart) overall incidence of adm declined, however longer survival and exposure to environmental risk factors have increased the incidence of non adm (adm) such as hodgkin's lymphoma (hl). since haart has improved overall survival substantially, the aim of chemotherapy should be complete remission rather than palliation with careful consideration of drug interactions and side of haart. between and a total of patients were detected hiv positive. twenty-one of these patients were diagnosed with a malignancy and patients referred to our department with a hematologic malignancy were evaluated retrospectively. diagnosis, stage, treatment, survival data were recorded. haart during chemotherapy, nadir cd count and cd count at diagnosis of malignancy was evaluated. four patients were diagnosed with high grade b cell lymphoma, patients with primary central nervous system lymphoma (pcnsl), patient with hl and patient with multiple myeloma (mm). all patients were male and median age at diagnosis was . ( - ). hiv seropositivity was identified during evaluation of malignancy in both pcnsl patients. median duration of hiv seropositivity before diagnosis of malignancy was months for the remaining patients. patient characteristics, treatment modification and cd counts are summarized in tables and . lymphoma was fatal in and the cause of death was identified as lymphoma progression in all patients including one patient diagnosed with hodgkin's lymphoma. a patient presented with multiple plazmositomas was diagnosed with multiple myeloma is currently receiving induction treatment together with haart. hiv related lymphoma patients frequently present with extra nodal disease, incidence of central nervous system involvement is also higher and prognostic score tends to be in the intermediate or high-risk groups. prognosis is also worse than hiv negative population. degree of immunosuppression is implicated and the duration of immunosuppression is directly correlated with the risk of developing lymphoma rather than hiv itself. haart allowed the use of aggressive chemotherapy since it improved immune system and decreased infectious complications. multiple myeloma is a rare neoplasm observed in hiv infection and the treatment is based on data obtained from hiv negative patients. treatment of such patients as well as lymphomas should take into consideration the toxic effects of haart combined with chemotherapy. since hiv positive [p ] patients are excluded from most studies, there are no guidelines to direct treatment and avoid toxicities. drug interactions should be monitored closely and modifications should be made accordingly. interruption of haart may not be mandatory since studies have shown safety of continuation of haart during chemotherapy. for newly diagnosed hiv and malignancy, careful clinical and laboratory evaluation should be made before postponing haart until after chemotherapy. disclosure of conflict of interest: none. the outcome of hdct and asct in refractory hodgkin lymphoma (r-hl) is not as encouraging as in relapsed hl. ten years ago we analyzed and reported outcomes of asct in our r-hl patients, however the follow-up was short. now we a reporting long term outcomes in r-hl after asct in one of the largest numbers reported to date. between and , patients with hl who underwent hdc and asct for r-hl in adult medical oncology (age years) were identified. r-hl is defined as partial response (pr), no response (nr), stable disease (sd), progressive disease (pd), relapsing within months (relapse o m) of finishing the planned (chemotherapy + radiation therapy (xrt)) treatment or refractory to salvage chemotherapy. kaplan-meier (km) method was used to estimate progression free survival (pfs) and overall survival (os) from the day of asct while progression is defined as progression of disease, relapse and death from any cause. all percentages are rounded to nearest. patients underwent hdc and asct during - and of them met the criteria of r-hl. male ( %), female ( %), median age at diagnosis was . years ( - years) and at asct was years ( - years). initial therapy was abvd in ( . %), mopp/copp alternating with abv or abvd in ( %) and others in ( %). ( %) had xrt after initial chemotherapy. response to initial chemo + xrt was pr in ( %), pd in ( %), cr in ( %) ( / relapsed within months and others have refractory relapse) and no response in ( %) and others in ( %). prior to salvage chemotherapy, ( %) had stage iii-iv, ( %) extra-nodal involvement, ( %) bulky disease and ( %) had b symptoms, spleen involvement in ( %), performance status , in ( %). eshap was used as first line salvage in ( %) or rd line ( %). post salvage / prior to hdc and asct disease status was pr in ( %), cr in ( %) and nr/sd in ( %). ( %) patients had a fdg-pet scan prior to asct, ( %) were in cr. beam was used as conditioning regimen. median follow-up for all alive patients is months ( - ) from asct. response rate post asct: cr in ( . %), pr in ( %), nr/sd in ( . %) and pd in ( . %) patients, others /unknown in ( . %). ( %) patients had xrt post auto-sct. type of first post hdc auto-sct event was no event in ( %), persistent disease in ( %), pd in ( %), relapsed disease in ( %), treatment related mortality in ( %) and died of other cause ( %). at last follow-up in november , patients ( %) are alive with no disease, ( %) alive with disease, ( %) died of disease and ( %) died of treatment related mortality or other causes. for entire group, km estimated median os is months, , , , and year survival is %: %: %: %: % respectively. median pfs is . month, , , , and year pfs is . %: . %: %: %: % respectively. we are reporting a very high risk group of patients with a very long follow-up. in patients with r-hl, eshap + beam combination resulted in high response rate ( . %). these remissions are durable. a year os survival of greater than % in our population is higher than most reports with similar numbers. although our cohort has a year os survival of %, % patients have either relapsed or died underscoring need for improvements in the management refractory hl. [p ] disclosure of conflict of interest: none. here we update the previously reported results of our reduced-intensity conditioning (ric) allo-hsct experimental program, initiated in . as of november , in our centre patients underwent ric allo-hsct. donors were hla-identical sibling in , fullymatched unrelated in , or -mismatch-unrelated in and haploidentical relative in . median age was years (range: - ). all patients ( m and f) had stage iib/iv refractory mf (n = ) or refractory ss (n = ). median number of previous treatment lines was (range: - ). source of stem cells was peripheral blood in patients and bone marrow in . median time from diagnosis to hsct was months (range: - ). conditioning included flu/ctx/tbi , pentostatin +tbi and flu/mel in case of hla-identical or unrelated donor, whereas the tt/flu/ctx/tbi regimen was used in the haplo setting. gvhd prophylaxis included csa/mmf in all patients, with the addition of atg in cases with unrelated donor and post-transplant ctx ( mg/kg giorni + e + ) in cases with haploidentical donor. full donor chimerism was obtained in / of the evaluable patients, in a median time of months (range: - ). grade ii-iv acute gvhd occurred in patients ( %), while grade iii-iv was observed in patients ( %). chronic gvhd occurred in patients ( %), being extensive in ( %), all transplanted from hla-identical sibling (no atg). following transplantation, a complete remission (cr) was achieved in out of the evaluable patients ( %), of whom experienced relapse at + and + months, respectively. transplant-related death occurred in patients ( %), of whom were in cr. out of the patients who did not achieve cr, died from progressive disease (median follow-up of months, range: - ), from a secondary malignancy and is still alive with disease months after transplant. of note, all pts who died in progression had chemoresistant disease at time of transplant. at the last follow-up, patients were alive and ( %) maintained cr after a median time of months (range: - ). in the whole population, the -year overall survival was % ( % ci - ) and the -year disease-free survival (dfs) was % ( % ci - ). however, when mf and ss were analysed separately, -yrs dfs were % ( % ci - ) and % ( % ci - ), respectively (figure) . apart from diagnosis, outcome appeared to be primarily associated with the disease status at transplantation, with a -yr dfs of % in the group of patients (n = ) who were in cr before starting the conditioning. after a median follow-up longer than years, we confirm the efficacy of ric allo-hsct as a powerful therapeutic strategy in inducing and maintaining remission in selected patients with chemosensitive advanced-stage ctcl, with results particularly encouraging in ss. [p ] disclosure of conflict of interest: none. outcomes of allogeneic hematopoietic stem cell transplantation for hodgkin lymphomas: a retrospective multicenter experience of the rete ematologica pugliese (rep) f gaudio , p mazza , am carella , d pastore , g pisapia , a mele , p galieni , n cascavilla , g specchia and v pavone hematology, university of bari, bari, italy; hematology, ospedale "san giuseppe moscati", taranto, italy; hematology, ospedale "casa sollievo della sofferenza", san giovanni rotondo, fg, italy and hematology, ospedale "cardinale panico", tricase, le, italy; hematology, ospedale "c. g. mazzoni", ascoli piceno, italy hodgkin's lymphoma (hl) is a potentially curable disease, and modern therapy is expected to successfully cure more than % of the patients. second-line salvage high-dose chemotherapy and autologous stem cell transplantation (sct) have an established role in the management of refractory and relapsed hl, leading to long-lasting responses in approximately % of relapsed patients and a minority of refractory patients. patients progressing after intensive treatments, such as autologous sct, have a very poor outcome. allogeneic sct represents the only strategy with a curative potential for these patients; this study reports a retrospective multicenter experience of the rete ematologica pugliese (rep) over the past years aiming to define the impact of patient, disease, and transplant-related characteristics on outcomes. patients with histologically confirmed diagnosis of hl who received allogeneic sct from to were retrospectively studied. the median age was years (range: - years) and ( %) were male. the majority of patients ( %) had had a prior autologous sct. at the time of allogeneic sct, ( %) patients had a chemosensitive disease and ( %) were chemorefractory. most ( %) patients received reduced-intensity conditioning, % received matched sibling donor and % matched-unrelated donor grafts. the disease status at day post-transplant was reported in out of evaluable patients. of the patients with chemosensitive disease, ( %) achieved a cr, ( %) had a pr or stable disease and ( %) had progressive disease. of the patients with chemorefractory disease achieved a cr ( %), had a pr or stable disease ( %) and ( %) had progressive disease. although the overall survival has improved significantly in mantle cell lymphoma (mcl) according to advanced treatment options, relapsed or refractory disease remains a challenge. recently, lots of targeted agents actively have been tried clinical studies and adapted to clinical practice in indolent lymphoma. however, the role of frontline autologous hematopoietic stem cell transplantation (auto-hsct) has not been fully understood in patients with mcl, compared with a few impressive published data about auto-hsct as salvage treatment option for patients with relapsed mcl. so, we retrospectively evaluated consecutive patients diagnosed mcl, and compared the clinical outcomes of high-dose chemotherapy followed by auto-hsct and conventional chemotherapy alone. between january and december , consecutive patients with newly diagnosed with mcl at catholic blood and marrow transplantation center in south korea were included in this study. all of the patients received high-dose cytarabine-containing regimen or chop with/without rituximab regimen for induction therapy regardless of transplant eligibility. the treatment approach in our institution for patients was based on the physician discretion for transplant eligibility or ineligibility that depend on patient age, comorbidities, and disease status. seventy patients were included in the analysis. initial chemotherapy regimens were consisted of chop (n = , %), r-chop (n = , %), r-hypercvad (n = , %), and hypercavd (n = , %). demographics and disease characteristics of both groups are shown in table . patients received auto-hsct were superior s overall survival (os; p = . ) and progression-free survival (pfs; po . ). the subgroup analysis according to high-risk of mcl international prognostic index (mipi) or bone marrow involvement was performed. between the two treatment arms among the high-risk mcl group, the clinical parameters were not different. the high-risk mcl patients with frontline auto-hsct showed superior os (p = . ) and pfs (po . ) compared with conventional chemotherapy alone. although mcl is classified within indolent lymphoma, frontline auto-hsct can be considered for patients diagnosed with mcl in the group of high-risk mipi or bm involvement with the favorable survival outcomes. disclosure of conflict of interest: none. nasal type extranodal nk/t-cell lymphoma (enktl) is a very rare and agressive malignancy characterized by a poor outcome. current standard therapy is not yet established. the role of high dose therapy followed by haematopoietic stem cell transplantation (hsct) is still controversial. we evaluated the outcomes of all the enktl patients undergoing hsct in a multicenter analysis on patients registered by the société francophone de greffe de moelle et de thérapie cellulaire (sfgm-tc) and compared them with a population of french patients who received chemotherapy alone. sixty four enktl ( males and females) received hsct, including allogeneic (allosct) and autologous transplantations (autosct). median age at the time of hsct was years (range: to years). overall, % of the patients presented with disseminated disease ( % and % in the allosct and autosct, respectively), % were in complete response (cr) at the time of hsct ( % and % in allosct and autosct groups, respectively) and % had received l-asparaginase regimen prior to hsct ( % and % in allosct and autosct groups, respectively). five ( %) and ( %) patients of the allosct and autosct groups underwent upfront hsct therapy, respectively. four patients received tandem autologous/ allogeneic transplants. in allosct, stem cell source was a matched related donor in patients, an unrelated donor in patients and an umbilical cord blood in patients. reduced intensity conditioning regimens (based on fludarabine-busulfan combination) and beam regimen were used in % and % of patients from the allosct and autosct groups, respectively. median overall survival for the whole cohort was . months (range: to months). the -year non-relapse mortality was . % and . % in the allosct and autosct groups, respectively (p = . ). the -year overall survival (os) and progression free survival (pfs) were . % and . % in the autosct and . % and . % in the allosct group, s respectively ( figure a) . the absence of cr prior to hsct was associated with a poor prognosis (p = . ). as compared to allosct, autosct resulted in a better outcome in patients who didn't achieve cr before transplant (p = . ) and tended to have better outcome in high pink risk score (figure b-c) . finally, at years pfs and os of patients who have been treated by chemotherapy alone (ct) (n = ) or followed by allosct (n = ) or autosct (n = ) in cr were %, % and %, % and %, %, respectively ( figure d ). in this french cohort, more patients received autologous hsct in upfront therapy than allogeneic hsct. in cr , there is no evidence suggesting that transplantation is associated to a better outcome than chemotherapy alone. however, a precise matching based on the pink score will be evaluated to ensure that patients who were intensified were not of worst prognosis. in refractory patients there is also no clear advantage to perform allosct when compared to autosct. however, in relapsing disease after ct or autosct allosct, allowed to obtained durable control of the disease. disclosure of conflict of interest: none. high relapse rate is one of concerns for allo-sct in pts with relapsed/refractory aggressive lymphoma. an optimal conditioning regimen designed for aggressive lymphoma may reduce relapse, especially during early post-transplantation period. however, it is not established yet. results of a german phase study of allo-sct with conditioning regimen of fludarabine, busulfan ( mg/kg po or . mg/kg iv), and cyclophosphamide with or without post-transplantation rituximab for relapsed/refractory aggressive lymphoma suggested the role of myeloablative busulfan-containing regimen in reducing relapse rate in pts with aggressive lymphoma. based on these results, we conducted a single institution prospective study to explore feasibility of the bmf regimen consisted of full-dose busulfan, melphalan, and fludarabine in pts with relapsed/refractory aggressive lymphoma (umin ). patients with aggressive lymphoma who achieved at least sd with salvage chemotherapy after experiencing either pd during first-line therapy, early relapse ( o mo) after firstline therapy, late relapse (≥ mo) but refractory to salvage therapy, relapse after auto-sct,; age - ; ecog ps of - ; and without severe organ dysfunction were eligible. donor source could be / matched related or unrelated donor pb/bm or cb with ≤ antigen mismatch; the bfm regimen was consisted of busulfan . mg/kg iv, fludarabine mg/m , and melphalan mg/m (yamamoto h. bbmt ). gvhd prophylaxis was csa + mtx (related pb), tac + mtx (unrelated bm), and tac + mmf (cb). primary end point of the study was survival with engraftment at day , and secondary end points were engraftment rate at day ; nrm and relapse rate at day and y; progression free survival (pfs), overall survival (os), and gvhd at y. protocol was approved by irb and written ic was obtained from all pts. twelve pts (male , female ) with a median age of y ( - ) were enrolled. ps was - in pts. diagnosis were dlbcl (n = ), transformed fl (n = ), enktcl (n = ), ptcl (n = ), and aitl (n = ). median number of previous line of therapy was . ( - ) and pts had failed previous auto-sct. diseases status at transplantation was cr (n = ), pr (n = ), and sd (n = ). donor source was cb (n = ), unrelated bm (n = ), and related pb (n = ). survival with engraftment at day , primary endpoint of the study, was achieved in %. neutrophil engraftment was achieved at a median of day ( - ). full donor chimerism at day was achieved in all of the pts evaluated. two pts developed vod which was manageable. with a median follow-up of mo, pts had progression of lymphoma at , , mo. five pts died and cause of death were progression of lymphoma in , interstitial pneumonitis in (at mo), systemic adenovirus infection in (at mo), and agvhd in (at mo). os and pfs at y were % and %, respectively. relapse and nrm rates were % and % (day ), and % and % ( y), respectively. agvhd of grade ii-iv was observed in / pts and pts developed limited cgvhd. this prospective study shows that allo-sct using myeloablative conditioning regimen with full-dose busulfan, melphalan, and fludarabine for relapsed/refractory aggressive lymphoma is feasible and deserves further evaluation. disclosure of conflict of interest: none. for patients with advanced ctcl, the allogeneic hsct seems to be curative with graft versus lymphoma effect playing a major therapeutical role. in this retrospective study, patients with a median age of years (range: - ) affected by ctcl underwent allogeneic hsct after a median of (range: - ) lines of chemotherapy, including autologous transplant for of them. the median time from diagnosis to hsct was months (range: - ). the diagnoses were: sezary syndrome (ss, n = ). mycosis fungoides (mf, n = ), primary cutaneous cd + lymphoma (n = ), panniculitis-like t-cell lymphoma (n = ), nk t cell lymphoma (n = ). at time of hsct, patients ( . %) were in complete remission (cr), ( . %) in partial remission (pr) and ( . %) had active disease. the patients were transplanted from an hla-identical (n = ), mismatched (n = ) or haploidentical (n = ) sibling, from matched unrelated donor (n = ) or from a single cord blood unit (n = ). different pre-transplant regimens were used as myeloablative (mac) in (th-bu-flu, n = ; bu-cy, n = ) or as reduced intensity (ric) in (th-flu-cy, n = ; th-bu-flu, n = ). al patients engrafted for neutrophils at a median of days (range: - ) and patients engrafted for platelets at a median of days (range: - ). acute gvhd was of grade -i in patients and ii-iv in ( . %). skin was the most common organ involved. five of evaluable patients experienced chronic gvhd which was mild in and severe in . at a median of months (range: - ), patients died ( mac and ric) because of gvhd (n = ), vod (n = ), pneumonia (n = ) or multiorgan failure (n = ). all patients surviving at months from transplant were in cr. only patients prepared with a ric (n = ) relapsed respectively at , , and months from hsct. these patients received dli associated or not to chemotherapy. three achieved cr, which remained stable in , while one patient died in cr from post dli acute gvhd. one patient (nk-t cell) not achieving cr is still alive with active disease. for all patients the median survival was months (range - ). with a median follow up of months (range: - ), patients ( mac, ric) are alive, in cr and with active disease. at years, the os was ± %; at years dfs was ± %. according with the median time ( months) from diagnosis to transplant, the -year os was ± % for patients transplanted early and ± % for the others (p o . ), while dfs was respectively s ± % and ± % (p o . ). despite the small number of patients, our results confirm the high susceptibility of ctcl to the graft versus lymphoma effect and point out the time to transplant as a crucial prognostic factors for the outcome. finally, the long-term follow up of our series strongly supports hsct for the cure of ctcl. disclosure of conflict of interest: none. recently, a new prognostic score, the nccn-international prognostic index (ipi) has been developed to stratify patients affected by diffuse large b cell lymphoma (dlbcl), and in high-intermediate and high risk groups the survival was equal or less than %. the aim of this analysis was to evaluate the outcome of a cohort of dlbcl patients undergoing high dose chemotherapy (hdc) as consolidation following first line chemo-immunotherapy, after their re-classification according to the nccn-ipi. we performed a retrospective study on patients diagnosed with dlbcl, with a high/intermediate or high-risk disease according to the ipi ( - ), who received upfront hdc with asct, in institutions. the patients were then re-stratified according to the nccn-ipi and arbitrarily classified in groups: low risk (nccn-ipi ≤ ) and high risk (nccn-ipi ≥ ). the pre-transplantation disease status was assessed by positron emission tomography (pet) or computed tomography (ct). the primary endpoints were non-relapse mortality, progression-free survival (pfs), overall survival (os) and relapse risk. the estimated -year pfs for all patients was . % ( % confidence interval [ci] . - . ) and the -year os was . % ( % ci . - . ). of these patients, had a low risk ipi score (ipi = ) and were considered high risk (ipi ≥ ). subsequently, the whole cohort was re-stratified according to the nccn-ipi: patients were allocated to the high-risk (nccn-ipi ≥ ) group, and to the low-risk group (nccn-ipi ≤ ). the analyses were then carried out for both groups. the -year pfs was . % ( % ci . - . ) in the low-risk group and . % ( % ci, . - . ) in the highrisk group (po . ), whereas the -year os was . % ( % ci . - ) in the low-risk group and . % ( % ci . - . ) in the high-risk group (p = . ). the significant difference in os and pfs between the two groups was mainly due to the cumulative incidence of relapse at years (graph ): . % ( % ci . - . ) in the low-risk group and . % ( % ci . - . ) in the high-risk group (po . ). non-relapse mortality was comparable in both cohorts: % ( % ci . - . ) for all patients. figure : cumulative incidence of relapse following hdc and according to nccn-ipi. patients affected by high-risk dlbcl still have an unsatisfactory prognosis after treatment with conventional therapy regimens, even in the rituximab era. the -year os and pfs in patients with nccn-ipi score ≥ range: from % to % and from % to % respectively . although this is a retrospective analysis subject to all related biases, our results suggest that upfront intensive therapy with autologous stem cell transplantation may significantly improve the outcome of these patients compared to conventional chemotherapy. the role of hdc in the treatment of dlbcl is controversial. however, new entities or new risk stratifications, as the one reported here, could allow to identify high risk subpopulations that could benefit from this approach. enteropathy-associated t-cell lymphoma (eatl) is an exceedingly rare and often rapidly fatal subtype of peripheral t-cell lymphoma, arising from intraepithelial lymphocytes. eatl type i is associated with celiac disease; type ii occurs in patients without inflammatory pre-conditions (according to who classification now called monomorphic epitheliotropic intestinal t-cell lymphoma (meitl)). surgical debulking and anthracyclinebased chemotherapy (ctx) followed by high-dose chemotherapy (hdctx) and autologous cell rescue (asct) are pursued when possible in this often malnourished and frail patient cohort. yet, even with intensive consolidation relapse occurs in - % of patients. the value of allogeneic hematopoietic cell transplantation (hct) is not clarified as of today due to limited reports. here, we report on a patient with meitl who was rescued with an allo-hct for his nd relapse following prior asct. moreover, we summarize the available literature on the use and outcomes of allo-hct for eatl and meitl. a y old man with spontaneous intestinal perforation was diagnosed with meitl following emergency partial resection of the small intestine. histology revealed infiltration by monotonous medium-sized lymphocytes with abnormal immunophenotype (cd +, cd +, cd +, cd -, cd -, tia- +) consistent with type ii eatl. post-surgical f-fdg pet-ct scan showed abnormal uptake in gastric antrum and pyloric region but no other manifestations. ctx with cho(e)p ( × ) followed by beam hdctx and asct was performed and achieved a complete remission (cr ). however, m post asct disease relapsed and was treated with × dhap, and × dhaox. cr was achieved after the rd cycle of salvage therapy. due to anthracyclineinduced cardiopathy allo-hct could not be performed at that time. m after completion of salvage therapy, disease relapsed again, and was progressive under pralatrexat treatment ( cycle, infusions). by then cardiac function had recovered and therapy was switched to dose-reduced mini-beam ( × ). in cr reduced intensity conditioning (ric; fludarabine, busulfan, atg) and allogeneic hct from a matched sibling donor was performed. ciclosporin a (csa) and mycophenolate mofetil (mmf) were given as gvhd prophylaxis. prompt engraftment in blood (day+ ) and full donor chimerism in the marrow (d+ ) were achieved. immunosuppression was tapered and discontinued on d+ (mmf) and d+ (csa), respectively. f-fdg pet-ct scan at m post-hct showed cr, but at m relapse was suspected (under work-up). only few cases of patients with eatl/meitl treated with allo-hct are reported in the literature (n = , table ), and the value of this highly aggressive therapy is not clear at this point. of note, the patients listed in table were given allo-hct instead of asct. long-term complete remission (cr) could be achieved in / patients, while patients suffered from early relapse and died of the disease (n = before d+ post allo-hct). asct following surgery and ctx appears to cure - % of patients in available series. no treatment concept is available for relapse following asct, and no published data are available for allo-hct for relapse post asct. the disease is exceedingly rare and is afflicted with very poor outcomes. therefore, patients given this aggressive treatment should be reported, even when treatment outcomes are not positive. disclosure of conflict of interest: none. strong graft versus lymphoma effects with low toxicicty of haploidentical hematopoietic stem cell transplantation comparing with hla-identical in t cell lymphomas: a retrospective multicenter study s bramanti, r devillier, s fuerst, b reda, a granata, s harbi, c faucher, i legrand, a santoro, d blaise and l castagna istituto clinico humanitas rozzano consolidation treatment of relapsed/ refractory t-nhl with allogeneic stem cell transplant (sct) is considered a curative options but few patients manage to undergo this procedure, due to the highly refractory nature of the disease. the primary aim of this work is to evaluate the gvl effects among t-nhl with both hla identical and haploidentical donors. we have retrospectively analized the long term outcome of consecutive patients affected by t-nhl, received hlaidentical or t-cell replete haplo-sct with pt-cy, in european centers, between february and october . the patients received nonmyeloablative (nmac) or reduced intensity (ric) conditioning regimen. gvhd prophylaxis consisted of mg/kg of pt-cy (day + and + ) in haplo setting and atg plus cyclosporine a in the hla identical setting. patients characteristics were reported in the table . no differences were founded in the two groups . most of the patients were transplanted in complete remissions but only as consolidation of first line. no graft failure occurred. the cumulative incidence of acute gvhd grade - was % in the haplo setting vs % in the hla id .extensive chronic gvhd was seen in % of haplo, and in % in the hla id . patients had cmv reactivation, hemorrhagic cystitis, and ebv reactivation. after a median follow-up of years os was % and % and pfs was % and % in the haplo vs hla id group see figure . nrm was % in haplo setting and % in hla identical one. the years cir is % and % in haplo and hla id setting respectively. this study confirm a strong anti-lymphoma effect of allo hsct without prohibitive toxicities. haplo-hsct with pt-cy shows low rate of cgvhd in a contest of poor prognosis t-nhl patients. [p ] disclosure of conflict of interest: none. ibrutinib is the first-in class bruton tyrosine kinase inhibitor that has been approved for the treatment of relapsed mantle cell lymphoma. however, despite the high response rate of % including % of complete response, the median duration of response is relatively short with an overall survival of % at months (wang ml, et al, n engl j. med ). we report a single experience of patients with relapsed mcl who underwent allogeneic stem cell transplant (allosct) after ibrutinib monotherapy salvage. all patients had previous autologous stem-cell transplantation (asct) before and were given ibrutinib at a dose of mg daily after the second or subsequent relapse. all patients had to be at least in pr according to cheson criteria before allosct. patients had an unrelated / ( ) or / ( ) allo-sct from peripheral hematopoietic stem cells after a reduced conditioning regimen with busilvex, fludarabine and antithymocyte globulin in association with zevalin according to our recent published phase study protocol (bouabdallah k, et al. ann oncol ). graft versus host disease (gvhd) prophylaxis consisted on ciclosporine and methotrexate. patients ( m/ f) were aged from to years and received between and previous chemotherapy regimens including asct in their last treatment strategy before introduction of ibrutinib. all patients had extensive disease with gastric involvement in patients and pulmonary localization in patient. median time between diagnosis and ibrutinib introduction was years ( - ) and the median time between asct and allosct was years ( ) ( ) . median duration of ibrutinib treatment was months ( - ) and it was stopped one week before proceeding to allo-sct. it was not planned to restart btk inhibitor after transplant. patients were assessed for response after at least months of treatment with ibrutinib. at time of evaluation, all patients were in complete ( ) or very good partial response ( ) before allosct. the patient in partial response had % tumor reduction with persistent gastric ulcer where histology examination shows cd + but negative cycline d lymphoid cells. all patients engrafted (median duration of pnn o g/l = days ( - ) and median duration of platelets o g/l = days ( - )) with fulldonor chimerism at month. one patient had a grade ii cutaneous chronic gvhd (cgvhd) with favorable outcome and developed months later a bronchopulmonary obstruction syndrome related to cgvhd. with a median follow-up of months ( - ) after allo-sct, all patients are alive in cr. one patient, in complete metabolic response before transplant had a gastric relapse months later but achieved again a cr months after reintroduction of ibrutinib. after the first case reported by furtado m et al (leuk lymphoma ), we report here additional cases with longer follow-up after allogeneic transplantation. the excellent tumor control after treatment with ibrutinib together with a very good outcome after allosct should drive to consider this approach in young patients with mcl relapse after asct. disclosure of conflict of interest: none. autologous hematopoietic stem cell transplantation (autosct) is considered the standard approach for high risk or relapsed/ refractory non-hodgkin and hodgkin lymphoma. although a large variety of conditioning regimens are available, including the widely used beam (carmustine, etoposide, cytarabine, melphalan), there is no consensus regarding a standard approach. in the context of carmustine shortage, we have chosen to replace it by thiotepa. however, clinical data about thiotepa-based autosct conditioning are still sparse, except some retrospective data for primary central nervous system lymphoma. thus, we designed a multicenter prospective study (nct ) to assess the efficacy and toxicity of a team (thiotepa, etoposide, cytarabine, melphalan) conditioning regimen. team regimen consisted in total dose thiotepa of mg/kg on day- ; etoposide mg/m / h and cytarabine mg/m / h (day- to - ); melphalan mg/m on day- . patients underwent autosct with team conditioning, and were included in this analysis if they have fullfilled the following criteria: age older than years, biopsy-proven hodgkin or non-hodgkin lymphoma, hiv seronegative, and first autosct. thirty-three male and nine female with a median age of years (range: - ) were analyzed thus far. karnofsky score was g/l was days (range: - ). of note, patients received thrombopoietic agents after engraftment because of persisting thrombocytopenia. the most significant regimen-related toxicities were mucositis in % of patients (median grade = , range: - ) and diarrhea in % of patients (median grade = , range: - ). other non-hematologic grade adverse events occurred in patients ( %) and no grade adverse events were observed. central line-associated bloodstream infection occurred in patients ( %). surprisingly, / evaluable patients ( %) developed human herpesvirus reactivation. only patients required intensive care unit transfer. the median duration of hospital stay was days (range: - ). after a median follow-up of months (range: - ), the non-relapse mortality (nrm) was %. only one patient relapsed of refractory aitl months after autosct and died month after. the estimated -year overall survival and progression-free survival were % and %, respectively. a team conditioning regimen seems to be a safe and valid platform in autosct for patients with high-risk or relapsed/ refractory lymphoma. although mucositis and diarrhea were frequent, there were no grade adverse events and no deaths related to the treatment. updated results with updated followup will be presented. disclosure of conflict of interest: none. the cell of origin has no prognostic impact on high-dose chemotherapy with r-beam and autologous stem cell transplant for diffuse large b cell lymphoma s lozano cerrada, r saliba, s srour, s ahmed, c hosing, r champlin and y nieto university of texas, md anderson cancer center, department of stem cell transplantation and cellular therapy diffuse large b-cell lymphoma (dlbcl) is a biologically heterogeneous disease that can be classified according to its cell-of-origin (coo). the germinal center b-cell (gcb) subtype has better outcome with frontline r-chop than the activated b cell (abc) subtype. however, the prognosis of these two types of dlbcl after high-dose chemotherapy and autologous stem cell transplant (asct) is less clear. the purpose of our study was to evaluate progression-free survival (pfs), event-free survival (efs) and overall survival (os) in a cohort of dlbcl patients treated with r-beam (rituximab, carmustine, etoposide, cytarabine, melphalan) and asct according to coo. we have the dicep regimen effectively reverses the poor outcome for lymphoma patients with suboptimal response or failure post st salvage treatment p kaloyannidis the outcome of patients (pts) with refractory hodgkin's (hl) and non-hodgkin lymphomas (nhl) post st salvage treatment (salv ) is considered poor. the published data, have shown extremely low survival rates ( - %) even after nd salvage treatment (salv ) followed by autologous stem cell transplantation (asct), due to the low response rates post salv and the high relapse rates post asct, confirming that the management of these pts remains a major challenge. we herein evaluated the dicep regimen [dose intesified cyclophoshamide ( gr/m ), etoposide ( mg/m ) and cisplatin ( mg/m ), days - ] as a salv treatment, in terms of safety and efficacy regarding disease response and stem cell mobilization/collection. moreover, we evaluated pts' long term outcome post asct. we retrospectively analyzed the data of ( hl, nhl) pts, with a median age of ( - ) yrs. twenty-one had suboptimal response ( % reduction): and minor response (≤ % reduction): ). three pts had stable disease while experienced progression. overall / pts underwent asct after a median of days (range: - ) post dicep. no pt was considered ineligible for the asct due to unacceptable toxicity post dicep; did not undergo asct because of progressive (n = ) or stable (n = ) disease. the -yr overall survival (os) was % for the whole cohort of pts ( % for hl and % for nhl, p = ns) while the -yr progression free survival (pfs) from dicep administration (± asct) was % ( % for hl and % for nhl p = ns). in particular, for the autografted pts, the -yr os was % ( % for hl, % nhl p = ns) and the -yr pfs was similar, % ( % for hl, % for nhl, p = ns) our data demonstrate that dicep is an effective salvage regimen with acceptable toxicity and no negative impact on the cd + collection. the promising response rates post dicep in combination with the very encouraging pfs rates achieved post asct, in this unfavorable and heavily pretreated group of patients, strongly support the rationale for using dicep as st line salvage regimen in selected pts in order to proceed to a successful asct. for the treatment of aggressive lymphoma, high dose chemotherapy followed by autologous stem cell transplant (asct) is an important component. however, the role of upfront asct in patients with diffuse large b cell lymphoma (dlbcl) is still controversial. furthermore, there is currently no consensus on a single best conditioning regimen for asct in patients with dlbcl. we retrospectively analyzed the records of patients with dlbcl who underwent upfront asct in state of complete remission (cr) or partial remission (pr) from institutions in korea. we evaluated the outcomes and prognostic factors of upfront asct in patients with dlbcl. we compared the outcomes of most widely used two conditioning regimens for asct; carmustine based regimens and busulfan containing regimens. total patients ( . %) achieved cr after asct and overall response rate (orr) was . %. with median follow up of months, patients ( . %) had progression or relapse. the -year overall survival (os) rates and progression free survival (pfs) rates were % and %, respectively. infection events were found in patients ( . %) and treatment related mortality was . %. these outcomes were comparable with the results of previous other studies. cox multivariate analysis for os showed that eastern cooperative oncology group performance status (ecog ps) ≥ (p = . ) and rituximab based induction therapy (p = . ) were significant prognostic factors. in addition, the following factors were significantly associated with pfs in multivariate analysis; female (p = . ), ps ≥ (p = . ) elevated β -microglobulin (p = . ), failure to achieve cr with induction chemotherapy (p = . ), carmustine based conditioning regimen (p = . ) and melphalan based conditioning regimen (p = . ). there were no significant differences in os and pfs according to stage, b symptom, bulky disease, high lactate dehydrogenase, bone marrow involvement, high or high-intermediate international prognostic index (ipi), absolute lymphocyte count and absolute monocyte count. therefore, it is considered that upfront asct can overcome the poor prognosis in patients with advanced stage or high risk ipi. in the analysis with conditioning regimen, neutrophil and platelet engraftment were slower in the carmustine group compared to the busulfan group. there were no significant differences in os between busulfan group and carmustine groups with -year os rates of . % and . %, respectively (p = . ). pfs at years was . % in busulfan group versus . % in carmustine group (p = . ). however, carmustine based conditioning regimen was poor prognostic factors for pfs in multivariate [p ] s analysis (p = . ). in subgroup analysis, busulfan group had significantly higher pfs compared to the carmustine group especially in female patients ( . months vs. months, p = . ), with b symptom ( . months vs. . months, p = . ) and abnormal serum ldh level ( . months vs. . months, p = . ). the outcomes of upfront asct in patients with dlbcl after induction therapy were acceptable. it is considerable in selected high risk patients who achieve cr with induction treatment, and have good performance status at diagnosis. in cases of conditioning regimen, busulfan based regimen resulted in improved outcomes compared with carmustine based regimen especially in patients with disseminated disease or female patients. disclosure of conflict of interest: none. no heavy chain was present in %). the predominant light chain was kappa ( %). patients had bence-jones positive myeloma. received bortezomib as induction therapy before transplant. we analyzed overall survival (os) and progression-free survival (pfs) in groups of patients. we separated the groups according to improvement in grade of response from preasct to postasct. the post-asct grade of response was measured months after asct. the os and pfs were estimated by the kaplan-meier method. pfs was measured from diagnosis to disease relapse and os was measured from diagnosis to death by any cause. results by subgroups of patients are detailed in table . median os and pfs of the whole group was years and months, respectively. if we analyze groups only by their grade of response before asct we find the following results: rc ( years os rate . %, median pfs months); pr/vgpr (median os . years and pfs months); sd/progression (median os . years and pfs months). according grade of response after asct, instead: rc ( years os rate %, median pfs: months); pr/vgpr (median os , years, and pfs months); sd/progression (median os . years and pfs months). in our experience, the grade of response before asct is a capital predicting factor for patients os and pfs. patient in cr before asct that preserve it after transplant, have a median pfs of months, the years os rate being . %. patients in situation of progression after asct have a very dismal prognosis (median os . years, pfs: months), however, patients who change from sd/progression to pr after asct have a median pfs of months and a os of . years. comparing these results we observe that this second group is particularly benefited by transplant. autologous peripheral blood hematopoietic stem cell transplantation in elderly patients with multiple myeloma as a standard therapeutic procedure. is it feasible? a single-center experience l cadievski, s genadieva stavric, z stojanoski, a pivkova veljanovska, d miloska, b kocoski, l cevreska and b georgievski university clinic of hematology, department for hematopoietic stem cell transplantation, university ss. cyril and methodius, skoje, republic of macedonia autologous peripheral blood stem cell transplantation (pbsct) represents a standard therapeutic approach in the treatment protocol of myeloma patients. it is known that multiple myeloma is a hematological disease that is a characteristic for the older population. autologous pbsct ideally should be performed in every myeloma patient, but with the elderly myeloma patients the procedure might be risky if know the possible comorbidities, or the possibility of the body to fully compensate the side effects of the conditioning regimen, the procedure or its possible complications. we present our experience in using high dose conditioning with melphalan mg/m followed by autologous pbsct for elderly myeloma patients, using the age limit od years. our retrospective analysis of our data during years of experience, shows that we have performed autologous pbsct on patients with myeloma at the age of or older. males ( . %), and female ( . %). patients ( %), were diagnosed with igg type myeloma, patients ( %) with iga myeloma, and patient ( %) with light chain myeloma. median age of the patients was . years ( - ). all patients were initially treated with cy-thal-dex regimen. in ( %) patients complete response (cr) was achieved, in ( %) very good partial response (vgpr), and in ( %), partial response (pr). in all patients the mobilisation of hematopoietic stem cells was performed with g-csf, and a median of apheresis procedures were performed, and the average number of collected cells was . × /kg tt mononuclear cells (range: . - . ). days to confirmed engraftment in our group of patients was . (range: - ). the number of blood transfusions was on average . (range: - ), and the number of transfusion of thrombocytes . units (range: - ). in the majority of patients, mainly after the year (that represents patients of the whole group), we used noncryopreserved hematopoietic stem cells, kept under the temperature of c, for median of days, thus avoiding the toxicity of dmso. additionally, we used central venous catheter inserted in the femoral vein for apheresis and application of the stem cells afterwards. the day after, the catheter was removed, thus avoiding catheter associated infections. all patients received standard infectious prophylaxis with fluconasole mg/daily, ciprofloxacin mg/ two times daily, acyclovir mg/ three times daily, cefixime mg/once daily, and ursodeoxycholic acid for vod prevention. no serious infectious complications were reported. our transplant related mortality was %. in the group with noncryopreserved stem cells no graft failure was reported. in two patients we even performed tandem autologous pbsct with no major complications. of the group of patients, the majority, patients ( %), had hta as comorbidity, ( %) with cardiomyopathy, and ( %) with inserted prosthetic aortic valves. three patients ( %) have died because of relapse of the disease. our oldest patents were and years old, and are still alive year posttransplant, in cr. we can conclude the performing autologous pbsct in elderly myeloma patients can be safe and effective therapeutic option, but with careful selection of the patients, balancing the risk profile of the patient and the benefit, or the risk of the procedure. affective supportive care, monitoring and reducing the risk of complications is an imperative to a good result. disclosure of conflict of interest: none. autologous stem cell transplantation program for patients with multiple myeloma in an outpatient setting k lisenko , s sauer , g egerer , j schmier , m witzens-harig , a schmitt , ad ho , h goldschmidt , , j hillengass and p wuchter , department of medicine v, heidelberg university hospital, heidelberg, germany; national center for tumor diseases heidelberg (nct), heidelberg university, heidelberg, germany and institute of transfusion medicine and immunology, mannheim, german red cross blood service baden-württemberg-hessen, medical faculty mannheim, heidelberg university, germany the first and second authors contributed equally. high-dose chemotherapy with melphalan and autologous blood stem cell transplantation (absct) for treatment of symptomatic multiple myeloma (mm) is performed in the usa and canada mostly on an outpatient basis, whereas in germany and western europe an inpatient setting is the standard. we report on a german single-centre program to offer the procedure on an outpatient basis to selected patients. major inclusion and exclusion criteria for eligibility were defined as follows: patients had to be able to reach the hospital within minutes, had reliable support from their family at home, had an ecog performance score of - and were willing and able to comply with the demands of the program. patients with severe co-morbidities were not included. all patients were treated on our outpatients' clinic and examined on daily visits by a team of physicians. feedback from patients was obtained by means of a questionnaire. from september to september , patients with mm stage iiia were enrolled. all engrafted within the expected time range: (median time to leukocyte , /μl and neutrophil recovery μ/l: days; median time to platelet recovery /nl: days, /nl: days). twenty patients ( %) had an episode of neutropenic fever but only in patients ( %) blood cultures were found to be positive. there occurred no cases of infection with multiresistent bacteria. although rather liberal criteria for hospital admission were applied, of patients ( %) could be treated entirely on an outpatient basis. eight patients ( %) were temporarily admitted for inpatient treatment with a median duration of . days (range: - days), mainly because of neutropenic fever. no severe adverse events occurred. feedback from patients revealed a high level of satisfaction with the outpatient setting. high-dose chemotherapy and absct on an outpatient basis is safe and feasible if conducted in a comprehensive surveillance program. the feedback from patients was very positive, thus encouraging further continuation and expansion of the program. disclosure of conflict of interest: none. high dose of melphalan (bor-mel). we retrospectively analyzed patients with mm who underwent asct between january and march . in these patients, conditioning regimen consisted of a high dose of melphalan ( - mg/ m ) intravenously on day - and two doses of intravenous bortezomib at . mg/m administered on days − and + . this cohort was compared with patients underwent asct between and , conditioned with high dose of melphalan alone. response rate was evaluated according to imwg criteria. all patients were evaluated after induction therapy and months after asct. all patients were followed until death or reference date (november, ). results: patients' demographics and baseline disease-related characteristics are shown in table . [p ] no difference was found in terms of neutrophil and platelet engrafment, hospitalization days (p= . ) and use of mechanical invasive ventilation (p= . ). bor-mel regimen did not enhance severity of preexisting peripheral neuropathy (pn) in any patients, and only one presented de novo grade pn. non relapsed mortality was . % and % in the bor-mel and mel cohorts, respectively (p= . ). complete response rate after transplant was significantly better in the bor-mel cohort than in the mel cohort ( . % vs. . %; p= . ) ( figure b) . when the analysis was restricted to patients who received bortezomib-based therapy, this difference was also statistically significant ( . % vs. . %; p= . ) ( figure d ). median of follow-up was months in the bor-mel vs. months in the mel cohort. no difference was found in terms of overall survival (os) and progression free survival (pfs) between both groups. for all patients, a post-transplant deeper response was associated with better os and pfs (p= . and p o . , respectively). our results are in line with previous studies demonstrating that bortezomib combined with melphalan is a well tolerated conditioning regimen and may enhance the response rate after transplant, even in patients receiving bortezomib in the induction therapy. these results should be confirmed in a randomized trial. for newly diagnosed patients (pts) with multiple myeloma (mm), the triple-agent induction treatment based on bortezomib plus dexamethasone in combination with cyclophosphamide (vcd) or lenalidomide (vrd) represent extremely reliable regimens, which in combination with early autologous stem cell transplantation (asct) result in high response rates and prolonged long-term outcomes. however, though both regimens are widely used, there are extremely limited studies that compare the vrd vs. vcd in terms of safety and efficacy. in the present study we compared the outcomes of newly diagnosed mm pts who received induction treatment vrd (n = ) or vcd (n = ) and proceeded early to asct. the vrd and vcd pts groups were similar regarding age at diagnosis ( vs. ys, p = ns), interval between diagnosis-asct ( , vs. , months, p = ns) and maintenance treatment post asct ( vs. pts, p = ns). per revised international scoring system (riss), the vrd-group had slightly more advanced disease (stage i: , stage ii: and stage iii: ), compared to vcd-group (stage i: , stage ii: and stage iii: ), however this difference was not statistical significant. the conditioning regimen consisted of single agent melphalan: mg/m . the t-test, kaplan-meir and cox regression were utilized for the statistical analysis. following a median of cycles of treatment (range: - for vrd vs. - for vcd, p = ns), in the vrd-group pts achieved complete remission (cr), pts very good partial remission (vgpr ≥ % reduction of m-band) and pt partial remission (pr: - % reduction of m-band) while in the vcd-group cr: , vgpr: and pr: pts (p = ns). the toxicities in terms of peripheral neuropathy, myelosuppression, liver and renal function were well tolerated and no patient discontinued treatment due to severe side effects. the -yr overall survival (os) was % for the vrdgroup vs. % for the vcd-group; nevertheless, the difference was not significant due to the size sample of the pt groups. the stage at diagnosis, the disease status pre-asct and the maintenance post-asct did not influence the os. interestingly, the -yr progression free survival (pfs) was significantly superior for patients who had been induced with the vrd regimen ( % vs. % p = . ) and for patients who achieved cr or vgpr before asct (pfs: %) while no pts with pr pre-asct was progression-free yrs post asct (p = . ). in multivariate analysis, only the cr or vgpr status before asct favorably affected the long term pfs. our results are in line with the limited published data from other studies with larger series of patients. in our study, very low disease burden before asct proven to be an independent factor for prolonged pfs. taking into consideration that vrd resulted in more cr or vgpr status, it is reasonable to conclude that vrd is a highly effective regimen and could be first treatment choice for newly diagnosed mm patients who are fit for early asct post induction. lenalidomide cohort and in the maintenance bortezomib cohort. baseline characteristics and outcome data were obtained via chart review. the primary outcome was pfs. the secondary outcomes were overall survival (os) and treatment-related toxicities. the median follow-up time was months. median time to death ( . years vs . , p = . ) and median time to progression ( . years vs . , p = . ) were not significantly different in the maintenance lenalidomide cohort compared to the maintenance bortezomib cohort. in the multivariate analysis, pfs was worse in patients at international staging system (iss) stage at diagnosis compared to those at iss stages and (hr, . ; % ci, . to . ; p = . ) and worse in patients with less than very good partial response (vgpr) to last prior therapy compared to those with a response to prior therapy of at least vgpr (hr, . ; % ci, . to . ; p = . ) [see figure ]. pfs was improved in patients with more than two years of maintenance therapy compared to those with less than two s years of maintenance therapy (hr, . ; % ci, . - . ; po . ), but this result does not account for patients who ended maintenance therapy due to disease progression. os was worse in patients at iss stage at diagnosis compared to those at iss stages and (hr, . ; % ci, . to . ; p = . ). peripheral neuropathy was more common in the bortezomib cohort ( % vs %, p o . ), while cytopenias were more common in the lenalidomide cohort ( % vs %, po . ). figure kaplan-meier curve for pfs for the maintenance lenalidomide group versus the maintenance bortezomib group by log-rank test (p = . ). lenalidomide and bortezomib maintenance after transplantation have equal efficacy in prolonging progression-free and overall survival in patients with multiple myeloma. iss stage significantly affects time to progression and overall survival, and response to last prior therapy affects time to progression. length of maintenance therapy may be a significant predictor and warrants further analysis. these findings suggest that both lenalidomide and bortezomib are acceptable maintenance therapy options for post-transplantation multiple myeloma patients. autologous stem cells transplantation (auto-hct) is an accepted method in multiple myeloma (mm) patients, but usually it is not curative. the issue of allogeneic hematopoietic stem cells transplantation (allo-hct) is challenging yet for myeloma. we investigated allo-hct in mm and compared with auto-hct. in this retrospective study, we recruited patients from january to january ( ( . %) patients in autologous group and ( . %) in allogeneic group). we performed allogeneic hct with peripheral blood stem cells source in our center for patients who are relatively young (less than years old) with good performance, have match sibling donor and accepted allogeneic hct. the conditioning regimens in autologous group was melphalan mg/m only and in allogeneic groups was fludarabine mg/m plus melphalan mg/m in consequent days. gvhd prophylaxis consisted of methotrexate and cyclosporine. the outcomes then compared between two groups using log-rank and gray tests and cox proportional hazard regression. the median follow-up in the autologous and allogeneic group was . months. three years disease-free survival of auto-hct was . % (ci: . %, . %) and . % (ci: . %, . %) for allo-hct patients (p value = . ). three years overall survival of auto-hct was . % (ci: . %, . %) and . % (ci: . %, . %) for allo-hct patients (p value = . ) showing no significant statistical difference between two groups. mortality rate was . % for auto-hct and for allo-hct was . %. the most common cause of death between two groups was relapse of primary disease. three year relapse incidence was . % (ci: . %, . %) for allo-hct and . % (ci: . %, . %) for auto-hct (gray's test p value = . ). the three year trm incidence was . % (ci: . %, . %) and . % (ci: . %, . %) in allogeneic and autologous patients respectively (gray's test p value = . ). despite there was no statistically significant difference between two groups in terms of os but dfs and relapse incidence was meaningfully better in allogeneic group. so, perhaps the reason of non-significant os improvement in allogeneic group is higher early death due to higher trm. we suggest that this study needs longer follow up to see whether allo-hct resulted in os improvement. disclosure of conflict of interest: none. myeloablative allogeneic hematopoietic stem cell transplantation from unrelated donors for patients with relapsed or refractory multiple myeloma n tsukada , s shingaki, m ikeda, t ishida and k suzuki division of hematology, japanese red cross medical center allogeneic hematopoietic stem cell transplantation (allo-sct) for patients with multiple myeloma (mm) is increasing in number despite in the era of novel agents, especially as a second line treatment and beyond. it has been reported that allo-sct for patients with mm resulted in high incidence of treatment related mortality (trm). high incidence of disease relapse is also a major problem especially after reducedintensity stem cell transplantation (rist). it is an important issue to reduce the incidence of trm while preventing disease relapse. the use of stem cells from unrelated donors is required for those without hla-matched sibling donors. the purpose of this study is to evaluate the feasibility of an intensified conditioning regimen incorporating both mg/ m of melphalan and gy of total body irradiation (tbi), followed by allo-sct from unrelated donors for patients with relapsed or refractory mm. we retrospectively analyzed eight consecutive patients who received allo-sct from unrelated donors with the conditioning regimen including gy of tbi, fludarabine mg/m for five days, and melphalan mg/m between april and july at the japanese red cross medical center. six patients received unrelated bone marrow transplantation (bmt) and two patients received cord blood transplantation (cbt). graft-versus-host disease (gvhd) prophylaxis was consisted of tacrolimus and short term methotrexate. the median age at allo-sct, the time from diagnosis of myeloma to allo-sct, and the numbers of prior treatment lines were . years (range: - years), . months (range: - months), and . lines (range: - lines), respectively. five patients are female. no episode of either grade ≥ iii toxicity or non-relapsed mortality was documented during the median follow-up period of over two years. cumulative incidence of grade ≥ ii acute and severe chronic graftversus-host disease were . % ( % confidence interval [ci] . %- . %) at days and . % ( % ci . %- . %) at days, respectively. probabilities of progression-free survival and overall survival were . % ( % ci . %- . %) and . % ( % ci . %- . %), at years, respectively. the results suggest that allo-sct conditioned with this intensified regimen may be tolerable for patients with relapsed or refractory mm. disclosure of conflict of interest: none. the role of allogeneic stem cell transplantation (allosct) in the era of novel myeloma drugs remains controversial. it is the only curative treatment option but non-relapse mortality makes the decision making difficult as opposed to achievements with autologous sct and new mm drugs by which the median survival is nowadays nearing years. aim of this study was retrospectively evaluate the outcome of allosct for mm performed at our institute, including evaluation of factors affecting survival. all consecutive patients allotransplanted for mm between and were included. the data were collected from our transplant registry. frequencies and medians were produced as appropriate. kaplan-meier method was used to calculate os and pfs and log rank test for comparisons. univariate analysis for factors affecting survival was performed with cox proportional hazard model. median age of all patients was ( - ) years. half of the patients had igg myeloma, % had iss score (score available for patients), and % had high-risk cytogenetics (data available for patients). response to treatment at sct was at least vgpr in % of patients, and transplant timing was early (within months from dg) in % of patients. sibling donors were used in % and muds in % of transplants, and conditioning was ma for % and ric for another % of patients. acgvhd grade - occurred in % and grade - in % of patients; % of patients had extensive chrgvhd. posttransplant cr rate was %. % of pattients have relapsed after allosct, and % are alive with the median follow-up of , years. non-relapse mortality has been % ( % until , % since then). the median survival of patients up to age of years is . years vs . years for patients years (n = ) with survival plateau after years at % level. transplant period, cytogenetics, donor type, conditioning intensity or occurrence of chrgvhd had no statistical impact on survival. significant differences in os were observed between disease status at scr vgpr vs o months from dg vs later), grade of acgvhd - vs - , and best resonse post-transplant cr vs not less than cr. the respective differences for pfs were in sct timing, grade of chrgvhd, and best post-transplant response. in univariate cox regression analysis the only significant factors for os were severity of acgvhd and cr vs other responses after sct, and for pfs allosct timing, severity of chrgvhd, and best response to sct. with allosct ca. % of mm patients can be cured but at the cost of high non-relapse mortality. the occurrence of grade - acgvhd and less than cr response to sct predict poor survival. considering the increasing survival expectations with modern standard therapy for mm, allosct may be recommended for younger patients with high-risk features, and allosct should be done early in the disease course. disclosure of conflict of interest: none. angiogenesis plays an important role in the pathophysiology of hematological malignancies including plasma cell myeloma (pcm). microrna- (mir- ) is overexpressed and displays oncogenic activity in cancers. however, little is known about the role of mir- in pcm. the aim of the present study is to examine the expression level of peripheral mir- in pcm patients and to determine its role in angiogenesis. vegf serum levels and mir- in pbmcs was measured in patients with pcm directly before melphalan mg/m followed by autologous hematopoietic stem cell transplantation (auto-hsct) and months after hsct; and healthy controls. the study population was divided into two groups after therapy: responders (stringent complete response, complete response, very good partial response, partial response) and nonresponders (stable disease, progressive disease). gene expression of mir- was quantified by sybr green real-time fluorescent quantitative pcr. further tube formation of huvecs and vegf secretion was measured in mir- mimic or inhibitor transfected human plasma cell myeloma cell lines h and rpmi- . the expression level of mir- was significantly increased ( . ± . versus . ± . ; p o . ) in pbmcs of pcm patients compared with healthy controls. further, serum vegf levels were increased in pcm patients ( ± pg/ml versus ± pg/ml in normal controls; p o . ). after auto-hsct, the expression level of mir- was significantly different in responders compared to nonresponders. responders had a lower expression of mir- compared to non-responders. further, serum vegf levels decreased in responders to auto-hsct compared to nonresponders. vegf expression was increased in the supernatant from mir- mimic transfected human pcm cell lines h and rpmi- compared with the negative control, while s vegf was decreased in the mir- inhibitor transfected cell lines. the angiogenic ability of huvecs was increased under pretreatment with the supernatant from h and rpmi- cells transfected with mir- mimic compared with negative controls and decreased when pretreated with mir- inhibitor transfected cells (fig. ) . this study demonstrated that mir- was upregulated in pcm patients. responders to auto-hsct had a decrease of mir- expression and vegf levels. further, mir- regulated angiogenesis. therefore inactivation of mir- or activation of its target gene may be a potential therapeutic approach in pcm. fig. : in vitro matrigel tube formation assay. (i), normal control (ii, iii), mir- mimic transfected h cells (iv), mir- mimic transfected rpmi- cells (v), mir- inhibitor transfected h cells. original magnification × . disclosure of conflict of interest: none. [p ] pilot study of busulfan/thiotepa as conditioning regimen followed by allografting and post transplantation cyclophosphamide in advanced relapsed myeloma patients c wolschke, e klyuchnikov, d janson, m heinzelmann, m christopeit, f ayuk and n kröger university medical center hamburg-eppendorf despite the significant improvement in outcomes has been observed for myeloma patients, the disease still remains incurable. due to limitations, such as trm and gvhd, the role of allogeneic stem cell transplantation as salvage therapy in this setting remains unclear. in present pilot study we provide data on the use of post cyclophosphamide (ptcy) as gvhd prophylaxis after a busulfan/thiotepa based conditioning regimen in patients who relapsed after autologous stem cell transplantation. between / and / myeloma patients (male n = , female n = ) with a median age of years (range: - ) (pts), who relapsed after autologous stem cell transplantation received allogeneic stem transplantation with with ptcy as gvhd prophylaxis after busulfan ( . mg/kg for age y and . mg/kg for age years) and thiotepa ( mg/ m )and for haploidentical and mmud additional fludarabin ( mg/m ). all pts. were relapsed after one or two autologous stem cell transplantations. donors were haploidentical (n = ), mmud (n = ), mud (n = ) and hla-identical sibling (n = ). stem cell source was pbsc (n = ) or bm (n = ). all patients received cyclophosphamide mg/kg of body weight on day + and + , which was in pts (n = mrd, n = mud) the only gvhd prophylaxis, while patients with mmud and haploidentical donor received also cyclosporine a from day + and mmf (until day ) and patients (mrd and mud) received additional cyclosporine. we observed no primary or secondary graft failure. the median time for neutrophil and platelet engraftment was (range: - ) and days (range: - ), respectively. major toxicities grade and were: renal (n = ) and mucositis (n = ). major infectious complications were: cmv: n = cmv-reactivations (n = ), sepsis (n = ), pneumonia (n = ) rsv-(n = ) and hsv (n = ). acute gvhd grade ii to iv and ii/iv was noted in % and %, respectively and mainly seen in patients with cyclophosphamide as single gvhd prophylaxis. remission rate were n = complete remission, n = vgpr, n = partial remission, n = n.a. after a median follow up of months pts progressed and patients (n = relapse, n = trm) died. the year pfs was % (n = ). busulfan/thiotepa is an active conditioning regimen for advanced relapsed myeloma patients. post cyclophosphamide might increase anti-myeloma activity, but as single gvhd prophylaxis it causes significant agvhd in mrd and mud and additional immunosuppressive agents such as cyclosporine should be added. disclosure of conflict of interest: none. magnetic resonance imaging (mri) for multiple myeloma (mm) is a sensitive, non-invasive and non-toxic method for detecting myeloma lesions. the goal of the study was to assess whether quantitative mri metrics can detect treatment response and replacement of neoplastic cells by fat marrow. the study was hipaa-compliant and irb-approved. we retrospectively identified all patients who achieved a complete response (cr) after induction therapy between and . inclusion criteria for the study was total spine mri imaging at diagnosis and after achieving cr. cr was determined using the imwg criteria. spinal vertebrae t through l were outlined with imagej software. fractures and lesions were excluded. images were analyzed using histogram-based (entropy, skewness, kurtosis) and texture-based statistics. a two-sided t-test was used to compare quantitative mri metrics from before therapy and after achieving cr. cox regression was used to explore the association between progression free survival (pfs) and change in each quantitative mri metric based on a median split. pfs was defined as the time from the second mri to death or progression of disease. nineteen patients met the above criteria. median age was . years (range: . - . ). majority of patients ( %) were male. majority of patients had iss stage disease ( . %) and standard-risk cytogenetics ( . %). an induction regimen containing an imid and/or a proteasome inhibitor was commonly used ( . %). all patients received an autologous stem cell transplant (asct) consisting of high dose melphalan followed by autologous stem cell rescue. three patients received a planned second asct. seven patients ( . %) were in cr before asct. nine patients ( . %) were treated with imid maintenance after planned initial therapy. median time to repeat mri imaging after cr was months (range: . - . ). mean change in measurements of kurtosis, skewness, entropy and texture analyses are shown in table . no significant change was detected between preand post-cr mri. furthermore, no significant association was seen between the change in any quantitative metric and pfs. [p ] despite promising results by other groups, we could not find a significant association between quantitative t image analysis and cr or pfs. there was heterogeneity in the time of repeat mri imaging which may have limited our ability to study interval change. although no definitive conclusions can be made from this small sample, correlation between pfs and kurtosis or texture d may be promising and should be investigated in a larger group prospectively. multiple myeloma (mm) treatment (tx) has evolved in recent years. solid data on the impact of new tx on patient (pt) outcomes outside clinical trials, however, is lacking. this study aimed at investigating tx practices, pt journeys, and outcomes in the real-world in countries with different access to new tx. the study was conducted between / and / in bulgaria, croatia, czech republic, poland, romania, and slovakia. it consisted of a cross-sectional (x) and a retrospective (r) phase. for the x-phase, investigators included all symptomatic mm pts seen during a -week counting phase to provide a snapshot of where in the pathway pts were at a given moment. for r-phase, investigators collected data on current and past tx, including symptoms, dosages, administration schedule, tx durations, tx interruption, reasons for change/discontinuation, and tx response. pts were selected in reverse chronological order with a quota of a maximum of pts who completed first-line ( l) tx within the past months (mo), pts in second-line ( l) and pts in third or higher lines ( +l). pts included in the x-phase could also be included in the r-phase, if they met the respective inclusion criteria. in total, physicians included pts in the x-and physicians included pts in the r-phase. in the x-phase, % of pts were o , % were - , and % were years; the median time since diagnosis was mo. % of pts were currently undergoing tx, % were previously treated and % had never been treated. of currently-treated pts, % received l, % l, % l and % +l. in the r-phase, % of pts were o years. of pts receiving l, % continued to l, % to l, % to l and % to l. of the % of pts eligible for stem cell transplantation (sct), % ( = % of all pts) received sct at l; these proportions were similar across countries. the most frequently-used regimens in l and l were bortezomib-based ( % and %, respectively), in l and +l lenalidomide-based ( % and %, respectively). median duration of l was mo, followed by a median disease-free interval (dfi) of mo. median dfi was longer in pts with sct than in those without ( . mo vs . mo). time to progression (ttp) decreased with later tx lines, from median mo at l to mo at l. depth of response, as assessed by the treating physician, decreased with each additional line of tx: % of pts achieved at least very good partial response (≥ vgpr) in l, while only % achieved ≥ vgpr at +l. ttp was longer in pts with better response levels: in l, median ttp for pts with ≥ vgpr was mo versus mo for pts with mo for pts with ovgpr. the most common ( ≥ %, all grades) adverse events (aes) and co-morbidities in l were anemia ( %), thrombocytopenia ( %), neutropenia ( %), neuropathy ( %), and fatigue ( %). these aes disrupted treatment in % in l, % in l and % in +l. the study found that of sct eligible pts, only slightly more than half were transplanted. poorer outcomes and increasing ae incidence with each tx line highlight the challenges of mm tx. information on real-world pt management may be valuable for physicians to plan their tx strategies and can provide input for health economic evaluations of existing and new tx. disclosure of conflict of interest: daniel coriu declares to have received consulting fees or other remuneration (payment) from novartis, amgen, pfizer, takeda, janssen. ivan spicka declares to have received research grants from celgene, consulting fees or other remuneration (payment) from bms, takeda, celgene, janssen-cilag, and amgen, and to be a member of the speakers bureau of celgene, janssen-cilag, amgen, and bms. zdenka stefanikova declares to have received consulting fees or other remuneration (payment) from amgen, celgene, and takeda and be a member of the speakers bureau of amgen, celgene, and takeda. daniela niepel, krisztian szabolcs toka, and paul schoen are amgen employees and hold amgen stock. dominik dytfeld, and georgi mihaylov have nothing to declare. safety and efficacy of autologous stem cell transplantation in elderly patients with multiple myeloma t maia , c marini , p medeiros , e aguiar , j cancela pires , r bergantim , f trigo and je guimarães , hematology department, centro hospitalar de são joão and faculty of medicine, university of porto autologous stem cell transplantation (asct) is considered standard treatment for multiple myeloma (mm) patients under the age of years, but its safety and efficacy still uncertain for patients over this age. retrospective analysis from one single centre concerning mm patients under, equal or over years who underwent asct between january/ and july/ . it was also compared to - years old mm patients diagnosed in this period of time who were not transplanted. we analysed a total of patients, of which underwent asct. onehundred-and-six of the transplanted patients were aged years or less (median , iqr years), patients were aged more than years (median , iqr years) and patients were non transplanted (median , iqr ). the conditioning regimen for younger patients who underwent asct consisted mainly of melphalan mg/m (mel ) while half of the elder patients received melphalan mg/m (mel ). regarding transplant-related myelotoxicity there were no statistical differences between patients aged years or less and over years old, however the first group needed less days of g-csf (p = . ). non-hematopoietic toxicity measured by infections and mucositis was not influenced by age. patients years conditioned with mel had more days of aplasia (p = . ), greater need of g-csf (p = . ) and transfusional support (p = . ) than patients ≤ years. there were no differences on non-hematopoietic toxicity. in the elderly group, patients conditioned with mel presented more aplasia days (po . ), higher grade of mucositis (p = . ) and more days of iv antibiotics (p = . ) than those transplanted with reduced dose of melphalan. comorbidities had no effect on transplant-related toxicity, either by age or by dose of melphalan. days of hospitalization and post-transplant complications did not differ according to age group. transplant related mortality was % at day posttransplant. survival after transplant in patients years old or under vs older patients (median follow-up time, months), was not influenced by age (os, mo vs mo, p = . ; pfs, mo vs mo, p = . ). regarding the non-transplanted elderly group, these are patients with more renal disease (p = . ) and poorer performance status (p = . ) than the transplanted cohort. there is also higher cytogenetic risk (p = . ). induction regimens were similar in transplanted group and non-transplanted group years old, and response to first line therapy (before asct of transplanted group) revealed no differences. infections were the most common complication in both groups. transplanted patients needed less days of hospitalization (p = . ). comparing the long term outcome of these two groups, survival curves of the elderly patients transplanted were clearly superior to the nontransplanted (os, mo vs mo, p o . ; pfs, mo vs mo, p o . ) although one has to consider that the non-transplant group has worse features than the elderly transplant group. transplantation in the elderly still debatable but this study shows that it might bring benefit. globally, transplant related toxicity is not influenced by age. regarding dose of melphalan, higher dose in elderly patients has higher toxicity, without apparent benefit in survival. therefore, age should not restrict the access to asct, but instead selection must be based on individual clinical and functional status. disclosure of conflict of interest: none. second autologous stem cell transplantation for relapse after allografting in multiple myeloma using cd + selected donor cells without immunosuppression p novak number of patients receiving a second allogeneic stem cell transplantation (sct) in europe is increasing despite high treatment related mortality (trm). in multiple myeloma only very few reports of second allogeneic sct exist with limited number of patients and substantial mortality. while in most hematological malignancies, the donor cell chimerism is dropping down if patients are relapsing, in myeloma donor cell chimerism remains complete despite relapse. to reduce trm we thought that full donor cell chimerism may allow us to perform a second high dose busulfan based chemotherapy followed by "autologous transplantation" after stem cell mobilization and collection. however, because two consecutive patients failed to collect sufficient cd + cells for an autologous transplantation even with plerixafor, we used donor t cell depleted cd + selected cells and transplanted those patients in an "autologous" fashion without any immunosuppression. to enhance graft-versus-myeloma effect, we added donor lymphocyte infusion (dli) at day . we report here on myeloma patients with a median age of years (range: - ) who relapsed after allogeneic sct and underwent a second "autologous" sct with cd + selected donor cells. all patients had received one (n = ) or two (n = ) autologous sct before . allografting. patients received an upfront auto-allo protocol and patients received . allogeneic sct as a salvage therapy. % of patients received a reduced intensity melphalan based conditioning regimen for . allogeneic sct and the median pfs was months (range: - ). before . allograft patients had received overall a median of (range: - ) treatment lines. at the time of . allogeneic sct all patients had a full or nearly full donor cell chimerism and remission status was very good partial remission (n = ), partial remission (n = ), stable disease (n = ), progressive disease (n = ). % of patients received a myeloablative busulfan based conditioning regimen and all received cd + selected stem cells with a median number of . × /kg cd + cells (range: . - . ) and × /kg cd + cells (range: . - ). engraftment was noted in % at a median of days (range: - ). no further graft-versus-host disease (gvhd) prophylaxis was performed and no acute gvhd (agvhd) was observed. according to treatment plan, patients received escalating dli around day + , starting with a median dose of × /kg (range: . - ) in combination with lenalidomide maintenance in patients. patients experienced agvhd ii-iv after dli. two patients had a severe gvhd (grade iii) which resolved completely after steroid therapy. no nonrelapse mortality after sct and dli was observed. after a median follow up of months (range: - ) the median pfs was months (range: - ) which translates into a pfs for all patients of % at year and % at years. median os was months (range: - ) and an os of % at years and % at years was observed. for patients with advanced multiple myeloma relapsing after allografting, a second "autologous" sct with cd + selected donor cells without immunosuppression followed by dli is an encouraging treatment option with low toxicity. disclosure of conflict of interest: none. second autologous stem cell transplantation as treatment option for relapsed patients with multiple myeloma: a single center experience (cic ) p ganeva, y petrov , m mincheff , i tonev , m guenova, l gartcheva , a michova , g arnaudov and g mihaylov ya. petrov; m. mincheff; i. tonev; l. garcheva; a. michova; g. arnaudov and g. mihaylov the use of modern therapies such as bortezomib, lenalidomide, thalidomide coupled with upfront high-dose therapy and autologous stem cell transplantation (asct) has resulted in improved survival in patients with newly diagnosed multiple myeloma (mm). the role of second asct as salvage therapy for relapse is unclear because of the availability of new agents to treat progression in multiple myeloma (mm). as the treatment options for management of patients with relapsed mm has become increasingly complex, physicians must consider both disease-and patient-related factors when choosing the appropriate therapeutic approach, with the goal of improving efficacy while minimizing toxicity. we retrospectively reviewed all mm patients who received a second asct as salvage therapy at our center from to december . for this period we performed transplants for mm patients. twenty five ( . %) patients received second asct ( patients were relapsed) and for patients asct was performed as tandem transplant. we analyzed only second asct for relapse. the median time to relapse after first transplant was . months (range: - months). all patients received reinduction therapy before the second asct. conditioning was performed with melphalan with two different doses ( mg/m and mg/m ). the median age at second transplant was . years (range: - years), and female/man ratio was / . median interval between first and second asct was . months (range: - months). we have no observed early deaths. until now ( %) patients are dead because of progression disease. response rate was assessed three months after asct, nine ( %) patients achieved vgpr, three ( . %) patients achieved at least a partial response, three ( . %) had sd and three ( . %) progressed despite salvage asct.median overall survival (os) was . months ( relapse ≥ months = . ; ≤ months = ). second asct is a feasible and safe option for salvage therapy in mm, especially in bulgaria where the possibility of using novel agents such as carfilzomib, lenalidomide, daratumomab for relapsed patients is limited to clinical trials, because of no reimbursement. the best results were observed in patients whose time to progression was more than months after first asct. advances in treatment of multiple myeloma (mm) has improved overall survival in these patients (pts). a steady increase in the risk of secondary malignancies has been reported over the last decades in mm survivors. estimated incidence of secondary acute myelogenous leukemia or myelodysplastic syndrome (t-mds/aml) after treatment with alkylating agents is %- . % per year - years after primary chemotherapy. no specific risk factor has been recognized, but genetic instability, natural history of the disease as well as induction therapy and autologous stem cell transplantation (hct) have been implicated. recently, novel anti-myeloma treatments have been linked with an increase in secondary malignancies, but no solid relationship has been established yet. in a retrospective study, we analyzed the incidence of secondary malignancies (t-aml/mds and solid tumors) in patients suffering from mm who had undergone autologous hct using high-dose melphalan conditioning regimen in our bmt unit. study population consisted of consecutive pts with median age of years ( - ), . % of them being male, who were transplanted during a period of years . type of myeloma was igg/a/d in %, . % and . % respectively, while . % was light chain and % nonsecretory. the majority of pts presented with k light chain myeloma ( . %). there was almost equal distribution between iss stages i and ii ( %/ . %) and only . % were diagnosed with advanced stage myeloma. most pts received two lines of chemotherapy ( %) and all of them more than one. treatment regimens before autologous hct included vad ( pts), bortezomib-based ( pts), dcep ( pts) and rd ( pts) and pts received radiotherapy. chemotherapy administration for mobilization was used in pts ( . %). conditioning regimen before autologous hct consisted of high-dose melphalan ( mg/m ) and in case of renal insufficiency mg/m . incidence of a secondary malignancy was . % after a median follow up period of months. t-aml /mds was diagnosed in ( . %) pts and ( . %) were diagnosed with breast and lung cancer respectively. pts diagnosed with secondary malignancy were previously exposed in induction therapy to melphalan ( ), vad ( ), bortezomib ( ), high-dose cyclophosphamide as mobilization treatment ( ) and radiotherapy ( ) . cytogenetic analysis was available in patients diagnosed with t-mds/aml and the majority ( / ) presented complex karyotype. abnormalities mainly observed were deletions and insertions in chromosomes , , . pts with secondary malignancies had an overall survival of months ( - ), however, after malignancy diagnosis, survival was very poor, four months only . secondary malignancies in pts with multiple myeloma after autologous hct occur with a substantial frequency and have a dismal prognosis. the role of novel treatment agents has to be elucidated. further studies are needed to identify new risk factors and develop better surveillance strategies. [p ] disclosure of conflict of interest: none. survival analysis after allogeneic hematopoietic stem cell transplantation in patients diagnosed with multiple myeloma: a single center experience p patricia hernandez* , r maria calbacho , a laura posada , g fabio augusto ruiz , r anabelle chinea hospital universitario nuestra señora de candelaria, santa cruz de tenerife (spain); hospital universitario ramón y cajal, madrid (spain) and hospital universitario cruces, barakaldo (spain) allogeneic hematopoietic stem cell transplantation (allohsct) may provide long term remission cures for patients diagnosed with high-risk multiple myeloma. however, its use is limited since it has a high rate of treatment-related mortality (trm), and because its efficacy compared to autologous hsct is not fully established. we studied patients that underwent allohsct between - . population characteristics are in table . all patients were treated at least with one prior therapy lines ( ) ( ) ( ) ( ) ( ) , all including autohsct ( . % underwent prior autohsct). % had or prior therapy lines. of them received bortezomib as part of treatment regimens. donor characteristics: non-related; hla-identical. gvhd prophylaxis: methotrexate plus a calcineurine-inhibitor: cyclosporine and tacrolimus. median follow-up . months ( . - . ), average was . months. seven patients died ( . %); because of progression ( . %), and ( . %) due to trm, including infections and immediate complications of transplantation, such as toxicities, icu admission and agvhd: infections: cmv reactivations, invasive fungal and bacterial infections. disease status: patients were in cr prior to allohsct. of them maintained it after. remaining patients died before disease was evaluated. seven patients were in pr prior to transplant, and reached cr after allohsct. one had progressive disease and reached cr after the procedure. two had stable disease and progressed after allo; one of them is in cr after additional therapy lines, and the other one died months after due to it. donor characteristics: hla-identical sibling donors: . % ( hla-mismatch, passed away . months after allo due to trm). one of the nonrelated donors, had an hla-mismatch, and died months after allohsct due to trm, the other one is alive after months. gvhd: ( . %) developed agvhd and of them maintained it chronically. two suffer from cgvhd, plus that initiated it as agvhd. were refractory to steroids. longterm survivors: patients had overcome three years after allohsct. they were among and years old at the time transplant was performed. none of them received bortezomib as part of therapy protocols for the disease. all had therapy lines prior allograft. were submitted to prior autologous hsct. relapse: patients relapsed after allohsct ( %, median time to relapse . months), being alive % at the end of the study. allogeneic hsct is associated with a high incidence of nrm and a low incidence of relapse. rates of acute and chronic gvhd are high. in our cohort, besides that more than % are alive until now, they suffer from extensive chronic gvhd and are in need of treatment. long-term survival may be related with patient factors such as young age, but also low tumor burden, or less prior therapy lines; in our group there are no differences in this aspect. studies including high-risk abnormal cytogenetics should help to define which patients are best candidates to allohsct. high-dose melphalan followed by autologous haematopoietic cell transplantation (ahct) remains the standard of care for eligible multiple myeloma (mm) patients. the majority of patients in clinical practice and trials receive a melphalan dose of mg/m (mel ), but a reduced dose of mg/m (mel ) is often used in patients perceived to be unable to tolerate mel . it remains to be determined whether this considerable dose difference results in different clinical outcomes. we therefore analysed patients with mm who underwent a first single mel or mel -conditioned ahct between january and december . all patients were included in the calm study, an analysis of a prospectively defined cohort of patients with data reported retrospectively to the ebmt, covering ahcts for mm and lymphoma. patients in the mel group were older than patients in the mel group at the time of ahct (median years [range: - ] vs years ; p o. ). compared to the mel group (n = , . %), fewer patients in the mel group (n = , . %) were overweight or obese ( . % vs . %; p = . ). compared to the mel group, more patients in the mel group had received proteasome inhibitor-containing induction therapy ( . % vs . %; p = . ), had a karnofsky score of ≤ ( . % vs . %; p = . ), and were transplanted in less than pr ( . % vs . %; p = . ). overall survival (os) from the time of ahct was not significantly different between the mel and mel group ( - significantly different between mel and mel ( days in both groups for neutrophil recovery; vs days for platelet recovery). however, late neutrophil recovery was noted in a small proportion of patients in the mel group. neutrophil recovery days post ahct was not observed in any engrafting patient in the mel group, but occurred in ( . %) engrafting patients in the mel group (p = . ). a cox proportional hazards model that included melphalan dose, age, and remission status at ahct showed that melphalan dose had no effect on os, pfs and relapse risk. the findings suggest that mel is not inferior to mel in younger and older mm patients and may reduce the risk of delayed haematological recovery in some patients. further analyses in relevant subgroups such as patients with high-risk features or renal impairment are required. disclosure of conflict of interest: none. high-dose therapy (hdt) followed by autologous stem cell transplantation (asct) remains the standard of care for patients younger than years of age with multiple myeloma (mm). different agents are being used to control the disease before asct, including the older thalidomide based combination or the newer bortezomib and lenalidomide based combination. the relation between the initial induction regimen and outcomes after asct is not completely clear. to evaluate the effect of different induction regimens on asct outcome, we retropsectively evaluated the outcomes of a low cost older regimen of thalidomide based combination in doublets or triplets with newer novel agents like bortizomib or lenalidomide based combination in a low resources country in transplant-elegible patients with multiple myeloma who underwent autologous stem cell transplantation at king hussein cancer center bmt program we retrospectively reviewed the files of patients diagnosed with mm from january till december , who received induction treatment followed by hdt and asct and followed up in a single institution. we compared the effects of different induction regimens, disease stage, and remission status before transplantation on over-all survival (os), event free survival (efs) and progression free survival (pfs) using kaplan meier curves. a total of patients were included, ( . %) of them received thalidomide based induction (group ) and ( . %) received bortezomib and lenalidomide based induction (group ). patients also offered no consolidation nor maintenance therapy. ( . %) patients were stage i, ( . %) stage ii and ( %) were stage iii. stage was not documented for ( , %) of cases. ( . %) were in complete remission (cr) and ( . %) were in partial remission (pr). the estimated -year os for the whole group was . %. there was no statistically significant difference between both groups in regards to initial iss stage of disease (p = . ) or cr status before asct. patients ( . %) in group achieved complete remission ( cr ) or very good partial response (vgpr), while ( . %) patient in group achieved cr or vgpr. there was no statistically significant difference between group and group in -years os ( -year os was % vs %, p = . ), efs ( . % vs . %, p = . ) or pfs ( . % vs . , p = . ). the use of an old, low-cost, thalidomide-based regimen in a low-resources country achieved a favorable transplantation outcomes in patients with multiple myeloma who received hdt and asct. double autologous stem cell transplantation (asct) is a useful treatment for multiple myeloma (mm) patients. we can make the second asct ( asct) without reinduction treatment (tandem regimen) or after a reinduction treatment after first asct ( asct) relapse (salvage regimen). we have conducted a retrospective study over mm patients undergoing a double asct performed in our centre from to . we have compared the different conditioning regimens used, and if there are any difference between tandem or salvage asct. we do not use maintenance treatment systematically. characteristics of patients and conditioning regimens in table . the overall survivals (os) of our patients are months (m) from treatment start till last control. the most important prognostic factors are the duration of the progression free survival (pfs) after asct (hr: . ( . - . ); p = . ), and the use of bumel like conditioning regimen at the asct or at the asct vs another conditioning regimens (hr: . ( . - . ); p = . ). today there are patients alive ( %), but only ( %) are free of mm now. the patients who were treated with tandem have a little better os than salvage patients ( m vs m; p = . . not significative). patients at tandem group who received different conditioning regimen at the asct and at the asct live more time than patients treated only with melphalan (mel ) at both asct. at salvage group the duration of pfs after asct is better than the pfs after asct ( m vs m). the use of the same conditioning regimen at the both asct has worse results than if we use different treatment. patients who were treated with s bumel at the asct or asct have better os than patients treated with cbv or mel . patients who not responded to reinduction treatment before asct have worst pfs after asct (rc: m, response; m and not response; only m). attention is drawn to the fact that patients who received bumel at asct have large os, but they are very few ( ) patients. only one patient has died during the asct, and was a patient of salvage group treated with bumel. double autologous transplantation continues to be a useful treatment despite the new mm treatments, and allows to prolonged the os. tandem asct probably is a useful treatment in high risk mm patients. salvage treatment is most useful in patients with a large pfs after asct, and good response to reinduction treatment. although mel continue to be the standard conditioning regimen for asct in mm patients, we have observed that patients treated with different conditioning regimen at asct and asct have better prognostic, and bumel has the best results in our serie. disclosure of conflict of interest: none. allogeneic haematopoietic stem cell transplantation (allo-hsct) is an effective treatment for myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). the prognosis of elderly patients with mds and aml after chemotherapy is poor. allo-hsct is feasible in these patients; however the management of elderly patients with mds and aml for allo-hsct is difficult. we performed a retrospective survey of allo-hsct for elderly patients with mds and aml in our institution. we retrospectively analyzed the records of elderly patients ≥ years with mds and aml who underwent allo-hsct in our hospital between january and december . in this study, we assessed the ipss-r (revised international prognosis scoring system) cytogenetic score and the ipss-r score against the outcome of elderly mds and aml patients who treated with allo-hsct. fifty-one elderly patients with mds and aml were treated with allo-hsct in our institution, patients with mds ( with mds overt aml) and with de novo aml. ages ranged from to years (median ), patients were female and were male. there was a history of malignant disease in patients. according to the ipss-r cytogenetic scores of mds patients, patients fell in the good risk group, were in the intermediate risk group, were in the poor risk group, and were in the very poor risk group. regarding the ipss-r score, patient fell in the low risk group, in the intermediate risk group, in the high risk group, and in the very high risk group. sixteen patients were in st complete remission (cr), patient was in nd cr, patients were in partial remission, and patients were not in remission (nr) upon administration of allo-hsct. all patients received a reduced intensity conditioning regimen. patients [p ] were treated with fludarabine (flu), melphalan and low dose tbi-containing regimens; patients were treated with flu, intravenous busulfan and low dose tbi; and one patient was treated with flu, cyclophosphamide and low dose tli. graftversus-host disease (gvhd) prophylaxis consisted of tacrolimus plus methotrexate in patients, and tacrolimus, methotrexate and mycophenolate mofetil in patients. thirty-four patients received anti-thymocyte globulin (atg). the donor source was sibling bone marrow (bm) in patient, sibling peripheral blood stem cell in , unrelated bm in and unrelated cord blood in . relapse-free survival (rfs) and overall survival (os) were . % ( % confidence interval (ci): . - . %) and . % ( % ci: . - . %) at year, . % ( % ci: . - . %) and . % ( % ci: . - . %) at years, respectively (figure .) . in this study, patients died before engraftment. non-relapse mortality (nrm) was . % at day . twenty-five patients developed chronic gvhd ( patients limited and extensive). the causes of death were disease progression ( patients), treatment-related mortality ( patients), infection ( patients) and other causes ( patients). we suggest that many elderly allo-hsct patients with mds and aml were in the very poor risk group when the ipss-r cytogenetics score was assigned, in the very high risk group when the ipss-r score was assigned and nr upon administration of allo-hsct. rfs and os were . % and . % at years, respectively. there is a need for novel treatment strategies to manage elderly mds and aml patients for allo-hsct. [p ] disclosure of conflict of interest: none. counting bone marrow blasts as a percentage of nonerythroid cells provides superior risk stratification for mds patients with erythroid predominance a sun , y yu , t zhang , q wang , d liu and s chen the first affiliated hospital of soochow university, jiangsu institute of hematology, suzhou, china patients with erythroid predominance (≥ % erythroblasts, mds-erythroid) compose a significant proportion of patients with mds. the erythroid/myeloid subtype was divided from the aml category into mds-erythroid by the who classification of myeloid neoplasms. at that time, there was no consensus on a more appropriate way of enumerating bone marrow (bm) blasts from tncs or necs in mds-erythroid patients. to clarify these questions, mds patients were retrospectively analyzed in our center. mds-erythoid was observed in . % of patients ( / ), and these patients had similar clinico-pathological features and overall survival, with cases of mds with o % encs. by calculating the percentage of bm blasts from necs, of patients ( . %) with mds-erythroid who were diagnosed within who subtypes without excess blasts (eb) were moved into higherrisk categories and showed shorter os than those who remained in the initial categories (p = . ). recalculating the international prognostic scoring system-revised (ipss-r) by enumerating blasts from necs, of ( . %) mdserythroid patients with relatively lower risk were re-classified as higher-risk and had significantly poorer survival than those who remained in the lower-risk category (p = . ). this was especially true for the intermediate risk group that was stratified by ipss-r (unchanged patients vs. shifted patients, p = . ). however, the impact of enumerating bm blasts from necs on classification and prognostication was not evident in all mds patients. in conclusion, our results suggested that enumerating the percentage of bm blasts from necs significantly improved the prognostic assessment of mds-erythroid, especially for patients within the intermediate risk group stratified by ipss-r. disclosure of conflict of interest: none. myelodisplastic syndrome (mds) is a group of clonal and heterogeneous diseases, characterized by ineffective hematopoesis. the incidence of mds is about % of all blood disorders in children, approximately % of them develops acute leukemia. allogeneic hematopoietic stem cell transplantation (allo-hsct) is effective curative treatment of mds in children, but depends on disease status, type of clonal chromosomal abnormalities presented at the time of allo-hsct and graft quality. the aim of this study: to analyze the influence of graft quality on the outcome of childhood mds after allo-hsct. allo-hsct were performed in patients (pts) p hypomethylating agents vs. allogeneic sct in elderly patients with advanced myelodysplastic syndromes: a single center study j cermak, a vitek, m markova-Šťastná, j soukupova-maaloufova and p cetkovsky institute of hematology and blood transfusion, prague, czech republic a group of patients older than years of age with myelodysplastic syndrome (mds) raeb ii or with acute myeloid leukemia with multilineage dysplasia with less than % of bone marrow blasts (mds raeb-t according to the fab classification) was treated with hypomethylating agents (hma) and the results were compared to those obtained in an age and diagnosis matched group of patients who underwent allogeneic stem cell transplantation (sct). in the hma group, patients received azacytidine (vidaza) in the dose of mg/ m × every days and patients were treated with decitabine (dacogen) in the dose of mg/m × every days. median number of cycles administered was . (range: - ). in the transplanted group, patients were transplanted upfront and patients were pretretated with combination chemotherapy, patients received myeloablative conditioning and patients were transplanted after reduced conditioning regimen. a hematologic response to hma (cr,pr, hematologic improvement) was observed in patients ( . %), cr was achieved in patients ( . %). in sct group, engraftment was achieved in out of patients, patients died after sct ( on complications related to sct, patients relapsed). no difference in year survival was observed between both the groups ( . % for hma vs. . % for sct), however, median overall survival (os) was . months in hma treated group compared to . months in sct group (p = . ). in a recent analysis performed at months after starting the treatment, patients treated with hma ( . %) and transplanted patients ( . %) were alive, patients in hma group and patients in sct group relapsed. estimated years survival was . % in sct group and only . % in hma group (p = . ). no significant differences in results and adverse effects of treatment were observed between patients aged - years and those older than years in both hma and sct groups. our results confirm previous observations showing that despite a promising effect of hma resulting in hematologic response in more than % of elderly patients with advanced mds, allogeneic sct still represents the only potentially curative treatment connected with long-term survival in a significant number of patients even in this mds patients subgroup. disclosure of conflict of interest: none. immunophenotypic assessment of erythroid dysplasia by a simplified cocktail in myelodysplastic syndromes in taiwan c-c li , p-f weng , c-t lin , j-l tang hypomethylating agent (hma) is commonly used as a bridge therapy to prevent leukemic transformation prior to selection of a donor for allogeneic stem cell transplantation (sct) in patients with myelodysplastic syndrome (mds), and showed low toxicity. although its roles are known, the underlying genetics and clonal dynamics upon hma treatment has not been systematically examined using serial samples, especially in allogeneic stem cell transplantation (sct) setting. in this study, we performed targeted serial sequencing on bone marrow samples from mds patients treated with hma for bridging of allogeneic sct. to perform targeted deep sequencing, bm mononuclear cells before hma treatment and, and fractionated t-cell samples (cd + fraction) as controls were taken before hma treatment. analysis of genetic mutations were performed using targeted resequencing by illumina hiseq (sureselect custom probe set targeting [p ] entire exon regions of a myeloid panel consisting of genes). all patients received hma (decitabine: , azacitidine: ), and the median number of cycles was four (range: - ). the overall response rate for hma pre-treatment was %: there were four cases of complete remission (cr) ( %), six cases of marrow cr ( %), and two cases of hematologic improvement ( %). targeted sequencing revealed mutations in patients ( / , %) with median of mutations per patient (range: - ). mutated genes were then grouped into biological pathways, defined in the cancer genome atlas (tcga) aml study. the most frequent biological pathway at diagnosis was dna methylation ( %), followed by activated signaling ( %), chromatin modifiers ( %), tumor suppressors ( %), spliceosome ( %), cohesin complex ( %), npm ( %), and myeloid transcription factors (tfs) ( %). when assessing the difference in pattern of variant allele frequency (vaf), we found the significant reduction of vafs in four ( %) patients after hma. with a median follow-up of . months, -year overall survival (os) were . % ( % ci, . - . ). there was no significant difference in os according to the presence of mutations in each biological mutational pathway (all, p . ). to identify prognostic value of mutational dynamics, we subclassified the change of variant allele frequencies (vafs) after median fourth cycles of hma [no mutated or reduction of vafs ( patients) vs. stable or increased ( patients)]. however, there was no significant association between the dynamic of mutation and os (p = . ). these data show that hma using as bridge therapy for allogeneic sct in mds patients is insufficient to achieve the sufficient molecular responses and, mutational pathway and dynamics may not prognostic in this clinical setting. to clearly demonstrate the role of hma pre-treatment in mds, systematic assessment on a larger cohort is necessary. disclosure of conflict of interest: none. any role of high-dose chemotherapy in mediastinal nonseminoma germ cell tumors? p pedrazzoli, s secondino, a necchi , f lanza and g rosti istituto nazionale tumori, milano and ospedale santa maria delle croci, ravenna among germ cell tumors, primary mediastinal nonseminoma germ-cell tumors (pmnsgcts) have the poorest outcome with -year overall survival ranging from to %. indeed, the presence of mediastinal location defines per se a "poor prognosis" category according to the igcccg classification. this clinically and biologically distinct disease entity is associated with lower complete response rates to chemotherapy (ct), high rates of relapse and disappointing results from salvage ct. current standard first line treatment for patients with mediastinal primary location is still four cycles of peb, as for all igcccg poor-prognosis patients. we have reviewed available data present in the literature, including recommendations and expert opinions, on the use of high-dose chemotherapy (hdc) with autologous stem cell support in pmnsgcts. the use of hdc as both early intensification (that is, first-line setting) and at disease recurrence (salvage setting) have been reported in small cohorts of patients. according to the largest retrospective comparison, it has been suggested that hdc, given up-front, may produce a % to % absolute improvement in survival compared with standard dose ct. studies of the ebmt suggest that responsive disease after induction therapy may have a better outcome. mediastinal primary had salvage rates by hdct of less than % based on an international multicenter analysis and an ebmt study. the use of hdct in pmnsgcts warrants further investigation, preferably with the use of modern hdct strategies (that is, multiple carboplatin and etoposide courses). while hdc cannot be routinely recommended in pmnsgcts, selected patients with chemosensitive disease may benefit from early intensification. a retrospective analysis evaluating the large ebmt database is ongoing; results will be presented at the meeting. disclosure of conflict of interest: none. high dose therapy and autologous stem cell transplantation in gynaecological malignancies: a monocentric retrospective study m nderlita , i vergote and d dierickx university hospitals leuven, department of gynaecology; university hospitals leuven and department of hematology high-dose chemotherapy (hdt) followed by autologous stem cell transplantation (asct) has been established as a treatment option in many relapsed hematologic malignancies. however, in spite of many small trials, there still is no proven role for this treatment in solid tumors including most gynaecological epithelial carcinomas. however, in some recurrent non-epithelial ovarian cancers, such as sex cord stromal tumors, germ cell tumors, neuroendocrine gynaecological tumors and gestational trophoblastic disease, some studies suggest a possible role for hdt followed by asct. we performed a monocentric retrospective descriptive analysis of all patients diagnosed with gynaecological malignancies and treated with hdct followed by asct in our center. clinical, laboratory, pathological and imaging data were collected and analysed, together with information on treatment and outcome. eleven patients were included in this analysis, with a median age of years (range: - ) at time of diagnosis. eight patients suffered from ovarian neoplasia. at time of diagnoses patients showed metastatic disease. first line therapy consisted of surgery (n = ), chemotherapy (n = ) or a combination of both (n = ). median time to progression after first line therapy was . months (range: - ) with a median time between primary diagnosis and start hdt of . months (range: - ). three patients underwent single ast, whereas the other patients had a tandem ast, with a median time of months between first and second hdt (range: - ). treatment related toxicity was manageable, although there was treatment-related death. at last follow up patients ( %) were still alive with a median follow up of . years (range: . - . ) after last asct for all patients. of the deceased patients died with progressive disease. although the number of patients is very small, this retrospective study shows that hdt and asct is feasible in heavily pretreated patients with relapsed/refractory gynecological malignancies, although further studies are mandatory for optimal selection of patients, histological subtype and timing of hdt during the disease course. disclosure of conflict of interest: none. the human endogenous retroviruses (hervs) are remnants of ancient exogenous retroviral infections of the humans: they represent about % of the human genome . the basic genes of hervs are group-specific antigen (gag), polymerase (pol) and envelope (env); there are also two regulatory regions, long terminal repeat (ltr), located at ' and ' ends. several reports have shown that hervs may play a role in the development of autoimmune diseases, such as multiple sclerosis . additionally the existence of a strong relationship between hervs expression and cancer, based on the mrna expression profile of hervs in normal and cancer tissues has been suggested . the increased level of expression level of herv-h in colorectal cancer (crc), a major cause of cancer death worldwide has been already shown. the aim of the study was to analyse the expressions of env genes of herv-r, herv-h, herv-k and herv-p in the peripheral blood mononuclear cells (pbmcs), in the tumor and in the adjacent normal tissues of colorectal cancer patients. a group of control composed by pbmcs from healthy subjects was also included. rna was isolated from the biological samples and a reverse transcription assay was conducted. quantitative real time pcr was performed to evaluate the expression of the hervs env gene. all the env genes were related to the expression of an housekeeping gene, gapdh. the quantification was carried out using comparative ct method and the difference between the levels of env gene expression in pbmcs, cancer and adjacent normal tissue was given by fold difference. fold difference values were relative to a calibrator: first the pbmcs of patients and then pbmcs of control healthy group. Δct values were analysed using the paired sample t-test, followed by a bonferroni correction. higher levels of expression of herv-h, herv-k and in particular herv-p were found in tumor tissues, as compared to pbmcs and to adjacent normal tissues of patients, with an increase of -, -and folds, respectively. the Δct distribution of herv-h, herv-k and herv-p in cancer tissues were statistically significant (po . ) ( table ). the expression of herv-h, herv-k and herv-p env gene resulted increased in the colorectal tumor tissues also when compared with the pbmcs of the healthy controls ( -, -and -folds, respectively). the Δct distribution of herv-h, herv-k and herv-p in tumor tissues were statistically significant ( ρ < . ). no difference of expression was observed between pbmcs of healthy controls, pbmcs and normal adjacent tissues of patients (figure ). hervs env gene expression cannot be used as a diagnostic biomarker, but it is conceivable that hervs are directly involved in the pathogenic process of cell transformation and, if the protein expression will be demonstrated, the protein of hervs env gene could be the target for new immunotherapy strategies against colorectal cancer. [p ] disclosure of conflict of interest: none. a biosimiliar g-csf filgrastim is as effective as a reference drug however itis not as cost effectiveas it supposed to be and by the way no impact on the health care system m kurt yüksel, g pekcan, u sahin, s bozdag, s toprak, p topcuoglu, o arslan, g gurman and m beksac ankara university school of medicine biosimiliars are up to times the size of small molecule generic drugs and far more structurally complex. additionally biosimiliars are manufactured in living cell lines using processes that cannot be exactly replicated from one manufacturer to the next. a biosimiliar cannot be identical to its reference biologic drug. with billion dollars in global sales of biologic medicines anticipated to go off patent by .this lead to fast production of biosimiliar drugs. besides, it is expected that biosimiliar drugs will be more cost effective than the reference drugs and will have a meaningful impact on health care systems around the world. aim: to compare biosimiliarfilgrastim (leucostim) with two reference g-csf filgrastim (neupogen) and lenograstim(granocyte) in the context of safety, efficacy and cost effectivity. records of patients with multiplmyeloma(mm) whom underwent autologous stem cell transplantation(asct) and received g-csf sc mikrogram/kg/day from +day until engraftment were [p ] retrospectively evaluated mm patients were treated with high dose melphalan and asct at the ankara university school of medicine bone marrow transplantation unit between and . the median age was ( - years) with % male. patients were divided into three groups (n = ) whom received reference filgrastim (neupogen), lenograstim (granocyte) and biosimiliarfilgrastim (leucostim): groups a, b and c respectively. the total cost of each g-csf in dollars was calculated by one package of g-csf multiplied by total used days . chi-square, mann-whitney u and kruskal-wallis tests were used for analyses of variance. the percentage of patients who received melphalan mg/m were% , , in groups a, b, c respectively (p = . ).there was no statistically significant difference between the engraftment day of neutrophil and ; platelet and in the groups. (p = . , p = . , p = . , p = . respectively) themedian numbers of g-csf administered days were ( - ), ( - ), ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in groups a, b, c, respectively .eventhough there was no statistical difference between the numbers of days( p = . ), the total cost in dollars was statistically difference between a vs b and c vs b (both p o . ) and there was no statistical difference between a vs c (p = . ), total cost in dollars as follows: $ ( - $), $( - $) and $( - $) for the group a, b and c respectively. our results demonstrate that biosimiliar gcsf leucostim is highly similar to existing licenced biologic products in turkey with no clinically meaningful difference interms of safety and efficacy. on the other hand it as a biosimiliardoes not have a meaningful impact on the cost savings to the health care system as expected when compared with reference filgrastim. disclosure of conflict of interest: none. in this study, we investigated the roles of prx ii, one of critical peroxidases besides catalase and gpxs, in cml primary cells at diagnosis and remission while patients were treated with sti (signal transduction inhibitor) and tested the same roles in imatinib(im) sensitive ph+ cell lines and resistant cell lines as well. newly diagnosed cml cells, im resistant k cells and parental k cells were treated stis and analyzed western blot assay to detect bcr-abl, phosphorylated bcr-abl and prx protein expression level. we added n-acetylcysteine ( - mm, hr) to k cells to show antioxidant effect of imatinib and analyzed dcf-da detection for intracellular ros level and western blot for prx protein level. mtt assay was performed to detect cell death by nac time-dependent treatment of mm nac( , , , hr). imatinib resistant k cells were established by treatment of gradual increment of imatinib. we also repeatedly investigated the effects of im therapy using prxii overexpressed k cells by transfection. at diagnosis of cml, ros level was elevated and prx ii was either absent or significantly suppressed. as ph chromosomes were decreased with stis, suppressed or absent prxs levels were restored to the level of normal individuals. these findings were also inversely correlated with the level of ph chromosomes in the cases of disease progression and re-remission with further treatment. when sti were treated in ph positive cell line, we found deceased cell survival and ros level by mtt assay and dcf-da methods respectively, but elevated prx ii by western blot. by the treatment of nac into ph+ cell lines, the level of dcf-da was decreased and mtt level was down, but prx ii level was elevated. interestingly, the level of bcr-abl oncogene were decreased in prx ii tranfected cells. meanwhile, we observed that prx ii restoration was mild or weak in imatinib resistant k- , which we established in our lab. the importance of the roles of ros and its prx ii, antioxydant enzymes in cml is further established by our work. our finding may contribute to find a new pathway on which tkis are working besides the mechanisms of atp binding competitively, blocking the binding of abl-bcr kinase to the substrate resulting apoptosis of ph+ cells. furthermore, our finding may be useful to overcome the stis resistant cml in the clinics in the future. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation for the treatment of mucopolysaccharidosis f shunqiao, s xiaodong and l junhui department of hematology, capital institute of pediatrics, china mucopolysaccharidosis (mps) is a lysosomal storage disorder caused by deficient activity of the iduronate-sulfatase.this leads to accumulation of glycosaminoglycans(gags) in the lysosomes of various cells,which causes progressive multisystem involvement with ensuing death.the aim of this study was to exploit the effect of treatment with allogenic hematopoietic stem cell transplantation and administration of high doses of cyclophosphamide early after haplobmt in these cases. we retrospectively reviewed data from mps patients ( cases mps ii, and case mps i). the two mps ii patients were -month-old and -month-old boy and the mps i patient is a -month-old girl at the time of transplantation. the reduced-intensity of bu+flu conditioning regimen in allo-hsct for these patients was as follows: busulfan mg/kg at - days before transplantion,fludarabine mg/m at - days before transplantion.graft-versus-host disease(gvhd) prophylaxis:rabbit antithymocyte globulin . mg/kg daily at - days before transplantation,shortcourse methotrexate,posttransplantation high-dose cyclophosphamide on days + and + was followed by mycophenolate mofetil and cyclosporine.the donors all were their hlahaploidentical father. these three patients' neutrophil engraftment occurred on + d, + d and + d after transplantation respectively, platelet engraftment occurred on day + d, + d and + d after transplantation respectively.complete donor type engraftment was confirmed by short tandem repeat-polymerase chain reaction(str-pcr) on day after transplantation. no regimen-related toxicity occurred,gvhd and graft failure were not observed. month after transplantation, the activity of the iduronate-sulfatase was increased to normal. the motion of metacarpophalangeal joints ameliorated, regression of hepatosplenomegaly, the neurocognitive function improved. allogeneic hematopoietic stem cell transplantation is an effective measure to treat patient with mps at least mps ii and mps i. the reduced-intensity conditioning regimen was helpful to decrease the regimen-related toxicity. posttransplant cyclophosphamide approach successfully used and reduced the incidence of gvhd. this study aimed to evaluate the feasibility of alternative donor hematopoietic stem cell transplantation (hsct) using busulfan, fludarabine, and thymoglobulin conditioning for patients with chronic granulomatous disease (cgd) who lack an hla-matched familial donor. medical records of consecutive patients who received alternative donor hsct between may and may were reviewed, and the transplant-related outcome measures were analyzed retrospectively. the donor source was unrelated peripheral blood (pb) in , unrelated cord blood (cb) in , and haploidentical father in patients. only transplants ( / allele-matched unrelated pb) were hla-matched according to current standards relevant to the donor type. the conditioning regimen was uniform; fludarabine mg/m on days - to - , busulfan . mg/kg/d (or mg/m /d) on days - to - , and thymoglobulin . mg/kg/d on days - to - (or on days - to - in cb recipients). all but one patient were male and their median age at transplantation was . y (range: . - . ). one patient who received a cord blood graft suffered from primary engraftment failure, while the other patients were successfully engrafted with their chimerism levels ranging from % to % (median %) at month post-transplant. the median days to neutrophil and platelet engraftment were . (range: - ) and (range: - ), respectively. among the patients engrafted, one patient experienced secondary graft failure which was rescued by a second transplantation. the remaining one patient who failed to engraft was also rescued with a haploidentical graft from his mother. eight patients ( %) developed cmv antigenemia, and one of those patients developed cmv hepatitis. three patients developed grade acute gvhd which were manageable. one patient who developed grade hepatic gvhd eventually died. two patients developed extensive chronic gvhd, but became free of immunosuppressants after a complete resolution in one and with remaining stable mild joint contractures in the other. including patients who were rescued by additional transplantation, patients are alive with their latest chimerism levels ranging from . % to % (median %). the estimated -y overall survival rate was . % with a median follow-up of months (range: - ). even though the majority of our cohort underwent a mismatched transplantation, the survival rate was excellent. while conditioning with busulfan, fludarabine, and thymoglobulin seems feasible for alternative donor hsct in patients with cgd, special attention needs to be payed on cmv infection and severe gvhd which might offset the high survival rate. disclosure of conflict of interest: none. diarrhea is a common infectious complication in patients who had hematopoetic stem cell transplantation so, we aimed to detect entamoeba histolytica ratio before engraftment, amoung patients who had diarrhea after periferic hematopoetic stem cell transplantation (phsct) in our clinic. allogenic phsct patients had a median age of (range: - ) and autolog phsct patients had a median age of (range: - ). diarrhea is described as an abnormal increase in the frequency ( or more times per day), volume or liquidity of stools. we based upon this description in this study. we made stool examination in the first day of diarrhea. as stool examination, we used direct microscopic evaluation and adhesin antigen test specific for e.hystolytica with enzyme linked immunosorbent assay (elisa), e. histolytica ii, techlab, blacksburg, usa). we accepted e.hystolytica positivity as detecting cyst or/and trophosoit in stool and antigen test positivity at the same time. in our study, of patients had diarrhea in the first days of phsct. diarrhea was found in of in autologous phsct patients (% ), of patients in allogenic phsct with non-myeloablative conditioning regiment (% ) and of patients in allo phsct with myeloablative conditioning regiment (% ). diarrhea occured at + th day of transplantation and the median duration of diarrhea was days. e. histolytica positivity was found of patients ( %) who underwent phsct within first days of transplantation. infection is an important mortality and morbidity factor for patients who had hematopoetic stem cell transplantation, when especially before engrafment (between - days). autologous phsct patients were elderly, with poor self-care and low socioeconomic status individuals. e. hystolytica is a frequent pathogen in posttransplant diarrhea at endemic regions. prophylactic metronidasole treatment should be used routinely for autologous phsct as in allogenic phsct. patients and companions sholud be tested for e.hystolytica before autologus/allogenic phsct in endemic regions. prophylactic treatment for amebiasis and scanning patient/companions could be a part of solution for post phsct diarrhea. despite the emergence of disease modifying therapies (dmts) for multiple sclerosis (ms) a cohort of patients with aggressive disease have ongoing progression/relapse, associated with progressive disability. autologous haematopoietic stem cell transplantation (ahsct) has been used worldwide for aggressive ms with inflammatory changes on mri. we update on a uk single centre experience of ahsct in ms. a retrospective audit of ahsct performed for ms from to at uk centre (king's college hospital) was undertaken. patients were selected for transplantation based on persistent clinical relapses (relapsing-remitting ms) or secondary progressive neurological disability with mri lesion activity despite use of at least dmt. primary progressive patients were also eligible if new/active mri lesions were demonstrable. followup included clinical evaluation, edss assessment and mri scanning. we report our preliminary findings. as of november , patients ( female, male, rrms, spms, ppms) had received ahsct. mean age at transplant was . years (range: - ). the mean baseline edss was . (range: . - . ). patients underwent cyclophosphamide/atg conditioning, while received beam/atg. whilst conditioning and stem cell infusion were well tolerated there was a high rate of infections, with / patients developing a culture confirmed infection. reactivation of ebv and cmv were observed in a number of patients ( and , respectively) while a number of delayed herpes zoster infections were also seen ( cases of shingles and of disseminated varicella infection in patients who had previously experienced it in childhood). median follow-up was for days ( - ). of patients with a formal month assessment (n = ), had a stable edss, had an improved score (median improvement . , range: . - . ) and had a deterioration in their score (median . , range: . - . ). at months (n = ), had a stable edss, had an improved score (median . , range: . - ) and had a deterioration in score (median . , range: . - . ). at months, two patients assessed both had improvements in edss scores (median , range: . - . ). for patients who underwent mri at month follow-up (n = ), had a stable lesion load, demonstrated improvement in lesions and had a new lesion (the remaining mri was difficult to read due to a high baseline lesion load in this patient). patients had mri's at months; were stable and demonstrated a reduction in lesion load. to date, no patients have developed secondary malignancies or autoimmune diseases. of patients with followup data, / rrms patients experienced suspected clinical relapses following hsct-only one had a new lesion on mri (with no gadolinium enhancement). of the received steroids to treat these relapses (it is unclear if the remaining patient received treatment). patient tried a new disease modifying therapy ( dose of rituximab) following hsct. ahsct in this cohort was feasible with universal mobilisation and harvest. whilst conditioning and stem cell infusion were well tolerated, there was a high rate of infectious complications in the neutropenic phase. however, the transplant related mortality was % despite significant levels of disability amongst this patient cohort. ahsct remains a treatment option to be further investigated in this difficult cohort of patients. disclosure of conflict of interest: none. peripheral blood (pb) stem cells (scs) mobilized with g-csf are the first-choice source for allogeneic transplantation. we carried out a prospective study on healthy donors (hds), to identify donor characteristics that could influence the effectiveness of mobilization with special focus on the value of the basal cd + cell count. sibling hds were analyzed in a prospective study. we tested somatic variables (sex, age, weight, height, volemia) and, basal blood counts (white blood cell, peripheral blood mononuclear cell, platelets, hematocrit, hemoglobin, cd + cell). hds received g-csf subcutaneously at a dose of μg/kg day. two different determinations of cd + cells were done in each donor: baseline (before g-csf administration) and in pb on the morning of the fifth day (after g-csf administration). consecutive hds ( males) with a median age of years were enrolled. the mean value of cd +on day was . cells/μl, while the median value was . cells/μl. we performed two multivariate analyses either by using median regression (to predict the median value of cd +on day ) according to the values of cd + at baseline, the first adjusted by gender, age and blood volume and the second by gender, age and bmi. results of both models indicate that from basal cd + values o = to values ranging between and cells/μl, predicted median values of cd + on day significantly increase, from . to . cells/μl for model adjusted by blood volume, and from . to cells/μl for model adjusted by bmi. baseline, pb cd + cell count correlated with the effectiveness of allogeneic pbscs mobilization and could be useful to plan the collection. disclosure of conflict of interest: none. comparison of efficacy between chemotherapy plus granulocyte colony stimulating factor (g-csf) and chemotherapy plus g-csf and granulocyte-macrophage colony stimulating factor (gm-csf) for mobilization of peripheral blood stem cells (pbsc) and hematological recovery post-transplantation in patients with multiple myeloma (mm). a retrospective study of autologous peripheral blood stem cell (apbsc) mobilization data of mm patients who treated with chemotherapy plus g-csf or chemotherapy plus g-csf and gm-csf from may to july . the mobilization efficacy and hematopoietic recovery were analyzed. a total of stem cell mobilizations were performed in mm patients. in the univariate analysis, successful collection rate of single harvest in female and in patients at iss stage iii, r-iss ii/iii and chemotherapy plus g-csf was lower(po . ). however, age(≦ yrs vs yrs), subtype, d-s staging (i+ii vs iii), cycles of chemotherapy before mobilization (≦ cycles vs cycles), disease phase before mobilization (pr vs cr) and interval diagnosismobilization (≦ months vs months) were not correlated with the cd + cell collection and successful mobilization rate(p＞ . ). in the multivariate model, rate of successful mobilization in patients who received chemotherapy plus g-csf+gm-csf mobilization regimen was high (or = . , %ci . - . ). the effect of mobilization regimen remained significantly (p = . ). all patients successfully underwent hematopoietic reconstruction without transplantation-related mortality. chemotherapy plus g-csf +gm-csf mobilization regimen can significantly increase the effect of apbsc mobilization and ensure the reconstruction of hematopoietic function after transplantation. this mobilization regimen is a safe and effective method of mobilizing apbsc. disclosure of conflict of interest: none. clinical efficacy of bk virus specific t-cells in treatment of severe refractory hemorrhagic cystitis after hla haploidentical transplantation om pello , a bradshaw , a innes , s-a finn , s uddin, e bray , e olavarria, jf apperley and j pavlu centre for haematology, imperial college at hammersmith hospital, london, uk and department of clinical haematology, hammersmith hospital, imperial college healthcare nhs trust, london, united kingdom hemorrhagic cystitis caused by bk virus (bkv) is a significant complication of allogeneic hematopoietic cell transplantation (hct). it is particularly common in the setting of hla haploidentical transplantation and can be challenging to manage. here we present a post haploidentical hct patient who developed severe bkv haemorrhagic cystitis resistant to standard therapy and who responded to adoptive transfer of donor t cells enriched with anti-bkv specific cells. a year old man underwent hct for acute myeloid leukaemia with inversion of chromosome and monosomy of chromosome while in first complete remission. as he had no related or unrelated hla identical donor, cells from his hla haploidentical sister were used. on day + of this procedure he developed haemorrhagic cystitis. supportive treatment was initiated and cystoscopy showed diffuse bleeding from his urinary bladder with blood clots. urine pcr for bkv showed . billion copies/ml. despite bladder irrigation, local therapy to s bladder mucosa and intravenous hydration, he failed to improve, so treatment with weekly intravenous cidofovir was initiated on day + . his symptoms improved, but on day + he again deteriorated on weekly infusions of cidofovir. his immunosuppression was slowly tapered off without any graft versus host disease (gvhd) but without any significant effect on his hemorrhagic cystitis. he underwent bladder diathermy, was treated with intravesicular hyaluronate and with intravenous cidofovir, but continued to have frank haematuria with blood clots and significant lower abdominal pain. although there was no evidence of obstruction his renal function deteriorated on cidofovir therapy. hence we elected to trial adoptive anti bkv therapy. a leukoapheretic lymphocyte collection was used to prepare an anti-bkv t cell enriched product using the clinimacs prodigy and the cytokine capture system from miltenyi biotec. the eluted product contained % and % of cd + and cd + lymphocytes expressing ifng+ respectively and the cd +/cd + dose adoptively transferred on day + of transplantation was . × /kg. in vivo expansion of anti-bkv t cells in the patient was analysed weekly for the first month using the research grade peptivators bkv lt and bkv vp and the rapid cytokine inspector (cd /cd t cell) kit. bk viral load was monitored by pcr in urine samples twice weekly. ifng+ anti-bkv reactive t cells were undetectable in the patient for the first two weeks after adoptively transfer of donor t cells. twenty days after the adoptive transfer an increase in the cd + ifng+ population was observed, in response to the bkv vp peptivator. this observation correlated in time with a substantial decrease of the urine bkv viremia from . million copies/ml to copies/ml and a complete resolution of patient's symptoms. no gvhd, recurrence of urinary symptoms or any other problems have been observed to date (day + of transplantation, + days after the adoptive transfer). we are not aware of any other reports of successful adoptive anti bkv cellular therapy. our experience suggests safety and efficiency of the use of anti-bkv t cell enriched products using the clinimacs prodigy and the ifng capture system in hla haploidentical hct where bkv cystitis constitutes a significant complication. this opens the possibility of further clinical trials. disclosure of conflict of interest: none. haploidentical donor (hd) has been used as an alternative stem cell source when patients do not have a hla-matched related or unrelated donor. to overcome the hla barrier, haploidentical stem cell transplantation (haplosct) using post-transplantation cyclophosphamide (ptcy) has been conducted. here, we compared the clinical outcomes of haplosct using ptcy with those of unrelated donor transplantation. eighty-two patients ( from hd and from unrelated donor [ud]) who underwent allogeneic hematopoietic stem cell transplantation (hsct) in seoul national university children's hospital from january to june , were analyzed. there were no significant differences between hd and ud patients with respect to median age of patients, sex distribution, and diagnosis [ . %], p = . ). the conditioning regimen of haplosct included targeted busulfan, fludarabine and cyclophosphamide using ptcy, tacrolimus and mycophenolate mofetil for graftversus-host disease (gvhd) prophylaxis. all patients showed engraftment except for a patient who underwent unrelated hsct. neutrophil engraftment of ud was faster than hd (median days versus . days, respectively, po . ). however, there was no significant difference of platelet engraftment. incidences of complications, such as hepatic venoocclusive disease, cmv infection, and hepatic dysfunction, between both groups, were comparable, except hemorrhagic cystitis (hd: . %, ud: . %, p = . ). moreover, cumulative incidence of acute gvhd (hd: . %, ud: . %, p = . ), severe chronic gvhd (hd: . %, ud: . %, p = . ), relapse (hd: . %, ud: . %, p = . ) and non-relapse mortality (hd: %, ud: . %, p = . ) were not significantly different. the overall and event-free survival of hd and ud were . % vs . % (p = . ) and . % vs . % (p = . ), respectively. the clinical outcomes of haplosct using ptcy were comparable with those of ud, and a trend of lower cumulative incidence of severe chronic gvhd and non-relapse mortality was encouraging. it could be a promising alternative therapeutic option in pediatric hsct. disclosure of conflict of interest: none. , median number of apheresis procedures was , ( - ), median amount od dmso infused ml ( - ). time to engraftment was median days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . statistical comparison between cryopreserved pbsc grafts and bm showed benefit for pbsc in the terms (po . ) of faster engraftment, less infective complications, less transfusion support and less hospital stay. in patients ( %) dmso related events were not registered during graft administration. in patients ( %) mild to moderate dmso related events were registered, as nausea in patients ( . %), vomitus in patients ( %), tachycardia in ( . %), hematuria in patients ( %) and patients ( . %) with bradycardia, hypotension, fever and high temperature during graft infusion. cryopreservation of stem cells is a feasible procedure at our institution. there are some issues that have to be improved. the process is standardized with achieved engraftment in all transplanted patients. disclosure of conflict of interest: none. effectivity of a fludarabine based conditioning regimen in allogenic hematopoietic stem cell tranplantation for patients with severe aplastic anemia and over twenty years old p mustafa , , k melya pelin , s handan haydaroglu and g ilknur gaziantep university, faculty of medicine, department of internal medicine; hematology and bone marrow tranplantation unit, gaziantep, turkey severe aplastic anemia (saa) is an anemia with bone marrow hypocellularity and caused by hematopoietic stem cell failure ( ) . allogenic periferic hematopoietic stem cell transplantation (aphsct) is a curative treatment choice ( ) . although cyclophosphamide (cyc) and anti thymocyte globulin(atg) is accepted as standart conditioning regimen, especially for patients with high rejection risk, using fludarabin (fu) based regimens show increased successful engraftment ratio with minimal toxic side effects ( ) . to the study, saa patiens who were transplanted from hla matched sibling donors between the years - were included. the patients comprised of male (% ) and female (% ). median age was (range: - ). the median time from diagnosis to transplantation was (range: - ) months. conditioning regimen consisted of cyc ( mg/m ), fu ( mg/m ), atg (fresenius rabbit, mg/kg). the median dose of stem cells was × stem cell/kg (range: - ). methotrexate ( mg/ m given four days) and cyclosporine (cyca) ( - mg/kg given months) were applied for graft versus host disease (gvhd) prophylaxis. all patients ecog performance status were good ( - ). prior to transplantation only one of the patients received atg-csa, the others received only supportive treatment. after aphsct, neutrophil engraftment was occured at a median of days (range: - ) and thrombocyte engraftment was occured at a median of days (range: - ). posttransplant graft failure was observed in only one patient at tenth month and this patient had aphsct again from the same donor with the same conditioning regimen. acute gvhd didnot occur in any patient. the (% ) of patients had common chronic skin/oral mucosa gvhd. these patients received methylprednisolone (mp) and/or mycophenolate mofetil (mmf) in addition to the cyclosporine treatment. extracorporal photopheresis was applied to the two patients with chronic gvhd. all chronic gvhd patients had complete response to the immunsupresive treatment with a median follow up time months (range: - ). one patient died from sepsis. at year overall-survival rate was %. fu based conditioning regimen in aphsct with young saa patients has favorable results. fu based regimen might be a gold-standard treatment in the future. cgvhd; tgfb-induced factor homeobox , interleukin receptor gamma, tetra trico peptide repeat domain , carbonic anhydrase i, serpin peptidase inhibitor clade a and myod family inhibitor. we established a -gene model (myod family inhibitor, tgfb-induced factor homeobox , tetra trico peptide repeat domain ) to diagnose cgvhd. our -gene model showed . % sensitivity, . % specificity, . % precision, . % accuracy and . % roc area in diagnosing cgvhd. tgfb-induced factor homeobox increased in expression after rituximab treatment in responders. myod family inhibitor was found to be able to predict rituximab responses in steroid-refractory cgvhd patients. we could demonstrate that gene expression studies were useful in the diagnosis of cgvhd after allo-hsct. we developed a -gene model to diagnose cgvhd. hematopoietic stem cell transplantation (sct) is physically and psychosocially demanding. however, exercise interventions may have positive impact on sentiment and psychological well-being in patients undergoing sct. we report on a prospective, randomized study comparing the influence of a multimedia sensor-based practice with classical physiotherapeutic treatment (pt) on psychological aspects and quality of life (qol). patients undergoing sct were randomized into the control group (n = ) receiving pt or the experimental group exercising on the nintendo-wii (n = ). patients of both groups performed the exercises under the supervision of a physiotherapist and completed the functional assessment of cancer therapy -bone marrow transplantation (fact-bmt), hospital anxiety and depression scale (hads-d) and distress thermometer at the date of hospital admission (t ) and on day (t ), (t ) and (t ) after sct. questionnaires were completed by the participants independently and without supervision. groups were compared using the mann-whitney u-test. a p value o . was considered statistically significant. the median age of patients was years in the control group and years in the experimental group. results of fact-bmt generally showed a decline of the qol domains measured on t and t and a raise at t in both groups. physical well-being (pwb) showed the strongest fluctuation of all domains. it declined significantly between t -t in both groups (pt p = . , wii p = . ), followed by a significant increase between t -t (both groups p = . ). however, only in wii-group results of pwb at t ranked significantly above t (p = . ). highest scores were proved for social and emotional well-being (swb/ewb) in both groups. in wii-group ewb increased significantly between t -t (p = . ) and ranked above pt-group at all times. functional well-being (fwb) scored lowest in both groups at all times. the score of bone marrow transplant scale (bmts), the second lowest score in both groups, was always higher in wii-group. the level of distress was comparable between both groups. however, at t distress increased above the cut-off level of in both groups (wii-group p = . , pt-group p = . ). this was accompanied by an increase of anxiety (p = . ) and depression (p = . ) in the pt-group, while both parameters decreased in the wii-group (p = . and p = . ), respectively. anxiety in intervention group , / , / , /, at t /t /t /t stayed below standard group , / , / , / , at all times. depression averaged out at , / , / , / , in physiotherapy group and , / , / , / , in wii-group. to the best of our knowledge, this is the first study to show that exergaming using the nintendo-wii is feasible in the immediate phase after hsct. exergaming may be regarded as beneficial since our data indicate less psychological distress and higher qol in sct recipients exercising with nintendo-wii. therefore, it may be used in addition to pt. disclosure of conflict of interest: none. acute graft versus host disease (agvhd) is the most frequent and serious complication following haematopoietic stem cell transplantation (hsct), with a high mortality rate. a clearer understanding of the molecular pathogenesis may allow for improved therapeutic options or guide personalised prophylactic protocols. circulating micrornas are expressed in body fluids and have recently been associated with the etiology of agvhd, but global expression profiling in a hsct setting is lacking. this study profiled expression of n = mature micrornas in patient serum, using the nanostring platform, to identify micrornas that were dysregulated at agvhd diagnosis. selected micrornas (n = ) were replicated in independent cohorts of serum samples taken at agvhd diagnosis (n = ) and prior to disease onset (day post-hsct, n = ) to assess their prognostic potential. sera from patients without agvhd were used as controls. dysregulated micrornas were investigated in silico for predicted networks and mrna targets. profiling identified micrornas that were differentially expressed at agvhd diagnosis. mir- a (p = . ), mir- b- p (p = . ), mir- - p (p = . ), mir- a (p = . ), mir- a (p = . ) and mir- a (p = . ) were significantly verified in an independent cohort (n = ). mir- a (p = . ), mir- a (p = . ), mir- (p = . ), mir- a (p = ), mir- b ( . ) and mir- (p = . ) were differentially expressed days post-hsct in patients who later developed agvhd (n = ). high mir- b expression was associated with improved overall survival (os) (p = . ), while high mir- a and mir- b- p were associated with lower rates of non-relapse mortality (p = . and p = . ) and improved os (p = . and p = . ). pathway analysis associated the candidate micrornas with haematological and inflammatory disease. circulating biofluid micrornas are dysregulated at agvhd onset and have the capacity to act as prognostic and diagnostic biomarkers. their differential expression in serum suggests a role for circulatory micrornas in agvhd pathology, which warrants further investigation. disclosure of conflict of interest: none. factors associated with medication adherence amongst allogeneic hematopoietic stem cell transplantation recipients j lehrer , e brissot , , a ruggeri , , r dulery , a vekhoff , g battipaglia , f giannotti , c fernandez , , , m mohty , and m antignac ap-hp, hôpital saint-antoine, service de pharmacie, paris, f- ; service d'hématologie et de thérapie cellulaire, hôpital saint antoine, assistance publique-hôpitaux de paris; sorbonne patients with median ages of years (range: - years) between december and march , which including case of primary hlh (homozygous missense mutation in unc d: n = ; homozygous missense mutation in prf : n = ; heterozygous missense mutation in prf in the combination with hemizygous missense mutation in sh d a: n = ; mutation in rab a: n = ; mutation in itk: n = ). cases of unknown causes hlh, cases of lympgoma -hlh (nk/t-cell lymphoma: n = , primary γδt cell lymphoma in skin: n = ; subcutaneous panniculitis-like t cell lymphoma: n = ; primary t cell lymphoma in skin: n = ) and cases of ebv associated hlh. patients achieved cr+pr before hsct, and patients nr. patients were transplanted from hla-haploidentical family donors, from hla-identical sibling donors, and from a matched unrelated donor. conditioning regimen include tbi and non-tbi. the median overall survival rate was . % with a median survival time of months (range: - months). os of primary hlh is . %, os of unknown causes hlh is %, os of lymphoma-associated hlh is %, os of ebv-hlh is . %. os of cr+pr is . %, os of nr is . % patients without engraftment died because of graft failure and toxicity of conditioning regimen. patients with engraftment died. of those, patient died of hsct-associated tma, patient died of grade iv agvdh, patients died of relapsed hlh or organ failure as results from unsuccessful treatment of the progressively elevated ebv-dna load. patient died of tumor relapse, and patient died of infection. acute gvdh occurred in patients with grade i-ii agvdh in patients and grade iii-iv agvdh in patients; chronic gvdh occurred in patients. patients achieved completed chimerism, patients appeared with mixed chimerism,and patient presented with graft failure. of ebv-hlh with engraftment, reactivated ebv infection was found in ( %) with the whole blood ebv-dna load at - copy numbers per ml. ptld occurred in patients confirmed by pathology. after reduced immunosuppressors, negative result of ebv infection was obtained while patients developed gvdh. for ebv-hlh, patients who carry with ebv loading ebvdna ≤ copies/ml before transplantation, overall survival rate was significantly higher than that of ebvdna copies/ml (po . ); who achieved cr+pr os was significantly higher than that of nr (po . ); who range: from diagnosis to transplantion ≤ months os was significantly higher than that of months (po . ). allogeneic hematopoietic stem cell is an effective method for primary hlh and lymphoma-hlh, ebv-hlh,even haploid transplantation. the remission status before transplantation is decisive for the prognosis. disclosure of conflict of interest: none. hepatic veno-occlusive disease after allogeneic hematopoietic stem cell transplantation in a single centre: revised diagnosis and incidence according to new ebmt classification s santarone, a natale, p olioso, g papalinetti, t bonfini, p accorsi, s angelini, g iannetti, m di ianni and p di bartolomeo dipartimento di ematologia, medicina trasfusionale e biotecnologie, bmt center, ospedale civile, pescara, italy sinusoidal obstruction syndrome, also known as venoocclusive disease (sos/vod), is a potentially life threatening complication that can develop early after hematopoietic cell transplantation (hct). in this study we retrospectively investigated the incidence, risk factors and outcomes of sos/vod in transplants, performed in patients between march and may , on the basis of the new diagnostic criteria and classification of the ebmt. the patient's median age was years ( to ). of them, were males and females. patients received one transplant and two transplants. a diagnosis of hematological malignant and nonmalignant disease was present in and cases, respectively. the disease risk at hct was standard in cases, intermediate in and high in . an hla identical sibling donor was used in in cases, an unrelated donor in and a haploidentical family donor in . conditioning was myeloablative in transplants and at reduced intensity in . source of hematopoietic stem cells was bone marrow in transplants and peripheral blood in . we did not limit the diagnosis of sos/vod to the classical days after hct, but all suspicious cases appearing in the first days were evaluated. sos/vod was diagnosed in cases, of which in the first days after transplant and between day and (median day ). their main clinical characteristics are shown in table . the severity of sos/vod was mild in patients ( %), moderate in ( %), severe in ( %) and very severe in ( %). the cumulative incidence (ci) of sos/vod was . + . %. among the most relevant variables studied in univariate analysis (recipient age and gender, ferritin level at hct, type of hematological disease, disease risk at hct, type of donor, number of transplants, time of transplant, drugs used in the conditioning regimen, intensity of the conditioning regimen, source of stem cells), there was no factor with an adverse impact on sos/vod incidence. of patients with diagnosis of sos/vod, ( %) died. sos/vod was the main cause of death in patients and a relevant contributing cause of death in . of relevance, of patients ( %) with severe sos/vod and of patients ( %) with very severe sos/vod died, whereas only one patient with moderate sos/vod died and no patient with mild sos/vod died. among patients with sos/vod, received defibrotide therapy and the best supportive available therapy. defibrotide was given for a median of consecutive days (range: to ), starting at day post-hct (range: to ) with a median total bilirubin level of , mg/dl (range: . - . ). the -year overall survival (os) of patients treated with defibrotide was better as compared to that of patients who received the supportive therapy ( % versus %) although the difference doesn't reach the significance (p = . ). the occurrence of sos/vod does influence significantly the -yr os considering that it was + . % for patients without sos/vod and + % for patients with sos/vod (p = . ). in conclusion, the new ebmt diagnostic and severity criteria for sos/vod has been very useful in identifying patients with severe and very severe forms of this complication. if validated in prospective studies, these criteria will allow an earlier selection of patients requiring immediate therapeutic intervention. [p ] disclosure of conflict of interest: none. the prognosis of patients with newly diagnosed ewing's sarcoma family of tumors (esft) has improved significantly over the last few decades. nonetheless, the long-term survival is still below % patients with high risk features.the role of s high dose chemotherapy and autologous stem cell transplantation (hdct and asct) for high risk and relapsed esft was analyzed. a retrospective medical chart review was done on patients with efst who underwent hdct and asct between september and january at seoul national university children's hospital. indications for hdct and asct included metastasis at diagnosis, bulky primary tumor ( ml), axial/ central primary site, and relapsed disease. single hdct and asct was performed in the earlier period, and the regimen was changed from mec (melphalan, etoposide, carboplatin), to topothiocarbo (topotecan, thiotepa, carboplatin), and to bumel (busulfan, melphalan). tandem hdct and asct was performed in the recent period, st hdct with bumel and nd hdct with modified mec (melphalan, etoposide, carboplatin). twenty-one patients who were diagnosed with esft at a median age of . years old underwent conventional chemotherapy, radiation therapy and/or surgery and received hdct and asct in complete response (n = ) or partial response (n = ). the overall survival of the patients was . % at median . years and the event free survival (efs) of the patients was . % at median . years from the last asct. the efs of the patients who underwent single hdct and asct with mec (n = ), topothiocarbo (n = ), and bumel (n = ) was . %, . % and % respectively. the efs of the patients who underwent tandem hdct and asct (n = ) was . %. seven patients relapsed at median . months from the last asct. despite further treatment, patients died of disease and patients are currently alive without disease. one patient developed acute myeloid leukemia at . months from the last asct and is currently alive without disease after additional chemotherapy, hla-haploidentical stem cell transplantation and donor lymphocyte infusions. one patient died of transplantation-related mortality due to septic shock and lung infection. hdct and asct may be a promising treatment option for patients with high risk or relapsed esft. further refinements may be needed to identify the optimal regimen and number of hdct and asct. disclosure of conflict of interest: none. post transplant cyclophosphamide (pt-cy) has expanded the use of unmanipulated haploidentical grafts which have a high hla disparity between host and donor. one of the consequences of hla disparity is the development of engraftment syndrome (es). this is an immunological reaction characterized by non-infectious fever and skin erythema that develops after neutrophil engraftment. es resembles an infectious process but treatment involves the use of high dose steroids. our hypothesis is that pts undergoing haploidentical transplants (haplo) with pt-cy should have a high rate of es given the high hla disparity between donor and recipient. objectives: to determine the incidence, symptoms, morbidity and mortality of es in patients undergoing haplo with pt-cy at our institution. retrospective analysis of patients with highrisk hematological diseases undergoing haplo with pt-cy at clinica santa maria between november and august . es was diagnosed using the spitzer criteria ( ). es was diagnosed if pts met major criteria or major plus minor criterion. symptoms could occur prior to or after neutrophil engraftment (neutrophils over cells / ul). all patients signed informed consent and the study was reviewed by our institutional review board. patients received haploidentical grafts ( table ) . all patients had neutrophil engraftment at a median of days. / patients ( %) had symptoms that met criteria for es ( table ). / were transferred to icu due to hypoxemia and patient died after diagnosis of es. / pts were treated steroids. all patients received broad spectrum antibiotics during the febrile period and neutropenia. blood cultures, ebv and cmv pcr were negative in all es pts. there were no significant differences in hospital stay or one-year overall survival (os) between patients who developed and pts who did not develop es (median vs. days respectively, p = . ; one-year os % vs. %, p = . , respectively). es is a frequent complication in patients undergoing hsct haplo with pt-cy. the incidence of es in our study was higher when compared to historical full match related donors series and lower when compared to cord blood transplant studies ( ) there was no increased morbidity and mortality associated with es diagnosis. prompt institution of steroids is recommended in es patients after ruling out an underlying infectious process to avoid further complications. haploidentical allogenic hematopoietic stem cell transplants (haplo-hsct) is an alternative transplant procedure for patients with hematologic malignancies that are in need of transplant and do not have a compatible donor. due to the broad hla disparity, the haplo-hsct can be performed with t-cell depletion and megadose of cd +. alternatively haplo-hsct can be performed with non t-cell depleted transplants (t-replete) either in combination with anti-thymoglobuline serum (atg) or post-transplant cyclophosphamide (pt-cy) as gvhd prophylaxis strategy. center effect is a known risk factor for outcomes of haplo-hsct in both t-cell depleted (tcd) and t-replete settings. however, many centers tend to specialize in one gvhd prophylaxis strategy making it difficult to differentiate the treatment effect from the center effect. the objective was to investigate the role of center effects in gvhd prevention strategy, on leukemia-free survival (lfs) and overall survival (os) in a population of adult patients with acute leukemia receiving haplo-hsct. a retrospective multicenter study was conducted on patients reported to the ebmt registry. inclusion criteria were: age years, lymphoblastic or myeloid acute leukemia (all or aml) in first or second complete remission (cr or cr ), receiving a haplo-hsct between and . in this population (n = ), in order to assess the interaction between center and gvhd prevention treatment, we then included in the study selected patients from the centers that had performed more than % of both tcd and t-replete haplo-hsct during the study period. center effects on the outcomes consisted of ) center effect on the baseline risk of event and ) interaction between center and strategy of gvhd prevention. these center effects were estimated using cox mixed-effects models and tested using permutation tests. all models were adjusted on age, cmv statuses, disease (all or aml), secondary leukemia, previous autologous transplant, disease status (cr or cr ), peripheral blood vs. bone marrow transplant, conditioning regimen. after selection, patients were available across centers in europe. one hundred and one ( %) patients received tcd, t-replete haplo-hsct ( ( %) using atg and ( %) using pt-cy). overall, ( %) patients had aml. there were ( %) peripheral blood transplants in the tcd group and ( %) in t-replete. median follow-up was . years. in adjusted analyses, without accounting for center effect, t-replete tended to be associated with better lfs (hazard ratio (hr): . ( %ci . - . ), p = . ) and os (hr = . ( %ci . - . ), p = . ). when center effects were included, there was significant heterogeneity across centers on the baseline risk of both outcomes (lfs: p = . and os: p = . ). when accounting for interaction between center by strategy for gvhd prevention, the effect of t-replete vs. tcd on the outcomes did vary across centers (p = . and p = . for interactions in lfs and os, respectively) ( figure ). we found an interaction between center and strategy for gvhd prevention on outcomes of patients who received a haplo-hsct. the difference between the strategies (tcd or t-replete) varied across centers, in size and direction. this could be in part related to the increase in expertise with each technique in some centers and with the different management of complications, such as infections-related and relapse. disclosure of conflict of interest: none. adherence included recognition of spuriously high levels (typically from contaminated lines) and delayed dose adjustment due to late reporting of levels by the laboratory. the most common cause of unjustifiable non-adherence was failure to increase the dose in response to a low level. inadequate or excessive dose adjustments may be due to lack of experience or unfamiliarity with the sop. two interventions were launched with the aim of improving adherence to the sop for therapeutic tacrolimus dosing. firstly, to provide a rapid and user-friendly calculation method, we developed a mobile phone application (tacrocalc, a dose calculator based upon the sop algorithm) for android and ios devices using python and swift, respectively. secondly, to reduce the number of spuriously high levels, all nurses responsible for specimen collection participated in an educational module delivered by medical and senior nursing staff. key messages included the need to: use only the dedicated colour-coded tacrolimus lumen to infuse iv tacrolimus; avoid sampling from this lumen; sample peripherally when other lumens are known to be contaminated (reasons for this are being explored); suspend infusion of iv tacrolimus minutes before taking a level; send only immediately pre-dose levels for oral tacrolimus. initial re-audit of episodes post intervention (data collection is ongoing) demonstrated a % increase in sop adherence (p = . ; fisher's exact test), with no cases of unjustifiable non-adherence and a significant reduction in spuriously high levels. in conclusion, the use of tacrocalc by doctors and the implementation of targeted teaching for nurses dramatically improved adherence to the tacrolimus sop. this should ultimately improve therapeutic dosing whilst avoiding toxicity, which may result in better transplant outcomes. tacrocalc is now being adapted to include an option for paediatric dosing, with the potential to dose related medications such as cyclosporine. disclosure of conflict of interest: none. king's college hospital, imperial healthcare, charing cross hospital and imperial healthcare, hammersmith hospital managed with calcium and vitamin d alone in / cases ( %) and together with bisphosphonates in / ( %). osteoporosis was managed with bisphosphonates ± calcium/ vit d in / and with calcium/vit d alone in / . / indicated that they would give bisphosphonates in the absence of osteoporosis, if a patient with osteopenia was receiving long term steroids. dissemination and implementation of existing guidance on investigation and managing low bmd post hct appeared to be poor amongst respondents to our survey. routine dxa scanning was underused; the trigger for dxa in the context of steroids is inappropriately high at many centres at mg/kg daily for months; in established osteoporosis, bisphosphonates were used less frequently than would be anticipated. these findings may reflect the limited data on which current recommendations have been made, or the large number of non-transplant guidelines for investigating and managing low bmd which confound management of this post-hct patient group. hematopoietic stem cell transplantation (hsct) still remains as the most efficient therapy for adult patients with acute myeloid leukemia. for older patients and those lacking a hlacompatible donor, autologous hematopoietic stem cell transplantation (auto-hsct) is a valid therapeutic option. authors aimed for determining the effect of auto-hsct for acute myeloid leukemia patients and analyze group of patients who underwent auto-hsct. the study has been set as a retrospective single center study. clinical information included age, gender, aml type and cytogenetic risk. pretransplantation treatment, mobilization and conditioning were analyzed and thus subsequently authors used kaplan and meier method to calculate the actuarial overall survival rate. table describes patients' characteristics. majority of patients received similar induction therapy based on combination of cytarabine and anthracycline. timespan from the diagnosis to auto-hsct varied from days to days, median was days. seventy ( , %) patients received a preparative regimen consisting of busulfan at mg/kg orally, four times daily for days for a total dose of mg/kg administered on day - through day - and melphalan - mg/m intravenously for over hours on day - . patients achieved an absolute neutrophile count (anc) of ≥ . × /l in between to days; median was days. patients achieved not transfused platelet count ≥ × /l in between to days; median was days. median of patients' discharge from hospital was days (range: from to days) since auto-hsct. hundred day mortality after autologous transplant was at . % ( / ). on the date of our evaluation (april , ), patients were alive and in continued cr. the relapse rate was . % ( patients) and patients ( . %) were lost from follow-up. the -year overall survival (os) was . %, so the target median of overall survival has not been reached. [p ] the development of dyslipidaemia is commonly observed after haematopoietic stem cell transplantation (hsct). few data are available concerning lipid profiles over a long followup period or with regard to the different transplantation types (autologous vs. allogeneic) or the effect of multiple transplantations on the development of dyslipidaemia. a retrospective, single center cohort study including adult patients ( years) who underwent hsct at the university hospital basel s - and who survived ≥ days was performed. patients with at least a baseline lipid measurement were included (n = ) and grouped according to the type of their first hsct (autologous or allogeneic). for the examination of the effect of subsequent hscts, patients with consecutive transplantations of the same type were included and other patients were censored when a different transplantation type was performed. serial lipid profiles (total-, ldl-and hdl-cholesterol and triglycerides) before and after transplantation were examined. of the patients, underwent a first, and of these at least one subsequent autologous hsct. underwent a first, and of these at least one subsequent allogeneic hsct. median age of patients at autologous hsct was y (iqr - ) and y ( - ) at allogeneic hsct. % and % were males, median bmi pre-transplant was ( - ) and ( - ). the majority of patients underwent s intensive conditioning before hsct. median follow-up time was . years in the autologous and . years in the allogeneic group, with a maximum follow up time of . and . years, respectively. table shows the number and percentage of patients with dyslipidaemia ( st autologous and allogeneic transplants). the distribution of exact total cholesterol values along with comparisons with baseline measurements according to group are presented in the figure . *% based on number of measurements available total, ldl-and hdl-cholesterol and tg increased within months of transplantation, regardless whether autologous or allogenic transplantation or a first or a subsequent transplantation was performed. the percentage of patients with dyslipidaemia accordingly rose significantly within months of transplantation and persisted throughout follow-up. although patients undergoing an autologous hsct presented with higher baseline values of total cholesterol, a significantly greater increase post-transplant was observed after allogeneic hsct. first and subsequent transplantations seem to behave similarly with respect to changes in lipid profiles. disclosure of conflict of interest: none. nuremberg, erlangen, germany; department of cancer immunology, institute for cancer research, oslo university hospital, radiumhospital, oslo, norway; kg jebsen center for cancer immunotherapy, institute of clinical medicine, university of oslo, oslo, norway; department of haematology and oncology, university hospital of the goethe university, frankfurt, germany and childrens hospital, goethe university, frankfurt, germany natural killer (nk) cells are lymphocytes of the innate immunity with a potent anti-tumor capacity. in tumor patients, such as multiple myeloma (mm) patients, an elevated number of nk cells correlates with a higher overall survival (os) rate. our study adressed nk cells characteristics and anti-tumor ability in mm patients. especially cytotoxicity of patientderived, cytokine-stimulated nk cells against mm cells has been analyzed at various time points (at diagnosis, before/ after chemotherapy and/or auto-sct). nk cells from patients were analyzed by facs after pbmcs isolation via ficoll separation at different time points: tp , before the start of high dose chemotherapy (hdc)/auto-sct; tp , after early leukocyte recovery (leukocytes /μl) and tp : at least weeks after tp . for testing nk cell cytotoxicity against mm cells, nk cells were purified via negative selection and expanded in vitro for - weeks in low doses il- and il- . nk cells were divided into the cd ++ cd − or cd + and cd + cd ++ subsets. while the major nk cell subset at tp was the cd + cd ++ nk cell subpopulation ( . %), after leukocyte recovery at tp cd ++ cd − /+ nk cells were the main subsets (cd − : . %; cd + : . %). we further evaluated the nk cell function upon tumor interaction at the defined time points. cd ++ cd − nk cells were the main subset to produce ifn-γ upon interaction with k cells at all different time points. the percentage of ifn-γ-positive cd + + cd − nk cells was slightly decreased at tp compared to tp but significantly increased from tp to tp (p-value: . ). similarly, mip- β-and cd a-positive cd ++ cd − cells remained constant between tp and tp , whereas their percentages increased from tp to tp [p-values: . (mip β) and . (cd a)]. moreover, in a small group of mm patients, we isolated nk cells and expanded them for - weeks prior to the functional assays. as expected, the expansion rate was reduced after chemotherapy compared to nk cells from healthy controls, but the patients nk cells increased their ability to kill mm cells due to the ex vivo cytokine expansion. conclusion: our data demonstrate that nk cells have an altered phenotype and function after hdc/auto-sct. remarkably, these nk cells were able to secrete cytokines and still displayed cytotoxic capacity against different types of tumor cells. however, as the proliferative capacity of nk cells seemed to be reduced following chemotherapy, innovative nk cell therapeutic approaches further improve the patients nk cell activity by an ex vivo cytokine stimulation procedure. finally, we suggest that an additive cell therapy with cytokinestimulated autologeous nk cells might improve the outcome of mm patients. lymphoid and myeloid acute leukemia are the most frequent type of cancer and the most frequent cause of cancer related death in children. relapse and refractory disease are the main clinical problems that current therapies are still unable to solve. one of the main nk cell activating receptors is nk cell group d (nkg d). nkg d receptor recognizes human mica/ulbp - ligands. these nkg d ligands are expressed in leukemia cells and constitute suitable targets for immunotherapy. the expression of nkg d ligands was analyzed in peripheral blood mononuclear cells from pediatric patients suffering from acute leukemia ( acute myeloid leukemia, b cell acute lymphoid leukemia and t cell acute lymphoid leukemia), as well as in leukemia cell lines (k , rs - , jurkat, nalm- , molt- , reh and cem), by flow cytometry using specific monoclonal antibodies directed against mica, micab, ulbp- , ulbp- , ulbp- and ulbp- , and by quantitative pcr using taqman probes. peripheral blood mononuclear cells from healthy donors were labeled with cd ra microbeads and depleted using automacs device. the hl i r-mndanticd bbz lentiviral vector was derived from the clinical vector cl i r-ef a-hgcopt but contained the extracellular domain of nkg d, the hinge region of cd a and the signaling domains of - bb and cd -z. the cassette was driven by mnd promoter. viral supernatant was produced by transient transfection of hek t cells with the vector genome plasmid and lentiviral packaging helper plasmids pcagg-hivgpco, pcagg-vsvg and pcag -rtr . cytogenetic studies and array comparative genomic hybridization were performed to analyze the genetic stability of lentiviral-transduced memory t cells. the in vitro cytotoxicity of cd ra − t cells against leukemia cells, healthy pbmc and mesenchymal stem cells (msc) was evaluated by performing conventional -hour europium-tda release assays or by flow cytometry using cfse and aad labeling of target cells. nkg dl were heterogeneously expressed in leukemia primary cells and cell lines. for b cell all primary samples, we found expression of mica/b, mica and ulbp decreased in refractory disease compared to remission (p = . , p = . and p = . , respectively). lentiviral transduction of nkg d- - bb-cd z markedly increased nkg d surface expression in cd ra − memory t cells, which became consistently more cytotoxic than untransduced cells against leukemia cells. additionally, no chromosomal aberrations nor cytotoxic activity against healthy pbmc or mesenchymal stem cells was observed in nkg d car expressing t cells. our results demonstrate nkg d-car redirected cd ramemory t cells target nkg dl expressing leukemia cells in vitro and could be a promising and safe immunotherapeutic approach for acute leukemia patients. peripheral blood stem cell mobilization and collection from elderly patients (≥ years) with multiple myeloma: a single center experience g cengiz seval , sk toprak , s civriz bozdag , m kurt yuksel , p topcuoglu , o arslan , m ozcan , t demirer , g gurman , h akan , m beksac and o ilhan clinic of hematology, yildirim beyazit university yenimahalle education and research hospital and department of hematology, ankara university school of medicine high-dose melphalan followed by autologous hematopoietic cell transplantation (auto hsct) has become the standard procedure for patients with symptomatic multiple myeloma (mm). the ability to mobilize stem cells from healthy donors shows little deterioration with age, the influence of patients' age on auto hsct is uncertain and studies in patients' ≥ years are scarce. severe studies specific to mm have failed to show an independent effect of patient age on cd + mobilization. we retrospectively compared myeloma patients below the age of with patients above years of age, analyzing cd mobilization into peripheral blood and the number of leukapheresis needed to collect at least one single stem cell graft. material and methods: from february through april , data from myeloma patients below the age of were compared to myeloma patients above years of age. all these data were obtained from the ankara university faculty of medicine center for therapeutic apheresis and written informed consent was signed according to our institution regulations. most of the patients received only gcsf at a dose of μg/kg bw twice-daily s.c. until stem cell procurement. patients underwent further pbsc collections until we obtained the target dose cd + cells/μl blood. a maximum of collections were performed in the first mobilization; if the cell dose was not achieved, we submitted patients to a second mobilization. fifty two of patients were above years of age (median age , range: - ) and patients were below the age of (median age , range: - ). baseline characteristics of the older and younger patient cohorts are summarized in table . mobilization regimens for the younger patient population were cyclophosphamide based (n: ), g-csf only (n: ) and +plerixafor (n: ). mobilization in the older population was with cyclophosphamide based (n: ), g-csf only (n: ) and +plerixafor (n = ). the chemotherapy regimens were not statistically different between both age groups. there were no significant statistical differences in time from diagnosis to mobilization, number of prior therapies or disease status between both patient groups. the number of cd + circulating cells before scheduled leukapheresis was mean . cells/μl (median cells/ μl, range: - ; sem ± . ) in all patients (including patients who failed mobilization). our data support the observation that after a standard mobilization regimen with anti-myeloma chemotherapy and once-daily growth factor support, patients above years of age show an impaired cd mobilization into peripheral blood compared to a younger population. this can be overcome by an increased number of leukaphereses. still the number of progenitor cells in the actual graft is inferior compared to the younger population. [p ] disclosure of conflict of interest: none. donor and/or recipient citomegalovirus (cmv) seropositivity has been associated with a poor overall survival (os) in patients who have received an allogeneic hematopoietic stem cell transplantation (allohsct). in comparison with seronegative donors, hsct from seropositive donors has been associated with decreased disease-free survival (dfs) and increased non-relapse-related mortality (nrm). we analyzed the prognostic impact of cmv serology status (donor/ recipient) in patients diagnosed with acute leukemia (al) [p ] s who had received an allohsct in our institution. retrospective unicentric study of patients diagnosed with al between and who received allohsct.the following outcomes were studies: os, dfs, and cumulated incidences of relapse (ri), nrm, acute graft-versus host disease (agvhd) and chronic gvhd (cgvhd). the series included patients ( males, females), median age of years . al type: ( %) all, ( %) aml. type of transplant: ( %) related donor, ( %) unrelated donor and ( %) unrelated umbilical cord blood. the majority, ( %), received myeloablative conditioning. stem cells source: peripheral blood ( %), cord blood ( %) and bone marrow ( %). cmv serology status: positive receptor ( %), negative receptor cases ( %); positive donor ( %), negative donor ( %). serology status combinations (d/r): +/+ ( %), +/ − ( %), − / − ( %), − /+ ( %). patients developed agvhd and ( %) cgvhd. the impact of donor/recipient cmv serology status on os, dfs, ri, nrm and incidence of agvhd and cgvhd for the overall series is reported in table . no statistically significant differences were detected in any of the analyzed variables. in this study, donor/recipient cmv serology showed no influence on the analyzed variables os, dfs, al relapse, nrm, acute and chronic gvhd. however, the sample size limits the validity of the results. disclosure of conflict of interest: none. supported in part with the grants pi / from fondo de investigaciones sanitarias and rd / / from rticc, instituto carlos iii and sgr (gre), generalitat de catalunya, spain. petersburg, russia during the last two decades ahsct has been used as a treatment option for ms with promising outcomes. qol is an important outcome of ms treatment. its assessment gives the patient's perspective on the overall effect of treatment. we aimed to study qol in ms patients before and after ahsct and search the value of the data obtained for decision-making. a total of patients with different types of ms were enrolled in the study: mean age- (range- - ) years old; male/ female- / ; mean edss- . (range: . - . ). all patients were treated by ahsct. reduced-intensity beam-like conditioning was used (bcnu mg/m , etoposide mg/m , ara-c mg/m and melphalan mg/m ). mean follow-up was months (range: - months). qol was assessed using generic questionnaire sf- . for comparisons t-test for independent samples or mann-whitney test was used. qol parameters in ms patients at months after ahsct improved in comparison to base-line: physical functioning- . vs . , role-physical functioning- . . further qol improvement was registered at long-term follow-up: integral qol index exhibited . at long-term follow-up as compared to . at base-line. qol improvement was more dramatic in relapsing-remitting ms than in progressive ms. we found a significant increase of all eight sf- scales in a year posttransplant as compared with base-line in relapsing-remitting ms patients (po . ). in progressive ms patients statistically significant improvement was registered for six out of eight sf- scales (except bodily pain and role-emotional functioning) (p o . ). improved qol parameters were preserved over the entire study period in all the patients who did not have disease progression or relapse. in conclusion, qol monitoring in ms patients after ahsct provides clinicians with the unique information regarding the changes in physical, psychological and social well-being of patients who have been treated with this new treatment modality. it allows to evaluate risks/ benefits of ms patients undergoing ahsct and might influence decision-making. further studies are needed to examine the trajectory of qol changes in this patient population to better define treatment outcomes after ahsct. disclosure of conflict of interest: none. pediatric patients with leukocyte adhesion deficiency type-i (lad-i), a rare autosomal recessive primary immunodeficiency disorder, experience severe and recurrent lifethreatening bacterial infections. allogeneic haematopoietic stem cell transplantation (hsct) offers the possibility of curative therapy although the conditioning regimen used for hsct in lad-i is still a controversial issue. this study provides evaluation of outcome of the lad-i pediatric patients who underwent reduced-intensity conditioning (ric) hsct. twenty four patients ( female) with severe lad-i who received hscts between februay and september at our center were enrolled. the median age at hsct was months (range: months- years). patients received bone marrow (n = ), peripheral blood progenitor cells (n = ) or umbilical cord blood grafts (n = ) from hla-matched related donors (n = ), mismatched related or unrelated donors (n = ), unrelated fully matched donors (n = ) and haploidentical relative donors (n = ). ric regimen was provided with fludarabine, melphalan and anti-thymocyte immunoglobulin. cyclosporine a and prednisolon were used as graft-versus-host disease (gvhd) prophylaxis. engraftment occurred in / , of which one patient experienced graft rejection.the median times to neutrophil and platelet engraftments were days (range: - days) and days (range: - days), respectively. with a median follow-up of months (range: - months), overall survival (os) was . %.the main causes of death were gvhd and infection. acute gvhd occurred in ten patients ( grade i-ii, grade iii-iv) and patients also developed chronic gvhd. there were no significant differences in acute gvhd occurence and also os regarding to the stem cell sources. at this time, patients with full chimerism and patients with mixed chimerism are alive and disease free. conclusion: hsct offers long term benefit in lad- and should be considered as an early therapeutic option if a suitable hla-matched stem cell donation is available. as pretransplant infections in primary immunodeficient patients especially those affected by lad- lead to rise in mortality rate, ric regimen is found to be safe and mixed donor chimersim appears sufficient to prevent significant symptoms. disclosure of conflict of interest: none. tregs based immunotherapy may be beneficial in several immune mediated diseases including graft versus host disease (gvhd). the possibility of cryopreserving tregs might lead to the administration of multiple doses, thus potentially increasing their efficacy in chronic diseases. however, there are few and controversial data on the functionality of tregs after cryopreservation. here, we evaluated the phenotype and the inhibitory capacity of thawed tregs. tregs were purified from leukapheresis of normal donors (n = ) by double immunomagnetic depletion (cd and cd ) followed by cd enrichment using the clinimacs system (miltenyi biotec) under gmp condition. the cells were cryopreserved in saline solution containing % human serum albumin (hsa) and % dmso with a controlled-rate freezing. cell viability was assessed by -aad staining. number/phenotype and function were evaluated on fresh and thawed tregs. cryopreserved autologous t effector (teff) cells were used in mlr assays. before cryopreservation the tregs enriched product mean viability was ± % and the mean percentage of cd +cd +cd +cd low and cd +cd +cd +cd lowfoxp + cells was ± % and ± %, respectively. we then analysed the tregs enriched product after thawing. mean viability of thawed tregs, by -aad staining, was ± %. the viable tregs were almost totally cd +cd + ( ± %). the mean percentage of cd +cd +cd low and cd +cd +cd lowfoxp + thawed cells was ± % and ± % respectively. the contaminant cells present in the treg enriched product were mostly cd +cd +cd + (around %). we further characterized the phenotype of the cd +cd +cd low population. this population was almost totally foxp + ( ± %) and expressed selected markers at various degree (cd l ( ± %), cd s ( ± %), cd ra+ ( ± %), hla-dr+ ( ± %), ccr + ( ± %), cd d ( ± %), cd + ( ± . %), cd +cd + ( ± %). notably, viable thawed tregs were able to induce inhibition of autologous teff cells in a : tregs:teff ratio as freshly isolated tregs: ± % (thawed) vs ± % (fresh) of inhibiton (p . ). in conclusion, here we demonstrated that thawed tregs from healthy donors mantain a stable phenotype. in addition, in our hands tregs show good suppressive ability after thawing despite lower expression of cd l and cd s relapsed and refractory malignant b cell diseases: evidence for therapeutic efficacy via subcutaneous administration of anti-cd × anti-cd antibody lymphomun r buhmann, p ruf, j hess, h lindhofer, u jacob and m dreyling the trifunctional antibody anti-cd × anti-cd lymphomun represents a chimeric immunoglobulin scaffold (mouse igg a/ rat igg b) with promising treatment outcome in patients suffering from malignant b cell diseases. by changing the lymphomun administration route from intravenous (i.v.) to subcutaneous (s.c.) the proinflammatory cytokine-mediated side effects were considerably slighter and generally welltolerated. most importantly, s.c. lymphomun showed outstanding responses in b cell depletion even in the absence of elevated cytokine levels (e.g. il- ) that are required for cytotoxic t cell activation. in summary, the clinical tolerability of s.c. lymphomun may result in a considerable improvement of the subjective well-being and in enhanced mobility due to decreased pain symptomatology. intestinal microbiota disruption is associated with acute gastrointestinal (gi) gvhd and inferior outcome in patients after allogeneic stem cell transplantation (asct). the wide use of systemic broad spectrum antibiotics adds a further risk factor contributing to major microbiota shifts. here, in a retrospective analysis of patients undergoing asct at the regensburg university medical center we assessed the relative expression of paneth cell antimicrobial peptides (amps) in human intestinal biopsies in relation to acute gi gvhd and systemic antibiotic treatment. the relative expression of paneth cell amps was significantly higher in biopsies of the upper gi tract than in the lower gi tract for reg α (p ≤ . ), human defensin (hd) (p ≤ . ) and hd (p ≤ . ). regarding the distribution of paneth cell amps in the gi tract we observed significantly higher expressions of all three paneth cell amps in the duodenum, jejunum and ileum compared to the stomach, colon and rectum (po . , figure ). in the presence of acute gi gvhd, paneth cell amps reacted contrarily in the upper and lower gi tract: we observed a decrease of hd , hd and reg α in the upper gi tract (p ≤ . ), similarly paneth cell count dropped in case of severe gi gvhd stage - (po . ). however in the lower gi tract severe acute gi gvhd was associated with an increase of paneth cell amps (p ≤ . ). initiation of additional systemic antibiotic treatment prior to day after asct correlated with a significantly higher expression of hd (p = . ) and reg α (p = . ) in intestinal biopsies compared to patients without or with initiation of systemic antibiosis after day . however, no significant differences were found in terms of hd expression in intestinal biopsies and start of systemic antibiotic therapy. the expressions of hd , hd and reg α in intestinal biopsies seem to respond to major microbiota disruptions caused by acute gi gvhd or systemic antibiotic treatment. while observations in the upper gi tract seem to reflect paneth cell damage, the relative increase in the lower gi tract may indicate inflammatory induction of amps in colonic epithelial cells in the course of gvhd. [p ] disclosure of conflict of interest: none. patients ( %) were in complete remission at the time of pcy haplo-sct. hematopoietic cell transplantation-comorbidity index was ≥ in patients ( %). thirteen patients ( %) received non-myeloablative conditioning regimen (as baltimore schema, luznik et al. bbmt ) prior to haplosct while remaining patients received busulfan-based regimen. all patients were given pcy and both csa and mmf as gvhd prophylaxis. day+ cumulative incidence of grade to and to acute gvhd was % and %. -year cumulative incidence of chronic gvhd was %. the cumulative incidence of non-relapse mortality and relapse at years were % and %, respectively. with a median follow up of months (range: - ), -year progression-free and overall survivals were % and %, respectively. disease status at the time of haplosct was a major determinant for outcome. indeed, year nrm and os were % and % in patients transplanted with active disease, respectively, while corresponding values in patients transplanted in cr were % (p = . ) and % (p = . ), respectively ( figure a and b) . we can conclude that in selected patients who could be candidate for second transplantation, haplosct is feasible and may represent a curative option. the overall incidence of relapse of % is promising in this situation for which no alternative for cure is available, with relatively good survival in patients transplanted in cr. however, the very high nrm ( %) in refractory patients should make us consider second transplant with caution in this setting. for these patients, specific developments are needed to avoid procedure-related toxicity. [p ] disclosure of conflict of interest: none. secondary solid tumors following hematopoietic cell transplantation for thalassemia major a natale, s santarone, a meloni, a pepe, m di ianni, s angelini, p di bartolomeo dipartimento di ematologia, medicina trasfusionale e biotecnologie-ospedale civile, pescara, italy secondary solid tumors (sst) have been described after hct, in particular for patients affected by hematologic malignancies. there is limited information about the incidence of sst following hct for thalassemia major (tm). the aim of this study was to determine the incidence of sst in patients with tm who received hct in our center between and . patients survived more than years after hct and were enrolled in the study. of them, were males and females. their median age at time of hct was years ( - ). as conditioning regimen, they received busulfan ( mg/kg) and cyclophosphamide ( mg/kg). the gvhd prophylaxis included cyclosporine and methotrexate. all patients received bone marrow cells from an hla identical donor. at time of this report, patients were cured, whereas patients rejected their graft and are now under regular transfusion treatment. overall, the median follow-up after hct was years ( - ). seven patients developed a malignancy . to years (median . years) after hct including carcinomas of the tongue, oral squamous cell carcinoma, colorectal cancer, thyroid carcinoma, carcinoma of the uterine cervix, and parotid carcinoma. the -yr cumulative incidence (ci) of developing sst was + . %. all patients underwent surgical resection of the tumor and in addition of them received chemotherapy and/or radiotherapy. of relevance, the patients with cancer of the oral cavity were affected by severe chronic gvhd with buccal cavity involvement. patients ( with parotid and with tongue carcinoma) died of tumor progression and are living. we compared these results with case control populations. first of all, we investigated the occurrence of solid tumors in the individuals ( males, median age years at time of marrow donation), who served as stem cell donors for hct. one donor developed breast cancer years after marrow donation at age of . the -yr ci of developing solid tumor for donors was . + . % with a statistically significant difference (p = . ) as compared to that of transplanted patients. the second case control population consisted of patients affected by tm treated with transfusions and iron chelation. the matching technique applied was based on the variables age and sex. one control per case (transplanted patient) was randomly selected from the miot (myocardial iron overload in thalassemia) registry and matched by sex and age with the transplanted patient population. patients developed an hepatocellular carcinoma (hcc) at age of and years, respectively. one patient died and one is living. using the event rate measure, we observed an event rate of . at years for the transplant group and . for the nontransplant group (p = . ). this study shows that the magnitude of increased risk of sst is twofold to threefold for patients treated with hct as compared with an age-and sex matched nontransplant tm patients or with stem cell donors. notably, among the transplanted patients we didn't observe any case of hcc, which is one of the most frequent solid tumor in nontransplant tm patients, whereas we observed cases of head/neck cancers. in our series, cgvhd seems to be a strong risk factor in the development of new solid tumors. patients with cgvhd, especially those with involvement of the oral cavity, must receive a very long careful monitoring and surveillance in order to prevent the development of secondary cancers. disclosure of conflict of interest: none. sequential treatment with bortezomib plus thalidomide plus dexamethasone followed by autologous hematopoietic stem cell transplantation (hsct); consolidation and maintenance therapy in patients with multiple myeloma a bachiri , ma bekadja , s talhi , s abderrahmani , h ouldjeriouat and r bouhass department of hematology, hmru oran, oran, algeria; department of hematology and cell therapy, ehu st november, oran, algeria and department of hematology and cell therapy, oran, algeria the management of multiple myeloma (mm) has been significantly improved in recent years in young patients, where ahsct and advent of new molecules was introduced as first line treatment. the sequential treatment (induction followed by autologous hematopoietic stem cell transplantation; consolidation and maintenance therapy) has increased rates response (cr and vgpr) as well as the overall survival. our purpose was to assess the efficacy and adverse effects of sequential treatment with vtd chemotherapy and autologous hsct followed by consolidation and maintenance therapy. in a prospective multicenter study, we evaluated this mm management strategy at oran, in two hematology centers. patients aged under years with de novo mm, were treated with induction included: bortezomib ( . mg/m , d -d -d -d ), thalidomide ( mg/ m d -d ) and dexamethasone ( mg, d -d ; d -d ). a total of to cycles where delivered every days. autologous stem cell was mobilized using g-csf alone ( μg/kg/day for days). leukapheresis to harvest stem cells were performed on day - and - . the conditioning regimen consisted of melphalan mg/m . a consolidation phase was initiated two months later with the same protocol (vtd), followed by a maintenance treatment with thalidomide mg/day given orally for months. this study was done over a -years period (january -december ). fifty patients were included. they include women and men (sex ratio = . ). the median age at diagnosis was years ( - ). according to durie salmon staging, % of patients were in stage iii, while % were in stages iii according to iss staging. the monoclonal component was igg in % of patients. postinduction overall response rate in the eligible patients was %, including % vgpr and % cr/ and % pr rates. the median of cd + rate was . x /kg ( . to ). all patients had engraftment on the median of day (range; to ) and platelet transfusion independence on the median of day (range; to ). there was no graft failure. one patient died following the procedure (trm). posttransplantation on day , cr and at least vgpr remained significantly higher ( %). in the evaluable patients, the estimated os at months was %, the estimated dfs at months was % and the pfs at months was . %. at the / / , ( %) patients are alive and ( %) without disease activity after a median follow-up of months (range; - ). the main hematological toxicities post transpland (grade / ) were thrombocytopenia ( %), neutropenia ( %), and anemia ( %). the most frequently observed nonhematological toxicities (all grades) included peripheral neuropathy ( %). our experience suggests that the sequential protocol used in first line produce a better outcome with fewer adverse events and is an interesting therapeutic option in term of efficacy and tolerance. disclosure of conflict of interest: none. micrornas are small, non-coding single-stranded rnas and regulate approximately % of all genes by repressing translation. they are present in bodily fluids, where they are protected from rnase-mediated degradation by encapsulation into extracellular vesicles (evs) and demonstrate a novel capacity to regulate the cellular differentiation of blood cells and immune function. candidate micrornas mir- , mir-- , mir- * and mir- have previously been associated with acute graft versus host disease (agvhd) in posthematopoietic stem cell transplant (hsct) patient plasma. however, validation in independent cohorts is necessary, and their presence within extracellular vesicles (evs) has not been explored. microrna expression was evaluated in a prognostic cohort (n = ) of day (d ) post-hsct patient serum samples by taqman qrt-pcr. further assessment in an independent cohort of serum samples taken at the time of agvhd diagnosis was also performed. expression was also assessed in serum evs at sequential time points (pre-hsct, d , d and d ) and an independent verification cohort of d serum samples by ev isolation, rna extraction and taqman qrt-pcr analysis. this study replicated elevated serum expression of mir- (po . ), mir- (p = . ), mir- * (p o . ) and mir- (p = . ) in agvhd, in a prognostic cohort of d post-hsct patient samples (n = ). expression was also associated with disease severity. further analysis at agvhd diagnosis in an independent cohort (n = ) confirmed high expression of mir- (p = . ), mir- (p = . ) and mir- * (p = . ) at disease onset. investigation of microrna expression patterns during early hsct at sequential time points (pre-hsct to d ) identified elevated micrornas at d post-hsct in all transplant patients. in a novel investigation of microrna expression in serum evs (n = ), mir- (p = . ), mir- (p = . ) and mir- * (p = . ) levels were lower at d in patients who later developed agvhd, and this was replicated for mir- (p = . ) and mir- (p = . ) (n = ). comparing serum to circulating evs, at d patients remaining agvhd-free had significantly higher expression of mir- (p = . ), mir- (p o . ) and mir- * (p = . ) in the ev fraction. results validate the capacity for circulating serum mir- , mir- and mir- * to act as diagnostic and prognostic biomarkers for agvhd. novel findings of differential expression between whole serum and the ev compartment prior to disease onset suggest a role for ev micrornas in the biology of agvhd, which warrants further investigation. disclosure of conflict of interest: none. prior data indicate similar outcomes after transplants from hla-haplotype-matched relatives, hla-idntical siblings and hla-matched unrelated donors. we used our dataset to answer a clinically important question: who is the best donor for a person with acute leukemia. we analyzed data from persons with acute leukaemia in st complete remission treated in a prospective, multi-centre study. patients were randomly divided into training (n = ) and validation (n = ) sets. consecutive subjects received a transplant from an hla-haplotype-matched relative (n = ) or an hlaidentical sibling (n = ). -year leukaemia-free survivals (lfss) were % ( % confidence interval [ci], , %) and % ( , %; p = . ). the multivariate model identified major risk factors for transplant-related-mortality (trm): older donor/recipient age (donor years/recipient years; hazard ratio [hr] = . ; [ . , . ]; p = . ), female-to-male transplants (hr = . ; [ . , . ; p = . ) and donor-recipient abo major-mismatch transplants (hr = . [ . , . ; p = . ). a risk score was developed based on these three features. trms were % ( , %), % ( , %) and % ( , %) for subjects with scores of - , and (p o . ). year lfs were % ( , %), % ( , %), and % ( , %; p = . ). the risk score was validated in an independent cohort. in recipients years, lfss were % and % (p = . ) after transplants from identical-sibling or children. our data confirm donor source or degree of hla-disparity is not significantly correlated with transplant outcomes. selection of the best donor needs to consider donor-recipient age, sex-matching and abo-incompatibility amongst persons with acute leukemia receiving transplants from family members. [p ] disclosure of conflict of interest: none. synergistic effect of kir ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation for treatment of myeloid malignancies d cardozo, a marangon, r da silva, fj aranha, j visentainer, s bonon, s costa, e miranda, c souza and f guimarães. the lack of one or more hla class i alleles, whose protein products are the ligands for kir receptors, has been exploited as a prognostic factor for the outcome of patients with haematological malignancies treated by haematopoietic stem cell transplantation (hsct). although it has been accepted that kir-hla interactions may influence the outcome of the hlamismatched hsct, there is no consensus regarding the settings of hla-matched transplantation. there are studies that have reported either benefits, or no effects, under the influence of inhibitory kir-hla interactions. additionally, certain activating kirs and/or reactivation of cytomegalovirus (cmv) infection have been reported to affect the outcome of hla-matched transplantation. the goal of this study was to evaluate the influence of kir-hla genotypes on the outcome of patients undergoing treatment for haematological malignancies by non-t-depleted lymphocyte haematopoietic stem cell transplantation (hsct) from hla-matched sibling donors. the prospective study was conducted at the center of hematology, university of campinas, and patients and their donors were followed up from to . kir and hla class i genes were genotyped and patients grouped based on the presence of kir ligands combined with kir genotype of their respective donors. patients with all kir ligands present (n = ) had a significantly higher (p = . ) incidence of acute graft-versus-host-disease (gvhd) than patients with one or more kir ligands missing (n = ). the overall survival following transplantation of patients with myeloid malignancies (n = ) was significantly higher (p = . ) in the group with one or more kir ligands missing (n = ) than in the group with all ligands present (n = ). presence of kir ds was associated with a worsening of hsct outcome while reactivation of cytomegalovirus (cmv) infection improved the outcome of patients with one or more kir ligands missing. our results indicate that kir-hla interactions affect the outcome of the hla-matched transplantation, particularly in patients with myeloid malignancies. disclosure of conflict of interest: none. p = . ), lower disease-free survival (p = . and p = . ) and lower overall survival (p = . and p = . ). one-year cir of the above two groups were . ± . % vs. . ± . % in mrd negative and positive patients, respectively (p = . ). in addition, those who had consistent positive mrd prior to hlamatched sibling hsct showed even worse outcomes compared to patients without pre-mrd. unmanipulated haploidentical hsct might have the stronger graft-versus-leukemia effect compared to hla-matched sibling hsct. it suggested that those who received unmanipulated haploidentical hsct with pre-mrd might not need more intensive relapse intervention after transplantation. disclosure of conflict of interest: none. the retrospective study of allogeneic hematopoietic cell transplantation for patients with mixed-phenotype acute leukemia in toranomon hospital, japan in the real clinical setting, however, there are substantial number of patients who can not achieve cr after chemotherapy. we conducted a retrospective study including such patients to elucidate the outcome of allogeneic hct in toranomon hospital, japan. we studied the patients with mpal diagnosed from july to september . mpal was diagnosed according to who classification in . from june , we examined cytoplasmic myelo-peroxydase (cmpo) routinely for flowcytometric analysis in all the patients, to distinguish mpal from acute lymphoblastic leukemia (all). we included the patients who were diagnosed as mpal in toranomon hospital, regardless of their diagnosis or clinical course in the previous hospitals. a total of mpal patients underwent their first allogeneic hct with related bone marrow or peripheral blood stem cells (r-bm/pb) (n = ), unrelated bone marrow (u-bm) (n = ), and unrelated umbilical cord blood (u-cb)(n = ). the median patient age was years (range: - ). the immunophenotype of leukemia cells included cases of b and myeloid (b/my) ( %) and cases of t and myeloid (t/my) cell lineage( %).eleven patients( %) harbored philadelphia chromosome. the remission induction chemotherapy was performed with all-type regimens in patients, and acute myeloid leukemia (aml)type regimens in of patients, patients( %) were not in cr at the time of transplantation. myeloablative conditioning (mac) regimens were used in pantients( %). the -year overall survival (os) rate was . % ( % confidence interval (ci), . - . %). to identify the factors that influenced os, we performed univariate analysis and compared the following pre-transplantation factors: age at the time of transplantation ( o vs. = years), committed immunophenotype (b/my vs.t/my), karyotype (philadelphia chromosome (ph vs.non-ph), disease status at the time of transplantation (cr vs.non-cr), donor cell source (r-bm/pb vs.u-bm vs.u-cb, cb vs.non-cb), and conditioning regimen (mac vs.reduced intensity conditioning). cr at the time of transplantation was extracted as a significant predictive factor for the better os( -year os; cr vs. non-cr, . % ( % ci, . - . %) vs. . % ( % ci, . - . %), p = . ). the cumulative incidence of relapse rate (rr) at years after transplantation was . % ( % ci, . - . %). to identify the factors that influenced relapse rate, we performed univariate analysis and compared pretransplantation factors same as above. harboring philadelphia chromosome was extracted as a significant predictive factor for lower relapse rate ( -year rr; ph vs.non-ph, . %( % ci, . - . %) vs. . %( % ci, . - . %), p = . ). the older patients(p = . ) and the patients in cr (p = . ) also showed a trend towards lower relapse rate. allogeneic hct provided . % of -year os for mpal patients in cr at the time of transplantation. on the other hand, for patients not in cr, year os was approximately %. the use of tyrosine kinase inhibitors along with chemotherapy before transplantation might prevent relapse after transplantation in mpal patients with ph chromosome. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (allo-hsct) is a standard of treatment for many patients with hematological malignancies. however, the disease relapse and graft failure after first allo-hsct ( st allo-hsct) lead to poor outcomes almost in all cases. second allo-hsct ( nd allo-hsct) is one of primary options that can decrease the mortality in this group of patients. here we report our experience of patients who underwent nd allo-hsct. the aim of the study was to estimate a clinical efficiency and practicability of nd allo-hsct. we included patients ( males/ females) with acute myeloid leukemia (aml, n = ), acute lymphoblastic leukemia (all, n = ) and myeloproliferative disease (mpd, n = ) who underwent nd allo-hsct for relapse ( , %) or graft failure ( , %) from the same (n = ) or another donor (n = ) between november and october . median age was years (range: - years). three ( %) patients had a matched related donor (mrd), nine ( %) patients had a matched unrelated donor (mud) and three ( %) patients had a mismatched unrelated (mmud) at the second transplant. to evaluate time gap affecting outcomes all patients were divided into two groups: who underwent nd allo-hsct in more/less than months after st allo-hsct. in "less than months" group three patients were re-transplanted for relapse and one-for graft failure, in other group there were seven patients who received nd allo-hsct for disease relapse and four-for graft failure. fisher's exact test were performed to exclude probability of imbalance between groups (p . ). median of overall survival (os) and disease-free survival (dfs) after nd allo-hsct was . months and . months respectively. (see figure a , c) two patients ( . %) developed graft failure and three relapsed ( %). acute graft-versus host disease (agvhd) incidence was extremely low as . % (n = ) even despite use of mud/mmud in % of cases. mortality rate were . % in a group of nd allo-hsct. it should be noted that only ( %) patients died because of disease progression. five patients ( . %) died in complete remission due to severe infections or previous toxicity (e.g. heart failure). the effect of donor change on dfs was not significant (p = , ). our statistical analysis reveal significantly differences in os in patient with long-term interval ( months) between st and nd allo-hsct. median of os in patients who underwent nd allo-hsct in more/less than months after st allo-hsct was , vs , months respectively. (see figure b , d) for hazard ratio (hr) estimation mantel-haneszel approach were used hr for group who were transplanted in less than months from st allo-hsct was . , ( % ci, . s to . , p = . ). as for dfs difference was not significant (p = . ). according to our analysis, performing nd allo-hsct in a period less than months after st allo-hsct seemed not very reasonable due to extremely high mortality even in young patients (hr- . , p = . ). as for "more than months" group it can be considered even despite hla-disparity between donor-recipient pair due to extremely low agvhd rate ( . %). donor change was not associated with better outcome (p = . ) disclosure of conflict of interest: none. hemopoietic stem cell transplantation (hsct) is an effective treatment for many hematologic disorders, and globally over procedures/year are performed in more than countries. however, not all the countries have enough resources and expertise to establish an hsct program, and patients are often forced to emigrate for transplantation, with heavy social and economic consequences. in the year the iuc (an italian ngo) identified the hiwa cancer hospital (hch) in sulaymaniya (iraqi kurdistan) as a possible site for the establishment of a new hsct transplant center. a hsct expert from italy (mi) following a visit to the hch, reported a positive conclusion on the feasibility of an hsct project. this was mainly due to the fact that many of the required technologies were already available at hch, including a -bed positivepressure, hepa-filtered-air clinical unit, last-generation cell separators and a well equipped hla laboratory. following this preliminary survey, a capacity building project was rapidly made and submitted to the italian agency for development cooperation, that approved and funded it in march with the specific aim to cure thalassemia patients either of kurdistan and of the refugees population from syria and other parts of iraq. in april , the joint italian and kurdish team started the project. a first autologous transplant was done in june followed by more autografts (overall, myeloma and lymphoma patients). in october, following appropriate downstaging, a first low-risk thalassemia patient was allografted from her hla-id sibling, followed by more patients. all the patients engrafted promptly, with one death occurring on day + with acute cardiac failure and a major toxicity recorded in a single patient (nhl, severe enterocolitis with perforation) that was successfully treated. the full process for the start-up included the following activities developed during -month time: ( ) s of transplants, the hch group also submitted to ebmt an application for full membership, that was promptly approved. in all this project, the italian counterpart provided over highly-experienced volunteer specialists (physicians, nurses, technicians and one physicist), each with a specific mission plan. despite the many difficulties and obstacles encountered, the clinical results obtained so far appear encouraging, though there is still need to furtherly support the hch in order to make it totally independent. following this intervention, the hch is the only one center performing both auto and allo hsct not only in the iraqi kurdistan region, but also in all the iraqi nation. we conclude that international cooperation may be fruitful also in the field of high-technology medicine, and may contribute to improve the capabilities of centers even in critical geographic areas, representing a valuable instrument also to implement nation-to-nation scientific exchanges. disclosure of conflict of interest: none. the use of plerixafor with g-csf in conditioning regimen for hematopoietic stem cell transplantations with tcr alpha/beta and cd depletion of graft in wiscott-aldrich syndrome patients: a single-center experience b dmitry , l alexandra , s larisa , g elena , s irina , t pavel , k rimma , n galina , m michael and m alexei grade acute gvhd (agvhd) was % ( pts). no pt experienced a grade agvhd. three patients presented a limited form of chronic gvhd ( %). incidence of oral mucositis and gastrointestinal/liver toxicity has been extremely low in this population of patients, even in those with active disease and heavily treated at the time of transplant. eight out of fifteen pts ( %) are alive with a median follow-up of months (range: - m). seven ( %) are in cytogenetic/molecular remission. six out the eight patients who were transplanted in cr or cr are alive ( %), while two out the seven patients who were transplanted in advanced phase are alive ( %). in this preliminary clinical experience, we find that unmanipulated haploidentical transplants with post-transplant cyclophosphamide are a valid alternative and have outcome comparable to unrelated and match sibling transplants, in pts with hematologic malignancies. advanced disease is the only adverse factor for diseasefree survival. we therefore consider this therapeutic option when a match sibling or a / ag mud donor is not immediately available. disclosure of conflict of interest: none. autism spectrum disorder (asd) is a group of neurodevelopmental disorders characterized by impaired social communication and interactions with restricted and repetitive behaviors. although asd is suspected to have either heritable or sporadic genetic basis, its fundamental etiology and pathogenesis are poorly understood. recently researchers have suggested that stem cells have therapeutic potential for asd. wharton's jelly-derived msc (wj-msc) from third-party donors (tpd) have high proliferation and differentiation potential. this cell population has also non-immunogenic and immunomodulatory properties, thus seem to be a promising treatment stem cell source. the polish stem cell bank (pbkm) has provided wj-msc for clinical application in a medical therapeutic experiment for children with asd. twenty-three patients (pts) with asd aged from to . / (median age: years and months), after bioethical committee approval, received intravenous injections of wj-msc, obtained from tpd. the cells were previously collected from healthy newborns, then processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapour. wj-msc immunophenotype was confirmed using flow cytometry assay. the pts received from to injections in intervals from to weeks. the average cell dose per infusion was . × ^ /kg of body weight (bw). each pt was examined by the same neurologist at the day of infusion. comorbidities present in some patients: unspecified speech disturbances, flaccid paralysis, flaccid tetraplegia, unspecified encephalopathy, epilepsy, sensorineural hearing loss. one patient was diagnosed with comorbidities: conductive hearing loss and intellectual disability. almost % of pts, after their treatment with wj-msc, revealed positive changes in neurological examination. an improvement in speech was observed in pts and improvement of cognitive functions ensued in pts. what is more, % of children showed progress in self-reliance, social interactions and improved their ability to concentrate. there was a reduction of aggressive behavior in pts and pts have experienced better quality of sleep. there was only one adverse event after wj-msc infusions -psychomotor agitation occurred in hours after the administration. five follow-ups have not yet been completed. the administration of thirdparty donor wj-msc seems to be safe and efficient procedure with promising preliminary results in patients with asd. hematopoietic stem cell transplantation between red cell incompatible donor-recipient pairs red blood cell depletion from bone marrow and peripheral blood buffy coat: a comparison of two new and three established technologies human bone marrow processing using a new continuous-flow cell separation device disclosure of conflict of interest: none. references . zama d, et al. gut microbiota and hematopoietic stem cell transplantation: where do we stand? bmt the kyoto encyclopedia of genes and genomes-kegg metabolites produced by commensal bacteria promote peripheral regulatory t-cell generation disclosure of conflict of interest: none antifungal prophylaxis in hematopoietic stem cell transplant recipients: the unfinished tale of imperfect success guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective differences in aspergillus-specific immune recovery between t-cell-replete and t-cell-depleted hematopoietic transplants toxoplasmosis following allogeneic hematopoietic stem cell transplantation diagnosis of toxoplasmosis after allogeneic stem cell transplantation: results of dna detection and serological techniques implementation of molecular surveillance after a cluster of fatal toxoplasmosis at neighboring transplant centers management of high blood pressure genes for blood pressure a prospective studyon the predictive value of plasma bk virus-dna load for hemorrhagic cystitis in pediatric patients after stem cell translantation cidofovir for bk virusassociated hemorrhagic cystitis:a retrospective study hemorrhagic cystitis after bone marrow transplantation bcsh/bsbmt guideline: diagnosis and management of veno-occlusive disease (sinusoidal obstruction syndrome) following haematopoietic stem cell transplantation drug safety evaluation of defibrotide defibrotide for prophylaxis of hepatic veno-occlusive disease in paediatric haemopoietic stem-cell transplantation: an open-label, phase , randomised controlled trial safety and effects of prophylactic defibrotide for sinusoidal obstruction syndrome in hematopoietic stem cell transplantation disclosure of conflict of interest: none university children's hospital basel, division of paediatric oncology/haematology late complications subcommittee of translated related complications and quality of life wp; clinic of paediatric haemato-oncology, department of women's and children's health, university of padova, italy; department of surgery, division of transplantation division of blood and marrow transplantation, the children's hospital at westmead ovarian function after bone marrow transplantation during childhood pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning haploidentical hematopoietic transplantation:current status and future perspectives t-cell replete haploidentical donor transplantation using post-transplant cy: an emerging standard-of-care option for patients who lack an hla-identical sibling donor hematopoietic stem cell transplantation in thalassemia major and sickle cell disease: indications and management recommendations from an international expert panel allogeneic stem cell transplantation for thalassemia major killer-cell immunoglobulin-like receptors reactivity and outcome of stem cell transplant kir b haplotype donors confer a reduced risk for relapse after haploidentical transplantation in children with all kir/hla interactions negatively affect rituximab-but not ga (obinutuzumab)-induced antibody-dependent cellular cytotoxicity reduction of minimal residual disease in pediatric b-lineage acute lymphoblastic leukemia by an fcoptimized cd antibody diagnoses: hodgkin's lymphoma(hl)- pts (refractory ; relapsed ); non-hodgkin's lymphoma(nhl disease status before asct: st (after refractority prior radiotherapy to the mediastinum - / ( . %); heavily pretreated patients with advanced disease (x lines previous treatment) / ( . %). grafts: pbsc - / pts with median of cd +cells- ccnu dose: / pts mg/m ; pt mg/m . engraftment: anc> cells/mkl: median=d+ ( ÷ ), / pts. plt> cells/ mkl: median=d+ ( ÷ ), / pts. full engrafted / pts / pt required a short-term mechanical ventilation ( of them died because of lung infection ad d+ and d+ ) aeruginosa associated sepsis on a background of graft failure); pts ( . %)-d+ and d+ (pulmonary toxicities +infection; both had prior mediastinal radiotherapy). relapse/ progression after asct- / pts ( . %), of them died. pt achieved secondary mds (diagnosed . mo after asct). for this group of pts with relapsed/refractory lymphomas (n= ) -year os= for nhl(n= ) efs= . (se ± . ) lomustine-containing conditioning regimen cem (lomustine, etoposide, melphalan) is effective and feasible in autologous stem cell transplant efficacy and toxicity of a ccnu-containing high-dose chemotherapy regimen followed by autologous hematopoietic cell transplantation in relapsed or refractory hodgkin's disease champlin re reduced-toxicity conditioning therapy with allogeneic stem cell transplantation for acute leukemia idarubicin-intensified bucy conditioning regimen improved survival in high-risk acute myeloid, but not lymphocytic leukemia patients undergoing allogeneic hematopoietic stem cell transplantation: a retrospective comparative study comparison of outcomes of idarubicin intensified tbi-cy and traditional tbi-cy conditioning regimen for high-risk acute lymphoblastic leukemia undergoing allogeneic hematopoietic stem cell transplantation: a single center experience inhibition of cd (il- r alpha) expression and t-cell proliferation by polyclonal anti-thymocyte globulins csf-primed bone marrow transplantation for patients with high-risk hematologic malignancies in an exploratory analysis, os after hct appeared to be longer in the cpx- arm in both age groups. these results suggest that cpx- may provide an effective bridge to successful transplant for a high-risk subgroup of aml patients. support: celator pharmaceuticals, inc., a subsidiary of jazz pharmaceuticals plc consulting ambit biosciences, amphivena therapeutics, ariad, astellas pharma sunesis, tolero; institutional research funding abbvie chiarella and louie: employment celator/jazz; stock jazz pharmaceuticals plc. hoering disclosure of conflict of interest: none. inkt-/nk-/cik-cell (subsets) are important for immunesurveillance. antibody b targets the vα -jα -invariant-t-cell-receptor (tcr) in the cdr -region, which is semiinvariantly rearranged in inkt-cells. we characterized: i.) inkt-/nk-/cik-subsets in pb-samples from healthy donors (n = subsets under stimulation with dendritic-cells of leukemic origin (dc leu ), generated from aml-blasts in mononuclear cells(mnc) and whole-blood (wb, containing soluble/cellular components of pts' pb) with 'cocktails' (dc-generating-methods/kits). . ) compared to healthy mnc (significantly) lower proportions of inkt-cells comparable correlations were seen in adultall-and cll-pts. . ) we quantified inkt-/nk-/cik-subsets before/after mixed-lymphocytecultures (mlc) of t-cell-enriched immune-reactive cells stimulated with mnc/wb (with or without pretreatment 'cocktails' inducing blasts' conversion to dc leu ) from aml-pts. our findings show, that )inkt-/nk-/cik-cells increase after mlc independent of the stimulator-cells-suspension (under the influence of il- ); ) pretreatment of mnc/wb-blasts with 'cocktails' increases inkt-counts and induces a shift in the composition of inkt-/nk-/cik-subsets after mlc, that might correlate with an improved antileukemic potential; ) individual samples showed varying, however higher inkt-, cik-cell-counts after pretreatment with different (especially prostaglandin-containing) 'cocktails'; ) dc-/inkt-/nk-/cikcells-values after mlc were comparable in physiological hypoxia vs normoxia; ) in cases with antileukemic blast-lytic activity after mlc t-/inkt-/nk-/cik-cells were significantly increased-pointing to an involvement of these cells in antileukemic reactions. in summary: ( ) healthy mnc present with significantly higher inkt-/nk-/cik-cells compared to aml/all/cll-leukemic mnc. ( ) subtypes of inkt-cells differ in healthy vs leukemic samples, resembling a shift in the composition of inkt-cells. ( ) amounts of inkt-/ nk-/cik-cells in aml/all/cll-mnc-samples correlate with prognosis. ( ) 'cocktail'-treated aml-blasts (resulting in dc leu ) lead to a shift in t-,inkt-/nk-/cik-cell-counts/compositions, what correlates with improved antileukemic activity against aml-blastspointing to a cross-talk of these cells. proportions of inkt-/ nk-/cik-cells management of philadelphia chromosome-positive acute lymphoblastic leukemia (ph+ all) outcome of allogeneic stem cell transplantation for aml and myelodysplastic syndrome in elderly patients (⩾ years) comorbidity-age index: a clinical measure of biologic age before allogeneic hematopoietic cell transplantation high rate of hematological responses to sorafenib in flt -itd acute myeloid leukemia relapsed after allogeneic hematopoietic stem cell transplantation phase i trial of maintenance sorafenib after allogeneic hematopoietic stem cell transplantation for fms-like tyrosine kinase internal tandem duplication acute myeloid leukemia haematopoietic cell transplantation with and without sorafenib maintenance for patients with flt -itd acute myeloid leukaemia in first complete remission quantitative monitoring of minimal residual disease (mrd) after sct was performed by four-colour flow cytometry and/or real time pcr. the median time of neutrophil engraftment (above . × e /l) was days, % of pts ( / ) engrafted, one patient died in aplazia. non-relapse mortality (nrm) after year and years was % ( / ) and % ( / ). causes of death were refractory gvhd (n = ), infection (n = ) and multiorgan failure (n = ). incidence of acute gvhd was evaluated in pts: % ( / ) of pts had gvhd (grade i+ii in pts, grade iii in pts). incidence of chronic gvhd was evaluated in pts, % ( / ) of pts had gvhd with median follow-up from sct months (range: - ), % of all pts ( / ) were alive ( in remission of cll with mrd negativity, with relapse), pts died ( from nrm, from cll relapse/progression), relapses ( %; / ) occurred. sequential use of chemotherapy and ric regimen with allogeneic sct is safety and effective treatment of high-risk cll with reponse rate % and low nrm. progression-free survival and overall survival at years from sct were % and % department of hematology, hemostasis, oncology and stem cell transplantation hannover deutsche klinik für diagnostik helios klinik wiesbaden, germany; imperial college london at hammersmith hospital du cane road centre for haematology london disclosure of conflict of interest: none. references . sibon d, brice p. optimal treatment for relapsing patients with at our institution, pr-hl is defined as partial response (pr), no response (nr), stable disease (sd), progressive disease (pd), relapsing within months of finishing the planned treatment. progression free (pfs) and overall survival (os) from the day of auto-sct was estimated by kaplan-meier (km) method. from to , patients with aethera trial criteria were identified. male ( %), female ( %), median age at diagnosis: yrs ( - ), at auto-sct: . yrs ( . - )( % o yrs). initial chemo: abvd in ( %). ( %) had radiation therapy (xrt) after initial chemo. response to initial chemo + xrt was refractory disease: ( %), relapse between - months: ( %) and relapse after months: ( %) aethera had % stable disease before sct vs we have only %. aethera months pfs ( % control arm, % brentuximab arm, investigator assessment) and our . % is not much different. despite having similar selection criteria, our median pfs is higher than both aethera trial placebo and experimental arm. clinically, rate of progression in both studies are very high and comparable at months. given the very high cost of this drug and while waiting for survival fifty-nine ( %) and ( %) patients had relapsed and primary refractory chemosensitive dlbcl, respectively. secondary ipi was - in ( %) patients, in ( %) patients and - in ( %) patients. fifty-one ( %) and ( % patients had gcb and abc tumors, respectively. abc patients received more prior lines of chemotherapy than gcb patients ( % vs % received lines of chemotherapy, p = . ). the rest of characteristics were equally distributed between both groups (table ) disclosure of conflict of interest: none disclosure of conflict of interest: none. p upfront autologous stem cell transplantation in patients with diffuse large b cell lymphoma: focused on risk factors for survival and conditioning regimens ds kim association between complete response and outcomes in transplant-eligible myeloma patients in the era of novel agents e jantunen and v varmavuo department of medicine disclosure of conflict of interest: none. and hematology department lenalidomide after stem-cell transplantation for multiple myeloma bortezomib induction and maintenance treatment in patients with newly diagnosed multiple myeloma: results of the randomized phase iii hovon- /gmmg-hd trial disclosure of conflict of interest: none we performed a retrospective study to investigate survival outcomes and toxicities of l maintenance therapy compared with b maintenance in mm patients post-ahct. this study included patients who received ahct for mm between and after induction with l-or b-based therapy. all patients received ahct within months of mm diagnosis and received melphalan mg/m conditioning. patients who received tandem transplantations (autologous or allogeneic) were excluded. only patients initiating maintenance therapy within months post-ahct were included. maintenance therapy was defined as monotherapy with either l or b. the primary outcome was pfs. secondary outcomes were overall survival (os) and treatment-related toxicities. patients received l maintenance and b maintenance post-ahct. at baseline there were no differences in iss stage, ds stage or cytogenetic risk between maintenance cohorts. at time of analysis, % (n = ) receiving l maintenance and % (n = ) on b maintenance experienced disease progression. median time to progression ( . vs . yrs, p = . ) was not significantly different between cohorts. by multivariable analysis, choice of maintenance (l vs b) was not significant for pfs or os. variables significant for improved pfs were iss stage i disease response improved while on maintenance in % (n = ) with l and % (n = ) with b. median os was not statistically different between maintenance cohorts ( . vs . yrs, p = . ). iss stage i/ii vs iii while cytopenias were more common in the l cohort ( % vs %, p o . ). the median follow-up time for survivors was months. these findings suggest that both lenalidomide and bortezomib are equivocal maintenance therapy options for post-transplantation mm patients. choice of maintenance therapy post-ahct for mm did not demonstrate a difference in survival outcomes. based on these data, maintenance choice should be guided by patient specific anticipated tolerance rather than drug type alone. iss stage and post-ahct disease response continue to be significant predictors for outcomes. toxicities recorded on maintenance were as anticipated. length of maintenance therapy may be a significant predictor and warrants further analysis. the analysis was underpowered to disclosure of conflict of interest: none. p real-world multiple myeloma management practice patterns and outcomes in six central and eastern european countries d coriu , d dytfeld , d niepel , i spicka second autologous stem cell transplantation as salvage therapy for multiple myeloma: impact on progression free survival and overall survival second autologous stem cell transplantation: an effective therapy for relapsed multiple myeloma second auto asct for treatment of relapsed multiple myeloma the role of second autografts in the management of myeloma at first relapse moving beyond autologous transplantation in multiple myeloma ebmt data office bortezomib-based versus nonbortezomib-based induction treatment before autologous stem-cell transplantation in patients with previously untreated multiple myeloma: a meta-analysis of phase iii randomized, controlled trials first-line therapy with thalidomide and dexamethasone in preparation for autologous stem cell transplantation for multiple myeloma mechanism of action of bortezomib and the new proteasome inhibitors on myeloma cells and the bone microenvironment: impact on myeloma-induced alterations of bone remodeling boys; girls) with following mds types: refractory cytopenia of childhood- ( %), refractory anemia with excess blasts - pts ( %), refractory anemia with excess blasts in transformation- pts ( %), juvenile myelomonocytic leukemia in pts ( %). the median of age was years ( - years) mac consisted busulfan (bu) mg/kg + cyclophosphamide mg/kg. ric included fludarabine (flu) mg/m + melphalan (mel) mg/m , flu - mg/m + bu mg/ kg. the bone marrow (bm) was used in pts ( %), peripheral blood stem cells (pbsc) in pts ( %), combination of bm and pbsc in pts ( %). -years overall survival (os) was % os was in pbsc group - %; bm group- %, combination of bm and pbsc- % there were two cases of mds, eb- , although erythroid aberrancy can not be found, fc did disclose significant aberrancy on myelomonocytic lineages. on the other hand, all the normal control bm samples revealed no any erythoid phenotypic abnormality. our study suggests this simplified cocktail of -tube, -color, fc is very sensitive and useful in the assessment of erythroid phenotypic abnormalities in mds we analyzed consecutive patients ( % were female, median age of (range: - ) allografted for mds (median ebmt risk score of , median disease risk index of intermediate risk) over a -year period ( - ) with mac conditioning for % and ric for % pfs ± %, grfs ± %, ri ± % and trm similarly, there was not difference between tdep and non tdep patients for -years pfs ( ± % and ± %, p value . ), -years gfrs ( ± vs ± , p value . ) (graph), -years ri ( ± % and ± %, p value . ) and -years trm ( ± % and ± %, p value . ). finally, tdep had no significant impact on -years grade - agvhd when compared to the non tdep ( ± % and ± %, p value . ). it had not either on -years cgvhd ( ± % and ± %, p value . ). our study shows that tdep is feasible on patients undergoing hsct for mds disclosure of conflict of interest: none. p mutational pathway and dynamics may not be prognostic in patients with myelodysplastic syndrome receiving hypomethylating agent pre-treatment for allogeneic stem cell transplantation republic of korea; department of computer science; the donnelly center for cellular and biomolecular research amebiazis after bone marrow transplantation use of a five-agent gvhd prevention regimen in recipients of unrelated donor marrow impact of age on outcomes after bone marrow transplantation for acquired aplastic anemia using hla-matched sibling donors treatment of acquired severe aplastic anemia: bone marrow transplantation compared with immunosuppressive therapy-the european group for blood and marrow transplantation experience disclosure of conflict of interest: none. leukemia, myelodisplastic syndrome, juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia s bondarenko hla-mismatched unrelated (n = , %), and haploidentical (n = , %) donors. response was achieved in % (n = ) of pts after - (median ) courses of hma therapy: complete remission (cr) in ( %), partial remission (pr) in ( %) of pts. stabilization (s) was documented in ( %) pts, in ( %) pts there was disease progression (p) after beginning of hma therapy mismanaging the gift of life: noncompliance in the context of adult stem cell transplantation l'adhésion thérapeutique et at. des lieux en allogreffe de cellules souches hématopoïétiques (csh) dans des services de pédiatrie et d'adulte. rapport de la sfgm-tc predictive validity of a medication adherence measure in an outpatient setting data is limited to small case series, transplant registries and a single prospective multicenter observational study. here we report our institutional experience with auto-hct in patients with hrl. twenty patients with hrl [non-hodgkin = ( %), hodgkin = ( %)] and treatable hiv infection underwent hdt consisting of carmustine, etoposide, cytarabine and melphalan (beam) followed by peripheral blood auto-hct from / to / . in cases rituximab was administered as part of the preparative regimen. patient-, disease-, and transplant-related characteristics are summarized in table . median age was years (range: - ). the median follow-up for surviving patients was months (range: - ) abbreviation: n: number of patients; m: male gender; auto-hct: autologous hematopoietic cell transplant; nos: not otherwise specified; dlbc: diffuse large b-cell lymphoma ara-c), melphalan; cr : first complete remission; cr :second complete remission disclosure of conflict of interest: none. p incidence of secondary primary malignancies (spm) in patients with multiple myeloma m curly , g laurent and k nicolaus city of hope igm ( . %), lines of induction regimens prior to hsct one in pts ( %), two in pts ( . %), in pts ( . %), and missing in pts ( %). induction regimens included imids and proteasome inhibitor (pi)s with alkylating agents in pts ( . %), imids and pis with no alkylating agents in ( . %), and alkylating agents with no imids or pis in ( . %) and missing data in ( %). radiotherapy was used pre hsct in pts ( . %), no radiation in pts ( %) and missing data in ( . %). plerixafor (p) was administered mostly for poor hsc mobilization as defined by the centers number of hsc collected o × in pts ( . %), - in pts ( . %), × in pts ( %), and data missing in ( %). the number of cd + hsc infused o × in pts ( %), - × in pts ( % , × in pts ( %), and missing in ( %). a total of pts developed spm with cumulative incidence of . % ( %ci . , . ) at mo. data are missing in pts ( %) use of radiotherapy, type of induction, hsc cell dose did not influence the cumulative conflict of interest: f. sahebi, none declared, s. iacobelli, none declared, l. koster none declared l. gardaret none declared, n. kroger received research fund from sanofi, curly morris, none declared p interaction between center effect and strategy for gvhd prophylaxis on outcome of t-cell depleted and t-cell replete haploidentical transplant inserm u ecstra team expanding transplant options to patients over years-improved outcome after reduced intensity conditioning mismatched-unrelated donor transplantation for patients with acute myeloid leukemia: a report from the acute leukemia working party of the ebmt nkg d ligands in tumor immunity comprehensive analysis of nkg d ligand expression and release in leukemia: implications for nkg d-mediated nk cell responses nkg d cars as therapy for cancer russian federation high incidence of mixed chimerism with impaired graft function remains a significant issue in patients with wiskott-aldrich syndrome (was) after hsct. simultaneous use of plerixafor with g-scf is efficient in inducing stem cell release and opening of bone marrow niches. the use of plerixafor/g-csf in conditioning demonstrates better levels of donor chimerism in patients with acute myeloid leukemia. we report our experience of plerixafor/g-csf usage in patients with was as an addition to myeloablateive conditioning to improve stem cell engraftment p = , . events were considered: death in patients, graft rejection in patients, mixed myeloid chimerism (less than % donor) in patients. median time of event was , months after hsct ( . - . ) all patients are alive, median fu is months, range: . - . . patients had acute gvhd: -grade (gut), -grade (skin), in both cases resolved after a short course of steroids. all patients had more than % donor chimerism monthly till the time of last fu. the comparison of peripheral blood chimerism (% of donor cells) in was patients transplanted with and without plerixafor/g-csf in conditioning is shown (figure ). the additional use of plerixafor with g-csf references . moratto et al disclosure of conflict of interest: none. is undesirable. fifteen pts ( males, females, median age , range: - years) with high risk hematologic malignancies ( acute myeloid leukemia n. , %; acute lymphoblastic leukemia n , % pretransplant conditioning regimen consisted of thiotepa mg/kg in two days, busulfan . mg/kg in three days, and fludarabine. source of stem cells was g-csf stimulated bone marrow in all. dose of marrow nucleated cells and cd + were . (range: . - . ) × /kg and . (range: . - . ) × /kg respectively. post-transplant cyclophosphamide at mg/kg/ day was given on days and after transplantation, together with cyclosporine (starting at day − until day posttransplant) and mycofenolate (from day + to day + ) modeling autism spectrum disorders with human neurons autism spectrum disorders neurobiology and genetics of autism: a developmental perspective. the development of autism: perspectives from theory and research wharton's jelly-derived mesenchymal stem cells treatment in children with cerebral palsy: our second preliminary results of the clinical application in poland a mucha , k kosterna , m chroscinska-krawczyk , m kotarska , k mitosek-szewczyk , m murzyn the polish stem cell bank cases application potential of bone marrow mesenchymal stem cell (bmscs) based tissueengineering for spinal cord defect repair in rat fetuses with spina bifida aperta sensory neuron differentiation potential of in utero mesenchymal stem cell transplantation in rat fetuses with spina bifida aperta: sensory neuron differentiation of in utero mscs analysis of post allo-hct relapse in acute leukaemia patients, a comparative on second allo-hct and donor lymphocyte infusions g orti , j sanz , i garcia-cadenas , i sanchez-ortega , mj jimenez , p barba , c ferra , r parody , j sierra , ma sanz , s querol and d valcarcel hospital universitari vall d´hebron; hospital universitario la fe; hospital de sant pau i la santa creu; hospital duran i reynals ico, hospital germans trias i pujol ico; hospital germans trias i pujol and banc de sang i teixits acute leukaemia relapse after allogeneic hematopoietic cell transplantation (allo-hct) associates poor prognosis. in this scenario, lowering the tumour burden prior to a second allo-hct ( nd allo-hct) or donor lymphocyte infusions (dli) is essential to improve survival. thus, patients that respond to chemotherapy and subsequently receive a dli or nd allo-hct appear to associate better outcomes compared to patients receiving only chemotherapy, but data regarding this particular group of patients is lacking. we retrospectively analysed a cohort of post allo-hct relapsed acute leukaemia patients, who, after tumour reduction, were treated with either a nd allo-hct or dli. data was collected from centers, patients were consecutively included from to . patients were treated to reduce the tumour burden and received the nd allo-hct or dli on morphological remission or postchemotherapy aplasia. patients ( %) were diagnosed with aml and ( %) with all. patients ( %) underwent nd allo-hct and ( %) received dli. median patient age was ( - ) years. the median follow-up was ( - ) days. since data regarding time from first allo-hct to relapse was unavailable, we calculated the time from allo-hct to nd allo-hct or dli (time to nd allo-hct or dli). median time to nd allo-hct/dli was ( - ) days, and was days and days for nd allo-hct and dli respectively (p = . ). regarding the dli group, the median dli dose was . x / cd + ( . - x ) cells and the mean number of infused dli was . /patient. one-year os was % (se ± %). in os univariate analysis, longer time to nd allo-hct/dli associated better survival rates (p = . ). the -year dfs was % (se ± %). a longer time to nd allo-hct/dli (p = . ) and nd allo-hct compared to dli (p = . ) (figure ) associated better dfs. the -year nrm was % (se ± %). univariate analysis identified pb as stem cell source as linked to better nrm (p = . ). the -year relapse incidence (ri) was % (se ± %). ri univariate analysis related longer time to nd allo-hct/dli (p = . ) to lower ri. on os multivariate analysis, longer time to nd allo-hct/dli was associated to better survival (p = . ). this association was also observed on dfs multivariate analysis (p = . ). table summarizes nd allo-hct and dli univariate analysis. grade ii-iv acute gvhd was diagnosed in ( %) and ( %) patients post nd allo-hct and dli, respectively. chronic gvhd was diagnosed in ( extensive) and patients after a nd allo-hct and dli, respectively. in this study, longer time to nd allo-hct/dli associated better dfs. nd allo-hct (compared to dli) associated better dfs on univariate analysis, but this association was not observed on multivariate analysis. of note, the nd allo-hct group included more patients with longer time to nd allo-hct/dli. this might be explained by nd allo-hct patients relapsing later or by the fact that the preparation of a nd allo-hct might require longer time than dli. results of this analysis warrant further study with larger number of patients.advancing age is associated with worse prognosis in acute myeloid leukemia (aml). intensive induction chemotherapy in patients aged ⩾ years results in lower aml remission rates with increased induction mortality vs younger patients. cpx- is a liposomal formulation of cytarabine and daunorubicin encapsulated at a : molar ratio. a phase iii, randomized, open-label study of cpx- vs + (cytarabine and daunorubicin) in newly diagnosed older patients with high-risk secondary aml showed superior survival in the cpx- arm (hazard ratio . ; p = . ). in that trial, eligible patients went on to allogeneic hematopoietic cell transplantation (hct). an exploratory analysis of those patients by age strata is reported here. patients aged to years with newly p number, composition and/or antileukemic activity of (dc-stimulated) invariant nkt-, nk-and cik-cells is predictive for outcome of patients with aml, all and cll cl boeck # , dc amberger # , f doraneh-gard , w sutanto , t guenther , j schmohl , f schuster , h salih , f babor , a borkhardt myelofibrosis (mf) is a hematolgic malignancy which is characterised by extramedullary hematopoiesis due to bone marrow fibrosis resulting in spleno-and/or hepatomegaly. allogeneic stem cell transplantation (allo-hsct) is the only curative treatment for mf but is associated with therapy related morbidity and mortality. retrospective studies suggested an increase of liver toxcicity in mf patients in comparison to other diseases following allo-hsct. the aim of this prospective study was to evaluate the impact of liver stiffness measured by transient elastography (fibroscan) on liver toxicity after allo-hsct. between and we included patients (male %, female %) who underwent allo-hsct due to primary mf( %), postpv/et-mf ( %) or mf in transformation ( %). the median age of the patients was y@@@ears (range: - ). conditioning regimen was mainly busulfan based reduced intensity. all patients received atg. gvhd prophylaxis was csa/mmf in all patients. stem cell source was peripheral blood in % and bone marrow in % of the patients. donor sources were as follows: mrd ( %), mud ( %) and haploidentical relative ( %). fibroscan was performed prior to conditioning. elevated liver enzymes, bilirubin above the normal value or the onset of veno-occlusive disesae (vod) from the time of conditioning start and within the first post-transplant days were considered as indicators for liver toxicity. the median stiffness of the liver measured by fibroscan on the day before conditioning treatment start was . kpa (range: . - . ). six patients ( %) had prior liver diseases such as cirrhosis (n = ), viral hepatitis (n = ), steatosis (n = ), or vod (n = ). the median onset of liver toxicity was day (range: − until + ). the median bilirubin level of all patients was mg/dl (range: - ). the median ap level was u/l (range: - ), the median ggt level was u/l (range: - ), the median alt level was u/l (range: and the median ast level was u/l (range: - ). the pearson-test revealed a positive correlation between liver stiffness and the elevation of the ap (r = . , p = . ) and ggt levels (r = . , p = . ). the comparison of the median maximum enzyme and bilirubin levels is shown in table . in two patients who developed severe vod requiering defibrotide, the liver stiffness level was . kpa and . kpa, respectively. the patient with the highest stiffness level ( . kpa) developed acute gvhd of the liver, which completely resolved after steroid treatment. only one of those five patients who had stiffness levels kpa died due to liver toxcity and concurrent septic shock, he suffered from viral hepatitis prior to transplantation. liver stiffness measured by transient elastography (fibroscan) positively correlates with the elevation of the cholestatic enzymes ap and ggt in myelofibrosis patients after allo-hsct and may predict liver toxicity. disclosure of conflict of interest: none.[p ]in the era of tyrosine kinase inhibitors (tki) as superior first line treatment in the therapy of cml, the concept of allogeneic hsct has been pushed to the role of salvage therapy. to date, data on allogeneic hsct after tki-therapy are scarcely available. in this study, we report single center data on the outcome of cml patients, for the most part pretreated with tki, who underwent allogeneic hsct between and with a follow-up of months to years. upon obtaining written informed consent patients diagnosed with bcr-abl-positive cml and patients with bcr-abl-negative atypical cml were included in this analysis. the majority of patients underwent myeloablative conditioning regimen. the median age at time of hsct was years with a range: from to years. twenty-one patients were transplanted from a matched related donor, and received stem cell grafts from an unrelated hla-compatible donor. / patients received tki-therapy before transplantation, patients received more than tki prior to hsct. / patients were treated with interferon prior to hsct. twenty-two patients were transplanted due to acceleration or blast crisis. twenty-six patients received an allogeneic hsct in chronic phase (cp, n = ) or complete hematologic (chr, n = ) or cytogenetic remission (ccyr, n = ). kinase domain mutations could be identified in seven patients including t i-mutation in four patients. seven patients showed "major route" cytogenetic aberrations. next to advanced disease status, tki intolerance (n = ) and tki resistance (n = ) were the main indications for hsct after . after a median follow up of years and months, those patients transplanted in cp, chr or ccyr showed an overall survival (os) of %. / patients died in remission and two patients died after cml relapse. after none of the patients transplanted in cp, chr or ccyr died or relapsed so far, with a median follow-up of days. all of these patients received tki therapy prior to transplant. twenty-two patients transplanted in advanced stage cml (bc and ap) had after a median follow up of years an os of %. the difference between survival curves is significant (log rank test p = . ; hr . , % ci of ratio . - . ). prior to transplantation of these patients received a tki-therapy. in this group, four patients died due to cml relapse, one died after development of donor cell leukemia and five patients died in remission. one patient with atypical cml was transplanted in bc and died of progressive disease shortly after transplantation. the other three patients with atypical cml were transplanted in cp-phase. with a median follow-up of days these patients are in ongoing remission. even in times of tkitherapy allogeneic hsct remains a successful and safe therapy option for cml patients with tki intolerance or resistance. patients transplanted in cp or complete remission had an excellent long-term outcome. allogeneic hsct should be considered in tki resistance or intolerance before the development of blast crisis. despite tki therapy, overall survival deteriorates in patients with advanced disease. however, this treatment modality can improve survival rates substantially compared to other available therapies. tkimaintenance therapy could be a possible strategy to prevent cml relapse, although randomized data on tki-maintenance therapy after allogeneic hsct are still lacking.[p ]disclosure of conflict of interest: none. use of first or second generation tki for cml after allogeneic hematopoietic stem cell transplantation: a study by the cmwp of the ebmt y chalandon, s iacobelli , j hoek , l koster , l volin , j finke , jj cornelissen , i yakoub-agha patients (pts) relapsing with cml after allogeneic hematopoietic stem cell transplantation (allohsct) may be treated with tki and/or dli. as nowadays the majority of cml pts would have received at least imatinib prior to transplantation, we were interested in analizing (a) the type of tki used after allohsct, (b) the indication for tki treatment, (c) the outcome of this treatment and d) the temporal relationship with dli if given. pts received tki after first allogeneic hsct for cml. transplants had been performed in cp , n = , ap, n = or for more advanced disease (bc/ cp , n = ) from hla identical siblings (n = ) or ud (n = ) between and . tki given prior to transplant was imatinib (n = ), dasatinib (n = ), nilotinib (n = ), bosutinib (n = ) and ponatinib (n = ). median age at transplant was ( . - ) years, pts ( %) were male. tki post allohsct were given between and . first tki given was either imatinib (n = ), dasatinib (n = ), nilotinib (n = ), bosutinib (n = ) or ponatinib ( ). the indications for tki therapy were the same as for transplantation (n = ), for relapse/progression/ persistent disease (n = ), for prophylaxis/pre-emptive (n = ), planned (n = ), others (n = ) and missing (n = ). median follow-up from start of tki was ( - ) months. the median time interval from transplant to tki was ( . - ) months. it was longer for tki given for relapse/progression with ( - ) months and shorter for tki given for prophylaxis/pre-emptive with . ( . haematopoietic cell transplant (hct) is the only curative approach for scd. due to concerns regarding the toxicities associated with myeloablative conditioning regimens in adults, a non-myeloablative protocol was developed by hsieh et al. (national institutes of health, nih protocol). the use of this novel regimen was able to achieve a curative degree of mixed donor chimerisms with minimal transplant-related complications. the alberta children's hospital (ach) has adopted this conditioning regimen in children due to the efficacy and low rates of toxicities published by the nih group. with generally lower rates of gvhd in younger recipients, our group had no reason to believe rates of toxicities would be greater in a younger population with fewer comorbidities secondary to scd than those described in the nih cohort. to our knowledge, there is no published literature describing the utilization of the nih protocol in a paediatric population. we describe our experience in children with scd who underwent matched sibling donor (msd) peripheral blood hct using nih protocol. this retrospective cohort describes outcomes of msd hct in children with scd who underwent hct with the nih conditioning regimen between - . a total of potential subjects were identified. eight subjects have consented to the analysis to date. msds with either normal haemoglobin or sickle cell trait were considered appropriate for donation. the transplant procedure: the conditioning regimen consisted of alemtuzumab . mg/kg/dose administered subcutaneously daily for five days (days − to day − ). patients received a tbi dose of cgy on day - , with testicular shielding for male recipients. gvhd prophylaxis consisted of a sirolimus load of mg/ m /dose (po) on day − , followed by mg/m /dose once a day starting on day . unmanipulated peripheral blood stem cells were infused on day . sirolimus was used for gvhd prophylaxis post-hct and continued until at least one year. weaning of sirolimus was initiated no earlier than year post-hct and if donor t-cell chimerisms were greater than %. institutional supportive care protocols for scd hct were followed. patients were eligible for early discharge post-hct even prior to neutrophil engraftment. eight patients ( f, m) have been registered on this retrospective study. follow-up ranges from to months post-hct. there were no failed stem cell mobilizations. all patients had donor neutrophil engraftment at a median of days. all patients are currently alive. there have been no cases of graft failure to date and no sickling crises post-hct. one patient has dropping myeloid chimerisms but still % donor. no cases of veno-occlusive disease, idiopathic pneumonia syndrome, cerebral hemorrhage, pres, or posttransplant lymphoproliferative disease were observed. three cases of cytomegalovirus (cmv) reactivation required pre-emptive therapy. only one patient did not initiate sirolimus weaning at year post-hct due to donor t-cell chimerisms of %; this patient is months post-hct and is likely to start weaning sirolimus soon. there have been no cases of acute or chronic graft-versus-host disease. nonmyeloablative conditioning regimen is safe and effective as curative therapy for scd. long-term follow-up of these children to assess organ function post-hct is underway. disclosure of conflict of interest: none.the number of new hiv/aids cases has been declining in developed countries, whereas it is still increasing in japan, with the cumulative number reaching , as of june , . hiv infection is associated with an increased risk of hematological malignancies such as non-hodgkin lymphoma (nhl). autologous hematopoietic cell transplantation (auto-hct) is a treatment option for hiv-infected patients with nhl and multiple myeloma (mm). however, the prognosis after auto-hct in hiv-infected japanese patients remains unclear. the aim of this study is to evaluate the effect of hiv infection on transplant outcomes after auto-hct in japan. using the national database of the japan society for hematopoietic cell transplantation, we retrospectively evaluated patients with nhl (n = ) and mm (n = ) who underwent their first auto-hct between and . presence of hiv antibodyperipheral t-cell lymphomas (ptcl) comprise a heterogeneous group of diseases among which ptcl-not otherwise specified (ptcl-nos) represents the most common histology. patients with ptcl are typically offered high-dose chemotherapy followed by autologous hematopoietic cell transplantation (auto-hct) as front-line consolidation. allogeneic hct (allo-hct) is generally offered in the relapsed setting; however, in selected cases it is also offered as front-line consolidation. no randomized controlled trial (rct) have been performed to date comparing offering an allo-hct versus other treatment modalities either in the front-line or in the relapsed setting. thus, we performed this systematic review/meta-analysis to assess the totality of evidence pertaining to the role of allo-hct in ptcl. search of the literature was undertaken via pubmed and web of science from inception until september , . no search limits were applied but studies presented only in abstract form were excluded. data were collected on treatment benefits (complete remission (cr), progression-free survival (pfs), overall survival (os)) and harms (non-relapse mortality (nrm), grade ii-iv acute graft-versus-host disease (gvhd), and chronic gvhd). the search identified references; however, only studies ( in front-line (n = pts), in relapsed/refractory setting (n = pts)) were eligible based on our inclusion criteria and had extractable data. three studies included both frontline and relapsed/ refractory cases but data for certain outcomes were reported separately. the median follow-up time for studies evaluating allo-hct in the front-line or relapsed/refractory setting ranged from - months and - months, respectively. in the front-line setting, allo-hct resulted in cr rates of % (( %ci = - %), studies, n = pts), pfs rate of % (( % ci = - %), studies, n = pts), and os rate of % (( % ci = - %), studies, n = pts). nrm rate was % (( % ci = - %), studies, n = pts). acute (grade ii-iv) and chronic gvhd rates were % ( % ci = - %), studies, n = pts) and % ( % ci = - %), studies, n = pts), respectively. in the relapsed/refractory setting, allo-hct resulted in cr rates of % (( % ci = - %), studies, n = pts), pfs rate of % (( % ci = - %), studies, n = pts), and os rate of % (( % ci = - %), studies, n = pts). nrm rate was % (( % ci = - %), studies, n = pts). acute (grade ii-iv) and chronic gvhd rates were % ( % ci = - %), studies, n = pts) and % ( % ci = - %), studies, n = pts), respectively. notwithstanding the need to perform a rct to compare the efficacy of allo-hct versus auto-hct as front-line consolidation in ptcl, the results of this systematic review/meta-analysis show very encouraging os rates of % following allo-hct. moreover, allo-hct also offers an encouraging os rate of % in patients with ptcl in the relapsed/refractory setting. the higher nrm rate in the relapsed/refractory setting probably reflects the adverse effect of a higher number of prior prescribed therapies. one of the limitations of our analysis is the inability to analyze outcomes for individual histologic subtypes due to the aggregate nature of the published data. disclosure of conflict of interest: none. high-risk patients with relapse or refractory hodgkin lymphoma do significantly better after hdc auto-sct compared to control arm of aethera trial. mature results from a cohort of patients s akhtar, s rauf, tam elhassan and i maghfoor king faisal specialist hospital and research center, riyadh, kingdom of saudi arabia brentuximab vedotin use in hodgkin lymphoma (hl) patients who had hdc auto-sct has been reported to improve progression free survival (pfs) but not the overall survival (os) in a phase trial (lancet ; : - ). in this trial, after hdc auto-sct, high risk hl patients were randomized to receive placebo (control gp) vs brentuximab (experimental gp) as consolidation therapy. we are reporting our experience of patients with similar selection criteria as control gp. hl patients z yrs who received hdc auto-sct with similar selection criteria as defined in aethera trial were identified that is, patients had at least one of the following risk advanced lymphomas still represent a therapeutic challenge and allo-hsct is among treatment options. between march and august , seventy-three patients (pts) affected by r/r lymphomas ( nhl and hl) underwent an allo-hsct after a treosulfan-based conditioning regimen and sirolimus as calcineurin-inhibitor-free prophylaxis of gvhd. six pts received a mrd, pts a mud, and pts a haplo unmanipulated pbsc. at allo-sct pts were in cr, pts were in pr, and pts had sd/pd; sixty patients underwent autologous sct before allo-hsct. hct-ci was evaluable for pts, had a score ≥ . thirty-three pts received treosulfan and fludarabine reduced toxicity conditioning regimen (rtc) and intensification with other alkylating agent or with gy total body irradiation was added on the remaining pts (myeloablative conditioning, mac). all pts received a backbone gvhd prophylaxis with sirolimus and mycophenolate mofetil; atg or pt-cy or both were added in , , and pts respectively. median numbers of infused cd +/kg and cd +/kg were . × (range: . - . ) and . × (range: . - . ), respectively. median follow-up was months (range: - ); median time to neutrophil ≥ . × /l was days, and days to platelet ≥ × /l. sixteen out of patients with pre-transplant active disease obtained a cr after treosulfan conditioning; nine of them ( hl and b-nhl) achieved durable cr without post transplant treatment. oneand -years os was % and %, pfs was % and % at and years respectively; cumulative incidence of relapse/ progression was % and % at and years. grfs was % and % at and years, respectively. transplant related mortality (trm) was % at days, % at year and for the entire follow-up. the -day cumulative incidence (ci) of agvhd grade ≥ was % and ci of agvhd grade ≥ was %; ci of moderate to severe cgvhd was % at years and for the entire follow-up. no differences in ci of agvhd or cgvhd were found if pts were stratified according to donor type, but ci of moderate-severe cgvhd was significantly higher in pts after mac regimens (p o . ). as expected, the outcome of pts in cr was significantly better compared with active disease, in terms of os (p = . ), pfs (p = . ), ri (p = . ). in multivariate analysis, intensity of conditioning regimen (rtc vs mac), gvhd prophylaxis (use of atg, pt-cy or none), donor sex and age at allo-sct did not impact the transplant outcomes; both os and pfs were reduced by active disease at allo-hsct (hr = . , ci % . - . , p = . and hr = . , ci % . - . , p = . , respectively) and by nhl histology (hr = . , ci % . - . , p = . and hr = . , ci % . - . , p = . , respectively); grfs and ri were impacted only by active disease (hr = . , ci % . - . and hr = . , ci % . - . , p = . , respectively). allo-hsct after treosulfan conditioning and sirolimus gvhd prophylaxis is feasible even in heavily pretreated pts affected by lymphomas. complete remission status before transplant remains crucial for better outcomes and in the era of new targeted treatments should be pursued. disclosure of conflict of interest: none.university of eastern finland, kuopio, finland and department of medicine, kymenlaakso central hospital, kotka, finland autologous stem cell transplantation continues to have an important role in the treatment of patients with multiple myeloma (mm). in mm patients the most commonly used mobilization method is granulocyte-colony stimulating factor (g-csf) ± cyclophosphamide (cy). generally, up to - % patients mobilize poorly with these methods and plerixafor may be used to enhance mobilization. the most important parameter of graft quality has usually been the number of cd + cells, but there are also significant numbers of other cell subsets in the grafts and they may also be of special interest in regard to post-transplant recovery and outcome. for example, a higher number of lymphocytes and nk cells in the grafts has been associated with improved lymphocyte as well as nk cell recovery, respectively. the mobilization methods used seem to affect the graft composition. however, there is currently no prospective data on the effects of plerixafor on the graft composition, post-transplant hematological and immune recovery or outcome in patients with mm. altogether eighty-seven patients with mm were included into this prospective study. seventy-seven patients were mobilized with g-csf ± cy (control group) and ten patients received also plerixafor due to poor mobilization (plerixafor group). in the control group / ( %) and in the plerixafor group / ( %) of patients were mobilized with g-csf+cy (p = . ). there were no statistically significant differences between the groups according to age, gender, paraprotein type, initial iss, induction therapy used or disease status at the time of mobilization. by imwg risk stratification, there were more high risk patients in the plerixafor group ( / vs. / , p = . ). cryopreserved graft samples were analyzed with flow cytometry for t and b cells (cd /cd /cd /cd ) as well as for nk cells (cd /cd +cd ). also, cd + cell subclasses were analyzed (cd /cd /cd ). complete blood counts were evaluated at + days, , , and months posttransplant. to evaluate immune reconstitution, flow cytometry of lymphocyte subsets (t, b, nk) was performed in a subset of patients at , and months after the graft infusion using the same antibody panel as for graft analysis. there were no significant differences between the groups in the number of cd + cells in the grafts. also, the median number of aphereses was two in the both groups (p = . ). the proportion of the more primitive cd + cells (cd + cd + cd -) was significantly higher in the plerixafor group (p = . ). in addition, the number of various lymphocyte subsets analysed was significantly higher in plerixafor group table ). there were no statistically significant differences in the course of hematological recovery. the recovery of blood cd +cd + t cells was significantly faster in the plerixafor group at one at three moths post-transplant. there was no significant difference in the progression-free survival (pfs) (log rank, p = . ) with the median follow-up time of days in the plerixafor group and days in the control group ( . ). in the present study plerixafor added to g-csf ± cy seemed to significantly alter the cellular composition of autologous blood grafts in poorly mobilizing mm patients. hematological recovery was comparable but the cd +cd + t lymphocyte recovery was faster in the plerixafor group. the pfs was comparable between the groups. disclosure of conflict of interest: dr. valtola has received honoraria from sanofi and jansen-cilag. dr. silvennoinen has received a research grant from celgene and janssen, honoraria from genzyme and sanofi and participated in advisory board organized by amgen, janssen and takeda. dr. siitonen has received honoraria from amgen and celgene. dr. jantunen has received honoraria from genzyme, amgen and sanofi and has participated in eu leadership meeting organized by genzyme as well as medical advisory board meeting organized by genzyme and amgen. dr. varmavuo has received consultancy fees from abbvie, roche, celgene, amgen and sanofi. the other authors declare no conflicts of interest. bortezomib after high-dose melphalan as conditioning regimen before autologous stem cell transplantation in patients with multiple myeloma: a comparison with the historical conditioning regimen with melphalan alone ga ferini; ja arbelbide; al basquiera; e nucifora; n schutz; v otero; d fantl hospital italiano d buenos aires, buenos aires, argentinahigh dose of melphalan followed by autologous stem cell transplantation (asct) is the standard of care for younger patients with multiple myeloma (mm). to enhance the efficacy of the conditioning regimen, the intergroupe francophone du myelome added bortezomib to melphalan showing improved response rates, without significant toxicity. bortezomib has shown synergistic effects with melphalan, mainly if the bortezomib is administered hours after the melphalan. since , we have changed our conditioning regimen for patients with mm undergoing asct by adding bortezomib toallogeneic stem cell transplantation (allosct) is a potencially curative option for patients with multiple myeloma (mm). despite the improvement of reduced-intensity-conditioning (ric), transplant-related mortality (trm) remains high. there is no consensus on which graft versus host disease (gvhd) prophylaxis regimen is superior. some studies have suggested that tacrolimus-based prophylaxis is more effective than cyclosporine (csa) in terms of lower incidence of severe acute gvhd (agvhd), with no impact on overall survival (os). herein, efficacy and toxicity between two gvhd prophylaxis regimens is analyzed. we retrospectively analyzed patients (pts) with relapsed mm who received allosct ric in the period from to in a single centre (table ) . population: age, years ( - ); median follow-up: months ( - ). conditioning regimen: allo-ric (fludarabine + busulfan or melphalan regimens) and % was bortezomib-based in the tacrolimus group. donor: matched related ( pts), unrelated ( ), mismatch unrelated ( ) and haploidentical ( ) donor. gvhd prophylaxis: all patients received a short course of methotrexate + csa ( pts, %) or tacrolimus ( pts, %). complete response at transplant was % at csa group and % at tacrolimus group. all pts underwent toxicity related to chemotherapy (mainly mucositis and neutropenic fever) with organ impairment (renal or liver) in % tacrolimus arm as well as pts in csa group. the incidence of agvhd was % and . % in tacrolimus and cyclosporine groups, respectively (p = . ). grade iii-iv agvhd were reported in pts ( %,tacrolimus) and pts ( %, cyclosporine), with severe gastrointestinal and liver involvement. glucocorticoid resistance was observed in % in both groups. patients with refractory agvhd received other immunosuppressive therapies: more than second-lines agents ( ) ( ) ( ) ( ) were necessary in fifty percent of pts in both groups to control gvhd. two patients had to interrupt tacrolimus due to neurological toxicity and suspected thrombotic thrombocytopenic purpura. no patients had to discontinue treatment in the csa arm because of toxicity. the -months os was . % ( % in tacrolimus vs . % in csa (p = . )) and the -months was . %. a total of pts died because of gvhd. during follow-up, only patients relapsed ( and months after allosct, respectively) in csa group. no relapse were seen in tacro group. in our experience, no significant differences were observed between both calcineurin inhibitor in terms of os, toxicity and gvhd incidence. an explanation could be our small number of patients. allosct is an effective therapy for selected patients but it is associated with high rates of gvhd and trm. a long-termsafety and effective prophylactic regimen is necessary as main objective.[p ]disclosure of conflict of interest: none. several parameters, including early lymphocyte, neutrophil, platelet recovery, and infused dose of cd + cells, have been associated with clinical outcome of patients with haematological malignancies. however, their prognostic significance remains uncertain. the aim of current study was to evaluate prognostic significance of clinical and laboratory parameters that might influence survival after autologous stem cell transplantation (asct) in hodgkin lymphoma (hl) and multiple myeloma (mm). this retrospective study included a total of with hl and mm patients (median age years, years, respectively) who underwent asct between november and june . hl patients were conditioned with beam ( . %) and cbv ( . %) regimen, while mm patients received conditioning with high dose of melphalan. high ips (international prognostic score) at diagnosis had . % hl patients and high iss (international scoring system) had . % of mm patients, of which . % had renal impairment. the average of transplanted cd + cells in hl patients was . × /kg (range: - . × /kg), and . × /kg (range: - . × / kg) in mm patients. after asct, favourable treatment response (partial/complete remission) achieved . % hl patients, of whom . % had infused o × /kg cd + cells. median time to recovery of absolute lymphocyte count × /l or greater (alc ) was days (range: - days), recovery of absolute neutrophil count ≥ × /l (anc ) was (range: - days), and platelet recovery ≥ × /l (plt ) was days (range: - days). after asct, . % mm patients achieved favourable treatment response, of whom . % had infused cd + cell dose . × /kg. median time to alc was days (range: - days), anc was (range: - days), and plt was days (range: - days). median follow up of patients with hl was months, while after asct, median event free survival (efs) was months, and overall survival (os) was months. treatment response after asct strongly influenced both efs and os after asct (po . ). in patients who achieved favourable treatment response, os and efs after asct were influenced by infused cd + cell dose (o × /kg vs. ≥ × /kg), prolonged recovery of alc by day+ , plt by day + , and achieving of anc by day + (po . ). multivariate analysis among significant variables showed that infused cd + cell dose was the most important parameter that influenced os and efs (po . ). median follow up of mm patients was months, while after asct, median efs was months and os was months. regarding patients who achieved favourable treatment response, os and efs after asct were influenced by the presence of renal impairment, infused cd + cell dose (≤ . × /kg vs. . × /kg) and plt recovery by day + (po . ). among these significant parameters, multivariate analysis pointed out infused cd + cell dose as the most important parameter that influenced both os and efs (po . ). these data suggest that number of infused cd + cells is an independent factor that may contribute to outcome of patients with hl and mm. disclosure of conflict of interest: none. high-dose therapy with autologous stem cell transplantation (asct) has become the treatment of choice for symptomatic eligible patients with multiple myeloma (mm). we studied an induction regimen of cyclophosphamide, bortezomib and dexamethasone (cybord) and showed rapid and deep responses after cycles in patients with newly diagnosed mm and we subsequently done asct with melphalan (mel) conditioning. cost is the major limiting factor in developing world.all the drugs used are generic brands manufactured in india. a total of mm patients (median age: . years, % male and % female) were transplanted between and . in all, patients had igg kappa- ( %), igg lambda- ( %), iga lambda- ( %), iga kappa- ( %), kappa light chain ( %), lambda light chain ( %) patients. prior to autograft, all cases had received cybord with generic medicines. median time diagnosis to asct was . months ( to months). stem cell mobilization was done with g-csf alone in ( %), g-csf plus plerixafor in ( %) and chemo mobilization in ( %) patients. all patients received asct support after conditioning with mg/m generic melphalan alone (dose adjustment was done according to renal status). all patients received thalidomide maintenance from march . bortezomib used was manufactured by dr. reddy's lab, hyderabad and melphalan used was manufactured by emcure pharmaceuticals, pune, india. patients from to received cyclophosphamide, vincristine, adriamycin and dexamethasone (cvad) protocol of originator medicines followed by originator melphalan conditioning and asct (cvad-mel-asct). at the time of autograft, ( %) of patients were in complete remission, ( %) in partial remission, ( %) very good pr. median day of engraftment was for neutrophils and for platelet. transplant related mortality was % ( / ) out of which died of infection and deaths of cardiac events. the pfs and os rates were % and % at median follow up of . months. patients who were treated with cvad-mel-asct had efs of % at yrs and % at yrs. cost of bortezomib showed significant difference, generic was usd where as for originator drug was usd for cycles of chemotherapy. cost of melphalan also showed difference with usd for generic and usd for originator drug. generic cybord showed excellent response rate and allows excellent stem cell collection and transplantation which can further consolidate response and improve outcome. cybord induction and melphalan conditioning with generic medicines can be considered a standard regimen for transplant-eligible patients with newly diagnosed mm in resource constraint situation. generic cybord-mel-asct is more cost effective than originator cvad-mel-asct. generic medicines produced in india are of good quality and cost effective. this study needs long term follow up to assess survival parameters at a median. disclosure of conflict of interest: none. and an extra copy of one or more odd-numbered chromosomes and as intermediate risk(ir) if they had t( ; ) or del( ) (q).overall survival (os) and relapse-free survival (rfs) were calculated from the time of allo hsct and auto hsct on day , from diagnosis to death or disease progression. the median age at presentation was . (range: - ) years, and ( . %) were men. at a median follow-up time of months, % were alive. of the patients with available fish samples underwent auto hsct. patients ( . %) achieved cr and patients ( . %) relapsed. of the patients who had received allo hsct, five patients ( . %) achieved cr and five patients ( . %) remained alive. in patients who received auto hsct, the risk of relapse was % less than those never transplanted (p = . ), but the difference was not significant in patients who received allo hsct. the relapse-free survival in hr patients was months (po . ), in ir was months (p o . ) and in sr was . months (p o . ). in transplant patients, rfs in hr patients was . times more than sr group (po . ) and in ir group was . times more than sr (p o . ). the survival time in transplant patients was significantly better than non-transplanted patients (p o . ). the median overall survival (os) in hr patients was . months, in standard risk group months and in sr patients was months. cytogenetic abnormalities detected by fish are of significant value in classification, risk stratification and management of patients with mm. we can use cytogenetic data to provide prognostic information and also to guide management and clinical practice. these data indicate that autologous stem cell transplantation could potentially be of benefit to myeloma patients. disclosure of conflict of interest: none. chronic graft-vs-host disease (cgvhd) is the most troublesome complication developing after allogeneic hematopoietic stem cell transplantation (allo-hsct). diagnosis of cgvhd has largely been based on clinical features only. we previously reported gene expression profiles in patients with cgvhd after allo-hsct. we extended our study to develop a molecular diagnostic method of cgvhd. we selected six most commonly expressed genes from the former dna expression study. and, a home-made -gene pcr array were used to evaluate gene expression profiles in the peripheral blood mononuclear cells of patients given allo-hsct ( cgvhd patients, non-cgvhd patients) and normal controls. the gene expressions of the allo-transplanted patients were compared to those of the stem cell donors. sybr green qpcr and multiplexqpcr were performed to confirm the usefulness of the selected genes in the diagnosis of cgvhd. infogainattributeeal and ranker were used to develop a gene model to diagnose cgvhd. k-nearest neighbor model and weka classifiers lazy ibk module were applied to evaluate the performance of the gene model. in another steroid-refractory cgvhd patients ( responders, non-responders), the gene expression changes were analysed using our -gene pcr array before and days after rituximab treatment. we identified six genes most accurately delineating cgvhd patients from those without treatments after allogeneic hematopoietic stem cell transplantation (hsct) are long and constraining for patients. medical adherence in hsct patients is of major concern in daily practice but it has been not yet described. , the aims of our study were to evaluate treatment adherence and to identify factors associated with adherence behaviors. an observational single-center study was based on self-reported questionnaires completed by patients in a hematology day hospital between november and july . the patientreported adherence was evaluated using the eight-item morisky medication adherence scale (mmas- ). , individual item scores were summed: patients with a score of / were considered as good adherents to medication whereas a poor adherence referred to a score under . among the latter, medium adherence ranged to a score of - , while a score of o was considered low adherence. socio-demographic and medical characteristics were collected by health records. a univariate model was used to evaluate if some of patients' characteristics were associated with adherence. statistical analysis was performed using r software (version . . - - rstudio, inc). fifty-six patients were included in the current study. median age at transplantation was years (range: - years). diagnosis were aml (n = ), all (n = ), myelofibrosis (n = ) and other hematological diseases (n = ). patients received a hsct from a related donor ( haploidentical). myeloablative conditioning was used in patients and reduced intensity regimen in patients. a total of . % ( / ) of the patients were poor adherent according to mmas- . among these patients, / were low adherent and / were medium adherent. the results of univariate analysis showed that a poor adherence was associated with a longer time since hsct and discharge at home. however elderly patients, patients treated with cyclosporine and patients with daily hydration at home were associated with a better adherence (po . ). our study presents the first data on adherence among patients undergoing hsct. risk factors associated with a poor adherence have been identified in order to determine patients' profiles that will benefit more from interventions to improve adherence. particular attention has to be paid to younger patients. efforts to establish a regular follow-up of these patients are needed in order to sustain patients in the treatment adherence to prevent the occurrence of severe complications. we studied the effect of basic fibroblast growth factor (fgfb) and dexamethason on expansion and immune modulation of mscs in patients with lymphomas. mscs were generated from bone marrow aspirates obtained from the patients with hodgkin's lymphoma (hl; n = ) and non-hodgkin's lymphoma (nhl; n = ). the adherent fraction of marrow aspirate was cultivated with/without the basic fibroblast growth factor (fgf-b, ng/ml) or dexamethason ( − М or − М) to reach - % confluence. then mscs were passaged with accutase and used for experiments after - passages. the number of msc precursors (cfu-f) in bone marrow of lymphoma patients was found to be significantly decreased both in patients with nhl ( ± , p o . ) or hl ( ± , p o . ). the time until - % confluence was significantly increased and took on average ± days (vs . ± . in donors). finally, the immunosuppressive ability of patient msc was significantly lowered and was only registered at the high concentrations of mscs ( : and : ). the expansion of patient mscs was significantly promoted with fgfb resulting in a significant decrease of primary cell cultivation (from . ± . to . ± . days; p = . ) and a statistically significant twofold increase in the number of cells received at the first passaging. in addition, in cultures with fgfb there was a decrease in the relative amount of resting mscs and a threefold increase of cycled cells in cd + mscs. dexamethasone has also provided a moderate stimulating effect on the msc growth. in fact, the use of − М of dexamethasone resulted in the increase of the cell yield by . times and of − М-by . times. however, fgfb and dexamethasone differed in their effect on the msc ability to inhibit the proliferative response of t lymphocytes upon stimulation with mitogens or alloantigens. indeed, fgfb failed to correct the impaired immunosuppressive activity of patient mscs, and median percentage of suppression still remained lowered- % vs % without fgfb. in contrast to fgfb, dexamethason could increase the immunosuppressive activity of patient mscs by . times (in dose of − М) and by . times (for − М). our data indicate that fgfb and dexamethasone used during the generation of mscs exert a stimulating effect on the msc expansion. in contrast to fgfb, dexamethasone, in the broad range: of doses, was able to enhance the suppressive properties of mscs that are initially reduced in patients with lymphoma. these findings suggest the existence of at least two mechanisms of impairments in immunoregulatory function of mscs in lymphomas-dependent and independent of the msc proliferation. disclosure of conflict of interest: none. free nonabsorbable antibacterial digestive decontamination is associated with a low incidence of gastrointestinal acute gvhd and better gvhd-free/ relapse-free survival (grfs) in the atg-based conditioning regimens nabil yafour commonly antibacterial prophylaxis based of oral no absorbable antibiotic such as (neomycin colistin, gentamicin, vancomycin) used before and after engraftment, other fluoroquinolone such as levofloxacine were recently used to prevent invasive infection. however the exact interaction with gastrointestinal acute graft versus host disease (gi-agvhd) remains unclear. the objective of this study was to evaluate a novel composite endpoint of gvhd-free/relapse-free survival (grfs), in which events include grades - gi agvhd, chronic gvhd requiring systemic therapy, relapse, or death in atg based-conditioning regimens, with free no absorbable antibacterial digestive decontamination prophylaxis. a total of evaluable consecutive patients with hematological disease were included in period of february to mai . patients with malignancies disease (n = ) received myeloablative conditioning regimens plus atg ( mg/kg); including once daily busulfan ( mg/m ,- d to - d, iv ) + fludarabine ( mg/m /d, - d to - d, iv) (aml = , all = , cml = ) or melphalan ( mg/m , d- , iv) (all = ). six patients received cy/atg for saa. gvh prophylaxis consisted to; ciclosporine a (csa) + mtx. csa was maintain levels between - ng/ml and tapered at the discretion of the treating physician. all patients were received peripheral blood stem cells (pbsc) graft from a matched related donor. since december levofloxacine and voriconazole was administered as antibacterial and antifungal prophylaxis. diagnostic, clinical grading and treatment of gi-agvhd and gi-cgvhd were performed according to established criteria and nih recommendations. probability of grfs was estimate by kaplan-meier method. median age was years (range: - ). median dose of cd + and cd + cell doses were . × (range: . - ) and . × (range: , - , ). the median time to neutrophil and platelet recovery were days (range: - ) and days (range: - ) respectively. at time of transplant / ( %) had an intestinal colonization with extended-spectrum betalactamase (esbl) producing bacteria. only / ( %) developed infectious diarrhea during the period of transplant. incidence of grade iii/iv gi-agvhd and gi-cgvhd requiring systemic therapy were % and % respectively. for patients with malignancies diseases (n = ), ( %) were alive at a median follow up of months (range: - ). incidence of relapse, disease free survival rates were %, % respectively. the grfs rate as defined previously was % at months. these results confirm that free no absorbable antibacterial digestive decontamination and atg-based conditioning regimens were associated with very low incidence of gi-gvhd and better grfs in patients with malignancies diseases. diverse bacterial populations of the gastrointestinal tract remain important factors to promote immune tolerance after allogeneic sct. disclosure of conflict of interest: none. g-csf primed hla haploidentical transplantation from maternal or collateral donor using atg plus reduced dose of posttransplantation cyclophosphamide: results of a phase ii prospective trial y wang , x-j huang peking university people's hospital, peking university institute of hematologythe transplantation milieu using granulocyte colony-stimulating factor (g-csf), and anti-thymocyte globulin (atg) for hlahaplotype-mismatched transplants from related donors has resulted in favourable outcomes with low transplant-related mortality (trm), without increased relapse rate. however, in this transplant modality, the poorer outcome owing to high incidence of graft-versus host disease (gvhd) related to maternal donor or collateral donor remains a concern. meanwhile the use of post-transplant cyclophosphamide (pt/cy) in recent years appears to be protective against severe acute and chronic gvhd. we performed a prospective pilot study of hla haploidentical stem cell transplantation (sct) from maternal or collateral donors with intensified conditioning including g-csf and atg, followed by two lower doses of pt/cy ( . mg/kg × doses). outcomes were compared with those of controls from matched-pair analysis who undergone haploidentical sct from other donors than mother or collateral relatives at the same time period. a total of patients with myelodysplastic syndrome (mds) or leukaemia undergoing haploidenticla sct from maternal or collateral donors were enrolled in the study. incidence of grade ii-iv and grade iii-iv acute gvhd at day were comparable between the study group and the control group ( . % vs. . %, p = . ; . % vs. . %, p = . ). incidence of cmv and ebv reactivation at day were also comparable between the study group and the control group ( . % vs. . %, p = . ; . % vs. . %, p = . ). after a median follow-up of days and days, the incidence of trm and relapse at year were comparable between the study group and the control group ( . % vs. . %, p = . ; . % vs. . %, p = . ); the probability of overall survival and lfs at year were comparable between the study group and the control group ( . % vs. . %; p = . ; . % vs. . %, p = . ). in conclusion, conditioning with atg and low-dose pt/ cy might be a feasible option for patients undergoing hla haploidentical, t-cell replete sct from maternal or collateral donors. trial registration: the study is registered at www. clinicaltrial.gov as nct . disclosure of conflict of interest: none. hematopoetic stem cell transplantation (hct) is a lifesaving treatment option for eligible patients with hematological malignancies. hct is inherently associated with a risk of nonrelapse mortality that varies greatly depending on transplant and patient characteristics. the assessment of the risk of complications and mortality before the procedure is extremely important. the hct comorbidity index (hct-ci) introduced by sorror m. is one of the tools proved to predict hct outcomes and was shown to be significant in various disease and hct settings. the objective is to evaluate hct-ci index of hct recipients, determine impact of different variables on ci score, particularly those, showing pulmonary and cardiac function. data of hct-ci of autologous (auto) and allogeneic (allo) hct recipients, transplanted during period january -october were analyzed. impact of pulmonary and cardiac function values on ci score was evaluated: dlco (diffusing capacity of the lung for carbon monoxide), fev (forced expiratory volume) and ef (cardiac ejection fraction) are parameters, reflecting pulmonary and cardiac function, which values are included into hct-ci score. the statistical data analysis was conducted using spss program. the differences were considered statistically significant at p ≤ . . records of allo and auto hct recipients, transplanted during . - . in vilnius university hospital were revised. median age of allo hct and auto hsc recipients was ( - ) and ( - ) years respectively. main indication for allo hct was acute myeloid leukemia ( %) patients and for auto hct -multiple myeloma ( . %) patients. hct-ci was completely calculated (no values missing) in allo and auto hct recipients. only patients with available complete hct-ci data were further analyzed. hct-ci in hct recipients was as shown in table . hct-ci score o was calculated in ( . %) and ≥ in ( . %) allo hct recipients. hct-ci score o was calculated in ( . %) and ≥ in ( . %) auto hct recipients. hct-ci score did not differ statistically significant between male and female recipients in both hct categories as well as in different age groups of patients (below and above years in allo and below and above years in auto hct). dlco was found to be below normal values (o %) in ( . %) allo hct and in ( . %) auto hct recipients. fev was less affected and found to be lower % in ( . %) allo hct and in ( . %) auto hct recipients. ef below % detected in ( , %) allo hct and in ( . %) auto hct recipients. low dlco was found to cause the greatest impact on hct-ci score and was statistically significantly associated with higher hct-ci (po . ). the most common hct-ci in both hct groups was score . dlco was found to be below normal ranges in relatively large patient group and had the greatest impact on hct-ci score. further studies on reasons of pulmonary function impairment and it's impact on hct outcomes are warranted.[p ]disclosure of conflict of interest: none. haemophagocytic lymphohistiocytosis (hlh), a life-threatening hyper-inflammation syndrome, is classified into primary and secondary forms. primary hlh is caused by gene mutations resulting in impaired cytotoxicity of natural killer (nk) cells and cytotoxic t lymphocytes (ctls). secondary hlh arises in the setting of autoimmunity, infection, malignancy, or less commonly, may be idiopathic. treatment of hlh has two major goals: halting the triggering event and controlling the overactive immune system. however, patients with primary or recurrent secondary hlh should subsequently undergo allogeneic hct for long lasting disease remission. we retrospectively evaluated hematopoietic stem cell transplantation (hsct) might be a valid treatment option for adults suffering from aggressive t-cell malignancies providing long term disease control. since a suitable hla-matched donor cannot be identified for all patients (pts) in need for transplantation, alternative donors graft sources such as related hla-haploidentical donors are considered. through introduction of t-cell-replete (tcr) hlahaploidentical transplantation (haplo-hsct) using post transplantation cyclophosphamide (ptcy) successful treatment with low non-relapse mortality rate (nrm) has been observed in lymphoma patients (luznik et al., bmt, ) . however, less data are available on the outcome of this haplo-approach in the treatment of t-cell malignancies, in particular when disease is refractory. we retrospectively evaluated the outcome of haplo-hsct using tcr grafts and ptcy in pts with peripheral t-cell lymphoma treated between and at our institution (t-nhl = , t-all = ; male n = ; median age: years). disease was refractory/active at time of transplantation in pts, while one had achieved second cr. all patients received at least prior treatment lines and one patient failed previous allogeneic transplantation. while fludarabine and cyclophosphamide served as backbone for conditioning, pts received a tbi-based and a drug-based conditioning regimen which was myeloablative in %. if disease was active at time of haplo-hsct, a sequential therapeutic concept was performed involving intensive chemotherapy (clofarabine n = ) shortly preceding conditioning (zoellner ak et al., bmt, ) . post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mycophenolate mofetil in all patients. graft source was bone marrow in pts. no primary graft rejection occurred; / pts engrafted, one died early in aplasia. neutrophil/platelet engraftment was achieved at a median of (range: - ) and (range: - ) days, respectively. acute gvhd grade ii-iii was observed in pts, whereas no patient developed grade iv agvhd. mild chronic gvhd occurred in one patient. % of the pts developed grade ii-iii treatment-related toxicities most commonly diarrhea ( %) and mucositis ( %); grade iv toxicity (mucositis) was observed in one patient only. no vod occurred. cmv reactivated in / pts at risk, whereas no ptld was seen. proven invasive aspergillosis was diagnosed in one patient. at day + seven pts achieved cr. pts relapsed and died (relapse n = , infection n = ). -year nrm was %. at a median follow up of months (range: - ) the estimated -year and -year overall survival (os) and progression-free survival (pfs) were %/ % and %/ %, respectively. three pts received haploidentical dlt pre-emptively (n = ) and therapeutic (n = ), leading to sustained cr in two, while no severe gvhd occurred. sequential therapy in the setting of tcr haplo-hsct using ptcy as gvhd prophylaxis is feasible, well tolerated and shows low rates of gvhd and acceptable nrm in patients with relapsed/refractory t-cell lymphoma/ leukemia providing an effective anti-lymphoma/leukemic activity. thus, we suggest that intensified tcr hapo-hsct using ptcy should be considered as an alternative for patients suffering from aggressive t-cell malignancies, lacking hlamatched donors. disclosure of conflict of interest: none. tacrolimus is a calcineurin inhibitor increasingly used as immunosuppression following allogeneic stem cell transplantation; maintenance of therapeutic serum levels is essential to reduce the risk of graft rejection and graft versus host disease. however, tacrolimus can be associated with serious side effects and potential drug interactions. regular monitoring of serum levels and appropriate dose adjustment is essential to ensure therapeutic levels and to avoid toxicity. in our adult bmt unit, an established standard operating procedure (sop) provides a prescriptive dosing algorithm for: (i) initiation of tacrolimus therapy; (ii) conversion between iv and oral routes; (iii) dose adjustment based upon tacrolimus serum level and interacting medications. we performed an audit assessing adherence to the sop dosing algorithm. inpatient tacrolimus dosing episodes from five consecutive haploidentical transplants were retrospectively analysed. episodes were excluded due to insufficient records. for the remaining episodes, tacrolimus serum levels and corresponding doses were identified. the response of the medical team to each serum level was compared with the sop dosing recommendation. to account for sensible rounding of doses, a margin of error of ± % was permitted. adherence to sop dosing was %. non-adherence to the sop ( %) was subcategorised as justifiable ( %) or unjustifiable ( %). justifiable non-autologous hsct is currently being explored for its efficacy and safety in the treatment of multiple sclerosis (ms). as more experience is gained in treating this cohort, treatment related mortality has steadily improved although the procedure still carries a degree of risk. ebv reactivation is well described in allogenic stem cell transplants although less so in autologous transplantation. we investigated the frequency of ebv reactivation in patients with ms undergoing autologous hsct at a single uk site. patients underwent autologous hsct for treatment of ms at king's college hospital between feb and aug . all were mobilised with cyclophosphamide g/ m and g-csf. were conditioned with cyclophosphamide and atg, and one with beam/atg. previous exposure to ebv (ebv igg) was assessed prior to transplant and local posttransplant ebv monitoring was performed on whole blood samples by means of quantitative pcr in patients. data was collected retrospectively. all ( %) patients were positive for ebv igg pre-transplant. overall, samples were tested for monitoring post-transplant. ( . %) patients demonstrated positive pcr post-transplant on local testing with one further patient being negative on local tests but later becoming positive on testing in their parent hospital (full results unavailable). of these , the median time to positive testing post-transplant was days ( - ). maximal ebv dna titre was reached at a median time of days post-transplant ( - ) with a mean maximum titre of . log ( . - . ). patients experienced symptomatic reactivation with an associated large paraproteinemia. one of these developed hyper-viscosity requiring plasma exchange and developed neurological symptoms mimicking an ms relapse (max ebv titre of . log). this patient received rituximab and ebv level is declining, the other was observed carefully but developed right leg weakness which is slowly improving. the patient with raised ebv at their parent hospital also received rituximab (unclear if this reactivation was symptomatic). we have developed a protocol to pre-emptively treat ebv reactivation with rituximab once a log titre is reached and one patient has so far been treated according to this. of the patients with locally confirmed reactivation who did not receive rituximab, ( %) self-resolved at a median time of days ( - ), ( . %) have ongoing re-activation ( with improving, with stable titres) and ( . %) have not had any local bloods performed ≥ months. patient with selflimiting reactivation later had a further positive titre ( days post-transplant and days post initial resolution). ebv reactivation appears to be common in patients with ms in the first months post autologous hsct. unlike in other patient groups such as aplastic anaemia patients receiving allogeneic transplants it can cause significant neurological symptoms which may be confused with ms relapse. the mechanism of this reactivation is probably related to atg administration but may be exacerbated by prior immune suppression in this heavily pre-treated group, the majority of whom have received highly active disease modifying therapies in the past. these results demonstrate the importance of monitoring for ebv reactivation following autologous hsct and the consideration of pre-emptive therapy. disclosure of conflict of interest: none. reduced bone mineral density (bmd) is a well recognised complication of hct. guidelines recommend scanning by dual energy x-ray absorptiometry (dxa) one year after transplant in all hct patients or else specific groups of high risk patients. , it is recognised that both dose and duration of steroids are risk factors for low bmd and it is recommended that prednisolone doses greater than or equal to mg/day for more than months should prompt a dxa scan. for patients with osteopenia it is recommended that calcium/vitamin d supplements are given together with lifestyle advice including diet, smoking cessation and weight bearing exercise. in this survey we have investigated the current practise in investigating and managing bone health in the context of hct. a survey was sent to all centres including countries registered with ebmt as of november . centres replied from countries. response numbers to each question were variable and are indicated by the denominators. / used a national guideline to guide their practise, and / used an international guideline. no single guideline was quoted more than once. low testosterone has been demonstrated to be an independent determinant of endothelial (dys)function in men. graftversus-host disease (gvhd) is a major contributor to nonrelapse mortality (nrm) after allogeneic stem cell transplantation (allosct). vulnerability of the recipients' endothelial cell system is a novel concept to explain why a proportion of patients with acute gvhd fail to respond to escalating immunosuppressive therapy and ultimately succumb to gvhd and related complications. this retrospective study investigated the prognostic impact of pre-transplant testosterone levels on nrm after allosct in male patients. between and , a total of male patients undergoing allosct at heidelberg university (median age years) provided informed consent to participate in this observational study (training cohort). a total of patients ( %) received transplants from related donors (rd). diagnoses were aml ( %), mds ( %), lymphoid malignancies ( %) and multiple myeloma ( %) . a total of patients ( %) received statin treatment post allosct as per institutional standard policy. for validation, an independent patient cohort of men allografted for aml and mds (median age years, % rd, no statin treatment) at essen university was analysed. pretransplant serum samples were collected between and months before allosct and cryopreserved at − °c. testosterone and suppressor of tumorigenicity- (st ) levels were measured by radioimmunoassay and elisa, respectively. median pre-transplant testosterone level in the training and validation cohort was . nmol/l (range: . - . nmol/l) and . nmol/l ( . - . nmol/l), respectively. in the training cohort, lower pre-transplant testosterone as continuous variable was associated with shorter os (p = . ). lower testosterone levels showed a trend towards higher nrm (p = . ) and a significant association with nrm after onset of acute gvhd (p = . ). multivariate analysis confirmed lower pre-transplant testosterone levels as a significant predictor of an increased nrm risk after gvhd onset (p = . ). in the subgroup of patients not receiving statins post-transplant, lower testosterone levels were associated with increased incidence of transplant-associated microangiopathy (p = . ), and, in addition, with higher pre-transplant st levels indicating endothelial vulnerability. in the validation cohort, similar results with regard to overall survival (os, p = . ), nrm (p = . ), nrm after acute gvhd onset (p = . ) in univariate analysis, and to nrm after gvhd onset (p = . ) in multivariable analysis could be observed. the association of pre-transplant testosterone levels (in quartiles) and incidence of nrm after gvhd onset in the training and validation cohort is depicted in figure a and b, respectively. our study suggests that low pre-transplant testosterone is associated with serological and clinical evidence for endothelial damage and is an independent risk factor for a fatal outcome of gvhd. prospective studies in the allosct setting investigating testosterone and testosterone supplementation in deficient patients are highly warranted. disclosure of conflict of interest: none. nk cells anti-tumor ability in multiple myeloma patients s tognarelli , , b jacobs , , , i von metzler , h serve , p bader, t klingebiel , a mackensen and e ullrich , department of pediatric stem cell transplantation and immunology, childrens hospital, goethe university, frankfurt, germany; cellular immunology, loewe centre for cell and gene therapy, goethe university, frankfurt, germany; department of hematology and oncology, university hospital erlangen-busulfan is one of essential drugs for hematopoietic stem cell transplantation (hsct). because of its narrow therapeutic range: targeted busulfan using therapeutic drug monitoring (tdm) has been used. generally, the initial dose of busulfan is determined by patients' body surface area as mg/m except for infants ( mg/m ). however, pharmacokinetic evidence of these initial doses is scarce. therefore, we investigated the full pharmacokinetics of busulfan in infant and child, and attempted to validate that these initial doses are acceptable. one hundred ninety-five pediatric patients undergoing hsct using four-day targeted busulfan were enrolled. of them, patients received hsct when their age was ≤ year old (infant group [ig]), and patients received when - years old (toddler group [tg]). the remaining patients were defined as a child group (cg). busulfan was administered intravenously once daily for consecutive days. tg and cg received mg/m as the first dose, and ig received mg/m . using daily tdm, we adjusted the next dose of busulfan. target daily and total area under the curve (auc) were μg*h/l/day and - μg × h/l, respectively. median first-day busulfan auc of ig, tg, and cg were , and μg × h/l, respectively, which was significantly different (p = . ). however, there was no significant difference in median total busulfan auc (ig; , tg; , and cg; μg × h/l, respectively, p = . ). the coefficient of variance (cv) of four-day busulfan aucs in ig and cg was similar (median cv: . % and . %, respectively), whereas cv of tg was . %. in sub-analysis of tg and cg who received equally mg/m as the first dose, there was an inverse correlation between age and first-day busulfan auc (r = − . , p = . ), as well as between age and cv of four-day busulfan aucs (r = − . , p = . ). initial busulfan dose as mg/m for infant could be acceptable in aspect of first-day auc and cv of four-day busulfan aucs. however, higher first-day auc and cv were shown in tg. although target total busulfan auc could be achieved safely by tdm, we suggest that reduction of initial dose less than mg/m is also necessary to patients with - years old to lower the relatively higher first-day auc. taken together, tdm is highly recommended to reduce busulfan toxicity, especially in younger children. disclosure of conflict of interest: none. post-induction treatment strategy of acute myeloid leukemia (aml) is currently driven by european leukemia net (eln) risk assessment at diagnosis. if it is well established that patients belonging to favourable-risk group can be treated with chemotherapy and/or autologous stem cell transplantation (sct) and that those belonging to the unfavourable-risk group should be addressed to allogeneic (allo) sct, for patients included in the intermediate-risk groups the best post-induction treatment has not been established yet. we report here a years ( - ) allo-sct single center experience in aml patients. median age was years (range: - ), %, %, % and % were grouped in the eln favourable, intermediate-i, intermediate-ii and unfavourable risk category, respectively and % of the patients were allotransplanted in advanced disease-phase ( nd complete remission). half of the patients received a sibling hla compatible donor, % of the cases received peripheral blood stem cells and half of the patients received a myeloablative conditioning regimen. graft versus host disease prophylaxis was conventionally based on cyclosporine and shor-course methotrexate, with the addition of antilymphocyte immunoglobulin in case of matched unrelated donor. the clinical and transplant characteristics of the patients according to the eln-risk group were well balanced. with a median follow up of months (range: - months), the projected years overall survival (os) and disease free survival (dfs) is % ( % ci: - %) and % ( % ci: - %). the median os and dfs in favourable/intermediate-i vs intermediate-ii/unfavourable is . and . months ( figure a ; p = , ) vs and , months ( figure b ; p = . ). the relapse rate (rr) and the non relapse mortality (nrm) at two years are % ( % ci: - %), and % ( % ci: - %), respectively. non differences were observed comparing the years rr and the years nrm of patients in the favourable/intermediate-i vs intermediate-ii/unfavourable eln risk group ( % vs %; p = . and % vs %; p = . ). interestingly, the percentage of patients allotransplanted in advanced phase of the disease was higher in those included in low/intermediate-i with respect to intermediate-ii/unfavourable eln-risk group ( % vs %; p = . ). our data suggest that allo-sct can cure approximately - % of aml patients, with no difference within the eln risk groups. disease recurrence remains the major problem and this is highly correlated to the percentage of patients in advanced phase of the disease at transplant, particularly in eln favourable/intermediate-i patients. we are currently collecting the data on minimal residual disease (mrd) status of these patients during chemotherapy and before transplant using moelcular biology on target genes and/or multiparametric flow cytometry on leukemia associated immunophenotype, in order to assess if the prognosis of these patients may be refine by the prospective application of mrd data.[p ]disclosure of conflict of interest: none. outcome of allogeneic stem cell transplantation for patients with high-risk acute leukemia according to donor type and graft-versus-host disease prophylaxis s lindner, t berg , j riemann , s ajib , z jedlickova , s gueller , f lang , a sackmann , , n goekbuget , , h martin , a bacigalupo , h serve , and g bug department of medicine ii, hematology and oncology, university hospital frankfurt, goethe university, germany; s german cancer consortium (dktk), german cancer research center, heidelberg, germany and università cattolica del sacro cuore, fondazione policlinico universitario gemelli, roma, italyin high-risk acute leukemia (hr-al), allogeneic hematopoietic stem cell transplantation (hsct) is the only potentially curative treatment. increasingly, hsct is being performed utilizing alternative donors. we retrospectively analyzed the outcome of consecutive patients (pts) with hr-al (aml/all, n = / ) undergoing first allogeneic hsct in our transplant unit between / and / according to donor type and graft-versus-host disease (gvhd) prophylaxis: in the matched related donor group (mrd, n = ), hsct was performed with standard immunosuppression (is), that is, calcineurin inhibitor (cni) plus methotrexate or mycophenolate mofetil (mmf). for / hla-allele matched unrelated donors ( / mud, n = ) or / hla-allele mud ( / mud, n = ) we used is and anti-thymocyte globulin (atg fresenius/neovii). hsct with a haploidentical family donor or an / hla-allele mismatched unrelated donor was performed using is with cni plus mmf and post-transplant cyclophosphamide (pt-cy, n = ). a myeloablative (n = ) or reduced-intensity (n = ) conditioning regimen was applied in complete remission (cr, n = ) or active disease (n = ). pts had a median age of years (range: - ) and hematopoietic cell transplantation comorbidity index of (range: - ). patient and treatment characteristics were well balanced between the groups except for a higher percentage of pts transplanted in cr in the pt-cy group ( % vs. - %, p = . ). peripheral blood stem cells were preferred for mrd, / mud and / mud ( %, % and %, respectively) and bone marrow for % of pt-cy based hsct. all pts engrafted. with a median follow-up of months (range: - ), probability of overall survival (os) at years was ± % for the mrd, ± % for the / mud, ± % for the / mud and ± % for the pt-cy group, without significant differences (p = . ). however, the probability of achieving the combined endpoint gvhd-and relapse-free survival (grfs) at years varied significantly between the groups (mrd ± %, / mud ± %, / mud ± % and pt-cy ± %, po . ), reflecting the high cumulative incidence (ci) of chronic moderate and severe gvhd at year in the mrd ( ± %) as opposed to the other groups ( / mud ± %, / mud ± % and pt-cy ± %, p o . ). of note, donor type had no impact on ci of transplant-related mortality (trm) at years ( ± %), acute gvhd g - at day + ( ± %) or leukemic relapse at years ( ± %). overall, aml pts years of age had a significantly inferior relapse-free survival compared to younger pts ( ± % vs. ± %, respectively, p o . ) without a higher ci of trm (p = . ). median time to aml relapse was months. our results suggest that pt-cy-based alternative donor hsct is safe in hr-al pts and provides a solid basis for a randomized clinical trial comparing hsct from haploidentical family donors and / mud, currently in preparation. while os did not vary between groups, grfs was dismal after mrd transplants without atg, due to high rates of severe chronic gvhd, consistent with published data. as leukemic relapse remains the major cause for treatment failure especially in elderly pts, maintenance strategies using novel drugs or cellular therapies are warranted. disclosure of conflict of interest: none. relapse following hematopoietic stem cell transplant (hsct) is the leading indication for a second transplant in patients with malignant disease. hsct has been shown to be superior to chemotherapy alone or palliative measures in these patients. for non-malignant disease a second transplant may be considered for graft failure after first transplant. data regarding the outcome of a second hsct for non-malignant disease is scarce. we retrospectively analyzed patients who underwent a second hsct, for survival and toxicity data. twentynine patients (age - years) who received a second hsct at our institution during - were included in the analysis. thirteen patients had an underlying malignancy and patients were transplanted for non-malignant indications, including inborn errors of metabolism, non-malignant hematologic diseases and immune deficiency. median follow up was months (range: - ). there were deaths ( %) in the malignant group, ( %) were due to disease relapse and ( %) were transplant related. fifty percent of deaths occurred within the first year following the second hsct. in the non-malignant group there were deaths ( %), of which ( %) were attributed to the underlying disease and ( %) were transplant related. all deaths but one occurred within the first year post hsct. treatment related mortality following second hsct is higher compared to first transplant. the higher survival rate in the non-malignant group suggests that transplant following graft failure should be considered ins patients with otherwise incurable underlying disease. though the outcome for patients with relapse of malignant disease following hsct is poor, a second transplant may benefit a subset of these patients. attempts to achieve complete remission prior to transplant should be made to improve outcome. due to the small number of patients in our cohort, further multi-center trials are needed. disclosure of conflict of interest: none. disseminated bcg infection (bcg-osis) is a rare but most serious complication in vaccinized especially immunocompromised children. severe combined immunodeficiency disorder (scid) is probably the commonest primary immunodeficiency associated with bcg-osis, though there is no such definitive data as most of the cases described in literature are in the form of reports. hematopoietic stem cell transplantation (hsct) is a life-saving treatment for patients with scid, especially if therapy is instituted early, prior to onset of infections.as bcg vaccine is routinely given to all iranian children at birth, the likelihood of having an active infection at the time of transplant would be significantly high. the main objective of this study was to evaluate the outcomes of hsct in scid patients with disseminated bcg infection . sixteen scid patients underwent hsct in our center since to , of which nine patients ( male, female) were enrolled in this analysis. all the patients had received bcg vaccination according to the national vaccination protocol, and had undergone anti-tuberculosis (tb) treatment prior to transplant due to disseminated bcg infection. the mean age at hsct was . months (range: - months). patients received bone marrow (n = ), peripheral blood progenitor cells (n = ) or umbilical cord blood grafts (n = ) from hla-matched related donors (n = ) and mismatched unrelated donors (n = ). three patients received unconditioned matched sibling donor transplants and ric regimen was provided with fludarabine, melphalan and rabbit anti-thymocyte immunoglobulin (thymoglobulin) in others . cyclosporine a and prednisolon were used as graft-versus-host disease (gvhd) prophylaxis. they also continued to receive anti-tb treatment. all patients but one engrafted. the median times to neutrophil and platelet engraftments were days (range: - ), and days (range: - ), respectively. engraftment with full chimerism ( %) occurred in patients and the other patients had mixed chimerism. with a median follow-up of months (range: - months), overall survival was . %. the main cause of death was disseminated bcg infection.three out of patients who achieved engraftment, developed acute gvhd (grade i-ii), while one patient developed extensive chronic gvhd. although anti-tb treatment continued, tuberculous dactylitis occurred in patients post-hsct that were successfully treated. on last post-hsct follow-up, patients with full chimerism and with mixed chimerism are alive and disease free. scid is called as a pediatric emergency as it invariably leads to fatality in infancy without early aggressive therapy and hsct. in hsct recipients, the impaired cellular immunity renders these patients more susceptible to infection. as previous reports suggest, our study demonstrates that with appropriate anti-tb cover, immunological reconstitution with complete recovery from bcg infection can be achieved by early hsct. disclosure of conflict of interest: none. paraproteinemia occurrence after allogeneic hematopoietic stem cell transplant as a possible marker for chronic gvhd onset f monaco , s tamiazzo , f dallavalle , l calcagno , m pini and m ladetto hematology and transfusion medicine, azienda ospedaliera ss. antonio e biagio e cesare arrigo, alessandria, italy transient monoclonal gammopathy is commonly reported after solid organ or stem cells transplant (sct) for hematologic malignancies. however the clinical significance of a paraproteinemia appearance is not fully understood, because the attempts to correlate its effect on survival rates, graft versus host disease (gvhd) occurrence and viral reactivations have led to controversial results. starting from these reports we decided to evaluate among our allogeneic transplanted patients the incidence of m-component and its possible relationship with chronic gvhd. one-hundred and one patients undergoing allosct at the hematology unit of alessandria (italy) between and were evaluated. % of patients were male and % were females. pretransplantation diagnosis included: acute myeloid leukaemia/ high-risk myelodisplastic sindromes ( %), acute lymphoblastic leukaemia ( %), lymphoproliferative disorders ( %) and other less common malignancies ( %) . patients with multiple myeloma were excluded from the study. all patients had, at least, two pre-transplantation serum electrophoresis with no evidence of pre-existing monoclonal component. serum electrophoresis was scheduled to be performed at , and days and years after transplantation. forty-nine patients were submitted to allo-sct from a sibling donor and from a matched related donor (mud); in vivo t-cell depletion with anti-thymocyte globulin was used in patients. thirty-four patients relapsed after allosct, ( %) developed chronic gvhd and patients ( %) are currently alive at the last follow-up. posttransplantation follow up ranged from to days with a median of days. paraproteins were detected in out of patients ( %), being monoclonal in patients, and bi or tri-clonal in the remaining cases; the immunoglobulin subclass most commonly observed was igg. ten-year overall survival of the whole population was %; splitting the population in two cohorts (with or without paraproteinemia) we did not detect any statistical differences in overall survival, gvhd development and relapse incidence at + and + days posttransplant; viceversa, after days, a statistically significant difference was observed in chronic gvhd occurrence in patients with or without paraproteinemia ( % vs %, respectively, po . ). ten-year overall survival curves were significantly better in patients with paraproteinemia as compared with the paraprotein-free group ( % vs %, p = . ), and an even more evident significance was seen in ten-year relapse free survival curves ( % for patients with paraprotein vs % for patients without paraprotein, p = . ). monoclonal gammopathy, also in our experience, is frequent following allo-sct. we observed a strong correlation between the occurrence of paraproteinemia, chronic gvhd and a significantly better overall and relapse-free survival. recently many evidences showed that b cells are involved in the pathogenesis of chronic gvhd (cgvhd) and anti-b-cell therapy has been suggested for the treatment of cgvhd. we speculate that the presence of a monoclonal gammopathy after allogeneic transplant is expression of the activation of the b-cell compartment. a prospective study with a larger population should be considered, in order to confirm our results and assay post-transplantation monoclonal gammopathy as an early marker for gvhd development. disclosure of conflict of interest: none.inherited bone marrow failure (ibmf) syndromes are rare pediatric disorders that characteristically associate physical abnormalities, progressive bone marrow failure and predisposition to cancer. the most common of these disorders is fanconi anemia (fa). stem cell transplantation (sct) using related or unrelated donors are the only curative therapeutically approach when severe marrow failure is established. the aim of the study was to analyze the results of sct for patients with ibmfs in a single center. we performed a retrospective study in pediatric patients with ibmf admitted in pediatric hematology and bone marrow transplant department, fundeni clinical institute between january and september . diagnosis and severityof ibmfs were established based on hematological results, bone marrow biopsy and clinical findings. genetic testing for ibmfs is not currently available in our country. indication for sct was established when patients developed moderate/severe aplastic anemia and became transfusion dependent.in case of dba, sct indication was established for steroid resistant disease. the donors were selected from family members or unrelated donors, / matched.the conditioning regimens used were reduced intensity (fludarabine - mg/m , cy mg/kg, f-atg mg/kg) for af, dc and myeloablative (busulfan i.v., fludarabine mg/m , thiotepa mg/kg, f-atg mg/kg) for dba. gvhd prophylaxis consisted of standard methotrexate and csa/tacrolimus. all parents signed informed consent forms. in our center, between and , patients with ibmf were diagnosed: ( %) patients with fa, ( %) patients with diamond blackfan anemia (dba), ( %) patients with diskeratosis congenita (dc), and ( %) patients with not classifiable ibmfs. the patient data is available in table . seven out of patients ( %) performed sct procedures: sibling patients ( patients with af, patient with dc), mud patients ( patients with af, patient with dba). all patients ( %) engrafted for pmn (median = , range: - days) and platelet (median , range: - days). / ( %) presented reactivation of cmv and received valganciclovir, / developed cmv disease (encephalitis and pneumonia), / ( %) developed bkv cystitis and required extensive hydration and levofloxacin. / ( %) developed grade i-ii skin acute gvhd day + , which responded to topical treatment and low dose of corticosteroids. / ( %) developed grade iii intestinal acute gvhd, which responded to high-dose corticosteroids. / ( %) developed grade iv intestinal chronic gvhd (day + ), without response to high-dose corticosteroids, mmf and later died on day + , due to infectious complications (severe pulmonary and cerebral aspergillosis). / patients ( %) are alive, with % donor chimerism / ( %) or stable mixed chimerism / ( %). median follow-up for sct patients was days ( days- y mo). conclusions in our study we observed a low incidence of severe complications associated with low mortality rate ( %) . sct is a procedure that associates multiple risk situations, but it remains the only curative cytomegalovirus (cmv) infections remain a significant cause of morbidity and mortality in patients whose immune systems are compromised, including hematopoietic stem cell transplant (hsct) recipients. although the adoptive transfer of third party cmv-specific t cells has proven both safe and clinically beneficial in treating even drug-refractory infections/disease, broader implementation and commercialization of this strategy has been hampered by (i) the postulated need for extensive cell banks generated from donors representing diverse hla profiles, and (ii) lack of large scale t cell manufacturing processes. to address these limitations we have developed a proprietary decision tool (cytomatch™) to identify a small panel of healthy donors who should provide almost universal hla coverage; and optimized a simple, scalable manufacturing process to generate large numbers of cmv-specific t cells. to assess the robustness of our strategy we generated a bank of cmv-specific t cells (viralym-c™) from carefully selected healthy donors. the lines were polyclonal, comprising both cd + ( . ± . %) and cd + ( . ± . %) t cells, expressed central cd ro+/cd l+ ( . ± . %) and effector memory markers cd ro+/cd l-( . ± . %), and were specific for the immunodominant cmv antigens ie and pp (ie : ± ; pp ± sfc/ × , n = ). a fixed-dose ( × cells/m ) phase i clinical trial was subsequently initiated to test the safety and efficacy of these "ready to administer" t cells in pediatric and adult hsct recipients with drug-refractory cmv infections. using our bank of just lines, we have identified a suitable line for of patients screened. of these, patients have been treated with viralym-c cells; received a single infusion and patient required infusions for sustained benefit. there were no immediate infusion-related toxicities; and despite the hla disparity between the viralym-c™ lines and the patients infused, there were no cases of de novo or recurrent graft versus host disease (gvhd). based on viral load (measured by quantitative pcr) and/or symptom resolution, viralym-c cells controlled infections in all patients with complete (cr) and partial responses (pr) achieved within weeks of infusion. one patient with cmv retinitis had complete resolution of symptoms following viralym-c™ infusion. our results demonstrate the feasibility, preliminary safety and efficacy of "ready to administer" viralym-c™ cells that have been generated from a small panel of healthy, eligible cmv seropositive donors identified by our decision support tool. these data suggest that cost-effective, broadly applicable t cell anti-viral therapy may be feasible for patients following hsct and potentially other conditions. disclosure of conflict of interest: drs. juan vera, ann leen and brett giroir hold equity and drs. ifigeneia tzannou, sunitha kakarla are employed by viracyte. haploidentical stem cell transplantation (hsct) protocols utilizing ex vivo t-cell depleted grafts have been proven efficient in preventing graft versus host disease (gvhd), but cause a delay in early t-cell recovery that increases the risk of graft rejection, leukemia relapse and viral infections. conventional donor lymphocyte infusion (dli) after hsct transplantation is conditioned because of the high prevalence of gvhd even with low dose of t cells. here we present preliminary data of escalating cd ro+ memory t cells as dli in three patients that received a selective graft depleted of naïve (cd ra+) t-cells. three children that were transplanted following nonmyeloablative conditioning regimen with a graft consisting of cd + and cd ra-cells, with mixed chimerism, lymphopenic and viral/opportunistic infections and minimal residual disease positive before hsct received dose scalating cryopreserved haploidentical cd ra-memory t cell starting with a initial dose of × /kg, until a maximal dose of × /kg with a days interval. we infused products with a naïve ( ra+) t-cell dose less than × /kg with . % purity of cd + cd ro+ memory t-cells in all cases. all infusions were well tolerated without any side effect during infusions neither gvhd. following the dli, a progressive increase in t cell counts was observed. our preliminary data suggest that dose escalating of haploidentical memory t cells ( ro+) as dli provides a safety platform, even with high dose of t cells ( × /kg), for adoptive immunotherapy in haploidentical ra+ depleted grafts with no gvhd complications, and allows an increase in t cell reconstitution. however, efficacy of this strategy requires longer studies. relapse after allogeneic hematopoietic stem cell transplantation (allohsct) remains a major therapeutic challenge: outcome is very poor, without curative option in most cases. second allohsct may be considered in few selected patients because of anticipated limitations: ( ) donor availability; ( ) high toxicity due to previous treatments; ( ) low efficacy considering the very advanced disease situation. we hypothesized that the use of post transplantation cyclophosphamide (pcy) haplo-sct after relapse following allohsct may deal in part with these limitations. in particular, the presence of full haplotype hla mismatch could provide a decisive antileukemic effect relative to alloreactivity. in absence of large series in this setting, we report here the outcome after haplosct for patients who relapse after a first allohsct. we retrospectively studied adult patients, who received a second pcy haplo-sct for hematological malignancies. patients were treated between and . the objective was to assess both the feasibility and the efficacy of haplosct in this setting. twenty seven patients were included: median time between first allohsct and relapse was months (range: - ). median age at second transplantation was years old (range: - ). most of patients had acute myeloid leukemia (n = , %) or hodgkin lymphoma (n = patients, %). fifteen the impact of minimal residual disease and its kinetics prior to different types of allogeneic hematopoietic stem cell transplantation on clinical outcomes in patients with acute myeloid leukemia z xiao-su , , l yan-rong , yan-hong , p xu-ying , qian-jiang , hao-jiang , lan-ping, xu , xiao-hui zhang , , yu-wang , , h xiao-jun , and c ying-jun this study investigated the impact of minimal residual disease (mrd) and its kinetics prior to different types of allogeneic hematopoietic stem cell transplantation (hsct) on clinical outcomes in patients with acute myeloid leukemia (aml) in complete remission (n = ). patients who received unmanipulated haploidentical hsct and patients who received hla-matched sibling hsct were enrolled. mrd measured using -color flow cytometry (fcm) at fixed time points before transplantation was retrospectively analyzed. the patients were divided into four groups based on mrd kinetics before transplantation: consistent negative, positive to negative, negative to positive and consistent positive. during the follow-up, total twenty ( . %) patients underwent relapse. through unique variate analysis, none of mrd status at various time points before unmaipulated haploidentical transplantation was associated with clinical outcomes, as well as the dynamic change of mrd before hsct (p . ), although the patients with consistent positive mrd before hsct seemed to have a relatively higher incidence of relapse (p = . ). one-year cumulative incidence of relapse (cir) were . ± . % vs. . ± . % in mrd consistent negative and consistent positive groups, respectively (p = . ). however, patients with positive mrd after the second chemotherapy or pre-mrd before hla-matched sibling hsct showed a significant poor outcomes including higher cir (p = . and both neuroblastoma (nrb) and rhabdomyosarcoma (rms) in childhood are the aggressive malignant disease with higher mortality. this paper aims to study the efficacy of autologous peripheral blood stem cell transplantation (apbsct) in the treatment of high risk advanced nrb and rms. patients with high-risk stage iv nrb and patients with advanced childhood rms were treated by apbsct in our hospital from october of to may of . in the subgroup of nrb patients, patients got complete remission (cr) and patient got cru while patients had tumor residual disease after intensive induction therapy before asct. the median age was . ( - ) years old. primary sites of the tumors included submaxilla (n = ), cervical (n = ), adrenal gland (n = )and retroperitoneal (n = ). the conditioning regimen consisted of busulfan and melphalan (busulfan mg/kg × d, melphalan mg/m x d) or cem regimen (carboplatin mg/m × d, etoposide mg/m × d, cyclophosphamide mg/ m × d); the pathology of stage iii childhood rms patients was embryonal rhabdomyosarcoma. there were cases in cr and case in partial remission (pr). the median age was . ( - ) years old. primary sites of the tumors included bladder (n = ), left forearm (n = ), retroperitoneal (n = ), pelvic (n = ) s and talus (n = ). the conditioning regimen consisted of melphalan, cyclophosphamide and dactinomycin (melphalan mg/m × d, cyclophosphamide mg/m × d, dactinomycin . mg/kg × d).there were double apbsct cases (nrb n = , rms n = ). all the relapse patients were treated with chemotherapy and radiation therapy. all the patients successfully underwent mobilization, collection and reinfusion. the time of hematopoietic reconstitution was ( . ± ) days, no severe toxicity was observed, no transplant-related death was found. with a median follow-up of . ( - ) months, one of the patients was lost to follow-up. in the subgroup of nrb patients (n = ): the -year event-free survival and total survival rate of all patients were . % and . %, respectively. the survival time of no recurrence was significantly different between the double transplantation group and single transplantation group (p o . ). in the subgroup of rms patients (n = ), patient died, patients live without pd( patients had double apbsct), patients suffered recurrence but still alive. apbsct achieved good outcome in patients with high risk advanced nrb and rms. transplantation-related toxicities were tolerable. double apbsct significantly improved the depth of remission. disclosure of conflict of interest: none. transplantation outcomes of a once-daily intravenous busulfan and fludarabine conditioning for allogeneic hematopoietic stem cell transplantation in pediatric aml and high risk mds: single center experience in korea y-t lim, e-j yang and k-m park department of pediatrics, pusan national university children's hospital, yangsan, koreathere have recently been some reports suggesting that oncedaily intravenous busulfan as a conditioning regimen for hematopoietic stem cell transplantation (hsct) possibly reduces the toxicities without influencing the clinical outcome as compared with the traditional times daily dosage schedule. but until recently there has been little research and limited data available on the safety and efficacy of oncedaily intravenous busulfan and fludarabine in pediatric allogeneic hsct. we report the outcomes for allogeneic hsc recipients, evaluating engraftment status, regimen related toxicities (rrt), and event free survivals (efs) after use of oncedaily intravenous busulfan and fludarabine conditioning for allogeneic hsct in children with aml and high risk mds in a single pediatric center of korea. from january to december , aml and high risk mds children who received once daily iv busulfan/fludarabine based conditioning regimen for allogeneic hsct were reviewed, bu/flu ± atg consist of intravenous fludarabine ( mg/m ) and busulfan ( ~ mg/m , once daily iv) on days - to - , and antithymocyte globulin (atg) ( mg/kg) on days - to - . all patients received tacrolimus and mini-dose methotrexate ( mg/m( )) for graft versus host disease (gvhd) prophylaxis. boys and girls were enrolled with median age of . years (range: . - . years). the median period from diagnosis to transplantation was months (range: - months). more than half of the patients had a matched sibling donor (n = , %), % patients (n = ) had a matched unrelated donor, % patients (n = ) had a mismatched unrelated donor, and the remaining patient had a mismatched family donor. as a stem cell source, peripheral blood stem cells (pbsc) were cases ( %), bone marrow and cord blood were cases in each. the median follow-up for patients was months. the median number of infused total nucleated cells and cd + cells except cord blood transplantation were . × ( )/kg and . × ( )/kg. all patients including who received cord blood were successfully engrafted. the median time to absolute neutrophil count (anc) recovery (anc × ( )/l) and platelet recovery (platelet , × ( )/l) were days, days in each. the incidence of acute gvhd was . %, while severe grade iii/iv gvhd was observed in only patient ( . %). there were only two cases ( . %) of extensive chronic gvhd in this study. transplant-related toxicities were acceptable, there was no case with cns toxicity, eleven patients ( . %) developed grade ii,iii mucositis and grade i-iii hepatic toxicity in twenty four ( . %), but transient. there was clinically diagnosed veno-occlusive disease (vod), but most recovered by fluid restriction and diuretics. nine patients ( %) showed positive cytomegalovirus (cmv) antigen/pcr but only one patient developed cmv colitis. eight patients died: due to relapse/disease progression, due to extensive chronic gvhd. the -year efs and overall survival were . % and . % respectively. at year, the cumulative incidence of relapse was . %. overall, once-daily intravenous busulfan and fludarabine was less toxic and effective as conditioning regimen in aml and high risk mds patients undergoing allogeneic transplantation in children. disclosure of conflict of interest: none. haploidentical stem cell transplantation from unmanipulated graft has becoming a practiced option for high risk hematological malignancies who lack a matched related or unrelated donor. lack of a matched sibling or unrelated donor (mud) can be a significant barrier to allogeneic transplantation in patients who stand to benefit from this procedure. hlahaploidentical donors are readily available for nearly all such patients. haplo transplantation has inherent advantages over mud transplantation including the lower cost of graft acquisition, greater availability of donors for ethnic minorities, and immediate access to the donor in patients in whom delay cerebral palsy (cp) is a heterogeneous group of conditions that result in permanent motor disability. it may occur due to perinatal hypoxic insults, developmental brain abnormalities, genetic diseases, traumatic or infectious causes. in general the condition is non-progressive, but improvement over time is rarely seen. various treatment methods have been used for the management of this disorder. however, there has been no absolute cure for cp. the ultimate goal of stem cells therapy is to use the regenerative capacity of the stem cells causing a formation of new tissues to replace the damaged tissue. the polish stem cell bank (pbkm) has provided wharton's jellyderived msc (wj-msc) for medical therapeutic experiment application in children with cp. wj-msc from third party donors were administered to patients (pts) with cp aged from . / to . / (median age: years and month). twenty two pts have received infusions intravenously (i.v.), pt intrathecally (i.t.), and pts via both routes (first i.v., next i.t.). the cells were previously collected from healthy newborns, processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapour. msc immunophenotype was confirmed using flow cytometry assay. the pts have received from to infusions in intervals from weeks to months. median i.t. dose was × cells per infusion, while median i.v. dose was × cells/kg of body weight per infusion. each patient has been examined by the same neurologist at the day of each infusion and the result of examination has been described in a follow-up. twelve patients were diagnosed with epilepsy as comorbidity. eighteen pts ( %) showed positive changes in neurological examination after their treatment with wj-msc. almost half of the children experienced improvement of cognitive functions ( out of pts). muscle tension was reduced in pts. improvement in the ability to concentrate, better contact with others and improved social interactions were observed in % of pts. correction of motility was noticed in pts, pts have experienced better quality of sleep. in cases there has been a reduction in the number of epileptic seizures ( pt even discontinued some of his medicines). there were no s noticeable changes in neurological examination of patients. seven follow-up forms have been not received yet. the experiment data provide evidence that third-party donor wj-msc are suitable and efficient stem cells for treatment in patients with cp. however further and more extensive examination, with a greater number of patients is needed, which will be beneficial for far-reaching results. spina bifida (sb) is a congenital malformation resulting from failure of fusion in the spinal neural tube during embryogenesis. despite surgical repair of the defect, most patients who survive with spina bifida have multiple system damage due to neuron deficiency in the spinal cord. it has been confirmed that the mesenchymal stem cells (mscs) have the ability to survive, migrate and differentiate into cells of a neural lineage. wharton's jelly-derived mscs (wj-mscs) from third-party donors have high proliferation and differentiation potential along with non-immunogenic features, thus seem to be a promising stem cell source. the polish stem cell bank (pbkm) has provided wj-msc for clinical application in a medical therapeutic experiment for children with sb. eleven patients (pts) were qualified for administration of wj-mscs. three pts have been waiting so far for their therapy after bioethical committee approval. seven pts were in the middle of stem cell therapy (after or injections), pt had finished one cycle of stem cell therapy ( injections -ijs) and resumed therapy by administering a first dose of wj-mscs. the cells were previously collected from healthy newborns, processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapors. six pts have received infusions intravenously (median dose: . × /kg body weight per infusion), and pt was given injection of × cells intrathecally. each patient has been examined by the same neurologist at the day of each infusion and the result of examination has been written in a follow-up. there were pts, who received at least doses of wj-msc, and all of them showed positive changes in neurological examination. the important improvement, declared by pts, was in areas: pronunciation and/or self-reliance ( pts), movement of arms and/or legs ( pts), quality of life ( pts), core stabilization ( pt). only one adverse event occurred after third injection of wj-msc: pt had nausea and a fever. in case of other pts it was too early to provide reliable feedback. the transplantation of wj-mscs could stimulate the mscs to differentiate towards sensory neurons. this could be one of the reasons of observed improvement of many vital functions in patients, after mscs treatment. this approach might have value in the experimental treatment of sensory neuron deficiency in spina bifida.